Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

McGregor, E. B., K. R. Solomon, and M.L. Hanson. 2008. Effects of planting system design on

the toxicological sensitivity ofMyriophyllum spicatum and Elodea canadensis to atrazine.

Chemosphere. 73(3): 249-260. (MRID 48261124)

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 17,2009

Purpose of Study:

This study examines the potential effects of atrazine on aquatic plants (macrophytes M spicatum

and E. canadensis) grown individually and in population and community-level assemblages in

large outdoor microcosms.

Summary of Study Findings

Methods:

The study used 15 outdoor microcosms located at the University of Guelph Microcosm Facility

at the Guelph Turfgrass Institute, Ontario, Canada. Each microcosm was about 1.2 m deep, 3.9

m in diameter and 12,000 L in volume. Microcosms were lined with black food-grade polyvinyl

chloride and were sunk into the ground so the tops were flush with ground level. Water was

supplied from an adjacent spring-fed irrigation pond (62 x 62 x 4 m deep). Thirty-five plastic

propagation trays (52 x 25 x 7 cm) filled with an amended sediment mix were added to the

bottom of each microcosm. The sediment was a 1: 1:1 mixture ofsand, loam, and organic matter

and covered approximately 38% ofthe floor of each microcosm. Sediment carbon analysis was

determined by combustion using a Leco Carbon Analyzer (LECO SC444). Total carbon content

was 7.0%, inorganic carbon was 1.0%, and organic carbon was 6.0%.

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1II1II1III

2087808
Water was circulated between the irrigation pond and microcosms at a rate of 12,000 L per day

for 25 days prior to atrazine treatment to reduce variability between and within the microcosms.

Six pots ofM spicatum (plants obtained from several on-site untreated microcosms) were

planted in each microcosm during the initialization time, and were not used for effects

assessment. Circulation ended two days prior to treatment (July 16, 2006). The macrophytes

used for effects assessment (see below) were added to the microcosms on July 17,2006, one day

prior to treatment. Treatment occurred on July 18,2006 and the experiment was terminated on

August 29,2006 (42 days).

Five planting designs were used: individual plants, population assemblages (low and high

density), and community assemblages (low and high densities). Each microcosm contained

plants potted in each ofthese arrangements.

For individual planting designs, M spicatum L. (Haloragaceae) and E. canadensis Michx.

(Hydrocharitaceae) were obtained from an on-site untreated microcosm whose populations were

originally obtained from a local reservoir (Guelph Correctional Facility, Guelph, Ontario). Each

plant was grown in an individual 164 mL cone-tainer (a plastic planting tube, 21 cm long with

3.8 cm internal diameter, bottom hole plugged with cotton pad) held in a 96-well planting tray.

Cone-tainers were filled with the same amended sediment used in the microcosms and were

soaked for several days in irrigation pond water to allow settling. Apical shoots, without side

roots or shoots, of each plant were cut to 5 cm prior to planting. Each shoot was planted 2 cm

deep and surrounded by 0.5-1 cm of Turface (Applied Industrial Materials, Buffalo Grove,

Illinois) to secure it. Six plants per species were placed evenly spaced in each tray. One tray

was placed in the center of each microcosm to provide maximum sunlight and reduce edge

effects (total number oftrays per microcosm containing individually potted plants was not

stated). On the day before atrazine application, 10 plant shoots per species were evaluated as a

baseline, and on days 14, 28, and 42, two plants from each species per microcosm were sampled

at random.

For assemblage planting designs, mixed (two species) and monocultures ofM spicatum and E.

canadensis were grown in plastic sterilite containers (34.9 x 20.6 x 32.1 cm). Containers were

filled with sediment and prepared for planting like the cone-tainers. Monocultures were grown

at two densities: low-density consisted of nine plants per container (one plant per 80 cm

2 in a 3x3

grid configuration) and high-density consisted of 18 plants per container (one plant per 40 cm

2 in

a 6x3 configuration). Two-species mixed planting designs were also grown at two densities: low

density was nine plants per species (18 plants total in a 6x3 configuration) and high density was

18 plants per species (configuration not provided). Species type was alternated in each row of

plants. A total of 18 sterilite containers (3 containers per assemblage design) were added to each

microcosm; evenly spaced around the cone-tainer trays in the microcosm center. Assemblage

planting designs (monoculture and mixed) were sampled on days 14,28, and 42 after treatment.

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After sampling, plants were transported to the laboratory in water from their respective

microcosm and immediately measured for biomass (wet and dry) of both roots and shoots.

Chlorophyll a was measured by ethanol extraction with an Ultraspec 3100 pro UVNisible

Spectrophotometer using methods described by Nusch (1980). Biomasses of assemblages of

plants were normalized to a per-plant basis to allow comparison with individually grown plants.

Microcosms were treated with either 0 (controls), 25,50, 100, or 250 Ilg/L atrazine (n=3

microcosms per treatment). Treatments were randomly assigned to microcosms. Atrazine (96%

a.i., technical grade) was dissolved in 50 mL acetone in amber bottles, shaken, and added to the

microcosms while agitating with a stirrer to promote mixing. Control microcosms received the

same volume of acetone.

Water samples for atrazine analysis and water quality determination were collected using a metal

depth-integrated water column sampler. A composite sample was created from at least four

randomly selected locations in each microcosm, and stored at 4°C until analysis (500 mL

aliquots were stored for water quality). Water sampling occurred on days -1, 1,4, 7, 14,28, and

42.

Water quality measurements were temperature, dissolved oxygen (DO), pH, alkalinity, hardness,

and conductivity. Maximum and minimum temperatures were measured daily Monday through

Friday at a water depth of20 cm. Point measurements oftemperatures and DO were made daily

at 50 cm depth with a HANNA HI-9143 meter. DO was not measured on the last two days of

the study due to equipment failure. Water hardness, alkalinity and conductivity were measured

on days -1, 21, and 42. pH was measured weekly. Hardness and alkalinity were determined

with standard methods and kits by Hach. pH and conductivity were measured with an Accumet

Research AR20 pH/Conductivity meter. Photosynthetically active radiation (PAR) was

measured weekly with a LI-185A Li-Cor QuantumiRadiometerlPhotometer at midday on clear

sunny days. Readings were normalized by taking them at the surface of each microcosm, at the

surface in a glass jar, and at a depth of 60 cm in a glass jar.

Atrazine concentrations were analyzed by enzyme linked immunosorbent assay (ELISA), using

the Abraxis Atrazine Assay Kit. Samples were prepared for analysis by bringing them to room

temperature and centrifuging 10 mL aliquots at 805 g for 10 min, then diluting them so

concentrations would fall within the range ofthe standard response curve (0.1-5 Ilg/L).

Calibration curves were calculated with an Ohmicron RPA-I photometric analyzer. Analyses

were performed within 48 hours ofsampling, generally within 24 hours. Time-weighted

averages at each treatment level for the three replicates were calculated.

For each monitored water quality variable and PAR, a one-way analysis of variance (ANOVA)

was performed to identify significant differences among treatments using SigmaStat 3.5.

ANOVA assumptions were tested with the Kolmogorov-Smimov test for normality ofresiduals

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and the Levene median test for homogeneous variance. When significance was found (p < 0.05),

means were compared with the control using Dunnett's test (a = 0.05).

Plant toxicity (change in biomass endpoints) was evaluated using regression techniques

described in Stephenson et al. (2000). Macrophyte data for all treatments was modeled in

SigmaPlot 2000 with linear, logistic, and hormetic equations. The best-fit models were selected

by visual examination ofthe plots and the corrected r

2 (1 - [residual sum ofsquares]/[corrected

sum ofsquaresD. When biomass endpoints showed a significant concentration response (p <

0.05), effective concentrations ECIO, EC2s, and ECso (10%, 25%, and 50% difference between

atrazine treatments and controls in measured endpoints) were calculated along with their

confidence intervals using proc NUN in SAS v9.1. Only new growth data were used to assess

effects via regression analysis: mean biomass data on each sampling day was subtracted from

mean biomass data from the previous sampling day.

Relative growth rates (RGR) were calculated from wet and dry biomass of control plants

according to the following equation:

RGR = [In(W2) -In(Wd]/(t2 - tl)

where WI and W2 are plant weights at time tl and t2. RGRs were compared for all planting

designs (individual, monoculture at high and low density, mixed culture at high and low density)

for samples on days 14,28, and 42 using one-way ANOVA (a = 0.05) in SigmaStat 3.5 and

Tukey's test for pairwise comparisons when significance was found.

Interactions between atrazine concentration and planting design factors on biomass endpoints

were assessed with two-way ANOVA (a = 0.05) using proc mixed in SAS v9.1. ANOVA

assumptions ofresidual normality and homogeneous variance were assessed using the ShapiroWilk test (proc univariate normal) and by graphical interpretation ofresidual plots. Non-normal

data were transformed by natural log or square root. When significant interactions between

atrazine and planting design were detected, the "slice" command (not further described) was

used to see how atrazine effects changed with planting design. When main factor effects were

significant but interaction effect was not significant, means between pairs oftest designs (e.g.

individual vs. population) were compared using orthogonal contrasts (procedure not further

described).

Results:

Time weighted average (TWA) atrazine concentrations for the 42-day atrazine exposure were

close to nominal: control microcosms (O.O±O.O J,!g/L), 25 J,!g/L microcosms (24.5±2.5 J,!g/L), 50

J,!g/L microcosms (49.5±2.8 J,!g/L), 100 J,!g/L microcosms (104.0±5.6 J,!g/L), and 250 J,!g/L

microcosms (247.5±12.2 J,!g/L). Measured TWAs were used as the exposure level in data

analysis.

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Average values for water quality parameters over all sampling days in the course ofthe study are

provided in Table 1. No significant (p < 0.05) differences from controls were detected with

Dunnett's test for multiple comparisons, except for minimum temperature and DO. Minimum

temperature post-treatment, though significantly different in treatments, showed no

concentration-response trend. Dissolved oxygen was significantly decreased at the two highest

treatment levels compared to controls. Figure 1 shows trends in DO at each treatment level over

the experiment.

For plant toxicity, nonlinear regression analysis showed significant concentration-response

relationships for over 90% ofthe sampling times, planting designs, and biomass endpoints

examined, and EClO, EC2S, and ECso were calculated in most cases (Table 2 and Table 3).

Effective concentration (ECx) represented a treatment related decrease in all endpoints except for

chlorophyll a in individually grown plants. For individually grown M. spicatum (days 14,28,

42) and E. canadensis, chlorophyll increased with increasing atrazine concentrations; therefore,

ECIO, EC2s, and ECso represent 10%,25%, and 50% increases for these endpoints only. Box

plots of ECso estimates are shown in Figure 2. The study authors do not state which biomass

endpoints were plotted, however, the study reviewer assumes that data from all biomass

endpoints were combined into each box plot.

Relative growth rates (RGR) ofM. spicatum and E. canadensis in control microcosms are

presented in Table 4. RGRs were similar among the different planting designs except for the

significant difference (p<0.05) in E. canadensis on days 28 and 42. Pairwise comparison

showed that the RGRwet ofindividually grown E. canadensis was significantly lower (p<0.05)

than those of plants grown in high and low density model populations. RGR data is not presented

for treated microcosms.

Atrazine had a significant (p<0.05) effect on plant wet and dry biomass at all time points (Table

5). This analysis did not examine atrazine effects from individual treatment concentrations.

Plant biomass was significantly affected by planting design in about 60% ofthe endpoints

(plant/date/biomass). Generally, there were no significant interactions between atrazine

concentration and planting design on wet or dry biomass. The exceptions were day 14 wet shoot

biomass ofM spicatum, wet shoot and total wet biomass ofE. canadensis on day 28, and all

biomass endpoints ofE. canadensis on day 42. The "slice" command was applied to assess how

atrazine effects varied over planting treatments. The study authors reported that the biomass of

individually grown E. canadensis was not significantly different in each atrazine treatment;

however, the biomass of plants grown in assemblages was significantly different in each atrazine

treatment. The study authors also reported that orthogonal contrasts comparing mean biomass

measures between planting designs showed few significant differences between planting designs,

and no consistent trends across biomass endpoints for planting systems that showed statistically

significant different means. No further information or data was provided by the study authors

regarding the 'slice" command and orthogonal contrast results.

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The study authors mention that individually grown E. canadensis were grazed upon in some

control microcosms. They do not indicate if grazing occurred in any ofthe treatment

microcosms. Plants did not grow at all in the 25 Ilg/L atrazine treated microcosms. The study

authors suggest that the lack of growth may have been due to the selection of low-quality plants

or the chance occurrence of an herbivore. Due to these issues, the study authors suggest they may

have used insufficient replication. No further information is provided. They also note that

relative growth rates ofthe macrophytes were significantly lower than those ofthe same species

from previous investigations conducted at the same experimental site over similar experimental

lengths.

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Table 1. Chemical and physical parameters of the microcosms averaged over the duration ofthe 42-day

experiment, plus pre-treatment values. The values presented are mean ± standard deviation of

measurements over all sampling times, calculated from the mean of3 replicate microcosms at each sampling

date. pH values were converted to corresponding hydrogen ion concentrations for statistics calculations.

Alkalinity and hardness are measured as mglL of CaCOJ• PAR=photosynthetically active radiation. (study

table 1)

ConductTreatment Minimum temperature Maximum temperature Dissolved oxygen pH Alkalinity Hardness ivity PAR

(JlgIL) (0C) (0C) (mgIL) (n=7) (mgIL) (mg/L) (JlS/cm) (mE2

/s)

Pre- (n=4) Post- (n=28) Pre- (n=4) Post- (n=28) Pre- (n=4) Post- (n=25) (n=3) (n=3) (n=3) (n=4)

Control 22.8 ±2.9 22.2±2.2 25.8 ± 1.8 25 ± 2.4 10.3 ± 0.4 11.4 ± 1.5 8.7 ± 0.4 177 ± 30 305 ± 18 657 ± 26 503 ± 107

25 22.4 ± 2.8 24.4 ± 2.3 * 25.5 ± 2.3 25.2 ± 2.3 10.4 ± 0.4 11.6± 1.6 8.8 ± 0.4 167 ± 34 295 ± 30 630 ± 17 510± 113

50 22.6±2.7 24.1 ± 2.0 * 26.2 ± 1.7 25.3 ± 2 10.4 ± 0.3 II ± 1.6 8.7 ± 0.5 161 ± 44 295 ± 33 647±15 577 ± 134

100 22.7 ± 2.4 22.1 ± 2.3 25.7±2.6 25.2±2.1 10.4 ± 0.4 9 ± 1.4 * 8.5 ± 0.6 184 ± 13 311±17 703 ±29 513 ± 148

250 22.5 ± 2.3 22 ± 2.1 26.3 ± 1.8 25.6±2 10.3 ± 0.3 7 ± 1.7 * 8.3 ± 0.5 199± 5 333 ±22 727±41 510±142

*Mean significantly different from control mean (p <0.05) by Dunnett's test.

Figure 1. Dissolved oxygen (mg/L) concentrations at a depth of 50 cm in aquatic microcosms prior to

treatment and post treatment with various levels of atrazine over the 42-day exposure duration. (study figure

1)

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Table 2. For Elodea canadensis, effective concentrations (llglL) of atrazine required to cause a decrease in the

endpoints of interest by 10%,25%, and 50% (ECIO, EC2S, and ECso) with associated 95% confidence

intervals, Chlorophyll a endpoint EC's on day 28 and 42 (not day 14) are those required to cause an increase

(110%,125%,150%). EC's were calculated from the best-fit model for endpoints that showed a significant

concentration-response relationship (model type, equation, and corrected r

2 are shown). CI = confidence

interval; Ind = individually grown plants; Popl = population low-density; Pop2 = population high density;

Coml = community low density; Com2 = community high density. (study table 2)

Day Endpoint Planting design EClO (95% CI) EC,5 (95% CI) EC50 (95% CI) Model Parameters Corrected r>

14 Chioro-a Ind nc nc nc nc nc

Wet root Ind 41.0 (0, 173.3) 58.3 (0, 182.9) 82.8 (0, 196.0) Logistic b = 3.1298; t = 0.0219; x = 82.8 0.86

Popl 0.4 (0, 4.8) 1.6 (0, 139) 6.2 (0, 35.2) Logistic b = 0.7988; t = 0.0205; x = 6.2 0.98

Pop2 5.5 (0, 17.8) 12.7 (0, 30.9) 29.3 (5.9, 526) Logistic b = 1.3159; t = 0.0137; x = 29.3 0.96

Coml 8.5 (0, 61.9) 230 (0,115.8) 627 (0, 217.2) Logistic b = 1.0972; t = 0.0202; x = 62.7 0.59

Com2 22.8 (8.6, 37.1) 57.1 (21.6,92.6) 114.2 (43.2,185.3) Linear b = 0.0104; x = 114.2 0.35

Dry root Ind 29.3 (0, 599) 73.2 (0, 1497) 146.5 (0,299.4) Linear b = 0.0027; x = 146.5 0.45

Popl 0.4 (0, 69) 1.3 (0, 18.3) 4.6 (0, 43.3) Logistic b = 0.8571; t = 0.0024; x = 4.6 098

Pop2 39 (0, 17.3) 10.8 (0,34.7) 30.2 (0,66.1) Logistic b = 1.0728; t = 0.0012; x = 30.2 0.85

Coml nc nc 0.001 (0,0.4) Logistic b = 0.0739; t = 0.0027; x = 0.001 0.42

Com2 22.5 (9.8, 35.2) 56.3 (24.5, 88.0) 112.5 (490, 176.0) Linear b = 0.0011; x = 112.5 0.48

Wet shoot Ind 0.006 (0, 0.4) 0.4 (0, 15.4) 23.2 (0,296.9) Logistic b = 0.2673; t = 0.2283; x = 23.2 0.81

Popl 1.2 (0, 6.0) 5.1 (0, 18.3) 21.8 (0, 49.6) Logistic b = 0.7592; t = 0.2623; x = 21.8 088

Pop2 25.9 (0, 71.4) 45.2 (0, 97.7) 78.9 (18.9, 138.8) Logistic b = 1.9757; t = 0.1777; x = 78.9 0.91

Coml 7.1 (0, 53.2) 209 (0, 107.2) 61.9 (0, 214.2) Logistic b = 1.0126; t = 0.1002; x = 61.9 0.72

Com2 47.9(0,146.7) 61.7 (0,151.0) 79.5 (2.3, 156.7) Logistic b = 4.3312; t = 0.0481; x = 79.5 0.97

Dry shoot Ind 8.6 (0, 379) 19.5 (0, 62.5) 44.2 (0, 1038) Logistic b = 1.3462; t = 0.0165; x = 44.2 0.86

Popl 17.1 (0,37.2) 21.5 (7.5, 35.6) 272 (13.7, 40.7) Logistic b = 4.7348; t = 0.0109; x = 27.2 0.99

Pop2 28.0 (0, 99.5) 35.0 (0, 92.2) 43.7 (8.3, 79.2) Logistic b =4.9281; t=0.0098; x=43.7 0.99

Coml 9.1 (0, 52.0) 18.2 (0, 74.2) 36.2 (0, 105.1) Logistic b = 0.0034; t = 0.0042; x = 36.2 0.39

Com2 27.2 (0, 126.1) 32.8 (0, 117.3) 396 (0, 103.0) Logistic b = 5.8223; t = 0.0040; x = 39.6 0.99

28 Chloro-a Ind nc 82.9 (5.0, 160.8) 165.8 (10.1, 321.5) Linear b = 1.3424; x = 165.8 0.74

Wet root Ind nc nc nc nc nc

Popl 9.0 (0, 36.2) 13.4 (0, 39.6) 20.0 (0.7, 39.3) Logistic b =2.7465; t= 0.1791; x=20.0 0.99

Pop2 11.8 (0, 27.1) 16.4 (3.4, 29.4) 22.7(14.4,311) Logistic b = 3.3517; t = 0.1533; x = 227 099

Coml 30.9 (0, 88.7) 48.7(0, 109.1) 76.6 (14.0, 139.2) Logistic b = 2.4223; t = 0.0504; x = 76.6 0.88

Com2 20.2 (9.9, 30.5) 50.5 (24.7, 76.3) 100.9 (49.3, 152.5) Linear b = 0.0612; x = 100.9 0.38

Dry root Ind nc nc nc nc nc

Popl 5.8 (0, 326) 9.6 (0, 39.7) 158 (0, 43.2) Logistic b = 2.1900; t = 0.0164; x = 15.8 0.99

Pop2 10.5 (0,26.8) 15.1 (0.4,29.7) 21.6 (11.6, 31.6) Logistic b = 3.0466; t = 0.0135; x = 21.6 099

Coml 33.0 (0, 70.0) 44.8 (11.6, 78.0) 61.0 (27.1,948) Logistic b = 3.5708; t = 0.0050; x = 61.0 0.96

Com2 5.1 (0,22.3) 9.7 (0, 31.4) 18.4 (0, 40.5) Logistic b = 1.7023; t = 0.0134; x = 18.4 099

Wet shoot Ind 4.8EClO (0, 15EC7) 0.00011 (0,0.02) 247 (0, 546.4) Logistic b = 0.0891; t = 0.3655; x = 247 0.83

Popl 4.4 (0, 16.023) 9.5 (0,25.9) 20.3 (1.6,39.1) Logistic b = 1.4347; t = 1.3464; x = 20.3 0.99

Pop2 9.0 (0, 18.0) 16.4 (58, 27.1) 300 (18.6, 41.4) Logistic b = 1.8250; t = 1.1458; x = 30.0 099

Coml nc nc nc nc nc

Com2 10.1 (0,36.5) 20.8 (0, 56.1) 427 (0, 88.0) Logistic b = 1.5291; t = 0.2607; x = 42.7 0.97

Dry shoot Ind 30.0 (1.3,58.7) 75.0 (3.4, 146.7) 150.1 (6.7,293.5) Linear b = 0.0418; x 150.1 0.97

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Day Endpoint Planting design ECIQ (95% CI) EC25 (95% CI) EC50 (95% CI) Model Parameters Corrected [2

Popl 4.5 (0,21.4) 9.1 (0,31.6) 18.3 (0,42.4) Logistic b = 1.5743; t = 0.1172; x = 18.3 0.99

Pop2 7.3 (0, 18.3) 12.8 (0.3, 25.3) 22.6 (10.9, 34.3) Logistic b = 1.9342; t = 0.1209; x =22.6 0.99

Coml nc nc nc nc nc

Com2 10.5 (0,26.0) 19.4 (0.68, 38.0) 35.9 (14.4, 57.3) Logistic b = 1.7821; t = 0.0464; x = 35.9 0.97

42 Chloro-a Ind nc 80.0 (8.5,151.4) 159.9 (17.0, 302.9) Linear b = 1.1121; x = 159.9 0.54

Wet root Ind nc nc nc nc nc

Popl 6.2 (0, 31.8) 10.4 (0, 39.2) 17.6 (0, 43.4) Logistic b =2.0955; t = 0.8724; x = 17.6 0.99

Pop2 19.4 (1.7, 37.0) 27.4 (10.5, 44.2) 38.6 (22.7,54.5) Logistic b =3.1936; t = 0.4066; x =38.6 0.99

Coml 20.8 (0, 54.1) 29.1 (0,60.2) 40.6 (12.5, 68.7) Logistic b =3.2826; t = 0.1600; x =40.6 0.99

Com2 4.2 (0, 19.0) 9.8 (0, 32.4) 23.1 (0, 51.3) Logistic b = 1.2907; t = 0.1462; x =23.1 0.98

Dry root Ind nc nc nc nc nc

Popl 5.3 (0,30.7) 9.3 (0, 39.7) 16.6 (0, 45.6) Logistic b = 1.9213; t = 0.0864; x = 16.6 0.99

Pop2 5.9 (0, 19.4) 11.4 (0, 28.6) 22.3 (4.4,40.1) Logistic b = 1.6491; t = 0.058; x = 22.3 0.98

Coml 21.0 (8.9, 33.0) 52.4 (22.3, 82.6) 104.8 (44.6,165.1) Linear b = 0.0222; x = 104.8 0.46

Com2 4.1 (0, 16.5) 9.5 (0,28.4) 22.3 (0, 45.8) Logistic b = 1.2950; t = 0.0215; x =22.3 0.99

Wet shoot Ind nc nc nc nc nc

Popl 6.8 (0, 23.6) 14.5 (0,377) 31.1 (2.7, 59.6) Logistic b = 1.4420; t =2.7193; x = 31.1 0.97

Pop2 nc nc 62.6 (42.3,82.9) Logistic b =3.5941; t = 1.6819; x = 0.96

Coml 39.1 (0, 140.1) 59.9 (0, 160.3) 91.9 (0, 189.1) Logistic b =2.5733; t =0.4981; x =91.9 0.99

Com2 23.3 (10.8, 35.8) 58.2 (27.0, 89.4) 116.4 (54.1,178.8) Linear b = 0.3625; x = 116.4 0.67

Dry shoot Ind nc nc nc nc nc

Popl 2.4 (0, 14.1) 7.0 (0, 29.6) 20.8 (0,55.7) Logistic b = 1.0140; t = 0.334; x =20.8 0.87

Pop2 17.8 (4.5, 31.2) 30.0 (15.1, 44.9) 50.4 (33.7, 67.1) Logistic b =2.1156; t = 0.2582; x = 50.4 0.95

Coml 28.6 (0, 105.1) 47.9 (0, 132.4) 80.1 (0, 172.5) Logistic b =2.1365; t = 0.1012; x = 80.1 0.97

Com2 2.4 (0, 13.2) 8.0 (0, 31.2) 26.9 (0, 67.3) Logistic b =0.9047; t = 0.1039; x =26.9 0.83

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Table 3. For Myriophyllum spicatum, effective concentrations (I!g/L) of atrazine required to cause a decrease

in the endpoints of interest by 10%,25%, and 50% (EClO, EC2s, and ECso) with associated 95% confidence

intervals, Chlorophyll a endpoint EC's are those required to cause an increase (110%, 125%, 150%). EC's

were calculated from the best-fit model for endpoints that showed a significant concentration-response

relationship (model type, equation, and corrected r

2 are shown). CI = confidence interval; Ind = individually

grown plants; Pop1 = population low-density; Pop2 = population high density; Com1 = community low

density; Com2 = community high density. (study table 3)

Day Endpoint Planting design ECIO (95% CI) EC25 (95% CI) EC50 (95% CI) Model Parameters Corrected r'

14 Chloro-a Ind 29.9 (20.9, 39.0) 74.8 (52.2, 97.5) 149.7 (1043,195.0) Linear b = 0.6449; x = 149.7 0.99

Wet root Ind 21.8 (0, 52.8) 36.0 (1.8, 70.1) 59.4 (21.7, 97.1) Logistic b=2.1893; t=0.7622; x= 59.4 0.92

Popl 18.4 (0,472) 323 (0, 65.9) 56.7 (17.5, 95.8) Logistic b = 1.9567; t = 0.5426; x = 56.7 0.82

Pop2 23.9 (0, 506) 39.1 (10.1,68.1) 63.9 (32.2, 95.6) Logistic b = 2.2337; t = 0.489; x = 63.9 0.9

Coml 15.1 (0,30.2) 26.5 (8.9,44.1) 465 (26.1, 669) Logistic b = 1.9487; t = 0.444; x = 46.5 0.97

Com2 24.7 (0, 55.1) 35.7 (73, 64.1) 51.5 (25.0, 77.9) Logistic b = 2.9887; t = 0.4183; x = 51.5 0.74

Dry root Ind 27.7 (0, 75.2) 43.7 (0, 93.7) 69.0 (16.1,121.8) Logistic b = 2.4067; t = 0.0417; x = 69.0 0.88

Popl 26.5 (12.9,40.2) 663 (32.1, 100.5) 132.7 (64.3, 201.0) Linear b = 00275; x = 132.7 0.5

Pop2 256 (0, 53.2) 41.7 (11.8, 71.5) 67.8 (35.4, 100.2) Logistic b = 2.2532; t = 0.0298; x = 67.8 0.9

Coml 13.4 (0, 29.5) 23.4 (5.0, 41.9) 40.9 (20.0, 61.8) Logistic b = 1.9724; t = 0.0264; x = 40.9 0.99

Com2 240 (12.0, 36.0) 59.9 (29.9, 90.0) 120.0 (59.8, 180.0) Linear b = 0.0204; x = 120.0 0.47

Wet shoot Ind 75.4 (0, 196.8) 188.6 (0,492.0) 377.2 (0, 984.0) Linear b = 0.5696; x = 377.2 03

Popl 277 (13.2, 42.2) 693 (33.1, 105.4) 138.5 (66.2,210.8) Linear b = 1.2575; x = 1385 0.29

Pop2 26.1 (0,65.4) 50.2 (0.7, 99.6) 96.4 (34.8, 157.9) Logistic b = 1.6825; t = 1.0424; x = 96.4 0.79

Coml 12.4 (0, 37.5) 36.8 (0, 83.0) 109.4 (23.9, 1949) Logistic b = 1.0079; t = 1.2698; x = 109.4 0.97

Com2 261 (15.1,37.1) 653 (37.7,928) 130.5 (75.4, 1857) Linear b = 0.8469; x = 130.5 0.69

Dry shoot Ind 357 (72, 643) 893 (17.9, 160.7) 178.6 (35.8, 321.4) Linear b = 0.0673; x = 178.6 0.43

Popl 24.0 (15.2, 32.7) 599 (38.0, 81.8) 119.8 (76.1,163.5) Linear b = 0.1006; x = 119.8 0.51

Pop2 27.6 (0, 62.617) 427 (6.5, 79.0) 66.2 (283, 104.2) Logistic b = 2.5083; t = 0.1008; x = 66.2 0.82

Coml 3.6 (0, 13.4) 14.1 (0,38.0) 55.8 (5.0, 106.6) Logistic b = 0.7975; t = 0.1139; x = 55.8 0.92

Com2 18.7 (U, 363) 31.7 (11.9, 51.6) 53.9 (31.5, 763) Logistic b = 2.0750; t = 0.0853; x = 53.9 0.88

28 Chloro-a Ind 21.0 (15.1, 26.9) 52.4 (37.6, 67.2) 104.9 (753, 134.5) Linear b = 1.0226; x = 104.8 0.94

Wet root Ind 6.4 (0, 143) 14.6 (2.9, 263) 33.5 (18.1,48.9) Logistic b = 1.3271; t = 2.2309; x = 33.5 0.94

Popl 22.2 (7.0, 375) 55.5 (17.4, 93.6) 111.0 (34.8, 1873) Linear b = 0.8680; x = 111.0 0.25

Pop2 171 (0,509) 25.7 (0, 59.6) 38.7 (47,72.7) Logistic b = 2.6884; t = 1.3293; x = 387 0.99

Coml 21.9 (1.6, 42.1) 26.4 (7.4, 45.4) 31.8 (8.9,54.7) Logistic b = 5.8401; t = 1.3301; x = 31.8 0.99

Com2 233 (12.2, 34.4) 58.2 (30.4, 860) 116.5 (60.9,172.1) Linear b = 0.4201; x = 116.5 0.46

Dry root Ind 1.7 (0, 10.2) 65 (0, 273) 25.2 (0, 65.4) Logistic b = 0.8142; t = 0.2445; x = 25.2 0.87

Popl 2U (45,37.7) 528 (11.4, 94.1) 105.5 (22.7, 1883) Linear b = 0.0841, x = 105.5 OJ7

Pop2 24.4 (0, 75.8) 31.2 (0, 76.4) 399 (38, 75.9) Logistic b =4.4753; t = 0 1333; x = 39.9 0.99

Coml 18.2 (0, 46.2) 22.4 (4.0, 40.8) 276 (6.9, 483) Logistic b = 5.2685; t = 0.168; x = 27.6 0.99

Com2 3.4 (0, 393) 73 (0, 58.9) 15.5 (0, 75.7) Logistic b=1.4515;t=0.1619;x=155 0.99

Wet shoot Ind 303 (17.6, 429) 75.6 (44.0, 107.3) 151.3 (88.0,2145) Linear b = U559; x = 151.3 0.61

Popl 240 (62, 41.8) 600 (15.5,1046) 120.1 (31.0,209.2) Linear b = 1.9232; x = 120.1 031

Pop2 377 (0, 1393) 57.2 (0,1581) 86.9 (0, 1844) Logistic b = 2.6307; t = 1.2715; x = 869 0.96

Coml 268 (6.1,47.5) 670 (152,1188) 133.9 (30.3, 2375) Linear b = 1.7578; x = 1339 0.18

- 10-
Day Endpoint Planting design ECIO (95% CI) EC25 (95% CI) EC50 (95% CI) Model Parameters Corrected r

Com2 25.9 (10.9, 40.9) 64.7 (27.2,102.3) 129.5 (54.4,204.6) Linear b = 1.0324; x = 129.5 033

Dry shoot Ind 4.8 (0, 17.6) 14.2 (0, 38.6) 42.5 (1.6, 83.5) Logistic b = 1.0025; t = 0.1960; x = 42.5 0.78

Popl 22.6 (6.6,38.5) 56.4 (16.6,96.3) 112.8 (33.1, 192.5) Linear b = 0.2842; x = 112.8 0.43

Pop2 43.3 (0, 143.6) 57.8 (0, 150.9) 77.2 (0, 163.1) Logistic b = 3.7953; t = 0.1551; x = 77.2 0.96

Coml 21.0 (9.8, 323) 52.6 (24.5, 80.6) 105.1 (49.0, 161.3) Linear b = 0.2354; x = 105.1 0.44

Com2 35.8 (0, 107.5) 51.3 (0, 120.8) 73.5 (5.1,141.8) Logistic b = 3.0615; t = 0.1749; x = 73.5 0.71

42 Chloro-a Ind 24.6 (9.4, 39.8) 61.5 (23.5, 99.4) 122.9 (47.1,198.8) Linear b = 0.8665; x = 122.0 0.79

Wet root Ind 25.0 (8.9 41.2) 62.6 (22.2 103.0) 125.2 (44.4 206.0) Linear b = 1.3906; x = 125,2 0.57

Popl nc nc 28.2 (0, 2363) Hormetic b=-0,0513; t=0.5160; 0.56

h = -0.0447; x = 28.2

Pop2 nc nc 35.6 (0, 251.6) Hormetic b = 1.0022; t = 0.8844; 0.53

h = -0.0447; x = 35.6

Coml 20.8 (0, 54.1) 29.1 (0, 60.2) 40.6 (12.5, 68.7) Logistic b = 3.2826; t = 0.16; x = 40.6 0.99

Com2 22.9(3.7,42.1 57.2 (93, 105.2) 114.4 (18.5, 210.4) Linear b = 1.0049; x = 114.4 0.63

Dry root Ind 22.9 (4.6, 41.2) 57.2 (11.4,103.1) 114.5 (22.8,206.1) Linear b = 0.1934; x = 114.5 0.43

Popl nc nc 24.6 (0, 435.0) Hormetic b = 1.0073; t = 0.0924; 032

h = -0.0409; x = 24.6

Pop2 nc nc nc nc nc

Coml 21.0 (8.9, 33.0) 52.4 (223, 82.6) 104.8 (44.6,165.1) Linear b = 0.0222; x = 104.8 0.46

Com2 423 (0, 121.0) 52.1 (0, 122.9) 64.0 (0, 154.7) Logistic b = 5.3061; t = 0.1674; x = 64.0 0.95

Wet shoot Ind 34.8 (9.4, 60.1) 86.9 (23.5, 1503) 173.8 (47,0, 300.5) Linear b = 1.0201; x= 173.8 0.41

Popl nc nc 53.1 (0 157.4) Hormetic b = 0.8781; t = 1.4204; 0.12

h = -0.0731; x = 53.1

Pop2 nc nc nc nc nc

Coml 36.0 (0, 136.1) 52.1 (0, 149.8) 75.5 (0, 170.8) Logistic b = 2.9648; t = 03721; x = 75,5 0.99

Com2 25.8 (1.9, 49.7) 64.5 (4.7, 124.2) 129.0 (9.5,248.4) Linear b = 1.2199; x = 129.0 0.6

Dry shoot Ind 13.6 (0, 32.5) 22,7 (2.0, 43.4) 37.8 (15.4, 603) Logistic b = 2.148; t = 0.2596; x = 37.8 0.88

Popl 21.3 (5.2, 37.4) 53.3 (130, 93.5) 106.6 (26.0,187.1) Linear b = 0.2862; x = 106.6 0.16

Pop2 nc nc 54.8 (0, 136.8) Hormetic b = 0.9117; t = 0.1119; 0.71

h = -0.0759; x = 54.8

Coml 26.8 (0, 101.5) 45.1 (0, 128.5) 75.9 (0, 168.2) Logistic b =2.1099; t = 0.1317; x = 75.9 0.97

Com2 23.4 (63 40.5) 58.4 (15.7,101.2) 116.9 (31.3, 202.5) Linear b = 0.1899; x = 116.9 0.65

- 11 -
:.

100

150

300

250

200

3:50

8 50

O"'-------.---.,..---.---I----.----'----r---....------.---.,..---r-----'-O

40

80

160 .,-----------__-----,-----------------r 400

Day 14 - E. canadensis Day 14 - M. spicaflJ 140

120

.~ 100 o

Ii?

U

W

:0-

ro

1jl

:;!:

,rod Fopl Pcp2 Coml Com2 'rod Popl Pop2 Coml Com2

120

100

BO

60

40

20 I=:=J t::::::J 20

O.l...----.---.---..,......--.___--.---..l----r----r----.-----r----.---LO

40

60

160,..--------=---=-=---=----:---.....----------------,- 160

Day 28 - E. canadensis Day 28 - M. spicatu

140 140

120 -J

ll00

l/'l 80

u

w

>-

-el

Ind Popl Pop2 Com1 Cem2 100 Pop1 Pap2 Cem1 Com2

40

120

100

140,.----------:=------:-=--=-----:---:-r------------------,- 200

Day 42 - E. canadensis Day 42 - M. spicalu lBO

160

140

120

100

BO

60

40

20

0.l...----.---.---...--_.___----r---...I.---.,..--~--.___-__r--~----l0

Ind Pop1 Pop2 Com! Com2 jrod Papl Pop2 Coml Cam2

Figure 2. Box plots of effective concentrations of atrazine resulting in 50% differences in macrophyte growth

compared to controls (ECso). Boxes range from 25th to 75th percentiles, whiskers from 10th to 90th percentiles,

and solid line within the box represents the median. Ind = individually grown plants; Popl = low-density

population; Pop2 = high density population; Coml = low density community; Com2 = high density

community. The growth endpoint (wet or dry roots or shoots) plotted is not provided. The study reviewer

assumes that each box plot represents data from all biomass endpoints. (study figure 2)

- 12 -
Table 4. Relative growth rates of E. canadensis and M. spicatum grown in control microcosms. Values are

mean (n=3) ± standard deviation. P-values are from results of one-way ANOVA. Post-hoc pairwise

comparisons with Tukey's test were performed for significant differences as determined by ANOVA (p <

0.05). (study table 4)

Low-density High-density Low-density High-density

Endpoint Individual population population community community p-Value

E. canadensis

Day 14 RGRwet 0.060 ± 0.057 0.082 ± 0.008 0.061 ± 0.021 0.040 ± 0.036 0.025 ± 0.013 0.301

RGRdry 0.041 ± 0.023 0.033 ± 0.017 0.027 ± 0.022 0.017 ± 0.016 0.014 ± 0.011 0.414

Day 28 RGRwet 0.049 ± 0.016 0.088±0.017 * 0.085 ± 0.005 * 0.019 ± 0.015 0.047 ± 0.011 <0.001

RGRdry 0.033 ± 0.036 0.066 ± 0.023 0.069 ± 0.012 0.014 ± 0.013 0.047 ± 0.006 0.029

Day 42 RGRwet 0.029 ± 0.025 0.077 ± 0.015 * 0.066 ± 0.002 * 0.040 ± 0.014 0.040 ± 0.008 0.014

RGRdry 0.034 ± 0.031 0.069 ± 0.015 0.066 ± 0.003 0.041 ± 0.018 0.045 ± 0.008 0.14

M spicatum

Day 14 RGRwet 0.126 ± 0.009 0.132±0.018 0.132 ± 0.003 0.141 ± 0.023 0.120±0.022 0.619

RGRdry 0.112 ± 0.013 0.118 ± 0.020 0.120 ± 0.003 0.129 ± 0.022 0.111 ± 0.018 0.692

Day 28 RGRwet 0.096 ± 0.005 0.063 ± 0.038 0.079 ± 0.027 0.084 ± 0.028 0.061 ± 0.016 0.452

RGRdry 0.102 ± 0.006 0.067 ± 0.044 0.078 ± 0.033 0.094 ± 0.030 0.079 ± 0.033 0.683

Day 42 RGRwet 0.047 ± 0.021 0.048 ± 0.024 0.045 ± 0.016 0.023 ± 0.014 0.041 ± 0.026 0.596

RGRdry ** 0.067 ± 0.006 0.053 ± 0.023 0.050 ± 0.020 0.037 ± 0.018 0.052 ± 0.026 0.518

* Significant (p<0.05) difference from individually grown plants (pairwise comparison) using Tukey's test.

** Data that did not meet the assumption of equal variance were LN transformed..

- 13 -
Table 5. Main and interactive effects of atrazine and planting design (Pdesign) on biomass endpoints for E.

canadensis and M. spicatum, as determined by a mixed-model two-way ANOVA.

Main effects Interaction

Dataset Atrazine Pdesign Atrazine*Pdesign

p-value p-value p-value

E. canadensis

Day 14 Wet root 0.0028* 0.3139 0.8018

Wet shoot 0.0008* 0.0099* 0.9771

Total wet 0.0005* 0.0167* 0.9846

Dry root 0.0132* 0.0401 * 0.3133

Dry shoot <.0001 * 0.0074* 0.8526

Total dry 0.0299* 0.0299 0.9173

Day 28 Wetroot' 0.0005* 0.9502 0.3693

Wet shoot <.0001 * <.0001 * 0.0014*

Total wet <.0001 * <.0001 * 0.0042*

Dry root' 0.0003* 0.8053 0.49

Dry shoot <.0001 * 0.5531 0.2189

Total dry <.0001 * 0.6569 0.2669

Day 42 Wet root' <.0001 * <.0001 * 0.0009*

Wet shoot 0.0004* <.0001 * 0.0001*

Total wet 0.0003* <.0001 * 0.0002*

Dry root' 0.0003* 0.0020* 0.0135*

Dry shoot 0.0002* <.0001 * 0.0174*

Total dry 0.0002* <.0001 * 0.0144*

M spicatum

Day 14

Day 28

Day 42

Wet root <.0001 * 0.0010* 0.9694

Wet shoot <.0001 * 0.0001 * 0.0113*

Total wet <.0001 * 0.0482* 0.5595

Dry root <.0001 * 0.0011 * 0.9326

Dry shoot <.0001 * 0.0907 0.083

Total dry <.0001 * 0.0654 0.3964

Wet root 0.0003* <.0001* 0.0817

Wet shoot 0.0029* 0.219 0.3389

Total wet 0.0006* 0.0582 0.3159

Dry root' 0.0110* 0.0019* 0.5266

Dry shoot 0.0007* 0.6833 0.4465

Total dry 0.0016* 0.2752 0.4536

Wet root' 0.0001* 0.0187* 0.7913

Wet shoot 0.0138* 0.0048* 0.181

Total wet 0.0032* 0.0003* 0.2286

Dry root' 0.0001 * 0.0187* 0.7913

Dry shoot 0.0001* 0.1855 0.1697

Total dry 0.0006* 0.0073* 0.6759

• Dataset does not meet ANOVA assumptions ofresidual normality and homogeneous variance

* Statistically significant (p<0.05)

- 14 -
Data Summary Table

Time Weighted Did

Nominal Average Recovery Magnitude of Atrazine Day ofStudy Atrazine Occur-if Effect Effect relative to Concentration Effect Concentration

so, when Comments

controls (Ilg/L) Occurred from Day Oto (see When Effect comment)? Occurred

No effect 251lg/L Not applicable Not applicable Not Effect magnitudes

applicable estimated from

figure 1.

No effect 50llgiL Not applicable Not applicable Not

applicable

No statistics were

104.0±5.6 provided

35% decrease 100llgiL Day 1

IlglL' No comparing

treatment and

control at each

sampling date.

Decrease in However, the study

dissolved authors compared

the control and

oxygen treatment

microcosm DO

measurements

50% decrease 250 flglL Day 1 247.5±12.2 No averaged over the

IlgIL' entire study. For

that comparison,

there was a

significant

difference (p<0.05)

for the 100 and 250

IlgIL treatment

levels.

1 TWA calculated by study authors for the entrre 42-day expenmental penod.

- 15 -
Reviewer Comments:

Data analysis and statistical procedures are, for the most part, described completely and are

appropriate for the aims ofthe study. Exceptions were minor, and include not enough

information provided on the "slice" command or on orthogonal contrast procedures, and lack of

information on which biomass endpoint was used for the ECso box plots.

In general, effects at each tested atrazine concentration could not be determined due to the way

in which data and analyses were presented. For instance, for the ANOVA analysis presented in

Table 5, the effects of atrazine were analyzed without regard to atrazine concentration (i.e., all

treatment levels were combined). Additionally, biomass data were not presented so effects at

each tested atrazine concentration level could not be determined by the reviewer. Regression

results and effect concentrations are provided in detail, but the ECxs ofthe biomass endpoints

were highly variable, thus cannot be considered reliable for establishing effect concentrations.

Specifically, there was great overlap among confidence intervals for ECI0, EC25, and EC50.

Furthermore, in many cases the ECx was not bounded by the treatment concentration range and

the lower bound 95% confidence interval was 0 ~glL.

Although the microcosms were multispecies, endpoints except dissolved oxygen were presented

for effects on individual macrophyte species rather than community-level effects.

Dissolved oxygen measurements were a function of photosynthesis by all macrophytes in a

microcosm. In addition, any phytoplankton in the microcosms would have contributed to

photosynthesis as well. The study authors do not report information about phytoplankton in the

microcosms, however it is likely that they were present given that the microcosms were outdoors

and were filled with water from an irrigation pond. Therefore, effects of atrazine on both

macrophytes and phytoplankton would have contributed to the observed effects on dissolved

oxygen.

Several experimental factors discussed by the study authors may have confounded the

interpretation of atrazine as the causal stressor or limit the ability to detect effects of atrazine.

For instance, macrophyte growth in the control microcosms was lower than expected by the

study authors and led to reduced biomass accumulation. They suggested a number offactors that

may have contributed to the low growth. As a consequence, low biomass accumulation likely

limited the ability to detect effects of atrazine on growth-based endpoints (root and shoot

weight). Other issues were herbivore grazing ofsome microcosms but not others and no growth

in the 25 ~g/L microcosms.

- 16 -
Description of Use in Document: Qualitative (except effective concentration data is invalid

due to high variability, single species effects, and factors confounding interpretation of atrazine

as the causal stressor)

Rationale for Use: Microcosm experiments contribute to the weight of evidence regarding

effects of atrazine on aquatic plant communities.

Limitations of Study: Data and analyses were not presented in a way in which effects from

each atrazine treatment concentration could be determined. Biomass data for each atrazine

treatment level was not presented (regression results are provided instead). There was high

variability in the ECx values. Several factors may have limited the ability to detect effects of

atrazine as the causal stressor: selective herbivory, no growth in the 25 /lg/L atrazine treatment,

and overall low growth in all microcosms. Except for dissolved oxygen; endpoints were

presented for effects on individual macrophyte species rather than community-level effects.

Experimental design and presentation ofthe results focused on planting system design effects.

Primary Reviewer: Sara H. Hendrix, M.S., Subcontracting Scientist, Cambridge

Environmental Inc.

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 1t7,t:tJ~~

Tertiary Reviewer: Brian Anderson, Biologist, EPA ~:Ir r /1;jL--

- 17 -