NITROBENZENE
Ambient Water Quality Criteria
Criteria and Standards Division
Office of Water Planning and Standards
U.S. Environmental Protection Agency
Washington, D. C. 20460
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CRITERION DOCUMENT
NITROBENZENE
CRITERIA
Aquatic Life
For nitrobenzene the criterion to protect freshwater aquatic
life as derived using the Guidelines is 480 ug/1 as a 24-hour
average and the concentration should not exceed 1,100 ug/1 at any
time.
The data base for saltwater aquatic life is insufficient to
allow use of the Guidelines. The following recommendation is
inferred from toxicity data for freshwater organisms.
For nitrobenzene the criterion to protect saltwater aquatic
life as derived using procedures other than the Guidelines is 53
ug/1 as a 24-hour average and the concentration should not exceed
120 ug/1 at any time.
Human Health
For the prevention of adverse effects due to the organo-
leptic properties of nitrobenzene in water, the criterion is 30
ug/i.
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Introduction
Nitrobenzene is produced for industrial use by the nitra-
tion of benzene with nitric and sulfuric acids. Estimates of
annual nitrobenzene production range from 200 to over 700 mil-
lion pounds (Dorigan and Hushon, 1976; Lu and Metcalf, 1975).
The principal use of nitrobenzene is for reduction to aniline,
which is widely used as an ingredient for dyes, rubber, and
medicinals (McGraw-Hill, 1971; Kirk and Othmer, 1967). The
commercial applications of nitrobenzene are: reduction to
aniline (97 percent), solvent for Friedel-Crafts reaction,
metal polishes, shoe black, perfume, dye intermediates, crys-
tallizing solvent for some substances, and as a combustible
propellent (Dorigan and Hushon, 1976).
Nitrobenzene is stored in closed containers and is not
usually released to the open air. Atmospheric contamination
is usually prevented in plants manufacturing or using nitro-
benzene by the use of activated charcoal absorbers or a car-
bon dioxide blanket. There is no industrial monitoring of
nitrobenzene in the atmosphere. The greatest loss of nitro-
benzene during production (estimated as eight million pounds
annually) occurs at the acid extraction step in the purifica-
tion of the crude reaction mixture, when nitrobenzene is lost
to the effluent wash (Dorigan and Hushon, 1976). Thus, the
greatest exposure to nitrobenzene occurs inside plants and
most cases of chronic nitrobenzene exposure in man are nitro-
benzene workers. Today plant levels of nitrobenzene are usu-
ally kept below the threshold limit value (TLV) of 5 mb/m^
(Goldstein, 1975; Am. Conf. Gov. Ind. Hyg., 1977) but much
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higher levels have been reported in the past (Pacseri and
Magos, 1958). Nitrobenzene may also form spontaneously in the
atmosphere from the photochemical reaction of benzene with
oxides of nitrogen.
Nitrobenzene, also known as nitrobenzol, essence of mir-
bane, and oil of mirbane, is a pale yellow oily liquid with an
almond-like odor (Kirk and Othmer, 1967). The color of the
liquid varies from pale yellow to yellowish brown depending on
the purity of the compound (Kirk and Othmer, 1967). In the
solid state it forms bright yellow crystals. Nitrobenzene,
CgH5NC>2, has a molecular weight of 123.11 g.
The physical properties of nitrobenzene are as follows:
a boiling point of 210° to 211°C at 760 mm Hg, a melting point
of 6°C, a density of 1.205 at 15°C, a refractive index of
1.5529, and a flash point of 89°C (Stecher, 1968). It is
steam volatile (Stecher, 1968) and at 25°C nitrobenzene has a
vapor pressure of 0.340 nun Hg (Jordan, 1954).
Nitrobenzene is miscible with most organic solvents, such
as ethanol, diethyl ether, acetone, and benzene (Kirk and
Othmer, 1967). It is slightly soluble in water, 0.1 per 100
parts of water (1,000 mg/1) at 20°C (Kirk and Othmer, 1967).
In aqueous solutions, nitrobenzene has a sweet taste (Kirk and
Othmer, 1967).
Nitrobenzene undergoes substitution reactions but re-
quires more vigorous conditions than does benzene. Substitu-
tion takes place at either the meta (3) position or the ortho-
(2) para-(4) positions depending on the physical conditions
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(Kirk and Othmer, 1967). Nitrobenzene undergoes photoreduc-
tion when irradiated with ultraviolet light in organic sol-
vents that contain abstractable hydrogen atoms (Barltrop and
Bunce, 1968).
Nitrobenzene is a fairly strong oxidizing agent (Kirk and
Othmer, 1967; Millar and Springfield, 1966). Since the com-
pound can act as an oxidizing agent in the presence of aqueous
solutions of alkali hydroxides, it has the capability of oxi-
dizing compounds containing free phenolic hydroxyl groups
without effectively changing these groups (Millar and Spring-
field, 1966). Nitrobenzene is reactive and will undergo
nitration, halogenation, and sulfonation by the same methods
used for benzene. However, these reactions are unlikely to
occur in environmental conditions.
The reduction of nitrobenzene to aniline probably out-
ranks all other uses of nitrobenzene as an industrial chemical
(Kirk and Othmer, 1967). The di- and the trinitrobenzenes are
used in military and industrial explosives. The great toxi-
city of nitrobenzene impairs its usefulness as an organic sol-
vent. It is readily absorbed by contact with the skin, inha-
lation of the vapor, or by ingestion. The absorption of
nitrobenzene into the body produces cyanosis (Kirk and Othmer,
1967).
Nitrobenzene has been found to have a metabolic turnover
slow enough to result in distinct accumulation under condi-
tions of daily exposure (Piotrowski, 1967). Complications
could arise from the possible accumulation of either nitro-
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benzene or p-nitrophenol. The half-life of the excretion of
the p-nitrophenol is approximately 60 hours (Salmowa, et al.
1963).
The toxicological data on the effects of nitrobenzene are
limited primarily to mammalian, especially human, studies and
case histories. There are few data on the toxic effects of
nitrobenzene to aquatic organisms. A freshwater fish acute
value for nitrobenzene was found to be 42,600 ug/1 with a
chronic value of more than 16,000 ug/1. A saltwater acute
value was 58,539 ug/1. In the case of mammals, nitrobenzene
is highly toxic when ingested, inhaled, or absorbed through
the skin. Exposure by any of these routes can result in head-
aches, drowsiness, nausea, vomiting, and methemoglobinemia
with cyanosis.
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REFERENCES
American Conference of Governmental Industrial Hygenists.
1977. Documentation of the threshold limit value for sub-
stances in workroon air. Cincinnati, Ohio.
Barltrop, A.J., and N.J. Bunce. 1968. Organic photochemis-
try, Part 4. The photochemical reduction of nitre-compounds.
Jour. Chem. Soc. Sec. C. 12: 1467.
Dorigan, J., and J. Hushon. 1976. Air pollution assessment
of nitrobenzene. U.S. Environ. Prot. Agency.
Goldstein, I. 1975. Studies on MAC values of nitro- and
ammo-derivatives of aromatic hydrocarbons. Adverse Effects
Environ. Chem. Psychotropic Drugs 1: 153.
Jordan, T.E. 1954. Vapor pressure of organic compounds.
Interscience Publishers, Inc., New York.
Kirk, R.E., and D.F. Othmer. 1967. Kirk-Othmer Encyclopedia
of Chemical Technology. 2nd ed. John Wiley and Sons, Inc.,
New York.
Lu, P.Y., and R. Metcalf. 1975. Environmental fate and bio-
degradability of benzene derivatives as studies in a model
aquatic ecosystem. Environ. Health Perspect. 19: 269.
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McGraw-Hill. 1971. Encyclopedia of science and technology.
McGraw-Hill Book Co., New York.
Millar, I.T., and H.D. Springfield, eds. 1966. Sidgwick's
organic chemistry of nitrogen. 3rd ed. Clarendon Press,
Oxford.
Pacseri, I., and L. Magos. 1958. Determination of the mea-
sure of exposure to aromatic nitro and amino compounds.
Jour. Hyg. Epidemiol. Microbiol. Immunol. 2: 92.
Piotrowski, J. 1967. Further investigations on the evalua-
tion of exposure to nitrobenzene. Br. Jour. Ind. Med. 24:
41.
Salmowa, J., et al. 1963. Evaluation of exposure to nitro-
benzene. Br. Jour. Ind. Med. 20: 41.
Stecher, P.G. , ed. 1968. The Merck Index. 8th ed. Merck and
Co., Inc., Rahway, N.J.
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AQUATIC LIFE TOXICOLOGY*
FRESHWATER ORGANISMS
Introduction
Static tests with the bluegill, Daphnia magna, and the alga,
Selenastrum capricornutum, indicate little difference in sensi-
tivity with no 50 percent effect concentrations lower than 27,000
ug/1. An embryo-larval test with the fathead minnow demonstrated
no adverse effects at the highest test concentration.
Acute Toxicity
The 96-hour LC50 for the bluegill is 42,600 ug/1 (Table 1)
and, after adjustment for test methods and species sensitivity,
this result provides a Final Fish Acute Value of 6,000 ug/1. The
Final Invertebrate Acute Value (1,100 ug/l)f based on a 48-hour
EC50 of 27,000 ug/1 for Daphnia magna, is lower and, therefore, it
becomes the Final Acute Value.
*The reader is referred to the Guidelines for Deriving Water
Quality Criteria for the Protection of Aquatic Life [43 FR 21506
(May 18, 1978) and 43 FR 29028 (July 5, 1978)] in order to better
understand the following discussion and recommendation. The fol-
lowing tables contain the appropriate data that were found in the
literature, and at the bottom of each table are the calculations
for deriving various measures of toxicity as described in the
Guidelines.
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Chronic Toxicity
No adverse effects were observed during an embryo-larval test
with the fathead minnow at test concentrations of nitrobenzene as ,
high as 32,000 ug/1 (Table 3). After division by the sensitivity
factor (6.7) a Final Fish Chronic Value of greater than 2,400 ug/1
is obtained. This also becomes the Final Chronic Value since
there are no reported results with invertebrate species and EC50
results with an alga are higher. A criterion may be derived in
the absence of a precise chronic value by using 0.44 times the
Final Acute Value (1,100 ug/1) at which concentration no adverse
effects were observed with the fathead minnow enbryo-larval test.
Plant Effects
The 96-hour EC50 values for reduction of cell numbers and
inhibition of chlorophyll a_ in the alga, Selenastrum capricornutum
are 42,800 and 44,100 vg/1, respectively (Table 4).
Residues
No measured steady-state bioconcentration factor (BCF) is
available for nitrobenzene. A BCF can be estimated using the
octanol-water partition coefficient of 71. This coefficient is
used to derive an estimated BCF of 15 for aquatic organisms that
contain about 8 percent lipids. If it is known that the diet of
the wildlife of concern contains a significantly different lipid
content, an appropriate adjustment in the estimated BCF should be
made.
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CRITERION FORMULATION
Freshwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
Final Fish Acute Value = 6,000 ug/1
Final Invertebrate Acute Value = 1,100 ug/1
Final Acute Value = 1,100 ug/1
Final Fish Chronic Value = greater than 2,400 ug/1
Final Invertebrate Chronic Value = not available
Final Plant Value = 43,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = greater than 2,400 ug/1
0.44 x Final Acute Value = 480 ug/1
*
The maximum concentration of nitrobenzene is the Final ^cute
Value of 1,100 ug/1 and the 24-hour average concentration is 0.44
times the Final Acute Value. No important adverse effects on
freshwater aquatic organisms have been reported to be caused by
concentrations lower than the 24-hour average concentration.
CRITERION: For nitrobenzene the criterion to protect
freshwater aquatic life as derived using the Guidelines is 480
ug/1 as a 24-hour average and the concentration should not exceed
1,100 ug/1 at any time.
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Table 1 Freshwater fifah acute values for nitrobenzene (U.S. EPA, 1978)
Adjusted
Bloaseay Teat Tine LC50 LC50
Method* Cong.** (hrtQ (uq/l> (uq/1)
Blueglll, S U 96 42.600 23.300
Lepomis macrochlrua
* S = static
** U " unmeasured
Geometric mean of adjusted values - 23,300 pg/1 33,300 m fi(0oo ,lg/i
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Table 2. Freshwater Invertebrate acute values for nitrobenzene (U.S. EPA, 1978)
fiiodssay Test rime
Hctnou* Cone,** (firs')
LC50
Adjusted
LCbO
Cladoceran,
Daphnia ma en a
S U 48 27.000 22,900
* S = static
** U = unmeasured
Geometric mean of adjusted values » 22,900 tig/1 22A?°Q - 1,100 t>g/l
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> 7
CTi
Table 3. Freshwater fish chronic values for nitrobenzene (U.S. EPA, 1978)
Chronic
Limits Value
organism Test* lug/i) (uq/1)
Fathead minnow. E-L >32.000 >16,000
Pimephales promelas
* E-L = embryo-larval
Geometric mean of cl
Lowest chronic value •» >16,000 Mg/1
Geometric mean of chronic values - >16,000 pg/l —&~T~ ™ >2,AOO Mg/1
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Table 4. Freshwater plant effects for nitrobenzene (U.S. EPA, 1978)
Concentration
Organism Eftect (ug/l>
Alga. EC50 96-hr 44.100
Selenaatrum chlorophyll a
capricornutum
Alga. EC50 96-hr 42.800
Selenastrum cell numbers
caprtcornutum
Lowest plant value •» 42,800 Mg/1
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SALTWATER ORGANISMS
Introduction
Static acute tests with the sheepshead minnow and Mysidopsis
bahia indicate the latter is much more sensitive to nitrobenzene.
Adverse effects were observed on the saltwater alga at concentra-
tions slightly higher than the LC50 for the mysid shrimp.
Acute Toxicity
The 96-hour LC50 value for the sheepshead minnow is 58,539
ug/1 and, after adjustment for test methods and species sensi-
tivity, this results in the Final Fish Acute Value'of 8,700 ug/1
(Table 5).
As stated earlier, the mysid shrimp is more sensitive with a
96-hour LC50 of 6,676 ug/1 (Table 6). The Final Invertebrate
Acute Value derived from this test is 120 ug/1; this concentration
also becomes the Final Acute Value.
Chronic Toxicity
No chronic tests have been reported on the adverse effects of
nitrobenzene on saltwater organisms.
Plant Effects
The cell numbers of Skeletonema costatum were reduced by 50
percent at a concentration of 9,650 ug/1 (Table 7). Chlorophyll a_
was equally inhibited at a concentration of 10,300 ug/1- The
lower of these two results is the Final Plant Value.
Residues
No measured steady-state bioconcentration factor (BCF) is
available for nitrobenzene. A BCF can be estimated using the
octanol-water partition coefficient of 71. This coefficient is
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used to derive an estimated BCF of 15 for aquatic organisms that
contain about eight percent lipids. If it is known that the diet
of the wildlife of concern contains a significantly different
lipid content, an appropriate adjustment in the estimated BCF
should be made.
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CRITERION FORMULATION
Saltwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
Final Fish Acute Value = 8,700 ug/1
Final Invertebrate Acute Value = 120 ug/1
Final Acute Value = 120 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 9,700 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 9,700 u9/l
0.44 x Final Acute Value = 53 ug/1
No saltwater criterion can be derived for nitrobenzene using
the Guidelines because no Final Chronic Value for either fish or
invertebrate species or a good substitute for either value is
available.
Results obtained with nitrobenzene and freshwater organisms
indicate how a criterion may be estimated.
For nitrobenzene and freshwater organisms 0.44 times the
Final Acute Value is less than the Final Chronic Value which is
derived from an embryo-larval test with the fathead minnow.
Therefore, it seems reasonable to estimate a criterion for nitro-
benzene and saltwater organisms using 0.44 times the Final Acute
Value.
The maximum concentration of nitrobenzene is the Final Acute
Value of 120 ug/1 and the estimated 24-hour average concentration
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is 0.44 times the Final Acute Value. No important adverse effects
on saltwater aquatic organisms have been reported to be caused by
concentrations lower than the 24-hour average concentration.
CRITERION: For nitrobenzene the criterion to protect
saltwater aquatic life as derived using procedures other than the
Guidelines is 53 ug/1 as a 24-hour average and the concentration
should not exceed 120 ug/1 at any time.
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Table 5. Marine fish acute values for nitrobenzene (U.S. EPA, 1978)
Adjusted
Bioaaeay Teat Time LC50 LC50
Method* gone.** (hre) tug/l>
Sheepshead minnow, S U 96 58,539 32,004
Cyprinodon variegatus
* S = static
*A II = unmeasured
Geometric mean of adjusted values «• 32,004 ~TL7— " 8,700 Mg/1
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00
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M
U)
Table 6 Marine Invertebrate acute values for nitrobenzene (U.S. EPA, 1978)
Soi^aisa
Mysid shrimp,
Mysldopaia bahia
bioassay Test Time
Metiiou* Cone.** (nrs.)
S U 96
LCSO
(uq/i)
6,676
Adjusted
LCbO
lun/ll
5.654
* S = static
** U •» unmeasured
Geometric mean of adjusted values » 5,654 vg/l
- 120 Mg/1
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Table 7 Marine plant effects for nitrobenzene (U.S EPA, 1978)
Concentration
Organism Effect fug/lj
Alga. EC50 96-hr 9.650
SUcletonema costaturn cell numbers
Alga, EC50 96-hr 10.300
Skeletonema coatatum chlorophyll a
Lowest plant value - 9,650 pg/1
00
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NITROBENZENE
REFERENCES
U.S. EPA. 1978. In-depth studies on health and environmental
impacts of selected water pollutants. Contract No. 68-01-4646,
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NITROBENZENE
Mammalian Toxicology and Human Health Effects
EXPOSURE
Introduction
Nitrobenzene, a pale yellow liquid at room temperature
with a characteristic bitter almond aroma, is also known as
oil of mirbane, nitrobenzol, and artificial bitter almond
oil. It is produced for industrial use by the nitration of
benzene with nitric and sulfuric acids. Estimates of annual
nitrobenzene production range from 200 to over 700 million
pounds (Dorigan and Hushon, 1976; Lu and Metcalf, 1975). The
principal use of nitrobenzene is for reduction to aniline,
which is widely used as an ingredient for dyes, rubber, and
medicinals. The commercial applications of nitrobenzene are:
reduction to aniline (97 percent), solvent for Friedel-
Crafts reaction, metal polishes, shoe black, perfumes, dye
intermediates, crystallizing solvent for some substances, and
as a combustible propellant (Dorigan and Hushon, 1976).
Nitrobenzene is stored in closed containers and not
usually released to the open air. Atmospheric contamination
is usually prevented in plants manufacturing or using nitro-
benzene by the use of activated charcoal absorbers or a car-
bon dioxide blanket. There is no industrial monitoring of
nitrobenzene in the atmosphere. The greatest loss of nitro-
benzene during production (estimated as eight million pounds
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annually) occurs at the acid extraction step in the purifica-
tion of the crude reaction mixture, when nitrobenzene is lost
to the effluent wash (Dorigan and Hushon, 1976). Thus the
greatest exposure to nitrobenzene occurs inside plants and
most cases of chronic nitrobenzene exposure in man are nitro-
benzene workers. Today plant levels of nitrobenzene are us-
ually kept below the threshold limit value (TLV) of 5 mg/m3
(Goldstein, 1975; TLV, 1978) but much higher levels have been
reported in the past (Pacseri and Magos, 1958). Nitrobenzene
may also form spontaneously in the atmosphere from the photo-
chemical reaction of benzene with oxides of nitrogen; the
symptoms of nitrobenzene poisoning are similar to the symp-
toms experienced by victims of Japanese photochemical smog
(Dorigan and Hushon, 1976).
Nitrobenzene can be detected for monitoring purposes by
colorimetric reaction, or by collection on a charcoal filter,
extraction, reduction to aniline, and production of a colored
product by diazotization of the aniline. These methods can
detect 1.0 to 500 mg/m3 (0.2 to 100 ppm) of nitrobenzene
(Dorigan and Hushon, 1976). Nitrobenzene in waste water can
be measured by gas chromatography (Austern, et al.) 1975).
Exposure of workers to nitrobenzene is monitored by urinary
levels of p-nitrophenol (Piotrowski, 1967) and p-aminophenol
(Pacseri and Magos, 1958).
The liquid nitrobenzene has a very low solubility in
water, although it is a good organic solvent. It also has a
low volatility, does not readily react with light, and is
non-corrosive. Nitrobenzene has a low volatility but a high
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specific gravity, so that the fumes can accumulate at floor
level in production plants. Some of the common derivatives
of nitrobenzene (besides aniline) are dinitrobenzene, nitro-
benzene-sulfonic acid, and nitrochlorobenzene. There are
many other derivatives of nitrobenzene, and many of them are
very hazardous to man as toxic agents, mutagens, and car-
cinogens.
Some of the physical and chemical properties of nitro-
benzene are summarized in Table 1.
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TABLE 1
Properties of Nitrobenzene (Dorigan and Hushon, 1976)
Formula: C6 H5 NO2 °r/JJ\.N02
Molecular weight:
Freezing point:
Boiling point:
Water solubility:
Soluble in:
Vapor pressure:
Vapor density:
Log partition co-efficient:
Density:
Flash point:
Autoignition temp:
Fire hazard (N.F.P.A.):
Viscosity:
Detection level of charac-
teristic bitter almond
odor:
123.11
5.6 - 5.7°C
210.9°C at 760 torr
0.1 - 0.2 gm/100 ml at 20°C
1.0 gm/100 ml at 100°C
ethanol, diethyl ether, acetone,
benzene, lipids
0.284 mmHg at 25°C
600 mmHg at 200°C
4.24 (air = 1.0)
hexane/water - 3.18 at 24.4°C
1.199 gm/ml at 25°C
87.8°C
482.2°C
medium; fire can be extinguished^ by
water, foam, CO^r or dry chemicals
1.682 cp at 30°C
10~4 mmoles/1
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Ingestion from Water
Nitrobenzene can be released into waste water from pro-
duction plants as the result of losses during the production
of nitrobenzene, aniline, and dyestuffs. The solubility of
nitrobenzene is low, and it produces a detectable odor in
water at a concentration as low as 0.03 mg/1 (Austern, et al.
1975), so that large amounts can not readily accumulate un-
noticed. Levels of nitrobenzene in waste water are monitored
by plants producing and using the chemical but nitrobenzene
levels in city water systems are usually too low to measure
(Pierce, 1979). Nitrobenzene in water from an industrial
spill is removed by treatment with activated charcoal.
There are no data available on mammalian toxicity Of
nitrobenzene ingested in drinking water.
Ingestion from Foods
There are reports of nitrobenzene poisoning resulting
from its uses as false almond oil in baking, rubbing on the
gums to ease toothache, contamination of alcoholic drinks,
and contamination of food (Nabarro, 1948). Leader (1932)
reported a case of nitrobenzene poisoning in a child who was
given "oil of almonds" for relief of a cold. Acute nitro-
benzene poisoning has occurred from ingestion of denatured
alcohol (Donovan, 1920; Wirtschafter and Wolpaw, 1944).
These cases are typical of accidental nitrobenzene ingestion.
Nitrobenzene is not an approved food additive (Dorigan and
Hushon, 1976).
A bioconcentration factor (BCF) relates the concentra-
tion of a chemical in water to the concentration in aquatic
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organisms, but BCF's are not available for the edible por-
tions of all four major groups of aquatic organisms consumed
in the United States. Since data indicate that the BCF for
lipid-soluble compounds is proportional to percent lipids,
BCF's can be adjusted to edible portions using data on per-
cent lipids and the amounts of various species consumed by
Americans. A recent survey on fish and shellfish consumption
in the United States (Cordle, et al. 1978) found that the per
capita consumption is 18.7 g/day. From the data on the 19
major species identified in the survey and data on the fat
content of the edible portion of these species (Sidwell, et
al. 1974), the relative consumption of the four major groups
and the weighted average percent lipids for each group can be
calculated:
Consumption Weighted Average
Group (Percent) Percent Lipids
Freshwater fishes 12 4.8
Saltwater fishes 61 2.3
Saltwater molluscs 9 1.2
Saltwater decapods 18 1.2
Using the percentages for consumption and lipids for each of
these groups, the weighted average percent lipids is 2.3 for
consumed fish and shellfish.
No measured steady-state bioconcentration factor (BCF)
is available for nitrobenzene, but the equation "Log BCF =
0.76 Log P - 0.23" can be used (Veith, et al. Manuscript) to
estimate the BCF for aquatic organisms that contain about
eight percent lipids from the octanol-water partition coeffi-
cient (P). Based on an octanol-water partition coefficient
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of 71, the steady-state bioconcentration factor for nitroben-
zene is estimated to be 15. An adjustment factor of 2.3/8.0
= 0.2875 can be used to adjust the estimated BCF from the 8.0
percent lipids on which the equation is based to the 2.3 per-
cent lipids that is the weighted average for consumed fish
and shellfish. Thus, the weighted average bioconcentration
factor for nitrobenzene and the edible portion of all aquatic
organisms consumed by Americans is calculated to be 15 x
0.2875 = 4.3.
Inhalation
Nitrobenzene is readily absorbed through the lungs with
retention of up to 80 percent (Piotrowski, 1967). There are
reports of nitrobenzene poisoning from inhalation of an ex-
terminator spray for bedbugs which was sprayed on a child's
mattress (Stevenson and Forbes, 1942; Nabarro, 1948). Poi-
sonings have also resulted from inhaled nitrobenzene used as
a scent in perfume and soap (Dorigan and Hushon, 1976).
Chronic and acute poisoning from exposure to nitrobenzene
fumes in production plants are well documented (Dorigan and
Hushon, 1976; Browning, 1950; Zeligs, 1929; Hamilton, 1919),
but since nitrobenzene is also absorbed through the skin, in-
dustrial poisoning cannot be attributed to inhalation alone.
A worker exposed to the TLV for nitrobenzene of 5 mg/m^
would absorb 18 mg/day through the lungs (Piotrowski, 1967).
Dermal
Nitrobenzene is highly fat-soluble and can be absorbed
through the skin at rates as high as 2 mg/cm2/hr (Dorigan
and Hushon, 1976). Medical literature contains many reports
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of poisonings from absorption of nitrobenzene in shoe dyes
and laundry marking ink. These reports were common during
the 19th century and the first half of this century.
Poisoning following the wearing of newly-dyed wet shoes
was reported in 1900 (Levin, 1927). The poisoning can result
from nitrobenzene or aniline, both of which were used in shoe
dyes and which cause the same toxic symptoms. There have
been reports of cases of shoe dye poisoning in an army camp
(Levin, 1927) , in children who were given freshly dyed shoes
(Zeitoun, 1959; Graves, 1928; Levin, 1927), and in adults.
Generally the affected people are brought to the physician's
attention with symptoms of dizziness, bluish color of lips
and nails (cyanosis), headache, and sometimes coma. All
these sypmptoms are due to methemoglobin formation from the
absorbed nitrobenzene or aniline.
Cyanosis and poisoning of newborns who came in contact
with diapers or pads containing marking ink were very common.
Generally this occurred when the diapers or pads were freshly
stamped by the hospital laundry (Etteldorf, 1951; Ramsay and
Harvey, 1959; MacMath and Apley, 1954; Zeligs, 1929; Rayner,
1886). Often the imprint of the ink could be seen on the
infant's skin. Removal of the diaper or pad and thorough
washing of the skin usually reduced toxic symptoms, although
methylene blue and ascorbic acid have also been used to re-
lieve cyanosis. The toxicity is often more severe in prema-
ture infants who are in an incubator and surrounded by fumes
as well as the dye on the cloth (Etteldorf, 1951). Washing
C-8
-------
of the marked diapers or pads before their use removes the
hazard of absorption of nitrobenzene or aniline from the
ink.
In Egypt, "pure bitter almond oil" (a mixture of two to
ten percent nitrobenzene and 90 to 98 percent cottonseed
oil) has been rubbed on babies to remove crusts from the skin
and to protect the children from other diseases. Zeitoun
(1959) reports cases of nitrobenzene poisoning seen in Alex-
andria hospitals as a result of this practice.
Hamilton (1919) reported a case of chronic nitrobenzene
poisoning in a woman who used it as a cleaning fluid for many
years. The continuous dermal absorption caused her to exper-
ience symptoms of multiple neuritis, extreme indigestion and
hemorrhages of the larynx and pharynx.
Dermal absorption of nitrobenzene is the cause of many
of the chronic and acute toxic effects seen in nitrobenzene
workers (inhalation also accounts for industrial toxicity al-
though the routes of exposure often cannot be distinguished).
The amount of cutaneous absorption is a function of the am-
bient concentration, the amount of clothing worn, and the
relative humidity (high humidity increases absorption) (Dori-
gan and Hushon, 1976). A worker exposed to the TLV of 5 mg/-
m^ could absorb up to 25 n*g per day; one-third of that amount
would pass through the skin of a clothed man (Piotrowski,
1967). Pacseri and Magos (1958) measured ambient nitroben-
zene in industrial plants and found levels of up to eight
times the current TLV.
C-9
-------
Hamilton (1919) reported a case of acute, fatal, nitro-
benzene poisoning that resulted from a soap factory worker
spilling "oil of mirbane" on his clothes. Immediate removal
of the contaminated clothing would probably have prevented
his death.
There are reports of acute and chronic poisoning due to
skin absorption of dinitrobenzene by workers in munitions and
nitrobenzene plants. Dinitrobenzene is believed to be much
more toxic than nitrobenzene (Maiden, 1907). Ishihara, et
al. (1976) reported a case of poisoning where a worker
handled a cleaning mixture containing 0.5 percent dinitroben-
zene. The worker wore gloves, but the dinitrobenzene pene-
trated through the gloves to cause acute symptoms of methemo-
globinemia and hemolytic jaundice. Re^sek (1947) described
dinitrobenzene diffusion through the skin of munitions
workers. Some of these workers who had chronic dinitroben-
zene poisoning experienced an acute crisis after exposure to
sun or drinking alcohol (beer). Alcohol ingestion or chronic
alcoholism can also lower the lethal or toxic dose of nitro-
benzene (Dorigan and Hushon, 1976). This acute reaction
could occur as late as six weeks after toxic symptoms dis-
appeared .
Although there are many literature references dealing
with occupational exposure to nitrobenzene, there are few, if
any, of nitrobenzene exposure resulting from water intake.
Therefore, data derived from occupational exposure were
= ~,ployed to develop information for establishing the water
<- 5_^_v criterion in tnis document.
C-10
-------
PHARMACOKINETICS
Absorption
Nitrobenzene absorption can occur by all possible
routes, but it takes place mainly through the respiratory
tract and skin. At the TLV of 5/mg/m3, a nitrobenzene
worker can absorb 18 mg/day through the lungs and 7 mg/day
through the skin (Piotrowski, 1967). On the average, 80 per-
cent of the nitrobenzene vapors are retained in the human
respiratory tract (Piotrowski, 1977).
Nitrobenzene, as liquid and vapor, will pass directly
through the skin. The rate of vapor absorption depends on
the air concentration, ranging from 1 mg/hr at 5 mg/m3 con-
centration to 9 mg/hr at 20 mg/m3. Air temperature does
not affect the absorption rate, but an increase of relative
humidity from 33 to 67 percent will increase the absorption
rate by 40 percent. Work clothes reduce cutaneous absorption
of nitrobenzene vapors by 20 percent (Piotrowski, 1977).
Maximal cutaneous absorption of liquid nitrobenzene is
0.2 to 3 mg/cm^/hr depending on skin temperature. Elevated
skin temperature will increase absorption. Absorption will
decrease with duration of contact. Cutaneous absorption can
be significant in industry, since contamination of skin and
clothes of dye manufacture workers may reach levels of 2 and
25 mg/cm2, respectively (Piotrowski, 1977).
In view of this high level of absorption from lungs and
skin, individual protection of exposed workers is justified.
Distribution
Upon entry into the body, nitrobenzene enters the blood
C-ll
-------
stream. Here it reacts with the hemoglobin to form its oxi-
dation product, methemoglobin. Methemoglobin has a reduced
affinity for oxygen, and the reduced oxygen carrying capacity
of the blood is the cause of most of the toxic effects of
nitrobenzene, including its lethality. Methemoglobin levels
from nitrobenzene have ranged from 0.6 gm/100 ml in indus-
trial chronic exposure to 10 gm/100 ml in acute poisoning
(Pacseri and Magos, 1958; Myslak, et al. 1971). The normal
methemoglobin level is 0.5 gm/100 ml. Under normal condi-
tions methemoglobin will slowly be reduced to oxyhemoglobin,
the normal form of blood hemoglobin.
Pacseri and Magos (1958) have demonstrated that sulfhe-
moglobin is also formed in the blood after chronic exposure
to nitrobenzene. They found average sulfhemoglobin levels in
nitrobenzene workers of 0.27 gm/100 ml (compared to the upper
limit of normal of 0.18 gm/100 ml). Pacseri postulated that
since blood sulfhemoglobin disappears more slowly than methe-
moglobin, it is a more sensitive indicator of nitrobenzene
exposure. Sulfhemoglobin may be more specific than sensitive
because methemoglobin is normally found in the blood but
sulfhemoglobin is not.
Uehleke (1964) measured the velocity of methemoglobin
formation from nitrobenzene in cats. He found the rate to be
variable and not related to the blood concentration of nitro-
benzene, although the methemoglobin formation velocity was
maximal in each animal at the time of highest blood concen-
tration of nitrobenzene. He also found that metabolites of
nitrobenzene are able to oxidize hemoglobin. Methemoglobin
formation from nitrobenzene has also been demonstrated
C-12
-------
in vitro (Dorigan and Hushon, 1976, cited from von Oettingen,
1941; Kusumoto and Nakajima, 1970).
Further indications of the presence of nitrobenzene in
the blood are the production of hemolytic anemia after acute
exposure (Harrison, 1977) and the alteration of the sodium
and potassium permeability of erythrocytes by derivatives of
nitrobenzene (Cooke, et al. 1968).
Nitrobenzene is very lipid soluble, with an oil to water
partition coefficient of 800. In a rat study the ratio of
concentration of nitrobenzene in adipose tissue versus blood
in internal organs and muscle was approxiraatley 10:1 one hour
after an intravenous dose (Piotrowski, 1977). Rabbits intu-
bated with 0.25 ml of nitrobenzene had 50 percent of the com-
pound accumulated unchanged in tissues within two days after
the intubation (Dorigan and Hushon, 1976).
Dresbach and Chandler (1918) have shown cerebellar dis-
turbance in dogs and birds from nitrobenzene vapors, although
they found blood changes as described above to be the predom-
inant effects in other mammals they tested. A histologic
study attributed these effects to changes in the Purkinje
cells of the cerebellum. Reports of the effect of nitroben-
zene on the liver vary from description of liver damage from
accumulated nitrobenzene (Dorigan and Hushon, 1976) to the
statement that nitrobenzene does not cause severe renal nor
liver damage (Goldstein, 1975). Goldwater (1947) has de-
scribed hyperplasia of the erythropoietic centers of the bone
marrow in workers chronically exposed to nitrobenzene, but he
C-13
-------
concluded that the hyperplasia is a secondary result of the
hemolytic effect of the compound. Makotchenko and Akhmetov
(1972) observed secretory changes of the adrenal cortex of
guinea pigs given nitrobenzene every other day at a dose of
0.2 gm/kg for six months.
Metabolism
Available information on nitrobenzene metabolism is
based on animal experiments and fragmentary human data.
There are two main metabolic pathways: 1) reduction to
aniline followed by hydroxylation to aminophenols, and 2)
direct hydroxylation of nitrobenzene to form nitrophenols.
Further reduction of nitrophenols to aminophenols may also
occur (Piotrowski, 1977). The rate of nitrobenzene metabo-
lism is independent of the dose in later stages of acute or
chronic intoxication. This can cause its accumulation in
highly lipid tissues (Dorigan and Hushon, 1976).
The reduction of nitrobenzene to aniline occurs via the
unstable intermediates, nitrosobenzene and phenyl hydro-
xylamine, both of which are toxic and have pronounced methe-
moglobinemic capacity. The reactions occur in the cytoplas-
mic and microsomal fractions of liver cells by the nitro- re-
ductase enzyme system (Pouts and Brodie, 1957). This enzyme
system is active in mice, guinea pigs, and rabbits, and is
less active in rats and dogs. The aniline is then excreted
as an acetyl derivative or hydroxylated and excreted as an
aminophenal. Reddy, et al. (1976) showed that the gut flora
C-14
-------
of rats was needed for the reduction of nitrobenzene and sub-
sequent methemoglobin formation.
The hydroxylation of nitrobenzene to nitrophenols does
not occur in the microsomal fraction. The reaction proceeds
via peroxidase in the presence of oxygen (Piotrowski, 1977).
Robinson, et al. (1951) studied nitrobenzene metabolism
in the rabbit using ^C labeled material. The main meta-
bolic product found was p-aminophenol (35 percent) which was
formed via phenylhydroxylamine. Seven phenols and aniline
were detected as metabolite.s within 48 hours of a dose of 150
to 200 mgAg body weight of nitrobenzene. Nitrobenzene was
retained somewhat in the rabbits; its metabolites were de-
tected in urine one week after dosing. Little unchanged
nitrobenzene was excreted in the urine. The major urinary
metabolites were p-aminophenol, nitrophenols, and nitro-
catechol. These constituted 55 percent of the urinary
metabolites and were excreted conjugated with sulfuric and
glucuronic acids. About one percent of the dose was expired
as radiolabeled carbon dioxide.
Yamada (1958) studied nitrobenzene metabolism in rabbits
in a three-month subcutaneous exposure study. He found that
urinary excretion of detoxification products varied in the
early stage of exposure, but did not in the later stages.
The reduction and hydroxylation pathways all became depressed
during the later stages of this chronic poisoning study.
Parke (1956) reports metabolites of nitrobenzene iso-
lated four to five days after administering 0.25 mg/kg orally
as a single dose in the rabbit (see Table 2) .
C-15
-------
TABLE 2
Metabolic Fate of a Single Oral Dose (0.25 g/kg.) of [14C] Nitrobenzene
in the Rabbit During 4-5 Days After Dosing (Parke, 1956)
Metabolite
Percentage of Dose (average)
Respiratory CO2
Nitrobenzene
Aniline
o-N i trophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Nitrocatechol
Nitroquinol
p-N i tropheny1
Mercapturic acid
(Total urinary radio-
activity)
Metabolized nitrobenzene
in feces
Metabolized nitrobenzene
in tissues
Total accounted for
1 "I
0.6* h-2 in expired air
0.4+_J '
0.1
9
9
3
4 I 58 in
31 urine
0.7
0.1
0.3
w^
(58)
9§
15-20
85-90%
60 total
* 0.5% in the expired air and <0.1% in the urine.
+ 0.3% in the urine and <0.1% in the expired air.
§ 6% of the dose was present in the feces as p-aminophenol.
An investigation of the metabolism of^"* C nitrobenzene
in the cattle tick, Boophilus microplusf and spider, Nephia
plumipes, was done by Holder and Wilcox (1973). They found
that the tick metabolized nitrobenzene to nitrophenol and
aniline whereas no free phenols were found as metabolites in
the spider. Aniline was the major metabolic product in both
species.
Nitrobenzene in water can be degraded by some bacteria,
such as Azobacter agilis, if present in sufficiently/small
C-16
-------
amounts. Nitrobenzene tends to inhibit its own degradation
at concentrations above 0.02 to 0.03 mg/1 (Dorigan and
Hushon, 1976; Lu and Metcalf, 1975).
Lu and Metcalf (1975) studied nitrobenzene in a model
aquatic ecosystem to assess biodegradation and biomagnifi-
cation. The ecosystem consisted of green filamentous algae,
Oedogonium cardiacium, snails, Physa, water fleas, Daphnia
magna, mosquito larvae, Culex quinquifasciatus, and mosquito
fish, Gambusia affinis, under controlled atmospheric condi-
tions. 0.005 to 0.5 mg/m3 (0.01 to 0.1 ppm) of 14C-labeled
nitrobenzene was added to the water and animals were removed
for analysis after 24 to 48 hours. The radiolabeled metabo-
lites were extracted and separated by thin layer chroma-
tography. The distribution of nitrobenzene and its degrada-
tion products is listed in Table 3.
C-17
-------
TABLE 3
Distribution of Nitrobenzene and Degradation Products in Model Aquatic Ecosystem (Lu and Metcalf, 1975)
o
i
M
OQ
Nitrobenzene equivalents, ppm
Total 14C
Nitrobenzene
Aniline
Acetanilide
Aminophenolsk
Nitrophenolsk
Polar
Unextractable
Rfa
0.72
0.60
0.35
0.20
0.10
0.0
H20
0.53755
0.50681
0.01262
0.00180
0.00106
0.00466
0.00896
0.00164
Oedognoium
(alga)
0.0690
0.0162
0.0032
0.0160
0.0080
0.0016
0.0240
—
Daphnia
(daphnia)
0.1812
0.0709
0.0079
-
0.0315
0.0394
0.0315
—
Culex
(mosquito)
0.5860
0.3952
0.0272
0.0272
-
0.1226
0.0138
-
Physa
(snail)
0.6807
0.3886
0.0169
0.0169
-
0.2190
0.0393
—
Gambusia
(fish)
4.9541
4.0088
0.2963
0.3527
0.0986
0.0847
0.1130
—
a TLC with benzene:acetone:Skellysolve B (bp 60-68°C):diethylamine=65:25:25:5 (v/v).
b The isomers could not be separated reliably because of small amounts and similar Rf values
-------
Nitrobenzene was neither stored nor ecologicaly magni-
fied. It was reduced to aniline in all organisms, acetylated
in fish and water extracts only, and hydroxylated to nitro-
phenols by mosquito Larvae and snails. The metabolites of
nitrobenzene formed by the different organisms are illus-
trated in Figure 1.
I
n
n
. '
l_Li
•a. £.«• !_£!_
Figure 1: Relative detoxication capacities of key organisms
of a model aquatic ecosystem following treatment with radio-
active nitrobenzene (Lu and Metcalf, 1975).
Excretion
In man the primary known excretion products of nitroben-
zene are p-aminophenol and p-nitrophenol which appear in the
urine after chronic or acute exposure. In experimental in-
halation exposure to nitrobenzene, p-nitrophenol was formed
with the efficiency of 6 to 21 percent. The efficiency of
C-19
-------
p-aminophenol formation is estimated from observation of
acute poisoning cases where the molar ratio of excreted
p-nitrophenol to p-aminophenol is two to one, since p-amino-
phenol is not formed at a detectable level in short subacute
exposure (Piotrowski, 1977).
Ikeda and Kita (1964) measured the urinary excretion of
p-nitrophenol and p-aminophenol in a patient admitted to a
hospital with toxic symptoms resulting from a 17-month
chronic industrial exposure to nitrobenzene. The results of
their study are shown in Figure 2, which demonstrates that
the rate of excretion of the two metabolites parallels the
level of methemoglobin in blood. The authors exposed five
adult rats to a nitrobenzene vapor of 125 mg/m^ (25 ppm)
for eight hours and measured the subsequent excretion of
p-aminophenol and p-nitrophenol. The results are shown in
Figure 3. The urinary excretion ratio of p-aminophenol and
p-nitrophenol corresponded to their findings in the human
case.
Studies of nitrobenzene concentrations in the blood
of an acutely exposed person indicate that the compound re-
mains in the human body for a prolonged period of time.
Similar observations have been made from excretion of the two
urinary metabolites in patients treated for acute or subacute
poisoning. The excretion coefficient of urinary p-nitro-
phenol, followed for three weeks, is about 0.008 per hour.
Metabolic transformation and excretion of nitrobenzene in man
is slower by an order of magnitude than in rats or rabbits
(Piotrowski, 1977).
C-20
-------
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Figure 2. Changes in the levels of total hemoglobin and methae-
moglobin in blood and of p-nitrophenol and p-aminophenol in
urine. The usual daily volume of urine was about 1 litre.
Figure 3. Excretion of p-nitrophenol and p-aminophenol in the
urine of rats exposed to nitrobenzene.
C-21
-------
Because of the slow rate of nitrobenzene metabolism in
man, the concentration of p-nitrophenol in the urine in-
creases for about four days during exposure and the concen-
tration on the first day is only about 40 percent of the peak
value. An estimate of the mean daily dose of nitrobenzene in
chronic industrial exposure can be obtained by the measure-
ment of urinary p-nitrophenol in specimens taken on each of
the last three days of the work week. The level of. nitroben-
zene exposure can be approximated using the formula y =
O.lSz, where y is the daily excretion of urinary p-nitro-
phenol in mg/day and z is the mean daily dose of absorbed
nitrobenzene in mg (Piotrowski, 1967). The extended systemic
retention and slow excretion of metabolites of nitrobenzene
in man is determined by the low rate of metabolic transforma-
tion (reduction and hydroxylation) of the nitrobenzene it-
self. The conjugation and excretion of the metabolites,
p-nitrophenol and p-aminophenol, is rapid (Piotrowski,
1977).
The urinary metabolites in man account for only 20 to 30
percent of the nitrobenzene dose; the fate of the rest of the
metabolites is not known (Piotrowski, 1977). Parke (1956)
studied 1*C - nitrobenzene metabolism in rabbits and was able
to account for 85 to 90 percent of the dose which was admin-
istered by intubation. One percent of the nitrobenzene was
exhaled as CC>2 in air, and 0.6 percent was exhaled as un-
changed nitrobenzene. Fifty-eight percent of the dose ap-
peared as urinary metabolites, p-aminophenol, nitrophenols,
aminophenols, nitrocatechols, and aniline. Thirty percent of
C-22
-------
of 71, the steady-state bioconcentration factor for nitroben-
zene is estimated to be 15. An adjustment factor of 2.3/8.0
= 0.2875 can be used to adjust the estimated BCF from the 8.0
percent lipids on which the equation is based to the 2.3 per-
cent lipids that is the weighted average for consumed fish
and shellfish. Thus, the weighted average bioconcentration
factor for nitrobenzene and the edible portion of all aquatic
organisms consumed by Americans is calculated to be 15 x
0.2875 = 4.3.
Inhalation
Nitrobenzene is readily absorbed through the lungs with
retention of up to 80 percent (Piotrowski, 1967). There are
reports of nitrobenzene poisoning from inhalation of an ex-
terminator spray for bedbugs which was sprayed on a child's
mattress (Stevenson and Forbes, 1942; Nabarro, 1948). Poi-
sonings have also resulted from inhaled nitrobenzene used as
a scent in perfume and soap (Dorigan and Hushon, 1976).
Chronic and acute poisoning from exposure to nitrobenzene
fumes in production plants are well documented (Dorigan and
Hushon, 1976; Browning, 1950; Zeligs, 1929; Hamilton, 1919),
but since nitrobenzene is also absorbed through the skin, in-
dustrial poisoning cannot be attributed to inhalation alone.
A worker exposed to the TLV for nitrobenzene of 5 mg/m3
would absorb 18 mg/day through the lungs (Piotrowski, 1967).
Dermal
Nitrobenzene is highly fat-soluble and can be absorbed
through the skin at rates as high as 2 mg/cm2/hr (Dorigan
and Hushon, 1976). Medical literature contains many reports
C-7
-------
of poisonings from absorption of nitrobenzene in shoe dyes
and laundry marking ink. These reports were common during
the 19th century and the first half of this century.
Poisoning following the wearing of newly-dyed wet snoes
was reported in 1900 (Levin, 1927). The poisoning can result
from nitrobenzene or aniline, both of which were used in shoe
dyes and which cause the same toxic symptoms. There have
been reports of cases of shoe dye poisoning in an army camp
(Levin, 1927), in children who were given freshly dyed shoes
(Zeitoun, 1959; Graves, 1928; Levin, 1927), and in adults,
Generally the affected people are brought to the physician's
attention with symptoms of dizziness, bluish color of lips
and nails (cyanosis), headache, and sometimes coma. All
these sypmptoms are due to methemoglobin formation from the
absorbed nitrobenzene or aniline.
Cyanosis and poisoning of newborns who came in contact
with diapers or pads containing marking ink were very common.
Generally this occurred when the diapers or pads were freshly
stamped by the hospital laundry (Etteldorf, 1951; Ramsay and
Harvey, 1959; MacMath and Apley, 1954; Zeligs, 1929; Rayner,
1886). Often the imprint of the ink could be seen on the
infant's skin. Removal of the diaper or pad and thorough
washing of the skin usually reduced toxic symptoms, although
methylene blue and ascorbic acid have also been used to re-
lieve cyanosis. The toxicity is often more severe in prema-
ture infants who are in an incubator and surrounded by fumes
as well as the dye on the cloth (Etteldorf, 1951). Washing
C-8
-------
of the marked diapers or pads before their use removes the
hazard of absorption of nitrobenzene or aniline from the
ink.
In Egypt, "pure bitter almond oil" (a mixture of two to
ten percent nitrobenzene and 90 to 98 percent cottonseed
oil) has been rubbed on babies to remove crusts from the skin
and to protect the children from other diseases. Zeitoun
(1959) reports cases of nitrobenzene poisoning seen in Alex-
andria hospitals as a result of this practice.
Hamilton (1919) reported a case of chronic nitrobenzene
poisoning in a woman who used it as a cleaning fluid for many
years. The continuous dermal absorption caused her to exper-
ience symptoms of multiple neuritis, extreme indigestion and
hemorrhages of the larynx and pharynx.
Dermal absorption of nitrobenzene is the cause of many
of the chronic and acute toxic effects seen in nitrobenzene
workers (inhalation also accounts for industrial toxicity al-
though the routes of exposure often cannot be distinguished).
The amount of cutaneous absorption is a function of the am-
bient concentration, the amount of clothing worn, and the
relative humidity (high humidity increases absorption) (Dori-
gan and Hushon, 1976). A worker exposed to the TLV of 5 mg/-
m-* could absorb up to 25 irg per day; one-third of that amount
would pass through the skin of a clothed man (Piotrowski,
1967). Pacseri and Magos (1958) measured ambient nitroben-
zene in industrial plants and found levels of up to eight
times the current TLV.
C-9
-------
Hamilton (1919) reported a case of acute, fatal, nitro-
benzene poisoning that resulted from a soap factory worker
spilling "oil of mirbane" on his clothes. Immediate removal
of the contaminated clothing would probably have prevented
his death.
There are reports of acute and chronic poisoning due to
skin absorption of dinitrobenzene by workers in munitions and
nitrobenzene plants. Dinitrobenzene is believed to be much
more toxic than nitrobenzene (Maiden, 1907). Ishihara, et
al. (1976) reported a case of poisoning where a worker
handled a cleaning mixture containing 0.5 percent dinitroben-
zene. The worker wore gloves, but the dinitrobenzene pene-
trated through the gloves to cause acute symptoms of methemo-
globinemia and hemolytic jaundice. Rejsek (1947) described
dinitrobenzene diffusion through the skin of munitions
workers. Some of these workers who had chronic dinitroben-
zene poisoning experienced an acute crisis after exposure to
sun or drinking alcohol (beer). Alcohol ingestion or chronic
alcoholism can also lower the lethal or toxic dose of nitro-
benzene (Dorigan and Hushon, 1976). This acute reaction
could occur as late as six weeks after toxic symptoms dis-
appeared.
Although there are many literature references dealing
with occupational exposure to nitrobenzene, there are few, if
any, of nitrobenzene exposure resulting from water intake.
Therefore, data derived from occupational exposure were
^•"iployed to develop information for establishing the water
•.2_i_v criterion in tnis document.
C-10
-------
PHARMACOKINETICS
Absorption
Nitrobenzene absorption can occur by all possible
routes, but it takes place mainly through the respiratory
tract and skin. At the TLV of S/mg/m3f a nitrobenzene
worker can absorb 18 mg/day through the lungs and 7 ing/day
through the skin (Piotrowski, 1967). On the average, 80 per-
cent of the nitrobenzene vapors are retained in the human
•
respiratory tract (Piotrowski, 1977).
Nitrobenzene, as liquid and vapor, will pass directly
through the skin. The rate of vapor absorption depends on
the air concentration, ranging from 1 mg/hr at 5 mg/m3 con-
centration to 9 mg/hr at 20 mg/m3. Air temperature does
not affect the absorption rate, but an increase of relative
humidity from 33 to 67 percent will increase the absorption
rate by 40 percent. Work clothes reduce cutaneous absorption
of nitrobenzene vapors by 20 percent (Piotrowski, 1977).
Maximal cutaneous absorption of liquid nitrobenzene is
0.2 to 3 mg/cm^/hr depending on skin temperature. Elevated
skin temperature will increase absorption. Absorption will
decrease with duration of contact. Cutaneous absorption can
be significant in industry, since contamination of skin and
clothes of dye manufacture workers may reach levels of 2 and
25 mg/cm2, respectively (Piotrowski, 1977).
In view of this high level of absorption from lungs and
skin, individual protection of exposed workers is justified.
Distribution
Upon entry into the body, nitrobenzene enters the blood
C-ll
-------
stream. Here it reacts with the hemoglobin to form its oxi-
dation product, methemoglobin. Methemoglobin has a reduced
affinity for oxygen, and the reduced oxygen carrying capacity
of the blood is the cause of most of the toxic effects of
nitrobenzene, including its lethality. Methemoglobin levels
from nitrobenzene have ranged from 0.6 gm/100 ml in indus-
trial chronic exposure to 10 gm/100 ml in acute poisoning
(Pacseri and Magos, 1958; Myslak, et al. 1971). The normal
methemoglobin level is 0.5 gm/100 ml. Under normal condi-
tions methemoglobin will slowly be reduced to oxyhemoglobin,
the normal form of blood hemoglobin.
Pacseri and Magos (1958) have demonstrated that sulfhe-
moglobin is also formed in the blood after chronic exposure
to nitrobenzene. They found average sulfhemoglobin levels in
nitrobenzene workers of 0.27 gm/100 ml (compared to the upper
limit of normal of 0.18 gm/100 ml). Pacseri postulated that
since blood sulfhemoglobin disappears more slowly than methe-
moglobin, it is a more sensitive indicator of nitrobenzene
exposure. Sulfhemoglobin may be more specific than sensitive
because methemoglobin is normally found in the blood but
sulfhemoglobin is not.
Uehleke (1964) measured the velocity of methemoglobin
formation from nitrobenzene in cats. He found the rate to be
variable and not related to the blood concentration of nitro-
benzene, although the methemoglobin formation velocity was
maximal in each animal at the time of highest blood concen-
tration of nitrobenzene. He also found that metabolites of
nitrobenzene are able to oxidize hemoglobin. Methemoglobin
formation from nitrobenzene has also been demonstrated
C-12
-------
in vitro (Dorigan and Hushon, 1976, cited from von Oettingen,
1941; Kusumoto and Nakajima, 1970).
Further indications of the presence of nitrobenzene in
the blood are the production of hemolytic anemia after acute
exposure (Harrison, 1977) and the alteration of the sodium
and potassium permeability of erythrbcytes by derivatives of
nitrobenzene (Cooke, et al. 1968).
Nitrobenzene is very lipid soluble, with an oil to water
partition coefficient of 800. In a rat study the ratio of
concentration of nitrobenzene in adipose tissue versus blood
in internal organs and muscle was approximatley 10:1 one hour
after an intravenous dose (Piotrowski, 1977). Rabbits intu-
bated with 0*25 ml of nitrobenzene had 50 percent of the com-
pound accumulated unchanged in tissues within two days after
the intubation (Dorigan and Hushon, 1976).
Dresbach and Chandler (1918) have shown cerebellar dis-
turbance in dogs and birds from nitrobenzene vapors, although
they found blood changes as described above to be the predom-
inant effects in other mammals they tested. A histologic
study attributed these effects to changes in the Purkinje
cells of the cerebellum. Reports of the effect of nitroben-
zene on the liver vary from description of liver damage from
accumulated nitrobenzene (Dorigan and Hushon, 1976) to the
statement that nitrobenzene does not cause severe renal nor
liver damage (Goldstein, 1975). Goldwater (1947) has de-
scribed hyperplasia of the erythropoietic centers of the bone
marrow in workers chronically exposed to nitrobenzene, but he
C-13
-------
concluded that the hyperplasia is a secondary result of the
hemolytic effect of the compound. Makotchenko and Akhmetov
(1972) observed secretory changes of the adrenal cortex of
guinea pigs given nitrobenzene every other day at a dose of
0.2 gin/kg for six months.
Metabolism
Available information on nitrobenzene metabolism is
based on animal experiments and fragmentary human data.
There are two main metabolic pathways: 1) reduction to
aniline followed by hydroxylation to aminophenols, and 2)
direct hydroxylation of nitrobenzene to form nitrophenols.
Further reduction of nitrophenols to aminophenols may also
occur (Piotrowski, 1977). The rate of nitrobenzene metabo-
lism is independent of the dose in later stages of acute or
chronic intoxication. This can cause its accumulation in
highly lipid tissues (Dorigan and Hushon, 1976).
The reduction of nitrobenzene to aniline occurs via the
unstable intermediates, nitrosobenzene and phenyl hydro-
xylamine, both of which are toxic and have pronounced methe-
moglobinemic capacity. The reactions occur in the cytoplas-
mic and microsomal fractions of liver cells by the nitro- re-
ductase enzyme system (Fouts and Brodie, 1957). This enzyme
system is active in mice, guinea pigs, and rabbits, and is
less active in rats and dogs. The aniline is then excreted
as an acetyl derivative or hydroxylated and excreted as an
aminophenal. Reddy, et al. (1976) showed that the gut flora
C-14
-------
of rats was needed for the reduction of nitrobenzene and sub-
sequent methemoglobin formation.
The hydroxylation of nitrobenzene to nitrophenols does
not occur in the microsomal fraction. The reaction proceeds
via peroxidase in the presence of oxygen (Piotrowski, 1977).
Robinson, et al. (1951) studied nitrobenzene metabolism
in the rabbit using l^c labeled material. The main meta-
bolic product found was p-aminophenol (35 percent) which was
formed via phenylhydroxylamine. Seven phenols and aniline
were detected as metabolites within 48 hours of a dose of 150
to 200 mg/kg body weight of nitrobenzene. Nitrobenzene was
retained somewhat in the rabbits; its metabolites were de-
tected in urine one week after dosing. Little unchanged
nitrobenzene was excreted in the urine. The major urinary
metabolites were p-aminophenol, nitrophenols, and nitro-
catechol. These constituted 55 percent of the urinary
metabolites and were excreted conjugated with sulfuric and
glucuronic acids. About one percent of the dose was expired
as radiolabeled carbon dioxide.
Yamada (1958) studied nitrobenzene metabolism in rabbits
in a three-month subcutaneous exposure study. He found that
urinary excretion of detoxification products varied in the
early stage of exposure, but did not in the later stages.
The reduction and hydroxylation pathways all became depressed
during the later stages of this chronic poisoning study.
Parke (1956) reports metabolites of nitrobenzene iso-
lated four to five days after administering 0.25 mg/kg orally
as a single dose in the rabbit (see Table 2).
C-15
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TABLE 2
Metabolic Fate of a Single Oral Dose (0.25 g/kg.) of [14C] Nitrobenzene
in the Rabbit During 4-5 Days After Dosing (Parke, 1956)
Metabolite
Percentage of Dose (average)
Respiratory CO2
Nitrobenzene
Aniline
o-Nitrophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Nitrocatechol
Nitroquinol
p-Nifcrophenyl
Mercapturic acid
(Total urinary radio-
activity)
Metabolized nitrobenzene
in feces
Metabolized nitrobenzene
in tissues
Total accounted for
1 ~j
0.6* j-2 in expired
air
0.4+.J
0.1
9
9
3
4
31
0.7
0.1
0.3
(58)
9§
15-20
85-90%
60 total
58 in
urine
* 0.5% in the expired air and <0.1% in the urine.
+ 0.3% in the urine and <0.1% in the expired air.
§ 6% of the dose was present in the feces as p-aminophenol.
An investigation of the metabolism ofl^ c nitrobenzene
in the cattle tick, Boophilus microplus, and spider, Nephia
plumipes, was done by Holder and Wilcox (1973). They found
that the tick metabolized nitrobenzene to nitrophenol and
aniline whereas no free phenols were found as metabolites in
the spider. Aniline was the major metabolic product in both
species.
Nitrobenzene in water can be degraded by some bacteria,
such as Azobacter agilis, if present in sufficiently/small
C-16
-------
amounts. Nitrobenzene tends to inhibit its own degradation
at concentrations above 0.02 to 0.03 mg/1 (Dorigan and
Hushon, 1976; Lu and Metcalf, 1975).
Lu and Metcalf (1975) studied nitrobenzene in a model
aquatic ecosystem to assess biodegradation and biomagnifi-
cation. The ecosystem consisted of green filamentous algae,
Oedogonium cardiacium, snails, Physa, water fleas, Daphnia
magna, mosquito larvae, Culex quinquifasciatus, and mosquito
fish, Gambusia affinis, under controlled atmospheric condi-
tions. 0.005 to 0.5 mg/m3 (0.01 to 0.1 ppm) of 14C-labeled
nitrobenzene was added to the water and animals were removed
for analysis after 24 to 48 hours. The radiolabeled metabo-
lites were extracted and separated by thin layer chroma-
tography. The distribution of nitrobenzene and its degrada-
tion products is listed in Table 3.
C-17
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TABLE 3
Distribution of Nitrobenzene and Degradation Products in Model Aquatic Ecosystem (Lu and Metcalf, 1975)
n
i
M
00
Nitrobenzene equivalents, ppm
Total 14C
Nitrobenzene
Aniline
Acetanilide
Aminophenolsk
Nitrophenols*3
Polar
Unextractable
Rfa
0.72
0.60
0.35
0.20
0.10
0.0
H20
0.53755
0.50681
0.01262
0.00180
0.00106
0.00466
0.00896
0.00164
Oedognoium
(alga)
0.0690
0.0162
0.0032
0.0160
0.0080
0.0016
0.0240
—
Daphnia
(daphnia)
0.1812
0.0709
0.0079
-
0.0315
0.0394
0.0315
—
Culex
(mosquito)
0.5860
0.3952
0.0272
0.0272
-
0.1226
0.0138
-
Physa
(snail)
0.6807
0.3886
0.0169
0.0169
-
0.2190
0.0393
—
Gambusia
(fish)
4.9541
4.0088
0.2963
0.3527
0.0986
0.0847
0.1130
-
a TLC with benzene:acetone:Skellysolve B (bp 60-68°C):diethylamine=65:25:25:5 (v/v).
b The isomers could not be separated reliably because of small amounts and similar Rf values
-------
Nitrobenzene was neither stored nor ecologicaly magni-
fied. It was reduced to aniline in all organisms, acetylated
in fish and water extracts only, and hydroxylated to nitro-
phenols by mosquito Larvae and snails. The metabolites of
nitrobenzene formed by the different organisms are illus-
trated in Figure 1.
LadL
n
.a.,
-*S
Figure 1: Relative detoxication capacities of key organisms
of a model aquatic ecosystem following treatment with radio-
active nitrobenzene (Lu and Metcalf, 1975).
Excretion
In man the primary known excretion products of nitroben-
zene are p-aminophenol and p-nitrophenol which appear in the
urine after chronic or acute exposure. In experimental in-
halation exposure to nitrobenzene, p-nitrophenol was formed
with the efficiency of 6 to 21 percent. The efficiency of
C-19
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p-aminophenol formation is estimated from observation of
acute poisoning cases where the molar ratio of excreted
p-nitrophenol to p-aminophenol is two to one, since p-amino-
phenol is not formed at a detectable level in short subacute
exposure (Piotrowski, 1977).
Ikeda and Kita (1964) measured the urinary excretion of
p-nitrophenol and p-aminophenol in a patient admitted to a
hospital with toxic symptoms resulting from a 17-month
chronic industrial exposure to nitrobenzene. The results of
their study are shown in Figure 2, which demonstrates that
the rate of excretion of the two metabolites parallels the
level of methemoglobin in blood. The authors exposed five
adult rats to a nitrobenzene vapor of 125 mg/m^ (25 ppm)
for eight hours and measured the subsequent excretion of
p-aminophenol and p-nitrophenol. The results are shown in
Figure 3. The urinary excretion ratio of p-aminophenol and
p-nitrophenol corresponded to their findings in the human
case.
Studies of nitrobenzene concentrations in the blood
of an acutely exposed person indicate that the compound re-
mains in the human body for a prolonged period of time.
Similar observations have been made from excretion of the two
urinary metabolites in patients treated for acute or subacute
poisoning. The excretion coefficient of urinary p-nitro-
phenol, followed for three weeks, is about 0.008 per hour.
Metabolic transformation and excretion of nitrobenzene in man
is slower by an order of magnitude than in rats or rabbits
(Piotrowski, 1977).
C-20
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s is ao
SEPTEMBER
O
|
^ Z
t. a
g:
P
Figure 2. Changes in the levels of total hemoglobin and methae-
moglobin in blood and of p-nitrophenol and p-aminophenol in
urine. The usual daily volume of urine was about 1 litre.
3000,
Figure 3. Excretion of p-nitrophenol and p-aminophenol in the
urine of rats exposed to nitrobenzene.
C-21
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Because of the slow rate of nitrobenzene metabolism in
man, the concentration of p-nitrophenol in the urine in-
creases for about four days during exposure and the concen-
tration on the first day is only about 40 percent of the peak
value. An estimate of the mean daily dose of nitrobenzene in
chronic industrial exposure can be obtained by the measure-
ment of urinary p-nitrophenol in specimens taken on each of
the last three days of the work week. The level of nitroben-
zene exposure can be approximated using the formula y =
0.18z, where y is the daily excretion of urinary p-nitro-
phenol in mg/day and z is the mean daily dose of absorbed
nitrobenzene in mg (Piotrowski, 1967). The extended systemic
retention and slow excretion of metabolites of nitrobenzene
in man is determined by the low rate of metabolic transforma-
tion (reduction and hydroxylation) of the nitrobenzene it-
self. The conjugation and excretion of the metabolites,
p-nitrophenol and p-aminophenol, is rapid (Piotrowski,
1977).
The urinary metabolites in man account for only 20 to 30
percent of the nitrobenzene dose; the fate of the rest of the
metabolites is not known (Piotrowski, 1977). Parke (1956)
studied l^c _ nitrobenzene metabolism in rabbits and was able
to account for 85 to 90 percent of the dose which was admin-
istered by intubation. One percent of the nitrobenzene was
exhaled as CC>2 m air, and 0.6 percent was exhaled as un-
changed nitrobenzene. Fifty-eight percent of the dose ap-
peared as urinary metabolites, p-aminophenol, nitrophenols,
aminophenols, nitrocatechols, and aniline. "Thirty percent of
C-22
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the nitrobenzene was still in the rabbit tissue four to five
days after dosing, and nine percent of the nitrobenzene
metabolizes was in the feces.
Urinary p-nitrophenol in man is determined after hydrol-
ysis of the conjugated metabolites. Analytical methodology
(of which there are several methods) involves removal of in-
terfering color substances, hydrolysis, extraction of
p-nitrophenol, re-extraction into an aqueous system, reduc-
tion to a p-aminophenol, and reaction to indophenol, which is
a blue colored product. The sensitivity is 5 ug per sample
(Piotrowski, 1977).
EFFECTS
Acute, Sub-acute, and Chronic Toxicity
Acute exposure to nitrobenzene can occur from accidental
or suicidal ingestion of the liquid nitrobenzene or ingestion
as false bitter almond oil in food or medicine. Cutaneous
absorption causing acute toxic reactions can result from
wearing wet, freshly dyed shoes (Levin, 1927), marking ink
used on diapers or protective pads (Etteldorf, 1951), use of
soap or skin oil containing nitrobenzene (Zeitoun, 1959), or
an untreated spill of nitrobenzene on the skin in an indus-
trial plant (Hamilton, 1919). The fatal dose of nitrobenzene
in humans varies widely; values from less than 1 ml to over
400 ml have been reported (Wirtschafter and Wolpaw, 1944)»
Chronic toxic effects in man generally result from industrial
exposure to vapors that are absorbed through the lungs or the
skin. One case of chronic toxicity was reported in a woman
who used nitrobenzene as a cleaning solution for many years
(Hamilton, 1919).
C-23
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Symptoms of chronic occupational nitrobenzene absorption
are cyanosis, methemoglobinemia, jaundice, anemia, sulfhemo-
globinemia, presence of Heinz bodies in the erythrocytes,
dark colored urine, and the presence of nitrobenzene metabo-
lites (e.g. nitrophenol) in the urine (Pacseri and Magos,
1958; Hamilton, 1919; Wuertz, et al. 1964; Browning, 1950;
Maiden, 1907; Piotrowski, 1967).
The symptoms of dinitrobenzene poisoning include those
found in nitrobenzene toxicity as well as abdominal pain,
weakness, enlarged liver, and basophilic granulations of red
corpuscles (Beritic, 1956; Maiden, 1907). Dinitrobenzene
poisoning also causes unequal responses in different exposed
workers.
The outstanding symptom of acute nitrobenzene poisoning
is cyanosis as a result of methemoglobin formation (up to 80
percent) (Piotrowski, 1967). If the cyanosis is severe or
prolonged the patient will go into coma and may die. Often
anemia is seen a week or two after acute poisoning as a re-
sult of the hemolytic effect of nitrobenzene (Stevenson and
Forbes, 1942). Suicidal ingestion of nitrobenzene has been
reported (Nabarro, 1948; Leinoff, 1936; Myslak, et al. 1971),
and it has also been used unsuccessfully to induce abortion
(Nabarro, 1948; Dorigan and Hushon, 1976). Harrison (1977)
reported a case of poisoning from an aniline-nitrobenzene
mixture which was accidentally ingested from a pipette by a
chemistry student. The mortality due to ingested nitroben-
zene in the above cases was variable, depending on the health
C-24
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of the patients and the treatments they received. Common
treatments include gavage, transfusions, oxygen therapy,
methylene blue, ascorbic acid, and toluidine blue. Treatment
is usually directed to reduce the methemoglobinemia which is
the immediate effect, and often the cause of death in nitro-
benzene poisoning. Death has resulted from intake of less
than 1 ml of nitrobenzene (Wirtschafter and Wolpaw, 1944).
Some of the reported toxicity values are summarized in
Table 4 (Fairchild, 1977). The term LDLo designates the
lowest reported lethal dose and TDLo is the lowest published
toxic dose.
C-25
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TABLE 4
Acute Toxicity Values (Fairchild, 1977)
Animal Route Toxic Dose
woman oral TDLo: 200 mg/kg
human oral LDLo: 5 mg/kg
rat oral LD50: 640 mg/kg
rat skin LD50: 2100 mg/kg
rat intraper. LD50: 640 mg/kg
rat subcutan. LDLo: 800 mg/kg
mouse subcutan. LDLo: 286 mg/kg
dog oral LDLo: 750 mg/kg
dog intraven. LDLo: 150 mg/kg
cat oral LDLo: 2000 mg/kg
cat skin LDLo: 25 mg/kg
rabbit oral LDLo: 700 mg/kg
rabbit skin LDLo: 600 mg/kg
guinea pig intraper. LDLo: 500 mg/kg
Aquatic toxicity at 96 hours - 10-100 mg/1 (ppm).
C-26
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Levin (1927) demonstrated in vivo production of methemo-
globin by nitrobenzene in dogs, cats, and rats, but not in
guinea pigs or rabbits. Dresbach and Chandler (1918) found
that nitrobenzene fumes caused cerebellar disturbances in
dogs and birds, while blood changes were the principal toxic
effects in other mammals they studied. Reddy, et al. (1976)
reported a delay in methemoglobin formation in germ free rats
by nitrobenzene and postulated that the gut flora of rats was
responsible for the reduction (in vivo) and methemoglobin
forming capacity of nitrobenzene. Shimkin (1939) measured
the toxicity of nitrobenzene in mice when absorbed through
the skin. He found the minimum lethal dose to be 0.0004 ml/
gm body weight by a subcutaneous route of administration.
The nitrobenzene caused respiratory failure, reduction of the
white blood cell count, and liver pathology in the mice.
Yamada (1958) did a chronic toxicity study in rabbits
that received a subcutaneous dose of 840 mg/kg body weight
per day for three months. He found a decrease in erythrocyte
number and hemoglobin content early in the exposure. These
values increased during the three months but did not return
to normal levels. Urinary excretion of detoxification pro-
ducts was variable in the early stages of the exposure, but
then all the detoxification reactions (reduction, hydroxyla-
tion, and acetylation) were depressed. As a result of these
observations, Yamada divided this response in the rabbit into
three stages: initial response, resistance, and exhaustion.
C-27
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The effects of subacute nitrobenzene exposure in rats
were studied by Kulinskaya (1974). Vasilenko and Zvezdai
(1972) measured blood changes and found suIfhemoglobin
formation to be the most regular and persistant change noted.
Increased methemoglobin levels with Heinz body formation and
anemia were also seen.
The cytotoxicity of nitrobenzene to cultured Erlich-
Landschutz diploid (ELD) cells was measured by Holmberg and
Malmfors (1974). They found no significant increase in cell
injury after five hours incubation with nitrobenzene. How-
ever, a 3M nitrobenzene solution reduced cell proliferation
by 50 percent in cultured hamster cells (Raleigh, et al.
1973). Nitrobenzene increases oxygen consumption by cultured
cells (Biaglow and Jacobson, 1977), and its derivatives are
used to sensitize malignant cells in vitro for radiation
(Chapman, et al. 1974). They suggest that this effect is due
to radical oxidation and increased cellular damage.
Nitrobenzene derivatives have a wide variety of toxic
effects. 1-Chloro - 2, 4 dinitrobenzene (DNCB) is a well
known skin sensitizer in guinea pigs, mice, and man (Hama-
guchi, et al. 1972; Jansen and Bleumink, 1970; Maurer, et al.
1975; Weigand and Gaylor, 1974; Noonan and Halliday, 1978).
Cooke, et al. (1968) showed that nitrobenzene derivatives
react with cell membranes to alter sodium-potassium con-
ductance, and sometimes affect action potentials of nerve
cells.
C-28
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M-dinitrobenzene is a potent methemoglobin former, and
is more toxic than nitrobenzene (Ishihara, et al. 1976;
Pankow, et al. 1975). Pentachloronitrobenzene (PCNB) is a
common fungicide with varying toxic effects in different
mammalian species (Courtney, et al. 1976).
Some of the toxic effects of nitrobenzene are summarized
in Table 5 and Apendix A (Dorigan and Hushon, 1976).
C-29
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TABLE 5
Toxicological Effects of Nitrobenzene
Organism Route
Human Inhalation
Inhalation
Inhalation
Oral
Oral
Rabbit Cutaneous
absorption
Oral
Oral
Exposure
0.2-0.5 mg/1
(40-100 ppm)
0.129 mg/m3
6-30 ug/1
333 ml
0.4 ml
0.7 gm/kg
700 mg/kg
600 mg
Exposure
Time
ca. 6 hrs.
-
6 hrs.
Single
Single
Single (?)
Single
Single
Oral
50 mgAg
Single
Response
Slight effects, e.g. headache,
fatigue.
Threshold level for electroen-
cephalograph distrubance.
Retained 80% of vapor in lungs.
Urinary excretion of p-nitro-
phenol (max. in 2 hrs. still
detected after 100 hrs.).
Max. dose with recovery report-
ed (folowing severe symptoms)
Mm. lethal dose reported.
After 52 hrs.: lethal
Lethal dose
Dizziness, loss of reflexes,
methemoglobinemia, congestion
of brain tissue-12 hrs.-death.
Tissue degeneration, especially
heart, liver, kidney.
Source
von Oettingen,
1941.
Andreeshcheva,
1964.
Salmowa, et al.
1963.
von Oettingen,
1941.
von Oettingen, 1941
von Oettingen,
1941.
Stecher, 1968
Chandler, 1919.
Papageorgiou &
Argoudelis, 1973.
-------
TABI,E 5 (Continued)
Organism
Guinea
pig
Rat
o
i
u>
Cat
Dog
Route
Inhalation
Oral
Oral
Exposure
Exposure
Time
Saturated air 2-5 hrs.
(0.04 vol. %)
ca. 3 gm
ca 1.2 gm
Inhalation 5 mg/m3
Single
Single
8 hrs.
Inhalation ca. 0.03mg/ Daily up to
m3 98 days
Inhalation 0.06-0.1 mg/ 70-82 days
m3
Oral
Inhalation
Oral
Oral
0.6 gm/kg
Single
Saturated air 2-5 hrs.
(0.04 vol. %)
2.4 gm
2.4 gm
Single
Single
Response
Source
Death following tremors, paraly- Chandler, 1919,
sis of hind legs.
0.5 hrs.: tremors, faint heart-
beats, labored respiration
2 hrs.: death.
Immediately motionless, then
complete recovery.
Metabolites excreted in 3 days.
Increased ability to form sulf-
hemoglobin in preference to
methemoglobin.
Cerebellar disturbances, in-
flamed internal organs.
Inhalation 0.008 mg/m3 73 days No effect.
LD50
Death following tremors, pa-
ralysis of hind legs.
Death in 12-24 hrs.
1 hr.: vomiting, then sleepy
continuing for 6 hrs.
104 hrs.: death.
Chandler, 1919.
Chandler, 1919.
Ikeda and Kita,
1964
Andreeshcheva,
1970.
Khanin, 1969.
Andreeshcheva.
1970.
Smyth, et al. 1969
Chandler, 1919.
von Oettingen, 1941;
Chandler, 1919.
Chandler, 1919.
-------
Synergism and/or Antagonism
Alcohol has a synergistic effect on nitrobenzene poison-
ing. Ingestion of an alcoholic beverage has induced immedi-
ate acute toxic symptoms, including coma, in a worker recov-
ered from the effects of chronic nitrobenzene exposure. Al-
cohol ingestion or chronic alcoholism can lower the lethal or
toxic dose of nitrobenzene (Dorigan and Hushon, 1976). In
subchronic dinitrobenzene poisoning, drinking of one beer or
exposure to sun can bring on an acute crisis as late as six
weeks after the disappearance of other symptoms (Rejsek,
1947). Therefore, workers exposed to subacute levels of
nitrobenzene or dinitrobenzene should be aware of the pos-
sible deleterious synergistic effects of alcoholic beverages
and sunlight.
Smyth, et al. (1969) studied the synergistic action be-
tween nitrobenzene and 27 other industrial chemicals by intu-
bation in rats. Most of the compounds tested did not alter
the LD50. In another study, ingestion of 2 to 20 ml of
ethanol increased the severity of reaction to a 0.1 ml intra-
venous dose of nitrobenzene in rabbits. This observation
agrees with the clinical data on the synergism of ethanol and
nitrobenzene (Dorigan and Hushon, 1976).
Kaplan, et al. (1974) studied the effect of caffeine,
an inducer of microsomal enzymes, on methemoglobin formation
by nitrobenzene in rats. Methemoglobin was formed and then
decreased in induced animals. The increased microsomal en-
zyme level increased the rate of metabolism and excretion of
nitrobenzene and thus caused a rapid decline of methemoglobin
levels.
C-32
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Teratogenecity
There is a paucity of information on the teratogenic
effects of nitrobenzene. In one study (Kazanina, 1968b), 125
mg/kg was administered subcutaneously to pregnant rats during
preimplantation and placentation periods. Delay of embryo-
genesis, alteration of normal placentation, and abnormalities
in the fetuses were observed. Gross morphogenic defects were
seen in four of 30 fetuses examined.
Changes in the tissues of the chorion and placenta of
pregnant women who worked in the production of a rubber
catalyst that used nitrobenzene were observed. No mention
was made of the effects on fetal development or viability
(Dorigan and Hushon, 1976). Menstrual disturbances after
chronic nitrobenzene exposure have been reported.
Garg, et al. (1976) tested substituted nitrobenzene
derivatives for their ability to inhibit pregnancy in albino
rats. Two of the compounds tested (p-methoxy and p-ethoxy
derivatives) inhibited implantation and pregnancy 100 percent
when administered on days one through seven after impregna-
tion.
The available data, although sketchy, indicate that
women who are or wish to become pregnant should avoid expo-
sure to nitrobenzene. Further studies of nitrobenzene tera-
togenicity in mammals are needed.
Mutagenicity
Chiu, et al. (1978) tested nitrobenzene and 53 com-
mercially available ion, heterocyclic + aliphatic nitro
C-33
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compounds for mutagenicity using the Ames Salmonella
typhimurium TA 98 and TA 100. They reported that 34 of the
53 compounds tested were mutagenic. Nitrobenzene was not
found to be mutagenic.
Trinitrobenzene was mutagenic in two in vitro assays,
the Ames Salmonella microsome assay, and the mitotic recom-
bination assay in yeast (Simmon, 1977). Other nitrobenzene
derivatives have demonstrated mutagenicity in in vitro
assays, so that the mutagenicity of nitrobenzene is still in
question and additional work is needed in this area.
Carcinogenicity
The available literature does not demonstrate the car-
cinogenicity of nitrobenzene, although it is suspect (Dorigan
and Hushon, 1976). This is another aspect of nitrobenzene
effects that needs more research.
Some nitrobenzene derivatives have demonstrated carcino-
genic capacities. Pentachloronitrobenzene (PCNB) induced
hepatomas and papillomas in mice (Courtney, et al. 1976).
4.
1 - Fluoro -2,4- dinitrobenzene (DNFB) was demon-
strated by Bock, et al. (1969) to be a promoter of skin tu-
mors in mice, although it does not induce them when admin-
istered alone.
C-34
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CRITERION FORMULATION
Existing Guidelines and Standards
The maximum allowable concentration of nitrobenzene in
air in industrial plants is 5 mg/m^. This value was set by
the joint ILO/WHO Committee on Occupational Health in 1975
(Goldstein, 1975). The OSHA (Occupational Safety and Health
Administration) standard for nitrobenzene in air is 5 mg/m^
(1 ppm) set in 1977 (Am. Conf. Gov. Ind. Hyg., 1977). This
is also the threshold limit value (TLV) in Germany and Sweden
while the TLV in the USSR is 3 mg/m^ (Dorigan and Hushon,
1976).
There are no standards for nitrobenzene levels in water.
Nitrobenzene was not listed among the substances for which a
maximum concentration has been set.
Current Levels of Exposure
A worker exposed to the current occupational standard of
5 mg/m^ (1 ppm) nitrooenzene for an eight-hour work day
would absorb approximately 24 mg by inhalation and 9 mg
cutaneously. The maximum eight-hour uptake would be 33 mg,
which is less than the "reasonable safe" level of 35 mg/day
(Dorigan and Hushon, 1976). Doses of up to 70 mg/day have
been reported for factory workers and up to 80 mg/day have
been reported in a dye stuff factory in England (Dorigan and
Hushon, 1976, citing Piotrowski, 1967).
Nitrobenzene can be a contaminant in industrial waste
water, and companies utilizing or making nitrobenzene are re-
quired to monitor its level in their effluent waste (Pierce,
1979). The minimum detectable level of nitrobenzene in
C-35
-------
drinking water by gas chromatography is 0.7 ng (Austern, et
al. 1975).
Nitrobenzene may be vented to the atmosphere. The vents
are usually equipped with absorbers or scrubbers, but some
nitrobenzene vapors can escape. Atmospheric nitrobenzene
levels outside a plant are not monitored by industry. Since
inner plant levels are below the TLV of 5 mg/m^ (i ppm) and
nitrobenzene vapors accumulate at the floor level due to
their high density, the external air nitrobenzene concentra-
tions are expected to be very low (Dorigan and Hushon, 1976).
Special Group at Risk
Workers in plants producing or using nitrobenzene have
the greatest risk of toxic exposure. At the current TLV
level of 5 mg/m^ (1 ppm) a worker could absorb as much as
33 mg/day. This is enough to produce symptoms of chronic
toxicity in some susceptible individuals (Dorigan and Hushon,
1976). The amount of nitrobenzene absorbed by a worker via
inhalation and cutaneous absorption can be estimated from the
level of total (free and conjugated) p-nitrophenol in urine
as described by Piotrowski (1977).
Due to the current widespread use of disposable diapers
and underpads in hospitals, nitrobenzene poisoning in infants
from laundry marking dyes is no longer a problem.
Pregnant women may be especially at risk with respect to
nitrobenzene as with many other chemical compounds, due to
transplacental passage of the agent. Individuals with
glucose-6- phosphate dehydrogenase deficiency may also be
C-36
-------
special risk groups (Calabrese, et al. 1977; Djerassi, et al.
1975). Additionally, because alcohol ingestion or chronic
alcoholism can lower the lethal or toxic dose of nitrobenzene
(Rejsek, 1947; von Oettingen, 1941), individuals consuming
alcoholic beverages may be at risk.
Basis and Derivation of Criterion
Because there are little or no data available on the
toxicity of nitrobenzene ingested in drinking water, or on
the teratogenic, mutagenic, or carcinogenic effects of nitro-
benzene in general, experimental testing is necessary before
an oral ingestion based criterion can be derived. It is
recommended that testing in these areas of toxicity be imple-
mented so that the effects of nitrobenzene on mammals may be
better understood.
Using the methodology of Stokinger and Woodward (1958),
a water quality criteria (WQC) is derived using the organo-
leptic level and the TLV.
Organoleptic Level: minimum detectable odor level in
water is 0.03 mg/1 = 30 ug/1.
Assuming a daily intake of 2 liters of water, the total
intake of nitrobenzene based on this criteria would be 60
micrograms/day. Recommended WQC = 30 ug/1.
A calculation of the percentage of exposure attributable
to fish and shellfish products is not applicable to a cri-
terion based upon organoleptic effects. Since an organolep-
tic effect is not based on a toxicological assessment, it
C-37
-------
would be inappropriate to apportion a percentage of exposure
to the consumption of toxicologically contaminated fish.
TLV: TLV = 5 mg/m3; air intake = 10 m3/day; assume
80 percent absorption:
(5 mg/m3) x (10 m3/day) x (0.8) = 40 mg/day
average over seven days:
40 mg/day x 5/7 = 29 mg/day
Assuming 100 percent gastrointestinal absorption of
nitrobenzene and consuming 2 liters of water daily and 18.7
grams of contaminated fish having a bioconcentration factor
of 4.3, would result in a maximum permissible concentration
of 13.9 mg/1 for the ingested water:
29 mg/day = ,
2 liters + (4.3 x 0.0187) x 1.0 *'* g/
WQC using TLV = 13.9 mg/1
Since the WQC using TLV is well above the detectable
odor level of nitrobenzene, water containing this concentra-
tion of nitrobenzene would not be esthetically acceptable for
drinking. Even though the limitations of using organoleptic
data as a basis for establishing a WQC are recognized, it is
recommended that a WQC of 30 ug/1 be established at the
present time. This level may be altered as more data are
developed upon which to calculate a WQC.
The analysis and recommendations generated in this docu-
ment are based on the literature available to date. If
future reports indicate that nitrobenzene may be carcino-
genic, mutagenic or teratogenic, a reassessment of the WQC
will be necessary.
C-38
-------
APPENDIX A
Toxiological Effects of Nitrobenzene
Organism
Human
n
i
Ul
VD
Route
Inhalation
Exposure
Inhalation Poor ventila-
tion
Inhalation
0.2-0.5 mg/1
(40-100 ppm)
Exposure
Time
8 hrs./day for 17
mos. factory worker
8 hrs./day for 1.5
mos. factory worker
paint firm
8 hrs./day for 4.5
mos.
ca. 6 hrs,
Inhalation 0.129 mg/m3
Inhalation "Large" amounts
poor ventila-
tion
Inhalation Acute
Response
Cyanosis, headache, fatigue methemoglob-
inemia (Ikeda and Kita, 1964).
Cyanosis, headache, fatigue, methemoglob-
inemia, liver damage, hypotension (Ikeda
and Kita, 1964).
Above plus: liver and spleen enlarged and
tender, hyperalgesia in extremeties
(Ikeda and Kita, 1964).
Slight effects, e.g. headache, fatigue
(von Oettingen, 1941).
Threshold level for electroencephalograph
disturbance (Andreeshcheva, 1964).
Hospitalized:
2 - vertigo, coma, cyanosis
3 - labored breathing, urine with
7 - almond odor, methemoglobinemia
recovery after 1 mo. (Ravault, et al.
1946).
Burning throat, nausea, vomiting, gastro-
intestinal disturbances, cold skin,
livid face, cyanosis (von Oettingen,
1941).
-------
APPENDIX A (Continued)
Organism Route Exposure
Human Inhalation -
n
i
Inhalation 6-30 u.g/1
Inhalation -
Inhalation -
Inhalation
Inhalation Acute
Exposure
Time
Nitrobenzene factory
worker
6 hrs.
Factory worker (rub-
ber accelerator)
Factory worker
(glass, porcelain)
Industrial exposure
Factory worker
(filled containers
with nitrobenzene)
Response
Intermittent symptoms: cyanosis, pallor
and jaundice, pharyngeal congestion,
headache, changes in blood cell composi-
tion (increased polynuclears and eosino-
phils (von Oettingen, 1941).
Retained 80% of vapor in lungs, urinary
excretion of p-nitrophenol (maximum in
2 hrs., still detected after 100 hrs.)
(Salmowa, et al. 1963).
Pregnant women: thickening of tissue in
blood vessels, decreased placental ab-
sorption, necrosis in placental tissue
(Ferster, 1970).
Changes in bone marrow, increased lymphoid
cell production, impairment of copper me-
tabolism and certain iron-containing
enzymes (Yordanova, et al. 1971).
Disturbance of motor impulses
(Zenk, 1970).
14 days: cyanosis, headache, backache,
stomach ache, vomiting
ca. 21 days: drank beer and fell uncon-
scious, cyanosis, dilated pupils, re-
tarded respiration, weak pulse
1 yr.: intelligence dimmed
2 yrs.: emaciated, atrophied muscles
3 yrs.: memory failed
6 yrs.: loss of perception of time and
space (Korsakoff's syndrome)
(Chandler, 1919).
-------
APPENDIX A (Continued)
Organism
Human
o
i
Rabbit
Route
Cutaneous
absorp-
tion
Cutaneous
absorp-
tion
Cutaneous
absorp-
tion
Oral
Oral
Oral
Exposure
Dye used in
diaper stamps
Shoe dye
0.5% by
weight in
paper
Exposure
Time
333 ml
0.4 ml
Subcuta- 0.8 mg/kg
neous
injection
Subcuta- 10-14 mg/kg
neous
injection
Cutaneous 0.7 gm/kg
absorp-
tion
Intraperi- 0.5gm/kg
toneal
injection
ca. 7 hrs.
(Handled carbon
paper)
From human milk
Single
Single
Daily
Single
Single
Single
Response
Babies: cyanosis, rapid pulse, shallow res-
piration, vomiting, convulsions, recovery
in 24 hrs. (von Oettingen, 1941).
Unconsciousness after consumption of alco-
hol beverages, death (Chandler, 1919).
Dermatitis (Calan and Connor, 1972).
Nurselings became cyanotic, recovery in 24
hrs. (mothers ate almond cake artifici-
ally flavored with nitrobenzene)
(Dollinger, 1949).
Maximum dose with recovery reported fol-
lowing severe symptoms (von Oettingen,
1941).
Minimum lethal dose reported (von
Oettingen, 1941).
Maximum dose not causing death (Yamada,
1958).
Minimum dose producing observable effects;
slow and lasting methemoglobinemia
(von Oettingen, 1941).
After 52 hrs.: lethal (von Oettingen,
1941).
Reduced blood pressure and myocardial
glycogen level (Labunski, 1972).
-------
APPENDIX A (Continued)
Organism
Rabbit
Route
Exposure
n
i
Guinea
pig
Intraven- 0.1 gm
ous
Oral
Oral
1 mg/kg
0.1 mg/kg
Inhalation Saturated air
(0.04 vol. %)
Inhalation
Subcuta-
neous
0.2 gm/kg
Exposure
Time
Daily or every 5
days
Oral
Oral
Oral
Oral
Oral
Oral
9 gm
4.8 gm
700 mg/kg
600 mg
300 mg
50 mg/kg
4 dose
15 ml
Single
Single
Single
Single
Single
Single
Single
2-5 hrs.
2-3 hrs.
Every other day for
6 mos.
Response
Simultaneous doses of 2-20 ml ethanol in-
creased severity of poisoning (Matsumara
and Yoshida, 1959).
Convulsions, death (von Oettingen, 1941;
Chandler, 1919).v
Lethal instantly (von Oettingen, 1941;
Chandler, 1919).
Lethal dose (Stecher, 1968).
Dizziness, loss of reflexes, methemo-
globinemia, congestion of brain tissue -
12 hrs. - death (Chandler, 1919).
Fatigue for 1 week (Parke, 1956).
Tissue degeneration, especially heart,
liver, kidney (Papageorgiou and
Argoudelis, 1973).
Lowered hemoglobin, erthyrocytes and
lymphocytes; increased leucocytes
(Kazakova, 1956).
Threshold toxic dose (Kazakova, 1956).
Death following tremors, paralysis of
hind legs (Chandler, 1919).
Death (Chandler, 1919).
Hemolytic anemia, loss of weight, de-
creased motor activity, fluxes in
urinary excretion of 17-hydroxy-cortico-
steroids (Porter-SiIber chromogens)
(Makotchenko and Akhmetov, 1972).
-------
APPENDIX A (Continued)
Organism Route Exposure
ca. 3 gm
Exposure
Time
Guinea Oral
pig
Oral
Oral
Oral
o
i
Rat
ca. 1.2 gm
50 mg/kg
1 mg/kg
Oral 0.1 mg/kg
Inhalation 5 mg/m3
Single
Single
1 year
Single
Single
8 hrs.
Inhalation ca. 0.03 mg/m3 Daily, up to 98
days
Inhalation 0.006-0.1 mg/ 70-82 days
m3
Inhalation 0.008 mg/m3 73 days
Oral 0.6 gm/kg Single
Intraperi- 0.8gm/kg Single
toneal
infection
Response
0.5 hrs.: tremors, faint heartbeats,
labored respiration
2 hrs.: death (Chandler, 1919).
Immediately motionless, then complete
recovery (Chandler, 1919).
Tissue degeneration, especially heart,
liver, kidney (Kazakova, 1956).
Lowered hemoglobin, erythrocytes,
lymphocytes; increased leucocytes
(Kazakova, 1956).
Threshold toxic dose (Kazakova, 1956).
Metabolites excreted in 3 days (Ikeda
and Kita, 1964).
Increased ability to form sulfhemoglobin
in preference to methemoglobin
(Andreeshcheva, 1970).
Cerebellar disturbances, inflamed inter-
nal organs (Khanin, 1969).
No effect (Andreeshcheva, 1964).
LD50 (Smyth, et al. (1969).
Lethal (Magos and Sziza, 1958).
-------
APPENDIX A (Continued)
Organism
Rat
o
i
Mouse
Route
Subcuta-
neous
injection
Subcuta-
neous
injection
Subcuta-
neous
injection
Subcuta-
neous
injection
Exposure
640 mgAg
300 mg/kg
200 mgAg
or
100 mgAg
125 mgAg
Exposure
Time
Single
Single
Single
Daily for 10 days
Single
Subcuta- 100-200 mgAg Single
neous
injection
Cutaneous 480
absorption
Intrap&ri- 1.23 gm/kg Single
jtoneal
injection
Intraperi- 1 gm/kg Single
toneal
Response
Blood catalase activity decreased contin-
uously over 96 hrs. (Goldstein and
Popovici, 1959).
LD (14 days) - methemoglobinemia, anemia,
sulfhemoglobinemia (Brown, et al. 1975).
Methemoglobinemia, sulfhemoglobinemia,
anemia (Zvezdai, 1972).
Delayed embryogenesis, abnormal fetal
development and embryo death, changes
in polysaccharide composition of pla-
centa (Kazanina, 1967, 1968a,c).
Sulfhemoglobin (most regular and persis-
tent form of hemoglobin) nitroxyhemo-
globin, increased methemoglobin
(Vasilenko and Zvezdai, 1972).
30 min.: prostrate, motionless
24 hrs.: death (von Oettingen, 1941).
40 min.: 67% dead (Smith, et al. 1967).
10-15 min.: incoordination, comatose,
shallow respiration
Several hrs.: regained coordination
Immediately before death: lost coordina-
tion again, respiratory arrest
48 hrs.: death (Smith, et al. 1967)
-------
APPENDIX A (Continued)
Organism
Mouse
Cat
n
i
•fc.
Cn
Dog
Route Exposure
Intraperi- 20 mgAg Single
toneal
injection
Intraperi- 12.3 mgA9 Single
toneal
injection
Inhalation Saturated air 2-5 hrs.
(0.04 vol, %)
Inhalation - 2-3 hrs.
Oral 2»4 gm Single
Inhalation "Thick vapor" 1.5 hrs.
Intravenous 0015-0-25 gm/ Single
injection kg
Exposure
Time
Oral
Oral
2808 gm plus 2 doses, 0.5 hrs,
6 gm apart
24 gm
Single
Response
Lethal dose (Brown, et al. 1975).
10 min.s 4.2% methemoglobin formed
(Smith, et al. 1967).
Death following tremors, paralysis of
hind legs (Chandler, 1919).
Death
Death in 12-24 hrs„ (von Oettingen, 1941;
Chandler, 1919).
Complete anesthesia and sleep (Chandler,
1919)o
Minimum lethal dose - lowered blood pres-
sure, pulse rate increased then decreased
respiration stimulated until paralyzed
(von Oettingen, 1941).
Immediate; agitation, then motionless
1 hr.: convulsions, then motionless
4,5 hrs.; tremors, hind legs paralyzed
18 hrso: death (Chandler, 1919).
Few hrs.: "stupid"
12 hrs.; deep coma, slow respiration,
lowered skin temperature, stomach
strongly alkaline (Chandler, 1919).
-------
APPENDIX A (Continued)
Organism
Dog
o
i
Chicken
Pigeon
Oral
Oral
Oral
Oral
Oral
Inhalation
Exposure
2.4 gm
1.2 gm
2.4 gm
Exposure
Time
Single
0.75-1.0 gm/kg Single
0.5-0.7 gm/kg Single
Daily
Single
Single
1 hr.
2-3 hrs.
Response
1 hr.: vomiting, then sleep continuing
for 6 hrs.
6 hrs.: appeared normal
15-68 hrs.: rigid muscles
104 hrs.: death (Chandler, 1919).
Minimum lethal dose (von Oettingen, 1941).
Salivation, unrest, dizziness, tremors,
increased pulse rate, sometimes con-
vulsions (Chandler, 1919).
Formed methemoglobin continuously at
"certain" concentration (Hashimoto, 1958)
Unsteady gait, recovery
(Chandler, 1919).
Immediately unconscious
12 hrs.: death (Chandler, 1919).
No effects
Death (Chandler, 1919).
-------
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