### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Leboulanger, 

## c., F. Rimet, M. Heme de Lacotte and A. Berard. 2001. Effects ofatrazine and nicosulfuron on freshwater microalgae. Environment International. 26: 131-135. (MRID 48261121)


### 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 ofa Level ofConcern (LOC).



### Date ofReview: 9/8/09


### Purpose ofStudy: This study examines the potential effects ofatrazine and nicosulfuron onphytoplankton communities in experimental outdoormicrocosms and in vitro 

### monocultures.


### Summary ofStudy Findings: The monoculture experiments will be mentioned, although in limited detail, in orderto focus on the community microcosm experiments. The review focuses on atrazine treatments (nicosulfuron was notreviewed).

Several other studies by the same authors reported data from the same set ofexperiments. Among those studies there is both overlap and difference interms ofthe experimental dates. The studies relatedto this review are Berard et aI., 1999a; Berard etaI., 1999b; Berard and Benninghoff, 2001; and Seguin et aI., 2001b. Among those studies, there is both overlap and difference in the 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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# 11111111111 2087865Methods: Two types ofexperiments were conducted: 1) 

### in vitro monocultures offour different strains of freshwaterphytoplankton and 2) natural phytoplankton (mixed species) communities in outdoor microcosms.



### In vitro monocultures. Four strains offreshwater phytoplankton were utilized (Table 1). The cyanobacteria, chlorophyte, and diatom strains were cultured in Z, L-C and DV media, respectively, in 250-mL borosilicate, glass Erlenmeyer flasks. Initial biomass at inoculation was not reported. Cultures were maintained at 20 ± 1DC on a 16:8 hour light:dark cycle with irradiance at 60 f.!mol/m2/s. Details were not reported regarding purity of the atrazine test compound, test concentrations utilized, number of treated replicates, and the inclusion of untreated controls.

Growth was monitored daily for 11 days via optical density at 650 nm (OD65o) after standardization to chlorophyll 

### a content and cell density for each species. Growth rates were estimatedusing the logistic growthrate equation ofHutchinson (1978).

For C. 

### vulgaris and N. 

### accomoda, the effect ofatrazine (10 f.!g/L) onphotosynthetic capacity was assessed using the 14C incorporation technique ofLewis and Smith (1983). The phytoplankton were acclimated at low light intensity (30 f.!mol/m2/s), with photosynthetic capacity measured for 30 minutes at 0 to**~500** f.!mol/m2/s (Figure 1). Values for a, 

## pand Proax were determined using the spreadsheets of Walsby (1997). No additional details regarding measurement of photosynthetic capacity were provided.

Natural phytoplankton communities in outdoormicrocosms. One experimentwas conducted in June 1997; specific dates were notreported. Source waterwas obtained from Lake Geneva. Each microcosm consisted ofa 5-L bottle containing 2.5 L oflake water with phytoplanktonplus 2.5 L offiltered (0.8-f.!m) lake water. Outdoor installation ofthe microcosms was described elsewhere (Berard 

### etal., 1999a). Any acclimation ofthe microcosms prior to use was not reported. Five microcosms were treated with atrazine (purity and product notreported) at 10 f.!g/L, with an additional five microcosms maintained as untreated controls. Use ofa solventwas notreported but a solvent was not used in Berard 

### etal., 1999a. Phytoplankton growth was monitored daily, for 11 days, via chlorophyll-a fluorescence measurements. Irradiance above the water surface and watertemperature at I-m depth in the lake were monitored; intervals and methods were not specified.

Water samples (50 mL) were taken from each microcosm at the beginning and the end ofthe experiments (day 11). Samples were treated with Lugol's iodine to kill and stain the cells, then cells were allowed to settle for 24 hours. Phytoplankton community composition (taxonomy and abundance) were determined by counting dominant species (counting accuracy ± 10%) using a
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### reverse-phase microscope. A definition of"dominant species" was notprovided, however, in otherpapers by the same authors ithas been defined as >100 individuals/ml.
Comparisons ofthe density ofeach dominant species and overall community composition betweenthe treated and control microcosms were analyzed using the Mann-Whitney nonparametric test (no p-values provided) and correspondence analysis (ADE-4 software), respectively. Chlorophyll a 

### concentrations were compared with the Student's test (p=0.05).

Results: In vitro 

### monocultures. For the atrazine treated cultures, calculated EC50 values ranged from 42 to 104 Ilg/L and the NOECs were <10 Ilg/L for all four strains (Table 

### 2).

In the presence of atrazine (10 Ilg/L), a and 

## Pparameters for photosynthetic capacity of 

### C. vulgaris 

### were significantly decreased (p 

### value not reported), as compared to the control, but Pmax 

### was unaffected (Figure 1). 

### Photosynthetic activity ofN. accomoda 

### in atrazine treated cultures was not affected underthe conditions ofthe experiment (30 minutes, 0to**~500** 

### Ilmol/m2/s).

Natural phytoplankton in outdoormicrocosms. During the experiment, watertemperatures (l-m depth) ranged from 16.5°C to 20°C and solarirradiance above the water surface ranged from 475 to 2,750 J/cm2 /day. In atrazine (10 Ilg/L) treated microcosms, biomass (measuredby chlorophyll a 

### fluorescence) increased from 15 Ilg/L at day 0 to 70 Ilg/L at 8-11 days and was slightly enhanced as comparedto the control microcosms (Figure 

### 2). The authors state that 'whereas there was no strong differences betweentreatments, the three sets were significantly different (Student's test, P

### = 0.05)". This suggests thatthe authors found a significant difference among the three groups compared simultaneously, butto the reviewers' knowledge, the Student's test is only for comparison oftwo groups. Without datait is unclear whatthe authors meant and specifically ifthere was a significant difference between atrazine treatments and controls.

Atthe beginning ofthe experiment, the majortaxonomic groups were (in diminishing order) pennate diatoms> cryptophyceae > chlorophyceae> xanthophyceae > chrysophyceae > centric diatoms> cyanobacteria (Figure 

### 3). At experimenttermination,**~30%** 

### ofthe total numberof dominant species were inhibited,**~12%** 

### were stimulated and**~58%** 

### were unaffected in the atrazine treated microcosms, as compared to controls (Mann-Whitney non-parametric test, level ofsignificance was not stated) (Figure 

### 5). Differences at the species level (Figure 

### 5) were not readily apparent atthe broaderlevel ofresolution (majortaxonomic groups) presented in Figure 

### 3. Figure 3 

### shows a difference betweenthe controls and atrazine treatments in the relative abundance ofcryptophyceae, xanthophyceae, and chrysophyceae, althoughreplicate variability is not reported and it is unclear ifthese differences are significant. There was a large shiftin community composition in both the atrazine treated and control microcosms from the beginning ofthe experimentto experimenttermination: pennate diatom populations increased, most other taxonomic groups decreased but were still present, and two (cyanobacteria and chlorophyceae) - 3 -

### were no longerpresent at testtermination. Correspondence analysis was used as another method oflooking at differences inthe treatments and controls based on community composition data, although the authors did not elaborate onthe analysis orhow itwas performed. Atrazine treated microcosms and control microcosms separated mainly on axis 2 ofthe ordination diagram (Figure 4).

Table 1 Freshwater phytoplankton used for in vitro monocultures. Organism Source

Isolated from Lake Geneva. Isolated from Lake Geneva. Culture Collection ofAlgae and Protozoa (Ambleside, U.K.). Oscillatoria limnetica CCAP 1459/18 (cyanobacteria). Culture Collection ofAlgae and Protozoa (Ambleside, U.K.). Pseudokirchneriella subcapitata CCAP 278/4 (chlorophyte; formerly Selenastrum capricornutum). Chlorella vulgaris SHL 108b (chlorophyte). Navicula accomoda SHL 107 (diatom).

Table 2. EC50 and NOEC for atrazine1,2. Species Oscillatoria limnetica Navicula accomoda Chlorella vulgaris EC50 (Ilg/L) 52 104 42 <10 <10 <10 Pseudokirchneriellasubcapitata 98 <10 1 No observed effect concentration. 2 Values calculated using growth rate (11), estimated from logistic equation ofHutchinson (1978),

except forP. subcapitata which was determined using total biomass.

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### irradiance (Jlmol photons/ml/s) Figure 1. Photosynthesis irradiance curves for Chlorella vulgaris acclimated at low light intensity (30 /lmol/m2 /s). Control (solid circles, bold line) and treated (open squares, dashed line) differ significantly on (l and parameters, but Pmax was not affected. Photosynthetic activity expressed as percent ofthe maximum activity ofthe controls. (study figure 1).

80 T I 70

# t

..-.. 60 50

..... ........ l:3 40 Q, 30 

## -r 2 0 :2 20 

### u 10 0 I----f0 2 4 6 8 10 Days 

### ofexperiment Figure 2. Growth in natural phytoplankton microcosms using chlorophyll a fluorescence: control (solid circles), atrazine treated (open squares), and nicosulfuron treated (solid triangles). (study figure 2 ).

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# 5 6

Figure 3. Taxonomic composition and abundance ofmajor taxa for natural phytoplankton microcosms at the beginning ofthe experiment (A) and at experimenttermination: control (B), atrazine treated (C), and nicosulfuron treated (D). Pennate diatoms (1), cryptophyceae (2), chlorophyceae (3), xanthophyceae (4), chrysophyceae (5), centric diatoms (6) and cyanobacteria (7). (study figure 3).

- 6 -Axis 2 16.2%

Figure 4. Correspondence analysis offinal phytoplankton communities in microcosms. Communities represented as percent ofthe total density for each algal species; controls (T), atrazine treated (A) and nicosulfuron treated (N). (study figure 4).



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### 50

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... en 

### 10 

# o No effect Inhibition

# r?1 Atrazine



### _ Nicosulfuron

Stimulation Figure 5. Number ofalgal species unaffected, inhibited or stimulated by test compounds relative to controls (not stated but assumed at end ofexperiment). (study figure 5).

- 7 -Data Summary Table: Naturalphytoplankton communities in outdoormicrocosms Time Weighted Magnitude of Nominal Day of Study Average Did Effect Effect Atrazine Effect Atrazine Recovery Comments relative to Concentration Concentration Occur-if controls ()Jg/L) Occurred from Day Oto so, when? When Effect Occurred 1 Magnitude of effects estimated from Figure 2. It is assumed that mean values are Increased plotted. It is phytoplankton Not uncertain ifthe community biomass Up to**~5%** 10 J.lg/L**~8** days Unknown applicable atrazine treatments (chlorophyll a are significantly fluorescence) different from the controls. This is not considered an effect. Magnitude of effects estimated from Figure 3. Cryptophyceae, chrysophyceae, and xanthophyceae appearto differ in controls and atrazine Change in Up to**~50%** treatments. phytoplankton reduction for No (effects No indication if community some groups Day 11 (end of observed at effects were composition 10 J.lg/L Unknown (up to**~100%** experiment) end of statistically (relative abundance increase in experiment) significant. ofmajortaxonomic groups) one group) As presented, the data appearto be a measure of relative abundance, that is, data do not indicate how actual abundance ofindividual taxonomic groups changed (i.e. - 8 -Time Weighted Magnitude of Nominal Day of Study Average Did Effect Effect Atrazine Effect Atrazine Recovery Comments relative to Concentration Concentration Occur-if controls (pg/L) Occurred from Day 0 to so, when? When Effect Occurred!
relative abundance might change without a change in actual abundance ofa given group). Magnitude of effects estimated from Figure 5 (i.e., # affected**~30%of** species/total # dominant species presented Change in species were No (effects in figure 5). phytoplankton inhibited 10**~gIL** Day 11 (end of Unknown observed at Level of community species**(~12%** of experiment) end of significance used composition dominant experiment) to determine effect species were (inhibition, stimulated) stimulation, orno effect) on each species is unknown. Atrazme concentratIOn was not measured durmg the experIment.

- 9 -Reviewer Comments: This study also investigated the potential effects ofnicosulfuron onphytoplankton in experimental outdoor microcosms and in vitro monoculture.

The source ofthe atrazine used in the experiments (product andpurity) was not reported.

The microcosms were filled with water from Lake Geneva. It is not stated whetherthe lake waterwas tested to establish that it was not contaminated with any pollutants. Anotherreviewed paperby the same authors (Berard et aI. 1999a) reports that atrazine has been detected in the euphotic zone ofLake Geneva (0.03-0.04 Ilg/L). Ifpollutants were present, they could confound the results ofthis study.

Installation ofthe microcosms priorto use was referenced (Berard etai., 1999a), butnot presented in this article. Any acclimation ofthe microcosms prior to use was not reported. The microcosms did not contain a sediment layer.

There is no indicationthat water in the microcosms was analyzed for atrazine following treatment.

The study authors state that some measurements were made atthe "beginning ofthe experiment". It is unclear ifthese measurements were made pre- orpost-treatment.

The study authors do not report that water quality was monitored in the microcosms. However, in other reviewed studies by the same authors there is datapresented for June 97, the month/year that experiments were performed in this study (Berard et aI., 1999a, Berard et aI., 1999b, Berard and Benninghoff, 2001, Seguin et aI., 2001b). It is uncertain ifthat data is associated with the microcosms presented in this study.

As amicrocosm 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.

A seemingly large shiftin community composition was observed over the 11 day experimentin the control treatments as evidenced by comparing the initial composition (day 0) to that in the controls on day 11 (Figure 3). This raises some concern about attributing the community shifts in the atrazine treatments (relative to the control) to the effects ofatrazine alone when other factors were clearly contributing to the observed shifts in composition.

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-The treatmentwas limitedto one test concentration (10 )lg atrazine/L).

Nicosulfuron treatments are included in the correspondence analysis, thus any atrazine effects relative to the controls are confounded by their inclusion.

The statistical results on chlorophyll a are difficultto interpret. It is not clear ifatrazine treatments were significantly different from controls given the study authors statement; "the three sets were significantly different (Student's test, P

### = 0.05)". This may mean thatthere was a difference among atrazine, nicosulfuron, and control compared simultaneously or a difference between eachpair ofgroups. 

### Description ofUse in Document: Qualitative



### Rationale for Use: The study contributes to the weight ofevidence regarding effects ofatrazine on aquatic plant communities.



### Limitations ofStudy: Several deficiencies existed with report ofthe methodology for the outdoormicrocosm investigations. No effortwas made to document whether other chemicals (including atrazine) were present in the treatment and/or control microcosms priorto experiment initiation. Water used in the microcosms was from Lake Geneva whichhas documented atrazine contamination. Installation ofthe microcosms and any acclimation interval priorto use were not detailed. Water quality may nothave been assessed, but was reported for the same experimental date in other studies by these authors. Atrazine treatmentconcentrations were not confirmed or monitored in the microcosms following treatment. There was apotential for bottle effects due to the small size ofthe microcosm.


### Primary Reviewer: Lynne Binari, M.S., subcontractorto Cambridge Environmental Inc. 

### Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA

# 1/lf,t~;t{wd Tertiary Reviewer: AnitaPease, M.S., Senior Biologist, EPA 

# Mtl5t- "(\3/1 \

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