### Open Literature Review Summary
Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Detenbeck, N.E., R. Hermanutz, K. Allen, and M.e. Swift. 1996. Fate and effects ofthe herbicide atrazine in flow-through wetland mesocosms. Environ. Toxico!. Chern. 15:937-946 (MRID 47543610).


### Purpose ofReview: Determinepotential 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 ofa Level ofConcern (LOC).



### Date ofReview: November 24,2009


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental flow-through wetlands (wetland mesocosms).


### Summary ofStudy Findings:

Methods: Mesocosms were the downstream wetlandportions offour ofthe eight outdoor experimental streams located atthe EPA Monticello Ecological Research Station (MERS) in Monticello, Minnesota, which are provided with water from the Mississippi River. The wetlands were originally constructed in 1987 by diverting about 90% ofthe flow from the lowerportion ofhalfofthe streams into 203-m-Iong flow-through mesocosms, each constructedto have four (30 m x 3 m x 0.8 m) pools and four (30 m x 2 m x 0.15 m) riffles in alternating sequence, and planted with 

### Typha latifola and 

### Zizania aquatica. By the time ofthe study (year notprovided), organic debris had accumulated in the riffles, vertebrate grazers including tadpoles 

### (Ranapipiens) and fathead minnows 

### (Pimephalespromelas) had naturally colonized the wetlands, and natural succession had resulted in dominantplants including: cattail 

### (Typha spp.), reed canary grass 

### (Phalaris arundinacea), and wild rice 

### (Zizania aquatica, fourth pools only) in the emergent community; waterweed 

### (Elodea canadensis), pondweed 

### (Potamogeton spp.), and coontail 

### (Ceratophyllum demersum) in the submersed community; and duckweed 

### (Lemna 

# '111111111 2087826

### minor) 

### in the floating-leaved community. Flow in the mesocosm wetlands was about 76 Umin. (No diagram ofexperimental setup is provided.)

Two ofthe wetlands were used as treatment mesocosms and two were maintained as controls. Treatment involved a steppedregime offour increasing atrazine concentrations: 15 j1g/L from May 18 to June 1 25 j1g/L from June 2 to July 15 50 j1g/L from July 16 to August 17 75 Ilg/L from August 18 to September4 Concentrations listed above are nominal. Treatmentwas administeredusing a stock solution made from well water and 30 mg/L commercial grade atrazine (AAtrex Nine-G, 85% active ingredient, water-dispensible granule; Ciba-Geigy, Greensboro, North Carolina). (No further details provided). According to the study authors, the treatment regime was designedto allow a series ofin situbioassays to be conducted at a series of increasing concentrations until LOEC concentrations could be found for aparticular endpoint; the design was not intended to mimic atypical time series for atrazine exposure in anatural aquatic ecosystem in an agricultural setting.

Water samples were collected weekly from the upperreach, middle reach, and outlet of each wetland and analyzed for atrazine. Standard solutions were made with Supelco (Bellafonte, Pennsylvania) standardreference solution. Samples were processed by concentrating with a C18 SPE column and eluting with ethyl acetate isooctane, and analyzed with an 

### HP 5840 gas chromatograph with a fused silica capillary column (25 m x 

### 0.32 mm internal diameter) and helium as the carrier gas, column temperature starting at 85°C for 1 minute and increasing to 250°C at 10°C/min. Average spike recoveries were 106% with arange of78-127% (n=24).
Temperature andpH (Coming CI07 meter), dissolved oxygen (Winkler method), and specific conductivity (Beckman RB4-250) were monitored weekly at early morning and midafternoon in the middle ofthe secondpool ofeach mesocosm. Nutrients were also analyzed: water samples were collectedweekly, filtered at 0.45 Il immediately, and stored frozen for nitrogen andphosphorus orat4°C (following addition ofphosphoric acid at 0.1 % v/v) for carbon. Nutrients were analyzed using aLatchet Quikchem AE autoanalyzer (ascorbic acid method for soluble reactive phosphorus, salicylatehypochlorite method for ammonium, orcadmiumreduction techniques for nitrate 

### + nitrite). Total or dissolved nitrogen andphosphorus were measured following an alkaline persulfate digestion offiltered and unfiltered samples, and dissolved and total organic carbon were analyzed using an Ionics carbon analyzer.

Atrazine concentrations were measured in three replicate sediment samples, collected from the thirdpool ofeach mesocosm priorto atrazine exposure and atthe end ofeach exposure regime using an Ekman dredge. Sediment samples were centrifugedto remove pore water, dried at 75°C, extractedwith 90% methanol, and analyzed using the same

2method as the water samples. Atrazine concentrations measured in sedimentwere compared to first-order model predictions based on the water concentration and the organic carbon content ofthe sediments (model will notbe described here, but is described in the original article). The top 3 or4 cm ofreplicate cores from the middle pool ofeach wetland was removed and analyzed for bulkdensity (n=3) and organic content (n=5). Following the disappearance ofmetaphytic mats ofalgae and detritus in treated wetlands during August, the depth ofdetritus accumulation overthe gravel substrate was measured in five random locations ofthe secondriffle ofeach mesocosm.
Periphyton was colonized on thin acetate strips (23 cm2) attached to the upper surface of a concrete brick for exposure periods of5 to 12 days during each exposure regime. In the 50- and 75-/lg/L tests, precolonized strips were placedinside individual glass tubes during 2 or 5 day exposures in test mesocosms, with halfofthe tubes (n=4 or 5) covered at both ends with Nitex netting (80-/lm mesh) to exclude grazers and the otherhalfleft open-ended. At the end ofeach exposure period, periphyton strips were analyzed for chlorophyll biomass and ash-free dry weight, and chlorophyll 

### a, b, and c, phaeophytin, and carotenoid concentrations were calculated from absorbance measurements (sources are cited for more information on the methods used for these measurements, but details are notprovided in the article).

Various bioassays were constructedto assess the different effects ofatrazine on biota during the four exposure regimes. Nutrient enrichment assays to observe combined effects ofnutrients and atrazine on periphyton were conducted during the 15- and 25- /lg/L exposure regimes, using the inverted pot method and addition ofNH4, N03, P04, and N03+P04 (methods and results will not be described in detail in this review). Periphyton assays testing for atrazine tolerance and resistance were conducted by incubating periphyton strips in filtered control water orfilterep treatment water andthen placing the strips in either control ortreatment f!lesocosms (methods and results will not be described in detail in this review).
Multiple vegetation bioassays were conducted. Cattails, potted in 1O-cm-diameter clay pots and grown from seeds germinated from a single seed spike of

### T. lati/ola, were tested for atrazine effects in the 15-, 25-, and 50-/lg/L exposure regimes. Seedlings were grown in the laboratory and subsequently in a riffle ofa MERS stream notused for the wetland mesocosms (growth time notprovided). On May 19, 14 plants were placed in the second pool ofeach mesocosm, with the soil surface 3-5 cm below the water surface. Number and length ofleaves, browning ofleaftips, and wet and dry weights ofabove-ground and below-ground biomass were measured on July 7 (6-7 plants permesocosm) for the lower exposures and on August 12 (5-7 plants permesocosm) for the 50-/lg/L exposure regime. In anotherbioassay, 5-cm-long shoots of

### Elodea canadensis (including the terminal bud) were collected from a MERS stream not used by the wetland mesocosms, and ten plant segments were suspended from large-mesh polypropylene bags in eitherthe second riffle or secondpool for 11, 12, and 19 days during the 25-, 50-, and 75-/lg/L exposure regimes, respectively. Initial and final wet and dry weights and main plus lateral shoot

3lengths were determined for plants in each test. Similartesting was performed on 5.5- cm-long shoots of

### Ceratophyllum demersum, with ten plants perwetland exposed for 8 days during the 50-f.lg/L regime, and eightplants per wetland exposed for 6 days during the 75-f.lg/L regime, and initial and final dry weights and lengths measured.

In addition to the bioassays, vegetative cover and vigor was qualitatively observed throughoutthe experiment. Afterearly senescence ofwild rice was observed in the treated wetlands, five plants were selected "randomly" from the fourth pool ofeach mesocosm, all leaves ofwidth at least 4 cm were removed and pooled for each mesocosm, and five leaves were "randomly" selected from the pool for analysis. The leaves were analyzedby cutting a 5-cm-long section from the middle ofeach leaf(20 to 25 cm2), and extracting and analyzing for chlorophyll 

### a using absorbance measurements as with periphyton.
Leopard frog tadpoles 

### (Ranapipiens; mean length 49 ± 5 mm, mean weight 1.3 ± 0.3 g) were collected in one stream on June 4, upstream ofatrazine inputs. In each wetland, 20 tadpoles were placed in wooden-frame cages (1.22 m long x 0.91 m wide x 0.91 m deep, covered with 3 mm mesh plastic screen), which were placed in the second pool-third riffle interface ofeach wetland in 30-cm-deep water, and the tadpoles were allowed to feed ad libitum. Endpoints measured after41 days included stage ofmetamorphosis and survival.

Juvenile fathead minnows 

### (Pimephalespromelas, mean length 25 ± 2 mm, mean weight 0.12 ± 0.04 g) were obtained from the EPA Environmental Research Laboratory in Duluth and tested atthe first pool-second riffle interface in cages as described above. Fish were acclimated to river water in aholding tank atMERS for 2 days priorto placement in cages. A random sample was collected for testing in each mesocosm, for 10 days in the 50-f.lg/L exposure regime (June 30 to August 10, n=25) and for 13 days in the 75-f.lg/L exposure regime (n=22, dates not provided). Endpoints included minnow survival, length, and weight.

Additional bioassays were conductedto testthe combined effects ofsunlight and atrazine on animals 

### (Daphnia magna and fathead minnow larvae). Animals were incubated in plasticjars with two holes cut on each side and covered with 80-f.lm mesh for flowthrough. Jars were suspended from clear acrylic racks at a depth such thatthey contained 100 mL water. Eachjarheld 10 animals, each rack held fivejars, and each mesocosm had one rack in the sunlight and one rack in full shade. No food beyond what could flow through the mesh was provided in any ofthese bioassays.


### Daphnia magna bioassays were conducted from May 20-22 (15 f.lg/L atrazine) and June 9-11 (25 f.lg/L atrazine). 

### D. magna were obtained from the stock culture maintained at the University ofMinnesota Cooperative Fisheries and Wildlife Unit, St. Paul,

4

### Minnesota. Gravid females were obtained one day priorto the first experiment and cultured atthe MERS between the first and second bioassay, and cultures were fed with yeast and ceraphyll provided by the Fisheries and Wildlife Unit. Forboth experiments, known neonates less than 24 hours oldhadto be mixedwith similarly sized individuals ofunknown age due to insufficientknown neonates available. Exceptforthe in situ nature oftheD. magnabioassays, methods followed EPA's 1989 guidance for estimating chronic toxicity ofeffluents to freshwater organisms (EPA/600/4-89/001).

Fathead minnow bioassays were conducted from June 1-8 and 18-25 (25 Ilg/L atrazine), July 17-25 and July 27-August 3 (50 Ilg/L), and August 18-25 (75 Ilg/L). Minnow larvae were obtained from the culture facility atthe EPA Environmental Research Laboratory in Duluth. Newly hatched larvae were picked up midmorning ofthe experimentinitiation day andtransportedto MERS (3 hours away), where they were allowedto acclimate to MERS waters atwetland temperatures for afew hours. "Almostnever" was mortality noted during this transport and acclimation process. Larvae were sorted into test containers with apipette (choosing only healthy-looking individuals) andputin the wetlands by 1700hron the day ofexperimentinitiation.

A summary ofall experiments performed, with concentrations tested and exposure durations, is providedin Table 1.

Statistical analyses ofthe results were performedusing univariate ormultivariate analysis ofvariance (ANOVA orMANOVA), as described in Table 2. All significance testing was conducted using an alpha level of0.05 unless otherwise stated (however, for clarity, p-values will be stated for all significance assertions in the review). In all analyses, data were tested for normality with the Shapiro-Wilks test andthen for homogeneity of variance using Bartlett's test. Datafailing normality andhomogeneity ofvariance assumptions were log-transformed, exceptforpercent survival data, which were arcsine and square-roottransformed.



### Results: Atrazine concentrations in the control wetlands were 0.69 Ilg/L on average, most likely reflected existing background atrazine inthe Mississippi River from agricultural runoff. Atrazine concentrations atthe outflow from the wetlandmesocosms were 78-96% ofthe concentrations atthe head ofthe wetlands. Sedimentconcentrations were 18-43% lower thanpredictedby the Freundlich sorption equilibrium model. See Table 3 for a summary. (Anothertable is provided in the article to comparepredicted 

### vs. measurement-based degradation rates and half-lives, butis not reproduced here. Measurement-basedhalflives were 8.4 and 14.2 days in the two treatmentmesocosms; predictedhalf-lives were about 3 times greater.)

5

### In periphyton bioassays, gross productivity was significantly (p<0.05) reduced in atrazine-treated mesocosms at 15 Ilg/L, and specific GPP (GPP/Ilg chlorophyll a) 

### was reduced at the highest exposure level (75 Ilg/L). Respiration was significantly (p<0.05) reduced at 25 Ilg/L atrazine and significantly (p<0.05) increased at 75 Ilg/L atrazine. Chlorophyll 

### a and ash-free dry weight were not affected by any atrazine treatments.

In the combination studies ofnutrient additions and atrazine treatment on periphyton, periphyton was found to be significantly (p<0.05) stimulated by nitrogen additions, and atrazine was found to be more effective in reducing carotenoid/chlorophyll a 

### ratios under phosphate enrichment (Figure 1). Atrazine levels of 15 and 25 Ilg/L were tested. The authors suggestthat this is evidence the mesocosms were nitrogen-limited, and that the severity ofthis limitation determined some sensitivity ofthe periphyton to the atrazine, for example as the community composition shifted to greens and blue-greens, which are more sensitive to atrazine even at these relatively low concentrations.

There was a significantreduction in specific NPP (mg DO/Ilg chlorophyll a) 

### at atrazine exposure levels 25 Ilg/L forperiphyton strips incubated in treated versus control water. Resistance andtolerance studies found thatperiphyton cultured in atrazine-treated water (50 Ilg/L) experienced less biomass reduction than control water cultures when placed in 50 Ilg/L treatment mesocosms, suggesting community-level resistance. See Figure 2. At the highest treatment level, specific NPP (mg DO/Ilg chlorophyll a) 

### was significantly (p<0.05) lower forperiphyton thathad been incubated in atrazine-treated water, regardless ofplacement in control or treatment mesocosms. However,this could have partly occurred because chlorophyll biomass per strip was higher in atrazine-treated wetlands (significance results or datanotprovided). The authors suggestthatthis may have been due to decreased metaphyton in the treatment mesocosms, which was visibly noticeable in the 75 Ilg/L mesocosms as opposedto the controls, although this was not directly measured.

Maximum DO in 15 Ilg/L atrazine wetlands was significantly (p<0.05) lowerthan in control wetlands (5.6 

### vs. 7.3 mg/L DO), consistent with the decrease in periphyton gross productivity. Nutrientconcentrations atthe midpoint ofthe wetlands went from no significant difference (two-sided test) priorto atrazine addition, to significant differences (p<0.05, one-sided test) in: ammonium and total dissolvedphosphorus at 15 Ilg/L atrazine; total dissolved nitrogen at 25 and 50 Ilg/L atrazine; and orthophosphate at 50 Ilg/L (all were significantly higher intreatment mesocosms). In the 75 Ilg/L treatment, ammonium and orthophosphate levels were higher in treatment mesocosms by over 50%, but differences were not significant due to high variability in the two replicates. See Figure 3 for dataplots. The authors describe these results as indicating that atrazine effects cannotbe mitigated by increasednutrient supply, and observe thatthe results are consistent with decreased periphyton productivity and nitrogen-limited growth.

6

### In the macrophyte bioassays, no significanteffects ofatrazine on cattail or

### Elodea biomass or stem length were found, althoughresults were limitedby high withintreatment variability. For coontails, a significant(p<0.05).multivariate effect of50 I!g/L atrazine on stem length and dry weightwas found, although individual effects on the two endpoints were not significant. Plant segments grown in treated wetlands were longer and lighter (greater length/weightratios) than those from controls. Additionally, wild rice 

### (Zizania aquatica) plants were more senescent in treatmentwetlands by August 12, the end ofthe 50 I!g/L exposure regime, and chlorophyll 

### a content ofleaves collected on August 25 were 25% ofthose from control wetlands.

No significanteffects ofatrazine on fathead minnow ortadpole growth ordevelopment were observed. Tadpole developmentwas accelerated (p<O.10), but inter-treatment variability was high: percent adult appearance was 11% in controls, 26-42% in treatments, and length ofback legs was 2.7-3.1 in controls, 3.5-3.8 in treatments. The authors note thatthe lack ofeffects in theirexperiment may havebeen due to artifacts from particular aspects oftheir experimental design, including the stocking protocol (bypassing effects on spawning habitat), the cages (protection from predation), andthe frog species used 

### (Rana catesbiana, for example, is more sensitive than their

### Rana pipiens).

The

### Daphnia magna and fathead minnow larvae bioassays testing combined effects of sunlight and atrazine found no significanteffects ofeithervariable individually orin combination, with the exception ofsignificant(p<0.05) atrazine effects on

### D. magna found at 15 I!g/L atrazine. The authors believe thatthe effectwas due to the synergistic impact oflow levels ofatrazine and limited food supply early inthe season.

Table 1. Endpoints measured in control and atrazine-treated wetland mesocosms. With the exception ofmeasurements on detritial accumulation and senescence (browning) ofZizania aquatica (wild rice), measurements were made on organisms colonized orgrown outside ofmesocosms, then incubated in mesocosms during specific treatment regimes. (study table 1)**Eco~ystem** component Endpointmeasured Concentration Exposure tested (days) Nutrients Watercolumn concentration 15-75 fJg/L 14-43 Detritus Accumulation 75 fJg/L a 106 Periphyton Biomass 15-75 fJg/L 5-12 (with/without grazers) 2-5 (with/without nutrientenhancement) 28 Productivity (02production) 9-27 Atrazine resistance 9-27 Submerged macrophytes Growth (wet and dry weight, length) Elodea canadensis 15-75 fJg/L 11-19 Ceratophyllum demersum 50-75 fJg/L 6-8 Emergentmacrophytes Growth (wet and dry weight), browning Typha lati/ola 15-25 fJg/L 19 Zizania aquatica IS-50 fJg/L 83 75 fJg/L a 106

7Animals Daphnia magna Survival 15-25 /lg/L 2 Ranapipiens tadpoles Metamorphosis, growth, survival 15-25 /lg/L 41 Pimephalespromelas Growth, survival 50-75 /lg/L 10-13 larvae Survival 25-75 ).lg/L 7

a Cumulative effect, 13 days (15 /lg/L) +43 days (25 /lg/L) + 32 days (50 /lg/L) + 17 days (75 /lg/L)

Table 2. Statistical tests used in analysis ofexperimental results. (study table 2) Independent variables Dependentvariable(s) Test/design I Atrazine Pretreatment nutrients I-way MANOYA Atrazine Nutrients Repeated-measures ANOYAs Atrazine Periphyton biomass (pigment 3-way MANOYA Nitrate concentrations on clay pots) Phosphate Atrazine Periphyton biomass (pigment 2-way MANOYA Nutrient addition concentrations on clay pots) Atrazine x nutrient Atrazine (colonization Periphyton chlorophyll a; NPP; NPP//lg I-way ANOYA exposure = incubation chlorophyll a; GPP; GPP//lg chlorophyll exposure) a; respiration Atrazine Periphyton productivity and respiration Mixed-model 3-way MANOYA: Fixed effects =colonization, productivity treatments Random effects =replicate Atrazine Typha above-ground biomass and below- MANOYA ground biomass Atrazine Elodea stem length and biomass MANOYA Atrazine Ceratophyllum stem length and biomass MANOYA Atrazine Daphnia survival Split plot/ANOYA Sunlight Atrazine Fathead minnow survival Splitplot/ANOYA Sunlight ANOYA = analysIs ofvanance GPP = gross primary productivity MANOYA = multivariate analysis ofvariance NPP = netprimary productivity



### 8Table 3. Mean measured and predicted atrazine concentrations in surfacewater, sedimentpore water, and bulk sedimentoftreated wetland mesocosm channels. Predicted sediment concentrations were based on the Freundlich adsorption equation and regressions ofparameters to organic carbon content ofsediments, following Kadlec and Alvord (1993) (study reference 17, see study for details). (study table 3)
Nominal concentration ofatrazine in watercolumn 15/.tg/L 25/.tg/L 50/.tg/L 75/.tg/L Mean surface water atrazine concentrations (/-lg/L) Upper 17.9 ± 1.9 26.2 ± 1.3 53.5 ± 7.0 85.0 ± 12.2 Middle 16.0 ± 0.2 26.4 ± 0.7 47.3 ± 0.7 78.5 ± 4.6 Lower 15.4±0.6 22.7 ± 2.7 47.8 ± 2.0 73.9 ± 5.6 Measured pore water atrazine 0.87 2.6 10.8 concentrations (/-lg/L) Measured bulk sediment atrazine concentration (/-lg/g dry 0.33 0.88 0.81 weight) Predicted bulk sediment atrazine concentration (/-lg/g dry 0.58 1.07 1.43 weight)



### « 1.00 -.J 0.90 :r: 0 0.80 C> ::l. 0.70 -.. Ul 0 0.60 0 :z 0.50 UJ l- 

### OAD 0 

### a: 0.30 

### « () 0.20 ::> 0.10 

### a.. Ul 0.00 ::l. NulrierH: C C p P N N N+P N+P Alrazine: C A C A C A C A

Figure 1. Mean ratio ofcarotenoids/chlorophyll a (llSPU/llg chi a) in periphyton on control (C) or nutrient-enhanced (N=N03, P=P04) inverted clay pots in control (C) or atrazine-treated (A) mesocosms. Bars represent standard deviation for each treatment combination. The interaction

9between atrazine treatment and phosphate enrichmentis significant (p<0.05). Nominal atrazine levels were 15-25 /lg/L. (study figure 1)

0040

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### ex: I- (I) co ::> 0.24

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Figure 2. Gross primary productivity (GPP)/periphyton substrate (mg DO/h/strip) for each combination ofmesocosm source and water source treatment (C == control, A == 50 /lg/L atrazine). Periphyton that had been growing in control mesocosms experienced a signilicantIy greater reduction in GPP when incubated in treatmentwaterthan did periphyton that had beengrowing in treated mesocosms (p<0.05). (study figure 2)

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Figure 3. Plots ofmeasured (a) soluble reactive phosphate (mg P04-P/L) and (b) ammonium (mg NH4-N/L) in control (C) and atrazine·treated (A) wetland mesocosms. Repeated·measures ANOVAs showed significant (p<0.05) treatmenteffects ofatrazine on phosphate during the 50 IlglL atrazine exposure regime (July 16-August7) and on ammonium during the 15 Ilg/L exposure regime (May 18- June 1). Dashed lines indicate new treatment regimes. (study figure 3)

11

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

- if (J-lg/L) Occurred Atrazine so, when Concentration (see from Day 0 to comment)? I

When Effect Occurred NR/reduced 151lg/L 9-27/NR 16/lg/L nol? Periphyton (p<0.05) gross no effect 25 Ilg/L NA 26.4 Ilg/L NA From study productivity no effect 50 ug/L NA 47.3 ug/L NA text, p.941.

no effect 75 Ilg/L NA 78.5 ug/L NA no effect IS ug/L NA 16 ug/L NA NR/reduced 251lg/L 9-27/NR 26.4/lg/L no/? Periphyton (0<0.05) From study respiration no effect 50 ug/L NA 47.3 ug/L NA text, p.941. NR/increased 75 /lg/L 9-27/NR 78.5/lg/L no/? (0<0.05) No effect IS Ilg/L 9-27/NR 16 ug/L nol? Netprimary NR/reduced 251lg/L NA 26.4 /lg/L NA (p<0.05) production NR/reduced From study (mg DOI/lg (0<0.05) 50llg/L NA 47.3 /lg/L NA text, p.942. chlorophyll a) NR/reduced (0<0.05) 751lg/L NA 78.5/lg/L NA Periphyton no effect IS Ilg/L NA 16 /lg/L NA biomass (chI no effect 25 ug/L NA 26.4 ug/L NA From study a, ash-free dry no effect 50 /lg/L NA 47.3 /lg/L NA text, p.941. weight) no effect 75 ug/L NA 78.5 ug/L NA 23% Dissolved decrease 151lg/L NR 16/lg/L nol? oxygen (0<0.05) From study (maximum no effect 25 ug/L NR 23.8 ug/L NA text, p.943. conc'n) no effect 50 /lg/L NR 32.3 /lg/L NA no effect 75**~lg/L** NR 39.7 ug/L NA 15% 151lg/L**~8** 16/lg/L partial by Increase in dav 45 Magnitudes phosphatel no effect 25 /lg/L NA 23.8 /lg/L NA estimated P04 (mg P/L) 40% (0<0.05) 50 112:/L**~85** 32.3/lg/L no from Fig.3 50% 75 J,lg/L 106 39.7 ug/L no/? 50% (p<0.05) 151lg/L**~8** 16 )..lg/L partial by Increase in dav 30 Magnitudes ammomum no effect 25 J,lg/L NA 23.8 /lg/L NA estimated (mgNIL) 100% 50 112:/L**~85** 32.3 ug/L no from Fig.3 250% 75 ug/L 106 39.7 ug/L no/? Typha no effect IS /lg/L NA 23.8/lg/L NA no effect 25 ug/L NA 32.3 ug/L NA From study biomass no effect 50 )..lg/L NA 39.7 )..lg/L NA text, p.944.
Elodea no effect IS /lg/L NA 16 /lg/L NA biomass and no effect 25**~lg/L** NA 26.4 ug/L NA From study stem length no effect 50 ug/L NA 47.3 ug/L NA text, p.944. no effect 75 Ilg/L NA 78.5 /lg/L NA

12Effect Magnitude of Nominal Dayof Time Did Comments Effectrelative Atrazine Study Weighted Recovery to controls Concentration Effect Average Occur- 

### if (f.1g/ L

) Occurred Atrazine so, when Concentration (see from Day 0to comment)?

When Effect Occurred Ceratophyllum NR/elongation 50 !!g/L 8 47.3 !!g/L no/? From study stem observed text, p.944. elongation no effect 75 IJg/L NA 78.5 IJg/L NA Zizania senescence 75% reduction 15-75 !!g/L 99 78.5 !!g/L From study (chlorophyll a in chi a no text, p.944. content) Rana tadpole**~300%** development increase 15-25**~lg/L** 41 23.8 !!g/L NA From study (% adult (p<0.10) text, p.944. appearance) Rana tadpole development 18% increase 15-25 !!g/L 41 23.8 !!g/L NA From study (length of (p<0.10) text, p.944. back legs) Pimephales no effect 15 !!g/L NA 16 !!g/L >10 days From study growth and survival no effect 75 !!g/L NA 78.5 !!g/L >13 days text, p.944. Daphnia NR/decrease 15 !!g/L 2 16 !!g/L no/? From study (p<O.05) survival no effect 25 !!g/L NA 26.4 !!g/L NA text, p.945. Pimephales no effect 25 IJg/L NA 26.4 IJg/L NA From study no effect 50 !!g/L NA 47.3 !!g/L NA larvae survival no effect 75 !!g/L NA 78.5 !!g/L NA text, p.945. Note: NR=Not Reported, NA=NotApplIcable, no/?=effects observed at end ofexperiment Bolded values are those datapoints specified in the "Cosm Data Points and Exposure Profiles" spreadsheet.



### Time weighted averages are takenfrom Table 3. When a description ofthe relative location ofthe experiment in the mesocosm is notprovided, the "Middle" number from Table 3 is taken (assuming this means the middle reach ofthe 203-m-long channel). For experiments/endpoints affected by the whole time-series ofconcentrations 

### (i.e., water quality parameters, some macrophyte endpoints, and

### Ranapipiens development), timeweighted averages ofthe average concentration for each exposure regime were computed as shown below.

TWA cone. 224 16.0 1135.2 23.8 1513.6 32.3 1334.5 39.7 ("Middle") avg cone duration (d) d*conc 1-Jun 16 14 15-Jul 26.4 43 17-Aug 47.3 32

4-Sep 78.5 17 18-May

2-Jun 16-Jul 18-Aug

13

### Reviewer Comments: The mostglaring deficiency ofthe study is that atrazine was detected in the control wetlands at an average concentration of0.69 Ilg/L. The authors attribute the contaminated controls to existing background atrazine in the Mississippi River from agricultural runoff. Moreover, statistical tests were limited by the small numberof replicates (only 2 controls and 2 treatments), and often the within-treatment variabilities were quite high. Given the lack ofa suitable control, low number ofreplication, and within-treatment variability, the results ofthe study should be interpreted with caution. In addition, it appears thatthere were sequential additions ofatrazine to the same system, ratherthan to separate, individual wetlands. Therefore, it is unclear whethermeasured values represent peak or average concentrations across the exposure profile.

The study was complex, encompassing many experiments. As such, and given the space constraints ofthe paper, there were certain details missing from discussions ofcertain experiments andresults, as noted throughout the review and in the Data Summary Table. The flow-through mesocosms were semi-natural andprovided an opportunity to observe effects in a controlled, yetpotentiallyrealistic environment. However, most ofthe experiments involved cultured organisms placed inthe mesocosms for relatively brief periods oftime, generally in enclosures, which preventedthe occurrence ofecosystemscale interactions. Forexample, the tadpole bioassays, as the authors themselves point out, are limited by their short duration, the provision oftadpoles from laboratory stock (so spawning habitat effects couldnot be observed), andthe use ofcages to prevent predation effects. There are few benefits to using semi-natural mesocosms in such experiments, since similarconditions could be created in much simpler artificial systems. Longer-term effects, such as water quality changes andthe early senescence ofwild rice plants, are difficult to interpretbecause ofthe step-up in concentration overthe growing season.
Despite these limitations, the authors provide evidence that atrazine effects on aquatic biota (particularly periphyton) couldbe significantly modified by feedbacks with variables like nutrient availability, presence ofdetritus and/ormetaphyton, and seasonality.
Description ofUse 

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

Rationale for Use: The study contributes to the weight-of-evidence by demonstrating effects ofatrazine onperiphyton metabolism, despite issues related to contaminated controls, low replication, andhigh within-treatmentvariability.

Limitations ofStudy: Atrazine was detected in the control samples and only two treatmentreplicates and two controls were used; therefore, the power ofthe statistical

14tests was weak and the ability to detecttreatment-related effects based on comparison with an atrazine-contaminated control is confounded. 

### In addition, it appears thatthere were sequential additions ofatrazine to the same system, ratherthan to separate, individual wetlands. Therefore, it is unclear whethermeasured values representpeak or average concentrations across the exposure profile.

Most experiments were ofshort duration and involved enclosures and pre-cultured organisms, thus preventing observation ofecosystem-level effects, despite the apparently semi-natural state ofthe wetland mesocosms.


### Primary Reviewer: SaraHendrix, 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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