Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Carney, C.E. 1983. The effects of atrazine and grass carp on freshwater communities.

Thesis. University of Kansas, Lawrence, Kansas (MRID 47543604).

Purpose of Review:

Determine 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 of Concern (LOC).

Date of Review:

November 24, 2009

Purpose of Study:

This study examines the potential effects of atrazine on macrophyte communities in

experimental ponds and in selected farm ponds with a history of atrazine use. The effects

of grass carp (Ctenopharyngodon idella Val.) on macrophyte communities are also

considered alone and in different combinations with atrazine.

Summary of Study Findings:

Methods: The thesis is broken into four parts: Experimental Pond Studies; In Situ

Chamber Experiment; Farm Pond Study; and Emergent Macrophyte Survey. Portions of

the study appear elsewhere: everything except the farm pond study is also described in

deNoyeIles et at. 1989 (described as "the 80S-day study" in the review), and parts ofthe

"experimental pond study" methods and results are also described by Dewey 1986 and

deNoyelles et al. 1994.

Experimental Pond Studies

Ten experimental ponds with surface areas of 0.045 ha each were constructed in 1977

and 1979 at the Nelson Environmental Study Area, ten miles north of Lawrence, Kansas.

By the summer of 1981, the ponds had developed "similar though sparse" (no further

description provided) macrophyte communities. In "the summer of 1981" (no exact dates

provided), the ponds were drained and then filled from a common reservoir (no

1

111••••111

2087813
description ofthe reservoir water or of how much time elapsed between draining and refilling provided). "At the beginning ofthe study" (again, no date provided) the ponds

were stocked with fish: 10 gizzard shad (Dorosoma cepadianum Lesueur, 30-40 em

long), 50 bluegill (Lepomis macrochirus Rafinesque, 3-7 cm long), and 20 channel

catfish (lctalurus punctatus Rafinesque, 10-15 cm long). Atrazine was added (no date

provided) at concentrations of 20 ~g/L or 100 ~g/L "to selected ponds" (no further details

or descriptions oftreatment vs. control selection) using either reagent or commercial

grade atrazine, and additionally a 200 ~g/L combined treatment pond was created.

Commercial atrazine was CO-OP liquid? 41 % active ingredient, EPA registration number

1990-318. In 1982 (no specific date provided) all ponds were converted to reagent grade

atrazine only, after no differences were noted between commercial and reagent grade

treatments at the same treatment level (no explanation of how exactly "no differences"

were determined). The two commercial grade 20 ~g/L ponds became 500 ~g/L ponds,

and the I00 ~g/L commercial pond and the 200 ~g/L combined treatment pond both

became 200 ~g/L ponds with reagent treatment. In May 1982, 4 grass carp of length 18-

23 cm were added to each pond, and roughly 10% of each pond in the southwest comer,

where cattail was less dense, was fenced offfrom the grass carp using I-inch poultry

mesh. The goal ofthe fence was to allow comparison of grass carp plus atrazine effects

on macrophytes with effects of atrazine alone.

A modified grapnel method, using a common garden rake on September 25, 1981 and a

smaller, 8-pronged rake with recurved 4-cm prongs on an extendable pole in 1982, was

used to sample the submerged macrophyte community with minimum disturbance to the

system. In 1981, at ten points around the shore, the rake was extended from shore, pulled

along the substrate for one meter, and brought to shore. Samples were scored on a scale

of 0 to 4 based on visual judgment, 0 being an empty rake and 4 being at maximum

capacity, and added to find a total pond score between 0 and 40. The thesis states that

"[a]nce the total macrophyte sample was scored, it was di vided into the species

comprising that total" but it is not stated how relative frequencies were measured. In

1982, the method changed so that instead of 10 samples, there were 8: four "random"

samples within the fenced sector and 4 samples outside the fenced sector. (No further

description of the spatial distribution ofsampling points was provided). In 1982,

macrophytes were sampled on June 30, August 4, September 2, and September 30.

Water chemistry parameters were monitored in 1982, including 'IDS, pH, turbidity, total

alkalinity, and planktonic chlorophyll-a, using integrated water columns. Atrazine levels

were analyzed from the water column samples by the EPA lab in Corvallis, Oregon.

Selected results were statistically analyzed using ANOVA (no details on exact methods

or grouping definitions).

In Situ Chamber Experiment

An in situ experiment was performed in four ofthe experimental ponds from July 26 to

August 31, 1982, with one control, one 20 ~g/L, one 100 ~g/L, and one 500 ~g/L chosen

"randomly". In each pond, two baskets containing ten segments of either Najas

guadalupensis (Spreng.) Magnus or Chara globularis Thuill. (representing vascular and

2
non-vascular macrophytes) were suspended 25-30 em from the surface. Each plant

segment was inside a perforated plexiglass chamber and began the experiment with five

nodes. Only terminal segments were used. Plant material was gathered from the Nelson

Area reservoir. "Periodically" (no details on frequency provided) the chambers were

cleaned (no description of cleaning method provided) and relative growth ofthe plant

segments assessed. Growth was measured as average number of nodes per segment,

rather than as segment length, to assure that cell division rather than cell elongation was

being measured.

Farm Pond Study

Twenty farm ponds in northeastern Kansas, all within 50 miles of Lawrence, were

located. These ponds did not contain grass carp or other biological control organisms.

Ten ofthe ponds received no herbicide runoff and slight to no use by cattle, while ten of

the ponds were adjacent to com or milo fields with a history oftriazine use (no

description of farm use histories were provided). With a method similar to that described

for the experimental ponds studies, the small extendable rake was used to sample

submerged macrophytes, and samples were taken at 10 points around the ponds to

determine a macrophyte score. Ponds were sampled for submerged macrophytes as well

as for water chemistry during July and August, 1982. Results were statistically analyzed

using ANOYA.

Emergent Macrophyte Survey

It was observed that the cattails seemed brown in the atrazine-treated ponds as opposed to

the control ponds; therefore, a survey ofthe effects of atrazine on the dominant emergent

macrophytes was conducted to quantify the observation. (The dominant emergent

macrophytes were Typha latifolia L. and Typha angustifolia L.) On September 30, 1982,

a lOin. by lOin, quadrat was placed at 10 "random" points within cattail stands in each

pond (no further details on the spatial distribution ofthe quadrats was provided), and the

total number of green and brown leaves within each quadrat were counted.

Results:

A table of atrazine concentrations over the 1982 season is provided in the thesis, but not

referenced anywhere in the text. See the Excel spreadsheet "Carney 1983 data.xls" (sheet

"atrazine cones") associated with this review for details.

Experimental Pond Studies

On September 25, 1981, total macrophyte cover decreased slightly at increasing atrazine

levels. Although total macrophyte cover was roughly equal in the 20 Ilg/L to the 100

Ilg/L treatments), the shift in community composition was dramatic. In the control ponds

and 20 Ilg/L treatment ponds, the macrophyte community mostly consisted of vascular

species, and at the >20 Ilg/L treatment levels the non-vascular plants (particularly C.

globularis) were more prevalent.
See Table 1 for a summary of macrophyte score observations estimated from thesis

Figures I and 2. The original figures had error bars to show the range of values among

replicates at each treatment level, and Table I notes whether the range among the treated

ponds had any overlap with the range among the control ponds. Overlap with the control

range is an indirect, ad hoc indicator ofstatistical significance ifthere was overlap,

differences are almost certainly not statistically significant, especially given the small

number ofreplicates.

On June 30, 1982, there was a clear decline in macrophyte levels as atrazine levels

increased. The pond sectors with grass carp had less than halfthe macrophyte scores of

the pond sectors without grass carp, with a sharpened reduction of macrophyte score over

increasing atrazine treatment. Water temperatures were cool, only about 15°C. Within

the fenced-off pond sectors, macrophyte community composition varied with atrazine

treatment levels, though between-replicate variations were large at control levels.

By August 4, water temperatures had increased to 25-20°C, there was a greater difference

between macrophyte levels in pond areas with grass carp and in the fenced-off sectors,

and submerged plants were totally absent in the 200 flg/L and 500 flg/L treatment ponds.

The 100 flg/L treatment ponds had experienced considerable growth within the fenced

comers.

By September 2, only the control ponds had any submerged macrophytes outside ofthe

fenced sectors. Additionally, some reddened and etiolated C. globularis appeared in the

200 flg/L treatment ponds, although it was absent from the controls.

On the final sampling day, September 30, only minor changes in species composition

were noted. P. pusillus reappeared and C. globularis increased in the 20 Ilg/L treatment

ponds.

See Table 2 and Table 3 for summaries ofthe 1982 results, estimated from thesis Figures

3 through 10. In all atrazine treatment levels with grass carp, there is no overlap ofthe

range in macrophyte score with the range among controls; however, overlap in the range

ofscores was observed in between controls and all 20 Ilg/L treatment levels from areas

without grass carp.

A statistical analysis (implicitly on the 1982 data, although this is not clearly stated)

using ANOVA found that "the submergent macrophyte community changed significantly

across the atrazine gradient (p-value < 0.00 I) being much denser in the fenced areas

within control and 20 flg/L ponds" but it is unclear on exactly which subset of data the

analysis was performed (only in fenced offsectors?). Two-way ANOVA showed

positive synergism between grass carp and atrazine in reducing submerged macrophytes

(p-value < 0.001), although there was no statistically significant reduction from grass

carp alone (which may be due to the sample size of only two control ponds). Among the

water chemistry parameters, turbidity changed significantly between atrazine treatments

(p-value < 0.05) but there was no clear trend (it was lower in control, 20 flg/L treatment,

and 500 flg/L treatment ponds). Total dissolved solids (TOS) "displayed significant

4
differences" (at p-value < 0.05) "and were lower in the control ponds than elsewhere" it

is unclear whether a significant trend occurred, and the grouping definition upon which

"significant differences" were found is not explicitly stated. The author describes the

TDS differences as resulting from macrophyte activity to uptake dissolved nutrients,

resulting in less TDS in ponds with more macrophytes.

In Situ Chamber Experiment

At the end ofthe in situ chamber experiment, N. guadalupensis had experienced

considerable growth in the control and 20 Ilg/L treatments ponds, little growth in the 100

Ilg/L treatment ponds, and no growth in the 500 Ilg/L treatment ponds. Decay occurred

at the high concentration levels (no further details given). For C. globularis, on the other

hand, very little growth occurred at lower atrazine levels (the author also states that there

was "high mortality") whereas there was more growth at the higher atrazine levels. The

author attributes the C. globularis results to a sensitivity to epiphytic algae, which was

prevalent in the low atrazine ponds but was reduced in the high atrazine ponds (entirely

absent in the 500 Ilg/L treatment ponds). See Table 4 for a summary ofthe results,

estimated from thesis Figures 11 and 12.

Farm Pond Study

In the farm pond study, there were clear differences in the submerged macrophyte

community between the "control" ponds (those not receiving agricultural runoff) and the

ponds receiving triazines-containing runoff Three water chemistry parameters were

significantly higher in the runoff-receiving ponds than in the controls: TDS (p-value <

0.10), total alkalinity (p-value < 0.10), and turbidity (p-value < O. 10). The turbidity

results are speculated by the author to result from silt loading, which may have also been

causative in reducing macrophyte cover, while the TDS and alkalinity results are believed

to be caused by macrophyte uptake of dissolved nutrients. Macrophytes were common in

all control ponds, but ofthe ten agricultural ponds, six had no macrophytes to speak of

and four had only sparse communities - statistical analysis found that macrophytes were

significantly more abundant in control ponds at p-value < 0.001. See Table 6 for a list of

macrophyte species observed and their overall prevalence by pond type and Table 5 for a

summary of the macrophyte cover data estimated from thesis Figure 13.

Emergent Macrophyte Survey

In the control ponds, 90-95% ofthe Typha spp. leaves were green, while in the 500 Ilg/L

treatment ponds, only about 30% ofthe leaves were green. See Table 7 for a summary

estimated from thesis Figure 14.

Table 1. Macrophyte sampling data from 9/25/1981 (thesis Figures 1-2). Maximum Macrophyte

Score =40.

Nominal

Atrazine Mean

Macrophyte

Score ("MS")

Any

overlap

with

control

MeanMS

(vascular)

5

Any

overlap

with

control

MeanMS

(Charophytes)
range? range?

0 27 ]4

]9 yes 14 yes 5 yes

21 4 yes ]9

4 N/A 0 N/A 4

means "not treatment level

Table 2. Submergent macrophyte data from the 1982 season (thesis Figures 3, 5, 7, and 9).

Maximum Macrophyte Score == 16.

Nominal Any l

Sampling Atrazine l\1ean overlap

Date Macrophyte with I ....<7 Score control

range'? .

0 9 -

6/30/] 982 20 4 no j

(grass 100 I no

! 200 0 no

I 500 0 no

0 13

20 II I IJU/ j Y15L I yes

100 3 No

200 2 no

500 ] no

0 6

982 20 I no

100 0 no

200 0 no

i 500 0 no

i 0 13

982 20 ]2 yes

grass 100 6 no

carp) 200 0 no

500 0 no

0 11 - r------

9/2/1982 20 I 0 no

(grass 100 0 no

carp) 200 0 no

500 0 no

0 15 -

20 14 yes

100 10 no

200 1 no !

500 i 0 no ,

6
9/3011982

(no grass

carp)

Table 3. Species-specific submergent macrophyte data from the 1982 season (fenced-off pond sectors

only; thesis Figures 4, 6, 8, and 10). Maximum total (combined) macrophyte score 16.

Nominal Any Any Any

Sampling Atrazine Mean overlap Mean MS(N. averla MeanMS overlap

Date rf MS(p. with guadalupensis) p with (C. with

w'v, pusilJus) control control globularis) control

range? range? range?

I 0 8 0 -

6/30/1982 20 I 2 yes 9 yes 0 yes

(no grass l~ 100 I 0 yes 0 no 3 no

carp) I

200 I 0 yes 0 no 1 no

500 0 yes 0 no 0 yes .~

0 0 -

0 yes

8/4/1982 (no yes 6 no

grass carp)

0 yes 0 yes

I i 500 no yes 0 yes

I

no no

9/2/1982 (no 0 no no 10 no

grass carp) i

i 200 0 i no no yes

I 500 0 no yes

0 -

982 I yes 7 yes 6

(no grass I ! yes

200 0 yes 0 no I yes

500 i yes yes

7
Table 4. Results of 37-day in situ chamber experiment (Figures 11-12 in thesis). Node numbers

represent growth.

Mean Number

olNodes (C.

globularis)

Any

overlap

with

control

range?

Table 5. Results of farm pond study submergent macrophyte survey. Maximum Macrophyte Score

=40.

Type

Table 6. Species-specific composition ofsubmergent macrophyte communities aggregated by pond

type in the farm pond study.

Percent Composition by !

i

Pond Type

c. !

r""fvn'

i Runoff- ,

Najas , .1 7

Potamogeton

I ,

Pfoliosus 0 12.5

P. , 0 12.5

Ceratophyllum

Chara , .u 6.7 0

! C braunii ),6

Table 7. Results of emergent macrophyte survey of September 30, 1982.

8
Nominal Any

Atrazine Percent overlap

Concentration Green with

(lig/L)

0

ZO 78 no

100 68 no

200 50 no

500

Data Summary Table:

Effect Magnitude of Nominal Day of Time Weighted Did Comments

Effect relative Atrazine Study Average Recovery

to controls Concentrati Effect Atrazine Occur- if

on (Jig/I) I Occurred Concentration so, when

from Day 0 to (see

I

When Effect comment)?

Occurred i

Emergent 15-20% +20 Ilg/L Day 1192 14.8 Ilg/L3 Data estimated macrophyte from thesis cover (% 25-30% ! 100 IlgiL Day 119 79.3 Ilg/L

green leaves I [zoo Ilg/L

no figure 14

45-50% Day 119 i 173.7 Ilg/L (review Table on Typha _•... I

spp.)! 65-70% I 500 "giL 1Day 119 453.8 Ilg/L 7). ~.__.

I See Submerged See I See deNa elle See See See

macrophyte deNoyelles I deNoyelies 198~ deNoyelies deNoyelles deNoyelies

cover4 1989 review 1989 "view ' I "view 1989 review 1989 review revIew

Submerged 90-95% un~o~~ Farm ponds,

macrophyte (significantly (farms with not k not

cover (farm less at . . un nown no experimental ~Ii~~~e~se apphcable I ponds) p<O.OOI) I data. - .._~-~.~. .•..-. ~'- •.•.... .. "-'-~-

I It is unclear whether the emergent macrophyte study performed by Carney is included somehow within

the emergent macrophyte data described in deNoyelles et aI., 1989. It does not appear to be the case, but if

it is, then this information is included within the deNoyelies 1989 review data summary table.

2 The date of the survey is known; however, the date of atrazine addition (Day 0) is unknown and estimated

from the table of atrazine measurements (table I ofthesis) assuming that the first day of atrazine

measurement was the day of application, since measured concentrations were roughly 100% of nominal on

that day.

3 Repeated applications of atrazine within the 1982 season are not mentioned in the thesis, and are therefore

assumed to not take place after the initial application. See "TWAs" sheet in "Carney 1983 data.xls" for

details.

4 Appears to be equivalent to or significantly overlapping with data presented in deNoyelles et aI., 1989.

Selected endpoints are specified in the deNoyelles ct a!. 1989 study revicw. However, TWAs and days of

study cffects occurred were not calculable fore deNoyclles et al. 1989 but may be calculable from the

information in this thesis. See "Carney 1983 data.xls".
Ma,gmtude of

N. guactalupensis growth

(number of

nodes)

C globularis

growth

(number of

nodes) 600% (+) 500 jlg/L

no

no

effects is

relative to nonagricultural

ponds. Data

estimated from

thesis figure

13 (review

____+-'-Tab~~~~_

Data estimated

from thesis

figure 11

(review Table

4).

Data estimated

from thesis

figure 12

(review Table

4). Notc that

effects are all

Time weighted averages were calculated based on thesis Table 1, which was not

referenced within the body ofthe thesis and was thus difficult to interpret. Chemographs

were derived assuming that 6/3/82, the first day when atrazine concentrations in the

ponds were measured, was the day of atrazine treatment, and that there were no

subsequent atrazine applications during the 1982 season (through September). A linear

decline in atrazine concentration over time was assumed; therefore, Time Weighted

Averages were calculated as simple averages of Day 0 and Effect Day concentrations.

For further details and calculations, see "Carney 1983 data.xls".

Nominal Day 0 Effect Day Time

Atrazine Weighted Experiment Concentration Effect Day Concentration Concentratio (~g/L) (~g/L) Average

n (~tg/L) (~g/L)

1982 ponds 20 18.78 119 10.73 14.76

1982 ponds 100 93.65 119 64.86 79.26

1982 ponds 200 198.9 119 148.50 173.70

1982 ponds 500 501.75 119 405.94 453.84

in situ chamber 20 15.19 36 12.76 13.98

in situ chamber 100 80.83 36 72.12 76.47

in situ chamber 500 459.08 36 430.09 444.58

5 Day 0, the day the in situ chamber experiment began, is Day 53 of the experimental pond study. This fact

is used to derive TWAs.

10
Reviewer Comments: Many details of methods implemented within this study are

absent or vague.

An atrazine concentration table, with concentrations at all treatment levels in all ponds on

two days in the summer in 1982, is presented as Table 1 ofthe thesis without any

explanation or interpretation in the text. The exact date of atrazine application as well as

any subsequent treatments during 1982 is unclear. As for the 1981 study, no information

is provided on the atrazine application dates. In both seasons, there is no discussion of

the methods of atrazine application; therefore it is unclear whether atrazine was evenly

distributed or fully dissolved, or whether other factors affecting atrazine concentrations

within the ponds may have interfered with the results.

The exact conditions ofthe ponds and any preexisting differences among them at the start

ofthe study in 1981 are not discussed. The dates and details of how ponds were drained

and refilled are not addressed. Similarly, it is stated that for 1982, reagent atrazine was

used because "no differences" were noted between commercial and reagent atrazine

ponds during the 1981 season. However, it is not stated whether this was determined

through measurements or statistics rather than subjective judgment. Overall, the

conditions in the experimental ponds at the start ofthe 1982 season appear rather

uncontrolled; it is possible that the "control" ponds are not reliable controls ifthey had

different initial conditions than some or all ofthe treated ponds.

The macrophyte scoring method appears subjective in several ways. First, it is not

described how the 10 or 8 (four inside the fenced sector, four outside) points around the

shore of each pond were selected, apart from describing the selection process as

"random". Were sets of points selected equidistantly? How can we be sure there was not

repeated sampling ofthe same spot, biasing the measurements low? Also, it is not stated

how the species specific breakdown ofthe macrophyte score was calculated. Were plants

separated and weighed? Individuals counted? Or was it based on visual judgment by

looking at the rake? The species-specific results are impossible to interpret without this

information.

For the in situ chamber experiment, the frequency and method ofthe chamber cleaning is

not described. The frequency and efficacy ofthe cleaning may be very important

because the low atrazine ponds had high algal epiphyte growth, and epiphyte covering the

chamber could interfere with the growth ofthe plant segments inside.

In the farm pond study, there is no information on how triazine use histories at the control

vs. agricultural runoff-receiving ponds were determined, and what exactly they are. The

farm pond study results, while dramatic, cannot be interpreted without more information

on triazine types, use frequencies and quantities, watershed hydrology, other pond usage,

and exposure concentrations oftriazines within the control and "runoff-receiving" ponds..

In the emergent macrophyte survey, the spatial distribution ofthe quadrats is unknown.

Ten lOin by lOin quadrats would fill about 7 square feet ofthe pond surface, or roughly

11
0.14% of the total surface area of a 0.045-ha pond. Therefore, the survey might not be

representative of all the cattails in each pond.

Finally, the use of ANOVA for statistical analysis is so poorly described that the

statistical results are almost meaningless. It is not stated exactly how different groups are

defined (for example, were all pond samples combined at one treatment level, or were

only fenced-off area samples used?). Furthermore, ANOVA comprises a range of

statistical methods to be applied under various circumstances and assumptions. The

statement that ANOVA found significance at a certain p-value is meaningless without

knowing whether the tests were applied properly. If ANOVA methods were applied

improperly (i.e. the assumptions were violated), the results are incorrect.

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

Rationale for Use: The study has some utility for demonstrating that atrazine caused

reduction in the macrophyte community with corresponding changes in species

composition leading to domination by Chlorophytes at levels of 100 ug/L. However,

exposure was not sufficiently characterized, and baseline aquatic community

assembledges were not documented prior to study initiation.

Limitations of Study: The study design was hypothesis-generating in nature, intending

to highlight the potential for different kinds of community-level effects to occur rather

than to accurately formulate effects and magnitudes under tightly controlled conditions.

It succeeds in the former by presenting qualitatively interesting information, but fails in

the latter by presenting data that are difficult to accurately interpret. The study results are

not appropriate for quantitative prediction due to the apparently uncontrolled

experimental conditions, subjective measurement methods, and lack of key details in the

descriptions of methods and results.

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. The study switched test chemical from formulated product to

technical grade atrazine after the first year and also switched exposure concentrations for

several of the ponds, i.e, two of the lowest exposures at 20 llg/L were increased to 500

llg/L while one pond formerly at 100 llg/L was increased to 200 llg/L. Atrazine

degradate concentrations were not reported. Large grazing animals (grass carp) were

unequally distributed across the studies due to predation; thus, there was likely

differential grazing in the study ponds. Controls exhibited wide variability and given that

there were only two controls for each of the two studies, the ability of the study to detect

treatment effects is limited.

12
Primary Reviewer: Sara Hendrix, M.S., StaffScientist, Cambridge Environmental. Inc

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

Effects Division, U.S. Environmental Protection Agency.

13