Trent R. Lewis, Mary O. Amdur,
              Martin D. Fritzhand,
               Kirby I. Campbell

         Division of Health Effects Research
        National Environmental Research Center
        Research Triangle Park, North Carolina
                  July 1972

The Aft series of reports is issued by the Environmental Protection
Agency to report the results of scientific and engineering studies,  and
information of general interest in the field of air pollution.   Information
presented in this series includes coverage of intramural activities
involving air pollution research and control technology and of coopera-
tive programs and studies conducted in conjunction with state and local
agencies, research institutes, and industrial organizations.  Copies of
AP reports are available free of charge to Federal employees,  current
contractors and grantees, and nonprofit organizations - as  supplies
permit - from the Air Pollution Technical Information Center,  Environ-
mental Protection Agency,  Research Triangle Park, North Carolina
                      Publication Number AP-111





         Sulfuric  Acid Mist Particle Size	    3
         Exposure Duration	    5

         Conclusions	    6

         Sulfuric  Acid Mist Particle Size	    7
         Sulfur-Containing Particulate Aerosols	    10
         Particulates in Combination with Sulfur Dioxide	    13
         Hygroscopic Properties of Sulfuric Acid	    15
         Conclusions	    16

         Detection by Subjects	    19
         Effects of Humidity, Acidity, and Particle Size on
         Subject  Response	    20
         Effects of Sulfuric Acid Mist on Respiratory
         Retention	    21
         Conclusions	    23
         Optical Chronaxy	    25
         Dark Adaptation	    26
         Continuous  Electroencephalography	    28

         Conditioned Reflexes	    28

         Conclusions	    29

SUMMARY	    31


    TOXICITY IN MAN	    33


                      LIST OF FIGURES

Figure                                                       Page
   1     Effect of Particle Size on Airway Response to
          1 mg/m  Zinc Ammonium Sulfate Aerosol	11

   2     Dose-Response  Curve of Zinc Ammonium Sulfate
          Aerosol for Different Particle Sizes	12

   3     Comparison of Irritant Potency of Sulfur Dioxide
          and Sulfur-Containing Particulates	13
                       LIST OF TABLES

 Table                                                        Page

   1      Optical Chronaxy on Ninth Minute of Exposure of
          One Subject to Test Atmospheres ..........    26

   2      Eye Sensitivity to Light in Three Subjects During
          Twentieth Minute  of Dark Adaptation  ........    27
         Number of Trials with H^SO^. Plus Light Required
          for Appearance of Conditioned Reflex  ........    30

         Threshold Aerosol Doses Required For  Sensory Response . 30


      Studies of atmospheric chemistry have shown that under certain

conditions sulfur dioxide can be converted to sulfuric  acid and particu-

late sulfates.  Additional aerometric and toxicologic research is needed

to supplement what little is known of the actual atmospheric concentra-

tions and chemical forms present under varying meteorological condi-

tions and of the toxicologic significance of this group of compounds

under different ecological conditions.  Most available animal data,

exclusive of data from mortality studies, relate to alterations of pul-

monary function during and following exposure to  sulfuric acid  and

particulate sulfates.   Although results of pulmonary function studies in

animals  exposed to these substances cannot be extrapolated  simply and

directly  to predict human responses, two significant toxicologic princi-

ples related to the assessment of health hazards presented by such air-

borne contaminants have been demonstrated.  First, data on mass con-

centration alone are  an insufficient basis on which to predict irritant

potency because particle size  plays  an important role in determining

the irritant potency of sulfur oxide particles (submicron particles being

more irritant).  Second, particulate oxidation products  of sulfur dioxide

are generally much more potent irritants than the sulfur dioxide gas

per se.

      Although sulfuric acid is known to be a much greater irritant than

sulfur dioxide to man,  the combined effect of particle size and  concen-

tration of sulfuric acid mist on exposed human subjects is still undeter-

mined.   Industrial- and episode-exposure studies  have implicated long-

and short-term exposure to sulfuric acid in the  etiology of acute and/or

chronic respiratory disease.   Russian scientists have studied the thres-

hold concentrations of oxides of sulfur that elicit certain sensory


     Other factors that affect the toxicity of particulate  oxidation pro-
ducts of sulfur dioxide,  such as retention, site of deposition, hygro-
scopic nature of the products, and the subject's breathing pattern are
Key Words:  toxicology, air pollution,  sulfuric acid, sulfates, human,
             animal,  respiratory,  neurosensory




                DECAY PRODUCTS


     Studies of atmospheric chemistry have shown that a portion of the
sulfur dioxide (302) emitted into the atmosphere undergoes oxidation,
leading, eventually, to the formation of sulfuric  acid (H  SO ) and sul-
fate particulates.  Studies in experimental toxicology demonstrate that
some of these particulate oxidation products have a greater irritant
potential than sulfur dioxide itself.  Clearly then, air quality standards
for sulfur dioxide should be  set with due  consideration for the potential
sulfur dioxide has,  under certain atmospheric conditions, for forming
its more hazardous derivatives.
     The acute air pollution episodes that occurred in Donora,  Penn-
sylvania, and London,  England, precipitated intensive research into
the toxicological and physiological effects of sulfur dioxide and  certain
of its decay products.  In spite of this research,  most of which was
conducted between,1950 and  1963,  many questions regarding health
problems related to atmospheric oxides of sulfur remain unanswered.
To consolidate the information that is available,  this report presents a
review of the literature on the  toxicology of some of the  sulfur dioxide
decay products, and, in one  section, presents recent data on the com-
parative toxicity of those decay products.


     Experimental studies that use mortality and pathology as the
criteria of response by necessity involve the exposure of animals to
pollutant concentrations far above those occurring in the atmosphere
in order to produce measurable effects.  Data from such studies cannot,
without some misgivings, be extrapolated to give practical guidance in
setting safe levels for those air pollutants.  Such studies, however, are
usually the initial step in investigating the toxicity of a substance.
     Considerable species differences  have been observed in the
response of small laboratory animals exposed to sulfuric acid mist.
Treon  et al.   exposed small numbers of several species to H2SO4 mist,
95 percent of the particles of -which were smaller than 2 micrometers
(jim) in diameter. Using concentrations ranging from 87 to 1600 milli-
grams per cubic meter (mg/m3), they found increasing sensitivity as
shown:  rabbits  < rats < mice < guinea pigs.   Guinea pigs died of expo-
sures not lethal for other species; when exposed to concentrations sub-
lethal to them, they had proportionately greater  respiratory irritation
and lung pathology than the other species studied.  The results of
Mathur^ also corroborated that guinea pigs  are  less resistant than mice
to sulfuric acid  mist.
Sulfuric Acid Mist Particle Size
     Questioning the influence of aerosol particle size on response,
Amdur et al. ^ began by determining the 8-hour  LCso* of solfuric acid
particles with a mass median diameter (MMD) of 1 (im.  The LC5Q was
18 mg/m3 for 1- to 2-month-old guinea pigs and 50 mg/m3 for 18-month-
old animals. In subsequent studies,  sulfuric acid mists  of 2.7-(j.m-MMD
particles proved more toxic than those  of 0.8-fi.m-MMD  particles and
*LC50 is the concentration lethal for 50 percent of the experimental

the smaller material was more toxic at 0° C than at room temperature. *
These data indicate that the age of the animal, particle size, and tem-
perature all influence lethality.
     Other variables have been found to influence the toxicity of sulfuric
acid:  the presence of smoke^  resulted in increased mortality and the
presence of aerosolized ammonium carbonate-* diminished the effects of
the acid.  The increase in mortality was attributed to the larger size of
the toxic  particles, which resulted from the adsorption of the acid, upon
the smoke particles.  The decrease in effects was attributed to  chemical
neutralization of the sulfuric acid.
     Amdur et al.   and Pattle et al.  both examined the gross and
microscopic pathology  of the lungs of exposed guinea pigs and agreed
in their findings.  Animals that died after exposures to H2SO4 of less
than 2 hours had grossly distended and emphysematous lungs, but no
other serious lesions.   The cause  of death appeared to be asphyxia-
tion caused by bronchoconstriction and laryngeal spasm.  The lungs of
animals that died after longer  exposures had gross pathological lesions,
including capillary engorgement and hemorrhage.   '   It has been sug-
gested that these changes represent sequelae to the combined effects of
anoxia and increased intrathoracic pressure caused by bronchoconstric-
tion and laryngeal spasm.   The lungs of animals that survived  the
longer exposures showed spotty areas of old hemorrhage and some
areas of consolidation,  especially around the hilar regions.  Although
such exposures do not  give rise to death,  during or afterwards, the
damage is repaired only very  slowly.
      Amdur et al. 3 reported that, when the H2SO4 concentration was
8 mg/m3, extending the exposure time to 72 hours  did not increase
mortality beyond the amount observed in 8-hour exposures.  Pathologi-
cal examination of the lungs,  however, revealed that the longer  expo-
sures greatly increased the damage.  In the 8-hour exposures,  damage
was limited to thickening of the alveolar walls and consolidation of lung
tissue (CT*=64 mg/m3-hr). In animals exposed for 72 hours (CT=576
mg/m3-hr),  damage was greatly enhanced.  These workers postulated
*CT is the product of time and concentration.

4                                   TOXICOLOGY - S02 DECAY PRODUCTS

 that sulfuric acid mist has two distinct toxic actions.  It promotes
 laryngeal and bronchial  spasms,  causing immediate death by an action
 related primarily to concentration and individual sensitivity,  and it
 also produces parenchymal lung damage, an effect that is more  depend-
 ent upon total dosage.  Pattle et al. 5 pointed out that the  lung damage
 probably results from the direct irritant action of acid mist deposited
 on the lung tissue and that such deposition and  irritation may be the
 mechanism by which the mist causes death in animals more resistant
 to bronchial-laryngeal spasm than the guinea pig.  This mechanism
 may account, as well, for many of the marked differences in mortality
 observed by early investigators who used different exposure concen-
 Exposure Duration
       Thomas et al. " exposed guinea pigs to sulfuric acid mist for
 periods of 18 to  140 days.  They used three particle sizes (0. 6,  0. 9,
 and 9. 0 fim)  in concentrations  of 4 mg/m3  or less, except for occasional
 accidental higher exposures. ,The coarse particles produced lesions in
 the upper respiratory tract and slight edema in the larynx and trachea,
 with less mucus  in the major bronchi.   The mists composed of  0. 9-^m
 particles produced greater lung damage  than either the 0.6- or 9. 0-|o.m
 particles.  Exposure of  young  animals to 0. 9-nrn mists produced
 slightly edematous lungs and,  rarely, capillary hemorrhage.  Some
 increase in desquamated epithelial cells was observed in the minor
 but not in the major bronchi; the  epithelium appeared intact.  Major and
 minor bronchi both had less mucus than  controls.  The authors con-
 cluded that the guinea pig could tolerate  continuous exposure to levels
 of about 2 mg/m  sulfuric acid mist for  more than 3 months with only
 minor effects.
      Bushtueva  '  exposed guinea pigs to  2 mg/m  sulfuric acid mist
 of unspecified particle size for 5 days.   The animals developed edema
 and thickening of alveolar walls.  Exposures to 1 mg/m3  for 1 to 2
 •weeks produced slight catarrh in the mucous membranes  of the trachea
 and bronchi,  and slightly defined but widely distributed interstitial pro-
 liferation accompanied by round-lymphoid-cell infiltration around the

Toxicity in Experimental Animals                                           5

blood vessels and bronchi.   These latter changes seemed to be progres-
sive, for they were more highly developed 2 to 3 months after exposure.
A concentration of 0. 5 mg/m  produced only a slight "lung irritation. "

      Sulfuric acid is  a respiratory irritant, and, when present in
sufficient concentration, it will cause the death of  exposed animals.
The guinea pig is the  most sensitive  species among the common small
laboratory animals,  followed in decreasing  order by mice,  rats, and
rabbits.  The guinea pig's greater sensitivity probably results from its
greater susceptibility to bronchial and laryngeal spasm. Although  the
enhanced sensitivity makes  this animal useful for some studies of
the  irritant potential  of low concentrations  of sulfuric acid, the results
must be applied judiciously when extrapolation is made to the effects
 of sulfuric acid  air pollution on human health.  In all cases,  con-
 centration has been shown to be a more important factor in sulfuric
 acid-produced death than duration of exposure.
      Sulfuric acid also causes pathological lesions in the lungs after
much less than lethal exposures.  Effective, short exposures (up to
 72 hours) produce a degree of lung damage related to the arithmetic
product of concentration and time (CT) rather than to the concentration
alone.  Such damage  is slow to repair. The fact that concentrations
below 2 mg/m^  produce only minor lung damage after  extended expo-
 sures (about 3 months)  indicates that the CT-damage relationship
observed in 8- and 72-hour exposures cannot be extrapolated directly
to long-term effects of low  concentrations.
      Data  clearly indicate that the particle  size of sulfuric acid mist
is also an important factor  in determining its irritant  potency.  When
the  criterion was mortality resulting from an 8-hour exposure, a mist
of larger particles (2. 7 (im versus 0. 8 (im)  was more  toxic.  When
pathological lesions resulting from long-term exposures were the
criterion of response, the effect of 0. 9-fj.m  particles was greater than
that of either 0. 6- or 9. 0-|j.m particles.  The effect of particle size
will be  discussed further in the next  section.
                                      TOXICOLOGY - S02 DECAY PRODUCTS

      Methods like the ones discussed in this  section can be used to
evaluate the effects of low  concentrations of irritants that do not
usually produce lung damage or cause death.  In other words, they can
be used to examine the response of experimental animals to air pollu-
tants at concentrations known to occur in urban atmospheres.

Sulfuric Acid Mist Particle Size
      Employing unanesthetized and spontaneously breathing guinea
pigs, Amdur      measured the effect of sulfuric acid mist on pulmo-
nary function by the following parameters:  flow resistance of the lungs
and  airways; compliance (a measure of the elasticity of the lungs); tidal
volume (the volume of each breath);  respiratory frequency; and minute
volume (tidal volume times respiratory rate).  These data also per-
mitted her to make rough estimates  of elastic-resistive work and the
total work of breathing.
      Amdur used concentrations ranging from 2 to 40 mg/m  and
particles of 0.8,  2.5, and  7.0 ^m (MMD).  Experimental results
indicated that the  size of particles in the aerosol is critical to both the
nature and degree of the irritant response elicited.   Results  of these
experiments made clear that data obtained from exposure to high con-
centrations could  not have  been extrapolated to indicate,  in a valid
manner,  the reaction to lower concentrations ,  since irritant potency
varied with both concentration and particle size of the sulfuric  acid
mist.  This fact underlines the need for,  and the importance of,
methods that can directly assess responses to low concentrations of
      The 7. 0-fim-MMD particles, at a concentration of 30 mg/m ,
produced a statistically significant increase in flow resistance  but no
other detectable changes in respiration.  Few particles of this  size
would be  expected  to penetrate beyond the nasal passages, so that the
observed increase in flow resistance was probably restricted to the
upper respiratory tract, especially since the  other  parameters measured
were not  altered.
Toxicity in Experimental Animals

     The 0. 8-nm-MMD particles produced an increase in resistance,
accompanied by a proportionally lesser decrease in compliance, an
increase in elastic and resistive work,  and, hence,  an increase in the
total work of breathing.  These changes were statistically significant
at the lowest concentration tested (1. 9 mg/m^).  Onset of response was
prompt and resembled the pattern of changes observed after inhalation
of sulfur dioxide.  Changes in pulmonary function such as these are
consistent with narrow air passages caused by bronchoconstriction,
mucosal swelling,  or  increased mucosal secretion.

     The 2. 5-^m-MMD particles also increased flow resistance at all
concentrations examined (2. 3 to 44  mg/m-).  The relative toxicity of
the  0. 8- and 2. 5-|j.m-MMD particles, evaluated by increased pulmonary
flow resistance,  was highly dependent upon concentration; however, at
high concentrations, the larger particles elicited a much greater re-
sponse, whereas at low concentrations the smaller particles gave the
greater response,  again demonstrating that data obtained from  expo-
sures to high concentrations could not be used to predict accurately the
responses to low concentrations. The increased toxicity of 2.5-^m-
as compared with 0. 8-(j.m-MM-D particles at high concentrations con-
firmed the earlier finding of Pattle et al.

      Further comparison of data for the two particle sizes suggested
a difference in physiological action, presumably related to the  site
where the particles were deposited in the respiratory system.  Response
to the 2. 5-jim-MMD particles, but  not to the 0. 8-|J.m-MMD particles,
was slow to develop,   occurring only after 40 to 45 minutes of exposure.
This delay suggested  the possibility that the different responses were
mediated by different  mechanisms,  a possibility that was confirmed
both by the nature  of the  changes in the mechanical properties and by
the  post-mortem appearance  of the  lungs.

     The alterations in mechanical properties that occurred indicated
that large sulfuric  acid particles produced a complete obstruction  of
some air  passages,- whereas small  particles caused a narrowing of the
airways but not complete obstruction.  The gross pathology of the  lungs
                                     TOXICOLOGY - SO, DECAY PRODUCTS

was consistent with data derived from pulmonary function tests.  Ani-
mals exposed to the high concentration of 2. 5 -|J.m-MMD particles devel-
oped extensive areas of atelectasis, frequently involving whole  lobes.
In addition, their lungs  were edematous,  as demonstrated by a  signifi-
cant increase in the ratio of lung weight to body weight.  These findings
suggested that high concentrations of larger particles,  which probably
are deposited in the vicinity of the major  bronchi, produce severe
local irritation of the larger  air passages and cause swelling and
increased exudation of fluid,  which eventually lead to complete

      The pulmonary flow-resistance values were slow to return to
pre-exposure levels after the end of a 1-hour exposure.  Animals
exposed to  15 mg/m or more still showed elevated resistance  2  hours
after exposure had ended.  This slow return to normal has been found
consistently after exposure to irritant aerosols  or to the combination
of irritant gas and inert particulate material. '•'•' ^'  The reasonable
explanation for the slower recovery from exposure to an aerosol  as
opposed to  an irritant gas is  apparently the fact that the particles,
having been deposited on the  respiratory tract,  continue  to exert  their
irritant  effect.   The irritant  gases,  on the other hand,  are cleared
from the lung when exposure ceases, and the respiratory system
returns  to normal functioning rather rapidly unless the exposure  con-
centration has been extremely high.  The enhancement that results from
combining inert aerosols and irritant gases appears to be mediated
through  the formation of an irritant aerosol,  '    which could account
for the prolonged response to such  exposures.

      Lewis et al. ^> ^  studied the  effects of chronic H2SO4-aerosol
exposure on canine pulmonary function.  Animals were exposed for
621 days to 0.5-(xm-MMD HzSO4 particles,  at a  concentration of 0. 8
mg/m^,  and pulmonary function tests were performed  225 days and
621 days following the onset of exposure.  At 225 days, diffusion
capacity had decreased.  After 621  days of exposure, average carbon
monoxide diffusion capacity and residual lung volume were  reduced and
Toxicity in Experimental Animals

total pulmonary resistance was increased.  Post-mortem examination
showed that the net lung volume was reduced.

Sulfur-Containing Particulate Aerosols

      Amdur and Corn'-  studied the irritant potency of zinc ammonium
sulfate,  zinc  sulfate,  and ammonium sulfate, and found a difference in
the potencies as judged by increases in flow resistance  resulting from
exposure to 1 mg/m  concentrations having an MMD particle size of
0.29 him.
      Zinc sulfate exhibited about half the irritant potency of zinc
ammonium sulfate.  Ammonium sulfate was the least irritant of the
three compounds,  producing a response about one-third to one-fourth
that observed from exposure to an equal concentration of  zinc ammonium
      In further studies of the effect of particle  size on the irritant
potency of zinc ammonium sulfate, four MMD sizes were used:  0.29,
 0.51,  0.74,  and 1. 40 (J,m.  Concentrations  ranged from 0. 25 to 3. 60
mg/m  .  Figure 1 shows the results obtained when the concentration
 of aerosol was held constant at about 1 mg/m and the particle size was
varied.   Particle size by weight is indicated in micrometers.  This
method for calculating mass-size is explained by Amdur and Corn.
 From the curve it is evident that the irritant potency, as  judged by the
increase in flow resistance produced by the 1-hour exposure,  increased
 as the particle size decreased over the range of sizes examined.
Amdur and Corn1" concluded that the greater irritant potency of the
 smaller particles  could represent the reaction to an increased number
of pinpoint stimuli produced by the greater number of particles present
in the more finely dispersed material; to the differing depth of penetra-
tion into the peripheral areas of the lung; or to a combination of these

      From data available for several concentrations of zinc ammonium
sulfate at various particle sizes, Amdur and Corn prepared a family of
dose-response curves.  As Figure  2 shows, not only was  the response
                                     TOXICOLOGY - S02 DECAY PRODUCTS

  100 —
                                J	LJ
                   0.05     0.10             0.50     1.0
                       AEROSOL MEAN SIZE BY WEIGHT, micrometers
 Figure 1. Effect of particle size on airway response to 1 mg/m^ zinc ammonium sulfate
 aerosol. Numerals beside each point indicate number of animals.  Data and calculation
 of mean size by weight from Amdur and Corn.16

 to a given concentration greater as the particle  size decreased, but the

 slope  of the dose-response curves was also steeper.   This means that

 a small increase in mass concentration produced a larger increment in

 biological response as the particle size decreased, re-emphasizing

 the fact that if  a compound is dispersed as particulate material a

 measure  of mass concentration alone is not sufficient to  permit useful

 assessment of  its irritant potency.

      Nadel et al.l'' studied the effect of aerosols of histamine and of

 zinc ammonium sulfate on anesthetized,  artificially ventilated cats.

 The aerosols were submicrometer in size and the concentrations of

 zinc ammonium sulfate were 40 to 50 mg/m  .  The sulfate aerosol

 produced a physiological response similar to that produced by hista-

 mine,  but lesser in degree.  The response to a  3-minute inhalation

 included increased pulmonary resistance, decreased pulmonary com-

 pliance, and increased end-expiratory transpulmonary pressure. As
Toxicity in Experimental Animals

a  60
                         1.2     1.6     2.0     2.4

                            ZINC AMMONIUM SULFATE, mg/m3
                                                                   3.6  4.0
 Figure 2.  Dose-response curve of zinc ammonium sulfate aerosol for different particle
 sizes. Numerals beside each point indicate number of animals.  Data from Amdur and
 correlation with the physiological studies,  the authors made anatomical

 studies after rapidly freezing the lungs in the open chest.  These showed

 that bronchioles up to 400 (im were narrower in exposed than in control

 animals and that the mucosa was longitudinally furrowed.  Bronchi and

 bronchioles larger than 400 ^im were not significantly different from

 controls.  The authors noted the  similarity between the action of hista-

 mine and of zinc ammonium sulfate aerosol.  In regard to extrapolating

 the results they concluded,  "It remains to be demonstrated that acute

 changes reported here at aerosol concentrations of 40 to 50 mg/m  are

exaggerated but similar in nature to those produced, if any,  at lower

concentrations1'.    Follow-up work is clearly needed in this field.

      In the course of examining the effect of aerosols on response to

sulfur dioxide,  Amdur and Underbill18 examined the irritant potency

of several other sulfates.  They reported that 1 mg/m3 ferric sulfate
                                      TOXICOLOGY - S02 DECAY PRODUCTS

 [^62(304)3] produced a 77-percent increase in flow resistance during

 a 1-hour  exposure, which would definitely indicate that this substance

 is an irritant. On the other hand,  ferrous sulfate (FeSO4) and manganese

 sulfate  (MnSO/j)  at the same concentration produced no detectable

 alteration in resistance.  Not all sulfates, then,  are irritant in nature,

 and the sulfate ion per se is not known to  have irritant potency.

 Particulates  in Combination with Sulfur Dioxide

       Because data from atmospheric chemistry  studies have indicated

 that sulfuric acid and particulate sulfates  are formed from sulfur

 dioxide emitted to the atmosphere, it is essential that the comparative

 irritant potency  of sulfur dioxide gas and  its  chemical reaction pro-

 ducts be examined.  A long-term research program undertaken by

 Amdur  et al.  has provided data on the effects of  sulfur dioxide gas and

 several particulate oxidation products on the  guinea pig.   The authors

 are using the  same physiological techniques for all the pollutants, which

 permits direct comparison of the irritant  potency of these compounds.

 Their data   are summarized in Figure 3.



   !  50
  UJ »,
  ^t 20

7 -
     0.01    0,02      0.05     0.1     0.2       0.5

                          SULFUR EQUIVALENTS,

                           SULFUR DIOXIDE, ppm
 Figure 3 . Comparison of irritant potency of sulfur dioxide and sulfur-containing particulates.
 Numerals beside each point indicate number of animals.  Data from Amdur.19
Toxicity in Experimental Animals

     Even a casual glance indicates clearly that an equivalent amount
of sulfur present as sulfur dioxide gas produces  a lesser irritant
response than either  the particulate sulfates or sulfuric acid.  One
part per million of SO  is the equivalent of  1. 3 mg S/m3.   When pre-
sent as sulfur dioxide gas,  this amount of sulfur increases flow
resistance by about 15 percent.  If,  through reaction in the atmosphere,
this  sulfur equivalent were  converted to 0. 7-|j.m-MMD sulfuric acid
mist, it could produce an increase  in resistance of  about 60 percent,
a fourfold potentiation of irritant response.   If, on the other hand, that
same amount of sulfur were converted to zinc ammonium  sulfate parti-
cles of 0. 3-|j.m MMD, resistance would increase about 300 percent, a
twentyfold  potentiation.  Such complete conversion in  the atmosphere
is of course highly unlikely, but an oxidation of 10 percent can occur
readily. Ten percent of  1 ppm sulfur dioxide has an equivalent sulfur
content of 0. 13 mg/mj,  and if this  were present as an irritant sulfate
with a particle size of 0. 3 |am,  it would increase flow resistance about
45 percent.  If the response to the particulate sulfate  and  the response
to the  remaining sulfur dioxide (which increased resistance about  15
percent) were purely additive, one  could estimate a total increase in
pulmonary flow resistance  of approximately 60 percent, an important
fourfold potentiation  in irritant response.  The response to such com-
binations is more than additive,  1">'-(J however, so that the overall irri-
tant  effect  •would  be even greater than these figures  indicate.   These
data clearly suggest  that a  toxicological response to sulfur dioxide gas
alone is not a sufficient basis for assessing its role in air pollution.

      The overall problem of the effect of inert aerosols on physiolog-
ical  responses to irritant gases  will not be  discussed  in detail here.
It has been reviewed  in the air quality criteria documents for  both sul-
fur oxides    and  particulate material,   and is discussed in a recent
                  23                       18
review by Amdur.    Amdur and Underhill    reported the results of a
study on the effects of various aerosols on the response to sulfur  dioxide
gas.  Their findings  indicated that the degree to  which the response to
sulfur dioxide was potentiated by an aerosol was determined by the
solubility of sulfur dioxide in the liquid droplet and  by the extent to
14                                   TOXICOLOGY - S02 DECAY PRODUCTS

which it was oxidized to sulfuric acid.  The  importance of solubility
was suggested by the  fact that the potentiating ability of aerosols of
sodium chloride,  potassium chloride, and ammonium thiocyanate could
be related, in a reasonable manner,  to the solubility of sulfur dioxide
in solutions of these salts.  Further  evidence for the role of solubility
came from the observation that none  of the various solid, dry aeroools
that were tested potentiated the response to sulfur dioxide.   These
included spectrographic carbon, activated coconut charcoal, iron oxide
fume, triphenyl phosphate, fly ash,  and manganese dioxide.  None of
these substances,  at concentrations of 8 mg/m  or above, produced  a
detectable effect on pulmonary flow resistance or potentiated response
to levels of sulfur dioxide  ranging from  1 to 100 ppm. In contrast,
aerosols of soluble salts of ferrous iron, manganese, or vanadium at
concentrations of 0. 7 to 1 mg/m  potentiated response to sulfur dioxide
about threefold.  At significant humidity levels like those in the respi-
ratory tract,  these substances would not only form droplets,  but would
also absorb sulfur dioxide; they are known to be agents that favor the
oxidation of sulfur dioxide  to the more potent sulfuric acid.  The pro-
perties of  these and other  sulfur compounds have been studied thoroughly
by many researchers, who have found that the toxicity of the compounds
is directly related to  the metal ion itself rather than  to the attached
sulfur radical.  These compounds will not be reviewed here,  but a
relevant bibliography is appended.

Hygroscopic Properties of Sulfuric Acid

      Another toxicologic aspect of sulfuric acid aerosol inhalation is
related to  its  hygroscopic nature.  Even when sulfuric acid  particles
are inhaled in relatively dry air, they will expand within the respiratory
system by water absorption because inspired ambient air becomes satu-
rated with  water drawn through  the nose, mouth,  and pharynx.  Since
large particles are retained with greater efficiency, more hygroscopic
particles,  such as sulfuric acid and water-soluble sulfate aerosols, are
deposited than non-hygroscopic  materials of the same size.  Depending
on other factors,  physiologic response may, but need not,  be enhanced
Toxicity in Experimental Animals                                          15

 in proportion to the increase in percentage retained because of expan-
 sion in situ.
      In the case of hygroscopic aerosols, the original size of the
 parent particle will also determine the subsequent size of the hydrated
 particle,  and, hence, the site of its retention along the respiratory
 system.  Larger particles will have a tendency to be trapped along the
 upper respiratory airways (nasopharnygeal), where their relative
 toxicity is usually lessened and from which removal is more  rapid.
 Increases in particulate  size from the nares  to the alveoli are difficult
 to quantitate in vivo; therefore, studies of the effects of hygroscopic
 particles have been limited largely to empirical models, in which
 alterations in,  or potentiation of, physiologic responses are related to
 variations in physicochemical properties of the particulate matter; for
 example,  size, chemical composition,  solubility,  catalytic converti-
 bility, and moisture absorbability.
      When interpreting physiologic  responses to aerosols of  a sulfurous
 nature, assessing their toxic potential,  and relating aerosols to models
 of respiratory deposition, one must not lose  sight of the fact that these
 phenomena are influenced by the hygroscopic nature of the particles.

      The studies discussed above cannot be extrapolated simply and
 directly to predict the human response to particular substances.  They
 do provide useful information regarding the nature of mechanisms  by
 which effects are produced; however,  they may lend more insight into
 responses of the sensitive segment rather than of the robust healthy
 segment  of the population.
      For practical considerations in using these experiments as indi-
 rect guidance in air pollution research, two critical points emerge.
 The first is the extreme importance of the particle size  to the irritant
 potency of particulate products of sulfur dioxide.  The most important
 size is in the submicron range.   Data on mass concentration alone pro-
 vide an insufficient basis  for predicting irritant potency  of particulate
 products  of sulfur dioxide.

16                                   TOXICOLOGY - S02 DECAY PRODUCTS

      The second important point is that the particulate oxidation pro-

ducts of sulfur dioxide generally have much greater irritant potency

than the parent sulfur dioxide gas,  as indicated by the comparative

data in Figure 3 as well as by increased response to sulfur dioxide

in the presence of particulate material known to dissolve the gas and/

or catalyze its conversion to sulfuric acid  or sulfates.  Particle sizes

of oxidative products of sulfur dioxide are, in part,  affected by the

hygroscopic properties of the products; consequently, physiologic

responses and pulmonary deposition of inhaled particulates may be

significantly altered by moisture in the atmosphere  and within the

respiratory tract.  Atmospheric levels of sulfur  dioxide and their

health implications  should therefore be assessed in terms of the

potential for formation of more  irritant substances.  To do otherwise

is to miss the toxicological point and seriously becloud important
Toxicity in Experimental Animals                                          17

                     TOXICITY IN  MAN

      Only limited research has been done on the effects of sulfuric acid

on human pulmonary function,  and none on the effects on man of the

various sulfates that have irritant potency for the guinea pig.  Studies

that regard both physiological methodology and characterization of the

aerosol particle size with meticulous attention are badly needed. The

exposure  of human volunteers to low concentrations of H2SC>4 mist, to

define its toxicological effects,  has involved two divergent, though

complementary, branches of research.  One  branch has concerned

itself with the effects of H^SCJj aerosols upon the normal physiological

responses of the central nervous  system:  the role of H2SO4 mist and

its interrelationship with sensory modalities  and cerebral cortical

function.  These studies are reported exclusively in the Russian litera-

ture.   The other branch of research has been concerned primarily with

the irritative effects  of H2SO4 aerosols and the changes in respiratory

function that occur subsequent to exposure to concentrations that

approximate  the allowable industrial standards  set by the governments

of the United States24'25 and the Union of Soviet Socialist Republics. 26



Detection  by  Subjects

     In 1913, Dorsch   studied workers  in the  battery room of a

German telephone exchange. He found  that H2SO4 mist was only

rarely detected by the employees  when  the concentration was below

0.5 mg/m3.  They complained of  slight nose  and throat irritation at

2. 0 mg/m3,  distinct  discomfort at 3 to 4 mg/m3, and marked discom-
                                                               ? Q

fort when the concentration rose to  6. 8 mg H2SO4/m3 .  Bushtueva

exposed ten volunteers  to H2SO4 aerosols with the  following results.

When exposed to concentrations less than 1. 1 mg/m3,  the volunteers

compained of ticklish,  scratchy throats; when exposed to levels  rang-

ing from 1. 1 to 2. 4 mg/m3,  all subjects  noticed considerable

irritation at the base of the esophagus and 40 percent of them experi-
enced some irritation of the eye mucosa.   When the aerosol level was
raised to 2. 4 to 6. 0 mg/m3, all subjects  experienced acute  irritation
of the mucous membranes, a pronounced  reflex cough, and irritation
of the eye mucosa.  The threshold concentration was approximately
0. 7 mg/m3 (0. 60 to 0. 85 mg/m3).  In that study,  the particle  size  of
the acid mist was not stated. Amdur2° was unable to detect responses
in subjects exposed to aerosol levels less than 1 mg/m3, using odor,
taste, or irritancy as parameters,  although all 15 of her subjects
detected the aerosol when the level was raised to  3 mg/m ,  noting
that it "felt like breathing dusty air. "  Some  subjects found 5 mg/m3
very objectionable, but others less so,  and, for all  subjects, a deep
breath of this concentration usually produced coughing.  In another
study, Bushtueva3^ found  that the presence of 0. 4 mg H2SO4/m3. even
when combined with 1 mg  SO2/m ,  was below the  sensitivity threshold
of all subjects.  The fact that the results  reported in each of these
papers  are remarkably similar to Dorsch's^^ original description of
H2SO4 mist irritancy, indicates that the dose-response curve for
human beings has  remained quite stable during the past 60 years of
experimentation and,  in contrast to animal data,  appears to be
physiologically predictable in almost all instances.

Effects of Humidity, Acidity, and Particle  Size on Subject Response
      Sim   studied the response of healthy males, aged 18 to  46, to
r^SO,^ mist.  He noted that at a given concentration a mist of larger
particle size and lower acidity,  produced under conditions of high
humidity, was more  irritant than a mist produced under lower humid-
ity at the same  concentration, but with  smaller particles and higher
acidity.  Pulmonary air-flow resistance was 35 to 100 percent greater
than normal when the volunteers were exposed to  a  ION H2SC>4 aerosol
of 1-p.m-MMD particles at a concentration of 39 mg H2SC>4/m  ,  a
temperature of 18.4° C,  and humidity of 61 percent.  Altering the
aerosol to 25° C,  91 percent humidity,  acidity of  4N H2SO4, and
particle size of 1. 5 (xm evoked  intense  coughing,  lacrimation, and

20                                   TOXICOLOGY - SO, DECAY PRODUCTS

rhinorrhea,  which continued throughout the exposure.  Coughing
decreased somewhat after the first 10 minutes of exposure and airway
resistance ranged from 43  to 150 percent greater than normal.  Adding
magnesium oxide particulate to the exposure air did not alter the irri-
tancy, although addition of  ammonia toward the end of the H2SC>4
exposure resulted in rapid  and complete disappearance of all symptoms,
thus corroborating Pattle's5 observations  on guinea pigs.  Toyama33
also correlated airway resistance with particle size.  In an aerosol  of
4. 6-(im particles,  a. mist concentration of 0. 8 to 1.4 mg/m3 increased
airway resistance 36. 5 percent in 24 healthy males; as an aerosol of
1. 8-|_im particles,  a mist concentration of 0. 01 to 0. 1 mg/m3  increased
airway resistance 17. 9 percent.  The latter concentrations appear
questionable, since 0.01 to  0. 1 mg H^SO^/m   are  concentrations far
below the reported thresholds for any physiological response.

      Hamilton32 stated in 1948 that the site of action for H2SO4 fumes
is the upper respiratory tract,  "that this action is  not severe,  and that
no instance of bronchitis [is]. . .traceable to such fumes. "  Sim3-!
described,  however, how he, himself, became increasingly sensitive
to the mist during 10 months of exposure,  developing a moderately
severe but extremely persistent bronchitis, which  immediately
exacerbated into uncomfortable periods of coughing and wheezing that
occurred whenever he was exposed to H2SC>4 mist.
 Effects of Sulfuric  Acid Mist  on Respiratory Retention
      Apparent increases in the respiratory rate of human subjects
 exposed to H2SO4 irritant were described in reports by Bushtueva, 2°
 Sim, 31 and Amdur.    The  increase is not suprising, for Wilson34
 in 1948 showed that the retention of inhaled aerosol particles decreased
 as the respiratory rate increased.   Morando3^ briefly (for 15 minutes)
 exposed normal human subjects not accustomed to inhaling HzSO4
 fumes to concentrations varying from 0. 35 to 5. 0 mg/m . Even at
 concentrations not subjectively detected by the  volunteers (less than
 0.5  to 0. 7 mg/m3), the respiratory rate increased rapidly and remained
Toxicity in Man                                                        21

elevated for several minutes after exposure.  Amdur^9 exposed fifteen
subjects to 0.35 to 0. 5 mg/m3 and noted that the respiratory rate
increased about 30 percent above  control values; the maximum inspira-
tory and expiratory flow rates decreased by about 20 percent; and tidal
volume increased by about 28 percent.   These changes occurred during
the first 3 minutes of exposure and were maintained throughout the 15-
minute exposure period.  Values returned to pre -exposure levels  within
15 minutes after the exposure ended.  During the first minute after expo-
sure,  the tidal volume rose above and then declined to control levels.
      These experiments do not provide the physiological evidence
necessary to indicate conclusively that bronchoconstriction was the
response to H2SC>4 mist, although, the data  suggest that this was the case.
Bushtueva  found clear-cut reflex changes in respiratory rhythm and
amplitude when she gave volunteers multiple exposures to an
of 1.8 to 2. 0 mg/m3. At concentrations of 1. 0 to 1. 1 mg/m , changes
in respiration were also noted,  though rhythm and amplitude were
decidely lower. She was unable to demonstrate respiratory changes
when the mist concentration was below 1. 0 mg/m3, but this inability
may have reflected differences  in the  degree of "aerosol dispersion. "
      As the respiratory rate  increases,  the percentage of H2SO4 mist
retained by the respiratory tract is definitely reduced.  The change in
respiratory rhythm and amplitude may be a reflex protective mechanism
that prevents the retention of  the irritant, ^9, 34 thereby decreasing its
toxic  effects.   By  measuring the total volume of air breathed, and the
concentration of the H2SO4 aerosol in the mixing chamber and in ex-
haled air, Amdur^° was able  to show  that average retention of inhaled
H2S<-)4 varied from 50 to 87 percent with exposure concentrations of
0.4 to 1.0 mg/m  . She gave  two possible reasons for such relatively
high retention.   First, the aerosol levels used in the  experiment in-
creased the respiratory rate by only 35 percent (5 to  7 breaths per
minute), certainly not enough to reduce retention far  below baseline
values; and,  second,  the strong affinity of H2SC>4 droplets for moist
surfaces, such as the mucous membranes of the respiratory tract,
may have enhanced retention.
22                                   TOXICOLOGY - SO? DECAY PRODUCTS

     As noted earlier, the respiratory rate and pulmonary air-flow

resistance of human volunteers rose rapidly as exposure concentrations

of H2SO4 aerosol were increased.   Symptomatically, bronchospasm

was also evident in the healthy subjects,  presenting a kaleidoscopic

picture of wheezing,  increased secretions, dyspnea,  spasmodic cough-

ing,  and occasional chest pains.   The intensity and combinations of the

symptoms depended upon the aerosol concentration and length of

exposure.     Two observer-subjects   reported occurrence  of chronic

bronchitis during long-term intermittent exposure to H2SC>4 aerosol.

     Bronchospasm, increased upper-respiratory-tract secretions,

and a rapid  respiratory rate were consistently found, along with eleva-

ted pulmonary air-flow resistance,  in volunteers exposed to mist levels

greater than 0. 35 to 0. 5 mg/m  .  All of the subjects involved in pre-

vious experiments were young healthy adults who could easily compen-

sate for the increased resistance imposed on their  breathing. Elevated

airway resistance severely embarrasses breathing in individuals already

burdened by cardiorespiratory deficiencies, however, making it more

difficult to obtain needed oxygen.  Moreover, bronchospasms and

increases in secretory and respiratory rates impair gaseous exchange

in the lungs, and carbon dioxide  accumulates,  which causes respiratory

acidosis.  An acidotic condition superimposed on a restricted oxygen

intake  explains many of the effects seen in pulmonary cripples when

they are exposed to rapidly rising concentrations of t^SO^ aerosols,

above threshold levels.


     Research data concerning the  effects of aerosol particle size,

temperature, or humidity on the human response to H2SC>4 mist

exposure  are scarce.  The few long-term human-exposure studies  have

been limited, essentially,  to epidemiological surveys.  Ethical  con-

siderations  preclude  achieving the requirements of controlled long-

term,  experimental,  human exposure as  such. Intensive industrial-

exposure investigations, rather than controlled laboratory experiments,

may then be  the only acceptable source of needed information.   Chronic
Toxicity in Man                                                       23

bronchitis did develop over the course of at least one long-term study,
however, and various case histories have reported the development of

acute and/or chronic respiratory disease after patients endured short-

or long-term exposure to H2SO4 aeros.ols. ^°>  '

     Definitive data are lacking, but exposure to low concentrations of
sulfuric acid appears to  increase pulmonary flow resistance in human
subjects.  Determining the minimum concentration at which such

changes might be expected to occur, which is possible  for SO2. is
impossible for H2SO4-  Unfortunately, most experiments on the general

complex of sulfur -containing pollutants have dealt with the less toxic
sulfur dioxide. Additional work, to be most meaningful, must be con-
ducted by a design that incorporates the refinements of both physiolog-
ical methodology and aerosol technology.  A failure in either of these

areas would  severely limit the value  of the data.


     In the U. S. S. R. , air pollutant limits have been rigidly set by the
standard,  "that the ambient air should not contain odors to be imposed
on the population against its wishes". ..."  With this  standard as a

guideline, the Russians in the middle fifties  conducted  studies to limit

SC>2 and H2SC>4 in the air to acceptable levels.  The limits finally

selected were based on the subjective reactions of human volunteers

exposed to either SC>2 or H2SO4-  The perception threshold for odor
and mucosal irritation was about 1. 5 mg SO2/m3 and about 0. 5 mg

          .   Subsequent to these studies, in 1957, the  Russian govern-
ment established the allowable single concentrations of SC>2 and

at 0. 5 and 0. 3 mg/m3,  respectively   levels one-half to  one-third the
threshold values. *26, 40
#The term threshold concentration applies to that level of H SO
 aerosol or SC>2 gas that is just perceptible as either odor or irritation.
 This concentration lies between 0. 6 to 0. 85 mg/m  for the aerosol,
 and between 1. 6 to 2. 8 mg/m3 for the gas. 26> 28> 30> 40> 41 Subthres-
 hold implies that the level of exposure cannot be detected either by
 odor or by irritation.
24                                   TOXICOLOGY - S02 DECAY PRODUCTS

      Between 1956 and 1962, Russian scientists attempted to define
 that level of SO2 and/or H2SO4 that could trigger responses in specific
 receptors of the respiratory system (including the nose).  They exposed
 subjects  simultaneously to subthreshold concentrations of SO2 and H2SC>4
 in an effort to detect responses in the central nervous system (brain).
 The respiratory receptors, particularly those in the olfactory area,
 have a well-recognized sensitivity to the  action of minimal concentra-
 tions of chemical substances that is partially explained by the fact that
 they are located where  the outside air initially comes in direct contact
 with the internal organism.

      The  varied responses to H2SC>4 exposure discussed in this section,
 although not strictly within the realm of classical toxicology,  are
 included because the data are closely related to the biological considera-
 tions of this review.  The practical  ramifications and toxicological
 implications of the subsequent neurophysiological responses have not
 been adequately explored.  In addition, one must exercise caution in
 interpreting such data,  since no replicative  studies have been made to
 confirm the reported neurophysiological responses,  and informative
 descriptions of experimental conditions have been less than adequate.

 Optical Chronaxy

     Alterations in optical chronaxy* following stimulation of the
 respiratory receptors were examined extensively in hopes of  finding
 the allowable limits of atmospheric  pollutants. No change in  optical

 chronaxy was noted when the legal maximum concentrations of SO2
 and/or H2SC>4  aerosol were administered to volunteers (for H2SO4,
*Weak electrical current applied to the eyeball that produces the sensa-
 tion of a flash of light.  Each subject has a maximum intensity of stim-
 ulation below which he does not perceive light.  The intensity thres-
 hold, expressed in units of electrical potential (i. e. , volts) is called
 the rheobase.  Chronaxy is the electrical current times twice the
 intensity that produces the rheobase.  A stimulating current of two
 rheobases causes light sensation only when it is sufficiently prolonged.
 This value, that is, the time threshold necessary for the appearance
 of light sensation,  is called optical chronaxy.2°
Toxicity in Man                                                        25

0.3 mg/m ; for SC>2, 0.5 mg/m3); nor when subthreshold concentra-
tions were administered (for t^SC^, 0. 6 mg/m3; for SC>2,  1.2 mg/m3)
(Table 1). 30 Exposure to levels of SC>2 or H2SO4 beyond threshold
consistently elicited increases in the optical chronaxy.   When the two
irritants were combined, such that the concentration of both was equal
to or greater than the subthreshold concentration,  the optical chronaxy
value indicated an additive or synergistic  depression of central nervous
system excitation.  These data were not sufficient to determine whether
the primary response came from the optic cortex itself, or whether a
response in the olfactory zone caused a secondary inhibitory effect in
the visual cortex,  for no changes were detected until concentrations
exceeded the odor threshold.  Ryazanov   has postulated the latter
mechanism as a possible  explanation for the increased optical chronaxy
                       TO TEST ATMOSPHERES3' 3°
Test gas
Fresh air
S02 and H2S04
SO- and H2S04
S02 and H2S04
S09 and H0SO,,
2 24
Concentration, mg/m
1.5 to 1.8
1.0 to 1.2
0.73 to 0.75
0.60 to 0.63
1.2 and 0.6
0.8 and 0.6
1.2 and 0.4
1.5 and 0.75
Average optical chronaxy, yF
0.61 ± 0.0037
0.80 t 0.0015
0.62 ± 0.0070
0.70 + 0.0090
0.62 + 0.0058
0.71 ± 0.0120
0.71 1 0.0120 (sic)
0.61 t 0.0100
0.98 + 0.0280
 Results  of  tests on one subject, Madame B.

Dark Adaptation
      Information based on optical chronaxy was substantiated by studies
of dark adaptation.  The Hagel adaptometer, an instrument that meas-
ures changes in light sensitivity of the visual organ while the subject is
in darkness, was used,  and test subjects were exposed to several con-
centrations of SC>2 and/or H2SO4 aerosol.   The average sensitivity of
subjects to  light  was computed from a curve illustrating changes in dark

adaptation with time.   The raw data (in relative units) in Table 23°
show a marked similarity to the data obtained with optical chronaxy
techniques.  Subthreshold concentrations had no effect on dark adapta-
tion,  but levels exceeding threshold reduced adaptation activity in the
visual cortex.   '   '    According to Ryazanov, 2  this effect can also
be explained as the result of a primary reflexive response in the
olfactory zone that leads to  a short period of  optical cortex inhibition,
which is followed by another, prolonged period of depressed activity in
the optical cortex secondary to the influence of reticular formation.26'43
The second component of this diphasic response was noted in some
subjects when their light sensitivity was found markedly depressed 50
to 60  minutes after the dark-adaptation curves had shown post-exposure
return toward normal.



Concentration of
gas added, mg/m3
Madame D
Madame P
Madame V
Average light

33,400 + 1,
33,200 ± 1,
41,100 + 1,
42,400 t
54,600 t 1,

54,300 1 2,
55,500 ± 1,
69,300 + 5,
62,850 + 3,
82,750 t 1,

42,133 +
41,767 +
56,200 t 3,
54,700 t 1,
68,967 t 3,

0 (sic)


Toxicity in Man

Continuous Electroencephalography

      The Russian scientists next turned to continuous monitoring of

the cerebral cortex by electroencephalography (EEC), directly record-

ing the state of central nervous system function during exposure to SO2

                       40  43  44
and/or H2SO4 aerosols.   '   '    Mucous membrane irritation,

secondary to the  sulfur irritation, innervates the trigeminal nerve,

while odor perception results from direct excitation of the olfactory

receptors.  Stimulation of any receptor organs is followed by alpha-

rhythm (low-frequency, high-amplitude EEC signal) suppression or

desynchronization.  Individuals with  well-defined alpha rhythms were

selected as subjects and the criterion of response was defined as the

onset of  electrical desynchronization,  or alpha-rhythm suppression.

Exposing subjects to subthreshold concentrations elicited no electro-

encephalographic changes,  nor did application of both SC>2 and H2SO4

at maximal allowable concentrations elicit alpha-rhythm desynchroni-

zation, at either  beginning or end of  exposure.  Desynchronization was

observed only when threshold doses  of H2SC>4 mist and/or SC>2 were

used; then it lasted for 1 to 2 seconds.  The H2SO4 aerosol changed

alpha rhythms only when the concentration was  sufficient to be noted

subjectively by the volunteers.  Subthreshold  concentrations, not

subjectively perceived, did not appear to alter the central nervous

system signals,  and, at higher concentrations,  the crude subjective

indices - odor perception and mucosal irritation - apparently correlated

well with the onset of alterations  in the central nervous system. Sub-

jective evidence  of cortical response was  found only when the concen-

trations  were sufficient to affect the  course of the other reflex effects

(i. e. ,  optical chronaxy and dark adaptation),  so that subjective

responses apparently were  valid as objective  indices of the cortical

effects of the irritants.

Conditioned Reflexes

      An additional physiological concept is referred to in the Russian

literature,  although apparently not discussed  at all in American toxico-

                                      45                  4.1
logical studies.   According to Gershuni   and Dubrovskaya,    cortical
28                                    TOXICOLOGY - SO? DECAY PRODUCTS

reactions may occur upon exposure to certain subthreshold (subsensory)

excitations.  This may play an essential role in toxicological and

health effects.   If cortical reactions to subthreshold levels do occur,

one must conclude that to be acceptable ambient pollutant levels must

not only be below the level of odor perception and irritancy but also

below the threshold of subconscious  reflex reactions,  particularly

those capable of altering the functional state of the cerebrum.    The

electrocortical conditioned reflex is a central nervous system phenom-

enon elicited only after a succession of repeated,  conditioned reflex

trials.  As indicated previously,  temporary desynchronization  of the

alpha rhythm occurs each time a subject is given a strong light signal.

When light is excluded, the  alpha rhythm is restored.  Experimentally,

a. system is established by which a subject inhales, for 10 seconds, a

known subthreshold concentration of SO2 gas and/or H2SO4 aerosol

that by itself does not cause the alpha  rhythm to desynchronize.  He

then receives a light over a 5- to 10-second interval.  After repeating

this combination (irritant plus light) several or many  times,  desynchro-

nization begins to appear before the light; that is,  apparently in

response to the unperceived odor.  The unperceived odor thus appears

to become the  conditioning stimulus, generating the so-called condi-

tioned electrocortical reflex.  "  Through this reflex,  EEG alpha-wave

desynchronization occurred when individuals were exposed to an H£SO4

aerosol  concentration of  0. 4 mg/m , an SC>2 concentration of 0. 6

mg/m , or to  a combination of either  0. 15 mg H2SC>4/m plus  0. 5 mg

SO2/m3, or 0. 3 mg H2SO4/m3 plus  0. 25 mg SC>2/m3. 40' 43> 44  All of

these concentrations are below the subjective sensory threshold.  With

respect to H2SO4,  the conditioned reflex was usually developed in the

course of 19 to 23 trials  with H2SO4 aerosol plus  light (Table 3).

Remarkable as it may seem, the complex neurological process result-

ing in disruption of cortical rhythm was initiated by a subjectively non-

perceivable odor.  °


      The Russian data are concerned  wholly with the  effects  of H2SO4

aerosols upon  the interrelationship between sensory modalities and
Toxicity in Man                                                        29

            Table 3.  NUMBER OF TRIALS WITH ^04 PLUS LIGHT


H2S04 concentration,
0.6 to 0.75
 cerebral cortical function.  Those concentrations of H2SO4 mist that

 produce subjective sensations (odor and mucous membrane irritation)

 were invariably associated with objective evidence of  central nervous

 system depression.  Subjective,  subthreshold,  acid mist concentra-

 tions were not associated with depression of either  optical chronaxy,

 dark adaptation, or electroencephalographic patterns.  Only when the

 electrocortical conditioned reflex technique was employed did sub-

 threshold levels result in detectable central nervous system changes.

 This is evidence that a chemical stimulator, unable to initiate an

 electroencephalographic response when used in subthreshold doses by

 itself, can stimulate the central nervous system and change the irri-

 tability of cortical cells  of the optical and other analytic or cognitive

 centers when it is used as a conditioning stimulus.     Table 4 sum-

 marizes  the sensory-neurologic dose-response data just described.


                         RESPONSE44 (mg/m3)
                                         Threshold dose
Perception of odor and
irritation of mucosa
Suppression of dark
Elevation of optical chronaxy
Disruption of alpha rhythms
Conditioning of electro-
cortical reflex
0.6 to 0.85
0.63 to 0.73
1.6 to 2.8
H2S04 + S02
0.3 + 0.5
0.3 + 0.5
0.6 + 1.2
0.3 + 0.5
0.15 + 0.5
0.3 + 0.25


      Studies of atmospheric  chemistry have shown that under certain

conditions sulfur dioxide can be converted to sulfuric acid and particu-

late sulfates.  Air quality criteria and standards for sulfur dioxide

should, therefore, take into account the irritant potency of these

oxidation products, recognizing that both sulfur dioxide and its

degradation products can react further with other air pollutant consti-

tuents. Additional aerometric and toxicologic research is needed to

supplement what  little  is known of the actual existence in the

atmosphere of this group of compounds and of  their toxicology.


      Sulfuric acid is a respiratory irritant that in sufficient concentra-

tion will cause the death of exposed animals.   The guinea pig is  the

most sensitive species among the common small laboratory animals,

followed in decreasing order  by mice, rats,  and rabbits.  The greater

sensitivity of the guinea pig results from its greater susceptibility to

bronchial and laryngeal spasm, a characteristic that makes the  animal

useful for some studies of the irritant potential of low concentrations

of sulfuric acid,  but that makes extrapolation of the results  obtained  of

questionable value in predicting the health effects of sulfuric acid air

pollution in man.  Experiments to date have shown that concentration

is a more important factor than duration of exposure in producing

death in animals.

      Sulfuric acid also produces pathological changes in lungs.   The

degree of lung damage observed after effective, short exposures (up to

72 hours) is related to the mathematical product of  concentration and

time (CT) rather than to concentration alone.  Sulfuric acid damage is

repaired slowly.  Concentrations below  2 mg/m3 produce only minor

luhg damage after  extended exposures (about 3 months),  which indicates

that the CT relationship observed in 8- and 72-hour exposures cannot
be extrapolated directly to long-term effects of low concentrations.
      The particle size of sulfuric acid mist seems to be another impor-
tant factor in determining its irritant potency.  When the" potency crite-
rion was mortality resulting from an 8-hour exposure, a mist of larger
particles  (2. 7 |J.m versus 0. 8 |J.m) was more toxic.  When pathological
lung damage resulting from long-term exposures was the criterion, the
effect of 0. 9-(J.m particles was greater than that of either 0. 6- or  9-|J.m
      Studies of pulmonary function in animals exposed to these sub-
stances cannot be extrapolated simply and directly to predict the human
response.  They are, however, providing useful information regarding
the nature of mechanisms by which effects are produced, and may yet
lend insight into responses that are more analogous to the responses  of
sensitive  individuals in the population than.of healthy.  For practical
considerations, these experiments present two principles for indirect
guidance in the  area of air pollution research.  First,  because particle
size plays an important role in determining irritant potency of sulfur
oxide particulates (submicron particles being more irritant),  data on
mass concentration alone are insufficient for predicting irritant potency.
The second important point is that the particulate oxidation products of
sulfur dioxide have  a generally much greater irritant potency than
sulfur dioxide gas per se.  This is indicated by comparative  data as
well as  by increases in response to sulfur dioxide in the presence of
particulate materials that are known to dissolve the gas and/or to cat-
alyze its conversion to sulfuric acid or sulfates.  Along with factors
such as the original particle size, presence of other materials in the
inhaled  air, the subject's breathing pattern, and the site of particle
deposition,  the hygroscopic nature of sulfuric acid and certain sulfate
aerosols affects physiologic responses to their inhalation.  Atmospheric
levels of sulfur dioxide and the  implication .of these levels for health
should thus  be assessed in terms of their potential for forming more
irritant substances.  To  do otherwise would be  to miss the toxicological
point  and  seriously becloud important issues.

32                                    TOXICOLOGY - S02 DECAY PRODUCTS

      The combined effects of particle size and concentration of
mist on exposed human subjects have still not been determined.  The
effects of temperature and humidity on the response in humans toH2SO4
have not been satisfactorily  examined either.  Long-term human expo-
sure information is,  by necessity,  limited to epidemiological surveys.
Since controlled long-term experimental exposure of humans cannot
ethically be performed,  industrial and episodic exposures are the only
sources of needed information and should be investigated.  One long-
term study and various case histories have  reported the development of
acute and/or chronic respiratory disease following short- and long-term
exposure to 1^230^ aerosol.
     Although definitive data are lacking, animal studies  indicate that
sulfuric acid should increase pulmonary flow resistance in human
subjects.  The concentration at which such changes might be expected to
occur is,  however, impossible  to determine.  Regrettably,  the bulk of
the experiments relating to the  general complex of sulfur-containing
pollutants has dealt with the  less  irritating sulfur dioxide.  More
research should now be directed to the other components of the complex.
     Russian scientists have been almost entirely concerned with the
effects of H2SO4 aerosols  upon sensory modalities,  cerebral cortical
function, and their interrelationships.  Concentrations of  H2SO4 mist
that resulted in subjective sensory stimulation (odor and mucous mem-
brane irritation) were found to be associated invariably with objective
evidence of central nervous  system depression.  Subjectively deter-
mined subthreshold concentrations  of acid mist were not associated
with depression of either optical chronaxy,  dark adaptation, or electro-
encephalographic alterations.  Only when the electrocortical conditioned
reflex was used did sensory subthreshold levels produce central nervous
system stimulation.  This has been taken as evidence  that, when used
as a conditioning stimulus, a chemical stimulator that is unable to
initiate an electroencephalographic response when used in subthreshold
doses by itself can stimulate the central nervous system and change the
irritability of cortical cells  of the optical and other  analytic or cognitive

Summary                                                              33


 1.  Treon,  J.  F.  et al.   Toxicity of Sulfuric Acid Mist.  A.M. A. Arch.
    Ind.  Hyg. Occup.  Med.   2^:716-734,  July-December 1950.
 2.  Mathur, K. and P. Olmstread.   Inhalation Toxicity of Sulfuric Acid
    Mist: Animal Experiment Study of the Problem.  Unpublished
    thesis,  Harvard School of Public Health, Boston, Mass.  September

 3.  Amdur, M. O. , R. Z.  Schulz,  and P. Drinker.  Toxicity of Sul-
    furic Acid  Mist to Guinea Pigs.  A.M. A. Arch. Ind.  Hyg. Occup.
    Med. 5_:318-329,  April 1952.

 4.  Salem,  H.  and H.  Cullumbine.  Kerosene Smoke and Atmospheric
    Pollutants.  Arch. Environ. Health.  2j641-647,  June 1961.

 5.  Pattle,  R.  E., F. Burgess, and H. Cullumbine.  The Effects of
    Cold Environment and of Ammonia on the Toxicity of Sulfuric Acid
    Mist to  Guinea Pigs.  J. Pathol. Bacteriol.  72_:219-232, July-
    October 1956.
 6.  Thomas, M. D. , R.  H. Hendricks, F. D.  Gunne, and J. Critch-
    low.  Prolonged Exposure of Guinea Pigs to Sulfuric Acid Aerosol.
    A.M. A. Arch Ind. Health.  J7:70-80,  January  1958.

 7.  Bushtueva,  K.  A.  Toxicity of H2SO4 Aerosol.   Gig.  i Sanit. 22:17-
    22, 1957.  In: U.S.S. R. Literature on Air  Pollution and Related
    Occupational Diseases.  A Survey,  Vol. I,  Levine,  B.  S. (trans. )
    U.S. Dept.  of Commerce, Office of Technical Services,  Washing-
    ton,  D.  C., January  I960.  p. 63-66.

 8.  Bushtueva,  K.  A.  Experimental Studies on the Effect of Low
    Oxides  of Sulfur Concentrations on the Animal Organism.  In:
    Limits  of Allowable Concentrations of Atmospheric Pollutants,
    Book 5, Ryazanov, V. A.  (ed. )  and Levine, B. S. (trans.).  U.S.
    Dept. of Commerce,  Office  of Technical Services,  Washington,
    D. C. ,  March  1963.  p. 92-102.

 9.  Amdur, M. O. and J. Mead. A Method for Studying the Mechani-
    cal Properties of the  Lungs  of Unanesthetized Animals.   Applica-
    tion to the  Study of Respiratory Irritants.   In:  Proc. Third
    National Air Pollution Symposium, Pasadena,  California, p.  150-
    159, April 18-20,  1955.
10.  Amdur, M. O. and J. Mead. Mechanics of Respiration in Unanes-
    thetized Guinea Pigs.  Am.  J. Physiol.   192_(2):364-368,  1958.
11.  Amdur, M. O.  The Respiratory Response of Guinea Pigs to Sul-
    furic Acid  Mist.  A.M. A. Arch. Ind. Health.  18 :407-414,
    November  1958.

12.  Amdur,  M. O.  The Effect of Aerosols on the Response to Irritant
    Gases.  In: Inhaled Particles and Vapors, Davies,  C. N.  (ed. ).
    Proc.  Intl. Symposium,  Oxford, March 29 - April 1, I960. Per-
    gamon Press,  Oxford, 1961.  p. 281-292.

13.  Amdur,  M. O.   The Response of Guinea Pigs to Inhalation of For-
    maldehyde and Formic Acid Alone and With a Sodium Chloride
    Aerosol.  Int.  J.  Air  Poll.  3:201-220, October I960.

14.  Lewis, T. R.,  K. I.  Campbell,, and T. R. Vaughan.  Effects  on
    Canine Pulmonary Function via Induced NO, Impairment, Particu-
    late Interaction, and ,Subsequent SOX.  Arch. Environ. Health.
    18:596-601, April 1969.
15.  Lewis, T. R.  Unpublished  results.

16.  Amdur,  M. O,  and M. Corn.  The  Irritant Potency of Zinc Am-
    monium  Sulfate of Different Particle Sizes.  Am. Ind. Hyg.
    Assoc. J. 24:326-333, July-August 1963.
17.  Nadel, J.  A. ,  et_ aL   Location and Mechanism of Airway Constric-
    tion after Inhalation of Histamine Aerosol  and Inorganic Sulfate
    Aerosol.  In:  Proc. Second International Symposium on  Inhaled
    Particles and Vapors, Davies,  C. N.  (ed. ).   Pergamon Press,
    Oxford,  1966.' p.  55.

18.  Amdur,  M. O.  and D. Underbill.  The Effect of Various Aerosols
    on the Response of Guinea Pigs to Sulfur Dioxide.  Arch.  Environ.
    Health.  16^:460-468, April 1968.
19.  Amdur,  M. O.  The Impact of Air Pollutants on Physiologic
    Responses of the  Respiratory Tract.   Proc.  Am. Philosophical
    Soc.   .U(l):3-8,  February 1970.
20. Amdur,  M. O.  The Influence of Aerosols Upon the Respiratory
    Response of Guinea Pigs to Sulfur Dioxide. Am. Ind. Hyg. Assoc.
    Quart.  ^8:149-155, June 1957.

21. Air Quality Criteria for Sulfur Oxides. U.S. Dept. of Health,
    Education,  and  Welfare,  Public Health Service,  National Air Pol-
    lution Control Admin.  , Durham,  N. C. Pub.  No. AP-50.  January
22. Air Quality Criteria for Particulate Matter.   U.S.  Dept. of Health,
    Education,  and  Welfare,  Public Health Service,  National Air Pol-
    lution Control Admin.  , Durham,  N. C. Pub.  No. AP-49.  January
23. Amdur,  M. O.  Toxicologic Appraisal of Particulate Matter,
    Oxides of Sulfur,  and  Sulfuric Acid.  J.  Air Poll. Control Assn.
    .1^:638-644, September 1969.

24. Threshold Limit Values for 1957,  adopted at the 19th Annual
    Meeting  of the  American Conference of Governmental Industrial
    Hygienists, St.  Louis, April 20-23, 1957.  A.M. A.  Arch. Ind.
    Health.  16:261-265,   1957.
36                                   TOXICOLOGY - S02 DECAY PRODUCTS

25. Amdur,  M. O.  Reports on Tentative Ambient Air Standards for
    SO2 and HzSO-l.  Ann.  Occup. Hyg.  3_:71-83,  February 1961.

26. Ryazanov, V.  Sensory Physiology as the Basis for Air Quality
    Standards.  Arch.  Environ. Health.   5^:480-494,  November  1962.

27. Dorsch, R.  The Pollution of the Air in the Storage Battery Room
    and its Surroundings by Sulfuric Acid.  Unpublished dissertation,
    Wurzberg, Germany, 1913.

28. Bushtueva,  K. A.  Determination of the Limit of Allowable  Con-
    centrations of H2SO4 in Atmospheric Air.  In: Limits of Allowable
    Concentrations of Atmospheric Pollutants, Book 3,  Ryazanov, V.
    A.  (ed. ) and Levine, B.  S.  (trans.).  U.S.  Dept.  of Commerce,
    Office of Technical Services, Washington, D.  C. ,  1957. p.  20-36.

29. Amdur,  M. O. , L. Silverman, and P. Drinker.   Inhalation of
    H2SC>4 Mist by Human Subjects. A.M. A.  Arch. Ind. Hyg.  Occup.
    Med.  6^:305-313,  October  1952.

30. Bushtueva,  K. A.  Threshold Reflex Effect of SOz and H2SO4
    Aerosols Simultaneously Present in the Air.  In: Limits of Allow-
    able Concentrations of Atmospheric Pollutants, Book 4,  Ryazanov,
    V. A.  (ed. ) and Levine,  B.  S.  (trans.).   U.S. Dept. of Commerce,
    Office of Technical Services, Washington, D.  C.,  1961. p.  72-79.
31. Sim,  V.  and R. Pattle.   Effect of Possible Smog Irritants on
    Human Subjects.   JAMA.  165:1908-1913, December 1957.

32. Hamilton, A.  et al.  Industrial Toxicology,  Second Edition. New
    York,  Paul B. Hoeber,  Inc., 1949.
33. Toyama,  T.  and K. Nakamura.  Synergistic Response of Hydrogen
    Peroxide Aerosols and SO2 to Pulmonary Airway Resistance.  Ind.
    Health.  2_:34-45,  March 1964.

34. Wilson,  I. and V. LaMer.  The Retention of Aerosol Particles in
    the Human Respiratory Tract as a Function of Particle Radium.
    J. Ind.  Hyg. and Toxicol.  -3£(5):265-280, September 1948.

35. Morando, A.  Experimental and Clinical Contribution to Human
    Pathology Due to Sulfuric Acid Fumes.  Med.  Lavoro.  47(10):557-
    561, October  1956.
36. Banyai,  A.  Pulmonary Hazards of Air  Pollution.  Arch. Environ.
    Health.  3_:396-403, October 1961.
37. Stokinger, H.  Toxicologic Interactions of Mixtures of Air  Pollu-
    tants.  Review of Recent Developments.   Internat. J. Air  Pollution.
    2_:313-326, February I960.
38. Goldman, A.  and W. T.  Hill.   Chronic  Broncho Pulmonary Disease
    Due to Inhalation of H2SO4 Fume. A.M. A.  Arch.  Ind. Hyg. Occup.
    Med.  8^:205-211,  September 1953.
39. Rosmanith, J. Onemocneni Dychocich Cest v Zomestnancu ve
    Vyrobne Hyseling Rirove (Respiratory Disease in Workers Produc-
    ing H2SO4). ev Oddeleni Chorob z Povolozi KUN 2 a. Oddeleni Hyg.

     Proce KHGS, Ogtrava.  Prague,  Pracovn I. Lekarstvi, 1957.
     p. 411-416.

40.  Ryazanov, V.  New Data on Maximum Allowable Concentrations of
     Pollutants in the Air in the USSR.  In:  London Proc. of the Diamond
     Jubilee International Clean Air Conference,  National Society for
     Clean Air, 1960.  p. 175.
41.  Bushtueva, K. A. et al.  Electroencephlographic Determinations
     of Threshold Reflex Effect of Atmospheric Pollutants.  Gig. i
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42.  Dubrovskaya, F.  Hygienic Evaluation of  Pollution of  Atmospheric
     Air of a Large City with Sulfur Dioxide.   In: Limits of Allowable
     Concentrations of Atmospheric Pollutants, Book 3,  Ryazanov,  V.
     A. (ed. ) and Levine, B. S. (trans.).  U.S. Dept. of Commerce,
     Office of  Technical Services, Washington, D. C. ,  1957.  p. 37-51.
43.  Magoun,  H.  The Waking Brain:  The Role of the Reticular System
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     Thomas,  1958.

44.  Epstein,  D.  Detection and Prevention of  Atmospheric Pollution in
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45.  Bushtueva, K. A.  New Studies of the Effect of SO2  and H2SO4
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38                                   TOXICOLOGY - S02 DECAY PRODUCTS




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 2.  Cember, H. e_t al.  Elimination of Radioactive Barium Sulfate
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 3.  Cember, H. , J. A. Watson,  and M. E.  Novak.  The Influence of
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