EPA-600/3-76-099
November 1976
Ecological Research Series
TOXICITY OF FOUR PESTICIDES TO
WATER FLEAS AND FATHEAD MINNOWS
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
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-099
November 1976
TOXICITY OF FOUR PESTICIDES TO WATER FLEAS
AND FATHEAD MINNOWS
Acute and Chronic Toxicity of Acrolein, Heptachlor,
Endosulfan, and Trifluralin to the Water Flea (Daphnia magna)
and the Fathead Minnow (Pimephales promelas)
by
Kenneth J. Macek, Mark A. Lindberg, Scott Sauter,
Kenneth S. Buxton, Patricia A. Costa
E G & G, Bionomics
Wareham, Massachusetts 02571
Contract 68-01-0738
Project Officer
A. Jarvinen
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH MJtfNESftP* 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.
11
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FOREWORD
Our nation's freshwaters are vital for all animals and plants,
yet our diverse uses of water for recreation, food, energy,
transportation, and industry physically and chemically alter
lakes, rivers, and stream. Such alterations threaten terrestrial
organisms,as well as those living in water. The Environmental
Research Laboratory in Duluth, Minnesota develops methods, conducts
laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution
affects aquatic life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes
through use of models
—to measure bioaccumulation of pollutants in aquatic
organisms that are consumed by other animals, including
man
This report determines the effect of four pesticides on two species
of aquatic organisms.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
111
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ABSTRACT
Water fleas (Daphnia magna) and fathead minnows (Pimephales
promelas) were chronically exposed to various concentrations
of acrolein, heptachlor, endosulfan and trifluralin in
separate flowing water systems. Exposures were through
two complete life cycles for Daphnia and through at least
one complete life cycle for fathead minnows.
Maximum acceptable toxicant concentrations (MATC's) for each
pesticide for both species were estimated by measuring
survival, growth and reproduction success as indicators of
toxic effects. The MATC for acrolein was estimated to be
>16.9 and <33.6 pg/1 for daphnids and >11.4 and <41.7 for
fathead minnows, those estimated for heptachlor were
>12.5 and <25.0 pg/1 for daphnids and >0.86 and <1.84 pg/1
for fathead minnows, those estimated for endosulfan were
>2.7 and <7.0 pg/1 for daphnids and >0.2 and <0.4 pg/1 for
fathead minnows, and those estimated for trifluralin were
>2.4 and <7.2 jug/1 for daphnids and >1.9 and <5.1 pg/1 for
fathead minnows.
This report was submitted in fulfillment of Contract No.
68-01-0738 by E G & G, Bionomics, under the sponsorship
of the Environmental Protection Agency. Work was completed
in December, 1974.
IV
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CONTENTS
SECTION PAGE
I Introduction 1
II Conclusions 5
III Recommendations 6
IV Materials and Methods 8
Exposure Systems 8
Acute Toxicity Procedures 10
Chemical Methodology 11
Statistics 21
Chronic Exposure 21
Daphnia magna 21
PimephaJ.es promelas 22
V Results 24
Acute Bioassays 24
Water Chemistry 24
Chronic Exposure 29
Daphnia magna to acrolein 29
Daphnia magna to heptachlor 29
Daphnia magna to endosulfan 32
Daphnia magna to trifluralin 33
Pimepha'les promelas to acrolein 35
Pimephales promelas to heptachlor 38
Pimephales promelas to endosulfan 38
Pimephales promelas to trifluralin 42
Residue Analysis 45
Calculation of Application Factors 48
VI Discussion 50
VII References 53
v
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TABLES
NO. PAGE
1 Chemical analysis of the diluent water utilized
during chronic exposures of Daphnia magna and
fathead minnows to acrolein, neptacnior,endosulfan
and trifluralin. 9
2 Recovery of heptachlor and heptachlor epoxide from
quality control water samples. 15
3 Recovery of heptachlor and heptachlor epoxide from
quality control tissue samples. 17
4 Recovery of endosulfan from quality control water
samples. 19
5 Recovery of trifluralin from quality control
water samples. 20
6 Recovery of trifluralin from quality control tissue
samples. 21
7 Mean and range of measured concentrations of
hardness, alkalinity, acidity, dissolved oxygen and
pH from water samples taken periodically during
chronic exposure of Daphnia magna to four pesticides. 25
8 Mean and range of measured concentrations of
hardness, alkalinity, acidity, dissolved oxygen and
pH from water samples taken periodically during
chronic exposure of fathead minnows (Pimephales
promelas) to four pesticides. 26
9 Nominal and mean measured concentrations of acrolein,
endosulfan and trifluralin in water during
continuous exposure of Daphnia magna. 27
10 Nominal and mean measured concentrations of acrolein,
heptachlor, endosulfan and trifluralin during
chronic exposure of fathead minnows (Pimephales
promelas).
11 Mean percent survival of Daphnia magna continuously
exposed to acrolein for 64 days. 30
12 Mean production of young per female Daphnia magna
continuously exposed to acrolein for 64 days. 30
13 Mean percent survival of Daphnia magna continuously
exposed to heptachlor for 64 days. 31
vi
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TABLES
NO. PAGE
14 Mean production of young per female Daphnia magna
continuously exposed to heptachlor for 64 days. 31
15 Mean percent survival of Daphnia magna continuously
exposed to endosulfan for 64 days. 32
16 Mean production of young per female Daphnia magna
continuously exposed to endosulfan for 64 days. 33
17 Mean percent survival of Daphnia magna continuously
exposed to trifluralin for 64 days. 34
18 Mean production of young per female Daphnia magna
continuously exposed to trifluralin for 64 days. 34
19 Survival and growth of fathead minnows (Pimephales
promelas) after 30 and 60 days, and 35 weeks
continuous exposure to acrolein. 26
20 Sexual development, spawning, hatchability of eggs,
and survival and growth of offspring after 30 and
60 days, for fathead minnows (Pimephales promelas)
continuously exposed to acrolein. 37
21 Survival and growth of fathead minnows (Pimephales
promelas) after 30 days, 60 days and 40 weeks
continuous exposure to heptachlor. 39
22 Sexual development, spawning, hatchability of eggs,
and survival and growth of offspring after 30 and
60 days, for fathead minnows (Pimephales promelas)
continuously exposed to heptachlor. 40
23 Survival and growth of fathead minnows (Pimephales
promelas) after 30 days, 60 days and 40 weeks
continuous exposure to endosulfan. 4^
24 Sexual development, spawning, hatchability of eggs,
and survival and growth of offspring after 30 and
60 days, for fathead minnows (Pimephales promelas)
continuously exposed to endosulfan. 43
25 Survival and growth of fathead minnows (Pimephales
promelas) after 30 days, 60 days and 61 weeks
continuous exposure to trifluralin.
vn
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TABLES
No. PAGE
26 Spawning results, egg hatchability, and survival and
growth of fathead minnow (Pimephales promelas)
fry after 30 and 60 days exposure to various
concentrations of trifluralin. 46
27 Mean measured concentrations of heptachlor (+
epoxide) and trifluralin in water (ug/1) and in
the eviscerated carcass (/ug/kg) of fathead minnows
(Pimephales promelas) continuously exposed to the
pesticides in separate systems. 47
28 Acute and chronic toxicity values (ug/1) for the water
flea (Daphnia magna) and calculated application
factors for each pesticide. 48
29 Acute and chronic toxicity values (pg/1) for the
fathead minnow (Pimephales promelas) and calculated
application factors for each pesticide. 49
Vlll
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ACKNOWLEDGEMENTS
During the period of performance of this research effort
we were fortunate to have laboratory assistance from Steve
Ells and Emily Dionne who maintained exposure systems and
were significantly involved with various aspects of fish
chronics, from Gerry LeBlanc who provided expertise
in performing the invertebrate chronics, and from Ronni
Krasny and Joanna Enos who performed chemical analyses,
and to Dr. Sam R. Petrocelli for contructively reviewing
the final report.
Finally, our sincere appreciation is extended to Mr.
Alfred Jarvinen, Project Officer, Environmental Protection
Agency, National Water Quality Laboratory, Duluth, Minnesota
for his guidance, patience, and contractual advice during
the performance of these studies.
IX
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SECTION I
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 biological effect of chemicals. 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., 1976a,
1976b). The "Laboratory Fish Production Index (LFPI)" as
defined by Mount and Stephan (1967) reflects toxic effects
on reproduction, growth, spawning behavior, egg mortality,
and fry survival. The highest observed toxicant concentration
that has no effect on these parameters during continuous
chronic exposure is termed the maximum acceptable toxicant
concentration (MATC).
Based on available information on occurrence and persistence
in the environment, relative toxicity to fishes and aquatic
invertebrates, current uses and use patterns, and/or total
amount of chemical applied annually, four pesticides were
selected for intensive investigation to evaluate the potential
chronic effects of these chemicals on aquatic organisms.
Acrolein (acrylic aldehyde) is a herbicide used in bodies of
water such as lakes, rivers, canals and irrigation ditches
for controlling submerged vegetation and floating plants
(Overbeck, 1962). As an example of acrolein's volatility,
Battelle (1970) reported that a concentration of 0.7 mg/1
of this herbicide in the water of treated irrigation ditches
flowing at a rate of approximately 130 cubic feet per second
decreased by 98% over a 19 mile stretch at a temperature of
64°F. At a lower and less typical temperature of 48°F, the
loss was only 62% over a distance of 27 miles. Burdick et
al. (1964) investigated the toxicity of acrolein to brown
trout and bluegills by a procedure approximating the concentration
to be expected from a single field application under certain
conditions. They reported the 24-hour LC50 of acrolein for
trout and bluegills to be 46 and 75 >ig/l, respectively.
They also observed that the threshold for mortality of both
species was below the concentration recommended for controlling
aquatic vegetation. Louder and McCoy (1962) reported that
24-hour LC50 values for acrolein and largemouth bass, bluegills,
bowfin, mosquito fish and fathead minnows ranged from 62-
183 jig/1. Little additional information on the effects of
acrolein on aquatic organisms is available.
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Heptachlor (1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-tetrahydro-
4,7-endo-methanoindene) is an organochlorine insecticide which
has been reported to be widely distributed in both terrestial
and aquatic environments. The occurrence, translocation,
persistence and degradation of heptachlor in soils and crops
have been adequately documented (Bruce and Decker, 1966;
King et al., 1966; Lichtenstein and Schulz, 1965; and
Lichtenstein et al., 1970). Harris and Sans (1972) have
described the behavior of the primary metabolite, heptachlor
epoxide, in soil. Lichtenberg et al. (1969) have reported
on the occurrence of heptachlor and its epoxide in surface
waters in the United States; Eichelberger and Lichtenberg
(1971) have described the persistence of these compounds
in river water, observing that no measurable degradation
or chemical change was evident for heptachlor epoxide
in raw river water during an eight week period. Henderson
et al. (1969) summarized the results of a national monitoring
program which consisted of the analysis of 590 composite
fish samples collected from 50 stations over a 2-year
period. They reported finding heptachlor and heptachlor
epoxide at concentrations ranging from 0.01-8.4 mg/kg in
32% of the samples. The results of this national monitoring
program were updated (Henderson et al., 1971) and similar
observations were reported. Hannon et al. (1970) investigated
the distribution of pesticides in water, bottom sediments,
zooplankton, algae, crayfish, aquatic insects and fishes.
They reported that measurable concentrations of heptachlor
and heptachlor epoxide were present in the majority of
sample types.
Much research has been conducted on the acute toxicity of
heptachlor to aquatic organisms and the effect of water
quality on acute toxicity. Sanders and Cope (1966) determined
a 48-hour EC50 (95% confidence limits) of 42 (21-63) jug/1
for Daphnia pulex exposed to heptachlor. Frear and Boyd
(1967) corroborated this estimate of toxicity of heptachlor to
daphnids by reporting a 48-hour LC50 of 52 ug/1 for Daphnia magna
exposed to heptachlor and 120 ^ig/1 for the same species exposed
to heptachlor epoxide. Sanders and Cope(1968) reported the
96-hour LC50 of heptachlor for stonefly naiads (Pteronarcys)
was 1.1(0.8-1.4) jug/1. The acute toxicity of heptachlor has
been reported for freshwater fishes (Henderson et al., 1959)
and estuarine fishes (Eisler, 1970) , with 96-hour LC50 values
ranging from 26-320 jag/1 for freshwater fishes. Bridges
(1965) investigated the effects of time and temperature on
the toxicity of heptachlor to redear sunfish and Eisler
(1970) evaluated the effects of temperature, salinity, and
pH on the toxicity of heptachlor to the estuarine killifish.
The available information on the potential chronic effects
of heptachlor to aquatic organisms apparently is limited to
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a report by Andrews et al. (1966) describing studies in
which bluegills were exposed to heptachlor in water and food
for 56 days and effects of exposure on growth, survival and
cell structure were observed.
Endosulfan (6,7,8,9,10,10-Hexachloro-l,5,5a,6,9,9a-hexahydro-
6,9-methano-2,4,3-benzodioxathiepin 3-oxide) is an organo-
chlorine insecticide for which little information on distribution
and persistence in aquatic ecosystems exists. Greve and
Wit (1971) reported that endosulfan was present at a
concentration of 70 pg/1 in water sampled from the Rhine
River after a massive fish kill. Information on the toxicity
of endosulfan to aquatic organisms is limited to the assess-
ment of acute toxicity and generally indicates that endosulfan
is one of the most toxic of these types of chemicals to
aquatic organisms. Sanders and Cope (1968) reported the
96-hour LC50 for stonefly naiads to be 2.3(1.6-4.3) pg/1.
Pickering and Henderson (1966) reported the 96-hour LC50
of endosulfan to fishes to be ca 3 pg/1. Schoettger (1970)
reported the 96-hour LC50 for rainbow trout and white suckers
tested at 10°C to be 0.3 pg/1 and 2.5 pg/1, respectively -
He also reported the 48-hour LC50 for Daphnia magna tested
at 19°c was 68 pg/1.
Trifluralin (e^,-(, at, -Trif luoro-2, 6-dinitro-N,N-dipropyl-
p-toluidine) is a herbicide which has had widespread use
in agriculture. Although little information on distribution
and persistence in aquatic ecosystems is available, its
acute toxicity to aquatic organisms has been investigated
extensively. Sanders (1970) has reported the following 48-
hour LC50 values for aquatic invertebrates and trifluralin:
Daphnia magna, 560 pg/1; the seed shrimp Cypridopis vidua,
250 pg/1; the scud Gammarus fasciatus, 1800 pg/1;the sowbug
Asellus brevicaudus, 2000 pg/1;the grass shrimp Palaemonetes
kadiakensis, 1200 pg/1; and the crayfish Orconectes sp.,
50,000 pg/1. Similar ranges of toxicity have been reported
for other aquatic invertebrates (Sanders and Cope, 1966; 1968).
Macek et al. (1969) reported the 96-hour LC50 (95% confidence
limits) for trifluralin and bluegills was 190(160-230) pg/1
at 12.7°C and 47(40-55) pg/1 at 23.8°C. These investigators
reported that comparable values for rainbow trout were
210(182-240) pg/1 at 1.6°C and 42(38-46) pg/1 at 12.7°C.
Parks and Worth (1965) reported 96-hour LC50 value for tri-
fluralin and bluegills was 58 pg/1, that for fathead minnows
was 93 pg/1, and that for goldfish was 585 pg/1. Information
on the effects of longer exposure of aquatic organisms to
trifluralin appears to be limited to the studies of Lawrence
(1966). He reported that fathead minnows exposed in plastic
pools containing soil treated with trifluralin at rates up
to 16 Ibs./acre exhibited no mortality during four weeks
observation.
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In view of the lack of definitive information on the potential
chronic toxicity of these four pesticides to aquatic
organisms, an investigation was designed to estimate the
MATC for each chemical and Daphnia magna and fathead minnows.
A reason for selecting both an invertebrate and a fish is
that susceptibility of fishes to a chemical should be compared
to the susceptibility of fish food organisms to that same
chemical. It was recently reported (Macek et a_l. , 1976a)
that populations of essential fish food organisms may be
significantly reduced by chronic exposure to concentrations
of lindane which would apparently not directly affect fishes.
Therefore, an understanding of the susceptibility of both
fishes and fish food organisms to chemicals is necessary in
establishing realistic and meaningful water quality criteria
and standards.
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SECTION II
CONCLUSIONS
Fathead minnows were equally or more sensitive than were
daphnids in both acute and chronic exposures to the four
pesticides tested. Acute toxicity estimates (LCSO's) for
daphnids and fathead minnows exposed to acrolein were 57
and 84 yg/1, respectively; heptachlor, 78 and 7 yg/1;
endosulfan, 166 and 0.86 yg/1; trifluralin, 193 and 115 yg/1.
MATC's estimated from the results of chronic exposures of
daphnids and fathead minnows to acrolein were nearly identical
for these two species (>16.9<33.6 yg/1 and >11.4<41.7 yg/1,
respectively). MATC's estimated for trifluralin were also similar
for both species (>2.4<7.2 yg/1 and >1.9<3.1 yg/1 for daphnids
and fathead minnows, respectively). However, for heptachlor
and endosulfan, there was an order of magnitude difference
between MATC's estimated for daphnids and those for fathead
minnows, with the fish appearing to be more sensitve than the
daphnids to these two pesticides.
Residues of heptachlor and its epoxide measured in the carcasses
of fathead minnows indicated that this pesticide significantly
bioconcentrated (20,OOOX) in fish tissues as a result of aqueous
exposure. Trifluralin, on the other hand, exhibited bioconcen-
tration factors by less an order of magnitude than those
calculated for heptachlor and fathead minnows. For both
pesticides, bioconcentration of residues by fathead minnows was
directly proportional to exposure concentrations and was linear
over the range of concentrations tested.
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SECTION III
RECOMMENDATIONS
Results of the present study indicate that an application
factor of 0.01 is appropriate for D_. magna exposed to
trifluralin. In general, water quality criteria for
pesticides are established "by multiplying the acute toxicity
values for the more sensitve species... by an application
factor of 0.01 except where an experimentallly derived
application factor is indicated" (N.A.S., N.A.E., 1973). When
calculations are made from the data of the present study the
theoretical results for these three exposures correspond
reasonably well to the empirically-derived MATC limits. That
is, for D. magna, if the 48-hour LC50 of 166 yg/1 of endosulfan
is multiplied by 0.01, the resulting recommended maximum
concentration of endosulfan in water is estimated to be
1.66 yg/1, a concentration which compares favorably with the
experimentally determined MATC limits of >2.7<7.0 yg/1.
Similarly, with theoretical maximum concentration of trifluralin
with respect to D_. magna of 1.93 yg/1 corresponds to determined
MATC limits of >2.4<7.2 yg/1. For P. promelas, these two
estimates are 1.15 and >1.9<5.1 yg/r, respectively.
Exposures of both species to acrolein and heptachlor and of
fathead minows to endosulfan resulted in calculated application
factors which were an order of magnitude higher (0.1) than
those utilized to establish water quality criteria (0.01).
Therefore, use of the established application factor of 0.01
would result in maximum allowable concentrations of these
pesticides in water which should not adversely affect these
aquatic organisms.
With regard to the protocol used in the performance of chronic
exposures with fathead minnows based on observations made during
this study, several recommendations follow:
A reduced level of spawning activity was observed among several
groups of fathead minnows in which the number of males was
approximately equal to or greater than the number of females.
Conversely, a high level of spawning activity was observed
among groups of fathead minnows in which survival was reduced
and only a few fish remained. Based on these observations
we suggest that the number of mature fathead minnows in the
spawning chamber be reduced to two males and four females per
chamber.
We also feel that adjusting photoperiod to follow Evansville
daylengths tends to retard early sexually maturing fathead
minnows and we recommend that a constant 16 hour light, 8 hour
dark photoperiod be used during the entire chronic exposure of
fathead minnows.
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Due to poor survival rate which we observed among several fry
groups as well as extreme variability in size of fish in the
same group, we recommend reducing the number of fathead minnow
fry from a total of forty per growth chamber to twenty per
growth chamber.
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SECTION IV
MATERIALS AND METHODS
Acute bioassay procedures were generally those recommended
in Standard Methods for the Examination of Water and
Wastewater(APHA 1971)^Chronic testing procedures for Daphnia
magna were determined through communications between Bionomics
staff members and personnel at the National Water Quality
Laboratory. The methodology for chronic testing of fathead
minnows generally followed the recommended bioassay procedure
issued by the Environmental Protection Agency's National
Water Quality Laboratory, Duluth, Minnesota (Bioassay Committee,
1971) .
EXPOSURE SYSTEMS
Proportional diluters (Mount and Brungs, 1967) , with a dilution
factor of 0.5 and a syringe injector, delivered the test
water and toxicant to the mixing chamber, mixing cells, and
ultimately to the test chambers. Five concentrations of
toxicant and a control flowed to mixing containers and into
separate glass delivery tubes leading to the replicate test
chambers.
In the test systems for Daphnia magna, glass battery jars,
17 cm tall and 13.5 cm in diameter, served as the exposure
aquaria and baffles were inserted in each to minimize
turbulence from influent water. Water depth in each aquarium
was 14 cm at which height water passed through a screened
outlet. Total volume of exposure solution was 2.0 liters.
Quadruplicate aquaria were provided for each toxicant
concentration and the diluter was modified to include food
cells which delivered a measured amount of food along with
the toxicant and diluent water.
Duplicate glass aquaria 30.5 x 90 x 30.5 cm tall were provided
for each toxicant concentration in the fathead minnow
exposures. Water depth in each aquarium was 15 cm allowing
a total volume of 41 liters and the flow rate to each aquarium
was equal to 7 tank volumes per 24 hours. Each aquarium was
subdivided to provide space for two larval growth chambers
25.0 x 20.5 x 12.5 cm, with a water depth of 15 cm. A 40
mesh stainless steel screen was affixed to one end of each
growth chamber to allow water to flow out while retaining
the young fish. Test water was delivered directly to the
test chamber and growth chambers through a glass, flow-
splitting chamber calibrated to keep the flow rate equal in
all 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 1.
TABLE 1. CHEMICAL ANALYSIS OF THE DILUENT WATER UTILIZED
DURING CHRONIC EXPOSURES OF Daphnia magna AND
FATHEAD MINNOWS TO ACROLEIN, HEPTACHLOR, ENDOSULFAN
AND TRIFLURALIN
Parameter
Calcium
Magnesium
Potassium
Sulfate
Nitrate
Nitrite
Ammonia
Phenol
Chlorine
Concentration
(mg/1)
6.0
2.1
1.1
11.6
<0.05
<0.05
0.01
<0.001
<0.01
Parameter
Chloride
Fluoride
Cyanide
Iron
Copper
Zinc
Cadmium
Chromium
Lead
Concentration
(mg/1)
17.6
0.5
<0.005
<0.01
0.004
0.01
0.001
<0.001
<0.01
Diluent water was delivered through PVC pipes to the exposure
systems. Test chambers in the fathead minnow units were
maintained in recirculating water baths. Water circulating
in the water baths was heated by two immersion coil heaters
controlled by a mercury column thermoregulator which maintained
a temperature of 25 + 1°C. Prior to entering experimental
units for Daphnia, diluent water was delivered to a stainless
steel headbox where immersion coil heaters and thermoregulators
maintained a temperature of 19 +_ 1°C. In addition, ultra-
violet lights (24 watt) were placed over the headbox and
water cells of each diluter to minimize the introduction of
fungus and pathogens into the test system.
Illumination was provided by a combination of Durotest
(Optima FS) and wide spectrum Grow Lux fluorescent lights
located centrally above the test chambers in all experimental
units. A constant photoperiod was controlled by an automatic
timer in the Daphnia experiments. The photoperiod for fathead
minnows followed the normal daylight hours of Evansville,
Indiana (average U.S. daylength) starting with the daylength
for December 1st (Evansville) on day one of each experiment.
Photoperiod was adjusted the first and fifteenth of each month.
Experimental units used in all exposures were screened with
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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 Daphnia
magna and the four pesticides to estimate the 48-hour LC50
and its 95% confidence interval. Five daphnids <24 hours
old in each of four replicate containers of each concentration
were exposed at 20 +_ 1°C. A linear regression equation was
calculated after converting test concentrations and
corresponding percent mortalities into logarithms and
probits, respectively, and this equation was utilized to
estimate the 48-hour LCSO's and confidence intervals.
Acute toxicity studies with fathead minnows and the four
pesticides were conducted in intermittent-flow water systems
using proportional diluters (Mount and Brungs, 1967). The
incipient LC50 was estimated when no additional significant
mortality (<10%) of the test organisms was observed at any
concentration during a 48-hour period. At this time, a
linear regression equation was calculated by converting test
concentrations and corresponding mortalities into logarithms
and probits, respectively. This equation was utilized to
estimate the incipient LC50 and 95% confidence interval.
The chronic exposure concentrations for both Daphnia and
fathead minnows were selected by evaluating observed mortality
and no-effect concentrations from the acute studies. The
48-hour LC50 for Daphnia and the incipient LC50 for fathead
minnows were used to estimate application factors describing
the relationship between acute and chronic toxicity.
Initially, pesticide concentrations and water quality parameters
were monitored in each aquarium each week to establish
that the concentrations and water quality characteristics
were constant with minimum variability. After the determin-
ation of the actual concentrations and characteristics in
the experimental systems a minimum monitoring effort was
conducted to measure variability and detect changes in
established means. Stock solutions of heptachlor, endosulfan,
and trifluralin (98%, 99%, and 97% a.i., respectively) in
Nanograde acetone were delivered to the dilution water from
50 ml glass syringes with stainless steel needles. The
solvent (acetone) was not added to the control water in
any of the chronic exposures and the amount added to the
highest pesticide concentrations did not exceed 43 mg/1
in the Daphnia exposures and 12 mg/1 in the fathead minnow
exposures. Stock solutions of acrolein (99% a.i.) in
100% ethanol were prepared prior to the filling of each
10 ml glass syringe which delivered the solution to the
10
-------
dilution water. The solvent (ethanol) was not added to
the control water and the amount added to the highest
acrolein concentration was 3.6 mg/1 in the Daphnia exposure
and 1 mg/1 in the fathead minnow exposure. Generally,
pesticide concentrations were determined once each week
during each of the chronic exposures by taking water samples
from one replicate of each concentration.
CHEMICAL METHODOLOGY
During the chronic exposures, total hardness, alkalinity,
pH and acidity were generally measured bi-weekly in the
control and one pesticide concentration according to
Standard Methods for the Examination of Water and Wastewat-^r
(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.
Acrolein (IUC name: propenal, C^R^O) in water was analyzed
by adding 2,4-dinitrophenylhydrazine (2,4-DNPH) to form the
acroleinrhydrazone complex, extracting the colored complex
with benzene and measuring the color intensity spectro-
photometrically at 365 nm. The method was modified from
Battelle (1970) .
A solution of 2,4-dinitrophenylhydrazine (2,4-DNPH), ACS
grade, was prepared by adding about 500 ml of distilled
water to 167 ml of concentrated hydrochloric acid in a
1000 ml volumetric flask. To this mixture we added 0.792 g
of 2,4-DNPH, diluted to 1000 ml with distilled water and
stirred until the solids were dissolved. The solution was
stored in darkness and fresh solutions were prepared
weekly. Amber-glass bottles (3.7 liters), equipped with
Teflon-lined caps, were prepared for sample collection by
washing the bottles with laboratory detergent, rinsing with
tap and then distilled water. The bottles were rinsed
twice with 30 ml of benzene, once with methanol, and
finally twice with distilled water.
Immediately before sampling, 40 ml of 2,4-DNPH reagent was
added to each bottle. Exactly 2000 ml of water was added
to each sample bottle, the bottle was capped and swirled
to mix the solution. After 1 hour, 900 ml of sample was
added to each of two 1-liter separatory funnels equipped
with Teflon stopcocks. Each portion was extracted with 25
ml of benzene, followed by a 15 ml benzene extraction,
allowing sufficient time after shaking (ca 5-10 min.) for
11
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phase separation. The benzene (ca 80 ml) from the 1800 ml
water extraction was combined in a 500 ml separatory funnel
(with Teflon stopcock) and extracted twice with 100 ml
portions of saturated NaCl, discarding the wash. The
benzene was then extracted once with 100 ml of sodium hydroxide
(3N), shaking for 15 seconds. A longer extraction time was
unnecessary and resulted in emulsions which were difficult
to break. The sodium hydroxide wash was discarded and the
benzene was passed through a 10 mm I.D. column containing
about 50 mm of anhydrous sodium sulfate. The column eluate
was collected in a 100 ml volumetric flask. The extracts
were diluted to 100 ml with benzene and the absorbance
measured at 365 nm utilizing a Coleman Model 6/35 UV/
VIS/NIR spectrophotometer with 25 mm cuvettes.
A blank was analyzed concurrently with each batch of aquaria
water samples and the blank absorbance was subtracted from
each sample absorbance reading to obtain the corrected
absorbance used to calculate the acrolein concentration. The
blanks used to correct various acrolein concentrations
were as follows:
Sample to be analyzed
Well water supply
Acrolein diluter
delivery tube samples
Acrolein aquaria samples
Blank used for correction
Reagent blank using distilled
water
Well water supply at the
diluter location
Acrolein control aquaria
samples
On July 12, 1973, the well water supply was analyzed for any
substance (aldehyde or ketone) which might react with 2,4-
DNPH to present a positive interference in the colorimetric
determination of acrolein. The analysis was conducted by
the method of additions, namely by analyzing well water
containing 0, 8.4, 42, and 105 ng/liter of added acrolein.
The results show that the well water contained 9 ng/liter of
interfering compounds, calculated as acrolein. This
concentration of contaminant remained constant throughout
the study as documented by well water analyses performed
concurrently with each batch of diluter water samples.
The data presented have been corrected for the observed
background. All acrolein results were calculated from a
standard graph of ppb acrolein versus absorbance at 365 nm.
Data generated from the following standards showed only
mild deviation from Beer's Law. The minimum concentration
detectable by this method was 3 ng/liter.
12
-------
jjg/1 Acrolein Absorbance
4.0 0.050
8.4 0.105
42.0 0.396
105.0 0.766
Heptachlor (IUC name: 1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-
tetrahydro-4,7-endo-methanoindene) and its degradation
product, heptachlor epoxide (IUC name: 1,4,5,6,7,8,8-
Heptachloro-2,3-epoxy-3a,4,7,7a-tetrahydro-4,7-methanoindan)
were analyzed from water and fish tissue by gas/liquid
chromatography using electron capture detection.
Water samples (400-500 ml) were collected in amber glass
bottles, equipped with Teflon-lined caps. The entire content
of the sample bottle was measured and added to a 500 ml
separatory funnel, equipped with a Teflon stopcock. Then
ca 50 ml of nanograde methylene chloride was added to the
bottle, the cap replaced, and the bottle shaken to extract
any pesticide which may have been adsorbed onto the glass
surface from the aqueous phase. The solvent was transferred
quantitatively from the bottle to the separatory funnel and
the water sample was extracted with this solvent portion.
After phase separation, the methylene chloride was drained
into a 250 ml Griffin beaker. The water was extracted a
second time with 30 ml of methylene chloride, which was
added to the first portion of solvent.
The combined solvent was passed through a 20 mm I.D. glass
column, equipped with a glass-frit and containing 50 mm of
anhydrous sodium sulfate. When the initial solvent had almost
reached the top surface of the sodium sulfate, an additional
portion of fresh methylene chloride (ca 30 ml) was added
to rinse any pesticide residue from the column walls and
the sodium sulfate. The solvent was added to a 500 ml
Kuderna-Danish evaporator as it dripped from the sodium
sulfate column. The evaporator was lowered and the tip of
the column was rinsed (with fresh solvent) into the evaporator
The Kuderna-Danish evaporator was fitted with a three-ball
Snyder column and the solvent was concentrated to ca 3 ml
over a steam bath at 70°C. The evaporator was cooled to room
temperature and the solvent was transferred to a 15 ml
centrifuge tube (Pyrex 8064 or equivalent), rinsing with
fresh methylene chloride. The remaining solvent was
evaporated to dryness at room temperature, using a steam of
clean dry air. Immediately after the last trace of
methylene chloride had evaporated, a known volume (1-10 ml)
of Nanograde hexane was added to dissolve the residue and
an aliquot was withdrawn for analysis of heptachlor and its
13
-------
epoxide by gas chromatography.
The gas chromatographic operating conditions were:
Instrument: Tracor Model MT-550 Dual Column
Detector: 15mC Ni*>3 electron capture
Column: 2 m x 2 mm I.D. glass column packed with 80/100 mesh
Supelcoport coated with 5% (by weight) of DC-200.
The column support was coated according to the
fluidized drying technique of Kruppa et al. (1967),
using a packing dryer supplied by the CRC Co.,
Addison, 111.
Gas flow: 35 cc/minute
Column: 35 cc nitrogen/minute at 27 psi column pressure
Detector: 60 cc nitrogen/minute
Temperature:
Inlet: 215°C Outlet: 210°C
Column: 190°C Detector: 290 C
Recorder: Corning Model 841, 0-1 mv full-scale
Response: lxlO~-'-^g of heptachlor and 5x10 g of heptachlor
epoxide gave half-scale recorder pen response
using an electrometer attenuation of 1.6x10"^
amperes.
Prior to the analysis of each batch of water samples, ca.
100 ug of heptachlor and the epoxide were injected to
saturate the column. After a stable baseline was obtained
(ca 1 hr.) a series of heptachlor and heptachlor epoxide
standards (Chem Service Cat#78 and 700, respectively) was
chromatographed and a graph was constructed of nanograms
injected versus the recorder response in millimeters of
peak height. The nanograms of pesticide represented by
sample peak heights were read from the graph and the results
were calculated. A calibration graph was constructed prior
to the analysis of each batch of heptachlor water samples;
furthermore, during the period of chromatographic analysis,
standard solutions of known weight were analyzed after
every ten sample extracts to verify the stability of instrument
response throughout the day.
Quality control samples were manufactured with well water
which contained known weights of heptachlor and heptachlor
epoxide, added to the water using a small volume (0.1-0.5 ml)
of Nanograde acetone as a co-solvent. The results of the
percentage recoveries and standard deviations obtained
(Table 2) show that no loss of the two compounds occurred
during sample preparation.
14
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TABLE 2. RECOVERY OF HEPTACHLOR AND HEPTACHLOR EPOXIDE
FROM QUALITY CONTROL WATER SAMPLES
Cone . added Oug/1 )
H.a H.E.b
1.0 1.0
10 10
Water
volume
(ml)
400
400
Percentage
recovery
H.
96
103
98
97
100
(Average) 99 + 2.6°
102
94
99
96
105
(Average) 99 + 4.3
H.E.
98
96
106
103
95
100 + 4.7
106
96
102
94
98
99 + 4.8
H. denotes heptachlor
3H.E. denotes heptachlor epoxide
'Standard deviation
Fish tissues were extracted for heptachlor and heptachlor
epoxide residues using a column technique modified from a
procedure described by Hesselberg and Johnson (1972) . These
authors ground fish tissue in a Waring Blendor along with
dry ice, stored the tissue at -20°C for 8-12 hours, and then
proceeded with the solvent extraction step. E G & G, Bionomics
used Tekmar analytical mill, Model A-10, featuring internal
metal tubing which circulates a liquid (from an external unit)
around the grinding chamber to raise or lower the temperature.
Sodium sulfate was purified by heating it for 16 hours at
450°C, cooling, and storing the compound in clean, all-glass
bottles. Anhydrous sodium sulfate (ca 5 times the tissue
weight) was added to the grinding chamber, followed by 2.0
grams of Celite 545, and finally the fish tissue. Coolant
was circulated through the mill until the temperature was
minus 50°C, and the tissue was homogenized by grinding at
20,000 rpm for 30 seconds. The resultant fine powder was
quantitatively transferred to a chromatographic column
(20 x 400 mm) and eluted with 250 ml of 50 percent ethyl
ether in petroleum ether. The eluate was collected (1-2
15
-------
ml/min.) in a 500 ml Kuderna-Danish evaporator, then a
three-ball Snyder column was installed and the solvent was
evaporated to a volume of ca 2-4 ml over a water bath at
70°C.
A large portion of extraneous materials, e.g., lipids, fats, and
oils, were removed from the tissue extract by performing the
hexane-acetonitrile partition clean-up procedure described
by the U.S. Department of the Interior (1972). The resultant
acetonitrile solution of the clean tissue extract was placed
in a Kuderna-Danish evaporator, equipped with a three-ball Snyder
column and concentrated to ca 3 ml over a steam bath. The
evaporator was cooled to ambient temperature and the solvent
was quantitatively transferred to a 15 ml graduated centrifuge
tube equipped with a metal-lined screw cap. The solvent
was adjusted to the volume required for analysis by evaporating
the solvent at ambient temperature, using a stream of clean
dry air, and then diluting to volume with Nanograde hexane.
An aliquot of the extract was analyzed by gas chromatography
with the following instrumental conditions:
Instrument: Tracer Model MT-550
Detector: 15 mC Ni*>3 electron capture
Column: 2 m x 2 mm ID glass containing 5% Dexsil 300 GC on
80/100 mesh Supelcoport packing. The column was
prepared according to the fluidized drying method
of Kruppa et al. (1967) .
Gas Flows: 35 cc/min.
Detector: 60 cc He/min.
Column: 30 cc He/min at 18 psi back pressure
Recorder: Corninq Model 841, 0-1 mV full-scale
Response: lxlO~10g of heptachlor and 2xlO~^^g of heptachlor
epoxide eluted in 4.85 and 9.77 minutes, respectively;
and gave half-scale recorder pen deflection at an
electrometer attenuation of 1.6x10"^ amperes.
The Dexsil 300 GC column was chosen for its extreme stability
at high temperatures. After analyzing ca ten tissue extracts,
the column temperature was elevated to 300°C overnight. The
next morning a large amount of heptachlor and heptachlor
epoxide (100 jag) was injected and the column temperature was
reduced to 210 C. Several injections of identical weights
(ca 0.1 ng) of the two standard compounds were made and
identical peak heights for all injections was assumed to
indicate column saturation. At this point the analysis of
another batch of tissue extracts proceeded. In this manner,
the need for more exhaustive clean-up of tissue was negated
and a greater number of tissue extracts could be chromatographed
with no apparent change in sensitivity or retention time of
heptachlor or heptachlor epoxide.
16
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Table 3 lists the percentage recovery and standard deviation
of heptachlor and heptachlor epoxide from quality control
samples using fathead minnow tissue known to be free from
naturally-occurring residues of either compound. According
to the U.S. Department of the interior (1972), the tissue
extract was dissolved in 30 ml of hexane (saturated with
acetonitrile) and extracted twice with two 30 ml portions
of acetonitrile (saturated with hexane). The combined
acetonitrile extracts were evaporated, as previously described,
and the extract was dissolved in hexane. The low percentage
recovery of both compounds was assumed to be due to the above
clean-up procedure, in that: 1) only two extractions of the
hexane phase were specified by this method; and 2) evaporation
of the acetonitrile (b.p. 81.6°C) may cause some loss of
these compounds.
Table 3. RECOVERY OF HEPTACHLOR AND HEPTACHLOR EPOXIDE FROM
QUALITY CONTROL TISSUE SAMPLESa
Weight
H.b
1.0
1.0
1.0
1.0
added Oug)
H.E.C
1.0
1.0
1.0
1.0
Tissue Weight
(g)
1.776
1.674
1.776
1.758
Percentage
recovery
H.
38
40
38
36
Average 38+1.6
H.E.
39
30
30
29
32+4.7
Quality control samples were manufactured using fathead
minnows fillets
H. denotes heptachlor
CH.E. denotes heptachlor epoxide
Standard deviation
The data were calculated using standard graphs as previously
described for the analysis of heptachlor and its epoxide
from water. The calibration graphs were also prepared prior
to analyzing a particular batch of tissue samples. Results
of analysis of fish tissues are corrected for recovery.
Endosulfan (IUC name: 6,7,8,9,10,10-hexachloro-l,5,5a,6,9,9a-
hexahydro-6,9-methano-2,4,3-benzodioxathiepin 3-oxide) was
analyzed from water by the extraction procedure and gas
17
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chromatographic analysis previously described for heptachlor,
Standard reference material was supplied by the Niagara
Chemical Division, FMC Corp. Middleport, N.Y. and was
identified as follows:
Compound Batch No. Purity %
Thiodan ether D188-87 100
Thiodan alcohol D188-85 100
Thiodan Ia ME-J264 99.9
. ME-J264 99.9
Thiodan II ME-J265 95.5
ME-J265 95.5
Thiodan sulfate MR-C747 100
Numeral (I) denotes the lower melting (
-------
amount of the compound in hexane and analyzing the solution
by gas chromatography. The compound was determined to be
100 percent active containing 60.0 and 40.0 percent of
isomers I and II, respectively.
TABLE 4. RECOVERY OF ENDOSULFAN FROM QUALITY CONTROL WATER
SAMPLES
Concentration
added (pg/1)
0.025
0.125
Water volume
(ml)
2000
2000
Percentage
recovery
92
108
104
100
96
Average 100 - 6.3a
87
100
96
93
94
Average 94 - 4.7
Standard deviation
On several test days (random), namely days 0, 5, 107; the
aquaria water from the highest endosulfan exposure (0.6 pg/1)
was examined for the presence of degradation products.
Endosulfan ether, alcohol, and sulfate were not detected
in any of the water samples analyzed.
Endosulfan containing isomers I and II at a 60:40 ratio was
used to manufacture the quality control samples in well
water. The analysis of these samples revealed the same
ratio of isomers present on the chromatogram, indicating
no difference in the percentage recovery of the two isomers.
The recovery of endosulfan was considered to be complete
and no correction factor was applied to the calculation of
results. Peak heights of endosulfan I and II extracted from
water were compared to analytically pure endosulfan standards
containing known weights and ratios of isomers I and II.
The concentrations of isomers I and II were summed and the
data were presented as total endosulfan.
19
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Trifluralin (,j, -\ , i -Trifluoro-2,6-dinitro-N,N-dipropyl-
p-toluidine) was analyzed from water and fish tissue by the
methodology previously described for heptachlor. Technical
trifluralin, (Lot No. 5GB70, 99 percent active, from Eli-Lilly
and Co., Indianapolis, Ind.) was used to calibrate the gas
chromatograph prior to and during the analysis of trifluralin
samples. Using an electrometer attenuation of 1.6xlO~"
amperes, 0.05 nanograms of trifluralin produced half scale
recorder response, eluting from the column in 1.80 minutes.
The extreme response of trifluralin to electron capture detection
enabled a minimum detectable concentration of 0.08 pg/liter,
when extracting a 65 ml volume of water. The minimum
detectable concentration of trifluralin from fish tissue, due
to the low percentage recovery and the interference of fats
and oils which survive the clean-up procedure was 0.10 jag/gnu
The percentage recovery of trifluralin from well water is
listed in Table 5 and from fish tissue in Table 6. Reported
concentrations of trifluralin in water and fish were calculated
by multiplying the raw data by factors of 1.20 and 5.55,
respectively -
TABLE 5. RECOVERY OF TRIFLURALIN FROM QUALITY CONTROL WATER
SAMPLES
Concentration
added (pg/1)
0.025
0.125
Water volume
(ml)
400
65
Percentage
recovery
84
84
76
81
75
Average 80 + 4.3a
78
87
85
77
88
Average 83 + 5.1
Standard deviation
20
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TABLE 6. RECOVERY OF TRIFLURALIN FROM QUALITY CONTROL
TISSUE3 SAMPLES
Concentration
added (jag/gm)
0.563
0.597
0.678
0.569
0.651
Tissue
weight (g)
1.776
1.674
1.476
1.758
1.536
Percentage
recovery
18
21
17
19
15
Average 18 + 2.2
whole fathead minnows
Standard deviation
STATISTICS
Measured biological parameters from replicate containers during
chronic exposure were averaged and subjected to analysis of
variance according to Steel and Torrie (1960) . When
treatment effects were indicated, the means of these effects
were subjected to Duncan's Multiple Range Test to determine
which treatments were statistically different. All differences
were considered statistically significant at a probability
of P=.05.
CHRONIC EXPOSURE
Daphnia magna
Laboratory stocks of Daphnia magna were obtained from the
University of New Hampshire, Durham, N.H., and successfully
cultured in the laboratory according to the methods of Biesinger
and Christensen (1972). Typically, 10 daphnids (<24 hours old)
were placed into each of four replicate experimental units,
resulting in a total of 40 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 Marriotte bottle via a
volumetric delivery system to a mixing chamber during 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.
21
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Survival and reproduction of Daphnia 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 were
randomly selected from each chamber to begin the second
generation exposure. The same procedures were followed for
the second and third generation, after which the experiment
was terminated.
Pimephales promelas
Chronic exposures of fathead minnows with endosulfan and
trifluralin began in September 1972, with heptachlor in
April 1973, and with acrolein in June 1973. Fathead minnows,
obtained as eggs from the Newtown Fish Toxicology Laboratory,
Newtown, Ohio., were 20 and 26 days old on day one of the
endosulfan and trifluralin chronic exposures, respectively.
Fathead minnows from brood stock at the Aquatic Toxicology
Laboratory of Bionomics, EG&G, Inc., Wareham, Massachusetts
were used to initiate chronic exposures with heptachlor and
acrolein. Test fish were one day old for heptachlor and 27
days old for the acrolein chronic exposure. In each test,
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). After 60 days exposure the number of
fish in each test chamber was impartially 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 external parasites on a few fish in the
trifluralin and endosulfan chronic exposures prompted the
use of flush treatments with a combination of malachite green
and formalin (25 pi/liter of formalin containing 3.7 g/1
malachite green crystals). These treatments were concentrated
between days 115 and 130 in each of the chronic exposures and
all tanks were treated equally whether or not parasites
were found.
Due to unequal numbers of males in each tank, some of the
male fish were removed after secondary sexual characteristics
were well developed. Five spawning sites of halved, 3 inch
transite drain tiles had been placed in each tank when fish
22
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were released from growth chambers after 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 water by means of an egg cup
and a rocker arm apparatus (Mount, 1968) . Dead eggs were
removed and counted each day until hatching was completed
(3-5 days at 20°C). 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 80% live hatch were placed in the respective growth
chambers. Cumulative mortality and total length of live
fish were determined at 30 and 60 days photographically
(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
was determined for each fish and three samples of eviscerated
fish per concentration were retained for residue analysis in
the tests with trifluralin and heptachlor.
23
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SECTION V
RESULTS
ACUTE BIOASSAYS
The analysis of the results of acute static bioassays with
Daphnia magna indicated the 48-hour LC50 (95% confidence
interval) was 57 (20-99) ;ug/l for acrolein, 78 (46-113) pg/1
for heptachlor, 166 (126-219) >ig/l for endosulfan, and 193
(115-327) pg/1 for trifluralin. Toxicity induced mortality
was observed with daphnids after 48-hour exposure to 50 pg/1
of acrolein, 50 pg/1 of heptachlor, 150 pg/1 of endosulfan and
100 pg/1 of trifluralin. Based on these data, the highest nominal
concentration selected for investigation during chronic
exposure was 60 pg/1 of acrolein, 50 pg/1 of heptachlor, 100
pg/1 of endosulfan and 100 pg/1 of trifluralin.
A 6 day continuous-flow bioassay conducted at 25 + 1°C with
51 day old fathead minnows indicated the incipient LC50 of
acrolein to this species was 84 (54-130) pg/1. A 10 day
continuous-flow bioassay conducted at 25 + 1°C with 60 day
old fathead minnows resulted in an estimated incipient LC50
of 7.0 (5.7-8.5) pg/1 of heptachlor. The incipient LC50 of
endosulfan for fathead minnows was estimated to be 0.86
(0.52-1.4) pg/1 based on a 7 day continuous-flow bioassay
with 53 day old fathead minnows. The incipient LC50 of
trifluralin for fathead minnows was estimated to be 115
(48-211) pg/1 based on a 12 day continuous-flow bioassay
with 44 day old fathead minnows. Based on these data, the
highest nominal concentration selected for investigation
during chronic exposure of fathead minnows was 89 pg/1 of
acrolein, 4 pg/1 of heptachlor, 0.60 pg/1 of endosulfan and
20 pg/1 of trifluralin.
WATER CHEMISTRY
The results of the chemical analysis of water samples taken
during the various chronic exposures indicate that hardness,
alkalinity, acidity, dissolved oxygen and pH varied minimally
both within any one chronic exposure and between chronics.
Statistical analysis showed no significant differences for
any of the above parameters between treatments within a chronic;
therefore, only means and ranges for the various parameters
are presented (Tables 7 and 8).
The results of gas chromatographic analyses of water samples
taken periodically during chronic exposure of Daphnia magna
to the three of the four pesticides are presented in Table 9.
During the heptachlor experiment, contamination of most of the
water samples with interfering trace organics in sufficient
quantities to preclude the quantitation of heptachlor was
encountered. After considerable investigation of test water,
24
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TABLE 7 MEAN AND RANGE OF MEASURED CONCENTRATIONS OF HARDNESS, ALKALINITY, ACIDITY,
DISSOLVED OXYGEN AND pH FROM WATER SAMPLES TAKEN PERIODICALLY DURING CHRONIC
EXPOSURE OF Daphnia magna TO FOUR PESTICIDES
Pesticide
ACROLEIN
Mean - S.D.b
Range
(N)
HEPTACHLOR
Mean - S . D .
Range
(N)
ENDOSULFAN
Mean - S . D .
Range
(N)
TRIFLURALIN
Mean - S . D .
Range
(N)
Concentration (mg/1)
Hardness
35 - 2.1
29 - 38
(5)
37 - 2.5
35 - 42
(4)
35 - 3.1
30 - 39
(5)
37 - 2.3
34 - 39
(5)
Alkalinity
33 i 2,1
30 - 34
(5)
34 - 2 6
32 - 39
(4)
32 - 3.6
26 - 35
(5)
35 - 2.8
31 - 37
(5)
Acidity
4.4 - 0.8
3.8 - 4.8
(5)
4.7 - 1.8
3,9 - 5.2
(4)
4.6 - 1.5
3.9 - 5.0
(5)
4.4 - 0.9
3.8 - 4.7
(5)
D.O.
7.5 - 0.6
6.5 - 8.6
(29)
6.9 - 1.5
4.1 - 8.8
(28)
7.0 - 0.6
6.4 - 8.0
(24)
6.6 - 1.0
4.6 - 8.7
(22)
PH
7.0 - 7.3
(5)
6.8 - 7.2
(5)
6.8 - 7.1
(5)
6.8 - 7.2
(5)
tsj
en
Dissolved oxygen
^Standard deviation
-------
TABLE 8. MEAN AND RANGE OF MEASURED CONCENTRATIONS OF HARDNESS, ALKALINITY, ACIDITY,
DISSOLVED OXYGEN AND pH FROM WATER SAMPLES TAKEN PERIODICALLY DURING
CHRONIC EXPOSURE OF FATHEAD MINNOWS (Pimephales promelas) TO FOUR
PESTICIDES
<7\
Pesticide
ACROLEIN
Mean - S.D.b
Range
(N)
HEPTACHLOR
Mean - S.D.
Range
(N)
ENDOSULFAN
Mean - S.D.
Range
(N)
TRIFLURALIN
Mean - S.D.
Range
(N)
Concentration (mg/1)
Hardness
32 - 4.7
23 - 39
(20)
33 - 5.1
21 - 40
(14)
35 - 3.4
31 - 40
(12)
33 - 4.5
23 - 39
(12)
Alkalinity
30 - 3.7
20 - 35
(20)
32 - 3.8
21 - 36
(14)
28 - 3.4
22 - 31
(12)
27 - 2.6
23 - 31
(12)
Acidity
3.6 - 1.5
1.9 - 7.0
(20)
4.6 - 1.6
2.9 - 6.0
(16)
4.4 - 1.5
2.9 - 6.7
(12)
4.2 - 1.5
2.0 - 7.0
(12)
D.07
8.2 - 1.3
4.9 - 9.6
(798)
7.5 - 1.2
4.3 - 9.6
(888)
7.9 - 1.0
4.8 - 9.4
(1386)
7.8 - 1.0
5.9 - 9.5
(1356)
pH
6.6 - 6.8
(20)
6.6 - 7.0
(16)
6.7 - 7.3
(12)
6.6 - 7.2
(12)
Dissolved oxygen
'standard deviation
-------
TABLE 9. NOMINAL AND MEAN MEASURED CONCENTRATIONS OF ACROLEIN,
ENDOSULFAN AND TRIFLURALIN IN WATER DURING CONTINUOUS
EXPOSURE OF Daphnia magna
Compound
ACROLEIN
ENSOSULFAN
TRIFLURALIN
Nominal
Concentration
(Jig/1)
60
30
15
8
4
100
50
25
12
6
100
50
25
12
6
Measured Concentration (pg/1)
Mean + S.D.*1
42.7 + 4.3
33.6 + 7.8
16.9 + 4.2
7.1 + 5.8
3.2 + 0.7
79.7 +28.6
37.7 +12.5
15.3 + 4.0
7.0 + 4.4
2.7 + 2.9
52.7 +10.7
25.6 + 4.7
14.0 + 3.7
7.2 + 2.0
2.4 + 1.3
Range
41-47
24-35
15-21
6-14
3-4
47-100
25-50
13-20
2-10
1-6
37-60
21-32
10-20
4-9
1-4
# Samples
8
8
8
8
8
8
8
8
8
8
6
6
6
6
6
Standard deviation
glassware, extraction solvents, column components, etc. we
determined that the batch of acetone solvent utilized to
prepare the heptachlor stock solution was contaminated with
unknown interfering trace organics. Unable to separate heptachlor
from these contaminants by various clean-up techniques, we have
reported all data for Daphnia on the basis of nominal
concentrations.
The results of gas chromatographic analysis of water samples
taken periodically during the chronic exposure of fathead
minnows to the four pesticides studies are presented in Table
10. For endosulfan and trifluralin, mean measured
concentrations were fairly consistent and varied only slightly
from nominal. The mean measured acrolein and heptachlor
concentrations were generally less than half of nominal.
Although we detected heptachlor epoxide in water samples
suggesting degradation in the system, observations of the
epoxide were intermittent and sporadic. Rather, we believe
the loss of approximately half of the heptachlor in the
system was due to adsorption of the chemical into glass, food,
and/or fecal material, or to incorporation into fish tissues,
or to both factors. During the period from day 210 to the end of
27
-------
TABLE 10. NOMINAL AND MEAN MEASURED CONCENTRATIONS OF ACROLEIN,
HEPTACHLOR, ENDOSULFAN AND TRIFLURALIN DURING
CHRONIC EXPOSURE OF FATHEAD MINNOWS (Pimephales
promelas)
Compound
ACROLEIN
HEPTACHLOR
ENDOSULFAN
TRIFLURALIN
Nominal
Cone, (jug/1)
89
45
22
11
5
4.0
2.0
1.0
0.5
0.25
0.60
0.30
0.15
0.08
0.04
20.0
10.0
5.0
2.5
1.2
Measured concentration (jag/1)
Mean - S.D.a
41.7 - 35.8
20.8 - 15.5
11.4 - 8.3
6.4 - 3.6
4.6 - 2.7
1.84 - 0.64
0.86 - 0.29
0.43 - 0.20
0.20 - 0.07
0.11 - 0.03
0.40 - 0.16
0.20 - 0.08
0.10 - 0.03
0.06 - 0.03
0.04 - 0.03
16.5 - 14.0
8.2 - 6.2
5.1 - 4.2
1.9 - 2.1
1.5 - 1.2
Range
9-140
5-60
3-32
2-13
1-10
1.2-2.3
0.72-1.2
0.29-0.74
0.17-0.27
0.07-0.14
0.21-0.85
0.05-0.39
0.04-0.17
0.02-0.13
0.01-0.12
3.6 - 50
1.9 - 28
1.4 - 18
0.37-5.5
0.40-4.2
# Samples
28
12
15
13
15
28
18
28
18
18
58
29
29
28
30
56
28
33
28
30
Standard deviation
28
-------
the heptachlor fathead minnow chronic we experienced
interferences in the analysis of water samples for heptachlor
similar to those previously described. Despite significant
effort, we were unable to identify the source of these
interferences during the chronic. We have subsequently identified
contamination of the solvent system as the likely cause of
these difficulties. Acrolein is a very unstable compound and
is readily oxidized; also it is extremely volatile (boiling
point 48°C). Thus it is not surprising that measured
concentrations were significantly lower than nominal.
CHRONIC EXPOSURE
Daphnia magna to acrolein
Statistical analysis of data on the survival of Daphnia magna
continuously exposed to acrolein for 64 days indicated
significant differences due to treatment. During the first
generation, continuous exposure to mean measured concentrations
of 42.7 and 33.6 ug/1 of acrolein for 22 days significantly
reduced survival of daphnids (Table 11). During the second
generation the effect on survival was cumulative for daphnids
exposed to 33.6 ug/1 of acrolein. The apparent reduction
in survival of second generation Daphnia exposed to 16.9 ug/1
of acrolein on day 43, was not consistent with observations on
the third generation where survival at this concentration
was equal to that of the controls. Production of young
during the first generation was extremely variable but
generally indicated no significant reductions which could
be correlated to chemical treatments (Table 12). Production
of young during generations two and three was less variable
than during generation one and indicated that no significant
differences existed among treatments where reproduction occurred.
Based on the observed effects of acrolein on survival of
Daphnia magna continuously exposed through three generations
of development, the estimated maximum acceptable toxicant
concentration for this species is >16.9 <33.6 jag/1.
Daphnia magna to heptachlor
Although we recognize the contamination of the solvent system
could possibly have affected the daphnids, for purposes of
discussion all experimental observations which appear dose-
related are presumed to be the effect of heptachlor in water.
Survival of Daphnia magna continuously exposed to nominal
concentrations of 50 and 25 jag/1 of heptachlor for 64 days was
reduced when compared to controls (Table 13). This effect
was observed in all three generations of daphnids exposed
to 50 pg/1, but only among the second and third generation
exposed to 25 pg/1. Survival among daphnids continuously
29
-------
TABLE 11. MEAN PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO ACROLEIN FOR 64 DAYS
Mean
Measured
Cone, (ug/1)
42.7
33.6
16.9
7.1
3.2
Control
GENERATION
b I
DAY
8
93
55
68
95
100
98
15
86
53
60
86
75
93
22
0
28
60
83
63
83
GENERATION
II
DAY
29
-
3
60
75
70
95
36
-
2
53
73
60
83
43
-
2
35
70
58
80
GENERATION
III
DAY
50
-
2
98
100
73
98
58
-
2
83
93
70
88
64
-
2
78
78
65
78
Each 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
TABLE 12. MEAN PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO ACROLEIN FOR 64 DAYS.
Mean
Measured
Cone, (ug/1)
42.7
33.6
16.9
7.1
3.2
Control
GENERATION^ I
DAY
15 22
5
1 8
16 21
14 16
2 14
10 10
GENERATION II
DAY
36 43
-
15 29
15 12
14 22
11 15
11 20
GENERATION III
DAY
57
-
17
17
12
13
16
64
-
22
15
14
14
17
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
30
-------
TABLE 13. MEANa PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO HEPTACHLOR FOR 64 DAYS
Nominal
Cone, (ug/1)
50.0
25.0
12.5
6.25
3.12
Control
GENERATION I
DAY
8 15 22
88 65 28
100 95 78
95 93 90
93 93 88
100 85 80
100 83 80
GENERATION
II
DAY
29 36
24 23
94 55
94 88
94 90
62 50
93 88
43
15
53
85
65
50
80
GENERATION
III
DAY
50 57
23 23
68 65
100 95
75 70
75 55
95 90
64
13
40
75
48
40
73
Each 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
exposed to 12.5 jig/1 heptachlor was not significantly different
from controls. Although survival of daphnids exposed to the
two lowest concentrations of heptachlor appeared to be reduced,
in view of the survival observed among daphnids continuously
exposed to 12.5 jag/1 we do not consider these observations to be
pesticide related (Table 13). Analysis of variance of data
related to production of young per femare Daphnia magna during
generations one, two, and three indicates no significant
differences due to treatment (Table 14).
TABLE 14. MEAN3 PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO HEPTACHLOR FOR 64 DAYS
Nominal
Cone, (pg/1)
50.0
25.0
12.5
6.25
3.12
Control
GENERATION I
DAY
15 22
24 63
18 44
34 60
30 54
46 42
33 52
GENERATION II
DAY
36 43
28 26
22 42
31 34
24 46
33 28
29 29
GENERATION III
DAY
57 64
44 36
41 59
41 52
32 44
40 21
41 47
Each value represents the mean of four replicates
'Duration of exposure for generations I, II, and III were days
1-22, 22-43, and 43-64, respectively
31
-------
Based on the observed effects of heptachlor on survival and
reproduction or Daphnia magna continuously exposed through
three generations, the estimated maximum acceptable toxicant
concentration for this species, based on nominal concentrations,
is >12.5 <25 pg/1.
Daphnia magna to endosulfan
Statistical analysis of data on survival of Daphnia continuously
exposed to endosulfan for 64 days indicated significant
differences due to treatment. Exposure to mean measured
concentrations of 79.7, 37.7, 15.3 and 7.0 pg/1 endosulfan for
22 days, significantly reduced survival of daphnids (Table 15) .
This effect on survival was cumulative, with survival of second
generation daphnids being significantly less than that of the
first generation. After one week of exposure of the third
generation, effects on survival again appeared to be cumulative
at these concentrations and was lower than during the first
week of the previous two generations. The poor survival of
third generation daphnids in the control and 2.7 pg/1 endosulfan
treatment after two and three weeks precludes drawing valid
conclusions about cumulative effects of exposure to this
concentration on survival between generations two and three.
TABLE 15. MEANa PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO ENDOSULFAN FOR 64 DAYS
Mean
Measured
Cone, (pg/1)
79.7
37-7
15.3
7.0
2.7
Control
GENERATION
bi
DAY
8
93
95
80
85
98
100
15
58
48
63
83
98
100
22
38
45
30
55
88
100
GENERATION
II
DAY
29
83
90
95
73
95
93
36
0
8
3
8
90
90
43
0
8
3
8
73
85
GENERATION
III
DAY
50
0
5
28
28
100
80
57
0
5
23
20
8
44
64
0
5
23
15
0
15
Each 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
Production of young during the first generation of Daphnia
exposed to endosulfan indicates no significant differences
which can be correlated to toxicant concentration (Table 16).
32
-------
TABLE 16. MEAN PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO ENDOSULFAN FOR 64 DAYS
Mean
Measured
Cone, (jag/1)
79.7
37.7
15.3
7.0
2.7
Control
GENERATION I
DAY
15 22
7 17
7 23
4 15
6 9
13 12
8 12
GENERATION II
DAY
36 43
-
5 2
23 43
18 19
19 24
17 34
GENERATION III
DAY
57 64
-
5 12
14 14
15 21
oc o
6 17
Each 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
f^
Mortality of animals was apparently due to fungal growth in
these containers
During the second generation, production of young was significantly
lower among daphnids exposed to 37.7 jag/1 endosulfan than in
controls and all other treatments. The previously mentioned
poor survival of third generation daphnids in controls and
2.7^ig/l endosulfan is reflected also in production of young
and prohibits any firm conclusions about third generation
reproductive effects.
Based on the observed effects of endosulfan on survival of
Daphnia magna continuously exposed through two generations,
the estimated MATC for this species is >2.7 <7.0 jig/1.
Daphnia magna to trifluralin
Significant differences due to treatment were indicated from
statistical analysis of data on the survival of Daphnia
continuously exposed to trifluralin for 64 days. Continuous
exposure to 52.7, 25.6, 14.0, and 7.2 jag/1 trif luralin
significantly reduced survival of daphnids with survival
being cumulatively less from generation to generation
(Table 17) .
Production of young per Daphnia during the first two
generations was significantly reduced during exposure to
52.7, 25.6 and 14.0 >ig/l of trif luralin on days 22 and 38
(Table 18). On day 15 of generation one, production of young
33
-------
TABLE 17. MEAN PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO TRIFLURALIN FOR 64 DAYS
Mean
Measured
Cone, (ug/1)
52.7
25.6
14.0
7.2
2.4
Control
GENERATION
b I
DAY
8
90
90
88
90
98
100
15
70
90
83
45
93
85
22
38
48
65
43
90
85
GENERATION
II
DAY
29
35
20
85
50
90
95
38
0
5
43
23
88
98
43
0
2
13
20
50
80
GENERATION
III
DAY
50
0
0
38
8
93
80°
57
0
0
35
8
93
80
64
0
0
15
0
75
75
Each value represents the mean of four replicates
DDuration of exposure for generations I, II, and III were days
1-22, 22-43, 43-64,respectively
i
'On day 47, two control containers over flowed with loss of
animals. Mean survival is for remaining two replicates
TABLE 18. MEAN PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO TRIFLURALIN FOR 64 DAYS
Mean
Measured
Cone, (pg/1)
52. 7
25.6
14.0
7.2
2.4
Control
GENERATION^ I
DAY
15 22
3 5
15 12
11 15
11 25
18 30
13 29
GENERATION II
DAY
38 43
0 0
8 0
18 9
32 15
31 8
30 13
GENERATION III
DAY
57 64
0 0
0 0
16 2
16 0
23 5
12 9
Each 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
34
-------
.was reduced only for Daphnia in 52.7 pg/1 indicating possible
cumulative effects on reproduction. On days 43, 57 and 64,
no further differences could be attributed to toxicant
concentration.
Based on the observed effects of trifluralin on survival
of Daphnia magna continuously exposed through three
generations, the estimated MATC for this species is >2.4
<7.2 jug/1.
Pimephales promelas to acrolein
Survival and growth of fathead minnows exposed to acrolein
for 30 and 60 days were similar for all treatments (Table
19). With the exception of the A replicate of 20.8 ug/1
where fish were lost due to a diluter malfunction, no
significant differences in survival and growth were observed
after 35 weeks exposure to acrolein.
Spawning of fathead minnows occurred in all acrolein treatments
and the controls between test days 130 and 212. Spawning
activity was generally below what has been observed in
other chronic exposures (Table 20). With the exception of
the B replicate of 20.8 jig/1 (where no spawning was observed)
no significant differences were observed in number of
spawnings per female, number of eggs spawned per female, number
of eggs per spawn, and the percent hatchability of eggs
which were spawned. The complete lack of spawning activity
in 20.8 jig/1 acrolein may be related to the diluter malfunction
which resulted in the death of fish in the A replicate of
this concentration. The malfunction resulted in the emptying
of the entire mixing chamber volume through the chemical
cell corresponding to this concentration. It is not known how
many times this occurred during an overnight period but the
measured concentration during that week's sampling interval
was 60 jig/1 or approximately 20 pg/1 higher than the mean
measured concentration for the highest treatment level. We
believe that the concentration of acrolein rose high enough
during this short period to kill the fish in A replicate
and severely stressed the fish in the B replicate resulting in
their failure to spawn. The fact that fathead minnows in the
highest treatment level (41.7 jag/1) survived and spawned as well
as controls points to this malfunction in the dilution system
as the cause for the poor performance among fish in the second
highest treatment.
No second generation (Fj) fathead minnows were exposed in the
20.8 jig/1 of acrolein treatment. Two larval groups exposed
to 41.7 /ag/1 of acrolein for 60 days experienced nearly complete
mortality (2% survival), and survival was significantly less than
35
-------
TABLE 19. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephal.es promelas) AFTER 30 AND
60 DAYS, AND 35 WEEKS CONTINUOUS EXPOSURE TO ACROLEIN "
Mean measured acrolein concentration (jjq/1)
Item
30 DAYS
% Survival3
Total Length
(mm)
S.D.
60 DAYS
% Survival
Total Length
(mm)
S.D.
35 WEEKS
% Survival
«•/*
#
-------
TABLE 20. SEXUAL DEVELOPMENT, SPAWNING, HATCHABILITY OF EGGS, AND SURVIVAL AND GROWTH
OF OFFSPRING AFTER 30 AND 60 DAYS, FOR FATHEAD MINNOWS (Pimephales promelas)
CONTINUOUSLY EXPOSED TO ACROLEIN
Mean measured acrolein concentration (ug/1)
Item
# ?
spawns/ 9
eggs/9
eggs/spawn
% Hatch-
ability a
(N)
30 DAYS
% Survival
Total Length
(mm)
S.D. b
60 DAYS
% Survival
Total Length
(mm)
S.D.
# larval
groups c
41.7 ppb
A B
10 8
1 1
106 86
118 99
83 72
(4) (1)
2
23
2
37
2 0
20.8
A B
0 4
0
0
0 0
: :
-
0 0
11.4
A B
6 8
1 2
147 222
110 136
90 72
(6) (6)
37 29
17 19
2.3 2.8
37 27
29 24
4 2 3.3
2 2
6.4
A B
5 6
5 1
503 108
101 93
90 84
(10) (2)
62 35
15 14
3.7 2.5
43 35
24 24
5.9 3.8
3 1
4.6
A B
7 8
2 2
150 241
81 107
83 85
(6) (9)
61 44
18 16
3.9 3.7
50 44
26 23
5,3 5.0
2 2
Control
A B
9 12
1 1
140 74
105 88
91 92
(5) (5)
59 45
18 15
3.2 2.9
49 40
24 24
4.5 5.8
2 2
oo
Hatchability samples contained 50 one day old embryos
)
Standard deviation
Each larval group contained 40 one day old larvae
-------
that of controls and lower concentration treatments.
Based on the survival of newly hatched fry during the
continuous exposure of fathead minnows to various concentrations
of acrolein, the estimated maximum acceptable toxicant
concentration of acrolein for this species is >11.4
<41.7 jag/1.
Pimephales promelas to heptachlor
Continuous exposure for 30 days to concentrations of heptachlor
as high as 1.84 jug/1 had no significant effect on survival
and growth of fathead minnows (Table 21). After 60 days
exposure, survival was significantly reduced for fathead
minnows exposed to 1.84 pg/1 of heptachlor; no fish survived
the 30-60 day exposure interval. After 40 weeks of exposure
no significant differences were observed in survival and
growth of fathead minnows exposed to the other concentrations
of heptachlor and controls.
Spawning of fathead minnows occurred in all heptachlor treatments
and controls between test days 141 and 261. The number of
spawns per female and number of eggs per female, were
similar for all tanks with surviving fish with the exception of
the A replicate of 0.86jug/l (Table 22). In this tank the
number of spawns per female and eggs per female was
significantly increased above all others. Parental fish in
this tank experienced only 40% survival after 40 weeks and it
is interesting that the few remaining fish exhibited a high
level spawning activity. Possibly the reduced competition
for spawning territory caused by reduced number of surviving
fish was responsible for this phenomenon. No significant
differences existed in hatchability of eggs from fish exposed
to concentrations of heptachlor as high as 0.86 jag/1.
Statistical analysis indicated no significant differences in
percent survival or growth of F^ fathead minnows after 30 and
60 days of continuous exposure to heptachlor concentrations
as high as 0.86 jug/1.
Based on these data the estimated maximum acceptable toxicant
concentration of heptachlor for this species is >0.86<1.84 M9/1•
Pimephales promelas to endosulfan
Continuous exposure for 60 days to concentrations of endosulfan
as high as 0.40 jug/l had no significant effect on the survival
and growth of fathead minnows (Table 23). However, during
the period between test days 117-145 all fish expired in both
duplicate tanks receiving a mean measured concentration of
0.40 jig/1 of endosulfan. At this time fish were one month from
the onset of spawning and were undergoing rapid development
38
-------
TABLE 21. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) AFTER 30 DAYS,
60 DAYS AND 40 WEEKS CONTINUOUS EXPOSURE TO HEPTACHLOR
Mean measured heptachlor concentration (/ug/1)
Item
30 DAYS
% Survival3
Total Length
(mm)
S.D.b
60 DAYS
% Survival
Total Length
(mm)
S.D.
40 WEEKS
% Survival0
*/*
Total Length
(mm)
vo
Survival based on 40 fish per duplicate
Standard deviation
°Survival based on 15 fish per duplicate after thinning at 60 days
-------
TABLE 22. SEXUAL DEVELOPMENT, SPAWNING, HATCHABILITY OF EGGS, AND SURVIVAL AND GROWTH
OF OFFSPRING AFTER 30 AND 60 DAYS, FOR FATHEAD MINNOWS (Pimephales promelas)
CONTINUOUSLY EXPOSED TO HEPTACHLOR
Mean measured heptachlor concentration (ug/1)
Item
# 5
spawns/?
eggs/?
eggs/spawn
% Hatchability
# samples
hatch
30 DAYS
% Survival
Total Length
,(mm)
S.D.b
60 DAYS
% Survival
Total Length
(mm)
S.D.
* fr*c
groups
1.84
A B
0 0
a
0 0
-
0 0
0.86
A B
4 9
21 5
3582 659
173 129
90 89
26 21
77 50
16 17
2.5 3.3
77 50
25 29
4.3 5 . 8
4 4
0.43
A B
8 7
5 6
691 781
145 124
79 86
17 14
77 90
19 17
2.6 2.7
73 87
28 26
4.5 5.6
4 4
0.20
A B
8 7
5 4
580 830
122 215
87 88
10 8
70 54
16 16
2.4 3.3
61 50
24 24
4.0 5.0
4 4
0.11
A B
9 7
4 2
559 162
140 95
87 85
7 6
74 78
15 16
3.2 1.9
68 78
25 24
4.5 3.0
4 4
Control
A B
5 11
5 6
495 898
108 154
88 88
12 25
65 68
17 16
3.3 2.6
64 68
22 25
4.6 3.7
4 4
Hatchability samples contained 50 one day old embryos
3
Standard deviation
Each larval group contained 40 one day old larvae
-------
TABLE 23. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) AFTER 30 DAYS
60 DAYS AND 40 WEEKS CONTINUOUS EXPOSURE TO ENDOSULFAN
Item
30 DAYS
% Survival a
Mean Total
Length (nun)
S.D.5
60 DAYS
% Survival
Mean Total
Length (nun)
S.D.
40 WEEKS
% Survival0
-------
of secondary sex characteristics. Analysis of variance of
percent survival after 40 weeks for fish in all remaining
treatments indicated no significant differences due to treatment
We observed an infestation of fish by external parasites in
the B replicate of the 0.04 pg/1 treatment which we conclude
contributed to the decreased survival observed in this
experimental unit. Analysis of variance of total lengths and
total weights of fatheads after 40 weeks exposure also revealed
no significant differences due to treatment.
Spawning of fathead minnows began in most tanks with surviving
adults between test days 142-162. The only exception was
duplicate B of 0.04 jag/1 i-n which no spawning occurred.
Presumably, this lack of spawning was associated with the
effects of the disease observed. Mean number of spawns and
eggs per female fathead minnow in each duplicate tank were
highly variable and precluded sensitive statistical analysis
(Table 24). It is worth noting that spawning activity was
increased for fathead minnows in the highest concentration
of heptachlor with surviving adults (previously discussed
chronic exposure) as it was in the chronic for fatheads exposed
to 0.20 jug/1 of endosulfan (also the highest exposure level
with surviving adults) . Analysis of variance revealed no
significant differences due to treatment in the number of
eggs per spawn or in egg hatchability for tanks where spawning
had occurred . Three separate groups of eggs from control
spawns were incubated in 0.40/ag/l endosulfan and only 1 percent
of these eggs hatched successfully.
Analysis of variance of percent survival and mean total lengths
of FI fathead minnows after 30 and 60 days of exposure indicated
no significant differences due to exposure to 0.20 jug/1 and
less .
Based on these data derived from continuous exposure of fathead
minnows to various concentrations of endosulfan, the maximum
acceptable toxicant concentration for this species is estimated
to be >0.20<0.40
Pimephales promelas to trifluralin
Analysis of variance of percent survival and total lengths of
fathead minnows after 30 days exposure to trifluralin indicated
no significant differences due to treatment (Table 25) . After
60 days all fish continuously exposed to a mean measured
concentration of 16.5 jig/1 trifluralin had died. No significant
differences in percent survival or total length at 60 days existed
between any of the other treatments and controls . During the
period between test days 125-158 all fish expired in duplicate
tanks receiving a mean measured concentration of 8.2 pg/1 of
42
-------
TABLE 24. SEXUAL DEVELOPMENT, SPAWNING, HATCHABILITY OF EGGS, AND SURVIVAL AND
GROWTH OF OFFSPRING AFTER 30 AND 60 DAYS, FOR FATHEAD MINNOWS (Pimephales
promelas) CONTINUOUSLY EXPOSED TO ENDOSULFAN
Mean measured endosulfan concentration Qug/1)
Item
# 9
Mean spawns/?
Mean eggs/?
Mean eggs/ spawn
% Hatchabilitya
# samples
hatch
30 DAYS
% Survival
Mean Total
Length (mm)
S.D.C
60 DAYS
% Survival
Mean Total
Length (mm)
S.D.
# fry groups'3
0.40
A B
0 0
-
- -
-
]b
3 0
— -
-
— —
- —
-
— —
0 0
0.20
A B
7 4
10 19
968 1538
93 80
74 82
23 14
70 79
18 15
4.2 2.9
66 64
25 25
5.3 5,2
3 4
0.10
A B
9 5
7 5
559 278
76 60
87 83
17 6
83 71
16 18
2.7 3.9
81 61
23 27
4.1 5.4
4 4
0.06
A B
7 8
8 9
709 575
89 67
84 94
24 13
80 68
18 16
2.6 3.3
60 68
25 25
2.9 4.8
4 4
0.04
A B
5 0
2 0
104 0
58
86
4 0
74
17
3.9
68
23
4.1
2 0
Control
A B
7 6
4 7
490 543
126 79
82 84
21 17
68 87
18 18
3.1 3.7
64 82
25 24
1.8 5.5
4 5
10
Hatchability samples contained 50 one day old embryos
)
Control eggs transferred to this experimental unit
^
Standard deviation
Each larval group contained 40 one day old larvae
-------
TABLE 25. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) AFTER 30 DAYS,
60 DAYS AND 61 WEEKS CONTINUOUS EXPOSURE TO TRIFLURALIN
Mean measured trifluralin concentration (yg/1)
Item
30 DAYS
% Survival
Total Length
(mm)
S.D.a
60 DAYS
% Survival
Total Length
(mm)
S.D.
61 WEEKS
% Survival0
rf/9
Extra ff
removed
Total Length
(mm)
cf
$
Total Weight
(g)
rf
9
16.5
A B
23 87
19 20
6.2 5.0
0 0
-
- -
— _
-
-
— —
- -
— —
- —
8.2
A B
90 93
24 22
7.3 5.0
85 93
26 26
6.2 5.2
0 0
-
-
- —
-
— —
— —
5.1
A B
,
93 100
20 21
5.1 5.4
93 100
24 25
6.4 5.8
13 47
1/1 3/4
0 0
60 60
53 58
2.0 3.0
1.6 1.5
1.9
A B
100 97
22 20
5.2 4.3
100 95
26 26
5.2 5.2
80 67
5/7 5/5
0 0
66 55
56 57
3,6 2.2
1.7 1.6
1.5
A B
100 97
17 18
4.2 3.8
100 93
22 22
4.7 4.3
73 73
6/4 3/6
1 2
65 63
49 53
3.3 3.3
1.5 1.8
Control
A B
100 100
21 21
4.3 3.5
100 100
23 26
5.1 5.0
67 100
3/3 5/7
4 3
60 63
61 58
3.7 2.8
2.0 1,9
a.
Standard deviation
Survival based on 40 fish per duplicate
Survival based on 15 fish per duplicate after thinning at 60 days
-------
trifluralin. At this time the fish were approximately one
month away from the start of the spawning period and were
undergoing development of secondary sex characteristics.
During the period between test days 163-263 more than half
of the fish died in tanks receiving 5.1 ug/1 trifluralin.
Surviving adults exposed to 5.1 pg/1 trifluralin did not begin
spawning until after day 263, approximately 100 days later than
fish in controls and those exposed to 1.9 pg/1. Due to this
delay, and the generally low level of spawning activity
observed during the chronic exposure, the maximum photoperiod
was extended approximately two months in an attempt to induce
further spawning. Survival of adult fish at termination
(61 weeks) was significantly reduced among fish exposed to a
concentration of 5.1;ug/l of trifluralin. Analysis of variance
indicated no significant differences due to treatment in
total length or total weight of fathead minnows after 61
weeks exposure.
Little spawning activity occurred in any tanks with surviving
adult fathead minnows in spite of the extended maximum
photoperiod. What little spawning occurred was very erratic
and precluded valid statistical analysis (Table 26). Percent
hatchability of the few egg groups incubated was similar in
all remaining concentrations of trifluralin and indicated
that there were no significant differences due to treatment.
Percent survival and total lengths of FI fathead minnows
in the few places where growth chambers were started indicated
that continuous exposure to 5.1 pg/1 trifluralin had no effect
on these parameters when compared with controls. This would
be anticipated considering that FQ fathead minnows exposed to
5.1 ;ag/l did not experience reduction in survival until after
163 days of exposure and this period of mortality probably
corresponded with secondary sexual development.
Based on these data derived from continuous exposure of
fathead minnows to various concentrations of trifluralin,
the maximum acceptable toxicant concentration for this species
is >1.95<5.1 pg/1.
RESIDUE ANALYSIS
Three samples of pooled eviscerated carcasses of terminated
adult fathead minnows in each concentration of trifluralin
and heptachlor with surviving adults were analyzed to determine
residues in the fish. Results of these analyses (Table 27)
indicate that the amount of trifluralin residue accumulated
is approximately 1000X the concentration in water, is directly
proportional to the level of exposure, and that this response
is essentially linear over the range of concentrations tested.
45
-------
TABLE 26. SPAWNING RESULTS, EGG HATCHABILITY, AND SURVIVAL AND GROWTH OF FATHEAD
MINNOW (Pimephales promelas) FRY AFTER 30 AND 60 DAYS EXPOSURE TO VARIOUS
CONCENTRATIONS OF TRIFLURALIN
Item
# ? spawning
Mean spawning/
9
Mean eggs
spawned/ 2
Mean eggs/
spawn
% Hatchabilitya
# Hatchability
samples
30 DAYS
% Survival
Mean Total
Length (mm)
5.0.°
60 DAYS
% Survival
Mean Total
Length (mm)
S.D.
# fry groups0
Mean measured trifluralin concentration (jaq/1)
16.5
A B
0 0
0 0
0 0
8.2
A B
0 0
0 0
0 0
5.1
A B
5 4
3 7
225 432
63 60
89 73
83 60
16 19
3.5 3,2
62 59
21 28
5.4 4.8
2 2
1.9
A B
8 6
2 2
64 104
36 42
95 75
56
19
2.1
56
28
4.6
3 0
1.5
A B
5 6
0 1
0 20
20
-
-
0 0
Control
A B
3 7
2 3
55 229
23 76
94 88
84
17
3.6
74
22
5.7
0 2
o\
Hatchability samples contained 50 one day old embryos
Standard deviation
•^
'Each larval group contained 40 one day old larvae
-------
TABLE 27. MEAN MEASURED CONCENTRATIONS OF HEPTACHLOR ( + EPOXIDE) AND TRIFLURALIN IN
WATER (pg/1) AND IN THE EVISCERATED CARCASS (pg/kg) OF FATHEAD MINNOWS
(Pimephales promelas) CONTINUOUSLY EXPOSED TO THE PESTICIDES IN SEPARATE SYSTEMS
Tank Number
Item
HEPTACHLOR
(276 days)
water
Carcass
Bioconcentra-
tion factor (X)
TRIFLURALIN
(425 days)
water
Carcass
Bioconcentra-
tion factor (X)
1
1.84
*
*
16.5
*
*
2
0.86
17,733
- 6752
20,620
8.2
*
*
3
0,43
10,240
- 6841
23,814
5.1
4,900
- 3500
961
4
0.25
4 250
± 577
17,000
1.9
2,533
- 1401
1,333
5
0.11
2,450
- 1131
22,273
1.5
1,333
± 351
889
Control
250
± 62
-
293
i 94
-
All fish died during exposure
-------
.Heptachlor residues in fish are presented as the combined
residues of heptachlor and heptachlor epoxide. Heptachlor
epoxide residues in fish generally constituted 10-24 percent
of the total residue. The amount of heptachlor residue
accumulated is approximately 20,OOOX the concentration in
the water and the residue appears proportional to the level
of exposure with the response apparently linear over the
range of concentrations tested.
CALCULATION OF APPLICATION FACTORS
Estimated LC50 values, maximum acceptable toxicant concentrations,
and the application factors derived therefrom are summarized
for the four pesticides and the two species undergoing chronic
exposures (Table 28, 29). Application factors determining the
relationship between the acute and chronic toxicity of each
compound for Daphnia magna, are calculated using the MATC
and the 48-hour LC50. Similar application factors are
calculated for fathead minnows utilizing the MATC and the
incipient LC50 which Eaton (1970) suggests to be a better
measure of acute toxicity for this type of calculation.
In the acrolein, heptachlor, and trifluralin exposures
application factors for Daphnia were similar to those
calculated for fathead minnows. However, the application
factor calculated for Daphnia exposed to endosulfan is an
order of magnitude lower than that calculated for fathead
minnows and endosulfan.
TABLE 28. ACUTE AND CHRONIC TOXICITY VALUES FOR THE WATER
FLEA (Daphnia magna) AND CALCULATED APPLICATION
FACTORS FOR EACH PESTICIDE
Pesticide
Acrolein
Heptachlor
Endosulfan
Trifluralin
48-hour LC50
Oug/1)
57
(20-99)
78
(46-113)
166
(126-219)
193
(115-327)
MATC
(ug/1)
>16.9<33.6
>12.5<25.0
>2.7<7.0
>2.4<7.2
Limits on Application
factors (jug/1)
0.30-0.59
0.16-0.32
0.016-0.042
0.012-0.037
48
-------
TABLE 29. ACUTE AND CHRONIC TOXICITY VALUES FOR THE FATHEAD
MINNOW (Pimephales promelas) AND CALCULATED
APPLICATION FACTORS FOR EACH PESTICIDE
Pesticide
Ac role in
Heptachlor
Endosulfan
Trif luralin
Incipient LC50
(pg/D
84
(54-130)
7.02
(5.76-8.54)
0.86
(0.52-1.40
115
(48-211)
MATC
>11.4<41.7
>0.86<1.84
>0.20<0.40
>1.9<5.1
Limits on Application
factors (>ig/l)
0.14-0.50
0.12-0.26
0.23-0.47
0.017-0.044
49
-------
SECTION VI
DISCUSSION
The incipient LC50 of acrolein to fathead minnows (84 pg/1)
which we determined in a flowing bioassay is in basic
agreement with the values which have been previously
reported. Burdick et al. (1964) reported 24-hour LC50
values of 46 pg/1 of acrolein for brown trout and 79 pg/1
for bluegill. Alabaster (1969) reported a 24-hour TL^
of 140 pg/1 of acrolein for harlequin fish. Louder and
McCoy (1962) reported a narrow range of acrolein toxicity with
five species of warmwater fishes including the fathead minnow
with 24-hour TI^ values ranging from 62-183 pg/1. It is
not surprising that our LC50 value in a 6-day flowing
study agrees with 24-hour static values because the
majority of mortalities observed in the 6-day test occurred
within the first 48 hours of exposure. No comparable
toxicity data were found for invertebrates and
acrolein with which to compare our results for Daphnia
magna.
The 48-hour LC50 of 78 pg/1 of heptachlor which we determined
for Daphnia magna is higher than the 48-hour LC50 values of
42 and 47 pg/1 which Sanders and Cope (1966) reported for
Daphnia pulex and Simocephalus serrulatus, respectively.
It appears that temperature as well as species differences
are responsible for the discrepancy since our value was
determined at 19°C while Sanders and Cope determined their
values at 15.5°C. These authors provide the evidence that
probably temperature caused the difference by reporting a 48-hour
LC50 of 80 pg/1 for Simocephalus serrulatus exposed to
heptachlor at 70°F or 21uc.This value is essentially identical
to the 78 pg/1 which we determined for Daphnia maqna at 19°C,
and is a two fold increase over the value which the authors
reported for the same species at the lower temperature.
As might be expected the incipient LC50 of heptachlor to
fathead minnow (7.0 pg/1) which we determined in a 10 day
flowing bioassay is much lower than the 96-hour TI^ of
130 pg/1 which Henderson et al. (1960) determined for the
same species. The much smaller size of our fish, longer
exposure period, and continuous renewel of heptachlor in
the flowing system all contribute to the lower value. In
our prelininary investigations a 96-hour static exposure of
fathead minnows resulted in a calculated LC50 of 157 >ig/l
which is in good agreement with the 130 pg/1 value reported
by Henderson et al.
50
-------
The 48-hour LC50 of endosulfan for Daphnia magna which we
estimated at 166 p.g/1, is approximately three times greater
than the 62 pg/1 reported by Schoettger (1970) for a similar
exposure of the same species. A possible explanation for
the discrepancy could be the age of the test organisms.
Daphnids in our acute studies were <24 hour old and Schoettger
does not specify age of Daphnia magna which he used. The
incipient LC50 of endosulfan which we determined for fathead
minnows (0.86 ug/1) is lower than two previously reported
acute values, again to be expected due to the constant
flow and longer duration of our study. Pickering and Henderson
(1966) estimated the 96-hour TLm of thiodan (registered
tradename of endosulfan) to bluegills as 3.3 >ig/l/ while
Schoettger (1970) estimated the 120-hour LC50 to white suckers
to be 2.1 jag/1. Fathead minnows in both our acute and
chronic exposures proved to be much more susceptable to
endosulfan than were Daphnia magna. Schoettger (1970)
reported a general tendency of most aquatic invertebrates
to be less sensitive than fish to thiodan (endosulfan).
The 48-hour LC50 of 193 jug/1 of trifluralin which we calculated
for Daphnia magna is 2-3 times lower than the 560 pg/1 which
Sanders (1970) reported for the same species in a similar
exposure to trifluralin. Our value is in better agreement
with the 240 pg/1 trifluralin which was reported by Sanders
and Cope (1966) as the 48-hour EC50 for Daphnia pulex.
The incipient LC50 of trifluralin to fathead minnows we
estimated as 115 ;ig/l is in fair agreement with Park and
Worth (1965) who report a 96-hour LC50 of 93.4 ug/1 of trifluralin
with the same species. Trifluralin seems to be similar to
endosulfan in that invertebrate species appear less sensitive
than fish species.
*
The bioconcentration factors (i.e. 17,000-23,000 X) in fish
continuously exposed to heptachlor are consistent with those
reported for other organochlorine compounds such as DDT
(Macek and Korn,1970), dieldrin (Bionomics, E G & G, Inc.,
unpublished data), hexachlorbenzene (Parrish et al., 1975), Aroclor
1254 (Hansen et al., 1971), toxaphene (Mayer et al., 1975)
and tetrachlorobiphenyl (Branson et al., 1974). The
bioconcentration factors for all of these compounds, including
heptachlor, are generally 1-2 orders of magnitude higher than
those determined for other types of agricultural chemicals
(Macek et al. , 1975) .
The observed residues in the tissues of fish continuously
exposed to concentrations of heptachlor which appear to be
safe for fathead minnow (i.e. <1.0 pg/1) are similar to
concentrations of heptachlor residues which Henderson et al.
51
-------
(1969, 1971) reported in field collected samples. This
observation suggests that the concentrations of heptachlor
residues occurring in many natural waters which these authors
sampled are probably below the MATC for fathead minnows.
However, in view of the variability among the estimated limits
for the MATC for lindane and aquatic organisms (Macek et al.,
1975) it is conceivable that some subtle harmful effects 3ue
to chronic exposure to heptachlor residues may be occurring in
contaminated surface waters. Since similar information on the
occurrence of endosulfan and trifluralin residues in natural
waters is not readily available, an assessment of the hazard of
chronic exposure of aquatic organisms to these compounds is
not possible. However, the relatively high toxicity of these
compounds to aquatic species suggests the need for some monitoring
information to quantify existing levels of endosulfan and
trifluralin residues in surface waters.
52
-------
SECTION VII
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53
-------
Burdick, G.E., H.J. Dean, and E.T. Harris. 1964. Toxicity of
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57
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TECHNICAL REPORT DATA
(Mease read fusjnietions on the /rirnv before completing)
I REPORT NO
EPA-600/3-76-099
4. TITLE ANDSUBTITLE
TOXICITY OF FOUR PESTICIDES TO WATER FLEAS AND
FATHEAD MINNOWS*
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
November 1976
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
Kenneth J. Macek, Mark A. Lindberg, Scott
Sauter, Kenneth S. Buxton, and Patricia A. Cosia
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
EG & G Bionomics
790 Main Street
Wareham, Massachusetts 02571
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
Contract
68-01-0738
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
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
15. SUPPLEMENTARY NOTES *subtitle—Acute and Chronic Toxicity of Acrolein,
Heptachlor, Endosulfan, and Trifluralin to the Water Flea (Daphnia magna)
and the Fathead Minnow (Pimephales promelas)
16. ABSTRACT
Maximum acceptable toxicant concentrations (MATC's) for each pesticide
for both species were estimated by measuring survival, growth and
reproduction success as indicators of toxic effects. The MATC for
acrolein was estimated to be >16.9 and <33.6 yg/1 for daphnids and >11.4
,?nd < 41.7 for fathead minnows, those estimated for heptachlor were
>12.5 and <25.0 yg/1 for daphnids and >0.86 and <1.84 yg/1 for fathead
minnows, those estimated for endosulfan were >2.7 and <7.0 yg/1
for daphnids and >0.2 and <0.4 yg/1 for fathead minnows, and those
estimated for trifluralin were >2.4 and <7.2 yg/1 for daphnids and >1.9
and <5.1 yg/1 for fathead minnows.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Pesticides*
Toxicity*
Bioassay*
Acroleins
Minnows
Daphnia
Heptachlor
b.IDENTIFIERS/OPEN ENDED TERMS
Acute toxicity
Chronic toxicity
Endosulfan
Trifluralin
Aquatic organisms
c. COS AT l Held/Group
6F
7C
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
68
20. SECURITY CLASS (Tills page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
58
•i, US GOVUWttNl PKINIINCOfTlCt 1J7S— 757-056/M35
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