EPA-600/1-77-033
June 1977
Environmental Health Effects Research Series
THE METABOLISM OF NALED
INHALED BY RATS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into 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)
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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
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clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-77-033
June 1977
THE METABOLISM OF NALED
INHALED BY RATS
Peter E. Berteau and Robert E. Chiles
Naval Blosciences Laboratory
University of California
Naval Supply Center, Oakland, CA 9^625
Interagency Agreement No.
IAG-D5-0697
Project Officer
Ronald L. Baron
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N..C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environ-
mental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for
use.
11
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FOREWORD
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, environmental carcinogenesis
and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory develops and revises 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 preparing 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.
Insecticides are often toxic to man and other animals as
well as to insects. When insecticides are applied as fine mists,
the material may be inhaled more readily than when applied as
coarse droplets. Naled has been found to be more toxic to rats
when breathed than when swallowed. This document reports
results of studies that indicate the most probable cause of the
increased toxicity is the more rapid entry of naled into body
fluids when breathed than when swallowed, rather than the
transformation of the material into a more toxic substance.
H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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PREFACE
With the increasing concern regarding hazards to workers
applying aerosols of pesticide formulations in the field, a great-
er importance is being placed upon a knowledge of the toxicity
of these pesticides when they are inhaled. This concern is becom-
ing even more manifest with the increasing use of ultra-low volume
techniques of spraying in which the aerosols generated contain a
greater number of particles in the respirable range (0.5-5.0 ym
diameter) than do aerosols generated when conventional sprays are
used.
Because of the difficult and varied parameters involved in
determining a true inhalation LD50, expressed in mg/kg body-weight,
an assessment of the toxic hazard of pesticide aerosols is often
based upon the oral LD50 which is more easily determined.
In a report entitled "Studies on the Effects of Particle
Size on the Toxicity of Insecticide Aerosols" (Berteau et al.,
1976, (included as Appendix A to this report), we listed inhalation
LD50 values in mg/kg body weight of certain pesticides to rodents
and compared these values with LD50 values obtained when animals
received the material orally. The methodologies by which we
obtained these inhalation LD50 values are described in that report.
In the case of one of the insecticides that we studied,
naled, the inhalation LD50 was about one-twentieth that of the
oral LD50 value (i.e., the pesticide formulation was about 20
times more toxic to rats when administered as a small particle
aerosol than when fed by gastric intubation).
This report attempts to examine one suggested
hypothesis that might account for this increased toxicity; namely
that the pesticide is metabolized to a more toxic material to a
greater extent in the respiratory than in the gastrointestinal
tract.
To enhance the understanding of the procedures outlined in
this report and to generate a better awareness of the significance
of the results, it is .suggested that the report be read in
conjunction with the Appendix.
IV
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ABSTRACT
Naled (DibromvJ) was prepared with a carbon label in the
1-ethyl position. The labeled compound was administered in
appropriate formulation vehicles to female rats by the inhalation,
oral or intraperitoneal routes. Treated animals were either
placed in metabolism cages and their excreta and expiration of
radioactivity monitored during 48 hours, or they were quickly
dissected after sacrifice, the lungs and stomach extracted with
ether and metabolic breakdown products analyzed for 14-C products
by thin layer chromatography. Some animals were also used to
determine the deposition and early distribution of inhaled naled.
Urinary levels of radioactivity were higher when animals inhaled
the compound than when it was administered by the other routes.
Levels deposited in the lung were very low and this fact limited
the generation of analytical data on the metabolic changes.
However, no evidence was provided to indicate that there was a
preferential metabolism to the debrominated form of naled (di-
chlorvos) when the compound was inhaled contrasted to other
roxites of administration.
This report was submitted as a supplementary report to the
U.S. Army Final Report for Contract MIPR-4962, entitled "Studies
of Effects of Particle Size on the Toxicity of Insecticide .
Aerosols", September, 1976, by the Naval Biosciences Laboratory
under the partial sponsorship of the U.S. Environmental Pro-
tection Agency. This report covers a period from October, 1975
to April, 1976 and work was completed as of February, 1977.
v
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CONTENTS
Page
Foreword iii
Preface . . . . , iv
Abstract ..... v
List of Figures vii
Tables ix
Acknowledgments x
Introduction . 1
Materials and Methods 3
Results 6
Discussion 9
Conclusion 10
References . 11
Figures 13
Tables 23
Technical Report Data Sheet 26
VI
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FIGURES
Number
1-11:
1
2
3
4
5
6
7
8
9
10
11
12 - 18:
12
.13
14
Pag<
Relationships between Rf values and radio-
active levels (cpm) from thin layer chromato-
grams of extracted lungs (L) and stomachs (S)
from female rats administered [1-l^C-ethyl]
naled by three specified routes; inhalation
studies refer to whole body exposure unless
otherwise stated.
Tracheal intubation (diesel oil) 13
Gastric intubation (diesel oil) 13
Inhalation (diesel oil) (rat 1) ^4
Inhalation (diesel oil) (rat 2) 14
Inhalation (diesel oil) (rat 3) 15
Inhalation (diesel oil) (head only) .... 15
Inhalation (xylene) (rat 1) 15
Inhalation (xylene) (rat 2) 15
Inhalation (xylene) (rat 3) ........ 17
IP injection (diesel oil) (rat 1) ...... \~j
IP injection (diesel oil) (rat 2) 18
Cumulative excretion (percent) of radioactivity
in urine and expiration in breath, with time
(hr) from female rats administered [l-l^c-ethyl]
naled by three specified routes (upper curves
refer to expired C02; lower curves to urine).
Gastric intubation (diesel oil) (rat 1) ... 19
Gastric intubation (diesel oil) (rat 2) ... 19
Inhalation (xvlene) 20
vii
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Number Page
15 Inhalation (diesel oil) 20
16 Inhalation (diesel oil)(head only) 21
17 IP injection (diesel oil) (rat 1) 21
18 IP injection (diesel oil) 22
viii
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TABLES
Number
1 Recovery of radioactivity over a 48-hour
period after individual female rats received
[1-l^C-ethyl] naled 23
2 Percentage of total recovered radioactivity
found in organs, feces, urine and expired
C02 of individual rats receiving [l-14C-ethyl]
naled 24
3 Deposition and early distribution of inhaled
[l-14c-ethyl] naled 25
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ACKNOWLEDGMENTS
The helpful assistance of Drs. W.A. Been and R.L. Dimmick
is greatly appreciated. The assistance of Chevron Chemical
Company, Richmond, California, in providing technical grade and
analytical samples of naled and of Shell Chemical Company, San
Ramon, California for an analytical sample of Dichlorvos is also
greatly appreciated. Research was supported by the U.S. Army
Medical Research and Development Command, Washington, D.C. 20314
under contract No. MIPR 4962 funded through the U.S. Environmental
Protection Agency.
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INTRODUCTION
Workers applying pesticides in the field are exposed to
those materials most frequently via the dermal and inhalation
routes. Of course, some inhaled material is swallowed, and
unless there is evidence to the contrary, then known oral LD50
values (mg/kg) are suitable criteria for estimating safe exposure
levels. In at least one instance, however, it has been demon-
strated that the measured inhalation LD50 value (mg/kg) is mark-
edly less than the equivalent oral value. Is this increased
toxicity solely because the toxic portion of the formulation was
absorbed more rapidly through the lung alveoli than through the
stomach lining, or is it an indirect result of some other mech-
anism peculiar to lung physiology such as preferential formation
of a more toxic product by enzymes or surface active agents, or
is it a direct result of chemical action interfering with lung
function?
We have studied these questions with one pesticide, naled,
which has been shown to be about 20 times more toxic when admin-
istered as small, airborne particles to female rats than when
administered by intubation.
A common breakdown product of naled is known to be dichlor-
vos (DDVP) ; debromination to this material can result from expos-
ure to sulfhydryl groups (Casida, 1972) . The dichlorvos may be
further broken down to dichloroacetaldehyde and dimethyl phos-
phoric acid (Hodgson and Casida, 1962) or naled may be hydro lyzed
directly to bromodichloroacetaldehyde (Gasida e_t al. , 1962) . The
chemical nature of these reactions is shown in the following
scheme:
CH30
J >P - 0 - C -
CH,0 I
Br
naled dichlorvos
0 n n n n n
CH,0X( V i1 CH.Q W jf V1
J>P-OH + H-C-C-C1 J/P-OH + H-C-C
CH3° Br CH3° H
dimethyl phosphoric bromodichloro- dichloroacetaldehyde
acid acetaldehyde
1
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In this study we have largely been concerned with the formation
of dichlorvos because this is the only toxic metabolite encoun-
tered in this system and, based upon oral administration, has an
oral LD50 value of 80 mg/kg; i.e., at least twice as toxic as
naled. It was therefore our hypothesis that preferential forma-
tion of dichlorvos in the lung when naled was inhaled might
account for the increased toxicity. This report is a study of
the early metabolism of naled to rats when the compound is admin-
istered by the oral, inhalation or intraperitoneal routes.
Studies have also been made on the fate of the administered naled
in the rat during the 48 hour period after it received the com-
pound by each of the three routes of administration, in order to
compare the relative rates of metabolic breakdown.
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MATERIALS AND METHODS
The preparation of naled, labeled with -^carbon in the 1-
ethyl position was contracted out to New England Nuclear Corp-
oration. They used Burton's method (1971) to prepare (1-^C-
vinyl) dichlorvos from a Perkow reaction of dimethyl phosphite
and labeled chloral. The dichlorvos was then brominated to form
the labeled naled. A radiochromatographic tracing provided by
the New England Nuclear Corporation verified that total radio-
chemical impurities separated by the TLC system devised by them
were less than 170. We determined by thin-layer chromatography
(see below) that the sample was free of dichlorvos and therefore
deemed suitable for our work. Plates used were 250 urn prepared
silica gel GF plates (Analtech, Inc., 75 Blue Hen Drive, Newark,
Delaware 19711). A total of 1.342 (6.1 mCi) was provided, of
specific activity 1.73 mCi/mM.
A suitable thin-layer system of solvents was devised for
separating naled and dichlorvos. Analytical grade samples of
these compounds provided by Chevron Chemical Company (naled) or
Shell Chemical Company (dichlorvos) were used. Typically 10 yg
in 1 yl of chloroform were applied to the plate. Initially, a
system reported in the literature for separating various organo-
phosphorus compounds was used (cyclohexane; acetone; chloroform,
70:25:5 v/v -- Getz and Wheeler, 1968) or a system devised by
New England Nuclear Corporation (unpublished information) hexane;
acetone (7:3 v/v). The latter system gave lower total separation
but a little less streaking. Eventually a system consisting of
benzene: acetone (9:1 v/v) was developed which gave Rf values of
0.51 for naled and 0.40 for dichlorvos.
Unlabeled naled and dichlorvos (Vapona^-' , provided by the
Shell Chemical Company) were visualized on chromatograms by spray-
ing with a general reagent for detecting organophosphorus com-
pounds. This spray was a 270 solution of 4-p_-(nitrobenzyl) pyri-
dine in acetone. The plate was then heated for 10 minutes at
110° and again sprayed with a 107o solution of tetraethylenepent-
amine in acetone (Getz and Wheeler, 1968). Both naled and di-
chlorvos appeared as blue spots; dichlorvos was the more sensi-
tive compound to the spray. With labeled naled, visualization
was achieved by autoradiography -- plates were exposed to X-ray
film (Kodak BB-54) for at least 48 hrs and developed. The
relative concentrations of the naled and any dichlorvos or other
radioactive impurities that might be present were then determined
by use of a Photovolt TLC densitometer. (Photovolt, 1115 Broadway,
New York, N.Y. 10010). Where levels of radioactivity were
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encountered which were too low to measure by densitometry, 1-cm
sections of the plate were scraped off, placed in scintillation
vials, active material eluted with methanol, a "cocktail" pre-
pared with a suitable scintillation fluid, and the activity was
counted using a Packard or Beckman scintillation counter.
Comparative metabolic studies using female Sprague Dawley
rats (normally 200 - 250 g in weight), obtained from Charles River
Breeding Laboratories, were performed in the following manner:
(1) Lung and stomach fortification
After the test rat was sacrificed, the stomach and
lungs were dissected, fortified with 100 yl of a solution contain-
ing 6.1 mCi of [l-14C-ethyl] naled in 10 ml of benzene, homogen-
ized with 3 ml of normal saline, saturated with anhydrous sodium
sulfate, extracted with 3 x 3 ml portions of ether, separated,
made up to 10 ml, and 100 yl portions were spotted on plates and
eluted in the manner described above.
(2) Intratracheal instillation
A rat was anesthetized with 0.2 ml of Pentasol injected
subcutaneously plus an additional 0.1 ml 5 min afterwards. The
neck was shaved, the trachea exposed and 9.2 yCi of labeled naled
in 0.1 ml of diesel oil containing a little benzene was injected
into the trachea.. The rat died in 2-3 minutes. Lungs and stomach
were dissected out and extracted with ether as in the procedure
described above. Of the ether solution, 50 yl was spotted.
(3) Inhalation
Eight rats were exposed either whole-body or constrained
in head-only exposure units (e.g., Hoben, et al., 1976) in a
modified Henderson chamber (Dimmick and HatchT~1969) to an aerosol
generated by atomizing a solution of [1-14-C-ethyl] naled in a
suitable formulation vehicle. Exposure time was 36 minutes.
Typically the solution in the atomizer contained 1.83 ml of the
labeled naled dissolved in 20 ml of xylene or diesel oil contain-
ing a little benzene.
Mass median diameter and geometric standard deviation of
aerosols were determined with an Andersen (1958) sampler operated
at 28.3 1/min connected at the mid-point of the exposure chamber.
Size analysis was made whenever there was a change in solvent or
concentration of active ingredient. Concentration of aerosol
was determined by withdrawing measured amounts of air through
membrane filters and either noting the weight change or digesting
the filters and determining the amounts by counting the radio-
activity.
After exposure, one animal was removed alive and placed in
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an all-glass metabolism cage (Stanford Glass Blowing Laboratories,
Inc., Stanford, California) for 48 hours where expired C02, urine
and feces were collected at intervals. Fluid for collection of
C02 was 2 methoxyethanol:ethanolamine (2:1 v/v) (Krishna and
Casida, 1966) placed in collection towers and changed at intervals
not longer than 8 hrs. The other animals were all sacrificed
(C02 inhalation) then the lungs and stomach of three of them were
dissected out and extracted with ether, for TLC determinations
of unchanged naled and dichlorvos, using the procedure described
in (1) for fortified lungs and stomach. The remaining four ani-
mals were used for a deposition study in the manner previously
conducted with l-14c-heptadecane (Berteau and Biermann, 1977) also
described in the companion report. Exposure runs were made with
[1-14-C-ethyl] naled in diesel oil as well as with xylene because
this solvent more closely paralleled formulations used in the
field. Another recommended adjuvant for formulating naled was
soya-bean oil. Unfortunately, preliminary studies revealed that
lipids such as vegetable oils interfered with TLC elution; large
"haIf-moon-shaped" spots with indeterminate Rf values were found.
This type of interference had been previously noted (Getz and
Wheeler, 1968).
(4) Oral and intraperitoneal administration
Similar metabolic studies were conducted on rats which
received diesel oil solutions of [1-l^C-ethyl] naled by oral or
intraperitoneal routes. In these studies each of two rats were
administered either 46 uCi or 9.7 yCi of labeled naled in 0.5 ml
of diesel oil intragastrically; or two rats received 23 yCi in
0.25 ml in diesel oil, intraperitoneally.
(5) Control runs
For additional controls the inhalation LD50 of dichlor-
vos in soya-bean oil was determined as previously described
(Berteau et al., 1976) (to be compared with that of naled), and
naled (Dib~rbm 14 concentrate) was also atomized and collected in
impinger fluid (xylene) for TLC analysis to determine whether
atomization influenced the stability of naled.
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RESULTS
Based upon the Rf values of naled and dichlorvos, TLC analy-
sis revealed no conversion to dichlorvos when a typical formula-
tion of naled in xylene was atomized and the aerosol collected
in impinger fluid. The inhalation LD50 value of dichlorvos (1070
Vapona(STin soya-bean oil) was 4.0 mg/kg; i.e., the compound was
about twice as toxic as a similar formulation of naled. (Berteau,
et aJL , 1976)
Mass median diameter (HMD) of typical aerosols used in these
studies was 1.8 - 2.0 ym with a geometric standard deviation of
2.0. The use of a volatile solvent such as xylene did not signi-
ficantly reduce the HMD of the aerosol from that of diesel oil
alone. Particle size data used to determine HMD values have been
reported in some detail elsewhere (Berteau e£ al^.,1976; Berteau
and Biermann, 1974).
(1) Lung and stomach fortification
Based upon the Rf values for naled and dichlorvos re-
portedly previously (page 3 ) , mean percentages with 957o confi-
dence intervals of the two compounds determined when lungs or
stomachs of rats were fortified with naled were:
Organ Naled Dichlorvos
Stomach 73.0 + 14.4 26.8 + 14.0
Lung 16.3+18.9 37.3+25.5
The percentage of dichlorvos is not significantly higher in
the lung than in the stomach t = 0.99 (p = 0.3 - 0.4) (Student's
t test) . ~
(2) Intratracheal instillation
Figure 1 represents a plot of Rf values (1 cm segments)
vs. cpm of a thin-layer chromatogram of extracts from intra-
tracheal instillation of [l-^^C-ethyl] naled.
(3) Distribution of [l-^C-ethyl] naled when administered
by the oral, inhalation or intraperitoneal routes.
Aerosol concentrations of radioactivity ranged from
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2.9 x 10" cpm/1 to 0.55 x 10^ cpm/1, depending upon the formu-
lation. Table 1 lists the percentage recovery of radioactivity
when [l-l^C-ethyl] naled in either xylene or diesel oil was admin-
istered to rats by the three different routes. In order to pro-
vide an indication of the relative distribution of total recovered
radioactivity in Table 2 the percentage of the total recovered
radioactivity in tissues and excretions is reported. The only
significant differences in metabolism were the higher urinary
excretion of radioactivity from inhalation contrasted to oral
administration in both solvents, and the lower organ retention
from a xylene aerosol contrasted to one generated from diesel oil.
(4) Deposition and early distribution of inhaled
[l-l-^C-ethyl] naled.
Table 3 lists percentages of the material deposited
in the respiratory tract and translocated to the gastrointestinal
tract immediately following inhalation administration of naled
to the rat. Significantly (p <0.1, t = 5.436) lower levels in
lung were encountered when the" formulation vehicle was xylene
than when diesel oil was used.
(5) Comparative formation of dichlorvos when haled was
administered to groups of two or three rats by the
oral, inhalation or intraperitoneal routes.
Figures 1 and 2 represent the thin layer chromatogra-
phic plots of material found in the lungs and stomachs of rats
that had received naled by intratracheal instillation and tra-
cheal intubation respectively. Figures 3, 4 and 5 represent
similar results from each of three rats which were exposed whole-
body to the aerosol in diesel oil. Figure 6 represents the same
chromatogram resulting from head-only exposure in diesel oil.
Figures 7, 8 and 9 are similar results generated when the expos-
ure was to the whole body in xylene. The results from intra-
peritoneal administration are expressed in Figures 10 and 11.
Some apparently unchanged naled was present but significant
amounts of a compound with an Rf value corresponding to that of
dichlorvos was found in both lungs and stomachs of rats which
inhaled naled. In addition, a metabolite of very low Rf value
(i.e., highly polar) was sometimes found (but not always) in the
lung.
(6) Rate of excretion of radioactivity in urine and
expired air.
Cumulative excretion (percent) of radioactivity in
urine and expiration in breath, with time, from rats which re-
ceived the radiolabeled naled by the various routes are expressed
as follows: Figures 12 and 13 (gastric intubation in diesel oil);
Fig-ore 14 (inhalation in xylene) ; Figures 15 and 16 (inhalation
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in diesel oil, whole body and head-only, respectively), and
Figures 17 and 18 (intraperitoneal injection). Each figure
represents the results obtained from a single female rat monitored
in the metabolism cage after it had received the insecticide. It
is evident that radioactivity appears much more rapidly in the
expired air of animals which inhaled the material, than in air
from animals exposed by the other routes.
8
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DISCUSSION
LD50 values for four pesticides have been cited in the
companion report (Berteau et al., 1976) when the pesticide is
administered in various formulations to female rats by various
routes.
In that report we noted the 20-fold variation between oral
and inhalation toxicity of naled and observed, in contrast, that
another insecticide, chlorpyrifos (Dursban©), showed little
difference between the oral and inhalation LD50 based upon the
same considerations.
In order to try to explain the phenomenon previously en-
countered with naled, showing increased toxicity of inhaled naled
when compared to the intubative toxicity, it was suggested that
selective metabolism to a more toxic material may occur to a
greater extent in the lung than in the stomach.
Our data now indicate that there is clearly a greater amount
of radioactivity excreted in urine and expired air and less re-
tained in organs and tissues when xylene rather than diesel oil
is used as a formulation vehicle (Tables 1 and 2). This obser-
vation may explain the fact that naled is more acutely toxic if
the inhaled pesticide is formulated in xylene rather than a vege-
table oil such as soya-bean oil (Berteau and Been, unpublished
observations); i.e., it is more rapidly absorbed into the animal.
Except in one intraperitoneal administration, total recovery of
radioactivity was better than 7070; levels encountered in urine
were distinctly higher when the compound was inhaled than when
it was administered by the oral or intraperitoneal routes. Urinary
metabolites are expected to be carboxylic acids such as dichloro-
acetic acid or bromodichloroacetic acid whereas that found in the
absorbant is probably carbon dioxide -- the end product of oxida-
tive metabolism.
Thus it appears that the breakdown is less complete when
naled is inhaled, although levels in expired air were not sub-
stantially lower. The existence of such an incomplete breakdown
may shed some light on the existence of a higher inhalation than
oral or intraperitoneal toxicity, although from Figures 12 - 16,
it is evident that radioactivity appears more rapidly in both
urine and expired air when the pesticide is administered by the
inhalation route, indicating a more rapid entry into the blood
stream from this route. The corrosive effect of naled in the
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lung has already been noted and limited pathological data have
been reported (Berteau et al., 1976). This corrosive effect may
partly explain the rapicfentry into the blood stream. This obser-
vation may again partially explain the high inhalation toxicity
of naled. However, it is recognized that these variations may
result from different absorption characteristics between the two
routes of administration rather than, or in addition to any
metabolic differences.
When we consider Figures 1-11, peaks corresponding to
the Rf values of both naled and dichlorvos are often present but
the very low levels found in the lung ( of Table 3), when naled
is inhaled, prevent a quantitative comparison of the two com-
pounds. The only significant differences between the chromato-
grams of lungs or stomach of inhaled versus oral administration
is the presence of a material of low Rf in the lungs. This com-
pound may very well be a carboxylic acid such as is excreted in
the urine. The low levels of radioactivity found in lungs and
other organs after [1-l^C-ethyl] naled is inhaled may also be
encountered with other compounds and may explain the dearth of
information on inhalation metabolism.
Although preferential metabolism to dichlorvos has not been
demonstrated to occur in the lung on the basis of thin layer
chromatography, attention must be drawn to the very rapid meta-
bolism of dichlorvos in animals (e.g. Page, et^ al. , 1970) and,
thus, it is recognized that any dichlorvos present may be too
transitory in nature to measure chemically and yet may remain
long enough to exert an increased toxic effect. In addition, it
has been demonstrated (Blair, e_t al. , 1970) that rates and routes
of metabolic breakdown are very similar when dichlorvos is admin-
istered to rats either by the oral or inhalation routes, thus
delayed breakdown of dichlorvos after inhalation compared to oral
administration would not explain the increased toxicity of naled.
CONCLUSION
In conclusion, evidence of rapid entry into the animal's
system when naled is inhaled provides a possible explanation for
its high toxicity whereas we have not generated any data indica-
tive of preferential metabolism to dichlorvos. This rapid entry
may result from the corrosive action of naled on the lungs.
10
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REFERENCES
1. Andersen, A. New sampler for the collection, sizing and
enumeration of viable airborne particles. J. Bacteriol.
76.:471-484, 1958.
2. Berteau, P.E. and A.H. Biermann. 1974. Investigations of the
toxicity of aerosols generated from formulations of
four pesticides. Appendix II. Treatment of data from
Andersen samples„ Semi-Annual Report to U.S. Army
Research and Development Command, Washington, D.C. 20314.
3. Berteau, P.E. and A.H. Biermann. Respiratory deposition and
early distribution of inhaled vegetable oil aerosols in
mice and rats. Toxicol. Appl. Pharmacol. 39:in press
1977.
4. Berteau, P.E., W.A. Been, and R.L. Dimmlck. Studies of
effects of particle size on the toxicity of insecticide
aerosols. Final Report to U.S. Army Research and
Development Command, Fort Detrick, Frederick, Maryland.
1976. 75 pp.
5. Blair, D., D.H. Hutson, and B.A. Pickening. The comparative
metabolism of (vinyl-^C) Vapona in rats after inhalation
exposure and oral ingestion of the compound. Unpublished
draft summary from Shell Chemical Company. Cited in
"1970 Evaluations of some Pesticide Residues in Food."
The Monographs, pp 124. Food Agric. Org. UN; Wld. Hlth.
Org. Review, 1971.
6. Burton, W.B. Synthesis of 2,2-dichlorovinyl dimethyl phos-
phate labeled with 14c, 36C1, and 32P. J. Agr. Food
Chem. 19:869-871, 1971.
7. Casida, J.E. Chemistry and metabolism of terminal residues
of organophosphorus compounds and carbamates, pg.295, in
Pesticide Chemistry, Proc. 2nd Internat. IUPAC Congress
Vol. VI, Tahori, A.S., Ed. Gordon and Breach, London.
1972.
11
-------
8. Casida-7 J.E., L. McBridey and-R-;P-. Nied^ruieier; Metabolism
of 2, 2-dichlorovinyi dimethyl phosphate in relation
to residues in milk and mammalian tissues. J. Agr.
Food Chenu lQt.370-377r 196.2^
9. Dimmick, R.I. and M.T. Hatch. Dynamic aerosols and dynamic
aerosol chambers, in An Introduction to Experimental
Aerobiology, Dimmick, R.L., and A.B. ATcers, Eds./
Wiley-Interscience, New York. 1969. pp. 181-183.
10-. Getzr, M.E. and H\G~. Wheeler-. Thin layer chroma to graphy of"
organophosphorus insecticides with several adsorbents
and ternary solvent- ay stems.- J. As sac. Qffic-. Anal.
Chem.' '51:1101-1107, 1968.
11. Guyton, A.C. Measurement of the respiratory volumes of
laboratory animals. Am. J. Physiol. 150:70-77, 1947.
12. Hoben, H.J., S.A. Ching, L.J. Casarett. A study of the
inhalation of pentachlorophenol by rats. Part II. A
new inhalation exposure system for high doses in
short exposure time. Bull. Environ. Cont. Toxicol.
15:86-92, 1976.
13. Hodgson.,- E._,- and_JLJ
-------
u>
2000 -i
1600-
1200-
Q_
O
350-
280-
210 -J
CD
•—i
X
S
Q_
O
70-
35-
IIII I I
200 004 0.6 008 1.0
^ t • • • t
i i i i i i i i i i
0.2 0.4 0,6 0.8 1.0
Rf value
Fig. 1. Tracheal intubation (diesel oil) Fig. 2. Gastric intubation (diesel oil)
Figures 1 -11: Relationships between Rf values and radioactive levels (cpm) from thin layer
chromatograms of extracted lungs (L) and stomachs (S) from female rats
administered [l C-ethyO naled by three specified routes; inhalation studies refer to whole
body exposure unless otherwise stated.
-------
Q_
O
150 n
120 -
90 ^
60-
30 -
Q_
O
250 -i
200-
150-
100-
50
0.2 0.4 0:6 0,8
0.2
0,4 Or6 0,8 1.CJ
Fig. 3. Inhalation
(rat))
Fig. 4. Inhalation (dies^l oil) (rat 2)
-------
Q_
O
200 -I
160 -
120 -
80 -
40
1001
Q_
O
jI i r i I i i i
0,2 0.4 0.6 0.8 100
20
0.2 0.4 0.6 0,8 1.0
value
Fig. 50 Inhalation (diesel oil) (rat 3)
Fig. 6. Inhalation (diesel oil) (head only)
-------
150 i
150 i
I r I »
0.2 0.4 0.6 0.8 1.0
r i
0.2 0.4 0.6 0.8 1.0
value
Fig. 7. Inhalation (xylene) (rat 1)
Fig. 8. Inhalation (xylene) (rat 2)
-------
200-
160-
120 J
S 100
80-
40 H
Q_
O
0.2 0.4 0.6 0.8 1.0
0.2 004 0.6 0.8 1.0
value
Fig. 9. Inhalation (xylene) (rat 3)
Fig. 10. IP injection (diesel oil) (rat 1)
-------
00
Q_
O
40
0.2 0.4 0.6 0.8 1.0
Rf value
Fig0 11. IP injection (diesel oil) (rat 2)
-------
40
30 -
> on
rz 20 -
10 -
30 -
r- 20 -
10 -
I I I
I I I I I I I
12 24 36 48
12 24 36 48
TIME (HOUR)
Fig. 12e Gastric intubation (diesel oil) (rat 1) Fig. 13, Gastric intubation (diesel oil) (rat 2)
Figures 12 -18: Cumulative excretion (percent) of radioactivity in urine and expiration in
breath, with time (hr) from female rats administered O14C-ethyQ naled
by three specified routes (upper curves refer to expired C02; lower curves to urine)0
-------
40 n
30 -
LU
3 20
o
10
II I ii i i
12 24 36 48
UJ
o
10
I I I I I I I
12 24 36 48
TIME (HOUR)
Fig. 14. Inhalation (xylene) Fig. 15. Inhalation (diesel oil)
-------
40.
O
40.
30-
= 20-
10-
I I I I I 1 I I
12 24 36 48
i r i I i I i I
12 24 36 48
TIME (HOUR)
Fig. 16. Inhaltion (diesel oil) (head only) Fig. 17. IP injection (diesel oil) (rat 1)
-------
CO
I I I I 1 I I I
12 24 36 48
TIME (HOUR)
Fig, 18. IP injection (diesel oil)
-------
TABLE 1. Recovery of radioactivity over a 48-hour period after individual female
rats received [l-14c-ethyi] rialeci.
Percentage Recovered
Xylene
Inhalation
Whole body
Bladder
Blood
Cecum
Carcass
Colon
Fata
Head
Heart
Kidney
Liver
Lung
Muscle
Spleen
Small
intestine
Stomach
Total Organs
Feces
Urine
Expired C02
Total
Recovery
0
0
0
0
0
1
0
1
0
0
0
4
0
29
39
74
.
--
.14
—
.16
.48
.27
—
.15
.07
.11
.33
.03
.92
.17
.83
.44
.70
.66
.00
Inhalation
Whole body Head
0
0
1
1
0
9
0
2
0
1
0
20
2
26
38
87
__
—
.35
—
.85
.86
.95
—
.34
.84
.27
.47
.09
.73
.43
.18
.29
.02
.93
<
.42b
0
1
4
3
1
14
0
3
0
2
0
32
2
22
33
90
only
__
—
.21
—
.41
.48
.13
—
.13
.28
.86
.75
.20
.61
.47
.53
.62
.20
.09
•t
.44b
Diesel Oil
Rat
_
-
0.
-
1.
0.
1.
-
0.
12.
0.
2.
0.
2.
0.
22.
1.
14.
34.
73.
Oral
1 Rat 2
_
_
69
-
26
84
29
-
65
02
27
50
14
51
58
75
20
57
07
19
0.02
__
1.85
—
1.19
2.87
3.19
—
0.94
13.51
0.36
2.54
0.18
2.85
1.12
30.62
2.97
13.63
38.61
85.83
Rat 1
0.01
3.03
0.65
—
0.44
0.57
1.34
—
0.58
6.98
0.18
1.90
0.13
1.40
0.53
17.74
1.96
7.85
24.01
51.56
LP
Rat 2
0.02
3.22
0.80
11.20
0.73
0.80
2.25
0.13
0.65
12.45
0.25
0.61
0.12
1.53
0.35
35.13
1.68
14.87
35.00
86.68
a Based upon approximate values obtained from headless carcasses of four rats of similar
sex and strain (fat, 1070; muscle 1570) .
b Amount administered was based upon aerosol concentration, duration of exposure, and
respiratory minute volume (0.65 ml/min/g for a rat) (Guyton, 1947).
-------
TABLE 2. Percentage of total recovered radioactivity, found in_organs, feces, urine and
expired C02 of individual rats receiving [T-l^C-ethvJJ naled}-.
Percentages of total recovered, route
Compartment
Organs and
Tissue
Feces
Urine
Expired C02
Xylene
Inhalation
Whole body
6.47
0.59
39.80
53.14
Diesel Oil
Inhalation
Whole body
23.08
2.62
29.76
44.53
Head only
35.96
2.90
24.54
36.58
Oral ip
Rat 1
31.08
1.64
19.91
47.37
Rat 2
35.67
3.46
15.88
44.98
Rat 1
34.40
3.80
15.22
46.57
Rat 2
40.53
1.94
17.16
40.38
See text for information regarding aerosol particle size, levels of pesticide administered
or aerosol concentration and duration of exposure.
-------
TABLE 3. Deposition and early distribution of inhaled [1-^C-ethyl] naled,
Tissue
or
Organ
Head
Lung
Trachea
Stomach
Esophagus
Duodenum
Percentage deposition with 9570 confidence intervals
xylene formulation diesel oil formulation
0
0
o
0
0
.96
.09
.03
.15
.04
~
± °
+ 0
± °
t °
± °
~ ~
.66
.04
.04
.14
.03
0
0
0
0
0
--
.37 +
.10 +
.50 +
.23 +
.71 +
0.
0.
0.
0.
0.
14
03
30
32
49
TOTAL
1.27 + 0.89
1.90 + 1.05
-------
APPENDIX
STUDIES OF EFFECTS OF PARTICLE SIZE
ON THE TOXICITY OF INSECTICIDE AEROSOLS
26
-------
Studies of Effects of Particle Size on the Toxicity of Insecticide
Aerosols
Summary and Principal Accomplishments
The purpose of this project was to determine whether the in-
halation toxicity of four selected pesticides was greater for animals
than the oral toxicity, and whether particle size played an important
role in the toxicity. Pesticides studied were chlorpyrifos, malathion,
naled and resmethrin.
To accomplish these purposes, we did the following:
1. Modified a Henderson chamber to allow effective exposure
of animals to small-particle aerosols of pesticides in
the 0.8 ym to 5 ym range.
2. Conducted 193 aerosol exposures of either rats or mice.
3. Measured the retention of aerosols in rats and mice of
the particle size being used.
4. Determined acute inhalation LCtSO values, calculated
inhalation LD50 values in mg/kg body weight for
two pesticides with small-particle aerosols, and found the
two other pesticides were not toxic enough to establish
inhalation LD50's under feasible exposure conditions.
To determine these values, use was made of the aerosol
concentration in air, time of exposure, respiratory minute
volume of the animal and retention and early distribution
of a tracer aerosol of comparable particle size.
5. Devised and built an exposure chamber, and a dispenser
to allow tests to be conducted with large-particle aero-
sols (13pm - 20 ym diameter). Ancillary to these tests,
perfected a method for measuring this size range, and
produced inexpensive holding units for head-only exposure
of rats.
6. Conducted tests with the above-mentioned aerosol to show
that large particles (18 y m - 20 ym) were less toxic than
small particles in the case of one pesticide.
7. Provided supplementary data to show that there is pro-
bably a concentration-per-particle effect that can influ-
ence calculated inhalation LD50 values; and that particles
in the 2 ym - 5 y m range may be about as toxic as particles
less than 2 ym.
8. Conducted tests on exposed animals to correlate dosages
with extent of changes of certain biochemical parameters,
such as plasma cholinesterase depression and changes in
whole blood serotonin or glutathione.
27
-------
TABLE OF CONTENTS
DD Form 1473, Report Documentation
Title Page- .,...,..,.,.-, , .,.,.... 1
Summary and Principal Accomplishments ..,....,.. 2
Table of Contents '."-..; 1 3
List of Appendices 4
List of Figures and Tables 6
Foreword 8
Materials and Methods 9
1. Animal exposure techniques 9
1.1 Inhalation exposure 9
1.1.1 Small particles (0.8-5 ym) 3 ym HMD . .... .9
1.1.2 Medium sized particles (ca. 8.0 ym MMD) ... .9
1.1.3 Large particles (13-20 ym MMD) 12
1.2 Oral exposure 12
1.3 Intraperitoneal exposure. 12
2. Parameters of toxicity 12
2.1 Mortality and visible signs 12
2.2 Biochemical parameters 13
2.2.1 Cholinesterase determinations 13
2.2.2 Serotonin determinations 13
2.2.3 Glutathione determinations- • - .13
3. Particle size determinations 13
4. Determination of whole-body retention of aerosols • -14
5. Miscellaneous determinations 14
Results 15
1. Retention of small particle aerosols in animals . . . 15
2. Toxicity data 15
28
-------
2.1 Mortality ...,.,.,.-, 15
2.1,1 Comparison of oral and inhalation administration 15
2.1.2 Effect of particle size and concentration on
toxicity • < 18
2.2 Biochemical parameters of toxicity 25
2.2.1 Cholinesterase depression 25
2.2.2 Whole blood serotonin levels 28
2.2.3 Whole blood glutathione levels . . 28
3. Miscellaneous determinations 32
3.1 Comparison of military and industrial formulations 32
3.2 Electric charge on particle deposition 32
3.3 Effect of xylene on toxicity of chlorpyrifos • • 32
Discussion .35
Conclusions 41
Acknowledgment 43
Literature Cited 44
APPENDICES:
Appendix I Small Particle Aerosol Exposure .48
1. Generation of small particle aerosols
(0.8-5.0 ym) 3 ym MMD ........ .48
2, Exposure to small particle aerosols • .48
3. Concentration and dosage measurements -50
4. Particle size measurement • • • •' • • -51
Appendix II Large Particle Aerosol Exposure 54
1. Generation of large particle aerosols
(13 to 20 ym MMD) .54
2. Exposure to large particle aerosols. . 55
3. Concentration and dosage measurements. 59
4. Particle size measurements. ..... .61
Appendix III Plasma Cholinesterase Determinations 63
Appendix IV Whole Blood Serotonin Determination 66
Appendix V Whole Blood Glutathione Determination 67
Appendix VI Determination of Whole Body Retention of Aerosols
68
29
-------
Appendix VII Miscellaneous Determinations • • • -..••• • • -73
1. Analysis for chlorpyrifos 73
2. Comparison of formulations 73
3. Partial dispersion of aerosol charge • -73
4. Effect of xylene on the toxicity of
chlorpyrifos ?74
5, Particle screening 74
Distribution List 75
30
-------
TABLES AND FIGURES
Table Page
1 Commercial formulations of insecticides used
in this study 10
2 Mean percentage, with 9570 confidence intervals, of
retention in various tissues of total calculated inhaled
aerosol mass in mice and rats 16
3 Conditions of inhalation exposure of mice and rats
to aerosols of four insecticide formulations . . . . 17
4 Inhalation and oral toxicities of four pesticides
to female mice and rats 23
5 Effect of particle size on mortality and plasma
cholinesterase upon groups of eight female rats
exposed head only to naled aerosols . . .24
6 The effect of xylene on the inhalation toxicity of
chlorpyrifos to mice 33
7 Effect on plasma cholinesterase after exposure of .64
mice to chlorpyrifos aerosol (particle size HMD 2 um)
8 Effect on plasma cholinesterase after exposure of
rats to naled aerosol (particle size HMD 18-20 um). .65
9 Percentage retention in various organs of total
calculated inhaled aerosol in female mice • • . . . .71
10 Percentage retention in various organs of total
calculated inhaled aerosol in female rats • 72
Figure
1 Chemical structures of insecticides under study . . .11
2 Dose response curves relating to oral administration
of four insecticides to female mice. 19
3 Dose response curves relating to inhalation admin-
istration of two insecticides to female mice . . . .20
4 Dose response curves relating to inhalation admin-
istration of three insecticides to female rats ... 21
5 Dose response curves relating to oral administration
of two insecticides to female rats 22
31
-------
Figure
6 Relationship between plasma cholinesterase depression
and inhalation dose of three insecticides (female mice) 26
7 Plasma cholinesterase recovery in mice following
inhalation administration of a given dose of three
insecticides , .27
8 Blood serotonin levels following inhalation exposure
of rats to a given dose of naled and to a soya-bean
oil control. 29
9 Blood serotonin levels following inhalation exposure
of rats to given doses of resmethrin aerosol and its
adjuvant panasol 30
10 Blood serotonin levels following inhalation exposure
of rats to given doses of chlorpyrifos and malathion
aerosols 31
11 Schematic of Renderson-type chamber ... 49
12 Size distribution of aerosols from Wells atomizer,
10% Dibrom in soya-bean oil.. . 53
13 Conceptual sketch of spinning disk atomizer ..... 56
14 Conceptual sketch of modified exposure chamber. ... 58
15 Size distribution of aerosols from spinning disk and
from atomizer 60
32
-------
FOREWORD
The U,S, Army is considering the use of ultra-low volume (ULV)
sprayers in the place of conventional foggers for several pesticide
application programs. The impact which smaller, more concentrated'
pesticide aerosols have on non-target organisms (including man) must
be considered for each potential application. This study was initia-
ted to provide information from which the application of ULV tech-
niques for specific pesticides could be determined. The primary .
objective was to determine the relative toxicological properties of
four insecticides when test animals*were exposed to "respirable" and
"ncn-respirable" aerosols. Secondary objectives were to compare the
toxicological properties of these pesticides after animals had been
exposed by different routes (principally oral and inhalation) and to
attempt to define the mechanisms that might have caused differences
in the resulting data.
Assessment of toxicological hazards to non-target organisms
when pesticides are applied in the field has largely been based upon
reported information on the LD50 values for these pesticides. These
values are available for almost all pesticide chemicals based upon
the toxicity when animals received oral doses. Such values may have
been used by regulatory agencies to assess the hazard to field workers.
However, the exposure of such persons must largely be by the inhala-
tion route; thus, there is need to know the true inhalation dose that
such an individual might receive. Inhalation toxicity is convention-
ally expressed as a Cxt value or LC50 x t, i.e., the lethal concentration
(c) of the chemical in the air times the time (t) of exposure. Such
a term is largely empirical and is of little use to enable valid com-
parisons to be made with reported values of oral, intraperitoneal,
intravenous, etc., toxicity where the animals can be assumed to have .
received all the amount of chemical administered.
In this report, therefore, we have expanded the term "LD50" to
include measures of inhalation toxicity expressed as mg/kg body-weight.
Such an expression is in keeping with the terminology encountered in
some recent toxicological reports (e.g., McFarland, 1975; Hoben, et
al., 1976). To enable such an expression to be made, in addition to
tKe Ct value, we must also know the respiratory minute volume of the
animal, preferably under the conditions of exposure and also the retention
and early distribution of the inhaled air or aerosol in the whole body.
For respiratory minute volumes we have used the values reported
by Guyton (1947) which were allegedly measured under resting condi-
tions. However, in order to perform such measurements, the animal
must be placed in a plethysmograph which, by its very restrictive
nature, induces considerable stress. It is therefore our opinion
that the values reported by Guyton are accurate enough to be appli-
cable to our conditions of exposure.
~
In conducting the research described in this report, the investi-
gators adhered to the "Guide for Laboratory Animal Facilities and Care"
as promulgated by the Committee on the Guide for Laboratory Animal
Resources, National Academy of Sciences - National Research Council.
33
-------
For retention values (of the total inhaled) we have, used our
ovm experimental data based upon the levels of an inert radio-
labeled tracer found in body organs of animals after they have
inhaled measured levels of the aerosol. The tracer was formula-
ted in vegetable oil, used as a vehicle for many of our exposures.
MATERIALS AND METHODS
For these studies four insecticides of current importance to
the U.S. Army for field studies were examined in laboratory
animals. These chemicals were the three organophosphorus insecti-
cides, chlorpyrifos (Dursbai^ , malathion and naled (Dibror/e}>,
and the pyrethroid resmethrin*. Details of the formulations are
given in Table 1. Chemical structures are shown in Figure 1.
1. Animaj._ exposure techniques
All exposures reported were performed with female mice
(NAMRU strain, 20-30 g weight) or female Sprague Dawley
rats (250-350 g),
1.1 Inhalation exposure
1.1.1. Small particles (0.8-5 urn) 3 ym HMD
Small particle aerosols were generated from formu-
lations of pesticide chemica'ls dispersed from
two refluxing Wells atomizers operated in parallel.
The aerosols were conducted into a rectangular
chamber (Henderson, 1952) containing 8-16 female
mice or 8 female rats. Doses were varied by vary-
ing the exposure time. Details of the exposure
methods and calculation of the dose are given in
Appendix 1.
1.1.2. Medium sized particles (ca. 8.0 urn HMD)
Attempts to generate aerosols of this particle size
range were without success. Further details of
these .attempts are given in Appendix 2.
* When referring to the insecticide chemical, generic names
will henceforth be used throughout the text. Certain formulations
may be identified by the use of trade names (e.g., Dibrom 14 concen-
trate) . Use of such names does not imply endorsement of the pro-
ducts by either the sponsoring agencies or by the University of
California.
-------
Table 1. Commercial formulations of insecticides used in this study
Pesticide
chemical
Formulation
adj uvant
Trade Name
Concentration of
active ingredient'
Chlorpyrifos
malathion
xylene
'inert ingredients'
Dow Mosquito
Fogging Concentrate
(Dursban®)
malathion
65% w/w
957o technical grade
rialed
aromatic hydrocarbons
Dibrom 14
concentrate
87% w/w
oo
VJ1
r.esmethrin
Panasol AN 2
SBP - 1382
40% w/w
Concentrations vary slightly with each batch formulated.
-------
^i
Cl
chlorpyrifos
OJ
cr\
P-O-C-
Br
noled
Ci
C-C!
I •
CH«
Figure 1. Chemical structures of insecticides under study
-------
1.1.3 Large particles ( IS - 2.0 ym MMD)
A modification of the May (1949) air-driven spinning
top was used to generate aerosols in this particular
size range. However, to obtain sufficient aerosol
concentration, a larger, electrically-driven spin-
ning disk was used. The particles were allowed to
descend under a lightly generated air flow to the
animals (8 rats) placed in head-only exposure hold-
ers. Details of the generation of these aerosols
and method of exposure are also given in Appendix 2.
1.2 Oral exposure
The procedure was essentially the same as that described
by Gaines (1968), Mice were sized so that groups of 10
or 16 did not vary by more than 1 g in weight. In the
case of rats a weight variation of 10 g or less was
acceptable for animals of 250 g or over. Solutions of
the pesticide were prepared in vegetable oil (soya-bean
oil or peanut oil) such that 0.5 ml or 0.1 ml contained
the desired dosage. The solution was administered by
oral intubation using a blunt-edged syringe needle.
Mortality was observed over a 14-day post-exposure period.
1,3 Intraperitoneal exposure
In order for a valid comparison of other routes of admin-
•istration with the inhalation route to be made, an intra-
peritoneal LD50 was obtained by injecting various doses
of Dibrom 14 concentrate diluted in 0.5 ml of 1,2-propylene
glycol into the peritoneal cavity. Five dose levels were
used, the maximum concentration being 2.75 w/v. Mortality
was normally rapid after we injected the animals; however,
surviving animals were held for observation for 14 days.
2, Parameters of toxicity
2.1 Mortality and visible signs
After animals had been exposed for selected times to
measured aerosol masses, the number of dead animals was
noted and live animals were housed for 14 days to deter-
mine delayed mortality. A similar follow-up was made
when oral and intraperitoneal routes were tested. The
LD50 values and 9570 confidence intervals for all routes
of administration were determined by plotting the per-
centage mortality against dose and using the statistical
37
-------
method of Litchfield and Wilcoxon (1949).
In the case, of the organophosphorus insecticides, symp-
toms signifying eholinergic effects were noted.
2•^ Biochemical parameters
2.2.1 C ho lines terajBe determinations
Plasma cholinesterase determinations were made on
mice or rats exposed to organophosphorus insecti-
cides. Blood was obtained from the eye (mouse) or
tail (mouse or rat) for determination before and
after exposure to the insecticide aerosols. The
method used was essentially that of Wolfsie and
Winter (1952) , which is a microadaptation of the
potentiometric method of Michel (1949). Details
of the method of determination are given in Appendix
3, The ratio of ApH/hr values after and before
exposure, multiplied by 100, gives a figure which
is indicative of cholinesterase depression, and is
expressed as cholinesterase percentage of pre- . ..
exposure value. A figure of 80% or less was con-
sidered significant evidence of eholinergic activity,
2.2.2 Serotonin determinations
Serotonin elevation in whole blood has been reported
to be elevated in animals exposed to certain pesti-
cides (e.g., Shilina, 1973). The method of deter-
mining the level in whole blood of rats or mice,
before and after inhalation exposure to the insecti-
cide aerosol, is essentially the modification by
Krueger et al. (1963) of the method of Udenfriend
et a_l. (r955y. Details are given in Appendix 4.
2.2.3 Glutathione determinations
Attempts were made to measure the effect of insecti-
cide aerosol exposure on whole blood glutathione in
mice or rats. Details are given in Appendix 5.
3 . Par tide Size Determinations:
With the small particle aerosols (3 urn HMD) the six stage
Andersen sampler,, operated at 1 ft3/min and backed by a
0.8 urn Millipore" filter after the final stage, proved use-
ful for obtaining the particle size distribution. For
larger particles the sampler was modified and calibrated
. for use at a lower flow rate. In Appendix 6 we have dis-
cussed the use of the sampler for various particle size
aerosols.
Millipore Corporation, Bedford, Massachusetts
38
-------
4 . Determination of who lebod y retention of aerosol s
As explained in the Introduction section, in order to
determine the true inhalation dose of a chemical (and hence
calculate an inhalation LD50 value in tng/kg body-weight)
it is necessary to know, among other terms, the percentage
of inhaled aerosol which is retained in various organs
of the animal. To make these measurements, mice or rats
were exposed in a rectangular Henderson chamber to a
typical formulation vehicle (soya-bean oil) containing
a small amount of a relatively inert radio-labeled tracer
(1-l^C-heptadecane) . Details of the exposure method
and calculation of the retention in the organs and tissues
examined are given in Appendix 6.
5 . Miscellaneous determinations
Because of the varied nature of the experimental work
described above, a number of observations were made
which encouraged us to perform several ancillary experi-
ments, some of which provide a peripheral, although
interesting, corollary to this study. Among these were
(a) modifying methods of analysis sometimes used to deter-
mine the purity or the concentration of pesticides in
aerosols; (b) the effect of a radioactive source (krypton")
on the dispersion of an electrical charge on an aerosol;
(c) the effect of xylene on the inhalation toxicity of
chlorpyrifos, and (d) the effect of "stripping" of the
particles above a certain size in the small particle
aerosols. Details of the experimental procedures involved
in these studies are given in Appendix 7.
39
-------
RESULTS
1. Retention of small particle aerosols in animal;?
Table 2 summarizes the retention levels of small
particle aerosols of soya-bean oil in various organs in
mice and rats. Although the majority (84.1%) of the
calculated inhaled material was retained in the' mouse,
only 28.37, was recovered in the rat. The significance
of this difference between the. two species is discussed
below.
2. Toxicity data
2.1 Mortality
2.1.1 Comparison of oral and inhalation administration
In Table 3 , the general conditions of exposure to
animals in the Henderson chamber are given. For
acute exposure, times in excess of two hours were
generally avoided, except when there was no indi-
cation of mortality, as in the case of malathion.
In all these exposures in the Henderson chamber
the aerosol concentrations were quite high (4-8
mg/1) but below those at which appreciable particle
coagulation might be expected to occur.
Table 4 lists the oral and inhalation toxicity
values obtained from all insecticides when animals
were exposed to given formulations of the insecti-
cides of MMD about 2 ym. The obvious increase in
toxicity of naled to rats when administered by the
inhalation route as contrasted to the oral route
is discussed below. Because we were unable to kill
mice with malathion or resmethrin aerosols, we
did not attempt to expose rats to these pesticides.
There was evidence that the adjuvant .used in the
standard formulation (4070 resmethrin in Panasol --
an alkyl naphthalene compound) was more toxic than
the insecticidal ingredient. Nine of 10 mice ex-
posed for five hours to an. aerosol of 4070. material
died (total aerosol concentration: 5.92 mg/1.) where-
as 16 out: of 16 mice died when exposed for five
hours to an aerosol of pure Panasol (aerosol con-
centration 6.13 mg/1). Attempts were made to formu-
late resmethrin in solvents other than Panasol
(xylene, 1,2-propylene glycol or soya-bean oil) but
the compounds were either insufficiently soluble or
the solvent was too toxic to be useful.
-------
Table 2 .
Mean percentage, with 95% confidence intervals, of retention in various
o
tissues of total calculated inhaled aerosol mass in mice and rats
Exposure method
Tissue
Head
Lung
Trachea
Stomach
Esophagus
Duodenum
Total
Mouse '
Whole body Head only
9.1 + 3.4
3.8 + 0.46 4.6 + 0.86
0.87+ 0.91 1.2 + 0.91
59 +30 40 + 13
4.7 + 9.7 1.4 + 0.75
7.0 + 5.8
84+22 47 + 13d
b,c
Rat
Whole body
3.1+0.1
9.9 + 2.7
0.16+ 0.07
13 + 7.7
0.39+ 0.47
1.9 + 1.3
28 + 9.3
aMass median diameter, 2.1 pn.
Females only.
CEight animals per group exposed.
Concentrations in head and duodenum were not determined.
-------
TABLE 3.
CONDITIONS OF INHALATION EXPOSURE OF MICE AND RATS.
TO AEROSOLS3 OF FOUR INSECTICIDE FORMULATIONS
Number of
Duration
Aerosol
V
!v
/ •
j.
Pesticide
Species Chemical
Mouse chlorpyrifos
Mouse nialathion
Mouse naled
Mouse resmethrin
Hat chlorpyrifos
' 'E.at naled
" Sir.all particle aerosols
Formulation
65% in xylene
95% technical grade
10 or 20% Dibrom 14
cone, in soya-bean
40% in xylene
65% in xylene
10% Dibro;?. 14 cone.
in soya-bean oil
exposure to No. of animals of exposure Concentration
determine LD^Q ' per exposure (range) (irdn) (range) (mg/1)
6 16 27 - 50 6.6 - 7.9
1 16 300 6.9
8 10 72-111 5.6-6.1
oil
2 16 96 - 177 6.5 - 6.8
5 8 60 - ISO 5.9 - 7.5
5 8 .48-61 4.7-5.5
only (MMD - 2 ym) - see Table 5.
C\]
-=r
-------
Figures 2-5 are typical dose-response data resulting
from the administration of these insecticide formula-
tions to mice or rats by either the oral or inhala-
tion route.
For our own information, a limited number of tissue
samples of animals exposed to naled were collected
to assess the possibility of correlating toxicity
with pathological lesions. Primary lesions in ex-
posed animals were those of pulmonary congestion
and serous edema. Pulmonary lesions were most
severe in animals exposed to naled dissolved in
organic solvents, but were not readily distinguish-
able from those lungs of animals exposed to the
organic solvent alone.
Congestion and edema appeared less severe in animals
exposed to naled dissolved in soya-bean oil but
appeared to increase in severity with the increase
in concentration of naled in the soya-bean oil.
Further experiments would be required to verify
fully this relationship.
2.1.2 Effect of particle size and concentration on toxicity
The inhalation LD50 for rats exposed to small parti-
cle aerosols (MMD 2.1 ym) of naled, formulated as
107o W/Y of Dibrom 14 concentrate in soya-bean oil,
was 7.7 mg/kg (7.2 - 8.4) (Table 4 ). The inhala-
tion LD50 for rats exposed to small particle aero-
sols (MMD 2.1 ym) of undiluted Dibrom 14 concentrate
was 3.1 mg/kg (2.5 - 4.0). The purpose of obtaining
the latter value was to compare, on a more equal
basis, changes in the LD50 value for particles
13 - 20 ym in diameter, where we had to use the
Dibrom 14 concentrate to obtain any death of exposed
rats. That value was >12.4 mg/kg (25% mortality
at this level; Table 5 ). Thus, large particles of
the concentrate must be about half as toxic (mg/kg
basis) as small particles of the 10% formulation,
but about 4 times less toxic than small particles
of the concentrate. One attempt was made to generate
a large particle aerosol with 65% chlorpyrifos in
xylene (Dow Mosquito Fogging Agent); no rats died
following 147 min exposure to a concentration of
0.45 mg/1 (estimated dose 12 . 2 mg/kg) ; however these
particles were smaller (MMD 8.0 ym) than aerosols
from naled.
-------
99-1
90 i
o
70 -
o
-| 50
QJ
O_
30 -
10 -
1 -
10
Dose response curves relating to oral administration of four
insecticides to female mice.
•chiorpyrifos
naled
malathion
/ jb resmethrin
Mr-^'
50 100 500 1,000
Dose, (mg/lcg)
5,000 10,000
Figure 2.
-------
4=-
VJ1
Dose response curves relating to inhalation administration of two
insecticides to female mice.
99 n
90-
70-1
|r 50
03
fe 30
o>
en
ra
•*—*
a
o>
o
10 -
1 -
10
naled
chlorpyrifos
50 100 500 1,000
Dose(mg/kg)
Figure 3.
-------
99 i
Dose response curves relating to inhalation administration of three
insecticides to female rats.
ON
CD
CD
£
CD
Q-
90
70
50 4
30
10
naled
dichlorvos
chlorpyrifos
1
0
10
50 100
Dose, (mg/kg)
500 1,000
Figure 4.
-------
Dose response curves relating to oral administration of two
insecticides to female rats.
99-i
90-
Ifyo
50 H
CD
CT
03
0130
OL>
O
D_
10.
1 -
10
chlorpyrifos
50 100
Dose, (mg/kg)
500 1000
Figure 5.
-------
TABLE '».
INHALATION' AND ORAL TOXICITIES OF FOUR PESTICIDES TO FEMALE MICE AND RATS
Toxicity (with 95% confidence intervals)
:o
Species
Mouse
Mouse
Mouse
Mouse
Rat
Rat
Pesticide3
Chemical
chlorpyrif os
malathion
naled
resnethrin0
chlorpyrifos
naledd'e
Inhalation
LD50
rag /kg
257(228-291)
>2080
156(141-174)
272-665
135 (98-186)
7. 7f (7.2-8
toxicityb
LCt-.Q
mg .min/m
2450 (2170-2760)
19,700
1480 (1340-1660)
2580-6330
7340 (5330-10,000)
.4) 419 (391-457)
Oral toxicity
Potency ratio
LD5Q Oral LD50 / Inhal. LD5Q
mg/kg
152 (143-162)
1680 (1631-1730)
222 (209-235)
1390 (1135-1703)
169 (146-196)
160 (131-195)
0.59
'
1.42
-
1.25
20.8
a See Table 3 for formulations used in inhalation exposures: For oral toxicity determinations the technical
grade pesticide cheTdcal was formulated in peanut oil.
b Based upon minute volumes of 1.25 ml/min/g (mouse) or 0.65 ml/min/g (rat) (Guyton, 1947).
c The toxicity of xylene in which resmethrin was formulated made an accurate determination, of the inhalation
LD5Q impossible (see text).
« Dibrom 14 concentrate (87% naled in aromatic hydrocarbons) when used alone appeared to be more toxic (see
Table 5).
e Intraperitoneal LD5Q 35.0 mg/kg (31.8 - 38.5).
f The inhalation LD5Q of aerosols of the metabolite dichlorvos (using 10% Vapona '•£ in soya-bean oil) was
4.03 tng/kg (3.35 - 4.53) for the rat. LCt50 - 219 (182-263).
-------
TABLE 5.
EFFECT OF PARTICLE SIZE ON MORTALITY AND PLASMA CHOLINESTERASE UPON GROUPS OF EIGHT
FEMALE RATS EXPOSED HEAD ONLY TO NALED AEROSOLS3
VD
Dose
mg/kg
2.36
3.71
5.16
10.10
12.22
12 . 35
Duration of Mean aerosol
exposure Concentration MMD Percentage
min mg/1 yin mortality
12
15
21
65
61
60
1.24
1.56
1.55
0.98
1.26
1.29
2.1
2.1
2.1
13C
18-20°
18-20°
12.5
87.5
87.5
25
25
25
plasma cholinesterase
Percentage of pre-exposure value
Mean Standard deviation
69
73
42
48
55
45
15
6.0
8.0
5.3
11
13
a Dibroml4 concentrate; 86.2% naled in aromatic hydrocarbons
k Based upon 28.3% whole body retention and a breathing rate of 0.65 ml/min/g
(Guyton, 1947). .
c Approximately 7% of the particles were below 5 urn in diameter
-------
For our own purpor.es, limited experiment's with
small particle nn-osols °f nalocl, from which a
fraction of larger particles had been removed
(screened aerosols), were conducted. When rats
were exposed to aerosols of 1070 w/v Dibrom 14
concentrate in soya-bean oil (where particles
above either 2 ym or 3 ym were removed) for the
same length of time that caused death of rats
when no particles were removed, no rats died.
When the exposure was continued for twice that
time (essentially doubling the dosage) no rats
died during or immediately after the exposure,
but the animals became markedly agitated, and
fought so intensely that it was necessary to
house each of them in individual cages. Two
days later all rats were dead.
Again, for our own purpose, rats were exposed to
aerosols of 2070 w/v of Dibroin 14 concentrate from
which particles above 2 vim had been removed. In
this test, a measured dosage of 7.0 mg/kg killed
62.5% of the animals -- a figure comparable to
the LD50 of 7.7 mg/kg, with the 10% formulation
and "unscreened" aerosols. These data provide
some evidence that particles 2 - 5 j.tm are about
as toxic, on a mass basis, as particles 2 ym and
less, and that small differences in the mass dis-
tribution of particle sizes in these ranges would
not be expected to significantly influence toxicity
measurements.
2.2 Biochemical parameters of toxicity
2.2.1 Cholinesterase depression
Plasma cholinesterase depression in mice was most
marked after inhalation of aerosols of chlorpyrifos,
less marked in the case of naled, and of doubtful
significance with malathion. The effect of particle
size on mortality incidence and plasma cholinester-
ase depression when rats are exposed to naled is
given in Table 5 . Figure 6 shows the relationship
of cholinesterase depression to dosage (mg/kg) after
mice were exposed to the three organophosphorus
insecticides under study; recovery of cholinesterase
from mice exposed to given doses of each of the
insecticides is indicated in Figure 7.
. 50
-------
Relationship between plasma cholinesterase depression and inhalation dose
of three insecticides (female mice).
S 100
fO
CD
to
o
o.
X
CD
I
O
<_
Q.
v—
O
CD
cn
03
CD
O
i_
CD
Q.
CD"
(/i
03
V-
CD
^—'
t/1
CD
90-
80
70
60
50
40
30
20
10
0.1
naled
maSathion
0.5 1
5 10
Dose mg/kg
50 100
500 1000
Figure 6.
-------
Plasma choline-.ierase rcco.^y in liiice following imi. 1 .
\
9
i
10
I
11
1 •
12
i
13
1
14
52
-------
2.2.2 Whole blood serotonin levels^
The effect of exposure of rats to either control
or to insecticide aerosols upon whole blood sero-
tonin levels are given in Figures 8 - 10 . These
graphs also show the recovery times until return
to pre-exposure values. Only chlorpyrifos and
soya-bean oil alone appeared to produce no effect
upon serotonin levels. With naled, a biphasic
response was evident, and with malathion and res-
methrin, exposure clearly elicited an increase in
whole blood serotonin.
Levels of serotonin in rats exposed
to resmethrin are complicated by the fact that its
adjuvant, Panasol, also affected the serotonin levels
Although Panasol appears to be more toxic than
resmethrin to rodents, elevation of serotonin
levels was far more prolonged when resmethrin was
present in the formulation. The results of this
work have already been reported (Berteau and Deen,
1976). Attempts to obtain meaningful data on
whole blood serotonin levels in mice exposed to
insecticide aerosols were precluded because of
the extreme variation in pre-exposure levels in the
strain used (NAMRU).
2.2.3. Whole blood glutathione' levels
In a typical run with eight female mice, mean and
957o confidence intervals for whole blood gluta-
thione (tail vein incision) was 2.72 + 0.45 mM
SH~/1; the same values in a run with rats was more
variable vis. 3.74 + 1.43 mH SH~/1. Inhalation
exposure of mice to both naled aerosol (207=, w/v
Dibrom 14 concentrate in soya-bean oil; dose 56.0
mg/kg) or to chlorpyrifos aerosol (6570 w/v chlor-
pyrifos in xylene; dose 325 mg/kg) produced no
significant change in blood glutathione. However,
after we exposed rats to naled aerosol (1070 w/v
Dibrom 14 concentrate in soya-bean oil; dose 4.76
mg/kg) there was a slightly significant (p <0.05;
p >0.02) decrease in blood glutathione. These
three runs provided the only meaningful data that
we generated in our studies with glutathione. In
general the resxilts were so erratic that further
studies were not made. The pesticide exposure
seemed to produce some obscuration of the color
produced by the reagent.
53
-------
7-,
"CT 6-
p
<5 A^
LoJ
Q
O
o -
3
o )
« z-ii
o
1-*
naled (6.9 mg/kg)
i;
Tf"~'
11
1
1
T
•-1C
i
control
02468 1012141618202224 1
HOURS
2 3
WEEKS
BLOOD SEROTONIN LEVELS FOLLOWING INHALATION EXPOSURE
OF RATS TO A GIVEN DOSE OF NALED AND TO A SOYA BEAN OIL
CONTROL
Figure 8.
-------
VJ1
7n
6-
LU
O
o
3J
4'
24
QQ
LU
O 1 „.
resmethrin (48 mg/kg)
Panasol (115 mg/kg)
T T
*I i I r i
02468 1012141618202224 ' 1
HOURS
2 3
WEEKS
BLOOD SEROTONIN LEVELS FOLLOWING INHALATION EXPOSURE
OF RATS TO GIVEN DOSES OF RESMETHRIN AEROSOL AND ITS
ADJUVANT PANASOL
Figure 9.
-------
Ul
CTv
"5s
3.
py
O
LlJ
a
o
q
CO
J_l
o
§i
6-
r-
3 -
4 It
3J
2-
1
1-
i
^
"^-^
™ ^ ^
^*v^
^
_T- "--?
• • ! .
9
\ /^U ]~
i en ic
-S--J
.j i
i
S m
•i
f
02468 1012141618202224
chlorpyrifos (52 mg/kg)
malathion (93 mg/kg)
1
HOURS WEEKS
BLOOD SEROTONIN LEVELS FOLLOWING INHALATION
EXPOSURE OF RATS TO GIVEN DOSES OF CHLORPYRIFOS
AND MALATHION AEROSOLS.
Figure 10.
-------
3. Miscellaneous determinations
3.1 Coroparison of military and industrial formulations.
Based upon the gas chrornatographic tracings, the military
and industrial formulations of chlorpyrifos and malathion
were identical. In the case of naled there appeared to
be some difference in the solvent used but the concen-
tration of active ingredient was approximately the same.
The formulations of resmethrin are entirely different
and the military formulation appeared to contain less
of the active ingredient. We have been given to under-
stand (Larson, personal communication) that a standard
military formula exists only for malathion and this
formulation has shov/n to be the same as that supplied
by industry and is almost pure material.
3.2 Electric charge on particle deposition
A high voltage (2000 volts) applied between two aluminum
disks produced a significant increase in deposition of
a soya-bean aerosol on the disks; this deposition was
decreased when a 85krypton source was inserted in the
line. The sum of the weights of aerosol deposited on
all disks (less that deposited when no charge was applied)
with and without the radioactive material indicated
that the efficiency of dispersion of charge is about 3870.
The relevance of these results to actual field conditions
is doubtful because of the excessively high voltage ap-
plied; however, the effect of electrical charge is too
small to be a severe limitation for defining toxicological
properties expected under field conditions.
3.3 Effect of xylene on toxicity of chlorpyrifos
Table 6 gives the results of the experiment to determine
if there is an effect of xylene on the toxicity of chlor-
pyrifos. Although there was some increase in particle
size following atomization of the formulations containing
xylene, the range of MMD's for all exposures where xylene con-
centration was increased was 1.0 - 3.0 ym. Based upon
the tabulated results, as the concentration of xylene
was increased (at the expense of chlorpyrifos) there was
some evidence of increased toxicity contrasted with the
same dose of chlorpyrifos that we achieved by increasing
the exposure time. Although there was no ultimate mor-
tality after two-hour exposure of mice to pure xylene,
the animals were obviously under very considerable stress
and their breathing appeared difficult. Longer exposure
57
-------
TA1U.E 6.
THE EFFECT OF XXLENE ON HIE INHALATION TOXIC1TY OF CHLORPYRIFOS TO MICE.
Cone, of
Chlorpyrif os
7,
63
63
63
63
63
63
63
63
31.5
16
6
Cone, of
xylene
%
11
11
18.5
26
37
37
37
37
68.5
84
94
Cone, of
Soya-bean
oil %
26
26
18.5
11
0
0
0
0
0
0
0
Exposure
time
min.
120
90
120
120*
120
90
60
50
97
120
60
Dose of Mean cholin-
chlorpyrifos ester ase % of
Mortality pre-exposure
mg/kg % value
561
283
513
530
743
555
426
289
583
418
93
25
12.5
62.5
25
87.5
62.5
37.5
0
12.5
87.5
0
11.5
10.5
11.0
11.5
14.6
10.8
11.5
11.1
12.9
10.7
. —
*0ne atomizer cut out after about 100 minutes.
58
-------
time to pure xylene produced mortality; 13/16 mice
died after three hours exposure to atomized xylene
and 4/8 rats died in a similar three-hour exposure.
In such runs, the level of xylene actually encountered
in the aerosol form (based upon levels found in the
sampling filter) as distinct from vapor, varied con-
siderably due to the temperature in the exposure
chamber.
59
-------
DISCUSSION
Effective laboratory studies of the inhalation toxicity of
pesticide formulations dispersed as aerosols present certain pro-
blems not always present in inhalation studies of toxic vapors.
The. two most important factors are, first, that particles possess
inertial properties (functions of size and density of particles)
that limit the immediate transport of particles into the deeper
recesses of the lungs; the breathing rate and the structure of
the entire respiratory system,therefore, also limits transport.
Inertial properties also limit the amount deposited in the res-
piratory tree -- some inhaled particles are exhaled.
Deposition for the purpose of this report is considered to
be a transitory process. In most cases deposition must be mea-
sured by analysis of whole-body retention. Retention, however,
is a temporal process in that material deposited in the respira-
tory apparatus may be absorbed or swallowed and then excreted,
or it may be metabolized and lost as vapor, or the material may
be transported to other tissues where it may remain for an indeter-
minate period. The rate of this process is both species-dependent
and is distributed within the individuals of a species population.
In addition, irritability of the substance may influence breathing
rate, depth of deposition, etc.
A second factor is that, in practice, pesticides are formula-
ted in a carrier vehicle; there is a concentration-per-particle
effect that influences the absolute amount of pesticide deposited
at a given site. Obviously, part of this effect is influenced
by rate of solvent evaporation during aerosolization. It is not
unreasonable to suggest that, at the deposition site, there is some
innate capacity for the pesticide to be absorbed into the blood
stream, and that this rate of absorption could be modified by the
absolute amount of material at the site.
These problems of measurement of deposition, retention and
excretion of inhaled aerosols are well known (e.g., Hatch and Gross,
1964).. We have outlined them here to indicate that we are aware
of the difficulties and that we have contributed additional, un-
supported effort toward attempting to solve some of these problems
beyond the original scope of the work.
Retention values of materials in the respiratory tree have
been reported to vary from as low as 0.6370 in mice, using virus
suspensions in agar media (Young e.t_ a_l., 3974), to 5070 in man,
monkey and guinea pig (Hatch and Gross, 1964), or about 6070 in
man (Muir, 1972)- In one report a retention level of 20Z is given
based upon a mathematical model (Anon., 1966). It has been gener-
ally accepted that larger particles tend to be deposited in the
upper respiratory tract and eventually swallowed, thus entering
60
-------
the esophagus and stomach, Goldberg and Leif (1950), using a
particle size Mi-ID 1.2 yrn, exposed mice to an aerosol of 32p_iabeled
?as~.(iy:!:e.lAa. bL?J?Jr.i:! anc^ found that of the total material retained,
30'%'~wa"s~Tn the respiratory tree and 707, in the gastrointestinal
tract
Vie have found no reports on the retention of vegetable oil
aerosols in the body; such information should be of use in deter-
mining the extent of retention of formulations of insecticides,
and thus provide an indication of the hazard to the field workers.
In our work, as indicated in Table. 2 , when mice were exposed
"whole body" the majority of aerosolized material found its way
into the gastrointestinal tract (mean inhaled level in the stomach,
esophagus and duodenum, 70.47, out of a total of 84.1% recovered).
When the animals were prevented from grooming and the major part
of their bodies was protected from exposure to the aerosol, there
was a slightly significant (p_ = 0.02 - 0.05) reduction in retention
in the stomach; however, in this run, measurements of material in
the head and duodenum were not made. Thus, it must be assumed
that whereas grooming is responsible for some of the entry of the
material into the gastrointestinal tract, a very significant portion
of the inhaled material must have been temporarily deposited in
the upper respiratory tract and then swallowed; about 1070 of the
total aerosol retained was actually retained in the head, presumably
in the nares and nasal turbinates.
The most significant difference in retention patterns was' the
markedly lower retention (_p_= <0.001), the higher lung retention
Cp__<0.001), lower head retention (p_ <0.01) and lower gastrointes-
tinal retention (_p <0.001) in the rat contrasted to the mouse.
This might be explained by considering the initial sites of deposi-
tion of the aerosol. At the chamber aerosol concentration encount-
ered, ca. 4 x 106 particles /cm-^, some coagulation of particles
can be expected, and coagulation is markedly increased when an
aerosol is allowed to pass through an orifice (Dimmick, e_t al. ,
1975). With the mouse, the small diameter of the glottis (<0.5 mm).
may act as such an orifice, creating turbulence and causing particles
to coagulate and be immediately deposited in the trachea and even-
tually swallowed. With the rat, the diameter of the glottis is
larger (2,0 mm) j thus, the particles will be expected to coagulate
at a lesser extent, and more will find their way into the lung of
the rat. Further, we have estimated that the small diameter of
the mouse trachea, ca. 1.0 mm, and the small radii of curvature
before and after the glottal orifice, causes some particles of 1 ym
diameter and greater to be impacted in the trachea immediately pos-
terior to the glottis (230 cm/sec linear velocity), whereas the
respiratory tree of the rat (tracheal diameter 2-3 mm) allows an
increased passage of particles 1 ym or less (90 cm/sec linear vel-
ocity) . Only about 167o of the mass of aerosol particles were lym
or less but, as noted, 5070 were 2.1 ym or larger. For this reason,
61
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an approximate twofold correction could be applied to percentage
of lung retention but we have not chosen to do so because the abso-
lute values shown in Table 2 are the best values avail-
able for estimations of lung dosages under our experimental condi-
tions .
It should be noted that, with the work of Goldberg and Leif
(1950) the mean particle size was smaller (] iim) than the HMD re-
ported here and consequently, in our work, an increased portion
of the aerosol could be expected to impact onto nasal and tracheal
surfaces of the mouse, thus explaining, in part, the lower fract-
ional lung retention and higher head retention that we encountered.
The low total retention in the rats, as compared to mice, cannot
be explained on these grounds. It is possible that the respiratory
minute volumes under the conditions of exposure may be different
than the values determined by Guyton (194/).
It is also recognized that the high aerosol concentration in
the exposure chamber (4 - 5 mg/1) may have had some effect on the
breathing rate leading to minute volume data different from those
reported by Guyton (1947). However, in order for an animal to
survive, the oxygen intake must at least be basal, which for the
mouse is approximately half the resting value (basal, 1600 mnP of
02/g/hr;resting 3600 mm3 of 02/g/hr; Dittmer and Grebe, 1958). The
same fact would be true when pesticides are formulated in an oil
medium; here, a possible irritant action may alter the respiratory
minute volume, but again, the basal value for oxygen intake would
have to be exceeded. Since the oxygen intake is proportional to
the breathing rate in the same atmosphere, in computing pesticide
toxicity on the basis of the. retention values reported in Table
2, the maximal error in inhalation LD50 values, expressed
as milligrams per kilogram of body weight, would not be greater
than a factor of 2, and probably much less, because the animals
did not become hypoxic.
In relating the significance of these studies to the hazard to
man of exposure to toxic aerosols, it must be noted that, in the
case of man, the tracheal diameter will be much larger (20 - 27 mm;
Pappagianis, 1969) and thus, at the concentration examined, the
percentage retention in the lung may be considerably greater.
From the information reported in Table 4, naled is approximately
21 times more toxic to the rat by the inhalation route than by the
oral route. With chlorpyrifos there is only slight increase in
toxicity when inhalation exposure is employed and, in view of the
assumptions made in calculating inhalation toxicity values, it is
doubtful if this increase is significant; mice displayed far less
difference between oral and inhalation LD50 values. In fact,
chlorpyrifos appears to be more toxic to mice by the oral route
than the inhalation route. These facts may be explained by the
62
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observation discussed above, that with small-particle aerosols,
retention in the lung was higher in rats than in mice.
With the small-particle aerosols it is recognized that the
LD50 values include some input from absorption through the skin
and stomach because whole body exposure was used. However, the
toxic effects of such additional absorption would be minimally
significant due to the clearly more rapid effect of absorption
from the 'lung alveoli.
Because of the high inhalation toxicity of naled (but not
chlorpyrifos), factors other than rapid absorption into the blood-
stream were considered. For this reason, the intraperitoneal LD50
for rats to naled was determined. Because of the extensive vas-
cularity within the peritoneal cavity, absorption by this route
involved essentially instantaneous entry into the bloodstream.
Unfortunately, it was not possible to use the same solvent as was
used for inhalation studies. But the solvent used, 1,2-propylene
glycol, is known to have a low toxicity upon entering the blood-
stream. The ip LD50 value of naled to rats of 35 mg/kg given in
a foot note to Table 4 is still markedly higher than the inhala-
tion LD50 of 7.7 mg/kg. The latter figure is based upon 28.370
retention of material inhaled; even if the retention were 100%,
the inhalation LD50 would be about 27 mg/kg (still less than the
ip figure). In addition, increase in the respiratory minute volume
by a factor of 4-5 times would be necessary for the inhalation
LD50 to approach the ip value. Such a change in ventilation was
not observed.
Rapid entry of naled into the bloodstream may not be the sole
reason for the high acute inhalation toxicity of this compound;
the corrosive effect of naled on the lung may, in part, be a cause.
The higher inhalation toxicity of naled (based on levels of active
ingredient) when applied as the concentrate, rather than diluted
10% w/v in soya-bean oil, lends credence to this hypothesis. How-
ever, mere corrosive action would not explain the fact that the
rats often displayed marked cholinergic symptoms and experienced
acute deaths. The possibility exists that naled might be meta-
bolized to an even more toxic material to a greater degree in the
lung than in the stomach. Such a material might be dichlorvos
(the debrominated derivative of naled which is known to be a trans-
formation product of naled in the presence of compounds containing
sulfhydryl groups^ e.g., cysteine or glutathione). The existence
of large quantities of such compounds in the lung particularly
increases this possibility. Studies are being conducted to deter-
mine the in vivo and in vitro metabolism of naled when administered
both into the stomach~i[bral) or into the lung (inhalation) . The
results of this work will be given in a separate report.
Plasma cholinesterase determinations in the mouse revealed that
inhalation doses of chlorpyrifos far below those needed to induce
63
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mortality produced very significant: lowering of the enzyme level.
This phenomenon is less marked with naled where the dose needed
to kill some animals more closely paralleled that needed to pro-
duce a significant lowering of cholinesterase. Pre-exposure
plasma cholinesterase levels are rapidly achieved in those animals
which survived exposure to naled, but. are reached more slowly
after exposure to chlorpyrifos. One reason may be that chlorpyri-
fos is known to be retained to a limited .extent in body fat (Smith
ej: a 1. , 1967) and thus, small but: toxic amounts may be released
In'to~tne blood stream for a few days following exposure.
The effect of malathion on cholinesterase depression was highly
variable; in one five-hour exposure, depression was barely signi-
ficant- (Figure 6); however, in another two-hour exposure, the de-
pression was 45% of the pre-exposure value. This variation may be
related to the level of circulating aliesterases. These enzymes
are known to detoxify malathion by hydrolyzing the ethoxycarbonyl
group of the succinic acid moiety (Cook et al., 1958; DuBois et al.,
1968).
The intention of this work was to determine the effect in
animals of acute inhalation exposure of pesticide formulations
such as might be used in ULV applications. Thus, the highest
possible aerosol concentration which would not result in excessive
collision of particles in the air and the shortest, reasonable
exposure time ( to reduce animal stress) were used. Where inhala-
tion LD50 values have been determined, exposure times were not
above three hours (Table 3 ). Such exposure conditions have
arbitrarily been defined by us as acute conditions although it is
recognized that there is no widely accepted definition of what
constitutes an acute aerosol exposure.
In ULV work with ground sprayers, the HMD of the aerosols has
been reported to be around 10 yrn. However, such aerosols are highly
heterodisperse and a significant number of particles in the 1-3 urn
will be encountered. Naled is considered to be a relatively non-
toxic pesticide based upon its reported oral LD50 values to rats; •
manufacturer's figure 430 mg/kg (Kenaga and End, 1974); Gaines
(1968) reports 250 mg/kg to male rats; we foxmd 1.60 mg/kg to female
rats and 222 mg/kg to female mice. Based upon whole body retention
after inhalation of aerosols, we found inhalation LD50 values of
156 mg/kg for mice but 7.7 mg/kg for rats. Thus, if these animal
data were to be used to assess the potential hazard to humans, we
would regard naled as one of the more toxic pesticides when there
is appreciable inhalation exposure. It is our impression, however,
that the high aerosol concentration of naled used in these studies
(ca. 5 g/m3) would seldom be encountered by field personnel for a
period of time long enough for serious inhalation dosages to occur.
A major purpose of this study, namely to determine whether there
is an effect of particle size upon toxicity of aerosols of insecticide
64
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femulations, has been fulfilled only in the case of one insecti-
cide chemical, naled. The other three insecticides were not
toxic enough to enahle large particle aerosols to induce mortality.
Table 5 clearly indicates, however, that naled is less toxic when
inhaled as particles in the 18 - 20 ym MMD range than as small
(2 yrn) particles, although a true inhalation LD50 for the larger
particle size was not determined. Due to the lower aerosol con-
centration (mg/1 of air) in the "large particle" chamber than in
the Henderson chamber, it was necessary to make repeat runs with
the small particles, but with the aerosol diluted with secondary
air to make sure that the decreased toxicity of the larger particles
was not some function of decreased rate of administration. Thus,
the first three values listed in Table 5 refer to experiments
conducted under these conditions and explain the apparent contra-
diction with the data reported in Table 4 . To compare the decreased
toxicity of larger particles with these data is probably more valid
than'to' compare them with the noted LD50 of concentrated aerosols.
Depression of plasma cholinesterase did not parallel dose or mort-
ality incidence. This fact lends further credence to the hypothe-
sis that lung corrosion may play a large part in the high inhala-
tion toxicity of naled. In fact, after exposure to aerosols of
the concentrated naled (Dibrom 14 concentrate) rats that had died
displayed blood stained mucus at the nose and mouth.
The purpose of the experiments where larger particles were
stripped from standard aerosols was to determine whether the very
small particles (< 2 ym) were more toxic than those in the 2-5 ym
diameter range. The rationale was that when animals were exposed
for the same time and with the same formulation as in runs used to
determine LD50 values for naled, but with larger particles removed,
then about the same number of animals should die if the smaller
particles were the more toxic because of their enhanced penetration.
No animals died under this circumstance, but when runs with "screened"
aerosols were prolonged, the LD50 (mg/kg basis) was about the same
as with unscreened aerosols. These data indicate, therefore, that
particles in the size range of 5 - 0.8 ym are equally toxic within
the limits of our measurements.
We have demonstrated, at least in the case of one pesticide,
what had been predicted; that toxicity decreased when aerosol particle
size increased above the 5 ym range, so the principle is established.
In ULV sprays, although the MMD will .be above this range, a greater
number of particles will be below 5 ym than will be encountered with
conventional mists. These particles will remain aloft downwind from
the sprayers to a greater extent than large particles, especially
in the calm air situations normally preferred during pesticide
spraying. In this case, diffusion would be the major factor in
decreasing aerosol concentration, and could present an increased
hazard of some undetermined extent to a poorly protected worker.
65
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CONCLUSIONS
Conclusions drawn from the 3Ladies follow.
1. The authors consider that inhalation toxicity data of
particulate material are more meaningful if LU50 values
are expressed as mg/kg body-weight rather than expressed
from LC50 x time. A valid comparison of inhalation
toxicity and toxicity from other routes of administra-
tion can be made only by use of mg/kg dosage measurements
Vie have shown that the determination of inhalation LD50
values (mg/kg basis) can be made with a precision of
about + 10%. The accuracy may depend on particle size,
concentration of toxic substances per particle, animal
species and irritant qualities that affect breathing
rate.
2. The inhalation toxicity in rats of aerosols in the size
range of 0.8 to 5 urn (2 ym HMD) made from 107U w/v Dibrom
14 concentrate in soya-bean oil was 7.7 mg/kg (+0.6
mg/kg) whereas the oral toxicity of the same material
was 160 mg/kg (+ 30 mg/kg); thus, this material is about
21 times more toxic for rats by the inhalation route
than by the oral route.
3. The inhalation toxicity in mice, with conditions as in
2, above, was 156 mg/kg (+ 15 mg/kg) whereas the oral
toxicity was 222 mg/kg (jh 13 mg/kg); thus, this material
is only slightly more toxic for mice by the inhalation
route than by the oral route.
4. The inhalation toxicity in rats of aerosols in the size
range 0.8 to 5 ym (2 ym MMD) made from 6570 w/w of chlor-
pyrifos in xylene was 135 mg/kg (+ 34 mg/kg) whereas the
oral toxicity was 169 mg/kg (+ 25 mg/kg); thus, this
material is not significantly more toxic for rats by the
inhalation route than by the oral route.
5. The inhalation toxicity in mice, with conditions as in
4 (above) was 257 mg/kg (+ 31 mg/kg) whereas the oral
toxicity was 152 mg/kg (+ 15 mg/kg); thus, this material
is slightly less toxic for mice by the inhalation route
than by the oral route.
6. Increasing the median particle sizes of naled aerosols
(Dibrom 14 concentrate) from 2 ym MMD to 13 - 20 ym MMD
significantly reduced the inha.lntJ.on toxicity to rats.
With chlorpyrifos the concentration obtained with the
larger particles was not sufficient to induce mortality
66
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and no conclusion can be made on the impact or particle size
for this pesticide. However, the inference is clear that
if a pesticide is more toxic when inhaled in the form of
small particles than it is by the oral route, then the
larger particles of that formulation will be less toxic
than the smaller ones, on a mg/kg basis.
7. When the concentration of naled in the formulation
aerosolized was increased from 15% w/w to 87% w/w its
apparent inhalation toxicity was increased (from LD50
7.7 + 0.6 mg/kg to 3.1 + 0.7 mg/kg) when comparable
doses were adminstered.
8. Primary lesions in animals exposed to naled aerosols
were those of pulmonary congestion and serous edema.
These lesions appeared to be less severe on the basis
of the dose of the insecticide chemical administered
in animals exposed to the diluted formulations of naled
than those exposed to the concentrate.
9. In the mouse, inhalation doses of chlorpyrifos far below
those needed to induce mortality produced very signifi-
cant lowering of plasma cholinesterase. This phenomenon
was less marked with naled, where the dose needed to
kill some animals more closely paralled that needed
to produce a significant lowering of cholinesterase.
Malathion produced erratic results relative to plasma
cholinesterase depression.
10. Re-establishment of normal plasma cholinesterase levels
in surviving mice exposed to naled areosols v/as rapid,
but was slower in the case of chlorpyrifos.
11. Inhalation by rats of sublethal doses of aerosols of
all the insecticides tested, except chlorpyrifos, caused
elevation of whole blood serotonin levels. This ele-
vation was not observed in animals after they inhaled
vegetable oil aerosols. The response varied. In the
case of naled, there was biphasic response; initially
there was lowering of serotonin, then elevation was
observed.
12. Whole body retention of a vegetable oil aerosol was
markedly higher in the mouse than in the rat (84.170
versus 28.3%). However, rats retained approximately
three times (9.93% versus 3.77%) the inhaled dose of
the aerosol in the lung than mice did. Most of the
aerosol inhaled by mice was either retained in the head
or was swallowed and appeared in the stomach. This
phenomenon was less marked with rats.
67
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13. Observations on the effects of naled to pulmonary tissue
after animals underwent acute exposures indicates the
inadequacy of using oral toxicity data to estimate acute
aerosol exposure hazards, For naled a different mode
of toxicity or change to a different chemical form may
be responsible for the effects observed after inhalation
by animals.
ACKNOWLEDGMENTS
The authors are particularly indebted to Dr. A.II. Biermann
for designing many of the aerosol exposure experiments; other
persons whose help and advice have been most appreciated are
Mr. M.A. Chatigny, Drs. H.E. Guard, R.J. Heckly, and H. Wolochow.
The technical assistance of Messrs. R.E. Chiles, W.L. Firestone
and Ms. G. Lam is also gratefully acknowledged.
Research was supported (in part) by the U.S. Army Medical
Research and Development Command, Washington, D.C. 20314,
under Contract No. MIPR No. 5962.
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Beutler, E., Duron, D. and Kelly, B.M. 1963. Improved method for
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APPENDICES
72
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APPENDIX I
SMALL PARTICLE AEROSOL EXPOSURE
^ •• Generation oj: small p_article__ aerosols (0.8 - 5.0 ym) 3 ym MMD
A number of atomizers are available for generating small parti-
cle aerosols. Most rely on the principle of using a jet to gener-
ate the aerosol. This aerosol is then allowed to impact upon
baffles and so remove the large particles. In our work we made-
use of the glass Wells type atomizer (DeOme, et al. 1944) which
essentially consists of a glass bulb enclosing a twin-fluid, peri-
pheral, refluxing stainless steel jet. Details of the operation
are given by Dimmick (1969). Larger particles are impacted onto
the inner surface of the bulb, then returned to the reservoir of
fluid to be re-atomized. With vegetable oils, we were able to
obtain aerosols of MMD 1.8 to 2.2 ym and a geometric standard devi-
ation about 2.0 ym. To obtain a relatively high rate of flow
through the exposure chamber, to alleviate excessive increase in
temperature of the animals, and to generate a concentration of
aerosol sufficient to obtain results within reasonable exposure
times, we used two Wells atomizers connected in parallel, each
of which contained about 25 ml of the fluid to be aerosolized.
2. Exposure to small particle aerosols
Aerosols of diluted or undiluted pesticide formulations gener-
ated as described above were allowed to enter a modified 14.6 liter
volume Henderson apparatus (Henderson, 1952; Speck and Wolochow,
1957) using an atomizer pressure of 15 lb/in2 and maintaining
the chamber under slightly negative pressure. Typical relative
humidity (wet bulb) was 56%. A detailed description of this appara-
tus has been described (Dimmick and Hatch, 1969). Essentially,
it consists of a dynamic system in which aerosol is continuously
generated, allowed to enter a closed chamber in which the pressure
is held slightly negative (ca. 2 cm water), moved through the
chamber containing the animals at a rate of 18.6 1/min, sampled
at some convenient point using Millipore* filters and the "used"
aerosol disposed of by combustion (see Fig. 11 ).
In a typical run, the selected insecticide was placed into
the atomizers which were then affixed to the input of a modified
Henderson chamber (see Fig. 11). Animals were placed in cages,
the cages positioned in the chamber and the transparent cover was
sealed in place. The stainless steel cages were perforated on
all sides, and compartmented to hold either eight mice or four
rats. They were cages previously used for exposure of animals to
bacteriological aerosols and had been shown not to influence the
Millipore Corporation, Bedford, Massachussetts
73
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12.7 cm
2cm ID
copper
tube
0 manometer
n
-------
average flow or homogeniety of aerosols passing through the Hen-
derson chamber. Atomizers were started simultaneously with the
air exhaust system in a manner practiced to insure a slight
negative pressure within the chamber. Samples were taken at
appropriate times and animals were visually monitored for signs
of acute distress or death. The exposure xras prolonged according
to the purpose of the test. At that time the atomizers were
turned off and either clean air (if the animals were to be held
for observation) or carbon dioxide gas (for retention studies)
was admitted for five minutes, and the animals were removed.
3. Concentration and dosage measurements
To measure the concentration of pesticide chemical contained
in the air, Millipore filter samples were taken periodically,
usually from a side port in the Henderson chamber but, in some
cases, downstream.
A 47 mm diameter, 0.8 pm pore size filter was used at a flow
rate of 3.4 1/min controlled by a critical orifice attached to
the vacuum line (for downstream samples the flow rate was 18 - 20
1/min.). To sample, a manual relief valve was opened, the sampling
started, and the relief valve was closed while simultaneously
adjusting the vacuum control valve to maintain the needed inter-
nal, negative pressure - an operation that required 3 to 4 sec.
Sampling time was 1.0 or 1.5 min for side samples or 0.5 min for
downstream samples. The difference in weight of the filter before
and after sampling gave an approximate* value for the total amount
of pesticide formulation that was collected on the filter in the
given time. A more accurate value could be obtained by extracting
the filter with a solvent such as hexane, making up to a known
volume and chemically analyzing for the pesticide. An analytical
procedure for chlorpyrifos is described elsewhere (see Appendix 7).
The flow rate during sampling was measured with a rotometer. The
concentration of pesticide in the aerosol is then given by
Concentration (mg/1) =
Weight of pesticide chemical on the filter (mg)
Sample flow rate (1/min) x sample time (min)
The dose administered to the animals is then calculated assum-
ing approximately 8470 of the total inhaled in the mouse and 2870 in
the rat is retained, (see Appendix 6 and Results), and that the
respiratory minute volume for a mouse is 1.25 ml/min/g and for a
An assumption was made that the concentration of pesticide chemi-
cals impacted on the filter was the same as that in the formula-
tion atomized. This would be true only in the case of non-volatile
formulations (e.g., those in soya-bean oil.).
75
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rat, 0.65 ml/min/g (Guyton, 1947). Dose (mg/kg) - sample concen-
tration in aerosol (mg/1) x time (rain) x respiratory minute
volume (ml/min/g) x Percentage lung retention
100
After removal from the chamber the animals were normally ob-
served for a period of two weeks to determine mortality.
4. Particle size measurement
In studies with bacterial aerosols in this laboratory, size an-
.alysis is done by sampling with Andersen samplers (Andersen, 1958)
that were modified to accept plastic Petri plates. This sampler
is a multi-stage sieve impactor, and has been shown to be an
efficient and accurate sampler for particles in the 0.8 ym to 8 ym,
aerodynamic, diameter range.
The modification was to insert pegs in each stage that.would
raise the plate levels to compensate for the difference between
the thickness of prescribed glass plates and plastic plates. A
modification made for mass analysis in this project was the addi-
tion of a thick, aluminum plate that rested on these pegs. The
plate then served to raise aluminum Petri plates (trimmed to lower
the top edge to an appropriate level) so that the inner, bottom
surface was within the 1:5 hole-to-distance ratio prescribed by
Ranz and Wong (1952) for the most effective impaction. Mass an-
alysis of the material collected per stage was done by weight
difference of the Petri plates before and after sampling. The
cumulative data, as percentage of total collected, was plotted on
log-probability paper to obtain a 50 percentile value, which is
equal to the Mass Median Diameter (HMD),. A final modification
was the provision of thin (0.5 mm) disks that rested on the bottom
of the Petri plates to serve as collecting surfaces. In some in-
stances, plates were washed with aliquots of hexane and the col-
lected material measured by chemical methods.
At the request of the sponsoring agency, the collecting and sizing
capabilities of the Andersen sampler, using an aerosol of 1% aqueous
ammonium fluorescein, was compared to counting and sizing particles
collected on Nuclcporc v^ filters. The amount collected on each
stage of the sampler was determined by f luorometr.i c analysis, and
count and size incn.surcrnents of particles on tlio filter were made
from scanning microscopic photographs. The HMD in both instances
was 1.1 ym and the percentages in each size range differed by only
-_h 3%. The accuracy and replicability of the sampler nppears to
be ('.•xcellent.
n: —
Nucleporc Corporation, Pleasanton, California
76
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Typical insecticide aerosol data are shown in Figure 12. Curve
A is the cumulative size distribution of soya-bean aerosol emerging
directly from a Wells atomizer. Curve B represents a recent sample
of aerosol from the exposure chamber and may be contrasted with
Curve C, taken from data initially collected and reported by Berteau
and Biermann (1974), who used a downstream-filter as a terminal
stage for the sampler. It is evident that the distributions of
particle sizes are essentially log-normal and that passing an
aerosol from the atomizer to the Henderson chamber removed some
of the larger particles, although the median diameters did not
change significantly.
77
-------
Mass collected
per stage,
%
cumulative
normal
distribution
99-
90-
70.
50
30-
10-
1-
D
A
B
C
50 30 20
10 532 1 .8
DIAMETER, MICRONS
Figure 12.
Size distribution of aerosols from Wells atomizer,
10% dibrom in soya-bean oil.
A. Directly from atomizer exit tube
B. In exposure chamber, 1975
C. From previous data (Berteau and Biermann, 1974)
D. Spinning disk, 25,000 rpm, chlorpyrifos
78
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APPENDIX II
LARGE PARTICLE AEROSOL EXPOSURE
1. Generation of large particle aerosols (13 to 20 yirn HMD)
To generate aerosols having particles in a defined, narrow
size range, appropriate sizing methods must be available. The
sizing methods, however, need an aerosol of the required size
for calibration. Hence, the following is presented in more or
less chronological order because at the start of the project we
had neither of the methodologies and development of both pro-
gressed simultaneously.
We had anticipated that the May (1949) version of the air-
driven spinning top would create particles in the desired range
of 20 yrn. Indeed, a slight modification of the top design yielded
a stable generator, and aerosols of soya-bean oil in the 10 to 40
Vim range were produced. Two problems became apparent. The first
was that sizing by microscopic examination of particles allowed
to settle on slides (which we had intended to use) was not success-
ful because the large particles of the oil tended to wet the glass
and to spread, so the observed circular spots of material were
larger than the droplets in air. A correction factor could have
been devised as is commonly done in field sampling, but not within
the time limitations of the contract. More than a dozen types of
surface materials were tested including TeflonD and various types
of silicone oils; none was satisfactory. The data did show, how-
ever, that the predominant numbers of particles were larger than
15 ym. We then attempted to measure the size by the rate of fall
of particles in a vertical glass tube; particles were observed by
forward-angle, light-scatter. Data so collected indicated that
the particles were in the 20-40 ym range but to have obtained a
representative sample would have been too tedious and time-consuming
for our purpose.
The second problem was that measurements of dispersed mass,
under conditions of maximal top efficiency that produced the larger
particles, showed that it would be impossible to obtain an airborne
concentration sufficiently high to achieve the needed dosage level
in reasonable exposure times. We decided to test a motor-driven,
disk dispenser, and an ultrasonic method.
There are commercial ultrasonic dispersion units available.
One is the Mist-O-Ger© generator used in inhalation therapy. A
test of soya-bean oil by the manufacturer was unsuccessful. Besides
being expensive, commercially available, motor-driven disk units
did not fit the requirements of our chamber operation.
We obtained a Black and Decker die-grinder rated at 37,000 rpm,
and also machined a stainless steel disk 10 cm in diameter that
79
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would fit the chuck. The top of the disk was hand-ground to a
velvet finish and a 2.5 mm hole 2.5 mm deep, was made in the center
The hole permitted liquid to be fed onto the disk via a small plas-
tic tube and avoided the difficult problem of the precise position-
ing of a metallic feed tube as with the air-driven top.
The grinder was enclosed in a copper jacket, and a plastic
shield around the disk was provided to remove satellite particles.
By applying an inward airflow to the shield, smaller particles
were selectively removed. Speed of the disk, regulated by a
variable transformer, was shown to be directly related to applied
voltage. At 25,000 rpm a vibration developed that was severe
enough to restrict practical usage of the disk above that speed.
A sketch of the disk assembly is shown in Fig. 13.
In attempts to generate particles in the 8 to 10 ym range, the
disk had to be operated at speeds higher than 25,000 rmp. Figure 12,
curve D, shows that the distribution of particles from this test
was not log-normal and was greater than in other aerosols. How-
ever, the noise level inside the chamber was unacceptable in the
sense that the measured level above background was over 100 deci-
bels, and would undoubtedly have caused additional stress to
animals exposed to such levels. No additional attempts to create
8 ym MMD aerosols were initiated.
2. Exposure to large particle aerosols
Because of the inertial properties of large particles (greater
than 10 pm) it is necessary that both sampling and animal exposure
be done in such a way that particles are encouraged to always
travel "downwards" and not to be rapidly diverted into bends in
ducts that would remove particles. For exposure of rabbits to parti-
cles 10 ym or larger, we had designed and built an exposure chamber
that conformed to these principles. The aerosol dispenser was
located in the top of a chamber that had inward sloping walls lead-
ing to a restricted bottom area that would allow the exposure of
heads of animals to the aerosol falling from the top compartment.
This arrangement allowed the principal mass of the aerosol to drift
downward so no excessive removal of large particles occurred.
The chamber was a plywood box 107 x 119 crn with an inner inverted
pyramid that terminated at the bottom in a cube 2.3 cm per side where
heads of animals could be inserted. A dispersion chamber,38 cm per
side, of PlexiglasQy, and having a door that could be sealed, was cen-
trally located at the top. Below the animal chamber another small
pyramid terminated in a 4 cm ID brass tee and a valve through which
air could be transported. A similar valve and tee system was lo-
cated above the dispersion chamber, thus, air could be directed
80
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Air in
\
Air in
Metal jacket
Air out
Vacuum inlet
"Spider" support
Cooling
Air in
Power cord
Figure 13. Conceptual sketch of spinning disk atomizer.
81
-------
either upward or downward within the chamber. All inner walls,
except. Plexiglas, were painted v:ith two coats of epoxy resin.
The floor area outside the exposuvre chamber contained holes, and
provision was made to transport air through the holes in an up-
ward direction to dissipate body-heat of animals when their heads
were constrained within the inner chamber. A simplified schematic
illustration of the chamber is shown in Figure 14.
The chamber was modified later, at the request of the spon-
soring agency, to permit exposure of rats rather than rabbits.
The description of the rat-holding units is included in this section.
As shown in the Figure, the sampling port was located directly
above the holding units, and the 2 cm diam. sample tube was posi-
tioned as close to the vertical as practicable, again conforming
to the principle that large particles can be transported efficiently
only in downward direction.
The first test of the chamber was with the air-driven top. In
that test we had positioned plastic, honeycomb sheets beneath the
dispenser and under the animal exposure space to encourage laminar
air flow, which was in the upward direction but slow enough so
particles 10 ym and larger would fall downward. Even with the
limited output of the small top, it. was evident that the honeycomb
material collected a sufficient mass of aerosolized soya-bean oil
to eventually cause dripping to occur directly on the heads of
animals in the exposure space. The upper honeycomb was removed.
The first test of the disk disperser revealed another problem.
The space at the top of the chamber had been designed to accommo-
date the air-driven top. The larger disk created an extensive air
turbulence and although the "ring" of particles formed at the
"stopping distance" (Sinclair, 1950) was clearly visible, particles
were being s'wept around the dispersion chamber so violently that
excess impaction occurred on tubing, wires and walls, causing
additional dripping. Consequently, a drip-pan was positioned be-
neath the dispersion chamber, as shown in Figure 14. As a result
the feed rate had to be increased and the air flow made slightly
downward. The latter increased the number of smaller particles
reaching the animals.
Another problem was noise generated by the disk. This
alleviated by mounting the spider on rubber pads. However, this
caused the disk to absorb some of the vibrational frequency. We
mounted a strobe Inmp near the disk and could observe waves of
fluid moving outward on the disk. Ideally, this should have been
a uniform sheet. The motion undoubtedly caused additional hetero-
geneity in the size of the particles produced. These factors were
not of sufficient magnitude to influence the exposure data more
than other factors; had the LD50 values been near those of the
82
-------
air inlet
pressure Jock
at this point
Sampling Configuration
! at point X
Air holes for animal
ventilation
to vacuum
Transparent viewpoint
Figure 14. Conceptual sketch of modified exposure chamber.
83
-------
smaller particles we would have been concerned, but since data
show that the larger particles,as generated in this system,
were obviously less toxic than the smaller ones, the only result
is that mono-disperse, larger particles may actually be somewhat
less toxic than reported here. The size distribution (Figure 15)
shoxtfs, however, that only 7% of the mass of particles were 5 ym in
diameter or less, hence, the difference in reported values could
not be less than 7% as toxic, even if one assumed 100% retention
of the mass in the smaller size range.
Animal containers for head-only exposure.^were similar to those
described by Hoben e_t al.. (1976) . A PlaytexJV* bottle holder and
cap was used to restrain the animal with a. No. 11 stopper insert-
ed in the bottom to prevent th animal from changing position.
To withdraw blood from the animal during exposure, a V-shaped slot
was made in the stopper to expose the tail. The original nurser
nipple was modified to act as a latex rubber gasket between the
plastic cap, containing a hole through which the head protruded,
and the base of the holder. The opposed longitudinal slots,
originally intended for observation of the formula level, provided
adequate ventilation to prevent overheating.
When used for large-particle exposures, containers were intro-
duced into the inner, bottom portion of the chamber through a
heavy rubber diaphragm, and remained in position by friction alone.
This allowed variable exposure time, access to the tail for bleed-
ing, and access to the main body of the animal for monitoring as
well as allowing animals to be introduced or withdrawn as single
units without affecting chamber conditions.
When these containers were used in the Henderson chamber,
head-only exposure, a solid stopper was used and the body ports
were closed with tape. Apparently, heat was readily dissipated
to the air stream as overheating did not occur during exposures
as long as three hours.
This modification has provided a convenient, semi-disposable,
head-only exposure device for a unit cost of less than one dollar.
3. Concentration and dosage measurements
In operation, air was withdrawn from the bottom of the chamber
at the rate of 3 1/uiin, or a linear, downward velocity of about
6 cm/min at the animals' heads. Because of the volume of the
chamber, the aerosol concentration increased during the first 3.5
minutes of dispersion. Consequently the. dosage was estimated from
integrated analysis of the buildup. Values of aerosol concentration
'international Playtex Corporation, Dover, Delaware.
-------
cumulative
normal
distribution
99 H
90
70-
Mass collected
per stage, 59
10
1-
50 30 20 10 5
Diameter.
3 2
I .8
Figure 15.
0: Size distribution of aerosols from spinning
disk, 87% naled, run at 16 K rpm except
A (bottom set) = 23 K rpm.
B: Size distribution of aerosols from atomizer,
stripped by 2-stage Andersen sampler. There
were no particles larger than 2.2 ym diameter.
Vertical arrows indicate Mass Median Diameters
(MMD).
85
-------
for the three successful aerosols of large particles listed below
were single, simultaneous s.amplci; and hence differ slightly from
those reported in Table- 5, which are integrated values. Pesti-
cide was fed to the disk by a constant-head, gravity-feed device.
The volume dispersed and the amount collected in the drip-pan was
measured after each run.
Concentration and dosage were determined and calculated by a
procedure similar to that used for small particles. During samp-
ling the Millipore filter was attached to the chamber in the same
position shown by the Andersen Sampler in Fig. 14. The sample flow
rate was normally 3.5 1/min and the duration of sampling 1.0 min.
Operational Data for Large-Particle. Aerosols
Volume applied to disk, ml
Volume collected in pan, ml
Dispersed, ml
mg/1 air, by filter sampler
mg/1, .by Andersen sampler
MMD, ym
Run 1
276
137
159
1.2
1.2
13
Run 2
300
145
155
•0.9
0.8
18
Run 3
327
166
161
1.2
1.1
20
These values show that we were able to operate the chamber in
a reproducible manner and that particle "behavior" within the cham-
ber was as we intended. During exposure of rats, a light beam from
a microscope lamp was directed through the exposure space so parti-
cles were made visible by light-scatter. The particles were seen
to be descending and appeared to be uniformly dispersed within the
space, and remained so throughout each run.
4. Particle size measurements
While constructing and testing the disk, we also tested the
use of an Andersen sampler at flow rates lower than 1 ft^/min to
determine whether this device could be used to sample particles
larger than it had been designed for, and we found this procedure
was possible. To calibrate this flow rate we created aerosols
composed of a mixture of 1 pm, 8 vim, and 15 urn diameter, uniform
polystyrene particles (Dow Chemical Co.). These aerosols were
dispersed by the air-driven top, because we found only the 1 \im
particles escaped from a Wells-type atomizer housing. We found
that at sampling rates less than 10 1/min the distribution per
stage was too broad ( 8 ym particles were found on stages 2, 3, 4
and a few on 5) to be useful, but at 12.5 1/min, 90% of the 8 ym
particles were collected by stages 2 and 3, 98% of 15 yra particles
were on stage 1 (27o on stage 2) and about 1070 of the 1 ym particles
were on stage 6. From these data a calibration curve for the flow
86
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rate of 12.5 1/min (an empty impinger served as a critical orifice)
was produced.
When the sampler was first used to size aerosols from the disk,
we observed that a significant portion of the sample had impacted
on the sieve above the first stage of the sampler and was not
reaching the first collecting plate, so we constructed a pre-impact-
ion disk that was 2 cm larger in diameter than the entrance tubing
to the sampler and located 3 mm below the opening. In use, the
mass collected on this disk was added to that collected on the
first stage to provide a mass datum for "15 ym or larger".
Figure 15 shows data collected from runs made with the disk.
The method is sensitive enough to show differences in aerosols pro-
duced at different disk speeds. Since data from all six stages
were within good agreement to a log-normal distribution (which is
the generally assumed variability of particles produced from liquids
by application of shear forces ) the pre-impaction stage proved
to be an effective device. Data shown in Figure 35 was obtained
during exposure of rats to those aerosols.
87
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APPENDIX III
PLASMA CHOLINESTERASE DETERMINATIONS
The method used for determining plasma cholinesterase was
essentially that of Wolfsie and Winter (3952) which is a micro-
adaptation of the electrometric method of Michel (1949) . A
heparinized melting point capillary, 1.5 - 2.0 x 100 mm, was
carefully inserted into the medial canthus of the eye of a white .
mouse. The orbital venus plexus was punctured and blood entered
the capillary. In the case of a rat, the tail vein was incised
with a scalpel and the blood released allowed to enter a capillary.
When the tube was almost full, one end was sealed by insertion
into clay'f. The capillary was centrifuged at. 12,500 rpm for
three minutes when the red cells and plasma separated. The tube
was then cleanly cut and 0.02 ml of plasma drawn into a Sahli
pipette, The contents of this pipette were then discharged into
1.0 ml of distilled water contained in a suitable vial and the
pipette was rinsed three times with the water. To the diluted
plasma solution was added 1.0 ml of barbital buffer for plasma,
pH = 8.00 (Michel, 1949) and the vial incubated at 25°C in a water
bath for about 10 minutes. After this time 0.2 ml of 0.11 M acetyl-
choline chloride solution was added and the pH read immediately
to the nearest. 0.01 units using a combination electrode*"*. The
solution was then allowed to incubate at 25°C for exactly one hour
to permit the enzymatic hydrolysis of the substrate. After this
time the pH was read again and the cholinesterase level in the
plasma expressed as ApH/hour. Certain correction factors tabu-
lated by Michel (1949) to correct for nonenzymatic hydrolysis of
the substrate, and corrections for variations in ApH/hour with
pH were not normally applied since they were negligible compared
to the drop in pH encountered. For a normal, healthy, non-exposed
mouse the ApH/hour was about 2.00 units, for the rat, the figure
was much lower, about 0.25 units.
After animals were exposed to aerosols of organophosphorus
insecticides their blood was again withdrawn within an hour of
terminating exposure and the procedure for determination of cho-
linesterase repeated. The ratio of the pH/hour values after and
before exposure multiplied by 100 gives a figure which is indi-
cative of cholinesterase depression and is expressed as cholin-
esterase percentage of pre-exposure value. A value of 80% or less
was considered significant evidence of cholinergic activity.
Examples of typical data generated when mice or rats were exposed
to small or large particle aerosols of two insecticides are shown
in Table 7 and 8.
Seal-case, Clay Adams, Parsippany, N.J.
Leeds and Northrup battery operated pH meter
-------
TABLE 7.
EFFECT ON PLASMA CHOLIXESTERASE AFTER
EXPOSURE OF MICE TO CHLORPYRIFOS AEROSOL
(PARTICLE SIZE KMD 2pm)
Formulation: 65% chlorpyrifos in xylene
Aerosol concentration: 7.87 mg/1
Exposure Time: 35 rain.
Dose: 223.7 ing/kg
Mouse
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Mean
S.D,.
Weight
33
31
29
33
31
31
30
28
30
32
32
30
32
33
33
32
Pre-exposure
pH1 pH2
7.83
7.88
7.88
7.85
7.86
7.88
7.84
7.89
7.87
7.90
7.86
7.90
7.90
7.90
7.93
7.87
5.47
5.62
5.68
5.85
5.67
5.61
5.86
5.82
5.70
5.73
5.75
5.89
5.63
5.74
5.79
6.03
Post-e>:
pHj_
7.90
7.88
7.86
7.76
7.84
7.88
7.87
7.73
7.81
7.87
7.88
7.85
7.86
7.78
7.71
7.63
:posure
pH2
7.52
7.54
7.58
7.44
7.52
7.54
7.59
7.49
7.55
7.57
7.73
7.60
7.60
7.63
7.51
7.48
A pH/hr
Pre post
2.36
2.26
2.20
2.00
2.19
2.27
1.98
2.07
2.17
2.17
2.11
2.01
2.27
2.16
2.14
1.84
0.38
0.34
0.28
0.32
0.32
0.34
0.28
0.24
0.26
0.30
0.15
0.25
0.26
0.15
0.20
0.15
Cholines terase
% of pre-
exposure value
16.1
15.0
12.7
16.0
14.6
15.0
14.1
11.6
12.0
13.8
7.1
12.4
11.5
6.9
9.3
8.2
12.3
3.0
-------
TAttLK 8.
EFFECT ON PLASMA CHOLI.NESTERASE
AFTER EXPOSURE OF RATS TO NALED
AEROSOL (PARTICLE SIZE, HMD 18 - 20pm)
Formulation: Dibrom 14 concentrate
Aerosol concentration: 1.3 mg/1
Exposure time: 60 nun.
Dose: 12.4 nig/kg
Mortality: 2/8
0
Rat
No.
1
2
3
4
5
6
7
8
Mean
S. D.
Weight
g>
259
269
298
295
280
292
284
290
Pre-exposure Post-exposure A pH/hr
pH^ pllo pHi p}\2 pre post
7.90 7.49 7.80 7.55 0.41 0.25
7.92 7.02 7.87 7.43 0.90 0.44
7.93 7,23 7.91 7.65 0.70 0.26
7.91 6.97 7.93 7.69 0.94 0.24
7.89 7.10 7.98 7.69 0.79 0.24
7.93 7.36 7.89 7.64 0.57 0.25
7.94 7.41 7.89 7.59 0.53 0.30
7.93 7.51 7.91 7.68 0.42 0.23
Cholinesterase
% of pre-
exposure value
60.9
49.0
37.2
25.5
30.4
43.9
56.6
54.8
44.8
12.8
90
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APPENDIX IV
WHOLE BLOOD SEROTONIN DETERMINATION
Female white mice of the NAMRU strain weighing 30 - 40 g, or
female white Sprague-Dawley rats* weighing 250 - 350 g were util-
ized for these studies. Blood from the mice was obtained from the
orbital venus plexus by insertion of a capillary tube into the
medial canthus of the eye. With rats, blood was obtained by in-
cision of the tail. Exactly 20 M! was drawn into a Sahli pipette.
The method for determining serotonin was essentially the modification
by Krueger e_t al. (1963) of the method of Undenfriend et al. (1955).
To 25 ml of a T70 solution of ethylenediaminetetraacetTc acid was
dissolved 0.75 g of L-ascorbic acid. (This solution must be used
within 2 hours of its preparation). For an experiment involving 8
animals, 1 ml of this solution was placed in each of 8 stoppered
centrifuge tubes, the 20 p.1 contents of the Sahli pipette discharged
into the solution and the pipettes rinsed 3 times with the solution.
To each tube was added 0.5 ml of borate buffer (pH 9.5) which had
previously been saturated with n-butanol and sodium chloride. The
solution was shaken briefly, 1.5 g of sodium chloride added followed
by 3.0 ml of n-butanol saturated with water. The tubes were stoppered
and mechanically shaken vigorously for 10 minutes. Eight other tubes
were prepared containing 4.0 ml of n-heptane saturated with water and
1.5 ml of 0.1 N hydrochloric acid. After shaking was complete we
centrifuged the tubes for five minutes, removed 2 ml of the butanol
top layer and added that to the heptane-hydrochloric acid mixture in
the other tubes. These tubes were then shaken vigorously for five
minutes, the heptane layer aspirated and the serotonin level in the
aqueous lower layer read with a spectrophotofluorometer** at 295 nm
excitation and 335 nm fluorescence. The fluorescence range was
scanned from 300-350 nm. A standard curve was prepared each day using
the same procedure except that in place of the blood samples we added
20, 40, 60, 80, and 100 ul of a solution containing 2.5 /jg/ml sero-
tonin (equivalent to 0.05, 0.1, 0.15, 0.2 and 0.25 p.g of serotonin).
A blank sample containing no serotonin was also prepared. A stock
solution containing 10 /xg/ml of serotonin was prepared weekly by dis-
solving 11.5 mg of serotonin creatinine sulfate complex*** in 500 ml
of 0.1 N hydrochloric acid. The n-butanol and n-heptane were "chrom-
atoquality" grade.
The animals (normally 8 in number) were exposed in the Henderson
chamber, the aerosol being generated from two Wells atomizers in
lei. Concentration and dose were obtained from sampling on Milli-
pore filters as described in Appendix 1, Section 3.
* Charles River Laboratories
** Fluorispec Model SF-1, Baird Atomic Inc., Cambridge, Mass.
***Sigma Chemical Company, St. Louis, Mo.
91
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APPENDIX V
WHOLE BLOOD GLUT ATHION_E__ DETERMINATION
The method used was adapted from the methods of Ellman (1959) .
Blood was withdrawn from mice or rats by tail incision; 2 pi was
added to 0.9 ml of distilled .water and 1.0 ml of phosphate or
Hepes buffer, pH 8.0, was added. In each of two Beckman 1 cm
cuvettes was placed 3 ml of this solution. Two-hundredths ml
(0.02 ml) of a solution containing 31J . & mg of 5,5' dithiobis-2-
nitrohenxoic acid'"' in 10 ml of phosphate buffer (pll 7.0) was added
to the blood sample. The absorbance from the red color developed
was measured zeroing the other cuvette with the untreated portion.
Results may be expressed as mM of SH"'/1. of blood from the formula
c - 36.8 A
where c = concentration
and A = absorbance
The constant (36.8) was verified by plotting a standard curve from
a known solution of glutathione.
The lack of consistency of this method is described in the
Discussion section. Attempts to use the modification of Beutler
et al. (1963) were without success.
*Aldrich Chemical Company, Milwaukee, Wisconsin
92
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APPENDIX VI
DETERMINATION OF WHOLE/ BODY RETENTION OF AEROSOLS
To determine an inhalation dose of an aerosol in an animal in
terms of mg/kg body—weight, it is necessary to know the respiratory
minute volume of the animal under the conditions of exposure and
also the fractional retention of the material breathed. The former
figure was not determined in this study,.but the generally accepted
values of Guyton (1947) applicable to resting conditions was used.
In the case of retention, however, the values reported (see Dis-
cussion) vary so greatly with the materials used that we decided
to determine values using the major adjuvant of our formulations,
namely, soya-bean oil. To do this procedure, use was made of a
radioactively labeled tracer. Initially 1- ^C-dodecanol* was used
but due to the possibility that this compound might be metabolized
and the label lost as ^C-carbon dioxide a more inert tracer was
substituted. Most of the recent retention studies have now been
made with l-^C-heptadecane.* To a 50 ml volumetric flask was
added 0.5 mCi of 1-^C heptadecane in about 0.15 ml of benzene.
About 5 ml of unlabeled heptadecane was added and the solution made
up to volume with soya-bean oil.
Sixteen mice'or eight rats were placed in the compartmented .cages
of the exposure chamber and were exposed as described above. At
least 25 ml of soya-bean oil containing the 1-l^C-heptadecane was
placed in each of two atomizers, the aerosol generated, and animals
were exposed for about 20 minutes. During that time two or three
filter samples were taken on 0.8 /^.m, 47 mm diameter Millipore
filters. The aerosol leaving the chamber was passed through two
impingers which removed almost all particles and the residual was
passed through a filter before the air was transmitted to the
vacuum system. After the exposure was complete, atomizers were
turned off and, simultaneously, carbon dioxide gas was allowed to
enter the chamber from a cylinder. By this means the animals were
sacrificed as rapidly as possible after exposure to the radioactive
aerosol. With mice, death resulted in less than one minute; with
rats, slightly longer. Carbon dioxide was allowed to pass for five
minutes and then the chamber was air-washed for a further five
minutes. The animals were removed and lungs, trachea, stomach,
esophagus and duodenum were dissected out. Heads were also removed.
In some cases a portion of liver and a blood sample were taken.
Lungs of mice were divided into three to five portions, the stomach
into two and the trachea and esophagus were used whole. Each portion
of tissue was placed in a plastic disposable scintillation vial and
digested by standing for 24 hours with Unisolttissue solubilizer.
With rats known fractions of lungs and stomach ( approximately
100 mg) were digested whole with 1 ml of tissue solubilizer. Fur
*Purchased from ICN Isotope and Nuclear Division, Irvine, California.
tlsolab Inc., Akron, Ohio.
93
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was removed from the heads and they were digested 1-4 days in tis- •
sue solubilizer (10 ml. for mice or 50 ml for rats) and after di-
gestion the material was made up to ten times its volume with Unisol
complement. Filter samples were digested with 2 ml of methanol. To
the tissue material in the vials (except the heads), 0.5 ml of
methanol and 10 ml of Unisol complement (scintillation- fluid) was
added for each ml in the vial. For the heads 10 ml portions were
placed in the vials. Three drops of 30% hydrogen peroxide were
added to the finished cocktail to remove color and diminish quench-
ing. Materials in the vials were counted in a Packard Tri Carb
scintillation counter set to maximum gain for *-^C . In one run a
Beckman LS 250 scintillation counter was used. To determine quench-
ing for the material in each vial, 1 ml of a standard solution con-
taining known counts was added to each vial containing tissue, then
if
Actual counts _
^ Counts with quenching
we could determine the true (unquenched) counts for each vial from
the equation:
(Counts in vial after addition of standard) x q =
Standard + (Counts in vial before addition of standard) x q.
This last term in this equation is the desired result. The radio-
active count of the aerosol was calculated from the equation:
Aerosol concentration (cpm/ 1H - Mgari count s/min on_the
r '
- __ _
Sample fkiwrate (1/min) x sample time (mm)
Then, for an animal mass m,
Total com breathed = Aerosol concentration x m x breathing rate (ml/rniri/g ) x time
1000 of exposure (min)
The actual corrected cpm in each vial was summarized for each organ;
then:
Percentage retention in organ = £EnLin_organ_(corrected) x 100
cpm breathed
The mean background radioactivity war, determined for each run
and this amount was subtracted from each vial reading. For liver
samples, the whole organ was weighed and a weighed portion only (ca.
100 mg) digested. VJith blood, the quenching was too great because
of the heme present, so only plasma was counted.
-------
At the end of the exposure, the Henderson chamber was closed, the
aerosol regenerated and the size distribution determined using an
Anderson sampler. PJ.ates were extracted 3 times with hexane, the
extracts made up to 10 ml and counted. The proportion of counts on
each stage was then utilized to determine the size distribution as
described above.
Tables 9 and 10 give the respective levels retained in organs or
tissues in a given population of mice or rats.
95
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TABLE 9.
PERCENTAGE RETENTION IN VARIOUS ORGANS OF TOTAL CALCULATED INHALED AEROSOL3 IN FEMALE MICE
Mouse no.'
Head
Lung
Percentage retention in tissue0
Trachea Esophagus Stomach
Duodenum
f Soya-bean oil containing 1-^C-heptadecane; aerosol of MMD 2.1ym
D Mean weight of mice, 30.5g
c Based on respiratory minute volume 1.25 ml/min/g (Guyton 1967)
Incomplete and anomolous results led to rejection of data on this animal
e Mean values and 95% confidence intervals following head only exposure were: lung, 4.6 +_ 0.86;
trachea, 1.2 + 0.91; stomach, 39.8 + 13.1; esophagus, 1.4 + 0.75; total (excluding head and
duodenum), 46.9+13.0. (See Discussion).
Total
1
2
3
4
5
6
7d
8
Mean6
95% conf. int.
2.47
7.28
10.50
12.84
12.80
7.21
-
10.30
9.06
±3.43
3.99
4.26
3.90
4.18
3.07
3.01
-
3.98
3.77
+0.46
1.34
2.85
0.50
0.06
0.91
0.33
-
0.08
0.87
+0.91
0.44
0.21
1.10
1.02
0.62
0.90
-
28.33
4.66
+9.67
90.85
53.62
102.96
37.01
75.79
36.14
-
15.21
58.80
+29.68
1.33
14.20
3.98
12.10
2.62
14.20
-
0.24
6.95
+5.80
100.42
82.42
122.94
67.21
95.31
61.79
-
58.13
84.10
+21.93
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TABLE 10.
PERCENTAGE RETENTION IN VARIOUS ORGANS OF TOTAL CALCULATED INHALED AEROSOL*1 IN FEMALE RATS
Rat no.b
Head
Lung
Percentage retention in tissue
Trachea Esophagus Stomach
Duodenum
Total
1
2
3
4
5
6
7
8
Mean
95% conf. int.
5.53
2.05
3.91
3.27
2.41
1.62
2.63
3.43
3.10
1.03
8.24
7.15
10.91
10.42
8.96
7.02
9.71
17.02
9.93
2.67
0.09
0.18
0.33
0.10
0.09
0.10
0.17
0.19
0.16
0.07
0.29
0.04
1.34
1.15
0.06
0.01
0.11
0.11
0.39
0.47
14.44
11.10
16.24
33.04
4.45
11.70
6.20
5.57
12.84
7.70
1.59
0.87
0.90
3.15
2.12
0.95
0.36
5.12
1.88
1.32
30.18
21.39
33.63
51.13
18.09
21.40
19.18
31.44
28.31
9.19
a Soya-bean oil containing l-^C-heptadecane; aerosol of HMD 2.1ym
b Mean weight of rats 364 + 37 g
c Based on respiratory minute volume 0.65 ml/min/g (Guyton 1947)
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APPENDIX VII
MISCELLANEOUS DETERMINATIONS
1. Analysis for chlorpyrifos
This method was essentially the same as that described by Dow
(1972) except that hexane was used as a solvent instead of methanol
because the latter material destroyed Millipore filters during
extraction. A standard curve was prepared by accurately weighing
125 mg of an analytical sample of chlorpyrifos'', dissolving in
hexane and making up to 250 ml with analytical grade hexane.
Dilutions of 0.2 ml, 0.5 ml, 1.0 and 2.0 ml were made up to 10 ml
with hexane. These solutions then comprised concentrations of 1.0,
2.5, 5.0 and 10.0 mg/100 ml. The absorbance was read at 289 nm
against a hexane blank using a Bookman DB spectrophotometer. Values
were plotted on linear graph paper to give the standard curve.
Beer's Law was well observed over the range of 1 to 5 mg/100 ml.
2. Comparison of formulations
Samples of pesticide formulations for use in the toxicological
studies discussed were normally obtained direct from the manufact-
urer. However, military formulations were obtained from Aberdecri___
Proving Ground, Maryland, and it: was desired to know if the_ forrilu"^
lations were the same. For this purpose, 0.1 yl of the~Torm'ulation
was injected into a Hewlett Packard F and M Scientific 700 lab-
oratory gas chromatograph having a Teflon column of dimensions
50 x 0;32 cm., containing 15% SE 54 on 100/120 gas chrom Q. The
injector temperature was 140°C, detector 180°C and the column oven
temperature was programmed from 100 - 200 C. The tracings were
compared for each sample injected.
3. Partial dispersion of aerosol charge
}n order to try to disperse any electrical charge that may
have 'been present on particles of oil aerosols generated in Wells
atomizers, we used a krypton^S source. To determine the effective-
ness of this procedure, an apparatus was prepared such that an
aerosol was allowed to pass between two pairs of electrically charged
aluminum disk plates. The plates were weighed and then the aerosol
was allowed to pass for 6 hours with no charge applied. A constant
vacuum was applied to draw the aerosol through. Initially no charge
was applied. The plates were weighed at the end of the experiment.
The experiment was then repeated with the charge of 2000 volts
applied with and without a krypton85 source in the line. The dist-
ance between the mouth of the atomizer and the plates was kept the
^-
The analytical sample of chlorpyrifos was kindly supplied by the
Dow Chemical Company.
98
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same for each, experiment. The efficiency of the radioactive source
in dispersing the charge was determined by the reduction in v;eight
of the plates when the source was present,
^• Effect of xylene on the toxicity of chlorpyrifos
One of the standard formulations of chlorpyrifos is a 657, w/v
solution of the active ingredient in xylene. In early inhalation
exposure runs there was some indication that chlorpyrifos may be-
come more toxic when its formulation was further diluted with
xylene. To examine this possibility, technical grade unformulated
chlorpyrifos x\ras used. The compound was then formulated by us,
using various concentrations of xylene and a nontoxic vegetable
oil adjuvant (soya-bean oil) , These formulations were atomized
and eight mice exposed in the Henderson chamber, as previously
described, to the small particle aerosols generated. Cholines-
terase measurements were made and 14-day mortality recorded.
Andersen samples before and after atomizing for the given exposure
time were taken for most runs.
5. Particle screening
The LD50 values reported for naled using Wells atomizers indi-
cated that about 1.0% of the particles were 4 ym or larger, although
the MMD was 2 ym. To determine if the toxicity of a 10% w/v solu-
tion of Dibrom 14 concentrate in soya-bean oil would increase if
these particles were removed, four runs were conducted in the Hen-
derson chamber. In these runs, before allowing the aerosol to
enter the chamber, a number 3 or a number 4 stage of the Andersen
sampler was placed in the aerosol line to screen particles 3 ytn
and larger, or 2 ym and larger, respectively. Sampling for aerosol
size and density, and determination of toxicity, was as described
previously.
99
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-033
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
THE METABOLISM OF NALED INHALED BY RATS
5. REPORT DATE
June 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Peter E. Berteau and Robert E. Chiles
8. PERFORMING ORGANIZATION [REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Naval Biosciences Laboratory
University of California
Naval Supply Center, Oakland, CA 94625
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
IAG-D5-0697
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development'
U.S. Environmental Protection Agency
Washington, D.C. 20460
13.TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA 6nn/n
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Naled (Dibrom(R)) was prepared with a 14 carbon label in the 1-ethyl position.
The labeled compound was administered in appropriate formulation vehicles to female
rats by the inhalation, oral or intraperitoneal routes. Treated animals were either
placed in metabolism cages and their excreta and expiration of radioactivity
monitored during 48 hours, or they were quickly dissected after sacrifice, the lungs
and stomach extracted with ether and metabolic breakdown products analyzed-fer-J4-£-
products by thin layer chromatography. Some animals were also used to determine the
deposition and early distribution of onhaled naled. Urinary levels of radioactivity
were higher when animals inhaled the compound than when it was administered by the
other routes. Levels deposited in the lung were very low and this fact limited the
generation of analytical data on the metabolic changes. However, no evidence was
provided to indicate that there was a preferential metabolism to the debrominated
form of naled (dichlorvos) when the compound was inhaled contrasted to other routes
of administration.
t7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c.. COS AT I Field/Group
insecticides
respiration
toxicity
rats
tests
metabolism
Naled
Dibrom(R)
06 F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
no
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
100
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