1 

Open Literature Review Summary 

Chemical Name: Atrazine 

PC Code: 080803 

ECOTOX Record Number and Citation: 

Pannard, A., B. Le Rouzic, F. Binet. 2009. Response of phytoplankton community to 

low-dose atrazine exposure combined with phosphorous fluctuations. Arch. Environ. 

Contam. Toxicol 57: 50-59. 

Purpose of Review (DP Barcode or Litigation): 

Review for inclusion in the CELOC calculation 

Date of Review: 3/16/16 

Summary of Study Findings: 

This study investigated the response of microalgal dynamics to long-term exposure (7-

weeks) to increasing atrazine doses, which were non-inhibiting during short-term 

exposure (a two days). The experiment was performed on a controlled phytoplankton 

community, sampled from the natural environment and maintained in semi-continuous 

culture. The dynamics of the phytoplankton community, exposed to a gradient of atrazine 

doses in the short or long term, were examined from photosynthetic activity (short-term 

exposure) or from photosynthetic activity, biomass, and community structure (long-term 

exposure).

Atrazine (TGAI) was used for short- and long-term exposure experiments. An aqueous 

atrazine solution consisting of 1.2 ug mL-1 was prepared by dissolving atrazine in 

distilled water using sonication without any organic solvent. 

The natural plankton community was collected by sampling 40 liters of water from a 

freshwater wetland in Brittany (Pleine-Fougeres, France) in autumn 2001. 

Macrozooplankton community was removed by filtration. 

Short-Term Sensitivity of the Phytoplankton Community to Atrazine

Short-term experiments (48 h of atrazine exposure) were first performed to determine the 

non-inhibiting doses to test in the long-term atrazine exposure experiment. Five nominal 

atrazine doses (controls and 0.05, 0.5, 5.0, and 50 ug/L) were tested on the community. 

Phytoplankton photosynthetic activity was monitored (five replicates per atrazine dose) 

from the rate of 14C incorporation after 1, 16, 22, 40, and 46 h of atrazine exposure in 

borosilicate glass vessels. These were incubated with 14C bicarbonates for 1 h in a 

temperature-controlled incubator, under light and temperature conditions similar to those 

for the initial phytoplankton community. The EC50 and EC10, which correspond to the 
2 

inhibitory doses reducing 50% or 10% of the biological activity, respectively, were 

calculated by adjusting the model of Bailer and Oris (1993; 1997) to the incorporation 

rates according to atrazine dose. 

Design of the Long-Term Atrazine Exposure Experiments

The initial community was first starved by reducing nutrient inputs and making it 

phosphorus deficient a few weeks before starting the long-term experiment. Phosphorus 

was then resupplied to obtain medium input (52.6 ug P-PO4 3- L-1) from the day of 

atrazine input (day 0) onward. 

The community was exposed to atrazine for 7 weeks in 500-mL borosilicate Erlenmeyer 

bottles. Two hundred forty milliliters of the phytoplankton community was placed in 

each of the 16 bottles, with four replicates per dose tested (controls and nominal doses: 

0.1, 1.0, and 10 ug atrazine/L). Semi-continuous culture conditions were maintained 

(discontinuous fresh input once a week), and similar atrazine doses were added weekly to 

each medium input to prevent dilution. Given that the atrazine half-life in a water column 

is between 80 and 150 days (Klassen and Kadoum 1979), the atrazine dose was assumed 

to remain almost constant throughout the 7 weeks of the experiment, i.e., more than 80% 

of the initial dose at the lowest half-life calculation (day 48). The phytoplankton biomass 

was measured by chlorophyll a concentration (Lorenzen 1967) and the biological activity 

was measured by carbon dioxide incorporation weekly. Taxonomic composition was 

determined at the beginning of the experiment, then after 1, 4, and 7 weeks of exposure. 

Fluctuations in the biomass of the coexisting species were evaluated by counting cells 

and colonies under a microscope using a Nageotte chamber and expressing the 

concentrations as cell units per milliliter for single-cell species and colony cell units per 

milliliter for colonial species (Aphanocapsa sp., Oscillatoria sp.). 

Dominance within the community was characterized by calculating Simpson’s index D of 

diversity 

Statistical Analysis 

The nonparametric two-factor statistical analysis of Friedman was used to test the effects 

of atrazine doses and time exposures on the rate of carbon dioxide incorporation (for 

short- and long-term experiments) and the evolution of biomass. A chi-square analysis 

was applied to test the homogeneity of community structures at different times and doses. 

The hypothesis that atrazine has no effect on the evolution of species densities was tested 

in a single-factor (dose) or two-factor (time and dose) analysis of variance. 

Lastly, a Kruskall-Wallis analysis was applied to test the effects of atrazine on Simpson’s 

index of diversity. In all statistical tests used, the probability level up to which the effect 

is significant was p<0.05. A factorial correspondence analysis, using software R was 

performed to see whether atrazine exposure was a predominant factor responsible for the 

final composition and structure of the phytoplankton assemblage. 
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Results Summary: 

Short-Term Sensitivity of the Community to Atrazine Exposure 

Atrazine-treated samples showed significantly lower biological activity than controls (S = 

42.58, 4 df, p<0.001). Communities exposed to 50 ug/L atrazine were inhibited after as 

little as 1 h with a 30% to 50% decrease in carbon incorporation (Fig. 1a). Results with 

longer exposure times, 16, 22, 40, and 46 h, confirmed this atrazine toxicity level (Fig. 

1a). The calculated EC50 was 60.7 ± 4.0 ug/L. 

 
4 

Long-Term Atrazine Exposure 

Biological Activity and Biomass 

The changes in primary production over time, based on the amounts of incorporated 

carbon dioxide, followed the same pattern regardless of atrazine treatment (Fig. 2). 

According to the author, carbon incorporation after 3 weeks decreased significantly 

compared to the control followed by potential recovery at six weeks (judged by 
5 

reviewer). For measured chlorophyll a, there were no significant differences observed 

between controls and treated communities (Fig. 2c). In the first period, increasing growth 

rates (C absorption rates) were significant following nutrient resupply (still available 

even 1 week after P-enriched medium input), which induced progressive biomass 

accumulation (chlorophyll-a abundance). In the second period, i.e., after 4 weeks, the 

growth rate decreased with accelerated nutrient impoverishment due to the higher 

phytoplankton biomass. Whereas biomass production exceeded loss due to weekly 

culture removal in the first period, it simply compensated this loss during the second 

period, as the phytoplankton biomass remained almost stable. 

Community Structure and Dominance 

The major impact on community structure, evaluated as a comparison of the initial and 

final densities of enumerated cells or colonies, was attributed to the biomass response 
6 

following P resupply (Table 1). Modification of the community structure due to low-dose 

atrazine exposure was also shown after 7 weeks of exposure, with a clear atrazine dose 

effect (Fig. 3). The first axis explained more than 61% of the variance of the data set. 

Going from left to right, the ordination on axis F1 differentiated the control communities 

from the atrazine exposed communities, at doses of 10, 1, and 0.1 ug/L, although the 

effect of the 0.1 ug/L dose showed high variability. 

It was concluded that community structure was significantly affected by 7 weeks of 

exposure to a dose of atrazine as low as 10 ug/L or less. In accordance with the 

correspondence analysis, significant changes in algal density occurred depending on the 

atrazine dose and taxonomic group (Table 1). 
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Further evaluation by the reviewers illustrate the percent reduction in cell densities 

(species, genus, and community-level) between the controls and treatment groups. 

Table 2. Percent Reduction of Treatment Final Cell Densities Compared to Controls

Treatment 

Skeletonema 

bibraianum 

Chlorolobion 

sp. 

Ankistrodesmus 

falcatus 

Chlorella 

sp. 

Oocystis 

sp. 

Unidentified 

chlorophyte 

Aphanocapsa 

sp. 

Oscillatoria 

limetica 

Total 

(Community) 

Density 

0.1 28 73 71 18 69 43 40 79 62 

1 -10 39 37 69 60 43 27 40 50 

10 22 54 66 73 78 56 33 49 67 

Bold values indicate significant difference versus the control. Biological significance without statistical 

significance described by the authors are italicized. 

A shift in community structure was also reflected in changes to dominance and diversity, 

as demonstrated by changes in Simpson’s index of diversity (1 – D) (Fig. 5). After 7 

weeks of exposure, (1 – D) was significantly higher in the community exposed to atrazine 

than in the controls (H = 9.2, 3 df, p = 0.027). This higher diversity accounted for a lower 

dominance in the atrazine treatments. The (1 – D) value was proportional to the atrazine 

dose, demonstrating a consistent dose-response effect. 
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Reviewer Conclusions: 

The authors present effects of atrazine on the phytoplankton assemblage, in terms of 

primary production and community structure for all tested concentrations (NOAEC <0.1 

ug/L). Their inclusion of phosphorous limitation as a variable in the study adds 

complexity to interpreting the results, as the nutrient limitation followed by the addition 

of phosphorous resulted in dramatic changes in abundance for all tested species 

independent of atrazine addition. Nonetheless, the data provided in Tables 1 and 2, and 

Figures 3 and 5, suggest that even low doses of atrazine were impacting the abundance 

of several tested species compared to those in the control community. 

The summary table below includes only those endpoints for use in the calculation of the 

CELOC, thus excludes the results from the short term experiment (Fig 1 and Fig 2). 

Data Summary Table for the Long Term Exposure Experiment 

Effect Magnitude of Effect 

relative to controls 

Nominal 

Atrazine 

Concentration 

(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 

Chlorophyll a No Effect 0.1 NA Not calculable NA 

Chlorophyll a No Effect 1.0 NA Not calculable NA 

Chlorophyll a No Effect 10 NA Not calculable NA 

Carbon 

incorporation 

~47% ↓ week 2, 

~18% ↓ week 3 

~25% ↓ week 4 

~62% ↓ week 5 

0.1 7-14 Not calculable Recovery 

week 6 

Difficult to 

interpret 

significance with 

available 

information. 
9 

Carbon 

incorporation 

~47% ↓ week 2, 

~12% ↓ week 3 

~33% ↓ week 4 

~23% ↓ week 5 

1.0 7-14 Not calculable Recovery 

week 6 

Difficult to 

interpret 

significance with 

available 

information. 

Carbon 

incorporation 

~47% ↓ week 2, 

~12% ↓ week 3 

~17% ↓ week 4 

~38% ↓ week 5 

10 7-14 Not calculable Recovery 

week 6 

Difficult to 

interpret 

significance with 

available 

information. 

Cell Density, 

Individual 

Phytoplankton 

Species and 

Communitylevel

Chlorolobion sp. (73% ↓) Ankistrodesmus 

falcatus (71 %

↓)

Oocystis sp. (69 %↓)

Unidentified 

chlorophyta (43%↓)

Aphanocapsa sp. (40 %↓)

Oscillatoria limnetica 

(79%↓)

Community (62%↓)

0.1 49 Not calculable No 

Recovery 

Effects only 

reported in terms 

of final 

measurement. 

Concentrations 

held constant by 

weekly additions 

of compound. 

Cell Density, 

Individual 

Phytoplankton 

Species and 

Communitylevel

Chlorolobion sp. (39% ↓) Ankistrodesmus 

falcatus (37 %

↓)

Oocystis sp. (69 %↓)

Chlorella sp. (60%↓)

Unidentified 

chlorophyta (43%↓)

Aphanocapsa sp. (27 %↓)

Oscillatoria limnetica 

(40%↓)

Community (50%↓)

1.0 49 Not calculable No 

Recovery 

Effects only 

reported in terms 

of final 

measurement

Cell Density, 

Individual 

Phytoplankton 

Species and 

Communitylevel 

Chlorolobion sp. (54% ↓) Ankistrodesmus 

falcatus (66 %

↓)

Oocystis sp. (78 %↓)

Chlorella sp. (73%↓)

Unidentified 

chlorophyta (56%↓)

Aphanocapsa sp. (33 %↓)

Oscillatoria limnetica 

(49%↓)

Community (67%↓)

10 49 Not calculable No 

Recovery 

Effects only 

reported in terms 

of final 

measurement

Community 

Structure

Changes in Simpson’s 

index of diversity and 

CCA results (shift in 

composition) 

0.1 49 Not calculable No 

Recovery 

An increase in the 

Simpsons index 

as well as a shift 

in the 

composition 

(changes in the 

relative 

abundance of the 

same species) in a 

dose response 

manner indicates 

Community 

Structure

Changes in Simpson’s 

index of diversity and 

CCA results (shift in 

composition) 

1.0 49 Not calculable No 

Recovery 

Community 

Structure

Changes in Simpson’s 

index of diversity and 

10 49 Not calculable No 

Recovery 
10 

CCA results (shift in 

composition)

that the 

concentrations 

tested affected the 

community 

structure leading 

to more variable 

communities than 

the controls and 

that the 

dominance in the 

community had 

shifted. 

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

Rationale for Use: 

This study demonstrates clear effects on multiple aspects of plant communities after the 

initiation of exposure to atrazine. While both the short and long-term experiments 

demonstrated effects, only the long-term experiments is applicable for use in the CELOC 

estimation. Effects reported for carbon incorporation suggest biologically significant 

reductions in primary productivity across all concentrations with recovery by week 6, 

however chlorophyll a did not reflect the same reductions across treatments. Statistical 

and biologically significant effects were noted for biomass (cell density) and different 

measures of community structure at the tested concentrations of 0.1, 1.0, and 10 ug/L. 

Limitations of Study: 

The method used in this study refreshed (weekly) each test solution and control with an 

aliquot of medium plus test material or medium only, respectively. The authors assumed 

that this maintained a relatively constant concentration at the intended nominal dose 

concentration based on the study duration and published half-lives of atrazine. They 

report greater than 80% recovery at the end of the study. Therefore, the reviewer 

assumed constant exposures at nominal concentrations. 

Primary Reviewer: 

Frank T. Farruggia, Ph.D. Biologist ERB3 

Secondary Reviewer:

Michael Lowit, Ph.D., Ecologist ERB1 

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 11:31:35 -04'00'

2016.03.24 13:44:12 -04'00'