Open Literature Review Summary

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

ECOTOX Record Number and Citation: Seguin, F., J. C. Druart, and R. Le Cohu. (2001). "Effects ofatrazine andnicosulfuron on periphytic diatom communities in freshwater outdoor lentic mesocosms." Annales De Limnologie-International Journal ofLimnology 37(1): 3-8. (MRID 48273501)
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: 9/17/09
Summary ofStudy Findings
Purpose: This study examines the potential effects ofatrazine to aquatic plants (periphyton) in experimental ponds (mesocosms).

Methods: Mesocosms were constructed at the INRA (InstitutNational de laRecherche Agronomique, Rennes, France) experimental platform. The mesocosms consisted of 15 identical circulartanks which were 3.2 min diameter, 1.2 m high, and had capacity approximately 5000 L, with a funnel-shaped base for ease ofdraining. Tank construction material, ambient conditions, and other aspects ofthe environmental conditions are not described, but are likely similarto those of the mesocosm experiment described in the lead author's other 2001 publication (Arch Env Contam & Toxicol, also reviewed).

The mesocosms were filled with a 7 cm layer ofnatural sediment from a local uncontaminated pond, and then a mixture oftap water and pond waterto a depth of0.7 m. Pond water contained phytoplankton and zooplankton, and was pumped from and into the pelagic zone. Details regarding homogenization, transport and storage, physical and biological characteristics, precise pond and tap water mixture proportions, and tap water quality, are notprovided. Water nutrient - 1 - 

# III1IIIIII 2087838levels measured in the mesocosms showed an orthophosphate concentration of0.0065 mg 

### P/L and inorganic nitrogen level of0.51 mg NIL. It is not stated whetherthese are averages, or when these values were measured. Temperature in the mesocosms ranged from l4.4°C to 24.8°C, with an average of 18°C. Again, it is not stated when these values were measured, or whetherthey are averaged over time.
Aquatic macrophytes 

### (Glyceria maxima) were introduced intwo concentric circles meant to mimic stillwater fish spawning habitat. Artificial substrata (glass slides) were placed in a holder and installed at constantdepth 30 cm below water surface, around the middle ofthe water column. Number ofslides and design ofholder are not described. Colonization ofthe slides and equilibration ofthe aquatic ecosystemtook place for 1 month priorto treatment.

Mesocosms were treated with nominal concentrations of0 (control), 2**~g/L,** and 30**~g/L** atrazine. Mesocosms treated with 2 nominal concentrations nicosulfuran (2**~g/L,** and 30**~g/L)** were also studied; however the results ofthe nicosulfuran experiments are not discussed further in this review. Three replicates were used at each treatment level including controls. The form and application method ofthe atrazine, use ofsolvent, or analytical testing for the presence of atrazine or other contaminants in the controls were not described. The study authors indicated that the experiment lasted for two months, although graphical data are presented for only 40 days after addition ofatrazine to the pond mesocosms.
The authors state that 4 randomly selected glass slides were collected on days -1 (1 day pretreatment), 1, 7, 17, and 37 after treatment. The graphical results show data for 7-day intervals, and the results are described inthe body text for day 40.
Periphyton was sampled by scraping algae from the surface ofthe four slides with a razor blade. Halfofeach sample was filtered through a Whatman GFIC glass fiber filter, then chlorophyll 

### a was extracted in 90% acetone for 24 hours at 4°C according to Millerioux (1975; "Comparaison des methods spectophotometriques pour Ie calcuI des pigments phytoplanctoniques") and measured according to the spectrophotometric methods ofJeffrey & Humphrey (1975; "New spectophotometric equations for determining chlorophylls a, b, CI, and C2 in higherplants, algae and natural plankton"). The rest ofthe sample was preserved in 5% formalin. A subsample of the preserved solution was counted (no species identification) by the Utermohl method with an inverted microscope. Another subsample was mounted in a high refactive index medium (Naphrax, Northern Biological Supplies Ltd., UK; RI=1.74) for microscopy, and about 400 frustules per sample were identified. Frustule size ofcommon diatoms were measured as an indicator ofmorphology. Periphytonbiomass was derived from chlorophyll 

### a measurements (calculations not described). Biomass was then compared between the control and individual treatments on each sampling day using a Mann-Whitney test.

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### Results: Periphyton chlorophyll 

### a concentration ranged from 2.8 to approximately 9**~g/cm2** over the duration ofthe experimentinthe controls and atrazine treatment groups. There was a significant increase in chlorophyll 

### a concentration in mesocosms containing 30**~g/L** as compared to the control on Day 40 (see 

### Figure 1; p-values not cited). Additionally, the chlorophyll 

### aper individual was significantly higher in the 30**~g/L** atrazine treatment groups as compared to the controls (see 

### Figure 2; no p-values or confidence levels are reported).

The algal density examination showed thatperiphyton consisted of99% pennate diatoms. The authors state thatthere were no significant differences between any atrazine treatment and controls relative to algal density. Although it is assumed that the Mann-Whitney testwas also used to determine statistical significance, the associated p-values are not provided (see 

### Figure 3).

The periphyton community consisted mainly of

### Achnanthidium minutissimum (Klitz) Czarnecki. Other species were found at low percentages, including 

### Cymbella minuta (Hilse), 

### Gomphomema angustatum (Klitz) and 

### Gomphonemaparvulum (Klitz). Exactpercentages are notprovided. The authors state thatthere was no "noticeable" size effect or valve distortion in

### Achnanthidium minutissimum due to atrazine exposure 

### (Figure 4). Statistical tests are notprovided.

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### .....c -&-a

...... A

.... n 

### -+-N

2S17 l&n 1111 4fl 27/6 10 9 8 1 6 S 4 3 2 1 

## O+--....,...--,..........-

.......---r--..---

2016 Figure 1. Chlorophyll a concentrations. C =controls, a =low atrazine treatment (2 JlglL), A=high atrazine treatment (30 JlglL). Asterisks (*) denote significant differences from controls as detected by the MannWhitney test. No p-values are provided. Symbols represent averages over3 replicates. (study Figure 2).

0,025 

# •

1817 

## un 2116**O-+---..,.-~-r-----.----r---.,..--** 20/6 Figure 2. Chlorophyll a concentrations expressed per individual. Symbol definitions are shown in Figure 1 above. Asterisks (*) indicate significant differences from controls; no statistical test or p-values are cited. Symbols represent averages over 3 replicates. (study Figure 5).

- 4 -2000 !!OO 1600 

# i' 1400

.....C 

## -g .200**~a** :.=, 1000 

### -+-A 

# f 800 ..... n 600 ...... N 400 200**O+--..,.,--"'"""T--r--,--.....,....~** 20/6 27/6 4n lin 1817 2sn Figure 3. Periphyton cell densities. C =controls, a =low atrazine treatment (2 IlglL), A=high atrazine treatment (30 IlglL). Symbols represent averages over 3 replicates. (study Figure 3).



## ....c

...... 8

...... ,,11

........ D -+-y..
12.1 12.0

......- ......- ......----.........--.-......--..- 1816 25/6 217 9n 1617 23n 12.7 12,6 12,S

### ~~~~~~ .......... 12,4 

## i

Figure 4. Lengths of Achnanthidiumminutissimum. C =controls, a =low atrazine treatment (2 IlglL), A= high atrazine treatment (30 IlglL). Symbols represent averages over 3 replicates. (Study Figure 4).

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### Data Summary Table.

Magllltude ofmcrease III penphyton chlorophyll a concentratIOn are estImatedbased on vlsualmterpolatIOn of data provided in Figure 1 ofthis review. Time Weighted Did Nominal Average Recovery Magnitude of Day of Study Atrazine Effect Effectrelative to Atrazine Effect Concentration Occur-if Comments controlsl Concentration Occurred from Day Oto so, when (pg/L) (see When Effect comment)? Occurred
Not available

- Effect at the 30 14% (increase- 21lglL Day40 no Not reported IlgIL treatment non-significant) measurements level was also significant when Periphyton normalizedto chlorophyll a density (review fig. concentration 61 % (significant Not available

- 2). Differences increase as 301lg/L Day 40 Not reported were significantly compared to the no differentby Manncontrol) measurements Whitney test; pvalue not stated. See review fig. I Unclear which 21lg/ L statistical test was Periphyton Not applied; no No effect Not applicable Not applicable significant density applicable differences are 301lg/L asserted, no pvalues provided. See review fig. 3. Diatom length 21lg/L Unclear whether ofdominant No effect Not applicable Not applicable Not statistical tests were species 30llgiL applicable applied. See review fig. 4.
1

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### Reviewer Comments: The study used adequate replication and attempted to create reasonably ecologically complex mesocosms through use 

### ofmacrophytes and natural inocula. However, the results are difficultto interpretbecause there were numerous shortfalls inthe presentation ofthe methods. The basic environment ofthe mesocosms was not described

- for example, whetherthey were indoor or outdoor. 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 statedthat a "mixture" ofpond andtap water was used to fill the mesocosms, with no proportions provided or discussion ofhow the mixing was performed. Water nutrient levels and temperatures are cited without description ofwhen and where the measurements were taken, or what averaging was used. In addition, no otherwater quality parameters were reported. The periphyton substrataholders are not described, nor are the total number ofslides thatthey hold. For statistical tests, no p-values are provided, and it is unclear whetherthe Mann-Whitney test was applied to all datasets orjustto chlorophyll 

### a measurements. The study describes periphyton sampling occurring on 5 days (Day -1, 1, 7, 17, and 37); however, the study figures show 7-day sampling days intervals, and its results are described for Day 40.
Perhaps most significantly, no information is provided on the percentage a.i. or formulation type ofatrazine orhow it was applied. There was no mention ofsolventuse; therefore, it was assumedthat solvent controls were not used. Additionally, no effortwas made to document whether other chemicals (including atrazine) were present inthe treatment and/or control ponds priorto study initiation. Although nominal atrazine concentrations were provided, no measurement ofatrazine exposure concentrations overthe duration ofthe study was conducted.
Although no exactpercentages were provided for the initial relative distribution ofdominant species in the periphyton community, the authors state that

### Achnanthidium minutissimum was by far the dominant species and other species were notpresent in significantnumbers. As such, the study amounts to a single-species study, and arealistic periphyton community was notbeing tested. The authors mentionthat the mesocosms may have developed the single-species communities because the inocola came from a natural pond's pelagic zone, where periphyton may be expectedto be sparse. Another factor may be that one month was insufficient colonizationtime for development ofa diverse periphyton community.

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### Secondary Reviewer: Catherine Aubee, Biologist, u.S. EPA 

### Description ofUse in Document: Qualitative



### Rationale for Use: The study contributes to the weight ofevidence regarding effects ofatrazine on aquatic plantcommunities including periphyton.



### Limitations ofStudy: Despite adequate replication at 3 mesocosms per atrazine treatment level, the study is limited by inadequate presentation ofwater quality parameters, methods and statistical analyses, lack ofinformation on atrazine treatmenttechnique and concentrations, and the effectively single-species periphyton communities which would fail to show ecosystem-level impacts. 

### In addition, it is unclear whether controls were contaminated with atrazine and/or other contaminants. 

### Primary Reviewer: SaraH. Hendrix, M.S., subcontractorto Cambridge Environmental Inc. 

### "\ J '2: 

### LC':r i) ,J,L;J / 

### Tertiary Reviewer: Anita Pease, M.S., SeniorBiologist, u.S. EPA

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