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 of plankton

communities in experimental ponds to atrazine, the most heavily used pesticide in the

United States. Ecology. 63 (5):1285-1293 (MRID 47543607).

Purpose of Review:

Determine the potential effects of atrazine on an aquatic community. Endpoints from this

study contribute to the weight-of-evidence regarding effects of atrazine on aquatic plant

communities and associated determination of a Level ofConcern (LOC).

Date of Review:

November 24, 2009

Purpose of Study:

This study examines the potential effects of atrazine to aquatic plants and animals in

experimental ponds (mesocosms).

Summary of Study Findings:

Methods: Methods are also described in deNoyelles et at., 1989 (described as "the 136-

day study" in that review), Kettle et at., 1987, and deNoyelles and Kettle 1980 (which

provides the greatest level of detail on most, but not all, methods and results).

In July 1979, six 0.045-ha ponds were drained and refilled with natural, planktoncontaining water from an adjacent reservoir fed by well water. (The experimental pond

setup and history is not described.) The purpose of draining and refilling the ponds was

to increase their similarity prior to atrazine addition. Subsequently (no specific dates

provided), fish were added to each pond: 50 bluegill sunfish of average length 85 mm, 20

channel catfish of average length 116 mm, and 7 gizzard shad of average 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

treatment ponds had nominal concentrations of 20 ~g/L, and the other two treatment

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1I1IIIIIII

2087810
ponds had nominal concentrations of 500 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 after that 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 of Agricultural and Food Chemistry

26:45-50). Water samples were taken from a walkway over each pond (distance from

shore not provided) with a column sampler at the end of a 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 per pond) 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 but not 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, but that silt particles 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 NaH14C03 in 125-mL glass stoppered bottles in a laboratory growth chamber.

Growth chamber conditions reproduced the 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 of Schindler et aI., 1972 (Journal ofthe Fisheries Research Board

ofCanada 29: 1627-1631) and counts per minute 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 similar treatment. 14C uptake

methods were as described above, but for 24 hours incubation instead of 4 hours.

Fluorescence was measured with a Turner Modell 0 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 itself was not fluorescent.) Phytoplankton for

these tests was taken from control as well as treatment ponds at various times, as

described in the results.

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A culture experiment was performed with the algae Cryptomonas marssonii Skuja and

Oocystis sp., the former isolated on day 60 from a 500 )lg/L treatment pond and grown in

the absence of atrazine for several months, the latter isolated (date not provided) from a

control pond and grown in the absence of atrazine for 19 days. Growth occurred in a

soil-water medium per Starr, 1964 (American Journal of Botany 51: 1013-1 044), 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 at beginning and end); however, only the zooplankton showed a treatment

response. Elsewhere it is stated that "filamentous algae and macrophytes remained

sparse in all ponds." Further details are not provided.

Zooplankton species samples were taken as vertical net hauls at night from the walkways

(no details on distance from shore or time of night). One sample was taken with a 49-cm

diameter, 157-)lm mesh net by straining about 400 L of pond water, and organisms were

counted in three 0.5 mL subsamples ofthe concentrated sample, giving an overall

detection limit of 0.07 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 a pond (specific pond not

stated) prior to atrazine addition. On the day after addition, offspring (0-24 hours old)

from several adults were placed individually into 100-mL plexiglass 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 throughout the lives of all the individuals to

count and remove offspring and record mortality. For examination, cylinders were

removed from the pond for 1 hour and held in buckets ofthe pond water, then in an

adjacent field laboratory each cylinder was opened in a dish of pond water and observed

under a microscope. Growth was estimated by taking one set of length measurements

(top of head to base of posterior 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 experiment were 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 of 20 and 500 )lg/L, or

used without atrazine for the controls. Observations were taken every 48 hours as

described for the in situ experiment, and each original individual was then placed into a

container with freshly prepared water.

Statistical techniques are not specifically introduced or described, and appear to be the

same as those used in deNoyelles and Kettle 1980 (Student-Newman-Keuls test, here

denoted SNK). Occasionally other techniques are employed and described in table notes.

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Results: Some results are exactly as described in deNoyelles and Kettle, 1980, and others

are described differently.

Turbidity, light penetration, 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 lower than 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 treatment ponds was only about 20% that ofthe 500 /lg/L ponds. No direct

data or statistical analyses are provided. The authors note that dissolved oxygen also

declined about 1-3 mg/L lower at both pond bottom and surface in the 500 /lg/L

treatment ponds compared to control ponds around the same time period as the pH

decline, and the 20 /lg/L treatment ponds showed similar declines, but only about 20% of

the effect in the 500 /lg/L treatment ponds. Again, no data or statistics are provided. The

study authors attribute treatment related changes in pH and DO to the effects of atrazine

on phytoplankton photosynthesis.

Four-hour 14C uptake by the phytoplankton communities was inhibited at both atrazine

treatment levels, and the decline in the 500 /lg/L treatment ponds was found to be

significant at p-value < 0.05 (SNK) and declined to <5% of pretreatment 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 somewhat recovered (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 but recovered by day 7 and remained recovered through day 63. See Figure 1

for a graph ofthe biomass and 14C uptake over time compared to the atrazine

concentration over this time frame. Between day 63 and 136, biomass and 14C uptake

were measured twice and both times the control ponds' levels were similar to the 20 /lg/L

treatment ponds' levels and twice those ofthe 500 /lg/L treatment ponds.

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 pond phytoplankton, 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. For phytoplankton from the 20 /lg/L treatment ponds,

fluorescence increases were only significant (p-value < 0.05, SNK) at atrazine

concentrations of 5 /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 of 20 and 500 /lg/L were

similar to control pond phytoplankton responses (insignificant since p-value > 0.05, test

not stated). For phytoplankton from the 500 /lg/L treatment ponds, only an atrazine

addition of 500 /lg/L produced a significant (p-value < 0.05, SNK) fluorescence response,

but the response was much weaker than the control pond response (no statistics provided,

but see Table 1). 14C uptake decreases were significantly different from those ofthe

4
control pond phytoplankton (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 but none 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 treatment ponds, but its abundance in 20 flg/L treatment ponds was similar

to the controls. Species declining less dramatically in the 500 flg/L treatment ponds and

apparently not declining in the other ponds 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 increased by day 15, including the

flagellates Mallomonas (especially M pseudocoronata Prescott) and Cryptomonas

(especially C. marssonii and C. erosa Ehrenberg) which represented over 95% ofthe

phytoplankton biomass in both 500 flg/L treatment ponds by day 15. These species

increased between day 2 and day 15 in the 20 flg/L treatment ponds, making up about

half ofthe total phytoplankton biomass at the peak oftheir increase, but their abundances

remained relatively unchanged in the control ponds. Finally, species present in all ponds

and occasionally abundant at both treatment levels were Dinobryon divergens v.

irregularis G. M. Smith, Rhodomonas pusilla (Bachm.) Javorn, Synedra acus Kutz, S.

radians Kutz, and Uroglenopsis Americana Catkins. After day 63 there were not

noticeable community composition changes in any ofthe ponds. See Figure 2 for a time

series ofselected species abundances over time.

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 1,000 rotifers per liter

(no table or figure provided). Tropocyclops prasinus mexicanus in the 500 flg/L

treatment ponds 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 of atrazine without declines in food supply found

no response to atrazine level. .

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Table 1. Fluorescence increases of pond 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 treatment level. (study table 1, but this 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 of pond phytoplankton communities (sampled on various field

experiment days) over 24 h to additions of atrazine. 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 (SNK test WIth 5-10 replicates for each

addition to a pond community sample)

Addition Day Control 20 flg/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 of Cyptomonas 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

6
Initial

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 t test comparing final values oftreatment and control for each species, with 10 replicate

cultures.

Table 4. Reproduction and growth of Simocephalus serrulatus (in situ experiment) compared to

phytoplankton biomass and 14C uptake. Growth was measured on day 10. (study table 4)

Simocephalus Phytoplankton

Atrazine

addition Young per Biomass* 14C uptake*

Pond (Jig/L) per day [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 Spearman rank method. All pairings significantly

correlated at p<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 or food availability.

7
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4

!;i <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 after 4-hour incubation (middle), and phytoplankton live biomass in Coulter counter 6-64

/-lm diameter particle 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).

8
103r-----:-~--_,_--:__----_,.-------__.

control 20 ug/L 500 ug/L

20 60

....".. " " ..

60 0

"--vr" /rf'''' .

I

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20 40

DAYS

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20 40 60 0

Figure 2. Abundance of selected phytoplankton species in each of the 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 of 2-10 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

9
Effect Magnitude of Nominal Dayaf 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 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 of deNoyelles et aI., 1989.

Time weighted average atrazine concentrations were visually estimated from the graph in

Figure 1. Since linear decline was graphed, the estimated atrazine concentration on Day

5 (midpoint of Day 0 and Day 10) was used as TWA. No calculations were required.

Reviewer Comments:

This study was a repackaged version ofthe results described in deNoyelles and Kettle,

1980. Since it is a journal paper, it is much briefer and more focused then the 1980

report. Many methodological details were omitted, in particular the 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 important piece of added information from this study is the graph of

atrazine concentrations over time (Figure I). The graph shows which dates atrazine

measurements occurred and what the concentrations were on those dates, which could

allow better estimation oftime weighted average concentrations in the data summary

table endpoints.

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The statistics in the paper are mostly the same as in the 1980 report, and the same

problems with the SNK test apply (see deNoyelles and Kettle, 1980 reviewer comments).

Some new statistical analyses were performed and look more appropriate, but these new

analyses were not relevant for Data Summary Table endpoints.

Description of Use in Document (QUAL, QUAN, INV): Qualitative

Rationale for Use: The data reported as part ofthe study has some utility in that it

confirms that atrazine concentrations of 20 and 500 Ilg/L result in significant declines of

phytoplankton biomass and 14C uptake within several days. Decreased phytoplankton

growth was followed by successional changes leading to the establishment of

phytoplankton species that are more resistant to atrazine. Laboratory studies indicated

that resistant species may occur at atrazine concentrations as low as 1 to 5 Ilg/L; however

the duration and magnitude ofresistant species succession is unclear. The study authors

also report that grazing zooplankton influenced the biomass and persistence ofresistant

phytoplankton species.

Limitations of Study: 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 unclear presentation ofsome methods and results (including

possibly inappropriate use ofthe SNK test). In addition, no effort was made to

document whether other chemicals (including atrazine) were present in the treatment

and/or control ponds prior to study initiation. Pre-sampling was not conducted to

determine whether aquatic communities were similar to one another prior to study

initiation. Controls exhibited wide variability and given that there were only two controls

and two replicates for each treatment level, the ability ofthe study to detect treatmentrelated effects is limited. Given these limitations, the quantitative results should not be

generalized. It is possible that the experiment's data reliability could be improved by

combining the atrazine concentrations provided in this study with the raw data provided

in deNoyelles and Kettle, 1980.

Primary Reviewer: Sara Hendrix, M.S., Staff Scientist, Cambridge Environmental Inc

Secondary Reviewer: Anita Pease, M.S., Senior Biologist, Environmental Fate and

Effects Division, U.S. Environmental Protection Agency.

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