United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/8-86f'0£6F
January 1993
&EPA
Health Assessment
Document for
Hydrogen Sulfide
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EPA/600/8-86/026F
January 1993
Health Assessment Document
for Hydrogen Sulfide
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
11
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PREFACE
The Office of Health and Environmental Assessment has prepared this health
assessment to serve as a source document for U.S. Environmental Protection Agency use.
In the development of the assessment document, the scientific literature has been
inventoried, key studies have been evaluated, and summary/conclusions have been prepared
so that the chemical's toxicity and related characteristics are qualitatively identified. The
relevant literature for this document has been reviewed through July 1992. Observed effect
levels and other measures of exposure-response relationships are discussed, where
appropriate, so that the nature of the adverse health responses is placed in perspective with
observed environmental levels.
Any information regarding sources, emissions, ambient air concentrations, and public
exposure has been included only to give the reader a preliminary indication of the potential
presence of this substance in the ambient air. While the available information is presented as
accurately as possible, it is acknowledged to be limited and dependent in many instances on
assumption rather than specific data. This information is not intended, nor should it be used,
to support any conclusions regarding risk to public health.
in
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ABSTRACT
Hydrogen sulfide (H2S) is a highly toxic gas that is immediately lethal in
concentrations greater than 2,000 ppm. Lethality appears to be due to anoxia in brain and
heart tissues resulting from the interaction of H2S with the cellular enzyme cytochrome
c oxidase. Inhibition of this enzyme halts oxidative metabolism, which is the primary energy
source for cells. Another toxic endpoint is the irritation of the mucous membranes,
particularly those of the respiratory tract and the eyes. Pulmonary edema occurs at sublethal
concentrations (250 to 500 ppm) in which sufficient exposure occurs before consciousness is
lost. Pulmonary edema also has been reported after long-term exposure to levels as low as
50 ppm. Concentrations above 50 ppm can cause initial loss of coronary reflex, changes in
visual acuity, and perception of blue or rainbow colors around lights, followed by very
painful inflammation, with ulceration in severe cases. Subchronic studies with mice have
shown that exposure to 80 ppm causes nasal lesions.
Olfactory sensation is lost at 150 to 200 ppm; hence, the characteristic odor of rotten
eggs is not sufficient to warn of lethal exposure. At concentrations equal to or less than
150 ppm, symptoms such as the inability to think logically and incoherence have been
reported. Recovered victims of exposure report neurologic symptoms such as headache,
fatigue, irritability, vertigo, and loss of libido. Long-term effects are similar to those caused
by anoxia due to exposure to other toxic agents such as carbon monoxide. Hydrogen sulfide
is not a cumulative poison. No mutagenic, carcinogenic, reproductive, or teratogenic effects
have been reported in the literature.
IV
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CONTENTS
DISCLAIMER
PREFACE
ABSTRACT
LIST OF TABLES
LIST OF FIGURES
AUTHORS, CONTRIBUTORS, AND REVIEWERS
1. SUMMARY AND CONCLUSIONS
1.1 BACKGROUND INFORMATION . .
1.2 METABOLISM AND TOXICITY . .
1.3 RECOMMENDATIONS
REFERENCES
2. PHYSICAL AND CHEMICAL PROPERTIES
REFERENCES
3. MEASUREMENT AND ANALYSIS
REFERENCES
4. SOURCES
4.1 NATURAL OCCURRENCE
4.2 PRODUCTION SOURCES
4.3 ATMOSPHERIC TRANSPORT AND ENVIRONMENTAL
FATE
REFERENCES
5. ECOLOGICAL EFFECTS
5.1 INTRODUCTION
5.2 EFFECTS ON HIGHER PLANTS
5.3 EFFECTS ON ALGAE AND BACTERIA
5.4 EFFECTS ON AQUATIC ANIMALS
5.5 EFFECT ON WILDLIFE
REFERENCES
6. EXPOSURE TO HYDROGEN SULFIDE
6.1 INTRODUCTION
6.2 AMBIENT CONCENTRATIONS
6.3 OCCUPATIONAL CONCENTRATIONS
REFERENCES
11
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iv
vii
viii
ix
1-1
1-1
1-2
1-5
1-6
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2-2
3-1
3-4
4-1
4-1
4-1
4-3
4-6
5-1
5-1
5-1
5-5
5-5
5-10
5-12
6-1
6-1
6-1
6-3
6-6
v
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CONTENTS (cont'd)
7. METABOLIC FATE AND DISPOSITION
7.1 ABSORPTION . .
7.2 METABOLISM AND PHARMACOKINETICS
7.3 EXCRETION .
REFERENCES
8. TOXICITY
8.1 ANIMAL EFFECTS
8.1.1 Introduction
8.1.2 Effects Associated with Acute Exposure . . . .
8.1.3 Effects Associated with Repeated Exposure . .
8.1.4 Chronic Toxicity
8.1.5 Effects on Respiration Control Receptors ...
8.1.6 Cellular Mechanism(s) of Toxicity
8.1.7 Summary of Effects on Laboratory and
Domesticated Animals
8.2 HUMAN HEALTH EFFECTS
8.2.1 Toxic Effects Associated with Acute Exposure
8.2.2 Epidemiological Studies
8.2.3 Summary of Human Health Effects
REFERENCES
9. CARCINOGENICITY
REFERENCES
10. MUTAGENICITY
REFERENCES
11. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS . ,
REFERENCES
12. CONCENTRATION-RESPONSE ASSESSMENT
REFERENCES
7-1
7-1
7-2
7-9
7-10
8-1
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8-1
8-1
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8-18
8-19
8-20
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8-24
8-32
8-36
8-38
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10-2
11-1
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VI
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Number
4-1
6-1
6-2
8-1
8-2
8-3
8-4
LIST OF TABLES
Occupations with Potential Exposure to Hydrogen Sulfide
Atmospheric Hydrogen Sulfide Concentrations
Ambient Air Quality Standards for Hydrogen Sulfide . .
Acute Toxicity Values in Laboratory Animals
Clinical Features After Hydrogen Sulfide Exposure ....
Clinical Findings Recorded
Effects of Exposure in Humans at Various Concentrations
in Air
Page
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6-4
8-2
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8-29
8-37
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LIST OF FIGURES
Number
4-1
7-1
The sulfur cycle
Metabolism of hydrogen sulfide
Page
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
This document was prepared in The Office of Health and Environmental Assessment
(OHEA) located in the Office of Research and Development (ORD).
The author and original project manager was Dr. Harriet M. Ammann, Environmental
Criteria and Assessment Office, U.S. Environmental Protection Agency, Research Triangle
Park, NC. The current project manager and scientific editor is Mr. Mark Greenberg,
Environmental Criteria and Assessment Office, U.S. Environmental Protection Agency,
Research Triangle Park, NC, 27711.
Earlier drafts of this document were updated and revised by Dynamac Corporation:
Dr. Aisar Atrakchi, Mr. Ed Odenkirchen, and Ms. Dawn Webb (authors); Dr. Nicolas P.
Hajjar (reviewer); and Ms. Karen Swetlow (technical editor). In 1986, an external review
draft was made available via a notice in the Federal Register. Comments received were
reviewed and incorporated where appropriate.
Technical assistance within the Environmental Criteria and Assessment Office was
provided by: Ms. Frances Bradow; Mr. Doug Fennell; Ms. Ruby Griffin; Mr. Allen Hoyt;
Ms. Barbara Kearney; Dr. Dennis Kotchmar, M.D.; Ms. Emily Lee; Ms. Diane Ray; and
Ms. Donna Wicker.
Technical assistance was also provided by ManTech Environmental Technology, Inc.,
Mr. John Barton, Mr. John Bennett, Ms. Lynette Cradle, Ms. Kathryn Flynn, Ms. Jorja
Followill, Ms. Miriam Gattis, Ms. Lorrie Godley, Ms. Wendy Lloyd, Ms. Varetta Powell,
Mr. Derrick Stout, Ms. Patricia Tierney, and Ms. Jane Winn-Thompson.
Technical assistance was also provided by Ms. Karen Guenette, Ms. Susan McDonald,
Ms. Carol Rankin, and Ms. Deborah Staves of Research Information Organizers.
The following individuals participated in a peer review of an earlier draft of this
document and contributed valuable comments and suggestions.
Mr. Chris Alexander
Dynamac Corporation
HHORockvillePike
Rockville, MD 20852
Dr. Joseph J. Bufalini
Atmospheric Sciences Research Laboratory
U.S. Environmental Protection Agency
(MD-54)
Research Triangle Park, NC 27711
Dr. James S. Bus
Chemical Industry Institute of Toxicology
P.O. Box 12137
Research Triangle Park, NC 27711
Dr. Doyle Graham
Head, Neuropathology Department
Duke University Medical School
Durham, NC 27705
IX
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (cont'd)
Dr. Alex Herbert
University of Alberta
6104 Clinical Sciences Building
Alberta, Canada T6G ZE1
Dr. James A. Popp
Chemical Industry Institute of Toxicology
P.O. Box 12137
Research Triangle Park, NC 27709
Dr. Michael G. Prior
Alberta Environment Centre
Box 4000 Vegreville
TOB 4LO Alberta, Canada
Dr. Charles Rothwell
Dynamac Corporation
11140RockvillePike
Rockville, MD 20852
Dr. C. Ray Thompson
University of California, Riverside
Riverside, CA 92521
Dr. Lawrence Valcovic
Office of Health and Environmental Assessment
Reproductive Effects Assessment Group
U.S. Environmental Protection Agency
(RD-689)
Washington, DC 20460
Dr. Benjamin Van Duuren
New York Environmental Health Center
550 First Avenue
New York, NY 10016
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1. SUMMARY AND CONCLUSIONS
1.1 BACKGROUND INFORMATION
Hydrogen sulfide (H2S) is a colorless gas. At low concentrations it has a characteristic
obnoxious odor similar to that of rotten eggs. Its molecular weight is 34.08, and it is heavier
than air. It is flammable in air, can explode, and can be ignited by static discharge. It burns
with a pale blue flame, and its combustion products are sulfur dioxide and water. Hydrogen
sulfide is the only thermodynamically stable, binary, sulfur-hydrogen compound that occurs
frequently in nature; because of its relative lack of hydrogen bonding, it is a gas under
normal conditions. It is soluble in water and in a number of organic compounds.
Produced in nature primarily through the decomposition of organic material by bacteria,
H2S is also a constituent of natural gas, petroleum, sulfur deposits, volcanic gases, and sulfur
springs. Such natural sources constitute approximately 90% of the air emissions of H2S,
which have been estimated to be 90 to 100 million tons annually.
Industrial sources and other anthropogenic activities contribute about 10% to the total
air burden of H2S. In the United States, 125,000 employees in 73 industries are potentially
exposed to H2S, according to the National Institute of Occupational Safety and Health. The
gas is used mainly as an intermediate and reagent in the preparation of other compounds of
reduced sulfur. It is also a by-product of many industrial processes that release it into the
atmosphere. Generally, it is not found in high concentrations in the ambient air. Occasional
catastrophic releases in processing and transport have exposed the general public to
concentrations high enough to elicit toxic symptoms and death.
Hydrogen sulfide is oxidized by photochemically-generated free radicals, especially by
hydroxyl radicals. It has a half-life in air typically ranging from 12 to 37 h, but this varies
depending on the presence of photoactive pollutants and temperature, so that seasonal and
geographic differences in concentrations are found. The half-life in air can exceed
37 h during very cold and dry winter conditions.
Ambient levels of H2S measured over short time intervals (e.g., 1 to 8 h) tend to be
ij
low, in the range of 0.001 mg/m (0.00072 ppm). Pollution episodes have reached levels of
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nearly 0.5 mg/m (0.358 ppm) in severe cases, and accidental releases such as well blowouts
1-1
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rt
have produced levels as high as 14.3 mg/m (10.26 ppm). At least one release in Poza Rica,
Mexico, emitted lethal levels of gas.
Ecologic effects have been studied primarily with bacteria and naturally generated H2S
(i.e., geothermally produced). Ambient levels generated by anthropogenic sources are well
below those known to cause symptoms of injury to higher plants. Hydrogen sulfide can act
as a nutrient sulfur source in sulfur-deficient plants. Hydrogen sulfide in water, generated
through decay, can be damaging to plants such as rice. Fish can be injured by high sulfide
levels; the toxicity is similar to that shown in mammals, including humans. However,
several marine macroinvertebrates are capable of tolerating long exposures to high
concentrations of H2S. This is explained by two protective mechanisms, one described in the
tubeworm, Riftia pachyptila, in which a sulfide-binding protein acts as a sulfide trap and
prevents it from spontaneous oxidation and subsequent inhibition of the respiratory chain
enzymes, and the second described in the clam, Solemya reidi, in which the mitochondria
possess a capability to oxidize sulfide to thiosulfate. The latter can function as an energy
source for the bacterial symbionts in the host clam gill tissue.
Effects on wildlife have not been demonstrated from ambient H2S levels, although high
levels from accidental releases can be lethal.
1.2 METABOLISM AND TOXICITY
Hydrogen sulfide is an extremely toxic gas and is a leading cause of sudden death in the
workplace. The cellular mechanism of toxicity is like that of cyanide, reversibly inhibiting
the respiratory enzyme, cytochrpme c oxidase.
Absorption of H2S through the skin is limited, but absorption through the nasal and
lung mucosa occurs readily. Hydrogen sulfide is not considered to be a cumulative poison,
since it is fairly rapidly oxidized to sulfates and excreted by the kidneys. Thiosulfate is also
a product of H2S oxidation. Hydrogen sulfide is distributed to various organs such as the
lung and the brain.
The immediate effect of inhaling H2S at concentrations of 1,000 to 2,000 ppm (1>390 to
2,780 mg/m3) for a few minutes are unconsciousness and respiratory paralysis, which may
lead to death due to inhibition of the respiratory center of the brain. Inhalation of only 1 or
1-2
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2 breaths of air containing 5,000 ppm (7,000 mg/m3) H2S causes unconsciousness.
At concentrations of 500 to 1,000 ppm (695 to 1,390 mg/m3), respiratory paralysis is
preceded by a period of rapid breathing or hyperpnea, and death will result unless the victim
is removed from the contaminated area and given artificial ventilation.
At concentrations between 250 and 500 ppm (347 to 695 mg/m3), the gas is extremely
irritating to the mucous membranes of the respiratory tract and the eyes. Pulmonary edema,
which can be life-threatening, almost always occurs. Prolonged exposure to the gas at
concentrations above 50 ppm (70 mg/m3) can result in pulmonary edema, although dryness
and inflammation of the epithelia of the entire respiratory tract are more common. The
epithelia of the eye, especially of the conjunctiva and the cornea, are similarly affected,
resulting in "sore eye" or "gas eye" characterized by inflammation, lacrimation, and
mucopurulent exudate; in some cases, permanent scarring of the cornea occurs after
ulceration.
It is a fallacy to assume that the obnoxious odor of H2S (like that of rotten eggs) will
give warning of the presence of the gas; this occurs only at low concentrations. The odor
threshold in humans is low (0.1 to 0.2 ppm; 0.14 to 0.28 mg/m3), but at levels of 150 to
250 ppm (208 to 347 mg/m3), the olfactory sense is lost. Those recovering from potentially
lethal exposures recall either no smell at all or a "sweetish" smell before losing
consciousness. Similarly, it should not be assumed that pain from the irritant effect,
especially in the eyes, will warn of dangerous exposure, since the gas anesthetizes the nerve
endings in these mucous membranes.
The levels of gas that produce these severe effects generally have not been encountered
in the ambient air or even in the workplace. Limited ambient air-monitoring data for various
U.S. locations, obtained prior to 1965, indicated maximum concentrations of less than 1 ppm
(1.4 mg/m3) when measured over short time intervals (e.g., <8 h) (see Table 6-1). Routine
measurements of the concentrations of H2S in ambient air were not made by the National Air
Sampling Network, and more recent monitoring information, which could aid in establishing
current ambient exposure levels, does not exist in the published literature.
It is during only catastrophic releases or failures of containment processes that the
public is exposed to the high concentrations of H2S gas (>50 ppm; 70 mg/m3) that have
been associated with chronic or acute pathological changes. During such accidents, there is
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often loss of life. Such an accident occurred in 1950 in Poza Rica, Mexico, when a flare
burning off H2S at a natural gas desulfurization plant failed. The nearby community was
inundated with gas for 20 min. As a result, 320 people were hospitalized; of these, 22 died.
After the Lodgepole, Canada, gas well blowout, ambient exposure levels of gas reached
o
15 ppm (21 mg/m ), and the exposed population complained of eye and respiratory irritation.
No long-term effects were recorded, and affected people and animals recovered completely.
Physicians reporting on recovered victims indicate that neurological and cardiological
lesions persist after high-level exposure, but no clear-cut sulfide toxicity has been implicated.
The damage has not been differentiated from that which occurs as a result of anoxia or
ischemia of the brain or heart. While there are also clear indications of damage to the eighth
cranial nerve and its associated central nervous system (CNS) connections, manifested as
disturbances in balance and gait, these too may be the result of anoxia rather than direct
sulfide toxicity.
The available literature contains insufficient human data on chronic exposure to low-
level concentrations of H2S. However, a health survey conducted by Dales et al. (1989) on
Canadian residents living downwind from two natural gas refineries at Pincher Creek,
Alberta, Canada, clearly indicated the health hazards associated with chronic low-level
exposure to sour gas, which may contain very high H2S levels. Children showed respiratory
symptoms, whereas no physiological changes were noted among adults.
The effects attributed to chronic low-level exposure (< 10 ppm; 14 mg/m3), such as
headache, fatigue, dizziness, irritability, and loss of libido, may also result from single or
recurring high-level exposures. Other workplace factors such as high humidity, temperatures,
noise levels, and work-shift effects have not been ruled out.
Animal toxicity studies showed that acute and repeated exposures to H2S can affect
various tissues, such as brain, lungs, nose, and heart. In a 90-day inhalation study B6C3F1
mice were exposed to levels of 10, 30, and 80 ppm (14, 42, and 111 mg/m3) H2S. The only
exposure-related histopathological lesion was inflammation of the nasal mucosa in animals
from the high exposure group. A similar study with Sprague-Dawley and Fischer-344 rats at
levels of 10, 30, and 80 ppm (14, 42, and 111 mg/m3) revealed no histopathological
abnormalities of the nasal tract. In female Sprague-Dawley rats of the high-exposure group,
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mean body weights were <90% of controls and brain weight was significantly reduced in
males of this group. These observations suggest that 80 ppm (111 mg/m3) is a LOAEL.
Farm animals exposed to 10 to 15 ppm (14 to 21 mg/m3) H2S during the Lodgepole gas
well blowout in 1982 experienced nasal and eye irritation, coughing, decreased food intake,
diarrhea, and bloody stool and urine. Similar effects were seen in cattle and horses during
the Drummond gas well blowout near Claresholm, Alberta, Canada, in 1984. No data were
available on chronic exposure of farm animals to H2S.
It is not possible to unequivocally state that mutagenic, carcinogenic, teratogenic, or
reproductive effects do not occur because data are insufficient.
1.3 RECOMMENDATIONS
The acute toxicity associated with exposure to H2S is clearly established, but false
assumptions about the recognition of danger by odor need to be dispelled, and adequate
information for dealing with catastrophic accidents needs to be promulgated.
There is a clear need for epidemiologic studies of long-term, low-level exposures of
populations near or involved in industries producing H2S. Neurological examination
followups of H2S accident victims are also imperative. Studies that resolve questions of
genotoxicity and carcinogenicity also need to be performed, and reproductive effects in
animals need to be evaluated.
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REFERENCES
Dales, R. E.; Spitzer, W. O.; Suissa, S.; Schechter, M. T.; Tousignant, P.; Steinmetz, N. (1989) Respiratory
health of a population living downwind from natural gas refineries. Am. Rev. Respir. Dis. 139: 595-600.
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2. PHYSICAL AND CHEMICAL PROPERTIES
Hydrogen sulfide (H2S) is a colorless gas, heavier than air under conditions of standard
temperature and pressure (specific gravity = 1.192), with a characteristic offensive odor, like
that of rotten eggs, at low concentrations. Its molecular weight is 34.08 (Weast, 1982). It is
flammable in air, burns with a pale blue flame, and is oxidized to sulfur dioxide (SO2). Its
autoignition temperature is 260 °C, with explosive limits of 4.3 and 46% by volume. The
gas has flammability limits from 44% to 4.0% (National Fire Protection Association, 1978).
It may be ignited by static discharge (Manufacturing Chemists Association, 1968). Its
combustion products are water and SO2 (Compressed Gas Association, 1981). Hydrogen
sulfide is soluble in water (437 mL/100 mL at 0 °C, and 186 mL/100 mL at 40 °C) (Weast,
1982), which may be important from a health viewpoint. It is also soluble in ethanol, carbon
disulfide (Weast, 1982), and a number of other organic solvents including ether, glycerol,
and solutions of amines, alkali carbonates, bicarbonates, and hydrosulfides (National Research
Council, 1977). The vapor pressure of H2S is 18.75 x 105 Pa at 20 °C and 23.9 x 105 Pa
at 30 °C. Its melting point is -85.5 °C, and its boiling point is -60.3 °C (Macaluso,
rj
1969). In air, 1 ppm (w/v) of H2S is equivalent to 1.4 mg/m .
Hydrogen sulfide can be oxidized by a number of oxidizing agents. The type of
reaction and its rate are dependent on the nature and type of the oxidizing agent involved.
Principal products of these reactions are SO2, sulfuric acid (H2SO4), and elemental sulfur.
Reaction with oxides of nitrogen in the atmosphere can result in the formation of SO2 and/or
H2SO4; in water the primary product is elemental sulfur. Interaction with photochemically
produced oxidants and hydroxyl radicals («OH) and ozone produces SO2, with further
/^
oxidation eventually producing H2SO4 and/or sulfate ion (SOI").
Hydrogen sulfide is the only thermodynamically stable binary sulfur-hydrogen
compound that occurs frequently in nature. It is the sulfur analogue to water. Because of the
relative lack of hydrogen bonding, it exists as a gas under normal conditions. However, it is
easily liquefied by reduced temperature or increased pressure. The liquid is colorless, with a
viscosity one-hundredth that of water (Bailar et al., 1973).
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REFERENCES
Bailar, J. C., et al. (1973) Comprehensive inorganic chemistry. Oxford, United Kingdom: Pergamon Press.
Compressed Gas Association, Inc. (1981) Handbook of compressed gases. 2nd ed. New York, NY:
Van Norstrand Reinhold Company.
Macaluso, P. (1969) Hydrogen sulfide. In: Mark, H. F.; McKetta, J. J.; Othmer, D. F., eds. Kirk-Othmer
encyclopedia of chemical technology. 2nd ed. John Wiley & Sons, Inc.; pp. 375-389.
Manufacturing Chemists Association. (1968) Chemical safety data sheet. Washington, DC: Manufacturing
Chemists Association.
National Fire Protection Association. (1978) Fire protection guide on hazardous materials. 7th ed. Boston, MA:
National Fire Protection Association.
National Research Council. (1979) Hydrogen sulfide. Baltimore, MD: University Park Press.
Weast, R. C., ed. (1982) CRC handbook of chemistry and physics. 62nd ed. Cleveland, OH: Chemical Rubber
Company.
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3. MEASUREMENT AND ANALYSIS
A number of sampling and analytical techniques used for the measurement of hydrogen
sulfide (H2S) in ambient air and occupational settings are available. Low concentrations of
H2S in ambient air are measured in field samples using paper or tiles impregnated with lead
acetate, which darkens with exposure. The detectable range of concentrations is 0.11 to
1.1 ppm (0.15 to 1.5 mg/m3). The color of the exposed samplers fades with exposure to
turbulent air and light. However, the use of lead acetate filter tape in continuous volume air
samplers is questionable because of fading that is due not only to interaction with light but
also to exposure to any oxidant (Sanderson et al., 1966). Tapes impregnated with mercuric
chloride do not fade (Pare, 1966), but sulfur dioxide in the air may change its sensitivity to
H9S (Dubois and Monkman, 1966). A standard reference method for the testing of H2S in
ambient air utilizes gas chromatography with a photoionization detector. This method can
detect concentrations below 0.7 ppb (0.001 mg/m3) without preconcentration (Environment
Canada, 1984).
A combination of gas chromatographic analysis and flame photometer detection is a
dynamic system for sampling sulfur-containing gases, including H2S, in ambient air. The
system's sensitivity depends on a number of variables, including the material on which the
sample is collected and the handling of the sample as it goes through the gas chromatograph.
fy
The detection limits for this method range from 3.5 to 93 ppb (0.005 to 0.13 mg/nr) (Pecsar
and Hartmann, 1971).
Adams and Koppe (1967) developed a technique using a gas chromatograph coupled
with a microcoulometric bromine filtration cell to determine H2S emitted into the air from
o
kraft paper mills. Concentrations down to a lower limit of 11 ppb (0.015 mg/m ) can be
measured on electronic titration equipment developed by Thoen et al. (1968).
Concentrations of 0.05 to 1 ppm (0.07 to 1.4 mg/m3) H2S in the air can be determined
by trapping the gas in an aqueous sodium hydroxide solution using an ascorbic acid adsorber,
and titrating the resulting sulfide ion with a standard cadmium sulfide solution using a sulfide
ion-selective electrode as an indicator (Ehman, 1976).
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However, the most sensitive method for the determination of H2S in ambient air was
reported by Natusch et al. (1972). It involves the use of fluorescence and has a sensitivity of
0.1 ppt (0.0002 ^g/m3).
Occupational exposure can be sampled via the use of personal samplers or monitors, and
samples can be taken intermittently or continuously. A number of commercially available
H2S monitors were evaluated for their suitability in continual monitoring situations and their
ability to measure concentrations (Smith and Shulman, 1988). The monitors used metal oxide
semiconductor sensors and employed a one- or two-point calibration with linearization to
obtain concentration data from the sensors. Performance information evaluated included
long-term zero and span stability, response time, and the effects of various temperatures,
humidities, and interferences on response. Because of good zero stability, all monitors could
be used to warn industrial employees of dangerous levels of H2S. However, to be useful for
concentration measurement, a two-point calibration monitor with electronic linearization
would be required. It was noted that the faster the response time, the greater the dependence
on humidity control. Detection limits for these monitors correspond to the exposure
standards and recommended occupational guidelines for H2S.
The National Research Council of Canada (1981) cited two analytical techniques used in
industry to determine worker exposure to H2S. One method employed a chemical reaction
with N, A/'-dimethyl-p-phenylenediamine and ferric chloride to form methylene blue, which is
spectrophotometrically measured,for H2S. The limits of detection are 0.7 to 72 ppb
(0.001 to 0.1 mg/m3) air (more concentrated samples must be diluted). This method is
considered to be the most accurate means of determining H2S in air and water. The other
technique uses iodometric titration, which has a detection limit of 0.5 ppm
(0.7 mg/m3)/30 L of air sampled.
A micromethod for the determination of H2S was described by Delwiche (1960). It is a
refinement of a method developed by Winkler (1913) and involves the precipitation of
colloidal lead sulfide, stabilization and particle size control with gum arabic, and
quantification in a photoelectric colorimeter. The sulfide is calculated by direct reference to a
standard curve prepared by the use of crystalline lead acetate or lead nitrite as a secondary
standard. This method is simple, convenient, and rapid, and it is accurate in the range of
0.01 to 0.1 fj.g H2S. An improvement on this method was made by Kruszyna et al. (1975).
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Standard methods are available for the determination of H2S and mercaptal sulfur in
natural gas and H2S and SO2 in industrial aromatic hydrocarbons; however, no details are
available (American Society for Testing and Materials, 1981). Procedures for the analysis of
H2S in biosamples have been described by Goodwin et al. (1989).
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REFERENCES
Adams, D. F.; Koppe, R. K. (1967) Direct GLC coulometric analysis of kraft mill gases. J. Air Pollut. Control
Assoc. 17: 161-165.
American Society for Testing and Materials. (1981) Standard test method for hydrogen sulfide and mercaptan
sulfur in natural gas (cadmium sulfate iodometric titration method). In: Annual book of ASTM standards.
Philadelphia, PA: American Society for Testing and Materials; designation: D 2385 - 81.
Delwiche, E. A. (1960) A micromethod for the determination of hydrogen sulfide. Anal. Biochem. 1: 397-401.
Dubois, L.; Monkman, J.L. (1966) The analyses of airborne pollutants, background papers prepared for the
national conference on pollution and our environment. Montreal, Canada: Canadian Council of Resource
Ministers.
Ehman, D. L. (1976) Determination of parts-per-billion levels of hydrogen sulfide in air by potentiometric
titration with a sulfide ion-selective electrode as an indicator. Anal. Chem. 48: 918-920.
Environment Canada. (1984) Standard reference method for ambient testing: hydrogen sulphide. Alberta, AB,
Canada: Environmental Protection Service, Technical Services Branch; report EPS 1/SRM/l.
Goodwin, L. R.; Francom, D.; Warenycia, M. W. (1989) Hydrogen sulphide analysis in biosamples by
gas-permeable membrane and ion chromatography. In: Prior, M. G.; Roth, S. H.; Green, F. H. Y.;
Hulbert, W. C.; Reiffenstein, R., eds. Proceedings of the international conference on hydrogen sulphide
toxicity; June; Banff, AB, Canada. Edmonton, AB, Canada: The Sulphide Research Network; pp. 29-40.
Goodwin, L. R.; Francom, D.; Dieken, F. P.; Taylor, J. D.; Warenycia, M. W.; Reiffenstein, R. J.;
Dowling, G. (1989) Determination of sulfide in brain tissue by gas dialysis/ion chromatography:
postmortem studies and two case reports. J. Anal. Toxicol. 13: 105-109.
Kruszyna, H.; Kruszyna, R.; Smith, R. P. (1975) Calibration of a turbidimetric assay for sulfide. Anal.
Biochem. 69: 643-645.
National Research Council of Canada. ;(1981) Hydrogen sulfide in the atmospheric environment: scientific criteria
for assessing its effects on environmental quality. Ottawa, Canada: National Research Council of Canada:
Panel on Hydrogen Sulfide; publication no. 18467.
Natusch, D. F. S.; Klonis, H. B.; Axelrod, H. D.; Teck, R. J.; Lodge, J. P., Jr. (1972) Sensitive method for
measurement of atmospheric hydrogen sulfide. Anal. Chem. 44: 2067-2070.
Pare, J. P. (1966) A new tape reagent for the determination of hydrogen sulfide in air. J. Air Pollut. Control
Assoc. 16: 325-327.
Pecsar, R. E.; Hartmann, C. H. (1971) Automated gas chromatographic analysis of sulfur pollutants. Anal.
Instrum. 9: H-2-1 - H-2-14.
Sanderson, H. P.; Thomas, R.; Katz, M. (1966) Limitations of the lead acetate impregnated paper tape method
for hydrogen sulfide. J. Air Pollut. Control Assoc. 16: 328-330.
Smith, J. P.; Shulman, S. A. (1988) An evaluation of H2S continuous monitors using metal oxide semiconductor
sensors. Appl. Ind. Hyg. 3: 214-221.
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Thoen, G. N.; DeHaas, G. G.; Austin, R. R. (1968) Instrumentation for quantitative measurement of sulfur
compounds in kraft gases. Tappi J. 51: 246-249.
Winkler, L. W. (1913) Z. Anal. Chem. 52: 641.
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4. SOURCES
4.1 NATURAL OCCURRENCE
Hydrogen sulfide (H2S) is produced in nature primarily through the decomposition of
organic material by bacteria. It develops in stagnant water that is low in oxygen content,
such as bogs, swamps, and polluted water (Denmead, 1962; Dixon and Lodge, 1965;
Alexander, 1974; Barrett and Clark, 1987). The gas also occurs as a natural constituent of
natural gas, petroleum, sulfur deposits, volcanic gases, and sulfur springs. Natural sources
constitute approximately 90% of the atmospheric burden of H2S. This has been estimated to
be ,90 to 100 million tons, of which 60 to 80 million are produced annually from land sources
and approximately 30 million tons from aquatic areas (Urone, 1976). Ambient air
concentrations of H9S due to natural sources are estimated to be between 0.11 and 0.33 ppb
(0.15 and 0.46 Mg/ni3) (Miner, 1969).
4.2 PRODUCTION SOURCES
Industrial processes and other anthropogenic sources contribute approximately 10% of
the air burden of H2S. The National Institute for Occupational Safety and Health (1977) lists
73 industries that emit H9S (Table 4-1). The gas is used mainly as an intermediate and
reagent in the preparation of other compounds of reduced sulfur. Processing operations in
kraft paper mills and manufacturers of viscose rayon and polyethylene and polyester resins
release H2S to the air. Petroleum refineries, natural gas plants, petrochemical plants, coke
oven plants, iron smelters, food processing plants, tanneries, heavy water processing plants,
and a variety of metal alloy manufacturers release H2S as a by-product.
Hydrogen sulfide found in natural gas may be present in concentrations ranging from
1.5 to 90%. It must be removed prior to use of the natural gas for heating or power
production. It is .an important source of elemental sulfur. Natural gas is usually sold only
when the H2S content is less than < 16.4 ppm (23 mg/m3), but some H2S can escape during
the transport and processing of natural gas (Miner, 1969).
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TABLE 4-1. OCCUPATIONS WITH POTENTIAL EXPOSURE TO
HYDROGEN SULFBDE
Animal fat and oil processors
Animal manure removers
Artificial-flavor makers
Asphalt storage workers
Barium carbonate makers
Barium salt makers
Blast furnace workers
Brewery workers
Bromide-brine workers
Cable splicers l
Caisson workers
Carbon disulfide makers
Cellophane makers
Chemical laboratory workers, teachers,
students
Cistern cleaners
Citrus root fumigators
Coal gasification workers
Coke oven workers
Copper-ore sulfidizers
Depilatory makers
Dyemakers
Excavators
Felt makers
Fermentation process workers
Fertilizer makers
Fishing and fish-processing workers
Fur dressers
Geothennal-power drilling and production workers
Gluemakers
Silk makers
Slaughterhouse workers
Smelting workers
Soapmakers
Sugarbeet and sugarcane processors
Sulfur spa workers
Sulfur products processors
Synthetic-fiber makers
Gold-ore workers
Heavy-metal precipitators
Heavy-water manufacturers
Hydrochloric acid purifiers
Hydrogen sulfide production and sales workers
Landfill workers
Lead ore sulfidizers
Lead removers
Lithographers
Lithopone makers
Livestock farmers
Manhole and trench workers
Metallurgists
Miners
Natural gas production and processing workers
Painters using polysulfide caulking compounds
Papermakers
Petroleum production and refinery workers
Phosphate purifiers
Photoengravers
Pipeline maintenance workers
Pyrite burners
Rayon makers
Refrigerant makers
Rubber and plastics processors
Septic tank cleaners
Sewage treatment plant workers
Sewer workers
Sheepdippers
Tank gagers
Tannery workers
Textiles printers
Thiophene makers
Tunnel workers
Well diggers and cleaners
Wool pullers
Source: National Institute for Occupational Safety and Health (1977).
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Processing of high-sulfur coal and oil can also result in the release of H2S. Crude oil
stock of 20,000 barrels may form up to 50 tons of H2S (Miner, 1969). Gebhart and
Andersen (1988) measured and evaluated ambient concentrations of H2S in and around two
oil wells in western North Dakota. The effectiveness of flaring exhaust gases to reduce
ground-level H2S concentrations was also studied. Measured concentrations ranged from
^
0.002 to 0.671 ppm (0.08 to 0.9 mg/m ). It was found that flaring exhaust gases reduced
the range of H2S concentrations to 0.002 to 0.097 ppm (0.08 to 0.13 mg/m3).
Combustion of sulfur-contaminated fuels releases some H2S to the atmosphere,
a problem that industries have generally mitigated by both decreasing the sulfur content of
fuels and by catalytically oxidizing the H2S. In automobiles, the latter method is used, but is
circumvented when carburetors and/or catalytic converters are not functioning properly.
Agriculture, too, is a source of H2S, particularly in large feedlot or barn operations,
where bacteria produce the gas in manure piles and tanks, and in settling ponds. Some fatal
cases of H2S poisoning have occurred in connection with the processing of manure and with
work associated with human sewage treatment and latrines. Deaths have been reported in
pigs and cattle following the emptying of slurry (manure) tanks, when agitation releases toxic
gases (McAllister and McQuitty, 1965; Lawson and McAllister, 1966; Clarke and Clarke,
1975).
Most cases of acute toxicity occur in accidental or episodic releases associated with
leaks from storage tanks or processing equipment, or in transfer or transport of the gas or
mixtures containing the gas. (See Chapter 8: Toxicity).
4.3 ATMOSPHERIC TRANSPORT AND ENVIRONMENTAL FATE
The lifetime of H2S is affected by ambient temperature and other atmospheric variables,
including humidity, sunshine, and presence of other pollutants. The decreased temperatures
and decreased levels of »OH in northern regions in winter increase the residence time of H9S
in air (Bottenheim and Strausz, 1980).
Studies of photo-oxidation by Cox and Sandalls (1974) and Stuhl (1974) concluded that
free radicals such as »O and »OH generated photochemically were of importance in oxidizing
H2S. Rate constants for the reaction of H2S with »OH, ranging from < 10"13 to
4-3
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10"10 cm3 mole'V1, were used to derive a lifetime for H2S in the troposphere ranging from
12 to 27 h (Sprung, 1977; Eggleton and Cox, 1978; Wine et al., 1981; Servant and Delaport,
1982).
Robinson and Robbins (1970), using data from other researchers, estimated that the
surface-catalyzed reactions of H2S with ozone (O3) are sufficiently rapid to cause H2S to
have a mean residence time in the troposphere from 2 h in urban areas to about 2 days in
more remote, unpolluted areas. However, Hales et al. (1974) suggest such catalysis is
negligible.
Spedding and Cope (1984) carried out a limited number of experiments at ground level
in a geothermal plume, in both summer and winter, and concluded that atmospheric lifetimes
of H2S oxidation to sulfur dioxide (SO2) were less than those deduced in the laboratory
reactions of H2S with «OH. They proposed that at least one other mechanism that occurs in
the dark when »OH is not present is responsible for H2S oxidation. Their calculated lifetime
for H2S in air was about 10 h.
Studies by Becker et al. (1975) and Hales et al. (1974) show that homogeneous
reactions of H2S with O3 are very slow, and can be considered negligible when compared to
reaction with »OH (Sprung, 1977). Becker et al. (1975) calculated the rate constants for the
or\ "5
hypothetical bimolecular reaction at kx = <2 x 10 cm/molecule/s. The authors state:
"This number reflects the technically limited accuracy in measuring slow reaction rates at
sufficiently low reactant concentrations to exclude chain processes rather than a true
bimolecular rate constant, k, which may still be substantially lower."
Microorganisms in soil and water are involved in oxidation-reduction reactions, which
oxidize H2S to elemental sulfur (see Chapter 5). Members of the genera Beggiatoa,
Thioploca, and Thiotrix function in transition zones between aerobic and anaerobic conditions
where both molecular oxygen arid H2S are found (National Research Council, 1977). Joshi
and Hollis (1977) described how Beggiatoa protects rice plants from the inhibitory effects of
H2S that accumulates in the soil (see Chapter 5). Other genera such as Thiobacterium,
Macromonas, Tliiovulum, and Tliiospira also interact at interfaces of water containing oxygen
and water containing H2S, but since these organisms have not been isolated in pure culture,
their specific role is less well understood. Some photosynthetic bacteria oxidize H2S to
elemental sulfur. Members of the families Chlorobiaceae and Chromatiaceae (purple sulfur
4-4
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bacteria) are obligate aerobes, phototropic, and are found in waters with high H2S
concentrations (National Research Council, 1977). The interactions of these organisms form
part of the global sulfur cycle, which is diagrammed in Figure 4-1.
Volcanoes and Burning Sulfur
Figure 4-1. The sulfur cycle.
Source: National Research Council (1977).
Hydrogen sulfide is oxidized by microbes to elemental sulfur, and finally to sulfate,
which is chemically relatively stable. Sulfate can be taken up by plants and incorporated into
plant protein, which in turn is incorporated into animal protein by herbivorous animals, and
on through the food web by carnivores. Decay of plant and animal material releases H2S
again through the action of decay microorganisms; some strictly anaerobic sulfate-reducing
bacteria can also reduce sulfate directly to H2S.
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REFERENCES
Alexander, M. (1974) Microbial formation of environmental pollutants. Adv. Appl. Microbiol. 18: 1-73.
Barrett, E. L.; Clark, M. A. (1987) Tetrathionate reduction and production of hydrogen sulfide from thiosulfate.
Microbiol. Rev. 51: 192-205.
Becker, K. H.; Inocencio, M. A.; Schurath, U. (1975) The reaction of ozone with hydrogen sulfide and its
organic derivatives. In: Benson, S. W., ed. Proceedings of the symposium on chemical kinetics data for
the upper and lower atmosphere; September 1974; Warrenton, VA. Int. J. Chem. Kinet. Symp. no.
1: 205-220.
Bottenheim, J. W.; Strausz, O. P. (1980) Gas-phase chemistry of clean air at 55° N latitude. Environ. Sci.
Technol. 14: 709-718.
Clarke, E. G. C.; Clarke, M. L. (1975) Veterinary toxicoloay. 1st ed. Baltimore, MD: Williams and Wilkins
Co.
Cox, R. A.; Sandalls, F. J. (1974) The photo-oxidation of hydrogen sulphide and dimethyl sulphide in air.
Atmos. Environ. 8: 1269-1281.
Denmead, C. F. (1962) Air pollution by hydrogen sulfide from a shallow polluted tidal inlet, Auckland, New
Zealand. In: Proceedings of the; first technical session of the clean air conference, University of New
South Wales, Auckland, New Zealand.
Dixon, J. P.; Lodge, J. P. (1965) Air conservation report reflects national concern. Science (Washington, DC)
148: 1060-1066.
Eggleton, A. E. J.; Cox, R. A. (1978) Homogeneous oxidation of sulphur compounds in the atmosphere. Atmos.
Environ. 12: 227-230.
Gebhart, D. H.; Andersen, S. R. (1988) Exposure to hydrogen sulfide in the vicinity of oil and gas wells.
Presented at: 81st annual meeting of the Air Pollution Control Association; June; Dallas, TX. Pittsburgh,
PA: Air Pollution Control Association; paper no. 88-95B.7,
Hales, J. M.; Wilkes, J. O.; York, J. L. (1974) Tellus 26: 277.
Joshi, M. M.; Hollis, J. P. (1977) Interaction of Begglatoa and rice plant: detoxification of hydrogen sulfide in
the rice rhizosphere. Science (Washington, DC) 195: 179-180.
Lawson, G. H. K.; McAllister, J. V. S. (1966) Toxic gases from slurry. Vet. Rec. 79: 274.
McAllister, J. V. S.; McQuitty. (1965) Release of gases from slurry. Rec. Agric. Res. 14: 73-78.
Miner, S. (1969) Preliminary air pollution survey of hydrogen sulfide: a literature review. Raleigh, NC:
U.S. Department of Health, Education, and Welfare, National Air Pollution Control Administration;
publication no. APTD 69-37. Available from: NTIS, Springfield, VA; PB82-243288.
National Institute for Occupational Safety and Health. (1977) NIOSH Criteria for a recommended
standard....occupational exposure to hydrogen sulfide. Cincinnati, OH: U.S. Department of Health,
Education, and Welfare, National Institute for Occupational Safety and Health; DHEW (NIOSH)
publication no. 77-158.
National Research Council. (1979) Hydrogen sulfide. Baltimore, MD: University Park Press.
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Robinson, E.; Robbins, R. C. (1970) Gaseous sulfur pollutants from urban and natural sources. J. Air Pollut.
Control Assoc. 20: 233-235.
Servant, J.; Delapart, M. (1982) Daily variations of the H2S content in atmospheric air at ground-level in France.
Atmos. Environ. 16: 1047-1052.
Spedding, D. J.; Cope, D. M. (1984) Field measurements of hydrogen sulphide oxidation. Atmos. Environ.
18: 1791-1795.
Sprung, J. L. (1977) Tropospheric oxidation H2S. Adv. Environ. Sci. Technol. 7: 263-278.
Stuhl, F. (1974) Determination of the rate constant for the reaction OH + H2S by a pulsed photolysis-resonance
fluorescence method. Ber. Bunsen Ges. Phys. Chem. 78: 230-232.
Urone, P. (1976) Reduced sulfur compounds. In: Stern, A. C., ed. Air pollution, v. 1. 3rd ed. New York, NY:
Academic Press; pp. 53-55.
Wine, P. H.; Kreutter, N. M.; Gump, C. A.; Ravishankara, A. R. (1981) Kinetics of OH reactions with the
atmospheric sulfur compounds H2S, CH3SH, CH3SCH3, and CH3SSCH3. J. Phys. Chem. 85: 2660-2665.
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5. ECOLOGICAL EFFECTS
5.1 INTRODUCTION
Available data on the ecological effects of hydrogen sulfide (H2S) relate more directly
to bacteriological or geothermal sources than to anthropogenic sources. Hence, more
information is available about the effects on plants and animals coming into contact with H2S
through soil and water than through air.
5.2 EFFECTS ON HIGHER PLANTS
Ambient levels of H2S are well below those known to cause signs of injury to higher
plants (National Research Council of Canada, 1977). Field injury of plants generally has not
been reported from ambient exposures. A report from a gas well blowout in Alberta,
Canada, in which monitored H9S concentrations ranged from 5 to 10 ppm (7 to 14 mg/m3)
for several hours, with higher peak exposures, indicated the possibility for effects on
vegetation. Alfalfa and hay crops in the exposure area were reported to have as low as
one-half to one-third of their normal yield. No comparisons with unexposed croplands were
made, and the effects of seasonal parameters such as moisture and temperature were not ruled
out. It must be noted that the blowout occurred in winter; therefore, growing field crops
were not affected (Lodgepole Blowout Inquiry Panel, 1984).
Relatively few air exposure or fumigation experiments have been done with higher
plants. McCallan et al. (1936) and Benedict and Breen (1955) conducted short-term, high-
exposure fumigation studies on 29 vegetation and 10 weed species, respectively.
In McCallan's study, plants were exposed for 5 h in the middle of the day to concentrations
ranging from 20 to 400 ppm (28 to 560 mg/m3) H2S. A wide range of injury was seen;
eight species showed no injury at 400 ppm (560 mg/m3), and other species displayed visible
injury at less than 40 ppm (56 mg/m3). Young, growing tissues were the most susceptible to
injury. Benedict and Breen (1955) fumigated 10 species of weeds, 3 to 6 weeks of age, with
100 to 500 ppm (140 to 700 mg/m3) H2S for 4 h. They also observed species differences in
susceptibility to injury and noted that younger plants were more sensitive to damage than
5-1
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older ones. Both studies indicated that increases in temperature and dry soil exacerbated the
damage.
The damage to young shoots and leaves consisted of scorching; basal and marginal
scorching of the next oldest leaves were also observed (Heck et al., 1970). Mature leaves
were unaffected. Heck et al. (1970) provided a table that divides 38 selected plants into
sensitive, intermediate, and resistant groupings. Included among plants sensitive to H2S are
kidney bean (Phaseolus vulgaris L.), buckwheat (Fagopyrum esculentum Moench), clover
(Trifoliwn sp.), cucumber (Cucumis sativus L.), soybean, (Glycine max. Merr.), tobacco
(Nicotiana glauca Grah. and Nicotiana tabacum L.), and tomato (Lycopersicon esculentum
Mill.). Among intermediately sensitive plants are Kentucky blue grass (Poa pratensis L.),
pepper (Cupisium futescens L.), and rose (Rosa sp.). Some plants resistant to the effects of
H7S are apple (Maluspumila Mill.), cherry (Prunus serotina Ehrhe.), mustard (Brassica
campestris L.), and strawberry (Fragaria sp.).
Thompson and Kats (1978) fumigated various crop and forest plants in continuous,
long-term exposure experiments. Two procedures using concentrations of 0, 0.03, 3.0, and
30 ppm (0, 0.04, 4 and 40 mg/m3) or 0, 0.03, 1.0, and 3.0 ppm (0, 0.04, 1.4, and
4 mg/m3) were employed. Alfalfa was exposed for 28 to 35 days, grapes for 117 or
145 days, and ponderosa pine for 76 days. In contrast to the low sensitivity to H2S shown
by plants in the high-concentration, short-term exposures conducted by McCallan et al.
(1936) and Benedict and Breen (1955), plants exposed to very low concentrations of H2S
over long periods of time showed considerably more damage (Thompson and Kats, 1978).
For instance, alfalfa (Medicago saliva L.) suffered visible leaf lesions after 5 days of
*2 *3
exposure to 3 ppm (4 mg/m ) H2S, but no damage was seen at 0.03 ppm (0.04 mg/m ).
The alfalfa yield, which is normally cut and regrown in farming practice, was reduced at
3 ppm and 0.3 ppm (4 and 0.4 mg/m3), but not at 0.01 ppm (0.014 mg/m3); exposure to
0.03 ppm (0.04 mg/m3) significantly increased yields. Seedless grapes (Vitis vinifera L.)
; O
suffered severe damage at 3 ppm (4 mg/m ) and easily detectable damage at 0.3 ppm
(0.4 mg/m3). Ponderosa pine (Pinus ponderosa) showed no visible effect until 4 to 6 weeks
of exposure at 3 ppm (4 mg/m3); at 8 weeks, defoliation occurred. At 0.3 ppm
(0.4 mg/m3), tip burn occurred after 8 weeks. No effect was seen at 0.03 ppm
5-2
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o
(0.04 mg/m ). The exposed plants accumulated sulfur in leaves, although pine did less than
alfalfa or grape, perhaps because of lower normal growth rates.
California buckeye (Aesculus California), sugarbeet (Beta vulgaris), and lettuce (Lactuca
sativd) were resistant to damage, and actually the latter two species exhibited .considerable
growth stimulation at 0.3 ppm (0.4 mg/m3). As indicated in repeat experiments, temperature
variation might play a role in differential growth rates. Buckeye was exposed for 117 days,
sugar beets for 123 or 134 days, and lettuce for 59, 88, or 96 days.
Airborne SO2 has been shown to contribute to the nutrition of plants, especially those
grown in sulfur-deficient soils. Faller and Linser (1972), using H2S in addition to SO2,
confirmed the findings of earlier researchers regarding this phenomenon. In the H2S
experiments, Faller and Linser exposed mature, flowering, and viable seed-bearing
sunflowers growing in a sulfur-free nutrient solution to 3 weeks of H2S fumigation ranging
from "a few" ppm to 200 ppm (280 mg/m3). Growth of all parts of the plants was
stimulated very significantly over that of the sulfur-deficient controls; the stem alone
approximately doubled in height. The sulfur content in all plants was elevated above that of
controls; this result has not been previously-observed in nutrient experiments with SO2.
Gas uptake in plants occurs primarily through the stomata, which can be opened or
closed in response to changes in environmental conditions (e.g., illumination, humidity, and
perhaps pollutant concentrations). The cell surface available for gas exchange within leaves
can be considerably larger than the external leaf surface, which is covered with cuticle and,
therefore, not permeable to gas. For example, the lilac leaf has 6 to 8 times the external
surface internally, while the bluegreen eucalyptus has 31.3 times the surface area internally
(Turrell, 1936). Closure of stomata can, therefore, reduce gaseous uptake dramatically and
perhaps protect against short-term, high-level exposure (Hosker and Lindberg, 1982).
Conversely, stomatal opening can increase gas uptake which may constitute a nutrient effect.
Closure of stomata in response to air pollution ("smog") was observed by Mansfield and
Heath (1963). Sulfur dioxide, in concentrations as low as 0.05 ppm (0.07 mg/m3), decreases
stomatal resistance (indicating opening of stomata), but higher concentrations do not cause a
corresponding decrease in resistance (Biscoe et al., 1973). The possible effects of H2S on
stomatal opening or closing have not been investigated. Taylor et al. (1983) measured the
flux of sulfur-containing gases to vegetation, however. Using bush bean (Phaseolus vulgaris)
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and soybean (Glytine max), they showed that internal flux, through stomata, was less for H2S
than SO2 but greater for H2S than carbonyl sulfide, methyl mercaptan, or carbon disulfide.
No direct effect on stomatal function could be deduced from these experiments.
Uptake of sulfide from soil and water has been studied far more extensively than air
uptake, since this can represent plant toxicity in soils that are waterlogged or raised in water
(e.g., rice). The sulfide found in soils and water results more from bacterial action during
the decay of plant and animal protein than from any anthropogenic source of air pollution.
Ford (1973) reported that citrus trees in poorly drained areas of Florida suffered root injury
at a threshold concentration of 2.8 mg/L aqueous sulfide after 5 days of exposure. Several
investigators have examined the effect of disulfide on rice (Oryza sativa L.). Hollis and his
co-workers (Pitts et al., 1972; Allam and Hollis, 1972; Joshi et al., 1975; Joshi and Hollis,
1977) found that 1 mg/L of sulfide inhibited nutrient uptake, oxygen release, and phosphate
uptake by rice seedlings. Some varieties, however, showed enhanced nutrient uptake with
exposure to 0.05 mg/L of sulfide. It was learned that the presence of the bacterium
Beggiatoa in the soil prevented the toxic effect of H2S, while the rice seedlings' presence
symbiotically enhanced the survival of the bacterium. Beggiatoa oxidizes H2S (Joshi and
Hollis, 1977). Respiration in rice roots was investigated by Allam and Hollis (1972).
Increasing H2S concentrations were found to increasingly inhibit respiration, so that 0.1 mg/L
inhibited respiration 14%, while 3.2 mg/L inhibited this function 25.6%. Assays of root
homogenates were made after 3 to 6 h of exposure to 0.1 to 3.2 mg/L sulfide. Assayed
enzymes that showed inhibition of respiration included ascorbic acid oxidase, polyphenol
oxidase, catalase, peroxidase, and cytochrome c oxidase. Of these, cytochrome c oxidase
was most dramatically inhibited. Forty percent inhibition was measured after a 6-h root
exposure to 0.1 mg/L sulfide. This evidence is consistent with the known mode of toxicity
of H2S, which is inhibition of metal-containing enzymes, most specifically cytochrome
c oxidase, the final electron acceptor of the respiratory chain. When it is incapable of
accepting electrons, electron transport along the entire cytochrome chain stops, thereby
halting oxidative respiration.
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5.3 EFFECTS ON ALGAE AND BACTERIA
Other plant communities in the ecosystem are also affected by H2S in natural waters.
Czurda (1941) found that some species and strains of algae were inhibited by 1 to 2 mg/L
sulfide, while others seemed unaffected at concentrations of 8 to 16 mg/L. He found that
effects on various physiologic functions such as cell division, respiration, uptake of nutrients
and anaerobic respiration were variably affected in different species of algae. Nakamura
(1938) delineated enzyme inhibition in two species of algae, Pinnularia sp. and
Oscillatoria sp. Concentrations of sulfide of 0.1 mM (3.2 mg/L) completely inhibited
catalase in both species and stimulated oxygen uptake in darkness. Photosynthetic oxygen
production was strongly inhibited even at 0.01 mM (0.32 mg/L), while CO2 fixation was
unaffected. Cell division was slightly inhibited by 1.0 mM (32 mg/L) in Oscillatoria, and
was stimulated twofold in Pinnularia.
The role of bacteria in the sulfur cycle, both in the evolution of H2S during decay
processes and in the oxidation of sulfide to sulfate, is discussed in Section 4.3, Atmospheric
Transport and Fate.
5.4 EFFECTS ON AQUATIC ANIMALS
The effect of dissolved H2S gas and dissociated hydrosulfide ion (HS~) has been
examined in a number of studies of aquatic organisms. In typical seawater with a pH of
about 8, less than 4% of the sulfide pool exists as H2S; in sediments of pH 7, about 50%
(Vetter and Bagarinao, 1989). A variety of studies have shown that HS" does enter aquatic
species and is rapidly oxidized to thiosulfate and other products (Vetter et al., 1987; Vetter
and Bagarinao, 1989; Bagarinao and Vetter, 1989).
Hydrogen sulfide is highly toxic to several fish species. Broderius and Smith (1976)
reported the effect of H2S, HS" ion, and pH variation on the lethal concentration for 50% of
the test organisms (LC50) in the fathead minnow (Pinephales promelas). The 96-h LC50
values for dissolved H2S gas decreased linearly from 57.3 /ng/L to 14.9 /*g/L, with pH
increases ranging from 7.1 to 8.7. The more alkaline the pH, the more H2S, which is a
weak acid, dissociates. Undissociated H2S is thought to be the primary toxic sulfur species
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that interacts with respiratory enzymes, so the increase in toxicity indicated by the decreased
LC50 seems paradoxical. Ions are transported across membranes such as lung epithelia less
readily than neutral chemical species. However, transport across the gill surface of fish
involves a complex ion exchange mechanism for ridding fish blood of CO2 in the form of
bicarbonate ion (HCOj), formed through the action of the enzyme carbonic anhydrase, which
is found in gill tissue. The authors (Broderius and Smith, 1976) suggest that acidic
microenvironments at the gill surface may re-form the undissociated H2S, which is easily
transported. It is equally plausible to assume that HS" exchanges for HCO^ through the ion
exchange transport system, which normally involves a chloride ion, and that the hydrogen ion
(H+) released from the cleavage of carbonic acid (H2CO3) by carbonic anhydrase associates
with HS" within the cell to re-form undissociated H2S. The 96-h LC50 values of the
dissolved HS" ion increased linearly from 64.0 to 780.1 /Lig/L with increasing pH ranging
from 6.5 to 8.7. The data for the HS" ion are straightforward: the more alkaline the pH, the
more HS" ion forms; therefore the transport rate and the resulting toxicity are lower.
Cleland and Kingsbury (1977) reported that the bluegill Lepomis macrochirus was
adversely affected at 1 ^g/L dissolved H2S. A 96-h exposure study of northern pike, Esox
lucius, by the same authors, reported an LC50 ranging between 17 and 32 pcg/L H2S.
Walleye eggs (Stizostedion vitreum vitreuni) would not hatch at concentrations of 0.02 to
0.7 /ig/L. Smith (1978) exposed several species of freshwater fish to low concentrations of
H2S and determined no-effect levels of ~5 /zg/L for all the exposed fish. The 96-h LC50
values for the various fish species ranged from 25 to 145 jtig/L. The author recommended a
2-jtig/L H2S concentration as a safe limit for freshwater fish. Smith and Oseid (1972) also
investigated H9S effects on walleye eggs and fry in 96-h exposure studies. The LC50 values
they report are 74 to 87 /ig/L for eggs and 7 /*g/L for fry. Reynolds and Haines (1980)
exposed newly hatched brown trout to H2S in concentrations ranging from 2 to 13 /Lcg/L for
periods of 8 to 22 days. In contrast to the damaging effect mentioned in other studies, these
authors reported that the survival rate increased in fry exposed to concentrations of 2 to
5 fig/L H2S, and that the exposed group's growth was enhanced by 50 to 200%.
Colby and Smith (1967) investigated the effect of H2S generated by paper fiber sludge
deposits ("mats") on the survival of walleye (Stizostedion vitreum vitreum Mitchill) eggs and
fry, and on the amphipod crustacean Gammarus pseudolimnaeus in field and laboratory
5-6
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investigations. In the field studies, green eggs (36- and 48-h postfertilization) and eyed eggs
(2-weeks postfertilization) were placed on paper fiber sludge mats (five stations) and normal
river bottom (three stations) in which pH, dissolved .oxygen, and dissolved sulfide varied.
Exposure times for two separate experiments were 6 and 13 days, to 5,800 eggs and
3,300 eggs, respectively. The latter study was followed by a survival-through-hatching study
on 14-day-old eggs. The lowest survival for green eggs occurred when the dissolved oxygen
concentration dropped below 3.0 ppm and dissolved sulfide reached a concentration of
0.58 mg/L. The mortality rate for eyed eggs and sac-fry was 100% after a 6-day exposure to
the highest dissolved sulfide concentration of 0.14 mg/L. At 0.28 mg/L, all eyed eggs and
sac fry died within 2 days. Green eggs (3 and 4 days old) showed greater tolerance to
dissolved sulfide when oxygen concentrations in the water were higher. At 5.6 ppm
dissolved oxygen, little mortality was noted at 0.08 and 0.20 mg/L dissolved sulfide; at
0.34 mg/L, 98% died after 6 days; and at 0.52 mg/L, 100% died within 72 h. In contrast,
at 8.3 ppm dissolved oxygen, up to 96% of eggs exposed to 0.09, 0.21, and 0.27 mg/L
survived the experiment. At 0.47 mg/L dissolved sulfide, mortality was 97% within 5 days.
In laboratory investigations, gammarids (Gammarus pseudolimnaeus) were intolerant to
dissolved sulfide concentrations of 0.16 to 0.36 mg/L, especially at low dissolved oxygen
concentrations (1.2 to 1.3 ppm). They were far more tolerant to similar sulfide
concentrations when dissolved oxygen was 5.0 to 5.1 ppm.
Torrans and Clemens (1982) noted in their work with channel catfish (Ictalurus
punctatus) that not only oxygen, but also temperature had an effect on H2S toxicity. They
investigated possible reasons for mortality of catfish during harvesting, when the black,
malodorous sediment of pond bottoms is disturbed (and H2S is released into the water).
Harvesting usually occurs in the summer, when water temperatures are higher and dissolved
oxygen is lower, and when transport over distances exposes fish to heat. The catfish were
implanted with electrodes in the opercular muscle and near the heart so that ventilation and
heart rates could be monitored. Acute exposure (0.5 mg/L H2S for 1 min at 20 °C) resulted
in an initial stimulation of heart rate and amplitude of ventilatory movement. Heart rate
increased from a resting rate of 88 to 128 beats/min (b.p.m.), while ventilation rate decreased
from 140 to 128 cycles/min (c.p.m), but with greater amplitude of opercular movement.
After 5 min of exposure, the heart rate decreased to 60 b.p.m.; ventilation rate decreased to
5-7
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88 c.p.m., and both became shallow and irregular. After 6 min and 40 s of exposure, the
opercular muscle went into a state of tetany and ventilation ceased. When the fish were
returned to freshwater after 8 min of exposure, the opercular muscle showed occasional
spasms, and ventilation was not restored, although the heart continued to beat with a steadily
decreasing rate for 1 h. The effect of H2S in vivo and in vitro on cytochrome c oxidase and
on blood lactate levels was determined and is discussed in detail in Chapter 7, Section 7.2,
Metabolism and Pharmacokinetics. Fish exposed so that brain cytochrome c oxidase was
inhibited 50% recovered full enzyme activity 6 h after they were returned to freshwater,
showing that inhibition is reversible and noncumulative.
Channel catfish and fathead minnows (Pimephales promelas) exposed to 20 mg/L total
dissolved sulfide at 20 °C, pH 8.0 (1.0 mg/L H2S) were removed from the solution when
respiration ceased, and their tissues were assayed for cytochrome c oxidase activity. For the
fathead minnow, enzyme activity varied from control levels in the testes to 55 % inhibition in
the kidney. In the channel catfish, the inhibition ranged from 28% for brain to 66% for
heart. The enzyme in the gill was affected before the brain and was inhibited to a greater
extent. Blood lactic acid levels rose, indicating active anaerobic metabolism. The time
course for recovery from H2S poisoning was determined; the enzyme returned to normal
levels 6 h after fish were returned to freshwater.
In subchronic toxicity studies with Gammarus pseudolimnaeus (gammarids), the
maximum safe level determined for 65-, 95-, and 105-day exposures was 2 /*g total sulfide/L,
while the 96-h LC50 was determined to be 20 jug total sulfide/L (Oseid and Smith, 1974).
Chronic studies on juvenile and adult bluegills (Lepomis macrochirus) demonstrated a
no-effect level of 2 jug/L H2S, but minnows, suckers, amphipods, and some aquatic insects
did show toxic effects at levels slightly higher than this limit (Smith et al., 1976; Smith,
1978).
In 1972, it was proposed that a water quality criterion for undissociated H2S should be
set at 2 /ig/L for fish and other aquatic life in both fresh and marine waters (Cleland and
Kingsbury, 1977). The National Academy of Sciences-National Academy of Engineering,
Environmental Studies Board had earlier recommended such a standard for freshwater
organisms, but proposed 10 jug H2S/L as a standard for marine life.
5-8
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Bagarinao and Vetter (1989) evaluated sulfide toxicity in ten species of shallow-water
marine fish and found a wide range of tolerance. Tidal-marsh fishes (e.g., killifish and
mudsucker) show high tolerance, while those from open coastal areas (e.g., northern
anchovy) have low tolerance. Sulfide concentrations in various marine habitats were
reviewed by Vetter and Bagarinao, 1989.
While the majority of aquatic organisms tested have exhibited very low tolerance for
H2S, it must be noted that several marine macroinvertebrates are capable of withstanding
long-term exposure to the compound at concentrations above expected lethal limits.
Examples of these organisms include the gutless protobranch clam (Solemya reidi), which is
found almost exclusively in habitats such as sewage outfalls and pulp-mill effluent zones
(Reid, 1980); the acoel turbellarians (Solenofinomorpha funilis and Pseudohaplogonaria sp.)
and the gastrotrich (Dolichodasys carolinensis) inhabiting the reduced zone of sediments
(Powell et al., 1980); and inhabitants of oceanic hydrothermal vent areas such as the
brachyuran crab (Bythograea thermydrori), the vestimentiferan tubeworm (Riftia pachyptila),
the vesicomyid clam (Calyptogena magnified), and the mussel (Bathymodiolus thermophilus)
(Williams, 1980; Cavanaugh etal., 1981; Felbeck et al., 1981; Cavanaugh, 1983). Research
on the organisms inhabiting deep ocean volcanic fumaroles has shown symbiotic relationships
with chemolithoautotrophic bacteria (Cavanaugh, 1983), which exploit the H7S of the vent
effluent to synthesize reduced carbon and nitrogen compounds used for the nutrition of the
symbionts and the host animal (Felbeck, 1981; Felbeck et al., 1981).
A focus of further research into hydrothermal vent symbiotic organisms has been the
ability of the host organism to withstand the comparatively high levels of H2S. Two
protective mechanisms have been described. The first mechanism was noted in the blood of
the vestimentiferan tubeworm R. pachyptila, which contains a sulfide-binding protein that
functions as a sulfide carrier (Arp and Childress, 1983). When bound to this protein, the
sulfide is stabilized against spontaneous oxidation and, consequently, does not disrupt aerobic
respiration (Powell et al., 1987). This transport system enables the tubeworm to transport
sulfide from ambient seawater to organs containing symbiotic bacteria without endangering
electron transport in the host organism's mitochondria. The second mechanism has been
proposed based on work with the gutless protobranch clam, 5". reidi. The mitochondria of
Solemya possess a sulfide-oxidizing capability that is linked to oxidative phosphorylation
5-9
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(ATP synthesis) (Powell and Somero, 1986; Powell et al., 1987). The major product of
Soleinya sulfide oxidation is thipsulfate, which is proposed to function as an energy source
for bacterial symbionts in the host clam gill tissue.
Marine macroinvertebrates lacking chemolithoautotrophic symbionts also exhibit a
similar sulfide detoxification mechanism. The foraging predatory brachyuran crab
(B. thermydrori) achieves protection from sulfide toxicity through a detoxification system
located in the hepatopancreas (Vetter et al., 1987). The initial step in this process is the
oxidation of sulfide by a sulfide oxidase enzyme to produce thiosulfate. However, it is not
known whether this sulfide oxidation process is ATP-generating. The investigators concluded
that crabs are sensitive to sulfide, but tolerances vary between habitats.
The presence of a variety of avoidance mechanisms for H2S toxicity combined with
their expression in different phyla suggests that H2S tolerance may be more widespread
among marine organisms than presently thought.
Some research suggests that a defense mechanism of some marine animals against
sulfide toxicity may be resistance of hemoglobin to reaction with sulfide. In marine animals,
mitochondria! sulfide oxidation appears to be the primary defense mechanism for protecting
cytochrome c oxidase against sulfide. This area has been reviewed by Vetter and Bagarinao
(1989).
5.5 EFFECT ON WILDLIFE
Very few studies exist that attempt to measure natural or accidental exposure of wildlife
to H2S, or to determine its effects. One investigation by Siegel et al. (1986) examined the
ambient levels of H2S at Sulphur Bay Wildlife area on Lake Rotorua, New Zealand, where
shore and water birds are exposed to H2S of geothermal origin in concentrations of 0.125 to
3.90 ppm (0.17 to 5.4 mg/m3). The authors state that exposure of these birds is higher than
would be expected for humans at these concentrations because small birds have a higher
oxygen utilization rate and, therefore, a higher ventilation rate than mammals of human size.
Populations in this wildlife area have nevertheless thrived, as indicated by the increasing
number of nests found for several species in the preserve. No other parameters of exposure
were measured on either a population level or an individual level.
5-10
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An attempt to determine the effect of exposure to fumes from a gas well blowout in
Alberta, Canada, on wildlife was made by the Canadian Wildlife Service (New Norway
Scientific Committee, 1974). A flight over the well and surrounding area the day of the
mishap to examine the lakes and larger sloughs for any evidence of dead or distressed
waterfowl, and over the areas between lakes, draws, and valleys to search for dead deer, did
not reveal any ill or dead wildlife. At the time of the blowout, all young fowl had reached
flying size, so both young and adults tending them could fly from the contaminated area.
A next-day overflight in the downwind area showed no dead or distressed birds, and the
distribution and activity of all birds seen appeared normal. Monitoring at various sites ranged
from 0 ppm (8 h) to as high as 0.02 ppm (1 h), although higher concentrations were probable
at time of release.
The gas well blowout that occurred at Lodgepole, Alberta, Canada, was investigated by
a board of inquiry. During the blowout, three moose and a raven were found dead near the
well site. Cause of death was not established. Animal track surveys indicated that large
ungulates such as elk were avoiding the immediate well-site area during the winter of the
blowout, but that they moved in normal patterns throughout the nearby forested areas,
conforming to those seen in surveys conducted in 1981. A small mammal survey conducted
by Alberta Fish and Wildlife in the cleared and perimeter areas of the well-site determined a
shift in species composition but no significant changes in numbers. Local residents said that
birds and small wild mammals disappeared from the area following the blowout. At times,
concentrations between 5 and 10 ppm (7 to 14 mg/m3) H2S were measured at various sites in
the area (Lodgepole Blowout-Inquiry Panel, 1984). Averaging times are unknown.
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6. EXPOSURE TO HYDROGEN SULFIDE
6.1 INTRODUCTION
Hydrogen sulfide (H2S) is a leading cause of sudden death in the workplace (Ellenhorn
and Barceloux, 1988). The National Institute for Occupational Safety and Health (NIOSH)
(1977) lists 73 categories of workers with potential for exposure to H2S (see Table 4-1).
Among those with the greatest likelihood of hazard are natural gas drillers, processors, and
producers; petroleum production and refinery workers; kraft pulp industry, coke oven, blast
furnace, and smelter workers; coal gasification workers; heavy water manufacturers; synthetic
fiber and rayon makers; pipeline maintenance workers; miners; livestock farmers and manure
processors; sewage treatment plant workers; sugarbeet processing workers; and tannery
workers (National Institute for Occupational Safety and Health, 1977).
Ambient concentrations of H2S tend to be low, constituting an odor nuisance.
Populations around sulfide-producing industries have been exposed to accidental releases of
widely varying concentrations, ranging from levels that caused malaise to higher levels that
were lethal.
6.2 AMBIENT CONCENTRATIONS
Ambient levels of H2S are not routinely measured. Examples of average and maximum
atmospheric concentrations of H2S found in various U.S. geographical locations before 1965
are listed in Table 6-1. More recent data on ambient levels of H2S in the United States were
not found in the published, literature. Motor vehicles, especially those whose carburetors
and/or catalytic converters are functioning improperly, are one source of concern for
contribution to the H2S air burden (Harvey, 1983).
Elevated ambient concentrations in two recorded episodes, one in the Great Kanawha
River Valley, WV, in 1950, and one in Terre Haute, IN, in 1964, were reported as 0.3 and
=0.33 ppm (0.41 mg/m3 and =0.46 mg/m3) (averaging times unknown), respectively (West
Virginia Department of Health, 1952; U.S. Department of Health, Education, and Welfare,
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TABLE 6-1. ATMOSPHERIC HYDROGEN SULFEDE CONCENTRATIONS (mg/m3)a
Location
New York City, NY
1956-1961
1962
Elizabeth, NJ
August-October 1963
Hamilton Township, NJ
May-October 1962
Woodbridge Township, NJ
April-May 1961
Greater Johnstown Area, PA
1963
Winston-Salem, NC
November-December 1962
Lewiston-Clarkston Area,
North Lewiston, ID
Near pulp mill, 1962
Great Kanawha River Valley, WV
Industrial area
February 1950- August 1951
Camas, WA
1962
Santa Barbara, CA
1949-1954
St. Louis, MO
1964
Terre Haute, IN
May-June 1964
Averageb
0.001
0.001
0.001
0.001
0.001
0.003
0.003
0.003-0.092
0.001
0.002-0.006
Maximum
0.013
0.006
0.247
0.049
0.305
0.210
0.011
0.037
0.410
0.006
1.4
0.094
> 0.460
a1.4 mg/m3 =* 1 ppm.
"Averaging times not stated.
Source: Miner (1969).
6-2
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1964). In the Terre Haute incident, levels of H2S measured at a nearby lagoon ranged from
o
2 to 8 ppm (2.8 to 11.2 mg/m ) (1-h averaging time).
During the Lodgepole oil well blowout in the foothills of Alberta, Canada, in 1982,
transient levels of up to 14.5 ppm H2S (averaging times unknown) were detected in
communities'located 20 km from the site. The maximum concentration detected in the city
of Edmonton, 130 km away, where the odor level was substantial even at concentrations well
under the peak, was 0.52 ppm (0.73 mg/m3) (Lodgepole Blowout Inquiry Panel, 1984).
Rotorua, New Zealand, is a major recreational and sports center for travelers from all
over the world. The proximity of the city to an active geothermal system is evident from the
widespread use of this energy source and the prevailing odor of H2S. Ambient
t^
concentrations ranging from 0.005 to 1.9 ppm (0.007 to 2.6 mg/m ) (averaging times ranged
from 10 to 60 min) have been measured. A preliminary study revealed no evidence of health
impairment (Siegel et al., 1986).
The states reported to have ambient air quality standards for H2S are identified in
Table 6-2.
6.3 OCCUPATIONAL CONCENTRATIONS
In the United States alone, H2S has been cited as a potential hazard in 73 occupations in
which approximately 125,000 employees are subject to exposure (National Institute for
Occupational Safety and Health, 1977) (see Table 4-1). Low-level concentrations routinely
occur in certain industries such as viscose rayon production, pulp processing, oil refining,
and gas and oil well operation. In all such occupations, potentially hazardous gases such as
carbon disulfide, mercaptans, SO2, and diverse hydrocarbons form a mixture with H2S, and
individual effects of these pollutants have been difficult to delineate. Information regarding
effects from exposure to low concentrations is scant and is often confounded by the presence
of other gases in the work environment.
In 1977, NIOSH recommended a ceiling limit of 15 mg/m3 or approximately 10 ppm
H2S for 10 min, for up to a 10-h work shift in a 40-h work week (NIOSH, 1977). The
/
present Threshold Limit Value (TLV) for H2S, expressed as a Time-Weigh ted Average
6-3
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TABLE 6-2. AMBIENT ABR QUALITY STANDARDS FOR HYDROGEN SULFTOE
State
California
Connecticut
Kentucky
Massachusetts
Minnesota
Missouri
Montana
Nevada
New York
North Dakota
Pennsylvania
Rhode Island
Texas
Virginia
Hawaii
Delaware
Indiana
Concentration (ppm)
0.03
0.2
0.01
0.014
0.05a
0.03b
0.5a
0.03b
0.05C
0.24
0.01
0.20d
0.10C
0.10
0.01
0.08
; 0.16
0.04
0.03
0.05
Average Time (hours)
1
8
1
24
0.5
0.5
0.5
0.5
1
8
1
1
24
1
1
0.5
24
1
1
1
"Not to be exceeded more than two times/year.
bNot to be exceeded more than two times/five consecutive days.
"Not to be exceeded more than one time/year.
dNot to be exceeded more than one time/month.
Source: Environmental Reporter (1991).
(TWA), is 10 ppm (-14 mg/m3) (ACGIH, 1989). (Threshold Limit Value is set by the
American Conference of Governmental Industrial Hygienists for exposure of healthy workers
8 h/day, 40 h/week. The TLV for short-term exposure limit (STEL), which represents the
maximum concentration to which workers may be exposed for up to 15 min, is 15 ppm
6-4
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n
(-21 mg/m ). Accidental exposures of workers and the general population have occurred in
which the levels were much higher, sometimes by several orders of magnitude. For example,
in Poza Rica, Mexico, in 1950, an accidental release of H2S from an absorption unit in a
natural gas refining plant killed 22 people and hospitalized 320 more in the nearby
community, even though the release lasted only 20 to 25 min (McCabe and Clayton, 1952).
6-5
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REFERENCES
American Conference of Governmental Industrial Hygienists. (1989) Threshold limit values and biological
exposure indices for 1988-1989. Cincinnati, OH: American Conference of Governmental Industrial
Hygienists.
Ellenhom, M. J.; Barceloux, D. G. (1988) Medical toxicology. - New York, NY: Elsevier Science Publishing
Co.
Environmental Reporter. (1991) State air laws. Washington, DC: The Bureau of National Affairs, Inc.; v. I-V.
Harvey, C. A. (1983) Determination of a range of concern for mobile source emissions of hydrogen sulfide.
Ann Arbor, MI: U.S. Environmental Protection Agency, Office of Mobile Sources; EPA technical report
no. EPA/AA/TSS/83-7.
Lodgepole Blowout Inquiry Panel. (1984) Lodgepole blowout inquiry phase I report to the lieutenant governor
in council with respect to an inquiry held into the blowout of the well, Arusco Dome Brazean River
13-12-48-12. Calgary, Alberta, Canada: Energy Resource Conservation Board; report no. D 84-9.
McCabe, L. C.; Clayton, G. D. (1952) Air pollution by hydrogen sulfide in Poza Rica, Mexico: an evaluation
of the incident of Nov. 24, 1950. AMA Arch. Ind. Hyg. Occup. Med. 6: 199-213.
Miner, S. (1969) Preliminary air pollution survey of hydrogen sulfide: a literature review. Raleigh, NC:
U.S. Department of Health, Education, and Welfare, National Air Pollution Control Administration;
publication no. APTD 69-37. Available from: NTIS, Springfield, VA; PB82-243288.
National Institute for Occupational Safety and Health. (1977) NIOSH Criteria for a recommended
standard occupational exposure to hydrogen sulfide. Cincinnati, OH: U.S. Department of Health,
Education, and Welfare, National Institute for Occupational Safety and Health; DHEW (NIOSH)
publication no. 77-158.
Siegel, S. M.; Penny, P.; Siegel, B. Z.; Penny, D. (1986) Atmospheric hydrogen sulfide levels at the Sulphur
Bay wildlife area, Lake Rotorua, New Zealand. Water Air Soil Pollut. 28: 385-391.
U.S. Department of Health, Education, and Welfare. (1964) The air pollution situation in Terre Haute, Indiana
with special reference to the hydrogen sulfide incident of May-June, 1964. Terre Haute, IN: Public
Health Service, Division of Air Pollution.
West Virginia Department of Health; Kettering Laboratory. (1952) Atmospheric pollution in the Great Kanawha
River Valley industrial area. Cincinnati, OH: University of Cincinnati.
6-6
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7. METABOLIC FATE AND DISPOSITION
7.1 ABSORPTION
The most common route of entry for hydrogen sulfide (H2S) is the lung.
Experimentally, sodium sulfide (Na2S) has been injected intravascularly or intraperitoneally,
or instilled orally by gavage, to determine its distribution and fate in tissues as well as its
metabolism. Absorption of H2S through the skin is limited. Exposure of large areas of skin
to pure H2S was lethal in guinea pigs after 45 min but did not affect dogs (Walton and
Witherspoon, 1925). In rabbits, exposure of the entire body, except the head, allowed a
qualitative detection of H2S in expired air (Laug and Draize, 1942). Absorption through the
tympanic membrane of workers wearing respirators was not a significant route of toxicity
(Ronk and White, 1985).
In aqueous solution, for instance in body fluids, H2S has two acid dissociation constants
and can thus exist as the hydrosulfide anion (HS~) and as the sulfide anion (S2~). The pKa
for Step 1 is 7.04; and the pKa for Step 2 is 11.96 (in solutions 0.01N to 0.1N at 18 °C).
H2S
HS"
H"
-2-
(1)
(2)
At human physiologic pH and temperature of 7.4 and 37 °C, respectively, about one-third of
the total sulfide exists as undissociated H2S, about two-thirds as HS", and minuscule amounts
r\
as S ". Since unionized small molecules tend to diffuse across membranes more readily than
ionized molecules do, it is likely that H2S is absorbed more rapidly than the negatively
charged ions. Absorption of H2S in protozoans occurred more rapidly than the ionic species
(Beerman, 1924). Absorption of H2S from the peritoneal cavity of mice occurred more
rapidly with an acidic carrier, which prevented sulfide ion formation, than in an alkaline
carrier, which enhanced ion formation (Smith and Abbanat, 1966).
7-1
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7.2 METABOLISM AND PHARMACOKINETICS
Hydrogen sulfide can be metabolized via three pathways: (1) oxidation to sulfate,
(2) methylation, and (3) reaction with metallic ion or disulfide-containing proteins
(Figure 7-1) (Beauchamp et al., 1984). Oxidation and methylation represent means of
detoxification, while the interaction with essential proteins, particularly the iron-containing
proteins of the respiratory chain, is largely responsible for the toxic actions of the gas.
The oxidation of sulfide to sulfate has been studied for nearly 40 years and is not as yet
precisely defined. While early in vitro studies with liver and kidney preparations postulated
intermediates such as free sulfur, polythionates, and thiosulfate, Garabedian (1945a,b)
proposed that sulfide oxidase enzymatically catalyzed the oxidation of sulfide. Baxter et al.
(1958) and Baxter and Van Reen (1958) confirmed the existence of a liver sulfide oxidase.
The observation was made by Sorbo (1958) that heme catalyzed sulfide oxidation to
thiosulfate. Several studies were initiated to determine the precise site of sulfide oxidation.
35S-Sodium sulfide incubated in vitro with blood rapidly bound to blood proteins (Curtis
et al., 1972). It was demonstrated too that this route of oxidation worked very slowly and
was insufficient to account for high levels of sulfate formation in living systems. Other
in vitro experiments (Bartholomew et al., 1980) showed that thiosulfate was the major
oxidation product of sulfide in liver mitochondria, and that this could then be converted to
sulfate by sulfide oxidase, which has been purified from rat and dog liver and kidney
(MacLeod et al., 1961a,b). The precise location for major oxidation of sulfide in vivo has
not been unequivocally established, but the liver is the most probable site.
The lung participates little in metabolism of sulfide to sulfate. Using whole-body
o c
autoradiography after intraperitoneal injection or gavage instillation of S-sulfide, Curtis
et al. (1972) showed that while the lung accumulated 35S-sulfide, very little was converted to
radioactively labeled sulfate. This confirms the work of MacLeod et al. (1961a) that sulfide
oxidase is absent in lung tissue.
Whole-body autoradiography of young male M.R.C. hooded rats, following
intraperitoneal injection of 35S-sulfide and 35S-sulfate and sacrifice of animals at time
intervals ranging from 3 min to 6 h after injection, showed that the label widely distributed
and accumulated in tissues, including the gastrointestinal tract and cartilage. The uptake into
bones indicated that oxidation to sulfate occurred prior to incorporation into
7-2
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Thiol-s-Methyi- Metalloproteins
Transferase (Fe, Cu)
CH3SH*
Thiol-s-Meihyl-
Transferase
CHgSCHg
: >• 1 Methemoglobin,
Disutfide- Ferritin
Containing „ _
Proteins SCytochrome
Oxidase
3 Catalase,
Peroxidase
Suca'nic Dehydrogenase
Polysulfide
[intermediates!
Sulfide
Oxidase
S20|
(Thiosulfate)
GSH _
"HS + SO;
\/
Y GSH Reduction
A,,
NADP
Sulfite
Oxidase
so?
Figure 7-1. Metabolism of hydrogen sulfide.
Source: Beauchamp et al. (1984).
Reaction
Consequences
Detoxification
Toxicity
Toxicity (?)
Toxicity (?)
7-3
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mucopolysaccharides. In addition to these tissues and lung, radioactive label also
accumulated in brain tissue and persisted there up to 20 min after sulfide injection (Curtis
etal., 1972).
Further attempts to identify the locale of sulfide oxidation were made by Bartholomew
et al. (1980) using 35S-sulfide and isolated, living, perfused rat livers, lungs, and kidneys.
These experiments confirmed the plasma binding of sulfide (up to 90% bound) and the lack
of sulfate formation in the lung. Release from carrier proteins in plasma and volatilization of
sulfide to H2S occurred, and 32% of the administered dose was lost from the blood through
the lung. Sulfide remaining in the blood was oxidized slowly, possibly within red cells.
The same experiments with kidney confirmed the findings of Curtis et al. (1972) that
sulfate was the major radioactive component in renal vein blood and urine, and that the
kidney can oxidize sulfide. Bartholomew et al. (1980) found a mechanism for rapid
oxidation of sulfide in liver mitochondria, which worked rapidly but only at low sulfide
concentrations that did not inhibit cytochrome c oxidase activity. Studies with isolated rat
liver perfused with heparinized homologous blood to which (a) Na235S in phosphate buffer
and (b) Na235S and unlabeled thiosulfate in buffer were added showed significant metabolism
of the sulfide to sulfate. After perfusion for 15 min in experiment (a) above, 70% of the
radioactively labeled sulfur was associated with sulfate, and the percentage increased to 82%
after 2 h of perfusion. In experiment (b) above, 54% of the radioactive sulfur was found in
thiosulfate after 15 min of perfusion, with 22% 35S in sulfate. After 30 min, the amount of
label present in thiosulfate had decreased to about 30%, while that in sulfate had increased to
about 46%. At the end of 2 h perfusion time, only 13% of the label remained in the
unreacted sulfide, and no radioactivity could be detected in thiosulfate. The work of these'
researchers confirmed the earlier work by MacLeod et al. (1961a,b) and Koj et al. (1967),
which found that thiosulfate is a major oxidation product of sulfide and that thiosulfate was
oxidized to sulfate in mitochondria. They proposed that glutathione (GSH) mediated
thiosulfate oxidation according to the following equations:
(1) (S • S03)2' + 2GSH
2- Vz0
(2) SO3
(3) 2HS'
HS" + HSO4" + GSSG (oxidized glutathione)
20
2'
(S • S03)' + H20
7-4
-------
MacLeod et al. (1961 a,b) suggested that sulfide oxidase converted the sulfide
intermediate to sulfate.
Weisiger and Jakoby (1979) have identified an.enzyme, thiol-S-methyltransferase, which
catalyzes the methylation of H2S to methanethiol (CH3SH), then dimethylsulfide (CH3SCH3).
The authors regarded this methylation as a means of detoxification because both products are
less toxic than H2S. The enzyme is found primarily in gut mucosa and liver, and may thus
serve to detoxify absorbed H2S that was produced by anaerobic bacteria in the intestinal tract.
The role of this enzyme in the detoxification of inhaled H2S has not been determined.
Reaction of H2S with metallic ion-containing protein is considered its major mechanism
of toxicity (Smith and Gosselin, 1979). Chance and Schoener (1966) had found sulfide to be
a stable inhibitor of mitochondrial heme-containing cytochrome enzymes, which are involved
in oxidative metabolism. Cytochrome c oxidase is the last enzyme in this complex of the
cytochrome chain that transfers electrons to oxygen as the final electron acceptor, combining
them with hydrogen ions to form water. In the presence of H2S, transfer of electrons to
oxygen cannot occur, all electron transport down the chain is stopped, and oxidative
metabolism, which is the primary energy source for mammalian cells, stops. Work by
Wever et al. (1975), Nicholls (1975), Nicholls et al. (1976), Smith et al. (1977), and Smith
and Gosselin (1979) showed that H9S causes chemical reduction of one of the hemes of this
enzyme, preventing electron transfer to oxygen. Chance and Schoener (1966) found that H7S
inhibits cytochrome c oxidase slightly more potently than does hydrogen cyanide (HCN), but
the mechanism of action appears to be similar. Smith et al. (1977) also conducted in vitro
experiments using sub-mitochondrial particles prepared from beef heart. They confirmed that
sulfide is a more potent inhibitor of cytochrome c oxidase than is cyanide. Nicholls (1975)
showed similar results and determined the lq for H2S to be -0.02 ^iM.
Inhibition of cytochrome c oxidase through in vivo and in vitro experiments, and
recovery from inhibition, was shown by Torrans and Clemens (1982) in channel catfish
(Ictalurus punctatus), in addition to measurement of some physiologic parameters (see
Chapter 5). Both fathead minnows (Pimephales promelas) and channel catfish were exposed
to 1.0 mg/L H2S (20 mg/L total sulfide) at 20 °C, water pH 8.0. Individual fish were
removed from the sulfide solution when ventilation ceased (13 to 23 min for the channel
catfish and 9 to 15 min for the fathead minnows), and tissues were removed for
7-5
-------
homogenization and assay of enzyme activity. Cytochrome c oxidase activities in the fathead
minnows ranged from control levels in testes to 55% inhibition in kidney. In the channel
catfish, the brain enzyme was inhibited 28% and heart enzyme 66%. Hydrogen sulfide
(unionized) affected the catfish brain and gill cytochrome c oxidase more than dissolved
sulfide ion. When fish were exposed to 0.1 mg/L H2S at 10 °C, brain enzyme was not
affected, even at 30 min exposure, but gill enzyme was inhibited 15% after 5 min and 39%
after 30 min exposure. At 0.3 mg/L H2S, brain enzyme activity was reduced by 25%, and at
0.5 mg/L brain enzyme activity was inhibited 56%, while gill enzyme activity was reduced
by 48% after 5 min exposure. The latter was the maximum effect at that concentration and
coincided with ventilatory arrest. Temperature had a great effect on enzyme activity of fish
exposed in vivo. Channel catfish exposed at 20 °C to 0.1 mg/L H2S showed enzyme
inhibition similar to those exposed to 0.5 mg/L at 10 °C. Thus, after 10 min of exposure to
0.1 mg/L H2S, brain cytochrome c oxidase activity was 58% reduced, while gill enzyme was
41% decreased; after 20 min, brain enzyme was 40% reduced, while gill enzyme was
reduced 33%; after 30 min, brain enzyme was 40% reduced, and gill enzyme was 26%
reduced. Blood lactate levels increased as cytochrome c oxidase levels decreased, indicating
high levels of anaerobic metabolism, and the fish became rapidly fatigued. High levels of
methemoglobin induced by pre-exposing fish to nitrite sulutions reduced the degree of
cytochrome c oxidase inhibition produced upon exposure to H2S.
Torrans and Clemens (1982) also measured in vitro cytochrome c oxidase inhibition by
sulfide. Even very low concentrations inhibited the enzyme in tissue homogenates. Catfish
- ' rj
brain-homogenate cytochrome c oxidase activity was decreased 18% at 10 M H2S, 64% at
10"6M H2S, and 100% at 10"4M H2S. Effects were similar for fathead minnow brain-
homogenate. The pH of the solution influenced dissociation of H2S and consequently its
toxicity. At pH 5, and 10"6M, 98% of the H2S is unionized, and greatest inhibition (65.4%)
occurs. As the pH of 7.04 was approached, inhibition decreased and more sulfide ion
formed, and at pH 7.5 only 14% H2S remained unionized, and enzyme inhibition decreased
to 45.7%. The reaction was reversible, as was also shown in vivo, and showed competitive
kinetics.
Since the effect of H7S poisoning is to deprive the cellular cytochrome chain of oxygen,
those cells having the highest oxygen requirement are most rapidly and severely affected.
7-6
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Nerve tissue and cardiac tissue have large oxygen demands and show the first effects of H2S
toxicity. Warenycia et al. (1989) reported on several case studies in which brain stem levels
of sulfide were measured shortly after the fatalities. Analysis indicated sulfide levels of about
1 fj.g/g tissue compared to a normal human brain stem concentration of about 0.7 ^g/g. Such
elevated levels were used to establish cause of death.
Besides cytochrome c oxidase, other metallo-proteins also react with H9S. When these
are enzymes, perturbations of other pathways may occur, although this effect would be nearly
overshadowed by the cessation of oxidative metabolism. Interactions of H2S with horseradish
peroxidase (Wieland and Sutter, 1928), potato polyphenol oxidase (Keilin, 1928), and
catalase (Stern, 1932) produced inhibition of these enzymes, but the importance of these
reactions to detoxification has not been further explored. Tenhunen et al. (1983) assayed
in vitro enzyme activity for heme synthetase, and 6-amino-levulinic acid synthetase (ALA-S)
from human venous blood. These enzymes are part of the pathway in the synthesis of
protoporphyrin, which is a precursor of heme. In 17 workers exposed to H?S and
methylmercaptan, these enzymes showed decreased activity when assayed. Erythrocyte and
protoporphyrin concentration in seven of these cases were below the control range. In the
in vitro experiments, both H2S and sulfide anion inhibited heme synthetase and ALA-S
synthetase. These results may be of importance for their indication of a possible additional
pathologic mechanism for H2S poisoning, as well as a means of assessing worker exposure
and/or health. However, it must be noted that the in vitro concentrations used to produce
inhibition were considerably higher (3.4 to 10 mmol/L) than the concentrations that workers
exposed to low levels would experience.
Jappinen (1989) evaluated 21 cases of acute H2S poisoning (< 10 min) that occurred in
sulfate pulp mills. In 6/21 cases, blood samples were collected in less than 2 h and changes
in heme metabolism were assessed. A decrease in ALA-S activity was most prominent when
blood sulfide concentrations were more than 100 /ug/L. Initial mean levels of heme synthase
and protoporphyrin in blood from these six individuals were also lower than mean control
values. Levels continued to be lower than controls 1 mo after the acute poisoning episode.
Hydrogen sulfide can act as a reducing agent for disulfide bridges in proteins. Such
change in protein structure has been proposed as an explanation for H?S inhibition of succinic
7-7
-------
dehydrogenase (Hayden, 1989). Whether inhibition of this enzyme has a role in the toxicity
of H2S has not been elucidated.
Reaction of H9S with methemoglobin constitutes a pathway for detoxification, resulting
in the formation of sulfmethemoglobin. Smith et al. (1977) using submitochondrial particles
from beef heart in vitro, showed that methemoglobin relieved the inhibition of cytochrome
c oxidase by H9S by re-initiating the oxidation of ferricytochrome c. They also indicated that
the undissociated H2S is a more potent inhibitor of the enzyme than the hydrosulfide anion.
This is in agreement with findings related to HCN and hydrogen azide molecules. Similar to
the work by Scheler and Kabisch (1963), Smith and Gosselin (1966) pretreated mice with
sodium nitrite. Nitrite causes the formation of methemoglobin. Smith and Gosselin (1966)
also preinjected mice intraperitoneally with human methemoglobin prior to injection of
sodium sulfide. Both injected nitrite and methemoglobin protected the mice from death from
subsequent injections of sodium sulfide.
Detoxification may also take place via interaction of pyruvate with H2S (Dulaney and
Hume, 1988). These investigators found that administration of pyruvic acid to mice prior to
ip sodium sulfide injection reduced sulfide-induced mortality.
Beck et al. (1982, 1983) demonstrated an anesthetic-like effect of both H2S and HCN at
concentrations ranging from 5,300 ppm (7,420 mg/m3) to 99% pure H2S on isolated nerve
preparations from the frog Ram. pipiens. Changes in membrane function led them to suggest
not only an inhibition of cytochrome c oxidase, but also a conformational H2S or HS"
induced change in membrane proteins, which they suggest might account for some of the
evidence of permanent nerve damage seen in some recovered victims of H2S poisoning. The
exposure concentrations used far exceed those from which victims usually recover, however.
Such possible change in membrane protein conformation has not been further investigated.
Other explanations for permanent nerve damage are equally plausible or more so. Examples
of phenomena that have been explored include nerve cell damage as a result of anoxia (Yap
and Spector, 1965; Yanagihara, 1976; Elovaara et al., 1978; Savolainen et al., 1980; Metter
and Yanagihara, 1979) and damage done by ischemia following anoxia.
7-8
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7.3 EXCRETION
While H2S usually enters via the lung, this organ can also serve in an excretory
capacity. Evans (1967), working with cats, showed that some of the sulfide from injected
sodium sulfide was exhaled. The percentage eliminated depended on the site of injection, and
the variation in injection site was related to a variation in the length of time that sulfide was
free in the blood. Zero to 37% of H2S and NaHS injected into the abdominal aorta was
eliminated through the lung, while 26.5% was exhaled when sulfide was injected into the
external jugular vein. The external jugular joins the vena cava, and blood flowing through it
enters the pulmonary circulation almost immediately. There is little time for interaction of
sulfide with blood components, or with organs whose tissues can metabolize H2S, before it is
exchanged in the lung. The abdominal aorta, in contrast, is near the beginning of the
systemic circulation, and sulfide injected here has to make a full circuit of the vascular
system before reaching the lung. Curtis et al. (1972) demonstrated clearly that sulfide binds
to plasma proteins, primarily the albumin fraction, until it is oxidized to sulfate and excreted
in the urine. The bound sulfide would not be exhaled.
Sulfate is the end-product of oxidation and is excreted in the urine (Curtis et al., 1972).
A small amount of sulfide is oxidized to sulfate by sulfide oxidase, and is eliminated in the
bile, appearing in the feces for excretion. The sulfate that is not excreted is widely
distributed in tissues and incorporated into tissue proteins, as shown through autoradiography
and other radioactive tracer methodology by Curtis et al. (1972).
The principal fate of injected sulfide is oxidation to sulfate and excretion in urine
(Curtis et al., 1972). Sodium 35S-sulfide administered intravenously to rats resulted in 45%
of the radioactively labeled sulfur appearing in the urine as sulfate within the first 6 h after
injection. Only small amounts (4.7 to 5.0%) appeared in the bile, indicating that the liver is
not a major site of excretion.
Similarly, intragastric administration of 35S (1.66 mg of sulfide sulfur) into rats showed
that sulfate, both as inorganic and ethereal sulfur, was mainly excreted in urine.
Radioactivity was also high in bone marrow. The significance of the latter finding was
unclear (Dziewiatkowski, 1945).
7-9
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Baxter, C. F.; Van Keen, R. (1958) Some aspects of sulfide oxidation by rat-liver preparations. Biochim.
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Beck, J. F.; Donini, J. C.; Maneckjee, A. (1983) The influence of sulfide and cyanide on axonal function.
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Laug, E. P.; Draize, J. H. (1942) The percutaneous absorption of ammonium hydrogen sulfide and hydrogen
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Savolainen, H.; Tenhunen, R.; Elovaara, E.; Tossavainen, A. (1980) Cumulative biochemical effects of repeated
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Smith, R. P.; Abbanat, R. A. (1966) Protective effect of oxidized glutathione in acute sulfide poisoning.
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Appl. Pharmacol. 8: 159-172.
Smith, R. P.; Gosselin, R. E. (1979) Hydrogen sulfide poisoning. J. Occup. Med. 21: 93-97.
Smith, L.; Kruszyna, H.; Smith, R. P. (1977) The effect of methemoglogin on the inhibition of cytochrome
c oxidase by cyanide, sulfide or azide. Biochem. Pharmacol. 26: 2247-2250.
Sorbo, B. (1958) On the formation of thiosulfate from inorganic sulfide by liver tissue and heme compounds.
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Stern, K. G. (1932) Ueber die Hemmungstypen und den Mechanismus der katalatischen Reaktion. 3. Mitteilung
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on catalysis]. Hoppe-Seylers Z. Physiol. Chem. 209: 176-206.
Tenhunen, R.; Savolainen, H.; Jappinen, P. (1983) Changes in haem synthesis associated with occupational
exposure to organic and inorganic sulphides. Clin. Sci. 64: 187-191.
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46: 422-432.
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8. TOXICITY
8.1 ANIMAL EFFECTS
8.1.1 Introduction
Studies involving laboratory animal species have used inhalation exposures to hydrogen
sulfide (H2S), administration of sodium bisulfide (NaHS) and sodium sulfide (Na2S) by other
routes of exposure, and oral gavage in which test solutions were preparing by bubbling H2S
through water. It should be noted that administration by gavage and other noninhalation
routes of exposure does not permit determination of direct effects on the respiratory tract and
does not allow estimation of inhaled concentration responses on the brain, principal target
organs for H2S. Thus, the focus of this section is upon studies involving inhalation of H2S.
While most of the toxicological observations described in the following studies are
specifically attributable to H2S poisoning, some effects are also characteristic of brain anoxia.
The number and kind of cellular changes, as well as the enzymatic changes, delineated in
tissues of animals exposed to low levels of H2S correlate very closely with those observed in
animals recovering from anoxia episodes (Yap and Spector, 1965; Yanagihara, 1976;
Elovaara et al., 1978; Savolainen et al., 1980). While there is some evidence of other
enzymes that play a role in cellular dysfunction, inhibition of cytochrome c oxidase is a
singularly important event since tissues with the highest oxygen demand, such as neural and
cardiac tissues, sustain the most rapid, consequential, and permanent damage.
8.1.2 Effects Associated with Acute Exposure
Some LC50 (concentration that is lethal to 50% of test animals) values for the exposure
of laboratory animals to H2S are presented in Table 8-1. Acute toxicity values for Na2S and
HS" are intended for comparison.
Lopez (1989) carried out a study to determine if an acute lethal exposure of H2S for
less than 5 min is capable of producing life-threatening pulmonary edema similar to that
observed in persons killed by accidental exposure to H2S. Male Fischer 344 (F344) rats
(10 per group) exposed to > 1,438 ppm (>2,000 mg/m3) developed severe dyspnea and died
8-1
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8-2
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within 5 min. Postmortem examination revealed severe edema characterized by a massive
accumulation of fluid in the interstitium and bronchoalveolar spaces. Prior et al. (1989) also
reported pulmonary edema in groups of 10 male F344 rats exposed to 1,000, 1,142, 1,428,
or 2,000 ppm (1,400, 1,588, 1,986, or 2,781 mg/m3) H2S for 7 min. Responses were
categorized into three stages. The first stage included avoidance of irritant gas, hyperactivity,
and lethargy; the second included unconsciousness and a change in the breathing patterns; and
the third was characterized by shallow breathing and gasping. Pulmonary edema developed
in all animals.
To compare pulmonary injury induced by inhalation of H2S to that caused by NaHS
injection, Lopez et al. (1989) exposed Sprague-Dawley (SD) rats to 1,655 ppm
(2,301 mg/m3) H2S, or injected them intraperitoneally with 30 mg/kg NaHS. All rats in
both treatments died within 3 min, but only those exposed to H9S showed severe respiratory
distress, severe dyspnea, and presence of frothy fluids coming out of the nose and mouth.
These signs were indicative of pulmonary edema and were confirmed by postmortem
examination of the trachea and lungs of the rats exposed to H9S.
Lopez et al. (1987) studied both the biochemical and cytological changes induced by
H2S inhalation in rats. Male F344 rats were exposed to 0, 10, 200, or 400 ppm (0, 14,
278, or 556 mg/m3) H2S for 4 h and killed at 1-, 20-, or 44-h postexposure. Both nasal and
bronchoalveolar lavage (BAL) fluids were obtained to determine enzyme activities and
epithelial cell morphology as markers of cellular injury. At 400 ppm (556 mg/m3), H9S
caused a marked but transient 320% increase in lactate dehydrogenase found in the nasal
lavage, and a significant increase (up to 90%) in alkaline phosphatase and aldehyde
dehydrogenase in BAL. Such elevations are markers of cell death. In addition, BAL protein
concentrations used as markers for changes in membrane permeability were elevated by more
than 3,000% at 44-h postexposure in rats exposed to 400 ppm (556 mg/m3) H2S. This
increase in BAL protein concentrations together with a 933 % increase in gamma glutamyl
transpeptidase (GOT) enzyme activity were indicators of edema. Exposure of rats to 10 or
200 ppm (14 or 278 mg/m3) for 4 h did not cause these changes in BAL. The only
significant changes caused by lower concentrations (10 and 200 ppm; 14 and 278 mg/m3)
were a 139 and 483% increase, respectively, in the cellularity of the nasal lavage fluid.
Gamma GT has been found in the sputum of individuals with cariogenic pulmonary edema,
8-3
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chronic bronchitis, or pulmonary embolism (Rosalki, 1975). The origin of H2S-induced
pulmonary edema was suggested to result from permeability as opposed to hydrostatic or
neurogenic factors (Lopez et al., 1988b).
Male F344 rats were exposed to 0, 9.6, 198, or 387 ppm (0, 13, 275, or 538 mg/m3)
H2S for 4 h, and four levels of the nasal cavity were examined histologically at 1, 18, and
44 h after exposure (Lopez et al., 1988b). Necrosis and exfoliation of respiratory and
olfactory mucosal cells, but not squamous epithelial cells was observed in rats exposed to
387 ppm (542 mg/m3). No nasal lesions were seen in the controls or the two lower exposure
levels. Male F344 rats (4 rats/exposure level) were exposed to 0, 83, or 439 ppm (0, 115,
or 610 mg/m3) for 4 h (Lopez et al., 1988b). In rats exposed to 83 ppm (115 mg/m3), only
mild perivascular edema was observed. Rats exposed to 439 ppm (610 mg/m3) had marked
perivascular and alveolar edema, and bronchioles contained polymorphonuclear leukocytes,
proteinaceous fluid, fibrin, and exfoliated cells. Necrosis of bronchiolar ciliated cells and
hyperplasia of alveolar Type II cells was also observed in this group. Nasal structures were
not examined. These changes, as well as pulmonary edema and fibrinocellular alveolitis,
were reversible.
The nasal lesions were confined to specific areas of the nasal cavity (Lopez et al., 1986,
1987, 1988a,b). The most damage was observed in the lateral wall of the nasal turbinates in
both F344 and Long Evans rats. Moreover, pulmonary lymphatics were distended and
lympharrhexia was observed in the thymus and lymph nodes (Lopez et al., 1986, 1988b).
Khan et al. (1990) reported no mortalities in F344 male rats exposed to 10 to 400 ppm
(14 to 556 mg/m3) H9S for 4 h. Mortality only occurred at levels above 500 ppm
(695 mg/m3). There were no adverse clinical signs in the 10, 50, and 200 groups
(14, 70, and 278 mg/m3); at 400 ppm (556 mg/m3), lethargy was observed immediately
following exposure. Exposure to sublethal concentrations (50 to 400 ppm; 70 to 556 mg/m3)
produced marked and highly significant depressions in the activities of cytochrome c oxidase
and succinate oxidase complexes of the respiratory chain. The inhibition of cytochrome
c oxidase activity in lungs was most severe (>90%) in rats that died from acute exposure to
>500 ppm (>695 mg/m3) H2S. In rats exposed to 200 and 400 ppm (278 to 556 mg/m3),
a marked recovery in cytochrome c oxidase activity of lungs was observed at 24- and 48-h
postexposure. In vitro studies with rat lung mitochondria showed that low concentrations of
8-4
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sulfide also caused a similar and selective inhibition of cytochrome c oxidase activity. This
effect was reversed upon removal of sulfide either by washing or by oxidation with
methaemoglobin.
Khan et al. (1987a) reported a 65% decrease in cytochrome c oxidase and 50% decrease
in succinate oxidase activities in lung mitochondria from F344 rats exposed to 500 ppm
(695 mg/m3) H2S for 2 h. Exposure to the same level for 4 h resulted in 50% mortality and
a 90 to 95% decline in cytochrome c oxidase activity in the lung mitochondria of dead
animals. In surviving rats, cytochrome c oxidase activity remained 60% depressed
48 h postexposure; however, full recovery was evident within 2 weeks. Khan et al. (1987b)
showed that various sulfur-containing compounds such as S2" inhibited enzyme activities in
bovine erythrocytes in a dose-dependent manner. At a concentration of 10 mM, superoxide
dismutase activity was inhibited by 48%; at 0.2 mM, the activity of catalase was inhibited by
25.7%.
Khan et al. (1991) exposed male F344 rats (6/group) to 0, 50, 200, and 400 ppm
(0, 70, 278, and 556 mg/m3) H2S for 4 h. After anesthesia and exsanguination, lungs were
lavaged and alveolar macrophages (AM) were collected. Although treatment had no effect on
basal respiratory rates of AM, a significant decrease in cell viability was observed in the high
exposure group.
Green et al. (1991) investigated the effect of acute exposure of H2S on lung surfactant
in F344 rats. Decreases in surfactant could increase fluid transport to lungs resulting in
reduction in gas exchange and could result in increased surface tension within the lungs.
Groups of 6 male rats were exposed for 4 h to actual concentrations of 194 and 290 ppm
(270 and 403 mg/m3). Controls were exposed to filtered air. At 1-h postexposure, animals
were sacrificed and the right lung was lavaged. Samples of the left lung were assessed by
light microscopy.
Exposure to 194 ppm (270 mg/m3) produced no adverse clinical signs or visible gross
changes in the lungs. There was, however, a statistically significant increase in protein and
lactic dehydrogenase in BAL compared to controls. Microscopic evaluation revealed focal
areas of perivascular edema. Animals exposed to 290 ppm (403 mg/m3) were visibly stressed
during and immediately after exposure. At necropsy, lungs exhibited focal areas of red
8-5
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atelectasis, and alveolar edema with substantial perivascular and peribronchial interstitial
edema.
The minimum surface tension values for BAL from controls and animals exposed to
194 ppm (270 mg/m3) H7S were nearly identical. By contrast, exposure to 290 ppm
(403 mg/m3) H2S resulted in a substantial increase in minimum surface tension and lowered
stability. The effects of H2S on surfactant activity were considered to be due to the leakage
of serum proteins into the alveoli.
Prior et al. (1988) examined the exposure-time relationships in male and female SD,
Long Evans, and F344 rats. All three strains were exposed to H^S for 2, 4, or 6 h. There
was a significant, sex-related difference in mortality and body weight loss; the males were
more sensitive (30%) than females (20%) in all three strains. Body weight loss was also
increased proportionately to H2S concentration. For every 10-ppm increase in exposure
concentration, the body weight loss increased by 0.21 g greater in males than in females and
was different between strains (F344 < SD < Long Evans). Marked differences were seen
in LC50/LC10 ratios for the length of exposure: 587/549 ppm for 2 h, 501/422 ppm for 4 h,
and 335/299 ppm for 6 h. These results are in agreement with those reported by Lopez et al.
(1987), which indicate that once H2S reaches its threshold, pulmonary edema and death
ensue.
Substance P (SP), an endogenous neurotransmitter, has a protective role in H2S
poisoning (Prior et al., 1990). Rats were injected with capsaicin, the irritant found in red
pepper that depletes the body of SP and other tachykinins, and subsequently exposed to
400 ppm H9S for 4 h. There was 100% mortality, but no mortalities in control rats injected
with saline. Animals depleted of SP exhibited a significantly greater degree of bronchial
epithelial cell exfoliation and ulceration following H2S exposure. These results with
capsaicin suggest a role for afferent C-fibers in pulmonary defense against H2S exposure.
Husain (1976) and Husain and Zaidi (1977) investigated various enzyme activities in
H2S-treated lung homogenates from albino rats. Homogenates were exposed to H2S for
1 h prior to measurement of enzyme activities. At 18 ppm (25 mg/m3), H2S inhibited acid
phosphatase, alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic
transaminase, and ATPase by 16.8, 11.0, 25.9, 15.9, and 13.3%, respectively. As the H2S
concentration increased, the inhibition of these enzymes also increased. Fructose
8-6
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1,6-diphosphate aldolase activity was unaffected by H2S, while arginase activity was
stimulated with increasing gas concentrations. The authors postulated that metallo-sulfate
complexes are formed from the interaction with H9S, and that H2S also combines with the
enzyme cofactor pyridoxal phosphate in the case of the transaminases. Such interactions with
enzymes other than cytochrome c oxidase could contribute to possible cumulative cellular
damage from either long-term, low-level, or repeated exposure to H9S gas. However, direct
evidence for the formation of such complexes is lacking. Exposure of rabbits to 72 ppm
(100 mg/m3) H2S for 30 min/day, for 7, 10, and 14 days resulted in relative increases in
activities of acid and alkaline phosphatase, ATPase, and deoxyribonuclease-II in lungs (Jonek
and Konecki, 1966).
Yang and Hulbert (1990) observed that exposure of guinea pigs for up to 1 h to H2S
concentrations up to 500 ppm (695 mg/m3) did not alter airway resistance or dynamic
compliance. However, there was an increase in airway responsiveness to methacholine in
animals exposed to 300 and 500 ppm (417 and 695 mg/m3). Airway responsiveness to
methacholine was also increased when Cam-Hartley guinea pigs were exposed to 100 ppm
(139 mg/m3) H2S for 1 h (Hulbert et al., 1989).
Cralley (1942) found a correlation between the irritation of rabbit throats by H2S and
other irritant gases, and the suppression of mucociliary activity of the trachea. Exposure of
rabbits to 400 and 600 ppm (556 and 834 mg/m3) for 10 and 5 min, respectively, resulted in
the cessation of ciliary motility without recovery in air.
Haggard et al. (1922) demonstrated the dramatic lethal effect of H9S, as well as striking
differences in the dose response, when dogs were exposed to concentrations of 0.05, 0.1, and
0.3% H2S by volume (500, 1,000, and 3,000 ppm, respectively). At 500 ppm (695 mg/m3)
(considered to be the minimal lethal concentration), the respiratory rate of the animals
showed a slight, progressive decrease. The depth of respiratory rate was also progressively
depressed. Death resulted from pulmonary edema after many hours (not reported) of
exposure. At 1,000 ppm (1,400 mg/m3), death ensued within 15 to 20 min of exposure.
Respiration was immediately stimulated as the dogs inhaled the gas, which led to strong
•hyperpnea; this was followed by cessation of breathing and death. When the concentration of
H2S was increased to 3,000 ppm (4,200 mg/m3), respiratory arrest occurred after a few
gasps.
8-7
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When dogs were injected iy with NajS, pulmonary edema was not observed (Haggard
et al., 1922), but dogs exhibited immediate hyperpneic breathing. This was followed by
variable periods of apnea, which was relieved by artificial ventilation. Haggard et al. (1925)
indicated that vagotomy eliminated the stimulatory effects of H2S on respiration.
Inhalation of H2S, leading to elevated sulfide levels in brain tissue, has been reported to
result in histopathological damage and changes in neurotransmitter levels which alone or
together may be responsible for the CNS effects observed in humans.
Citing a Norwegian report (Savolainen, 1982), the World Health Organization (1987)
reported that acute H^S intoxication caused brain edema, as well as degeneration and necrosis
of the cerebral cortex and the basal ganglia in rhesus monkeys. Exposure parameters were
not specified. Effects of H2S on brain tissue of monkeys were also reported by Lund and
Wieland (1966).
Lund and Wieland (1966) found that inhalation of H2S resulted in pronounced
histopathological changes in brain tissues of rhesus monkeys. A lethal exposure of one
monkey to 500 ppm (695 mg/m3) H2S for 35 min caused no pathologic changes in fixed and
stained tissue sections of brain, kidneys, adrenal glands, or heart. However, necropsy
revealed a severely hyperemic liver and dilation of the blood vessels. The second monkey
was exposed to 500 ppm (695 mg/m3) H2S for 35 min until breathing ceased; it was revived,
exposed again until it lost consciousness, and then revived. At 5 days postexposure, it was
sacrificed and the tissues were examined. Histologic examination of the brain revealed spotty
regions of altered cells and a noticeable vascularization in the region of the basal ganglia, in
the upper parts of the putamen, and on the caudate nucleus. The lesions characteristically
had newly formed capillaries and increased glial formation. The cortex of the occipital lobe
was altered, with lamellar separations between the lower layers of cortex. The smaller blood
vessels of the cortex were hyperemic. Necrosis of the parenchymal cells of the cortex was
evident. No pathologic lesions were seen in tissues other than the brain. The liver, however,
was severely hyperemic. The third monkey was exposed in similar fashion; however,
exposure was interrupted after 22 min. Spontaneous respiration never ceased, but the
monkey was somnolent, ataxic, anorexic, and relatively immobile, and exhibited
uncoordinated movements. The animal showed only slight improvement and was sacrificed
after 10 days. Examination of the brain showed damage in the basal ganglia, an increase in
8-8
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glia, and spotty lesions of the cortex in the parietal and occipital lobes. There was a decrease
in Purkinje cells in the cerebellum. No pathologic lesions of the kidneys, adrenals, heart, or
liver were seen.
Administration of NaHS by ip injection of SD rats (10 or 30 mg/kg) resulted in
increases in amino acid levels in brainstem tissue, while other brain regions were unaffected.
Since some of these amino acids (e.g., taurine) may be involved in neuronal control of
breathing, it was speculated that alteration of amino acid neurotransmitter levels may result in
H2S-related arrest of the respiratory drive (Kombian et al., 1988). Taurine has been shown
to depress respiration upon intraventricular administration (Mueller et al., 1982; Wessberg
et al., 1983). However, decreases in amino acid (including taurine) levels in brainstems of
SD rats, but not ICR mice, following repeated ip dosing with NaHS, was reported by
Reiffenstein and Warenycia (1987). Warenycia et al. (1989c), using patch clamp studies of
neuroblastoma cells, found that addition of taurine or cysteic acid in the presence of NaHS
resulted in reversible abolition of inward sodium currents. Neither compound alone had any
effect. The sulfhydryl agents, /5-mercaptoethanol and dithiothreitol also reversibly abolished
sodium currents.
Warenycia et al. (1989a) measured the brain sulfide levels in rats following inhalation
of H9S or ip injection of NaHS. Male SD rats either were exposed to 1,650 ppm
rj
(2,294 mg/m ) H2S until death (time to death, 4.9 min ± 1.4 min) or injected ip with
various concentrations of NaHS with corresponding sulfide levels ranging from 7.5 to
50 mg/kg and sacrificed 2 min later. The brainstem and cortex of control rats were found to
contain endogenous levels of sulfide; corresponding values were 1.26 and 1.66 jug/g.
Injection of NaHS resulted in sulfide levels of 4.42 and 4.76 jtg/g in the brainstem and
cortex, respectively. Analysis of the brainstem, cerebellum, hippocampus, striatum, and
cortex revealed a high correlation between brain sulfide levels and corresponding NaHS dose
levels. Brain sulfide levels in rats inhaling H2S were approximately 10% of those expected
from the dose administered. These low amounts reflect either the extreme lethality of H9S or
the probable metabolism and possible formation of nonlabile species of sulfide. Subcellular
fractionation clearly demonstrated that H2S exposure resulted in sulfide uptake into nerve
cells, as evidenced by an increase of sulfide content in the synaptosomes, mitochondria, and
myelin. It was concluded that the brainstem selectively accumulates H2S and that this
8-9
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preferential accumulation may, in part, account for the lethal action of H2S on respiratory
centers (Warenycia et al., 1987).
Warenycia et al. (1989b) found that administration of NaHS to rats at 30 mg/kg resulted
in significant increases in regional catecholamine levels in brain. This dose was described as
2 X LD50. The hippocampus, striatum, and brainstem all showed increases in nor-adrenaline
and adrenaline. In vitro studies showed that NaHS inhibited monoamine oxidase.
Elovaara et al. (1978) demonstrated a marked decrease in mouse brain protein synthesis
after a 2-h exposure to 100 ppm (139 mg/m3) H2S, as evidenced by a decrease in 14C-leucine
incorporation. In subsequent experiments, Savolainen et al. (1980) found that this decrease
in protein synthesis correlated with an increasing inhibition of cerebral cytochrome c oxidase
when mice were repeatedly exposed to 100 ppm (139 mg/m3) H2S for 2 h at 4-day intervals.
Nicholls (1975) showed that H2S forms a heme-sulfide complex, which is very slow to
dissociate (Kj ~ 0.02 pM for H2S). Repeated exposure to the gas would cause increasing
numbers of complexes to form, resulting in less oxidative metabolism in the affected cells.
The limiting factor in recovery would be the rate of synthesis of new heme (Shanley et al.,
1977). While these studies indicate a cumulative effect on the brain resulting from H2S
exposure, similar damage is seen as a result of anoxic episodes (Yap and Specter, 1965;
Yanagihara, 1976). In anoxia, there is a decrease in protein synthesis as well as RNA
synthesis, and a decrease in the formation of polyribosomal complexes (Yanagihara, 1976).
Higuchi (1977) studied the effects of exposure to H2S on rat behavior. Rats were
exposed to concentrations of 100 to 500 ppm (139 to 695 mg/m3) H2S. At 200 ppm
(278 mg/m3), there was an immediate inhibition of discriminated avoidance response;
at 300 to 500 ppm (417 to 695 mg/m3), the Sidman-type avoidance response was also
inhibited.
Exposure of guinea pigs to 20 ppm (28 mg/m3) H2S, 1 h/day, for 11 days was shown
to cause significant reduction in total lipids (14 to 34%) and phospholipids (11 to 21%) of the
cerebral hemisphere and brainstem tissues (Haider et al., 1979, 1980). Levels of amino acids
were not determined. The associated increase (18%) in malonaldehyde in the cerebral
hemisphere suggest peroxidation of polyunsaturated lipids. Exposure of rabbits to 72 ppm
(100 mg/m3) H2S for 1 h/day, for 2 days resulted in a reduction in adenosine triphosphatase
(ATPase) and alkaline phosphatase in brain tissue (Kosmider and Zajusz, 1966).
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Dahne and co-workers (1983) examined the brains of eight cattle whose survival time
after H2S poisoning ranged from 18 h to 10 days. Histological examination of the brain
disclosed spotty regions of neuronal necrosis with vascular proliferation and gliosis in the
basal ganglia. Laminar necrosis of the cerebral cortex was also noted, particularly in the
occipital cortex. Up to 60 h after intoxication, bilaterally symmetrical lesions were seen in
the dorsal neocortex and, to a somewhat lesser degree, in the cornu Ammonis of the
hippocampus, the lateral geniculate nucleus, the globus pallidus, the caudate nucleus, and the
cerebellar Purkinje cell layer. These lesions were characterized by eosinophilic neuronal
necrosis and astrocytic edema, and were accompanied by low-grade edema of the white
matter. After 10-days postexposure, the lesions had progressed to laminar necrosis with
resorption of necrotic tissue by macrophages. The lesions described in this study are similar
to those seen in systemic hypoxia and in intoxications that impair tissue utilization of oxygen,
such as carbon monoxide poisoning.
Doses in the range of 20 /zmol/kg Na2S injected intravenously into cats caused
immediate hyperpnea, which was often followed by permanent respiratory arrest (Evans,
1967). If the carotid sinus region was locally anesthetized, the hyperpnea did not occur;
however, in a single trial where the sulfide was injected into the ascending aorta allowing
interaction with the aortic chemoreceptors, hyperpnea still occurred.
Hays et al. (1972) exposed cows, goats, and mice to various concentrations of H2S.
Each animal served as its own control. The LC50 for mice is reported in Table 8-1. Body
weight and food and water consumption were measured in all animals. Rectal temperature
was measured in mice and goats, heart rate in goats and cows, and milk production in cows.
Plasma cortisol concentration in goats and carbonic anhydrase activity and phenobarbital
sleeping time in mice were also recorded. Goats were individually exposed, but data were
pooled in experimental or control groups of three to five animals.
All goats exhibited an initial decrease in food and water consumption; one goat/group
developed a fever and one goat exposed to 100 ppm (139 mg/m3) died after 19 h of
exposure. All goats exhibited eye irritation, coughing, shivering, and a reduction in urinary
output. Some goats exhibited an increased heart and/or respiratory rate; a 50% mean
increase in plasma cortisol levels was observed in animals exposed to 100 ppm (139 mg/m3)
H2S. Cows exposed to 20 to 50 ppm (28 to 69 mg/m3) exhibited lacrimation and discomfort,
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and an alteration in normal body function. No change in milk production was observed.
It was suggested that exposure to low levels of H2S for short periods of time results in
irritation and facilitates the establishment of bacterial infection.
Limited information suggests that H2S decreases the ability of animals or humans to
withstand infection. Rogers and Perm (1981) exposed male Long Evans rats in nose-only
exposure chambers to 45 ppm (63 mg/m3) H2S for 2, 4, or 6 h. Immediately following
exposure, rats were anesthetized and challenged with a 30-min staphylococcal (coagulase
negative Staphylococcus epidermidis) aerosol through a nose-only exposure chamber. Rats
were sacrificed at 30 min (time 0), 3-h, and 6-h postbacterial challenge. Exsanguinated lungs
were homogenized, plated, and grown on a selective growth medium for staphylococci, arid
colonies were counted. Rats exposed for 4 h to H2S had 6.5-fold greater percent colony-
forming units (CPU) than controls, while the 6-h H2S-exposed group had a 52-fold greater
percent CPU than controls. Since there was no evidence of pulmonary edema to promote
bacterial growth, it was concluded that H2S significantly affected the antibacterial system of
the rats by impairing AMs.
8.1.3 Effects Associated with Repeated Exposure
Hulbert et al. (1989) exposed F344 rats (9/sex/group) to 1, 10, or 100 ppm (1.4, 14, or
139 mg/m3) H2S, 8 h/day, 5 days/week for 5 weeks. The only significant histopathological
difference was an increase in trachea! 'ciliated cells in animals from the 100 ppm (139 mg/m3)
group. Measurement of airway resistance and dynamic compliance after animals were
anesthetized indicated there was no effect of exposure on these parameters. However, some
exposed rats in each group responded to a 10-fold lower dose of methacholine.
In 90-day inhalation toxicity studies conducted for the Chemical Industry Institute of
Toxicology on SD rats (Toxigenics, 1983c), F344 rats (Toxigenics, 1983b) and B6C3F1 mice
(Toxigenics, 1983a), animals (15/sex/exposure group) were exposed to 0, 10.1, 30.5, or
80 ppm (0, 14, 42, or 111 mg/m3) H2S for 6 h/day, 5 days/week.
This highly detailed study included neurologic function tests assessing posture, gait,
and tone of facial muscles, and examined pupillary, palpebral, extensor thrust, and
crossed-extensor thrust reflexes, before and after exposure. Eyes were examined with both a
monocular ophthalmoscope and a slit-lamp biomicroscope at the end of the exposure period.
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Extensive clinical pathology included blood volume, appearance, urine specific gravity,
protein, pH, ketone, and glucose. Hematologic parameters and serum chemistry parameters
were determined. Detailed necropsy examination was performed, individual major organs
were excised, and tissues were collected and examined microscopically. These included the
brain (cerebellum and two levels of cerebrum, medulla, optic nerve), spinal cord (cervical,
thoracic, and lumbar—two sections each), peripheral nerves (sciatic and anterior tibial, with
remaining sciatic nerve removed and stored in buffered formalin), eyes, pituitary, thyroid,
parathyroid, salivary glands (submaxillary), heart, lungs (four levels), spleen, liver, pancreas,
adrenals, lymph nodes (mesenteric and mandibular), kidneys, bladder (inflated with
formalin), lacrimal glands, ovaries, uterus, oviducts, vagina, cervix, stomach, small intestine
(duodenum, jejunum, ileum), large intestine (large and small colon and caecum), skeletal
muscle (thigh), skin, mammary glands (males and females), bone (femur), bone marrow
(smear and section), aorta, ear canal with zymbal gland, nasal turbinates (four levels),
trachea, testes, epididymis, esophagus, thymus, prostate, seminal vesicle, and any gross
lesion(s).
In addition, a neurological study was performed on the two strains of rats. Male and
female rats (5/sex/strain) from each exposure and control group were used. Following
anesthesia with sodium pentobarbital, rats were perfused with glutaraldehyde and the intact
animal was then refrigerated at approximately 4 °C overnight. The right and left sciatic
nerve and their branches were dissected together with specimens of the cervical and lumbar
spinal cord and placed in a 4% glutaraldehyde solution. Specimens were examined by
routine light microscopy for evidence of pathologic change. The control and highest
exposure groups were examined initially. If changes were detected, lower exposure groups
were examined.
In mice, the only exposure-related histopathological lesion was inflammation of the
nasal mucosa in the anterior segments of the nose which was observed in 8/9 male mice and
in 7/9 female mice in the group exposed to 80 ppm (111 mg/m3). This lesion was also
present in two high dose mice that died during the course of the study. The lesion was
generally minimal to mild in severity and was located in the anterior portion of the nasal
structures, primarily in the squamous portion of the nasal mucosa, but extending to areas
covered by respiratory epithelium. This lesion was not observed in any animals in the other
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exposure groups. Thus, 80 ppm (111 mg/m3) is considered a LOAEL for nasal inflammation
in mice, while 30.5 ppm (42 mg/m3) is the corresponding NOAEL.
Significant reductions in body weight gain were noted in all exposure groups at various
times during the study. Decreased weight gain in animals exposed to 80 ppm (111 mg/m )
occurred consistently in both male (approximately 90% of control during last 7 weeks of
study) and female (<90% of control during last 3 weeks of study) mice.
A significant reduction in body weight gain was noted in all rats exposed to 80 ppm
(111 mg/m3). In F344 and male SD rats, mean body weights were never <93% of control.
In female SD rats, the effect on body weight was statistically significant at various time
points in all exposed groups, but mean body weight in the 80 ppm (111 mg/m3) group was
<90% of the control groups during most of the study. Statistically significant changes in
absolute kidney, liver, and spleen weight were also observed in the male rats exposed to
80 ppm (111 mg/m3), but no differences were apparent when organ weights were normalized
to body weight. Brain weight was significantly reduced in the male SD rats in the high-
exposure group and slightly, but not significantly reduced in females (Toxigenics, 1983c).
There were no exposure-related clinical signs in rats. Neurologic function examinations
yielded negative results. Blood volume, appearance, occult blood, urine specific gravity,
protein, pH, ketone, and glucose values were all normal. Ophthalmoscopic examination,
hematology, serum chemistry parameters and urinalysis were also normal. Histopathological
examination, which included four sections of the nasal turbinates, revealed no abnormalities
in comparison with controls.
The effects of a 3-mo exposure of rats to H2S were reported by Fyn-Djui (1959).
Groups of 10 male white rats were exposed to 0, 0.14, or 7.14 ppm (0, 0.2, or 10 mg/m3)
H2S, 12 h/day. Body weights were measured, and motor function was observed. At study
termination, gross necropsy was performed on two rats/group.
Exposure to 0.14 ppm (0.19 mg/m3) produced no changes in body weight; however,
motor chronaxy changes were observed. At 7.14 ppm (9.9 mg/m3), a decrease in body
weight was seen and similar changes in motor chronaxy were noted. It was suggested that
these fluctuations in the extensor and flexor chronaxy were the result of a cerebral cortex
effect and were indicative of changes in the functional state of the brain. Necropsy of rats
exposed to 7.14 ppm (9.9 mg/m3) revealed irritation of the tracheal and bronchial mucosa;
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less pronounced irritation was observed in animals exposed to 0.14 ppm (0.19 mg/m3).
Brain cortex changes in the animals at 7.14 ppm (9.9 mg/m3) consisted of prickled,
thickened, and swollen dendrites.
Significant weight loss was observed in monkeys, rats, and mice exposed to 20 ppm
(28 mg/m3) for 3 mo; however, CNS disorders were not evident (Sandage, 1961). When
exposed to concentrations of 20-25 ppm (28 to 35 mg/m3) H2S for 150 days, one rabbit lost
weight and four others exhibited variable weight gain. Gamma albumin was also increased
(Kuwai, 1960).
Wakatsuki (1959) exposed groups of rabbits (number, sex, and strain not reported) to
100 ppm (139 mg/m3) H2S, 300 ppm (417 mg/m3) carbon disulfide (CS2), or a combination
of the two gases, 30 min/day for 4 mo. Clinical observations made during the study and
continuing for 4 mo postexposure included general conditions, body weight, peripheral blood
picture, serum calcium, blood specific gravity, total serum protein, and serum protein
fraction.
Rabbits exposed only to H2S exhibited comparatively slight changes and no measureable
abnormal findings in general condition, body weight, number of erythrocytes, serum calcium,
total serum protein, and serum protein fraction. Slight changes such as oligochromemia,
reticulocytosis, leucopenia, decrease of pseudoacidophilic cells, relative lymphocytosis, and
an increase in toxic granules were observed; however, recovery was complete within 4 mo.
Rabbits exposed to a combination of the two gases exhibited more pronounced effects, and
complete recovery was not observed.
Kosmider et al. (1967) exposed rabbits to 71 ppm (100 mg/m3) for 1 to 5 h (until they
lost consciousness) or for 0.5 h/day for 5 days. Electrocardiograms revealed disorders of
repolarization in acutely exposed animals. Repeated exposure resulted in arrhythmias in the
form of ventricular extrasystoles, bigeminal rhythms, and disorders of ventricular
repolarization manifested as flattened T-waves. When animals with H2S-induced arrhythmias
were treated with calcium-binding compounds, such as sodium citrate, normal rhythms were
restored. Arrhythmias returned in several instances, and repeated doses of sodium citrate had
to be used after several hours to restore physiologic rhythms.
Kosmider et al. (1967) followed these experiments with histochemical studies.
Fragments from the apical region of the heart and heart vasculature were examined for the
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activity of two enzymes. They found that ATP phosphohydrolase activity in blood vessels
and the sarcolemma of the heart muscle cells was decreased in exposed animals as compared
with controls. Nicotinamide adenine dinucleotide phosphate, reduced (NADPH)
oxidoreductase activity in heart;muscle cells and vascular endothelium was likewise reduced.
It is not possible to distinguish whether these effects result directly from H2S toxicity on the
cells examined or whether they are secondary effects of H2S poisoning of the whole animal.
The authors state that these effects are the result of H2S action directly on the heart.
Changes in activity of these enzymes affected the active transport of sodium and
potassium ions in the heart muscle cells and the walls of blood vessels. These changes led to
changes in concentrations of these ions across heart cell membranes, which in turn caused
changes in electrical activity. These changes can account for the observed differences in
rhythm and repolarization in the experimental animals. The significance of these observations
is that changes in heart function may be the direct response of the heart cells to H2S
exposure, rather than a secondary response elicited by the action of the nervous system on the
heart. Since other enzyme activities were not measured arid in vitro enzyme assays were not
done, it is unclear whether the decrease in activities is directly attributable to action of H2S
on the enzymes, or to interference with oxidative metabolism by the gas.
Lowering of alkaline phosphatase and succinate dehydrogenase in heart tissue was
reported in rabbits exposed to 72 ppm (100 mg/m3), 1 h/day, for 7 or 14 days (Dwornicki,
1979).
A series of studies conducted by Renne et al. (1980) and reported in preliminary form
investigated the potential toxic and synergistic effects of exposure to geothermal effluents
(H2S and ammonia). In the first study, groups of 10 rats and 10 guinea pigs (sex, strain, and
number not reported) were exposed for 7 days to 100 ppm (139 mg/m3) H2S, 250 ppm
(174 mg/m3) NH3, or a combination of the two gases. Complete necropsies were performed
on all animals. No significant histopathological lesions or clinical pathological alterations
were observed.
A subsequent 7-day exposure to 220 ppm (306 mg/m3) H2S, 250 ppm (174 mg/m3)
NH3, or a combination of the two gases resulted in a significant increase in the incidence of
respiratory tract lesions in guinea pigs. Mild interstitial pneumonitis was observed in 70% of
guinea pigs exposed to a combination of H2S and NH3 compared to 30% of controls and a
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40% incidence in groups exposed to either gas alone. An increased incidence of mild acute
suppurative tracheitis and laryngitis and mild chronic nephritis was observed in all groups.
The increased incidence of respiratory tract lesions appeared to be treatment-related;
however, the significance of the increased incidence of chronic nephritis was not known.
Curtis et al. (1975) exposed groups of three pigs (sex and strain not reported) to
8.5 ppm (12 mg/m3) H2S, 24 h/day for 17 days, or 2 ppm (2.8 mg/m3) H2S in combination
with 50 ppm (35 mg/m3) NH3, 24 h/day for 19 days. No statistically significant changes in
body weight gain or respiratory tract structure were observed.
The 1982 Lodgepole, Alberta, Canada, gas well blowout exposed farm animals to levels
of 10 to 15 ppm (14 to 21 mg/m3) H2S as well as to other gaseous constituents of the well
effluent. Members of the community described problems in cattle, pigs, horses, and
household pets. They noted that the animals exhibited runny noses and eyes, coughing, and
decreased food intake. Most cattle in the exposed area were affected, young animals showing
more severe signs of irritation of mucous membranes than old. Residents also indicated that
some animals exhibited diarrhea, red stools, red urine, and decreased weight gain. A local
veterinarian and members of five families described an almost total disappearance of small
wild animals and birds; these did not reappear for a "long time" after the blowout had been
controlled (Lodgepole Blowout Inquiry Panel, 1984; Herbert, 1985).
The Alberta Environmental Centre staff reported some "significant" changes in the
activity of certain enzymes in the blood of cattle exposed to emissions from the Lodgepole
blowout. The changes were not characterized further. The enzymes superoxide dismutase,
glutathione peroxidase, glucose-6-phosphate-dehydrogenase, acetylcholine esterase, and
aspartase aminotransferase were found to be involved in the detoxification of H2S or
otherwise affected by it (Beck, 1985). The changes appeared to be transient and reversible,
and their importance and possible relationship to clinical disease in the exposed animals are
not known (Harris, 1986).
Calves continually exposed to 20 or 150 ppm (28 or 208 mg/m3) H2S for 7 days
exhibited a number of clinical signs (Nordstrum, 1975; Nordstrum and McQuitty, 1976).
At 20 ppm, toxic effects included distress, lethargy, restlessness, occasional diarrhea and
vomiting, coughing, irregular respiration and dyspnea, photophobia, keratitis, corneal
opacity, nasal irritation, and epistaxis. These signs were also observed at 150 ppm
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(208 mg/m3); in addition, calves exhibited severe keratoconjunctivitis, clinical blindness,
reduced food and water consumption, and elevated temperature.
Similar findings of eye and respiratory irritation in cattle and horses were reported by a
veterinarian following a well blowout in 1984 (Drummond 6-30 Sour Gas Well Blowout).
The Alberta Environment Centre and Alberta Agriculture staff conducted followup research
of the livestock on 16 farms beginning the day following the blowout and continuing over the
next 3 mo. Owners of livestock were contacted a year later to determine if any unusual
health problems had occurred. Immediate complaints following the blowout generally
consisted of irritation of ocular and respiratory membranes, respiratory disease (pneumonia),
reduced exercise tolerance, and reproductive failure. The investigation team concluded that
eye and respiratory irritation could be attributed to exposure to the wellhead emissions and
may have made animals more susceptible to the effects of infective keratoid conjunctivitis
(pinkeye) and infective respiratory disease (pneumonia). Decreased exercise tolerance of
horses, and loss of weight, condition, and appetite may have been caused by exposure to
gases. No consistent patterns of animal disease could be identified. Hydrogen sulfide
concentrations ranged from 0.01 to 3.50 ppm, (0.014 mg/m3 to 4.90 mg/m3), with a mean
concentration over the 4 days of the episode of 0.36 ± 0.57 ppm (0.51 + 0.80 mg/m3)
(Alberta Agriculture, 1986).
The effects of H2S have also been examined in a study involving oral gavage with
solutions prepared by bubbling H2S through water (Arthur D. Little, Inc., 1987).
Sprague-Dawley rats (20/sex/dose) were administered solutions containing 1.0, 3.5, or
7.0 mg/kg/day H2S, once daily, 7 days/week, for 89 days. The principal findings were
dose-related clinical signs of restlessness (males) and salivation (females). These signs
occurred also in the low exposure group. Significant increases in mortality occurred in the
high dose group (males only). There were no adverse, dose-related histopathological findings
in the respiratory tract or other organs.
8.1.4 Chronic Toxicity
No chronic toxicity studies were found in the available literature.
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8.1.5 Effects on Respiration Control Receptors
Carotid sinus chemoreceptors (carotid bodies) may play a role in stimulating the
ventilatory reflex upon interaction with blood sulfide at sublethal levels. Both the rate and
depth of ventilation increase to the point of hypernea. Heymans et al. (1931, 1932) showed
that injecting a small amount of Na2S into the common carotid artery of dogs resulted in
hyperpnea. After denervation of the sinus by transection of the sinus nerve, larger doses of
sulfide had no immediate effect on respiration, and the late effect was respiratory depression.
Injection of Na2S into the internal carotid or vertebral arteries had the same effect as
denervation. The sulfide would be diluted by the general circulation and metabolized before
it reached the chemoreceptors.
These results were confirmed by the use of cross-perfusion techniques, in which isolated
carotid sinuses of a recipient dog received the entire blood supply from a donor dog
(Heymans et al., 1931, 1932). Sodium sulfide injected into the recipient dog's general
circulation elicited no stimulatory effect on respiration, since the carotid chemoreceptors were
not part of its circulation. However, the donor dog, whose blood perfused the recipient's
chemoreceptors, elicited the response when injected systematically with Na-,8. A similar,
although secondary, response was observed with the aortic chemoreceptors (Heymans and
Neil, 1958).
However, the effect on carotid and aortic bodies seems inconsistent with the depressant
effect on the central nervous system (CNS). Early researchers of this phenomenon did not
offer an explanation for this seeming contradiction, yet clearly ascertained that it existed
(Haggard et al., 1922; Heymans et al., 1931, 1932; Evans, 1967). It is possible to resolve
this paradox if the normal function of the carotid and aortic bodies is examined together with
the cellular effect of H2S.
The physiological function of the reflexes associated with the chemosensors of the
carotid and aortic bodies is to maintain a ventilation rate and depth that is adequate for
supplying tissue cells with oxygen. The chemosensors are primarily sensitive to the partial
pressure of oxygen (pO2), or oxygen tension, in blood flowing through the carotid sinuses
and the aortic arch. Under normal conditions, no oxygen is removed from the blood before
it reaches these vessels; therefore, the pO2 is between 100 and 104 mmHg and the
hemoglobin is saturated with oxygen. Oxygen tension must decrease considerably for the
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reflexive increase in ventilation to be activated. The carotid and aortic chemosensors do not
respond with rapid impulse firing until the pO2 falls into the range between 30 and 60 mmHg
(Biscoe, 1971). Such a decrease normally occurs only with hypotension if the systolic
arterial blood pressure falls below 80 mmHg. When the-oxygen tension falls together with
blood pressure, the chemosensors, in concert with the baro- or pressure sensors in the same
blood vessels, initiate reflexes to increase the rate and depth of ventilation and blood
pressure; this can lead to restoration of normal pO2 under normal circumstances.
This same response is seen in sublethal H2S poisoning; however, it also inhibits neural
function. Hydrogen sulfide most rapidly affects the intracellular imtochondrial enzyme
cytochrome c oxidase, interfering with the transfer of electrons and hydrogen ions to oxygen,
thus blocking oxidative metabolism. Cells that are dependent on oxidative metabolism,
and/or those having a high oxygen demand such as those of the nervous system or the heart,
would be most rapidly and severely affected. In the case of the carotid and aortic
chemoreceptors, halting of oxidative metabolism has the same effect as a decrease in oxygen
supply. As oxidative metabolism in these highly sensitive nerve endings ceases, they respond
with rapid-fire impulses to the respiratory centers, initiating the reflexive increase in rate and
depth of ventilation. Reflexive hyperpnea is therefore a logical consequence of the inhibition
of cytochrome c oxidase in the chemosensors of the carotid and aortic bodies by H2S
(Ammann, 1986).
8.1.6 Cellular Mechanism(s) of Toxicity
Reiffenstein (1989) investigated the cellular mechanism(s) of sulfide intoxication in rat
brains using neurochemical and neurophysiological approaches. Several "model" systems
were utilized, including the in vitro hippocampal slice (considered to be the best model for
studying human "knockdown" seen in H2S poisoning) and the in vivo iontophoresis of HS"
onto single hippocampal pyramidal cells to test the effects of H2S on the rates of
spontaneously firing neurons. Within 2 min of an intraperitoneal injection of 10 or 30 mg/kg
NaHS (corresponding to LD30 and LD99 doses), there .were significant increases in brainstem
aspartate, glutamate, taurine, gamma aminobenzoic acid (GABA), and alanine
neurotransmitter concentrations. However, no changes were found in the cerebral cortex,
striatum, and hippocampus. At the low dose, aspartate (an excitatory neurotransmitter) and
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glycine (an inhibitory neurotransmitter) levels were decreased in the cerebellum, and the
glutamine (precursor of glutamate) level was elevated in the brainstem. In contrast to these
findings, Kombian et al. (1989) using the push-pull perfusion technique, observed no changes
in brainstem glutamate, aspartate, glycine, or GABA levels following a 15-mg/kg
intraperitoneal injection of NaHS. However, this dose caused a delayed decrease in the
release of glutamine to 61% of the control (p < 0.05). At 3 j«g/mL NaHS (the physiological
level is around 2 ^g/mL), there was a 62% decrease in the glycine level when compared to
controls. Such a decline in the concentrations of an inhibitory neurotransmitter in the
brainstem area can lead to unopposed excitatory events and final loss of respiratory drive.
At the LD99, only 5-hydroxytryptamine (5HT) and dopamine levels were increased in
the brainstem, whereas epinephrine and norepinephrine levels were increased in the
hippocampus and striatum (Reiffenstein, 1989). In another study, Reiffenstein et al. (1988)
found that the levels of the latter neurotransmitters together with dopamine were higher than
controls in the brainstem region. It was suggested that the HS"-induced increase in
catecholamine levels was due to the inhibition of monoamine oxidase (MAO) enzyme
function. A similar conclusion was derived in the 1989 study where H2S at high doses
inhibited MAO in vivo as well as in vitro. Dithiothreitol at 0.1 mM was able to restore
enzyme activity by over 400% (Warenycia et al., 1989).
In vivo iontophoresis of 30 to 50 nA HS" onto hippocampal pyramidal cells blocked the
spontaneous firing of these cells; however, very low doses (2 to 10 nA) gave the opposite
results (i.e., the firing rate was increased). In in vitro studies using intracellular
microelectrode recordings from CA1 pyramidal cells, 27 to 200 /*M NaHS caused dose-
dependent membrane hyperpolarizations and a decrease in membrane resistance; more
hyperpolarizations were seen after washout. Similar findings were reported by Baldelli et al.
(1989). The mechanism(s) of H2S inhibition of neuronal activity in hippocampus, the site of
retrograde amnesia in humans, may result from the suppression of synaptic input and direct
membrane hyperpolarization. Similar mechanisms may also be operative in the brainstem
region, which is the site of cardiovascular respiratory centers.
Both Reiffenstein (1989) and Kombian et al. (1988) used the sucrose gap junction
technique to study the electrical properties of frog sympathetic ganglia. Nicotine
(0.01 mM)-induced membrane depolarizations were not affected by NaHS; however, NaHS
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significantly potentiated the muscarine-induced and epinephrine-induced hyperpolarizations
when compared to controls. In addition, NaHS alone caused membrane depolarizations. The
activity of the Na-K-ATPase was not directly affected by NaHS; the only change observed
was potentiation upon washout.
Patch clamping of mouse neuroblastoma cells showed that NaHS, in the presence of
either taurine or cysteine amino acids, resulted in the reversible inhibition of sodium channel
currents (opening of these channels initiates the membrane action potential). None of these
compounds had any effect by itself. Inhibition of these channels may be responsible for the
loss of respiratory drive due to H2S poisoning (Warenycia et al., 1989; Reiffenstein, 1989).
Another mechanism of H2S toxicity is via free radical generation. Beck et al. (1981)
showed that H2S in vitro underwent rapid oxidation, production of H2O2, and oxygen
utilization. Khan et al. (1987a) indicated that the H2S stimulation of superoxide anion
generation by xanthine oxidase and free radical production, and the direct inhibition of
/-\
various free radical scavenging enzymes such as glutathione in vivo by Sz~ and other sulfur-
containing compounds, can be deleterious and cytotoxic.
8.1.7 Summary of Effects on Laboratory and Domesticated Animals
The effects of H2S inhalation on a variety of animal species have been investigated.
The types of effects across species are similar and principally involve the respiratory tract and
brain. Acute exposures (e.g., 4 h or less) of rats or monkeys to levels of about 500 ppm
(695 mg/cu.m) or greater were found to cause mortality. Exposure of rats to 300 ppm
(417 mg/cu.m) resulted in visible stress, and 200 ppm (278 mg/cu.m) was associated with
lung edema and other changes. The most pronounced clinical signs preceding death in rats
and monkeys were respiratory distress and histological lesions of the respiratory tract and
brain. Acute exposures of rats to sublethal levels as low as 50 ppm have resulted in
decreased activities of cytochrome c oxidase, a respiratory chain enzyme essential for oxygen
utilization at the cellular level. Severely decreased activity of cytochrome c oxidase was
found in rats that succumbed to levels of 500 ppm (695 mg/cu.m) H2S and greater. In mice
exposed to 100 ppm (139 mg/cu.m) H2S for 2 h at 4-day intervals, protein synthesis in brain
decreased and was correlated with increasing inhibition of cytochrome c oxidase. Some
studies with rabbits suggest that acute, repeated exposure to 100 ppm (139 mg/cu.m) H2S
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caused heart arrhythmias. It is not clear if H2S has a direct effect on the heart or if these
effects are secondary to poisoning.
A 90-day repeated inhalation study with SD and F344 rats in which an extensive
neurological and histopathological examination was performed did not reveal any abnormal
neurological function at the highest concentration (80 ppm; 111 mg/cu.m). The
histopathological examination, which included four sections of the nasal turbinates, revealed
no abnormalities compared to controls. In B6C3F1 mice, the only exposure-related
histopathological lesion was inflammation of the nasal mucosa in the 80-ppm (111 mg/cu.m)
group. There were no indications of adverse effects of any kind in both species at exposure
levels of 10 and 30 ppm (14 and 42 mg/cu.m).
Domesticated animals may be more sensitive to H2S than rodents. Cows exposed to
20 to 50 ppm (28 to 70 mg/m3) exhibited lacrimation and discomfort, but no apparent effect
on milk production. In cattle exposed to unknown, but lethal levels of H2S, necrosis of the
cerebral cortex was observed. More numerous clinical signs, including blindness, have been
found in calves exposed continually to 20 or 150 ppm (28 or 208 mg/cu.m) for 7 days. One
goat exposed to 100 ppm (139 mg/cu.m) for 19 h died; all goats exhibited eye irritation,
coughing, and shivering while some goats exhibited increased heart and/or respiratory rate.
There are no studies involving long-term H2S inhalation.
8.2 HUMAN HEALTH EFFECTS
Hydrogen sulfide (H2S) poisoning attracted the interest of a number of research
scientists during the 19th century (see the review by Mitchell and Davenport, 1924). The
characteristic respiratory excitation caused by both inhalation of the gas and injections of H9S
and sodium sulfide were described by the mid-1800s. Also known was the high lethality of
H2S, its ability to cause respiratory arrest, its irritant effect, and the efficacy of removing
victims from the contaminated environment and reviving them with artificial ventilation
(Lehmann, 1892).
Probably the most widespread and common complaint of persons exposed to low
concentrations of H2S for short or extended periods of time are those related to odor.
An extensive discussion on the psychological and esthetic aspects of odor in general, and
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specifically applying to the odor of H2S, is included in the National Research Council (1977)
monograph on H2S. Hydrogen sulfide has a lower limit for detection of odor of 0.003 to
0.02 ppm (0.004 to 0.03 mg/cu.m). At concentrations up to 30 ppm (42 mg/cu.m), H2S has
an odor like that of rotten eggs; at 30 ppm (42 mg/cu.m) the odor is sweet or sickeningly
sweet. At 100 ppm (139 mg/cu.m) and above, H2S quickly fatigues the sense of smell; at
concentrations approaching 150 ppm (208 mg/cu.m), H2S apparently abolishes odor sensation
by anesthetizing the olfactory nerve (Indiana Air Pollution Control Board, 1964). People
who have survived exposure to sudden, high concentrations reported either no awareness of
odor at all, or a sickening sweet smell before loss of consciousness. The assumption that
odor will warn of life-threatening levels of H2S is unwarranted, since instantaneously
introduced doses > 150 ppm (208 mg/cu.m) are not perceived at all (Ahlborg, 1951).
Ruth (1986), in his review, indicated an odor threshold range of 0.0007 to
0.014 mg/m3 (< 1 to 10 ppb) with an irritant level of 14 mg/m3 (10 ppm). An earlier
review by Amoore and Hautala (1983) listed the odor threshold at 8 ppb (0.012 mg/m3).
Fyn-Djui (1959) reported a minimum perceptible threshold of 0.012 mg/m3 (8 ppb).
A similar odor threshold range was identified by The World Health Organization (1987).
8.2.1 Toxic Effects Associated with Acute Exposure
Acute exposures of 500 to 2,000 ppm (695 to 2,781 mg/m3) for seconds or minutes
primarily targets the nervous system, although other tissues with high oxygen demand,
particularly the heart, are also affected. Symptoms include fatigue, dizziness, intense
anxiety, loss of olfactory function, collapse, respiratory and cardiac failure, and death.
Usually acute intoxication occurs from a single, massive exposure of 2,000 ppm
(2,781 mg/m3) or more, and unconsciousness occurs within a few seconds, without
significant warning or pain. Unconsciousness, termed "knock-down" by workers, is almost
immediately followed by respiratory paralysis, and after that by a short period of tonic
convulsions (Yant, 1930). The heart continues to beat for several minutes. Death occurs
unless the victim is removed from the contaminated area and artificial ventilation is
immediately initiated. Pettigrew (1976) reports that 26 persons died from exposure to
unspecified concentrations of H2S between October 1, 1974, and April 28, 1976, in the high-
sulfur oil fields of Wyoming and Texas. At times, victims exposed to less massive
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concentrations will recover spontaneously provided they have been removed from
contamination. Other instances of fatalities due to H2S overexposure were reported by
Campanya et al. (1989), Osbern and Crapo (1981), Sanz et al. (1990), and McDonald and
Mclntosh (1951).
If the victim is not removed from the gaseous environment and given artificial
ventilation, spontaneous recovery of ventilation may not occur and death may ensue. Even if
ventilation does resume, asphyxia will eventually occur with continued exposure if the victim
remains in the contaminated environment. Animal data indicate that this is due to
inactivation of cellular respiration, specifically the reversible inhibition of cytochrome
c oxidase, as described previously. According to Haggard (1921), breathing is never
spontaneously restored after respiratory paralysis occurs from H9S exposure, and death from
asphyxia will ensue. Rescuers must know that a self-contained breathing apparatus is
absolutely vital if contaminated areas are to be entered. Many potential rescuers have
succumbed, together with victims of H2S exposure who might have been saved, because they
were unaware of the lethality and rapid, overwhelming action of this toxic gas (Kleinfeld
et al., 1964; Adelson and Sunshine, 1966; Simson and Simpson, 1971; Smith and Gosselin,
1979). Occasionally there are some lingering effects, such as nystagmus and disturbances of
equilibrium, suggesting ototoxic effects, and changes in gait, speech, or arm movement,
suggesting motor involvement. Changes in electrocardiogram (ECG) and myocardial
infarction have been reported; these persistent effects may result from prolonged hypoxia
rather than direct exposure to H2S.
Lethal H2S poisoning exerts its effects directly on the nervous system. If the
concentration of the gas is sufficiently high, the respiratory center of the brain ceases
functioning and breathing stops. At lower concentrations (between 500 and 1,000 ppm;
••>
695 and 1,390 mg/m ), the respiration controls in the carotid body are stimulated, and
hyperpnea, followed by apnea, results from the instigation of the normal autonomic reflex.
Asphyxiation from H2S results on the cellular level as the gas inhibits cytochrome c oxidase
and prevents the utilization of oxygen by cells in a manner similar to the action of HCN.
Only the uncombined, unoxidized form of the gas in the bloodstream exerts these effects.
Hydrogen sulfide is not considered to be a cumulative poison because it is rapidly oxidized to
harmless sulfates, which can be readily eliminated from the body. Hence, its respiratory/
8-25
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asphyxiation role occurs only at higher concentrations, where the effect is rapid and often
fatal.
Instances of permanent neurological damage resulting from acute poisoning have been
described (Aufdermaur and Toenz, 1970; Matsuo et al., 1979; Arnold et al., 1985).
Included among the signs are prolonged coma, convulsions, increased tonus with extensor
spasms, and negative Babinski's sign (Matsuo et al., 1979). Fatigue, somnolence, headache,
irritability, insomnia, anxiety, poor memory, loss of olfactory function, and loss of libido
were reported in recovered victims (Ahlborg, 1951; Poda, 1966; Arnold et al., 1985; Illinois
Institute for Environmental Quality, 1974). Also described are changes in gait, nystagmus,
vertigo, and other indications of toxicity to the eighth cranial nerve (vestibulocochlear nerve)
and its associated CNS structures (Ahlborg, 1951). Computerized axial tomography (CAT
scan) performed on a victim of acute poisoning (Matsuo et al., 1979) and postmortem
examination of brain tissue of victims suggest cereberal lesions characteristic of cerebral
anoxia rather than any specific neurotoxicity by H2S (Lund and Wieland, 1966).
Changes in heart rhythms and electrocardiograms after acute H2S poisoning have been
reported by several physicians (Drews, 1940; Krekel, 1964; Arnold et al., 1985). While
cardiac muscle, like the nervous tissue, has a high oxygen demand and is highly sensitive to
anoxic damage, there is a suggestion by Kosmider et al. (1967) that specific enzyme damage
may result from H2S poisoning.
Workers exposed to H2S concentrations between 500 to 1,000 ppm (695 and
o
1,390 mg/m ) exhibit a period of extremely rapid breathing or hyperpnea. From a practical
standpoint, this can increase the inhaled dose of gas, resulting in increased damage.
Kaipainen (1954) reported a case of a worker shoveling manure who was found unconscious
and contracted convulsions. He had transient ECG abnormalities similar to myocardial
infarction, dilated pupils that were responsive to light, negative Babinski signs, and blood
pressure of 240/140 mmHg. Venesection was carried out at 12-h postexposure, and blood
pressure was lowered to 95/80 mmHg. One day later the patient was incoherent, unable to
answer questions, almost unconscious, and showed muscle spasticity. By 45 days later, the
patient was normal except for dizziness. The author stated that convulsions usually occur at
small doses of H2S inhalation, and they are preceded by giddiness, accelerated breathing, and
finally narcosis. No convulsions prevail at higher doses (no levels were specified).
8-26
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Experience with H2S poisoning in the fossil fuel fields of Alberta, Canada, has been
reviewed for the period 1969-73 by Burnett et al. (1977) and for 1979-83 by Arnold et al.
(1985). These were retrospective studies based on the files of the Compensation Board and
the files of the Medical Services Branch, Worker's Health and Safety, Calgary, Alberta,
Canada; therefore, only complaints for which medical attention was sought were considered.
The records contained no neurological followups. Burnett et al. (1977) examined 173 cases,
among which 6% fatalities occurred. In the 250 cases considered by Arnold et al. (1985),
the fatality rate was 2.8% (7 cases). The symptoms of acute toxicity that emerge from all of
these reports include immediate respiratory paralysis and collapse at very high concentrations
o
(>2,000 ppm; 2,781 mg/nr), and collapse and apnea preceded by a period of hyperpnea at
sublethal concentrations (500 to 1,000 ppm; 695 to 1,390 mg/m3). The sequelae of
poisoning in victims who are resuscitated vary, probably as a result of initial effect, time and
intensity of exposure, and length of anoxia to vital tissues.
Recovery from acute intoxication is usually rapid and complete. Symptoms varying in
nature and severity develop soon after acute poisonings and persist for different durations.
In reviewing a number of cases, Poda (1966) described a syndrome including nervousness,
nausea, headache, insomnia, and a dry, nonproductive cough that lasted for 1 to 3 days.
Gaitonde et al. (1987) reported a case in which a 20-mo-old child was exposed for nearly a
year to measured H2S levels of 0.6 ppm (0.83 mg/m3) or less. Upon hospital admission,
ataxia, choreoathetosis and dystonia were observed. Tomograms of the brain showed striking
bilateral areas of attenuation in the basal ganglia and in some of the surrounding white
matter. A repeat brain scan at 10 weeks showed complete resolution. There was no
respiratory disease. No abnormalities were found upon neurophysiological investigation.
After 10 weeks, ataxia resolved. A muscle biopsy specimen taken one month after
presentation showed normal mitochondrial structure and function. Burnett et al. (1977) listed
the frequency of complaints of 173 poisoning victims in Alberta who sought medical attention
(Table 8-2). In an extension of the work of Burnett et al. (1977), Arnold et al. (1985) listed
the frequency of complaints of 250 medical claims in Alberta (Table 8-3). The most
frequently reported complaints include unconsciousness, nausea, vomiting, and headache.
The poisoning cases reviewed by Ahlborg (1951) differ somewhat from some of these
descriptions in that sequelae of acute intoxication appeared shortly after initial acute exposure
8-27
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TABLE 8-2. CLINICAL FEATURES AFTER HYDROGEN SULFIDE EXPOSURE
Feature
Loss of consciousness
Disequilibrium
Nausea/vomiting
Headache
Sore throat/cough
Conjunctivitis
Weakness of extremities
Dyspnea
Convulsion
Pulmonary edema
Cyanosis
Hemoptysis
At
Accident
Site
74
17
13
9
8
5
4
3
3-
—
1
1
Observed Frequency
At
Physician's
Office
__a
—
28
25
9
9
—
13
--
—
~
--
(%)
At
Emergency
Room
16
29
22
16
14
11
4
--
6
20
11
—
aNot reported.
Source: Burnett et al. (1977).
and persisted for approximately 1.5 mo. In one case, symptoms still evident after 3 years
included drowsiness, fatigue, headache, lack of initiative, irritability, anxiety, poor memory,
and decreased libido. These patients also displayed symptoms indicating damage to the
eighth cranial nerve (vestibulocochlear), such as vertigo, nystagmus, and disturbances of
equilibrium.
Some of Ahlborg's cases had been previously exposed. Other reports in which such
sequelae as well as damage to other vital tissues such as the heart were recorded involved
lengthy periods of anoxia due to paralyzed respiration (Kapainen, 1954; Hurwitz and Taylor,
1954; Kemper, 1966). Since H2S is rapidly metabolized and does not persist in the body of
8-28
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TABLE 8-3. CLINICAL FINDINGS RECORDED
Sign or Symptom
Unconsciousness
Headache
Nausea/vomiting
Dyspnea
Disequilibrium
Conjunctivitis
Sore throat/cough
Felt ill
Neuropsychological
Extremity weakness
Chest pain
Pulmonary edema
Bradycardia
Convulsion
Cyanosis
Hemoptysis
Frequency of
Notation
135
65
62
57
54
46
41
31
20
19
18
14
10
5
3
1
Percentage
54.0
26.0
24.8
22.8
21.6
18.4
16.4
12.4.
8.0
7.6
7.2
5.6
4.0
2.0
1.2
0.4
Source: Arnold et al. (1985).
recovering victims, it is generally thought that persistent neurologic or cardiac effects are the
result of anoxia to these tissues rather than a specific effect of sulfide damage.
Biesold et al. (1977) performed an electron microscopic examination of several regions
of lung tissue excised from a 7-year-old boy who died 24 h after being exposed to H2S
vapors from an old-fashioned farm latrine. A severe alveolar and interstitial edema of the
hemorrhagic type was found. Analysis of the structural elements of the alveolar septa gave
evidence of a direct toxic effect of H2S on the endothelial and epithelial barrier of the alveoli,
which permitted plasma and blood cells to infiltrate the interstitial and alveolar space. There
was widespread damage to the squamous epithelium, resulting in partial denudation of the
8-29
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basal membrane. Indications of endothelial gaps were found, and these were often covered
with microthrombi.
At concentrations of H2S ranging from 100 to 1,000 ppm (139 to 1,390 mg/m3), the
respiratory tract and the eyes are the target organs. Respiratory paralysis prevails as a result
of stimulation of carotid body chemoreceptors by H2S, causing hyperpnea followed by apnea.
This phase of poisoning is critical and requires initiation of artificial respiration to prevent
death by asphyxiation (National Research Council, 1979). The respiratory symptoms include
bronchitis, rhinitis, pharyngitis and laryngitis. The eyes are seriously affected and symptoms
include lacrimation, hyperemia, retro-orbital aching, blepharospasm, distorted and blurred
vision, photophobia, and illusion of rainbow colors around lights. Also, conjunctivitis,
keratitis, cornea! ulceration, and temporary loss of vision have been reported (Illinois Institute
for Environmental Quality, 1974; National Research Council, 1979). The systemic effects of
H2S poisoning are headaches, fatigue, irritability, insomnia, mild depression and
gastrointestinal disturbances.
Rochat (1923) described lesions of the cornea, observed by slit-lamp illumination, of
workers in a sugarbeet processing plant. Similar lesions were also seen by Barthelemy (1939)
and Masure (1950) in H2S:exposed viscose rayon workers and in workers of the gas industry
(Carson, 1963). Painful soreness with severe photophobia and tears that burned "the cheeks"
were the symptoms reported by Howes (1944) in his investigation of an outbreak of eye
problems in a tannery. Nesswetha (1969) described the progression of lesions of the eye in
his review of thousands of H2S exposure histories of viscose rayon workers. In this review,
coexposure to CS2 was suggested to play an important role by lowering the ocular
sensitization threshold to H2S. Slight, grayish opacity with petechial stippling of the
superficial cell layer of the cornea can be observed upon slit lamp examination. The lesions
are due to swelling and blistering of the epithelial cells rather than to cellular infiltration.
As the injury progresses, vacuoles form in the cells, which burst and produce epithelial
defects that spread and join to form larger and very painful ulcers on the corneal surface.
Concomitant with the progress of the corneal keratitis is an inflammation of the conjunctiva,
which becomes reddened. The lesions generally heal without permanent damage, except in
very extreme exposures in which the erosion of the corneal surface can leave scars. Injury to
the eyes is generally restricted to the cornea and conjunctiva. Nesswetha reported that
8-30
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severity of damage increased with H2S exposure concentration. Subjective symptoms most
commonly described are fogging or blurring of vision, the perception of colored, rainbow-
like rings around lights, tearing, sensation of foreign bodies in the eye, photophobia, pain in
and behind the eyes, everted eyelids, and blepharospasm. All the above-named authors agree
that ocular problems are the earliest symptoms observed in subchronic H2S exposure, and that
they appear before any complaints of respiratory difficulties are made.
Exposure to H2S can cause loss of the cornea! reflex and anesthetize the surface of the
eye, so that pain and irritation may not be immediately felt upon exposure. Vision is often
affected first, with changes ranging from perceived halos or rainbows around lights to "blue"
and blurred vision. Damage to the conjunctiva and cornea! epithelium (apparently reversible,
except with repeated insult) results in "sore eye" or "gas eye," an intensely painful
manifestation of inflammation that occurs after the initial loss of sensation passes, which is
accompanied by visual changes. In severe form, actual ulceration of the cornea occurs,
leading to scar formation and permanent impairment of vision. An instance of H2S
keratoconjunctivitis was described by Luck and Kaye (1989).
Not only are the mucous membranes of the eye affected, but H2S can also affect the
respiratory tract; effects include bronchitis, rhinitis, pharyngitis, and laryngitis (Yant, 1930;
Barthelemy, 1939; Milby, 1962; Arnold etal., 1985).
Acute health effects associated with a release, over a 2-day period in 1987, of sulfur
compounds was assessed by a symptom prevalence questionnaire (Haahtela et al., 1992).
A questionnaire was administered to residents in 29 households 10 days after high exposure to
H2S was measured. Symptom prevalence was compared to that reported when the same
households were administered the questionnaire about 4 mo later. Forty-five individuals
responded to both questionnaires, and the only significant finding was an increased incidence
of breathlessness. Levels were as high as 0.1 ppb (0.135 jitg/m3). The 24-h averages for the
2-day exposure were 0.02 and 0.03 ppb (0.035 and 0.043 mg/m3). It is difficult to attribute
H2S as the cause of breathlessness. Mesityl oxide, a sensory irritant, was also reported to be
present in the air because of its odor. Levels were not measured. The SO2 concentration
o
was about 3 jug/m both during exposure and long after exposure had ceased.
At concentrations lower than the odor threshold value of H2S, Ryazanov (1962)
reported subtle responses such as alterations in the, rate and amplitude of respiration,
8-31
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contraction of vocal cord and bronchial muscles, variation in vascular smooth muscle tone,
change in optical chronaxy, change in light sensitivity of dark-adapted eyes, inability of the
cerebrum to maintain an assimilated rhythm, and establishment of an electrocortical reflex
that could lead to reduction in CNS performance. However, the significance of these
findings are unclear as this study has not been replicated.
Vicas and Green (1989) reported the accidental H2S poisoning of three workers.
Patient A, a 32-year-old male, opened a valve and collapsed unconscious. Patient B, a
24-year-old male, collapsed while attempting to rescue him. Patient C, a 34-year-old male,
tried to assist the other two and collapsed. Patient B exhibited marked central cyanosis and
pulmonary edema; he became asystolic and was pronounced dead on arrival at the hospital.
At autopsy, brain sulfide levels were elevated and changes were consistent with hypoxia.
Patient A was given oxygen and ventilatory support. He developed seizures, became
comatose, and died on the seventh day. Patient C showed progressive neurologic
improvement and was discharged with some impairment of visual geometric detail.
Bhambhani and Single (1991) evaluated the effect of exercise (bicycle ergometer) on
oxygen uptake and lactic acid production in volunteers. Sixteen males were randomly
exposed to 0, 0.5, 2.0, and 5.0 ppm (0, 0.7, 2.8, and 6.9 mg/m3) H2S on four separate
occasions. Only exposure to 5 ppm (6.9 mg/m3) H2S resulted in a significantly higher
maximum oxygen uptake. This concentration also resulted in a significant increase in lactic
acid production, but did not affect exercise capacity.
8.2.2 Epidemiological Studies
The National Research Council (1979) defines chronic intoxication in terms of the
effects observed as a result of intermittent exposure to low to intermediate concentrations of
H2S in the range of 50 to 100 ppm (70 to 140 mg/m3). The Illinois Institute for
Environmental Quality (1974) describes chronic poisoning as a prolonged exhibition of
symptoms, which results either from an extended single exposure or repeated, short exposures
that do not produce symptoms typical of acute or subchronic poisoning. The symptoms
generally are described as lingering, behavioral, and neurasthenic in nature and include
fatigue, lack of initiative, mental depression, inability to concentrate, and abnormal peripheral
reflexes indicative of CNS depression. Other symptoms include local irritation of the eyes
8-32
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and respiratory tract, bradycardia, cold sweats, chills, gastrointestinal disturbances, sleep
disorders, and headaches (Vigil, 1979).
At concentrations between 10 and 20 ppm (14 to 28 mg/m3), exposure over time may
cause irritation of the mucous membranes of the respiratory tract and the eyes. Still
unresolved, however, is the debate concerning whether or not "chronic poisoning" exists as a
pathologic entity or is a subjective response to an obnoxious odorant. It is also not clear if
the reported signs and symptoms result from continuous low-level exposure or occur from
damage done by isolated (and usually unmeasured) peak high-level exposure. Confounding
factors include the following: all occupational studies performed with low-level, chronic
exposure also involve exposure to other toxic gases such as SO2, carbon disulfide (CS2),
mercaptans, sulfuric acid mist, and mixtures of volatile organic compounds that individually,
or in aggregate, elicit similar complaints; the working conditions (e.g., night work, high
humidity, and temperature) present many variables.
Ahlborg (1951) studied five cases in the shale oil industry thought to involve chronic
H2S poisoning. The frequency of neurasthenic symptoms such as loss of appetite, poor
memory, dizziness, irritability, itching, headache, and fatigue was greater among the group
of exposed workers. The author could not determine whether these symptoms resulted from
the H2S exposure or from the stressful environment.
Similar symptoms were reported by Barthelemy (1939) and Rubin and Arieff (1945) in
studies in the viscose rayon industry. These workers were exposed to mixtures in which CS2
predominated, but which also contained H2S. These researchers also could not separate the
indicated symptoms from work stress, nor could they attribute them to H2S exposure
exclusively. Glebova (1950) reported that infants who were exposed to H2S emanating from
their mother's clothing during breastfeeding showed a spectrum of signs and symptoms. The
mothers worked in an artificial silk factory where they were exposed to H2S and CS2. When
the mothers were moved away from the H2S-contaminated areas, their infants' symptoms
cleared. Concentrations of 0.02 to 0.04 ppm (0.028 to 0.055 mg/m3) H2S were measured
during breastfeeding times. No attempts to measure CS2 were made. Affected babies
showed poor or retarded development, low weight gain, and listlessness. Some also showed
lack of animation, anemia, paleness, regurgitation after feedings, and gastrointestinal distress.
Susceptibility to infectious disease was also increased. The methods in this Russian study
8-33
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were not clearly delineated, and no control population comparisons were made. The effects
described were not adequately-related to H2S exposure, and effects from other toxic agents,
work conditions, or other confounding factors were not ruled out. Consequently, attribution
of observed effects to H2S should be viewed with strong reservations.
Jappinen et al. (1990) evaluated respiratory function in a cohort of 26 male pulp mill
workers exposed daily to H2S at levels "usually below" 10 ppm (14 mg/m3). Most
measurements were between 2 and 7 ppm (2.8 and 9.7 mg/m3) with a range of 1 to 11 ppm
(1.4 to 15.3 mg/m3). The mean duration of exposure was not reported. It appears that
spirograms were obtained before daily exposure and 30 min after workplace exposure.
Results were compared to predicted values from the Finnish general population. Although no
significant changes on pulmonary function or bronchial responsiveness (as measured by
histamine challenge) were observed, this study design has low power to detect alterations of
pulmonary function parameters and should not be construed as a negative finding. The
investigators also exposed 10 asthmatics (3 men and 7 women) in a laboratory exposure
setting in which individuals were exposed to 2 ppm (2.8 mg/m3) H2S for 30 min. Subjects
rapidly became accustomed to the odor, and 3 of 10 subjects complained of headache after
exposure. There were no significant effects on pulmonary function parameters and no
clinical symptoms were observed.
There are some data that suggest that exposure to H2S and organic sulfides may lead to
increases in cardiovascular and coronary mortality. Such an association was reported by
Jappinen and Tola (1990) in male pulp mill workers. In this cohort (4,179 person-years), the
overall mortality was slightly increased over that expected. Cardiovascular and coronary
death were significantly elevated over expected deaths (standard mortality ratio •= 150).
In those with an exposure > 5 years and with a follow-up period of > 15 years, there were
22 observed cardiovascular deaths (12.7 expected) and 14 observed coronary deaths
(8.7 expected). Differences in smoking habits were reported not to explain these findings,
although the proportion of smokers was 80% higher than in other cohorts examined.
Kangas et al. (1984) investigated the results of H2S, methyl mercaptan, and dimethyl
disulfide exposure in 10 different cellulose mills in Finland. Concentrations ranged from
0 to 20 ppm (0 to 27.8 mg/m3) H2S, 0 to 15 ppm methyl mercaptan, and 0 to 1.5 ppm
dimethyl disulfide; SO9 concentrations reached 20 ppm in some locations. Exposed workers
8-34
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reported headaches and decreased ability to concentrate more often than matched controls.
Sick leave was also used more frequently among the exposed groups than among controls.
Ferris et al. (1979), Chan-Yeung et al. (1980), and Higashi et al. (1983) examined
respiratory effects in workers in a single pulp and paper mill in the United States, in a single
mill in Canada, and in 18 viscose rayon plants in Japan, respectively. Ferris et al. found no
significant mortality or morbidity for respiratory symptoms or illness in his study.
No increases in respiratory symptoms were found by Chan-Yeung et al. (1980), and Higashi
et al. (1983) did not detect increases in respiratory symptoms or decreases in pulmonary
function in their study populations. It should be noted that the workers in these studies were
exposed to a mixture of potentially hazardous compounds. The levels of H2S measured in
these exposures were very low: <4 ppm (<5.6 mg/m3) (Ferris et al., 1979); <0.2 ppm
(<0.28 mg/m3), with mean of 0.05 ppm (0.07 mg/m3) (Chan-Yeung et al., 1980); and an
average of 3 ppm (4.2 mg/m3) (0.3 to 7.8 ppm, range) (Higashi et al., 1983).
In contrast to the above studies, Dales et al. (1989) found increased respiratory
symptoms in the younger vs. the older population living in a 300-mi2 area downwind from
natural gas resources. Symptoms included irritation and inflammation the respiratory
mucosa. These natural gas plants may contain high levels of H2S. However, it is not
possible to determine if H2S solely contributed to these findings since sulfur oxide particulate
matter was present. The authors concluded that attention should be placed on health effects
induced by chronic low-level exposure to H2S and other sulfurated compounds.
Tenhunen et al. (1983) investigated the effect of worker exposure to H7S and methyl
mercaptan on heme synthesis (heme forms part of the hemoglobin complex). Venous blood
was collected from 17 workers in pulp production where the 8-h time-weighted average H2S
concentrations ranged from 0.05 to 5.2 ppm (0.07 to 7.2 mg/m3), with methylmercaptan
ranging from 0.7 to > 1.0 ppm TWA and dimethyl sulfide ranging from 0.03 to 3.2 ppm.
Enzymes in the heme synthesis pathway (a-amino-levulinic acid synthetase and heme
synthetase) showed decreased activities in eight and six cases, respectively. Erythrocyte
protoporphyrin (a precursor of heme) was decreased in seven cases. None of the workers
had clinical anemia. The authors attributed these changes to H2S exposure, but were not able
to establish whether repeated peak or continuous low-level exposure occurred. No unusual
complaints were recorded for any workers in the test or control groups.
8-35
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Dysfunction of the vestibular portion of the vestibulocochlear nerve and its associated
CNS connections has also been reported in some cases of exposure to H2S. Characteristic
symptoms include dizziness, loss of equilibrium, nystagmus, and disturbances of gait or
movement; such symptoms occur as a result of exposure to concentrations of 2,500 ppm
(700 mg/m3) H2S (Poda, 1966; Arnold et al., 1985). Exposure to H2S has been associated
with falls causing secondary injury and death, and may be attributed, in part, to this
neurologic effect (Arnold et al., 1985).
8.2.3 Summary of Human Health Effects
At sufficiently high concentrations (> 1,000 ppm; 1,390 mg/cu.m), H2S is rapidly fatal
to humans, causing respiratory paralysis and apparent inhibition of cellular respiration.
At levels between 500 and 1,000 ppm (695 and 1,390 mg/cu.m), a period of rapid breathing
(hyperpnea) is followed by cessation of breathing (apnea) and death. Damage to organs and
the nervous system can result from the anoxia caused by the depression of cellular
metabolism at levels above 250 ppm. At lower concentrations (50 to 100 ppm; 70 to
139 mg/cu.m), the immediate and prolonged effects are irritation with inflammation of
mucous membranes, particularly of the eye and the respiratory tract. Pneumonitis can result
in pulmonary edema and can be a threat to life. Though ambient concentrations tend to be
below those considered harmful to human health, no long-term, low-level epidemiological
studies have been performed to determine whether H2S causes pulmonary changes similar to
those caused by other irritant gases such as oxides of nitrogen and sulfur. At very low
concentrations, offensiveness of odor, with mostly subjective reactions to stench, is the
dominant effect. Also, neurological symptions such as visual hallucination, and short-term
memory loss have been recently reported at levels thought to be not harmful. More studies
are required to assess H2S toxicity at very low concentrations. Essentially, no human health
data and practically no experimental data on long-term exposures at low levels exist.
No epidemiological studies relating to cancer, teratogenesis, or reproductive effects have been
performed.
8-36
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TABLE 8-4. EFFECTS OF EXPOSURE IN HUMANS AT VARIOUS
CONCENTRATIONS IN AIR
Clinical Effect
Level of Hydrogen Sulfide
ppm mg/m3
Reference
Odor perception threshold
Offensive odor of rotten eggs
Offensive odor (sickening sweet)
Occupational Exposure Limit (O.E.L.)
Serious eye injury
Olfactory paralysis
Pulmonary edema, threat to life
Strong nervous stimulation of
respiration
Respiratory paralysis, immediate
collapse, death
0.003-0.02 0.004-0.028 Indiana Air Pollution Control Board
(1964)
Ahlborg (1951)
National Research Council (1977)
National Research Council (1977)
National Research Council (1977)
420-700 National Research Council (1977)
700-1,400 National Research Council (1977)
1,000-2,000 1,400-2,800 National Research Council (1977)
<30
>30
10
'10
50-100
150-200
300-500
500-1,000
<42
>42
14
14
70-14C
210-35'
420-701
700-1, 4(
8-37
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9. CARCINOGENICITY
No long-term chronic studies for carcinogenic effects have been done with hydrogen
sulfide (H2S). Weisburger et al. (1981) conducted a long-term bioassay of the toxicity and
cancer-causing potential of a number of industrial chemicals, including sodium sulfide.
Sodium bisulfide was administered by gavage to Charles River-CD rats (26 males and
26 females per treatment group) at doses of 9 or 18 mg/kg, in the presence and absence of a
1% thyroid extract (to guard against possible thyroid gland impairment by sulfide). Doses
were administered twice a week for 56 weeks and two to three times a week for the
remaining 22 weeks. After the 78 weeks of treatment, the animals were observed for
26 weeks and then sacrificed. No statistically significant evidence of carcinogenicity was
found in the treatment groups, although the low survivability in groups treated with thyroid
extract made the results ambiguous. The dose ranges tested for sodium bisulfide alone,
which caused some lethality in males at the low dose at 52 weeks, appeared to achieve the
maximum tolerated dose. However, an insufficient number of animals survived treatment,
which precludes definitive judgments about carcinogenic potential. Because of the lack of
adequate animal test data, this compound is placed in Category D, based on the weight-of-
evidence criteria in the U.S. Environmental Protection Agency's Carcinogen Risk Assessment
Guidelines issued in August 1986. A Category D ranking means that the available data are
inadequate to assess a chemical's carcinogenic potential.
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REFERENCES
Weisburger, E. K.; Ulland, B. M.; Nam, J.; Gart, J. J.; Weisburger, J. H. (1981) Carcinogenicity tests of
certain environmental and industrial chemicals. JNCI J. Natl. Cancer Inst. 67: 75-88.
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10. MUTAGENICITY
Using the Ames test with Salmonella typhimurium TA 1535, Gocke et al. (1981) found
evidence of weak mutagenicity as shown by the number of revertants to wild type for this
mutant strain of bacteria, which grows only in the absence of histidine. Addition of the S-9
microsomal fraction from the liver of Aroclor-pretreated rats abolished the effect. Since only
a single tester strain was used and cytotoxic records were not provided, and since there may
have been confounding effects introduced by different growth media, it cannot be
unequivocally stated that evidence of mutagenicity by H2S exists.
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REFERENCES
Gocke, E.; King, M.-T.; Eckhardt, K.; Wild, D. (1981) Mutagenicity of cosmetics ingredients licensed by the
European Communities. Mutat. Res. 90: 91-109.
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11. REPRODUCTIVE AND DEVELOPMENTAL
EFFECTS
No data on human developmental effects of inhaled H2S were found, but based on the
limited information available in animals, H2S appears to have potential to alter normal
developmental processes.
Saillenfait et al. (1989) exposed pregnant Sprague-Dawley (SD) rats (7 to 9/group) to
0, 50, 100, or 150 ppm (0, 70, 139, or 208 mg/m3) H2S 6 h/day during Gestational Days 6
to 20. Maternal body weight gain was significantly reduced at 150 ppm (208 mg/m3) and
fetal body weight was slightly (4 to 7%) reduced in all exposed groups. In dams exposed to
100 or 150 ppm, reduced absolute weight gain and increased implantations and increased live
fetuses were observed. No external anomalies were observed in any of the treatment groups.
In a follow-up experiment, 23 pregnant females were exposed to 100 ppm (139 mg/m3) for
6 h/day on Gestation Days 6 to 20. Fetal weights, number of live and dead fetuses, number
of implantation sites and resorptions and external malformation were recorded. No maternal
toxicity or adverse effects on the developing embryo or fetus was observed. Twenty litters
(278 fetuses) were examined for anomalies.
Studies by Hannah and colleagues suggest that H2S has the potential to alter normal
CNS patterns in the developing fetus. Roth and Hannah (1989) exposed pregnant SD rats to
75 ppm (104 mg/m3) H2S for 7 h/day from Gestational Day 5 to Postnatal Day 21.
Randomly selected pups (8 exposed and 8 control) were sacrificed at Postnatal Days 7, 14,
and 21. Population densities of Purkinje and granule cells in the cerebellum were quantitated
on Days 7 and 14. There was a 20% increase in the density of surviving Purkinje cells along
the primary fissure, but no significant change in the mean number of granule cells. Exposure
to H2S also produced alterations in amino acid levels in the cerebellum. Brain levels of
aspartate, glutamate, taurine, and GABA were significantly reduced below control levels by
Postnatal Day 21. At Postnatal Day 7, taurine was elevated at 125% of control values. The
consequence of these alterations are unknown. In Purkinje cells from the Day 21 group,
alterations in both dendritic architecture and growth process was observed.
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When pups from SD rats exposed to 50 ppm (70 mg/m3) H2S for 7 h/day from
Gestational Day 6 to Postpartum Day 21 were examined, brain levels of taurine were elevated
above controls at Day 7 but had returned to normal by Postpartum Day 21 (Hannah et al.,
1990). At exposure levels of 20 and 50 ppm (28 and 70'mg/m3) H2S during gestation and
postpartum, pups from SD rats exhibited severe alterations in the architecture and growth
characterizes (i.e., nonrandom) of the Purkmje cell dendritic fields.
Hayden et al. (1990) also evaluated the effects of H2S exposure on development of
SD rats. Pregnant rats were exposed to H2S concentrations of 0, 20, 50, or 75 ppm (0, 28,
70, or 104 mg/m3) from Gestational Day 6 to Postpartum Day 21. Developmental and
reproductive data were generated from 6 maternal rats in the 50 and 75 ppm (70 and
104 mg/m3) groups and 12 rats in the 20 ppm group. In the control group, 24 maternal rats
were evaluated. Culling took place in maternal rats at Postpartum Day 1. There was no
treatment-related effect on maternal body weight gain (8 to 15 rats/treatment group). The
most significant observation in dams was a treatment-related increase in parturition time over
matched controls. Liver cholesterol was significantly elevated in dams from 75 ppm at
Postpartum Day 21. There were no treatment-related effects on litter size, viability, sex
ratio, eyelid opening, or surface righting. Neonatal pup development parameters were
monitored on Postpartum Days 1, 7, 14, and 21. At Day 1, litters were culled to reduce
litter size to 12 pups. At 20 pprn (28 mg/m3), hair development and pinna detachment was
significantly accelerated. Hair development in the 50 ppm (70 mg/m3), but not 75 ppm
(104 mg/m3), also was accelerated.
Andrew et al (1980) investigated the effect of exposure of 220 ppm (306 mg/m3) H2S
on spermatogenesis in Wistar rats. A group of 10 rats was exposed for 3 h/day for 7 days.
Controls were exposed to air and a positive control was dosed with triethylenemelamine.
Parameters evaluated in females included fertility, corpora lutea, total implants, and dead
implants. There were no effects of exposure on these parameters.
These investigators also examined the effect of H2S exposure on prenatal development.
Pregnant Wistar rats were exposed to 220 ppm (306 mg/m3) for 3 h/day, 5 days/week, either
throughout gestation (1 to 18 days) or during a portion of organogenesis (day 7 to 11 or
day 12 to 16). At day 21, animals were examined for dead implants, fetal malformations,
and growth retardation. There was no evidence of maternal toxicity or embryotoxicity.
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There was a high (23%) incidence of wavy ribs in the group exposed throughout gestation.
It was not clear to the authors if it was solely due to treatment or was genetic in origin for
this particular group of animals.
There have been no two-generation studies which have examined the role of H2S on
brain development and CNS functioning.
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REFERENCES
Andrew, F. D.; Renne, R. A.; Cannon, W. C. (1980) Reproductive toxicity testing for effects of H2S in rats.
In: Pacific Northwest Laboratory annual report for 1979 to the DOE Assistant Secretary for
Environment: part 1, biomedical sciences. Richland, WA: U.S. Department of Energy, Pacific
Northwest Laboratory; pp. 276-278; report no. PNL-3300 PTI.
Hayden, L. J.; Goeden, H.; Roth, S. H. (1990) Growth and development in the rat during sub-chronic exposure
to low levels of hydrogen sulfide. Toxicol. Ind. Health 6: 389-401.
Higuchi, Y.; Fukamachi, M. (1977) Behavioral studies on toxicity of hydrogen sulfide by means of conditioned
avoidance response in rats. FOLIA Pharmacol. Jpn. 73(3): 307-320.
Renne, R. A.; McDonald, K. E. (1980) Toxic effects of geothermal effluents: acute and subacute inhalation
toxicology of hydrogen sulfide and ammonia in rodents. In: Pacific Northwest Laboratory annual report
for 1979 to the DOE Assistant Secretary for Environment: part 1, biomedical sciences. Richland, WA:
U.S. Department of Energy, Pacific Northwest Laboratory; p. 275; report no. PNL-3300 PTI.
Roth, S. H.; Hannah, R. S. (1989) The neurophysiological effects of low concentrations of hydrogen sulphide on
the developing and mature nervous systems. In: Prior, M. G.; Roth, S.; Green, F. H. Y.; Hulbert,
W. C.; Reiffenstein, R., eds. Proceedings of international conference on hydrogen sulphide toxicity;
June; Banff, AB, Canada. Edmonton, AB, Canada: The Sulphide Research Network; pp. 139-156.
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12. CONCENTRATION-RESPONSE ASSESSMENT
The lexicological data base has been reviewed and an inhalation reference concentration
(RfC) was verified by the U.S. Environmental Protection Agency (EPA) RfD/RfC Work
Group on June 21, 1990. The documentation is available via the Integrated Risk Information
System (IRIS) (U.S. Environmental Protection Agency, 1991). The Integrated Risk
Information System is an on-line data base containing EPA risk assessment results and
regulatory information. An RfC is defined as an estimate, with uncertainty spanning perhaps
an order of magnitude of a daily exposure to the human population (including sensitive
subgroups) which is likely to be without adverse effects during a lifetime (U.S.
Environmental Protection Agency,. 1990). The derivation of the RfC is based on a complete
review of the toxicological literature and encompasses adjustments for exposure duration and
dosimetry and utilizes uncertainty factors account for specific extrapolations between the
population in which the effect was observed and the human population. The critical, usually
the most sensitive, effect is the focus of the RfC derivation and for this effect the
no-observed-adverse-effect level (NOAEL), or lowest-observed-adverse-effect level (LOAEL)
if a NOAEL is not available, is identified. Detailed discussion concerning these issues can be
found in U.S. Environmental Protection Agency, 1990.
The RfC for H2S is 9E-4 mg/m3 and was derived from the NOAEL for inflammation of
the nasal mucosa in mice (Toxigenics, 1983c). Details concerning this critical effect and
other aspects of the study are discussed in Chapter 8. Since the RfC may change due to
evaluation of additional data, the reader is referred to IRIS for the most current information
regarding the RfC for H2S.
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REFERENCES
IRIS, Integrated Risk Information System [data base]. (1991) On-line. Washington, DC: U.S. Environmental
Protection Agency, Office of Health and Environmental Assessment, Office of Research and
Development.
U.S. Environmental Protection Agency. (1990) Interim methodology for development of inhalation reference
concentrations. Research Triangle Park, NC: Office of Health and Environmental Assessment,
Environmental Criteria and Assessment Office; EPA report no. EPA-600/8-88-066F. Available from:
NTIS, Springfield, VA; PB90-145723.
12-2
* US. GOVERNMENT PRINTING OFFICE: 1993-750-002/60,153
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