### 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 ofLakt: Genevaphytoplankton community structure to atrazine. Arch. Hydrobiol. 145(3):277- 295. 

### (MP"tP**i.t(~1..bH63)** 

### Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study were also used to parameterize the Comprehensive Aquatic System Model (CASM) and establish the model-associatedLOC for atrazine.



### Date ofReview: December 18, 2008


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants in lake enclosures (outdoormicrocosms).


### Summary ofStudy 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 orderto 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 of1995, 1996, 1997, and 1998. The authors reference 1996 and 1998 reports that documented background atrazine concentrations of0.03 to 0.04 )..tg/L in the euphotic zone ofthe whole lake. For microcosms, phytoplankton communities were sampled from the middle ofLake Geneva, from the euphotic layer, using ahand pump to avoid cell damage. Samples were filtered through a 200 )..tm mesh andremaining zooplankton were killedby oxygen depletion via bubbling pure nitrogen through the samples for 1hour. A preliminary experiment (results not shown in the paper) showed 

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### that the oxygen depletion did not affect the phytoplankton growth. The resulting samples representedphytoplankton inocula for the microcosms.

Microcosms were 5 LPyrex bottles. They were initialized with 2.5 Lofplankton suspension (inocula) and 2.5 L ofnatural lake water from the same sampling location, that had been filtered through a 0.9 11m polycarbonate membrane filter. Nutrient concentrations were measured atthe beginning ofthe experiments. Water samples were laken lhruugh a silil;ull l;CtlJ 

### <inaglass ana silic0il tubing. The

### ii1iCtGC0SiilS 'vv'crc 

### continuously aerated to prevent sedimentation and depletion ofcarbonates. Microcosms were placed in a quiet area ofLake Geneva at adepth of1.5 m, undernatural 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 ofcontrols 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 of400 individuals per dominant species (more than 100 individuals per mL), which gave an accuracy ofabout ±

### 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. Therelative densities (percentages) ofthe inoculaalgal classes (except for Bacillariophiceae, which was separated into pennate and centric diatoms) were analyzed by correspondence analysis. The density ofeach dominant species in the contaminated communities was compared to control using the Mann-Whitney non-parametric test (also calledthe Wilcoxon rank-sum test, which tests the null hypothesis thatthe 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 overreplicate communities. Significance ofBCI differences was assessedusing 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 formertwo were part ofthe authors' existing laboratory culture collection isolated from Lake Geneva, and the lattertwo 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 ofyear and under different environmental conditions in Lake Geneva. Figure 2 shows the seasonal pattern ofSecchi 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 ofalgal 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

- inparticular, the experiment ofApril 1997 began during the first days ofzooplankton grazingjust afterthe 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 closestto the middle ofthe spring bloom, had depleted nutrients comparedto other experiments. June 1995 andJune 1996 experiments started during the "clearwater" phase. The inocula communities for these experiments had low biomass and were dominated by Chlorophyceae 

### (Chlorella vulgaris, Elakatothrixgelatinosa, and some

### Eudorina elegans) and cryptophytes. Silica concentrations were higher in these experiments because ofnutrientrecycling inducedby intense grazing. June 1997 and July 1995 experiments started atthe end ofthe clear waterphase, 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 clearwater experiments. August and Septemberexperiments 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 summerand early autumn experiments. lrradiance was highest during the summer and lowest during the fall, with intermediate irradiance in the spring.

3The 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 ofincreased 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 significanttreatment 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 ofthe results is provided in the original article but is not reproduced here.
Beginning 

### vs. end ofexperimentbiomass 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.)

43 to 5 replicates pertreatment (depending on experiment) 

### quietzone of lake 

### Geneva

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Figure 1. Design ofthe outdoor microcosms in Lake Geneva.



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### & Figure 2. Secchi transparency measured in Lake Geneva by lnstitut National de Recherche Agronomique, Station d'Hydrobiologie Lacustre. Starting dates ofthe twelve microcosm experiments shown by arrows.

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Figure 3. Correspondence analysis ofinoculum characteristics ofthe 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.

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Figure 4. Bray-Curtis indices ofthe 12 microcosm experiments. The Bray-Curtis index was calculated for the mean density ofreplicate communities, and the significance ofdifference between control and treatmentwas 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 testwas 95%. *: C-C/C-A Mann-Whitney test was 90%.

8Table 1. Biomass and diversity ofthe 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 ofmicrocosm cultures. Duration ofeach 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



### 9Table 2. Response ofthe 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 ofeach experiment: +: greater density ofindividuals than in controls; 0: no significant effect; -: lower density ofindividuals 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%) Mougcotiagracillima 0 0 0 () +(89%)

-_._----

10Table 3. Biomass (chlorophyll a concentration) and diversity (Shannon diversity index in bits per individual) calculated atthe end ofeach ofthe 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 DidRecovery Comments of

Effect 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=NotReported



### Reviewer Comments: Some aspects ofinterpretation were made difficult by differences in methods and presentations usedby 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 difficultto 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 ofresults. Some species, which showed no reaction to atrazine in the laboratory, were stimulated by indirect effects ofatrazine in situ, namely the unequal stress the contaminantplaced on their competitors. ChIarella vulgaris, 

### on the otherhand, almost always significantly declined with atrazine treatment, and responded similarly in the laboratory.

12

### The microcosms were affected by artifacts ofthe sealedbottles, as the authors note. In the bottles, diversity tends to collapse overtime. Also, they note that itwas difficultto take uniform samples, particularly for filamentous orcolony-forming species.
Description ofUse in Document (QUAL, QUAN, INV): QUAL

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. 

### atrazine on aquatic plantcommunities.

Limitations ofStudy: Study effects were difficultto quantify forthe Data Summary Table due to the wide range ofeffects observed in the different experiments. The study design was scientifically sound.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc
SecondaryReviewer: Brian Anderson, U.S. EPA

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