Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Hamala, l.A. and H.P. Kollig. 1985. The effects ofatrazine on periphyton communities

in controlled laboratory ecosystems. Chemosphere. 14:1391-1408 (MRID 47543615).

Purpose of Review:

Determine potential effects of atrazine on 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 of Concern (LOC).

Date of Review:

November 24,2009

Purpose of Study:

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

community) in experimental laboratory flow-through chambers (microcosms).

Summary of Study Findings:

Methods:

The experiment was conducted at the EPA's Aquatic Ecosystem Simulator (AEcoS), a

laboratory chamber system built in a 19.5 m long, 0.46 m wide, 0.51 m deep, "U"-shaped

flume. The flume was divided into 4 independent flow channels (A, B, C, and D), each

further subdivided into four 250-L microcosms (1, 2, 3, and 4), which were set up in flow

sequence so that the effluent from an upstream microcosm passed over a weir into the

next downstream microcosm. The flume was made of Plexiglass and lined with 4-mil

Teflon film. Uniform mixing in each microcosm was achieved with longitudinally

suspended paddlewheels. Deionized water was gravity fed into the upstream microcosm

at a flow of250 L/day. Water temperature was maintained at 15°C. A photoperiod of 12

hours light, 12 hours dark was maintained with fluorescent lights at 8608 lux. The

microcosms AI, Bl, Cl, and Dl were used for initial nutrient introduction (via peristaltic

pumps) and additional nutrients were added to A2, B2, C2, and D2. See Table 1 for

nutrient concentrations. The second set of microcosms (A2, B2, C2, D2) were the set

sampled for the experiment, two as controls and two as treatments at a single treatment

concentration.

'1.1.11111

2087840
Periphyton communities were established in the microcosms by inoculating and then

cross-inoculating each with composite tow and littoral sediment samples from local

northeast Georgia ponds that were not expected to have background atrazine exposure

(no further information on pond sources provided). Teflon-covered plastic strips (10 x 30

cm) were suspended along the sides of each microcosm for 3 different 14-day

colonizations, with sampling on days 44, 59, and 80 after inoculation. Microcosms were

allowed to be colonized and reach steady state for 43 days, by which time dissolved

oxygen diurnal cycles had become similar, with concentrations from 7-13 mg/L,

indicating functional stability.

Atrazine was added to the microcosms A2 and D2 on the 44th day after periphyton

inoculation. First each microcosm was "loaded" to a concentration of 100 Ilg/L, and then

this concentration was maintained with a constant-head flask and dripper input system

calibrated to deliver 2 mUmin. The atrazine spiking solution was prepared by dissolving

technical grade atrazine (96.5% active ingredient, Ciba-Geigy Corporation) in methanol

in a volumetric flask. A portion ofthis stock solution was added to an Erlenmeyer flask,

the methanol was evaporated with dry nitrogen gas, and distilled water was added and

stirred on a magnetic stirring plate for 24 hours. Samples were taken periodically and

analyzed to confirm complete dissolution ofthe atrazine. Atrazine input to the treatment

microcosms was stopped on the 59th day after periphyton inoculation (Day 14 after

addition), and the microcosms were allowed to recover for 21 days (until Day 35 after

addition). (Note that although the article numbers days beginning from periphyton

inoculation, the review will count Day 0 as the day of atrazine addition.) Triplicate water

samples were taken for atrazine analysis every 3 days with methylenechloride using an

ultrasonic Politron apparatus, and analyzed using a Tracor 900-78S HPLC equipped with

Partisil ODS-2 column and water-methanol mobile phase. Atrazine concentrations

during the exposure period averaged 89.6 ± 3.3 Ilg/L in microcosm A2 and 96.9 Ilg/L ±

6.0 Ilg/L in microcosm D2.

Perophyton strips were sampled on Day -1, 14, and 35 after atrazine addition (one day

before treatment began, at the end ofthe treatment period, and at end of a 21-day

recovery period), in each case after 14-day colonization periods. A known strip area was

scraped into a beaker, the sample was brought up to a known volume with distilled water

and blended at high speed for 5 seconds, and aliquots ofthe resultant slurry were

dispensed for algal enumeration, chlorophyll a content analysis, and determination of

ash-free dry weight (AFDW).

For identification and enumeration, algal samples were preserved with diluted acid

Lugol's solution and examined with a Zeiss invertoscope 0 microscope with brightfield

and phase contrast capabilities. Enumeration, excluding diatoms, was based on the

inverted microscope method of Utermohl. Density (excluding diatoms) was estimated

from counting at least 400 algal units at 640 x , units being unicells, coenobia, palmellas,

colonies, or filaments. For diatoms, permanent slides were prepared by "cleaning" the

2
sample using Van der Werff (1958)'s organic digestion procedure, drying a diluted

aliquot on a round coverslip, and mounting the coverslip onto a microscope slide with

Hyrax medium. Diatom density was then estimated by counting at least 400 diatom

valves at 1000x. A total of 33 algal taxa were identified in the experiment (see Table 2).

Algal species diversity (H ') was determined with the Shannon-Weiner equation, and

changes in algal community structure were evaluated with the SIMI index, which has a

maximum of 1 when species composition and average relative densities are identical and

owhen they are completely different. (The study cites sources for these measures, but

does not define them within the article.)

Algal biomass as chlorophyll a was analyzed with a Turner Design Modell 0 fluorometer

and corrected for phaeophytin a. Samples were extracted with 90% acetone within two

hours of collection using a B. Braun cell homogenizer. Total community biomass as

AFDW was measured according to the American Public Health Association's 1981

standard methods.

Dissolved oxygen (DO) in the microcosms was measured hourly using an Orbisphere

Model 2708 monitor interfaced with a computerized system. Community metabolism

measures (net and gross productivity, nighttime and total respiration) were calculated

from the hourly DO changes with a computerized modification of Odum's (1956) mass

balance equation for open, flowing water systems (no further description provided).

A nested analysis of variance was used to test significance of differences between

controls and treatments on each periphyton sampling day in the following variables: total

algal density, algal division density, chlorophyll a, and AFDW. All algal densities were

transformed by 10glO(X+1) prior to statistical analysis (the purpose ofthis transformation

is not explained, normality tests ofraw and transformed data are not mentioned, and

specific ANOVA techniques are not described). Community metabolism results were

averaged within replicate treatments and plotted over time without any statistical

analysis.

Results:

By the end ofthe pretreatment period (43 days after inoculation), all colonizable surfaces

in the microcosms were covered with a dense mat of periphyton. Plankton were not a

significant part ofsystem biomass due to the flow-through nature ofthe microcosms (this

was confirmed by unpublished data). Microinvertebrates in the periphyton mat were

sparse except for rotifers and nematodes. Periphyton communities were dominated by

(average relative abundances follow in parentheses) Chlorophyta (71 %), Bacillariophyta

(19%), Cryptophyta (3%), and Cyanophyta (2%). See Figure 1. Dominant species were

Chlamydomonas spp. (27%), Stigeoclonium lubricum holdfast cells (23%), Characium

ambiguum (7%), Chlorella sp. (7%), Fragillaria crotonensis (17%), Cryptomonas ovata

(3%), and Chroococcus minor (2%). Community biomass (chlorophyll a and AFDW)

before treatment was within the range observed during previous AEcoS experiments, and

3
algal diversity estimates indicated a relatively complex, mature periphyton community

(see Table 3 pretreatment values). No significant differences existed between control and

treatment microcosms prior to atrazine addition (see Table 3 and Figure 2).

After 14 days of atrazine treatment at nominal levels of 100 !Jg/L, community

metabolism was profoundly disrupted relative to controls. Net productivity decreased

within hours of atrazine addition and continued to decrease throughout the exposure

period, falling to negative values by Day 10 after addition. The effect on respiration was

much weaker than the effect on productivity, with no consistent change in nighttime

respiration during the first week after atrazine addition. During the second week,

transient increases in nighttime respiration of 8-34% over controls were noted, which the

authors attribute to a delayed increase in heterotrophic activity. The P/R ratio decreased

to 0.63 immediately after atrazine addition and continued to decline until atrazine inputs

ceased, with a minimum of 0.29 on Day 13 after addition. See Figure 2 for time-series

plots of net productivity, dark period respiration, and P/R.

Community structure changes on periphyton strips that were colonized during the

treatment period were observed, including decreased algal density and diversity, altered

species composition, and reduced biomass accumulation in treatments relative to

controls. Total algal and Chlorophyta average density were significantly different from

controls (p :s 0.05), decreasing to 23% and 15% of control values respectively. See Table

3. Green algal species with large average density decreases included Ankistrodesmus

faleatus (11 % of control value), Chlamydomonas spp. (16%), Cosmarium reinsehii

(10%), Scenedesmus dimorphus (1 %), Straurastrum manfeldtii (10%), and Stigeoclonium

lubricum holdfast cells (15%). See Table 4. The Bacillariophyta and Cryptophyta were

minor components ofthe periphyton community in both controls and treatments (see

Figure 1). Substantial decreases in density were observed for these divisions, however,

compared to control values (see Table 3). Chroocoecus minor, a small unicellular

Cyanophyte, increased to 224% of control values (Table 4) and accounted for the large

increase in Cyanophyta relative abundance in treatment microcosms (Figure 1). As Table

4 shows, individual algal species varied considerably in their response to atrazine level of

100 !Jg/L for 14 days; while most declined in cell density, sometimes dramatically so, a

few species increased. The authors note that these effects were perhaps characteristic of

natural assemblages and could not necessarily have been predicted from single species

bioassays. The algal diversity also decreased with treatment relative to controls: H' was

2.35 in treatments and 2.77 in controls, and total number ofspecies was 18 in treatments

and 24 in controls. SIMI (based on all observed taxa) was 0.64 in treatment relative to

controls (indicating dissimilarity), although only 3.3% ofthe total relative abundance in

control microcosms were not observed in the treatment microcosms. The authors

interpret this as meaning that atrazine eliminated some ofthe rarer algal species, and

changed the relative abundances ofthe dominant species. See Table 3 for summary data.

Chlorophyll a and AFDW decreased to 8% and 52% of control values, respectively,

during the treatment, but sample variation among the two replicates was high and so the

4
differences were found to be insignificant. However, since chlorophyll a was apparently

more affected by atrazine treatment than AFDW, the authors state that either much ofthe

AFDW biomass was nonalgal (note that nematodes and rotifers were present in the

periphyton mats), or else the per-cell chlorophyll a content declined due to treatment. In

either case, although the results are not statistically significant, the authors conclude that

atrazine did reduce the overall algal growth rate.

During the 21-day recovery period, in which no atrazine inputs were applied to the

treatment microcosms (complete washout occurred within 4 days in the sediment-free

microcosms), daily net productivity recovered steadily until it was similar to control

values on Day 31 after atrazine addition. In nighttime respiration, there was a transient

decrease (82.5-86% of control values on Days 15-16 after atrazine addition) which came

back to within 90% of control values after Day 16. P/R ratio recovered similarly to net

productivity, achieving a value of 1.04 on Day 31. See Figure 2. The authors suggest

that the fairly rapid recovery in these parameters indicate that atrazine's primary effect is

algistatic and not algicidal in nature.

In contrast to the community metabolism measures, periphyton community structure

measures showed little to partial recovery during the recovery period. Significant (p :s

0.05) differences were still found in the total algal average density (25% of controls) and

total Chlorophyta average density (25% of controls). See Table 3. Some green algal

species increased as percentages of control abundances compared to the treatment period

(Characium ambiguum, Cosmarium reinschii, Stigeoclonium lubricum), while others

remained low (A nkistrodesmusfalcatus, Scenedesmus dimorphus, Staurastrum

manfeldii). See Table 4. The total average density ofBacillariophyta increased in

treatment microcosms to 74% of control values, due primarily to a large increase in

Nitzschia palea, which comprised 96% oftotal Bacillariophyta (Table 4). Total average

density ofCryptophyta remained low in treatment microcosms (8% of control values)

while that ofCyanophyta decreased to percentage of control values seen in the

pretreatment period (calculated from Table 3 data). Algal diversity increased

substantially to become similar to or higher than control and pretreatment values (Table

3), but the SIMI value remained low at 0.60, indicating that community composition

remained dissimilar from controls. The authors note that it seems some ofthe rare

species were able to reestablish themselves, but the relative abundances ofthe dominant

species remained substantially different.

Chlorophyll a and AFDW community biomass estimates in treatment microcosms were

25% and 33%, respectively, ofthe control values. In comparison with the treatment

period, chlorophyll a increased and AFDW decreased (Table 3). The authors interpret

the results to indicate that chlorophyll a per algal cell increased during the recovery

period, although total algal density remained lower.

5
At one point, the authors tangentially describe unpublished data showing that, after a full

recovery post-treatment, the periphyton communities did not appear to develop atrazine

resistance when again exposed to 14 days of 100 /lg/L atrazine.

Table 1. Elemental concentrations of nutrient input to microcosms AI, 81, Ct, and D1.

Macronutrient m /L Micronutrient

P 0.01 B

Si 5.88 Mn

HC03 75 Zn

Mg 2.67 Fe

K 3.02 Co

NOrN 0.083 Cu

Ca 9.65 Mo

Na 37.96

S04 13.91

Cl 38.08 EDTA

Additional inputs to A2, B2, C2, and D2:

NOrN 0.747

P 0.09

C 4

/L

30.8

100

14.9

31.4

0.33

0.0035

2.75

440

Table 2. Algal species identified during the experiment.

Cyanophyta

Chroococcus minor (Kuetz.) Naegeli

Oscillatoria subbrevis Schmidle

BacilJariophyta

Achnanthes minutissima Kutz.

Cymbella minuta Hilse ex. Rabh.

Gomphonema gracile Ehr. emend. V.H.

Gomphonema parvulum (Kutz.)

Fragilaria erotonensis Kitton

Navicula minima Grun.

Nitzschia palea (Kutz.) W. Sm.

Synedra delicatissima W. Sm.

Synedra socia WalJace

Synedra ulna (Nitz.) Ehr.

Cryptophyta

Cryptomonas ovata Ehr.

Chlorophyta

Ankistrodesmusfalcatus (Corda) Ralfs

Chaetosphaeridium globosum (Nordst.) Klebahn

Characium ambiguum Hem1ann

Chlamydomonas spp.

Chlorella sp.

Coelastrum mieroporum Naegeli

Cosmarium meneghinii Breb

Cosmarium reinschii

Cosmarium undulatum

Mougeotia sp.

Oedogonium sp.

Scenedesmus abundans (Kirch) Chodat

Scenedesmus bijuga (Turp.) Lagerheim

Scenedesmus denticulatus Lagerheim

Scenedesmus dimorphus (Turp.) Kuetzing

Spirogyra sp.

Staurastrum manfeldtii

Staurastrum paradoxum Meyen

Stigeoclonium lubricum (Dillw.) Kuetzing

6
Ulothrix zonata (Weber and Mohr) Kuetzing

Table 3. Periphyton community structure estimates after each ·atrazine exposure period

(pretreatment, treatment, recovery). Mean values (n=6, 2 replicate microcosms, 3 replicate water

samples from each) and standard deviations (SD) are shown. Values in bold are significantly

different than control values (p 0.05).

Pretreatment Treatment Recovery

Mean SD Mean SD Mean SD

Algal Density (unitslcm2

)

Total Algae Control 8.76E+05 1.09E+05 4.83E+06 1.01E+06 8.13E+06 2.45E+06

Treatment 8.84E+05 1.55E+05 1.01E+06 2.19E+05 2.03E+06 7.06E+05

Chlorophyta Control 7.25E+05 6.70E+04 4.19E+06 8.00E+05 5.74E+06 5.39E+05

Treatment 6.13E+05 2.24E+04 6.16E+05 1.46E+05 1.46E+06 5.56E+05

Bacillariophyta Control 1.16E+05 7.07E+04 9.31E+04 6.59E+04 3.78E+05 3.93E+05

Treatment 2.l7E+05 6.24E+04 1.81E+04 6.72E+03 2.84E+05 1.44E+05

Cyanophyta Control 1.40E+04 l.79E+04 2.00E+05 I.72E+05 1.73E+05 6.75E+04

Treatment l.76E+04 2.77E+04 4.32E+05 6.55E+04 1.44E+05 2.48E+04

Cryptophyta Control 2.06E+04 9.06E+03 3.46E+05 3.IOE+05 1.85E+06 1.93E+06

Treatment 3.64E+04 1.04E+04 3.04E+04 2.32E+04 1.41E+05 7.17E+04

SIMI (similarity) Trt. vs. Crl. 0.88 0.64 0.6

H' (diversity) Control 3.18 2.77 2.88

Treatment 3.26 2.35 3.26

Total # Species Control 22 24 25

Treatment 21 18 22

Chl. a (flglcm2

)

Control 3.22 0.86 3.89 1.55 3.92 1.33

Treatment 3.89 1.05 0.3 0.04 0.98 0.47

AFDW (mglcm2

)

Control 0.3 0.03 0.27 0.08 0.33 0.13

Treatment 0.27 0.04 0.14 0.08 0.11 0.03

7
Table 4. Average algal species densities, as percentages of control values, for dominant species on

periphyton strips after each atrazine exposure period. * denotes that the species was not observed in

either control or treatment microcosms. Blank values for Synedra ulna appear in the original article.

Pretreatment Treatment Recovery

126 224 90

132 11 16

93 0 118

117 16 38

56 44 78

97 0 * 131 10 46

133 1 3

86 10 17

268 53 110

56 15 76

60 29 128

203 34 7

122 25 110

164 87 32

Species

Bluegreen

Chroococcus minor

Green

Ankistrodesmusfalcatus

Characium ambiguum

Chlamydomonas spp.

Chlamydomonas spp. (palmella stage)

Chlorella sp.

Cosmarium reinschii

Scenedesmus dimorphus

Staurastrum manfeldtii

Stigeoclonium lubricum

S. lubricum holdfast cells

Diatoms

Achnanthes minutissima

Fragilaria crotonensis

Nitzschia palea

Synedra socia

Synedra ulna

Cryptophyta

Cryptomonas ovata

8

177 9 8
Pre- Treatment

Treatment Recovery

"'-

o

t':' 80 "-

60

40

20

o

c 'I c c T

Figure 1. Average relative abundance of algal divisions (% of total) in Control (C) and Treatment

(T) microcosms before, during, and 21 days after atrazine treatment at 100 IlglL for 14 days.

9
(b)

'I ,., I /\' ,I

f

150.0 I I I ar-j I J I I I I I (i', i , iIi I ' I

36 40 '" 48 52 56 60 64 68 72 76 80

Time (days)

300.0

o

:.:: 1-0 275.0

""J:

.~ C\l

Co .... 250.0 CIl""'... ll.l_

0::""'... N Z25.0 "t:IO

.8 CIl

1i> 200.0

Cl. c

S

:t 175.0

to

Cl

(a)

I

I-~"

'\,.,....J

36 4Q '" 48 52 56 60 64 68 72 76 80

Time (days)

o

S 0.0

:t -50.0

-100.0

400.0

350.0

1-0 >,J: 300.0 ....

':;N 250.0~ ......... t:.::- 200.0

:::l""'... "t:I N 150.0

0 0 1-0 100.0

c.. CIl

50.0 ....

Cl)

Z

1.50 (c) -'- Control lLicrocosms --- Treatment Kicrocosm.s

0.75

0.50

Fia=e 2. Average Net Productivity (2&).

Dark Period Respiration (2b). and PjR Ratio1

(2.:) in Control and Treatment lLicrocO:mls.

I Gross Productivity (GP) 1) P R = . I TotAl Respiration (TR)

where GP - Net Productivity + Dark

Period Respiration and TR = 2 (Dark

Period Respiration)

'-~ 1

I

II I I I... f

L f " \,.'\,J v

'_, I

t '---''Jf 0.25

1.25

o 1.00

....

--Il:l

c::

c::

""'...

c..

0.00 I i Ti-j i I I I I I ",riO, iIi I I I ' I

36 40 '" 48 52 56 60 64 68 72 76 80

Time (days)

Figure 2. See caption in lower right subplot area. Atrazine input began on "Day 45" (called Day 0 in the review) and ended 14 days later.

10
Data Summary Table:

E.ffect Magnitude of Nominal Day ofStudy Time Did Comments

Effect relative Atrazine Effect Weighted Recovery

to controls Concentration Occurred Average Occur - if

(j1g/L) Atrazine so, when

Concentration (see

from Day 0 to comment)?

When Effect

Occurred

Not during From Fig. 2

Decrease in treatment, and text p.

net

100% but full 1398.

community (became 100/-lg/L Day 6 93.3 J..lg/L recovery Recovery

productivity negative) after -16 days

treatment postended. treatment.

Decrease in Day 14 Partial at

total (effect end of21-

number of 25% 100/-lg/L measured at 93.3 J..lg/L day From Table

end of 3. periphyton treatment recovery

species period) period

Decrease in Day 14

biomass of (effect

periphyton 50% 100/-lg/L measured at 93.3 J..lg/L No From Table

end of 3. (ash-free treatment dry weight) period)

Change in 35% Day 14

algal species (different (effect From Fig. 1,

composition Chlorophyta 100/-lg/L measured at 93.3 J..lg/L No significance

(decrease in cell density end of from Table

Chlorophyta with p < treatment 3.

abundance) 0.05) period)

Day 14

Decrease in 75% (effect

total algal (different 100 J..lg/L measured at 93.3 J..lg/L No From Table

end of 3. density with p < 0.05) treatment

period)

Decrease in Day 14 Yes, at

periphyton (effect end of21- measured at From Table algal 15% 100 J..lg/L end of 93.3 J..lg/L day 3. diversity treatment recovery

(H') period) period

Balded values are those data pomts speCIfied m the "Cosm Data Pomts and Exposure Profiles" spreadsheet.

Time weighted averages are the average of 89.6 Ilg/L and 96.9 Ilg/L, cited on p.I394 as

the average measured atrazine concentrations in the two microcosms over the treatment

period. Since application was continuous and no further concentration data were

provided, the time weighted average on Day 6 was assumed equal to that of Day 14.

11
Reviewer Comments:

This was a well-controlled laboratory microcosm study with periphyton. Results were

presented clearly and relatively completely. Although the laboratory system was not very

realistic, it provides a reasonable test bed for the limited question of direct effects of

atrazine on the periphyton community. Since the microcosms are not complete with drift

plankton and other communities that would be found in a natural aquatic ecosystem,

indirect ecosystem-level effects cannot occur in this experiment. The experimental

design was further limited in that it used only two replicates and one treatment level, the

atrazine treatment lasted only two weeks, and most measurements (besides DO for

community metabolism) were taken infrequently.

Some aspects ofthe methodology could have been further described. The natural source

ofthe inoculant periphyton was not described in detail. The equations used for

community metabolism were not defined, nor was their implementation on the computer

system described; likewise, the community diversity and similarity measures were not

defined within the article. The explanation ofthe statistical analyses was vague and left

unclear the interpretation ofthe "significant differences" that were found, although with

only two replicates, it would have been difficult to detect significant differences in any

case.

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

Rationale for Use: The study demonstrates that atrazine has direct and indirect effects

on periphyton community structure at constant exposure concentrations of 100 Ilg/L.

Community productivity was immediately and severely inhibited during the 14-d

exposure period; however, the recovery was fairly rapid. Changes in community

structure were evidenced by decreased algal density and diversity, altered species

composition, and reduced rates of biomass accumulation. Little to no recovery in

community structure was observed during the 21-day recovery period.

Limitations of Study: Only two replicates and one test concentration were used in this

study, community composition and biomass measurements were infrequent (there was

only one "treatment" measurement day), and the laboratory microcosm design precluded

any ecosystem-level effects. In addition, it is unclear whether the source ofthe

periphyton communities taken from the northeast Georgia ponds had been exposed to

background levels of atrazine and/or other pesticides However, direct effects of

continuous 100 Ilg/L atrazine on the periphyton community metabolism and algal cell

density were obvious.

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

12
Secondary Reviewer: Anita Pease, M.S., Senior Biologist, Environmental Fate and

Effects Division, U.S. Environmental Protection Agency.

~!l~/11

13