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 of atrazine and nicosulfuron on

periphytic diatom communities in freshwater outdoor lentic mesocosms." Annales De

Limnologie-International Journal of Limnology 37(1): 3-8. (MRID 48273501)

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this study

contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant communities

and associated determination of a Level ofConcern (LOC).

Date of Review: 9/17/09

Summary of Study Findings

Purpose:

This study examines the potential effects of atrazine to aquatic plants (periphyton) in

experimental ponds (mesocosms).

Methods:

Mesocosms were constructed at the INRA (Institut National de la Recherche Agronomique,

Rennes, France) experimental platform. The mesocosms consisted of 15 identical circular tanks

which were 3.2 min diameter, 1.2 m high, and had capacity approximately 5000 L, with a

funnel-shaped base for ease of draining. Tank construction material, ambient conditions, and

other aspects ofthe environmental conditions are not described, but are likely similar to 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 of natural sediment from a local uncontaminated

pond, and then a mixture oftap water and pond water to a depth of 0.7 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 not provided. Water nutrient

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III1IIIIII

2087838
levels measured in the mesocosms showed an orthophosphate concentration of 0.0065 mg P/L

and inorganic nitrogen level of 0.51 mg NIL. It is not stated whether these 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 whether they

are averaged over time.

Aquatic macrophytes (Glyceria maxima) were introduced in two concentric circles meant to

mimic stillwater fish spawning habitat. Artificial substrata (glass slides) were placed in a holder

and installed at constant depth 30 cm below water surface, around the middle ofthe water

column. Number ofslides and design of holder are not described. Colonization ofthe slides and

equilibration ofthe aquatic ecosystem took place for 1 month prior to treatment.

Mesocosms were treated with nominal concentrations of 0 (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 of atrazine 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 in the body text for day 40.

Periphyton was sampled by scraping algae from the surface ofthe four slides with a razor blade.

Half of each 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 higher plants, 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 of common diatoms were measured as an

indicator of morphology. Periphyton biomass 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 experiment in the 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 a per

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 that periphyton consisted of 99% pennate diatoms. The

authors state that there were no significant differences between any atrazine treatment and

controls relative to algal density. Although it is assumed that the Mann-Whitney test was also

used to determine statistical significance, the associated p-values are not provided (see Figure

3).

The periphyton community consisted mainly ofAchnanthidium minutissimum (Klitz) Czarnecki.

Other species were found at low percentages, including Cymbella minuta (Hilse), Gomphomema

angustatum (Klitz) and Gomphonema parvulum (Klitz). Exact percentages are not provided.

The authors state that there was no "noticeable" size effect or valve distortion in Achnanthidium

minutissimum due to atrazine exposure (Figure 4). Statistical tests are not provided.

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9

8

1

6

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4

3

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O+--....,...--,..........-.......---r--..---

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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 over 3 replicates. (study Figure 2).

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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).

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2000

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1600

i' 1400 .....C

-g .200 ~a

:.=, 1000 -+-A

f 800 ..... n

600 ......N

400

200

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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).

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......8

...... ,,11

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12.0 ......- ......- ......----.........--.-......--..-

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Figure 4. Lengths ofAchnanthidium minutissimum. 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 estImated based on vlsualmterpolatIOn of

data provided in Figure 1 ofthis review.

Time Weighted Did

Nominal Average Recovery Magnitude of Day ofStudy Atrazine

Effect Effect relative 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 Day 40 no Not reported IlgIL treatment

non-significant) measurements level was also

significant when

Periphyton normalized to

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 different by 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

of dominant 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 difficult to

interpret because there were numerous shortfalls in the presentation ofthe methods. The basic

environment ofthe mesocosms was not described - for example, whether they were indoor or

outdoor. The construction material ofthe mesocosms is not provided, nor is the location and

environment ofthe pond or ponds from which sediment and water were sampled to construct the

mesocosms. It is stated that a "mixture" of pond and tap water was used to fill the mesocosms,

with no proportions provided or discussion of how the mixing was performed. Water nutrient

levels and temperatures are cited without description of when and where the measurements were

taken, or what averaging was used. In addition, no other water quality parameters were reported.

The periphyton substrata holders are not described, nor are the total number ofslides that they

hold. For statistical tests, no p-values are provided, and it is unclear whether the Mann-Whitney

test was applied to all datasets or just to 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

of atrazine or how it was applied. There was no mention of solvent use; therefore, it was

assumed that solvent controls were not used. Additionally, no effort was made to document

whether other chemicals (including atrazine) were present in the treatment and/or control ponds

prior to study initiation. Although nominal atrazine concentrations were provided, no

measurement of atrazine exposure concentrations over the duration ofthe study was conducted.

Although no exact percentages were provided for the initial relative distribution of dominant

species in the periphyton community, the authors state that Achnanthidium minutissimum was by

far the dominant species and other species were not present in significant numbers. As such, the

study amounts to a single-species study, and a realistic periphyton community was not being

tested. The authors mention that the mesocosms may have developed the single-species

communities because the inocola came from a natural pond's pelagic zone, where periphyton

may be expected to be sparse. Another factor may be that one month was insufficient

colonization time for development of a diverse periphyton community.

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

Description of Use in Document: Qualitative

Rationale for Use: The study contributes to the weight of evidence regarding effects of atrazine

on aquatic plant communities including periphyton.

Limitations of Study: Despite adequate replication at 3 mesocosms per atrazine treatment level,

the study is limited by inadequate presentation of water quality parameters, methods and

statistical analyses, lack of information on atrazine treatment technique 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: Sara H. Hendrix, M.S., subcontractor to Cambridge Environmental Inc.

"\ J '2: LC':r i),J,L;J

/

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, u.S. EPA M?}t

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