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 of atrazine and

nicosulfuron on freshwater microalgae. Environment International. 26: 131-135. (MRID

48261121)

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: 9/8/09

Purpose of Study:

This study examines the potential effects of atrazine and nicosulfuron on phytoplankton

communities in experimental outdoor microcosms and in vitro monocultures.

Summary of Study Findings:

The monoculture experiments will be mentioned, although in limited detail, in order to focus on

the community microcosm experiments. The review focuses on atrazine treatments

(nicosulfuron was not reviewed).

Several other studies by the same authors reported data from the same set of experiments.

Among those studies there is both overlap and difference in terms ofthe experimental dates. The

studies related to this review are Berard et aI., 1999a; Berard et aI., 1999b; Berard and

Benninghoff, 2001; and Seguin et aI., 2001 b. 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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Methods:

Two types of experiments were conducted: 1) in vitro monocultures offour different strains of

freshwater phytoplankton and 2) natural phytoplankton (mixed species) communities in outdoor

microcosms.

In vitro monocultures. Four strains of freshwater 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

estimated using the logistic growth rate equation of Hutchinson (1978).

For C. vulgaris and N. accomoda, the effect of atrazine (10 f.!g/L) on photosynthetic capacity was

assessed using the 14C incorporation technique of Lewis 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 outdoor microcosms. One experiment was conducted in

June 1997; specific dates were not reported. Source water was obtained from Lake Geneva.

Each microcosm consisted of a 5-L bottle containing 2.5 L oflake water with phytoplankton plus

2.5 L offiltered (0.8-f.!m) lake water. Outdoor installation ofthe microcosms was described

elsewhere (Berard et al., 1999a). Any acclimation ofthe microcosms prior to use was not

reported. Five microcosms were treated with atrazine (purity and product not reported) at 10

f.!g/L, with an additional five microcosms maintained as untreated controls. Use of a solvent was

not reported but a solvent was not used in Berard et al., 1999a. Phytoplankton growth was

monitored daily, for 11 days, via chlorophyll-a fluorescence measurements. Irradiance above the

water surface and water temperature 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 not provided, however, in

other papers by the same authors it has been defined as >100 individuals/ml.

Comparisons ofthe density of each dominant species and overall community composition

between the 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 of N. accomoda in atrazine treated cultures

was not affected under the conditions ofthe experiment (30 minutes, 0 to ~500 Ilmol/m2/s).

Natural phytoplankton in outdoor microcosms. During the experiment, water temperatures (l-m

depth) ranged from 16.5°C to 20°C and solar irradiance above the water surface ranged from 475

to 2,750 J/cm2/day. In atrazine (10 Ilg/L) treated microcosms, biomass (measured by chlorophyll

a fluorescence) increased from 15 Ilg/L at day 0 to 70 Ilg/L at 8-11 days and was slightly

enhanced as compared to the control microcosms (Figure 2). The authors state that 'whereas

there was no strong differences between treatments, the three sets were significantly different

(Student's test, P = 0.05)". This suggests that the authors found a significant difference among

the three groups compared simultaneously, but to the reviewers' knowledge, the Student's test is

only for comparison oftwo groups. Without data it is unclear what the authors meant and

specifically ifthere was a significant difference between atrazine treatments and controls.

At the beginning ofthe experiment, the major taxonomic groups were (in diminishing order)

pennate diatoms> cryptophyceae > chlorophyceae> xanthophyceae > chrysophyceae > centric

diatoms> cyanobacteria (Figure 3). At experiment termination, ~30% ofthe total number of

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 at the broader level ofresolution (major taxonomic groups) presented in Figure

3. Figure 3 shows a difference between the controls and atrazine treatments in the relative

abundance of cryptophyceae, xanthophyceae, and chrysophyceae, although replicate variability

is not reported and it is unclear ifthese differences are significant. There was a large shift in

community composition in both the atrazine treated and control microcosms from the beginning

ofthe experiment to experiment termination: pennate diatom populations increased, most other

taxonomic groups decreased but were still present, and two (cyanobacteria and chlorophyceae)

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were no longer present at test termination. Correspondence analysis was used as another method

oflooking at differences in the treatments and controls based on community composition data,

although the authors did not elaborate on the analysis or how it was 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 of Algae and Protozoa (Ambleside, U.K.).

Oscillatoria limnetica CCAP 1459/18 (cyanobacteria). Culture Collection of Algae 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

Pseudokirchneriella subcapitata 98 <10

1 No observed effect concentration.

2 Values calculated using growth rate (11), estimated from logistic equation of Hutchinson (1978),

except for P. subcapitata which was determined using total biomass.

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-til 120 , ...

.-

S 100 I

......

]

--C 80

.->.--('3

(,) 60

(,) .-....

0 40 .d....

c:

20 til

0

-

0

..c

0

a 200 400 600

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 of the maximum

activity of the controls. (study figure 1).

80 T

I

70 t ..-.. 60

........ 50 .....

l:3

40

Q,

30 -r 2

0

:2

u 20

10

0 I----f0 2 4 6 8 10

Days of experiment

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 of major taxa for natural phytoplankton microcosms at the

beginning ofthe experiment (A) and at experiment termination: 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).

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Axis 2

16.2%

Figure 4. Correspondence analysis of final phytoplankton communities in microcosms. Communities

represented as percent of the total density for each algal species; controls (T), atrazine treated (A) and

nicosulfuron treated (N). (study figure 4).

60

50

... 40

.8

e

30

.-(j

c.. QJ ...

"0

en

10

o

No effect Inhibition

r?1 Atrazine

_ Nicosulfuron

Stimulation

Figure 5. Number of algal species unaffected, inhibited or stimulated by test compounds relative to controls

(not stated but assumed at end of experiment). (study figure 5).

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Data Summary Table: Natural phytoplankton communities in outdoor microcosms

Time

Weighted

Magnitude of Nominal Day ofStudy 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

appear to 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. ofmajor taxonomic

groups) one group) As presented, the

data appear to be a

measure of

relative

abundance, that is,

data do not

indicate how

actual abundance

ofindividual

taxonomic groups

changed (i.e.

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Time

Weighted

Magnitude of Nominal Day ofStudy 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 of a

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, or no

effect) on each

species is

unknown.

Atrazme concentratIOn was not measured durmg the experIment.

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Reviewer Comments:

This study also investigated the potential effects of nicosulfuron on phytoplankton in

experimental outdoor microcosms and in vitro monoculture.

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

The microcosms were filled with water from Lake Geneva. It is not stated whether the lake

water was tested to establish that it was not contaminated with any pollutants. Another reviewed

paper by the same authors (Berard et aI. 1999a) reports that atrazine has been detected in the

euphotic zone of Lake Geneva (0.03-0.04 Ilg/L). If pollutants were present, they could confound

the results ofthis study.

Installation ofthe microcosms prior to use was referenced (Berard et ai., 1999a), but not

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 indication that water in the microcosms was analyzed for atrazine following

treatment.

The study authors state that some measurements were made at the "beginning ofthe

experiment". It is unclear ifthese measurements were made pre- or post-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 data presented 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 if that data is associated with the

microcosms presented in this study.

As a microcosm 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 shift in community composition was observed over the 11 day experiment in

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 of atrazine alone when other

factors were clearly contributing to the observed shifts in composition.

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The treatment was limited to 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 difficult to interpret. It is not clear if atrazine

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 that there was a

difference among atrazine, nicosulfuron, and control compared simultaneously or a difference

between each pair of groups.

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.

Limitations of Study: Several deficiencies existed with report ofthe methodology for the

outdoor microcosm investigations. No effort was made to document whether other chemicals

(including atrazine) were present in the treatment and/or control microcosms prior to experiment

initiation. Water used in the microcosms was from Lake Geneva which has documented atrazine

contamination. Installation ofthe microcosms and any acclimation interval prior to use were not

detailed. Water quality may not have been assessed, but was reported for the same experimental

date in other studies by these authors. Atrazine treatment concentrations were not confirmed or

monitored in the microcosms following treatment. There was a potential for bottle effects due to

the small size ofthe microcosm.

Primary Reviewer: Lynne Binari, M.S., subcontractor to Cambridge Environmental Inc.

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 1/lf,t~;t{wd

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA Mtl5t- "(\ 3/1 \

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