### Open Literature Review Summary

Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: Johnson, B.T. 1986. Potential impact ofselected agricultural chemical contaminants on a northern prairie wetland: A microcosm evaluation. Environ. Toxico!. Chem. 5:473- 

### 485. 

## (mt.io**qt5''l',~ ~D?»)**  

### Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study were also used to parameterize the Comprehensive Aquatic System Model (CASM) and establish the model-associated LOC for atrazine.



### Date ofReview: January 14,2009


### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in laboratory microcosms.


### Summary ofStudy Findings:

Methods: This study examined the effects of3 differentherbicides (including atrazine) and 3 different insecticides, in anested experimental design. Also, some animal toxicity experiments were conductedto examine the effects ofthe insecticides in particular. 

### In this review, only the methods pertaining to atrazine effects on the microcosms will be fully described; methods notrelevantto atrazine will be omitted, and methods tangentiallyrelevantto atrazine orconducted withoutthe microcosms will be briefly summarized.
Experiments were conducted in microcosms consisting ofsediment, water, plants (algae and macrophytes), and animals (planktonic and benthic invertebrates) in 4 L glassjars. Eachjar was filled with 3.8 L ofwater and hydrosoil (sediment) at aratio of9:1 by volume, and covered with a circularglass plate (15 cm diameter) to reduce waterloss. Each plate had a central hole, about 5 mm in diameter, for gas exchange. Microcosms were randomly located on abench in a 7 ft x 15 ft x 7 

### ft environmental chamber (Panel



# ' ••111.1.1 2087856Key, Earth City, MO) maintained at 20±1°C, with a ceiling bank oft1uorescent lights (Gro-Iux) providing 1,400 lux in a 16 hour light8 hour dark photoperiod. These conditions were called "standard conditions".

Soil matter (sediment from a permanent wetland and topsoil from an adjacent agricultural field) came from Clay County Waterfowl Production Area, about 21 km southeast of Vermillion, South Dakota. Sediments, portions ofoverlying water, and field soil were removed in the spring and fall and transported to the laboratory (Columbia National Fisheries Research Laboratory [CNFRLJ) within 48 hours. Topsoil contained 2.6% organic matter and 0.22% Kjeldahl nitrogen, and wetland sediment contained 9.8% organic matterand 0.79% Kjeldahl nitrogen. Soybeans and corn were grown in the fields around the wetland site, but gas-liquid chromatographic-mass spectrophotometric analyses ofthe wetland samples found no detectable pesticide residues.

Microcosm water was a mixture ofstandard reconstituted water (EPA, 1975, 

### Alethodfor acute toxicity tests withfish, macroinvertebrates) andamphibians) and natural wetland water in a ratio of8: I by volume. Reconstituted water had the following amounts ofsalts per 10 L: 0.48 g NaHCOJ, 0.30 g CaS04, 0.30 g MgS04, and 0.02 g KCI. The pH, alkalinity, and conductivity were 7.2 and 35 mg/L (as CaC03) and 150 Ilmhos/cm, respectively. Water loss due to sampling was replenished with reconstituted water, but replenishments neverexceeded 0.5% (v/v) ofthe total unit volume. Evaporative water loss was replaced weekly with deionized water from a Millipore reverse osmosis unit (Millipore Corporation, Bedford, Massachusetts), which had pH 6.0, alkalinity and total hardness 10 and 1.0 mg/L (as CaCOJ), and conductivity about 1.0 Ilmhos/cm.

After allowing one week for the sediments to settle and the waterto clear, three types of macrophytes were added to each microcosm: 

### Lemma sp. (duckweed, surface floating, 10 plants of**~50** mg each), 

### Ceratophyllum sp. (coontail, nonrooting, submergent, 15 cm segment of 109), and 

### Elodea sp. (American Elodea, rooting, submergent, 15 cm segment of5 g). 

### Elodea plants were weighted with a small segment ofglass tubing to hasten rooting in the sediment. Natural communities ofinvertebrates and algae then developed within each microcosm, with patches ofbluegreen and filamentous green algae visible on the water surface within 1 week. Cladocerans, ostracods, copepods, rotifers, and tubedwelling chironomids (attached onjar sides) appeared within several weeks. Before treatments, microcosms were cross-seeded to attain uniform ecosystem compositions. Microcosms were allowed to equilibrate for about 2 weeks before treatment with the pesticides. (Timeline is summarized as in the article; it is unclear from when the 2 week equilibration time is counted.)

Pesticides applied in the experiment were: atrazine, trifluraIin, triallate, carbofuran, [onofos, and phorate (the first three being herbicides and the lastthree being insecticides). Atrazine was Aatrex, 85.5% active ingredient wettable powder from**Ciba~** Geigy Corporation, with a solubility of70 mg/L at 20°e. Each ofthe six pesticides were

2

### tested at three different concentrations, 10, 100, and 1,000 flg/L, 

### which were introduced by stock solutions in acetone orwater based on % 

### active ingredient, mixing with 1.0 g of topsoil in alSO m1. beaker and allowing to sit for one hour, adding 50 mLreconstituted water, and then gently stirring with a glass rod and slowlypouring the total slurry into the water surface ofthe microcosm. The microcosm waterwas not mixed. Control treatments received the soil and carrier solvent. Treatments were made once and not repeated, and the experiments were terminated after 30 days. (No atrazine concentration measurements weretaken in the microcosms.)

Each microcosm series consisted offour treatments with fourreplicates pertreatment, with one treatmentbeing the control. All measurements ofspecific variables weretaken in triplicate unless otherwise stated. Most samples were taken weekly. Datawere analyzed in a split-plot design, in which the linear statistical model containedthe main effect at each treatment, replicates within treatment, weeks, and the interaction of treatment x 

### week, using ANOVA as implemented in SAS. Within-treatmentreplicates were used as errors to testthe treatmenteffect, and the residual mean square was used as the error to testthe effects ofweek and treatment x 

### week. Simple one-way ANOVA was used to analyze variables measured only atthe end ofthe experiment. Ifthe overall F value was significant atp

## :s 0.05, Fisher's least-significant-difference testwas used to find mean differences. Samples were tested for homogeneity ofvariance.

48-hour EC50 values were determined for the insecticides using standard acute toxicity tests with

### Daphnia magna and 

### Chironomus riparius; forthe herbicides, published EC50 datawere confirmed by performing bracketing experiments above and below the reported EC50. Additionally, tests were performedto evaluate directpesticide effects on daphnid growth andreproduction: a microcosm acute toxicitytest (14 or 30 days) with first-instar «24 hours old) 

### D. magna and early fourth-instar 

### C. riparius larvae using standard toxicity testmethods; a viability testwherein 25 mature (>2,500 flm 

### long), gravid daphnids were addedto each microcosm48 hours before treatment, with the population monitored throughoutthe study and replenishedwhen less than 5 organisms were observed; and a7-day chronic toxicity test (not in microcosms). (Further details on these experiments will notbe summarized here, but are in the article.)
Phytoplankton toxicitytests assessed the short-term growth inhibition of

### Selenastrium capricornutum in microcosm water. Actively growing algae were concentratedby centrifugation andresuspended, and 1 mg (dry weight) ofthese cells was placed into a 125 mL Erlenmeyer flask containing 50 mL ofamicrocosm's water. An algal bioassay was conducted according to the methods ofa 1978 EPA report on the protocol for bottle tests with this alga (EPA-600/9-78-0

### 18). The flasks were harvested after 7 days and the dry weight biomass ofthe algal cells was determined gravimetrically with tared Gelman glass fiber filters.



### 3Water quality measurements (dissolved oxygen, conductivity, pH, hardness, and alkalinity) and total organic content ofthe sediments were determined using American Public Health Association (1980) standard methods. Total nitrogen and total phosphorus concentrations in the water and sedimentwere measured with a Technicon Auto Analyzer II (Technicon Inc., Tarrytown, New York) by a colorimetric method afterpersulfate digestion.

Gross primary productivity and community respiration wcre estimated from diurnal changes in dissolved oxygen in the microcosms, overtwo days, following the methods of McConnell (1962, 

### Productivity relations in carboy microcosms, Limnol. Oceanogr.). The glass coverofthe microcosms was removed 1 day before and during sampling days. The three-point diurnal measurements were made weekly.

Total macrophyte biomass was measured at 30 days. All macrophytes were collected from each microcosm, washed to remove soil, dried for several days at 50°C, cooled, and weighed, to determine total macrophyte dry weightbiomass.

Sediment microbial activity bioassays were conducted by removing 

### 25.0 g (wet weight) ofhydrosoil and 25 mL ofwater from amicrocosm and mixing in 1,000 Ilg/L pesticide with a magnetic stirrer, with tests beginning after I hour ofincubation at 20°e. Further tests at 100 and 10 Ilg/L were only to be conducted ifthe initial tests showed at leasta 25% deviation from controls. Respiration (oxygen consumption), electronic transfer system (ETS) activity, monophosphotase activity, and mineralization ofglucose were tested using published methods. Oxygen readings were taken every 15 minutes for I hour with a Gibson differential respirometerusing a constant pressure manometric technique. ETS activity was measured with the 2-(p-iodophenyl)-3-(p-phenyl-5-phenyl) tetrazolium chloride (INT) method, with an INTmixture (6.0 mL microcosm water, 2.0 mL 0.2% INT, 1.0 mL 0.1 % sodium citrate) blended into I mL ofsedimentslurry in a 50 mL centrifuge tube with a vortex mixer, the mixture incubated under nitrogen gas in a stainless steel anaerobic containerfor 1hourat 28°C, and the INT-tormazan contentof the solids measured as follows. The INT-formazan was extracted with 25 mL 95% ethanol in a vortex mixer until no red-colored formazan was beingremoved, the ethanol batches were mixed, and an aliquot was centrifuged at 10,000g at 3°C for 5 minutes and analyzed with a Spectronic 100 spectrophotometerwith a flow-through cell (Bausch and Lomb, Rochester, New York) at480 nm. Absorbance values were corrected with controls and compared to an INT-formazan standard curve. Monophosphotase activity was measured with amodifiedp-nitrophenol (PNP) method, with a PNP mixture (7.0 mL bufferof0.05 M tris(hydroxymethyl)-aminomethane [THAM] at pH 8.0 and 2 mL of 1% PNP) added to 1 mL ofsediment slurry in a 50 mL centrifuge tube, the mixture incubated at 35°C for 1.5 hours while mixing every 15 minutes on a vortex stirrer, and the PNP contentofthe solids measured analyzed as follows. The uptake was terminatedby adding 10 mL ofa chilled acetone:THAM solution (l: 1 by volume, pH 12) and placing the samples on crushed icc, tben the samples were centrifuged at 10,000 g and 3°C tor about 5 minutes and spectrophotometrical1y analyzed at 410 nm. Absorbance values

4were corrected to controls (types ofcontrols were hydrosoil with substrate, which were not incubated, and hydrosoil without substrate, which were incubated). Glucose uptake was measured using a 14C-labeled glucose (uniformly labeled, 300 mCi/mM, Pathfinder, Inc., 81. Louis, Missouri). ] mL ofsediment slurry was added to a sterile 50 mL Erlenmeyer flask containing 10 mL ofmicrocosm water, 0.1 ).lCi of14C-Iabeled glucose, and 25 ).lg nonlabeled glucose, and the flasks were tightly sealed with arubber stopper and incubated for] hour at 20°C in the dark. Labeled CO2 (corresponding to mineralized glucose) was captured in a 1.0 mL volume of0.2 N 

### KOt! in a suspended glass vial, the reaction was stopped by acidifying the mixture with 0.1 N HCl andplacing on crushed ice, and samples were refrigerated overnight at 2°C for increasedrecovery. The total volume ofthe KOH trapping solution was analyzed with liquid scintillation counting, after mixing with a 15 mL Fluoralloy:Triton X-I00 scintillation cocktail (14: 1 by volume) (cocktail and scintillation counterby Beckman Instrument Inc., Fullerton, California). Values were corrected for counting efficiency, quench, and background radiation.

Results: This study examined the effects of3 different herbicides (including atrazine) and 3 different insecticides, in a nested experimental design. Also, some animal toxicity experiments were conducted to examine the effects ofthe insecticides in particular. In this review, only the results pertaining to atrazine effects on the microcosms will be fully described; results not relevant to atrazine will be omitted, andresults tangentiallyrelevant to atrazinc or conducted outside the microcosms will be briefly summarized.

The published EC50 values for atrazine toxicity to D. 

### magna and C. 

### riparius were confirmed; EC50 values were 3,600 and 1,000 ).lg/L, respectively. Other animal experiments found no evidence ofatrazine influence on daphnid survival, growth, or reproduction (same population of] was established posttreatment in all treatment levels, including controls), no acute toxicity effects on daphnids or chironomids after 14 and 30 day exposure periods, and no effects ofchronic exposure at 10 or 1,000 Ilg/L on daphnid adult or instar survival. Forthe 7-day chronic toxicity test, the treatment levels of0, 10, and 1,000 ).lg/L atrazine had: 90% gravid females at all treatment levels; the number of adults >1,500).lm long were 37.5 ± 5.4,46.7 ± 9.0, and 45.3 ± 6.0, respectively; and the number ofinstars <1,500).lm long were 28.7 ± 13.5,20.5 ± 1.7, and 20.0 ± 5.3, respectively; there were no significant differences at pSO.05. (Numbers are means ± standard deviations with fourreplicates.)
Phytoplankton lethality tests found that atrazine exposure of 1,000 ).lg/L resulted in significant (psO.05) biomass reduction. Dry weight algal biomasses, with acute exposure after 30 days in the microcosm, were (mean ± standard deviation with four replicates): 5.8 ± 0.8 mg at 0 Jlg/L 6.9 ± 0.5 mg at 10 Ilg/L 6.6 

### ± 0.9 mg at 100 Jlg/L



### 53.7 ± 0.5 mg at 1,000 flg/L

Gross primary productivity (GPP) gradually increased overtime in most microcosms (Figure 1). There was some initially wide variation within treatments which the author attributes to differences in macrophyte establishment. Immediately following atrazine treatment, there was a decline in GPP, and dissolved oxygen and community respiration decreased as well. Reductions in GPP relative to controls were 23, 32, and 91% in the 10, 100, and 1,000 flg/L treatments, respectively. The 10 and 100 flg/L treatments recovered at 7 days and remained similarto controls until the experiment ended, butthe 1,000 flg/L treatmentneverrecovered.
Macrophyte biomass was not affectedby 30 days ofatrazine exposure at 10 or 100 flg/L, but there were significant (p:S;0.05) reductions ofabout 50% in the 1,000 flg/L treatments. Dry weight total biomass results were (mean ± standard deviation with four replicates): 1.64 ± 0.56 g at 0 flg/L 1.94±0.71 gat 10 flg/L 1.45 0.20 gat 100 flg/L 0.86 ± 0.24 g at 1,000 flg/L

Waterquality measurements showed some response to atrazine treatment. Conductivity in the 1,000 flg/L treatments was increased about 20% over controls andpretreatment values (Figure 2); othertreatment levels showedno significant (p:S;0.05) differences from controls. Additionally, pH was lower in the 1,000 flg/L treatment microcosms, and on 3 ofthe 4 posttreatment weeks was significantly (p:S;0.05) different from control (Figure 3). Again, othertreatment levels showed no significant differences. Alkalinity measurements at 30 days were doubled in the 1,000 flg/L treatments compared to the controls, a significant (p:S;0.05) difference, but the othertreatments did nothave significant differences. Final (30 day) alkalinity measurements (in mg as CaC03) were (mean ± standard deviation with four replicates): 104.0± 27 at 0 flg/L 96.0 ± 46 at 10 flg/L 142.7 ± 65 at 100 flg/L 229.0 ± 39 at 1,000 flg/L The author describes apossible mechanism for the microcosm feedbacks resulting in these water quality changes. Total nitrogen (TN) generally increased overtime in all microcosms, by about 28% overthe 30 days ofthe experiments, with no noticeable effects due to atrazine treatment. On the otherhand, total phosphorus (TP) tended to decrease about 50% overthattime period, in controls and at 10 and 100 flg/L atrazine treatment levels. At 1,000 flg/L atrazine, 

### TP remained similarto pretreatment levels. Total organic carbon in the microcosm sediments showed no significant (p:S;0.05)

6differences between treatments and controls atthe end ofthe 30 day experiments, with a range in dry weightofabout 8-12%.

Microbial activity measures were not affected significantly (p:S0.05) by atrazine exposure. Only 1,000 /lg/L tests were performed. Uptake rates ofINT-F (2-(Piodophenyl)-3-(p-phenyl-5-phenyl) tetrazolium-formazan, for determining electronic transfer system activity), PNP (p-nitrophenol, for determining monophosphatase activity), O2, and glucose were measured per gram dry weight ofhydrosoil (except for glucose uptake rates, which are expressed per gram wet weight hydrosoil). Results were (mean ± standard deviation with four replicates): /lg INT-F/g/hr: 2,616 ± 87 (treatment); 2,615 ± 162 (control) /lg PNP/g/hr: 517 ± 28 (treatment); 481 ± 38 (control) IlL Oz/g/hr: 90 ± 13 (treatment); 89 ± 9 (control) /lg glucose/g/hr: 2.6 ± 0.3 (treatment); 2.7 ± 0.4 (control)



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Figure 1. Influence ofatrazine on gross primary productivity. All values are means offour replicates. Asterisks denote significant differences**(p~0.05)** from control; mean differences determined by ANOVA, based on a nested design. Nominal atrazine concentrations were control (open circle, 0), 10 IJ.g/l, (solid square, _),100 prg/l, (open square, D), and 1,000 prg/L (solid circle, e). Day0 denotes the day ofpesticide treatment.



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350 

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Figure 2. lnfluence ofatrazine on conductivity. All values are means offour replicates. Asterisks denote significant differences**(p~0.05)** from control; mean differences determined by ANOVA, based on a nested design. Nominal atrazine concentrations were control (open circle, 0), 10 f1g/L (solid square, _),100 f1g/L (open square, 0), and 1,000f1g/L (solidcircle, .). Day

# °denotes the day of pesticide treatment.

9

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Figure 3. Influence ofatrazine on pH. Allvalues are means offour replicates. Asterisks denote significant differences (p:::S0.05) from control; mean differences determined by ANOVA, based on a nested design. Nominal atrazine concentrations were control (open circle, 0),10 Itg/L (solid square, _),100 /lglL (open square, 0), and 1,000 Itg/L (solid circle, .). Day 0denotes the day ofpesticide treatment.

10From study Table 5. 

### Data Summary Table: P-values are shown for significant difference from controls. Effect Magnitude of Nominal Dayof Study Time Did Comments Effect relative Atrazine Effect Weighted Recovery to controls Concentration Occurred Average Occur

- if (pg/L) Atrazine so, when Concentration (see ! from Day 0to comment)? I When Effect 

## j v



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# ~~~~~6~~t-----+-1.0--f.l.-g.I-L-_---l--N-A-_-~.-j---.----i Effectwas macrophyte No effect 100 NA evaluated at biomass ( >0.05) end ofstudy. I 50% (p<0.05 1.000 ug/L Dav 30 NA

Decrease**L..~~~<0.05** .l.QJl:.g/L Day 2 NA NA

, From Fig.l

### ;~~~sar 32% (p>0.05 lQQ..gg/L II Day 2 

## I**~~ ~I** **N~A** I and text p. Yt"t 91% (.p:S0.05) 1,000 II

## giL Day") , No 480. roduc IVI y' I'" Change In f-N'o effect II NA- 

# k

A

- - NA-----+----------J 10 IIgll From study daphnid 1 (p>0.05) I'" j 

# I f-.- -----+'-----.- Table 3 and reproduction No effect 

### I 1000- 

# ,Z 

# INA NA

### ~NA textp. 479. 

### * (p>0.05)

## .---~g 

# --=t ----+--------1 No effect 10 IL NA NA NA Change in (p>0.05) IJ,g

### ---~---**-._~--=E-** 

# ----- From study sediment No effect ,I NA

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# I NA and textp. conductivitv 100 IIglL NA I NA 

## ') I (p>0.05) i I'" I L- ---1 481. 20% (p:SO.02..L+ 1,000 Ilg /L 

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### I estimated

### >_0.05t_~ __!g 

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. 1 from Fig.3.

_____ ..10% (p<0.05) i 1,000 J.lglL

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### I NA**(p>O.O~** J.l

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# 1:0%**~~ ~-·---1·**  NA

- - EffeotW",!' Table 6. ( <0.05) 

### I 1,000 J.lg/L Day 30 I measured at p- I

. _-L..--. I I end ofstudy I**~----_'-:** Note: NRc=Not Reported, NA=NotApplicable

a Atrazine concentrations were not measured; therefore, time-weighted average values were not detelmined.

11

### No atrazine concentration measurements were taken in the microcosms, so the actual concentrations are unknown. Time weighted averages were assumed equal to nominal due to the lackofinformation.



### Reviewer Comments: This study provided good replication, with four replicates ateach ofthree treatment levels plus four controls. A wide range ofmeasurements were taken on various components ofthe microcosm systems. However, the study is strongly limitedby lack of atrazine concentration data. The atrazine was added as partofa soil slurry, which was not mixed into the microcosms but allowed to naturally settle from the top, in an attempt to recreate natural runoffconditions. The methodology is also likely to result in atrazine concentrations much differentthan nominal, probably smallerconcentrations due to adsorption to the soil particles in the slurry. This may be why so little effect was found from the 10 and particularly the 100 Ilg/L atrazine treatments.

As with any microcosm study, there are limitations to how realistic the laboratory conditions could be in recreating a natural aquatic environment. The microcosms in this study contained areasonable setofaquatic ecosystem components, including macrophytes, but still may notbe complex enough to stand in for areal pond. One ofthe primary shortcomings is the ill-described, apparently somewhat short acclimatization process. It

### does not appearthatthe microcosms had reached steady state when the experiment commenced; for example, the authornotes that some within-treatment variability in OPP was probably dueto differences in macrophyte establishment.


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

Rationale for Use: Study evaluated relevant endpoints and contributes to the weight ofevidence regarding potential effects ofAtrazine on aquatic plants. However, atrazine concentrations were not measured.



### Limitations ofStudy: No atrazine concentrations were measured, but areprobably far from nominal due to surface-only application within a soil slurry. Microcosms may be too simplified to apply to natural environments, and may nothave reached steady state by the start ofthe experiment.


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


### Secondary Reviewer: Brian Anderson

12