»EPA
United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Criteria and Standards Division
Washington DC 20460
EPA 440/5-80-061
October 1980
c.)
Ambient
Water Quality
Criteria for
Nitrobenzene
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AMBIENT WATER QUALITY CRITERIA FOR
NITROBENZENE
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
ii
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
David J. Hansen, ERL-Gulf Breeze
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects:
Karl Gabriel (author)
Medical College of Pennsylvania
Steven D. Lutkenhoff (doc. mgr.)
ECAO-Cin
U.S. Environmental Protection Agency
Si Duk Lee (doc. mgr.), ECAO-Cin
U.S. Environmental Protection Agency
Patrick Durkin
Syracuse Research Corporation
Sherwin Kevy
Children's Hospital Medical Center
David J. McKee, ECAO-RTP
U.S. Environmental Protection Agency
Alan B. Rubin
U.S. Environmental Protection Agency
James Withey
Health and Welfare, Canada
John Autian
University of Tennessee
J. P. Bercz, HERL
U.S. Environmental Protection Agency
Richard Carchman
Medical College of Virginia
Thomas J. Haley
National Center for Toxicological Res,
Van Kozak
University of Wisconsin
V.M. Sadagopa Ramanujam
University of Texas Medical Branch
Carl Smith
University of Cincinnati
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, P. Gray, R. Rubinstein.
iv
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TABLE OF CONTENTS
Page
Criteria Summary
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Chronic Toxicity B-l
Plant Effects B-2
Summary B-2
Criteria B-2
References B-7
Mammalian Toxicology and Human Health Effects C-l
Introduction C-l
Exposure C-2
Ingestion from Water C-2
Ingestion from Food C-4
Inhalation C-5
Dermal C-6
Pharmacokinetics C-8
Absorption C-8
Distribution C-9
Metabolism C-ll
Excretion C-l4
Effects C-l9
Acute, Subacute, and Chronic Toxicity C-l9
Synergism and/or Antagonism C-24
Teratogenicity C-24
Mutagenicity C-25
Carcinogenicity C-25
Criteria Formulation C-27
Existing Guidelines and Standards C-27
Current Levels of Exposure C-27
Special Groups at Risk C-28
Basis and Derivation of Criterion C-28
References C-31
Appendix C-45
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CRITERIA DOCUMENT
NITROBENZENE
CRITERIA
Aquatic Life
The available data for nitrobenzene indicate that acute toxicity to
freshwater aquatic life occurs at concentrations as low as 27,000 wg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No definitive data are available concerning the chronic
toxicity of nitrobenzene to sensitive freshwater aquatic life.
The available data for nitrobenzene indicate that acute toxicity to
saltwater aquatic life occurs at concentrations as low as 6,680 wg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No definitive data are available concerning the chronic
toxicity of nitrobenzene to sensitive saltwater aquatic life.
Human Health
For comparison purposes, two approaches were used to derive criterion
levels for nitrobenzene. Based on available toxicity data, for the protec-
tion of public health, the derived level is 19.8 mg/1. Using available
organoleptic data, for controlling undesirable taste and odor qualities of
ambient water, the estimated level is 30 ug/1. It should be recognized that
organoleptic data as a basis for establishing a water quality criterion have
limitations and have no demonstrated relationship to potential adverse human
health effects.
VI
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INTRODUCTION
Nitrobenzene 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 ani-
line, 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,
crystallizing solvent for some substances, and as a combustible propellant
(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 nitrobenzene by the use of activated charcoal ab-
sorbers or a carbon dioxide blanket. There is no industrial monitoring of
nitrobenzene in the atmosphere. The greatest loss of nitrobenzene during
production (estimated as eight million pounds annually) occurs at the acid
extraction step in the purification 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 nitrobenzene workers. Today plant
levels of nitrobenzene are usually kept below the threshold limit value
(TLV) of 5 mg/m3 [Goldstein, 1975; American Conference of Governmental
Industrial Hygienists (ACGIH), 1977] but much higher levels have been re-
ported in the oast (Pacseri and Magos, 1958). Nitrobenzene may also form
spontaneously in the atmosphere from the photochemical reaction of benzene
with oxides of nitrogen.
A-l
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Nitrobenzene, also known as nitrobenzol, essence of mirbane, 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 compounds (Kirk and Othmer, 1967). In
the solid state it forms bright yellow crystals. Nitrobenzene,
C6H5N02» has a modular 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 mm 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 requires more vigorous
conditions than does benzene. Substitution takes place at either the
meta-(3) position or the ortho-(2) or para-(4) positions depending on the
physical conditions (Kirk and Othmer, 1967). Nitrobenzene undergoes photo-
reduction when irradiated with ultraviolet light in organic solvents 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 compound can act as an oxidizing
agent in the presence of aqueous solutions of alkali hydroxides, it has the
capability of oxidizing compounds containing free phenolic hydroxyl groups
without effectively changing these groups (Millar and Springfield, 1966).
A-2
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Nitrobenzene is rective and will undergo nitration, halogenation, and sulfo-
nation by the same methods used for benzene. However, these reactions are
unlikely to occur in environmental conditions.
The reduction of nitrobenzene to aniline probably outranks 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.
A-3
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REFERENCES
American Conference of Governmental Industrial Hygienists. 1977. Documen-
tation of the threshold limit value for substances in workroom air. Cincin-
nati, Ohio.
Barltrop, A.J. and N.J. Bunce. 1968. Organic photochemistry, Part 4. The
photochemical reduction of nitro-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 ami no-derivatives
of aromatic hydrocarbons. Adverse Effects Environ. Chem. Psych. Drugs.
1: 153.
Jordan, I.E. 1954. Vapor Pressure of Organic Compounds. Interscience Pub-
lishers, Inc., New York.
Kirk, R.E. and D.F. Othmer (eds.) 1967. Kirk-Othmer Encyclopedia of Chemi-
cal Technology. 2nd ed. John Wiley and Sons, Inc., New York.
Lu, P.Y. and R. Metcalf. 1975. Environmental fate and biodegradability of
benzene derivatives as studies in a model aquatic ecosystem. Environ.
Health Perspect. 19: 269.
A-4
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McGraw-Hill. 1971. Encyclopedia of Science and Technology. McGraw-Hill
Book Co., New York.
Millar, I.T. and H.O. Springfield (eds.) 1966. Sidgwick's Organic Chemis-
try of Nitrogen. 3rd ed. Clarendon Press, Oxford.
Pacseri, I. and L. Magos. 1958. Determination of the measure of exposure
to aromatic nitro and amino compounds. Jour. Hyg. Epidemiol. Microbiol.
Immunol. 2: 92.
Stecher, P.G. (ed.) 1968. The Merck Index. 8th ed. Merck and Co., Inc.,
Rahway, New Jersey.
A-5
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Aquatic Life Toxicology*
INTRODUCTION
Static tests with the bluegill, Daphnia magna, and the alga, Selenastrum
capricornutum. indicate little difference in sensitivity with no 50 percent
effect concentration lower than 27,000 yg/1. An embryo-larval test with the
fathead minnow demonstrated no adverse effects at the highest test concen-
tration of 32,000 yg/1.
Static acute tests with the sheepshead minnow and Mysidopsis bahia indi-
cate that the latter is much more sensitive to nitrobenzene. Adverse ef-
fects were observed on a saltwater alga at concentrations slightly higher
than the LC5Q for the mysid shrimp.
EFFECTS
Acute Toxicity
The 48-hour EC5Q for Daphnia magna and the 96-hour LC50 for the
bluegill are 27,000 and 42,600 ug/1, respectively (Table 1).
The saltwater species are comparable to the freshwater species in their
sensitivity to nitrobenzene. The mysid shrimp LC5Q is 6,680 wg/l (Table
1) and the LC5Q for the sheepshead minnow is 58,600 »g/l.
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 2).
*The reader is referred to the Guidelines for Deriving Water Quality Crite-
ria for the Protection of Aquatic Life and Its Uses in order to better
understand the following discussion and recommendation. The following
tables contain the appropriate data that were found in the literature, and
at the bottom of each table are calculations for deriving various measures
of toxicity as described in the Guidelines.
B-l
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Plant Effects
The 96-hour EC5Q values for reduction of cell numbers and inhibition
of chlorophyll £ in the freshwater alga, Selenastrum capricornutum, are
42,800 and 44,100 yg/1, respectively (Table 3).
The cell numbers of Skeletonema costatum were reduced by 50 percent at a
concentration of 9,650 yg/1 (Table 3). Chlorophyll a was equally inhibited
at a concentration of 10,300 yg/1.
Summary
The acute 50 percent effect levels of Daphnia magna and the bluegill
were 27,000 and 42,600 yg/1, respectively. No effects on fathead minnow
embryos or larvae were observed at concentrations as high as 32$000 yg/1. A
freshwater alga was of similar sensitivity with an EC5Q value for chloro-
phyll a of 44,100 yg/1.
Ninety-six-hour LC5Q values were 6,680 and 58,600 ug/1 for the mysid
shrimp and sheepshead minnow, respectively. The EC5Q for cell numbers of
a saltwater alga was 9,650 yg/1.
CRITERIA
The available data for nitrobenzene indicate that acute toxicity to
freshwater aquatic life occurs at concentrations as low as 27,000 yg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No definitive data are available concerning the chronic
toxicity of nitrobenzene to sensitive freshwater aquatic life.
The available data for nitrobenzene indicate that acute toxicity to
saltwater aquatic life occurs at concentrations as low as 6,680 yg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are avialable concerning the chronic toxicity of
nitrobenzene to sensitive saltwater aquatic life.
B-2
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Table 1. Acute values for nitrobenzene (U.S. EPA, 1978)
Species
Cladoceran,
Daphnla magna
B 1 ueg 1 1 1 ,
Lepomls macrochlrus
LC50/EC50
Method* (uo/l)
FRESHWATER SPECIES
S, U 27,000
S, U 42,600
Species Acute
Value (ug/l)
27,000
42,600
SALTWATER SPECIES
I
U)
Mysld shrimp,
Mysldopsls bah I a
Sheepshead minnow,
Cyprlnodon varlegatus
S, U
S, U
6,680
58,600
6,680
58,600
* S = static, U = unmeasured
No Final Acute Values are calculable since the minimum data base
requirements are not met.
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Table 2. Chronic values for nitrobenzene (U.S. EPA, 1978)
Chronic
LlMlts ¥•!••
Species Method* (pg/l) (tig/1)
FRESHWATER SPECIES
Fathead minnow, E-L >32,000
Plmephales promelas
* E-L = embryo-larva I
No acute-chronic ratio Is calculable.
CO
I
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Table 3. Plant values for nitrobenzene (U.S. EPA, 1976)
Result
Species Effect (ug/D
FRESHWATER SPECIES
Alga, 96-hr EC50 44,100
Selenastrum capr I cornutum ch lorophy 11 _a_
Alga, 96-hr EC50 42,800
SeIenastrum caprIcornutum cell numbers
SALTWATER SPECIES
Alga, 96-hr EC50 9,650
Skeletonema costatum cell numbers
CO
I Alga, 96-hr EC50 10,300
{Jt Skeletonema costatum chlorophyll^
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REFERENCES
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. U.S. Environ. Prot. Agency, Contract No.
68-01-4646.
B-6
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Mammalian Toxicology and Human Health Effects
INTRODUCTION
Nitrobenzene, a pale yellow liquid at room temperature with a character-
istic bitter almond aroma, is also known as oil of mirbane, nitrobenzol, and
artificial bitter almond oil. It is produced for industrial use by the ni-
tration of benzene with nitric and sulfuric acids. Estimates of annual ni-
trobenzene 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, me-
tal polishes, shoe black, perfumes, dye intermediates, crystallizing sol-
vent, and as a combustible propellant (Dorigan and Hushon, 1976).
Nitrobenzene is stored in closed containers and not usually released to
the open air. In plants manufacturing or using nitrobenzene, atmospheric
contamination is usually prevented by the use of activated charcoal absorb-
ers or a carbon dioxide blanket. There is no industrial monitoring of ni-
trobenzene in the atmosphere. The greatest loss of nitrobenzene during pro-
duction (estimated as eight million pounds annually) occurs at the acid ex-
traction step in the purification of the crude reaction mixture, when nitro-
benzene is lost to the effluent wash (Dorigan and Hushon, 1976). Thus, the
greatest exposure to nitrobenzene occurs inside plants, while most cases of
chronic nitrobenzene exposure in man involve nitrobenzene workers. Today,
plant levels of nitrobenzene are usually kept below the threshold limit
value (TLV) of 5 mg/nr* [Goldstein, 1975; American Conference of Governmen-
tal Industrial Hygienists (ACGIH), 1977] but much higher levels have been
reported in the past (Pacseri and Magos, 1958). Nitrobenzene may also form
C-l
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spontaneously in the atmosphere from the photochemical reaction of benzene
with oxides of nitrogen; the symptoms of nitrobenzene poisoning are similar
to the symptoms experienced by victims of Japanese photochemical smog (Dori-
gan 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 ani-
line. These methods can detect nitrobenzene from 1.0 to 500 mg/m3 (0.2 to
100 ppm) (Dorigan and Hushon, 1976). Nitrobenzene in wastewater can be mea-
sured 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).
Some of the physical and chemical properties of nitrobenzene are summar-
ized in Table 1. Common derivatives of nitrobenzene (besides aniline) are
dinitrobenzene, nitrobenzene-sulfonic acid, and nitrochlorobenzene. There
are many other derivatives of nitrobenzene, and many of them are very hazar-
dous to man as toxic agents, mutagens, and carcinogens.
EXPOSURE
Ingestion from Water
Nitrobenzene can be released into wastewater from production plants as
the result of losses during the production of nitrobenzene, aniline, or dye-
stuffs. 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;
U.S. EPA, 1970; Alekseeva, 1964), so that large amounts can not readily ac-
cumulate unnoticed. Levels of nitrobenzene in wastewater are monitored by
plants producing and using the chemical but nitrobenzene levels in city
C-2
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TABLE 1
Properties of Nitrobenzene
Formula:
Molecular weight:
Freezing point:
Boiling point:
Water solubility:
Soluble in:
Vapor pressure:
Vapor density:
Log partition co-efficient:
Density:
Flash point:
Autoignition temp:
Viscosity:
Detection level of character-
istic bitter almond odor:
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
1.682 cp at 30°C
10~4 mmoles/1
*Source: Dorigan and Hushon, 1976
c-3
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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 in-
gested in drinking water.
Ingestion from Food
There are reports of nitrobenzene poisoning resulting from its uses as
false almond oil in baking, rubbing on the gums to ease toothache, contami-
nation 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 nitrobenzene poisoning
has also occurred from ingestion of denatured alcohol (Donovan, 1920; Wirt-
schafter and Wolpaw, 1944). These cases are typical of accidental nitroben-
zene ingestion. Nitrobenzene is not an approved food additive (Dorigan and
Hushon, 1976).
A bioconcentration factor (BCF) relates the concentration of a chemical
in aquatic animals to the concentration in the water in which they live.
The steady-state BCFs for a lipid-soluble compound in the tissues of various
aquatic animals seem to be proportional to the percent lipid in the tissue.
Thus, the per capita ingestion of a lipid-soluble chemical can be estimated
from the per capita consumption of fish and shellfish, the weighted average
percent lipids of consumed fish and shellfish, and a steady-state BCF for
the chemical.
Data from a recent survey on fish and shellfish consumption in the
United States were analyzed by SRI International (U.S. EPA, 1980). These
data were used to estimate that the per capita consumption of freshwater and
estuarine fish and shellfish in the United States is 6.5 g/day (Stephan,
C-4
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1980). In addition, these data were used with data on the fat content of
the edible portion of the same species to estimate that the weighted average
percent lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
No measured steady-state bioconcentration factor (BCF) is available for
nitrobenzene, but the equation "Log BCF = (0.85 Log P) - 0.70" can be used
(Veith et al., 1979) to estimate the BCF for aquatic organisms that contain
about 7.6 percent lipids (Veith, 1980) from the octanol/water partition co-
efficient (P). Based on an average measured log P value of 1.84 (Hansch and
Leo, 1979; Dec, et al., Manuscript), the steady-state bioconcentration fac-
tor for nitrobenzene is estimated to be 7.31. An adjustment factor of
3.0/7.6 = 0.395 can be used to adjust the estimated BCF from the 7.6 percent
lipids on which the equation is based to the 3.0 percent lipids that is the
weighted average for consumed fish and shellfish. Thus, the weighted aver-
age bioconcentration factor for nitrobenzene and the edible portion of all
aquatic organisms consumed by Americans is calculated to be 7.31 x 0.395 =
2.89.
Inhalation
Nitrobenzene is readily absorbed through the lungs with retention of up
to 80 percent (Piotrowski, 1967). There are reports of nitrobenzene poison-
ing from inhalation of an exterminator spray for bedbugs which was sprayed
on a child's mattress (Stevenson and Forbes, 1942; Nabarro, 1948). Poison-
ings have also resulted from inhaled nitrobenzene used as a scent in perfume
and soap (Dorigan and Hushon, 1976). Chronic and acute poisonings from ex-
posure to nitrobenzene vapor in production plants are well documented (Dori-
gan and Hushon, 1976; Browning, 1950; Zeligs, 1929; Hamilton, 1919), but
since nitrobenzene is also absorbed through the skin, industrial poisoning
cannot be attributed to inhalation alone. A worker exposed to nitrobenzene
C-5
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at 5 mg/m3, the current Occupational Safety and Health Administration
(OSHA) standard (40 CFR 1910.1000), would absorb 18 ing/day through the lungs
in 6 hours (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 lit-
erature contains many reports 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.
There have been reports of cases of shoe dye poisoning in an army camp
(Levin, 1927), and in children who were given freshly dyed shoes (Zeitoun,
1959; Graves, 1928; Levin, 1927). The most frequent signs and symptoms were
dizziness, bluish color of lips and nails (cyanosis), headache, and some-
times coma.
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; Ray-
ner, 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 relieve cyanosis. The toxicity is often more severe in prema-
ture infants who are in an incubator and exposed to the vapor as well as to
the dye on the cloth (Etteldorf, 1951). Washing of the marked diapers or
pads before their use removes the hazard of absorption of nitrobenzene or
aniline from the ink.
C-6
-------
In Egypt, "pure bitter almond oil" (a mixture of 2 to 10 percent nitro-
benzene 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) reported cases of nitrobenzene poisoning seen in Alexandria
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 experience 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 ac-
counts for industrial toxicity although the routes of exposure often cannot
be distinguished). The amount of cutaneous absorption is a function of the
ambient concentration, the amount of clothing worn, and the relative humidi-
ty (high humidity increases absorption) (Dorigan and Hushon, 1976). A
worker exposed to the current OSHA standard (40 CFR 1910.1000), 5 mg/m ,
could absorb up to 25 rug in six hours, and one-third of that amount would
pass through the skin of a clothed man (Piotrowski, 1967). Pacseri and
Magos (1958) measured ambient nitrobenzene in industrial plants and found
levels of up to eight times the current limit.
Hamilton (1919) reported a case of acute, fatal, nitrobenzene 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. Dinitro-
benzene is believed to be much more toxic than nitrobenzene (Maiden, 1907).
C-7
-------
Ishihara, et al. (1976) reported a case of poisoning where a worker handled
a cleaning mixture containing 0.5 percent dinitrobenzene. The worker wore
gloves, but the dinitrobenzene penetrated the gloves to cause acute symptoms
of methemog1obinemia and hemolytic jaundice. Rejsek (1947) described dini-
trobenzene diffusion through the skin of munitions workers. Some of these
workers with chronic dinitrobenzene 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 nitrobenzene
(Dorigan and Hushon, 1976). This acute reaction could occur as late as six
weeks after toxic symptoms disappeared.
Although there are many literature references dealing with occupational
exposure to nitrobenzene, there are few, if any, reports of nitrobenzene ex-
posure resulting from water- intake. Therefore, data derived from occupa-
tional exposure will be used to develop information for establishing the
water quality criterion in this document.
PHARMACOKINETICS
Absorption
Nitrobenzene absorption can occur by all possible routes, but it takes
place mainly through the respiratory tract and skin. At 5 mg/m3, a nitro-
benzene worker can absorb 18 mg through the lungs and 7 mg through the skin
in 6 hours (Piotrowski, 1967). On the average, 80 percent of the nitro-
benzene vapor is 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 concentration to 9 mg/hr at 20 mg/m3. Air tempera-
ture does not affect the absorption rate, but an increase of relative
humidity from 33 to 67 percent will increase the absorption rate by 40
C-8
-------
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/cnr/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
the skin and clothing of dye manufacture workers may reach levels of 2 and
P
25 mg/cm , respectively (Piotrowski, 1977).
Distribution
Upon entry into the body, nitrobenzene enters the bloodstream, where it
reacts with the hemoglobin to form its oxidation product, methemoglobin.
Methemoglobin has a reduced affinity for oxygen, and the reduced oxygen car-
rying capacity of the blood is the cause of most of the toxic effects of
nitrobenzene, including its lethality. Methemoglobin levels from nitroben-
zene have ranged from 0.6 gm/100 ml in industrial 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 conditions
methemoglobin will slowly be reduced to oxyhemoglobin, the normal form of
blood hemoglobin.
Pacseri and Magos (1958) have demonstrated that sulfhemoglobin is also
formed in the blood after chronic exposure to nitrobenzene. In nitrobenzene
workers, they found average sulfhemoglobin levels of 0.27 gm/100 ml (com-
pared to the upper limit of normal of 0.18 gm/100 ml). Pacseri postulated
that since blood sulfhemoglobin disappears more slowly than methemoglobin,
it is a more sensitive indicator of nitrobenzene exposure. Sulfhemoglobin
may be more specific than sensitive because methemoglobin is normally found
in the blood whereas sulfhemoglobin is not.
C-9
-------
Uehleke (1964) measured the velocity of methemoglobin formation from ni-
trobenzene in cats. He found the rate to be variable and not related to the
blood concentration of nitrobenzene, although the methemoglobin formation
velocity was maximal in each animal at the time of highest blood concentra-
tion of nitrobenzene. He also found that metabolites of nitrobenzene are
able to oxidize hemoglobin. Methemoglobin formation from nitrobenzene has
also been demonstrated j£ vitro (Dorigan and Hushon, 1976, 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 de-
rivatives of nitrobenzene (Cooke, et al. 1968).
Nitrobenzene is very li>id soluble, with an oil to water partition coef-
ficient of 800. In a rat study, the ratio of the concentration of nitroben-
zene in adipose tissue versus blood in internal organs and muscle was ap-
proximately 10:1 one hour after an intravenous administration (Piotrowski,
1977). Rabbits intubated with 0.25 ml of nitrobenzene had 50 percent of the
compound accumulated unchanged in tissues within two days after the intuba-
tion (Dorigan and Hushon, 1976).
Dresbach and Chandler (1918) have shown cerebellar disturbances in dogs
and birds exposed to nitrobenzene vapor. A histologic study attributed
these effects to changes in the Purkinje cells of the cerebellum. Reports
of the effect of nitrobenzene 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 or liver damage
(Goldstein, 1975). Goldwater (1947) has described hyperplasia of the ery-
thropoietic centers of the bone marrow in workers chronically exposed to ni-
C-10
-------
trobenzene, but he concluded that the hyperplasia is a secondary result of
the hemolytic effect of the compound. Makotchenko and Akhmetov (1972) ob-
served secretory changes of the adrenal cortex of guinea pigs given nitro-
benzene every other day at a dose of 0.2 gm/kg for six months.
Metabolism
Available information on nitrobenzene metabolism is based on animal ex-
periments and fragmentary human data. There are two main metabolic path-
ways: (1) reduction to aniline followed hy 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 metabolism is independent of the dose in later
stages of acute or chronic intoxication. This can cause its accumulation in
high-lipid tissues (Dorigan and Hushon, 1976).
The reduction of nitrobenzene to aniline occurs via the unstable inter-
mediates, nitrosobenzene and phenyl hydroxylamine, both of which are toxic
and have pronounced methemoglobinemic capacity. The reactions occur in the
cytoplasmic and microsomal fractions of liver cells by the nitro-reductase
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 ex-
creted as an aminophenol. Reddy, et al. (1976) showed that the gut flora of
rats was needed for the reduction of nitrobenzene and subsequent methemoglo-
bin formation.
The hydroxylation of nitrobenzene to nitrophenols does not occur in the
microsomal fraction. The reaction proceeds via a peroxidase in the presence
of oxygen (Piotrowski, 1977).
Robinson, et al. (1951) studied nitrobenzene metabolism in the rabbit
using *4C-labeled material. The main metabolic product found was p-amino-
C-ll
-------
phenol (35 percent) which was formed via pheny1hydroxylamine. 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 detected in urine one week after
dosing. Little unchanged nitrobenzene was excreted in the urine. The major
urinary metabolites were p-aminophenol, nitrophenols, and nitrocatechol.
These constituted 55 percent of the urinary metabolites and were excreted
conjugated with sulfuric and glucuronic acids. About 1 percent of the dose
was expired as radiolabeled carbon dioxide.
Yamada (1958) studied nitrobenzene metabolism in rabbits in a 3-month
subcutaneous exposure study. He found that urinary excretion of detoxifica-
tion 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 isolated four to five
days after administering 0.25 mg/kg orally as a single dose in the rabbit
(Table 2).
An investigation of the metabolism of 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
inthe spider. Aniline was the major metabolic product in both species.
Nitrobenzene, if present in sufficiently small amounts in water, can be
degraded by some bacteria, such as Azobacter agilis. Nitrobenzene tends to
inhibit its own degradation at concentrations above 0.02 to 0.03 mg/1 (Dor-
igan and Hushon, 1976; Lu and Metcalf, 1975).
Lu and Metcalf (1975) studied nitrobenzene in a model aquatic ecosystem
to assess biodegradation and biomagnification. The ecosystem consisted of
C-12
-------
TABLE 2
Metabolic Fate of a Single Oral Dose (0.25 g/kg) of [l^c] Nitrobenzene
in the Rabbit During 4-5 Days After Dosing3
Metabolite
Respiratory C02
Nitrobenzene
Aniline
o-Nitrophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Nitrocatechol
Nitroquinol
p-Nitrophenyl
Mercapturic acid
(Total urinary radio-
activity)
Metabolized nitrobenzene
in feces
Metabolized nitrobenzene
in tissues
Total accounted for
Percentage of Dose (average)
1
0.6* • 2 in expired air
0.4+
0.1
9
9
3
4
31
0.7
0.1
0.3
60 total
-58 in
urine
(58)
9**
15-20
85-90%
aSource: Parke, 1956
* 0.5% in the urine and 0.1% in the expired air.
+ 0.3* in the urine and 0.1% in the expired air.
** 6% of the dose was present in the feces as p-aminophenol.
C-13
-------
green filamentous algae, Oedogonium cardiacium. snails, Physa, water fleas,
DaPhnia rcagna, mosquito larvae, Culex quinquifasciatus. and mosquito fish,
Gambusia affinis, under controlled atmospheric conditions. 14C-labeled
nitrobenzene 0.005 to 0.5 mg/m3 (0.01 to 0.1 ppm) was added to the water
and animals were removed for analysis after 24 to 48 hours. The radiolabel-
ed metabolites were extracted and separated by thin layer chromatography.
The distribution of nitrobenzene and its degradation products is listed in
Table 3.
Nitrobenzene was neither stored nor ecologicaly magnified, but was re-
duced to aniline in all organisms, acetylated in fish and water extracts
only, and hydroxylated to nitrophenols by mosquito larvae and snails. The
metabolites of nitrobenzene formed by the different organisms are illustra-
ted in Figure 1.
Excretion
In man, the primary known excretion products of nitrobenzene are p-ami-
nophenol and p-nitrophenol which appear in the urine after chronic or acute
exposure. In experimental inhalation exposure to nitrobenzene, p-nitrophe-
nol was formed with the efficiency of 6 to 21 percent. The efficiency of
p-aminophenol formation is estimated *rom observation of acute poisoning
cases where the molar ratio of excreted p-nitrophenol to p-aminophenol is
two to one, since p-aminophenol 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 methemoglo-
bin in blood. The authors exposed five adult rats to nitrobenzene vapor at
C-14
-------
TABLE 3
Distribution of Nitrobenzene and Degradation Products in Model Aquatic Ecosystem*
o
i
«-•
tn
Nitrobenzene equivalents, ppm
Total 14C
Nitrobenzene
An i 1 i ne
Aminophenolsb
Nitrophenols13
Polar
Unextractable
Rfa
0.72
0.60
0.20
0.10
0.0
H20
0.53755
0.50681
0.01262
0.00106
0.00466
0.00896
0.00164
Oedqgnoium
(alga)
0.0690
0.0162
0.0032
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
-
0.1226
0.0138
—
Physa
(snail)
0.6807
0.3886
0.0169
-
0.2190
0.0393
^
Gambusia
(fish)
4.9541
4.0088
0.3527
0.0986
0.0847
0.1130
"
*Source: Lu and Metcalf, 1975
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.
-------
o
z
IU
X
0.
o ^
I
a
•8
O
ii
il
o
200O
1500
1000-
500-
HOURS
FIGURE I
Relative detoxication capacities of key organisms of a model aquatic
ecosystem following treatment with radioactive nitrobenzene.
Source: Lu and Metcalf, 1975
C-16
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3 C
<" O
HI
I
0.
O
Si
>_. in
9
II
o
IT
HOSPITAL DAYS
10 15 20
r~. 500-
400-
300-
200-
100-
-16
I /f \
15 20
SEPTEMBER
* 2
25
I
m
O
O
o
CD
2
D
o
O
m
o" i
o >
m
^
o
o
(—
o
03
FIGURE 2
Changes in the levels of total hemoglobin and methemoglobin in blood and
of p-nitrophenol and p-aminophenol in urine. The usual daily volume of
urine was about 1 litre.
Source: Ikeda and Kita, 1964
C-17
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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 ex-
posed person indicate that the compound remains in the human body for a pro-
longed 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-nitrophenol, followed for
three weeks, is about 0.008 per hour. Metabolic transformation and excre-
tion of nitrobenzene in humans is slower by an order of magnitude than in
rats or rabbits (Piotrowski, 1977).
Because of the slow rate of nitrobenzene metabolism by humans, the con-
centration of p-nitrophenol in the urine increases for about four days
during exposure and the concentration on the first day is only about 40 per-
cent of the peak value. An estimate of the mean daily dose of nitrobenzene
in chronic industrial exposure can be obtained by the measurement of urinary
p-nitrophenol in specimens taken on each of the last three days of the work
week. The level of nitrobenzene exposure can be approximated using the for-
mula y = 0.18z, where y is the daily excretion of urinary p-nitrophenol in
mg/day and z is the mean daily dose of absorbed nitrobenzene in mg (Pio-
trowski, 1967). The extended systemic retention and slow excretion of meta-
bolites of nitrobenzene in man is determined by the low rate of metabolic
transformation (reduction and hydroxylation) of the nitrobenzene itself.
The conjugation and excretion of the metabolites, p-nitrophenol and p-amino-
phenol, 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
C-18
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100
ao
o
60
c
o
o
« 40
0.
20
Parent
[ | Nitrophenois
NO2
Reduced CAniline}Dj| Aminophenois
Conjugated
Oedogonium Oaphnia
Culex
FIGURE 3
Physa
Gambusia
Excretion of p-nitrophenol and p-aminophenol in the urine of rats ex-
posed to nitrobenzene.
Source: Ikeda and Kita, 1964
C-19
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(Piotrowski, 1977). Parke (1956) studied 14C-nitrobenzene metabolism in
rabbits and was able to account for 85 to 90 percent of the dose which was
administered by intubation. One percent of the nitrobenzene was exhaled as
C02 in air, and 0.6 percent was exhaled as unchanged nitrobenzene. Fifty-
eight percent of the dose appeared as urinary metabolites, p-aminophenol,
nitrophenols, aminophenols, nitrocatechols, and aniline. Thirty percent of
the nitrobenzene remained in the rabbit tissue four to five days after
dosing, and nine percent of the nitrobenzene metabolites were in the feces.
Urinary p-nitrophenol in man is determined after hydrolysis of the con-
jugated metabolites. Analytical methodology (of which there are several
methods) involves removal of interfering color substances, hydrolysis, ex-
traction of p-nitrophenol, re-extraction into an aqueous system, reduction
to a p-aminophenol, and reaction to indophenol, which is a blue colored pro-
duct. The sensitivity is 5 vg per sample (Piotrowski, 1977).
EFFECTS
Acute, Subacute, and Chronic Toxicity
Acute exposure to nitrobenzene can occur from accidental or suicidal in-
gestion 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); use of on diapers
or protective pads (Etteldorf, 1951); use of soap or skin oil containing ni-
trobenzene (Zeitoun, 1959); or from an untreated spill of nitrobenzene on
the skin in an industrial plant (Hamilton, 1919). The fatal dose of nitro-
benzene 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 vapor that is absorbed
C-20
-------
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).
Symptoms of chronic occupational nitrobenzene absorption are cyanosis,
methemoglobinemia, jaundice, anemia, sulfhemoglobinemia, presence of Heinz
bodies in the erythrocytes, dark colored urine, and the presence of nitro-
benzene metabolites (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 nitro-
benzene 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 result
of the hemolytic effect of nitrobenzene (Stevenson and Forbes, 1942). Sui-
cidal ingestion of nitrobenzene has been reported (Nabarro, 1948; Leinoff,
1936; Myslak, et al. 1971), and the compound has also been used unsuccess-
fully to induce abortion (Nabarro, 1948; Dorigan and Hushon, 1976). Harri-
son (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 nitrobenzene in the above cases was variable, de-
pending on the health 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
C-21
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reduce the methemoglobinemia which is the immediate effect, and often the
cause of death, in nitrobenzene poisoning. Death has resulted from intake
of less than one ml of nitrobenzene (Wirtschafter and Wolpaw, 1944).
Some of the reported toxicity values are summarized in Table 4 (Fair-
child, 1977). The term LDLo designates the lowest reported lethal dose
and TDLo 1S the lowest published toxic dose.
Levin (1927) demonstrated jn_ vivo production of methemoglobin by nitro-
benzene in dogs, cats, and rats, but not in guinea pigs or rabbits. Ores-
bach 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 de-
lay in methemoglobin formation in germ free rats by nitrobenzene and postu-
lated that the gut flora of rats was responsible for the reduction of Qn_
\nvp_) 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 products was variable
in the early stages of the exposure, but then all the detoxification reac-
tions (reduction, hydroxylation, 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-22
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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 nitro-
benzene. However, a 3M nitrobenzene solution reduced cell proliferation by
50 percent in cultured hamster cells (Raleigh, et al. 1973). Oxygen con-
sumption by cultured cells is increased by nitrobenzene (Biaglow and Jacob-
son, 1977). Its derivatives are used to sensitize malignant cells In vitro
to radiation effects (Chapman, et al. 1974). The authors suggest that this
effect was 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 humans (Hamaguchi, 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 conductance, and sometimes affect action poten-
tials of nerve cells.
m-Dinitrobenzene is a potent methemoglobin former, and is more toxic
than nitrobenzene (Ishihara, et al. 1976; Pankow, et al. 1975). Pentachlo-
ronitrobenzene (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 Appendix A
(Dorigan and Hushon, 1976).
C-23
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TABLE 4
Acute Toxicity Values*
Animal
Human (female)
Human
Rat
Rat
Rat
Rat
Mouse
Dog
Dog
Cat
Cat
Rabbit
Rabbit
Guinea pig
Route
oral
oral
oral
skin
i.p.
s.c.
s.c.
oral
i.v.
oral
skin
oral
skin
i.p.
Toxic
TD
LDLo:
LD50:
Dose
200 mg/kg
5 mg/kg
640 mg/kg
LD5Q: 2,100 mg/kg
LD50:
LDLo:
LDLo'
LV
LDLo:
in. o
Lo' '
LDLO:
LDLO:
LDLO:
LDLO:
640 mg/kg
800 mg/kg
286 mg/kg
750 mg/kg
150 mg/kg
000 mg/kg
25 g/kg
700 mg/kg
600 mg/kg
500 mg/kg
*Source: Fairchild, 1977
Aquatic toxicity: Tl_m at 96 hours: 10-100 mg/1 (ppm).
C-24
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Synergism and/or Antagonism
Alcohol has a synergistic effect on nitrobenzene poisoning. Ingestion
of an alcoholic beverage induced immediate acute toxic symptoms, including
coma, in a worker who had apparently recovered from the effects of chronic
nitrobenzene exposure. Alcohol 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).
Smyth, et al. (1969) studied the synergistic action between nitrobenzene
and 27 other industrial chemicals by intubation in rats. Most of the com-
pounds tested did not alter the LD5Q. In another study, ingestion of 2 to
20 ml of ethanol increased the severity of reaction to a 0.1 ml intravenous
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 mi-
crosomal enzymes, on methemoglobin formation by nitrobenzene in rats.
Methemoglobin was formed and then decreased in induced animals. The in-
creased microsomal enzyme level increased the rate of metabolism and excre-
tion of nitrobenzene and thus caused a rapid decline of methemoglobin levels.
Teratogem'city
There is a paucity of information on the teratogenic effects of nitro-
benzene. In one study (Kazanina, 1968b), 125 mg/kg was administered subcu-
taneously to pregnant rats during preimplantation and placentation periods.
Delay of embryogenesis, alteration of normal placentation, and abnormalities
in the fetuses were observed. Gross morphogenic defects were seen in four
of 30 fetuses examined.
C-25
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Changes in the tissues of the chorion and placenta of pregnant women who
used nitrobenzene in the production of a rubber catalyst were observed. No
mention was made of the effects on fetal development or viability (Dorigan
and Hushon, 1976). Menstrual disturbances after chronic nitrobenzene expo-
sure have also 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 100 per-
cent when administered on days one through seven after impregnation.
The available data, although sketchy, indicate that women who are or
wish to become pregnant should avoid exposure to nitrobenzene. Further
studies of nitrobenzene teratogenicity in mammals are needed.
Mutagem'city
Chiu, et al. (1978) tested nitrobenzene and 53 commercially available
heterocyclic and aliphatic nitro- compounds for mutagenicity using the Ames
Salmonella typhimurium strains 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 j_n vitro assays, the Ames
Salmonella microsomal assay and the mitotic recombination assay in yeast
(Simmon, et al. 1977). Other nitrobenzene derivatives have demonstrated mu-
tagenicity in j£ vitro assays, so that the mutagenicity of nitrobenzene is
still in question and additional work is needed in this area.
Careinogenicity
The available literature does not demonstrate the carcinogenicity of ni-
trobenzene, however, some nitrobenzene derivatives have demonstrated carcin-
ogenic capacities. For example, pentachloronitrobenzene (PCNB) has induced
hepatomas and papillomas in mice (Courtney, et al. 1976).
C-26
-------
1-Fluoro-2,4-dinitrobenzene (DNFB) was demonstrated by Bock, et al.
(1969) to be a promoter of skin tumors in mice, although it does not induce
them when administered alone.
Carcinogenic activity is frequently a general characteristic of struc-
turally related compounds (Arcos and Argus, 1974). Because of the struc-
tural similarity of nitrobenzene to the above nitrobenzene derivatives, ni-
trobenzene should be regarded as a suspect carcinogen. The same conclusion,
based on more circumspect reasoning, was reached by Dorigan and Hushon
(1976). This suspicion, while strong enough to warrant the testing of ni-
trobenzene for carcinogenicity, is not sufficiently strong to recommend a
criterion based on carcinogenicity.
C-27
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CRITERION FORMULATION
Existing Guidelines and Standards
The maximum allowable concentration of nitrobenzene in air in industrial
plants is 5 mg/m3. This value was set by the joint ILO/WHO Committee on
Occupational Health in 1975 (Goldstein, 1975). The OSHA standard for nitro-
benzene in air is 5 mg/m3 (1 ppm) set in 1977 (40 CFR 1910.1000). This is
also the limit in Germany and Sweden while the exposure limit in the USSR is
3 mg/m3 (Dorigan and Hushon, 1976).
There are no standards for nitrobenzene levels in water. Nitrobenzene
was not listed among the substances for which a maximum water concentration
has been set.
Current Levels of Exposure
Extrapolating from Piotrowski's exposure data, a worker exposed to the
current occupational standard of 5 mg/m3 (1 ppm) nitrobenzene 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 (Piotrowski,
1967).
Nitrobenzene can be a contaminant in industrial wastewater, and compa-
nies utilizing or producing nitrobenzene are required to monitor its level
in their effluent waste. Using gas chromatography the minimum detectable
level of nitrobenzene in drinking water is 0.7 ng (Austern, et al. 1975).
Nitrobenzene may be vented to the atmosphere. The vents are usually e-
quipped with absorbers or scrubbers, but some nitrobenzene vapor can
escape. Atmospheric nitrobenzene levels outside a plant are not monitored
C-28
-------
3
by industry. Since inner plant levels are below the standard of 5 mg/m
(1 ppm) and nitrobenzene vapor accumulates at the floor level due to its
high density, the external air nitrobenzene concentrations are expected to
be very low (Dorigan and Hushon, 1976).
Special Groups at Risk
Workers in plants producing or using nitrobenzene have the greatest risk
of toxic exposure. At the current OSHA standard of 5 mg/m3 (1 ppm), a
worker could absorb as much as 33 mg/day. This is enough to produce symp-
toms of chronic toxicity in some susceptible individuals (Dorigan and
Hushon, 1976). The amount of nitrobenzene absorbed by a worker via inhala-
tion 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, in
most cases, has been studied and corrected.
Pregnant women may be especially at risk with respect to nitrobenzene
due to transplacental passage of the agent. Individuals with glucose-6-
phosphate dehydrogenase deficiency may also be at special risk (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 bever-
ages may be at increased risk.
Basis and Derivation of Criterion
There are no established standards for nitrobenzene in water. Because
there are little or no data available on the toxicity of nitrobenzene in-
gested in drinking water, or on the teratogenic, mutagenic, or carcinogenic
effects of nitrobenzene in general, experimental testing is necessary before
C-29
-------
a criterion can be derived from oral ingestion data. It is recommended that
testing in these areas of toxicity be implemented so that the effects of ni-
trobenzene on mammals may be better understood.
Until more toxicological data on oral ingestion in animals are gener-
ated, criterion levels must be estimated from occupational exposure data and
from organoleptic data. As reported, nitrobenzene produces a detectable
odor in water at a threshold (lowest discernible concentration) of 30 yg/l
(Austern, et al . 1975; U.S. EPA, 1970; Alekseeva, 1964). It should be
noted, however, that this criterion level is based on aesthetics rather than
health effects.
A water quality criterion (WQC) can be derived from the Threshold Limit
Value (TLV) of 5 mg/m3. This can be done by estimating the total daily
dose allowed by the TLV from both inhalation and dermal exposure. An inha-
lation absorption coefficient of 0.8 will be used based on data provided by
Piotrowski (1967, 1977). Assuming an air intake of 10 m3/work day, the
portion of allowable dose by inhalation is 40 mg (5 mg/m3 x 10 m3/work
day x 0.8). The portion of the allowable dose by dermal exposure can be
calculated from the 7:18 ratio of dermal: inhalation exposure estimated by
Piotrowski (1967, 1977), i.e., 7/18 x 40 mg/work day » 16 mg/work day. Thus
the total allowable dose per work day is 56 mg (40 mg + 16 mg). The allow-
able daily intake (ADI) can be calculated by adjusting for a 5/7 day work
week, i.e., 56 mg/work day x 5/7 = 40 mg/day.
Assuming 100 percent gastrointestinal absorption of nitrobenzene, a
daily water consumption of 2 liters, a daily fish consumption of 0.0065 kg,
and a bioconcentration factor of 2.89, the water quality criterion is:
40
2 liters + (2.89 x 0.0065)
= 19.8 mg/1
C-30
-------
Since the WQC using the TLV is well above the detectable odor level of
nitrobenzene, water containing this concentration of nitrobenzene would not
be aesthetically 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 tfg/l 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 document are based on
the literature available to date. If future reports indicate that nitroben-
zene may be carcinogenic, mutagenic or teratogenic, a reassessment of the
WQC will be necessary.
C-31
-------
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C-45
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APPENDIX
Toxicological Effects of Nitrobenzene
Organism Route
Human Inhalation
Inhalation
Exposure
Inhalation
Inhalation
Inhalation
Inhalation
Poor ventilation
0.2-0.5 mg/1
(40-100 ppm)
0.129 mg/m3
"Large" amounts
poor ventilation
Acute
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 hours.
Response
Cyanosis, headache, fatigue methe-
moglobinemia (Ikeda and Kita,
1964).
Cyanosis, headache, fatigue, methe-
globinemia, liver damage, hypoten-
sion (Ikeda and Kita, 1964).
Above plus: liver and spleen en-
larged and tender, hyperalgesia
in extremeties (Ikeda and Kita,
1964).
Slight effects, e.g., headache,
fatigue (von Oettingen, 1941).
Threshold level for electroen-
cephalograph disturbance
(Andreeshcheva, 1964).
Hospitalized:
Day i _ fatigue, headache, asthma
2 - vertigo, coma, cyanosis
3 - labored breathing, urine
with almond odor
7 - methemoglobinemia recovery
after 1 mo. (Ravault,
et al. 1946).
Burning throat, nausea, vomiting,
gastrointestinal disturbances,
cold skin, livid face, cyanosis
(von Oettingen, 1941).
-------
APPENDIX (Continued)
Organism Route
Human Inhalation
Exposure
Inhalation
6-30 yg/l
o
Inhalation
Inhalation
Inhalation
Inhalation
Acute
Exposure Time
Nitrobenzene fac-
tory 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 composition (increased
polynuclears and eosinophils (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 tis-
sue in blood vessels, decreased
placental absorption, necrosis in
placental tissue (Ferster, 1970).
Changes in bone marrow, increased
lymphoid cell production, impair-
ment of copper metabolism and cer-
tain iron-containing enzymes
(Yordanova, et al. 1971).
Disturbance of motor impulses
(Zenk, 1970).
14 days: cyanosis, headache, back-
ache, stomach ache, vomiting
ca. 21 days: drank beer and fell
unconscious, cyanosis, dilated pu-
pils, retarded respiration, weak
pulse
1 yr.: intelligence dimmed
2 yrs.: emaciated, atrophied
muscles
-------
APPENDIX (Continued)
Organism
Human
Route
Exposure
Exposure Time
o
00
Cutaneous
absorption
Cutaneous
absorption
Cutaneous
absorption
Oral
Oral
Oral
Dye used in
diaper stamps
Shoe dye
0.5% by
weight in
paper
333 ml
0.4 ml
ca. 7 hrs.
(Handled carbon
paper
From human milk
Single
Single
Response
3 yrs.: memory failed
6 yrs: loss of perception of time
and space (Korsakoff's syn-
drome) (Chandler, 1919).
Babies: cyanosis, rapid pulse,
shallow respiration, vomiting,
convulsions, recovery in 24 hrs.
(von Oettingen, 1941).
Unconsciousness after consumption
of alcoholic beverages, death
(Chandler, 1919).
Dermatitis
(Calan and Connor, 1972)
Nurselings became cyanotic, recov-
ery in 24 hrs. (mothers ate al-
mond cake artificially flavored
with nitrobenzene)
(Dollinger, 1949).
Maximum dose with recovery report-
ed following severe symptoms
(von Oettingen, 1941).
Minimum lethal dose reported
(von Oettingen, 1941).
Rabbit
Subcutaneous
injection
0.8 mg/kg
Daily for 3 mo.
Maximum dose not causing death
(Yamada, 1958).
-------
APPENDIX (Continued)
Organism
Rabbit
Route
Subcutaneous
injection
Cutaneous
absorption
Exposure
10-14 mg/kg
700 mg/kg
Exposure Time
Single
Single
Intraperitoneal
injection
Intravenous
500 mg/kg
100 mg
Single
Daily or every 5
days
n
JL Oral
>£>
Oral
Oral
Oral
Oral
Oral
9 gm
4.8 gm
700 mg/kg
600 mg
300 mg
50 mg/kg
4 dose:
15 mil
Single
Single
Single
Single
Single
Response
Minimum dose producing observable
effects; slow and lasting methe-
moglobinemia (von Oettingen, 1941)
After 52 hrs.: lethal
(von Oettingen, 1941)
Reduced blood pressure and myocar-
dial glycogen level
(Labunski, 1972).
Simultaneous doses of 2-20 ml etha-
nol increased severity of poison-
ing (Matsumara and Yoshida, 1959).
Convulsions, death (von Oettingen,
1941; Chandler, 1919).
Lethal instantly (von Oettingen,
1941; Chandler, 1919).
Lethal dose (Stecher, 1968).
Dizziness, loss of reflexes, methe-
moglobinemia, 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)
-------
APPENDIX (Continued)
Organism Route
Rabbit Oral
Oral
Exposure
1 mg/kg
0.1 mg/kg
Exposure Time
Single
Single
Response
Lowered hemoglobin, erthyrocytes
and lymphocytes; increased leuco-
cytes (Kazakova, 1956).
Threshold toxic dose
(Kazakova, 1956)
Guinea
pig
Inhalation
Subcutaneous
o
en
O
Oral
Oral
Oral
Oral
Oral
Saturated air
(0.04 vol. %)
0.2 gm/kg
ca. 3 gm
ca. 1.2 gm
50 mg/kg
1 mg/kg
0.1 mg/kg
2-5 hrs.
Every other day
for 6 mos.
Single
Single
1 year
Single
Single
Death following tremors, paralysis
of hind legs (Chandler, 1919).
Hemolytic anemia, loss of weight,
decreased motor activity, fluxes
in urinary excretion of 17-hydroxy-
corticosteroids (Porter-SiIber
chromogens)
(Makotchenko and Akhmetov, 1972).
0.5 hrs: tremors, faint heartbeats,
labored respiration
2 hrs: death (Chandler, 1919).
Immediately motionless, then com-
plete recovery (Chandler, 1919).
Tissue degeneration, especially
heart, liver, kidney
(Kazakova, 1956).
Lowered hemoglobin, erythrocytes,
lymphocytes; increased leucocytes
(Kazakova, 1956).
Threshold toxic dose (Kazakova,
1956).
-------
APPENDIX (Continued)
Organism Route
Rat Inhalation
Inhalation
Inhalation
o
en
Exposure
5 mg/m3
Exposure Time
8 hrs.
ca. 0.03 mg/m3 Daily, up to
98 days
0.06-0.1 mg/m3 70-82 days
Inhalation
Oral
Intraperitoneal
0.008 mg/m3
600 mg/kg
800 mg/kg
73 days
Single
Single
injection
Subcutaneous
injection
Subcutaneous
injection
Subcutaneous
injection
Subcutaneous
injection
640 mg/kg
300 mg/kg
200 mg/kg
or
100 mg/kg
125 mg/kg
Single
Single
Single
Daily for 10 days
Single
Response
Metabolites excreted in 3 days
(Ikeda and Kita, 1964).
Increased ability to form sulfhemo-
globin in preference to methemo-
globin (Andreeshcheva, 1970).
Cerebellar disturbances, inflamed
internal organs (Khanin, 1969).
No effect (Andreeshcheva, 1964).
LD50 (Smyth, et al. 1969).
Lethal (Magos and Sziza, 1958).
Blood catalase activity decreased
continuously over 96 hrs.
(Goldstein and Popovici, 1959).
LD (14 days) - methemoglobinemia,
anemia, sulfhemoglobinemia
(Brown, et al. 1975).
Methemoglobinemia, sulfhemoglobin-
emia, anemia
(Zvezdai, 1972).
Delayed embryogenesis, abnormal
fetal development and embryo death
changes in polysaccharide composi-
tion of placenta
(Kazanina, 1967, 1968a,c).
-------
APPENDIX (Continued)
Organism
Rat
Route
Subcutaneous
injection
Exposure
100-200 mg/kg
Exposure Time
Single
Response
SuIfhemoglobin (most regular and
persistent form of hemoglobin) ni-
troxyhemoglobin, increased methe-
moglobin (Vasilenko and Zvezdai,
1972).
Mouse
o
i
Ul
to
Cutaneous
absorption
480 mg/kg
Intraperitoneal 1.23 gm/kg
injection
Intraperitoneal 1 gm/kg
Intraperitoneal
injection
Intraperitoneal
injection
20 mg/kg
12.3 mg/kg
Single
Single
Single
Single
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
coordination again, respiratory
arrest
48 hrs: death (Smith, et al. 1967).
Lethal dose
(Brown, et al. 1975).
10 min.: 4.2% methemoglobin
formed
(Smith, et al. 1967).
Cat
Inhalation
Inhalation
Oral
Saturated air
(0.04 vol. %)
2.4 gm
2-5 hrs.
2-3 hrs.
Single
Death following tremors, paralysis
of hind legs (Chandler, 1919).
Death
Death in 12-24 hrs. (von Oettingen,
1941; Chandler, 1919).
-------
APPENDIX (Continued)
Organism
Dog
Route
Inhalation
Intravenous
injection
Oral
o
en
CO
Oral
Oral
Oral
Oral
Exposure Exposure Time
"Thick vapor" 1.5 hrs.
150-250 mg/kg
28.8 gm plus
6 gm
24 gm
2.4 gm
500-700 mg/kg
Single
2 doses, 0.5 hrs,
apart
Single
Single
750-1000 mg/kg Single
Single
Response
Complete anesthesia and sleep
(Chandler, 1919).
Minimum lethal dose - lowered blood
pressure, pulse rate increased
then decreased; respiration stimu-
lated until paralyzed
(von Oettingen, 1941).
Immediate: agitation, then motion-
less
1 hr.: convulsions, then motionless
4.5 hrs.: tremors, hind legs para-
lyzed
18 hrs.: death (Chandler, 1919).
Few hrs.: "stupid"
12 hrs.: deep coma, slow respira-
tion, lowered skin temperature,
stomach strongly alkaline
(Chandler, 1919).
1 hr: vomiting, then sleep continu-
ing 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, tre-
mors, increased pulse rate, some-
times convulsions (Chandler, 1919)
-------
APPENDIX (Continued)
Organism Route
Dog Oral
Exposure
Exposure Time
Daily
Response
Formed methemoglobin continuously
at "certain" concentration
(Hashimoto, 1958).
Chicken Oral
Oral
1.2 gm
2.4 gm
Single
Single
Unsteady gait, recovery
(Chandler, 1919).
Immediately unconscious
12 hrs.: death (Chandler, 1919),
Pigeon
Inhalation
1 hr.
2-3 hrs.
No effects
Death (Chandler, 1919).
o
01
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