Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Pratt, l.R., N.J. Bowers, B.R. Niederlehrer, and J. Cairns, Jr. 1988. Effects of atrazine on

freshwater microbial communities. Arch. Environ. Contam. Toxicol. 17:449-457.

Ct1'\ t "(t~ l(S S"i l.-f )

Purpose of Review:

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

study were also used to parameterize the Comprehensive Aquatic System Model (CASM)

and establish the model-associated LOC for atrazine.

Date of Review:

December 10, 2008

Purpose of Study:

This study examines the potential effects of atrazine to aquatic microbial communities in

artificial streams (microcosms).

Summary of Study Findings:

Methods:

21 microcosms were constructed in high-density polyurethane tubs (35 cm x 28 cm x 15

cm), each with an inlet tube at one end and three drain holes at the other end. The inlet

tube delivered a mixture of diluent and toxicant with a flow rate giving approximately 7

turnovers ofthe 7 L test volume per day. Three replicates were used at each treatment

level: diluent control, solvent (methanol) control, and five concentrations of atrazine.

Diluent water, consisting oftap water (source: New River, Virginia; hardness -70 ppm

CaC03, alkalinity -45 ppm CaC03, pH -8.4) dechlorinated by activated charcoal

filtration, flowed from a head box into a flow splitter and 21 mixing chambers. Each

mixing chamber was also fed by a toxicant reservoir containing appropriate

concentrations of atrazine, diluent, and solvent for each microcosm. Each microcosm

was then fed by the test solution flowing out ofits mixing chamber. (No diagram

provided.) Lighting was from Vita Lite bulbs (CRI>90, Duro Test Corp.) located -30 cm

above the microcosms (intensity -5000 lux). Lights were set to a 16 hours light, 8 hours

dark photoperiod. Water temperature was uncontrolled and ranged from 13.5°C to 15°C.

1

11111111111

2087818
Atrazine added to the microcosms was technical grade (98.6%, FL851205, Ciba-Geigy),

while analytical standards were made from analytical grade atrazine (98.8%, Code #S85-

0653-3, Ciba-Geigy), Primary stock was made by dissolving 200.84 mg of atrazine in

100 ml of methanol and diluting in distilled water to I L. Secondary stocks were made

from primary stock by diluting with diluent water. Both primary and secondary stocks

were made on day 0 and again on day 10. Nominal atrazine concentrations in the

microcosms were 3, 10, 30, 100, and 300 Ilg/L. The solvent control test solutions were

made with methanol at a concentration of0.015% in diluent water.

Atrazine concentrations in the microcosms were measured on days 0, 10, and 21. For

these measurements, approximately 500 mL oftest solution were collected from each

microcosm and filtered through a 0.45-llm GA-6 Metricel membrane filter (Gelman).

Atrazine was extracted from 250 mg offiltered water using C18 SPE columns (IT.

Baker) and eluted from the columns with 2.5 mL ofHPLC grade acetonitrile (ACN) for a

100-fold increase in concentration. These samples were then stored in glass vials with

Teflon caps at 4°C for analysis the next day. Standards were extracted the same way for

each analysis. Samples were analyzed with a high performance liquid chromatograph

and Beckman Ultrasphere ODS columns (5 Ilm mesh, 4.6 mm ID x 250 mm). A 67:33

water:ACN solution was used to elute the atrazine at 1.5 mLimin. Absorbance at 220 nm

was used to monitor peak height, and concentration was calculated from standards.

Precision (based on replicate measures) had a coefficient of variation of2.04%, and

average recovery was 104.1 %. The minimum detectable level of atrazine with the

technique was 0.5 Ilg/L.

Test microbial communities were collected on polyurethane foam substrates (6.0 cm x

5.0 cm x 4.0 cm) in Pandapas Pond, Montgomery County, Virginia. (Pond, watershed,

surrounding land use, etc. not described.) Communities included at least 150-200 species

of bacteria, protozoa, algae, fungi, and small metazoans. The substrates were placed in

the pond and collected 14 days later (no further description of date or location of

sampling). In each microcosm, two polyurethane foam substrates were placed as species

sources, and five barren substrates ("islands") were also placed to provide colonizable

habitat. (Spatial arrangement not described.) One ofthe island substrates was removed

from each microcosm after 3, 7, 14, and 21 days and examined for protozoan species

richness. Three reference substrates were examined prior to the start ofthe test to

deteITI;line initial species richness, and at the end ofthe experiment, the species sources

were examined to evaluate survival.

Each substrate was sampled by placing it in a randomly numbered sterile Whirl-Pak®

bag, squeezing to remove as much ofthe contents as possible, and discarding the

substrate. Samples were allowed to settle and then "repeatedly" subsampled and

examined at 200-400x for identification of protozoa to the lowest possible taxonomic unit

(usually species, sometimes genus). (Time allowed for settling and number/size of

2
subsamples not provided.) The total number oftaxa was recorded and then matched with

the original sample using the random ID number.

Water quality measurements (temperature, pH, dissolved oxygen, and conductivity) were

taken in each microcosm once per week, about one hour after the lights came on and

before substrate collection. Additionally, water hardness and alkalinity were measured

weekly in the diluent control, solvent control, and the highest test concentration. Water

quality measurements followed the protocol of USEPA, Methods for the Chemical

Analysis ofWater and Wastewater, 1985. On Day 21, several non-taxonomic variables

were evaluated. Biomass was estimated as protein concentration. Samples were

concentrated by centrifugation (1500 rpm for 10 minutes), protein was extracted with 0.5

N NaOH, and then protein concentrations in the extract was measured following the

method of Bradford (1976), A rapid and sensitive methodfor the quantitation of

microgram quantities ofprotein, utilizing the principle ofprotein-dye binding, Anal

Biochem 72. Aliquots of each sample were fixed to pH<2 with trace pure HN03 for

analysis of calcium, potassium, and magnesium (again following USEPA, 1985). Metals

were analyzed by flame atomic absorption spectrophotometry on a Perkin-Elmer model

603 spectrophotometer. Chlorophyll a was extracted in 90% acetone overnight and

concentrations detennined by the trichromatic method (following the American Public

Health Association's 1985 standards).

For statistical analysis of protozoan colonization of initially barren substrates, data was

fitted to the MacArthur-Wilson equilibrium model, given by:

S =Seq (1 - e-Gr )

where S is the number ofspecies at time t, Seq is the equilibrium number ofspecies, and

G is the colonization rate. Parameters Seq and G were estimated using nonlinear least

squares regression, and then the curves for different treatment concentrations were

compared using dummy variable analysis (factors chosen for dummy variables are not

described). For each sampling day available, species numbers as well as non-taxonomic

variables were compared using analysis of variance (ANaYA) (exact procedure not

described). When ANaYA found significant differences at p<0.05, multiple

comparisons were made using Fisher's LSD. No-observable-effects concentrations and

lowest-observable-effects concentrations were also defined, but will not be described in

the results because they are trivial.

Results:

Atrazine levels remained stable during the 21-day study period, though concentrations

were slightly different than nominal (see Table 1). Water chemistry (pH, temperature,

hardness, alkalinity, conductivity) remained in the nonnal range for dechlorinated tap

water.

3
Nonlinear regression was adequate to describe protozoan colonization ofthe

polyurethane foam substrates. Results showed some significant treatment effects, but

treatment as well as time effects were not consistent (see Figure 1). Low atrazine

treatment microcosms «30 Ilg/L nominal concentrations) experienced increased species

numbers on days 14 and 21. The two highest atrazine treatments (l00 and 300 Ilg/L

nominal concentrations) showed initial decreases, but by day 21 the species number

decrease was only significant at the highest treatment level. Colonization curves were

significantly different among treatments (F12,70 = 11.02, p<O.OOOI; it is not clear to what

test these statistics refer). Seq was significantly greater at <30 Ilg/L nominal atrazine

concentrations and significantly lower at 300 Ilg/L nominal. G was significantly lower at

all treatment levels except at the highest test concentration (300 Ilg/L nominal). See

Table 2. At the end ofthe experiment, there were no differences in survival rates on the

source substrates.

Non-taxonomic measurements taken on Day 21 showed some significant effects. Protein

and chlorophyll a levels were significantly increased in the two lowest atrazine

treatments (3 and 10 Ilg/L nominal), but was significantly decreased in the highest

treatments (300 Ilg/L nominal). Midmorning dissolved oxygen, an indicator of primary

productivity, was significantly decreased in treatments with 30 Ilg/L nominal

concentrations or higher. For the metals, magnesium and calcium also were significantly

decreased in treatments with 30 Ilg/L nominal concentrations or higher, while potassium

was only reduced in the highest treatments(300 Ilg/L nominal). See Table 3 and Table 4

for the summary data.

Table 1. Measured atrazine concentrations in microcosms. Means and standard deviations (SD) are

shown. (study table 1)

Nominal Measured atrazine concentration (gg/L)

concentration Day 0 Day 10 Day 21 Time Mean

(gg/L) Mean SO Mean SO Mean SO Mean SO

Control <0.5 <0.5 <0.5 <0.5

Solvent Control <0.5 <0.5 <0.5 <0.5

3 3 0.11 3.6 0.33 3.1 0 3.2 0.34

10 9.2 0.23 11.3 0.58 9.6 1.1 10 1.15

30 29.7 2.31 33.3 1.34 33.1 4.82 32 3.26

100 102 8.02 102 10.4 126 14.4 110 15.6

300 324 8.02 336 17 350 14.4 337 16.4

4
Table 2. Effects of atrazine on estimated MacArthur-Wilson colonization parameters: equilibrium

species number (Se,/) and colonization rate (G). Parameter values are based on nonlinear regression

fits to the protozoan species number data. Asymptotic standard errors (SE) are also provided.

Values significantly different from control are marked with (*). (study table 2)

Nominal

concentration

(gg/L) Seq Seq SE G GSE

Control 33.2 2.34 0.349 0.104

Solvent Control 34.4 1.92 0.309 0.068 ..

j 42.4 * 2.65 0.167 * 0.03

10 52.6 * 5.06 0.122 * 0.028

30 61.7 * 12.26 0.081 * 0.031

100 34.8 3.05 0.138* 0.031

300 24.1 * 1.18 0.266 0.048

Table 3. Non-taxonomic measures: data from Day 21, means and standard deviations (SD). Values

significantly different from control according to one-way ANOVA are marked with (*). (study table

3, part 1 of 2)

Nominal Dissolved oxygen

concentration Protein (mg/L) Chlorophyll (ug/L) (mgL!J

(gg/L) Mean SD Mean SD Mean SD

Control 35.7 7.4 1326 491.8 9.23 0.6

Solvent Control 31.9 8 732 241.5 8.67 0.81

3 49.7 * 11 2247.3 * 711.3 8.9 0.1

10 57.2 * 20.6 2356 * 1330.8 7.47 1.63

30 47 5.3 1037 216.1 6* 1.08

100 42.7 2.28 538.7 50.4 6.3 * 1.55

300 21.2 * 5.1 88.3 * 124.1 6.73 * 0.15

Table 4. Non-taxonomic measures: data from Day 21, means and standard deviations (SD). Values

significantly different from control according to one-way ANOVA are marked with (*). (study table

3, part 2 of 2)

Nominal

concentration Potassium (mg/L) Magnesium (mg/L) Calcium (mg/L)

(gg/L) Mean SD Mean SD Mean SD

Control 2.04 0.44 3.82 0.36 24.1 2.19

Solvent Control 2.04 0.54 3.69 0.49 23 0.29

3 1.73 0.1 4.17 0.45 23 0.29

10 2.56 0.9 4.17 0.45 21.8 0.82

30 2.17 0.52 3.15 * 0.06 20.2 * 0.29

100 1.66 0.11 3.45* 0.2 22.7 * 2.5

300 1.14 * 0.19 3 * 0.11 20 * 0

5
3 7 14

Day.

21

3.2 ug/L 110

•

337

Figure 1. Colonization of polyurethane foam substrates by protozoa over time. Curves were fitted

using the MacArthur-Wilson equilibrium model. Points marked with asterisks are significantly

different from controls. Average (not nominal) concentration for each treatment level is provided in

the upper left corner of each subplot.

6
Data Summary Table:

Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect Atrazine Effect Weighted Recovery

relative to Concentration Occurred Average Occur- if

controls (pg/L) (measurements Atrazine so. when

only reported Concentration (see

for Day 21) from Day 0 to comment)?

When Effect

Occurred

No stat. sig.

adverse 3 I!g/L NA 3.2 1!g!L NA Magnitudes effect

No stat. sig. estimated

Number of adverse 10 I!g/L NA 10 I!g/L NA from Fig. 1.

protozoa effect (*) indicates

species in No stat. sig. significant

difference.

new adverse 30 I!g/L NA 32 I!g/L NA Increases colonies effect

No stat. sig. were

observed at 3 adverse 100 I!g/L NA 110 I!g/L NA to 30 ug/L. effect

15% * 300 lJ,g/L Day 21 337 lJ,g/L NA

No stat. sig. NA

adverse 3 I!g/L 3.2 I!g/L NA Magnitudes

effect calculated

No stat. sig. NA from table 3.

adverse 10 I!g/L 10 1!g!L NA (*) indicates

Protein effect significant

biomass of No stat. sig. NA difference.

protozoa adverse 30 I!g/L 32 I!g/L NA Significant

effect increases

No stat. sig. NA were

adverse 100 I!g/L 110 I!g/L NA observed at 3

effect and 10 ug/L.

40% * 300 lJ,g/L Day 21 337 lJ,g/L NA

No stat. sig. NA Magnitudes

adverse 3 I!g/L 3.2 I!g/L NA calculated

effect from table 3.

No stat. sig. NA (*) indicates

adverse 10 I!g/L 10 I!g/L NA significant

Chlorophyll effect difference.

a of algal No stat. sig. NA Significant

protozoa adverse 30 I!g/L 32 I!g/L NA increases

effect were

60% 100 lJ,g!L Day 21 110 ug/l NA observed at

the lowest

90%* 300 I!g/L Day21 337 I!g/L NA two test

levels.

Primary No stat. sig. Magnitudes

productivity adverse 3 I!g/L NA 3.2 I!g/L NA calculated

(mid- effect from table 3.

7
Effect Magnitude of Nominal Day ofStudy Time Did Comments

Effect Atrazine Effect Weighted Recovery

relative to Concentration Occurred Average Occur- if

controls (pg/L) (measurements Atrazine so, when

only reported Concentration (see

for Day 21) from Day 0 to comment)?

When Effect

Occurred

morning No stat. sig. (*) indicates

dissolved adverse 10 !!g/L NA 10 !!g/L NA significant

oxygen) effect difference.

35% * 30 !!g/L Dav21 32 ug/L NA

35% * 100 )..lg/L Day 21 110 )..lg/L NA

30% * 300 ug/L Day 21 337 )..lg/L NA

No stat. sig. NA

adverse 3 !!g/L 3.2 !!g/L NA

effect

No stat. sig. NA Magnitudes adverse 10 !!g/L 10 !!g/L NA calculated effect from table 3. Potassium No stat. sig. NA (*) indicates adverse 30 !!g/L 32 !!g/L NA significant effect

No stat. sig. NA difference.

adverse 100 !!g/L 110!!g/L NA

effect

45% * 300 )..lg/L Day 21 337 ug/L NA

No stat. sig. NA

adverse 3 !!g/L 3.2 !!g/L NA

Magnitudes effect

No stat. sig. NA calculated

from table 3. Magnesium adverse 10 !!g/L 10 !!g/L NA (*) indicates effect

20% * 30 ug/L Dav21 32 ug/L NA significant

10% * 100 )..lg/L Day 21 110 )..lg/L NA difference.

20% * 300 )..lg/L Day 21 337 Ug/L NA

No stat. sig. NA

adverse 3 !!g/L 3.2 !!g/L NA

effect Magnitudes

No stat. sig. NA calculated

adverse 10 !!g/L 10 !!g/L NA from table 3. Calcium effect (*) indicates

15% * 30 !!g/L Day 21 32 !!g/L NA significant

difference.

5% 100 !!g/L Day 21 110 !!g/L NA

15% * 300 !!g!L Day 21 337 )..lg/L NA

Note: NA=Not AvaIlable (effect observed on last day ofexpenment)

Time weighted averages are taken directly from study table 1 (time means).

Reviewer Comments:

8
This was a well-controlled microcosm study with reasonable replication, five levels of

treatment, and both diluent and solvent controls. Methods are generally well described

and followed published standards, although more detail on the statistical methods and

exactly when they were applied would have been useful. Also, the conditions ofthe pond

from where the protozoa colonies were collected were not described; background

exposure to atrazine may therefore be possible.

Protozoa came from a pond, but their colonization in a running water scenario was

examined. The incoming water was dechlorinated tap water, devoid ofthe nutrients of

natural water, and the substrate was likewise unnatural. The ecosystem under study

consisted entirely of protozoa (Aufwuchs) that had attached to polyurethane substrates in

the pond. By not including larger plants and animals, the ecosystem structure was greatly

simplified from that of a natural ecosystem, and therefore the effects noted may not be

applicable to a more complete ecosystem structure.

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

Rationale for Use: Study contributes to the weight of evidence of potential effects of

atrazine to aquatic ecosystems.

Limitations of Study: This microcosm study had good replication and methods.

However, the focus was protozoa colonies on artificial polyurethane substrates, watered

by dechlorinated tap water. These conditions are highly simplified from a natural

ecosystem structure, and so results may not be easily generalized.

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

Secondary Reviewer: Brian Anderson, U.S. EPAIfL il/!'/If

9