Open Literature Review Summary

Chemical Name: Atrazine

CAS No: 1912-24-9

ECOTOX Record Number and Citation:

Lynch, T.R., RE. Johnson, and W.J. Adams. 1985. Impact of atrazine and

hexachlorobiphenyl on the structure and function of model stream ecosystems. Environ.

Toxicol. Chern. 4:399-413. (rvte..\Y.) i..(t>Lf3>S12. ")

Purpose of Review:

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

study were also used to parameterize the Comprehensive Aquatic System Model (CASM)

and establish the model-associated LOC for atrazine.

Date of Review:

December 5,2008

Purpose of Study:

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

experimental streams (lotic mesocosms).

Summary of Study Findings:

Methods: Experiments were conducted with a PCB isomer (2,4,5,2',4',5'-

hexachlorobiphenyl) as well as atrazine. Only the methods associated with atrazine

experiments will be described here.

Laboratory streams were constructed from existing concrete troughs in a former fish

hatchery owned by the State ofMichigan. The streams consisted oftwo sections: an

upper constant-head reservoir (3.96 m x 0.58 m x 0.27 m), and an identically sized lower

portion that served as the stream channel. Discharge in the stream channel was

maintained at 60 L/min by gate valves (water depth vs. velocity not described). Water

diverted from the adjacent spring-fed woodland stream, Cheny Creek (no further

description ofstream order or watershed characteristics), was gravity-fed into the

reservoirs after screening out large debris (mesh size not provided). Stream effluent was

filtered through activated carbon and polyurethane foam filters before discharge.

,•••••••11

2087809
Water temperature was not adjusted and so followed the natural diurnal and annual

fluctuations ofthe creek, ranging from a maximum of 15.5°C in May to a minimum of

0.5°C in December. Water quality parameters also changed over time, ranging from 9.0

to 11.2 mg 02/L (dissolved oxygen), from 164 to 202 mg CaC03/L (total alkalinity -

units are as appear in the article, though there appears to be a typo), and from 182 to 217

mg CaC03/L (hardness). Stream channels were illuminated by "high-intensity" coolwhite fluorescent lamps suspended above the streams (luminance/irradiance and exact

spatial parameters not described). Photoperiod, controlled by an automatic timer, was

adjusted every two weeks to correspond to natural time-varying day-length.

The laboratory stream channels were lined with natural substrate to a depth of 5 to 10 cm.

Substrate was transferred in June 1975 from Buckhorn Creek, Mecosta County,

Michigan, a natural creek located about 1 km from the hatchery. Cheny Creek substrates

were not available because the creek had been channelized and lined with concrete in the

vicinity ofthe hatchery. One year was allowed for biological colonization ofthe

laboratory streams by the organisms incidentally introduced with the substrate and those

drifting in the incoming water supply. Experiments took place over the second year

(1976-1977).

Two replicate streams were treated with atrazine in four 30-day phases - autumn, winter,

spring, and summer - each followed by a 60-day recovery phase (except for the summer

treatment, which had no recovery). Treatment was conducted with technical-grade

atrazine (Ciba-Geigy, Greensboro, North Carolina) stock solution dissolved in dimethyl

sulfoxide (DMSO). One stream designated as control received DMSO only. Syringe

pumps delivered stock solutions to turbulent mixing chambers as the lower end ofthe

reservoir troughs (see Figure 1). Nominal atrazine concentration in the treatment streams

was 25 jlg/L. Solvent concentration in both treatment and control streams was 0.69

jlL/L. In each seasonal treatment cycle, data collected included "chemical residues in

water, sediments, invertebrates, fish and plants" (not further described in the paper's

methods or results presentations); species composition and abundance ofthe

macroinvertebrate benthos and drift; periphyton growth; and primary production and

community respiration rates.

Benthic invertebrates were sampled in both pre-treatment and treatment phases ofthe 2-

year investigation. Two samples per stream were taken at 45-day intervals during the

pretreatment year, and three samples per stream were taken at 30-day intervals during the

treatment year. Sampled positions were determined using a grid system and a table of

random numbers (grid spacing and underlying distribution ofrandom numbers not

described). A 10.2 cm diameter core sample was removed from each sample location and

preserved immediately in 10% (v/v) formalin. Organisms were later separated from the

substrate material by sucrose flotation and examination ofthe substrate under

magnification. Organisms were preserved in 70% alcohol and later identified and

enumerated. Subsequently, the organisms were oven-dried at 50°C for 12 hours, cooled,

and the dry weight determined. The Shannon diversity index and species equitability

2
index (not defined within the article, although citations are provided) was computed for

each sample. Pretreatment data was pooled over all six streams (including the three

streams testing PCB isomer effects).

Drift invertebrates were sampled on days I, 5, 10,20, and 29 ofthe treatment phases and

days 14,42, and 60 ofthe recovery phases, and species composition and abundance

determined. Drift traps were 10-cm lengths of 10.2 cm diameter polyvinylchloride pipe,

with one end screened with 0.5-mm Nytex mesh. The traps were inserted into a wooden

weir at the downstream end of each stream that had two 10.2 cm diameter openings, so

that all drifting organisms were captured as the entire stream discharge passed through

the traps. At the time ofthis sampling, incoming drifting organisms were removed from

source water using traps attached to water supply pipes at the head of each stream. Drift

traps were changed every 4 hours (except 2400-0800 hours) and collections totaled over

the 24-hour day. Organisms were sorted (no details on sorting procedure), preserved in

70% ethanol, and later identified and enumerated.

Periphyton growth was measured on four glass microscope slides (25 mm x 75 mm) held

in a rack, vertically in the stream just above the bottom, with the long axis ofthe slide

parallel to the flow ofthe water. Slides were sampled (removed) at 4-day intervals for 28

days prior to the start of each treatment and for an additional 28 days during treatment.

After removal, the slides were dried on a hot plate and the periphyton removed by

scraping with a razor blade into tared aluminum pans. The samples were oven-dried at

105°C for 24 hours, cooled, and weighed to determine dry weight. Then the samples

were combusted at 500°C for 1 hour, cooled, and reweighed to determine ash-free dry

weight. Loss on ignition was considered to be accrued organic material.

Gross primary productivity and community respiration rates were determined from

diurnal changes in dissolved oxygen using photosynthesis-respiration chambers, as

described by Maki and Johnson (1976), Evaluation ofa toxicant on the metabolism of

model stream communities, J. Fish. Res. Board Can. Three chamber bottoms were each

buried at I-m intervals in the substrate of each stream at the time the streams were first

established. For measurement, the chamber top was placed over the chamber bottom, and

the chamber filled with stream water until air bubbles were gone. A dissolved-oxygen

probe was then inserted into a port on the upper surface ofthe chamber. Daily rates of

primary productivity and respiration were determined using the method of Whitworth and

Lane (1969), Effects oftoxicants on community metabolism in pools, Linmol. Oceanog.

Results were calculated as mg 02 produced or consumed per m

2

per day, based on the

area-to-volume relationship ofthe chamber. (Chamber set-up and calculations are not

further described.) Data from three chambers were averaged to yield a mean value for

each stream on each measurement day: days 2, 4, 8, 15, 24, and 29 during the treatment

phase, and days 20, 42,54, and 60 ofthe recovery phase, for the autumn period; and days

26 and 30 ofthe treatment phases and days 20, 40, and 60 ofthe recovery phases during

the winter, spring, and summer periods.

3
Statistics were computed using the ANOVA (analysis of variance) subprogram ofSPSS

(Statistical Package for the Social Sciences) software. Effects of atrazine and time

(month oftreatment or recovery) on the community-level variables were initially tested

with two-way ANOVA, with data from the four seasonal periods analyzed separately.

Specific differences were also evaluated using a priori orthogonal contrasts and Duncan's

multiple range test. Student's t tests were used to determine significance of differences

over annual means for pretreatment and treatment years.

Results: Experiments were conducted with a PCB isomer (2,4,5,2',4',5'-

hexachlorobiphenyl) as well as atrazine. Only the results associated with atrazine

experiments will be described here. Note that some figures will display HCBP

(2,4,5,2',4',5'-hexachlorobiphenyl) results as well as atrazine results.

The benthic macroinvertebrate community was dominated by scraper and gatherer functional

groups, including 23 taxa from 9 orders (list provided in the article, but is not reproduced here). The

number of taxa per sample, organisms per square meter, and standing biomass were generally

higher during the treatment year than in the pretreatment year, while species diversity and

equitability decreased during the treatment year. None ofthese differences were statistically

significant at p-value < 0.05. See

Table 1. Monthly mean values for benthic invertebrate counts, biomass, and species

diversity are shown in Figure 2 through Figure 4. The equitability index is not shown,

but it was similar to the diversity index (positively correlated with r = 0.87, p<O.OO 1).

Two-way ANOVA for each ofthe four seasonal periods was performed, testing effects of

month of measurement and of atrazine treatment. Monthly changes were often

significantly different at p-value < 0.05, but significant treatment effects were found only

in the spring season. Differences between treatments were further evaluated using a

priori orthogonal contrasts fOf 100 separate comparisons (for atrazine). Two significant

effects were found due to atrazine: in equitability in Winter Month 3 (p<0.029), and in

taxa per sample in Spring Month 3 (p<O.OOI). The authors note that, given a set of 100

random comparisons, five significant results are expected due to chance alone at a =

0.05; therefore, they concluded that this result shows a strong lack of evidence for

significant impacts oftreatment on community-level variables.

In the invertebrate drift samples, pulses were noted in all streams during the initial 24

hours of autumn and summer treatments (see Figure 5) (drift samples were not taken in

the first 24 hours during winter and spring treatments). The increase was mainly driven

by increases in Baetis mayflies. Only in summer were drift measurements notably higher

in the atrazine streams vs. the control (DMSO-treated) stream. (Statistical tests and their

results are not described.)

No significant effects of atrazine treatment on periphyton production were noted. Four

slides were collected from each stream on each collection day, and daily means for these

four slides were used as replicates in two-way ANOVA. The different streams' samples

4
from the same collection day, as well as accrued or cumulative biomass during each

treatment phase, were also compared using Duncan's multiple range test. See Figure 6.

No significant effects of atrazine treatment on gross primary productivity or community

respiration were noted (see Figure 7). Two-way ANOYA for each treatment period

found no significant effects oftreatment, and the primary productivity to respiration ratio

(PIR ratio) was particularly constant over treatments vs. control (see Figure 8).

Table 1. Annual means and standard deviations (over 9 months in pretreatment year, 10 months in

treatment year) for selected community variables based on monthly means (of two samples in

pretreatment year, three samples in treatment year). No results were statistically different from

pretreatment values at p<0.05. (from study table 2)

Stream Taxa/sample Organisms/ Biomass Diversity Equitability m2 (g dry wt/m2

)

Pretreatment Mean 9.23 9,757 8.64 2.30 0.57

(1-6 pooled) Std Dey 1.47 6,301 3.92 0.47 0.13

4 (DMSO + Mean 9.57 12,901 9.15 2.24 0.52

atrazine) Std Dey 0.97 4,290 1.50 0.20 0.08

5 (DMSO) Mean 9.37 15,494 9.15 2.31 0.48

Std Dey 1.52 8,404 2.19 0.31 0.13

6 (DMSO + Mean 9.17 13,025 11.23 2.18 0.49

atrazine) Std Dey 0.95 4,637 5.18 0.19 0.08

5
Slfeom ResefllOl,

WOIe< Sl4lQly

Figure 1. Diagram of one pair of laboratory model streams. (study figure 1)

• • CONTROLS • __ u_o HeBP

0-- • --0 AT RAZINE

300

w

200

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<t

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.....

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2:

en

z

<t

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AS ONOJ FMAMJ JA SONDJ

(1975) I (l976) I

TIM E

F M A M J

(1977)

Figure 2. Mean number of benthic macroinvertebrates per sample. Each data point for the

pretreatment year is the mean of 12 samples (2 per stream x 6 streams). Each data point for the

6
treatment year is the mean ofsix samples (3 per stream x 2 streams). Shaded bars indicate the

timing and duration of the treatment phases (study figure 2)

:.:-:':~.~-::

;;H[~

:un~; ::ill:::n:

t~t.~!~·]

• • CONTROLS ............ HCBP

e>ooo-. --0 ATRAZINE

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(19761 I ·(1977)

TIME

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Figure 3. Mean standing biomass of macroinvertebrates in the benthos of the laboratory streams.

Each data point for the pretreatment year is the mean of 12 samples (2 per stream x 6 streams).

Each data point for the treatment year is the mean ofsix samples (3 per stream x 2 streams). Shaded

bars indicate the timing and duration of the treatment phases (study figure 3).

;';:::'.. ...

F M A M J J A SON 0 J F M A M J

(l976) I (/977)

TIME

• • CONTROLS ------. HeBP 0--.--0 ATRAZINE

ASONOJ

(1975) I

2.6

2.8

7
Figure 4. Mean species diversity of benthic macroinvertebrates. Each data point for the

pretreatment year is the mean of 12 samples (2 per stream x 6 streams). Each data point for the

treatment year is the mean ofsix samples (3 per stream x 2 streams). Shaded bars indicate the

timing and duration of the treatment phases (study figure 4)

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b

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2

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Figure 5. Macroinvertebrate drift in the DMSO-treated control and atrazine-treated streams.

Treated stream values are the means of pooled data for the two streams. Shaded bars indicate the

timing and duration of the treatment phases (study figure 6).

8
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Figure 6. Temporal changes in the mean standing biomass (dry wt) of periphyton in the atrazinetreated streams and DMSO-treated control stream (upper figure). Lower figure is shown to provide

legend and time-axis labels; other information in the lower plot may be ignored. Mean values are

shown, with error bars indicating ±1 standard error, and are calculated from 4 measurements per

stream, made after 20, 24, and 28 days of treatment and after 50, 54, and 58 days of depuration.

(study figure 7)

9
PRODUCTION RESPIRATION

CONTROL' • 0 0

TREATEO ..------.. [)ouu- __ -O

1400

1200

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ATRAZINE

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Figure 7. Gross primary production and community respiration in the DMSO-treated control and

atrazine-treated streams. Shaded areas indicate treatment phases. (study figure 9)

ATRAZINE CONTROL' • TREATEDl>---uC

, , " '5 "" J , , I

AM JJ A SO N OJ F M A M J

TIME

Figure 8. Temporal changes in the ratio of gross primary production to community respiration (P/R)

in the DMSO-treated control and atrazine-treated streams. Shaded areas indicate treatment phases.

See above figure for time axis labels. (from study figure 10 - lower figure has been edited out of the

original figure, except for the time axis and labels)

10
Data Summary Table:

Effect Magnitude Nominal Day of Time Did Comments

ofEffect Atrazine Study Weighted Recovery

relative to Concentration Effect Average Occur- if

controls (~gfL) Occurred Atrazine so, when

Concentration (see

from Day 0 to comment)?

When Effect

Occurred

N/A - Atrazine Lack of

Periphyton concentrations effect is

biomass no effect 25 J.lg/L No effect were not no described

measured in article

text

N/A - Atrazine Lack of

Gross primary concentrations effect is

productivity no effect 25 J.lg/L No effect were not no described

in article measured

text

N/A - Atrazine Lack of

Community concentrations effect is

no effect 25 J.lg/L No effect were not no described respiration measured in article

text

Benthic N/A - Atrazine Lack of

macroinvertebrate concentrations effect is

community no effect 25 J.lg/L No effect were not no described

(multiple measured in article

variables) text

N/A - Atrazine Lack of

Total drifting concentrations effect is

organisms' no effect 25 J.lg/L No effect were not no described

measured in article

text

Drift ofBaetis N/A - Atrazine Effect concentrations nymphs (no.l24 no effect 25 J.lg/L No effect were not no attributed

hoursi measured to solvent.

Note: NA=Not ApplIcable

Time weighted averages were not computed. A syringe pump provided atrazine to

achieve a constant stream water concentration during treatment phases. Although no

atrazine was added during recovery phases, it was assumed that effects (or lack thereof)

were most informative when defined over the treatment phases. No atrazine

concentration measurements were provided, so nominal concentrations are assumed.

Reviewer Comments:

I Ibid.

2 Ibid.

11
This was a limited study that examined one atrazine concentration (25 j.!g/L) and used'

two treatment stream replicates and a single control stream. A solvent control stream was

used, and the solvent (DMSO) appeared to cause significant effects, possibly

outweighing the effects ofthe small amount of atrazine (although interannual effects due

only to time may have been more responsible for the treatment vs. pretreatment changes).

The authors did not find any statistically significant effects, but these results are limited

by the small number ofreplicate streams. To some extent the small numbers statistics

problems were well handled by taking multiple samples per stream, but the stream-bystream variability was not well explored by this study. Initial condition differences

among the streams (i.e. after the pretreatment year, before the first treatment) were not

discussed.

The design and set-up ofthe artificial streams was mixed in its applicability to natural

lotic systems. Allowing a full year for colonization increases the realism ofthe streams.

Additionally, the use offour treatment phases over the four seasons was informative in

showing the strong time-sensitivity ofthe measured ecological endpoints. However, the

streams necessarily underwent trophic modifications due to the laboratory set-up: the

authors note that regulation ofthe discharge led to increases in filamentous algae, and

that detritus like leaves was screened out ofthe systems, which may impact certain

heterotrophs.

Some detaiis ofthe set-up and analysis were not described: the water depth in the stream

channels; the watershed(s), land use, etc. ofCheny Creek and Buckhorn Creek; the

lighting intensity and spatial arrangement; the "chemical residues" which were apparently

measured for water, sediments, invertebrates, fish, and plants, but are not described in the

results; the definitions of community indices used; and the complete description ofthe

methods used to calculate productivity and respiration.

The figures appeared to provide a reasonably complete presentation ofthe data, but the

reviewer's copy ofthe original article was not entirely legible (shaded areas did not scan

properly).

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

Rationale for Use: The study utility was limited by the deficiencies described below.

However, the results provide some qualitative evidence that effects of atrazine on streams

biota at low concentrations are weak compared to seasonal effects.

12
Limitations of Study: The study used only two treatment streams and one control

stream. Additionally, the use of DMSO as a solvent could be a confounder. However,

results provide some qualitative evidence that effects of atrazine on streams biota at 25

flg/L concentrations are weak compared to seasonal effects.

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

Secondary Reviewer: Brian Anderson, U.S.EPA/IlL fI/J/!(

13