### Open Literature Review Summary
Chemical Name: Atrazine 

### CAS No: 1912-24-9



### ECOTOX Record Number and Citation: deNoyelles, F., Jr., W.D. Kettle, and D.E. Sinn. 1982. The responses ofplankton communities in experimental ponds to atrazine, the most heavily used pesticide in the United States. Ecology. 63 (5):1285-1293 (MRID 47543607).


### 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 ofa Level ofConcern (LOC).



### Date ofReview: November 24, 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 

### etat., 1989 (described as "the 136- day study" in thatreview), Kettle 

### et

### at., 1987, and deNoyelles and Kettle 1980 (which provides the greatest level ofdetail on most, but not all, methods and results).

In July 1979, six 0.045-haponds were drained and refilled with natural, planktoncontaining water from an adjacentreservoir fed by well water. (The experimental pond setup and history is not described.) The purpose ofdraining and refilling the ponds was to increase their similarity priorto atrazine addition. Subsequently (no specific dates provided), fish were added to each pond: 50 bluegill sunfish ofaverage length 85 mm, 20 channel catfish ofaverage length 116 mm, and 7 gizzard shad ofaverage length 185 mm, representing predators, benthic omnivores, and filtering omnivores, respectively. Atrazine in a commercially available formulation (CO-OP liquid, 41 % active ingredient, EPA registration number 1990-318) was added on July 24 to four ofthe six ponds. Two treatmentponds had nominal concentrations of20**~g/L,** and the othertwo treatment

1 

# 1I1IIIIIII 2087810ponds had nominal concentrations of500 J.lg/L, with two ponds maintained as controls. (No details provided on atrazine application methods.)

Ponds were intensely monitored for the first 63 days after atrazine addition, and sampled monthly afterthat until day 136. Physical and chemical conditions monitored were pond temperature and light profiles, turbidity, pH, alkalinity, and dissolved oxygen. Atrazine concentration was analyzed seven times between day 2 and day 92, according to the procedures ofKadoum and Mock, 1978 (Journal ofAgricultural and Food Chemistry 26:45-50). Water samples were taken from a walkway over each pond (distance from shore notprovided) with a column sampler at the end ofa

### 2A-m pole: three samples were collected from each pond, and two water columns were taken for each sample. The samples were also used to measure phytoplankton.
Samples (one sample perpond) for phytoplankton species distribution measurements were preserved in acid Lugol's, concentrated in settling chambers, and counted with a Wild inverted microscope. Biomass was estimated from particle counts in seven size intervals (from 8 J.lm to 64 J.lm, to include phytoplankton butnot silt) from a Model ZB Coulter counter, using part ofthe composited 3 water samples collected for water chemistry. Microscopic sample observations on each sampling date verified that most phytoplankton species were included in the particle counts, butthat siltparticles were not. Detection limits for species distribution measurements were 2-10 organisms/mL.

Carbon uptake by phytoplankton was measured by incubating column samples for 4 hours with NaH 14C03 in 125-mL glass stopperedbottles in a laboratory growth chamber. Growth chamber conditions reproducedthe pond surface temperature (temperature not stated) and light conditions at I-m depth (bottles rotated around circular fluorescent lamps at 150 J.lE/m2/s). After incubation, samples were processed using the acidification and bubbling method ofSchindler etaI., 1972 (Journal ofthe Fisheries Research Board ofCanada 29: 1627-1631) and counts perminute were used directly in the carbon uptake comparIsons.
Phytoplankton fluorescence experiments in laboratory growth chambers were conducted to observe changes in fluorescence response before atrazine addition and then 2 minutes after atrazine addition (concentrations of 1,5,20, and 500 J.lg/L after addition), and 14C uptake responses were observed after 24 hours with similartreatment. 14C uptake methods were as described above, but for 24 hours incubation instead of4 hours. Fluorescence was measured with a TurnerModell0 Rack-mount Fluorometer (Turner Designs, Mountain View, California; F4T5 blue lamp R466 infrared sensitive photomultiplier with Corning 5-60, 3-66, and 2-64 filters) calibrated against extracted chlorophyll 

### a standards. (The atrazine itselfwas not fluorescent.) Phytoplankton for these tests was taken from control as well as treatmentponds at various times, as described in the results.

2A culture experimentwas performed with the algae Cryptomonas marssonii Skuja and Oocystis sp., the former isolated on day 60 from a 500 )lg/L treatmentpond and grown in the absence ofatrazine for several months, the latter isolated (date notprovided) from a control pond and grown in the absence ofatrazine for 19 days. Growth occurred in a soil-watermedium per Starr, 1964 (American Journal ofBotany 51: 1013-1044), at 20°C and 150 )lE/m2/s. 10 replicate cultures were used.

The authors state that macrophytes, insects, amphibians, and fish were monitored (biomass atbeginning and end); however, only the zooplankton showed atreatment response. Elsewhere it is statedthat "filamentous algae and macrophytes remained sparse in all ponds." Furtherdetails are notprovided.
Zooplankton species samples were taken as vertical nethauls at night from the walkways (no details on distance from shore ortime ofnight). One sample was taken with a49-cm diameter, 157-)lm mesh netby straining about400 L ofpond water, and organisms were counted in three 0.5 mL subsamples ofthe concentrated sample, giving an overall detection limit of0.07 organisms/L. Copepod nauplii and rotifers were counted in the phytoplankton samples by settling 100 mL and counting the whole assemblage.

The cladoceran Simocephalus serrulatus (Koch.) was measured for growth and reproduction. Several Simocephalus adults were collected from apond (specific pond not stated) priorto atrazine addition. On the day after addition, offspring (0-24 hours old) from several adults were placed individually into 100-mLplexiglass cylinders covered at both ends with 153-)lm mesh nylon netting. Ten such cylinders were placed in each pond at 1 meter depth and examined every 2 days throughoutthe lives ofall the individuals to count and remove offspring and record mortality. Forexamination, cylinders were removed from the pond for 1 hour andheld in buckets ofthe pond water, then in an adjacent field laboratory each cylinderwas opened in adish ofpond water and observed under a microscope. Growth was estimatedby taking one set oflength measurements (top ofheadto base ofposterior spine) on day 10. Additionally, Simocephalus was incubated in the laboratory to isolate direct effects from atrazine. Young from the same group used for the in-situ experimentwere incubated in groups of 10, three replicate groups at each treatment level, in closed 300-mL containers in the same growth chamber and light conditions used in the 14C uptake measurements (described above). Water for the containers was control pond water filtered through an 86-)lm mesh to remove larger zooplankton and mixed with atrazine to achieve concentrations of20 and 500 )lg/L, or used without atrazine for the controls. Observations were taken every 48 hours as described forthe in situ experiment, and each original individual was then placed into a containerwith freshly prepared water.

Statistical techniques are not specifically introduced ordescribed, and appearto be the same as those used in deNoyelles and Kettle 1980 (Student-Newman-Keuls test, here denoted SNK). Occasionally othertechniques are employed and described in table notes.

3Results: Some results are exactly as described in deNoyelles and Kettle, 1980, and others are described differently.
Turbidity, lightpenetration, temperatures, and total alkalinity in the ponds did not appear affected by atrazine treatment. The authors state that the pH in the 500 

### /lg/L treatment ponds were 0.3 pH units lowerthan the control pond values for the first few weeks after addition, when 14C uptake by the phytoplankton was low (see below), while the decline in the 20 

### /lg/L treatmentponds was only about 20% that ofthe 500 

### /lg/L ponds. No direct data or statistical analyses areprovided. The authors note that dissolved oxygen also declined about 1-3 

### mg/L lower at both pondbottom and surface in the 500 

### /lg/L treatmentponds compared to control ponds around the same time period as the pH decline, and the 20 

### /lg/L treatmentponds showed similardeclines, but only about 20% of the effect in the 500 

### /lg/L treatmentponds. Again, no data or statistics are provided. The study authors attribute treatmentrelated changes in pH and DO to the effects ofatrazine on phytoplankton photosynthesis.

Four-hour 14C uptake by the phytoplankton communities was inhibited atboth atrazine treatment levels, and the decline in the 500 

### /lg/L treatmentponds was found to be significant atp-value < 0.05 (SNK) and declined to <5% ofpretreatment or control pond values. Live phytoplankton biomass also declined significantly (p-value < 0.05, test not stated) at the same time. Although these levels never appeared to fully recover, after day 24 they seemed somewhatrecovered (no statistics provided). In the 20 

### /lg/L treatment pond, 14C uptake and biomass were significantly (p-value < 0.05, test not stated) declined on day 2 butrecovered by day 7 and remained recoveredthrough day 63. See Figure 1 for agraph ofthe biomass and 14C uptake overtime comparedto the atrazine concentration overthis time frame. Between day 63 and 136, biomass and 14C uptake were measured twice and both times the control ponds' levels were similarto the 20 

### /lg/L treatmentponds' levels and twice those ofthe 500 

### /lg/L treatmentponds.
Phytoplankton collected from all ponds at various times during the study (2nd and 3rd months) and exposed to atrazine in the laboratory showed fluorescence increases and 14C uptake decreases. For control pondphytoplankton, fluorescence increases were significant (p-value < 0.05, SNK with 10 replicates) with atrazine additions as low as I 

### /lg/L, and 14C uptake decreases were significant (p-value < 0.05, SNK) with atrazine additions as low at 5 

### /lg/L. Forphytoplankton from the 20 

### /lg/L treatmentponds, fluorescence increases were only significant (p-value < 0.05, SNK) at atrazine concentrations of5 

### /lg/L or above, and the increases that did occur were significantly (pvalue < 0.05, test not stated) less intense than the responses ofthe control pond phytoplankton. The 14C uptake responses at atrazine treatments of20 and 500 

### /lg/L were similarto control pond phytoplankton responses (insignificant since p-value > 0.05, test not stated). Forphytoplankton from the 500 

### /lg/L treatment ponds, only an atrazine addition of500 

### /lg/L produced a significant (p-value < 0.05, SNK) fluorescence response, butthe response was much weakerthan the control pond response (no statistics provided, but see Table 1). 14C uptake decreases were significantly different from those ofthe

4control pondphytoplankton (p-value < 0.05, test not stated) and appeared much weaker. See Table I and Table 2 for the data. (These tables are copied from deNoyelles and Kettle 1980 because they contain exactly the same data.)

In the culture experiment, the phytoplankton species from the 500 flg/L ponds showed less response to atrazine than the species from the control pond, which the authors assert indicated some resistance to atrazine in the former species butnone in the latter. See Table 3 for the data and statistical results.
Phytoplankton community species composition was altered by atrazine treatment. Peridinium inconspicuum Lemm., a dinoflagellate, was inhibited almost completely in the 500 flg/L treatmentponds, but its abundance in 20 flg/L treatmentponds was similar to the controls. Species declining less dramatically in the 500 flg/L treatmentponds and apparently not declining in the otherponds were Coelastrum spp., Dictyosphaerium pulchellum Wood, Glenodinium sp., Oocystis spp., Scenedesmus spp., Staurastrum tetracerum Ralfs, and Tetraedron minimum (A. Braun) Hansgirg. Other dominant species in the 500 flg/L ponds declined by day 7 but increasedby day 15, including the flagellates Mallomonas (especiallyM pseudocoronata Prescott) and Cryptomonas (especially 

### C. marssonii and 

### C. erosa Ehrenberg) which represented over95% ofthe phytoplankton biomass in both 500 flg/L treatmentponds by day 15. These species increased between day 2 and day 15 in the 20 flg/L treatmentponds, making up about halfofthe total phytoplankton biomass atthe peakoftheir increase, buttheir abundances remained relatively unchanged in the control ponds. Finally, species present in all ponds and occasionally abundant at both treatment levels wereDinobryon divergens v. irregularis G. M. Smith, Rhodomonaspusilla (Bachm.) Javorn, Synedra acus Kutz, 

### S. radians Kutz, and UroglenopsisAmericana Catkins. Afterday 63 there were not noticeable community composition changes in any ofthe ponds. See Figure 2 for atime series ofselected species abundances overtime.
Zooplankton abundance before atrazine addition was high and similar in all ponds, with 100 adult crustaceans per liter, 1,000 copepod nauplii per liter, and 1,000rotifers perliter (no table or figure provided). Tropocyclopsprasinus mexicanus in the 500 flg/L treatmentponds declined from 70% oftotal zooplankton biomass before atrazine addition to <25% within 14 days (no table or figure provided). The in situ experiments with Simocephalus serralutus showed that their growth and reproduction declines were perfectly correlated with declines in the phytoplankton food supply

- growth with phytoplankton biomass, reproduction with 14C uptake (Table 4); laboratory experiments where they experienced elevated levels ofatrazine without declines in food supply found no response to atrazine level.

.

5Table 1. Fluorescence increases ofpond phytoplankton communities following 2 minute exposure to fresh atrazine addition on field experiment days 91 and 93. Values are % increases in fluorescence after the exposure (compared to pre-addition fluorescences); some values are decreases (-). Individual values shown for the two ponds at each treatmentlevel. (study table 1, butthis table is copied from deNoyelles and Kettle 1980 review, table 7 - data are exactly the same)

Addition Control 20 flg/L 500 flg/L Control 1.1 -0.9 -0.2 -0.8 -0.7 -0.3 I /lg/L 4.3* 3.4* -0.3 0 -0.3 0 5 /lg/L 11.1 * 7.1 * 1.7* 2.2* -1 -0.4 20/lg/L 36.5* 22.7* 8.1 * 11.1* -1.2 -0.7 500/lg/L 148.8* 124.0* 58.2* 65.9* 11.3* 9.6* * significantly different from non-addition/control results at p<0.05 (SNK test with 10 replicates for each addition to a community pond sample)

Table 2. 14C uptake (CPM) responses ofpond phytoplankton communities (sampled on various field experiment days) over 24 h to additions ofatrazine. Values are % declines in 14C uptake after the exposure (compared to pre-addition uptake); some values are increases (+). Individual values shown for the two ponds at each treatment level. (study table 2, but this table is copied from deNoyelles and Kettle 1980 review, table 8 - data are exactly the same)

* significantly different from non-addItIOn/control results at p<0.05 (SNKtest WIth 5-10 replicates for each addition to apond community sample)

Addition Day Control 20flg/L 500 flg/L

1 /lg/L 85 3.25+ 98 7.0+ 0.5 49 5.4* 5 /lg/L 83 4.1 98 6.9* 6.7* 49 12.4* 56 18.0* 2.4 21.2* 

## I 20.2* 3.3 [ I 20/lg/L 70 19.5* 13.9* 10.6* 

## I 14.4* 1.8+ 

### I 0.6 85 11.8* 49 83.1 * 500/lg/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*

..

Table 3. Biomass changes in cultures ofCyptomonas marssonii (500 Ilg/L pond isolate) and Oocystis sp. (control pond isolate) incubated with atrazine at 500 Ilg/L for 19 days. Biomass expressed as micrograms fresh mass per liter, calculated from microscope cell counts and cell volume measurements. (study table 3) Cryptomonas Control Atrazine Oocystis Control Atrazine

6Initial Mean 60 60 216 216 Range 58-62 58-62 158-274 158-274 Final

Mean 5,829 2,915 25,120 149 Range 1,011-16,686 76-6,136 7,770-47,073 17-767

SE 1648 650 4142 73 t* 1.644 (p<0.2) 6.028 (p<O.OOl) 

### * Two-tailed ttest comparing final values oftreatment and control for each species, with 10 replicate cultures.

Table 4. Reproduction and growth ofSimocephalus serrulatus (in situ experiment) compared to phytoplankton biomass and 14C uptake. Growth was measured on day 10. (study table 4) Simocephalus Phytoplankton Atrazine addition Youngper Biomass

### * 14C uptake* Pond (Jig/L) perday [A] Growth [B] (mg/L) [C] (cpm) [D]

2 500 0.81 1.09 4.2 28.7 3 500 2.5 1.229 6.1 39 1 20 4.06 1.429 14.3 522.3 5 20 2.87 1.36 12.4 376.3 4 

# ° 2.96 1.349 10 422 6 0 6.7 1.52 36.2 1395.7 Comparisons (r/) AC BC AD BD 0.94 1 1 0.97 

### * BIOmass and 14C uptake were averaged for the perIod dUrIng whIch at least 90% ofthe young were recorded.

t Correlation coefficients were calculated using the Spearmanrank method. All pairings significantly correlated atp<0.05 (critical value for significance was rs = 0.885; two-tailed test, n = 6). Life span averaged from 11 to 16 days and did not correlate with either atrazine treatment orfood availability.

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## <31_~O~-::_::_::_=_i:""=_::_=_=_=_:L:_:O~~~**==::::;:==:::i:=~O=~I=OJ** o 10 Figure 1. Atrazine concentration in raw pond water (bottom), phytoplankton 14C uptake in counts per minute after4-hour incubation (middle), and phytoplankton live biomass in Coulter counter 6-64 /-lm diameterparticle counts (bottom). Treatment means are plotted with vertical bars indicating range between the two ponds at 0 /-lg/L controls (solid line), 20 /-lg/L (dashed line), and 500 /-lg/L (dotted line).

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60 0 40 20 Figure 2. Abundance ofselected phytoplankton species in each ofthe two control, two 20 /lglL, and two 500 /lglL ponds (solid and dotted lines represent replicate ponds). Cells were counted microscopically with a detection limit of2-10 cells/mL.

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

- 

### if controls (JIg/I) Atrazine so, when Concentration (see from Day 0 to comment)?

When Effect Occurred Phytoplankton productivity (14C uptake decline)! Phytoplankton productivity (fluorescence decline) 1

9Effect Magnitude of Nominal Dayaf Time Did Comments Effect Atrazine StudyEffect 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 I Change in 

## I phytoplankton species compostionI Change in zooplankton (decline in cyclopoid copepods) Decreased Simocephalus reproduction Decreased 3% 2Ollg/L 10 1911g/L NR Calculated Simocephalus from growth 20% 5OOllg/L 10 4851lg/L NR review Table 4 Note: NR=Not Reported I See Data Summary Table in review ofdeNoyelles et aI., 1989.
Time weighted average atrazine concentrations were visually estimated from the graph in Figure 1. Since lineardecline was graphed, the estimated atrazine concentration on Day 5 (midpoint ofDay 0 and Day 10) was used as TWA. No calculations were required.

Reviewer Comments: This study was arepackaged version ofthe results described in deNoyelles and Kettle, 1980. Since it is ajournal paper, it is much briefer and more focused then the 1980 report. Many methodological details were omitted, in particularthe experimental pond system setup and history, atrazine application methods, location ofthe walkways in the pond (distance from shore that samples were taken), temperature in the 14C uptake growth chamber, and the date that 

### Oocystis was isolated for the culture experiment. Because of the paper's focus on phytoplankton, the methods and results for macrophytes, insects, amphibians, fish, and filamentous algae were not described, although they were briefly alluded to. Also, detailed results ofthe physical and chemical pond monitoring were not provided.
However, an importantpiece ofadded information from this study is the graph of atrazine concentrations overtime (Figure I). The graph shows which dates atrazine measurements occurred and whatthe concentrations were on those dates, which could allow betterestimation oftime weighted average concentrations in the data summary table endpoints.

10

### The statistics in the paper are mostly the same as in the 1980report, and the same problems with the SNKtest apply (see deNoyelles and Kettle, 1980 reviewer comments). Some new statistical analyses were performed and lookmore appropriate, butthese new analyses were notrelevant for Data Summary Table endpoints.
Description ofUse in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: 

### The datareported as part ofthe study has some utility in that it confirms that atrazine concentrations of20 and 500 Ilg/Lresult in significantdeclines of phytoplankton biomass and 14

### C uptake within several days. Decreasedphytoplankton growth was followed by successional changes leading to the establishment of phytoplankton species that are more resistant to atrazine. Laboratory studies indicated thatresistant species may occur at atrazine concentrations as low as 1to 5 Ilg/L; however the duration and magnitude ofresistant species succession is unclear. The study authors also reportthat grazing zooplankton influenced the biomass and persistence ofresistant phytoplankton species.

Limitations ofStudy: 

### Similarly to the deNoyelles and Kettle, 1980 report, the main limitations ofthe study are small samples (two replicates at each oftwo treatment levels plus two controls) and unclearpresentation ofsome methods andresults (including possibly inappropriate use ofthe SNKtest). 

### In addition, no effort was made to document whether other chemicals (including atrazine) were present in the treatment and/or control ponds priorto study initiation. Pre-sampling was not conducted to determine whether aquatic communities were similarto one anotherpriorto study initiation. Controls exhibited wide variability and given thatthere were only two controls and two replicates for each treatment level, the ability ofthe study to detecttreatmentrelated effects is limited. Given these limitations, the quantitative results should notbe generalized. It 

### is possible thatthe experiment's datareliability could be improved by combining the atrazine concentrations provided in this study withthe raw data provided in deNoyelles and Kettle, 1980.
Primary 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.

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