Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Seguin, F., C. Leboulanger, F. Rimet, J.C. Druart, and A. Berard. 2001. Effects ofatrazine and nicosulfuron onphytoplankton in systems ofincreasing complexity. Archives ofEnvironmental Contamination and Toxicology. 40(2): 198-208. (MRID 48261134)
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 plantcommunities and associated determination ofa Level ofConcern (LOC).

Date ofReview: September24, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine and nicosulfuron to aquatic plants (phytoplankton) in single-species microplates, indoor microcosms, outdoor enclosure microcosms, and experimental ponds (mesocosms).
Summary ofStudy Findings: This review focuses on the effects ofatrazine (nicosulfuron was notreviewed).

Forthe outdoormicrocosm experiments, several otherreviewed studies by the same authors reported datafrom the same setofexperiments. 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, the statistical analyses, and so on. The specific differences among all the studies are too numerous to adequately summarize inthe DER ofeach study, therefore the reader is referred to the individual DERS. The studies related to this review are Berard et aI., 1999a, Berard et aI., 1999b, Berard and Benninghoff, 2001, and Leboulanger et aI., 2001.

- 1 - 

# '1.1.11111 2087837Methods: 

### Four distinct experiments are described in the study. Methods and results are reviewedprimarily for the microcosm and mesocosm experiments, with the single-species tests summarized in brief.


### Single-Species Tests Several phytoplankton species were cultivated in clearpolystyrene microplates under 12: 12 light:dark cycles with approximately 100-150**~E/mz** 

### Is light intensity atthe lid. Growth was monitored daily with a Dynex MRX CE97 spectrophotometerto measure OD65o
. 

### Cultures in the exponential growthphase were diluted (106 

### cells/ml) and then grown for atleast one week in the microplate before treatment. Atrazine (98%, Greyhound Service, UK) was prepared inpure acetone, with a final concentration ofacetone of0.05% in all treatments and controls. Six replicates were used at each of 10 atrazine treatment levels (0**~g/L** 

### to 256**~g/L,** 

### exacttreatment levels between were notreported). The cell density after 96 hours ofgrowth (posttreatment) was used to calculate the EC50 value. Sigmoid dose-response curves were fitted to the data using the least-square method.

Indoor

### Microcosms Glass bottles (500 mL) were filled completely with plankton-containing natural water collected from the light-saturated layer ofLake Genevain December 1999. Grazers and large particles were removed bypassing the waterthrough a**250-~m** 

### net and nitrogen was bubbledthrough to kill any remaining heterotrophs. The systems were kept in controlled chambers with a 12:12 light:dark cycle (200**~E/mz/s)** 

### and constanttemperature of12°C. Samples were enriched with soluble P, N, and Si to achieve nutrient concentrations of0.02 mg P/L (soluble reactive phosphorus), 0.60 mg N/L (inorganic nitrogen), and 1.3 mg SiOz/L (silicates). These conditions were meant to simulate the conditions ofearly spring in the lake. Formore description ofthe protocol, the study cites Berard (1996, Effects offour organic solvents on natural phytoplankton assemblages, consequences for ecotoxicological experiments onherbicides, Bull. Environ. Contam. Toxicol. 57:183-190). Before the atrazine treatment, the bottles were sampled for cell identification and counting. Five bottles were solvent controls (solvent and concentrationnot provided) and nine bottles eachreceived a different atrazine treatmentranging from nominal concentrations of0.1**~g1L** 

### to 100**~g1L** 

### (otherthan 50**~g1L,** 

### the six otherintermediate nominal concentrations were notprovided). It 

### is not stated whetherthe atrazine product and solventused in the single-species tests was also used for this experiment. Phytoplankton growth was measured daily as in vivo fluorescence, Taxonomy and chlorophyll a were measured atthe end ofthe experiments (experiment duration not described).

Outdoor 

### (Enclosure) Microcosms A series ofsix outdoor experiments were performed during the spring and summer of 1997 and 1998. Samples were collected in the center ofLake Geneva. Previously publishedbackground - 2 -atrazine concentrations were 0.03-0.04 

### /lg/L in the euphotic zone ofthe whole lake for the period from 1995 to 1997. In each experiment, 5 Lbottles were filled with 2.5 L oflake water as nutrient stock and 2.5 L ofnatural phytoplankton sample (not described how these were obtained orprocessed), then treated as described for the indoormicrocosms to remove heterotrophs. The bottles were sampledpriorto treatmentto measure nutrients, cell counts, chlorophyll 

### a, and cell types (Table 1 and Figure 1). Five bottles were treated with 10 

### /lg/L atrazine, and five bottles were treated with solventblanks (controls). It is not stated whetherthe atrazine product and solventused inthe single-species tests was also used for this experiment. The bottles were placed 1 m belowthe lake surface nearthe shore. They were monitored daily for in vivo fluorescence and final taxonomy and biomass were determined atthe end ofeach experiment (exposure duration varied by sampling date from 10 to 21 days, see Table 1).



### Mesocosms The mesocosm experimentwas conducted in circular outdoortanks atthe ENSAR campus (Ecole Nationale Superieure d'Agronomie de Rennes, France). Each tank was 3.2 m in diameter, 1.2 m high, and held about 5,000 L, with a funnel-shaped base. The tanks were sunk about 0.4 m into the ground to obtain stable temperatures. Nutrientconcentrations inthe mesocosms were 0.0065 mg 

### P/L orthophosphate and 0.51 mg

### NIL inorganic nitrogen (not stated whenthese measurements were taken orhow they were averaged). Temperatures in the mesocosms ranged from 14.4°C to 24.8°C with an average of18°C (not stated when measurements were taken). The mesocosms were filled with 5 cm ofnatural sediment from a local uncontaminated pond. The tanks were filled with a mixture oftap water and water from a nearby pond at a depth of0.7 m. The pond water containedphytoplankton and zooplankton (not stated ifthis pondhad atrazine contamination). Many methodological details were notprovided, for example regarding homogenization, transport and storage, physical and biological characteristics, precise pond and tap watermixture proportions, and tap waterquality. Aquatic macrophytes (Glyceria maxima) were introduced in two concentric circles, with identical numbers ofGlyceria in each mesocosm, and were meantto mimic stillwaterfish spawning habitat.

The mesocosms were allowed to develop for 3 weeks before atrazine treatment. Atrazine was dissolved with acetonitride, which was believedto be less toxic than acetone. Solvent concentration inthe tanks was not provided. Three replicates were treated at eachtreatment level: 0 /lg/L (solvent control), 2 

### /lgIL, and 30

### /lg/L atrazine. Phytoplankton was sampled with a homemade sampler. Samples were obtained from several points in each experimental unit and were pooled for analysis. One liter ofsample was filtered through a Whatman GFICfilter for chlorophyll 

### a determination. Chlorophyll was extractedby incubation for 24 h at 4°C in 90% acetone, and then chlorophyll 

### a was measured spectrophotometrically

Effects ofatrazine onphotosynthetic efficiency was assessed by calculating 

### P/I curve parameters in one control mesocosm and one 30

### /lg/L treatmentmesocosm, using the method ofLewis and Smith (1980). Thethree parameters, a,**~,** and Pmax, were calculated priorto treatment, immediately aftertreatment, and on day 11 aftertreatment.

- 3 -

### Species Composition andStatistics Samples were taken pre-treatment, during exposure, and atthe end ofthe microcosm and mesocosm experiments to identify and countphytoplankton cells. A 50 mL aliquot of'algal suspension' was taken from eachtank. Lugol was added to kill and stain cells. After cells were allowed to settle for 24 h, they were examinedunder areverse phase microscope for identification and enumeration. The abundance ofthe dominant species was estimated ±10%. The density ofeach dominant species (definedby study authors as 

### >100 individuals/mL) in the treatment groups was comparedto that ofthe controls using the Mann-Whitney test (p-value not reported). Compositional differences betweentreatment and control communities were described using the Bray-Curtis dissimilarity index (BCl). BCls were calculated for the mean densities inreplicate communities, and significant differences in community composition between atrazine treatment andthe control were assessed by applying the Mann-Whitney testto the within-control BCl versus the control-treatmentBCl. Forthe indoormicrocosm study, a correspondence analysis betweentreatment levels and the controls was performed with the ADE program for multivariate analysis, with a data matrix defined by the total density ofthe dominant species. The correspondence analysis was not adequately explained, it is unclearhow the control communities were included in the analysis, and nicosulfuron treatments were included inthe analysis.

- 4 -Table L Biomass and diversity ofthe inoculum used in the outdoor microcosm experiments, initial nutrient concentrations, temperature and surface irradiance throughout the experiments, and study duration (study table 1)
June '97 Aug. '97 April '98 May '98 June '98 July '98 Sept. '98 Chlorophyll a (JigIL) 1.15 2.05 1.75 3.23 1.57 2.01 6.83 H' (bits/individual) 2.2 1.80 1.40 1.14 2.11 2.94 1.9 SRP (mg P/L) 0.003 0.001 0.018 0.003 0.001 0.001 0.000 DIN (mg N/L) 0.38 0.207 0.55 0.42 0.45 0.037 0.156 Silica (mg Si02/L) 0.70 0.1 1.08 0.12 0.28 0.54 0.14 Mean temperature (0C) 19.3 23.9 9.3 14.2 17.5 22.0 17.4 Minimum 18.1 21.2 4.4 10.8 15.2 18.8 15.3 Maximum 20.5 25.2 12.4 20.7 20.6 24.1 20.7 Mean surface 1,038 883 616 1,242 866 751 546 irradiance (Jimol/m2/s) Minimum 466 305 70 863 254 107 223 Maximum 1,485 1,149 892 1,505 1,456 1,274 906 Duration 11 14 10 21 13 16 16 Notes: H' = Shannon diversity index; SRP = soluble reactive phosphorus

- 5 -Xanlhoph)'ccae Zygnematophyccae Chlorophyceae: BaciIJariophyceae(penn..tcs) Bacillariophyceae(centrales) Cluysophyceae Cryptopbyc:eae Dinophyuae Cyanobacteria

Figure 1. Class-level composition ofalgal communities in outdoor microcosms before treatment in each experiment, expressed as % ofthe total phytoplankton identified. (study figure 1)



### Results:


### Single-Species Tests EC50 values determined by the single-species tests are presented in Table 2. According to the study authors, the results were similarto previously published EC50 values. In general, diatoms were less sensitive than chlorophytes. The most sensitive species tested was Chlorella vulgaris 

### (strain isolated from Lake Geneva; EC50 = 

### 4.3 Ilg/L) and the least sensitive species tested was Nitszchia 

### sp. (strain isolated from Lake Geneva; EC50 = 

### 412Ilg/L).

IndoorMicrocosms The microcosmtreated with 50 llg/L atrazine was lost during the experiment. According to the study authors, no concentration-related differences in in vivo 

### fluorescence or chlorophyll a 

### parameters were seen atthe end ofthe experiment (datanot shown). The correspondence analysis showed some variation in community structure with atrazine treatment level (Figure 2).

- 6 -OutdoorMicrocosms Depending on the experiment, some significant differences in chlorophyll biomass (as measured by in vivo fluorescence) were seen inthe 10 Ilg/L atrazine treatmentmicrocosms. Depending on the experiment, chlorophyll inthe atrazine treatment increased, decreased, orwas not significantly different from the controls (Table 3). Community composition ofthe atrazine treated communities, as assessed by the Bray-Curtis index (reported as similarity according to the study authors), was significantly differentthan the control communities for two ofthe experimental dates (Table 3). Forthe abundance ofindividual species (those thatthe study authors defined as "dominant", i.e. 

### > 100 cells/ml), most were not affected by atrazine treatment**(~52),** some were stimulated**(~22),** and some were inhibited**(~12)** comparedto the controls at the end ofthe experiment 

### (Figure 3).

Mesocosms Phytoplankton biomass was low in all mesocosms (maximum of4.5 Ilg/L after 17 days), with no differences among controls and treatments according to the study authors 

### (Figure 4). Succession occurred during the experimentin all mesocosms 

### (Figure 5), the reviewer assumes the average ofall mesocosms is plotted). According to the study authors, succession was similar in treated and control tanks with some difference intiming and proportion. The chlorophyte 

### C. vulgaris dominated the initial assemblages but decreased overtime. Overthe experiment, the cryptophytes Rhodomonas minuta and Cryptomonas ovata increased as did total chlorophyll a. By the end ofthe experiment, the chrysophyte Ochromonas sp. was increasing. Datawas not provided for the specific species.
Bray-Curtis indices between treatments and controls showed a dose effect according to the study authors, with indices generally smallerfor the higher atrazine concentration (30 Ilg/L). BCI is reported as similarity according to the study authors. The differences were significant (MannWhitney test; p-value notprovided) on the following days and treatment levels: on days 2 and 7 post-treatment at 2 Ilg/L atrazine, and on 2, 10, 17, 23, and 27 post-treatmentat 30 Ilg/L atrazine 

### (Figure 6).

Atrazine treatment mesocosms had significant differences from controls inthe density ofmajor taxonomic groups (Table 4). Significantdifferences occurred on different days for differenttaxa and differenttreatment levels. The hightreatment level (30 Ilg atrazine /L) showed more significantdifferences from the control thanthe low treatment level (2 Ilg atrazine/L). Cryptophytes were the only taxonomic group that did not differ from controls at either treatment level.

P/I curve parameters are provided in 

### Table 5. Pmax was lowerin the atrazine treatment mesocosmthan inthe control on day 1 and higher on day 10. The otherparameters showedthe same trend.

- 7 -Table 2. Calculated EC50 and LOECs for atrazine from 96-hour microplate assays for each strain tested. (from study table 3) Class Strain EC50 (llg/L atrazine) LOEC (llglL atrazine) C. vulgaris Leman 4.3 3 C. vulgaris Villaumur 172 128 Chlorophyceae P. subcapitata 118 64 S. acutus 45 32 A·formosa 261 10 N. accomoda 164 50 Bacillariophyceae Nitszchia sp. 412 50**~N~tzschia** sp "t-avieu1a sp

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# !_-~~,------j--_J Figure 2. Correspondence analysis ofphytoplankton communities at the end ofthe indoor microcosm experiment. Points labeled A<#> represent atrazine treatments, Al =0.1 IlglL though A9 = 100 IlgIL and N<#> represent nicosulfuron treatments, Nl =0.1 IlglL though N9 = 100 IlglL, and T was not defined. (study figure 2)
- 8 -Table 3. Atrazine effect on chlorophyll biomass (in vivo fluorescence) and Bray-Curtis indices during the 7 outdoor microcosm experiments. +: Treatments significantly higher in biomass than controls during 2 or more days (Student's T test with 5 replicates, 95% confidence); -: Treatments significantly lower in biomass than controls during 2 or more days (Student's T test with 5 replicates, 95% confidence). *: Bray-Curtis index calculated between controls and treatments significantly different from within-controls BCI (MannWhitney test, no p-value or confidence provided). (study table 4) 

### Experiment Atrazine Effect Bel 

### (controls/atrazine) June '97 

### + 0.61 

## * August '97 

### + 0.95 April '98

- 

### 0.86 May '98 0 0.93 June '98 0 0.86 July '98 0 0.89 

## * September '98

- 

### 0.81

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### !! 60 .! J so

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inhibition Figure 3. Response ofdominant phytoplankton species to 10 JiglL atrazine (Mann-Whitney tests for the seven experiments, p-value not reported) measured at the end ofeach outdoor microcosm experiment. (study figure 3)

- 9 -4.S 4.0 .3.5 ,;;- 3.0. 

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### ......c**~a** -+-A

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Figure 4. Chlorophyll a content ofthe mesocosms. C: control, a: 2JigIL atrazine, A: 30 JiglL atrazine, n: 2JigIL nicosulfuron, N: 30 JiglL nicosulfuron. (study figure 4)**Chlult~h)** 1.'1:,1

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.....**"\~** u (>11% 1(10%

Figure 5. Class-level taxonomic composition ofalgal communities in mesocosms at the different sampling periods, expressed in % oftotal phytoplankton identified. The study reviewer assumes composition represents average ofall cosms. (study figure 5)

Table 4. Significant responses ofdominant taxonomic groups to atrazine treatment in mesocosms (MannWhitney test on each sampling day; no p-value provided). <: decreased density relative to controls; >: increased density relative to controls; 0: no significant effect. (study table 5)
Class 2J1g/L 30 Jig/L Bacillariophyceae 0 > days 2,10 Cryptophyceae 0 0 Chlorophyceae < day 17 < day 17,23 Chrysophyceae > day 40 > day 40

- 10-Table 5. Parameters ofthe P/I curves obtained in one control and one atrazine treatment mesocosm (30 flglL). Units are: Pm =flmol COz/mg chi a/h, u=flmol C/mg chi a/h/(flmol photons/mz/s), J3 = same as u. (study table 6)

Day Type Pm u B -1 (pre-treatment) Start 274 1.722 -0.081 Day 1 Control 392 2.201 0 Atrazine (30 IlgIL) 292 1.807 -0.028 Day 10 Control 184 2.193 -0.173 Atrazine (30 Ilg/L) 194 2.901 -0.140

- 11

-1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00

1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 

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Fig. 6. Bray-Curtis indices ofthe differentphytoplankton samples in the mesocosms. The Bel 

### were calculated for the mean density of replicate communities. and 

### the significance of difference between control and 

### treated was tested with 

### a Mann-Whitney test between indices calculated among 

### controls (C-C) and indices calculated be

### tween controls and 

### tests (C-A).*: (C-AlC·C) 

### Mann-Whitney 

### test (90 

### %) (study figure 6)

- 12

-Data Summary Table:
Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Atrazine Atrazine Occur-if Effect Effectrelative Concentration Effect Concentration so, when Comments to controls (pg/L) Occurred from Day Oto (see When Effect comment)? Occurred
0.1 Ilg/L Indoor microcosm experiments. 6 other intermediate Study authors concentrations, statementthat not defined there was no effect Change in (no data or phytoplankton statistics biomass (in vivo No effect Not applicable Not applicable Not provided), no fluorescence and applicable replication, chlorophyll a unknown parameters) intermediate 100ilgIL concentrations except 50 Ilg/L treatment which was lost during the experiment, study duration unknown

- 13

-Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Effect Effectrelative Atrazine Effect Atrazine Occur- if Comments Concentration Concentration so, when to controls (pg/L) Occurred from Day Oto (see When Effect comment)? Occurred
Outdoor microcosms Response varied Increases over controls seen in (increase, June and August decrease, and no effect) among 7 1997 (on 2 ormore outdoor days), decreases in Change in microcosm Unknown April and Sept. phytoplankton experiments 10 IlgIL Unknown (not Unknown (no (date effects 1998 (on two or biomass (in vivo occurring in provided) measurements) observed not more days), no fluorescence) different years reported) effect in May, and seasons June, July 1998. (magnitude of Differences effect significantby unknown). Student's ttest, 5 replicates, 95% confidence (table 3)

.



### • Outdoor microcosms Response varied BCI ofcontrols among 7 significantly outdoor different from BCI Change in microcosm calculatedbetween phytoplankton experiments Unknown controls and community occurring in Unknown (not Unknown (no (date effects treatments by composition different years 10 Ilg/L provided) measurements) observed not Mann-Whitney and seasons (Bray-Curtis (magnitude of reported) test (no p-value index!) provided) for June effectunknown 1997 (BCI = 0.61) as within- and July 1998 control BCI not (BCI = 0.89). No provided) within-control BCls provided (table 3).

- 14

-Time Weighted Did Nominal Day of Study Average Recovery Magnitude of Atrazine Atrazine Occur- 

### if Comments Effect Effectrelative Concentration Effect Concentration so, when to controls (pg/L) Occurred fromDayOto (see When Effect comment)? Occurred
Outdoor microcosms Differences based on Mann-Whitney Pooled data test (no p-value Varied (-12 from end of provided) species experiments No (effects Change in species decreased, -22 (varied from Unknown (no observed at abundance species 10 !lg/L 10 to 21 days measurements) end of Number ofspecies increased,

- 52 for different experiments) estimated from species no experimental Figure 3. effect) dates)
Based on 'dominant species, i.e., >100 cells/ml)
2 !lg/L Mesocosm experiments (Figure 4).
Change in Not Study authors state phytoplankton No effect Not applicable Not applicable applicable thatthere was no biomass 30 !lgIL statistical (chlorophyll a) difference between treatments and controIs (statistical test and p-value not provided)
Change in Unknown (within-control Day 2 Yes-Day Results for phytoplankton BCI not 2 !lg/L 10 community mesocosms composition provided) (Figure 6) (Bray-Curtis Differences are index calculated Unknown (no significantby between measurements) Mann-Whitney Unknown treatments and (within-control test (no p-values controls 30 !lg/L Day 2 No provided). BCI not compared to BCI provided) Based on calculated within- controls) I statements by the study authors, the

- 15

-Time Weighted Did Nominal Day of Study Average Recovery Magnitude of Atrazine Atrazine Occur- 

### if Comments Effect Effectrelative Concentration Effect Concentration so, when to controls (I'g/L) Occurred from Day Oto (see When Effect comment)? Occurred
reviewer assumes that BCI is expressed as similarity on the yaxis in Figure 6. Despite the significant difference between treatment/control BCI and withincontrol BCI, the effects for the 2 Jlg atrazine/L do not appear to be treatmentrelated. This is because the treatment!controI BCI on d+2 and d+7 is nearly the same as that prior to treatment (d-l). The significant difference on these dates may be due to increased similarity among control communities.
Not No effect 2 Jlg/L Not applicable applicable Mesocosm study Change in Increase Unknown (no (table 4) Bacillariophyceae (unknown measurements) Not Significant density magnitude) 30 Jlg/L Day 2 and 10 applicable differences detected with Transient effect Mann-Whitney (see comment) test (no p-values 2Jlg/L provided). Change in Unknown (no Not No density values Cryptophyceae No effect Not applicable measurements) applicable are provided. density 30 Jlg/L

- 16

-Time Weighted Did Nominal Day of Study Average Recovery Magnitude of Atrazine Atrazine Occur-if Effect Effectrelative Effect Concentration so, when Comments Concentration to controls (",giL) Occurred from Day Oto (see When Effect comment)? Occurred
Decrease With the limited (unknown data provided itis Not difficultto assess magnitude) 2 !1g/L Day 17 applicable the likelihood that Change in Transienteffect Unknown (no these are treatment Chlorophyceae (see comment) measurements) related effects. density Given available Decrease Yes

- by information, the (unknown 30 !1g/L Day 17,23 day 27 reviewerjudges magnitude) that at least some ofthe effects were Increase likely transient and (unknown 2!1g!L Day 40 nottreatment magnitude) related due to No (effects temporal Change in Unknown (no observed at variability or when Chrysophyceae measurements) end of an effect occurred density Increase experiment) on only a single (unknown 30 !1g!L Day 40 day during the magnitude) experiment.

Mesocosm Decreased experiment (table 5) maximum Unknown (no Yes- by photosynthetic 25% decrease 30 !1g!L Day 1 measurements) Day 10 1 replicate only, efficiency (P/I measurements curve) onlytaken on days 1 and 10.

1 It was not clearwhat level oftaxonomic resolution was used to calculate BCI.

- 17

-Reviewer Comments:

The study has a broad focus, with arange ofexperiments examining effects ofboth atrazine and nicosulfuron. Due to the broad scope, some data and methodological details ofinterest for this review were notprovided. The data summary table for this study has many gaps because ofthe overall lack ofinformation.

The indoor microcosm experiments had no replication, intermediate nominal concentrations of atrazine treatments were notprovided, and the duration ofthe experiment was not stated. The outdoormicrocosms had strong replication (5 replicates), but only one treatment level (lO flg/L). The small size ofthe containers used for the microcosm experiments may have introduced container edge effects. A complete description ofinstallation ofthe indoor and outdoor microcosms priorto use was referenced (Berard et aI., 1996 and Berard et aI., 1999a, respectively), but not presented in this article. Any acclimation ofthe microcosms priorto treatment was notreported. Microcosms did not contain a sediment layer. Waterused for the microcosm experiments was from Lake Geneva which has known contamination with atrazine. Microcosm experiments used water collected from Lake Geneva at different times ofthe year, thus any background contamination may have varied among the experiments.

The construction material ofthe mesocosms is notprovided, nor is the location and environment ofthe pond orponds from which sediment and water were sampledto constructthe mesocosms. It is stated that a "mixture" ofpond and tap water was used to fill the mesocosms, with no proportions provided or discussion ofhow the mixing was performed. It is unknown ifwater used for the mesocosms was contaminated with atrazine.

Waternutrient levels and temperatures are cited without description ofwhen and where the measurements were taken, or what averaging was used.

It is unclear whetherthe same grade and brand ofatrazine was used in all the experiments; it is only specified for the single-species microplate tests.

The p-value is notreported for many statistical tests and in some cases the statistical test which was used is notreported.

Actual atrazine concentrations are unknown (not measured) and controls were not measured for contamination with atrazine. Also, the mesocosms as well as both types ofmicrocosms may have been affected by experimental artifacts, including enclosure effects, lowbiomass and nutrient levels, and (particularly in the mesocosms) strong successional dynamics that may have confounded effects ofatrazine. Only 3 weeks were allowed for colonizationin the mesocosms,

- 18

-which may have been insufficient for attaining areasonably steady-state system priorto treatment.

Limitedresults were presented for many ofthe endpoints.

The study authors stated thatthe Bray-Curtis dissimilarity index was used, but its implementation was not well-described, which may leadto some misinterpretation. Since BCI is never defined with an equation, it is unknown exactly which version ofit is being used, what level oftaxa are being assessed, and so on. In some cases, the study authors referto the BCI as a similarity index orjust as an index; therefore, it is unclearwhethervalues presented inthe paper represent dissimilarity or similarity (presumably I-dissimilarity). For example, the study authors state that a dose effect on BCI was found in the mesocosm experiment, and thatthe indices were lower withhighertreatment; ifthe index is dissimilarity as stated inthe methods, than this would seem to indicate greater similarity betweentreatment and control athighertreatment levels. This appears to be a counterintuitive result. The reviewer assumes thatBCI indices presentedby the study authors are expressed as similarity; however neitherthe method northe results are discussed in enough detail to be certainhow BCI results shouldbe interpreted.

The PII analysis was based on only one treatment mesocosm and one control mesocosm (no replication).

The correspondence analysis (indoormicrocosm experiment) was based on one replicate at each concentration. Atrazine-only effects are confounded by inclusion ofnicosulfurontreatments in the analysis. It is not clearhowthe control data was used in this analysis. Because ofthese issues, there is no basis determining treatmentrelated effects

.. 

### Description ofUse 

### in Document: Qualitative (mesocosm experiments expectphotosynthetic activity, outdoormicrocosms), 

### Invalid (indoormicrocosm, photosynthetic activity for mesocosm experiments)



### Rationale for Use: Qualitative

- Mesocosm experiments and outdoormicrocosm experiments contribute to the weight ofevidence regarding effects ofatrazine on aquatic plantcommunities.

Invalid

- For indoormicrocosm experiments the duration ofexposure is unknown, there was no replication oftreatments, and six ofthe nine treatment doses are unknown. Forthe mesocosm experiments, there was no replication for photosynthetic activity measurements.

- 19

-Limitations ofStudy: 

### All experiments lack complete description ofthe methods, atrazine concentrationmeasurements, p-values for "significant" statistical results, andpresentation of sufficient data for many endpoints. The mesocosm experimentresults may have been affected by strong successional dynamics (perhaps due to insufficient acclimation time priorto treatment). Solvent concentration was not stated for the mesocosm experiment. No replication was used for photosynthetic activity measurements (mesocosms). The indoor and outdoor microcosm experiments may be affected by bottle effects. Water used for the microcosm experiments was from Lake Genevawhich has documented atrazine contamination. It 

### is unknown ifthe pond waterused for the mesocosm experiments was contaminatedwith atrazine. Solvent and the concentrationused were not stated for indoor oroutdoor microcosm experiments. The indoormicrocosm experimenthad no replication, failed to state all the nominal concentrations used for the atrazine treatments, and the duration ofexposure was not stated.
Primary Reviewer: 

### Sara H. Hendrix, M.S., Subcontracting Scientist, Cambridge Environmental Inc.

-1· " Secondary Reviewer: 

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

# /;J1f,{;-k/c,~.M/7f Tertiary Reviewer: 

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

### I1.tt~**~(t3/11**

- 20-