Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Numberand Citation: Nystrom, R, M. Paulsson, K. Almgren, and H. Blank. 2000. Evaluation ofthe capacity for development ofatrazine tolerance in periphyton from a Swedish freshwater site as determinedby inhibition ofphotosynthesis and sulfolipid synthesis. Environmental Toxicology and Chemistry. 19: 1324-1331. (MRID 48261128)
Purpose ofReview: Determine potential effects ofatrazine on an aquatic comnumity. 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 5, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine 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/minuteusing an air-driven Teflon-membrane pump and flow-distributor. The source water was pumped from the GotaAlv River ata depth of about 0.5-m. Aquariawere maintained on a natural daylightphotoperiod (on average, 17:7 hour light:darkcycle) using fluorescent light with an irradiance of 120 Ilmol/m2/s atthe 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, concentrationnot specified), and Milli-Q water (9.8 L). Eight ofthe aquariawere continuously treated (flowrate 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 2087819treatmentconcentration/aquarium) using a peristaltic pump. The fmal co-solvent(acetone) concentrationwas 0.01 % (v:v). The remaining four aquariawere maintained as controls continuously treated with acetone (0.01 %, v:v). Each aquarium contained water-mixing equipment (not further described) to ensure instantmixing oftreatment solutions andriver water andto 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 notreported. Samples were filtered (glass fiber, Whatman type GF/F) and then extracted using solidphase C18 cartridges (Accubond SPE, 6-mL, 500 mg). Loading and elutionprocedures were not described. Atrazine was quantified using reverse-phase HPLC; methods were referenced (Durand and Barcelo, 1990; Guasch etal., 1998) butnotdescribed.
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, longterm effects ofatrazine onperiphyton photosynthetic activity, sulfolipid synthesis, thymidine, and adenine incorporation, chlorophyll a content, dry weight, and species composition were assessed as describedbelow. Additionally, periphyton was assessed for development oftoleranceto 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. Atthe end ofthe 4 week exposure, glass disks with periphyton were sampled from three aquaria each day: one randomly selected control aquaria andtwo randomly selected atrazine treated aquaria. Periphyton disks were stored in unfilteredriver 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 addedto the periphyton samples (disks) inthe 20-mL vials. For short-termtoxicity experiments, an equal volume ofatrazine solution (double concentration stock solutions in GF/F filtered river water) was addedto the vials in a geometric concentration series (test concentrations and exposure interval were not reported). An aliquot (100 ilL) ofaqueous H14C03
- solution (adjustedto 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 atthe same temperature as the aquaria and illuminated viafluorescent light from below with an irradiance of80 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 ofair. 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 addedto the periphyton samples (disks) in the 20-mL vials. Short-term toxicity experiments were conducted as described above with an exposure interval of24 hours. To determine sulfolipid synthesis, 35S04- was added at 10 

### -2-JlCi/mL. Samples were incubated for 16 hours in a waterbath at 17°C under continuous illumination from above atthe 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 deionizedwater and the rinse water was filtered through GFIC filters. Filters were thenrinsed with deionized waterto reduce the amount ofunincorporated 35S04-. Filters and the glass disks were stored frozen (-18°C) until extraction. Lipids were extractedby adding methanol (2 x 1 mL) followed by chlorofonn (2 x 1 mL). The extractwas then washed by adding deionized water (4-4.5 mL), centrifugingthe resultant solution, and removing the top layer. The procedure was repeated eighttimes. The washed organic phase (chlorofonn-lipid extract) was evaporatedto dryness, andthe resulting residues were analyzed using LSC. Fonnaldehyde blanks were used to estimate unincorporated 35S0 4
-andthe control values were subtracted from the final results.
Periphytonlipids were analyzed by one-dimensional thin layer chromatography (TLC) on nonnal-phase plates (silica gel) developed withchlorofonn: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 chloroplastlipid, was used as areference standardbecause itwas presumedto be the majorperiphyton 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 sodiumborate) was added to kill the samples. Samples were extracted with 4MNaOH (final concentration 0.25M) at 60°C for 1 hour, placed on ice, treated with ice-cold trichloroacetic acid (TCA, final concentration 22%) for2:30 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 thenneutralized withNaOH andthe waterwas evaporated. Radioactivity was measured using LSC with a dual"radiolabel program for 14C and 3H. Chlorophyll 

### a. Periphyton samples (disks) weretreated with dimethylsulfoxide (DMSO, 1 mL) and stored frozen until extraction. Samples were extracted at 60°C in darkness for 30 minutes, thentreated with 90% acetone, and analyzed spectrophotometrically (method ofJeffrey and Mumphrey, 1975). Periphyton recovered on Plexiglas plates (117 cm2) detached from the water"mixing equipment was also analyzed for chlorophyll 

### a. The periphytonfilm was scraped from aplate into river water (80 mL) and homogenized via shaking. An aliquot(volume notreported) ofthe periphyton suspensionwas centrifuged. The supernatantwas removed, DMSO (l mL) was added, and the sample was stored frozen. Extractionand 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 atroomtemperature, thenweighed, thenheatedto 450°C overnight and reweighedto 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 videotapedto facilitate identification. Species identifications were conducted by counting 50 randomly chosen fields at a magnification of 1,000x using aphase-contrast microscope. Relative abundance ofeach 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 notreported ifsimilarity was based onjust species or species and groups ofspecies as reported in table 2.

Statistical analyses. Datawere analyzed for variance heterogeneity (Cochrans's test) and normal distribution (X2 

### test), then, ifnecessary, were IOi0g transformedto meetthe assumptions for parametric tests. For long-term effects tests, the interceptbetweenthe linearregression line (FIG.P v. 5.0 software) and the average control value wasjudgedto be the no effect concentration (NEC) as an estimate ofthe long-termno observed effect concentration (NOEC). The 95% confidence limits ofthe regressions were calculated according to 

### Dixon and Massey (1985). For short-termtoxicity tests, lowest observed effect concentration (LOEC) values were estimated using an analysis ofvariance (ANOVA). When ANOVA was statistically significant (p<0.05), eitherDunnett's test (equal sample sizes) orBonferroni'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 neitherpresented, nordiscussed.
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:SQDGratio decreased with increasing atrazine at concentrations :-::0.32 fJM 

### (Figure 4).
Periphyton dry weight gradually decreased to a maximum of-30% ofcontrol periphyton at 1.0 fJM atrazine exposure but was only -20% and -10% ofcontrol 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 of5% difference from controls) between the 0.10 and 1 l-lM (22-216 l-lgIL) atrazine treatment concentrations. The study authors reportthatthe 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 bacteriawith 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 difficultto interpret because EC50 values were 1.9-2.2 fJM for the periphyton from the four control microcosms (no priorexposure). For sulfolipid synthesis, periphyton communities previously exposedto 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 of0.56 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.



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0.10 0.32 1.0 3.2 long-term atrazine expolure (JolM) Figure 1. Long-term effects ofatrazine on chlorophyll acontentofperiphyton on glass disks (1.5 cm2) expressed as a percentage ofthe control. The average control chlorophyll acontent (100 %) was 2.6 ,..g1disk. Values are mean ofthree samples from each microcosm with standard deviation (vertical bars). Dotted lines indicate 95% confidence limits. Linearregression included all treatments, except 0.056 ,..M. (Figure 1 of study)

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### 1.0 3.2 LOtIg-term atrazine exposure (I'M) Figure 2. Long-term effects ofatrazine on chlorophyll a contentand 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 ,..g11.5cm:! and the average AFDW was 0.93 mgl1.5 cm2• Open square = chlorophyll a, solid square

### =AFDW. Values are mean ofthree samples from each microcosm with standard deviation (vertical bars). (Figure 4 from study) -6-

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### Itrufne exposure (JIM} Figure 3. Specific photosynthetic and sulfolipid synthetic activity (dpm/p.g chlorophyll acontent) expressed as a percentage ofthe control. Values are mean activity foreach microcosm. Solid square

### =photosynthetic activity and open square

### =sulfolipid synthetic activity. Average specific activity in controls was 51,000 dpmlp.g chlorophylla and 6,700 dpmlp.g chlorophylla for photosynthesis and sulfolipid synthesis, respectively. The average incorporation rates were 27 nmol carbonJp.g chlorophylla in 1 hour and 46 pmol sulfatelp.g chlorophyll a in 16 hours. (Figure 2 from study)



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### 11M Figure4. Ratio ofperiphyton 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)



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### atrazln. .xposur. [pM} Figure 5. Long-term effects ofatrazine on periphyton community structureexpressed as Bray-Curtis similarity. Similarity indices were calculated from the relative abundance ofspecies as related to the controls, generating four differentindices pertreatment and three percontrol aquarium. Dotted lines indicate 95% confidence limits. Linear regression included all treatments, except 1.8 and 3.2 11M. (Figure 6 from study)

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0.10 0.32 1.0 3.2 Long-term atrazine exposure**(~M)** Figure 7. Long-term effects ofatrazineon periphyton community tolerance. EC50 values for short-term inhibition ofphotosynthesis (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 ofatrazine 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 Shortterm 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 effectconcentration. 3 Lowestobserved effect concentration. 4 Halfmaximal effective concentration.

Table 2. Number ofperipbyton species and relative abundance ofperiphyton species or groups ofspecies observed after 4 weeks ofatrazine 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 

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### ata ummary a 

### e: Time Weighted Average Magnitude of NominalAtrazine Day of Atraz;ne DidRecovery Effect Effectrelative Concentration StudyEffect 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 SSNR 4• 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) errorbars 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 SSNR 4

. Responses chlorophyll a 216 j.lg/L were considered (per area, N0 3 likely 'no effect' Plexiglas whentreatment plate) -9O%to-95% errorbars decrease 388 and 690 j.lg/L :::::4 weeks Not calculable overlappedwith 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 SSNR 4• Responses weightof were considered periphyton likely 'no effect' (per area, when treatment Plexiglas No effect 690 j.lg/L :::::4 weeks Not calculable Not applicable errorbars plate) overlapped with 

# I J those ofthe controls.5Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- if so, 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 SSNR 4• 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 

# I Not ","cul.bl, specific stimulation I SSNR 4

. Responses sulfolipid -150% No 3 were considered synthesis stimulation 3881J.g/L 2':4 weeks Not calculable likely 'no effect' activity whenthetreatment**(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 SSNR 4• Responses were considered Change in likely 'no effect' community when treatment structure errorbars (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 consideredan effect

- 12
-Time Weighted Average Magnitude oj NominalAtrazine Day oj Atrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- 

### if so, Comments to controls (pgILi Occurretf jromDayOto when? When Effect Occu"ed giventhe variability in the control microcosms (70% similarto 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** Notcalculable 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• Responses

VarIOUS decrease (taxa \ 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 overlappedwith that ofcontrols.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

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### I The study authors reported that measured atrazine concentrations were substantially lowerthan the nominal concentrations; two- to three-fold lower atthe 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 andtwo atrazine-treated

microcosms were randomly sampled per day). Sampling dates for specific treatments and controls were not reported. 3 Effect observed at end ofexperiment. 4 Statistical significance oftreatmentcompared to controls was notreported. In addition, reviewer cannotjudge significance ofeffect because there was no replication ofatrazine-treated microcosms. SResponses were considered likely 'no effect' when the treatmentresponse 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 NominalAtrazine Day oj iAtrazine DidRecovery Effect Effectrelative Concentration Study Effect Concentration Occur

- 

### if so, Comments to controls (pglL/ Occurrecr from Day 0 to when? 

# I de"'"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 overlappedwith that ofcontrols.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 whetherthe river waterwas tested to establishthat

## ii was not contaminated with any pollutants. Results from analysis of water from untreated control microcosms were neither presented nordiscussed.

The microcosms did notcontaina sedimentlayer.

As amicrocosm study, there may be little relevance oftheresults to natural systems, despite the natural source ofthe communities, giventhe small size ofthe microcosms (22-L aquaria) and lack ofa sedimentlayer.

Microcosm water was not monitored for water quality. Temperature was reportedly monitored, butthe datawere not presented.

The purity ofthe atrazine testcompound used was notreported.
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 ofmeasured atrazine in the microcosmwatermay 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/orquantitative data) were notpresented.

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 thatperiphyton 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 orthrough respiration. 

### Description ofUsein Document: Invalid


### Rationale for Use: Only a single microcosm was treated at each atrazine testconcentration. Although a sufficientnumber oftreatments were used for regression based analyses, the endpoints generated from those analyses are not relevant forthe current analysis ofatrazine. Quantitative results for atrazine in the treated microcosmwaterwere notpresented, butreported as being "substantially" lowerthanthe nominal concentrations at all test levels. Test concentrations and replication used for the short-termtoxicity (tolerance) experiments were not reported.


### Limitations ofStudy: No effort was made to document whether otherchemicals were present in the treatment and/or control microcosms priorto study initiation. The microcosms did not contain a sedimentlayer. Microcosm waterwas not monitored for water quality; temperature was monitored butdata was not reported. Purity ofthe testcompound was not reported. Only a single microcosm was treated at each atrazine testconcentration, Quantitative results for atrazine inthe treated microcosm water were not presented, butreported as being substantially lowerthan the nominal concentrations at all test levels. Althoughreportedly measured, no results from analysis ofwater from untreated control microcosms were presented. Source water used in the microcosms was from a river and so contamination was possible, in addition, aflow through microcosm design was used so contamination, ifany, may have varied during the experiment. Test concentrations used for the short-termtoxicity (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

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# c4J

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