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 of Review:

Determine potential effects of atrazine on an aquatic community. Endpoints from this

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant

communities and associated determination of a Level ofConcern (LOC).

Date of Review:

November 24,2009

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants and animals in

experimental flow-through wetlands (wetland mesocosms).

Summary of Study Findings:

Methods:

Mesocosms were the downstream wetland portions offour ofthe eight outdoor

experimental streams located at the 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 lower portion of half ofthe streams into 203-m-Iong flow-through

mesocosms, each constructed to 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 not provided), organic debris had accumulated

in the riffles, vertebrate grazers including tadpoles (Rana pipiens) and fathead minnows

(Pimephales promelas) had naturally colonized the wetlands, and natural succession had

resulted in dominant plants 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 of experimental setup is provided.)

Two ofthe wetlands were used as treatment mesocosms and two were maintained as

controls. Treatment involved a stepped regime 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 September 4

Concentrations listed above are nominal. Treatment was administered using 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 designed to allow a series of in situ bioassays to be conducted at a series of

increasing concentrations until LOEC concentrations could be found for a particular

endpoint; the design was not intended to mimic a typical time series for atrazine exposure

in a natural aquatic ecosystem in an agricultural setting.

Water samples were collected weekly from the upper reach, middle reach, and outlet of

each wetland and analyzed for atrazine. Standard solutions were made with Supelco

(Bellafonte, Pennsylvania) standard reference 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 a range of78-127% (n=24).

Temperature and pH (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 second pool of each mesocosm. Nutrients were also

analyzed: water samples were collected weekly, filtered at 0.45 Il immediately, and

stored frozen for nitrogen and phosphorus or at 4°C (following addition of phosphoric

acid at 0.1 % v/v) for carbon. Nutrients were analyzed using a Latchet Quikchem AE

autoanalyzer (ascorbic acid method for soluble reactive phosphorus, salicylatehypochlorite method for ammonium, or cadmium reduction techniques for nitrate +

nitrite). Total or dissolved nitrogen and phosphorus were measured following an alkaline

persulfate digestion of filtered 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 third pool of each mesocosm prior to atrazine exposure and at the end of each

exposure regime using an Ekman dredge. Sediment samples were centrifuged to remove

pore water, dried at 75°C, extracted with 90% methanol, and analyzed using the same

2
method as the water samples. Atrazine concentrations measured in sediment were

compared to first-order model predictions based on the water concentration and the

organic carbon content ofthe sediments (model will not be described here, but is

described in the original article). The top 3 or 4 cm ofreplicate cores from the middle

pool of each wetland was removed and analyzed for bulk density (n=3) and organic

content (n=5). Following the disappearance of metaphytic mats of algae and detritus in

treated wetlands during August, the depth of detritus accumulation over the gravel

substrate was measured in five random locations ofthe second riffle of each mesocosm.

Periphyton was colonized on thin acetate strips (23 cm

2

) attached to the upper surface of

a concrete brick for exposure periods of 5 to 12 days during each exposure regime. In the

50- and 75-/lg/L tests, precolonized strips were placed inside individual glass tubes

during 2 or 5 day exposures in test mesocosms, with half ofthe tubes (n=4 or 5) covered

at both ends with Nitex netting (80-/lm mesh) to exclude grazers and the other half left

open-ended. At the end of each 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 not provided in the article).

Various bioassays were constructed to assess the different effects of atrazine on biota

during the four exposure regimes. Nutrient enrichment assays to observe combined

effects of nutrients 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 or filterep treatment water and then

placing the strips in either control or treatment 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 of a MERS stream not used for the wetland

mesocosms (growth time not provided). On May 19, 14 plants were placed in the second

pool of each mesocosm, with the soil surface 3-5 cm below the water surface. Number

and length of leaves, browning of leaftips, and wet and dry weights of above-ground and

below-ground biomass were measured on July 7 (6-7 plants per mesocosm) for the lower

exposures and on August 12 (5-7 plants per mesocosm) for the 50-/lg/L exposure regime.

In another bioassay, 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 either the second riffle

or second pool 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

3
lengths were determined for plants in each test. Similar testing was performed on 5.5-

cm-long shoots of Ceratophyllum demersum, with ten plants per wetland exposed for 8

days during the 50-f.lg/L regime, and eight plants 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

throughout the experiment. After early senescence of wild rice was observed in the

treated wetlands, five plants were selected "randomly" from the fourth pool of each

mesocosm, all leaves of width 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 analyzed by cutting a 5-cm-long section from the middle of each leaf (20 to

25 cm

2

), and extracting and analyzing for chlorophyll a using absorbance measurements

as with periphyton.

Leopard frog tadpoles (Rana pipiens; mean length 49 ± 5 mm, mean weight 1.3 ± 0.3 g)

were collected in one stream on June 4, upstream of atrazine 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 of each wetland in 30-cm-deep water, and the tadpoles were allowed to

feed ad libitum. Endpoints measured after 41 days included stage of metamorphosis and

survival.

Juvenile fathead minnows (Pimephales promelas, mean length 25 ± 2 mm, mean weight

0.12 ± 0.04 g) were obtained from the EPA Environmental Research Laboratory in

Duluth and tested at the first pool-second riffle interface in cages as described above.

Fish were acclimated to river water in a holding tank at MERS for 2 days prior to

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 conducted to test the combined effects ofsunlight and atrazine

on animals (Daphnia magna and fathead minnow larvae). Animals were incubated in

plastic jars 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 that they contained

100 mL water. Each jar held 10 animals, each rack held five jars, 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 prior to the first experiment and

cultured at the MERS between the first and second bioassay, and cultures were fed with

yeast and ceraphyll provided by the Fisheries and Wildlife Unit. For both experiments,

known neonates less than 24 hours old had to be mixed with similarly sized individuals

of unknown age due to insufficient known neonates available. Except for the in situ

nature ofthe D. magna bioassays, methods followed EPA's 1989 guidance for estimating

chronic toxicity of effluents 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 at the EPA Environmental Research Laboratory in

Duluth. Newly hatched larvae were picked up midmorning ofthe experiment initiation

day and transported to MERS (3 hours away), where they were allowed to acclimate to

MERS waters at wetland temperatures for a few hours. "Almost never" was mortality

noted during this transport and acclimation process. Larvae were sorted into test

containers with a pipette (choosing only healthy-looking individuals) and put in the

wetlands by 1700 hr on the day of experiment initiation.

A summary of all experiments performed, with concentrations tested and exposure

durations, is provided in Table 1.

Statistical analyses ofthe results were performed using univariate or multivariate analysis

of variance (ANOVA or MANOVA), as described in Table 2. All significance testing

was conducted using an alpha level of 0.05 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 and then for homogeneity of

variance using Bartlett's test. Data failing normality and homogeneity of variance

assumptions were log-transformed, except for percent survival data, which were arcsine

and square-root transformed.

Results:

Atrazine concentrations in the control wetlands were 0.69 Ilg/L on average, most likely

reflected existing background atrazine in the Mississippi River from agricultural runoff.

Atrazine concentrations at the outflow from the wetland mesocosms were 78-96% ofthe

concentrations at the head ofthe wetlands. Sediment concentrations were 18-43% lower

than predicted by the Freundlich sorption equilibrium model. See Table 3 for a summary.

(Another table is provided in the article to compare predicted vs. measurement-based

degradation rates and half-lives, but is not reproduced here. Measurement-based halflives were 8.4 and 14.2 days in the two treatment mesocosms; predicted half-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 of nutrient 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 suggest that 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 significant reduction in specific NPP (mg DO/Ilg chlorophyll a) at atrazine

exposure levels 25 Ilg/L for periphyton strips incubated in treated versus control water.

Resistance and tolerance studies found that periphyton 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 for periphyton that had been incubated in atrazine-treated water,

regardless of placement 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 data not provided). The authors suggest that this may

have been due to decreased metaphyton in the treatment mesocosms, which was visibly

noticeable in the 75 Ilg/L mesocosms as opposed to the controls, although this was not

directly measured.

Maximum DO in 15 Ilg/L atrazine wetlands was significantly (p<0.05) lower than in

control wetlands (5.6 vs. 7.3 mg/L DO), consistent with the decrease in periphyton gross

productivity. Nutrient concentrations at the midpoint ofthe wetlands went from no

significant difference (two-sided test) prior to atrazine addition, to significant differences

(p<0.05, one-sided test) in: ammonium and total dissolved phosphorus 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 in treatment 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 data plots. The authors describe these results as indicating that atrazine

effects cannot be mitigated by increased nutrient supply, and observe that the results are

consistent with decreased periphyton productivity and nitrogen-limited growth.

6
In the macrophyte bioassays, no significant effects of atrazine on cattail or Elodea

biomass or stem length were found, although results were limited by high withintreatment variability. For coontails, a significant (p<0.05).multivariate effect of 50 I!g/L

atrazine on stem length and dry weight was found, although individual effects on the two

endpoints were not significant. Plant segments grown in treated wetlands were longer

and lighter (greater length/weight ratios) than those from controls. Additionally, wild

rice (Zizania aquatica) plants were more senescent in treatment wetlands by August 12,

the end ofthe 50 I!g/L exposure regime, and chlorophyll a content of leaves collected on

August 25 were 25% ofthose from control wetlands.

No significant effects of atrazine on fathead minnow or tadpole growth or development

were observed. Tadpole development was accelerated (p<O.1 0), but inter-treatment

variability was high: percent adult appearance was 11 % in controls, 26-42% in

treatments, and length of back legs was 2.7-3.1 in controls, 3.5-3.8 in treatments. The

authors note that the lack of effects in their experiment may have been due to artifacts

from particular aspects oftheir experimental design, including the stocking protocol

(bypassing effects on spawning habitat), the cages (protection from predation), and the

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 significant effects of either variable individually or in

combination, with the exception ofsignificant (p<0.05) atrazine effects on D. magna

found at 15 I!g/L atrazine. The authors believe that the effect was due to the synergistic

impact of low levels of atrazine and limited food supply early in the season.

Table 1. Endpoints measured in control and atrazine-treated wetland mesocosms. With the

exception of measurements on detritial accumulation and senescence (browning) of Zizania aquatica

(wild rice), measurements were made on organisms colonized or grown outside ofmesocosms, then

incubated in mesocosms during specific treatment regimes. (study table 1)

Eco~ystem component Endpoint measured Concentration Exposure

tested (days)

Nutrients Water column 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 nutrient enhancement) 28

Productivity (02 production) 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

Emergent macrophytes 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

7
Animals

Daphnia magna Survival 15-25 /lg/L 2

Rana pipiens tadpoles Metamorphosis, growth, survival 15-25 /lg/L 41

Pimephales promelas 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 of experimental results. (study table 2)

Independent variables Dependent variable(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 Split plot/ANOYA

Sunlight

ANOYA = analysIs ofvanance

GPP = gross primary productivity

MANOYA = multivariate analysis of variance

NPP = net primary productivity

8
Table 3. Mean measured and predicted atrazine concentrations in surface water, sediment pore

water, and bulk sediment of treated wetland mesocosm channels. Predicted sediment concentrations

were based on the Freundlich adsorption equation and regressions of parameters to organic carbon

content ofsediments, following Kadlec and Alvord (1993) (study reference 17, see study for details).

(study table 3)

Nominal concentration of atrazine in water column

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

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:r: 0.90

0

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:z 0.50

UJ

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«

() 0.20

::>

a.. 0.10

Ul

::l. 0.00

NulrierH: C C p P N N N+P N+P

Alrazine: C A C A C A C A

Figure 1. Mean ratio of carotenoids/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

9
between atrazine treatment and phosphate enrichment is significant (p<0.05). Nominal atrazine

levels were 15-25 /lg/L. (study figure 1)

0040

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Fcrip"ylon SOUI'GU: co"trol

Figure 2. Gross primary productivity (GPP)/periphyton substrate (mg DO/h/strip) for each

combination of mesocosm 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 treatment water than did periphyton that had been growing in

treated mesocosms (p<0.05). (study figure 2)

10
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Figure 3. Plots of measured (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) treatment effects of atrazine on phosphate during the 50 IlglL atrazine

exposure regime (July 16-August 7) 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)?

When I 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?

Net primary 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

(mg NIL) 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

12
Effect Magnitude of Nominal Day of Time Did Comments

Effect relative Atrazine Study Weighted Recovery

to controls Concentration Effect Average Occur- if

(f.1g/L) Occurred Atrazine so, when

Concentration (see

from Day 0 to 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=Not ApplIcable, no/?=effects observed at end of experiment

Bolded values are those data points specified in the "Cosm Data Points and Exposure Profiles" spreadsheet.

Time weighted averages are taken from Table 3. When a description ofthe relative

location ofthe experiment in the mesocosm is not provided, 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 of concentrations (i.e., water

quality parameters, some macrophyte endpoints, and Rana pipiens 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 most glaring deficiency ofthe study is that atrazine was detected in the control

wetlands at an average concentration of 0.69 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 number of

replicates (only 2 controls and 2 treatments), and often the within-treatment variabilities

were quite high. Given the lack of a suitable control, low number ofreplication, and

within-treatment variability, the results ofthe study should be interpreted with caution.

In addition, it appears that there were sequential additions of atrazine to the same system,

rather than to separate, individual wetlands. Therefore, it is unclear whether measured

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 of certain

experiments and results, as noted throughout the review and in the Data Summary Table.

The flow-through mesocosms were semi-natural and provided an opportunity to observe

effects in a controlled, yet potentially realistic environment. However, most ofthe

experiments involved cultured organisms placed in the mesocosms for relatively brief

periods oftime, generally in enclosures, which prevented the occurrence of ecosystemscale interactions. For example, 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 could not be observed), and the use of cages to prevent

predation effects. There are few benefits to using semi-natural mesocosms in such

experiments, since similar conditions could be created in much simpler artificial systems.

Longer-term effects, such as water quality changes and the early senescence of wild rice

plants, are difficult to interpret because ofthe step-up in concentration over the growing

season.

Despite these limitations, the authors provide evidence that atrazine effects on aquatic

biota (particularly periphyton) could be significantly modified by feedbacks with

variables like nutrient availability, presence of detritus and/or metaphyton, and

seasonality.

Description of Use in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: The study contributes to the weight-of-evidence by demonstrating

effects of atrazine on periphyton metabolism, despite issues related to contaminated

controls, low replication, and high within-treatment variability.

Limitations of Study: Atrazine was detected in the control samples and only two

treatment replicates and two controls were used; therefore, the power ofthe statistical

14
tests was weak and the ability to detect treatment-related effects based on comparison

with an atrazine-contaminated control is confounded. In addition, it appears that there

were sequential additions of atrazine to the same system, rather than to separate,

individual wetlands. Therefore, it is unclear whether measured values represent peak or

average concentrations across the exposure profile.

Most experiments were ofshort duration and involved enclosures and pre-cultured

organisms, thus preventing observation of ecosystem-level effects, despite the apparently

semi-natural state ofthe wetland mesocosms.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Anita Pease, M.S., Senior Biologist, Environmental Fate and

Effects Division, U.S. Environmental Protection Agency.

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