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Mode of Action of
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The Nervous System
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Toxaphene
EP 600/1
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i 1
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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 nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The nine series are-
1. Environmental Health Effects Research
2. Environmental Protection Technology
3 Ecological Research
4. Environmental Monitoring
5 Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8 "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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MODE OF ACTION OF CYCLODIENE INSECTICIDES:
THE NERVOUS SYSTEM INFLUENCED BY TOXAPHENE
by
Larry A. Crowder
Department of Entomology
University of Arizona
Tucson, Arizona 85721
Project No. R-804351
Project Officer
Dr. Robert Lewis
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, N.C. 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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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 the use.
ii
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FOREWARD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of existing and new man-made environmental hazards is necessary
for the establishment of sound regulatory policy. These regulations
serve to enhance the quality of our environment in order to promote the
public health and welfare and the productive capacity of our Nation's
population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
The report discusses the mode of action, excretion, metabolism, and
behavioral effects of toxaphene and combinations of toxaphene, methyl
parathion, and/or chlordimeform in various insects, the mouse and the rat.
F. G. Hueter, Ph.D.
Acting Director
Health Effects Research Laboratory
iii
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ABSTRACT
This report contains information concerning the mode of action, excretion,
metabolism, and behavioral effects of toxaphene and combinations of toxa-
phene, methyl parathion, and/or chlordimeform in various insects, the mouse
and rat.
By employing radioisotopes of ions involved in neural functioning, toxaphene
was shown to alter ionic movements to produce an increase of internal K
J i
and Ca within the central nervous systems of the American cockroach,
Periplaneta americana. Toxaphene increased the levels of cAMP and cGMP
in tissues of the cockroach, Leucophaea maderae. These increases were
also observed in tissues of mice poisoned with toxaphene, with the highest
levels occurring in mice displaying advanced symptoms of poisoning. ATPase
enzymes were inhibited by toxaphene in tissues of £. americana and the
mouse; inhibition of Na+-K+ AXPase and Mg"^ ATPase was observed in mouse
tissues both in vivo and in vitro.
Rat pups peri- or postnatally exposed to sublethal doses of toxaphene and
methyl parathion in combination or alone showed few significant changes in
motor skills, behavior, or learning ability as measured by a simple T-maze.
Combining chlordimeform and/or methyl parathion with ^^Cl-toxaphene pro-
duced some significant changes in the amount of Cl recovered in feces
and the amount deposited in tissues of orally-dosed mice. Toxaphene and
toxaphene-methyl parathion in combination with ^C-chlordimeform resulted
in lower recoveries of ^C in tissues of mice than from those dosed with
l^C-chlordimeform alone. Toxs.phene also decreased the quantity of para-
nitrophenol excreted in the urine of mice dosed with a toxaphene-methyl
parathion combination.
Dosage-mortality studies indicated that toxaphene does not potentiate
methyl parathion in several hemipteran predators or lepidopterous pests
under laboratory conditions. Methyl parathion was more toxic to the
predators than target insects.
This report was submitted in fulfillment of Project Number R-804351 by
the University of Arizona under the (partial) sponsorship of the Environ-
mental Protection Agency. Work was completed as of July 1, 1979.
IV
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CONTENTS
Foreward iii
Abstract iv
List of Figures ix
List of Tables xi
Acknowledgements xv
Sections
I Conclusions 1
II Recommendations ..... 2
III Introduction 3
IV Methods and Materials 5
A Effects of Toxaphene on the Nervous System 5
1. Ion Movements in the Nervous System of the
American Cockroach 5
a. Experimental Animals and Dissection Procedures . 5
b. Ions and Solutions 5
c. Experimental 6
(1) Uptake of Ions 6
(2) Efflux of Ions 6
d. Radioassay 7
2. Cyclic Nucleotides in Several Tissues of the
Cockroach and Mouse 7
a. Animals 7
b. Experimental Design 7
c. Assay Procedure 8
3. ATPase in Several Tissues of the Cockroach and
Mouse 8
a. Experimental Animals 8
b. Experimental Design 9
(1) In Vivo Experiment 1 9
(2) In Vivo Experiment II 9
(3) In Vitro Experiments 9
c. Tissue Collection and Homogenate Preparation . . 9
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Page
d. Electron Microscopy 10
e. Enzyme Assay 10
(1) In Vivo Experiment 1 10
(2) In Vivo Experiment II 11
(3) In Vitro Experiments 11
f. Statistical Analysis 11
4. Neonatal Development and Postnatal Learning
in the Rat 11
a. Perinatal Exposure to Toxaphene-Methyl Parathion,
and Methyl Parathion 11
(1) Maternal Behavior 12
(2) Grasp-Hold Reflex 12
(3) Startle Response 12
(4) Placing Reflex 12
(5) Righting Reflex 12
(6) Open Field Test 13
(7) Maze Learning Transfer 13
b. Peri- and Postnatal Exposure to Toxaphene 13
B Toxaphene and Methyl Parathion Against Non-Target and
Target Insects 15
1. Non-Target Insects 15
2. Target Insects 16
a. Lepidopterous Larvae 16
b. Houseflies 17
C Fate of Toxaphene, Chlordimeform, and Methyl Parathion
in the Mouse 17
1. Toxaphene 17
2. Chlordimeform 19
3. Methyl Parathion 19
V Discussion
A Changes in the Nervous System Influenced by Toxaphene. . . 20
1. Ion Movements in the Nervous System of the
American Cockroach . 20
a. Methodology. 20
(1) 24Na+ 20
(2) 36C1~ 20
(3) 42K+ 21
(4) 45Ca^ 28
vi
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,b. Relationship of the Observed Changes to Neural
Activity 33
c. Differences Between Nerve Sections 34
2. Cyclic Nucleotides in Several Tissues of the
Cockroach and Mouse 34
3. ATPases in Several Tissues of the Cockroach
and Mouse 40
a. Electron Microscopy 40
b. In Vivo Experiments 41
c. In Vitro Experiment 42
(1) Mouse 42
(2) .P. americana 42
(3) Regressions 48
4. Neonatal Development and Postnatal Learning in the Rat 48
a. Perinatal Exposure to Toxaphene-Methyl Parathion
and Methyl Parathion 48
(1) Pup Mortality 48
(2) Coordination Tests 51
(3) Open Field Testing 51
(4) Maze Learning Transfer 51
b. Peri- and Postnatal Exposure to Toxaphene 56
(1) Coordination Tests 56
(2) Maze Learning Transfer 56
B Toxaphene and Methyl Parathion Against Non-Target
and Target Insects 56
1. Non-Target Insects 56
2. Target Insects 67
a. Lepidopterous Larvae 67
b. Houseflies 67
C Fate of Toxaphene, Methyl Parathion, and Chlordimeform
in the Mouse 72
1. Excretion: Toxaphene 72
2, Excretion: Chlordimeform 76
3. Excretion: Methyl Parathion 76
4. Tissue Retention: Toxaphene 76
5. Tissue Retention: Chlordimeform 81
VII
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Page
VI References 83
VII List of Publications 88
VIII Glossary 89
viii
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FIGURES
.No. Page
1 T-Maze employed in learning transfer analysis of rats
postnatally exposed to toxaphene, and perinatally exposed
to toxaphene, methyl parathion, and toxaphene: methyl
parathion 14
42 +
2 Rates of K flux measured from the brain section of
the central nervous system of P_. americana exposed to
10 M toxaphene in vitro 24
42 +
3 Rates of K flux measured from the thoracic section of
the central nervous system of I\ americana exposed to
10 M toxaphene in vitro 25
42 +
4 Rates of K flux measured from the abdominal section
of the central nervous system of _P. americana exposed to
10 M toxaphene in vitro 26
45 -H-
5 Rates of Ca flux measured from the brain section of
the central nervous system of P_. americana exposed to
10 M toxaphene in vitro 29
45 ++
6 Rates of Ca flux measured from the thoracic section
of the central nervous system of _P. americana exposed to
10 M toxaphene in vitro 30
7 Rates of Ca flux measured from the abdominal section
of the central nervous system of JP. americana exposed to
10 M toxaphene in vitro 31
8 Mean hang time (grasp-hold reflex) of rat pups perinatal-
ly exposed to methyl parathion 53
9 Mean hang time (grasp-hold reflex) of rat pups perinatal-
ly exposed to methyl parathion:toxaphene (1:2) 53
10 Activity in the open field test by rat pups perinatally
exposed to methyl parathion 54
11 Number of boluses deposited in the open field by rat pups
perinatally exposed to methyl parathion 54
12 Results of maze learning transfer by rats perinatally ex-
posed to methyl parathion 55
IX
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No. Page
13 Results of maze learning transfer by rats perinatally ex-
posed to methyl parathion and toxaphene (1:2) 55
14 Mean hang time of rat pups perinatally exposed to toxaphene. 57
15 Results of maze learning transfer by rats postnatally ex-
posed to toxaphene 58
16 Results of maze learning transfer by rats perinatally ex-
posed to toxaphene 58
x
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TABLES
No. Page
1 Two-Way Analysis of Variance for Control vs. Exposed
Sections. F Values for Uptake and Efflux of Ions by Each
Nerve Section 21
2 Results of T-Test Evaluation of Sodium-24 Movements Between
Control and Exposed Nerve Sections at Each Time Internal. 22
3 Results of T-Test Evaluation of Chlorine-36 Movements Bet-
ween Control and Exposed Nerve Sections at Each Time In-
terval 23
4 Results of T-Test Evaluation of Potassium-42 Movements
Between Control and Exposed Nerve Sections at Each Time
Interval 27
5 Results of T-Test Evaluation of Calcium-45 Movements Bet-
ween Control and Exposed Nerve Sections at Each Time In-
terval 32
6 Cyclic Nucleotide Concentrations in Several Tissues of a
Laboratory Culture of the Cockroach, L_. maderae, as Deter-
mined by Competitive Protein-Binding Assay 35
7 Cyclic Nucleotide Concentrations, Determined by Compete-
tive Protein-Binding Assay, of the Cockroach, L_. maderae
Treated with 207 ug/Animal of Toxaphene 36
8 Cyclic AMP Concentrations, Determined by Competitive Pro-
tein-Binding Assay, in Several Tissues of the Male Mouse
Orally Dosed with 112 mg/kg of Toxaphene 37
9 Cyclic GMP Concentrations, Determined by Competitive Pro-
tein-Binding Assay, in Several Tissues of the Male Mouse
Orally Dosed with 112 mg/kg of Toxaphene 38
10 Means of Total ATPase Specific Activity + S.E. in Kidney
and Liver of Control and Toxaphene-Dosed Mice Used in In
Vivo Experiment I 41
11 Percentage of Inhibition of ATPase Activities Due to
Toxaphene (112 mg/kg) in Mice In Vivo 41
XI
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No. Page
+ + | |
12 Means of Na -K and Mg ATPase Specific Activities in
Kidney, Brain and Liver From Control and Toxaphene-Dosed
Mice Used in In Vivo Experiment II 43
_1_ _1_ i I
13 Means of Na -K and Mg ATPase Specific Activities in
Homogenates of Kidney, Brain and Liver, from Mice, Sub-
jected In Vitro to Various Concentrations of Toxaphene. . 44
14 Percentage of Reduction or Increase in Activity of the
ATPase of Mouse Tissues Tested in Response to Ethanol
and Various Concentrations of Toxaphene In Vitro 45
15 Means of Na -K and Mg ATPase Specific Activities in
Homogenates of P_. americana CNS and Malpighian Tubules
Subjected In Vitro to Various Concentrations of Toxaphene 46
16 Percentage of Reduction or Increase in ATPase Activity
of P_. americana Tissues Tested in Response to Ethanol
and Various Concentrations of Toxaphene In Vitro. . . - . - . 47
17 Significance of Linear and Linear-Log Regressions of
the Different ATPases with the Treatments 49
18 Prediction Equations of Linear Log Regression Between
Tissues of the Mouse and P_. americana and the Four Toxa-
phene Concentrations 50
19 Results of Coordinatior Tests Performed on Rat Pups Peri-
natally Exposed to Methyl Parathion, and Methyl Parathion:
Toxaphene (1:2) 52
20 Results of Coordinatior. Tests Performed on Rat Pups Peri-
natally Exposed to Toxaphene 57
21 Methyl Parathion LD-50 Values (yg/g) at 24 Hr for Preda-
tors from Several Areas, in Arizona in 1977 60
22 Toxaphene LD-50 Values (Ug/g) at 24 Hr for Predators from
Several Areas in Arizona in 1977 61
23 Methyl Parathion LD-50 Values (yg/g) at 24 Hr for Preda-
tors from Several Areas in Arizona in 1978 62
24 Methyl Parathion LD-50 Values (yg/g) at 48 Hr for Preda-
tors from Several Areas: in Arizona in 1978 63
25 Methyl Parathion LD-50 Values (yg/g) at 48 Hr for Preda-
tors from Several Areas in Arizona in 1978. After an Ap-
parent Anomalous Value was Removed from the Original Data
Set 64
xii
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No. Page
26 Methyl Parathion LD-50 Values (yg/g) at 48 Hr for Preda-
tors from Several Areas in Arizona in 1978. After an
Apparent Anomalous Value was Removed from the Original
Data Set 64
27 Dosage-Mortality After 48 Hr of Yuma Predators in 1978
Using Methyl Parathion (MP) at the LC-50 Level (ng/insect),
Toxaphene (T) at Twice the Methyl Parathion Concentration,
and a 1:2 Methyl Parathion:Toxaphene Mixture (MP-T) ... 66
28 Two Sample T-Test at the 95% Confidence Level on 1978
Predators from Yuma Comparing Methyl Parathion (MP) at
the LC-50 Level (ng/insect), Toxaphene (T) at Twice the
Methyl Parathion Concentrations, and a 1:2 Methyl Para-
thion: Toxaphene Mixture (MP-T) 68
29 Methyl Parathion LD-50 Values (yg/g) for Laboratory
Strains o±. Lepidopterous Larvae and Houseflies 69
30 Dosage-Mortality After 48 Hr of Lepidopterous Larvae and
Houseflies using Methyl Parathion (MP) at the LC-50 Level
(ng/insect), Toxaphene (T) at Twice the Methyl Parathion
Concentration, and a 1:2 Methyl Parathion:Toxaphene Mix-
ture (MP-T) 70
31 Two Sample T-Test at the 95% Confidence Level on Lepidop-
terous Larvae and Houseflies Comparing Methyl Parathion
(MP) at the LC-50 Level (ng/insect), Toxaphene (T) at
Twice the Methyl Parathion Concentrations, and a 1:2
Methyl Parathion:Toxaphene Mixture (MP-T) 71
32 Mortality from Excretion-Retention Studies 73
33 Mortality of Mice from Combinations of Toxaphene and
Methyl Parathion 74
O £
34 Recovery of Cl from Urine and Feces of Male Mice Orally
n r
dosed with Cl-Toxaphene 75
O f
35 Recovery of Cl from Urine and Feces of Female Mice
Orally dosed with Cl-Toxaphene 75
14
36 Recovery of C from Urine and Feces of Male Mice Orally
14
Dosed with C-Chlordimeform 77
37 Retention of Cl by Tissues of Male Mice Orally Dosed
o £
with Cl-Toxaphene 78
xin
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No. Page
O £
38 Retention of Cl by Tissues of Male Mice 8 and 33 Days
o/:
After a Redose of Cl--Toxaphene 79
39 Comparitive Retention of Cl by Tissues of Male and Fe-
o /
male Mice Orally Dosed with Cl-Toxaphene 80
14
40 Retention of C by Tissues of Male Mice Orally Dosed with
14
C-Chlordimeform 82
xiv
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ACKNOWLEDGEMENTS
Acknowledgement is made to Keith D. Butler, Kamal M. Fattah, Gregory C.
Lanzaro, Daniel J. Pape, and Roy S. Whitson for aiding in the conduct
of these studies. Appreciation is also extended to Dr. George W. Ware
for his support of this project. Special thanks go to Anna Collins
for her patience in typing this manuscript and lettering the figures.
This investigation is indebted to Dr. Robert Lewis, Project Officer,
for his continued interest and amiable attitude.
xv
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SECTION I
CONCLUSIONS
Toxaphene increased the internal levels of K+ and Ca , increased cAMP
and cGMP quantities, but inhibited ATPases in the central nervous system
of the cockroach. These changes may or may not be interrelated. In-
creases in cAMP and cGMP, as well as the inhibition of ATPases, were also
observed in other tissues of the cockroach and in tissues of the mouse.
Oral exposure to sublethal doses of toxaphene appeared not to cause im-
pairment of learning abilities of adult rats, as well as when the exposure
was received perinatally. Perinatal exposure to toxaphene, methyl para-
thion, or a combination of both produced only minor changes in motor skills
and behavior of -he neonates.
Combining toxaphene with methyl parathion did not potentiate the toxicity
of methyl parathion under laboratory conditions. This was true for vari-
ous lepidopterous pests and hernipteran predators. The toxicity of methyl
parathion with or without toxaphene was much greater to the predators
than the pests.
Combinations of toxaphene, methyl parathion and/or chlordimeform caused
only slight differences in excretion and tissue retention of the individual
insecticides in mice. Important differences were (1) methyl parathion
and/or chlordimeform combined with toxaphene increased quantities of
toxaphene retained by some tissues, and (2) toxaphene reduced the amount
of para-nitrophenol (a major metabolite of methyl parathion) excreted in
the urine.
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SECTION II
RECOMMENDATIONS
Although many data have been collected on several facets of the use and
properties of toxaphene, many important questions still remain. The most
striking questions are toxaphene's exact mode and site of action, as well
as it's metabolism. Although the continued use of toxaphene is currently
being questioned, answers to the above questions might still increase our
knowledge of the compounds that have been released into the environment
and eventually become incorporated into the food chain.
Indications were obtained tha: toxaphene does not interfere with the learn-
ing ability of adult rats but produces slight changes in motor functions
and behavior of rats exposed serinatally. These changes were not drastic,
but indicated the need to pursue these questions to determine the potential
effect of toxaphene on the behavior and neuro-muscular coordination of
mammals chronically exposed.
The use of toxaphene in combination with methyl parathion, and/or chlor-
dimeform on crops should be farther examined. The toxicity of methyl
parathion appeared not to be potentiated by addition of toxaphene, but
such combinations might restrict the metabolism, excretion, and tissue
retention of other insecticides in mammals.
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SECTION III
INTRODUCTION
Among the chlorinated hydrocarbon insecticides, toxaphene (ClriH1ACl0,
ID lu o
chlorinated camphene with a chlorine content of 67-69%), is of importance
and interest because of its tremendous use on cotton. Information re-
garding this compounds mode of action, excretion, and metabolism in
animal systems is imperative to better evaluate the effects on man and
other non-target species. Unfortunately, there is a paucity of such
information with which to make a proper judgement concerning toxaphene's
continued usage.
Although it is generally accepted that toxaphene acts on the nervous
system, a precise locus of action has not been defined. LaLonde and
Brown observed the action of toxaphene on sensory nerves of Periplaneta
americana (L.) isolated from the central nervous system. Following a
latent period, toxaphene generated 100 mV spikes for 30 min. Intense
forms of activity were also observed in isolated nerve preparations from
2
this same species treated with toxaphene in a study by Dary and Crowder .
Latent periods between introduction of toxaphene and onset of intense
activity decreased as the concentrations were increased. Wang and Mat-
3
sumura observed toxaphene neurotoxicity; it appeared to be more highly
neurotoxic (threshold concentration 6x10 M) than was indicated by in
vivo toxicity.
There is considerable evidence suggesting cyclic AMP (adenosine 3', 5'-
cyclic monophosphate) and ATPases (adenosine triphosphatase enzymes) are
involved in neural transmission. The transport of Na and K ions across
the membrane through the active transport mechanism is thought to be
4
controlled by ATPase (Skou ). This led to investigations of ATPase as
the mediator of insecticide action on ion fluxes, dominated by the research
of Koch5, Koch et al. , Desaiah and Koch7' '9, and Yap et al. They
demonstrated that ATPase was inhibited by DDT, cyclodienes, and toxaphene
in numerous tissues of a variety of animals.
Cyclic nucleotides have been shown to be involved in nerve function (Rail
11 12
and Gilman , and Bloom ) and as mediators of the active transport mecha-
-------
1 O
nism (Horwitz and Eaton ) The potential relationships between insecti-
cides and the action of cyclic AMP has been explored by several investiga-
tors (Casida and Maddrell , Kacew and Singhal ' , and Rojakovick and
March17).
But the effects of insecticides, and specifically toxaphene, on cyclic
nucleotides and ATPase activity are still unresolved. It was hypothesized
that toxaphene may interfere with transport mechanisms in nerve membranes
thus causing the effects observed electrophysiologically. Since these
transport mechanisms are related to cyclic nucleotides and ATPase, the
objectives of the project were to:
(1) Measure ionic fluxes across nerve membranes in cyclodiene-
and toxaphene-treated animals.
(2) Examine the effects of cyclodienes and toxaphene on cAMP, cGMP,
adenyl cyclase, guanyl cyclase, and ATPase in nerves.
(3) Determine the effects of cyclodienes and toxaphene on synaptic
and axonal transmission.
To generate further information about toxaphene, an additional ob-
j ective was to:
(4) Determine the uptake, metabolism, and excretion of toxaphene
in combination with methyl parathion and chlordimeform.
-------
SECTION IV
METHODS AND MATERIALS
A. EFFECTS OF TOXAPHENE ON THE NERVOUS SYSTEM
1. Ion Movements in the Nervous System of the American Cockroach
a. Experimental Animals and Dissection Procedures
American cockroaches were maintained in the rearing facilities of the
Department of Entomology, University of Arizona. The cockroaches were
©
fed Purina^Dog Chow coated with honey and glycerin (12:1:1, v/v),
with water supplied ad libitum. Conditions within the rearing facility
were: 24 + 3° C, 30 to 60% relative humidity, and a 9:15 hr light-
dark cycle.
Adult male cockroaches were removed from the colony and anesthetized
with carbon dioxide. The head, legs, and wings were clipped from the
body, and a dorsal, longitudinal incision made along the length of the
body. The gut, fat body, and reproductive organs were removed, and
three drops of saline were placed in the body cavity to prevent desic-
cation of the ventral nerve cord (VNC). The VNC was severed at the
cereal nerves, posterior to the sixth abdominal ganglion, and between
the metathoracic and first abdominal ganglia. Nerve sections produced
by these cuts were designated: thoracic (TH), consisting of the 3
thoracic ganglia and connectives, and abdominal (AB), consisting of the
6 abdominal ganglia and connectives. The third section, brain (BR),
consisted of the ganglia from the head capsule.
The head was cut into two halves, pulled apart with forceps, and BR
dissected from the head capsule, often without the suboesophageal
ganglion. Therefore, BR consisted of the 3 major lobes (proto-, deuto-,
and tritocerebrum), and in some cases, the suboesophageal ganglion.
b. Ions and Solutions
45 ++
Four radioactive ions were employed: calcium-45 ( Ca , ICN Chemical
o r
and Radioisotope Division, Irvine, Calif.), chlorine-36 ( Cl , New
42 +
England Nuclear, Boston, Mass.), potassium-42 ( K ), and sodium-24
( Na ). The K and Na were produced in a Triga Mark I reactor by
the Nuclear Engineering Department, University of Arizona.
1 O
The saline of Yamasaki and Narahashi was used throughout this study,
-------
and prepared with demineralized distilled water, with a final pH of 7.4.
c. Experimental -
19
The methods employed were adapted from those of Matsumura and O'Brien ,
20 21 22
Hayashi and Matsumura , Treherne , and Eldefrawi and O'Brien . Exposure
of the nerve sections to toxaphene was entirely in vitro. The experiment
was divided into 2 main areas of evaluation: uptake and efflux of ions.
(1).Uptake of Ions
For uptake measurements, 10 o.f each CNS section (BR, TH, and AB) were used
at each of the 7 time periods for exposed and a like number for control
evaluations of each ion. Once dissected, TH and AB sections were pulled
across a piece of filter paper to remove extraneous fat body, tracheae,
and saline. The third section, BR, was placed directly into the saline.
Once dissected, BR, TH, and AB were placed into scintillation vials con-
taining 1.0 ml of saline composed of 1 of the 4 radioactive ions. The
-2
saline had 1.0 ul of 10 M technical grade toxaphene (batch number X16189-
49) in acetone added, for a final concentration of 10 M toxaphene for
exposed samples. This concentration was selected because it had been used
19 20
for DDT and dieldrin in similar experiments '
Nerve sections were incubated in the radiolabelled saline for the following
time periods: 1, 2.5, 5, 10, 15, 30 and 60 min. Following the prescribed
incubation period, nerve sections were removed from the saline and pulled
along the vial wall to remove as much extraneous saline as possible. Each
section was then transferred into a preweighed dry scintillation vial,
reweighed, and 1.0 ml of TS-J tissue solubilizer (Research Products Inter-
national Corp., Elk Grove Village, 111.) added to each for digestion.
(2).Efflux of Ions
Ten of each CNS section (BR, TH, and AB) were used for the entire evalua-
tion of control as well as exposed efflux of each ion. Dissected nerve
sections were placed into sci-ntillation vials containing 1.0 ml of radio-
labelled saline for 30 min. This saline contained neither acetone nor
toxaphene. Following the incubation period, BR, TH, and AB were pulled
along the vial wall to remove excess saline and passed through a series
of 1.0 ml rinses of non-labelled saline containing either 10 M toxaphene
for exposed samples, or 1.0 Jl of acetone for controls. The sections
were allowed to remain in each rinse vial for a specified time interval,
then transferred to the next without being pulled along the vial wall.
-------
The different rinses represented the amount effluxed after: 1, 2.5, 5, 10,
15, 30 and 60 min. The nerve sections were pulled along the wall of the
last rinse vial and placed into individual, dry scintillation vials. Efflux
data were expressed as the percentage of radioactivity remaining within the
sections over time. Percentages were based upon the sum of rinse values
for each section plus that remaining within the section. This represented
the total sum of the activity within the section at the beginning of efflux
measurements. Each rinse value, added to those before it and divided by
the total sum, resulted in the percent efflux up to that time period. Each
/«j
section had 1.0 ml of TS-I^ tissue solubilizer added for digestion.
d. Radioassay
Radioassay was performed on a Nuclear Chicago Liquid Scintillation Spectro-
photometer (Model 6822). To each of the digested nerve samples from uptake
and efflux measurements, 10 ml of scintillation cocktail were added. The
cocktail was composed of: 4.0 grams PPO and 0.05 grams POPOP (Research
Products International Corp.) per liter toluene. Rinses from efflux
measurements had 10 ml of a cocktail for aqueous samples, consisting of
4.0 grams PPO and 0.05 grams POPOP per liter of a 2:1 mixture of toluene
^)
and the surfactant Triton X-lOu^ added to each. All samples were assayed
for 10 min for a counting efficiency of >90 percent.
2. Cyclic Nucleotides in Several Tissues of the Cockroach and Mouse
a. Animals
Male and female cockroaches, Leucophaea maderae (Fab.), were maintained as
a mixed laboratory population with free access to water and food consisting
of honey and glycerin-coated dog chow. Male Swiss mice (Charles River Lab-
oratories, Inc.) were maintained on a diet of rat chow and water ad libitum.
The rearing room was maintained as previously described.
b. Experimental Design
Adult cockroaches were injected with 207 yg/animal of toxaphene in 0.05 cc
mineral oil under the third tergite, and held for 24 to 168 hr without food
and water for sacrifice of 2 males and 2 females at each of 8 time intervals.
Controls, injected with 0.05 cc mineral oil, and untreated individuals were
also held without food and water for 24 to 168 hr. One male and 1 female
from each of these latter 2 groups of insects were sacrificed at each of the
8 time intervals. Additionally, cockroaches (15 males and 15 females) from
the laboratory stock were sacrificed immediately to determine normal cAMP
-------
and cGMP levels. Brain, VNC, Malpighian tubules, gastric caeca, fat body,
rectum, and testes or ovaries were dissected from the sacrificed individuals
and frozen immediately to be assayed for cAMP and cGMP. Tissues were analyzed
separately on an individual animal basis.
Fifteen-week old male mice weighing approximately 37 g were orally dosed
©
with 112 mg/kg of toxaphene in 0.16 cc Mazola corn oil and held with free
access to food and water. Controls were dosed with 0.16 cc of corn oil.
Five treated and 5 control mice were sacrificed at each time period: 15, 45,
90, and 240 min. The 240 min treated sample included 2 mice that died bet-
ween 110-145 min. Brain, spiral cord, liver, kidney, and testes were dissect-
ed from the animals and immediately frozen to be assayed for cAMP and cGMP.
Tissues were analyzed separately on an individual animal basis.
c. Assay Procedure
A single sample containing approximately 10 mg of a frozen tissue was homo-
genized with 1 ml 5% (w/v) TCA. The homogenate was centrifuged at 0°C, 4000
G for 15 min. The supernate was extracted with 5 ml of acidified ether
(3 ml 0.1 N HC1/100 ml ethyl ether). The aqueous phase was air-dried in a
stream of compressed air and the residue reconstituted with 1 ml Tris-EDTA
buffer, pH 7.5
Competitive protein-binding assay kits for cAMP and cGMP (Diagnostic Products
Corp., Culver City, CA. 90230) were used following extraction of the tissues.
The assay procedure consisted of the competition of ( H)-cyclic nucleotide
and unlabelled cyclic nucleotide for binding sites on a binding protein.
Unbound nucleotides were separated from bound by adsorption onto dextran-
coated charcoal. Bound radiolabelled nucleotide was then radioassayed.
Cyclic nucleotide concentrations were determined from standard curves pre-
pared from serial dilutions o:: stock standards contained in the assay kits.
The curves, 3 replicates for cAMP and 4 replicates for cGMP (1 replicate for
each kit), were analyzed by weighted linear regression and plotted using the
TO O/ 0 R O £.
logit log method (Rodbard and Lewald , Felman and Rodbard , and Rodbard ' )
27
Protein concentrations were determined by the method of Lowry et al. employ-
ing a standard curve prepared in triplicate with bovine albumin serum. Un-
knowns were calculated from the respective standard curve equations.
3. ATPases in Several Tissues of the Cockroach and Mouse
a. Experimental Animals
Mice used for in vivo experiment I were male Swiss mice. Male Swiss Webster
8
-------
mice used in both in vivo experiment II and the in vitro investigations were
obtained from the University of Arizona Department of Veterinary Science.
Adult male cockroaches, Periplaneta americana (L.), were removed from the
Department's cultures and held separately in the rearing room.
b. Experimental Design
(1) In vivo Experiment I
Male mice were used at 20-30 weeks of age and weighing approximately 38 g.
Eighteen mice, each constituting a replicate, were dosed by oral gavage with
112 mg/kg of toxaphene in 0.16 cc of corn oil. Mice surviving the dose were
sacrificed 60-90 min later during the second symptom of the poisoning as de-
scribed in the cyclic nucleotide study, i.e. hyperactivity indicated by
kicking, chewing, and coordinated jerky movements with rapid breathing tremors.
If death occurred within the first hour, mice were immediately processed.
Controls (N = 18, 1 mouse/replicate) were dosed with 0.16 cc corn oil and
sacrificed 60-90 min later.
(2) In vivo Experiment II
Male mice were employed when 9-11 weeks old and weighing approximately 28 g.
Treated (N = 35, 1 mouse/replicate) and the control mice (N = 28, 1 mouse/
replicate) were exposed to toxaphene and corn oil, respectively, in the
manner described above.
(3) In vitro Experiments
Twenty mice (each constituting a replicate) were used to study the effects
of in vitro toxaphene exposure on mouse tissues. Each tissue was subjected
to 4 treatments and 2 controls: 10 M, 10 M, 10 M and 10 M toxaphene; 10
yl ethanol and untreated controls. Three subsamples were run for each treat-
ment in each of the 3 tissues. Tissues from 3 cockroaches were combined to con-
stitute a replicate. Fourteen replicates were performed in the manner
described above for mice.
c. Tissue Collection and Homogenate Preparation
Brain, whole liver and the right kidney were dissected immediately after
sacrificing the mouse, washed with ice cold homogenization solution (pH 7.6)
containing 0.25 M sucrose, 10 mM Tris (hydroxymethyl) aminomethane and 0.5
O Q
mM EDTA (ethylene diamine tetraacetic acid) (Clark and Nicklas ), and cut
into small pieces with scissors. Each tissue was then transferred to a pre
weighed beaker containing 2 ml ice cold homogenization solution and weighed.
The Malpighian tubules and central nervous system were dissected from adult
male cockroaches and transferred immediately to ice cold homogenization
solutions.
9
-------
The method used in tissue fractionation was after Koch All tissues
were homogenized by a motor-criven homogenizer at 500 rpm for 2 min. The
liver was homogenized in 15 ml homogenization solution, brain and kidney
in 10 ml each, and cockroach tissues in 5 ml each. The whole homogenate
was centrifuged for 10 min, with 2 washings, at 0°C and 900 G in a Sorvall
RC2-B refrigerated centrifuge-.. The supernate from cockroach tissues was
retained as the homogenate fraction. Mouse tissues were further centrifuged
for 20 min at 0 C and 13,000 G; the resulting pellet was resuspended in
homogenization solution to a volume of 5 ml and used as the homogenate fraction.
Glassware used in the fractionation procedure was maintained ice cold using
crushed ice.
d. Electron Microscopy
The pellets of homogenate fractions for liver, brain and kidney were prepared
as previously described and immediately fixed in 0.1 M sodium cacodylate buf-
fer (pH 7.4) containing 2% glutaraldehyde for 2 hr. The tissues were post
fixed in the same buffer containing 2% Os0, (osmium tetroxide) and dehydrated
in ethanol. They were than embedded in Epon 812 and sectioned with a Sorvall
MT-2 microtome. The sections were examined on a Philips EM 200 electron
microscope at a magnification of 11,600.
e. Enzyme Assay
(1) In vivo Experiment I
The reaction mixture was prepared as a stock solution and kept refrigerated.
It contained (as final concentrations in the stock solution) 5mM ATP (adenosine
5'-triphosphate, disodium salt; Eastman Kodak Co.), 5 mM Mg (as Cl salt;
from Merck Chemical Co.), 90 mM Na+, 22 mM K+ (both Na and K as Cl salts; Math-
eson Coleman and Bell Manufacturing Chemists), 118 mM imidazole buffer (pH
7.5; J. T. Baker Co.), 0.19 nM NADH (diphosphopyridine nucleotide "reduced
NADH disodium"; ICN Pharmaceuticals, Inc.), 0.5 mM PEP (phosphoenol pyruvate;
Sigma Chemical Co.) and 3 units of pyruvate kinase (ICN Pharmaceuticals, Inc.)
Another stock reaction mixture was prepared as above except for the addition of
1 mM ouabain (ICN Pharmaceuticals, Inc.). Ouabain is a cardiac glycoside which
-i- + 29
specifically inhibits the Na -K ATPase (Mcllwain ). One hundred microliters
of each homogenate fraction were added to 3 ml of reaction mixture. Enzyme
activities were measured at 37 C for 40 min using a water bath. Stoppered 12
ml centrifuge tubes were used as reaction vessels. The reaction was stopped
by addition of 100 yl ice cold 30% TCA (trichloroacetic acid).
10
-------
(2) In vivo Experiment II
The enzyme activities were determined by the procedures of Skou '
The reaction mixture (3 ml) contained, in final concentrations, 5mM ATP,
5 mMMg^, 90 mM Na+, 19 mM K+, 120 mM imidazole buffer (pH 7.5), and 100
yl homogenate fraction. Ouabain (where added) was 3 mM and ImM for mouse
and cockroach tissues, respectively. The reaction was stopped by the
addition of 200 ml ice cold 30% TCA and the enzyme activity measured at
37°C for 30 min.
(3) In vitro Experiments
The reaction mixture used was identical to that of in vivo experiment II
except for the addition of toxaphene. Toxaphene stock solutions were
prepared in ethanol in such a way that when added in 10 pi aliquots would
give a final concentration of 10 M, 10 M, 10 M or 10 M toxaphene in
3 ml reaction mixture.
-H-
Mg - ATPase activity was measured in the presence of Ouabain in the
reaction mixture. Na -K ATPase activity was calculated as the difference
t |
between total ATPase and Mg -ATPase measured activities. ATPase activities
were obtained in terms of quantities of inorganic phosphate (Pi) released
during the reaction. One milliliter of the sample was treated according to
32
the method of Ohnishi et al. for ATPase and read at 750X in a Bausch and
Lomb Spectronic 20. The Pi concentration of the sample was then determined
from a standard curve with concentrations ranging from 0.001-0.1 mg Pi.
This method was found unreliable for concentrations above 0.1 mg Pi. Protein
27
concentrations of the samples were determined after Lowry et al.
f. Statistical Analysis
Enzyme activities were calculated for all experiments as mgs Pi released/
mg protein/hr. A one-way ANOVA followed by mean separations (SNK procedure)
was performed on data at an alpha level of 0.05.
(4) Neonatal Development and Postnatal Learning in the Rat
a. Perinatal Exposure to Toxaphene-Methyl Parathion and Methyl Parathion
Inseminated, adult female rats (Sprague-Dawley) were obtained from Hilltop
Lab Animals (Chadworth, Calif.). Beginning on day 7 of pregnancy and con-
tinuing through the fifteenth, the experimental females received daily oral
(gavage) doses of methyl parathion (MP) or methyl parathion and toxaphene
(MP-T). The doses administered were 1.0 mg/kg MP and 1.0 mg/kg MP + 2.0
mg/kg toxaphene in 0.1 ml corn oil. Control females were dosed under the
same regime with 0.1 ml corn oil. The rats were caged in pairs until 2 or
3 days prior to parturition then caged individually. The animals had access
11
-------
to food (Wayne's Lab BLOX^ and water ad libitum.
Once all females had given birth, the pups were distributed among the fe-
males in a group (control or experimental) so that each had 10 pups. Once
the pups were 3 days old and until they reached 15 days, daily measurements
of weight and mortality were recorded for each litter.
(1) Maternal Behavior - Beginning on day 5 postpartum, maternal behavior
33
was observed by measuring retrieval time (Paulsen et al. ). Following
weighing, pups were scattered about the bottom of the cage and the amount of
time necessary for the mothers to initiate the return of the pups to their
nests was recorded.
Behavioral tests to measure neonatal development commenced on the seventh
34
day postpartum and consisted of the grasp-hold reflex (Davenport and Gonzalez )
35
righting reflex (Ibid.)> startle reflex (Eayrs and Lishman ) and placing
reflex (Ibid.)- Beginning at 18 days of age, the pups were tested in the open-
O £
field test for emotionality (Candland and Nagy ). They were tested for
maze learning transfer upon reaching an age of 66-68 days. Description of
these tests follows:
(2) Grasp-Hold Reflex - Each pup received 3 daily tests of grasp-hold reflex
ability on days 7 through 15. In this test a 0.5 mm diameter wire was sus-
pended 15 cm above a bed of wood shavings. Each pup was "hung" on the wire
by its forepaws, released, and the length of time the pup held on to the wire
before falling was measured.
(3) Startle Response - Each pup was held upright and a sharp clicking sound
was made behind its head. Tne age at which the first response occurred was
recorded, as well as the age at which 90% of the litter scored a positive
response.
(4) Placing Reflex - The pups chin was touched to a 0.5 mm wire, suspended
directly in front of it. The first appearance of the response was recorded
at the age at which the animal first placed either forepaw on the wire on
either side of its chin. This test was performed concurrently with grasp-
hold.
(5) Righting Reflex - Each pup received 3 daily righting reflex trials be-
ginning on day 7. On each trial, the pup was held by the head and tail end
upside down and dropped, approximately 35 cm onto a bed of wood shavings.
The response was scored positive when the pup landed on all fours. The age
at which the first positive response occurred, and the age at which 90% of
the trials by the litter were positive were recorded.
12
-------
(6). Open Field lest - The open field consisted of a circular area, 1.8 m
in diameter marked off in 11.3 cm. squares. The field was contained by a
69 cm high wall constructed of unpainted corrugated aluminum roofing. A
single 200 W light was suspended 76 cm above the center of the field. Pups
were tested in the open field at the following ages: 18, 23, 30, 44, 54 and
65 days of age. Each pup was placed individually into the open field and
observed for a period of 10 min. Counts were made of the numbers of squares
crossed and the numbers of boluses deposited in defecation during each 10
min period. Each pup was tested only once at each of the appropriate ages.
(7). Maze Learning Transfer - When the pups were 55 days old, 5 males and 5
females from each group (control, experimental) were randomly selected for
maze learning ability. These rats had the food removed from their cages,
and were fed for 1 hr each day in a T-shaped maze (Fig. 1) to condition
them to associate feeding with the maze. By the time the actual test began,
they were at approximately 85% of their weight when the conditioning began,
and remained within this range throughout the evaluation. When they were
between 66 and 68 days old the actual testing began.
Each rat was placed in the start box and allowed to travel to the T junction
where it had to make a decision of turning to the right or the left. A one-
way swinging gate insured that a decision was made. If the rat turned to the
right (arbitrarily established as the first correct direction) it received
2 food pellets (Noyes 45 mg) but nothing for a left turn. Each rat received
10 trials per day, then allowed to feed ad lib following the trials, as well
as all during the week-end.
The criterion for "learning" was established as 9 out of 10 consecutive
correct trials. Once a rat had met the criterion, the "correct" side of the
maze was reversed to measure "learning transfer". Again the number of trials
necessary to meet the criterion in this new direction were recorded. This
alternation of "correct" side occurred 3 more times, so that each rat had
met the "learning" criterion 3 times to the right, and twice to the left.
b. Peri- and Postnatal Exposure to Toxaphene
Rats of the postnatal group (5-6 wk adult males and females) were dosed
daily by oral gavage with 6 mg/kg toxaphene (T) on 0.1 ml corn oil for 21
days. The perinatal group (14 wk females) received daily oral doses of 6 mg/kg
in 0.1 ml corn oil from day 7 following introduction of males until parturi-
tion. Controls of both groups were dosed on the same schedules with 0.1 ml
corn oil.
13
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(Scale: 1/10 actual size)
Figure 1. T-Maze employed in learning transfer analysis of rats
postnatally exposed to toxaphene, and perinatally exposed
to toxaphene, methyl parathion, and toxaphene:methyl
parathion. A. Start box. B. One way swinging gates
through which the rat must pass to ensure that a choice
was made.
14
-------
Once the pups of the perinatal group weire 7 days old, hehayiora.l tests
consisting of the grasp-hold reflex, righting reflex, and startle reflex
were initiated as previously described. When rats of both the perinatal
and postnatal groups were between 14-16 weeks old, they were tested for
"learning" and "learning transfer" abilities as described above.
B. TOXAPHENE AND METHYL PARATHION AGAINST NON-TARGET AND TARGET INSECTS
1. Non-Target Insects
Several adult hemipteran predators were tested from three different areas in
Southern Arizona. During the summer of 1977, Nabis spp., Geocoris spp., and
Orius tristicolor White were collected from alfalfa fields at University of
Arizona Agricultural Experiment Stations in Tucson, Safford, and Yuma with a
sweep net. Individual Nabis spp. and Geocoris spp. or several 0_. tristicolor
were placed in 10 dram glass vials with snap-on plastic caps and transported
in an ice chest to prevent dehydration and overheating. After return to
the laboratory, the vials were removed from the ice chest and warmed to room
temperature. A small square of paper toweling (approx. 2x2 cm) with a
drop of approx. 0.5 M sucrose solution (just enough to moisten the paper) was
placed into each vial for the insects to feed upon.
After being held in the laboratory overnight at room temperature, the insects
were dosed by topical application. The insects were anesthetized with carbon
dioxide. Solutions of methyl parathion or toxaphene were then applied to
37
the dorsal surface of the thorax after the technique of Hopkins et al. , using
a motor-driven microapplictor. All Nabis spp. and Geocoris spp. received 0.25
pi of a specific concentration of insecticide dissolved in acetone while (5.
tristicolor received 0.10 pi. Controls were either untreated or dosed with
acetone in an amount comparable to the treatments. The insects were returned
to their vials and held in the laboratory at room temperature after dosing.
Mortality was determined by probing the insects to observe a lack of reaction
or movement. Mortality values were obtained at 24 hr and based on 10-30
insects in groups of 10 per concentration. LD-50 values (yg insecticide/g
insect) were determined for each insecticide by logarithm-probit analysis
38 39
(Finney ' ) and compared statistically.
A similar procedure was used during the summer of 1978. Nabis alternatus
Parshley, 11. americoferus Carayon, Ceocoris punctipes (Say), (5. pallens
Stal, and C). tristicolor were collected from the same alfalfa fields as used
in 1977.
15
-------
However, a D-Vacr was employed because it was less injurious than a sweep
net. Nabis spp. were placed singly in each vial to avoid cannibalism.
Since no aggression was observed among Geocoris spp. or (). feristicolor, up
to 5 Geocoris spp. or 10 (3. tristicolor were placed in each vial. Paper
toweling and sucrose solution were placed in the vials as previously described.
The day following collection each insect was dosed topically with the ap-
propriate amount of a specific concentration of MP, T, or MP-T mixture
(1:2). After determining the MP LC-50 levels (ng insecticide/insect), each
species was dosed with MP at or near the LC-50 level, T twice that of the
MP, and a 1:2 MP-T mixture. Nabis. spp. and Geocoris spp. received 0.25 pi
of solution while 0. tristicolor received 0.10 yl. Controls were untreated
or dosed with acetone in the appropriate amount. After dosing, one Nabis
spp., 5 Geocoris spp., or 10 0. tristicolor were placed in each vial. Mortal-
ity values were obtained at 24 and 48 hr. Depending on the abundance of each
species, the values were based on 10-80 insects in groups of 10 per concentra-
tion for MP, and 10-90 insects in groups of 10 for T and MP-T.
2. Target Insects
a. Lepidopterous Larvae
Newly hatched larvae of the tobacco budworm, Heliothis virescens (Fab.);
corn earworm, E. zea (Boddie); beet armyworm, Spodoptera exigua (Hubner); and
salt marsh caterpillar, Estigmene acrea Drury, were obtained from the USDA
40
Cotton Insects Laboratory in Tucson. A modified Shorey and Hale diet was
used to rear the larvae. Cups containing the larvae were held in a humidified
environator at 27 C with a 15:9 hr light:dark cycle until the larvae attained
the proper size.
Third instar larvae weighing 15-30 mg (10-20 mg for S^ exigua) were selected.
Either 20% of the larvae to be dosed or at least 10 individuals, were weighed
and the average weight calculated. JH. virescens and H. zea larvae were placed
individually into 30 ml clear plastic cups filled halfway with medium and
enclosed with paper lids. Plastic petri dishes containing medium, prepared by
first slanting the dishes and filling halfway with medium, were used to treat
15 S. exigua or 10 E. acrea per dish. E. acrea were anesthetized with CO
2_
prior to dosing to slow their rapid movements. Larvae were dosed by topical
application to the thorax with 0.5 yl of MP, T, or MP-T (1:2) as described
for non-target insects and returned to the environator. Controls were either
untreated or dosed with acetone. Mortality values were obtained at 24 and 48
hr and based on 40 insects in groups of 10 per concentration.
16
-------
b. Houseflies
For reference material, houseflies, Musca domestica L., were obtained from
the laboratory culture of the Department of Entomology at the University
of Arizona, Tucson.
At 4-5 days of age, flies were sexed under CO anesthesia. Female flies
were dosed by topical application on the thorax with 1.0 pi of MP, T or
MP-T as previously described. After dosing, the flies were placed in tall
cylindrical paper containers (17 x 9 cm) fitted with screen lids and kept
at room temperature. A sugar cube and small glass vial filled with water
and fitted with a cotton plug were provided to each cage. Mortality values,
were obtained at 24 and 48 hr and based on 75 insects in groups of 25 for each
concentration.
C. FATE OF TOXAPHENE, CHLORDIMEFORM AND METHYL PARATHION IN THE MOUSE
To determine if combinations of MP-T-chlordimeform (C) affect the excretion,
retention, or mortality of any of the individual compounds when orally fed
to mice, the following experiment was conducted in 3 parts: (1) Toxaphene -
T alone and in combination with the other compounds. The combinations were
T-MP, T-C, and T-MP-C. (2) Chlordimeform - C-T, C-MP, and C-T-MP. (3) Methyl-
Parathion - MP and MP-T.
Mortality data were collected from the excretion-retention dosing of each
part. Additional mortality dosing was performed for each combination to
obtain 3 replicates of 12 each.
1. Toxaphene
Male Swiss mice, 16-17 weeks, and an average of 39.98 g were orally dosed via
a syringe and feeding tube attached to a motor driven microapplicator with 1.0
QV:
mg (25 mg/kg) Cl-Toxaphene (42 yCi/g) in 0.16 ml corn oil. For the T-MP
combination, the dose included 0.5 mg MP (12.5 mg/kg), the T-C included
0.125 mg C (3.2 mg/kg), and T-MP-C had 0.5 mg MP and 0.125 mg C added to the
1.0 mg T.
For excretion studies, 3 replicates of 4 mice each were placed into glass
metabolism chambers following dosing. The metabolism cages provided for
separate collection of urine and feces, and were maintained in an air condition-
ed environment (22-25°C, 50% RH; L:D - 11:13). The animals were provided lab-
oratory chow and water ad libitum.
Urine and feces were collected and mortality counts made at 3, 6, 12, 24, 48,
72, 96, 168, and 192 hr. Feces and urine were stored at 20 C to await further
17
-------
analysis. One mouse from eacii replicate was sacrificed at 192 hr and frozen
to by analyzed for tissue retention. The remaining mice in each replicate
were given an additional dose1; these animals were referred to as "redosed".
Urine and feces were collected in the previously described manner. Likewise,
1 mouse from each replicate of redosed animals was sacrificed at the last
sample time and retained for tissue storage determination. Another 3 mice
from each treatment were helc. until the 33rd day following redose and sac-
rificed to obtain tissue retention data at that point. At each sampling
period cages were rinsed to remove any urine residues; the rinses were frozen
until analyzed.
Feces samples were thawed, air dried, weighed, and ground to a powder, and
2, 50 mg subsamples digested in 3 ml of TS-I^tissue solubilizer using heat
to aid digestion. Bleaching was accomplished with 0.3 ml benzoyl peroxide
(lg/5 ml toluene). Tissues (brain, liver, lipid, kidney, skeletal muscle,
©
and testes) were weighed and 50 mg subsamples digested in 3 ml of TS-I^
radioassay was performed. Quench was corrected using the external standard
method. Female mice, 16-17 weeks, average weight 29.41 g, were orally dosed
in the same manner and at the saire levels as the males except with non-label-
led T and their lower weights resulted in the following dosages: T (34 mg/kg),
MP (17 mg/kg) and C (4.3 mg/sg). Also ovaries were substituted for testes.
Twelve mice were used for ea^h treatment (T, T-MP, T-C, T-MP-C and control)
and placed in holding cages. Urine and feces were not collected, but mortal-
ity counts were taken at 3, 6, 12, 24, 48, 72, 96, 168, arid 192 hr. After 192
hr all were redosed.
Following this mortality study, the 12 controls and the 12 T-dosed females
were dosed with 1,0 mg of the radio-labelled T and placed in metabolism
cages. This gave a group of 12 mice that had not received T and a group that
was receiving its third dose. Urine and feces were collected at 24, 48, 72,
and 96 hr for both groups, and also at 168 and 192 hr for the single dosed
group. The tri-dosed group was sacrificed at 96 hr and 3 carcasses retained
for tissue analysis. After 192 hr the remaining group was redosed, and urine
and feces collections made at 24, 48, 72, and 96 hr, and 3 carcasses retained
for tissue analysis. Therefore urine, feces, and tissues were available from
mice which received single, double, and triple doses of T. Cages were rinsed
at every collection with acetone arid 1:1 mixture of water: methanol.
18
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2, Chlord imeform
All methods employed and dosages applied in this part of the experiment
were almost identical with those used for male mice in part (1) Toxaphene.
14
The only difference was in the dosing combinations and the use of C-
chlordimeform (15.3 pCi/mg; Ciba-Geigy Corp.)- The dosing combinations
were C, C-T, C-MP, and C-T-MP.
3. Methyl Parathion
Methods employed and dosages applied were the same as previously described
with the following exceptions. Only MP and T-MP combinations were eval-
uated, with neither compound being radio-labelled. Urine and feces were
collected at 3, 6, 12, 48, 72, and 96 hr only, and no redose was made.
Four mice from each treatment were retained for tissue analysis, and only
liver, kidney, and lipid were sampled.
Urine was analyzed for para-nitrophenol (PNP) by electron capture gas chroma-
41
tography following the complete method of Cranmer . Feces and tissues were
extracted in hexane, dried through sodium sulfate and concentrated to 1.0 ml.
Lipid samples were partitioned between acetonitrile and hexane (4:1) after
the initial hexane extraction, dried, reconstituted in hexane, dried through
sodium sulfate, and then concentrated to 1.0 ml. Analysis for MP in feces and
tissues is being evaluated by flame photometric gas chromotography.
In addition to the mortality data collected from the excretion-retention
studies, the possible potentiation of MP by T was studied in this evaluation.
Male mice received doses of one half the LD (56T and 16MP mg/kg) and a 2:1
combination based upon the MP one half LD (32T and 16 MP mg/kg). The groups
were: T (56 mg/kg), T (32 mg/kg), T (56) + MP (16 mg/kg), T (32) + MP (16 mg/kg),
MP (16 mg/kg), and corn oil-dosed controls. Each group consisted of 4 repli-
cates of 12 (48 total/dose). Mortality counts were made up to 48 hr.
19
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SECTION V
DISCUSSION
A. CHANGES IN THE NERVOUS SYSTEM INFLUENCED BY TOXAPHENE
1. Ion Movements in the Nervous System of the American Cockroach
a. Methodology -
A primary criticism of the procedures employed is that by evaluating ion fluxes
in vitro, innumerable openings are created for the exchange of ions when con-
42
nectives and peripheral nerves are severed (O'Brien ). Therefore, this pro-
cedure does not distinguish between fluxes through these openings and those
through the natural channels of the extraneuronal and axonal membranes. This
would be a valid point if the observed changes were presented in absolute
terms. The results herein, although based upon numerical data, describe sig-
nificant changes relative to controls under the same circumstances. Therefore,
regardless of the numerical values, toxaphene did cause significant changes
in some ion movements. However, it must be reemphasized that the values pre-
sented for uptake and efflux should not be considered as representative of the
actual values for ionic movements that would occur in the intact nervous system.
24 + 24 +
(1) Na - Overall, there were no changes in Na movements (Table 1). Re-
24 +
lative uptake of Na between control and toxaphene-exposed sections was very
inconsistent. However, evaluation of each time interval uncovered a signifi-
cant reduction at 5 min for AB, and 10 min for all 3 sections (Table 2).
24 + 24 +
Efflux of Na also displayed few changes. Na efflux was slightly acceler-
ated, becoming significant for AB and TH by 60 min. BR displayed no significant
changes (Table 2).
(2) Cl - As with Na , overall there were no changes in Cl movements
(Table 1). However, evaluation of each time period showed the relative uptake
o/: _
of Cl by toxaphene-exposed BR as elevated over controls between 2.5 and 10
min, but lower after 60 min, with no changes in TH and AB (Table 3), Efflux of
36
Cl from toxaphene-exposed BR was significant only at the 15 min sampling
period, with no changes in TH or AB (Table 3).
20
-------
/ O L / O i
(3) K - Movements of K were significantly altered by toxaphene in all
42 +
but TH (Table 1). Differences in uptake of K by toxaphene-exposed BR and
AB occurred after 30 min. All 3 sections displayed inconsistent rates of up-
take for the first 10 to 15 min; then exposed sections became accelerated
over controls through the remainder of the evaluation (Fig. 2a, 3a, 4a).
TABLE 1. TWO-WAY ANALYSIS OF VARIANCE FOR CONTROL VS. EXPOSED SECTIONS. F
VALUES FOR UPTAKE AND EFFLUX OF IONS BY EACH NERVE SECTION. (*)
INDICATES SIGNIFICANT DIFFERENCES AT THE 0.05 LEVEL. VALUES WERE
SIGNIFICANT IF GREATER THAN 3.92
M
Na
42K+
36cr
45
Ca
Uptake
Brain
Thoracic
Abdominal
0.36
0.87
3.37
12.90*
1.83
7.02*
0.01
2.22
0.27
15.72*
19.54*
16.61*
Efflux
Brain
Thoracic
Abdominal
0.69
0.71
0.41
6.19*
2.51
9.29*
0.01
0.08
3.02
0.14
8.50*
18.01*
42, +
The influence of toxaphene upon K. efflux was similar to that for uptake,
in that BR and AB displayed significant changes, while TH did not (Fig. 2b,
3b, 4b). This decrease in efflux became significant for BR after 30 min,
whereas AB displayed a decrease throughout the entire evaluation (Table 4).
21
-------
TABLE 2. RESULTS OF T-TEST EVALUATION OF SODIUM-24 MOVEMENTS BE-
TWEEN CONTROL AND EXPOSED NERVE SECTIONS AT EACH TIME IN-
TERVAL
Uptake (rain)
1
2,5
5
10
15
30
60
Efflux (min)
1
2,5
5
10
15
30
60
Brain
NS
NS
NS
S
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Thoracic
NS
NS
NS
S
NS
NS
NS
NS
NS
NS
NS
NS
NS
S
Abdominal
NS
NS
S
S
NS
NS
NS
NS
NS
NS
NS
NS
NS
S
Significance determined at the 0.05 level. (S) significant.
significant
(NS) not
22
-------
TABLE 3. RESULTS OF T-TEST EVALUATION OF CHLORINE-36 MOVEMENTS BE-
TWEEN CONTROL AND EXPOSED NERVE SECTIONS AT EACH TIME IN-
TERVAL
Uptake (rain)
1
2.5
5
10
15
30
60
Efflux (rain)
1
2.5
5
10
15
30
60
Brain
NS
S
s
S
NS
NS
S
NS
NS
NS
NS
S
NS
NS
Thoracic
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Abdominal
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Significance determined at the 0.05 level. (S) significant. (NS) not
significant
23
-------
w
en
M
H
O
W
PM
8
7
6
5
4
3
2
1
0
ILL
1 5
J L
10 15
30
TIME (rain)
60
O
H
z
W
Pi
>
II
H
H
2:
w
w
100
90
80
70
60
50
40
30
20
10
0
CONTROL
TOXAPHENE
"EXPOSED"
1 I I
1 5
Fig. 2
I
10 15
30
TIME (min)
60
/ O J_
Rates of K flux measured from the brain section of
the central nervous system of £. americana exposed to
10~5^ toxaphene in vitro.
a. Uptake.
b. Efflux.
24
-------
w
CO
CO
H
O
PLI
O
S
12
11
10
9
8
7
6
5
4
3
2
1
0
I I I
. CONTROL.
XOXAFHENE.
EXPOSED
10 15
30
60
TIME (min)
B
H
H
2
W
W
P-.
100
90
80
70
60
50
40
30
20
10
0
CONTROL
I I 1
10 15
30
TIME (min)
60
Fig. 3
42 +
Rates of K flux measured from the thoracic section
of the central nervous system of P_. americana exposed
to 10~->M toxaphene in vitro.
a. Uptake.
B. Efflux.
25
-------
w
co
M
H
O
o
(^
w
CM
TOXAPHENE
* EXPOSED
TIME (min)
Pi
H
M
M
H
H
Z
w
w
CL,
100
90
80
70
60
50
40
30
20
10
0
CONTROL
TOXAPHENE
1 5
Fig.
10 15
30
60
TIME (min)
42 +
Rates oi: K flux measured from the abdominal section
of the central nervous system of P_. am eric ana exposed
to 10-5M toxaphene in vitro.
a.
b.
Uptake.
Efflux.
26
-------
TABLE 4. RESULTS OF T-TEST EVALUATION OF POTASSIUM-42 MOVEMENTS BE-
TWEEN CONTROL AND EXPOSED NERVE SECTIONS AT EACH TIME IN-
TERVAL
Uptake (min)
1
2.5
5
10
15
30
60
Efflux (min)
1
2.5
5
10
15
30
60
Brain
NS
NS
NS
NS
NS
S
S
NS
NS
NS
NS
NS
S
S
Thoracic
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Abdominal
NS
NS
NS
NS
NS
S
S '
S
S
S
S
S
S
S
Significance determined at the 0.05 level. (S) significant. (NS) not
significant
27
-------
The effect of toxaphene upon K. movements was opposite that induced by DDT
/ O _I_
or dieldrin. Toxaphene caused an increase of internal K levels while DDT
and dieldrin were reported as causing reductions of same (Matsumura and
19 ? 0
O'Brien , and Hayashi and Matsumura ). Both DDT and dieldrin induced a
42 +
decrease of internal K levels with in vivo preparations, while few changes
were evident from tissues exposed in vitro.
A possible explanation for the increase of internal K levels by toxaphene
might be the inhibition of a Na -K ATPase. It was reported by Desaiah and
Koch that toxaphene inhibited this enzyme in catfish brain. This could re-
42 +
suit in a reduction of K efflux as was observed herein. If uptake pro-
ceeded as normal in all samples, but efflux was reduced by toxaphene, it is
42 +
possible that an accumulation, of K would occur in these samples, giving
the illusion of increased uptake (Fig. 2a, 3a, 4a).
45 ++ 45 ++
(4) Ca - Of the ions investigated, Ca displayed the most acute
changes as a result of toxaphene exposure (Table 1). During the initial 10
min of evaluation, uptake by exposed sections was greatly accelerated over
controls (Fig. 5a, 6a, 7a). This difference became significant between 5
and 10 min for AB and BR, and within 10 min for TH (Table 5).
45 +
Between 10 and 15 min, Ca uptake displayed an abrupt and dramatic reduc-
tion. Following this reduction the difference in uptake between control and
exposed sections increased, becoming significant by 60 min for all 3 sections.
45 ++
Toxaphene was responsible for a significant drop in Ca efflux from all but
BR (Table 1). A significant decrease in efflux from AB occurred throughout
the entire 60 min (Fig. 7b), while TH displayed significant reduction from 2.5
min through the remainder of the evaluation (Fig. 6b). The increase in uptake
and decreased efflux would lead to significant increases in the internal levels
of ^Ca"-.
45 -H-
Changes in Ca movements due to toxaphene, bore little resemblance to those
changes induced by DDT or dieldrin. Dieldrin was reported as causing a net
45 ++
reduction of internal Ca levels, while DDT was reported as reducing efflux
19
in vivo, yet increasing it ±n vitro (Matsumura and O'Brien , and Hayashi and
Matsumura ).
45 -H-
Involvement of cyclic AMP in the uptake of Ca is possible. Functioning of
cyclic AMP, and the enzyme responsible for its breakdown (phosphodiesterase),
28
-------
w
w
CO
M
H
PM
a
g
S
o
pi
w
^
cd
o
i
i-
0
a
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
^
II
TOXAPHENE,
"EXPOSED ""
1 5 10 15
30
TIME (min)
60
o
2
S
>
H
II
M
H
W
U
Fd
P4
100
90
80
70
60
50
40
30
20
10
0
I I I
. CONTROL.
TOXAPHENE
Fig
10 15
45 H
Rates of Ca
30
TIME (min)
60
5 Rates of "tJCa^r flux measured from the brain section
of the central nervous system of £. americana exposed
to 10~5M toxaphene jln vitro.
a. Uptake.
b. Efflux.
29
-------
w
en
M
H
O
o
pi
w
u
r
fn
o
s
'J j .1 A
30
TIME (min)
o
M
S3
£
M
H
O
O
II
O
H
Z
W
w
PM
CONTROL
TIME (min)
45 -H-
Fig. 6 Rates of Ca flux measured from the thoracic section
of the central nervous system of ]?. am eric ana exposed to
10~^M toxaphene in vitro.
a. Uptake.
b. Efflux.
30
-------
w
CO
CO
H
O
o
w
PM
18
17
16
15
14
13
12
11
10
9
8
7
6
o
H
H
O
W
U
W
PH
100
90
80
70
60
50
40
30
20
10
0
CONTROL
TOXAPHENE_
EXPOSED
10 15
30
TIME (min)
60
45 ++
Figure 7. Rates of Ca flux measured from the abdominal section
of the central nervous system of P_. americana exposed to
10~^ M toxaphene in vitro.
a. Uptake.
b. Efflux.
31
-------
TABLE 5. RESULTS OF T-TEST EVALUATION OF CALCIUM-45 MOVEMENTS BE-
TWEEN CONTROL AND EXPOSED NERVE SECTIONS AT EACH TIME
INTERVAL
Uptake (rain)
1
2,5
5
10
15
30
60
Efflux (min)
I
2,5
5
10
15
30
60
Brain
NS
NS
S
s
NS
NS
S
NS
NS
NS
NS
NS
NS
NS
Thoracic
NS
NS
NS
S
NS
NS
S
NS
S
S
S
S
S
S
Abdominal
NS
NS
S
S
NS
NS
S
S
S
S
S
S
S
S
Significance determined at: the 0.05 level. (S) significant. (NS) not
significant
32
-------
have been observed to be Ca dependent (Rasmussen , and Kakiuehi et al. ).
j I
Increases in cyclic AMP have been observed to be associated with Ca uptake
43
(Rasmussen ) while brain and nerve cord of _L. maderae displayed significant
increases in cyclic AMP after exposure to toxaphene in vivo (Butler and Crow-
45 45 ++
der ). Therefore, increased uptake of Ca may be in response to this ele-
vation of cyclic AMP by toxaphene.
b. Relationship of the Observed Changes to Neural Activity
The initial effect of toxaphene may have been upon the membranes of the glial
and perineural cells and the ion regulation controlled by them. Dary and
2
Crowder observed that isolated abdominal nerve sections of 7_. americana ex-
posed to 10 M toxaphene, displayed an average latent period of 26 min before
intense activity was observed electrophysiologically. However, changes in ion
movements by toxaphene became significant before this time, especially efflux
, 42 + , 45,, -H-.
of K and Ca
Therefore, the initial response to toxaphene by the neurons may be due to the
altered ionic concentrations within the extracellular spaces. The concentra-
+ H | 4- _
tions of K and Ca would have been greater, while those of Na and Cl
could have been unchanged or possibly reduced. These concentration changes
could account for the nerve activity observed with toxaphene exposure. For
example, high internal concentrations of K would lead to a reduction in the
1 8
resting potential (Yamasaki and Narahashi ). Such a lowering of the resting
potential could make the neuron more receptive to a stimulus, and able to pro-
duce high frequency activity, as has been observed during the early stages of
1 2
toxaphene poisoning (Lalonde and Brown , Dary and Crowder , and Wang and
3 ++
Matsumura ). Elevated Ca concentrations could stabilize the membrane a-
46
gainst this depolarization (Ulbricht ), enabling the nerve to continue func-
tioning rather than leading to conduction block due to the high K levels.
Since there was no overall significant change in Na and Cl movements, it
is possible that these ions play only a small part in the initial disruption
of the CNS following toxaphene exposure. However, they may have a role in
this process during the later stages of poisoning.
The processes involved in ion movement changes brought about by toxaphene are
45
uncertain. The stimulation of cyclic AMP production (Butler and Crowder ),
or the inhibition of a Na -K ATPase (Desaiah and Koch ) by toxaphene might
explain the increases and decreases of the ions as observed. However, these
two processes may not be mutually exclusive, as ouabain (inhibitor of Na -K
ATPase) and high K levels have been observed to increase cyclic AMP levels
33
-------
47 +
in brain slices of guinea pigs (Shimizu et al ). Also, high levels of K
1,1 i I
and Ca have been observed to inhibit Na -K ATPase of crab peripheral nerve
(Skou ). Therefore, stimulation of cyclic AMP formation by toxaphene could
I i i _l_
accelerate Ca uptake, resulting in the indirect inhibition of Na -K ATPase,
or an increase of internal K by blockage of Na -K ATPase could stimulate
cyclic AMP production. Either would result in an increase of internal concen-
[ i i i
trations of Ca and K and a reduction of Na , as was observed in this study.
c. Differences Between Nerve Sections
The difference in response to toxaphene by the 3 nerve sections was primarily
in the time it took for one section to display symptoms, relative to the others.
The earliest and most numerous significant changes occurred with AB. Its
large surface to volume ratio may be responsible for this difference.
2. Cyclic Nucleotides in Several Tissues of the Cockroach and Mouse
Male and female cockroaches selected at random from a laboratory population
were compared for differences in cAMP and cGMP levels of various tissues (Table
6). Cyclic AMP was significantly greater in males for the gastric caeca and
rectum, and cGMP was significantly greater in testes than ovaries. The greatest
concentration of cAMP was found in Malpighian tubules, which was the only tissue
significantly different from other tissues for either sex. There was no sig-
nificant difference between tissues for cGMP for either sex.
Cyclic AMP (3.0-36.0 picomoles/mg protein) and cGMP (1.7-3.9 picomoles/mg pro-
tein) found in all tissues of untreated cockroaches were comparable to concentra-
48
tions of these eye Lie nucleotides in vertebrates (Robinson , Ferrendelli et al.
49,50,51^ Hepp et al<52^ and Steiner et a!.53). Cyclic GMP was less than cAMP,
54
as was also the case with mice (Tables 8 and 9). Fallen and Wyatt working with
male reproductive organs of crickets, showed that cyclic nucleotide concentrations,
except for Acheta domesticus L. used by Ishikawa et al. , were comparable to
those in vertebrates. Levels reported herein for L_. maderae agreed with the re-
54 ~
suits of Fallen and Wyatt
Analysis of variance showed no significant differences between untreated and oil
control levels of cAMP and cGMP for any tissue, or between cAMP and cGMP controls
(Table 7). Cyclic nucleotide levels in tissues were compared between oil control
and T-treated cockroaches, "-treated cockroaches produced a large amount of var-
iability in cyclic nucleotide levels; however, significant differences were still
observed between T-treated and control samples. Symptoms of poisoning from T
were not noted during the course of the experiments.
34
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The greatest and most significant increase in cAMP in all tissues samples
occurred at 24 hr after T treatment. Following this time cAMP fluctuated un-
til 144 hr after treatment, when cAMP again increased significantly. Cyclic
GMP was slightly increased over controls at 24 hr after T treatment, with only
brain, nerve cord, and fat body being significant. Cyclic GMP continued to
fluctuate until 168 hr after treatment, when cGMP increased in all tissues
sampled.
Analyzed over time, cyclic nucleotide levels for a particular tissue w=>.re
found not to be significantly different between time intervals; therefore,
values for that tissue for all time intervals were pooled. On this basis
(Table 7) it was found that in the presence of T, cAMP increased in all tissues,
significantly in gastric caeca and malpighian tubules, and cGMP increased sig-
nificantly in all treated tissues except fat body. Since the presence of T
was associated with increased levels of both cAMP and cGMP in tissues of the
cockroach, both nucleotides might be involved in the mode of action or metabo-
lism of T by the insect.
T directly or indirectly increased cAMP and cGMP in malpighian tubules. This
14
result supports the idea of Casida and Maddrell that insecticide poisoning
may increase malpighian tubule secretion through the mediating action of cAMP.
They reported that insect diuretic hormone induced adenyl cyclase at the re-
ceptor sites in malpighian tubules resulting in increased levels of cAMP.
Rojakovick and March tested several insecticides in vitro on the cockroach,
Gromphadorhina portentosa, brain adenyl cyclase and phosphodiesterase enzymes.
They could not demonstrate to any degree of certainty, whether insecticides
have any direct effect on the enzymes. However, it appears that the presence
of T in vivo has a direct or indirect relationship with cyclic nucleotide
levels in several tissues of L^. maderae.
Cyclic AMP and cGMP in several tissues of the mouse were compared between oil
controls and T-treatments, the latter being subdivided into observable sympto-
matic and asymptomatic groups (Tables 8 and 9). These comparisons were made
at 4 time periods and correlated with symptoms. At 15 min, no symptoms were
observed. At 45 min, the first symptom appeared to be mild, exhibited by mice
lying quietly in a prone position with labored breathing. At 90 min after treat-
ment the symptoms consisted of hyperactivity illustrated by kicking, chewing,
and uncoordinated body movements. The third symptom, lasting only 2-5 min and
appearing 100-180 min after being dosed, was expressed by mice lying on their
39
-------
backs in convulsions, leading immediately to death or reversal to the first
symptom. Mice that died from the dose were combined with those sacrificed
at 240 min.
Cyclic nucleotide levels for a particular tissue of controls were not sig-
nificantly different between time intervals, Asymptomatic mice were not
significantly different from controls at any time, Cyclic AMP was increased
significantly over controls in all symptomatic tissues (Table 8), but cGMP
was increased significantly only in symptomatic testes (Table 9), It appear-
ed that T poisoning was associated with increased levels of endogenous cAMP,
but not with cGMP, Fifteen min after application of T, no symptoms were ob-
served and no difference between T-treated and controls was found. At 45
min there was a slight, but non-significant, increase in cAMP in nervous
tissue (brain and spinal cord) and liver, However, there was a significant
increase in testicle cAMP and cGMP. The greatest symptomatic reaction to
T occurred at 90 min, This time also reppresented the greatest, and signifi-
cant, increase in cAMP in all tissues examined. At 240 min cyclic nucleo-
tide levels were not significantly different from controls.
Increased hepatic and renal cAMP in the presence of T was similar to the
effect found by Kacew and Singhal ' for cyclodienes and DDT, They reported
that in vitro and J.n vivo administration of insecticides to rats increased
cAMP levels and adenyl cyclase activity. Increased cAMP associated with the
presence of T appeared to be related to the degree of symptomatic poisoning.
This might implicate cAMP in the mode of action or metabolism of T by the
mammal as well as the insect.
3. ATPases in Several Tissues of the Cockroach and Mouse
a. Electron Microscopy
Electron micrographs of kidney, brain and liver homogenates were taken to in-
sure that the homogenate fractions were rich in plasma membrane vesicles. The
micrographs demonstrated this point, The kidney homogenate was the richest
in membrane vesicles and had the lowest protein content per ml. The brain
homogenate fraction appeared to contain larger vesicles than the kidney homo-
genate and some structures sinilar to what Koch termed nerve endings. The
liver fraction had the highest protein content, but the lowest specific activi-
ty for ATPases, It was densely contaminated with blood cells and other cellular
debris. This fraction was very rich in mitochondria.
40
-------
TABLE 10. MEANS OF TOTAL ATPase SPECIFIC ACTIVITY + S.E. IN KIDNEY AND LIVER
OF CONTROL AND TOXAPHENE-DOSED MICE USED IN IN VIVO EXPERIMENT I.
SPECIFIC ACTIVITY EXPRESSED IN yM Pi/mg PROTEIN/HR.
Kidney Brain Liver
Control (corn oil) 66.61 + 1.07b* 36.12 + 0.64b 10.78 + 0.23b
Toxaphene (LD,.n)** 45.01 + 1.71a 31.57 + 0.30a 9.26 + 0.15b
* Means followed by the same letter in each tissue are not significantly dif-
ferent (LSD, a= 0.05)
** 112 rag/kg of toxaphene
TABLE 11. PERCENTAGE OF INHIBITION OF ATPase ACTIVITIES DUE TO TOXAPHENE (112
mg/kg) IN MICE IN VIVO.
_1_ _i_ | _i_
Total ATPase Na -K ATPase Mg ATPase
Experiment I
Kidney 32.4
Brain 12.6
Liver 14.1
Experiment II
Kidney 14.0 12.6 15.0
Brain 5.2 0.6 8.8
Liver 7.5 0.2 10.6
b. In vivo Experiments
In in vivo experiment I, homogenates from T-treated mice exhibited significant
decreases in total ATPase activity of the kidney, brain and liver (Table 10).
The LD5Q dose (112 mg/kg) did not display 50% inhibition of the activity; the
highest inhibition reached was 32.5% in kidney homogenates (Table 11).
41
-------
In in vivo experiment II, Na -K ATPase was inhibited significantly only in
.1-1-
the kidney, and the Mg ATPase was inhibited significantly in all three
tissues (Table 12). The highest inhibition percentage was observed in the
kidney for both Na+-K+ and Mg"1"*" ATPases, 12.6% and 15.0% respectively (Table
I j
11). In general, the Mg ATPase activity was more sensitive to T than the
Na -K ATPase in all tissues.
The mice used in experiment 1 were observed to have lower specific ATPase
activities in all tissues, and their ATPases were more vulnerable to inhibi-
tion by T than of those used in experiment II. However, the LD dose did
not exhibit more than 32.4% inhibition,
c. In Vitro Experiment
(1) Mouse - The solvent control treatment (10 pi ethanol)"reduced the activity
significantly in all ATPases tested except the Na -K ATPase of the brain, and
I i,
displayed an insignificant stimulation of the liver Mg ATPase (Table 13).
The highest inhibition due to ethanol was in the Na -K ATPase of liver (10.9%)
I
and the only stimulation was a 2.2% increase in Mg ATPase of the same tissue
(Table 14).
Na -K ATPase activity was significantly inhibited only in the kidney homo-
-4
genates by all levels of T (Table 13). The highest concentration (10 M) ex-
hibited the highest degree of inhibition (with a maximum of 44%) and the low-
est concentration produced the lowest inhibition (Table 14), Na -K ATPase
activity was not inhibited in the brain homogenates. In liver homogenates the
activity was variable in response to T with a significant reduction caused by
the 10 M concentration, while higher concentrations (10 M and 10 M) inflicted
no significant reduction (Tables 13 and 14).
i i
Mg ATPase activity was significantly reduced by all T concentrations in both
the kidney and the brain (Tables 13 and 14). In the liver homogenates only the
_| i
highest concentration exhibited a significant reduction in Mg ATPase activity
(Table 13). The total ATPase activity was inhibited 28.9%, 6.0% and 14.7% in
kidney, brain and liver, respectively, by the highest concentration which pro-
duced the highest degree of inhibition.
(2) I\ americana - The solvent control treatment (ethanol) inhibited the act-
ivity in Na -K ATPase of the CNS (Table 15). Otherwise, it did not have a
significant effect (Table 16) with the exception of the total ATPase activity
_l I
of the Malpighian tubules which showed a slight stimulation of the Mg ATPase
activity by 9.6% (Tables 15 and 16).
42
-------
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44
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TABLE 14. PERCENTAGE OF REDUCTION OR INCREASE IN ACTIVITY OF THE
ATPase OF MOUSE TISSUES TESTED IN RESPONSE TO ETHANOL
AND VARIOUS CONCENTRATIONS OF TOXAPHENE IN VITRO.
MINUS AND PLUS SIGNS INDICATE REDUCTION AND INCREASE,
RESPECTIVELY.
Tissue
Kidney
Brain
Liver
ATPase Solvent*
activity control
Total -5.9
Na+-K+ -5.1
Mg -6.5
Total -2.9
Na+-K+ -0.1
Mg -5.1
Total -0.2
Na+-K+ -10.9
Mg +2.2
Toxaphene concentration
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-44.0
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-24.6
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-24.5
-11.6
-2.9
-2.8
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-4.3
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(M)
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* 10 yl ethanol
45
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46
-------
TABLE 16. PERCENTAGE OF REDUCTION OR INCREASE IN ATPase ACTIVITY OF P^.
AMERICANA TISSUES TESTED IN RESPONSE TO ETHANOL AND VARIOUS
CONCENTRATIONS OF TOXAPHENE IN VITRO.
MINUS AND PLUS SIGNS
INDICATE REDUCTION AND INCREASE, RESPECTIVELY.
ATPase Solvent*
Tissue activity control
CNS Total -9.8
Na+-K+ -14.1
Kg"1"1" -1.9
Malpighian tubules Total +2.4
Na+-K+ -4 . 9
Mg +9.6
Toxaphene concentrations (M)
ID'4
-47.8
-58.9
-29.2
-26.8
-52.6
+0.1
io-5
-34.5
-48.7
-8.2
-19.9
-42.4
+3.9
io-6
-34.0
-48.4
-6.8
+1.1
-3.3
+9.2
ID"7
-12.5
-17.9
-3.2
+9.6
+8.1
+13.1
* 10 pi ethanol
47
-------
Na -K ATPase activity of the CNS was significantly reduced by all T concen-
trations used (Table 15) . The: 10 '4 concentration produced the highest in-
hibition (58.9%); 10~5 and 10 were equipotent at 48.7% and 48.4%, respec-
tively; and the lowest concentration (10~ M) resulted in 17.9% inhibition
(Table 16). In Malpighian tubules, the Na -K ATPase activity was significant-
4 5 6
ly reduced by 10 and 10 M; was not affected by 10~ ; and was significantly
stimulated by 10 M concentration (Table 15).
-I...I / C
Mg ATPase activity of the CNS was reduced by 10 and 10~ M. In Malpighian
ji
tubules, Mg ATPase activity was not inhibited but displayed a trend of stim-
ulation which became significant at 10~ M. However, the initial increase in
this activity in this tissue was as high as 9.6% due to ethanol alone (Table
16). The higher dose (10~ M) inflicted a significant reduction on the stim-
ulation by ethanol (Tables 15 and 16). The lower doses were unable to reverse
the ethanol effect.
(3) Regressions - Two regression analyses were conducted to determine the sig-
nificance of the dose response of each ATPase activity in each tissue of both
animals. The linear regression included the solvent treatment (ethanol) as the
zero T treatment, but the linear log regression was made for the four T con-
centrations only. The significance of dose response based upon both regressions
is presented in Table 17.
Table 18 lists the prediction equations for the activity of the ATPase against
log of T concentrations. The linear regression indicated that there was a
dose response in all ATPases of the kidney, malpighian tubules, total and Na -K
ATPases of the CNS and the Na+-K+ ATPase of the liver. With linear logiQ re-
i -*
gressions (where data for control treatment was omitted) only the Na -K ATPase
of the brain did not show a significant dose response to T.
4. Neonatal Development and Postnatal Learning in the Rat
a. Perinatal Exposure to Toxaphene - Methyl Parathion and Methyl Parathion
(1) Pup Mortality - Mortality of rat pups was significantly greater among litters
that had been perinatally exposed to the insecticides. Total percent mortalities
for 15 days postpartum were: 30% for pups exposed to MP vs. 10% for controls,
and 76.7% for MP-T pups vs. 2'.3.3% for controls. Perinatal exposure to MP or
MP-T caused no significant difference in the mean total weight gained by the
rat pups from day 5 through day 15 postpartum. Mortality of rats pups perinatally
exposed to MP and MP-T may or may not be directly attributed to the insecticides.
Although no significant differences were observed in the analysis of retrieval
times by the mothers, the mortality may have been as a result of poor mothering
48
-------
TABLE 17. SIGNIFICANCE OF LINEAR AND LINEAR-LOG REGRESSIONS OF THE DIF-
FERENT ATPases WITH THE TREATMENTS. S = SIGNIFICANT,
NS = NOT SIGNIFICANT.
Tissue
Kidney
Brain
Liver
CNS
Malpighian tubules
ATPase
activity
Total
Na+-K+
Mg"
Total
Na+-K+
vr ++
Mg
Total
Na+-K+
Mg^
Total
Na+-K+
Mg"
Total
Na+-K+
Mg++
Linear
regression
S
S
S
NS
NS
NS
NS
S
NS
S
S
NS
S
S
S
Linear-log
regression
S
S
S
S
NS
S
S
S
S
S
S
S
S
S
S
49
-------
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50
-------
by the exposed females. During the first 3 days of retrieval testing (pups
5-7 days old), exposed mothers took longer (although not statistically sig-
nificant) to retrieve the pups than the control mothers. This is important,
as at this age the pups are rather dependent on the mother for retrieval to
be fed. After 1-2 days of insufficient feeding, the pups became very weak and
died. Therefore a change in maternal behavior brought about by exposure to
MP or MP-T may have been responsible for the mortality.
(2) Coordination Tests - Results of the righting, startle, and placement re-
flex tests are presented in Table 19. The only significant change from peri-
natal exposure was an extension of the time required by litters exposed to
MP-T to successfully master the righting reflex. The results of the grasp-
hold reflex also uncovered no significant differences. MP litters were ob-
served to grasp-hold for a slightly shorter period of time as they aged com-
pared to controls (Fig. 8) but the MP-T litters displayed no such trend
(Fig. 9).
(3) Open Field Testing - This test for emotionality indicated a possible
change due to MP exposure but not with MP-T. MP rats moved about the open
field more than the controls, and the number of defecations (boluses) were
more constant (Figures 10 and 11). Activity by MP rats was significantly
higher only at 23 and 54 days. It is not clear in the literature whether
high activity means high or low emotionality, but it does appear that MP
caused some change in the emotionality of the neonates.
The effect of perinatal exposure to MP-T on emotionality of the rat pups was
very small. Activity in the open field was almost indistinguishable between
MP-T and control rat pups. There appeared to be a slight difference in the
number of boluses deposited by each group, but the patterns were so variable
that no inferences could be drawn.
(4) Maze Learning Transfer - There were no overall nor consistent differences
in maze learning or transfer as a result of perinatal exposure to MP or MP-T.
However, as with the emotionality tests, MP rats displayed greater differences
over controls than did MP-T animals.
The MP group required about 2 X as much time to meet the first criterion as
the controls (Fig. 12). However, the exposed group took longer to master
the transfer to the left. The groups then handled the next right about the
same, but the MP rats again had difficulty transferring to the second left
as well as the final transfer back to the right.
51
-------
TABLE 19. RESULTS OF COORDINATION TESTS PERFORMED ON RAT PUPS
PERINATALLY EXPOSED TO METHYL PARATHION, AND METHYL
PARATHION: TOXAPHENE (1:2)
Methyl Parathionc
Experiment
start
1
finish
Methyl Parathion:Toxaphene
start finish
Righting Reflex
Control
Experimental
13. 3a
14. 5a
18. 7a
18. Oa
14. Oa
16. Oa
17. 7a
22. Ob
Startle Reflex
Control 11. Oa H.7a
Experimental 11.5a 12.5a
Placement Reflex
Control 9.7a 14.Oa
Experimental 10.Oa 13.5a
11.3a
13. Oa
7.0a
7.3a
12.7a
14. Oa
14.3a
13.7a
1
Values followed by the same letter are not significantly different at
the 0.05 level.
Mean number of days for observation of first correct response.
?
Mean number of days for the daily number of correct responses of each
litter to equal or exceed 90%.
52
-------
o
0)
CO
H
O
O
(U
M
l-l
H
O
25
20
15
10
. CONTROL .
EXPOSED
I
I
I
I
I
I
7 8 9 10 11 12 13 14 15
AGE OF PUPS (days)
Fig. 8. Mean hang time (grasp-hold reflex) of rat pups
perinatally exposed to methyl parathion.
25
20
15
10
.CONTROL .
EXPOSED
1
9 10 11 12 13
AGE OF PUPS (days)
14
15
Fig. 9 Mean hang time (grasp-hold reflex) of rat pups
perinatally exposed to methyl parathion: toxaphene
(1:2).
53
-------
Q
W
w
CO
§
o
1/1
o
o
500U-
450-
400-
350
300
250 _
10
CONTROL
EXPERIMENTAL
\
20
30
40
AGE (DAYS)
50
60
Fig. 10 Activity in the open field test by rat pups peri-
natally exposed to methyl parathion.
M
O
CO
w
O
O
CONTROL
Fig,
10
20
30
40
50
60
70
AGE (DAYS)
11 Number of boluses deposited in the open field by rat
pups perinatally exposed to methyl parathion.
54
-------
s iz
o
o H
H Pi
w
CO H
PM
50
30
20
10
RIGHT
LEFT
[]] CONTROL
RIGHT
EXPERIMENTAL
LEFT
RIGHT
DIRECTION OF CORRECT RESPONSE
Fig. 12 Results of maze learning transfer by rats perinatally
exposed to methyJ parathion.
60
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w
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o
gs 40
w
CO H
iJ H
H u 30
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RIGHT
LEFT
RIGHT
LEFT
RIGHT
DIRECTION OF CORRECT RESPONSE
Fig. 13 Results of rnaze learning transfer by rats perinatally
exposed to methyl parathion and toxaphene (1:2)
55
-------
Since the MP group performed about as well on the first and second evaluation
to the right, it may be inferred that their learning ability was not severely
impaired, but perhaps the ability to transfer this knowledge (i.e. turn to
the left) was.
Rats perinatally exposed to MP-T did not appear to be impaired in maze learn-
ing or transfer. As with the MP group, MP-T rats performed as well as controls
on the first criterion to the right (Fig. 13).
b. Peri- and Posnatal Exposure to Toxaphene
(1) Coordination Tests - Perinatal exposure to T caused no significant dif-
ferences in grasp-hold (Fig. 14), startle, or the initiation of the righting
reflex. However, there was a significant difference observed in the length
of time required for 907» of the trials to be positive by T litters; it re-
quired more time for these pups to master the skills than the controls
(Table 20).
(2) Maze Learning Transfer - There were no significant differences in maze
learning and transfer of this learning between postnatally exposed and control
rats (Fig. 15). Perinatally exposed rats did not display any difference in
learning or transfer as compared to controls (Fig. 16). The early patterns
were similar in both exposed groups. Both groups mastered the first criterion
to the right earlier than controls, and later on the first left. For the
last 3 changes the postnatal group improved on each change, while the peri-
natal and controls of both groups had more difficulty with the second left
than with the remaining right turns. But this was offset by controls
mastering the second right before the exposed rats. The final 2 turns were
handled easily and about equally by both groups.
In the maze evaluation of both MP and MP-T, controls of both displayed an
overall reduction in the number o£ trials for meeting each consecutive
criterion. This would be expected, for in learning one criterion, trans-
fer of this learning would ma.ke later learning easier. However, the exposed
rats did not display this trend.
B. TOXAPHENE AND METHYL PARATHION AGAINST NON-TARGET AND TARGET INSECTS
1. Non-Target Insects
In 1977 the number of predators collected from each location (Tucson, Safford,
and Yuma) varied considerably and in some cases few or none of particular species
were obtained. Almost all MP LD5Q values (^g insecticide/g insect) at 24 hr
56
-------
o
CD
to
H
O
35
30
25
20
15
10
CONTROL
EXPERIMENTAL
10
11
12
13
14
15
AGE (days)
Fig. 14 Mean hang time of rat pups perinatally exposed to
toxaphene.
Table 20. RESULTS OF COORDINATION TESTS PERFORMED ON RAT PUPS
PERINATALLY EXPOSED TO TOXAPHENE.
Experiment
Righting Reflex
Control
Experimental
Startle Reflex
Control
Experimental
Start
13. 5a
14. Oa
10. 5a
11. Oa
2
Finish
15. 5a
18. 5b
11. Oa
11. 5a
a. Values followed by the same letter are not significantly
different at the 0.05 level.
1. Mean number of days for observation of first correct re-
sponse.
2. Mean number of days for the daily number of correct re-
sponses of each litter to equal or exceed 90%.
57
-------
H
W
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W
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i-J M
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tu _
b
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CD <
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50
40
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20
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m
%:
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x'5;
y',y
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I 1 CONTROL
IP EXPERIMENTAL
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K::;:
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§f.:x
1
X-X*
K-X'
SS;
RIGHT
LEFT
RIGHT
LEFT
RIGHT
DIRECTION OF CORRECT RESPONSE
Fig. 15 Results of maze learning transfer by rats postnatally
exposed to toxaphene.
80
70
w
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H Pi
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Crt H ,._.
J M 50
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^ EXPERIMENTAL
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"x"i
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1
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RIGHT
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RIGHT
LEFT
RIGHT
DIRECTION OF CORRECT RESPONSE
Fig. 16 Results of maze learning transfer by rats perinatally
exposed to toxaphene.
58
-------
were obtained and are presented in Table 21. Tucson and Safford Nabis spp.
displayed nearly the same LD-50 values of 1.65 and 1.56, respectively. Yuma
Nabis spp. produced a non-significant regression due to the few insects tested
and the erratic results, but the estimated LD-50 was 1.01. Tucson and Safford
Georcoris spp. also displayed similar LD-50 values with 1.95 and 2.34 respective-
ly, while the Yuma population produced a non-significant regression with an
estimated LD-50 of 2.85. Too few (3. tristicolor were collected from Safford
and Yuma for tests and the Tucson population results were too erratic for a
significant regression, but the estimated LD-50 for the Tucson population was
0.68.
The LD-50 values at 24 hr for T are presented in Table 22. Attempts to estab-
lish dosage-mortality values for T all failed to give significant regressions.
Only Tucson Nabis spp; Tucson and Yuma Geocoris spp; and Safford (). tristicolor
were tested to an extent to give LD-50 estimates of over 10,000 for Nabis spp.
and 282, 89, and 90 for the Geocoris sp. populations and Q. tristicolor, re-
spectively.
The 1978 MP LD-50 values at 24 and 48 hr are presented in Tables 23 and 24,
respectively. JSL alternatus, _N. americoferus, and Q. tristicolor displayed
the highest intraspecific values for the Yuma populations. The values for jG.
pallens were higher for Tucson than Yuma populations. These intraspecific
differences were not significant between locations. When comparing the species,
generally G^. pallens displayed the highest LD-50 values (except for Yuma _N.
alternatus) followed by Cr. punctipes and Jtt. alternatus, N_. americoferus, and ().
tristicolor.
At 24 hr, only Safford IJ. alternatus displayed a significant regression with
an LD-50 of 1.39. Tucson and Yuma populations displayed non-significant re-
gressions, with estimated LD-50 values of 1.81 and 2.62, respectively. It
was observed that in each set of data for the Tucson and Yuma populations
there was a value that appeared to be an anomaly. If the respective values
were removed, significant regressions were obtained (Table 25) and the LD-50
values became 1.38 and 2.19, respectively. At 48 hr, Safford had the only
significant regression with an LD-50 i>f 1.01, while the LD-50 estimate was 1.63
for the Tucson population and 2.12 for the Yuma population. If the anomalous
values removed from the 24 hr values were also removed at 48 hr (Table 26) the
Yuma population displayed a significant regression arid identical LD-50 value of
2.12, while the.Tucson population failed to produce a significant regression
and the estimated LD-50 dropped to 1.25
59
-------
TABLE 21. METHYL PARATHION LD-50 VALUES (yg/g) AT 24 HR FOR PREDATORS
FROM SEVERAL AREAS IN ARIZONA IN 1977
Insect
Nabis spp.
Geocoris spp.
Orius tristicolor
Adult average weight
Area
Collected
Tucson
Safford
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
s (mg) : Nabis
LD-502
1.65
1.56
(1.01)
1.95
2.34
(2.85)
(0.68)
spp. 7 .48,
95%
Fiducial Limits Slope
1.33 - 2.74 3.90
1.19 - 2.23 7.10
1.64
0.28 - 2.81 2.45
1.51 - 3.13 4.47
2.41
16.25
Geocoris spp. 3. 28, 0.
tristicolor 0.15.
"Values in parentheses are estimated values due to non-significant
regressions.
60
-------
TABLE 22. TOXAPHENE LD-50 VALUES (pg/g) AT 24 HR FOR PREDATORS FROM
SEVERAL AREAS IN ARIZONA IN 1977
Insect
Nabis spp.
Geocoris spp.
Orius tristicolor
Adult average weights
Area 95%
Collected LD-50 Fiducial Limits Slope
Tucson ( > 10, 000) 0.12
Safford
Yuma ___ ___ ___
Tucson (282) 1.32
Safford ___
Yuma (89) 0.76
Tucson ___ ___ ___
Safford (90) 1.65
Yuma ___ ___ ___
(mg) : Nabis spp. 7. 48, Geocoris spp, 3. 28, 0.
tristicolor 0.15.
"All values are estimated values due to non-significant regressions.
61
-------
TABLE 23. METHYL PARATHION LD-50 VALUES (pg/g) AT 24 HR FOR
PREDATORS FROM SEVERAL, AREAS IN ARIZONA IN 1978
Insect
Nabis alternatus
Nabis americof erus
Geocoris pallens
Geocoris punctipes
Orius tristicolor
Area Collected
Tucson
Safford
Yuma
Tuc son
Saffotrd
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
2 95%
LD-50 Fiducial Limits
(1.81)
1.39 0.90 - 1.70
(2.62)
(0.97)
(1.37)
(1.61)
(2.43)
2.13 1.82 - 2.40
1.93 1.56 - 2.19
(0.60)
(0.73)
(0.91)
Adult average weights (mg) : N. alternatus 6.31, N. americoferus
G. pallens 3.28, G.
punctipes 3.72,
0. tristicolor 0.2.
Slope
2.85
4.10
2.81
3.71
13.83
2.59
21.03
4.67
3.91
4.15
0.51
2.64
10.48,
"Values in parentheses are estimated values due to non-significant regressions.
62
-------
TABLE 24. METHYL PARATHION LD-50 VALUES (yg/g) AT 48 HR FOR
PREDATORS FROM SEVERAL AREAS IN ARIZONA IN 1978
Insect
Nabis alternatus
Nabis americoferus
Geocoris pallens
Geocoris punctipes
Orius tristicolor
Area Collected
Tucson
Safford
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
Tucson
Safford
Yuma
2 95%
LD-50 Fiducial Limits
(1.63)
1.01 0.003 - 1.30.
(2.12)
(0.69)
(1.29)
(1.15)
(2.40)
2.14 1.60 - 2.54
1.85 1.40 - 2.09
(0.45)
(0.35)
(0.66)
Adult average weights (mg) : N. alternatus 6.31, N. americoferus
G. pallens 3.28, G.
punctipes 3.72,
0. tristicolor 0.2.
Slope
1.60
3.57
9.16
2.60
10.57
1.41
21.75
4.07
;
4.45
0.82
13.27
2.28
10.48,
"Values in parentheses are estimated values due to non-significant regressions.
63
-------
TABLE 25. METHYL PARATHION LD-50 VALUES (pg/g) AT 24 HR FOR PREDATORS
FROM SEVERAL AREAS IN ARIZONA IN 1978. AFTER AN APPARENT
ANOMALOUS VALUE WAS REMOVED FROM THE ORIGINAL DATA SET
Insect
Nabis alternatus
Orius tristicolor
Area Collected
Tucson
Yuma
Tucson
Safford
Yuma
LD-50
1.
2.
0.
0.
0.
38
19
50
30
75
95%
Fiducial Limits
1.
2.
0.
0.
0.
23
04
42
26
60
- 1.
- 2.
- 0.
- 0.
- 13
81
42
87
36
.04
Slope
8
8
5
8
4
.34
.17
.29
.46
.44
Adult average weights (mg) : _N. alternatus 6.31, (). tristicolor 0.2
TABLE 26. METHYL PARATHION LD-50 VALUES (yg/g) AT 48 HR FOR PREDATORS
FROM SEVERAL AREAS IN ARIZONA IN 1978. AFTER AN APPARENT
ANOMALOUS VALUE WAS REMOVED FROM THE ORIGINAL DATA SET
Insect
Nabis alternatas
Orius tristicolor
Area Collected
Tucson
Yuma
Tucson
Safford
Yuma
LD-502
(1.25)
2.12
(0.40)
(0.34)
0.60
95%
Fiducial Limits Slope
7.90
1.13 - 4.02 9.31
1.05
9.96
0.49 - 0.69 4.53
1
Adult average weights (mg): _N. alternatus 6.31, CL tristicolor 0.2
"Values in parentheses are estimated values due to non-significant regressions
64
-------
There were no significant regressions obtained from any data set for _N. americo-
ferus. The estimated LD-50 values at 24 hr were lowest for the Tucson population
(0.97), slightly higher for Safford (1.37), and highest for Yuma (1.61). At 48
hr the Tucson population displayed the lowest estimated LD-50 of 0.69, Yuma dis-
played a value of 1.15, and the Safford value was 1.29. In all cases the 48 hr
value was less than the 24 hr value.
With G_. pallens, Yuma populations produced the only significant regression at both
24 and 48 hr, those from Tucson displayed non-significant regressions, and no
values could be obtained from Safford due to the few insects caught there. At 24
hr, the Yuma population had an LD-50 of 2.13, while the estimated value for Tucson
was 2.43. However, the Yuma LD-50 increased to 2.14 at 48 hr and the Tucson esti-
mated value dropped to 2.40.
Although few CJ. punctipes were found in either Tucson or Safford and no LD-50 value
was obtained from these areas, they were plentiful in Yuma and significant regres-
sions were obtained at 24 and 48 hr. The LD-50 was observed to be 1.93, at 24 hr
and 1.85 at 48 hr.
There were no significant regressions for (3. tristicolor since the dosage-mortality
values were so erratic. The estimated LD-50 values at 24 hr were 0.60 for Tucson,
0.73 for Safford, and 0.91 for Yuma. At 48 hr, the estimated values were 0.45,
0.35, and 0.66 for Tucson, Safford, and Yuma, respectively. As with N^. alternatus,
there was at least one anomalous value in the data of each population, but it could
not be validly removed. Removal of the appropriate value would produce significant re-
gressions for each population at 24 hr, but only for the Yuma population at 48 hr.
The LD-50 values at 24 hr were 0.50 for Tucson, 0.30 for Safford, and 0.75 for Yuma.
At 48 hr, the LD-50 was 0.60 for Yuma and an estimated 0.40 for Tucson and 0.34
for Safford.
The dosage-mortality results of MP and T alone and in combination are presented in
Table 27. Only the predators from Yuma were examined since enough insects of each
species were collected to allow several replications for each test. In all cases
T produced less mortality than MP alone or the MP-T mixture, except for (). tristicolor
which had high mortalities with T and low mortalities with MP-T. There does not
appear to be a discernible difference between MP alone and MP-T (except for Ch tris-
ticolor) ; therefore, a two sample t-test was performed to test for significance bet-
65
-------
TABLE 27. DOSAGE-MORTALITY AFTER 48 HR OF YUMA PREDATORS IN 1978 USING
METHYL PARATHION (MP) AT THE LC-50 LEVEL (ng/INSECT), TOXA-
PHENE (T) AT TWICE THE METHYL PARATHION CONCENTRATION, AND
A 1:2 METHYL PARATHION:TOXAPHENE MIXTURE (MP-T)
Insect
Nabis alternatus
Nabis americoferus
Geocoris pallens
Geocoris punctipes
Orius tristicolor
Treatment
(Dose In ng)
MP(13)
T(26)
MP(13)-T(26)
MP(15)
T(30)
MP(15)-T(30)
MP(8)
T(16)
MP(8)-T(16)
MP(8)
1(16)
MP(8)-T(16)
MP(0.13)
1(0.26)
MP(0.13)-T(0.26)
Number of
Replications
4
2
3
4
4
5
3
4
5
3
7
9
6
5
5
Average1
Mortality (%)
46.4
-9.3
44.3
47.9
7.4
56.0
44.3
37.3
47.1
44.3
-13.7
45.4
56.2
51.4
10.7
Negative mortality values result from mortality being less than controls.
66
-------
ween the percent mortalities of MP and MP-T, T and MP-T, and MP and T (Table 28).
With both Nabis species and (J. punctipes MP and MP-T did not produce significantly
different mortalities. Although (*. pallens displayed lower mortalities from T
than from MP or MP-T, they were not significantly different. (D. tristicolor dis-
played abnormally high mortalities from T and very low mortalities from MP-T,
resulting in MP and T not being significantly different while MP-T was significant-
ly less than either insecticide alone.
2. Target Insects
a. Lepidopterous Larvae
The MP LD-50 values for lepidopterous larvae are presented in Table 29. 11. virescens,
II zea, and ^. exigua proved to be more susceptible to MP than I£. acrea. The values
for II. virescens and II. zea were extremely close although H. zea appeared slightly
more resistant, but not significantly by comparison of the fiducial limits. MP was
significantly more toxic to J5. exigua, but significantly less toxic to E^. acrea in
comparison to the Heliothis species.
At 24 hr, the MP LD-50 values for II. virescens, II. zea and 5^. exigua were 9.70,
10.38, and 3.47, respectively. The values at 48 hr were only slightly different at
9.08, 9.57, and 3.75, respectively. Although a significant regression was not ob-
tained at any time for E. acrea, the dosages required were significantly greater
than for any other insect tested. The estimated LD-50 values at 24 and 48 hr were
91 and 72, respectively.
A dosage-mortality study with MP, T, and MP-T was performed on the larvae as described
previously for the predators. The results, presented in Table 30, indicate T was
less toxic than MP or MP-T, except for ^. exigua at 48 hr. A two sample t-test
(Table 31) demonstrated no significant differences between MP and MP-T for lepidop-
terous larvae. _H. virescens displayed a significant difference between MP and T at
48 hr. T mortality increased in _S_. exigua at 48 hr, while MP mortality decreased
with no significant differences between them or MP-T ,
b. Houseflies
Houseflies were observed to be very susceptible to MP (Table 28), and were signifi-
cantly more susceptible than S^. exigua when the fiducial limits were compared. Al-
though the houseflies were used to perfect dosing techniques, they also were used
as a reference and were more susceptible than the lepidopterous larvae, but about
equally as susceptible as the predators. At 24 hr the LD-50 was 1.04 and dropped to
0.87 at 48 hr.
67
-------
TABLE 28. TWO SAMPLE T-TEST AT THE 95% CONFIDENCE LEVEL ON 1978
PREDATORS FROM YUMA COMPARING METHYL PARATHION (MP)
AT THE LC-50 LEVEL (ng/INSECT), TOXAPHENE (T) AT
TWICE THE METHYL PARATHION CONCENTRATIONS, AND A 1:2
METHYL PARATHION:TOXAPHENE MIXTURE (MP-T). S -
SIGNIFICANT, NS - NOT SIGNIFICANT
Insect
Nabis alternatus
Nab is americof erus
Geocoris pallens
Geocoris punctipes
Orius tristicolor
Concen-
tration
of MP (rig)
13
15
8
8
0.130
Time
After
Dosing
(hr)
24
48
24
48
24
48
24
48
24
48
HP vs.
MP-T
NS
NS
NS
NS
NS
NS
NS
NS
S
S
T vs.
MP-T
S
S
S
S
NS
NS
S
S
S
S
MP vs.
T
S
S
S
S
NS
NS
S
S
NS
NS
68
-------
TABLE 29. METHYL PARATHION LD-50 VALUES (yg/g) FOR LABORATORY STRAINS
OF LEPIDOPTEROUS LARVAE AND HOUSEFLIES.
Insect
Heliothis virescens
Heliothis zea
Spodoptera exigua
Estigmene acrea
Musca domestica
Average larval weights
15.28, E. acrea 23.02;
Time After
Dosing (hr)
24
48
24
.48
24
48
24
48
24
48
LD-502
9.70
9.08
10.38
9.57
3.47
3.75
(91)
(72)
1.02
0.87
(mg) : H. virescens 21.91,
average adult
weight (mg)
95%
Fiducial Limits
8.78 - 11.60
8.35 - 10.19
9.14 - 12.39
8.57 - 10.92
2.91 - 4.20
3.17 - 4.47
0.94 - 1.13
0.83 - 0.91
H. zea 20.84, S.
: M. domestica 26
Slope
4.32
5.04
3.79
4.40
3.24
4.60
2.52
2.77
5.82
9.02
exigua
.45
"Values in parentheses are estimated values due to nonsignificant regressions.
69
-------
TABLE 30. DOSAGE-MORTALITY AFTER 48 HR OF LEPIDOPTEROUS LARVAE AND
HOUSEFLIES USING METHYL PARATHION (MP) AT THE LC-50 LEVEL (ng/
INSECT), TOXAPHENE (T) AT TWICE THE METHYL PARATHION CONCEN-
TRATION, AND A 1:2 METHYL PARATHION:TOXAPHENE MIXTURE (MP-T)
Insect
Heliothis virescens
Heliothis zea
£5podoptera exigua
Estigmene acrea
Musca domestica
Treatment Number of
(Dose in ng) Replications
MP(180)
T(360)
MP(180)-T(360)
MP(200)
T(400)
MP(200)-T(400)
MP(50)
T(100)
MP(50)-T(100)
MP(3000)
T(6000)
MP(3000)-T(6000)
MP(25)
T(50)
MP(25)-T(50)
4
4
4
4
4
4
4
4
4
4
4
4
3
3
3
Average1
Mortality (%)
25.0
0
15.0
55.0
0
47.5
28.0
28.0
47.0
65.0
0
45.0
65.3
10.7
84.0
1
Negative mortality values result from mortality being less than controls.
70
-------
TABLE 31. TWO SAMPLE T-TEST AT THE 95% CONFIDENCE LEVEL ON LEPIDOP-
TEROUS LARVAE AND HOUSEFLIES COMPARING METHYL PARATHION
(MP) AT THE LC-50 LEVEL (ng/INSECT), TOXAPHENE (T) AT
TWICE THE METHYL PARATHION CONCENTRATIONS, AND A 1:2
METHYL PARATHION:TOXAPHENE MIXTURE (MP-T). S - SIGNIFI-
CANT, NS - NOT SIGNIFICANT
Insect
Heliothis virescens
Heliothis zea
Spodoptera exigua
Estigmene acrea
Musca domestica
Concen-
tration
of MP (ng)
180
200
50
3000
25
Time
After
Dosing
(hr)
24
48
24
48
24
48
24
48
24
48
MP vs.
MP-T
NS
NS
NS
NS
NS
NS
NS
NS
NS
S
T vs.
MP-T
NS
NS
S
S
S
NS
S
S
S
S
MP vs.
T
NS
S
S
S
S
NS
S
S
S
S
71
-------
,Dosage-mortality studies with MP, X, and MP-T were also performed on the houseflies'
and are presented in Table 29. Although T toxicity was low, the MP-T mixture ap-
peared to be slightly more toxic than MP alone. Results of a two sample t-test
are presented in Table 30. At 24 hr no significant differences between MP and
MP-T were observed, while significant differences between T and MP-T and MP and
T were. The same was true at 48 hr, except for MP vs MP-T due to the increased
toxicity of MP-T. The houseflies were the only insects to display a significant
difference between MP and MP-T.
C. FATE OF TOXAPHENE, METHYL PARATHION, AND CHLOPvDIMEFOKM IN THE HOUSE
Mortality data from the excretion-retention studies indicated that mortality occur-
red only in treatments containing raethyl parathion, i.e., MP, T-MP, MP-C, and
T-MP-C (Table 32). All mortality occurred within 3 hr of dosing. It appeared that
MP toxicity was slightly enhanced by C combination, and slightly lowered by T, but
none of these differences were significant. No female mortality was observed with
any treatment.
The second mortality study using only MP and T combinations verified the above in
that no potentiation of MP was produced by T. As before, T actually lowered the
mortality (not significantly) even though applied in combination at 2 and 3.5 X that
of MP (Table 33).
1. Excretion:Toxaphene
o /:
Overall, the excretion of Cl (represents T and/or its metabolites recovered follow-
O f.
ing an oral dose of Cltoxaphene) was not altered by combinations of C and/or
MP. There were no significant differences in the total amount of Cl excreted in
the urine with any combination, or at either dose (Table 34).
Q £
Total excretion of Cl via the feces was significantly altered by the C and MP
combinations (Table 34) but a consistent pattern was not established. Following the
initial dose more Cl was excreted in the feces when combined with C yet less
O £
when combined with MP and C-MP. Following the redose Cl excretion was lowered
when combined with MP but no change when combined with C or C-MP.
o r
Excretion of Cl by female mice (Table 35) was lower (significantly via feces as
well as total excretion) following a dose 7 days after the first. However, those
o/:
females which received a. third dose 14 days after a redose excreted Cl at essen-
tially the same levels as those which had only received one dose.
72
-------
TABLE 32. MORTALITY FROM EXCRETION-RETENTION STUDIES. DOSAGES
PER MOUSE WERE: 1.0 mg TOXAPHENE (T) 0.5 mg METHYL
PARATHION1 (MP), AND 0.125 mg CHLORDIMEFORM(C). MEAN
MORTALITY BASED UPON 3 REPLICATES OF 12 .
(percent mortality)
3
Treatment X Mortality (%)
T 0 a
T-MP 20.29 b
T-MP-C 29.17 b
T-C 0 a
MP 25.50 b
MP-C 29.17 b
C 0 a
48 of 60 dosed at 0.59 rag/mouse,
^-MP-C and MP-C, 2 replicates of 12 and MP, 5 replicates of 12.
3
Means followed by the same letter are not significantly different at
the 0.05 level.
73
-------
TABLE 33. MORTALITY OF MICE FROM COMBINATIONS OF TOXAPHENE AND
METHYL PARATHION. MEAN % MORTALITY BASED ON 4 REPLICATES
EACH CONTAINING, 12 MICE
(percent mortality)
Treatment - (rag/kg)
T (32)
T (56)
MP (16)
T (32) - MP (16)
T (56) - MP (16)
X Mortality (%)b
0
0
34.09
20.83
29.17
a
a
b
b
b
a
T - toxaphene; MP - methyl parathion
Means followed by the same letter are not significantly different at the
0.05 level.
74
-------
TABLE 34. RECOVERY OF Cl FROM URINE AND FECES OF MALE MICE
ORALLY DOSED WITH 36C1-TOXAPHENE. SIGNIFICANCE IS
IN RELATION TO T AND THOSE VALUES WITHIN THE SAME
DOSE ONLY
(percent recovery)
TREATMENT3
T T-C
Urine
Initial Dose 18.15a
Redose 28.00a
Feces
Initial Dose 42.45a
Redose 58.76a
25
17
54
46
.Ola
.36a
.70a
.97a
T-MP
21.22a
23.39a
31.49b
46.83a
T-MP-C
17.50a
25.88a
27.52b
57.16a
o
Values followed by the same letter are not significantly different at
the 0.05 level.
T-toxaphene, 25 mg/kg; MP-methyl parathion, 12.5 mg/kg, C-chlordimeform,
3.2 mg/kg.
TABLE 35. RECOVERY OF Cl FROM URINE AND FECES OF FEMALE MICE ORALLY
DOSED WITH 36C1-TOXAPHENE. TOTALS REPRESENT THE EXCRETION
THROUGH 96 HR ONLY.
(percent recovery)
Urine
Feces
Excretion (urine + feces)
0
Single Dose
14.87a
34.57a
49.44a
Redose
11.85a
17.20b
29.05b
Triple Dose
19.71a
31.00a
50.71a
o
Values followed by the same letter are not significantly different at
the 0.05 level.
75
-------
,2. Excretion:Chlordimeform
Excretion of C (represents C and/or its metabolites recovered following an oral
dose of C-chlordimeform) was not affected by combinations of T and/or MP. The
14
only significant change was a lowering of C excretion when combined with MP in
the urine following a redose (Table 36). Although the difference between some
values were large, the large variance resulted in non-significant differences.
3. Excretion;Methyl Parathion
The excretion of para-nitrophenol (PNP), a metabolite of MP, was lowered by the
addition of T. The mean total amount of PNP sampled from urine 96 hr after dosing
was 172.5 yg from MP-mice, and 67.5 yg from those treated with MP-T. Analysis of
feces is ongoing for the presence of MP and methyl paraoxon. Little of either has
been found and analysis of other metabolites may be necessary.
4. Tissue Retention:Toxaphene
Retention of Cl in several tissues of male mice is reported in Table 37. Lipid
was the only tissue which displayed no significant changes as a result of combining
O f
T with C and/or MP. Adding C to T led to more Cl being deposited in the brain,
O £
muscle and testes. The T-MP combination resulted in significantly more Cl in
brain, and less in kidney, and liver. When both C and MP were added to T the re-
sults were similar to T-MP; brain had significantly more, and kidney and liver had
less Cl than T alone.,
n f
In general the amount of Cl retained by tissues increased after a redose. Tissues
o £
from mice sacrificed 33 days after the redose had Cl levels nearer those of single
O £
dosed mice. Following a redose, Cl concentrations were significantly elevated in
kidney by T-C and T-MP-C combinations and higher in muscle by T-MP and T-C, yet was
significantly lower in lipid from T-C and T-MP-C (Table 38). Thirty three days after
the redose, T-C still led to reduced " Cl levels in lipid, while T-MP and T-MP-C in-
duced higher levels in the liver.
O £ *3 £.
After a single dose of Cl-toxaphene, female mice accumulated more Cl in brain,
muscle, and gonads than did males (Table 39). Both sexes were dosed with 1.0 mg/
mouse, but the effective dose was 25 ing/kg for males and 34 mg/kg for females, which
may explain the higher level of tissue retention. Following a redose, the measured
levels were still higher than those from redosed males, yet none of the differences
were significant.
76
-------
TABLE 36. RECOVERY OF C FROM URINE AND FECES OF MALE MICE ORALLY
DOSED WITH 14C-CHLORDIMEFORM. SIGNIFICANCE IS IN RELATION
TO C AND THOSE VALUES WITHIN THE SAME DOSE ONLY.
(percent recovery)
Urine
Initial Dose
Redose
Feces
Initial Dose
Redose
cb
31.87a
37.17a
74.96a
49.66a
TREATMENT21
C-T
28.07a
26.40a
77.45a
50.00a
C-MP
38.23a
19.82b
75.60a
42.97a
C-T-MP
30.65a
23.14a
53.23a
66.19a
Values followed by the same letter are not significantly different at
the 0.05 level
Cchlordimeform, 3.2 mg/kg; MP-methyl parathion, 12.5 mg/kg, T-toxaphene,
25 mg/kg
77
-------
TABLE 37. RETENTION OF CL BY TISSUES OF MALE MICE, ORALLY DOSED
WITH 36ci-TOXAPHENE. SIGNIFICANCE IS IN RELATION TO T
(ppm 36C1)
Treatment Brain
Ta 0.19a
T-C 1.80b
T-MP 0.54b
T-MP-C 0.67b
T-toxaphene, 25 mg/kg
3.2 mg/kg.
Kidney
0.72a
1.26a
0.36b
0.29b
; MP-methyl
TISSUEb
Lipid
10.61a
11.67a
9.90a
10.85a
parathion,
Liver Muscle Testes
0.54a 1.18a 0.75a
0.91a 3.81b 4.16b
0.32b 0.47a 0.66a
0.35b 0.49a 0.48a
12.5 mg/kg; C-chlordimeform,
Means followed by the same letter are not significantly different at the
the 0.05 level, ANOVA.
78
-------
TABLE 38. RETENTION OF Cl BY TISSUES OF MALE MICE 8 AND 33 DAYS
AFTER A REDOSE OF 36Ci TOXAPHENE. SIGNIFICANCE IS IN RE-
LATION TO T OF THAT TISSUE ONLY.
(ppm 36C1)
Treatment Brain
8C
Tb 2.59a
T-C 2.12a
T-MP 3.20a
T-MP-C 2.15a
33
T l.Sla
T-C 1.17a
T-MP 1.44a
T-MP-C 2.51a
Kidney
0.32a
1.42b
0.54a
1.22b
0.49a
0.69a
0.39a
0.79a
*3
Tissue
Lipid
16
7
16
2
3
1
3
3
.96a
.91b
.69a
.79b
.60a
.96b
.17a
.17a
Liver
0.73a
2.14a
1.94a
0.63a
0.39a
0.32a
1.02b
1.12b
Muscle
1.96a
2.76b
3.73b
1.96a
1.48a
1.48a
1.38a
2.52a
Testes
2.
2.
2.
2.
2.
1.
2.
2.
64a
27a
81a
68a
16a
64a
37a
41a
Values followed by the same letter are not significantly different at the
0.05 level.
T-toxaphene, 25 mg/kg; MP-methyl parathion, 12.5 mg/kg, C-chlordimeform
3.2 mg/kg.
"Days after receiving redose.
79
-------
TABLE 39. COMPARATIVE RETENTION OF Cl BY TISSUES OF MALE AND
FEMALE MICE ORALLY DOSED WITH 36C1-TOXAPHENE. SIGNIFICANCE
IS IN RELATION TO EACH DOSE AND WITHIN EACH TISSUE ONLY.
(ppm 36C1)
Dose Brain
Single Dose
Male 0.19a
Female 2.17b
Redose
Male 2.59a
Female 3 . 93a
Kidney
0.72a
1.22a
0.32a
2.46a
TISSUE3
Lipid Liver Muscle
10.61a 0.54a 1.18a
10.96a 0.40a 3.63b
16.96a 0.73a 1.96a
13.74a 0.87a 2.03a
Gonads
0.75a
3.15b
2.64a
4.58a
Values followed by the same letter are not significantly different at the
0.05 level.
80
-------
5. Tissue Retention:Chlordimeform
14
Combining T and/or MP with C resulted in lowering C deposition in some tissues
14
(Table 40). Addition of MP led to a drop of C levels in the liver, while MP,
14
T, and both together when added to C caused less C to be retained in lipid,
muscle, and the testes.
Following the redose, C-T led to only one significant change by increasing the
14 14
level of C in the testes. C-MP also increased C levels in testes as well as
in the liver. When all 3 insecticides were combined, significant increases in
14
C levels occurred in the testes, liver, and lipid.
81
-------
14
TABLE 40. RETENTION OF C BY TISSUES OF MALE MICE ORALLY DOSED WITH
^C-CHLORDIMEFORM. SIGNIFICANCE IS IN RELATION TO C, WITH-
IN EACH TISSUE, AND WITHIN EACH DOSE ONLY
(ppm
Treatment
Initial Dose
cb
C-T
C-MP
C-T-MP
Redose
C
C-T
C-MP
C-T-MP
Brain
0.56a
0.40a
0.38a
0.48a
0.17a
0.49a
0.39a
0.42a
TISSUE3
Kidney Lipid
0.75a
O.Sla
0.51a
0.72a
0.42a
0.29a
0.57a
0.47a
0.25a
O.QSb
0.09b
0.12b
0.16a
0.14a
0.32a
0.38b
Liver
0.56a
0.52a
0.33b
0.44a
0.65a
0.60a
1.03b
1.15b
Muscle
0.36a
0.19b
0.21b
0.19b
0.23a
O.lla
0.27a
0.33a
Testes
0.60a
0.32b
0.19b
0.17b
O.lOa
0.22b
0.36b
0.40b
Values followed by the same letter are not significantly different at the
0.05 level.
C-chlordimeform, 3.2 mg/kg; MP-methyl parathion, 12.5 mg/kg; T-toxaphene,
25 mg/kg.
82
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SECTION VI
REFERENCES
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553, March 1979.
3. Wang, C.M., and F. Matsumura. Relationship Between the Neurotoxicity
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+ +
4. Skou, J.C. Enzymatic Basis for Active Transport of Na and K Across
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1975.
11. Rail, T., and A.G. Oilman. The Role of Cyclic AMP in the Nervous
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12. Bloom, F.E. The Role of Cyclic Nucleotides in Central Synaptic
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13. Horwitz, B.A., and M. Eaton. The Effect of Adrenergic Agonists and
Cyclic AMP on the Na /K ATPase Activity of Brown Adipose Tissue.
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14. Casida, J.E., and S.H.P. Maddrell. Diuretic Hormone Release on
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1(1): 71-83, March 1971.
15. Kacew, S., and R.L. Sirighal. Influence of p, p'- DDT, <= -Chlordane,
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1363-1371, November 1973.
16. Kacew, S., and R.L. Sirighal. Role of Cyclic Adenosine 3':5'- Mono-
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17. Rajakovick, A.S., and R.B. March. Insecticide Cyclic Nucleotide In-
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February 1976.
18. Yamasaki, T., and T. Narahashi. The Effects of Potassium and Sodium
Ions on the Resting and Action Potentials of the Cockroach Giant Axon.
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19. Matsumura, F., and R.D. O'Brien. Insecticide Reaction with Nerve.
Interactions of DDT with Components of American Cockroach Nerve. J
Agr Food Chem. 14(1): 39-43, January 1966.
20. 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.
21. Treherne, J.E. The Movements of Sodium Ions in the Isolated Abdominal
Nerve Cord of the Cockroach, Periplaneta americana. J Expl Biol (London).
38(3): 629-636, September 1961.
22. Eldefrawi, M.E., and R.D. O'Brien. Permeability of the Abdominal Nerve
Cord of the American Cockroach to Fatty Acids. J Insect Physiol ' (London),
12(9): 1133-1142, September 1966.
84
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23. Rodbard, D., and J.E. Lewald. Computer Analysis of Radioligand
Assay and Radioimmunoassay Data. Acta Endocrinol Suppl (Copenhagen)
147: 79-103, March 1970.
24. Felman, H., and D. Rodbard. Mathematical Theory of Radioimmunoassay.
Principles of Competitive Protein Binding Assays. J.P. Lippincott Co.,
Philadelphia, Pa., 1971. p. 89-107.
25. Rodbard, D. Statistical Aspects of Radioimmunoassays. Principles of
Competitive Protein Binding Assays. J.P. Lippincott Co., Philadelphia,
Pa., 1971. p. 204-259.
26. Rodbard, D. Statistical Quality Control and Routine Data Processing for
Radioimmunoassay and Immunoradiometric Assays. Clin Chem. 20(10): 1255-
1270, October 1974.
27. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. Protein
Measurement with the Folin Phenol Reagent. J Biol Chem. 193(1): 265-275,
November 1951.
28. Clark, J.B., and W.J. Nicklas. The Metabolism of Rat Brain Mitochondria.
Preparation and Characterization. J Biol Chem. 245(18): 4724-4731, Sep-
tember 1970.
29. Mcllwain, H. Chemical Exploration of the Brain. Elsevier, Amsterdam,
1963. p. 154.
30. Skou, J.C. The Influence of Some Cations on an ATPase from Peripheral
Nerves. Biochim Biophys Acta (Amsterdam). 23(2): 394-401, February 1957.
31. Skou, J.C. Enzymatic Aspects of Active Linked Transport of Na and K
Through the Cell Membrane. Prog Biophys Molec Biol. 14(1): 131-166,
April 1964.
32. Ohnishi, T., R.S. Gall, and M.L. Mayer. An Improved Assay of Inorganic
Phosphate in the Presence of Extralabile Phosphate Compounds: Application
to the ATPase Assay in the Presence of Phosphocreatine. Anal Biochem.
69(1): 261-267, November 1975.
33. Paulsen, K., V.J. Adesso, and J.J. Porter. DDT: Effects on Maternal
Behavior. Bull Psychonomic Soc. 5(2): 117-119, February 1975.
34. Davenport, J.N., and L.M. Gonzalez. Neonatal Thyroxine Stimulation in
Rats: Accelerated Behavioral Maturation and Subsequent Learning Deficit.
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35. Eayrs, J.T., and W.A. Lishman. The Maturation of Behaviour in Hypothy-
roidism and Starvation. Anim Behav (London). 3(1): 17-24, January 1955.
36. Candland, O.K., and Z.M. Nagy. The Open Field: Some Comparative Data.
Ann N.Y. Acad Sci. 159(3): 831-851, July 1969.
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37. Hopkins, A.R., H.M. Taft, and W. James. Reference LD-50 Values for
Some Insecticides Against the Boll Weevil. J Econ Entomol. 68(2):
189-192, April 1975.
38. Finney, D.J. The Adjustment for a Natural Response Rate in Probit
Analysis. Ann Appl Biol. 36(2): 187-195, June 1949.
39. Finney, D.J. Probit Analysis. 2nd ed, Cambridge University Press,
Cambridge, 1962. pp. 318.
40. Shorey, H.H., and R.L. Hale. Mass-Rearing of the Larvae of Nine
Noctuid Species on a Simple Artifical Medium. J Econ Entomol. 58(3):
522-524, June 1965.
41. Cranmer, M. Determination of p-Nitrophenol in Human Urine. Bull
Environ Contam Toxicol. 5(4): 329-332, July 1970.
42. O'Brien, R.D. Mode of Action of Insecticides. Ann Rev Entomol. 11(1):
369-402, January 1966.
43. Rasmussen, H. Cell Communication, Calcium Ion, and Cyclic Adenosine
Monophosphate. Sci. 170(3956): 404-412, October 1970.
44. Kakiuchi, S., R. Yamazaki, and Y. Teshima. Regulation of Brain Phos-
I I i [
phodiesterase Activity: Ca Plus Mg - Dependant Phosphodiesterase and
Its Activation Factor from Rat Brain. Adv Cyclic Nucleotide Res., Raven
Press, New York, N.Y., 1972. Vol 1, p. 455-477.
45. Butler, K.D., and L.A. Crowder. Increased Cyclic Nucleotides in Several
Tissues of the Cockroach and Mouse Following Treatment with Toxaphene.
Pest Biochem Physiol. 7(5): 474-480, October, 1977.
46. Ulbricht, W. Die ZeitlLche Verlauf Der Kalium-Depolarisation Der
Schurringsmembran bei verschiedenen Calcium-Konzentration und Anodischer
Polarisation. Pfliigers Archiv (Berlin). 277(3): 270-284, July 1963.
47. Shimizu, H., C.R. Creveling, and J.W. Daly. Cyclic Adenosine 3',5'-Mono-
phosphate Formation in 3rain Slices: Stimulation by Batrachotoxin, Ouabain,
Veratridine, and Potassium Ions. Molec Pharm. 6(2): 184-188, March 1970.
48. Robinson, G.A. Cyclic AMP. Academic Press, New York, N.Y., 1971, pp 531.
49. Ferrendelli, J.A., A.L. Steiner, D.B. McDougal, and D.M. Kipnis. The
Effect of Oxotremorine and Atropine on cGMP and cAMP Levels in Mouse
Cerebral Cortex and Cerebellum. Biochem Biophys Res Comm. 41(4): 1061-
1067, November 1970.
50. Ferrendelli, J.A., D.A. Kinscherf, and D.M. Kipnis. Effects of Amphetamine,
Chlorpromazine, and Reserpine on Cyclic GMP and Cyclic AMP Levels in Mouse
Cerebellum. Biochem Biophys Res Comm. 46(6): 2114-2120, March 1972.
86
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51. Ferrendelli, J.A., E.H. Rubin, and D.A. Kinscherf. Influence of Div-
alent Cations on Regulation of Cyclic GMP and Cyclic AMP Levels in
Brain Tissue. J. Neurochem. 26(4): 741-748, April 1976.
52. Hepp, K.D., R. Edel, and 0. Wieland. Hormone Action on Liver Adenyl
Cyclase Activity. The Effects of Glucagon and Fluoride on a Particu-
late Preparation from Rat and Mouse Liver. Eur J. Biochem (Berlin).
17(1): 171-177, November 1970.
53. Steiner, A.L., J.A. Ferrendelli, and D.M. Kipnis. Radioimmunoassay
for Cyclic Nucleotides. III. Effect of Ischemia , Changes During De-
velopment and Regional Distribution of Adenosine 3',5'-Monophosphate
and Guanosine 3',5'-Monophosphate in Mouse Brain. J Biol Chem. 247(4):
1121-1124, February 1972.
54. Fallon, A.M., and G.R. Wyatt. Cyclic Guanosine 3',5'-Monophosphate.
High Levels in the Male Accessary Gland of Acheta domesticus and Related
Crickets. Biochim Biophys Acta (Amsterdam). 411(2): 173-185, December
1975.
55. Ishikawa, E., S. Ishikawa, J.W. Davis, and E.W. Sutherland. Determination
of Guanosine 3',5'-Monophosphate in Tissues and of Guanyl Cyclase in Rat
Intestine. J Biol Chem. 244(23): 6371-6376, December 1969.
87
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SECTION VII
LIST OF PUBLICATIONS
Butler, K.D., and L.A. Crowder. Increased Cyclic Nucleotides in Several
Tissues of the Cockroach and Mouse Following Treatment with Toxaphene.
Pest Biochem Physiol. 7(5): 474-480, October 1977.
o r
Dary, C.C., and L.A.. Crowder. Uptake of Cl-Toxaphene in the Cock-
roach, Leucophaea maderae (Fab.). Bull Environ Contain Toxicol. 18(6)
766-772, December 1977.
Dary, C.C., and L.A. Crowder. Bioelectrical Activity of Isolated Ventral
Nerve Cords of the American Cockroach, Periplaneta americana (L.), Treat-
ed with Toxaphene. Bull Environ Contain Toxicol. 21(4/5): 548-553,
March 1979.
Whitson, Roy S., and L.A. Crowder. Ion Movements in the Nervous System
of the American Cockroach, Periplaneta americana (L.), Influenced by
Toxaphene. J Environ Sci Health Pt B. 14(5): 545-562, August 1979.
88
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SECTION VIII
GLOSSARY
AB - Abbreviation of abdominal, refers to abdominal section of central nervous
system.
Active Transport - Movement of ions and molecules across cell membranes by ex-
pending energy.
Adenyl Cyclase - Enzyme which converts ATP to cAMP.
Aldrin - 1, 2, 3, 4, 10, 10-hexachloro-l, 4, 4a, 5, 8, 8a-hexahydro-endo-exo-
1, 4:5, 8 - dimethanonaphthalene.
Asymptomatic - Not displaying symptoms.
ATPase - Adenosine triphosphatase enzymes.
Axonal - Associated with axons (extensions of nerve cells which transmit im-
pulses) .
BR - Abbreviation of brain, refers to brain section of central nervous system.
C_ - Abbreviation of chlordimeform.
45 -H-
Ca - Symbol for radioisotope of calcium.
cAMP - Cyclic adenosine 3',5'-monophosphate.
Cereal Nerve - Nerve leading from the cerci (sensory structures on the posterior
of insects) of the cockroach into the ventral nerve cord.
cGMP - Cyclic guanosine 3',5'-monophosphate.
Chlordimeform - _N' - (4-chloro-o-tolyl)-N^ N-dimethylformamidine.
Q £
Cl - Symbol for radioisotope of chloride ion.
C-MP - Abbreviation of chlordimeform, methyl parathion mixture.
CNS - Abbreviation of central nervous system.
Cyclic Nucleotides - High energy phosphate compounds, i.e. cAMP, cGMP.
Cyclodiene Insecticide - Any one of a group of compounds, derived by the Diels-
Alder reaction in which hexachlorocyclopentadiene is one of the reactants.
89
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DDT - l,l,l-trichloro-2,2-bis(p-chlorophenyl) ethane.
- Apparatus used to collect insects by vacuum action.
Dieldrin - 1,2,3,4,10,1 O-hexachloro-6 , 7-epoxy-l ,4,4a,5,6,7,8, 8a-octahydro-
endo-exo-1 ,4 : 5,8-dimethanonaphthalene.
Efflux - Diffusion outward.
Extraneuronal - Outside or surrounding the nervous system.
Fat Body - Diffuse "tissue" in insects below the epidermis and around the gut,
which serves as a store of glycogen, protein, and plays an active part in meta-
bolism.
Ganglia - Small solid masses of nervous tissue containing numerous cell bodies.
Gastric Caeca - Tubular extensions of the fore part of the midgut of insects.
Hemipteran - Refers to insects of the order Hemiptera (true bugs).
In Vitro - Experimentation on a tissue or organ removed from the animal.
In Vivo - Experimentation on the whole living animal.
Icm - Atom or group of atoms that Carries a positive or negative charge.
Ionic Flux - Movement of ions across a membrane.
42 +
K - Symbol for radioisotope of potassium.
Larvae - (sing, larva) Immature stages of insects displaying complete metamor-
phosis.
LC-50 - Lethal concentration., median (lethal for 50% of inoculated group) , dose
per animal (mg/animal)
LD-50 - Lethal dose, median ( lethal for 50% of inoculated group) , dose per unit
weight (mg/g) .
Lepidopterous - Refers to insects of the order Lepidoptera (moths and butterflies)
Malphighian Tubules - Tubular glands, excretory in function.
Methyl-parathion - 0,0-Dimethyl 0-p-nitrophenyl phosphorothioate,
i
Mg - Symbol for magnesium.
Mitochondria - Microscopic bodies occurring in the cytoplasm of a cell, and re-
sponsible for energy production,
Mode of Action - Method by which a compound exerts its effects.
90
-------
MP - Abbreviation of methyl parathion.
mV - Millivolt, 10~ volts.
yCi - Microcurie; unit of radioactivity containing 10 curies or 37,000
disintegrations per second.
24 +
Na - Symbol for radioisotope of sodium.
Neonatal - New born.
Non-Target Insects - Insects not intended to be killed by insecticides.
Ouabain - Cardiac glycoside obtained from seeds of Strophanthus gratus,
specifically inhibits Na -K ATPase, C H 0
Partitioned - Separation of substances between solvents of different polarities.
Parturition - birth
Perinatal - About the time of birth, in this instance, prior to birth.
Phosphodiesterase - Enzyme responsible for the breakdown of cyclic nucleotides.
Pi - Symbol for inorganic phosphate.
Plasma Membrane Vesicles - Small spheres of cell membrane formed when the cell
membrane is fractured.
POPOP - Phosphor used in liquid scintillation cocktails, p-bis-2-(5-phenyloxa-
zolyl)-benzene.
Postnatal - Following birth.
Postpartum - Following birth.
ppm - parts per million.
PPO - Phosphor used in liquid scintillation cocktails, 2-5-diphenyloxazole.
Radiolabelled - Compound in which one or more of the elements of it's structure
are radioactive.
Scintillation - Method of quantifying radioactivity by counting the flashes of
light given off by a phosphor excited by ionizing radiation.
Scintillation Cocktail - Solution of phosphors used to evaluate radioactivity.
Site of Action - Location in a tissue or organ where a compound exerts it's effect.
Spp - Species,
Suboesophageal Ganglion - Ganglion located at the base of the arthropod head,
lying below the esophagus.
91
-------
Supernate - Fluid portion of material which has been separated by centri-
fugation.
Symptomatic - Displaying symptoms.
T^ - Abbreviation of toxaphene.
Target Insects - Insects which are intended to be killed by insecticides.
T-C(C-T) - Abbreviation of toxaphene, chlordimeform combination.
TH - Abbreviation of thoracic, refers to thoracic section of the central nervous
system.
T-MP(MP-T) - Abbreviation of toxaphene, methyl parathion combination.
T-MP-C(C-T-MP) - Abbreviation of toxaphene, methyl parathion, chlordimeform
combination.
Topical Dose - A dose which is applied externally.
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.
VNC - Abbreviation of ventral nerve cord.
92
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA- 600/1-80-003
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Mode of Action of Cyclodiene Insecticides:
The Nervous System Influenced by Toxaphene
5. REPORT DATE
January 1980_
6. PERI-OMMiiMG ORGANIZATION CODE
7. AUTHOR(S)
Larry A. Crowder
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Entomology
University of Arizona
Tucson, Arizona 85721
10. PROGRAM ELEMENT NO.
11.CONTRACT/GRANT NO.
R-804351
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
U. S. Environmental Protection Agency
Office of Research and Monitoring
Washington, DC 20460
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A study was made concerning the mode of action, excretion, metabolism, and
behavioral effects of toxaphene and combinations of toxaphene, methyl parathion,
and/or chlordimeform in various insects, the mouse and rat. toxaphene (1)
altered ionic movements within the central nervous system of the cockroach,
Periplaneta americana, (2) increased levels of cAMP and cGMP in tissues of
the cockroach, Leucophaea maderae, and the mouse, and (3) inhibited ATPase
enzymes in tissues of P. americana and the mouse. Rat pups perinatally exposed
to sublethal doses of Toxaphene and methyl parathion showed few significant
changes in motor skills, behavior, or learning ability. Postnatal exposure
to toxaphene did lead to an impairment of learning ability in adult rats as
measured by a simple T-maze. Combining chlordimeform and methyl parathion with
36C1 recovered in feces and the amount deposited in tissues of orally-dosed mice.
Combining toxaphene with methyl parathion did not potentiate the toxicity of
methyl parathion under laboratory conditions to various hemipteran predators
and lepidopterous pests.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Toxicology
Poisons
Insecticides
Pesticides
Hazardous Materials
Physiological Effects
Chlorine organic compounds
Toxaphene
Cyclodiene
Chlorinated camphene
organophosphate
methyl parathion
formamidine
chlordimeform
06, A.M.T
13. DISTRIBUTION STATEMENT
Release Unlimited
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF P.-VGES
108
20. SECURITY CLASS (Thispage/
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
93
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