EPA-600/3-76-047
May 1976
Ecological Research Series
CHRONIC TOXICITY OF ATRAZINE TO
SELECTED AQUATIC INVERTEBRATES AND FISHES
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
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This document is available to the public through the National Technical informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-047
May 1976
CHRONIC TOXICITY OF ATRAZINE TO
SELECTED AQUATIC INVERTEBRATES AND FISHES
by
Kenneth J. Macek
Kenneth S. Buxton
Scott Sauter
Sarah Gnilka
Jerry W. Dean
Bionomics, EG§G Inc.
Wareham, Massachusetts 02571
Contract No. 68-01-0092
68-01-1844
Project Officer
John Eaton
Environmental Research Laboratory
Duluth, Minnesota 55804
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory-Duluth, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the
U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or
recommendation for use.
ii
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ABSTRACT
Representatives of the aquatic invertebrate species of water
flea (Daphnia magna), midge (Chironomus tentans), and scud
(Gammarus fasciatus); and the fish species "bluegill (Lepomis
macrochirus)» fathead minnow (Pimephales promelas), and
"brook trout (Salvelinus fontinalis) were chronically exposed
to various concentrations of atrazine in separate flowing-
water systems.
Maximum acceptable toxicant concentrations (MATC) of atrazine
for the selected species in soft water were estimated using
survival, growth, and reproduction as indicators of toxic
effects. The MATC was estimated to be between 0.11 and 0.23
mg/1 for midges, between 0.14 and 0.25 mg/1 for water fleas,
and between 0.06 and 0.1*1- for the scud. For fishes the MATC
was estimated to be between 0.09 and 0.50 mg/1 for bluegills,
between 0.21 and 0.52 mg/1 for fathead minnows, and between
0.06 and 0.12 mg/1 for brook trout. The incipient-LC50 for
fishes and the 48-hour LC50 for invertebrates was estimated
from acute exposures and was used to calculate application
factors (MATC limits/LC50). For aquatic invertebrates and
atrazine the estimated application factors were between
0.15 and 0.32 for midges, between 0.02 and 0.04 for water
flea, and between 0.01 and 0.02 for scud. Application
factors were estimated between 0.01 and 0.0? for bluegills,
between 0.01 and 0.03 for fathead minnows, and between 0.01
and 0.02 for brook trout.
This report was submitted in fulfillment of Project Number
18050 HQH, Contracts 68-01-0092 and 68-01-1844, under the
sponsorship of the Water Quality Office, Environmental
Protection Agency.
in
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CONTENTS
SECTION PAGE
I Conclusions 1
II Recommendations 3
III Introduction 4
IV Methods and Materials 6
Chronic Exposure Systems 6
Acute Toxicity Procedures 9
Chemical Methods 10
Statistics 13
Chronic Exposure 13
Chironomus tentans 13
Daphnia magna 14
Gammarus fasciatus 14
Lepomis""macrochirus 15
Pimephales promelas 16
Salve linus fontinaTis 17
V Results " 20
Acute Bioassays 20
Water Chemistry 21
Chronic Exposure 21
Chironomus tentans 21
Daphnia magna 25
Gammarus fasciatus 26
Lepomis macrochirus 28
^Pimephales promelas 31
Salvelinus fontinaTis 34
Residue Analysis37
Calculation of Application Factors 39
VI Discussion 41
VII References 46
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TABLES
PAGE
1. Physical characteristics of test chambers
utilized to evaluate the chronic toxicity of
atrazine to fishes and fish food organisms. 7
2. Chemical analysis of the diluent water utilized
during chronic aqueous exposure of fishes and
aquatic invertebrates to atrazine. 8
3. Percent recovery of added atrazine from water
(|ig/l) and whole fish (fig/g). 12
Jf; Mean and range of measured concentrations of
hardness, alkalinity, acidity, dissolved oxygen
and pH (range only) from water samples taken
periodically during chronic exposure of aquatic
invertebrates and fishes to atrazine. 22
5» Nominal and measured atrazine concentration (mg/1)
in water during chronic exposure of aquatic
invertebrates and fishes to atrazine. 23
6. Summary of the effect of various concentrations
of atrazine on hatchability, pupation and
emergence of Chironomus tentans continuously
exposed for two generations. 24
7« Mean percent survival of Daphnia magna continuously
exposed to atrazine for 64 days. 25
8. Mean production of young per female Daphnia magna
continuously exposed to atrazine for 64 days. 26
9- Survival and reproductive success of GamTnarus
fasciatus exposed to atrazine for 17 weeks. 27
10. Survival and growth during 6 and 18 months, and
results of spawning activity of bluegill
(Lepomis macrochirus) continuously exposed to
atrazine. 29
11. Hatchability, survival, and growth of bluegill
(Lepomis macrochirus) fry exposed to atrazine. 31
VI
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PAGE
12. Survival and growth of fathead minnows
(Pimephales -promelas) exposed 30 days, 60
days, and 43 weeks to various atrazine
concentrations. 32
13. Spawning results, egg hatchability, survival
and growth of offspring after 30 and 60 days,
for fathead minnows (Pimephales promelas)
continuously exposed to atrazine. 33
1*K Mean increase in length and weight of yearling
brook trout (Salvelinus fontinalis) during 90
and 306 days continuous exposure to atrazine. 35
15. Results of spawning activity of yearling brook
trout (Salvelinus fontinalis) during continuous
exposure to atrazine. 36
16. Survival and growth of second generation brook
trout (Salvelinus fontinalis) during the first
90 days of development of fry continuously
exposed to atrazine. 38
17. Summary of concentrations of atrazine (mg/1)
producing acute and chronic toxicity to aquatic
species, and calculated application factors
describing the relationship between acute and
chronic toxicity (MATC/LC50). 40
vu
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ACKNOWLEDGEMENTS
During the period of time devoted to this research effort,
we were fortunate to have laboratory assistance from Mark
Lindberg and Gerald LeBlanc, who maintained exposure systems
and were significantly involved with routine aspects of the
fish chronics. Gratitude is extended to Krogh Derr, Pat Costa
and Karen Frey for their involvement and expertise in conducting
the invertebrate chronics. Also, our appreciation is directed to
Mr. Curtis Hutchinson for construction of all diluter systems
utilized throughout this study, and to Rod Parrish and Dr. Sam
Petrocelli for constructively reviewing the final report.
We are grateful to Mr. Richard Griffith, Northeast Regional
Director, U.S. Bureau of Sport Fisheries, Boston, Mass, for
his assistance in obtaining disease-free brook trout used in
the chronic exposure and to Mr. Quentin Pickering, Newtown Fish
Toxicology Station, EPA, Cincinnati, Ohio, for providing fathead
minnow eggs used in the study.
Finally, our sincere appreciation is extended to Mr. John Eaton,
Project Officer, Environmental Protection Agency, Environmental
Research Laboratory-Duluth, Duluth, Minnesota, for his guidance
and constructive advice during the performance of these studies.
Vlll
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SECTION I
CONCLUSIONS
Estimates (LC50) of the acute toxicity of atrazine generally
ranged from 4.9-15.0 mg/1 for all the species tested except
the midges for which the estimated 48 hour LC50 was 0.?2 mg/1.
All six species were similar in their susceptibility to
chronic exposure to atrazine with the lower limit of the
estimated MATC concentrations for all species ranging from
0.06-0.21 mg/1.
Continuous exposure of chironomids through two successive
generations to a mean measured concentration of 0.23 mg/1
atrazine resulted in reduced hatching success, larval
mortality, developmental retardation, and a reduction in the
number of organisms pupating and emergingD
Continuous exposure of daphnids to atrazine through three
successive generations indicates that reproduction is a
much more sensitive measure of species susceptibility than
is survival during acute or chronic exposures. Although
continuous exposure to a mean measured atrazine concentration
of 1.15 mg/1 had no significant effect on survival, continuous
exposure to 0.25 mg/1 significantly reduced production of
progeny.
Morphological development of FI gammarids is a more sensitive
indication of species susceptibility to chronic exposure to
atrazine than is survival. Although continuous exposure of
parental gammarids to 0.49 mg/1 atrazine for 119 days had no
effect on survival, and continuous exposure to 0.14 mg/1 for
30 days had no effect on survival of FI gammarids, the number
of FI gammarids exposed to 0.14 mg/1 atrazine which success-
fully developed to the seventh instar form was reduced 25$
when compared to lower treatments and controls.
Growth of brook trout is the most sensitive indication of
species susceptibility to chronic exposure to atrazine.
Although continuous exposure to a mean measured concentration
of 0.72 mg/1 atrazine for 44 weeks had no effect on survival
of parental fish, continuous exposure during the period to
0.12 mg/1 atrazine significantly reduced growth of fry when
compared to lower treatments and controls.
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The data indicate the tendency for all three fishes to
bioconcentrate atrazine from water is very low when compared
to other pesticides. Chemical analysis of fish tissue
samples indicated that residue concentrations after prolonged
exposure were below minimum detectable limits for all the
fishes analyzed.
The similarity of the limits of the application factors for
each of the fishes, and two of the three invertebrates,
tested supports the general validity of the application
factor concept.
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SECTION II
RECOMMENDATIONS
Midges (Chironomus tentans) were more susceptible to atrazine
than any of the other species studied. A variety of sublethal
effects were observed. These observations, combined with the
relatively short life history of midges, and the adaptability
of these organisms to the laboratory, suggests the species as
a desirable bioassay organism for evaluating the chronic
toxicity of chemicals to aquatic organisms.
Concerning chronic toxicity studies with bluegill, we feel
that more information must be developed on the laboratory
conditions necessary to induce and maintain spawning activity
and on the procedures necessary to handle and feed newly
hatched bluegill fry, in order to allow successful completion
of bluegill chronic toxicity studies.
Based on the lack of an acceptable level of spawning activity
among several groups of fathead minnows in which the number
of males was essentially equal to or greater than the number
of females, we suggest that care be taken throughout a
fathead minnow chronic to insure that during the spawning
period the ratio of females to males is greater than 1.5*1.
We recommend that the fathead minnow be considered the fish
species of choice, among those tested, for chronic toxicity
bioassays. This recommendation is based on the fact that an
adequate level of spawning activity can be induced under
laboratory conditions, successful handling of eggs and larvae
is possible, and excellent survival of fry is obtained. The
combination of these factors provides opportunities for
statistical and biological evaluation of possible toxic
effects due to chronic exposure to chemicals. Finally,
studies with this species represent the only "true chronic"
(at least one complete life history) of the fishes studied.
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SECTION III
INTRODUCTION
The current concern regarding -the protection of aquatic life
in surface waters has prompted evaluation of the effects of
chemicals on aquatic invertebrates and fishes. Much of the
toxicological research with aquatic biota has been limited
to the development of acute toxicity values as a measure of
the effect of chemicals on the biota. More recently,
utilization of the chronic exposure of fishes and aquatic
invertebrates to chemicals has received particular attention
due to the numerous parameters that can be evaluated as
indices of toxic effects (Mount, 1968; Eaton, 1970; Arthur,
1970; McKim and Benoit, 1971; Arthur et al., 1973; Macek
et al., 1975) The "Laboratory Fish Production Index
TfTpfD" as defined by Mount and Stephan (1967) reflects toxic
effects on reproduction, growth, spawning behavior, egg
hatchability, and fry survival. Somewhere between the highest
observed toxicant concentration that has no effect on these
parameters during continuous chronic exposure and the lowest
effect concentration is a theoretical value termed the maximum
acceptable toxicant concentration (MATC).
The occurrence and persistence of chlorinated hydrocarbon
insecticides and heavy metals in surface waters has resulted
in widespread interest in the effect of these chemicals in
aquatic ecosystems. As a result, much information on the
effect of these materials on aquatic organisms has been
generated (Cope, 1966; Johnson, 1968; Eislejr, 1973). However,
muah less is known of the effects of less persistent
chemicals despite the fact that many of these are utilized
extensively in agriculture. One of the most widely utilized
of these non-persistent chemicals is the herbicide atrazine
(2-chloro-4-ethlyamino-6-isopropylamino-s-triazine) with more
than 100 million pounds being applied annually to agricultural
lands in the United States (Hall .et al., 1972).
Past studies with atrazine have been few and consisted of
short-term aqueous exposures which generated acute toxicity
values (Walker, 1964; JWPCA, 1968; Sanders, 1969). Due
to the extensive use of this chemical and lack of information
concerning its long-term, subtle effects on aquatic organisms,
this study was undertaken to determine the MATC of atrazine
for selected aquatic invertebrates and fishes based on
continuous chronic exposure. The organisms selected for this
research effort were:
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INVERTEBRATES
1. Chironomus tentans (Chironomidae, midge)
2. Daphnia magna (Cladocera. water flea)
3. Gammarus fasciatus (Gammaridae, scud)
FISHES
1. Lepomis macrochirus (Centrarchidae, bluegill)
2. Pimephales promelas (Cyprinidae, fathead minnow)
3« Salvelinus fontinalis (Salmonidae, brook trout)
A reason for selecting both invertebrate and fishes is that
the susceptibility of fishes to a chemical should be compared
to the susceptibility of fish-food organisms to that same
chemical. An understanding of both these phenomena will be
invaluable in establishing realistic and meaningful water
quality criteria and standards.
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SECTION IV
MATERIALS AND METHODS
The methodology for chronic testing of fishes generally
followed the recommended bioassay procedures issued by the
Environmental Protection Agency1s National Water Quality
Laboratory, Duluth, Minnesota (Bioassay Committee 1971a,
19?lb, 19?lc). Acute bioassay procedures were bascially
those recommended in Standard Methods for the Examination of
Water and Wastewater (APHA, 1971)•Chronic testing procedures
for invertebrates were determined through communication of
Bionomics staff members with personnel at the National Water
Quality Laboratory.
CHRONIC EXPOSURE SYSTEMS
Proportional diluters (Mount and Brungs, 196?)» with a
dilution factor of 0.5 and a syringe injector, delivered the
test water and toxicant, dissolved in dimethyl sulfoxide, to
the mixing chamber, mixing cells, and ultimately to the test
chambers. Five atrazine concentrations and a control flowed
to mixing containers and into separate glass delivery tubes
leading to the replicate test chambers. In the test system
for Daphnia magna, baffles were inserted in each of the
quadruplicate test chambers to minimize turbulence of influent
water. The diluter was also modified to include food cells
which delivered a measured amount of food along with the
toxicant and diluent water. All other exposure systems
utilized duplicate test chambers of varying construction and
flow rates as summarized in Table 1.
Two growth chambers 21 x 20 x 15 cm (height x length x width)
with a water depth of 16 cm were provided for the young Gammarus
and received test water at a flow rate equal to that in the
adult test chambers. Fathead minnow test chambers were
subdivided to provide space for two growth chambers 25 x 20.5
x 12.5 cm, and test chambers for brook trout contained a
shelf which supported two growth chambers 25 x 25 x 12.5 cm
with a water depth of 12.5 cm. Water was delivered directly
to both the adult test chamber and growth chambers through
a glass, flow-splitting chamber calibrated to provide equal
flow rate to all chambers. The bluegill test chambers
supported two 18 x 28 x 12 cm growth chambers with a water
depth of 10.5 cm, and air was utilized to pump water from the
test chamber to the growth chambers at a rate of 6 chamber
volumes every 2*4- hours. All fish growth chambers had ^0-mesh
stainless steel screen affixed to one end to allow water to
flow out while retaining the young fish- Bluegill and brook
trout test chambers were aerated with oil-free air in order
to maintain dissolved oxygen levels above 60 percent saturation.
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TABLE 1. PHYSICAL CHARACTERISTICS OF TEST CHAMBERS UTILIZED TO EVALUATE THE CHRONIC
TOXICITY OF ATRAZINE TO FISHES AND FISH FOOD ORGANISMS
Species
Chironoraus
tentans
Daphnia
magna
Gammarus
fasciatus
Lepomis
macrochirus
Pimephales
promelas
Salvelinus
fpntinali s
Material
( shape )
glass
(rectangular)
glass
(cylindrical)
glass
(rectangular)
stainless steel
(rectangular)
glass
(rectangular)
stainless steel
(rectangular)
Dimensions3
(cm)
21x26x18
17x13.5
25x40x21
40x180x30.5
30.5x90x30.5
40x90x30.5
Water
Depth
(cm)
16,0
14.0
19.0
30.5
15.0
30.5
Volume
(liters)
7-5
1.8
16.0
168.0
41.0
84.0
Flow Rate
(tank vol./
24 hours)
6
2
3
7
7
7
Dimensions are height x length x width for rectangular chambers and height x diameter
for cylindrical chambers.
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Diluent water was pumped from a 400 foot subterranean well to
a cement holding tank. Results of the chemical analysis of
the diluent water are summarized in Table 2.
TABLE 2. CHEMICAL ANALYSIS OF THE DILUENT WATER UTILIZED
DURING CHRONIC AQUEOUS EXPOSURE OF FISHES AND
AQUATIC INVERTEBRATES TO ATRAZINE
Parameter
Calcium
Magnesium
Potassium
Sulfate
Nitrate
Nitrite
Ammonia
Phenol
Chlorine
mg/liter
6.0
2.1
1.1
11.6
<0.05
<0.05
<0.1
<0.001
<0.01
Parameter
Chloride
Fluoride
Cyanide
Iron
Copper
Zinc
Cadimum
Chromium
Lead
mg/liter
17.6
0.5
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Chironomus. In addition, ultra-violet lights (24 watt) were
placed over the headbox and water cells of each invertebrate
diluter to minimize the introduction of fungus and pathogens
into the test system.
Illumination was provided by a combination of Duro-tast
(Optima FS) and wide-spectrum Grow-Lux fluorescent lights
fixed centrally above the test chambers in all experimental
units. Incandescent bulbs (100 watt) simulated a 15-minute
dawn or dusk light intensity change (Drummond and Dawson,
1970). A constant 16-hour light, 8-hour dark photoperiod
was controlled by an automatic timer in the Chironomus and
Daphnia experiments.
The photoperiod for Gammarus, bluegills, and brook trout
followed the normal daylight hours of Evansville, Indiana
(representative of U.S. daylength), and was adjusted the
first and fifteenth of each month. The photoperiod for
fathead minnows followed Evansville daylengths but was
started with the daylength for December 1st on day one of
the experiment (Nov. 11). All experimental units were
screened with black polyethylene curtains to prevent
unnecessary disturbance of test organisms and the influence
of extraneous lighting on the intended photoperiod.
ACUTE TOXICITY PROCEDURES
Static acute toxicity bioassays were conducted with inverte-
brates and atrazine to estimate the 48-hour LC50 and its 95$
confidence interval. Five organisms in three or four
replicate containers at each concentration were exposed at
20 ± 1°C. First instar Chironomus tentans, Gammarus fasciatus,
and <24-hour old Daphnia magna were the organisms exposed.
A linear regression equation was calculated after converting
test concentrations and corresponding percent mortalities to
logarithms and probits, respectively, and this equation was
utilized to estimate the 48-hour LC50's and confidence
intervals.
Acute toxicity studies with trout and bluegill were conducted
in flow-through systems using proportional diluters (Mount
and Brungs, 1967). The incipient LC50 was estimated when
no additional significant (>10$) mortality of the test
organisms was observed for 48 hours among fishes exposed to
any concentration. At this time, the exposure was terminated
and a linear regression equation was calculated by converting
atrazine concentrations and corresponding mortalities into
logarithms and probits, respectively. This equation was
utilized to estimate the incipient LC50 and 95$ confidence
interval. Due to the solubility of atrazine in water, a
static bioassay was used to estimate the acute toxicity of
atrazine to fathead minnows.
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The chronic exposure concentrations for both invertebrates
and fishes were selected by evaluating observed mortality
and no-effect concentrations from acute studies. The ^8-hour
LC50 values for invertebrates and the incipient LC50 values
for fishes (except minnows) were used to estimate application
factors describing the relationship between acute and
chronic toxicity.
CHEMICAL METHODS
Toxicant concentrations and basic water quality characteristics
were initially monitored in each aquarium each week to
establish that the toxicant concentrations and water quality
characteristics were constant with minimum variability.
After the determination of these concentrations and parameters
in the experimental systems, a minimum monitoring effort was
conducted to measure variability and detect changes from the
established means. Generally, atrazine concentrations were
determined once each week during each of the chronic exposures
by talcing 500 ml water samples from each aquarium. Stock
solutions of. atrazine (9*J$ a.i., Ciba-Geigy Chemical Co.) in
nanograde dimethyl sulfoxide (DMSO) were delivered to the
dilution water from 50 ml glass syringes through stainless
steel needles. The solvent (DMSO) was not added to the
control water in any of the chronic exposures and the amount
added to the highest atrazine concentration of each chronic
test ranged from 12 p.g/1 during the brook trout chronic to
65 fig/1 during the Daphnia chronic. In each case, these
concentrations were determined to be less than 1/500 of the
48-hour or 96-hour LC50 of the solvent for each species.
Water samples were taken in amber-glass bottles with teflon-
lined caps. The volume of water was accurately measured and
transferred to a separatory funnel. The sample bottle was
rinsed with 50 ml of methylene chloride to remove any adsorbed
atrazine and this solvent was added to the separatory funnel
containing the water sample. The separatory funnel was shaken
for two minutes to extract atrazine from the water, allowed
to stand until phase separation occurred, and the solvent
drained into a beaker. The extraction was repeated three
times using 25 ml of methylene chloride per extraction and
the extracts combined in the beaker.
The solvent extract was passed through a 20 x 50 mm column of
sodium sulfate and the column was rinsed with an additional
50 ml portion of methylene chloride. The solvent was
evaporated to approximately 5 ml on a stream bath, quantita-
10
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tively transferred to 15 ml centrifuge tubes with methylene
chloride, and evaporated to dryness using a gentle stream of
clean, dry air at 40°C. The extract was immediately dissolved
in an accurately measured volume of hexane and a portion was
withdrawn and analyzed "by gas/liquid chromatography under the
following conditions!
Instrument: Beckman Model 45 GC gas/liquid chromatograph
Detector: Helium-arc electron capture
Column: 183 x 2 mm I.D. glass containing 5% Dexsil on 80/100
mesh Supelcoport
Carrier gas: High purity helium at 55cc/min.
Detector gas: High purity helium at HOcc/min. and carbon
dioxide at 3cc/min.
Inlet: 225°C, Column: 200°C, Outlet: 240°C, Detector: 26o°C
Recorder: Imv full-scale, chart speed 1.3 cm/min.
Atrazine was eluted in 4.2 minutes, and 10 ng gave full-scale
pen deflection at an electrometer attenuation of 8 x 10~10
amperes. Linearity of response was not assumed during the
study, rather a graph of peak height versus ng of atrazine
injected was constructed prior to analyzing each batch of
atrazine samples. The amount of atrazine in each sample was
then determined by graphical interpolation. The recovery of
atrazine from spiked water samples using the above methods
was essentially quantitative and no adjustments were neces-
sary for recovery (Table 3)-
Fish tissue was extracted by the column method of Hesselberg
and Johnson (1972). The extraction solvent was diethyl ether.
The extract was analyzed by gas/ liquid chromatography using
conditions previously described for water analysis. No clean-
up of the fish extract was performed prior to analysis. The
chromatographic column (5fo Dexsil 300 GC) was cleansed of
contamination by fish oils and other extractable interferences
by heating overnight at 325°C. The next morning the tempera-
ture was reduced to 250°C, and injection of atrazine was made
to condition the column, and finally the oven temperature was
lowered to 200°C and atrazine analyses were resumed with no
change in efficiency. The recovery of atrazine from spiked
tissue samples was again essentially quantitative (Table 3)«
11
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TABLE 3, PERCENT RECOVERY OF ADDED ATRAZINE FROM WATER
(|ig/l) AND WHOLE FISH (|ig/g)
Sample Type
Water
Whole Fish
Added
Cone.
(ppm)
0.05
0.05
0.05
0.50
0.50
0.50
5.0
5.0
5.0
0.380
0.494
0.392
3.82
3-52
3.49
Measured
Cone.
(ppm)
0.052
0.049
0.050
0.46
0.51
0.50
4.6
4.9
5.0
0.374
0.488
0.384
3.94
3.44
3.46
Recovery (%}
104
98
100
92
102
100
92
98
100
98.4 ± 4.1a
98
99
98
101
98
99
98.8 ± l.la
Standard deviation.
During the chronic exposures, total hardness, alkalinity,
pH and acidity were generally measured bi-weekly in the
control and one atrazine concentration according to Standard
Methods for the Examination of Water and Wastewater (APHA,
1971).Temperature and dissolved oxygen concentrations were
measured in selected tanks each day using a YSI dissolved
oxygen meter with a combined oxygen-temperature probe.
12
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STATISTICS
Means of the biological parameters measured in each duplicate
container during chronic exposure were subjected to
analysis of variance according to Steel and Torrie (1960).
When treatment effects were indicated by analysis of variance,
the means of these effects were subjected to Duncan's Multiple
Range Test (1955) to determine which treatments were different
from the controls. Statistical significance was accepted at P=.Q5»
CHRONIC EXPOSURE
Chironomus tentans
Stock cultures of Chironomus tentans were obtained from
Michigan State University, East Lansing, Michigan. Cultures
were maintained in 60 1 glass aquaria utilizing a substrate
prepared by placing 50 grams of paper hand towel (Nibroc,
Brown Co.) in a blender with 5 grams of high protein chicken
feed and 1 liter of water to cover the mixture. The mixture
was homogenized for three to five minutes. This substrate
was used to cover the bottom of each aquarium to a depth of
1.5 cm. Emergent adults were allowed to mate within the
screened aquaria and provided a ready supply of egg masses.
Egg masses were selected from the stock cultures according to
uniformity of age, and one egg cup of 100 eggs was suspended
in each of the replicate experimental aquaria. Egg cups were
made from 20 ml glass vials with the bottoms removed and
replaced with ^0 mesh nylon screen. After hatching was com-
pleted in all treatments (3-5 days), the number of surviving
first instar larvae were counted and carefully placed on the
substrate.
After 26 to 29 days, the number of emergent adults, pupal
exuviae, and dead pupae were observed and recorded. Mating
of adults was observed, and egg masses produced by mating
pairs in each replicate aquaria were collected and utilized
to conduct second generation exposures according to the above
methods. At the initiation of the second generation exposure,
new substrate was placed in the experimental aquaria which
would receive first instar larvae. The number of emergent
adults, pupal exuviae, and dead pupae was observed in each
replicate concentration and the experiment terminated.
13
-------
Da-phnia magna
Laboratory stocks of Daphnia magna were obtained from the
University of New Hampshire, Durham, New Hampshire, and
successfully cultured in the laboratory according to the
methods of Biesinger and Christensen (1972).
Typically, 10 daphnids (<2^ hours old) were placed into each
of four replicate experimental units, resulting in a total
of *K> animals per concentration. A food supply consisting of
trout starter and dry-powdered Cerophyl (2:1) was prepared
in an aqueous suspension (12.5 mg/ml) and delivered from a
Mariotte bottle via a volumetric delivery system to a mix-
ing chamber each diluter cycle. The diluted food suspension
was subsequently transferred to the food cells from which
25 ml (0.1 mg/ml) were delivered to each test container
during each diluter cycle.
Survival and reproduction of daphnids were recorded after
one, two and three weeks. Reproduction was measured by
recording the number of young in each experimental chamber
weekly and discarding the progeny after weeks one and two.
At the end of the third week, the number of original animals
remaining was recorded, the specimens discarded, and 10
daphnids (<2k hours old) were randomly selected from each
chamber to begin the second generation exposure. The
above procedures were followed for the second and third
generation, after which the experiment was terminated.
Gatmnarus fasciatus
The test organisms were collected in October 1972 from the
raceway outlets at the National Fish Hatchery, North
Attleboro, Massachusetts. Sexually mature adults were
acclimated at 17 + 1°C in the laboratory for a period of
three weeks. After the acclimation period, females were
isolated and their progeny collected. When sufficient
1 to 22-day old gammarids were available, the chronic
exposure to atrazine was initiated by placing 30 specimens
into each replicate aquarium. During the exposure, the
gammarids were fed pre-soaked maple leaves, water cress, and
Elodea. Brine shrimp (Artemia) nauplii and Daphnia were
also fed to the gammarids weekly.
Survival was recorded once each month by siphoning
contents of each test chamber into a pan and counting the
gammarids. At the onset of reproduction, all adult chambers
were checked daily for gravid females which were then
14
-------
isolated individually in ^00 ml beakers containing the
corresponding test solutions. Some females were maintained
for up to 28 days in "beakers with the test solution being
replaced every 48 hours with fresh test solution from the
appropriate experimental aquarium. Twenty to fifty young
collected from isolated females in the same adult chamber
on the same day were placed in the respective larval
growth chamber and exposure continued for 30 days. At the
end of 30 days, survival and growth were recorded by the
procedure of Clemens (1950). After a period of 1? weeks, the
exposure of the original gammarids to atrazine was terminated.
Lepomis macrochirus
In December 1971» chronic exposure of bluegills to atrazine
was initiated using 7 to 10 cm fish obtained from a commercial
hatchery in Wisconsin. Bluegills were acclimated to the test
water for three months, after which twenty fish were randomly
distributed to each test chamber. Bluegills were fed ad
libitum the largest commercially prepared trout pellet which
they would take twice daily. All tanks were siphoned twice
weekly to remove fecal material, excess food, and detritus,
and were brushed when algal growth became excessive. Total
length and weight of each individual fish was measured at the
initiation of exposure, after 90 days, and at thinning (185
days) using 100 mg/1 of tricaine methanesulfonate (MS-222) to
lightly anesthetize the fish.
During the first year, when secondary sex characteristics
were well developed, fish in each tank were separated into
groups of males, females and undeveloped fish, using the shape
of the urogenital opening as the main criterion for sexual
differentiation (McComish, 1968). Sexually mature fish were
randomly reduced to 3 males and 7 females per duplicate tank
and all other fish were discarded after examination of gonadal
development and measurement of total length and weight. At
this time, two spawning substrates similar to those described
by Eaton (1970), were placed into each duplicate tank.
Substrates were 30.5 x 40 x 5 cm with a bowl-shaped depres-
sion 25 cm in diameter and 4 cm deep. Although '"-egills
developed secondary sex characteristics and exhibioed
territorial behavior characteristic of spawning bluegill
populations under natural conditions, no spawning occurred
among either exposed or control fish during the first year.
Consequently, substrates were removed and the exposure of
bluegills to atrazine was continued a second year, repeating
the photoperiod schedule.
15
-------
During the second year of bluegill exposure, when males
demonstrated territorial behavior, the substrates were
replaced in the tanks and total length and weight was
determined for each fish. Substrates were checked for eggs
after 1:00 p.m. each day and eggs were brushed loose from
the substrate under a stream of test water. Eggs were also
siphoned from the tank bottom after each spawn. Two
hundred eggs were randomly selected from each spawn and
placed in an egg cup which oscillated in the respective test
water by means of a rocker arm apparatus (Mount,^1968). Eggs
from each spawn were counted volumetrically by first counting
a subsample of exactly 1 ml and multiplying that number by
the total measured volume (ml) of spawned eggs. Eggs were
allowed to settle for two minutes in the graduated cylinder
before measuring. Hatchability was expressed as the percent
of live fry hatching out of 200 eggs.
Thirty bluegill fry from the earliest two spawnings in each
tank with at least 25 percent live hatch were placed in the
growth chambers. Fry from all other spawns were discarded
after hatchability was determined. Fry in the growth chambers
were fed live zooplankton from mixed laboratory cultures of
copepods, rotifers, protozoans, and brine shrimp ad libitum
three times daily. Cumulative mortality and total lengths of
fry were determined after 30, 60 and 90 days using the photo-
graphic method of McKim and Benoit (1971). Parental bluegills
were sacrificed after spawning had ceased in all tanks for
three weeks. Total length, total weight, sex, and gonadal
condition were determined for each fish and samples of muscle
were retained for residue analysis.
Pimephales promelas
Chronic exposure of fathead minnows to atrazine began in
September 19?1 with 18-day old fish obtained as eggs from the
Newtown Fish Toxicology Station, Newtown, Ohio. Forty fish
were randomly distributed to each test chamber. Cumulative
mortality and total length of live fish were determined after
30 and 60 days using the photographic method of McKim and
Benoit (1971). At 60 days, the number of fish in each test
chamber was randomly reduced to fifteen. Fathead minnows
were fed ad libitum twice daily with a commercially prepared
trout starter food which was supplemented with daphnids and
brine shrimp nauplii. All tanks were siphoned twice weekly
to remove fecal material, excess food, and detritus, and were
brushed when algal growth became excessive.
The discovery of bacteria and external parasites on a few
16
-------
fish prompted the use of flush treatments of tetracycline
hydrochloride (**> mg/1 active ingredient) and a combination
of malachite green and formalin (25 (il/l of formalin contain-
ing 3-7 g/1 malachite green crystals). These treatments
were concentrated between days 70 and 129 of the experiment.
In order to establish the appropriate ratio of male fish to
female fish, some of the males were removed after secondary
sexual characteristics were well developed. Five spawning
sites of halved, 3-inch transite drain tiles were placed in
each tank when fish were released from growth chambers at 60
days. The tiles were placed concave surface down at locations
that minimized the chance of encounters by separate egg-
guarding males. When spawning began, eggs were removed and
counted after 1:00 p.m. each day. Fifty eggs from each spawn
were oscillated in their corresponding test waters by means
of an egg cup and a rocker arm apparatus (Mount, 1968). Dead
eggs were removed and counted each day until hatching was
completeo (3-5 days at 25PC). Percent hatch was based on the
number of live fry from 50 eggs.
Forty fry from the earliest two spawns in each tank with at
least QOyo live hatch were placed in the respective growth
chambers. Cumulative mortality and total length of live fish
were determined at 30 and 60 days by the photographic method
of McKim and Benoit (1971). Fry from all other spawns were
discarded unless a growth chamber was later made available
by termination of 60-day old fry. Finely ground starter
food and brine shrimp nauplii were fed three times daily to
fry in the growth chambers.
Parental fish were sacrificed after all spawning had ceased
for one week. Total length, weight, sex and gonadal condition
were determined for each fish and three samples per concentra-
tion of eviscerated fish were retained for residue analysis.
Salvelinus fontinalis
Chronic exposure of brook trout to atrazine began in May 1972,
with yearling fish obtained from the National Fish Hatchery,
Manchester, New Hampshire. Distribution of fish to the test
chambers was delayed by the discovery of external parasites
on a few fish. The parasites were controlled by flush
treatments of malachite green and formalin (25 (J-l/1 of formalin
containing 3*7 g/1 of malachite green). These treatments were
repeated shortly after the distribution of 15 fish to each
duplicate tank to insure control of the parasites. Brook trout-
were fed twice daily with a measured ration of the largest
trout pellet they would take. The feeding rate was based on a
17
-------
percentage of the initial average biomass per tank and was
adjusted after 90 days and 136 days when total weights of
fish were again measured. Feeding rates were from a New
York State fish hatchery feeding chart (Deuel et al., 1952).
Total length and weight of individual fish were measured at
the initiation of exposure and after 90 days exposure,
utilizing 100 mg/1 of tricaine methanesulfonate (MS-222) to
quiet the fish during measuring. All tanks were siphoned
twice weekly to remove fecal material, excess food, and
detritus, and were brushed when algal growth became excessive.
Secondary sexual characteristics were observed on test day
210 and males, females and undeveloped fish were identified
in each tank and the number of sexually mature fish was
randomly reduced to 2 males and A- females per tank. All
other fish were discarded after being measured and examined
for gpnadal condition. At this time, two stainless steel
spawning substrates, similar to those described by Benoit
(197^)i were placed in each duplicate tank. The bottom of
these box-like substrates (25 x 25 x 15 cm) contained a
gridwork of 2.5 cm cubicles made of 20-mesh stainless steel
screen to retain the eggs where they were deposited. A
25 x 25 cm square of 4-mesh stainless steel screen was placed
on top of the egg retainer and silicone adhesive was used to
attach 1.3 - 2.5 cm diameter stream gravel 2 cm apart on the
screen.
Substrates were checked after 1:00 p.m. each day and eggs
were recovered by removing the gravel screen and egg retainer
and pipetting the eggs into a pan containing the test water.
Eggs from each spawn were counted and two egg cups containing
a group of 50 and 100 eggs, respectively were attached to the
rocker apparatus (Mount, 1968) for incubation in their respec-
tive test aquaria. Dead eggs were removed and recorded daily
and the number of fry hatching each day in the incubation cups
with 50 eggs was recorded. After 15 days, the group of 100
eggs was removed and the number of eggs developing a neural
keel was observed to determine percent fertilization.
Hatchability (percent of 50 eggs) and mean time to hatch
(degree days) were determined.
Twenty' five fry from the earliest two spwans in each tank
with at least 50$ live hatch were placed in the respective
growth chambers. Fry from all other spawns were discarded
after determining percent hatchability. Cumulative mortality
and total length of live fry were determined after 30, 60
and 90 days using the photographic method of McKim and Benoit
(1971). Brook trout fry were fed five times daily ad libitum
with a commercially prepared trout starter food.
18
-------
After spawning had ceased in all tanks for a period of three
weeks, parental fish were removed and the condition of the
gonads was observed. Total lengths and weights were deter-
mined for each individual fish "before obtaining muscle
samples for residue analysis and discarding the remaining
fish.
19
-------
SECTION V
RESULTS
ACUTE BIOASSAYS
Analyses of the results of acute static bioassays with the
invertebrate species indicated the 48-hour LC50 (95/J confidence
interval) of atrazine for Chironomus, Daphnia, and Gammarus
was 0.72 (0.36-1.44), 6.9 (.X2-8.1J, and X7 (3.6-8.0) mg/1,
respectively. Mortality was observed with Chironomus exposed
to 0.5 mg/1, with Daphnia exposed to 3.0 mg/1, and with Gammarus
exposed to 2.4 mg/1. Based on these data, the highest nominal
concentrations selected for the chronic exposures were 2.0 mg/1
(Chironomus), 1.2 mg/1 Daphnia and 1.0 mg/1 (Gammarus).
A 7-day continuous-flow bioassay conducted at 19 ± 1°C with
6.5-gram bluegill indicated the 96-hour LC50 for atrazine and
bluegill was >8.0 mg/1, and the incipient LC50 was 6.7 (5.4-8.4)
mg/1. Fathead minnows were not killed in 8 days by 8 mg/1, the
highest concentration of atrazine that could be maintained in
test chambers in a flow-through test. A 120-hour acute bioassay
conducted at 19 ± 1°C with 1.8-gram fathead minnows and utilizing
the 24-hour renewal technique indicated that both the 96-hour
and incipient LC50 values were 15 (11-20) mg/1. An 8-day
continuous-flow bioassay at 13 ± 1°C with 52-gram brook trout
indicated the 96-hour LC50 for atrazine and brook trout was
6.3 (4.1-9.7) mg/1, and the incipient LC50 was 4.9 (4.0-6.0)
mg/1. In each of these tests, fish appeared darkened and stressed
at concentrations as low as 1.4 mg/1. In order to select
concentrations for chronic studies which would include safe
concentrations, other acute studies were performed on each species.
Bluegill (15 g) exposed to a nominal concentration of 0.5 mg/1
atrazine for 28 days in a flow-through test became lethargic,
fed poorly, and exhibited partial loss of equilibrium. Toxicity
was dose-related since effects occurred earlier and were more
severe at higher concentrations and no effects were observed at
lower concentrations nor in the controls. We observed 25 percent
mortality among fathead minnow fry (approximately 3-5 day old)
statically exposed to a measured concentration of 0.52 mg/1
atrazine for 96 hours. Mortality was dose-related with more
occurring at higher concentrations and none at lower ones or in
the controls. Based on these data, the highest nominal concentra-
tions selected for chronic exposures were 0.1 mg/1 (bluegill),
0.25 mg/1 (fathead minnow) and 1.0 mg/1 (brook trout).
20
-------
WATER CHEMISTRY
The results of the chemical analyses of water samples taken
during the various chronic exposures indicate that hardness,
alkalinity, acidity, pH and dissolved oxygen varied minimally
within any one chronic exposure. Statistical analysis showed
no significant differences for any of the a"bove parameters
between treatments within a chronic; therefore, only means
and ranges for the various parameters are presented (Table 4).
The results of gas chromatographic analyses of water samples
taken periodically during chronic exposure of the fishes and
aquatic invertebrates to atrazine indicated that mean measured
atrazine concentrations closely approximated nominal concen-
trations (Table 5).
CHRONIC EXPOSURE
Chironomus tentans
The continuous exposure of first generation chironomid eggs to
mean measured concentrations of atrazine as high as 1.33 mg/1
had no significant effect on hatchability (Table 6). However,
about 6o>% mortality was observed among first ins tar larvae
developing from eggs exposed to 1.33 rog/1 atrazine. Further-
more those first instar larvae surviving exposure to this
concentration of atrazine never developed beyond late first
instar-early second instar during 37 days exposure. Larvae
hatched from eggs exposed to a mean measured atrazine concen-
tration of 0.?8 mg/1 did not experience significant first
instar larval mortality but the majority of these larvae never
developed beyond late second instar-early third instar.
Larvae that experienced developmental retardation when com-
pared to controls were considered affected by chemical exposure,
and no assessment of the potential for emergence of these
retarded forms was made. All data regarding pupation and
emergence represent comparable data for all groups during 37
days exposure since pupation and emergence were completed
among control organisms within that time. Based on these
criteria and an analysis of variance of comparable data, we
observed a significant reduction in the number of adults
emerging among those groups exposed to mean measured atrazine
concentrations of 0.42 and 0.23 mg/1. No significant differ-
ences in hatchability, survival, pupation and emergence were
observed between midges exposed to a mean measured atrazine
concentration of 0.12 mg/1 and controls during the first
generation exposure (Table 6).
21
-------
to
TABLE 4, MEAN
DISS(
DURI
•••••l^^^^^^^^^^^^^^^^^™^™
Species
Chironomus
tentans
Mean 1 S.D,
Range
# of Samples
Daphnia
magna
Mean ± S.D.
Range
# of Samples
Gammarus
fasciatus
Mean ± S.D.
Range
# of Samples
Lepomis
macrochirus
Mean 1 S.D.
Range
# of Samples
Pimephales
promelas
Mean ± S.D.
Range
# of samples
Salvelinus
fontinalis
Mean ± S.D.
Range
# of Samples
AND RANGE OF MEASURED CONCENTRATIONS OP HARDNESS,. ALKALINITY, ACIDITY,
)LVED OXYGEN AND pH (RANGE ONLY) FROM WATER SAMPLES TAKEN PERIODICALLY
G CHRONIC EXPOSURE OF AQUATIC INVERTEBRATES AND FISHES TO ATRAZINE
Hardness
(mg/1)
'Ws!
5
32.2±0.81
(27-35)
18
34.710.53
(29-39)
42
33.9±^.5
(25-^1)
20
36.2±2.3
(33-40)
13
35.7±3.^
(30-43)
16
Alkalinity
(mg/1)
39.0±2.5
(36-4o1
5
30.3±1.0
(24-35)
18
32.810.75
(27-46)
42
32.7±3.7
(34-46)
20
34.6±2.7
(27-48)
!3
3ll'2m
16
Acidity
(mg/1)
4.8±1.3
(4.3-5.D
5
4.1±0.45
(1,9-6.0)
18
4.4+0.44
(1.9-9.6)
42
4.7±1.8
(2.9-9.0)
24
4.4±1.3
(2.9-6.7)
8
4.5±1.8
(1.9-7.7)
20
D.O,
(mg/1)
6.8±0.5
(5.8-8.2)
29
6.710.23
(6.4-7.3)
10
8.4-0.11
(6.0-9.9)
162
6.310.8
(3.6-9.D
1764
6.910.7
(3.4-10.2)
238
8.110.8
(5.9-H.2
1176
PH
(6.8-7.2
16
(6.3-7.5)
45
(6.4-7.2)
120
(6.3-7.4)
30
(6.5-7.3)
14
(6.4-7.4)
-------
TABLE 5. NOMINAL AND MEASURED ATRAZINE CONCENTRATIONS (mg/1)
IN WATER DURING CHRONIC EXPOSURE OF AQUATIC
INVERTEBRATES AND FISHES TO ATRAZINE
Species and
Nomin. Cone.
Chironorrais
tentans
2.00
1.00
0.50
0.25
0.12
Daphnia
magna
1.20
0.60
0.30
0.15
0.08
Gainmarus
fasciatus
1.00
0.50
0.25
0.12
0.06
Lepomis
macrochirus
0.100
0.050
0.025
0.012
0.006
Pimephales
0.250
0.125
0.062
0.031
0.015
Salve linus
fontinalis
1.00
0.05
0.25
0.12
0.06
Measured Concentration mg/
Mean S.D.
1.33 ± 0.930
0.78 ± 0.310
0.42 ± 0.170
0.23 ± 0.062
0.11 ± 0.025
1.15 ± 0.370
0.55 ± 0.122
0.25 ± 0.057
0.14± 0.036
0.06 ± 0.012
0.94 ± 0.167
0.49 ± 0.208
0.24 ± 0.069
0.14 ± 0.029
0.06 ± 0.011
0.095 ± 0.026
0.049 ± 0.011
0.025 ± 0.008
0.014 ± 0.004
0.008 ± 0.004
0.213 ± 0.075
0.112 ± 0.044
0.054 ± 0.017
0.033 ± 0.016
0.015 ± 0.008
0.72 ± 0.278
0.45 ± 0.156
0.24 ± 0.042
0.12 ± 0.039
0.065 ± 0.013
Range
0.69 - 3.3
0.24 - 2.8
0.13 - 0.63
0.15 - 0.33
0.082- 0.19
0.41 - 1.70
0.35 - 0.79
0.14 - 0.35
0.08 - 0.21
0.05 - 0.09
0.50 - 1.30
0.29 - 0.80
0.16 - 0.53
0.08 - 0.21
0.03 - 0.08
0.034 - 0.17
0.031 - 0.084
0.011 - 0.053
0.005 - 0.031
0.004 - 0.022
0.11 - 0.41
0.05 - 0.23
0.031 - 0.084
0.011 - 0.057
0.004 - 0.045
0.18 - 1.10
0.12 - 0.66
0.17 - 0.32
0.03 - 0.20
0.021- 0.081
iter
of Samples
20
20
19
20
20
21
21
18
15
16
40
40
38
40
35
106
63
58
58
57
« p
35
31
28
35
30
/ /-
46
28
23
28
21
23
-------
TABLE 6. SUMMARY OF THE EFFECT OF VARIOUS CONCENTRATIONS OF
ATRAZINE ON HATCHABILITY, PUPATION AND EMERGENCE
OF Chironomus tentans CONTINUOUSLY EXPOSED FOR
TWO GENERATIONS
Mean measured
atrazine cone.
(mg/1)
Control
0.12
0.23
0.42
0.78
1.33
Rep
A
B
A
B
A
B
A
B
A
B
A
B
Generation
I
%Ea.tch Pupa Adult
91
93
90
86
90
91
86
90
86
84
92
89
95
91
83
75
63
31
0
oa
°b
0D
89
93
90
80
70
59
30
26
-
_
-
—
Generation
foEatch
90
83
95
90
89
93
70
63
-
_
-
••
II
Pupa Adult
87
83
93
81
71
69
11
29
-
_
-
••
87
80
89
90
65
56
10
26
-
—
-
—
aOnly late second-instar larvae present after 37 days
continuous exposure.
bOnly late first-instar larvae present after 38 days
continuous exposure.
Egg masses for the second generation exposure were obtained
from those groups where first generation adults emerged
during the same period as the controls (day 33-36 of exposure)
Continuous exposure of second generation eggs to 0.42 mg/1 of
atrazine significantly reduced hatchability of eggs. Reduced
hatchability was not observed during the first generation
exposure to 0.42 mg/1 and is indicative of a cumulative
effect of atrazine on the midges during the exposure of
successive generations. As was observed during the-first
generation exposure, continuous exposure of midges to 0.42
and 0.23 mg/1 of atrazine significantly reduced the number
of midges pupating and emerging. The data do not indicate
a cumulative toxic effect on these processes due to exposure
to these two concentrations of atrazine.
The results of continuous exposure to CJ. tentans to mean
measured concentrations of atrazine of 0.23 mg/1 and greater
through two successive generations resulted in reduced
hatching success, increased larval mortality, developmental
retardation, or reduction in the number of organisms pupating
and emerging. Exposure to 0.11 mg/1 atrazine had no observ-
able effect on the growth and development of these organisms
24
-------
when compared to control groups. Based on these data, the
maximum acceptable toxicant concentration of atrazine for
C. tentans is estimated to be >0.11 and <0.23 mg/1.
Daphnia mgana
Statistical analysis of the mean percent survival of original
Daphnia from each of three successive generations indicated
that continuous exposure to mean measured concentrations of
atrazine as high as 1.15 mg/1 had no significant effect on
survival (Table 7)- Also, these data indicate that no
atrazine-induced cumulative effects on mortality were observed
in second or third generation daphnids.
TABLE 7. MEANa PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO ATRAZINE FOR 64 DAYS
Mean
cone
measured
. (mg/1)
Control
0.06
0.14
0.25
0.55
1.15
Generation
I
Day
8
89
81
84
78
68
88
15
61
72
75
75
5^
85
70
49
51
65
60
34
82
Generation
29
92
95
82
84
64
91
Day
38
88
92
79
82
56
89
II
43
84
91
78
80
52
71
Generation
III
Day
50
69
70
39
78
50
82
57
65
64
36
72
45
76
65
55
55
36
59
44
74
aEach value represents the mean of four replicates.
^Duration of exposure for generations, I. II and III were
days 1-22, 22-43, 43-64, respectively.
Analysis of variance indicated that the mean number of young
produced per female daphnid during the first generation was
significantly reduced by exposure to atrazine (Table 8).
Continuous exposure of the first generation Daphnia to mean
measured concentrations of 1.15. 0.55, and 0.25 mg/1 atrazine
significantly reduced the production of young. Although
reproduction among second and third generation daphnids exposed
to these concentrations was generally less than other groups,
variability in the data precluded ascribing statistical
significance to these observations.
We are, at this time, unable to explain the declining rate of
progeny production observed in every experimental group_
during the successive generation experiment. It is obviously
not toxicant related, but is apparently related to inadequacies
in the laboratory life-support systems.
25
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TABLE 8. MEANa PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO ATRAZINE FOR 64 DAYS
Mean measured
cone, (mg/1)
Control
0.06
0.14
0.25
0.55
1.15
Generati on I
Day
15 22
16.3 40.0
24.4 33.0
19.0 30.5
14.6 18.3
18.8 24.8
13.7 8.0
Generation II
Day
36 43
10.8 15.8
6.3 12.2
4.9 14.4
3.6 10.1
8.8 10 . 1
3.7 13-2
Generation III
Day
57 67
16.6 11.8
6.3 9.7
10.4 14.9
4.8 3.3
6.6 8.8
6.3 8.2
aEach value represents the mean of four replicates.
Duration of exposure for generations I, II, and III were days
1-22, 22-43, 43-64, respectively.
The results of this research indicate that reproduction of
Daphnia mgana is a much more sensitive measure of species
susceptibility to atrazine than is survival. Some of the
adult daphnids in each generation were males, and data on
reproduction was adjusted to represent that portion of the
population, in each experimental unit, directly involved in
production of progeny. The incidence of males induced from
parthenogenetic forms appeared to be higher in gr'oups exposed
to atrazine than in the control groups. Hutchinson (1967)
considered the presence of males in test populations a reac-
tion to an environmental stress, which atrazine may be in this
case.
Based on the statistical analysis of the survival and repro-
ductive success of daphnids continuously exposed to atrazine
for three successive generations, the maximum acceptable
toxicant concentration of this chemical for Daphnia magna is
estimated to be >0.l4 and <0.25 mg/1.
Gammarus fasciatus
Statistical analysis indicated that survival of original
gammarids continuously exposed to a mean measured concentra-
tion of 0.94 mg/1 atrazine for 30 days was significantly less
than all other experimental groups (Table 9). No significant
difference between survival of control groups and gammarids
continously exposed to 0.49 mg/1 for 119 days was observed.
26
-------
TABLE 9. PERCENT SURVIVAL AND REPRODUCTIVE SUCCESS OF Gammarus fasciatus EXPOSED TO
Adult Survival
(*)
Day 30
60
91
119
Total # young
Survival after
30 days W
Mean Measured Atrazine Concentration (mg/1)
0.94
A B
1*0 40
20 18
16 16
4 10
-
-
0.4-9
A B
58 60
30 42
24 32
24 14
18 1
22 0
0.24
A B
64 82
32 60
24 56
14 34
33 40
52 38
0.14
A B
56 66
26 44
16 34
12 32
39 43
82 65
0.06
A B
74 66
26 58
16 42
16 32
56 14
86 43
Control
A B
64 74
24 38
30 30
12 18
18
33
to
-------
Complete reproductive impairment of surviving adult gammarids
was observed among groups exposed to 0.94 mg/1 atrazine, and
the number of progeny produced per female appeared reduced
among groups exposed to 0.4-9 mg/1 atrazine. Additionally,
the survival during the first 30 days of development of
young produced by gammarids exposed to 0.4-9 and 0.24- mg/1
atrazine appeared to be less than that of young produced by
gammarids exposed to 0.14 and 0.06 mg/1 atrazine (Table 9).
The growth of second generation gammarids during the 30 day
period following shedding by the females was significantly
reduced by continuous exposure to concentrations of atrazine
as low as 0.14- mg/1. None of the young organisms exposed to
0.4-9 mg/1 atrazine developed beyond sixth ins tar stage
and half of them never developed beyond the fifth instar. Of
the young gammarids continuously exposed to 0.24 mg/1 and 0.14
mg/1 atrazine, only 75?£ developed to seventh instar, while
100# of the control organisms and 93^ of those exposed to
0.06 mg/1 developed to seventh instar.
The small number of young gammarids produced among control
groups was due to the fact that in these aquaria males
greatly outnumbered females. Consequently, we occasionally
observed moribund gravid females apparently due to continued
pursuit by various males and repeated copulation and separa-
tion. Also, nearly 6o# of the gravid females isolated during
the experiment failed to yield young gammarids.
Based on a statistical evaluation of the above data, and
consideration of the effects of continuous exposure to
atrazine on the survival and reproductive potential of adult
gammarids, and on the morphological development of second
generation gammarids, we estimate the maximum acceptable
toxicant concentration of atrazine for Gammarus fasciatus is
>0.06 and <0.l4 mg/1.
Lepomis macrochirus
Survival and growth of bluegill exposed to atrazine for 6
and 18 months was similar for all concentrations (Table 10).
Spawning activity occurred between days 594 and 716 of
exposure. Although spawning activity was too sporadic to be
conclusive, the fact that egg numbers in the highest concen-
tration (.095 mg/1) were comparable to the controls suggests
that atrazine had no effect on spawning within the range of
concentrations tested. An attempt was made to induce
further spawning from bluegill by administering interperitoneal
injections of carp pituitary extract on test day 716. All
28
-------
TABLE 10.
SURVIVAL AND GROWTH DURING EXPOSURES OF 6 AND 18 MONTHS, AND RESULTS OF
SPAWNING ACTIVITY OF BLUEGILLa(Lepomis macrochirus) CONTINUOUSLY EXPOSED
TO ATRAZINE
Item
6 MONTHS
Survival (%}
Total Length
(mm)
Total Weight (g)
18 MONTHS
o
Survival (%}
*/9
Total Length
(mm)
(f
?
Total Weight(g)
^
s
# of Spawnings
Eggs/ ?
Eggs/Spawn
Mean Measured Atrazine Concentration (mg/liter)
0.095
A B
100 85
124 129
35 4o
90 80
3/6 3/5
185 199
157 153
120 146
77 69
4 3
12920 2432
19380 4053
0.049
A B
95 80
127 128
40 40
90 100
3/6 5/5
200 193
155 153
165 14?
62 63
o 4
- 732?-
9164
0.025
A B
loo 95
126 128
37 38
100 90
3/7 2/7
172 179
158 166
102 112
73 86
8 0
5153
4509
0.014
A B
95 100
129 130
41 40
100 100
3/7 4/6
192 192
158 161
143 138
74 76
7 8
6703 8218
6703 6163
0.008
A B
95 85
130 126
41 37
loo 70
3/7 2/5
198 199
156 165
146 163
72 78
0 7
- 15254
- 10895
Control
A B
75
133 1
43
90 1
3/6 3,
182 2
160 1
122 1
76
36890 5
32668 20
to
co
95
41
72
2
initial length and weight 100 mm, 15 g.
bBased on 10 fish per aquarium after thinning on day 185 of exposure.
-------
fish were injected with 0.5-1.0 cc of a 200 [ig/100 ml
solution of carp pituitary in physiological saline. The
injections failed to induce the desired increase in spawning
and may have been administered too late in the spawning period
to be effective. Parental fish were sacrificed on test day
736 and samples of muscle were retained for residue analysis.
The percent of eggs successfully hatching among groups of
eggs exposed to each of the four highest concentrations of
atrazine tested was similar to that observed among control
groups and indicates that continuous exposure to mean
measured concentrations of atrazine as high as 0.095 nig/1
did not significantly affect hatchability of eggs (Table 11).
Survival of bluegill fry was significantly lower during 30
days exposure to 0.095 and 0.0^-9 mg/1 than to the other
three concentrations of atrazine, suggesting a toxicant
related effect. Unfortunately, survival of fry in control
groups was very low and it is impossible to confirm whether
fry survival was toxicant related. Survival of fry in all
groups during this period appeared to be related to an early
acceptance of the appropriate available food rather than
toxicant related. This interpretation is based on the
observation that survival in all groups was excellent during
the final 60 days of exposure when feeding habits became
well established and food was readily accepted.
Total lengths of bluegill fry at 30 days did not vary
significantly between controls and any concentration. Total
lengths at 60 and 90 days were significantly lower among
fish exposed to 0.0^9 mg/1 of atrazine, however these were
the only fry groups remaining in the experiment when a
heater malfunction caused a decrease in temperature in the
experimental system. The malfunction could not be corrected
soon enough to bring growth rate of these fish back to a
rate comparable with other groups. Total lengths of bluegill
fry in all other tanks were similar after 60 and 90 days
exposure, and we conclude that continuous exposure of
bluegill fry to atrazine concentrations as high as 0.095 mg/1
for 90 days had no significant effect on growth (Table 11).
Based on the data obtained from continuous exposure, it
appears that the maximum acceptable toxicant concentration is
greater than the highest measured atrazine concentration
TO.095 mg/1) to which bluegill were exposed during the chronic
test. Since we observed loss of equilibrium among bluegill
continuously exposed to 0.50 mg/1 atrazine for 28 days, the
maximum acceptable toxicant concentration of atrazine for
this species is estimated to be between 0.095 and 0.50 mg/1.
30
-------
TABLE 11.
HATCHABILITY, SURVIVAL, AND GROWTH OF BLUEGILL
(Lepomis macrochirus) FRY EXPOSED TO ATRAZINE
ITEM
Mean Measured Atrazine Concentration (mg/1)
0.095
HatchabilityW 91
# of egg groups 4
Survival ($)
30 days
60 days
90 days
Mean Total Len^
+ S.D.
30 days
60 days
90 days
# of fry
groups01
18
18
18
gth
20±2.9
30±3.1
40±5.9
1
0.049
90
3
22
20
20
17±1.8C
18±2 . 0
18±2 . 0
1
0.025
87
6
45
45
42
22±3.2
30±6.o
38±8.0
2
0.014
81
12
36
35
35
20±3 . 2
30+3.1
38±4.9
4
0.008
66
3
48
46
43
21±2.4
25±3-7
35±3-5
2
Control
84
7
22
22
22
18±2.0
29±3-l
40±7.2
2
Each group contained 200 one day old eggs.
Standard deviation.
cFry groups exposed to lower water temperature due to heater
malfuncti on.
Each group contained 50 one day old fry.
Pimephales promelas
Statistical analysis of data on survival and growth of
fathead minnows after 30 and 60 days exposure to atrazine
indicated that survival was significantly less and growth
significantly greater among controls than all treated
groups (Table 12). We believe these to be related
phenomena. Although no explanation for the lower survival
among controls exists, we suggest the greater growth among
fish remaining in these tanks is related to reduced
competition for food. This hypothesis is supported by the
observation that during the period of exposure from 9-43
weeks no significant differences in growth and survival
among any of the experimental groups were evident.
Spawning activity of fathead minnows in all experimental
groups occurred between days 164 and 280. Virtually no
spawning activity occurred among fathead minnows exposed to
0.033 mg/1 atrazine, and spawning among fish exposed to
0.213 mg/1 in replicate A was reduced (Table 13). These
were the only groups in which the number of males was equal
31
-------
TABLE 12. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) EXPOSED 30
DAYS, 60 DAYS, AND 43 WEEKS TO VARIOUS ATRAZINE CONCENTRATIONS
Item
?0 DAYS
Survival (7°)
Total Length
(mm)
(S.D.)
60 DAYS j.
Survival (fo)°
Total Length
(mm)
(S.D.)
43 WEEKS
Survival W
#rf removed
rf/? at term.
Total Length
(mm)
Total Weight
(S)
Mean Measured Atrazine Concentration mg/liter
0.213
A B
86 89
16 a 13
(3.9)a(2.1)
80 86
21 22
(3.2) (3.3)
93 6?
0 1
8/6 3/6
66 63
54 52
2.4 2.0
1.8 1.8
0.112
A B
86 86
16 14
(3.8) (3.5)
71 69
18 21
(3.9) (4.8]
87 67
0 1
3/10 3/6
66 70
51 54
2.7 3.3
l.o 1.5
0.054
A B
80 69
14 13
(3.2) (3.8)
69 66
19 20
(3.6) (4.0)
80 80
1 1
4/7 2/9
63 65
48 54
2.5 2.5
0.9 1.3
0.033
A B
100 97
14 13
(2.8)(3.5
90 70
20 21
(3.5M5.5:
87 80
2 3
5/6 4/5
64 69
57 53
2.4 2.8
1.5 1.0
0.015
A B
63 83
14 13
(4.5) (3.8)
57 67
19 21
(6.5) (5.D
80 87
0 3
4/8 4/6
62 69
50 55
1.8 3.0
1.3 1.3
Control
A B
60 47
20 19
(4.2) (3.9)
60 47
24 26
(4.2) (4.1)
93 93
0 2
5/9 2/9
64 60
52 55
2.4 2.0
1.1 1.3
W
to
aStandard Deviation,
^Survival based on 40 fish/duplicate tank.
cSurvival based on 15 fish/duplicate tank after
thinning on day 60 of exposure.
-------
CO
rpA-RTT? T* SPAWNING RESULTS, EGG HATCHABILITY, SURVIVAL AND GROWTH OF OFFSPRING
TABLE 13. DAWNING ^ULiS^UU £Ai ^^ ^^ (pimephales promelas) CONTINUOUSLY
EXPOSED TO ATRAZINE
Item
Spawnings/?
Eggs
spawned/ ?
Eggs/Spawn
% Hatcha
(N)
30 DAYS
Survival (%}
Length Xmm)
(S.D.)b
60 DAYS
Survival W
Length (mm)
(S.D.)
# fry groups
Mean Measured Atrazine Concentration
0.213
A B
1.6 7.8
133 1208
100 154
82 76
5 27
56 64
12 10
(2.3) (2.7!
45 28
17 21
,(2.9) (3.7)
' 1 3
0.112
A B
5.5 10.8
757 1255
138 116
77 81
22 27
68 73
11 10
(1.5) (2.5
57 69
17 16
(3-3) (4.5
2 4
0.054
A B
3.5 5.3
399 1160
133 217
74 87
8 23
65 60
13 12
(2.6) (2.3
48 55
20 19
(3.8) (3.6
2 2
0.033
A B
0.2 0
7.5 0
45 0
0 0
0 0
mg/liter
0.015
A B
4.3 7.7
450 862
120 112
76 84
11 22
50 69
11 11
(1.8) (2.71
25 67
15 18
(4.0) (4.4!
2 4
Control
A B
3.5 9.9
446 1284
143 130
83 74
19 37
62 89
9 10
(1.7) (2.1)
62 84
16 19
(4.0) (4.5)
3 2
aEach sample contained 50 one day old eggs.
Standard Deviation.
°Each group contained 40 one day old fry.
-------
to or greater than the number of females and suggests that
such sex ratios may encourage competition among males and
discourage spawning by females. With the exception of these
groups, there were no significant differences in the mean
number of eggs produced per female, and the mean number of
eggs per spawn indicating that continuous exposure to atrazine
had no effects on these parameters. Hatchability, survival
and growth of second generation fathead minnows through 30
and 60 days was similar among all treatments.
The available evidence, based on the chronic exposure of
fathead minnows to atrazine, indicates that the maximum
acceptable toxicant concentration is greater than 0.213 mg/1-
Since we observed 25$ mortality among 3 "to 5 day old fathead
minnow fry during 96 hours exposure to 0.8? mg/1 atrazine in
a static system, the estimated maximum acceptable toxicant
concentration of atrazine for the fathead minnow is
between 0.21 and 0.8? mg/1.
Salvelinus fontinalis
Continuous exposure for 44 weeks to mean measured atrazine
concentrations as high as 0.72 mg/1 had no significant effect
on survival of brook trout. Continuous exposure to 0.72,
0.45, and 0.24 mg/1 atrazine for 90 days significantly reduced
the weight and total length of brook trout when compared to
controls. Total lengths and weights of brook trout fry were
significantly reduced by 306 days exposure to concentrations
of 0.72, 0.45, 0.24 and 0.12 mg/1 atrazine (Table 14). Brook
trout in these tanks appeared lethargic when compared to fish
in the controls and those exposed to 0.065 mg/1 atrazine, and
did not feed as well.
Spawning activity of yearling brook trout in all experimental
units was delayed approximately one month due to a malfunction
in the lighting system which exposed fish in all tanks to dim
lighting during scheduled night hours. After correcting the
problem, spawning occurred in all tanks between days 222 and
302 of exposure. Total number of eggs spawned, number of eggs
per female, and percent fertilization and hatchability appeared
to be unaffected by exposure to all concentrations of atrazine.
Variability between replicate tanks of the same concentration
precluded ascribing statistical significance to values which
appeared reduced at higher atrazine concentrations (Table 15).
The percentage of eggs developing a neural keel after 15
days was extremely variable between replicate tanks of the
34
-------
TABLE 14. MEAN INCREASE IN LENGTH AND WEIGHT OF YEARLING BROOK TROUT (Salvelinus
fontinalis) DURING 90 AND 306 DAYS CONTINUOUS EXPOSURE TO ATRAZINE
Item
90 DAYS
Mean increase
in length
(mm)
Mean increase
in weight (g)
306 DAYS
Mean increase
in length
(mm)
Mean increase
in weight (g)
Mean Measured Atrazine Concentration mg/liter
0.72
A B
10 5
22 8
70 57
149 122
0.45
A B
10 16
20 36
51 70
100 157
0.24
^A B
12 16
24 39
54 64
106 155
0.12
A B
18 22
4o 46
66 78
153 180
0.065
A B
17 24
44 52
87 86
214 204
Control
A B
27 26
60 61
101 92
253 216
CO
-------
TABLE 15. RESULTS OF SPAWNING ACTIVITY OF YEARLING BROOK TROUT (Salvelinus
DURING CONTINUOUS EXPOSURE TO ATRAZINE ^axveiinue
Item
# V*
# spavming
# spawns/ 9
Total # eggs
spawned
# eggs
spawned/?
Neural keel
developed(^)
Incubation tim
(degree days)
Mean Hatchabil
(*)
(N)a
Mean Measured Atrazine Concentration mg/1
A B
2/4 2/4
4 2
2 2
1485 589
371 295
26 40
e
38?
ity
58 8
1 1
U.M-5
A B
2/4 3/3
4 2
1 2
531 814
133 40?
0 83
405
48
0 3
0.24
A B
2/4 1/3
4 3
2 3
1790 1710
448 570
74 63
432
67 26
5 2
0.12
A B
2/4 3/3
4 3
2 2
1606 1506
402 502
27 66
432
62 78
1 3
0.065
A B
2/4 2/4
4 4
2 2
1412 1782
353 446
65 50
450
25 34
2 2
Control
A B
3/3 2/4
2 4
2 3
1122 1492
561 373
70 41
441
65 37
2 1
Indicates the number of groups of 50
developed a neural keel.
eggs in which at least half successfully
-------
same treatment but indicated that about half of the eggs
spawned successfully developed to this stage. Incubation in
degree-days was significantly reduced for brook trout eggs
spawned and incubated in 0.72 and 0.45 mg/1 of atrazine.
Survival of brook trout fry after 30 days exposure was
similar for all treatments. After 60 and 90 days exposure,
survival of fry was reduced for brook trout exposed to
0.?2, 0.4-5 and 0.24 mg/1 of atrazine (Table 16).
As was observed during the first generation exposure, mean
length and weight of fry, including those from unexposed
parents, was significantly reduced after 90 days exposure
to 0.72, 0.45 and 0.24 mg/1 of atrazine.
As in parental fish, the lethargy and reduction in feeding
activity appeared to be the underlying causes for the
measured response.
Based on the available evidence relating to pesticide-induced
reduction in growth of yearling brook trout, we estimate that
the maximum acceptable toxicant concentration of atrazine for
brook trout is between 0.065 and 0.12 mg/1.
RESIDUE ANALYSIS
Three samples of muscle tissue from adult bluegill and brook
trout taken at the end of the exposure period were analyzed to
determine the concentration of atrazine residues in fishes
exposed to the highest concentration of atrazine in each chronic,
The small size of the individual fathead minnows prompted the
use of three samples of pooled eviscerated carcasses of
adults exposed to the highest concentration of atrazine. In
all cases the results indicated that the concentrations of
atrazine in tissues were below the minimum detectable limits.
Brook trout exposed to a mean measured concentration of 0.74
mg/1 atrazine for 44 weeks contained <0.20 mg/kg atrazine in
the muscle tissue. Bluegill exposed to 0.094 mg/1 for 78
weeks contained <0.20 mg/kg atrazine in the muscle. Fathead
minnows exposed to 0.21 mg/1 atrazine for 43 weeks contained
<1.7 mg/kg atrazine in the eviscerated carcass. These data
clearly indicate that the fishes tested do not bioconcentrate
atrazine significantly.
37
-------
TABLE 16. SURVIVAL AND GROWTH OF SECOND GENERATION BROOK TROUT (Salvelinus fontinalis)
DURING THE FIRST 90 DAYS DEVELOPMENT OF FRY CONTINUOUSLY EXPOSED TO ATRAZINE
1
Item
Mean Measured Atrazine Concentration mg/1
0.72
A B
a ID
No. groups 1 3
0.45
A B
lb 3
Survival W 1
30 days
60 days
90 days
Mean Lengt
(mm)
30 days
60 days
90 days
91 95 P-00 85
9 39
4 15
i
21 23
22 25
25 28
Mean Weight
(g)
90 days
0.10 0.11
68 2?
48 9
24 20
26 23
29 28
0.14 0.11
0.24
A B
2 2
86 92
72 16
32 8
21 20
24 22
27 25
0.11 0.10
0.12
A B
2 2
78 96
32 76
22 58
21 23
25 27
31 33
0.20 0.28
0.065
A B
2 2
84 84
66 52
46 38
22 21
26 24
34 32
0.30 0.22
Control
A B
2 2
96 88
80 74-
52 50
24 25
27 27
3^ 35
0.27 0.30
CO
00
a
Fry groups contained 25 one day old fry.
'Fry from unexposed parents.
-------
CALCULATION OF APPLICATION FACTORS
A summary of the estimated LC50 value, the maximum acceptable
toxicant concentration, and the application factor derived
therefrom for atrazine and all species studied is presented
(Table 1?). The MATC for each species except bluegill and
fathead minnows is estimated between the highest mean measured
concentration having no significant effect and the lowest
mean measured concentration significantly affecting the
organism during chronic exposure. The MATC for bluegill
and fathead minnows is estimated between the highest mean
measured concentration applied during chronic exposure and
the minimum concentration which produced harmful effects
during acute exposure. Application factors describing the
relationship between the acute and chronic toxicity of
atrazine for the species studied are calculated using
concentrations bracketing the MATC and the 48-hour LC50 for
invertebrates. Similar application factors are calculated
for fishes utilizing the values bracketing the MATC and the
incipient LC50 which Eaton (1970) suggests to be a better
measure of acute toxicity for this type of calculation.
Application factors are similar for five of the six species
tested. Application factors for the fishes are remarkably
similar. However, the factors calculated for one of the
invertebrates (midges) are an order of magnitude greater
than those calculated for the fishes, and the other two
invertebrates.
39
-------
TABLE 1?. SUMMARY OF CONCENTRATIONS OF ATRAZINE (mg/1)
PRODUCING ACUTE AND CHRONIC TOXICITY TO AQUATIC
SPECIES, AND CALCULATED APPLICATION FACTORS
DESCRIBING THE RELATIONSHIP BETWEEN ACUTE AND
CHRONIC TOXICITY (MATC/LC50)
Species
Chironomus
tentans
Daphnia
magna
Garomarus
fasciatus
Lepomis
macrochirus
Pimephales
promelas
Salvelinus
fontinalis
Common
Name
midge
water flea
scud
bluegill
fathead
minnow
brook
trout
LC50a
0.?2 ,
(0.36-1.4)°
6.9
(5.2-8.1)
5-7
(3-6-8.0)
6.7
(5.4-8.4)
15
(11-20)
4.9
(4.0-6.0)
MATC
>.1K.23
>.14<.25
>.06<.14
>.io<.5o
>.2K.52
>.06<.12
Limits on
application
factor
0.15&0.32
0.02&0.04
0.01&0.02
0.01&0.07
0.01&0.03
0.01&0.02
48-hour LC50 for invertebrates, incipient LC50 for fishes.
confidence interval.
40
-------
SECTION VI
DISCUSSION
The estimated 48-hour LC50 value of 6.9 mg/1 determined for
Daphnia is higher than the previously estimated value of
3.6 mg/1 (FWPCA, 1968). The difference may be attributable
to any number of variables (e.g. diluent water quality, age
of test organisms, temperature, etc.). No previous estimates
of the acute toxicity of atrazine to chironomids and
gammarids are available.
The 96-hour LC50 of atrazine to rainbow trout (Salmo gairdneri),
based on the results of a static bioassay, was previously
estimated to be 12.6 mg/1 (FWPCA, 1968). The difference
between this value and our 4.9 mg/1 estimated incipient LC50
for brook trout is probably due to species differences and/or
the fact that the rainbow trout were tested under static
conditions while the brook trout were tested in a dynamic
system. Our estimate of the acute LC50 of 6.7 mg/1 for
bluegill compares favorably with a previously estimated 96-
hour LC50 of about 6 mg/1, (i.e. 12 mg/1 50$ Wettable Powder
Formulation) reported by Walker (1964,
The results of these investigations indicated that the three
invertebrate species were very similar in their susceptibility
to chronic exposure to atrazine despite the fact that there
were order of magnitude differences in their susceptibility
to acute exposure. It is interesting to note that this
similarity existed despite the fact that the MATC for each
species was estimated on the basis of different criteria.
The most sensitive criterion for determining the effects of_
atrazine on these invertebrates was developmental retardation
for Chironomus, production of young for Daphnia, and survival
of young for Gammarus.
The fishes tested were not only similar to each other in
their susceptibility to chronic exposure to atrazine but also
were remarkably similar to the invertebrates studied. Only
one other study has been reported describing the susceptibility
of the above species to chronic exposure to a pesticide
(Macek et al., 1975). In that study, we found that although
the estimated MATC of lindane to the fishes tested were similar,
the MATC estimates for two of the three invertebrates tested
were significantly lower.
Except for Gammarus, little other previous information is
available to provide a basis for making generalizations
about the relative susceptibility of fishes and invertebrates
to chronic exposure to chemicals in water. However, much of
41
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the previously reported information leads one to the conclusion
that invertebrates generally are more susceptible to chronic
exposure than fishes. Pickering and Thatcher (1970) reported
the MATC of LAS for fathead minnows was >0.63 <1.2 mg/1,
while Arthur (1970) reported the MATC of LAS for Gammarus
•pseudolimnaeus to be >0.2 10.6 <18.6 p.g/1, while Arthur and Leonard (1970) reported
the MATC of copper in soft water for G. pseudolimnaeus was
>4.6 <8.0 indicating again that the invertebrate species may
be at least 2 times more susceptible than the minnow. More
recently, Arthur et al. (1973) have reported that gammarids
may be 3 times more susceptible to chronic exposure to NTA
than were fathead minnows.
Only one similar chronic toxicity study with bluegill has
been previously described (Eaton, 1970). In comparing the
data from our study with those previously reported, it
appears that the bluegill in our study were significantly
larger and older than those in Eaton's work. As a result,
when spawning did occur in our study, we observed more spawns
per female and greater egg production per female than reported
in the previous work. It is extremely unfortunate that the
factors influential in stimulating or inhibiting spawning
activity in our study could not be adequately identified.
The percent hatch of bluegill eggs which we observed
(generally >8Cf5) was comparable to that observed by Eaton
(generally>72$). As in our previous study with lindane
(Macek et al. 1975) we experienced difficulty in maintaining
and feeding bluegill fry during the initial 30-day developmental
period. We attribute this primarily to an inability to
provide adequate amounts of suitable food forms during the
various stages of fry development. Eaton (1970) experienced
the same difficulties as described above and suggested that
lack of suitable food was the primary factor responsible for
poor fry survival. Before additional effort is expended to
investigate chronic toxicity of chemicals to bluegill,
definitive information to determine what are suitable food
sources for developing bluegill fry, and how adequate numbers
of these can be effectively made available to the fry, must
be developed.
The results of our investigation of the chronic toxicity of
atrazine to fathead minnows, though not without its problems,
reinforces our opinion that the species provides the best
opportunity for evaluating chronic toxicity of chemicals to
freshwater fishes. Certainly more work has -been reported with
42
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this species than any other (Mount, 1968; Brungs, 1969? Mount
& Stephan, 1969; Hermanutz et al. 1973) and procedures for
utilizing this species in long-term laboratory assays are
well defined. The results of our fathead minnow chronic
study in terms of performance of the test organisms are
excellent and indicate again that subtle long-term effects of
chemicals can be evaluated using this technique.
The number of spawnings per fathead minnow female, the number
of eggs per spawn, the percent hatchability, and the survival
of fry during the first 60 days development are as good or
better than that generally observed in previous studies.
In view of these facts, and the fact that the fathead minnow
procedure represents the only "true chronic bioassay" of the
three species tested (i.e. at least one complete life cycle)
we feel strongly that fathead minnows should be one of the
freshwater fish species of choice until methods suitable for
using other species are sufficiently defined.
We feel the brook trout offers significant potential for
use in "partial chronic bioassays". Generally the numbers
of fish participating in spawning and the number of eggs
spawned were adequate. Although admittedly variable, the
percent fertilization of eggs, the percent hatch and survival
through the first 30 days (to swim-up) were generally good.
We feel that valid information can be generated utilizing
this species and these procedures. However, obvious critical
areas in the performance of such assays exist. The accidental
exposure of parent brook trout to increased day length at a
time which coincided with the period of late garnetogenesis
and the onset of spawning activity verified Henderson*s (1963)
observation that functional maturity in brook trout is delayed
or inhibited by increasing day length. Additionally we
suggest that separate systems, free of light, be provided
for egg incubation as Letritz (1959) has suggested that both
trout and salmon eggs are sensitive to direct light.
We also suspect that the decreased survival of brook trout
fry observed in all groups during the period 30-90 days
after hatch may be related to the stress associated with
handling fry at 30 and 60 days to count and measure them.
We suggest that a single measurement after 60 days exposure
would provide the same useful information regarding
potential effects of chemicals on second generation trout
yet would minimize possible adverse effects of handling.
We have recently conducted several similar studies with
brook trout eggs and fry and achieved excellent success
in hatching and fry survival by shielding the developing
43
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eggs from light and minimizing handling of fry. We have
observed that continuous exposure of developing brook trout
eggs to 0.72 and 0.^5 mg/1 atrazine significantly reduced
incubation time (degree-days). A similar phenomenon has
recently been reported by Halter and Johnson (197*0. These
investigators observed that exposure of coho salmon to
Aroclor 125^- resulted in premature hatching of eggs.
The results of the analysis of fish tissue samples to
determine the concentration of atrazine residues showed
that residues in fish tissues were below minimum detectable
limits in all cases. Certainly such findings preclude
concern over bioconcentration of atrazine by fishes, a
subject of considerable concern for other types of pesti-
cides. These data are, in fact, consistent with other
studies that we have conducted which suggest that biocon-
centration of triazine herbicides by fish is relatively
low (BionomicSfunpublished data).
During the last two years, we have had the opportunity to
investigate the bioconcentration of some 50 pesticides by
bluegill during a minimum of 30 days continuous exposure
(Bionomics, unpublished data). With all but three of the
pesticides studied, we observed bioconcentration factors of
at least 10X, and with more than half of those studied we
observed bioconcentration factors >50X. The absence of
detectable concentrations of atrazine residues from our
experiments suggest that fishes do not bio concentrate
atrazine to the extent they do many pesticides. Certainly,
bioconcentration of atrazine does not appear as important
a phenomenon as it appears to be for certain chemicals
which have recently been the cause of great concern among
regulatory agencies and environmentalists. For example,
Macek and Korn (1970) reported brook trout continuously
exposed to 3 ng/1 DDT for 120 days concentrated the chemical
approximately 8500X. Dr. R. Reinert, U.S.D.I., Great Lakes
Research Lab., Ann Arbor, Michigan has found that rainbow
trout bi©concentrate methylmercury by as much as 8000X
(personal communication). Hansen et al. (1971) reported
marine fish exposed to polychlorinated biphenyls concentrated
the material 30,OOOX. Mayer et al. (1975) reported brook
trout exposed continuously to toxaphene concentrate that
chemical 5,000-16,OOOX.
The value of estimating the MATC for a chemical and an
aquatic organism is clearly demonstrated in the case of
atrazine. All of the previously reported acute toxicity
44
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information suggested response to atrazine exposure in the
range of 5-10 mg/1 (Walker, 1964; FWPCA, 1968; Sanders,
1969). Based on field observation Walker (1964) suggested
that atrazine concentrations of 0.5-1.0 mg/1 had no effect
on fishes and only temporarily affected benthic forms.
Hiltibrand (196?) suggested that 10 mg/1 atrazine had no
discernible effect on bluegill and green sunfish fry during
8 days post-hatch exposure. All of these data suggest safe
concentrations of atrazine generally an order of magnitude
greater than the estimated MATC values for the six species
tested in chronic studies. Although the time and effort is
considerably greater in developing and estimating MATC
values for chemicals based on chronic exposure, it appears
to be the only currently available method for generating the
information required to establish meaningful and realistic
water quality criteria.
Mount and Stephan (196?) have reported the utilization of
application factors to estimate chronic toxicity of
chemicals to fish based on acute toxicity data, and consider-
able information based on the chronic exposure of a variety
of fishes to pesticides and heavy metals supports this
hypothesis. The similarity of the limits of the application
factors for each of the fishes and two of the three
invertebrates tested with atrazine lends confidence to the
general validity of the application factor concept.
45
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SECTION VII
REFERENCES
American Public Health Association, 1971. Standard Methods
for the Examination of Water and Wastewater, 13th ed.
Am. Public Health Assoc. , New York, 874 p.
Arthur, J. W. , 1970. Chronic effects of linear alkylate
sulfonate detergent on Gammarus pseudolimnaeus ,
Campeloma decisum and Physa Integra. Water Research
4:251-257.
Arthur, J. W. , A. E. Lemke, V. R. Mattson and B. J. Halligan,
1973. Toxicity of sodium nitrilo triacetate (NTA) to the
fathead minnow and amphipod in soft water. Water Research
8:187-193.
Arthur. J. W, and E. N. Leonard, 1970. Effects of copper on
Gammarus pseudolimnaeus , Physa integra and Campeloma
decisum "in soft water. J. Fish. Res. Bd. Canada 27:
1277-1283.
Benoit, D. A. , 1974. Artificial laboratory spawning substrate
for brook trout (Salvelinus f ontinalis , Mitchill).
Trans. Am. Fish. Soc. 103:144-145.
Biesinger, K. E. and G. M. Christensen, 1972. Effects of
various metals on survival, growth, reproduction, and
metabolism of Daphnia magna. J. Fish. Res. Bd. Canada
29:1691-1700.
Bioassay Committee, 1971a. Recommended bioassay procedures
for fathead minnow (Pimephales promelas, Rafinesque)
chronic tests. National Water Quality Laboratory, Duluth,
Minnesota, 13 p.
Bioassay Committee, 1971b. Recommended bioassay procedures
for bluegill (Lepomis macrochirus , Rafinesque) partial
chronic tests. National Water Quality Laboratory, Duluth,
Minnesota. 12 p.
Bioassay Committee, 1971c. Recommended bioassay procedures
for brook trout (Salvelinus f ontinalis , Mitchill) partial
chronic tests. National Water Quality Laboratory, Duluth,
Minnesota, 12 p.
Brungs, W. A., 1969. Chronic toxicity of zinc to the fathead
minnow Pin
Fish. Soc,
46
minnow Pimephales promelas, Rafinesque. Trans. Am.
3oc. 98:272-2791
-------
Clemens, H. P., 1950. Life cycle and ecology of Garmnarus
fasciatus, Say. Ohio State Univ., Franz TheoTStone
Inst.Hydrobio-Contrib. 12. 63 p.
Cope, 0. B., 1966. Contamination of the freshwater
ecosystem by pesticides. J. Appl. Ecol. 3 (Suppl.):
33-44.
Deuel, C. R., D. C. Haskell, D. R. Brockway and 0. R. Kingsbury,
1952. New York State fish hatchery feeding chart, N.Y.
State Conserv. Dept. Fish. Res. Bull. No. 3. 26 p.
Drummond, R. A. and W. F. Daws on, 1970. An inexpensive
method for simulating diel patterns of lighting in the
laboratory. Trans. Am. Fish. Soc., 99:434-435.
Duncan, D. B., 1955. Multiple range and multple F-tests.
Biometrics 11:1-42.
Eaton, J. G., 1970. Chronic malathion toxicity to the
bluegill (Lepomis macrochirus, Rafinesque). Water
Research 4:b/3-b«4.
Eisler, R., 1973. Annotated bibliography on biological
effects of metals in aquatic environments. EPA,
Ecol. Res. Ser0 EPA-R3-73-007, Washington, D.C., 287 p.
FWPCA, 1968. Water Quality Criteria. Report of the National
Tech. Adm. Cotran. to Seer, of the Interior Fed. Water
Poll. Contr. Adm. U.S.D.I., 234 p.
Hall, J. K., M. Pawlus and E. R. Higgins, 1972. Losses of
atrazine in run-off water and soil sediment. J.
Environ. Qual. 1:172-176.
Halter, M. T. and H. E. Johnson, 1974. Acute toxicities of
polychlorinated biphenyl (PCB) and DDT alone and in
combination to early life stages of coho salmon
(Oncorhyncus kisutch). J. Fish. Res. Bd. Canada
31:1543-1547.
Hansen, D. J., P. R. Parrish, J. I. Lowe. A. J. Wilson, Jr.
and P. D. Wilson, 1971. Chronic toxicity, uptake and
retention of Aroclor 1254 in two estuarine fishes.
Bull. Environ. Contam. Toxicol. 6:113-119.
Henderson, N. E., 1963. Influence of light and temperature
on the reproductive cycle of the eastern brook trout,
Salveliiius fontinalis. J. Fish. Res. Bd. Canada
20:859-897.
47
-------
Hermanutz, R. 0., L. H. Mueller amd K. D. Kempfert, 1973.
Cap tan toxicity to fathead minnows (Pimephales
promelas), bluegills (Lepomis macrochirusj and brook
trout (Salvelinus fontinalisj.J. Fish. Res. Bd.
Canada 30:1811-1817;
Hesselberg, R. J. and J. L. Johnson, 1972. Column extraction
of pesticide from fish food and mud. Bull. Environ.
Contam. Toxicol. 7:115-120.
Hiltibrand, R. C., 1967. Effects of some herbicides on
fertilized fish eggs and fry. Trans. Am. Fish. Soc.
96:414-416.
Hutchinson, G. E., 1967. A Treatise on Limnology, Vol. 2
John Wiley & Sons, New York, 1115 p.
Johnson, D. W., 1968. Pesticides and fishes - A review of
selected literature. Trans. Am. Fish. Soc. 97:398-424.
Leitritz, E., 1959. Trout and salmon culture. State of
California, Dept. Fish & Game, Fish Bull. No. 107,
169 p.
Macek, K. J. and S. Korn, 1970. Significance of the food
chain in DDT accumulation by fish. J. Fish. Res. Bd.
Canada 27:1496-1498.
Macek, K. J., K. S. Buxton, S. K. Derr, J. W. Dean and S. Sauter,
1975. Chronic toxicity of lindane to selected aquatic
invertebrates and fishes. EPA, Ecol. Res. Series
(In Press).
Mayer, R. L., P. M. Merhle and W. P. Dwyer, 1975. Toxaphene
effects on reproduction, growth and mortality of brook
trout. EPA, Ecol. Res. Series (In Press).
McComish, T. S., 1968. Sexual differentiation of bluegills
by the urogenital opening. Prog. Fish. Cult. 29:28
McKim, J. M. and D. A. Benoit, 1971. Effects of long-term
exposures to copper on survival, growth, and reproduction
of brook trout (Salvelinus fontinalis). J. Fish. Res.
Bd. Canada 28:655-662.
Mount, D. I., 1968. Chronic toxicity of copper to fathead
minnows (Pimephales promelas, Rafinesque). Water
Research 2:215-223.
48
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Mount, D. I. and W. A. Bnings, 1967. A simplified dosing
apparatus for fish toxicological studies. Water Research
1:21-29.
Mount, D. I. and C. E. Stephen, 1967. A method for establishing
acceptable toxicant limits for fish-malathion and the
butoxyethanol ester of 2,4-D. Trans. Am. Fish. Soc.
96:185-193.
Mount, D. I. and C. E. Stephan, 1969. Chronic toxicity of
copper to the fathead minnow (Pimephales promelas; in
soft water. J. Fish. Res. Bd. Canada 25:2449-2457.
Pickering, Q. H. and T. 0. Thatcher, 1970. The chronic toxicity
of linear alkylate sulfonate (LAS) to Pimephales
promelas, Rafinesque. J. Water Pollut. Control Fed. 42:
243-254.
Sanders, H. 0., 1969. Toxicity of pesticides to the crustacean
Gammarus lacustris. U.S. Bur. Sport. Fish Wild, Tech. Rep.
25. 18 p.
Steel, R. G. D. and J. H. Torrie, 1960. Principles and procedures
of statistics. McGraw Hill, New York. 481 p.
Walker, C. A., 1964. Simazine and other s-triazine compounds
on aquatic herbicides in fish habitats. Weeds 12:134-139.
49
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-047
3. RECIPIENT'S ACCESSIOf+NO.
4. TITLE AND SUBTITLE
Chronic Toxicity of Atrazine to Selected Aquatic
Invertebrates and Fishes
5. REPORT DATE
May 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kenneth J. Macek, Kenneth S. Buxton, Scott Sauter,
Sarah Gnilka, Jerry W. Dean
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Bionomics
EG & 6 Inc.
790 Main Street
Wareham, Massachusetts 02571
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
68-01-0092 and 68-01-1844
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Duluth
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/ORD
5. SUPPLEMENTARY NOTES
6. ABSTRACT
Representatives of the aquatic invertebrate species of water flea (Daphnia
magna), midge (Chironomus tentans),' and scud (Gammarus fasciatus); and the fish
species bluegill (Lepomis macrochirus), fathead minnow (Pimephales promelas),
and brook trout (Salvelinus fontinalis) were chronically exposed to various
concentrations of atrazine in separate flowing-water systems.
Maximum acceptable toxicant concentrations (MATC) of atrazine for the
selected species in soft water were estimated using survival, growth, and
reproduction as indicators of toxic effects. The MATC was estimated to be between
0.11 and 0.23 mg/1 for midges, between 0.14 and 0.25 mg/1 for water fleas, and
between 0.06 and 0.14 for the scud. For fishes the MATC was estimated to be
between 0.09 and 0.50 mg/1 for bluegills, between 0.21 and 0.52 mg/1 for fathead
minnows, and between 0.06 and 0.12 mg/1 for brook trout. The incipient-LC50 for
fishes and the 48-hour LC50 for invertebrates was estimated from acute exposures
and was used to calculate application factors (MATC/LC50). For aquatic invertebrate
and atrazine the estimated application factors were between 0.15 and 0.32 for
midges, between 0.02 and 0.04 for water flea, and between 0.01 and 0.02 for scud.
Application factors were estimated between 0.01 and 0.07 for bluegills, between
0.01 and 0.03 for fathead minnows, and between 0.01 and 0.02 for brook trout.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Toxicity
Fishes
Invertebrates
Pesticides
Chronic toxicity
Tissue residues
Flowing water system
Application factors
Atrazine
06T
8. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (This Report I
Unclassified
21. NO. OF PAGES
58
20. SE
22. PRICE
EPA Form 2220-1 (9-73)
50
^- U.S. GOVEMMENT HUNTING OFFICE: 1976-657-695/5'i'tI Region No. 5-11
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