Open Literature Review Summary

Chemical Name: 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 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 of Concern (LOC).

Date of Review: October 21, 2009

Purpose of Study:

This study examines the potential effects of atrazine, prometryn, and isoproturon on periphyton

communities in static-renewal indoor microcosms. Observed pollution-inducted community

tolerance (PICT) responses were then compared to species-sensitivity distributions (SSDs)

obtained from the US EPA ECOTOX database.

Summary of Study Findings:

This review focuses on the effects from atrazine treatments.

Methods:

All experiments were conducted from November 2002 to March 2003 (not reported 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 on plexiglas racks,

to serve as surface substrates for periphyton. The aquaria were 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 of 130-150 /-lmol/m2

/s. Aquaria water was continuously mixed (10 cm/s) with

paddles. Any acclimation ofthe microcosms, to allow periphyton to colonize the disks, prior to

treatment was not reported.

- 1 -

IIII.I.IBI

2087863
Eight aquaria were 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, with two 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 at the respective application

concentration. Following 14 days of exposure, long term effects of atrazine on periphyton

chlorophyll a content and diatom species composition were determined as described below.

Additionally, after 14 days of exposure, development oftolerance was assessed through reexposure of collected periphyton communities to atrazine for a short interval and then effects on

photosynthetic activity (short-term toxicity) were determined as described below. For the

photosynthetic activity experiments, analyses of communities 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

et al. (1994).

Diatom species composition. Periphyton samples (disks), in replicates offive, were preserved

and analyzed according to the method of Krammer 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% hydrogen peroxide solution for ~24 hours until the solution was

colorless. Species were identified under a light microscope based on the siliceous exoskeleton.

Relative distribution ofthe most dominant species was determined by counting at least 100

individuals per species. The seven most 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 individually placed in scintillation vials

containing 4 mL control water, then treated with atrazine in a geometric concentration series

(dilution factor 1:2) at nominal concentrations of 0.0075-2 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". It is unclear whether the experimental

design for the short term test used controls (untreated and treated with DMSO) and atrazine

treatments for all microcosm communities (controls and atrazine treated) or if communities from

control aquarium (untreated and treated with DMSO) served as controls during the short-term

tests. All treatments were performed in triplicate. 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 sensor positioned

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 saturation pulse. Initial experiments determined that yields

remained constant with actinic light exposure intervals exceeding 2.5 minutes.

Relative inhibition offluorescence yield at 665 nm, as compared to the control (unclear which

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 information restricted to micro- and macro-algae. Median effective

concentrations were used for exposure intervals of~l day. Invalid or unspecific data points (e.g.

tests stating a phylum or functional group, such as phytoplankton) were omitted. When more

than one toxicity value was present for the same species, geometric means were calculated.

Effect parameters were held to growth-related endpoints (overall growth, population changes,

changes in chlorophyll a or biomass). Empirical SSDs were constructed according to the method

of Solomon et al. (1996), with toxicity data plotted cumulatively. To determine cumulative

frequency distributions, percentages were calculated as follows:

[n + l]/[N + 1], where

n = rank number ofthe datum point and

N = total number of points in the dataset.

Results:

Chlorophyll a concentrations varied among the 0.0075-0.25 mg/L atrazine treated microcosms

after 14 days of exposure but appeared to remain at levels similar to the controls (Table 1). The

study authors did not distinguish the untreated (negative) control from the DMSO control. At the

0.5 and 1 mglL treatment concentrations, chlorophyll a was decreased 60% and 72%,

- 3 -
respectively, as compared to the average of all the controls (Table 1). According to the study

authors, there was large variation within the microcosms (data not provided).

Diatoms comprised the majority (>80%, 37 species) ofthe periphyton algae in the microcosms.

In atrazine treated microcosms, species numbers were similar to controls for microcosms treated

at 0.0075-0.25 mg/L and the number of diatom species decreased 62% in the microcosms treated

with 0.5-1 mg/L as compared to the average of all control microcosms (Table 1). A noticeable

shift in diatom community structure also occurred at the highest atrazine treatment

concentrations after 14 days of exposure; Nitzschia capitellata increased from :S~5% of diatom

species at 0-0.13 mg/L exposure to comprising ~40% at 0.25 mg/L and ~95% at the 0.5 and 1

mg/L concentrations (Figure 1). Shannon-Weaver biodiversity values were 0.94-1.7 for

untreated controls, 0.76-1.56 for periphyton treated at 0.0075-0.25 mg atrazine/L, then decreased

to 0.14-0.15 at the 0.5 and 1 mg atrazine/L treatment concentrations (Table 1).

For one of the 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 of the 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 at the 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 of photosynthetic activity

(~5-20%) at each treatment concentration (0.0015-2 mg/L), as compared to periphyton from one

of the control microcosms not previously exposed to atrazine (Figure 2a). Data for the 0.0075

mg/L short-term treatment was not presented.

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 of SSDs 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% of the cumulative fraction of affected

species represented in the SSD. EC50 values of the more tolerant communities increased to

levels ofthe more tolerant species. Sensitivities of pre-exposed communities shifted to the right

end ofthe SSD, covering 85% of affected species represented in the SSD

The study authors suggested that a consistent relationship between the algal SSDs and maximally

induced community tolerance after atrazine exposure could be determined such that the right end

of the 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 that the SSDs would be good predictors ofthe

effects of PSII herbicides, like atrazine, on freshwater microphytobentic communities and

predicted no-effect concentrations derived from the SSDs.

Table 1. Periphyton structural characteristics (chlorophyll a, diatom species, and Shannon-Weaver diversity)

followin2 14 days of exposure 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) NA1 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 of diatoms

40

o

20

r!J Nitzschia capitellata

mFragilaria 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 of diatom species following 14 days of exposure 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,., aft8f pre-expos;ure

(b)

0.3

0,4

":'-.....

~O.2

--

uP,

W 0,1

(a)

.-. 100 • untreated

• pnHJKjXlliufe (0.125 mg L" atfeztne} j eo

C

>. 60 B

{ 40 •

(5 • IIl.A.

§ 20 Xis ~....rf 0 ~:;;;""'_._-....,r--.......................-rrr-.........--.

0.01

Figure 2. Short-term inhibition of photosynthetic 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

lfilO.8

b 0.6

i 0.4 t0.2

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 of induced tolerance (range of pollution-induced

community tolerance after long term exposure). (study figure 3)

- 7 -
Data Summary Table: Periphyton in static-renewal, indoor microcosms

Time

Weighted

Magnitude Nominal Average Did

ofEffect Atrazine Day ofStudy Atrazine Recovery Effect relative to Concentration Effect Concentratio Occur-if Comments

controls (pgIL) Occurred nfromDayO so, when?

to When

Effect

Occurred

~25% 15 Magnitude of effects

decrease estimated from Figure 2;

SSNR1

,2.

~10

30 decrease Effects data represents a

~40% single control microcosm

Decreased decrease 60 (not stated if negative or

photosynthesis; DMSO treated)

short-term (1 ~50% 125 hour) toxicity decrease Not Not No data provided 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 of all control

after day 14) (untreated) microcosms was

130 Ilg/L. The lowest

~70-80% 500, 1000, reported EC50 of all

decrease 2000 microcosms was 113 Ilg/L

(this was a control

microcosm, unknown if

negative or DMSO treated).

~5% 15,30 decrease

Decreased ~20% 60

photosynthesis; decrease Magnitude of effects

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 of effects

biomass stimulation Not Experiment calculated from results in

(chlorophyll a,

14 calculable terminated Table 1 using average of all +8% at 14 days Ilg/disk) stimulation 15 control microcosms;

- 8 -
Time

Weighted

Magnitude Nominal Average Did Day ofStudy Atrazine

Effect ofEffect Atrazine Effect Concentratio Recovery Comments relative to Concentration Occur-if

controls (llglLj 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 of effects

calculated from results in

32-47% 15,30 Table 1 using average of all

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 of effects decrease calculated from results in

Change in 37% 30

Table 1 using average of all

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 not reported.

2 Only one microcosm per treatment.

3 Magnitude of effects were 63%, 17%,30% and 41% decreases at the 0.03, 0.06, 0.13 and 0.25 mg/L treatments,

respectively.

- 9 -
Reviewer Comments:

This study also examines the potential effects of prometryn and isoproturon on periphyton.

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 allow periphyton colonization, prior to treatment was not

reported.

As an indoor microcosm study, there may be little relevance ofthe results to natural systems,

despite the natural source ofthe communities, given the small size ofthe microcosms (20-L

aquaria) and lack of a 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 nor monitored in the 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 3.1 0 (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.

Except for 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 of photosynthesis (EC50) between communities from the negative

and DMSO control microcosms (no data nor statistics were provided to support this statement).

- 10 -
Both of the 'control' communities (with or without prior DMSO exposure) were subsequently

exposed to DMSO (1 mIll) during the short-term tests, complicating the interpretation of whether

DMSO had an effect on the EC50 of photosynthetic inhibition.

During the short-term toxicity experiments, identification ofthe 'control' was unclear in both the

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 of control versus the other (the

study reviewer made assumptions when possible).

The authors report high within-microcosm variation for chlorophyll a.

Description of Use in Document: Invalid

Rationale for Use: There was only a single microcosm (i.e. no replication) per treatment level.

No statistics were provided. DMSO was used as solvent (l mIll) at a concentration 100% higher

than the criteria for study acceptance «0.5 mIll, static-renewal testing), thus the toxic effe<;ts of

atrazine may have been confounded by the addition ofsolvent. Although negative and DMSO

control microcosms were used, the authors did not report ifthere were differences between the

two types of controls for chI a, species numbers, or biodiversity, thus effects due to DMSO could

not be discounted by the reviewer. The use of controls is unclear for the short-term toxicity tests.

Limitations of Study: There were numerous reporting and measuring deficiencies. No effort

was made to document whether other chemicals (including atrazine) were present in the

treatment and/or control microcosms prior to study initiation. The microcosms did not contain a

sediment layer. Water chemistry ofthe microcosms was not monitored either prior to or

following treatment. Only a single microcosm was treated at each atrazine test concentration.

Nominal atrazine concentrations were not confirmed and concentrations were not monitored 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-microcosm variability for at least one endpoint (chlorophyll

a). It is unclear what controls were used in the short-term toxicity tests and subsequent analysis.

Primary Reviewer: Lynne Binari, M.S., Subcontractor to Cambridge Environmental Inc.

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 1/ff;ttJ/~

Tertiary Reviewer: Anita Pease, M.S., Senior Biologist, EPA f{1f5( {g ll3 / I\

- 11 -