### Open Literature Review Summary
Chemical Name: Atrazine 

### CAS No: 1912-24-9


### ECOTOX Record Number and Citation: deNoyelles, F. Jr., and W.D. Kettle. 1980. Herbicides in Kansas waters

- evaluations of effects ofagricultural runoffand aquatic weed control on aquatic food chains. ContributionNumber219, Kansas WaterResources Research Institute, University of Kansas, Lawrence, Kansas (MRID 47543605). 

### Purpose ofReview: Determine the potential effects ofatrazine on an aquatic community. Endpoints from this study contribute to the weight-of-evidence regarding effects ofatrazine on aquatic plant communities and associated determination ofaLevel ofConcern (LaC).


### Date ofReview: November24, 2009 

### Purpose ofStudy: This study examines the potential effects ofatrazine to aquatic plants and animals in experimental ponds (mesocosms). 

### Summary ofStudy Findings:
Methods: Methods are also described in deNoyelles et al. 1989 (described as "the 136- day study" in thatreview) and in Kettle et al. 1987; however, this study provides the most detail.


### PondExperiment An experimental pond system consisting offourteen 0.045-ha ponds (2 divided into quadrants), 3 smallerponds, and aholding pondto receive toxic outflowwas constructed at the University ofKansas Nelson Environmental Studies area, 16 km north of Lawrence, Kansas, and completed in November 1977. A nearby 0.33-hareservoir, maintained by well water, was the water source for filling the ponds. None ofthe ponds, including the reservoir, received watershed runoff. See Figure 1 for adepiction ofthe experimental pond setup. Aftercompletion, the ponds were maintained undisturbedwith awaterdepth of2 muntil the first week ofJuly 1979, when they were drained. On July

1 

# 1II1IIIIII 2087811

### 13, six ofthe ponds were filled to 2.1 m (470 m3 ) with natural water including plankton from the reservoir. Thepurpose ofdraining and refilling was to allow the ponds to begin the experiment with similarwaterquality and plankton communities. On July 18 and 19, the following fish were addedto each pond: 50 bluegill sunfish 

### (Lepomis macrochirus, a plankton and insectpredator, average length 80-90 mm), 20 channel catfish 

### (Ictalurus punctatus, abenthic omnivore, average length 116 mm), and 7 gizzard shad 

### (Dorosoma cepedianum, a filtering omnivore, average length 181-189 mm).
On July 24, atrazine in commercial liquid form (CO-OP liquid atrazine, EPA reg. # 

### 1990- 318, 40.8% active atrazine ingredient), was added to four ofthe ponds to achieve nominal atrazine concentrations of20 

### !-!g/L (two ponds) and 500 

### !-!g/L (the othertwo ponds), with the lasttwo ponds maintained as controls. No further atrazine applications orother manipulations occurred overthe course ofthe experimentuntil December 3,136 days after atrazine addition, when the ponds were drained to remove the fish, thereby ending the experiment. The only source ofnew waterto the ponds was rainfall, and by December 3 the pond depths had declinedby about 20-40 cm. Monitoring ofthe ponds began four days priorto atrazine addition and continued until the end ofthe experiment. Pondtemperature and lightprofiles, turbidity, conductivity, pH, alkalinity, and dissolved oxygen were monitored: temperature, light and oxygen measurements were taken atdiscrete depths, while otherparameters were measured from column sample (sampler loweredto within 8 cm ofthe pondbottom). Two water columns were taken for each sample, and 3 samples were taken perpond, all from the western 1/3 

### ofthe pond where there was awalkway extending overeach pond. Although the aforementionedpondparameters were measured 1 and 4 days priorto atrazine addition as well as on days 2,7,10,15,24,31,42,58,63,84,91, and 114 after addition, atrazine concentrations were only measured on day 114 (from column samples). Atrazine concentrations were 75% of"original" (more likely, ofnominal) levels on Day 114. Biological parameters were measured on the full setofmeasurementdays described above. Samples forphytoplankton counts were collected as columns (as described above) andpreserved with acid Lugol's. Organisms were concentrated in settling chambers and counted with aWild inverted microscope. Some species-specific counts were made, as will be described in the results. The species-specific counts used one sampleperpond and had detection limits of2 to 10 organisms/mL. Algal biomass was estimated from column samples with fluorometry. A TurnerModell0 Rack-Mount Fluorometercalibrated against extracted chlorophyll a 

### standards was operated with a F4T5 blue lamp for excitation at400-500 mm with aR466 infrared sensitive multiplier, using filters ofCorning 5-60 (color specification) for excitation, Corning 3-66 for reference, and Corning 2-64 for emission. Carbon uptake by phytoplankton was measuredusing incubation withNaH14C03, with 6 columns perpond for each sample. Water samples were measured out into 100 mL aliquots in 125 mL glass-stoppered bottles, with 14C added at a concentration of20 

### !-!Ci/L. The bottles were incubated either

2

### in a control pond at 0.5 m and 1 m depths for fourhours (no mixing), orin alaboratory environmental chamber set atpond temperature, attached on aframe which rotatedthem around circular fluorescent lamps providing constantmixing and a light intensity of150 /lE/m2

### /s (similarto that ofapond atdepth 1-1.5 m on a clearday). Immediately after incubation, samples were processed for scintillation counting using a Packard Model 2425 Tri-carb liquid scintillation spectrometer, usingthe acidification andbubbling method described by Schindleretal., J. Fish. Res. Bol. Canada (29) pp. 1627-1631, 1972. Countsperminute values from the incubated samples were used directly ratherthan converting them to productivity values. Zooplankton were sampledby vertical net hauls at night, when they were more uniformly distributed by depth. From the pond walkway, one sample was taken with a49 cm diameter, 157 /lm mesh net and two samples with a 30 cm diameter, 20 /lm mesh net. Samples were preservedby adding an equal volume of95% ethanol, 2% formalin solution. The large net samples were counted for IOdates (Days 2, 7, 10, 15, 24, 31,42, and 84 after atrazine addition and the two dates priorto addition), and the other samples were counted infrequently to validate the large net samples. Each large net sample represented 400 liters ofpond waterbased onthe haul depth and was concentratedto a40 mL sample. Three 0.5 mL subsamples were counted for all organisms. The detection limit was about 1 organism per 15 liters, 0.07 organisms per liter.
The fish stocked in the ponds were monitored for mortality during the experimentby retrieving dead individuals. At the end, whenponds were drained, the fish were retrieved, killed in formalin, transferredto water for length and weight measurement, and then preserved in 70% ethanol for laterdissection. Otherorganisms monitored inthe ponds were filamentous algae and macrophytes 

### (Najas, Chara), with weekly visual observations, and benthic invertebrates, with 4 dredge samples perpondtaken atthe end ofthe experiment.


### Short Term andLaboratoryExperiments Phytoplankton collected as column samples from control ponds orthe reservoirwere exposedto atrazine (usingthe same commercial product as appliedto the treatment ponds) in the laboratory so that short-term changes in 14C uptake and fluorescence could be measured. (Othercommercial herbicides were also tested, butthe results will notbe described in this review.) Nominal treatment concentrations were 1,5,20, and 500 /lg/L. For 14C uptake measurements, atrazine was added to the 125 mL bottles and the bottles were incubated for 24 hours in continuous 150 /lE/m2

### /s light, using the rotating rack and followed by scintillation counting as previously described. Responses were expressed as percentage decreases in counts perminute after atrazine addition and compared to nonaddition controls. For fluorescence measurements, the fluorometer described above was used. An initial fluorescence reading was taken, followed by addition ofatrazine, and

3

### another fluorescence reading taken 2 minutes later. Percentage increases I 

### were recorded in terms ofbefore vs. 

### aftermeasurements and comparedwith controls. To facilitate such comparisons, samples were all handled identically, especially by keeping them underthe same low (0.1 /lE/m2

### /s) lightconditions before and during the atrazine exposure. (In some experiments, the fluorescence responses were measured on the samples prepared for 14

### C uptake measurementjustbefore their 24 hour incubation.)
Several relatedtypes ofshort-termphytoplankton experiments were conducted additionally. 

### In one, phytoplankton samples from atrazine-treated ponds were taken and again exposed to varying levels offresh atrazine, to compare responses (14

### C uptake and fluorescence) ofpreviously exposed organisms to those with no priorexposure. Several such experiments were run, afterDay 50. 

### In another experiment, control pond phytoplankton were exposed to filtered treatmentpond water: atDay 97, unfiltered water from each control pond was mixed with an equal volume offiltered water from a different control pond and one treatmentpond ateach ofthe two treatment levels and the percentage increase in fluorescence after addition was recorded. (All filtered waters had the same fluorescence values.) Another set ofphytoplankton resistance experiments involved cultures ofphytoplankton from a control pond and a 500 /lg/L pond taken on Day 60, which grew for 45 days in the absence ofatrazine. Organisms from one culture from eachpond were placed in filtered (0.45 /l HA millipore filter) control pond water, followed by atrazine additions of20 and 500 /lg/L, and fluorescence readings recorded as percentage increases overpre-addition readings. Additionally, the cultures were exposed to filtered water from each control and 500 /lg/L pondby adding 1 mL ofculture to 50 mL offiltered pond water. 14C 

### uptake and fluorescence responses were measured as described above and recorded as percentage increases ordecreases compared to values recorded for exposure to filtered control pond water.
Short-term experiments were conducted withNajas 

### macrophytes. Two 30 cm diameter plastic soil trays, resting on the pondbottom at a depth of2.1 m, were eachplanted with several individuals (each about 20 cm long and all totaling 12 g in mass), in each pond. Theplants were checked for mortality at days 14,31, and 63. (It is not statedwhen the plants were placed inthe ponds, butitwill be assumed that it was on Day 0.)


### In zooplankton experiments, life tables were constructed for 2 species ofcladocerans (Simocephalus serralaturs 

### andDaphniapulex), 

### using young producedby several adult females kept in culture in the laboratory. (The cultures had been startedearlier in the summerby isolating Simocephalu 

### from the reservoir andDaphnia 

### from anearby lake.) The young used to start each life table were 12 ± 

### 12 hours old andhad sizes 0.74 mm for Daphnia 

### and 0.61 mm for Simocephalus. 

### To begin each in situ life table, 10 young of each species were individually placed in 100 mL plexiglass cylinders, which could be

I The authors note that fluorescence increases after atrazine addition are indicative ofthe physiological action ofatrazine in blocking electrontransport in photosynthesis, since the absorbed light energy must be released as fluorescence energy ifit is successfully blocked. Forthis reason they suggestthat fluorescence values in the presence ofatrazine notbe directly interpreted as algal biomass estimates.


### 4opened into two sections, with 153 /lm nylon netting overthe ends. Concurrent microscopic counts ofphytoplankton showed that all common species were less than 50 /lm in diameter and couldreadily pass through the netting. 20 cylinders were placed in each ofthe 6 ponds on day 2 ofthe field experiment, and every 2 days mortality was counted and offspring counted andremoved. Forthese procedures, cylinders were removed inside buckets ofwater and each cylinderwas opened in a dish ofwaterfor observation via microscope, which tookplace at a nearby field lab. The original animals returned to suspension in the pond within 1 hour afterneverleaving their ownpond water or cylinder. Upon return, the cylindernetting was brushed clean and the cylinder flushed by gentle agitation in the pond. Observations continueduntil all animals were dead.
Additional laboratory life tables were constructedusing only

### Simocephalus, starting on Day 28 ofthe field experiment. They were started using young as described above, exceptthey were placed in 300 mL glass stopperedbottles, using water from a control pond filtered through an 86 /lm mesh net which filtered out cladocerans but not phytoplankton. Atrazine concentrations of0, 20, and 500 /lg/L were created in the bottles (presumably using the same commercial atrazine describedpreviously, although this is not explicitly stated). The bottles were kept at 25°C, similarto pondtemperature, and on a 15 hour light, 9 hour dark cycle (light sources and intensity not described). Animals were examined (mortality and offspring recorded) every 2 days by removing the original individuals and placedthem in freshly prepared water (filtered control pond water with atrazine added, as described above), so they were not as subject to changing atrazine concentrations orphytoplankton assemblages as the in situ animals. Finally, short-term experiments were conductedwith benthic invertebrates: 

### Anallagma civile (damsel fly nymph), 

### Musculium transversum (clam), and

### Lirceus garmani (isopod). 

### 10Anallagma were suspended in each pond in aperforated 2 Lplexiglass box for 96 hours beginning on day 2 to observe mortality. 

### Musculium were studied in the same way, and additionally were observed over a longertime periodbeginning on day 15 and lasting forthe next 35 days, with the latterexperiment involving each clam placed in aplexiglass cylinderas used for the in situ zooplankton experiment, with 10 suspended in each pond, and mortality and size observed "periodically over26 days" (the statement seems inconsistentwith the 35 day duration, and is vague as to measurement frequency). 

### Musculium and

### Lirceus were studied in the laboratory to determine a lethal concentration ofatrazine over a 24 hour exposure (concentrations used were 0.02, 0.50, 20, 40, 60, and 80 mg/L).
Statistical analyses are not introduced in the text, butp-values cited in the tables are footnoted with "significantly different (p<0.05)

... using Student-Newmann-Keul's test" or with one-way ANOVA. No further description ofimplementation is provided. All footnotes in the review tables are slightly rewritten versions ofthose in the study, with Student-Newmann-Keul's test written as "SNK" test.



### 5,I.

... ,**."~**

Figure 1. The Experimental Pond Facility at the University ofKansas. From westto east: reservoir pond, 14 experimental ponds (2 divided), 3 smallerexperimental ponds (south) and a holding pond to receive toxic outflow from the experimental ponds. The six ponds used in this study are located in the first row east ofthe reservoir, and were used as (from north to south): control, 20 /lg/L, control, 500 /lg/L, (divided pond not used), 500 /lg/L, 20 /lg/L.



### Results:


### PondExperiment Physical and chemical conditions in the ponds did not dramatically change with atrazine treatments. The authors note several weak effects, but performed no statistical tests, and reviewer examination ofraw data did not find anything noteworthy. The authors note thatthe slight changes they describe appeardue to changes in phytoplankton photosynthesis and macrophyte growth, notto direct effects ofatrazine. Strong but ultimately transient effects ofatrazine on phytoplankton biomass and productivity were observed in the 500 )..lg/L treatmentponds, while the 20 )..lg/L treatment ponds actually showed some increases over control pond biomass, and only abriefeffect

6

### onproductivity (14

### C uptake). 14

### C uptake declined significantly (p-value 

### < 0.05) attwo days in ponds atboth treatment levels. These direct effects ofatrazine onphytoplankton photosynthesis were borne outby the short-term experiments, described below. See Table 1 andTable 2 for the data. Phytoplankton community compositions in the ponds were dramatically alteredby atrazine treatment, although no statistical analysis was performed on these data. 

### Tetraedren minimum and

### Peridinium inconspicuum decreased to near absence in the 500 Ilg/L treatmentponds, butdid notnoticeably decline in the 20 Ilg/L treatmentponds. Most other species 

### (Mallomonas spp., 

### Cryptomonas spp.) initially declined in the 500 Ilg/L treatmentponds, butwere able to recoverby day 24; these species actually experienced increases inthe 20 Ilg/L treatmentponds atthe sametime as the declines in the 500 Ilg/L treatmentponds. One species, 

### Dinobryon divergens var. 

### Schauiuslandii, was absent from all ponds in the beginning butpresentin all ponds by day 31. Two 

### Synedra species, 

### S. acus and

### S. radians, were present atthe beginning and did not decline with the 500 Ilg/L atrazine addition. 

### In the control ponds, species distributions remained. relatively stable overthe time when many species declined in the 500 Ilg/L treatment ponds. See Table 3, Table 4 andTable 5 for the data.2


### Zooplanktonresponses to atrazine treatmentwere not extreme (no statistical analyses were performed on these data), but adecline in the cyclopoid copepodpopulation was notable in the 500 Ilg/L treatmentponds. 

### Diaphanosoma didnot show a similardecline. 

### Chydorus measurements were very low in all ponds. Other species, presentbutin numbers too small to count, did not experience noticeable population changes. See Table 6 for cyclopoid copepod and

### Diaphanosoma data.
Fish inthe experimental ponds developed growth andreproductive differences at different atrazine treatments, butno mortality differences (over 80% oforiginal fish survived to the end ofthe experiment). Growth was measuredby mean length ofeach species atthe end comparedto the beginning ofthe experiment. At the beginning, the mean lengths in the differentponds were within 5% ofeach for

### Lepomis and 2% ofeach otherfor

### Dorsoma (Ictalurus was not sized atthe beginning, butthe fish were obtained from ahatchery andhadbeen noted to all be 116 mm long). Mean growths by pond ranged from 16-26 mm for

### Lepomis, 27-48 mm for

### Dorosoma, and 36-47 mm for 

### Ictalurus. There was "no significantdifference" (no statistical test orp-value cited) in growth in the 20 Ilg/L ponds compared with control ponds, butreducedgrowth was noted in the 500 Ilg/L treatmentponds: decreased growth comparedto growth in control ponds was 26% for

### Lepomis, 31 % for

### Dorosoma, and 16% for

### Ictalurus. The only species to reproduce was 

### Lepomis. The young (atthe fry stage of20-40 mm atthe end ofthe study) were retrieved during the pond drainageprocess by repeated seining while the pond was

2The authors also note declines in Kirchneriella lunaris, Glenodinium sp., Scenedesmus spp., Coelastrum spp., Dictyosphaerium sp. and Staurastrum sp. in the 500 flg/L ponds, whileRhodomonaspusilla and Uroglenopsis americana were present and occasionally abundant in these ponds. These species were not counted in detail and so no data are presented.
7

### 0.1 m deep until no more were being caught. The difference in numberofyoung was dramatic, with an average of 1376 young in the control ponds comparedto averages of59 and 53 in the 20 and 500 

### /lg/L treatmentponds respectively. (No statistical analysis provided.) The adult fish were dissected to observe gonad morphology, butno differences were found associated with atrazine level. 3



### Filamentous algae and macrophytes remained rare in all ponds throughoutthe study. Dredge samples ofbenthic invertebrates did not find any differences in distribution of chironimidae and mayfly nymphs (the dominant members ofthe benthic insect community). (No data are provided.)


### Short Term andLaboratoryExperiments In phytoplankton communities notpreviously exposedto atrazine, significant (p-value < 0.05) increases in fluorescence occurredwithin 2 minutes ofexposure to only 1

### /lg/L atrazine (increase of3% to 4%), rising to 7-11 % at 5

### /lg/L, while for 20 and 500 

### /lg/L levels the increases were 30% and 130%. See Table 7. Subsequently, decreases in 14

### C uptake after 24 hours were significant (p-value < 

### 0.05) atthe 5

### /lg/L level (decreases of 4% to 7% undercontrol uptake rates), while for 20 and 500 

### /lg/L atrazine the decreases were 10-20% and 80-90%. See Table 8. Fluorescence responses were consistent with the development ofatrazine tolerance, with control pondphytoplankton sensitive to even 1

### /lg/L, butno significantincrease in the 20 

### /lg/L pond phytoplankton at that level. At higherconcentrations ofadded atrazine (5, 20, and 500 

### /lg/L) both 20

### /lg/L and control pondphytoplankton had significantresponses (p-value < 

### 0.05), butthe response intensities were significantly different (SNKtest; p-value < 0.05) betweenthe control pond and 20

### /lg/L pondphytoplankton. The phytoplankton from the 500

### /lg/L pond showedno significantresponses to atrazine additions below 500 

### /lg/L, and the response to 500

### /lg/L atrazine was only 10% ofthe response ofthe control pondphytoplankton. 14

### C uptake responses, onthe otherhand, were similarin the control pond and the 20 

### /lg/L pondphytoplankton, butthe 500 

### /lg/L pondphytoplankton showedweakerresponses (no statistical comparisons provided). Atmost atrazine concentration additions tested, there was a significant (p-value < 

### 0.05) decline in 14C 

### uptake, as noted in Table 8.
The authors note, however, thatthe resultsjustdescribed may be due more to the continually oralready provoked responses from the atrazine in the treated ponds than to actual resistance. This suspicion was exploredby performing a very similar experiment, exceptthat filtered water from the treatedponds was used instead ofatrazine added in the lab. As Table 9 shows (compare to Table 7), the fluorescence increases from the treated pond waterwere comparable to those from adding fresh atrazine. To further explore the issue, on day 105 phytoplankton were removed from the 500

### /lg/L treatmentpond water by gentle filtration followed by resuspension in control pond water, and less than 24 hours laterwere exposed to atrazine and tested for fluorescence and 14

### Cuptake responses.

3 These effects were clearly unexplained atthe time afthis study, but were taken up again in the Kettle et al. 1987 study (see thatreview).
8

### (This experimentwas notdescribed in the methods section ofthe study.) Theresulting reductions in 14

### C uptake and increases in fluorescence were only about 15%, much smallerthan the fluorescence increases and 14C 

### uptake decreases previously found (results nottabulated, no statistical analysis). Therefore, the culture experiments were performed. Thephytoplankton taken from a 500 )lg/Ltreatmentpond on day 60 and cultured for45 days, as opposedto the phytoplankton similarly cultured from a control pond, had significantly less ofaresponse to 500 )lg/Lpond water orfresh atrazine addition (p-value < 0.05). See Table 10. The authors interpretthe cultureresults as showing thatphytoplankton resistance to atrazine did indeed develop.


### In the

### Najas macrophyte experiment, where

### Najas individuals collected from nearby ponds were placed in each pond, all plants in the 500 )lg/L treatmentponds were dead after 14 days ofexposure. 

### In the 20 )lg/L treatmentponds, visual estimates ofbiomass found reductions ofabout 50% relative to control ponds, butthe plants survived atthis relative abundance throughoutthe study.
Life tables produced for

### Daphniapulex and 

### Simocephalus serrulatus showed thatboth species experienced significant (p-value < 

### 0.05) reductions in the number ofyoung produced and in daily production ofyoung in the 500 )lg/Ltreatmentponds. 

### In the 20 )lg/L treatmentponds, 

### Daphnia experienced similarreductions while 

### Simocephalus did nothave significanteffects. Additionally, 

### Daphnia had a significantly (p-value < 0.05) shorter lifespan in the 500 )lg/L treatmentponds. See Table 11. The life tables developed for 

### Simocephalus in the laboratory, where there were no food limitations (as opposedto the in situ environment), showedno significantresponses to concentrations of 500 )lg/L atrazine. Again, see Table 11

.

. The invertebrate tests where

### Anallagma and 

### Musulium were suspended in the ponds for

96 hours found no mortality. 

### Musculium growth in the ponds for 35 days also showedno response to atrazine (no dataprovided). 

### In the lab, no mortality responses were found for 

### Musculium or

### Lirceus garmani up to concentrations ofatrazine up to 80 mg/L.

9Table 1. Fluorescence values in Ilg chIa per liter for pond phytoplankton communities. Each value is a mean from recordings of3 column samples. Individual values shown for the two ponds at each treatment level. (from study table 3) Days since Date Atrazine Control 20f-lg/L 500f-lg/L Application 7/20/1979 -4 14.1 11.6 12.3 14.9 12 11.3 7/23/1979 -1 11.6 13.8 10.9 14.6 11.4 8.8 7/26/1979 2 10.8 14.5 12.2 18.7 8.1 8.2 7/31/1979 7 9.9 17.3 14.1 6 2.1 4.9 8/3/1979 10 6.2 20.6 8.7 14.5 3.4 3.9 8/8/1979 15 4.9 5.3 11.9 14.7 7.2 1.8 8/17/1979 24 8.3 9.9 17.5 16.8 7.3 19.1 8/24/1979 31 8.4 10.9 10.3 12.8 10.3 21 9/4/1979 42 6.2 5.6 9.6 26.4 4.8 3.1 9/18/1979 56 5.6 7.4 13.3 18.5 8.1 7.5 9/20/1979 58 5.6 7.7 12.7 20.3 6 6.5 9/25/1979 63 5.2 8.9 13.3 20.2 6.9 9.9 10/2/1979 70 6.2 7.1 17.7 22.4 8.4 8.1 10/16/1979 84 9.2 8.1 15.3 22.7 12.4 5.9 10/23/1979 91 9.4 10.6 19.4 20.3 14 7 11/15/1979 114 6.9 5.9 9.4 15 14.1 2.9 Average 7.34 9.99 13.24 17.81 8.08 7.84 afterDay 0

Table 2. 14C uptake in counts per minute (CPM) for pond phytoplankton communities after 4 h incubation in the lab. Each value is a mean of4 samples and is corrected for dark 14C uptake by subtracting dark bottle CPM. Individual values shown for the two ponds at each treatment level. Changes from July 23 to 25 were significantly different (p<O.05; SNK test) from the control ponds at both atrazine treatment levels. (from study table 4) Days since Date Atrazine Control 20flg/L 500f-lg/L Application 7/20/1979 -4 589 635 643 673 553 560 7/23/1979 -1 411 697 555 466 463 415 7/26/1979 2 405 673 345 368 20 20 7/31/1979 7 601 2122 1019 140 12 44 8/3/1979 10 260 1392 322 518 54 66 8/8/1979 15 299 277 403 479 154 26 8/17/1979 24 168 468 341 364 235 575 8/24/1979 31 269 499 331 406 245 501 9/4/1979 42 267 366 422 746 166 101 9/25/1979 63 239 492 497 514 163 208 10/23/1979 91 262 368 471 429 140 118 11/15/1979 114 136 111 128 145 131 35 Average 291 677 428 411 132 169 afterDay 0

10Table3. Distribution ofTetraedron minimum (A. Braun) Hansgirg. andPeridinium inconspicuum Lemm. in ponds during the field experiment. Values are in cells/mL with blanks (-) below the detection limits of10 and 2cells/mL, respectively. Individual values are shown for the two ponds at each treatment level. (from study table 6)
Tetraedron Peridinium Days since Date Atrazine Control 20flg/L 500 flg/L Control 20 flg/L 500 flg/L Application 7/20/1979 -4



### - 50



### - 2 2

- 271 198 286 314 193 305 7/23/1979 -1 2 62

- 2 2



### - 100 88 143 133 62 114 7/26/1979 2 2 86 2 2 5 2 76 52 90 93 10 19 7/31/1979 7

- 133 7 2



### - - 93 14 14 45



### - - 8/3/1979 10



### - 225 67 31

- - 136 36 76 45

- - 8/8/1979 15 76 324 281 433 5



### - 105 38 112 60



### - - 8/17/1979 24 671 3392 640 3861

- - 136 29 26 33



### - - 8/24/1979 31 240 2303 566 5498



### - - 69 26 57 40

- - 9/4/1979 42 105 1352 847 12916

- - 50



### - 14 62

- - 9/25/1979 63 81 607 2235 6341

- - 14 2

- 17



### - - 10/16/1979 84 17 495 1297 2073



### - - - - - - - - 11/15/1979 114 64 438 2292 471 7



### - - - - 

### - - -

11Table 4. Distribution ofMallomonaspseudocoronata Prescott andMallomonas spp. (smaller species, unidentifiable due to preservation) in ponds during the field experiment. Values are in cells/mL with blanks (-) below the detection limit of2 cells/mL. Individual values shown for the two ponds at each treatmentlevel. (from study table 7)
Mallomonas Mallomonas spp. Days since Date Atrazine Control 20Jig/L 500Jig/L Control 20 Jig/L 500 Jig/L Application 7120/1979 -4 2

- 2 2 7 5



### - 2

- - 2



### - 7/23/1979 -1 21 7 60 26 29 2 7 7 31 7 2 5 7/26/1979 2 21 5 105 24 17



### - 5



### - 14

- 

### - - 7/31/1979 7 24 10 1449 88

- 

### - - 

### - 405 12

- - 8/3/1979 10 40 502 2 1019



### - - 7 33 224 167 12

- 8/8/1979 15 64 5



### - 2372

- 7 55 55 657 39 155 31 8/1711979 24 10

- - 

### - 2 2421 45 5 62 21 990 3156 8/24/1979 31

- - 2

- 39 1147 5

- 5

- 951 2754 9/4/1979 42

- - - 2 245 129

- 

### - - 

### - 294

- 9/25/1979 63 2



### - - - - - 12

- - 62

- 2 10/16/1979 84



### - - - 

### - - - 157

- - 39 10 29 11/15/1979 114

- - - - 

### - - 67

- 

### - 2 2 12

12Table 5. Distribution ofCryptomonas marssonii Skuja andSynedra spp. in ponds during the field experiment. Values are in cells/mL with blanks (-) below the detection limit of2 cells/mL. Individual values shown for the two ponds ateach treatmentlevel. (from study table 8) Cryptomonas Synedra Days since Date Atrazine Control 20j.lg/L 500j.lg/L Control 20j.lg/L 500j.lg/L Application 7/2011979 -4 114 21 86 60 38 17 426 136 314 350 364 366 7/2311979 -1 105 12 83 154 48 58 171 48 129 202 231 217 7/26/1979 2 50 12 50 88 10



### - 86 63 95 160 131 119 7/31/1979 7 62 10 302 119



### - 131 214 36 114 40 ""



### - .).) 8/3/1979 10 112 12 516 288 2



### - 21 252 150 157 26 33 8/8/1979 15 69 17 452 107 426 2 24 214 793 250 52 10 8/17/1979 24 57 167 36 79 198 86 152 378 171 57 143 21 8/24/1979 31 36 112 121 62 445 228 93 140 150 55 33 44 9/4/1979 42 40 38 447 224 419 583 64 98 202 39 5 133 9/25/1979 63 74 50 129 217 312 183 5 67 12 262 74

- 10/16/1979 84 250 76 69 271 240 95 5 110 5 148 81 5 11/15/1979 114 317 12 93 226 754 24 881 48 10 1285 60



### -

13Table 6. Distribution ofzooplanktonDiaphanosoma brachyurum and cycloid copepods (species notdistinguished) in ponds during the field experiment. Values are in organisms/L, with plus signs (+) denotingvalues between 0.07 and 1 organisms/L and blanks (-) for values below the detection limit of0.07 organisms/L. Individualvalues shown for the two ponds at each treatment level. (from study table 14) Diaphanosoma Cyclopoida spp. Days since Date Atrazine Control 20 flg/L 500 flg/L Control 20 flg/L 500 flg/L Application 7/20/1979 -4 14 9 11 8 7 3 59 104 67 62 53 55 7/23/1979 -1 37 45 49 33 25 5 83 96 95 62 59 90 7/26/1979 2 50 119 145 93 83 7 58 95 112 65 57 37 7/31/1979 7 101 354 298 403 236 39 39 56 61 33 5 9 8/3/1979 10 197 262 91 224 119 85 33 11 22 60 1 4 8/8/1979 15 135 70 4 133 110 191 13 1 1 11
- 3 8/17/1979 24 + 1 + 130 + 106 + 2 + 1 + 1 8/24/1979 31 + + - 110



### - 154 + 1 + 3 + 10 9/4/1979 42 + + + 51



### - 70 + 1 + 4 + 3 10/16/1979 84

- + 

### - 1 + 

### - + + + 5 + +

14Table 7. Fluorescence increases ofpond phytoplankton communities following 2 minute exposure to fresh atrazine addition on field experimentdays 91 and 93. Values are % increases in fluorescence afterthe exposure (compared to pre-addition fluorescences); some values are decreases (-). Individual values shown for the two ponds at each treatment level. (from study table 9)

* signIficantly different from non-addItIOn/control results atp<0.05 (SNKtest wIth 10 replicates for each addition to a community pond sample)

Addition Control 20J)g/L 500 J)g/L Control 1.1 -0.9 -0.2 -0.8 -0.7 -0.3 1 f.tg/L 4.3* 3.4* -0.3 0 -0.3 0 5 f.tg/L 11.1 * 7.1 * 1.7* 2.2* -1 -0.4 20 f.tg/L 36.5* 22.7* 8.1* 11.1 * -1.2 -0.7 500 f.tg/L 148.8* 124.0* 58.2* 65.9* 11.3* 9.6*

..

Table 8. 14C uptake (CPM) responses ofpond phytoplankton communities (sampled on various field experiment days) over24 h to additions ofatrazine. Values are % declines in I4C uptake after the exposure (compared to pre-addition uptake); some values are increases (+). Individual values shown for the two ponds ateach treatmentlevel. (from study table 10)

Addition Day Control 20J)g/L 500pg/L

If.tglL 85 3.25+ 98 7.0+ 0.5 49 5.4* 5 f.tg/L 83 4.1 98 6.9* 6.7* 49 12.4* 20 f.tg/L 56 18.0* 2.4 21.2* 

## I 20.2* 3.3 

## I 1 70 19.5* 13.9* 10.6* 

## I 14.4* 1.8+ 

## I 0.6 85 11.8* 49 83.1 * 500 f.tg/L 56 86.8* 87.4* 89.6* 

## I 88.0* 20.8* 

## I 0.9 70 90.3* 88.2* 89.7* 

## I 87.6* 18.7* I 9.5 85 91.6* * significantly different from non-addition/control results atp<0.05 (SNKtest with 5-10replicates for each addition to apond community sample)

15Table 9. Fluorescence increases ofpond phytoplankton communities exposed to an equal volume of filtered water from anotherpond on field experiment day 97. Values are % increases in fluorescence after 2 minutes ofexposure to the otherpond's water (compared to fluorescence after exposure to their own pond's water). Individual values shown for the two ponds at each treatment level. (from study table 11)

Addition Control 2.7 Control 2.1 20**~lg/L** 22.6* 13.2* 500**~g/L** 91.0* 100.9* * significantly dIfferent atp<0.05 (SNKtest with 10 replicates for each addition to apond community sample)

Table 10. Fluorescence increase and 14C uptake decrease ofphytoplankton cultures isolated after 60 days from control and 500 !Jg/L treatment ponds. Cultures grew for45 days in the absence of atrazine and were then exposed to filtered water from the 500 !Jg/L treatment ponds as well as additions of20 and 500 !Jg/Lfresh atrazine in filtered control pond water. Values are % changes from responses with exposure to filtered control pond water. Individual values shown for the two 500 !JglLtreatmentponds. (from study table 12)
% increase in % decrease in C-14 fluorescence uptake Control 500 flg/L Control 500 flg/L Treatment Pond Pond Pond Pond Culture Culture Culture Culture 500**~g/L** 106.3 24.2 59.9 32.4

(Pond) 129.2 58.8 69.1 28.8 20**~g/L** 21.8 7.1 500**~g/L** 105.5 48.6 Note: all responses from 500**~g/L** cultures are slgmficantly different (p<0.05, one-way ANOVA with 4 replicates for eachtreatmentto aculture) from those ofcontrol pond cultures.

16Table 11. Reproductive and longevity responses of2 species ofc1adocerans (Simocephalus serrulatus and Daphniapulex) exposed to atrazine in situ and in the laboratory. Each value is the mean for 10 individuals for each species ateach treatment level observed every 2days throughout their lifespan. (from study table 15)**Young/~ Young/~/day**  Lifespan (days) in situ (began Day 2) Control 23.72 1.99 11.88 Daphnia 20/lg/L 6.35* 0.7* 9.06 500/lg/L 6.53* 0.87* 7.53* Control 52.4 4.72 11.1 Simocephalus 20/lg/L 47.9 3.5 13.7 500/lg/L 22.7* 1.72* 13.2 Laboratory (began Day 28) Control 9.63 0.64 15.1 Simocephalus 20/lg/L 8.93 0.65 13.8 500/lg/L 9.7 0.69 14.11 * sIgnificantly dIfferent from control responses (p<0.05, SNK test)



### Data Summary Table: Effect Magnitude of Nominal Dayof Time Did Comments Effect Atrazine Study Effect Weighted Recovery relative to Concentration Occurred Average Occur

- if controls (flg/L) Atrazine so, when Concentration (see from Day 0 to comment)?

When Effect Occurred Phytoplankton 25% 20/lg/L 7 19.8/lg/L Yes Calculated biomass (Day IS) from review (fluorescence 35% 500/lg/L 2 498.8/lg/L Yes Table 1. decline)! (DaY IS) Phytoplankton 33% 20/lg/L 2 20.0/lg/L Yes Calculated productivity (Day IS) from review 

### C

4Cuptake >95% 500/lg/L 2 498.9/lg/L Yes Table 2. decline)1 (Day 24) Change in phytoplankton species composition2 Change in 0 20/lgiL No effect Not Not Calculated zooplankton applicable applicable from review (decline in Yes Table 6. cyclopoid 85% 500/lg/L 7 496.2/lg/L (Day 31) copepods) Decreased 0 20/lg/L No effect Not Not Effects fish growth applicable applicable noted at end
17Effect Magnitude of Nominal Day of Time Did Comments Effect Atrazine StudyEffect Weighted Recovery relative to Concentration Occurred Average Occur- 

### if controls (fig/L) Atrazine so, when Concentration (see from Day () to comment)?

When Effect Occurred (Lepomis) 26% 5OOllg/L 136 425.4 Ilg/L No of Decreased 0 2Ollg/L No effect Not Not experiment. fish growth applicable applicable (Dorosoma) 31% 5001lg/L 136 425.4 Ilg/L No Decreased 0 20 flg/L No effect Not Not fish growth applicable applicable (lctalurus) 16% 5001lg/L 136 425.4 Ilg/L No Decreased Lepomis reproduction 50% 20 J.1l!/L 14 19.7 Jlg/L No 50% value is based on Submerged visual macrophyte 100% (all estimates. survival 500 J.lg/L 14 492.3Ilg/L No Assumes trays (Najas) died) were placed in ponds on Day O. Decreased 70% 2Ollg/L 15 (est.) 19.6 flg/L Not Life tables applicable began on Day Daphnia Not 2 ofthe field reproduction 70% 500 flg/L 15 (est.) 489.6 flg/L applicable experiment Decreased 5-10% 20 flg/L 15 (est.) 19.6 flg/L Not and lasted until applicable all individuals Simocephalus Not died reproduction 55% 500 flg/L IS (est.) 489.6 flg/L applicable (estimated to Decreased 25% 20 flg/L 15 (est.) 19.6Ilg/L Not be 15 days applicable later). Daphnia Not lifespan 35% 5OOllg/L 15 (est.) 489.6 flg/L applicable Balded values are those datapomts specIfied m the "Cosm Data Pomts and Exposure Profiles" spreadsheet. I Similarendpoints appear in the "Cosm Data Points and Exposure Profiles" spreadsheet. 2 See Data Summary Table in review ofdeNoyelles et aI., 1989. Time weighted average concentrations are estimated from the only datapointprovided on atrazine concentrations

- that atrazine levels measured in the ponds on Day 114 were 75% ofthe "original" levels (which are assumed equal to the nominal concentrations). Lineardecline is assumed overtime to estimate atrazine concentrations onrelevant days ofthe study,4 and time weighted averages are calculated as simple averages ofthe initial and effect day concentrations. See the Excel-linked table below, and "deNoyelles & Kettle 1980 tables.xls" for details.

4 The linear decline estimates a 70% reduction by the last day ofthe study (day 136), which is in agreement with the value cited in Kettle et al. 1987 (see that review).
18TWA Calculations:
Nominal Initial Effect TWA

Conc. Initial Conc. Effect Day Conc. (fJg/L) Day (f!g/L) Day Conc. (fJg/L) (llQ/L) 20 0 20 2 19.9 20.0 500 500 497.8 498.9 20 0 20 7 19.7 19.8 500 500 492.3 496.2 20 0 20 14 19.4 19.7 500 500 484.6 492.3 20 2 19.9 17 19.3 19.6 500 497.8 481.4 489.6 20 0 20 136 14.0 17.0 500 500 350.9 425.4



### Reviewer Comments: This study provides some descriptions ofmethods that are applicable to other studies, but are not described in those other studies (deNoyelles et al. 1989, Kettle et al. 1987, Carney 1983, etc.). Primarily, the hydrological setup ofthe experimental pond facility is described in much more detail, with a site drawing, and aclear statementthat the ponds receive no watershed runoffand so could notbe affected by local herbicide runoff.
The atrazine concentrations in the experimental ponds overtime remains highly uncertain. Only apercentage of"original" concentrations remaining on Day 114, which presumably is applicable to both the 20 and 500 Ilg/L treatments, is reported. 

### In addition, an assumption is made thatthe concentrations on Day 0 were in the same as nominal (experience shows thatthis is unlikely to have actually occurred). Given the lack of atrazine concentration measurements, the time weighted average concentrations are uncertain and shouldnot be used as exact, known values.


### In the algal biomass andproductivity measurements, measurements were made with reasonable frequency and are tabulated in detail, in terms ofthe more directquantities of counts perminute 14C uptake and fluorescence readings. As such, they are not as subject to uncertainty in interpretation as would be calculated productivity orbiomass values. However, in the "percentage increases" and "compared to controls" relative assessments, there is inconsistency in the definitions ofthese comparisons which makes interpretation difficult (See remarkbelow on statistics as well.)


### In the animal and macrophyte experiments, which are important for the purposes ofthis literature review, some important details ofthe methods are missing. It is not stated on what day the soil trays were placed in the ponds for the macrophyte growth experiment.

19It is not stated thatthe same commercial atrazine usedthroughoutthe study was used to constructthe laboratory life tables for

### Simocephalus. There is inconsistency in the description ofthe timing ofthe benthic invertebrate experiments (did they last 35 days or 26 days?), although there was no mortality in this experiment. Many results were presented without any statistical analysis. (Given the small number of samples and exploratory nature ofthe study, this may have been reasonable, however.) Physical and chemical parameterchanges, fish growth differences, bluegill reproduction declines, and no differences in dredged invertebrate populations were all discussed without any reference to statistical tests for significance.

The statistics that are presented are impossible to interpret and perhaps wrong. The explanations are too briefand unclearto determine how the SNKtest was implemented and whether itwas done so correctly (afterANOYA; groups defined by treatment level; all 3 groups considered in the same analysis). As amultiple comparison test, the SNK test shouldhave been done to test for differences among the 3 treatment level groups, but the results are described as showing difference from the control, as ifatwo group comparison had been done. An SNKtest is inappropriate for a simple two group comparison. Often when an SNKtest was cited, anumber ofreplicates was given which hadneverbeenpresented in the text ortable description and seemed odd. (Are the values in the table then sample means? Why is this not stated? See Table 7 - Table 9.) Overall the impression is giventhatthe statistics were notproperly done. In any case, they were notproperly explained.

In general, the report is presented in a surprisingly nonlinear fashion for an experimental study. There were difficulties in locating the results for each experiment described in the methods. Inparticular, the laboratory tests for phytoplankton resistance seemed ad hoc in both execution and in terms ofthe results presentation. With such a large study and wide range ofexperiments performed, organizedpresentation is essential for conveying what happened in a comprehensible fashion and was too often found lacking. Description ofUse in Document (QUAL, QUAN, INV): Qualitative
Rationale for Use: The study provides further details regarding the experimental design ofponds used in mescosm studies presented in deNoyelles et al. 1989 (described as "the 136-day study" in thatreview) and in Kettle et al. 1987. Although the study results are weakenedby the small number ofreplicates andtreatments andby a lack ofinformation aboutthe atrazine concentration overtime, the study provides reasonable evidence that macrophyte cover is reduced by atrazine exposure even at 20 llg/L concentrations. In addition, shifts in phytoplankton species composition are apparent at atrazine concentrations of500 llg/L

20

### Limitations ofStudy: This was an early, exploratory mesocosm study which identified many important aquatic community responses to atrazine. The main limitations ofthe study are lack ofatrazine concentration data, small sample (two replicates at each oftwo treatment levels plus two controls), andunclearpresentation ofsome methods and results (including unclear statistics). Because ofthese limitations, the quantitative results should notbe generalized. However, alarge amount ofrelatively unprocessed data is provided, in addition to site descriptions, which could help fill in some ofthe information gaps in related studies describing this 136-day experiment orothers taking place atthe University ofKansas experimental ponds system. 

### Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental Inc 

### Secondary Reviewer: AnitaPease, M.S., SeniorBiologist, Environmental Fate and Effects Division, U.S. Environmental ProtectionAgency.

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