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

ECOTOX Record Number and Citation: Kosinski, RJ. 1984. The effects ofterrestrial herbicides on the community structure**~f** 

### streamperiphyton. Environ: Pollut. (Series A). 36: 165-189. 

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

Date ofReview: October 22, 2008
Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants (periphyton) in experimental streams (lotic mesocosms).
Summary ofStudy Findings:

Methods: Two sets ofexperiments were conducted in 1981 and 1982. In each year, experimental design was afactorial combination of

## aor0.01 mg/kg (0 or 10 /lg/L) of "chronic" atrazine treatmentduring initial colonization, and 0, 0.1, 1.0, or 10 mg/kg (0, 100, 1,000, or 10,000 /lg/L) "acute" atrazine treatment 3 to 4 weeks after initial colonization. (The factorial design also includedthree otherpesticides, butthis review will be limitedto the methods andresults for the atrazine experiments.)

The 1981 experiments used apparently pristine water from Minter Spring, southern Brazos County, Texas. The spring water was almost anoxic andhadtemperature nearly constant at around 21°C, mean specific conductance at 25°C of54.6 mS/m, alkalinity of 71 mg CaC03

### /L, 404 /lg/L nitrate, and 21 /lg/L solublereactive phosphate (SRP). (No information on frequency ortiming ofwater quality measurements.) Cyanobacteria (mostlyAnabaena), 

### diatoms (Nitzschia, Rhopalodia, 

### andNavicula), 

### and afew species of protozoans, rotifers, ostracods, and chironomids were presenton the walls orin the water.

1 

# 111111.111 2087870The author states that it is "improbable" that algae in the spring had prior atrazine exposure (no further discussion or evidence oftesting for background levels).

The 1982 experiments used water from the Little Brazos River, western Brazos County, Texas

- an intensively farmed cotton and sorghum region. The riverreceived high volumes ofrunofffrom adjacent fields, as evidenced by the gully erosion patterns in the watershed terrain. Water and algae were taken from the hardpan clay riffle at abridge that crosses the river. Atthe sampling point, the river was about 30 cm deep and 7-10 m wide (no information on flow rate or discharge). Dense growth of

### Cladophora glomerata (chlorophyll 

### a measured to be 250 mg/m2) was observed, as well as a "diverse invertebrate and fish community" (no further details). Waterquality was highly mineralized and nutrient-rich. Mean specific conductance was 95.5 mS/m, alkalinity was 153 mg CaC03/L, and nitrate and SRP were 1716 and 78 Ilg/L, respectively. Dissolved oxygen varied between 75-150% ofsaturation. (No information on frequency ortiming ofwaterquality measurements was reported.)
Experiments were performed in 32 artificial streams (4 herbicides x 4 acute treatments x 2 chronic treatments, 1 replicate for each combination), located on a flat roofon the Texas A&M University campus. Each stream consisted ofa wooden trough, 2.44 m long and 12.6 cm wide, lined with plastic and filled with source waterto a depth of4 cm, with apump at one end circulating waterthrough a garden hose beneath the trough and back through the channel at a velocity of3 cm/s. The substrate for colonization was 1-2 cm diameter autoclaved, streamworn pebbles, which lined the middle 1.4 m ofeach stream. A heavy glass plate with four horizontal microscope slides attached to it with arubber band was placedbetween the pebble zone and the pump in each artificial stream. Translucentfiberglass covers protectedthe streams against excessive evaporation and the elements, and automatic reservoirs filled with source waterkept the stream depths constant. Twice a week, 2 liters (15% ofthe stream volume) were discarded and replaced with source water, and atrazine levels were broughtto nominal concentrations atthis time. (No further details were reported on age and handling ofsource water or on materials used;) Water-algae suspensions harvested from the source spring orriver (no dates provided) were homogenized in a blenderto break up large filaments, and each stream received 

### 100 mL ofhomogenate for colonization. The colonization tookplace over 3-4 weeks (no specific number ofdays cited) inside the artificial streams, with the "chronic" herbicide treatment levels maintained by adding more herbicide with each 15% water replacement. Aftercolonization, (apparently single) "acute" treatments were applied using commercial atrazine (Aatrex 80 WP) atnominal concentrations based on the active ingredient concentration in the commercial preparation (specific application method and dates not stated).

The rate ofatrazine decay in the streams was determined via water samples from both control and 10 mg/kg (10,000 Ilg/L) streams at time lags ofa few minutes, 1 day, 3 days, and 7 days after acute treatment application, which were frozen until analysis. Analysis was performed atthe TAMU Agricultural Analytic Service by a nitrogen-phosphorus

2detectormethod modified from Purkayastha and Cochrane, J. 

### agric. FoodChern. 21, 1973.
Algal samples were taken each week (exceptthe week following acute treatment application) by removing one colonization slide "from two streams per chronic x acute treatment" (the meaning ofthis is unclear, since the study states there were 32 streams total, which would leave one replicate for each herbicide/chronic/acute treatment combination in the factorial experimental design). 

### In the week following acute treatment in 1982, six slides perchronic x acute treatmentwere harvested to allow between-slide comparisons within a stream (again, the meaning of"perchronic x acute treatment" is unclear and this appears inconsistent, since there are only 4 slides total per stream and one replicate at each herbicide/chronic/acute combination). Organisms from the upper side ofeach slide were scraped into 

### "6:3: 1 preservative" (probably water:ethanol:formalin, and apparently kept in some kind ofstorage vials, butdetails are notprovided). Forcell counting, storage vial material was diluted and duplicate Sedgewick-Raftercells were made, and on each cell 10 fields were counted at 400 x on an Olympus inverted microscope. 

### In 1982, since aggregated growths like cyanobacterial mats were common, large filaments and mat fragments were counted in an additional 10 fields per cell at 40 x. For unicells and "modular" colonies ofdefinite size, such as 

### Scenedesmus, the counting unit was individuals or colonies. For spreading colonies, such as 

### Nostoc, the counting unitwas the number ofoccupied second-order squares ofa Whipple grid. For filaments, the counting unit was length in terms ofthe size ofa second-order square. The volume ofcellular contents in each counting unit was determinedby geometric approximation (no details on primitive shapes orvolumes used), and final results were expressed as 11m3 biovolume permm2ofslide surface. Nondiatoms were identified from wetmounts ofpreserved material at400 x and 1000 x, while diatoms were identified at 1000 x from Hyrax mounts ofcleaned frustules. Nine references were used for identifications (not listed here).

Statistical analyses were performed on only the 15 most common species in each experiment in 1981 and the 20 most common in 1982. The experimental design and number ofreplicates limitedthe multivariate analysis to 7 dependent variables, so seven "species groups" were defined, sometimes on taxonomic grounds and sometimes on less strict associations (such as "filamentous diatoms"). See Table 1 for the species groups defined in 1981 and 1982. The species group variables representthe sum ofthe biovolumes ofthe component species. These variables were transformed by In(x+1) and subjectedto univariate ANOYA, as well as multivariate ANOYA (MANOYA) to process several response variables at once and track correlations between them (implemented via the GLM procedure in SAS software). Significance was determined atthe 0.05 level by Pillai's trace, and computed eigenvectors were used for aprincipal components analysis ofselected data sets. Univariate ANOYA was additionally performed on ShannonWeiner diversity indices computed from the total biovolumes ofthe 15 or20 dominant species.

3

### Results: Only the results for atrazine will be presented.

The half-life ofatrazine in the artificial streams was calculated as 3.2 days, and no interferences from plasticizers or other non-herbicide contaminants were detected in the control samples.

Some effects did notyield significantresults and so were notpresented. Only main effects ratherthan interactions (between chronic and acute treatments) were presented due to the absence ofinteraction. The Shannon-Weinerindex ranged from 0.721 to 2.385 but varied in response to succession ratherthan treatment, and therefore, was not further discussed.

The authors claim that "between-stream error" was the main source ofvariation within a treatment, since multivariate ANOYA found it significant 38 out of44 times, while 3 of the 4 tests on within-stream slides found insignificantresults.

See Table 2through Table 8 for the results ofthe atrazine comparisons. Weeks are designated as weeks before (-) or after (+) 

### application ofacute atrazine treatment, with Week 0 datataken one day afterthe application. The dominant species groups in 1981 were NITZ and FILDIA, with FILDIA as the second dominantgroup replaced by RHO at Week 2. Species abbreviations are defined in Table 1. In 1982, OTHDIA and ACHNAN dominated up to Week 0, whereas in Weeks 1-3 the dominant species groups were ACHNAN and then RHO. Overall, no clearpattern suggesting resistance was found, and for the mostpartthere were no significantmultivariate effects from treatment. Different 

### streams were found to have different communities (significant multivariate effects, p<0.05 with MANOYA), but these effects were not significantly associated with different 

### treatments (with few exceptions). Exceptions were chronic effects during Week 0 in 1981 (p=0.038) and Week 3 in 1982 (p=0.032), acute effects during Week 1 in 1982 (p=0.026), and chronic x acute effects during Week 3 of 1982 (p=0.027). The author does not suggest apattern to these effects. Univariate effects on specific species groups during specific weeks were found, particularly in 1982 when acute treatment effects were found also as opposedtojustchronic effects. It 

### is unclearhow much the lack ofeffects (orofpatterns therein) were due to the small number ofreplicates, from experiment design as well as laboratory mistakes in 1981; see Reviewer Comments.
The authornotes thatreductions in algal crop in the two highesttreatment streams were obvious to the eye andpersisted for the duration ofthe experiment. The observation was confirmedby the results ofthe principal components analysis, shown in Figure 1 in which treatments with different community structures would be expected to occupy different areas ofthe plot. Figure 1 shows data from Week 1 of1982, the time at which the differences were most apparent.

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Figure 1. Principal components analysis ofthe algal counts one week after acute atrazine addition in 1982. Principal components (PCl, PC2) are linearcombinations ofthe species group densities which maximize separation between the acute treatment levels. Polygons enclose the points from the 0, 0.1, 1, and 10 mg/kg (0,100,1,000, and 10,000 p.lg/L) treatments. PCl and PC2 explained 89.4% and 7.1%, respectively, ofthe separation between the acute treatments.

Table 1. Composition ofspecies group variables in each yearofexperiments (from study tables 1-2)
1981 CALO NFILBGS GREENS FILDIA NAVSYN NUZ RHO 1982 PHOR SCENCLA CENTRLES ACHNAN OTHDIA NITZ RHO Calothrix sp. Chroococcus sp., Rhabdoderma sp. Scenedesmus sp., S. bijuga alternans, Stichococcus bacillaris Melosira sp., Navicula confervacea Navicula cocconeiformis, Synedra delicatissima Nitzschia ignorata, N. palea, N. palea tenuissima Rhopalodiagibba ventricosa, R. Gibberula

Phormidium minnesotense, P. tenue Cladophora glomerata, Scenedesmus quadricauda Biddulphia laevis, Cyclotella meneghiniana, Melosira italica, M italica tenuissima Achnanthes sp., A. lanceolata dubia, Cocconeisplacentula lineate Amphora veneta, Diploneis elliptica, Gomphonema angustatum intermedium, G. parvulum, Surirella ovata Nitzschia ignorata, N. linearis, N. Palea Rhopalodiagibba, R. gibba ventricosa



### 5Table 2. 1981 log-transformed Iln(x+I)] weekly mean total biovolumes in llm3/mm2. Week 1 and 3 data are missing due to loss ofsamples in a lab accident. Slide collection error required combining the lower two and higher two acute treatment results (from study table 3)
Week Atrazine Treatment -2 -1 0 1 2 3

Acute

Acute Ch ' Control 12.137 12.857 13.502 13.438 romc

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### _ Control + 100 f.lg/L 12.400 12.097 13.403 14.017 1,000 + 10,000 f.lg/L 12.287 13.070 13.296 13.570

Table 3. 1982 log-transformed [In(x+1)] weekly mean total biovolumes in llm3/mm2. Different superscript letters denote significant differences (no p-value provided) by Duncan's multiple range test (from study table 3)
Week Atrazine Treatment -2 -1 0 1 2 3 h

. Control 12.885 12.155 13.170 a 13.382 13.948 14.492



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_ Control 11.913 12.080 13.721 a 14.662 a 14.899 ab 15.467 a 100 f.lg/L 11.737 11.778 13.099 a 14.837 a 14.674 ab 14.916 a 1,000f.lg/L 11.638 11.432 12.068 b 13.168 b 13.028 ab 13.767 ab 1O,000f.lg/L 11.929 12.122 12.196 b 11.353 c 12.605 b 12.272 b

Table 4. 1981 p-values for multivariate effects on 7 species groups ofdifferent independentvariables (MANOVA). Values in bold indicate significance at p < 0.05. Week 1 and 3 data are missing due to loss ofsamples in a lab accident. (from study table 3)
Week

Independent Variable -2 -1 0 1 2 3 Chronic treatment 0.169 0.381 0.038 0.636 Acute treatment 0.800 0.334 0.761 0.720 Chronic x Acute 0.113 0.792 0.844 0.315 Streams 0.052 0.002 0.001 0.001

Table 5. 1982 p-values for multivariate effects on 7 species groups ofdifferent independent variables (MANOVA). Values in bold indicate significance at p < 0.05. (from study table 3)
Week Independent Variable -2 -1 0 1 2 3 Chronic treatment 0.284 0.238 0.554 0.190 0.068 0.032 Acute treatment 0.761 0.811 0.103 0.026 0.384 0.340 Chronic x Acute 0.522 0.849 0.840 0.274 0.426 0.027 Streams 0.001 0.001 0.005 0.027 0.005 0.007 Slides 0.001

6Table 6. 1981 log-transformed [In(x+l)) mean biovolumes (/l-m3 /mmZ ) ofspecies groups that showed statistically significant univariate effects (ANOYA, p<O.OS) from chronic atrazine treatment. (There were no significant univariate effects from acute treatment in 1981.) (from study table 1 and 4)

Week

-2 -2 -I 

## o Variable NFILBGS NAVSYN RHO RHO TreatmentMeans Control 10t-JglL

1.867 4.804 1.245 5.384 7.958 2.755 9.841 3.472 Species mostaffected Chroococcus sp. Synedra delicatissima Rhopalodiagibba ventricosa, R. gibberula Rhopalodiagibberula

Table 7. 1982 log-transformed [In(x+l») mean biovolumes (/l-m3 /mmZ ) ofspecies groups that showed statistically significant univariate effects (ANOYA, p<O.OS) from chronic atrazine treatment. (from study table 1 and 4)

Week

-2 -1 

## o o o 2 2 Variable RHO PHOR PHOR NITZ RHO PHOR NITZ TreatmentMeans Control 10fJglL 

## o 2.834 6.748 1.968 8.443 4.212 10.37 7.713 8.715 2.306 8.542 5.054 11.318 6.602 Species mostaffected Rhopalodiagibba, R. gibba ventricosa Phormidium minnesotense Phormidium minnesotense, P. tenue Nitzschia ignorata Rhopalodiagibba Phormidium minnesotense, P. tenue Nitzschiapalea

7Table 8. 1982 log-transformed [In(x+1)) mean biovolumes (/-lm3/mm2) ofspecies groups that showed statistically significant univariate effects (ANOVA, p<O.05) from acute atrazine treatment. (from study table 1 and 4)
TreatmentMeans

Week Variable Control 100pglL 1,000flglL 10,000flg!L Species mostaffected

0 PHOR 8.585 8.694 5.367 2.663 Phormidium minnesotense, P. tenue 0 RHO 9.984 4.769 5.826 1.462 Rhopalodiagibba, R. gibba ventricosa CNTRLES 4.635 2.945 2.624 1.609 Biddulphia laevis ACHNAN ] 1.546 12.439 11.062 9.573 Achnanthes sp. RHO 14.350 14.426 9.414 3.683 Rhopalodiagibba, R. gibba ventricosa I OTHDlA 10.730 10.854 9.653 7.295 Amphora veneta 2 PHOR 10.970 5.997 3.773 6.755 Phormidium minnesotense 2 SCENCLA 10.833 7.494 0 3.984 Cladophora glomerata 2 ACHNAN 11.836 12.109 11.184 11.039 Achnanthes sp., A. lanceolata dubia 3 SCENCLA 9.632 8.614 0 0 Cladophoraglomerata 3 RHO 15.142 14.417 11.396 1.549 Rhopalodiagibba, R. gibba ventricosa Biddulphia laevis, 3 CNTRLES 4.120 2.683 0 0 Cyclotella meneghiniana



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

- if ion (fig/L) Atrazine so, when Concentratio (see nFomDay 0 comment) ? to When Effect Occurred no effect (I% Maximum increase, not sig. 100 I-lg/L N/A 53 I-lg/L effects at I diff. from no week. No control) recovery within Reduction in 10% (sig. diff. 3 weeks after periphyton from all other 1,000 I-lg/L Day 7 528/lg/L Yes, Day addition unless biovolume treatments) 14 otherwise noted. 20-25% (sig. No p-value 10,000 provided. See diff. from all I-lg/L Day 7 5,284 I-lg/L no review table 3 other treatments) for source data. Change in Maximum periphyton no cleareffect 100/lg/L NA 53 I-lg/L no effects at I

8Effect Magnitude of Nominal Day of Study Time Did Comments Effect relative to Atrazine Effect Weighted Recovery controls Concentrat Occurred Average Occur

- 

### if ion (Jlg/L) Atrazine so, when Concentratio (see nfromDay 0 comment)? to When Effect Occurred community week. No composition Some effect 1,000 Ilg/L Day 7 528 Ilg/L No recovery within 3 weeks after addition. Source qualitative Cleareffect 10,000 Day 7 5,284 Ilg/L No magnitudes: PCI Ilg/L in Figure I;MANOYApvalue was 0.026. 

### Time weighted averages were calculated using the only atrazine concentration informationprovided: the half-life ofatrazine in the artificial streams was calculated at 3.2 days. Daily concentrations were computedbased on the assumption ofinitial concentrations equal to nominal levels, and exponential decay with 3.2 day half-life. Time weighted averages on Day 7 were simple averages ofthe computed concentrations on Days 0-7. See the Excel-linked table below for calculations. Reviewer Comments: This study suffers from deficiencies in methodology and presentation that limit the usefulness ofits results. However, details on study methodology were reported in several publications including the following (all ofwhich were reviewed):

• Moorhead, D.L. and RJ. Kosinski. 1986. Effect ofatrazine on the productivity ofartificial stream algal communities. Bull. Environ. Contam. Toxicol. 37:330- 336.

• Kosinki, RJ., 

### and M.G. Merkle. 1984. The effectoffour terrestrial herbicides on the productivity ofartificial stream algal communities. 1. 

### Environ. Qual. 13:75- 82.



### Details on the study design were reported inthese publications that were not reported in the currentpublication. Details from all publications thatreported study design details from the same series ofstudies are included in this review.

The study methodology deficiencies include problems with the data aggregation method, and with the lack ofinitial steady state in the communities under study. Lab and sampling errors also interfered with the analyses (see Table 3).

9

### Statistics were all performed on species groups. Aggregation could introduce misclassification errors, and effects to sensitive species within groups may not be detected.

The final methodological issue is that succession dynamics appeared strong enough to possibly overwhelm any treatment effects, as suggested by the Shannon-Weiner indices (which the authordoes not discuss in detail). Succession couldbe a strong confounder. Some ofthe observed changes inthe periphyton community could result from the interaction oftreatmentwith succession dynamics or even from succession alone, due to insufficienthomogenization ofthe initial colonization material. The artificial streams should have been at something closerto a steady state in terms ofthe community dynamics when the acute treatments were applied, in orderto separate out the succession and treatment effects.

In terms ofpresentation, several importantpieces ofinformation were unclear ormissing from the study. Background atrazine in source waterwas not analyzed. Determination of background atrazine levels would allow for more accurate interpretation ofthe results. No schedule or dates were provided for exactly when sampling occurred, when waterwas replaced in the artificial streams, orwhen herbicide was applied. No p-value was provided for the Duncan's multiple range test results, although this multiple comparison method is not recommended due to its tendency to provide inflated estimates·of significance. The "species most affected" in Tables 6-8 had no specific method presented for determining them, so it is unclearwhatthese species really represent. Finally, the description ofperiphyton sampling from the artificial streams was apparently inconsistent: itwas not clearifthere were only 1 orfour replicates at each herbicide/chronic/acute treatment combination level. However, a subsequentpublication based on the same data and methods provided additional clarifying information (Kosinski and Merkle (1984). There was also some errorin the description ofthe sampling protocol.

Since most ofthe methodological issues would bias the study results towards the null, the fact that some statistically significanteffects were found is suggestive ofactual treatment effects. However, the magnitude ofeffects determined in the study are likely underestimates ofeffects because ofthe aforementioned issues.
Description ofUse in Document (QUAL, QUAN, INV): QUAL

Rationale for Use: The study deficiencies limitthe utility ofthis study. However, the study appears sufficientto contribute to the weight ofevidence regarding the concentration-responserelationship ofatrazine and aquatic plants. Datawere obtained from several additional publications to allow for an evaluation ofstudy adequacy.

10

### Limitations ofStudy:

The study methodology deficiencies includeproblems with the data aggregation method, and with the lack ofinitial steady state in the communities under study. Lab and sampling errors also interferedwith the analyses. In addition, source waterfor controls was not evaluated for the presence ofatrazine, and there were several reporting deficiencies Primary Reviewer: SaraHendrix, M.S., StaffScientist, Cambridge Environmental Inc 

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### Secondary Reviewer: Brian Anderson, U.S. EPA

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