Open Literature Review Summary
Chemical Name: Atrazine CAS No: 1912-24-9
ECOTOX Record Number and Citation: Hoagland, K. D., R. W. Drenner, J. D. Smith, and D. R. Cross. 1993. Freshwater community responses to mixtures of agricultural pesticides: effects of atrazine and bifenthrin. Environmental Toxicology and Chemistry. 12: 627-637. (MRlD 48261118) 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 plantcommunities and associated determination ofa Level ofConcern (LOC).

Date ofReview: September 3, 2009 Purpose ofStudy: This study examines the potential effects ofatrazine andbifenthrin on aquatic plants and animals in experimental tanks (mesocosms). Summary ofStudy Findings: This review focuses on treatments effects ofatrazine.

Methods: The study was conducted in two phases. Phase 1 (addition oflow dose applications) was initiated May 22, 1990, and lasted 14 days, and phase 2 (addition ofhigh dose applications to the same tanks used inphase 1) was initiatedJune 12, 1990, and lasted 14 days (community effects reported for day 7 only). Bothphases utilized six experimental tanks as controls and atrazineonly treatments (12 othertanks had treatments ofbifenthrin only orbifenthrinplus atrazine), each a 5,500-L fiberglass tank (1.8-m diameter, 2.2-m height), located atthe Texas Christian University (FortWorth, Texas) outdoor mesocosm facility. The tanks were filled (water depth, volume notprovided), on May 6, 1990, with water containing natural phytoplankton assemblages from apond (0.7 ha) located adjacentto the mesocosm facility and were continuously mixed throughoutthe study using an airlift mixer system (pond was sampled 'near - 1 - 

# 11111••111 2087857

### the shore', no details onhowpond water was distributed to the tanks). The tanks were stocked with bluegill sunfish (Lepomis macrochirus, 

### 10 fish pertank) electrofished from the same pond, and supplemented with zooplankton collected from a local reservoir (not further detailed). Throughoutthe study, all ponds were amended every other day with 10% ofinitial ambienttotal phosphorus and total nitrogen to prevent nutrient depletion. Tanks were allowedto acclimate for two weeks before adding pesticide.

Forbothphases, atrazine was applied via soil slurry to simulate storm run-off. Formulated atrazine (Hi-Yield, 40.8% a.i. w/v) was combined with 150 mL deionized water and 0.5 L of sandy loam soil (58.3% sand, 19.6% silt, 22.1 % clay, 2.17% organic matter) then,justpriorto treatment, was slurried with 125 mL ofpond water. The treated slurry was evenly distributed across the surface waterin the tanks viathe airlift mixing system, with turbidity appearing uniform within 10 minutes. Untreated slurried soil (no pesticide additions) was added to control tanks inthe same manner.
A factorial design was used withtreatments containing three levels ofatrazine andthree levels of bifenthrinresulting in nine treatment combinations. Phase 1 consisted ofduplicate control tanks and duplicate atrazine-treated tanks atnominal concentrations of0 f.!g/L, 20 f.!g/L and 200 f.!g/L. Forphase 2, the duplicate tanks treated during phase 1 at 0 f.!g/L, 20 f.!g/L and 200 f.!g/L were increased to 250 f.!g/L and 1,500 f.!g/L, respectively. Bifenthrin was added at nominal concentrations of0, 20, and 200 ng/L (phase 1) and 0,20, and 1600 ng/L (phase 2). Withthis design, there were four atrazine-only treatments (20, 200, 250, and 1500 f.!g/L).
Atrazine concentrations were measured at 1 hour and 12 days after the initial application (phase 1), and at 1 hour and 14 days after the second application (phase 2). Water samples were taken just below the surface near the tank center. Atrazine was extracted (method not detailed) and analyzed by gas chromatography equipped with a 63

### N electron-capture detector. The limit of detection was 5 f.!g/L.
Tanks were sampled for water quality, phytoplankton, and zooplanktontwo days priorto pesticide treatm,ent (phase 1), seven and 14 days post-treatment inphase 1, and seven days posttreatment in phase 2. Tanks were sampled for water chemistry (dissolved oxygen, total phosphorus, total nitrogen, pH andturbidity) by commontechniques. Primary productivity was measuredusing the light:darkbottle, dissolved oxygenmethod (sampling technique unspecified). Algal chlorophyll was determined using a chloroform-methanol extractionmethod (sampling technique unspecified). Watertemperature and fish mortality were monitored daily. Zooplankton samples (2 L) were collected from the surface at three locations in each tank (6 L total per tank), filtered (54-f.!m mesh Nitex netting) and the filtrate preserved with 5% sucroseformalin (w/v). Organisms were identified to genus and counted using a Wildco counting wheel and Wild M5 dissecting microscope. - 2 -

### Phytoplankton samples (250 mL) were collected, without filtration, just below the surface near the tank center and preserved with 0.5% Lugol's iodine. For community monitoring, subsamples (4 mL) were settled overnight, then ten random fields were counted using a Wild M-40 inverted microscope. Standing stock of plankton community components (nano-, micro- and macroplankton) were assessed via size fractionation by passing the sample through filters ofdecreasing porosity (200, 20 and 2 IJ.m), with the resultant material retained on the filters analyzed for total phosphorus by acidpersulfate hydrolysis and the orthophosphate malachite green method. Response variables were analyzed using analysis ofvariance (ANOVA) on individual sample dates with SYSTAT v4.0. Treatment effects were analyzed on a date-by-date basis to determine when significant effects occurred. Aprobability value (p) ::SO. 

### 1 level ofsignificance was selected to reduce the probability iftype II errors (accepting a false null hypothesis, i.e., no effect).
Results: 

### All statistical tests presented in this section are based on ANOVA results as provided by the study authors. These statistics do not distinguish between the atrazine treatment levels. Additionally, the statistical analysis was based on all atrazine treated tanks, including tanks treated with both atrazine and bifenthrin. Statements about differences between atrazine-only treatments and the control (data from only one control was presented by the authors

- the second was dropped due to bifenthrin contamination) are based on estimates by the reviewer from the study figures and have no statistical basis (i.e., no statistical tests were presented regarding differences between atrazine-only treatments and the control; furthermore, except for measurements of atrazine, no information about variation between treatment replicates was presented).
The reviewer concurs withthe study authors' statementthat community changes observed during phase 2 were the combinedresult ofeffects from phase 1 plus the subsequentincreasedtreatment levels ofatrazine.
At the 20 and 200 IJ.g/L applications rates (phase 1), measured atrazine concentrations in the atrazine-only tanks were 48-85% and 55-95% ofnominal, respectively, at 1 hour, and 35-105% and 80-85% of nominal, respectively, at 12 days (Table 1). 

### At the 250 and 1,5001J.g/L applications rates (phase 2), measured atrazine concentrations were 148-196% and 120-180% of nominal, respectively, at 1 hour, and 144-200% and 73-93% ofnominal, respectively, at day 14 (after the start ofphase 2). In the control tanks (no atrazine or bifenthrin added), measured atrazine concentrations were <5 IJ.g/L (n 

### = 2) at 1 hour after the initial application (phase 1), but were not monitored thereafter. Note that community and water quality measurements are plotted for only control tank#2 in Figures 1,2,3, 

### and 4. - 3 -

### Turbidity was reduced at day 7 during both phases, but in the higher atrazine levels only (atrazine-only treatments). Total phosphorus was reduced at day 7 for both the high and low atrazine conditions (atrazine-only treatments), but this effect was only observed in phase 2 (Figure 2). Total nitrogen was significantly increased at all intervals (ANOVA), but the increases may have been due to the nitrogen present in atrazine. Water pH was 7.8-8.0 in the treated tanks at all intervals, but increased in the control tank from pH 7.9 at days 7 and 14 of phase 1 to almost pH 9 at day 7 ofphase 2 (Figure 2). Chlorophyll was significantly affected by the effects ofatrazine during bothphases (ANOVA: phase 1, p=0.061 and phase 2, p=O.OOl); the study authors concludedthat "recovery" had occurred after 14 days in phase 1 based on ANOVA results. During phase 1, effects ofatrazineonly treatments on chlorophyll were not evident while reductions were evident for treatments with both atrazine and bifenthrin (Figure 1). 

### During phase 2, chlorophyll was reduced relative to the control in the atrazine-only treatments. Atrazine had no significant effect onprimary productivity or green colonies during phase 1, but both were significantly (ANOVA: p=O.OOl) affected at 7 days following treatment inphase 2. In phase 1, atrazine-only treatments showed an apparentreduction inprimary productivity relative to the control (Figure 1). 

### In phase 2 atrazine-on1y treatments, primary productivity and green colonies were reduced at low (250 Ilg/L) andhigh applications (1500 Ilg/L) in excess of80%, relative to controls (Figure 1). There were no significanteffects (ANOVA) to diatoms or green unicells at any interval. In atrazineonly treatments, there was an apparent decrease in greenunicell density 14 days following treatment in phase 1 (Figure 1). Particulate phosphorus (2-20 11m fraction) was significantly (ANOVA: p=0.022) affected by atrazine during phase 1. Phosphorus concentrations inthe atrazine-only treatments were greater than or equal to the control (Figure 3). During phase 2, significant atrazine effects were evident again (ANOVA: p=0.069), with> 60% reductions inparticulate phosphorus (2-20 11m) 

### relative to the control (Figure 3). The 20-200 11mfraction was significantly (ANOVA: p=0.054) affected during phase 1 (14 days following treatment), butnot during phase 2 (ANOVA: p=0.112). Phosphorus concentrations inthe atrazine-only treatments were less than the control (Figure 3: phase 1, day 14; phase 2, day 7). There were no significant effects onthe >200 11m fraction following treatment at during phase 1, butthere were significant (ANOVA: p=O.OOl) effects during phase 2. Phosphorus concentrations inthe atrazine-only treatments were less than the control (Figure 3: phase 1, day 7; phase 2, day 7). There were significant atrazine treatment effects (ANOVA) on zooplankton densities (Bosmina, copepod nauplii, and rotifers) with the exception ofcyclopoid copepodids (Figure 4). Inphase 1 atrazine-on1y treatments, rotifer densities in atrazine-only treatments were lowerthan the control (14 days following treatment), copepod naup1ii densities were lowerinthe high-atrazine treatment (200 Ilg/L) at all intervals, while Bosmina and cyclopoid copepodids densities appear similarto the control (Figure 4). No fish mortality was reported inthe atrazine-only treated tanks.

-4-

### PHASE 1 PHASE 2 Day 7 Day 14 PRIMARY PRODUCTIVITY

... 0.28., ., % 0.875 0.158 0.08b 0.718 boO'258 l\oI • 0.581 0.28'" 

### o 0.0'"

........... 

# : .".. '

# .. Day 7

CHLOROPHYLL
8°bO.278

.... 0.081**~"'O ~"lIot**  0.188 &20
- ......**0'18'"** '.. 0.531**_~** 0.52'"**~-- 0'008**  0.001 ;:;•• 0.030



### 1 1\ 0

.... 35**l\** 0.523 ! \ 0

.... 88 

## ! \ I 

# L O.001 0.001 0.001 

# •...'. ']. 0.831**0'255** 0

.... 80



## ... ::-- DIATOMS 8"'} 0.173
25- 0

. 702

...**~~** 0.880 1 ••' 

# .'
IX W 

# ...

.... GREEN COLONIES 

# ;**::L~::~: Ll~** **~:~::** Q. 0

.... 87 I." 0.725 5 

### U O. ..,4'
>C 

# L O 3 0 08 0.371

N L H 0

.... 85 0.803 0.328 

## .. ,"If. 1c \ 

# :)X

'" \



# ".

N L H BIFENTHRIN GREEN UNICELLS

## 8~0'855 0.558

... 0

.... 82 2 ••••.
N L H IX W m ::::E :;:) z

........... No Atrazine

--- Low Atrazlne
• High Atrazine Figure 1. Mean responses ofprimary productivity, chlorophyll and phytoplankton to atrazine and bifenthrin additions; atrazine only results above "N" on x-axis. Probability values from ANOVA ofbifenthrin (top), atrazine (middle) and their interaction effects (bottom) are given to the right ofeach graph, for days 7 and 14 ofphase 1 and day 7 ofphase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N = no bifenthrin; L =low bifenthrin; H =high bifenthrin. Figure from Hoagland etal. 1993. - 5 -

## PHASE 1 PHASE 2 Day 7 Day 14 Day 7 

### TOTAL PHOSPHORUS 0.010 0.346 0.315 0.036 0.055

....... 0.814 

### ti·

..

## fi 0.833 0.929 0.351

10



## ....
.... t1l :10 6 

### TOTAL NITROGEN 0.150 0.080 0.531 0.014 0.008 0.615 

# ............

.....+..... 0.018 0.001 0.387


### pH 9.0l 0.261 

## 1 0.206 

## l

.... 0.453 8.0 

## ..+ ..... 0.211 0.105 0.001 % 

# •••••• • 0.500 0.738 0.017 Q, 7.9 

# ..,



# .....

# -.

4 ::)IZ 2 

### TURBIDITY 0.001 0.077 0.041

N L H 0.001 0.004 0.012

N L H BIFENTHRIN N L H 0.001 0.277 0.610

............ No Atrazine

- .... - Low Atrazine
• High Atrazlne Figure 2. Mean responses ofIimnological parameters to atrazine and bifenthrin additions; atrazine only results above "N" on x-axis. Probability values from ANOVA ofbifenthrin (top), atrazine (middle) and their interaction effects (bottom) are given to the right ofeach graph, for days 7 and 14 ofphase 1 and day 7 of phase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N=no bifenthrin; L =low bifenthrin; H = high bifenthrin. Figure from Hoagland etal. 1993.

- 6 -

# PHASE 1 

# PHASE 2 Day 7 Day 14 Day 7 PHOSPHORUS (>200 pm) 0.007 0.002 

# \ 0.001 4 0.895 0.812 0.001 -J 0.583 0.001



### ..... 0.184 Q 2



### PHOSPHORUS (20-200 pm) 0.038 

# •• 0.232 0.001 7 0.395

### .::.~ 0.054 0.112 -J

..... 5 0.376 0.608 0.052 Q 

## .....• 

# ' 3 

# ••••
PHOSPHORUS (2-20 pm)

-J 10

..... Q 5
N L H 0.311 0.022 0.151 "10 0.114

, 0.885 

# tt:4" 0.911 

# ••••
N L H BIFENTHRIN**"1>~** 

## '.'. 

# ::x

N L H 0.051 0.089 0.287



## .....+ No Atrazine

- - Low Atrazlne
• High Atrazine Figure 3. Mean responses ofparticulate phosphorus size fractions to atrazine and bifenthrin additions; atrazine only results above "N" on x-axis. Probability values from ANOVA ofbifenthrin (top), atrazine (middle) and their interaction effects (bottom) are given to the right ofeach graph, for days 7 and 14 ofphase 1 and day 7 ofphase 2. Initial concentrations/densities are indicated by an arrow on y-axis. N = no bifenthrin; L 

### =low bifenthrin; H = high bifenthrin. Figure from Hoagland etal. 1993.

- 7 -

# PHASE 1 

# PHASE 2 Day 7 Day 

### 14 Day 7 BOSMINA 1 0.001 0.001 

# , 0.001 0.952 0.002 

# \ 0.001 0.5 0.451 0.018 0.001 \


# .... '\


### CYCLOPOID COPEPODIDS 0.2 0.001 0.014 0.441 

### a: 0.917 0.524 0.238 w 0.1 0.946 0.706 

# •.....•.... 0.679 

# ... -J

...... 

### cz: W Q. t') 

### COPE POD NAUPLII 0 

### ,.. 0.001 

# t=--. 0.137 0.020 2 

# , 0.010 '. 

# " 0.005 

# ..'\ 

# .. 0.330 a: 

# ....... w 0.123 

# ...... 0.955 

# •• 0.703 ID 1 

## ,\ '\ ;:) 1 

### z

0.078 0.013 0.380

N L H 0.488 0.032 0.415 

# ........**.~.** ".:4 

# ..'

N L H BIFENTHRIN 0.004 0.015 0.083



### N L H



### .....+ No Atrazlne

- - Low Atrazlne
• High Atrazlne 5 10

Fig. 4. Mean responses ofzooplankton to atrazine and bifenthrin additions. Probability values from ANOYA of bifenthrin(top)andatrazine(middle) effectsandtheirinteractioneffects (bottom)aregiventotherightofeachgraph. fordays 7and 14ofphase 1andday7ofphase2. Off-scaleinitialdensities areindicatedbyanupward-pointing arrow. N = no bifenthrin; L = low bifenthrin; H = high bifenthrin.



### - 8 -Table 1. Atrazine concentrations in experimental mesocosms (adapted from Table 1 ofstudy). Atrazine-only (Le. no bifenthrin) treatment tanks are presented. Atrazine (J,12:1L) Phase 1 Phase 2 Tank Hour Day Mean- MeanTreatment No. 1b, c 12 measured Hour 1b Day 14 measured Control (no atrazine or bifenthrin) 1e <5 NA



### --- <5 NA



## --- 2 <5 NA



### --- <5 NA



## --- Low 

### atrazine3 13 17 21 19 490 500 495 11 9.5 6.9 8.2 370 360 365 AVG: 14 AVG: 430 High atrazine 3 15 110 170 140 2700 1100 1900 7 190 160 175 1800 1400 1600 AVG: 158 AVG: 1750

a Low tanks treated atnommal concentratIOns of20 Ilg/L (phase 1) and and 250 IlgIL (phase 2). HIgh tanks treated atnominal concentrations of200 IlglL (phase 1) and 1,500 Ilg/L (phase 2). b Hour 1 is post-treatment ofpesticide.

CThe initial date ofapplication appears to have beenMay 22, 1990, and not May 25, 1990, as presented in the article

table (pp. 628-629). d Table reported 10 days posttreatment, reviewer assumed day 12 (see reviewer comments)
e Forthis tank, community data were notpresentednor used for analysis due to contamination with bifenthrin. NA= Not analyzed

- 9 -Data Summary Table: 

### Dataare summarized for phase 1 experiments. Phasetwo experiments are not included because community changes observed during phase 2 were the combinedresult ofeffects from phase 1 plus the subsequentincreasedtreatment levels ofatrazine.
Time Weighted Magnitude of Nominal Day of Study AverageAtrazine Did Effect Effectrelative to Atrazine Effect Concentration Recovery Comments controlsI Concentration Occurred from Day Oto Occur-if (pglLl When Effect so, when? Occurretf Magnitude of effects estimated from Figure 1. No statistical tests**~40%** (decrease) 20 11giL 7 14 Ilg/L Day 14 orraw data showing experimental variability were available for this effect. Primary productivity Magnitude of effects estimated from Figure 1. No statistical tests -40% (decrease) 2OOllg/L 7 158 Ilg/L No orraw data showing experimental variability were available for this effect.
-19% (increase) 20llgiL 7 141lg/L Not Magnitude of applicable effects estimated from Figure I.
No statistical tests or raw data showing experimental Chlorophyll variability were 1581lg/L Not available for this -19% (decrease) 2OOIlg!L 7 applicable effect. This is considered not likely a treatment related effectgiven the increase at low atrazine



### - 10

-Time Weighted Magnitude of Nominal Day of Study AverageAtrazine Did Atrazine Concentration Recovery Effect Effectrelative to Concentration Effect from Day Oto Occur-if Comments controli (pglLl Occurred When Effect so, when? Occurretf
concentrations and equal decrease at high atrazine concentrations.**~220%** 20 11giL 7 141lgIL Not Magnitude of (increase) applicable effects estimated from Figure 1. No statistical tests Diatoms orraw data (density)**~I50%** Not showing (increase) 200llgiL 7 158 Ilg/L applicable experimental variability were available for this effect. No observed 2Ollg/L Not Not applicable Not Magnitude of effect applicable applicable effects estimated from Figure 1. No statistical tests Green colonies orraw data (density) No observed Not Not showing effect 2OOllg/L applicable Not applicable applicable experimental variability were available for this effect.**~50%** (decrease) 20 11gIL 14 14 11giL Notreported Magnitude of effects estimated from Figure 1. No statistical tests Green unicells orraw data (density)**~75%** (decrease) 200llgiL 14 158 IlgIL Notreported showing experimental variability were available for this effect. Phosphorus**~45%** (decrease) 20llgiL 7 14 IlgiL 14 Magnitude of (standing effects estimated stock of>200 from Figure 3. 11m size- No statistical tests fractionof**~20%** (decrease) 2OOllg/L 7 1581lg/L 14 orraw data plankton showing community) 4 experimental



### - 11

-Time Weighted Magnitude of Nominal Day of Study AverageAtrazine Did Effect Effectrelative to Atrazine Effect Concentration Recovery Comments controlsl Concentration Occurred from Day Oto Occur-if (llg IL

/ When Effect so, when? Occurretf variability were available for this effect. -20% (decrease) 20 jlg/L 14 14 jlg/L Notreported Magnitude of Phosphorus effects estimated (standing from Figure 3. stock of20- 200 jlm size- No statistics or fraction of**~50%** (decrease) 200 jlg/L 14 158 jlglL Not reported experimental plankton variability data was community) 4 available for this effect.**~15%** (increase) 20 jlg/L 7 14 jlg/L Not Magnitude of Phosphorus applicable effects estimated (standing from Figure 3. stock of2-20 jlm size- No statistics or fraction of Not experimental plankton**~38%** (increase) 200 jlg/L 7 158 jlg/L applicable variability data was community)4 available for this effect. No effect 20 jlg/L Not Not applicable Not Magnitude of Bosmina observed applicable applicable effects estimated (density) from Figure 4. No effect 200 jlglL Not Not applicable Not observed applicable applicable No effect 20 jlg/L Not Not applicable Not Cyclopoid observed applicable applicable Magnitude of copepodids effects estimated (density) No effect 200 jlglL Not Not applicable Not from Figure 4. observed applicable applicable

13% (increase) 20 jlglL 7 Not applicable Not Magnitude of applicable effects estimated from Figure 4. Copepod nauplii No statistics or (density) experimental**~57%** (decrease) 200 jlglL 14 158 jlglL Notreported variability data was available for this effect.
Rotifers -38% (decrease) 20 jlg/L 14 14 jlg/L Notreported Magnitude of

- 12

-All effects reported mtable are for atrazme-only expenmental treatments (l.e. WIthout addItIOn ofbifenthnn) 2 Tanks treated atnominal concentrations of20 Ilg/L and 200 1lg!L for Phase 1, and at 250 1lg!L and 1,500 Ilg/L for

Phase 2. 3Average concentration from day 0 (1 hourpost-treatment) to day 12 (day 12 assumed by reviewer

- see reviewer comments). 4Plankton community comprised ofphytoplankton and zooplankton. Time Weighted Magnitude of Nominal Day of Study AverageAtrazine Did Effect Effectrelative to Atrazine Effect Concentration Recovery Comments controls} Concentration Occurred from Day Oto Occur-if (flgIL/ When Effect so, when? Occurretf (density) Notreported effects estimated from Figure 4. No statistics or**~50%** (decrease) 200llg/L 14 158 Ilg/L experimental variability data was available for this effect.
No results reported for atrazine-only treatments, and the None reported at authors did not Bluegill any atrazine- reportthatthere mortality only treatment was mortality for level these treatments, so it is assumed that no mortality occurred

.



### . .

- 13
-

### Reviewer Comments: This study also investigated the individual effects ofbifenthrin and the combined effects of atrazine plus bifenthrin onthe aquatic community.
The study authors provide sound statistical analysis; however, the results ofthose analyses are of limited use. This is because the results of ANOVA did not distinguish between atrazine treatment levels. Furthermore, the analyses also did not distinguish between treatments that contained atrazine-only and those that contained atrazine plus bifenthrin, that is, atrazine effects as reported in the study were based on all treatments that contained atrazine including those that contained bifenthrin as well. All statistical tests reported in the results section ofthis review are based on ANOVA as provided by the study authors. Atrazine-only effects summarized in this review (data summary table and results section) are estimates based on data in study figures of atrazine-only treatments and have no statistical basis regarding differences between treatments and the control. Additionally, except for measurements ofatrazine, no information was provided aboutreplicate variation ofcommunity variables (e.g. chlorophyll).
The experimental tanks were filled with water from anatural pond (0.7 ha). It

### is not stated whetherthe natural pond water was tested to establishthat it was not contaminated with any pollutants. Ifthis were the case, it could potentially leadto false conclusions. Furthermore, zooplankton were obtained from a different water source (anearby reservoir). It

### is unclear if water from the reservoir was added to the tanks and, ifso, ifit was contaminated withpesticides. Determinations for atrazine in the control ortreatmenttanks priorto study initiationwere not provided. The control tanks were analyzed for atrazine at 1 hour after initial application but were notmonitoredthereafter during phase I orphase 2. The authors reportthe atrazine concentration inthe control ponds was <5**~g/L,** 

### which is the limit ofdetection ofthe method theyusedto measure atrazine. The limit ofdetection is afairly high concentration and was 25% ofthe lowest concentration tested (20**~g/L).** 

### Bifenthrin contamination in one ofthe control tanks was sufficientlyhigh enough for the study authors to exclude it from analysis. Given all this, there is little evidenceto discountthe potential for atrazine contamination, particularly relative to the lowesttreatment concentration.

It 

### is unclear how many days afterthe initial pesticide application that atrazine was measured (phase I). The authors report that it was both 10 and 12 days throughoutthe paper. Based onthe actual dates provided, the reviewer assumed thatthe measurement was made 12 days after the first application.
The tanks were allowed to equilibrate for 16 days priorto treatment, and there was no indication thatthe tanks contained sediment otherthan the sediment slurry used as the test compound vehicle. The tank systems were also continually mixed throughout the study.

- 14

-Tank water was not monitored hardness. Temperature (daily) and dissolved oxygen (weekly) were reportedly monitored; however, no results were provided. Meteorological conditions were not monitored. Atrazine concentrations were only determined at two intervals following each application. Methods used to extract atrazine from the water samples were not described. Ponds were sampled for water parameters, phytoplankton and zooplankton only at two intervals after the initial application (phase 1) and at a single interval afterthe second application (phase 2)

. Phytoplankton were not identified at the species level to assess any changes in species composition. As the study authors noted, any effects which occurred in phase 2 ofthe study were a product ofboth the residual phase 1 effects and increased atrazine treatment levels, and, therefore, are oflimited value. Only one control tank was used for analysis (and presented in figures) as the second control was excluded due to contamination withbifenthrin. 

### Description ofUse 

### in Document: Invalid


### Rationale for Use: Data from only one control pond was presented due to bifenthrin contamination in the second replicate, thus there is increased uncertainty about the apparent effects due to atrazine based on differences between the control tank and the treatment tanks. The study authors provide sound statistical analysis; however, the results are of limited use. This is because the results of ANOVA did not distinguish between atrazine treatment levels. Furthermore, the analyses also did not distinguish between treatments that contained atrazine only and those that**containe~** atrazine plus bifenthrin. Atrazine effects reported by the study authors were based on all treatments that contained atrazine, including those that contained bifenthrin as well. Therefore, there is no statistical basis for the reviewer to establish atrazine-only treatment effects (additionally, the variationbetweenreplicates is unknown).


### Limitations ofStudy: There were numerous deficiencies in the measuring andreporting of experimental conditions. No effort was made to document whether other chemicals (including atrazine) were present in the treatment and/or control tanks priorto study initiation (i.e., experimental addition ofpesticides). The tanks were equilibrated for 16 days prior to treatment, and, apparently, contained no sediment layerto bufferthe system. It is not clearhow similarthe tank communities were to one anotherpriorto pesticide application

- only amean value is reported for eachparametermeasured (e.g. chlorophyll). Meteorological conditions were not monitored. Additionally, itwould have been useful to identify plankton atthe species level to assess any changes in species composition. The study had a factorial designthat included treatments containing both atrazine and bifenthrin. The statistical analysis performed was sound but did not examine the simple effects ofatrazine-only treatments. Furthermore, the statistics provided did not distinguish between atrazine treatment levels. Therefore the statistics provided

- 15

-were oflimited use. Datafrom only one control pond was presented due to contamination in the second replicate, thus there is even greateruncertainty aboutthe apparenttreatment effects due to atrazine based on differences between the control tank and the treatment tanks. Community changes observed during phase 2 were the combinedresult ofeffects from phase I plus the subsequent increasedtreatment levels ofatrazine, thus effects due to phase 2 treatments cannot be isolated. 

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


### "**.~** 

### Secondary Reviewer: Michael Lowit, Ph.D., Ecologist, EPA 

# '/!t{iWJ:"1-1,M;I v
. 

### Tertiary Reviewer: BrianAnderson, Biologist, EPA

- 16

-