-------
TABLE OF CONTENTS
Page
PREFACE v
Maps of the USSR
Orientation vii
Climatic, Soil and Vegetation Zones viii
Major Economic Areas ix
Major Industrial Centers x
Principal Centers of Ferrous Metallurgy and Main
Iron Ore Deposits xi
Principal Centers of Non-Ferrous Metallurgy and
Distribution of Most Important Deposits of
^on-Ferrous Metal Ores xii
Principal Centers of the Chemical Industry and of
the Textile Indus try xiii
Principal Centers of Wood-Working, Paper, and Food
Industries xiv
Main Mining Centers xv
Principal Electric Power Stations and Power Systems xvi
BIOLOGICAL EFFECT OF LOW CARBON DIOXIDE CONCENTRATIONS
0. V. Yeliseyeva 1
HYGIENIC EVALUATION OF POLLUTION OF THE ATMOSPHERE
WITH HEXAMETHYLENEDIAMINE
A. Ye. Kulakov 18
CONTAMINATION OF ATMOSPHERIC AIR WITH MALEIC ANHYDRIDE
AND ITS HYGIENIC EVALUATION
K. V. Grigor'yeva 30
BIOLOGICAL EFFECT OF LOW NITROBENZENE CONCENTRATIONS
- IN ATMOSPHERIC AIR
N. G. Andreyeshcheva 44
LOW CONCENTRATIONS OF UNSATURATED HYDROCARBONS OF THE
ETHYLENE SERIES IN AIR AROUND PETROCHEMICAL COMPLEXES
Candidates of Medical Sciences M. L. Krasovitskaya
and L. K. Malyarova 57
111-
-------
Page
THRESHOLD CONCENTRATIONS OF ACETOPHENONE DURING SHORT-
AND LONG-TERM INHALATION
Candidate of Medical Sciences N. B. Imasheva 79
CYCLOHEXANOL AND CYCLOHEXANONE IN ATMOSPHERIC AIR AND
THEIR HYGIENIC SIGNIFICANCE
A. A. Dobrinskiy 94
ON THE COMBINED ACTION OF THREE MINERAL ACIDS
Candidate of Medical Sciences V. P. Melekhina 106
TOXICITY OF SULFUR OXIDES UNDER CONDITIONS OF LONG-TERM
CONTINUOUS EXPOSURE
Decent K. A. Bushtuyeva 114
BIOLOGICAL ACTION OF LOW MANGANESE CONCENTRATIONS DURING
INHALATIONAL EXPOSURE
Candidates of Medical Sciences F. V. Dokuchayeva
and N. N. Skvortsova 139
MAXIMUM PERMISSIBLE CONCENTRATIONS OF NOXIOUS SUBSTANCES
IN THE ATMOSPHERIC AIR OF POPULATED AREAS
Professor V. A. Ryazanov and Professor M. S. Gol'dberg .... 149
IV
-------
PREFACE
The present volume consists of reports dealing with a number of
investigations conducted in the Soviet Union at its various public health
institutes and in departments of public health of some universities.
The great strides in the development of industrial chemistry in the
country has stimulated studies of the biological effects of chemical air
pollutants as well as studies dealing with public health implications of
these pollutants. Such studies assume an ever-increasing importance in the
Soviet Union.
The results of these investigations provide a basis for the establish-
ment of a nexv series of maximum permissible concentrations for new toxic
substances in the atmospheric air, and constitute the scientific criteria
for assessing the degree of pollution. They also form the foundation for
a number of ameliorative sanitation measures for control of atmospheric
pollution in populated areas.
The material included in this volume deals with the response of the
organism',
(1) to lci'7 concentrations of chemical air pollutants around major
petrochemical complexes that are now under intensive development;
(2) to low concentrations of air pollutants around chemical plants
manufacturing as raw material for nylon production the salts of adipic acid
and hexamethylenediamine and also salts of sebaic acid and hexamethylene-
diamine; and
(3) to the various concentrations of air pollutants around organic
synthesis planes and other facilities of the new chemical industry.
Methods of determination of low concentrations of various substances in the
atmospheric air are also described in some of the papers of this volume.
Much of the background material presented in the prefaces to the pre-
ceding volumes of this series is also relevant to the present volume.
Some background information on the distribution of the Soviet industry's
production machine may be of interest in connection with that country's present
and potential pollution problems and investigations. The planned distribu-
tion of production in the Soviet Union favors effective exploitation of the
natural resources of the USSR, especially in its eastern areas where enormous
V
-------
natural resources are concentrated, and has led to the creation of large
industrial centers and complexes of heavy industry in many of the country's
economic areas (see page ix). The many diverse climatic conditions of the
country and its major economic areas as well as the geographical distribu-
tion of the Soviet Union's principal industrial and mining centers and of
its principal electric power stations and power systems can be seen from
the various maps presented as background material in this volume.
Public awareness of the environmental crisis and the pollution problem
has been greatly stimulated in the USSR by the description, in the local
press, of such phenomena as dirty urban air, polluted rivers, ravaged
forests and public parks, and poisoned wildlife as well as by the revealing
of the causes of these conditions. In the Soviet Union, like in the West,
pollution now poses for the leaders of the country some fundamental choices
between the economics of production, on one hand, and the progressively
worsening living conditions, on the other. There appears to be, at present,
a greater appreciation and a better understanding of the immense problems
of air and water pollution on the part of the urban and rural administrative
agencies. As a result of a mounting demand for the maintenance of a high
quality physical environment, protective measures against the pollution
threat are gradually taking shape in the USSR and much relevant air pollution
research data are being developed in the various industrial regions of that
country.
It is hoped that the papers selected for presentation in this volume
will be conducive to a better appreciation of some of the air pollution
investigations conducted in the USSR. As the editor of this volume I wish
to thank my co-workers in the Air Pollution Section of the Institute for
their valuable assistance.
M. Y. Nuttonson
July 1971
-------
ADMINISTRATIVE DWSKWS
lift
I. M.S.FS.R
Kjrelofmntih S-S.lt.
Estonun S.S.R-
.
Lithuanian S S R
Whi't Bir.no S S R
Ukrainian & S R.
Moldavian S S.R.
.
10 A/mer.art S.S.R.
11. Azwbaydihin S.S.R
12 KuahhS-SR.
13. UA*k &S.R
14. TurknMnS.lR.
15. T»0zh.k S.S.R.
16 KirgtzS.S.R.
A.S.S.R.
A. KomiASSR
a. iwmumwnASSR
C MartysMy* ASSR
0 Chntshskiy* ASSR
E MotthMhayi ASSfl
r. TlUrsiuyj ASSR
G. 8«nl«r«Kay« ASSR
H." D«K«tansk«yi ASSR
J. S«vw(^O»«tmiti«yi AS
R. lUbcrdinftkjyi ASSR
L AbUwnkaya ASSR
yi ASSR
« ASSR
P. Soryit Mongo1 ***/» ASSR
Q. Y*mitsk«yi ASSR
-------
CLIMATIC ZONES AND REGIONS* OF THE USSR
00 ICO MO
o r,oo ,000 .
v-?'^?^;-:.^,' --.^rARCTIC
--:r ^ -
Zones: I-arctic, II-subarctic, Ill-temperate, IV-subtroplcal
Regions: 1-polar, 2-Atlantic, 3-East Siberian, A-Pacific, 5-Atlantic,
6-Siberian, 7-Pacific, 8-Atlantic-arctic, 9-Atlantic-continental forests,
10-continental forests West Siberian, 11-continental forests East Siberian,
12-monsoon forests, 13-Pacific forests, U-Atlantic-continental steppe,
15-continental steppe West Siberian, 16-mountainous Altay and Sayan,
17-mountainous Northern Caucasus,, 18-continental desert Central Asian,
19-mountainous Tyan-Shan, 20-western Transcaucasian, 21-eastern Transcau-
casian, 22-mountainous Transcaucasian highlands, 23-desert south-Turanian,
24-mountainous Pamir-Alay
(After B. P. Alisov, "Climate of The USSR", Moscow 1956)
SOIL AND VEGETATION ZONES IN THE U.S.S.R.
. -...'.- ..-. . y •...•'•, 'f»A" '^-VVvViV v-W^ » >
•t/r+f:*&*•£::& •^*S$^S&$&.
9km
viii
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 1969)
-------
MAJOR ECONOMIC AREAS 0F THE U.S.S.R.
•VI Volga
VII Ur.l,
VIII We,l Siberian
IX Eat) Slbarian
X Far Eastern
XI Bailie
XII Soulh-WesUrn
XIII Donals-Dniapar
XIV Southern
XV Tranicaucaiian
XVI Kazakhtlan
XVII Central
XVIII By.loru»!an
ntral Chnrnoiam
Iga-Vyalka
" Caucasian
PLANNED DISTRIBUTION OF INDUSTRIAL PRODUCTION IN ORDER
TO BRING IT CLOSER TO RAW MATERIAL AND FUEL SOURCES
An example of the planned distribution of industrial production in the USSR is the creation of large
industrial centers and complexes of heavy industry in many of the country's economic areas: the North-West
(Kirovsk, Kandalaksha, Vorkuta), the Urals (Magnitogorsk, Chelyabinsk, Nizhny Tagil), Western and Eastern
Siberia (Novosibirsk, Novokuznetsk, Kemerovo, Krasnoyarsk, Irkutsk, Bratsk), Kazakhstan (Karaganda, Rudny,
Balkhash, Dzhezkazgan).
Large'industrial systems are being created - Kustanai, Pavlodar-Ekibastuz, Achinsk-Krasnoyarsk,
Bratsk-Taishet and a number of others. Ferrous and non-ferrous metallurgy, pulp and paper, hydrolysis and
saw-milling industries are being established in the Bratsk-Taishet industrial system. The Achinsk-Kras-
noyarsk industrial system is becoming one of the largest centers of aluminum and chemical industries, and
production of ferrous metals, cellulose, paper, and oil products.
Construction of the third metallurgical base has been launched in Siberia, and a new base of ferrous
metallurgy, using the enormous local iron and coal resources, has been created in Kazakhstan. A high-
capacity power system is being organized in the same areas. Non-ferrous metallurgy is being further
developed in Kazakhstan, Central Asia and in Transbaikal areas. The pulp and paper, as well as the timber,
industries are being developed at a fast rate in the forest areas of Siberia and the Far East.
Ferrous metallurgy is also developing in the European part of the country by utilizing the enormous
iron ore resources of the Kursk Magnetic Anomaly and the Ukrainian deposits. Large new production systems
are under construction in the North-West, along the Volga, in the Northern Caucasus and the Ukraine.
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 1969)
ix
-------
THE MAJOR INDUSTRIAL CENTERS OF THE USSR
Mam centres ol ferrous metallurgy
" " " non-ferrous metallurgy
O Centres ol chemical industry
(After A. Efimov, "Soviet Industry", Moscow 1968)
-------
PTUNfTPAT, rENTRBS DP T?F
Karaganda
AAlasu "
Complete cycle m«l«llurgy
Steel tmelling and mel«l
foiling
Smelling of ferro«ltoy»
Mining oft
A iron orei
Q coking eo*I
nwnganete ore'l
I IAIN IRON ORE DEPOSITS IN THE U.
(After A. Lavrishchev, "Economic Geography of
the U.S.S.R.", Moscow 1969)
Xi
-------
PRINCIPAL CENTERS OF NON-FERROUS METALLURGY IN THE U.S.S.R.
Metallurgy:
copper ® Icoil
(iluininium ($i nickel
O line O I'"
DISTRIBUTION OF MOST IMPORTANT DEPOSITS OF NON-FERROUS I-^ETAL ORES
xii
(After A. Lavrishohev, "Economic Geography
of the U.S.S.R.", Moscow 1969)
-------
PRINCIPAL CENTERS OF THE CHEMICAL INDUSTRY IN THE U.S.S.R.
O Chemical indu
Oil-relining in
O Production of
O Production
PRINCIPAL CENTERS OF THE TEXTILE INDUSTRY IN THE U.S.S.R.
urei on (he map thow:
I Vyihny 6 Rlhrv I? Vithug.
Volochnk 7 Ot)h« 13 Ibili!,
Linen cndull.y
© Woollen induvliy
O Silk
O Olhcr br«nch« ol Ihl lnliU induiliy
3 f-lov ? Klinliy IS NiAK«
4 Kalinin 10 Sniolvntk 16 M«*gelan
S Ivanovo 11 Bryanik 17Nogin\k
xiii
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 1969)
-------
PRINCIPAL CENTERS OF WOOD-WORKING AND PAPER INDUSTRIES IN THE U.S.S.R.
Industry:
Timber-sawing and wood-working
© Paper
O Principal lumbering areas 500 o JM tOOOk
' ' •
PRINCIPAL CELTTEy'S OF THE FOOr INDUSTPY IN THE T7..S.3.P.
40 tt> 60 100 HO HO UP
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 19&9)
xiv
-------
THE MAIN MINING CENTERS OF THE USSR
Oil refining
Oil pipes
Gas pipes
Power italiom
(After A. Efimov, "Soviet Industry", Moscow 1968)
-------
PRINCIPAL ELECTRIC POWER STATIONS AND POWER SYSTEMS IN THE U.S.S.R.
Principal Electric Power Stations
Thermal Hydro-power
If. jjt in operation
j*j J* under construction
and planned
Groupi ol electric
power stationi
Operating afomic electric power stations
Areas ol operation of single power grids
European parl of the U.S.S.R.
Cenlia! Siberia
Areas of operation ol integrated power grids
Northern Kazakhstan
Central Asia.
North-Wesf '
and Weil
Caucasus
(Geolermicheskaya)
omsomoIsk-on-Amur
'.v.v •.. .-i TT/^
-,, . .. I»T •', O-> J-A:P=A-H
Figures indicate following power siations:
9 Dnieprodzerzhinsk 17 Shatura
10 Dmeproges 18 EleklrogorslE
II Kalhovka I? Ivankovo
' Baltic
2 Narva
JKegum aova vanovo ,s »„,
4 Plavinas 12 Slarobeshevsk 20 The 22nd C.P.S.U. CongreM "6 Varzob
5 Novaya Byelorusitc/a 13 Zuyevikaya HEPS on the Volga 77. Toklogul
ftDubossary 14 Shterovka 2t The Lenin HEPS on (he Volga 28 Alamedi
7 Kanev 15 Krasnodar 22 Chardarinskaya 360 0
SKremenchug 16 Kashira 23 Chtrchik-Bozsu
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 19&9)
-------
BIOLOGICAL EFFECT OF LOW CARBON DIOXIDE CONCENTRATIONS*
0. V. Yeliseyeva
Fourth Main Administration, Ministry of Public Health of the USSR and
Communal Hygiene Department, Central Institute for Advanced Training of Physicians
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 7-27, (1966).
Studies conducted by Pettenkofer have shown that the amount of COj can
be used to estimate the purity of air in dwellings and the degree of their
aeration. Pettenkofer proposed a hygienic norm of tt^ for dwellings of
0.07%, and Flugge, 0.1%. A 0.1% C02 content in dwellings is still the
hygienic limit at the present time. Although this limit has not been phys-
iologically substantiated, it has played a positive role in practice and
is still used for calculating the required air exchange in closed spaces,
and serves as the criterion for evaluating the purity of room air and the
operation of ventilating systems.
Numerous studies conducted on animals and people have shown that an
increase in the C02 content of inhaled air does not leave the organism
unaffected and may cause substantial shifts in its condition. The central
nervous system is particularly sensitive to excess carbon dioxide.
Despite the fact that there has been a considerable number of studies
dealing with the influence of C02 on the various physiological functions
of the organism, they were all made by using large concentrations and are
desultory in character. Of greatest interest in this respect are the
studies of V. P. Zagryadskiy, Z. K. Sulimo-Samuylo, P. F. Vokhmyaninov
and 0. Yu. Sidorov (1961). The authors studied the influence of relatively
low C02 concentrations on man in short- and long-term exposure. The COj
content in the inhaled air ranged from 1 to 2%. Breathing with the gas
mixture lasted from five hours to ten days. The authors note that an
increase in the C02 content causes first of all a change in the activity
of the respiratory and cardiovascular systems. When the stay in the
chamber was brief, the respiration rate increased or remained unchanged,
and after ten days there was a tendency for the respiration rate to slow
down. An increase in pulmonary ventilation due to a considerable deepen-
ing of the respiratory movements was noted. There was also observed a
lasting spasm of the peripheral vessels, a considerable slowing down of
the frequency of cardiac contractions, and an increase of the minimum
pressure by 10~15 mm Hg. After a prolonged exposure, changes in the elec-
trocardiogram were observed, i.e., an increase of the P-Q, QRST intervals,
indicating a certain slowing down of the conduction functions.
* The physiological studies were directed by Candidate of Medical Sciences A. D. Semenko.
- 1-
-------
Short-term exposure, to CCL impaired the trace conditioned reflexes
to time, the percentage of erroneous reactions in the study of motor
conditioned reflexes increased, and so did the number of erroneous answers.
The subjects could not sustain an active attention for six minutes and
began to doze off. A long exposure to carbon dioxide in a concentration
of 1.5-2% was associated with lability of the functions of the motor and
visual systems, impairment of motor coordination, high emotional excita-
bility, restless sleep, irritability, and marked drowsiness in the morning
hours. There were up-and-down fluctuations of working capacity and a
considerable increase in the number of errors in the course of work.
Changes in the electroencephalogram were very slight. It is important to
note that the change of the physiological functions of the organism was
observed not only during exposure to atmosphere with a higher CC^ content,
but also during the period of the aftereffect on passing to the conditions
of a normal gaseous medium. The longer the organism was exposed to con-
ditions of higher C0~ content, the higher was the carbon dioxide concentra-
tion, and the longer was the duration of the aftereffect period.
In the course of the last few decades, the C0? content of atmospheric
air in large cities has been increasing during the winter months. Thus,
in Moscow, the mean daily concentration at that time may reach 0.07%, and
the single concentrations are still higher (K. A. Bushtuyeva, V. A. Ryazanov,
Ye. F. Popova, 1956), this being explained by a markedly increased amount
of burned fuel. Under these conditions, it becomes impossible to maintain
the CC>2 concentration in the air of dwellings at the level of Pettenkofer
and Flugge's norm. The question arises, to what extent is this norm valid
under changing living conditions? Actually, Pettenkofer's suggestion was
based on the idea of using carbon dioxide as an indirect index for the
pollution of air of residential and public buildings by volatile products
of human metabolism contained in exhaled air, respiration, and malodorous
gases from body surface and clothing. In a modern city, where CO 9 is gen-
erated chiefly by the combustion of fuel, it loses the meaning of an in-
direct sanitary index. For this reason, the observance of this norm for
the air of residential and public buildings is not justified.
To insist on the observance of this norm is to require a decrease in
the concentration of carbon dioxide in the surrounding atmosphere, which
would involve extremely costly measures aimed at decreasing the discharge
of CC>2. This could be done only if carbon dioxide as such were proven to
have undesirable hygienic effects on man in concentrations such as 0.1%.
However, as we have seen above, data on the physiological action of ($2
concentrations below 1% are lacking in the literature.
For these reasons, we undertook a study of the influence of CC^ in
concentrations of 0.5% and below on the respiratory function, the cardio-
vascular system, and the electrical activity of the human cortex. The
methods of pneumography, rheovasography and electroencephalography were
used for this purpose.
- 2 -
-------
Our studies were conducted on healthy persons 19 to 45 years of age.
A constant, predetermined CO 2 concentration was maintained during the
entire investigation.
The source of gas was a cylinder of liquid carbon dioxide at a pres-
sure of 36-46 atm, from which carbon dioxide flowed through a reducing
valve, a float-type flowmeter, and a capillary rheometer into a mixing
tank. Pure air was also fed into the latter at a constant rate of 20 1/min.
The gas mixture was then fed into a cylinder whose contents the subject
breathed.
The lines carrying the air consisted of glass and Duralumin tubes.
The air supply was purified with silica gel, activated carbon, and a
cotton filter.
The actual concentrations of carbon dioxide in the cylinder were
determined by a spectrophotometric method that we developed (M. D. Manita,
0. V. Yeliseyeva, T. V. Dyshko, 1964), both before and after the experiment.
The apparatus that we used insured an adequate constancy of the con-
centrations. During the entire observations, the subject remained in an
electrically and acoustically insulated room, sitting in a comfortable
position in a soft chair. A cylinder continuously supplied with pure air
or with air of a known carbon dioxide concentration was placed at the
height of his face. The apparatus for dispensing the gas fnr for record-
ing the functions studied and the experimenter were located in an adjoin-
ing room.
The examinations were conducted with a background of rhythmic photic
stimulations of varying frequency, duration and intensity, generated by a
photostimulator built by the Kaiser Co. The lamp was placed before the
subject's eyes at an optimum distance determined for each person individ-
ually. This distance was kept constant during the period of all the
experiments. The intensity of the rhythmic photic stimulation was changed
every five seconds from 0.1 to 0.6 Joule. The entire series of stimulations
lasted 40 sec in the following sequence (eight 5-second stimulations in
Joules): 0.2, 0.6, 0.2, 0.1, 0.2, 0.6, 0.2, 0.1. The subject sat quitely
in the room and signaled the change in light intensity by slightly depress-
ing a button. This movement was recorded on a myogram. The method made
it possible to control and maintain the subject's wakefulness, otherwise
they-usually sank into a drowsy state toward the end of the experiment.
The conditions were identical in all the tests. The main tests were con-
ducted after repeated training. After the subjects had become accustomed
to the experimental situation, we recorded the curves during inhalation of
pure air. This served as the background against which the influence of
corresponding carbon dioxide concentrations was studied. Because we studied
low concentrations (from 0.1 to 0.5%), the pure air was regularly checked
- 3 -
-------
for its carbon dioxide content. The CCU concentration did not exceed 0.05%
and amounted to an average of 0.04%. During the entire experiment we alter-
nated the recordings of the inhalation of different concentrations of CO 2
and pure air.
Numerous investigations, particularly those of Holden and co-workers,
established that the physiological action of carbon dioxide depends on the
partial pressure of this gas. A 0.23% increase of its content in alveolar
air when the body is at rest leads to a 100% increase in the ventilation
volume. A 0.01% decrease or increase of COo content in alveolar air causes
a corresponding 5% average decrease or increase of the ventilation volume
(Holden and Priestley^ 1937). Thus there is a very significant dependence
of the change of pulmonary ventilation on the (X^ pressure in alveolar air.
An increase in pulmonary ventilation is a compensatory reaction of the
organism designed to decrease the carbon dioxide content of alveolar air
when it increases in the inhaled air.
Ye. M. Berkovich (1939) has shown that the inhalation of €62 starting
at a concentration of 1% increases the respiratory capacity, extends the
duration of both breathing phases, and slightly decreases the respiration
rate.
In the light of modern physiological-concepts (Heymans and Cordier',*
1940; M. V. Sergiyevskiy, 1950, 1961; M. Ye. Marshak, 1961, and others),
carbon dioxide alters normal breathing by acting not on the breathing
center directly, but primarily on the vascular receptor zones and the cor-
tex of the cerebral hemispheres. Of particularly great importance in the
stimulation of breathing, along with the receptors of the lungs, are the
chemoreceptors located at the point of branching of the common carotid.
As the C0~ content increases, stimulation of the chemoreceptors of the
aortal-carotid zone takes place in the blood. By reflex action, this
causes a deepening and a certain acceleration of breathing.
In addition, it has been shown (Holden and Priestley, 1937) that the
nerve cells of the respiratory center can be stimulated by carbon dioxide
present in the tissue fluid bathing them, into which it penetrates from
the blood. Thus, carbon dioxide acts reflexly on the respiratory center
by stimulating the chemoreceptors, and also automatically through the
blood.
•To study the influence of C0~ on the function of external respiration,
we selected seven persons aged 19 to 45, nonsmokers, without a change in
the nasopharynx or upper respiratory tract. The respiratory movements were
recorded by using the method of pneumography with a cuff attached to the
frontal surface of the chest.
The duration of a single examination of different persons varied from
20 to 24 minutes. During the first five minutes the subject breathed pure
* [Translator's note: Kholden and Pristli, according to the transliteration of Russian reference.]
** r " " Geymans and Kord'ye, according to the transliteration of Russian reference.-]
- 4 -
-------
air, then, without his noticing it (by turning a three-way stopcock) a
mixture with a given (XU concentration was supplied to the cylinder, which
he also breathed for five minutes. Pure air was then fed into the cylinder
again. The instant of start and finish of the gas supply was marked on
the tape by means of an electric marker connected to a switch.
The data obtained were treated according to the rules of variation
statistics with calculation of the confidence factor. Changes with a
confidence factor of no less than 95% were considered statistically
significant.
The treatment was used in two periods: the first two recordings
during inhalation of pure air for five minutes were taken as the "back-
ground", and the next three recordings during inhalation of a given CC>2
concentration for five minutes as the "effect". The period of the
"aftereffect" during inhalation of pure air was divided into the "close
aftereffect" (3 recordings) and "distant aftereffect". In each curve,
the depth and frequency of respiration were calculated. Results of the
determinations were expressed in percent. The average level of the first
two ("background") determinations were taken as 100%.
The action of each concentration studied on the respiration was
repeated two to three times on the same subject.
Two C02 concentrations were studied, 0.5 and 0.1%. The 0.5% concen-
tration was found to be active for all seven subjects. In all of them,
a decrease in the amplitude of the respiratory movements was noted. How-
ever, in different persons the statistically reliable changes were not
the same and were manifested in different periods: in subjects S. and Ch.
the changes took place during the "effect" period and were preserved dur-
ing the "aftereffect" period. The same results during the "aftereffect"
period were obtained in subjects 0., R., Z. and M. In subject A, changes
were observed only during the "aftereffect" period (Table 1).
Table 1
Changes in the Amplitude of Respiratory Movements (in $) During
Inhalation of CC>2 in 0.5$ Concentration
Subject
S.
0.
R.
Z.
A.
M.
Ch.
Periods of Observation
Background
100
100
100
100
100
100
100
Exposure
91,6 (;i)
87,0
92,5
94, -1
89,0
98.2
77,1 (b)
Aftereffect
Close 1 Distant
88,3
58.9 (b)
7-1,35 (a)
74,8 (a)
8-!, 5 (a)
83,1 (a)
60,6 (c)
70,1 (c)
74,7 (c)
72/5 (c)
71,1 (c)
99,'J
—
Note. Confidence factor: a - 95$; b - 99$; c - 99.9$
-------
At a C02 concentration of 0.1%, statistically reliable changes
(also in the decreasing direction) in respiration amplitude were obtained
in five out of six subjects (Table 2).
As an illustration, we shall cite the dynamics of changes in the
respiration amplitude in percent for subject 0. during inhalation of
different COo concentrations (Fig. 1).
Table 2
Changes in the Amplitude of Respiratory Movements (in percent)
During Inhalation of C02 in 0.1$ Concentration
Subjects
G.
0.
R. .
Z.
Zh.
M.
Periods of Observations
Background
100
100
100
99,9
99,9
100
Exposure
90,7
88,7
9-1, <1
95,2
79,4 (c)
91,2
Aftereffect
Close
96,3
66,9 (c)
78,8 (a)
90.5
71,6 (b)
80,2(c)
Distant
96,0
75.2 (1.)
78,6 (b)
85,7 (c)
88,6
—
Note: a, b, c - see Table 1.
As far as the respiration rate is concerned, and also the inspiration-
expiration ratios, they did not undergo any noticeable changes in any of
the subjects. Thus, carbon dioxide in concentrations of 0.5 and 0.1%
against a background of inhalation of pure air causes changes only in the
depth of breathing.
The data which we obtained disagre with the literature data to some
extent. However, such discrepancies in the results are readily explained
by the fact that our examinations involved the use of considerably lower
carbon dioxide concentrations than those used by the authors of the above
studies.
Both the excitability of the respiratory center and the sensitivity
of the receptors accepting carbon dioxide as an adequate stimulus display
substantial differences in the effect of different concentrations. This
is necessarily reflected in the characteristics of external respiration
responses to the action of carbon dioxide.
In order to study the influence of carbon dioxide on the state of the
peripheral circulation in man, we made use of the rheovasography method.
As we know, high frequency curves for studying the fluctuations of
the heart and blood vessels were first used in 1932 by Atzler and Lehmann.
In the Soviet Union, the first investigators to develop a method of study
- 6 -
-------
of blood circulation by means of high frequency currents were A. A. Kedrov
and A. I. Naumenko (1941, 1949, 1954), who validated it theoretically and
applied it in experiments on animals and in observations on people in the
study of intracranial blood circulation.
The method of rheovasography was used for several years at the Insti-
tute of Therapy of the USSR Academy of Medical Sciences in the study of
cardiovascular diseases. The rheovasography method consisted essentially
of the fact that an electrical current of high frequency encounters a
certain resistance on passing through a portion of live tissue. The mag-
nitude of this resistance is affected by the degree of blood filling and
the rate of blood flow in the given portion of tissue (A. A. Kedrov and
A. I. Naumenko, 1949). The more the blood circulation rate changes per
unit time (positive and negative acceleration of the blood flow), the
wider the range over which the resistance of the portion of live tissue
to the electric current changes. After a cardiac systole, the rate of
:>>
t,
o
wo-
so-
.10-
70-
60
SO
•a
3
4->
a.
a
\
/
\J
/ W 12 II If II
Periods of experiment
Fig. 1. Change in the amplitude of
respiratory movements in subject 0.
during inhalation of different C02 con-
centrations.
1 - pure air; 2 - 0.1%; J> - 0.5$;
AB - period of gas supply.
blood flow always increases (positive, pulse acceleration) during a
certain period of time. The rate then slows down. After a certain time
interval following the systole, under the influence of the elastic
force of the vessels, a new increase of the rate of blood flow takes
place (positive vascular acceleration). The rate then slows down again
and reaches the level it had before the start of the systole. The blood
filling also changes correspondingly. Such changes in any portion of
live tissue arise regularly during the period of each cardiac cycle and
are recorded by means of rheovasography. This method is marked by a high
sensitivity. In serious disorders of the peripheral blood circulation,
the data of rheovasography always reflect the existing hemodynamic dis-
turbances in a more differentiated manner (V. A. Karelin, 1957). The same
- 7 -
-------
complexes are repeated on a rheovasogram. Each complex consists of one
large and two small spikes. The large spike has a steep rising edge
and a gentler descending edge. It is generally recognized (V. A. Karelin,
1957; A. S. Loginov and Yu. T. Pushkar', 1962) that the large spike is a
reflection of the systolic acceleration of the blood flow. In time, it
almost coincides with the ventricular complex of the electrocardiogram.
The small spikes appear after the systole in time and may correspond to
an acceleration of the blood flow as a result of the elastic force of
the vessels (positive vascular acceleration).
Our studies were made by means of an RG1-0.1 type rheograph whose
output was connected to the recorder of a Kaiser 8-channel electroen-
cephalograph. Seven people aged 18 to 45 were selected for the experiment.
The rheovasogram was recorded from the right arm, the electrode being
placed on the skin of the shoulder and forearm. The recording was made
in the morning hours, always at the same time.
Each experiment lasted 20-24 minutes. The rheovasogram was recorded
10-12 times every two minutes. The first two recordings were made during
inhalation of pure air and were taken as the background; the next three
recordings, during the period of exposure, which lasted five minutes; and
the remaining recordings, during the period of the aftereffect, when the
subject breathed pure air again. During treatment of the data, the after-
effect was divided into two periods: three recordings pertained to the
"close aftereffect", and the remaining ones, to the period of the "distant
aftereffect".
To analyze the rheovasography data, we measured the sizes of the
rising and falling edges of the large spike and second spike. The results
of the determinations were expressed in percent of the average of the
first two "background" determinations.
Data from the study of the influence of 0.5% carbon dioxide on the
rheovasographic indices are listed in Table 3.
The data of Table 3 show that carbon dioxide in 0.5% concentration
caused distinct and significant changes in blood filling of the vessels
in all seven subjects. The index of the influence of carbon dioxide was
an increase in the size of the rising and falling edges of the large
spike and also of the second spike. Only subject S. responded to carbon
dioxide inhalation with a decrease in the size of the descending edge of
.the large spike of the rheovasogram.
In the majority of subjects, the action of carbon dioxide manifested
itself already during the period of exposure. Changes in the rheovasogram
were stable in character and were preserved during the entire period of
- 8 -
-------
the aftereffect.
Table 3
Changes of Complexes (in $) of the Rheovasogram Under the
Influence of Carbon Dioxide in 0.5$ Concentration.
Sub-
ject
R.
Z.
O-i
•A.,
•o.
.B.
C.
Complexes of
Rheovasogram .
Rising edge
Descending edge
Second spike
Rising edge
Bescending edge
Second spike
Rising edge
Descending edge
'Second spike
Rising edge
Descending edge
Second spike
Rising edge
•Descending edge
Second spike
Rising edge
Descending edge
Second spike
Rising edge
Descending edge
Second spike
Periods of Observations
Back-
ground
100,0
-100,0
100,2
99,9
100,0
99,9
100,3
100,0
100,1
99,9
99,9
100,0
99,6
100,0
100.0
100,0
100,0
100,2
100,0
100,0
100.0
Exposure
117,9(b)
113,3(a)
138,3
102,']
95.8
100,2
114,7
102,4
103,7
98,3
104,3
9G,2(c)
120,6(c)
117,2(a)
133",8(a)
112,6(a)
109,4 (a)
100,0
110,9
93,S(a)
98,9
Aftereffect
Close
131, l(c)
I20,4(b)
179,0(a)
112.8(b)
104,1 (a)
112,2
142, 3(c)
111, 5(c)
133, 6 (a)
109,8
105,5
M4,7(a)
123,3(c)
12l,5(c)
I34.8(a)
126, 0(b)
119.7(1))
129,1
H3,9(c)
80,0(c)
10S.9
Distant
135, 0(c)
116,2(a)
203, 2(c)
119,5(a)
109,5
111,2
151, 5(c)
120, l(c)
158, 5(c)
98,5
100,7
99,4
I24,5(c)
118, 4(b)
129, 7(a)
125,8(c)
126. 2(b)
109.1 (a)
112,l(c)
75.4(c)
103,8
Note: a, b, c - see Table 1.
Similar but less distinct results were obtained in a study of carbon
dioxide in 0.1% concentration. The changes were statistically reliable
in 5 out of 7 subjects (Table 4).
As is evident from Table A, the inhalation of carbon dioxide in
0.1% concentration caused an increase of all three indices of the rheovas-
ogram. These data show beyond any doubt that even a carbon dioxide
concentration as low as 0.1% is capable of affecting the state of the
-------
blood flow in healthy people. Fig. 2 shows graphs of the changes in the
size of rheovasogram complexes for subject 0.
Table 4
Changes in Rheovasogram Complexes (in percent)
Under the Influence of Carbon Dioxide in 0.1% Concentration.
Subject
R.
. Zh.
2.
'0.
A.
Rheovasogram Complexes
Rising edge
Falling edge
•Seconfl spike
Rising edge
Falling edge
Second spike
Rising edge
Falling edge
Second spike
Rising edge
Falling edge
Second spike'
Rising edge
Falling edge
Second spike
Periods of Observations
Back-
ground
100,0
100,0
100,0
98,5
100,0
100,0
100,0
100,0
100,5
100,0
100,0
100,0
99,9
97,0
99,9
Exposure
10-1,8
104,9(b)
103,0
11-1,3'
109,0(a)
115.8
99,6
102,0
95, -1
109,6
103,3(a)
98,7
„ 107,6
101,1
131, -1
Aftereffect
Close
111, 7(a)
lll,l(n)
115.3
127,5(c)
110,5(b)
137, l(c)
96,5
97,9
75,5(c)
108,3
101.0
84,0
109,3
103,1
117,5(a)
Distant"
115, 7(c)
114, 7(b)
126,8(c)
120,2(c)
106. l(b)
132,3(c)
95,3
97,7
79,3(b)
120,5(a)
108,2(c)
76,0
96,4
89,8
123,3
Note: a, b, c - see Table 1.
ittS-
U>
2,
o
HI
c
v 100
"O
o.
a
so-
r\
i
i
f o e s to i! iii if is
Periods of experiment
Fig, 2.. Change in. rheovasogram complexes (rising edge)
during inhalation of different 0)3 concentrations in
subject 0. (Reliability of changes 95$).
Notation same as in Fig. 1.
- 10 -
-------
The responses of the electrical activity of the brain to the inhal-
ation of increased carbon dioxide concentrations were studied by Bremer
and Thomas (1936), S. I. Subbotnik and P. I. Shpil'berg (1946), and
others. They established that the inhalation of increased carbon dioxide
concentrations leads to the development of the desynchronization response.
I. S. Repin (1960) observed desynchronization in rabbits in all parts of
the central nervous system during their inhalation of up to 5% CC^.
Similar data were obtained by Yu. N. Ivanov (1962).
The effect of carbon dioxide concentration below 1% was not studied.
In recent years, practical sanitary investigations have made increas-
ingly broader use of methods based on the recording of changes in the
electrical activity of the brain under the influence of low concentrations
of noxious substances. These methods have found wide applications in the
studies performed in the Department of Communal-Hygiene of the Central
Institute for Advanced Training of Physicians.
We undertook the task of following the changes in the electrical
activity of the human brain during inhalation of carbon dioxide in con-
centrations below 1%.
Three electroencephalographic methods were used for this purpose:
method of development of the electrocortical conditioned reflex, method
of rhythm assimilation, and method of reinforcement of the intrinsic
potentials of the brain.
The experimental conditions were identical in all the tests.
The tests were made on an 8-channel encephalograph with a Kaiser
photostimulator. The electroencephalograms were taken by the unipolar
method (in combination with the bipolar method) off the occipital,
parietal, and temporal areas of the head.
At the same time, the time mark, frequency and intensity of rhythmic
photic stimulations and the time of the gas supply were recorded.
The recording of biocurrents was made on the same channels with the
same degree of sensitivity, and the electrodes were placed on the same
areas. The subjects were asked to participate in the experiment only
during the morning hours.
For each subject, training recordings of electroencephalographic
curves were first made for one month. Afterward, the curves were recorded
during inhalation of pure air, then with a given carbon dioxide concentra-
tion.
- 11 -
-------
The method of development of the electrocortical conditioned reflex
was first used in hygienic studies by K. A. Bushtuyeva, Ye. F. Polezhayev
and A. D. Semenenko (1960). This method makes it possible to reveal fine
functional changes in the cerebral cortex arising under the influence of
various external factors. We studied the development of the electrocorti-
cal conditioned reflex in five healthy persons with a well-defined a rhythm.
The inhalation of the gas lasted 15 seconds, and during the next five
seconds was reinforced with intermittent light. The conditioned reflex
changes in electroencephalography with reinforcement with rhythmic light
have been described by R. A. Pavlygina and V. S. Rusinov (1960) and also
by Pavlygina (1960). In order to exclude the development of the conditioned
reflex to time, each new pairing of the action of the gas and light was
given at different time intervals.
We expected that in the case of formation of a temporal relationship
to the inhalation of definite carbon dioxide concentrations, the imposed
rhythm should appear before the rhythmic light was turned on.
However, the 0.5% carbon dioxide concentration did not cause the
formation of an electrocortical conditioned reflex in any of the 5 subjects.
The number of pairings reached 23-25. Concentrations of 2-2.5% also failed
to cause the appearance of an imposable rhythm before the light was turned
on.
The second electroencephalographic method used for the determination
of thresholds of the reflex action of carbon dioxide - rhythm assimilation -
was proposed by A. G. Kopylov (1956).
The method as modified by us has been described by A. D. Semenenko
(1963) and was used in studies conducted at the communal hygiene department
of the Central Institute for the Advanced Training of Physicians by
V. I. Filatova (1962), B. Mukhitov (1962), N. B. Imasheva (1963), V. A. Chizh-
ikov (1963), and P. G. Tkachev (1963).
Two subjects with a well-defined a rhythm and a satisfactory assimilation
of the rhythm in the range of usual frequencies were selected for the experi-
ments. For each, a frequency of flickering light was chosen which differed
(was greater or smaller) from "his" a rhythm. An optimum distance for each
subject was then established between the subject and the photostimulator
lamp; the maximum total amplitude of rhythm assimilation for a large inten-
sity of flashes (0.6 Joule) and the minimum amplitude for the lower intensity
of flashes (0.1 Joule) were then observed at this distance. The frequency
of the Flickering light and the distance of the lamp were kept unchanged
during all the experiments. The entire series of light stimulations lasted
40 seconds. The intensity of the rhythmic photic stimulus was changed every
5 seconds from 0.1 to 0.6 Joule.
- 12 -
-------
The recording of the assimilation curves was repeated every 2 minutes
for 20 minutes. The subjects breathed pure air during this entire period.
In studies with gas, the first two recordings of the curves were made
during inhalation of pure air, the next three recordings during the period
of inhalation of the gas, and the following ones, 5 seconds after cessation
of the supply of carbon dioxide, then every 2 minutes until the reestablish-
ment of the initial level of the assimilated rhythm or the appearance of a
tendency toward its reestablishment.
The electroencephalograms obtained were analyzed from the change in
the amplitude of 'the assimilated rhythm, expressed in microvolts. To cal-
culate the amplitude, oscillations exactly correspodning to the frequency
of the applied light pulses were recorded first, then the amplitude of the
recorded assimilated rhythm was measured in millimeters for each intensity
separately and the total amplitude was measured for every 40 seconds of
rhythmic photic stimulations. The total amplitude of the assimilated
rhythm based on all the intensities for the entire period of the experiment
was calculated in percent of the average total amplitude obtained from
the results of the first two curves of rhythm assimilation recorded in
the second and fourth minutes of the examination, and taken as 100%. The
results of the examinations are given in Fig. 3 and 4.
A carbon dioxide concentration of 0.5% was found to be the active
concentration for both subjects.
In subject R. T., there was noted a well-defined tendency toward an
increase in the amplitude of the assimilated rhythm, although statistically
reliable changes appeared only during the period of the aftereffect (the
reliability of the changes was 99%).
Somewhat different results were obtained in subject I. A., in whom
the action of the gas was manifested in the second minute of inhalation
(reliability of changes 95%) and was also characterized by an increase in
the total amplitude of the assimilated rhythm. When using the third method
of study of the electrical activity of the brain, we employed the frequency
of the rhythmic light stimulus corresponding to the frequency of the intrin-
sic potentials of the subjects' brain. The calculation of the total ampli-
tude of the reinforced rhythm was made by means of a special electronic
device, an integrator built by engineer B. N. Balashov.
The study of the reinforcement of potentials of the intrinsic rhythm
during inhalation of low carbon dioxide concentration was made on seven
practically healthy people aged 19-45. The experimental conditions and
the treatment of the results were analogous to the ones described above.
- 1-3 -
-------
tso-
170-
a ISO-
1>»
t -ISO-
•o
CO
• H
H ^"
t/1
10 //#-
o
«, 110-
•o
| wo-
o.
B
***
• -• — '
%
i
I
. I
j
/••*
,'
"X'V,^
/ \
/ \
r \>
i \
» \
i \
1 \
"^ — . — '—
•* ~ i i 1 r 1 1 1 1 1 1 —
2
-------
The total amplitude was expressed in percent of the three initial
changes taken as the background. Statistical treatment of the results
was carried out in comparison with data for inhalation of pure air in the
following periods of observations: "background", "exposure", "close after-
effect" and "distant aftereffect" (Table 5).
Table 5
Changes in the Amplitude of Reinforced Rhythm (in percent)
Under the Influence of Inhalation of the Carbon Dioxide Concentrations Studied
Sub-
ject
UK.
V.
0.
A.
,
H.
N.
E. •
C02 concentra-
tions studied
Mcncc 0,05
0,1 - '
0,5
Mcncc 0,05
0,1
0,5
Aicncc 0,05
0,5
Mcncc 0,05
0,1
0.5
Aicncc 0,05
0,1
0,5
Mcncc 0,05
0,1
0,5
Mcncc 0,05
0,1
0,5
Periods of Observations
Back-
ground
101,4
98,8
102,7
99,1
100,0
98,5
99,2
99,4
102,3
100,0
101,3
99,1
100,0
93,0
102.5
100,1
101,0
100,5
99,2
100,0
Exposune.
102,4 '-.
96,2
95,0
99,3
104,9 (b)
89, 6 (b)
100,7 ..
96,5
105,1
104,0
97 ,-7
96,9
108,2 -
83,2 (b)
101.9
108,3
94 , 7
100,1
103,7
92,6 (b)
Aftereffect
Close
95,9
97.7
87,7
101,5
105,6
91,5 (c)
104,1
103,3
102,5
107,7
99,5
91.6
106.6
74,5 (b)
107,9
116,0
93,5 (a)
102,1
106,4
89,r(c)
Distant
100,8
92,0 (a)
89, 7 (a)
101,3
110,0
•96,8 (a)
109,0
103,9
104,4
108,7
' 97,3
94.3
124,5 (a)
79,5 (b)
102,9
103,4
82.9 (c)
99.0 '
106,9 (a)
87,8
Notft: o, b, c - see Table 1.
From Table 5 it is apparent that carbon dioxide in 0.5% concentration
affected five of the seven subjects tested. This effect consisted in a
decrease of the amplitude of reinforced rhythm (Fig. 5). The 0.1% concen-
tration caused an increase in the amplitude of decreased rhythm in four
out of seven subjects (in 3 during the period of "distant aftereffect" and
in 1 during the period of exposure).
Thus, it may be concluded that carbon dioxide in 0.5 and 0.1% concen-
trations is capable of influencing the functional state of the cerebral
cortex.
- 15 -
-------
•o
§
o
J=
<«+>
o>.
s.
0) fc.
•O
3
%
•no -
:SO-
70-
a
^b=^—
/'
.*s
s
^^~
\
xx' •
/'** \ '^ ^
2 in 0.5% concentration.
3. The data obtained strongly suggest that single C02 concentrations
in the air'of residential and public buildings should not exceed 0.1%, and
average daily concentrations 0.05%, independent of the source of C09.
- 16
-------
LITERATURE CITED
BcpKOiiii-i 1Z. M.- ii3iio.n. wypn. CCCP. 1939, T. XXVI. n. 4.
CT|). 408—419.
I) y in T y c n a K. A., II o .'i c x< a c u E. ., C c M c n c n i< o A A. fur-
ii can. 1960, A? I, CTp. 57—61.
FcfiM.iiic K. n KopAbc A. 13 KII.: Ai.ixnTC.nbiii.ifi ucmp M.—J\.,
1940. 170 CTp.
Hnaiiou 10. II. ii3iio;i. wypn. CCCP. 1962, T. 48 A° 3
CTj). 279—289.
H M .1 in c D a II. B. Hir. n can. 1963, Ar° 2, crp. 3—8.
KapeJMiii B. A. Xiipypnia. 1957, A1? I, erp. 34—37.
KcApon A. A. IVIMII. MCA. 1941, T. 19, Ar° 1, crp. 71—80.
KcApou A. A. » HayneiiKo A. M. ixaniui y 'ic^oi>ci;a. M., 1961, 251 CTJI.
M a I> uu a K M. E. MaTCpiiaflu o (JiyMKHiioiin.ni.nofi opraiiii3annn Abixa-
TC^i.noro uciiTpa. B KII.: Honoc n (|)ii3iiojionni u naTO^onui
niifi. HOA pCA. B. B. napima. M., 1961, crp. 140—141.
MyxiiTOB D. M. Fur. n can. 1962, Ar2 6; CTp. 16—24.
D a n n u v n u a P. A. 3/iciopMai;nii CT»o^a MOSIM
iipn co'iCTaiiiiii auyi;a c PIITMHMCCKHM CIICTOM. B KII.: 3i;cncpiiMCii-
Ta.ni,HOC iisyiciuic aaKoiiOMCpnocTcfi Kopw Co.ibujiix no^yuiapnfl. M.,
1950, cTp. 39-48.
IT a n .n 1.1 r n n a P. A. n P y c 11 n o n B. C. YcjionnopctpJicKTOpiian
nepccrpofiKa KopKODofi PIITMIIKII y 'ic^oncKa iipn co'iCTaiiiiii siiyKa c
pnrMii'iccKUM cncTOM. B KII.: 3KcnepiiMCiiTa;ibiioc myicniic 3aii;iiuOTiii.ix. /McAi'ii.i, 1950, 362 crp.
C c p r H c n c K n ii AV B. Ilonuc Aainn.ic o pcry^nunn Awxannn. B KM.:
Ilonoc n (|)ii3iio.noriin u iiaro-nonm Auxainm. PloA pcA. B. 13. I'lapn-
iia. A\.. 1961, CTp. 188-190.
C y 6 0 o T n n K C. H., I.IJ n n /i i> C c p r n. H. 3.ncKTpo3iiuc(l>a;iorpa-
(Jiii'jcc.Kiic ncc;ic;ionai!ii)i D.niiiiiinsi Ko.nc6aiiiiii yponnn caxapa, KIIC/IO-
POAH ii yr.nci n .T a TO n a B. M. Tiir. n can. 1962, A» II, CTp. 3—8.
XO^ACII Ji. >i;. C. n ripiicT.Tii AM;. P. B KM.: Awxamie. BIIOMC.V
riis. 1937, 441 crp.
MioioiKon B. A. Tur. n can. 1963, As 6, CT|). 8—15.
A (7. Icr II. u. Lchmann C.. Arbcitsphysiologic. 1932, Bd. 5, H. 6,
' S. 636—681.
B r e in c r P., Thomas I. Action dc 1'anoxcmic, dc 1'hypcrcapnic
ct dc I'acapnic sur I'activilc clcctriquc du cortex cerebral. Sociclc
de Biologic, 1936. CXXI1I, N 36. p. 1256-1261.
- 17 -
-------
HYGIENIC EVALUATION OF POLLUTION OF THE ATMOSPHERE
WITH HEXAMETHYLENEDIAMINE
A. Ye. Kulakov
Institute of General and Communal Hygiene im. A. N. Sysio,
Academy of Medical Sciences, USSR
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 28-41, (1966).
Hexamethylenediamine was first prepared by V. Solonin in 1896. It is
used in modern industry chiefly as an intermediate in the production of HA
or HS salts (hexamethylenediamine adipate or sebacate) from which the
polyamide resin nylon, used as the material for a synthetic fiber of the
same name and for various plastic products, is prepared by poly condensation.
As an intermediate, hexamethylenediamine is used in the production of
poly ure thanes , from which a special rubber and plastics are obtained. As a
hardener, it finds applications in the production of epoxy resins and
certain pigments. Hexamethylenediamine is also used in the production of a
chemical antifoaming agent. In industry , 'hexamethylenediamine is obtained
from adipic acid. According to the data of A. P. Martynova (1957) and
V. S. Filatova et al. (1958), hexamethylenediamine is observed in substantial
concentrations in the air of plants producing nylon and HA salts. Our
attempts to find data on atmospheric pollution with hexamethylenediamine in
the literature have been unsuccessful.
Hexamethylenediamine, H2N(CH2)gNH2 , is a colorless crystalline substance
with an unpleasant odor. The melting point is 42° (41-39°C.), and the boil-
ing point, 204-205°C. It volatilizes readily. The volatility at 65°C. is
22.2 mg/1, and amounts to 1980 mg/1 at 186°C. Hexamethlenediamine dissolves
very well in water, alcochols, acetone, more sparingly in benzene, and poorly
in ether. It is a strong organic base (stronger than ammonia). From air,
hexamethylenediamine strongly absorbs water and carbon dioxide, forming with
the latter a carbonate quite soluble in water. When stored in contact with
air, hexamethylenediamine oxidizes and darkens. It keeps well in aqueous
and alcohol solutions.
'In chemical structure, hexamethylenediamine is similar to such poisons
as cadaverine and putrescine, which are formed during putrefaction of pro-
teins. Its toxic effect has been little strudied, and the available data
pertain. chief ly to the action of high concentrations. The studies of
Hindelbrandt [cited by Curtius, Clemm (1900), Ceresa, Blasiis (1950)],
V. S. Filatova et al. (1951), and A. P. Martynova (1957) have shown that
hexamethylenediamine has a general resorptive action which is manifested in
- 18 -
-------
an impairment of the permeability of fine and medium blood vessels,
various dystrophic and degenerative changes in internal organs (lungs,
liver, kidneys, adrenals, heart muscle, brain, bone marrow, stomach,
and intestine). It has a depressing effect on the central nervous system,
lowers the arterial pressure, and changes the morphological composition
of the blood. The inhalation of hexamethylenediamine causes strong head-
ache (S. I. Vol'fkovich et al., 1959). When deposited on the skin even
in small concentrations, it causes dry and wet necroses. During storage
in open air, the toxic action of hexamethylenediamine gradually disappears.
If humidity reaches the carbonate formed with air, the toxic action on
the skin is reestablished (V. S. Filatova et al., 1951). In low concen-
trations, hexamethylenediamine has a sensitizing effect on human skin,
causing dermatitides and eczemas (Duvernevil, Buisson, 1952; F. P. Odintsova,
'1961; L. P. Tsirkunov, 1963, M. D. Bagnova, 1962). We observed four cases
of such sensitizing action on sensitive persons, three of whom were in con-
tact with hexamethylenediamine for two to four weeks, and one for two days.
Gipstein (1941), Anderson (1950), Rein, Rogin (1950), Themine (1955),
Zawahry (1956), Morris (1960), and M. I. Leonenko (1962) indicate the
ability of certain nylon products to sensitize susceptible people and cause
such dermatitides and eczemas.
However, Fanburg (1940), Dobkevitch,-Baer (1947), Sibney, Jennes
(1951), Calnon, Wilson (1956), and Janson (1959) do not share this opinion.
In their view, dermatitides and eczemas are not caused by nylon, but by
the dyes used in the nylon products, and properly treated nylon does not
have any sensitizing action.
Last et al. (1950) , Schweisheimer (1948) and others successfully used
certain nylon products in surgical practice (catheters, suture material)
and hold that nylon products have no toxic effects. However, Openheimer
et al. (1953) observed a reticulum cell sarcoma around a nylon film which
had been sutured into rats.
The basis of the mechanism of the toxic action of hexamethylenediamine
is the ability of nono- and diamines to "free" bound histamine and possibly
also other biologically active substances (Paton, 1957). This apparently
explains the similarity of the toxic action of hexamethylenediamine and
histamine, as well as the ability of the former to exert a sensitizing
influence on susceptible people.
Taking into account the industrial importance of hexamethylenediamine
and the possibility of contamination of atmospheric air with this compound,
we undertook a study of certain aspects of the biological action of hexa-
methylenediamine, and on this basis attempted to propose its maximum per-
missible concentration in atmospheric air.
- 19 -
-------
.3 C
We determined hexamethylenediamine in air by means of a colorimetric
method based on its reaction with dinitrochlorobenzene, proposed in 1957
by V. I. Kuznetsov, Z. M. Pimenova and A. P. Martynova. The reaction
forms 1.6-bis-aminohexane, which dissolves in chloroform and gives a
yellow color. Hexamethylenediamine is absorbed in cotton wetted with
distilled water. Up to 15 liters of air was collected at the rate of
3 liters per minute. The sensitivity of the method was 0.0025 mg per ml
of solution; ammonia does not interfere when present in amounts up to
5 mg. This method, suitable for the determination of hexamethylenediamine
in industrial quarters, was modified by us under the supervision of
N. G. Polezhayev in order to increase its sensitivity. In the new modifi-
cation, the air was passed through two absorbers with a porous plate No. 1,
20-25 mm in diameter. The absorbing solution used was a 0.1 N solution of
sulfuric acid (10 ml). For the analysis, 300 liters of air was collected
at a rate of 1-2 liters per minute.
The reaction of hexamethylenediamine with dinitrochlorobenzene took
place in alkaline medium with heating for 30 min. in a water bath. After
cooling, the compound formed (1.6-bis-aminohexane) was extracted by agita-
ting in acid medium with 0.5 ml of chloroform for 20 minutes. Using the
intensity of the yellow color of chloroform, the sample was compared with
a standard scale. The sensitivity of the method was 0.0003 mg in the sample.
0.5 ml of ammonia gives a yellow coloration corresponding to 0.0003 mg of
hexame thylenediamine.
In order to establish the threshold of the reflex effect of hexamethyl-
enediamine on people, we carried out the determination of the threshold of
olfactory sensation by using a method recommended by the Committee on Sani-
tary Protection of Atmospheric Air (V. A. Ryazanov, K. A. Bushtuyeva,
Yu. V. Novikov, 1957).
The study was made on 19 people whom an otorhinolaryngologist pronounced
healthy for this type of test. A total of 273 determinations of odor with
concentrations of 0.0110, 0.0074, 0.0041, 0.0032 and 0.0027 mg/m3 were made.
Table 1
Threshold of Olfactory Sensation
of Hexamethylenediamine.
Number of
Subjects
5
12
2
Quantity of
OBservations
69
1C9
35
Concentration of
Hexataethylenediamine, rng/b^
Threshold
0,007-1
0.0011 •
0.0032
Subthreshold
0.00 11
0.0032
0.0027
- 20-—
-------
As is evident from Table 1, the threshold of olfactory sensation
for the most sensitive persons of this group was a concentration of
0.0032 mg/m3. The concentration of 0.0027 mg/m^ was not perceptible
to any of the subjects tested.
As we know, studies made by G. V. Gershuni et al. have shown that
a low intensity factor may give rise in the organism to responses whose
strength does not reach the capacity to involve mechanisms causing
conscious perception, i.e., sensation, but which can be objectively
recorded by means of highly sensitive methods of investigation. One
such method, which we used, consists in studying the light sensitivity
of the eye in the course of dark adaptation during simultaneous inhalation
of low concentrations of chemical substances. This method has been vali-
dated by the studies of several authors. The light sensitivity was deter-
mined by means of an ADM adaptometer in three persons ranging in age from
21 to 26 years and having a smell threshold of 0.0041 mg/m3.
In subject S. (Table 2, Fig. 1), concentrations of 0.0041 and 0.0032
mg/m^ caused a marked depression of the light sensitivity of the eye over
the entire course of the experiment after the gas mixture was supplied.
The 0.0027 mg/m^ concentration caused a much slighter depression, statis-
tically reliable only in the 20th minute. The 0.0017 mg/m3 concentration
was found to be inactive: the curve representing the change of the light
sensitivity of the eye almost coincided with the curve obtained during
inhalation of pure air.
Table 2
Change in the Light Sensitivity of the Eye in the Course of Dark
Adaptation During Inhalation of Different Hexamethy-lenediamina
Concentrations.
(Average values in percent at 15thjninute of experiment).
Subject
T.
S.
N.
S
'erica of
Experi-
ment,
Minutes
20
25
30
40
20
25
30
-10
20
25
30
40
Pura Air
198
2GI
301
320
119
129
M2
150
17.1
201
212
219
Concentrations, mg/m'
O.OO'H
337(1))
38 1 (a)
442(b)
4-l5(a)
85(c)
99(b)
103(b)
I05(c)
10100
17-1(0)
178(0)
200(0)
0,0032
319(b)
405(0)
-109(0) .
49G(a)
9-l(c)
9G(n)
103(b)
107(c)
168(0)
197(0)
21(0)
2M(0)
0.0027
.
293(.i)
335(0)
359(0)
389(0)
105(n)
11-1(0)
127(0)
M3(0)
—
—
—
• — .•
0.0017
201(0)
277(0)
316(0)
353(0)
119(0) .
•125(0)
13-1(0)
142(0)
—
— .
—
—
Note. Confidence factor: a - 95$, b - 99#, c - 99#, 0 - unreliable
- 21 -
-------
minutes
1. Change of the light sensitivity of the eye in subject
T. during inhalation of hexamethylenediatnine.
1 - pure air; 2 - concentration' of 0.0017 mg/m5;
3 - 0.0027 ng/m?; k - 0.0032 rrg/m3; 5 - O.OOkl ng/m3.
In subject T., on the contrary, the 0.0041 and 0.0032 mg/m^ concen-
trations caused a marked reliable increase of the light sensitivity of
the eye at the 20th minute of the experiment. A reliable but slighter
increase in the light sensitivity of the eye at the 20th minute was also
noted at the 0.0027 mg/m^ concentration. The 0.0017 mg/m3 concentration
was found to be inactive.
In the third subject, N. , the 0.0041 mg/m^ concentration at the
20th minute caused a reliable depression -of the light sensitivity of the
eye,- while the 0.0032 mg/m^ concentration proved inactive.
Thus, the threshold of the reflex effect of hexamethylenediamine on
the light sensitivity of the eye for the two subjects corresponded to the
0.0027 mg/m^ concentration, and the subthreshold concentration was 0.0017
- 22 -
-------
In order to establish the lowest subsensorial threshold of the reflex
effect of hexamethylenediamine, we also used the method of study of the
electrical activity of the brain. A modification of this method was used
in the form of reinforcement of the intrinsic rhythm with a flashing light.
This method was first employed by A. D. Semenenko and B. N. Balashev in
hygienic studies (1963).
The subject was placed in a weakly illuminated (1.5 Ix) screened
electroencephalographic chamber. At a distance of 0.7-2 m from his eyes
was placed the flash lamp of a photostimulator that was turned on for
20 seconds every minute, the light intensity being changed every 5 seconds.
The flicker frequency of the light of the flash lamp was close to the sub-
ject's ot-rhythm frequency. In the intervals separating the switching on
of the flickering light, the subject limbered up for 20-25 seconds (move-
ment in the chair), to keep his general muscle tone, then a sound generator
whose frequency varied from 200 to 2000 Hz was turned on for 10 seconds.
During the test, the subject closely followed the variation of the intensity
of the light flashes and sound pitch, which he noted by squeezing a pressure-
sensitive detector held in his hand. The subject could be seen by means of
a mirror through a window in the chamber. The examination lasted 13-14
minutes. During the examination, an electroencephalogram was recorded; for
each lead of the latter, the integrated energy of the brain potentials was
recorded every 5 seconds on separate channels by means of a multichannel
integrator of Balashev's design. The pulses of the pressure-sensitive
gauge, which the experimenter used to follow the responses of the subject
to changes in the photic and acoustic stimuli, were recorded on a separate
channel. An electroencephalogram segment obtained while the flash lamp
was on was subjected to analysis. Each subject underwent a preliminary
training.
The physiological fluctuations over the course of the examination were
insignificant. The cylinder whose contents were breathed by the subject
was continually supplied with pure air. The gas mixture of known concen-
tration was connected to pure air at the 4th minute of the test and was cut
off after 6 minutes. All the switching operations were performed in another
room out of the subject's sight. Data obtained during inhalation of pure
air and a certain hexamethylenediamine concentration were compared in
relative units and subjected to variational statistical treatment. The
examination was made with a 16-channel electroencephalograph of the Galileo
Co. The recording was made from both hemispheres with uni- and bipolar
leads, and statistical treatment was applied to data obtained with occipito-
temporal leads. Four subjects having a smell threshold of 0.0041 mg/m3 par-
ticipated in the experiments. A total of 64 examinations were performed.
Three hexamethylenediamine concentrations were studied: 0.0027, 0.0017
and 0.0011 mg/m3. The 0.0027 mg/m3 concentration caused a decrease in the
energy of the brain potentials in all four subjects (Table 3 and Figs. 2 and
-. 23 -
-------
3). In subject T. the decrease in the energy of brain biocurrents was
noted in both hemispheres, but the most statistically significant changes
were observed in the left hemisphere. In the remaining subjects significant
changes were observed in one hemisphere only.
The 0.0017 mg/m^ concentration caused a decrease in the energy of
brain potentials in only three subjects, and in subject T. reliable changes
also occurred in both hemispheres. On the whole, the decrease in the energy
of brain potentials for the 0.0017 mg/m^ concentration was less pronounced
than for the 0.0027 mg/m^ concentration. The 0.0011 mg/m3 concentration
did not cause significant changes in the electrical activity of the brain
in any of the subjects, although in T. and S. there was noted a tendency
toward a decrease in the energy of biocurrents in the right cerebral hemis-
phere at the 7th-9th minute of the test. However, this decrease did not
reach a statistically significant value.
In our investigations, we were able to subject the data obtained from
the two hemispheres to separate statistical treatments. The experiments
showed that low hexamethylenediamine concentrations caused unequal changes
in the electrical activity in different cerebral hemispheres of one and the
same subject. The 0.0027 mg/nH concentration caused the largest decrease
in the energy of potentials in the left cerebral hemisphere, and the 0.0017
mg/m^ concentration, in the right hemisphere. The unequal effect of hexa-
methylenediamine on the electrical activity of the cerebral hemisphere was
also observed in other subjects.
B •
too
•
o
in '
a
90-
tu
•a
3
O.
a
«<
70-
-.0
~f~
9
10 -U //minutes
Fig. 2. Change in the electrical activity of the brain in subject
T. during inhalation of hexamethylenediamine (right hemisphere).
1 - pure air; 2~- concentration, 0.0011 rag/m'; 3 - 0.0017 mg/m';
k - 0.0027 mg/m5; AB - period of gas supply.
- 24 -
-------
The literature contains indications of the presence of a temporary
interhemispheric asymmetry in some healthy persons under normal conditions
(Ye. A. Zhirmunskaya, 1963). It is possible that the interhemispheric
asymmetries manifest themselves more markedly under the influence of low-
intensity factors.
f/a . •
g 100
30-
SO-
70-
o.
a
../
'.?
// /minutes'
Fig. 3. Change in the electrical activity of the brain in subject
T. during inhalation of hexamethylenediamine (left hemisphere).
Notatioi) same as in Fig. 2.
Thus, the threshold of change in the electrical activity of the brain
in the most sensitive persons during inhalation of hexamethylenediamine
corresponds to a concentration level of 0.0071 mg/m^.
On the basis of the data obtained, we recommend as the highest single
maximum permissible concentration of hexamethylenediamine in atmospheric
air a level of 0.001 mg/m^, which is below the threshold of action according
to the most sensitive method of investigation (Table 4).
We studied the contamination of atmospheric air with hexamethylenediamine
around a plant producing HA salt. In addition to hexamethylenediamine, the
discharges of the plant also contained ammonia, which in high concentrations
interferes with the determination of hexamethylenediamine, and for this
reason we determined both of these substances simultaneously. Ammonia was
considered in the calculation of the hexamethylenediamine concentration.
- 25 -
-------
Table 5
Change in the Energy of Brain Potentials During Inhalation of Different Hexamethylenediamine Concentrations
(Average Values in Percent Relative to the'lst, 2nd and 3rd Minutes of the Experiment).
•Subject
T.. '
s.
M." .
R..
Period of
Experiment,
Minutes
4-6
7-9
10—12
4—6
7—9
10—12
4—6
7-9
10—12
4-6 '
7-9
10—12'
Right Hemisphere
Pure Air,
99
96
' 93
• 95
97
95
102
102
100
101 -
99
95 '
0.0027
mg/m^
98(0)
89(o)
' 80(a)
100(0)
9S(o) •
84(o)
102(0)
103(0)
97(o
8S(a)
82(c
80(b)
Pure Air
94
98
85
93
' 106 '
92
105
108
107 •
101
94
93
0.0017
mg/m3
88(0)
84(c)
72(o)
92(o)
S9(a)
84(o)
100(o)
100(0)
95(a)
97(o)
95(o)
94(o)
o.boii
mg/m3
99(o)
9l(o)
90(o)
96(o)
98(o)
95(o)
102(o)
106(o)
10l'(o)
93(o)
. 90(o)
85(o)
Left Hemisphere
Pure Air
104 '
98
95
107
112
107 •
106
114 ;
105 •
' 104 .
' 102
99
0.0027
mg/m3
86(a)
78(a)
76(a)
100(o)
• 93(c)
89(a)
97(a)
100(b)
96(o)
99(o)
95(o)
94(0)
Pure Air
94
99
87
j 98
97
85
103
100
. 104
il04
101-
102
0,00!7
Eg/m^
93(0)
86(a)
76(o)
101(o)
92(o)
86(0)
9S(o)
95(o)
94(o)
106(0)
104(o)
99(o)
0.001]
mg/nP
97(o)
94(o)
90(o)
9S(o)
95(o)
92(o)
98(o)
96(o)
96(o)
102(o)
101(o)
100(o)
Note. Confidence factor: a - 99£i b - 99%, c - 99%, o - unreliable.
-------
A'
Table k
Thresholds of the reflex effect of
hexamethylenediamine on the human organism
during inhalation
Threshold
"~ ~
Olfactory sensitivity
Effort on _ the light
sensitivity of the eye
_ Effect on the electrical
activity of the brain
loncentration in rag/i
Minimum
Active
0,0032
0.0027
0,0017
Maximum
Inactive
0,0027
0,0017
0,0011
The collection of samples was started at a distance of 300 m on the leeward
side of the plant, since at a closer distance the odor of hexamethylenedia-
mine was clearly perceptible.
The results of the study of pollution of atmospheric air are shown
in Table 5.
Table 5
Contamination of Atmospheric Air with Hexamethylenediamine
around an HA Plant
Distance
From
Source, m
300
500
Number of
Samples
Total
2.1
56
Posi-
tive
15
I
Concentration,
rng/m'
Maximum
0.012
—
Aver-
age
o.ooi'i
0,0010
Distribution of _
Concentrations, rag/m?
Above
0.01
1
—
O.OI-
0.006
3
—
0.005-
0.002
7
—
0.001
and
below
4
1
The atmospheric air around the plant at a distance of 300 m is con-
taminated with hexamethylenediamine in concentrations several times greater
than the highest single maximum permissible concentration that we are pro-
posing. At a distance of 500 m, of the 56 collected samples, hexamethylene-
diamine was observed in only one, and only in the amount of 0.001 mg/m^.
Since other chemical compounds are also discharged by the HA plant
into the atmospheric air, the problem of the width of the sanitary protective
zone can be solved only on the basis of a hygienic evaluation of the entire
group of chemical substances discharged into the atmosphere.
-.-.27 -
-------
Conclusions
1. The threshold of olfactory perception of hexamethylenediamine
in the most sensitive persons is 0.0032 mg/m3. The 0.0027 mg/m3 concen-
tration is imperceptible.
2. The threshold of the reflex effect of hexamethylenediamine on
the light sensitivity of the eye is 0.0027 mg/m3. The 0.0017 mg/m3 con-
centration did not cause any significant changes.
3. The threshold of the reflex effect of hexamethylenediamine on
the electrical activity of the brain corresponds to 0.0017 mg/m3. The
0.0011 mg/m3 concentration is the subthreshold level.
4. On the basis of the data obtained, a highest single maximum
permissible concentration of hexamethylenediamine of 0.001 mg/m3 is
proposed.
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S c h w e i s h c i ni c r \V. Rayon Textile Atonlhly. 1948. v. 29. N. 2,
-p. 79. . . .
Pa ton W. Pharinacol. Rev.. 1957, v. 9,-N. 2. p. 269-328. .
Sidney W., Jcnnes M. Mcd. Surg., 1951, v. 20, N. 6. p. 272.
The mi me P. Bull. Soc. franc. Derm. Syph., 1955, t. 62, N. 4. p. 452.
Zawahr.y M. U. Brit. J. Derm., 1956, v. 63, N. 2, p. 59-60.
- 29--
-------
CONTAMINATION OF ATMOSPHERIC AIR WITH MALEIC ANHYDRIDE
AND ITS HYGIENIC EVALUATION
K. V. Grigor'yeva
Ukrainian Scientific Research Institute of Communal Hygiene
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 42-57, (1966).
Maleic anhydride, C^j^O-j, is a white crystalline powder with a fairly
pungent odor, boils at 202°C. and sublimes. It is used as the raw material
for the preparation of valuable lacquer-paints, and plasticizers in the
production of artificial resins.
Maleic anhydride is obtained from the oxidation of benzene, furfural,
or any other hydrocarbons of the paraffin and olefin series with more than
four carbon atoms. The method of preparation of maleic anhydride from waste
products of phthalic anhydride formed by catalytic oxidation of naphthalene
by atmospheric oxygen at high temperature- has come into widespread use.
In this process, 7-15% of maleic anhydride is formed. The mixture of vapors
of phthalic and maleic anhydrides, naphthoquinone , carbon monoxide and water
is fed into special units for condensation of phthalic anhydride on cooling.
The uncondensed vapor of all these substances form the tail gases, which
are then fed into a scrubber. Maleic anhydride is obtained from the wash
waters by evaporation.
The residual gases after the scrubber, consisting of vapors and aerosols
of maleic and phthalic anhydrides and naphthoquinone, are discharged into
the atmosphere.
The toxic properties of maleic anhydride have been described in very
few papers. Acute poisoning is characterized by an irritating effect on
the mucous membranes of the eye and upper respiratory tract (N. V. Lazarev,
1954).
Menschick (1955) studied the health of workers over the course of
20 months since the start of operation of equipment for synthesizing phthalic
anhydride. The air of the plant was contaminated with a number of substances
including phthalic and maleic anhydrides, naphthoquinone, and carbon monoxide.
The author found a significant decline in the health of the workers, manifested
in disorders of the nervous system (fits of excitation, tetanic state, sen-
sitivity impairments in the area of the trigeminal nerve) , hypersecretory
excitation of the stomach, increased permeability of the stomach wall and
- 30 -
-------
vessel walls, anginal pains, decrease of systolic and diastolic pressures
(of central origin), irritation of the mucous membrane of the eye (con-
junctivitis), blood effusion in the nasal cellular tissue up to and includ-
ing nasal hemorrhages, and ulcerations of the mucosa of the larynx, pharynx,
trachea, and bronchi, blood changes (normochromic anemias, leucocytoses,
thrombopenia), a sharp decrease in the content of vitamin C in the organism,
and impairment of the phosphorus and calcium metabolism.
Marsico and Rozero (1957) cite nine cases of acute poisoning with
maleic anhydride in loaders during its loading. The symptoms of poisoning
are characterized primarily by the irritant action of the poison: an acute
irritation of the mucous membranes of the eye, nose, and upper respiratory
tract.
Angielski and Rogulski (1960) describe the aminoaciduria and decrease
of protein and protein-free SH groups in the kidneys of animals which had
received maleic anhydride in subcutaneous injections in doses of 250-300
mg/kg.
M. P. Slyusar' and I. A. Cherkasov (1961), while administering maleic
anhydride to experimental animals by inhalation, found a considerable
weight decrease, a drop in vitamin C content of the tissues, and an increase
of total protein in the blood serum. The-authors observed the earliest and
most substantial changes in a study of the phagocytic activity of neutro-
philes.
These few studies show that maleic anhydride is a toxic substance.
However, there are no data on its content in atmospheric air, the effect
of low concentrations on man has not been studied, and no maximum permis-
sible concentration has been established for the atmospheric air of popu-
lated areas.
Considering these facts and the necessity of expanding the production
of maleic and phthalic anhydride for the national economy, the Committee
on Sanitary Protection of Atmospheric Air charged the Ukrainian Institute
of Communal Hygiene with the problem of investigating the contamination of
the atmosphere with maleic anhydride in the surroundings of a chemical
plant and with submitting a hygienic evaluation of the values obtained.
The study of the atmosphere was made by using a polarographic method
developed by I. B. Kogan (1958) and slightly modified by us. Maleic
anhydride was trapped from air on an FPP-15 filter, then in two sequentially
connected absorbing containers filled with 10 ml of a 0.1 N solution of
hydrochloric acid. The polarography was carried out after dissolving the
maleic anhydride collected on the filter in the liquid from the first
absorber. The half-wave potential of maleic anhydride is 0.69. The sensi-
tivity of the method is 1 mg per ml. The accuracy of the method is ^10-15%.
- 31 -
-------
Phthalic anhydride, benzole acid and a-naphthoquinone do not interfere
with the determination. The atmospheric air around the plant complex
was collected at distances of 500, 1000, and 2000 m from it. Two series
of samplings were carried out. The results of the study are listed in
Table 1.
The highest maleic anhydride concentrations were found at a distance
of 500 m from the plant, while at the distance of 2000 m the maleic
anhydride content in all 28 samples was below the sensitivity limit of the
method.
The second stage of the study consisted in an experimental validation
of the maximum permissible concentration of maleic anhydride in atmospheric
air. This was done by using the methods recommended by the Committee on
Sanitary Protection of Atmospheric Air (V. A. Ryazanov, K. A. Bushtuyeva,
and Yu. V. Novikov, 1957).
An experimental mixture of vapors and aerosols of maleic anhydride
was obtained by heating it in distillation flasks on a water bath at 37°C.
To solve the problem of the highest single maximum permissible concen-
tration of maleic anhydride, the threshold of smell and its irritating
effect on the upper respiratory tract as well as the threshold of the
reflex effect on the functional state of the cerebral cortex were determined.
To determine the threshold of olfactory sensation and irritating' effect,
753 determinations were made with the 12 subjects. The results obtained
are given in Table 2.
Table 1
Content of Maleic Anhydride in Air Samples Collected
in the Area of the Chemical Complex
Distance .
From Sounce
. of :
Discharges, m
Number
of Tests
Number of
Positive
Samples''
Concentration, mg/m5 .
Maximum . .
Average
First Serites
1000
2ono
16
9
2,6
0,29
•- . ' . Second Series.
• '500 •
1 000
2000
78
37
19
29
11
• . —
. 0,06
0.015
0,007
.0,001-
During the inhalation of maleic anhydride in concentrations from 8 to
11 mg/m^, a strong reflex cough, buring in the laryngeal part of the pharynx,
and lachrymation in the majority of subjects, were observed.
- 32 -
-------
A concentration from 5 to 7 mg/m^ causes the perception of odor and
irritation of the upper respiratory tract.
Maleic anhydride in concentrations from 2 to 3 mg/m-' has an insignifi-
cant odor and a slight irritating effect.
Table 2
Thresholds of Perception of Odor and Irritating Effect
of Maleic Anhydride.
Number
nf
Subjects
r
2
1
1
- 1
1 '
5
Concentration, mg/ni3
Smell
Minimum
Perceptible
1,7 •
,6
.5
• ,4
.4
,4
,3
Imper-
ceptible
,6
,5
,4
,3
,3
,3
.2
Irritating Effect •
Minimum
Perceptible
.5
' .2
,1
,4
,1
.0
,0
Imper-
ceptible
1.4
1.1
1.0
1,2
1.0
0,9
0.9
A concentration of 1.3 mg/m^ is the odor threshold for the most
sensitive persons. Symptoms of the irritating action are of major sig-
nificance here. The subjects note dryness and tickling in the lower part of
the pharynx. The threshold concentration for the irritating effect was found
to be 1 mg/m^. It gives a sensation of freshness and a barely perceptible
tickling.
The next stage of the study was the determination of the influence of
low maleic anhydride concentrations on the reflex change of the light sen-
sitivity of the eye.
A number of authors (F. I. Dubrovskaya, K. A. Bushtuyeva, M. T. Takhirov,
N. F. Izmerov, G. I. Solomin, and others) have demonstrated that this method
permits the recording of the change in the cerebral cortex caused by stimu-
lation of the reflexogenic zones of respiratory organs by slight concentra-
tions of chemical substances. The light sensitivity of the eye was studied
with the aid of an ADM adaptometer. The threshold of the irritating effect
of maleic anhydride was first determined in three subjects. The course of
dark adaptation was studied while supplying pure air and a gaseous mixture
containing 6.2, 1.65 and 0.85 mg/m-^ of maleic anhydride. Each concentration
was studied no fewer than three times. A total of 78 determinations were
made. The results of the examination are given in Table 3.
The 6.2 and 1.65 mg/m^ concentrations caused a reliable change of the
light sensitivity of the eye in all the subjects during the 20th minute in
comparison with pure air. The 0.85 mg/m3 concentration was found to be
inactive.
- 33 -
-------
Table 3
I
to
Average Values of the Lighjt Sensitivity of the Eye in Relative Units
During Inhalation of Different Maleic Anhydride Concentrations.
Subject
M.
K. '
T.
15th Minute
Pure
Air
58500
81250
51020
Maleic Anhydride
0.85
64700
66466
48400
1.65
54766
62760
57 966
6.2
59200
59550
—
•- 20th Minute
Pure
Air
73380
94550
61360
"1'Sleic Anhydride
0.85
75433
60633
60433
1.65
53500 (a)
67 620 (a)
113333(a)
6.2
55900 (a)
72 850 (c)
—
25th Minute
Pure
Air
82220
104450
77 180
Maleic Anhydride
0.85.
83800
95 166
73333
1.65
63566
75380 (a)
120566
6.2
81 100
74 350 (c)
—
Note. Confidence factor: a - 99#, b - 99$, c - 99.956.
-------
y.
' no-
ii o-
100-
so-
to-
£ 10-
w-
JO-
20-
W
W 15 20
30 Tine'
Fig. 1. Effect of maleic anhydride on the.
light sensitivity of the eye in subject T.
1 - pure air; 2 - concentration, 0.85 mg/rn^
3 - 1.65 mg/m3;
in
I
too-
so
so
w-
60-
so-
30-
10
Fig
1 -
10
15
20
/ IU I) W Si . -
-------
Dark adaptation curves for two subjects illustrate the influence of
maleic anhydride on the light sensitivity of the eye (Figs. 1, 2).
Thus, the threshold of the effect of maleic anhydride on the light
sensitivity of the eye is 1.65 mg/m3, and 0.85 mg/m3 is a subthreshold
concentration. Assuming a certain storage coefficient, one can recommend
0.5 mg/m3 as the highest single maximum permissible concentration of maleic
anhydride.
To study the effect of low maleic anhydride concentrations under
conditions of an extended, continuous experiment on laboratory animals,
a round-the-clock exposure of white rats was conducted for 70 days.
The experiments involved 50 male rats weighing 90 to 120 g. The
exposure was carried out in 5 chambers containing 10 rats each.
The first group of rats was subjected to the action of maleic
anhydride in 0.03 mg/m3 concentration, the second to 0.08 mg/m3, the
third to 0.34 mg/m3, the fourth to 0.8 mg/m3, and the fifth was the con-
trol group. The exposure was made in two series: 1) with 0.08, 0.34, and
0.8 mg/m3 concentrations; 2) with 0.03 mg/m3 concentration. Each series
had its own control. The effect of maleic anhydride in different concen-
trations is described without separation into series.
During the course of the exposure, air samples were taken from the
chambers almost daily. The concentrations in the chambers varied between
the following limits: in the first group, from traces to 0.042 mg/m3, in
the second from traces to 0.2 mg/m3, in the third from traces to 0.78 mg/m3,
and in the fourth from 0.3 to 1.56 mg/m3.
The fluctuations of the concentrations are shown in Fig. 3.
Table 4
Weight of Animals Subjected to Chronic Action of Maleic Anhydride.
Avfwrage
Y.'eight of
Animal
Before tfee
start of
exposure
During
exposure
Group and Concen-
tration in mg/ra'3
Control
First (0.03)
• Control i
Second (0.08)
Third (0.34)
Fourth (0.8)
Control
. First (0.03)
Control
Second (0.08)
Third (0.54)
Fourth (0.08)
M ± m
158. 2*12. 8
160,6 + 5,6
229. -1 ±8
212,8+9,7
•216.7 + 8,8
211,5*10,6
212 + 8,0
213 + 5,8
250,0+3.74
2'I5.2±
-------
mg/m
0.2-
'o.i-
3
A A /^ /I -.
x_/v^v v sr
To evaluate the toxic effect of maleic anhydride, the general con-
dition of the animals, their weight, morphological composition of the
blood, content of SH groups in the blood serum and phagocytic activity
of neutrophiles were followed during the experiment. At the end of the
exposure, anatomic pathological analyses of the animals' organs were
made.
0,0! mg/m3
0 10 20. 30 i/0 SO 63 70days
Fig. 3. Fluctuations of maleic anhydridb
concentrations in the second group.,
In the course of the experiment, maleic anhydride showed a substan-
tial influence on the overall condition and weight dynamics of animals
of the third group (0.34 mg/m^) and particularly the fourth group (0.8
mg/m3). In the second (0.08 mg/m^) and first (0.03 mg/m3) groups, no
changes were observed. Since the first days of contact with the substance,
the animals of the fourth group were agitated, constantly sniffed, sneezed,
and rubbed their noses with their paws. A few days later the rats calmed
down and were sluggish, failing to become animated even during feeding.
They ate less than the animals in the other groups.
The weight change in animals of the third and fourth groups was
particularly pronounced during the period of experimental starving.
After 5 days of underfeeding (the rats received 1/5 of the usual ration
during this period) they lost more weight than the others, and the restor-
ation of their weight was subsequently less rapid (Figs. 4, 5, Table 4).
Grams
JOO-
m-
2SO-
??o-
200-
'W-
160-
ItiO-
120
Fig. 4. Average weight of rats of the
second, tt)ird, fourth and control groups
of the experiment during the period of
exposure.
1 - control group; 2 - second group ,
(0.08 mg/m3); 3 - third group (0.34 mg/nr);
k - fourth group (0.8 mg/m'); AB - experi-
mental starving.
Exposure
- 37 -
-------
Grams
• no-
2?0-
200-
IJ0-
ieo-
x-'
A ff.
Fig. 5. Average weight of rats of the
first and control groups during the period
of exposure.
1 - control group; 2 - first group (0.03 mg
AB - experimental starving.
Exposure
Observation of the state of the morphological composition of the
blood did not show any significant changes in the amount of hemoglobin
in animals of any groups. The number of erythrocytes decreased signifi-
cantly in animals of the fourth group (0.8 mg/m^).
20000-
16000-
11000-
1000
t/000-
2000
Background Exposure
Fig. 6. Average number of leucocytes in rats
of the second, third, fourth and control
groups during the period of exposure.
Notation same as in Fig. A.
As far as the number of leucocytes is concerned, stable leucocytosis
was observed in animals of the third and fourth groups during the period
of exposure (Fig. 7).
No such effect was observed in rats of the second group (concentration
0.08 mg/m3), but they showed an unstable leucocytic reaction. This was
particularly apparent after ten days' starving.
- 38 -
-------
MOO-
12000-
1000-
4000-
2000-
Background Exposure
Fig. 7. Average number of leucocytes in
rats of the first and control groups during
the period of exposure.
Notation same as in Fig. 5.
In rats of the first (0.03 mg/m^) and control groups, the number of
leucocytes remained unchanged over the course of the experiment (Table 5,
Figs. 6, 7).
One of the indices characterizing the influence of external factors
on the organism is the change of its immunobiological reactivity, in
particular, the phagocytic activity of the neutrophiles. Recently, this
test has begun to be used for the validation of maximum permissible con-
centrations of noxious chemicals (M. P. Slyusar' and I. A. Cherkasov, 1961)
Table 5
Number of Leucocytes in White Rats Subjected to
Chronic Action of Maleic Anhydride.
Average ••
Number of .
Leucocytes
Prior to
the start
of
exposure
During^
Exposure
Group. and. Concen-. .
tration in mg/m?
Control
Firsf (0.05)
Control
Secorid (0.08)
Third (0.54)
Fourth (0.8)
Control
First (0.03)
Control
Second (0.08)
Third (0.34)
Fourth (O.e)
M±m
13 150±IOOO
11 727±518
12320 d-616
12 150+5-11
12000-1-3-16
10C90 + 8-1-1
1 4 049 + 803 ,
14204 + 335
12960 + 516
16120+935
18465 ±873
20033 + 9S8
t
Confidence
Factor
i
1,26
0,1
0,3
1,5
0,1
2,9
5,4
6,2
L
99%
99,9°/o
99,9°i
We determined the opsono-phagocytic numbers (number of microbes
absorbed by one leucocyte) and the opsono-phagocytic activity of leuco-
.cytes (percent ratio of active neutrophiles to the total number of counted
neotrophiles) (Table 6).
As is evident from Table 6, the phagocytic activity of leucocytes in
animals of the third and fourth groups (0.34 and 0.8 mg/m3) decreased.
- 39 -
-------
In rats of the first and second group (0.03 and 0.08 mg/nH) , the
phagocytic activity of leucocytes did not differ in any way from the
control.
According to our data, the opsono-phagocytic numbers in animals of
all the groups remained unchanged during the period of the chronic
experiment.
Protein macromolecules carry on their surface a large number of polar
groups including mercapto groups. When toxic substances enter the organ-
ism, they react with these groups. Blocking of an SH group decreases the
protein activity. At the same time, the SH groups have a considerable
influence on the activity of the enzymes participating in protein, fat,
and carbohydrate metabolism. This in turn indicates the great influence
of a given functional group of proteins on the generation, intensity and
direction of the various physiological processes (B. N. Gol'dshteyn, 1955).
Table 6
Opsono-phagocytic Activity of Leucocytes in White Rats
Subjected to Chronic Action of Maleic Anhydride.
Average Activity
of Leucocytes
Before the start
of exposure
During
.'exposure
-
Group and Concen-
tration'' in mg/m5_
' Control
First (0.03)
Control*
Second (0.08)
Third (0.3A)
Fourth (0.8)
Control
First (0.03)
Control
Second (0.08)
Third (0.3*0
Fourth (0.8)
M ± in
75,6 + 4,3
73,5+1,9
82,0-1-1,3
79,4±2,8
8-1,
-------
no appreciable changes of any kind could be demonstrated in the content
of active SH groups as compared to the control group, so that in a repeat
experiment with the lowest concentration, 0.03 mg/m^ (first group), this
test was not employed. Blocking of the SH groups occurred only in animals
of the fourth group (0.8 mg/m3) .
Table 7
Change in the Height of Polarographic Waves of Protein-Free
Filtrate' of Blood Serum in Rats Subjected to the Chronic
Action of Maleic Anhydride.
Average height-, of broup and Concen-
polarographib waves pration in mg/m*
1
.During
exposure
Control
First (0.08)
Third (0.54)
Fourth (0.8)
/'/ ± m
7.2 + 0,16
7,5±0,19
7,4 + 0,14
5,3±0,18
/
1.2
1.2
1,9
7,9
Confi-
dence
Factor
99,976
In animals of the fourth group (0.8 mg/m^), histomorphological
analyses made after completion of the exposure showed a vague atrophy
of the nasal mucosa and inflammatory changes in the lungs, primarily
around the medium and fine bronchi. In the remaining groups as compared
to the control, no appreciable changes could be established.
Thus, continuous chronic exposure of white rats to maleic anhydride
for 70 days has shown that the 0.08 mg/m^ concentration has a marked
effect on the experimental animals, causing a weight lag, a decrease in
the number of erythrocytes and neutrophile activity, an increase in the
number of leucocytes, a decrease in the activity of SH groups of blood
serum proteins, and inflammatory changes in the lungs. The 0.34 mg/m3
concentration showed a lesser effect on the animals during the same period
of time. Only a weight lag, a decrease in the phagocytic activity of
neutrophiles and an increase in the number of leucocytes were observed.
The 0.08 mg/m3 concentration caused only an unstable leucocytic reaction
in rats of this group. Maleic anhydride in 0.03 mg/m3 concentration
caused no changes of any kind in the experimental animals during the course
of exposure.
Comparison of the experimental data with indices of the content of
the substance, detected in the surroundings of the chemical complex under
consideration during the first series of studies, showed maleic anhydride
to be present in amounts substantially exceeding the single maximum per-
missible concentration of maleic anhydride which we have proposed. This
•was noted during a period when for all practical purposes the purification
equipment was not operating. High-efficiency scrubbers with a degree of
purification of 98-99% (M. Kh. Tsipenyuk and Yu. Ye. Tsipenyuk, 1963) have
now been installed, which purify the discharge gases of the phthalic pro-
duction, and the highest maximum single concentrations of maleic anhydride
- 41 -
-------
at a distance of 500 m from the source of discharge do not exceed our
recommended maximum permissible value of 0.5 mg/m3.
Conclusions
1. The threshold of smell of maleic anhydride in the most sensitive
persons is 1.3 mg/m3, and the threshold of irritating action is 1 mg/m3.
The imperceptible concentration for these studies was determined at
0.9 mg/m3.
2. The sub threshold concentration for the reflex effect on the
cerebral cortex was found to be 0.85 mg/m3.
3. The highest single maximum permissible concentration of maleic
anhydride in atmospheric air which can be recommended is 0.5 mg/m3.
4. In a round-the-clock exposure for 70 days, maleic anhydride in
0.8 mg/m3 concentration causes the following changes in white rats:
decrease of the number of erythrocytes and activity of the neutrophiles,
increase in the number of leucocytes, decrease in the activity of SH
groups of blood serum proteins, weight lag, and inflammatory changes in
the lungs. A 0.34 mg/m3 concentration causes a decrease in the activity
of the neutrophiles, an increase in the number of leucocytes, and a weight
lag in the animals. A concentration of 0.08 mg/m3 of maleic anhydride is
the threshold value for its toxic effect on the organism of warm blooded
animals, and a concentration of 0.03 mg/m3 is the sub threshold value.
5. The average daily maximum permissible concentration of maleic
anhydride in atmospheric air which can be recommended is 0.05 mg/m3.
=- 42 -
-------
LITERATURE CITED
A ii a T o it c K a n B C 1'nnicna rpy;ia n npotpsaGo-ncBamtfl, 1961, A1? 5,
CT|). 37—41.
F o n bflm TC ii M 13. H. O njiiiuimii cy.ii.cjJni.ipiMbiiux rpynn na Cno-
Jiorii'iccKiic cnoiicTiia TKaiiciiux 6e/iKou. K.MCB. 1955.
Koran M; B. Hir. n can., I95S, N° 7.
Jl a 3 a p c n H. B. Bpc,ain.ic uemccTua D upoMUUJJiciiiiocTH. M. I. foe-
xiiMii3AaT. Jl., 1954.
P n 3 a n o D B. A. Caiiiiiapnan oxpana aiMoctpcpnoro uo3,T.yxa. M.,
1958.
P n 3 a n o 11 B. A., B y iu T y e is a K. A., H o B n K o n K). B. K MCTO-
AIIKC SKcncpiiMCiiTa^LiMoro oOociiODaniisi npcjic.Tbiio AonycTis.Moii KOII-
UCiiTpamiii aTMOC(j)Cpnbix 3arpn3iiciiiifi. B KII.: ripc.ic.Tbiio Aoiiycrii-
MWC aTMOC(])epiihie 3arpn3iieiiiin. M., 1957, D. 3, crp. 117—151.
C^iocapb M. PI. n Ll e p K a c o D H A. K uonpocy o TOKCIIMCCKOU
xapaKTepiicTiiKe ii riiniciiiiMccKo.M iiopMitponaiiini Ma.nemiOBoro an-
rn;ipii;va. Marcpna/ibi naymion ceccnn no TOKciiKO.iornn BUCOKOMO.IC-
Kyjisipnbix cocAnnciinfi, 1961.
C o A o M n ii f. H. rMriienn'iecKafi oueiiKa flnnn.na KBK 3arpH3niiTC.in
aTMoc(J)cpnoro BOSAyxa. B KII.: npCAC.ibiio AonycTii.Mbie Konuenipa-
mni 3TMOc(pcpnbix aarpnaiieiiiinT A1., 1962, D. 6, crp. 146—163.
LJ, n n e n 10 K M. X., U. n n e n 10 K 10. E. Fur. n can., 1963, As I.
crp. 66.
JI n o B c K n ii R. H. Kapinna Kponn n ec K.nnnii'iccKoc siiauciicc. foe-
Mcana/iar VCCP, KHCD, 1957.
Angielski J., Rogulski. Acta biochim. polon., 1960, v. 8,
N. 2—3, p. 269.
Marsico F., Rozero G. Folia med.. 1957, N. 4.
M c n s c h i c k H. Arch. Gewerbepathologic und Gewerbehygicne.,
1955, N. 13.
- 4-3 -
-------
BIOLOGICAL EFFECT OF LOW NITROBENZENE CONCENTRATIONS
IN ATMOSPHERIC AIR
N. G. Andreyeshcheva
Communal Hygiene Department, Central Institute
for Advanced Training 'of Physicians
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 58-73, (1966).
Nitrobenzene, CgH5N02 , is the simplest nitro compound of the aromatic
series. It is a pale yellow liquid with a bitter almond odor. The boiling
point is 210. 8°C. , the specific gravity 1.21, and the melting point 5.7°C.
Nitrobenzene dissolves readily in fats, alcohol, and ether and is insoluble
in acids and alkalis. It is prepared by nitrating benzene.
Nitrobenzene is widely employed in aniline dye plants in the production
of artificial aniline dyes, explosives, drugs, etc. During the current
seven-year plan (1959-1965), the output of aniline dyes will double, thereby
sharply increasing the production and consumption of nitrobenzene.
The toxic properties of nitrobenzene as a result of the action of
large doses on the organism are marked (V. K. Navrotskiy, 1955; K. I. Morozova,
1934; Laubender, Schwamb, 1951; Goldstein, Popovici, 1958; P. Sulamit, 1960,
1962).
The authors administered nitrobenzene chiefly subcutaneously and
perorally, most frequently once. Its effect on the blood hemoglobin is the
most pronounced. It involves the formation of methemoglobinemia followed
by sulfhemoglobinemia; the activity of the blood catalase and peroxidase
decreases, Heinz bodies are formed intensively, and the number of erythrocytes
and reticulocytes decreases.
The chronic effect of nitrobenzene in small doses, chiefly on the
secretory function of the stomach and partly on the blood, was studied only
with peroral introduction by M. I. Kazakova (1954) and G. A. Kalashnikova
(1955).
The lack of data on the toxicity of small doses of nitrobenzene during
long-term inhalational penetration into the organixm or data on its contam-
ination of atmospheric air has led to a study of the biological effect of
low nitrobenzene concentrations and of its hygienic importance as an air
pollutant.
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The experimental part of the study was carried out by using a method
of determination of low nitrobenzene concentrations in atmospheric air
proposed by M. V. Alekseyeva. The method is based on the absorption of
nitrobenzene by a nitrating mixture. This forms dinitrobenzene, which is
determined from the purple color formed by its reaction with a ketone or
alkali. The sensitivity of the method is 0.25 mg in a volume of 2 ml.
We selected the optimum conditions for complete nitration of low
nitrobenzene concentrations (50 minutes in a boiling water bath). The
nitrobenzene concentrations were measured in a photoelectrocolorimeter
with a green filter or a spectrophotometer at a wavelength of 560-570 my
after first recording the calibration curves.
We started the validation of the highest single maximum permissible
concentration of nitrobenzene in atmospheric air, in accordance with the
recommendation of the Committee on Sanitary Protection of Atmospheric Air,
from the determination of the threshold of olfactory sensation. The exper-
iment involved the participation of 29 practically healthy people aged 17-35,
Table 1
Results of Determination of Olfactory Sensation Threshold
of Nitrobenzene.
Number of Subjects
4
2
7 '
8
4
1
1
1
1
Nitrobenzene Concentration, ms/m^ .
Threshold
0,0182
0,023
0,027
0,031
0,037
0,0-15
0,057
0,070
0,09-1
Subthreshold
0,0169
0,018
0,020
0,024
0.028
0,032
0,011
0,055
• 0,069
The olfactory sensation was tested once every 24 hours. A total of
1252 determinations were made, and it was found that in the most sensitive
persons, the minimum perceptible concentration of nitrobenzene was
0.0182 mg/m3 (Table 1).
'In the works of several authors (K. A. Bushtuyeva, 1957; F. I. Dubrov-
skaya, 1957; M. M. Plotnikova, 1957, and others), the dark adaptation method
was used for studying the threshold of reflex action. We used this method
in three cases. The smell of subthreshold concentrations of nitrobenzene
affected the light sensitivity of the visual system in all three subjects.
Results of the experiment are listed in Table 2.
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Table 2
Results of Determination of the Change in the Light Sensitivity
of the Eye During Inhalation of Nitrobenzene.
Subject
S. ,
G.
V.
Concentration, mg/m5
Light Sensitivity
Threshold
0,0157
0,0167
. 0,0168
Subthreshold
0,0118
0,01-1 '1
0, 01-14
Olfactory Sensation
Threshold '
' 0,0182
0,0182
'0,0230
Subthreshold
0,0169
0,0169
0,0190
In one of the three subjects, a nitrobenzene concentration of
0.0157 mg/m^ caused a reliable decrease of the light sensitivity of the
eye which may be considered the threshold sensitivity in this method
(Fig. 1). The method of dark adaptation made it possible to reveal the
effect of Subthreshold nitrobenzene concentrations for olfactory sensation
on the functional activity of the central nervous system. Electroencephal-
ographic studies were also made to determine the reflex effect of nitro-
benzene.
In hygienic practice, the study of brain biocurrents was started by
using the method of development of the electrocortical conditioned reflex
(K. A. Bushtuyeva, Ye. F. Polezhayev, and A. D. Semenenko, 1960; V. A. Gof-
mekler, 1961, and others).
In subsequent studies use was also made of the method of functional
loading with calculation of the amplitude of the assimilated rhythm to
light stimulation (A. D. Semenenko, 1963; N. B. Imasheva, 1963; V. I. Fila-
tova, 1962, and others).
We used the electroencephalographic method of reinforcement of
intrinsic brain potentials under conditions of functional loading with
flickering light of varying intensity, proposed by A. D. Semenenko. The
method is based on the ability of the central nervous system to produce a
reinforcement of the intrinsic potentials in response to the action of a
rhythmic stimulus.
The observations were made on six practically healthy people 20-35
years, of age who had not previously been in contact with the chemical
substances.
The electrical activity of the brain was recorded on a Kaiser 8-channel
electroencephalograph with a photostimulator. The biocurrents were taken
off the temporal and occipital areas uni- and bipolarly. Simultaneously,
the respiration and a myogram were recorded.
- 46 -
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S W IS 20 ?5 JO tO. _
Time of examination, minutes
Fig. 1. Change irv;the light ..sensitivity
of the eye during inhalation of nitrobenzene
in subject S.
1 - pure air; 2 - concentration, 0.0118
ng/ra?; 5 - 0.0157 rag/in3; k - 0.0169 mg/ra?
Stimulation by rhythmic light with a frequency equal to that of the
intrinsic potentials of the brain (10-12 seconds) and with different
intensities was carried out every 2 minutes for 45 seconds. During this
period, the intensity of the flash of the photostimulator lamp was changed
9 times.
One of the channels of the instrument was connected to a Balashov
integrator, which made it possible to obtain the total energy output in
the form of 9 spikes measured in millimeters during processing of the
cu rve.
• After establishing the background (obtaining curves during inhalation
of pure air), nitrobenzene was supplied in concentrations below the value
of the threshold of smell. The inhalation of the gas lasted 6 minutes and
was carried out from the 8-th to the 14-th minute of the experiment. The
first 6 minutes of the recording on the day of gas inhalation constituted
the background for the given day of the examination. After disconnecting
the gas, the recording of the curves was continued in the same sequence up
- 47 -
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to the 24-26th minute of the experiment, until a tendency to restore the
initial level of intrinsic rhythm appeared. The experiments with inhal-
ation of nitrobenzene were alternated with the inhalation of pure air.
Concentrations of 0.0129 and of 0.008 mg/m^ were investigated. The
0.0129 mg/m3 nitrobenzene concentration was found to be active in five
subjects, while the 0.008 mg/m^ concentration was the subthreshold value
for them. One subject was found to be less sensitive (Table 3).
Table -3
Results of Study of Reflex Effect of Nitrobenzene
by Functional Electroencephalography.
Subject
•
Kh. -
G.
I.
L.
A.
Ya.'
Corfcentration; mg/ra^
El'eatroencephalography
Threshold
0,0129
0,0129
0,0129
0,0129
—
0,0129
Sub-
threshold
0,008
0,008
0,008
0,008
0,0129
0,008
Olfactory Sensation
Threshold
0,0177
0,0182
0,027
0,027
0,0182
—
Sub-
threshold
0,0160
0,0169
0,02
0,02
0,0169
—
Analysis of the electroencephalographic curves established that in one
subject (Kh.) after a 6-minute inhalation of nitrobenzene, no restoration
of the rhythm and energy occurred, not only during the 24 minutes of the
experiment, but also 40-50 minutes after the gas was disconnected during
a 20-30 minute rest. Changes in the curve of this subject (Fig. 2) took
place in the 10th minute of the experiment, 2 minutes after the gas was
supplied, were then reinforced in the 14th minute, when the gas was dis-
connected, and during the following 2 minutes. The confidence factor of
the changes reached 99.9%.
w /? i,
AB - period of''gas inhalation.
- 48 -
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As a result of the analysis of the curves, we came to the conclusion
that the method employed in the study of the reflex effect of atmospheric
pollutants is highly sensitive.
On the basis of studies of the threshold of olfactory sensation of
nitrobenzene, its influence on the light sensitivity of the visual system,
and a study of the reflex effect of nitrobenzene on the electrical activity
of the human brain, we recommend the highest single maximum permissible
concentration of nitrobenzene in atmospheric air of populated areas as
0.008 mg/m3, the subthreshold concentration according to the most sensitive
method (Table 4).
Table 4
Results of Study of the Reflex Effect
of Nitrobenzene.
Threshold
.Olfactory sensation
Effect on light sensi-
tivity of the eye
Effect on electrical
actiVity of cerebral
cortex
Concentration, mg/m^
Minimum
Active
0.0182
0,0157
0,0129
Maximum
Inactive
0,0169
0,0118
0,008
To substantiate the average daily maximum permissible concentration
of nitrobenzene, a round-the-clock dynamic exposure of white rats was
carried out for 73 days.
Four groups of male white rats with 18 in each group were exposed.
In the first group, the nitrobenzene concentration was 0.8 * 0.022 mg/m.3,
in the second 0.08 * 0.01 mg/m3, in the third 0.008 * 0.0008 mg/m3, and
the fourth group was the control.
It is known from the literature that nitrobenzene affects chiefly the
blood. However, the clinical picture of poisoning with nitrobenzene indi-
cates the possibility of selective injury to the central nervous system
(Ya. I. Kukhta et al., 1934).
This enabled us to select the following tests characterizing the
influence on the central nervous system as the chief indicators of the
effect of nitrobenzene on the organism: behavior and weight of the animals,
motor chronaxy of the antagonist muscles of the shin, cholinesterase
activity, and on the blood - total hemoglobin, methemoglobin, sulfhemoglobin
and oxyhemoglobin, Heinz bodies, and histopathological data.
- 49 -
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The investigation lasted 101 days. During the first week of exposure,
irritation of the upper respiratory tract (head cold, sneezing) and the
appearance of a serous crust on the eyelids were observed in rats of the
first group (0.8 mg/m^). A few days later, these symptoms passed. The
animals were active in all groups except the first, in which a periodic
sluggishness of the rats was noted.
No statistically reliable changes in the weight of the animals of
all three groups were observed as compared with the control group.
The central nervous system under normal conditions has a subordinate
influence on the neuromuscular system, as expressed in a correct ratio of
the chronaxias of the extensors and flexors. When the process of inhibi-
tion arises in the cerebral cortex, this ratio changes and even becomes
inverted (Chao Cheng-ch'i, 1961; V. A. Gofmekler, 1961, and others).
We studied the chronaxy and rheobase of antagonist muscles of the
hind leg of the rat by means of an ISE-01 electronic pulse chronaximeter
once every ten days in six rats of each group during the first half of
exposure and in five rats during the second half.
An inverse ratio of the chronaxies of antagonist muscles in rats of
the first group (0.8 mg/m^) occurred on the llth day and was preserved
during the entire exposure. In rats of the second group (0.08 mg/m3), the
inversion of the ratio was observed on the 21st day and was also preserved
during the entire period of the experiment. A disturbance of the correct
ratio of the chronaxies of antagonist muscles occurred as a result of an
increase in the chronaxy of the flexors (the confidence factor of the
changes reached 95-99%). The reestablishment of the correct ratio of
chronaxies was observed after ten days of the recovery period. No change
of the ratios occurred in the third group (Fig. 3).
During the development of inhibitory processes in the centers, an
accumulation of acetylcholine takes place in the tissues. An increase in
the activity of cholinesterase is one of the mechanisms of compensation
for this disturbance (D. Ye. Al'pern, 1958, and others).
The study of cholinesterase activity of whole blood was made every
fortnight by using A. A. Pokrovskiy's chemical method (1953) modified by
A. P. Martynova (1957).
The nitrobenzene concentration of 0.8 mg/m-^ (first group) caused in
the rats a statistically reliable increase of cholinesterase activity
(99.9%) on the 12th day of exposure, and the 0.08 mg/m3 concentration
(second group), starting on the 39th day of exposure. During the recovery
period, the cholinesterase activity returned to the initial level in both
the first and second groups. The cholinesterase activity in-rats of the
- 50 -
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third group (0.008 mg/rn^) was similar to that of the control group over
the course of the entire study (Fig. 4).
IS63
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Dates of investigation
Fig. 3. Ratio of chronaxies of antagonist muscles during'"
chronic exposure to nitrobenzene.
1 - first group (0.08 mg/rn'); 2 -'second group (0.08 mg/m^
3 - third group (0.008 mg/m5); k - fourth group, control;
AB - period of exposure.
The disturbance of the correct ratio of chronaxies of antagonist
muscles and the increase in the cholinesterase activity of the blood indi-
cates the presence of inhibitory processes in the central nervous system,
arising from the influence of nitrobenzene vapors.
Minutes ^ ff
JksE-
30-
• 20-
10-
iU3
Fig. 4.
Date of investigation
Activity of cholinesterase of whole blood in rats
during chronic exposure to nitrobenzene.
Notation same as ih Fig.- 3.
- 51 -
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Of late, the literature increasingly emphasizes the view that the
basis for the various disturbances during poisoning with aromatic
nitroamino derivatives are changes in respiratory enzymes. The formation
of methemoglobin itself is attributed to the depression of certain
intraerythrocytic oxidation-reduction enzyme systems whose normal activity
consists in reducing methemoglobin to active hemoglobin (P. Sulamit et al.,
1960).
Sulfhemoglobin may be formed as an intermediate in the formation of
bile pigments from hemoglobin.
To measure the total hemoglobin, methemoglobin, Sulfhemoglobin and
oxyhemoglobin (every 10 days), we chose the method of determination of
hemoglobin with a photoelectrocolorimeter (Evelyn, Malloy, 1938). The
quantity of blood employed was decreased to 0.05 ml. We did the determ-
ination with an SF-5 spectrophotometer.
In rats of all groups, the total hemoglobin before exposure ranged
from 10.307 to 13.453 g per 100 ml of blood; there was up to 1.63%
methemoglobin of the total hemoglobin, up to 0.28% Sulfhemoglobin, and
up to 98.0-98.95% oxyhemoglobin of the total hemoglobin.
During exposure in rats of the first group (0.8 mg/m3), on the 19th
day of exposure, as the methemoglobin increased from 4.20 to 12.65%, there
was a sharp decrease in the amount of total hemoglobin (7.565-9.252 g per
100 ml of blood) and oxyhemoglobin (87.01-95.27%). In rats of the second
group (0.08 mg/nH), the changes were similar in nature but somewhat less
pronounced.
Starting with the 48th day of exposure, an increase of Sulfhemoglobin
to 1.91% with a sharp decrease of methemoglobin were noted in animals of
the first group. At the same time, the total hemoglobin slightly increased.
In the second group on the 58th day of exposure, Sulfhemoglobin increased
up to 1.40% in some rats. These changes were preserved until the end of
the exposure (Fig. 5).
Restoration of the amount of hemoglobins to the original level occurred
on the 12th day after completion of the exposure. In rats of the third
group, the content of hemoglobins did not exceed the limits of fluctuations
of these indicators in animals of the control group (see Fig. 5).
Isolated Heinz bodies appeared on the 48th day of exposure in rats of
the second group only (0.8 mg/m^).
Thus, nitrobenzene in concentrations of 0.8 and 0.08 mg/nH during
long-term round-the-clock penetration of the organism of white rats decreases
the quantity of total hemoglobin and oxyhemoglobin, causes methemoglobinemia,
and subsequently sulfhemoglobinemia. A nitrobenzene concentration of
- 52 -
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0.008 mg/m3 was shown to be inactive by all the tests, and we therefore
propose it as the average daily maximum permissible concentration in
atmospheric air.
g/100 ml of blood
Ah"
I!
J
1
1.2
E
E
;-H
MM
hi
i/ i/ 1^ I*
Fig. 5. Content of total hemoglobin (i), methemoglobin (ll) and'
sulfhemoglobin (ill) of the blood in rats during chronic exposure
to nitrobenzene.
Notation same as in Fig. 5.
On the basis of the experimental studies, we attempted to follow the
process of action of nitrobenzene on the organism, in particular, on the
hemoglobin system, during long-term inhalation of low concentrations. The
extent of the action is indicated by sulfhemoglobin, whose quantity in-
creased not only in percent ratio to the total hemoglobin but also in
absolute terms, in milligrams per 100 ml of blood.
.- 53 -
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Methemoglobinemia and sulfhemoglobinemia constitute one of the
characteristic biochemical manifestations of nitrobenzene intoxication.
However, before the appearance of changes in the blood, there are earlier
changes in the central nervous system, as indicated by data obtained from
the study of the motor chronaxy of antagonist muscles, and of cholinester-
ase of whole blood.
Apparently, by indirectly altering the processes of cellular metab-
olism, nitrobenzene impairs the tissue respiration of the brain and of
other internal organs, subsequently causing blood hypoxia as a result of
the transformation of part of the hemoglobin into methemoglobin and
sulfhemoglobin and of hemolysis of the erythrocytes.
The ability of nitrobenzene during long-term penetration into the
organism in concentrations of 0.8 and 0.08 mg/m^ to impair the vital
functions of cells of the central nervous system and the respiration of
organs and tissues, and to alter the state of the enzyme system in the
blood of rats indicates that the action of the poison directly affects
the "body chemistry" I. P. Pavlov always kept in mind, particularly when
dealing with the activity of the central nervous system, which he viewed
as a function of the biochemical processes of the organism, while at the
same time distinguishing its coordinating role in all the processes taking
place in the organism.
In a hygienic evaluation of the pollution of the atmosphere with
nitrobenzene around an experimental plant that used this chemical as the
raw material, we collected samples of atmospheric air during the spring-
summer period of 1963. The discharges of the plant studied contained
vapors of nitrobenzene, nitrochlorobenzene and benzene. In order to
carry out a separate determination of these substances, we developed a
method of determination of nitrochlorobenzene with measurement on a
spectrophotometer.
The peak for the measurement of nitrochlorobenzene absorbed in dis-
tilled water (0.5 1/min) was at a wavelength of 207-208 my with a slit
opening of about 1.9. The sensitivity of the method was 0.25 yg per ml.
Benzene was also measured on the spectrophotometer by using the
method of Alekseyeva et al. (1963). Nitrobenzene was determined colori-
metrically by Alekseyeva's method, as indicated above.
We determined nitrobenzene in concentrations exceeding our proposed
highest single maximum permissible concentration (0.008 mg/m^) at a
distance of up to 200 m, and in lower concentrations at a distance of up
to 300 m (Table 5).
-.54 -
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Table, 5
Pollution of Atmospheric Air with Nitrobenzene Around
One of the Plants.
Distance
From Source
of Discharge
150
200
250
270
300
Number -of
Samples
Collected
27
34
37
34
25
, Maximum
Nitrobenzene
Concentra-
tion) rng/m5'
0,0957
0,0'139
0.008 J,
0,0060
• Distribution of Concentra-
tions (mgj/m*) of nitro-
benzene in the samples
0.14 — 0.01
27
28 -
0.011-
0,003
1
1
Below
0.008
1
11
6
Nitrochlorobenzene was determined at a distance of 100-300 m in con-
centrations of 0.4-0.08 mg/mA Benzene was not observed in any of the
samples.
Results of the laboratory studies were used by the plant for the
introduction of changes in the flowsheet of the planned operations.
Conclusions
1. Nitrobenzene has a high toxic activity which injures first the
central nervous system, then the blood.
2. On the basis of a study of the threshold of olfactory sensation
of nitrobenzene, the reflex effect of its vapors on the light sensitivity
of the visual system and on the electrical activity of the brain, we rec-
ommend that 0.008 mg/m3 be established as the highest single maximum per-
missible concentration of nitrobenzene in the atmospheric air of populated
areas.
3. A chronic action of nitrobenzene vapors in concentrations of
0.8 and 0.08 mg/m^ caused an impairment in the correct ratio of chronaxies
of antagonist muscles, an increase in the cholinesterase activity of whole
blood, a decrease in total hemoglobin and oxyhemoglobin, and an increase
of methemoglobin and sulfhemoglobin.
'4. As the average daily maximum permissible concentration of nitro-
benzene in atmospheric air of populated areas, we propose the concentration
of 0.008 mg/m3, which did not cause any significant changes during chronic
round-the-clock exposure.
5. Atmospheric air is contaminated with nitrobenzene vapors up to a
distance of 250 m around one of the plants that use it as the raw material.
- 55 -
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LITERATURE CITED
caa M. B., Kpu.ioBa H. A., XpycTa.icna B. A.
Fur. H can., 1963, N° 1, crp. 32.
• Ajibiicpn J\. E. B/iiinime iicpniiofi CIICTC.MU na aKTimiiocib xo.imi-
acTcpasiJ H xo.iiiiicprii'iccKyio pcaKumo Oo.ibitoro oprannsMa. B KH.:
CoupcMciiiibic uonpocu iicpoiiaMa D tjwsiio.iormi H namnoniH. MCA-
. ni3. M., 1958. crp. 45— -16.
B y tu T y c n .1 K. A. AlaTcpiia-nbi K ycTanon.ieiinio iipc,ae./iMio AOnycTii-
MOM KoimeiiTpnumt asposo-nn cepiiou KHOIOTU D atMoccjjepiiOM BOS-
Ayxe. B c6.: Hpc/icvibHO AonycTiiMbie KoimeiiTpamiii atMOccpepiiux
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ByiiJTycna K. A., no.noKacB E. O., CCMCIICIIKO A. Jl.
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crp. 57—61.
P o <[> M c K J\ c p B. A. Marcpnaflbi K ooociiouaiiiiio npcjic^biio Aony-
CTiiMbix KoiiUciiTpauiiii aueTaron B aT,\ioc(j)cpiiOM B03Ayxc. B co.:
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JlyOpoBCKan O. H. riiriicinmccKaH oneiihca sarpnaiiciinocTii aiMO-
c(J.'epiioro no;tAyxa 6o;iL.tiioro ropoAa ccpiiiicrbiM rasoM. B c6.: flpe-
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AiiTopcij)Cp;iT. Hcp.Mb, 1954.
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Cy.'ia.MiiT 11. n Ap. Hcc.iCAOiiaiiiic TOKCII'ICCKOFO AciiCTRiin
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LOW CONCENTRATIONS OF UNSATURATED HYDROCARBONS OF THE ETHYLENE SERIES
IN AIR AROUND PETROCHEMICAL COMPLEXES
Candidate of Medical Sciences M. L. Krasovitskaya and L. K. Malyarova
Ufa Institute of Hygiene and Professional Diseases
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 74-100, (1966).
Thus far, hygienists have been working with the concept of the "sum"
of hydrocarbons, which includes saturated, unsaturated, aromatic and chlor-
inated hydrocarbons. Because of the great variety of hydrocarbons in atmos-
pheric air, the variability of their composition and their diverse toxicity,
this overall definition excludes the possibility of a hygienic evaluation
of their contents in the atmosphere.
The object of the present study was to separate from the "sum" the
hydrocarbons of the ethylene series, to establish their content in atmos-
pheric air, and to study the effect of low concentrations of ethylene,
propylene, and butylenes on man. A hygienic evaluation of hydrocarbons of
the ethylene series is of particular interest in areas with a large petroleum
refining and petrochemical industry polluting atmospheric air with unsatu-
rated hydrocarbons (cracking gas contains approximately 20-25% propylene,
up to 12-15% butylenes, 10-12% ethylene, 5-6% C5 hydrocarbons).
Unsaturated hydrocarbons of the ethylene series up to C^ inclusive
are gaseous substances, and those beginning with C5, liquids. All these
hydrocarbons have a narcotic effect, which, however, is observed only at
high concentrations, on the order of several tens of volume percent. The
strength of the effect increases with the number of carbon atoms. In
addition to the narcotic effect, olefins, starting with butylenes, cause
an irritation of the respiratory tract. The threshold of smell for butylene
(N. V. Lazarev, 1954) is 0.59 mg/m^. Compounds with branched chains had a
weaker effect than normal olefins. According to the Gor'kiy Institute of
Labor Hygiene and Professional Diseases, the maximum permissible concen-
tration of olefin vapors in the plants is 50 mg/m3, and for butylenes, the
maximum permissible concentration is 100 mg/m3.
In the literature available to us, there are no data on the effect of
low concentrations of individual hydrocarbons on the human organism and
its higher nervous activity. Studies dealing with the action of hydrocarbons
on the human organism pertain to the sum of saturated and unsaturated hydro-
carbons and refer to concentrations existing in the air of industrial quar-
ters (R. S. Ostrovskaya, 1961; S. Kh. Nikolov, 1961; S. Kh. Karimova, 1961;
- 57 -
-------
R. F. Gabitova, 1960; B. A. Shekhtman, 1929, 1961, and others). No studies
have been found dealing with the influence of microconcentrations of
ethylene, propylene, and butylenes on the human or animal organism during
administration by inhalation.
The hygienic substantiation of the maximum permissible concentrations
of ethylenic hydrocarbons in the atmosphere consists in the experimental
investigation of the effect of low concentrations of ethylene, propylene,
and butylenes on the physiological reactions of the human organism.
To perform the experiment, propylene and butylenes were obtained in
the laboratory by dehydration of the corresponding alcohols - isopropyl
and n-butyl alcohols - over active aluminum oxide at 375-400°C. (for pro-
pylene) and 350°C. (for butylenes). The alcohols were passed at a rate of
800-900 ml per liter of catalyst per hour. The purity of the gases obtained
was continually checked with a KhT-2M chromathermograph'. The content of
pure gases varied between 95.5 and 99.5%. The petroleum refinery supplied
ethylene of 99.9% purity.
To substantiate the highest single maximum permissible concentration,
the olfactory threshold of these substances and the influence of their
low concentrations on the optical chronaxy and dark adaptation were deter-
mined, and the threshold concentrations causing the formation of the
electrocortical conditioned reflex during inhalation were established.
To obtain constant concentrations of the hydrocarbons studied, use
was made of a method recommended by the Committee on the Sanitary Protec-
tion of Atmospheric Air. The gaseous mixture for the experiment was ob-
tained by mixing a known concentration of the gas studied with pure air
in certain ratios.* The two air streams were controlled by stopcocks and
checked with flow meters. The actual gas content prior to the experiment
was determined with a titrimetric gas analyzer. The constancy of the
concentration was checked for three days for each substance in the course
of 4-6 hours (Table 1).
In eleven persons who showed no changes in the olfactory organs, the
threshold of smell for butylene, propylene, and ethylene was determined.
A total of 536 observations were made. Each concentration was checked an
average of three times. The thresholds of olfactory sensation are given
in Table 2.
* The gas was dispensed with a stopcock. A hermetically sealed cylinder was evacuated with a
vacuum purap to a certain reduced' pressure. The stopcock was rinsed with the substance being dispensed
and was then connected to the evacuated cylinder. A certain volume of gas contained in the stopcock
was washed out with pure air until the pressure was equalized. The gas-air mixture obtained was
agitated by means of glass rods on a shaker.
- 58 -
-------
Table 1
Constancy of the Concentration in the Experimental Air Mixture.
Substance
Butylenes
Propylene
Ethylene
Days of
Observa.-
t.i on
1
2
' 3
1
2 •
3
1
2
3
Concentration in mg/rn? after 2 hours
3,12
' 2.16
3,71
5,0
2, OS
7,02
2.0
11,3
3,2-1
3,12
1,99
. 3,18
4,75'
2,6
6,75
2,0
11,6
3,2-1
3,18
1,92
3,64
4,68
2,15
7,25
2,5
11,3
3,18
3,18
3,64
As is evident from Table 2, the thresholds of olfactory sensation for
butylenes, propylene, and ethylene are similar: in the most sensitive
persons they amount to 15.A, 17.3 and 20 mg/m^ respectively.
Table 2
Results of Determination of the Threshold of Olfactory Sensation
for Unsaturated Hydrocarbons (C2-C4)
Number of "
. Subjects 1
Minimum Perceptible
Concentration, mg/m3\
Imperceptible
Concentration, mg/m*
1
2
6
2
Ethylene
20.0
22,0
26,3
Propylene
17,3
18,6 ..
22,7
24.0
Butylenes
15.4
17,3
21,4
19,0
20,0
22,0
15,1
17,3
18,6
22,7
14,3
15.4
17,3
The optical chronaxy was determined in 3-4 subjects. During the
first 3-4 days, training of the subjects was carried out for each sub-
stance. The rheobase and chronaxy were established six times with 3-minute
intervals during the course of the experiment. After the third measurement,
the subject was given a gas of known concentration or pure air to breathe.
- 59 -
-------
Three examinations were made with each concentration. The threshold for
the determination of the rheobase and chronaxy was the first sensation
of phosphene.
The results of the study are listed in Tables 3, 4 and 5.
Table J>
Change of Optical Chronaxy'Before and After Inhalation of Ethylene
(in percent at 0 minute).
Concentration,
mg/m3
Before Inhalation ''I
After Inhalation
Time of Experiment, minutes
0
3 6 |
9 | 12
15
Subject Ye.
Pure Air •
9.6
13,9
19,0
100
100
100
100
- 101
101
101
100
101
101
101
101
102
102
67fc)
66(c)
101
103
100
68(c)
101
100
100
100
Subject T.
Pure Air
9.6
13,9
19,0
100
100
100
100
100
98
100
102
98
98
101
99
98
98
101
129(b)
98
98
101
100
98
98
101
100.
Subject R.
100
100
100
100
99
100
99
101
99
100
100
100
90
99
40(c)
134(a)
100 '
99
80(c).
125
99
99
100
94
Pure Air
9.6'
13.9
19,0
Note.. ^Confidence' factor-a - 95$, b - 99$, c - 99.9$.
The change of the rheobase was noted in a study of the effect of
ethylene in a concentration of 19 mg/m^ in subject Ye. In the remain-
ing cases, the rheobase remained without changes.
Thus, the thresholds of the reflex effect of ethylene, propylene
and butylenes on the optical chronaxy are at the same level and amount
to 13.9-14.3 mg/m3 (Fig. 1 and 2). A trace response during inhalation
of threshold concentrations occurred in the 12th minute during the action
of propylene (subject G.) and ethylene (subject T.).
The method of dark adaptation was also used to establish the thres-
hold of the reflex effect of the hydrocarbons studied.
The examinations were conducted with an ADM adaptometer. The dark
adaptation curves were obtained with three subjects for each substance
- 60 -
-------
Table 4
Change of Optical Chronaxy Before and After Inhalation of Propylene
(in percent.at 0 minute).
Concentration,!.
• mg/m5 •
Before
Inhal
jtion 1
• Time
0
1
3
of
6 '
After
Experiment
I •
1
j
Inhalation
minutes
12 I
15 .
Subject Ye.
Pure' Air
9,6
' M.3
20.7
Pure Air
9,6
M.3
20,7
Pure Air
9.6
M,2
20,7
100
• 100
100
100
101
100
100
. 1GO
101
100
100
100
102 •
100
95(a)
7C(c).
101
101 •
100
. S9(b)
101
100
100
96
Subject G.
100
100
100
IOC
100
100
100
100
99
100
100
100
99
100
163(c)
192(c)
99
101
118(a)
101
99
100
100
100
Subject 0.
100
100
100
100
100
100
100
100
101
100
100
100
101
100
66,5(b)
139(c)
101
100
93
100
100
100
100
100
Note: Confidence factor: a - 95$, b - 99$, c - 99.*•
studied. During the first few days, the subjects were trained, then the
initial curve of dark adaptation was recorded during inhalation of pure
air. Subsequently, a definite hydrocarbon concentration was studied every
three days. Results of the action of the substances studied on the light
sensitivity of the eye are shown in Table 6.
Thus, a concentration of 14 mg/m3 of ethylene, propylene, and buty-
lenes caused a change of the light sensitivity in all the subjects. In
two cases, the action of this concentration caused a decrease of the
light sensitivity (in subject G. during inhalation of propylene and in
subject M. under the influence of butylenes). In the remaining cases,
a rise of the dark adaptation curve was noted (Fig. 3). The 11 mg/m^ con-
centration did not act on all of the subjects either. Thus, the threshold
of the reflex effect of the hydrocarbons studied on the course of the dark
adaptation curve lies below the threshold of smell and amounts to 11 mg/rn^.
The 8 mg/m3 concentration was found inactive for all the hydrocarbons studied.
The next stage of our study was the determination of the action of ethylene,
propylene, and butylenes on the development of the electrocortical con-
ditioned reflex.*
* The"electrical activity of the brain was studied in collaboration with Yu. A. Terekhov. and
K.' I. Sukhotina, members of the scientific staff of the physiology laboratory.
- 61'-
-------
Table 5
Change- of Optical Chrpnaxy Before and After Inhalation of Butylenes
'in percent at 0 minute).
Concentration,
Before Inhalation
After Inhalatipn
Time of Experiment, minutes
12
Subject Ye.
fture Air
10,4
14,0
20,0
100
100
100
100
101
100
99
100
101
99
100
100
102
99
71 (a)
52(c)
101
100
99
93
101
100
100
99
Subject G.
Pure Air
10,4
' 14,0
20,0
100
100
100
100
100
100
100
100
99
100
100
100
99
100
1 10(c)
1 2 1 (b)
99
100
100
105
99
102
100
100
Subject 0.
Pure Air
10,4
. 14,0
20,0
100
100
100
100
100
101
99
. 100
101
102
100
100
101
102
16'l(c)
lG5(c)
101
102 •
100
100
100
99
100
100
Subject R.
Pure Air
. .10,4
14,0
•. 20,0
100
100
100
100
100
100
100
100
100
101
100
100
103
100
150(a)
147(h)
100
100 ..
• 100
100
100
101
100
. 100
Note. Confidence Factor: a - 95$, b - 99$, c - 99.9$.
ftf
100-
>> sol
7ff-
\\ /:
\\ , //
\\ //
\\//»
V v' •
Fig. 1. Change of optical chronaxy
during inhalation of butylenes in
subject Ye.
1 - pure air; 2 - concentration of
butylenes. 10.4 mg/m5; 3-14 ng/m5;
4-20 mg/m5.
D.
o
60-
V
J 6. 9
Time, minutes
- 62 -
-------
The examinations were made on a 2-channel encephalograph of domestic
manufacture using the method of Bushtuyeva, Polezhayev and Semenenko
(1960). Results of the determination of the threshold of"influence of
the hydrocarbons on the electrical activity of the brain are given in
Table 7.
A
uo-
HO-
no-
100-
so-
30-
70-
60-
50-
6
F
" 1 NV
_«=.•=.-«=,*_-»/ . ,_] ,\ ,
\ ^ f\
\ ' /
\ /
\ i
\ !
\ '•
. \i
3 6 3 12 IS
Time, minutes
Fig-. 2. Change of optical chronaxy
during inhalation of ethylene in
subject T.
1 - pure air; 2 - ethylene concentra-
tion |9.6 mg/m5; 3 - 13.9 mg/m3;
't - 19 ns/m3.
Table 6
Average Values of Light Sensitivity of the Eye During Inhalation of
Different Concentrations of Hydrocarbons of the Ethylene Series
in Percent of 15th Minute.
Subject
20th Minute
- Pure
Air
Concentration, mg/m'
u.o
11.0
8,0
25th Minute
Pure
Air •
Concentration, mg/m3
14.0
n.o .
8.0
Ethylsne
.G.
M.
G.
M.,
D.
161
116
129
161
HG
129
161
146
124
252(a)
288(a)
182(b)
126
156
148
129
191
221
149
29G(c)
239
214(b)
Propylene
lO-l(c)
234 (c)
215(c)
156
199(b)
153
152
135
192
221
149
100(b)
190(a)
193(a)
Butyle-nes
234(c)
120(b)
150(b)
187(b)
175(b)
121
158
152
129
192
221
177
265(b)
1 G5(c)
130(c)
210(a)
244 i
155
25S(c)
230
220(a)
237(c)
23-1 (c)
166
201
238
140
ISO
213
154
205
204
177
Note. Confidence Factor: a - 95%; b - 99%; c - 99.9%.
- 63 -
-------
wo]
350
JOO-
•r! 100\
•150
so-
8 5 .10 15 20 25 30
Time, minutes
-------
The conditioned reflex was usually developed at the 4-9th pairing,
more seldom at the 11-14 th. The threshold of reflex change of the
electrical activity of the brain lies at the level of 4.4 mg/m^ for all
the gases (Fig. 4, 5). Combined data from the study of the reflex effect
of ethylene, propylene, and butylene on the receptors of the respiratory
organs are listed in Table 8.
Table 8
Threshold Concentrations of Ethylene, Propylene and Butylenes
Acting on Receptors of Respiratory Organs.
Threshold '
'Sensation of smell
Effects on electrical excitability
.- (optical chronaxy)
Effects on light sensitivity
Effects onielectrical activity of
the brain.
Fhresbcld Concent-rations, mg/m^
Ethylene
20.0
13,9
11,0
4,4
Propylene
17,3
14,2
11,0
4,4
Butylenes
15.4
M,0
11.0
4.4
' A^$$|^^
•'A11 '
* Ajfej^1/ %...;fo&^^
Fig. 4. Electroencephalogram of subject M. Conditioned-reflex desynchron-
ization occurred during inhalation of propylene in 4.4 mg/ra5 concentration
(at 7th, pairing).
1 - electroencephalogram of the left occipital area; 2 - right occipital
area; a - mark indicating when light was turned on; B - mark indicating
supply of gas.
- 65 -
-------
*^ty^^
*,«rf$tyV*,Vft^
Fig. 5. Electroencephalogram for Subject M. No conditioned-reflex desyji-
chronization occurred during inhalation of propylene in 3.3 mg/m3 concen-
tration (19th pairing).
Notation same as in Fig. k.
The results obtained made it possible to propose 3 mg/m.3 as the
highest single maximum permissible concentration of ethylene, propylenes,
and butylenes.
In view of the fact that the hydrocarbons which we studied are
constantly present in the atmosphere, the threshold of their effect on the
electrical activity of the brain was determined for their combined presence
(Table 9).
Table 9
Combined Action of Ethylene, Propylene, and Butylenes on the
Development of the Electrocortical Conditioned Reflex.
C on cent rst ions of
ethylene + propyl-
er.-a + butylenes,
mg/m*
1.1-rl. 1 + 1,1
2, H- 1,1-1- 1,1
1,1-1-2,1-1-1,3
l.H-1, 1-1-2,1
. 3.2 + 3,3+3.2
Total con-
centration
in fractions
of maximum
permissible
value
1,0",
. -1,3
1.3
1,3
3.0
Subject
M.
_
' +
+
+
+•
A.
+• ••
+
•^
+
_
K.
'•
-------
mixture. Each series involved five groups of 15 white male rats each
(Table 10).
Fig. 6. Electroencephalogram of subject If. No conditioned reflex desynchronization of
the rhythm occurred during inhalation of ethylene, propylene, and butylenes (total con-
centration in fractions of maximum permissible value equal to unity) (l5th pairing).
Notation same as^in Fig. 4.
i$^M^®A^
ifpwv^
•8
Fig. 7. Electroencephalogram of subject M. Conditioned-reflex desynchronization of the
rhythm during inhalation of ethylene, propylene and butylenes (total concentration inJ
fractions of maximum permissible value equal to 1.3) (llth pairing).
Notation same as in Fig. 4.
o
The 3 mg/m concentration is the highest single maximum permissible
concentration that we are proposing, and 100 mg/m3 is the maximum per-
missible concentration of butylene in the air of industrial quarters.
The exposure of each series of animals lasted 70 days.
'The gases for the experiment were prepared and their purity was
checked in the same manner as in the first part of the study. The obser-
vations were made by using the following tests: weight and behavior of
the animal, ratio of the chronaxies of antagonist muscles, blood pressure,
number of blood leucocytes and their phagocytic activity, whole blood
- 67 -
-------
cholinesterase, and fluorescent* microscopy of leucocytes.
Table 10
Concentrations of Substances Studied in the Chambers,
Series
I
11
Concentra-
tion
Assumed
Actual
Concentra-
tion
Assumed
Actual
Concentrate [in, mj/m^
First Second
Group 1 . GrouP
(Butyl- i(Propy^-
enes) '1 ene;
100,0
104,3 +
1.0
13.0
3,12 +
0,02
100,0
103, 1±
1.3
3.0
3,13 +
0,02
Third
Gr.oup
(Eihyl-
ene)
100,0
104,1 +
0,7
3,0
3,18±
0,03
Fourth
Group (mix-
ture of eth-
ylene, pro-
pylene and_
Dutyienes)
33.»,of
103.1 +0.5
^
1.0 of each
Ctotal of
3.12 + 0.02
Fifth
Group
(control)
i °
0,24+0,1
: 0
0
The animals in each chamber were divided into two subgroups: the ratio
of chronaxies of antagonist muscles and the blood pressure were determined
in the first group (8 rats), whereas in the second (7 rats), the number of
leucocytes, their phagocytic activity, and whole blood cholinesterase were
determined and fluorescent analysis of the leucocytes was performed. At the
end of the exposures, some of the animals of each group were given the
"swimming test", and the remaining ones were sacrificed. The organs of the
sacrificed animals were then subjected to histopathological analysis. The
choice of the above methods was determined by the nature of the action of
the substances studied (narcotics) .
The ratio of chronaxies of antagonist muscles is the most sensitive
test reflecting the functional shifts in the central nervous system. The
present day understanding of the physiological essence of chronaxy has per-
mitted the use of chronaximetry as a method for the investigation of the
functional state of the nervous system in the study of the chronic action
of low concentrations for the standardization of atmospheric pollutants
(V. P. Melekhina, 1962; V. A. Gofmekler, 1961, and others).
*Editor's note: For the Russian use of" the terms "luminescept" and "luminescence" in this paper,
we have substituted "fluorescent" and "fluorescence", on tha basis of the definitions of these terms.
- 68 -
-------
Cholinesterase. The physiological role of cholinesterase is closely
related to the activity of the central nervous system. The mechanism of
action of narcotics on the central nervous system may be attributed to an
impairment of the processes of chemical transfer of stimulation in the
brain synapses. Judging from the investigations of M. Ya. Mikhel'son,
the inhibition of cholinesterase by the narcotic plays an important part
in the mechanism of nerve-type narcosis in the broadly accepted cholinergic
mechanism of synaptic transfer of nerve impulses. According to this
author's data, with certain ratios of the enzyme to acetylcholine, narcotic
concentrations of various substances decrease the cholinesterase activity
by 50-70%. A parallelism has been established between the strength of
action of 16 different narcotics and their concentrations, which depress
cholinesterase to the same extent (N. V. Lazarev, 1961).
Blood Pressure. The hypotensive action of hydrocarbons has been
established by numerous authors in experiments on animals and clinical
observations of people in contact with these substances under industrial
conditions (R. F. Gabitova, 1960; Z. Sh. Zagidullin, 1960; R. S. Ostrovskaya,
1961; L. I. Geller et al. , 1963, and others).
Our observations of animals (white rats) exposed to natural conditions
in the immediate vicinity of petroleum refineries (1963) established a
statistically reliable lower level of blood pressure in rats of this group
as compared with the control (in the 13-17th week of the experiment).
A considerably larger (by a factor of 2-1/2 - 6) number of cases of
hypotonia occurs in children who have lived for 5 years in the vicinity of
petroleum refineries, as compared with the control group.
Phagocytic activity. The literature contains indications of a decrease
of immunobiological activity under the influence of the gas factor (A. N.
Volkova, 1959; P. A. Zhilova, 1959; D. G. Pel'ts, 1960).
A decrease in the phagocytic activity of the blood under the influence
of atmospheric pollutants has been noted by E. A. Sarkisyants (1959),
M. L. Krasovitskaya, L. K. Malyarova, and T. S. Zaporozhets (1963).
Fluorescent microscopy is one of the methods for determining early
quantitative and qualitative changes of the formed elements of white blood
corpuscles. The use of this method is based on the ability of nucleic acid
to exhibit fluorescence on combining with the fluorochromes of the acridine
series: the nuclear nucleoproteins of degenerating cells combine with
acridine dyes differently than do the nucleoproteins of uninjured cells, as
is reflected in the character of their fluorescence (M. P. Meysel' 'and
V. A. Sondak, 1956).
Normally, the nuclei of white cells fluoresce with an emerald-green
color. When the synthesis is disturbed, and there is depolymerization of
-- 69 -
-------
the nuclear deoxyribonucleic acid, the nuclei begin to flucresce with a
red or orange color. The method of fluorescent microscopy of white blood
corpuscles for the evaluation of the effect of low concentrations of chem-
ical substances was first used by A. D. Semenenko at the department of
communal hygiene of the Central Institute for Advanced Training of Physi-
cians .
Fluorescent microscopy was successfully used by M. I. Gusev and
K. N. Chelikanov (1963) in the study of amylene in chronic experiments on
animals.
The "swimming" test for endurance (adapted for toxicology by M. I.
Rylova, 1959) was used in our studies because it involved the mobilization
of all the resources of the organism. This in our view should be an
integral indicator. During our previous investigations on animals under
natural conditions, this test yielded demonstrative results.
During the entire course of the two series of exposures, all the rats
were healthy and active, and their behavior did not differ from that of
the controls. Starting on the 48th day of exposure, all the animals re-
ceived a scorbutigenic diet for ten days. A statistically reliable weight
decrease was then observed in rats of the first and second groups in the
first series of experiments. At the end of the exposures, the weight of
all the animals was at the same level.
Measurements of the chronaxies of antagonist muscles were made by
means of an ISE-01 electronic pulse stimualtor. The chronaxy was deter-
mined in eight rats of each group under identical conditions and at the
same time. The character of the change in the ratio of the chronaxies
of antagonist muscles over the course of the exposures is shown in Tables
11 and 12.
Table 11
Ratio of Chronaxies of Antagonist Muscles in Rats of Series I.
Grotip
•First
Second
Xhird
Fourth
" Fifth '
Before
Exposure
1,7±0,15
1,9+0, M
1.6±0,I4
1,7 + 0,1.5
I,6±0.15
Day of Experiment
17th
l,5±0.1(b)
I.G±0,23
2.0±0,25
2.0±0,2S
2,0+1.2
35th
0.9 + 0.22(b)
1.3±0,31(a)
l,2±0,2S(a)
!,-l±0,12(a)
2,3+0,28
52nd
1,0 +
0,!7(b)
1.0+
0,2'l(a)
1.1+0,25
1,1+0,26
1,6+0,1
68th
0,7-1-
0,13(c)
1,0±
0,09(c)
0,9±
0,13(c)
0.9 +
O.ll(c)
1,6±0.02
Note. Confidence factor: a - 95$; b - 99#5 c - 99«9#.
- 70 -
-------
P.OS
fl.pt-
0.0J-
0,0?-
0,01-
// J5 i?
Days of
Exposure Exposure
Jo
Before
Fig.- 8. Motor chronaxy of antag-
onist muscles in rats of series I.
I, II, III, IV, V - exposure groups;
solid line denotes the chronaxy of
extensors, dashed line, that of flexors;
AB - period of exposure.
ff.M-
O.SJ-
0,0!-
P.OI-
0.03-
0.0?-
0.01-
o
Si
\ff.ffj-
$0,0?-
'•%O.OI-
\
0.03:
0,0?
0,01-
O.OJ-
0,0?
e.st-
w
f~-
7 -^
Jo
Before
Exposur
x^^^
,*••"""""" — — ^^
f • t
IS Ji J? (
Days of
5 Exposure
Fig. 9. Motor chronaxy of
antagonist muscles in rats
of series II.
Notation same as in Fig. 8.
- 71 -
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In the course of the exposure, the ratios of the chronaxies of
flexors and extensors in experimental rats of series I underwent a change:
over the course of the experiment, these indicators were found to converge,
and toward the end of the exposure their inverse ratio was established in
the first, third, and fourth groups. In rats of the second group, the
chronaxies of the flexors and extensors were found to be equal toward the
end of the experiment.
Table 12
Ratios of Chronaxies of Antagonist Muscles in Rats of Series II.
Group
First
Second
Third
Fourth •
Fifth
Before
Exposure
1.4-1-0.3
1.35 + 0,15
1.6±0,3
1,3 + 0.29
1,5 + 0.22
Day of Experiment
ITtti -
1,2 + 0.2
1,3+0,18
1.4 + 0.1
1,8±0,11
1,3-1-0,14
35th
1.4±0,15
I,9±0.11
1,4±0,18
1,6±0.11
1,3±0,15
52nd
1,2 + 0, IS
1,5±0,15
1,4 + 0.13
1,3±0,1
1,6 ±0.08
68th
l.C + 0.1
1, -1 + 0, 18
1,4±0.15
1,3+0.1
1,6+0,1
In the control rats, the ratio of chronaxies of antagonist muscles
did not change substantially during the course of the experiment and
ranged from 1.6 to 2.3. In rats of all the groups of series II, the
ratios of chronaxies of antagonist muscles remained within the limits of
the physiological norm.
The difference in the changes of the rheobase of the extensors and
flexors was insignificant, i.e., statistically unreliable in all the groups,
The chronaxy of antagonist muscles in rats of series I according to groups
is shown in Fig. 8.
The blood pressure was determined in eight rats of each group before
and during exposure under identical experimental conditions, using the
method of Martynova (1956). Results of the investigations are given in
Tables 13 and 14.
Table 13
Level of Blood Pressure in Rats of Series I.
Group
-First
Second
Third
Fourth
Fifth
Before
Exposure
100 + 3,0
10-! + -!, 2
10-1 + 3,0
105+1,4
102±2,1
•Day of Experiment
18th
98±1,7
99+1,2
GO + 2,1
90+3.0
104 ±5,9
35th
97 + 3,4
99+14,0
S8+4,2(a)
88+3, 6(a)
103±4.4
52nd
85 + 3, l(b) '
90 + 2, i(a)
8S±2,l(b)
S4 + 2,3(c)
98±0,C
69th' '
7Cf3,5(c)
S0+3,5(b)
7Gj-2,l(b)
79 + 3, 3(b)
98+2,9
Note. Confidence factor: a - 95$; b - 99$; c - 99.9$.
-_72 -
-------
Table 14
Blood Pressure Level in Rats of-Series II,
Group
First
Second
Third .
Fourth
Fifth
Before
Exposure
103 -i- 1,2
105+1,2
I0-l±l,2
106±0,9
106+1,3
Day of Experiment
18th
107+1,9
104±1,3
107±1,4
105±1,3
107±1,5
35th
109 ±1,8
106+0,9
105 + 0,8
110 + 2,0
103±1,5
52nd
10C + 2.8
109 + 2,1
10G + 0.9
109±1,7
108±3,4
68th
lOGi-1,1
101+1,5
106+1,4
106+1,4
10S±1.1
The blood pressure in rats of series I before exposure and in the
first two weeks did not appreciably differ from that of the controls.
From the middle of the exposure time, a statistically reliable decrease
was noted in the third and fourth groups. From the 52nd day of the exper-
iment, the change of this indicator was already established in all the
animals of this series. The blood pressure level in the control was with-
in the limits of physiological norm over the entire course of the experiment.
In rats of series II, the blood pressure level in all the groups (both
experimental and control) was the same and corresponded to the physiological
norm during the entire period of observations.
To study the activity of whole blood cholinesterase, we used a
potentiometric method of determination (M. I. Goshev, 1958) modified by
Dmitriyeva. The results obtained are given in Tables 15 and 16.
Table 15
Activity of Cholinesterase of Whole Blood in Rats of Series I
(.in Hicromoles of CH^OOH).
Group
First
Second
Third
Fourth
Fifth
Before
Exposure
2,19 + 0,21
2,30 + 0,37
2,01 ±0,19
2,63-1:0,3
2,35 ±0,35
Dav of Experiment
19th
2,81 ±0,22
2,35-1:0,23
1,92.10,21
2,5G±0,27
2,51 ±0,34
36th
1,84 + 0,25
2,02 + 0,13
1,82 + 0,09
2,09±0,19
2,2 +0,13
53rd
l,51±0,09(c)
2,01±0,1
l,70 + 0,09(c)
2,0 + 0,15
2,32±0,3
70th
l,32±0,04(c)
2,01+0,09
l,5-1.0,l(c)
l,77±0,07(c)
2,1±0,01
Note. Confidence factor: a - 95%, b - 99$, c - 99.c
Thus, in series I of the experiment, the enzymatic activity of the
blood before exposure and during the first half of the experiment was main-
tained at the same level in the animals of the five groups. A statistically
reliable depression of cholinesterase activity was observed on the 53rd day
of exposure in rats of the first and third groups. At the end of the experi-
ment, a statistically reliable decrease of cholinesterase activity as com-
pared with the control was observed in rats of the first, third and fourth
groups.
- 73 -
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Table 16
Activity of Whole Blood Cholinesterase in Rats of Series II
(in Micrqmoles of CH£OOH).
Group
First
Second
Third
Fourth
Fifth
Before
Exposure
2,3±0,15
1,8+0,007
1,8G±0,007
1,92±0,007
1,98±0,15
Day of Experiment
19th
2,20 ±0,20
1,72 ±0,08
1,83 + 0,10
1,96 ±0,07
1,97+0,20
37th
2,28+0,18
1,66±0,10
1.73 + 0,10
1,97+0,11
2,03±0,12
53rd
2,15 + 0,18
1,75±0,11
1,77+0,19
1,99±0,10
2,09±0,13
70th
2,08 + 0,17
1,82 + 0,08
1,83±0,09
2,0 +0,09
2,11±0,1G
No statistically reliable difference in cholinesterase activity was
established in experiments of series II as compared with the control.
The leucocytes were counted by the standard method. No statistically
reliable difference in the number of leucocytes was established during the
course of the experiments in any of the rats of series I and II as com-
pared with the control.
In the determination of the phagocytic activity of the blood (D. G.
Pel'ts, 1954), the percentage of phagocytizing neutrophiles and the
phagocytic number were taken into account. In series I of the experiments,
two reliable results were obtained in the study of phagocytic activity:
in rats of the second group (growth of the phagocytic number on the 19th
day of exposure) and of the first, second and third groups (on the 36th
day). No changes of the phagocytic number and percentage of phagocytizing
neutrophiles were observed in animals of series II.
In the fluorescent microscopy of white blood cells, no differences in
the content of leucocytes whose nuclei had an orange color were observed
between rats of the experimental and control groups of both series.
The "swimming" test was conducted after completion of the exposure.
No reliable differences were established in the duration of swimming
in rats of all the groups. The indicator of this test within each group
was marked by a great variability.
No pathologic changes in the internal organs were observed in the
autopsy of animals of series I and II.
Thus, the studied exposure to ethylene, propylene, butylenes, and a
combination of these substances showed that a concentration of 3 mg/m^ of
each of these gases and their mixture is inactive under chronic experimental
conditions and can be recommended as the average daily maximum permissible value.
- 74 -
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To determine the hydrocarbons in atmospheric air, a method of hydro-
carbon analysis developed in the laboratory and involving the use of a
KhT-2M chromathermograph was employed.
The developed method made it possible to establish the actual content
of unsaturated hydrocarbons in atmospheric air. The samples were collected
within a 2.5 km radius of petroleum refineries. Single and average daily
concentrations were determined. A total of 140 analyses were performed.
The results are shown in Table 17.
Table 17
Pollution of Atmospheric Air vdth Unsaturated Hydrocarbons Within
a 2.5 km Radius from Petroleum Refineries.
•frydrooarbons
Ethylene
Propylene
Butylene
Araylenes
Concentrations, ng/m5
Single
Maximum
-1,97
3.5
4,15
3,68
Average
2,31
1,37
2,43
2,13
Average Daily
Maximum
2,37
1,60
1,08
1.97
Thus, the highest single concentrations of each of the hydrocarbons
which we determined at the indicated distance are slightly higher than
the maximum permissible concentrations. The highest content of hydro-
carbons when present together exceeds the proposed maximum permissible
value severalfold.
Conclusions
1. The hydrocarbon gases ethylene, propylene, and butylene have
similar thresholds of olfactory sensation and thresholds of reflex effect
on the human organism.
The threshold of smell for butylenes, propylene, and ethylene for
the most sensitive persons amounts to 15.4, 17.3 and 20 mg/m^ respectively.
The threshold of reflex effect determined by the method of chronaxi-
metry was 13.9-14.2 mg/m3.
The threshold of the effect on the reflex change of the light sensi-
tivity of the eye was 11 mg/m3.
- 75 -
-------
The threshold concentration causing the formation of the electro-
cortical conditioned reflex was found to be 4.4 mg/m3. The maximum
inactive concentration was 3.3 mg/m^.
2. The maximum permissible concentrations of unsaturated hydrocarbons
of the ethylene series should lie below the threshold of olfactory sen-
sation, the thresholds of their action on the electrical excitability
and light sensitivity of the eye, and also below the threshold of formation
of the electrocortical conditioned reflex.
In this connection, the highest single maximum permissible concentra-
tion in atmospheric air which can be recommended is 3 mg/m3 for ethylene,
propylene, and butylene.
3. During the combined action of ethylene, propylene and butylenes,
a simple summation of the effect of these substances is observed. The
combined action of these hydrocarbons in concentrations equal to the pro-
posed maximum single values causes a change in the electrical activity of
the brain. Similar changes were obtained in all combinations of these
substances at which their sum exceeded the proposed maximum permissible
concentration (3 mg/m^).
The highest single maximum permissible concentration for hydrocarbon
gases of the ethylene series when present together should be 3 mg/m^.
4. The hydrocarbon gases - ethylene, propylene, and butylenes - in a
concentration of 100 mg/m3 during chronic, round-the-clock action on animals
cause changes in the functional state of the central nervous system, i.e.,
an impairment of the subordinate influence on the chronaxy of antagonist
muscles, depress the cholinesterase activity, and have a marked hypotensive
effect.
During the exposure, the number of leucocytes did not change, and there
were no differences in the content of leucocytes, whose nuclei were orange
in color. Changes in the phagocytic activity of the blood were not regular
in character and cannot therefore be correlated with the action of the
hydrocarbons.
The duration of swimming of the rats in all the experimental groups
was much shorter than in the control, but the great scatter of this indi-
cator within the group has led to a statistical unreliability of these
results.
5. Ethylene, propylene, and butylenes in concentration of 100 mg/m^
under conditions of a chronic experiment have the same strength of action
on the organism. The existing fluctuations in the strength of action of
these substances are not distinctly expressed in the various tests and are
- 76 -
-------
therefore attributed to the individual sensitivity of the experimental
animals.
6. During the combined action of ethylene, propylene, and butylenes,
a simple summation of the action of these substances is observed in a
chronic experiment. Changes in the animals during the isolated action
of ethylene, propylene, and butylenes in concentration of 100 mg/m3 of each
substance were analogous to the changes taking place during exposure to a
combination of these substances with the same total concentration.
7. Exposure of the animals to the hydrocarbons in concentration of
3 mg/m^ has no effect on the behavior and weight of the animals, on the
blood pressure level and ratio of the chronaxies of the antagonist muscles,
on the cholinesterase activity, or on the number and phagocytic activity
of leucocytes, does not injure the nuclear nucleoproteins (according to
the data of fluorescent microscopy), and is not reflected in the results
of the "swimming" test.
8. The highest single maximum permissible concentration of these
hydrocarbon gases which we are proposing, 3 mg/nP, is inactive in a
chronic experiment and can also be recommended as the average daily con-
centration.
LITERATURE CITED
A.ncKcccna M. B. OupCAe.iciiiic aT.MOCcliepiibix 3arpH3i[ciiHH. Mc;t-
•ma, M., 1903, crp. 122—126.
B y in T y c B a I\. A., Fl o .1 c >K a c D E. d>., CCMCIICIIXO A. R.
Fur. it can., 1900, As I, cip. 57—01.
B y in T y c u a K. A., Ilo.ic/Knco E. ., CCMCIICKKO A. fl.
0II3H0.1. wypn. CCCP, 1900, T. 4, crp. 452—457.
Bo.iKoua A. !•!. Fur. u can., 1959, j\» 1, cip. 80—32.
T a O.n Ton a P. (1). MaTcpiia.ib; K H3yi;cmiio xponii'iecKoii miTOKCiixa-
iinn npoAyKT.T.Mii MMoroccpniicToii iie(J)Tii. B Co.: Fiinicun rpjvia n
o.xpana 3Aopoiibsi pr.Oo'inx i:c(l)Tnnoi'[ u ne<[iTexiiM!i'iccKpf! npouuui-
.ICMIIOCTII. Vc|)a, 1960, T. I, crp. 107—127.
Fc/i.ncp'Jl. H., 13 c.i o M u TUC u 5.
crp. 3—8.
-- 77 -
-------
,U c M c i; T i, c r, a At. H. Aiin.nna yr.ncBOAOpo.T.iiw.x rasou. M.— JI., foe-
TOMTCXII3A.1T, 1S59.
>Kii.ion a H. A. fur. i; can., 1959, M- 12, cyp. 18—23.
3 a r n A y .'i •'! n ii 3. HI. B/innniic GauimipcKiix copiiiicri-ix iic;pTcii i;a
cppAC'iiio-coeyAiicTyio cucrcMy paoo'iiix, saiinibix Ha AOOUMC n nope-
P.IUOTKC iix. Ii c6.: rimicua rpy.ia n oxpaiia j.iopom.K pnoo'inx v,
llCtjmmofl H ]!CCj>TCXI!Mll'lQC!;Oli MpOMbllU.'iCIIHOCTM. Vt^a, I9GO, T. 1.
crp. 139—151.
K ;i p n M o » a A. X. Cocroniinc iicpiuiofi ciicic.Mbi n noKovopwc duo-
xiiMii'iccKiic noKci3aTC.'i:-i y paGo'iiix, aannrux ri npoii3iio;icTne cirnv-
Tii'ieckoro cnnpT.'i. B KII.: Ata-repiia.ni,i iiayiiioii nOii(|>cpcii!ini!, nocrsn-
ui.c;ino}"i i;ciipociiM niriiciiu rpy/'ia, iipo([)ccci!oiia.iiiuoii n.Tro.rio"iiii i!
iipo.M[.iiLi.'iciiiioii TOKciiKo.ionn: n iicf|n-jnioii n nc(|)Tc\iiMi:'iVcii< c u T. C., C o u o .1 c n A. C.
XpoM:iTorpai|)ii>iccKiiii MCTO;I pan.iCJii.iioro o:ipc;ic.iciuiti yr.neno;iopo-
AOII u noxiyxc. 1'iir. n can., l'.)G2, A\> 7, crp. 27—32.
K p a c o n ii u K ;i n M. ,'!., A\ a A n p o n n Jl. K., 3 a n o |> o >K c n T. C.
riinic'iiM'iccKnji onciiiwi jiirpimiciiiiM aT.Moc(J)Cjnioro no3/iyxa i)i,iupo-
ca.Mii iiciliTcct'popai'aTiJuaioiniix- n XII.MIIMCCKIIX npc/uipmmu'i. A\;ITC-
pii:i.'ii,i pcciiyu.niii!3iiciiiiii. B cO.: npc/ic-nwio AOiiycTii.Mi.ic KoiiuciiTpauiiii
aTMOC(Jicpiiux 3arp>i3iicinu"i. Mc;ini3, 19G2, ». VI.
M n K o.i oo C. X. K nonpocy o xpoiui'iccKOM uosACiicrnini yr.nciio.io-
poAOH na oprani!3M lie.TOue;
-------
THRESHOLD CONCENTRATIONS OF ACETOPHENONE
DURING SHORT- AND LONG-TERM INHALATION
Candidate of Medical Sciences
N. B. Imasheva
Department of Communal Hygiene, Central Institute for Advanced Training of
Physicians, and Department of General Hygiene, Kuybyshev Medical Institute
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 101-118, (1966).
A rapid industrial development is associated with the discharge into
atmospheric air of new chemical substances whose biological effects on man
are unknown and whose maximum permissible concentrations have not been
established. One such chemical is acetophenone , which is the subject of
our study.
Acetophenone is used in organic synthesis in the chemico-pharmaceutical
industry, the perfume industry, etc. However, the consumption of acetophenone
in these industries is not large, and no high acetophenone contents in the
discharges should therefore be expected. The chief source of contamination
of atmospheric air with acetophenone is the production of acetone and phenol
by the cumene process. A product of side reactions, acetophenone is present
in atmospheric air in equal amounts with the starting materials and end
products, and frequently surpasses them in quantity (M. I. Fongauz, 1958;
V. P. Melekhina et al. , 1961). However, no data on the effect of low concen-
trations of acetophenone on the human organism have been reported thus far
in the Soviet or foreign literature. We therefore undertook the task of
making a hygienic evaluation of acetophenone as an atmospheric contaminant.
Acetophenone CgHsO (methyl phenyl ketone) is an organic compound, an
aliphatic-aromatic ketone. It has a characteristic pungent odor resembling
that of the bird cherry. Its melting point is 19.6°C. and boiling point
202. 9°C. Acetophenone is sparingly soluble in water, but dissolves well
in ethyl alcohol, diethyl ether and other organic solvents. Methyl phenyl
ketone is quite reactive: it can be nitrated, sulfonated, gives all the
reactions with mercuric chloride, ammonium chloride, and phosphoric and
picric acids. The chlorination product of acetophenone, chloroacetophenone ,
is a very strong lachrymator.
To determine low acetophenone concentrations in atmospheric air, we
initially used Khrustaleva's method, based on the property of acetophenone
of giving with metadinitrobenzene a pink color in an alcohol medium in the
presence of an alkali. However, the sensitivity of this method, 1 yg/2 ml,
proved inadequate. For this reason, a spectrophotometric method of determ-
ination of acetophenone was developed under the supervision of M. D. Manita.
The method is based on a selective absorption of certain rays of the light
source passed through a layer of the liquid under study. In addition to a
simple qualitative method in which photographs of spectra of the solution
- 79 -
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of the absorbing substance and pure solvent are compared, use may be made
of a photometer to measure the fractions of the light flux which are
absorbed at different wavelengths, and the data obtained can be used to
plot a curve, characteristic of the given substance, of the dependence of
the absorption intensity on the wavelength.
In a quantitative determination by the spectral method, one must
check to see whether the substance obeys Beer.'s law, according to which
the absorption is proportional to the number of molecules in the path of
the beam, i.e., to the concentration of the substance. Studies made with
acetophenone along these lines have shown that Beer's law is obeyed in
work with low concentrations of an alcohol solution of acetophenone. Its
absorption spectra in ethyl alcohol are characterized by a rather high
optical density. Thus, at a maximum A of 244 my, the log of the molar
extinction coefficient is 4.2, which corresponds to the number 15,850.
Such a coefficient permits the observation of small amounts of acetophenone
in alcohol solution (Gilem and Shtern, 1957). On the basis of theoretical
calculations, a solution of acetophenone in ethyl alcohol in 1 mg/1
(1 Mg/ml) concentration is characterized by an optical density of 0.130
(quotient obtained by dividing the molar coefficient 15,850 by a millimole
of acetophenone 120,144). The optical density of an alcohol solution of
acetophenone, determined by means of an SF-4 spectrophotometer, was found
to be 0.128 in our case. Commercial acetophenone contains only 97% of
pure acetophenone, and therefore the optical density of our standard
solution was 0.130.
To determine acetophenone vapors, the air was collected in a modified
Zaytsev absorber containing AMS brand silica gel, at a rate of 4 1/min.
Five ml of ethyl alcohol was then poured over the adsorbent, decanted after
30 min, and examined in the spectrophotometer. The standard used was ethyl
alcohol which had been in contact, with 2 g of silica gel in order to create
identical conditions for the sample and the standard. The optical density of
the solution thus obtained was used to determine the amount of acetophenone
by means of a calibration graph, then its concentration was calculated from
the formula
A x 5 x 1000
where A is the optical density and V is the volume of air in liters drawn
through the absorber. This method made it possible to raise the sensitivity
of the determination of acetophenone to 0.25 yg/ml.
In the medical literature, acetophenone is mentioned in the 1870-1880's,
when Dujardin-Beaumetz* and Barde proposed it under the name "hypnone" as a
soporific. However, studies of several authors (Hirt, 1886; Rohfenbiller,
1887; Seifert, 1887) showed that it is not always possible to achieve the
soporific effect of hypnone, and that the use of the compound in high doses
involves a certain risk: the blood pressure of the patients drops, the
* [Translator's note: Dyuzharde-Bomets, according to the transliteration of Russian reference.]
- 80 -
-------
breathing rhythm is altered, the pulse weakens and fatigue increases
(Laborde, 1885, S. S. Kamenskiy, 1889). A study of the literature deal-
ing with the effect of hypnone reveals contradictions in the interpreta-
tion of the problems of action of acetophenone. We have been unable to
find any indications of a study of the threshold of action of acetophenone
on the human and animal organism either in the domestic or in foreign
literature.
The presence of the characteristic pungent odor of acetophenone
enabled us to start our investigations from a certain threshold of olfac-
tory sensation. To produce specific concentrations of acetophenone vapors,
we used an apparatus recommended by the Committee on the Sanitary Pro-
tection of Atmospheric Air.
We determined the threshold of olfactory sensation of acetophenone
in 18 practically healthy people 18 to 60 years old. The effect of a
single concentration was observed during the day; pauses between the
experiments lasted over four hours. An individual sensation threshold was
established for each subject, and the threshold concentration was considered
to be that which the subject could correctly identify no fewer than twice
out of three determinations. A total of 455 determinations were made.
Table 1
Threshold of Olfactory Sensation of
Acetophenone Vapors.
Number of
Subjects
1
2
H
. 1
Concentration, ms/m*
Minimum
Perceptible
0.08
0.027
0,02
0,01
Imperceptible
0,05
0,02
0,01
0,008
As can be seen from Table 1, the threshold of olfactory sensation
in the most sensitive persons is 0.01 mg/mX The 0.008 mg/m3 concentra-
tion was found to be the sub threshold value.
The stimulation of one perception system inevitably results in a
change in the functions of the other perception systems. This is explained
by the fact that after stimulating the receptors of one system, the impulses
reach the cortex, where they produce an excitation center which radiates
over the cortex and thus encompasses other areas. This situation also
applies to the olfactory system. Several authors have shown that the stimu-
lation of the olfactory system by ammonium hydroxide, bergamot oil, and
pyridine in toluene causes an increase in the light sensitivity of the eye
- 81 -
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(K. Kh. Kekcheyev, 0. L. Matyushenko, 1936; P. 0. Makarov, 1958).
In hygienic practice, researchers have observed the action of sub-
threshold odor concentrations of various substances on the light sensi-
tivity of the eye (F. I. Dubrovskaya, 1957; Yu. G. Fel'dman, 1960, and
others).
We also studied this phenomenon in three subjects by means of an
ADM adaptometer in a darkened room. Pure air or acetophenone of known
concentration was supplied to a cylinder for 10 minutes until the first
determination of the light sensitivity. The observation lasted no fewer
than three days with each concentration. Acetophenone in 0.02 mg/m^
concentration was found to cause a decrease in the light sensitivity of
the eye in all three subjects. The 0.015 mg/m^ concentration led to a
decrease in light sensitivity in two subjects, but it was less pronounced
than during the action of a higher concentration, and in one subject, to
an increase of the sensitivity. Acetophenone in 0.01 mg/m3 concentration
caused a change in the light sensitivity of only one subject; the 0.007
mg/m3 concentration was inactive. Table 2 lists data on the light sensi-
tivity of the eye during the course of the study.
Fig. 1 shows data on one of the subjects. The changes occurring under
the influence of inhalation of acetophenone vapors in the brain itself
were then investigated. This was done by using the method of functional
loading, which is based on the property of living tissue of reproducing a
certain number of electric oscillations (N. Ye. Vvedenskiy, 1952).
Under natural conditions, afferent impulses from various exteroceptors
and interoceptors continually reach the central nervous system. Consequently,
the use of rhythmic stimuli in the study of the functional capacity of the
central nervous system is natural and expedient.
"In the lability parameter we had a perfectly independent characteristic
of the functional capacity of the tissue; this parameter reaches deeper
into the intimate essence of the processes taking place and predicts the
functional shifts taking place more accurately than temporal parameters
(chronaxy, etc.) aimed at characterizing a single excitation wave" (L. V.
Latmazanova, 1938).
Recently, many studies have appeared dealing with the ability of
cortical elements to rearrange their rhythmics to correspond to a given
frequency (V. S. Rusinov, 1957; N. V. Golikov, 1956, and others). To eval-
uate the functional state of the brain, the electroencephalographic method
was made to include functional tests allowing for changes in the electroen-
cephalogram in response to afferent stimuli. They are based on the follow-
ing electrophysiological law: any excitation is associated with an electrical
effect, which constitutes one of the aspects of the complex process of
- 82 -
-------
excitation. The presence of the constant activity of the cortex inter-
feres with the study of the biocurrents at rest. However, the use of
a load, for example, a rhythmic photic stimulus, may give rise to pro-
cesses taking place in latent form in the organism. The rearrangement
1/E
(.in thousands)
?50-
?oo-
c
-------
Table 2
Change in the Light Sensitivity of the Eye After Inhalation of Acetophencne Vapors.
Acetophenone
v*
1 ' Light Sensitivity of the Eye During Observation
Concentration,
='m k minute
5 minutes
10
minutes
.15f
minutes
20
minutes
25 1 30
minutes j minutes
i
40
minutes
Subject N.
00
.p-
Pure Ai.r
0,02
0,015
0,010
0,007
Pure Air
0,02
0,015
0,010
26,8
12,3
12,2
20,1
25,6
987
376
359
1 170
'. 448
7 835 .
4 350
3 590
6 520
8 273
14 650
4 583
6 033
8 080
15 167
16 200
5857
6 72G
10 740
17 166
19 600
6 037
7 262
11 333
21 100
25 000
G ISO
7 426
11 600
23 933
22 900
6 323
7 604
11 900
24 433
Subject '-T.
22,8 •
8,0
29,9
22 ,' 3
2 930
2 113
2 930
2 680
13167
6530
8 533
1
-------
the fourth minute. Prior to the administration of the experimental mixture,
the background was recorded two to three times, then the mixture of pure
air and acetophenone was supplied for five minutes. In the fourth minute the
light stimulus was turned on, and further determinations were made as in
the case of supply of pure air. The entire experiment lasted 40-50 minutes.
To achieve a better objectivity of the results of the electroencephalographic
study, we recorded the action potentials by using a method proposed by the
British scientists Lovell and Dosset*(cited by G. N. Speranskiy, Yu. M.
Pratusevich and N. M. Korzh, 1960). The synchronization factor, expressed
as the ratio of the quantity of synchronized (coinciding) oscillations to
the quantity of light stimuli supplied during 5 seconds, was calculated in
percent from the formula
Ps
Ks = p- x 100,
*n
where Ks is the synchronization factor, Ps is the quantity of synchronized
oscillations, and Pn is the quantity of light pulses supplied.
At the same time, the energy of each synchronized potential (either
in millimeters of the height reached by the pen of the electroencephalograph,
or in microvolts) and the height of the synchronized spike multiplied by
the calibration factor with which the recording of the electroencephalogram
was made were calculated. The total energy of assimilated rhythms during
the action of the light stimulus in the form of rhythmic light flickerings
lasting 45 seconds was also determined.
Studies of the acetophenone concentrations (0.007 and 0.003 mg/rn-^)
did not reveal any distinct change in the synchronization factor in any of
the subjects. As far as the total energy of the rhythm assimilated by the
brain during inhalation of acetophenone vapors in 0.007 mg/nH concentration,
there occurred a 35-40% statistically reliable decrease of this energy in
all the subjects in the 2nd-6th minute. The 0.003 mg/m3 concentration did
not cause any changes. Such phenomena were also observed in healthy people
(V. V. Artem'yev, 1962; Ye. N. Sokolov, 1962). Fig. 2 graphically shows
the dynamics of change in the amplitude of the assimilated rhythm (energy)
under the influence of inhalation of acetophenone vapors in two subjects in
whom changes were observed in the 2nd minute after the gas mixture was
supplied.
Thus, the threshold of action of acetophenone on the electrical activity
of the brain is 0.007 mg/m^, whereas the thresholds of smell and change of
the light sensitivity of the eye are 0.01 mg/m^. The 0.003 mg/m^ concentra-
tion was found to be inactive, as indicated by all the tests, and can be
recommended as the highest single maximum permissible value.
Experimental studies of the effect of acetophenone on the organisms of
animals were made in the 1870's - 1880's simultaneously with clinical
* [Translator's note: Lovel1 and Doset, according to the transliteration of Russian reference. J
- 85 -
-------
observations of the effects of hypnone. The experiments were conducted
on frogs, cats, guinea pigs and monkeys. Acetophenone was injected sub-
cutaneously and intravenously, and also introduced into the stomach.
However, all these studies were made with relatively high concentrations
and had the character of acute experiments (S. S. Kamenskiy et al.).
Mairet and Combemalle (1885) attempted to carry out a dynamic exposure
of the animals, and for this purpose placed two cats, a guinea pig, and
a rabbit in a wooden box with a volume of 175 dm^ with slits and kept
them there for 1 hour and 10 minutes. An atomizer was used to introduce
a mixture of 3 g of acetophenone, 10 ml of alcohol and 200 ml of water
into the box during this entire period. Muscular weakness and paretic
symptoms were noted in all the animals. Unfortunately, it is impossible
to use the authors' data for an accurate calculation of the concentration
to which the animals were exposed. There is no doubt that this concen-
tration was high. No data are available on the influence of low aceto-
phenone concentrations during a long-term exposure.
Background
Fig. 2. Dynamics of change in the amplitude of assimilated
rhythm during the action of acetophenone vapors. Average data
for subjects V. and A.
AB - 4 minutes of inhalation of gas; BC - period in minutes after
inhalation of gas. 1 - inhalation of pure air; 2 - acetophenone
concentration, 0.003 mg/nP; 3 - acetophenone concentration,
0.007 n^/m3.
To study the chronic action of acetophenone, a round-the-clock
exposure of white rats to acetophenone in concentrations of 0.007 and
0.07 mg/m^ was carried out for 70 days. The 45 males selected, weighing
60-70 g, were divided into three groups. The first group was exposed to
acetophenone in 0.007 mg/m3 concentration, the second to 0.07 mg/m3, and
the third was the control. The acetophenone content in the first chamber
- 86 -
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was an average of 0.007 + 0.0008 mg/m^, and in the second chamber,
0.076 + 0.002 mg/m3.
The influence of acetophenone on the organism was evaluated by
observing the behavior and weight change of the animals and studying
the chronaxy of antagonist muscles, the cholinesterase activity, and
protein fractions of the blood serum.
No peculiarities of any kind were observed in the behavior of the
animals during the entire course of the chronic experiment. The rats
were active and gained weight equally in all groups. The weight was
measured every ten days.
Numerous authors who have studied the ratios of chronaxies of
antagonist muscles have shown that this parameter may be used as a cri-
terion for the effect of noxious substances on the central nervous system,
as expressed in a decrease of subordination (Yu. M. Uflyand, 1941;
0. V. Verzilova, 1947; F. S. Trop, 1958).
The hygienic literature contains a report of a decrease in the in-
fluence of the cerebral cortex on the level of motor chronaxy of antag-
onist muscles during long-term exposure to vapors of noxious substances
in low concentrations (V. A. Gofmekler, 1961; Yu. G. Fel'dman, 1960;
G. I. Solomin, 1961, and others).
We determined the motor chronaxy by means of an electronic pulse
stimulator, model ISE-01, once every 10 days. Fig. 3 shows that the curve
of the chronaxy of extensors in rats of the first (0.007 mg/m^) and con-
trol groups is higher than the curve of chronaxy of flexors during the
entire course of observation, i.e., a normal ratio of chronaxies of the
antagonist muscles exists. In the second group (0.07 mg/m^), however,
by the end of the second month of exposure, an inverse ratio of the
chronaxies of flexors and extensors occurred.
In our study, we decided to follow the change of cholinesterase
activity as well. A sharp change of this activity is observed in many
diseases, and also when the organism is acted upon by chemical substances.
Investigators note that chemical substances (hydrogen sulfide, tetraethyl
lead, Dowtherm, phenol, etc.) in high concentrations cause an inactivation
of the enzyme, which may be reversible or irreversible, depending on the
degree to which the activity of the enzyme has been depressed. The
hygienic literature also contains studies indicating a change of cholines-
terase activity even during exposure to noxious substances in low concen-
trations (G. I. Solomin, 1961, R. Ubaydullayev, 1962, and others).
To determine the cholinesterase activity, we used the chemical method
of Pokrovskiy (1953) modified by A. P. Martynova (1957). The method is
- 87 -
-------
based on a determination of the time required for a change in the color
of the indicator to occur as a result of the pH shift during hydrolysis
of acetylcholine.
0.15-\
Dates -of examinations
Fig. 3. Average data on the chronaxy of
antagonist muscles in rats during exposure
to acetophenone.
1 - first group (0.007 mg/m3); II - second
group (0.07 mg/m?); III - control
1 - extensors; 2 - flexors; AB - period of
exposure.
The change of cholinesterase activity was observed on five rats of
each group, once every two weeks. During exposure, a change in the activ-
ity of the enzyme was observed in rats of the second group (0.07 mg/m3)
toward the second month of exposure, but these changes were not the same.
Thus, in three rats of this group (Fig. 4), there was a 22% depression
of cholinesterase activity (as compared with the background of this group
and the control group). A reverse reaction was observed in one rat,
i.e., a 45% increase in cholinesterase activity.
Acetophenone in 0.007 mg/m^ concentration (first group) did not
cause any statistically reliable changes of cholinesterase activity under
these conditions.
- 88 -
-------
in
01
so-
in
o
JO-
o
o
CO
X.
>.» tt
'^^^B^S^2Z77^^^SZSZZ^S^^l^
- — ^ -*.„,'!
'VII
Dates of examinations
Fig. 4. Influence of acetophenone on the cholinesterase
activity of whole blood of white rats.
Notation same as in Fig. 3.
Recent literature contains abundant material pertaining to the study
of blood proteins. As we know, serum proteins possessing the same mobili-
ty in an electric field can be fractionated into albumins and ex, 6 and y
globulins. In various diseases, and also., under the influence of unfavor-
able external factors, both the total amount of protein and the ratio of
the protein fractions in the blood undergo a change.
In the practice of hygienic standardization of noxious substances in
atmospheric air, the method of study of the ratio of protein fractions in
the blood was first used by R. Ubaydullayev (1961). The author explained
that furfural in 10 mg/nH concentration in long-term action causes a marked
change in the ratio of the protein fractions of the blood: the content of
albumins decreases by 33-40% when compared with the initial level. When
furfural was present in 0.33 mg/m3 concentration, the changes were less
definite in character. Only a concentration of 0.052 mg/m3 was found to
be inactive under the same conditions.
We determined the total amount of protein by means of an RPL-2
refractometer, and the protein fractions by paper electrophoresis. The
phoregrams obtained, colored with an acid blue-black dye, were cut into
fractions and eluted with a 0.1 N solution of sodium hydroxide, then sub-
jected to colorimetric analysis on FEK-N-57. The sum of the figures
obtained from the colorimetric analysis of each individual fraction was
taken as 100, then the percent content of each fraction was correspond-
ingly calculated.
To check the investigation, five rats of each group were selected.
The blood sample was taken prior to feeding, once every 20 days. No
--89 -
-------
changes of total protein were observed, but the ratios of the protein
fractions underwent a change. In rats of the second group (0.07 mg/m3),
a decrease in albumins to 24-35% was observed by the end of the first
month of exposure as compared with the initial level (38.7-48.9%) and
with rats of the control group. The amount of globulins increased cor
respondingly. At the start of the second month of exposure, a sharp
decrease of the albumin - a globulin ratio was noted. Only on the 20th
day after the end of exposure had the protein indicators of rats of this
group reverted to their initial values.
In rats of the first group (0.007 mg/m^), no changes in the protein
fractions were observed as compared with the control group (Fig. 5).
Some of the rats from each group were sacrificed after the completion
of exposure for the purpose of histopathological analysis. Changes in
the form of congestion of the cardiac vessels and a pronounced dystrophy
of the liver were observed only in rats of the second group (0.07 mg/m^).
100
IS
(ft
a
•H
I*
o *•
rH in
a c >
SO
:>o
JS
^Wrt
t»i
^i^fe!
m
Sf
31
iPM
^w/\--::
Kin- Kin
% %
Kin KIH Kin
Dates of Tests
Fig. 5. Change in the ratio of albumins and globulins in
the blood of white rats during exposure to acetophenone. ,
I - first group (0.007 mg/m3); II - second group (0.07 mg/nr);
K - control group
On the basis of the results of the studies, we can recommend an average
daily maximum permissible concentration of acetophenone in atmospheric air
at the level of the highest single concentration, i.e., 0.003 mg/m.3.
We also determined acetophenone in air around the plant section pro-
ducing acetone and phenol by the cumene process at a synthetic alcohol
plant. Acetophenone was formed as the product of a side reaction. The
sources of its discharge into the atmosphere are sections for the produc-
tion and decomposition of isopropylbenzene hydroperoxide and for process-
ing of the by-products. The discharges seep through leaks in the equipment,
packing glands, flanged joints, during the collection of samples, and
through the system of local ventilation. In addition to acetophenone, many
other substances reach the atmospheric air.
- 90 -
-------
In June-July 1962, we collected single samples of atmospheric air
around this plant at a distance of 100 to 2000 m. Acetophenone in the
samples was determined colorimetrically. Results of analyses of the
air samples are given in Table 3.
Table 3
Single Concentrations of Acetophenone in Atmospheric Air
Around a Synthetic.Alcohol Plant.
Distance from
_ Source of
Discharges, m
100
kOO
800
1,200
1,600
2,000
Number of Samples
Total
3
•26
28
25
26
25
Of these,
above the
sgnsitivity
of the method
3
25
18
11
6
0
Maximum
Concentration, mg/m-5
0,023
0,028
0,016
0,007
0,007
On the basis of the laboratory data obtained for the contamination
of atmospheric air with acetophenone, the administration of the plant was
given specific recommendations for improving the surrounding air reservoir.
We believe, however, that this is only a temporary measure.
The enterprise constitutes a large source of contamination of atmos-
pheric air x^ith various chemical substances. The only radical means of
purifying the surrounding medium would be the development of new technolog-
ical processes.
Conclusions
1. The threshold of olfactory sensation of acetophenone and its
reflex effect on the light sensitivity of the eye lie at a level of
0.01 mg/m3.
2. The threshold of the reflex effect on the electrical activity of
the brain in the most sensitive persons is 0.007 mg/m^.
' 3. Acetophenone in 0.07 mg/m^ concentration during chronic exposure
of experimental animals causes changes that are both functional (disturbance
of the ratio of chronaxies of antagonist muscles, and of protein fractions
of the blood, depression or activation of the enzyme cholinesterase) and
organic (pathological changes in internal organs).
- 91 -
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4. Under the same conditions, acetophenone in 0.007 mg/m^ concen-
tration does not cause any changes in the organism.
5. On the basis of the study, we are proposing a highest single and
average daily maximum permissible concentration of acetophenone of
0.003 mg/m3.
LITERATURE CITED
A.iCKCCcna M. B., K p 1.1 .'i o i> a H. A., X p y c T a ;i c u a B. A.
OllpCAC.>1CIMIC npO.MC>lICT MiiCTiiTyia runicim IIMCIIII
<1>. . 3piiCM.ina, 19GO.
A-;I ;i A »< a ;i o u a H. A. McA-'ieiniuc S.ICKTPHMCCKIIC iiOTCiiuna.'ii,! n
ro.iomiOM Mosry. Ha.'i.'AH CCCP, M., 1962, crp. 71—79.
A |) T e M i> c n B. B. HeiiOTOpuc oco6ciuiocTii uuauamiux s.iCKTpii'ie-
CKIIX noTciiiuia.'ioi) Kopu GO.TMUIIX iio.iyinapiiii. Octionnwc uonpocu
3JiCKT|>o(])ii3iio.noriiii uciiTpa.TMio:"! nepDiioi": CIICTCMM. MSA. AM VCCP,
KMCU, 1962.
B n c A c n c K n fi H. E. CooTiioiiicniiii MO/i\Ay PIITMIIMCCKIIMH npouccca-
MII n (JiyiiKiinoiiii.'ibiiori aKTimiiocTLio iiosOy>K,iciiiioro ncpiiiio-Miiiinc'i-
iioro viinapaTa. I'lo.Tiioc coflpaiinc co'iiniCMiiri. /!., 1952, T. 3,
crp. 8-1—97.
B c p 3 ii .'i o ii a 0 B. Dio.i.i. 3KCiicp. Cuo.n. n MC.H., 1947, T. 23, u. G,
CT|). 411—412.
FO^IIKOIJ II. B. ii3iio.'ioni'iccKiic OCHOFIW Tcopmi
rpa(])ini. B KM.: Bonpocw rcopiiu n iipaKTHKii
(j)ini. Jl.. I95G, crp. 3—26. •
f o (]) M c K .'i c p B. A. MaTCpna.ni>! K o6ocno!):niiiio iipc.'ie.ni.no ;iony-
cniMoii KoiiuciiTpaiuHi ancraron n aTMOc(])cpno.\i uo3,ayxc. /.luce.
Kan/i. M., IOGI.
/I y C p o D c K a ii O. H. riiniciiii'iccKnn oncin;a aarpnaiiciiiiocTii
ncwiyxa CoJii.moro ropo;ia ccpiiiicrwM raaoM. /.Ui^c. i. <1>. 3piicMana, 1961, crp. 16.
- 92 -
-------
n o K p o » c K >i ft A. A. BOCII.-MCA. >Kypn., 1953, A'» 9, cip. 9—13.
Co KO.HO 11 E. II. IlpiipoAa (J)Oiionou PIIT.MIIKM Kopu 6o.ni.uinx iio.iy-
Uiapllfl OCMOUIlliIC DOIlpOCbl 3jlCKTpO(p!!3HO.rIOriil! UCUTpa.nMIOf! MCpB-
iioii ciiCTe.Mbi. HBA. AM YCCP, Kiica, 19G2, cip. 157—188.
C M o .1 n n JI. M. O6 ycnoeiiini piiTwa CHCTOHUX MC.ibKaniiii npi: o-iaro-
BO.M iioBpoKAeimii iia iicpiic])cpiiii. PctjjcpaTu AOK.ia;ion 5-ii Koiujic-
pciiiliiii MO.IOAUX yiienij.x MiicTiiryTa iiopMa.nbiiofi n naro.noni'iccKoi'i
(|)M3i!0.ioriiii. M., 1959, crp. 71—72.
Cucpn nc Kiifi f. H., nparyccuii'i 10. M. flAH CCCP, 1961,
T. 139, n. 13, crp. 759—762. . .
Tpon . C. XponaiiCHsi iicpiicjicpii'iccKiix iicpiiiio-Mi.iuic'iiiLix-npnuo-
pon i! neiiTpa.nLiiLi.x AiinraTC;iMii,ix iiciiponon npH OTpiiii.iciiini >KII-
noviiiiix cepoyr.ncpOAOM, M-
no AOiiycTiiMofi KoiiuciiTpaiiiiii (Jiypcjiypo/in B aT.MOcc|)cpnoM »03Ayxc.
7-lncc. KniiA. M., 1962. . •;•
V (|) i\ r. ii A 10.-M. Teop:in n npaKTiixn xpoiiaKcii.Mcrpi'in. .0., 1941.
<;) c n i, A M :s " 10. f. fiir. 'ii can., 1960, ^?> 5, cip. 3—7.
«1> o ii r a y.3 A\. M. riiriicua rpy/ia n nponaiioACTue 4'Ciio.T.T if auero-
iia ' KyMo.nbiiwM cnocoCo.M. OT'ICT MiiCTiiTyra rni'iiciiu IIMCIIII
. ci>: SpiicMana. M., 1959, crp. 100.
I" ii n i\ c M A., 11.1 rep E. 3.ncKT|)oiniiiic ciicurpbi nor.noLH.cnnn opraiui-
'ICCKIIX • cocAiuiciiiiii. H3A. iinocTpaitiioii ^iiTCparypLi. .M., 1957,
a p. 5—331. • • " •
Lal>or
-------
CYCLOHEXANOL AND CYCLOREXANONE IN ATMOSPHERIC AIR
AND THEIR HYGIENIC SIGNIFICANCE
A. A. Dobrinskiy
Department of Coirjnunal Hygiene, Central Institute for
Advanced Training of Physicians
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 119-132, (1966).
The main object of our study was to establish the maximum permissible
concentrations in atmospheric air for two substances: cyclohexanol and
cyclohexanone. The present report presents data for the validation of the
highest single maximum permissible concentrations of these substances.
Both substances are good solvents and find broad applications in such
branches of industry as the production of leather, lacquers, dyes, and
other synthetic materials. Cyclohexanol and cyclohexanone are mainly used
as starting materials and intermediate products for syntheses. In particu-
lar, they are widely employed as intermediates in the production of capro-
lactam and adipic acid, which are the monomers in the production of the
synthetic fibers caprone and nylon.
In 1958, 40% of all the polyamide resins were obtained from capro-
lactam. The preparation of this product is one of the large-tonnage pro-
cesses in present-day industrial organic synthesis.
When cyclohexanol and cyclohexanone are used in industry, they may
escape into atmospheric air. Thus, in the production of caprolactam, the
operational cycles are carried out at high temperatures, which in the pro-
cesses of synthesis of intermediate products promotes the escape of cyclo-
hexanol and cyclohexanone into the ventilation air, etc.
Both substances studied belong to the group of aliphatic hydrocarbons
of the cyclohexane series. One of them (cyclohexanol) is an alcohol and
the other a ketone. Both are colorless liquids with similar physicochem-
ical constants (Table 1).
These indices demonstrate to some extent the similarity of the
toxicological properties, since a definite relationship has been established
between the structure of many organic compounds and their biological effect
(N. V. Lazarev, 1944; A. A. Golubev, Ye. I. Lyublina, 1962).
- 94 -
-------
Table 1
Physicochemical Constants of Cyclohexanol and Cyclohexanone.
No.
1
2
3
4
5
6
Constants
Molecular weight
Specific gravity
Boiling point
Ignition point
Temperature coefficient
in deg/mm at 760 ram Hg
Vapor pressure in mm Hg,
Refractive index for the
sodium D line 50°/15°
of vapor pressure
141. 4° /1 2. 5"
wavelength of
Cyclohexanone
100.1
0.962
160.1°
67.8°
0.045
400
1.461
Cyclohexenol
98.1
0.947
157°
64°
0.046
400
1.451
The methods of preparation of cyclohexanol and cyclohexanone have
been worked out on a large industrial scale and are carried out in a single
production cycle. The product thus obtained contains both substances.
Various means are used to separate the mixture and isolate cyclohexanol
and cyclohexanone. The starting product -in the different methods of pre-
paration may be phenol, cyclohexane, nitrocyclohexane, eyelonexylamine,
nitrobenzene, or benzene (M. M. Shumilina et al., 1959; Polhar, Sugino
Kiichiro et al., 1957; D. R. Cova, 1961).
Data on the toxicological characteristics of these substances are
scarce. Cyclohexanol and cyclohexanone are capable of irritating the upper
respiratory tract and of having a narcotic effect in high concentrations.
Inhalation of their vapors leads to disorders of the central nervous system
and changes in the picture of the peripheral blood. Both substances or
products of their transformation are excreted with the urine in the form of
compounds with sulfates, causing the excretion of bound sulfites. Cyclo-
hexanone has a marked damaging effect on the blood vessels and liver, a
depressing effect on the function of the adrenal cortex, and an effect on
the thyroid gland (G. V. Lobovikova and A. A. Preobrazhenskaya, 1950;
G. V. Lobovikova, 1954; A. P. Voronin, 1958; Treon, Crutchfield, Kitzmiller,
1943; Nelson, Ege, 1943, 1945).
• Cyclohexanone has a pronounced chronoconcentration effect (A. P. Voronin,
1958). This is confirmed by a decrease in the threshold concentrations of
the gas when the exposure is extended.
In practice, the main routes by which these substances reach the
organism are the upper respiratory tract and the skin. The majority of the
authors who have studied the influence of cyclohexanol and cyclohexanone on
- 95 -
-------
the organism have tested high concentrations of these substances.
In short-term experiments with penetration into the organism of
experimental animals by inhalation, G. V. Lobovikova tested cyclohexanol
concentrations above 250 rng/m-^, and cyclohexanone concentrations above
160 mg/m^. In A. P. Voronin's experiments, the cyclohexanone concentra-
tions exceeded 500 mg/m^.
Under chronic action conditions, the concentrations were also high:
for cyclohexanol, 400 mg/m3, and for cyclohexanone, 100 mg/m^ (G. V.
Lobovikova) and 2000 mg/m^ (A. P. Voronin).
The influence of cyclohexanol and cyclohexanone on the higher nervous
activity of animals has been the subject of several studies. Lobovikova
established that cyclohexanone vapors in 100-400 mg/m3 concentration under
conditions of chronic administration and with an exposure of 3-6 hours by
inhalation caused changes in the conditioned reflex activity of white
rats.
Voronin describes disturbances of the conditioned reflex activity of
rabbits (the study was made by using Ye. I. Lyublina's method) with an
average cyclohexanone concentration of 4000 mg/m3.
V. A. Savelova, A. S. Bruk and N. V. Klimkina (1962) observed that
the threshold dose in the study by the conditioned reflex method (rats)
was 0.04 mg/kg of cyclohexanol.
Foreign authors (Treon et al.) believe cyclohexanol to be more toxic
than cyclohexanone.
Lobovikova studied the toxicity of these substances when administered
via the gastrointestinal tract. She established that cyclohexanone causes
intoxication symptoms in lower doses.
The maximum permissible concentration for plant sections was determined
to be 10 mg/m3 for cyclohexanone and was not determined for cyclohexanol.
We have been unable to find any data on the contamination of atmospheric
air by these substances in the domestic or foreign literature.
•Q. M. Gavruseyko and A. S. Maslennikov (1950) in a survey of production
department of a caprolactam plant observed concentrations of cyclohexanone
from 1.5 to 10 mg/m^ and cyclohexanol from 0.4 to 2.8 rng/m^ in working areas
in the dehydrogenation section, and up to 14 mg/m3 of cyclohexanone and up
to 16 mg/m^ of cyclohexanol in the fractionation section. These concentra-
tions caused intoxication symptoms in the workers. Headache was the most
frequent complaint, and various autonomic disturbances were noted. The
- 96 -
-------
prime objective symptom was a tendency to develop hypochromic anemia
(cited by 0. M. Gavruseyko and A. S. Maslennikov). The authors ascribe
the principal effect to cyclohexanone.
Thus, there are data on the toxic effect of relatively high concen-
trations of cyclohexanol and cyclohexanone in both the domestic and for-
eign literature. The investigations were carried out only in acute
experiments or under conditions of chronic action with exposure lasting
up to 8 hours.
We have not found in the literature any data on the thresholds of
action of these substances, which could have been used for validating their
maximum permissible concentrations. Concentrations of the substances at
the level of those studied are lacking not only for atmospheric air but
also for the air of industrial buildings.
All of this prompted us to carry out studies that would permit the
validation of the maximum permissible concentrations of cyclohexanol and
cyclohexanone in atmospheric air. Colorimetric methods proposed by
A. S. Maslennikov (1960) were used in our studies for determining cyclo-
hexanol and cyclohexanone in air.
The method of determination of cyclohexanone is based on its conden-
sation with furfural and the formation of furfurylidenecyclohexanone. In
an acid medium, the compound turns a crimson color. The sensitivity of
the method is 2 yg/ml.
The method of determination of cyclohexanol is based on its reaction
with p-dimethylaminobenzaldehyde. In the presence of sulfuric acid, the
compound colors the solution a cherry red. The sensitivity of the method
is 5 pg/ml. The methods are nonspecific, designed for the determination
of these substances when the latter are separately present in air. How-
ever, in the experimental validation of the maximum permissible concentra-
tions, the work was done xvith each substance individually.
In view of the inadequate sensitivity of the methods described, they
were modified under the direction of M. V. Alekseyeva. A spectrophoto-
metric determination was worked out in the visible region of the spectrum
with an absorption maximum for cyclohexanol at a wavelength of 510 my, and
for cyclohexanone at 550 my, which made it possible to increase the sensi-
tivity of the methods of determination substantially (to 0.05 yg/ml for
cyclohexanone and to 0.4 yg/ml for cyclohexanol).
This increase in sensitivity was achieved by measuring the optical
density of the solutions in special glass cells with a length of 42 mm
and by modifying the method of collection of air samples.
- 97 -
-------
In the collection of air samples for cyclohexanone, the absorbing
solution used consisted of purified distilled ethyl alcohol mixed with
distilled water in the proportion of 1:2 in the amount of 3 ml. Absorb-
ers with porous plate No. 2 placed in melting ice were employed. At a
rate of sample collection of 0.5 1/min, no breakthrough into the second
absorber occurred within the sensitivity limits of the method.
During the collection of air samples for cyclohexanol, the condi-
tions were the same, but the amount of the absorbing solution was reduced
to 3 ml.
In substantiating the highest single maximum permissible concentra-
tions of cyclohexanol and cyclohexanone, we used the methods recommended
by the Committee on Sanitary Protection of Atmospheric Air. The thres-
holds of olfactory sensation of the two substances, and the reflex change
of the light sensitivity of the eyes under the influence of low cyclohex-
anone concentrations were determined, and the influence of low concentra-
tions of both substances on the electrical activity of the brain was
studied by the method of reinforcement of the intrinsic brain potentials
and by the method of development of the electrocortical conditioned reflex.
The threshold of olfactory sensation was determined for cyclohexanol
in 22, and for cyclohexanone in 15 practically healthy persons. Results
of the determination are listed in Table 2.
Table 2
Thresholds of Olfactory Sensation for Cyclohexanol
and Cyclohexanone.
Number cf Subjects
• Concentration, mg/m?
Minimum
Perceptible
Imperceptible
Cyclohexanol
1
9
8
3
1
3,26
0,66
0,52
0,35
' 0.2-1
1,71
0,54
0,35
0,25
0,20
Gyclohexanone
2
3
1
6
3
3,0
1,19
0,77
0,37
0,21
1,21
0,80
0,41
0,23
0,14
- 98 -
-------
It is evident from Table 2 that the threshold for the most sensitive
persons is a vapor concentration of 0.24 mg/m3 for cyclohexanol and 0.21
mg/m3 for cyclohexanone. Concentrations whose odor was imperceptible were
0.20 mg/m3 for cyclohexanol and 0.14 mg/m3 for cyclohexanone. In order to
substantiate the thresholds of the reflex effect on the functional state
of the nervous system by the method of dark adaptation, considering the
similarity of the thresholds of olfactory sensation of the two substances,
we decided to experiment with cyclohexanone only, which has the lower
threshold of olfactory sensation.
Three people participated in the study. The threshold of olfactory
sensation in one of them was 0.21 mg/m3, and in the other two, 0.37 mg/m3.
The investigations were carried out with an ADM adaptometer by using a
standard procedure. The active concentrations were considered to be those
at which the difference of the average values of the light sensitivity
during inhalation of pure air and of the gas concentration studied was
statistically reliable (Table 3).
Table J>
Light Sensitivity of the Eyes in the 20th and 25th minutes
of Adaptation During Inhalation of Cyclohexanone Vapors
in Percent of 15th MS&te.
Subject
L.
. B.
K.
Minute
of.
Examin-
ation
20-si
25->i
20-si
25 -si
20-si
25-si
Pure
Air
119.3
163,1
128,5
197,0
125,0
1-18,4
Cyclohexanone Concentration, mg/m3
0,11
132,0
151,0
MS, 3
198,0
1-13,0
167,0
/
1,57
1,2
3,24 '
0,2
4,3
0,4
o.oc
116,0
152,3
131,3
197,6
126,3
M5.3
/
0,7'
1,1
0,6
0.3
0,6
1,3
Table 3 shows that statistically reliable changes in the light sensi-
tivity of the eyes occurred only in the 20th minute of dark adaptation, i.e.
immediately following the inhalation of the gas, at a cyclohexanone concen-
tration of 0.11 mg/m3. This cyclohexanone concentration was found to be the
threshold value according to the method employed. A concentration of 0.06
mg/m3 was found to be inactive.
In order to substantiate the highest single maximum permissible concen-
trations of the various chemical substances in atmospheric air, a number of
researchers (K. A. Bushtuyeva, Ye. F. Polezhayev, A. D. Semenenko, 1960;
V. A. Gofmekler, 1960; Yu. G. Fel'dman, 1960; Li Sheng, 1961; G. I. Solomin,
1961; R. U. Ubaydullayev, 1962; B. M. Mukhitov, 1962; D. G. Odoshashvili,
1963, and others) successfully used the method of development of the
- 99 -
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electrocortical conditioned reflex. Low concentrations of the chemical
substances which had no subjective influence on the olfactory system
caused distinct changes in the electrical activity of the brain.
Of late, there has been an increasing emphasis in electrophysiology
on a trend dealing with the bioelectric indicators under conditions where
a functional load is applied to the brain. The methods used are those of
rhythmic stimulation (V. Ye. Mayorchik and B. G. Spirin, 1951; N. I. Zis-
lina, 1955; A. G. Kopylov, 1956, 1960, and others), and also the method of
change in the electrical activity of the brain during the action of a
rhythmic photic stimulus of increasing brightness ("responsiveness curves,"
M. N. Livanov, 1944). In these tests, the characteristics of the encepha-
lographic responses to stimuli are used to evaluate the indexes of the
condition of the brain.
The method of electroencephalography with functional loading as applied
to the determination of permissible concentrations of atmospheric pollutants
was developed by A. D. Semenenko (1963) and used by a number of researchers
(N. B. Imasheva, 1963; T. G. Tkachev, 1963; V. A. Chizhikov, 1963).
To elucidate the influence of low concentrations of cyclohexanol and
cyclohexanone on the electrical activity of the brain, we used two methods:
the method of development of the electrocortical conditioned reflex, and the
method of electroencephalography with functional loading. The electroencepha-
lographic studies were made with a Kaiser 8-channel electroencephalograph with
an electrophotostimulator.
During the examination, the subject was placed in a state of relative
rest in a special electrically shielded room equipped for these purposes.
During the devebopment of the electrocortical conditioned reflex, the
unconditioned stimulus was light causing desynchronization of the a rhythm,
and the conditioned stimulus was the inhalation of various concentrations
of cyclohexanol and cyclohexanone. The gas was supplied to the subject for
15 seconds, and the last five seconds v?ere reinforced with light. The con-
ditioned reflex was considered formed and the concentration of the gas active
if desynchronization of the a rhythm occurred after a series of pairings
before the light was turned on.
In the second method, the electrical activity of the subject's brain
was recorded during exposure to intermittant light of changing intensity.
The intensity of the flickering light changed every five seconds within a
range from 0.1 to 0.6 Joule over the course of 25 seconds of recording of
the reinforced rhythm curve. The reinforced rhythm curves were recorded
every minute. After the third minute of the examination, a given concen-
tration of the gas studied was mixed in with the pure air fed into the
cylinder and breathed by the subject. After the sixth minute, the gas
supply was discontinued, and a period of close and distant aftereffect was
- 100 -
-------
observed. Usually, 11-12 reinforced rhythm curves were recorded. Thus,
the entire time of the study was divided into four periods: I - background,
II - action of the gas, III - close aftereffect, IV - distant aftereffect.
Each period lasted 3 minutes.
Each curve was analyzed from the change of the amplitude of the
intrinsic rhythm. For convenience in carrying out the comparative analysis,
the total amplitude for all the intensities was expressed in percent. The
average amplitude of the first three reinforced rhythm curves was taken as
100%.
The amplitude of reinforced rhythm in the gas experiment was compared
with the amplitude of assimilated rhythm in experiments with pure air.
Data for different periods of the study were also compared. The results
were subjected to statistical treatment. The tests with cyclohexanone
involved six people and those with cyclohexanol, five people. The results
are given in Tables 4 and 5.
Table 4
Effect of Cyclohexanol on the Electrical Activity of the Brain.
Subject
V.
T. .•
S.
L.
M. '
Method
Rhythm
Reinforcement '
» >
» V
£o/uiitioned Reflex
j t
Concentration, mg/m*
0.2
+
+
+
0
0
" 0.14
—
0
-h
+
0.11
— •
—
+
+
O.OG
0
0
0
—
"
Notation: + action; - absence of action; 0 - not studied.
Table 5
Effect of'Cyclohexanone on the Electrical Activity of the Brain.
Subj-ect
± •• •
N.
V. '
R.
L.
M.
Method
Rhythm
Reinforcement
» »
» >
» »
Conditioned Reflex
» »
Concentration, mg/m?
0.09
• -f-
—
+
+
0
0
o.oc
—
0
—
_
-f-
-(-
0.0'!
0
0
0'
0
—
• —
Notation same as in Table 4.
- 101 -
-------
In the study of the influence of cyclohexanol vapor inhalation on
the change of the electrical activity of the brain, concentrations of 0.2,
0.14, 0.11 and 0.06 mg/m3 were investigated.
In the study by the method of reinforcement of intrinsic brain
potentials, a gas concentration of 0.2 mg/m^ was found to be the threshold
value for three people. In two out of three subjects, the gas in this
concentration caused a decrease (Fig. 1) and in one person, an increase in
the amplitude of the imposed rhythm. In 0.11 mg/nP concentration, the gas
did not cause any change in the electrical activity of the brain in any of
the subjects.
In the study with the method of development of the electrocortical
conditioned reflex, a cyclohexanol concentration of 0.11 mg/m3 was found
to be the threshold value in both subjects. A concentration of 0.06 mg/m^
was inactive.
•H C
i—( -H
Q. fl)
;io-
100-
30-
80-
70
Fig-
1 -
j 1 1 1 1 i 1 1 ' 1 1 i—
/ 2 3 * 5 S 7 S S 10 II J2 '
tj.r.e of examination,, minutes
1. Effect of cyclohexanol on the electrical activity of the
brain in subject V.
pure air; 2 - concentration, 0.11 mg/m3; 3 - 0.14 mg/m ,
4 - 0.20 mg/m'.
o
•o
0> GJ
•a o
3 t,
w-
x^-
30-
10-
\ X '7
v X/
• t
I2'31 5
"'-""" \
1 X-
i i i i
S 7 S S
£$*»*
W II 11
time of examination, minutes
Fig. 2. Effect of cyclohexanone on the electrical activity of the
brain in subject I.
1 - pure air; 2 - concentration, 0.06 mg/m5; 3 - C.09 mg/m'.
- 102 -
-------
In the study of the influence of inhalation of cyclohexanone vapors
on the electrical activity of the brain, three concentrations were inves-
tigated: 0.09, 0.06 and 0.04 mg/nH. In the investigation by the method
of reinforcement of intrinsic brain potentials, a concentration of 0.09
mg/m3 was found to be the threshold value for three people. In two persons
this concentration caused a decrease and in one, an increase (Fig. 2) in
the amplitude of the imposed rhythm. The 0.09 mg/m^ gas concentration was
found to be inactive for one subject.
Cyclohexanone in 0.06 mg/m^ concentration caused no change in the
electrical activity of the brain in any of the subjects. The electrocorti-
cal conditioned reflex was formed with the 0.06 mg/m3 concentration in both
subjects. In this method, a gas concentration of 0.04 mg/m3 was found to
be inactive.
Table 6
Thresholds of Action of Cyclohexanol and Cyclohexanone on Man.
Substance
Cyclo-
hexanol
Cyclo-
hexanone
Olfactory
Sensation
Thres-
hold
0.2k
0.21
Subthres-
hold
0.20
0.14
Light Sensitivity
of the Eye
Thres-
hold
Not
Deter-
mined
0.11
Subth res-
hold
Not
Deter-
mined
0.06
Electroencephalography
Rhythm
Reinforcement
Thres-
hold
0.20
0.09
Sub-
thres-
hold
0.14
0.06
Conditioned
Reflex
Thres-
hold
0.11
0.06
1
Sub-
thres-
hold
0.06
0.04
Highest
Maximum Per-
missible
Concentration,
mg/m5
0.06
0.04
Thus, the concentrations of 0.06 mg/m^ of cyclohexanol and 0.04 mg/m^
of cyclohexanone did not cause a change in the electrical activity of the
brain in any of the subjects. Comparative data on the thresholds of action
of the two substances are listed in Table 6.
Conclusions
1. The threshold of olfactory sensation in the most sensitive persons
is 0:24 mg/m.3 for cyclohexanol and 0.21 mg/m^ for cyclohexanone.
2. The threshold of change in the light sensitivity of the eyes during
inhalation of cyclohexanone vapors lies at a level of 0.11 mg/m.3.
3. The threshold of the reflex effect on the electrical activity of
the brain for cyclohexanol lies at a level of 0.11 mg/m^ and for cyclohexanone
- 103 -
-------
at a level of 0.06 mg/m^.
4. Both substances caused a change in the electrical activity of
the brain in lower concentrations when studied by the method of develop-
ment of the electrocortical conditioned reflex than when studied by the
method of reinforcement of intrinsic brain potentials.
5. Cyclohexanone has lower thresholds of olfactory sensation and
of effect on the electrical activity of the brain.
6. The highest single maximum permissible concentrations in atmos-
pheric air v/hich we recommend are 0.04 mg/m3 for cyclohexanone and 0.06
mg/m^ for cyclohexanol.
LITERATURE CITED
G y m T y c n a K. A., FI o .1 c >i< a e u E. cl>., C c M e ii e n K o A. J3,. Fur.
ii can., I960, JY M c K Ji c p B. A. fur. n can., 1960, ,\b •!, crp. 9.
3 ii c Ji ii u a H. 1-1. ^KypuaJi ni.iciucfi ncpniioii ACfiTo.:u.!!OCTii ic.iciin
H. n. llar.jiona, 1955, T. V, n. 5, cip.'677.
M M a in c D a I-I. B. Tur. n can., 1953, JVs 2, crp. 3.
]\ o ii i.i .M o r. A. T. OueiiKa (jiyiiimnoiin.nwioro COCTO;HI:M ro.ioimoro
Moara :.:'CTO;IO.M ajicnTpoanucebaJiorpaijiii'iccKiix Kpnni.ix ycnocimn p;;r-
r,ia. B KM.: BOMJIOCL.; rcopini 11 upaKTiiiti: 3.icnTpo3!mc(l)a.norpn('pi!ii.
• .n.,'l95G, cip. 90,
K o ii M.Ji o n A. F. Mc'io/i. a:iCKTp03i!HCf|>a.'!orpa(!>!iliocKi!.\ Kpiioux ycuo-
cinisi pin-Ma nnn iisyiciuifi ^:yiiKunona.i!.noro cocioHir.in rojioniioro
v.oara 'ICJICIICK.T. B KII.: Bonpocbi 3.ncKTpot[ni3iio.ioniii n siiuciji.i.io-
rpacl>iiii. M.—JL, 19GO, 41.
JI a 3 a p c n II. B. HcajicuTpoJiiiTi,!. OIIUT Ciio.noro-fpissiiKO-xiiMiiiocKof:
1IX CMCTCMaTHKH. JI., 19;i4.
JI n n a i! o n M. H. Kpnuwc a.icKTpii'iccKofi pcai;T!:r,iiocr:i Kopu ro.nor,-
noro Moura >KIICOTIIIJX n Mc.noncKa n nop.Mc n naTO-ioriiM. l-Isncciii!!
AH CCCP. CepiiJi Ciio.ioniii. 19-H, .K° G. cip. 331, 339.
JI n Ulan.. Fur. n can., 1061, A*; 8, CTp. 11.
JI o 6 Q u n K o r. a f. B. ripiiMciiciiiic Mctoaa yc.io::iiu.\ po(p.'icKcon ,a.ir:
OUCIIKIl TOKCIS'ICCKIIX KOllHCHTpaiUli'l IlpOMWlll.'ICIIMI.-IX HAOI1. Tc3liCW
AOKJia/ion MO.iOAi.ix naynihix paCoTiiiiKOB. rop'uKiii'i. 195'1.
JI o 6 o ii i! !•: o ii r. T. B., fl p c o 0 p a >u c n c K a n A-. A. K uonpocy oo
OIICIIKC To;
-------
M ;i c n c n n n K o n A. C. OiipeAc.nciuic uiiK-'iorciccaiio/ia. OII|)CAC.:ICH:IC
Ui:i;.'ioivi«Mi:on:i. B KM.: Oiipc.ac.nciinc Dpc;im.ix UCIUCCTU u no:i,iyxc
npoi!nno;;.CT[ii;iiiii>i.s iio.MOinciiiii'i. I'opi.Kiifi, I9GO.
My xii TOD !j. Al. Alaiop:ia.ii,i K oGociioiianmo upc/uvibiio AOiiycTii-
.MOM KoiiiicHTpauiiii (j)cno.ia u nT.\ioc(|)cpiio.M iioa.'iyxc. J^ncc. i:aiiA.
Al., 1902.
OAOiiiniiiuii.il! /.I. f. AlaTCpiia.iu K oOociionnmno npeAC.-u-iio AO-
iiyciiiMoi'i Komeinpamm MCTii.ni c u n. F. Biio.ioni'iccKoe ACMCTIIHC Ma^i.ix KOMUciiTpam'.ri aim-
Aiinn n riiriicnii'iccKoc 3iia
-------
ON THE COMBINED ACTION OF THREE MINERAL ACIDS
Candidate of Medical Sciences V. P. Melekhina
Department of Communal Hygiene, Central Institute for Advanced Training of Physicians
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 133-141, (1966).
The study of the combined action of various environmental factors has
attracted the attention of many hygienists. The necessity of studies of
this type has been emphasized in resolutions of the 13th and 14th congresses
of hygienists.
In recent years, in connection with the rapid growth of the chemical
industry in the Soviet Union, studies in the area of hygienic standard-
ization of a series of combinations of atmospheric pollutants have been
considerably expanded. The combinations of sulfur dioxide and sulfuric
acid aerosol (K. A. Bushtuyeva, 1961), chlorine and hydrogen chloride
(V. M. Styazhkin, 1962), and carbon disulfide and hydrogen sulfide
(B. K. Baykov, 1963) have been studied. The authors showed that in the
range of low concentrations which may occur in the air of populated areas,
the character of the action of two atmospheric pollutants is determined as
a total or partial summation. However, the insufficient number of studies
in this area does not yet permit one to state that any combination of
atmospheric pollutants will be characterized by an additive synergism.
The object of the present study was to establish the nature of the
combined action of three mineral acids - sulfuric, nitric and hydrochloric.
The study of this combination is of practical importance, since the source
of their joint discharge into the atmosphere are chemical complexes and
inorganic fertilizer plants whose number and capacity (particularly the
fertilizer plants) are increasing considerably in the Soviet Union. The
combination under consideration is also of major scientific interest,
since all three acids are strong electrolytes and in dilute solutions
dissociate completely into hydrogen ions and the corresponding acid resi-
due. The more dilute the acid, the less its specific properties are mani-
fested and the more manifest are the properties common to all the acids,
due to the hydrogen ions. In atmospheric air at low concentrations of
these acids, we encountered those properties of the acids which are
characteristic of dilute solutions. In this connection, the question arises
whether mineral acids have an effect due solely to the hydrogen ion and
whether they should therefore be standardized on the basis of this indicator
alone. For example, can the concentration of acids be expressed in milli-
grams of hydrogen ion per m3 of air? If one could obtain a confirmation of
this hypothesis, the control of the purity of atmospheric air in populated
areas would be considerably simplified.
- 106 -
-------
To achieve the stated objective, we used methods widely employed in
the hygienic standardization of maximum permissible concentrations of
atmospheric pollutants. These were the methods of determination of the
threshold of olfactory sensation and the very sensitive method of dark
adaptation.
The experiments were conducted by using a procedure recommended by
the Committee on the Sanitary Protection of Atmospheric Air.
In determining the precise concentrations of the volatile acids in
the experiment, xve could not use their determination from the correspond-
ing anions, since in addition to the anions of these acids, anions of their
corresponding salts are also present in air and are therefore determined.
On the other hand, we found it impossible to use the microtitrimetric method
of determination of hydrochloric acid (M. V. Alekseyeva, 1963) because of its
inadequate sensitivity. For this reason, in collaboration with Candidate of
Biological Sciences M. D. Manita, we worked out a spectrophotometric method
of determination of mineral acids in the presence of their salts. The method
is based on the measurement of the optical density of colored aqueous solu-
tions of the acids in the presence of a 0.01% alcohol solution of methyl red.
The color depends on the pH of these solutions in the range from 5.3 to 4.3,
vrtiich corresponds to a concentration of hydrochloric acid from 0.18 to 1.83
yg/ml, nitric acid from 0.31 to 3.15 yg/ml, and sulfuric acid from 0.24 to
2.4 )Jg/ml. The sensitivity of the determination of hydrochloric acid was
0.18 yg/ml, sulfuric acid 0.24 yg/ml, and nitric acid 0.31 yg/ml.
Other acids and alkalis interfere with the determination. Sulfuric
acid in amounts up to 1 mg/m-^ and carbon dioxide in amounts up to 0.05
vol. % do not interfere.
Optimum conditions were selected for the spectrophotometric analysis:
the aqueous solutions of the acid were determined in the visible region of
the spectrum at Ymax = 530 my in a cell with a thickness of 1 cm. A cali-
bration graph was plotted for each acid.
The samples were collected by using a procedure recommended by
M. V. Alekseyeva, i.e., into double-distilled water in a V-shaped absorber
with a porous plate at a rate of 0.5-1 1/min.
In starting the work we considered the fact that sulfuric and hydro-
chloric acids had been standardized for the atmosphere of populated areas
(0.3 and 0.05 mg/m^). We had to check the data obtained earlier and in
the case of discrepancies, to introduce corrections. The influence of
low concentrations of nitric acid had not been studied and its maximum
permissible concentration for atmospheric air had not been established.
- 107 -
-------
We began the study of the effect of low concentrations of the acids
on the human organism with the determination of their thresholds of
olfactory sensation. The observations were made on 16 people 17 to 36 years
of age. A total of over 800 tests were conducted (including 415 for the
determination of the threshold of olfactory sensation of hydrochloric acid,
243 for nitric acid, and 148 for sulfuric acid). Results of the determina-
tion are listed in Table 1.
Thus, the thresholds of olfactory sensation for the most sensitive
persons were found to be 0.70 mg/m3 for nitric acid, 0.39 mg/m3 for hydro-
chloric acid, and 0.60 mg/m3 for sulfuric acid.
Table 1
Thresholds of Olfactory Sensation of Mineral Acids.
Number of
Subjects
Threshold Concentrations
ng/nr
' H+, mg/m3
Subthreshold Concentrations
tng/m5
H+, mg/m5
Nitric Acid
1
3
7
I
1
1 .
1
0.70
0,80
0,85
0,90
. 1,07
1,25
. 1.50
0,01099
0.01256
0,01334-
0.01413
0.01679
0.01962
0,02355
0,60 :
0,70'
0,70]
0.85
0,85
1.07
" 1,40]
0,00942
0.01099
0,01099
0,01334
0,0133-!
0,01679
0,02198
Hydrochloric Acid
3
7
3
0.39
0.40
0,45
0,01053 .
0,01080
0,01215
0,30
0,32
0,40
0,0081
0,0086
0.010S
Sulfuric Acid
2
8
1
0,60
0.74
0,81
0,0120
0,0148
0.0162
0,43
0,00
0,74
0, 0096
0,0120
0,0148
The thresholds were equal in hydrogen ion concentrations: for nitric
acid - 0.01099 mg/m3, for hydrochloric acid - 0.01053 mg/m3, and for
sulfuric acid - 0.0120 mg/m3. As is evident from the data cited, the figures
defining the thresholds of olfactory sensation in hydrogen ion concentrations
are rather close to each other.
The subthreshold concentrations of the acids were found to be as
follows: for nitric acid 0.60 mg/m3, for hydrochloric acid 0.30 mg/m3, and
for sulfuric acid 0.43 mg/m3; in hydrogen ion concentrations this corresponds
to 0.00942, 0.0081, and 0.0096 mg/m3. Essentially, it may be said that these
figures are of the same order.
- 108 -
-------
The results of our studies differ from the data on the threshold of
olfactory sensation of hydrochloric acid obtained earlier by Ye. V.
Yelfimova. Unfortunately, we do not know the reason for this, but we can
surmise that a factor is our use of a more sensitive method of determina-
tion of the acids.
The threshold concentrations of olfactory sensation of sulfuric acid
are essentially no different from the existing literature data (K. A. Bush-
tuyeva, 1961). Having determined the thresholds of olfactory sensation
separately for each acid, we undertook the determination of the thresholds
in different combinations. Initially, we studied the threshold of olfactory
sensation of a combination of the too acids - nitric and hydrochloric.
Twelve people participated in the experiments. A total of 86 determinations
were made. Nine combinations were studied. The results are given in Table
2. The concentrations of the acids are expressed in fractions of the thres-
hold concentrations for each acid observed and are summed up.
All the perceptible concentrations in terms of the hydrogen ions were
above 0.01 mg/m^. All the imperceptible combinations in terms of the
hydrogen ion xvere found to be below 0.01 mg/m3. Analyzing the data from
the standpoint of the combined action of the acids we can state that we
are dealing with a simple summation.
Table 2
Thresholds of Olfactory Sensation of the Sura of the Two Acids
(Nitric and Hydrochloric). . .
Subject
N.
M.
S.
G.
E.
T.
B.
K.
L.
.YiU
• B.
. A.
Threshold. Concentrations
Sum of
Fractions
1,17
LOG
LOG
• 0,97
0,99
0,99 .
1,0
1,1-1
•1,10
0,99
1,0
0,99
Sum of Acids
in H+tmg/m?)
0,0176-!
0,01201
0,01283
0,01283
0,01283
0,0120.1
0,01283
0,01332
0,01283
0-, 01 20-1
0,0120-1
0,01282
Subthreshold Concentrations
Sura of
Fractions
0,SG
0,76
0.9S
0,G9
0,76
0,76
0,93
0,71
0,71
0,76 '
0,77
Sum of Acids
in H+Cmg/V)
0.0083G3'
0,003353 •
0,0120-10 •
0,008363 '
0,008353
0,008363
0,008363
0,008-165
0,003-1 65
0,008363
0,008363
0,69 t 0,008363
t
The olfactory sensation threshold of the sum of the three acids was
determined on nine people. A total of 83 observations were made and ten
pairings of different combinations of the three acids were studied. The
results are given in Table 3.
- 109 -
-------
Table 3
.Olfactory Sensation Threshold of the Sum of the Three Acids.
Subject
N.
M.
S.
Ch.
.E.
T.
B.
K.
L. '
Threshold Combinations
Sum of
Fractions
0,98
1,14
1,1-1
1,10
1,06
1.13
1.01
1.16
0,99
Sun of Acids
in
H* (mg/m3)
0,01103
0,01555
0,01555
0,01565
0,01555
0,01555
0,01268
0,01573
0,01268
Subthreshold Combinations
Sum of
Fractions
0,96
0,93
0,93
0,78
0,88
0.81
0,88
0,96
0,94
Sum of Acids
in
H* (mg/ra5)-
0,01101
0,01187
0,01187
0,01103
0,01187
0,01103
0,01103
0,01187
0,00870
It is clear from Table 3 that a perceptible combination of acids was
that in which the sum of the fractions of threshold concentrations exceeded
unity. All the imperceptible combinations were those in which the sum was
less than unity. On the basis of the data obtained it may be concluded
that the threshold concentration of the sum of the three acids for the most
sensitive persons in terms of the hydrogen ion is 0.01103 mg/m3.
Thus, the threshold concentrations obtained, expressed in hydrogen ions
of the individual acids, as well as of combinations of two and three acids,
are almost at the same level. This is to say, the principal physiological
effect in terms of the threshold of olfactory sensation is due to the con-
centration of hydrogen ions in the solutions of these acids.
The next stage of the present study consisted in investigating the
reflex effect of the mineral acids on the light sensitivity of the eye.
The tests were conducted on three people, 17, 22 and 32 years old.
The thresholds of olfactory sensation of all three acids were deter-
mined for two subjects, and that of sulfuric acid only was determined for
the third subject (Table A).
The investigations of dark adaptation were started with the determina-
tion of the individual thresholds for each acid separately, and then for
their mixture. The estimate of the influence of the total acids was also
expressed in fractions of the threshold values. The gas was inhaled start-
ing with the 15th minute of adaptation for 4 1/2 minutes. As a result of
the study, the thresholds listed in Table 6 were established.
- 110 -
-------
Table 4
Thresholds of Olfactory Sensation of Acids in Persons
Participating in Adaptometric Tests.
Subject
T.
K.
R.
Threshold Concentrations of Acids
Nitric
mg/m5
0.85
0.80
H4', ng/m5
0.01354
0.01256
Hydrochloric
mg/ra^
0.40
0.40
H+, mg/m5
0.0108
0.0108
Sulfuric
mg/m^ H+, mg/m5
0.74 0.0148
0.74 0.0148
0.73 0.0146
Comparing the results of the determination of the thresholds of
olfactory sensation and thresholds of the reflex effect (Tables 4 and 5),
one can observe that they are essentially quite similar. In this respect
our results are in complete agreement with the data obtained earlier in a
study of the acids by other authors (K. A. Bushtuyeva, 1961; Ye. V. Yelfi-
mova, 1962; V. M. Styazhkin, 1962).
Table 5
Threshold Concentrations of Acids Determined by the
Dark Adaptation Method.
Concentrations of Acids
Subject
T.
K.
R.
Nitric
mg/m5
0,SGT(c)
0,85 (a)
0,84 (b)
H+, n)g/m3
0.0135
0,0133
0,0131
Hydrochloric
mg/ta'
0,10 (b)
0,40 (a)
0,42 (b)
H+, rag/m?
0,0108
0,0108
0,0113
Sulfuric
rag/m3
0,80 (c)
0,81 (b)
0,73 (b)
•!+, mg/m^
0,0160
0,0161
0,0146
Note. Confidence factor: a - 95$, b - 99$, c - 99-9$.
We then studied the influence of inhalation of different combinations
of the three acids on the dark adaptation. The effect was estimated in
the same manner as was indicated in the determination of the threshold of
olfactory sensation. The data obtained are listed in Table 6.
The active combination of acids was found to be the one in which the
sum of the fractions of the threshold concentrations exceeded unity.
- Ill -
-------
Table 6
Effect of Inhalation of the Sum of the Three Acids on Dark
Adaptation in the 20th Minute of the Experiment.
Subject
T.
R.
K.
Note. Con
Sum of Acid Fractions
of Threshold Values > 1
Light Sensi-
tivity in
Percent of
Background
130 (a)
78 (c)
140.4 (p)
fidence factor
Concentra-
tion of Sum
of Acids in
H+, mg/mJ
0.02070
0.02018
0.01908
a - 95*, b
Sum of Acid Fractions
of Threshold Values < 1
Light Sensi-
tivity in
Percent of
Background
117.5
94.4
97
- 99%, c - 99.9?!
Concentra-
tion of Sum
of Acids in
H+, mg/m3
0.01057
0.01158
0.00625
;.
Conclusions
1. The established thresholds of olfactory sensation of nitric,
sulfuric and hydrochloric acids are respectively equal to 0.70, 0.60 and
0.40 mg/m3, which in terms of the hydrogen ion per cubic meter corresponds
to 0.01099, 0.01203 and 0.01053 mg/m3.
2. A mixture of two and three acids is perceived when the sum of the
fractions of their threshold values exceeds unity. The perceptible con-
centrations expressed in hydrogen ions per cubic meter are above 0.01
mg/m^.
3. The threshold concentrations of the reflex effect, studied by
the dark adaptation method, lie at the level of the threshold values, or
slightly above, for olfactory sensation. The combined action manifests
itself as a type of additive synergism.
4. Our data are in good agreement with the assumption that the reflex
effect of mineral acids (nitric, sulfuric and hydrochloric) in the atmos-
pheric air of populated areas is determined by the concentration of the
hydrogen ions.
5. The results of the studies make it possible to propose that the
contamination of atmospheric air by the mineral acids which we studied be
evaluated on the basis of the concentration of hydrogen ions alone, inde-
pendently of the anionic residue of the acid.
6. The highest single maximum permissible concentration of the sum
of these acids and separately for each acid in terms of the hydrogen ion
should not exceed 0.010 mg/m.3.
- 112 -
-------
LITERATURE CITED
A .1 c K c c c o a M. B. Onpc.ae.icni!c x.iopiicroro ucaopo.ia —
co.iMiiofi Kiic.noTiii Onpc,o.e:icni!c aTMOCtbcpm.ix sarpfniieimfi, 1963,
CTJ). 28—31.
B a ii K o ii 5. K. Fur. n can., 1963",^ 3, crp. 3—S.
b y 111 T y c D a K. A. Honwe Aamibic o pctji.ncKTOpiio.M .iciicTiiim cepiii'.-
croro rnaa n a3po?o.'in ccpiiofi KIIC.IOTU na "ic.noneKa. ripc.T.c.ii.!!o .T.O-
nycTii.Muc Kciinciiipnu.1111 ar.MOCijicpiiiiix sarpjisneinifi, 19G1, ». 5,
^cip. 118—126. . '
E .1 <}) n M o D a E. B. Marepna.Tb! K nii'iiciiii'iecKori onciiiic
co.:i5inoii Kiic.iOTii! (x.iopiiCToro noAOpo.ia) ic
HHTe.na. npejc.iMio .lonyciiiMMc KoimcitTpatuiii aiMoccJicpnux sarpus-
nciiiifi, 1962, n. 6, crp. 31— 49.
M a 11 n T a M. A., Me.TexiniaB.il. Tur. 11 can., 196-!, ». 3,
crp. 53—56. •
CTO/KXIIII B. M. Maicpna.'iH K riiriicni!'iccKo:.iy oCociionaiuiio npc-.
Ao.ibiio AonycTiiMux Konnciirpaniiii x.nopa n x.iopiicroro noAopo.ia
npii nx COUMCCTIIOK npiicyrcTnnii B aiMOcAcpnoM no3,iyxc. FlpCAC.-.h-
110 AonycTiiMLie Koiinciirpaumi arMocibepHux snrpjisiicniii'i, 1962, B. 6,
crp. 96-105.
- 113 -
-------
TOXICITY OF SULFUR OXIDES UNDER CONDITIONS
OF LONG-TERM CONTINUOUS EXPOSURE
Decent K. A. Bushtuyeva
Department of Communal Hygiene,
Central Institute for Advanced Training of Physicians
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 142-172, (1966).
This paper presents materials and results of studies with various con-
centrations of sulfur dioxide, sulfuric acid aerosol, and their combinations
under conditions of extended continuous exposure. The studies were made on
white rats in seven series of experiments. A total of 120 animals, with 15
in each series and the control group, were used. The sulfuric acid aerosol
used in our experiments had a particle size of less than 2 y. The following
tests were utilized to study the resorptive effect of sulfur oxides: behavior
and weight of the animals, ratio of chronaxias of antagonistic muscles, whole
blood cholinesterase, serum protein fractions, content of coproporphyrins in
the animals' urine, and pathomorphological studies following the completion
of exposure.
Tests x^ith sulfur dioxide. The toxicity of sulfur dioxide has been
dealt with in many studies, but the majority of the latter cannot be dis-
cussed in this present paper, since they deal with very high concentrations.
They will therefore be omitted here.
We studied the resorptive effect of sulfur dioxide on white rats in
two series of experiments under conditions of continuous exposure in the
course of 65 days. The actual concentrations for each series were (M i CJ)
in series I, 4.86 ± 1.26 mg/m3, and in series II, 8.53 — 2.1 mg/m3. The
air samples were taken from the chambers and analyzed daily. The indicated
sulfur dioxide concentrations did not affect the behavior or weight of the
animals.
The chronaxia of the antagonist muscles of the right hind limb was
examined every ten days according to the usual procedure with the aid of
an ISE-01 electronic stimualtor. This method has been widely adopted in
the practice of hygienic studies for the substantiation of maximum permis-
sible concentrations (R. V. Borisenkova, 1954; Chzhao Chzhen-tsi, 1959;
V. A. Gofmekler, 1961; Yu. G. Fel'dman, 1962; V. A. Chizhikov, 1963, and
others) as the method for studying the state of the central nervous system.
Data on the ratio of chronaxias of extensors to flexors are presented
- 114 -
-------
in Fig. 1. As we know, the normal ratio, which is a relatively constant
value ranging from 1.15 to 1.30, does not always remain greater than
unity. Under the influence of various factors, these normal ratios may
change, and in the presence of deep shifts, become inverted and fall below
unity.
As is evident from Fig. 1, sulfur dioxide in the concentrations studied
affects the central nervous system. The changes in the presence of the
8.53 mg/m^ sulfur dioxide concentration were more pronounced, more constant,
and statistically more reliable. Recovery of the initial ratio was observed
only 17 days after the completion of exposure. At a lower concentration
(4.86 mg/m^) , the changes were less constant and rapidly returned to the
initial values after the exposure was discontinued.
in >.J'
CO
3 '•'•
§ ...
t-, '•'
s:
° >,#•
t«*
• H
•*•* • 03-
cU ' •
ce .
J^-7^
-..-*""-^""
.
x\
~^c;r,*V^£<^"1S"
C
;
>^*^P._- — — .
*•*' / "^ '
/
-^^
"**C
Time of observation
Fig. 1. Ratio of chronaxias of antagonist muscles in animals
exposed to sulfur dioxide.
AB - period of exposure; 1 - control group, 2 - series I (4.86
irg/m3); 3 - series II (8.53 mg/m5); a, b, c - confidence factor
of the changes (respectively 95, 99 and 99.9$).
We studied the activity of whole blood cholinesterase by Pokrovskiy's
method (1953) modified by Martynova (1957). Recently, this method has
also been widely adopted in studies of the influence of chemical substances,
and has proven highly sensitive and reliable (G. I. Solomin, 1961; R. Ubay-
dullayev, 1961, and others). The cholinesterase activity was determined in
five rats of each series and control group at 15-day intervals (Table 1).
A decrease in the time of decomposition of acetylcholine chloride and
hence, an increase in cholinest.erase activity was observed in both series
as early as the first determination follox^ing the start of exposure, i.e.,
on the 13th day. This increase grew during the exposure and reached its
highest values toward its end. The recovery of cholinesterase activity was
gradual and reached the background values only on the 17th day following
the end of exposure.
-------
Table 1
Time of Decomposition of Acetylcholine Chloride of Whole Blood of
Rats Exposed'to Sulfur-Dioxide.
Date of
Investigation
Background on the basis of 17
•determinations '...
Exposure. 23/1V
7/V
2!/V
4/VI
Recovery period 18/VI
29/VI
Concentration, mg/ra'
series I
4.86
41,1
35,6 (a)
35,4 (c)
31,8 (c)
25,8 (c)
38,8 (a)
40,4 •
series II
8.55 "
41,4
32,0 (c)
32,6 (c)
25,7 (c)
24.2 (c)
3G.2 (c)
40,6
Note.
. a -
and ff., the
*, c - 99.$
confidence factor of the changes is:
as compared with the background.
The protein fractions of the blood serum were determined by paper
electrophoresis followed by recording of the phoregrams on photographic
paper and weighing of the sections of individual fractions. The percent
content of each fraction was determined from the weight. The determina-
tion was made every 20 days in five rats'of each group (Table 2).
Table 2
Protein Fractions of the Blood Serum in Percent During Exposure
' ' to'Sulfur Dioxide.
Period of
Observation
Background
Exposure
Recovery
Fraction
A Ibumins •
° - Globulins
" • -Globulins
7 • Globulins
Albumins
a - 'Globulins
?' ~ -Globulins
7 ' Globulins
Albumins
a - Globulins
? - Globulins
7 " Globulins
Concentration, mg/rn^
Control
40,20
18,1
24,1
17,6
40,4.
' 17,7
25,5
16,9
38,2
18,7
25,6
18,4
Series I
4.86
40,25
18,1
24,1
17,6
35,8 (b)
17,5
27,0 (b)
19,6 (a)
35,3 (c)
19,5
24,6
20,5
Series II
8.55
40,26
18,1
24,1
17,6
37,8
14,8 (b)
28,6 (c)
18,7
32,2 (b)
19,4
26,8
21,6
In the control group of animals, there were no reliable changes during
any of the periods as compared with the background, whereas under the in-
fluence of sulfur dioxide, a decrease of the percent content of albumins and
a corresponding increase of the globulin fractions, particularly $ globulins,
took place.
- 116 -
-------
To evaluate the degree of influence of different sulfur dioxide con-
centrations on the protein fractions, Table 3 lists data during the exposure
and recovery periods in percent of the background concentrations. The table
shows that at the 4.86 mg/rn^ concentration changes are observed in the per-
cent content of albumin, (3 globulin, and y globulin fractions, whereas at
the 8.53 mg/my concentration there is a shift in all the fractions.
Table 3
Change in Protein Fractions Under the Influence of Sulfur Dioxide
in Percent of Original Values._
Series
1(4.86 ms/m3)
11(8,53 mg/m3)
Exposure
Albu-
mins
11,1
6,2
Globulins
a
0
18,3
P
12,0
18,6
•••
11.3
6,2
Recovery Period
Albu-.
mins
12, '1
20.0
Globulins
a
0
0
p
0
0
{
0
22,7
The last index, which we used to evaluate the effect of sulfur dioxide,
was the excretion of coproporphyrins in the urine. As an indicator of the
general biological shift in the organism, this method has lately begun to
be extensively applied to the substantiation of maximum permissible concen-
trations of atmospheric pollutants (G. I. Solomin, 1962; B. G. Odoshashyili,
1963; V. A. Chizhikov, 1963; P. G. Tkachev, 1963).
The coproporphyrins. were determined in a 24-hour portion of urine
collected from five rats of each series every ten days. A quantitative
determination was carried out by using a spectrophotometric method
(Yu. K. Smirnov, M. I. Gusev, 1956).
The data obtained are shown in Fig. 2, where data on the excretion of
coproporphyrins by animals of the control group (M i. 3a) are crosshatched.
As can be seen from Fig. 2, the 8.53 rng/m-^ concentration of sulfur dioxide
causes an increase in the content of coproporphyrins excreted in the urine
as early as four weeks after the start of exposure. At the 4.86 mg/m^ sul-
fur dioxide concentration, this increase is already observed 1 1/2 months
from the start of exposure. The change in the content of coproporphyrins
in both series was found to be unstable, and disappeared on the 9th day after
the exposure was discontinued.
Autopsy of animals subjected to the action of sulfur dioxide in 4.86
mg/m-* concentration immediately after exposure did not show any pathologico-
anatomical changes. The internal organs and brain of the rats were sub-
jected to histopathological analysis. Microscopic examination of the lungs
of all the rats of this series showed the presence of small foci of inter-
stitial pneumonia and of emphysema in the adjoining areas (Fig. 3), a slight
swelling of the collagenous fibers in the adventitia of the blood vessels
- 117 -
-------
and bronchi, and in some cases a catarrhal-desquamative bronchitis. The
elastic fibers in the stroma of the lung were evenly distributed over the
entire cross section. Substantial changes were observed in the trachea of
the animals: desquamation of the mucous membrane, and lumps of mucopurulent
content in the lumen of the trachea. Examination of the cerebral cortex
revealed a subdued hyperchromatism of individual pyramidal neurons of layer
V of the cortex, turbidity of the cytoplasm, and small foci of gliosis. The
cerebellum showed angularity and wrinkling of the Purkinje cells. Golgi
staining in the top layers of the cortex, primarily in layer III, showed the
apical dendrites of certain pyramidal neurons to be devoid of spinelike
processes and to have swellings and beaded thickenings. Neurons arranged in
rows were unchanged. Such changes were absent in animals sacrificed at the
end of the recovery period.
to
8
Time of Observation
Fig. 2. Content of coproporphyrins in the urine of experimental
animals exposed to the action of sulfur dioxide.
Notation same as in Fig. 1.
Autopsy of animals subjected to the action of sulfur dioxide in
8.53 mg/m3 concentration, sacrificed immediately after the exposure was com-
pleted, did not show any macroscopic changes. Small foci of pneumonia and
hyperemia of the spleen were found in rats killed at the end of the recovery
period.
Microscopic examination of the lungs showed areas of interstitial
pneumonia, emphysema in the adjoining areas, and catarrhal-desquamative
bronchitis. The elastic fibers in the injured areas of the lung frequently
appeared broken, coiled and spiral. The collagenous fibers in the adventitia
of the blood vessels and bronchi were swollen. Toward the end of the recovery
period, the number of histiocytes filled with the pigment increased. In the
- 118-
-------
trachea, there was necrosis and desquamation of the mucosa over a large
area (Fig. 4), and hyperplasia of the mucosa with papillomatosis. The
lumen of the trachea had a purulent content and blood clots. There was
hyperplasia of the lymphatic follicles of the submucosa.
f
Fig. 3. Lung of white rat No. 5 of- series I. Diffuse
thickening of interalyeolar septa caused by infiltration
of cellular elements into them. Staining with hematoxylin-
eosin. Magnification 20x5.
The liver had areas of dystrophy, and the heart, fine and isolated
blood extravasations in the myocardium. No changes were established in the
kidneys or other organs. In the cerebral cortex, the cloudiness of the
cytoplasm and hyperchromatism of certain pyramidal neurons of layers II and
V of the cortex were noted. There were isolated and small foci of gliosis,
and, in the cerebellum, areas of fallout and hyperchromatism of the Purkinje
cells. The medulla oblongata had no special characteristics.
When Golgi staining was used, individual neurons whose apical dendrites
were devoid of spines and had beaded thickenings and swellings (Fig. 5) were
found among the cells of different layers more often than in animals of
series I. In animals sacrificed at the end of the recovery period, the
changes were considerably evened out.
Studies with sulfuric acid aerosol. The high toxicity of sulfuric acid
aerosol as compared with that of sulfur dioxide has been demonstrated by
several investigators (Treon et al., 1949; Amdur, 1953, and others).
Our early work (K. A. Bushtuyeva, 1956, 1961) indicates that changes in
the pulmonary tissue such as interstitial pneumonia with a change in the
content of histamine are observed at a sulfuric acid aerosol concentration of
- 129 -
-------
Fig. 4. Trachea of white rat No. 24 from series II.
Necrosis and desquamation of mucosa over a large area.
Staining with hematoxylin-eosin. Magnification -
- magnifying glass.
1 mg/m3 and after a continuous exposure of 120 hours. When the exposure lasted
one month, the initial symptoms of irritation of the pulmonary tissue are also
observed at a concentration as low as 0.1 mg/m3.
-
?;'-*
Fig. 5. Brain of rat No. 17 of series II. Apical
dendrites of neurons are devoid of spines and have
beaded thickenings and swellings. Golgi staining.
Magnification 40x10.
The present investigations were conducted in two series on white rats.
The actual concentrations of sulfuric acid aerosol in the chambers were
1.04 ± 0.057 mg/m3 (series III) and 1.8 ± 0.162 mg/m3 (series IV). The same
tests as in the preceding series I and II were employed.
- 120 -
-------
Data on the weight of the animals did not shoxj any substantial changes.
Results of the study of the ratio of chronaxies of antagonist muscles
for animals of series III and IV are shown in Fig. 6. A disturbance of
the normal ratio under the influence of sulfuric acid aerosol occurred in
animals of both series only a month after the start of exposure. In animals
of series III, it is preferable to speak of a convergence of the chronaxies,
and in those of series IV, of an inversion of the normal ratio. Results of
the determination of cholinesterase activity in animals of series III and
IV are presented in Table 4.
in
CO
•H
X
cc
c
o ,
[_
c.
o
«-.
0
o
•H
CO
fr.
/.<"•
'./•
» <7
/?/-
^/-
N-v'' Xx^
•vv . ^. — _^_ ,: _—•*,,
''*•*£''' ""-5'
v"x,-'' \ /
b 'v'
i
/ "~^
/ *'
/ f' I
/
'
Time of observation
Fig. 6. Ratio of chronaxias of antagonist muscles in animals
exposed to sulfuric acid aerosol. ,
AB - period of exposure; 1 - series III (1.04 mg/m ); 2 - series
IV (1.8 mg/m5); a, b, c - confidence factor of the changes
(95, 99 and 99.9$ respectively).
Thus, sulfuric acid aerosol in the concentrations studied, like sulfur
dioxide, causes a decrease in the time of decomposition of acetylcholine
chloride, i.e., an increase in cholinesterase activity. The recovery of the
activity took place only six days after the completion of exposure.
Table 4
Time of Decomposition of Acetylcholine Chloride of Whole Blood of
Rats F-xposed to Sulfuric. _Acid .Aerosol.
Date of
Background on the
4 determinations
Exposure
Recovery period
Investigation
basis of
23/1V
7/V
21 IV.
4 IV I
18/Vi
29/VI
Concentrations^ mg/m'
Series in
' 1.0
41.8
-10,6
37,4 (c)
36, S (c)
34,0 (c)
•12,0
40,0
Series IV
1.8 '
40,5
37,8 (c)
33,6 (n)
30,0 (c)
30,0 (c)
41.0
40.0
- 121 -
-------
Results of a study of the total blood serum protein in animals of
series III and IV showed no statistically reliable changes when compared
with the background data. The ratio of the protein fractions changed
significantly, however. At the same time, there was a decrease in the
percent content of albumins and an increase in $ globulins without a
change in the other fractions (Table 5).
Table 5
Protein Fractions of the Blood Serum (in Percent) During Exposure
to Sulfuric Acid Aerosol.
Period of
. Observation
Background
Exposure
Fractions
a -
P-
7'-
a -
P-
Recovery period
7-
a -
P-
7-
Albumins
Globulins
Globulins
Globulins
Albumins
Globulins
.Globulins
•Globulins
Albumins
Globulins
Globulins
Globulins
Concentrations, mg/m
Series III
1.0
'10,26
18,1
2-1,1
17,6
3.1 , 2 (c)
18,6
27, 3 (a)
18,7
3G,5
17,5
27,1
18,9
Series IV
1.3
40.26
18,1
2-1,1
17,6
33,6 (c)
17,7
29, 1 (c)
19,6
33, 8 (c)
20,05
26,2
19,95
Table 6
Change in the Albumin-(3 ^Globulin Ratio During Exposure to
Sulfuric Acid Aerosol.
Series
HI. )
IV J
Background
1.G7
Exposure
. I,lS-l(c)
1,181 (c)
Recovery Period
1,3-16
1,380
As is evident from Table 6, a considerable decrease of the albumin-6-
globulin ratio takes place under the influence of Sulfuric acid aerosol.
Interesting results were obtained in experiments on animals of series
III and IV with administration of coproporphyrins (Fig. 7). The amount of
coproporphyrins is expressed in micrograms per 100 g of animal weight. The
interval within the range of the physiological norm has been crosshatched.
As is evident from the data cited, sulfuric acid aerosol in 1.8
concentration (series IV) caused a marked increase in the amount of copro-
porphyrins only seven days after the start of exposure. At the end of the
first month, the amount of coproporphyrins reached 3.139 yg per 100 g for
an average norm of 0.305 yg per 100 g. However, this index gradually
- 122 -
-------
decreased, and 1 1/2 months later corresponded to the physiological norm.
A fresh increase was observed toward the end of the exposure. This course
was approximately repeated by the curve of excretion of coproporphyrins
for series III as well, but a sharp increase was first observed only one
month and eight days after the start of exposure.
/ B '
o
0
T 0,6-
0.2-
^// % % 7/y % ?S/v ?/V, % % %
Time of observation
Fig. 7. Content of coproporphyrins in the urine of rats
exposed to sulfuric acid aerosol.
Notation same as in Fig. 6.
A pathomorphological analysis of animals of series III, sacrificed
both immediately after exposure and at the end of the recovery period,
showed the presence of focal pneumonia in the lungs and congestion of the
spleen.
Microscopic analysis of the lungs revealed interstitial pneumonia
with swelling of collagenous fibers of interstitial tissue, bronchi, and
adventitia of the blood vessels, catarrhal bronchitis, and emphysema of the
lungs. The elastic fibers were severely loosened and, in areas of emphy-
sema, fragmented. No changes were noted in the trachea.
In the kidneys, there were dystrophic changes in the epithelium of
the convoluted tubules and wrinkling of the glomeruli. The liver had small
areas of dystrophy and degeneration with signs of venous congestion. There
were fine myocardial extravasations of blood. The spleen showed a hyperemia
of the pulp and a marked hemosiderosis.
- 1.23 -
-------
The cerebral cortex had focal hyperchromatism of some pyramidal neurons
of layers II and V. Fine foci of gliosis and fallout of cells of Ammon's
horn and their replacement by microglia were observed. In the cerebellum,
there was fallout and wrinkling of the Purkinje cells.
Golgi staining showed, among pyramidal neurons of layers III and V,
certain neurons having beaded enlargements and swellings with a lack of
spines, but they were few in number. Unchanged neurons with even dendrite
contours covered with spines were observed more frequently.
A pathomorphological examination of animals of series IV, sacrificed
both immediately after exposure and at the end of the recovery period revealed
large hemorrhagic foci in the lungs and hyperemia of the spleen.
Microscopic examinations of organs of animals sacrificed both immediately
after exposure and at the end of the recovery period gave approximately the
same results.
In the lungs, there was observed a large-focus and interstitial
pneumonia consisting in a diffuse enlargement of the interalveolar septa
as a result of accumulation of histiocytes, lymphocytes, and a small number
of polymorphonuclear leucocytes. The lumina of the alveoli were empty, the
collagenous fibers of the interstitial tissue were swollen, the elastic
tissue was markedly loosened and consisted of a tangled network with breaks
of the fibers in areas of emphysema (Fig. 8). The obliterated alveoli were
threaded with elastic and argyrophil fibers (Fig. 9). There was venous con-
gestion and catarrhal-desquamative bronchitis. In the kidneys, liver and
heart, the changes were similar to those in animals of series III.
•KV:
'
'*'/.•• •''''"' ""^i
' ' ('.".•> • ,
Fig. 8. Lung of white rat No. 60 from series IV.
Elastic tissue is loosened and consists of a matted
network with rupture of the fibers in areas of
emphysema. Weigert staining. Magnification 40 x 10.
-JL24 -
-------
Examination of the cerebral cortex showed hyperchromatism of neurons
of layers II and V of the cortex. There was focalgliosis, wrinkling and
serious damage to neurons shown by Nissl staining. The cerebellum showed
wrinkling and fallout of Purkinje cells. There was focalgliosis in the
medulla oblongata. Golgi staining showed some neurons whose apical dendrites
were devoid of spines and had beaded enlargements and swellings (Fig. 10).
The main mass of neurons contained spines on the dendrites.
:?$
ft
Fig. 9. Lung of rat No. 58 from series IV. Obliterated
alveoli are threaded with argyrophil fibers. Tibor -
Papp staining. Magnification 40 x 10.
Fig. 10._ Brain of_rat No. 60 from series IV. Neuron
whose apical dendrite has beaded enlargements and swell-
ings. No spines are present. Golgi staining.
Magnification 40 x 10.
- 125 -
-------
Studies of the combined action of sulfur oxides. We studied three
combinations of sulfur oxides. In the first combination (series V), a
sulfur dioxide concentration of about 5 mg/m^ was used (4.79 — 1.51 mg/m3)
x^ith a sulfuric acid aerosol concentration of 1 mg/m^ (0.92 i 0.211 mg/m3) .
This combination should have been helpful in evaluating the nature of the
combined resorptive action of sulfur oxides. The other two combinations
(series VI and VII) made it possible to substantiate and evaluate the
existing maximum permissible concentrations (average daily values) of sulfur
oxide present together. Therefore, the nature of the combined action will
be evaluated first on the basis of materials of series V, and then the
results of the study in series VI and VII will be assessed.
The first combination of sulfur oxides had no effect on the general
state and weight of the experimental animals, either in absolute indices
of weight or as expressed in percent. Data on the ratio of chronaxies of
antagonist muscles are shown in Table 7.
Table 7
Ratio of Chronaxies of Antagonist Muscles in Animals of Series V,
Dates of
Background
Exposure
Recovery period
Investigation
G/I1I
1/IV
- 22/IV
1/V
13/V
2 1/V
31/V
7/VI
12/VI
21/IV
29/VI
Ratio of Chronaxies
1,19.1
1,104
1,115
1,11-1
1,108
1,013 (a) •
0,9-lS (a)
O.S65 (c) '
0,870 (c)
0,80-1 (c)
1,110
The data cited show that the combined action of sulfur dioxide after
one month and 10 days causes the chronaxies to become similar, and then
the ratio of the chronaxies becomes reversed. Comparison of the data cited
with the effect of each individual sulfur oxide (Fig. 1 and 6) shows that
the combined action leads to a greater inversion of the chronaxies, as shown
by lower values of the ratio and also the presence of an inverse ratio in
the first week of the recovery period. This is characteristic of all five
rats-of series V. A normal ratio prevailed during the isolated action of
each oxide during this period.
To compare the action of sulfur oxides, we calculated the percent
decrease of the ratio of chronaxies during the periods of exposure and the
recovery period for each oxide from double concentrations and their combina-
tion (Fig. 11). The ratio of chronaxies in the control group was taken as
- 126 -
-------
Fig. 11. Comparative evaluation of the
action of sulfur oxides on the ratio of
chronaxies of antagonist muscles in
percent in enimals of series I, II, III,
IV and V.
1, 4 - sulfur dioxide in concentrations of
4.86 and 8.53 icg/m5 respectively; 2, 5 - sul-
furic acid aerosol in concentrations of 1 and
1.8 mg/m3 respectively; 3 - combination of
sulfur oxides in concentrations of 4.79 ng/m*
of sulfur dioxide and 0.92 mg/ra^ of sulfuric
acid aerosol.
100%. It is evident from a comparison of these data that the total decrease
of this index when each oxide is used separately (9.25 JL 8.8 = 18.05) exceeds
the decrease in the ratio of chronaxies during inhalation of the mixture of
the same concentrations (12%) by slightly less than a factor of 2. We com-
pared the change of the ratio during inhalation of the combination (series V)
with 1/2 the sum of the changes in the ratio of double concentrations of
sulfur oxides (series II and IV). In such a comparison, one-half the sum of
the percent decrease of the chronaxies for double concentrations of each
oxide (2A.25%) is approximately equal to the change in the ratio of chron-
axies during the combined action of sulfur oxide (12%) in series V of the
study. These data show that the inhalation of a mixture of sulfur oxides
causes a change in the ratio of chronaxies corresponding to a simple summation.
Results of the investigation of cholinesterase activity of the blood
of rats in all five series of the exposure are shown in Fig. 12.
It is obvious from the data obtained that during inhalation of the
mixture of sulfur oxides, the decomposition time of acetylcholine chloride
is shortest, i.e., the increase in cholinesterase activity is most pronounced
At the same time, during the two weeks of the recovery period, there was a
statistically reliable although slight increase in activity.
Fig. 13 gives comparative data on the increase of cholinesterase
activity (in percent of the initial value) in the period of exposure and
recovery, i.e., those periods during which the changes were observed. These
data indicate a significant increase of the effect during inhalation of the
mixture of sulfur oxides as compared with the sum of the effects during the
- 127 -
-------
Time of observation
Fig. 12. Time of decomposition of acetylcholine chloride
during different exposures to the action of sulfur oxides.
1 - series I (sulfur dioxide) - 4.86 mg/m3; 2 - series II
(sulfur dioxide) - 8.53 mg/rP; 3 - series III (sulfuric
acid aerosol - 1.04 mg/np;; 4 - series IV (sulfuric acid
aerosol -1.8 mg/m5); 5 - series V (sulfur dioxide -
4.79 rag/iiP, sulfuric acid aerosol - 0.92 rag/m'); AB - period
of exposure.
action of each of them individually (33.3% for series V versus 15.8% + 6.8%
= 22.6%). The effect of increase of cholinesterase activity in the first
case substantially exceeds the effect for the double concentrations of each
oxide and is very close to their sum.
33,3* .)
w
«
I/.SS'-
Fig. 13. Comparative evaluation of the
effect of sulfur oxides on the increase
of cholinesterase activity in average
percentages for the period of exposure
and the recovery period.
1, 4 - sulfuric acid aerosol in concen-
trations of 1.04 and 1.8 mg/m3 respectively;
2, 5 - sulfur dioxide in concentrations of
4.86 and 8.53 rag/mf respectively; 3 - combin-
ation of sulfur oxides in concentrations of 4.79
mg/ip5 of sulfur dioxide and 0.92 mg/m? of sul-
furic acid aerosol.
In a study of the total blood serum protein, we did not obtain any
reliable changes during the period of exposure to the combination of sulfur
oxides. However, during the recovery period, the increase in the content
of total protein was statistically significant. Thus, whereas during the
period of background measurements the total protein content was 7.67 mg %,
during the exposure period it increased to 7.85 mg %, and during the
recovery period climbed to 8.10 mg %.
- 128 -
-------
Comparison of the results obtained with data for sulfur dioxide
(series I) and sulfuric acid aerosol (series III) in animals of these
series did not show any reliable changes in the content of total protein
of the blood serum.
Thus, the combination of sulfur oxides causes greater changes in this
index than does each oxide individually. The protein fractions of the
blood serum for animals of series V are listed in Table 8.
Table 8
Protein Fractions of the Blood Serum (in percent) During Inhala-
tion of fixture of Sulfur Oxides (Series V).
Protein Fraction
Albumins
o - Globulins
P - Globulins
7 - Globulins
Periods of Observations
Background
'10,26
18,1
24.1
17,6
Exposure
35,6 (c)
16,2 (b)
29,0 (c)
19,2 (b)
Recovery
Period
32, 6 (c)
19, T
28,5'(b)
19,7 (a)
As can be seen from Table 8, a shift takes place in all the protein
fractions under the influence of the mixture of sulfur oxides. At the same
time, there is a decrease in the percentage of albumins and a globulins
with a sharp increase of 8 and y globulins.
An evaluation of the character of the reaction to sulfur oxides when
the latter are present together is shown in Table 9. Statistically signifi-
cant changes in the various fractions in all the series are indicated as a
percent change relative to the background data. The plus sign marks an
increase of the content, and the minus sign marks a decrease.
Table 9
Comparative Evaluation of Changes in the Protein Fractions in
Percent Under the Influence of Sulfur Oxides.
Series
1
111
Sum
V
11
IV
Sum
• Concentrations
mK/m? •
SO,
4.86
—
4,79
8,53
—
H:SO,
—
1,04
0,92
—
1,8
Albumins
— 11.1
-15,1
-26,2
-11,6
— 6,2
— 16,6
—22,8
Globulins
a
0
0
0
— 10,5
— 18,3
0
— 18,3
q '
-i-13,6
-i-13,2
+ 26, S
H-19,8
-MS, 6
-i-20,7
-'•39,o
v
1
-i-11.3
0
-•-11,3
-1-9.1
0
0
0
- 129 -
-------
From Table 9 it may be concluded that each concentration of sulfur
dioxide causes a particular reaction: a low concentration (4.86 mg/m3)
decreases the percent content of albumins during the period of exposure
and increases it for 6 globulins; a double concentration causes a smaller
decrease of the percent content of albumins, but decreases it for a glob-
ulins as well, without changing the percentage content of y globulins. A
change in the latter in the presence of a double concentration of sulfur
dioxide is manifested only during the recovery period. Sulfuric acid
aerosol causes more regular shifts in the protein fractions, decreasing
the percent content of albumins and increasing the content of 6 globulins
when it is present in both lower and higher concentration.
In a comparative evaluation of the combined action of sulfur oxides,
attention is drawn to the great similarity of the response reaction with
double concentrations of each oxide. Thus, one-half the sum of double
concentrations for each oxide is nearly equal in effect to the combination
of their half-concentrations. This is particularly apparent in the case
of a and (3 globulins: for the combination during the period of exposure,
there was a 10.5% decrease in a globulins versus !"• 3^ = 9.15% for double
2
concentrations. The same is observed in the ratio of 6 globulins: 19.8%
versus 19.6% respectively. A good agreement is given by this calculation
for albumins and $ globulins during the recovery period. This analysis of
the material on the protein fractions leads to the conclusion that there is
a simple summation of the effects during inhalation of a mixture of sulfur
oxides. It would be difficult to expect a higher degree of agreement be-
tween the results when the experimentation is performed on a living organism.
Results of the determination of coproporphyrins in the urine of animals
of series V are shown in Fig. 14. An increase in the amount of copropor-
phyrins in the urine was already observed seven days after the start of
exposure. No such rapid reaction was noted in animals of series I (sulfur
dioxide - 4.86 mg/m3) and series III (sulfuric acid aerosol - 1 mg/m3).
When each oxide was used separately, the first reaction was manifested
1 1/2 months after the start of exposure in animals of series I and one
month and eight days in animals of series III. In series with double con-
centrations of sulfur oxides, a reaction was already observed one week after
the start of exposure in animals of series IV (sulfuric acid aerosol) -
1.8 mg/m3).
.After comparing the data on coproporphyrins in all 5 series, x^e came
to the conclusion that their excretion in the urine during the action of
the mixture of sulfur oxides is approximately the same as for isolated
action, but the first reaction appears earlier.
Focal pneumonia and hyperemia of the spleen were established by
pathologico-anatomical autopsy of animals of series V both after the exposure
and at the end of the recovery period.
- 130 -
-------
Time of observation
Fig. 14. Content of coproporphyrins in the urine of animals
of series V.
1 - control group; 2 - combination of sulfur oxides; AB - period
of exposure.
Microscopic examination of organs of the animals, sacrificed immediately
after exposure and at the end of the recovery period, revealed approximately
identical changes. Large-focus and diffuse interstitial pneumonia was dis-
covered in the lungs. There was catarrhal-desquamative bronchitis and
catarrhal-purulent tracheitis with hyperplasia of the mucosa of the trachea
(Fig. 15). There were peribronchial and perivascular edema and sclerosis,
and an irregular thickening of the vessel wall. The elastic framework was
diffuse, and the fibers were frequently broken (Figs. 16, 17). The pneumonic
segments were threaded with a tightly woven elastic network. The histiocytes
in the lungs of rats of this series had short, teratic processes, larded with
tiny lumps and coarse grains of pigment (Fig. 18).
I
Fig. 15. Trachea of rat No. 68 from series V. Catarrhal-
purulent tracheitis with hyperplasia of the mucosa. Stain-
ing with hematoxylin-eosin. Magnification 10 x 10.
- 131 -
-------
iT'SSs.^y-. i -"^V^ |w';:^wv-
v -•• "••• . • •-: ;,.
tig-a
PI Vlll »
Fig. 16. Lung of rat No. 66 from series V. Diffuse
elastic framework, frequently broken and ceiled fibers.
Weigert staining. Magnification 40 x 10. '
Examination of the cerebral cortex revealed areas of wrinkling and
hyperchromatism of pyramidal neurons of layers II and V of the cortex.
There was fallout of cells of Ammon's horn and their replacement with
microglia. There were perivascular cuffs, consisting of round cells, edema
in the lower parts of the brain, hyperchromatism in the cerebellum, and
angularity of Purkinje cells (Fig. 19). In the medulla oblongata, the con-
tours of large neurons were smooth. There was gliosis and neuron ophagi a.
In Golgi staining, among normal neurons with well-defined spines on
their dendrites, there are fairly frequent neurons whose apical dendrites
are devoid of spines and have beaded enlargements and swellings (Fig. 20) .
Such changes disappear toward the end of the recovery period.
Two other combinations of sulfur oxides (series VI and VII) were
studied in order to check the existing maximum permissible (average daily)
concentrations of sulfur oxides when present together. These two series of
studies were carried out in different periods. The actual concentrations
were (M ± a) , in series VI: sulfur dioxide 0.182 - 0.067 mg/m3, and sulfuric
acid aerosol 0.104 — 0.042 mg/m^. In series VII, the concentrations were:
sulfur dioxide 0.101 ± 0.25 mg/m3, and sulfuric acid aerosol 0.099 ± 0.008
-* (versus the given concentrations of 0.1 mg/m^ for each oxide).
We did not observe any influence of the combinations studied on the
behavior and weight of the animals. The weight gain of the animals was the
same in both the experimental and control groups.
- 132 -
-------
Results of the study of the ratio of chronaxies of antagonist muscles
for animals of series VI are given in Fig. 21. A normal ratio of chronaxies
in animals of series VI was preserved during the first month and half of
exposure. However, at the end of the second month of exposure, a converg-
ence began which proved to be statistically reliable on 7/VI. The week after
the completion of exposure, the ratio of chronaxies reached the background
values. Thus, the combined action of sulfur oxides in concentrations close
to the maximum average daily values for each oxide manifests itself after
two months of continuous round-the-clock exposure, causing a convergence of
the chronaxies. Despite the fact that statistically significant changes
were obtained during only one period of observation, we are forced to
acknowledge this combination as being active on the central nervous system.
The conclusion drawn from group data is supported by analysis of material
on the individual rats of this series.
teSvt^*
Fig. 17. Lung of rat No. 71 from series V.
Ruptures of argyrophil fibers. Tibor-Papp stain-
ing. Magnification 40 x 10.
" ;•'•>•
•.:. '
Fig. 18. Lung of rat No. 64 from series V. Histio-
oytes with short teratic processes filled with small
lumps and coarse grains of pigment. Beletskiy stain-
ing. Magnification 20 x 10.
- 133 -
-------
Vjp; - *•;•"— --J- ^^~r--n ••--•»••"-
1, ' ':; •:, • '.."&.
<&'•>'&'&*.
% $$
.fe: v
i
Fig. 19. Cerebellum of rat No. 74 from series V.
Fallout, hyperchromatism and angularity of Purkinje
cells. Staining with hexatoxylin-eosin. Magnifica-
tion 40 x 10.
V
Fig. 20. Cerebral cortex of rat No. 6l from series V.
Neuron whose apical dendrites are devoid of spines and
have beaded enlargements and swellings. Golgi staining.
Magnification 40 x 10.
- 134 ~
-------
o
o
-p
Time of observation
Fig. 21. Ratio of chronaxies of antagonist muscles in animals
of series VI.
AB - period of exposure; 1 - control group; 2 - series VI;
c - confidence factor of the change, 99.9$.
In animals of series VII, the normal ratio of chronaxies was preserved
during the entire course of the observation. There was not a single case
of convergence or crossing of the chronaxies in the individual animals,
despite the fact that the exposure was extended to 75 days.
Results of determination of cholinesterase activity in animals of
series VI showed that the second combination of sulfur oxides causes a
statistically reliable decrease in the decomposition time of acetylcholine
chloride, i.e., an increase in cholinesterase activity. This increase in
cholinesterase activity also manifests itself only at the end of the second
month of exposure. Analysis of the material for individual rats shows a
decrease in decomposition time to 30 minutes (or 40 minutes in background
measurements) .
No changes in cholinesterase activity in animals of series VII were
observed, either in average indices or in the analysis of the material for
the individual animals.
No statistically reliable changes in the content of total protein were
observed in animals of series VI or VII.
Results of the study of the ratio of protein fractions of the blood
serum in animals of series VI are presented in Table 10. Data for the
period of exposure are divided into two groups: the first includes materials
for 4 and 22/V, i.e. , the first 1 1/2 months of exposure, and the second for
6/VI; i.e., the end of the second month of exposure.
From Table 10 it is evident that reliable changes in the ratio of pro-
tein fractions were observed only at the end of the second month in a
single fraction, the 3 globulins. The percent content of albumins also
decreased, but not in all the rats, so that the average value of the differ-
^ence proved to be statistically insignificant. During the recovery period,
-------
the decrease of the percent content of albumins was reliable, without
appreciable deviations in any of the globulin fractions.
Table 10
Protein Fractions of Blood Serum in Percent in Animals of Series VI.
Protein Fractions
a -
P-
7 -
Albumins
Globulins
Globulins
.Globulins
Back-
ground
-10,26
18,1
24,1
17,6
Exposure
4 and
22/V
39,7
17,9
2-!, 9
17,5
6/VI
35,0
18,7
29,4 (c)
16,9
Recovery
Period
• 35,0 (a)
1S.S
24,7
21.4
The combined action of sulfur oxides in concentrations of 0.1 mg/m^
each did not cause any changes in the ratio of the protein fractions
(Table 11).
The last index which we used for the evaluation of the effects of the
investigated concentrations of sulfur oxides was the content of copropor-
phyrins in the urine of the experimental animals. The sulfur oxides in
combinations of series VI and VII did not influence the excretion of copro-
porphyrins. Nor were any changes observed in the histopathological examin-
ination of the internal organs and brain of animals of these series.
Summing up the results of the study with the last two combinations of
sulfur oxides (series VI and VII), one can conclude that the inhalation of
a mixture of sulfur oxides in concentrations close to the average daily
maximum permissible ones for atmospheric air is not without effect under
conditions of prolonged continuous action. This is demonstrated by a de-
crease in the subordinational influence of the central nervous system,
manifested by a convergence of the chronaxies of the antagonist muscles,
and also by an increase in cholinesterase activity and a change in the
protein fractions of the blood serum, chiefly as a result of an increase in
the percent content of 8 globulins. All these disturbances occurred at the
end of the second month of exposure. The above suggests that the combined
presence of sulfur dioxide and sulfuric acid aerosol in the atmospheric air
of populated areas is not permissible in concentrations at the level of the
existing maximum permissible average daily concentrations for each oxide.
At the same time, the combined presence of sulfur dioxide and sulfuric acid
aerosol in concentrations of 0.1 mg/m^ each, when the period of exposure is
extended to 75 days, turns out to have no effect on the animals. If this
concentration is expressed in fractions of the maximum permissible average
daily values for each oxide, the sum is 1.66 (0.1:0.15 + 0.1:0.1).
- 136 -
-------
Table 11
Protein Fractions of the Blood Serum in Percent in Animals of Series VII.
Group of
Animals •
Control
Series VII. .
Background
Albu-
mins
41,5
41,0
Globulins
a
19,9
19,2
?
2-1,1
2-1,9
. T '
14,5
14,9
Exposure
Albu-
mins
39,4
40,2
Globulins'
a
19,0
17,7
P
26,0
25,8
7
15,3
16,2
The results of the last series of the experiment on the combined action
of sulfur oxides indicate that the joint presence of sulfur dioxide and
sulfuric acid serosol in the atmospheric air of populated areas (average
daily concentrations) may be evaluated by using a formula derived from the
principle of simple summation:
m
where a is the observed sulfur dioxide concentration; m is its maximum per-
missible (average daily) concentration; b is the observed concentration of
sulfuric acid aerosol, and n is the maximum permissible average daily con-
centration.
Conclusions
On the basis of the above, it may be concluded that a long-term
continuous inhalation of a mixture of sulfur oxides enhances the effect of
the influence of each individual oxide according to additive synergism.
Therefore, the joint presence of sulfur dioxide and sulfuric acid aerosol
in the atmospheric of populated areas (average daily concentrations) should
be estimated by using a formula derived from the principle of simple
summation.
-..137 -
-------
LITERATURE CITED
B o p n cc ii K o 13 a P. B. Tiir. n can., 1954, Al> 1, crp. 2-1—29.
byiuTycnn K. A. Fur. n can., 1956, jY° 2, crp. 17—22.
B y m T y c u a K. A. 3KciiepiiMCi!Ta.'ibiii.jc Aaiim.ic o B.innnnn Ma.iux
KoiiucnTpniiiiri OXIIC.IOB ccpu iia opramnM WHUOTIIUX. B cC.: I'lpc-
AC.Hbiio AOiiycTii.Muc KOimcHTpaniin aTMOCc|)cpnu.\ 3.irp 1,
cip. 35—37.
C o .n o M ii n I'. M. riiniciiii'iccxasi OUCIIKH Aiiim.ia Ka:c sarpnainiTC.nfi
ar.Moccjicpiioro iios.iyxa. B cG.: FlpeAC/ii'iio AoiiycriiMUC Koiiuciirpa-
HUM imioc(|)cpin>ix 3arpn3iiciu:ii. 1962, c. 6, M6—164.
TKa'icn n. T. Fur u can., 1963, A» 4, crp. 3—11.
V 0 a ii A y .n n a e n P. Sarpsmiciiiic ar.MocmepHoro sosAyxa (Jn'ptbypj-
AO.M n cro riinicini'jccKcTj! onciiKa. B co.: npCAC.ni>!io AOiiycTiiMi,:c
KoiinciiTpauiii: aT.voc(!)ep!».!x 3arp«3nciinii. 1961, 7, crp. 11—31.
C.M L A M a i! 1O. P. AUCTOII K.TK aT.\iocc[)Cp;ioe sarpcsiieiine. B co.:
ripcAC.Tbiio Aonyciii.Mi.ie KOHncin-pauini aiMocipepMux sarpjuiiciiiii!.
1962, n. 6, crp. 109—127.
ll >K a o M >K e n • mi. fur. n can., 1959, B. 10, ci-p. 7—12.
M n >i< n K o n B. A. MaTCpiia.ibi K oCociioiiaiiiiio npc,T.e.TL>!io AOiiycin-
Mbix KoiinciiTpamn'i io.nyii.nciiAii>i3ou>iai!aTa B arMoccjicpiiOM B03Ayxe.
Tnr. 11 can., 1963, .Nj 6, crp. 8--15. • . •
T r c o n J., D u t r a F., C a p p e 1 J., S i g u o n M., J o u n k e r \V.
• Arch.'Induslr. hyg., 1950, N 2, p. 716—734
Amdtir M. Pub!. Hlth. rep.. 1953. May. p. 474.
- 138 -
-------
BIOLOGICAL ACTION OF LOW MANGANESE CONCENTRATIONS
DURING INHALATIONAL EXPOSURE
Candidates of Medical Sciences
F. V. Dokuchayeva and N. N. Skvortsova
A. N. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences, USSR
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 173-184, (1966).
The preceding paper* reported on the contamination of atmospheric air
with industrial discharges of manganese and their influence on the health
of the population, and also on the intratracheal administration to white
rats of low doses of manganese approximately corresponding to the maximum
permissible value for plant buildings.
The present paper discusses the results of chronic inhalational
exposure of white rats to low manganese concentrations.
The experimental validation of maximum permissible concentrations of
dust materials involves certain difficulties because of the lack of the
means for providing stable and low concentrations in the exposure chambers.
In addition, the dust frequently has neither an odor nor an irritating effect.
For this reason, the establishment of its maximum permissible content is
possibly only on the basis of a general toxic effect during an extended
inhalational exposure.
We used a dust chamber with a volume of ^0.8 m3, patterned after a
model chamber built by the Kiev Institute of Labor Hygiene and Professional
Diseases. A modification of the Erman dust generator made it possible to
produce in the chamber a manganese dust concentration of 0.3 and 0.03 mg/m ,
i.e., 275 and 2750 times lower than in the author's experiments.
The experiments were conducted on white rats of pure Wistar strain in
two series: in the first (nine males and 16 females) with the 0.3 mg/m^
concentration (maximum permissible value for plant buildings) and in the
second (12 males and 16 females) with the 0.03 mg/m-* concentration (highest
single maximum permissible value for atmospheric air). Each series had its
own group of control rats. Because of the presence of only one exposure
*V. F. Dokuchayeva and N. N. Skvortsova. Contamination of atmospheric air with manganese compounds
and their influence on the organism. In: Maximum permissible concentrations of atmospheric con-
taminants. Prof. V. A. Ryazanov, ed., 1962, Vol. VI.
- 139 -
-------
chamber (characterized by a large bulk), the exposure of the two series of
animals x^as carried out in sequence (Table.l).
Table 1
Summary Table of Experiments
Experiment
Series and
• Date of •
Exposure
I
15/VII 1960 r.—
20/1 I9G1 r.
11
27/IH-l/XI
196! r.
III.
8/IX 19G1 r. —
7/1 1 1962 r.
No. of rats
Males
/ 9
12
8
Fe-
males
16
16
—
".inOo concen-
tration,
mg/m3,
M ± m
0,302 + 0,054
0,033 + 0,002
4,12 + 0,181
Control
No. of rats
Hales
9
15
6
Fe-
males
16
15
—
In the course
of the exper-
iment, total
no. of animals
sacrificed
13
8
1
The tests employed included a dynamic observation of the weight of
the animals, a study of the red blood, histopathological examination of
the lungs, and certain biochemical tests (porphyrin metabolism, cholin-
esterase activity of the blood, the ratio-of blood serum protein fractions,
and ascorbic acid content of the liver and adrenal glands).
To check the adequacy of the biochemical tests chosen, another series
of experiments (III) was carried out which included eight experimental and
six control male rats - a positive control group exposed in an adapted
gas chamber to higher manganese concentrations, on the order of 3-6 mg/m^
(average concentration 4.12 mg/m3), i.e., 100 to 200 times higher than the
maximum permissible value for atmospheric air.
Manganese dioxide powder (95% Mn02) predried at 100°C. and ground,
containing an average of 82% of particles measuring less than 5 y, was
used for the exposure of animals.
The manganese concentrations in the chambers were checked by taking
samples every hour (the collection of each sample lasted one hour).
The manganese content was determined by using a method modified by
the air hygiene section of the A. N. Sysin Institute of General and Communal
Hygiene, Academy of Medical Sciences, USSR.*
"•Instructions for the Organization of the Study of Atmospheric Pollution. Uedgiz, 1963, p. 123.
- 140. -
-------
The exposure was carried out for 5-6 hours daily on working days only.
The total duration of the exposure for series I (0.3 mg/m3) was six months,
for series II (0.03 mg/m3) seven months, and for series III (3-6 mg/m3)
five months.
The analyses showed that samples with concentrations of 0.15 to 0.45
mg/m3 of series I comprised 89.4%, with more than half of the total number
of samples (53.5%) consisting of Mn02 concentrations in the range of 0.25-
0.35 mg/m3. Samples with higher concentrations were generally observed
only in the course of 61 hours out of 872, i.e., in 7% of the cases. The
average of all 872 samples was 0.302 Jl 0.054 mg/m3 Mn02-
The scatter of the concentrations in magnitude for the seven months of
exposure of series II animals was approximately the same. The largest number
of hourly samples (915, or 89.1%) ranged from 0.015 to 0.045 mg/m3. The
average hourly concentration of MnC>2 in these groups of concentrations was
0.027 mg/m3, and the average of all 1027 samples was 0.033 ± 0.002 mg/m3.
Manganese concentrations in the 100-liter gas chamber also corresponded to
the specified conditions of the experiment, the average of all 545 samples
being 4.12 ± 0.181 mg/m3.
As we know, two Mn02 concentrations - the highest single value
(0.03 mg/m3) and the average daily value (0.01 mg/m3) - have been confirmed
as tentative maximum permissible values for atmospheric air. These concen-
.trations were established by calculations on the basis.of the maximum per-
missible concentration of manganese for the air of plant buildings, a value
also determined without experimental verification.
It was not possible to establish the highest maximum permissible con-
centration of manganese experimentally, since manganese compounds have no
odor and no irritating effect. Cases of acute poisoning with manganese
under industrial conditions are not known either. However, even very slight
amounts of manganese, as will be shown below, are capable of causing certain
reactions in the organism. In intensity of toxic action, manganese may be
classified with a certain approximation among such industrial poisons as
lead and mercury (A. F. Makarchenko).
For this reason, the noxiousness of manganese concentrations which may
be present in atmospheric air should be evaluated only on the basis of their
general toxic effects, checked by experiments on animals, and the value of
the maximum permissible concentration of manganese should be determined in
the form of a daily average, as was recommended by V. A. Ryazanov for sub-
stances having a general toxic effect.
We did not have the technical facilities available for round-the-clock
exposure, and therefore conducted the exposure for 5-6 hours a day. How-
ever, since our experiments were chronic in character, they can be used as
- 141 -
-------
material for substantiating the average daily maximum permissible concen-
tration.
Occupational pathology data indicate a high toxicity of manganese
compounds, which are capable of accumulating in the organism and causing
ill effects. The toxic influence of manganese does not manifest itself
immediately, but only as a result of more or less lasting contact with
it (V. S. Surat, A. P. Sapozhnikov and A. P. Shilova, 1936; E. A. Drogichina,
1948; G. N. Cherepanova, 1946; V. A. Mikhaylov and F. S. Trop, 1953; Couper,
1937, and others).
The most frequent manifestations of manganese intoxication under
industrial conditions are lesions of the striopallidal system of the brain
and lesions of the respiratory organs (pneumonia, pneumonoconioses, chronic
bronchitis, laryngitis, etc.) (Z. I. Bakradze, 1923; Buttner, Lenz, 1937;
M. E. Machabeli, 1957, and others).
In a medical survey of children in the vicinity of industrial discharges
we also observed a high disease rate, particularly pneumonias and diseases
of the upper respiratory tract, i.e., the demonstrated shifts in the state
of health of the child population are similar to the changes developing in
workers under industrial conditions.
For this reason, the chief criterion in series I of the animals was
the histopathological examination of the lungs.* The results of the exam-
ination after six months of exposure showed that the histopathological
changes in the lungs of males of series I could not be evaluated as being
the consequence of exposure, since some changes were frequently observed
in the majority of the control animals.
In contrast, examinations of the female rats showed an adverse effect
of manganese in 0.3 mg/m^ concentration on the respiratory tract. A con-
sequence of this effect was chiefly the appearance of inflammatory changes
in the pulmonary tissue and also the appearance of circulatory disorders
in the vascular system of the lungs and the development of moderate symptoms
of peribronchial and perivascular sclerosis of the pulmonary tissue. Changes
of this kind were noted in the experimental group in all 15 female rats,
whereas they occurred in only four out of 13 control rats.
The statistically reliable difference between the number of lesions
of the lungs in female rats in the experimental and control groups sug-
gests that the manganese concentration of the order of the maximum per-
missible value for plant buildings is active on the organism of female rats
during chronic inhalational exposure.
The histopathological examination was made by Candidate of Medical Sciences Yu. N. Solov'yev.
- 142 -
-------
No changes of any kind were observed in the weight (the rats were
x^eighed twice a month) and in the state of red blood (the number of
erythrocytes and the hemoglobin were determined at the start, in the middle
and at the end of exposure).
fcc O
f. 60
§"8!
O. .-< •
O
«
D. :
O
O
1.?
'
i,o\ • .
«/
B.i-
n L
0.2
^\,
.
^/ \
/ A V>
/ — / \
"*•*"""
S 6 7 S S 10 II 12 13 n
Number of determinations
Excretion of coproporphyrin \vith urine during inhalational
exposure of rats to manganese.
1 - concentration, 4.12 ng/m3; 2 - control; AB - period of
exposure.
According to the data of biochemical investigations, exposure to the
4.12 mg/m3 concentration of MnC>2 (series III) increased the cholinesterase
activity of the blood and caused the effect of porphyrinuria in the experi-
mental animals (Table 2 and Figure).
In series II of experiments, no changes were observed in the porphyrin
metabolism. The same applies to the determination of ascorbic acid in the
liver and adrenals (using the method of the Vitaminology Institute) , in the
content of which no shifts were observed, in contrast to the data of Rudra
(1944), N. V. Tatarinova (1951), and A. M. Birenbaum (1957), who observed
such shifts when manganese was administered with food.
Table 2
Activity of Blood Cholinesterase in Rats Exposed to the 4.12 mg/m'1
Concentration of unOg {series .III), Minute_s_.
-
Wales
rimental
rol
Date of Determination
25/Vll 19CI r.
30
30'
G.'IX
35
35
9,'X
35
35
17/XI
30
30
30/1 1052 r.
17
22
G.'ll
21
27
In a study of the composition of protein fractions of the blood*
••The determination of the protein fractions was carried out by Doctor of Medical Sciences A. A. Tyurina
and Candidate of Medical Sciences L. I. Kulikova.
- 143
-------
in experimental female rats of series II, changes were observed in the ratio
of the content of protein fractions, i.e., an increase in the amount of
globulins and a decrease in the amount of albumins. In the males, such
changes were not detected even when concentrations such as 4.12 mg/rn-^ of
MnC>2 were employed (Table 3).
At the end of the experiment, a histopathological examination of the
pulmonary tissue was also performed on animals of series II. Preparations
of the lungs were stained with hematoxylin-eosin and hematoxylin picrofuchsin
according to Van Gieson*and argyrophil fibers were developed by Bielschowsky's**
method. In addition, the preparations were stained with toluidine blue, and
the reaction was carried out according to Hotchkiss (the MacManus reaction).
The examination of the experimental group of males of series II in two
out of 12 animals showed definite inflammatory phenomena in the lungs
(purulent bronchitis, chronic abscessing bronchopneumonia). No signs of
gross pathology were observed in the remaining animals of this group. How-
ever, accumulations of macrophages containing lumps of brown dust pigment
were found in the peribronchial lymphoid tissue and under the pleura in the
majority of animals of this group. In some cases, vague signs of desquamation
of the alveoli and bronchial epithelium were observed along with manifesta-
tions of a certain increase in the number of collagenous fibers in the
peribronchial zones and in the stroma of lymphatic nodes of the hilum of the
lung.
In the experimental group of females, gross inflammatory changes of the
lungs were observed in six rats out of 14. The remaining changes were
analogous to those found in the male rats of this group. In almost all the
cases, minor accumulations of dust macrophages were also observed without
any sings of reaction among the surrounding tissue components.
Among the 14 male rats of the control group, inflammatory changes were
noted in four cases, but they were less definite than in the experimental
group. In the control group of females, inflammation signs in the respira-
tory organs were observed in only two rats out of 13.
In Hotchkiss staining and staining with toluidine blue, as well as in
the development of argyophil fibers according to Bielschowsky, no fundamental
structural changes were detected in the lungs of either the experimental or
the control animals.
Comparing these materials of the histological examination with similar
data obtained from the exposure of animals of series I (0.3 mg/m^), one can
note that in the groups of males being compared, the number of animals with
inflammatory phenomena in the lungs in the experimental and control groups
was approximately the same (Table 4), and in any case does not permit one to
consider any kind of relationship between the appearance of inflammatory
** [Translator's note: Van Gizon, Bilishov, Khochkis, according to the transliteration of Russian reference. ]
- 144 -
-------
Table 3
Protein Composition of Blood Serum (in percentages) in Animals of Series II and III During Exposure
to Manganese Dioxide.
I
h-"
JS
I
Mn02
Concen-
tration,
mg/m5
0.033
(Series II)
4.12
(Series III)
Group of
Animals
Experimental
Females
Control Females
Expf-imental
Males
Control Males
Experimental
Kales
Control Males
Total
Protein
8,53
8,34
7,49
G.8G
. 8,46
8,52
Globulins
'(
12,70
12, 44
13,98
14,24
15,20
14,12
P
20,86
19,39
25' 08
25,74
19,42.
21,15
a
11,86
10,39
13,07
11,52
15,03
13,84
Total Amount
of
Globulins
45,49
42,57
52,56
51,91
49,66
49,11
Total
Amount of
Albumins
54,24
57,46
47,84
48,15
50,59
50,98
Albumin-
Globulin
Ratio
1,19
1,35
O',91
0,93
1,02
1.04
-------
processes in the experimental males and the penetration of manganese into
the respiratory tract.
Table 4
Number of Animals with Changes in the Lungs.
Series
1
II .-
Ill
I'r.Og
Concen-
tration,
mg/m?
0 302
0 033
4 12 .
Males'
Experimental Control
4 out of 8
2 out of 12
5 out of 8
7 out of 9
4 out of 14
J> out of 6
Females
Experimental
15 out of 15
6 out of 14
—
Control-
4 out of IJ,
2 out of 13
—
In experimental females of series II, inflammatory changes were observed
in six rats out of 14 (somewhat more frequently than in the control) , but this
difference proved to be statistically unreliable, and therefore a manganese
concentration of 0.03 mg/m3 cannot be considered to be active on the respira-
tory organs of female rats.
However, during inhalational exposure to the 0.03 mg/m^ manganese con-
centration for seven months, the pulmonary tissue of the animals, indepen-
dently of the sex, showed a deposition of particles of manganese dioxide
aerosol, which was absorbed more slowly than other oxide compounds of mangan-
ese, but was also excreted more slowly (E. N. Levina, 1962). This fact is
alarming if one takes into account the cumulative properties of manganese.
It is possible that during a longer exposure, even such low manganese concen-
trations may prove to be active on the pulmonary tissue when the lability of
the organism is greater.
Moreover, inhalational exposure of animals of both sexes to 0.3 and
0.03 mg/m3 concentrations also shoxved that the organism of female rats is
more sensitive to the action of low doses of manganese than that of male
rats. The high sensitivity of females to manganese has also been observed
in intratracheal administration.
A study of the red blood in series II of the experiment (0-03
did not reveal any changes in the hemoglobin content or in the number of
erythrocytes , either in the males or in the females. In series III (4.12
mg/m3) did not reveal any changes in the hemoglobin content or in the number
of erythrocytes, either in the males or in the females. In series III
(4.12 mg/m^) , toward the end of the fifth month of exposure, a statistically
reliable decrease in the number of erythrocytes was observed in the experi-
mental rats as compared with the controls. Toward the end of exposure, the
amount of erythrocytes in the experimental group increased to 106.5% of the
initial value, and in the control, to 113.4%. The difference in the hemoglobin
-.146 -
-------
content and magnitude of the color index was not significant.
Dynamic observation of the weight of the animals showed that in series
III there was no substantial difference in the weight of the experimental
and control animals, exposed to the 0.03 mg/m3 manganese concentration as in
the group of females of series II. In the group of males (0.03 mg/rn-^) , for
the same average weight at the start of the experiment, the curve of the
average weight of experimental males starting from the end of the first month
of exposure runs all the way to the end of the experiment above the curve of
the control animals. At the same time, the difference in the average weight
in the fifth month and at the end of the exposure becomes statistically
reliable.
Conclusions
1. The inhalational exposure of white rats to an average concentration
of 0.302 mg/nH of MnO? for 5-6 hours a day for six months causes changes in
the lungs of female rats manifested in inflammatory phenomena in the pulmon-
ary tissue, the onset of circulatory disorders in the vascular system of the
lungs, and the development of moderate symptoms of peribronchial and peri-
vascular sclerosis of the pulmonary tissue.
2. An average concentration of 0.033 mg/nH of Mn02 during inhalational
exposure for 5-6 hours a day for seven months proves to be active on the
composition of the protein fractions of the blood serum in female rats,
causing a change in the ratio of the content of albumins and globulins.
At the same concentration, the lungs of animals of both sexes show
dust particles of manganese dioxide, and in male rats there is observed a
tendency toward a gradual and more rapid weight increase as compared with
the control animals.
3. In the inhalation exposure of male rats to an average concentration
of 4.12 mg/m^ of MnC>2 in the course of five months (group of positive con-
trol), the phenomena of prophyrinuria, increase of cholinesterase activity
of the blood and decrease of the number of erythrocytes at the end of the
exposure were observed.
4. Our data suggest that the reaction of white rats to the action of
manganese may be different depending on the sex of the animals. For this
reason, the experiments should be conducted on animals of both sexes, since
the reactivity of the females to the action of toxic substances may be greater
than that of males.
5. A further experimental study of even lower doses of manganese
administered in round-the-clock inhalational exposure is necessary.
147
-------
LITERATURE CITED
BaKpa,i3e 3. II. Tiiniciia Tpyaa, 1923, ,V° 10—11, 40.
B i! p e 11 6 a y M A. M. Vsp. CMOXIIM. JKyp.n., 1957, XXIX, .Y° 1, crp. 113.
JloKyMaena B. ., CxnopuoBa H. H. Sarpr.aiieiMie ar.Moc^ep-
noro DODjiyxa coeaniiSiiiisiMii Mapraiuia n MX B.inninie na opra:i:i3M.
B CO.: ripCAC.TbilO .lOHVCTH.MUe KOIUlCIITpailliil aTMOCt^CpHUX 33rpr3-
iicinifi. M.. 19G2. u. 6, c'rp. 62.
JH. p o r ;i M ii u a 3. A. OT.ia.icinmc noc.ne.iCTmsfi n icp.'niih np» IIMTO;;-
cni;a!u:n MapraimcM. B cu.: Bonpocu ninicm,: rpy.ir. n npcvJ)L>cc;:o-
li.i.-ibiiux 3cj\ n M. 3. Tnr. n can., 1957, .V? 'i, crp. 29.
PK 3 a n on B. A. OCIIOUIIHC iipinmiinb! ruriiCMii'iccKoro iiopMiiponamin
aTMOc(j)C[);ii>ix aarpsisiiciiin'i. B cG.: ripc.'ic.'ibiio AOiiycniMuc KOI;HCMT-
paunii atMOCcjicpiiui.x 3arp»3MciiiiM. M.,_i952, D. 1, crp. 9.
C y p a T B. C., C a n o >i< n n K o n A. Fl.; Ill n n o n a A. TI. KasancK.
MCA. >nypii., 1936, j\? 2, cip. H9.
TarapnnoBa H. B. Bonpocw niiraiiim, 1951, jYs 1, crp. G6.
Mcpcnanoua T. H. BCCTHIIK A MM CCCP, 19-16, B. 5, crp. 27.
3pMan M. M. fur. n can., 1959, jV« 7, crp. 75.
B u 1 1 n e r M. E., Lenz E.. Arch. Gewcrbepath. u. Gewcrbchyg.,
1957, Bd. 7.
Co u per M. J. chim. mcd. de pharrnac., de tpxicol. ct revue des
nouvcllcs sclentif. nation, ct etrangercs. Paris. 1837, v. 3. Seric II,
p. 233. ' . '
Rudra M. N. Nature. 1944, v. 153. p. 743. . • . .
- 148 -
-------
MAXIMUM PERMISSIBLE CONCENTRATIONS OF NOXIOUS SUBSTANCES
IN THE ATMOSPHERIC AIR OF POPULATED AREAS*
From "Biologicheskoe deystvie i gigienicheskoe znachenie atmosfernykh
zagryazneniy". Pod redaktsiey Prof. V. A. Ryazanova i Prof. M. S. Gol'dberga.
Izdatel'stvo "Meditsina" Moskva, p. 185-187, (1966).
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Contaminant
Acrolein
Amyl acetate
Amylene
Aniline
Acetone
Acetophenone
Benzene
Low-sulfur gasoline in
terms of C
Butyl acetate
Butylene
Vinyl acetate
Hexamethylenediamine
Isopropylbenzene hydroperoxide
Dichlo roe thane
Dimethyl
Maximum Permissible
Concentrations mg/m.3
Maximum Single
0.30
0.10
1.5
0.05
0.35
0.003
2.4
5.0
0.1
3.0
0.2
0.001
0.007
3.0
0.03
Daily Average
0.10
0.10
1.5
0.03
0.35
0.003
0.80
1.5
0.1
3.0
0.2
-
-
1.0
0.03
Approved by the Assistant Chief Physician of Public Health of the USSR on Kay 26, 1964.
- 149 -
-------
(Cont'd)
No.
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Contaminant
Dow therm
Isopropylbenzene
Methanol
Methyl acetate
Maleic anhydride
Methyl methacrylate
Manganese and its compounds
(in terms of Mn02)
Arsenic [inorganic compounds
except hydrogen arsenide
(in terms of As) ]
Nitrobenzene
Carbon monoxide
Nitrogen oxides (in terms
of N205)
Propylene
Non toxic dust
Metallic mercury
Soot (carbon black)
Lead and its compounds
(except tetraethyl-lead)
Lead sulfide
Hydrogen sulfide
Maximum Permissible
Concentrations mg/m^
Maximum Single
0.01
0.014
1.5
0.07
0.5
0.1
-
"
0.008
6.0
0/3
3.0
0.5
-
0.15
-
-
0.008
Daily Average
0.01
-
0.5
0.07
0.05
0.1
0.01
0.03
0.008
1.0
0.1
3.0
0.15
0.0003
0.05
0.0007
0.0017
0.008
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(Cont'd)
No.
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Contaminant
Sulfur dioxide
Sulfuric acid
Carbon disulfide
Sulfuric (in concentration
of H ions)
Hydrochloric
Nitric
Styrene
Formaldehyde
Toluylene diisocyanate
Phosphoric anhydride
Fluorine compounds (in terms
of F)
Phenol
Furfural
Chlorine
Chlorobenzene
Hydrogen chloride
Chloroprene (2-chloro-l,
3-butadiene)
Hexavalent chromium (in
terms of Cr03)
Maximum Permissible
Concentrations mg/m3
Maximum Single
0.5
0.3
0.03
0.01
0.01
0.01
0.003
0.035
0.05
0.15
0.03
0.01
0.05
0.10
0.1
0.05
0.25
0.0015
Daily Average
0.15
0.1
0.01
-
-
-
0.003
0.012
0.02
0.05
0.01
0.01
0.05
0.03
0.1
0.015
0.08
0.0015
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(Cont'd)
No.
52
53
54
55
Contaminant
Cyclohexanol
Cyclohexanone
Ethyl acetate
Ethylene
Maximum Permissible
Concentrations mg/m^
Maximum Single
0.06
0.04
0.1
3.0
Daily Average
-
0.1
3.0
Remarks. 1. When several substances characterized by a complete summation
of action are present together in atmospheric air, the sum of their concen-
trations as calculated by the formula below should not exceed 1.
x = a + b_ + _c_ etc>
m m2 m^
where x is the unknown total concentration; .§., b., £. are the concentrations
m m2 mo
of the substance being determined divided by the corresponding maximum per-
missible concentration for isolated action.
Substances characterized by such action include the following groups:
a) sulfur dioxide and sulfuric acid aerosol;
b) hydrogen sulfide, carbon disulfide and Dowtherm;
c) isopropylbenzene and isopropylbenzene hydroperoxide;
d) ethylene, propylene, butylene and amylene;
e) strong mineral acids (sulfuric, hydrochloric and nitric) in terms
of the concentration of H ions.
2. When hydrogen sulfide and carbon disulfide are present together in
atmospheric air, the maximum permissible concentrations for each of them
individually are preserved.
3. Lists of indexes of maximum permissible concentrations of noxious
substances in the atmospheric air of populated areas, approved on 14 February
1961 for Nos. 221-61 on 13 April 1962, and since 20 June 1963 for Nos . 442-63
should be considered as no longer in force.
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