### 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 envir onment 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 collecte d 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 Phytopl ankton Community to Atrazine Short-term experiments (48 h of atrazine expos ure) were first performed to determine the non-inhibiting doses to test in the long-ter m 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 14 

### C incorporation after 1, 16, 22, 40, and 46 h of atrazine exposure in borosilicate glass vessels. These were incubated with 14 

### C bicarbonates for 1 h in a temperature-controlled incubator, under light a nd 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 starti ng the long-term experiment. Phosphorus was then resupplied to obtain medium input (52.6 ug P-PO 4 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 th e 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 simi lar 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 pe r milliliter for single-cell sp ecies 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 experiment s) and the evolution of biomass. A chi-square analysis was applied to test the homogeneity of communi ty 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.

### 3 Results Summary:

Short-Term Sensitivity of the Comm unity 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 th e second period, i.e., after 4 weeks, the growth rate decreased with accelerated nutrient impoverishment due to the higher phytoplankton biomass. Whereas biomass pr oduction 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, eval uated as a comparison of the initial and final densities of enumerated cells or coloni es, 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 ).

### 7 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.

### 8 Reviewer Conclusions:

The authors present effects of atrazine on the phytoplankton assemblage, in terms of primary production and community structure fo r 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 th e 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 int erpret 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 andCommunitylevel 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 andCommunitylevel 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 andCommunitylevel 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 C hanges 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 no ted 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'



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