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 of atrazine and

nicosulfuron on phytoplankton in systems ofincreasing complexity. Archives of Environmental

Contamination and Toxicology. 40(2): 198-208. (MRID 48261134)

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:

September 24, 2009

Purpose of Study:

This study examines the potential effects of atrazine and nicosulfuron to aquatic plants

(phytoplankton) in single-species microplates, indoor microcosms, outdoor enclosure

microcosms, and experimental ponds (mesocosms).

Summary of Study Findings:

This review focuses on the effects of atrazine (nicosulfuron was not reviewed).

For the outdoor microcosm experiments, several other reviewed 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 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 in the DER of each

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

2087837
Methods:

Four distinct experiments are described in the study. Methods and results are reviewed primarily

for the microcosm and mesocosm experiments, with the single-species tests summarized in brief.

Single-Species Tests

Several phytoplankton species were cultivated in clear polystyrene microplates under 12: 12

light:dark cycles with approximately 100-150 ~E/mzIs light intensity at the lid. Growth was

monitored daily with a Dynex MRX CE97 spectrophotometer to measure OD65o. Cultures in the

exponential growth phase were diluted (106 cells/ml) and then grown for at least one week in the

microplate before treatment. Atrazine (98%, Greyhound Service, UK) was prepared in pure

acetone, with a final concentration of acetone of 0.05% in all treatments and controls. Six

replicates were used at each of 10 atrazine treatment levels (0 ~g/L to 256 ~g/L, exact treatment

levels between were not reported). The cell density after 96 hours of growth (post treatment)

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 of Lake Geneva in December 1999. Grazers and large particles

were removed by passing the water through a 250-~m net and nitrogen was bubbled through to

kill any remaining heterotrophs. The systems were kept in controlled chambers with a 12:12

light:dark cycle (200 ~E/mz/s) and constant temperature of 12°C. Samples were enriched with

soluble P, N, and Si to achieve nutrient concentrations of 0.02 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 of early spring in the lake. For more description ofthe

protocol, the study cites Berard (1996, Effects offour organic solvents on natural phytoplankton

assemblages, consequences for ecotoxicological experiments on herbicides, 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 concentration not

provided) and nine bottles each received a different atrazine treatment ranging from nominal

concentrations of 0.1 ~g1L to 100 ~g1L (other than 50 ~g1L, the six other intermediate nominal

concentrations were not provided). It is not stated whether the atrazine product and solvent used

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 at the 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 of Lake Geneva. Previously published background

- 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 L bottles were filled with 2.5 L oflake water as

nutrient stock and 2.5 L of natural phytoplankton sample (not described how these were obtained

or processed), then treated as described for the indoor microcosms to remove heterotrophs. The

bottles were sampled prior to treatment to 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 solvent blanks (controls). It is not stated whether the atrazine product and

solvent used in the single-species tests was also used for this experiment. The bottles were

placed 1 m below the lake surface near the shore. They were monitored daily for in vivo

fluorescence and final taxonomy and biomass were determined at the end of each experiment

(exposure duration varied by sampling date from 10 to 21 days, see Table 1).

Mesocosms

The mesocosm experiment was conducted in circular outdoor tanks at the 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. Nutrient concentrations in the mesocosms were

0.0065 mg P/L orthophosphate and 0.51 mg NIL inorganic nitrogen (not stated when these

measurements were taken or how they were averaged). Temperatures in the mesocosms ranged

from 14.4°C to 24.8°C with an average of 18°C (not stated when measurements were taken).

The mesocosms were filled with 5 cm of natural sediment from a local uncontaminated pond.

The tanks were filled with a mixture oftap water and water from a nearby pond at a depth of 0.7

m. The pond water contained phytoplankton and zooplankton (not stated ifthis pond had

atrazine contamination). Many methodological details were not provided, for example

regarding homogenization, transport and storage, physical and biological characteristics, precise

pond and tap water mixture proportions, and tap water quality. Aquatic macrophytes (Glyceria

maxima) were introduced in two concentric circles, with identical numbers of Glyceria in each

mesocosm, and were meant to mimic stillwater fish spawning habitat.

The mesocosms were allowed to develop for 3 weeks before atrazine treatment. Atrazine was

dissolved with acetonitride, which was believed to be less toxic than acetone. Solvent

concentration in the tanks was not provided. Three replicates were treated at each treatment

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 GFIC filter for

chlorophyll a determination. Chlorophyll was extracted by incubation for 24 h at 4°C in 90%

acetone, and then chlorophyll a was measured spectrophotometrically

Effects of atrazine on photosynthetic efficiency was assessed by calculating P/I curve parameters

in one control mesocosm and one 30 /lg/L treatment mesocosm, using the method of Lewis and

Smith (1980). The three parameters, a, ~, and Pmax, were calculated prior to treatment,

immediately after treatment, and on day 11 after treatment.

- 3 -
Species Composition and Statistics

Samples were taken pre-treatment, during exposure, and at the end ofthe microcosm and

mesocosm experiments to identify and count phytoplankton cells. A 50 mL aliquot of 'algal

suspension' was taken from each tank. Lugol was added to kill and stain cells. After cells were

allowed to settle for 24 h, they were examined under a reverse phase microscope for

identification and enumeration. The abundance ofthe dominant species was estimated ±10%.

The density of each dominant species (defined by study authors as >100 individuals/mL) in the

treatment groups was compared to that ofthe controls using the Mann-Whitney test (p-value not

reported). Compositional differences between treatment and control communities were

described using the Bray-Curtis dissimilarity index (BCl). BCls were calculated for the mean

densities in replicate communities, and significant differences in community composition

between atrazine treatment and the control were assessed by applying the Mann-Whitney test to

the within-control BCl versus the control-treatment BCl. For the indoor microcosm study, a

correspondence analysis between treatment 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 unclear how the control

communities were included in the analysis, and nicosulfuron treatments were included in the

analysis.

- 4 -
Table L Biomass and diversity of the 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 of algal communities in outdoor microcosms before treatment in each

experiment, expressed as % of the 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 similar to 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).

Indoor Microcosms

The microcosm treated 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 at the end ofthe experiment (data not shown). The correspondence

analysis showed some variation in community structure with atrazine treatment level (Figure 2).

- 6 -
Outdoor Microcosms

Depending on the experiment, some significant differences in chlorophyll biomass (as measured

by in vivo fluorescence) were seen in the 10 Ilg/L atrazine treatment microcosms. Depending on

the experiment, chlorophyll in the atrazine treatment increased, decreased, or was 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 different than the control communities for two ofthe

experimental dates (Table 3). For the abundance ofindividual species (those that the 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) compared to the controls at

the end ofthe experiment (Figure 3).

Mesocosms

Phytoplankton biomass was low in all mesocosms (maximum of 4.5 Ilg/L after 17 days), with no

differences among controls and treatments according to the study authors (Figure 4). Succession

occurred during the experiment in all mesocosms (Figure 5), the reviewer assumes the average

of all mesocosms is plotted). According to the study authors, succession was similar in treated

and control tanks with some difference in timing and proportion. The chlorophyte C. vulgaris

dominated the initial assemblages but decreased over time. Over the 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. Data was 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 smaller for 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 not provided) 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-treatment at 30 Ilg/L atrazine

(Figure 6).

Atrazine treatment mesocosms had significant differences from controls in the density ofmajor

taxonomic groups (Table 4). Significant differences occurred on different days for different taxa

and different treatment levels. The high treatment level (30 Ilg atrazine /L) showed more

significant differences from the control than the 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 lower in the atrazine treatment

mesocosm than in the control on day 1 and higher on day 10. The other parameters showed the

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

ri --Axia2"- -- P8 %ot variance)

I

J'

I

I .16 Aster:'ooella tomosa I

aKa i 1

I -AS -... oml.la-!!-r_' •

1

Axl." . " ·allL.

o

{68 % of varlancej--·St-- us II. -:a.~~.. .....

_Xl ~'mi.Ju.lta:var nanncplanctica

I ~Cld1.r:aralas sp • I· ! .~ ~~~

I f"-"~ I

r

u f1agelatesl~ .

.~=_.- J I

!_-~~,------j--_J Figure 2. Correspondence analysis of phytoplankton communities at the end of the 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

70

!! 60

.!

J so

40

1:! 30

20 •

10

o

A matla~ine

f?Z.I nicOSlMuron

inhibition

Figure 3. Response of dominant phytoplankton species to 10 JiglL atrazine (Mann-Whitney tests for the

seven experiments, p-value not reported) measured at the end of each outdoor microcosm experiment. (study

figure 3)

- 9 -
4.S

4.0

.3.5

,;;-

3.0.

.s 2.$

S 2,G

'fi U

1,0

0.5

0.0 ..-,... ~r--.,-__-T--r--....---.----,

~.... ''''~ ",'\ Jo.,..<e'l:l :'\ , "\ \> 1;1< <;> ~.:'" ~\ ~'l. 't/''' ~~

......c ~a

-+-A

.......n

......N

Figure 4. Chlorophyll a content of the 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..

Cry pL\lphr~~.1':

BaciIlariophyceac

Chrysophyc:ellC:

CyatlObaC:leria

-r-" I"'"" ,..., r- r- ..." - ru

IlIIl u I..t- ..... "\~

(>11%

1(10%

Figure 5. Class-level taxonomic composition of algal communities in mesocosms at the different sampling

periods, expressed in % of total phytoplankton identified. The study reviewer assumes composition

represents average of all cosms. (study figure 5)

Table 4. Significant responses of dominant 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 of the 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

*

a: high concentrations

*

*

b: low concentrations

*

*

*

*

I

Fig. 6. Bray-Curtis indices of the different phytoplankton 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 between 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 ofStudy Average Recovery

Atrazine Atrazine Occur-if Effect Effect relative 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, statement that

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 ofStudy Average Recovery

Effect Effect relative 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 or more

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 significant by

unknown). Student's t test, 5

replicates, 95%

confidence (table

3).

• Outdoor

microcosms

Response varied BCI of controls

among 7 significantly

outdoor different from BCI

Change in microcosm calculated between

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

effect unknown 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 ofStudy Average Recovery Magnitude of Atrazine Atrazine Occur- if Effect Effect relative Comments 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 Varied (-12 test (no p-value 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) species no estimated from

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 that there 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 phytoplankton Results for BCI not 2 !lg/L 10 community mesocosms

composition provided) (Figure 6)

(Bray-Curtis Differences are index calculated Unknown (no significant by between measurements) Unknown Mann-Whitney treatments and

(within-control test (no p-values controls 30 !lg/L Day 2 No BCI not provided). compared to BCI

provided) calculated within- Based on

controls) I statements by the

study authors, the

- 15 -
Time

Weighted Did

Nominal Day ofStudy Average Recovery Magnitude of Atrazine Atrazine Occur- if Effect Effect relative Comments 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

treatment related.

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

Transient detected with effect

Mann-Whitney (see comment)

test (no p-values

2Jlg/L provided). Change in Unknown (no Not

Cryptophyceae No effect Not applicable No density values measurements) applicable density 30 Jlg/L are provided.

- 16 -
Time

Weighted Did

Nominal Day ofStudy Average Recovery Magnitude of Atrazine Atrazine Occur-if Effect Effect relative 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 it is

Not magnitude) difficult to assess 2 !1g/L Day 17 applicable Change the likelihood that in Transient effect 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 reviewer judges magnitude) that at least some

ofthe effects were

Increase likely transient and

(unknown 2!1g!L Day 40 not treatment

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

maximum 5) Unknown (no Yes- by photosynthetic 25% decrease 30 !1g!L Day 1 measurements) Day 10 1 replicate only, efficiency (P/I measurements

curve) only taken on days

1 and 10.

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

- 17 -
Reviewer Comments:

The study has a broad focus, with a range of experiments examining effects of both atrazine and

nicosulfuron. Due to the broad scope, some data and methodological details ofinterest for this

review were not provided. 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 not provided, and the duration ofthe experiment was not stated. The

outdoor microcosms 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 prior to use was referenced (Berard et aI., 1996 and Berard et aI., 1999a,

respectively), but not presented in this article. Any acclimation ofthe microcosms prior to

treatment was not reported. Microcosms did not contain a sediment layer. Water used 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 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. It is unknown if water

used for the mesocosms was contaminated with atrazine.

Water nutrient levels and temperatures are cited without description of when and where the

measurements were taken, or what averaging was used.

It is unclear whether the same grade and brand of atrazine was used in all the experiments; it is

only specified for the single-species microplate tests.

The p-value is not reported for many statistical tests and in some cases the statistical test which

was used is not reported.

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, low biomass and

nutrient levels, and (particularly in the mesocosms) strong successional dynamics that may have

confounded effects of atrazine. Only 3 weeks were allowed for colonization in the mesocosms,

- 18 -
which may have been insufficient for attaining a reasonably steady-state system prior to

treatment.

Limited results were presented for many ofthe endpoints.

The study authors stated that the Bray-Curtis dissimilarity index was used, but its

implementation was not well-described, which may lead to 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 refer to the BCI as a

similarity index or just as an index; therefore, it is unclear whether values presented in the 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 that the indices were

lower with higher treatment; ifthe index is dissimilarity as stated in the methods, than this would

seem to indicate greater similarity between treatment and control at higher treatment levels. This

appears to be a counterintuitive result. The reviewer assumes that BCI indices presented by the

study authors are expressed as similarity; however neither the method nor the results are

discussed in enough detail to be certain how BCI results should be interpreted.

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

replication).

The correspondence analysis (indoor microcosm experiment) was based on one replicate at each

concentration. Atrazine-only effects are confounded by inclusion of nicosulfuron treatments in

the analysis. It is not clear how the control data was used in this analysis. Because ofthese

issues, there is no basis determining treatment related effects..

Description of Use in Document: Qualitative (mesocosm experiments expect photosynthetic

activity, outdoor microcosms), Invalid (indoor microcosm, photosynthetic activity for mesocosm

experiments)

Rationale for Use:

Qualitative - Mesocosm experiments and outdoor microcosm experiments contribute to the

weight of evidence regarding effects of atrazine on aquatic plant communities.

Invalid - For indoor microcosm experiments the duration of exposure is unknown, there was no

replication oftreatments, and six ofthe nine treatment doses are unknown. For the mesocosm

experiments, there was no replication for photosynthetic activity measurements.

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Limitations of Study: All experiments lack complete description ofthe methods, atrazine

concentration measurements, p-values for "significant" statistical results, and presentation of

sufficient data for many endpoints. The mesocosm experiment results may have been affected

by strong successional dynamics (perhaps due to insufficient acclimation time prior to

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 Geneva which has documented atrazine contamination. It is

unknown ifthe pond water used for the mesocosm experiments was contaminated with atrazine.

Solvent and the concentration used were not stated for indoor or outdoor microcosm

experiments. The indoor microcosm experiment had no replication, failed to state all the

nominal concentrations used for the atrazine treatments, and the duration of exposure 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: Anita Pease, M.S., Senior Biologist, EPA I1.tt~ ~(t3/11

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