### Open Literature Review Summary
Chemical Name: Atrazine CAS No: 1912-24-9
ECOTOXRecord Number and Citation: DeNovelles, Jr., F., W. D. Kettle, C. 

### H. Fromm, M. F. Moffett, and S. L. 

### Dewey. 1989. 

### Use of Experimental Ponds to Assess the Effects of a Pesticide on the Aquatic Environment. Department of Systematics and Ecology, University of Kansas. Entomological Society of America. Miscellaneous Publications No. 75: 41

- 56 (MRID 47543608). Purpose ofReview (DP Barcode or Litigation): Determinepotential effects ofatrazine on an aquatic community. Endpoints from this study contributeto the weight-of-evidenceregarding effects ofatrazine on aquaticplantcommunities and associated determination ofaLevel ofConcern (LOC).
Date ofReview: November24,2009 Summary ofStudy Findings Purpose: This study examines thepotential effects ofatrazine to aquatic plants and animals in experimental ponds (mesocosms).
Methods: Two studies with experimentalponds were conducted. The first studybeganin July of1979, lasted 136 days, and used six 0.04-ha (0.099 acres) experimental ponds filled to adepth of2.1 meters (6.9 ft) representing 0.68 acre-feetofwater. The second study began inJune 1981, lasted for 805 days, andutilized ten 0.04-haexperimental ponds filled to adepth of2.1 meters. All ponds were located atthe University ofKansas Nelson Environmental StudyAreanear Lawrence, KS.
Prior to any treatment with atrazine, all ponds were allowed an undisturbed year in order to effectively establish a benthic plant and animal community. Then, the ponds were drained and immediately refilled to a depth of2.1 meters from an adjacent OA-ha pond maintained by well water.

- 1
- 

# II1IIIIIII 2087828The ponds were stocked with 10 gizzard shad 

### (Dorosoma cepedianum; 15.2 cm total length (TL», 20 channel catfish 

### (Ictalurus punctatus; 9.4 cm TL) and 50 bluegill sunfish 

### (Lepomis macrochirus; 5.1 cm TL) one weekafter filling and a few weeks before atrazine was added. The same three species were used in both the l36-day and 80S-day studies. In addition, grass carp (N=4 perpond) 

### (Ctenopharyngodon idella; 18

- 23 cm) were added to the 10 ponds ofthe 805- day study as amacrophyte grazerafterthe first year. Adult fish ofall species were blocked from the southwest comer ofeach pond (approx. 5% ofthe pond area) using a 2.5-cm mesh poultry fence in orderto separatethe effects ofatrazine on macrophytes from grazing by grass carp.
In the 136-day study, a commercial grade (formulated) atrazine (CO-OP Liquid Atrazine®; 41 % active ingredient) was diluted in 20 liters ofpond water and then dispensed through a hose moved throughout the pond. In the 80S-day study, a suspension ofreagent (technical) grade atrazine (97% ai) was dispensed by adding suspending atrazine powder in 4-liter volume portions into thepondsjustbelow the surface ofthe waterfrom a boat. Treatment solutions were passed through fine screens so as to finely disperse particles for rapid dissolution.
The 136-day study consisted of two control ponds and two atrazine-treated ponds each at nominal concentrations of20llg/L and 500 Ilg/L. The 80S-day study started with two control ponds, four ponds with 20 Ilg/L concentration (two formulated product, two technical grade), three ponds with 100 Ilg/L concentration, (one formulated product, two technical grade), and one pond with 200 Ilg/L concentration (formulated product and technical grade combined in a 1: 1 ratio) for the first year beginning in May of 1981. In the second 80S-day study, ponds were retreated at the beginning ofthe second and third years. Beginning in the second year ofthe study, formulated product was replaced with technical grade atrazine. The two ponds formerly treated with formulated product at 20 Ilg/L were increased to 500**~tg/L;** the one pond formerly treated with formulated product at 100 Ilg/L was increased to 200 Ilg/L, and the other pond treated at 200 Ilg/L with a 1: 1 mix offormulated and technical grade was thereaftertreated with technical grade alone at 200 Ilg/L. The paper does not state what schedule was used for 'measuring atrazine concentrations; however, Figure 2 ofthe study suggests that samples may havebeen collected atroughly 3-wkintervals. Temperature, oxygen, and light penetration profiles, along with measurements of turbidity, alkalinity, pH, and nutrient conditions for nitrogen and phosphorus were measured weekly one month per pond prior to atrazine treatment then once or twice monthly through the following Spring. Water quality samples were taken from water samples collected within 8 cm ofthe pond floor from a walkway which extended one-third ofthe way toward the middle ofeach pond.
Tolerance ofphytoplankton to atrazine was determined in a laboratory by incubating water samples withphytoplankton from all ponds for 24 hours with and withoutan additional 100 Ilg/L of atrazine, while being gently rotated for mixing. Relative abundance of submersed macrophytes was estimated using a modified version of the grapnel method where a rake

- 2 -attached to long pole is pulled along I m ofthe pond bottom and the contents are brought to the shore for identification. Relative abundance ofemergent macrophytes (cattails) was determined by observing aerial photographstaken in August and Septemberof 1982 and 1983. Values were expressed as a percentage ofshoreline covered. Browning ofcattail blades was determined by examining ten 25-cm quadrants within a cattail stand and counting the number ofbrown and green blades. Phytoplankton production, chlorophyl biomass and species abundance were determined from watercolumn samples. Zooplankton was sampled with one vertical net haul taken at night using a IS7-llm mesh net with a 29-cm opening. Insects were sampled every week from May through Septemberin 1982 and 1983 using emergence traps. These values were expressed as total number captured per sample. The 136-day study followed only the phytoplankton and zooplankton responses and did not include detailed macrophyte and insect analysis whereas these parameters were measured in the 80S-day study.
Fish and tadpole abundance was sampledby lowering theponds to adepth of0.75 meters. The waterwas drained through afine screen to ensurethatall organisms were captured for sampling. Afterthis, eachpond was completelydrained to capture anyremaining organisms.
Results


### Water Quality Alkalinity in the ponds ranged from 100 240 

### mg/L [as CaC03]; differences in alkalinity between ponds was generally less than 60 

### mg/L. There were "some" declines in both dissolved oxygen (DO) and pH over the first 2 weeks ofatrazine addition in both the 136- and 80S-day studies that the authors attributed to decreased photosynthesis. In the highest atrazine test concentration (500 flg/L), DO is reportedto haveremained above

### 4.5 mg/Lthroughoutthe study, and ponds did not differ by more than 1 pH unit with pH values ranging between 7 9 during May

- September. Water temperatures ranged from 20°C to 30°C during the May -September sampling period. Turbidity was the only parameter that is reported to have been "highly" correlated with atrazine concentration (r=0.82); turbidity increased with increasing atrazine concentrations.

### Atrazine Concentration Initially measured atrazine concentrations were within 10% ofnominal. By 6 months measured concentrations had declinedto 70% ofnominal andby 12 months, atrazine had declinedto 25% ofnominal in the**805~day** study (Figure 1). A similarrate ofdecline was reported for atrazine inthe 136-day study

.



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..... 5-0-;-'01. 0-1.- 75'-0--6-'-0 o..J Time (!lays) Figure 1. Measured atrazineconcentrations for the 805-d study. Values plotted as treatmentmeans standard error. Slashes along xaxis indicatetimes ofatrazine treatments. Figure from DeNoyelles etai. 1989. i()

### Table 

### 1. Summary ofEffects ofAtrazine in Aquatic Community. Time Weighted Did Magnitude of Nominal Dayof Study Average Recovery Effect Effectrelative to Atrazine Effect Atrazine Occur if Comments Concentration Concentration so, when controls (pg/L) Occurred fromDayOto (see When Effect comment)? Occurred Recovery was not specified atthis level, but assumed to be 2::3 weeks, given the lower magnitude ofeffect and recovery at the 50% (p$O.OS; higher 136-d and 80S-d 20**~g/L** :S3 weeks 16-20**~g/L** Yes concentrations. studies) Range provided for TWA (up to 3 weeks); further detail was not Phytoplankton provided and this treatmentwas not biomass and graphically production represented in ('4C uptake) Figure 3, below. (80S-d) 100**~g/L** 0 100**~g/L** Yes Recovery 2::3 weeks
Recovery2::2 weeks; determined**~90%** (80S-d) 200**~g/L** 0 200**~glL** Yes from Figure 3 in the study (and below).

I**~90%** (136-d i 

# IYes I i500**~g/L** 0 500**~g/L** Recovery 2::3 weeks I and 80S-d) 

# I I L ._ ..

## _ .. 

# INot "po,"d 120

## ~g/L Notreported Notcalculable Not Reported forthe reported 136-d study; p Change in ' "

..**"_.~._~-~** values forthis endpointwere not phytoplankton reported. species composition Not reported 500**~glL** Notreported Notcalculable Not There was a shift reported away from 

### I dominantgreen 

### I algae and 

## I

- 5-- 6 - Time Weighted Did Magnitude of Nominal Day of Study Average Recovery Effect Effectrelativeto Atrazine Effect· Atrazine Occur- 

### if Concentration Comments controls Occurred Concentration so, when (pg/L) from Day 0to (see When Effect comment)?

! Occurred 

## I

.._-_._-- dinoflagellates to flagellated algae and golden algae. Tolerance was evident. 

## I
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I reported Not calculable Magnitude of effects estimated !75-80% reported from Figure 7 in the study document (Figure 2 below) and is relative to pre-treatment Emergent abundances; macrophyte No includes areas with
cover (i.e., and withoutgrass Typha) carp.**~90%** 5OOIlg/L Not reported Not calculable Results are from the 80S-d study and effects were statistically significantat p:SO.OS. 50% - no grass Magnitude of carp 20 f.1g/L N. •"f-'UI ,"'U Notcalculable effects estimated carp from Figure 7 in the study document and is relative to carp pre-treatment Submerged 100 JIg/L Notreported ""I

.." l"h1

.. abundances (Figure macrophyte 100%

- grass No 2 below).
cover carp I (with or I Results are from

grass 200llg/L Natreported Not calculable the 80S-d study and carp) effects were statistically 100% (with or Not INot calculable I significantat withoutgrass 

## In<i1 n.:; !Effect Magnitude of Effectrelative to controls 

## iNominal Atrazine Concentration (llg IL) Day of Study Effect Occurred Time Weighted Average Atrazine Concentration from Day0to

When Effect Occurred Did Recovery Occur- 

### if so, when (fee comment)? Comments

Balded llues are those data BiologicalData 

## I**spe'~lrlied·1** in the "Cosm DataPoints and Exposure**PIVIIIC~"** spreadsheet.

There was a statistically significant (p<0.05) decrease In photosynthesis measured In phytoplankton in ponds treated with atrazine. According to the author, these plants are directly affected by atrazine, which inhibits the electron transport in photosystem II by inactivating electron transport carrierproteins upon occupying a specific binding site in the protein structure ofthe chloroplast. Potentially, this reduction in photosynthesis couldresultin a decrease in total biomass ofthe aquatic system. According to the study authors, ponds treated with 20 jlg/L did not develop communities of phytoplankton with tolerance to higher concentrations ofatrazine as evidencedby initial declines in biomass in ]982 when 500 jlg/L was addedtotwo ponds with concentrations of20 jlg/L). Phytoplankton declined in biomass and production at all concentrations during the first three weeks ofexposure in both studies. Phytoplankton declines of50% occurred at 20 jlg/L while there was nearcomplete cessation ofcarbon intake and biomass declines to 10% ofcontrol pond levels in higher (500 ug/L) atrazine treatment concentrations. However, after that three week period, the biomass and productionrecovered, andreturnedto the levels ofthe control pondatall treatment concentrations. It was determined that all persisting phytoplankton were tolerant ofor resistant to the herbicide.
In the 136-day study, changes in species composition were recorded with a shift away from the originally dominant green algae (Chlorophyceae) and dinoflagellates (Dinophyceae) to flagel1ated algae (Cryptophyceae) and golden algae (Chrysophyceae). According to the authors, the ponds receiving atrazine at all concentrations became dominated by organisms resistant to or tolerant to atrazine exposure. Phytoplankton from ponds treated with 100 J.lg/L returned to control pond levels ofcarbon intake within 3 weeks and did not exhibit a decline with the next treatment ofatrazine the following year. However, the pond treated with 20 J.lg/L exhibited an initial decline following the secondyear oftreatmentwhen atrazine concentrations were raisedto 500jlg/L. According to the study authors, the decline in carbon uptake following addition ofthe 

### - 7

-500 Ilg/L dose indicates that tolerant communities were not present in the pond initially treated with 20 Ilg/L atrazine. Phytoplankton collected over the 80S-day study were exposed to an additional 100 /lg/L of atrazine and incubated for 24 hours. Phytoplankton from all atrazine-treated ponds exhibited less reduction in carbon uptake than control pond phytoplankton. According to the study authors, this result provides further evidence ofthe shift toward more tolerant species ofphytoplankton exposed to atrazine.
The authors concluded that direct toxicity ofatrazine appears to be minimal to animals for the concentrations tested in the mesocosms from this study. There were "some declines" in numbers ofgrazing zooplankton following the initial declines in phytoplankton biomass in the 136-d study, but the zooplankton returned to control pond levels within three weeks; these declines were attributed to food availability and not to the direct effect ofatrazine. However, because of the direct effects on photosynthetic organisms, there may be indirect effects on animals that consume these organisms.
The biomass of channel catfish at the end of the 80S-day study was unaffected by atrazine treatments. Grass carp were lost from one control pond and one 500 /lg/L treatment after the second year ofthe study. Grass carp biomass declined at >100 Ilg/L; according to the author, grazing by grass carp had a greater effect on macrophyte abundance with increasing atrazine concentration. Percentage ofcattail cover, submersed macrophyte relative abundance, tadpole final biomass, emergent insect numbers, grass carp and bluegill final biomass were significantly affected (p<O.OS) by the atrazine treatment (Figure 1). These measurement endpoints showed significant (p<0.10) negative lineartrends with increasing atrazine concentrations. Only cattails in the area where grass carp were present were not affected. Declines in biomass ofsubmersed macrophytes were most evident at atrazine concentrations 2100 Jlg/L.



### - 8

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Figure 2. Depiction ofpercentage ofcattail cover, submersed macrophyte relative abundance, tadpole final biomass, emverging insectnumbers, grass carp final biomass and bluegill final biomass in ponds treated with 0,20,100,200 and 500 f.1g atrazine/L in 80S-day study. Figurefrom DeNoyelles etal. 1989.

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- 500Ug/L

...... 1982



## o 10 20 30 

## o 10 20 0 10 20 30 DAYS 

## o 10 20 30
Fig. 3. Cllrbcm-14 uptake by sumt}le, ofphytoplankton communities li'ommntrol (solid line) and atrH:t.ine lrented (doHcJ line) ponds for the first 30 tI ofexposure in the 136-d study lIlI(1 the first 25 lind 35 d ofexpOlmre in years 1 and 2, respectively, ofthe 805-d study. 

### rn 1982, the 100- and 200·fJ.W1ltt>rpllnds shown here r<.'Crlved their second year's dOSt'. lind 500 fLg/liter wns lidded to tWIl previolls 20'fLwliter ponds, CllrbOlHlptllkc is plotted as lhe ll1j;>an (;-loI counts per minute (cpm) from four samples from each pond.

Declines in tadpole biomass, primarily bullfrogs 

### (Rana catesbiana) were evident by the end of the 80S-day study even at the lowest atrazine concentration of20 I-lg/L; however, the report does not state the extent ofthe decline.


### Reviewer Comments
The area surrounding the study sites is not completely characterized in terms ofprevious chemical treatments. However, a companion study by DeNoyelles and Kettle (1980) indicates that the ponds received no watershed runoffand, therefore, could not be affected by local herbiciderunoff.
It is stated that prior to atrazine addition, the experimental ponds were drained and then immediately refilled with water from an adjacent OA-hapond maintained by well water. It is not stated whether this water was tested to ensure that it was not contaminated with any pollutants.

- 10

-Ifthis were the case, it could potentially lead to false conclusions. No analyses were conducted to either qualitatively or quantitatively describe whether atrazine or other pesticides/chemicals were present in the control or treatment ponds prior to study initiation. The study does not mention whetherany atrazine degradates were measured.
Ponds were allowed to equilibrate/acclimate for an entire year prior to use; however, the ponds were drainedjust prior to initiating the study. 

### It is unclear what was accomplished during the acclimation period since draining the ponds would likely have disrupted the aquatic community in spite ofthe authors' beliefthat it "probably does not alterthe already established benthic plant and animal communities". The methods section does not state how much time was allowed for the aquatic community to re-establish itselfafterdraining; however, the text implies that 3 weeks may have elapsed. Ifthe ponds were to be drained priorto the study, then information could have been collected on vertebrate, invertebrate, and aquatic plant communities and this information couldhave in turn been usedto compare recoverydata.
In the first study, atrazine was already dissolved in the concentrated stock solution; however, in the second study, atrazine powder was suspended and not dissolved. 

### It is uncertain whetherthe atrazine in the second study ever went into solution. Although the suspension was passed through a fine-mesh screen to ensure that the compound was dissolved, it is possible that the screen acted as a filter that prevented larger suspended particles from entering the pond ingredient. According to the authors, measured concentrations differed by less than 10% of nominal [at study initiation] and were 70 and 25% of nominal after 

### 6 and 12 months, respectively.
In the second year ofthe multi-yearstudy, the researchers stopped using formulated product and relied on technical grade atrazine. Nominal treatment concentrations ofthe one pond treated with formulated product at 100 JlglL were increased to 200 Jlg/L with technical grade, while the two ponds at nominal treatments of20 Jlg/L with formulated product were increased to 500 JlglL with technical grade in the second and third years ofthe study. Presumably then, the study consisted oftwo ponds each at 20, 100, 200, and 500 Jlg/L for the last two years ofthe study with all ofthe ponds treated with technical grade atrazine. Thus, test chemical and exposure concentrations for three ofthe testponds changedin the middle ofthe study.
With only two replicates at each treatment, the study has a low power to detect any treatment effects shouldthey occur.
Based on the caption to Figure 2 ofthepaper, a "small amount" ofatrazine was requiredto bring the treatmentponds up to the nominal concentrations in the third year ofthe study. This implies that each ofthe ponds was treated to raise the treatment concentrations back to nominal rather than retreating each the ponds at the original rate. Thus, it is uncertain how much atrazine was addedto each ofthe ponds in the second andthird years ofthe study.

- 11

-It is unclear from the longer second study whether ponds were retreated using the same method for dispersing the chemical as that used in the first year.
The basis for the stocking rates offish used in the study is not discussed in the paper. Rather, the readeris referred to an earlierpublication by DeNoyelles.
The methods used for measuring phytoplankton productivity, chlorophyll biomass and species abundance are not described in thepaper. The frequency ofthese analyses is not discussed. Considering that ponds were in close proximity to one another and were all filled with wellderived water from adjacent ponds, there was a relatively large range in alkalinity (100

- 240 mg/L). It is unclear why these differences in alkalinity existed; however, such differences could impact primary productivity and confound interpretation ofthe study results since differences in productivity could influence dissolvedoxygen, pH andturbidity. Although the authors report that dissolved oxygen (DO) concentrations did not drop below 4.5 mg/L, this measurement was for surface water where diffusion would have been greatest. More reliable measures ofDO wouldhave been at several depths in the watercolumn. Phytoplankton in treated ponds recovered to levels that were comparable to controls after three weeks, and the authors hypothesize that the recovery was due to the growth ofalgae that were tolerant to atrazine given that atrazine levels had not declined substantially in the treated ponds. In the shorter 136-d study, changes in species composition were recorded with a shift from originally dominant Chlorophyceae (green algae) and Dinophyceae (dinoflagellates) to Cryptophyceae (flagellated algae) and Chrysophyceae (golden algae). However, in the 805-d study, no data were provided on the species ofphytoplankton present and whether any species was displaced by another. Replacement ofone species by another could have marked effects on aquatic communities where foraging species may notbe as resilientto such changes in theirdiet.
It is mentioned in the study that tadpoles were collected trom the pond; however, no data on the potential effects ofatrazine on the tadpoles are provided; it is only stated that atrazine had a minimal direct effect on animals, and that reduction in tadpole biomass is likely due to loss of macrophyterefuges as feeding sites.
Due to loss ofgrass carp in one ofthe control ponds and one ofthe 500**~g/L** treatment ponds, the level ofgrazing by these herbivores was disproportionate across the study. According to the authors, the fish were lost due to predation. This suggests that the study environment was not particularly stable.

- 12

-There is very little that can be concluded with absolute certainty from this study. While it is good in theory, there are far too many inconsistencies in the methods and procedures. Many of the processes are not explained, and it remains uncertain how reliable measurements were obtained. Performing experiments based on mesocosms is clearly important to determine the effects ofpesticides on organisms, but in orderto glean any significant information a study will have to be more tightly controlled. This is further evidenced by the fact that bullfrogs had moved into the ponds andwere one ofthetreatmenteffects noted in the study. Description ofUse in Document(QUAL, QUAN, INV): Qualitative
Rationale for Use: Exposure was not sufficiently characterized, and baseline aquatic community assembledges were not documented prior to study initiation. The study has some utility for demonstrating that aquatic communities were not permanently impaired at a functional level, i.e., aquatic systems recovered, in terms ofabundance; however, there may have been effects on species diversity as plant species more tolerantto atrazinereportedly displaced less tolerant species.
Limitations of Study: No effort was made to document whether other chemicals (including atrazine) were present in the treatment and/or control ponds prior to study initiation. Presampling 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 afterthe first year and also switched exposure concentrations for several ofthe ponds, i.

### e, two ofthe lowest exposures at 20 !1g/L were increased to 500 !1g/L while one pond formerly at 100 !1g/L was increased to 200 !1g/L. Atrazine degradate concentrations were notreported. Phytoplankton and zooplankton werenot identified atthe species level in orderto determine whether more tolerant species had displaced less tolerant species. Large grazing animals (grass carp) were unequally distributed across the studies due to predation; thus, there was likely differential grazing in the study ponds. Additionally, bullfrogs were immigrated into the ponds during the course of the study, and were not likely evenly distributed across treatments; thus, predation by the bullfrogs would have been different across treatments. Controls exhibited wide variability and given that there were only two controls for each ofthe two studies, the ability ofthe study to detecttreatmenteffects is limited. Primary Reviewer: Joseph Gandolfi, Intern Biologist, Environmental Fate and Effects Division, U. S. Environmental Protection Agency. Secondary Reviewers: Thomas Steeger, Ph.D., SeniorBiologist, Environmental.**F.~te** and**Effect~** Division, U

### :..§. I Environmental Protection Agency.

### ~]~J .. /J...-. 

# <4**t_~** '.--- &6/11
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- 13
-Anita Pease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. EnvironmentalProtectionAgency.

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