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

ECOTOX Record Number and Citation: Jurgensen, T.A. and K.D. Hoagland. 1990. Effects ofshort-tenn pulses ofatrazine on attached algal communities in a small stream. Arch. Environ. Contam. Toxicol. 19:617- 623. (MRID 47543504)
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 LaC for atrazine.

Date ofReview: October 16,2008
Purpose ofStudy: This study examines the potential effects ofatrazine to attached aquatic plants in stream enclosures.
Summary ofStudy Findings:

Methods: The study was conducted in a spring-fed stream, OtterCreek, in westernNebraska near Lake McConaughy (a map is provided in the studybutnotreproducedhere). The stream watershed was hillyrangeland with sandy soils, used primarily for cattle grazing. Background levels ofatrazine were not detected in the stream in analyses ofthree streamwater samples taken atthe beginning, middle, and end ofthe 6-week study period. The stream pH was 8.0, with total alkalinity of63 

### mglL as CaC03, and anitrate-nitrogen concentration of0.64 mg/L. Atthe study site, the stream was 2-3 m wide with an average depth of30-40 cm and typical flow velocity ranging from 0.5 to 1 m/s.
Unglazed baked clay square tiles, 7.6 cm long on each side, were used as periphylon substrates. Eighteen tiles were attached to each treatmenttray, consisting ofaplexiglass plate anchored to the stream bottom. Five treatment trays were used at each ofthree


# III11IIIII 2087855replicate sites, with an experimental control (no enclosure), atreatment control (enclosed but with 0 jlg/L atrazine), and treatments at 2 Ilg/L, 30 Ilg/L, and 100 Ilg/L atrazine.
Initially, the trays were leftto colonize for two weeks priorto the first treatment (on June 21, 1985), andthen enclosures were attached to the 4 enclosure trays (excluding the experimental control tray) at each site. Treatmentenclosures consisted ofplexiglass boxes (30.5 x 25.2 x 46.6 cm high), open on top and bottom, attached to the trays with neoprene seals to prevent leakage and weighted to prevent movement with the current. The height ofthe boxes was sufficient to extend above the water surface and keep the trays isolated from the surrounding stream. Atrazine (98%, Supelco, Houston, TX) was dissolved in deionized water and added to enclosures to achieve the desired nominal concentration, including the control enclosure that received only deionized water. The enclosures were maintained for 24 hours and then removed. On Day 14, a second 24- hour atrazine exposure was applied.
Periphyton were sampled 2, 7, and 14 days afterthe end ofeach atrazine application period. For each tray, 2 tiles were removed and scraped with arazorblade, with the resulting algal suspensions preserved in 6:3: 1 water:ethanol:formalin. These samples were analyzed for cell counts and total biomass. Cell counts ofcommon and abundant algal taxa (comprising at least 35 of500 diatom frustules and 10% oftotal algal density, based on preliminary counts) were conductedusing a Palmercell at400 x magnification with an Olympus model FH microscope. Living and nonliving cells were tallied separately. Permanent slides were also made, using Hyrax mounting medium to facilitate diatom identifications. Total biomass was determined as ash-free dry weight (AFDW) modified fromAmericanPublicHealthAssociation, 1981, Standard methods for the examination ofwater and wastewater (APHA 1981).

A 200 mL water sample was taken from two ofthe 100 jlg/L treatment enclosures at the end ofeach 24 hour exposure period for atrazine analysis. The samples were extracted with pesticide-quality methylene chloride (J.T. Bake Chem. Co.) within 8 hours after collection by shaking twice, for 2 minutes, in a separatory funnel and drawing offthe methylene chloride. Extracts were stored at4°C and later concentratedto 10 mL in a Kudema-Danish evaporator with a 3-ball Snydercolumn, treated with 10 mL ofhexane, and concentrated to 5 mL (following APHA 1981). Extracts were analyzed for atrazine using aTracormodel 560 gas chromatograph, a Hall 700A electrolytic conductivity detector in the nitrogen mode, and a glass column packed with 35 STAP on 80-100 CWHP. Standards were made from 98% pure atrazine (Shell, Pesticides Division). The method had a detection limit ofabout 50 jlg/L. Likely actual atrazine concentrations for the 2 jlg/L and 30 jlg/L nominal treatments were extrapolated from the 100 Ilg/L results.

Statistical analyses were performed on the periphyton biomass and live algal cell density using 3-way ANOYA with repeated measures to account for the autocorrelation ofstream samples over time. Sample sizes, especially afterthe second 24-hour atrazine exposure,



### 2were smallerthan expected due to loss of"several" sample trays "due to storm runoff' (no further details provided). Because ofthis problem, data were analyzed for collections made afterthe first exposure (3 sampling dates) and for collections made afterboth exposures (6 sampling dates), ratherthan after each exposure. Additionally, regressions wereperformed on AFDWs, ash weights, total living algal densities, and nonliving diatom densities.

Results: Atrazine concentrations measured in the nominal 100 Jlg/L enclosures atthe end ofthe 24 hour sampling period ranged from 58 to 93 Jlg/L, with an average of77.5 Jlg/L. The authors state that, assuming similar atrazine losses from the othertreatment level enclosures, the nominal 2 Jlg/L treatments would have atrazine concentrations of 1.2-1.9 Jlg/L, and the nominal 30 Jlg/L treatments would have atrazine concentrations of 17-28 Jlg/L. (Averages, assuming 77.5% ofnominal, would be 1.55 Jlg/L and 22.5 Jlg/L in the nominal 2 and 30 Jlg/L treatments, respectively.)

Biomass declined in all treatments overtime, butno significant differences were found between the four atrazine treatments (including the treatment control). As shown in Table 1, 3-way ANOVA found significant effects oftime (p-value 0.05 forthe first 3 sample dates, p-value == 0.0004 taking all 6 sample dates) on total biomass, butno significant effects oftreatment (p-value = 0.73,0.60). The experimental control and treatmentcontrol had significant differences, particularly in the total biomass compared on all 6 sample dates (p-value of0.01) (seeTable 2). The experimental control, treatment control, and 100 Jlg/L treatment dynamics oftotal biomass as well as Oscillatoria live cells andNitzschiapalea live cells are graphed in Figure 1, Figure 2, and Figure 3, respectively. Total biomass ofexperimental and treatment controls are also tabulated in Table 4.

Oscillatoria sp. was the most abundant alga, andNitzschiapalea was the dominant diatom. Green unicells and the common diatoms Fragilariapinnata andNavicula luzonensis were also examined. None ofthe species showed any significanttreatment effects (Table 1), exceptpossiblyFragilariapinnata on the first three sample dates (pvalue == 0.07).

Sediment (measured as ash remaining after incineration oforganic material) increased overtime in all ofthe treatments, in rough correspondence with biomass decline (Table 3). The authors attribute the biomass decline overtime on the tiles to sediment deposition, which they qualitatively argue (citing related workby otherauthors) is a much stronger effecton the attached stream community than the atrazinetreatments used in theirexperiment. These assertions are notquantitatively analyzed. (The authors present lists ofreferences on maximum concentrations ofatrazine residues in surface waters and on atrazine exposure studies for algae, butthey are notreproduced in this review.)



### 32



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30 Time (days) Figure 1.**Ash~free** dry weight biomass (mg/cm2) ofattached stream communities on tile substrates in experimental control, 0Jig/L treatment control, and 100 ",gIL atrazine enclosures. Vertical bars represent standard errors; arrows on time axis represent onset of24-hour atrazine treatment periods.

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## at 20 30 Time (days) Figure 2. Oscillatoria live cell densities (cells/mm2) on tile substrates in control, 0 ",gIL treatment control, and 100Jig/L atrazine enclosures. Vertical bars represent standard errors; arrows on time axis representonset of24-hour atrazine treatment periods.

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### of 10 20 30 Time (days) Figure 3. Nitzschiapalea live cell densities (cells/mm2) on tile substrates in control, 0J1g/Ltreatment control, and 100J1g/L atrazine enclosures. Vertical bars represent standard errors; arrows on time axis represent onset of24-hour atrazine treatment periods.

Table 1. P-values from 3-way ANOVA for comparison ofbiomass and live cell densities offour atrazine treatments (0, 2, 30, and 100 J1g/L) with respect to treatment, time, and treatment effects with time (Trt. x Time) for the first three sampling dates (only the first atrazine treatment) and all six sample dates (including both atrazine treatments). (study table 1)
First 3 sample dates Treatment Time Oscillatoria sp. 0.82 0.97 0.39 0.50 0.03 0.67 Nitzschiapalea (Klitz) 0.17 0.1 I 0.92 0.70 0.002 0.85 Greenunicells 0.42 0.2I 0.76 0.44 0.09 0.90 Fragilariapinnata Ehr. 0.07 0.22 0.90 0.1 I 0.03 0.92 Navicula luzonensis Hust. 0.25 0.62 0.91 0.71 0.81 0.57 Total biomass 0.73 0.05 0.76 0.6 0.0004 0.88

Table 2. P-values from 3-way ANOVA for comparison ofbiomass and live cell densities offour experimental control and treatment control (0J.1g1L) with respect to treatment, time, and treatment effects with time (Trt. x Time) for the first three sampling dates (only the first atrazine treatment) and all six sample dates (including both atrazine treatments). (study table 4)

Oscillatoria sp. Nitzschiapalea (Klitz) 0.31 0.05 0.04 0.30 0.63 0.8 I 0.03 0.03 0.21 0.06 0.65 0.60
0.01Table 3. Ash (mg/cm2 ) remaining afterorganic materialwas incinerated during ash-free dry weight analysis. Residual ash consisted mostly of inorganic sedimentdeposited on the periphyton communities. Percentages are the fraction oftotal dry weight composed ofash. (study table 2)

2 ug/L 100ug/L Sample Community Standard % Standard % Standard % Standard %

date a e Mean Deviation Ash Mean Mean Ash 25-lun 17 days 6.7 1.5 85 25 3.2 95 48 34 95 28 14 94 30-lun 22 days 8.1 2.9 83 23 9.3 95 16 9.3 88 25 26 91 7-Jul 29 days II 0.2 91 16 9.9 95 36 50 92 18 1.6 96 10-Jul 32 days 23 4.1 95 20 1.4 96 18 8.2 93 49 

### * 97 15-Jul 37 days 45 38 91 19 5.3 96 27 15 97 58 22-Jul 44 days 16 1.6 97 28 29 91 34 2.4 99 89

Table 4. Ash-free dry weight biomass (mg/cm2) from the experimental controls (no enclosure) and treatment controls (0 ug/L) for the six sample dates. (study table 3)
Treatment C..antral

Sample

date Mean Standard Deviation Mean Standard Deviation 25-Jun 1.05 0.52 1.16 0.23 30-lun 1.11 0.33 1.6 0.53 7-Jul 0.44 0.26 1.l5 0.58 10-Jul 0.09 

### * 1.l7 0.35 15-lul 0.01 

### * 1.13 0.6 22-lul 0.15 

### * 0.42 0.21 

### *No standard deviation due to loss ofreplicates

6Data Summary Table: E.1fect Magnitude Nominal Day of Study Time Weighted Did Comments of Effect Atrazine Effect Average Recovery relative to Concentration Occurred Atrazine Occurcontrols (flg/ L) 

# I Concentration ifso, Overthe Study when (.,ee 

# +
Dun"'an comment) 

### I 

# IL- Total 2 "giL (short-

# ~j gIL Over all biomass,**~tenn** pulse)_**__~~____** j sample dates, live eell 30 "gIL (short- Not , I 7-"giL--j p-value for densities of No effect -,<,nlU'u!,,) ti"hle 

# I**~,** "g NIA tr,"ment misc. 100 !!g/L (short- PP effectontotal**abundant~** tenn pulse) 5.2 !!g/L 

### I biomass was**~hy~n** _ -- : 0.60 --------'- Average exposure concentrations overthe study duration are included in thepreceding table.

Reviewer Comments: The study conditions were apparently not sufficiently controlled, as evidenced by the disappearance ofmultiple trays. Perhaps more importantly, the details ofthe events surrounding the trays being carried off(storm conditions, timing, exactly which trays were lost, etc.) were not clearly discussed. Forexample, the number ofreplicates changed overtime, buthow orwhen replicate numbers changed was notreported. The only information that trays were missing is in Table 3 and Table 4 when standard deviations cannot be reported. It is not clearhow the runoffevent leading to the missing trays affected the study, but it could have had significant impact. Taking the authors' own assertions about the importance ofsedimenteffects, the sedimenttransport ofa storm event could have had amultitude ofeffects on the streambed periphyton, including scouring them out, burying them, or carrying away the sediment built up overtime. Overall, the issue ofthe storm that apparently tookplace during the experimentreceived insufficientdiscussion and may have confounded the results.

Theresults presented are lacking in certain details. No regression results appearin the study, despite the description in the methods section. No sedimentresults are provided for the experimental controls, and no statistical analysis is used to test correspondence between sediment and biomass. The authors' points about the importance ofsediment are thus notwell supported by the results presented. Also, the presentation ofthe sample date timing in the methods section is confusing. Forexample, the methods section states that the first sample was taken 2 days afterthe end ofthe first enclosure period (so 3 days afterthe beginning ofenclosure), and thatthe first atrazine treatment began June 21. However, as shown in Table 3 and Table 4, the first sample date was June 25, notJune 24 as would be expected. These inconsistencies confuse time weighted average calculations.Thereasons for and implications ofsignificantdifferences between the experimental controls and the treatment controls are not discussed. However, the fact that this was the single most significantresult found for treatment effects (p-value of0.01 for treatment effects on total biomass over all sample dates) shows thatthere musthave been amajor impacton the periphyton community simply from imposing 24-hourenclosures in deionized water. This calls into question whetherthe atrazine treatment results are applicable to a more realistic flow-through situation.
Also, the detection limit was 50 

### ug/L. Therefore, the actual test levels atthe nominal 2 

### ug/L and 30

### ug/L nominal test concentrations are unknown. The measured:nominal ratio observed at the 100 

### ug/L nominal test concentration was applied to the 2 

### ug/L and 30 ug/L test levels to estimate a "measured" exposure value.
Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: Study results may have been confounded by a storm event and by the high detection limit ofthe analytical methods used in this study. However, the study contributes to the weight ofevidence ofpotential atrazine effects.

Limitations ofStudy: This study examinedpotential impacts ofshort-term (24-hour) pulses on atrazine on streambed periphyton communities, which did not create noticeable effects. However, the study is limited by: 1) the occurrence ofa storm event during the study, which decreased the number ofreplicates and couldhave overshadowed any subtle atrazine impacts; and 2) the finding ofsignificant differences between the treatment controls (enclosed with 0 

### Jlg/L atrazine treatment) and experimental controls (no enclosure), which points to the artificial nature ofthe atrazine treatmentregime. Also, time weighted average concentrations are highly uncertain, particularly in the 2 ug/L and 30 ug/L nominal levels where atrazine concentrations were not measured. Also, the detection limit in this study was 50 

### ug/L; therefore, atrazine may have been present at background concentrations <50 

### ug/L.
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc
Secondary Reviewer: Brian Anderson j}7//1

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