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 of agricultural runoff and aquatic weed control on aquatic food chains.

Contribution Number 219, Kansas Water Resources Research Institute, University of

Kansas, Lawrence, Kansas (MRID 47543605).

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 (LaC).

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 al. 1989 (described as "the 136-

day study" in that review) and in Kettle et al. 1987; however, this study provides the most

detail.

Pond Experiment

An experimental pond system consisting of fourteen 0.045-ha ponds (2 divided into

quadrants), 3 smaller ponds, and a holding pond to receive toxic outflow was constructed

at the University of Kansas Nelson Environmental Studies area, 16 km north of

Lawrence, Kansas, and completed in November 1977. A nearby 0.33-ha reservoir,

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 a depiction ofthe

experimental pond setup. After completion, the ponds were maintained undisturbed with

a water depth of 2 m until 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 m

3

) with natural water including plankton

from the reservoir. The purpose of draining and refilling was to allow the ponds to begin

the experiment with similar water quality and plankton communities. On July 18 and 19,

the following fish were added to each pond: 50 bluegill sunfish (Lepomis macrochirus, a

plankton and insect predator, average length 80-90 mm), 20 channel catfish (Ictalurus

punctatus, a benthic 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 of 20 !-!g/L (two ponds) and 500 !-!g/L (the other two ponds), with

the last two ponds maintained as controls. No further atrazine applications or other

manipulations occurred over the course ofthe experiment until December 3,136 days

after atrazine addition, when the ponds were drained to remove the fish, thereby ending

the experiment. The only source of new water to the ponds was rainfall, and by

December 3 the pond depths had declined by about 20-40 cm.

Monitoring ofthe ponds began four days prior to atrazine addition and continued until the

end ofthe experiment. Pond temperature and light profiles, turbidity, conductivity, pH,

alkalinity, and dissolved oxygen were monitored: temperature, light and oxygen

measurements were taken at discrete depths, while other parameters were measured from

column sample (sampler lowered to within 8 cm of the pond bottom). Two water

columns were taken for each sample, and 3 samples were taken per pond, all from the

western 1/3 ofthe pond where there was a walkway extending over each pond. Although

the aforementioned pond parameters were measured 1 and 4 days prior to 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, of nominal) levels on Day

114.

Biological parameters were measured on the full set of measurement days described

above. Samples for phytoplankton counts were collected as columns (as described

above) and preserved with acid Lugol's. Organisms were concentrated in settling

chambers and counted with a Wild inverted microscope. Some species-specific counts

were made, as will be described in the results. The species-specific counts used one

sample per pond and had detection limits of 2 to 10 organisms/mL. Algal biomass was

estimated from column samples with fluorometry. A Turner Modell 0 Rack-Mount

Fluorometer calibrated against extracted chlorophyll a standards was operated with a

F4T5 blue lamp for excitation at 400-500 mm with a R466 infrared sensitive multiplier,

using filters of Corning 5-60 (color specification) for excitation, Corning 3-66 for

reference, and Corning 2-64 for emission. Carbon uptake by phytoplankton was

measured using incubation with NaH14C03, with 6 columns per pond 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 four hours (no mixing), or in a laboratory

environmental chamber set at pond temperature, attached on a frame which rotated them

around circular fluorescent lamps providing constant mixing and a light intensity of 150

/lE/m2/s (similar to that ofa pond at depth 1-1.5 m on a clear day). Immediately after

incubation, samples were processed for scintillation counting using a Packard Model

2425 Tri-carb liquid scintillation spectrometer, using the acidification and bubbling

method described by Schindler et al., J. Fish. Res. Bol. Canada (29) pp. 1627-1631, 1972.

Counts per minute values from the incubated samples were used directly rather than

converting them to productivity values.

Zooplankton were sampled by vertical net hauls at night, when they were more uniformly

distributed by depth. From the pond walkway, one sample was taken with a 49 cm

diameter, 157 /lm mesh net and two samples with a 30 cm diameter, 20 /lm mesh net.

Samples were preserved by 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 prior to addition), and the other samples

were counted infrequently to validate the large net samples. Each large net sample

represented 400 liters of pond water based on the haul depth and was concentrated to a 40

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 experiment by

retrieving dead individuals. At the end, when ponds were drained, the fish were

retrieved, killed in formalin, transferred to water for length and weight measurement, and

then preserved in 70% ethanol for later dissection. Other organisms monitored in the

ponds were filamentous algae and macrophytes (Najas, Chara), with weekly visual

observations, and benthic invertebrates, with 4 dredge samples per pond taken at the end

ofthe experiment.

Short Term and Laboratory Experiments

Phytoplankton collected as column samples from control ponds or the reservoir were

exposed to atrazine (using the same commercial product as applied to the treatment

ponds) in the laboratory so that short-term changes in 14C uptake and fluorescence could

be measured. (Other commercial herbicides were also tested, but the results will not be

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 per minute 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 of atrazine, and

3
another fluorescence reading taken 2 minutes later. Percentage increasesI were recorded

in terms of before vs. after measurements and compared with controls. To facilitate such

comparisons, samples were all handled identically, especially by keeping them under the

same low (0.1 /lE/m2/s) light conditions before and during the atrazine exposure. (In

some experiments, the fluorescence responses were measured on the samples prepared

for 14C uptake measurement just before their 24 hour incubation.)

Several related types ofshort-term phytoplankton experiments were conducted

additionally. In one, phytoplankton samples from atrazine-treated ponds were taken and

again exposed to varying levels of fresh atrazine, to compare responses (14C uptake and

fluorescence) of previously exposed organisms to those with no prior exposure. Several

such experiments were run, after Day 50. In another experiment, control pond

phytoplankton were exposed to filtered treatment pond water: at Day 97, unfiltered water

from each control pond was mixed with an equal volume of filtered water from a

different control pond and one treatment pond at each ofthe two treatment levels and the

percentage increase in fluorescence after addition was recorded. (All filtered waters had

the same fluorescence values.) Another set of phytoplankton resistance experiments

involved cultures of phytoplankton from a control pond and a 500 /lg/L pond taken on

Day 60, which grew for 45 days in the absence of atrazine. Organisms from one culture

from each pond 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 over pre-addition readings. Additionally, the cultures were exposed

to filtered water from each control and 500 /lg/L pond by adding 1 mL of culture to 50

mL of filtered pond water. 14C uptake and fluorescence responses were measured as

described above and recorded as percentage increases or decreases compared to values

recorded for exposure to filtered control pond water.

Short-term experiments were conducted with Najas macrophytes. Two 30 cm diameter

plastic soil trays, resting on the pond bottom at a depth of 2.1 m, were each planted with

several individuals (each about 20 cm long and all totaling 12 g in mass), in each pond.

The plants were checked for mortality at days 14,31, and 63. (It is not stated when the

plants were placed in the ponds, but it will be assumed that it was on Day 0.)

In zooplankton experiments, life tables were constructed for 2 species of cladocerans

(Simocephalus serralaturs and Daphnia pulex), using young produced by several adult

females kept in culture in the laboratory. (The cultures had been started earlier in the

summer by isolating Simocephalu from the reservoir and Daphnia from a nearby lake.)

The young used to start each life table were 12 ± 12 hours old and had 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 of atrazine in blocking electron transport in photosynthesis, since the absorbed light energy must be

released as fluorescence energy if it is successfully blocked. For this reason they suggest that fluorescence

values in the presence of atrazine not be directly interpreted as algal biomass estimates.

4
opened into two sections, with 153 /lm nylon netting over the ends. Concurrent

microscopic counts of phytoplankton showed that all common species were less than 50

/lm in diameter and could readily 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 and removed. For these procedures, cylinders were

removed inside buckets of water and each cylinder was opened in a dish of water for

observation via microscope, which took place at a nearby field lab. The original animals

returned to suspension in the pond within 1 hour after never leaving their own pond water

or cylinder. Upon return, the cylinder netting was brushed clean and the cylinder flushed

by gentle agitation in the pond. Observations continued until all animals were dead.

Additional laboratory life tables were constructed using only Simocephalus, starting on

Day 28 ofthe field experiment. They were started using young as described above,

except they were placed in 300 mL glass stoppered bottles, using water from a control

pond filtered through an 86 /lm mesh net which filtered out cladocerans but not

phytoplankton. Atrazine concentrations of 0, 20, and 500 /lg/L were created in the

bottles (presumably using the same commercial atrazine described previously, although

this is not explicitly stated). The bottles were kept at 25°C, similar to pond temperature,

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 placed them in freshly prepared water (filtered control pond

water with atrazine added, as described above), so they were not as subject to changing

atrazine concentrations or phytoplankton assemblages as the in situ animals.

Finally, short-term experiments were conducted with benthic invertebrates: Anallagma

civile (damsel fly nymph), Musculium transversum (clam), and Lirceus garmani (isopod).

10 Anallagma were suspended in each pond in a perforated 2 L plexiglass box for 96

hours beginning on day 2 to observe mortality. Musculium were studied in the same way,

and additionally were observed over a longer time period beginning on day 15 and lasting

for the next 35 days, with the latter experiment involving each clam placed in a plexiglass

cylinder as used for the in situ zooplankton experiment, with 10 suspended in each pond,

and mortality and size observed "periodically over 26 days" (the statement seems

inconsistent with the 35 day duration, and is vague as to measurement frequency).

Musculium and Lirceus were studied in the laboratory to determine a lethal concentration

of atrazine 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, but p-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 of implementation 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 of Kansas. From west to east: reservoir

pond, 14 experimental ponds (2 divided), 3 smaller experimental 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 of the 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:

Pond Experiment

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

that the slight changes they describe appear due to changes in phytoplankton

photosynthesis and macrophyte growth, not to direct effects of atrazine.

Strong but ultimately transient effects of atrazine on phytoplankton biomass and

productivity were observed in the 500 )..lg/L treatment ponds, while the 20 )..lg/L treatment

ponds actually showed some increases over control pond biomass, and only a brief effect

6
on productivity (14C uptake). 14C uptake declined significantly (p-value < 0.05) at two

days in ponds at both treatment levels. These direct effects of atrazine on phytoplankton

photosynthesis were borne out by the short-term experiments, described below. See

Table 1 and Table 2 for the data.

Phytoplankton community compositions in the ponds were dramatically altered by

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 treatment ponds, but did not noticeably decline in the 20 Ilg/L treatment ponds.

Most other species (Mallomonas spp., Cryptomonas spp.) initially declined in the 500

Ilg/L treatment ponds, but were able to recover by day 24; these species actually

experienced increases in the 20 Ilg/L treatment ponds at the same time as the declines in

the 500 Ilg/L treatment ponds. One species, Dinobryon divergens var. Schauiuslandii,

was absent from all ponds in the beginning but present in all ponds by day 31. Two

Synedra species, S. acus and S. radians, were present at the beginning and did not decline

with the 500 Ilg/L atrazine addition. In the control ponds, species distributions remained.

relatively stable over the time when many species declined in the 500 Ilg/L treatment

ponds. See Table 3, Table 4 and Table 5 for the data. 2

Zooplankton responses to atrazine treatment were not extreme (no statistical analyses

were performed on these data), but a decline in the cyclopoid copepod population was

notable in the 500 Ilg/L treatment ponds. Diaphanosoma did not show a similar decline.

Chydorus measurements were very low in all ponds. Other species, present but in

numbers too small to count, did not experience noticeable population changes. See Table

6 for cyclopoid copepod and Diaphanosoma data.

Fish in the experimental ponds developed growth and reproductive differences at

different atrazine treatments, but no mortality differences (over 80% of original fish

survived to the end ofthe experiment). Growth was measured by mean length of each

species at the end compared to the beginning ofthe experiment. At the beginning, the

mean lengths in the different ponds were within 5% of each for Lepomis and 2% of each

other for Dorsoma (Ictalurus was not sized at the beginning, but the fish were obtained

from a hatchery and had been 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 significant difference" (no statistical test or p-value cited) in

growth in the 20 Ilg/L ponds compared with control ponds, but reduced growth was noted

in the 500 Ilg/L treatment ponds: decreased growth compared to growth in control ponds

was 26% for Lepomis, 31 % for Dorosoma, and 16% for Ictalurus. The only species to

reproduce was Lepomis. The young (at the fry stage of20-40 mm at the end ofthe study)

were retrieved during the pond drainage process by repeated seining while the pond was

2 The authors also note declines in Kirchneriella lunaris, Glenodinium sp., Scenedesmus spp., Coelastrum

spp., Dictyosphaerium sp. and Staurastrum sp. in the 500 flg/L ponds, while Rhodomonas pusilla 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 number of young was

dramatic, with an average of 1376 young in the control ponds compared to averages of 59

and 53 in the 20 and 500 /lg/L treatment ponds respectively. (No statistical analysis

provided.) The adult fish were dissected to observe gonad morphology, but no

differences were found associated with atrazine level. 3

Filamentous algae and macrophytes remained rare in all ponds throughout the study.

Dredge samples of benthic 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 and Laboratory Experiments

In phytoplankton communities not previously exposed to atrazine, significant (p-value <

0.05) increases in fluorescence occurred within 2 minutes of exposure to only 1 /lg/L

atrazine (increase of 3% 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 14C

uptake after 24 hours were significant (p-value < 0.05) at the 5 /lg/L level (decreases of

4% to 7% under control 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 of atrazine tolerance, with control pond phytoplankton sensitive to even

1 /lg/L, but no significant increase in the 20 /lg/L pond phytoplankton at that level. At

higher concentrations of added atrazine (5, 20, and 500 /lg/L) both 20 /lg/L and control

pond phytoplankton had significant responses (p-value < 0.05), but the response

intensities were significantly different (SNK test; p-value < 0.05) between the control

pond and 20 /lg/L pond phytoplankton. The phytoplankton from the 500 /lg/L pond

showed no significant responses to atrazine additions below 500 /lg/L, and the response

to 500 /lg/L atrazine was only 10% ofthe response ofthe control pond phytoplankton.

14C uptake responses, on the other hand, were similar in the control pond and the 20 /lg/L

pond phytoplankton, but the 500 /lg/L pond phytoplankton showed weaker responses (no

statistical comparisons provided). At most 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, that the results just described may be due more to the

continually or already provoked responses from the atrazine in the treated ponds than to

actual resistance. This suspicion was explored by performing a very similar experiment,

except that filtered water from the treated ponds was used instead of atrazine added in the

lab. As Table 9 shows (compare to Table 7), the fluorescence increases from the treated

pond water were comparable to those from adding fresh atrazine. To further explore the

issue, on day 105 phytoplankton were removed from the 500 /lg/L treatment pond water

by gentle filtration followed by resuspension in control pond water, and less than 24

hours later were exposed to atrazine and tested for fluorescence and 14C uptake responses.

3 These effects were clearly unexplained at the time afthis study, but were taken up again in the Kettle et

al. 1987 study (see that review).

8
(This experiment was not described in the methods section ofthe study.) The resulting

reductions in 14C uptake and increases in fluorescence were only about 15%, much

smaller than the fluorescence increases and 14C uptake decreases previously found

(results not tabulated, no statistical analysis). Therefore, the culture experiments were

performed. The phytoplankton taken from a 500 )lg/L treatment pond on day 60 and

cultured for 45 days, as opposed to the phytoplankton similarly cultured from a control

pond, had significantly less of a response to 500 )lg/L pond water or fresh atrazine

addition (p-value < 0.05). See Table 10. The authors interpret the culture results as

showing that phytoplankton 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 treatment ponds were dead

after 14 days of exposure. In the 20 )lg/L treatment ponds, visual estimates of biomass

found reductions of about 50% relative to control ponds, but the plants survived at this

relative abundance throughout the study.

Life tables produced for Daphnia pulex and Simocephalus serrulatus showed that both

species experienced significant (p-value < 0.05) reductions in the number of young

produced and in daily production of young in the 500 )lg/L treatment ponds. In the 20

)lg/L treatment ponds, Daphnia experienced similar reductions while Simocephalus did

not have significant effects. Additionally, Daphnia had a significantly (p-value < 0.05)

shorter lifespan in the 500 )lg/L treatment ponds. See Table 11. The life tables

developed for Simocephalus in the laboratory, where there were no food limitations (as

opposed to the in situ environment), showed no significant responses 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 showed no

response to atrazine (no data provided). In the lab, no mortality responses were found for

Musculium or Lirceus garmani up to concentrations of atrazine up to 80 mg/L.

9
Table 1. Fluorescence values in Ilg chI a per liter for pond phytoplankton communities. Each value

is a mean from recordings of 3 column samples. Individual values shown for the two ponds at each

treatment level. (from study table 3)

Days since

Date Atrazine Control 20 f-lg/L 500 f-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 after Day 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 of 4 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 20 flg/L 500 f-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 after Day 0

10
Table 3. Distribution of Tetraedron minimum (A. Braun) Hansgirg. and Peridinium inconspicuum Lemm. in ponds during the field experiment. Values

are in cells/mL with blanks (-) below the detection limits of 10 and 2 cells/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 20 flg/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 - - - - - - -

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Table 4. Distribution ofMallomonas pseudocoronata Prescott and Mallomonas spp. (smaller species, unidentifiable due to preservation) in ponds during

the field experiment. Values are in cells/mL with blanks (-) below the detection limit of 2 cells/mL. Individual values shown for the two ponds at each

treatment level. (from study table 7)

Mallomonas Mallomonas spp.

Days since

Date Atrazine Control 20 Jig/L 500 Jig/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

12
Table 5. Distribution of Cryptomonas marssonii Skuja and Synedra spp. in ponds during the field experiment. Values are in cells/mL with blanks (-)

below the detection limit of 2 cells/mL. Individual values shown for the two ponds at each treatment level. (from study table 8)

Cryptomonas Synedra

Days since

Date Atrazine Control 20 j.lg/L 500 j.lg/L Control 20 j.lg/L 500 j.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 -

13
Table 6. Distribution of zooplankton Diaphanosoma brachyurum and cycloid copepods (species not distinguished) in ponds during the field experiment.

Values are in organisms/L, with plus signs (+) denoting values between 0.07 and 1 organisms/L and blanks (-) for values below the detection limit of 0.07

organisms/L. Individual values 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 + +

14
Table 7. 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. (from study table 9)

* signIficantly different from non-addItIOn/control results at p<0.05 (SNK test wIth 10 replicates for each

addition to a community pond sample)

Addition Control 20 J)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 of pond phytoplankton communities (sampled on various field

experiment days) over 24 h to additions of atrazine. 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 at each treatment level. (from study table 10)

Addition Day Control 20 J)g/L 500 pg/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 at p<0.05 (SNK test with 5- 10 replicates for each

addition to a pond community sample)

15
Table 9. Fluorescence increases of pond phytoplankton communities exposed to an equal volume of

filtered water from another pond on field experiment day 97. Values are % increases in fluorescence

after 2 minutes of exposure to the other pond'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 at p<0.05 (SNK test with 10 replicates for each addition to a pond community

sample)

Table 10. Fluorescence increase and 14C uptake decrease of phytoplankton cultures isolated after 60

days from control and 500 !Jg/L treatment ponds. Cultures grew for 45 days in the absence of

atrazine and were then exposed to filtered water from the 500 !Jg/L treatment ponds as well as

additions of 20 and 500 !Jg/L fresh 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 !JglL treatment ponds. (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 each treatment to a culture) from those of control pond cultures.

16
Table 11. Reproductive and longevity responses of 2 species of c1adocerans (Simocephalus serrulatus

and Daphnia pulex) exposed to atrazine in situ and in the laboratory. Each value is the mean for 10

individuals for each species at each treatment level observed every 2 days 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 Day of 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

C4C uptake >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

17
Effect Magnitude of Nominal Day of Time Did Comments

Effect Atrazine Study Effect 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 data pomts specIfied m the "Cosm Data Pomts and Exposure Profiles" spreadsheet.

I Similar endpoints appear in the "Cosm Data Points and Exposure Profiles" spreadsheet.

2 See Data Summary Table in review of deNoyelles et aI., 1989.

Time weighted average concentrations are estimated from the only data point provided 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).

Linear decline is assumed over time to estimate atrazine concentrations on relevant 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).

18
TWA 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 of methods 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 a clear statement that the ponds

receive no watershed runoff and so could not be affected by local herbicide runoff.

The atrazine concentrations in the experimental ponds over time remains highly

uncertain. Only a percentage 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 that the concentrations on Day 0 were in the same as nominal

(experience shows that this is unlikely to have actually occurred). Given the lack of

atrazine concentration measurements, the time weighted average concentrations are

uncertain and should not be used as exact, known values.

In the algal biomass and productivity measurements, measurements were made with

reasonable frequency and are tabulated in detail, in terms ofthe more direct quantities of

counts per minute 14C uptake and fluorescence readings. As such, they are not as subject

to uncertainty in interpretation as would be calculated productivity or biomass 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 remark below 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.

19
It is not stated that the same commercial atrazine used throughout the study was used to

construct the 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 parameter changes, 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 brief and unclear to determine how the SNK test was implemented

and whether it was done so correctly (after ANOYA; groups defined by treatment level;

all 3 groups considered in the same analysis). As a multiple comparison test, the SNK

test should have been done to test for differences among the 3 treatment level groups, but

the results are described as showing difference from the control, as if a two group

comparison had been done. An SNK test is inappropriate for a simple two group

comparison. Often when an SNK test was cited, a number ofreplicates was given which

had never been presented in the text or table 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 given that the statistics were not properly done. In any case, they were

not properly 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. In particular, 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 of experiments performed, organized presentation is essential for conveying what

happened in a comprehensible fashion and was too often found lacking.

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

Rationale for Use: The study provides further details regarding the experimental design

of ponds used in mescosm studies presented in deNoyelles et al. 1989 (described as "the

136-day study" in that review) and in Kettle et al. 1987. Although the study results are

weakened by the small number ofreplicates and treatments and by a lack of information

about the atrazine concentration over time, 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 of 500 llg/L

20
Limitations of Study: This was an early, exploratory mesocosm study which identified

many important aquatic community responses to atrazine. The main limitations ofthe

study are lack of atrazine concentration data, small sample (two replicates at each oftwo

treatment levels plus two controls), and unclear presentation ofsome methods and results

(including unclear statistics). Because ofthese limitations, the quantitative results should

not be generalized. However, a large 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 or others taking place at the University

of Kansas experimental ponds system.

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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