EPA-600/1-76-008
January 1976
Environmental Health Effects Research Series
MODE OF ACTION OF CYCLODIENE INSECTICIDES
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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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 ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies re-
lating to the tolerances of man for unhealthful substances or
conditions. This work is generally assessed from a medical view-
point, including physiological or psychological studies. In ad-
dition to toxicology and other medical specialities, study areas
include biomedical instrumentation and health research techniques
utilizing animals - but always with intended application to human
health measures.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/1-76-008
January 1976
MODE OF ACTION OF CYCLODIENE INSECTICIDES
by
Larry A. Crowder
Department of Entomology
University of Arizona
Tucson, Arizona 85721
R-800384
Project Officer
Ronald L. Baron
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
Research Triangle Park, North Carolina 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.
ii
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ABSTRACT
This report contains information concerning the mode of action, excretion,
and metabolism of the cyclodiene insecticides. Toxaphene was the primary
candidate for investigation with major emphasis on the mammalian system.
36
Excretion of Cl-toxaphene was studied in the laboratory rat. Upon
extraction, most of the radioactivity occurred in the water fractions of
urine and feces as ionic chloride, indicating considerable metabolism of
toxaphene. Only minimal storage appeared to occur.
Uptake of radioactivity in several tissues of Leucophaea maderae was deter-
36 ~~~~~~~~
mined after injections of Cl-toxaphene. In subcellular particles of
ventral nerve cord and brain, significant levels of 36C1 occurred in the
larger cell fragments; microsomes were also labelled. Ventral nerve cords
of L. maderae and Periplaneta americana showed increased activity when
exposed to toxaphene.
The toxicity of toxaphene to Gambusia affinis was divided into 5 stages,
and the residue level at each stage was determined. Metabolic alteration
of toxaphene appeared to be minimal. Excretion was not observed.
This report was submitted in partial fulfillment of a grant (R-800384) to
the University of Arizona from the Environmental Protection Agency.
iii
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CONTENTS
Page
Abstract iii
List of Figures vi
List of Tables viii
Acknowledgements J-x
Sections
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Methods and Materials 5
A Fate of Toxaphene in the Rat 5
1. Uptake and Excretion Experiments 5
a. Experimental Design 5
b. Analytical Procedures 5
2. Metabolism by the Liver 6
B Fate of Toxaphene in Cockroaches 6
1. Uptake in the American Cockroach,
Periplaneta americana 6
2. Uptake in Leucophaea maderae 7
a. Distribution Following an Injected
Dose 7
b. .In Vitro Studies of Distribution
in Nerve 7
C Uptake of Cl-Toxaphene in Mosquitofish 10
1. Selection of a Gambusia Population 10
2. Preparation of Solutions 10
3. Extraction Techniques 10
4. Radioassay 10
5. Toxicity Experiments 11
6. Uptake Experiments 11
7. Excretion Experiments 11
iv
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Page
8. Partitioning of Metabolites 12
D Effect of Toxaphene on the Nervous System 12
1. Toxicity Experiments 12
2. Electrophysiological Studies of
Cockroach Nerves 12
3. Effect on Ion Fluxes in Cockroach
Nerves 13
V Discussion 14
A Fate of Toxaphene in the Rat 14
1. Uptake and Excretion 14
2. Metabolism by the Liver 26
B Fate of Toxaphene in Cockroaches 26
1. Uptake in the Americana Cockroach,
Periplaneta americana 26
2. Uptake in Leucophaea maderae 29
C Uptake of Cl-Toxaphene in Mosquitofish 37
D Effects of Toxaphene on the Nervous System 53
1. Electrophysiological Studies of
Cockroach Nerves S3
2. Effect on Ion Fluxes in Cockroach
•Nerves 55
VI References 65
VII List of Publications 69
VIII Glossary 70
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Administered a Single Dose and Redose of Cl-
FIGURES
No. Page
1 Flow Sheet Representing Preparation of Residue
for Gas-Chroraatographic Analysis 8
2 Accumulative Excretion of Radioactivity in Rats
Administe
Toxaphene
3 Relative Percent Recovery of Cl in Excretion
of Rats Following Cl-Toxaphene 17
4 Ionic Cl Excretion in Water Fractions of the
Rat Feces and Urine Following Cl-Toxaphene
(20 mg/kg) 23
3fi
5 Total Uptake of Cl by Rat Tissues and Organs
36
Following a Single Dose of Cl-Toxaphene (24 rag/
kg) 25
36
6 Passage of Cl Through the Intestinal Tract into
the Feces of Rats Following Cl-Toxaphene (24 mg/
kg) 27
7 Penetration of Cl-Toxaphene into the American
Cockroach 31
36
8 Uptake of Cl-Toxaphene in Nerve Cords of
Leucophaea maderae Incubated Jin Vitro 32
9 Percent Recovery in Rinse Series Relative to
Cl-Toxaphene Uptake in Nerve Cords of Leucophaea
maderae Incubated Jin Vivo 33
10 LCcn Determination for Gambusia affinis After 20
3\) "^ ' ~~^~~'
Hours Exposure 40
11 LT,n Determination for Gambusia affinis at 2000
36
ppb Cl-Toxaphene 41
12 Percent Mortality of Gambusia affinis as a Function
of Exposure to 2000 ppb Toxaphene 42
13 Uptake of Cl-Toxaphene in Gambusia affinis as a
Function of Exposure to 2 ppm Cl-Toxaphene 43
14 Uptake of Cl-Toxaphene for Large and Small Gambus ia
affinis as a Function of time 46
VI
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No. Page
"~"~"" 36
IS Body Load of Cl-Toxaphene and TR Residues in
Gambusia affinis as a Function of Time in Fresh
Water 4?
16 Mean Body Burden of Cl-Toxaphene and TR Residues
in Gambusia affinis at Each Stage of the Toxicity
Syndrome 50
17 LDcn (^8 hour) of Toxaphene on Leucophaea maderae 34
18 Oscillograph of Spontaneous Nerve Activity from
Ventral Nerve Cord of Leucophaea maderae 56
19 Oscillograph of Nerve Activity Immediately Fol-
lowing a Dose of 2,1 mg Toxaphene on the Ventral
Nerve Cord of Leucophaea maderae 57
36
20 Uptake of Cl in Abdominal Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
10~7M Toxaphene 59
21 Uptake of Cl in Thoracic Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
10 M Toxaphene 60
22 Uptake of Cl in Abdominal Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
10"5M Toxaphene 61
36
23 Uptake of Cl in Thoracic Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
10~5M Toxaphene 62
24 Uptake of Cl in Abdominal Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
-4
10 M Toxaphene 63
36
25 Uptake of Cl in Thoracic Segment of Ventral
Nerve Cord of Periplaneta americana Exposed to
10 M Toxaphene 64
vii
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TABLES
No. Page
36
1 Excretion of Cl in Urine and Feces of Rats
36
Following Cl-Toxaphene IS
2 Hexane-Water Extraction of Rat Feces Following
Cl-Toxaphene 19
3 Hexane-Water Extraction of Rat Urine Following
36
Cl-Toxaphene 20
4 Cl-Ionic and Non-Ionic Composition of Rat Feces
16
(Water Fraction) Following Cl-Toxaphene 21
36
5 Cl-Ionic and Non-ionic Composition of Rat Urine
(Water Fraction) Following Cl-Toxaphene 22
6 Uptake of Radioactivity in Various Rat Tissues
and Organs Following a Single Dose of Cl-
Toxaphene 24
7 Radioactivity Recovered From Rat Liver Homogenates
and Supernatants Incubated With Cl-Toxaphene 28
16
8 Penetration of Cl-Toxaphene into the American
Cockroach 30
36
9 Uptake of Cl in Leucophaea maderae Ventral
Nerve Cords After Injection with 175.4 p,g Cl-
Toxaphene per Insect 34
O/l
10 Distribution of Cl-Toxaphene in Tissues of
Leucophaea maderae 35
11 Toxaphene Recovered From Leucophaea maderae In-
jected with 175.9 ^g Cl-Toxaphene per Cockroach 36
12 Recovery of Cl-Toxaphene in Nerve Tissue of
Leucophaea maderae 38
13 Results of Regression Analysis for the Uptake
36
and Excretion of Cl-Toxaphene by Gambusia
affinis 44
14 Metabolic Partitioning of Toxaphene in Gambusia
affinis 48
O£
15 Cl-Toxaphene and TR Residues and Toxicity
Symptoms Exhibited at Each Hour in Gambusia affinisSl
Vlll
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ACKNOWLEDGEMENTS
Acknowledgement is made Co Curt C. Dary, Edward F. Dindal, Robert A.
Schaper, and Roy S. Whitson for aiding the conduct of these studies.
Appreciation is also extended to Dr. George W. Ware for his support of
this project. Special thanks go to Martha A. Castillo for her patience
in typing this manuscript, and Hazel C. Tinsley in lettering the figures.
This investigation is indebted to Dr. Ronald L. Baron, Project Officer,
for his valuable advice and guidance.
IX
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SECTION I
CONCLUSIONS
In Che rat, approximately one-half of an oral dose of Coxaphene (20 mg/kg)
is excreted within 9 days. Most of that excreted occurs as ionic chloride,
indicating considerable metabolism of toxaphene. It is suggested that
other organelles besides microsomes may be involved in this metabolism.
Only minimal storage occurs 1 day following treatment. A second dose given
on the 9th day is excreted in a similar manner.
Toxaphene accumulates in ventral nerve cords of Leucophaea maderae. The
large amount retained after rinsing indicates penetration or binding of
toxaphene by the nerve cord. Toxaphene localizes in the larger cell frag-
ments, e.g., fragments of nerve sheath, nuclei, and unbroken cells. Micro-
somes are also labelled.
Overt symptoms of toxaphene poisoning for L. maderae follow a pattern similar
to that reported for other cyclodienes. At the moribund stage, the ventral
cord gives evidence of endogenous activity. Ventral nerve cords of
Periplaneta americana show increased nerve activity when exposed to toxaphene,
Nerves affected by toxaphene show prolonged volleys of spikes (13-24 spikes
per burst and 8-12 bursts per minute) until apparent death of the nerve.
Sorption of toxaphene into Gambitsia affinis is a linear function with respect
to time. Excretion is not observed. Metabolic alteration of toxaphene
appears to be minimal. Differences in individual mortality appear to be
due to differences in uptake rather than in ability to tolerate particular
body loads of toxaphene.
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SECTION II
RECOMMENDATIONS
1. Currently, Coxaphene is commonly used in combination with methyl-
parathion, and more recently with methyl-parathion and chlordimeform. It
is recommended that it be determined whether the uptake, metabolism, storage,
and excretion of toxaphene in the mammal is influenced by these other
insecticides. Furthermore, experiments should be conducted to quantitate
the potentiation, if any, of one insecticide upon another in these combina-
tions. This would be important in determining efficacy in insects and
potential hazards in various non-target organisms.
2. Specific localization of cyclodienes and formation of nerve-cyclodiene
complexes was studied principally in insects during this project. This
work should be continued to include the mammalian systems. By measuring
the rate of nerve-cyclodiene complex formation, the time course and amounts
complexed could then be correlated with electrophysiological and ionic events.
3. It is recommended that studies of the effect of cyclodienes on ion fluxes
in nerve tissue continue and be expanded to include mammalian nerve. Dur-
ing this project, toxaphene's effect on spontaneous nerve activity was
studied; however, single-cell electrophysiological recordings of its effect
on individual neurons is necessary to help elucidate the mode of action.
Furthermore, it is suggested that ion fluxes (influx and efflux) in the
nervous system be measured with radioactive potassium, sodium, calcium,
and chloride. Only chloride influx was examined in this project. Related
to toxaphene's current usage in combination with other insecticides pre-
viously described, the effect of toxaphene on ion fluxes in nerve tissue
should also be explored.
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SECTION III
INTRODUCTION
Toxaphene (chlorinated camptiene with a chlorine concent of 67-69%) is
the major chlorinated hydrocarbon insecticide used on cotton. Studies on
the mode of action of toxaphene are of primary importance to the EPA and
other regulatory agencies, and as such this insecticide was selected for
investigation.
As is the case with other chlorinated hydrocarbon insecticides, the mode
of action of toxaphene and other cyclodienes has not been elucidated. What
little is known concerning their mode of action has been presented in reviews
by Oahm , Winteringham and Lewis , Roan and Hopkins , O'Brien ' , and
more recently by Brooks . Most of these authors concurred that cyclodienes
probably act by interfering with nerve transmission rather than as enzyme
inhibitors.
Cyclodiene insecticides are believed to have a mode of action similar to
that of other chlorinated hydrocarbon insecticides, e.g., DDT and lindane;
neurophysiological evidence indicates action on the central nervous system
78 9
(Lalonde and Brown , Wang and Matsumura , Shankland and Schroeder ). It
has been suggested that some cyclodienes must be metabolized into a toxic
g
form before they have pronounced neurotoxic effects (Wang and Matsumura ).
No theory has been put forth for the mode of action of cyclodiene insecti-
cides, but three attractive hypotheses have been generated for DDT and
lindane (Mullins , Matsumura and O'Brien ' , and Holan ). Experimen-
tation centered around these theories might aid in elucidating the mode of
action of cyclodienes.
It was hypothesized that cyclodienes may interfere with transport mechanisms
in membranes. Interactions with nerve membranes could lead to alterations
in ionic transport across the membrane and, therefore, result in electrical
potential modifications. By employing techniques of electrophysiology,
potentials and ionic fluxes could be determined in relationship to cyclo-
diene poisoning. Additionally, a knowledge of uptake, metabolism, and
excretion of cyclodienes could be considered along with electrophysiological
and symptomological studies.
-------
The objectives of Che project were to study:
(1) Metabolism and excretion of cyclodienes in various animals.
(2) Specific localization of cyclodienes in various tissues with special
emphasis on the nervous system.
(3) Nerve-cyclodiene complexes as related to nerve disruption following
acute poisoning.
(4) Ionic fluxes across nerve membranes from cyclodiene-treated animals.
-------
SECTION IV
METHODS AND MATERIALS
A. FATE OF TOXAFHENE IN THE RAT
1. Uptake and Excretion Experiments
a. Experimental Design -
Thirty-day-old albino rats (HolCzmann Co.)» weighing an average of 114 g,
were deprived of food 24 hrs prior to dosage. Twenty mg/kg of technical
36
grade Cl-toxaphene (42 ^Ci/g; Hercules, Inc.) in 0.5 ml of a peanut
oil-gum acacia solution was orally administered via a stomach tube. Controls
were dosed with 0.5 ml of the peanut oil-gum acacia solution. In each of
2 experiments, 3 treated and 3 control rats were placed into glass metabo-
lism chambers, which provided for separate collection of urine and feces
14
(Halladay ). Another group of treated and control animals were held for
organ and tissue sampling at 9 time intervals; 3 treated and 1 control were
used at each interval. On the ninth day, 3 treated and 3 control rats
were given an additional dose of 20 mg/kg; these animals were referred to
as "redosed". All holding and metabolism cages were maintained in an air
conditioned environment (22-25°C; 50% KH; L:D - 11:13). The animals were
provided Purina Laboratory Chow and water _ad_ libitum.
Urine and feces were collected daily, weighed, and stored at 0°C to await
further analysis. At scheduled time intervals, rats were sacrificed and
their organs and tissues excised, weighed, and stored at 0°C. Additionally,
the animals employed in the excretion experiment were sacrificed for organs
and tissue samples at the end of 9 and 20 days. Blood obtained from heart
punctures was immediately centrifuged at 3,000 rpm for 5 minutes in a refrig-
erated superspeed Sorvall centrifuge, model RC2-B, to precipitate cellular
matter.
b. Analytical Procedures -
Feces were thawed, air-dried, ground to a powder, and 1 g samples extracted
with 25 ml each of hexane and water. Urine samples were also extracted
with hexane and water. Aliquots of all extracts were then digested and
solubilized in NCS® (Amersham/Searle Corp.) and Triton X-100, using heat
to aid digestion. Tissue samples were thawed, minced with scissors, and
homogenized with NCS^ over heat.
5
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36
Determination of Cl in urine and feces water extracts was accomplished
with acidification (3-4 drops of 1 H HNO.J followed by precipitation
(several drops of O.SM AgNO^). After centrifugation, the precipitant was
discarded and the procedure repeated until AgNO, saturation was attained.
36
The supernatants were radioassayed to determine non-ionic Cl; the ionic
•J£ Q£ O£
Cl was then calculated by subtracting non-ionic Cl from total Cl.
Toluene based fluor (5 g PPO and 0.06 g POPOP/1 toluene) was added to all
samples. Radioassay was performed on a dual channel (Nuclear-Chicago
Model t>822) liquid scintillation spectrophotometer. Quench was corrected
using the external standard method.
2. Metabolism by the Liver
Adult male rats were sacrificed and their livers removed. A 3 g portion
of each liver was placed in 30 ml of ice-cold 0.25 M sucrose and homogenized.
The homogenate was centrifuged at ca. 12,000 x G for 10 min to obtain the
microsomal supernatant.
One ml samples of homogenate and microsomal supernatant (90 mg tissue/ml)
were placed in reaction vessels for incubation. The following was added
to each vessel: 0.05 ml glucose-6-phosphate dehydrogenase (0.1 rag/ml in
water), 1 ml glucose-6-phosphate (4.67 mg/ml in 50 mM phosphate buffer),
0.085 ml of 2.7 mM KC1, 0.125 ml NADP (8 mg/ml in 50 mM phosphate buffer),
O£
and 8 ml of phosphate buffer. One ^1 of Cl-toxaphene (50 p,g) in acetone
was added to the vessels of both samples; 1 M-l acetone was used as the
control. The vessels were incubated for 15 hours in a shaking water bath
at 37°C. Following incubation, samples were extracted twice with hexane
and water. Precipitated tissue was removed and digested with 2.5 ml of
All samples were then radioassayed.
B. FATE OF TOXAPHENE IN COCKROACHES
1. Uptake in the American Cockroach, Periplaneta americana
~ 26
P. americana cockroaches were topically dosed with 75 M-g of Cl-toxaphene
in 1 p,l acetone. At each of several time intervals, cockroaches were
weighed and rinsed with acetone. Memolymph was extracted according to
the method of Sternberg and Corrigan . Remaining cockroach carcasses
16
were homogenized in insect saline (Yamasaki and Narahashi ) and filtered
through 2 layers of cheesecloth. Samples of the homogenates as well as
the heraolymph were prepared for scintillation counting and radioactivity
6
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determined. Quench was corrected via the external standard method.
2. Uptake in Leucophaea maderae
a. Distribution Following an Injected Dose -
Adult male L. maderae. 2-6 weeks post final molt, were injected with 0.05 cc
toxaphene in mineral oil (175*4 ^g/dose) in the third abdominal segment
between tergites. All punctures were sealed with paraffin to avoid the
loss of the injected dose.
Controls consisting of both non-injected and insects injected with 0.05 cc
mineral oil were prepared. Test or control insects to be dissected within
24 hours were not provided with nourishment. Water was given to insects
sacrificed at 48, 72, 96, and 120 hours after injection.
Following incubation periods of 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours,
insect hemolymph was collected by the method of Sternberg and Corrigan ,
weighed, and then prepared for scintillation counting. Each insect was then
dissected for removal of fat body, entire alimentary canal, and abdominal
nerve cord. Weights of all tissues were recorded and each was prepared for
radioassay.
To determine the nature of the radio-labelled material found, tissues of
asymptomatic and symptomatic L. maderae were gas-chromatographed following
extraction and clean-up. This included tissues which were prepared for
radioassay and freshly prepared samples. Fresh samples were handled in
the same manner as above, but time of incubation and tissue preparation
differed (Fig. 1). The ethyl ether layer was also prepared for scintil-
lation counting to determine Cl content.
b. In Vitro Studies of Distribution in Nerve -
Six trials were performed using 3 ventral nerve cords of L. maderae. 2nd
to 6th ganglia--a total of 104 in all for each concentration of toxaphene,
-2 -7
6 concentrations in all, 10 - 10 M. Following dissection, 3 nerve
cords were weighed and placed in each of the center wells of Warburg flasks
containing a 0.5 ml concentration of toxaphene dissolved in mineral oil.
The Warburg flasks were incubated on a shaker for 24 hours at 37°C. Flasks
were also prepared for controls as above with 3 cords incubated in 0.5 ml
mineral oil.
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CENTRIFUGE
Insect
10 min @ 650 rpm
1 ml aliquot
hemolymph
I
1 ml acetone
agitate 1 min
0.2 ml water
agitate 1 min
I
5.0 ml ethyl ether
agitate 5 min
1 hour wait
Dissection
tissue
Homogenize
0.1 ml water/g tissue
0.25 ml acetone/g tissue
agitate 1 min
10 ml ethyl ether
agitate 5 min
1 hour wait
extract
extract
r
Water
Layer
I
Dry
0.23 ml
I
Solubilize
NCS®
I
Count
Ethyl
ether
layer
1
Dry
0.25 ml
I
50 ml
Hexane
I
Dry
0.25 ml
I
Sodium
Sulphate
Analyze
Gas Chromatography
I
Water
Layer
I
Dry
0.25 ml
I
Solubilize
NCS
I
Count
Ethyl
ether
layer
Dry
0.25 ml
I
5.0 ml
Hexane
I
Dry
0.25 ml
I
Sodium
Sulphate
I
Analyze
Gas Chromatography
Figure 1. Flow sheet representing preparation of residue of insect
tissue for gas-chromatographic analysis.
-------
After incubation, the cords were removed from the flasks and sent through
a series of 4 rinses at different time intervals for each rinse: rapid
0-5 sec, 100 sec, 10 min, and 20 win; one ml insect saline (Yamasaki and
Narahashi ) was used for each rinse. The cords were again weighed at the
20 min rinse.
Rinse vials, ambient solutions left in the Warburg flasks, and nerve cords
were then prepared for scintillation counting. Contents of rinse vials
were concentrated, then solubilized in NCff^. Nerve cords were homogenized
in ground glass tissue grinders with 1.0 ml NC^ until a clear liquid was
formed. Ambient solutions were quickly transferred to scintillation vials
for radioassay.
Homogenized nerves from male L. maderae were assayed for toxaphene bind-
ing in a way similar to that for whole nerves. Nerve cords were homogenized
in 3 ml phosphate buffer pH 7.4 and then incubated for 24 hours with Cl-
toxaphene. Following incubation, the homogenate was centrifuged for 10
min at 1,000 x G. The precipitate was washed 3 times and radioassayed;
the rinses and supernatant were also radioassayed.
In a related experiment, male L. maderae were removed from the colony and
their head, legs, and wings were removed, with the head being retained for
brain removal. The body was opened dorsally, and viscera removed. Two or
3 drops of insect saline were then added to the body cavity to keep the
nerve cord both alive and moist. The nerve was further exposed by clean-
ing from it all fat body, trachea, and muscle. The nerve cord, from the
3rd thoracic to the 6th abdominal ganglia, and brain were removed. The
isolated tissues were placed in 5 ml of ice-cold 0.25M sucrose solution and
homogenized, with the homogenate kept at or below 4 C. The homogenates
36
were then transferred to centrifuge tubes. Cl-toxaphene in acetone,
giving a final concentration of 4.14 p>g/ml or 10 M, was added to each
homogenate. Controls were treated with equal volumes of acetone. Incu-
bation occurred at room temperature (24 C) for one hour, after which the
reaction was stopped by immersion in an ice bath. Homogenates were then
centrifuged at 20,000 x G for 45 min to settle all but the microsomes.
The supernatant was decanted, the sediment resuspended, recentrifuged, and
the supernatant added to the first. The combined supernatants were then
centrifuged at 100,000 x G for 45 min. The sediment containing micro-
somes from the 20,000 x G centrifugation was resuspended in 0.25M sucrose
and placed on a discontinuous density gradient consisting of 0.8, 1.0,
-------
1.2, 1.5, and 1.8 M sucrose layers, and centrifuged at 90,000 x G for 2
hours. The various sediments were removed, dried, and radioassayed.
C. UPTAKE OF 6C1-TOXAPHENE IN MOSQUITOFISH
1. Selection of Gambusia Population
G. affinis were collected from a total of 5 locations in Pima and Final
County, Arizona. Four were eliminated due to their high susceptibility
to toxaphene or the seasonable instability of their environment. Fish for
this research were obtained from a sewage oxidation pond located in Tucson,
Arizona. Fish were maintained in the laboratory in aged tap water for 24
hours prior to testing. Fish used in experiments were selected by randomly
dip-netting fish from the stock containers. The sex of individual fish
was not recordedi Mean weight was 1.082 + S.D. of 0.627 g.
2. Preparation of Solutions
Solutions used for mortality experiments to screen populations of G. affinis
were prepared by the appropriate aqueous dilution of a 1% acetone-based
36
stock solution of Cl-toxaphene (0.042 p,Ci/mg) in a method described by
Boyde and Ferguson . All other experimental solutions were prepared by
-2 36
appropriate aqueous dilution of a 10 M solution of Cl-toxaphene in re-
distilled acetone. Experimental solutions employed tap water which was
aged for at least 24 hours at room temperature prior to use. A solvent
control test was conducted at the rate of 2 ml redistilled acetone per 1
of water and resulted in 0% mortality after 48 hours. This concentration
of solvent was roughly 5 times as high as in the highest experimental
solution. All experiments were conducted at room temperature.
3. Extraction Techniques
Fish were homogenized in 40 ml of redistilled acetone in a ground glass
tissue grinder. After grinding, the extract was filtered through filter
paper, evaporated to dryness, and redissolved in 0.25 ml of distilled
water and 0.25 ml of hexane. Extraction of 10 fish topically dosed with
0.1 ml of Cl-toxaphene solution prior to extraction resulted in an
average recovery rate of 96.01 + S.D. of 5.28%. A recovery of 96% was
used in the calculation of all experimental data.
4. Radioassay
Fluor used in preliminary experiments was toluene based containing 0.06 g
POPOP and 5.0 g PPOlnone 1 of reagent grade toluene. Bray's solution, a
10
-------
dioxane based fluor, was employed in all other experiments. Bray's solution
contains 60 g naphthalene, 10 ml methanol, 20 ml ethylene glycol, 4 g PPO,
0.2 g POPOPandone 1 reagent grade dioxane. This fluor was stored in dark-
ness until use and, due to the instability of dioxane, added to samples
immediately before counting.
5. Toxicity Experiments
On the basis of preliminary tests, it was found that fish exposed to lethal
concentrations of toxaphene exhibited a particular chain of toxic symptoms.
This chain was divided into 5 characteristic stages as follows:
STAGE DESCRIPTION
1 Fish apparently healthy
2 Fish swimming at surface, often swimming
perpendicularly into the side of the
aquarium.
3 Fish losing equilibrium; no longer swim-
ming in horizontal attitude. Tail-end
down, swimming against sides of container;
sometimes rolling-over as they swim.
4 Fish prostrate on bottom of aquarium;
gills ventilating rapidly; occasional
darting behavior.
5 Death as indicated by the cessation of
gill movements.
To correlate the amount of toxaphene residues with these symptoms, G. affinis
were placed in 5 1 aquaria containing 2 ppra of Cl-toxaphene. Samples
were taken during the next 10 hours so that there were 10 fish sampled at
each of the 5 toxic stages. Samples were rinsed in tap water and analyzed
as previously described.
6. Uptake Experiments
Two 5 1 aquaria were set up with aged tap water containing 2 ppm of Cl-
toxaphene. G. affinis were introduced to each aquarium at 0800 and
sampled hourly for 8 hours. Each sample consisted of 10 fish which were
rinsed with fresh tap water and frozen individually for subsequent extraction.
At the time of sampling, the toxic stage of each fish was recorded. This
experiment was duplicated.
7. Excretion Experiments
Two 5 1 aquaria were set up as in the uptake experiments and fish introduced
at 0800. After 8 hours exposure to 2 ppm of Cl-toxaphene, fish were
transferred to 5 1 aquaria containing untreated, aged tap water. Dead
11
-------
fish were removed, rinsed in tap water, and frozen for subsequent analysis.
Also at the time of transfer, a sample of 10 fish was collected, rinsed,
and frozen. Samples were taken every hour until all fish were removed.
At the time of sampling, the toxic symptoms of each fish were recorded.
This experiment was duplicated.
8. Partitioning of Metabolites
36
A total of 34 fish killed by an 8 hour exposure to 2 ppm Cl-toxaphene
was used to characterize the composition of radioactive fish. Fish were
homogenized in 400 ml acetone. Samples of this homogenate were preapred
for radioassay. Two hundred ml of homogenate were evaporated to dryness
and partitioned in 100 ml distilled water, and 100 ml of hexane added to
the nonpolar fraction. Aliquots of both fractions were evaporated to
dryness, redissolved in 0.25 ml distilled water and 0.25 ml hexane, and
prepared for radioassay.
D. EFFECT OF TOXAPHENE ON THE NERVOUS SYSTEM
1. Toxicity Experiments
LDC,., LC,_, and LTC. studies were made using male adult L. maderae. The
3U jO jU — "
animals were treated by injection. After treatment, they were kept in
Mason jars with adequate food and water. Depending on the test, 2 varia-
bles were kept constant. For the LD-Q and LC , times varied from 24-120
hours. The LT,Q was made from the most lethal dose and concentration found
from the LD,Q and LC,Q. Control insects were injected similarly to the
test animals and maintained as above.
2. Electrophysiological Studies of Cockroach Nerves
Electrophysiological studies were made using an extracellular suction elec-
18
trode system (Florey and Kriebal ). Glass capillary tubes, fashioned to
approximate the outer diameter of a length of ventral nerve cord, were
used in drawing the nerve in contact with a Ag-AgCl cathode through a
saline bridge. Nervous activity was observed on a Tektronix Type 5103 Dual
Beam Storage Oscilloscope amplified by a Grass P-5 Type Preamplifier. A
Ag-AgCl rod, 0.5 mm diameter and 3.0 cm long, was immersed in a saline
bathing solution and used as the ground. The nerves were bathed in insect
saline, pH 7.1> (Yamasaki and Narahashi ) contained in a polyurethane well,
2.0 x 2.0 x 6.0 cm. Temperature of the saline varied between 22-26°C.
Frequency of spikes and bursts of spikes were measured using a Tektronix
DC502 Frequency Counter. Spike and burst intervals were observed on a
Mentor N-750 spike analyzer.
12
-------
Nerve cords were obtained from male £. americana. A length of ventral
nerve cord including the 3rd through 6th abdominal ganglia was used in
the preparations.
Perfusion of insect saline at 1.0 ml/min into the cell was continuous.
Temperature and pH of the saline were observed during each preparation.
Toxaphene dissolved in mineral oil was used throughout the~studies. Five
concentrations were used: 10 ,10 ,10 ,10 , and 10 M. Toxaphene
was introduced in close approximation to the nerve by injection from a 22
gauge Yale needle (0.05 ml at all concentrations).
Specimens were observed for spontaneous nerve activity before pharma-
cological tests were made. Mineral oil was introduced as a control before
testing the effects of toxaphene. Activity of a nerve was observed from
the time of dissection until pharmacological tests were concluded.
3. Effect on Ion Fluxes in Cockroach Nerves
Uptake of chloride ion by abdominal and thoracic segments of ventral nerve
cord of P. americana was studied. Adult male cockroaches were removed
from the colony, the body opened dorsally, and the viscera removed to expose
the ventral nerve cord. The nerve cord was then cut between the 3rd thoracic
and 1st abdominal ganglia. Both segments were removed and placed in ice-
cold saline (Yamasaki and Narahashi ). Once all the nerve segments were
removed, they were transferred to Cl-labelled saline, with or without
toxaphene, and allowed to incubate for various times at room temperature
(24 C). Following incubation, the segments were weighed, solubilized with
, and radioassayed.
13
-------
SECTION V
DISCUSSION
A. FATE OF TOXAPHENE IN THE RAT
1. Uptake and Excretion
Due to the paucity of information concerning the fate of toxaphene in the
mammalian system, the following study was undertaken. Reported herein
are routes and rates of excretion as well as amount and loci of accumulation
in various tissues of male rats administered an oral dose of Cl-toxaphene.
36
Average excretion of radioactivity derived from Cl-toxaphene, represented
as percent of administered dose is reported in Table 1. During 9 days,
52.6% of a single dose was excreted. About one-half of this occurred the
first day. Approximately 30% was excreted in urine while 70% appeared in
feces. Excretion on an accumulative basis is presented in Fig. 5; feces
equilibrated between 2-3 days while urine excretion continued an upward
trend. Feces have also been reported as the major excretion route for dieldrin
19 20 21
(Matthews et al. ) and mirex (Mehendale et al. and Gibson et al. ) in
rats. However, the amount of toxaphene excretion in urine demonstrated herein
was greater than observed with either dieldrin or mirex.
36
Following a second dose, urine played a greater role in excreting Cl
than with the single dose (Table 1). The peak of excretion in feces
appeared delayed about 1 day longer than in the single dose; here it took
2 days for 50% of the excretion to occur. Again similar trends were noted--
excretion in feces equilibrated early while urine continued an upward trend
(Fig. 2). Based upon total recovery expressed as 100%, urine increased
while feces decreased (Fig. 3). On the third day, relative percents of
urine and feces were equal. This importance of urine excretion during the
later days was evident in both single-dosed and redosed animals. It was
observed that less of the toxaphene dose was excreted in redosed than single-
21
dosed. This contrasts to the report by Gibson et al. that redosed rats
eliminated 25% of an administered dose of mirex as opposed to 18% for
single-dosed.
14
-------
Table 1. EXCRETION OF Cl IN URINE AND FECES OF RATS
FOLLOWING Cl - TOXAPHENE, 20mg/kg
(percent administered dose)
Single Dose
Day
1
2
3
4
5
6
7
8
9
Total1
Total0
Urine
1.46
3.20
2.89
2.35
1.82
1.19
1.15
0.54
0.72
15.3
29.1%
Feces
23.95
7.45
1.25
1.10
1.06
1.23
0.69
0.27
0.31
37.3
70.9%
Total
25.4
10.6
4.1
3.5
2.9
2.4
1.8
0.8
1.0
52.6
100%
Redose
Day
10
11
12
13
14
15
16
17
18
19
20
Urine
1.81
3.55
2.26
3.08
3.09
1.77
1.07
0.97
1.31
0.73
0.42
20.1
46.7%
Feces
6.00
11.60
1.40
1.10
1.20
0.60
0.40
0.40
0.20
0
0
22.9
53.3%
Total
7.8
15.2
3.7
4.2
4.3
2.4
1.5
1.4
1.5
0.7
0.4
43.0
100%
The single-dosed animals were redosed with 20 mg/kg on the 9th day.
Percent administered dose.
"Percent recovered dose expressed as 100%.
15
-------
c
(U
o
4 Or-
30
ECES —SINGLE DOSE
o
ra
o
UJ
C£.
Lul
s:
o
20
« 10
— REDOSE
""*••—••-««
URINE —REDOSE
INGLE DOSE
I I
TIME, days
Figure 2. Accumulative excretion of radioactivity in rats
administered a single dose and redose of 36C1-
toxaphene.
16
-------
lOOi—
S 80
-------
36
In both single-dose and redose experiments, 90% of Che Cl was recovered
in water fractions of feces (Table 2). Likewise in urine, only a small
amount of radioactivity was observed in the hexane fractions (Table 3).
Because radioactivity was found in the water fractions, it appeared that
a considerable amount of toxaphene metabolism had resulted; therefore,
the water and hexane fractions were analyzed for ionic chloride (Tables
4 and 5). 68.2% of the radioactivity excreted by single-dosed rats via
feces existed as ionic Cl; the amount in redosed animals was somewhat less.
Ifi
In urine, ionic Cl increased from 76.2% in single-dosed to 90.2% in redosed
rats; this resulted from a decrease of non-ionic chloride found in urine-
36
water fractions of redosed animals. Total ionic Cl excretion in combined
feces and urine was less in redosed rats. By comparing the individual feces
36
and urine accumulative ionic Cl excretion (Fig. 4), it appears that this
lower amount in redosed rats resulted from a smaller excretion by feces.
This anaylsis emphasizes the considerable degree of toxaphene metabolism
occurring in rats following an oral dose.
Tissues and organs of rats, following a redose of Cl-toxaphene, retained
6.0%. By comparing redosed and single-dosed rats, the manner in which the
first dose was concentrated compared to the second was determined. Here
it was demonstrated that redosed rats contained 0.55% less in selected tis-
sues; that is equivalent to almost 20% less dose retention than in single-
treated rats.
Uptake of radioactivity in various tissues over a period from 3 hours to
20 days is tabulated in Table 6. In almost all cases, the greatest concen-
tration was found at 12 hours followed by a rapid decrease which is in
22
close agreement to the 6 hour peak reported by Lamb et al. for dieldrin
in pheasant tissues. Blood cells exhibited a peak at 3 days. Less than
10% of the administered dose remained after 1 day (Fig. 5). This differs
from that found with mirex in rats where 34% of a dose was retained in
tissues and organs after 7 days (Mehendale et al. ). Most of the large
concentration up to day 1 could be accounted for by the amount in the sto-
mach. With respect to the nervous system, brain tissues did not concentrate
an extraordinary amount of radioactivity. Fat storage appeared non-significant;
23
this agrees with the investigation of Bateman et al. for sheep and steers.
18
-------
Table 2. HEXANE - WATER EXTRACTION OF RAT FECES FOLLOWING
Cl - TOXAPHENE, 20 rag/kg
(percent administered dose)
5
Single Dose
Day
1
2
3
4
5
6
7
8
9
Totalb
Total0
iexane
1.55
2.15
0.30
0.13
0.08
0.01
0.01
0.08
0.06
4.37
11. 8%
Water
22.4
5.3
0.95
0.97
0.98
1.22
0.68
0.19
0.25
32.9
88.27,
Total
23.95
7.45
1.25
1. 10
1.06
1.23
0.69
0.27
0.31
37.3
1002
a b
Redose*'0
Day
10
11
12
13
14
15
16
17
18
19
20
Hexane
0.38
1.28
0.13
0.05
0.01
0
0
0
0
0
0
1.85
8.3%
Water
5.62
10.29
1.27
1.06
1.16
0.01
0.38
0.36
0.25
0.01
0
21.0
91.7%
Total
6.0
11.6
1.4
1.1
1.2
0.6
0.4
0.4
0.2
0
0
22.9
100%
The single dose animals were redosed on the 9th day with 20 mg/kg.
Expressed as % administered dose.
c
Percent recovered dose expressed as 100%.
19
-------
Table 3. HEXANE - WATER EXTRACTION OF RAT URINE FOLLOWING
Cl - TOXAPHENE, 20 mg/kg
(percent administered dose)
b
Single dose
Day
1
2
3
4
5
6
7
8
9
Total1
Hexane
0.023
0.004
0.004
0.002
0.001
0.002
0.014
0.001
0.002
0.05
Totano.31
Water
1.42
3.19
2.88
2.35
1.82
1.19
1.14
0.54
0.719
15.2
96.71
Total
1.46
3.20
2.89
2.35
1.82
1.19
1.15
0.54
0.719
15.7
1001
o K
Redosea>D
Day
10
11
12
13
14
15
16
17
18
19
20
Hexane
0.031
0.006
0.007
0.007
0.003
0.001
0.003
0.002
0.003
0
0
0.1
0.51
Water
1.778
3.544
2.257
3.068
3.086
1.767
1.072
0.964
1.307
0.733
0.421
20.00
99.51
Total
1.81
3.55
2.26
3.08
3.09
1.77
1.07
0.97
1.31
0.73
0.42
20.1
1001
Single dosed animals redosed on the 9th day with 20 mg/kg.
Percent administered dose.
cPercent recovered dose expressed as 100%.
20
-------
Table 4. Cl - IONIC AND NOW-ION1C COMPOSITION OF
RAT FECES (WATER FRACTION) FOLLOWING Cl -
TOXAPHENE, 20 mg/kg
(percent admiaistered dose)
Single Dose
Day
1
2
3
4
5
6
7
8
9
b
Total
Total0
Ionic
13.4
7.1
1.1
1.4
1.4
2.4
1.2
0.2
0.7
28.5
76. 2%
Non- Ionic
3.0
3.7
0.9
0.4
0.4
0
0.2
0.2
0.1
8.9
23.82
Total
16.4
10.8
2.0
1.8
1.8
2.4
1.4
0.4
0.6
37.4
100%
Redose
Day
10
11
12
13
14
15
16
17
18
19
20
Ionic
4.5
7.0
0.4
0.7
1.1
0.4
0.4
0.1
0.3
0
0
14.9
70.3%
Non- Ionic
1.1
3.3
0.9
0.4
0.1
0.2
0
0.3
0
0
0
6.3
29.8%
Total
5.6
10.3
1.3
1.1
1.2
0.6
0.4
0.4
0.3
0
0
21.2
100%
Single dosed rats redosed with 20 mg/kg on 9th day.
Percent administered dose.
"Percent recovered dose expressed as 100%.
21
-------
36
Table 5. Cl - IONIC AND NON-IONIC COMPOSITION,OF
RAT URINE (WATER FRACTION) FOLLOWING Cl -
TOXAPHENE, 20 mg/kg
(percent administered dose)
Single Dose
Day
1
2
3
4
5
6
7
8
9
Totalb
Total6
Ionic
-0.36
4.4
3.6
3.7
2.6
1.9
0.5
0.9
1.1
18.3
76.2*
Non- Ionic
2.5
0
0.8
-0.1
0.8
0.5
0.5
0.3
0.3
5.6
23.3%
Total
2.2
4.4
4.4
3.6
3.4
2.4
1.0
1.2
1.4
24.0
100%
Redosea
Day
10
11
12
13
14
15
16
17
18
19
20
Ionic
1.2
3.2
2.3
2.9
2.9
1.5
0.9
0.9
1.3
0.7
-0.3
17.5
90.71
Non- Ionic
0.6
0.3
0
0.2
0.2
0.3
0.2
0.1
0
0
-0.1
1.8
9.31
Total
1.8
3.5
2.3
3.1
3.1
1.8
1.1
1.0
1.3
0.7
-0.4
19.3
1001
Single dosed animals redosed with 20 mg/kg on 9th day.
[>
Percent administered dose.
%
"Percent recovered dose expressed as 1001.
22
-------
40i—
-------
Table 6. UPTAKE OF RADIOACTIVITY IN VARIOUS RAT-TISSUES
AND ORGANS FOLLOWING A SINGLE-DOSE OF Cl -
TOXAPHENE, 24 mg/kg
(percent administered dose)
Tissue -o^
Kidney
Muscle
Fat
Testes
Brain
Blood Cells
Blood
Supernatant
Liver
First 2 cm
Small Intes-
tine
Last 2 cm
Small
Intestine
Large
Intestine
Esophagus
Spleen
Stomach
Total
3/24
0.05
0.93
0.14
0.02
0.03
3.1
0.64
0.33
0.06
0.10
0.19
0.04
0.04
3.70
9.37
6/23
0.13
1.6
0.15
0.08
0.06
0
1.20
1.10
0.34
0.34
0.60
0
0.06
18.6
24.26
12/24
0.43
5.3
0.86
0.28
0.23
0
2.35
2.33
0.34
0.28
1.20
0.04
0.08
77.20
90.90
1
0.10
1.3
0.57
0.06
0.05
0.06
1.30
0.50
0.05
0.13
0.19
0.03
0.05
2.00
6.39
2
0.03
0
0.31
0.04
0.04
0.90
0.60
0.31
0.01
0.01
0.08
0.01
0.02
0.63
2.99
3
0.03
0.65
0.18
0.03
0
2.6
0.36
0
0
0.15
0.02
0.01
0
0.61
4.64
5
0.01
2.4
0.18
0.03
0
0
0
0.01
0
0
0.03
0.02
0.03
0.39
3.10
7
0
0.40
0.02
0.02
0
0
0.18
. 0
0
0
0.04
0
0.24
0.16
1.06
9
0.03
0.14
3.65
0.06
0.01
1.10
0.09
0.48
0.84
0
0
0.03
0.06
0.12
6.57
20
0
0.81
0.03
0
0
1.17
0.06
0
0.09
0
0
0
0
0
2.16
24
-------
100
QJ
U
(U
Q.
to
o
o
CO
40
TIII i—r I
6 12
123456789
TIME, days
Figure 5. Total uptake of 36C1 by rat tissues and organs
following a single dose of 3^C1-toxaphene
(24 mg/kg).
25
-------
36
Passage of Cl through the intestinal tract required about 1 day (Fig. 6).
The peak in small intestine occurred at 6 hours; no differences were noted
between the first 2 cm and last 2 cm segments. At 12 hours, or an additional
6 hours, large intestine contained the bulk of Cl. The amount passing
into feces increased rapidly and peaked at 2-3 days.
2. Metabolism by the Liver
This experiment was designed to determine whether toxaphene is metabolized
24
by rat liver. Detoxification of toxaphene by liver was reported by Conley
as evidenced by excretion of ethereal sulfate and glucuronate. Since the
microsomal system of liver is important in detoxification and degradation
of drugs, this work compared metabolism of toxaphene by a whole liver homogenate
to that of a microsomal supernatant from liver.
Percent radioactivity recovered in each fraction appears in Table 7. Infer-
ring that radioactivity found in water fractions represented toxaphene
metabolism, it can be seen that whole liver homogenate metabolized about
2 1/2 times the amount metabolized by microsomal supernatant. Thus, it
would seem that although the microsomal fraction was responsible for some
of the toxaphene metabolism, there was another organelle(s) which may be
important to the degradation of this insecticide.
B. FATE OF TOXAPHENE IN COCKROACHES
1. Uptake in the American Cockroach, Periplaneta americana
Radioisotopic studies were performed in cockroaches to observe if Cl-
toxaphene accumulated about the ventral nerve cord and in other tissues.
Substantial amounts of radiolabelling associated with a single tissue
might indicate a site of action. This site of action could be in the ven-
2s
tral nerve cord if toxaphene behaves like the cyclodienes (Sun et al. ).
If radiolabelling were found concentrated in peripheral areas of the insect
26
body, a possible site of action like that of DDT might be indicated (Cochran ,
27 28
Webster , and Weiant ). However, possibly due to penetration difficulties,
metabolism translocation by the hemolymph and/or excretion and storage,
toxaphene may never reach its site of action as the parent molecule or an
active metabolite. To this end, insects showing symptoms of poisoning must
be studied along with asymptomatic animals. From this approach, plus electro-
physiological studies and symptomology of poisoned insects, a well defined
picture may be illustrated showing toxaphene activity in L. maderae and P_.
26
americana.
-------
4.6i—
10
4.4
4.2
•FECES
QJ
U
i.
\
OJ
Q.
UJ
0
C3
UJ
DC
UJ
1-
~
,_)
s
a
^j
1.2
1 .0
0.8
0.6
0.4
0.2
/ ^^
'l I >v
Ml X
— / \ I X
' v X
— / V X
/ /V*_i LARGE INTESTINE X
-' / '' X
1 / t X
/ / \ ^nd SMALL INTESTINE X
-^-. i \ >/^ X
' Jk. 1 * >^ X
1 /*4 \ /S Jst SMALL INTESTINE >
/^*"^^^ *-- / ^
0 "2 1 o ^ . r-
*— " """^s^^
^^^
(. ( ^1
r •» o
24 24
4
TIME, days
Figure 6. Passage of 36C] through the intestinal tract into the feces of rats
following 36Cl-toxaphene (24mg/kg).
8
-------
Table 7. RADIOACTIVITY RECOVERED FROM RAT-LIVER HOMOGENATES
AND SUPERNATANTS INCUBATED WITH Cl - TOXAPHENE
(percent administered doae)
~~^ — -^Fraction
Sample ^"^-^^^
Homogenate :
Mean Recovery
TL of Total Recovered
% of Total Extracted
Supernatant :
Mean Recovery
% of Total Recovered
% of Total Extracted
Hexane
32.66
38.87
77.49
45.29
54.25
91.88
Hater
9.49
11.29
22.51.
4.00
4.79
8.12
Precipitate
42.70
50.82
~
34.19
40.95
—
Total
84.02
100.00
100.00
83.49
100.00
100.00
28
-------
Amount of Coxaphene uptake up to 48 hours is reported in Table 8. Approx-
imately 1/3 of the applied dose had penetrated by 24 hours; however, this
represents only 50% of the LD5Q dose. Cockroaches were moribund at 48
hours and contained 63 tig toxaphene, or 84% of the applied dose.
Figure 7 relates amount of penetrating dose to its occurrence in nemolymph
up to 24 hours. The concentration in hemolymph appeared to plateau at ca.
4 hours while total penetration plateaued between 4 and 8 hours. Hemolymph
maintained 24.09 ± S.D. 3.2% of the total toxaphene occurring throughout
the 48 hour period.
2. Uptake in Leucophaea maderae
An insecticide binding mechanism associated with the membrane of nerve cells
appears to be a generally recognized phenomenon applicable to the whole
29
family of chlorinated hydrocarbons (Matsumura and Hayashi ). Studies have
shown that dieldrin complexes with components of nerves (Matsumura and
29 30 31 32 33
Hayashi ' , Telford and Matsumura ' ; Sellers and Guthrie , Jakubowski
34
and Crowder ). These studies may be applicable to other cyclodienes.
There appeared to be uptake by the ventral nerve cord Ln vitro. Cl-toxaphene
increased as a function of concentration (Fig. 8). Rinsing did remove
substantial amounts of radiolabelled material, but after 20 min nerve cords
retained significant amounts (Fig. 9). The four-fold difference in
radioactivity of the nerves above that found in the final rinse might indicate
penetration or binding of toxaphene by the nerve cord. Radioactivity accu-
mulated about the nerve cord of L. maderae incubated .in vivo (Tables 9 and
10).
Radioactivity occurred in the greatest concentration at 48 hours (Table 9).
This period of time corresponded to the symptom in the poisoning syndrome
where the insects were prostrate with leg movements. At 120 hours insects
which exhibited symptoms of poisoning accumulated more Cl than did asymp-
tomatic cockroaches (Table 10). Gas-chromatographs of nervous tissue residue
showed no differences from the toxaphene standard.
Occurrence of Cl in several other tissues of L. maderae is shown in Table
11. Radioactivity did not appear to accumulate in hemolymph in any regu-
lar pattern. The greatest concentration occurred at 6 hours. Radioactivity
was not found in excreta until 48 hours after injection. The average amount
occurring between 48 and 120 hours was 271.0M,g/g excrement.
29
-------
Table 8. PENETRATION OF &C1 - TOXAPHENE INTO THE AMERICAN COCKROACH
Time,
hours
\
I
2
4
8
24
48
Hemolymph
Percent of fl
^.B applied dose
1.11
2.79
2. IS
S.08
4.57
6.04
11.09
1.48
3.72
2.87
6.77
6.09
8.05
14.79
Ca
tLS
8.84
8.00
5.47
7.82
11.75
12.97
51.56
re as s
Percent of
applied dose
11.78
10.67
7.29
10.42
15.66
17.29
68.75
Total
Percent of
Uj5 applied dose
± SE
8.37
10.13
6.35
15.23
23.67
25.63
62.66
11.16 + 4.0
13.51 ± 5.8
8.46 + 3.4
20.31 + 7.7
31.56 + 6.5
34.17 ± 4.3
83.54 +11.7
75
dose.
30
-------
1 2
Figure 7
4 8
TIME, hours
Penetration of 36C1-toxaphene into the
American cockroach.
24
31
-------
2000
1000
800
" 600
o
01
en
o 200
o
o>
I 100
u_ 80
60
40
•»
LU
LlJ
£ 20
X
o
G 10
VO n
CO O
I
1
I
1
10"5 10" 103 102 1U1
TOXAPHENE, molar
Figure 8. Uptake of 36Cl-toxaphene in nerve cords of
Leucophaea maderae incubated iji vitro.
32
-------
50
c
Ol
o
S-
01
Q.
o
o
40
30
20
10
1
I
I
1
600
1200
5 100
TIME, seconds
Figure 9. Percent recovery in rinse series relative to 36r,i-toxaphene
uptake in nerve cords of Leucophaea maderae incubated iji vitro.
33
-------
Table 9. UPTAKE OF Cl IN LEUCOPHAEA MADERAE
VENTRAL NERVE CORDS AFTER INJECTION
WITH 175.95 >ig Cl • TOXAPHENE PER
INSECT
(tig/nig tissue)
36
Time, hours Cl-toxaphene
2
4
6
8
12
24
48
72
96
120
0.696
0.941
0.350
0.134
0.365
0.317
2.390
0.311
0.383
0.185
34
-------
Table 10. DISTRIBUTION OF 36C1 - TOXAPHENE IN TISSUES
OF LEUCOPHAEA MADERAE
No. of Hour Average,
Tissue Insects Disposition Time ne/K
Hemo lymph
Nerve cord
Fat body
Alimentary
canal
75
21
12
126
27
12
75
21
12
75
21
6
A
A
S
A
A
S
A
A
S
A
A
S
<120
120
120
<120
120
120
<120
120
120
<120
120
120
193.88
67.02
563.50
654.00
184.59
268.71
263.89
309.62
299.03
272.97
260.72
305.02
a A • Asymptomatic, S • Symptomatic
b Average of values recorded at 2, 4, 6, 8, 12, 24, 48, 72,
and 96 hours.
35
-------
Table 11. TOXAPHENE RECOVERED FROM LEUCOPHAEA MADERAE INJECTED WITH 175.95 p.g
PER COCKROACH --
tissue)
36
Cl - TOXAPHENE
•—•^Time , hours
Tissue "--^
Hemolymph
Fat body
Gut
2
6.88
190.65
234.75
4
126.75
155.80
453.70
6
908.75
18.09
238.15
8
2.15
1120.57
246.75
12
245.40
110.00
109.70
24
147.75
162.99
181.12
48
5.84
196.70
115.40
72
259.17
303.82
565.60
96
42.20
114.40
311.60
120
67.02
309.62
260.72
u>
-------
Binding patterns of toxaphene to subcellular components of insect nerve
31
and brain were studied essentially by the methods of Telford and Matsumura .
Percentages of the total amount of Cl-toxaphene added, and found in each
fraction, are given in Table 12. Fractions were identified as A., A., A.,
A., Ac, A, and MIC. A. through A, were identified by the following descrip-
456 1 31
tions given by Telford and Matsumura with MIC referring to microsomes:
A. consisted of cell membranes, and smaller particles of ca. 12
M- in diameter. A. also had cell membranes, but larger and more
electron dense than those found in the A layer. Small pieces
of nerve ending particles containing synaptic vesicles were also
observed. A- was primarily pinched-off nerve endings containing
synaptic vesicles, and a few mitochondria. The A, layer had nerve
endings of a more electron dense nature than the two previous layers,
and more free mitochondria occurred here. Fragments of nerve sheath
appeared in this layer from the nerve cord homogenate. Fraction
A had some mitochondria, nuclei, and fragments of sheath from the
nerve cord homogenate. The A, layer contained nuclei, large pieces
o
of tissue, and large pieces of nerve sheath from the nerve cord
homogenate.
Results showed that most of the toxaphene was found associated with the
heaviest (nuclei and nerve sheath) fraction and the lightest (microsomes)
of both tissues. It is interesting to note that sub-fractions of the
brain homogenate showed higher amounts of toxaphene than those of nerve
tissue, except for the A. (cell membrane) and microsomal fractions. It
may indicate that subcellular fractions of brain showed a higher binding
affinity for toxaphene than those of nerve tissues.
C. UPTAKE OF 36C1-TOXAPHENE BY MOSQUITOFISH
High susceptibility of fish and discovery of resistant strains could aid
the study of toxaphene*s mode of action. Although there is information
on toxicity and residues, there has been little work done to quantify actual
uptake of toxaphene from the aquatic medium.
Uptake and excretion of related insecticides have been investigated in
35
mosquitofish. Ferguson et al. demonstrated existence of processes
of uptake and excretion for endrin, but did not quantify these processes.
The major source of endrin uptake was contaminated water rather than accu-
mulation through the food chain. Endrin was also released into water
by contaminated fish which indicated some type of excretion mechanism.
37
-------
Table 12. RECOVERY OF Cl - TOXAPHENB IN NERVE TISSUE OF
LEUCOPHAEA MADERAE
(percent administered dose)
ac t ion
Tissue
A
-A5
Microsomes
Nerve
Brain
1.6
1.1
0.4
0.8
0.3
1.3
0.8
1.9
0.6
1.3
5.0
9.7
17.3
10.3
Description of fractions given in text.
38
-------
O£ 'I'J OQ
Wells and Yarbrough ' and Yarbrough and Wells studied retention of DDT,
aldrin, dieldrin, and endrin in resistant and susceptible mosquitofish.
Using radioactive tagging, they demonstrated that cell membranes of resistant
fish bind more insecticide than membranes of susceptible fish. These results
suggested that resistance is in part the result of a membrane barrier in
resistant fish.
Quantification of the amount of toxicant actually absorbed is important for
investigations into the mode of action of these insecticides. The present
research was undertaken to quantify uptake of toxaphene by mosquitofish and
relate these data to the toxicity syndrome.
A total of 100 fish was used to determine the 24 hour LC_0 (Fig. 10). Since
future experiments were to be conducted over short time periods, the purpose
of this testing was to determine appropriate experimental concentrations
rather than to define the lethal concentration of a particular population
of fish. Thus the LC_0 represents a 24 hour time period. The LC.g as
determined by a best fit line is approximately 860 ppb. Extrapolating from
this curve, 2 ppm was selected as the appropriate experimental concentration
for testing fish. Fig. 11 illustrates the LT~0 for oxidation pond Gambusia
at 2 ppm of toxaphene to be 12 hours. Since both uptake and excretion were
to be studied, 8 hours was selected as the optimum period for absorption
of this concentration level. Fig. 12 shows that at periods longer than 8
hours mortality increases rapidly, making excretion results impossible to
interpret in moribund fish.
Fig. 13 represents results of the uptake experiments with mean concentration
36
of Cl-toxaphene and Tr residues for each trial plotted over time. Regres-
sion analysis of each trial resulted in linear uptake equations (Table 13).
These were significant at the 0.01 level.
Total recovery was calculated on the basis of uptake experiments. In each
O£
case, 9.67 mg of Cl-toxaphene was added to the test solutions. For the
2 uptake experiments, average total recovery was 0.046 mg, or 0.47%.
Presumably the remainder was left in the water or adsorbed on the test
containers. The computer program used in these experiments calculated the
number of mg of toxaphene represented by the scintillation counts per
sample and also per g of fish. This value is misleading as it represents
not only toxaphene present but also any metabolites of toxaphene which
contain radioactive chlorine. 39
-------
0)
u
-------
o»
<_>
i-
-------
7 Or—
02468
TIME, hours
Figure 12. Percent mortality of Gambusia affini s as a
function of exposure to 2000 ppb toxaphene
42
-------
0.6
0.5
I 0.4
OH
0.3
:r
o.
X
° 0.2
ro
0.1
EXPERIMENT #1
EXPERIMENT #2
I I I I 1 I I I
23456
TIME, hours
8
Figure 13. Uptake of
Gambus ia
36
C1 -toxaphene in
i as a function
of exposure to 2 ppm 36C1 -toxaphene.
43
-------
Table 13. RESULTS OF REGRESSION ANALYSIS FOR THE UPTAKE AND EXCRETION OF
Jo
Cl - TOXAPHENE BY GAMBUSIA AFFINIS
Experimental
group
Uptake A
Uptake B
Excretion A
Excretion B
Regression
equation
Y - 0.00068 + 0.00058 (X)
Y - 0.00071 + 0.00052 (X)
Y - 0.00601 -I- 0.00004 (X)
Y » 0.00471 + 0.00002 (X)
r
0.76266 *
0.86208 *
0.04808
0.04089
*Signi£icant at the 0.01 level, Student t test.
-------
On the basis of these results, several observations could be made concerning
dynamics of toxaphene uptake by G. affinis. Uptake was a linear function
and was directly proportional to length of exposure. Fig* 14 plots uptake
by 2 different weight groups of fish, less than 800 mg and more than 1200
mg. The graph shows that at every point, small fish contained more Cl-
toxaphene per g of tissue than large fish. Mean body load of fish weighing
between 800 and 1200 mg in every case fell between the plots shown in Fig. 14.
Comparison between paired points revealed that fish weighing less than 800
mg acquired between 10-42% more residue than fish weighing more than 1200 mg.
36
Mean concentration of Cl-toxaphene and TR residue for each trial of the
excretion experiment was plotted over time (Fig. 15). Regression analysis
of data resulted in statistically nonsignificant excretion equations (Table
13). Calculation of confidence intervals about individual points indicated
that at the 0.05 level, there was no significant difference In body load
over time in either trial. Therefore, from these data, there was no indica-
tion of excretion during the first 6 hours following exposure.
Table 14 represents partitioning of fish extracts into water and hexane
fractions. This revealed that 88.7% of the radioactive chlorine was soluble
in the nonpolar phase.
Observation of the toxicity syndrome and characterization into 5 stages
were subjective processes. Interpretation of toxicity symptoms without a
knowledge of the mode of action of toxaphene was impossible although certain
behavior might suggest physiological correlates. The first stage of the
toxicity syndrome, and the most difficult to assess, was when fish began
to swim at the surface against the side of test containers. Swimming at
the surface is normal in water with low oxygen content and is also the
normal feeding position for G. affinis. Normal fish, however, retreated
from the surface when the aquarium was approached whereas poisoned fish re-
mained at the surface. Subsequent toxicity stages were all marked by rapid
gill ventilation which further suggested respiratory involvement. The
third toxicity stage was characterized by fish swimming against the side of
aquaria, but with some loss of equilibrium which was evidenced by sinking
of the posterior end so that fish attempted to swim up towards the surface.
Finally, fish lost their ability to maintain normal dorsal-ventral orienta-
tion and rolled to the side. At stage 4 fish sank to the bottom and were
prostrate with rapidly ventilating gills. At this stage, there was occasional
45
-------
0.7r—
E
Q.
Q.
QL
Of.
0.6
0.5
0.4
0.3
X
o
\—
I
o 0.2
VO
CO
0.1
FISH WT.
FISH WT.
800 MG.
1200 MG.
I I 1 I I I I I
Figure 14,
2345678
TIME, hours
Uptake of Cl-toxaphene for
large and small Gambusla affinis
as a function of time.
46
-------
0.8
0.7
0.6
O.
P 0.5
OS
OC
\o
CO
0.4
— !
to
o
CL
X
X
o
C£.
=
O
00
A N = 61
B N = 83
i i i i i i i
0123456
TIME, hours
Figure 15. Body load of 36C1-toxaphene and TR
residues in Gambusia affi ni s as a
function of time in fresh water.
-------
Table 14. METABOLITE PARTITIONING OF TOXAFHENE IN GAMBUSIA AFFINIS*
(M-g/g tissue)
36
Cl-Toxaphene
and TR per sample
Percent of total
QQ O£
Cl-Toxaphene
and TR/g of fish
Hexane fraction
0.676
88.700
0.586
Water fraction
0.084
11.300
0.072
Total
0.760
100.000
0.658
Average Cl-Toxaphene and TR residues control • 0.68 + 0.01 p,g.
-------
darting behavior, until death which was identified by the cessation of gill
movements.
36
Fig. 16 indicates average body load of Cl-toxaphene and TR residues at
each toxicity symptom. By the time fish exhibited the first toxicity
response to toxaphene, rising to the surface of the water, they had already
sorbed 90.3% of the average fatal residue. Fish which were characterized
as normal had accumulated 35% of the fatal residue. In stages 3, 4, and
5, fish showed obvious signs of toxicity.
Average residue per g of fish and toxicity symptom exhibited at each hour
are shown in Table IS. Fish progressed through toxicity symptoms at
approximately the same rate until the eighth hour. This was the point on
the mortality curve (Fig. 12) where mortality increased rapidly. At this
point it was possible to examine differences in body load between fish
with identical exposure times, but which exhibited different toxicity symptoms.
The onset of a particular toxicity stage was directly proportional to body
load.
Since fish were processed whole in these experiments, it was impossible to
determine what portion of these residues had been absorbed into particular
organs and what portion was simply adsorbed to scales and fins. Informa-
tion concerning toxicity symptoms is perhaps most important when •considering
in vitro studies into the mode of action of toxaphene in fish. Data show
that earlies toxicity symptoms were visible when toxaphene content was as
low as 0.2 ppm and this was in a toxaphene resistant population. In non-
resistant populations, where the LCSQ is around 30 ppb, toxic symptoms
would presumably be apparent with even lower body loads.
Compared to 36 hour LD^.s reported by Boyde and Ferguson , G. affinis used
in these experiments were highly resistant. The highest resistance reported
by these workers was 480 ppb while nonresistant populations had LDSQs
around 10 ppb.
Due to virtual insolubility of toxaphene in water, its presence in any aquatic
medium is either as a suspension or else adheres to particles in the water.
Thus under experimental conditions, uptake by fish may be due to 1) adsorption
of toxaphene to the body of fish, 2) simple diffusion of toxaphene into fish,
and 3) active processes of absorption into fish. On the basis of adsorption
alone, it would be expected that toxaphene residues of whole fish would
49
-------
0.7
0.6
o>
o>
*0.5
to
LU
0
1— 1
£ 0.4
as:
i—
0
** 0.3
LU
z
LU
Q.
I 0.2
i
o
£>
*>
0.1
—
r— i
Figure 16.
12345
TOXICITY STAGE
Mean body burden of Cl-toxaphene and
TR residues in Gambusia affinis at each
stage of the toxicity syndrome.
50
-------
Table 15. Cl - TOXAPHENE AND TR RESIDUES AND TOXICITY SYMPTOMS EXHIBITED AT EACH HOUR
IN GAMBUSIA AFFINIS
-------
increase as a function of time. All fish of the same weight or body sur-
face would theoretically have the same body load at any given time. Table
15 shows, however, that any time when more than one toxic symptom was
exhibited, mean body load was different for fish showing different symptoms,
and that amount of residue present was consistent with severity of the
symptom. Since exposure time was equal for all fish, different body loads
at various toxicity stages appeared to reflect differences in rates of up-
take. If adsorption or diffusion were solely responsible for uptake, there
should be no differences in body load at any given .time regardless of toxic
symptom; thus, active processes of absorption appeared to be implicated in
the uptake process. This agrees with the conclusions of Ferguson et al.
concerning endrin uptake in G. affinis.
Partitioning of fish extracts revealed that 88.7% of the radioactive chlorine
was recovered from the nonpolar fraction. Recovery rates, however, for the
hexane/water partitioning procedure similarly averaged 87.62%. Thus, there
was apparently minimal metabolic alteration in the toxicant after a period
of 8 hours. Dehydrochlorination has been reported as a method of toxaphene
39
metabolism in rats (Ohsawa et al. ). If this were the case in fish, one
would also expect fish to excrete chloride. The excretion data showed no
evidence of excretion within 8 hours; this correlated with lack of toxa-
35
phene metabolism during that period. Likewise, Ferguson et al. found no
•
evidence of metabolic or chemical alteration of endrin in their studies
with G. affinis.
Insecticide resistance in fish is not completely understood at this time.
35
Ferguson et al. attributed resistance to increased physiological tolerance
rather than differences in rates of uptake. Data presented here showed
that onset of toxicity symptoms varied within any given population, and
that onset of particular toxicity symptoms was directly proportional to
body load. Thus within this population, differences in toxic response were
due to different body loads rather than different tolerances of a particular
toxicant level.
Observations of the progression of toxicity symptoms in G. affinis suggested
that intoxication involved an increased oxygen requirement. The first stage
of toxicity was marked by fish swinning at the surface of water which is
typical for these fish in water of low oxygen content. Swimming toward the
52
-------
surface, coupled with decreased activity before finally sinking to the bot-
tom, suggested a depletion of energy. This was in spite of an apparent
increasing need for oxygen which was evidenced by rapid gill ventilation
35
which continued until death. Ferguson et al. noted increased oxygen
requirements for endrin-poisoned fish, but related them to increased activity
that characterized endrin poisoning. With toxaphene, activity decreased
with onset of toxicity symptoms while the requirement for oxygen appeared
to increase.
The very sharp rise in mortality at 8 hours observed in the mortality curve
(Fig. 12) coupled with similarity of body loads at toxicity stages 3-5
suggested that mortality was due to a very critical level of toxaphene at
the site of action. If several different sites of action were involved,
one would expect each to be affected at slightly different concentrations.
This would tend to flatten the mortality curve. It was suggested, therefore,
that the response of G. affinis to toxaphene was due primarily to toxicity
mechanisms acting at one site.
0. EFFECT OF TOXAPHENE ON THE NERVOUS SYSTEM
1. Electrophysiological Studies of Cockroach Nerves
LD_n studies indicated poor insecticidal activity of toxaphene toward the
cockroach L. maderae (Fig. 17). As the dose was increased, symptoms of
poisoning occurred more rapidly.
Symptoms appeared similar to those of other chlorinated hydrocarbon insecti-
cides (e.g., dieldrin, DDT, and lindane). Following injection of toxaphene
at high concentrations (10 - 10 M), insects would run in circles at the
bottom of their circular confine. Later, they would become prostrate on
their dorsum with legs and other appendages still active. Animals observed
displaying this behavior were recorded as "prostrate and kicking". This
sort of activity was followed by a purely prostrate condition with only
maxillary palps and antennae active. Death was recorded when the insect
was prostrate, totally inactive, and would not respond to stimuli.
When examined, insects considered dead did have active hearts with irregular
and faint beats. Insects dissected during the "prostrate and kicking" phase
possessed hearts with rhythmic normal beating rates. Those cockroaches
observed as prostrate had irregular heart beats but at a nearly normal rate.
53
-------
99.5
99.0
98.0
95.0
| 90.0
o
- 80.0
>-
3 7°-°
g 60.0
s:
£ 50.0
I 40.0
°" 30.0
20.0
10.0
5.0
J I I I
1
J I L_L
102
8 103 2
DOSE, ug/g
8 101*
Figure 17.
LD50 (48 hour) of toxaphene on Leucopheae maderae
-------
Control cockroaches showed no symptoms of poisoning and all specimens
survived. Dissected insects showed normal heart activity.
In electrophysiological studies of ]?. americana. there appeared to be 3
phases of nervous activity following dissection. The first phase was
characterized by bursts of high intensity spikes that were irregular in
rhythm and varying in spike amplitudes and frequency (Fig. 18). This
first phase of spontaneous activity appeared to be a form of dissection
shock lasting an average of 67 sec from the time of contact with the
electrode. The second phase was a period of recovery from dissection.
This period showed activity of an irregular form. Spikes of varying ampli-
tude could be observed throughout this period. These spikes ranged in
amplitude from 0.02 volts to 0.12 volts. No bursts of spikes were observed.
The third phase was a period of stabilization usually occurring 2 hours from
contact with the electrode. It was marked by strictly baseline activity
where nerve impulses were too weak to be seen over the interference at
0.1 volt/division. This period was where testing of pharmacological agents
began.
Mineral oil (0.05 ml) used as a control appeared to have little effect on
nerve activity. When applied directly on the nerve, it covered the nerve
making good contact, but produced little effect.
Toxaphene in mineral oil at doses of 10 - 10 H had immediate effects on
the nerve (Fig. 19). There appeared to be "buzz saw" effects similar to
DDT, spikes started at low amplitudes (0.02 volts) and frequency and then
built to high amplitudes and very high frequency. This was unlike dissection
shock in that these bursts occurred rhythmically. Each individual nerve
appeared to react differently; some appeared very susceptible, others resis-
-4 -5
tant. Toxaphene at lower concentrations (10 and 10 M) had little effect.
2. Effect on Ion Fluxes in Cockroach Nerves
Effect of chlorinated hydrocarbons on the central nervous system may be
that of disrupting normal fluctuations of ions across the neural membrane.
In particular, they may disrupt sodium and potassium ion exchanges. For
instance, DDT seems to accelerate the rate of potassium efflux (Matsumura
12
and O'Brien ), whereas dieldrin accelerates sodium influx (Hayashi and
40
Matsumura ). Although both eventually lead to an accumulation of sodium
ions within nerve, the way in which this is done is different, thus suggesting
55
-------
0.8 ,—
0.6
0.4
0.2
•*- -b
I
I
20 40 60
DIVISIONS
80
100
Figure 18. Oscillograph of spontaneous nerve activity
from ventral nerve cord of Periplaneta americana.
a. Trace showing spontaneous nerve activity.
b. Trace showing frequency recording of spikes
with discriminator setting of 0.1 volt
amplitude.
56
-------
o.8n—
0.6
0.4
0.2
rrA
r a
Figure 19.
J I
20
40 60
DIVISIONS
80
100
Oscillograph of nerve activity immediately
following a dose of 2.1 mg toxaphene on the
ventral nerve cord of Periplaneta americana.
b.
Trace showing activity following toxaphene
treatment.
Trace showing frequency recording of spikes
with a discriminator setting of 0.1 volt
amplitude.
57
-------
slight differences in their node of action. In this light, a study of
the effect of toxaphene on ionic fluxes of the insect central nervous system
was undertaken.
Preliminary work with chloride ion is reported herein. In order to find
a concentration of toxaphene and incubation times to use for these investi-
-7 -5 -4
gations, 10 ,10 , and 10 M toxaphene have been used. At each concentra-
tion, rate of uptake of chloride ion was measured by incubating nerve segments
from one minute to one hour, and plotting rate versus that of the control
(Fig. 20-25). Although the difference between control and treated in Fig. 20
appeared significant, extended incubation times (e.g., 2 and 3 hours) will
be necessary for other differences to become evident.
58
-------
Of.
o
IO
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
Figure 20.
CONTROL
TREATED
I I I I
10 15
30
TIME, minutes
60
Uptake of Cl in abdominal segment of ventral nerve
core of Periolaneta americana exposed to 10"7M
toxaphene
59
-------
CONTROL
TREATED
I I
10 15
30
TIME, minutes
60
Figure 21. Uptake of Cl in thoracic segment of ventral nerve cord
of Periplaneta americana exposed to 10"7M toxaphene.
60
-------
DC.
o
u
I
_
vo
200
190
130
170
160
150
140
130
120
100
90
80
70
60
50
40
30
20
10
I
\
15
CONTROL
TREATED
I
\
60
30
TIME, minutes
Figure 22. Uptake of 36C1 in abdominal segment of the ventral nerve
cord of Periplaneta americana, exposed to 10~5M toxaphene.
61
-------
Ol
LJ
a:
o
CJ
I
o
200
190
180
1/0
160
150
140
130
120
110
100
90
SO
70
60
50
40
30
20
10
CONTROL
TREATED
I
I
30
TIME, minutes
Figure 23. Uptake of 36C1 in thoracic segment of the ventral nerve
of Periplaneta amerlcana exposed to 10"5M toxaphene.
62
-------
LU
Q
C£.
O
o
i
V£>
ro
150
140
130
120
110
100
90
00
70
60
50
40
30
20
I I I I
10 15
36,
CONTROL
TREATED
I
30
TIME, minutes
60
Figure 24. Uptake of JOC1 in abdominal segment of the ventral
nerve cord of Periplaneta americana exposed to lO'
toxaphene.
63
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10 15
CONTROL
TREATED
+/
t /
I'
/
'*
40
30
20
__
—
—
—
1 1 1 1 1 1
30
TIME, minutes
60
Figure 25. Uptake of 36C1 in thoracic segment of the ventral nerve
cord of Periplaneta americana exposed to lO^M toxaphene.
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SECTION VI
REFERENCES
1. Dahn, P. A( The Mode of Action of Insecticides Exclusive of
Organic Phosphorus Compounds. Ann Rev Entomol. 2:247-260,
January 1975.
2. Winteringham, F. P. W., and S. E. Lewis. On the Mode of Action
of Insecticides. Ann Rev Entomol. 4:303-318, January 1959.
3. Roan, C. Co, and T. L. Hopkins. Node of Action of Insecticides.
Ann Rev Entomol. 6:333-347, January 1961.
4. O'Brien, R. D. Mode of Action of Insecticides. Ann Rev Entomol.
11:369-402, January 1966.
5. O'Brien, R. D. Insecticides. Action and Metabolism. New York,
.Academic Press, 1967. p. 136-140.
6. Brooks, Go T. Chlorinated Insecticides. Vol. 2:Biological and
Environmental Aspects. Cleveland, CRC Press, 1974. 197 p.
7. Lalonde, D. J. V., and A. W. A. Brown. The Effects of Insecticides
on the Action Potential of Insect Nerves. Can J Zool (Ottawa).
32(2):74-81, April 1954.
8. Wang, C. M., and F. Matsumura. Relationship Between the Neuro-
toxicity and In Vivo Toxicity of Certain Cyclodiene Insecticides
in the German Cockroach. J Econ Entomol. 63(6) :1731-1734,
December 1970.
9. Shankland, D. L., and M. E. Schroeder. Pharmacological Evidence
for a Discrete Neurotoxic Action of Dieldrin (HEOD) in the American
Cockroach Periplaneta americana (L). Pesticide Biochem Physiol.
3(l):77-86, March 1973.
10. Mull ins, L. J. Structure-Toxicity of Hexachlorocyclohexane Isomers.
Sci. 122(3159):118-119, July 1955.
11. Matsumura, F., and R. D. O'Brien. Insecticide Mode of Action.
Absorption and Binding of DDT by the Central Nervous System of the
American Cockroach. J Agr pood Chem. 14(l):36-39, January 1966.
65
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12. Matsumura, P., and R. D. O'Brien. Insecticide Reaction with Nerve.
Interactions of DDT with Components of American Cockroach Nerve.
J Agr Food Chen. 14(l):39-43, January 1966.
13. Holan, G. New Halocyclopropane Insecticides and the Mode of
Action. Nature (London). 221(5185):1025-1029, March 1969.
14. Halladay, S. C. An Inexpensive Metabolism Cage for Small Animals.
Bull Environ Contam Toxicol. 10(3):155-156, September 1973.
15. Sternburg, J., and J. Corrigan. Rapid Collection of Insect Blood.
J Bcon Bntomol. 52(3):538-539, June 1959. ~
16. Yaoasaki, T., and T. Narahashi. The Effect of Potassium and Sodium
Ions on the Resting and Action Potentials of the Cockroach Giant
Axon. J Insect Phyaiol (Oxford). 3(2): 146-158, May 1959.
17. Boyde, C., and D. E. Ferguson. Susceptibility and Resistance of
Mosquitofish to Several Insecticides. J Econ Entomol. 57(4):
430-431, August 1964.
18. FLorey, E., and M. D. Kriebal. A New Suction-Electrode System.
Camp Btochem Physiol (Oxford). 18(1):175-178, May 1966.
19. Matthews, H. B., J. D. McKioney, and G. W. Lucier. Dieldrin
Metabolism, Excretion, and Storage in Male and Female Rats.
J Agr Food Chem. 19(6):1244-1248, November 1971.
20. Mehendale, H. M., L. Fishbeln, M. Fields, and H. B. Matthews.
14
Fate of Mirex- C in the Rat and Plants. Bull Environ Contam
Toxicol. 8(4):200-207, October 1972.
21. Gibson, J. R., G. W. Ivie, and H. W. Dorough. Fate of Mirex and
It's Major Photodecomposition Product in Rats. J Agr Food Chem.
20(6):1246-1248, November 1972.
22. Lamb, D. W., Y. A. Griechus, and R. L. Linder. Distribution of
14
Dieldrin- c in Pheasant Tissues After a Single Administration.
J Agr Food Chem. 18(1):168-171, January 1970.
23. Bateman, G. Q., C. Biddulph, J. R. Harris, D. A. Greenwood, and
L. E. Harris. Toxaphene: Transmission Studies of Milk of Dairy
Cows Fed Toxaphene Treated Hay. J Agr Food Chem. 1(4):322-324,
May, 1953.
66
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24. Conley, B. E. Pharmacologic Properties of Toxaphene, a Chlorinated
Hydrocarbon Insecticide. J Amer Med Assoc. 149(12):1135-1137,
July 1952.
25. Sun, Y. P., C. H. Schaefer, and E. R. Johnson. Effects of Applica-
tion Methods on the Toxicity and Distribution of Dieldrin in House-
flies. J Econ Entomol. 60(4):1033-1037, August 1967.
26. Cochran, D. G. Susceptibility of Sexes of Periplaneta. J Econ
Entomol. 48(2) :131-133, April 1955.
27. Webster, E. J. An Autoradiographic Study of Invertebrate Uptake
of DET-C136. Ohio J Sci. 67:300-307, May 1967.
28. Weiant, E. A. Control of Spontaneous Activity in Certain Efferent
Nerve Fibers from the Metathoracic Ganglion of the Cockroach,
Periplaneta americana. Proc 10 Int Conge Entomol (Montreal).
2:81-82, December 1958.
29. Matsumura, P., and M. Hayashi. Dieldrin: Interaction with Nerve
Components of Cockroaches. Sci. 153(3737):757-759, August 1966.
30. Matsumura, P., and M. Hayashi. Comparative Mechanisms of Insecticide
Binding with Nerve Components of Insects and Mammals. Vol 25:Res
Rev. New York, Springer-Verlag, 1969. p. 265-273.
31. Telford, J. N., and P. Matsumura. Dieldrin Binding in Subcellular
Nerve Components of Cockroaches. An Electron Microscopic and Auto-
radiographic Study. J Econ Entomol. 63(3):795-800, June 1970.
32. Telford, J. N., and P. Matsumura. Electron Microscopic and Auto-
radiographic Studies on Distribution of Dieldrin in the Intact
Nerve Tissue of German Cockroaches. J Econ Entomol. 64(1):230-238,
February 1971.
33. Sellers, L. G., and P. E. Guthrie. Localization of Dieldrin in
Housefly Thoracic Ganglion by Electron Microscopic Autoradiography.
J Econ Entomol. 64(2):352-354, April 1971.
34. Jakubowski, T., and L. A. Crowder. Binding of Cl-Dieldrin Co
Suspected Target and Non-target Proteins In Vitro. Bull Environ
Contain Toxicol. 10(4):217-224, October 1973.
35. Ferguson, D. E., J. L. Ludke, and G. G. Murphy. Dynamics of Endrin
Uptake and Release by Resistant and Susceptible Strains of Mosquito-
fish. Trans Amer Fish Soc. 95(4):335-344, October 1966.
67
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36. Wells, H. R., and J. D. Yarbrough. In Vivo and In Vitro Retention
14 14 ~~ ~~
of C-Aldrin and c-Dieldrin in Cellular Fractions from Brain
and Liver Tissues of Insecticide Resistant and Susceptible Gambusia.
Tox Appl Pharmacol. 24:190-196, February 1972.
14
37. Wells, M. R. and J. D. Yarbrough. Epoxidation and Fate of C-
Aldrin in Insecticide Resistant and Susceptible Populations of
Mosquitofish, Gambusia affinis. J Agr Food Chen. 21(3):428-429,
May 1973.
38. Yarbrough, J. D., and M. R. Wells. Vertebrate Insecticide Resistance:
jn Vitro Endrin Effect on Succinic Dehydrogenase Activity on Bndrin
Resistant and Susceptible Mosquitofish. Bull Environ Gontam Toxicol.
6(2):171-176, March 1971.
39. Ohsawa, I., J. R. Knox, S. Khalifa, and J. E. Casida. Metabolic
Dechlorination of Toxaphene in Rats. J Agr Food Chem. 23(1):98-106,
January 1975.
40. Hayashi, M., and F. Matsumura. Insecticide Mode of Action. Effect
of Dieldrin on Ion Movement in the Nervous System of Periplaneta
americana and Blattella germanica cockroaches. J Agr Food Chem.
15(4):622-627, July 1967.
68
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SECTION VII
LIST OF PUBLICATIONS
36
Crowder, L. A., and E. F. Dindal. Fate of Cl-Toxaphene in Rats. Bull
Enviorn Contain Tox. .!2_:320-327, March 1974.
Schaper, R. A., and L. A. Crowder. Uptake of Cl-Toxaphene in Mosquito-
fish, Cambusia affinis. Bull Environ-Contain Tox. In Press.
69
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SECTION VIII
GLOSSARY
Abdominal Nerve Cord - The central nervous system of insects, which runs
along the ventral or abdominal surface.
Absorption - Take into the body.
Adsorption - Adhere to the body.
Ag - AgCl - Silver-silver chloride; used for fabricating electrodes employed
in electrophysiology.
AgN03 - Silver nitrate.
Aldrin - 1, 2, 3, 4, 10, 10-hexachloro-l, 4, 4a, 5, 8, 8a-hexahydro-endo-
exo-1, 4:5, 8 - dimethanonaphthalene.
Alimentary Canal - Internal tube from mouth to anus involved in digestion
of food.
Anesthesia - Agent causing loss of sensation, with or without loss of
consciousness.
Anti-Enzyme - Any compound which can inhibit or destroy an enzyme.
Cell Membrane - Extremely thin membrane which covers the surface of
animal cells.
Centrifuge - Machine using centrifugal force for separating substances
of different densities.
Chlordimeform - N* - (4-chloro-o-tolyl)-N, N-dimethylformamidine.
Cl - Radioactive chlorine.
C02 - Carbon dioxide.
CJBi - Counts per minute; unit of radioactivity measurement.
Cyclodiene Insecticide - Any one of a group of compounds, derived by the
Diels-Alder reaction in which hexachlorocyclopentadiene is one of the
reactants.
70
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Cytosol - Background fluid in which organelles are suspended in a cell.
DDT - l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane.
Dehydrochlorination - Removal of chlorine and hydrogen from a molecule.
Dieldrin - l,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-
endo-exo-l,4:5,8-dimethanonaphthalene.
Diffusion - Passing through Che body or medium.
Efflux - Diffusion outward.
Electrode - Conductor used to establish.electrical contact with a non-
metallic part of a circuit.
Electrophysiology - Area of physiology where irritable tissue, e.g.
muscle and nerve,is studied usually with electrical recording instru-
mentation.
Endrin - l,2,3,4,10,10-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-
endo-endo-l,4:5,8-dimethanonaphthalene.
Excretion - Removal of waste products from the body.
Fat Body - Diffuse "tissue" in insects below the epidermis and around
the gut, which serves as a store for fat, glycogen, protein, and plays
an active part in metabolism.
Fluor - Solution of phosphorescent compounds used for liquid scintillation.
Gas Chromatography - Method of separating compounds based upon their
volatility and movement on a stationary phase.
Memolymph - Blood of insects.
HNO., - Nitric acid.
Influx - Diffusion inward.
In Vitro - Experimentation on a tissue or organ removed from the animal.
In Vivo - Experimentation on the whole living animal.
Ion - Atom or group of atoms that carries a positive or negative charge.
Ionic Flux - Movement of ions across a membrane.
KC1 - Potassium chloride.
LC5Q - Concentration at which 50% mortality in a population occurs.
71
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LD-0 - Dose at which 50% mortality in a population occurs.
Lipophilic - Readily dissolvable in non-polar solvents.
LT.Q - Time at which 50% mortality in a population occurs.
M - Molar; a concentration in grains per liter in which one mole is
equivalent to one molecular weight of the substance.
mM - Milltmolar; 10~3M.
Metabolism - The breaking-down or building-up of compounds in the bio-
logical system.
Methyl-parathion - 0,0-Dimethyl 0-j>-nitrophenyl phosphorothioate*
uCi - Microcurie; unit of radioactivity containing 10 curies or
37,000 disintegrations per second.
Microsomes - Vesicles with attached ribosomes formed from the disrupted
endoplasmic reticulum.
Mirex - Dodecachlorooctahydro-l,3,4-metheno-2H-cyclobuto[cd] pentalene.
Mitochondria - Microscopic bodies occurring in the cytoplasm of a cell,
and responsible for energy production.
Mode of Action - Method by which a compound exerts its effects.
Moribund - Dying and usually motionless.
Mortality - Subject to death.
NADP - Nicotinamide adenine dinucleotide phosphate.
®
NCS - Tissue solubilizer for liquid scintillation.
Heurotoxicant - A compound which adversely affects the function of nerve
activity.
Nerve Impulse - Progressive alteration in the charges around a nerve
fiber that follows stimulation, and responsible for transmission.
Nerve Sheath - Structural, non-neural covering of the central nervous
system.
Non-Polar - Without any positive or negative charges.
Nuclei - (pi) Part of the cell which contains the chromosomes or genetic
material.
72
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Oscillograph - A recording from an instrument which exhibits alternating
current wave forms or other electrical oscillations.
Partitioning - Separation of substances into various phases or solvents.
j>H - Hydrogen ion concentration expressed as the negative logarithm.
Polar - Possessing a positive or negative charge.
POPQP - p-bis- 2-(5-phenyloxazolyl) -benzene.
PPB - Parts per billion.
PPM. - Parts per million.
PPQ - 2-5-Diphenyloxazole.
Quench - Suppression of the flashes of light during scintillation.
Radiolabelled - Compound in which one or more of the elements of it's
structure are radioactive.
Resistant - Animal or population not affected by, or tolerable to, a
poison, either partly or entirely so.
Saline - Solution of salts, often times used for the bathing of
animal tissues.
Scintillation - Method of quantifying radioactivity by counting the
flashes of light given off by a phosphor excited by ionizing radiation.
Site of Action - Location in a tissue or organ where a compound exerts
it's effect.
Spike - Long narrow peak observed on an oscilloscope corresponding
to a nerve impulse.
Spontaneous Activity - Nervous activity which occurs without stimulation.
Sorption - Process of taking-up or holding, including both absorbtion
and adsorption.
Synaptic Vesicle - Spheres found within the nerve endings, which contain
the chemical transmitter.
Tarsi - Segment (fifth to the base) of an insect leg.
Tergite - Thickened plate of cuticle on dorsal side of a segment of an
arthropod.
73
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Topical Dose - A dose which is applied externally.
Toxaphene - Substance which results when camphene is chlorinated to
contain 67-69% chlorine; average molecular weight - CinH._ClQ.
1U Lv o
Toxicity Syndrome - Sequence of symptoms following exposure to a poison.
TR • Toxaphene related*
Trachea - Cuticle lined tube conveying air from the spiracles to the
tissues in insects and other arthropods.
Triton X-100 - Emulsifier; alkyl phenoxy polyethoxy ethanol.
Uptake - Incorporate, or absorb into the body.
Viscera - Collective term for the organs of the body.
74
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TECHNICAL REPORT DATA
(P'lease read liiiUiifiiu'a en the rc\usc before com/ilcting)
I REPORT NO.
CPA-600/1-76-008
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
MODE OF ACTION OF CYCLODIENE INSECTICIDES
5. REPORT DATE
January 1976
6. PERFORMING ORGANIZATION CODE
7 AUTHORIS)
Larry A. Crowder
8. PERFORMING ORGANIZATION REPOHT NO.
9. PERFOFtMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Department of Entomology-
University of Arizona
Tucson, Arizona 85721
1EA078
NO.
R800384
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle ParR. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15 SUPPLEMENTARY NOTES
16. ABSTRACT
This report contains information concerning the mode of action, excretion, and
metabolism of the cyclodiene insecticides. Toxaphene was the primary candidate for
investigation with major emphasis on the mammalian system.
Excretion of 36Cl-toxaphene was studied in the laboratory rat. Upon extraction,
most of the radioactivity occurred in the water fractions of urine and feces as ionic
chloride, indicating considerable metabolism of toxaphene. Only minimal storage
appeared to occur.
Occurrence of radioactivity in several tissues of Leucqphaea maderae was deter-
mined after injections of 36ci-toxaphene. Uptake of 10-DM 3°C1-toxaphene in sub-
cellular particles of ventral nerve cord and brain was studied and showed significant
levels in the larger cell fragments; microsomes were also labelled.
The toxicity syndrome of toxaphene to Gambusia affinis was divided into 5 stages,
and the residue level at each stage v/as determined. Excretion was not observed.
Metabolic alteration of toxaphene appeared to be minimal. Differences in individual
mortality appeared to be due to differences in uptake rather than differences in
ability to tolerate particular body loads of toxaphene.
Ventral nerve cords of Periplaneta americana and L_. maderae showed increased
nerve activity as viewed electrophysiologically when exposed to toxaphene. Toxaphene
appeared to be a neurotoxicant.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b IDENTIFIERS/OPEN ENDED TERMS
c COSATI field/Group
*Pesticides
* Insecticides
*Toxicity
Metabolism
*Chloririe Organic Compounds
Chloronydrocarbons
Halohydrocarbons
Physiological effects
Toxaphene
Cyclodiene
Chlorinated camphene
06 A
Of; T
13 OlSTniUUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS I fli.f kciic>,l>
UNCLASSIFIED
21 NO OF FACES
84
20 SECURITY CLASS (Tint paf-cl
UNCLASSIFIED
22 PRICE
EPA Form 2220 1 (9-73)
75
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