### Open Literature Review Summary
Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Herman, D., 

### N:K7Kausnik,**and-K:R~Soiomon.** 

### N8°6-:-Impactofatr:fzine on periphyton.------- in freshwater enclosures and some ecological consequences. Can. J. Fish. Aquat. Sci. 43:1917-1925 (MRlD 47543502).
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: November24, 2009
Purpose ofStudy: This study examines the potential effects ofatrazineto aquatic plants (periphyton) in experimental lake enclosures (limnocorrals).
Summary ofStudy Findings:

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

### etal. (1987), Hamilton, 

### etal. (1988), and Hamilton, 

### etal. (1989).

Six enclosures (limnocorrals) were constructed in Lake St. George, a 10.3-ha mesotrophic lake in southern Ontario, Canada (two otherarticles headed by a co-author are cited for details). The enclosures were 5 m x 

### 5 m x 

### 5 m deep, and made ofa single piece ofyellow nylon-reinforcedpolyvinyl chloride (PVC), supported on the water surface by floats and anchored firmly in the sedimenton the bottom. Three limnocorrals were used as controls and three were treated with atrazine.

The enclosure walls were lowered on May 5, 1983, and remained in place until July 1984. Periphyton substrates were colonized for 3 weeks beforethe first atrazine treatment. Two atrazine treatments occurred, on June 1, 1983 and July 6, 1983 (Day 35). Atrazine (80% activeingredient, manufacturernotprovided) was applied to the water

1surface using abackpack spray system to give anominal concentration of 100jlg/Lwith each application. Complete mixing ofthe atrazine withinthe enclosures occurred within 24 hours. Atrazine concentrations measurements were carried out (described in a separate paper). Due to lack ofwater exchange with the lake fornutrientrenewal, weekly nutrient additions were applied to the enclosures at arate equivalentto 6.5 g N/m2 /yr (as sodium nitrate) and 0.5 g P/m2 /yr (as phosphoric acid).


### ------PeriphytOTcco+oni-zatiol1 :substrates-were**made-of-6:-§-Gin-x-4-Gm-s~r-ips-6Hhe-same~P-VGu-------­** material used for the enclosures, with two strips attached to aholderusing silicone glue. A holderwas made ofthree 2.5 cm x 10 cm microscope slides attached along their short edge to a 20 cm segment ofglass tubing using silicone glue. A sampling unit was created by tying four such holders togetherwith nylon string, spaced at 0.5, 1.5,2.5, and 3.5 m depths. Twelve sampling units were placed in each enclosure, and one unitwas randomly selected and extracted on each sampling day. Sampling occurred on Days 1, 5, 14,24,34,37,42,54,68,96;137, and 329.

Forperiphyton sampling, one strip from aholderat each depth was immediately preserved in Lugol's solution to beusedto direct enumeration. Using a scalpel, periphyton from the second strip was scraped into distilled water and mixing until homogeneous in a Waring blender. Aliquots oftheresulting suspension were analyzed for chlorophyll and organic matter content.

To determine organic matter content, samples were filtered onto apreashed, preweighed Whatman GFC (2.4-cm diameter) filter and dried at 70°C for about 24 hours, then weighed, the ashed at 500°C for 1 hour and reweighed. The dry weightminus the ashed weight gave the ash-free dry weight (AFDW), used as an estimate oforganic matter. For' chlorophyll analysis, the samples were filtered onto Whatman GFC filters and frozen until extraction. Chlorophyll extraction followed the published methods ofMarker, et

## at. (1980) and Arvola (1981), and spectrophotometric analysis ofchlorophyll 

### a with a Bausch and Lomb Spectronic 88 used the equations and procedure given by Nusch (1980) (see article for full citations; all are in peer-reviewedjoumals).
Periphyton algae enumeration followed the procedure ofLund, et

## at. (1958) (inverted microscope technique). The homogenized suspension samples were subsampled and allowed to settle for at least 12 hours in a 50 mL phytoplankton sedimentation chamber. At least 300 cells were countedper sample. Filamentous algae had been broken into shortchains by theblending, and individual cells were counted inthese. Forthe more intactCyanophyta filaments, number ofcells was estimatedusing filament length and average cell size. Length, width, and depth were measured on arandom selection of3-6 cells for each species, total species volume was calculated assumed a simple geometric solid form ofthe cells, and fresh weightbiomass was estimated from volume assuming a cell density equivalentto the density ofwater.

2, 

# ..

Periphyton photosynthetic activitywas measured in situ using 14C assimilation as an estimate ofnetprimary productivity. A microscope slide was attached on the edge ofa lid for a 250 mL glass incubation bottle, perpendicularto the inside surface, using silicone glue, and an injection portwas drilled through the lid and sealed with silicone glue. A 6.5 cm x 2.5 cm strip ofPVC was sealed to the slide, using silicone glue, for periphyton colonization, and each lid was attachedto holder, as describedpreviously. Fourholders were tied together forperiphyton sampling at 0.5, 1.5,2.5, and 3.5 m depths. 3 sets ofholders were suspended in each enclosure on May 6 and allowedto -----,c=oionize for 3Saays before the firs! productivity measurem-ent:-Be'(;cmse pro-ductivity measurements were onerous, only one control and on treatment enclosure were sampled overthree 3-day productivity sampling periods, two in the firstpulse and one afterthe second.

Forproductivity samples,water samples from the enclosures atthe appropriate depths were taken using a Kemmerer sampler and dispensed into 250 mL incubation bottles. Lids, with the attached periphyton substrate, were fitted to each bottle. The bottles were suspended at the sampling depths for 30 minutes to stabilize theirtemperature before being broughtto the surface, injected with 74 kBq of14C-Iabeled sodium bicarbonate (Amersham, Oakville, Ontario), and then lowered again and incubated for 2 hours. All incubations were carried out in the same controllimnocorral between 10:00 and 14:00. After incubation, the periphyton was scraped from the PVC strip and filtered onto a Millipore (45 /lm) filter, a drop of0.5 N HCI was added to eliminate any retained radiolabeled bicarbonate, andthe filters wereplace in a scintillation bottle containing 3 mL of2-methoxyethanol and 12 mL ofAquasol-2 (New EnglandNuclear). Radioactivity was measured in a PackardTri-Carb 460C liquid scintillation systemusing the sample channel ratio method to correct for quenching due to detritus or chlorophyll.
During oneperiphyton productivity measurement, phytoplankton samples from four depths were incubated for 3 hours in 500 mL bottle atthe same time as the periphyton, after injecting 111 kBq of14C

-Iabeled sodium bicarbonate through aportin the lid. The phytoplankton 14C assimilation was then measured as described for periphyton.
Lightreadings were taken with a Li-Cor spherical quantum sensor (Li-1935b) sensitiveto photosynthetically activeradiation (-400-700 nm) andrecorded on aLi-Cor (Li-51OB) data integrator. Carbon assimilation was calculatedusing methods described in the manual ofVollenweider (1969).

Waterquality measurements were taken weekly. Temperature and dissolved oxygen profiles were sampled using a YSI model 51B (Yellow Springs InstrumentCo.) dissolved oxygen meter. Water samples were collectedusing a depth integrated samplerand analyzed for Kjeldahl nitrogen, combined nitrate-nitrite nitrogen.(N03-N02-N), total phosphorus, dissolved inorganic carbon (DIC), andphytoplankton chlorophyll 

### a using the

3 

### - 

## -----standard methods ofEnvironment Canada, 1979. (Secchi depth measurementresults ar.e described in Table 1, butthe methods are not introduced.)
Analysis ofvariance (ANOVA) with pooled variance was used to analyze AFDW, chlorophyll, and carbon assimilationrates on each sampling day ormeasurementperiod, with sampling depth considered as a separate factor. For algal biomass, the means and standard errors were calculated for each sampling depth.

Results: The results presented in the article are relatedto the following articles: Hamilton, 

### etal. (1987), Hamilton, 

### etal. (1988), and Hamilton, 

### etal. (1989). The atrazine concentrations are also presented, in the mean, in Hamilton, 

### etal. (1989). Similarwater quality measures are provided in Hamilton, 

### etal. (1987) and are summarized differently (and more completely) in Hamilton, 

### etal. (1989). Many ofthe periphyton measures appear similarto those ofHamilton, 

### etal. (1987); however, the methods described in the 1987 article are different and appearto be separate (laterin the season) periphyton investigations within the same enclosures.

Atrazine measurements in the 3 treatment enclosures are shown in Figure 1. During the first pulse, concentrations ranged from 80-140 jJg/L, and afterthe secondpulse the concentration increased 20-30% in all enclosures ratherthan doubling. Water quality measures showedno significant differences between treatments and controls during the first pulse, butthroughoutthe secondpulse (before the next spring), treatments had significantly (no p-value) lower oxygen concentrations at 0.5 and 3.5 m depths, and significantly (no p-value) greater Secchi depth, DIC, and N03 -N02-N. Bythe following spring (Day 329), all parameters had recovered withthe exception ofN03-N02-N, which remained increased inthe treatment enclosures. See Table 1.

AFDW was greater in the controls than in the treatment enclosures, particularly afterthe secondpulse (see Figure 2). Differences were significant (no p-value) at 0.5 m depth on Day 34, and at 0.5 and 1.5 m depths on most subsequent sampling days.
Chlorophyll values showed no significant differences between treatments and controls until 19 days afterthe second atrazine application (Day 54), and afterthat, chlorophyll levels were consistently lowerin the treatment enclosures, though not all differences were significant (no p-value). See Figure 3. Periphyton organic matterdecreased with the sampling depth, butthere was no effect on depth on chlorophyll.
Algal groups in the periphyton'were the Cyanophyta (blue-green), Chlorophyta (green), and the Bacillariophyceae (diatoms). On the first sampling day, the p<mnate Bacillariophyceae dominated in most control and treatment enclosures (Table 2). Dominant species were

### Fragilaria crotonensis and 

### Synedra spp., with others also common including

### Achnanthes minutissima, Tabelariafenestrate, Cymbella cistula,

4

### Gomphonema angustatum, G. 

### truncatum, and

### Navicula radiosa. Afterthe spring bloom, the BaciIIariophyceae in both control and treatmentenclosures were dominated by 

### Achnanthes minutissima and 

### Gomphonema angustatum. Forthe rest ofthe summer, the Chlorophytagroup dominatedtotal algal biomass and cell numbers in both control and treatment communities. Chlorophytain the control enclosures were dominated by filamentous species, primarily 

### Oedogonium sp. and

### Bulbochaete sp. by biomass, and in the atrazine enclosures, 

### Coleochaete sp. was dominant. See Figure 4. The most drastic impact ofatrazine on the algal groups was on Cyanophyta. In the controls, 

### Oscillatoria ------s"'"'p".....-=a=n-:>aumctentille<:Cffagmentsoominateci, 

### witilToiypotftrlXsp. increasing in numiJer

## oy------- September. In the treatmentenclosures, Cyanophyta almost completely disappeared during the first pulse (Figure 5), despite no apparent impact on Chlorophytaor BaciIIariophyceae, which only decreased relative to controls on Day 42 and 96 (respectively), and then not at all depths. Cyanophyta in the treatmentenclosures remained absent for the remainder ofthe experiment. The following spring, (Day 329), Bacillariophyceae and Cyanophytahad increasedin relative abundance in the control enclosures, while in the atrazine treated enclosures Chlorophyta dominated biomass and Bacillariophyceae dominated total cell numbers. Overall biomass in the treatments did notrecoverto become similarto control levels.

Carbon assimilation measurements (Figure 6) showed no significantdifferences due to treatment in the first pulse, but significantly (no p-value) lowerrates were found during the secondpulse at 1.5 and 2.5 m. 0.5 m measurements during this time were not possible due to loss ofthe PVC strips from Days 47-49.
Periphyton and plankton chlorophyll and productivity measurements were comparedby expressing each on aper-enclosure basis (multiplying the mean value ofthe four sampling depths by the total wall surface area for the periphyton, and by the nominal volume ofthe enclosure forthe phytoplankton). Algae on the sediment surface were not sampled and are not included in the periphyton calculation. Phytoplankton chlorophyll levels were greatest in the early spring in both treatments and controls, while periphyton chlorophyll levels increased with time to reach amaximum atthe end ofthe summer. In both control and treatment enclosures, the periphyton chlorophyll values were greater than those ofthe phytoplankton after4-5 weeks ofcolonization. The loss ofthe 0.5 m PVC strips was compensated by averaging total enclosureproductivity overthe remaining depths. See Figure 7. Simultaneous productivity measurements are shown in Table 3, which according to the authors shows thatperiphyton productivity could equal orexceedthat ofphytoplankton after 10 weeks ofcolonization.

The authors mention that enclosures effects may include excessive periphyton buildup compared to natural lake conditions, and edge effects creating anonrandom distribution ofzooplankton.

5

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### 5

Figure 1. Atrazine concentration in the water ofthe three treated enclosures in the first and second atrazine pulses. Arrows indicate days ofatrazine applications (both nominally 100 IJ.glL). (study figure 1)

6

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Figure 2. Mean ash-free dry weight (AFDW) values for the periphyton in the control and atrazinetreated enclosures atfour depths in the first and second atrazine pulses. Error bars represent one standard error and arrows indicate days ofatrazine application. (study figure 2)

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Figure 3. Mean chlorophyll values for the periphyton in the control and atrazine-treated enclosures atfour depths in the first and second atrazine pulses. Error bars represent one standard errorand arrows indicate days ofatrazine application. (study figure 3)



### 8

## Atrazine

sp. sp, Tetrasporo Spirogyro Tefroedron mtl"limum OedogonLtJm sp. B Oedogonlum sp.A Mougeotio sp. Bufbochaete sp. Sf igeoelo nium polymorphum S. atfen uatum**~:J:-:"'"** »:-::-> 

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Figure4. Composition ofthe Chlorophyta community in the control and atrazine-treated enclosures in the first and second atrazine pulses. Composition is described using the mean percent contribution ofthe common Chlorophyta species (averaged overthe four depths) to total Chlorophyta biomass. Arrows indicate days ofatrazine treatment. (study figure 4)

9A. CHLOROPHYTA 

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Figure 5. Mean biomass values (natural log transformation) for the three periphytic algal groups in the control and atrazine-treated enclosures at four samplingdepths on six sampling days. Errorbars represent one standard errorfor the mean at each depth (n=3).**Atr~zine** treatment occurred on Day 

## oand 35. (study figure 5)

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Figure 6. Mean carbon assimilation rates for the control and atrazine-treated enclosures for two measurementperiods in the first atrazine pulse and one in the second pulse. Error bars represent one standard error. The second atrazine treatmentwas on Day 35. (study figure 6)



### 11.-......periphyton -phytoplankton

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Figure 7. Mean total chlorophyll values for periphyton and phytoplankton in tbe control and atrazine-treated enclosures from the day periphyton strips were placed in the enclosures. Errorbars represent one standard errorfor the mean on each sampling day (n=3). Arrows indicate days of atrazine treatment. (study figure 7)

12."

Table 1. Mean water chemical and physical values for the control enclosures (C) and the atrazinetreated enclosures (A) for the first and second atrazine pulse periods in 1983 and for the April 1984 sampling day. * = significant difference between the means (ANOVA) at the 95% confidence level (p-value < 0.05). (study table 1)
Pulse I (n=18) Pulse 2 (n=36) April 1984 (n=2) C A C A C A Oxygen (mgIL, 0.5 m depth) 9.26 9.45 9.90 7.80

### * 9.70 10.10

- 

## -- Oxygen (mg/L, 3.5 m depth) 5.67 5.45 6.54 5.20 

### * 9.70 10.10 Temperature (OC, 0.5 m depth) 18.7 19.1 23.0 23.0 6.0 6.2 Temperature (OC, 3.5 m depth) 10.5 10.0 18.6 18.3 6.0 6.2
Secchi depth (m) 3.2 3.5 3.3 4.4

### * 2.4 2.3
DIC (mg/L) 36.08 36.53 23.74 38.54 

### * 29.9 31.3
N03-NOr N (mgIL) 0.317 0.250 0.235 0.477 

### * 0.107 0.335
Total nitrogen (mg/L) 0.639 0.667 0.719 0.748 0.846 0.881
Total phosphorus (mgIL) 0.025 0.022 0.021 0.018 0.025 0.025

Table 2. Mean percentage total algal biomass and numbers for the three periphytic algal groups on six sampling days. Percentages were averaged overthe four sampling depths, and standard deviations (SD) (n=12) are also shown. The second atrazine application occurred on Day 35. (based on study table 4) Cyanophyta Chlorophyta Bacillariophyta Day Controls Atrazine Controls Atrazine Controls Atrazine Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD % biomass 1 1.3 2.3 2.5 4.5 21.1 20.5 24.8 21.2 77.7 19.3 72.7 19.9 34 1.7 2.6 <0.1 89.6 11.4 88.8 8.2 8.8 11.7 11.2 8.2 42 1.9 3.0 0 90.8 10.9 90.7 5.2 7.3 11.3 9.3 5.2 68 0.9 1.4 0 94.3 6.9 93.2 4.7 4.8 5.9 6.8 4.7 96 4.8 6.3 0 88.8 9.8 96.5 3.0 6.3 7.5 3.5 3.0 329 15.9 22.4 0 26.6 3.8 62.4 18.1 57.0 35.5 37.6 18.1 % numbers I 30.6 38.7 24.4 22.0 22.1 18.9 17.5 16.3 47.3 25.7 58.1 20.0 34 18.8 19.7 0.8 2.9 62.5 26.2 68.0 16.3 18.7 19.5 31.2 15.5 42 25.1 22.2 0 56.8 23.3 75.1 14.2 18.2 15.9 24.9 14.2 68 14.0 24.8 0 94.3 24.2 60.0 26.0 9.3 6.9 40.0 26.0 96 28.5 28.8 0 55.8 31.2 79.3 18.8 15.7 18.0 20.7 18.8 329 51.4 16.8 0 3.8 4.5 25.7 28.5 44.8 18.2 74.3 28.5

13Table 3. Total enclosure periphyton and phytoplankton netprimary productivity (mg C/hr/enclosure) for control and atrazine-treated enclosures in July (Days 47-49). Periphyton strips had colonized for 10 weeks from May 6untilJuly 18-20. (study table 3)
Controls Atrazine Periphyton Phytoplankton Periphyton Phytoplankton 1 2812 831 593 1251 :2**~2~** 4:7.4-.---603 !-OM 3 1017 2104 427 404 Mean 1719 1136 541 899

14

# · t .'.



### Data Summary Table: Effect Magnitude of Nominal Day of Time ])id Comments Effect Atrazine StudyEffect Weighted Recovery relative to Concentration Occurred Average Occur- if controls (pg/L) Atrazine so, when Concentration (see from Day 0 to comme'!t)?

When Effect Occurred Decrease in 10-20% Integrated l'eS='"IJay From dissolved (significant at 100-200/-lglL overDay 107.9/-lg/L 329 (next Table 1. oxygen p<0.05) 35-137 spring) Increase in 30% Integrated Yes-Day From Secchi depth (significantat 100-200 /-lgiL overDay 107.9/-lgiL 329 (next Table I. p<0.05) 35-137 spring) Increase in 60% Integrated Yes-Day dissolved From inorganic (significant at 100-200/-lgIL over Day 107.9/-lgiL 329 (next Table I. carbon p<0.05) 35-137 spring) Increase in 100% Integrated From nitrate-nitrite (significant at 100-200/-lgiL overDay 107.9/-lgIL No Table I. nitrogen p<0.05) 35-137 Decrease in -50% periphyton (significant From Fig.2 biomass at p<0.05 at 100-200 /-lgIL Day 36 105.2/-lgIL No and textp. (ash-free dry most depths 1920. weight) & days) Decrease in -50% (not From Fig.3 periphyton significant at 100-200 /-lgIL Day 54 116.6/-lgiL No and textp. chlorophyll p<0.05) 1920. Change in periphyton From Fig. community 100% 100-200 /-lgIL Day 42 110.7/-lglL No 5 & Table. composition 2. _(decrease in Cyanophyta) Decrease in periphyton 70% 100-200 /-lg/L Days 47-49 114.6/-lgIL NR From productivity Table 3. (NPP) Decrease in phytoplankton 20% 100-200 /-lgiL Days 47-49 114.6/-lgIL NR From productivity Table 3. (NPP) Note: NR=NotReported Bolded values are those datapoints specified in the "CosmData Points and Exposure Profiles" spreadsheet.



### Time weighted averages are computed from the information in Figure 1. The atrazine concentrations and sample days were estimated from the graph, then averaged overthe three treatment enclosures. A daily chemograph was constructed assuming linear change between each sample day. For extrapolation beyondthe last atrazine measurement day, a constantdecline atthe previous interval's rate was assumed, which resulted in negative

15concentrations afterDay 123; therefore concentrations after Day 123 were forced equal to zero. Time weighted averages were taken as the cumulative average since Day 0 for each day. Fortime-integrated effects, the time weighted averages were themselves averaged overthe days being integrated, i.e. Day 35-137, to construct an average TWA overthe appropriate interval.

See calculations in the linked-Excel spreadsheet, below. In the spreadsheet, treatment-**------a·v~erage-d-eon·een-tra'i-ons-on-s-a-mpl\.1** cl-ay,=, 'Vvere-yell-0vv

### h-i-gh-l-i-ghtecl-te-ea-se--i-n i-flt8F}301at:ieR,.,-.

-------

16**.~**

Cales:
Day Cone (,.,.) TWA INTEGRATED: 0 100.0 2-78 100 0 Days 3So137 1079 1 1028 2.78 101 Days 47-49 114.6 2 105.6 278 103 3 108.3 278 104 4 1111 278 106 5 113.9 278 107 6 116.7 -1.17 108 7 115.5 -117 109 8 114.3 -117 110 9 113.2 -117 110 10 1120 -117 110 11 110.8 -1_17 110 12 109.7 -117 110 13 108.5 -117 110 i

..**-'tu'i~--·u:1·i---I"'iu** 15 107.2 -0.11 110 16 107.1 -0.11 110 17 1070 -0.11 110 18 1069 -011 109 19 1068 -011 109 20 1067 -2.42 109 21 104 3 -242 109 22 101.8 -2.42 109 23 99.4 -2.42 108 24 97.0 -2.42 108 25 94.6 -2.42 107 26 922 -242 107 27 89.8 -2.42 106 28 87.3 300 105 29 90.3 3.00 105 30 93.3 3.00 105 31 96.3 3.00 104 32 99.3 3.00 104 33 102.3 3.00 104 34 105.3 17.50 104 35 122.8 17.50 105 36 140.3 1.00 106 37 141.3 1.00 107 38 142.i 1.00 107 39 143.3 1.00 108 40 144.3 1.00 109 41 145.3 1.00 110 42 146.3 -1.47 111 43 144.9 -1.47 112 44 143.4 -1.47 112 45 141.9 ·VH 113 48 140.4 -1.47 114 47 139.0 -1.47 114 48 137.5 -1.47 115 49 136.0 -1.47 115 50 134.6 -1.47 115 51 133.1 -1.47 116 52 131.6 -1.47 116 53 130.1 -1.47 116 54 128.7 -0.85 117 55 127.8 -0.85 117 56 127.0 -0.85 117 57 126.1 -0.85 117 58 125.3 -0.85 117 59 124.4 -0.85 117 60 1236 -0.85 117 61 1227 ·0.85 118 62 1219 -0.85 118 63 121.0 -0.85 118 64 1202 -0.85 118 65 119.3 -0.85 118 66 118.5 -0.85 118 67 117 6 -0.85 118 68 116.8 -0.85 118 69 1159 -0.65 118 70 115.1 -0.65 118 71 114.2 -0.85 118 72 113.4 -0.65 118 73 112.5 -0.85 118 74 111.7 -0.85 117 75 1108 -085 117 78 110.0 ·229 117 77 1077 ·229 117 78 105.4 -229 117 79 103.1 -229 117 80 100.8 ·229 117 81 98.5 -229 116 82 96.2 ·229 116 83 93.9 -2.29 116 84 91.7 ·2.29 116 85 69.4 -2.29 115 86 87.1 -2.29 115 87 84.8 -229 115 88 82.5 -2.29 114 89 80.2 -2.29 114 90 77.9 -2.29 113 91 75.8 -2.29 113 92 73.3 -2.29 113 93 71.0 -2.29 112 94 68.7 ·2.29 1;2 95 66.4 -2.29 111 96 64.1 -2.29 111 97 61.8 -2.29 110 98 59.5 -2.29 110 99 57.3 -2.29 109 100 55.0 -2.29 109 101 52.7 ·2.29 108 102 50.4 ·2.29 108 103 48.1 -2.29 107 104 45.8 -2.29 106 105 43.5 -2.29 106 17 106 41.2 -2.29 105 107 38.9 -2.29 105 108 36.6 -2.29 104 109 34.3 -229 103 110 32.0 -229 103**~.** ..



### Reviewer Comments: Compared to the otherperiphyton study from this experiment (Hamilton, 

### etal., 1989), this study was straightforwardin itpresentation ofmethods and results, and no severe methodological shortcomings were evident The descriptions ofthe methods used were terse, often relying on references for further description, but gave basic summaries ofall techniques. Like most ofthe various Hamilton, 

### etal., papers, Lake St. George is not**----~des_cr-i·becl-wel1~;_backgr6l:incl-lev·e-Js-q·f-atrazin'V in-t-he-Iake-are-fi0t-m.·eas·a:r:eg,a:r-1-a-t:h'e~-------­**  surrounding land use and atrazine runoffpotential is also not explained.

-The statistics used in the paper were insufficient in that tests for homogeneity ofvariance and specific ANOVA tests are not described, and "significant" results throughout the paperare not assignedp-values. One exception is Table 1, which apparently uses ap-value of

### 0.05, which can perhaps be assumed for the other "significance" statements as well. The authors attempt to compare periphyton andphytoplankton productivity on an enclosure scale; however, this analysis is not useful becausethe enclosures are artificial substrates that would notbe available in anatural lake setting.

The most fundamental problem with the Lake St. George enclosure experimentis the enclosures themselves. In this study, the PVC is described as "yellow", which implies that it may be opaque. Itis expected thatperiphyton colonization would result in opaque walls after colonization occurs. As the authors themselves mention, the periphyton buildup on the enclosure walls can lead to spatial variability in certain parameters (they mention zooplankton density). It is quite possible thatthe spatial patterns createdby the wall effects make the periphyton colonization success in the sampling unit substrates dependent on the sampling location. Sampling location may therefore be a confounding variable in the periphyton analyses, due to the nonrandom spatial patterns createdby opaque, periphyton-covered enclosure walls.


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

Rationale for Use: : Results confirmthat atrazine concentrations of 

### 100 Jlg/L inhibitedproduction ofperiphyton within limnocorrals and altered the compostion ofthe periphyton community.



### Limitations ofStudy: No p-values provided for "significant" results. Enclosure wall effects could confoundperiphyton analyses due to random locations ofthe sampling units within a spatially patterned environment.


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

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

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