### Open Literature Review Summary

ChemicalName: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Schmitt-Jansen, M. and R. Altenburger. 2005. Predicting and observing responses of algal communities to photosystem II-herbicide exposure using pollution-induced community tolerance and species-sensitivity distributions. Environmental Toxicology and Chemistry. 24: 304-312. (MRID 48261132)

. 

### Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofa Level ofConcern (LOC).



### Date ofReview: October 21, 2009


### Purpose ofStudy: This study examines the potential effects ofatrazine, prometryn, and isoproturon onperiphyton communities in static-renewal indoormicrocosms. Observedpollution-inducted community tolerance (PICT) responses were then comparedto species-sensitivity distributions (SSDs) obtained from the US EPA ECOTOX database.


### Summary ofStudy Findings: This review focuses onthe effects from atrazine treatments.



### Methods: All experiments were conducted from November 2002 to March 2003 (notreported when atrazine experiments were conducted). Twelve 20-L glass aquaria (microcosms) were filled with unfiltered water from a eutrophic pond (source water location and characteristics were not presented). Each aquarium held 100 glass disks (1.5-cm diameter), mounted onplexiglas racks, to serve as surface substrates forperiphyton. The aquariawere maintained in a climate chamber at 18-22°C on a 14: 10 hour lightdark cycle photoperiod using light (source not specified) with an irradiance of130-150 /-lmol/m2/s. Aquaria waterwas continuously mixed (10 cm/s) with paddles. Any acclimation ofthe microcosms, to allowperiphytonto colonize the disks, priorto treatment was not reported.
- 1 - 

# IIII.I.IBI 2087863Eight aquariawere treated with atrazine (purity >99.2%), in dimethyl sulfoxide (DMSO), at nominal concentrations from 0.0075-1 mg/L (0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.50, and 1.0 mglL). The final co-solvent (DMSO) concentration was 0.1 % (v:v). The remaining four aquaria were maintained as controls, withtwo aquaria treated with DMSO at 0.1 % v:v (solvent controls) and two aquaria receiving no treatments (untreated controls). At 7 days post-treatment, the water in each aquarium was entirely replaced and retreated atthe respective application concentration. Following 14 days ofexposure, long term effects ofatrazine on periphyton chlorophyll 

### a content and diatom species composition were determined as described below. Additionally, after 14 days ofexposure, development oftolerance was assessed through reexposure ofcollectedperiphyton communities to atrazine for a short interval and then effects on photosynthetic activity (short-term toxicity) were determined as described below. Forthe photosynthetic activity experiments, analyses ofcommunities from three aquariums (two atrazine treatment levels and one control) were performed each day, with all twelve aquaria (treated and controls) analyzed within 4 days. Atrazine concentrations were not measured during the experiment.
Chlorophyll 

### a. Periphyton samples (disks) were extracted with acetone (final concentration 90%) and sonication (5 minutes), then stored frozen (-80°C) in darkness for 24 hours. Extracts were filtered (O.2-llm) and analyzed by reverse-phase HPLC according to the method ofWoitke 

### etal. (1994).

Diatom species composition. Periphyton samples (disks), inreplicates offive, were preserved and analyzed according to the method ofKrammer and Lange-Bertalot (1986-1991). Samples were fixed with formaldehyde (final concentration 5%), then algal material was scraped from the disks and boiled in 30% hydrogenperoxide solution for**~24** hours until the solution was colorless. Species were identifiedunder a lightmicroscope based on the siliceous exoskeleton. Relative distribution ofthe most dominant species was determined by counting at least 100 individuals per species. The sevenmost dominant species were ranked according to relative abundance, with the remaining diatom species grouped together. Shannon-Weaver diversity (based on species richness and distribution) was calculated according to the method of Muhlenberg (1998).
Photosynthetic activity (short-term toxicity tests on pre-exposed communities from the microcosms). Periphyton samples (disks) were individuallyplaced in scintillation vials containing 4 mL control water, then treated with atrazine in a geometric concentration series (dilution factor 1:2) at nominal concentrations of0.0075-2 mglL. The final concentration ofthe co-solvent (DMSO) was 0.1 %. The study authors state that "samples without any contamination and samples containing DMSO served as controls". Itis unclear whetherthe experimental design for the shorttermtestused controls (untreated andtreated with DMSO) and atrazine treatments for all microcosm communities (controls and atrazine treated) or ifcommunities from control aquarium (untreated and treated with DMSO) served as controls during the short-term tests. All treatments were performed intriplicate. Prepared samples were maintained at in situ - 2 -light and temperature, as described above, with gentle agitation for 1 hour. Photosynthetic activity was then measured using a pulse-amplitude-modulated (PAM) fluorescence-based method, with quantum yield measurements taken at 665 nm using a fiberoptic sensorpositioned 1 mm above the sample. Samples were acclimated to darkness for 3 minutes, then illuminated for 3 minutes with actinic light at 111 Ilmol/m2/s. Operational quantum yield (Fv/F'm) was detected after applying a single saturationpulse. Initial experiments determinedthatyields remained constantwith actinic light exposure intervals exceeding 2.5 minutes.

Relative inhibition offluorescence yield at 665 nm, as comparedto the control (unclearwhich control), was calculated to model concentration-response relationships. Median effective concentration values were calculated using log-logistic analysis as follows: y 

### =A2

### + [AI

-A2]/[1 

### + (x/xof], where Al = maximum response, A2 

### = minimum response,


### x = concentration,
Xo = concentration at median efficacy, and p = slope. Analyses were performed using nonlinear least-squares fitting (Origin software).
Modeling Species-Sensitivity Distributions (SSDs). The US EPA ECOTOX database (www.epa.gov/excotox; update May 5, 2003) was used to gather atrazine toxicity values for aquatic organisms, with informationrestrictedto micro- and macro-algae. Median effective concentrations were used for exposure intervals**of~l** day. Invalid orunspecific datapoints (e.g. tests stating aphylum or functional group, such as phytoplankton) were omitted. Whenmore than one toxicity value was presentfor the same species, geometric means were calculated. Effectparameters were held to growth-related endpoints (overall growth, population changes, changes in chlorophyll a orbiomass). Empirical SSDs were constructed according to the method ofSolomon 

### etal. (1996), with toxicity dataplotted cumulatively. To determine cumulative frequency distributions, percentages were calculated as follows: [n 

### + l]/[N

### + 1], where

n = rank number ofthe datumpoint and N 

### =total number ofpoints in the dataset.

Results: Chlorophyll a concentrations varied among the 0.0075-0.25 mg/L atrazine treated microcosms after 14 days ofexposure but appearedto remain at levels similarto the controls (Table 1). The study authors did not distinguish the untreated (negative) control from the DMSO control. Atthe 0.5 and 1 mglL treatment concentrations, chlorophyll a was decreased 60% and 72%, 

### - 3 -respectively, as compared to the average ofall the controls (Table 1). According to the study authors, there was large variation withinthe microcosms (datanotprovided).

Diatoms comprised the majority (>80%, 37 species) ofthe periphyton algae inthe microcosms. In atrazine treated microcosms, species numbers were similarto controls for microcosms treated at 0.0075-0.25 mg/L and the number ofdiatom species decreased 62% inthe microcosms treated with 0.5-1 mg/L as compared to the average ofall control microcosms (Table 1). A noticeable shiftin diatom community structure also occurred atthe highest atrazine treatment concentrations after 14 days ofexposure; Nitzschia capitellata increased from**:S~5%** ofdiatom species at 0-0.13 mg/L exposure to comprising**~40%** at 0.25 mg/L and**~95%** atthe 0.5 and 1 mg/L concentrations (Figure 1). Shannon-Weaverbiodiversity values were 0.94-1.7 for untreated controls, 0.76-1.56 forperiphyton treated at 0.0075-0.25 mg atrazine/L, then decreased to 0.14-0.15 atthe 0.5 and 1 mg atrazine/L treatment concentrations (Table 1).

For one ofthe previously non-exposed periphyton control microcosms (not stated which one), inhibition of photosynthetic activity followed a dose-response pattern with**~25%** inhibition when treated with atrazine at 0.015 mg/L,**~50%** inhibition at 0.13 mg/L, and**~70-80%** inhibition at 0.5-2 mg/L (Figure 2a). No difference (not stated how determined) was observed in the EC50 values ofthe untreated and solvent blank controls (reviewer assumes these 'controls' were the communities previously unexposed to atrazine in the microcosms). Both control microcosm communities (untreated (negative) and DMSO treated) were exposed to DMSO in the short-term testing. EC50 values were 0.113 to 0.15 mg/L for untreated controls (difference between negative and DMSO treated controls not provided). Prior exposure to atrazine appeared to induce tolerance, but only atthe 0.125 mg/L treatment concentration (EC50 

### = 0.35 mg/L). EC50 values at higher pre-exposure levels (0.25-1.0 mg/L) were not presented. Re-exposure of periphyton previously treated at 0.125 mg/L showed less inhibition ofphotosynthetic activity**(~5-20%)** at each treatment concentration (0.0015-2 mg/L), as compared to periphyton from one ofthe control microcosms not previously exposed to atrazine (Figure 2a). Data for the 0.0075 mg/L short-termtreatmentwas notpresented.
Species-sensitivity distribution. The compiled SSD dataset (n = 169) included 44 species consisting of7 macrophyte species and 37 microalgal species (Figure 3). The microalgal dataset consisted of 17 chlorophytes, 10 diatoms, 8 cyanophytes, 1 chrysophyte, and 1 cryptophyte. SSDs were constructed for the three main microalgal groups; chlorophytes, diatoms, and cyanophytes. Reported EC50 values ranged from 0.012-1.274 mg/L, with the cyanophyte Pseudanabaena galeata the most sensitive and the chlorophyte Chlamydomonas moewusii the least sensitive. Median EC50 values were 0.078 mg/L for macrophytes, 0.075 mg/L for cyanophytes and 0.069 mg/L for chlorophytes; however, the median EC50 was higher for diatoms at 0.179 mg/L.

A comparison ofSSDs to the pre-exposed (pollution-induced community tolerance, PICT) and untreated natural community sensitivities showed that community EC50 values covered - 4 -concentration ranges similar to the single-species data (Figure 3). Median EC50 values of untreated (control) periphyton communities covered 55% ofthe cumulative fraction ofaffected species represented in the SSD. EC50 values ofthe more tolerant communities increased to levels ofthe more tolerant species. Sensitivities ofpre-exposed communities shifted to the right end ofthe SSD, covering 85% ofaffected species represented inthe SSD

The study authors suggestedthat a consistentrelationship betweenthe algal SSDs and maximally induced community tolerance after atrazine exposure could be determined suchthat the right end ofthe SSD, which corresponds to the maximum PICT, would represent a concentration range at which most plant cells are severely affected. In addition, the authors suggest that the correspondence with the periphyton community responses observed in the conducted microcosm experiments and the compiled algal SSDs indicate thatthe SSDs would be good predictors ofthe effects of PSII herbicides, like atrazine, on freshwater microphytobentic communities and predicted no-effectconcentrations derived from the SSDs.

Table 1. Periphyton structural characteristics (chlorophyll a, diatom species, and Shannon-Weaverdiversity) followin2 14 days ofexposure to atrazine at 0.0075-1 m2/L. (studv table 1) Atrazine (nominal mg/L) Parameter Controls 0.0075 0.015 0.03 0.06 0.125 0.25 0.50 1.0 Chlorophyll a (Ilg/disk) NA 1 1.46 0.77 1.36 2.27 1.29 0.44 0.99 0.84 0.71 0.48 0.34
Diatom species (no.) 22 12 10 9 17 9 7 15 15 16 5 5 Biodiversity 1.7 1.02 0.94 1.16 1.44 1.05 0.76 1.4 1.41 1.56 0.14 0.15 1 Not analyzed.

- 5 -

## • Nitzschia 

## parea [J 

## Gomphonema parvurlJm 

# mNavicula semi 

### nulum 

## o other

## species ofdiatoms

40



## o 20 

### r!J 

## Nitzschia capitellata 

# m Fragilaria 

## spec. IAl 

### Navicula 

### halophila Nitzschia archibaldii 100



### control 0.01 0.02 0.03 0.06 0.13 0.25 0.50 1.00
Figure 1. Relative distribution ofdiatom species following 14 days ofexposure to atrazine at 0.0075-1 mglL. (study figure 2)

0,1 atrazlne (mg L·l )**O.O+--~}r""""'i** ---.--................................".---

...... 0,01 0,1 pre-exposure with atrazine (mg L") 

## •

• • 

## • 

# • • • • untreated
• EC,., aft8fpre-expos;ure



### (b) 0.3 0,4



# - ":' .....**~O.2**


# -- uP, W 0,1

(a) .-. 100 • untreated

• pnHJKjXlliufe (0.125mg L" atfeztne} 

# j eo C >. 60 

## B { 40 • (5 • 

### IIl.A. § 20 

## X

## is**~....r** 

# f 0

## ~:;;;""'_._-....,r--

# .......................-rrr-.........--. 0.01

Figure 2. Short-term inhibition ofphotosynthetic activity (concentration-response relationships and loglogistic modeling) for an untreated microcosm and periphyton pre-exposed to atrazine at 0.125 mg/L for 14 days (a) and algal tolerance (EC50s) for an untreated microcosm and microcosms pre-exposed to atrazine at 0.0075-0.125 mg/L for 14 days (b). (study figure 1)

- 6 -1 

# • • 

## • I

0.1 

### EC50 

### atrazine <mg l"1) 

## • 

# r:>1 untreated 

## communities

• Pre-exposed communities

• Ecotox·database .•• 

## A



# .,., . 

# .
. 

## . 

# , . 

# • 

# .

' 

# ,. I

0.0**-I-r-----,,---,.......,"""""'...,....,...,~** 

### ........t.-
.... 0.01 

# (a) 

# 11.0 lfi 

# lO.8

b 0.6 

# i
0.4 

# t

0.2

Figure 3. Species-sensitivity distributions (SSDs) constructed with data from single-species tests using algal species derived from the US EPA ECOTOX database. Also depicted are median effective concentrations (EC50s) from community tests (controls) and the range ofinduced tolerance (range ofpollution-induced community tolerance after long term exposure). (study figure 3)

- 7 -Data Summary Table: 

### Periphyton in static-renewal indoormicrocosms , Time Weighted Magnitude Nominal Average Did of Effect Atrazine Day of Study Atrazine Recovery Effect relative to Concentration Effect Concentratio Occur-if Comments controls (pgIL) Occurred nfromDayO so, when? to When Effect Occurred**~25%** 15 Magnitude ofeffects decrease estimated from Figure 2; SSNR1,2.**~10** 30 decrease Effects datarepresents a**~40%** single controlmicrocosm Decreased decrease 60 (not stated ifnegative or photosynthesis; DMSO treated) short-term (1**~50%** 125 hour) toxicity decrease Not Not No dataprovided on controls using untreated 1 hour calculable applicable used for short-term tests or if periphyton**~60%** 250 they were performed. (control decrease microcosms Average EC50 ofall control after day 14) (untreated) microcosms was 130 Ilg/L. The lowest**~70-80%** 500, 1000, reported EC50 ofall decrease 2000 microcosms was 113 Ilg/L (this was a control microcosm, unknown if negative orDMSO treated).**~5%** 15,30 decrease
Decreased**~20%** 60 photosynthesis; decrease Magnitude ofeffects short-term (1**~30%** 125 estimated from Figure 2; hour) toxicity decrease Not Not SSNR1,2. following pre- 1 hour calculable applicable exposure to**~50%** 250 No data provided on controls atrazine at decrease used for short-term tests or if 0.125 mg/L for**~60%** they were performed. 14 days decrease 500, 1000**~80%** 2000 decrease Change in +90% 7.5 Magnitude ofeffects biomass stimulation Not Experiment calculated from results in (chlorophyll a, 14 calculable terminated Table 1 using average ofall +8% at 14 days Ilg/disk) stimulation 15 control microcosms;

- 8 -Time Weighted Magnitude Nominal Average Did Day of Study Atrazine Effect of Effect Atrazine Effect Concentratio Recovery Comments relative to Concentration Occur-if controls (llglL j Occurred nfromDayO so, when? to When Effect Occurred 17-63% 30,60,125, SSNR1,2, decrease3 250 60% Cannot distinguish between 500 negative and DMSO decrease controls. 72% 1000 decrease +28% 7,5 stimulation Magnitude ofeffects calculated from results in 32-47% 15,30 Table 1 using average ofall Change in decrease Experiment control microcosms; Not number of +13 to 14 calculable terminated SSNR1,2, diatom species +21% 60,125,250 at 14 days stimulation Cannot distinguish between negative and DMSO 62% 500,1000 controls. decrease 19% 7.5 increase 13% 15 Magnitude ofeffects decrease calculated from results in Change in 37% 30 Table 1 using average ofall decrease Experiment control microcosms; diatom diversity 14 Not terminated SSNR1,2. (Shannon- 16 to 17% calculable at 14 days Weaver) increase 60, 125 Cannot distinguish between 29% negative and DMSO increase 250 controls.
88% 500,1000 decrease 1 StatIstIcal sIgnIficance, as compared to controls, was notreported. 2 Only one microcosm pertreatment. 3 Magnitude ofeffects were 63%, 17%,30% and 41% decreases atthe 0.03, 0.06, 0.13 and 0.25 mg/L treatments,

respectively.

- 9 -Reviewer Comments: This study also examines the potential effects ofprometryn and isoproturon onperiphyton.
The microcosms were filled with water from a eutrophic pond which was not further described. It is not stated whether the pond water was tested to establish that it was not contaminated with any pollutants during the study period. If any atrazine were present in the controls, then observed effects relative to the controls may be misleading.
The microcosms did not contain a sediment layer. Any acclimation ofthe microcosms, to allowperiphyton colonization, prior to treatmentwas not reported.

As an indoormicrocosm study, there may be little relevance ofthe results to natural systems, despite the natural source ofthe communities, giventhe small size ofthe microcosms (20-L aquaria) and lack ofa sediment layer.

Water chemistry, including pH, turbidity, dissolved oxygen, alkalinity, total nitrogen, total phosphorus, conductivity and particulate and dissolved organic carbon, were not monitored in the micrpcosms. Only a single microcosm was treated at each atrazine test concentration.
Atrazine treatment concentrations were neither confirmed normonitored inthe microcosms following treatment. The study authors did not expect any significant deviations from the nominal concentrations based on Log

### Kow values (losses due to sorption to the biofilms) and modeling using the following software programs: EPI Suite v 3.10 (to assess expected stability over 7 days between water changes), WVOLWIN (to assess losses due to volatility), and BIOWIN (to assess losses due to biodegradation). Only diatoms were identified at the species level to assess any changes in species composition and community structure. Exceptfor the calculation ofEC50 values, no statistics were provided.
DMSO solvent was used at a concentration of 1 mIll in all experiments. Although a negative and a DMSO control were used in the microcosms, effects due to DMSO could not be discounted for chI a, species number, and biodiversity because the reported data did not distinguish among the control microcosms. For the short-term toxicity tests, the authors reported that there was "no difference" in the inhibition ofphotosynthesis (EC50) between communities from the negative and DMSO control microcosms (no data nor statistics were provided to support this statement).

- 10

-Both ofthe 'control' communities (with or without prior DMSO exposure) were subsequently exposedto DMSO (1 mIll) during the short-termtests, complicating the interpretation ofwhether DMSO had an effect onthe EC50 ofphotosynthetic inhibition. During the short-termtoxicity experiments, identification ofthe 'control' was unclear in boththe methods and in statements made by the study authors in the results. For the short-term toxicity tests, 'controls' discussed by the study authors were communities from microcosms not preexposed to atrazine. It is not clear if a second set of controls were used in the short-term experiments and, ifso, when the authors are referring to one level ofcontrol versus the other (the study reviewermade assumptions whenpossible).
The authors reporthigh within-microcosm variation for chlorophyll a. Description ofUse in Document: Invalid

Rationale for Use: There was only a single microcosm (i.e. no replication) pertreatmentlevel. No statistics were provided. DMSO was used as solvent (l mIll) at a concentration 100% higher than the criteriafor study acceptance «0.5 mIll, static-renewal testing), thus the toxic effe<;ts of atrazine may have been confoundedby the addition ofsolvent. Although negative and DMSO control microcosms were used, the authors did notreport ifthere were differences betweenthe two types ofcontrols for chI 

### a, species numbers, orbiodiversity, thus effects due to DMSO could not be discountedby the reviewer. The use ofcontrols is unclear for the short-termtoxicitytests.

Limitations ofStudy: There were numerous reporting and measuring deficiencies. No effort was made to document whether other chemicals (including atrazine) were present inthe treatment and/or control microcosms priorto study initiation. The microcosms did notcontain a sedimentlayer. Water chemistry ofthe microcosms was not monitored eitherpriorto or following treatment. Only a single microcosmwas treated at each atrazine test concentration. Nominal atrazine concentrations were not confirmed and concentrations were notmonitored in the microcosms following treatment. Changes in community structure were not assessed, except for diatom species. The concentration ofsolvent (DMSO) was relatively high. No statistics were performed. There was high within-microcosmvariability for at least one endpoint (chlorophyll a). Itis unclear what controls were used inthe short-termtoxicity tests and subsequent analysis.
Primary Reviewer: Lynne Binari, M.S., Subcontractorto Cambridge Environmental Inc. Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA

# 1/ff;ttJ**/~** Tertiary Reviewer: AnitaPease, M.S., SeniorBiologist, EPA 

# f{ 1f5( 

### {g 

### ll3/ I\

- 11

-