Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Gustavson, K. and Wangberg, S. 1995. Tolerance induction and succession in

microalgae communities exposed to copper and atrazine. Aquat. Toxico!. 32:283-302

(MRID 47543614).

Purpose of Review:

Determine the 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:

November 24, 2009

Purpose of Study:

This study examines the potential effects of atrazine to microalgae in experimental lake

enclosures. Specifically, the objectives ofthe study were to follow the succession of

species and tolerance of exposed phytoplankton communities and compare tolerance

levels in phytoplankton and periphyton communities.

Summary of Study Findings:

Methods: This study also examined effects of copper, and tolerance to zinc and diuron.

Methods and results described in this study review will be limited to those relevant for

the atrazine investigations.

The experiments took place over 20 days, August 8-29 (the year is not stated).

Enclosures were created out of closed-bottom, 3.5 m deep, 1.5 m diameter clear nontoxic polyethylene cylinders with 0.1 mm thick walls, each containing about 6 m3 of lake

water and attached to a pontoon bridge in Lake Bure, Denmark. Lake Bure is a

mesotrophic clear lake that is not fed by wastewater runoff. No further information about

the size of Lake Bure or the distance ofthe enclosures from shore was provided. Six

enclosures received atrazine at 3 treatment concentrations (2 replicates at each treatment

level) of 0.0046 /lM (1.0 /lg/L), 0.046 /lM (9.9 /lg/L), and 0.092 /lM (19.8 /lglL). Three

1111•••111

2087841
enclosures were controls. The atrazine (appears to be reagent grade: Pesternal, Riedel-de

Haen, MW == 215.6 g/mol) was dissolved in distilled water to 10 ppm with ultrasound and

no organic solvents. This solution was added slowly to the enclosures using a soft

silicone tube to distribute it evenly with depth. The positions oftreatment and control

enclosures on the pontoon were random.

Water samples were taken from the enclosures and the lake with a 3-m-Iong rigid tube

sampler of volume 2.3 L. Macrozooplankton were removed during the sampling by a

140 J.1m filter. Periphyton was sampled on submerged 1.5-cm2

-area glass discs in the

enclosures at a depth of0.5 m.

Tolerance measurement of phytoplankton and periphyton (in terms of J4C uptake after

additions of atrazine) was begun within 0.5 hours after sampling, in a mobile laboratory.

lO-mL scintillation vials, containing 9.9 mL lake/enclosure water and 0.1 mL of atrazine

test solution, were prepared; for periphyton, each vial contained a 1.5 cm

2 glass disc and

9.9 mL filtered (GF/C) lake water. The atrazine test solutions were prepared in distilled

water to achieve a geometric series of 15 nominal vial concentrations ranging from

0.00082 J.1M to 2.8 J.1M atrazine (about 0.2 J.1g/L to 603.9 J.1g/L), one single replicate for

each test concentration and five control replicates. A 50 J.1L (1 J.1Ci) aliquot of 14C_

bicarbonate e4C Agency, Denmark) was added to each vial. After a preincubation period

of I hour, the vials were incubated for 2 hours in clear acrylic holders, in situ, at a depth

giving irradiance of about 300 J.1E/m2

/s corresponding to light-saturated photosynthesis.

The non-fixed 14C was removed after acidification with hydrochloric acid. The biotic

fixed 14C was counted on a Beckmann Liquid scintillation spectrometer LS 1801 (the

authors say that abiotic 14C was always less than 1% - no details on how this was

determined were provided). Tolerance was determined by examining the dose-response

curve and determining the ECso value via log-linear interpolation after normalizing the

14C fixation to the mean ofthe 5 controls.

Phytoplankton biomass in water samples was measured as chlorophyll a, after extraction

in ethanol for 12 hours and spectrophotometry analysis. Phytoplankton community

composition and succession were determined by counting cells in Lugol-fixed samples

using an inverted microscope. Species composition was evaluated using the S0rensen

similarity index (the 1948 paper is cited, but the index is not defined in the text), and via

detrended canonical correspondence analysis (DCCA) using the software package

CANOCO (Ter Braak, 1988) after transforming the cell counts to square roots to increase

the influence ofthe less abundant species. Particulate organic carbon (PaC) and nitrogen

(PaN) were measured by dry combustion of particles retained on precombusted GF/C

filters in an Elemental Analyzer. Standards were run routinely after every four samples.

Surface quantum flux densities were measured with a LI-COR spherical quantum sensor

(model not provided). Nutrients were determined "according to conventional standard

methods" (no reference provided), and pH was measured in situ (no details provided).

2
Atrazine was analyzed in three replicate 100 mL water samples taken from each

enclosure on different days. Samples were liquid/liquid extracted twice using 100 mL of

dichlormethane, and the extracts then reduced to dryness in a rotary evaporator, dissolved

in 1 mL dichlormethane, and injected in the HPLC (Waters model 510 and a Waters UVdetector 440) using a Novapak C- I8 HPLC column with dichlormethane as the mobile

phase (1 mLlmin). Concentrations of atrazine were determined using peak integration

and standard curves constructed the same day as the analysis, and extraction blanks were

run routinely after each sample. The detection limit was 0.0001 IJ,M, and replicate

measurements had a 2% coefficient of variation.

Results: Only results relevant to atrazine will be described. It should be noted, however,

that the study focuses on the copper results, because more effects were seen with copper

than with atrazine.

Atrazine concentrations in the enclosures remained stable over the course ofthe

experiment, ranging from 0.005 to 0.003 IJ,M (0.6 to 1.1 IJ,g/L) for the low (0.0046 IJ,M or

1.0 Jlg/Lnominal) enclosures, from 0.046 to 0.069 IJ,M (9.9 to 14.9 IJ,g/L) for the medium

(0.046 IJ,M or 9.9 IJ,g/L nominal) enclosures, and 0.082 to 0.111 IJ,M (17.7 to 23.9 IJ,g/L)

for the high (0.092 11M or 19.8 IJ,g/L nominal) enclosures. (No further details are

provided.)

Atrazine tolerance in the phytoplankton communities was relatively constant throughout the

experiment, and additions of atrazine to the enclosures did not induce atrazine tolerance (no data

provided) (see Figure 1 for comparison of atrazine tolerance in lake vs. control enclosure

phytoplankton communities - they were very similar). Likewise, when the tolerance of periphyton

was measured 22 days after atrazine addition to the enclosures, there were no changes in periphyton

community tolerance based on prior atrazine exposure, or controls vs. lake samples. The ECso values

for atrazine were very similar in periphyton and phytoplankton (see

Table 1).

At the beginning ofthe experiment, chlorophyll concentrations in the enclosures were

about 10 llg chl-a/L. The addition of atrazine caused a small increase in the chlorophyll

concentrations after 2 weeks (no data or statistics provided). pH in the enclosures

increased as the experiment continued due to reduced water movement in the enclosures,

which resulted in reduced uptake of carbon dioxide from the atmosphere (see Figure 2).

POC/PON ratios became as low as 6.6 in the atrazine enclosures, whereas the control

enclosures had ratios 01'7.5 to 8.4 (no further details or data are provided). The

chlorophyll/POC ratio was about 30% higher in the high concentration atrazine

enclosures (no further details or data are provided). Nutrient concentrations remained

similar over all atrazine enclosures and the lake, and constant over time (no further

details or data are provided).
The number ofrecorded phytoplankton species was similar in all enclosures. In the

control enclosures, a succession was seen with increased numbers ofAnabaena ef

seheremtiev, Closterium aeutum, and Chrysoehromulina parva, and similar trends but

with stronger magnitudes were observed in the atrazine enclosures (no magnitudes or

statistics are provided). See Table 2 and Table 3 for species distribution data. The

communities from the atrazine enclosures, control enclosures, and the lake all had

Sorensen index between 0,593 and 0.957 (no further details or data are provided). DCCA

ordination could explain only 60% at most ofthe total variation in species composition,

but found that medium and high atrazine concentration enclosures had succession in the

same direction as the controls (see Figure 3).

Cladoeera and copepods were more abundant in the enclosures than in the lake due to

lack of predation, and enclosures with atrazine addition had larger numbers ofCladocera,

though the mean length ofthe zooplankton was not affected by atrazine level. There was

no noticeable decrease in zooplankton density across atrazine enclosures and controls.

(No further details or data are provided.)

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0.010 0.100 1.00 10.0

Anazin. concentf,tion (JlMJ

Figure 1. Atrazine tolerance of phytopJankton communities from Jake samples (solid line) and

control enclosures (dotted line), Jines representing the mean and error bars the standard deviations

over all samples and all days of measurement (13 data points tota)). Vertical dashed lines represent

nominal atrazine concentrations in the enclosures.

4
9.0

:J:

0-

8.5

8.0

12 18

Date in August

24 30

Figure 2. pH values in the enclosures. Solid squares =Lake; open circles with error bars = control,

low and medium atrazine treatment, and low copper treatment enclosures (mean and range);

triangle with dotted error bars high atrazine treatment enclosures (mean and range). Stars are

high copper concentration, and diamonds are not defined in the study.

2 --"""y----------------,

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A

N . . . . ••• . -

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0 t a.

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0

0

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Component 1
Figure 3. Ordination of a nCCA of phytoplankton community composition data. Relevant symbols

are the open circles (controls); solid squares (lake); triangles (medium atrazine treatments); and

inverted triangles (high atrazine treatments). All are clustered in the upper left corner of the plot.

Table 1. ECso values for phytoplankton and periphyton communities. Low atrazine treatments have

a nominal concentration of 1.0 /!g/L, medium treatments have 9.9 /!g/L, and high treatments have

19.8/!g/L. (from study Table 2)

Phytoplankton (29 Aug~

M atrazine atrazine

Periphyton (30 August)

M atrazine atrazine

Low Atrazine

Lake

Control

0.27 58.2

0.24 ± 0.Q35 a 51.8 ± 7.5 a

0.26 ± 0.021 b 56.1 ± 4.5 b

Medium Atrazine 0.23 ± 0.008 b 49.6 ± 1.7 b

0.27 0.0025 b 58.2 ± 0.5 b

mean from mean with n =3

b mean and deviation from mean of duplicates

0.24

0.25

0.35

0.15

0.3

51.8

53.9

75.5

32.4

64.7

Table 2. Number ofspecies in different systematic groups on August 28. It is assumed that the last

column heading on study Table 3 is a typo, and should read "A_high(a), A~high(b)". (from study

Table 3)

Systematic class Lake Controls

Cyanophyceae II 8 10 10 10 10 I 1

Bacillariophyceae 7 5 5 4 5 6 6

Eug1enophyceae 0 0 1 0 0 0 0

Chlorophyceae 18 17 22 22 22 18 22

Dinophyceae 2 2 2 1 I I 1

Cryptophyceae 3 3 3 3 3 3 3

Chrysophyceae 2 3 2 1 1 2 2

Prymnesiophyceae 2 2 2 2 2 2 2

Number of 46 40 47 43 44 42 47

Table 3. Cell numbers on August 28 for algal species that in some cases had more than 1000

cells/mL. "+" notation is not defined in the study, but reproduced here for completeness.

It is assumed that the last column heading on study table 4 is a typo, and should read "A~high(a),

A_high(b)", and that the second 28 Aug. heading should read "C_l, C_2". (from study Table 4)

Systematic class Lake Controls

Anabaena cf scheremetievi + 10,040 30,981 38,174 59,714

Lyngbya limnetica + 291 74 163 378

Monoraphidium cf + 8 + minutumlcirernale

Closterium cf acutum 565 423 914 1,747 672 1,396

A1ougeofia sp. 1,692 660 689 409 538 511

6
Rhodomonas minuta 906 83 130 5 44 172

Katablepharis sp. 302 + 630 398 135 216 378

Chrysochromulina parva 4,047 2,966 5,298 8,904 1,434 8,555 7,698

906 540 867 741 184 288 I 65

Data Summary Table:

E.lfect Magnitude Nominal Day ofStudy Time Weighted Did Comments

ofEjJect Atrazine EjJect Average Recovery

relative to Concentration Occurred Atrazine Occur- if

controls (f.Jg/L) Concentration so, when

from Day 0 to (see I When EjJect comment)

~t;;.'t·nkto ~o e~ecl_ 1.0 .glL

Occurred ? ~~-

not 0.9 IlgiL not

a applicable Study states

periphyton . ff t . 9 9 IL not 12.4 Ilg/L not lack of effect.

community no e ec . I1g . a applicable No data

tolerance to

no effect 19.811g/L not 20.81lg/L not provided.

atrazine applicable applicable -- Smail increase IIncrease in NRJ"small" NR Day 14 Not calculable NR after 2 weeks chlorophyll a stated; no data. ,..---- ---~- .~~.._-~~ ~~- ~------~~~

Decrease Study reports

ratio of only: 6.6 = low

particulate ·~15-20% NR NR Not calculable NR value for

organic treatments, vs.

carbon to p.o.

-t1

7.5 to 8.4 =

nitTogen control range~_

Increase in

chlorophyll to No further data

particulate 30% 119.8 jlg/L NR 20.8 11 giL NR or details

organic reported.

I carbon ratio I- I

not

no effect 1.0 IlgiL a Ikable 0.91lg/L applicable Study states (;:00' no effect 9.9 jJ.g/L I not 12.4 Ilg/L not lack of effect.

concentrations applicable applicable No data

no effect 19.8 jJ.g/L not 20.81lg/L not provided.

applicable applicable

Enhancement Qualitative of enclosurecaused statement

successional NR NR NR Not calculable NR based on

observation changes in and DCCA phytoplankton

~om~llnity ;* O'd;",tion I - ~~----~

Increase in No further data

Cladocera NR NR NR Not calculable NR or details

Mean hmnthc

09 IL -_. not--1 ~~~d~:~~t~ effect 1.0 not

. Ilg applicable Ilack of effect. density of

not not No data zooplankton effect 9.9 12.4 IlglL applicabl~rovided. _J

7
Note: NR=Not Reported

Balded values are those data points specified in the "Cosm Data Points and Exposure Prames" spreadsheet.

Since no time series of atrazine concentrations were provided, but the study stated that

atrazine concentrations did not change much over time, the midpoint ofthe range of

concentrations measured at each treatment level was used as the time-weighted average.

The minimum and maximum measured concentrations, in !lM, were cited in the study

text. Calculation oftime-weighted averages involved simply converting to units of !lg/L

and taking the average of the minimum and maximum. See below, and the linked Excel

spreadsheet.

11M J,lg/L midpoint of range::: TWA

atrazine atrazine (ug/L)

range -

low 0.005 1.1 0.9

0.003 0.6

rangemed 0.069 14.9 12.4

0.046 9.9

rangehigh 0.111 23.9 20.8

0.082 17.7

Reviewer Comments: Many factors limit the usefulness ofthis study for the purposes of

the atrazine literature review. Most obviously, since the authors observed much more

substantive impacts with copper addition, they focused primarily on the copper results at

the expense of a full presentation ofthe atrazine results.

The basic study design was limited by having only two controls at each treatment level,

and all treatment concentrations were low. The study lasted only a few weeks, and the

enclosures had strongly altered physical, chemical, and biological conditions (i.e. mixing

with depth, pH, and predation) which limit the generality ofthe results to naturallentic

systems.

ManY'details regarding both methods and results were not provided. The year ofthe

experiment is not stated, only the month and days. Very little information is provided

about the site and the general setup, including the lake size and surrounding land uses, the

distance ofthe enclosures and lake samples from the shore, and the relative locations of

treatment and control enclosures. It is not stated when or how abiotic 14C was determined

in the 14C uptake measurement protocol. There is no information on how nutrient levels

or pH were determined. Atrazine concentrations appear to be exhaustively measured;

however, the results are not shown, and only minimum and maximum values at each

8
~/t~!( l

treatment level are provided. Many results, both with and without reported effects, are

not described adequately (that is, with the use of data tables or graphs). These include

atrazine tolerance with prior atrazine exposure; increases in chlorophyll a and the

chlorophyll/POC ratio; decrease in the paC/paN ratio; lack of effects on nutrient

concentrations and zooplankton; and time-dependent phytoplankton community

composition (i.e. successional changes). The Data Summary Table has many missing

entries due to the lack of complete reporting on these results.

In terms of presentation, there were other problems with the study. The DCCA

(detrended canonical correspondence analysis) was not useful it was poorly described

and was able to account for only 60% ofthe total species abundance variability in the

first two components. Multiple figures and tables were not fully explained, that is,

symbols were presented in the tables for which no definition was available in the study

text or in the caption, and additionally some apparent typos in the tables interfered with

interpretation.

Description of Use in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: The results ofthe study show that atrazine caused no effect to

phytoplankton and periphyton community tolerance at concentrations::: 20 !!g/L.

Limitations of Study: The study is limited by only two replicates at each treatment level,

very low atrazine treatment concentrations (20 !!g/L or less) studied for a short period of

time (3 weeks), and physical/chemical/biological artifacts due to the Closed-bottom 6 m3

water volume enclosures. In addition, no effort was made to document whether atrazine

or other chemicals were present in lake water or enclosure controls. Moreover, very little

information about the atrazine effects (or lack thereof) was provided in the article. Other

limitations associated with this study are described in the Reviewer's Comments.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Anita Pease, M.S., Senior Biologist, Environmental Fate and

Effects Division, U.S. Environmental Protection Agency.