1 

Open Literature Review Summary 

Chemical Name: Atrazine 

PC Code: 080803 

ECOTOX Record Number and Citation: 

Baxter, L.R., D.L. Moor, P.K. Sibley, K.R. Solomon, M.L. Hanson. 2011. Atrazine does not 

affect algal biomass or snail populations in microcosm communities at environmentally relevant 

concentrations. Environmental toxicology and chemistry, vol. 30 (7): 1689-1696. 

Purpose of Review (DP Barcode or Litigation): 

Review for inclusion in the CELOC calculation 

Date of Review: 3/15/16 

Summary of Study and Findings: 

This study was conducted over 73 d at the University of Guelph Turfgrass Institute Microcosm 

Facility (Guelph, ON, Canada). Each 12,000-L microcosm is 1.2m deep and 3.9m in diameter, 

with an actual water depth of approximately 1m. The microcosms were lined with a black foodgrade polyvinyl chloride (PVC) pond liner, contained soil covering 44% of the bottom surface 

(14.5 % OC and 1.8% IC), and were filled with spring water from an adjacent irrigation pond. 

Water was circulated at 12,000 L/d among the microcosms for seven weeks. Circulation among 

the microcosms was stopped 3 days prior to treatment. 

 

Atrazine Treatment 

Technical grade atrazine (96% a.i.: Syngenta Crop Protection) was added in a random design 

with nominal concentrations of 0, 1, 10, 30, and 100 ug/L with 3 replicates per treatment level. 

Acetone was used as a solvent with a final concentration per microcosm at 0.001% acetone, 

controls also received solvent (no solvent free controls included). 

General chemistry included collections of total nitrogen and total phosphorous 

(d=1,7,14,28,49,70), total hardness and alkalinity (d=1,7,14,28,49,70), pH, conductivity, 

temperature and DO (Monday-Friday starting on d14). Photosynthetically active radiation (PAR) 

was measured mid-day (d=0, 8, 17, 38, 44, and 71) at a depth of 60 cm. 

Macrophytes 

Shoots (5-10 cm length) were added two weeks prior to treatment in 4 L pots. Each cosm 

received 3 pots with 6 shoots of Myriophyllum spicatum/pot and 3 pots with six shoots of Elodea 

canadensis per pot. Total biomass, wet and dry mass, for shoots (both species) and roots (M. 

spicatum only). 
2 

Phytoplankton and periphyton 

Periphyton substrates were added on the north side of each microcosm two weeks prior to 

treatment to allow colonization. A periphytometer (48 back-to-back microscope slides in a 

Plexiglas frame) was fixed at the same location in each microcosm and suspended just below the 

water surface. Additional periphyton measurements were taken using 30 x10 cm strips of PVC 

pond liner material hung from the edges of the microcosms and reaching a depth of 60 cm (or 2/3 

of the water depth). Use of periphytometers allowed quantification of periphytic growth at the 

surface and in the relative absence of snail grazing, while growth on the PVC pond liner was 

subject to conditions of increased water depth and grazing pressure. Analyses of phytoplankton 

and periphyton biomass as ash-free dry weight (AFDW) and chlorophyll a were performed on 

days 1, 4, 7, 14, 21, 28, 49, and 70. 

Phytoplankton samples were collected by filtering 1.5 L of microcosm water through Whatman 

glass fiber (GF/ C) filters (pre-combusted and pre-weighed for AFDW samples). Periphyton 

samples were collected by separately scraping each substrate and collecting the resulting slurry 

on a glass fiber filter as above. Three microscope slides were used for each of AFDW and 

chlorophyll a of periphyton at the surface (six slides total). Polyvinyl chloride liner strips were 

divided in half and each portion was scraped separately for AFDW and chlorophyll-a analysis. 

Chlorophyll-a filters were wrapped in aluminum foil and frozen until spectrophotometric 

analysis. Ash-free dry weight was analyzed immediately following filtration. 

Growth of filamentous algae masses in the microcosms was assessed weekly through a scoring 

system. Three people separately ranked the amount of filamentous algae in each microcosm on a 

scale of 1 to 3, where 1=no or low colonization, 2=some masses, 3=high colonization. 

Confidence in the assessment was given a score of 1 (low) to 10 (high). 

Snails 

Growth and fecundity of snails was tracked using four snail cages in each microcosm. The cages 

consisted of 35x25x25 cm translucent food-grade polypropylene containers, fitted with 200 

micron mesh covering four 6-cm diameter holes to allow for circulation of water through the 

containers. Lids on the containers were modified so that mesh fabric (2-mm mesh) covered 90% 

of the surface area to allow light penetration. Photosynthetically active radiation measurements 

inside and outside the cages indicated no significant shading. Floats were fixed near the top of 

each container such that the top 10 cm of each cage emerged above the water surface. One day 

before treatment, 10 adult marsh snails (Stagnicola elodes; 21.4x3.47 mm) were released in one 

cage per microcosm to track fecundity, and another 10 juveniles (6 to 9 mm) were placed in one 

cage per microcosm for quantification of growth rates. The same procedure was carried out 

concurrently in separate cages with the snail Physella spp. (adults 10.4x1.35 mm; juveniles <2 

mm). 

The shell length of caged juvenile snails was measured on days 15, 22, 38, 51, and 73 using 

calipers and the snails returned to the cages. Egg masses produced in the adult snail cages were 

removed and counted weekly, and the number of eggs per egg mass was recorded. In each 
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microcosm, 50 adult Physella spp. were also free-released to ensure a base population; however, 

these snails have been found to naturally inhabit the microcosms, so actual populations were 

expected to be much higher. Substrates for the enumeration of free-living snails were added two 

weeks prior to treatment. These consisted of 30x30 cm removable mats made from PVC pond 

liner, suspended at the same location in each microcosm. Average size and egg production rates 

of free-living snails were estimated from these substrates. Free-living snail size measurements 

were performed weekly starting at day 7 by removing the snail substrates and measuring shell 

sizes with calipers. The number of snail egg masses on each mat and the number of snail eggs 

per egg mass were counted and the egg masses were removed. Zooplankton traps as described in 

Boudreau et al. [see 33 in Baxter et al. 2011] were also used to quantify the free-living snail 

population on the bottom of the microcosms. These traps were deployed overnight on days 1, 4, 

7, 14, 21, 28, 36, 42, 49 and 65. Snails accumulating in the trap jar and funnel were washed into 

the sample jar, preserved in 70% ethanol, and enumerated. 

Data analysis 

Parameters were assessed individually by repeated-measures analysis of variance (ANOVA) 

(a=0.05) using the mixed procedure of SAS 9.1 for Windows with the Kenward-Rogers 

correction. Pretreatment measurements of biomass were used as baseline covariates for algal 

parameters and number of surviving snails per cage was used as a time-varying covariate for 

caged snail size. In each analysis the most appropriate heterogeneous variance structure was 

determined based on the lowest possible Akaike’s Information Criterion value. If a significant 

interaction was found, the slice command was used to identify the days exhibiting treatment 

differences and these were further assessed by one-way ANOVA. For size of caged juvenile 

snails, the treatment response prior to covariate inclusion was investigated by comparing 

regression parameters for mixed-model growth curves using one-way ANOVA. Filamentous 

algae scores were assessed by repeated-measures ANOVA on ranks using Sigmaplot 11.0 for 

Windows (Systat Software). Dunnett’s test (a=0.05) was used for all post-hoc analyses. Nominal 

concentrations of atrazine were used in all statistical analyses. 

Results 

Atrazine and general chemistry 

Concentrations of atrazine measured 1 h after treatment were within 10% of nominal values, and 

dissipation to 36% or less of initial concentrations occurred by day 70 (Table S1). 

Concentrations in control microcosms were below the limit of detection (0.05 ug/L). 
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Table S1. Mean actual concentration of atrazine with standard deviation (SD) for all measured 

time-points (1 h to 70 d) by nominal concentration in microcosms (n=3) after treatment at day 0, 

measured by enzyme-linked immunosorbant assay (limit of detection = 0.05 μg/L). ND = nondetect. 

Time Contro

l 

1μg/L (SD) 10μg/L (SD) 30μg/L (SD) 100μg/L (SD) 

1 h ND 1.21 (0.091) 11.30 (1.344) 28.43 (4.225) 100.57 (5.661) 

1-d ND 1.17 (0.076) 12.88 (0.980) 35.07 (6.543) 117.77 (11.580) 

4-d ND 1.03 (0.339) 9.55 (0.934) 24.93 (2.658) 75.70 (2.712) 

7-d ND 0.93 (0.130) 8.72 (1.553) 22.07 (2.159) 80.59 (4.662) 

14-d ND 0.80 (0.147) 8.40 (1.300) 25.40 (3.293) 88.20 (5.359) 

28-d ND 0.55 (0.156) 5.02 (1.028) 17.90 (2.152) 66.93 (10.826) 

49-d ND 0.40 (0.188) 5.70 (1.911) 14.80 (4.409) 64.40 (15.492) 

70-d ND 0.22 (0.083) 3.50 (1.272) 9.70 (2.458) 36.40 (8.794) 

No significant differences in water chemistry parameters including pH, dissolved oxygen, 

conductivity, temperature, hardness, alkalinity, and total nitrogen and phosphorus were found 

between treatments and controls, but all changed significantly with time. Although there were no 

differences between treatments, hypoxic conditions existed (<3 mg/L DO) on at least one day in 

13 of the 15 microcosms, and in seven microcosms this occurred on more than 20% of days posttreatment (Table 3). Because water chemistry measurements were taken between 8 and 9 AM 

after nighttime respiration, the measured values represent minimum values. Photosynthetically 

active radiation was not different between treatments, but was significantly affected by time, 

with an increase at d 38 and declining values afterward. Measurements of PAR 1m above the 

microcosms did not decline with time. 
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Macrophytes 

In the following summary some data that the authors indicated as not being statistically 

significant (e.g., wet weight of macrophytes and chlorophyll a for phytoplankton) are not 

discussed. The following summary is intended to identify the results that were reported as 

significant by the authors.

The authors report statistically significant declines in Myriophyllum spicatum shoot (~ 78%) and 

root (~ 77%) weights and in Elodea canadensis shoot (~81%) weights for the 100 ug/L treatment 

as compared to the controls. There were no statistically significant differences reported for the 1, 

10, or 30 ug/L treatment groups. However, the reviewer determined that biologically significant 

effects are reported for M. spicatum (Figure 2, Table 4) which shows an approximate 46% and 

19% reduction in shoot weight compared to the control for the 10 ug/L and 30 ug/L treatment 

groups, respectively. In addition, the reviewer determined that potentially biologically significant 

effects to M. spicatum root dry weight were reported at 1, 10, 30 and 100 ug/L (Figure 2, Table 

4). There is less certainty in the significance of the reductions in root dry weight as compared to 

the reductions in shoot weight. This reduced certainty stems from overlapping error bars across 

the doses (Figure 2). Lastly, effects to E. canadensis shoot weight were biologically significant 

only at 30 and 100 ug/L, with 17% and 81% reductions respectively. 
6 

 

Phytoplankton and Periphyton 

The authors report no treatment related reductions on periphyton or phytoplankton. It is difficult 

to interpret the variability for each treatment level from the presented data in Figure 3. 

Snails 

Based on the aforementioned potential effects of the high nutrient levels in the microcosms, the 

study results for snails are in question. The hypothesis that changes in the autotroph community 

from increasing atrazine levels cause increases in snail populations on a dose responsive manner 

cannot truly be tested in this study. If the uncertainty around high nutrient levels wasn’t present 

and the results do indeed reflect the pattern of the sail population effects in the presence of 

atrazine, then there would be some weight to place on this portion of the study. The authors 

report no chemical related changes in the snail populations. 

Table 4: Reviewer estimated mean dry weights and percent reduction (compared to 

controls) from Figure 2. 

Treatment (ug/L) M. spicatum shoot 

dry weight 

M. spicatum root dry 

weight 

E. canadensis shoot 

dry weight 

Control (0) 37 13 52 

1 42 (+14%) 7 (-46%) 51 (-2%)

10 20 (-46%) 6 (-54%) 60 (+15%)

30 30 (-19%) 9 (-31%) 43 (-17%)

100 8 (-78%) 3 (-77%) 10 (-81%)
7 

 
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Endpoints for use in the calculation of the CELOC are listed below in the summary table. 

Data Summary Table: 

Effect Magnitude 

of Effect 

relative to 

controls

Nominal 

Atrazine 

Concentr

ation 

(ug/L) 

Day of 

Study 

Effect 

Occurred 

Time Weighted 

Average 

Atrazine 

Concentration 

from Day 0 to 

when Effect 

Occurred

(ug/L) 

Did Recovery 

Occur – If so, 

when?

Comments 

Biomass, 

Macrophytes 

46% 

reduction in 

mean M. 

spicatum root 

dry weight, 

biologically 

significant 

(reviewer 

estimated 

magnitude 

and 

significance) 

1 NA 0.7 No. 

Endpoints 

measured at 

study 

termination 

Biomass, 

Macrophytes 

48% 

reduction in 

mean M. 

spicatum

shoot dry 

weight, 54% 

reduction in 

mean M. 

spicatum root 

dry weight, 

both 

biologically 

significant 

(reviewer 

estimated 

magnitude 

and 

significance) 

10 Unclear of 

initial 

event, 70-d 

used

7.4 

Biomass, 

Macrophytes 

~19 % 

reduction in 

mean M. 

spicatum

shoot dry 

weight, 31% 

reduction in 

mean M. 

spicatum root 

dry weight, 

17% 

30 Unclear of 

initial 

event, 70-d 

used

19.05 
9 

reduction in 

E. canadensis 

shoot dry 

weight, each 

biologically 

significant 

(reviewer 

estimated 

magnitude 

and 

significance) 

Biomass, 

Macrophytes 

reduction in 

mean M. 

spicatum

shoot (78%) 

root (77%) 

and in E. 

canadensis 

shoot (~81%) 

dry weights 

(reviewer 

estimated 

magnitude), 

Statistically 

and 

biologically 

significant 

(author

estimated 

significance) 

100 Unclear of 

initial 

event, 70-d 

used

68.5 

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

Rationale for Use: 

This study demonstrates clear effects on macrophyte plant communities after the initiation of 

exposure to atrazine. Statistical and biologically significant effects were noted for macrophyte 

biomass at the tested concentrations of 1, 10, 30, and 100 ug/L. 

Limitations of Study: 

Summary data and statistics for many endpoints captured throughout the duration of this study 

were not presented in the report. This is critically important because the authors report 

significance information for endpoints but only averages across treatments are represented in the 

figures so within treatment trends could not be verified. 

Although there was an acclimation period, it differed for the biotic groups (macrophytes, 

phytoplankton, periphyton, and snails). 

High within-treatment variability was observed, especially but not limited to periphyton and 

phytoplankton. This was apparent even on day 0. Variability in DO measurements (Table 3) 
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may be reflective of the high variability in phytoplankton and periphyton response but it is hard 

to tell. The authors indicate that it wasn’t a result of block effects. In reality it is difficult to judge 

the apparent variability in DO measurements because Table 3 just shows > or < 3 mg/L instead 

of actual concentrations. 

These were outdoor microcosms but there was no discussion of weather during the experimental 

phase or if/how water levels were maintained. 

No solvent-free control treatment was included. 

Primary Reviewer: 

Frank T. Farruggia, Ph.D. Biologist ERB3 

Secondary Reviewer:

Michael Lowit, Ph.D. Ecologist ERB1 

References: 

Baxter, L.R., P.K. Sibley, K.R. Solomon, M.L. Hanson. 2013. Interactions between atrazine and 

phosphorous in aquatic systems: Effects on phytoplankton and periphyton. Chemosphere 90: 

1069-1076. 

Digitally signed by FRANK FARRUGGIA 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=FRANK 

FARRUGGIA, dnQualifier=0000048865 

Date: 2016.03.24 11:29:43 -04'00'

2016.03.24 13:45:02 

-04'00'