EPA-600/1-76-005
January 1S76
Environmental Health Effects Research Series
IN-YITRO SCREEHING METHODS EVALUATING THE
NEUROTOXIC POTENTIAL OF PESTICIDES
Health Effects Research Laborator>
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
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EPA-600/1-76-005
January 1976
IN-VITRO SCREENING METHODS EVALUATING THE NEUROTOXIC
POTENTIAL OF PESTICIDES
by
Toshio Narahashi
Department of Physiology and Pharmacology
DuRe University Medical Center
Durham, North Carolina 27710
Contract EPA-68-02-1289
Project Officer
John A. Santolucito
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, 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.
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CONTENTS
Page
I. Introduction 1
II. Methods 1
III. Results 6
IV. Tables 18
V. Figures 23
tit
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I. INTRODUCTION
Toxic effects of a variety of pesticides and environmental agents have been
a matter of public concern during the past decade. Toxicity may appear in
different forms in terms of the symptoms of poisoning, the speed of action, and
the severity of effects. Certain pesticides and environmental agents are potent
neuropoisons, some are metabolic poisons, and some others are carcinogenic compound
In view of the increasing number of pesticides and environmental agents which
come into contact with public life, it is urgently needed to develop methods whereby
toxicity of such agents can be evaluated with a minimum amount of cost and time
and with a maximum degree of accuracy and reliability. This contract work was
aimed at developing such methods for neurotoxic pesticides and environmental agents.
Several isolated nerve and muscle preparations have been tested for their
suitability for such toxicity evaluation. It was found that the abdominal nerve
cord preparations isolated from the crayfish were far more suitable than other
preparations examined.
II. METHODS
Crayfish Abdominal Nerve Cord
Sources of animal.
The crayfish Procambarus clarkii were purchased from several animal
supply houses.
Maintenance of animal.
The crayfish were kept alive in kitchen plastic trays (about 12 x 9 x 5 inches)
at a room temperature of 22°C. Some of 6 to 10 crayfish were put in a tray
containing tap water in a depth of approximately 2 inches. No attempt was made
to feed them. Water was changed every other day. Some of the crayfish died within
a week after delivery. However, those which survived the first week usually lived
1
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for more than several weeks, and no difficulty was encountered in maintaining
constant supply to the laboratory work.
Isolation of the abdominal nerve cord.
All legs and claws were severed from their origin, and the crayfish was
mounted with the ventral side up on a wooden board (8 x 6 x 0.5 inches) by means
of small nails. The ventral cuticle of the abdomen was removed by means of
scissors. Care was taken not to damage the abdominal nerve cord which was
located immediately beneath the ventral cuticle. Then both ends of the
abdominal nerve cord were tied with silk threads. The threads were attached
to the outside of the first and last abdominal ganglia so as not to damage these
two ganglia. All branches originating from each ganglion were cut, and the
entire abdominal nerve cord was isolated without damaging any ganglion.
van Harreveld solution was often dropped onto the open abdomen to prevent the
nerve cord from drying up.
Nerve chamber.
Several types of nerve chambers were compared for their suitability for the
present purpose. The type which was finally adopted is diagrammatically shown
in Figure 1. It was made of plexiglass. The structure is so simple that it takes
only a few hours to make one.
The nerve chamber consists of three compartments. The central compartment
is equipped with a pair of wire recording electrodes. Hypodermic needles
(gauge 24) perfectly serve for this purpose. The central compartment is also
equipped with an inlet and an outlet for bathing medium. Each of the side
compartments is equipped with a hypodermic needle (gauge 24). This needle serves
to tie off the thread attached to the nerve preparation.
The isolated abdominal nerve cord was mounted on the electrodes in the central
compartment of the chamber, and the compartment was filled with van Harreveld
2
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solution. The entire chamber was covered by a glass plate to prevent the
nerve from drying up. When recording was to be made, the solution was drained
by suction through the outlet of the central compartment. The nerve cord was
now hung in air on the electrodes. This increased the external electrical
resistance between the two recording electrodes thereby increasing the amplitude
of the recorded action potentials.
Electronics recording equipment.
The isolated abdominal cord generated action potentials spontaneously.
They originated from the ganglia. The spontaneous discharges recorded as
described above were in the order of millivolts in amplitude and about 100 per
second in frequency. The amplitude and frequency were maintained fairly
constant for 3 hours or longer.
Spontaneous discharges from the isolated nerve cord were amplified by a
RC coupled amplifier and displayed on a cathode ray oscilloscope. An audio
monitor was often used for long-term experiments, and was very helpful in detecting
changes in frequency of discharges. Any oscilloscope equipped with an amplifier
could be used for this purpose with a gain of 0.2 - 1 mV/cm on the face of the
scope.
Spontaneous discharges displayed on an oscilloscope were photographed on
a film using the Nihon-Kohden Oscilloscope Camera. More convenient way to
acquire data was to use an electronic counter. The Tektronix DC-503 Electronic
Counter proved quite satisfactory for this purpose. The recorded action potentials
were amplified and fed into the counter to directly read the frequency of discharges.
Physiological saline solution.
van Harreveld solution (van Harreveld, 1936) was used as the physiological
saline solution for the crayfish nerve cord. Tris buffer was substituted for
bicarbonate buffer in the original solution. The solution used contained
3
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207.3 mM NaCl, 5.4 mM KC1, 13.0 mM CaCl2.2H20, 2.6 mM MgCl2.6H20, 1.9 mM Trizma
HC1, and 0.4 mM Trizman Base with a final pH adjusted to 7.55.
Frog Neuromuscular Junction.
Sources and maintenance of animal.
The frogs Rana pipiens were purchased from various animal houses, and were
kept in containers with dripping tap water. For the present purpose, small
frogs were preferred to large frogs, because the equilibrium of the muscle with
a test compound was reached sooner with small muscles than with large muscles.
Isolation of the sciatic nerve-sartorius muscle preparation.
The frog was decapitated, pithed with a fine needle, and skinned. The
sartorius muscle was isolated with a short length of the sciatic nerve attached.
Both ends of the muscle were tied with silk threads.
Muscle chamber.
The muscle chamber used for the frog sartorius was a glass cyclinder with
25 mm in diameter and 80 mm in height. A rubber stopper was fitted into the
bottom of the cylinder. A metal hook attached to the rubber stopper served to
fix one of the threads that tied the muscle. The other thread that tied the
muscle was connected with a force displacement transducer (Grass model FT03C).
Ringer's solution was introduced to the bottom of the chamber through a glass
capillary, while it was sucked up from the surface of the fluid through another
capillary. A pair of silver-silver chloride electrodes were placed on both
sides of the muscle for direct (muscle) stimulation. The nerve was stimulated
via a suction electrode which was made of a glass capillary with a spiral silver-
silver chloride wire outside and another spiral silver-silver chloride wire inside.
Stimulation and recording.
The nerve and the muscle were stimulated alternately at a frequency of 0.2HZ.
Supramaximal stimuli were used. Isometric contractions of the muscle were
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recorded on a Grass Polygraph (model 5D) via a transducer.
Physiological saline solution.
Ringer's solution was used to bath the frog nerve-muscle preparation.
I contained 115 mM NaCl, 2.5 mM KC1, 1.8 mM CaCl2.2H20, 2.15 mM Na2HP04.7H20, and
0.85 mM NaH2PCvH20 with a final pH adjusted to 7.2.
Crayfish Neuromuscular Junction.
Stimulation and recording.
A claw was severed from the crayfish. The tip of the one side of the claw
was chopped off and inserted into a plastic tubing which was in turn connected
to an Ag-AgCl wire via van Harreveld solution. Another Ag-AgCl wire
was placed near the proximal cut end of the claw. These two wires served for
direct muscle stimulation. A suction electrode similar to that used for the
frog preparation was used to stimulate the nerve. Isometric muscle contractions
were recorded on a Grass Polygraph via a transducer as in the case of the frog
muscle.
Test Compounds
Test compounds were dissolved in pure ethanol in a concentration of
1 x ICf'M to 1 x 10"2M to make up stock solutions. Immediately before use, the
stock, solution was diluted with physiological saline solution to give a desired
final concentration of the compound. Control experiments were performed to see
the effect of ethanol itself.
Temperature
All experiments were conducted at a room temperature of 22°C.
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III. RESULTS
Crayfish Abdominal Nerve Cord
Under normal conditions, the abdominal nerve cord elicited spontaneous
discharges at a frequency of 50-100/sec. The amplitude of action potentials
was of the order of millivolts. The action of a test compound may be manifested
by an increase or a decrease in the frequency of discharges. A biphasic effect
could also be observed, with a usual pattern of a transient increase followed
by a decrease in frequency. These effects were produced within 30-60 min after
introduction of a test compound in a manner dependent upon the concentration.
In the absence of test compounds, spontaneous discharges were maintained at a
constant level for as long as 3 hours.
In addition to pesticides and other environmental chemicals provided by the
EPA for test, three insecticides were tested as models, including p,p'-DDT,
allethrin and toxaphene.
Organophosphorus and carbamate insecticides.
The effects of allethrin, p,p'-DDT, toxaphene, and organophosphorus and
carbamate insecticides on the spontaneous discharges are summarized in Table 1.
Detials of the effect of each compound are described below.
Ethanol control.
Since all test compounds were dissolved in ethanol to make up stock solutions,
the effect of ethanol itself was examined first. Figure 2 illustrates the
frequency of spontaneous discharges of the abdominal nerve cord before and during
application of 1% ethanol (v/v) and after washing with ethanol-free van Harreveld
solution. Ethanol caused no appreciable effect on the frequency of discharges
over a period of 30 minutes. Thus it can be concluded that ethanol may be used
as a solvent as long as itsfinal concentration does not exceed 1%.
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p.p'-DDT.
p,p'-DDT has been known to stimulate the abdominal nerve cord of the
cockroach to increase discharge frequency (see Narahashi, 1971, for references).
DDT was also effective in stimulating the crayfish abdominal nerve cord as
shown in Figure 3. The effect was moderate with 1 x 10~5M DDT, and was not
reversed after washing with DDT-free solution. At a concentration of 1 x 10~"M,
the increase in discharge frequency was more pronounced, the level after a 30-min
application attaining approximately 300% of that of the control. The effect
was not reversed but rather augmented after washing with DDT-free solution.
Toxaphene.
Toxaphene was effective in increasing the frequency of spontaneous discharges
(Figure 4). The effects were almost the same with 1 x 10~5M and 1 x 10~"M,
toxaphene, and the frequency was elevated to approximately 200% of that of the
control.
Allethrin.
Allethrin and other pyrethroids have been known to stimulate and paralyze
the abdominal nerve cord preparations of the cockroach and crayfish (Narahashi ,1971;
Camougis and Davis, 1971; Burt and Goodchild, 1971a,b; Camougis, 1973). In the
present experiments, allethrin was found to be most potent among the compounds
tested to stimulate and paralyze the crayfish abdominal nerve cord (Figure 5A).
The results of typical experiments are illustrated in Figures 5B to 5E.
Even at a very low concentration of 1 x 10"8M, allethrin caused a sizable increase
in the frequency of discharges. Each dot in Figure 5 and in subsequent figures
for other chemicals represents single measurement of the frequency for a period
of either 0.2 sec or 1 sec, and the measurements were repeated as are shown by
several dots. Large scatters of dots at each time during the latter half
of application of 1 x 10~8M allethrin (Figure 5B) indicate periodic bursts of
high frequency discharges. Similar effects were obtained with 1 x 10 7M
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allethrin (Figure 5C).
At a concentration of 1 x 10~6M, the effect of allethrin was represented
by a transient increase in frequency followed by a decrease and an eventual
paralysis (Figure 50). The stimulation occurred very quickly in 5 min after
application. At a concentration of 1 x 10~5M, a transient increase in discharge
frequency was quickly followed by a complete paralysis (Figure 5E). Recovery
after washing with allethrin-free solution was very poor or absent regardless
of the concentration.
Carbofuran.
Carbofuran was the only test compound that exhibited a potent stimulating
action comparable to that of allethrin. At a concentration of 1 x 10~8M,
the frequency of discharges was steadily increased attaining a maximum in 15
min (Figure 6A,B). After that, the frequency tended to decrease over a period
of 45 min. Essentially the same effect was observed with 1 x 10~6M carbofuran
(Figure 6c). At a concentration of T x 10~5M, however, a depressive action was
observed after a transient increase in frequency (Figure 6D). At a concentration
of 1 x ICf'M, no clear-cut stimulation resulted, and the nerve cord was paralyzed
completely (Figure 6E). Recovery after washing with solution free of carbofuran
was incomplete (Figure 6E).
Carbaryl.
Carbaryl was not as potent as carbofuran or allethrin, but slightly
stimulated .the nerve cord at a concentration of 1 x 10~6M (Figure 7A). Although
the average frequency was not substantially increased, burst of high frequency
discharges occurred toward the end of a 60-min application of carbaryl as is
illustrated by a dot at a high frequency level. At a concentration of 1 x 10"5M,
the stimulating effect was much more pronounced and was followed by a tendency
toward paralysis (Figure 7B). At a concentration of 1 x lO'^M, an exceptionally
8
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large transient increase in discharge frequency was quickly followed by a
complete paralysis (Figure 7C). Partial recovery occurred after washing with
carbaryl-free solution.
Leptophos.
The effect of leptophos on the abdominal nerve cord was not particularly
striking. At a concentration of 1 x 10~5M, the frequency of discharges tended
to increase transiently (Figure 8A). At a concentration of 1 x ICf'M, a small
transient increase was followed by a gradual decline of frequency (Figure 8B).
Baygon (Propoxur).
Baygon was moderately effective in stimulating and paralyzing the
nerve cord. It had no effect on discharges at a concentration of 1 x 10~7M.
At a concentration of 1 x 10~6M, a transient and slight increase in discharge
frequency occurred without sign of paralysis (Figure 9A). At a concentration
of 1 x 10~5M, bursts of discharges became evident as shown by scattered dots in
Figure 9B. A large transient increase in frequency followed by a paralysis
occurred at a concentration of 1 x lO'^M (Figures 9C).
Ferbam.
The effect of ferbam at a concentration of 1 x 10~6M or 1 x 10~5M was not
obvious (Figure 10A). At a concentration of 1 x ICT^M, bursts of discharges were
produced as shown by scattered dots in Figure 10B.
Dichlofenthion.
Dichlofenthion had no appreciable effect on discharge frequency at a
concentration of 1 x 10"5M. It caused some bursts of discharges at a concentrator
of 1 x lO'^M, but the effect was never striking (Figure 11).
Monocrotophos.
Monocrotophos had no effect on discharges at a concentration of 1 x 10~6M.
It caused a small and transient increase in frequency at 1 x 10"5M, but the effect
9
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was not remarkable (Figure 12A). Only at a concentration of 1 x lO'^M, the
stimulating effect became obvious but was never striking (Figure 12B). There
was no sign of paralysis at any of these concentrations.
Dursban.
The effect of dursban at a concentration of 1 x 10~6M or 1 x 10~5M was
almost negligible (Figure 13A). At a concentration of 1 x 10~'*M, however, dursban
caused an increase in discharge frequency which was maintained over a period of
50 min (Figure 13B). Bursts of discharges also appeared.
Sex attractants, growth regulators and chlordimeform.
The effects of several insect sex attractants, insect growth regulators,
and chlordimeform on the spontaneous discharges of the crayfish abdominal cord
are summarized in Table 2. Examples of experiments with the insect growth
regulator Altosid and the codling moth sex attractant Codlemone are illustrated
in Figures 14 and 15, respectively.
It is clearly seen that all of the insect growth regulators and sex
attractants tested (Altosid, Orfamone, ZR777, cis-11 tetradecenyl acetate, TH 6040,
and Codlemone) have no effect on the spontaneous discharges of the crayfish
abdominal nerve cord even at a very high concentration of 1 x lO"1* g/ml. The
insecticide chlordimeform had a moderate stimulating effect at a concentration
of 1 x 10"" g/ml.
Frog Neuromuscular Junction.
The sciatic nerve-sartorius muscle preparation of the frog has been examined
for its usefulness for evaluating the neurotoxicity of various pesticides.
Since the neuromuscular junction of this preparation is nicotinic in nature, control
experiments were performed with a typical nicotinic blocking agent and a typical
anticholinesterase agent. d-Tubocurarine and edrophonium were used for this
purpose.
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However, it was found that this preparation was not as sensitive as the
crayfish abdominal nerve cord to various compounds. Therefore, only several
test compounds were selected for the test. The results are summarized in
Table 3.
d-Tubocurarine.
d-Tubocurarine blocked neuromuscular transmission completely at a concentration
of 5 x 10 6M. The contraction induced by muscle stimulation was suppressed only
slightly. The effects were slowly reversed after washing the preparation with
drug-free Ringer. Figure 16 illustrates an example of such an experiment. The
observed strong neuromuscular blocking action of d-tubocurarine is to be expected,
and the results indicate that the method used here is satisfactory for examining
the neuromuscular blocking action of nicotinic agents.
Edrophonium. .
The next control experiments were done using edrophonium which is known
to inhibit cholinesterases reversibly. A fairly high concentration was required
for edrophonium to block neuromuscular transmission, 1 x ICT1* M having virtually
no effect. Neuromuscular transmission was effectively blocked at a concentration
of 1 x 10~3 M (Table 3, Figure 17). No marked potentiation or prolongation of
muscle contraction was observed at any concentrations tested. The results indicate
that although this preparation responds to the neuromuscular blocking action of an
anticholinesterase in a predicted manner, it may not be suitable to check the
neuromuscular stimulating action of an anticholinesterase.
Allethrin.
Allethrin was also examined for the purpose of comparison. The muscle
contraction evoked by nerve stimulation was augmented somewhat at low concentrations
(10~6 M - 10~5M), whereas it was suppressed at higher concentrations (1-5 x lO^M)
(Figure 18, Table 3). The contraction induced by direct stimulation was also
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decreased to some extent at high concentrations. Thus the frog neuromuscular
preparation respondsto the action of allethrin, but the sensitivity falls short
of that of the crayfish nerve cord preparation, the difference being of the order
of 1000.
Carbofuran.
As described in a previous section, carbofuran was among the most potent
insecticides in stimulating and paralyzing the crayfish abdominal nerve cord
(Table 1). Therefore, carbofuran was tested on the neuromuscular preparation
of the frog. As is shown in Figure 19, the response to indirect nerve stimulation
was suppressed more effectively than that to direct muscle stimulation by 2.5 x 10 "*M
carbofuran. Data with various concentrations of carbofuran are given in Table 3.
When compared with the crayfish abdominal nerve cord, the frog neuromuscular
preparation is much less sensitive to carbofuran. The former preparation was
affected at a concentration of 1 x 10~8M, whereas the latter was affected only
when the concentration was increased to 1 x 10~5M, the difference in sensitivity
being 1000-fold.
Leptophos.
At concentrations up to 2.5 x lO'1* M leptophos had virtually no effect on
the muscle contraction as evoked by either indirect or direct stimulation (Figure
20, Table 3).
Dichlofenthion.
Dichlofenthion (1 x 10"3 M) had little or no effect on the muscle contraction
evoked by either direct or indirect stimulation (Figure 21) except one case where
the nerve evoked contraction was drastically reduced (Table 3).
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Crayfish Neuromuscular Junction.
The nerve-muscle preparation of the crayfish has been examined for its
usefulness for evaluating the neurotoxicity of various pesticides. Since the
neuromuscular junction of this preparation is not nicotinic but glutaminergic
in nature, different results are expected from those with the frog neuromuscular
junction. It was found that this preparation was particularly insensitive
to organophosphate and carbamate insecticides. It was, however, very sensitive
to allethrin.
The results are summarized in Table 4.
Allethrin.
Allethrin stimulated and paralyzed the crayfish neuromuscular preparation
very effectively (Figure 22). Even at a concentration of 1 x 10"8 M, it
augmented the muscle contractions produced by either nerve or muscule stimulation
(Table 4).
Edrophonium.
This anticholinesterase had little or no effect on the crayfish neuromuscular
junction at 1 x 10~" M. At a higher concentration of 1 x 10"3 M, it suppressed
the muscle response evoked by nerve stimulation, but had no effect on the response
produced by direct stimulation (Figure 23, Table 4).
Ca rbofu ran.
Despite a potent action on the crayfish abdominal nerve cord, carbofuran
(1 x lO"1* M) had no effect on the muscle responses evoked by either nerve or
muscle stimulation (Figure 24, Table 4).
Dichlofenthion.
At a concentration of 1 x 10~5M, dichlofenthion had no effect on the muscle
response evoked by either direct or indirect stimulation (Table 4). At a
concentration of 1 x 10"1* M, an increase in response was obtained in three cases,
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whereas a decrease in response was obtained in three other cases (Figure 25,
Table 4).
Leptdphos.
At concentrations up to 2.5 x 10~5M, leptophos had no effect on the muscle
response evoked by nerve stimulation (Figure 26, Table 4).
IV. DISCUSSION
It is clearly shown that the crayfish abdominal nerve cord preparation
is much more sensitive thanthefrog neuromuscular preparation to various
neurotoxic agents. Differences in sensitivity are particularly evident for
allethrin and carbofuran. The nerve cord is 1,000 times more sensitive than
the frog muscle to either of these chemicals. Although the crayfish neuromuscular
preparation is as sensitive to allethrin as the crayfish abdominal nerve cord,
it is not sensitive to organophosphate and carbamate insecticides. Thus, in
terms of sensitivity to various pesticides, the abdominal nerve cord of the
crayfish is ranked top among the preparations examined.
The crayfish abdominal nerve cord is easiest to handle among the preparations
used. The nerve cord is stable over a 3-hour period under normal conditions,
and the effects of various drugs are quite reproducible. Data are obtained in
the form of the frequency per unit time on a counter which is easy to operate.
Though not tried in this study, it is possible to convert the digital data into
an analog form which is in turn registered on a strip chart recorder. Thus data
are read in a digital or analog form, depending on the availability of equipment
and on the degree of need to quantitative values.
With appropriate investment, it is also possible to handle several preparations
at a time to speed up the analysis. For example, with 4 channels of recording
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and data acquisition system, 4 experiments can be made simultaneously. Each
channel is composed of a preamplifier, a counter, a digital-to-analog converter,
and a strip chart recorder. It is also possible to feed the data to a computer
for analysis.
The frog neuromuscular preparation is also easy to handle and data are
reproducible. However, it generally takes a longer time for the effect of a
drug to attain a steady state than with the crayfish abdominal nerve cord
preparation. The crayfish neuromuscular preparation is more difficult to
handle, because muscle response fluctuates more widely than that of the frog
muscle.
The crayfish is inexpensive and can be obtained all the year round. It
is easy to keep in the laboratory for a long time without special equipment.
Thus the crayfish is a highly suitable material in this respect also.
In view of these considerations, the abdominal nerve cord preparation from
the crayfish is clearly the most suitable material to evaluate neurotoxicity of
various pesticides and other environmental agents with reasonably simple equipment
and moderate costs.
V. SUMMARY AND CONCLUSION
The abdominal nerve cord preparation isolated from the crayfish is far
superior to the frog neuromuscular and crayfish neuromuscular preparations for
evaluation of neurotoxicity of various pesticides and other environmental agents.
Neurotoxicity is manifested as stimulation and/or paralysis of spontaneous discharges
of the nerve cords. The techniques involved in this experiment are rather simple,
and require only reasonable amounts of conventional electrophysiological equipment.
Thus such toxicity evaluation can be performed with a modest amount of expense.
More sophisticated and efficient data acquisition systems could be developed using
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basically the same techniques.
The order of potency of various insecticides in stimulating the crayfish
abdominal nerve cord is as follows: allethrin = carbofuran > carbaryl >Baygon
(propoxur) = p,p'-DDT = toxaphene> ferbam = monocrotophos=dursban^ chlordimeform
> leptophos * dichlofenthion. No neurotoxic effect is exerted by insect sex
attractants and insect growth regulators including Altosid, Orfamone, Codlemone,
ZR 777, cis-11 tetradecenyl acetate, and TH 6040.
Acknowledgements
The author wishes to express his sincere thanks to Mr. Edward M. Harris for
maintenance of electronics equipment, Miss Kendall Fullenwider for help in data
analysis, and Mrs. Gillian Cockerill, Mrs. Delilah Munday and Mrs. Frances Bateman
for secretarial assistance.
REFERENCES
Burt, P.E. and R. E. Goodchild. (1971a). The site of action of pyrethrin I
in the nervous system of the cockroach Periplaneta americana L.
Entomol. Exp. Appl. 14: 179-189.
Burt, P.E. and R. E. Goodchild. (1971b). The spread of topically applied
pyrethrin I from the cuticle to the central nervous system of the cockroach
Periplaneta americana. Entomol. Exp. Appl. J4_: 255-269.
Camougis, G. (1973). Mode of action of pyrethrum on arthropod nerves.
In: Pyrethrum. The Natural Insecticide. Ed. by J.E. Casida, Academic
Press, New York and London, p. 211-222.
Camougis, G. and W. M. Davis. (1971). A comparative study of the neuro-
pharmacological basis of action of pyrethrins. Pyrethrum Post. 11: 7-14.
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Narahashi, T. (1971). Effects of insecticides on excitable tissues.
Advances in Insect Physiology, Vol. 8, Ed. by J.W.L. Beament, J.E. Treherne
and V.B. Wigglesworth, Academic Press, London and New York, p. 1-93.
van Harreveld, A. (1936). A physiological solution for freshwater
crustaceans. Proc. Soc. Exp. Biol. Med. 34: 428-432.
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TABLE 1
SUMMARY OF THE EFFECTS OF ORGANOPHOSPHORUS AND CARBAMATE INSECTICIDES
ON THE FREQUENCY OF SPONTANEOUS DISCHARGES IN THE ABDOMINAL
NERVE CORD OF THE CRAYFISH
Compound
10"
Concentration (M)
10~7 10"6 10~5
10"
Allethrin
p,p'-DDT
Toxaphene
Carbofuran
Carbaryl
Leptophos
Baygon (Propoxur)
+
0
0
Ferbam
Dichlofenthion
Monocrotophos
Dursban
0
0
0
0
0
0
0
+
0
+
+
+ Increase in discharge frequency.
++ Drastic increase in discharge frequency.
- Drastic decrease in discharge frequency.
0 No change in discharge frequency.
"
++
'"} Increase followed by decrease.
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TABLE 2
SUMMARY OF THE EFFECTS OF SEX ATTRACTANTS, GROWTH REGULATORS
AND CHLORDIMEFORM ON THE FREQUENCY OF SPONTANEOUS DISCHARGES
IN THE ABDOMINAL NERVE CORD OF THE CRAYFISH
Compound
Altos id
Orfamone
Codlemone
ZR 777
Cis-11 tetradecenyl acetate
Chlordimeform
TH 6040
10"7
0
0
0
0
0
0
0
Concentration
10"6
0
0
0
0
0
0
0
(g/mi)
10"5 10'*
0 0
0 0
0 0
0 0
0 0
0 +
0 0
+ Increase in discharge frequency.
0 No charge in discharge frequency.
19
-------
EFFECTS OF PESTICIDES AND OTHER BLOCKING AGENTS ON THE FROG
MUSCLE CONTRACTION EVOKED BY DIRECT AND INDIRECT STIMULATIONS
Data are given in percentage of decrease (-) or increase (+) in contraction.
Two numbers for a given concentration indicate the initial and later changes,
N, nerve stimulation. M, muscle stimulation.
Compound
Concentration (M)
10"6 5 x 10"6 10"5 5 x 10"5 10"" 2.5 x 10"" 5 x 10~" 10"3 2 x 10~-3
d-Tubocurarine
12-16-74A N -97
M 0
12-16-74B N -100
M -32
12-26-74B N -97
M -29
12-23-74B N -100
M -40
Edrophonium
12-24-74A N
M
12-24-74B N
M
12-23-74B N
M
01-10-75A N
M
01-10-75B N
M
Carbofuran
12-23-74B N -25
M -23
12-27-74A N
M
12-18-74A N 0
M
12-26-74A N
M
01-09-75A N
M
01-08-75A N
M
0 -80
0 -42
0 -75
0 -18
-17
0
-22 -63
-22 -47
-70
-50
-24 -100
-22 -34
+15, -27
0
-21 -79
0
-12 -85
-25 -71
-70
-60
-55
-29
20
-------
TABLE 3 (Continued)
Compound
Leptophos
12-17-74A N
M
12-24-74B N
M
12-24-74A N
M
Dichlofenthion
12-26-74B N
M
12-26-74A N
M
01-09-75B N
M
12-24-74B N
M
12-16-75B N
M
Allethrin
12-23-74A N
M
01-09-75B N
M
01-10-75C N
M
12-16-75B N
M
Concentration (M)
10~6 5 x 10~6 10~5 5 x 10~5 10"" 2.5x10"" 5x10"" 10"3 2 x 10";
000
0 +4
0 -8
0 0 00
0 0 00
-5
+3, -30
0 +9+0
0 +7 -14
0 0
0 0
0 -90
0 -43
0
+33 -36 -77
0 -14 -22
+7, -36
-37
-64
-46
+16 0 0
21
-------
TABLE 4
EFFECTS OF PESTICIDES AND EDROPHONIUM ON THE CRAYFISH
MUSCLE CONTRACTION EVOKED BY NERVE AND MUSCLE STIMULATIONS
Compound
10"
10"
Concentration (M)
10"6 10"5 2.5 x 10"5 10"" 10"3
Allethrin N
M
Edrophonium N
M
Carbofuran N
M
Dichlofenthion N
M
0
0
0
0
+ or -*
+ or -*
Leptophos N
M
N Nerve stimulation.
M Muscle stimulation.
+ Increase in muscle contraction.
- Decrease in muscle contraction.
+,- Increase followed by decrease.
* Increase in 3 cases and decrease in other 3 cases.
22
-------
TOP VIEW
INR
R OUT
SIDE VIEW
JL
02 4 6 8 10
cm
Figure 1. Diagrammatic sketch of the cham-
ber used for the crayfish abdominal nerve
cord. IN, inlet of bathing medium; OUT,
outlet of bathing medium; R, recording
electrode; N, nerve.
!%EtOH
200r
O
-------
p,p'-DDT
400
o
0)
(/>
o-
CD
300
200
100
0
i
0
20
40
60
80
100 120
Time(min)
Figure 3. Effects of 1 x 1(T5M (circles) and 1 x 10~4M
(squares) p,pl-DDT on the frequency of spontaneous discharges
of the crayfish abdominal cord. Each point represents the
mean ^standard error (4 experiments).
400
300
o
-------
CONTROL
IXIO"6M
ALLETHRIN
5 min
60 min
I mV
50 msec
Figure 5A. Effects of allethrin on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
lxKT8M Allethrin
300
o
Lu
100-
10 20 30 40 5O 60
Time (min)
70 80 90 KX)
Figure 5B. Effects of allethrin on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
25
-------
I x IO"7M Allethrln
M,|!
i i i i i i 1*1 i
0 10 20 30 40 50 60 70 8C
Time (min)
Figure 5C. Effects of allethrin on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
Allethrin
300
u
o>
\ 200
it
100
4
»
10 20 30 40 50 60
Time (min)
70 8G 90 100
Figure 5D. Effects of allethrin on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
26
-------
300r
0200
cr
OJ
100
lxlO-5MAIIethrin
I 1
0 10 20 30 40 50
Time(min)
60 70
80
Figure 5E. Effects of allethrin on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
CONTROL
I X IO"6M
CARBOFURAN
30 min
50 msec
Figure 6A. Effects of carbofuran on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
27
-------
300
u
0>
$200
0>
100
;
1 x IO"8M Carbofuran
10 20 30 40 50 60
Time(min)
70
80 90
Figure 6B. Effects of carbofuran on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
40Or
3OO-
o
o>
<
cr
0)
200
IOO
r
OHt
I x IO"6M Carbofuran
:! :
*
10 20 30 40 50 60 70 80 90 100
Time(min)
Figure 6C. Effects of carbofuran on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
28
-------
200
u
0>
V)
0)
LL
'00
I x IO'4M Carbofuran
I 1
10 20 30 40 50 60 70 8O
Time(min)
Figure 6D. Effects of carbofuran on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
300
200
0)
100
10
I x IO~5M Carbofuran
* *-.
20 30 40
50 60 70
Time(min)
80 90 100 110
Figure 6E. Effects of carbofuran on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
29
-------
lxlO"6MCarbaryl
300r
o
o>
s!
200
100
0
: i
10
20 30 40
50 60 70
Time(min)
80 90 100 110
Figure 7A. Effects of carbaryl on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
400r
300
o
o>
£
200
100
I xlO'5M Carbaryl
r
i
0 10 20 30 40 50 60 70 80 90 IOO 110
Time(min)
Figure 7B. Effects of carbaryl on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
30
-------
700
600
5OO
<
cr
400
300
200
100
lxlO'4MCorboryl
10 20 30 40 5O 60 70 80
Time(min)
Figure 7C. Effects of carbaryl on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
lxlO'5M Leptophos
200
o
o>
\ (00
CT
it
n
i 1
» .
f : 1
i * ? i f * r
* f
llllllllflll
10 20 30 40 50 60 70 80 90 100 110
Time (min)
Figure 8A. Effects of leptophos on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
31
-------
200
o
81
100
lxlO"*M Leptophos
10 20 30 40
50 60 70
Time (min)
80 90 100
110
Figure 8B. Effects of leptophos on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
200
o
cr
cu
100
lxlO"6M Baygon
0 10 20 30 40 50
Time (min)
60 70
80
Figure 9A. Effects of Baygon (propoxur) on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
400
lxlO~5M Baygon
300-
n-200
it
100
0
->
1
0
k
^
* fc
"Is* i
! «
i t i i i
" r
M
F
ill ill
10 20 30 40 50 60 70 80 90 100 IK
Time (min )
Figure 9B. Effects of Baygon (propoxur) on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
32
-------
3°°r lxKT4M Baygon
200
I
100
0 10 20 30 40 50 60 70 80 90 100 110
Time(min)
Figure 9C. Effects of Baygon (propoxur) on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
lxlO"5M Ferbam
2OO
i
o
a>
x!
IOO
a>
i I i
0 10 20 30 40 50 60 70 8O 90 100 110
Time(min)
Figure 10A. Effects of ferbam on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
I xlO'4M Ferbam
300 |
a> 200
CT
100
"r i
.. . ! '
1 1 I
i
M #
:
0 10 20 30 40 50 60 70 80 90 100 110
Time(min)
Figure 10B. Effects of ferbam on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
33
-------
IxlO^M Dichlofenthion
o t-vyvy
$
£ 100
n
'» ' ; i
! ? ; ,
: :
: i
, r , * ,
10 20 30 40 50 60 70 80 90 100 110
Tlme(min)
Figure 11. Effects of dichlofenthion on the frequency of spontaneous dis-
charges of the crayfish abdominal nerve cord. Each point represents a
measurement during a period of 0.2 or 1 sec.
200
o
£
u_
'00
0
I x IO~5M Monocrotophos
r :
0 10 20 30 40 50 60 70 80 90 100 110
Time(min)
Figure 12A. Effects of monocrotophos on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
200r
100
LL
IxlO M Monocrotophos
i
*
i
10 20 30 40 50 60 70 80 90 100 110
TimeOnin )
Figure 12B. Effects of monocrotophos on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
34
-------
lx!0'5M Dursbon
u
0)
<
p- 100
I
10 20 30 40 50
Time (min)
60
70
80
Figure 13A. Effects of dursban on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
500r
400-
lx!CT4M Dursban
0)
100
1 £
0 10 20 30 40 50 60 70 80
Time (min)
90 100
Figure 13B. Effects of dursban on the frequency of spontaneous
discharges of the crayfish abdominal nerve cord. Each point represents
a measurement during a period of 0.2 or 1 sec.
35
-------
ALTOSID(g/ml)
200
o
0>
xlOO
o-
it
n
r|
'r
0 | IO'7
» c * !
i i
1
1-
io-6 1 io-5
r r fc " *
i i i
1 io
" tt
i
-4
I
1
1
1
i
40
80
120 min
Figure 14. Effects of Altosid on the frequency of spontaneous discharges
of the crayfish abdominal nerve cord. Each point represents a measurement
during a period of 0.2 or 1 sec.
CODLEMONE(g/ml)
200
o
9
£lOO
2
u_
n
rl 0 1 lO'7
'i * . , 1 ,
L 1
iii
1 io-6 1 io-5
: r i ' &
h ' -
III
1 io-
r 5 I
i
i
1
i.
i
40
80
120 min
Figure 15. Effects of Codlemone on the frequency of spontaneous discharges
of the crayfish abdominal nerve cord. Each point represents a measurement
during a period of 0.2 or 1 sec.
36
-------
5 x IO-6M
d-TUBOCURARINE
»
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I2-26-74B
WASHING
mm.
Figure 16. Effects of 5 x 10 M d-tubocurarine on the contractions of the
frog sartorius muscle evoked by nerve(N) and muscle(M) stimulations.
I x IO'4M
EDROPHONIUM
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I2-24-74B
I x IO"3M
EDROPHONIUM
N M
Imln.
Figure 17. Effects of 1 x 10 M edrophonium on the contractions of the frog
sartorius muscle evoked by nerve(N) and muscle(M) stimulations.
37
-------
N M
I x I(T5M
ALLETHRIN
t
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I2-23-74A
I x IO'4M
ALLETHRIN
SxlO^M
ALLETHRIN
WASHING
I
min.
10"5M,
1(T4M,
Figure 18. Effects of 1 x 10 X 1 x 10~X and 5 x 10'^M allethrin on the
contractions of the frog sartorius muscle evoked by nerve(N) and muscle(M)
stimulations.
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
1-8-75A
2.5 » ICT«M
CARBOFUWN
WASHING
I mln.
Figure 19. Effects of 2.5 x 10~^M carbofuran on the contractions of the
frog sartorius muscle evoked by nerve(N) and muscle(M) stimulations.
33
-------
i x icr5M
LEPTOPHOS
t
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I2-24-74A
WASHING
t
WASHING
I min.
Figure 20. Effects of 1 x 10~4M and 2.5 x 10~4M leptophos on the con-
tractions of the frog sartorius muscle evoked by nerve(N) and muscle(M)
stimulations.
FROG MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION , x (0-3M
I2-26-74A DICHLOFENTHION
' ,////,/ ,/
2xKT3M
DICHLOFENTHION
WASHING
t
I min.
Figure 21. Effects of 1 x 10~3M and 2 x l(r3M dichlofenthion on the contrac-
tions of the frog sartorius muscle evoked by nerve(N) and muscle(M) stimula-
tions.
39
-------
CRAYFISH MUSCLE
NERVE(N)AND
MUSCLE(M) STIMULATION
2-Z4-7SA
lxKT7M
ALLETHRIN
I min.
Figure 22. Effects of 1 x 10" M allethrin on the contractions of the cray-
fish muscle evoked by nerve(N) and muscle(M) stimulations.
CRAYFISH MUSCLE
NERVE STIMULATION
2-I8-75A
I x IO'4M
EDROPHONIUM
t
I x IO'3M
EDROPHONIUM
WASHING
I
'Og.
I min.
Figure 23. Effects of 1 x 10'4M and 1 x 1CT3M edrophonium on the contrac-
tions of the crayfish muscle evoked by nerve stimulations.
40
-------
I x icr4M
CARBOFURAN
I
CRAYFISH MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I-2-75B
N M
/ r>
WASHING
I
I min.
Figure 24. Effects of 1 x 10 M carbofuran on the contractions of the cray-
fish muscle evoked by nerve(N) and muscle(M) stimulations.
CRAYFISH MUSCLE
NERVE(N) AND
MUSCLE(M) STIMULATION
I-30-75B
I X ICT4M
DICHLOFENTHION
I
N M
WASHING
*
I20g.
I min.
Figure 25. Effects of 1 x 10"4M dichlofenthion on the contractions of the
crayfish muscle evoked by nerve(N) and muscle(M) stimulations.
CRAYFISH MUSCLE
NERVE STIMULATION
2-19-756
LEPTOPHOS
\
WASHING
I min.
Figure 26. Effects of 2.5 x 10'^M leptophos on the contractions of the cray-
fish muscle evoked by nerve stimulations.
41
-------
TECHNICAL REPORT DATA
fflease read Initriiciiuns on tlic reverie before completing}
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
IN-VITRO SCREENING METHODS EVALUATING
THE NEUROTOXIC POTENTIAL OF PESTICIDES.
5. REPORT DATE
January 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Toshio Narahashi
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Physiology and Pharmacology
Duke University Medical Center
Durham, North Carolina 27710
10. PROGFIAM ELEMENT NO.
11.
1EA078
EPA-68-02-1289
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The abdominal nerve cord preparation isolated from the crayfish is far superior
to the frog neuromuscular and crayfish neuromuscular preparations for evaluation of
neurotoxicity of various pesticides and other environmental agents. Neurotoxicity
is manifested as stimulation and/or paralysis of spontaneous discharges of the nerve
cords. The techniques involved in this experiment are rather simple, and require
only reasonable amounts of conventional electrophysiological equipment. Thus such
toxicity evaluation can be performed with a modest amount of expense. More sophis-
ticated and efficient data acquisition systems could be developed using basically
the same techniques.
The order of potency of various insecticides in stimulating the crayfish
abdominal nerve cord is as follows: allethrin - carbofuran > carbaryl > Baygon
(propoxur) = p,p'-DDT = toxaphene > ferbam * monocrotophos = dursban = chlordimeform
> leptophos - dichlofenthion. No neurotoxic effect is exerted by insect sex
attractants and insect growth regulators including:Altosid, Orfamone, Codlemone,
ZR 777, cis-11 tetradecenyl acetate, and TH 6040.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Toxicity
Nerve Cells
Pesticides
Evaluation
b.lCENTIFIERS/OPEN ENDED TERMS
c. COSATl Field/Group
06, A
06, F
06, T
!J. DISTRIBUTION STATEMENT
L
RELEASE TO PUBLIC
19. SECURITY CLASS (This KcpcrtJ
UNCLASSIFIED
21. NO. OF PAGES
45
20. SECURITY CLASS (This pa^i.-
UNCLASSIFIED
22. PRICE
Form .'220-1 (9-73)
42
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