### Open Literature Review Summary Chemical Name: Atrazine CAS No: 1912-24-9
ECOTOX Record Number and Citation: Lynch, T.R., RE. Johnson, and W.J. Adams. 1985. Impact ofatrazine and hexachlorobiphenyl on the structure and function ofmodel stream ecosystems. Environ. Toxicol. Chern. 4:399-413. 

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### .(t>Lf3>S12. ") 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: December 5,2008 Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental streams (lotic mesocosms). Summary ofStudy Findings:
Methods: Experiments were conducted with aPCB isomer (2,4,5,2',4',5'- hexachlorobiphenyl) as well as atrazine. Only the methods associated with atrazine experiments will be described here. Laboratory streams were constructed from existing concrete troughs in a former fish hatchery ownedby the State ofMichigan. The streams consisted oftwo sections: an upper constant-headreservoir (3.96 m x 

### 0.58 m x 

### 0.27 m), and an identically sized lower portion that served as the stream channel. Discharge in the stream channel was maintained at 60 L/min by gate valves (water depth 

### vs. velocity not described). Water diverted from the adjacent spring-fed woodland stream, Cheny Creek (no further description ofstream order orwatershed characteristics), was gravity-fed into the reservoirs after screening out large debris (mesh size notprovided). Stream effluentwas filtered through activated carbon and polyurethane foam filters before discharge.



# , •••••••11 2087809Watertemperature was not adjusted and so followed the natural diurnal and annual fluctuations ofthe creek, ranging from a maximum of 15.5°C in May to a minimum of 0.5°C in December. Water quality parameters also changed overtime, ranging from 9.0 to 11.2 mg 02/L (dissolved oxygen), from 164 to 202 mg CaC03/L (total alkalinity

- units are as appear in the article, though there appears to be atypo), and from 182 to 217 mg CaC03/L (hardness). Stream channels were illuminated by "high-intensity" coolwhite fluorescent lamps suspended above the streams (luminance/irradiance and exact spatial parameters not described). Photoperiod, controlled by an automatic timer, was adjusted every two weeks to correspond to natural time-varying day-length.
The laboratory stream channels were lined with natural substrate to adepth of5 to 10 cm. Substrate was transferred in June 1975 from Buckhorn Creek, Mecosta County, Michigan, anatural creek located about 1 km from the hatchery. Cheny Creek substrates were not available because the creek had been channelized and lined with concrete in the vicinity ofthe hatchery. One year was allowed for biological colonization ofthe laboratory streams by the organisms incidentally introduced with the substrate and those drifting in the incoming water supply. Experiments tookplace overthe second year (1976-1977).
Two replicate streams were treated with atrazine in four 30-day phases

- autumn, winter, spring, and summer- each followed by a 60-day recovery phase (except for the summer treatment, which had no recovery). Treatmentwas conducted with technical-grade atrazine (Ciba-Geigy, Greensboro, North Carolina) stock solution dissolved in dimethyl sulfoxide (DMSO). One stream designated as control received DMSO only. Syringe pumps delivered stock solutions to turbulent mixing chambers as the lower end ofthe reservoirtroughs (see Figure 1). Nominal atrazine concentration in the treatment streams was 25 jlg/L. Solvent concentration in both treatment and control streams was 0.69 jlL/L. In each seasonal treatment cycle, data collected included "chemical residues in water, sediments, invertebrates, fish and plants" (not further described in the paper's methods orresults presentations); species composition and abundance ofthe macroinvertebrate benthos and drift; periphyton growth; and primary production and community respiration rates.
Benthic invertebrates were sampled in both pre-treatment and treatmentphases ofthe 2- year investigation. Two samples per stream were taken at45-day intervals during the pretreatmentyear, and three samples per stream were taken at 30-day intervals during the treatment year. Sampledpositions were determined using a grid system and atable of random numbers (grid spacing and underlying distribution ofrandom numbers not described). A 10.2 cm diameter core sample was removed from each sample location and preserved immediately in 10% (v/v) formalin. Organisms were later separated from the substrate material by sucrose flotation and examination ofthe substrate under magnification. Organisms were preserved in 70% alcohol and later identified and enumerated. Subsequently, the organisms were oven-dried at 50°C for 12 hours, cooled, and the dry weight determined. The Shannon diversity index and species equitability

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### index (not definedwithin the article, although citations are provided) was computed for each sample. Pretreatment data was pooled overall six streams (including the three streams testing PCB isomereffects).
Driftinvertebrates were sampled on days I, 5, 10,20, and 29 ofthe treatmentphases and days 14,42, and 60 ofthe recovery phases, and species composition and abundance determined. Drifttraps were 10-cm lengths of10.2 cm diameterpolyvinylchloride pipe, with one end screened with 0.5-mmNytex mesh. The traps were inserted into awooden weir atthe downstream end ofeach stream that had two 10.2 cm diameter openings, so that all drifting organisms were captured as the entire stream discharge passedthrough the traps. Atthe time ofthis sampling, incoming drifting organisms were removed from source waterusing traps attachedto water supply pipes atthe head ofeach stream. Drift traps were changed every 4 hours (except 2400-0800 hours) and collections totaled over the 24-hourday. Organisms were sorted (no details on sorting procedure), preserved in 70% ethanol, and lateridentified and enumerated. Periphyton growth was measured on four glass microscope slides (25 mm x 

### 75 mm) held in arack, vertically in the streamjust above the bottom, with the long axis ofthe slide parallel to the flow ofthe water. Slides were sampled (removed) at4-day intervals for 28 days priorto the startofeach treatment and for an additional 28 days during treatment. Afterremoval, the slides were dried on ahot plate and the periphyton removed by scraping with arazorblade into tared aluminum pans. The samples were oven-dried at 105°C for 24 hours, cooled, and weighed to determine dry weight. Then the samples were combusted at 500°C for 1hour, cooled, and reweighed to determine ash-free dry weight. Loss on ignition was consideredto be accrued organic material.
Gross primary productivity and community respiration rates were determined from diurnal changes in dissolved oxygen using photosynthesis-respiration chambers, as describedby Maki and Johnson (1976), 

### Evaluation of a toxicanton the metabolism of modelstream communities, J. Fish. Res. Board Can. Three chamberbottoms were each buried at I-m intervals in the substrate ofeach stream atthe time the streams were first established. Formeasurement, the chambertop was placed overthe chamberbottom, and the chamber filled with stream wateruntil airbubbles were gone. A dissolved-oxygen probe was then inserted into aport on the upper surface ofthe chamber. Daily rates of primary productivity and respiration were determined using the method ofWhitworth and Lane (1969), 

### Effects of toxicants on community metabolism inpools, Linmol. Oceanog. Results were calculated as mg 02 

### produced or consumedperm2

### per day, based on the area-to-volume relationship ofthe chamber. (Chamber set-up and calculations are not further described.) Data from three chambers were averaged to yield a mean value for each stream on each measurement day: days 2, 4, 8, 15, 24, and 29 during the treatment phase, and days 20, 42,54, and 60 ofthe recovery phase, for the autumn period; and days 26 and 30 ofthe treatmentphases and days 20, 40, and 60 ofthe recovery phases during the winter, spring, and summerperiods.

3Statistics were computed using the ANOVA (analysis ofvariance) subprogram ofSPSS (Statistical Package for the Social Sciences) software. Effects ofatrazine and time (month oftreatment or recovery) on the community-level variables were initially tested with two-way ANOVA, with data from the four seasonal periods analyzed separately. Specific differences were also evaluated using apriori orthogonal contrasts and Duncan's multiple range test. Student's ttests were used to determine significance ofdifferences over annual means for pretreatment and treatment years.
Results: Experiments were conducted with a PCB isomer (2,4,5,2',4',5'- hexachlorobiphenyl) as well as atrazine. Only the results associated with atrazine experiments will be described here. Note that some figures will display HCBP (2,4,5,2',4',5'-hexachlorobiphenyl) results as well as atrazine results.

The benthic macroinvertebrate community was dominated by scraper and gatherer functional groups, including 23 taxa from 9 orders (list provided in the article, but is not reproduced here). The number oftaxa persample, organisms per square meter, and standing biomass were generally higherduring the treatmentyearthan in the pretreatmentyear, while species diversity and equitability decreased during the treatmentyear. None ofthese differences were statistically significant atp-value < 0.05. See Table 1. Monthly mean values for benthic invertebrate counts, biomass, and species diversity are shown in Figure 2 through Figure 4. The equitability index is not shown, but it was similarto the diversity index (positively correlated with r

### = 0.87, p<O.OO1). Two-way ANOVA for each ofthe four seasonal periods was performed, testing effects of month ofmeasurement and ofatrazine treatment. Monthly changes were often significantly different at p-value < 0.05, but significant treatment effects were found only in the spring season. Differences between treatments were further evaluated using a priori orthogonal contrasts fOf 100 separate comparisons (for atrazine). Two significant effects were found due to atrazine: in equitability in Winter Month 3 (p<0.029), and in taxa per sample in Spring Month 3 (p<O.OOI). The authors note that, given a set of 100 random comparisons, five significant results are expected due to chance alone at a 

### = 0.05; therefore, they concluded that this result shows a strong lack ofevidence for significant impacts oftreatment on community-level variables.

In the invertebrate drift samples, pulses were noted in all streams during the initial 24 hours ofautumn and summertreatments (see Figure 5) (drift samples were not taken in the first 24 hours during winter and spring treatments). The increase was mainly driven by increases in Baetis mayflies. Only in summer were drift measurements notably higher in the atrazine streams vs. the control (DMSO-treated) stream. (Statistical tests andtheir results are not described.)
No significant effects ofatrazine treatment on periphyton production were noted. Four slides were collected from each stream on each collection day, and daily means for these four slides were used as replicates in two-way ANOVA. The different streams' samples

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### from the same collection day, as well as accrued orcumulativebiomass during each treatment phase, were also comparedusing Duncan's multiple range test. See Figure 6.
No significanteffects ofatrazine treatment on gross primary productivity orcommunity respiration were noted (see Figure 7). Two-way ANOYA for each treatmentperiod found no significanteffects oftreatment, and the primary productivity to respiration ratio (PIRratio) was particularly constantovertreatments 

### vs. control (see Figure 8).
Table 1. Annual means and standard deviations (over9 months in pretreatmentyear, 10 months in treatmentyear) for selected communityvariables based on monthly means (oftwo samples in pretreatmentyear, three samples in treatmentyear). No results were statistically differentfrom pretreatmentvalues atp<0.05. (from study table 2)
Stream Taxa/sample Organisms/ Biomass Diversity Equitability m2 (g dry wt/m2) Pretreatment Mean 9.23 9,757 8.64 2.30 0.57 (1-6 pooled) Std Dey 1.47 6,301 3.92 0.47 0.13 4 (DMSO + Mean 9.57 12,901 9.15 2.24 0.52 atrazine) Std Dey 0.97 4,290 1.50 0.20 0.08 5 (DMSO) Mean 9.37 15,494 9.15 2.31 0.48 Std Dey 1.52 8,404 2.19 0.31 0.13 6 (DMSO + Mean 9.17 13,025 11.23 2.18 0.49 atrazine) Std Dey 0.95 4,637 5.18 0.19 0.08



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Figure 1. Diagram ofone pair oflaboratory model streams. (study figure 1)

• • CONTROLS

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## I TIM E F M A M J (1977) Figure 2. Mean number ofbenthic macroinvertebrates per sample. Each data pointfor the pretreatmentyear is the mean of 12 samples (2 perstream x 6 streams). Each data pointfor the

6treatment yearis the mean ofsix samples (3 per stream x 2 streams). Shaded bars indicate the timing and duration ofthe treatmentphases (study figure 2)**:.:-:':~.~-:: ;;H[~** **:un~;** ::ill:::n:**t~t.~!~·]**

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Figure 3. Mean standing biomass ofmacroinvertebrates in the benthos ofthe laboratory streams. Each data pointfor the pretreatmentyearis the mean of12 samples (2 per stream x 6 streams). Each data pointfor the treatment yearis the mean ofsix samples (3 per stream x 2 streams). Shaded bars indicate the timing and duration ofthe treatmentphases (study figure 3).

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7Figure 4. Mean species diversity ofbenthic macroinvertebrates. Each data pointfor the pretreatmentyearis the mean of 12 samples (2 per stream x 6 streams). Each data pointfor the treatmentyear is the mean ofsix samples (3 perstream x 2 streams). Shaded bars indicate the timing and duration ofthe treatment phases (study figure 4)

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Figure 5. Macroinvertebrate drift in the DMSO-treated control and atrazine-treated streams. Treated stream values are the means ofpooled data for the two streams. Shaded bars indicate the timing and duration ofthe treatment phases (study figure 6).

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... . A S 0 N 0 J F M A M J Months Figure 6. Temporal changes in the mean standing biomass (dry wt) ofperiphyton in the atrazinetreated streams and DMSO-treated control stream (upperfigure). Lowerfigure is shown to provide legend and time-axis labels; other information in the lowerplot may be ignored. Mean values are shown, with error bars indicating±1 standard error, and are calculated from 4 measurements per stream, made after20, 24, and 28 days oftreatment and after 50, 54, and 58 days ofdepuration. (study figure 7)

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### o N 0 J TI ME F A M J Figure 7. Gross primary production and community respiration in the DMSO-treated control and atrazine-treated streams. Shaded areas indicate treatment phases. (study figure 9)

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, , " '5 "" J , , I AM JJ A SO N OJ F M A M J TIME Figure 8. Temporal changes in the ratio ofgross primary production to community respiration (P/R) in the DMSO-treated control and atrazine-treated streams. Shaded areas indicate treatmentphases. See above figure for time axis labels. (from study figure 10

- lowerfigure has been edited out ofthe original figure, exceptfor the time axis and labels)

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### Data Summary Table: Effect Magnitude Nominal Day of Time Did Comments ofEffect Atrazine Study Weighted Recovery relative to Concentration Effect Average Occur- if controls**(~gfL)** Occurred Atrazine so, when Concentration (see from Day 0to comment)? When Effect Occurred N/ A

- Atrazine Lack of Periphyton concentrations effect is biomass no effect 25 J.lg/L No effect were not no described measured in article text N/A

- Atrazine Lack of Gross primary concentrations effect is productivity no effect 25 J.lg/L No effect were not no described in article measured text N/A

- Atrazine Lack of Community concentrations effect is no effect 25 J.lg/L No effect were not no described respiration measured in article text Benthic N/A

- Atrazine Lack of macroinvertebrate concentrations effect is community no effect 25 J.lg/L No effect were not no described (multiple measured in article variables) text N/A

- Atrazine Lack of Total drifting concentrations effect is organisms' no effect 25 J.lg/L No effect were not no described measured in article text Drift ofBaetis N/A

- Atrazine Effect concentrations nymphs (no.l24 no effect 25 J.lg/L No effect were not no attributed hoursi measured to solvent. Note: NA=Not ApplIcable


### Time weighted averages were not computed. A syringe pump provided atrazine to achieve aconstant stream waterconcentration during treatmentphases. Although no atrazine was added during recovery phases, itwas assumed that effects (orlackthereof) were most informative when defined overthe treatmentphases. No atrazine concentration measurements were provided, so nominal concentrations are assumed.
Reviewer Comments:

I Ibid. 2 Ibid.
11This was a limited study that examined one atrazine concentration (25 j.!g/L) and used' two treatment stream replicates and a single control stream. A solvent control stream was used, and the solvent (DMSO) appeared to cause significant effects, possibly outweighing the effects ofthe small amount ofatrazine (although interannual effects due only to time may have been more responsible for the treatment vs. pretreatment changes). The authors did not find any statistically significant effects, but these results are limited by the small number ofreplicate streams. To some extentthe small numbers statistics problems were well handled by taking multiple samples per stream, but the stream-bystream variability was not well explored by this study. Initial condition differences among the streams (i.e. afterthe pretreatmentyear, before the first treatment) were not discussed.
The design and set-up ofthe artificial streams was mixed in its applicability to natural lotic systems. Allowing a full year for colonization increases the realism ofthe streams. Additionally, the use offour treatment phases overthe four seasons was informative in showing the strong time-sensitivity ofthe measured ecological endpoints. However, the streams necessarily underwenttrophic modifications due to the laboratory set-up: the authors note that regulation ofthe discharge led to increases in filamentous algae, and that detritus like leaves was screened out ofthe systems, which may impact certain heterotrophs.
Some detaiis ofthe set-up and analysis were not described: the water depth in the stream channels; the watershed(s), land use, etc. ofCheny Creek and Buckhorn Creek; the lighting intensity and spatial arrangement; the "chemical residues" which were apparently measured for water, sediments, invertebrates, fish, and plants, but are not described in the results; the definitions ofcommunity indices used; and the complete description ofthe methods used to calculate productivity and respiration.
The figures appeared to provide areasonably complete presentation ofthe data, butthe reviewer's copy ofthe original article was not entirely legible (shaded areas did not scan properly). 

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


### Rationale for Use: The study utility was limited by the deficiencies described below. However, the results provide some qualitative evidence that effects ofatrazine on streams biota at low concentrations are weak compared to seasonal effects.

12Limitations ofStudy: The study used only two treatment streams and one control stream. Additionally, the use ofDMSO as a solvent could be a confounder. However, results provide some qualitative evidence that effects ofatrazine on streams biota at 25 flg/L concentrations are weak comparedto seasonal effects. Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc
Secondary Reviewer: Brian Anderson, U.S.

# EPA/IlL fI/J/!(

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