Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Gruessner, B. and M.e. Watzin. 1996. Response of aquatic communities from a

Vermont stream to environmentally realistic atrazine exposure in laboratory microcosms.

Environ. Toxico!. Chern. 15:410-419 (MRID47543613).

Purpose ofReview:

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 periphyton andbenthic

invertebrates from a Vermont stream and established in recirculating 120-L laboratory

microcosms. Measured endpoints included biomass ofthe attached algal community and

the functional and taxonomic composition ofthe benthic invertebrate community.

Summary of Study Findings:

Methods:

Collection

Stream organisms were collected from Patrick Brook in Hinesburg, Vermont, about 15

km south ofBurlington in the LaPlatte River watershed. The area is predominantly rural

(20% ofthe LaPlatte watershed is urbanlresidential, 40% is agricultural, and 40% is

forested). Patrick Brook drains a small lake. The collection site was about 5 km

northeast ofthe lake and about 0.5 km east ofthe confluence with the LaPlatte River.

The brook at the collection site averaged 1-2 m in width and was a mix ofshallow (5-15

em) riffles and 20-75 em pools. The collection site did not receive any runoff from land

where atrazine was in use. Collection occurred over 3 weeks in July 1993.

1111••••11

2087839
Artificial substrates were used to collect benthic invertebrates and periphyton. For the

benthic invertebrates, to reduce the high natural variability of benthic samples, collection

boxes with homogenized substrate were created and anchored to the stream bottom for 3

weeks to attract colonization. The substrate was a natural coarse (1-4 em diameter)

substrate, similar to that found at the site, which had been washed and air-dried to remove

organisms. The substrate had then been incubated for 2 weeks in the sun in a large,

shallow tank containing water from the stream site, to allow regrowth ofthe microbial

community. High density polyethylene 10 cm by 10 em by 7 cm boxes with I em

diameter holes cut into the sides were filled with the substrate. Each box had 13 holes on

2 ofthe sides, and 9 holes on the sides facing perpendicular to the streamflow, which

were wedged into the anchoring platform. The anchoring system was such that only the

top I to 2 em ofthe boxes were exposed above the surrounding streambed substrate.

After leaving the boxes anchored for 3 weeks arranged "randomly in a checkerboard

pattern", the boxes were collected by nesting each into an empty, intact box while still

submerged and fitting with a lid. The nested boxes were transported to the laboratory in

coolers.

For periphyton, glass microscope slides (2.5 em by 7.5 em) were anchored a few em

above the stream bottom with long axes vertical and faces perpendicular to the flow.

After being colonized for the same 3 weeks as the boxes, they were placed into plastic

slide boxes and kept moist during transport to the laboratory in coolers. The authors note

that the smooth surface may not have supported all periphyton species found at the site.

Laboratory microcosms

Six laboratory lotic microcosms were created, each consisting of a three-tank system with

a head tank above, a microcosm tank in the middle receiving gravitational flow from the

head tank, and a receiving tank below that drained the microcosm tank, with a

submersible pump (Little Giant Pump Co., Oklahoma City, Oklahoma) in the receiving

tank to circulate water back up to the head tank. See Figure 1 for a diagram. The

specially designed microcosm tanks were made of clear acrylic plastic (made by Vermont

Plastic Specialties of Williston, Vermont). The head and receiving tanks were 60-L tanks

made by Nalge Co. (Rochester, New York). The tanks were connected by polyurethane

tubing (Nalge Co.), which was chosen for stability in water and chemical resistance. The

flow rate was regulated by valves on the tank drains to maintain a water depth of 12 em

in the microcosms. A total of 120 L of water circulated through the tanks about 250

times per day. The microcosm tanks were covered with 500 jlm mesh netting to prevent

the escape of emerging adult insects. Water draining into the receiving tank was filtered

through a 500 jlm mesh funnel to capture any organisms in the flow.

All water used in the experiment was collected at the same site where the organisms were

collected, and passed through a 250 jlm sieve to remove macroorganisms and debris

before being added to.the tanks. Two full spectrum bulbs (Vita Lite, Duro Test

Corporation, Fairfield, New Jersey) in fluorescent light units were suspended above each

2
microcosm and set on a 14 hours on, 10 hours dark cycle (approximating the natural light

conditions at that time of year).

After transport to the laboratory, four "randomly selected" invertebrate collection boxes

were removed from their transport container and placed into each microcosm. One set of

four boxes was preserved in 10% formalin solution containing Rose Bengal to aid in

organism identification, and the composition ofthe benthic community from these four

boxes were used as "initial condition" estimates. Nine "randomly selected" periphyton

slides were also placed into each microcosm with the same orientation to the flow as they

had been placed in the field. Three slides were scraped, washed on glass fiber filters

(Type AlE, Gelman Sciences, Ann Arbor, Michigan) and frozen until chlorophyll a

analysis (described below). The algal biomass on these slides was used as "initial

condition" estimates.

The microcosms were allowed to rest and equilibrate for 24 hours, and then atrazine was

added to 3 ofthe 6 microcosms. Atrazine commercial product (AAtrex 90, Ciba-Giegy,

Greensboro, North Carolina) was dissolved into 1 L ofstreamwater and added to each

head tank to deliver a total concentration of 5 Ilg/L in the treatment systems. One liter of

uncontaminated streamwater was added to the control tanks. The day of initial treatment

was considered Day 1. 1 On Day 3, about 15% ofthe water was removed from each ofthe

treatment and control microcosms and replaced with fresh streamwater to ensure

adequate nutrient supply and waste removal from the systems, and to lower the atrazine

concentrations in the treatment microcosms. Then, on Days 6 and 7 about 50% ofthe

water in each system was replaced, and about 15% on Day 11. The exposure pattern was

designed to mimic the observed atrazine concentration dynamics in Vermont streams

receiving atrazine runoff occasionally.

Samplingandanarys~

Throughout the 2 week study, temperature and conductivity was measured daily (Model

33 SCT Meter, YSI Yellow Spring, Ohio). On Days 1,5, 10, and 14, three replicate

water samples were collected from each microcosm and analyzed for atrazine content by

enzyme immunoassay (Atrazine RaPID Assays, Ohmicron, Inc., Newtown,

Pennsylvania). Water samples from the collection site were also analyzed to verify that

the streamwater was free ofatrazine. On Days 1,3,5, 7, 9, 12, and 14, emerged adult

insects were collected from the mesh covers and water surface. On Days 1,5,9, and 14,

any material outside that collection boxes in the microcosm tank was collected on a 500

11m sieve and combined with the material from the receiving tank's filter. These samples

were called emigrant samples. (Day 1 emigrant and emerged insect samples were

collected prior to atrazine addition.) On Days 3, 7, and 14, periphyton slides were

removed from corresponding positions in each microcosm for determination of algal

biomass (see below). Finally, on Day 14, the invertebrate collection boxes were removed

I Throughout the review text, we will follow the study's definition of atrazine addition on Day I. However,

in the Data Summary Table, the day of atrazine addition will be considered Day O.

3
and all material from them preserved in 10% buffered formalin solution containing Rose

Bengal.

Attached algal biomass on the periphyton slides was estimated by analyzing the scraped

and washed (using glass fiber filters, as described above) material for chlorophyll a. The

filters were frozen for 1 to 3 weeks, depending on the time ofsampling, and extracted

overnight in 90% acetone at 4°C. Extracts were centrifuged at 500 rpm for 20 minutes,

decanted, and brought up to 5 mL. The chlorophyll a concentration ofthe extracts,

corrected" for the presence of pheophytin a, were determined with spectrophotometry as

described in Standard Method<;for the Examination ofWater and Wastewater, American

Public Health Association, 1989.

Preserved invertebrate samples (from collection boxes, emerged insects, and emigrant

samples) were washed over a 500 /lm mesh sieve to remove fine particles. Organisms

were picked out under a dissecting microscope. Organisms other than insects were

assigned to general taxonomical groups. Adult emerged insects were counted but not

further identified. Insects from the collection boxes and emigrant samples were

identified to genus when possible and categorized into functional groups based on

feeding behavior. The functional groups were collectors, predators, scrapers, and

shredders. The chironomid tribe Tanytarsini was counted as 10% scrapers and 90%

collectors based on the distribution in one box from each ofthe microcosms that were

identified to lower taxonomical levels. Since the functional classifications were not

highly accurate due to complex feeding habits of many insects, insects were also

classified as predators or nonpredators (consisting of collector, scraper, and shredder

groups).

Statistical analyses ofthe collection box data consisted primarily oftwo-level nested

ANOVA to test for differences between subsamples (collection boxes within in each

microcosm) and to separate that variability from the variation between treatment and

control microcosms. When within-microcosm differences were significant, ANOVA and

Tukey multiple comparisons were used to determine which microcosms were different

from each other. For the emerged and emigrant insect samples, two-way ANOVA was

used to test for effects oftreatment and of collection day on the number of organisms in

functional and major taxonomical groups. Similar analysis was applied to chlorophyll a

concentrations. When co1lection day effects were significant, Tukey multiple comparison

tests were applied to determine which days were significantly different. Treatment tank

atrazine concentrations on each sampling day were compared using one-way ANOVA

and Tukey multiple comparison. A log (n) or log (n+ I) transformation was needed to

achieve homogeneity of variances in some ofthe data sets (specific datasets that were so

transformed are not stated). If the variances could not be equalized this way, a Wilcoxon

or Kruskal-Wallis test was used for comparisons.

Results:

4
After 3 weeks of colonization in Patrick Brook, all microscope slides were covered with a

dense layer of periphyton. Although the slides visually appeared to have variable

amounts of periphyton gro\\-1h, the coefficient of variation in the chlorophyll a

concentrations ofthe three initial periphyton samples was less than 1%. In the collection

boxes, fine sediments had deposited within the coarse substrate and many organisms

were identified in the initial samples. In total during the experiment, 44 insect genera

were found, 7 insect tribes or subfamilies not further identified, and other organisms were

also found (gastropods, nematodes, oligochaetes, mollusks, and unknown pupae). The

study lists all invertebrates groups and their functional feeding group in an appendix (not

provided in this review). The functional group distribution ofthe initial benthic samples

is shown in Table 1. The coefficients of variation (CVs) are generally around 30-35%;

however, the shredders had CVs near 1 due to small numbers and were not included in

further analyses. Organisms not classified functionally, primarily oligochaete worms and

insect pupae, were about 7% ofthe total abundance. For the taxonomical classification of

all organisms representing more than 1% ofthe total abundance, the mean CV was about

60% (no data table provided).

Atrazine levels in the treatment microcosms were near the nominal level of 5 J.!g/L after

application and then dropped as expected (Table 2). Concentrations were not different

across the microcosms at each treatment level (no statistical test results provided), except

on Day 14 when microcosm 3 had significantly different and higher atrazine

concentrations than the other two (one-way ANOVA followed by Tukey test, p-value <

0.05). Temperature and conductivity readings were "consistent" (no statistics provided)

across all microcosms. Mean temperature ranged from 24.6 to 25.0°C and mean

conductivities ranged from 220.8 to 226.5 mho. The conductivities in the microcosms

throughout the study were similar to those recorded in Patrick Brook (no data or statistics

provided). The temperatures in the microcosms were approximately 5°C higher than that

in the stream.

Mean chlorophyll a concentrations did not differ significantly due to treatment (p-value

0.26), but the variability in the treatment microcosms appeared higher than in the

controls, especially later in the experiment (Figure 1). Chlorophyll a concentrations

differed significantly by collection day (p-value < 0.01; see Figure 1, where decreases

over time are clear, although there was no significant difference between Day 7 and Day

14). Using ANOVA with slides as replicates followed by Tukey multiple comparison

tests, chlorophyll a concentrations in microcosm 3 were found to be significantly

different from and lower than those in the other two treatment microcosms on Day 7 (pvalue < 0.03).

For organisms from the boxes collected at the end ofthe experiment, there was no

significant difference between treatments and controls in total number of organisms or in

the number of collectors, scrapers, predators, or non-predators (Table 3). The nested

ANOVA showed no differences in the numbers of organisms in each functional group

within the sets oftreatment and control microcosms, with one exception. The collectors

5
had significant differences within the treatment microcosms (p-value = 0.02).

Microcosm 3 was found to be significantly different than the other two treatment

microcosms (Tukey, p-value < 0.05) and was observed to contain a higher number of

collectors. Similarly, one genus (Paraleptophlebia spp., a collector) had significant

differences within the treatment group (ANOYA, p 0.0 I) and it was found that

microcosm 3 was significantly different from the other two (Tukey, p-value < 0.05).

There were no significant differences between treatments and controls in the numbers of

organisms categorized into taxonomical groups (Table 4).

For emigrant samples, there were no significant differences due to atrazine treatment on

the total or functional groups numbers of emigrants. There was, however, a significant

collection day effect. See Table 5 for statistics and Figure 3 for graphical illustrations.

Over time, predators appeared to become more abundant while nonpredators became less

abundant. Breaking abundances down by taxa (looking at the ten most abundant insect

taxa), again there Was no·significant treatment effect on abundances, but in many cases

there were significant collection day differences (see Table 6 for statistics and Figure 4

for graphical illustrations). The authors note the appearance of great variability in the

treatment microcosms (see Figure 4); they combined the emigrant samples with the

collection box samples to look at mean CYs across all functional and taxonomic groups

and determined that the mean CYs were about twice as high in the treatment microcosms

as the control microcosms (no data provided).

There were significant effects from treatment as well as collection day on the number of

emerged insects from the microcosms, but no significant interaction between the two

factors (see Table 7 for statistics). From Table 7, it is clear that by the end ofthe

experiment there were no longer significant treatment effects on emerged insect numbers.

Collection day results and significant differences are depicted in Figure 5.

Table 1. Mean number and percentage of organisms in the initial benthic invertebrate samples

(N=4) within each of the functional groups. (study Table 1)

Number of organisms Percent oftotal

Mean SD1 Cy2 Mean SD CY

Scrapers 54.3 19.8 35.13 ]4.13 7.39 52.29

Collectors 248.7 82.07 33 63.75 28.74 45.09

Shredders 3.75 3.59 95.84 0.85 0.64 75.63

Predators 55 20.02 36.39 13.97 6.36 45.54

Nonpredators 306.75 93.94 30.62 78.72 35.24 44.77

Other 27.25 7.5 27.52 7.31

389 115.23 29.62

SD = Standard deviation

2CY Coefficient of variation

6
Table 2. Atrazine concentrations (~g/L) in microcosms, three samples from each microcosm each

day. (study Table 2)

Day I.

Mean SE1

Day 5

Mean SE

DaylJ!

Mean SE

Daill

Mean SE

Treatment

Microcosm 1

Microcosm 2

.Microcosm 3

Control

Microcosm 1

Microcosm 2

4.73

4.82

4.68

ND2

ND

ND

0.084

0.084

0.19

3.54

3.65

3.48

ND

ND

ND

0.115

0.121

0.127

0.98

1.5

1.12

ND

ND

ND

0.023 1.11 0.046

0.121 1.12 0.Q35

0.075 1.35 0.046

NA3

NA

NA

1 SE Standard error

2 ND = Not detected at 0.05 Ilg/L

3 NA Not available samples contaminated after collection

Table 3. Mean number of organisms remaining in the collection boxes at the end of the experiment

and results of the statistical comparisons. (study Table 3)

-value

0.60 1

0.37 2

0.29 1

0.36 1

0.28 1

Control

Mean SD

86.7 9.53

283.3 17.92

141.33 12.72

372.33 14.9

Treatment

Mean SD

77.3 13.63

269.03 68.86

122 9.45

348.33 67.36

1 Nested ANOVA

2 Wilcoxon rank test

Scrapers

Collectors

Predators

Nonpredators

Total

7
Table 4. Mean number of collection box organisms in major insect taxa and other groups and the

results ofstatistical comparisons. (study Table 4)

Treatment Control

Mean SD Mean SD

Insect taxa

Stenelmis spp. ]4.33 2.85 13.67 1.76 0.59

Microtendipes spp. 8.67 4.18 11.67 2.76 0.58

Tanypodinae 76.33 6.33 92.33 10.84 0.27

Tanytarsini 109.67 22.17 120.33 3.95 0.66

Heptageniidae 33.33 9.94 38.33 6.84 0.81

Paraleptophlebia spp. 145.33 43.8 154.33 16.18 0.73

Helicopsyche spp. 16.33 5.81 19.67 1.2 0.32

Cernotina spp. 30.33 1.86 30.33 3.93 1.00

Other groups

Oligochaeta 29.67 lOA 19.67 4.67 0.43

Nematoda 0 0 2.33 1.2 0.12

Sphaeriidae 1.33 0.67 4 0.58 0.25

Gastropoda 18.33 9.7 9.33 3.76 0.73

Pupae 7.67 1.76 8 1.15 0.80

7.33 IA5 9 1.53 OA7

Table 5. Results oftwo-way ANOVA testing the effects of atrazine on the numbers of emigrants in

functional groups and on the total number of emigrants. (study Table 5)

T

Treatment Collection '" p-values p-values p-values

Collectors 0.65 <0.001 0.52

Scrapers 0.12 0.04 0.57

Predators 0.32 0.01 0.88

Nonpredators 0.9 <0.001 0.52

Total 0.09 <0.001 0.47

8
Table 6. Results of the statistical tests of the effects of atrazine on the numbers of organisms in

major taxa emigrating from the invertebrate collection boxes. (study Table 6)

0.43

0.67

0.75

0.85

0.75

0.17

0.68

0.29

0.9

0.98

Treatment x

Collection

p-values3

0.41

0.09

0.02

0.01

<0.01

<0.01

0.24

0.03

<0.01

<0.01

Collection

p-values2

0.21

0.39

0.79

0.91

0.67

0.59

0.38

0.46

0.56

0.64

Treatment

J:valuesl

Insect taxa

Stenelmis spp.

Microtendipes spp.

Polypedilum spp.

Orthocladius spp.

Tanypodinae

Tanytarsini

Baetidae

Paraleptophlebia spp.

Helicopsyche spp.

Nectop~yche spp.

Other groups

Planoboridae 0.33 0.79 NO

Physidae 0.57 <0.01 0.94

Pupae 0.93 0,01 0.53

I Two-way ANOYA, except for Wilcoxon rank test for Planoboridae

2 Two-way ANOYA, except for Kruskall-Wallis test for Planoboridae

3 Two-way ANOVA. ND Not determined

Table 7. Results of the statistical tests of the effects of atrazine on the number of adult emergent

insects in the microcosms. (study Table 7)

Two-wa ANOVA ANOVA

Treatment

Collection day

Treatment x collection

(interaction)

<0.01

<0.01

0.35

Day 1 0.17

Day 3 0.02

Day 5 0.02

Day 7 0.04

Day 9 0.09

Day 12 I

9
WATER LINE

HEAD TANK

...--A:::;;---- 11JBING

VALVE

TUBING

VALVE

TUBING

FILTER --..,-" I

RECEIVING

TANK -t--t1---;1----PUMP

Figure l. Schematic of a microcosm system as used in the experiment. The microcosm tank

measured 100 em long by 40 cm wide by 20 cm deep, and the head and receiving tanks were 70 cm

high and 40 cm in diameter.

a

3

...., E

j

t;j 2 ..

11/

8

u

III

.c

0

:i:

u

0 II

b

rn Treatment

o Control

c

Initial Day 3 Day 7 Day 14

Collection Day

10
a

as

1 0 ~:~::

0 ......+-'-'9--1.~~j'"':j~I--Iu::.l4-..l..

.. 40

., 30 0'>

o

Q 20

Control

EJ Treatment

o

b

Figure 2. Mean chlorophyll a concentrations and standard errors for algal biomass (N = 3).

Treatment and control means were not significantly different. Significant differences at p-value <

0.05 are indicated by different letters over the pairs of bars. Missing error bars are too small to be

depicted.

200

.!/l

i 150

2'

100

I 50 :::J

Z

Day 1 Day 5 Day 9 Day 14

Collection Day

Collectors

Day 1 Day 5 Day 9 Day 14

Collection Day

Scrapers

III 250 b

E

-2 200

a'" 150

100

!

E 50 :::>

z 0 ~,,+...L.J~--l.~:j-.L..t:;+...L.

15 b ..

c:

8. 10

(5

Day 1 Day 5 Day 9 Day 14

CoIJectlon Day

Predators

Day 1 Day 5 Day 9 Day 14

CoIJection Day

Totals

Day 1 Day 5 Day 9 Day 14

Collection Day

Nonpredators

Figure 3. Mean counts and standard errors for emigrant organisms in functional groups and the

total number of emigrants in the treatment and control microcosms (N=3). Treatment and control

means were no significantly different. Significant differences at p-value < 0.05 between collection

days are indicated by different letters over the pairs of boxes.

1]
Day 1 Day 5 Day 9 Day 14

Collection Day

po/ypedi/um Spp.

a

Day 1 Day 5 Day 9 Day 14

Collection Day

OrthocJadinae spp,

Day 1 Day 5 Day 9 Day 14

Collection Day

Microtendipes Spp.

E 15

'2'"

g'" 10

6 a a <II a E<II ';: 5 a

S,4

0 3 "0 2

.8

E 1 :::>

Z 0

Day 1 Day 5 Day 9 Day 14

Collection Day

Stene/mis spp.

Day 1 Day 5 Day 9 Day 14

Collection Day

Tanytarsini

[) Treatment 0 Control

....

o

i

z

5

b

Day 1 Day 5 Day 9 Day 14

Collection Day

Tanypodinae

IE 2S ab ." In

E 6 b a E In ." In

'2 20 'i! ';: S '" be '" '" g 4 15 0 0

"0 "0 "0 3 ... 10 ... .. Q,> 2 .J:l S C ..Q ..Q

E E E 1

2 ;:> ;:>

0 z z 0

Day 1 Day 5 Day 9 Day 14 Day 1 DayS Day 9 Day 14 Day 1 Day S Day 9 Day 14

Collection Day Collection Day Collection Day

Nectopsyche Spp, He/icopsyche Spp. Paraleptoph/ebia spp,

.. lit 60 C E30 a E 40 j <II a 50 <II 1: a <: .. 'E a 30 '" S, 20 a g E!l 40 0 be <5 - 20 a '0 30 0 "0 ... 20 i .. 10

10 .J:l

§ E 10 a ::l

z 0 z 0 z 0

Day1 DayS Day 9 Day 14 Day 1 Day 5 Day 9 Day 14 Day 1 Day S Day 9 Day 14

Collection Day Collection Day Collection Day

Planoboridae Physidae Pupae

Figure 4. Mean counts and standard errors for emigrant organisms in major taxa (>1% of total

abundance) in the treatment and control microcosms (N=3) on different days. Treatment and

control means were not significantly different. Significant differences at p-value < 0.05 between

collection days are indicated by different letters over the pairs of bars. Missing error bars are too

small to be depicted.

12
50 a ..

40 ·~t:~~~

i ab ::;:::::

;~~~m~ 30 :::=:::; QII ........ C5 ::::::::

i i 20

2:

1 0 .+."••••

;l!!!llil 0

Day 1 Day 3

a"

DayS

a"

~ay7

o Treatment

o Control

Day 9 Day 12 Day 14

CollectiOn Day

Figure 5. Mean counts and standard errors for emerged insect samples collected on different days

from treatment and control microcosms (N=3). Letters with * indiCate where treatment means

differed significantly from controls (p-value < 0.05). Significant differences at p-value < 0.05 between

collection days are indicated by ditIerent letters over the pairs of bars.

Data Summary Table:

Day of Comments

Study

Effect

Occurred

Nominal

Atrazine

Concentrat

ion (Jig/L)

Magnitude of

Effect relative

to controls

Effect Time Did

Weighted Recovery

Average Occur if

Atrazine so, when

Concentration (see

i . from Day () to comment)?

I I When Effect

~ri_~_ 1:~~~:-~-n-ill-~-a-n-t~-5-~-~---~:-~-:-~-:-:-tu-t~·-:-o-~-:-:-:-:-c-~-l-e~-n-o-t---~s-e-e-~-U-d-y-t-cr-t~

( 026) 14-day applicable p.414.

p= . experiment

not

applicable not applicable

no effect

throughout

14-day

experiment

5/lg/L

no significant

effect (p=0.28

in boxes,

p=0.09 for

total

emigrants)

Benthic

invertebrates

No significant

effects for

collection box

or emigrant

invertebrates,

or for

subdivisions by

functional

,L-_.. . -+- -+ ~----_tI------__if__---__if_g><.lr_:_o-u~p~sandtaxa

+100% Effect

(doubled) 2.6 !lg/L magnitude and

(average for Yes (Day (difference 5 /lg/L Day 0 timing

significant at Day 0 Day II) estimated from

0<0.01) I 10) I Figure5 I

Increase in

emerged adult

insects

I

Bolded values are those data pOints specIfied In the "Cosm Data POints and Exposure Profiles" spreadsheet.

13
Time weighted average calculations were complicated by the exposure pattern in the

microcosms, which involved replacing significant fractions ofthe total water with fresh

(uncontaminated) streamwater on specific days. On Day 3 (Day 2 if atrazine application

occurs on Day 0) and Day 11 (Day 10), 15% ofthe water was replaced; on Days 6 and 7

(5 and 6), 50% ofthe water was replaced. Because these replacements were substantial

and the time periods between replacements were short, it was assumed that

concentrations were constant in between each replacement, and reduced in a stair-step

fashion on the day of each replacement for calculation oftime weighted averages.

Calculations are shown in the table below, linked to an.Excel spreadsheet.

Cumulative

Atrazine Time

Cone. Weighted

Day (ug/L) Average

0 4.7 4.7

1 4.7 4.7

2 3.6 4.3

3 3.6 4.2

4 3.6 4.0

5 2.4 3.8

6 1.2 3.4

7 1.2 3.1

8 1.2 2.9

9 1.2 2.7

10 1.2 2.6

Reviewer Comments:

Overall, the study was well presented. Considering the space constraints, many data were

provided and methods and results were described in good detaiL The statistics were

almost totally comprehensive, with only a few minor gaps where no p-values were given.

The main limitation ofthe study is that there appears to be a problem with Treatment

Microcosm 3. Compared to the other treatment microcosms, this microcosm had

significantly higher atrazine concentrations on Day 14, significantly lower chlorophyll a

concentrations on Day 7, and more collector organisms in the boxes, particularly

Paraleptophlebia spp. This effect highlights some potential problems in the experimental

design.

Despite the authors' efforts to create homogenous initial conditions in the benthic

community collection boxes, there could have been significant variability in some

collection boxes due to stream microconditions during the 3-week colonization period.

The authors' reliance on "random selection" and "random placement" in the box

placement, both in the stream bed during colonization and in the distribution ofthe boxes

in the microcosm tanks, fails to address the potential for these effects. CVs appeared to

14
£(l3/l{

be substantial between the four boxes analyzed for initial conditions, and insufficient data

are provided to ensure that the study design is truly random variation or ifthere is some

pattern or categorical differences between boxes. For these reasons, it is possible that

Treatment Microcosm 3 may have received different initial benthic communities than the

other treatment and/or control microcosms.

Another possibility is that the experimental area was not well controlled. No details are

provided on the surroundings ofthe microcosm setup. In terms of interpreting the results,

the changes in Treatment Microcosm 3 would tend to exaggerate any effects of atrazine

addition, since it had higher atrazine concentrations.

Other potential limitations ofthe study are due to the collection system in the stream bed.

As the authors note, the microscope slides maybe too smooth for all types ofperiphyton

to colonize. The types of benthic invertebrates that can colonize the boxes may also be

limited. Overall, the communities collected and used in the microcosms are likely to be

much more simplified than a natural lotic system. The benthic invertebrate community

may have been unsustainable over time, predators became more abundant and

nonpredators less so, regardless oftreatment.

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

Rationale for Use: The results ofthis laboratory microcosm study show that atrazine

concentrations of:::5 Ilg/L do not impact periphyton and benthic invertebrates

communities collected from streams in Vermont.

Limitations of Study: Because one ofthe three treatment microcosms had several

significantly different results from the other two, it is likely that there were issues

associated with the experimental setup (dissimilar initial conditions or uncontrolled

laboratory environment). This issue resulted in higher atrazine levels in the dissimilar

treatment microcosm, which should have resulted in more pronounced average effects of

atrazine in the small sample; however, 2 ofthe 3 endpoints showed no effect. The noeffect results, though from a simplified laboratory system rather than a real ecosystem,

are likely to be reliable predictors of lotic systems and for the time period and exposure

pattern used in the study.

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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