Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Nystrom, R, M. Paulsson, K. Almgren, and H. Blank. 2000. Evaluation of the capacity for

development of atrazine tolerance in periphyton from a Swedish freshwater site as determined by

inhibition of photosynthesis and sulfolipid synthesis. Environmental Toxicology and Chemistry.

19: 1324-1331. (MRID 48261128)

Purpose of Review:

Determine potential effects of atrazine on an aquatic comnumity. Endpoints from this study

contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant communities

and associated determination of a Level ofConcern (LOC).

Date of Review: October 5, 2009

Purpose ofStudy:

This study examines the potential effects of atrazine on periphyton in experimental flow-through

microcosms.

Summary ofStudy Findings

Methods:

Twelve 22-L glass aquaria (microcosms) were continuously supplied with filtered (l-mm, nylon

net) water at a continuous flow rate of221 mL/minute using an air-driven Teflon-membrane

pump and flow-distributor. The source water was pumped from the Gota Alv River at a depth of

about 0.5-m. Aquaria were maintained on a natural daylight photoperiod (on average, 17:7 hour

light:dark cycle) using fluorescent light with an irradiance of 120 Ilmol/m2/s at the water surface.

Each aquarium held 170 glass disks (l.5-cm2

) mounted on polyethylene holders to serve as

surface substrates for periphyton.

Treatment solutions were prepared by combining atrazine (purity not reported), acetone (200

mL, concentration not specified), and Milli-Q water (9.8 L). Eight ofthe aquaria were

continuously treated (flow rate 1.0 mL/minute) with the atrazine solutions at eight nominal

concentrations in a geometric concentration series from 0.056-3.2 IlM (12-690 IlgIL, one

- 1 -

1111•••111

2087819
treatment concentration/aquarium) using a peristaltic pump. The fmal co-solvent (acetone)

concentration was 0.01 % (v:v). The remaining four aquaria were maintained as controls

continuously treated with acetone (0.01 %, v:v). Each aquarium contained water-mixing

equipment (not further described) to ensure instant mixing oftreatment solutions and river water

and to reduce any boundary layer.

Water samples (volume not specified) were taken from each ofthe treated and control aquaria

for atrazine analysis. Timing ofthe sampling was not reported. Samples were filtered (glass

fiber, Whatman type GF/F) and then extracted using solid phase C18 cartridges (Accubond SPE,

6-mL, 500 mg). Loading and elution procedures were not described. Atrazine was quantified

using reverse-phase HPLC; methods were referenced (Durand and Barcelo, 1990; Guasch et al.,

1998) but not described.

Periphyton bio-films were allowed to establish (colonize) on the glass disk substrates and

Plexiglas plates in the treated and control aquaria for one month, from June 17 to July 15, 1996.

Following the 4-week colonization interval, long term effects of atrazine on periphyton

photosynthetic activity, sulfolipid synthesis, thymidine, and adenine incorporation, chlorophyll a

content, dry weight, and species composition were assessed as described below. Additionally,

periphyton was assessed for development oftolerance to atrazine through re-exposure to atrazine

for a short interval (short-term toxicity). Short-term toxicity was based on effects to

photosynthetic activity and sulfolipid synthesis. At the end ofthe 4 week exposure, glass disks

with periphyton were sampled from three aquaria each day: one randomly selected control

aquaria and two randomly selected atrazine treated aquaria. Periphyton disks were stored in

unfiltered river water during handling. Atypical disks with uneven growth were discarded.

Disks were cleaned on the backside (not further described), placed in 20-mL glass vials, and

processed as described below.

Photosynthetic activity. Filtered (GF/F) river water (2 rnL) was added to the periphyton samples

(disks) in the 20-mL vials. For short-term toxicity experiments, an equal volume of atrazine

solution (double concentration stock solutions in GF/F filtered river water) was added to the vials

in a geometric concentration series (test concentrations and exposure interval were not reported).

An aliquot (100 ilL) of aqueous H14C03-solution (adjusted to pH 9 with O.lM NaOH) was added

to each vial at 0.25 IlCi/mL. Samples were incubated for 1 hour with continuous shaking in a

water bath maintained at the same temperature as the aquaria and illuminated via fluorescent

light from below with an irradiance of 80 Ilmollm2/s. Formaldehyde (2%, v:v) was added to the

samples to end incorporation, then concentrated acetic acid (200 ilL) was added and the samples

were dried at 60°C under a stream of air. Dried residues were treated with dimethylsulfoxide (1

mL) and analyzed for total radioactivity using LSC.

Synthesis ofsulfolipids. Filtered (GF/F) river water (l mL) was added to the periphyton samples

(disks) in the 20-mL vials. Short-term toxicity experiments were conducted as described above

with an exposure interval of 24 hours. To determine sulfolipid synthesis, 35S04- was added at 10

-2-
JlCi/mL. Samples were incubated for 16 hours in a water bath at 17°C under continuous

illumination from above at the same quality and irradiance as the photosynthesis tests.

Incorporation was stopped by shaking the samples to detach the periphyton and then filtering

(GF/C) the samples. Vials and glass disks were rinsed with deionized water and the rinse water

was filtered through GFIC filters. Filters were then rinsed with deionized water to reduce the

amount of unincorporated 35S04-. Filters and the glass disks were stored frozen (-18°C) until

extraction. Lipids were extracted by adding methanol (2 x 1 mL) followed by chlorofonn (2 x 1

mL). The extract was then washed by adding deionized water (4-4.5 mL), centrifuging the

resultant solution, and removing the top layer. The procedure was repeated eight times. The

washed organic phase (chlorofonn-lipid extract) was evaporated to dryness, and the resulting

residues were analyzed using LSC. Fonnaldehyde blanks were used to estimate unincorporated

35S0 4- and the control values were subtracted from the final results.

Periphyton lipids were analyzed by one-dimensional thin layer chromatography (TLC) on

nonnal-phase plates (silica gel) developed with chlorofonn:methanol:acetic acid:water

(85:15:10:3.5, v:v). Lipids were detected using a TLC-plate scanner and autoradiography.

Unlabeled sulfoquinovosyldiacylglycerol (SQDG), a chloroplast lipid, was used as a reference

standard because it was presumed to be the major periphyton sulfolipid.

Thymidine and adenine incorporation. Periphyton samples (disks) were treated with [U14C]adenine at 200nM (specific activity = 271 mCi/mmol), incubated 1.5 hours, then treated with

[methyl)H]thymidine at 20nM (specific activity = 83 ci/mmol), and incubated an additional 30

minutes (incubation conditions were not further described). Formaldehyde (2%, v:v, buffered

with sodium borate) was added to kill the samples. Samples were extracted with 4M NaOH

(final concentration 0.25M) at 60°C for 1 hour, placed on ice, treated with ice-cold

trichloroacetic acid (TCA, final concentration 22%) for 2:30 minutes, and finally filtered (0.22-

Jlm, nitrocellulose). Filters and vials were rinsed with ice-cold 5% TCA. Filters were then

rinsed with ice-cold 80% ethanol and dried. Precipitated macromolecules on the filters were

hydrolyzed with O.IM HCI at 80°C for 4 hours. HCI was then neutralized with NaOH and the

water was evaporated. Radioactivity was measured using LSC with a dual"radiolabel program

for 14C and 3H.

Chlorophyll a. Periphyton samples (disks) were treated with dimethylsulfoxide (DMSO, 1 mL)

and stored frozen until extraction. Samples were extracted at 60°C in darkness for 30 minutes,

then treated with 90% acetone, and analyzed spectrophotometrically (method ofJeffrey and

Mumphrey, 1975).

Periphyton recovered on Plexiglas plates (117 cm

2

) detached from the water"mixing equipment

was also analyzed for chlorophyll a. The periphyton film was scraped from a plate into river

water (80 mL) and homogenized via shaking. An aliquot (volume not reported) ofthe

periphyton suspension was centrifuged. The supernatant was removed, DMSO (l mL) was

added, and the sample was stored frozen. Extraction and analysis are described above.

- 3 -
Dry weight. Aliquots (3-5 mL) ofthe homogenized suspension ofperiphyton scraped from the

Plexiglas plates (described above) were filtered onto preweighed GFIF filters. Filters were dried

at room temperature, then weighed, then heated to 450°C overnight and reweighed to estimate

ash-free dry weight (AFDW).

Species composition. Periphyton samples (disks) were preserved in 70% ethanol and stored at

4°C in darkness. One sample per aquarium was videotaped to facilitate identification. Species

identifications were conducted by counting 50 randomly chosen fields at a magnification of

1,000x using a phase-contrast microscope. Relative abundance of each species or group of

species was scored as 1-50 depending on absence or presence in the 50 fields. Community

structure based on relative abundance was analyzed using the Bray-Curtis similarity index. It

was not reported ifsimilarity was based on just species or species and groups ofspecies as

reported in table 2.

Statistical analyses. Data were analyzed for variance heterogeneity (Cochrans's test) and normal

distribution (X2 test), then, if necessary, were IOi0g transformed to meet the assumptions for

parametric tests. For long-term effects tests, the intercept between the linear regression line

(FIG.P v. 5.0 software) and the average control value wasjudged to be the no effect

concentration (NEC) as an estimate ofthe long-term no observed effect concentration (NOEC).

The 95% confidence limits ofthe regressions were calculated according to Dixon and Massey

(1985). For short-term toxicity tests, lowest observed effect concentration (LOEC) values were

estimated using an analysis of variance (ANOVA). When ANOVA was statistically significant

(p<0.05), either Dunnett's test (equal sample sizes) or Bonferroni's test (unequal sample sizes)

was performed using Toxstat (v. 3.3).

Results:

Quantitative results for atrazine in the treated microcosm water were not presented. The study

authors reported that measured atrazine concentrations were "substantially" lower than the

nominal concentrations. At 0.056 J.1M, the lowest test concentration, measured atrazine was

reportedly two- to three-fold lower than the nominal concentration, with the difference between

measured and nominal increasing at the higher test concentrations. Results from analysis of

water from untreated control microcosms were neither presented, nor discussed.

Long-term effects. Periphyton chlorophyll a was gradually reduced with increasing atrazine

concentration relative to the controls on both the glass disks (Figure 1) and Plexiglas plates

(Figure 2). Specific photosynthetic activity and specific sulfolipid activity increased relative to

controls in atrazine treatments of ~216 J.1g/L (1 J.1M) and ~121 J.1g/L (0.56 J.1M), respectively

(Figure 3). The study authors report that chlorophyll a was reduced more than photosynthetic

activity at atrazme ~1.0 J.1M, resulting in increasing specific activity. Based on EC50 values,

-4-
photosynthetic activity was slightly more sensitive to atrazine than sulfolipid synthesis (Table

1).

For the sulfolipid analysis, 35sol- was reportedly incorporated into at least five different lipids.

The major lipid (Lipid I) was unidentified but was detennined not to be the chloroplast lipid

SQDG. The study authors had assumed that SQDG was the major sulfolipid ofperiphyton. The

Lipid 1:SQDG ratio decreased with increasing atrazine at concentrations :-::0.32 fJM (Figure 4).

Periphyton dry weight gradually decreased to a maximum of-30% of control periphyton at 1.0

fJM atrazine exposure but was only -20% and -10% of control periphyton at 1.8 and 3.2 fJM

atrazine exposure, respectively (Figure 2). The study authors suggested that the increase in dry

weight at the higher atrazine treatment levels indicated a shift to a community dominated by

heterotrophic microorganisms.

Total periphyton species was reduced at atrazine concentrations of 1.8 and 3.2 fJM (388 and 690

l-lgIL) but not at concentrations 1 l-lM (216 fJgIL) or lower (Table 2). Likewise, the relative

abundance of dominant species and groups of species showed a similar response to atrazine.

Two taxa, Mougeotia sp. A and cyanobacteria (chain forming), were exceptions which showed

reductions in relative abundance at atrazine concentrations as low as 0.32 fJM (69 fJgIL) and 0.18

fJM (39 l-lgIL), respectively. Changes in community structure were also detected with the BrayCurtis similarity index, primarily a decrease in similarity at the two highest atrazine

concentrations (Figure 5). There was a small gradual decrease in similarity (maximum of 5%

difference from controls) between the 0.10 and 1 l-lM (22-216 l-lgIL) atrazine treatment

concentrations. The study authors report that the trend was significant (p<0.00001).

Thymidine:adenine incorporation ratio increased with increasing atrazine concentration (Figure

6). The increasing ratio is suggestive that community structure shifted toward increasing

bacteria with increasing atrazine exposure.

Calculated EC50 and no effect concentrations are presented in Table 1 for all long-term

endpoints.

Short-tenn toxicity (tolerance) experiments. For photosynthetic activity, communities with prior

exposure to atrazine at 0.056-0.56 llM showed an increasing tolerance to atrazine re-exposure

with EC50 values increasing from 1.6-2.4 llM (p=0.002 for the trend); however, tolerance

decreased with prior exposure at:-::l llM atrazine (Figure 7). The results are difficult to interpret

because EC50 values were 1.9-2.2 fJM for the periphyton from the four control microcosms (no

prior exposure). For sulfolipid synthesis, periphyton communities previously exposed to atrazine

at 0.056-0.32 l-lM were generally more tolerant (higher EC50) than communities from most of

the control microcosms. Tolerance decreased (lower EC50) with increasing prior exposure

concentrations of 0.56 llM atrazine and higher (Figure 7). The results are difficult to interpret

- 5.;.
....

because there was relatively high variability observed in the EC50 values from the four control

microcosms.

-o

...

-c:

o

0140

....

o

120

100 -I-i~---~~T-----------l

15 ~----i----~:~Jt~~~~~-------------------

5 80 ". "

"& " ',. 80 '. '. . ' CIJ

0.. 40

0.10 0.32 1.0 3.2

long-term atrazine expolure (JolM)

Figure 1. Long-term effects of atrazine on chlorophyll a content of periphyton on glass disks (1.5 cm2

)

expressed as a percentage ofthe control. The average control chlorophyll a content (100 %) was 2.6 ,..g1disk.

Values are mean ofthree samples from each microcosm with standard deviation (vertical bars). Dotted lines

indicate 95% confidence limits. Linear regression included all treatments, except 0.056 ,..M. (Figure 1 of

study)

120 .,.

-(;

...

.. 100

c:

0

0 80

-0

&0

•

•

• 40 e

.2

c:'Q 20

0

o 0.10 0.32 1.0 3.2

LOtIg-term atrazine exposure (I'M)

Figure 2. Long-term effects of atrazine on chlorophyll a content and ash-free dry weight (AFDW) of

periphyton from Plexiglas plates expressed as a percentage ofthe control. The average control chlorophyll a

content (100 %) was 3.7 ,..g11.5 cm:! and the average AFDW was 0.93 mgl1.5 cm2

• Open square = chlorophyll

a, solid square =AFDW. Values are mean of three samples from each microcosm with standard deviation

(vertical bars). (Figure 4 from study)

-6-
! 400·

o..........-I"I-~--r-.- .....--r------r--....-.,.....

a •

•

D

• ! •

a •

o

I

0.10 0.32 1.0 3.2

Long-term Itrufne exposure (JIM}

Figure 3. Specific photosynthetic and sulfolipid synthetic activity (dpm/p.g chlorophyll a content) expressed

as a percentage ofthe control. Values are mean activity for each microcosm. Solid square =photosynthetic

activity and open square =sulfolipid synthetic activity. Average specific activity in controls was 51,000

dpmlp.g chlorophyll a and 6,700 dpmlp.g chlorophyll a for photosynthesis and sulfolipid synthesis,

respectively. The average incorporation rates were 27 nmol carbonJp.g chlorophyll a in 1 hour and 46 pmol

sulfatelp.g chlorophyll a in 16 hours. (Figure 2 from study)

i •

e

0

c

S

-.... '" --

l

a

- 2

31

J!-

=

...........

'"......'",

. ....... ._--~._--_. '~~ __ . --~~~------------------ ,

o .....,~II-__-,....-..---r--,.--r--_-'T"'"

o o.te 0.32 1.0 3.1

Lont-term .tr-" 'XJ:l'OSIft 11M

Figure 4. Ratio of periphyton major sulfolipid (Lipid 1) to thylakoid lipid sulfoqninovosyldiacylglycerol

(SQDG). Values are mean oftwo samples. Dotted lines indicate 950/. confidence limits. Linear regression

included all treatments eX(:ept 0.056 and 0.10 p.M. DPM =disintegrations per minute. (Figure 3 from study)

- 7 -
)C 80

"CJ

• - ... .... .Ii - ....-- - ... :::::::: ...

-... 60

::s:•

I .I

.. I • 40

-...

!i

0

...

I 20

•...

III

0

o 0.10 0.32 1.0 3.2

Long-t.rm atrazln. .xposur. [pM}

Figure 5. Long-term effects of atrazine on periphyton community structure expressed as Bray-Curtis

similarity. Similarity indices were calculated from the relative abundance ofspecies as related to the

controls, generating four different indices per treatment and three per control aquarium. Dotted lines

indicate 95% confidence limits. Linear regression included all treatments, except 1.8 and 3.2 11M. (Figure 6

from study)

c

'';:::

CI

...

.

.5•

.!

•

!

2

o -'-r---lll-__-r--......--r"-_-r--__r-...,..,

o 0.10 0.32 1.0 3.2

Long-term at,a•• expo.ure b.M)

Figure 6. Long-term effects of atrazine on short-term bacterial activity measured as thymidine incorporation

and total community activity measured as adenine incorporation. Values are mean of 3-5 samples from each

microcosm with standard deviation (vertical bars). Linear regression fitted to all treatments, Dotted lines

indicate 95% confidence limits. (Figure 5 from study)

8 -
•

r

2'

::L 8

0 '....' III

0

!!! 6

., .J

tl

c:

c

.. 4 !! "J c

...

.. • c:: 2 N

CI

....

'I(- 0

0

• \--!---~-:--- -. I

0.10 0.32 1.0 3.2

Long-term atrazine exposure (~M)

Figure 7. Long-term effects of atrazine on periphyton community tolerance. EC50 values for short-term

inhibition of photosynthesis (solid square) and sulfolipid synthesis (open square) plotted relative to long-term

atrazine exposure levels. Linear regression ofshort-term photosynthesis EC50 values included 0.056-0.56 J.1M

levels; dotted lines indicate 95% confidence limits. (Figure 7 from study)
Table 1. Short-term and long-term endpoints for the effects of atrazine on periphyton (Table 1 ofstudy)

Atrazine (f.1M)

Effects 95% CL< NEC <95% CLI NOEC2 LOEC3 EC504

Thymidine/adenine incorporation 0.0050 < 0.040 < 0.14

Short term

Photosynthesis

Sulfolipid synthesis

Long term

Chlorophyll a

Photosynthetic activity

Sulfolipid synthesis activity

Sulfolipid synthesis pattern

Bray-Curtis index

0.080 < 0.14 < 0.23

0.090 < 0.15 < 0.23

0.040 < 0.18 < 0.53

0.040 < 0.28 < 0.63

0.040 < 0.09 < 0.20

0.1-1.0

1.0-3.2

0.3-3.2

3.2-10

1.2-2.4

2.6-7.4

0.7

l.l

1.9

ND

ND

ND

No-effect concentration with 95% confidence limits.

2 No observed effect concentration.

3 Lowest observed effect concentration.

4 Halfmaximal effective concentration.

Table 2. Number of peripbyton species and relative abundance of periphyton species or groups ofspecies

observed after 4 weeks of atrazine exposure. The 0.056, 0.10, 0.18, 0.32, 0.56, 1, 1.8, and 3.2 pM nominal rates

correspond to 12,22,39,69,121,216,388, and 690 ~gIL, respectively (based on an atrazine m.w. of215.7

glmoJ). (Table 2 ofstudy)

.Alraziae (JLM)

Conttol 0.056 0.10 0.18 0.32 0.56 1 Ul .u

No. ofIIp!Cies 33 :!: 4- 31 - 5 28 :!: 4 30:: 2 29 :!: 3 2Sl :': I 28:,: 2 18 :': 6 16 :: 4

DomUlaat speciH or gmaps of

.tfclwDtdwrs lIti~illla 32 :!: 9 36:,: 7 36 :!: 9 38:!: 2 31 4 42 :! 7 35 :: 4 3 1 0 I

~fftIW" 14 :!: (\ 19 :': 2 11 :! 4 14:! 6 16 7 11:!: .2 17 4 12 !1 .2 :': 1

~.-. d. porwhmr 12:!: (\ 11 :': 5 13 :': 3 16:,: 5 !1 4 6:: 3 6 2 1 1 0 0

~sp. 4:!: 2 7 :! 3 7 :! 3 7::6 6 .2 5:': 3 4::::2 3 3 o:: I

s..-ra Sip. A 2S :: 8 28:::: 9 18 :': 18 25 :!: 2 21 4- 19:::: 2 16 :: 8 4 3 1.:: 1

.sy-iro sp. B 27 :t 7 37:':11 35 :: 13 37:! 9 34 6 30 :: 12 36 3 15 3 4:': 4

Tal»lJ4rit1.~ 46 :!: 3 46 :!: .2 46 4 48:!: .2 45 1 44 :: 4 40 3 26 4 9:::: 2

~Sip.A 11 ::!: 5 10:::: 6 8 5 16:': 6 3 .2 .2 :!: I 1 1: :::! 0 0 1 :::: 1

~sp.B 7 :': 5 8:,: 4 6:': 4 11::12 2 3 1 :': 1 1 1 1 1 0:.::0 e:v-bactma (c:hail:I.) 44 :!: 6 28 :! 24 40:,: 4 33 :!: 2 18 7 7 :! 6 2 3 1 1 0 0

(1ll1iceDuJ.) 4 :!: 2 13 _ 15 7 6 4-3 1 :2 j _ 6

1 - 1 0 0 .2 - .2

•Mean :: staIldard cIevia1:ion (l'l~...". = 12, "_=3).

- 10-
Data summary TabIe:

Time Weighted

Average

Magnitude of NominalAtrazine Day of Atraz;ne DidRecovery

Effect Effect relative Concentration Study Effect Concentration Occur ifso, Comments

to controls (pgIL;t Occurretf ftomDayOto when?

When Effect

Occurred

~20% decrease 12 Ilg/L :::::4 weeks Not calculable No 3 Magnitude of

Not calculable

effects estimated

No effect 22 and 39 Ilg/L :::::4 weeks Not applicable from Figure L

~35%to~0% ! No 3

69 and 121 Ilg/L :::::4 weeks INot calculable SSNR4

• Responses decrease

Change in were considered

chlorophyll a ~O% decrease 216 Ilg/L :::::4 weeks Not calculable N0 3 likely 'no effect'

(per glass N0 3 when treatment

disk) error bars

overlapped with

-85%to-90% 388 and 690 j.lg/L :::::4 weeks Not calculable 95% confidence

decrease limits ofthe four

control

microcosms.5

No effect 12, 22, 39, 69 Ilg/L :::::4 weeks Not calculable Not applicable Magnitude of

N0 3 effects estimated ~O% 121 j.lg/L :::::4 weeks Not calculable from Figure 2.

decrease

Change in

-70% decrease :::::4 weeks Not calculable N0 3 SSNR4

. Responses chlorophyll a 216 j.lg/L

were considered (per area, N0 3 likely 'no effect' Plexiglas when treatment plate) -9O%to-95% error bars

decrease 388 and 690 j.lg/L :::::4 weeks Not calculable overlapped with

those ofthe

controls.5

No effect 12,22,39,69 !J.g/L :::::4 weeks Not calculable Not applicable Magnitude of

effects estimated

-20 to 30% 121,216,388 fJg/L :::::4 weeks Not calculable N0 3 from Figure 2. Change in ash- decrease

free dry SSNR4

• Responses weight of

were considered periphyton likely 'no effect' (per area, when treatment Plexiglas No effect 690 j.lg/L :::::4 weeks Not calculable Not applicable error bars plate) overlapped with

I J

those ofthe

controls.5
Time Weighted

Average

Magnitude of Nominal Atrazine Day of Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur- ifso, Comments

to controls (pglLl OccuTTetf from Day Oto when?

When Effect

OccuTTed

No effect 112,22,39,69, 121 ;::4 weeks Not calculable INot applicable Magnitude of

~glL effects estimated

-50% IN0 3 from Figure 3.

2OOIlg/L 2':4 weeks Not calculable Change in stimulation SSNR4

• Responses specific

No 3 were considered photosynthetic -225% 3881lg/L 2':4 weeks Not calculable likely 'no effect'

activity stimulation

,-. when the treatment

(dpmllJ.g No 3 value overlapped chlorophyll a) with the range of -150%

stimulation 690 1J.g/L 2':4 weeks Not calculable values for the four

control

i microcosms.

No effect 12,22, 39, 69 Ilg/L 2':4 weeks Not calculable INot applicable Magnitude of

N0 3 effects estimated

I -50% to from Figure 3.

Change in -60% 121 and 200 Ilg/L 2':4 weeks INot ","cul.bl,

specific stimulation I SSNR4

. Responses

sulfolipid -150% No 3 were considered

synthesis stimulation 3881J.g/L 2':4 weeks Not calculable likely 'no effect'

activity when the treatment

(dpml~g No] value overlapped

chlorophyll a) -275% with the range of

stimulation 69OIlg/L 2':4 weeks Not calculable values for the four

control

I microcosms.

12,22, 39, 69, 121 I

INot applicable Magnitude of No effect 12':4 weeks Not calculable

1-30%d=a"

and200 ~g/L effects estimated

!No]

from Figure 5.

3881lg/L ?:.4weeks Not calculable

SSNR4

• Responses

were considered

Change in likely 'no effect'

community when treatment

structure

error bars

(Bray-Curtis I overlapped with

similarity 95% confidence

index) -40% decrease 690 ~glL 2':4 weeks Not calculable No> limits ofthe four

I controls

I microcosms.5

The 5% decreased

similarity at 200

IJ.glL was not

I considered an effect

- 12 -
Time Weighted

Average

Magnitude oj Nominal Atrazine Day oj Atrazine Did Recovery

Effect Effect relative Concentration Study Effect Concentration Occur- ifso, Comments

to controls (pgILi Occurretf jromDayOto when?

When Effect

Occu"ed

given the variability

in the control

microcosms (70%

similar to one

another).

No effect (all 12,22 ).1g/L ~4weeks Not calculable Not applicable taxa)

No effect (taxa N0 3

1-9,11)

39 Jlg/L ~4weeks Not calculable

25% decrease

(taxa 10)

No effect (taxa N0 3

1-7,9,11)

Up to 73% 69 ).1g/L ~4weeks Not calculable

decrease (taxa

8 and 10) :

Magnitude of

Change in No effect (taxa N0 3 effects calculated

relative 1-7,9, II) from results in

abundance 121 J.lg/L 2::4 weeks Not calculable Table 2.

I sco~of Up to 84%

SSNR4 VarIOUS decrease (taxa • Responses

\ taxonomic 8 and 10) were considered

groups No effect (taxa N0 3 likely 'no effect'

i (species and when treatment

I groups of 1-7,9, II) mean± SD

i species/ Up to 95% 215 J.lg/L ~4 weeks Not calculable overlapped with

that of controls.5

I decrease (taxa

8 and 10)

No effect (taxa N0 3

2,4,9)

Up to 100% 388 J.lg/L 2::4 weeks Not calculable

decrease (taxa

1,3,5-8, lOll)

No effect (taxa No 3

II) 690 Jlg/L 2::4 weeks Not calculable I

Up to 100%

I - 13 -
~---:-----':-_--:-::--'- -'--------:---- I I

The study authors reported that measured atrazine concentrations were substantially lower than the nominal

concentrations; two- to three-fold lower at the lowest concentration (0.056 fJ.M), with the difference increasing at

higher concentrations (p. 1329). The 0.056, 0.10, 0.18, 0.32, 0.56, 1, 1.8 and 3.2 fJ.M nominal rates correspond to

12,22,39,69, 121,216,388, and 690 fJ.g!L, respectively, based on an atrazine m.w. of215.7 glmo!.

2 Effects were determined beginning 4 weeks after initial exposure (one control and two atrazine-treated

microcosms were randomly sampled per day). Sampling dates for specific treatments and controls were not

reported.

3 Effect observed at end of experiment.

4 Statistical significance oftreatment compared to controls was not reported. In addition, reviewer cannot judge

significance of effect because there was no replication of atrazine-treated microcosms.

S Responses were considered likely 'no effect' when the treatment response overlapped with variation in the control

response. Error bars for treated microcosms and control microcosms represent within microcosm variation, not

replicate treatments. Replicate control microcosms were plotted individually. For some endpoints, 95% confidence

intervals were provided for the control microcosms.

6 (1) Achnanthes minutissima, (2) Diatoma tenius, (3) Gomphonema cf. parvulum, (4) Gomphonema sp., (5) Synedra

sp. A, (6) Synedra sp. B, (7) Tabellariaflocculosa, (8) Mougeotia sp. A, (9) Mougeotia sp. B, (10) Cyanobacteria

(chain), (11) Cyanobacteria (unicellular)

Time Weighted

Average

Magnitude oj Nominal Atrazine Day oj iAtrazine DidRecovery

Effect Effect relative Concentration Study Effect Concentration Occur - ifso, Comments

to controls (pglL/ Occurrecr from Day 0 to when?

Ide"'"ose (taxa

When Effect

._"_.

IOccurred

I I

~. I

I H

12,22,39,69, 121 Magnitude of No effect and200 fJ.g!L 2:4 weeks INot calculable Not applicable effects calculated

i--- from results in

Table 2

Number of SSNR4

, Responses periphyton were considered species 45% to 52% 388 and 690 fJ.g!L 2:4 weeks Not calculable N0 3 likely 'no effect' decrease

when treatment

mean± SD

I

overlapped with

that of controls.s

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Reviewer Comments:

The microcosms were filled with water from the Gata Alv River located in southern Sweden. It

is not stated whether the river water was tested to establish that ii was not contaminated with any

pollutants. Results from analysis of water from untreated control microcosms were neither

presented nor discussed.

The microcosms did not contain a sediment layer.

As a microcosm study, there may be little relevance ofthe results to natural systems, despite the

natural source ofthe communities, given the small size ofthe microcosms (22-L aquaria) and

lack of a sediment layer.

Microcosm water was not monitored for water quality. Temperature was reportedly monitored,

but the data were not presented.

The purity ofthe atrazine test compound used was not reported.

Quantitative results for atrazine in the treated microcosm water were not presented, but reported

as being substantially lower than the nominal concentrations at all test levels with the difference

between nominal and measured increasing at higher atrazine concentrations. At the lowest

atrazine concentration (12 J.t,g!L), the measured concentration was reported as 2-3 times lower

than nominal. The study authors suggested that low concentrations of measured atrazine in the

microcosm water may have been due to saturation ofthe SPE cartridges during extraction.

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

Quantitative results, either tabular or plotted, were not presented to support the reported EC50

and NEC values for the long-term effects of atrazine on photosynthetic activity and sulfolipid

synthesis.

At least five different lipids were reportedly detected by TLC; however, supporting

chromatographic data (chromatogram and/or quantitative data) were not presented.

Treatment concentrations used in the short-term tests were only described as "doubleconcentration stock solutions" applied in a "geometric concentration series".
The study authors reported that periphyton sulfolipid synthesis was examined because sulfolipids

are almost exclusively synthesized by photosynthetic microorganisms making it an alternative

method to measure autotrophic activity. In addition, they suggest that the method is possibly a

better determination of algal growth than short-term carbon fixation because it measures the

synthesis of membrane lipids whereas some photosynthetically fixed carbon may be lost as

exudates or through respiration.

Description of Use in Document: Invalid

Rationale for Use: Only a single microcosm was treated at each atrazine test concentration.

Although a sufficient number oftreatments were used for regression based analyses, the

endpoints generated from those analyses are not relevant for the current analysis of atrazine.

Quantitative results for atrazine in the treated microcosm water were not presented, but reported

as being "substantially" lower than the nominal concentrations at all test levels. Test

concentrations and replication used for the short-term toxicity (tolerance) experiments were not

reported.

Limitations of Study: No effort was made to document whether other chemicals were present in

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

contain a sediment layer. Microcosm water was not monitored for water quality; temperature

was monitored but data was not reported. Purity ofthe test compound was not reported. Only a

single microcosm was treated at each atrazine test concentration, Quantitative results for

atrazine in the treated microcosm water were not presented, but reported as being substantially

lower than the nominal concentrations at all test levels. Although reportedly measured, no

results from analysis of water from untreated control microcosms were presented. Source water

used in the microcosms was from a river and so contamination was possible, in addition, a flow

through microcosm design was used so contamination, if any, may have varied during the

experiment. Test concentrations used for the short-term toxicity (tolerance) experiments were

not adequately described.

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

Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA f/If~c4J

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