AMERICAN INSTITUTE OF CROP ECOLOGY
A RESEARCH ORGANIZATION DEVOTED TO PROBLEMS OF
PLANT ADAPTATION AND INTRODUCTION
WASHINGTON, D. C.
AICE" SURVEY OF USSR AIR POLLUTION LITERATURE
Volume XV
A THIRD COMPILATION OF TECHNICAL REPORTS
ON THE
BIOLOGICAL EFFECTS AND THE PUBLIC HEALTH ASPECTS
OF ATMOSPHERIC POLLUTANTS
Edited By
M. Y. Nuti
The material presented here is part of a survey of
USSR literature on air pollution
conducted by the Air Poll; ;on
AMERICAN INSTITUTE OF CROP ECOLOGY
This survey is being conducted under GRANT R 800878
(Formerly R01 APOO
OFFICE OF AIR PROGRAMS
he
U.S. ENVIRONMENTAL PROT
•AMERICAN INSTITUTE OF CROP ECOLf
809 DALE DRP
SILVER SPRING, MARYLAND 20910
-------
PUBLICATIONS of th. AMERICAN INSTITUTE OF CROP ECOLOGY
26
No.
I UKRAINE— Ecologicol Crop Geography cf the Ukraine and the
Ukrainian Agrc-CIJn-.atic Analogue! in North America
2 POLAND-Agricul rural CKnafoloay of Poland and IB Agro-
Climati*- Analogues in North America
3 C7?CHOSLOVAKlA-A3,icultural Climatology of Czechoslo-
vakia and Its Agro-Climatic Analogues in North Americc
YUGOSLAV!A-Agricultural Climotologyof Yugoslavia and Its
Agro-Climatic Analogues in North America
GREECE—Ecological Crop Geography of Greece and Irs Agro-
Climatic Analogues in Norm America
ALBANIA-Ecologicol Plant Geography of Albania, Irs Agri-
cultural Crops and Some North American Climatic Analoguei
7 CHINA—Ecological Crop Geography of China and Its Agro-
Climatic Analogues in North America
8 GERMANY-Ecologicol Crop Geography of Germany and Irs
Agro-Climatic Analogues in North America
*9 JAPAN (1)-Agricultural Climatology of Japan and Its Agro-
Climatic Analogues in North America
10 FINLAND-Ecological Crop Geography of Finland and Its Agro-
Climatic Analogues in North America
II SWEDEN-Agricultural Climatology of Sweden and Irs Agro-
Climatic Analogues in North America
12 NORWAY- Ecological Crop Geography of Norway and Its Agro-
Climolic Analogues in North America
13 SIBEftlA-Agricul rural Climatology of Siberia, Its Natural Belts,
and Agro-Climatic Analogues in North America
14 JAPAN (2)-Ecologicol Crop Geography and Field Practices of
Japan, Japan's Natural Vegetation, and Agro-Climatic
Analogues in North America
15 RYUKYU ISLANDS-Ecological Crop Geography and Field
Practices of the Ryukyu Islands, Natural Vegetation of the
Ryukyus, and Agro-Climatic Analogues in the Northern
Hemisphere
16 PHENOLOGY AND THERMAL ENVIRONMENT AS A MEANS
OF A PHYSIOLOGICAL CLASSIFICATION OF WHEAT
VARIETIES AND FOR PREDICTING MATURITY DATES OF
WHEAT
(Based on Data of Czechoslovakia and of Some Thermally
Analogous Areas of Czechoslovakia in the United States
Pacific Northwest)
17 WHEAT-CLIMATE RELATIONSHIPS AND THE USE OF PHE-
NOLOGY IN ASCERTAINING THE THERMAL AND PHO-
TOTHERMAL REQUIREMENTS OF WHEAT
(Based on Data of North America and Some Thermally Anal-
ogous Areas of North America in the Soviet Union and in
Finland)
18 A COMPARATIVE STUDY OF LOWER AND UPPER LIMITS OF
TEMPERATURE IN MEASURING THE VARIABILITY OF DAY-
DEGREE SUMMATIONS OF WHEAT, BARLEY, AND RYE
19 BARLEY-CLIMATE RELATIONSHIPS AND THE USE OF PHE-
NOLOGY IN ASCERTAINING THE THERMAI^AND PHO-
TOTHERMAL REQUIREMENTS OF BARLEY
20 RYE-CLIMATE RELATIONSHIPS AND THE USE OF PHENOL-
OGY IN ASCERTAINING THE THERMAL AND PHOTO-
THERMAL REQUIREMENTS OF RYE
21 AGRICULTURAL ECOLOGY IN SUBTROPICAL REGIONS
27. MOROCCO, ALGERIA, TUNISIA-Physicol Environment and
Aari culture
23 LIBYA and EGYPT-Physical Environment and Agriculture. . .
24 UNION OF SOUTH AFRICA-Physical Environment and Agri-
culture, With Special Reference to Winter-Rainfall Regions
25 AUSTRALIA-Physical Environment and Agriculture, With Spe-
cial Reference to Winter-Rainfall Regions
S. E. CALIFORNIA and S. W. ARIZONA-Phyiical Environment
and Agriculture of the Desert Regions
27 THAILAND—Physical Environment and Agriculture
28 BURMA-Physical Environment and Agriculture
28A BURMA-Diseases and Pests of Economic Plants
28B BURMA-Climote, Soils and Rice Culture (Supplementary In-
formation and a Bibl iography to Report 28)
29A VIETNAM, CAMBODIA, LAOS-rhysical Environment and
Agriculture
29B VIETNAM, CAMBODIA, LAOS-Diseoses and Pests of Economic
Plants
29C VIETNAM, CAMBODIA, LAOS-CItautological Data (Supple-
ment to Report 29A)
30A CENTRAL and SOUTH CHINA, HONG KONG, TAIWAN-
Physical Environment and Agriculture ...... $20.00*
306 CENTRAL and SOUTH CHINA, HONG KONG, TAIWAN-
Ma|or Plant Pests and Diseases
31 SOUTH CHINA-lts Agro-Climatic Analogues in Southeast Asia
32 SACRAMENTO-SAN JOAQUIN DELTA OF CALIFORNIA-
Physlcal Environment and Agriculture
33 GLOBAL AGROCLIMATIC ANALOGUES FOR THE RICE RE-
GIONS OF THE CONTINENTAL UNITED STATE
34 AGRO-CLIMATOLOGY AND GLOBAL AGROCLIMATIC
ANALOGUES OF THE CITRUS REGIONS OF THE CON-
TINENTAL UNITED STATES
35 GLOBAL AGROCLIMATIC ANALOGUES FOR THE SOUTH-
EASTERN ATLANTIC REGION OF THE CONTINENTAL
UNITED STATES
36 GLOBAL AGROCLIMATIC ANALOGUES FOR THE INTER-
MOUNTAIN REGION OF THE CONTINENTAL UNITED
STATES
37 GLOBAL AGROCLIMATIC ANALOGUES FOR THE NORTHERN
GREAT PLAINS REGION OF THE CONTINENTAL UNITED
STATES
38 GLOBAL AGROCLIMATIC ANALOGUES FOR THE MAYA-
GUEZ DISTRICT OF PUERTO RICO
39 RICE CULTURE and RICE-CLIMATE RELATIONSHIPS With Spe-
cial Reference to the United States Rice Areas and Their
Latitudinal and Theimal Analogues in Other Countries
40 E. WASHINGTON, IDAHO, and UTAH-Physieat Environment
and Agriculture
41 WASHINGTON, IDAHO, and UTAH—The Use of Phenology
in Ascertaining the Temperature Requirements of Wheat
Grown in Washington, Idaho, and Utah and in Same of
Their Agro-Climaficolly Analogous Areas in the Eastern
Hemisphere
42 NORTHERN GREAT PLAINS REGION—Preliminary Study of
Phenological Temperature Requirements of a Few Varieties
of Wheat Grown in the Northern Great Plains Region and in
Some Agro-Climotically Analogous Areas in the Eastern
Hemisphere
43 SOUTHEASTERN ATLANTIC REGION-Phenologlcal Temper-
ature Requirements of Some Winter Wheat Varieties Grown
in the Southeastern Atlantic Region of the United Stares and
in Several of Its Lotitudinally Analogous Areas of the Eastern
and Southern Hemispheres of Seasonally Similar Thermal
Conditions
44 ATMOSPHERIC AND METEOROLOGICAL ASPECTS OF AIR
POLLUTION—A Survey of USSR Air Pollution Literature
45 EFFECTS AND SYMPTOMS OF AIR POLLUTES ON VEGETA-
TION; RESISTANCE AND SUSCEPTIBILITY OF DIFFERENT
PLANT SPECIES IN VARIOUS HABITATS, IN RELATION TO
PLANT UTILIZATION FOR SHELTER BELTS AND AS BIO-
LOGICAL INDICATORS—A Survey of USSR Air Pollution
Literature
(Continued on inside of back cover)
-------
AICE-AIR-72-15
AICE* SURVEY OF USSR AIR POLLUTION LITERATURE
Volume XV
A THIRD COMPILATION OF TECHNICAL REPORTS
ON THE
BIOLOGICAL EFFECTS AND THE PUBLIC HEALTH ASPECTS
OF ATMOSPHERIC POLLUTANTS
Edited By
M. Y. Nuttonson
The material presented heie is part of a survey of
USSR literature on air pollution
conducted by the Air Pollution Section
AMERICAN INSTITUTE OF CROP ECOLOGY
This survey is being conducted under GRANT R 800878
(Formerly R01 AP 00786)
OFFICE OF AIR PROGRAMS
of the
U.S. ENVIRONMENTAL PROTECTION AGENCY
*AMERICAN INSTITUTE OF CROP ECOLOGY
809 DALE DRIVE
SILVER SPRING, MARYLAND 20910
1972
-------
TABLE OF CONTENTS
Page
PREFACE v
Maps of the USSR
Orientation vli
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
Non-Ferrous Metal Ores xil
Principal Centers of the Chemical Industry and of
the Textile Industry xiii
Principal Centers of Wood-Working* Paper, and Food
Industries xiv
Main Mining Centers xv
Principal Electric Power Stations and Power Systems xvi
MAXIMUM PERMISSIBLE CONCENTRATIONS OF NOXIOUS SUBSTANCES IN
THE ATMOSPHERIC AIR OF POPULATED AREAS
V. A. Ryazanov 1
SOME ASPECTS OF THE BIOLOGICAL EFFECT OF MICROCONCENTRATIONS OF
TWO CHLOROISOCYANATES
I. A. Zibireva 6
THE TOXICOLOGY OF LOW CONCENTRATIONS OF AROMATIC HYDROCARBONS
I. S. Gusev 19
i
CHRONIC ACTION OF LOW CONCENTRATIONS OF ACROLEIN IN AIR ON
THE ORGANISM
M. I. Gusev, I..S. Dronov, A. I. Svechnikova,
A. I. Golovina, and M. D. Grebenskova 35
STUDY OF THE REFLEX AND RESORPTIVE EFFECTS OF THIOPHENE
Sh. S. Khlkmatullayeva 46
SANITARY-TOXICOLOGICAL APPRAISAL OF THE COMBINED EFFECT OF A
MIXTURE OF BENZENE AND ACETOPHENONE VAPORS IN ATMOSPHERIC AIR
V. R. Tsulaya 57
iii
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Page
MAXIMUM PERMISSIBLE CONCENTRATIONS OF PHENOL AND ACETOPHENONE
PRESENT TOGETHER IN ATMOSPHERIC AIR
Yu. Ye. Korneyev 69
SANITARY EVALUATION OF THE COMBINED ACTION OF ACETONE AND
PHENOL IN ATMOSPHERIC AIR
U. G. Pogosyan 82
ON THE COMBINED EFFECT OF LOW CONCENTRATIONS OF ACETONE AND
ACETOPHENONE IN AIR ON THE ORGANISM OF MAN AND ANIMALS
N. Z. Tkach 98
ON THE COMPARATIVE TOXICITY OF BENZENE, TOLUENE, AND XYLENE
(STUDY OF THE REFLEX EFFECT)
I. S. Gusev 113
LITERATURE CITED IN 1968 PAPERS 124
LITERATURE CITED IN 1967 PAPERS 129
-------
PREFACE
The present volume constitutes the third* compilation of the USSR
technical reports on a number of investigations of the biological effects
of specific air pollutants. These investigations have been conducted at
various public health institutes and in departments of public health of
some of the universities of that country.
Industrial emissions of various air pollutants and their adverse
effect on human health have stimulated investigations of their biological
effects. The great strides in the development of industrial chemistry in
the USSR have called attention to the toxicological significance of the new
chemical air pollutants and made it imperative to conduct investigations as
to the biological effects of these chemical air pollutants. It also suggested
the need to conduct investigations dealing with the public health implications
of these pollutants.
The material included in this volume deals with the biological effects
of low concentrations of ambient chemical toxic substances such as
(1) a number of chemical compounds used in the production of new
herbicides,
(2) a number of aromatic hydrocarbons of industrial importance,
(3) those emitted by chemical and oil-chemical plants,
(4) a number of chemicals used in the production of plastics, dye-
stuffs, antioxLdants, pharmaceutical preparations, insecticides, etc.
The results of the above studies provide in the USSR a basis for the
establishment of a series of new maximum permissible concentrations for new
toxic substances in the atmospheric air and constitute the scientific criteria
for assessing the degree of pollution of the air medium. They also form the
foundation for a number of ameliorative sanitation measures to be undertaken.
Some background information
-------
country's economic areas (see page ix). The many diverse climatic con-
ditions of the country and its major economic areas as well as the geo-
graphical distribution 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.
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 1972
vi
-------
40 ' 65' SO ° 100 ' 120 " " WJ " 160 *17C
International Sound*
—— Autonomous R»ublic 'ASSfl
-------
CLIMATIC ZONES AND REGIONS* OF THE USSR
..CJ!..-,0,„-;.. - ^^T^-^ ARCTIC OCEAN r ..-v^xr.
v->
•~~>-vr">- xi:..#..-\ ''T*1^1 _^~'-
* %C:, x'<\ \- V.V*
- -
-'* V.^
^'\r\- >^v3
^^V/rO^i < -^
"X^ A fe^<
^w^*^?—
v^ :/sVrr^^f;^-1
&&&=&*•
•^M^c^^m
<• .,-,.
Zones: I-arctic, II-subarctic, Ill-temperate, IV-subtropical
Regions: 1-polar, 2-Atlantic, 3-East Siberian, 4-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, lA-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.
tA vi*:°/-
tf—-^.T-;s< ftt&Kffinl
-0%^fe^rowij
^^J Cenl«nvntal glacier F -^Erd For..-!
I.. ; J Tyd-i 1 1
viii
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow 1969)
-------
MAJOR ECONOMIC AREAS OF THE U.S.S.R.
^V^TV'JIP otMurmanik
Karaganda0
Semipal
Uil-Kame
VI Volga
VII Urali
VIII Weil Sibe
IX EAI| Siberi
X Far East
XI Baltic
XII Soulh-Weilern
XIII Doneli-Dniepcr
XIV Soulhern
XV Transcaucauan
XVI Kazakhstan
XVII Cenlral Asia,
XVIII Byelorussian
PLANNED DISTRIBlttlON 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-'rVect
(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-Taisnet and a number of others. Ferrojs 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
Main centres of ferrous metallurgy
" " " non-ferrous metallurgy
O Centres of chemical industry
(After A. Efimov, "Soviet Industry", Moscow 1968)
-------
PRINCIPAL CENTERS l)K FKfrkniN MFTAT T
WeltovsV Z.iboilali
Complete cycle me
Slecl imclling and
rolling
Smelling of forrooll
Mining ol:
iron orei
coking coal
MC oret
I1AI'! IRON ORE DEPOSITS IN TKE L'.S.S.S
(After A. Lavrishchev, "Economic Geography of
the U.S.S.R.", Moscow 1969)
xi
-------
PRINCIPAL CENTERS OF N03-FESROUS METALLURGY IN THE U.S.S.R.
^L^tjggfr
Metallurgy:
copper (5)
iiiununiuni
DISTRIBUTION OF MOST IMPORTANT DEPOSITS OF TJON-FERROUS :
-------
PRINCIPAL CENTERS OF THE CHEMICAL INDUSTRY IN THE U.S.S.R.
*\ £^ ^^-&&».'^ \
xiii
(After A. Lavrishchev, "Economic Geography
of the U.S.S.R.", Moscow
-------
PRINCIPAL CENTERS OF WOOD-WORKING AND PAPER INDUSTRIES IN THE U.S.S.R.
f. . K?v,d.?Jtnf dTD'Murmansk
Dnepropetrovsk /.'
.^>'V-;^' '•Ty'umen
Industry:
Timber-sawing and wood-wojliing
@ Roper
LJ? Piinclpal lumbering areas
-^ Forests
PPINCIPAL CE'-rTL'-S OF TIE FJOI INDUSTFZ O THE TT.S.S.P
(After A. Lavrishchev, "Economic GeoKraohv
of the U.S.S.R.", Moscow 1969) '
XIV
-------
THE MAIN MINING CENTERS OF THE USSR
Oil refining
Oil pipei
Gas pipes
Power llalioni
(After A. Efiraov, "Soviet Industry", Moscow 1968)
-------
PRINCIPAL ELECTRIC POWER STATIONS AND POWER SYSTEMS IK THE U.S.S.R.
Principal Electric Power Station*
Thermal Hydro-power
ijl in operation
-A- under conilfuction
a. 1 Uigmihaya_ K— I'
and planned
ot etetlric
power \iatiom
Operating atomic cleeinc power
Areas of operation of single power grids
European par! of Iho u S.S.R.
Central Siberia
Areas of operation of integrated power grids
Northern Kaiahhilan
Central Alia
Figures indicate lollowing power >iaiion>:
1 Bailie 9 Dnicprodnrthlntk 17 Shalura
2 Narva lODmeprogei IB Eleklrogorsk 24 Nur«k
JKegum UKakhovka 1? Ivankovo 25 Raqunlkaya
-------
MAXIMUM PERMISSIBLE CONCENTRATIONS OF NOXIOUS SUBSTANCES IN THE
ATMOSPHERIC AIR OF POPULATED AREAS*
(V. A, Ryazanov)
From Akademiya Meditslnakikh Nauk SSSR. "Biologicheskoe deystvle i
gigienicheskoe znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova.
Vypusk 11, Izdatel'stvo "Meditsina" Moskva, p. 201-204, (1968).
Pollutant
1
1. Nitrogen dioxide
2. Nitric acid (based on HN03 molecule
(based on hydrogen ion)
3. Acrolein
4. Alpha-methyls tyrene
5 . Alpha-naphthoquinone
6. Amyl acetate
7. Amylene
8. Ammonia
9. Aniline
0. Acetaldehyde
1. Acetone
2. Acetophenone
3. Benzene
.4. Gasoline (low-sulfur petroleum
gasoline in terms of "C")
.5. Shale gasoline (in terms of "C")
.6. Butane
.7. Butyl acetate ,
.8. Butyl ene
.9. Butyl alcohol
0. Butyl phosphate
1. Valeric acid
2. Vanadium pentoxide
Concentration, mg/m^
Maximum single
2
0,085
0,4
0,006
0,30
0.04
0.005
0,10
1,5
0.20
0,05
0.01
0,35
0.003
1.5
5,0
0,05
200.0
0.10
3,0
0.3
0,01
0.03
Mean daily
3
0,085
0.4
0.006
0.10
0.04
0.005
0,10
1.5
0.20
0.03
0.35
Ot003
0,8
1,5
0.05
0.10
3.0
— —
— —
0,01
0,002
* Approved by the Assistant Chief Public Health Physician of the USSR on 12 September 1967, No. 692-67.
- 1 -
-------
1
23. Vinyl acetate
24. Hexamethylenediamine
25. Bi vinyl
26. Diketene
27. Dime thylani line
28. Dimethyl sulfide
29. Dimethyl disulfide
30. Dimethylformamide
31 . Dowthenn
32. Dichloroethane
33 . 2, 3-Dichloro-l,4-naphthoquinone
34. Diethylamine
35. Isopropylbenzene
36. Isopropylbenzene hydroperoxide
37. Caprolactam (vapors, aerosol)
38. Caproic acid
39. Malathion
40. Xylene
41. Maleic anhydride (vapors, aerosol)
42. Manganese and its compounds (in
terms of MnO£)
43. Butyric acid
44. Mesidine
45. Methanol
46. Metaphos
47, Metachlorophenyl isocyanate
48. Methyl acrylate
49. Methyl acetate
50. Methyl mercaptan
51. Methyl methracylate
52. Monome thylani line
53. Arsenic (inorganic compounds other
than arsine, in terms of AS)
54. Nitrobenzene
55. Parachloroaniline
56. Parachlorophenyl isocyanate
57. Pentane
58. Pyridine
59. Prppylene
60. Propyl alcohol
61. Non toxic dust
62. Metallic mercury
2
0.20
0.001
3.0
0.007
0,0055
0.08
0.7
0,03
0.01
3.0
0.05
0.05
0.014
0.007
0.06
0.01
0.015
0.2
0,2
0.015
0.003
1.0
0.008
0,005
0.01
0.07
9-10-6
0.1
0.04
0.008
0.04
0, 0015
100. 0
0.08
3.0
0.3
0.5
3
0,20
0.001
1.0
0.03
0.01
1.0
0.05
0.05
0.014
0.007
0.06
0..005
0.2
0.05
0.01
0.01
•
0,5
G.,005
0.07
r
0..1
——
0..003
0.008
^__
0..0015
25. 0
0..08
3,0
0.15
0. 0003
— 2 —
-------
1
63. Soot (carbon black)
64. Lead and its compounds (other than
tetraethyl lead) in terms of Pb
65. Lead sulfide
66. Sulfuric acid (based on H2S04 molecule)
(based on hydrogen ion)
67. Sulfur dioxide
68. Hydrogen sulfide
69. Carbon di sulfide
70. Hydrochloric acid (based on HCl molecule
(based on hydrogen ion)
71. Styrene
72. Thiophene
73. Toluylene diisocyanate
74. Toluene
75. Trichioroethylene
76. Carbon monoxide
77. Acetic acid
78. Acetic anhydride
79. Phenol
80. Formaldehyde
81. Phosphoric anhydride
82. Phthalic anhydride (vapors, aerosol)
83. Fluorine compounds (in terms F)
Gaseous compounds (HF, SiF4)
Soluble inorganic fluorides (NaF,
Na2SiF6)
Sparingly soluble inorganic fluorides
(AlF3, Na3AlF3, CaF2)
In the combined presence of gaseous
fluorine and fluorine salts
84. Furfural
85. Chlorine '
86. Chlorobenzene
87. Chloropropene
88. Hexavalent chromium (in terms of Cr03)
89 . Cyclohexanol
90. Cyclohexanone
91. Carbon tetrachloride
92 . Epichlorhydrin
93. Ethanol
94. Ethyl acetate
2
0.15
0.3
0.006
0.5
0.008
0.03
) 0.2
0.006
0.003
0.6
0.05
0.6
4-0
3.0
0.2
0.1
0-01
0.035
0.15
0^10
0.02
0.03
0.2
0.03
0.05
0.10
0.10
0.10
0.0015
0.06
0.04
4.0
0.2
5.0
0.1
3
0.05
0,0007
0.0017
0.3
0.006
0.05
0.008
0.01
0.2
0.006
0,003
0.02
0.6
1.0
1.0
0.01
0.012
0.05
0.005
0.01
0,03
0,01
0.05
0.03
0.10
0.10
0.0015
0.06
On/
.04
___
0,2
5f\
.0
0.1
- 3 -
-------
1
95.
96.
Ethylene
Ethylene oxide
2
3.0
0.3
3
3.0
0.03
REMARKS
1. In the combined presence in atmospheric air of several substances
possessing a summation effect, the sum of their concentrations as calculated
by the formula below (§ 2) should not exceed 1 for:
a) acetone and phenol
b) sulfur dioxide and phenol
c) sulfur dioxide and nitrogen dioxide
d) sulfur dioxide and hydrogen fluoride
e) sulfur dioxide and sulfuric acid aerosol
f) hydrogen sulfide and dowtherm
g) isopropylbenzene and isopropylbenzene hydroperoxide
h) furfural, methanol and ethanol
i) strong mineral acids (sulfuric, hydrochloric and nitric) in
terms of the hydrogen ion concentration (H)
j) ethylene, propylene, butylene and amylene
should not exceed 1.3 for:
a) acetic acid and acetic anhydride
should not exceed 1.5 for:
a) acetone and acetophenone
b) benzene and acetophenone
c) phenol and acetophenone
2. Formula for the calculation:
m,
m,
where X is the unknown total concentration:
a b c is the concentration of the substance being determined,
1 * "I" ~divided by the corresponding maximum permissible con-
1 *- * centration for isolated action.
3. In the combined presence in atmospheric air of:
a) hydrogen sulfide and carbon disulfide
b)' carbon monoxide and sulfur dioxide
c) phthalic and maleic anhydrides and alpha-naphthoquinone, the
maximum permissible concentrations for each of them individually
are retained.
- 4 -
-------
4. In the combined presence in atmospheric air of parachlorophenyl
isocyanate and metachlorophenyl isocyanate, temporarily, until a method of
their isolated determination is developed, the standardization should be
made on the more toxic substance, i. e., parachlorophenyl isocyanate.
5. The maximum permissible concentrations of noxious substances in
the atmospheric air of populated areas as.formulated in December 1966
(No. 655-66), should be considered obsolete.
-------
SOME ASPECTS OF THE BIOLOGICAL EFFECT
OF MICROCONCENTRATIONS OF TWO CHLOROISOCYANATES
I. A. Zibireva
A. N. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences of the USSR.
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i
gigienicheskoe znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova.
Vypusk 11, Izdatel'stvo "Meditsina" Moskva, p. 91-107, (1968).
Chloroisocyanates (para- and metachlorophenyl isocyanates), high-molecular-
weight compounds that will be used in the production of a new herbicide, para-
chlorophenyldimethylurea, may occur as pollutants of the atmospheric air of
populated areas.*
The key element of these compounds is a benzene ring, two hydrogen atoms
of which are replaced by an isocyanate group (-NCO) and a chlorine atom (-C1):
the substances differ in the position of the isocyanate group (para isomers).
Parachlorophenyl isocyanate consists of white crystals with a pungent sweeetish
odor. Metachlorophenyl isocyanate is a colorless liquid with a similar odor.
Data on the toxicology of these compounds are very scarce. The toxicolog-
ical properties of metachlorophenyl isocyanate have not been studied at all.
The effect of parachlorophenyl isocyanate on the animal body has been studied
by I. N. Frolova (1962) at the level of the maximum permissible concentration
for the air of industrial buildings, 0.0001 mg/1 and higher. As was shown by
the studies, parachlorophenyl isocyanate is a highly toxic substance. When
it enters the body by inhalation, its fatal concentrations are expressed in
hundredths of a milligram per liter. The zone of the toxic effect is extremely
narrow. Parachlorophenyl isocyanate affects a number of the animal body
functions, causing dystrophic changes in the liver, kidneys, and heart muscle.
A number of authors have indicated the ability of substances of the iso-
cyanate group to act as allergens. Isocyanates are highly active compounds.
This leads to the assumption that they react with the body proteins to form
antigenic proteids (M. V. Gol'dblatt and Yu. Gol'dblatt, 1960). It should be
noted that aliphatic isocyanates (hexamethylene diisocyanates) are primarily
direct stimulants, whereas aromatic ones, which include para- and metachloro-
phenyl isocyanates, have a lesser stimulant effect and are more likely to cause
a sensitization.
*The
of the
'he study was made at the request of the Dzerzhinsk Branch of the State Scientific Research Institute
Nitrogen Industry and Organic Synthesis, Which initiated this project. '
- 6 -
-------
The toxicological properties of isocyanates have attracted the attention
of researchers. Aromatic isocyanates containing a chlorine atom in the ring
require a special study, since it is known that the appearance of a new chem-
ical group or atom in the molecule causes a change in the nature of the toxic
effect of a substance. The strength of the effect is considerably affected
by the spatial arrangement of the substituent radicals in the molecule, i.e.,
by the position isomerism (A. A. Golubev and V. Ya. Rusin, 1963).
The study of the nature of the effect of chloroisocyanates as possible
pollutants of atmospheric air is a very timely problem, since these com-
pounds have not yet been introduced into industrial production.
In order to establish the degree and character of the effect of micro-
concentrations of para- and metachlorophenyl isocyanates, we studied the
reflex responses produced by the stimulation of the human respiratory organs
by these substances, and their resorptive effect on the body of animals.
The results of the experiments were evaluated by the range method
(R. N. Biryukova, 1962) by considering the significance of the difference
between indices obtained under the influence of different concentrations of
chloroisocyanates and during inhalation of pure air (reflex effect) and the
differences between the indices of experimental and control groups of animals
(resorptive effect).
The content of chloroisocyanates in air was checked by using a modified
variant of the method developed by A. A. Belyakov for determining the content
of parachlorophenyl isocyanate in the air of industrial buildings. The
method is based on alkaline hydrolysis: parachlorophenyl isocyanate is con-
verted into parachloroaniline, the latter undergoes diazotization in a mixture
containing sodium nitrite and bromide, and azo coupling is carried out with
alpha-naphthol; a compound is thus formed which gives a pink color to the
solution. The color intensity is measured with a photoelectrocolorimeter.
The sensitivity of the method is 1 ug in the sample. Under the direction of
M. V. Alekseyeva, the sensitivity of the method was increased by introducing
changes into the relative proportions and volume of the reactants; in the
new modification, the method permits the detection of as little as 0.05 yg
of parachlorophenyl isocyanate in the sample. The procedure proved applicable
to the determination of metachloircphenyl isocyanate as well (with the same
sensitivity).
In the study of the reflex effect of chloroisocyanates on the human body,
the thresholds 'of olfactory perception were determined, and the influence of
microconcentrations of these substances on the light sensitivity of the eye
and the electrical activity of the human brain were studied by using physi-
ological methods employed for the standardization of atmospheric pollutants.
- 7 -
-------
As is evident from Table 1, parachlorophenyl isocyanate affects the
reflex responses of man in lower concentrations than metachlorophenyl
isocyanate. It is noteworthy that the odor subthreshold concentration of
metachlorophenyl isocyanate (0.008 mg/m3) does not alter the course of the
dark adaptation curve, in contrast to the first substance; the threshold
concentration of 0.010 mg/m3 is active. No such differences in the nature
of action of the substances were observed in the electroencephalographic
studies.
Table 1
Thresholds of Reflex Effect of Chloroisocyanates.
Substance
Parachlorophenyl
isocyanate . . .
Metachlorophenyl
isocyanate . . .
Olfactory
Perception
Light
Sensitivity
of the Eve
Electrical Activ-
ity of the Brain
Concentrations, mg/m5
Minimum
Perceptible
0,015
0,010
Maximum
Impercep-
tible
0,0064
0,008
1.5
0,0068
0,010
Maximum
Inactive
0,0029
0,008
1?
••-{ -H
s •*
0,0029
0,008
Maximum
Inactive
0,0015
0,005
As has been shown by many authors, the method of electroencephalography
permits a quantitative consideration of the change of the functional state
of the central nervous system during the action of low concentrations of
toxic substances, and is the most sensitive method for the study of the re-
flex effect of atmospheric pollutants on the human body. In our studies, we
used the "alpha-rhythm-burst response" technique developed by A. D. Semenenko.
A statistically significant depression of the alpha rhythm was caused by
Chloroisocyanates in concentrations that did not affect the light sensitivity
of the eye. Parachlorophenyl isocyanate did not change the electrical activ-
ity of the brain in a concentration of 0.0015 mg/m3, nor did metachlorophenyl
isoeyanate in a concentration of 0.005 mg/m3.
The method is objective, and the work of the experimenter is considerably
facilitated and more accurate when a biocurrent integrator is connected.
However, the duration of the experiment and possibly the excessive load on
the central nervous.system cause fatigue in the subject in the majority of
cases, and this has an adverse effect on the course of the experiment. We
therefore set up experiments in accordance with a shortened variant of the
"alpha-rhythm-burst response" technique proposed by Semenenko with a smaller
load for the subject. The duration of the test was cut by one-half, i.e.,I )
it lasted only 9 minutes. The entire experiment as usual consists of 18 qyclps,
each of which lasts half a minute, not one minute as before. The cycle begins
- 8 -
-------
with the supply of flickering light (13 seconds); during that time, the
light intensity is changed 2 to 3 times. This is followed by limbering
up (about 10 seconds), which is interrupted by a sound signal; during the
next 4-5 seconds the subject waits. The specified concentrations of the
substance are supplied in fractions, during the flickering light.
When this technique is employed, the experiment is set up under ob-
viously difficult conditions: the time of supply of the "gas" is shortened,
and the quantitative analysis of the curve is carried out in only 10 seconds.
If the effect of the substance studied can be obtained under these conditions,
it is probably more reliable, which proves the sensitivity of the method.
Chloroisocyanates in the same concentration caused a decrease in the amplitude
of the alpha rhythm that was sufficiently extensive and lasting to be con-
firmed statistically. The inactive concentrations were also in agreement.
These concentrations (0.0015 mg/m3. parachlorophenyl isocyanate and
0.005 mg/m^ metachlorophenyl isocyanate) are proposed as the highest single
maximum permissible concentrations for atmospheric air.
When the organism is exposed to different compounds, aspects of the
effect that are specific and characteristic of certain substances can. be
noted as well as a number of nonspecific changes in the state of the organism
that are of the same type for different poisons. When the effect of low and
microconcentrations of atmospheric pollutants is studied, its specific fea-
tures disappear, and nonspecific shifts, which are regarded as a straining
of the defensive reactions of the body in response to the action of the chem-
ical substances, assume a special importance. The mobilization of the de-
fenses indicates that the ambient medium does not match the sanitary level.
In order to determine the toxic effect of chloroisocyanates on the
animal body, a round-the-clock inhalational experiment was carried out on
white rats (males) for 80 days. The resorptive effect of para- and meta-
chlorophenyl isocyanates was studied in concentrations equal to the highest
single maximum permissible concentrations (0.0015 and 0.005 mg/m3 respectively)
and concentrations 20 times as high (0.03 and 0.1 mg/m3).
During the experiment, several indices were observed: ratio of the
rheobases and chrohaxies of antagonist muscles; excretion of coproporphyrin
with the urine (M. I. Gusev and Yu. K. Smirnov, 1960); electrophoretic
separation of the protein fractions of the blood serum (A. Ye. Gurvich,
1955) with refractometric determination of total protein; counting of the
absolute number .of eosinophils in peripheral blood (S. M. Bakman, 1958);
determination of neutral 17-ketosteroids in the urine; amperometric titration
of sulfhydryl groups in the blood serum. At the end pf the exposure and of
the recovery period, the ascorbic acid content was studied in the adrenal
glands, kidneys, brain, and liver by titrating with 2. 6-dichlorophenol
indophenol; pyruvic acid was determined in the liver by the Friedemann-Haugen
- 9 -
-------
method, the sulfhydryl groups were determined in the liver (N. N. Pushkina,
1963), and sialic acids in the blood serum were determined by the method
of Hess modified by Pushkina.
In the study of the effect of smaller concentrations (0.0015 mg/m^ para-
chlorophenyl isocyanate, 0.005 mg/m3 metachlorophenyl isocyanate), no statis-
tically significant difference in the indices of the experimental and control
groups of animals was found in any of the tests. These concentrations may be
recommended as the mean daily maximum permissible ones.
Let us consider the effect of large concentrations: 0.03 mg/m3 parachloro-
phenyl isocyanate and 0.1 mg/m3 metachlorophenyl isocyanate.
Electrophoretic analysis of the blood serum proteins (Fig. 1) revealed
an appreciable decrease of the albumin-globulin ratio (more marked and last-
ing in the presence of parachlorophenyl isocyanate). In addition, parachloro-
phenyl isocyanate is characterized by a statistically significant increase of
the total globulins owing to an increase of the gamma globulin content and, to
a lesser extent of the beta globulin fraction during the first half of the
exposure. The decrease of the albumin-globulin ratio during inhalation of
methylchlorophenyl isocyanate was due to the presence, on the one hand, of a
tendency toward a decrease in the amount of albumin, and on the other hand, to
an increase of globulins (in one case this increase was significant). In the
individual globulin fractions, only the increase of alpha globulin content
was found to be appreciable; during the action of parachlorophenyl isocyanate,
this increase appeared in the second half of the experiment, replacing the in-
crease of the gamma and beta globulin fractions. At the same time, a statis-
tically significant decrease of albumins was also observed.
S. Ya. Kaplanskiy (1962) has pointed out that when the body is acted
upon by harmful agents, instead of the albumins leaving the circulatory
system, the latter is penetrated by alpha globulins originating in the liver,
some of which have the same electrophoretic mobility as the corresponding
serum globulins. Similar changes in the ratio of protein fractions of the
blood serum apparently arise under the influence of metachlorophenyl iso-
cyanate and, during the second period of exposure, parachlorophenyl isocyanate.
As far as the increase in total globulins and particularly in the gamma
globulin fraction during the action of parachlorophenyl isocyanate is con-
cerned, it is known that allergic disorders always involve changes in the
protein fractions of the blood serum with a regular increase of individual
globulins (V. I. Pytskiy, 1963). Globulins are highly reactive proteins that
readily combine with various substances. Chemical substances of non-protein
nature causing allergy usually are not complete antigens and constitute de-
terminant groups, which acquire sensitizing properties only after combining
with the body proteins. General activation of protein synthesis is a pre-
requisite without which there can be no manufacture of a protein that is new
- 10 -
-------
for an organism and characterized by a specific affinity for a given antigen.
The majority of authors relate the formation of antibodies to gamma globulins,
which display a rate of displacement equal to that of antibodies in an elec-
tric field. In addition, it is well known that the production of antibodies
and gamma globulins is carried out by cells of the plasma series (P. F. Zdro-
dovskiy, 1961).
Parachlorophenyl isocyanate
too
80
120
120
I0°
s
o
#0
too
KO
too
Ba
k-
un 1
Albumin-globulin ratic
Exposure
Ibumin
lobulins
Metachlorophenyl isocyanate
lpha i
leta globulins
amma globulins
tecoyeiy
Penoc
A B A
55 74
/d
lit 29 44 55 74
18
Observation Time, Days
Fig. 1. Change in the ratios of protein fractions of the blood
serum during the action of parachlorophenyl isocyanate (0.03
mg/m?) and metachlorophenyl isocyanate (0.1 mg/m?).
1 - pure air; 2 - parachlorophenyl isocyanate, 0.03 mg/m';
3 - metachlorophenyl isocyanate, 0.1 mg/m*. The areas of sta-
tistically significant changes are crosshatched. Degree of
significance: a - 95.0#; b - 99.9%.
Under the influence of various factors, a change in the amount of
eosinophils in the blood takes place in the direction of both an increase
and a decrease of their number depending on the nature of the interaction.
Considering that the number of eosinophils in the blood is a variable
quantity subject to fluctuations in the course of a day (daily rhythm), the
blood of rats was taken under identical and constant conditions (before feed-
ing), preferably during the morning hours, when the number of eosinophils
was the lowest.
- 11 -
-------
The number of eosinophils in rats receiving metachlorophenyl isocyanate
did not differ from their number in the control animals during the entire
course of exposure. Parachlorophenyl isocyanate induced a statistically sig-
nificant increase of the eosinophil content of the blood. Eosinophilia (the
number of eosinophils was 42% higher than in animals of the control group)
corresponded in time to the increase of the gamma globulin fraction of the
blood serum and was absent in the second half of the exposure (Fig. 2). One
of the constant symptoms of allergy is eosinophilia, observable in the blood,
tissues, and mucous membranes. An increased bone marrow eosinophilopoiesis
is thought to take place during sensitization. Yu. F. Valiyev (1964) indicates
the simultaneous presence of a relatively distinct relation between the in-
crease in the phagocytic capacity of eosinophiles and their content in differ-
ent intracellular substances, including those formed during an allergic re-
action. It is possible that eosinophils active toward these substances are
transported with the blood stream toward the "shock" organs where the allergic
reaction becomes localized. In the lungs and spleen of the experimental ani-
mals, accumulations of eosinophilic leucocytes were observed which were not
found in the organs of the control animals.
In different experimental studies, the investigation of the. functional
state of the adrenal glands through observation of the excretion of neutral
17-ketosteroids with the urine is widely employed. At the start of exposure
to parachlorophenyl isocyanate, the content of 17-ketosteroids in the urine
of the experimental animals did not differ substantially from the background
indices (see Fig. 2). Later, simultaneously with a normalization of the con-
tent of the gamma globulin fraction and a certain decrease in the total glob-
ulins and in the number of eosinophils in the blood to the level of the con-
trol, there occurred an increase in the excretion of 17-ketosteroids with the
urine (the confidence factor of the changes was 99%). A change in the content
of 17-ketosteroids in the urine of rats during the action of metachlorophenyl
isocyanate occurred much earlier, although it was insignificant in the first
half of the experiment.
An intensified excretion of 17-ketosteroids indicates an increase in
the functional activity of the hypophyseal-adrenocortical system, which
characterizes stress reactions to harmful factors. According to the data of
F. M. Shleyfman (1961), the greatest changes in the functional state of
adrenal glands are observed precisely during the period of progressive adapta-
tion to an external irritant. A similar interpretation of the increased ex-
cretion of 17-ketosteroids may be entirely attributed to the action of meta-
chlorophenyl isocyanate.
During the action of parachlorophenyl isocyanate, the reaction of the
adrenal glands apparently has a more specific character and takes place ini
response to the sensitizing effect of the substance. As a result of the iti-
creased activity of the hypophyseal-adrenocortical system, there is an enhance-
ment of the excretion of gluco-corticoids, which, because of their antiallergic
- 12 -
-------
effect, depress the plasmacytic reaction (P. F. Zdrodovskiy, 1961, 1964) and
hence, antibody production, and depress eosinophilopoiesis. We tend to re-
gard the increase of the total globulins in the blood serum with a predomin-
ance of the gamma globulin fraction, eosinophilia and the superseding increase
of excretion of neutral 17-ketosteroids indicating an intense formation of
gluco-corticoids, as indications of a possible sensitizing effect of para-
chlorophenyl isocyanate (0.03 rng/m^). These indications disappear when the
120
6
CO
h
in s:
•a to
-p >>
8-8
-p O
Background
Exposure
Recovery period
51
66
10
Observation time, days
Fig. 2. Effect of chloroisocyanates on the level of eosinophiles
in peripheral blood and excretion of neutral 17-ketosteroids with
the urine. Notation same as in Fig. 1.
humoral medium in which the reaction between the antigen and the corresponding
antibodies takes place changes in a direction unfavorable to the occurrence of
sensitization processes. It should also be kept in mind that there is no
single test which could completely determine the allergic nature of the effect;
it becomes necessary to content oneself with only a set of positive data
(B. S. Preobrazhenskiy, 1964).
- 13 -
-------
We were interested in the supply of vitamins to the organism of the
experimental animals: in the adrenal glands, a decrease in the content
of ascorbic acid, which is involved in the oxidative transformation of
steroid hormones, is a manifestation of an increase in the functional activ-
ity of the adrenals; the process of antibody formation is sensitive to a de-
ficiency of vitamins. The content of ascorbic and pyruvic acids (the latter
may be used as an indicator of the level of vitamin B-^ in the body) was de-
termined in the organs of the rats. Results of the analyses are given in
Table 2.
Table 2
Change of Certain Biochemical Indices During the Action of Para-
chlorophenyl Isocyanate and Metachlorophenyl Isocyanate.
Biochemical Index
\Htamin C, ng$
Adrenal glands . .
Kidneys
Liver pyruvic
acid, mg % .
Sulfhydryl r
groups, yinoles . . .
^-Sialie acids of
Mijed serum, ng %
Control
(Pure Air)
525,4±17,0
18 8±1 4
39 4+1,7
29,4-1-2,9
1,9+0,6
0.90+.0.0!
152,8±4,7
Parachloro-
phenyl iso- •
cyanate. 0.03 .
ng/m3
332,2±24,7(c)
10 4+1 3 (b)
29 4±1,3 (b)
21 8±1.3 (a)
5,0+0,6(b)
0,53±0,ll(a)
263,0±28,0(b)
Metachlpro-
phenyl isocy- , ,
anate, 0.1 mg/nP
320, 1 + 21, 2 (c)
95+17 (b)
29,2+1,9 (b)
21,0±1,9 (a)
4,2+0,7 (a)
0,75±0,02(c)
229,6±19,3(b)
Remarks. 1. The table gives values of M + m. 2. Degree of sig-
nificance: a - 93#; b - 99*; c - 99.9#
Chloroisocyanates caused a decrease of vitamin C level in the adrenal
glands, kidneys, brain, and liver; this decrease was most pronounced in the
kidneys and adrenal glands (by 45.7-36.7%). Rats have the ability to syn-
thesize ascorbic acid in the course of intermediate metabolism in response
to the influence of unfavorable factors of the ambient medium (B. A. Lavrov
and B. I. Yanovskaya, 1956).
However, during a prolonged action of chloroisocyanate, the increased
biosynthesis of vitamin C evidently does not compensate for the greater de-
mand of the rat organism for this vitamin, and this results in a decrease of
the content of ascorbic acid not only in the adrenal glands but also in other
organs.
- 14 -
-------
An accumulation of pyruvic acid was observed in the liver. A delay in
the splitting of this acid can be used as an indication of hypovitaminosis
B-j^, since thiamine in the form of the pyrophosphate is a coenzyme partici-
pating in the oxidative decarboxylation of pyruvic acid. Z. S. Gershanovich
and A. I. Minkina (1951) found that vitamin B^ retards the oxidation of
ascorbic acid. A decrease of the thiamine level as a result of the action
of chloroisocyanates promotes the consumption of vitamin C in the rat organ-
ism even further.
These vitamins also share an affinity for the sulfhydryl groups of
proteins. Ascorbic acid is thought to be involved in the metabolism of
glutathione and other sulfhydryl groups. The presence of a certain amount
of sulfhydryl groups is necessary for the reduction of dehydroascorbie acid
to ascorbic acid. The enzyme system of oxidation and decarboxylation of py-
ruvic acid is considered to be a sulfhydryl enzyme. At the same time, all
enzyme poisons reacting with sulfhydryl groups substantially affect the activ-
ity of thiamine-containing enzymes (R. S. Vorob'yeva and S. V. Suvorov, 1961).
It should be kept in mind that the sensitivity of thiol enzymes to
various chemical agents apparently varies, so that the effect of each of them
is unique. Many are undoubtedly capable of reacting not only with thiol
radicals but also with other chemical groups, and this gives a distinctive
mark to the character of their effect (M. L. Belen'kiy and V. I. Rozengart,
1949). Para- and metachlorophenyl isocyanates cause a decrease in the amount
of sulfhydryl groups in the blood serum. A decrease in the content of sulf-
hydryl groups in the liver was also observed (see Table 2). It is possible
that chloroisocyanates interact with the reactive groups of proteins (sulf-
hydryl groups) and also with certain forms of thiamine.
Of late, attention has been focused increasingly on the study of carbo-
hydrate-protein complexes and products of their splitting. Most of the
techniques are based on the determination of the sialic acids (collective
name for derivatives of neuraminic acid) entering into their composition,
since sialic acids constitute the most reactogenic group of glycoproteins.
Glycoproteins include hormones, enzymes, enzyme inhibitors and activators,
and hormone carriers. Glycoproteins include many substances having a sero-
logical specificity (gamma globulins, complement components). The function
of sialic acids in the body has thus far received little study, but the avail-
able data indicate that they participate in defensive mechanisms against the
action of harmful agents (A. A. Titayev et al., 1964).
We used the determination of the content of sialic acids in the blood
serum as an indicator of the nonspecific changes resulting from the effect
of chemical substances on the rat organism. Chloroisocyanates caused a
70-50% statistically significant increase in the content of sialic acids
relative to the control (see Table 2).
- 15 -
-------
In substantiating the maximum permissible concentrations of atmospheric
pollutants, the excretion of coproporphyrins with the urine is frequently
studied as an indicator of general biological shifts in the body. A marked
decrease of the content of coproporphyrins in the urine took place under
the influence of chloroisocyanates. The confidence factor of the changes
caused by parachlorophenyl isocyanate was 99%, and metachlorophenyl isocya-
nate, 95%.
All the changes caused by the inhalation of chloroisocyanates were
associated with shifts in the values of the rheobases and chronaxies of the
extensors and flexors of the right rear shin (the determination was made by
using a standard technique with the aid of an ISE-01 electronic stimulator).
Against the background of an increase in the rheobase of the extensors, their
chronaxy was shortened; the rheobase of the flexors decreased slightly with-
out causing any appreciable changes of the chronaxy. These shifts lead to
a disturbance of the normal ratios of the rheobases and chronaxies of the
extensors and flexors: a change of the rheobases in opposite directions
causes an increase of their ratio in antagonist muscles; the chronaxies of
the extensors and flexors converge, causing an inversion of their ratio,
which falls below unity (Fig. 3). More pronounced and stable changes occur
under the influence of parachlorophenyl isocyanate.
According to the data of many authors (Yu. M. Uflyand, D. N. Markov,
A. N. Magnitskiy, and others), the action of various unfavorable factors
leads to an attenuation of the influence of the central nervous system on
the periphery, and this effects a change in the values of the rheobase and
chronaxy and an impairment of the interrelationships between antagonist
groups of muscles.
In the course of a three-week recovery period, disturbances in the
various systems of the experimental animals disappeared, indicating a
functional character of the shifts observed.
In summarizing, it may be stated that the action of large concentrations
of the substances studied gives rise to changes that indicate both a non-
specific action of the two chloroisocyanates and, to a certain extent, the
ability of parachlorophenyl isocyanates to cause a sensitization of the organism.
Symptoms of the allergic effect of parachlorophenyl isocyanate were ob-
served only during the first half of the exposure. It is probable that in a
concentration of 0.03 mg/m^, while stimulating the manufacture of antibodies,
this substance simultaneously stimulates mechanisms which depress antibody
formation and the allergic reaction. I. Ya. Uchitel1 and E. L. Khasman (1964)
suggested that the slight production of antibodies upon immunization of ani- i
mals with polysaccharide complexes that, like many allergens, are not complete
antigens, takes place because the doses of endotoxin capable of causing ^he
formation of antibodies are sufficiently large to give rise to a stress reaction
- 16 -
-------
that can depress the production of a specific protein. This type of endo-
toxin action is observed, for example, in rats, animals which are particu-
larly sensitive to the action of a stressor (P. F. Zdrodovskiy, 1964). This
is apparently why rats are resistant to the action of allergens.
Observation time, days
Fig. 5. Ratio of the rheobases and chronaxies of antagonist muscles
during exposure to chloroisocyanates. Notation same as in Fig. 1.
Parachlorophenyl isocyanate has a somewhat similar effect. It should
be remembered, however, that the intensity of the irritant is too low to
classify this substance as a stressor in the generally accepted sense. The
observed changes in the pattern of action of parachlorophenyl isocyanate in
the course of the experiment should apparently be attributed to the duration
and continuity of the effect. The fact that metachlorophenyl isocyanate has
no such specific action may be ascribed to its lower toxicity.
- 17 -
-------
Conclusions
1. The low level of the active concentrations indicates a high toxicity
of the chloroisocyanates. As shown by the results of the studies, parachloro-
phenyl isocyanate is the more toxic substance.
2. The toxic effect of both chloroisocyanates is chiefly manifested in
a straining of the defensive reactions of the body. During the action of
parachlorophenyl isocyanate (0.03 mg/m3), there are certain signs of aller-
gization of the animals, this being uncharacteristic of the meta isomer in
the concentrations studied. In experimental studies of the effect of atmos-
pheric pollutants, it is necessary to consider the possibility of a sensitiz-
ing action of chemical substances in low concentrations.
3. Differences in chemical structure are of unquestionable significance
in the degree and character of the action of microconcentrations of chloro-
isocyanates.
4. The highest single and mean daily maximum permissible concentrations
of para- and metachlorophenyl isocyanates are proposed at levels of 0.0015
and 0.005 mg/m3, respectively.
LITERATURE CITED
Note: References mentioned in this paper are to be found at the end of the
volume in the 1968 bibliography.
- 18 -
-------
THE TOXICOLOGY OF LOW CONCENTRATIONS OF AROMATIC HYDROCARBONS
I. S. Gusev
A. N. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences of the USSR
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i
gigienicheskoe znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova.
Vypusk 11, Izdatel'stvo "Meditsina" Moskva, p. 132-150, (1968).
The steady interest in benzene, toluene and xylene shown by toxicologists
and hygienists is due to the fact that these substances thus far have not lost
their industrial importance, possess a pronounced toxicity with a wide spectrum
of effects and, despite a large number of studies, remain far from adequately
studied. The toxicity of benzene, toluene and xylene has been studied (but not
at all to the same extent) in acute and subacute experiments on animals and also
in chronic experiments at the concentrations prevailing under industrial condi-
tions and exceeding the existing maximum permissible levels for plant shop areas.
The influence of low concentrations, i. e., concentrations equal to or below
the existing maximum permissible ones for industrial buildings, has not been
studied.
During the entire history of the toxicology of these substances, most
attention was concentrated on the investigation of benzene. Considering it
to be the most toxic of aromatic hydrocarbons of the benzene series, toxi-
cologists have aimed most of their investigations at low benzene concentrations.
Yu. V. Novikov (1957) studied the effect of benzene in concentrations
of 64 and 13 mg/nP during an exposure of 5% months, at the rate of six hours six
times a week. At the 64 mg/m concentrations, the animals showed functional
shifts in higher nervous activity; these shifts appeared during the 2nd-4th
month of exposure and gradually progressed. The effect was manifested in the
disinhibition of differentiating inhibition, extension of the latent period
of conditioned reflexes to a bell and loss of motor conditioned reflexes to
red light in rats of weak type, and also in the appearance of equalizing and
paradoxical phases in other rats. Changes in the central nervous system
were observed pathomorphologically. They were much less distinct in rats
of the second group. No distinct changes were found in the morphological
composition of the blood.
A. P. Volkova (1958) carried out a dynamic exposure of rabbits to benzene
in concentrations of 50 and 20 mg/m^ (three hours each in the course of three
months). During the action of the first concentration, leucopenia, throm-
bopenia, anemia and reticulocytosis were observed in the animals; the phagocytic
activity of the blood leucocytes decreased by 50%. At the 20 mg/m^ concen-
tration, a slight leucocytosis was observed in the blood of the animals; the
phagocytic activity of the blood decreased by 40%. Numerous hemorrhages were
- 19 -
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observed anatomicopathologically in the internal organs, and considerable ones
(as well as petechial hemorrages in the lungs) were found in the cortical and
medullary substance of the kidneys.
A. I. Korbakova, S. N. Kremneva, N. K. Kulagina and I. P. Ulanova (1960)
studied the effect of benzene concentrations of 20-40 mg/m^ in the course of
a 12-month chronic exposure (5% hours six times a week). The animals used
were cats, rabbits, and rats. In all the animals, the exposure caused undu-
latory and gradually progressive changes in the nervous system, and a tendency
toward a depression of cholinesterase activity. Changes in the blood appeared
after disorders in the nervous system and were manifested in a decrease of the
total amount of thrombocytes, a certain shift to the left at the end of the
exposure, and the appearance of young neutrophils.
The indicated studies are the only experimental ones on the effect of
low benzene concentrations. No experiments have been conducted on the effect
of low concentrations of toluene and xylene.
Another practically important and scientifically interesting problem is
that of the dependence of the degree and character of the toxicity of a sub-
stance on the change of its chemical structure, i. e., the problem of the
comparative toxicity of substances of a single series. Until now, the solution
of this problem has been approached from the standpoint of the effect of these
substances on the blood and blood-forming organs, this effect being considered
the most dangerous and specific. Thus, not one official classification of
chronic intoxication with benzene mentions changes in the nervous system. Most
often, the problems of the interrelationship of specific and nonspecific re-
actions in a comparative evaluation of the effect of benzene and its homologs
remained outside the scope of the researchers.
In recent years, papers dealing with the effect of the substances studied
on the central nervous system have been published quite frequently. Thus,
R. I. Yaroslavskaya (1952) noted that a decrease in the excitability of the
cerebral cortex during the action of benzene takes place before the develop-
ment of characteristic changes in the peripheral blood. G. E. Rozentsvit
(1954) pointed out that the early stages of chronic benzene intoxication
lead to disorders of the functional state of the cerebral cortex and of. the
cortical-subcortical neurodynamics. Disorders of the nervous system fre-
quently precede changes in the blood and often are the only sign of intoxica-
tion. N. V. Revnova (1965) cited the data of an observation of 100 patients,
in 19 of which the only manifestation of intoxication with benzene, toluene
and xylene were disorders of the nervous system functional in character and
defined as a neurasthenia syndrome with vegetative dysfunction.
The studies cited above (Yu. V. Novikov, 1957; A. I. Korbakova et al,
1960) also note an earlier and pronounced disturbance of the functions of
the central nervous system during the action of benzene. These data indicate
- 20 -
-------
a major importance of disorders of the activity of the nervous system in the
pathogenesis of occupational intoxications and the necessity of a compre-
hensive evaluation of all the changes arising under the influence of the
substances studied.
Changes in the fine structures of the central nervous system and
distortions of the biochemical processes give rise to disorders of the
physiological functions. That is why in the prophylaxis of intoxications it
is so important to study "nonspecific" reactions during exposure to low con-
centrations of toxic substances and to establish their thresholds.
The interest in the toxicology of low concentrations of chemical sub-
stances is explained by the possibility of not only their action on the
human body under industrial conditions, but also their penetration of atmos-
pheric air and action on the organisms of the population living in the
vicinity of the industrial plants. The effect of the poison depends not
so much on its concentration as on the duration of'its action. Consequently,
under the conditions prevailing in populated areas, the danger of contact
with low concentrations increases considerably. The mode of action of pol-
luted air of populated areas on the human body has been taken into account in
the practical standardization of the mean daily maximum permissible concen-
trations of atmospheric pollutants, which specifies that a prolonged round-
the-clock experiment must be carried out. The mean daily concentrations of
toluene and xylene have not been established thus far, and the mean daily
maximum permissible concentration of benzene requires an additional experi-
mental verification.
The object of the present study was to obtain experimental data on the
effect of benzene, toluene and xylene in concentrations below the existing
maximum permissible ones for industrial buildings (the maximum permissible
value for benzene is 20 mg/m^ and for'toluene and xylene, 50 mg/nH), to
evaluate them in a comparative manner, and also to obtain experimental material
for substantiating the maximum permissible concentrations of these substances
in the atmospheric air of populated areas.
To this end, we conducted a continuous 85-day exposure of 105 white male
rats divided into 7 groups with 15 animals in each group. Pure air was sup-
plied at a rate of 35 1/min into tjxe chamber of the first group of animals
(control), and air with an admixture of a certain amount of the substances
studied was supplied to the remaining groups. The average concentrations
in the chambers during the exposure and their fluctuations are shown in Table
1. The concentrations of the substances in the chambers were controlled
daily. The average concentrations of the substances during the exposure were
close to the calculated ones, with small indices of error of the arithmetic
means. The air samples were taken and analyzed by using the techniques of
M. V. Alekseyeva (1964).
- 21 -
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Table 1
Concentrations of.Bei
During
ne, Toluene and Xylene in the Chambers
.-the-Clock Exposure
Group
First
Second •
Third
Fourth
Fifth
Sixth
Seventh
Substance
Control
' > Benzene
' 1 Toluene
" } Xylene
Concentration, ng/m'
Calcu-
lated
1,5
15.0
0,6
15,0
0.2
15,0
Average for
the Period
of Exposure
1,49-4-0.024
15,07±0,233
0,59-1-0,009
14,65±0,1G2
0,234-0.006
14,60±0,168
Range
1.05—1,90
12,0—18,5
0,43—0,78
12.0—16,6
• 0,16—0,38
12,1—17,4
The concentrations of benzene (1.5 mg/m3), toluene (0.6 mg/m^), and xylene
(0.2 mg/m^) were taken at the level of subthreshold concentrations for the re-
Elex effect (I. S.'Gusev, 1965). These concentrations are found most frequently
Ln atmospheric air, and the study of the possibility of their resorptive effect
Ls of definite interest from the standpoint of a sanitary evaluation of the pol-
lution of air in populated areas.
The concentration of the substances studied,"15 mg/m3 (10 times the sub-
threshold value for the reflex effect of benzene), was taken at the same level
for the purpose of the clearest possible comparative evaluation of their toxic
affect.
Before the exposure, the animals were kept in chambers for two weeks to
get them to adapt to the conditions of the experiment and to record the back-
ground data on the indices of interest to us. The exposure was followed by
a one-month recovery period.
The animals were fed twice a day in accordance with the standards set
forth by the instructions of the Ministry of Health of the USSR,'with the daily
Introduction of cereals, meat, milk, vitamins, vegetables, bread, and feed
pellets. The animals received water in unlimited amounts.
In order to'reinforce the changes detected and to identify latent signs
af intoxications, during the'second half of exposure, a 15-day partial starving
af the animals was conducted, which is considered in toxicology as one of the
nethods of functional loading that'disturbs the existing systems of complex
reflex regulations. Cereals, meat, milk and bread were excluded from the ration.
rhe animals were fed once: feed pellets and vegetables.
In the course of the experiment, observations were made on the dynamics j
uf the weight and the general condition of the experimental animals. Considetf-
ing that the substances studied were poisons for the central nervous system
- 22 -
-------
and blood, we concentrated our attention on these functions. Analyses of the
ratios of motor chronaxies of antagonist muscles and of the cholinesterase
activity of whole blood served as indices of the effect on the central system.
The effect on the blood was evaluated from changes in the total number of
leucocytes and differential white count of the rats.
The results of all the observations were treated statistically by the
range method, with the'determination of the degree of significance of the
changes obtained (A. M«, Merkov, 1960).
We began the studies on healthy adult rats with an average weight of
220 g (the weight range from 180 to 260 g in the groups). During the exposure,
the external appearance of the experimental animals did not differ appreciably
from that of the controls. Animals of the third, fifth and seventh groups,
exposed to the substances studied in a'concentration'of 15 mg/m^, were char-
acterized by an increased excitability, restlessness, unwillingness to be
handled, and agressiveness, manifested particularly in rats of the seventh
group (xylene).
Animals in all the groups willingly accepted food and gained weight normal-
ly. They were weighed once every 20 days. The changes were evaluated from the
percent increase over the initial weight. In the first half of the exposure,
the weight of rats of all the groups did not differ from that of the controls.
During the period of experimental starving, animals of all the groups lost
weight; the most pronounced weight loss was observed in rats of the seventh
group. Subsequently, their weight lagged slightly behind the weight of the
control animals, but the data of the change were not statistically significant.
. f.SO -
Periods of experiment (in 10-day segments)
Fig. 1. Dynamics of ratios of motor chronaxies of antag-
onist muscles in animals of the second, fourth, sixth and
control groups.
1 - control group; 2 - .second group; 3 - fourth group;
4 -"sixth group
Muscular chronaxy is subject to cortical regulatory influences. Changes
of the motor chronaxy are due to disturbances of the functional state of the
- 23 -
-------
cerebral cortex and, in particular, of the motor analyzer of the cortex*
(U. Sh. Akhmerov, 1956).
The level of the ratios of motor chronaxies of antagonist muscles is
completely determined by central influences, and its change is regarded as
an attenuation of the subordinating influences of the cerebral cortex.
Our studies of the motor chronaxy and rheobase were carried out with
an ISE-01 pulsed electronic stimulator on five rats of each group, once
every ten days. These indices were determined on the flexors and extensors
of the right rear shin. The response was recorded from the twitch of the paw.
Average results of the ratios of chronaxies of antagonist muscles for the
groups of experimental animals were treated statistically with reference to
the analogous indices of the control (Table 2).
As is evident from this table, the average ratios of motor chronaxies
of antagonist muscles in animals of the second, fourth and sixth groups
during the action of subthreshold concentrations of benzene, toluene and
xylene in terms of the reflex effect did not differ appreciably from the
analogous indices of the control group. Statistical treatment of the material
did not show any significant changes in any of the groups (Fig. 1).
The action of the substances studied in concentrations of 15 mg/m^ caused
distinct and profound changes in the ratios of chronaxies. As early as the
20th day of exposure, an inverted ratio was observed in animals of the seventh
group, whereas a pronounced decrease of the ratios of chronaxies was noted in
animals of the fifth group. In rats of the third group (benzene), statis-
tically significant changes of these indices were noted ten days later. Sub-
sequently, these changes became accentuated, retaining high confidence factors
until the end of the exposure. During the partial experimental starving, the
degree of inversion of the ratios of chronaxies of antagonist muscles in the
groups of animals under consideration increased somewhat. After the starva-
tion, a certain increase of the ratios was observed in animals of the third
group, whereas a further accentuation of the changes took place in animals
of the fifth and seventh groups, particularly marked in animals of the latter
(Fig. 2). An interesting sequence was established during the recovery period.
A rapid recovery was observed in animals of the third group; as early as the
20th day of this period, the changes became insignificant, and toward the end
of the month the ratio of chronaxies returned to its original level. -The
indices under study returned to normal somewhat more slowly in rats of the
fifth group; the significance of the changes disappeared toward the end of
the month, but the level of the ratios still lagged behind the control. A very
slow recovery was noted in the seventh group of animals; an inverted ratio of
chronaxies with a high degree of significance of the detected changes was
preserved until the end of the recovery period.
* Editor's note: motor cortex.
- 24 -
-------
Table 2
Change in.the Ratios of Motor Chronaxies of Antagonist Muscles in the Course of.Chronic Exposure of Animals
. to Low Concentrations of _Benzene, Toluene, and Xylene. .
in
I
Group
First , .
Second . .
Third . .
Fourth
Fifth . . .
Sixth a .
Seventh . .
Average Values for Groups of Ratios of Chronaxies of Antagonist Muscles
Background
tv4^
1.33
l,28(o)
l,33(o)
l,27(o)
!,29(o)
l,29(o)
l,27(o)
•.": 2 ;•
1,27
l,26(o)
1.36(o)
l,2G(o>
Exnosure
3
1,33
1.34(o)
1^2(0)
l,32(o
1.30(o)! 1.29(0)
1,32(0)
1,30(0)
4
1,31
1,23(0)
1.24(o)
l,30(o)
l,08(b)
l,35(o)
0,98(b)
5
1,32
1.27(o)
0,88(b)
l,28(o)
0,82(c)
l,27(o)
0,73(c)
6
1,33
l,34(o
0,76 (c
l,33(o
0.74(c
l,32(o)
0,79 (c)
<
7
1,31
l,28(o)
0,70 (c)
l,30(o).
0.69(c)
l,27(o)
0,69(c)
8
1,32
l,33(o)
0.73 (c)
1.34(o)
0,63 (c)
l,26(o)
0.57(c)
Recovery
9
J.35
1 ,31 (o)
0,93(b)
l,33(o)
0.85(b)
'1 .35 (o)
0.63(c)
10
,32
,28(o)
,29(o)
.09 (a)
.30(0)
0,87 (b)
11
1,33
1.30(o)
—
l,20(o)
0.97(b)
Note. ,In this and the_following tables, the letters a, b, and c denote the confidence factors of the
data obtained, corresponding to the probability of an error of not more than 5, 1, and 0.1$ respectively.
o - not significant.
-------
covery period
123 US 6 7 6 9 10 11 12
Periods of experiment (in ten-day segments)
Fig. 2. Dynamics of ratios of motor chronaxies of antag-
onist '.muscles in animals of the third, fifth, seventh and
control groups.
1 - control group; 2 - third group; 5 - fifth group;
4 - seventh group.
The dynamics of tatios of motor chronaxies of antagonist muscles during
the action of benzene, toluene and xylene in concentrations of 15 mg/m3 indi-
cates the extent and manifestation of the changes taking place in the central
nervous system.
Choiinesterase is one of the antiregulatory factors participating in the
pathogenesis of all the vegetative disorders associated with an increased
formation of acetylcholine; the ability of the organism to'adapt is explained
by the humoral mechanism of self-regulation (D. Ye Al pern, 1958).
Studies of the cholinesterase activity of whole blood were made by using
the Fleischer-Pope photometric method (1954) modified by the department of
communal hygiene of the First Moscow Medical Institute im. M. I. Sechenov.
The modifications introduced into the Fleischer-Pope method concerned the
sequence of the control setup and the number of control samples.
Having observed that blood per se does not give a color in solutions,
the staff of the department proposed that duplication of controls with blood
for each of the experimental samples be excluded, and replaced with a single
control for a given experiment. The sequence of the subsequent procedure »
remained unchanged.
The observations were made on five rats of each group, twice a month over
- 26 -
-------
the course of the entire study. The blood was taken from the tail vein of
the animals. Average values of the cholinesterase activity of whole blood
according to groups of animals and the results of the statistical treatment
are presented in Table 3.
As is evident from this table, the cholinesterase activity showed marked
fluctuations in all the groups in the course of the exposure.
For greater clarity, the results of the study of cholinesterase activity
are given in percent of the control (Fig. 3).
No significant changes were detected in the statistical treatment of data
of the second, fourth and sixth groups. The cholinesterase activity of rats
of the third, fifth and seventh groups during the first half of the exposure
showed a depressant tendency that was statistically significant for the seventh
group only. During the second half of the exposure, particularly the starva-
tion period, an increase in cholinesterase activity significant only for rats
of the fifth group was observed.
Thus, the study of the cholinesterase activity of whole blood in the
course of round-the-clock exposure of the animals to benzene, toluene and
xylene did not yield any clear-cut data that would enable one to use the
effect of the substances studied on the central nervous system in a compara-
tive evaluation based on this index.
The lack of precise and definite results in no way indicates an absence
of the effect of benzene, toluene and xylene in a concentration of 15 mg/m^
on the state of the central nervous system. This method, based on the deter-
mination of total cholinesterases (specific and pseudo) cannot compare with
chronaximetry in sensitivity. In our view, a given intensity of the action
of the substances studied may affect the mediator factors of nervous stimula-
tion in animals of heightened sensitivity (even for a given duration of con-
tact), as illustrated by a marked and stable depression of cholinesterase
activity in rats Nos. 9 and 10 (seventh group). Benzene, toluene and xylene
are not specific inhibitors of cholinesterase and it is entirely possible
that a longer contact time is necessary for the manifestation of a more pro-
nounced action of these substances on the mediator functions.
I
Recent studies by A. P. Volfcbva (1958), A. I. Korbakova et al. (1960),
Kuhbock and Lachnit (1962), Deichmann and Villiam (1963) and others indicate
more frequent and earlier damage of the white blood cells during the action
of low benzene concentrations.
We determined the total number of leucocytes twice a month on five rats
of each group. The blood was taken from the tail vein by incision, asepsis
rules being observed. The blood was withdrawn by using N. M. Nikolayev's
sampling method. The counting was done in a Goryayev chamber.
- 27 -
-------
Table 3
Change in the Cholinesterase Activity of Whole Blood of Animals in the Course of Chronic Exposure to Low
Concentrations of Benzene, Toluene and Xylene,
10
00
Group
First .......
Third ' . . . \ . .
Fourth
Fifth .
Sixth '
Seventh .
Average Indices for Grouns According to Periods of Exneriment.
Background
1
204,6
217,4(0)
204,2(o)
192.8(0)
197,0(o)
231,4(0)
221..6 (o)
2
174,2 >
186,8(0)
188,0(6)
162,8(o)
184,8(0)
180,0(o)
lG3,6(o)
Exposure
3
170,8
170,2 (o)
172,8(0)
160,0(0)
173, 2 (o)
195,4 (o)
176,0(0)
4
225,0
231,4 (o
204,0 (o
194.4 (o
199,0 (o
198,6 (o
166,0 (A
)
5
200.0
208,6(o)
200,2(0
196,0(0
185,8(0
227,4(0
185,4 (o)
'6
162,8
175,8
174,6
173,0
223,6
207,0
184,0
o)
o)
o)
o!
(o)
7
242,8
250, 8(0)
233,2(o)
214,6(o)
250.8(o)
237.6(o)
215,2(o)
Recovery
S
233.3
210,0(oi
228,3(0)
200,0(o)
•228,3(0)
215,0(o)
183,3 (o\
-------
130
120
17/0.
a/00
I 80
3
4->
to
vatiOT
Exposure
/\
Star-
vation
\
\
Recovery
Period
{2345678
•Periods of experiment (15 days')
-——— Control group — Benzene
—•— Toluene ~ Xylene
Tig. 5. Change in the cholinesterase activity of
whole blood of animals in the course of round-the-
clock exposure.
1.- cholinesterase activity of animals of .the third,
fifth, seventh, and control groups; 2 - cholinesterase
activity of animals of the second, fourth, sixth, and
control groups.
Starvation
iCovery
/. Z 3 t 5 6- 7 8
Periods of experiment (two weeks)
Fig. 4. Total number of leucocytes in the peripheral
blood of animals of the second, fourth, sixth and
control groups. Notation same as in Fig. 1.
- 29 -
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Table 4
Change in the Total Number of Leucocytes in the Peripheral Blood of Animals During the Action of Low
Concentrations of Benzene, Toluene and Xylene.
Group
First
Second- • •
Third t
Fourth '
Fifth
Sixth
Average Indices for Groups AccordinR to Periods of Exceriment
Background Exposure
1
18280
17 750 (o)
18210(o)
18420
19610
17050
20 150
o)
3
nl
2 3
18800
20620(o
19460 o
20260(o
20550(o
19350(o
20 870 (o
20550(o)
18220(0)
17 930 (o)
20210(0)
19990(o)
18 130 (o)
19690(o)
4
19670
20050 (o
18210 (o
20780 (o
19 490 (o
20 100 (o
27890 (a
5
26530
24860
16690
26670
25000
25090
27060
?,
o)
°
0)
o)
6
21230
21 370 (o)
15460 (o)
•22420 (o)
17050 (o)
19630 (o)
27 830 (a)
7
24780
28 500 (o)
20140(0)
25 890 (o
25 270 (o
25 1 10 (o
36 030 (o
Recovery
8
26630
27 000 (o)
24266(0)
24416(o)
25 400 (o)
24 356 (o)
30 866 (o)
1
-------
Average indices of the total number of leucocytes according to groups
of animals for the period of chronic exposure and the results of the sta-
tistical treatment are listed in Table 4.
The total number of leucocytes in animals of the second, fourth, and
sixth groups which inhaled subthreshold concentrations in terms of the
reflex effect of the substances studied did not substantially differ from
the control in the course of exposure. No significant changes were detected
by the statistical treatment (Fig. 4).
In animals of the third, fifth, and seventh groups (at a concentration
of the substances studied of 15 mg/m3), these indices displayed some marked
changes. Thus, the total number of leucocytes in the peripheral blood of
animals of the third group had decreased by the 45th day; the significance
of the changes obtained was of degree B. During the period of experimental
starvation, these changes were somewhat accentuated, but their significance
was not confirmed statistically. Subsequently, adaptation of the white blood
cells to benzene was observed, with complete normalization by the 20th day
of the recovery period. In the fifth group of rats, no distinct statistically
significant changes were established in the total number of leucocytes during
the course of the experiment. During the starvation period there was noticed
a tendency toward a drop in their level. More persistent changes on the data
of the indices was observed in the animals of the seventh group. As early
as the end of the first month of exposure, a statistically significant increase
in the number of leucocytes was observed (degree A). Subsequently, the number
of leucocytes in the blood of animals of this group remained higher during
the starvation period as well, and the significance of the changes was confirmed
statistically. A marked increase in the number of leucocytes was noted during
the last period of exposure, but because of the large range of the indices in
the group itself, which we attributed to different individual sensitivities of
the rats to xylene, these changes were not statistically significant. By the
20th day of the recovery period, the total number of leucocytes in the blood
of rats of the seventh group had decreased markedly, but remained above these
indices for the control group (Fig. 5).
The collection of blood for studying its picture was made simultaneously
with that of blood for counting the total number of leucocytes, and on the
same animals. The analysis involved the use of the technique of N. 6. Alekseyev,
whereby the blood smear was stained first in a specially prepared solution of
Azur II, and the smears thus prepared were counterstained with the Romanovskiy-
Giemsa staining solution. In order to derive the picture, we counted units
of 200 cells over the entire surface of the specimen (continuous count method).
Analysis of the material obtained and statistical treatment of the data
according to the separate formed elements (absolute values) make it possible
to establish the following:
a) la the peripheral blood of animals of the second, fourth, and sixth
- 31 -
-------
groups, no clear-cut changes of the formed elements were noted;
6
Periods of experiment (2 weeks)
Fig. 5. Change in the total number of leucocytes in the
peripheral blood of animals of the third, fifth, seventh,
and control groups. Notation same as in Fig. 2.
b) Unstable leucopenia during the action of benzene in a. concentration
of 15 mg/m^ was accompanied by neutropenia with the appearance of staff cells,
a certain decrease in the number of eosinophils and monocytes, with a rela-
tively constant number of leucocytes.
c) An'extended action of toluene in a concenttation of 15 mg/m^ causes
neutropenia, the appearance of isolated staff cells, and relative lymphocytesis
in the blood of experimental rats.
d) Leucocytosis during the action of xylene in a'concentration of 15 mg/m
is associated with neutropenia, absolute lymphocytosis, an increase in the
number of monocytes, and the appearance of staff cells.
The above-indicated changes were not pronounced. The shift to the left
did not go beyond the appearance of isolated staff cells in the blood. We
regard the changes obtained as initial functional shifts. At this stage,
these changes were essentially compensated by the mobilization of defensive
reactions.
The results of pathomorphological studies constitute a valuable confir-
mation of the'effect of the substance studied, and aid in the determination
of its extent, nature and direction.
In order to carry out the pathomorphological studies, three rats of each
group'were killed by'decapitation on the last day of exposure. Their lungs,
heart, liver, spleen, kidneys, and brain were examined. Statistical treat-
ment of data on the relationship of the weight of the internal organs to the
total weight of the animals did not yield any significant changes.
- 32 -
-------
The results of pathomorphological studies* lead to the conclusion that
the concentrations of 1.5 mg/m3 benzene, 0.6 mg/m3 toluene and 0.2 mg/m3
xylene do not cause any perceptible morphological changes in the organs or
tissues of the experimental animals; benzene, toluene and xylene in a concen-
tration of 15 mg/m3 cause distinct changes of essentially the same type, at
the level functional shifts, Consisting in circulatory hemodynamic disorders
in the internal organs (lungs, liver, heart, kidneys, spleen, brain), a de-
crease in the glycogenic activity of the liver, its lipodystrophy, an increased
breakdown of erythrocytes in the spleen with deposition of hemosiderin, and
partial tigrolysis in the neurons of the brain.
Thus, the above-mentioned functional changes in animals exposed to benzene,
toluene and xylene in a concentration of 15 rag/m^ have received an ample patho-
morphological corroboration.
A unified approach to the evaluation of toxicity using the same highly
sensitive indices, single stages of investigation and methods of quantitative
evaluation of the effects observed enabled us to obtain data characterizing
the comparative toxicity of the substances studied.
Conclusions
1. A continuous 85-day exposure of white rats to benzene, toluene and
xylene in a concentration of 15 mg/m3 causes marked changes in the central
nervous system and blood, refl'ected pathomorphologically in the internal organs
of the experimental animals.
2. Most noticeable are changes in the central nervous system, which
occur before the changes in the blood and are more pronounced and more stable.
The degree of manifestation and stability of the changes in the central nervous
system increases with the number of methyl groups in the benzene ring.
3. We regard the changes obtained in the total number of leucocytes and
in the white count of the experimental animals (less stable for benzene and
toluene) as initial functional shifts. At the level in question, it is im-
possible to carry out a precise comparative evaluation of these changes.
4. There is no reason to regard benzene as more toxic than toluene or
xylene.
5. The concentrations of 0.6 mg/m^ toluene and 0.2 mg/m^ xylene, which
preclude the possibility of not only reflex reactions but also of resorptive
* The pathomorphological studies were carried out by the scientific collaborator 0. V. Kolbasova under
the direction of Doctor of Medical Sciences V. P. Osintseva, director of the laboratory of pathomorphological
studies of the A. N. Sysin Institute of General and Communal Hygiene of the Academy of Medical Sciences of
the USSR, both of whom the author sincerely thanks.
- 33 -
-------
action under conditions of a prolonged constant contact, may be recommended
as the mean daily maximum permissible values for atmospheric air.
6. For the time being, until the problem of the possibility of accumu-
lation during the action of low concentrations of benzene is solved, we
consider it advisable to leave its mean daily maximum permissible concentra-
tion at the earlier level, 0.8 mg/m3, which amounts to one-half the inactive
value which we have established.
LITERATURE CITED
Note: References mentioned in this paper are to be found at the end
of the volume in the 1968 bibliography.
- 34 -
-------
CHRONIC ACTION OF LOW CONCENTRATIONS OF ACROLEIN
IN AIR ON THE ORGANISM
Prof. M. I. Gusev, Decent I. S. Dronov, Decent A. I. Svechnikova,
A. I. Golovina, and M. D. Grebenskova
Department of General Hygiene, Rostov-on-Don Medical Institute
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i gigienicheskoe
znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova. Vypusk 10,
Izdatel'stvo "Meditsina" Moskva, p. 122-135, (1967).
In industry, acrolein* is prepared by oxidizing propylene over CuO or
by vapor phase condensation of acetaldehyde with formaldehyde in the presence
of Zn^CPO^)-. Considerable quantities of acrolein are used in the production
of glutaralaehyde and methionine.
Acrolein is a major product of glycerin synthesis. It escapes into the
atmosphere from plants where glycerin is subjected to a high temperature
(130-180 °C.): during the manufacture of oilcloth and linoleum, in the core
sections of foundry shops, in the production of insulation in the electrical
industry. In recent years the presence of acrolein in the exhaust gases of
motor transport has been demonstrated.
In atmospheric air and in the air of apartments around the Leningrad
Fat Plant, V. Z. Yas'kova found very high concentrations of acrolein which in
all probability were due to the use of a nonspecific method of determination
of acrolein.
M. M. Plotnikova (1960) determined acrolein in atmospheric air around a
chemical and an oil-chemical plant. At a distance of 100 m from the oil-
chemical plant, the highest concentrations were 2 mg/m3, in all the samples
the amount of acrolein was above the maximum permissible concentration
(0.3 mg/m3), and at a distance of 1000 m, the maximum concentrations were
0.64 mg/m3. Around the chemical plant (with the oil-boiling shop as the
source of pollution), at a distance of 200 m, the highest concentration was
20 mg/m3, and at 1500 m, 0.3 mg/m3.
The effect of acrolein on the human organism is manifested by a marked
irritation of the mucous membranes and a certain general toxic effect
(N. V. Lazarev, 1954). The maximum permissible concentration of acrolein
for shops is 0.002 mg/1.
* See article by M. M. Plotnikoya in the Collection "Maximum Permissible Concentrations of Atmospheric
Pollution", Prof. V. A. Ryazanov, Ed., I960, vol. 4.
- 35 -
-------
To validate the highest single maximum permissible concentration of
acrolein in atmospheric air, M. H. Plotnikova determined the odor thres-
hold. The minimum perceptible concentration was found to be 0.8 mg/m3.
The threshold of the reflex effect, determined by the method of optical
chronaxy, was 1.75 mg/m3, and that of the light sensitivity of the eye,
0.6 mg/m3. The highest single maximum permissible concentration of acrolein
in atmospheric air was established at a level of 0.3 mg/m3. The mean daily
maximum permissible concentration of acrolein (0.1 mg/m3), adopted on the
basis of a calculation, has not been experimentally validated thus far.
On the recommendation of the Committee on Sanitary Protection of
Atmospheric Air, we studied the effect of low acrolein concentrations on
the organism of experimental animals. A round-the-clock chronic exposure
of white rats in three chambers (with a capacity of 100 1 each) was carried
out for this purpose. Group IV was the control. The round-the-clock ex-
posure was carried out over the course of two months. There were 10 rats in
each chamber.
Acrolein in the air of the exposure chambers was determined by the
tryptophane method by D. P. Senderikhina, modified by M. M. Plotnikova.
The method is based on the fact that on reacting with acrolein, tryptophane
forms a stable violet color. The sensitivity of the method is 0.002 mg in
2 ml of solution. The specificity of the method is of no major significance
for our studies (M. V. Alekseyeva, 1962).
In the course of exposure in the first chamber (group I), the average
acrolein concentration was found to be 1.52 — 0.05 mg/m3 with fluctuations
from 2 to 1 mg/m3, in the second chamber (group II) 0.51 JL 0.02 mg/m3 with
fluctuations from 0.22 to 0.77 mg/m3, and in the third chamber (group III)
0.15 i 0.01 mg/m3 with fluctuations from 0.1 to 0.3 mg/m3. Group IV was
the control.
In order to validate the mean daily maximum permissible concentration
of acrolein in atmospheric air, the following factors were determined in the
experimental animals during the exposure: dynamics of weight and behavior;
conditioned reflex activity;.changes in the activity of whole blood cholin-
esterase; excretion of coproporphyrin with urine; percent content of the
number of fluorescent* leucocytes and histopathological changes in the organs
and tissues of animals which died from the effects of acrolein during exposure,
and in some of the rats sacrificed after the end of exposure.
At the end of the first week of exposure, the animals of group I
(1.52 mg/m3) had a sickly appearance, became sluggish and apathetic, and
their fur turned dull. The animals ate poorly. Their condition gradually
we have
* Editor's note: For the Russian use of the terns "luminescent" and "luminescence" in this paper.
ive substituted "fluorescent" and "fluorescence", on the oasis of the definitions of these terms.
- 36 -
-------
worsened, and for this reason the exposure in this chamber was discontinued
on the 24th day. During this period, all 5 rats in which the higher nervous
activity was studied died, and of the remaining animals in the experiment,
only two rats perished. No visible changes were observed in the behavior
and condition of rats of groups II, III and IV.
All the rats were weighed once a week during the exposure.
are shown in Table 1.
Table 1
The results
Group
I
II
III
IV
Acrolein
C oncentrat ion
ng/mS
1,52
0,51
0,15
Control
Exposure '
(end)
90,7 (a)'
114, 7 (b)
128,3.
129,1
Recovery
Period
108,7 (c)
121.7
128,9
134.8
Note. Degree of significance: a - 95$; b -
c - 99.9#
Comparison with the control is given. on the basis
of the condition on 24 April, date on which the exposure
was discontinued.
In animals of group I (1.52 mg/m^) , a rapid loss of weight was observed;
when it was necessary to discontinue the exposure, the weight had decreased
by 14% as compared with the initial weight. The weight decrease in animals
of this group also continued after the exposure was discontinued. At the end
of the first week of the recovery period, the weight loss was 25Z. The aver-
age weight of the rats reached its original value only at the end of the
5th week of the recovery period, then began to increase gradually.
Statistically significant weight changes in the direction of a decrease
were also observed in rats of group II (0.51 mg/m3).
There were no statistically significant weight changes in the experimental
animals of group III (0.15 mg/m^).
i
To establish the maximum permissible concentrations of chemical substances
in atmospheric air, a number of authors used the conditioned reflex method
(Yu. V. Novikov, N. F. Izmerov, M. I. Gusev and K. N. Chelikanov, Ya. G. Dvoksin,
V. N. Kursanov, and others). The studies showed that changes in the functions
of the cortex of the cerebral hemispheres take place very rapidly. Early signs
of disturbance of the higher nervous activity are phase states, facilitation
of differentiation, loss of certain reflexes and finally, disconnection of
all reflexes of the stereotype. In grave injuries, the natural conditioned
reflex to the appearance and odor of food disappears. The recovery period
may last a month and sometimes longer.
- 37 -
-------
Essentially, the change in the higher nervous activity during the action
of toxic substances is manifested in a steady weakening of the stimulating
and inhibiting processes followed by the development of protective inhibition.
The sensitivity of the method of conditioned reflexes is very high. The
maximum permissible concentrations of benzene, mercury, and lead for plant
shops earlier established in a study of their action on experimental animals
during a chronic exposure with the aid of the method of conditioned reflexes
were found to be many times greater than the threshold of their action.
However, the procedure used earlier for studying conditioned reflexes
in Kotlyarevskiy's chamber is extremely laborious and time-consuming. We
used an accelerated procedure for studying the higher nervous activity in
white rats, modified by Ya. G. Dvoskin (1961). A total of 18 white male
rats weighing from 64 to 102 g were used in the experiment; 5 each from groups
I, II, and III, and 3 from the control group. In all the animals, the stere-
otype was developed in the following sequence: bell, light; light, bell.
Before exposure Exposure
J t 7 3 II 3 S-J 10 >2 >5 17
§?do /
4Jma> •-
^&U> | £
Fig. 1. Effect of acrolein on the conditioned reflex
activity of rat No. 12 (concentration 1.52 ag/m5).
The most profound changes in higher nervous activity were observed in
rats of group I (1.52 mg/m3). After the start of exposure, the magnitude of
the motor conditioned response compared with the control decreased to both
the bell and light. This was followed by the appearance of phase states.
The conditioned reflex activity of one of the rats of this group is shown in
Fig. 1.
Changes in the conditioned reflex activity were also observed in rats
of group II (0.51 mg/m-*), but in a much less distinct form (Fig. 2).
In rats of group III (0.15 mg/m^) and in the control group, no changes
were detected in the conditioned reflex activity, as is evident from Figs, 3
and 4. These figures show that in animals of groups III and IV, the normal
strength relationships to the weak and strong stimuli and also the latent
period were preserved during the entire study.
- 38 -
-------
Before exposure Exposure Recovery period
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it was shown that a change in cholinesterase activity is caused by concen-
trations 10-20 times smaller than the maximum permissible ones for industrial
plants.
Magnitude of
atent motor con-
period, ditioned
sec response, mn
^O.'N.^^^^fe
Before exposure Exposure Recovery period
/ * 7 3 ii i s t w n is n is n n xnj* MJI to a *s tfsests* S7ss ft * s 1 10 n n n is st
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Fig. 4. Conditioned reflex activity of rat No. 5 (control group).
In our work, the activity of blood cholinesterase in white rats was
determined by Pokrovskiy's method (1953) modified by A. P. Martynova (1957).
Essentially, this method consists in the hydrolysis of acetylcholine,
determined by the time of change in the color of the indicator as a result
of the pH shift. The cholinesterase activity was determined once a week
in 20 rats (5 each from 4 groups). The results of the study are shown in
Table 2.
The time of acetylcholine hydrolysis in the control group was 38.7-40.3
minutes. In rats of group I (1.52 mg/m3), on the 15th day since the start
of exposure, a significant decrease in blood cholinesterase activity was
observed: The time of acetylcholine hydrolysis increased to 61 minutes.
On the 24th day, the exposure of the animals in this chamber was discontinued,
then the first study was made after 2 days, and the cholinesterase activity
was found to remain low. A return of the cholinesterase activity to normal
was recorded on the 2nd day after the exposure was discontinued.
A decrease in cholinesterase activity was also observed in rats of
group II (0.51 mg/m3). Statistically significant changes were recorded the
first time on the 34th day of exposure and reached a maximum on the 41st day
of the experiment. The time of acetylcholine hydrolysis began to decrease
gradually even before the exposure was discontinued, and reached normal
indices on the 10th day of the recovery period.
I
In animals of group III (0.15 mg/m3) and the control group, no changes
in the activity of whole blood cholinesterase were observed during the entire
experiment.
- 40 -
-------
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The mechanism of disturbance of
the porphyrin metabolism during the
action of toxic substances has been
inadequately studied thus far. How-
ever, many believe that this disturb-
ance takes place as a result of a
depression of enzyme systems which is
associated with a change in the cellular
metabolism in the nervous system, liver,
bone marrow, etc. (A. M. Chernyy,
R. B. Mogilevskaya, and others).
The amount of coproporphyrin
excreted with the urine during the
action of toxic substances in low con-
centrations decreases in some cases
(carbon monoxide, dowtherm, styrene,
dimethylformamide), and increases in
others (lead, pentene, toluylene
diisocyanate).
The study of porphyrin metabolism
in connection with the validation of
the maximum permissible concentrations
of chemical substances in atmospheric
air has been widely adopted, partly
because of the high sensitivity of this
method (M. I. Gusev, V. A. Chizhikov,
G. I. Solomin, D. G. Ododoshvili, and
others).
The coproporphyrin excreted with
the urine was determined spectrophoto-
metrically. The content of copropor-
phyrins was obtained from the optical
density of the maximum absorption at
402-403 my, determined with an SF-4
spectrophotometer. The urine was col-
lected simultaneously from 5 rats of
each group in the course of 24 hours,
once a week. A total of 56 analyses
were made, which amounted to 14 analyses
in each experimental group of animals.
The amount of coproporphyrin excreted
with the urine by the rats, in micro-
grams per 100 g of weight, is shown in
Table 3.
- 41 -
-------
In animals of group III (0.15 mg/m3) , no statistically significant
deviations in porphyrin metabolism were found as compared to the control.
In group II (0.51 rng/m-*), a statistically significant decrease of the amount
of coproporphyrin excreted with the urine was noted as early as the first
three weeks since the start of exposure. This decrease gradually became more
pronounced until the admission of acrolein to the chamber was discontinued.
In group I (1.52 mg/m^), before the time when it was necessary to dis-
continue the exposure, the amount of excreted coproporphyrin had a tendency
to increase, although this was found to be statistically insignificant. After
the exposure was discontinued, the porphyrin metabolism began to decrease
gradually, this decrease lasted for 5 weeks, and only in the 6th week of the
recovery period did the excretion of coproporphyrin gradually level off, and
in the 8th week reached the indices of the control group.
Table 3
Excretion of Coproporphyrin with the Urine in 24 Hours
(in micrograms per 100 g of weight).
Period of Study
From the. Start
of Exposure
First Three Weeks
Second Three Weeks
Third Three Weeks
Recovery Period
Concentration of Acrolein, rag/m3
Control
1,88
1,94
1,78
1,87
0.15
1,54
1,61
1,60
1,61
0,51
1,43 (a)
1,26 (b)
0,67 (c)
1,50
0.54
1,93
0,99 (c)1
0,72(c)
1,59
Note. Degree of significance: a - 95$; b - 99$; c - 99.9$.
1 Starting on 24 April the exposure was discontinued.
Observations of the early physicochemical and structural changes taking
place in the cells can be made by using the fluorescent microscopic method
(M. N. Meysel1 and A. V. Gutkina, 1953; M. N. Meysel1 and V. A. Sondak, 1954;
M. Ya. Khodas, 1954, and others). A. D. Semenenko (1963), who used the method
of induced (secondary) fluorescence, observed changes in the leucocytes of
blood taken from white rats in the course of chronic exposure to aniline in
concentrations of 0.03, 0.3, and 3 mg/m^.
In all three groups, a dependence was established between the manifesta-
tion of fluorescence and the concentration of the toxic substance, expressed
in the degree of change of the cells and time of its manifestation.
M. I. Gusev and K. N. Chelikanov (1963) used the fluorescent method of
study of blood leucocytes for an experimental validation of the mean daily
maximum permissible concentration of pentenes in atmospheric air. A statis-
tically significant increase in the number of fluorescent leucocytes was
- 42 -
-------
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established as compared with the control
group of the animals. This increase
occurred gradually, and the blood count
returned to the original norm even during
the period of exposure.
In our study, we determined the per-
cent content of fluorescent leucocytes in
the blood of rats during their exposure to
acrolein. For this purpose, the blood taken
was fluorochromed with acridine orange diluted
1:10,000, and examined under an ML-1 micro-
scope. The results are shown in Table 4.
In animals of group I (1.52 mg/m3),
an increase in the percent content of fluor-
escent leucocytes of the blood took place in
the very first week of exposure. Later it
decreased somewhat, but still remained high
as compared with the control. It should be
pointed out that it was difficult to take
the blood from rats of group I, because it
coagulated rapidly. This explains the lack
of data on the amount of fluorescent leucocytes
on some days of the study. The content of
fluorescent leucocytes remained high for
20 days after the forced discontinuation of
the exposure, but after 27 days of the re-
covery period the percent content of fluor-
escent leucocytes returned to normal, so that
further studies were discontinued.
In group II (0.51 mg/m3), there were
also statistically significant changes in
the percent content of fluorescent leucocytes,
which, starting with the first week of ex-
posure, continued to remain high until the
'end of the exposure period. After 11 days of
the recovery period, the number of fluores-
cent leucocytes began to decrease and reached
the normal indices.
In animals of group III (0.15 mg/m3),
for which there were no changes in the other
indices, a statistically significant increase
in the number of fluorescent leucocytes of
the blood as compared with the control group
- 43 -
-------
was observed 24 days after the start of exposure. Similar changes were also
observed by other investigators. For example, A, D. Semenenko (1963) re-
corded significant changes in the blood of rats during their chronic exposure
to aniline in a concentration of 0.03 mg/m^, whereas P. G. Tkachev (1963),
who used other methods of investigation, failed to observe any changes at
this concentration. This makes it necessary to postulate that the method of
fluorescent microscopy is very sensitive, so that it can be recommended for
use in the establishment of maximum permissible concentrations of chemical
substances in atmospheric air.
At the same time, it is necessary to study the nature of the process
whereby the content of fluorescent leucocytes in the blood increases when the
organism is acted upon by toxic substances in low concentrations.
After the chronic experiment was completed, some of the experimental
animals were subjected to an anatomico—pathologic examination..* Histological
examination of organs of animals in groups III and IV CO. 15 mg/m3 and control)
failed to detect any marked differences. Changes were found mainly after
the action of acrolein in concentrations of 0.51 and 1.52 mg/m3 (groups I and
II). Initial changes at the 0.51 mg/m3 concentration were manifested in the
proliferation of the columnar epithelium of the bronchi with hyperpreduction
of mucus and excessive infiltration of the bronchial walls by eosinophil
leucocytes.
In rats exposed to acrolein in the 1.52 mg/m3 concentration, there were
marked changes of an inflammatory nature, in the form of purulent panbronchitis,
bronchiolitis, and large-focus pneumonia (in one-half of the observations) in.
the respiratory organs, and complications of fibrous-purulent pleuritis in
two cases. In the internal organs (myocardium, liver), marked dystrophic
changes were observed in the form of granular and adipose dystrophy with small
areas of necrobiosis.
Conclusions
1. Prolonged inhalation of acrolein vapors in concentrations of 1.52-
0.51 mg/m3 by the experimental animals causes a weight loss, change of the
conditioned reflex activity, decrease of cholinesterase activity, change of
porphyrin metabolism in the direction of a decrease, and an increase in the
percentage of fluorescent leucocytes of the blood.
2. Under the same conditions of exposure, acrolein in a concentration of
0.15 mg/m3 has no effect on the organism of white rats with the exception of
an increase in the number of fluorescent leucocytes of the blood. The method
of induced (secondary) fluorescence permits a direct observation of the ' I
earliest physicochemical and structural changes in the cells, and can therefore
* The anatomico-pathologic examinations were performed by K. L. Volehenko.
- 44 -
-------
be recommended for use in the establishment of the mean daily maximum per-
missible concentrations of toxic substances in atmospheric air.
3. The mean daily maximum permissible concentration of acrolein in
atmospheric air established at the present time and equal to 0.1 mg/m3 is
at the level of the sub threshold concentration and can be left unchanged.
LITERATURE CITED
I
Note: References mentioned in this paper are to be found at the
end of this volume in the 1967 bibliography.
- 45 -
-------
STUDY OF THE REFLEX AND RESORPTIVE EFFECTS OF THIOPHENE
Sh. S. Khikmatullayeva
A. N. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences of the USSR
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i
gigienicheskoe znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova.
Vypusk 11, Izdatel'stvo "Meditsina" Moskva, p. 120-132, (1968).
Thiophene, a representative of a group of heterocyclic compounds, was
discovered in 1883. From the standpoint of physical properties, it is a
colorless liquid whose odor resembles that of benzene. In chemical properties,
thiophene is stable toward oxidizing agents, does not enter readily into addi-
tion reactions, and is easily nitrated and sulfonated.
Thiophene is used in the production of plastics, antioxidants, dyestuffs,
pharmaceutical preparations, insecticides, etc.
In its toxicological properties, thiophene is a nerve and blood poison
(N. V. Lazarev, 1954; McCord, 1931; Hultgren, 1926; Christomanos, 1930; A. G.
Mikhaylets and D. G. Pel'ts, 1964).
The extensive industrial use of thiophene, the possibility of its pollution
of atmospheric air, and the lack of literature data on the toxicity of its low
concentrations account for the relevance and timeliness of the present study.
To determine low thiophene concentrations, a spectrophotometric method was
developed (Ye. P. Aigina and G. S. Terekhova), whereby the optical density of
an alcohol solution of a thiophene mixture was measured at a wavelength of
231 ml* in quartz cells with a light path of 10 mm on an SF-4 instrument. The
sensitivity of the method was 0.5 yg/ml, and the accuracy, 4^5%.
To study the reflex effect of low thiophene concentrations, we carried
out physiological tests on people, widely employed in sanitary practice,
which consisted in the determination of the threshold of olfactory perception,
the light sensitivity of the eye (V. A. Ryazanov et al., 1957), and the bio-
electric activity of the human.cerebral cortex (K. A. Bushtuyeva, Ye. F.
Polezhayev, and A. D. Semenenko, 1960). The resorptive action of thiophene
was studied by means of physiological, hematological and biochemical tests
such as the ratio of chronaxies of antagonist muscles, absolute number of
leucocytes and the differential count, content of total protein and protein
fractions of the blood serum, sulfhydryl groups and sialic acids in the
blood serum by the method of Hess modified by N. N. Pushkina (1963), and
coproporphyrin in the urine.
- 46 -
-------
At the end of the exposure, we determined the ascorbic acid content in the
organs and sulfhydryl groups and pyruvic acid in the liver by the Friedemann-
Haugen method modified by Pushkina.
We began our experimental studies with the determination of the threshold
of olfactory perception in 21 persons with a normal sense of smell. For two
of the most sensitive persons of this group, the odor threshold of thiophene
was found to be 2.1 mg/m3, and the subthreshold concentration, 1.9 mg/m .
^oo
ISO
160
i 100
I
80
60
uo
5 10 15. 20
line, minutes
Pig. 1. Change in the light sensitivity of the eyes in
subject U. during inhalation of thiopiiene vapors.
1 - pure air; 2 - concentration, 2.1 mg/in^- 3 _ 1.4 og/n3;
4 - l.Olne/115; 5 - 0.8
The reflex effect of thiophene concentrations of imperceptible odor was
studied by using the dark adaptation method on three persons 19 to 40 years
of age.
The effect of four thiophene concentrations (2.1, 1.4, 1.0 and 0.8 mg/m3)
supplied from the 15th through the 20th minute of the test was studied. The
results of the experiment show that the first three thiophene concentrations
- 47 -
-------
had an effect on the light sensitivity of the subjects' eyes. In addition,
in subject A. we noted an increase of the light sensitivity upon inhalation
of thiophene vapors in concentrations of 2.1, 1.4 and 1.0 mg/nH, with a high
confidence factor of the changes obtained. In the two other persons we ob-
served a decrease of the light sensitivity of the eye in response to the same
concentrations (Fig. 1). The inhalation of thiophene vapors in a concentra-
tion of 0.8 mg/m3 did not cause any substantial changes in the light sensi-
tivity of the subjects1 eyes. Data on the light sensitivity of the eyes
during inhalation of thiophene are listed in Table 1.
Table 1
Change in the Light Sensitivity of the Eye in the Course of Dark
Adaptation During Inhalation of Different Thiophene Concentrations
(average values in percent of 19th minute).
Subject
A.
M.
Z.
Period,
Minutes .
20
25
30
40
20
25
30
40
20
•25
30
40
Pure Air
117.2
124.5
131.9
. 147.1
122,1
133,2
142,8
156,5
117.3
125.6
135.1
143,7"
. Thiophene Concentration, ng/m?
2.1
146, 3(b)
.211. 6 (c)
242, 8 (c)
263. l(c)
.91.0 (a)
97.2(o)
99.0 (a)
115.0(b)
82. 6 (c)
100,8 (c)
109,0 (a)
124. 2
-------
/ ^. 3 if 5 6 78 9 10-It 12 13 1U 15
minutes
mej mnues
Fig. 2. Change in the bioelectric activity of the cerebral
cortex in subject L. , during inhalation of thiophene vapors
( left Hemisphere ) .
1 - pure air; 2,- concentration, 0.8 ng/nr; 3 - 0.6 mg/rn'.
During the test, the subjects were exposed to a rhythmic light of dif-
ferent intensities (18 seconds), which was followed by a light muscular work-
out (25 seconds), a weak sound of variable frequency (10 seconds) and
expectation of the next light stimulus (7 seconds); these periods were re-
peated 15-18 times in each observation.
Changes of the brain biopotentials during inhalation of thiophene
vapors in a concentration of 0.8 mg/tn3 were analyzed by means of an automatic
integrator and were characterized by both a reinforcement and a depression
of the biopotentials. Statistical treatment of the data confirmed the signif-
icance of the changes, compared with the results obtained after the inhalation
of pure air.
We observed a reinforcement of the brain's biopotentials in subjects
Yu. and G., and a depression in L. (Fig. 2, Table 2).
I ,
Thiophene in a concentration of 0.6 rag/m5 had no significant effect on
the brain biopotentials of the subjects. Since this value is shown to be the
subthreshold concentration by the most sensitive test, we propose it as the
highest single maximum permissible concentration in atmospheric air.
Combined data from the study of the reflex effect are listed in Table 3.
- 49 -
-------
Table 2
Change in the Energy of the Brain's Biopotentials During Inhalation
of Different Thiophene Concentrations (average values in percent of
the first three minutes - background period).
0>
•1
L.
Y.U.
G.
in
•s"*"
1|
4—5
6—7
8—9
10-11
12—13
14—15
4-5
6-7 .
8-9
10-11
12—13
14—15
4—5.
6-7
8-9
10-11 '
12—13
14—15
Right Hemisphere
Pure
Air
100,4
101,0
102,4
99.8
100.7
102.8
98.2
96,8
99.10
99.6
97.2
99,2
103.4
105.0
102,1
102,9
105,9
111,6
°J*3
100,8(o)
104.8
103.1
90.4
103.2
102.5
97,3
100,5
99.2
94,3
95,9
101,2
109,6
115.8 (a)
107,7
111,6
110,0
107,4
0.6
100,0
99,6
103,2
102.8
99.2
100.8
101,8
96,4
103.2
101.8
100,2
103.5
97,7
101,8
103.2
103.2
105.2
104.1
left Hemisphere
Pure
Air
98,2
98.4
102,1
98.7
101,4
103,0
101,1
104,1
105.8
104,3
103,5
100,6
104,9
107.2
105,6
105,7
111,1
109,5
mg^
93,3
98,2" •
93,8 (a)
81,1 (a)
92,2
90,8 (b)'
109, 6 (a)
109,6
116,9 (a)
119.0(b)
111.3
113.5 (a)
99 2
10o',3(a)
101.3
98,0
101, 5 (a)
102.2
°'63
100,6
101,8
104,7
104,0
100.5
104.7
100,7
93,4
98,8
101,8
99,1
103,5
o
a
0
o
:°
t°
o)
3
0
0
100,8
103,7
98,2
101,2
102,8
96,4'
Note. Confidence factor: a - 99#; b - 99$; o - unreliable.
Table ~b
Reflex Effect of Thiophene on the
Human Organism.
Thiophene Concen-
tration, tng/m3
Olfactory Percep-
light Sensitivity of
the eye . ' . . .
Bioelectric activity
of the cerebral
cortex • . ••
threshold
2.1
1,0
0,8 .
Sub^
threshold
19
0,8
.,
0,6
- 50 -
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We studied the resorptive effects of thiophene in the course of round-the-
clock chronic exposure on 60 white male rats during 80 days. The animals were
divided into four groups: the first was exposed to a thiophene concentration
of 20 mg/m3, the second to 3 mg/m3, the third'to 0.6 mg/rn3, and the fourth
group was the control. During the experiment, observations were made every 15
days; the results were treated statistically.
The general state of the animals during the course of the experiment re-
mained unchanged. The rats were active and gained weight normally. The results
of treatment of the weight of the rats in per-
cent of the initial weight remained unreliable
compared with the control.
In the study of the chronaxy'of extensors
and flexors during the experiment, statisti-
cally reliable changes expressed in a conver-
gence of the chronaxies of flexors and extensors
were observed in rats of the first group (20
mg/m3). Further studies showed an inversion of
the ratios of chronaxies of flexors and exten-
sors in rats of the first group, and these
changes occurred before other indices, in the
third week of the experiment (Fig. 3). A res-
toration of the normal ratio of chronaxies of
antagonist muscles in this group of rats oc-
curred on the 26th day after the end of ex-
posure. We observed shifts in the leucocyte
content later than changes in the chronaxies
of antagonist muscles. Leucopenia occurred
in rats of the first group (20 mg/m3) as late
as the 6th week of exposure, and lymphopenia,
in the 8th. The restoration Of leucocytes
also occurred on the 26th day, and that of
lymphocytes, on the 10th day after the end of
the experiment (Fig. 4).
The total amount of blood serum proteins
in rats of all the groups did not show any
changes in the course of the experiment. De-
viations in the protein fractions which oc-
curred in the fourth week were observed only
in rats of the first group.
These changes were characterized by a de-
crease in the amotint of albumin, an increase
of gamma globulin, and'a decrease of the
albumin-globulin ratio, this being in agree-
ment with the data of a number of authors who
Exposure
Exposure
Exposure
Control
30/X/I 5/11 ft/Hi 13/lY
Date of exposure
Fig. 3. Disturbance of the
motor ohronaxy of antagonist
muscles in experimental rats
during exposure to thiophene
vapors.
1 - extensors; 2 - flexors.
- 51 -
-------
found changes in the fractional composition of the blood serum of people and
animals under the influence of harmful chemical agents (V. A. Chizhikov, 1964;
P. G. Tkachev, 1964; K. A. Bushtuyeva, 1966). Further studies of the relative
amounts of protein fractions in rats of the first group revealed changes in
the beta globulin fraction which took place in the sixth week of the experiment
and returned to normal at the end of the experiment. Changes in the relative
amounts of the protein fractions in rats of the first group were confirmed by
results of histopathological analyses. It is probable that the shift which we
observed in the relative amounts of protein fractions in rats were due to the
toxic effect of thiophene on the liver.
25/X/M965 25/1 9/H 25/lf fl/Hl28//1110/IV28/IY
• 1/1-1956
Date of study
Fig. 4. Number of leucocytes in the peripheral blood
of rats of different groups.
Group 1-20 mg/m^j Group H - 3 ng/m'; Group IH -
0.6 ng/niS? Group IV - control.
The content of sulfhydryl groups in the blood serum of rats of the first
group decreased in the fourth week. We attribute these changes to functional
shifts in the course of the processes of inhibition and stimulation of the
nervous system, brought about by the action of thiophene.
A reliable increase in the content of sialic acids in the blood serum.of
rats of the first group was detected at the end of the experiment. J
- 52 -
-------
In the third week of the experiment, a decrease in the excretion of copro-
porphyrin with the urine was established (Fig. 5), which may be regarded as a
consequence of a disturbance of the activity of the central nervous system
(Yu. K. Smirnov, 1957). More pronounced changes in the excretion of copro-
porphyrin with the urine of rats were observed'during the action of thiophene
in a concentration of 20 mg/m3 than at 3 tng/m3, and these changes lasted a
long time after the exposure.
0.6
0.7
h"
ai-H r>K
M)fl) U.O
0.3
0.1
5f[-1966 .
Date of study
Fig. 5. Dynamics of excretion of eoproporphyrin with rat urine
during exposure to thiopene vapors. Notation same as in Fig. 4.
One of the biochemical indicators of vitamin metabolism was the determi-
nation of ascorbic acid in the organs of the rats at the end of the experiment.
A disturbance of vitamin C metabolism indicates an unfavorable influence of'
atmospheric pollutants (Ye. F. Yelfimova, and N. N. Pushkina; V. A. Yakamees,
1966). We established a significant decrease in the content of ascorbic acid
in the brain, liver, kidneys and adrenal glands of animals of the first group.
The most pronounced changes were observed in the kidneys.
A decline in the amount of ascprbic acid, a decrease of the sulfhydryl
groups and an increase of pyruvic acid in the liver of animals of this group
correlated with histopathological data indicating a decrease of glycogen.
In rats of the second group (3 mg/m^), the changes developed more slowly.
Shifts in the ratio of chronaxies of antagonist muscles occurred as late as the
sixth week of the experiment. Leucopenia in the peripheral blood of rats of
this group was observed as late as the sixth week and was unstable. No change
was noted in the number of lymphocytes. The concentration of the protein frac-
tions of the blood serum in rats of the second group remained without any
appreciable changes over the course of the experiment, with the exception of
- 53 -
-------
the gamma globulin fraction, which increased in the fourth week and gave signif-
icant results by statistical treatment. These changes may be regarded as a
defensive-adaptive reaction of the body to the influence of a harmful agent,
in particular, thiophene (3 mg/m3). The increase in the content of gamma glob-
ulin may also be due to the activation of Kupffer's cells and plasma cells in
the organism of this group of rats (histopathological data).
The manifestation of the toxic effect of thiophene at the 3 mg/m3 concen-
tration on sulfhydryl groups of the blood serum of rats was confirmed statis-
tically and occurred later than in rats of the first group, i
week of the experiment.
e., in the sixth
A change in the amount of coproporphyrin in the urine was also observed
in this group of rats, but to a lesser extent and later than in rats of the
first group.
Table 4
Results of the Resorptive Effect of Thiophene on the Organism of
White Rats.
Indicator
Ratio of chronaxies of antagonist
Protein fractions:
globulins:
8
Albumin-globulin ratio
Sulfhydryl groups in blood
Sulfhydryl groups and pyruvic acid of
Sialic acids in blood serum
*.
Thiophene Concentration,
fflg/m?
20
+
"+
+
- +
+
+
+
,'+
+
+
+
+
3,0
+
.+.
+
+
+
+ -
+
+
0,6
— .
Control
—
Symbols: + reliable result, - unreliable result.
-54 -
-------
The ascorbic acid content of the internal organs of rats also underwent a
reliable decrease. Changes in pyruvic acid, sulfhydryl groups in the liver and
sialic acids in the blood serum were not reliable.
In the third group (thiophene concentration, 0.6 mg/m3) the results of the
studies differed little from the control and were not statistically reliable.
Combined data on the resorptive effect of thiophene on the experimental rats
are shown in Table 4.
Functional changes in rats of the first and second groups were confirmed
by histopathological studies, which showed the presence in the lungs of neutro-
phils, areas of desquamative bronchial epithelium, and proliferation of the ep-
ithelium of the bronchi in the lungs. In the liver,-an irregular engorgement
of the capillary network was noted, activated forms of Kupffer's cells were fre-
quently found in the lumen of the sinuses (round macrophages, and accumulation of
neutrophils in the lumen of large vessels), and lymphoid infiltration appeared
around the triads. There was a distinct decrease of glycogen over the periph-
ery of hepatic lobules. No such changes were found in rats of the third group.
Conclusions
1. The threshold concentration for olfactory perception of thiophene is
2.1 ing/m , and the subthreshold concentration, 1.9 mg/nH.
2. The thiophene concentration acting on the light sensitivity of the eyes
is below the threshold of olfactory perception and equal to 1 mg/m^, and the
inactive concentration is 0.8 mg/m3.
3. Changes in the bioelectric activity of the cerebral cortex of the sub-
ject were observed during inhalation of thiophene vapors in a concentration of
0.8 mg/m^; a concentration of 0.6 mg/m3 was found to be inactive.
4. A prolonged round-the-clock action of thiophene in concentrations of
20 and 3 mg/m3 gives rise to a series of physiological, hematological, bio-
chemical and pathomo-rphological changes in the body of the experimental animals.
The 0.6 mg/m3 thiophene concentration was found to be inactive.
/.
5. The highest single and mean daily concentrations of thiophene which
can be recommended are at a level of 0.6 mg/m3.
- 55 -
-------
LITERATURE CITED
Note: References mentioned in this paper are to be found at the
end of the volume in the 1968 bibliography.
- 56 -
-------
SANITARY-TOXICOLOGICAL APPRAISAL OF THE COMBINED EFFECT
OF A MIXTURE OF BENZENE AND ACETOPHENONE VAPORS
IN ATMOSPHERIC AIR
V. R. Tsulaya
A. R. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences of the USSR
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i
gigienicheskoe znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova.
Vypusk 11, Izdatel'stvo "Meditsina" Moskva, p. 107-120, (1968).
The toxic effect of benzene at and slightly above the level of the
maximum permissible concentration for plant shops was studied by A. P. Volkova
(1960), Gabor and Raucher (1960), A. I. Korbakova et al. (1965), and others.
The toxic effect of low concentrations of these substances was investigated
by Yu. V. Novikov (1956) and I. S. Gusev (1965).
In analyzing the data obtained by the different authors one can con-
clude that benzene in low concentrations has a predominant influence on the
central nervous system and causes a series of changes in the blood-forming
system, primarily in the white blood cells. A decrease in the number of
thrombocytes in the blood is considered to be one of the early signs of
benzene poisoning.
In a study of the effect of large concentrations of benzene in combin-
ation with chloroform and toluene, T. A. Shtessel' (1938) observed a simple
summation effect of their action. During the combined action of large con-
centrations of benzene and acetone, M. I. Ol'shanskiy and V. V. Likhacheva
(1935) noted an enhancement of the influence of the mixture. A prolonged
combined action of benzene and acetone in concentrations at and above the
level of those in plant shops was studied by N. A. Zhilova (1959). On the
basis of the experiments performed, the author holds that the benzene-acetone
mixture irritates the central nervous system and blood-forming organs much
more strongly than any of these substances does individually.
The effect of low concentrations of acetophenone, both during short-
term exposure of man and under conditions of a chronic experiment on animals,
was studied by N. B. Imasheva (1963), who observed marked functional changes
in the central nervous system and in the proportion of protein fractions of
the animal blood. During the combined action of low concentrations of aceto-
phenone with phenol (Yu. Ye. Korneyev, 1965) and acetophenone (N. Z. Tkach,
1965), the authors found a simple summation of the effects.
The literature contains no studies dealing with the combined action of
low concentrations of benzene and acetophenone discharged into atmospheric
- 57 -
-------
air during the production of synthetic phenol and acetone.
i/E
&5000
&5000
tsooo
15000
10
30
/5 ^0 25
Time, minutes
Fig. 1. Effect of low concentrations of a mixture of benzene
and acetophenone vapors on the light sensitivity of the eye
in subject Sh. Yu.
1 - pure air; 2 - gaseous mixture with a total concentration
index of 1.03; 3 - gaseous mixture with a total concentration
index of 0.75.
The object of the present paper was to study the nature of the biological
effects of a mixture of low benzene and acetophenone concentrations in atmos-
pheric air.
The character of the inhalational effect of this mixture on man was
evaluated by determining the thresholds of its olfactory perception and its
reflex effect on the light sensitivity of the eye and bioelectric activity of
the brain. These tests are based on the influence exerted on the bodily func-
tions by an extraneous stimulation focus arising in the central nervous system
during inhalation of the substances studied.
- 58 -
-------
115
90
1 2 3
5 5 7 6 9 10 11 12 13 Hi t5
Time, minutes
Fig. 2. Change in the bioelectric activity of the brain in
subject R. L. during inhalation of a mixture of benzene and
acetophenone vapors. Left hemisphere.
1 - pure air; 2 - gaseous mixture with n total concentration
index of 1.05; 3 - gaseous mixture with a total concentration
index of 0.88.
Table 1
Thresh9lds of the Reflex Effect for the Most Sensitive Persons
During the Combined Action of Benzene and Acetophenone.
Olfactory Perception
Light Sensitivity
of the Eye
Bioelectric Activity
of the Brain
-
P4->
a-o
1
O X
C 4)
O,T)
CO C
•P O
O.rf
EH-P
A IS
Ifi
Q) V
6r-IC
i.-S.H
'b'S-g
3eo S
S "4->
0§
aa-
4JO
OH
ho
on
Thr
tra
ffl G
•PO
O.H
II
I!
o>c
-p o
O-H
6-4 -p
2
^
Sx
&H
E-I-P
1,18
0,003<:
0,79
1,2
0,86
0,005
0,0035
0.75
1,1
0,0035
1,05
0.9
0,003
0,88
Note. Numerator of the fraction -? benzene, denominator - acetophenone.
- 59 -
-------
As is evident from Table 1 and also Figs. 1 and 2, all the tests showed
the active mixture of benzene and acetophenone vapors to be one whose total
concentration index in fractions of the threshold values for isolated action
of the ingredients was equal to unity; for the inactive mixture, this index
was below unity, i. e., the effect takes place in accordance with a simple
summation.
The most sensitive was found to be the electroencephalographic test
(minimum active mixture of benzene and acetophenone vapors in concentrations
of 1.1 and 0.0035 rag/m3 respectively).
In expressing the total content of the mixture employed in fractions
of the maximum permissible concentration of each component, it was found
that mixture I (minimal active) consists of:
1.1 . 0.0035 _ . , QQ
y-=- benzene + ~ nn?n acetophenone = 1.89,
and mixture II (maximum inactive) , of
0.9 . 0,003 . . , ,
•=— =• benzene + T^"™? acetophenone = 1.6
J. . D
The studies performed lead to the conclusion that when benzene and
acetophenone are simultaneously present in atmospheric air, their total con-
centration in fractions of the maximum permissible concentration of the sub-
stances for isolated action should not exceed 1.5.
Whereas the highest single maximum permissible concentrations are designed
to prevent a degree of atmospheric pollution which could cause reflex responses
through the irritation of receptors of the respiratory organs, the mean daily
standards prevent a chronic resorptive effect of toxic substances during pro-
longed inhalation.
During the action of very low concentrations of toxic substances under
conditions of a chronic experiment, the functional changes are basically
nonspecific in character and should be regarded as defensive-adaptive responses
(V. A. Ryazanov, 1964).
In order to evaluate the toxic effect of a mixture of benzene and aceto-
phenone vapors on the animal organism, the following factors were observed:
general state, behavior and dynamics of the weight of the animals, change of
the motor chronaxy of antagonist muscles, content of leucocytes, erythrocytes
and thrombocytes in the blood, differential white count, total content of nucleic
acids in the blood, and amount of 17-ketosteroids in the urine. At the end of
the experiment, some of the animals were killed and subjected to anatomicopatho-
logical and histological analyses.
The inhalational exposure of 45 white male rats was carried out continuous-
ly for 84 days. The rats were divided into three groups of 15 each.
- 60 -
-------
The first group of animals was subjected to the action of a mixture of
benzene and acetophenone vapors in a concentration 10 times the maximum in-
active mixture of substances used in the study of the reflex effect on the
biocurrents of the brain. On the average, the mixture consisted of 9 i 0.194
mg/m3 benzene and 0.030 - 0.0033 mg/m3 acetophenone.
The second group of animals inhaled a mixture at the level of the highest
inactive concentration in the electroencephalographic tests. On the average,
it contained 0.9 - 0.2 mg/m3 benzene and 0.003 - 0.00005 mg/m3 acetophenone.
The third group of animals served as the control.
To evaluate the functional state of the cortex of the large hemispheres,
the ratio of chronaxies of antagonist muscles was studied, whose change is
affected by central influences.
85/XI-19G5 3/XII?8/XIf 1S/J Z//H966 18/113/11115JRI28/111 ft/IV
Date of Test
Fig. 3. Ratio of chronaxies of antagonist muscles in rats
exposed to benzene and acetophenone vapors.
1 - first group (total concentration index 9.9)? 2 - second
group (total concentration index 0.9)j 3 - third group (control).
The motor chronaxy of antagonist mustles (extensors and flexors of the shin)
was studied in five rats of each group once every 15 days with the aid of an
ISE--01 electronic pulse stimulator.
The results of the tests, conducted in accordance with a standard pro-
cedure, are shown in Fig. 3, from which it is apparent that a change in the
ratio of chronaxies of antagonist muscles manifested itself very early in the
- 61 -
-------
first group of animals, on the 15th day of exposure, and remained below unity
during the entire period of exposure. This indicates a substantial effect of
mixture I, which caused functional changes in the central nervous system and
was manifested in a decrease of the subordinating influence of the center on
the ratio of chronaxies of antagonist muscles.
Considering the literature data on the specific effect of benzene on the
white blood cells, we studied the influence of this mixture of vapors on the
absolute content of leucocytes, the differential count, and the thrombocyte
content. As was shown by the results, the leucocyte content dropped consider-
ably in the first group, but the changes reached a statistically reliable
difference, compared with the control only in the third month of exposure
(Fig. 4).
A
13
f"
a m
8
Exposure.
Q/XII-1965 13/M25/XII20/I7/IH966 ZZ/U'12/ffI26/111if/17
Date of test
Fig. 4. Average content of leucocytes in the blood of
rats exposed to benzene and acetopnenone vapors.
Notation sane as in Fig. 5.
In the differential count, a slight shift to the left (increase in the
number of staff cells) and an appreciable decrease in the absolute number of
lymphocytes were observed at that time.
Toward the end of the second month of exposure, the content of thrombocytes
in the blood of rats of the first group dropped sharply (Fig. 5). No appreci-
able changes were found in the erythrocyte content.
As is evident from the results of the studies, changes in the peripheral
blood occurred much later than in the central nervous system. It may be '
postulated that this system, being the most sensitive to the action of a mix-
ture of two substances with a narcotic effect, played the major role in the
- 62 -
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change of the blood composition.
350
| 300
I
§
5 250
8
«
200
Recovery
Period
25/XIH965 20/1 7/1H96G 22/11 12/fIf 20/111 It/IV
Date of study
Fig._5. Average thrombooyte content in the blood of rats
of different groups. Notation same as in Fig. 3.
At the present time, a considerable amount of data has accumulated in
support of an important role of nucleic acids in various processes connected
with the manifestation of life and primarily with protein synthesis and the
growth and division of cells.
A series of physiological states of the organism are related to the
intensification of the synthesis and metabolism of nucleic acids. Particu-
larly intense is'the nucleic metabolism in processes of blood formation,
enzyme synthesis, and formation of protein secretions (S, S. Debov, 1954).
Many toxic substances acting on the body lead to a quantitative change
in the content of nucleic'acids in certain organs and media (Campbell and
Kosterlitz, 1952; Rambach, Moomav, Aet, and Cooper, 1952, and others).
A major part in the vital activity of the organism is played by the
defensive function of nucleic acid.
A. I. Oparin and T. N. Yevreinova (1947) showed that when ENA is added
to various proteins, their thermostability increases.
A. N. Belozerskiy (1944) held that the defensive function of DNA is
manifested in the fact that it strongly binds alkaline proteins of the type
of histones or protamines. It should be added that by forming such compounds,
a nucleic acid not only neutralizes alkaline proteins, but also strips them
- 63 -
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of their toxic properties.
Ehrich, Drabkin and Format! (1949) showed a relationship between nucleic
acids and the production of antibodies by mature plasma cells of lymphatic
nodes. Goret (1949) pointed out that the nucleoproteins of cells destroyed
in the body are a factor stimulating leucocytosis and phagocytosis.
To determine the total content of nucleic acids in the blood, we used
a method proposed by P. V. Simakov (1960). The recipe was based on the
general principles"of the method of determination of nucleic acids in bio-
logical substances, developed by A. S. Spirin (1958) and applied to blood.
The method is based on the extraction of nucleic acids with hot perchloric
acid, followed by the determination of nucleic phosphorus in the solution by
photometry at wavelengths of 270 and 290 my; the content of nucleic acids is
calculated from the concentration of nucleic phosphorus.
The determination was made in five rats (separately) of each group.
As is evident from Fig. 6, the content of nucleic acids in the blood
of rats of the first group increased substantially relative to the control
as early as the end of the first month of exposure and remained high until
the end of the period of exposure. The amount of these acids in the blood of
rats of the second group differed insignificantly from the data for the control.
On the 17th day of the recovery period, the content of nucleic acids in the
blood of rats of the first group decreased to the level of the control.
The increase in the amount of nucleic acids in the blood of rats of the
first group eras apparently due to an increase in the content of staff cells
in the blood, reflecting an intensification of the defensive function of the
organism in response to the action of the irritant.
It is well known that the penetration of young cells into the blood
causes a severalfold increase in the content of nucleic acids (Rurt, Murray
and Rossiter, 1951).
Thus, changes in the quantitative content of nucleic acids in the blood
were found to be a sensitive indicator of the functional shifts occurring in
the animal body under conditions of chronic exposure to microdoses of benzene
in combination with acetophenone.
Studies have shown (Yu. Ye. Korneyev, 1965j N. Z. Tkach, 1965) that the
determination of 17-ketosteroids in the urine is a sensitive test for evaluatr
ing the nonspecific effect of toxic substances on the functional state of the/
hypophyseal - adrenocortical system under the conditions of a chronic experi-'
ment.
- 64 -
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soo
13/Xff-f965s 25/Xfl 20/15/fH966a Z2/1I16/111 28/111 1tfIV
Date of study
Fig. 6. Average content of nucleic acids in the blood of
rats exposed to benzene and acetophenone vapors. Notation
same as in Fig. 3.
I
The analysis was carried out on the total urine of five rats of each
group.
Our studies showed that the content of 17-ketosteroids in the urine of
animals of the first group was higher than normal during almost the entire
course of exposure, but no significant difference in the results was obtained
relative to the control.
It should be noted that after the completion of exposure, all the
changes detected in the body of the experimental animals returned to normal
in a comparatively short period of time (16-18 days). Hence, the shifts ob-
served were relatively slight and functional in character.
No
a mixture
changes could be observed in animals of the second group,
e of 0.9 - 0.02 mg/m3 benzene and 0.03 - 0.00005 mg/m3 ac
which inhaled
acetophenone.
The internal organs and brain of the rats were subjected to histopatho-
logical analysis. In rats of the first group, a slight focal proliferation
of structures of the epithelium lining the bronchi was observed, apparently
formed in response to irritation by the inhaled vapors of benzene and aceto-
phenone. The parenchyma of the liver had dilated sinuses. The nuclei of
the liver cells retained their structure and only isolated ones were pyfcnotic.
Kupffery cells were markedly hypertrophied, indicating activation of the
reticuloendothelial system of the liver.
- 65 -
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Some metabolic processes of the liver were studied by means of histo-
chemical reactions. A marked decrease in the content of glycogen in the cells
was observed, indicating a certain decline in the functional activity of the
liver. In the determination of the content of RNA in the liver cells, an
intensive synthesis of which indicates a high biological activity of the organ,
a certain decrease of the pyroninophilic granules in the cytoplasm of the
liver cells was noted. The RNA granules were often irregularly distributed,
and the intensity of this reaction showed slight fluctuations in certain
animals.
In the cerebral cortex in animals of the first group, many nerve cells
were in a state of segmental chromatolysis. The chromatolysis observed in
the neurons of the cerebral cortex was reversible; this was demonstrated by
merely discontinuing the action of benzene and acetophenone vapors on the
body, whereupon the chromatolysis effects disappeared.
A study of the reaction for RNA in the nerve cells of the cerebral cortex
showed that the effect of a mixture of benzene (9 mg/m^) and acetophenone
(0.003 mg/nH) on the body leads to a decrease of RNA, compared with the control,
The following conclusion may be drawn from the results of the chronic
experiment. The inhalation of mixture I of substances by the animals caused
marked changes in the functional state of the central nervous system, mani-
fested in a change of the normal ratio of chronaxies of the antagonist
muscles. The changes detected were confirmed by studying the state of the
nerve cells of the cerebral cortex.
A normal ratio of chronaxies of antagonist muscles was observed during
the recovery period. Changes in the nerve cells of the cerebral cortex were
also reversible. Consequently, changes in the central nervous system were
only functional in character.
Much later than in the central nervous system, in the course of the ex-
periment, changes were detected in the peripheral blood (first group of rats),
manifested in leucopenia, absolute lymphopenia, a slight shift of the differen-
tial count to the left, and thrombocytopenia.
Another highly sensitive test was found to be the determination of the
total content of nucleic acid in the blood, whose amount during the exposure
was increased in rats of the first group, compared with the control.
No reliable change in the number of erythrocytes in the blood and 17-
ketosteroids in the urine could be obtained.
I
The changes detected in rats of the first group during the exposure re-i
turned to normal during the recovery period. In rats of the second group, no
reliable changes relative to the control were observed.
- 66 -
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Thus, mixture II of the substances was found to be Inactive toward the
organism of rats during a prolonged, continuous inhalation.
In order to study the pollution of atmospheric air with benzene and
acetophenone vapors, analyses were carried out around a typical industrial
plant producing synthetic phenol and acetone; they showed that at a distance
of 100 m from the source of discharge, the highest single total concentra-
tion of the mixture is 197 times the recommended norm, and at distances of
300 and 500 m, 21.8 and 6.28 times respectively (Table 2). At a distance of
1000 m, benzene and acetophenone were not detected in the atmosphere. Yu. Ye,
Korneyev, U. G. Pogosyan and N. Z. Tkach (1965) also failed to detect any
pollution of air with phenol and acetone at this distance.
Table 2
Pollution of Atmospheric Air with Benzene and Acetophenone (in milli-
grams per m3) Around a Synthetic Phenol and Acetone Plant.
Substance
Benzene
Acetophenone
Total concen-
tration above
proposed norm
(1.5)
Distance
From
Source
of Dis-
charge, m
. 100
300
500
1000
100
300
500
1000
100
300
500
Number of
Samples
16
26
25
25
11
17
26
25
.97 times greatc
6.28 " "
Limits of
fluctuations
2,1—4,73
0,030—1,18
0,010—0,18
Average
Concen-
tration
3.3
0,68
0,091
Negative Results
0,050—0,879
0,032—0.0967
0,0030—0,0286
0.258
0,0575
0,0076
Negative Results
r
It may be assumed that for the plant studied, the width of the sanitary
protective zone with respect to such leading components of atmospheric dis-
charge as benzene, phenol, acetone and acetophenone should be no less than
1 km.
- 67 -
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Conclusions
1. In a study of the reflex effect of a mixture of benzene and aceto-
phenone vapors on the human body, a complete summation of the effects of
their action was found; the total concentration index of the threshold mix-
tures is equal to 1.
2. Chronic round-the-clock exposure of rats for 84 days to a mixture
of benzene and acetophenone vapors in concentrations of 9.0 and 0.030 mg/nH
causes a series of statistically reliable functional shifts in the body of
the animals: in the central nervous system, blood system, and in the total
content of nucleic acids in the blood.
Pathomorphological changes of reversible nature in certain internal
organs were also observed.
3. Chronic exposure under the same conditions to a mixture of benzene
and acetophenone vapors in concentrations at the level of the maximum in-
active ones as determined by the electroencephalographic test failed to
cause any reliable shifts in the animal organism, compared with the control.
4. When benzene and acetophenone vapors are present together in atmos-
pheric air, the total concentration of the mixture in fractions of the maximum
permissible concentration of each of the substances should not exceed 1.5.
5. The sanitary protective zone between the boundary of synthetic phenol
and acetone plants and the boundary of the residential area should be at least
1 km wide.
LITERATURE CITED
Note: References mentioned in this paper are to be found at the end
of the volume in the 1968 bibliography.
- 68 -
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MAXIMUM PERMISSIBLE CONCENTRATIONS OF PHENOL AND ACETOPHENONE
PRESENT TOGETHER IN ATMOSPHERIC AIR
Aspirant (graduate student) Yu. Ye. Korneyev
A. N. Sysin Institute of General and Communal Hygiene,
Academy of Medical Sciences of the USSR
From Akademlya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i gigienicheskoe
znachenie atmosfernykh zagryazneniy". Red.'V. A. Ryazanova. Vypusk 10,
Izdatel'stvo "Meditsina" Moskva, p. 155-169, (1967).
The study of the complex effect of atmospheric pollutants on the organism
constitutes at the present time one of the newest trends in research on the
hygiene of atmospheric air. In this connection, we were assigned the objective
of studying the nature of the combined effects of phenol and acetophenone va-
pors on the organism.
In industry, the combined action of these compounds occurs when phenol and
acetone are produced by the cumene process, which'is associated with the dis-
charge of vapors of phenol, acetophenone, acetone, benzene, and other toxic sub-
stances into atmospheric air, i. e., substances that act simultaneously on the
human organism. No data in the literature are available on the combined action
of phenol and acetophenone. When several poisons, even of similar structure,
are acting, the result obtained may differ considerably from the action of each
of these substances taken individually, and is manifested in each case by a
complete or partial summation, potentiation, antagonism, or mutually independ-
ent action of the component parts of the mixture (N. S. Pravdin, 1929; N. V.
Lazarev, 1938; V. A. Ryazanov, 1954). Thus, only an organized experiment can
in each case provide a complete and exhaustive solution to this problem. Most
frequently, the action of toxic substances involves a simple summation of
their action, whose evaluation is made by using the general formula
X --A- + -L.
x - MA + MB '
where A and B are the concentrations of the investigated substances in atmos-
pheric air in milligrams per cubic meter; MA and MB are the maximum permis-
sible concentrations of these substances in milligrams per cubic meter for
their isolated action.
The highest single and mean daily maximum permissible concentrations in
atmospheric air for phenol were established at a level of 0.01 mg/m3 for phenol
and 0.03 mg/m3 for acetophenone (B. M. Mukhitov, 1962; N. B. Imasheva, 1963).
The toxicological properties of phenol have been described rather extensively
in the literature. The modes of its penetration into the organism are extreme-
ly varied. In studies of workers employed for long periods of time in plants
producing phenol, different authors have noted instances of acute and chronic
poisoning due to inhalation of phenol vapors. Most common are injuries to the
- 69 -
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central nervous system, followed by those to the cardiovascular system. The
activity of the gastrointestinal tract is disturbed, and there are disturb-
ances of vitamin metabolism (V. K. Navrotskiy, 1928; Z. E. Grigor'yev, 1953;
V. I. Petrov, I960).
In a study of workers exposed to phenol vapors, A. S. Stegniy and Ye. K.
D'yakonenko (1961) noted hypotonia, polyneuritic disturbances of sensitivity
and the absence of ventral reflexes, and insufficiency of convergence.
The toxicology of acetophenone has been insufficiently studied, and from
the standpoint of the aspect of interest to us, only the work of N. B. Imasheva
gives the most complete description of the action of low concentrations of
acetophenone on the organism.
Phenol has extensive applications in industry which are clearly not com-
parable to those of acetophenone, but the combined action of these two com-
pounds is indissolubly associated with the production of phenol and acetone
'by the cumene process, in which a steadily evolved by-production is aceto-
phenone, whose discharged amounts vary according to the demand for it, i. e.,
as the industry's demand for acetophenone decreases, the amount of its dis-
charges into the atmosphere increases. Thus, the action of phenol and aceto-
phenone on the organism under ordinary conditions does not occur in an iso-
lated manner, but rather there is a steady combined action whose nature we
studied in our experiments.
In determining phenol in atmospheric air, we used two methods: first,
determination of-phenol in the reaction with diazotized paranitroaniline,
whose reaction with phenol in a carbonic acid medium produces a color rang-
ing from yellowish-green to reddish-brown. The sensitivity of the method
is 0.2 yg in a volume of 5 ml. This was followed by the use of the more
sensitive method of determination of phenol with 4-aminoantipyrine (V. A.
Khrustaleva, 1962).
In the experiments performed, acetophenone was determined spectrophoto-
metrically (M. D. Manita, 1963). The method involves measurement of the op-
tical density of a solution of acetophenone in ethyl alcohol at a wavelength
of 244 my in a cell with a light path of 10 mm. The sensitivity of the method
is 0.25 yg in 1 ml. In studying the actual pollution of atmospheric air
around a plant producing synthetic phenol and acetone, we used a chemical
method of determination of acetophenone vapors (V. A. Khrustaleva, 1961). In
the study of the reflex effect of low concentrations of a phenol-acetophenone
mixture, we determined the threshold of olfactory perception of the mixture,
and studied its influence on the light sensitivity of the eye and the elec-
trical activity of the cerebral cortex. The observations were made on prap-
tically healthy people.
The determination of the odor threshold of the phenol-acetophenone mixture
- 70 -
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was made according to a procedure recommended by the Committee on Sanitary
Protection of Atmospheric Air. First, all the subjects were familiarized with
the odors of phenol and acetophenone, then the thresholds of olfactory percep-
tion of each of these substances were checked. A total of 22 persons partic-
ipated in these experiments. For two subjects, the odor threshold concentra-
tion of phenol was 0.017 mg/m3 (subthreshold concentration, 0.016 mg/m3); 14
subjects identified the minimum phenol concentration at a level of 0.022 mg/m3
(subthreshold concentration 0.017 mg/m3); in the remaining subjects, the odor
threshold was at higher levels. B. Mukhitov established the threshold of ol-
factory perception of phenol for the most sensitive persons at the level of
0.022 mg/m3.
The odor threshold of acetophenone for 10 subjects was found to be 0.010
mg/m3 (subliminal concentration 0.008 mg/m3). For 11 persons, the threshold
concentration was at a level of 0.0285 mg/m3 (subliminal concentration of
0.01 mg/m3). The odor threshold for one subject was above 0.03 mg/m3. The
threshold of olfactory perception of acetophenone for the most sensitive per-
sons was established by N. B. Imasheva at a level of 0.01 mg/m3.
The odor threshold of a phenol-acetophenone mixture was determined on 18
subjects. Of these, 6 persons who were the most sensitive in the tests per-
formed participated in experiments involving both isolated action of the com-
pounds and the mixture of phenol and acetophenone. A total of 752 determina-
tions were made. In studying the action of the mixture, its total concentra-
tion was expressed in fractions of the threshold concentrations according to
the given test. Studies involving the determination of the odor threshold of
the phenol-acetophenone mixture for the most sensitive persons gave concurrent
results.
The odor threshold for these persons was defined as 0.022 mg/m3 for phenol
and 0.01 mg/m3 for acetophenone.
The odor threshold mixture had a total concentration index of 0.99
,0.013 mg/rn^ 0.004 mg/m3N i. e., when low concentrations of phenol and
0.022 mg/mj + 0.01 mg/m3 '
acetophenone vapors are present together in atmospheric air, a complete sum-
mation of their action takes place.
Expressed in fractions of the thresholds for isolated action, the imper-
ceptible mixture had a total concentration index of 0.72.
The study of .the influence of low concentrations of the phenol-acetophenone
mixture on the light sensitivity of the eye was made on an ADM adaptometer
using a procedure recommended by the Committee on Sanitary Protection of
Atmospheric Air. We introduced some modifications of the design of the cylinder
and supplied air at a rate of 30-35 1/min. The increased velocity of air move-
ment was not perceived by the subjects. It was thus possible to make the volume
- 71 -
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of supplied air (500 ml/sec) equal to the volume of air inhaled by the per-
son at rest.
Adaptometric studies were conducted on three subjects. Two mixtures
of different concentrations of phenol and acetophenone were tested. A
total of 93 determinations were made. Each test lasted 45 minutes, and the
light sensitivity of the eyes was measured successively every 5 minutes.
Four tests were carried out with each gaseous mixture. The results were sub-
jected to statistical treatment.
In the adaptometric studies, our calculations were made with threshold
values obtained by B. M. Mukhitov and N, B. Imasheva for the isolated action
of phenol and acetophenone. The total concentration index of the first mix-
, ^ -i n [0.00747 mg/m3 , . ,. 0.00517 mg/m3 , . ..
ture was equal to 1.0 p/\ m cc 7~T~ Cphenol; + A m Ta4»— (acetophenone) ] ,
[u.UJ.->-> mg/nH u.ui mg/m
j .M, «. * 4.1. A • 4- 4- n -7-7 (0.0059 mg/m3 0.0039 mg/m3)
and that of the second mixture, to 0.77 ^, -mvv— i n + —R-TTT—' •> •*( .
' (0.0155 mg/m3 0.01 ing/m-*)
In thousands
ZtO
200
ISO
120
80
10
Minutes of study
Fig. 1. Curve of dark adaptation of the eyes in
subject T. H. during inhalation of gaseous mixture
of phenol and acetone.
1 - pure air; 2 - mixture with total concentration
index of 1.0: 3 - mixture with total concentration
index of 0.77.
In the studies performed, the first mixture caused statistically signifi-
cant changes in all three subjects. The second mixture caused no changes in
the course of the curve of dark adaptation of the eyes (Fig. 1).
- 72 -
-------
Thus, in this experiment as well as in the experiments involving the deter-
mination of the threshold of olfactory perception of the phenol-acetophenone
mixture, a complete summation of the action of the compounds studied was ob-
served again: the total concentration index of the minimum active mixture
was equal to 1.
We studied the influence of low concentrations of the phenol-acetophenone
mixture on the electrical activity of the cerebral cortex by using a procedure
developed for sanitary investigations by A. D. Semenenko. We used the method
of reinforcement of man's intrinsic alpha rhythm during successive stimulation
of the subject with intermittent light corresponding to the frequency of his
rhythm and to the procedure similarly described in the paper of U. G. Pogosyan.
The studies were conducted on three subjects (112 tests).
The leads were taken from each hemisphere from the occiput, temple, and
forehead. Two mixtures of gaseous concentrations of phenol and acetophenone
were tested. First mixture: phenol - nmcg 1^ = °-49
-------
100
80
Minutes of study
Fig. 2. Change in the electrical activity of the brain
in subject T. K. during inhalation of a gaseous mixture
of phenol and acetophenone.
Notation same as in Fig. 1; AB - period of inhalation of
gaseous mixture.
Group I - acetophenone (0.00171 mg/m^) + phenol (0.0062
The total concentration index of this mixture, expressed in fractions of
the existing mean daily maximum permissible concentrations, was 1.19.
Group II - acetophenone (0.01732 mg/m3) + phenol (0.0637 mg/m^).
The total concentration index of the mixture was 12.14.
Group III - control.
In addition, in groups IV and V, in collaboration with Graduate Student
N. Z. Tkach, we studied the effect of three compounds: acetophenone, phenol,
and acetone.
Group IV - acetophenone (0.00147 mg/m3) + phenol (0.0048 mg/m3) + acetone
(0.136 mg/m3).
The total concentration index of the mixture was 1.36.
Group V - acetophenone (0.01141 mg/m3) + phenol (0.04162 mg/m3) + acetone
(1.334 mg/m3).
The total concentration index of the mixture was 11.76.
- 74 -
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The studies were conducted on white male rats with an initial weight of
70-100 g. Fifteen animals were placed in each exposure chamber. The concen-
trations of the substances studied were checked daily during the 84 days of
exposure. During that time, observations were made on the general condition
of the rats, their weight, the cholinesterase activity, motor chronaxy of
antagonist muscles, porphyrin metabolism, number of eosinophils in peripheral
blood, and excretion of 17-ketosteroids and vitamin C with the urine.
The general condition of the animals and their weight in all the groups
did not differ in any way from those of animals of the control group.
We determined the cholinesterase activity by using a slightly modified
procedure of Fleisher, Pope and Spear (1954). A statistically significant de-
crease in cholinesterase activity was detected on the 4th day of exposure in
animals of groups II and V, exposed to high concentrations of the compounds
studied. The changes remained distinct until the end of the experiment, and
the cholinesterase activity returned to normal only during the recovery period.
In the animals of the remaining groups, no such disturbances arose (Fig. 3).
IS/I
Date of study
Fig. 3. Changes in cholinesterase activity in experimental animals
during inhalational exposure to mixtures of phenol, acetophenone,
and acetone.
1 - group I (mixture of phenol and acetophenone with total concen-
tration index of 1.19); 2 - group II (mixture of phenol and
acetophenone with total concentration index of 12.14); 3 - group
III (control); 4 - group IV (mixture of phenol, acetophenone, and
acetone with total concentration index of 1.36); 5 - group V
(mixture of phenol, acetophenone, and acetone with total concen
tration index of 11.76); AB - period of exposure.
- 75 -
-------
We were able to evaluate the functional state of the nervous system from
data on the motor chronaxy of antagonist muscles, which was studied in the
course of the chronic experiment. As we know, shifts in motor chronaxy, which
is a fairly mobile indicator of the excitability and changes under the in-
fluence of the slightest factors of the surrounding medium acting on the orga-
nism, accurately characterize the functional state of the organism. Exposure
of the animals to high concentrations of the substances studied (groups II and
V) had a considerable influence on the functional state of the nervous system
(Fig. 4). A change in motor chronaxy appeared in these groups toward the end
of the first month of exposure. There was no change in the remaining groups
of animals. At the end of the recovery period, the ratio of chronaxies re-
turned to normal.
Various intoxications and illnesses affecting the nervous system cause
disturbances in the porphyrin metabolism. The appearance of coproporphyrin
in the urine indicates incipient manifestations of a disturbance of nervous
regulation in the synthesis of hemoglobin (0. M. Chernyy and S. E. Krasovitskaya,
1951). In our studies, a significant increase in the excretion of copropor-
phyrin with the urine was observed in animals of groups II and V during three
months of exposure. During the recovery period, the excretion of copropor-
phyrin became normal (Fig. 5).
In the course of the chronic experiment, we also observed the excretion
of 17-ketosteroids with the urine. A leading role in the regulation of excre-
tion of steroid hormones of the adrenal cortex, which possess a marked reac-
tivity, is played by the nerous system, and the function of the cortical part
of the adrenals depends on the corticotrophic hormone of the anterior hypophy-
seal lobe, which in turn is controlled by the central nervous system (S. G.
Genes, 1955). The corticosteroid function is a response to a stimulus acting
on the organism. The study of 17-ketosteroids gives an idea of the disturbance
of the mechanisms controlling the function of the adrenal cortex. The adrenal
hormones influence the morphological composition of perpiheral blood. This
is particularly apparent in the eosinophils. Eosinopenia serves as an indi-
cator of the reinforcement of the function of the adrenal cortex (D. Ya.
Shchurygin, S. F. Murchakova, and N. A. Belov, 1957).
We therefore investigated the excretion of 17-ketosteroids and the number
of eosinophils in the blood during exposure of the rats. Changes in the excre-
tion of 17-ketosteroids with the urine were observed in animals of groups II
and V. It can be seen from Fig. 6 that an increased excretion of the hormones
in these groups corresponds to the entire period of the chronic experiment,
but statistically significant changes were obtained only in the third month of
exposure, which may be accounted for to some extent by the irregularity of the
excretion of 17-ketosteroids by the control group of the animals. The content
of 17-ketosteroids in the urine of animals of groups I and IV differed only
slightly from that of animals of the control group. The analyses were made
every 15 days at a fixed time.
- 76 -
-------
g 2
3
2
•8
ti '
CD
• H
O
•H
3s^
*^».
VJ
5
i _i
5/IX 2e/tt 12/X 22/X
mental animals during u
a/*t*4- /
* «
^•"TZXsssis**-^
•^
N-Jis— ~
/A/ ///// 21/11 I/XII
Dates of study
^*d
B
=-5?
/
/
• t
tf/Jtll it/Ml till
xies of extensors and flexors in experi-
ihalational exposure to mixtures of phenol,
,
Notation same as in Fig.
yg/100 g of weight
/I
0.5 -
I I j II p • | 1
gt/X 3/X 13/X 28/X 12/XI 27/XI /2/Xff 27/Xll H/l ft//
Dates of study
Fig. 5.. Excretion of coproporphyrin with the urine during
inhalational exposure of experimental animals to mixtures
of phenol, acetophenone, and acetone.
Notation same as in Fig. 3.
|ig/100 g of weight
15/X 30/X M/XI 29/XI ff/XII 27/Xll tS/l tg/l
Dates of study
Fig. 6. Excretion of 17-ketosteroids with urine during exposure of
experimental animals to mixture of phenol, acetophenone, and acetone.
Notation same as in Fig. 3.
- 77 -
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The dynamic study of the content of eosinophils in peripheral blood may
serve as one of the indicators of the reactivity of the organism. The most
diverse factors leading to the development of inhibition in the central nerv-
ous system cause a decrease of the absolute number of eosinophils in peripheral
blood (K. Kh. Kyrge, 1956). The eosinophils were counted in a Fuchs-Rosenthal
counting chamber (S. M. Bakman, 1958). A decrease in the number of eosinophils
in the peripheral blood of animals of groups II and V was observed. Eosinopenia,
confirmed statistically, was detected twice in the second and third months of
exposure (Fig. 7). No significant changes in the fluctuation of eosinophils
were observed in animals of the remaining groups.
The determination of the content of ascorbic acid in the urine of animals
of all the groups failed to show any changes as compared with the control.
Thus, inhalational exposure of experimental animals to a mixture of high
concentrations of phenol and acetophenone (group II) and their combination with
acetone (group V) clearly showed changes in cholinesterase activity, motor
chronaxy of antagonist muscles, porphyrin metabolism, excretion of 17-keto-
steroids with urine, and a drop in the eosinophil count in the peripheral blood
of the animals.
250
zoo
ISO
too
SO
«/U IS/X 31/X 15/XI JO/JC/ tS/XIt 18/1
Dates of study
Fig. 7. Fluctuations of eosinophils in the peripheral
blood of the animals during inhalational exposure to
mixtures of phenol, acetophenone, and acetone.
Notation sane as in Fig. 3.
- 78 -
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No changes in the organism of the animals during prolonged exposure were
caused by total concentrations of phenol and acetophenone at a level of 1.19
in fractions of the existing maximum permissible concentrations and of 1.36 in
the combination of these compounds with acetone.
Considering that the gap between our proposed highest single concentra-
tion of the phenol-acetophenone mixture'(1.5) and the investigated mean daily
concentrations (1.19 and 1.36) is small, and that the minimum resorptively
acting mixture lies at a level of 1.96, in accordance with the recommendation
of the Section on Sanitary Protection of Atmospheric Air of the Ail-Union
Problem Commission, we propose a mean daily concentration of the investigated
compounds at the same level as the highest single concentration, i. e., 1.5
in fractions of the maximum permissible concentrations for isolated action.
One of the sections of our work consisted in a study of the actual pollu-
tion of atmospheric air around a plant producing synthetic phenol and acetone,
and in a. sanitary evaluation of the data obtained. The samples were collected
in May 1964 on the leeward side of the plant at distances from 100 to 1000 m.
According to the data of the municipal sanitary-epidemiological station, the
plant discharges into the atmosphere 2.2 tons of pure phenol per year. The
discharges of acetophenone are not taken into account. At a distance of 500
m, the highest single concentrations of'phenol surpassed the maximum permis-
sible concentrations by a factor of 8.7, and those of acetophenone, by a
factor of 9.3 (Table 3). The total concentration of the investigated compounds
surpasses our recommended highest single concentration by a factor of 12. At
a distance of 1000m, neither of these compounds was observed in atmospheric
air. The width of the sanitary protective zone for this type of production
can be established only after analyzing the atmospheric air for the entire
assortment of the toxic substances discharged by the plant.
Table 3
Pollutioi) of Atmospheric Air Around a Plant
Producing Synthetic Phenol and Acetone •
Distance
From
Source, m
Number
-of
Samples
Of These,
Above .The
Sensitivity
of»the Method
Concentrations, mg/m'
Maximum
Average
•Phenol
100
300
500
1000
17
13
23
25
17
13
20
—
0,29
0.219
0,087
—
0,1642
0,1019
0.03417
—
Acetophenone
100
300
500
1000
11
17
26
25
/
11
. 17
12
0,879
0.0967
0.0286
—
0,258
0,0571
0,00764
—
- 79 -
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Conclusions
1. The odor threshold mixture of phenol and acetophenone for the most
sensitive persons is one of 0.013 mg/m3 phenol and 0.004 mg/nH acetophenone
with an index of total concentration in fractions of threshold concentrations
for isolated action equal to 1. A mixture of 0.01 mg/m3 phenol and 0.0026
mg/m3 acetophenone with a total concentration index of 0.72 is imperceptible.
2. The threshold mixture from the standpoint of the light sensitivity
of the eyes is one consisting of 0.00747 mg/m3 phenol and 0.00517 mg/m3 ace-
tophenone with a total concentration index of 1. The total concentration
index of the inactive mixture is 0.77.
3. A mixture of 0.00759 mg/m3 phenol and 0.00357 mg/m3 acetophenone
with a total concentration index of 1 is active with respect to the elec-
trical activity of the brain.
A mixture with a total concentration index of 0.77 is inactive.
4. The studies performed show that when phenol and acetophenone are
jointly present in atmospheric air, a complete summation of their action takes
place.
5. Since the maximum permissible'concentrations of phenol and acetophenone
were established with a certain margin, their highest single total concentra-
tion expressed in fractions of the corresponding maximum permissible concen-
trations should not exceed 1.5.
6. Exposure of experimental animals to phenol concentrations of 0.0637
mg/m3 and acetophenone concentrations of 0.01732 mg/m^ (total concentration
index 12.14) and to a mixture of phenol, acetophenone, and acetone with a
total concentration index of 11.765 revealed substantial changes in cholin-
esterase activity, motor chronaxy of antagonist muscles, porphyrin metab-
olism, and content of 17-ketosteroids in the urine of the animals, and
caused a marked eosinopenia.
7. A mixture of phenol and acetophenone and also their combination with
acetone does not cause any changes in the animal organism during a prolonged
exposure if their total concentration in fractions of the maximum permissible
values does not exceed 1.19 and 1.36 respectively.
8. The mean daily maximum permissible concentration of phenol and ace-
tophenone present simultaneously in atmospheric air may be recommended at the
same level as the highest single concentration.
This conclusion also applies to the combined action of the three compounds.
I /
9. The width of the sanitary protective zone for a plant producing syn-
thetic phenol and acetone can be established after a complete analysis of atmos-
pheric air for all of the toxic substances discharged by the plant.
- 80 -
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LITERATURE CITED
Note: References mentioned in this paper are to be found at the
end of the volume in the 1967 bibliography.
- 81 -
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SANITARY EVALUATION OF THE COMBINED ACTION OF
ACETONE AND PHENOL IN ATMOSPHERIC AIR
U. G. Pogosyan
A. N. Sysin Institue of General and Communal Hygiene,
Academy of Medical Sciences of the USSR
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i gigienicheskoe
znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova. Vypusk 10,
Izdatel'stvo "Meditsina" Moskva, p. 135-154, (1967).
Experimental studies of the biological effect of atmospheric pollutants
have been greatly expanded in the Soviet Union and have formed the basis for
the standardization of the maximum permissible content of noxious substances
in atmospheric air. A list of such norms currently specifies the maximum
permissible concentrations of 67 ingredients for isolated action.
However, the discharges of a single enterprise frequently contain several
noxious substances, and the surrounding atmosphere is polluted by several of
them simultaneously. The substances used in industry are very complex mix-
tures of various compounds; the different ingredients may react with each other
and form new, sometimes more toxic compounds. For example, a single populated
area frequently contains several different kinds of industrial plants discharg-
ing different substances, etc. For this reason, in addition to developing
further the studies of various ingredients on the organism, it is necessary to
investigate the action of their mixtures.
From the standpoint of their combined action, atmospheric pollutants have
been studied comparatively little. Research along these lines has shown that
when certain substances are simultaneously present in air, a complete summation
of their action is observed: such are sulfur dioxide and sulfuric acid aerosol
(K. A. Bushtuyeva, 1961); hydrogen sulfide, carbon disulfide and Dowtherm (Kh.
Kh. Mannanova, 1964); ethylene, propylene, butylene and amylene (M. L.
Krasovitskaya, 1964); isopropylbenzene and its hydroperoxide (G. I. Solomin,
1964); strong mineral acids (sulfuric, hydrochloric, and nitric) in concen-
trations of H ions (V. P. Melekhina, 1964). The results of these studies are
embodied in a table of maximum permissible concentrations of noxious substances
in the atmospheric air of populated areas, ratified at the end of 1965.
One of the most promising technological processes that have emerged in the
world industry of organic synthesis is the method of combined production of
acetone and phenol via isopropylbenzene hydroperoxide (cumene). It was first
developed and applied on an industrial scale in the Soviet Union in 1949. Be-
cause of the steadily increasing consumption of acetone and phenol in various
branches of the national economy, the cumene method of production of these
- 82 -
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compounds, which is the most economical method, is being widely adopted. Enter-
prises engaged in the combined production of acetone and phenol by the cumene
process are among those which can cause pollution of the atmosphere with nox-
ious substances and their combinations because of imperfections from the stand-
point of sanitary protection of the outside environment, imperfections in the
technological processes, and an inadequate development of methods of purifi-
cation of the discharges.
Studies have shown (V. P. Melekhina and M. A. Pinigin, 1961) that the
atmospheric air around a plant of combined acetone and phenol production is
polluted with acetone, phenol, and many other substances at a considerable
distance from the plant. The study of the combined action of acetone and
phenol in low concentrations is of definite practical and theoretical interest
because of the necessity of standardizing the content of these compounds in
atmospheric air when they are jointly present. Our study correlates data
obtained in an experimental study of this problem.
Acetone (dimethyl ketone) is an irritating and narcotic compound which has
the property of accumulating in the organism. Phenol (carbolic acid) is a
nerve poison which has a local irritating and cauterizing effect (N. V. Lazarev,
1951). In determining acetone and phenol in atmospheric air, we used a nephe-
lometric method for acetone (M. V. Alekseyeva, B. Ye. Andronov, S. S. Gurvits,
and A. S. Zhitkov, 1954) and a colorimetric method for phenol (V. A. Khrustaleva,
1962).
Acetone reacting with iodine in an alkaline medium forms a white suspen-
sion of iodoform, whose intensity is compared with a standard scale. The
sensitivity of the method is 0.001 mg in a volume of 4.5 ml. The method is
specific, and phenol does not interfere with the determination.
When phenol reacts with 4-aminoantipyrine in the presence of potassium
ferricyanide at pH 9.3, a pink color appears. The sensitivity of the method
is 0.0002 mg in a volume of 2 ml. Acetone does not interfere with the deter-
mination.
We began our study of the effect of low concentrations of acetone and
phenol on the human organism by determining the threshold of their olfactory
perception in a mixture, having first verified the already-established odor
thresholds of the separate substances in 18 persons.
The determination of the threshold of olfactory perception was performed
by using a standard procedure'(V. A. Ryazanov, K. A. Bushtuyeva, and Yu. V.
Novikov, 1957). Of 18 people, 5 were selected for whom the odor thresholds
of both acetone and phenol were the lowest and amounted to 1.1 mg/m3 for
the former compound and 0.022 mg/m3 for the latter; this confirms some
studies made earlier (Yu. G. Fel'dman, 1962; B. A. Mukhitov, 1963). The con-
centration of acetone and phenol in the determination of the threshold of
- 83 -
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olfactory perception of their mixture is given in Table 1.
Table 1
Concentration of Acetone and Phenol Corresponding to the Thres-
hold of Olfactory Perception of Their Mixture.
Concentration of Acetone and Phenol in Their Mixtur
ng/Di3
Acetone
i;i*
t.i
0,72
1,1
0,55
~P~
0,55
1.1
0,36
1.1
Phenol
0,022
0,022
0,011
0.022
0,011
0.022
0.008
0.022
0.011
0.022
tn Fractions of Threshold
for Isolated Action
Acetone
1,0
0,65
0,5
. 0,5
0,33
Phenol
1,0
0,5
0,5
0.36
0,5
i Total Concen-
tration in Fractions
of the Threshold
Concentration of each
Component Part
2,0
1,15
1,0
0.86
0,83
* Numerator - concentration studied; denominator - odor threshold
concentration for isolated action.
We shall illustrate the nature of the combined action of the substances
studied by using as an example the data obtained on the five most sensitive
persons (Table 2).
As can be seen from Table 2, the odor threshold of the acetone-phenol
mixture for the five most sensitive persons was established for an index of
1.0 of the total concentration in fractions of the threshold concentration for
each component part. The remaining 13 indicated concentrations were not per-
ceived by the subjects, which is quite natural, since they had a higher odor
threshold for both compounds taken both separately and in a mixture.
The perception threshold is not the limit of the physiological activity
of acting stimuli. Very frequently, without being accompanied by sensations,
stimulations cause definite physiological responses of so-called subliminal
character (G. V. Gershuni, 1949). This phenomenon was also confirmed in
studies with low concentrations of noxious substances in the inhaled air.
Studies made along these lines showed that subliminal concentrations of chem-
ical stimuli whose odor is imperceptible cause changes in the functional state
of different organs and systems, particularly the visual system, and the thres-
hold of these changes is slightly below the threshold established by the inter-
rogation method.
- 84 -
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Table 2
Thresholds of Olfactory Perception of an Acetone-
Phenol Mixture for the Most Sensitive Persons.
Total Concentration
in Fractions of
Threshold Concentra-
tion of Each
Comoonent Part
2,0
1.15.
1.0
0,86
0,83
Results of Tests
Perceive
5 Persons
5 »
5 »
Do Not
Perceive
5 Persons
5 »
The light sensitivity is an extremely delicate and labile function of the
visual system, whose level is affected by various factors in the surroundings.
There is special merit to the hypothesis, based on a series of studies (L. A;
Orbeli, 1934; A. V.'Lebedinskiy, 1935; K. Kh. Kekcheyev, 1946; P. P. Lazarev,
1947; S. V. Kravkov, 1950), that the light sensitivity of the eyes at the level
of total dark'adaptation reflects not only the processes taking place in the re-
ceptor itself, but is intimately tied up with the state of the visual centers
of the cerebral cortex and its stem part. Hence, a change in the level of the
light sensitivity of the eye under the influence of secondary stimuli should
be regarded as being a reflection of physiological shifts of not only periph-
eral but also central origin.
A study of the reflex effect of an acetone-phenol mixture on the light
sensitivity of the eyes under dark adaptation conditions was made by using an
ADM adaptometer according to a standard procedure on 3 out of 5 persons with
the most sensitive odor threshold. All three had a normal vision and a com-
paratively stable dark adaptation curve. In view of the fact that for the
most sensitive persons the odor threshold concentrations in our studies co-
incided with the concentrations established earlier (Yu. G. Fel'dman, 1962;
B. M. Mukhitov, 1963), in carrying out the adaptometric tests we used the
thresholds of the reflex effect of acetone and phenol on the light sensi-
tivity of the eyes already established by these authors.
The first combination with a total concentration index of 1.6 causes
statistically significant changes of the light sensitivity of the eyes in
all three subjects. The second combination with a total concentration index
of 1 caused statistically significant changes of the light sensitivity of
the eyes in only two persons. It proved inactive for the third subject.
An acetone-phenol mixture with a total concentration index of 0.85 was
found to be inactive.
- 85 -
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Table 3
Ratio of Acetone and Phenol Concentrations in the Determination
of the Threshold of Their Reflex Effect on the Lirht Sensitivity
of the Eye
Concentration of Acetone and Phenol in the Hixtupi*
In mg/ni'
Acetone
0.44*
0,55
0,27
0.55
0,22
0,55
Phenol
0.0125
0,0156
0,0078
0.0156
0.0070
0,0156
In Fractions of Threshold
for Isolated Action
Acetone
0.8
0.5
0.4
Phenol
0.8
0,5
0,45
Total_Concentra-
tion in
Fractions of
Threshold Concen
t rat ion of each
CoBpflHSnT part
1,6
1.0
0.85
* Numerator concentrations studied; denominator - threshold con-
centration for the given test for isolated action.
Thus, the threshold of the reflex effect of acetone and phenol on the
light sensitivity of the eye is at the level of a total concentration index
equal to 1. A mixture containing acetone and phenol in concentrations amount-
ing to less than 1 does not act on the light sensitivity of the eye.
The response of the light sensitivity of the eyes in our subjects to a
brief inhalation of acetone and phenol in a mixture was marked by individual
characteristics. Thus, in subjects R. L. and Ye. S., the'changes were mani-
fested in a decrease of the light sensitivity of the eyes, and in subject
G. L. the light sensitivity of the eyes increased.
In"studies involving the experimental standardization of atmospheric pol-
lutants, the method of electroencephalography has come into wide and justified
use. Being a highly sensitive indicator of the functional state of the nerv-
ous system, the electroencephalographic method best satisfies the requirements
arising in the solution of theoretical problems and in practical hygienic
standardization of noxious substances in atmospheric air.
We conducted a study of the reflex change of the electrical activity of
the brain during inhalation of a mixture of acetone and phenol on three sub-
jects by using a quantitative analysis of the reflex response of reinforce-
ment of the intrinsic alpha rhythm (A. D. Semenenko, B. N. Balashov, and Ye.
V. Arzamastsev, 1963).
The thresholds of the reflex effect of acetone and phenol on the energy
of the brain potentials were established at the level of 0.44 mg/m.3 (Yu. G.
- 86 -
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Fel'dman, 1962) and 0.0155 mg/m3 (B. M. Mukhitov, 1963) respectively. Our
tests utilized these threshold values, established on the basis of desyn-
chronization of the alpha rhythm during the development of the electrocortical
conditioned reflex. The tests were based on the use of the dependence of the
nature of the electroencephalogram on the functional state of the brain.
Principal attention was focused on the response of a burst of the alpha rhythm
and on the changes arising against the background of this burst as a result
of inhalation of an aceton-phenol mixture.
The stimulus used was light flickering rhythmically at a frequency equal
to the optimum frequency of the subject's alpha rhythm.
All the subjects underwent training experiments for 4 weeks in order to
make them develop a well-defined alpha rhythm and a definite dynamic stereo-
type. The main observations were begun after obtaining comparatively stable'
data during inhalation of pure air. This was accomplished by using standard,
stable conditions of observations. During the tests, the subject had elec-
trodes attached to the surface of his head in a faintly illuminated, sound-
proof chamber, rested in a comfortable semireclining position, and breathed
pure air supplied at a rate of 30 1/min from a cylinder. Two pickup units
recording the motor response to the stimuli were attached to the wrists.
All the subjects had been given preliminary instructions. 'The electroen-
cephalogram was recorded in the following sequence: first, the initial back-
ground 'was recorded while pure air was being supplied. Starting with the 4th
minute, the subject breathed the mixture studied for 6 minutes, then pure air
was again supplied for the next 6 minutes (recovery period). The entire ob-
servation lasted 18 minutes, including 3 minutes of'training. During each
minute, the rhythmic light was given for 18 seconds, and its intensity was
changed every'5 seconds. In the intervals separating the times when the light
was turned on, the subject made free movements in his chair (limbered up). A
sound whose intensity was also varied was then turned on for 10 seconds. After
the sound was discontinued, the subject waited for the light for 7 seconds.
During the entire study, the subject was in a state of active mental and
motor activity. This removed the possibility of the onset of phase states.
It is known that functional loads make it possible to identify very delicate
shifts arising in the tissues and Organs. Indeed, the functional'load we
used, the photic and sonic stimuli, their differentiated analysis, and the re-
sponse to these stimuli formed the background for the identification of the
shifts caused by the reflex effect of low concentrations of acetone and phenol
whose odor was imperceptible and which did not act on the light sensitivity
of the eye. These changes occurred against a background of the reinforced
intrinsic alpha rhythm of the subjects.
In our studies, the electric potentials were recorded bipolarly on a 16-
channel Galileo electroencephalograph from the frontal, temporal, and parietal
- 87 -
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regions and their combinations from both cerebral hemispheres. The subject's
response to changes in the intensity of the photic and sonic stimuli (pressing
on the piezoelectric pickup) was also recorded. The electroencephalogram of
each observation was analyzed on the basis of the amplitude of the reinforced
intrinsic alpha rhythm expressed in microvolts, i. e., from the integrated
energy of the brain potentials. To perform a comparative analysis of the re-
sults obtained, the total amplitude of the reinforced intrinsic alpha rhythm
was expressed in percent. The average total amplitude for the first 3 minutes
taken as the background was taken as 100%, and the total amplitudes in the re-
maining minutes were compared with it (Ai D. Semenenko, 1963). The results of
these studies were treated statistically, i. e., the degree of significance
of the shifts obtained were found. The ratio of the investigated and thres-
hold concentrations is given in Table 4.
Table 4
Ratio of Concentrations of Acetone and Phenol in the Determination
of the Threshold of Their Reflex Effect on the Electrical Activity
of the Brain
Concentration of Acetone and Phenol in the Mixture
In rag/m'
Acetone
0,22*
.0.44
0,17
0,44
Phenol
0,0078
0,0156
0,0068
0,0156
In Fractions of the
Threshold for Isolated
A rehi on
Acetone
0,5
0,39
• Phenol
0,5
0,44
Total Concentra-
tion in Fractions
of the Threshold
Concentration of
Each Component Part
1.0
•0,83
* Numerator-Concentration studied; denominator - threshold concen-
tration for the given test for isolated action.
A mixture with a total concentration index equal to 1 acts on the energy
of the brain potentials, causing statistically significant changes of the rein-
forced intrinsic alpha rhythm in all the subjects (Fig. 1).
A concentration of acetone and phenol with a total index of less than 1
has no effect on the electrical activity of the brain.
The data obtained from all three tests permit one to draw the conclusion
that low concentrations of acetone and phenol present together in the inhaled
air produce a complete summation of their action.
When the threshold concentrations obtained by the electroencephalographic
method are expressed in fractions of'the existing maximum permissible concern- j
trations of the separate ingredients, the minimum effective total concentration
is 1.41, and the maximum ineffective concentration, 1.16. Hence, in the joint
presence of acetone and phenol in atmospheric air, the sum of their concentrations
expressed in fractions of the maximum permissible ones should not exceed 1. /
- 88 -
-------
5 S 7 8 9 10 it 12 13 « f5
Minutes of study
Fig. 1. Change in the energy_of brain potentials in subject
R. L. under the influence of inhalation of an acetone-phenol
mixture.
1 - pure air; 2 - 0.22 ng/m' acetone and 0.0078 att/a? phenol;
3 - 0.17 n«/in3 acetone and 0.0068 ng/m? phenol.
To detect the resorptive effect of low concentrations of acetone and
phenol in a mixture for the purpose of validating their mean daily maximum
permissible concentration in atmospheric air, we carried out a chronic three-
month exposure of white rats. The 48 males selected for the experiment, with
an initial weight ranging from 90 to 110 g, were divided into three groups of
16 animals each.
Group I of the animals was exposed to a mixture of acetone and phenol in
concentrations 10 times greater than the maximum inactive mixture for brief
inhalation. On the average, it amounted to 1.657 - 0.0215 mg/m3 - acetone and
0.0545 - 0.0013 mg/m3 - phenol; the total concentration index expressed in
fractions of the maximum permissible concentrations of each ingredient for iso-
lated action was equal to 10.19.
Group II of the animals was exposed to a mixture in concentrations at the
level of the maximum inactive mixture for brief inhalation. On the average, it
amounted to 0.1735 i 0.00305 mg/m3 - acetone and 0.00516 i 0.0000769 mg/nH -
phenol; the total concentration index in the same fractions was equal to 1.012.
Group III of the animals served as the control.
During the entire course of exposure and of the recovery period, the ani-
mals were subjected to the same conditions. Pure air and air containing ace-
tone and phenol were supplied to the exposure chambers at a rate of 35 1/min.
The air from the chambers was checked daily for its content of acetone and
- 89 -
-------
phenol. To estimate the toxic effect, the general state and behavior of the
animals was followed over the course of the entire experiment. During the
entire period of exposure, this observation failed to detect any shifts in
these indices in animals of the experimental groups as compared with the con-
trol. No symptoms indicating a specific effect of acetone and phenol were
observed.
The animals were weighed every 20 days. Observation of the weight dy-
namics showed that animals of all the groups gained weight in approximately
the same manner, but toward the end of the exposure and of the recovery pe-
riod a slight weight lag in animals of the control group was noted.
During the chronic experiment, we studied the motor chronaxy of antag-
onist muscles and the activity of blood cholinesterase, and determined the
content of coproporphyrin and certain vitamins - C, Bl, 62 - and N-methyl-
nicotinamide in the daily urine.
In selecting the indicated tests for the study of the character of the
resorptive effect of low acetone and phenol concentrations on the organism
of the animals, we were guided by the assumption that the resorptive effect
of low concentrations of noxious substances consists of general, nonspecific
shifts that are sometimes of the same type and insignificant. As a rule,
these shifts were functional in character and should be regarded as defensive-
adaptive responses (V. A. Ryazanov, 1964). The participation of central nerv-
ous mechanisms in the formation of these shifts should be considered general
and obligatory.
One of the manifestations of the relationship between the nerve centers
and peripheral formations is subordination. It consists in the regulation of
the functional state of the periphery by higher centers. The removal of the
subordinating influence of the central nervous system immediately affects the
level of many functional characteristics of the neuromuscular apparatus, par-
ticularly its excitability, one of the indices of which is the chronaxy, i. e.,
the speed with which a physiological response to a stimulus takes place. In
this case, a substantial difference is found in antagonist muscles. It is known
that the centers of flexors have a higher excitability than those of extensors.
In chronaximetry, this is manifested in the fact that the former have a lower
chronaxy than the latter. In a normal relationship of the processes of stimula-
tion and inhibition in the cortex of the cerebral hemispheres, a normal ratio
of chronaxy of antagonist muscles is observed. It is usually larger than 1.
Under the influence of various environmental factors, this ratio changes so that
the chronaxies become similar or the ratio is inverted, and its coefficient be-
comes smaller than 1. This is a manifestation of a weakening of the subordi-
nating influence of the central nervous system, caused by the development of a
process of inhibition in the cerebral cortex. /
We studied the motor chronaxy of antagonist muscles in 5 rats of each
group once every 10 days. A graphic illustration .of the results obtained is
- 90 -
-------
given in Fig. 2, which shows that a normal ratio of the chronaxies of exten-
sors and flexors was observed in rats of group II and III. In contrast, in
animals of group I on the 45th day of exposure, there was a disturbance in
the normal ratio of chronaxies, i. e., the chronaxy of the flexors became
greater than that of the extensors. These changes were statistically sig-
nificant and lasted until the end of the exposure. At the end of the re-
covery period, the ratio of the chronaxy of extensors and flexors in animals
of group I returned to normal.
X
0.10
ff
020
0,15
0.10
5 13 25
35 45 SS 65 IS 85
Days of study
Fig. 2. Chronaxy of antagonist muscles in rats during
exposure to a mixture of acetone and phenol (group averages).
I, II and III - groups of animals; 1 - extensors; 2 - flexors;
AB - period of exposure.
The creation of a biological equilibrium between the requirements of the
organism and the activity of the enzyme systems constitutes one of the mech-
anisms by which the organism adapts to the living conditions. The biological
and chemical transformations in the organism take place with the participation
of enzyme systems. One of the enzymes is cholinesterase, which hydrolyzes
acetylcholine. Changes in the activity of cholinesterase and in the cholin-
ergic reactions of the blood are one of the indicators of the functional state
of nervous activity (D. Ye. Al'pern, 1958). Experimental studies made by D. L.
Pevzner (1954) showed that in normal relationships of the basic nervous pro-
cesses - stimulation and inhibition - the magnitude of cholinesterase activity
in the blood serum does not undergo any significant fluctuations. The preva-
lence of the inhibitory process in the cerebral cortex corresponds to a
- 91 -
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distinct decrease in cholinesterase activity.
The study of blood cholinesterase activity was carried out in 5 rats of
each group every 15 days using the procedure of J. Fleisher and E. Pope, modi-
fied by N. N. Pushkina and N. V. Klinikina (N. N. Pushkina, 1963). In rats of
group I on the 37th day of exposure, a statistically significant decrease in
cholinesterase activity (95% of the blood was observed as compared with the
control). This was not observed in animals of group II (Fig. 3).
The literature offers no data on the relative influence of low concen-
trations of acetone on the activity of blood cholinesterase. According to
the data of B. A. Mukhitov (1963), the use of low phenol concentrations causes
an increase in the activity of blood cholinesterase. Our studies showed a de-
crease in cholinesterase activity. This is possibly a manifestation of the
specific effect of acetone as a narcotic. It is known from the work of M. Ya.
Mikhel'son (1948) that narcotics decrease the activity of cholinesterase.
However, given the fact that low concentrations of the substances do not have
a specific effect, it is more correct to attribute the results obtained to the
nature of the combined action of acetone and phenol and to treat this as a
consequence of the formation of an inhibitory center in the cerebral cortex.
A substantial decrease in blood cholinesterase activity was observed only
briefly in our studies. Evidently, the compensatory mechanisms rapidly estab-
lished an equilibrium between the enzyme system and the requirements of the
organism produced by the action of the chemical stimulus, the acetone-phenol
mixture.
The mechanisms of transformation of energy in the organism of animals
and man are based on processes of biological oxidation, i. e., processes of
cellular respiration. Biological oxidation is an enzymatic process. The
enzymes participating in it constitute an intricate complex of proteins and
metalloporphyrins. They determine the respiratory function of the blood.
Thus, porphyrins as biologically active substances enter into the composi-
tion of hemoglobin, a pigment which is indispensable to the vital functions.
In the human organism, there occur a number of transformations of porphyrins,
including coproporphyrin with its two isomers, I and III.
The detection of porphyrins in the white matter of the brain and spinal
cord is of major interest for gaining an understanding of the nature of the
physiological processes occurring in the nervous system. According to a
plausible hypothesis, in areas of the central nervous system where the cyto-
chrome is either absent or present in insignificant amounts (white matter of
the brain), the oxidation processes are carried out with the aid of porphyrins;
and the fact that porphyrins are good hydrogen acceptors and donors should b^
considered experimentally proven. / /
Clinical observations and experimental studies by Yu. K. Smirnov (1953)
- 92 -
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lead to the assumption that porphyrin metabolism is one of the indicators
reflecting the functional state of the central nervous system: an inten-
sification of this metabolism takes place when the hypothalamic region of
the brain is stimulated. Inhibitory processes in the brain decrease its
functional activity and cause a decrease of porphyrin metabolism. All
this confirms the hypothesis that the determination of the content of por-
phyrin and its transformations in the daily urine may serve as an objective
test for evaluating the functional state of the central nervous system.
This explains the wide use of this test in recent years in experimental
studies aimed at validating the maximum permissible content of noxious sub-
stances in atmospheric air.
7 22 37 S3 71 IS
Days of study
Fig. 3. Dynamics of blood cholinesterase activity
in rats during exposure to an acetone-phenol mixture
(group averages).
1 - group I; 2 - group II; 3 - group IH; AB - period
of exposure.
We determined the coproporphyrin content in the combined urine of 5 rats
of each group once every 12 days, using the procedure of M. I. Gusev and Yu.
K. Smirnov (1960). The determinations showed that under the influence of
acetone and phenol, the porphyrin metabolism of animals of group I is depress-
ed. In our studies, statistically significant shifts in the porphyrin metab-
olism occurred during the 8th week of exposure. At the end of the recovery
period, the coproporphyrin content of the animals in group I continued to
remain low as compared with groups II and III (Fig. 4). Changes in the por-
phyrin metabolism of animals of group I should be explained in terms of a
disturbance of the normal relationship of the basic nervous processes as
- 93 -
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being a "physiological gauge" of the defense of the animal organism.
We followed the excretion of vitamins C, Bl, and 62, and N-methyInicotin-
amide with the urine. As we know, vitamins are catalysts for the biochemical
reactions taking place in the live cell. They participate in metabolism pri-
marily in the composition of the enzyme system. Thus the determination of the
content of vitamins may furnish additional information on the character of the
metabolic-enzymatic processes in the organism of experimental animals. The
existing literature data point to the observed disturbances of vitamin meta-
bolism in workers employed in the production of phenol and acetone by the
cumene process (N. N. Pushkina, 1964), who are exposed to their high concen-
trations in the air of plant shops.
21 33 tS 58 70 82
Days of studies
Fig. 4. Dynamics of excretion of coproporphyrin with
the urine in rats exposed to an acetone-phenol mixture.
Notation same as in Fig. 3
The content of these vitamins was determined in the combined daily urine
of 5 rats of each group every 12 days. Shifts in the excretion of the vita-
mins in animals of the experimental groups were slight and statistically in-
significant, and on the 80th day of exposure the content of vitamins in the
urine of animals of all the groups was approximately at the same level.
In histopathological studies of animals of group I, the following
characteristics were observed: circulatory disturbances in certain parentehym-
atous organs, edema in the stroma of villi of the small intestine, and change
in the permeability of the capillaries with development of moderate perivas-
cular and pericellular edema in the brain.
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Thus, the chronic experiment established that in group I of the animals,
acetone and phenol in concentrations 10 times greater than the highest Inactive
mixture for brief inhalation (1.657 mg/m3 acetone and 0.0545 mg/m3 phenol)
cause shifts indicating a response of the nervous system: disturbance of the
normal ratio of the chronaxy of antagonist muscles, decrease in blood cholin-
esterase activity, decrease in the content of coproporphyrin in the urine,
and also a number of slight anatomico-pathologic changes.
In group II, acetone and phenol in concentrations corresponding to the
highest inactive mixture in brief inhalation (0.1735 mg/m3 acetone and 0.00516
mg/mJ phenol) caused no changes in the organism of the animals under the con-
ditions of the chronic experiment.
A study of the resorptive action of the mixture of acetone and phenol also
indicates a complete summation of their effects. Hence, the mean daily maximum
permissxble concentration of acetone and phenol present together may be proposed
at the same level as the highest single concentration, i. e., the sum of their
fractions of the existing mean daily maximum permissible concentrations for
isolated action should not exceed 1.0.
We studied the atmospheric air around a synthetic phenol and acetone plant
during the spring of 1964. The air samples to be analyzed for acetone and
phenol content were collected on the leeward side at distances of 100, 300, 500
and 1000 m from the plant at a level of 1-1.5 m above ground. Single concentra-
tions of acetone and phenol were determined simultaneously. The results are
presented in Table 5 and 6.
Table 5
Single Concentrations of Acetone in Atmospheric Air Around a
Plant Producing Synthetic Phenoljand Acetone.
Distance
From Source
of Discharge
m
100
300
500
Number of
Samples
3
14
13
24
Number of
Samples Below
the Sensitiv-
ty of the Methq
24
Concentration, mg/m3
Maximum
L
2.19
0,714
Average
1.41
0,415
labie 6
Single 'Concentrations of Phenol in Atmospheric Air Around a Plant
Producing Synthetic Phenol and Acetone.
Distance
From Source
of Dis-
charge, m
100
300
500
1000
Number of
Samples
17
13.
23
25
Number of
Samples Below
the Sensitivit;
of the Method
_
—
3
25
Concentration, ag/m5
Maximum
0,323
0,2197
0.0869
—
Average
0,187
0,115
0,039
—
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As is evident from Table 5 and 6, at a distance of 100 m, the highest
acetone concentration surpassed its maximum permissible concentration (0.35
mg/m3) by a factor of 6, and at a distance of 300 m, by « factor of 2. Only
at a distance of 500 m was no acetone detected.
At a distance of 100 m from the source of discharge, the highest single
phenol concentration was 32 times as high as its maximum permissible concen-
tration (0.01 mg/m3); at 300 m it was 22 times as high, at 500 m, 8 times,
and only at a distance of 1000 m was the phenol concentration undetectable.
Of practical interest is a comparison of the data obtained with the maximum
permissible concentrations of acetone and phenol present together; expressed
in fractions of'the highest single maximum permissible concentrations for
isolated action, they should not exceed 1 in this case. The data presented
in Tables 5 and 6 show that a distance of 100 m, the total concentration of
acetone and phenol exceeds the maximum permissible value for their combined
presence by a factor of 38. At a distance of 300 m, this excess is 24-fold.
Thus, a study of the pollution of atmospheric air around a plant dis-
charging phenol and acetone simultaneously revealed a considerable pollution
of the ambient air, particularly with phenol, so that prophylactic measures
should be directed primarily at the removal of phenol vapors from the waste
gases.
A sanitary-protective zone for plants producing synthetic phenol and
acetone can be recommended only by taking into account the pollution of at-
mospheric air by other noxious substances discharged along with acetone and
phenol.
Conclusion
1. When a mixture of acetone and phenol acts on the sense of smell,
light sensitivity of the eye and electrical activity of the human brain, a
complete summation of the action of these compounds on the organism is ob-
served.
2. A biological effect of a mixture of acetone and phenol is observed
only when their total concentration expressed in fractions of the threshold
concentrations for isolated action amounts to less than 1.
3. When acetone and phenol are jointly present in atmospheric air,
their'highest single concentration should be at the biologically inactive
level, i. e., should not exceed 1 in fractions of the highest single maxi-
mum permissible concentrations for isolated action.
4. The mean daily maximum permissible concentration of an acetone-
phenol mixture in atmospheric air is recommended at the level of the highest
single concentration.
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LITERATURE CITED
Note: References mentioned in this paper are to be found at the
end of the volume in the 1968 bibliography.
- 97 -
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ON THE COMBINED EFFECT OF LOW CONCENTRATIONS OF ACETONE AND ACETOPHENONE
IN AIR ON THE ORGANISM OF MAN AND ANIMALS
Aspirant (graduate student) N. Z. Tkach
A. N. Sysin Institute of General and Communal Hygiene, Academy of Medical Sciences of the USSR
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvle i gigienicheskoe
znachenie atmosfernykh zagryazneniy". Red. V. A. Ryazanova. Vypusk 10,
Izdatel'stvo "Meditsina" Moskva, p. 170-186, (1967).
The rapid development of chemical industry is associated with the discharge
of large amounts of toxic substances into the atmosphere. The discharges of
a single industrial enterprise most often contain a whole series of noxious
substances.
One of the most frequent combinations of atmospheric pollutants are
acetone and acetophenone.
The main sources of pollution of atmospheric air by this combination are
enterprises that produce synthetic phenol and acetophenone simultaneously by
the cumene process. In this method, acetophenone is obtained as a product
of side reactions and is also always discharged into the atmosphere.
The combined action of atmospheric pollutants was first studied by
K. A. Bushtuyeva (1960) (sulfur dioxide and sulfuric acid aerosol), V. M. Sty-
azhkin (1962) (chlorine and hydrogen chloride), and B. K. Baykov (1963)
(carbon disulfide and hydrogen sulfide). In these studies, the authors
observed a complete or partial summation of the action of the compounds studied.
The object of our work was to study the effect of low concentrations of
acetone and acetophenone on the organism of man and experimental animals when
these compounds are jointly present.
Acetone and acetophenone are toxic substances affecting primarily the
central nervous system. The toxicology of acetone has been treated in a
large number of studies (A. Ye. Yefremov, 1929; N. F. Okuneva, 1930;
I. D. Mishenin, 1933; I. S. Tsitovich, 1935; P. M. Sukhov, 1935; N. V. Lazarev,
1954; Yu. G. Fel'dman, 1960; Ch. Khadnan' et al., 1962, and others).
Literature data on the toxicology of acetophenone are very scant (Laborde,
1885; S. S. Kamenskiy, 1889; Friedman and Maase, 1910; N. B. Imasheva, 1963,
and others). These investigations deal primarily with the study of the effect
of toxic concentrations of acetone and acetophenone. Only the work of , /
Yu. G. Fel'dman on acetone and that of N. B. Imasheva on acetophenone are de-
voted to the study of the influence of low concentrations in both brief con-
tact and under conditions of a chronic experiment.
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In the literature, we were unable to find any data on the combined
action of these ingredients.
M. I. Ol'shanskaya and V. V. Likhacheva (1935) studied the combined
action of acetone and benzene vapors on pigeons in concentrations of
25,000 and 10,000 mg/m3 respectively. The authors found a complete summa-
tion of the action of these compounds.
The mixture with the strongest effect proved to be one containing
equal amounts of benzene and acetone.
T. A. Shtessel1 (1937) also obtained a simple summation of the effect
in a 2- and 4-hour experiment on white mice with combinations of acetone
and methanol, acetone and ethyl ether, acetone and toluene, and acetone
and benzene. The criterion of the action of each compound taken individually
and in combination was the lateral position of the white mice.
A reinforcement of the toxic effect of acetone in combination with
benzene is indicated by Nischiama and Isami (1957). The authors found that
during the combined action of benzene and acetone, the leucocyte content of
the blood decreases even more than during the action of benzene or acetone
alone.
We began our experimental studies with the determination of the thres-
hold of olfactory perception of acetone and acetophenone taken separately,
then in combination.
The acetone was determined by the nephelometric method, whose sensi-
tivity is 1. ]ag in a volume of 4.5 ml. Acetophenone was determined spec-
trophotometrically, with a sensitivity of 0.25 yg in 1 ml.
The odor threshold of acetone was determined in 22 practically healthy
people 18 to 48 years old using a standard procedure (V. A. Byazanov,
K. A. Bushtuyeva, and Yu. V. Novikov, 1957). A total of 412 observations
were made with concentrations of 2.0, 1.654, 1.481, 1.096, and 0.80 mg/m3.
The results obtained from the data are listed in Table 1.
It is evident from Table 1 that for the most sensitive persons, the
odor threshold of acetone is 1.096 mg/m3, arid the subliminal concentration
is 0.8 mg/m3.
The odor threshold of acetophenone was determined in 16 persons; a
total of 216 determinations were made with 4 acetophenone concentrations.
The results of the observations are given in Table 2.
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Table 1
Determination of the Threshold of Olfactory
Perception of Acetone
Number of
Subjects
2
4
6
10
Concentration of
A r*at- nno mr /mo
Minimum
Perceptible
2,0
U654
1,481
1,096
Maximum
Imper-
ceptible
1,654
1,481
1,096
0,80
Table 2
Determination of the Threshold of
Olfactory Perception of Acetophenone
Number of
Concentration of
Acetophenone, mg/rn^
ouujeuts
3
6
7
Minimum
Perceptible
0,0251
0,0156
0,010
Maximum
Imperceptible
0,0156
0,010
0,008
The results of our studies are in accord with those of Yu. G. Fel'dman,
whose data indicate that the odor threshold of acetone is 1.1 mg/m3, and
with those of N. B. Imasheva, whose data show the odor thresholds of
acetophenone to be 0.01 mg/m3. After determining the separate odor thres-
holds, we studied the sensitivity of the olfactory system to mixtures of
different concentrations of acetone and acetophenone in the following com-
binations :
Mixture I - acetone + acetophenone - 1.096 mg/m3 +0.01 mg/m3.
Mixture II - acetone + acetophenone - 0.711 mg/m3 + 0.0061 mg/m3.
Mixture III - acetone + acetophenone - 0.565 mg/m3 + 0.005 mg/m3.
Mixture IV - acetone + acetophenone - 0.348 mg/m3 + 0.004 mg/m3.
A total of 265 determinations were made on 16 subjects. To obtain
comparable results, the concentrations of each ingredient were expressed in
fractions of their isolated thresholds. It is known that for a total concen-
tration index equal or close to 1, there is a complete summation of the effect:
for an index greater than 1 but smaller than 2, there is a partial summation;
and for an index of less than 1, potentiation.
r
Analysis of the results obtained from the studies showed that the con-
centration of acetone and acetophenone in the first combination (mixture I)
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was perceived by all 16 persons. The total concentration index of this
mixture ranged from 1.06 to 2.0. The majority of the subjects (12) also
identified the odor of the second mixture, in which the sum of the rela-
tive concentrations ranged from 1.03 to 1.26. Only the most sensitive
persons (5) perceived the third mixture, whose total concentration index
was 1.01 (Table 3).
Table !>
Determination of the Threshold of Olfactory Perception of Acetone
and Acetophenone in Combination
Mixture
Acetone dng/m3)
Acetophenone (mg/m3)
Sum of fractions of
individual threshold
Number of persons
perceiving the odor
Number, of persons no\
perceiving the odor
I
1,096
0,010
From 1,06
s to2,0
16
11
0,711
0,0061
Froml 1 03
To 1.26
12
4
III
0,565
0,005
1,01
5
IV
0,348-0,711
0,004-0,0061
FronO.67
*o 0,88
__
11 i 16
From Table 3 it is also apparent that the minimum perceptible con-
centrations in the mixture are 0.565 mg/m3 acetone and 0.005 mg/m.3
acetophenone for a sum of relative concentrations equal to 1.01. The
mixture is not perceived by its odor if the sura of the relative concen-
trations is less than 1.
Hence, in this case there is a complete summation of the action of
acetone and acetophenone. The summation effect was also found in the case
of other, less sensitive persons.
A study of the reflex effect of low concentrations of acetone and
acetophenone on the functional state of the central nervous system was
made by determining the light sensitivity of the eye under dark adaptation
conditions. This test, widely employed at the present time in the practice
of sanitary standardization of atmospheric pollutants, makes it possible
to record, by means of the reflex physiological reactions, the response to
the action of concentrations of toxic substances whose odor is imperceptible
but which change the functional state of the cerebral cortex.
A change in the course of the dark adaptation curve during the action
of concentrations of noxious substances of imperceptible odor was observed
by V. A. Gofmekler (1960), Yu. G. Fel'dman (1960), R. Ubaydullayev (1961),
and others.
In a study of the combined action of sulfur dioxide and sulfuric acid
aerosol, K. A. Bushtuyeva (1960) observed the phenomenon of physiological
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summation. She established the fact that an increase in the light sensi-
tivity of the eye as compared with the normal dark adaptation curve during
the combined action of these compounds is equal to the sum of the increases
during the combined action of these compounds is equal to the sum of the
increases during their individual action.
The phenomenon of partial summation was noted by B. K. Baykov (1963)
in a study of the action of carbon disulfide in combination with hydrogen
sulfide.
Our observations were made on three subjects, 24 to 27 years of age,
according to a standard procedure. Persons whose odor threshold was the
most sensitive participated in the experiment. A total of 42 tests were
conducted. The following mixtures were studied:
1. Acetone + acetophenone - 0.283 mg/m^ + 0.005 mg/m^.
2. Acetone + acetophenone - 0.223 mg/m3 + 0.0037 mg/m3.
The threshold of the reflex effect of acetone on the light sensitivity
of the eye for the most sensitive persons is 0.55 mg/m^ according to the
data of Yu. G. Fel'dman, and 0.01 mg/m^ according to those of I. B. Imasheva.
The total concentration index of the first mixture is 1.01, and of the second,
0.77.
Anaylsis of the results obtained showed that statistically significant
changes of the light sensitivity take place only during the action of the
first mixture, when the sum of the fractions of the thresholds is equal to
1.01.
In one subject, an increase in light sensitivity was noted after the
inhalation of the experimental mixture; in the second a decrease was ob-
served, and the third subject responded to the action of acetone and
acetophenone in the first combination first by an increase in light sensi-
tivity (in the 20th minute), and then by its sharp decrease in the 25th
minute with a return to the initial level at the end of the experiment.
The light sensitivity in the 20th minute of adaptation for all the subjects
is given in Table 4.
A mixture of lower concentrations of acetone and acetophenone for a
total concentration index of 0.77 was found to be inactive and failed to
cause any significant changes in light sensitivity as compared with pure
air.
Hence, a complete summation of the action of the compounds studied is
also observed in the adaptometric tests.
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Table 4
Data of Adaptometric Tests
Subject
Concentration,
irm?
Acetone +
Acetophenone
Light Sensitivity
in Relative Units
in 20th Minute &
Adaptation
S. R.
S. L.
Pure Air
0,283 + 0,005 '
0,223 + 0,0037
Pure Air
0,283 + 0,005
0,223 + 0,0037
Pure Air
0,283 + 0,005
0,223 + 0,0037
* Degree of significance:b -
81 475
103 050 (b)*
82 133 (o)
83 225
111 000 (b)
82 133 (o)
95 550
74 175 (c)
90 033 (o)
; o - not significant.
We also studied the effect of subliminal concentrations of acetone in
combination with acetophenone on the electrical activity of the brain by a
quantitative analysis, i.e., the reflex response of a burst of the alpha
rhythm of the human brain (A. D. Semenenko, 1964). An analogous procedure
was described in the articles of U. G. Pogosyan and Yu. S. Korneyev.
The study was conducted on three subjects on a Galileo polyphysiograph;
a total of 62 observations were made. The biocurrents were recorded from
the temporal and frontal lobes of both hemispheres and their combinations,
and also from the central-parietal part of the left hemisphere. Data obtained
from the temporal lobes, which were the most sensitive, were subjected to a
graphic statistical analysis. The following mixtures were studied:
1. Acetone + acetophenone - 0.22 mg/m3 + 0.0035 mg/m3.
2. Acetone + acetophenone - 0.18 mg/m3 + 0.0027 mg/m3.
In these studies we also used separate thresholds of action of acetone
(0.44 mg/m3) and acetophenone (0.007 mg/m3), established by Yu. G. Fel'dman
and N. B. Imasheva. The sum of the relative concentrations (in fractions
of threshold values) of the first mixture was equal to 1, and that of the
second mixture, to 0.78. The effect of each mixture was studied no fewer
than 4 times and was regularly alternated with the supply of pure air.
In our experiments, the first mixture of acetone and acetophenone caused
an increase in the energy of the brain potentials in two subjects, whereas in
the third, a decrease in the energy of the potentials was noted. At the
same time, changes were obtained which were characterized by a statistically
significant difference, compared with the control, in the 3rd and 4th minutes
of inhalation of the experimental mixture. The quantitative data obtained
- 103 -
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during inhalation of the second mixture did not differ appreciably from the
data obtained during inhalation of pure air.
Thus, the results of studies based on the most sensitive test also
confirm the phenomenon of complete summation of subliminal stimuli.
Expressing the data obtained from electroencephalography in fractions
of the existing maximum permissible concentrations of each component, we
see that the minimum active mixture amounts to 1 . 79 :
0.22 mg/m-j 0.0035 mg/mj _,,,,., ,, _
— — — - , T + n nno I T ~ U.OJ + l.lO -
0.35 mg/mj 0.003 mg/mj
, T n nno I
mg/mj 0.003 mg/
and the maximum inactive mixture amounts to 1.41:
0.18 mg/m3 0.0027 mg/m3 _0c1+09_141
0.35 mg/m3 + 0.003 mg/m3 ~ °'51 + °'9 ~ 1'41-
On the basis of these data one can postulate that the combined presence
of ace.tone and acetophenone in atmospheric air is permissible if the sum of
fractions of these concentrations of their existing maximum permissible values
for isolated action does not exceed 1.5.
In order to study the resorptive action of acetone in combination with
acetophenone, a chronic inhalational exposure of white male rats was carried
out in the course of 84 days. The tests were conducted on 45 rats with an
initial weight of 80-100 g, divided into three groups of 15 animals each.
The animals were exposed to mixtures of the indicated compounds in the
following average concentrations:
Group I - acetone + acetophenone 1.855 mg/m^ + 0.168 rng/m^.
Group II - acetone + acetophenone 0.192 mg/m^ + 0.00197 mg/m^.
Group III was the control.
Expressing the concentrations of the compounds in fractions of their
maximum permissible values for isolated action, we find that the sum of the
fractions of the first mixture is equal to 10.9, and that of the second,
to 1.2.
Conversion of the acetone and acetophenone concentrations of the second
mixture to the threshold concentrations for their isolated action based on
the most sensitive test (electroencephalography) shows that the sum of their
fractions is equal to 0.71. Hence, animals of group II were exposed to '
acetone and acetophenone concentrations indifferent for man, whereas animals
of group I were exposed to concentrations of these compounds which were
10 times as high.
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The action of the mixtures of these compounds was evaluated by observ-
ing the general state and weight of the animals; studying the motor chronaxy
of antagonist muscles; determining the cholinesterase activity, dynamics of
excretion of coproporphyrin, neutral 17-ketosteroids and vitamin C with the
urine, and determining the content of the absolute number of eosinophils in
peripheral blood.
During the exposure, rats of all the groups were active and gained
weight regularly.
In studying the functional state of the central nervous system in the
experimental animals during exposure to low concentrations of acetone and
acetophenone, we used the method of determination of the motor chronaxy of
antagonist muscles. The literature contains indications of a direct relation-
ship between the level of motor chronaxy and the functional state of the
central nervous system, which by virtue of subordinational relationships
regulates the activity of peripheral nerve apparatus as well as that of lower
nerve centers (Yu. M. Uflyand, 1941; A. N. Magnitskiy, 1948).
A manifestation of the subordinational influences of the brain is the
constancy of the ratio of the chronaxy of extensors and flexors, whose
numerical expression normally amounts to 1.5:1, 2:1, and 2.5:1.
The motor chronaxy was measured in 5 rats of each group three times a
month with an ISE-01 electronic pulse stimulator.
Starting with the 4th week of exposure, statistically significant
changes in the normal ratio of the chronaxy of extensors and flexors in
rats of group I took place chiefly as a result of a prolongation of the
chronaxy of flexors. Starting with the 6th week of exposure, a reverse
ratio of the chronaxies took place, following which the curves were at approx-
imately the same level and returned to the limits of the physiological norm
only during the recovery period.
In rats of group II, no such disturbances of the normal ratio of
chronaxy were observed over the course of the entire exposure, and there
were no reliable changes as compared with the control.
The changes which we detected in the normal ratio of chronaxies may be
regarded as a manifestation of inhibitory processes in the central nervous
system which encompass the complex system of cortical and subcortical
subordination centers.
The presence of inhibitory processes in the central nervous system of
the experimental animals during the action of acetone in combination with
acetophenone was confirmed by a depression of the activity of whole blood
cholinesterase of the animals.
- 105 -
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According to modern interpretations, the transfer of stimulation from
a nerve to a muscle is accomplished with the participation of the system
acetylcholine - cholinesterase. Here acetylcholine plays the part of a
mediator. An excessive accumulation of acetylcholine in the organism causes
a decrease in the lability of the nervous processes and the development of
a state of inhibition (I. N. Volkova, 1954; D. Ye. Al'pern, 1963).
A decrease in the activity of the enzyme was noted by several authors
during a prolonged action of various chemical agents (N. B. Imasheva, 1963;
V. A. Chizhikov, 1964; A. V. Mnatsakanyan, 1964, and others).
The cholinesterase activity was determined by the method of J. Fleisher
and E. P. Pope (1954). The method is based on a change in the intensity of
the color of the solution as a function of the amount of acetic acid formed
by the enzymatic cleavage of acetylcholine chloride. The color intensity is
determined on an FEK-M instrument.
The cholinesterase activity was determined in 5 rats of each group
twice a month; the blood was taken from the tail vein by incising the blood
vessel.
It is evident from Fig. 1 that as early as the end of the first month of
exposure, the rats of group I showed a considerable decrease in the activity
of the enzyme which lasted until the end of the second month. At the end of
exposure, the cholinesterase activity became somewhat normal in animals of
this group, but remained as before at a lower level than that of the control
group. The observed changes in cholinesterase activity are characterized by
a statistically significant difference (95-99%).
As far as the cholinesterase activity of animals of group II is con-
cerned, we did not observe any significant changes over the entire course
of the exposure.
We estimated the porphyrin metabolism of the experimental animals from
the dynamics of the excretion of porphyrin with the urine. The literature
contains indications of a direct connection between the porphyrin metabolism
and serious disturbances in the central nervous system (A. M. Charnyy and
S. E. Krasovitskaya, 1951). A. M. Charnyy postulated that the pbrphyrins may
participate in oxidation-reduction processes in areas of the brain where the
cytochrome and cytochrome oxidase are lacking.
The relationship between the excretion of porphyrins from the organism
and the activity of bone marrow erythropoesis was pointed out by Watson
(1941). In sanitary studies, the porphyrin metabolism was first investigated
by M. I. Gusev (1960). During the exposure of rabbits to lead oxide in ,a (;
concentration of 10 yg/m^ for 6 months, the author observed that the amount
of coproporphyrin in the urine in this group of animals increased twofold 'as
compared with the control. A disturbance of the porphyrin metabolism was
- 106 -
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also noted by other authors who studied low concentrations of atmospheric
pollutants in chronic experiments (G. I. Solomin, 1962; D. G. Odoshashvili,
1962; V. A. Chizhikov, 1964, and others).
too •
31/X 15/XI 30/XI 1S/XU IS/I
Date of study
Fig. 1. Change in the activity of blood cholines-
terase in rats of different groups.
1 - group I - acetone + acetophenone with total
index of 10.9: 2 -group p - acetone + acetophenone
with total index of 1.2? 3 - group III - control;
AB - period of exposure.
In our studies, we used this test with a quantitative determination
of coproporphyrin on an SF-4 spectrophotometer in the ultraviolet region
of the spectrum (M. I. Gusev and Yu. I. Smirnov, 1960). The daily urine
was collected in special receivers every 15 days from 5 rats of each group.
A total of 33 determinations were made, including 9 before the exposure
(background), 18 during the exposure, and 6 during the recovery period.
Analysis of the data obtained showed that the combined action of acetone and
acetophenone in concentrations of 1.855 and 0.0168 mg/m3 respectively caused
a definite shift in the porphyrin metabolism of animals of group I.
yg./100 g of weight
ff/lX 3/X
12JXI Z7/XI ttfXIl ff/Xtl 21/1
Dates of study
Fig. 2. Excretion of coproporphyrin with the
urine in rats of different groups through the
effects of acetone and acetophenone.
Notation same as in Fig. 1.
- 107 -
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It is evident from Fig. 2 that starting with the 4th week of exposure,
the rats of this group showed an increased excretion of coproporphyrin.
During the exposure, its content in the urine increased, and during the
second period reached a statistically significant difference as compared
with the control group (degree of significance, 95%). In group II, no
significant differences from the control were observed.
It is known that the activity of the anterior hypophyseal lobe - adrenal
cortex hormonal system is controlled by the central nervous system via the
hypothalamus.
It was found from literature data that the action of various chemical
compounds in high concentrations causes a nonspecific reaction in the anterior
lobe - adrenal cortex system; a manifestation of this reaction is a decrease
in the absolute quantity of eosinophils in the blood and an increase in the
content of neutral 17-ketosteroids in the urine (Ye. I. Spynu, 1962;
L. E. Corn, 1963).
We attempted to detect the presence of nonspecific reactions in the
hormonal system in response to the combined action of low concentrations
of acetone and acetophenone.
yg/100 g of weight
S -
tSjX 30/X M/JU 29/XI 13/XII 28/Xtt 12/1 ZO/I
Dates of study
Fig. 5. Content of neutral 17-ketosteroids in
the urine of rats of different groups.
Notation same as in Fig. 1.
The determination of 17-ketosteroids was made by using the procedure
of 0. M. Uvarovskaya (1956) followed by colorimetry on an FEK-M instrument
with a green filter. The studies were made twice a month, and a total of
24 determinations were performed. A graphical representation of the results
obtained is given in Fig. 3, which shows that during the first few days of
exposure, the amount of 17-ketosteroids in the urine of animals of group I
decreased slightly as compared with the control group. However, starting with
the 40th day of exposure, a sharp increase in the content of 17-ketosteroids
was noted in rats of group I. Whereas in rats of the control group the
amount of 17-ketosteroids during that period was 6.85 yg per 100 g of weight,
in rats of group I it was 17.41. The increased level of 17-ketosteroids was
- 108 -
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preserved until the 70th day of exposure, and at the end of exposure
approached the values of the control. The changes obtained were statis-
tically significant.
The fluctuations of 17-ketosteroids observed in the urine of group II
of animals did not differ significantly from the control. The eosinophils
of the blood were counted by using S. M. Bakman's method (1958). The blood
was taken from 5 rats of each group twice a month at a fixed time, and a
total of 99 analyses were performed.
In animals of group I after 10 days of exposure, the number of eosino-
phils increased somewhat, probably as a result of the initial response of
the animal organism to the action of acetone and acetophenone (Fig. 4).
Subsequently, a sharp decrease in the number of eosinophils was observed
which lasted with some fluctuations until the end of exposure. Here the
difference in the decrease of the concentration of eosinophils as compared
with the control group reached significant values (95%). The number of
eosinophils in rats of group II was close to that of the control, although
on the 55th day of exposure an increase was observed, but it did not reach
a statistically significant difference. An eosinopenic response was observed
by A. Ye. Kulakov (1965), who studied the effect of low concentrations of
hexamethylenediamine on the organism of animals in a chronic experiment.
tf/ee tff/jr 3t/t /t/xi 30/ti isitu ts/i
Dates of study
Fig. 4. Absolute number of eosinophils
in the blood of rats exposed to the com-
bined action of acetone and acetophenone
in low concentrations.
Notation sane as in Fig. 1.
Hence, the investigations performed indicate a high sensitivity of our
tests and permit the observation of a nonspecific response of the organism
to the adverse effect of atmospheric pollutants.
A study of the dynamics of excretion of vitamin C with the urine in
white rats over the entire course of exposure failed to reveal any disturb-
ances in the C vitamin balance of the experimental animals. The content of
- 109 -
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the vitamin in the urine of animals subjected to the exposure did not differ
appreciably from that of the control group.
Table 5
Single Concentrations of Acetone in Atmospheric Air Around a
Plant Producing Phenol and Acetone
Distance From
Source of
Discharges, m
100
300
500
Number of
Collected
Samples
14
13
24
Number of
Samples
Belpw.The
Sensitivity
of the Metnoc
24
Concentration, mg/rn^
Maximum
2,19
0,714
*
Average
1,41
0,415
On the basis of the completed studies, we propose a mean daily maximum
permissible concentration (in fractions of the existing maximum permissible
concentrations) of acetone and acetophenone jointly present in atmospheric
air at a level of 1.2. We are aware of the fact, however, that a wide
range of unstudied values separates the active (10.9) and inactive (1.2)
total concentration of acetone and acetophenone. The Section on Sanitary
Protection of Atmospheric Air deemed it possible to approve the mean daily
concentration of these compounds (in fractions of their maximum permissible
concentrations) at the level of the highest single concentration (1.5).
In order to determine the single concentrations of acetone and aceto-
phenone in atmospheric air around a plant producing synthetic phenol and
acetone, we collected 131 samples at distances from 100 to 1000 m from the
plant.
As is evident from Table 5, at a distance of 100-300 m from the plant,
atmospheric air is considerably polluted with acetone. Among the 27 samples
collected at this distance, concentrations substantially exceeding the maxi-
mum permissible value (0.35 mg/m3) were observed. Only at a distance of
500 m from the plant was no acetone found in any of the 24 samples.
Table 6
Single Concentrations of Acetophenone in Atmospheric Air Around
a Plant Producing Phenol and Acetone
Distance 'From
Source of
Discharges, ID
100
300
500
1000
Number of
Collected
jSamples
11
17
26
25
Number of
BtMle
Sensitivity
af the Itetha
9
25
Concentration, mg/m^
Maximum
0,877
0,097
0,028
Average
0,258
0,057
0,007
- 110 -
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It is evident from Table 6 that acetophenone is found in substantial
concentrations at a distance of 100-500 m from the plant, where its highest
concentrations exceed the maximum permissible value (0.003 mg/m3). At a
distance of 500 m, the maximum permissible concentration is exceeded in
46% of the samples. Only at a distance of 1000 m from the plant was no
acetophenone found in any of the collected samples. Consequently, the sani-
tary protective zone for this plant should be no smaller than 1000 m.
Conclusions
1. A determination of the thresholds of olfactory perception of
acetone and acetophenone acting individually made it possible to establish
that the minimum perceptible concentration of acetone for the most sensitive
persons is 1.096 rag/mS, and that of acetophenone, 0.01 mg/m3.
2. When acetone and acetophenone are acting in combination, their odors
exhibit a complete summation.
3. The reflex effect of acetone in combination with acetophenone on
the light sensitivity of the eye and electrical activity of the brain is
characterized by an action expressed in a complete summation.
A. Chronic round-the-clock exposure of white rats to a mixture of
acetone and acetophenone for a total concentration index (in fractions of
the maximum permissible values) equal to 10.9 causes definite functional
shifts in the organism of the experimental animals, manifested in a decrease
of the subordinational influence of the brain on the level of the motor
chronaxy of antagonist muscles, a depression of the activity of blood cholin-
esterase, an increase in the excretion of coproporphyrin with the urine, and
also the presence of a nonspecific response of the anterior hypophyseal lobe -
adrenal cortex system, which we evaluated from the increase in the content
of 17-ketosteroids in the urine and a decrease in the absolute number of
eosinophils in the blood. An acetone-acetophenone mixture with a total con-
centration index equal to 1.2 did not cause any significant changes in the
animal organism according to the selected tests.
5. In the combined presence of acetone and acetophenone in atmospheric
air, their highest single and mean daily concentration expressed in fractions
of the maximum permissible values for isolated action should not exceed 1.5.
- Ill -
-------
LITERATURE CITED
Note: References mentioned in this paper are to be found at the end
of this volume in the 1967 bibliography.
- 112 -
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ON THE COMPARATIVE TOXICITY OF BENZENE, TOLUENE, AND XYLENE
(STUDY OF THE REFLEX EFFECT)
I. S. Gusev
A. N. Sysin Institute of General and Communal Hygiene,
USSR Academy of Medical Sciences
From Akademiya Meditsinakikh Nauk SSSR. "Biologicheskoe deystvie i gigienicheskoe
znachenie atmosfernykh zagryazneniy". Red; V. A. Ryazanova. Vypusk 10,
Izdatel'stvo "Meditsina" Moskva, p. 96-108, (1967).
The long-known aromatic hydrocarbons benzene, toluene, and xylene are
still attracting the attention of toxicologists and hygienists. This is'be-
cause these compounds thus far have not lost their industrial importance,
possess a marked toxicity and a wide spectrum of action, there'is no unified
view of the comparative toxicity and mechanism of their action, and the low-
est parameters of their effect on the living organism have not been established.
Pure benzene, toluene, and xylene are colorless liquids with a specific
aromatic odor, and are poorly soluble in water. The boiling point of benzene
is 80.1°C., toluene 110.8°C, and zylene 140.59C (mixture of isomers). The
volatility compared to ether is 3 for benzene, 4.2 for toluene, and 13.5'for
zylene. They are good solvents of fats, resins, rubbers, essential oils, dyes,
and other organic compounds.
As solvents, the compounds under consideration find extensive applications
in various branches of industry. In addition, benzene is widely used in organic
syntheses'for the production of phenol, nitrobenzene, chlorobenzene, maleic
anhydride, and other compounds. Toluene is the starting material in the pro-
duction of explosives. Xylidenes and phthalic acids are obtained from xylene.
Because of their marked narcotic effect, benzene, toluene, and xylene
affect primarily the functions of the nervous system. Thus, G. E. Rozentsvit
(1954) emphasizes that functional disturbances of the central nervous system
precede morphological changes in the blood. Some investigators even go so far
as to consider the changes in other organs as consequences of damage to the
nervous and endocrine systems.
A change in the reflex activity of animals acted upon by various concen-
trations of benzene was observed by R. I. Yaroslavskaya and I.'M. Rozovskiy
(1952), Yu. V. Novikov (1957), A. I. Korbakova, S. N. Kremneva, N. K. Kulagina,
and I. P. Ulanova (1960).
In exposures of mice to xylene in a concentration of 550 mg/m3, A. A.
Golubev (1959) noted a lag in the rate of development of conditioned reflexes
and a decrease in the regularity of differentiated responses.
- 113 -
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Ye. N. Lyublina (1950) determined the minimum concentrations of benzene,
toluene, and xylene acting on the central nervous system of rabbits. For a
40 minute exposure, they were 300, 500, 1000 mg/m3 for benzene, 300, 500, and
1000 mg/m3 for toluene, and 100, 200, and 400 mg/m3 for xylene.
Concentrations disturbing the unconditioned reflex activity of rabbits
were 1500 for benzene, 1000 for toluene, and 750 mg/m3 for xylene (A. S.
Faustov, 1961). The author emphasizes that in contrast to benzene and toluene,
xylene causes a depression of the central nervous system.
The comparative evaluation of the toxicity of benzene, toluene, and xylene
is made by most authors on the basis of the action of these compounds on the
blood and blood-forming organs. From this standpoint, M. L. Mgebrov, N. D.
Rosenbaum, Lind, and others consider benzene to be more toxic. Others indi-
cate that chronic poisoning with toluene may be associated with a depression
of the activity of the bone marrow, as in the case of benzene. R. G. Leytes,
B. I. Martsinkovskiy and L. K. Khotsyanov hold that toluene and xylene act
like benzene, but that their action is associated with less pronounced changes
in the blood (0. N. Olimpiyeva, V. M. Retnev, and A. P. Rusinova, 1958).
The study and comparative evaluation of minimum concentrations of these
compounds on the organism are of interest from the standpoint of both toxi-
cology and sanitary protection of atmospheric air.
There are scant literature data on the pollution of atmospheric air with
vapors of benzene, toluene and xylene. The sources of atmospheric pollution
may be not only major coking plants and petroleum refineries (A. A. Itskovich
and V. A. Vinogradova, 1956; M. L. Krasovitskaya and T. S. Zaporozhets, 1961),
but also small enterprises and printing houses which use these compounds as
solvents (Yu. V. Novikov, 1956).
With their distinct, specific odor, benzene, toluene, and xylene are able
to act on the receptors of the upper respiratory tract and nasal cavities and
cause a series of reflex responses involving the nervous system and its high-
est stage, the cerebral cortex. "The odors and the reflexes that they cause
are by no means indifferent for the organism. Since they are reflected in the
cortex, they can produce time relationships and involve the most diverse pro-
cesses in their action" (S. V. Anichkov, 1952).
In recent years, a number of investigations have shown that chemical com-
pounds in concentrations coinciding with the threshold of olfactory perception
or slightly above it are able to cause such reflex responses as a change in the
vascular tone, rhythm and depth of respiratory movements, optimal chronaxy,
galvanic skin reflex, and the light sensitivity of the eye (K. A. Bushtuyeya,
1960; M. K. Borisova, 1960; M. T. Takhirov, 1960, and others). • '
On this basis, we undertook a study of the thresholds of olfactory ,
- 114 -
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perception in order to obtain a comparative evaluation of the effect of these
compounds. The thresholds of olfactory perception of benzene, toluene, and
xylene had already been studied. However, the studies were conducted by dif-
ferent experimenters using different procedures, and the threshold values for
the same substance differed considerably. Thus, N. V. Lazarev (1963) gave the
following thresholds of olfactory perception: benzene 5 mg/m3. toluene 2 mg/m3,
and xylene 0.8 mg/m3. According to the data of Yu. V. Novikov'(1956), the thres-
hold of olfactory perception of benzene is 3 mg/m3. Ch'en Yun-t'ai established
the threshold of olfactory perception of ortho-xylene at the level of 0.73
mg/m3 (1963).
We made a determination of the thresholds of olfactory perception accord-
ing to a procedure recommended by the Committee on Sanitary Protection of
Atmospheric Air (V. A. Ryazanov, K. A. Bushtuyeva, and Yu. V. Novikov, 1957).
The concentrations of benzene, toluene, and xylene were determined by
using M. V. Alekseyeva's procedure (1964). It is based on nitrating these com-
pounds, then extracting the nitro compounds with butanol. When a base is added
to the extract, the solution acquires a certain color (purple for benzene, orange
for toluene, and greenish-blue for meta-xylene). The color of the solutions
was measured colorimetrically. The determination was made with the aid of photo-
electrocolorinieter (FEKN-57) and calibration curve. The sensitivity of the
method was 0.5 Ug for benzene, 1 yg for toluene, and 2 yg for xylene in a volume
of 2 ml. The samples were collected before and after each observation, and the
fluctuations of the concentrations were insignificant.
Table 1
Results of Determination of the Olfactory
Perception Threshold for Benzene Vapors
Qtanber of
Subjects
8
3
2-
5
Concentration of Benzene, ng/m3
Minimum
Perceptible
>4,0
3,4
3.2
2,8
MaxiffillQ
Imperceptible
3,2
3,0
2,5
The threshold of olfactory perception of benzene was determined on 18
subjects. A total of 7 concentrations were studied, and 518 tests were con-
ducted. The results are shown in Table 1.
The first benzene concentration studied, 4 mg/m3, was imperceptible for
8 persons out of 18. The minimum perceptible concentration for the 5 most
sensitive persons was 2.8 mg/m3, and the maximum imperceptible concentration
was 2.5 mg/m3. Thus, the results of our studies confirm the data on the ol-
factory perception threshold obtained by Yu. V. Novikov in 1956 (3 mg/m3).
- 115 -
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Table 2
Kosiilts of Determination of the Olfactory
Perception Threshold for Toluene Vapors
Number of
Subjects
5
6
4
3
3
3
6
Concentration of Toluene, ing/m^
Minimum
Perceptible
>3,2
3,2
2,8
2,3
2,0
1,8
1,5
Maximum
Imperceptible
27»
2.3
2,0
1,8
1,5
1,27
The threshold of olfactory perception of toluene was studied on 30 sub-
jects, A total of 7 concentrations were studied, and 744 observations were
made, the results of which are presented in Table 2.
Of 30 persons, 5 did not perceive the toluene concentration studied - 3.2
mg/m3; the 1.5 mg/m3 concentration was found to be the minimum perceptible
value for the most sensitive 6 subjects. The maximum imperceptible concentra-
tion for the same subjects was 1.27 mg/m3. Thus, the threshold of olfactory
perception of toluene, 1.5 mg/m3, was found to be slightly below the threshold
given by N. V. Lazarev (2 mg/m3).
The determination of the threshold of olfactory perception of meta-xylene
was performed on 18 persons. Six concentrations were studied, and a total of
431 observations were made. The results of the experiment are shown in Table
3. It is evident from the latter that the minimum perceptible concentration of
meta-xylene for the foremost sensitive subject is 0.6 mg/m3, and the maximum
imperceptible concentration is 0.41 mg/m3. Hence, the threshold of olfactory
perception of meta-xylene (0.6 mg/m3) practically coincides with the threshold
for the ortho and meta isomers of xylene (0.73 mg/m3) obtained by Gh'en Yun-t'ai,
Thus, a lowering of the thresholds of olfactory perception was observed
from benzene to xylene. The increase in the number of methyl groups in the
benzene ring probably causes an intensification of the odor.
The next stage of the comparison of the toxicity of benzene, toluene, and
xylene were studies of the effect of their minimum concentrations on the elec-
trical activity of the cerebral cortex.
The rhythm of the electric potentials of the brain constitutes an elec-
tric manifestation of the processes occurring in different regions of/the
- 116 -
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cortex of the cerebral hemispheres and other formations of the central
nervous .system.
Table 3
Results of Determination of the Threshold
of Olfactory Perception of Xylene Vapors.
Number of
Subjects
2
3
5
4
4
Concentration of Xylene, mg/m'
Minimum
'erceptible
1,9
1,4
1,0
0,85
0,6
Maximum
Imperceptible
1,4
1,0
0,85
0,6
0,41
K. A. Bushtuyeva, Ye. F. Polezhayev, and A. D. Semenenko (1960) were
the first to apply the method of functional electroencephalography to the
study of the reflex effect of minimum concentrations of chemical agents.
They found that concentrations which are below the threshold of olfactory
perception and which as such do not produce any visible effect, can affect
the electrical activity of the cerebral cortex.
At the present time, several modifications of functional electroenceph-
alography are used to study the effect of minimum concentrations of chemical
substances on the central nervous system. In order to study the'effect of
odor subthreshold concentrations of benzene, toluene, and xylene, we chose
the method of quantitative analysis of the reflex response of the alpha
rhythm flare-up (A. D. Semenenko, 1963), which in our view better reflects
the dynamics of the processes occurring in the cerebral cortex. The method
is based on the phenomenon of reinforcement of the electric potentials by
various rhythmic stimuli whose frequency corresponds to the intrinsic rhythm
of the brain. Stimulation in rhythm with the brain potentials (trigger
stimulation) was proposed in 1949 by Walter and Shipton. N. P. Bekhtereva
and V. V. Usov (I960) noted that trigger stimulation may considerably rein-
force the alpha rhythm and produces a greater effectiveness of the functional
load.
In our studies, the stimulation was carried out by using a rhythmic
light whose intensity was changed every 5 seconds in order to increase the
functional load on the central nervous system. The study of the effect of
low concentrations of the compounds studied on the electrical activity of the
cerebral cortex was started after developing a synchronous and well-defined
alpha rhythm in the subject. The experiments were performed in a specially
equipped, shielded, and faintly illuminated chamber. The subject was in a
semireclining posture in a soft, easy chair. In front of his face was placed
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a cylinder through which pure air was supplied at a rate of 30 1/min, and at
the desired moment, this air was combined with vapors of the investigated com-
pound in a given concentration. The device for adjusting the supply of vapors,
the electroencephalograph, and the instruments controlling the stimulators
were located outside the chamber.
The study of the effect of each concentration consisted of 10 observa-
tions representing the alternation of the recording of biocurrents during in-
halation of the compound in a given concentration (five observations) and
during inhalation of pure air (five observations). A single observation con-
sisted of 18 one-minute cycles, each of which included the application of a
sound stimulus - 10 seconds, waiting for the light - 7 seconds, application
of photic stimulus - 18 seconds, and 25 seconds of active limbering up, done
by the subject in order to produce a stable rhythmic stereotype and to main-
tain the general tone.
The eighteen minutes of the experiment consisted of three minutes of
training, followed by three minutes forming the background for the given
observation, with the remaining 12 minutes devoted to the experiment proper.
During the administration of the compound, the "gas" was supplied for 6
minutes, and the recovery period lasted 6 minutes. The gas-air mixture was
supplied intermittently, beginning immediately before the sound was turned
on and ending when the photic stimulus was turned off.
Analysis of the recording of the biocurrents was based on the integrated
energy of the reinforced rhythm. The results of the tests were treated sta-
tistically and involved the determination of the confidence factor of the
changes obtained.
The reflex effect of benzene in concentrations of imperceptible odor on
the electrical activity of the cerebral cortex was studied on 5 subjects most
sensitive to the odor threshold. The biopotentials were recorded on a 16-
channel electroencephalograph of the Galileo Co. The biocurrents were taken
off the temporal, occipital, and frontal regions and their combinations,
bipolarly. A benzene concentration of 2 mg/m^ caused a reinforcement of the
electric potentials from the left temporo^occipital regions of the brain.
The dynamics of these changes can be followed from the data of Table.4.
It is evident from Table 4 that statistically significant changes of
electric potentials appear in the 3rd-4th minute of supply of the gas-air
mixture. The reliability of these changes increases by the 5th-6th minute
of supply of the gas-air mixture and disappears by the 5th-6th minute of
the recovery period. No significant changes were detected in the statis-
tical treatment of recordings from other regions of the brain. Inhalation
of benzene in a concentration of 1.5 mg/m^ by the subjects did not cause'aay
significant changes in the biopotentials of the brain.
- 118 -
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Table 4
Significance of Changes in the Electric Potentials of the Brain
During Inhalation of Benzene in a Concentration of 2 og/n?
Subject
A. S.
K. S.
II. Z.
N. V..
S. L.
Gas-Air Mixture
Minutes
1-2
0
0
0
0 -
0
3-4
b
a
a
c
b
5—6
C
C
b
o
c
Recovery Period
Minutes
1-2
a
o
0
b
c
3-4
a
0 '
c
b
c
5-6
O
0
0
O
0
Note. Degree of significance: a - 99$; b - 99$; c - 99.9$;
o - insignificant.
The change in the electric potentials of the brain in subject N. during
inhalation of different benzene concentrations is shown in Fig. 1.
/ Z 3 I S 6 7 8 9 10 11 12 13
15
Fig. 1. Change in the electric potentials of the
brain of subject N. during inhalation of various
benzene concentrations.
1 - pure air; 2 - concentration 2 rtg/m'; 3 - concentration
1.5
- 119 -
-------
The reflex effect of odor subthreshold concentrations of toluene on the
electrical activity of the cerebral cortex was studied on subjects with the
most sensitive odor threshold. The biocurrents were recorded from the fronto-
central and temporo-occipital regions. In a concentration of 1 mg/m3, toluene
caused a distinct, statistically significant reinforcement of the electric
potentials from the left fronto-occipital regions of the brain in all the sub-
jects. The reliability of these changes increased by the 5th-6th minute of
supply of the gas-air mixture and remained until the end of the recovery
period (Table 5).
In the remaining recordings, no changes in the electric potentials were
found during inhalation of 1 mg/m.3 of toluene and none were detected in any
recordings during inhalation of 0.6 mg/m3.
Table 5
Reliability of Changes in the Electric Potentials of the Brain
During Inhalation of Toluene in a Concentration of 1 ng/m3.
Subject
.
V. S.
N. V.
P. N.
S. L.
Gas_Air Mixture
Minutes
1—2
b
b
0
a
3-4
O
O
c
a
5-3
O
c
b
a
Recovery Period
Minutes
1—2
c
b
c
a
3—4
a
c
b
a
5-tt
O
C
a
c
Note. Degree of significances a - 99#J b - 99#5 c - 99.9$
o - insignificant.
no
105
wo
15
*-r*
• t 3 4 S e 7 8 9 tO' 11 12 13 « 15
Bin.
Fig. 2, Change in the electric potentials of the brain
of subject N. during_inhalation of different concentra-
tions of toluene.
1 - pure air; 2 - concentration 1 mg/m^; 3-0.6 ng/m^
- 120 -
-------
Data on the change in the electric potentials of the brain of subject N.
during inhalation of different concentrations of toluene are presented in Fig,
2. The reflex effect of xylene was also studied on 4 subjects with the most
sensitive odor threshold. The biocurrents were recorded from the fronto-
occipital region. The first xylene concentration studied, 0.32 mg/m3, caused
a distinct, statistically significant decrease of the electric potentials
from both fronto-occipital regions of the brain in all the subjects. These
changes were more pronounced in the left fronto-occipital region. The de-
gree of significance increased by the 5th-6th minute of supply of the gas-air
mixture and, without decreasing, was preserved until the end of the recovery
period (Table 6). A xylene concentration of 0.21 mg/m3 caused no changes
whatsoever. The change in the electric potentials of the brain of subject
N. during inhalation of different xylene concentrations is shown in Fig. 3.
Table 6
Significance of Changes in the Electric Potentials of the Brain
During Inhalation of Xylene in a Concentration of 0.32 fflg/ffl'.
Subject
A. K.
N. V.
PN •
S. L.
Gas-Air Mixture
Minutes
1-2
O
O
B
• b '
3-4
a
b
o
a
5-6
0
c
b
a
Recovery Period
Minutes
1-2
b
b
c
b
3-4 | 5-6
a
c
b
b
o
c
c
b
Note. Degree of significances a - 95$; b - 99& c - 99.9$;
o - insignificant.
.7.
105
100
35
SO
,
.
\ j*.
' AC^
-
t ,f
— — Xylene *
_^_ .•5*^1-
?ssr^r^s
\
"--x
••
3
f— _^ y^ .^^^^C
^T ^^V^^ **i
^yf^,,^ v
A /- — ,
V
i • i i i i
t i 3 t S 6 J 8 9 10 11 12 13 H 15
Kin.
Fig. !>, Change in the electric potentials of the
brain of subject N. during inhalation of different
concentrations of xylene.
1 - pure air; 2 - concentration 0.32 mg/»>5i 3 - con-
centration 0.21 rag/HP
- 121 -
-------
Table 7
Results of Study of the Threshold of Olfactory Perception and
Reflex Effect of Microconcentrations of Benzene, Toluene, and
Xylene on the Electrical Activity of the Brain.
Substance
izene
.uene
Lene
.Olfactory Perception
Reflex Effect on
Biocurrents of Brain
Concentration, mg/ra?
Threshold
2,8
1,5
0.6
Subthreshold
2,5
1.27
0.41
Threshold
2.0
1.0
0,32
Subthreshold
1,5
0.6
0,21 ..
Comparing the results of the studies (Table 7) and evaluating the charac-
ter of the action of microconcentrations of benzene, toluene and xylene on the
electrical activity of the cerebral cortex, one can reach the following con-
clusions:
1. The thresholds of olfactory perception are lowered as the number of
methyl groups in the benzene ring increases.
2. The magnitude of the threshold concentrations from the standpoint
of the effect on the electrical activity of the brain decreases from benzene
to xylene in proportion to the decrease of the thresholds of olfactory per-
ception.
3. Benzene and toluene reinforce the electric potentials; xylene has
the opposite effect, causing a marked depression of the electrical activity
of the cerebral cortex. The restoration of electrical activity of the brain
during the action of toluene and xylene takes place more slowly. «
4. Concentrations of 1.5 mg/m3 benzene, 0.6 mg/m3 toluene and 0.2 mg/m^
xylene are the Subthreshold ones for the effect on the electrical activity of
the brain and their odor is imperceptible; we therefore recommend them as the
highest single maximum permissible concentrations for atmospheric air. Our
proposal was examined and approved by the section on sanitary protection of
atmospheric air of the Ail-Union Problems Commission.
- 122 -
-------
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•AHOXHH n. K. npo6.ne.\ia ueiitpa u nepH(j>ep;ui B 4>H3iioj!oni» nepo-
" «ofi iBeaTe-ifaHociH. ropbKiift, 1935, crp. 162.
Ace T. B., BOJI -B. B. H ap. TpyAM -u 'MaTepita.ibi yKpajiHCKoro TO-
cyaapCTseHHoro HHCTHTyta pa6oKeu MeAKUintbt, 1926, -crp. It—55.
BaflKos B. >K. rnr. H can., 1953, cr-p. 3—8.'
DauKoe B. K. B KH.: npca.e.auHo aonycTHMbie xpHueiiTpauuit .win-
c«pepHbix 3arpH3neiiHH, 1964, e. 8,-crp. 127—138.
B a K a Ji e H H it K K. E. B KH.: Teaiicu 10-fi nayHHofi ceccmt Cacpi-
AOBCXoro nHCTHTyia ninieiibi ipyaa n npoifeccHOHaflbnoft naro.io-
FHH. CBCPAJIOBCK, 1960, crp. 59—60.
BaKMaH C. M. JIaCop. flb.io, 19S8, 5. 13—15:
BaxMan C. M. Bpa'i. ae.io, 1960, 11, 110—105.
BaJiaxoacK H i*i C. Ji. u B a .1 a XOBCKH fl H. C. Meto.ui x-i-
• MHMCCKoro ana.iina Kpoeii. M., 1953, crp. 723.
BcneHbKHH M. JI. 3/ieMCHTbi KOJiimecroeimofi OIICHKH (j>apMai:o.ici-
THiecxoro eip^eKTa. Hsjt. AH JlaTBiifloKOii CCP. Para, 1959.
BCCKOB C. fl. OCHOBU xiotmecKoii fexno.ioni'.H. M.. 1962.
BexTepesa H. -IT. H VCOB B. B. . TeaHCU AomiaAoe naymon
' KOHeppocn.ia-
BOB. fliicc. Kami. M-, 1952.
B op H COB a M. K- B KH.: npc.ic.ibtio AonycTuuue KonueRrpauici ai
• .jioobepHbix 3arpa3iieiiin"i,- 1960, B. 4, crp. 61—75.
ByuiTyesa K. A. FHrHeHti'iccKan oaestKa OKHOIOB cepw xaK atMOC-
epar, 1964.
- 129 -
-------
ByiuiycDa K. A. B KH.: npeae.ibno aonycruMbie KomittiTpamiu HT-
iiocijjcpnux 3arpfUHeii!!ii. M., 1961, crp. IIS—126.
By ui ryes a K. A. B «H.:-npeae.ibno aonycTHMwe KOiiucnrpa:aiii ar-
Moctpepnux aarpsQHCHKii. M.. 1960, B. 4, cip. 92—101.
ByuiTyesa K. A. .B KH.: Opeie-ibuo aonycuiMue KoaueiiTpamui ai-
Moclpepiiux 33rpH3iiei(iiii. M., 1962, B. VI.
ByuiTyesa K. A. B KH.: npe.ie.ibHo aonycrifMuc KOHneiiTpaun:i
aTMOCipepiibix sarpssHCHiifi. 1957. B. 3, cxp. 23—41.
ByuiiyeBa K. A., no.iex. K. B KH.: KpatKaa xtiMtiiecKan 3nu.iiKJione3.ua. M..
1961, T. I, cip. 90—91.
Bofinap 'A. O. Eiio^onmecKan ioiro.i. JKVDK. CCCP. 1954, 6, 691—696.
BoJib4>OBCKa H P. H. H " £1 a B W40B a T. H. CfiopmiK Ha\T-
•HHX pa6oy aa roflH OieHecTBeHHOH BOMHM. M., 1915, cxp. 155—159.
Bpeanwe iBemecrBa B npo.Mbiiii.ienHocTH. rioa pea. H. B. Jlasapeoa
JI., 1963, cTp. 90—114.
B a a o B A. M., B a r H o .B a M. J\., B a c H .1 b e B A. C., T1 y ui K H-
aa H. H. HTQiwaa MayqHan KoiicfiepeHmia MOCKOBCKOFO Haymo-
Hcoie^OBaTC-ibCKoro HHCTiiTyra KM. $. . SpiiCMaHa. 1961.
TeHAepceH, Xa rapa. B KH.: BpcjHbie rasu B npoMbiui.iciuio-
CTH. M.—JI.. 1960, crp. 109.
TCHCC C. F. HepanaH oncTe.\ia ti BHyrpeHHaji ceicpemts, 1955.
re4»Tep JI. H., UlyJibiMan E.'&., H o n H K. C ff.., n.n;ib-
Aiau H. H., SapeitKaa A. H. H K-iefin C. M. Tpy^u
BopOHeykCKoro .MeanmiHCKoro iiHCTiiTyta, 1935, T. V.
JiJieMA. H lilrepHE. S^cKrpOHHWc cneKrpw nor.nomcHiin
opranimecKiix coeaHHeHHfi. Ilep. c anrn. Flo.t pea. JI. A. B;UOMCK-
4>eJibAa. H3A- HiiocT-paHiiofi .niiTeparypM. M., 1957.
PCKHH E. JI. B KH.: Bonpocbi ninieiibi rpyma, npo4>naTOJiop!m H
flpOMblUMeHHOH HHTOKCIIKaUHH. CsepJ-lOBCK, 1959.
Tejib30H JI. 51., TepuiKOB fl. A. SpiitporpaMMbt KBK'MCTO.I
KniiHKiecKoro HccneaoBaHiw Kposii. H34. CHSupCKoro OT,ie.ieinm
AMH OGCP. KpacHoapcK. 1959.
aySep'MaH C. B. B tpvaax Bopone/Kcxoro iiea-ifUJWCKoro'n:i-
CTHTyta, 1957, T. XXIX, ctp. 31—34.
rojiiuHHCxaa M. T. j[3no.i. H
-------
FopuixoB C. H. O coapeMen.'iux jieio.i»x riinieiticiccKiix •HCC.IC.IO-
saiwii. M., 1964, crp. 9—24.
rocpMeK^ep B. A. Hir. n can.. I960. 4. 9—14.
I. cxpMex.iep B. A. B KII.: ripeje.iwio jionycTiiMuc KoimciiTpamiu
aiMoapepiibix sarpfoiteinifi. M., 1961, B. VIII.
Toui€B A. H. Bonp. MCA. .XIIMIIII. 1938, T. 4.
rpuroposa O. H. Bonp. oxp. Mai. n ACT. 1953, 10, 50, 55.
rpiiropOBa 0. ITT. 'Jexoc.iOBaiiKas iiejiiaTpim, 1957, 2, 233.
TpuropoBa O. FI., T KTO B T. H. Boup. oxp. Mar. 11 ACL, 1957.
1, 31, 34.
ipiiropoBa O. fl. Bonpocu oxp. jiar. H ACT., 1962, 9. 13, 17.
rpMuunyii Jl. Ji. Baibtiiiie 3O3mioii.iiut KPOBH n HX K.ninuiKO
HocrHsecKoe 3HaHeii(ie. M.. 1962.
TpoHc6epr E. Ul. XiiMii'iecKan npoMbiui-ienHOCTb, 1961, 7.
•I' y .1 b K o X. F. MaTopna.ibi caHinapHofi xapaKTepiiCTHKu np
CTBa Banaaiieoux coeAiiHcinu'i. ABTopei|>. Sana, nice., 1951.
FVCCB M. H. O 06.: ripeAe.ibiio AoiiyciiiMbie •KOimeiiTpamiu aiutoc-
(pepuux aarpaaiieHufc. M., 1960, crp. 7—38.
Tyces M. H. B «n.: lOpeae-ibHo aonycri»iue KOHueHtpamiii auMoc-
iJiepHux sarpaaHeHiifi. Meania. M., 1960, B. 4. crp. 26.
F y c e B M. H. H C M H p H o B K). H. B KII.: .Ilpe.aejibHo jonycriiMbife
KOHueHTpamm aTMoctbepHbix sarpasiieHiiii. M., -I960, B. 4, cip.
139-142
TVCCB M. H., Me.TiiK-aHO'B K. H. Fur. H can., 1963, 5, 3—8.
Jl n n T p H e B B. A- BIO.I.I. axcnep. 6110.1. n «ea., 1939, 8, 6.
ZI,MiiTp«eBa H. B. Jla6op. Ae.io, 1S63, 6.
Plryuoa A. A. B cS.: 'K.iHHiiKO-nirueHimecKiie iicc-ieiOBauns no TOK;
"cHieoKKM sewecTsaM, npiKMetifleuuM B IIOBUX npoiissoacTBax.
JI.. 1940, B. 2.
Crop OB A. n. Mop$OjioriiqecKiift aHa.nns xpoBit. Mcflrns. M., 1954.
E r o p o B K. B. Tpyaw 'AcrrpaxaHCKoro MeaimiiHCKoro IIHCTIITVT:I,
1958. T. 14. cip. 205.
EpeMHH E. n.. KacnapCKan 3. A. -BnoxiiMitn, 1950, T. 15, o. 2.
>Kryn II. B. JIa6op. zeno. .1962. 4. 23.
?K«yACxaH .P. M. -npeae-ibiio aonycrtiMue xonueHTpauHii SAOBH-
TUX napes, ra303 « nw.iH « aojayxe paooinx •noMeiueitiift, 1933,
B. 32, cxp. 52.
33Kp W€ BC« ii ft E. B., Bacn.ij>esa fl. T. JlroMimecueHTnaH
'MKKpocKonHa B K/iiiHiiKo-reMaToioni'iecKux iicc.ieflOBaHHH.x. Mea-
TH3. M.. 1963.
II-Ma mesa H. B. THI-. n can., 1963, 2, 3—8.
II UK OB HI A. A.. BiiHorpaaosa B. A. Tesncbt aox.iaaoB na-
VHHO& ceccnjt HoBocn6»ipoKOro !iayHHo-ncc.ie,ioBaTenbCKoro caim-
rapHOro .HiicrnTyTa 15—18 Maa"l966 r. HoBocii6npCK, 1956,
Kasac JI. H. Apxits o^Ta-iutaionni, 1925, T. 1, CTp. 505—526.
Ka.iaOyxoB H. H. y-cnexn cosp. 6110.1., 1940, 12. B. 1.
KaiieHCKiifi C. C. Mareptia.ibi A-I« (papMaKo.ionni aaero^enoHa.
/IHCC. AOKT. GFI6, 1889.
KanKaeo 3. A. Tur. H can., 1963. 12.
Koran A. X. Bro^n. 3Kcncp. 6110.1. H MCA.. 19=>9. 4, 8, 10. 109-
113
Koran H. 5. ri(Miip»rpa<|>u
-------
K o xc e B H ii K o B B. A., M e m e p c K ii A P. M. CoopeMciiHwc MCTO-
AM ana^.-raa 3.ieKTpo3Hue<}>a.iorpa.MM. Meanis'. At, 1953.
KO3.iOB H. B. ABTope4>e?aT anccepTamiH Ha coiicKanne yvenofi cie-
nemi aoKTopa MeAinumcKux nayx. Aliwcx. 1962.
K'ouoHOBa B. A., AKCCHOBO B. B. Far. 11 can., 1963, 7, 7—11.
KopGaxosa A. H., KpeMHesa C. H., Ky.ianiHa H. K.,
y Ji a n o B a H. n. B xn.: JIpoMMW.ieHHaH TOKCHKo.iornfl. Al
1960, crp. 232—238.
K o p o T K o B a KD. K. Teancw jox.ia.20B K>6iiJiefiHOH Haywofi cecc;i:i
HHCTHTyia ninieHM rpyaa H npotpsaSo-aeaaHiifi AMH CCCP, no-
• CBHIIieHHbte 40-.16THIO Be.lIIKOft OKTH6pbCKOfl COUHajIHCTiPieCKOH DC-
BOJIIOUHII, 16—21 Aek-a6pH. M. Ill, At, 1957.
K o p o T K o B a IO. K. BecTHiiK OTOp:uio.iap., :1959, 3, 69—72.
KoioTKOsa K). K. 'ripOMNui-isHHaa TOKCHKo-ioraa. M., I960, crp.
141—150. ,
KpaBKOB C. B. ii3;io.i. xypsr. CCCP, 1940, -28, 4, 313—322.
K p a c K H n a 'H. A., T y T p o a a H. At Tpyafai MOCKOSCKOTO HOVH-
BO-HCCflejosaTe-ibCKofo HiicrHTyra 3n;iaeM;io.ioniH w -MHSpo6iio-
JioriiH. HrM.\ryHo.ioniH n npo(pH.iaKTiiKa KHiuenHbix wnfieiaiHri. 1962,
B. 9, cip. 180—186.
KpacoBHUKan C. 9. Vcnexn cospeMeHKofi 6iio^oniH, 1951, 32. 1,
166—192. 23 (Zt H. UIvpurnH, C. 4>. Mvpqaxoaa, H. A. Beaos,
1957).'
KpacoBiiuKan M. JI., 3 a n o p o K e u T. C. B KH.: MaTepiia.iM
KOH(t)epeim;iii, nocBnmeHHOH BonpocaM ranieHu tpyj.i,
H npoMbiui.iemioH TOKCiiKO-ioraii IB Het})TsiHofi n
H TipoMNiu-ieHHOcTH 30 wan — 2 HIOHH 1961 r.
1961, crp. 88—91.
K). I"., BexTepeaaH. n.. TycejibKHKOB B. H..
KOJKCBHHKOB B. A., CennieHKOD B. T., VCOB B. B.
TexHHKa H MeroAiiKii 3JieKTpo3imc$aaorpa4>iiH. M-.—JI., 1963.
KpeiiHeoa C. H. H To^rcKaa At C. AlaTepiiajiH Hayniou cec-
CHH no TOKCHKO^OniH BblCOKOMO-ieKJMHpHKX COCflUHeHBH. 'At— .1..
196U cip. 60—61.
KyJiaKOB A. E. Tnr. a can., 1964, 4, 8—13.
K y Ji a K o B A. E. THF. H can., 1965, 5, 15—20.
KyuiaKOBCKiift O. C. JKypH. BWCUI. Heps. ACHT., 1954, 4, I.
137—140.
Kwpre K. X. npd6ji. 3HaoKpHHO.i. " ropMonoiep., 1956, 2, 4,
110—117.-
Jl a 3 a p e B H. B. OCHOBBI npoMbiinjjeHHofi TOKCHKO^OFHH, 1933.
JI as a pea H. B. HapKOTiiKit. JI., 1940, cxp. -188.
JT asa pen H. B. Bpeiiiwe BeutecTBa B npoMbiuuieHHOcru, 1934.
JI a 3 a p e B iH. B. BpeAiiue Bemecr-oa a npoMbiui.ieiiHOCTH, 1963.
JlapnoHOD JI. 4>. FHrHeiia rpyaa n TCXHIIKH oesonacnocTH, 1954. o.
JleBHH C. H. B Kit.: CBCpa-ioscKan oS-iacTKas KoiKJiepenuHfi ncspo-
uaTOJioroa, ncuxHarpoB n HeftpoxiipyproB, 1950.
JI e B H « a 3. H. CfiopmiK :pa6or TOKciiKO-ionmecKofi jia6opaTop;r-i
• iiHCTUTyxa furaeHu tpyaa n npo(p3a6o.ieBaHHfi, 1948, T. XII, Jli-
HHHrpaj.
JI e p H e p H. n., B p y c H .1 o B c K H ft E. C. A^JiepnmecKiie 30311110-
ipiuibHue 3a6o.iCBaBH5i. Kites, 1961.
JI H UI.3 H. B KH.: .npojeJibiio jonycTirxittc KOiiueinpau'iiH
HUX* 3arpH3iieii;iM. B. VII. At, 1963.
- 132 -
-------
Jin6.in)ia E. H. B KM.: 3>ap.\iaKo.iori!H ;r TOKcaxononm, 1950 13
3, crp. 33—37.
Jin 6.inn a E. H. B KII.: Hcc.ie,io3ati;m B oS-iacni npoMbitiiJieiiHoii
TOKCKKo.TOrmi. 1948. 12, 5, 74.
MatHHUKHfi A. H. Cy6opjninauiifl B HepBHOfi CHCTCMC it ee 3iia-
lemie B »3iiKa, 1956, T. 1.
awn. 3, crp. 262—274.
MepKOB JI. -M. 06iuan Teopun :i MeroaiiKa caHiiTapHO-CTaTticTH-
MecKoix) HCC-ieAOBattiia. Hsa. 2-e. M., 1963.
M H .1 n e p C. B. B co.: •npe.ie.ibuo jonyciiiMbie KoimeiiTpamtH HTMO-
oJiepHHx sarpHSHeinifi. Meanis, 1955.
Mnnaes A. A. C6op-iutK rpyaon KOH({)epe;miiii MaioAwx runieint-
CTOB ji camrrapHbtx Bpanefi. M., 1965.
M'HiHKHHa H. A. Tpyjw nayiHou ceccmt .leHiinrpa^CKoro Hayq:io-
HcaieAOBaTeJii>CKoro"irriCTiiTyTa nir. Tpyasi n npotJiaaOo^L-Baiinfi,
nocBnuiCHitofi ntoraM paOoTw aa 1956 r/Jl., 1958, cTp. 237—243.
" M H a u a K a « R K A. B. .B KM.: npeae.iwio aonyciiwiwe •Koi(ue:iTpau;ni
3arpa3HC!i;ifi. M., 1964, B. 8, crp. 89 — 118.
C. 'H. C6op:ii!K nayiuwx pafior MOCKOBCKDPO oo-
*acTHoro Haymio-HCMCAODaTe.ibCKoro caHiiTapHo-riiriiciiit'iecKof-,
MHCTHTyra. M.. 1948. T. 1, ctp. 82—124.
MyxHTOB B. M. Pur. n can.. .1962,6, 16—24.
MyXHToa B. M. B XH.: npMC.iuHO Aonycr.iMue KoimenTpai(;i»
aTMOC(j>epHux aarpHsiieunfi. B.. VII. M., 1963.
MbiTHHK II. ^. apMaKo.nonia n TOKciiKo.ionm. 1940, 3, 6, 39— 16.
HaBpouKJiii B. K. BCCTH. AMH CCCP, I960. 3, 57—67.
Haaapenxo B. A. Tpyaw CiioreoxiiMiiMecxoil -^a6opaTopii«i AH
CCCP, 1937. 4, 265—271.
HexoTOpwe npo6.ieMH ninieiibi tpjma a npo(pecciiOHa.ibHo8 nato-
JIOTHH. flop. pea. 3. •?. Mnpy3A3epa. Meano. M.. 1960.
HaKOJiaeiB H. M. COB. MCA. .1954. 18,4, 38.
HHOHTflJ»a M. B. B KH.: .Meroabi onpe.ic.ieilim BpeaHMx BemccTB
a BOSAyxe H apyntx cpe.iax. 4. I. M.. 1960, crp. 238—240.
HOBHKOB K). B.'B KII-: npe.w.ibuo ionycTHMbie xoimeHTpauHii at-
Moc4>epiiux sarpnsHenufi, 1957, *. 3, cip. 85—108.
OflouiaiJiBH-TH A. T. Tnr. H can., 1962, 4, 3—7.
OjniMnnesa O. H., PeTiiep B.-M., Pycnn oaa A. O. B KH.:
Bonpocu rariieuu tpyaa H npo^naTononia npH paeotax c oeuso-
jioy H ero roMO.iora.Mii. .HticTirryT yco9epuje»CT30Baiiiin apaiesi
KM. KHpoaa. Jl., 1958. B. 17. ctp. 8-22.
- 133-
-------
0.1 b ui a n c K n A iM. If. H Jl H x a i e B a B. B. Tpyau PocroaCKoro-
na-flony MeAiniimcKoro «iiCT;iTyra. 1935, 2, crp. 120—127.
O p .1 o B 'a A. A., M a 3 y H H H a T. H. COB. MOJ., I960, 9.
OCHOBW aiiAuKpiiiicjioriin. Floj pea. H. M. /Ipaaniina. M. . Me-
pexcuHCKoro. 1953.
n a B.I oe H. it rio-i.-ioe co6panne TpvjoB, 1949, T. 5, crp. 112—137
H crp. 142—153.
na.ibuea K). H. Fur. H can.. 1964, 2, 28—33.
OaxoMbmes A. >H. Fur. H can., 1960, 11, 77—84.
FI expos A. A. ycnexn XHMHII, 1953, 22, B. 8.
FleT-po-B B. H. Firr. H can., 1960, 2, 60—62.
nerpyflbKHii a A. M. -B KH.: HpaKTimecKan 6iioxiiMiin. M.,'1961,
crp. 120.
IlaoTHtucoBa M. M. B KH.: npeaenbHo jtonycruMbie KoimeiiTpa-
UHH aTMOc4>epHbi.x 3arpH3HeHHH. 'Yloa. pea. npoip. B. A. Pssaaosa,
1960, B. 4, crp. 75—91.
rioKpoBCKHH A. A. iBoeii. .Mea. KypR., 1953, 9, 61—65.
rioKpoBCKHfi B. A. ToKciiKo.ioni?! H runiciia npoiisBOjCTBa cmi-
TeTHiecKoro TcaywyKa. M., 1955.
II ox p o B c K H if B.* A. B r-pvjax 'BopOHe/KCKoro aeaimHHCKoro na-
CTHTyra, ,1957, T. XXIX. ' ,x
FloJieraeB M. H., Anapeesa H. A. fHr. H can. 1959, 6, 73.
n o n o B B. A. B KH.: •MaTepua.ibi Bcecotoanoft KOHcfjepeHmiM -no canu-
Tapaofi o.xpane a-nMoc^epnoro soa^yxa. M., 1964, crp. 32—33.
ITpaBiAMB H. C. O KOM6;iHHpOBaHHOM A6HCTBHH fliOB, 1929.
Jl peate-HeHC KH ft B. E. B KH.: PyKosoacTBo no KJiiiHimecKifM na.-
6oparopHbiM AcoiejiosaHiinM. M., .1960, crp. 249,
IlpoKoneHKO T. A. .B KH.: Bonpocu ninieHu Tpyaa, npo(J)naTOr
.loniH H npoM. TOKCiiKo.ionii(, Ceep^JioacK, 1958, T. 2,
crp. 244—254.
riviiiKHHa -H. H. £ KH.:'BHOXHMHiecKite-MeTOAbi HCCJieAOBaitiiH. M.,
" 1963, crp. 13.
PaccKaaosaT. B. Tpyaw OjeccKoro MCAHKHiicKoro iiHCTHTyra
MM. H. H. :IlHporo3a. 1958, «. 3, crp. 113—119.
Pe3HHK 3. B. Bpati. aeao, 1954, 7, 627—631.
P H n n T. X. TpvAbi OMCKOTO MeanmiHCKoro HHCTHryTa, K« 48. OMCK,
-1963.
Punn F. X.. MaTepna.ib: • .BcecorosHofi -KonipepeHiiiiii -no caHnra-pnoH
oxpane aiMoopepHoro sosayxa, 1964.
P o x K o B a B. M. FlaTo.iorHH, K.iHHHKa H TepanHH orpaB/ieHHfi.
/luce. JL. 4948.
POSCHUBIIT F. 3. Pe
-------
Pfl3HOB B. A. PVKOSCUCTBO no KOMMyna.ibHoii niniCHe, 1961 T. 1.
cip. 158—159. "
P H 3 a ii o B B. A. Fur. 11 can., 1949. 5, 3.
PH3anoB B. A. CamiTapnas oxpaiia aT.\iocn.iiia.ia AKaaeMim .Ha-
yK CCCP, ypa.ibCKiifl rocyaapCTBeHHbift ymiBepciiTeT, 1964.
Ce.iHHXjiii^ K. H. Tirr. it can., 1961, 10, 6—12.
CC-MC HC n« o A. A. .Pur. H can., 1961, 5, 3.
CeMCHe.HKO A. fl. Tiir. n can., 1963. 7, 49.
CeMCHCiHKO A. ZL. B iMarepiia-nax XIX MOCKOBCKOI'I naymio-npaK-
THHecKofi >KOKd>epenmiH no npo6.ieM3.\i npoMbiuj.ieuHafi niniciiu.
M.. 1S63.
CeMCHCHKO A. R. riepsan nooo-i/KCKasi naynian KOH(J)epeHmra ru-
rHCHKcroB. •Kyri6uiiieB, 1963, cip. 142—144!
CeueneHKO A. fl. Pur. H can., 1963, 7, 49.
CeMCH€«KO A. R. n Ba.iauiOB B. H. MaTepiia.nu KOK({)epeu-
UHH no HioraM Haynsibtx HCCJieaoBaiitifl 33 1963 r. HHCTirrvTa on-
mefl H KOMMyxaflbHOH nirHeHW HM. A. H. Cucntia AMH CCCP. M.,
1964, crp. 34—35.
C e p r e Ji b O. C.. K -i» M e H K o A. A. BCCTHHK peim-eHO-ionm H pa-
ano.norHH, 1957. 5, 76—81.
Ce p'e Hce H-P OH ui BH. £noxitMiiiecKiie aieroau Asinnioia n HC-
caeflOBaiiHfl. Mea. HJI iByxapecr, 1963. crp. 259.
C 64eii03 H. M^-IIaB.iOB H. n., BBeaeHCKiifi E. E. Oii-
3HOflOrH« .HepBHOfl CHCTOMU, 1952. CTp. 191.
C o K o Ji o D E. H. B <6.: OpiieHTHpoBomiwfl pe<|>.ieKC n opiieiiTiipo-
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meKHoft sonpocaM nirHeHH rpyaa, npo^naTo.ioniu n
aoft TOKCHKoaorHH B HeipTJinoa n -iieipTexnMjittecKof!
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46
47
THE SUSCEPTIBILITY OR RESISTANCE TO GAS
AND SMOKE OF VARIOUS ARBOREAL SPECIES
GROWN UNDER DIVERSE ENVIRONMENTAL
CONDITIONS IN A NUMBER OF INDUSTRIAL RE-
GIONS OF THE SOVIET UNION-A Survey of USSR
Air Pollution Literature
METEOROLOGICAL AND CHEMICAL ASPECTS
OF AIR POLLUTION; PROPAGATION AND DIS
PERSAL OF AIR POLLUTANTS IN A NUMBER OF
AREAS IN THE SOVIET UNION-A Survey of USSR
Air Pollution Literature
48 THE AGRICULTURAL REGIONS OF CHINA
EFFECTS OF METEOROLOGICAL CONDITIONS
AND RELIEF ON AIR POLLUTION; AIR CON-
TAMINANTS - THEIR CONCENTRATION
TRANSPORT, AND DISPERSAL- A Survey of USSR
Air Pollution Litereture
SO.
61.
AIR POLLUTION IN RELATION TO CERTAIN
ATMOSPHERIC AND METORO LOGI CAL
CONDITIONS' AND SOME OF THE METHODS
EMPLOYED IN THE SURVEY AND ANALYSIS
Of AIR POLLUTANTS- A Survey of USSR Air
Pollution Literature
MEASUREMENTS OF DISPERSAL AND
CONCENTRATION, IDENTIFICATION, AND
SANITARY EVALUATION OF VARIOUS AIR
POLLUTANTS, WITH SPECIAL REFERENCE TO
THE ENVIRONS OF ELECTRIC POWER PLANTS
AND FERROUS METALLURGICAL PLANTS
-A Survey of USSR Air Pollution Literature
53 GAS RESISTANCE OF PLANTS WITH SPECIAL
REFERENCE TO PLANT BIOCHEMISTRY AND TO
THE EFFECTS OF MINERAL NUTRITION A
Survey o< USSR Air Polution Literature
54 THE TOXIC COMPONENTS OF AUTOMOBILE
EXHAUST GASES: THEIR COMPOSITION UNDER
DIFFERENT OPERATING CONDITIONS, AND
METHODS OF REDUCING THEIR EMISSION - A
Survey of USSR Air Pollution Literature
55 A SECOND COMPILATION OF TECHNICAL
REPORTS ON THE BIOLOGICAL EFFECTS AND
THE PUBLIC HEALTH ASPECTS OF
ATMOSPHERIC POLLUTANTS - A Survey of USSR
Air Pollution Literature
66 TECHNICAL PAPERS FROM THE LENINGRAD
INTERNATIONAL SYMPOSIUM ON THE
METEOROLOGICAL ASPECTS OF ATMOSPHERIC
POLLUTION (PART I) - A Survey of USSR Air
Pollution Litereture
67 TECHNICAL PAPERS FROM THE LENINGRAD
INTERNATIONAL SYMPOSIUM ON THE
METEOROLOGICAL ASPECTS OF ATMOSPHERIC
POLLUTION (PART II) - A Survey o* USSR Air
Pollution Literature
58 TECHNICAL PAPERS FROM THE LENINGRAD
INTERNATIONAL AYMPOSIUM ON THE
METEOROLOGICAL ASPECTS OF ATMOSPHERIC
POLLUTION (PART III) - A Survey of USSR Air
Pollution Literature
52 A COMPILATION OF TECHNICAL REPORTS ON
THE BIOLOGICAL EFFECTS AND THE PUBLIC
HEALTH ASPECTS OF ATMOSPHERIC
POLLUTANTS - A Survey of USSR Air Pollution
Literature
59 A THIRD COMPILATION OF TECHNICAL
REPORTS ON THE BIOLOGICAL EFFECTS AND
THE PUBLIC HEALTH ASPECTS OF ATMOSPHER-
IC POLLUTANTS - A Survey of USSR Air Pollution
Litereture
Reprlnh from various periodical!:
A INTERNATIONAL COOPERATION IN CROP IMPROVEMENT
THROUGH THE UTILIZATION OF THE CONCEPT OF
AGROCUMATIC ANALOGUES
(the UM of Phenology, Meteorology and Geographical
latilude for Hie r\irpo»e» of Plant Introduction and *• Ex-
change of Impwed Plant Varieties B»tw»tn Voriow
Countriei.)
B SOME PRELIMINARY OBSERVATIONS OF PHENOUOGICAL
DATA AS A TOOL IN THE STUDY OF PHOTOPERIODIC
AND THERMAL REQUIREMENTS OF VARIOUS PLANT
MATERIAL
•C AGRO-CLIMATOLOGY AND CROP ECOLOGY OF THE
UKRAINE AND CUMATIC ANALOGUES IN NORTH
AMERICA
D AGRO-CLIMATOIOGY AND CROP ECOLOGY OF PALES-
TINE AND TKANSJORDAN AND CLIMATIC ANA-
LOGUES IN THE UNITED STATES
USSk-Som* Phyilcol and Agricultural ChoroctwUtiet of *•
DrovghtArea ondltiCltmoticAnalogvei ihould be ada>*»ed to the
American tnititute of Crop Ecology, 809 DaU
Drive, Silver Spring, Maryland 20910.
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