Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Downing, H. F., M. E. DeLorenzo, M. H. Fulton, G. 1. Scott, C. J. Madden, and J. R. Kucklick.

2004. Effects of the agricultural pesticides atrazine, chlorothalonil, and endosulfan on south

Florida microbial assemblages. Ecotoxicology. 13: 245-260. (MRID 48261110)

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: October 7,2009

Summary of Study Findings

Purpose of Study:

This study examines the potential effects of atrazine on phytoplankton and zooplankton collected

from two locations along a southern Florida canal. Chlorothaloni1 and endosulfan effects on the

aquatic microbial assemblages were investigated separately; those results are not reported here.

Methods:

Microbial assemblages (algae, bacteria and protozoa) were collected from two locations (Sites C

and E) along the C-111 canal, located in Miami-Dade County, Florida, during October (wet

season) 1999 and February (dry season) 2000 (Figure 1). Site C was surrounded on all sides by

agricultural fields used primarily for tomato production, while Site E, ~5 miles downstream from

Site C, was surrounded mostly by sawgrass marsh. Artificial substrates [polyurethane foam units

(PFUs), each 4 x 5 x 6 cm

3

], attached to a 2-m nylon rope via cable-ties, were positioned at a

depth of 30 cm below the water surface, forty-eight substrates per collection site.

Microorganisms were allowed to colonize the artificial substrates for 7 days (168 hours). The

substrates were then collected, beneath the water surface, into I-gallon, plastic jars; eight

substrates per jar. The jars, containing substrates plus canal water, were sealed and transported

on ice to the NOAAlNational Ocean Service (NOS) Charleston (South Carolina) Laboratory.

Prior to transport, microorganisms were "extracted" from some ofthe artificial substrates (PFUs,

number not specified) on site immediately after collection by gently squeezing the PFU contents

- 1 -

'••••••111 2087821
(~60 mL) into individual, sterile sample jars, with additional PFUs "extracted" following

transport to the laboratory. Canal water (24 L) was collected from each site into stainless steel

cans (size not specified) for use during atrazine exposure experiments. Additional canal surface

water (volume not specified) was collected from each site into l8-L stainless steel containers at

five unspecified intervals during the 7-day colonization period for atrazine analysis as described

below.

Atrazine exposure. Each test system consisted of a 600-mL glass beaker containing one

colonized artificial substrate (PFU) and 500 mL of filtered (0.22-llm, Gelman Maxi Capsule

Filter) canal water (respective to PFU collection site) amended with nutrients according to

ASTM (1996) standard protocols for freshwater medium preparation. Atrazine (purity 96%), in

acetone, was applied at 20 and 200 Ilg/L; final co-solvent concentration 0.01 %. Control systems

treated with acetone at 0.01% were also prepared. All treatments (0, 20 and 200 Ilg/L atrazine)

were replicated in triplicate. Following treatment, the beaker contents were stirred to mix.

Incubation conditions were not reported. 15 mL water samples were collected for atrazine

analysis randomly from the beakers at 0, 24, and 168 hours posttreatment. Artificial substrates

(PFUs) were removed at 24 and 168 hours posttreatment and the microorganisms collected

(extracted) as described above. Extracted microorganisms were homogenized by gently stirring,

then aliquots were removed and analyzed for chlorophyll a content, photosynthetic activity

(carbon assimilation), bacterial abundance, heterotrophic bacterial productivity (leucine

incorporation) and species composition as described below.

Atrazine in canal surface water. 2-3 Lwater samples were filtered (0.7-llm, 90-mm glass fiber,

Whatman type GF/F) under pressure, loaded onto pre-conditioned (methylene chloride, acetone)

0.5-g Isolute ENV+ solid phase extraction (SPE) cartridges, and stored frozen (temperature,

interval not reported) until elution. Atrazine residues were eluted with acetone, followed by

methylene chloride. Extracts were evaporated under nitrogen, then resulting residues were

reconstituted in hexane and, as needed, cleaned up via florisil micro-column. Samples were

again evaporated under nitrogen, with resulting residues reconstituted in isooctane and analyzed

using GC-MS with electron ionization (EI) under the following conditions: J&W Scientific DB5ms GC column (0.25 mm x 60 m, 0.25 11m film thickness), injection volume 2.0 ilL, flow rate

1.5 mL/min., velocity 32 cm/sec., collision gas methane, temperature program of 60°C for 1 min.

: ramp to 150°C at 25°C/min. : ramp to 240°C at 5°C/min. : ramp to 300°C at 15°C/min.: hold

for 3 min., total run time 29.60 min., monitoring m/z 200 and 215, with atrazine-d4 as an internal

standard (m/z 205); limit of detection was not reported.

Verification of atrazine treatment rates for exposure tests. The 0-, 24- and 168-hour test system

water samples (15 mL) were separately loaded onto pre-conditioned (methanol, deionized water)

l-g octadecyl (C18) SPE cartridges (Burdick and Jackson). After sample loading, the cartridge

was rinsed with water, dried under vacuum for 5 minutes and atrazine was eluted with ethyl

acetate. The resulting eluate was dried over anhydrous sodium sulfate (Na2S04), evaporated

under nitrogen, with the residues reconstituted in isooctane and analyzed by GC/MS-EI as

described above.

- 2 -
Chlorophyll a. Microorganism samples (10 mL) were filtered (GF/F), then the filters were

placed in vials with saturated magnesium carbonate (MgC03) solution (1 mL) and stored frozen

(-20°C, interval not specified) until analysis. Prior to analysis, samples were extracted with

acetone (final concentration 90%), shaken, refrigerated overnight, shaken and again refrigerated

overnight (total extraction interval 48 hours), then brought to room temperature in darkness and

analyzed using a fluorometer (Sequoia-Turner Model 450).

Phototrophic carbon assimilation (primary productivity). Microorganism samples (5 mL) were

treated with [14C]sodium bicarbonate (NaH14C03, concentration not reported), then maintained at

in situ light and temperature regimes (not further detailed) in an incubator for 24 hours.

Following incubation, the samples were filtered (0.45-~m, cellulose, nitrate), then the filters

were rinsed with filtered (O.2-~m) canal water (respective to microorganism collection site),

placed in scintillation vials with 10% HCI and allowed to sit in darkness overnight. Thereafter,

scintillation fluid was added and the samples were equilibrated overnight and analyzed for total

radioactivity using LSC.

Phytoplankton (microalgae) community composition. Changes in community based on photopigment composition were investigated using October 1999 microorganisms exposed to atrazine

(20 and 200 ~g/L) for 24 hours. Microorganism samples (10-20 mL) were filtered (25-mm,

GF/F) and the filters stored frozen (-80°C) until extraction. Samples were extracted with icecold acetone and sonication (1 minute), with resulting extracts stored frozen (-20°C) overnight.

The samples were centrifuged (2,795 x g, 10 minutes, 4°C), then an aliquot (1 mL) was filtered

(0.2-~m) into a nitrogen-filled, amber 1.8-mL HPLC vial and stored frozen (-80°C) until analysis

by reverse-phase HPLC under the following conditions: VYDAC 201TP C18 column (4.6 x 250

mm, 5 ~m particle size, 300 A pore size), methanolic mobile phases (Van Heukelem et al.,

1992), inline diode array detector (method not further described). Pigments were identified

based on comparisons of absorbance spectra and against reference standards.

Community composition was determined via identification of phototrophic taxa in

gluteraldehyde preserved samples (see bacterial abundance below) using an Olympus BX-50

epifluorescence microscope with magnification at 500.

Bacterial abundance was determined via acridine orange direct counts; method ofHobbie et al.

(1977) as modified by Kepner and Pratt (1994). Microorganism samples (5 mL) were preserved

with gluteraldehyde (final concentration 2%), then stored in darkness at 4°C until analysis.

Preserved samples were diluted with filtered (0.2-~m) deionized water, then stained, in darkness,

with acridine orange (1 00 ~g/L, final concentration) for 3 minutes. The stained samples were

filtered (0.2-~m Poretics PCTE black polycarbonate membrane), then bacterial counts were

conducted using an Olympus BX-50 epifluorescence microscope, with magnification at 1,250,

and equipped with a super high-pressure mercury lamp under blue light excitation (470-490 nm).

- 3 -
Leucine incorporation (heterotrophic bacterial productivity). Microorganism samples (5 mL)

were treated with eH]leucine (105 nmol/L, final concentration) and incubated in darkness at in

situ temperature (not further detailed) for 60 minutes. Addition of 50% trichloroacetic acid

(TCA) stopped leucine incorporation, and the samples were extracted in a hot water bath at 80°C

for 15 minutes. Extracted samples were cooled, then filtered (0.45-llm cellulose nitrate) under

low vacuum. Filters were rinsed with ice-cold 5% TCA, followed by ice-cold 80% ethanol, then

placed in scintillation vials, dissolved with ethyl acetate and analyzed for total radioactivity using

LSC.

Statistical analyses. Response values were analyzed for variance heterogeneity and normality

(Shapiro Wilk W test, JMP software) and, ifrequired, results were transformed to meet

assumptions of parametric tests. A multi-factor, multivariate analysis of variance (MANOVA)

was used to detect differences in microbial assemblages among seasons, collection sites,

treatments and length of exposure. Average number oftaxa per PFU were analyzed using twoway ANOVA. For both MANOVA and ANOVA analyses, a priori orthogonal contrasts were

used to detect significant differences between treatments and controls. Photo-pigment results

(normalized to chlorophyll a) and changes in relative abundance of dominant taxa groups were

analyzed using two-factor MANOVA, with collection site and treatment as main effects. Genera

were grouped as follows: (1) Bacillariophyta, (2) Cryptophyta, Chrysophyta and Dinophyta, (3)

Cyanophyta, (4) Heterotrophic protists, and (5) Chlorophyta and Euglenophyta.

Results:

In the canal water at sites C (agricultural site) and E (sawgrass marsh site), atrazine was detected

at 18.92 ± 1.20 ng/L and 24.5 ± 3.77 ng/L, respectively, in October 1999 (wet season), and at

32.68 ± 1.24 ng/L and 11.3 ± 1.01 ng/L, respectively, in February 2000 (dry season) indicating

the microbial assemblages were pre-exposed to atrazine during the 7-day colonization period

(Table 1).

During the acute exposure experiments, measured atrazine concentrations at the 20 Ilg/L

application rate were 119-124% of nominal at time 0, decreasing to 82-101% of nominal at 24

hours and 69-86% of nominal at 168 hours (Table 2). At the 200 Ilg/L application rate, atrazine

was detected at 56-92% of nominal at time 0, 59-69% of nominal at 24 hours and 41-48% of

nominal at 168 hours. Any analysis of water from the untreated control test systems was not

reported.

At the 200 Ilg/L treatment rate, atrazine significantly (p<0.05) reduced phytoplankton biomass

(~30-40%, as chlorophyll a) and primary productivity (~60%, as carbon assimilation). With the

exception of the February 2000 microorganisms, where biomass was decreased only ~5% after

24 hours (Figures 2-3), atrazine exposure reduced biomass and primary productivity regardless

- 4 -
of site (C or E), collection season for microbial assemblages (Oct. or Feb.), or exposure duration

(24 or 168 hours).

At the 20 ~g/L treatment rate, phytoplankton biomass and primary productivity were not

significantly decreased (:S~ 10%, p~0.05), when responses were pooled for site, season and length

of exposure (Figure 2). However, in a comparison of seasonal variability, phytoplankton

biomass collected in Oct. was significantly (p<0.05) decreased ~35%, as compared to controls,

after 168 hours of exposure, while biomass for phytoplankton collected in Feb. was stimulated

~+20% (not significantly different from control) after 168 hours (Figure 3).

Two-way MANOVA determined significant (p<O.OOOI) differences in microbial assemblages

between the two collection sites, with the artificial substrates colonized at site E (sawgrass marsh

site) having more taxa (n = 54), as compared to substrates colonized at site C (agricultural site, n

= 43, p. 254). Relative abundance of green algae (chlorophytes + euglenophytes) and

cyanobacteria taxa were higher at site C (30% and 23% oftaxa, respectively), than at site E (26%

and 15%, respectively). While diatoms and heterotroph taxa were higher at site E (33% and

17%, respectively), than at site C (28% and 12%, respectively).

Atrazine exposure significantly (p<0.05) decreased total taxa (phytoplankton + heterotrophs,

sites combined) 10% and ~25%, as compared to controls, after 24 hours at both the 20 and 200

~g/L treatment rates, respectively (Figure 4). However, the extent of the effects varied

according to collection site with total taxa decreased 14% and 36% for microorganisms from

sites C and E, respectively, at the 200 ~g/L treatment rate; site results for the 20 ~g/L rate were

not presented. Based on the reviewer's interpretation of Figure 4, diatoms, bluegreen algae,

green algae and brown algae were decreased ~20%, ~25%, ~30% and 75%, respectively, at the

200 ~g/L rate, as compared to controls, while diatoms, green algae and brown algae were

decreased ~15%, ~1O% and ~25%, respectively, at the 20 ~g/L rate with no effect to bluegreen

algae, and no effect to heterotrophs at either treatment rate. The study authors reported that

green algae and chrysophytes decreased 4% and 5%, respectively, at the 200 ~g/L rate, while

diatoms and heterotrophs each increased by 4%; however, that statement, specifically in regards

to diatoms, appears contradictory to the results depicted in Figure 4.

Pigment analysis found significant (p<0.0001, two-way MANOVA) differences in algal pigment

composition between the two collection sites, with fucoxanthin the most abundant pigment at

both sites; quantitative results were not presented. Concentrations (normalized to chlorophyll a)

of the pigments fucoxanthin, chlorophyll eland c2 were reported to be significantly higher (p

value not reported) at site E, while chlorophyll b was more abundant at site C. Concentrations of

pigments violaxanthin, lutein and canthazanthin were not reportedly significantly (p value not

reported) different between the two collection sites. Atrazine, at 200 ~g/L, decreased levels of

fucoxanthin (~20%) and increased chlorophyll b (~26%) in phytoplankton from site C, while

resulting in almost no change «10%) in pigment levels in phytoplankton from site E; however,

responses were reportedly not significantly different from controls (p value not reported).

- 5 -
Bacterial abundance was significantly decreased (~20%, p<0.05) at the 200 f.lg/L treatment rate,

when responses were pooled for site, season and length of exposure, but there was no observable

decrease at the 20 f.lg/L rate (Figure 2). Bacterial productivity (as eH]leucine incorporation) was

stimulated at both the 20 f.lg/L (~1 0-40%, not statistically significant, p2:0.05) and 200 f.lg/L

(~40-100%, statistically significant, p<0.05) treatment rates (Figures 2 and 5).

The study authors proposed that chronic exposure to "background" levels of atrazine in the canal

may have selected for species that were more resistant, resulting in the reduced responses seen in

these experiments at the lower 20 f.lg/L atrazine treatment rate.

- 6 -
Biscayne

Bay

(;1

Everglades

National Park

Atlantic

Ocean

'\) Bay ..

4 Legend " di>- • Sample Location

cYp " 8'" . Ruads

L! . 0 C-lll Canal

I 0 5 Q

()

-

CJ Land ! .<;1 i i .. Agriculture Kilometers CJ Water

Figure 1. Map ofsouthern Florida study area and sampling site locations (C and E). Figure 1 from Downing

etal.2004.

- 7 -
ChloWlhJltmil Lr,.,Il1Sl;;IhAlraJiol..'

('onlro! AlrilJ.ioi!

) 1·+,'.lf-1

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i

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o'! l·ndn"Jllar CnRlml

• = (\'Ol(\~; 0 =Lo"," ':('[}~'l!nl(-JIlnn: I?J = JlI~h ~\)I1(C'lItrijti'iXI

CIl 1"n>lhak'<lIl FndI",ulf""

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Control

:;; 2t1lHJ

is 161KI ."Jl

:~ JeIK)

<':

c

:-'1_.1

."J:

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f Ce'otro!

Figure 2. Mean responses (pooled for site, season and length of exposure) of chlorophyll a (l1g1L), primary

productivity (phototrophic carbon assimilation, mgIL/d), bacterial abundance (cells/mL), and bacterial

incorporation of [3H]leucine (CPM) to atrazine at 20 I1glL ("Low") and 200 I1gIL ("High"), with standard

deviation (vertical bars); "*,, = significantly (p<0.05) different from control. Low and High treatment rates

were 2 and 20 I1glL, respectively, for chlorothalonil, and 1 and 10 I1glL, respectively, for endosulfan. Figure 2

from Downing et al. 2004.

- 8 -
Oi: "'''~r 1<)')9 rd,rJ~ry ~(KJt)

'MI,O lu,. I,.v'K.,\,mlrill'iun

.!4 hour S hiJ:h (.'tttlL·rnltv.l1cIn

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41......

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':\t".,inr Chk.•r..tlllllonil ":ndmlilllrllQi ("om,trol: ·\trjlJ::int" ("hlclrolh..'"lIil Fnd~t.tf..n

\1'"

(fIlCh""r

.atUI

41ll1Jl--------1

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Figure 3. Mean responses (pooled for site) of chlorophyll a (/!glL) to atrazine at 20 /!g/L ("Low") and 200

/!glL ("High") at each incubation interval, with standard deviation (vertical bars); "*,, = significantly

(p<0.05) different from control. Top = 24 hr posttreatment, bottom = 168 hr. Low and High treatment rates

were 2 and 20 /!glL, respectively, for chlorothalonil, and 1 and 10 /!glL, respectively, for endosulfan. Figure 4

from Downing et al. 2004.

- 9 -
:;:

Chlol'othalonil

tt"l"rl>lrl>pbic htllio<t•

• ehr:-...,ph.) 1,.... (:r~·..l"..h.)I"'-

Oin''1tb}1l"< IIJTu"'n<1

E) Chl"ropIJ, 1(.... Eu~kn' ...h,l(",

l~n'I'IT"

o (:'01""1,11,1'" IhhJcl':rL',-n..

IIRl'fII"ri"l'h,w< ,di"t"m<. *

..:ndosulfan

---.~.._-- 1.<110

Alrll:r.in.,

C'lIIlr,,1 Lu"

(I

l!l

3:5

40

Figure 4. Average number of taxa found in each atrazine treatment (20 or 200 f.lg/L) after 24 hours of

exposure with microorganisms collected in October 1999 at both sites; "*" = significantly (p<0.05) different

from control. Low and High treatment rates were 2 and 20 f.lg/L, respectively, for chlorothalonil, and 1 and

10 f.lg/L, respectively, for endosulfan. Figure 4 from Downing et al. 2004.

- 10 -
Chh,rotlu;",,,il Lnam ulfan

o loll.' cl:r.:c-ntr.ui-I:f"'J

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Figure 5. Mean responses (pooled for site and season) of bacterial incorporation of [3H]leucine (CPM) to

atrazine at 20 p.g!L ("Low") and 200 p.glL ("High") at each incubation interval, with standard deviation

(vertical bars); ); "*,, = significantly (p<0.05) different from control. Top = 24 hr posttreatment, bottom =

168 hr. Low and High treatment rates were 2 and 20 p.g!L, respectively, for chlorothalonil, and 1 and 10

p.glL, respectively, for endosulfan. Figure 3 from Downing et al. 2004.

- 11 -
Table 1. Atrazine concentrations (ngIL, mean ± st. dev., n = 5) in canal surface water during I-week

microorganism colonization period (from Table I ofstudy).

Site Cl Site El

Season2 Mean ± St. Dev. (Range) Mean ± St. Dev. (Range)

Oct-99 (wet season) 18.92 ± 1.20 (17.2-20.0) 24.5 ± 3.77 (18.8-28.4)

Feb-OO (dry season) 32.68 ± 1.24 (31.2-33.9) 11.3 ± 1.01 (10.2-12.8)

1 Site C was surrounded on all sides by agricultural fields used primarily for tomato production, while Site E, ~5

miles downstream from Site C, was surrounded mostly by sawgrass marsh.

2 Florida agricultural pesticide application heaviest during dry season (December-April) and lightest during wet

season (May-November).

Table 2. Measured atrazine concentrations (flgIL, mean ± st. dev., n not specified) for treated microorganism

exposure experiments (from Table 4 ofstudy).

200 112 ± 25.2 117 ± 2.54 +4.0 81.3 ± 0.25 28.0

Season

Oct-99

Feb-OO

Nominal

(Jig/L)

20

200

20

Ohr

23.8 ± 6.14

184 ± 3.37

24.8 ± 3.80

24 hr

16.4 ± 0.12

137 ± 0.03

20.2 ± 0.27

% Decreasel

31.3

26.0

18.3

168 hr

13.7 ± 0.06

95.3 ± 2.29

17.1±0.49

% Decrease l

42.6

48.4

31.0

1 Decrease in average atrazine concentration from that detected at 0 hr, except for Feb-OO 200 Jig/L treated at 24

hours which was an increase from 0 hr.

- 12 -
Data Summary Table: Natural microbial assemblages incubated indoors (in open beakers)

Time Weighted

Magnitude Nominal Day ofStudy Average Atrazine Did

Effect ofEffect Atrazine Effect Concentration Recovery Comments relative to Concentration from Day Oto Occur-if

controls (p.g/L) Occurred When Effect so, when?

Occurred

Magnitude of effect

20 J.lg/L; Not applicable; estimated from

Phytoplankton No observed measured 23.8- pooled mean of Not applicable Not Figure 2. Effect not

biomass effect 24.8 J.lg/L 24- and 168- applicable statistically

(chlorophyll a, hour responses significant

J.lg/L); pooled (p2:0.05).

mean response Magnitude of effect

for site, season Not applicable; mean 111.9 J.lg/L1

; estimated from

and length of ~30% 200 J.lg/L; pooled mean of Experiment Figure 2.

exposure decrease measured 112- 24- and 168- Oct. 122.5 J.lg/L, terminated at Effect statistically 184J.lglL hour responses

Feb. 101.3 J.lg/L 168 hours. significant

(p<0.05).

Magnitude of effect

Primary 20 J.lg/L; Not applicable; estimated from

productivity No observed measured 23.8- pooled mean of Not applicable Not Figure 2. Effect not

(carbon effect 24.8 J.lg/L 24- and 168- applicable statistically

assimilation, hour responses significant

mg elL/day); (p2:0.05).

pooled mean Magnitude of effect

response for Not applicable; mean 111.9 J.lg/L; Experiment estimated from

site, season and ~60% 200 J.lg/L; pooled mean of Figure 2.

length of decrease measured 112- 24- and 168- Oct. 122.5 J.lg/L, terminated at Effect statistically

exposure 184 J.lg/L hour responses

Feb. 101.3 J.lg/L 168 hours. significant

(p<0.05).

20 J.lg/L; No (effect

~35% observed at Magnitude of Phytoplankton decrease measured 23.8 168 hours 15.8 J.lg/L end of effects estimated biomass J.lg/L experiment) from Figure 3. (chlorophyll a, Effects statistically J.lg/L); pooled 200 J.lg/L; significant for site (Oct 99) ~30% measured 184 24 hours 160.5 J.lg/L No (p<0.05). decrease J.lg/L

Magnitude of effect

20 J.lg/L; estimated from

Not Figure 3. Effect not

Phytoplankton No effect measured 24.8 Not applicable Not applicable applicable statistically

biomass J.lg/L significant

(chlorophyll a, (p2:0.05).

J.lg/L); pooled

No (effect for site (Feb 00) 200 J.lg/L; Magnitude of effect

~40% measured 112 168 hours 101.3 J.lg/L observed at estimated from

decrease end of Figure 3. Effect J.lg/L experiment). statistically

- 13 -
Time Weighted

Magnitude Nominal Day ofStudy Average Atrazine Did

Effect olEffect Atrazine Effect Concentration Recovery Comments relative to Concentration from Day Oto Occur-if

controls (pg/L) Occurred When Effect so, when?

Occurred

significant

(p<0.05).

~15% 2Ollg/L; Only interval

Bacillariophytes decrease measured 23.8 24 hours 20.1 Ilg/L analyzed. Magnitude of

(diatoms, Ilg/L effects estimated

average 2OOllg/L; from Figure 4;

number)2 ~20% measured 184 24 hours 160.5 Ilg/L Only interval SSNR3

.

decrease4

Ilg/L analyzed.

2Ollg/L; Not

Cyanophytes No effect measured 23.8 Not applicable Not applicable applicable Magnitude of

(b1uegreens, Ilg/L effects estimated

average 2OOllg/L; from Figure 4;

number)2 ~25% measured 184 24 hours 160.5Ilg/L Only interval SSNR.

decrease

Ilg/L analyzed.

~5% 2Ollg/L; Only interval

Chlorophytes + decrease measured 23.8 24 hours 20.1 IlglL analyzed. Magnitude of

Euglenophytes Ilg/L effects estimated

(greens, average 2OOllg/L; from Figure 4;

number)2 ~30% measured 184 24 hours 160.5 Ilg/L Only interval SSNR.

decrease4

Ilg/L analyzed.

Chrysophytes + ~25% 2Ollg/L; Only interval

Cryptophytes + decrease measured 23.8 24 hours 20.1 Ilg/L analyzed. Magnitude of

Dinophytes j..lg/L effects estimated

(browns, 2OOllg/L; from Figure 4;

average ~75% measured 184 24 hours 160.5 j..lg/L Only interval SSNR.

number)2 decrease4

j..lg/L analyzed.

~10% 20 j..lg/L; Only interval

Total decrease measured 23.8 24 hours 20.1 Ilg/L analyzed. Magnitude of

phytoplankton j..lg/L effects estimated

(average 200 j..lg/L; from Figure 4;

number)2 ~30% measured 184 24 hours 160.5 j..lg/L Only interval SSNR.

decrease j..lg/L analyzed.

20 j..lg/L; Only interval No effect measured 23.8 Not applicable Not applicable analyzed. Magnitude of Heterotrophs Ilg/L effects estimated (average from Figure 4; number)2 200 j..lg/L; Only interval SSNR. No effect4 measured 184 Not applicable Not applicable analyzed. Ilg/L

Total ~10% 20 j..lg/L; 24 hours 20.1 Ilg/L Only interval Magnitude of

- 14 -
Time Weighted

Magnitude Nominal Day ofStudy Average Atrazine Did

Effect ofEffect Atrazine Effect Concentration Recovery Comments relative to Concentration from Day Oto Occur-if

controls (pglL) Occurred When Effect so, when?

Occurred

microorganisms decrease measured 23.8 analyzed. effects estimated

(average Jlg/L from Figure 4.

number)2

200 JlglL;

Effects statistically

~25% Only interval significant

decrease5 measured 184 24 hours 160.5 JlglL analyzed. (p<0.05). JlglL

Magnitude of effect

20 JlglL; Not applicable; estimated from

Bacterial No observed pooled mean of Not Figure 2. Effect not

biomass effect measured 23.8- 24- and 168- Not applicable applicable statistically (abundance, 24.8 Jlg/L hour responses significant cells/mL); (p2:0.05). pooled mean

response for Magnitude of effect

site, season and

200 JlglL; Not applicable; mean 111.9 IlglL; Experiment estimated from

length of ~20% measured 112- pooled mean of Oct. 122.5 Jlg!L, terminated at Figure 2.

exposure decrease 184 Jlg/L 24- and 168- Feb. 101.3 Jlg!L 168 hours. Effect statistically

hour responses significant

(p<0.05).

Magnitude of effect

Bacterial 20 Jlg!L;

Not applicable; mean 17.5 JlglL; Experiment estimated from

productivity No effect measured 23.8- pooled mean of Oct. 15.8 Jlg!L, terminated at Figure 2. Effect not

24- and 168- statistically (leucine 24.8 Jlg/L hour responses

Feb. 19.2 Jlg/L 168 hours. significant incorporation, (p2:0.05). cpm); pooled

mean response Magnitude of effect

for site, season

200 JlglL; Not applicable; mean 111.9 1lg!L; Experiment from article text (p.

and length of ~46% measured 112- pooled mean of Oct. 122.5 Jlg!L, terminated at 253).

exposure stimulation 184 Jlg/L 24- and 168- Feb. 101.3 JlglL 168 hours. Effect statistically

hour responses significant (p<0.05,

Figure 2).

Magnitude of effect

20 JlglL; Experiment estimated from

Bacterial No effect measured 23.8- Not applicable Not applicable terminated at Figure 5. Effect not

productivity 24.8 JlglL 168 hours. statistically

(leucine significant

incorporation, (p2:0.05).

cpm); pooled Magnitude of effect

mean response

for site and 2OOIlg!L; mean 111.9 IlglL; Experiment estimated from

~100% Figure 5.

season stimulation measured 112- 168 hours Oct. 122.5 Jlg/L, terminated at Effect statistically 184 Jlg/L Feb. 101.3 JlglL 168 hours. significant

(p<0.05).

Mean ofTWAs for October 1999 and February 2000 expenment (attached Worksheet).

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2 Average numbers estimated from Figure 4. For Control: bacillariophytes (diatoms) = 15, cyanophytes

(bluegreens) = 8, chlorophytes + euglenophytes (greens) = 13, chrysophytes + cryptophytes + dinophytes

(browns) = 4 [yielding total phytoplankton = 40], heterotrophs = 7 [yielding total microorganisms = 47]. For

"Low" atrazine treatment: bacillariophytes = 13, cyanophytes = 8, chlorophytes + euglenophytes = 12,

chrysophytes + cryptophytes + dinophytes = 3 [yielding total phytoplankton = 36], heterotrophs = 7 [yielding total

microorganisms = 43]. For "High" atrazine treatment: bacillariophytes = 12, cyanophytes = 6, chlorophytes +

euglenophytes = 9, chrysophytes + cryptophytes + dinophytes = 1 [yielding total phytoplankton = 28],

heterotrophs = 7 [yielding total microorganisms = 35].

3 Statistical Significance, as compared to controls, was Not Reported.

4 The study authors reported that relative abundance of chlorophytes + euglenophytes (greens) and chrysopytes

decreased 4% and 5%, respectively, while diatoms and heterotrophs each increased by ~4% (p. 254); the reviewer

did not agree with this conclusion, based on the data shown in Figure 4.

5 Total microorganisms from sites C and E were reduced 14% and 36%, respectively, as compared to controls (p.

254).

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Reviewer Comments:

No analysis for atrazine or other pesticides in exposure water (i.e. water used for the

experiments) at test initiation was reported. In addition, negative controls were not utilized; only

solvent (acetone) controls were included. The study authors cite that pesticide application in

southern Florida is highest during the dry winter months (December-April), when primary

agricultural production occurs, and lightest during the rainy summer months (May-November).

However, the sampling periods (October and February) are not necessarily reflective of these

reported extremes.

There may be little relevance ofthe results from these experiments to natural systems, despite the

natural source of the communities, given that the microorganisms were incubated in 600 mL

beakers containing nutrient amended, filtered (0.22-llm) canal water under unspecified

conditions of light and temperature. During the 7-day colonization period, canal water was not

monitored for pH, temperature, turbidity, dissolved oxygen, alkalinity, total nitrogen, total

phosphorus, conductivity, and particulate and dissolved organic matter.

Phytoplankton and zooplankton were not identified at the species level to assess changes in

species composition.

This study also examines the potential effects of chlorothalonil and endosulfan on microbial

assemblages collected from the C-111 canal in southern Florida. Endosulfan was detected in the

canal water at sites C and E at 7.84 ± 3.64 ng/L (5.34-11.97 ng/L) and 0.76 ± 0.29 ng/L (0.30-

1.07 ng/L), respectively, in October 1999, and at 436 ± 509 ng/L (183-1,350 ng/L) and 16.4 ±

8.58 ng/L (7.35-27.5 ng/L), respectively, in February 2000. Canal water was also analyzed for

chlorothalonil; however, the analytical method did not provide adequate results. Tables 2 and 3

ofthe study were not included in this review because the statistical analyses pooled responses

from atrazine, chlorothalonil, and endosulfan exposure.

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: There were multiple deficiencies in the design, implementation, and

reporting ofthis study. No chemical analysis (atrazine or other pesticides) of exposure water or

the controls was conducted prior to study initiation. Atrazine and endosulfan were assumed to be

present, as they were detected in the canal water during the interval when microbial assemblages

were colonizing the artificial substrates. However, previously documented fluctuations in

measured pesticide concentrations in the water body reinforce the fact that the sample water

(used in experiments) concentrations may not have been reflective ofthe inconcurrently

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Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, U.S. EPA

measured surface water concentrations. Canal water was not monitored for pH, temperature,

turbidity, alkalinity, total nitrogen, total phosphorus, conductivity, or organic carbon during the

colonization period. No negative (solvent-free) controls were included. Incubation conditions

(light source, photoperiod, temperature) during the acute exposure experiments were not

described. Phytoplankton and zooplankton were not identified at the species level to assess

changes in species composition. Some results were presented as pooled data which combined

data from atrazine treatments, exposure durations, sites, and seasons. Finally, for some

endpoints, results were not reported for all experiments and exposure durations.

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

Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division

U.S. Environmental Protection Agency Ct~ 17c1,/,\,)..,'U/l\

I

AfuSi cP113/l \

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