Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

DeLorenzo, M. E., J. Lauth, P. L. Pennington, G. 1. Scott and P. E. Ross. 1999. Atrazine effects

on the microbial food web in tidal creek mesocosms. Aquatic Toxicology. 46: 241-251.

Purpose of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this study

contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant communities

and associated determination of a Level ofConcern (LOC).

Date of Review: 9/9/09

Summary of Study Findings

Purpose of Study: This study examines the potential effects of atrazine on phytoplankton and

zooplankton in experimental estuarine mesocosms.

Methods: To simulate non-point source atrazine exposure in an estuarine environment, three

compartmentalized mesocosm systems were set up inside a greenhouse and assigned one ofthree

treatment levels. Each mesocosm system consisted ofthree chambers (Units A, B and C),

connected via a "sub-tidal" channel. Each chamber contained a low, middle, and high zone to

simulate tidal creek conditions. Tidal flow of an unspecified rate and volume and a salinity

gradient of7-25% were simulated. Each system included a single exposure portal, connected to

the subtidal channel, in Unit A (Figure 1 below). Slope, dimensions, and total volume ofthe

mesocosms were not reported. Uncharacterized sediment, cordgrass (Spartina alterniflora) and

glasswort (Salicornia virginica) plants, oysters (Crassostrea virginica), mud snails (Nassarius

vibex), grass shrimp (Paleomonetes pugio), fiddler crabs (Uca sp.) and mummichog fish

(Fundulus heteroclitus) were collected from a reference marsh located on the western branch of

Leadenwah Creek, Wadmalaw Island, South Carolina, and transferred to the mesocosm units.

The acclimation interval for the prepared mesocosms prior to treatment was not reported, but

was a minimum of 3 days.

Three days prior to treatment, artificial substrates [polyurethane foam units (PFUs), each 4 x 5 x

6 cm

3

] were positioned in the sub-tidal channel of each mesocosm system to colonize

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microorganisms. Attached weights held the artificial substrates below the water level at "low

tide".

Atrazine (purity not reported) in acetone at 2.013 mg/mL was added to 56.6 L of freshwater in

Unit A of one mesocosm at 40 /lg/L and to Unit A of a second mesocosm at 160 /lgiL. The third

mesocosm system was maintained as a solvent control. All three mesocosms received an equal

volume of acetone (test solution vehicle) with a final concentration of 0.079 mLiL. The atrazine

was introduced into Unit A as an uncharacterized pulsed exposure at "ebb tide" and allowed to

mix through Units Band C via the subtidal channel with the incoming "high tide.". This

application method was designed to simulate a run-off event whereby a pesticide is introduced

into a tidal creek system, diluted by incoming tides, and transported away from the point of

entry.

Water samples (unreported volume) were collected for atrazine analysis immediately after

treatment (Time = 0) and at 48 hours posttreatment. Additional 10 mL water samples were

collected into a beaker from the sub-tidal channel of each mesocosm at 2 (48 hours), 5, and 9

days posttreatment for chlorophyll a analysis. Dissolved oxygen measurements were taken at 1

(24 hours), 2 (48 hours), and 9 days posttreatment.

Artificial substrates (PFUs) were collected from each mesocosm immediately prior to atrazine

treatment and at 24 and 48 hours posttreatment. Additionally, clean PFUs were placed in each

mesocosm 24 hours after treatment, then collected after 8 days of exposure (9 days

posttreatment). Microorganisms were collected by gently squeezing the PFU contents (~60 mL)

into individual, sterile sample cups.

Atrazine. Water samples were loaded onto solid-phase C18 (SEP) cartridges pre-conditioned

with methanol (two column volumes) followed by distilled, deionized water (one column

volume). After loading, the cartridge was rinsed with water (one column volume), dried by

drawing air through the cartridge (~5 minutes), then wrapped in aluminum foil and stored frozen

(-20°C, interval not specified). Atrazine was eluted with ethyl acetate (two column volumes) and

the resulting eluate concentrated (~0.5 mL) via TurboVap. The concentrated residues were

diluted with isooctane (~10 mL), re-concentrated (~0.5 mL), brought to volume (l mL) with

isooctane, then transferred to GC vials, capped and stored frozen (interval not specified) until

analysis. Atrazine was quantified by GC under the following conditions: J&W Scientific DB-5

5% pheny1:95% methy1-po1ysiloxane column (0.25 mm x 0.25 mm, 30 m length), injection

volume 2 IlL, sp1itless mode, average linear velocity 33 cm/s, carrier gas helium, nitrogenphosphorus detection (NPD), atrazine reference standard at 120 ng/mL; limit of detection was

not reported.

Phototrophic carbon assimilation. An aliquot (5 mL) of collected microorganisms (from PFUs)

were treated with [14C]sodium bicarbonate (NaH14C03), at 1 /lCi/mL, 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-/lm, cellulose, nitrate membrane), then the filters

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were rinsed with filtered (0.2-jlm) seawater, placed in scintillation vials with 10% HCl (1 mL)

and allowed to sit in darkness overnight. After which, scintillation fluid was added, the samples

were equilibrated for 4 hours, then analyzed for total radioactivity using LSC.

Chlorophyll a. 10 mL water or 3 mL microorganism samples were filtered (glass fiber, Type

GF/F), then the filters were placed in vials with 1 mL saturated magnesium carbonate (MgC03)

solution and stored frozen (-2°C, unspecified interval) until analysis. Prior to analysis, samples

were thawed, treated with acetone (9 mL), shaken, refrigerated overnight, shaken and again

refrigerated overnight, then brought to room temperature and analyzed using a fluorometer

(Sequoia-Turner Model 450) with excitation (NB440) and emission (SC665) filters.

Phototrophic (algal) bio-volume. Microorganism samples (5 mL) were preserved in 2% buffered

formalin (final concentration), then stored (interval not specified) at 4°C in darkness until

analysis. Aliquots (40 jlL) of diluted formalin preserved samples were analyzed for algal cells

by counting at 500 magnification. Algae were identified to genus where possible, and the

number of cells, average cell shape, and size were noted for each taxon. Phototrophic biovolumes (jlm3

) were estimated for each taxon using cell sizes and simple geometric shapes, then

multiplied by the total number of cells per taxon in each sample.

Bacterial abundance was determined via epifluorescence microscopy. Formalin preserved

samples (described above) were treated with chemical dispersant (Na3 PPi, 0.25 mL), sonicated,

diluted with filtered (0.2-jlm) seawater, then stained with acridine orange (100 mg/L, final

concentration) for 3 minutes. The stained samples were filtered (O.2-jlm black membrane), then

bacterial counts were conducted under oil immersion at 1,250 magnification using a Whipple

disc and Zeiss fluorescence microscope equipped with a super high-pressure mercury lamp with

a blue light filter (470-490 nm). Bacterial density was calculated using the following equation:

Bacterial density (cells/mL) = (Nx Af)/(d x Ag), where N = average number of cells counted, Af=

the effective area ofthe filter (mm\ Ag = area ofthe counting grid (mm2), and d = dilution

factor.

Ciliates and flagellates (heterotrophic). Microorganism samples (5 mL) were preserved with 0.5

mL Lugol's solution [acetic acid:Lugol's (50 mL:200 mL)], then diluted samples were counted

at 500 magnification using a Sedgewick-Rafter slide (1 mL). A minimum of200

organisms/sample were counted, with a standard error of <1 0%. Non-pigmented ciliates and

flagellates were sorted into size classes of <20 and >20 jlm. Taxonomy was not determined.

Response values from three PFUs were averaged for each treatment (0, 40 and 160 jlg/L), with

statistics calculated using MINITAB software. Pair-wise comparisons between treatments of

each endpoint measured (chlorophyll, bacterial abundance, etc.) were done by one-way analysis

of variance (ANOVA) for each time point (0,24 and 48 hours and 9 days).

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Results: At the low treatment rate (40 Ilg/L), measured atrazine concentrations were 121 %,

22.7% and 0.6% of nominal in Units A (entry point), B and C, respectively, at time O. At 48

hours, levels had reached 59.2%, 9.7% and 10.3% of nominal, respectively. Mean atrazine

levels in the low treatments decreased by 45.3% from 19.3 Ilg/L to 10.5 Ilg/L in 48 hours (Table

1 below). At the high application rate (160 Ilg/L), measured atrazine concentrations were 157%,

0.4% and <0.1% of nominal in Units A, Band C, respectively, at time O. At 48 hours, they had

reached 15.2%, 13.0% and 3.5% of nominal, respectively. Mean atrazine levels in the high

treatments decreased by 79.8% from 84.0 Ilg/L to 16.9 Ilg/L in 48 hours. In the untreated

(solvent) control mesocosm, atrazine was detected in the water at 0.097 Ilg/L at time 0 and at

0.233 Ilg/L at 48 hours. Detection limits were not reported.

One-way ANOVA detected no significant effects of atrazine treatment on dissolved oxygen

(DO)

All phototrophic parameters measured (chlorophyll a in water, chlorophyll a in organisms,

phototrophic assimilation and phototrophic bio-volume) were significantly decreased (p=S0.001 at

maximum effect) at both treatment levels (attached Worksheet), although carbon assimilation

was not measured beyond 48 hours. Chlorophyll a in the water and organisms were decreased

(p=S0.002) 77-79% and 46-47%, respectively, at 24 hours for both treatment rates, as compared to

the control mesocosm, and remained significantly (p<O.OO1) reduced for the remainder of the

study (attached Worksheet). Phototrophic assimilation was decreased (p=0.032) 39% and 62%

at the 40 Ilg/L and 160 Ilg/L rates, respectively, at 24 hours and was decreased (p<O.OOl) 72%

and 83%, respectively, at 48 hours, but was not monitored thereafter. Phototrophic bio-volume

was decreased 54-56% (p=O.OOI) at 24 hours for both treatment rates and remained significantly

(p=S0.001) reduced for the remainder ofthe study.

Twenty-eight algal taxa were identified from the colonized artificial substrates, with the majority

(twenty) ofthe identified taxa diatoms (Chrysophyta, Table 2 below). Atrazine exposure caused

noticeable changes in algal community composition with total taxa decreasing from twenty-eight

to twenty in the atrazine treated mesocosms. Total algal bio-volume was decreased 54-56% at

24 hours for both treatment rates, as compared to the control, and was decreased 54% and 73% at

the 40 Ilg/L and 160 Ilg/L rates, respectively, at 9 days; ANOVA analyses were not reported

(Table 3, below). Diatom, dinoflagellate, chlorophyte and cryptophyte bio-volume decreased in

atrazine treated mesocosms; however, cyanophyte bio-volume increased. The study authors

reported there were decreases in numbers of Chlamydomonas and Cryptomonas, with increases

in a small CymbeUa sp. and many coccoid blue-green algae; quantitative results were not

presented. Additionally, diatom Cylindrotheca sp. had fewer, smaller and more curved cells in

atrazine treated mesocosms.

Bacterial abundance was significantly decreased (p=O.OOI) at 48 hours for both treatment rates,

as compared to the control, but there were no significant differences at study termination (see

attached Worksheet). For protozoa, there were no significant effects on numbers of smaller

ciliates «20 Ilm) in atrazine treated mesocosms, as compared to the control, but larger ciliates

(>20 Ilm) were significantly increased at 48 hours (p=0.010) and 9 days (p=0.003) for both

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treatment rates. Smaller flagellates «20 /lm) were significantly increased (p=0.022-0.023) at 24

and 48 hours in atrazine treated mesocosms (both rates) and remained elevated (p=0.055) at 9

days. However, larger flagellates (>20 /lm) were significantly decreased (p=0.013-0.021) at 48

hours and 9 days for both treatment rates.

Unit A Unit 8 Unit C

( llig.h Marsh)

( Mid Marsh)

( Low MarSh)

( Channel )

[ rIig.h Marsh)

( Mid Marsh)

( Ltlw MarSh)

[ Channel}

( , lig.h Marsh

( Mid Marsh

( Low Maflih

( Dilution

Water

Toxicant Delivery Saltwater

Reservoir

Figure 1. Mesocosm unit design simulating an estuarine (tidal creek) ecosystem (Lauth et ai., 1996). Figure

from DeLorenzo et ai. 1999.

Table 1. Atrazine concentrations in experimental tidal mesocosms (from attached Workshee

Atrazine (I-Ig/L)

Mesocosm Unie Measured

Nominal

% Decrease l Time 0 48 hours

Control 0.097 0.223

A 40 48.544 23.681

B 9.072 3.861

C 0.243 4.101

Average 19.286 10.548 45.31

A 160 251.202 24.382

B 0.609 20.725

C 0.103 5.677

Average 83.971 16.928 79.84

t).

1 Decrease m average atrazme concentratIOn at 48 hours posttreatment.

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Table 2. Algal taxa identified in experimental mesocosms (from colonized artificial substrates) over the

course of the study; grouped as to phylum (Prescott, 1978, from Table 2 ofstudy).

Taxon

Chlorophyta

.r;.~lq'!!:x4?lr}?,!q~?P:m

Chyrsophyta

Amphiprora sp.

Asterionella sp.

Bacillaria sp.

Biddulphia sp.

Chaetoceros sp.

Coscinodiscus sp.

Cylindrotheca sp.

Cymbella sp.

Dinobryon sp.

Diploneis sp.

Fragilaria sp.

Gomphonema sp.

Licmophora sp.

Melosira sp.

Navicula sp.

Nitzschia sp.

Pleurosigma sp.

Rhizosolenia sp.

Sellaphora sp.

Stauroneis

Cryptophyta

.. .......... mr;.1)!Pt?lr}?'!q~?P:m . Cyanophyta

Cylindrical blue-green

Filamentous blue-green

Oscillatoria sp.

............ .§X'!~c.~?c.?c.c.1!~~P:mm Euglenophyta

§1![JI~'!C:?P:m

Pyrrhophyta

Gymnodinium sp.

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Table 3. Change in algal community composition; expressed as percent decrease in average bio-volume as

compared to control (i.e., magnitude of effect), except where noted as an increase (+) (from Table 3 ofstudy).

40 JlglL Treatment 160 Jlg/L Treatment

Phytoplankton 24 h 48 h 9 d1 24 h 48 h 9 d1

Diatoms 49.53 47.02 53.81 45.62 44.21 73.85

Dinoflagellates 89.27 88.58 100.00 91.07 96.43 100.00

Chlorophytes 20.00 78.15 82.05 47.90 83.19 100.00

Cryptophytes 76.74 72.09 36.46 88.37 100.00 89.99

Cyanophytes 64.67 +29.29 +52.49 31.33 +94.70 +87.02

Total algae 56.49 56.43 53.95 54.09 53.61 73.02

Artificial substrates placed III mesocosms at 24 hours posttreatment and removed after 8 days of exposure (9 days

posttreatment).

Daat Summary Tahie:

Time Weighted

Nominal Average Did

Magnitude ofEffect Atrazine Day ofStudy Atrazine Recovery

Effect Relative to Controls Concentration EffectFirst Concentration Occur- Comments

(p.gIL/ Detected from Day Oto IfSo,

When Effect When?

Occurred

Chlorophyll a 77%*--v 40 JlglL 2

in water p**<O.OOl

(mglL) 79%*--v 160 JlglL 2

Chlorophyll a 47%*--v 40 Jlg/L 1

Magnitude

of effects

in organisms

46%*--v

p=0.002 calculated

(mglL) 160 JlglL 1 from results Not calculable No

Phototrophic 39%*--v 40 JlglL 1 in Table 1

assimilation p=0.032 ofstudy

(mg C/L*h) 62%*--v 160 Jlg/L 1 (attached

Worksheet).

Phototrophic 56%*--v 40 Jlg/L 1

bio-volume p=O.OOl

(Jlm3 x 105

) 54%*--v 160 Jlg/L 1

Diatoms 50%--v 54%--v 40 JlglL 1 9

(average bio46%--v 74%--v

Magnitude

volume) 160 JlglL 1 9 of effects

89%--v IOO%--v

from Table

Dinoflagellates 40 JlglL 1 9 3 above;

(average bio91%--v IOO%--v Not calculable statistical

volume) 160 JlglL 1 9 No significance

Chlorophytes 20%--v 82%--v 40 Jlg/L 1 9 not

(average bio- reported.

volume) 48%--v 100%--v 160 Jlg/L 1 9 Day 9

values (on

Cryptophytes 77%--v 36%--v 40 Jlg/L 1 9 the right)

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Time Weighted

Nominal Average Did

Magnitude ofEffect Atrazine Day ofStudy Atrazine Recovery

Effect Relative to Controls Concentration EffectFirst Concentration Occur- Comments

(pgIL/ Detected from Day Oto IfSo,

When Effect When?

Occurred

(average bio- 88%-.It 90%-.It 160 Ilg/L 1 9 are from

volume) substrates

introduced

65%-.It 52%t 40 11giL 1 9 Yes to the Cyanophytes (Day 2) mesocosms (average bio- at 24h and volume) 31%-.It 87%t 160 Ilg/L 1 9 Yes

(Day 2) are rounded

from two

56%-.It 54%-.It 40llgIL 1 9 decimal

places.

Total algae

(average bio- No

volume) 54%-.It 73%-.It 160llg/L 1 9

1 For the nommal treatment levels of 40 and 160 IJ.g/L , mean (n=3) atrazme concentratiOns m the water column

decreased from 19.3 and 84.0 IJ.glL at time 0 to 10.5 and 16.9 IJ.g/L, respectively, at 48 hours. No further

measurements were taken.

* Reported to be statistically significant; level ofstatistical significance was not reported.

** Probability (p) values were not reported separately for treatment levels. Values could not be reproduced because

only means were presented.

Reviewer Comments:

1. The purity and source ofthe atrazine test compound used were not reported.

2. The tidal mesocosms were prepared using sediment, plants and animals obtained from a

natural reference marsh. It is not stated whether the natural marsh site was tested to establish

whether it was contaminated with pollutants. Atrazine was detected in the water ofthe

untreated control mesocosm at 0.097 ~g/L at time 0 and 0.233 ~g/L at 48 hours, suggesting

that the levels of atrazine detected in the treated mesocosms at time 0 could be due either to

the atrazine treatment or to prior contamination. The detection limits for atrazine analysis

were not reported.

3. No negative controls were included; only solvent controls were used.

4. Atrazine concentrations were only determined at two intervals (24 and 48 hours) following

application.

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5. The study authors noted that chlorophyll a and total biovolume in the control mesocosm

steadily increased over the 9-day study, indicating that the microorganism communities

(colonization) had not achieved equilibrium with the surrounding water prior to treatment.

6. Nine-day measurements were only reported for substrates introduced 24 hours after dosing

with atrazine.

7. Reportedly, previous experiments (Lauth et ai., 1996) using tidal mesocosms determined that

pesticide concentrations reached equilibrium in the water of each unit within 24 hours (two

tidal exchanges). As the study authors noted, applied atrazine had not reached equilibrium

between the three units in the treated mesocosms after 48 hours in this study.

8. The water of the tidal mesocosms was not monitored for pH, temperature, turbidity,

alkalinity, total nitrogen, total phosphorus, conductivity, or particulate and dissolved organic

carbon.

9. The mesocosm sediment was not analyzed for pesticide content, particle SIze, cation

exchange capacity, organic content, and pH prior to treatment.

10. Dimensions, total volume, and slope (to create low/middle/high zones) ofthe mesocosm

units were not reported.

11. Pairwise comparisons (one-way ANOVA) were reportedly used to compare each treatment

endpoint to the control. However, only one set ofp-values is reported (see Table 1).

12. The study authors reported that the diversity of the phytoplankton taxa colonized in the

control mesocosm of this study was similar to that observed. in a reference tidal creek

(DeLorenzo, unpublished data), but no supporting data were presented.

13. The initial microbial communities were dominated by diatoms. The relative abundance of

diatoms decreased throughout the study in the atrazine-treated units. The total number of

taxa identified also decreased from 28 to 20. The authors assert that the effects on substrates

introduced at 24 hours and examined at 9 days indicate decreased colonization ability in the

presence of atrazine. However, it is unknown whether the source microbial assemblages

were comparable across treatments at the time these substrates were introduced.

14. Overall, phytoplankton and larger flagellates decreased in abundance and productivity, while

bacteria, larger ciliates and smaller flagellates increased in abundance. However, dissolved

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oxygen (DO) levels in the atrazine-treated units was similar overall to DO in the control

units, which the authors suggest might indicate (l) a compensatory phototrophic response or

(2) that metabolic rates of the heterotrophic organisms may have decreased with decreased

oxygen production.

15. The study authors noted that atrazine had a significant effect on the microbial food web of

the experimental tidal mesocosms by influencing interactions between phytoplankton (algae),

bacteria, and grazers (heterotrophic protozoa). This may have occurred through direct effects

(phytoplankton), by influencing competitive interactions (bacteria), and/or by impacting the

heterotrophic food source food source (grazers).

16. The study authors also proposed that the trend toward smaller microorganism cell size in the

atrazine treated mesocosms has potential adaptive significance: It may allow the alga to

sustain its nutritional needs while photosynthesis is impaired, and/or it might reduce

adsorption ofthe pesticide via a decreasing cell surface area:volume ratio.

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

This study is considered Invalid (INV) for use in the risk assessment process.

Rationale for Use:

No quantitative exposure-response relationships could be inferred, as the system failed to reach

equilibrium during the study period and the treatments lacked sufficient replicates.

Limitations of Study: Numerous deficiencies existed with respect to the experimental design

and implementation ofthis study. No effort was made to document whether other chemicals

were present in the treatment and/or control mesocosms prior to study initiation, and analyses of

the control mesocosm indicate the systems may have been contaminated with atrazine prior to

treatment. The acclimation interval was insufficient to establish equilibrium-phase colonization

of microorganisms prior to treatment. Mesocosm sediment was not characterized, and the water

was not monitored for pH, temperature, turbidity, alkalinity, total nitrogen, total phosphorus,

conductivity, or organic carbon. Purity ofthe test compound was not reported. Frequency and

intensity of atrazine pulses was not described. An acetone solvent control (0.079 mLlL) was

used, but no separate negative control was established. Atrazine concentrations were only

determined at two intervals following application, and equilibrium ofthe applied test substance

between the three units of each treated mesocosm had not been achieved by the second sampling

interval at 48 hours post-treatment.

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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

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