PB 180 522T
i
U.S.S.R. LITERATURE ON AIR POLLUTION AND
RELATED OCCUPATIONAL DISEASES - VOLUME 17.
THE BIOLOGICAL EFFECTS AND HYGIENIC IMPOR-
TANCE OF ATMOSPHERIC POLLUTANTS - BOOK 10
B. S. Levine
i
B. S. Levine
Washington, D. C
1968
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U.S.S.R. LITERATURE ON AIR POLLUTION
AND RELATED OCCUPATIONAL
DISEASES
1 7
A .SURVEY
by B. 1. L«vin«, Ph. D.
THE BIOLOGICAL EFFECTS AND HYGIENIC IMPORTANCE
OF ATMOSPHERIC POLLUTANTS
BOOK 10
Processed by
CLEARINGHOUSE FOR FEDERAL SCIENTIFIC
AND TECHNICAL INFORMATION
U. S. DEPARTMENT OF COMMERCE
Springfield, Virginia
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The present English edition is a part of a survey conducted by
B. S. Levine, Ph. D.
of 3312 Northampton St. , N. W.
Washington, D. C. 20015
supported by PHS Research Grant AP - 00176,
awarded by the Division of Air Pollution
of the U. S. P. H. Service
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CONTENTS
Preface 1
Basic Problems of Sanitary Atmospheric Air Protection Z
New Experimental Material for the Determination of
Maximum Allowable Concentration of Hexamethylene-
diamine in the Air of Inhabited Areas 8
Hygienic Basis for the Determination of the Allowable Con-
centration Limit of Divinyl in Atmospheric Air 18
Toxicity of Ethylene Oxide in Low Concentrations 33
A Study of Hygienic Properties of Methanol as an Atmos-
pheric Air Pollutant 39
Atmospheric Air Pollution with V;'.pors of Hydrolytic Alcohol
and its Effect on the Organism 46
Biological Effects and Hygienic Evaluation of Air by Phthalic
Anhydride 54
Comparative Toxicity Studies of Benzene, Toluol, and Xylol
by the Reflex Activity Method 60
Experimental Data Proposed as a Basis for the Determination
of Maximal Allowable Ammonia Concentration in Atmos-
pheric Air 67
Chronic Effects on the Organism of Small Concentrations of
Acrylaldehyde in Air 77
Hygienic Evaluation of the Combined Effects of Acetone and
Phenol in Atmospheric Air 85
Determination of Allowable Maximum Concentrations of Phenol
and Acetophenone when Simultaneously present in Atmos-
pheric Air 97
Combined Effects of Low Acetone and Acetophenone Concentra-
tions in the Air of the Living Organism 108
- ii -
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Studies in the Standardization of Maximum Allowable Hydrogen
Fluoride Concentrations in the air of Inhabited Areas 118
Experimental Basis for the Determination of Maximum Allow-
able Concentration of Vanadium Pentoxide in Atmospheric Air 128
An Hygienic Standardization of Alphamethylstyrene in Atmos-
pheric Air 139
Appendix 146
- in <-
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PREFACE
The papers comprised in The Biological Effects and Hygienic Im-
portanca of Atmospheric Contaminants contain new data pertaining to the role
"expanding popular chemistry" played in the industrial history of the USSR.
Few data have been found in the literature %on the harm that the high-tonnage-
chemical-procefls industry can cause to the population. Whatever the branch
of industry, little is known of the toxicity of its emissions--especially in low
concentrations inhaled over a long time, or the methods of analyzing atmos-
pheric air. Consequently, it has not been determined in what concentrations
the new industrial emissions may be discharged into the atmosphere without
adversely affecting the population's health.
It is understandable that under such circumstances the planning or-
ganizations send in numerous inquiries to the scientific research institutes
engaged in problems of industrial environmental hygiene. Answers to re-
quests for such information presuppose availability of appropriate experi-
mental data on such questions as: •what is the danger of a given type of in-
dustry for human health, in -what combination and concentrations are harm-
ful compounds discharged into the atmosphere, what are the maximum toler-
able concentrations of such substances in the air, by what methods can these
contaminants be determined, what measures can be recommended to reduce
atmospheric pollutant concentrations, what sanitary zones can be considered
adequate for industry of a given type, etc.
Some of these questions can be answered by studies conducted in
some institutes, laboratories, and departments directed by the Project Com-
mission of the Science Section of the USSR Ministry of Public Health. Papers
presented in this collection are directly related to problems arising from the
above indicated need for new and up-to-date environmental sanitary-hygienic
information.
A. Ye. Kulakov investigated hexamethylenediamine, used in the
production of nylon.
G. Kh. Ripp presents results of his interest in air pollution caused
by divinyl, used in manufacturing synthetic divinyl-alpha-methyl-styrene
rubber.
T. Yuldashev for the first time discusses the effects of ethylene
oxide.
R. Ubaidullayev studied the hydrolytic phase of industry, dealing
with cotton by-products, a raw material important to the Uzbekistan industry.
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L. P. Slavgorodakiy has studied pollution by phthalic anhydride,
and its toxicity.
I. S. Gueev made a comparative evaluation of auch important com-
pounds as benzene, toluene, and xylene, which are widely used as organic
solvents and as raw material in synthesizing aromatic compounds.
M. I. Gusev and A. A. Minayev report on alpha-methylstyrene
in the air in the proximity of synthetic rubber plants.
U. G. Pogosyan, Yu. Ye. Korneyev, and N. Z. Tkach investi-
gated the production of acetone and phenol by the cumene method, a most
interesting product of chemical synthesis with diverse discharges under
the prevailing plant conditions.
M. S. Sadilova presents new and interesting studies on the effects
of hydrogen fluoride on animals when administered chronically in microcon-
centrations.
The collection also contains papers on the chronic effects of
acrylaldehyde, ammonia, and vanadium pentoxide.
BASIC PROBLEMS OF SANITARY
ATMOSPHERIC AIR PROTECTION
Prof. V. A. Ryasanov
History indicates that air pollution as a sanitary hygienic Droblem
was recognized about 100 years ago. The state of the atmosphere has since
changed greatly, and the sources and types of pollution have become complex,
and the problems to be solved are entirely different and immeasurably more
complicated. This writer is in disagreement with the viewpoint of our foreign
colleagues, who pessimistically view the air pollution problem and regard
the increasing levels of urban pollution as unavoidable, and believe that man-
kind must become inured to this "civilization disease". Years of observa-
tion and reflection lead to a different conclusion. However, the striking
transformations -which progressively occur in this complex problem must be
taken into consideration if a correct assessment of its tendencies is to be ar-
rived at.
During the 19th and the first half of the 20th century mineral fuels
were regarded as the principal source of air pollution due to the use in fire-
places, stoves, boilers, and electric stations, along with sulfuric acid, soot,
and ash because these were the most dispersed, the most universal, and also
the most harmful. By the middle of the present century the situation had be-
gun to change. Many countries, especially the USSR, have taken radical mea-
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sures to liquidate smoke-contaminated air: fireplaces and small boilers
were replaced by heat supplied from central power stations, efficient
coal-burning processes were developed which minimized the products
of incomplete combustion, ash catchers of high purification degree were
installed, the sulfur content of fuel wae lowered, and, finally, mineral
fuel was replaced by natural gas, both in large power stations and in homes;
industrial and domestic processes were electrified by replacing smoking
fuel with electric power. By the middle of the 20th century the problems of
"smoke1' in the real sense of the word was theoretically solved, and in many
cities of the economically developed countries it was solved in practice.
Newly developing nations should solve their "smoke" problem by introducing
district heat, gas, and electric facilities or centers. There are also other
auxiliary means.
However, in addition to the process of fuel combustion there are
other factors that play a known, though limited, role in air pollution, such
as the metallurgical industry's high-capacity hearths; however, such pollu-
tion sources, located in particular geographic areas, have a limited effect
since the pollutants did not extend beyond a certain environmental zone. The
nonferrous metallurgy has also created dangerous pollutant foci. As the
oxide deposits became exhausted toward the end of the 19th century, the
metallurgic industry was compelled to exploit low grade polysulfate contain-
ing orea, which led to severe sulfurous air pollution, which broke up the
ground protecting surface layer for milea around the nonferrous ore sources.
Polysulfide ores yield a dust of highly complex composition, some components
of which are highly toxic to man and animals. The pyrometallurgical process
transformed these extraneous metallic elements into smoke, thereby increas-
ing their dispersion and enhancing the dust danger, while oxidation of these
elements further increased their toxicity. With the advent of light metals
such as aluminum, magnesium, beryllium, etc., their harmful compounds
became known, such as fluorine compounds connected with the phenomenon
of fluorosis among children, and resins containing large amounts of 3, 4-
benzopyrene, and beryllium which caused in the nearby populated areas a
pathologic condiction known as berylliosis . However, the above mentioned
serious antihygienic conditions came into being as the result of the metal-
lurgical industry development and were looked upon as a local phenomena as
long as fuel combustion was a national problem. It is, therefore, understand-
able why smoke should compel the attention of all authorities concerned.
Metallurgical enterprises as sources of air pollution and the sani-
tary hygienic problems they created are of equal importance theoretically and
practically. Theoretically this problem can be solved by the judicious use
of raw material. Instead of being discharged into the atmosphere as vast
amounts of SOa generated in smelting, these concentrated gases should be
utilized as a raw material by plants producing sulfuric acid and plants
related to the nonferrous metallurgy. Instead of being used whole, poly-
metallic ore should undergo preliminary flotation for fine selective separa-
- 3 -
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tion, and each fraction should be agglomerated and smelted in separated
furnacea. Dust residues must be trapped with the aid of modern, glass
cloth filters. Metallurgical combines must be complexes of factories or
shops yielding a variety of products and utilizing, without waste, every-
thing of value in the ore. Furthermore, the pasties industry has been ra-
pidly forcing nonferrous metal products out of the consumer market because
of their inexpensive and simple production, their toughness and anticorro-
sive properties, and a number of other qualities which make them irreplace-
able, even though they first appeared in the role of "ersatz". The nonfer-
rous metals are not being replaced by plastics, to which the future belongs.
At the end of the first half of the 20th century automobile ex-
hausts resulting from liquid fuel came into being as a serious contaminant.
For example, in the USA the number of automobiles increased from 10 mil-
lion in 1920 to over five times that number in 1950, an increase of 40 mil-
lion in 30 years. In cities the automobile is a source of various harmful and
unpleasant waste products, such as carbon monoxide, hydrocarbons, alde-
hydes, resin compounds (some of them carcinogenic), products of the break-
down of antiknock agents, which contain finely dispersed vapor of lead oxide,
etc.
The most threatening factor in air pollution in the 20th century is
the rise of photochemical fog, which is entirely new in man's external en-
vironment. Such fog, or "smog" as it has been called, was first observed
in Los Angeles in 1945, where by 1950 it had become a serious problem.
Smog is formed as the result of photochemical reactions occurring in open
air during periods of sunshine. These reactions originate with the organic
substances present in automobile exhausts, principally the pentane and hex-
ane olefins with nitric oxides as a major contributor, with the resulting for-
mation of czone. In the presence of light and hydrocarbons, nitrogen perox-
ide is converted back to nitric oxide, and the reaction continues as long as
there is sunlight. Ozone also reacts with the olefins. As a result of com-
plex and diverse reactions that generate free radicals, ozonides, and perox-
ides, complexes of organic materials are formed in the atmosphere which
are highly active chemically, causing irritation of the mucous membrane of
the eye accompanied by lacrimation, loss of visibility, damage to plants, etc.
Lacrimation caused by photochemical smog is the most frequent complaint of
the population. No successful persistent attempts have been made to deter-
mine the connection between smog and its effects. It is possible that each ef-
fect is caused by a different chemical agent. A previously unknown compound,
peroxyacetylnitrate was isolated from the air, but it had not been established
that it was an active component of the Los Angeles smog. However, it has
been demonstrated that photochemical fog arose from air contaminated by
exhaust gases particularly in the presence of temperature inversion. Smog
formation in Los Angeles appears to be the result of unique conditions pre-
vailing there: the absence of public transportation, use of private individual
automobiles in the millions, the frequently occurring anticyclone weather, and
the constant intense solar radiation are some of such conditions. Recently
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smogs have appeared in other USA cities including San Francisco, Washing-
ton, New York, etc. This atmospheric condition has been gradually spread-
ing over other USA cities. It is reasonable to assume that the effect of
photochemical smog in addition to discomforting and disabling effects caused
more serious pathologic manifestations, frequently of a chronic nature. The
universal rise in automotive traffic progressively enhanced the harmful ef-
fects of exhaust gases so that the appearance of analogous photochemical fog
phenomena can be expected to appear in other countries. Soviet health
authorities should regard this with the consideration it deserves.
The A. N. Sysin Institule of General and Municipal (Public) Hy-
giene of the USSR Academy of Medical Sciences has initiated preliminary
surveys in this direction. Chemist V. A. Popov has perfected the phenol-
phtalein method used in the USA to determine oxidizers in the air. Tenta-
tive investigations in Moscow, Baku, and Batumi have shown that during the
summer, i. e. , when solar radiation is at its maximum, the products of
photochemical transformations of exhaust gasee can be discovered in the air
of the above-mentioned cities. Although the concentration of the oxidizere
is only about 1Z percent of those in the air of American cities, they rise
close to the critical tolerance established by the California standards. The
concentration of oxidizers increased with the number of automobiles, and
unless effective measures are taken to reduce the discharge of exhaust
gases, citiea in the USSR will come to experience smog conditions similar
to those occurring in the USA. The universal use of automotive transporta-
tion, its rapid and progressive growth in the number of auto vehicles, the
discharge of poisonous pollutant components into the air of populated areas,
the susceptibility of the components of these gases to undergo diverse photo-
chemical conversions in which highly reactive free radicals participate
point to atmospheric pollution by automobiles as an oncoming threatening dan-
ger from the viewpoint of modern hygiene. In this connection it must be kept
in mind that exhaust gases contained other ingredients, such as lead aerosols,
carbon monoxide, carcinogenic substances, etc. This leads sanitary hygien-
ists to the conclusion that air pollution by automobile exhausts constitutes a
problem equal in its urgency to that of air pollution caused by industrial com-
bustion, with which hygienists throughout the world occupied themselves
earlier. This problem of air pollution by automobile exhausts must occupy
a leading place in world's scientific and sanitation efforts. There are means
by which this problem can be solved by central regulation of center trans-
portation, proper maintenance of vehicle operation, extending public transit
beyond the city limits, converting automotive engines to condensed gas, and
converting automotive gasoline to electric engines. Research in the direc-
tion of such developments should be conducted on a great scale by engineers
and other scientists engaged in solving problems in interstellar space. In
this connection it should be noted that radioactive atmospheric air pollution
is a specialized field which is intimately connected with international poli-
tics .is excluded from this discussion. The attention here will be centered
on such phases of the problem which deal with the developments in advanced
- 5 -
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industrial chemistry, which brought into being the most acute and burning
hygienic questions of our times.
Chemistry has played an important role in hastening the rise of
the Communist society. The 23rd Congress of the CPSU examined the rapid
developmental rates of the chemical and petroleum industries. Chemistry
and its products extend into all aspects of manufacture and the total life of
the population. The manufacture of raw materials, intermediate products,
and finished articles from synthetics promises to become one of the most
widespread, complex, and massive sources of environmental pollution,
especially of the air, unless hygienists, chemists, and engineers investi-
gate these compounds for their characteristics, their biological effects, and
their danger to man; learn to isolate them by specific and highly sensitive
analytical methods; and prevent their appearance in the atmosphere in con-
centrations that have a deleterious effect on man.
Environmental pollution has become a universal problem. In March
1963 the World Health Organization in Geneva held a special international sym-
posium on microchemical pollution of the environment. According to this
symposium, hundreds of new compounds are synthesized throughout the
world each year, many of which find practical application and become a
source of environmental pollution. It would be very time-consuming to list
the areas in which the new chemical compounds find application, among
which are fertilizers, pesticides, defoliants, plastic articles used in ma-
chine building, home construction, and civic works; new fibers for clothing,
footwear, and other daily usage, packaging, cleaners, detergents, adhesives,
synthetic medicines, vitamins, fats, antioxidants; liquid fuel, additives, etc.
Most of these organic compounds have never been investigated for their toxi-
cological and physiological properties, nor have means been devised for de-
tecting them in complex mixtures and low concentrations. The possibility
of their combined effect, and of their spontaneous change in the open air in
some instances into carcinogenic substances and teratogenic effects, com-
plicates the problem considerably. In addition, engineers, builders and con-
sumers ask for data on the harmfulness of new substances which will be
used by workers on the job, the inhabitants near the plants, and the millions
of consumers of the new chemistry throughout the USSR. For this reason
the new developing chemistry must be the principal concern of hygienists,
particularly of those who deal with atmospheric hygiene. The problems are
complex and may appear insoluble. Therefore, all available resources must
be mobilized, the sequence of operations must be strictly planned, and man-
power must be properly mobilized, and all available auxiliary resources must
be directed toward strengthening the scientific teams which have already been
organized and approved. It is easier to achieve a breakthrough where quali-
fied personnel, modern equipment, and an experimental background have
built up than where all these have to be created anew. Therefore all re-
sources should be directed toward reinforcing already existing scientific
centers for the study of toxicology and chemistry, of new synthetic compounds
- 6 - \
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and the environmental pollutant associated with them, particularly air pollu-
tants. In this respect situations differ in different countries. In the USSR
the need is greatest in planning more active investigations of pertinent specif-
ic questions, to differentiate, in the first instance, between studies that are
absolutely essential for immediate practical results and those of more re-
mote interest. Students working for college degrees should be assigned
problems raised by modern chemical industries, especially those interested
in the development and production of new synthetics. Results obtained should
be published without undue delay so that hygienists and other health author-
ities could study them with all possible sense of priority. This applies par-
ticularly to organic compounds and substances which are highly volatile,
have a definite odor, and irritate the mucous membranes, especially of the
upper respiratory tract. They can be neutralized in the following ways:
1. Burnoff in special furnaces. Unfortunately, because of the
low concentrations of contaminants and the inadequate heat of combustion,
fuel has to be expended to burn them. The drawback of this method is the
destruction of valuable organic materials that could be extracted and used in
the national economy. On the other hand, -where these compounds cannot be
rendered harmless in any other way, this is an appropriate method.
Z. Ordinarily, compounds of this type can be easily condensed
and, therefore, lend themselves to low-temperature separation. After the
basic mass has been condensed, the remainder, which has no value, can be
burned.
3. These compounds can also be absorbed, with subsequent de-
sorption or elution of the products and regeneration of the filters. This
method of recuperation is widely used in practice to avoid discharges of or-
ganic compounds. By combining these three methods it is possible to achieve
satisfactory disposal of the most varied discharges of synthetic chemistry.
For materials with a low activity threshold, means must be found
of replacing them with less toxic compounds or of introducing a technology
based on essentially different methods, i. e. , methods which did not require
toxic materials. For example, the high-temperature heat conductor dynel,
which is widely used in synthetic chemistry and pollutes vast areas in the
manufacture of synthetic fibers, can be replaced by electric heating. The
appropriate technical institutes need to devote considerably more attention
to developing this advanced method in their planning.
By systematizing the different types of the air-pollution problems
related to plastic chemistry industries, it is possible to arrive at a means of
lessening or abolishing their pollution effects by burning residual discharges
or other suitable means based on the following two prerequisites:
1. Hygienists and toxicologists must actively participate in the
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activities in the work of industrial research institutes engaged in develop-
ing industrial synthetic products.
2. Maximum support must be given to studies of the biological
and toxicological effects of new chemical compounds introduced into produc-
tion, so that their critical concentration tolerances (MAC) could be deter-
mined as soon as possible, and specific and highly sensitive methods of de-
tection can be developed.
The foregoing makes it clear that the problem of air pollution
has become considerably more complex and difficult in its century-long
history, and has given birth to new questions different from the ones which
prevailed during the first half of the present century.
The principal sources of air pollution now are industries of the
new synthetic chemistry and automotive exhausts. The present volume is
devoted to discuseione of practical problems caused by automotive exhausts,
NEW EXPERIMENTAL MATERIAL FOR THE DETERMINATION
OF MAXIMUM ALLOWABLE CONCENTRATION OF
HEXAMETHYLENEDIAMINE IN THE AIR OF INHABITED AREAS
From the A. N. Sysin Institute of General and Municipal Hygiene of the
USSR Academy of Medical Sciences
Soviet and foreign literature dealing with the biological effects of
hexamethylenediamine (HMD) and on its effects on man is limited. Reported
studies \vere made using small numbers of experimental animals, and high
concentrations of the investigated substance. The most pertinent data found
in the literature relative to HMD effects on the respiratory tract were found
in two experimental papers published by V. S. Filatova, V. A. Russkikh, He.
V. Chernet, and T. G. Lapina in 1951, and by A. P. Martynova in 1957.
Analysis of HMD biological effect in acute and chronic inhalation experiments
has shown that it is a toxic substance having a general resorptive action re-
sulting in the constriction of the capillary and small blood vessels and in
-------
dystrophic changes in the lungs, liver, kidneys, spleen, heart, brain, spinal
cord, stomach, and intestines. HMD depresses effect on the central ner-
vous system, lowers arterial pressure, and changes the morphological com-
position of the blood. According to Martynova, this toxic effect occurs at
concentrations of 1 mg/m3 and higher. No lower concentrations have been
studied. A literature survey conducted by Paton in 1957 indicated that the
toxic action of HMD rested in the capacity of mono- and diamines and other
related chemical substances to liberate bound hystamines and other related
biologically active substances. Diamines and aminee of high carbon atom
numbers possessed this effect in the highest degree.
In a paper published in 1964 dealing with the reflex action of HMD
in man, the present author proposed 0.001 mg/m3 as the maximum allowable
single-exposure concentration in atmospheric air. The present paper pre-
sents results of animal inhalation tests of low HMD concentrations 24 hours
daily for 3 months. In this study 60 white male rats were divided into four
equal groups. Animals of Group 1 inhaled air containing HMD in concentra-
tions of 1 mg/m3 or the maximum allowable for industrial premises. Ani-
mals of Group II inhaled air containing HMD in concentrations of 0.64 mg/m3 ,
or the maximum single-exposure allowable concentration in atmospheric air.
The animala were checked during inhalation, also for a month before and af-
ter inhalation exposure by the following tests: weight, reticulocytes, leuco-
cytes, and eosinophile counts; luminescence and phagocytosis; Vi-antibody
titre after immunization against typhoid fever; motor chronaxy of the an-
tagonist muscles; and rate of coproporphyrine elimination. After the inhala-
tion period, internal organs were studied for macro and microhistological
changes .
Table 1 presents the data obtained from determining the HMD con-
centration on a daily basis.
lable I
UOrttE.ilrtsTIOr,;, OF Ht X AME Tri» L tNi_L I A« I >Jt IN IHE
CHA^btHb »N Ui/M3 JF CHABBEH AIH
fin (B« 1
n'~°J(J
I
II
III
1
M
1,086
0,043
0,0012
j
0.25
0,0159
0,0005
m
0,018
070014
0,00004
Tolal
Ana ly set
129
118
115
Animals were weighed once every 10 days. Animals of Group I
lost 5. 1% of their preexperimental weight in the course of inhalation ex-
posure, as shown by graphs presented in Figure 1.
The other two groups presented no differences in weight from the
controls. In her experiments Martynova also noticed that during HMD
-------
tos
wo
F'd
\17/X1I 28/XH 8/1 19/1 29/1 8111 18/11 28/111 8/111 IS/111/I IV
i '. boa/ wer^M OT eayen Rent»I an:nal& through
chru«a>OPfraconc«ntr4l»ona. -,
I «^/™3j V - 0,04 -ad/"3) 3 - 0-001 Pg/oJf
H - tlion Qir
inhalation by white rats 6 times
daily for 6 months in concentra-
tions of 10 and 1 mg /m3 the ex-
perimental animals lost weight
as compared with the controls.
Reticulocyte counts made
by the A. P. Yegorov method
showed that only 1 mg/m3 HMD
concentration induced after two
weeks a statistically reliable
(R = 0.99) increase in reticulo-
cyte count as compared with the
control group. However, two
weeks later the count returned
to its normal level, as shown by
curves in Figure 2.
The leucocyte count, de-
termined by the N. M. Nikolayev
method (1954), was practically
the same as of the control group.
It has been established
by V. N. Chernigovskaya, A. Ya.
Yaroshevskii (1953, 1957), K. Kh.
Kyrche (1956), et al, that the
eoeinophile level, like that of
other formed blood elements, de-
pended on the functional state of
the brain. Investigators such as
Thorn (1953), I. A. Eskin, G. M.
Vidavskaya (1956), Aschekenasy
(1957);!. A. Eskin, N. V. Mik-
haylova (1953), et al. , found that
the hormonal system, consisting
of the anterior pypophyseal lobe and the adrenal cortex, that controls the
hypothalemus, affected the eosinophile content of the peripheral blood. Re-
cent pathological investigations pointed to functional shifts in this hormone
system which effected changes in eosinophile counts in man and in experi-
mental animals when toxic substances were administered in concentrations
present in the air of industrial workrooms (Ye. I. Spynu, 1962; L. E. Gorn,
1963; et al.).
It has been shown that an allergic reaction raised the eosinophile
count in the peripheral blood, as a protective reaction, reflecting the eosino-
philes' capacity to reduce the stress of the allergic reaction by inactivating
the hystamines, heparines, and other biologically active substances the
.?.. No of ratJcuJocy t«s in the blood
• j thfi ^antod of chronic exptrtoenlal
iation of !o* henaoethyIoned^affline
concentrat tone
. 2 . 3. and ^ bane e& «r Ftj= l>
- 10 -
-------
number of which in free states increased sharply, as was shown by I. P.
Lepner, Ye. S. Brusilovakiy, 1961; L. D. Grishpun, 1962, etc. It is im-
portant that the effect of HMD on eoainophile count in the peripheral blood
under the conditions of this experiment be clearly understood. Using the
S. M. Bakman method (1958), the present author made absolute counts
during the first month of inhalation exposure once a week; and thereafter,
once fortnightly, totaling 223 blood analyses.
Jffff-
16IXI2Will II 9/1 17/f ?9/f f3/Il 27IIf I3IW7J///Vf/IV
Fie
Fig. 3 shows that af-
ter a week of inhalation ex-
posure all groups manifested
a drop in eosinophile counts,
and at the end of the second
week the rats of Group I re-
acted most profoundly; how-
ever, in comparison with the
controls they evidenced no
statistically reliable differen-
ces. For technical reasons
the interval of inhalation ex-
posure was interrupted for two
days (15 and 16 January) at the
end of the first month; 14-16
hours after its renewal a
sharp increase was noticed
in the amount of eosinophiles
in the blood of animals of
Groups I and II, particularly
in rat No. 3 of Group I and rat
No. 4 of Group II, whose counts before-and-after were respectively 474/1313
and 200/837 per m3 of blood. Subsequently the eosinophile count in the ani-
mals of the first two groups dropped to a constant level at the end of the ex-
periment with statistical R = 0.95. In these groups the eosinophile counts
dropped more than 50% in comparison with the initial counts, pointing to com-
pensatory functions (S. M. Bakman, I960; et al.), inherent in the hormonal
system (anterior hypophyseal lobe, adrenal cortex), under HMD in concentra-
tions of 1 and 0.4 mg/m3 . Animals of Group III had an eosinophile count of
0.0001 mg/m3, which was close to that of the controls. In other words, the
tests indicated a non-specific reaction of the organism to the negative effects
of microconcentrations of atmospheric contaminants. Many authors (M. N.
Meysel1, V. A. Sondak, 1956; S. S. Sergei1, A. A. Klimenko, 1957; M. Ya.
Khodas, 1959; Ye. B. Zakrzhevskiy, L. G. Vasil'yeva, 1936; et al.) have
shown that the nature of leucocyte luminescence during vital staining •with
acridine orange changed in blood diseases, or in ionizing radiation, the
leucocytes fluorescence changing from green to yellow, orange, and red as
a result of cellular physiochemical changes.
3. Absolute eoBmo^hilic count in the blood of
pen a-en til emails durirj rcricd of chronic lohal-
i jn of Ion he»«oetny I ened unine concmlrtt i ona.
I, 2, 3, end M aina »• m .c«j. I.
- 11 -
-------
A. D. Semenenko (1963) and P. G. Tkachev (1963) observed a
change in leucocyte luminescence following the inhalation of low aniline
concentrations. M. I. Gueev and K. H. Chelikanov (1963) noted the same
for amylene. For this reason the present author used acridine orange in
1:50,000 concentrations in a physiological solution. 0.02 ml of blood was
taken from the animal's tail vein and placed in a teat tube containing 0. 18
ml of the acridine solution. Then a single drop of fluorochromed blood
from the test tube was placed under a microscope in the usual manner. A
Reichert microscope was used for immediate examination at 5 x 60 magnifi-
cation. One hundred leucocytes were counted in each specimen, and the
percentage changes of leucocytes with illumination were calculated. Al-
together, 182 blood samples were thus examined.
Fig. 4 shows that after
a week's inhalation the Group I ani-
mals' leucocyte illumination per-
centage rose and then, toward the
beginning of the second month, re-
turned to the initial level. In some
animals the changed cells reached
21-25%. This increase was less
pronounced in animals of Group II
(9-11% in isolated cases), and re-
turned to normal after 3 weeks in-
halation. This phenomenon was
noted also by M. I. Gusev and K.N.
Chelikanov (1963). The increase in
the % of leucocytes with varying il-
lumination, observed in the case
of animals of Groups I and II, is
statistically reliable (R = 0.05 and
0.99). Yellow and orange tones
predominated, with red appearing only occasionally. Data of Group III dif-
fered little from those of the controls.
The protective and adaptive functions of the organism depend upon
its general physiological reactivity, which in turn is a function of many fac-
tors in relation to the external environment (physical, chemical, biological,
alimentary, etc.) which, in acting upon the organism, can alter its resistance
and susceptibility (A. D. Ado). The immunobiological reactivity of the or-
ganism can serve as a clue to its general biological reactivity, which in turn
can serve as a clue to specific and general resistance.
Studies in occupational pathology (T. V. Rasskazova, 1958; A. P.
Volkova, 1959; V. K. Havrotskiy, I960; A. I. Pakhomychev, I960; A. F.
Stoyanovskiy, 1961; et al.) indicated changes in organism's immunobiological
251X11 HI 911 nil 29/1 13111 231 III
ij- M. >'eii_enl of luminescent leucocytes dur-
,j Ihe (.er'ad of thruric inhalation of lo
ine -oncontr»t i one
satno as in Fig. I.
ID 2, 3, and
- 12 -
-------
reactivity under industrial conditions. The same is true of experimental
animals exposed to Inhalation of poisonous substances for prolonged intervale
(JSQ0 , COB , lead, tetraethyl lead, CCl^ , nitrobenzene, benzene, anilene,
etc.)- Depression of phagocytosis and of antibody formation, under the ef-
fects of harmful substances in concentrations at or exceeding the MAC levels
for indoor air under working conditions has been recorded, which induced
the present author to study the possibility of reducing the immunobiological
indices during chronic HMD microconcentrations inhalation.
In the study of phagocytosis a 24-hour culture of staphylocuccue in
a concentration of 500 million per ml was used. Equal quantities (0.06 ml)
of blood, in a 2% sodium citrate solution, and a microbe suspension were
used. The phagocytosis reaction lasted 20 min at 37°C. After centrifuga-
tion a leucocyte smear was made and stained by the Bimza-Romanovskii
method. One-hundred neutrophiles were counted along with the percentage
of active leucocytes (extent of phagocytosis) and the phagocytic index was
determined. This approach was suggested by V. S. Gostev and associates
in 1950. To determine the phagocyte activity of the leucocytes 204 blood
analyses were made, the first two a week apart, the remainder every two
•weeks.
The results indicated
that the percent of active leuco-
cytes in animals of Group 1
(1 mg/m3) dropped sharply af-
ter one week's inhalation of
HMD, while after two weeks
it began to rise to a level above
that of the control group. This
was followed by a new depres-
sion in phagocytic activity
which persisted to the end of
the inhalation period. The mo0£
pronounced change occurred in
rat No. 14 of Group I, pre-inocw-
lationphagocytic index of which
was 99.3-100; after a week of
inhalation it dropped to 55.1,
rose again to 92. 9 after two
weeks, and finally remained
stationary at 79.3. At the end
of the inhalation period the percent of active leucocytes in Group I animals
rapidly returned to the background level. However, such statistically re-
liable results are not less pronounced than the changes noted in Group II
animals (0.04 mg/m3).
ISM 2VXJI
lain fS/ffJ 2/lV II/IY
Phasjoc)li'- laiicccyt* activity on th« belli of
G indrcolcr in enptrtrecntiI tniftili during chroo«
ic onporiQontil irhilatiof ol Ion HB thf I en»d i «•! ne cwv-
cenlra I1 one
I, 2, 3, k, sane as in Fie. I.
D*aro» of reliability - e - 9bi, b - 9aJ, c - 99.9J
- 13 -
-------
2 •
I -
itiw
Fig. 6. Phogocjrlic laucoayto activity en tho bins of
intonoivo indicator In «nn>ri nonta! aniocfB during the
poricd of chronic oof^r i rc on to I irhalotien of Ion h«x«-
Qo thy I an«a i of i no cancantrtt leno
IB 2B 3, ana 4 BOBKJ as in Fig, )„
Fig. 6 shows that the
intensity of the phagocytic pro-
cess also underwent analogous
and reliable changes in the ani-
mals of Groups I and II. Animal
No. 14 in Group I again exhibited
the most pronounced changes.
Prior to inhalation the phago-
cytosis index was 7.4-7.9,
while after a week it dropped to
1.3. After two weeks it rose to
5.3, then dropped again to 2.5,
where it remained, with several
fluctuations, to the end of the ex-
periment. Changes in phagocytic
index in animals of Group III
(0.001 mg/m3) did not differ sub-
stantially from that of the con-
trols. On the basis of the above
it can be stated that the dynamics of the phagocytic process in all animals of
Groups I and II followed a generally regular pattern of depression. A rise
in phagocytic activity in the second inhalation week following a sharp depres-
sion indicated that the phagocytic process had undergone phase changes dur-
ing chronic inhalation of HMD by rats exposed to concentrations of 1 and 0.04
mg /m3 .
Experimental animals were vaccinated with Vi typhoid fever vac-
cine manufactured at the Moscow Scientific Research Institute for Epidemi-
ology and Microbiology. Since nothing was found in the literature describing
procedures for immunizing rats, the investigation was of an exploratory
character. Vaccination was initiated a month after HMD inhalation, when
changes could be expected in the organism's reactivity. The vaccine -was in-
jected subcutaneously into the hind leg paws. The first injection consisted
of ZOO million typhoid fever bacilli and 0.04 mg of typhoid fever Vi-antigen.
After a week the second and following injections consisted of 300 million
typhoid fever bacilli and 0.06 mg of Vi-antigen. After standing a day,
0. 5-0. 6 ml of blood was taken from the rats' vein and the serum was allowed
to separate from the clot at 34 C. The Vi-antibody titre was determined by
the passive hemoagglutination reaction recommended by N. A. Kraskina
and N. M. Gutorova in 19&2 Before the vaccine was administered the Vi-
antibody titre in the experimental animals was practically nil. A week later
Vi-antibody titre in the Group I animals was lower than in the controls, not
even a trace of it was found in rat No. 12 of Group I. A somewhat less ex-
pressed phenomenon was noted in rats of Group II.
A statistically reliable difference (R = 0.95) in the Vi-antibody
titre was noted only in animals of Group I a weak after the second inocula-
- 14 -
-------
tion. After injections at the end of the inhalation exposure and during the
recuperation period, the titre did not differ significantly from that of the
control group. Animals of Group III which inhaled the lowest HMD concen-
tration (0. 001 mg/m3), manifested no titre-levele differing from the con-
trols throughout the test period.
Studies of coproporphyrin elimination rate via. the urine were con-
ducted using an SF-4 spectrophotometer as recommended by M. I. Gusev,
Yu. I. Srmrnov, I960. Urine was collected daily in special containers every
10 days from rats of each group. Before inhalation exposure and during the
recuperation period, 3 examinations were made for each rat, and 9 during
the inhalation exposure. Fluctuations in the rate of coproporphyrin elimina-
tion via_ the urine of Group I animals during the HMD inhalation period point-
ed to a tendency toward reduction of its excretion rate on some days of the
fiyet half of the experimental inhalation; however, the difference was not
statistically significant. A reliable difference was observed during the re-
cuperation period, which was related chiefly to a drop in the control animala.
No substantial differences from the control animals were found in animals of
Groups I and II.
Results of motor chronany (ratio) tests showed that disturbance of
the normal ratio occurred in animals of Groups I and II a month after inhala-
tion exposure was initiated; it persisted at this level with certain fluctua-
tions for the next 40 days of the inhalation exposure period (Fig. 7), after
which the normal ratio was restored. A statistically reliable difference
was observed in animals of Groups I and II on the 50th day of inhalation
(R = 0. 99 in both groups). In animals of Group III the chronaxy ratio varied
•within the limits of physiological fluctuations.
Internal organis of 26 rats were examined by sacrificing them im-
mediately after inhalation, and 19 animals were sacrificed Z months later to
study latent effects.*
Disection of Group 1 animals (1 mg/m3) revealed a fatty depletion
of the hypodermic tissue. Examination of the liver disclosed venous plethora
(Fig. 8), vascular wall plasma infiltration and perivascular lymphoidal-
hystiocytic nodules. The liver glycogen content was stable, with a slight
reduction in isolated cases.
The miocardium showed the presence of individual or groups of
unevenly stained fibers on the background of venous plethora. The transverse
striations and nuclei were particularly marked. Only plethora and lymphoidal
^Doctor of Medical Sciences V. P. Osintsevaya supervised the pathomorpho-
logical examinations, which were conducted at the Pathomorphology Labora-
tory of the A. N. Sysin Institute of General and Municipal Hygiene of the USSR
Academy of Medical Sciences.
- 15 -
-------
infiltration along the tubules were found in the kidneys. Lipides were dis-
covered in all three zones of the intestines.
WJUWJUffl 19:1 29if Sit
29II 8IIII 161112(111
^•a> 7. Uu*cle »nt»9ist motor ehroo«)iy r»tio in •xptri
••ntal mi*«|s •Hooted to chrontc *>f^*ri ««ntt I >nh«l»tion
or h»*«««Uiy l«n«rtf«»ir« c«nc*ntr«t t«na
'» 2, 3, »nd U v»»« >« in
8. Liver ««ction of tni««l Mo. 2 of firoup I h«»»te»ylin
• t«m«d. tt*snif i cat ion 10 K 10.
The capillary brain
network of Group 1 animals
was slightly distended. Cells
with vacuolated cytoplasm,
with dispersal or thickening
of the Nisslevskiy matter
blocks and very occasional-
ly with cromatolysis were
found in the cerebral cor-
tex and subcortical nodes.
Cells with pericellular ede-
ma were also encountered
(Fig. 9).
The liver, miocardi-
um, kidneys, and brain of
the Group II animals (0.04
mg/m3) showed changes sim-
ilar to those found in Group
I, but in a less pronounced
degree. The quantity of in-
testinal lipides suffered a
reduction.
Examination conduct-
ed after 2 months of inhala-
tion exposure showed that
animals of Groups I and II
exhibited a purulent penu-
monia of a rather severe in-
tensity which rose with in-
crease in the HMD concen-
tration.
Organic changes in
animals which inhaled HMD
in concentrations of 1 and
0.04 mg/m3, manifested a
monotypical character both
in immunized and non-im-
munized rats. Follicles were
found more often in the spleen
of the immunized animals
than in the nonimmunized, and
- 16 -
-------
f'i, 9. Brtin •tction of tni»«l I of Group I, •tttnod by Ni*»%l
••tbod. Magnification 90 a (S.
their size was considerably larger. The number of plasmatic cells in the
immunized rats increased markedly, in connection with which the formation
of antibodies was observed. The morphology of the immune process in ani-
mals of Groups I and II (1 and 0.04 mg/m3) was less pronounced than in the
other two. Group III animals (0.001 mg/m3) exhibited no morphological
changes from those observed in the controls.
Results of the experiments point to the unfavorable effect of small
HMD concentrations (1.0 and 0.04 mg/m3), most strikingly in the loss of
weight in Group I animals, and in the increased number of reticulocytes.
Animals of Groups I and II also exhibited an eosinopenic response and an in-
crease in the percent of leucocytes with a change in the luminescence.
The results also show that such low concentrations of HMD can af-
fect the state of the organism's immunobiological reaction inducing depression
of the phagocytosis processes, of antibody formation, and other immuologi-
cal processes. Animals of Groups I and II also negatively affected the nor-
mal ratio of antagonistic-muscle chronaxy, as well as pathomorphological
changes in the internal organs.
The HMD concentration inhaled by animals of Group III (0.001/m3)
was the only one having no effect in any of the tests.
-17-
-------
CONCLUSIONS
1. Continuous inhalation of HMD in 1 mg/m3 concentration for a.
period of three months unfavorably affect the organism of experimental
animals inducing loss of body weight, raising the reticulocyte count, pro-
ducing eosinopena, increased leucocyte count, lowering the phagocytic
index and the rate of Vi-antibody productions, upsetting the normal motor
chronaxy ratio in the antagonistic muscles, and inducing pathomorphologi-
cal changes in the internal organs.
Similar but milder changes are caused by an HMD concentra-
tion of 0. 04 mg/m3 with the exception of loss of body weight and the num-
ber of reticulocytes.
2, In 0.001 mg/m3 concentration HMD administered under identi-
cal conditions haa no effect on the organism of the test animals.
3. The average daily maximum permissible concentration of
HMD in atmospheric air in inhabited areas can be the same as was previous-
ly proposed for the maximum single-exposure concentration, i.e., 0. 001
mg/
3
m
HYGIENIC BASIS FOR THE DETERMINATION OF THE ALLOWABLE
CONCENTRATION LIMIT OF DIVINYL IN ATMOSPHERIC AIR
G. Kh. Ripp
From the Department of General Hygiene of the
M. I. Kalinin Omsk Medical Institute
Divinyl (butadiene-1, 3) is a baaic ffionomer used in the production
of synthetic rubber. As a diene hydrocarbon with two conjugate double bonds
it polymerizes easily and lends itself to the manufacture of various kinds of
synthetic rubber. Under ordinary conditions divinyl is a gas having a most
unpleasant odor and a density of 1. 87; at a temperature of minus 4. 5° C
divinyl condenses into a colorless, transparent, volatile liquid. It is
weakly soluble in water, dissolves easily in acetone, benzene, and amylace-
- 18 -
-------
tate (A. A. Petrov, 1953) and less so in ethyl alcohol (P. Vataulik, I960).
i
V. A. Pakrovskiy (1955). V. S. Freydlin (1935), L. F. Larionov
(1954), E. N. Levina (1948), Henderson, Haggard (1930) and others charac-
terized high divinyl concentrations as narcotic and a mucous membrane and
hemogenic organic irritant. The maximum allowable concentration for in-
dustrial premises is 100 mg/m3 .
Disorders of the central nervous system such as rapid fatigue,
headache, drowsiness have been observed among divinyl workers. Rhinitis,
layngitis, pharyngitis, conjunctivitis, changes in the composition of the peri-
pheral blood, dyspeptic phenomena, drop in arterial pressure, etc. were
recorded by S. B. Glauberman (1957), L. I. Gefter, Ye. D. ShuTman (1935),
S. D. Polyak, N. I. Pil'man, A. I. Zaretskaya, andS. M. Kleyn(1935),
A. A. Yegunov (1940), E. N. LevLia (1948), A. A. Orlova and G. N. Mazu-
nia (I960), L. F. Larionov (1954). Most of the symptoms appeared after
contact with divinyl has been discontinued.
Animals suffering of chronic divinyl poisoning usually exhibit
slight increase in mucous cells of the epithelium of the bronchi, local micro-
cellular infiltration, emphysema of the lungs, hyperplasia of the bone mar-
row with an increase in immature forms, an increase of myeloid elements
in the red pulp of the spleen, and symptoms of irrigation in the lymphatic
nodes. (L. F. Larionov, 1954).
Most of the cited investigators described cases of industrial •work-
ers exposed to divinyl in heavy concentrations. No data -were found dealing
with the effects of low concentrations in atmospheric air. The widespread
use of divinyl by synthetic rubber industry prompted the present author to
study the effects of divinyl microconcentrations to determine a hygienic basis
for its maximal allowable concentration in atmospheric air, which is the pur-
pose of the present paper. The -work was conducted under the supervision of
Candidate of Biological Sciences M. D. Manit; and a spectrophotometric
method for determining microquantities of divinyl under experimental con-
ditions and in atmospheric air was developed, and the optimal parameters
for selecting air samples were determined. The method enabled to deter-
mine divinyl in atmospheric air under natural conditions in the presence of
other synthetic rubber production compounds such as alpha-methylstyrene,
isopropylbenzene, butane, and butylenes.
The threshold of odor perception was determined by a generally
employed method (V. A. Ryazanov, K. A. Bushtuyeva, Yu. A. Novikov,
1957). However, since under ordinary circumstances divinyl is a highly
volatile gas, certain additions to the method were made, related to gas
dosing in the system. An experimental mixture of gas and air in the re-
quired concentration was made in 40 liter steel cylinders (oxygen or nitro-
gen) by exhausting them and then filling them with a small amount of gas-
- 19 -
-------
eous divinyl. After being introduced into the cylinder the divinyl was rare-
fied and evenly mixed with a large volume of compressed air up to a pres-
sure of 20-30 atm or more. A constant divinyl concentration was main-
tained -when the mixture was taken from the cylinder through a reducing
valve. The divinyl concentration in the cylinder was computed; in the gas-
eous phase it was measured by an SF-4 spectrophotometer.'i By changing
the rate of mixture flow from the cylinder and rarefying it with pure air
forced into the cylinders by means of air blowers at a rate of 15-20 1/min,
it was possible to reach the divinyl concentration required for inhalation
in the experiments. This method of gas mixture feeding insured an even
and constant divinyl concentration in the cylinders. By using a reducing
valve to change the air-divinyl flow rate from the cylinder, it was possible
to obtain the necessary gas concentration in the system. The rate of the
gas mixture flow was determined by a rotating flow meter installed below
the reducing valve.
Sixteen persons 18 to 36 years of age having no deviations from
the normal state of the ear, throat, and nose were used in the odor per-
ception threshold tests. All volunteers were first familiarized with the
odor of divinyl. The acrid and unpleasant odor of the gas is easily dis-
tinguished and remembered. The divinyl concentration in the cylinders
was checked before and after each experiment. Altogether 336 tests were
run at divinyl concentrations of 6.0, 5.2, 4.8, 4.4, 4.2, 4.0, and 3. 8
mg/m3 . Results are recorded in Table 1.
Title I
J
No. of
obeervg-
t lone
6
6
3
1
Hinioa 1 per-
cmf. t i ve dp —
v my 1 coocnsi
MBK i IQA 1 non —
porcepl. di-
,,,ny| concn*.
in "g/o ' m "i9/B
4.0
4,2
4,4
4,8
3,8
4,0
4.2
4.4
0
Total ob-
e«rvit ione
150
126
48
12
The data show that the thresh-
old as established by the experiment
was 4 mg/m3 . Effect of divinyl on eye
sensitivity to light was studied by the
method recommended by the Commit-
tee on Sanitary Protection of Atmos-
pheric Air, using 4 persons aged 20-
25 with normal vision and odor percep-
tions. Sensitivity to light studies were
conducted after a 10-min disadaptation. The first 4-6 days the volunteer in-
haled pure air as described elsewhere; the following days divinyl was intro-
duced into the cylinder in concentrations below the olfactory threshold be-
tween the 15th and 20th minute with the subjects seated in a dark room. Test
persons were tested on three successive days to determine the effect of the
gaa in the above cited concentrations.
The first concentration, 4.2 mg/m elicited no odor perception
response; however, as in the case of 4. 0, all testers became more light-
sensitive on the 20th minute as compared with pure air. All results were
statistically significant.
- 20 -
-------
Two of the volunteers, (P.A.K. andU.A.F.), manifested high-
er sensitivity at 3. 8 mg/m3, but in only one of these was the increase
statistically signigicant. In the remaining cases the sensitivity change was
not statistically reliable, even with a certain change in the sensitivity curve.
No effect was detected at 3.6 mg/m3 gae concentration as shown in Fig. 1. ,
10
15 ZO _ 25
T\m» in 01nutti
Fig. I. Effect of divinyl inhalation IB different concentration*
, an eye sensitivity to lljht of f«««l« t»it p«r*on P.A.K.
I - Cle.n air; 2 - k,2 ng/»3| 3 - *,»i/"3l •• - 3.8 «g/»*,
5 - 3.6 •8/«3
The reflex-action threshold for eye sensitivity to light was, thus,
3. 8 mg /md , which is about the same as that for odor perception.
i
The central nervous system threshold was determined by generat-
ing an electrocortical conditioned reflex as described by K. A. Bushtuyeva,
Ye. F. Polezhayev, and A. D. Semenko in I960 using an electrocencephalo-
graph manufactured by the Kaiser Company, * and 24 test persons. Of these
2 men and 2 women aged 18-30 were selected having a clear alpha-rhythm
and well defined synchronization to light. All test volunteers-first partici-
pated in a similar experiment and in the course of their training gave good,
*This work was conducted at the Department of Municipal Hygiene of the Cen =
tral Institute of Physician Training under the direction of Doctor of Medical
Sciences K. A. Bushtuyeva.
- 21 -
-------
clear encephalogram readings. Tests were made daily at the same hour
for each individual.
The divinyl concentrations tested were 3. 8, 3.6, and 3. 0 mg /m3 .
At the first concentration two individuals developed a conditioned electrocor-
tical reflex; A. S. achieved ,'thla at the sixth session on the first day and at
the fifth on the second day. This concentration had an analogous effect on
the 3rd and 4th days, but the two lower concentrations did not provoke a
reflex in this test person. At the 5-17th sessions G. Kh. developed the re-
flex at 3.8 mg/m3, had a very slight reaction at 3.6, and none at 3. 0. R. P.
and G. G. developed a conditioned electrocortical reflex at 3.6 mg/m3 (Fig.
2) and none at concentration of 3. 0 mg/m3 (Fig. 3).
T«ble ?_
EYE SENSITIVITY TO LI&HT DURING EXPERIMENTAL OIVINTL IN-
HALATION (ON THt 20TH MINUTE OF INHALATION IN PfcHCENT OF
THE IbTH MINUTE OF INHALATIUN)
o &
W
a L.
0 i
C S
b.U.B.
D.E.N.
L
a
C
0)
o
128
III
112
123
Bg/n^ of di» my 1
4.2
X
156
129
176
222
* >s
c -Z
IT .
99,9
99,9
99,9
99, 9 i
4.0
j;
131
116
146
128
,*
C —
tr -
99,0
90,0
99,9
50,"0
3.8
*
\130
no
120
121
i~
C .1
IX .
75,0
50.0
98,0
50,0
3.6
X
128
112
-
* >.
• —
ac. .
o
No
oh»nge
No
^fi^^K^^^^^^^^
(>*-1V'*'«'-M"*V'V«M^»«itV^^
^^
Fig. 2. Electroencephalogram o.
6tn combination during 3
ogram of R, p. Conditioned reflan alphk-rhythB daaynchronllit ion
.6 Bg/D3 divjnT/. mhalitioni AC - inhalitien of I nveat i gated gia
»' oentriiioil ac _J,,tohing of lioht
onlittion on the
con-
- 22 -
-------
^
^J*>^>^vv^^ • * ~ M^to»»sr^ - v^
Fi£. 3. Eleclroenctphalograt of R. F. No condi t loned-ref !•« ilpht rhyth« d»Bynchroni lit IOR dur-
ing inhtlition of 3 eg/"3 of divinyl.AC tnd BC •••« •• In Fig. 2.
In other words, a. divinyl concentration of 3 rng/m3 had no ef-
fect on any of the test persons.
A comparison of thresholds determined for odor perception, eye
sensitivity to light, and electrical brain activity (4, 3. 8 and 3. 6 mg/m3 ,
respectively) leads to the assumption that 3.0 mg/m3 can be recommended
as the maximum single-exposure concentration of divinyl in atmospheric air.
For 81 days (19 May to 7 August 1964) -white rats were exposed
continuously to divinyl to determine its resorptive action under condition of
uninterrupted, prolonged inhalation, as well as to establish the average
daily maximum tolerance dose. Four chambers with a volume of 0.1 m3
(Fig, 4) were used. Air was run into the chambers at the rate of 30 1/min
by a VPP-4 timed air blower. Divinyl in gaseous form was added to pure
Four groups of rats -were used. Group 1 served as the control
(pure air), and the remaining three were exposed to concentrations of 1.03 ±
0.006, 3.08 ±0.006, and 30. 8 ± 0.75 mg/m3, respectively.
The divinyl concentrations in the chambers were checked daily by an
SF-4 spectrophotometer in a gas cell, as well as after passing through a solid
absorber. The temperature and humidity in the chambers were also checked
during the inhalation exposure. These indices were comparatively stable.
Animals were fed in the chambers twice daily and received a varied diet
(boiled meat, milk, groats, bread, oats, grass, vegetables, and fish oil
once a -week). The state of the animals and changes occurring during thepso-
longed exposure to divinyl was checked by a variety of teats. Results
- 23 -
-------
described in the text. Before initiating the test, the experimental animals
were quarantined and observed for 2 weeks and background studies were con-
ducted related to the indexes chosen as bases to evaluate the following changes
.
•f «MM»ur« ch««o«
trailing d*vic»a
Fij. *. 6«wr«l vtM of ••M»ur« ch««c«r» *ith O«M con-
rat) ing d*
Each chamber housed one group of 14-15 male rats of approximate-
ly the same weight. At the end of the inhalation exposure some animals were
sacrificed immediately by decapitation, the living were kept under observa-
tion for an additional 2 weeks. At the end of the recuperation period these
animals were also sacrificed and their organs studied histopathologically.
The following anatomical and functional changes were noted in the course of
inhalation exposure:
Animals' General Condition and Weight. During the preliminary
inhalation days Group IV animals were restless probably due to the stimu-
lating effects of the gas. This reaction was less noticeable in rats of Group
III. Animals of the first three groups appeared healthy and active; they ate
well throughout the exposure and no difference of any kind was noted in
their general condition. They gained weight. However, at the beginning
of the 3rd month rats of Group IV became visibly less active, their appetite
fell as did their rate of gain in body weight (Fig. 5). The weight differ-
ence reached statistically reliable values only at the end of the exposure
period. After the end of the inhalation exposure, Group IV rats gained
weight rapidly.
State of Peripheral Blood. The blood of 5 rats in each group was
examined every fortnight at a given time of the day. Blood was taken from
the tail vein. The parameters investigated were erythrocyte count, hemo-
- 24 -
•
-------
globin (in a photoelectric erythroht mometer), and reticulocyte count. The
red blood changee in the rats during the inhalation period were not pro-
nounced.
280'h
fij. i. Uynasica of »v«r»3» ••iyht r»ts. of different group*
in th» ceurM o( 24-hour divinyl inhalttlon.
I - Control groupj 2 Group ll| I BB/"3 «f divinrl; 3 -
Group HI, 3 "9/»3» "* - Group IV, 30 "9>
Leucocyte counta were made by the generally accepted method in-
cluding the leucocytic formula.
By the end of the inhalation period only rats of Group IV manifested
a alight leucocytoaia, which became statistically reliable only on the 80th day.
Rats with slight leucocytosis also manifested shifts in the percent content of
lymphocytes and stabnuclears, but theae differences were not statistically
reliable.
Activity of the Whole Blood Cholinesterase. Whole blood cholin-
esterase activity is related to the function of the mediators and unconditional-
ly affected the state of the central nervous system and the tranemittal of
nerve pulses from the muscles. Whole blood cholinesterase activity also
- 25 -
-------
caused rapid hydrolysis of acetylcholine, and its inactivation allowed the
latter substance to accumulate.
In the present study 5 rats from each group had been examined
twice before inhalation exposure and once every fortnight during exposure
by the Nicholle modification of the Limperos and Ranta method. This meth-
od is described by Merewether (I960). Visual determination of acetylcholine
hydrolysis time served as the index of cholinesterase activity.
50
iS
iO
JS
JO
25
20
P
F n
-
L J'
^^
0
r , or to
ho let i ixi
••"--. /
^f- ' -,'
X
— — """"-•" ' - i7'
7 22 38 SS '73 8L
Uaya of
tnhelBt i en
As. Fig. 6 shows,
\ changes in this activity were
\ significant only in animals
\ of Group IV. Statistically
reliable differences were
\ noted in this Group on the
V 7th, 38th, 56th, 73rd, and
•- V"? Rlstd a_v s nf ^xno s\irp Siib —
• • / sequently the indices re-
turned to almost the initial
values.
;
Accupor*.- Optical Properties of the
p»r7«}d Blood Serum. The capacity
Frg> 6. Diviryl affect on activity of •*!<>«« blood cholinea-
toroDo of different r«t groups during continuous 24-hour eon-
j i . . _ **
d i v i ny I inhalation. j«oe notationB as in Fi
5.
of different substances to
selectively absorb light in a
given spectral band and at
one or another •wavelength is characteristic also of blood serum. Kament-
akaya (I960) mentioned spectral data on the uv absorption by rabbit blood
serum in the region of 230 to 300 millimicron wavelength, with maximum
absorption at 275 and minimum at 250 millimicrons. This selective absorp-
tion is explained by the presence in rabbit serum of aromatic amino acids of
tyrosince (maximum wavelength, 280 millimicrons) and tryptophane (maxi-
mum wavelength, 275 millimicrons). Kamentskaya believed that the changes
in the absorption spectra of blood serum were related to the serum protein
change, particularly tyrosine and tryptophane. Analogous data were report-
ed by A. D. Semenko (1963), who studied the effects of low aniline concentra-
tions on the optical properties of blood serum in rate. It has also been noted
that the maximum and minimum serum absorption corresponded to 278 and
250 millimicrons, respectively.
The ratio of optical density at \ to that at X . fluctuated in
healthy rats from 1. 8 to 1. 93 (absorption index) .
Before being exposed to experimental inhalation 20 rats (5 from
each group) had been examined to determine the spectral characteristics of
their blood serum. Spectrophotometer SF-4 and quartz cuvettes with a layer
thickness of 10 mm. were used. The serum was diluted 500 times by physio-
- 26 -
-------
I
logical solution. The maximum and minimum were identical with '
Kamentgkaya's figures, but in the near uv (around 205-208 millimicrons) '
fchero was also considerable light absorption. This peak was not used in ,
the prooent otudy because 205 millimicrons is actually beyond the resolv-
ing power of the SF-4. The aerum-absorption index before inhalation
exposure was 1. 7-1. 85. Even on the 9th day of exposure 3 rats of Group IV
manifested a rise to 2. 07-2. 36, while on the 30th day the index for all rats
in £hio Group stood at 2.03-2.36, where it remained to the end of the in-
halation period, falling to a lower level during the recovery period. No
marked shifts in this index were noticed in the rats of the other three animal
groups.
Regardless of changes in the absorption index of Group IV animals,
the location of the absorption bands did not change. Evidently shifts occurred,
under the divinyl effects, in the content of aromatic amino acids, and these
ehifte altered the serum light absorption principally as a result of increased
optical densities at \ =278 millimicrons. The general intoxication of the
organism in connection as the result of divinyl inhalation by Group IV rats
should have led to an increase in the content of several amino acids in the
blood.*
The mechanism of a spectral shift clearly needs more detailed
investigation, but the high sensitivity of the spectrophotometric method of
studying optical properties of blood serum makes it possible to detect the
finest changes in its protein composition. The index remained high for 2
weeks of the recovery period and did not return to the initial levels. Among
rats of Groups II and III there were no significant shifts, through the inhala-
tion period, either in absorption band or in optical density. Thus, the
changes in optical blood serum properties observed in rats of Group IV point
to changes in the state of serum proteins, associated with the effects of
divinyl in 30 mg/m3 concentration. At the same time, the absence of change
in the other three groups indicated that divinyl concentrations of 1 and 3 mg/m
are inactive in this test situation.
Comparing the blood data brings out marked shifts in rats of Group
IV with respect to leucocyte count, drop in cholinesterase activity, and
changes in optical blood properties even in the absence of pronounced changes
in red blood cells.
Arterial Pressure. The possibility of evaluating the effect of
divinyl on the animal organism during continuous, prolonged inhalation ex-
posure is related to the previously described hypotensive activity of divinyl
*At the same time the effects of divinyl on the optical properties of the blood
were being investigated, T. I. Lyutikovaya and the present author conducted
a similar study in which white rats were inoculated with cracking-process
Here, too, there were marked shifts in the absorption index.
- 27
-------
under industrial conditions. A second monomer in the manufacture of syn-
thetic rubber also possessed this hypotensive activity- -alpha-methyl -
styrene (E. A. Kapkayev, 1963) The rats1 arterial pressure was determined
by an anemic method (V. M. Chernov, 1947; A. Kh. Kogan, 1959) in a caudal
piethysmograph with a glass cuff attached to the caudal root. Pressure was
recorded on a sphygmomanometer and oscillations on a water manometer.
Arterial pressure was determined in 5 rats of each group after the animals
were habituated to the experimental apparatus and the initial level of arterial
pressure was ascertained. Subsequent arterial pressure checks were made
during inhalation exposure once every 3 weeks during evening hours.
Arterial pressure was stable in all rat groups before inhalation
exposure, and for more than 2 months of the experimental exposure (Fig. 7).
However, on the 7Znd inhalation day the average pressure in Group IV drop-
ped from 90-93 mm Hg to 75-77; it remained at this level to the end of the
inhalation test and throughout the recovery period. Beginning with the 72nd
day this blood pressure drop was statistically reliable. No change was noted
in the arterial pressure of the other rat groups. Thus, according to this
test the effective divinyl concentration was 30/mg/m3 .
t
0
a
c
t
i
a
O
L.
ifc
L.
V
tf
r0<7
1
30
80
70
SO
\
ff)
•
-
.
•
1
^\. 3
\.^
'"•
-_ ,
~
fore Pun o4 of «»p«rl-
prl_ «ent«l inhalation
h«l»-l V
lion ^ . _
t i i ii
-
^ ^'^ —
Z
. •
- — .
• lion
Pxriod
10
52
72 SO
Dtyo
Fi£. 7,. Blood pre«Bur* chw»9«& ia r«4a of diff»rtult groups in
the course of continuous 2^-hour div my I inhcliiion. £&•• ne-
tationa aa in Fig. 5.
Animals' Overall Motor Activity. Evaluation of the divinyl effects
on the central nervous system and the prevailing stimulation or inhibition in
the cerebral cortex was made by the method of objective recording of total
motor activity based on the principles of actograph. The actographic method
has been employed in recording motor and conditioned reflex activity and in
studying the daily periodicity of the animal organism (A. D. Slonim, 1953;
N. I. Kalabukhov, 1940; Ya. B. Maksimovich, 1954; O. S. Kishakovskiy,
1954). Impulses were recorded by a kymograph connected with pneumatic
capsules, cushions, electromechanical counters, etc. Special organic
glass chambers were built with a sectioned bottom consisting of seven mov-
- 28 -
-------
able plates fastened in the center on semiaxes as shown in Fig. 8. When
the pressure on one half-plate exceeded 0. 5 C/01* » an electric contact un-
der the plate closed, making it possible to record every movement of the
animal in the chamber. The contacts were connected to an electromagnetic
mechanical counter in a 12v d.c. circuit. During the study the counter re-
corded all incident pulses.
Fig. 8. Ch««bor for th« «tuo> of toUl »otor •ctivity of •»-
Three such chambers were used, each of which housed a rat for 3
hours. Each rat was examined not less than 3 times weekly. Five rats of
each group were studied actographically. Before inhalation exposure, rats
were oriented to the equipment in the chambers over a period of several
days. On the 2nd or 3rd day the rats became adapted to the equipment, and
their behavior in the chamber became normalized. Only in the first 5-10 min.
after being placed in the chambers did the rats evidence any reaction to the
environmental change. In order to exclude even this, the counter was intro-
duced 15 min. after the animal was placed into the chamber. The background
of total motor activity was studied for 3-4 days, the average data during the
initial period was taken as 100 percent. Later, motor activity was totaled
by weekly inhalation periods for each animal group. Resulting data are pre-
sented in block form in Fig. 9. The degree of change (reduction) in motor
activity in rats of Group IV beginning on the 7th week of inhalation exposure
somewhat exceeded the changes in weight, general well being and arterial
pressure of the rats. The objective-recording method made it possible to
record these shifts earlier than they appeared in the previous investigations.
The fall in total motor activity affected rats of Group IV to the end of the in-
halation exposure and through the recovery period.
•
- 29 -
-------
no
120
'00
so
so
VJ
10
tl
• ff '•J-iTT
_ r
t
li_ v
%rr T '
||
)
1 P
r*n ' n r -
> ': ? R ' . ' ' '
- - f ;
i <
. : \ '
\z$$n
'''".' M ;
, •' ,; • ' iii
•M ! i
m HI
* *
*.
\ \
\ h,
• ' '•&¥•
m
* .
. / •
fejT
It''
9. Ch»r3«* in Motor activity of rat* during continuous 2
sur* to diyinyi'irh«l«tion, ov*lu«tod on th« o*ai> of «e«kly
ptriods «na in poroont of initial v*luo>.
(-IV »ni«»| group*} 0 - basic background) I - 12 exposure •««*ti
Ib - 2b ••*!(• of rocovory period.
The differences in Group IV (compared with the controls) are sta-
tistically reliable. Rats of Groups II and II manifested no significant changes
as compared with the control rats. According to this test, divinyl was effec-
tive only in a concentration of 30 mg/ma.
Results of Pathohistological Examination. * Sections of the liver,
kidneys, heart, spleen, brain, and bone marrow (femur) were used. In addi-
tion, the mucous membrane of the tip and pharynx of the nose were examined.
The specimens were embedded by the usual methods, sectioned
and stained with hematoxiline-eosine. Brain specimens were also embedded
in celloidine, sectioned and stained by the Nissl method. Results were as
follows: the nasal mucous membrane was not intact even in animals of the
control group, which manifested focal and in isolated instances, diffuse in-
filtration; the epithelium appeared normal. The recorded changes may have
been caused by factors in the external medium (mechanical effects in connec-
tion with the large number of animals in the chamber, air humidity, etc.).
Group III animals (3 mg/m3 manifested disintegration of the mucous-mem-
brane epithelemium more frequently than the control group. A greater dif-
ference was manifested by rats of Group IV (30 mg/m3), which showed pro-
nounced inflammatory infiltration of the mucous membrane. In 3 animals
the process was diffuse, with massive exudation from the nasal cavity.
The lungs of Group II rats did not differ from those of the controls.
Most rats of Group III exhibited a moderate intermediate pneumonia and small
atelectasis foci and emphysema against a background of well-developed lymph-
oidal tissue. There were small atelectasis foci and emphysema also well-
developed hyperplasial lymphoidal tissue in nearly all rats of Group IV.
Many of the animals exhibited obvious intermediate infiltration, perivascu-
lar edema, and interstitial pneumonia. No histopathological changes were
noted in the brain sections of any control or experimental rats. The same
was true of the bone marrow. Only animals in Group IV manifested
*V. V. Veselovaya investigated and described the microscopic specimens.
- 30 -
-------
hemoeiderin accumulation in the red pulp of the spleen.
k-
Many animals of Group IV exhibited thinning of the hepatic columne
with almost total absence of clear cells, which predominated in the liver of
the other three groups. It must be assumed that these cells were rich in
glycogen. Individual rate of Group IV showed edema of the endothelium of
the sinuses. Marked changes were noted in the kidneys of Group IV rats.
Similar, but less pronounced, processes were seen in rats of Group III,
appearing as swelling of the epithelium of the convoluted tubules and the
appearance of protein and individual hyaline cylinders in the tubule lumens.
Data obtained from all tests during rats' inhalation exposure,
pathological investigation excepted, divinyl in a concentration of 1 or 3
mg/m3 caused no noticeable changes in the general state of the animals,
significant results being obtained in most tests only at 30 mg /m3 of divinyl.
Morphological changes induced in animals exposed to the inhalation of
divinyl in concentraion of 3 mg/m3 indicate that the gas has a stimulating
effect on the mucous membranes of the respiratory tract, on the lungs, and
on the kidneys. A concentration of 1 mg/m3 was ineffective, and can be
recommended as the average daily concentration in atmospheric air.
In 1964 this author investigated divinyl as an atmospheric air pollut-
ant in the vicinity of a synthetic rubber plant which began to operate in 1963,
producing divinyl--alpha-methylstyrene rubber. At the present time these
two basic monomers are produced locally. Divinyl is produced by dehydra-
tion of butane released by an oil refinery. A rosin emulsifier is used for
polymerization. The following are potent sources of divinyl discharged
into the atmosphere: polymerization and monomer-distillation shops, shops
where divinyl is extracted from a copper-ammonia complex by chemiabsorp-
tion, and the divinyl storage shop.
The investigation •was conducted in the July-August period (170
samples) and November-December of 1964 (150 samples) at outside-air tem-
peratures of plus 25 to 30° C in the summer and minus 7 to 15° in the winter.
The samples were passed through a solid sorbent in U-shaped absorbers
with aqueous aspirators or pumps from PkhB.-54-MV equipment. During
summer months the divinyl concentration exceeded the proposed maximum
single-exposure tolerance level (3 mg/m3) at only three points: 50 and 70m
from the divinyl extraction shop (6.12 and 6 mg/m3) and 30 m from the mon-
omer distillation shop (3.31 mg/m ).
During the winter months excessive amounts were discovered only
at 20 m from the same shops (3. 2 and 3. 5 mg/m3). At 500 m the divinyl
concentration was 0.2-0.7 mg/m3 , while at a radius of 1500-2000 m there
•were no traces.
- 31 -
-------
The low boiling temperature of clivinyl (minus 4°C) and the high
temperature of the ambient air during the summer (20-30°) caused consider-
able escape of the gas from the shops. During the winter months divinyl con-
centration was lower at all sampling stations, In this connection special at-
tention should be focused on making all systems in the shops absolutely
leak-proof, particularly during the summer, to reduce the discharge of
divinyl into atmospheric air.
CONCLUSIONS
1. On the basis of the present study it is recommended that 3
mg/m be adopted as the maximum single-exposure concentration in atmos-
pheric air.
2. On the basis of results obtained in this study under conditions
of prolonged, chronic divinyl inhalation, it is suggested that 1 mg/m3 be
adopted as the permissible average daily concentration of divinyl in atmos-
pheric air.
3. To determine microquantities of divinyl in atmospheric air in
the presence of other products of synthetic rubber production , it is suggest-
ed that a spectrophotometric method be employed which retains the divinyl
on a solid sorbent with subsequent spectrophotometric determination of the
elated isooctane solution in anSF-4 spectrophotometer.
- 32 -
-------
TOXICITY OF ETHYLENE OXIDE
IN LOW CONCENTRATIONS
T. Yuldashev
From the Department of Municipal Hygiene of the
Central Institute of Post Graduate Medicine
and the Uzbek Scientific Research Institute
for Hygiene, Sanitation and Occupational Diseases
Ethylene oxide (oxirane) was discovered by A. Wuertz in 1857, it
is a colorless gas at room temperature having the odor of ether and a caustic
taste. At low temperatures it turns into a fluid which is more volatile than
either. Its boiling and freezing points are, respectively, 10.7 and - 1 11. 7° C .
In a 3 to 80 percent mixture with air it can be explosive. It is soluble in
•water, alcohol, ether, and other material containing free hydrogen atoms.
Ethylene oxide (EO) easily interacts with many organic and inorganic sub-
stances and is stable at high temperatures. There are two ways to pro-
duce EO: by the interaction of alkalies with ethylene chlorohydrin, and by
direct catalytic oxidation. EO is used in the organic-synthesis industry to
produce glycol and polyglycol esters, acrylonitrile, and acrylic acid esters
and their polymers, used in the manufacture of organic glass, synthetic fi-
bers ("orlon"), and synthetic rubber. In addition EO is used in the produc-
tion of textiles, lubricants, and plastic solvents; also as a pesticide, in
daily life, and as a disinfectant in medicine. EO is a highly poisonous nar-
cotic, which may be due to the formation in the organism, as a result of EO,
of formaldehyde or ethylene glycol with subsequent formation of oxalic acid.
It is also possible that EO combined with trimethylamine to form an active
acetylcholine, or it may react with the amino groups of proteins (N. V.
Lazarev, 1963) According to Lazarev a 10 min. exposure to an atmosphere
containing EO vapor will produce severe vomiting, dizziness, sweet tasfce
in the mouth, and - for several weeks thereafter - impairment of cardiac
activity. E. M. Bongard and V. F. Shlyapin (1958) examined the health of
workers coming in direct contact with EO. Acute EO poisoning occurred in
workers under emergency conditions, when the EO concentration became
high. Acute EO poisoning affected the nervous system and normal psycho-
logical functions. Soviet and foreign studies agreed regarding the toxicity
of relatively high EO concentrations; however, no biological effects of small
concentrations in atmospheric air were recorded in the literature. Using
the M. V. Alekseeva modification of the E. Sh. Gronsberg method, the
author conducted a study - first, for the determination of the odor percep-
tion threshold - of EO in atmospheric air.
Data in Table 1 show that the odor perception threshold concentra-
tion in the most sensitive subjects was 1. 5 mg/m3 , and the maximum odor
- 33 -
-------
nonperceptible, 1.0 mg/m5
Tib It I
RfiULTS OF LTHVLtNE OXIuc FnKtiHOLD ODJfi
DfcTthbllttTIOHi
Studies on EO effect on eye sen-
'eitivity to light showed that for the most
sensitive persons the minimum odor
perception threshold was 1 mg /m3 , while
the concentration of odor perception sub-
threshold was 0. 5 mg/m3 (Table 2,
Fig. 1).
The effect of EO on the electrical
activity of the cerebral cortex in mar
was studied using an 8 - lead Kaiser
EEG equipped with engineer B. N.
Balashev's integrator; the A. D. Semenko method was followed, by which
the subject's EEG was recorded, while a rhythmically pulsing light of op-
timum frequency and variable intensity (0.1-0.6 j) was used as the stimula-
tion. The EEG helps to determine the nature of amplication of the alpha-
rhythm's natural potential.
No. gf
obsor-
*0 t ions
2
3
3
2
^ 2
6
2
H.n .rce J
c on c rt • m |
1.5
1 ,9
2,7
3,5
4,3
5,4
tiara than 6.6
Max . nonpar -
percept i ble
concne . -
in 09 /m
1,0
1,5
1.9
2,7
3,5
4,3
— ,
1
Total ob-
servat ions
75
103
99
71
53
170
40
Tibia 2.
tTt SENilTIVBTY TO LIGHT CHANGE DURING EXPERIMENTAL
ETHYLtNE OXIDE INHALATIOB
- -- -
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t.B.
L.K.
a -
o
u a
« 9
rf
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0 •»"
1,5
1,5
2,7
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O -
205
327
159
2,4
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642
427
166
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i1
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£
312
445
133
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2,5
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178
389
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Hotei Rsl ubi I ityi • - 95i| b - 99}) c - 99.9)
After several tests with pure air, the subject, unknown to him, was given
inhalation of EO for 3 min. in a concentration below the odor perception
threshold; the effect of this concentration is judged by changes elicited in
the alpha-rhythm. Tests were made with EO in concentrations of Z. 3,
1.4, 0.65, and 0.3 mg/m". For the group of volunteers the threshold of
odor perception was 1. 9-2. 7 mg/m3 , while 0. 65 was the minimum concen-
tration which elicited changes in alpha-rhythm potential. A concentration
of 0. 3 mg/m3 was inactive (Fig. 2); it is here proposed as the maximum
single-exposure concentration in atmospheric air (Table 3).
Maximal allowable average daily concentrations were determined
by means of chronic experiments using 60 white male rats weighing 95-130 g.
Rats were divided into 4 groups of 15, three of which were exposed to con-
tinuous 83 days inhalation of the following EO concentrations in mg/m :
- 34 -
-------
I, 0.3 (the above recommended single-exposure tolerance); II, 0.03,111, 0.01;
and IV, control. The EO concentration in each chamber was checked daily.
The range of averages for Groups I, II, and III, respectively, was 0.31±
0.0069, 0.03±0.0016, and 0. 015 ± 0 . 0069 mg /m3 . The parameters used in
judging EO effects were: general behavior and body weight gain, effect
on chronaxy ratio of the antagonist muscles, change in whole blood chlo-
rides and residual nitrogen content, changes in blood serum protein fraction
picture, and the results of pathomorphological and pathohistological examin-
ation at the end of inhalation and the recovery periods.
to
1S 20 15
Ti«e in ainute*
30
f>3. I. Ethylene oiide effect on ev* leridtivity to light of
feule Z Jk.Kh.
I - Cltw air, 2 - 2.1* ajjm3; 3 - I "g/» j ** - 0.5 «g/B3
s'.
//J
» /'i7
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9ro5nd'f ^ j "•«»'•>>• Mri.d-
/ 2 J i, 5 6 7 __JI__ _9 - >0 n 12
TIB* in Binutee
Fiyt 2. Enhancement of initnl (orlginil rhy th
tiela dunoj ethylene o»ide inh«l«tio n by f
I - Clean «irf 2 _ I .Ij »g/«3j 3 - 0,65
4 - 0=3 »9/"3
13
ul« H.I.
- 35 -
-------
RG oc 1 1 on studied*
Odor porceptcefi
E jo sans' t r v i ty to
1 igM
Electrical ac 1 1 v i ty
dg/o-^ of a thjr 1 en«
oaico
Ihr.Bhold
ccncn.
1,5
1,0
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of cram cortea
Sub-
LW_nnt^«* 1
tnraQno i
concn.
1,0
0.5
0,3
Tab I• 3 , , r 11
UDLt OF Ht»uLU obiAiNEU br Animals of all groups were
OF tiHYLtNt cuiut tFFtu ON teAM'i, active throughout the inhalation
period and no statistically reliable
changes in weight were found. Five
rate of each group were examined
every 10 days, under identical condi-
tions and at the same hour of the day,
and to check on the hind leg muscles
chronaxy ratio; a response was re-
corded on an ISE-01 pulse chronaxy-
meter when the animal jerked its
foot. The muscle-antagonist chronaxy
ratio constitutes a sensitive test which reflected functional shifts in the cen-
tral nervous system when low concentrations of toxic substances were in-
haled (K. A. Bushtuyeva, 1961; V. A. Gofmekler, I960; Yu. G. Fel'dman,
1962; R. Ubaydullayev, 1961; V. A. Chizhikov, 1963; and others).
Rate of Group I (0. 3 mg/m^ experienced an inverse antagonist
ratio on the 8th week of exposure to inhalation (increase in flexor chronaxy)
which persisted to the end of the experimental inhalation period. These
changes reverted to normal during the recovery period. The statistical
reliability of the changes was 0. 95-0 . 999- No reliable changes were ob-
served in the antagonist ratio in rats of the other three groups (Fig. 3).
On the basis of data presented by P. V. Zimakov (1946) and
N. V. Lazarev (1963) indicating that EO affected the kidney function and the
mineral salts content in the blood, which is a reliable index of the harmful
activity of several unfavorable factors, a study was initiated to determine
the dynamics of chlorides and residual nitrogen in the whole blood of white
rats. Chlorides were determined in 5 rats of each group once every 15 days,
using the Levinson method based on the fact that chlorides yielded insoluble
silver chloride with silver nitrate. The proteinless filtrate of the blood,
containing the chlorides, is titrated by a solution of silver nitrate in the
presence of calcium chromate to a change of color.
In rats of Group I the change in blood chloride content appeared at
the end of the second month of inhalation exposure; it took the form of a
reduction in chloride concentration from 458. 3 mg percent of 375. 6 (controls:
469.5). Statistical reliability of these changes waa 0.999. The content of
chlorides returned to normal (Fig. 4) during the recovery period.
No reliable change in the blood chloride content was noted in rats of
Groups II, III, and IV. After 20 days the residual blood nitrogen was deter-
mined in 5 animals of each group; Convee cups were used in the chloride de-
termination, a method based on the fact that organic matter decomposed by
sulfuric acid in the presence of a catalyzer formed amonium sulfate.
- 36 -
-------
015
C.K
Of?
O.W
DOB
Q.OS
Exposure ptnod
Ammonia was set free by the addition
of an alkali. Sulfuric acid which did
not react with the ammonia, is ti-
trated with an alkali. The amount of
bound acid corresponded to the amount
of ammonia set free, which in turn cor-
responded to the amount of nitrogen.
A change in the residual-nitro-
ben content of the blood appeared in the
Group I rats during the latter half of
the second month of inhalation exposure
rising from the initial level of 36.4 mg%
to 56.3 and then returning to normal
level during the recovery period. The
control reading was 38.1. The reliabil-
ity of this change was 0.95.
Rats of Groups II and III exhibi-
ted no reliable change (Fig. 5). Paper
electrophoresis tests were made every
20 days to determine the effects of EO
on the protein fraction picture of the
blood serum uaing 5 rats of each group?
throughout the inhalation period no re-
Fig. 3. Huici. •ntajonut chron«iy ratio m «*- liable changes in the overall amount of
p.naent.l »ni.,lG .ff.ct.d by .thyl.n. wild*. ,& ..if ^4-4«T,B -art-Tr- ob-
i, n, in, .nd iv -group, .tudi.d, .oi.d eurv. protein and protein fractions were OD
- .Kt^.«-B, br,k«, i,n. «,rv.. . fluori. served. In summarizing the results, it
Zi
' 13 33 J/t 13 23 2/XI 12 2Z O/H/r
Study dates
tyw,
tf/f R/II
Study dal..
Dil«8 of
I -
. IJ. tthylen. ond. «ff.ot on .hoi. blood eKlorid«B f,3_ 5. Eff.ct of .Ihylen. o«id. on residue"
10 rile of diff.r.nt group! |n -no|. blood of rats of difUroot ercapD.
O.J og/B3j 2 - 0.03 Bg/«3} 3 _ o.OI «9/B3| >* - eon- MB. BB in Fig. >*.
trol
- 37 -
-------
can be stated that chronic 83-day exposure to EO inhalation of white rats
in a concentration of 0. 3 mg/m3 elicited pronounced changes in the an-
tagonist muscles chronaxy ratio and in the concentration of chlorides and
residual nitrogen in the blood. An EO concentration of 0. 03 mg/m3 eli-
cited no changes in any of the tests. Therefore, this concentration is
recommended as the average daily maximal concentration in atmospheric air.
To determine the extent, type, and intensity of air pollution, a
survey was conducted in the summer of 1967 in the proximity of the EO man-
ufacturing planta: single-exposure air samplea were collected at 50 to 500 m
from a leeward discharge source (Table 4).
ETHTLENt OXIUE AIR POLLUTION AROUND THE PLANT
-
H« tire
frgn dis-
charge
point
50
100
200
300
500
y_ a f
NO • 0 '
26
28
31
30
32
.
Ha.,..)
c«ncn.
0,262
0,230
0,231
0,258
O.I
Distribution of concentrations
In mgjm*
Mart Into
O.Z
8
3
5
6
—
0.2-0,15
17
10
11
16
" —
0.15—0,]
1
11
12
7
1
Less thin
O.I
4
3
1
31
Results of the study showed that EO air pollution around the plant
in an area of 500 m radius, the EO concentrations were substantially below
those recommended for a single-maximal exposure. Serious consideration
should be given the probability that if plant production were to be enhanced
by the introduction of new technology (e.g. , EO production by direct oxi-
dation), the intensity of atmospheric polution might considerably increase.
In this connection it must be borne in mind that in establishing boundaries
for the sanitary protection zone, the presence in the proximity of other
plants discharging air pollutants should be taken into account.
CONCLUSIONS
1. Ethylene oxide as a poison affected all body organs and systems,
principally the central nervous system.
2. The EO odor perception threshold for most sensitive test per-
sons was found to be 1. 5 mg/m3 ; threshold eye sensitivity to light 1. 0 mg/m3 ;
and threshold of electrical cerebral cortex activity 0. 65 mg/m3 ,
3. It is recommended that 0. 3 mg/m3 ef EO be adopted as the limit
of its concentration in atmospheric air.
- 38 -
-------
4. Chronic, Z4-hr. exposure of white rate to the inhalation of air
containing 0.3 mg/m3 of EO for 83 days produced changes, affected the ani-
mals' flexor-extensor chronaxy ratio and the chloride and residual nitrogen
blood contents.
5. It is suggested that the average daily maximal allowable EO
concentration in atmospheric air be sat at 0. 03 mg/m3 .
6. Atmospheric air pollution with EO has been demonstrated as
far as 500 m from the discharge source.
A STUDY OF HYGIENIC PROPERTIES OF METHANOL
AS AN ATMOSPHERIC AIR POLLUTANT
R . U b aydull ayev
From the A. N. Sysin Institute of General and
Municipal Hygiene, USSR Academy of Medical Sciences, and the
Uzbek Scientific Research Institute for Hygiene,
Sanitation, and Occupational Diseases
Methanol is produced synthetically and by hydrolysis in large
quantities. It is used -widely in the manufacture of organic dyes, pharma-
ceutical compounds, formaldehyde, and other chemicals. The main sourceQ
of methanol as an air pollutant are plants manufacturing paint, varnish, wood-
chemistry products, furniture making and some metal working plants.
Methanol is a nerve and blood vessels poison the effects of which are cumu-
lative. L. I. Kazas (1925) and V. M Rozhkova (1948) consider that the basic
poison effects of methanol came from its primary oxidation products in the
organism, viz. , formaldehyde, which arrested cell respiration and inhibit-
ed the oxidation functions of the body. The effects of low methanol concen-
trations on the living organism have been studied by Chiao Chen-Tsi (1959),
who experimentally determined the concentration of its odor perception
threshold (4.1 mg/m3) and threshold of reflex an eye sensitivity to light
(3.3 mg/m3); he also conducted chronic inhalation series with white rats
and methanol vapor in concentrations of 50 and 177 mg/m for 12-hr, daily
intervals for 3 months. These studies served as a basis for setting the
- 39 -
-------
maximum allowable single-exposure and maximal average-daily concentra-
tion limits in atmospheric air at 1. 5 and 0. 5 mg/m3 , respectively.
However, in setting the single-exposure concentration level the
authors did not employ the most sensitive index, which is electrical cerebral
cortex reflex activity and continuous 24 hr. inhalation exposure. In the
present paper error by omission was corrected by using the most modern
experimental procedures. Methanol concentration in the atmospheric air
was determined by the method of M. V. Alekseyeva (1963). First, the
threshold of methanol odor perception was determined in 25 test volunteers
18 to 40 years old. Nine methanol concentrations ranging from 3.9 to 12.2
mg/m° were used, and results reported in Table 1.
Table I
Data in the last line show that the
THHtiHoius OF wtTHANOL VAPGH ODOR minimum perceptible and maximum nonper-
concentrations were 4. 5
Nod. of
tool
Poroons
3
5
4
T
2
5
6
Minna! (jarcvp-
tibl* concn.
in iig'/n3
10.3
8,4
7 5
' > *•*
6,5
5.6-
4.5
Maximal nonperc«p-
tible eoncn. in
•B/-3
9.7
7,5
6 5
5,6
4,5
3.9
. r^
mg/m , respectively, closely approximating
the 4.1 mg/m3 recommended by Chiao Chen-
^3 J ^"^
Tai. The lowest methanol concentration
which upon inhalation affected eye sensitiv-
ity to light waa studied next, using an ADM
adaptometer and three test persons aged 18
to 25. Teats were made once daily on each
individual under identical conditions and at a standardized time of day.
The physiological background - i.e., the normal curve of eye
adaptation to the dark or sensitivity to light - was determined on 8 successive
days by 15- and 20-min. pure air inhalations. A 4.11 mg/m3 methanol con-
centration elicited a sharp change in the light subjects' eye sensitivity to
light, while only one individual reacted to a concentration of 3.52 mg/m3 ;
the inactive methanol concentration for this individual was 3.06 mg/m3
(Fig. 1).
Effect of low methanol concentrations
on the electrical cerebral cortex reflex ac-
tivity was investigated by the method of A. D.
Semenenko (1963) using an 8-lead EEG. Six
subjects most sensitive to olfactory stimuli
were selected for this test (Table 2) At
least 4 tests were made with each concen-
tration, and the methanol inhalation was al-
ternated by tests with pure air.
10 15 20 25
I toe in Bin.
£<£• I. Changes in »y» sensitivity to
light effected by methanol vapor inhala-
tion.
I - Clean air) 2 - 4.11 •q/B3I 3 - 3.53
•B/»3i ^ - 3.06
Results of this investigation showed
that methanol in 1.46 mg/m3 concentration
affected the alpha-rhythm amplitude; how-
ever, this was true in only 2 cases of the
- 40 -
-------
group which inhaled air containing methanol in concentration of 1.17 mg/m3
The 1.01 mg/m3 concentration proved inactive in all test persons. Results
nrp nhown In Table 3 indicating that the maximal methanol concentration of
single inhalation determined in earlier studies (1.5 mg/m3) needs to be
lowered to 1 mg/m3 .
Table 2
QtTHANOL YA^OH HEFLtX EFFECT OM ELECTRICAL
BHAIN CORTEX ALTIVITt
Toot
per song
G. K.
P. K.
T. A.
K. K.
L. T „
L. tl.
MB/" of nathtnol
1.4G
+
j_
+
+
1.17
+
Not in«y»li-
geleiT
+
Not investi-
gated
I.Ol
—
—
—
—
Remark i + t tt k i a t ice I Ijr reliable »nd - etetie-
ticelly non-rol title che/ig**
Tible 3
UETHANJL LifltbriOLu ODOH ANO HLFLLX EFFECT ON THE HUMAN
OR&ANISB
"
Inreahol d
Odor per-
capt ion
Eye ««ni 1 1 1 v > ty
to 1 1 jh t
E loctricel »ct ivi ty
of brain
cm-tea
According to / According to thie
Cn»0 Chan-Le* ' luthnr
Concantrtt ioni in ag/a
U in i Ba 1
•cl i ve
4,1
3,3
Not
•tudiad
HlIIBll
mtct ive
3,7
2,4
•iniael
ective
4,5
3,53
1. 17
B««iael
meet i»e
3,9
3,06
1. 01
the end of the experimental period the control
amount of weight.
In order to check the
average-daily expoaure limits
45 white male rats were sub-
jected to chronic, 24-hr., 90
day inhalation exposures.
The rats, which weighed 100
to ZOO g, were divided into
three groups of 15 specimens;
they were tested by following
methanol concentrations, in
mg/m3: Group I, 5 (some-
what higher than the odor per-
ception threshold); Group II,
0.5 (the existing average daily
maximum allowable concen-
tration); and Group III, con-
trols. The air temperature
and methanol concentrations
had been checked in each cham-
ber daily in the course of the
study. Average methanol con-
centration for Group I was
5. 31 ±0. 62 mg/m3 , for Group
II, 0. 57± . 059 mg/m3 . Dusr-
ing the inhalation period ani-
mals of all groups were
healthy, active, and gained m
body •weight. However, toward
animals lost some neglible
Once every 10 days 5 rats of each group were checked for theisr
motor chronaxy ratio. Prolonged inhalation of methanol vapors by rats in
Group I (5.3 mg/m3) manifested statistically reliable changes beginning with
the 6th week, also a paradoxical and inverse ratios of antagonist chronaxy.
At the end of the recovery period the chronaxy ratio of flexor and extensor
muscles returned to normal.
Rats of Group II manifested no changes in their chronaxy indices
(Fig. 2).
- 41 -
-------
n
I.B
1.5
c / 3
J *-2
**-
o /./
u
° to
-^
4
* as
as
0.1
/? ' 6
~\ ;
-V
c;^ /
^^
-
-
_
-
-
-
l
- -
^N^y/^\_x<^
\ x-/ / ^
^ i *
\ , / \
\ ' \
\ ' \
\ ' v
\ / \
\ ' x
\~"
Inhalation period
i i i i i i i i
\
V /
V" * 2
/
/
f
s
s
r
_
1 1
Dties of study
Fig- 2. Muscle an Ugoniats, chronaxy ratio changes in rata
effected by the inhalation of oethanol vipor. ,
A and B period of Inhalation. 1 - Clean airj 2 - 5.31 »y/«
3 - 0.37 ng/"3
M. I. Gusev (I960), K. A. Bushtuyeva (1964), B. M. Mukhitov
(1961) and others have noted changes in the excretion of coproporphyrin with
the urine of the animals during prolonged action of several harmful chemical
agents, a physical phenomenon which was carefully studied here. Daily
urine samples had been collected from 5 rats of each group into specially
made glass containers. Porphyrin was extracted from the urine by the Fish-
er method; the amount of coproporphyrin was determined once every two
weeks with anSF-4 spectrophotometer in the 400-410 millimicrons range.
The amount of coproporphyrin excreted via the urine of Group I rats per 100 g
of body weight dropped sharply beginning with the 7th week and then stayed
at this level for the duration of the inhalation period (Fig. 3). Normalization
set in on the 20th day of the recovery period. No statistically reliable changes
were observed in rats of Group II.
Whole blood cholinesterase activity was determined, colorimetrical-
ly by the A. A. Pokrovskiy (1953) method as modified by A. P. Martynova
(1957), using 5 rats of each group fortnightly. Before experimental inhalation
acetylcholine hydrolysis time averaged 38-39 min. Beginning with the 6th
week, hydrolysis time for rats of Group 1 rose to 41 min. , indicating a de-
crease in cholinesterase activity. By the end of the inhalation period the
time had risen to 43 min. (Fig. 4), after which it returned to normal. No
reliable changes were found in rats of Group II.
Reports found in the literature clearly indicate that total blood
protein and the blood protein fractions picture were affected by disease and
by different chemical substances (R. Ubaydullayev, 1961; V. A. Chizhikov,
- 42 -
-------
1964; P. G. Tkachcv, 1964; Granati, Sekavo, 1956; Mario, Carlo, 1957.
Therefore, the action of low methanol concentrations on protein fractions
of blood serum was studied experimentally by the method of paper elecO
trophoresis in 5 rata of each group. Blood was taken from the tail vein on
an empty stomach every 15 days.
tilt!
p/iv i/v H/V zs/v 8/vi 22/vi 6/rii
^^ ' Dittu of study
fit- 3. Coproporphrm* «li*inition »i» th« urin*
during B«th«nel »*por inhalction.
Notation* »••« •• in Fig. 2.
S//K
riti
JB
31.
Period of «»p»ri-
•antil lnh«l»tion
28/IV 13/V
7/VI 21/VI 5/VII 19/m
D*t»B of «tudy
Fig. U. Ch«ng«» in *ol« blood ch«( ioest«r»»» ictrv-
i ly in rtts foj loimg ••thinel vipor inhilition.
Notitioni •••• ••• In Fig. 2.
- 43 -
-------
Changes appeared 7 weeks after the beginning of inhalation in rats
of Group I Results showed that albumin content dropped and the amount of
beta- and gamma-globulins increased. Alpha-globulin content did not change.
The fractional protein picture in rals of Group II did not change (Table 4).
- -lib la It
TJTAL HrtQTEIN AND PROTEIN FRACTIONS IN BLOOD SERUM IN RAtV
CHRONICALLY EXPOSED TO IKHALATIO* OF BETHANOL VAPOR
1 nhal »t i on
period
before
exposure
On ttSlh in-
halation dty
-
On yoth
dav
vmj
[
On 20th
recovery
cay
Group
I
II
III '
Con-
trol
ii
in
Con- ,
trol
I
II
III
Con-
trol
I
II
III
Con-
trol
Total
proto in
9,02
9,31
8,91
8,79
9,01
9,Q2
9,17
9,16
8,89
9,27
9,05
8,79
Albu-
• m
41,8
44,8
45,1
34,7
44,1
43,6
32,8
42,0
42,9
39,4
42,2
44,2
(i lobu 1 ins
Alpha
24,6
23.2
23.3
24,0
21,5
20,9
23,3
21,8
21,8
25.3
20,2
20,2
B.ta
20,7
19,4
19.2
24,0
20,2
20,9
25.8
21,9
21,2
21,2
22,1
22,9
Ga«»a
12,9
12,6
12,4
IT. 3
14,2
14,6
18.1
M.3
14,1
-
14.1
15,5
12,7
Al bu»in/
Globu 1 in
rat 10
0,72
0,81
0,83
0,53
0,79
0,87
v 0,49
0,72
0,76
0.65
0.73
0,80
Trtl* 5
METHANOL VAPOR POLLUTED ATMOSrHERIC AIR IN THE ENVIRONS
OF U/BEKISTAN HtDHOLITIC PLANTS
Ue tfl re f roo
source of
di scharye
No. of
eanpl eb
Single concentration In
•9/"3
Haxiaal
UiniMl
No. of air aiiplaa
of conccntrat tona
1 and
h lyhtr
1 and
b.lo.
THE ANDI^HANSK PLANT
100
200
3OO
500
16
18
27
18
4
2,26
1.67
0,33
0.14
0.46
0.11
0.05
—
10
5
—
—
6
13
27
18
THE FERGANSK PLANT
100
200
300
500
17
15
25
19
4.49
2,2
0,83
0.3
0.89 "
0.1
0,11
—
16
9
—
—
1
6
25
19
THE Y-ANOYULLU PLANT
100
200
300
14
24
14
1,15
0.55
0,05
0,1
1
—
3
—
—
11
24
14
- 44 -
-------
No changes were observed in the total proteins. The results show that after
90 days only rats of Group I (5.31 mg/m3)had been affected. A methanol-
vapor concentration of 0. 57 mg/ma was inactive. Accordingly 0. 5 mg/m3
is recommended as the average daily maximal permissible concentration.
An air pollution survey was conducted with reference to methanol
in May and August 1962 in the proximity of the Andizhansk, Fergana, and
Yangiyul1 hydrolytic plants of Uzbekistan, leeward of the plants at distances
of 100 to 500 from the discharge point (Table 5).
Data in the table show that at 100 and 299 m from the first two
plants the methanol concentration in the air exceeded the allowable maximum
(1 mg/m3). At the third plant this excess was detected only at 100 m. At
greater distances from the plants all concentrations were less than 1 mg/m3 .
CONCLUSIONS
1. Methanol air pollution in the vicinity of hydrolytic plants was
detected up to 200 m from the plants.
2. The methanol odor perception threshold for highly sensitive indi-
viduals was determined at 4. 5 mp/m3 , of eye sensitivity to light at 3.35 mg /
m3 , and of effects on electrical brain activity at 1.17 mg/m3 .
3. It is, therefore, recommended that the maximum allowable
methanol single exposure concentration in atmospheric air should not ex-
ceed 1 mg/m3 .
4. Chronic, 24 hr. , 90-day inhalation of methanol in the air in a
concentration of 5.3 mg/m3 elicited in experimental rats changes in direct
antagonist chronaxy ratio, in whole blood cholinesterase activity, in the
elimination rate of coproporphyrin via the urine, and in the blood serum
protein fractions. A concentration of 0.57 mg/m3 proved inactive.
5. It is recommended that the average daily methanol concen-
tration in the air be set at 0.5 mg/m3 .
- 45 -
-------
ATMOSPHERIC AIR POLLUTION WITH VAPORS OF HYDROLYTIC
ALCOHOL AND ITS EFFECT ON THE ORGANISM
R. Ubaydullayev
From the A. N. Sysin Institute of General and
Municipal Hygiene, USSR Academy of Medical Sciences, and the
Uzbek Scientific Research Institute for Hygiene,
Sanitation, and Occupational Diseases
Hydrolytic alcohol is ethyl alcohol (ethanol) containing a variety
of acid admixtures in 0.036-0.13 g/li concentration computed on the basis of
acetic acid, unsaturated compounds in 0.07-0.89 g/li on the basis of allyl
alcohol, 3-6.2 g/li of methanol, 1.7-21.6 g/li of carbonyl computed on the
basis of acetaldehyde, 0.024 g/li of furfural and 2.3 g/li of higher spirits
computed as isobutyl alcohol. Normal ethanol is a transparent, colorless,
volatile fluid with a specific odor having a boiling point of 78. 4° C. It mixes
in all proportion with v/ater, ether, and chloroform, and dissolves mineral
salts, especially chlorides, nitrates, acetates, and some essential fatty oils.
Ethanol is produced normally by fermentation from such starch-containing
materials as potatoes, cereals, rice, or molasses. Synthetic ethanol is
produced from ethylene and acetylene.
The most widespread of the ethanol products is hydrolytic alcohol,
the manufacture of which is based on fermentation of hexose sugar obtained
from hexose-containing plants. Ethanol is used as a solvent in the manu-
facture of varnishes and polishes, in synthesizing many organic compounds,
as a raw material in the production of synthetic rubber by the Lebedev
method, as a fuel for internal-combustion engines, and preparing food, medi-
cines, and other items of the economy. Ethanol has narcotic properties and
can be classed as a poison. Chronic use or exposure to ethanol has caused
organic diseases of the nervous, digestive, and cardiovascular systems,
liver, etc.
However, no information was found in the literature on the effects
of low ethanol concentrations on man and animals as a result of vapor inhala-
tion. Its maximum permissible concentration in atmospheric air has also not
been established, and no data were found on air pollution around plants pro-
ducing or using ethanol. These phases of air pollution were studied by the
present author and discussed in this report.
Atmospheric air pollution with ethyl alcohol was determined by
I. A. Pinigina's method (1961), which is based on the interaction of alcohol
with a vanadium-hydroxyquinoline complex, which formed an orange dye.
The sensitivity of the method is 0.002 mg in 2. 5 ml. To obtain the required
ethanol-air mixture the air was passed through an S-shaped device filled with
- 46 -
-------
hydrolytic ethyl alcohol. For several days before the experiment was ini-
tiated the ethanol concentration constancy in the cylinder was checked and
found that its fluctuations were negligible. Threshold of odor perception
was tested on 25 persons aged 18 to 40. Altogether 385 readings with 7
concentrations between 6.3-14.8 mg/m3 were made. Results are summar-
ized in Table 1.
Ttble I
THRtiiHULtJ Ol/OR DE1ERM liUT I UN OF HYDhOLYTIC
tlHTL ALCOHOL
OB . 01 to&t
persona
I
' 7
7
4
6
Concentration, in mg/m^
Hininal
porcapt ib la
10,2
8.7
8.0
7.3
7.1
WBI 1 f'B t OOO —
p«rc«pt ibl*
9.4
' 8.0
7,3
7,0
6,3
Methanol effect on eye sensitivity to
light was checked next by the method of
adaptation to darkness. Forty-two tests were
made with persons aged 18-27 years and
methanol concentrations of 6.12, 6.97, and
8. 29 mg/m3 , The lowest concentration was
inactive in all four test persons; two respond-
ed to the middle concentration, and all re-
acted to the highest concentration (Table 2).
Table 2
THRESHOLD OF REFLEX CHANGES IN EYE SENSITIVITY TO LIGHT
EFFECTED faY THE INHALATION OF HYDHOLYTIC ETHYL ALCOHOL
VAPOR, ON THE JOTH MINUTE EXPRESSED IN PERCENT OF THE
ISTH MINUTE MAGNITUDES
Ti
• 61
p«raonB t
initials
1 . L.
6. t.
Ch.A.
b.V.
Clean
air
158.6 (o)
156,0 (o)
133,5 (o)
118.0 (o)
Coneen trail at in «J/»^
8,29
I94,8~(b)
226, 7 (b)
223,6 (c)
214. 4(b)
6.97
150,0 (o)
155,6 (o)
151,9 (b)
130,7 (b)
6,12
—
134,3 (o)
147,8 (o)
I P. .h
Thresh
old
8,29
8,29
6,9V
6,97
throah
eld
6.97
6.97
6.12
6.12
Haiti Reli*biIity d«gr.»i i - 95}, b - 99J, c - 99.9i
The effect of low ethanol concentrations on the electrical activity
of the cerebral cortex was studied by the A. D. Semenenko method (1963) of
reflex-elicited alpha-rhythm rises in man, induced by simultaneous flashing
of light stimulation at frequency approximating that of the rhythm and by a
sound of variable intensity. On the background of such functional loading
the natural alpha-rhythm became enhanced, and the nature of the curves re-
corded changed during inhalation of the gaseous mixture in active concen-
trations pointing to a change in the functional state of the nervous system
and in the cerebral cortex activity. Curves were recorded on a 8-lead EEC
manufactured by the Hungarian firm Orion Budapest. Test persons were
five of the most sensitive to olfactory stimulation. Tests were conducted
under identical conditions at the same time of the day. Four of five tests
were made with each of the two test concentrations of 4. 9 and 6.14 mg/ma .
Statistical analysis of the results indicated that in all instances the lower
- 47 -
-------
ethanol concentration was inactive and the higher waa active (Table 3).
Results are summarized in Table 4.
Tab IB 3
EFFECT OF LO* HYDHOLYTIC ETHYL ALCOHOL CONCENTRATIONS
ON THE ELECTRICAL ACTIVITY OF THE BRAIN CORTEX
Test persona
in i 1 1 at a
1.5,
D. «.
H. K.
L. B.
M. *.
6,1 wrlM'
H en i Bj)(ie >•
teft
4-
+
+
Right
+
+
—
+
4.9 urlM1
Hem aphar*
Left
—
—
—
Ri oht
—
—
—
N o t o i + BtiliBiicil ly »tynilic«nt| - »Ut i « : i c* I \y
nan-eignif kctnt,
Ttbli
EFFECT OF L0» HYDHOLYTIC ETHYL ALCOHOL CONCENTRATIONS
ON RESHIHATOHY RECEPTIVE OHGANi
On the basis of results ob-
tained with reflex reactions in man,
it is proposed that 5 mg/m3 of
methanol in atmospheric air be
adapted as the maximal allowable
single hydrolytic ethyl alcohol con-
centration in atmospheric air.
In the next experimental
series 45 white male rats weighing
100-120 g were divided into three
groups and exposed to 24-hr. , 90-
day methanol air inhalation, Rats
of Group II inhaled an ethanol-vapor
Functions
Odor perdition
Eye sens « t iv i ty to
1 iflht
£ Iscl rical activi Ly of
brain cortex
Concint rtt ion in ng/i
Thr,8holdT'Subthra*h-
, old
"7,1
6,9
6,1
6.3
6.1
4,9
esterase activity, total amount of protein
Resulting numerical data were processed
~__concentration of 5 mg/m ; rats of
Group 1-25 mg/m3 ; and rats of
Group III were the controls. Ethanol
effects were evaluated on the basis
of general behavior, gain or loss of
body weight, motor chronaxy change,
rate of coproporphrin elimination
via the urine, whole blood cholin-
, and the protein fractions picture.
statistically.
The experimental ethanol-air mixture was fed into the chambers at
a rate of (original sentence not complete), which V. A. Popov (1964) found to
create the most favorable conditions for the animals. Actual ethanol concen-
trations were 29.25±2.1 mg/mj for Group I and 5.59±0.45 for Group II.
During the inhalation period all animals were healthy, active, and gained body
•weight. However, rats of Group I manifested a negligible loss in body
w e i g ht.
Muscle antagonist chronaxy was determined at the same hour of day
and under identical conditions every 10 days in 5 rats of each group, employ-
ing an IES-01 pulse stimulator. Results are recorded in Table 5, Fig. 1. The
data show that discoordination of the normal chronaxy ratio of flexor and ex-
teneor muscles in rats of Group I appeared during the 6th week of inhalation
but the ratio returned to normal during the recovery period. Rats of Group II
exhibited no statistically reliable changes as compared with the controls.
- 48 -
-------
tiblt 5
MUSCLE ANTAGONISTS CHRONAXY RATIO IN RATS INHALING HYDRClLYHC
ETHYL ALCOHOL VAPOR
ft 1
rer i 008
Before a«(josore
During o«poBur«
*
Durinj r»cov«ry
t>«riod
Dales of
atudy
23/1 V
3/V
13/V
22/V
31/V
9/VI
19/VI
29/VI
9/VI I
18/VII
29/V1I
11/V1II
23/VIII
1/1X
Group B
I
II Contra) Go. Ill
E«ten»4r and ftemor ohrortaxy
ret IOB
,62 (o)
.29 (o)
,30 (o)
,55 (o)
.26 (o)
,37 (o)
0,82 (c)
0,93 (c)
0.91 (b)
1.1 (o)
0.91 (a)
0,79 (o)
1 .08 (o)
1.23 (o)
1.53 (o)
.33 (o)
,43 (o)
,50 (o)
,58 o)
,53 (o)
.42 (o)
.49 (o)
,35 (o)
,42 Jo)
.26 (o)
.56 (o)
.15 (o)
.09 (o)
,65
,45
.58
,54
.48
,53
.41
,54
,57
,44
.44
,39
1,22
1.23
Not
Statistical r« I lability i a - 93* j b - 99.9ij c - not
\
Z3/IV 3/V 13 22 31
Exparia. inhalation period
i i i i_
9/Vi 19 23 9IVII IS 29
Dates of aludy
^J I/it
Fid'. I -
anlagoniats ehronaiy ratio chanB«» «ff«cted by
inhalation of hydrolytic athyl alcohol vapor.
A - B - Inhalation p.nod; I - Clean air, 2 - 29.25 -a/a^l 3 -
Whole blood cholinesterase activity was tested in 5 rats of each
group by the A. A. Pokrovskiy (1953) method and A. P. Martynova (1957)
method. Blood was taken from the caudal vein once every 15 days. The.
average hydrolysis time was 37-39 min. (Table 6, Fig. 2). At the end of
the 6th week rats of Group 1 exhibited a drop in whole blood choline ate rase
activity, acetycholine time rose to 4Z min. However, at the end of the 9th
-------
week of inhalation it normalized, only to rise again during the 10th and llth
weeks. This increase was evidently related to the protective or defense
function of the animals' organism. After 20 days of recovery hydrolysis
rate returned to normal. The rats of Group II evidenced no changes as
compared with the control group.
Tail* 6
UlANGti IN RATi' IHOLINtiTEfiAbE ACTIVITY DURING HtDrtOLV-
TIC tlHfL ALCOHOL VAfOH INHALATION IN MINUTE* of AlETYL-
ChOLIN HtUHOUoJi,
- .
Pen oda
-
DaLea
-
Before I
eupoBura 1
1
n
h.
^
I
»
t
I
o
n
Recovery
- ^28/IV
- 13/V
24/V
• 7/VI
21/VI
5/V1I
L9/VII
2/VI1I
12/VII!
1/IX
GroupC
,
(29,2 m-iM1)
37,6 (o)
37.8 (o)
39,8 (o)
39,0 (o)
42.0 (c)
42. 2 (b)
39.8 (o)
•41,8 (B)
42,2 (a)
3S.7 (o)
M
C>.09 vrliO
37.0 (o)
38,8 (o)
34,4 (o)
39,2 (o)
38,6 (o)
38,8 (o)
37.4 (o)
37,8 (o)
37,8 (o)
38,0 (o)
Control
1 II
39,2
39,2
38,0
38,0
3fl 2
' 3S.8
38.8
38,0
37,6
37,4
1
Note ' R«l i«bi I i !/• deyroei » -
-------
Rate of coproporphrin elimination was checked fortnightly and re-
corded on the basis of 100 g of body weight. In rate of Group I the excretion
rate dropped sharply during the 6th week and remained at this level through
experimental inhalation period (Fig. 3). At the end of the recovery period
the level returned to normal. Animals of Group II manifested no statisti-
cally reliable changes.
27/IV
, itudy d«t«i
fig. 3. Chtnyti in r«t« of aoproporphvnn •lioinition vjj tht
urm* of rits inhaling hydrol/tic «tK«nal vtporT
Notations »••• »• in Fig. I.
Paper electrophoresis was employed in studying the effects of low
ethanol vapor concentrations on total protein and its fractional composition.
The blood was taken from the caudal vein on an empty stomach every 15 days.
At the end of the second month of inhalation the albumins in rats of Group I
fell and the gamma-globulin fraction increased. This increase waB statisti-
cally reliable and persisted to the end of the experiment. The albumin/globu-
lin coefficient did not return to normal during the recovery period (Table 7,
Fig 4) No changes were observed in rats of Group II. None of the experi-
mental animals showed any change in the total protein.
In summary it can be stated that continuous 90-day inhalation had a
marked effect on animals of Group I (29. 1±2 1) and none on those of Group II
(5 59±0.045 mg/m3). On this basis it is recommended that the allowable
maximum single-exposure concentration of methanol in atmospheric air be
set at 5 mg/m3 ; the same limit should be adapted for the average daily con-
centration of hydrolytic ethyl alcohol.
A study was made of the distribution of methanol (hydrolytic ethyl
alcohol) air pollution in the vicinity; 186 samples of air at 100, 200, and 300 m
from three hydrolysis plants in Uzbekistan, always on the leeward side of the
emission source, were collected. The samples were aspirated through two
- 51 -
-------
activated-coal absorbers. Air samples varied from 10-40 litera, aspirating
the air at the rate ol 1 li/min. , at air temperature of 20 to 35° C, relative
humidity from 25 to 70 percent, barometric preeeure from 730 to 750 mm
J-Jg, wmd velocity from 1 to 5 m/eec. Tibs T.*allLer was and a
( f
100 •
2 90
o
o
C
C
so •
I.Q
13/Y
10/YI 13/VI
Study ditaa
i/II
Fig. l<. Chtngn in blood ••run protein frictions of rtti Inhtl.
Ing hydroljrtio «th«nol vtpor
Notillona una ta In Fig. I.
T«bl* 7
CHANGES IN BLOOD SERUM TOTAL PROTEIN AM) fHOTEIN FhAC-
TIONi OF HATb UUrilNI, INHALI N& HYDROLYTIC ETHANOL VAHOH
- -
Period
Prior to
elhtnol
i nht 1 • 1 1 on
On 50lh m-
On yoth in-
halation day
urmj recovery
eriod
.
*
Groups
•I
II
III
trol
I
II
III
Con-
trol
I '
II
III .
Con-
trol
I
II
III
Con-
trol '
c
•^ 9
"a ~Z
•- i.
a
9,09
9.05
8,91
8,89
9,04
9,88
9,40
8,63
8,73
9,38
8,67
8,79
C
a
1
42,3
42,6
45,1
32,3
42,6
43,7
36,3
45,4
42,9
37.9
44,8
44,2
G lobul in*
All*.
23,8
22,9
22,9
24,6
21,6
20,8
23,4
21,1
21,8
24,0
21,1
19,8
beta
19,5
20,4
19.4
26,0
21,4
20,9
23,5
21, 1
21,1
22,6
21,8
23,4
(>•••»
14.4
14.1
12,6
17,1
14,4
14,6
16,8
12,4
14,2
'
15,5
12,3
12,6
e
0
-— c -
C - (J
r ~ *
? a -
a o e
< o> u
0.73
0,74
0,83
0,48
0,74
0,77
0,54
0,84
0.76
0,6.0
0,83
0.80
- 52 -
-------
ATMOSPHERIC AIR POLLUTION WITH HTBROLtllC ETriANOL VAPfH
IN THE ENVIRONS OP THE UZBEKISTAN PLANTS.
*et«r»
fro*
d i8ch«rg«
point
No. of
• ir
•••pl«i
Single con en e.
•n -g/.3 \
MtaiBil '
UmiMl
Nu>b«r of M»pl»i «ilh
indicated concne.
2 and
higher
1 \a 2
Baloi 1
100
200
300
100
200
300
100
200
26'
17
27
25
16
27
44
25
F«rg»ngl< plenl
2,79
0,16
0.018
0,55
Andiihanek plant
4,52
1,68
0,37
0.45
10
Yanguiyulak plant
2.2
0,5
10
2
10
II
17
5
14
26
10
25
Data in Table 8 show that emissions at 100 m or more from the
three plants under survey contained ethanol concentrations below the recom-
mended limit for single-exposure (5 mg /m3 ).
CONCLUSIONS
1. Air around the investigated Uzbek hydrolytic plants contained
negligible concentrations of hydrolytic ethyl alcohol.
Z. The present study showed that the thresholds of ethanol odor
perception in most sensitive persons was 7.1 mg/m3 ; eye sensitivity to lighfe
was 6.97 mg/m3, and of reflex effect on cerebral cortex activity 6.1 mg/m3.
The maximum inactive or subthreshold concentration in most sensitive per-
sons was 4.9 mg/m3 .
3. It is recommended that the maximum allowable single concen-
tration of hydrolytic ethyl alcohol in atmospheric air be set at the 5 mg/m
level.
4. Chronic, Z4-hr, 90-day inhalation by experimental rats of air
containing ethanol vapor in Z9.25 mg/m3 concentration affected the flexor
extensor muscle chronaxy ratio, the cholinesterase activity, the rate of
coproporphyrin elimination via the urine, and the ratios of blood-serum pro-
tein fractions. A 5. 59 mg /m3 ethanol concentration had no effect on the
organism.
53 -
-------
5. Results of chronic experiments with rate indicated that the max-
imal allowable average 24-hr. ethanol vapor concentration in atmospheric
air can be set at 5 mg /m3 .
BIOLOGICAL EFFECTS AND HYGIENIC EVALUATION
OF AIR BY PHTHALIC ANHYDRIDE
L. P. Slavgorodskiy
From the Ukrainian Scientific-Research
Institute of Communal Hygiene
Phthalic anhydride (PhA) is a benzene ortho-dicarboxylic acid
anhydride. Its boiling and melting points are 284.5 and 128-131° C respec-
tively; its odor resembles that of bitter almonds. It sublimates easily and
dis solves in water (0.6 g in 100 ml at 25° C). When dissolved in hot water it
converts to phthalic acid. It is also soluble in eulfuric acid, alcohol, ether,
and benzene. PhA sublimates in the form of flat, white often rhombic
crystals. PhA is used in many branches of industry. It is widely used in dye
production: phthaleins, rhodamines, indigo, anthranilic acid, derivatives of
anthraquinone, and phthalocyanines. Orthophothalic acid esters are used as
plasticizers in producing glyptal, polyester, and epoxy resins. It is aleo
used in the pharmaceutical and tanning industries, and because of its wide
reactive capacity it is used in chemical laboratories.
PhA can be manufactured in several ways, the most common in-
dustrial of which is based on oxidizing naphthaline in the presence of a cata-
lyzer at 350-500°C. The manufacture and use of PhA are factors associated
with air pollution. After cleaning in scrubbers, waste gases from PhA pro-
duction are discharged into the air, part of the PhA being discharged by the
plants' ventilators. No references were found in the literature dealing with
the existence of PhA in the air around industrial enterprises. Effects of
PhA on the human and animal organism have been studied previously; and
some authors (Friebel et al., 1956; L. A. Titunov, A. A. Denisenko, 1957;
Yu. K. Korotkova, 1957, I960; S. N. Kremneva, M. S. Tolgskaya, 1961)
investigated the toxicity of PhA when introduced into the gastroenteritic tract.
S. N. Kremneva and M. S. Tolgskaya (1961) administered a 2 percent emul-
- 54 -
-------
eion of PhA to narcotized white rats intratr ache ally. Doses in excess of 30
mg/kg were lethal, deat.i ensuing in a few hours or in the course of days.
Friebel, et al. (1956) investigated inhalation of PhA dust and vapor by guinea
pigs in concentrations of 644.5 mg/m3, respectively. These concentrations
irritated the conjunctiva and respiratory passages. An admixture of
naphthoquinone and maleic acid enhanced the irritating effect.
In 1959 Yu. K. Korotkova subjected 10 guinea pigs to the inhalatioa
of PhA dust (600 mg/m3) every day for 15 min. for 30 days. The concen-
tration was determined gravimetrically. The animals coughed and sneezed
during the inhalation tests; and after the first inhalation three of the animals
exhibited palpebral edema, conjunctival hyperemla, and nasal discharges.
Body weight loss was observed in a few cases. Dissection disclosed hyper-
emla of the mucous membrane of the trachea and of the bronchi accompanied
by fusing of the pleural surfaces and moderate plethora of the internal or-
gans. S. N. Kremneva and M. S. Tolgekaya (1961) conducted static injection
experiments for 6 months, 6 days a week, 3 hrs. a day, with PhA of 5-12
mg/m3 . At the end of the 6th month arterial pressure had dropped to 70
percent of its normal level. Eosinophilia developed in the majority of cases
in the course of injections. Results showed that a 1-2 mg/m3 concentration
was ineffective. The review shows that only high PhA concentrations were
studied for brief periods. The effect of low concentrations for 24 hr, peri-
ods has not been investigated. Furthermore, a maximal allowable limit of
1 mg/m of PhA has been set for industrial premises, none has been estab-
lished for atmospheric air.
The wide use of PhA by the industry, the insufficient study devoted
to its toxic properties, and the prevailing local air pollution sources prompted
the present author to study the problem of atmospheric air pollution with PhA.
The purpose of the present paper is to find an experimental basis for a maxi-
mal allowable PhA concentration in atmospheric air. Here as in other air
pollution studies, the thresholds of odor perceptions and of effects on eye
sensitivity to light were investigated first. The M. D. Manita spectrophoto-
meter was used in the study.
THRESHOLD OF FHTHALIC ANHYDRlDt ODOR PERCENT kOH
- - - .
Ho. of
testers
6
4
17
No. of odor
tests
~
100
66
_ i
Minimi percep-
tible COO C/l S.
in mg/m
0.32
. 0.53 .
0.72
Mined nonper—
captible concn.
in ag/»-3
0.22
0.32
0.53
- 55 -
-------
The odor perception thret hold was determined on the basis of 374
observations and 27 healthy test volunteers. Results are listed in Table 1,
and show that the highest nonactive PhA concentration range was 0.32-0.72
mg /m3.
Recent studies aimed at determining maximal single exposure con-
centration of PhA, established, by tests of eye sensitivity to light, 0.22
mg/m3 as the maximal allowable concentration. Effect on PhA on eye sensi-
tivity to light was investigated in cooperation with three test persons using
adaptometer ADM making determinations at 5 min. intervals. Results are
listed in Table 2. The 0. 96 mg/m3 concentration elicited statistically re-
liable changes on the 20th minute of dark adaptation of all test persons.
RESULTS OF ACAPTQUETRIl STUDIES
- --• ~ The 0.55 mg/m3 concentra-
Tlbu ^ . tions caused statistically reliable
In i uel 5
of
toBtars
EA.
•
P.
F.
MS/"3 of phth.l 10
•nhydrids
C 1 can 1 1 r
0,50—0,55
0,96—1,09
1,49—1 ,79
C lean a i r
0,50—0,55
0,96—1,09
1,64—1,79
Clean «ir
0,50—0,55
0,96—1 ,09
1,64—1,79
fy« ••nsitivity
to light on 20th
idtptition am.
23 533
21 066 (o)
30 700 (b)
v 45 899 (c)
41 330 .
39 933 (o)
59 433 (b)
77 100 (c)
21 067
2"! 333 (o)
35 367 (a)
41 633 (b)
N o t a i R«li«b.lliy digreei I - 95); b -
c - 99.yt; o - Not r*Ii»ble
changes on the 20th minute of adapta-
tion to the dark. The 0. 55 mg/m3
concentration was inactive. In two
of the three test persons 0. 72 mg/m3
of PhA was the threshold of odor
perception. No such test was made
with the third person.
On the basis of effects which
PhA yielded in the trigeminal tests,
it is recommended that 0.2 mg/m3
PhA .concentration in atmospheric
air be set as its allowable maximal
single concentration.
The allowable maximal aver-
age daily concentration was determin-
ed by using 60 white male rats en-
closed in 100-liter chambers into
which prefiltered air was run in at
the rate of 20-23 li/min. Rats were
exposed to 24 hour daily inhalations for 70 days. Rats were divided into four
groups which inhaled air containing the following PhA concentrations in mg/m
rats of Group I--1. 52; (misprint for 1. 32--B . S . L .); rats of Group II--0. 54;
of Group III--0.18; rats of Group IV served as controls. It should be noted
that the Group I concentration is close to the allowable level in industrial
premises; 0.18 is below the odor perception threshold and close to the recom-
mended single-exposure concentration; 0.54 is a concentration often en-
countered in the air at 500 m from PhA plants located in the proximity of in-
habited areas.
- 56 -
-------
During the chronic experiment records were kept of the rats' con-
dition, their body weight, muscle antagonisfs, motor chronaxy, cholinester-
aee activity, and the morphological composition of the blood. The behavior
and activity of the rats in the first three groups did not differ from that of
the controls. The rats in all groups gained weight.
It has been shown by many authors that changes in motor chro-
naxy ratios reflected changes in the functional state of the central nervous
syatem (Yu. N. Uflyand, 1941; A. N. Magnitskiy, 1948; A. F. Makarchenko,
1956). This index has become first in the order of studies conducted in
determining limits of allowable maximal air pollutant concentrations in at-
mospheric air. It was used for the purpose in the present studies. This
index was also used in experiments with animals, in 5 rats of each group
by a technique and procedure described in some of the preceding reports.
Deviations from the normal (control) muscle antagonist chronaxy ratios in
rats of Group I were noted on the 31st day of the experiment; two weeks
later chronaxy ratios returned to normal. Rats of Group I manifested less
pronounced changes, and those of Group III showed no chronaxy ratio
changes (Fig. lh,.
20 31 m _SO . SI 73
Tiae in ii»y»
Fio. I. Phthilic inhydrid* «ffeci on "uscU
nofor chronaxy of Group I r»ls (a), Groufj II (b), Gr
111 (c), ond Grnuj) IV rete (d). Solid lin» curves -
oBtonaora, brohon line curvao -
AB - Inhalation pond
- 57 .-
Group
-------
Previous studies have indicated that cholinesterase activity changed
in some pathological conditions of the organism, giving rise to an index util-
ized in arriving at hygienic norms. G. I. Solomin (1961), V. A. Chizhikov
(1963), F. I. Filatova (1962), and D. G. Odoshashvili (1962) demonstrated
that blood cholinesterase activity was affected by chemical substances.
The present author studied cholineeterase activity of whole blood
by the chemical method of A. A. Pokrovskiy (1953) as modified by A. P.
Marynova (1956). Cholinesterase activity was evaluated on the basis of
change in acetylcholine hydrolysis time. Rats of each group were examined
once every 2 weeks. On the 42nd day of administration, rats of Group I
experienced inhibition of cholinesterase activity, acetylcholine hydrolysis
time rising to 49 min. (from 41). Statistically reliable changes were noted
in rats of Group U, but they were less pronounced. Hydrolysis time in rats
of Group III did not vary from that of the controls (Fig. 2).
Evidence presented in the
literature that PhA affected the
hematopoetic system (K. Ye.
Bakaleynik, I960; S. N. Kremneva
and M. S. Tolgskaya, 1961; V. S.
Anatovskaya, 1961). This fact
served as the basis for analyzing
the morphological composition of
the blood. Determinations were
made of the amount of hemoglobin,
erythrocytes , thrombocytes , and
leucocytes. Changes in the throm-
bocyte count in rats of Groups I and
II appeared on the 70th day. No
substantial changes were found in
c 70
Q
c «
I so
J. 55
? SO
L */"
"C
5* ts
o 40
O -,
£1
cr
/
•
r ' t
i ^
/-"
X
^,-^i J»: •?
Sr-vC^^^-*^ ' ^
1 1 1 i
\
\
\
L *
~"*
1
Background
Naa study
IB
Phth*lic anhydride efl«ct on blood eholm«»-
t«n*a activity
- Group I; 2 - Group I 1; 3 - Group 111) 4 - Group
IV (Control). AB - tnhalition pvnod
leucocyte count. Hemoglobin and erythrocytes fluctuated within normal
limits. Thus, the results indicated that the chronic experiments brought
into evidence of changes in the motor -chronaxy ratios of the muscle antagon-
ists, cholinesterase activity, and thrombocyte counts in rats of Group I (1.32
mg/m3). Less pronounced changes appeared later , in the study in rats of
Group II. During the 2 -week recovery period these functions returned to
their normal levels. No deviations were noted in rats of Group III. Data of
the chronic experiments were processed statistically and confirmed the con-
clusions. On the basis of results obtained in this study it is recommended
that 0.2 mg/m3 should be adapted as the maximal allowable PhA in atmos-
pheric air. A survey was made of air pollution with PhA plants producing
it using the I. B. Kogan (1961) polarographic method and aspirations con-
taining 96° ethylol.
- 58 -
-------
The possibility of PhA vapors and aerosol escaping into the plant
surrounding atmosphere was checked. The plant produced 10,000 tons of PhA
per annum. Table 3 presents data on the level of pollution by PhA vapors and
aerosols. Results showed that only at 1000 m from the plant did the sample
concentrations fall below the 0.2 mg/m3 allowable concentration.
Table J
PHfHALIC ANHYDRIDE >-OLLUTIOW OF ATMOSPHERIC AlH IN THE
REGION OF THE CHEMICAL COMBINE
1
Metirs froil Ho. of co | .
• ourc. of |(|cl.d air
d i6chargo
250
500
1000
1 500
saaplao
17
37
32
10
Saaples mi In
pollution »- Utiisal
bo«e level concn.m
of odor per— Bg/»3
cept ion
9
22
17
- 3
0,134
0,652
0,1510
0,055
/3
con en .
0.031
0,064
0,08-4
0.017
For PhA producing plants the sanitary break zone should be not
lees than 1000 m. The zone proposed by construction planners (500 m) is of
insufficient width for plants with a production capacity of over 10, 000 T/year.
CONCLUSIONS
1. The threshold of phthalic anhydride odor perception is 0.32
mg/m3 for most sensitive persons; 0.22 mg/m3 is the eubthreshold con-
centration.
2. Analysis of the dark-adaptation curve for short-time inhalation
of PhA indicated that its activity threshold is 0. 92 mg/m3, while 0. 55 is a
subthreshold concentration.
3. 0.2 mg/m3 is recommended as the maximum single-exposure
concentration of PhA.
4. Chronic, 24 hr. , 70-day exposure to inhalation of vapors and
aerosols of a PhA in concentrations of 1.32 and 0.54 mg/m3 elicited statis-
tically reliable changes in the flexor and extensor chronaxy ratio in whole
blood cholinesterase activity and in the thrombocyte count. A 0.2 mg/m3
PhA concentration proved inactive and is recommended as the average daily
maximal concentration.
5. For reasons discussed in a preceding paragraph of this paper,
provision should be made for 1000 m sanitary break zones around plants built
for the production of 10, OOOT of PhA per annum.
- 59 -
-------
COMPARATIVE TOXICITY STUDIES OF BENZENE, TOLUOL, AND XYLOL
BY THE REFLEX ACTIVITY METHOD
I. S. Gusev
From the L. N. Sysin Institute of General and Communal Hygiene
of the USSR Academy of Medical Sciences
Aromatic hydrocarbons benzene, toluol, and xylol have been
studied by toxicologists and hygieniste for many years past. Despite the
facts that these substances have been and still are extensively used indus-
trially and that they all possessed broad toxic spectra, the information found
in the literature regarding their comparative toxicity, no allowable limits
have been established for their concentration in the living environment.
Pure benzenej toluol, and xylol are colorless liquids having specif-
ic aromatic odors, and all are poorly soluble in water. The boiling point of
benzene is 80.1°, of toluol--110. 8° , and of xylol--140. 5° . Benzene is three
times, toluol 4.2 times, and xylol 1.35 as volatile as ether. All are good
solvents of fats, resins, raw rubber, essential oils, and dyes and other
organic compounds. These compounds are widely used as solvents indiffer-
ent industries. Benzene is also w:dely used in organic synthesis of phenol,
nitrobenzene, chlorobenzene, maleic anhydride, and other compounds. Toluol
is a raw material used by the explosives industry. Xylol is used in the pro-
duction of xylidine and of phthalic acid.
Benzene, toluol, and xylol have a narcotic effect primarily on the
nervous system. Thus, G. E. Rozentsvit (1954) showed that functional disrup-
tions of the CNS led to morphological changes in the blood. Some investiga-
tors believe that changes in other organs were the causes of nervous and en-
docrine systems poisoning. Changes in reflex activity in animals at various
concentrations of benzene have been reported by R. I. Yaroslavskaya and I.
M. Rozovskiy (1952), Yu. V. Novikov (1957), A. I. Korbakova, S. I.
Kremneva, N. K. Kulagina and I. P. Ulanova (I960). A. A. Golubev (1959)
injected mice with xylol in 550 mg/m3 concentration and observed delays in
conditioned reflex reactions and delayed differentiation response. Ye. N.
Lyublina (1950) exposed rabbits for 40 min. to benzene, toluol, and xylol to
test their effect on the CNS. The concentrations were for benzene and
toluol 300, 400, 1000 mg/m3, and for xylol 100, 200, 400 mg/m3. Concen-
trations which broke down unconditioned reflexes in rabbits were: benzene
1500, toluol 1000, and xylol 750 mg/m3 . (A. S. Faustov, 1961). The author
showed that xylol, unlike benzene and toluol, acted as a CNS depressant.
- 60 -
-------
Comparative evaluation of the toxicity of benzene, toluol, and xylol
have been conducted by different authors based on their effects on blood and
the hematopoeitic organs. From this point of view (M. G. Mgebrov, N. D,
Rozenbaum, Lind and others) benzene was the more toxic. Others believed
that chronic toluol and benzene poisoning depressed the CNS activity equally.
R. G. Leytes, B. I. Martsinkovskiy and L. K. Khotsyanov are of the opin-
ion that toluol and xylol acted analogously to benzene, but produced signifi-
cantly fewer changes in the blood (O. N. Olimpiyeva, V. M. Retnev, A. P.
Rusinova, 1958). Studies and comparative evaluation of effects of the a e com-
pounds in minimal effective concentrations are of basic interest toxicological-
ly and from the standpoint of sanitary production of atmospheric air.
Scanty information was found in the literature on air pollution by
benzene, toluol, and xylol. Major production and processing industries are
not the only air pollution sources. Discharges from small industries also
played an important contributing part. Having specific odors, the com-
pounds under study affected nasal and upper respiratory receptors and eli-
cited series of reflex reactions in which higher nervous centers and the
cerebral cortex played a part. Odors and reflexes elicited by them are not
without sequential effects. AB cortical reflexes they have the capacity to
form temporary interconnections with a variety of processes (S. V. Anichkov,,
1952).
Recent studies have shown that threshold concentrations of chemi-
cals could induce reflex reactions such as change in vascular tonus, rhythms
and depth of pulmonary response, optimal chronaxy, skin-galvanic response
and eye sensitivity to light (K. A. Bushtuyeva, I960; M. K. Borisova, I960;
M. T. Takhirov, I960, and others), with or without accompanying recordable
cortical responses. Because of this it is possible to study the threshold of
odor perception of these compounds, which have not been studied before. Such
studies were made by different methods. Thus, N. V. Lazarev (1963) deter-
mined odor perceptions thresholds for benzene at 5 mg/m3 , for toluol at 2
mg/m3 , and for xylol at 0. 8 mg/m3 . According to Yu. V. Novikov (1956) the
odor perception threshold for benzene is 3 mg/m3 . Chen1 Yun'-tay set the
odor perception threshold of orthoxylene at 0.73 mg/m3 (1963).
The present author determined odor perception thresholds by the
method proposed by the Committee on Sanitary Protection of Atmospheric Air
(V. A. Ryazanov, K. A. Bushtuyeva, Yu. V. Novikov, 1957). Benzene, tol-
uol, and xylol concentrations -were determined by the method of M. V. Alek-
seyeva (1964). The method is based on the nitration of the compounds follow-
ed by the extraction of the nitrogen compounds with butanol. Upon the addi-
tion of alkali to the extract a color appeared (for benzene--violet; toluol--
orange; metaxylol-green-blue). The colored solutions were read colori-
metrically. Determinations were made with a FEKN-57 photoelectric colori-
meter, and results compared with calibrated standards. The sensitivity of
- 61 -
-------
the method is 0.5 microgramB for benzene, 1 microgram for toluol, and 2
micrograms for xylol with 2 ml samples.
DtTtHMINAl lUNt, OF
T.t.1. I
btNildt IHHtiHOLL) OUUH
nJ U f
tions
8
3
2
5
j
•J-J/B ot ooniane
H in iowl
percept ib IB
3,4
3,2
2.8
Mammtl non-
percept i b 1 e
3,2
3,0
2,5
The odor perception threshold was
determined with the cooperation of 18 test
persons. In all 7 concentrations and 518
observations were studied. ( Results are
listed in Table 1).
The first benzene concentration
studied was 4 mg/m3 ; 8 of the 18 test
persons found this concentration inactive;
the minimal perceptible concentration for 5 test persons was 2. 8 mg/m3 ,
and the maximal nonperceptible concentration was 2.5 mg/m3. Thus, re-
sults of the experiments confirmed those obtained by Yu. V. Novikov in 1956
(3 mg/m3).
Studies of odor perception thresholds for toluol •were made on 30
teat persons, making a total of 744 tests and using 7 concentrations. Re-
sults are shown in Table 2.
Table 2
DETLHUINATIGNt, OF TOLUUL VArtJR IHHt^M-
HOLU OL/OH k'E.RCEHTION
Five of the 30 test persons failed to
detect the odor of toluol in concentration of
3. 2 mg /m3 ; for 6 persons 1. 5 mg/m3 was
the minimum perceptible concentration for
most sensitive persons. The maximal non-
perceptible concentration for these persons
was 1.27 mg/m3. The odor perception
threshold of 1.5 mg/m3 for toluol agreed
with the data of N. V. Lazarev (2 mg/m3).
Tests for determination of meta-
lxylol odor perception threshold were made
on 18 test persons using 6 concentrations with 431 observations in all. Re-
sults are shown in Table 3. Data in Table 3 show that the minimal percepti-
ble concentration for the four most sensitive persons was 0.6 mg/m3, and
the maximal subthreshold concentration was 0.41 mg/m3 . Accordingly the
odor perception threshold of metaxylol (0. 6 mg/m3) is almost identical with
the concentration recorded by Chen1 Yun1-tay (0.73 mg/m3). Accordingly,
the drop in the odor perception threshold concentration from benzene to xylol
was already demonstrated inactive, that an increase in the number of methyl
groups enhanced the odor intensity in this chemical series.
No. of de-
tsrninat i ont
5
6
4
3
3
3
6
"9/»J of toluol "~
M - - --
ceptible perceptible
>3,2
3,2
2,8
2.3
2,0
1.8
1,5
2,8
2,3
2,0
1,8
1,5
1.27
- 62 -
-------
Ubls 3
DETERMINATION OF XTLOL VAPQH THRESHOLD
ODOH PERCEPTION IN MGIH
No. of
loots
2
3
5
' 4
4
Mg/|3 of rj lol
M i n i •• 1
parcvpt ib !•
1.9
1,4
1.0
0,85
0,6
Maxiiul nen-
p«rc«pt ib 1 a
1.4
1.0
0,85
0.6
0,41
The next study aimed at determin-
ing the effects of their minimal concentra-
tions affecting electrical activity of the
cerebral cortex. Corticopotential rhythms
reflected active processes in different sec-
tions of the cerebral hemispheres and other
adive phenomena in the CNS.
K. A. Bushtuyeva, Ye. F. Polez-
hayev, and A. D. Semenko (I960) first used
the functional electroencephalographic method in their study of responses to
reflexes evoked by minimal concentrations of chemicals. Their results
showed that eubthreshold odor perception concentrations alone elicited no
visible effects, but altered electrical activity of the cerebral cortex. Re-
cently, modifications of this method have been used in the study of effects
of minimal concentrations of chemicals on the functional activity of the CNS.
The present author determined the effect of different odor perception concen-
trations of benzene, toluol, and xylol by the method of quantitative analysis
of alpha-rhythm reflex responses (A. D. Semenko, 1963) which it was thougM
best reflected the course of the cerebral cortex processes. The method ia
based on the entrancement of biopotentials induced by disturbances in the
cortical electrical rhythms (trigger stimulation); it was proposed by Walter
and Shipton (1949). N. P. Bekhtereva and V. V. Uaov (I960) showed that
trigger stimulation strengthened alpha-rhythms and was highly effective at
high functional loads. In the present studies stimulation was induced by light
pulsations of regulated intensity applied at five-second intervals. The effect
of low concentrations of the substances on the elctrical brain activity had
been recorded after the pattern of alpha-rhythms in the test persons was es-
tablished. Experiments were conducted in a special dimly lit chamber. The
test person was seated in a semirecumbent position in a comfortable chair.
In front of him was a cylinder filled with fresh air which was released at the
rate of 30 li/min. ; at the proper moment the tested gas was mixed with the
fresh air at desired concentrations; appropriate regulating and controling in-
strumentation was used in such experiments.
Ten observation tests were made with each tested pollutant concen-
tration intermittently, five with the gas vapor and air and 5 with clean air.
Each teat consisted of 18 one-minute cycles consisting of 10 sec. of accoustical
stimulations, 7 seconds free from stimulation, 18 sec. of stimulation with
light and 25 sec. of active warm-up prior to recording simple rhythmic stero-
types and maintenance of general tonicity. Eighteen minutes of the experiment
consisted of 3 minutes of training, 3 minutes to set up the experiment, and the
remaining time for the experiment proper. Gaseous mixture was fed for 6
minutes, 6 minutes were given to recovery. The vapor-air mixture was re-
leased before initiating sound stimulation and ended with stimulation with
- 63 -
-------
Analysis of the biocurrent records was carried out by integrating
the energy of amplified rhythms. Results were processed statistically to
determine reliability index changes. Five observations were made to de-
termine concentration of subthreshold odor perception of benzene and its
effect on the elctrical cerebro-cortical activity in most sensitive persons.
Bipolar biocurrents were recorded on a 16-lead EGG manufactured by the
Galileo company, from the temporal, parietal, and frontal regions individual-
ly and in combinations. Benzene concentration of 2 mg/m produced en-
hanced electrical potentials from the left occipital-temporal part of the cere-
bral cortex. Dynamics of the indicated changes are shown in Table 4.
RELIABILITY OF ELECTRQPOTENTIAL BRAIN CHANGS EFFECTED BY THE IN-
HALATION OF AIR CONTAINING 2 ng/B3 ,0f BENZENE VAPOR
1 n 1 t I a 1 a
of
Tester*
A. S.
K. S.
a, i.
N. V.
S. L.
Air-gn »i»tur»
Hinutei
1-2
0
O
O
o
O
3—4
b
a
a
c
b
B— 8
C
C
b
0
c
Recovery period
Minute*
1—2
a
o
o
b
c
3-4
a
o
c
b
.c
B-6
O
0
o
0
o
N o I • i - R.I labilityi a - 931j b - 99%t o - 99.9$| 0 - Not
. re 11 ib le
Data in Table 4 indicate that statistically reliable data were re-
corded during the 3rd-4th minute after inhalation of the gas mixture was
initiated, increased by the 5-6th minute and disappeared by the 5-6th minute
of the recovery period. Statistical treatment of data recorded from other
parts of the brain showed no statistically reliable changes. Below the 1. 5
mg/m3 concentration, benzene produced no statistically reliable changes in
electrical brain activity. Changes in bioelectrical potentials in test person
N at different benzene concentrations are shown in Figure 1.
Studies of reflex response
to subthreshold odor-perception
toluol concentrations were made
with the cooperation of 4 test per-
sons highly sensitive to this com-
pound. The biocurrents were re-
corded from the frontal-central
and temporal-occipital regions.
Toluol concentrations of 1 mg/m
produced in all the subjects statis-
tically reliable rise in the biocur-
rents included in the left temper al-
occipital brain regions. The re-
liability of such increases rose to-
8 9 10 11 12 13 Ik IS
Fie. I. Changes in brain e lee trppotent i> I a of test
psreon affected by inhalation of different benzene
concentrations ^
I - Clean air; 2 - i»9/-3 °f b«n«ne, 3-1.5 "B/»
- 64 -
-------
ward the 5-6th minutes after the start of gas-air inhalation and persisted
to the end of the recovery period (Table 5).
RELIAfalLITY OF BRAIN ELECTKOPQTtHT ML CHA.VUES E
BY («£ JttHAMTIOM OF AIR CONTAINING I W/t»? OF TOLUOL
Ini tuls
of
ob oervad
V.6.
M.V.
P.N.
S=L.
Recovery period
HinuUt ;
1-2
b
b
0
a
3-4
o
0
c
a
5-G
O
c
b
a
Recovery P«Ho4 i
MTnut.B
1-2
c
b
c
a
3—4
a
c
b
a
5-G
o
c
a
c
Not*
0»gr«« of •tttiatiei ra I nb Hn ty i I - 9S|| b
o - 9y.9>j o - not r.llacle
No statistically reliable response was observed at 1.0 and 0.6
mg/mj concentrations of toluol.
Data on changes of cortical electropotentials in test person N at
different toluol concentrations are presented graphically in Fig. 2.
10 n a 13 it is
Fig. 2. brain e I ect ropo ten 11 » I ch«ny«» in FeMi* H
eficctod by the inhalation of air cpnt»inir>y dif-
ferent toluol v»(.or concentrations
I - CI.Bfl &,r, 2 - I oH/B3 of toluolj 3 - 0.6 «9/»
toluol vipor
Four test persons coopera-
ted in studies conducted for re-
flex effects manifested by different
xylol concentrations. Biocurrents
•were recorded from the temporal-
occipital regions. Xylol concen-
trations of 0.32 mg /m3 elicited
statistically reliable drops in the
electropotentiale in both temporal-
occipital hemispheres in all test
persons, the left hemisphere show-
ing the most pronounced effect. De-
~gree of reliability appeared at the
5-6th minute after initiation of gas-
air inhalation and lasted to the end
of the recovery period (Table 6).
4
STATISTICAL RELIABILITY OF BRAlH ELECTROPOTEMTIU. CHAKtES EFFECTED
BY THE INHALATION OF AIR CONIAInldt, O.ii BG/M3 OF XYLOL
Ini 1 1 a 1 B
of
test
persona -
A.K.
N.V.
P.It.
S.L.
bit air BiJiiure
•VtrKjt*«
1—2
O
O
B
b
3—1
a
b
0
a
5-6
O
c
b
a
neco*«*-y vmr • wu
U+ouV»a
1-2.
b
tjr
C
b
3-4
a
c
b
b
1 -_
5—6
O
C
c
b
no QJI
Statistical re I iabi I i ty i \ -
o - Not rol )«b lo
- 65 -
b -
c - 99.9J
-------
X
105
100
SS
90
vx
-
Xylol-
.. air in- — «.
-•^C", ,-'
N
i i i i i
^/-xx-x
V
.1111 __!__
, j 3 i. 5 5 7 8 9 10 I' 12 '3 '<• '5
Minute*
Fig. 3. Brain aleetropotinlitl th«na«» durirtd iHptl.
inh.ittion of different iylol eortc«ntr«lion« by "le
teater H
I - Cletn iirj 2 - 0.32 «9/"3l 3 - 0-21 mg/m
Results obtained in this study are listed in Table 7 for purposes
of comparison and evaluation of effects of benzene, toluol, and xylol micro-
concentrations on the electrical activity of the cerebral cortex.
. . T»bi«_7
STUDIES AND RESULTS OF ODO& I*ERCEPT (OM THRESHOLDS AJID Oj-
REFLEX EFFECTS OF BENZENE, TOLUOL, AND XTLOL COliCENTflArfONS
ON ELECTRICAL BRAIN ACTIVITY
£>ubst«nc«
B«niene
Toluo 1
Xy lol
Oder Perception
R«fl«* effect! on br«m
b loourrent*
Corte«ntrit ion in «fl/«3 ^ .
Threihol d
2.8
1.5
0.6
Sub threshold
2.5
1.27
0.41
Threefold
-2.0
1.0
0.32
Subthreihold
1.5
0.6
0,21
On this basis the present author proposes the following conclusions:
1. Odor perception thresholds decreased with increase in the num-
ber of methyl groups in the benzene ring.
2. Concentrations of odor perception thresholds used as indexes
in evaluating effects on cerebral electrical activity decreased benzene,
toluol, xylol in the following order:
3. Benzene and toluol enhanced the electrical potentials, while
xylol had the opposite effect, causing a decrease in the electrical activity
of the cerebral cortex. Return to normal electrical brain activity in toluol
and xylol poisoning proceeded slowly.
- 66 -
-------
4. 1.5 rng/rn3 of benzene, 0.6 mg/m3 of toluol, and 0.2 mg/m3
of xylol are subthreshold concentrations as shown by results of electrical
potential brain tests. These concentrations are recommended as maximal
permissible single-exposure concentrations for atmospheric air. This
recommendation was checked and approved by the Section of the Sanitary
Protection of Atmospheric Air of the Ail-Union Sanitary Study Committee.
EXPERIMENTAL DATA PROPOSED AS A BASIS
FOR THE DETERMINATION OF MAXIMAL ALLOWABLE
AMMONIA CONCENTRATION IN ATMOSPHERIC AIR
\
M. M. Sayfutdinov
From the F. F. Erisman
Moscow-Scientific-Research Institute of Hygiene
Ammonia is the simplest nitrogen-hydrogen compound; it is a
colorless gas with a sharp pungent odor and biting taste. It liquifies under
pressure or cooling. It is easily soluble in ether, alcohol and oils. It dis-
solves easily in large quantities in water, forming ammonium hydroxide.
It reacts actively with acids and other compounds. Ammonia is made most
commonly from nitrogen and hydrogen (I. D. Fotonich, 1956; S. A. Beskovp
196Z). It is a valuable nitrogen-containing liquid fertilizer, and is also
the basic raw material for the production of nitric acid and ammoniacal
fertilizers. Large quantities of it are used for the preparation of ammonium
hydroxide and ammonium chloride. Ammonia is "widely used in the refrigera-
tion technology, in soda production and in nitrate treatment of steel products.
Nitrate combines are the principal dischargers of ammonia into the
air; other sources include nitric acid and ammonium salt refrigeration plants,
coke oven gas and tanning plants as well as animal product processors. Ni-
trate combines and coking plants for ferrous metallurgy are most responsible
for this. Considerable atmospheric air pollution with ammonia comes from
the soil and open waterways. Literature survey disclosed scanty data re-
garding ammonia in the air surrounding such production plants and residen-
tial areas.
- 67 -
-------
V. A. Ryazanov (1961) found ammonia concentration between
20. 1-57 mg/m3 on the premises of one of the largest plants synthesizing
ammonia and 0,1-0.2 mg/m3 at a radius of 1 km from a large ammonia
plant. The average daily concentration at different points on the premises
varied between 0.216-3.309 mg/m3. At 2 km from the chemical plant, the
concentration of ammonia did not exceed 0.004-0.005 mg/m3 in the city air.
A study -was conducted cf the degree of atmospheric air pollution
•with ammonia around the Novolipets Metallurgical Factory, the largest
source of ammonia discharge into the atmospheric air. The factory grounds
house coking and nitrate plants. Ammonia is found in the carbon dioxide gas
and in the waste water of coke and chemical plants. Ammonia is found in
the air as a result of coke gas loss from leaks in gas conduits, scrubbers,
exhaust gases, etc. Ammonia gas also comes from open ammonia storage
and measuring tanks and from waste water storage reservoirs.
Ammonia is synthesized in nitrate production from coke oven gas,
discharged by the coke chemistry industry. Regardless of the closed techno-
logical system the gas escaped into the air during repairs, tank drainage,
transfer, etc.
Studies of the air around the Novolipets Metallurgical Combine
were conducted during September-October 1963 and May-June 1964 and 1965,
in connection with a study of the nitrate industry and the synthesis of liquid
ammonia. Four hundred air samples were collected on the leeward side of
the source at different distances and at 1. 5 meters above the ground.
Tab In I
AMUONIA POLLUTION OF JHL Airi iUHROUND IMC~ THE NOVOLIP-
SUK META^LURblCAL PLANT
i MoV»3 of •••onfi"
Mel«r» (ram 1963 r
pol lut ion
source
500
1 000
3000
5000
7000
10 000
Ha.in.l
1.40
3,5
2.25
1.0
0,20
A»«r«g«
0,31
0,5
0.45
0,16
0,09
>
1964 r
Mixiwtl
3,60
0.39
1,30
1,30
1 ,20
A««r«g«
0,62-
0,18
0,26
0,18
0,19
1965 r.
H«*l««l
4,68"
1,73
0V39
0.28
0,11
0,13
Avtrigt
2,19
0,91
0,24
0,22
0,16
0,12
Results of the studies shown in Table 1 indicate that the greatest
ammonia concentrations were found at 500 and 1000 m from the original
source. These concentrations rose to higher values when the plants were
in operation.
- 68 -
-------
Some authors studied air pollution around less prolific points of
ammonia discharge. Thus, Z. D. Markova (1941) found, in residential areas
of Rostov-on-Don, ammonia in 0.02-0.05 mg/m3 concentrations, whereas
in the city parks the concentration did not exceed 0. 02 mg/m3 . V. A.
Kononova and V. B. Aksenova (1963) found ammonia in 0.015-0.057 mg/m3
in the air around stock farms in a zone up to 150 m. According to American
authors, ammonia concentrations varied from 0.0058-0.0143 mg/m3 in
clean air areas of Chicago (cited by V. A. Ryazanov, 1961. The concentra-
tion of ammonia in clean air, according to Stoklaz, varied between 0. 02-0. 04
mg/m3, which according to Ryazanov is too high. Thus, the available data
suggest that, depending on the pollutant source, ammonia concentrations in
the air varied from 0.015-0.057 mg/m3, to areas where special pollutant
sources are nonexistent where the concentration varied between 0.003-0.005
mg /m3 .
Ammonia is an irritant gas, affecting primarily the mucous' mem-
branes of the respiratory tract and the central nervous system (CNS). Such
action is a consequence of ammonia's high solubility in the moisture coated
surfaces of the mucous membranes and its easy penetration into the blood
stream. Cases of acute ammonia poisoning have been described which oc-
curred as a result of storage tanks bursting or of ammonium hydroxide
spillage in everyday use (R . N. Vol'fovskaya and G. N. Davydova, 1945;
E. I. Lyublina, 1948; V. K. Trutnev and N. V. Velikorussova, 1955; K. V.
Yegorov, 1958, and others). R. N. Vol'fovskaya and G. N. Davydova (1945),
V. K. Trutnev, Lehmann (1886-1899), Horvath (1926-1929) and others classed
ammonia as a respiratory poison with invasive necrotic effects, and R. N.
Vol'fovskaya and G. N. Davydova were of the opinion that ammonia also had
resorptive properties. Damage to the nervous system resulted in loss of
consciousness and high excitation verging on incoherence. I. P. Pavlov
(1896) noted that ammonia administered to animals in 50 mg/kg doses in-
duced drowsiness and ataxia, and at 2k times that dose, tremors and death.
Studies conducted by V. V. Pravdich-Neminskiy (1958), Ye. A. Vladimirova
(1938), E. E. Kosyakov (1962), N. B. Kozlov (1962), E. E. Martinson (1962)
and others confirmed Pavlov's observations.
Investigations of Recine (1956) demonstrated that ammonia poison-
ing adversely affected the blood clotting process through its destructive ef-
fect on thrombin and residual nitrogen balance in the blood increases.
Increased ammonia concentration in the blood and tissues produced physico-
chemical and structural changes in tissue proteins, disrupted ion exchange
and acetylcholine metabolism, and lowering tissue respiration (N. B. Kozlov,
1963). Chronic effects of low gaseous ammonia concentrations elicited
catarrhal symptoms in the upper respiratory tract, conjunctivitis and a
lowering of body resistance to infectious diseases. City sewerage workers
who have been exposed to low ammonia and hydrogen sulfide concentrations
in the air were victims of chronic hypertrophic catarrh, leading to atrophy
(M.D. Ayzenberg, 1927; T. V. Ass, V. V. Vol. et al. , 1926). Abadi,
- 69 -
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Trakhta, and Trusso (cited by R. M. Zhmudskaya, 1933) found conjunctivitis
and destruction of palatal membranes in workers exposed for a long time to
small ammonia concentrations.
According to Henderson and Haggard (1930) the threshold ammonia
concentration perceived by man was 37 mg/m3 . The threshold concentration
of ammonia capable of eliciting reflex effects in man, according to T. M.
Alpatov (1964) is 22 mg/m3 . The maximum allowable ammonia concentration
in chronic exposure according to Leman (1896), Silverman, Whittenberger,
Muller (1949) is 69 mg/m3. The maximal allowable ammonia concentration
in industrial premises adopted in 1930 by the Moscow Institute of Labor Pro-
tection was 20 mg/m3. The number of sources which contributed ammonia
as an atmospheric air pollutant is great, yet few studies have been made to
determine the significance of this fact, and available data were judged to be
of contradictory character. The object of the present investigation was to
determine experimentally maximum allowable single average-daily toler-
able concentrations of ammonia in atmospheric air. Experiments were con-
ducted with a 25% solution of the gas.
Ammonia concentration in the air was determined colorimetrical-
ly by the Nessler method. The sensitivity of this method is 0.3 micrograms
of ammonia per sample. The maximum single ammonia concentration in the
air was determined by the odor perception threshold of the gas as recom-
mended by The Committee on Clean Air Protection (1957). Twenty-two
healthy persons, aged 17 to 48 years, participated, and 432 studies over a con-
centration range from 0.4 to 5,0 mg/mb were conducted.
- - -- - The threshold of am-
monia perception in the most
sensitive test persons was
0.5 mg/m3. Results of numer-
ous observations indicated
that ammonia elicited reflex
response in threshold odor
perception concentrations,
which originated in the opti-
cal analyzer and in the cere-
bral cortex response was taken as the index in the present study. Effects of
ammonia on the human body were determined with the aid of an adaptometer
ADM in cooperation of three test persons whose threshold of ammonia odor
perception was previously determined. Tests were conducted with 0. 65 and
0.51 mg /m3 concentrations for one test person and 0.51, 0.45 and 0.32
mg/m3 for the other two, based on the differences in the threshold detection
levels in the test persons. Effects of each concentration were observed at
least three times. Obtained data indicated that, ammonia in 0.45 mg/m3
concentration lowered eye sensitivity to light in two persons (D. N. and L.N.)
THHtbHOLD OF AMMONIA OuOfl PERCEPTION
Nuober of
est persons
2
7
9
4
No. of daler-
ainit i one
36
138
200
55
. J
Am aonn ooncn, in «g/»
M in i (Qtt 1
percept ib la
1.95
0.98
0.70
0,50
H«*IB»| non—
p«rc«pt ib !•
1,0
0,75
0,55
0.45
- 70 -
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while 0.32 mg/m3 concentration was inactive (Fig. 1). In the third test per-
son (Sh. L.), the threshold odor perception concentration which affected eye
sensitivity to light was 0.65 mg/m3 and the eubthreshold concentration was
0. 5 mg/m3 .
60 000 -
Fig. I
I - C!««n
Ey« tvnsitivily to light ehcngn in fexl*
D.N. •«f«ct*d Ly inhilition of ttr
conttimng difi«r«nt concn*. of •••oflit.
O.Mi ag/»3, t, _
- Anoonii 0.32 »g/»i 3 - Am
0.3 «g/«3
ooi«
The threshold ammonia con-
concentration which affected elec-
trical cerebral cortical activity was
determined by continuous recording
of the alpha-rhythm evoked by
flashing of light. The work was done
at the Department of Community Hy-
giene of the Central Postgraduate
Medical Institute with cooperation
of 5 test persons aged 18 to 24 whose
concentrations of threshold percep-
tion of ammonia odor had been pre-
viously determined. Changes in bio-
potentials were registered on an 8-
.lead "Kayzer" electroencephalo-
graph. Analysis of overall cere-
bral-cortical activity was made by
using a multilead Balashev (1964)
integrator. Biocurrents were regis-
tered by the bipolar method. Periodic light pulses were flashed from a photo-
stimulator at a frequency of 8 flashes per second and intensity of 0.1, 0.2,
and 0.6 i. Three tests -were made with ammonia concentration and addi-
tional 2 or 3 control runs with clean air. Total bioelectrical activity during
the entire period of optical stimulation was calculated by means of an in-
tegrator. Results were recorded in percentages of average activity level
computed for the first 3 minutes, which assigned the value of 100%.
In three test persons whose odor perception threshold was 0. 55
mg/m3, ammonia concentrations of 0.35 mg /m3 affected the cerebral-corti-
cal electrical activity and in two of the test persons (Kh. V. and K.V.) a
drop in total bioelectric activity occurred in the first minute, and in test
person D. M. , bioelectrical activity was enhanced. Concentrations of 0.22
mg/m3 produced no changes (Fig. 2) in any of the 3 test persons.
In two subjects (D. N. and I. N.) with thresholds of 0.76 mg/m3 ,
the activating concentration was 0.44 mg/m3 and the subthreshold concentra-
tion was 0.32 mg/m3. Clear-cut ammonia effects appeared in the fourth
minute followed by a weakening between the 5-6th minutes. A more pro-
nounced ammonia effect on the organism during the first exposure minute
can be also seen while determining ammonia odor perception threshold and
during the study of eye sensitivity to light.
- 71 -
-------
7.
HO
o too
so
BO
23656 7 8 9 10 n 12
H inutv*
Fig. 2. br&in potential amplitudes of fee«le K. V.
in tha course of inhaling air containing different
concentrations of anoonia vapor.
I - Clean air, 2 - Auon i a 0.22 »9/»3l 3 - A»«ooia
0.35 «9/«3
Zone betnen arrona p«rgod of air-^naon i« vapor in—
halalian
Thresholds of ammonia reflex activity are presented in Table 3.
Accordingly, the 0.2
THRESHOLD REFLEX EFFECT OF AMMONIA
Indoa investigated
Odor perception
Chonyee in eye aenai-
t ivi if to 1 i 3ht
Chon-jos in electrical
bre in ec 1 iv i ty
Concentration in "g/»
Threshold
0.50
0,45
0,35
bub threoho
0,45
0,32
0,22
Table 3 mg/m3 concentration which was
designated as the subthreshold on
the basis of the most sensitive
test results is here recommended
as the maximal allowable single
concentration in atmospheric air.
Determination of average
daily allowable concentration of
ammonia in atmospheric air was
studied by effects on animals un-
der conditions of chronic experiments. White rats were exposed daily for
24 hours to the inhalation of ammonia air mixtures in different
concentrations during an uninterrupted period of 84 days. Rats of Group I
inhaled air containing ammonia in 20 ±0.1 mg/m3 concentration which is the
maximal allowable concentration for air in industrial areas; Group II--
2. 0± 0. 61 mg/m3 ; and Group III-- 0.2 ±.0072 mg/m3 which is identical with
the concentration determined as the maximum allowable single concentra-
tion for the atmospheric air; Group IV, controls. Sixty white rats, weigh-
ing 105-150 were selected for the chronic inhalation experiment. Rats -were
housed in 100 liter chambers. Clear air containing gaseous ammonia in
known concentrations was continuously run into the chamber at the rate of
28-30 li/min. Air samples for analysis were taken daily.
In the course of the continuous chronic experiment the general
condition of the rats, their weight, latent reflex time, porphyrin metabolism,
and urinary ammonia content were recorded. Blood serum cholinesterase,
redox activity, luminescent leucocyte content, hemoglobin, erythrocytes and
nucleic acids were determined. At the end of the inhalation exposure and the
recovery period, some of the rats were sacrificed for post mortem study.
- 72 -
-------
Experimental results were evaluated statistically as described in some of the
preceding reports:
During the experimental period rats of all groups were healthy,
active, and maintained the normal rate of gain in body weight.
Data obtained on latent r eflex-activity time served as the basis for
evaluating functional state of the CNS effects produced by the chronic ammonia
inhalation experiment, as suggested by A. A. Minayev (1965), A. P. Fomin
(1965), and using the S. I. Gorshkov "chronoreflexogenometer" . Determina-
tions were made in sigmas of the time which elapsed between an electric
motor stimulus and reflex response to it.
Results of the study are shown in Table 4 and graphically in Fig. 3.
T.bli 1*
CHANGES IN LATENT REFLEX nut OF RATS
Pan ods
bffont i nh • 1 • t i on
.1st inhalation Banlh
2d inhilition Booth
3d inhalation Bonth
Recovery
period
Group
I
(20 Mr/M1)
71,4(0)
57,6(c)
66,3(o)
68,3(o)
68,0(o)
II
(2 MT/M1)
69,2(0)
66,4(o)
68,0(o)
68,4(0)
»68.8(c)
HI
(0.2 KT/M1)
70,4(0)
67,8(o)
67,6(o)
68,4(o)
68,0(o)
.IV
Control
.71,3
67,0
68,3
68.1
68,2
N o i e i i>t»tistical ral'abililfi c - 99.9$| o - not reliable
80
70
BO
50
I
/ 23 *• 5
Periods of study
Fiy. 3. Changes in latent period of ref I a» reipona* in rati
effected by the inhalation of air containing different
amaonia, concent rat i ona
I - uroup I (20 ng/B^i 2 - Group II ^2 By/B^)j 3 - Group
III ( 0.02 «g/«3); >) _ Group )V AB _ p.rlod of
ooporisentaI inhalation
- 73 -
-------
The time of latent motor reflex response diminished in rats of
Group I in the first month of the inhalation exposure to 20 mg/m3 , ammonia
concentration.
Blood cholinesterase activity was determined by the Fleischer-
Fauns method as modified by N. N. Pushkina and N. V. Klimkina (N. N.
Pushkina, 1963). Diminished cholinesterase activity was observed in rats
of Group I. No changes appeared in rats of the other group judged by the
graphic records in Fig. 4
H/XH 20/KII 30/XH 23'I IS/H 8/111 30/111 IZ/IV ^S/l' 5/v
' D«t«* ef atuo>
'F'ii- M- Chengca ,n blood chol ineateme activity or r»t« ef-
I fected by inhalation of air containing iMoflia
(Notation! •••• (B in Fig. 3)
Redox function of the blood serum was determined by a modified
Tumberg method (Yu. L. Anin, 1964) which is based on the methylene blue
color change in blood serum heated over a boiling water bath and recording
the time required for complete color disappearance. Complete disappear-
ance in the instances of rats of Groups I and II (Table 5, Fig. 5) proceeded
at a slower rate pointing to a drop in redox activity in the animal body.
IN THt HECQX FUNCTION IN BLOOU
OF RATS IK MINUTES,
T.blB 5
Groups
Periods
Prior to inhalation
Inhalation period
Recovery
period
(20 ur/M1)
10,53(0)
I9,IO(c)
12r66(o)
11
(2 MT u1)
10,93(o)
15,0(c)
12.73(o)
III
(0.2 3tr u1)
10p46(o) "
12,20(0)
12,63(o)
|V (Control
of IV Group)
10,66
12,05
12,66
Note - itatistical reliability - c - 99.9J} o - Statistically
not P.I lab!.
- 74 -
-------
20
if
16
z n
10
A
,'J// is/n a/in jo/m
D*t«B of study
2SJIV
Fig. 5. Effect of inaonii on the rtdox function of
r«t«' blood *«rvia
Notations otB« BB in Fig. 3 .
Luminescence under the microscope has been used as an index in
determining early qualitative changes in the morphology of white blood ele-
ments. The method is based on the capacity of nucleoproteins of degenerat-
ing cells to combine with acridine orange more than with the nucleoproteins
of newly generated cells (M. N. Meysel', V. A. Sondak, 1956). This phe-
nomenon was not observed in the present study. No changes were noted in
erythrocyte counts, hemoglobin, concentration, and in total nucleic acid
content.
Yu. K. Smirnov (1953) showed that stimulation of the nervous
system was followed by an increase in porphyrin metabolism and a retarded
return to normal. Gusev (I960) first included porphyrin metabolism studies
as a guiding index in evaluating the presence of lead in atmospheric air.
Other scientists, G. I. Solomin (1961), Li Shen (1961), V. I. Filatova (1961),
B. M. Mukhitov (1963) and others confirmed^ the high sensitivity of this
method. The rate of coproporphyrin elimination via the urine was deter-
mined spectrophotometrically. During the inhalation exposure, only rats
of Group I showed coproporphyrin changes (Fig. 6).
Results of chronic daily 24-hour exposure of -white rats to am-
monia-air inhalation in concentrations of 20 mg/m3 shortened reflex time,-
lowered blood cholinesterase activity and the redox function, and also in-
creased the rates of coproporphyrin and ammonia eliminations via the urine.
In 2 mg/mj concentrations ammonia lowered the redox function
of the blood serum. In 0.2 mg/m3 concentration in the air ammonia elicited
no physiological change in the experimental rats. Pathohistological studies
disclosed no significant changes in the internal organs and in the CNS in the
- 75 -
-------
experimental rats.
eg per 100 gr
of body ct.
USD-
9/ai IS/XII 25IXU 25/i IZ/li ll/ili 2/lY
0«to6 of »tudy
Fig. 6. Effftci of *ir-»Bi>oni» inh»l4tion of
proporfhynn ••tabeliu
Notations &«ne ts in Figure 3.
281 IV B/v
CONCLUSIONS
1. Many industries, especially metallurgical plants, having coke
oven gas and nitrate operations on the premises, constituted the chief source
of atmospheric air pollution with ammonia.
2. Subthreshold ammonia concentration which had no effect on the
cerebral cortex biopotentials is at the 0.2 mg/m3 level, which can be regard-
ed as its maximal allowable single concentration in atmospheric air.
3. Under conditions of chronic inhalation exposure such a concen-
tration proved toxicologically inactive. On this basis such ammonia concen-
tration can be recommended as its maximal allowable limit in atmospheric air.
- 76 -
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CHRONIC EFFECTS ON THE ORGANISM OF SMALL
CONCENTRATIONS OF ACRYLALDEHYDE IN AIR
Prof. M. I. Gusev, Instructor I. S. Dronov, Instructor
A. I. Svechnlkova, A. I. Golovina, and M. D. Grebenakova
Department of General Hygiene of the
Rostov-on-Don-Medical Institute
Acrylaldehyde (AAD)# is produced industrially by oxidizing propy-
lene over CuO or a vapor-phase condensation of acetylaldehyde with formal-
dehyde in the presence of Zia PQj . Much AAD is consumed in producing
glutaric aldehyde and methionine. AAD is an important product of glycer-
ine synthesis. It gets into the atmosphere when glycerine undergoes in-
dustrial treatment at high temperatures (130-180° C): in manufacturing lino-
leum and oilakin, in the core sections of foundries, and in manufacturing in-
sulation in the electrical engineering industry. It has been shown in recent
years that AAD is a constituent of automobile exhausts.
V. X. Yas'kova has found in the outside and indoor air around the
Leningrad Grease Combine high coricentrations of AAD which may have been
due to the use of a nonspecific analytical method. M. M. Plotnikova (I960)
made quantitative studies of AAD in atmospheric air around chemical and oil
plants. At a distance of 100 m from the latter the maximum concentrations
were 2 mg/m^ , and in all samples the amount of AAD exceeded the maximum
allowable 0. 3 mg /m3 , while at a distance of 1000 m in the maxima were 0. 65
mg/m . At a distance of 200 m from the drying-oil shop of a chemical plant
the maximum concentration was ZO mg/m3 ; at 1500 m, 0.3 mg/m3 . AAD acts
aa a strong mucous membrane and as a general nonspecific toxin (N. V. Laza-
rev, 1954). The maximum allowable AAD concentration for air of indoor work =
ing premises was set at 0. OOZ mg/li. Plotnikova used the threshold of odor
perception technic in determining the allowable maximal single exposure con-
centration of 0. 8 mg/m3 of acrylaldehyde in the air. The threshold of optical
chronaxy concentration was determined at 1. 75 mg/m3 , and for eye sensitiv-
ity to light it was 0. 6 mg/m3 . The maximal allowable single concentration of
acrylaldehyde in the air was found aa 0. 3 mg/ma . The maximal allowable 24-
hour single concentration was determined by a process of computation without
experimental verification, and was set at 0.1 mg/m3.
Effect of low acrylaldehyde concentrations in the air on the living
*Cf. the article of M. M. Plotnikova in the collection Maximum Tolerable
Concentrations of Atmospheric Pollutants, edited by Prof. V. A. Ryazanov,
I960, No. 4.
- 77 -
-------
organism was studied by the procedure recommended by the Committee for
the Sanitary Protection of Atmospheric Air, by exposing 40 white rats to
daily Z4-hour continuous inhalation of AD over a period of 60 days. The rata
were divided into 4 equal numbered groups; three of the groups were housed
in special inhalation chambers; animals of group 4 served ae controls.
Acrylaldehyde concentrations in the chambers were determined by the
Plotnikova modification of D. P. Senderikkhina's tryptophan method. In
reacting with tryptophan AAD formed a stable violet dye. The method is
accurate to 0. 002 mg in 2 ml of solution. In the present study specificity of
the method was not of importance (M. V. Alekseyeva, 1962).
The AAD concentrations in the chambers housing Groups I, II, and
III were correspondingly as follows in mg/m3 : 1. 52 i 05 with fluctuations from
1 to 2 mg/m3 ; 0. 51 ± 0.2 with fluctuations from 0.22 to 0.77; and 0.15 ±.0.01
with fluctuations from 0.1 to 0.3.
The following indexes were used in the study: changes in weight and
behavior, in conditioned-reflex activity; changes in whole-blood cholinesterase
activity, in rate of coproporphyrin elimination via the urine, in the percentage
of luminescent leucocytes, and pathological changes in the organs and tissues
of post mortem rats which died during the inhalation tests, or were sacrificed
at the end of the experiment. At the end of the first week rats of Group 1
(1. 52 mg/m3) appeared sickly, sluggish and apathetic, and the fur became
lusterless; they ate poorly. The condition of the rats became progressively
•worse, and inhalation was discontinued on the 24th day. Before this, 5 rats
(whose central nervous system was studied) died, and 2 died subsequently.
By contrast, the rats in the other three groups exhibited no changes in their
general behavior.
Table 1 summarizes changes in the weekly weights of each group.
table I
Group
I
II
III
IV
Acrolein in End of
nj/B-3' inne.le.tion
1
1.52
0,51
0.15
Conlrol
90.7 (a)'
114,7 (b)
128,3
129,1
Recovery
period
108.7 (c)
121,7
128.9
134.8
N o t e i Statistic.! re I i «b1111/1 • - 95ij b - 99*j c - 99.9|
~~" Coop.nson mth control on thebee.il, of condi t ion* prevei I my on
24/IV «hich correspond* lo the tl*» of lnh»le,tion dieeont inuelion .
Rats of Group 1 lost 14% of their initial weight in the course of
the abbreviated inhalation exposure; loss in body weight continued after
exposure to experimental inhalation was discontinued. By the end of the
- 78 -
-------
first week of the recuperation period the weight loss was 25%, and only toward
the end of the 5th week of the recovery did the average weight in this group
reach the initial level and continued to rise beyond it. Rats of Group II also
exhibited a statistically reliable weight loss, whereas rats of Group III
showed no statistically significant changes in their body weight.
Yu. V. Novikov, N. F. Izmerov, M. I. Gusev, and K. N. Chelli-
kanov; Ya. G. Dvoksin, V. N. Kursanov, and others applied the conditioned
?eflex method to determine the maximum allowable concentrations of chemi-
cals in atmospheric air. Their studies showed that changes in cerebral-
cortex functions appeared very rapidly. Early symptoms of impairment in
the higher nervous activity appear in the form of phase states, differential
disinhibition, falling out of certain reflexes, and finally, inclusion of all
stereotypical functions. Severe central nervous damage caused natural con-
ditioned optical and odor and taste perception reflexes to disappear. The
Festoration period in such cases may last a month or longer.
The chief symptom or indication of changes in the higher nervous
activity induced by toxic agents is constant relaxation of the stimulatory
and inhibitory processes, with subsequent development of protective inhibi-
tion. The high sensitivity of the conditioned-reflex method is indicated by
the fact that it was used in determining the activity threshold for benzene,
lead, and mercury considerably below those established by previous chronic-
inhalation experiments based on the evaluation indexes. However, the old
method of studying conditioned reflexes in the Kotlyarev chambers is elabor-
ate and time-consuming. In the present study of the higher nervous activity
in white rats an accelerated method was used in the form of the Ya. G.
Dvoskin modification (1961). Eighteen white rats weighing 64-102 g were
divided into three groups of 5 rats each and a control group of 3 rats. The
stereotype -was studied in the order of bell-light; light-bell.
Before Vifjpr
tir mh lilt ion
During ex^t I .
inhaI tl ion
9 II 3 i I 10 12 15 17 IS li ?
-------
Changes in CRA also appeared in rats of Group II (0.51 mg/m3),
but in a less pronounced form (Fig. 2).
before ex- - -- - —
pll . inhol ,
otloo ,- Inhalation period - i Recovery period
2 t 7 9 II J S \/ Iff I! IS 17 IS 22 H H llJ/t JS Jt tfHtft? SOttS* iJ S3 SI t f I 10 If H 17IS' tl
Fig. 2. 0.51 mg/m acrolem effect en conditioned refle« activity of rat No. 19.
No CRA changes had been observed in rats of Group in
(0.15 mg/m3) and IV (Control), which is clearly shown in Figs. 3 and 4:
throughout the inhalation exposure period these rats maintained the pro-
per strength correlation with weak and strong stimuli, aa well as in the
oatent period. As previously mentioned, the Group I rats died as a result
of AAD inhalation and it was impossible to study their behavior during
the recovery period. On the 10th day of discontinued inhalation the cere-
bral-cortex activity of rats in Group II returned to normal.
1
0
s
•D >-.
o *-
c -
o >
-z so
~B«fO
o .. 10
0 ° ?n
"a 0
i i /
: * o
a c
a.
Before e»pll. . Inhalation ._ Recovery
inhalation period period
? if 7 9 11 J J 8 10 It 15 17 IS ?? H tt ?3 Jii JS Jl W IJ US W SO S? St S7 SS 61 t f S 10 H Hi 17 IS I
S
T
^rf
s^
^
^-^L
^
s
V
•^
1 —
/
•^.
•x.
•f
k'
•^^
"-
*s
^
^^
•^^
•-
-.
/
h*«-
'-
>k
X?
/
^
•X
\
.V
0^\
t
s
-
F^
—
•
-
-
^^
X
r^
^
•*
*-
s k
r*~
^^
^
^
— H
c-^
Fly. 3. 0.15 ag/B of acrolem on conditioned refle* activity of rat No. 6.
&
1 G
C '
0 C
i 5 so
5 - M
I 2 w
s " ?o
••• ;
-, ;
1 ? 0
-~ I.
before flji— T — > — -^ —-~- ~ ^"' ~ ~ ~ ~~ "
pll. inhal- Inhalation -' -__ ._.""" "
tj)on psnod " • ' Recovery ptriod
, / * 7 f 11 J 3 i 10 If IS 17 IS ?? H ?S HJ* JS Jt fff IJ tS f) SOS! Jt S7SS 11 * f i 10 1! K 17 IS !l
^
«^
'
5^,
,/y
1
^
-*.
/'
^
—
-*£
~T
—
^p^
'
^'
***•
—
m
^
— *i
**•
V.
*r*
rS"
^_
x^_
/
,'
*
*k
-"
^
v.
^
>
**
•s.
L.
^*
-,
V
,,
— ^
s
s
S
s
s
^
/
j
^
**^
v
_
•-•
s
Jf
^^
••V
/
N(
s
•**,
,
FIE- ^• Conditioned refl«« activity of Control rat Ho. 5.
- 80 -
-------
It is generally believed that changes in whole-blood cholinester-
ase activity reflected disturbance in the functional state of the nervous
system (Ye. B. Babskiy and A. A. Kirillov, 1938; D. Ye. Al'pern, 1958)
and shifts in the functional state of the organism as a whole. Thia index
has been widely used in determining maximal allowable concentration of
chemical substances in the air ane has proved sensitive in a series of
studies conducted under the direction of V. A. Ryazanov by G. I. Solomin
(1961); D. G. Odoshashvili (1962), Li Shen (1961), R. U. Ubaydullayev (1962)c
P. G. Tkachev (1963), V. A. Chizhikov (1963), V. I. Filatov (1963), and
others; in fact, the new index limits were 10 to 20 times below the old
allowable maximal concentrations for industrial premises.
Cholinesterase activity was determined by using Pokrovskiy's
method (1953) as modified by A. P. Martynova (1957). The method is
based on the rate of acetylcholine hydrolysis as indicated by the rate of
color change due to change in the pH factor. CholinesteraBe activity was
determined weekly in 5 rats of each group. Results are shown in Table 2.
(Table 2 is shown on page 81).
Hydrolysis time in the control group was 38. 7-40.3 min; a
statistically reliable rise in the time to 61 min manifested itself in rats
of Group I on the 15th day of exposure to inhalation. Two days after the
experiment was terminated on the 24th day, cholinesterase activity was
still below normal. The initial level was not reached until the second day
following the sixty days of the regular inhalation period.
Statistically reliable changes in rate of Group II cholinesterase
activity first appeared on the 34th day of inhalation and reached a maximum
on the 41st day. Hydrolysis time began to drop even before the end of the
inhalation period and returned to normal on the 10th day of the recovery
period. No change in whole-blood cholinesterase activity was observed in
rats of Group III or control rats at any time.
The basic mechanism of the effect of toxic substances on por-
phyrin-metabolism has not been adequately investigated. It is widely be-
lieved, however, that it suppressed the enzyme systems and affected
cellular metabolism in the nervous system, liver, bone marrow, etc.
(A. M. Chernyy, R. B. Mogilevskaya, and others).
The rate of coproporphyrin elimination via the urine is lowered
by some toxic substances, such as carbon monoxide, dynel, styrene, di-
methylformamide, and increased in the presence of other substances, such
as lead, pentane, toluylene-diisocyanate.
- 81 -
-------
>
oT
r- in i/b —•
10 m o CTI
OOO O O)
-r en
TJ- CO OO
m CTI o*
O O CN
^r CM — en
•* in •«• to
OCM
O lO LH CO
Oi O O c
Changes in porphyrin metabolism
proved to be a useful and sensitive index;
it wse widely employed by M. I. Gusev, V. A.
Chizhikov, G. I. Solomin, D. G. Ododosh-
vili, -and others in establishing the limit of
allowable pollutant concentrations in atmos-
pheric air. Rate of coproporphyrin elimin-
ation via -the urine was determined spectro-
photometrically and by computing the optical
density of maximum absorption at 402-403
millimicrons. An SF-4 spectrophotometer
was used. 24-hour urine of 5 rats in each
group was collected once a week. Thus,
56 analyses were made, 14 from each group.
Table 3 shows the amount of coproporphyrin
extracted from the urine of the rats and re-
sults were recorded in micrograms per 100 g
of body weight.
Table 3
2^-HOUR COFHOPQHPHYRIN ELlfcHNATIDN ftI IH UfiINt IN
TERMS OF MICROSHAMS PER IDO & OF BODY
- - — - — —
Study periods f ran
i ih« I at i on in i tn-
1 1 on
F irst thre« *e«li(
Second three lacks
Third thr«» maataB
Recovery period
Acrolcin in nj/B
Ctfitrel
0.15
i
1,88
1,94
1,78
1,87
1.54
1.61
1,60
1,61
0.5]
1,43 (a)
1.26 (b)
0,67 (c)
1,50
0.54
1,93
0,99 (c)1
0,72 (c)^
1.59
N o t 9 i Ref i«bi I i ty degr«ei • -
b -
—"o o S
Ic 2M/IV inhalation diaconlinuetf
No reliable metabolic changes were
noted in rats of Group III; statistically re-
liable drop appeared in rats of Group II dur-
ing the first 3 weeks of experimental inhala-
tion. This reduction continued to the end of
the inhalation period.
Rata of Group I manifested a tendency
to increased elimination rate to the end of the experimental inhalation, al-
though this change was not statistically reliable. After inoculation was dis-
continued porphyrin metabolism began to drop gradually, which continued
until the 6th week of the recovery period, when the curve began to level out,
so that during the 8th week it coincided with the level of the control group.
- 82 -
-------
With the aid of luminescent-microscopy
according to M. N. Meysel1 and A. V. Gutkina
(1953), M. N. Myesel1 and H. A. Sondak (1954),
A. D. Semenenko (1963) was able to detect after
secondary stimulated luminescence, changes in
blood leucocytes in white rate subjected to
chronic aniline inhalation in concentrations of
0.03, 0.3, and 3 mg/m3 . Depending on the
concentration of the pollutant in the air, lumin-
escence was noted in rats of all three groups.
M. I. GusevandK. N. Chelikanov (1963)
used the leucocyte luminescence method in at-
tempting to determine daily allowable maximal
pentane concentrations in atmospheric air. A
statistically reliable increase in luminescent
leucocyte count was observed in their studies.
The leucocyte count increase persisted through
the inhalation period, but returned to normal
during the recovery period.
Table 4 summarizes the results of tests
made to determine, with an Ml-1 microscope,
the percent content of luminescent leucocytes in
the blood fluorochromed with acridine orange
diluted 1:10, 000. During the first week of ex-
perimental inhalation, rats of Group I (1. 52 mg/m3
exhibited a leucocyte increase. The count subse-
quently decreased to levels still above those of the
controls. It was difficult to get blood from rats
of Group I due to its rapid coagulation, which ex-
plains the gaps in leucocyte data on some days of
the experiment. The increased leucocyte counts
persisted for 20 days after the premature ter-
mination of the inhalation period; it returned to
normal 7 days later, after which no further tests
,were run.
Rats of Group II (0. 51 mg/m3) also mani-
fested a statistically reliable change in leucocyte
percentage during the first week, a change which
continued to the end of the experimental inhalation. After 11 days of re-
covery the leucocyte count began to drop and returned to normal.
Rats in Group III, which had shown no indications of being affect-
ed by AAD in any of the other tests, exhibited a statistically reliable ia-
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- 83 -
-------
crease in leucocyte percentage on the 24th day of inhalation. Other investi-
gators noted similar changes. For example, A. D. Semenko (1963) produced
reliable changes in the blood of rats chronically exposed to the inhalation of
aniline in a concentration of 0.03 mg/m3 , while P. G. Tkachev (1963), who
used different methods, detected no changes with the same concentration.
This disparity in results suggests that luminescent microscopy is a highly
sensitive method which can be used in determining the maximum allowable
concentration of chemical substances in atmospheric air. In this connec-
tion the necessity arises to study the nature of the process by which lumin-
escing-leucocyte count increased when toxic substances in low concentra-
tions affected the organism.
After the chronic experiment was ended some of the animals un-
derwent patho-anatomic study.* Histological examination of organ tissues
of animals in Groups III and IV revealed no noteworthy irregularities. The
first changes, at a concentration of 0.51 mg/m3 (Group II) appeared as pro-
liferation of the cylindrical epithelium of the bronchi with hyperproduction of
mucous and excessive infiltration of the bronchial walls by eosinophiles.
Rats subjected to the inhalation of air containing 1. 52 mg/m3 of AAD (Group
I) exhibited acute changes of an inflammatory nature: in the respiratory or-
gans in the form of purulent panbronchitis and bronchiolitie, and macrofocal
pneumonia in one half of the rats; and in two cases in the form of fibropuru-
lent pleurisy. In the internal organs (miocardium, liver) acute dystrophic
changes were noted in the form of granular and fatty dystrophy (degenera-
tion) with small sections of necrobiosis.
CONCLUSIONS
1. Prolonged inhalation of acrylaldehyde (AAD) vapor in 0.51 and
1.52 mg/m3 concentrations caused weight loss, changes in conditioned-re-
flex activity, a. reduction in cholinesterase activity, a drop in porphyrin
metabolism, and an increase in the percentage of luminescent leucocytes in
the blood of the experimental animals.
2. Aside from an increase in leucocytes, no changes were effect-
ed by the inhalation of 0.15 mg/m3 of AAD under identical circumstances.
The secondary luminescent method presents the opportunity to directly ob-
serve the earliest pliysiochemical and morphological changes in the cells.
Therefore, it can be recommended for determining the average daily maxi-
mum allowable concentration of a pollutant in atmospheric air.
3. The presently established maximal average daily allowable
concentration of AAD in atmospheric air (0.1 mg/m3) is below the threshold
of activity and may be retained without change.
^Conducted by K. L. Volchenko.
- 84 -
-------
HYGIENIC EVALUATION OF THE COMBINED EFFECTS
OF ACETONE AND PHENOL IN ATMOSPHERIC AIR
U. d Pogoayan
From the A. N. Sysin Institute of General and Municipal Hygiene
of the USSR Academy of Medical Sciences
Experimental studies of the biological effects of atmospheric
pollution found wide acceptance in the Soviet Union in determining hygienic
standards for maximum allowable harmful substances concentrations in
atmospheric air. At this writing, 67 such pollutants had their individual
maximal fixed. However, a single industrial plant will often discharge
iato the air a mixture of pollutants. Industry employs highly complex com-
binations, of compounds which can react with each other and form new, and
sometimes more toxic, compounds. In addition, an inhabited district may
have several different industries, each discharging into the air its type of
pollutant. For this reason, the continued investigation into the effects of
individual pollutants must be supplemented by studies of the effects of two
or more pollutants simultaneously present in the air.
While the literature dealing with such problems is scant, such
studies as have been made show that the combined effect of air pollutants
a, b, c. . .is as a rule the sum of their individual effects: e.g., sulfur
dioxide and the aerosol of sulfuric acid (K. A. Bushtuyeva, 1961) hydrogen
sulfide, carbon disulfide, and dynel ( Kh. Kh. Mannanova, 1964); ethylene,
propylene, butylene, and amylene (M. L. Krasovitskaya, 1964); isopropyl-
benzene and its hydroperoxides (G. I. Solomin, 1964); and strong mineral
acids (sulfuric, hydrochloric, and nitric) in concentrations of H-ions (V.P.
Melekhina, 1964). The results of these experiments are included in the
table of MAC of contaminants in atmospheric air which was approved at the
end of 1965.
Among the promising technological processes in the world's or-
gano-synthetic industry is the method of producing acetone and phenol simul-
taneously through isopropylbenzene hydroperoxide ( cumene). It was de-
veloped and first introduced on an industrial scale in the Soviet Union in
1949- In connection with the increasing demand for acetone and phenol In
different branches of the national economy, the cumene method, being the
most economical, has found widespread use; plants employing this method
can be regarded as future potential atmospheric air pollutants from the
standpoints of air protection because of the imperfection of the productions
processes involved and other similar factors.
- 85 -
-------
It haB been shown by V. P. Melekhina, M. A. Pinigin (1961) and
others that the air around a plant producing acetone and phenol simultaneous-
ly is polluted over extended distances by these two substances and others.
Therefore, investigating the combined effect of phenol and acetone in low
concentrations is of pressing theoretical and practical importance, es-
pecially in relation to determining limits of concentrations from the sani-
tary hygienic viewpoint.
Acetone (dimethylketone) belongs to the family of compounds
which possess cumulative irritant and narcotic properties. Phenol (carbol-
ic acid) is a nerve poison; it also has irritant and narcotic properties (N.
V. Lazarev, 1951). In this study the nephelometric method was used in
determining phenol and acetone in atmospheric air (M. V. Alekseyeva,
B. Ye. Andronov, S. S. Gurvits, A. S. Zhitkov, 1954) and the colorimetric
method for the former (V. A. Zhrustaleva, 1962). Acetone reacts with
iodine in an alkali medium to form a white suspension of iodoform the in-
tensity of which can be estimated by a standard scale. The sensitivity of
the method is 0. 001 mg in 4. 5 ml. The method is specific, and phenol
does not interfere with the determination.
The action of phenol with 4-aminoantipyrene in the presence of
potassium ferrocyanide at pH= 9- 3 forme a rose-colored dye. The sensi-
tivity of this method is 0.0002 mg in 2 ml. Acetone does not interfere with
the determination. The investigation began by determining odor percep-
tion concentration for the two chemicals individually, and then in combina-
tion. Eighteen test volunteers cooperated in this study.
The odor perception threshold concentration was determined by
using a generally accepted method (V. A. Ryazanov, K. A. Bushtuyeva,
Yu. V. Novikov, 1957). Five of the eighteen test persons whose odor per-
ception was the most sensitive (1.1 mg/m3) for phenol and 0.22 mg/m3 for
acetone. This was identical with results of previous investigations of Yu.
G. Fel'dman (1962), B. A. Mukhitov (1963). The concentrations employed
in first test are summarized in Table 1.
Table 2 presents results of odor perception threshold determina-
tions in the five most sensitive test persons; the threshold served as an in-
dex and showed that the threshold concentration of each component of the
combination was equal to 1.0. The remaining 13 testers were not sensitive
to these concentrations, which is entirely natural, since they had a higher
threshold of odor perception for each substance individually and in combina-
tion.
The odor perception threshold is not to be confused with the limit
of physiological activity of the irritants. Often, without being accompanied
by sensation, stimulants evoke definite physiological responses of a so-call-
- 86 -
-------
ed subsenory nature (G. V. Gershuni, 1949). This phenomenon has been I
confirmed in studies with low concentrations of harmful substances in in-
haled air. Investigations along this line have shown that subsenory concen-
trations of chemical irritants normally insensible by odor can cause changes
in the functional state of different organs and systems, particularly the
visual analyzer; and the threshold of such changes is considerably below that
of which the subject is aware.
ACETONE AND PHENOL CUNCENTHATI 0Mb CORRESPONDING TO
THRESHOLD OF ODOR PERCEPTION OF THEIR MIXTURES
Acelona ir>d ph«nol in •»tur»
•9/-3
Aoaiano
I.I"
1.1
0.72
1.1
0.55
I.I
0,55
1,1
0,36
I.I
Phanol
0,022
0.022
0,011
0,022
0.011
0,022
0.008
0.022
0.011
0,022
In pirta of individual
threshold parcaption
Action*
1.0
'o.65
0,5
0.5
0.33
Phono I
1.0
0,5
0.5
0,36
0,5
Suanary coo en . o*
ind iv i dual tub-
threaholdo of each
component
2.0
1.15
1,0
0,86
0,83
•In the numerator _ the concent rat i on jookad forj in n
threshold odor concentration of individual component.
nuifiLon -
" Table 2 ~
ODOR PEriCEPTlDN THRESHOLDS OF ACETONE PHENOL.
VAPOR PERCEPTION FOH PEHiONi OF MOST SEfiSI -
TIVE ODOR PERCEPTION
Eye sensitivity to light is a
fine and labile function of the visual
analyzer the level of which is affect-
ed by different factors in the external
environment. Particular attention
should be centered on the view based
on a series of studies by L. A. Orbeli
(1934), A. V. Lebedinskiy (1935), K.
Kh. Kekcheyev (1946), P. P. Lazarev
(1947), S. V. Kravkov (1950), accord-
ing to when eye sensitivity to light at the level of complete adaptation to the
dark reflected not only processes in the receptor itself but also the state
of the visual centers in the cerebral cortex and the brain stem. According-
ly, a change in the level (efficiency) of eye sensitivity to light as a result of
an irritant can be regarded as reflecting physiological shifts of both peri-
pheral and central origin.
threshold fractions
of each coupon »nt
part i
2,0
• 1.15
1.0
0,86
0.83
Parcapt i b la
5 taatara
5 •
5 i
Not
parcapt ib 1 a
5 taatara
5 , »
- 87 -'
-------
Selecting three of the five subjects who were most sensitive with
respect to odor perception and using an ADM adaptometer according to a
generally accepted method, the author investigated the reflex effect of an
acetone-phenol mixture on eye sensitivity to light under conditions of adapta-
tion to the dark. All three subjects had normal vision and a comparatively
stable dark-adaptation curve. In making the ADM test the fact was con-
sidered that the odor perception threshold of the selected most sensitive
testers coincided with that previously ascertained (Yu. G. Fel'dman, 1962;
B. M. Mukhitov, 1963) and as a point of departure adopted the same authors'
results for the thresholds of acetone and phenol reflex action on eye sensi-
tivity to light.
"._ '___'_ j T»tla 3
OF ACETONE AND PHENOL CONCENTRATIONS IN MIXTURES
Acatone and phenol cortCdntrtt ions in aivlure
In mj/o
Acetone
0,44*
0,55"-
0,27
0,55
0,22
0,55
Phenol
\
0,0125
0,0156
0,0078
0,0156
0,0070
0,0156
In fractions of indi-
vidual coeponant thres-
holds
Acetone
0.8
1
0,5 '
0,4
Phenol
0,8
0.5
0,45
Suoaary of frac-
tion! of in-
dividual coupon -
•fit's fractions
-1.6
•i.o
0.85
•In numerator - th« concentration leokvd fort In nonrator -
thrjeBrtold concentration nf individual 101100 indicated by th«
taBt.
The first combination, with a. total-concentration
index of 1.6, produced statistically reliable changes in all three testers;
•when the index dropped to 1.0, only two persons responded, and at 0. 85 none
reacted. Thus, it appears that the reflex-action threshold of acetone and
phenol in combination is a combined-concentration index of 1.0. Brief inhala-
tion of the combined vapors produced contrasting effects in the testers, the
eye sensitivity to light of R. L. and Ye. S. G. L. increasing. Electro-
encephalography (EEG) has been used broadly in experiments designed to
determine norms for atmospheric pollutants. The EEG method adequately
satisfies the requirements inherent in the theoretical and practical problems
of pollution standards, being a highly sensitive indicator of the nervous sys-
tem functional state. Reflexive changes in the electrical activity of the
brain of three testers' inhaling the acetone-phenol mixture were also noted
by making quantitative analysis of the natural alpha-rhythm amplification
(A. D. Semenko, B. N. Balashov, Ye. V. Arzamastsev, 1963).
The threshold concentration of acetone and phenol affecting energy
brain potentials have been set at 0.44 mg for the former (Yu. G. Fel'dman,
" "" -"88"-
-------
1962) and 0.15 mg/m3 for the latter (B . M. Mukhltov, 1963). These were
accepted as initial values in the present study, which were established from
deaynchronization of the alpha-rhythm in generating an electrocortical con-
ditioned reflex'. The present studies were baaed on the EEG dependence on
the functional state of the brain. Primary attention was given to the alpha-
rhythm peaks and to changes in the background of the peak as a result of
inhaling the mixture, using as the stimulant a rhythmically blinking light
with a frequency equal to the optimum for the test person's alpha-rhythm.
For four weeks all the test persons underwent training, exhibiting
well-defined alpha-rhythms and a definite dynamic sterotype. Basic obser-
vations began after it was noted that inhalation of clean air was accompanied
by only few fluctuations. This level was reached by conducting the observa-
tions under standard, nonvarying conditions. During the experiment the
test persons were housed in a dimly lit soundproof chamber, in a comfort-
able semirecumbent position, with electrodes fastened to the cranial sur-
face; they breathed from a cylinder through which 30 libers of clean air
passed per minute. Two pickups on the back of the hands recorded motor
responses to stimulation. The test persons were given prior instruction.
The EEC's were recorded in the following order: initial background with
clean air feed; after 4 min, a 6 min inhalation of the mixture, followeded by
a 6-min period of breathing clean air (recovery period). Each test lasted
18 min, including 3 min of conditioning. The light was flashed for 18 sec
every minute, its intensity changing every 5 sec. In the intervals between
flashes the testers voluntarily moved in the chair. Then sound of different
intensity was introduced for 10 sec. For 7 sec after the sound stopped test
persons waited for the light to go on.
Through the experiment the test persons were active mentally
and physically, which prevented the development of phase states. Functioa-
al loads can induce serious changes in tissues and organs, and the loads
used in this study in the form of light and sound, their differential analy-
sis, and responses evoked by such stimulators created a background which
brought out shifts caused by reflex action of low acetone and phenol concen-
trations not perceived by taste, and which had no effect on eye sensitivity
to light. These changes occurred against a background of amplified natural
alpha-rhythm. Electric potentials were recorded by a bipolar method, on
a 16-lead EEG manufactured by the Galileo company, from the frontal, tem-
poral, and parietal regions of both sides of the brain. Responses to changea
in the intensity of the light and sound stimulants, pressure on a piezo pickup,
were also recorded. The EEG of each test person was analyzed as to the
magnitude of amplified natural alpha-rhythm in microvolts, i. e. , the inte-
grated energy of brain potentials. For purposes of comparative analysis the
total amplitudes were expressed in percent, with 100 percent being the aver-
age total amplitude of the first 3 min (taken as the background) against
which the total amplitude of the remaining minutes was measured (A. D.
-- 89-- •
-------
Semenenko, 1963). The results of these tests were processed statistically,
and levels of reliability were determined for the shifts that occurred. Ra-
tios of the studied and threshold concentrations are shown in Table 4.
RATIOi OF ACETONE AND PHENOL CONCtNTRAHOwTlN DETER-
MINING THRESHOLDS OF THEIR REFLEX ACTIOH ON EL£CTrtlCAL
BRAIN ACTIVaTT
Coficsntrati on of tcetone phenol mature
In o^/Q3
Ace tone
0,22*
0,44
0,17
0,44
Phenol
0,0078
0,0156
0,0068
0,0156
In perte of indiviautl1
component thre&holde
Acetone
0,5
0.39
Phenol
0.5
0,44
In euenanzed
cpncn. af thresh-
old con en . parts
of individual oon-
ppnents
* 1.0 '
0,83
X
•In nuperator - the looked for concantratloni in denominator -'
threshold con ccntration of in aiviau Jf component threshold
The mixture which had ;i total concentration index equalling 1
caused statistically reliable changes in the enhanced natural alpha-rhythm
of all test persons as illustrated by Fig. 1.
i 5 6 7 8 9 _IO_ 11 .-12 .-13 « 15
Minutes of investigation
Fig. I. Changes in br»in energy potentials in ()e^-
son R. L. affected by the inhalation of acetone-
phanol vapor miture.
I - Clean air, 2 - 0.22 »g/"3 acetone plu* 0.007B
rng/o^ o* phenol | 0.17 »9/»^ o* acetoon and O.OOfctt
of phenol
Concentrations with an index of less than 1 had no effect on the
electrical activity of the brain.
Data of all 3 tests lead to the conclusion that the combined effect
of acetone and phenol in inhaled air is equal to the eummaxy effect of the
individual admixture components.
-------
If the threshold concentration determined by the EEG method is
expressed as a fraction of the existing maximal allowable concentrations of
the individual ingredients, the minimum effective total concentration will be
1.41; the maximum ineffective, 1.16. Accordingly, when acetone and phenol
are simultaneously present in atmospheric air, the sum of their concentra-
tions, expressed as a fraction of the allowable maximu, must not'exceed !„
To explain the resorptive action of low acetone and phenol concen-
trations in mixture, in determining their average daily permissible concen-
trations in air, 48 white male rate weighing 90-110 g were divided into 3
equal groups and subjected to 3-month chronic inhalation tests.
Rats of Group I were exposed to the inhalation of a mixture concen-
tration 10 times higher than the maximum ineffective mixture; inhalation ex-
periments were of short duration; the average was 1. 657 ± . OZ15 mg/m3 for
acetone and 0. 545 ± .0013 mg/m3 for phenol. The total concentration index
expressed as a fraction of the maximum allowable concentrations of the two
chemicals individually was 10.19.
In the case of Group II rats, the acetone-phenol mixture contained:
0.1735±0. 00305 mg/m3 of acetone and 0. 00516 ± 0. 0000769 mg/m3 phenol
and a concentration index of 1. 012 .
Rats of Group III served as controls.
During the inhalation and recovery periods the rats lived under
identical conditions. Fresh air and air containing the acetone-phenol mix-
tures •were run into the inhalation chambers at the rate of 35 li/min. Mix-
ture content in the chambers was checked daily. Observation of the gener-
al condition and behavior of the rats during the inhalation experiment re-
vealed no changes indicative of toxicity, and no symptoms indicating specific
effects of acetone and phenol inhalation. Rats were weighed every 20 days.
Rats of all 3 groups gained weight equally, but at the end of experiment and
during the recovery period the controls lagged slightly.
During the chronic experiments the investigation included motor
chronaxy of the antagonist muscles, blood cholinesterase activity, copro-
porphyrin concentration, and content of vitamins C, B! , B0 and N-methyl-
nicotine amide--in the daily urine.
In choosing these resorptive action tests the author was guided by
the assumption that this effect induced by small concentrations of injurious
substances accumulated from general, nonspecific, sometimes monotypical
and insignificant shifts which, as a rule, were functional and could be re-
garded as defense-adaptive responses (V. A. Ryazanov, 1964). Participa-
tion of central nervous mechanisms in the generation of these shifts must be
- 9~f- ~"
-------
considered general and obligatory.
One indication of a connection between the nerve centers and
peripheral formations is control of subordination, i.e. , regulation of the
functional state of the periphery by the higher centers, a phenomenon
which directly reflected in the level of many functional characteristics of
the nervous and muscular apparatus, particularly its stimulability, one of
the indexes of which is chronaxy: the rate of physiological response to
stimulation. Here the primary difference appears in motor chronaxy of
antagonistic muscles. The flexor centers are more easily stimulated than
those of the extensors, i.e., the former have lower chronaxy than the lat-
ter. Given a normal relationship between the processes of stimulation
and inhibition in the cerebral cortex, the proper ratio (as a rule greater
than 1) of antagonist chronaxy will persist. This ratio changes under the
effect of different environmental factors, the chronaxies converging, the
ratio tending to become inverted. A telling symptom here is a weakening
of the effect of the central nervous system, which is caused by develop-
ment of the inhibitory process in the brain cortex.
11 crosec.
am
aw
13 & 15 '5 55 65 75 S3
Dayt of investigation
Fig. 2. klUGcIt arttaaonilt* chronaa/ in rats affected
by inhalation of nr containing acetone and phenol
Qiatures (averages according to group*).
IP IB, and 191 — flat groups) I - Extensors, 2 — f laiore
AB - ExperiDantaI inhalation period
Motor chronaxy was checked once every 10 days in 5 rats from
each group. Curves in Fig. 2 show that rats of Group II and III exhibited
normal ratios, while those in Group I on the 45th day of inhalation began
to manifest a statistically reliable increase in correct flexor chronaxy as
against that of the extensors. At the end of the recovery period the
chronaxy ratio in rats of Group I returned to normal.
--92
-------
Establishing a biological balance bet-ween the demands of the or-
ganism and the enzyme system activity is one of the mechanisms of its
adaptations to the conditions of existence. The enzyme systems partici-
pate in the biological and chemical transformations in the organism. One
of these is cholinesterase, which hydrolized acetylcholine. Changes in the
cholinesterase activity and cholinergic blood reactions reflected the func-
tional state of nervous activity (D0 Ye. Al'pernD 1958). Experiments by
D. L. Pevsner (1954) showed that under conditions of normal relationships
among basic nervous processes, such as stimulation and inhibition,, the
amount of blood cholinesterase activity did not fluctuate significantly. The
predominance of the inhibitory process in the cerebral cortex corresponded
to a marked drop in cholineeterase activity.
Blood cholinesterase activity was checked every 15 days by the
method of J. Fleisher and E. Pope as modified by N. N. Pushkina and
N. V. Klimkina (Pushkina, 1963). This parameter was checked in 5 rats
from each group. On the 37th inhalation day a drop was noted in cholines-
tesase activity as indicated by R = 0.95. No such change was noted in
of Group II (Fig. 3).
§ 253
^ 2JD
c
^220
. \
\
-\
200 -
ISO
ISC
0
JC
,2
, 1
22 37 53 71 85
Invaot ijati on doya
f >9- 3. Oynooics of blood cholinoateroae cclivily
rat.e inhaling vopor oinlura of Qcoteme end phenol.
(Group Qvorajoo)
I - Group (; <; - Uroup II: tb — InhalotiBJi pariodo
No data were found in the
literature dealing with the rela-
tive effects of low acetone and
phenol concentrations on blood
cholinesterase activity. According
toB. A. Mukhitova (1963), small
phenol doses lead to increased
cholinesterase activity, while in
the present experiment the oppo-
site was true; this can be explain-
ed by the specific narcotic acetone
action. M. Ya. Mikhel'son chowed
that narcotics reduced cholinee£e£'-
ase activity. However, since low
concentrations had no specific ef-
fect, it would be more appropriate
to ascribe these results to the na-
ture of the combined acetone and
phenol action and regard this as
the result of an inhibitory focus
arising in the cerebral cortex.
A substantial drop in blood cholinesterase activity was observed
for brief periods in the course of the experiment. Obviously the compensa-
tory mechanisms rapidly established a balance between the enzyme system
and the requirements of the organism, caused by the effect of a chemical
-------
stimulant, the phenol-acetone mixture.
At the base of the energy conversion mechanisms in the living
organism lie the processes of biological oxidation, i.e., of cellular respir-
ation. Biological oxidation is an enzymic process, which is a complex of
proteins and metallic porphyrins which create the condition for the blood
respiratory function. Thus, porphyrins, as biologically active substances,
are elements of such a vitally important pigment as hemoglobin. The human
organism contains a series of porDhyrin conversions, among them, copropor-
phyrin with two of its isomers, I and III. Presence of porphyrins in the
•white matter of the brain and of the spinal cord is significant as a factor
controling the character of the nervous system physiological processes.
It is assumed that in these central nervous system regions where the
cytochrome is either absent or occurred in insignificant quantities (the
white matter of the brain) , porphyrins were responsible for the oxidizing
processes. The supposition that porphyrins are good hydrogen acceptors
and donors must be considered experimentally proved.
Clinical observations and experimental investigations of Yu. K.
Smirnov (1953) emphatically suggest that porphyrin metabolism can be re-
garded as one indicator of the functional state of the central nervous system:
enhanced porphyrin metabolism comes into play when the hypothalamic re-
gion of the brain is stimulated. Inhibitory processes in the brain reduced its
functional activity and lead to a lowered porphyrin metabolism. All this
supplants the as sumption that determination of changes in the daily porphy-
rin content can be used as an objective test in determining the functional
state of the central nervous system. For this reason the test has been used
widely in experimental studies of the maximal allowable toxic substance
concentrations in atmospheric air. With the aid of M. I. Gusev's and Yu. K.
Smirnov1 s method (I960), the present author determined the total copropor-
phyrin content of the urine of 5 rats in each group. Porphyrin metabolism
was below normal in rats of Group I; a statistically reliable change appeared
in the 8th week of inhalation. At the end of the recovery period the copro-
porphyrin level was lower than that of rats in Groups II and III (Fig. 4).
This decline must be explained as caused by disturbing the normal relation-
ship between the basic nervous processes, as a physiological "means" of
protecting the animals' organism.
Rates of vitamins C, Bx , Ba, and N-methylnicotine amide elimin-
ation with the urine were studied next. Vitamins are catalyzers of biochemi-
cal living cell reactions. They participate largely in metabolism, of sub-
stances which constitute the enzyme systems. Accordingly determining
vitamin content can offer supplementary evidence regarding the nature of
the metabolic-enzyme processes. Literature reports describe vitamin
metabolism disturbance among workers engaged in phenol and acetone
production by the cumene method (N. N. Pushkina, 1964) at high concen-
- 94 --"
-------
Zr 33 i5 S3 70
Inusst igat ion day a
82
trations of these substances in the shop air.
Vitamins were determined in Z4-hour urine specimens daily in
5 rate from each group at 12 day intervals. Shifts in vitamin elimination
by animals of the 3 groups were insignificant and statistically unreliable;
on the 80th inhalation day the level of all the groups was nearly the same.
Pathocytological tissue
examination of rats of Group I
showed vascular damage in some
parenchymatous organs, edema
of the stroma in the villi of the
small intestines, and a change in
capillary permeability with develop-
ment of moderate perivascula?
and pericellular edema in the
brain.
Thus, chronic exposure of
Group I rats to the inhalation of
air polluted with acetone and phemol
simultaneously in concentrations
(1.657 and 0.0545 mg/m3) ten
times in excess of the maximum ia=
active concentration of the mixture
for short-duration exposure produced shifts •which pointed to a reaction in
the nervous system, viz. , disturbance of the normal chronaxy ratio of the
muscle antagonists, reduction in blood cholinesterase activity, a decrease
in the rate of coproporphyrin elimination via the urine, and a series of
minor pathomorphological changes. A phenol-acetone mixture in a concen-
tration equal to the maximum active level (0.00516 and 0.1735 mg/m3, re-
spectively) produced no changes in rats of Group II.
Investigation of the acetone-phenol mixture resorptive effect also
pointed to a complete summation of their individual effects. Therefore, it
can be recommended that the maximum average-daily allowable concentra-
tions for phenol and acetone in mixture be set at the single-exposure level,
i. e. , the sum of their fractions of the existing average-daily maximal
allowable concentrations taken individually should not exceed 1.0.
In the spring of 1964 an investigation was made of the air around a
synthetic phenol-acetone plant. Samples were taken on the leeward side 100C
300, 500, and 1000 m from the plant, 1-1. 5 m above the ground. Results of
simultaneously determined single-exposure concentrations of phenol and
acetone are summarized in Tables 5 and 6.
ig.
M. R»te of coproporphyrin eli«il»ti3n VF'Q the
in rats durmy mha.la.li an of ace ton»-ph~irnol va-
por oiiture inhalation
"olatiunt uxa a» in Figure 3.
- 95 -
-------
Table 5
SINOLE ACETONE CONCENTRATION IN ATBOiPHEH 1C AIR AROUND
PLANTS, PRODUCING SYKTHETIC ACETOME AMD PHENOL
~
^Qlore froo
d i Bchtrge
source
100
300
500
Na. o' »ir
saup 1 QO
14
13
24
No. of BfttDp I ••
b«lo* nothod
sens 1 1 1 * i ty
_
—
24
Hg/.J
~
Manntl
2.19
0.714
—
Aver.9.
1.41
0,415
—
Tabla 6
ijINnLE PHENOL CONCENTRU IONS IN ATMUiPHLrtl C AIR IN
THE VILINITY OF PLANTS PHOUUUNG SYNTHETIC ACETONE
AND PHENOL
4«tara frora
dischirga
source
100
300
BOO
1 000
No. of air
Mmp 1 ea
17
13
23
25
No, of «ir
Btapll*
b»le« Mthod
• en»iliv'»y
—
3
25
"y/.3
«««n«i
0,323
0,2197
0.0869
—
Av»r»g«
0.187
0,115
0.039
—
At 100 and 300 m the acetone readings exceeded the maximum
allowable (0.35 mg/m3) by 6 and 2 times respectively; only at 500 m were
no traces of the pollutants found. For phenol the maximum allowable 0.01
mg/m3 concentration was exceeded at these three distances by multiples
of 32, 22, and 8 correspondingly, and only at 1000 m were no traces of
the pollutants found.
It is of some practical value to compare these data with the maxi-
mum allowable phenol and acetone concentrations when present as a mixture
in doses in defined fractions of the maximal single-exposure concentration
of each individually, the sum of which must not exceed 1. At 100 and 300 m
the phenolacetone concentration exceeded the maximum allowable combined
concentrations by factors of 38 and 24, respectively, indicating that the air
surrounding this plant was heavily polluted, especially with phenol. Pre-
ventive measures should, therefore, be instituted first by freeing the
gaseous emissions of phenol vapors.
The width of the sanitary protection felt around synthetic phenol-
acetone plants can be determined reliably only by taking into account the
effects of other gaseous pollutants discharged into the air by the plants.
- 96 -
-------
CONCLUSIONS
1. Effect of an acetone and phenol mixture acting on the odor-
perception organ, on eye aenaitivity to light, and the electric activity of
the brain, should be evaluated as ;he simple fractional aum of the indi-
vidual components' effects.
2. Biological effects of the phenol and acetone mixture will appea?
when the fractional summation of their individual allowable maximal will
exceed the value of 1.
3. The maximum permissible concentration of a phenol-acetome
mixture in atmospheric air should be set at the biologically inactive con-
centration level for single exposure where the fractional summation of the
acetone and phenol individual single allowable maximal levels did not ex-
ceed 1.
4. The average-daily allowable phenol and acetone combination
can be set at the maximum single-exposure level.
DETERMINATION OF ALLOWABLE MAXIMUM CONCENTRATIONS
OF PHENOL AND ACETOPHENONE WHEN
SIMULTANEOUSLY PRESENT IN ATMOSPHERIC APR
Yu. E. Korneyev
From the A. N. Sysin Institute of General and Municipal
Hygiene of the USSR Academy of Medical Sciences
The object of this study is to investigate the combined effect of
phenol (Ph) and acetophenone (APh) vapors on the human organism. Such
conbined effect occurs when the cumene method is employed in the produc-
tion of phenol and acetone (AC), during which vapors of Ph, APh, acetone,,
benzene, and other toxic substances are emitted into the atmosphere. The
effect of combinations of several of these poisone on the human organism
may differ from that when the same substances are inhaled individually:
- 97 ~-—-
-------
the result may be a total or partial summation, diminution or enhancement
of the effect (N. S. Pravdin, 1929; N. V. Lazarev, 1938; V. A. Ryazanov,
1954). Actual final effect can be determined by experimental study of each.
Most often the final effect is one of simple summation of the individual
effects which can be expressed by the following formula
X = A/M + B/M
A B
where A and B are the maximum allowable concentrations of the pollutant
in mg/m3 of air when they are present in the air individually.
The maximum allowable single-exposure and average-daily con-
centration for phenol and acetophenone in atmospheric air were set at 0.01
and 0.03 mg/m3, respectively (B. M. Mukhitov, 1962; N. B. Imasheva,
1963). The toxicity of phenol has been adequately described elsewhere. It
enters the organism in a variety of ways. Workers engaged in phenol pro-
duction frequently manifest acute and chronic poisoning as a result of in-
haling phenol vapors. The principal harm is first to the central nervous
system, followed by the cardiovascular system; the activity of the gastro-
intestinal tract is damaged, and ^itamin metabolism is impaired (V. K.
Navrotskiy, 1928; Z. E. Grigor'yt;v, 1953; V. I. Petrov, I960). A. S.
Stegniy and Ye. K. D'yakonenko (1961) noted that workers exposed to phenol
vapors developed hypotonia, polyneuritis, impairment of perception, ab-
sence of abdominal reflex, and inadequate convergence.
Little attention has been given to the toxic properties of phenol,
and in the field which is related to this study only N. B. Imaaheva's work
fully explains the effects of low concentrations on the organism. Although
phenol, which ia widely used in industry, is not comparable to acetophenone
which is given off as a byproduct. Under ordinary production conditions
phenol and acetophenone do not affect the organism individually, but have
a constant effect the nature of which has been investigated experimentally.
Two methods were used in determining phenol in atmospheric
air: In a reaction with diazotized p-nitroaniline, which in a carbonic acid
medium produced a yellow-green to red-brown color; this method has a
sensitivity of 0.2 micrograms in 5 ml; and a more sensitive method involv-
ing 4-aminoantipyrine (V. A. Khrustaleva, 1962). Acetophenone was de-
termined spectrophotometrically (M. D. Manita, 1963) by determining the
optical density of an acetophenone solution in ethyl alcohol at a wavelength
of 244 millimicrons in a'cell with a layer thickness of 10 mm. The sensi-
tivity of this method is 0.25 micrograms in 1 ml. The method of V. A.
Khrustaleva (1961) was used in studying actual air pollution around a plant
producing phenol and acetophenone. Effect of low phenol and acetophenone
concentrations simultaneously present in the air was investigated by the
- 98 -----
-------
methods of threshold of odor perception, eye sensitivity to light, and elec-
trical activity of the cerebral cortex. Experiments were conducted with
healthy persons. The threshold of phenol and acetophenone mixture odor
perception were determined by a method recommended by the Committee
on the Protection of Air Cleanliness. First, all subjects were familiarized
with the odor of phenol and acetophenone mixture after which their threshold
of odor perception to the individual mixture componenets was determined.
Twenty-two persons cooperated, of whom two had a phenol odor perception
of 0.017 mg/m3 and a subthreshold of 0.016 mg/m3 ; for 14 others the read-
ings were 0.022 and 0.017 mg/m3, respectively; while for the remaining
6 persons the threshold was high. B. Mukhitov set 0.022 mg/m3 as the
odor perception threshold for most sensitive persons. Ten persons had aa
average odor perception threshold of 0.010 mg/m3 (subsensory, 0.008
mg/m3); average readings for all others were 0.0285 and 0.01 mg/m3,
respectively; for one person the odor perception threshold lay above 0.03
§ ^
mg/m . N. B. Imasheva set the odor perception threshold of acetophenoae
at 0.01 mg/m3 for most sensitive persons.
Determination of odor perception threshold for phenol-acetophen-
one was conducted with the cooperation of 18 persons, 6 of whom were proved
to be most sensitive. Altogether 752 tests were made. The summation con-
centration of the phenol and acetophenone mixture was expressed as a frac-
tion of the threshold concentrations, as previously described. For most
sensitive individuals the results of the two sets of tests were identical, i.eos
a threshold of 0.022 mg/m3 for phenol and 0.01 for acetophenone.
The odor perception threshold concentration of the mixture had a
concentration summation of
0.013 3 0.004 . 3
+- mg/m'
i. e. , when simultaneously present in atmospheric air the effect of low
phenol and acetephenone concentrations is equal to the sum of their individual
effects .
Expressed in terms of fractions of the individual thresholds, the
undetectable mixture had summation concentration index of 0.72. The effect
of low phenol and acetophenone concentrations on eye sensitivity to light was
investigated using an ADM adaptometer and a method recommended by the
Clean Air Committee. Some changes were made in cylinder design and air
was run in at the rate of 30-35 li/min. The increased rate of air flow was
not perceived by the subjects. Volume of air flow (500 ml/sec) was general-
ly maintained at the level of normal breathing. Three test persons were
chosen for adaptometric studies. Two mix -concentrations were used) four
- W- "
-------
45-min tests were made with each mixture. Ninety-three readings were
taken. Eye sensitivity to light was checked every 5 min and the results
processed statistically.
B. M. Makhrtiov and N. B. Imasheva obtained the following con-
centration summation index in their studies of phenol and acetophenone
individually:
0.00747
1.0 0.0015
mg/m3 (PH) +
mg/m3 (AP) and 0.77
0.0059 0.0039
0.155 0.01
The first mixture effected a statistically reliable change in all
3 test persons, the second had no effect on the behavior and the dark-
adaptation curve (Fig. 1).
In thou-
sands
ZOO
ISO
I 120
80
As in the olfactory thresh-
old tests complete simple summa-
tion of the effects prevailed here:
the concentration summation in-
dex for the minimum effective
mixture was 1.0.
ID
of
20 25 JO
invest ig«11on.
The effect of low phenol and
acetophenone concentration mix-
tures on the cerebroccrtical ac-
tivity using (1) a method developed
by A. D. Semenko for hygienic
investigations which involving
amplification of the natural alpha-
rhythm, while successively stimu-
lating the subject with a flickering
light at a frequency fitted to that of
the subject's alpha-rhythm; and (2)
a method similar to that described
by U. G. Pogosyan. Altogether
112 tests were made on 3 test per-
sons. Frontal, lobal, and parietal
readings were taken from each
hemisphere. Two gaseous phenol
and acetophenone mixtures were used. The first was 0. 00759/0. 0156 mg/m3
for phenol = 0.49 of the individual threshold concentration, and for aceto-
phenone 0.0357/0.07 mg/m3 = 0.51, and a fractional-concentration summa-
tion index of 1.0. In the second mixture the proportions were 0.00584/
0.0156 (= 0. 374) and 0.00276/0.007 (=0.394), and an index of 0. 77 (Fig. 2).
Results of EEG tests were also computed by the Mukhitov-Imasheva threshold
method. The first mixture produced statistically reliable changes in all 3
test persons; the second, none, confirming the principle of simple fractional
Fig. I. Curve of eye »fUpl«tioo le dark of fe«alfe T.
1. during tha inhalation of tcetona—pheno I vapor BIX-
turo inh»lation.
I - Clean uirj 2 - Mixture »ith a sunettion concentra-
tion index o* I: 3 — buareutlon concentration index of
0.77
-------
summation of the phenol and acetophenone individual limiting concentrations
in atmospheric air.
so
Fig
T. H
3 e 9 12
Hinutea of inv»i t igtt ion
2. Gh»ng«i in •(•etrietl brain tctivily df F««aU
. afftctfrd by rnh»lit i on -
-------
Pollutant Concentration, in mg/m3
Index of Individual
Concentrations*
I Acetophenone 0. 0017, phenol 0.0062 1.19
II Acetophenone 0. 01732, phenol 0.0637 12.14
III Controls
IV Acetophenone 0.00147, phenol 0.0048, AC 0.136 1.36
V Acetophenone 0.01141, phenol 0.04162, AC 1.334 11.76
^Decimal fraction of existing maximum allowable average-daily con-
centrations .
Concentrations were checked daily. During the inhalation period
the general condition, weight, blood cholinesterase activity, motor antag-
onist chronaxy, porphyrin metabolism, eosinophile count in peripheral
blood, and rate of 17-ketosteroids and vitamin C elimination with the urine
were checked.
The four test groups differed in no way from the controls with
respect to weight and general condition.
A slightly modified Fleisher-Pope-Spear method (1954) was gener-
ally used in determining cholinesterase activity. Rats of Groups II and V
exhibited a statistically reliable reduction during the fourth week of inhala-
tion which continued to the end of the exposure period; only during the re-
covery period did the cholinesterase-activity level return to normal. No
negative effects were observed in the other groups (Fig. 3J_.
400
300
IS/IK
IS/t
IS/X/I
31/X IS/11 JO///
Days of etudy
FIJ. 3. Changes irt chol irtesUrkfce atiivi'ly !rt hxpMtriMnUI r»U
effected by the inhalation of air containing vapor •iilur&s of
phenol, acetophenone and ao«tone.
I - liroup I (Mixture o* phanol and acetophenone having a auaoary
concentration indea of I.IU)| 2 -Group It (vap«r fial^r; of
phenol «nd acetophenone having a sum^ry concanlrat too index o1
I2.I4)} 3 -Group III (Control)i >t -Grouf; IV (..ixtur.of phanol,
aeolophenone and aeetona sith a »«*»«nr concentration indax of
I.36J> S - Sroup V (laixture of phenol, acetophanon« h«ving a SUB-
•ary concentration index of ll.70)j AB - Eiiper i.snti I inhalation
per i od
- 102--- ----
-------
The nervous aystem condition was evaluated in the motor chronaxy
of basic (antagonist muscles) shifts in which constituted an active index of
stimulation states and which reflected changes in the external environment
and the functional state of the organism with high precision. Exposure of
the rats to high concentrations of the experimental gases (Groups II and V)
had a significant effect on the functional state of the nervous system (Fig.
4), and induced changes at the end of the first month which disappeared at
the end of the recovery period. The other groups manifested no changes in
chsonaxy ratio.
i/xi H/Jt/ rr/jri i/x/i n/ti/
Days of atudy
Fli- **. Satioa of fle*ar and aitncsor chrona*i»a in rato dufinfl
period* of inhalation of air containing * «i«lur» of phanol, icefo
pnenona, and acalont vapors.
Notations MBa •» in Fig. 3.
• crog/lOU g of b udy wight
1.0
if/It J/X IS/X tS/X 12/XI Z7/XI 12/ttll 27/lU
Dates of study ' '_
Fig. S. flat* of eoproporphyrin •liaination via the unn« dur,ng
tho inhalation of air containing a •i«tur« oT~ph«nolD oootophon-
on« and acaton* vapors
Notation* MM as in Fig. 3.
Some intoxications and illnesses cause damage to the nervous
system and lead to disturbed porphyrin metabolism. Elinination of copro-
porphyrijie with the urine is an early indication of impaired nerve regula-
tion in hemoglobin synthesis (O. M. Charnyy, S. E. Krasovitskaya, 1951).
In the course of a 3-month experimental inhalation test, the experimeatal
rats exhibited a statistically reliable increase in coprophyrin eliminatiom
via the urine.
-------
During the restoration period this parameter returned to normal (Fig. 5).
Records were also kept of 17-ketosteroids elimination via the
urine. The leading role in regulating excretion of steroid hormones of the
adrenal cortex, which possess a well-defined reactivity, is played by the
nervous system; the function of the adrenal cortex depends on a cortico-
tropic hormone of the anterior hypophysis, which in turn is regulated by
the central nervous system (S. G. Genes, 1955). The corticosteroid func-
tion responds to stimuli affecting the organism. Results of 17-ketosteroids
investigations reflect damage done to the mechanisms which regulated the
adrenal cortex function. This applies particularly to the eoeinophiles.
Eosinopenia indicates functional amplification in the adrenal cortex (D. Ya.
Belov, 1957).
MiorojC/100 g " . -
of body mi.
X/X
tl/XI H/XI 12/M Z7/XII
Da/i af invaat i git ion
19/1
FIJ. 6. 1at« of k«to«t«roida divination v|« th« urine of r»U
oipoacd to th« inhilition of «tr contain ing a • ••lure «f phoool.
«o«toph»noo», tnd tactom vtpori.
Notations «aae is in Figur* 3.
In this connection a study was made of 17-ketosterold secretion
and hematic eosinophile count. Rats of Groups II and V exhibited a change
in the first parameter throughout the inhalation period, which was statistical-
ly reliable only during the third month. This can be explained partly by the
fluctuating 17-ketosteroid elimination in the control group (Fig. 6). Groups
I and IV differed little from the controls. These tests were run every 15
days at the same hour of the day.
Daily intermittent analysis of eosinophile counts in the peripheral
blood can present to the investigator a picture of the organisms organic
reactivity. The most diverse effects leading to inhibition in the central
nervous system provoke a drop in absolute eosinophile count (K. Kh. Kyrge,
1956). Counts were made in a Fuchs-Rosenthal chamber (S. M. Bakman,
1958). Rats of Groups II and V exhibited a reduced eosinophile count in the
peripheral blood. Statistically significant eosinopenia appeared twice in the
second and third month of experimental inhalation (Fig. 7). No statistically
reliable changes were noted in the other groups.
-------
100
so •
S/IX
IS/X 31/X IS/11
Ottel of investigation
Fig. 7. FluekuitioriB irt p6ri(Jh»f»l blood eo
in rats subjected to tht inh»Ution of »lr containing
. inture of phenol, «o« kof>H«no*«, *nd »oeion* v«por«.
Notations »»t«e •• In Fig. 3-
Investigation of ascorbic acid content in the urine of the teat
animals revealed no deviations from the controls.
Thus, exposure of the rats to the inhalation of air containing
phenol and acetophenone (Group II) and also acetone (Group V) affected
cholinesterase activity, motor chronaxy of muscle antagonists, porphyrin
metabolism, excretion of 17-ketosteroids with the urine, and eosinophile
count in the peripheral blood. Fractional summation concentration of phenol
and acetophenone at 1.19 index level in relation to their individual MAC
and 1.36 when acetone was added provoked no organic changes even under
conditions of chronic inhalation.
Taking into account the small difference between the maximum
single-exposure to phenol-acetophenone concentration index here recommend-
ed (1.5) and the inactive average-daily concentrations investigation (1.19 and
1.36), as well as of the level of the minimum resorptively effective mixture
(1. 96), the present author accepts the recommendation of the Sanitary Air
Section of the All-Union Study Commission that the average-daily concen-
tration of the test gases be set the same as that for single exposure, viz. ,
1.5.
Part of the present study was devoted to an investigation of actual
degree of air pollution in the vicinity of a synthetic phenol and acetophenone
"- 105—
-------
plant and hygienic assessment of the resulting data. Samples were col-
lected in May 1964 on the leeward side of the plant at 100 to 1000 m. Ac-
cording to data of the municipal sanitation-and-epidemilogy station the
plant discharged 2.2 metric tons of pure phenol into the air annually.
Acetophenone discharges were not computed. At 500 m the maximum sin-
gle phenol concentration exceeded the allowable maximum by a factor of
8.7; those of acetophenone by a factor of 9. 3 (Table 3.) The combined
concentration was 12 time above the recommended level. No traces of
either gas were detected at 1000 m. The width of the sanitary break belt
around a plant of similar type cannot be established without taking into
account the entire complex of the toxic emissions.
Uble 3
ATMOSPHERIC AIR POLLUTION IN THt NtblON OF PHENOL AND
ACETONE PHUJUCTION
Maters fron
source of
pol lut ion
1
Nuabar of i
samp I 01 j
SvBplat abova
Mtnod of
•«n»i t Ivily
Conc«nlnrtioni in Bg/'
•axlisl
—
Phonol
100
300
500
1000
17
13
23
25
17
13
20
—
0,29
0,219
0.087
, —
0,1642
0.1019
0,03417
—
100 .
300
500
1000
11
17
26
25
U
17
12
0,879
0.0967
0.0286
—
0.258
0,0571
0,00764
—
CONCLUSIONS
1. The threshold odor perception concentration of the gas mix-
tures tested by most sensitive test persons was 0.013 mg/m3 for phenol +
0.004 mg/m3 for acetophenone with a fractional summation index of 1.0.
An index of 0. 72 (0. 01 mg/m3 phenol + 0. 0026 mg/m3 acetophenone was not
perceptible.
2-3. The threshold concentrations for eye sensitivity to light
in combination were as follows:
Fractional Concentrations Sum-
mation Index
Phenol Acetophenone Min. Effective Max Ineffective
Eye sensitivity
to light 0.00747 0.00517 mg/m3 1.0 0.77
Electrical Brain
Activity
0.00759 0.00357 mg/m3
- 106 -
1.0
0.77
-------
4. The effect of phenol and acetophenone simultaneously present
in the atmospheric air equals the simple sum of their individual effects.
5. Since the maximum allowable concentrations of phenol and
acetophenone were established with some leeway, their maximum allowable
combined single-exposure and average-daily concentrations, expressed as a
fractional summation of the individual limit concentrations, such sum should
not exceed 1.5. This also applies to a phenol-acetophenone-acetone mixture.
6. Inoculation of experimental animals with phenol-acetophenone
and with phenol-acetophenone-acetone which have fractional concentration
summation indices of 12.14 and 11.765, respectively, elicited significant
changes in cholinesterase activity, motor antagonist chronaxy, porphyrin
metabolieh, 17-ketosteroid content, and caused pronounced eosinopenia.
Y/hen the respective indices were lowered to 1.19 and 1.365 no guch eequelle
were noted.
i
7. The conbination of phenol and acetophenone, and their combina-
tion with acetone elicited no changes in the organism even upon prolonged
(chronic) exposure to the inhalation of air containing simultaneously vapors
of phenol and acetophenone or the two plus acetone when the fractional sum-
mation of their individual maximal allowable concentrations have indexes not
exceeding 1.19 and 1.36 correspondingly.
8. This evaluation principle is applicable to investigations deal-
ing with air containing three simultaneous pollutants.
9. The width of the sanitary protection zone in the case of plants
producing synthetic phenol and acetone should be recommended by taking into
account the total complex of pollutants emitted into the atmospheric air,
and not on the basis of discharged phenol, acetophenone and acetone alone.
- 107 -
-------
COMBINED EFFECTS OF LOW ACETONE AND ACETOPHENONE
CONCENTRATIONS IN THE AIR OF THE LIVING ORGANISM
N. Z. Tkach
From the A. N. Sysin Institute of General
and Municipal Hygiene of the
USSR Academy of Medical Sciences
The rapid development of the chemical industry has been accom-
panied by increased discharges into the atmosphere of a great volume of
toxic complexes of broad contaminant spectra. A most frequently encounter-
ed combination consists of acetone (AC) and acetophenone (AP), by products
of the cumene method for the production of synthetic phenol (PH) and ace-
tone (AC) simultaneously. Acetophenone in this case is a product of sec-
ondary reactions; it is also continuously discharged into the atmosphere.
Results of previous studies indicated that the combined effect of these
pollutants were equal to the fractional summation of the effects of the in-
dividual components: sulfur dioxide + sulfuric acid aerosol (K. A. Bush-
tuyeva, I960); chlorine + hydrogen chloride (V. M. Styazhkin, 1962); and
carbon disulfide + hydrogen sulfide (B. K. Baykov, 1963).
The purpose of the present study was to determine the effects of
low acetone and acetophenone concentrations on the living organism. Both
poisons attack principally the central nervous system. Considerable study
has been devoted to the toxicology of acetone (A. Ye. Yefremov, 1929; N. F.
Okuneva, 1930; I. D. Mishenin, 1933; I. S. Tsitovich, 1935; P. M. Sukhov,
1935; N. V. Lazarev, 1954; Yu. G. Fel'dman, I960; Ch. Khadnan1 et al.,
1962; and others), whereas the literature on acetophenone is inadequate
(Laborde, 1885; S. S. Kamenskiy, 1889; Friedman and Maase, 1910; N. B.
Imasheva, 1963; et al.) and deals with the effects of high toxic concentrations,
except the work of Fel'dman on acetone and Imasheva on acetophenone which
deal with low concentrations under conditions of brief and chronic exposure.
No studies were found in the literature which dealt with the combined effects
of acetone and acetophenone.
M. I. Ol'shanskaya and V. V. Likhacheva (1935) investigated the
combined effects of acetophenone and benzene vapors on doves in concen-
trations of 25,000 and 10,000 mg/m3. The authors investigated the frac-
tional summation of the individual effects and found the most powerful mix-
ture to be 1:1. T. A. Shtessel' (1937) conducted 2- and 4-hr experiments
with white mice by administering to them combinations of acetone and methyl
alcohol, acetone and ethyl ether, acetone and toluene, and acetone and ben-
zene and found that the principle of simple summation prevailed in all cases.
- I'ffff --'
-------
The critical concentration of individual or combined substance were re-
garded as the one which caused the experimental mice to assume a lying-
down lateral position.
Nischiama and Isami (1957) noted intensification of the toxic ef-
fects of acetone and benzene, and a leucocyte count drop to a greater de-
gree than when the two are administered individually. The experiment be-
gan by determining the threshold concentration of acetone and acetophenone
odor perception individually and in combination. Acetone was determined
nephelometrically, the' sensitivity of which was 1 microgram per 4.5 ml.
Acetophenone was determined by a spectrometric method of O.Z5 micrograms
per ml sensitivity.
The odor perception threshold concentration for acetone was
determined in 22 healthy persons aged 18-48 years, using the commonly
accepted method (V. A. Ryazanov, K. A. Buahtuyeva, Yu. V. Novikov,
1957). All told, 412 observations were made at concentrations of 2.0, 1.654,
1, 481, 1. 096, and 0. 80 mg/m3 . Results summarized in Table 1 show that
for the most sensitive test persons the threshold was 1.096, while 0. 80 was
the highest subthreshold concentration.
Tibia I
DETERMINATION OF ACtTONE THRESHOLD OF
ODOR PERCEPTION
No. of tost
person a
2
4
6
10
Mg/B^ of »c«toph«noo«
perc«pt i b 1 o
2,0
1,654
1,481
1.096
y»xiBil not
p«rc«pt ib le
1.654
1,481
1,096
0,80
Table 2
DETERMINATION OF THRESHOLD OF ACtTO-
PHENONE ODOR PERCEPTION
_ - -
No. of leil
persons
B9/-3 of .c.topn.n.n.
Hininil
1
3 ,-
6
7
0,0251
0,0156
0,010
Maximal not
percept ib !•
0,0156
0,010
0,008
In the present study four acetophenone concentrations were used
in 216 teats and 16 test persons. Results presented in Table 2 agree with
those of Fel'dman, who set the acetone threshold concentration at 1.1 mg/m3,
and Imasheva, who set the acetophenone threshold concentration at 0.01
mg /m3 .
Odor perception thresholds for mixtures of different concentra-
tions were investigated by conducting 265 tests on 16 persons. A frac-
tional concentration summation index equaling 1.0 indicated simple sum-
mations between 1 and 2 indicated partial summation; less than 1 reflected
an increase in the order of magnitude. Table 3 shows that the majority of
the test persons recognized the second mixture by its odor (fractional con-
centration summation index - 1.03-1.26) Only the most sensitive test per-
sons detected the third mixture (index = 1.01).
-109 -~
-------
Ttblfe
DETERMINATION OF THRESHOLD ODOR PtRCEPTIOM Of ACETONE
AND ACETOPHENONE VAPOR COMBINED
Mixture
Acetono («g/B-^)
Acetophenone (ag/n-^)
Fractional euaoation of
individual threshold
Nuober of teat persona
perceiving tha odor
Nunber of test persons
not percoivmg the odor
I
1.096
0,010
Or 1,06
0.0 2,0
16
—
II
0.711
0,0061
Or 1.03
Ao 1.26
-
12
4
III
0,565
0,005
1.01
5
ii
IV
0,348-0.711
0,004-0.0061
Or 0.67
AO 0.88
—
16
Data in Table 3 also show that the minimum perceptible mixture
is an acetone and acetophenone concnetration of 0.565 + 0.005 mg/m3 con-
centrations respectively, with a summation index of 1.01. The mixture
cannot be detected if the index is less than 1. In other words, the effect
of acetone and acetophenone is equal to the sum of their individual effects,
a phenomenon applicable to less sensitive test persons as well.
The functional state of thfe central nervous system was determined
by the eye sensitivity to light method as indicated by eye adaptation to dark.
This test is used widely in arriving at standards for atmospheric pollutants,
using physiological reflex reactions as indexes of effects of toxic sub-
stance concentrations not perceptible by odor but affect the physiological
state of the cortex. Results of animal behavior tests with subthreehold
pollutant concentrations were reported by V.A. Gofmekler (I960), Yu. G.
Fel'dman (I960), R. Ubaydullayev (1961), and others.
In her studies of combined effects of sulfur dioxide and sulfuric
aerosol acid, K. A. Bushtueva discovered the phenomenon of physiological
summation: when these compounds were administered simultaneously, eye
sensitivity to light enhanced to a degree, amounting to a simple summation
of the effects produced by the two contaminants individually.
B. K. Baykov (1963) described the fractional summation phenom-
enon in a study of the effects of combined carbon disulfide and hydrogen sul-
fide. The 3 test persons who cooperated in this study were most percep-
tive to odors ranging between 24 and 27 years of age. The standard method
was used with which 42 tests were made using two mixtures of AC + AP,
mg/m3 : 0. 283 + 0. 005, and 0. 223 + 0. 0037, corresponding to summation
concentration indices of 1.01 and 0.77, respectively. According to Fel'dman
and Imasheva, threshold concentrations of reflex action of acetone and aceto-
phenone on eye sensitivity to light for the most sensitive testers were 0.55
and 0. 01 mg/m3 . Only the first mixture index = 1. 01 was statistically signi-
cant, and the reaction" of the subjects were quite different. In one the
~J- no"-
-------
sensitivity rose; in another it dropped; in the latter it rose at first during
the 20th minute, then dropped sharply during the 25th minute; by the end of
the experiment, it returned to normal. Table 4 presents eye adaptation to
light data for the three test persons during the 20th minute.
ADePTOMETHIC
RE;JJLTji
Toot person 'e
ini \ i a 1 o
b. P.
b= L.
YD. 1.
«9/«3
Acatone-
ecat ophenone
C 1 een a p r
0.283 + 0,005
0.223 + 0,0037
Clean air
0,283 + 0,005
0,223-- 0,0037
C Ixan air
0.283 -|- 0,005
0.223 -J 0,0037
Eye oenoi t iv i ty to
1 ight on the 20th
adaptation Binutt
81 175
103 060 (b)*
82 133 (o)
83 225"
1 1 1 000 (b)
82 133 (o)
95 550
74 175 (c)
90 003 (o)
"Statistical reliability) b -
c - not reliable
The mixture of low ace-
tone and acetophenone coneen-
trations having a fractional con-
centration summation under 0. 77
proved physiologically inactive
and elicited no statistically signi-
ficant shifts or changes, indicating
that the principle of fractional con-
centration summation also pre-
vailed in adaptometric studies. The
investigation included a study of
the effect of subthreshold acetone
and acetophenone concentrations
jointly on the electrical cerebro-
cortical activity by the alpha-
rhythm method (A. D. Semenko, 1964). A similar method has been de-
scribed by U. G. Pogosyan and Yu. S. Korneyev. Tests were conducted
with cooperation of three persons using a "Galillo" polyphysiograph, mak-
ing 62 readings. Records were made of the temporal and frontal biocur-
rents of both hemispheres and their combination, and of the central parietal
part of the left hemisphere. A statistical plot was made of the temporal,
most sensitive data. Here, as before, two acetone + acetophenone mixtures
were tested (mg/m3): 0.22 + 0.0035, and 0 .18 + 0 . 0027 in which the
F el'dm an and Imasheva thresholds were 0. 44 and 0.007 mg/m3 , respec-
tively, and which correspond to fractional concentrations summation indices
of 1. 0 and 0.78. The effect of each mixture was tested 4' or more times
with regular intermittent clean air breathing intervals.
The first mixture increased the cerebral potential energy in two
test persons and decreased it in the third. Statistically significant changes
appeared on the 3rd and 4th inhalation minutes. Data obtained with the
second mixture did not differ significantly from those with clean air, show-
ing that the principle of simple fractional concentrations summation also
operated in the case of subthreshold stimulation. Accordingly, the frac-
tion summation index for the first acetone-acetophenone mixture of threshold
concentrations in terms of mg/m should be computed as follows:
0.22 0.0035
0.35 + 0.003
for the present mixture.
= 0. 63 + 1.16 = 1. 79 and
0.18 0.0027
0.35 + 0.003
= 0.51 + 0.9 = 1.41
'- 111 -
-------
On the basis of the above data, it can be suggested that the
maximum concentration summation index of AC and AP in atmospheric air
should not exceed 1.5.
The resorptive effects were studied using 45 white male rats
which •were divided into 3 equal groups and exposed to chronic inhalation
for 84 days. The initial weight of the rats ranged between 80-100 g. The
average concentrations of acetone .ind acetophenone in mg/m3 were as
follows:
Group I--1. 855i£). 168; Group II--0.192±00197; Group III- -Controls .
Indices of maximum allowable concentrations for rats inhaling mixture 1
was 10. 9 and rats inhaling mixture 2 was 1. 9- Adjusting the Group II index
on the basis of the thresholds of individual components and results of most
sensitive EEG test persons reduced the index to 0. 71. Accordingly, animals
of Group II inhaled a subthreshold mixture and Group I inhaled a mixture
10 times as concentrated.
Indices of effects in this study were general condition and weight;
motor antagonist chronaxy; cholinesterase activity; rate of coproporphyrin
elimination; neutral 17-ketosteroids, and Vitamin C elimination with the
urine; and changes in absolute eosinophile count in the peripheral blood.
In the course of the inhalation experiment rats in all groups were
active and gained weight uniformly. Yu. M. Uflyand (1941), A. N. Magnit-
skiy (1948) and others found that there was a direct relationship between the
functional state of the central nervous system and the motor chronaxy level.
Through subordinating relationships the former regulated the activity of
the peripheral nerve apparatus and of the deeper lying nerve centers. The
subordinating brain effect is reflected in the constancy of normal extensors
and flexors chronaxy ratios of 1. 5:1, 2:1, and 2. 5:1. Motor chronaxy was
checked in 5 rats of each group 3 times each month using an ISE-01 electron
pulse stimulator. Statistically reliable ratio changes appeared in rate of
Group I beginning with the 4th week, chiefly as a delay in the flexor chronaxy.
An inverse ratio appeared at the beginning of the 6th week, after which the
curve flattened out and returned to the physiological norm only during the
recovery period. In rats of Group II no such distortions -were noted. Group
I changes can be interpreted as reflecting inhibition in the central nervous
system, involving a complex system of cortical and subcortical subordina-
tion centers. The presence of these processes is confirmed by depression
of whole blood cholinesterase activity of the test animals.
According to modern concepts, stimulus transmission from a
nerve to a muscle involves the acetylcholin system, i.e. , cholinesterase.
- 112 -
-------
Here acetylcholin plays the part of mediator: an excessive accumulation of
it in the organism leads to reduced lability of nerve process and to develop-
ment of inhibition (I. N. Volkova, 1954; D. Ye. Al'pern, 1963).
Reductions in enzyme activity as a result of prolonged exposure
to various chemical compounds have been noted by several authors (N. B.
Imasheva, 1963; V. A. Chizhikov, 1964; A. V. Mnatsskanyan, 1964; et al.).
In this study cholinesterase activity was determined by the J. Fleischer
aKid E. P. Pope method (1954) based on changes in the intensity of solution
coloration as a result of changes in the amount of acetic acid formed during
acetylcholin chloride hydrolysis. Color intensity was determined photo-
electrocolorimetrically. Twice monthly, 5 rats of each group had their
cholinesterase activity checked by the usual tail vein method.
Curves in Fig. 1 show a
significant reduction in enzyme
activity which appeared by the end
of the first month of inhalation in
rats of Group I and continued to
the end of the second month. By
the end of the inhalation period a
trend toward normalization had
set in, but the level was still de-
pressed as compared with the con-
trols. Statistical reliability index
of these changes was 95-99 percent,,
No statistically reliable changes
were noted in rata of Group II
throughout experimental inhalations
periods.
2 100
tS/II
31/1 IS/11 JO/ft
ln»ai ligtlion dale*
15/1
Fig. I .
Blood ehaIinetteraaa activity cKtngee in rats
of d>ff»r»nl groups
I - Group I - Aceton* + ac» I opt>«non« of fractional »u«-
• •tion md«K 10.9; 2 - Grojp II - acetone + aoetoph«n-
orM of fractional sunaiation ind«» I .2j 3 - Group
Control,, AS - Period o» inhalation
III-
Porphyrin metabolism was evaluated on the basis "of its excretion
rate with the urine. A. M.. Charnyy and Se. E. Krasovitskaya (1951) noted
a direct relationship bet-ween porphyrin metabolism and serious disturbances
in the central nervous system. The first author suggested that the porphy-
rins may participate in the redox processes of those parts of the brain in
which cytochrome and cytochrome oxydase were missing. Watson (1941)
established a relationship between rate of porphyrin excretion from the
organism and erythropoetic activity in the bone marrow, and M. I. Gusen
(I960) -was first to introduce porphyrin metabolism in his hygienic studies.
For 6 months the present author administered lead oxide to rabbits in a 10
microg/m3 concentration and discovered that the amount of coproporphyriii
in the urine doubled as compared with the control animals. Disturbed por-
phyrin metabolism has also been noted by other authors engaged in chronic
administration of low concentrations of atmospheric pollutants (G. I. Solo-
min, 1962; D. G. Odoshashvili, 1962; V. A. Chizhikov, 1964; et al.).
- 113 -
-------
The present author used an SF-4 spectrophotometer In the UV region (M. L.
Gusev, and Yu. I. Smirnov, I960). Twenty-four hour urine specimens had
been collected every 15 days from 5 mice in each group, making 33 readings
--9 before, 18 during, and 6 after inhalation. Analysis of the results
showed that the combined effect of acetone and acetophenone in concentra-
tions of 1.855 and 0.0168 mg/m3 , respectively, caused a definite shift in
the porphyrin metabolism of Group I rats.
./1 OU 9 of body
0.2-
151IX 3/X 13/X 28/X 12/X/ 27/XI 12/XII 27/XII 21] 1
Investigation data* '
2. Rate of coproporph/nn elimination
Curves in Fig. 2 show that be-
ginning with the 4th week of experi-
mental inhalation, coproporphyrin
metabolism increased noticeably and
rose to a statistically significant level
during the second inhalation period
(R = 0.95). Rats of Group II exhibited
no substantial deviations from the con-
trols .
The hormonal system activity
encompassing the anterior lobe of the
\hs urino of rale Inhaling acetone + acetophen- , , , , ., , , ,
one vapor. ' hypophysis and the adrenal cortex is
Notations Sana as m Fi3. i. regulated by the central nervous system
through the hypothalmus . High concen-
trations of some chemical compounds elicited nonspecific response in this
system, namely reduction in the absolute eosinophile count in the blood and
an increase in neutral 17-ketosteroids in the urine (Ye. I. Spynu, 1962;
L. E. Corn, 1963). In this connection the present author attempted to find
nonspecific reactions as a result of low acetone and acetophenone concen-
trations. 17-ketosteroids were determined by the O. M. Uvarovakaya method
(1956) with check tests made twice monthly for a tetal of 24 ta§Cs.
Curves in Fig. 3 show that the 17-ketoateroid level dropped in rats of Group
I during the early days of the inhalation and sharply rose beginning with the
40th day. During this period the 17-ketosteroid level in the control group
was 6. 85 microg per 100 g of body weight, while in rats of Group I it was
17.41, a rise that continued until the 70th day, after which it returned to the
control levels. These changes were statistically reliable.
The fluctuations in 17-ketosteroid content in rats of Group II
did not differ substantially from those of the control group.
Eosinophile counts in the blood were made by the S. M. Bakman
(1958) method. Twice a month, at the same hour of the day, blood was taken
from 5 rats in each group; a total of 99 readings were made. After 10 days
of inhalation the eosinophile count in rats of Group I dropped somewhat,
apparently as a result of the initial reaction of the organism to acetone and
acetophenone (Fig. 4). It dropped sharply later to a lower fluctuating level
" - 114 ~-
-------
which persisted to the end of the inhalation experiment. The reliability in-
dex of the drop was 0. 95. The osinophile count in rats of Group II approxi-
mated that of the controls, although on the 55th day there was an increase
that was not statistically reliable. A. Ye. Kulakov (1965) also observed
eosinopenia in animals chronically receiving low concentrations of hexa-
methylenediamine.
• - zoo •
Uicrogr/l Ig of body vcight
IS/f JO/lt H/XI 29/XI 13/XH 28/111 12/1 20/1
Jnvcs11ya 11on datea
Fig. 3. Content of naulral |7-k«to«tyroi d» in
the urine of rtl» of diff«r«nt Groupi
Notation* aaa* aa Fig. I.
If/a IS/1 31/t It/Xl 30/n iS/tll IS/I
Dates of investigation
Fig. *J. Actual nuaiber of blood eoeino-
philai of rats axpoaaa to th» inhaliticn
of air containing aoiton* and aeetophe-
non« «n lo« concent ratlona
Motttion* aaa« aa m Fig. I.
Thus, the investigations point to the high sensitivity of the
tests employed and bring out the nonspecific organism reactions to unfavor-
able effects of atmospheric pollutants. Records of vitamin C elimination
rate through the experimental inhalation showed no statistical significant
changes; in other words, the vitamin C balance remained undisturbed.
Based on results of the foregoing teats it is recommended that 1.2 be
adopted as the maximum allowable fractional concentrations summation
index for acetone and acetophenone combination in atmospheric air.
However, in view of the wide range of unknown values between the thresh-
old (10.9) and subthreshold (1.2) concentrations the Committee Section on
The Protection of Air Cleanliness resolved to set the average-daily allow-
able index at the same level as the single-exposure, iyz. , 1.5.
In addition to above experiments with humans and animals a
survey was made of single acetone and acetophenone concentrations in
the air around a plant producing synthetic phenol and acetone, taking 131
samples at 100 to 1000 m from the plant.
Data in Table 5 show that at 100-300 m from the plant there was
considerable acetone air pollution: in 27 samples acetone concentrations
substantially exceeded the maximal allowable 0.35 mg/m3 . Only at 500
m were all 24 samples negative for acetone.
- 115 -
-------
liblt S
ACfTONt CONCENTRATION IN T*t *»H SUHHOUNBIIIU
PLAMTS WObUCIW PHENOL AKO ACtTONE
_
Molars f roo
discharge
point
100
300
500
No . of air
o*nplai
col lee tad
14
13
24
1 fc/.3
Air »»«plel
b«lo« oethod
aensi t i* i iy
—
24
V 9
-a.,..,
> 2,19
\ 0.7 14
v —
Average
1,41
0.415
—
Data in Table 6 show that acetophenone was present in significant
concentrations in samples collected at 100-500 m from the plant, the maxi-
mum concentrations exceeding the allowable maximum of 0.003 mg /m3 . At
500 m 46 percent of the samples exceed the allowable maximum. Only at
1000 m were all the air samples negative for acetophenone.
Table 6
SINGLE ACETOPHLNONE CONCENTRATIONS 11* ATHOiPHEH 1C AIR
AROUND PLANTS PROUUGIHS PtttSUL AND ACETONE
He tera f roro
di echirga
po int
100
300
500
1 000
Mi* f
no. or air
tiaples
col lictcd
M
17
26
25
- : = . 3
oample* b«- Mg/n
1 o* mmnt i-
tl¥ 1 t»
••Ihod
-
—
9
25
UaxUal
0.877
0,097
0,028
—
-
' Av«r«9*
0,258
0.057
0,007
Accordingly, the sanitary protection zone for thia type of manu-
facture must be not less than 1000 m wide.
CONCLUSIONS
1. On the basis of odor perception threshold, the minimum per-
ceptable acetone and acetophenone concentrations individually should be for
the most sensitive persons, 1. 096 and 0. 01 mg/m3 , respectively.
Z. Where acetone and acetophenone are simultaneously present in
an air sample the intensity of their combined odors is subject to simple
summation.
3. The reflex effect of acetone and acetophenone in simultaneous
combination on eye sensitivity to light and on electrical brain activity can
be expressed in terms of complete summation.
-------
4. Chronic 24-hour daily exposure of white rats to the inhalation
of air containing a mixture of acetone and acetophenone the fractional con-
centrations summation index of which is equal to 10. 9 will bring about func-
tional shifts in the organism of the experimental rats expressed as a lower-
ing in the muscle antagonist chronaxy control by the central nervous activity,
in the central control of cholinesterase activity of the blood, will enhance
the rate of coproporphyrin elimination via the urine, and will give rise to
many nonspecific reactions originating in the kidney cortex, probably due
to increased 17-ketosteroid content in the urine and reduced number of
blood eosinophiles. No physiological changes were noted in experimental
rats which inhaled air containing a mixture of acetone and acetophenone the
Bummation index of which was 1.2.
5. The maximal single and average 24-hour fractional concentra-
tions summation index of acetone and acetophenone simultaneously preseat
in atmospheric air must not exceed 1.5.
- 117
-------
STUDIES IN THE STANDARDIZATION OF MAXIMUM ALLOWABLE
HYDROGEN FLUORIDE CONCENTRATIONS IN THE AIR
OF INHABITED AREAS
M. S. Sadilova
From the Sverdlovak Institute of Industrial
Hygiene and Occupational Dieeasea
Many publications appeared in the Soviet and foreign literature
which deal with toxicology of fluorine (F) and the clinical course of indus-
trial fluorosis (Ye. Ya. Girskaya, 1959; et al.). While the effects of F on
drinking water have been given adequate treatment, the same cannot be said
for small concentrations of F dumped into the air as a result of industrial
emissions.
For the past several years this author investigated the levels of
atmospheric pollution near aluminum and cryolite plants in the Urals,
particularly the zonal distribution of F in the air at different heights (M. S.
Sadilova, 1958, 1959, 1964), accompanied by a survey of the health of chil-
dren who live in these areas. In F-industry districts the inhalation exhibit-
ed a general tendency toward illness and toward diseases of the respiratory
organs; the noted F-specific diseases were mottling the enamel of the
teeth, and increased F secretion via the urine. Children living near alumin-
um-producing plants had abnormally high F content in the bones. These ob-
servations showed that F in atmospheric air must be considered as an.im-
portant factor in the pathology of the juvenile population (M. S. Sadilova,
1957, 1962; A. F. Aksyuk and G. V. Bulychev, 1962). The continued growth
in the number of aluminum, cryolite, superphosphate, and other similar
plants made it imperative that the maximal allowable concentration of F in
the air be established.
S. V. Miller (1955) recommended such a level on the basis of
calculations, starting from the maximum allowable concentration of F in
drinking water (1. 5 mg/1), on the assumption that F compounds in water
and air were toxic in the same degree. Taking 1.5-2 liters as the amount of
drinking water consumed per day and 15-20 cu m as the amount of air passing
through the lungs every 24 hours, Miller calculated that the F concentration
in air ought not to exceed 0.15 mg /m3 . Considering the fact that F had an
unfavorable effect on living conditions in areas where industrial plants dis-
charged F, Miller recommended a maximal single concentration of 0.03
mg/m3 and an average 24-hour daily concentration of 0.01 mg/m3 . This
recommendation was adopted.
- ITS'"--
-------
The present author investigated the effects of hydrogen fluoride
(HF), which is the moat toxic compound contained in industrial discharges.
The effects of HF and F-containing dust (individually and in combination)
will be discussed later. The odor perception threshold for HF was deter-
mined experimentally by a method commonly employed in the Soviet Union.
Concentration constancy was checked by sampling the air before and after
a test.
A total of 672 tests was made with 17 persons of normal odor per-
ception. Test concentrations of 0.02 to 0.22 mg/m3 were used, and results
showed that the minimum perceptible concentration ranged between 0.03 and
0. 11; for the majority of the subjects (10) the threshold was 0.03. The
subthreshold concentration was 0.02 mg/m3 (Table 1).
T*bl9 I
DETERMINATION OF riYDHObtN FLUORIDt OOOR
THRESHOLD PERCEPTION
Teit
persons
1
1
2
3
10
No. of ob-
43
37
76
123 •
393 -
"9/-3
Threshold
0,111
0,106
0,052
0.042
0,030 '
Sublhreshold
0.051
0.054
0.042
0.032
0.020,
A determination was made next of HF effects on the central ner-
vous system by way of the upper respiratory tracts. Changes in eye sensi-
tivity to light were recorded in 3 test persons with normal vision. Result-
ing concentrations were 0. 02, 0.03, and 0. 06 mg/m3 . ' The latter two con-
centrations, and especially the last, produced a marked increase in light
sensitivity, while clean air and the lowest concentration proved inactive.
These results were statistically reliable. The light=sensitivity data for one
of the three test persons are presented in Fig. 1.
Thus, the odor perception threshold and the threshold of reflex
action of HF on the functional state of the cerebral cortex, as recorded
by the adaptometer method, were on the same level, viz., 0.03 mg/m3.
The results clearly point to the fact that HF belonged to substances having a
trigeminal effect. Animals were exposed to experimental inhalation of HF
unterruptedly for 5 months on specially constructed chambers. Hydrofluoric
acid vapor in appropriate concentrations in air was run into the inhalation
chambers at the rate of 18 li/min. The following HF concentrations were
used in the tests: 0.10, 0.03, and 0. 01 mg/m3 . By using a 40 percent
hydrofluoric acid as the stock solution, the above HF- air mixtures could be
- 119 -
-------
obtained by diluting the 40% etock solution according to the following ratios:
1:27, 1:500, and 1:40, 000, respectively. Air samples from the chambers
were collected in a cylinder with a No. 2 ashless membrane filter and sub-
sequently passed through two consecutive absorbers containing twice-dis-
tilled water. F content was determined by S, K. Chirkov's alizarin-thorium
method (1957), which has a. sensitivity of 0.001 mg/m.3 . HF- concent rations
were checked 5 times a day in chambers I and II, and 4 times a day in chamber
III. No significant fluctuations were observed.
10 IS 20 25 30
Fid* I. Eyo sensitivity to
wno inhaled clean air (I), air contai
of hydrogen (lugrida (?J, 0.03
ma/o-3 of hydroyen rlui
in on* t«at pareoq
' "Y,9.°2-i/-?
U0r7b. "'3ji
and a.06
Each chamber contained 19-22 white male rats 2 months old. HF
fluctuations were as follows (in mg/m3: Group I--0. lOiO. 002 j Group II--
0. 03±0. 0001; Group III--0. 01±0. 00009; Group IV--control.
The F content in the rats' diet was standard: wheat bread--0.13
mg %; black bread--0.36 mg %; cabbage--0.17 mg %; oat grain--0.17 mg %;
dry matter, mild--0.14 mg/li. The general condition, weight, and state of
peripheral blood were observed before and during the experiment. It was
noted that during the first inhalation month a large share of the Group II
rats became agitated, attacked each other, and behaved aggressively toward
the experimenter. Afterwards they returned to normal behavior. No changes
in weight were observed. Hemoglobin, erythrocytes, reticulocytes, leuco-
cytes, leucocyte formula of the peripheral blood did not substantially deviate
- 120 - ~"
-------
from the normal. Special attention was given to the functional state of the
central nervous system of the experimental animals, measuring it from the
conditioned reflexes and chronaxy. We also utilized neurohistological
methods. These tests were run on 8-11 animals in each group.
During the chronic experiments, special attention was paid to the
functional state of the central nervous system which was studied by the
method of condition reflexes and motor chronaxy supplemented by neurohisto-
logical studies. Rats in the Kotlyarev chamber were trained to two positive
conditioned reflexes, a bell and a red light, and one negative, a buzzer.
After the positive reflexes were established, a sterotype developed, con-
sisting of 7 signals in a given order: bell, light, light, bell, buzzer, bell,
light. The effect of F on conditioned reflex activity was judged by the rate
of reflex formation and reinforcement, the number of correct responses, and
*,be latency period duration. In developing the sterotype the state of differ-
entiating inhibition, successive inhibition, and the character of the strength
correlations of the reflexes to stimuli of varying strength were taken into
consideration. The chronic effects of HF in the two lower concentrations
were also studied against a background of added functional loading.
The results showed that serious disturbances in conditioned reflex
activity developed as a result of HF inhalation in 0.10 mg/m3 concentration
such as delayed reinforcement of positive reflexes, inadequate reflex sta-
bility, and prolonged latent period (Table 2).
T«b_!« 2
FORMATION AND STABILITY Of POSITIVE CGHDITIOHED REFltXtS IN
EXPERIMENTAL ANIMALS EXPO&EO TO THE. INHALATION OF HIUHOutt*
FLUORIDE
Con en
in
•9/-3
0.10
0.03
_ 0,01
Con-
trol
••(•put
l»a. j«m»s
M±m
1 -
M-fm
(
M±m
t
M±m
6» 1 1
AppȤr-|
• no*
of
r.f !••
4,2±0,5
1.6 (c)*
4,3±0.3
a,«(h)
3,3±0,1
l,6(o)
3.4±0,1
R«f la«
f •«•-
tion
15, 1 -t 2,9
3,6 (b)
9.5*2,3
?,l (*)
4,7±l,l
0,2 (o)
4,5±0.6
H.f U«
fill-
out
in I
6,3±0,8
5,7 (C)
5,5-MJ
2,3 (a)
1,3±0,4
0,2 (o)
1.4±0,3
LigKt
R.Tli* 1 ""•« '•»
ipp«ir-j f >»•-
•"«• | lion
5,7±0.8
4, 1 (c)
3.9±0.-l
2,3 (a)
2,4±0,2
0 (o)
2,4±0.2
38,1±7,6
4,3 (c)
11.3-4,5
1.5 (o)
4,3±1,0
0,1 (o)
4,5±0,9
q«ri««
fill-
out
in \
17,0±1,5
9.8 (c)
W±l,0
<-0 (b)
1.8±0,4
0,6 (o)
2.1±0.3
ralnbilityi » _ 95>» b - S^li o - 99.9J
"-'121 -
-------
In developing the stereotype in the experimental rats there
appeared instances of successive inhibition and disinhibition of the dif-
ferentiation in comparison with the groups of rats subjected to lower con-
centrations and with the controls. The force of the conditioned reflexes
stimulation and response reaction lost its impact as shown by the appear-
ance of compensating and paradoxical phases.
Tab IB 3
HYDROGEN FLUORIDE EFFECT ON CONDITIONED"REFLEX ACTIVITY OF
MENTAL ANIMALS IN THE COURit OF FOrtlAMON AND FIXATION OF CONCI-
TIONED REFLEXES
lnde»e»
.•tent period in
response to bel 1
n reaponM to
1 >ghl
Jisturbed Force
rat i on
Equal i /ing
phaae
ParadoH toal
phase
Rgf lax fall out
(n response to
sound of ba 1 1
In reaponae to
1 lyht f laeh
Sequent i • 1
inh i b i t 1 on
In response to
sound at boll
o f lash of
light
SUtia-
t ical
Inderne*
M t m
t
M± ra '
(
M ± m
I
M -t m
t
M + m
M ± m
I
M +m
t
M + m
I
Concentration in my/m
0,10
1,37 ±
0,03
16.7 (c)*
2,09±
0,05
M.5 (c)
.
3,3 ± [,8
1,3 (o)
6,3+1,40
4,5 (c)
3,0 + 0,45
6,6 (c)
13, 0±1. 2
10,4 (c)
23,3 +
. 4,02
5,7 (c)
31,0 + 5,
5.9 (c,
0,03
0,63 ±
0,04
2,0(o)
1,38 +
0,03
0,7(o)
-
3,0± 0,9
l,0(o)
2,7 + 0,9
3.0 (b)
Her
2,6 + 2,6
3.6 (b)
13,3 ±
3,3
3,9 (c)
17,7 +
4,7
3.3 (b)
0,01
0,69 ±
0,02
1 , 1 (o)
1,49±
0,03
1,8 (o)
Nona
Mono
0.11 ±
0,1
1.1 (o)
0,33 -
0, 18
0.1 (o)
0,33 ±
0, 18
0,6 (o)
0,77 ±
0 29
0,32 (o)
Controls
0,65 ±
0,03
1. 41 i
0,03
.8 ±0.7
No**
None
0,36 +
0.12
0,1±0,13
0,9± 0,3
"Statistical reliabilityi a - 954; b - 99|j c -
o — unreltable
Changes in conditioned reflex activity were also noted in rats
which inhaled 0.03 mg/m3 concentration of HF, especially in the presence
of functional load under such conditions there appeared an increase in the
number of conditioned reflex fall outs and in the number of successive
-------
inhibition cases. The 0.01 mg/m3 HF concentration elicited no changes in
conditioned reflex activity.
At the end of the month's recovery period the conditioned reflex
activity of Group I rats somewhat improved but remained different from the
controls, while that of Group II returned to normal. Results of muscle
antagonist chronaxy studies are presented graphically in Fig. 2.
S
L
C.
m
ao/s
0.010
nnnc
•
-«•— T-
1 1 1 1 1
f
f
2
i i
0010
/? nnf
UOtJJ
_ .^-s^ ^
1 I 1 1 1 L 1_
/ 2 3 4
Observation I i»a in
Fig. 2. Huicli int«goni»t» c*w-«na»y m r»t» during
lh« inhilalion of tir containing hydroyvn fluorio* in
iniBtli of Groups I, ll. III, ind IV.
I - •Bt«ntors| 2 - f l«»or»
AB - lnhal«tiofl p«riod
For rats of Groups III and IV the central nervous system control
functions remained within normal limits. In rats of Groups I and II changes
appeared immediately in the form of delayed response and as compensation,
and inverse antagonist chronaxy ratio. Chronaxy delay was especially char-
acteristic of the extensors (38.4 percent of cases as against 30. 8 for the
flexors). These results were statistically reliable.
- 123 - •-
-------
These changes in the central nervous system activity indicated
profound inhibition involving the complex system of cortical and subcortical
control centers. Rats of Groups 1 and II also exhibited statistically reli-
able depressed cholinesterase activity, an enzyme which transmits nerve
stimulation.
The neurohistological examination of the brain, conducted by O.K.
Shturkina of the Sverdlovsk Medical Institute, centered on the motor and sen-
sory analyzers, mainly the snapses and the state of the nerve cells. Rate
of Group I showed hyperemia of the membrane capillaries and of the brain
matter, perivascular edema, thickening of the dendritee with formation of
moniliform distensions, and disappearance of protoplasmic spines. The
nerve cells suffered structural changes: uneven coloration of the Nissel sub-
stance, partial lysis thereof, and thickening, swelling, and shriveling of the
protoplasm. In some nerve cells the nuclei had disappeared and neurono-
phagia appeared. During a 3-month recovery several changes of a compen-
satory nature were observed: destructive changes in the nerve cells were
less pronounced, and the spines on the dendrites became less clear. This
process was not fully compensated, because profound destructive changes
were found in individual nerve cells with dead nuclei. It is understandable
why recovery was incomplete in rats of Group I; the damage to their ner-
vous activity was more than functional in its effect.
De Eds (1936), Roholm (1937), Thomas, Wilson, De Eds (1935) are
of the opinion that changes in bone tissue, characteristic for fluorosis,
are the result of impaired calcium phosphate metabolism brought about by
fluoride inhibition of the enzyme phosphates. They suggested that this
enzyme participated in the processes of bone formation by liberating inor-
ganic phosphorus from phosphoric acid esters; the former, by joining a
bond with calcium ions, forms calcium phosphate, which is essential for
building bone tissues. Roholm (1937) attached basic importance to alkaline
phosphatase in these processes. However, the literature on the effects of
F on phosphatase activity presented contradictory data. The present in-
vestigation of alkaline phosphatase was conducted -with 6 rate from each
group. Its activity was determined by the method of G. K. Shlygin and S. Ya.
Mikhlin. After an initial rise in enzyme activity among rats of Groups I
and II the level dropped; the Group III level did not differ from that of the
controls.
Despite fluctuations in alkaline phosphatase activity at times
during inhalation, the inorganic phosphorus concentration in the blood was
constant and did not exceed normal limits. This is probably related to the
fact that phosphatase activity is not the only factor which supported the
phosphorus level. Even in a healthy organism this activity was not at a
constant intensity level, while inorganic-phosphorus concentration varied
within narrow limits. Probably the mechanism responsible for this latter
- "12"4 -
-------
circumstance is complex and diverse. It has been established that phosphates
migrated directly from the bone source to the blood immediately as the inor-
ganic phosphorus concentration of the latter dropped to a lower level.
References in the literature indicated that oseeua-phosphatase
activity in rate decreased under the effects of F; therefore, its activity
in the experimental animals was checked after experimental inhalation was
discontinued. The method of A. Bogdanskiy, with slight modifications
by Ye. P. Yeremin and Z. A. Kasparaekaya (1950) was used to determine
this activity in the tibia of 6-11 rats from each Group. The 3 HF concentra-
tions produced no changes.
_, _ Tibl»
UdTTLINu OF DENTAi. ENAMEL IN EXPERIMENTAL ANIMALS
NO. Or
atptl .
1 m c 1
I a B I
tniul •
19
20
-
22
-
Extent of d«»»ga
1
•
Group 1
Vary tliyht
Slight
Madiua
COOBI derib la
Group 1 1
Vary Blight
blight
Hediu*
ConB idartb 1 a
Group 1 1 1
Very slight
Si ight
Madiua
Considerable
Month* of aipoiur*
•
1
2
3
4 \ 5
Nuebar of mini* nth aottled
taath
, I
1
-
-
i
9
4
2
'
J
.
'
.
—
2
2
15
2
2
2
17
2
6
9
-
19
4
7
2
From the time that the clinical symptoms of fluorosis became
known, it was necessary to assume that one of its early signs was mottling
of dental enamel. A four-point system (Table 4) to evaluate tooth damage
was used in this study. Observations confirmed that mottling was in fact
an early specific sign of F-caused morphological changes. In white rats
the color of healthy teeth is yellowish, the teeth gleam and are reminescent
of amber. The teeth of five of the Group I rats after only a month's experi-
mental inhalation became transparent, whitened, and lusterless. The
severity of the damage increased as experimental inhalation continued, and
by the end of the 5th month all rats had experienced some damage: the upper
and lower front teeth looked like marble, were coarse and streaked, and
worn down. In rats of Group II these changes were less pronounced and oc-
- 125 -
-------
curred later.
At the end of the inhalation period, enamel erosion and prism
damage were discovered in Groups I and II, which also exhibited certain
changes in bone tissue. Rats of Group I developed stenosis of the meduallary
canal of the tubular bones, while the contours of the periostitis were uneven.
In the pelvic bones there was damage to Haversian system structure, osteo-
cyte lysis, and in the periosteum region there appeared focal resorption of
the bone matter with connective tissue proliferation. Bone lime had accumu-
lated nonuniformly. Changes in rats of Group II were less severe and were
localized primarily in the lumbar region of the backbone and in the bones of
the pelvis. Also noted were periostal unevenness, focal disorganization of
the Haversian system, and nonuniform lime deposits. The chantes found in
teeth and bone tissue are incontrovertible evidence of impairment of calcium
phosphate metabolism as a result of HF inhalation.
G. Khristiani ascribed considerable significance to the level of
F deposits in the skeleton and suggested that increased F content in the
bones is a symptom and diagnostic factor in the early recognition of F-in-
toxication. He also assumed that the severity of bone changes depended
on the degree of F deposition. The present author examined 6 rats from each
group after inhalation was discontinued to determine F deposition in the
teeth and in the femur. The teeth and bones were carefully freed of their
soft tissue, dried to a constant weight, and pulverized. 0.03 g of the powder
material was calcined and dissolved in 3 drops of hydrochloric acid solution,
passed through an ion-exchange column with anionite AV-17. The amount
of F elicited from the column was determined by the alizarintolyl method.
Examination showed that HF concentrations of 0.10 and 0.03 mg/m3 caused
F to accumulate in the organism, thus confirming results of Christiani's
experiment. (See Table 5, page 128).
F content in the urine was determined by an ion-exchange chroma-
tographic method first used for such investigations in the United States
(Harold, Nielsen, Logan, Utan, I960; Talvitie, Brewer). All groups exhib-
ited a substantially higher level of F in the urine than did the controls be-
ginning with the Znd month and lasting to the end of the experimental inhala-
tion period. It is of interest to note that in spite of Its high excretion level,
F also accumulated in the bones.
Histological examination of the internal organs of the Group I
rats after termination of the experimental inhalation, the upper respiratory
paths showed severe epithilial desquamation, vascular hyperemis, and focal
inflammatory lymphocytic, leucocytic, and histiocytic infiltration. Varying
degrees of severe focal inflammation of the mucous membrane of the nose
appeared in all animals, as well as bronchitis and peribronchitis . There
was hyperemia of the pulmonary and 2 cases of 5 focal pneumonia was noted.
- 126 - "
-------
Tiblt 4
FLUORIDE CONTENT IN DENTAL AND bONE HijSUti OF EiHEHIMtNTAL
Aniail
group
1
11
III
Control
f««th 1 Thi9hbon«
M±m
IIO,5±5,60
18,2±1,20
15,2±2,I4
12,2±0,98
1 1 M±m | 1
17.27 (c)*
3.87 (b)
1,27 (o)
121,3 + 8,70
51,4f3,20
28,9 + 2,83
23,3 + 4.0
9.57 (c)
5.12 (c)
I.M (o)
•St«ti»ticil rvlitbilityi b - 99J| c - 99.Mj 0 - Hot r«li»bl«
Protein and aliphatic dystrophy were discovered in the liverj in the kidneys,,
protein dystrophy and increased permeability of the arterioles were seen.
Plethora was observed in the vessels of the red spleen pulp, while reduction
in some lymphoidal follicles and hyperplasia of the reticular cells was found
in the white pulp. The walls of the central artery of the spleen had thicken-
ed as a result of plasmatic impregnation. Myocardial change was expressed
aa disappearance of transverse striation and disjunction of the myofibrilles .
The changes in rats of Group II were less pronounced. The nasal
mucous membrane was thinner in places, the cell structure could not be
clearly delineated, while in some cases, epithelial proliferation was ob-
served. Hypersecretion and desqramation of the epithelium were observed
in the bronchi. Plethora was found in the lungs and parenchymatous organs.
No hietological changes were found in rats of Group III.
CONCLUSIONS
1. The odor perception threshold and the threshold of reflex
action of HF in the human organism (the latter established by adaptation
to the dark) are both 0.03 mg/m3 .
2. Five-month experimental inhalation HF in concentrations
of 0.10 and 0.03 mg/m produced many changes in warm-blooded animals:
a. inhibition in the central nervous system, the higher
concentration leading to irreversible destructive changes in the nerve
cells;
b. a change in calcium phosphate metabolism;
c. F accumulation in the bones; and
d. histomorphological changes in teeth, bones, and internal
-.127--
-------
organs.
3. The extent of the changes varied with the HF concentration in
the inhaled air.
4. An HF concentration of 0.01 mg/m3 produced no changes in the
experimental animals.
5. The investigation had shown that:
a. an HF concentration of 0.15 mg/m cannot be regarded as
threshold of odor perceptive concentrations;
b. An HF concentration of 0.03 mg/m3 cannot be taken for
the maximum allowable single-exposure concentration;
c. the maximum single-exposure concentration of HF must
not exceed 0. 02 mg/m3 .
EXPERIMENTAL BASIS FOR THE DETERMINATION OF MAXIMUM
ALLOWABLE CONCENTRATION OF VANADIUM
PENTOXIDE IN ATMOSPHERIC AIR
V. M. Pazynich
From the Department of General Hygiene
Dnepropetrovsk Medical Institute
Air pollution by vanadium pentoxide aerosol (VPO) is caused by a
variety of technological processes used in several branches of the national
economy. The most significant of these is the metallurgical industry, where
ferro-vanadium and VPO are used as additives in producing alloy steels.
Because VPO has a comparatively low melting point (675° C), its vapors es-
cape into the air and in the form of an aerosol the particles of which run in
size of up to 2 microns (I. V. Roshchin, 1952, 1963). Therefore, vanadium
pentoxide pollution can affect not only industrical premises but also the
atmosphere, along with other stack emissions. Vanadium pentoxide aerosol
can pollute the air as a result of stack discharges omitted by boiler plants
and other systems operating on coal. The lower-grade coals have a high ash
content. According to V. A. Nazarenko (1937), the VPO content of ash in coal
- 1Z-8—
-------
and lignite ranges between 0.44 x 10 " and 9-3 xlCf a percent. In petroleum
fly aeh, particularly petroleum with a high sulfur content, VPO content
ranges between 5 to 17 percent (I. V. Roehchin, 1962; it can also be found
in aome of the heavy refining products, such aa lubricant ash, where its con-
tent may run from 0. 5 to 40 percent (Yu. P. Pal'teev, 1964). Thus, oil-
fueled heating installations and diesel motors can pollute the air through
their stack emissions and tailpipe discharges (Morette, 1959).
The metal industry development, the construction of powerful
thermo-electric stations, the growth of diesel-powered bus fleets in the
cities, etc., are probable sources of atmospheric pollution by VPO.
Many investigators called attention to this fact in the past few
years. Cholak, et al. (1952) found up to 0.2 micrograms of vanadium per
cubic meter of air in Cincinnati and its environs. Morette (1959) found 11
to 1922 micrograms per gram of street dust in Paris. Dohan (1961) dis-
covered in the air of five American cities vanadium concentrations of 0. 2
to 1.2 mg/m3 . The present author conducted studies in 1963-1964 in the
proximity of some large metallurgical plants and found vanadium in the
atmospheric air in 87 percent of the samples; vanadium pentoxide concen-
trations were as follows:
Distance from Source Micrograms of Vanadium
of Emission, meters Per Cubic Meter of Air
150 1.49*0.25
500 0.47±0.09
1000 0.35±0.39
1500 0.98±0.13
In eleven percent of the positive samples VPO concentrations rose
to 2 mg/m3 . According to data found in the literature, vanadium (and par-
ticularly its pentoxide) is not indifferent to the organism, causing irritation
of the respiratory paths, acute and chronic inflammation of the upper res-
piratory tract, and lung changes (I. V. Roshchin, 1962; Ye. I. Matantaeva,
1960; Kh. G. Gul'ko, 1951; Dutton, 1911; et al.). In addition, vanadium and
its compounds have negatively affected the nervous system (Ye. A. Mel1-
nikova, 1957; K. P. Selyankina, 1961;!. V. Roshchin, 1963) Dutton, 1911;
et al.), the blood (Kh. G. Gul'ko, 1951; Symanski, 1939; et al.), hemo-
dynamica (I. V. Roahchin, 1952), and metabolism (T. A. Prokopenko, 1958;
Lewis, 1959; et al.). Many authors have also noted changes in some internal
organs (Ya. B. Reznik, 1954; Molfino, 1939; et al.). According to Franke
and Moxson (1936, 1937) chronic exposure to vanadium compounds elicited
more toxic symptoms in the organism than exposure to arsenic.
---129"-" ""
-------
In addition, vanadium compounds in atmospheric air can act as
catalyzers of sulfur dioxide present in the air and convert it to sulfuric
acid aerosol (Tomas, 1959), which, according to K. A. Bushtuyeva (1961),
possessed more irritating properties than the initial sulfurous anhydride.
All this points to the fact that vanadium and its compounds are important
components of air pollution and should be taken into account by the munici-
pal departments of hygiene.
Among the various compounds, the most important is pent-
oxide, which is one of the most toxic vanadium compounds (I. V. Roshchin;
1952, 1963) it is volatile, can enter the atmosphere in relatively large
qxiantities and at greater frequency than others. Therefore, at the recom-
mendation of the Committee on Sanitary Protection of the Air, a study was
initiated to establish a hygienic basis for the determination of the maximum
average-daily concentration of VPO in atmospheric air. In this connection
a study was conducted in the summer of 1964 in which 33 rats weighing 110 -
130 g were exposed to the inhalation of vanadium pentoxlde continuously for
70 days. The VPO concentration for rats of Group I was 0. 027±0. 002
mg/m3 , which is 3. 7 times below the maximum concentration permitted
in industrial premises (0.1 mg/m3). In rats of Group II it was 0. 002±0. 00013
mg/m3 , i.e., close to the concentrations found in the air around metallur-
gical plants that were not using vanadium for special purposes. Rats of
Group III served as controls. Air was run into the three exposure chambers
of 100 liters capacity at the rate of 30-30 li/min, assuring adequate air ven-
tilation (V. A. Popov, 1964).
Vanadium pentoxide is most frequently encountered in air in the
form of a condensate. Therefore, it was melted in a tubular furnace to
generate the aerosol. The furnace temperature was stabilized at 800° C
with a specially designed thermostat to assure a constant rate of evaporation.
The finely dispersed aerosol •was fed into the chambers by pumping air into
the furnace at a rate of 2. 5-3 li/min. This set-up was kept apart from
the animal exposure chambers to prevent its noise from disturbing the ex-
perimental rats.
Eighteen air samples were collected from the chambers in the
course of the inhalation period by using AFA-KhA filters which were de-
composed in a mixture of concentrated nitric and sulfuric acids. An FEK-M
photoelectric colorimeter was used for the determination of VPO according
to the method of M. V. Nifontova (I960). The experimental results were pro-
cessed statistically. The rats' condition before, during, and after experi-
mental inhalation was recorded on the basis of their general condition, body
weight, motor chronaxy of the antagonist muscles, blood cholinesterase
activity, total protein, blood serum protein fractions, oxyhemoglobin in
venous blood, and luminescence of blood leucocytes. At the end of the in-
- 130 -
-------
halation period rate of oxygen absorption in granulated liver tissue was
determined by a Warburg device; a pathohietological examination of
some internal organs was also made. During the inhalation period rats of
all three groups gained weight and were active.
Motor chronaxy of muscle antagonists was included because V. A.
Ryazanov (1961) has shown it to be a sensitive test in determining maximum
allowable concentrations of toxic substances. AnISE-OL electrol pulse
stimulator was applied every 10 days, under identical conditions, to the
right hind shin. Data in Table 1 show that in rats of Group II chronaxy
remained unaffected, but in rats of Group I substantial changes occurred
during inhalation, such as a statistically reliable drop in extensor chronaxy
(0. 8 microsec on the average) and a more noticeable increase in flexor
chronaxy (4 microsec). Even more striking was the change in chronaxy
ratio of the antagonists in rats of Group I. As data in the Table show, an
inhibitory process developed in the central nervous system on the 20th day,
which as a result of the altered control function of the central nervous
system on the periphery (Yu. M. Uflyand, 1941) appeared in the form of
flexor and extensor chronaxy equalization. This process had become ag-
gravated on the 30th day developing an inverse ratio which persisted to the
end of the inhalation period. After 20 days of restoration the motor chro-
naxies, and the ratio between them, returned to their normal levels.
. Vanadium pentoxide effect on the nervous system, known from the
literature and confirmed by the chronaxy tests reported above, was the basis
for determining cholinesterase activity, the regulation of which is largely
a function of the nervous system, particularly of the cerebral cortex (D.
Ye. Al'pern, 1958, 1963). The assumption here is that if VPO affects the
central nervous system, the change might show up in the blood cholinesterase
activity.
The method of Hestrin (1951) was used to determine gammas of
acetylcholin in cholinesterase decomposition per minute. The results are
summarized in Table 2.
Only rats of Group I manifested statistically reliable decrease
in cholinesterase activity, which lasted through the restoration period.
The other two groups exhibited no statistically reliable changes during
any of the 10-day periods. Total protein and protein-fractions in the
blood serum were also determined.
Administering vanadium to the animal organism caused a drop in
the rate of protein synthesis (Snyder, Cornatzer, 1958) and an increase in
serum gamma-globulin (Joya, 1958). We used the micromethod of Kjel'dhal
to determine total blood serum protein in terms of gram-percent. Results
are presented in Table 3. _ ,.,,
-------
Table I
U>
Nl
MUSCLE ANTAGONISTS MOTOR CHRONAXT IN SATS DURING VANADIUM PENTOXIDE INHALATION PErflOD
1
01 3
a o
- L
Chroni»)r
of
• »0
In »«e.
i
roouy
f
• t«nw>n
!•• in LL
MC.
1
• t 10 !
mds*«B [
of
f laxorc
to
•MtCfl-
,ior«
(X 100)
I
II
111
I
II
HI
I
II
in
1 a
- ro M
+-* u u
fO — t»
*-, +J C
JO
Mxm
M ± in
MT HI
M+m
M+.m
M±m
M+m
M±ni
M + m
Tan day p»riodB of invest "9« lion
before inhalttion
1
31, OT
l,73(o)
24, 7±
3,51 (o)
28,, 0±
2,31
50, 0±
3,21 (o)
41,7 +
4.25(0)
43, 0±
1.73
63 +
0,89 (o)
61 +
2,41(o)
65 +
2,31
2
31, 7±
5,24(o)
26, 7±
4,25(o)
32, 0±
4.00
55.0 +
7 , 55 (o)
49,3 +
5,46(o)
45,7 +
3,93
62 +
l,73(o)
60 +
5,l3(o)
70 +_
3.53"
During inhilatron per p oa
1
2
i
35, 3 ±-
7,80(o)
28,3 +
4,91 (o)
23,3 +
2,73
39,7 +
7 , 06 (o)
45,0 +
7,77(o)
38,0 +
\4,5l
86 +
5,00(b)
64 +
6,90(o)
60+1.86
'
45,7 +
3,29(5)
34,7 +
7,84(o)
30,0 +
0.5S
45,0 +
3,79(o)
1 1', llo)
45, 3±
1.67
108 +
9, 40 (a)
60±
3 , 48 (o)
662f.2.70
- "
3
74,3 +
l,34(c)
30. 3-^
4,06(c)
32, 3J
3.67
44,3 +
5,55(o)
4fl,0i:
6,03(o)
49,0 +
3,21
172 +
19. 7 (b)
63±
4,06(o)
-66±
4.10
Ten d«y period of invea 1 1 gt I 'on
During inhalation period
4
' 78.3 +
7,69 (b)
26,7 +
3,18 (o)
33,3+0.89
41,0+ •
5,13 (o)
43,7 +
5,24 (o)
51.3+0,89
193 ±14. 5 (c)
63 +
5.70(o)
65+0.55
5
74,7 +
9,94 (a)
24.3-1-
3,67 (o)
31,0+1 ,00
27,7 +
3,85 (a)
41,7 +
8,11 (o)
49,0±3,79
270 +
30,0 (b)
60±
4.40 (o)
64 + 3,18
-
6
94,0 +
2,51 (c)
23,7 +
3,18 (o)
30,3+0,34
33,7 +
6,33 (o)
41,0 +
3,21 (o)
48,7+1 ,76
293 +
40,0 (b)
58 +
4,20 (o)
63+4,50
7
88,3 +
5,37(c)
24,0 +
3,61(o)
29,3 +
1,76
34,7 +
6,20 (o)
41,3 +
3,33(o)
46, OT
2,80
263 +
3I,8(b)
59 +
4,40(o)
64+1,53
-
Recovery period
1
38,5 +
0,50(a)
22 7 +
3,51(o)
32,5 +
0,50
34 .O-1-
7,00(o)
40,0 +
8,00(o)
48,7 +
4,51
118 +
72,0(o)
57 +
3.00(o)
68-5,50
2
32,5-;
l,50(o)
20 , 5 :
l,50(o)
29,5^
1 ,50
SI ,7-1
3,51 (o)
35,0 +
4,00
44.3 +
1.53
63 +
1 .00 (o)
61 +
10,0(o)
66+1,00.
Notci i,t»tiolic«l ralnbilityi
b -
0 -
rB''«bl«
-------
faLOOO CHOUNESTERASt ACTIVITY IN HAli IN GAMHA6 OF
ACETYLCHOLIM HYDROLtiED «H I )U. Of BLOOD PER MINUTE )
Perpod
EcTore
inhil it ion
During
inhtl at i on
R e c o* • ry
Slilit-
tictl
M± m
M ± m
M ± m
M««l gr^i*
{
1
120. 0±7, 20(o)*
98, 1 ±4, 95 (a)
84,0±4,62(a)
M
12l,7±7,45(o)
H9,4±3.33(o)
104,8±3,95(o)
III Control
Group
127,7^3,13
119,r±5,53
100,0±6.77
pan od
•SUtnticil r«l iibill tyi • - 95I| 0 - Mot r»li,bl«
Tibia 3
TOTAL til03U PROTEIN CHANdEb IP) fHITE RATi DURINU PERIOCi
OF VANALJIUH PLNTOXIOE IHHALATION
fariod
b»For«
mhtlat ion
Dvrina
inh»r»tion
S tB I 1 • —
Lie*)
ind«>«*
M ± m
M ± m
^,
l
6,OOiO,15(o)*
6,l7±0,03(b)
AniB«l group*
ll
4.80±0,73(o)
6,67±0,53(o)
1 1 1 Control
Group
5.75±0,00
6,65±0,05
•Statistical roli.b.lityi b -
0 - Not r«li«bl«
It was established that after 40 days of inhalation the seriim pro-
tein in rats of Group I was virtually at the same level as before the experi-
mental inhalation, while in rats of Groups II and III (controls) there was an
increase incident to the normal growth of the organism. S. D. Balakhovskiy
and I. S. Balakhovskiy (1953) are of the opinion that total blood protein in
young rats constituted 4. 8% of their body weight and 7. 2% in mature rats.
In other words, VPO inhalation inhibited protein synthesis in rats of Group I0
the difference from the controls being statistically reliable.
Blood serum protein fractions were determined by the method of
paper electrophoresis . Results are summarized in Table 4.
The albumin/globulin index remained relatively constant. Protein
fractions did not differ significantly from those of the controls, whereas in
rats of Group I there was a statistically reliable increase in globulin by the
40th day of inhalation, accompanied by a reliable globulin decrease,
The method of luminescent microscopy of leucocytes with vital
staining of the specimens had been used in establishing maximum tolerable
toxic concentrations of air pollutants (M. I. Gusev, K. N. Chelikanov, 1963).
~ - 133----
-------
Ttblt
PROTEIN FRACTION CONTENT IN BLOOD bEHUi OF HATS IM
Obeer-
vat ion
period
Before
inha 1 -
» 1 1 on
exposure
.
Inhala t ion
e«poBure
.
Recover/
t-er iod
•
Protein
f raot ion*
A Ibumns
a - , '-
gl obul ine
-
P -
gloou 1 ins
y .
glocul me
AIU/
glob.
co.ff .
Albunmt
ry -
„ globulins
P _
g 1 obul me
y _
g 1 obul in*
Alb./
"'**•/
jlob .
3 t wv ej
coaff .
A 1 bun in I
fy -
g [Qbul mi
AID./
glob.
cotff .
^
tical
At + m
M + m
M ± m
M ± m
M ± m
M ± m
M± m
M ± m
M± m
M ± m
M± m
M± m
M ± m
V - M + m
g 1 obul in*
Alb./
ylob.
coeff .
M ± m
Aniatl group
41, 9±
1 , 10 (o)
24,4 ±
2,l2(o)
21,0±
1 ,21 (o)
12,5 ±
l,16(o)
0.72± K-
0,03(o)
39. 8±
2,39(o)
23, 5 ±
1 ,02 (o)
18, 4±
0,13(a)
13,5-
1,49 (a)
0,67 ±
0,07(o)
37, 4 ±
l,28(o)
22, 9±
3,21 (o)
24. 7 ±
4.20(o)
15,1 ±
0,41(0)
-
0,60 ±
0.04 (o)
"
41,8 ±
1,41 (o)
23,3 ±
l,09(o)
22,4 ±
0,84(o)
12,6 ±
l,56(o)
0,72±
0,04(o)
40, 0±
0,67(o)
25,2 ±
0,47(o)
22,4 ±
0,99(o)
12,4 ±
1.56(0)
0,67 ±
0,03(o)
39.4 ±
5,04(o)
22, 9 ±
2.8fl(o)
24 , 1 "±
2.94(0)
13,7 ±
0.78(o)
0.66±
O.Ol(o)
III (Control)
41,0 ± 0.48
24,9 ± 0,14
20,9 ± 0,42
1,31 ± 0,08
0,70 ±0,02
41 ,5 ± 0,53
23.1 ± 2.51
23,7 ± 3,28
11,7 ± 0,24
0,71 ± 0,01
36,7 ± 1,40
26,7 1 1,85
22,9 ± 0,66
13.7 ± 2,60
0,59 ± 0,04
•Stitntic.l reli.bilUyi a - 95£) o - Not r.li.bl.
The present author-employed the supravital staining with acridine orange in
1-ZO, 000 dilution (Ye. B. Zakrzhevskiy and L. G. Vasil'yeva, 1963). Speci-
mens prepared in this way did not lose the advantages of vital staining and
at the same time prolonged the time during which the staining method could
be applied. Normal and changed leucocytes were differentiated by the lumin-
escence intensity of their nuclei. This method depends on the extent of
fluorochrome molecule polymerization (Ye. B. Zakrzhevskiy and L. G.
Vasil'yeva, 1963), which in turn depends on the condition of the DNA and RNA
molecules in the protoplasm and in the cell nucleus. Of considerable im-
portance is the degree of polymerization of the nucleic acids themselves, the
-------
nature of the complex bond with acridine orange, and the chemical struc-
ture of the nucleic acids. Nondenatured nucleic acids have a helical struc-
ture and emanate green luminescence, while denatured ones have the shape
of a single spiral and emanate red luminescence.
In the early stages of DNA reorganization in the cell nucleus the
luminescence is yellow instead of the normal green, which later changes to
orange and then to red. Normal blood has only occasional leucocytes with
altered luminescence, which must be related to their destruction.
Ubli 5
PERCENT Of LEUCOCYTES VI1H CHAM&ED LUMINESCENCE
)b»arv»l ion
p. nod
inFit 1 it ion
lnh« 1 »t ion
Afttr 10 -
20 dtyi
Mi dtyt
70 dtfi
pan od
Sttti*-
1 IC«I
«±.
Mim
M±m
Mf m
M± m
*ni««l group
'
l.25±0,70(o)
5, 75 + 0, 75 (b)
I3,75±3,84(a)
20,25±5,40(a)
I,25±0,63(o)
II
1,25±0,70(0)
2,00±0,82(o)
l,75±0.75(o)
l,25±0,25(o)
l,00±0,58(o)
1 1 1
Control
l,25±0,70
2.25±0,25
l,25±0,25
l,25±0,25
l,50±l,50
Notci Stiti.tlo.l r.liibllityi t - 9SJj b - 99
-------
Tiblt 6
PERCENT OF HEMOGLOBIN IN RATS" VENOUS BLOOD
Obs«rv»t ion
d.r
Before
inha |« t ion
1 nha 1 at ion
period
Recovery
piriod
Statis-
tical
indeias
f<\ ± m
M+ m
Mi m
Aniaal group
I
60, 0± 1
5I,5±2
63,0±1
,8(0)*
,9(b)
,0(o)
11
60,0L1, 8(0)
59,6+2,1(0)
62,0±2,0(o)
III
Control
60,0+ 1,8
61,2+1,3
59,012,0
•Statistical rel iabi I i ty i b - 99$| 0 -not re Ii«bI •
Because of a certain disunity character of the data it was neces-
ary to obtain the average oxyhemoglobin content in the blood of rats in all
three groups. Statistically reliable changes were found only in rats of
Group I; no such changes were observed in rats of Groups II and 111. The
oxyhemoglobin content in rats of Group I returned to normal after the 20th
day of the recovery period.
An experiment conducted by F. and M. Berngeym (A. O. Boykar,
1953) also sheds some light on the role of vanadium in the redox processes
in the organism. The authors showed that introduction of metavanadate and
vanadium acetate in amounts up to and including 10-20 g of vanadium en-
hanced tissue respiration by the granulated liver in higher animals, raised
oxygen demand by a factor of 10. At the end of the inhalation period tests
were conducted to determine the extent of VPO's effect on tissue respiration.
Eleven rats from the three groups were decapitated and their livers were re-
moved. Respiration of the granulated liver tissue was studied in a Warburg
device. The results are shown in Table 7.
Tab). 7
OXYGEN CONSUMtD BY 500 MG OF GROUND LIVER fN *AfiBURG
APPARATUS IN 20 MINUTES ClN MICROLITRE6)
Statis-
tical
Indicator
M±m
Anintl group
I
263.3+~14,9(a)
124%
ll
222,0+11,9(0)
104%
1 1 1
Control
212,3±4,26
100%
Note: Statistical reliability! b - 95|j o -not
re IiabIe
Oxygen demand in rats of Group I was 24 percent higher than in the
control rats, a substantially lower increase than the one reported by the
Berngeyms, in whose experiment vanadium compounds were added directly
to the granulated tissue in vitro; whereas in the present study the rats in-
-------
haled the pentoxide. Oxygen demand in rats of Group II did not differ re-
liably from the controls.
Histological examination of certain internal organs found patho-
logical changes only in rats of Group I. The lungs showed the presence of
acute stagnant vascular plethora, focal hemorrhage, and signs of panbron-
chitis; in the liver were found acute plethora of the central veins, surround-
ed by isolated small hemorrhages around them, individual infiltrates be-
tween the hepatic lobules, and signs of granular, cellular dystrophy. The
kidneys exhibited pronounced granular dystrophy of the epithelium of the
contorted tubicles, which in some sectors showed signs of necrosis. There
was acute myocardial vascular plethora surrounded by focal hemorrhages.
Results of all tests show that a vanadium pentoxide concentration
of 0.027 mg/m3 in the air produced in the chronic experiment functional and
morphological changes in the organism of the test animals; a concentration
of 0.002 mg/m3 had no such effect.
It should be noted that the two VPO concentrations differed by
the disparity between the two test concentrations (almost a factor of 10).
This author conducted in 1964 a second series of tests with an intermediate
concentration close to the inactive level, using 20 young white male rats
divided into a control group and a group subjected to continuous 40-day in-
halations of air containing 0.006±0.0056 mg/m3 of VPO.
Two tests were conducted in the first series to determine func-
tional changes- muscle antagonist chronaxy and leucocyte luminescence,
and records of body weight and general well being. Four rats from each
group were used in the motor chronaxy test. During the first month of
inhalation there were no statistically reliable changes in the chronaxies
but at the end of that month some small but significant shifts were found
in the chronaxy ratio of extensors and flexors.
Ubu 6
PERCtNT OF LtULQCTTEi HUH CHAN&EU LUMI NtSCENCE OF
NUCLtt (ifcWNO SERlEo)
Qbsarvit ion
period
Before inhalation
Inhilat ion
After 15 diy»
After 30 d*ys ,
After L|Q deye ;
After
Bt»rr»ti on
SUtii-
tical '
Indi-
cttorg
M±m
M + m
M±m
M±n,
AniB*l group
£»p*rl-
MOUI
0,67±0,33(o)
l,50 + 0,56(o)
3,17+1,25(0)
4,83±0.79(b)
Control
0.67 + 0.33
0,60 + 0.40
1,67±0,42
1.00±0.37
Not* i Statistical r»l i«bi I i ty i b -
lnbU
- 137 -
o - not r»-
-------
Leucocyte luminescence was studied in 6 rats from each group
(Table 8). During the first month there were no significant changes.
Initial shifts appear in the animal organism more clearly under
functional loading. With this in mind the rats were subjected to a 3^-day
fast during the 6th week of experimental inhalation giving them only water.
Tests made at the end of the fast revealed substantial changes in the func-
tional condition of the test animals. There was a statistically reliable
change in the chronaxy ratio of shin flexors and extensors, and the same
was true of leucocyte luminescence, a 5-fold shift occurring by compari-
son \vith the controls.
CONCLUSIONS
1. Chronic, 24 hr., 70-day inhalation by rats of air-polluted
with a VPO concentration of 0. 027 mg/m3 of vanadium pentoxide induces
substantial changes in the functional state of the organism, as well as
morphological changes in some organs.
2. A concentration of 0.006 mg/m3 produced slight functional
changes which with functional loading in the form of starvation became ag-
g r a v at e d.
3. A VPO concentration of 0.002 mg/m3 proved inactive.
4. On the basis of this investigation it is recommended that the
maximum average-daily allowable concentration for VPO in atmospheric
air be set at 0. 002 mg/m3 .
- 133 --
-------
AN HYGIENIC STANDARDIZATION OF
ALPHAMETHYLSTYRENE IN ATMOSPHERIC AIR
M. I. Gusev and A. A. Minayev
From the F. F. Erisman Moscow Scientific
Research Institute of Hygiene
Modern chemical industry is characterized by the progressively
increasing production of synthetic compounds and substances and by changes
and improvements in their production processes and quality. In the course
of the 1958-1965 seven-year period production of synthetic rubber increased
by a factor 3-4. In this connection it should be noted that a modern divinyl-
methylstyrene synthetic-rubber plant is capable of polluting the air around
it with alphamethylstyrene (AMS) vapors over a radius of 4000 m.
AMS is a homolog of benzene with one unsaturated bond in the side
chain. It is a colorless or slightly yellowish fluid having a boiling point of
162-163° C and a characteristic odor of naphthalene (N. V. Lazarev, 1963).
Some authors investigated its toxic effects in chronic experiments.
A. I. Korbakova (1961) and E. A. Kapkayeva (1963) discovered that
exposure to AMS vapors in concentrations of 10-40 and 5-50 mg/m3 for 5-6
hrs. daily over 6 months affected the functional state of the liver, kidneys,
and central nervous system of white rats. They also found slight changes
in the peripheral blood of animals exposed to 50 mg/m3 of AMS vapors.
T. A. Blinova (1953, 1954) found that different AMS inhalation parameters
(100 mg/m3, 6-7 hrs. daily for 30 days) had a myelotoxic effect in the peri-
pheral blood of rabbits; erythropenia, leucotosis with a small shift to the
left, monocytosis, and reduced reticulocyte count. Workers engaged in
synthetic rubber production chronic intoxication with AMS vapors plus di-
vinyl developed an asthenovegetative syndrome and cerebral asthenia of a
nonspecific type. Hypotonia and hypothermia have been noted in workers
at the end of a work week (E. A. Kapkayeva, 1963; Z. A. Yermolova et al. ,
1964).
The maximum average-daily allowable concentration of AMS vapors
in atmospheric air was determined next. Sixty white rats weighing 140-170 g
were divided into 4 groups of 15 rats each. Rats of Group I were exposed to
the inhalation of air containing 5 mg/m3 of AMS 24 hrs. daily for 90 con-
secutive days, which is the maximum allowable AMS concentration in indoor
working premises; for rats of Group II - 0. 5 mg/m3 ; and for rats of Group
III - 0. 05 mg/m3 , Group IV served as controls. Each group was divided
into three subgroups of five rats each for investigation of blood, biochemis -
-------
try, and physiology. Before experimental inhalation rats were observed
for 45 days of background study. The experimental inhalation period ex-
tended over three months followed by a one month's recovery period. Ex-
perimental data were processed statistically.
AMS effect on the functional state of the central nervous system
was evaluated on the basis of the latent period of the unconditioned defen-
sive reflex reaction of the time required for a stimulus to be transmitted
over a reflex area.
It has been established by Soviet and foreign authors (V. V.
Zakusov, 1957; S. I. Gorshkov, 1964; P. Tasaki, 1948) that the latent period
of an unconditioned reflex reaction in laboratory animals was a fine and
sensitive differentiation of functional changes in the central nervous system
activity resulting from chemical and physical stimuli, due to the fact that
central synapses and cells constituted the units in which the most pro-
nounced quantitative changes appeared.
According to S. I. Gorshkov (1962), "The latent period of a reflex
reaction reflected the functional state of an individual tissue at ,the moment
it generated a single excitory •wave, more characteristically than did
chronaxy. "
Precise evaluation of this reaction requires that the following
conditions be observed: simultaneous initial reading of latent-period from
the moment of stimulation and cessation of counter operation the moment a
response is generated, stimulation must be instantaneous; it must be in
effect only for the shortest imperative time. A reflexometer proposed by
Gorshkov (1964) satisfied these requirements (Fig. 1).
The following method was used in this study. The rat was placed
into the chamber with its extremities in contact with parallel metal plates.
The chamber cover and the counter were placed on the animal's spine. Af-
ter the time recorder was switched on a threshold-tension current was fed
in for the production of unconditioned electrocutaneous (painful) stimuli.
At the threshold tension the rat was startled, the switch on the cover was
shorted, the contact was broken, and the counter ceased to operate. The
time elapsed between the moment of the stimulus (place where the mobile
contact was placed) and the arrow on the counter stopped moving (deter-
mined on the scale of the device) is the (LPURDR) latent period of the un-
conditioned reflex defense response.
In one series of tests 5 readings were made with a single cur-
rent tension at intervals of 2-3 min. (to avoid trade effects). The small
differences in the readings •were regular and constituted a functional charac-
- 140 -
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teristic of the central nervous system. The arithmetic mean of all read.
ings is the LPURDR.
Fifl. I. ft«fl«xoton*Mt*r
• - Conputorj b - AniMl ch«»fe*r
The AMS-LPURDR tests were made weekly on 5 rats from each
group. During the 5th week rats of Group I (5 mg/m3) manifested statisti-
cally reliable depression of the functional state of the central nervous system,
expressed as an increase in LPURDR (Fig. 2). By the end of the recovery
period the latent time returned to normal. No changes were noted in rats
of Groups II and III.
Once every two weeks blood samples were taken to determine the
contents of nucleic acids, leucocytes, erythrocytes, and hemoglobin, and
changes in the leucocyte luminescence. Erythrocyte counts and hemoglobin
were determined by an erythrometer; leucocytes were counted in a Goryayev
chamber. The concentrations used in the tests caused no changes in the
three parameters employed.
References in the literature indicated that without replacing
other forms of microscopy (ordinary, phase-contrast, electron, interfer-
ence, UV, etc.), luminescent microscopy offered additional advantages
for biological and medical investigations (Ye. B. Zakrzhevskiy, L.. G.
Vasil'yeva, 1963). A. D. Semenko (1963) studied changes in leucocyte
coloration caused by atmospheric pollutants. M. I. Gusev and K. N.
Chekalinov (1963) were the first to apply this method in establishing hygienic
norms for toxic air pollutants, directing their efforts to pentane in a chronic
- 141 - -
.
•
1
-
.
-------
experiment with animals.
&UI 13 ft 2//V 3 IS 23 30 7/V It Zt 28
lnv«Btig(tion date*
18 25 ZfHI 3 IS 23 30
2.
I - i a
Chanj** m UUnt r»f]«« piriod tffieWby »l|>nftB«thy| -
ityrol
g/«J, yroup l} 2 - 0.5 »g/«3, group II, 3 - 0.05 «9/«3. group
11} 1* - Control group. AB - Inhilttion porlod.
In this connection the present investigation included fluro-
chromed vital liquid specimens, acridine orange in a physiological solu-
tion in 1:100, 000 dilution serving as the fluorochrome. An ON-18 lumines-
cent illuminator \vas used as the source of light stimulation.
Rats of Groups I and II manifested altered leucocyte staining
properties from normal green to yellow or orange-yellow, the changes in
Group I being more clearly defined and faster stained. Group in did not
differ from the controls (Fig. 3).
Attention of many investigators is at present being directed to
the nucleic acids, the biological functions of which is closely related to
protein biosynthesis, to the mechanisms of normal and pathological
growth and development, to heredity, and to biological specificity.
The nucleic acids are high-molecular organic compounds found
in all biological entities and cells of the organism, and are their constituent
element. However, up to the present no one has applied them to studies
similar to the present one. The present author employed the spectrophoto-
metric method of total quantitative determination of the nucleic acids (RNA
and DNA) proposed by A. S. Spirin (1958), in which nucleic acids are ex-
tracted with hot perchlorate and determined photometrically in the UV band
- 142 - -
-------
at two wavelengths (270 and 290 millimicrons). The procedure described
by P. V. Simonov in 1960-62 was followed in making quantitative blood
analyses.
A/Ill 7/V It/tr ll/IV 5/lV IS/V l/VI ^JS/W JO/VI ti/VII ' ?8/Vlf
Investigation datM , " . _ .
Fig. 3. Effect of ilphaattHyl ttyrol »tpor en Ihk no«t«r of
blood leueooyt«» •! Ih chuc««e».
Notctiofi* &••• •• in Fiy. 2.
7/ir Zl/iv
131* Z/VI tSlVI ..XI VI Hi VII
Invaitigit ion
Fig. M. Eff.ct of tlphtMthylityrol on th» content of
nucleic »cid«.
Notitioni MB* •• in Fig. 2.
281 VII
blood
The results, graphically presented in Fig. 4, show that by the
4th week of inhalation the Group I AMS concentration caused a drop in total
nucleic-acid content; after a brief remission a new reduction set in which
- 143 -
-------
did not return to normal during the recovery period. In Groups ,11 and III
the amount of nucleic acid in the rats' blood did not differ from the con-
trols.
AMS effect on porphynn metabolism was judged on the basis of
changes in the rate of copropprphyrin elimination via the daily urine by
5 rats, determined once every two weeks. The amount of coproporphyrin
was determined by the method described by M. I. Gusev and Yu. K. Smirnov
(I960).
Decrease in rate of coproporphyrin elimination via the urine was
noted on the 2d week of experimental inhalation; this decrease leveled off
in the course of the inhalation experiment; it remained above the initial
level even at the end of the recovery period. The change in Group II was
less pronounced, and Group III did not differ from the controls. Results
are summarized in'Fig. 5.
. 5.
Effect of • I phiM thy I oly rol vtfor on unn»ry
coproporphyrin.
Notations etmt *a in Fij. 2.
None of the three AMS concentrations used caused any changes in
the weight and behavior of the animals during the chronic experiment.
After experimental inhalation 5 rats in each group were sacri-
ficed. Pathomorphological and histological examinations of the organs show-
i *^
ed that an AMS concentration of 5 mg/m caused changes in the lungs, kid-
neys, heart, and liver.
On the basis of the foregoing the present author concludes-
1. Alphamethylstyrene vapors in concentration of 5 mg/m3 and
lower had no effect on the behavior, weight, and general blood picture
- 144 -
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(leucocyte, erythrocyte, hemoglobin count) of the test animals.
2. An AMS concentration of 5 mg/m3 depressed the central ner-
vous system (increased reflex time), altered the biochemical reactions (re-
duced the content of nucleic acids in the blood and of coproporphyrin in the
urine), and changed the luminescent properties of the leucocytes.
Functional changes were confirmed by pathohistological changes
in lungs, liver, heart, and kidneys.
3. In concentration of 0. 5 mg/m3 AMS altered only the lumines-
cent properties of the leucocytes, while 0.05 mg/m3 had no effect.
4. The maximum average tolerance allowable concentration of
AMS vapors in atmospheric air can be aet at the level of its accepted single-
exposure allowable concentration of 0. 04 mg/m3 which is the inactive level
in chronic inhalation exposure of the experimental animals.
- 145 -
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APPENDIX
MAXIMAL ALLOWABLE CONCENTRATIONS OF HARMFUL SUBSTANCES
IN ATMOSPHERIC AIR OF INHABITED ARtAS
Ml Allonblc
Concent rat Ion*
Naae 01 Air KOI lutant
_
1 . Acrolam
2. Alphaaalhy 1 styrol
3, Al phanaptKaquininona
14. A«y laetlata
5. A »y lana
6. A»onia '
7. Am 1 m
0. Aeatona
9. Acetophanona
10. Beniane _ ._
II. benzane, crude oil. lo» in S,
on tha basis of C
12. Ban»enaf ptala oilf on th«
b aft i a of C
13. butyl acalata
|ll. Butyl ana
15. Vyi"1/' »cetat*
16. Haxanethy 1 enaaiaaine
17. Hyeroparom da of I BOpropy 1 b«r>i«na
Smg|(
0,30
0,04 '
0.008
0,10
' 1.5
0.2
0,05
0,35
0,003
J'-5
"
o!o5
0.1
3.0
0,2
0.001
0,007
It). Divinyl 3,0
19. Dichloroathana
20. 0 iBcthy 1 f OTMBI da
21. D i ny 1
22. 1 aopropy Ibanzana
23. Nitric acid, HN03; on tha ba»ia
of H
2^. Sulfur.c acid, Hp£0L on tha
basis of H ' t
25. Hydrochloric acid, on tha
basi B of H
26. Xylo
27. Metanol
20. Itethy l*cat«ta
29. Halcin anhydrida
30. Methy laatacry lata
3,0
0,03
0,01
' 0,014
0.4
J),006
0,3
0,006
0,2
0,006
On
,2
i n
& ,u
0 07
V | V •
0.5
O.I
"ass.
0,10
0.04
0,10
1.5
—
0.03
0.35
0.003
0.80
i ^
1,5
0,05
0.1
3.0
'0.2
0,001
0,007
1 ,0
1,0
0,03
0,01
0.014
—
—
0.1
0,002
—
—
0 5
\J | *J
0,07
0.05
O.I
• Appro»«d by th« Chi«f Sanitary Offi««r of th« USSR, l«5.
- 146 -
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APPHDK COgT.'O*
Naae of Air Pal lulanl :
31. Manganea* and ita ooapouad.
in tara of HnuS
a.
32. Araenic .
llnorganio coBpounds)
(Hydrogen arsenide excluded)
33. Nitrobenzene
3M . Carbon lanovide
35. 0*jd«» •*, nitrogen, on the
Baaia of NjOa
IS." "5*ide of ethyTene
37. Pyndine
38. Propy leae
39. Dual, nontoMic
MO. VanadluB pentomde
Ml. Mercury, aatallic ,
M2. Sulfur dioiide
M3. Hydrogen culfide _ :
MM. Carbon biaulfida
MS. Lamp Blank
M6. Lead and Its compound a.
(tetraethy lead excluded) as Pb
M7. Lead »ulf ide
M8. Styrol
M9. Toluldma Iftoc/nate
50. Toluol
51. Foraaldehyda
52. PhoB.horlc anhydride
53. Phthalic anhydride
54. fluorine covpounds, computed on
the .baai* of F
55. HydroMuonde
36. Phenol
57. Furfurol
58. Chlorine
S9. Chlorobeniana
60, Chloroprene
b«bia «f CrCj
62. Epichlorohydrln
63. Ethenol hydrolyllo, oa th« b»8ie
of ethanol
6M. Ethylaeetata '.
65. Ethv lene ' , " ' •
67. Cyelohenanon
i
Con cent rat lona
Uaiieal
Single .
'
—
o.ooa
6.0
0,3
0,3
0.08
3,0
0.5
— "
^
0'.5
0,008
0,03
0.15
,
0,003
0,05 .
0,6
0,035
0, 15
0,,'2
0,03
0,02
0,01
0.05
0,1
0 1
0,25
0.0015
0.2
,
5,0
01
, i
3,0
0,06
0.04
' Average
Ojj -tlQU^T
4
0,01
0,003
0.008
1,0
0,1
0,03
0,08
3,0
0. 15
0,002
0.0003
0,15
0,008
0,01
0,05
0,0007
0,0017
0.003
0.02
0,012
0,05
— k f\
0,2
0,01
0,01
0,01
0.05
0,03
0.1
0,08
0.0015
0.2
5.0
01
,1
3.0
0.06
0,01
- 147 -
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Notes: - ' -
1. In cases of simultaneous presence-ln the air of more than one
pollutant the MAC of which are subject to the summation principle, the sum
of their concentrations must be computed by the formula discussed in Note 2.
The summation must not exceed 1 in the following combinations:
a) Sulfur dioxide and sulfuric acid aerosol;
b) Hydrogen sulfide, carbon disulfide and dinyl;
c) Isopropylene benzene and hydroperoxlde of isopropy-
lene benzene;
d) Ethylene, propylene, butylene and amylene;
e) Sulfuric acid, hydrochloric acid and nitric acid (on
the hydrogen ion basis);
f) Acetone and phenol;
g) Acetone and acetophenone.
2. Computation formula:
X = A /Mj. + B /Ma + C /Mg + D /M«, + etc. •
where X is the sought summation concentration and
A/Mi. + B/MSJ + C/Mg
are the concentrations of pollutant components divided
by their corresponding MAC.
3. Where the atmospheric air contained hydrogen sulfide and
carbon disulfide their corresponding integral MAC values and not their
fractional values should form the computation basis.
- -148 -
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