### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Hamilton, P.B., G.S. Jackson, N.K. Kaushik, K.R. Solomon, and G.L. Stephenson. 1988. The impact oftwo applications ofatrazine on theplankton communities ofin situ enclosures. Aquat. Toxico!. 13: 123-140 (MRID 47543617).



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



### Date ofReview: November 24, 2009



### Purpose ofStudy: This study examines the potential effects oftwo atrazine applications to aquatic plants and animals (including species interactions and community stability) in experimental lake enclosures (limnocorrals).



### Summary ofStudy Findings:

Methods: The basic experimental design ofthis study is also reported in Hamilton, 

### etal. (1987) (1983 study), Hamilton, 

### etal. (1988), andHerman, 

### etal. (1986).

Lake enclosures were constructed in Lake St. George, Ontario, Canada, a 10-ha mesotrophic dimictic lake surroundingby agricultural land. (Background levels of atrazine in the lake are not described.) The experimenttookplace during the ice-free time between May 1983 and April 1984. The six nylon-reinforced polyvinyl chloride (PVC) enclosures (5 m x 

### 5 m x 

### 4.5 m deep) hadbeenpreviously installed (two sources
are referenced for descriptions; itis notmentionedwhetherthe enclosures reach the lake bottom). Atrazine was applied (product grade, manufacturer, and number oftreatment enclosures used 

### vs. controls not mentioned, though the lattercanbe inferred as 3 controls and 3 treatments from some ofthe article's tables) intwo pulses of0.1 mg/L (100 !!g/L),

on June 1, 1983 and 35 days lateron July 6, 1983, by spraying the water surface with a



# III1II1III 2087860

### hand-pumped backpackapplicator (two sources are cited for details, one examiningthe mixing process in such applications in enclosures). The study ended 323 days afterthe first treatment. To maintain nutrient levels, HP03 

### and NaN03 

### were added weekly (Hamilton, 

### etai., 1987, is cited for details). One smallmouthbass was added to each enclosurepriorto the first atrazine application to control the planktivorous fish population (this addition is not mentioned in the 1986 or 1989 papers by Hamilton, 

### et ai.). The study was terminated 323 days afterthe first atrazine application.

Baseline samples were collected weekly beginning 3 weeks before the first atrazine treatment. Hamilton, 

### etai. (1987) is cited for waterquality and atrazine sampling and analysis method descriptions. Phytoplankton were collected using a 25 mm (outside diameter) x 

### 3.5 m (long) integratedplankton sampler, with 8 samples collected from each enclosure and combined andthoroughly mixed before subsampling. I 

### l5 mL subsamples

were taken and preserved with 1 mL ofLugol's solution, then counted in the laboratory using Utermohl's inverted microscope technique. A minimum of300 cells were counted from each subsample, and cell dimensions were measured for 1-9 individuals ofeach species. The cell counts and mean dimensions by species were used to estimate population numbers and total fresh weightbiomass ofeach species (a 1976 MSc thesis from the University ofWaterloo is cited for details). Zooplankton samples were collected (for description ofthe procedure, a 1986 paperwith two ofthe same coauthors

as the presentpaper is cited). There were 21 sampling dates in total.

Statistical analyses ofthe data for each sampling day were performedusing Student's t 

### test, with significance detected forp-values < 

### 0.05. When biological data were not meeting the assumptions ofnormality andhomogeneity ofvariance well, they were transformedby 10glO(X+1) (tests fornormality and homogeneity ofvariance are not described). Differences in the community composition between enclosures were comparedusing the Morista coefficient ofassociation (MCA) (a source is cited, butthe MCA is not defined in the article).

Results: The results presented in the article are notredundantwith any results described in the related articles, Hamilton, 

### etal. (1987) (later study), Hamilton, 

### etal. (1989), and Herman, 

### etai. (1986).



### Atrazine concentration measurement data are notprovided.

The phytoplankton community changed seasonally. In the spring (May), diatoms 

### (Cyclotella stelligera, Fragiiaria crotonensis, Synedra spp.) dominated, followed by a
summer (mid-June through September) shiftto green algae 

### (Tetraedron minimum, Oocystis iacustris, Eiakatothrixgeiatinosa), andtwo additional taxa 

### (Lagerheimia quadriseta, Sphaerocystis schroeteri) became prominentin the fall. Table 1 lists the dominanttaxa identified in the experiment.

2

### Figure 1 shows the total number ofalgal taxa found in the samples between Day -20 and Day 114. The control enclosures always hadmore algal taxathan the atrazine treatment enclosures, butthe differences were only significant (p<0.05) on Day 27,98, and 114. In terms oftotal cells numbers, the treatments were often lowerthan controls, butthe differences were never significant (Figure 2). Fresh weightbiomass, however, dropped 47-82% in the treatment enclosures afterthe second atrazine pulse, with significant (p<O.05) differences on day 77 (Figure 3).

The green algae were reduced after atrazine treatment (see Figure 2, Figure 3). Total green algaenumbers were significantly (p<0.05) reduced 12 and 26 days afterthe second atrazine application. Fresh weightbiomass was significantly (p<0.05) reduced on Day 27,49,63,77, and 114, with Chlorophytabiomass at levels >93% reduced compared with controls between Day 49-77, but recovery was noted the following spring (Day 323). Species-specific significant (p<0.05) declines were noted in: E. 

### gelatinosa (Days 13,27,63,77,98,114), S. 

### schroeteri (Days 27,77,114), 

### T minimum (Days 27, 49, 63, 114), and 0. 

### lacustris (Day 27). The typically summergrowing species L. 

### quadriseta

was delayed in its growth in the treatmentenclosures, establishingprominentpopulations about47 days laterthan in controls. No differences between control and atrazine exposed populations of

### Scenedesmus acutiformis were noted. See Figure4.

Dinoflagellate (Pyrrhophyta) numbers were reduced after atrazine exposure, with significant (p<0.05) differences on Day 1,49,63, and 77 (Figure 2). 

### Gymnodium spp.
were especially affected, with significantreductions in numbers on Day 13,27,49, and 77 (Figure 5). Dinoflagellatebiomass was significantly (p<0.05) lowerthan controls on Day49, 77, 98, and 114 (Figure 3). Generally, the dinoflagellates hadthe lowest numbers andbiomass ofany phytoplankton group, and there was high variability in their data.

The Chrysophytacommunity, excluding diatoms, consisted mostly ofsmall unidentifiable monads. Although theirbiomass andnumbers tendedto be lowerin the treatment enclosures, no significant differences were found (Figure 2 and Figure 3). Diatom (BaciIlariophyceae) cell densities were lowerin the treatment enclosures from Day 27-114, but again, no differences were significant (Figure 2). Reductions in diatom biomass, noted only afterthe second atrazine treatment, were significantfrom Day 49-77, butrecovered by Day 114. 

### C. stelligera, F. 

### crotonensis, and

### Synedra spp. were dominant Bacillariophyceae species; see Figure 4.

Cryptophyta, in contrast, briefly increased in the atrazine enclosures: numbers increased

on Day 13 (Figure 2), and biomass on Day 13 and 22, although no differences were observed afterthattime. 

### Rhodomonas minuta appearedresponsible forthe increase (Figure 5), with some contribution from 

### Cyptomonas erosa (including 

### C. erosa var.

3

### reflexa). The next spring, Day 323, the Cryptophyta levels were lowerin the treatment enclosures.

In zooplankton, the seasonal pattern ofrotifer growth was similarin control and treatment enclosures (Figure 6). 

### Kellicottia longispina was dominant in the spring, giving way to 

### Conochilus and

### Polyartha in the summer, which both remainedpersistentfor the rest of the study, though in the fall 

### Cohochilus spp. showed some decline and

### Kellicottia bostoniensis became prominent. Mean number ofRotiferataxa was generally somewhat lower in the treatment enclosures, butthe difference was only significant (p<0.05) on Day 63 (Figure 1). Ofthe microzooplankton, no differences were found in the speciesspecific numbers of

### Keratella cochlearis, K. 

### longspina, K. 

### bostoniensis, or 

### Chromogaster spp., 

### Polyartha spp. hadhigher numbers in the treatment enclosures on Day 62, and 

### Conochilus spp. increaseduntil Day 98 in the controls but only until Day 62 in the treatments, then declined significantly (p<0.05) in the treatments. See Figure 6.

The crustaceans, like the rotifers, had similar seasonal patterns in the control and atrazine treated enclosures. 

### Bosmina longirostris and

### Diacyclops bicuspidatus were dominant in the spring, while 

### Ceriodaphnia lacustris and

### Daphniagaleata mendotaebecame dominantfilter feeders in late June through the fall, and

### Diaptomus oregonensis was the dominant cyclopoid in June through late fall. Little effect ofatrazine treatmenton the number ofmacrozooplankton (Crustaceae) taxa, although the reduction from treatment
on Day 77 was significant (p<0.05) (Figure 1). Cladocerapopulations were consistently 46.6% lowerin the atrazine exposed enclosures afterDay 13, but no differences were significant. The reduced cladoceran numbers were due to significant (p<0.05) declines in 

### B. longirostris (Figure 6), which began showing reductions afterDay 13 and had significantreductions on Day48, 62, and 76. Atrazine did not have a noticeable effect on the total numberofcopepods, but

### D. oregonensis was consistently less abundant in treatmentenclosures after Day 48. Immature crustacea (copepodites andnauplii) were not initially affected by atrazine, but at Day 114 there were significantly fewer immature zooplankters in the treatment enclosures, and a similardifference was found the following spring (Day 323).

Morista coefficients ofassociation (MCA) are presented in Table 2 for the phytoplankton communities. The control corrals did not have very similarcommunities throughout the experiment, although they became somewhatmore similar from Day 77-114. Controls C2 and C3 had the most dissimilarity, while C 1 was initially similarto C2 (until Day 27) and then became more similarto C3 (Day 27-63). The atrazine treated enclosures had

more similarcommunity assemblages to each other, with A2 and A3 consistently similar until Day 114, and Al showing some divergencc after Day 63. Differences between controls and treatments manifested aftertreatment, with large differences after the second atrazine treatment especially (MCA from 0.21-0.36 afterDay 27), but differences after the first treatment and before the secondtreatment were less dramatic than within-control group differences.

4

### MCA for the microzooplankton (Rotifera) are presented in Table 3. These communities remained similaramong the control enclosures through Day49, with enclosure Cl diverging afterthat. The atrazine-treatedenclosures remained similarthroughoutthe experiment. There were no cleardifferences betweenthe atrazine-treated and control communities, since there was no consistenttrend, and lowermean MCAs occurred only
on Day 63, 98, and 125 (of0.64, 0.52, and 0.57, respectively)with atleastone enclosure being highly dissimilaron each ofthese days.

Macrozooplankton communities were variable in how similarthey were (Table 4). The controls were similaronly until Day I, while the atrazine-treatedenclosures were similar until Day 77, afterwhich A1 diverged from the othertreatments. The control and atrazine-treated enclosures differed from one anotherconsistently, though not dramatically, afterDay 63

.



## • 1 r" r I 1 I**~r-r--"r--Ti--"T--**

20 40 60 80 100 120 DAV$ AFTER TREAT

.....ENT

Figure 1. Mean number ofphytoplankton, rotifer, and crustacean species enumerated in sample counts. Vertical bars representone standard deviation for each sample date. Solid line=Controls, Dashed line=Atrazine treated enclosures. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 1)



### 5TorAL

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-20 -6 13 27 "'9 63 77 98 1I4323 ----1.1 I J I**j-+--A-"'+---S-'~A** DAyS AFTER TREATMEN T H9B31

Figure 2. Mean numbers oftotal phytoplankton, Chlorophyta (3-8 taxa), Chrysophyta (2-10 taxa), Bacillariophyceae (0-11 taxa), Cryptophyta (2-8 taxa), and Pyrrophyta (0-3 taxa). Solid bars=lake stations (not described in the article text), Blankbars=controls, Slashed bars=atrazine enclosures. Numbers are log transformed with errorbars representing 2 standard deviations. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 2)

630

.

V, l/',; <t :;**~,0-":** c) m**20-~**

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ID CR'rPTOPHYTA

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-20 -6 Il 27 491 lH 77

-- t.'--"_.•-t---J ---------l---J

__1\----1- ()n'l'S lonER 1 Rl:Ar"'tNI (''183J

Figure 3. Mean fresh weight biomass for total phytoplankton, Chlorophyta, Chrysophyta, Bacillariophyceae, Cryptophyta, and Pyrrophyta are presented for outside (solid), control (blank), and atrazine (slash) enclosures. Biomass data are log transformed with errorbars representing 2 standard deviations. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 3)



### 7CONTROL ATRAIINI::**f~M~~10~H~~~ GtLAlr~I~~** 

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Figure4. Mean numbers for the common phytoplankton (Elakatothrixgelatinosa, Sphaerocystis schroeteri, and Tetraedron minimum, top; Oocystis lacustris, Lagerheimia quadriseta, and Scenedesmus acutiformis, middle; and eyclotellastelligera, Fragillaria crotonensis, and Synedra spp., bottom). Vertical bars represent one standard deviation for each sample date. Control populations
are on the left and atrazine-exposed populations are on the right. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 4)

8

# :**~,t>-<~~+/i** '5
2 CONTROL ATRAZINE 

# ==**1iAd;~8;;;·t1fr.B'~** :>

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-20 0 30 GO SO t:?l) -Zll " :SO ftO 90 120



# .. * DAYS AFTEH

### TR~ATME~T

Figure 5. Mean numbers for the common phytoplankton (Cryptomonas erosa andRhodomonas minuta, top, and Peridinium sp. and Gymnodinium spp., bottom). Vertical bars represent one standard deviation for each sample date. Control populations are on the left and atrazine-exposed populations are on the right. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 5)

9Figure 6. Mean numbers for the common zooplankton (Keratella cochlearis, Kellicottia longispina, and Polyartha spp., top; Kellicottia bostoniensis, Chromogasterspp., and Conochilus spp., top-middle; Daphniagaleata mendotae, Bosmina longirostris, and Ceriodaphnia lacu.stris, bottom-middle; and Diacyclopsb. thomasi, Diaptomus oregonensis, andTropocyclopsp. mexicanus, bottom). Vertical bars represent one standard deviation for each sample date. Control populations are on the left and atrazine-exposed populations are on the right. Single arrow represents the first atrazine application, double arrow the second atrazine application. (study figure 6)

10Table 1. The common plankton taxa encountered throughoutthe experiment. a=very common, "statistically affected" after atrazine exposure, b="A weak statistical effect", and c=no apparent effect. (no significance results mentioned in original caption) (study table I)

Phytoplankton Green algae (Chlorophyta) Elakatothrixgelatinosa Wille a Lagerheimia quadriseta (Lemmermann) G.M. Smith b Ooeystis laeustris Chodat a Seenedesmus aeuttformis Schroeder b Sphaeroeystis sehroeteriChodat a Yellow flagellates (Chrysophyta) Bitriehia ehodatii(Reverdin) Chodat Chromulina minuta Doflein Dinobryon divergens Imhof Dinobryon soeiale Ehrenberg Mallomonas sp. Monosiga sp. Pseudokephyrion spp. Salpingoeeafrequentissima (Zacharias) Lemmermann Diatoms (Bacillariophyceae) Cyclotella stelligeri (Cleve et Grunow) Van Heurck b Fragilaria erotonensis Kitton b Synedra sp. b Cryptoflagellates (Cryptophyta) Cryptomonas caudata Schiller c Cryptomonas erosa Ehrenberg c Cryptomonas erosa var. rejlexa Marsson c Cryptomonas ovata Ehrenberg Rhodomonas minuta Skuja c Dinoflagellates (Pyrrophyta) Gymnodinium spp. a Peridinium cf. inconspieuum Lemmermann Zooplankton Rotifers (Rotifera) Chromogaster spp. Conochilus spp. c Kellicottia bostoniensis (Rousselet) c Kellicottia longispina Kellicott c Keratella cochlearis (Muller) c Cladocerans Bosmina longirostris (O.F. Muller) a Ceriodaphnia laeustris (Birge) c Copepods Daphniagaleata mendotae Birge c Diacyclops bicuspidatus thomasi S.A. Forbes c Diaptomus oregonensis Libbjeborg b Tropocyclopsprasinus mexicanus Kiefer c

11Table 2. Comparisons ofMorista coefficients ofassociation for the phytoplankton communitywithin the control (Ct, C2, C3) and atrazine (At, A2, A3) enclosures, and between control and atrazine enclosures (mean, minimum, and maximum shown). (study table II)

Control Atrazine Control vs. Atrazine Cn=9i Days CI-C2 CI-C3 C2-C3 AI-A2 AI-A3 A2-A3 Mean Min. Max.

-20 0.99 0.99 0.97 0.77 0.99 0.79 0.90 0.69 1.00 -6 0.64 0.57 0.85 0.97 0.94 0.93 0.85 0.6H 0.96 I 0.50 0.83 0.37 0.62 0.65 0.94 0.63 0.22 0.86 13 0.78 0.32 0.08 0.67 0.58 0.70 0.58 0.17 0.87 27 0.58 0.38 0.52 0.66 0.62 0.33 0.54 0.35 0.81 49 0.01 0.26 0.01 0.46 0.80 0.57 0.21 0.02 0.72 63 0.13 0.71 0.28 0.81 0.85 0.93 0.36 0.18 0.67 77 0.14 0.90 0.15 0.39 0.42 0.96 0.18 0.09 0.25 98 0.36 0.56 0.40 0.48 0.11 0.64 0.24 0.03 0.72 114 0.04 0.08 0.80 0.52 0.50 0.17 0.21 0.01 0.47

Table 3. Comparisons ofMorista coefficients ofassociation for the rotifera community within the control (Ct, C2, C3) and atrazine (At, A2, A3) enclosures, and between control and atrazine enclosures (mean, minimum, and maximum shown). (study table III)

Control Atrazine Control vs. Atrazine Cn=9i Days Ci-C2 Ci-C3 C2-C3 Ai-A2 Ai-A3 A2-A3 Mean Min. Max.

-20 0.98 1.00 0.96 0.99 0.94 0.90 0.95 0.85 0.99 -6 1.00 0.84 0.80 1.00 0.74 0.76 0.89 0.67 0.99 1 0.92 0.69 0.91 0.99 0.96 0.93 0.91 0.73 0.99 13 0.97 0.67 0.81 0.96 0.86 0.69 0.77 0.37 0.99 27 0.98 0.89 0.96 1.00 1.00 1.00 0.95 0.87 1.00 41 0.65 0.49 0.75 0.99 0.61 0.68 0.71 0.42 0.99 49 0.98 0.79 0.89 0.92 0.70 0.75 0.81 0.62 0.99 63 0.54 0.28 0.51 0.41 0.36 0.97 0.64 0.19 0.99 77 0.64 0.55 0.79 0.91 0.94 0.99 0.80 0.53 0.97 98 0.41 0.96 0.62 1.00 0.87 0.87 0.52 0.23 0.92 114 0.32 0.70 0.91 1.00 0.94 0.95 0.81 0.63 0.91 125 0.75 0.88 0.81 0.95 0.95 0.95 0.57 0.18 0.94 139 0.63 0.34 0.74 0.91 0.84 0.96 0.68 0.45 0.90

12Table 4. Comparisons ofMorista coefficients ofassociation for the crustacean community within the control (CI, C2, C3) and atrazine (AI, A2, A3) enclosures, and between control and atrazine enclosures (mean, minimum, and maximum shown). (study table IV)

Days Control Atrazine Controlvs. Atrazine (n=9) Ci-C2 Ci-C3 C2-C3 Ai-A2 Ai-A3 A2-A3 Mean Min. Max. -20 0.96 0.87 0.80 0.68 0.99 0.65 0.86 0.71 0.99 -6 0.86 0.78 0.99 0.94 0.99 0.89 0.91 0.70 0.99 1 0.89 0.87 0.91 0.99 0.95 0.93 0.94 0.88 1.00 13 0.31 1.00 0.31 0.98 0.90 0.85 0.78 0.34 1.00 27 0.67 0.95 0.47 0.93 0.73 0.61 0.75 0.43 0.97 41 0.90 0.52 0.69 0.87 0.90 0.99 0.80 0.48 0.98 49 0.96 0.62 0.78 0.88 0.96 0.83 0.70 0.16 0.96 63 0.92 0.33 0.56 0.70 0.53 0.68 0.68 0.22 0.96 77 0.40 0.64 0.86 0.34 0.36 0.42 0.47 0.08 0.90 98 0.72 0.81 0.61 0.13 0.10 0.92 0.46 0.07 0.88 114 0.94 0.29 0.48 0.16 0.07 0.99 0.55 0.06 0.94 125 0.90 0.62 0.51 0.04 0.03 0.01 0.39 0.02 0.84 139 0.58 0.90 0.71 0.31 0.08 0.94 0.49 0.20 0.88

13

### Data Summary Table: Effect Magnitude of Nominal Day of Time Did Comments Effect Atrazine StudyEffect Weighted Recovery relative to Concentration Occurred Average Occur

- 

### if controls (flg/L) Atrazine so. when Concentration**(~ee** from Day 0 to comment)?

When Effect Occurred Change in

, phytoplankton Yes (the community >93% following From text, composition (signiticant 100-200 I-lg/L Day 49 130 flg/L spring, p.128. (decrease in at p<0.05) green algae Day 323) biomass) Decrease in From total numberof 20% lOOllg/L Day 1 1001lg/L No Fig.l and phytoplankton text, p. taxa 126. Change in numberof No effect 100-200 flg/L NA NA NA From zooplankton Fig.l taxa Change in rotifer No effect 100-200 Ilg/L NA NA NA From community Table 3. composition Change in crustacean zooplankton 45% (not community statistically 100 Ilg/L Day 13 100 Ilg/L No From text, composition significant) p. 133 (decrease in Cladocera numbers) Decrease in NR From text, immature (significant 100-200 Ilg/L Day 114 169.6Ilg/L No zooplankton at p<0.05) p. 133
Note: NA=NotApplicable, NR=Not Reported Bolded values are those datapoints specified in the "Cosm DataPoints and Exposure Profiles" spreadsheet.

Since no atrazine measurements were reported, itwas assumedthatno degradation occurred and concentrations were always nominal (100 /-lg/L on Day 0, 200 /-lg/L starting Day 35). Time weighted average concentrations were then computed as the cumulative average over all elapsed days; see linked Excel table below. Note that Herman etaf. 1986 and Hamilton, etaf. 1989 papers provide atrazine concentrations and may be used to provide more specific TWA's.

14Day Cone TWA 0 100 100.0 1 100 1000 2 100 1000 3 100 100.0 4 100 1000 5 100 100.0 6 100 100.0 7 100 1000 8 100 1000 9 100 1000 10 100 100.0 11 100 1000 12 100 100.0 13 100 100.0 14 100 1000 15 100 1000 16 100 1000 17 100 1000 18 100 1000 19 100 100.0 20 100 1000 21 100 1000 22 100 1000 23 100 1000 24 100 1000 25 100 1000 26 100 100.0 27 100 100.0 28 100 1000 29 100 1000 30 100 100.0 31 100 100.0 32 100 1000 33 100 100.0 34 100 100.0 35 200 1028 36 200 105.4 37 200 1079 38 200 110.3 39 200 1125 40 200 114.6 41 200 1167 42 200 118.6 43 200 1205 44 200 122.2 45 200 1239 46 200 1255 47 200 127.1 48 200 1286 49 200 130.0 50 200 131.4 51 200 1327 52 200 1340 53 200 1352 54 200 136.4 55 200 1375 56 200 1386 57 200 1397 58 200 140.7 59 200 1417 60 200 142.6 61 200 1435 62 200 1444 63 200 1453 64 200 1462 65 200 1470 66 200 1478 67 200 1485 68 200 1493 69 200 1500 70 200 150.7 71 200 1514 72 200 1521 73 200 1527 74 200 1533 75 200 1539 76 200 154.5 77 200 155.1 78 200 1557 79 200 1563 80 200 1568 81 200 1573 82 200 1578 83 200 1583 84 200 1588 85 200 1593 86 200 1598 87 200 1602 88 200 1607 89 200 161 1 90 200 1615 91 200 162.0 92 200 162.4 93 200 162.8 94 200 1632 95 200 1635 96 200 1639 97 200 164,3 98 200 1646 99 200 165.0 100 200 1653 101 200 1657 102 200 1660 103 200 1663 104 200 1667 105 200 167.0 106 200 1673 107 200 1676 108 200 167.9 15 109 200 168.2 110 200 1685 111 200 1688 112 200 1690 113 200 1693

### Reviewer Comments: This study provided arelatively comprehensive examination ofthe phytoplankton and zooplankton communityresponses to the two pulses ofatrazine in the lake enclosures. As in the otherarticles on the same enclosure experiment, although only one atrazine treatment level is examined, 3 replicates for treatments and controls in a semi-natural experimental setting provide a somewhatreasonable sample size for statistical analysis (i.e., more than 2 replicates found in many ofthe 'cosm studies). In addition, it is unclear whether atrazine was present in the controls as no information was provided on surrounding landuse orbackgroundpesticide concentrations in Lake St. George where the enclosures were located. As inthe other studies publishedby Hamilton, there is some
cause for concern regarding enclosure effects. In this article, bargraphs ofbiomass and population numbers for the majorgroups ofplankton also seem to provide open-water sample analyses, which for the diatoms and dinoflagellates show much stronger differences between open and enclosed samples than between atrazine-exposed and nonexposed samples. Since, at least in these particularvariables, atrazine effects were outweighedby enclosure effects, otherparts ofthe study (which do not compare to openwater samples) may be compromisedby the confounding effects ofenclosure.

Anotherissueraised by information inthis study thatis notprovided in the other Hamilton, 

### etal., studies is highertrophic level effects causedby the presence of planktivorous fish. The methods state that one smallmouth bass was addedto each enclosure to control these populations; otherwise, there is no mention ofthe fish, and the other studies fail to mention fish anywhere. It 

### is unclear whetherthe enclosures were similarin terms ofthe number ofplanktivorous fish present during pre-treatment and whetherthe planktivorous population changed with treatment.

The following presentation deficiencies in the methods and results sections were noted:

no background atrazine concentrations are provided, norare data ofmeasured atrazine concentrations in thetreatment enclosures provided; zooplankton sampling was not described; the Moristacoefficient ofassociation was not defined; and the species list in Table 1 is incomplete, missing 

### Tetraedron minimum and

### Polyartha sp., which are both listed in the Figures (4 and 6, respectively) and inthe body text. In particular, the openwater samples displayed inthe bargraph figures, which provide insights into the potential limitations ofthe experimental methodology, are not described in the methods and so their data cannotbe precisely interpreted.



### Description ofUse in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: Results confirm a shift in phytoplankton community structure following two applications ofatrazine (35 days apart) at 100 Ilg/L. Significantdeclines in the Chlorophyta community were observed, beginning 13 days after atrazine

16application. Recovery ofthe phytoplantkon community structure did notoccuruntil 323 days afterthe first atrazine application.



### Limitations ofStudy: Enclosure effects, evidentin diatom and dinoflagellate populations (biomass, numbers), may have influenced othervariables for which no openwater samples were analyzed. Only one treatment level was tested andthere is a lackof data on the potential presence ofatrazine in the control enclosures. Highertrophic level endpoints were not analyzed, although feedbacks with planktivorous fish populations

were possible.



### Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc



### Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental Protection Agency.

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