Open Literature Review Summary

Chemical Name: Atrazine CAS No: 1912-24-9

ECOTOX Record Number and Citation: Carney, C.E. 1983. The effects ofatrazine and grass carp on freshwater communities. Thesis. University ofKansas, Lawrence, Kansas (MRID 47543604).
Purpose ofReview: Determine potential effects ofatrazine on an aquatic community. Endpoints from this study contributeto the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LOC).

Date ofReview: November 24, 2009
Purpose ofStudy: This study examines the potential effects ofatrazine on macrophyte communities in experimental ponds and in selected farm ponds with ahistory ofatrazine use. The effects ofgrass carp (Ctenopharyngodon idella Val.) on macrophyte communities are also considered alone and in different combinations with atrazine.
Summary ofStudy Findings:

Methods: The thesis is broken into four parts: Experimental Pond Studies; In Situ Chamber Experiment; Farm Pond Study; and Emergent Macrophyte Survey. Portions of the study appear elsewhere: everything exceptthe farm pond study is also described in deNoyeIles etat. 1989 (described as "the 80S-day study" in the review), andparts ofthe "experimental pond study" methods and results are also described by Dewey 1986 and deNoyelles et al. 1994.


### ExperimentalPondStudies Ten experimental ponds with surface areas of0.045 haeach were constructed in 1977 and 1979 at the Nelson Environmental Study Area, ten miles north ofLawrence, Kansas. Bythe summerof 1981, the ponds had developed "similarthough sparse" (no further description provided) macrophyte communities. In "the summer of1981" (no exactdates provided), the ponds were drained and then filled from acommonreservoir (no

1 

# 111••••111 2087813description ofthe reservoir water or ofhow much time elapsed between draining and refilling provided). "Atthe beginning ofthe study" (again, no date provided) the ponds were stocked with fish: 10 gizzard shad (Dorosoma cepadianum Lesueur, 30-40 em long), 50 bluegill (Lepomis macrochirus Rafinesque, 3-7 cm long), and 20 channel catfish (lctaluruspunctatus Rafinesque, 10-15 cm long). Atrazine was added (no date provided) at concentrations of20**~g/L** or 100**~g/L** "to selectedponds" (no further details or descriptions oftreatment vs. control selection) using eitherreagent or commercial grade atrazine, and additionally a 200**~g/L** combined treatmentpond was created. Commercial atrazine was CO-OP liquid? 41 % active ingredient, EPA registration number 1990-318. In 1982 (no specific date provided) all ponds were converted to reagentgrade atrazine only, after no differences were noted between commercial and reagent grade treatments at the same treatment level (no explanation ofhow exactly "no differences" were determined). The two commercial grade 20**~g/L** ponds became 500**~g/L** ponds, and the I00**~g/L** commercial pond and the 200**~g/L** combined treatment pond both became 200**~g/L** ponds with reagent treatment. In May 1982, 4 grass carp oflength 18- 23 cm were added to each pond, and roughly 10% ofeach pondin the southwestcomer, where cattail was less dense, was fenced offfrom the grass carp using I-inch poultry mesh. The goal ofthe fence was to allow comparison ofgrass carp plus atrazine effects on macrophytes with effects ofatrazine alone.
A modified grapnel method, using a common garden rake on September 25, 1981 and a smaller, 8-pronged rake with recurved 4-cmprongs on an extendable pole in 1982, was used to sample the submerged macrophyte community with minimum disturbance to the system. In 1981, at ten points aroundthe shore, the rake was extended from shore, pulled alongthe substrate for one meter, and brought to shore. Samples were scored on a scale of0 to 4 based on visualjudgment, 0 being an empty rake and 4 being atmaximum capacity, and addedto find atotal pond scorebetween 0 and 40. The thesis states that "[a]nce the total macrophyte sample was scored, it was divided into the species comprising that total" but it is not stated how relative frequencies were measured. In 1982, the method changed so that instead of 10 samples, there were 8: four "random" samples within the fenced sectorand 4 samples outside the fenced sector. (No further description ofthe spatial distribution ofsampling points was provided). In 1982, macrophytes were sampled on June 30, August 4, September2, and September 30.
Water chemistry parameters were monitored in 1982, including 'IDS, pH, turbidity, total alkalinity, and planktonic chlorophyll-a, using integrated watercolumns. Atrazine levels were analyzed from the water column samples by the EPA lab in Corvallis, Oregon.
Selectedresults were statistically analyzed using ANOVA (no details on exact methods or grouping definitions).
In Situ ChamberExperiment An in situ experiment was performed in four ofthe experimental ponds from July 26 to August 31, 1982, with one control, one 20**~g/L,** one 100**~g/L,** and one 500**~g/L** chosen "randomly". In each pond, two baskets containing ten segments ofeitherNajas guadalupensis (Spreng.) Magnus or Chara globularis Thuill. (representing vascularand



### 2non-vascular macrophytes) were suspended 25-30 em from the surface. Eachplant segment was inside a perforatedplexiglass chamber andbegan the experiment with five nodes. Only terminal segments were used. Plant material was gathered from the Nelson Areareservoir. "Periodically" (no details on frequency provided) the chambers were cleaned (no description ofcleaning method provided) and relative growth oftheplant segments assessed. Growth was measured as average number ofnodes per segment, ratherthan as segmentlength, to assure thatcell division rather than cell elongationwas being measured.
Farm PondStudy Twenty farm ponds in northeastern Kansas, all within 50 miles ofLawrence, were located. These ponds did not contain grass carp orotherbiological control organisms. Ten ofthe ponds received no herbicide runoffand slightto no use by cattle, while ten of the ponds were adjacentto com ormilo fields with ahistory oftriazine use (no description offarm use histories were provided). With a method similarto that described forthe experimental ponds studies, the small extendable rake was used to sample submerged macrophytes, and samples were taken at 10points around the ponds to determine amacrophyte score. Ponds were sampled for submerged macrophytes as well as for waterchemistry during July and August, 1982. Results were statistically analyzed using ANOYA. EmergentMacrophyte Survey Itwas observed that the cattails seemed brown in the atrazine-treated ponds as opposed to the control ponds; therefore, a survey ofthe effects ofatrazine on the dominant emergent macrophytes was conducted to quantify the observation. (The dominant emergent macrophytes were Typha latifolia L. and Typha angustifolia L.) On September 30, 1982, a lOin. by lOin, quadrat was placed at 10 "random" points within cattail stands in each pond (no further details on the spatial distribution ofthe quadrats was provided), and the total numberofgreen and brown leaves within each quadrat were counted. 

### Results: Atable ofatrazine concentrations overthe 1982 season is provided in the thesis, butnot referenced anywhere in the text. See the Excel spreadsheet "Carney 1983 data.xls" (sheet "atrazine cones") associated with this review for details.
ExperimentalPondStudies On September25, 1981, total macrophyte cover decreased slightly at increasing atrazine levels. Although total macrophyte cover was roughly equal in the 20 Ilg/L to the 100 Ilg/Ltreatments), the shift in community composition was dramatic. In the control ponds and 20 Ilg/L treatment ponds, the macrophyte community mostly consisted ofvascular species, and at the >20 Ilg/L treatmentlevels the non-vascularplants (particularly 

### C. globularis) were more prevalent.See Table 1 for a summary ofmacrophyte score observations estimated from thesis Figures I and 2. The original figures had errorbars to show therange ofvalues among replicates at each treatment level, and Table I notes whetherthe range among the treated ponds had any overlap with therange among the control ponds. Overlap with the control range is an indirect, ad hoc indicator ofstatistical significance ifthere was overlap, differences are almost certainly not statistically significant, especially given the small number ofreplicates.
On June 30, 1982, there was acleardecline in macrophyte levels as atrazine levels increased. The pond sectors with grass carp had less than halfthe macrophyte scores of the pond sectors withoutgrass carp, with a sharpenedreduction ofmacrophyte score over increasing atrazine treatment. Watertemperatures were cool, only about 15°C. Within the fenced-offpond sectors, macrophyte community composition variedwith atrazine treatment levels, though between-replicate variations were large at control levels. By August4, watertemperatures had increased to 25-20°C, there was a greaterdifference between macrophyte levels in pond areas with grass carp and in the fenced-offsectors, and submerged plants were totally absent in the 200 flg/L and 500 flg/L treatmentponds. The 100 flg/L treatmentponds had experienced considerable growth within the fenced comers. By September2, only the control ponds had any submerged macrophytes outside ofthe fenced sectors. Additionally, some reddened and etiolated 

### C. globularis appeared in the 200 flg/L treatmentponds, although it was absent from the controls.
On the final sampling day, September 30, only minor changes in species composition were noted. P. pusillus reappeared and 

### C. globularis increased in the 20 Ilg/L treatment ponds.
See Table 2 and Table 3 for summaries ofthe 1982 results, estimated from thesis Figures 3 through 10. In all atrazine treatment levels with grass carp, there is no overlap ofthe range in macrophyte score with the range among controls; however, overlap in therange ofscores was observedin between controls and all 20 Ilg/L treatment levels from areas withoutgrass carp. A statistical analysis (implicitly on the 1982 data, although this is not clearly stated) using ANOVA found that "the submergentmacrophyte community changed significantly across the atrazine gradient (p-value < 0.00I) being much denser in the fenced areas within control and 20 flg/L ponds" but itis unclear on exactly which subset ofdatathe analysis was performed (only in fenced offsectors?). Two-way ANOVA showed positive synergism between grass carp and atrazine in reducing submerged macrophytes (p-value < 0.001), althoughthere was no statistically significantreduction from grass carp alone (which may be due to the sample size ofonly two control ponds). Among the waterchemistry parameters, turbidity changed significantlybetween atrazine treatments (p-value < 0.05) butthere was no cleartrend (it was lower in control, 20 flg/L treatment, and 500 flg/L treatmentponds). Total dissolved solids (TOS) "displayed significant

4differences" (at p-value < 0.05) "and were lowerin the control ponds than elsewhere" it is unclear whether a significanttrend occurred, and the grouping definition upon which "significant differences" were found is not explicitly stated. The authordescribes the TDS differences as resulting from macrophyte activity to uptake dissolved nutrients, resulting in less TDS in ponds with more macrophytes.
In Situ ChamberExperiment At the end ofthe in situ chamberexperiment, N. guadalupensis had experienced considerable growth inthe control and 20 Ilg/Ltreatments ponds, little growth inthe 100 Ilg/L treatment ponds, andno growth in the 500 Ilg/L treatmentponds. Decay occurred at the high concentration levels (no further details given). For C. globularis, on the other hand, very little growth occurred at lower atrazine levels (the authoralso states that there was "high mortality") whereas there was more growth atthe higher atrazine levels. The author attributes the C. globularis results to a sensitivity to epiphytic algae, which was prevalent in the low atrazine ponds but was reduced inthe high atrazine ponds (entirely absent in the 500 Ilg/L treatment ponds). See Table 4 for a summary ofthe results, estimated from thesis Figures 11 and 12.
Farm PondStudy Inthe farm pond study, there were cleardifferences in the submerged macrophyte communitybetween the "control" ponds (those not receiving agricultural runoff) and the ponds receiving triazines-containing runoff Three water chemistry parameters were significantly higher in the runoff-receiving ponds than inthe controls: TDS (p-value < 0.10), total alkalinity (p-value < 0.10), and turbidity (p-value < O. 10). Theturbidity results are speculated by the author to result from silt loading, which may have also been causative in reducing macrophyte cover, while the TDS and alkalinity results are believed to be caused by macrophyteuptake ofdissolved nutrients. Macrophytes were common in all control ponds, but ofthe ten agricultural ponds, sixhad no macrophytes to speak of and four had only sparse communities

- statistical analysis found that macrophytes were significantly more abundant in control ponds at p-value 

### < 0.001. See Table 6 for alist of macrophyte species observed and their overall prevalenceby pond type and Table 5 for a summary ofthe macrophyte cover data estimated from thesis Figure 13. EmergentMacrophyte Survey In the control ponds, 90-95% ofthe Typha spp. leaves were green, while inthe 500 Ilg/L treatmentponds, only about 30% ofthe leaves were green. See Table 7 for a summary estimated from thesis Figure 14.

Table 1. Macrophyte sampling data from 9/25/1981 (thesis Figures 1-2). Maximum Macrophyte Score =40.
Nominal Atrazine Mean Macrophyte Score ("MS") Any overlap

with control MeanMS (vascular)

5 Any overlap

with control MeanMS (Charophytes)range? range?

0 27 ]4 ]9 yes 14 yes 5 yes 21 4 yes ]9 4 N/A 0 N/A 4
means "not treatmentlevel
Table 2. Submergent macrophyte data from the 1982 season (thesis Figures 3, 5, 7, and 9). Maximum Macrophyte Score == 16.

Nominal Any

# l
Sampling Atrazine l\1ean overlap

Date Macrophyte with 

### I

.... <7 Score control range'?

. 0 9



### - 6/30/]982 20 4 no j

(grass 100 I no

! 200 0 no I 500 0 no 0 13 20 I I I IJU/j Y15L I yes 100 3 No 200 2 no 500 ] no

0 6 982 20 I no 100 0 no 200 0 no i 500 0 no
i 0 13 982 20 ]2 yes grass 100 6 no carp) 200 0 no 500 0 no

0 11



### - r------ 0 9/2/1982 20 I no

(grass 100 0 no carp) 200 0 no 500 0 no

0 15



### - 20 14 yes 100 10 no 200 1 no ! 500 i 0 no ,

69/3011982 (no grass

carp)

Table 3. Species-specific submergent macrophyte data from the 1982 season (fenced-offpond sectors only; thesis Figures 4, 6, 8, and 10). Maximum total (combined) macrophyte score 16.

Nominal Any Any Any Sampling Atrazine Mean overlap Mean MS(N. averla MeanMS overlap

Date rf MS(p. with guadalupensis) p with (C. with w'v, pusilJus) control control globularis) control range? range? range? 

### I 0 8 0 

### - 6/30/1982 20 

### I 2 yes 9 yes 0 yes (no grass**l~** 100 

### I 0 yes 0 no 3 no

carp) I 200 

# I 0 0 1 yes no no 500 0 yes 0 no 0 yes**.~** 0 0



### - 0 yes 8/4/1982 (no yes 6 no grass carp) 0 yes 0 yes

I i 500 no yes 0 yes I

no no 9/2/1982 (no 0 no no 10 no grass carp) i i 200 0 i no no yes I 500 0 no yes 0



### - 982 I yes 7 yes 6 (no grass I ! yes 200 0 yes 0 no I yes 500 i yes yes

7Table 4. Results of37-day in situ chamberexperiment (Figures 11-12 in thesis). Node numbers representgrowth.

Mean Number olNodes (C. globularis) Any overlap

with control range?

Table 5. Results offarm pond study submergent macrophyte survey. Maximum Macrophyte Score =40.

Type

Table 6. Species-specific composition ofsubmergent macrophyte communities aggregated by pond type in the farm pond study.

Percent Composition by !

i PondType

c. ! r""fvn' i Runoff-

, Najas , .1 7 Potamogeton I , Pfoliosus 0 12.5 P. , 0 12.5 Ceratophyllum Chara , 6.7 0 .u

! C braunii ),6

Table 7. Results ofemergent macrophyte survey ofSeptember30, 1982.

8Nominal Any Atrazine Percent overlap Concentration Green with (lig/L)

0 ZO 78 no 100 68 no 200 50 no 500



### Data Summary Table: Effect Magnitude of Nominal Day of Time Weighted Did Comments Effect relative Atrazine Study Average Recovery to controls Concentrati Effect Atrazine Occur- if
on (Jig/I) 

## I Occurred Concentration so, when from Day 0 to (see 

# I When Effect comment)? Occurred 

### i Emergent 15-20% +20 Ilg/L Day 1192 14.8 Ilg/L3 Dataestimated macrophyte from thesis cover (% 25-30% ! 100 IlgiL Day 119 79.3 Ilg/L green leaves 

### I 

# [zoo Ilg/L no figure 14 45-50% Day 119 

## i 173.7 Ilg /L (review Table on Typha



## _•... I spp.)! 65-70% 

# I 500 "giL

# 1

Day 119 453.8 Ilg/L 7).**~.** 

## __. 

# I See Submerged See I See deNa elle See See See macrophyte deNoyelles I deNoyelies**198~** deNoyelies deNoyelles deNoyelies cover 4 1989 review 1989 "view '

# I

"view 1989 review 1989 review revIew Submerged 90-95%

# un~o~~ Farmponds, macrophyte (significantly (farms with not k not cover(farm less at . . un nown no experimental

### ~Ii~~~e**~se** apphcable 

# I ponds) p<O.OOI) 

### I data.

- ..**_~-~.~.** .•..-.**~'-** •.•.... ..**"-'-~-**

I It is unclear whetherthe emergent macrophyte study performed by Carney is included somehow within the emergent macrophyte data described in deNoyelles et aI., 1989. It does not appear to be the case, but if itis, then this information is included within the deNoyelies 1989 review data summary table. 2 The date ofthe survey is known; however, the date ofatrazine addition (Day 0) is unknown and estimated from the table ofatrazine measurements (table I ofthesis) assuming that the first day ofatrazine measurement was the day ofapplication, since measured concentrations were roughly 100% ofnominal on that day.
3 Repeated applications ofatrazine within the 1982 season are not mentioned in the thesis, and are therefore assumed to not take place after the initial application. See "TWAs" sheet in "Carney 1983 data.xls" for details.
4 Appears to be equivalentto or significantly overlapping with datapresented in deNoyelles et aI., 1989. Selected endpoints are specified in the deNoyelles ct a!. 1989 study revicw. However, TWAs and days of study cffects occurred were not calculable fore deNoyclles et al. 1989 but may be calculable from the information in this thesis. See "Carney 1983 data.xls".Ma,gmtude of

N. guactalupensis growth (number of nodes)

C globularis growth (numberof nodes) 600% (+) 500jlg/L no

no effects is relative to nonagricultural ponds. Data estimated from thesis figure 13 (review

____**+-'-Tab~~~~_** Data estimated from thesis figure 11 (review Table 4). Data estimated from thesis figure 12 (review Table 4). Notc that effects are all

Time weighted averages were calculatedbased on thesis Table 1, which was not referenced withinthe body ofthe thesis and was thus difficultto interpret. Chemographs were derived assuming that 6/3/82, the first day when atrazine concentrations in the ponds were measured, was the day ofatrazine treatment, and thatthere were no subsequent atrazine applications during the 1982 season (through September). A linear decline in atrazine concentration overtime was assumed; therefore, Time Weighted Averages were calculated as simple averages ofDay 0 and EffectDay concentrations. For further details and calculations, see "Carney 1983 data.xls".
Nominal Day 0 Effect Day Time Atrazine Weighted Experiment Concentration Effect Day Concentration Concentratio**(~g/L) (~g/L)**  Average
n**(~tg/L) (~g/L)**  1982ponds 20 18.78 119 10.73 14.76 1982 ponds 100 93.65 119 64.86 79.26 1982 ponds 200 198.9 119 148.50 173.70 1982 ponds 500 501.75 119 405.94 453.84 in situ chamber 20 15.19 36 12.76 13.98 in situ chamber 100 80.83 36 72.12 76.47 in situ chamber 500 459.08 36 430.09 444.58

5 Day 0, the day the in situ chamberexperimentbegan, is Day 53 ofthe experimental pond study. This fact is used to derive TWAs.

10Reviewer Comments: Many details ofmethods implemented within this study are absent or vague. An atrazine concentration table, with concentrations at all treatment levels in all ponds on two days in the summer in 1982, is presented as Table 1 ofthe thesis without any explanation or interpretation in the text. The exact date ofatrazine application as well as any subsequenttreatments during 1982 is unclear. As forthe 1981 study, no information is provided on the atrazine application dates. In both seasons, there is no discussion of the methods ofatrazine application; therefore it is unclearwhether atrazine was evenly distributed or fully dissolved, or whetherotherfactors affecting atrazine concentrations within the ponds may have interferedwith the results.
The exact conditions ofthe ponds and any preexisting differences among them atthe start ofthe study in 1981 are not discussed. The dates and details ofhow ponds were drained andrefilled are not addressed. Similarly, it is statedthat for 1982, reagent atrazine was used because "no differences" were notedbetween commercial and reagent atrazine ponds during the 1981 season. However, it is not stated whetherthis was determined through measurements or statistics rather than subjectivejudgment. Overall, the conditions in the experimental ponds atthe start ofthe 1982 season appearrather uncontrolled; it is possible thatthe "control" ponds are notreliable controls ifthey had different initial conditions than some orall ofthetreated ponds.

The macrophyte scoring method appears subjective in several ways. First, it is not describedhow the 10 or 8 (four inside the fenced sector, four outside) points around the shore ofeachpond were selected, apart from describing the selection process as "random". Were sets ofpoints selected equidistantly? How can we be sure there was not repeated sampling ofthe same spot, biasing the measurements low? Also, it is not stated how the species specific breakdown ofthe macrophyte score was calculated. Were plants separatedand weighed? Individuals counted? Orwas it based on visualjudgmentby looking at the rake? The species-specific results are impossible to interpret without this information.

Forthe in situ chamberexperiment, the frequency and method ofthe chambercleaning is not described. The frequency and efficacy ofthe cleaning may be very important because the low atrazineponds hadhigh algal epiphyte growth, and epiphyte covering the chamber could interfere withthe growth ofthe plant segments inside.

In the farm pond study, there is no information on how triazine use histories atthe control vs. agricultural runoff-receiving ponds were determined, and what exactly they are. The farm pond study results, while dramatic, cannot be interpretedwithout more information on triazine types, use frequencies and quantities, watershed hydrology, otherpond usage, and exposure concentrations oftriazines within the control and "runoff-receiving" ponds

..
In the emergent macrophyte survey, the spatial distribution ofthe quadrats is unknown. Ten lOin by lOin quadrats would fill about 7 square feet ofthe pond surface, orroughly

110.14% ofthe total surface area ofa 0.045-hapond. Therefore, the survey might notbe representative ofall the cattails in each pond. Finally, the use ofANOVA for statistical analysis is so poorly described thatthe statistical results are almostmeaningless. Itis not stated exactly how different groups are defined (for example, were all pond samples combined at one treatment level, or were only fenced-offarea samples used?). Furthermore, ANOVA comprises arange of statistical methods to be applied under various circumstances and assumptions. The statementthat ANOVA found significance at acertain p-value is meaningless without knowing whether the tests were applied properly. IfANOVA methods were applied improperly (i.e. the assumptions were violated), the results are incorrect. Description ofUse in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: The study has some utility for demonstrating that atrazine caused reduction in the macrophyte community with corresponding changes in species composition leading to domination by Chlorophytes at levels of 100 

### ug/L. However, exposure was not sufficiently characterized, and baseline aquatic community assembledges were notdocumented priorto study initiation.

Limitations ofStudy: The study design was hypothesis-generating in nature, intending to highlightthe potential for differentkinds ofcommunity-level effects to occurrather than to accurately formulate effects and magnitudes under tightly controlled conditions. It succeeds in the former by presenting qualitatively interesting information, but fails in the latterby presenting datathat are difficult to accurately interpret. The study results are not appropriate for quantitative prediction due to the apparently uncontrolled experimental conditions, subjective measurement methods, and lack ofkey details in the descriptions ofmethods and results.

No effort was made to document whether other chemicals (including atrazine) were present in the treatment and/or control ponds prior to study initiation. Pre-sampling was not conducted to determine whether aquatic communities were similar to one another prior to study initiation. The study switched test chemical from formulated product to technical grade atrazine after the first year and also switched exposure concentrations for several ofthe ponds, 

### i.e, two ofthe lowest exposures at 20 

### llg/L were increased to 500 

### llg/L while one pond formerly at 100 

### llg/L was increased to 200 

### llg/L. Atrazine degradate concentrations were not reported. Large grazing animals (grass carp) were unequally distributed across the studies due to predation; thus, there was likely differential grazing in the study ponds. Controls exhibited wide variability and given that there were only two controls for each ofthe two studies, the ability ofthe study to detect treatment effects is limited.

12Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental. Inc
Secondary Reviewer: Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental Protection Agency.

13