### Open Literature Review Summary

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

ECOTOX Record Number and Citation: Mufioz, 

### 1., M. Real, H. Guasch, E. Navarro, and S. Sabater. 2001. Effects of atrazine on periphyton under grazing pressure. Aquatic Toxicology. 55: 239-249. (MRID 48261125)
Purpose ofReview: Determinepotential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plantcommunities and associated determination ofaLevel ofConcern (LOC).

Date ofReview: October 5, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine onperiphyton, inthe presence and absence ofsnails 

### (Physella acuta), in indoor experimental flow-through microcosms.
Summary ofStudy Findings: Experiments including snails will be mentioned, although in limited detail, in orderto focus on the periphyton community experiments conducted inthe presence and absence ofatrazine alone.

Methods: Twelve indoor, once-through Perspex artificial channels (170 cmlong, 10 cm wide, 10 cm deep) were continuously suppliedwith waterto yield aflow rate of1 cm/second (90 L/hour) and water depth of 1.8-2.2 cm withinthe channels (microcosms). The source waterwas a well located 50 m from the test facility. The microcosms were maintained on a 8:16 hour lightdarkcycleusing 500W metal halide lamps with irradiance of140 IlE/m2/s atthe water surface. The floor ofeach channel (microcosm) was homogenously covered with 20 x 10 cm sections ofsand-blasted glass to serve as surface substrates for periphyton, with smaller 1.2 x 1.2 cm sections ofthe glass substrate interspersedbetweenthe larger sections to serve as sampling units.

To collectperiphyton, sections ofglass substrate were left in a stream (L'Avenc6) for 2 weeks; no additional details wereprovided. According to the study authors, this stream was "unpolluted". The colonized sections ofglass substrate were then introduced into the - 1 -

1 ••••

# 1.111 2087814

### microcosms and allowed to acclimate for 12 days priorto atrazine treatment. Snails 

### (Physella acuta) were collected from another local stream (Congost, NE Spain) and transported to the test facility in aerated containers. Selected snails (shell length 7-9 mm) were introduced into microcosms 48 hours priorto treatment (20 snails per microcosm, tissue dry wt. 0.9 g/m2

, 

### equivalent density 118 snails/m2). 

### A net (mesh size not specified) was placed at each microcosm out-flow to retain the snails.

The experimental design consisted offour treatment groups as follows: 1) untreated microcosms without snails (control), 2) atrazine treated microcosms without snails, 3) untreated microcosms with snails, and 4) atrazine treated microcosms with snails; with all groups replicated in triplicate. No solvent controls were utilized.

The treatment solution was prepared by dissolving powdered atrazine (2:90% purity, Makhteshim S.A., personal communicationl) in well water (1 L, concentration not specified) with acetone (0.5 mL, co-solvent final concentration 0.05%, v:v) and sonication for 10-15 minutes*. Six microcosms were continuouslytreated (flow rate 4 

### mL/minute) atthe microcosm in-put with atrazine, at a nominal concentration of14 flg/L, using a peristaltic pump.
Following treatment, oxygen saturation, temperature, conductivity and pH were measured in the microcosm water daily for up to 18 days in September, 1998. No additional experiment dates were specified. Soluble reactive phosphorus, nitrate (N-N03-), nitrite (N-N02-) 

### and ammonium (N-NH4 +) were measured inthe water atthree unspecified intervals.

At five unspecified intervals, water samples (volume not specified) from treated and control microcosms were analyzed for atrazine using the EnviroGard Triazine QuantiTube Test Kit (Millipore), with a detection limit of0.04 flg/L. Analysis methods were not further described.
Photosynthetic activity (carbon incorporation). Nine 1.2 x 1.2 cm sampling units with periphyton were randomly selected from each microcosm at days 0, 1,3,6, 12 and 18 posttreatmentand separately placed in vials with filtered (glass fiber, Whatmantype GF/F) 4 mL ofmicrocosm water. For each nine-sample unit set, five samples were used for light incubations, two for dark incubations and two were formaldehyde-killed to serve as controls. Samples were pre-incubated for 1 hour in an agitator underthe same temperature and light conditions as the microcosms, then

# e 4

C]bicarbonate (Hl4C03-) solution (specific activity 1 mCi/mL) was added at 1 flCi/vial and incubation continued underthe same conditions for 1 hour. Formaldehyde (2%, v:v) ceased

# e 4

C]-incorporation, then HCl (pH 2-3) was added and the samples evaporated for 8 hours on a hot-plate. Dried residues were treated with 1 mL dimethylsulfoxide and analyzed for total radioactivity using LSC.

1 Information obtainedviapersonal communication with Dr. Munoz on October 20, 2009 is provided in Attachment 1. 

### - 2 -

### Chlorophyll a. Five 1.2 x 1.2 cm sampling units withpeiiphytonwere randomly selectedfrom eachmicrocosm atthe same intervals as for the photosynthetic activity tests. Samples were extracted with 5 mL of90% acetone using sonication for 7-10 minutes in anice-bath, then analyzed spectrophotometrically (method ofJeffrey and Humphrey, 1975).

At study termination, extractedpigments were analyzed using reverse-phase HPLC (Hypersil ODS column, 250 mm, 5 flm particle size, i.d. not specified) withfluorescence detection (Kontron SFM detector). Chromatographic conditions were referenced (Mantoura and Llwewllyn, 1983), butnot described. Chlorophylls and carotenes were identifiedthrough comparisonofspecific extinction coefficients, retentiontimes and absorbance characteristics and againstreference standards (Sigma) ofchlorophylls a and bandu- and**~-carotenes** 

### (Foppen, 1971, Mantouraand Llewellyn, 1983, Rowan, 1989). Chlorophyll derivatives phaeophytin aand b, chlorophyllide a and b, andpheophorbides were also prepared (Sartory, 1985, Leavitt

### etal., 1989).
Species composition. Two 1.2 x 1.2 cm samplingunits withperiphytonwere randomly selected from eachmicrocosm at days 0, 3 and 18 posttreatment andpreservedin 4% formaldehyde solution. Periphytonwas detached via sonicationfor microscopic examination at a magnification of600. Periphytontaxawere assignedto four classes, crustose, prostrate, stalked orfilamentous, based onphysiognomy. Bio-volume was determined according to the method of Lowe and Laliberte (1996).

Snails. Initial average dry weight (70°C until constantweight) was determined for a subsset of snails from the same source as the treatment organisms. Individual dry weights were determined for eachtreatment organism at study termination. Snail numberin eachmicrocosmwas monitored daily. Dead snails were replacedwith snails ofthe same shell length. Snails found on the net atthe out-flow were removed andreturnedto the respective microcosminput. At study termination, number oflaid egg masses and number ofeggs permass were determined. No juveniles developed during the study.

Statistical analyses. Periphytonresponse values were analyzedby three-way analysis ofvariance (ANOVA) to detect differences betweentreatments with arepeated measures design (withtime as arepeated factor). When statistically significant (p<0.05), response values were analyzed using the Tukey Honest significantdifference (HSD) test. Differences inperiphytonpigment compositionbetweentreatments were analyzedbytwo-way ANOVA, while snail biomass changes were analyzed by one-wayANOVA.



### *Atrazine was detected in the source stream in aprior study (see Guasch et al. 2001).



### - 3 -

### Results: Overthe 18-day study, atrazine concentrations intreatedmicrocosms averaged (n = 22) 14.18 ± 

### 5.93 f.lg/L; 

### individual results were notpresented. Atrazine was not detected «0.04 f.lg/L) 

### in untreated control microcosms.
Physico-chemical parameters ofthe microcosm water remained constant throughout the study, with an average watertemperature of23 ± 

### 0.05°C, pH range of7.4-7.6 and conductivity of 1,900 f.lS/cm. 

### Mean phosphate (P-P04-), 

### nitrate and nitrite were 2.4 ± 

### 1.4 f.lg/L, 

### 15.5 ± 

### 0.83 mg/L 

### and 0.22 ± 

### 0.07 mg/L, 

### respectively, while ammoniawas not detected «0.01 mg/L).



### Microcosms without snails. Periphyton bio-volume increased significantly (p<0.05) in both control (untreated) and atrazine treated microcosms over the 18-day study, but was decreased**~40%** 

### in atrazine treated microcosms, as compared to the controls, at study termination (Figure 1). There were no significant differences in pigment composition between atrazine treated microcosms and the untreated controls at study termination (Table 1). Chlorophyll a concentration, photosynthetic activity (carbon incorporation) and photosynthetic efficiency (carbon incorporationperunit chlorophyll a) were decreased**~15%** 

### (day 12),**~15%** 

### (day 12) and**~40%** 

### (day 6), respectively, at intervals during the study (Figures 1 and 

### 2). However, there were no significant differences over time between atrazine treated microcosms and the untreated controls.

The periphyton community colonizing the microcosms was dominated by the diatoms Achnanthes lanceolata, Achnanthes minutissima and Nitzschia linearis, plus basal cells ofthe green algaStigeoclonium tenue (Table 2). At study termination, filamentous form periphyton in atrazine treated microcosms were decreased**~90%,** 

### as compared to controls, crustose forms**~40%,** 

### prostrate forms 10% and stalked forms**~5%.** 

### However, decreases were significant (p=0.02) only for the filamentous forms (Figure 3, Table 

### 3). Total periphyton abundance was decreased**~30%** 

### in atrazine treated microcosms at 18 days, as compared to controls (Table 

### 3, attached Worksheet). The densities oftwo species, Cocconeisplacentula and S. tenue (cells and filaments) were reported as significantly (p=0.02-0.04) increased in the atrazine treated microcosms (Table 2).

Microcosms with snails. Snail grazing alone resulted in decreases in periphyton chlorophyll a concentration (p<O.OOI), photosynthetic activity (p<O.OOOI), photosynthetic efficiency (p=0.047) and inhibition in the rate ofbio-volume growth, with the addition ofatrazine further enhancing those effects (Figures 1 and 

### 2) Grazing and atrazine exposure combined produced more-thanadditive effects with respect to carbon incorporation (p=0.02). Pigment composition was not significantly affectedby grazing either in the presence or absence ofatrazine (Table 1).

Snail grazing significantly reduced the abundance ofstalked (p=0.01), prostrate (p=0.008) and crustose (p=0.02) forms of periphyton with abundances of all forms, including filamentous, - 4 -

### further decreased in the presence of atrazine (Figure 

### 3). Grazing resulted in significant decreases in densities of

### A. minutissima (p=0.002), 

### Amphora sp. (p=0.02) and 

### C. placentula (p=0.04); whereas, grazing plus atrazine resulted in significantly lower densities for A. 

### lanceolata (p=0.0l), 

### A. minutissima (p=0.04), 

### Amphora sp. (p=0.04) and 

### Nitzschiafonticola (p=0.002). At studytermination (18 days), total periphyton abundances were decreased 56% and 77% in microcosms containing snails alone and snails plus atrazine, respectively, as comparedto untreated controls (no snails, no atrazine; Table2, attachedWorksheet).

There were no significant differences between microcosms treated with atrazine and untreated controls regarding snail biomass (p=0.9), number of escaped snails (p=0.70), number of egg masses (p=0.73) or number of eggs per mass (p=0.66). Mortality was near 10% for all microcosms, withno significantdifferences (p=0.09) betweentreatments.

- 5 -

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# 6 8 10 12 14 16 18 days Figure 1. Periphyton chlorophyll a (A) and bio-volume (B); mean values from three microcosms per treatment with standard deviation (vertical bars). Figure 1 from Munoz etal., 2001.



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# 6 8 10 12 14 16 18 days Figure 2. Photosynthetic activity (A) and photosynthetic efficiency (B); mean values from three microcosms pertreatment with standard deviation (vertical bars). Figure 2 from Munoz etal., 2001.



## - 7 -

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## 18 

# C Treatment Days Figure 3. Abundance ofperiphyton physiognomic classifications; mean values from three microcosms per treatment with standard deviation (vertical bars). A

### =atrazine treatment, AS 

### =atrazine 

### + snail, S 

### =snail treatment, C = control (no atrazine, no snail). Figure 3 from Munoz etal., 2001.

Table 1. Effect ofatrazine (14 IlglL), on periphyton pigmentcomposition in the presence and absence of grazing pressure after 18 days ofexposure, expressed as percentage ofrecovered (mean ± s.d., n = 3; from Table 1 ofstudy). Control Atrazine Snail Pigment (no atrazine, no snail) (no snail) (no atrazine) Atrazine + Snail Carotenoids 32.8 ± 0.5 33.4 ± 2.3 32.2 ± 0.8 31.9 ± 6.6 Chlorophyll a 56.8 ± 3.4 57.4 ± 4.8 51.8 ± 6.5 50.8 ± 6.7 Chlorophyll b 0.6 ± 0.2 0.9 ± 0.7 5.1±5.1 6.3 ± 5.4 Chlorophyll c 2.7 ± 0.4 2.3 ± 0.3 2.2 ± 0.7 1.9 ± 0.7 Degraded chlorophyll 10.1 ± 7.9 6 ± 3.1 8.6 ± 2.5 9.3 ± 5.2



### - 8 -Table 2. Effect ofatrazine (14 /!gIL) on periphyton species abundance in the presence and absence ofgrazing pressure, expressed as density x 103 cells/cm2 (mean ± s.d., n = 3; from Table 2 ofstudy). Day 3 Day 18 Atrazine 

### + Atrazine 

### + Taxa Day 0 Control Atrazine Snail Snail Control Atrazine Snail Snail Achnanthes Ianceolata Breb. 469 ± 321 316±390 32 ± 20 152 ± 92 127 ± 63 584±318 647 ± 230 578 ± 196 427 ± 450 Achnanthesminutissima Klitz. 1,224 ± 1,034 646 ± 858 817 ± 454 364 ± 470 196± 103 2,274 ± 1,689 203 ± 806 598 ± 750 385 ± 345 Amphora sp. 68 ± 52 142 ± 152 21 ± 13 28 ± 18 16 ±.28 158±151 117 ± 47 5±9 12 ± 21 Ceratoneis sp. 17 ± 35 0 0 0 0 0 0 0 0 Chroococcus sp. 60 ± 120 0 0 0 0 0 0 23 ±40 0 Cocconeisplacentula Ehr. 41 ± 20 31 ± 35 25 ± 33 20 ± 7 7±3 39 ± 35 70± 5 8 ± 14 0 Cosmarium sp. 0 0 0 4±7 0 15 ± 27 0 3±5 0 Dictyosphaerium sp. 135 ± 105 642 ± 516 373 ± 541 100 ± 73 32 ± 55 452 ±460 281 ± 427 178 ± 268 203 ± 158 Fragilaria capucina Desmazieres 87 ± 333 16 ± 16 28 ± 24 84 ± 75 0 8 ± 13 39 ± 36 23 ±40 8 ± 14 Fragilaria construens (Ehr.) Grun. 118±124 0 16 ± 18 20 ± 35 0 8 ± 15 0 12 ± 20 3±5 Melosira varians Agardh 4±8 5±9 0 0 0 0 0 0 0 Monoraphidium sp. 0 3±5 0 0 0 0 0 0 0 Navicula criptocephala Klitz. 8 ± 16 33 ± 16 12 ± 21 24±2 12 ± 6 54 ± 74 39 ± 67 0 12 ± 12 Nitzschiafonticola Grun. 79 ± 60 117 ± 140 22 ± 19 16 ± 18 4±5 70 ±46 78 ± 67 0 13 ± 12 Nitzchia linearis Smith 153±81 304 ± 350 432 ± 55 360 ± 427 25 ± 20 240 ± 336 764 ± 772 81 ± 141 40 ± 38 Scenedesmus sp. 7 ± 14 0 0 0 0 109 ± 117 0 11 ± 18 16 ± 28 Stigeoclonium tenue (Ag.) Klitz (basal cells) 220 ± 106 93 ± 161 103 ± 58 48 ± 55 23 ± 24 151 ± 506 109 ± 133 452 ± 274 184± 76 Stigeoclonium tenue (filaments) 59 ± 102 81±141 10 ± 18 256 ± 285 23 ± 27 1,875 ± 1,269 211 ± 253 746 ± 923 208 ± 145 Synedra ulna (Nitzsch.) Ehr. 21 ± 6 125 ± 135 222 ± 96 108 ± 32 12 ± 6 1,415 ±426 835 ± 49 571 ± 711 221±155 Total abundance 2,771 ± 1,809 2,555 ± 2,689 2,400 ± 42 1,584 ± 219 478 ± 178 7,451 ± 3,521 5,218 ± 725 3,289 ± 1,800 1,732 ± 929



### - 9 -Table 3. ANOVA probability (p) values (from Table 3 ofstudy). Factor Taxa/growth form Atrazine (A) Snail (S) Time (T) AxT SxT AxS AxSxT Achnanthes lanceolata NS J NS 0.002 NS NS 0.03 0.01 Achnanthes minutissima NS 0.002 0.008 NS NS NS 0.04 Amphora sp. NS 0.02 NS NS NS NS 0.04 Cocconeisplacentula 0.04 0.01 NS NS 0.04 0.001 NS Nitzschiajonticola NS NS NS NS NS NS 0.002 Stigeoclonium tenue (cells) 0.03 NS 0.003 NS 0.004 NS NS Stigeoclonium tenue (filaments) 0.02 NS 0.003 0.03 NS NS NS Filamentous 0.02 NS 0.003 0.03 NS NS NS Stalked NS 0.01 0.0001 NS 0.002 NS NS Prostrate NS 0.008 0.0001 NS 0.0022 0.0001 0.0003 Crustose NS 0.02 NS NS NS NS NS 1 Not significant.



### - 10

-

### Data Summary Table: Periphyton andgastropods in indoor,jlow-through microcosms; periphyton data representthe "atrazine on£v"conditions, in the absence of grazingpressure. Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration StudyEffect Concentration Occur

- 

### if so, Comments to controls (pg/L) Occurred from Day Oto when? When Effect Occurred Magnitude ofeffect estimated from Periphyton 14 11giL; Figure 1. chlorophyll a 15% decrease measured 14.18 ± 12 Not calculable No Effects not (llg/cm2) 5.93 11giL statistically significant (p=0.86). Periphytonbio- 141lg/L; Study Magnitude ofeffect -40% decrease measured 14.18 ± 18 Not calculable terminated at estimated from volume 5.93 Ilg/L day 18. Figure 1. SSNR1
• No, but Magnitude ofeffect Periphyton control estimated from 14 Ilg/L; decreased to Figure 2. photosynthetic 15% decrease measured 14.18 ± 12 Notcalculable level of Effects not activity 5.93 1lg/L atrazine statistically (Ilg C/cm2/hr) treated at day significant 18. (p=0.38). No, control Periphyton and atrazine photosynthetic 141lg/L; treated Magnitude ofeffect efficiency -40% decrease measured 14.18 ± 6 Not calculable decreasedto estimated from (Ilg CIllg 5.93 1lg/ L same level at Figure 2. SSNR. ch10rophy1l/hr) days 12 and 18. Magnitude ofeffect 141lg/L; Study estimated from Filamentous**~90%** decrease measured 14.18 ± 18 Not calculable terminated at Figure 3. periphyton 5.93 Ilg/L day 18. Effects statistically significant (p=0.02, Table 3). Magnitude ofeffect estimated from Stalked 14 11giL; Study Figure 3. periphyton -5% decrease measured 14.18 ± 18 Not calculable terminated at Effects not 5.93 11giL day 18. statistically significant (p>0.05, Table 3).



### - 11

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

- 

### if so, Comments to controls (pg/L) Occurred from Day Oto when? When Effect Occurred Magnitude ofeffect estimated from Prostrate 14 flg/L; Study Figure 3. periphyton 10% decrease measured 14.18± 18 Not calculable terminated at Effects not 5.93 flg/L day 18. statistically significant (p>0.05, Table 3). Magnitude ofeffect estimated from Crustose 14 flg/L; Study Figure 3. periphyton**~40%** decrease measured 14.18 ± 18 Not calculable terminated at Effects not 5.93 flg/L day 18. statistically significant (p>0.05, Table 3). Magnitude of effects calculated from results in Achnanthes 14 flg/L; Yes, day 18, Table 2 ofstudy lanceolata 90% decrease measured 14.18 ± 3 Not calculable (+11% (attached Breb. 5.93 flg/L stimulation). Worksheet). (abundance) Effects not statistically significant (p>O.05, Table 3). Magnitude ofeffect calculated from Achnanthes results in Table 2 of minutissima 14 flg/L; Study study (attached Klitz. 91 % decrease measured 14.18 ± 18 Not calculable terminated at Worksheet). (abundance) 5.93 flglL day 18. Effects not statistically significant (p>0.05, Table 3). Magnitude of effects calculated from results in 14 flg/L; Magnitude of Table 2 ofstudy Amphora sp. 85% decrease measured 14.18 ± 3 Not calculable effect26% (attached (abundance) 5.93 flg/L decrease at Worksheet). day 18. Effects not statistically significant (p>O.05, Table 3).



### - 12

-Time Weighted Average Magnitude of NominalAtrazine Day of Atrazine DidRecovery Effect Effectrelative Concentration StudyEffect Concentration Occur

- 

### if so, Comments to controls (pg/L) Occurred from Day Oto when? When Effect Occurred Magnitude of effects calculated from results in Cocconeis 14 flg/L; Yes, day 18, Table 2 ofstudy placentula Ehr. 19% decrease measured 14.18 ± 3 Not calculable (+79% (attached (abundance) 5.93 flg/L stimulation). Worksheet). Effects statistically significant (p=0.04, Table 3). Magnitude ofeffect Cosmarium sp. 14 flg/L; Study calculated from 100% decrease measured 14.18 ± 18 Notcalculable terminated at results in Table 2 of (abundance) 5.93 flg/L day 18. study (attached Worksheet). SSNR. Magnitude of 14 flg/L; Magnitude of effects calculated Dictyosphaerium 42% decrease measured 14.18 ± 3 Not calculable effect 38% from results in sp. (abundance) 5.93 flg/L decrease at Table 2 ofstudy day 18. (attached Worksheet). SSNR. Fragilaria Magnitude ofeffect capucina +388% 14 flg!L; Study calculated from measured 14.18 ± 18 Not calculable terminated at results in Table 2 of Desmazieres stimulation 5.93 flg/L day 18. study (attached (abundance) Worksheet). SSNR. Fragilaria Magnitude ofeffect contruens (Ehr.) 14 flg/L; Study calculated from 100% decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of Grun. 5.93 flg/L day 18. study (attached (abundance) Worksheet). SSNR. Magnitude ofeffect Melosira varians 14 flg/L; calculated from Agardh 100% decrease measured 14.18 ± 3 Not calculable No results in Table 2 of (abundance) 5.93 flg/L study (attached Worksheet). SSNR. Magnitude ofeffect Monoraphidium 14 flg/L; calculated from 100% decrease measured 14.18 ± 3 Not calculable No results in Table 2 of sp. (abundance) 5.93 flg/L study (attached Worksheet). SSNR.



### - 13

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

- 

### if so, Comments to controls (pg/L) Occurred from Day Oto when? When Effect Occurred Magnitude of Navicula 14 IlglL; Magnitude of effects calculated criptocephala 64% decrease measured 14.18 ± 3 Not calculable effect28% at from results in Klitz. 5.93 Ilg/L day 18. Table 2 ofstudy (abundance) (attached Worksheet). SSNR. Magnitude of effects calculated from results in Nitzschia 14 IlglL; Yes, day 18, Table 2 ofstudy fonticola Gron. 81% decrease measured 14.18 ± 3 Not calculable (+11% (attached (abundance) 5.93 IlgiL stimulation). Worksheet). Effects not statistically significant (p>0.05, Table 3). Magnitude of Nitzschia 14 Ilg/L; Study effects calculated +218% from results in linearis Smith stimulation measured 14.18 ± 18 Not calculable terminated at Table 2 ofstudy (abundance) 5.93 IlgiL day 18. (attached Worksheet). SSNR. Magnitude ofeffect Scenedesmus sp. 14 IlglL; Study calculated from 100% decrease measured 14.18± 18 Not calculable terminated at results in Table 2 of (abundance) 5.93 IlglL day 18. study (attached Worksheet). SSNR. Magnitude ofeffect Stigeoclonium calculated from results in Table 2 of tenue (Ag.) 14 IlglL; Study study (attached Klitz. (basal 28% decrease measured 14.18 ± 18 Not calculable terminated at cells, 5.93 IlgiL day 18. Worksheet). abundance) Effects statistically significant (p=0.03, Table 3). Magnitude of effects calculated Stigeoclonium 14 Ilg/L; No, magnitude from results in
tenue (filaments, 88% decrease measured 14.18 ± 3 Not calculable ofeffect 89% Table 2 ofstudy abundance) 5.93 Ilg/L decrease at (attached day 18. Worksheet). Effects statistically significant (p=0.02,



### - 14

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

- 

### if so, Comments to controls (pg/L) Occurred from Day Oto when? When Effect Occurred Table 3). Magnitude ofeffect Synedra ulna 141lg/L; Study calculated from (Nitzsch.) Ehr. 41 % decrease measured 14.18 ± 18 Not calculable terminated at results in Table 2 of (abundance) 5.93 IlgIL day 18. study (attached Worksheet). SSNR. Magnitude ofeffect Total periphyton 141lg/L; Study calculated from 30% decrease measured 14.18 ± 18 Notcalculable terminated at results in Table 2 of abundance 5.93 Ilg/L day 18. study (attached Worksheet). SSNR. There were no significant Snail (Physella differences in acuta) biomass, biomass (p=O.9), mortality, 14Ilg/L; Study mortality (p=0.09), number ofegg No effect measured 14.18 ± 18 Notcalculable terminated at number ofegg masses, number 5.93 IlgIL day 18. masses (p=O.73) or ofeggs per egg number ofeggs per mass egg mass (p=0.66), as comparedto controls. 1 SSNR = StatIstIcal sIgmficance, as comparedto controls, was notreported.



### - 15

-

### Reviewer Comments: 

### Although notreported as part ofthe study, the purity ofthe atrazine test compound was reported via email (see Attachment 1) to be greater than 90%. The source water for the flow-through microcosms was a well located 50 m from the test facility. Atrazine was not found inwater from untreated control microcosms withinthe detection limits ofthe test (>0.04 J.lg/L)

.. No othertests for chemical contaminants were described. Prior exposure of organisms to atrazine or other chemicals was not established or reported. Microcosm water was not monitored for turbidity, alkalinity, or particulate and dissolved organic carbon. Oxygen saturation was reportedly monitored; however, the datawere notpresented.

The concentration ofacetone used as solvent (0.5 mLlL) was 5 times higher than the maximum recommended solvent level of 0.1 mLiL in flow-through systems (see OPPTS 850.1000 guidelines for Special Considerations for Conducting Aquatic Laboratory Studies). In addition, the lack ofa solvent control confounds the ability to differentiate treatment-related effects from those that might due to the high solvent concentrations

..
The study was designed to test organismal response in the presence or absence ofatrazine and thus was limitedto one test concentration (14 J.lg atrazine/L).

The microcosms were maintained on a 8:16 hour light:dark cycle photoperiod, whereas 12:12 hourand 16:8 hourlightdarkcycles are consideredmore typical.
Description of

### Use in Document: 

### Invalid

Rationale for Invalid Study Classifcation: 

### The study is classified as invalidbecause the concentration ofacetone solventis 5 times higher thanthe maximum concentrationrecommended for flow-through systems. According to the established criteriafor acceptance andreview ofatrazine aquatic micro/mesocosm open literature studies, a study shouldbe classified as invalid ifthe amount ofsolvent in a flowthrough study exceeds 0.1 mLiL. In addition, the lackofa solventcontrol confounds the ability to discriminate treatment-related effects and adds further uncertaintyregarding the interpretation ofresults relative to anegative control withno added solvent.

Limitations ofStudy: 

### There were numerous reporting and measuring deficiencies. Purity ofthe test compoundwas notreported. As noted above, the most serious flaw ofthe study is the amount ofsolventinthe treatment groups (5 times abovethe recommended maximum concentrationfor flow-through systems) with no associated solventcontrol. No effortwas made to documentwhether chemicals other than atrazine were presentinthe treatment and/or control microcosms priorto study initiation. It

### was not established whetherthe natural periphyton and snails collected forthe study hadpriorexposure to atrazine. Waterquality monitoring was

- 16

-

### limited: microcosm waterwas nottested for turbidity, alkalinity, or organic carbon; dissolved oxygenwas monitored, butnotreported.


### Primary Reviewer: Lynne Binari, M.S., Subcontractorto Cambridge Environmental Inc. 

### Secondary Reviewer: Catherine Aubee, Biologist, Environmental Fate and Effects Division U.S. Environmental ProtectionAgency 

# G')t--. I; ,Jv,'YI,,,L 

## ,JOI/ Tertiary Reviewer: AnitaPease, M.S., SeniorBiologist, U.S. EPA

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### Attachment 1: Correspondencewith Lead Author (Dr. Isabel Munoz)

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History: Re: atrazine and aquatic grazers Isabel Munoz to: CatherineAubea This message has been replied to. 1012012009 11 :20 AM

Dear ('::at.herine, The a'C.r:azine used Has supplied by l1akhteshim S.A. a manuf3cture.r: er.treprise. Puridity was higher 0'0% but I do ne.t remember exactly the percentage. We**di~~olved** the atrazine in water by sonication ;10-15 min) in order to**a\~·Jid** tht:': effects of the**ac·~,t,:)ne.** \111'2 solve 3mall**q1Ja~'ltiti~.s** <Jf pcn..,Jer in 3. IJOO rnl o.f Hi.lli Q**wat~3l:. t';h\~n** **da~:** Bolved we add a**littlt:.~ nlO::'C~**  and ':=:0 011. It. was really hard but we have not Ihore channels to test the effect (If acetone alc

..ne. I hope I helped you .

.**~in("';e}·~ly,** 1"")-,,,1 Ht;:";"" iriqinal**M€ssag~** ----- FU.'m: <Aubee. Cather**inel~epamai** 1. epa. qov> To: <munoz l<pcrtho3. t·io. 'JI:

.. os> Sent: Wednesday, October 14, 2009 2:23 PM Sl.lbject: atrazine,and aquatic grazerE

> [lO;?,3.r IsaJ:>el, > > I dlO \'l.riting In Le9a.rd to the study de;3Cl:ibed :-n iiO\J.2:.. 2001 a.rticle > "Effects of atLaziu@ 0]1 peLiphyton u)ideL gIaziIlg**pres~ure«** (Aquatic > T,o;-;.icolo:;1Y). Can yc·u prcvide the .souJ:ce and purity" cf the pOPl"Joe,l; ;. 3tr3zir,e used in the**e:~periment7** r.lso, were the atrazine-free > tre:3.tment.s**pr~pared** \'lith cr**~"i** th,;ju't.**aceto~ie?**
> t-1any t.hank.'3, > '> Catherin.e Aubee > Biologist

- OPpiEFED > > Environmental Prctection Agency > ()ne Pot.ornac Yard

2777 S. Crystal Drive Arlington, VA 22202

> Mail Code: 7507-2

> Email:**aubee~catherine@epa.'-J()v** '-' I?hone: (·'03) 347-3029

Fa:..: ('103)**:;d)~;-t)204**

>



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