ORNL
Oak Ridge
National
Laboratory
Operated by
Union Carbide Corporation for the
Department of Energy
Oak Ridge, Tennessee 37830
ORNL/EIS-81
EPA
United States
Environmental Protection
Agency
Office of Research and Development
Health Effects Research Laboratory
Cincinnati, Ohio 45268
EPA-600/1-78-027
REVIEWS OF THE ENVIRONMENTAL
EFFECTS OF POLLUTANTS:
V Cyanide
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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ORNL/EIS-81
EPA-600/1-78-027
October 1978
REVIEWS OF THE ENVIRONMENTAL EFFECTS OF POLLUTANTS: V. CYANIDE
by
Leigh E. Towill, John S. Drury, Brad L. Whitfield,
Eric B. Lewis, Elizabeth L. Galyan, and Anna S. Hammons
Information Center Complex, Information Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
operated by
Union Carbide Corporation
for the
Department of Energy
Reviewer and Assessment Chapter Author
James L. Way
Washington State University
Pullman, Washington 99164
Interagency Agreement No. D5-0403
Project Officer
Jerry P. Stara
Office of Program Operations
Health Effects Research Laboratory
Cincinnati, Ohio 45268
October 1978
Prepared for
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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This report was prepared as an account of work sponsored by an agency
of the United States Government. Neither the United States Government nor
any agency thereof, nor any of their employees, contractors, subcontractors,
or their employees, makes any warranty, express or implied, nor assumes any
legal liability or responsibility for any third party's use or the results
of such use of any information, apparatus, product or process disclosed in
this report, nor represents that its use by such third party would not
infringe privately owned rights.
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
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CONTENTS
Figures vii
Tables ix
Foreword xi
Acknowledgments xiii
Abstract xv
1. Summary 1
1.1 Properties and Analysis 1
1.2 Environmental Occurrence 2
1.3 Biological Aspects in Microorganisms 3
1.4 Biological Aspects in Plants 4
1.5 Biological Aspects in Humans and Experimental Animals . . 5
1.5.1 Metabolism 5
1.5.2 Effects 7
1.6 Biological Aspects in Wild and Domestic Animals 8
1.7 Conclusions 9
2. Chemical and Physical Properties and Analysis 11
2.1 Summary 11
2.2 Physical and Chemical Properties 12
2.2.1 Hydrogen Cyanide and Salts 12
2.2.2 Cyanogen and Cyanogen Halides 18
2.2.3 Cyanates and Isocyanates 19
2.2.4 Thiocyanates 20
2.2.5 Nitriles and Isocyanides 21
2.2.6 Cyanohydrins 24
2.2.7 Cyanogenic Glycosides 24
2.2.8 Complex Cyanide Compounds 25
2.3 Analysis for Cyanides 26
2.3.1 Sampling and Sample Handling 26
2.3.2 Methods of Analysis 27
2.3.3 Comparison of Analytical Procedures 33
3. Biological Aspects in Microorganisms 40
3.1 Summary 40
3.2 Metabolism 40
3.2.1 Uptake 40
3.2.2 Biotransformation 42
3.3 Effects 57
3.3.1 Growth Effects 57
3.3.2 Metabolic Effects 59
4. Biological Aspects in Plants 77
4.1 Summary 77
4.2 Metabolism 78
4.2.1 Hydrocyanic Acid Incorporation 78
4.2.2 Cyanide Release from Plants 80
4.2.3 Cyanogenic Glycosides 80
4.2.4 Pseudocyanogenic Glycosides 87
4.2.5 Lathyrogenic Compounds 88
4.2.6 Glucosinolates (Thioglucosides) 89
4.2.7 Indoleacetonitrile 92
4.2.8 Cyanopyridine Alkaloids 93
4.2.9 Nitrile Herbicides 93
111
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IV
4.3 Effects 95
4.3.1 Cyanide and Respiration 95
4.3.2 Inhibition of Photosynthesis 98
4.3.3 Inhibition of Enzymes 98
4.3.4 Physiological Effects 99
5. Biological Aspects in Wild and Domestic Animals Ill
5.1 Summary Ill
5.2 Mammals Ill
5.2.1 Metabolism Ill
5.2.2 Toxic Effects Ill
5.3 Fish 112
5.3.1 Metabolism 112
5.3.2 Effects 112
5.4 Birds 121
5.4.1 Metabolism 121
5.4.2 Effects 121
5.5 Invertebrates 121
5.5.1 Metabolism 121
5.5.2 Effects 122
6. Biological Aspects in Humans 127
6.1 Summary 127
6.2 Metabolism 128
6.2.1 Uptake and Absorption 128
6.2.2 Transport and Distribution 130
6.2.3 Detoxification 133
6.2.4 Excretion 137
6.3 Effects 138
6.3.1 , Mechanism of Action 138
6.3.2 Acute Effects 138
6.3.3 Chronic Effects 139
6.3.4, Treatment for Cyanide Poisoning 147
7. Environmental Distribution and Transformation 164
7.1 Summary 164
7.2 Production and Usage 164
7.3 Sources 165
7.4 Distribution and. Transformation in the Environment. . . . 168
7.4.1 Distribution and Transformation in Soils 168
7.4.2 Distribution and Transformation in Water 170
7.4.3 Distribution and Transformation in Air 170
7.5 Waste Management 171
7.6 Biomagnification and Cycling 172
7.7 Cyanide in Foods 172
8. Environmental Assessment of Cyanide 177
8.1 Production, Uses, Transportation, and Potential
Environmental Contamination 177
8.1.1 Production 177
8.1.2 Preparations and Transportation 177
8.1.3 Uses 177
8.1.4 Potential Environmental Contamination 177
8.2 Environmental Persistence 179
8.2.1 Biomagnification and Cycling 179
8.2.2 Persistence in Foods 179
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V
8.2.3 Persistence in Soils 179
8.2.4 Persistence in Water 180
8.2.5 Persistence in Air 180
8.2.6 Waste Management 180
.3 Effects of Aquatic and Terrestrial Organisms . . 180
8.3.1 Aquatic Organisms 180
8.3.2 Terrestrial Organisms 181
.4 Effects on Human Health 182
8.4.1 Toxic Effects 182
8.4.2 Teratogenic, Mutagenic, and Carcinogenic Effects. . 183
8.4.3 Treatment 183
.5 Potential Health Hazards 184
8.5.1 Occupational 184
8.5.2 General Population 184
.6 Potential Environmental Hazards 185
.7 Regulations and Standards 185
.8 Cyanide Analysis 187
8.8.1 Biologic Sample 187
8.8.2 Analytical Methods 187
.9 Summary of Opinion and Projected Research Needs. ..... 188
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FIGURES
3.1 Amount of cyanide remaining in a medium containing 10 M
KCN per milligram of a strain of Baci,11us punrilus as a
function of time 41
3.2 Production of HCN during growth of a snow mold fungus in
shake culture. Free HCN collected by aeration, total
HCN by steam distillation 44
3.3 Regression line showing the molar ratio between added glycine
and cyanide recovered when whole bacteria were fed varying
amounts of glycine. Similar dots refer to experiments made
with the same bacterial preparation 45
3.4 Time course of cyanide production in response to growth of
Pseudomonas aeruginosa strain 9-D2 in 2% peptone at 37°C. . 46
3.5 Time-course production of cyanide by Chromobaetex'i-um
vLolaceum (in 1% peptone medium) in relation to growth. . . 47
3.6 Hypothetical pathways for cyanide formation from glycine. . . 48
3.7 A cyclic process for converting HCN to C02 which involves
glutamate, Y~amino butyric acid, succinic semialdehyde,
and 4-amino-4-cyanobutyric acid 53
3.8 Effect of pH on the degradation of cyanide by a cyanide-
resistant bacterium 54
3.9 Proposed pathway for the incorporation of cyanide by ChTomo-
baoterium violaoeim 55
3.10 Production of Xi*C02 and 15NH3 by a strain of Bac-illus pumilus
as a function of time in cells exposed to Kli*C15N 56
3.11 Inhibition of DNA synthesis by cyanide 63
4.1 Generalized biosynthetic pathway for cyanogenic glycosides. . 85
4.2 Concentration of hydrocyanic acid potential in plant parts
of Greenleaf Sudan grass (sorghum) 86
4.3 Scheme showing the metabolites of dichlobenil in bean leaves
after a five-day uptake of a 12 ppm solution via the roots
(R = biopolymer, e.g., polysaccharides) 94
5.1 Relationship between median lethal concentration (LC50) of
sodium cyanide as CN and exposure time for five species
of freshwater fish 113
vn
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viii
6.1 Fate of cyanide ion in the body 133
6.2 Rate of visual improvement per month in patients with tobacco
amblyopia treated with parenteral hydroxocobalamin or
cyanocobalamin 144
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TABLES
2.1 Physical properties of hydrogen cyanide 13
2.2 Physical properties of sodium cyanide 14
2.3 Physical properties of potassium cyanide 15
2.4 Physical properties of cyanogen 18
2.5 Physical properties of some cyanates 20
2.6 Physical properties of some important cyanides 22
2.7 Physical properties of acrylonitrile 23
2.8 Methods for determining cyanide 28
3.1 Cyanogenic fungi 43
3.2 Occurrence of cyanogenesis in bacteria 49
3.3 Occurrence of microorganisms in nitrile-fed Ohio River water
text oxidation systems 51
3.4 Comparative assimilation of H14CN by various fungi 52
3.5 Toxicity of cyanurates and cyanides to microorganisms .... 58
3.6 Compounds inhibiting both growth and division of
Sacohapomyoes oevewLs-iae 60
3.7 Effect of cyanide on bacterial respiratory chains 61
3.8 Influence of various pesticides on the yield of bacterial
mass of Asotobacter v-inelandi-i- and the synthesis of
nitrogenase enzyme complex 66
3.9 Extent of inhibition of specific activity of nitrogenase
enzyme complex of Azotobacter v-ineland-ii, by several
pesticides 67
4.1 Naturally occurring glycosides 82
4.2 Cyanide content of selected plants 87
4.3 Pseudocyanogenic glycosides 88
4.4 Glucosinolates in domesticated crucifer plants 90
4.5 Relative cyanide-insensitive respiration of higher plants . . 96
IX
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X
5.1 Toxicity of cyanurates and cyanides to fish 114
5.2 Estimation of the toxic proportion of each poison and the
total toxicity of the mixture for rainbow trout 120
5.3 Toxicity of cyanides and thiocyanates to aquatic inverte-
brates 123
6.1 Comparison of cyanide concentrations in tissues from
rabbits killed by HCN with concentrations in tissues from
rabbits killed with KCN 131
6.2 Cyanide levels in human tissues and fluids after fatal
cyanide poisoning 132
6.3 Rhodanese activity in tissues of the dog, rhesus monkey,
rabbit, and rat 134
6.4 Replacement of sodium thiosulfate by other sulfur-
containing compounds 135
6.5 Human response to inhaled cyanide and cyanide-containing
compounds 140
6.6 Animal response to inhaled cyanide and cyanide-containing
compounds 141
6.7 LD50 of compounds containing cyanide moiety after skin
absorption by rabbits and guinea pigs 142
6.8 Acute toxicity of cyanide and cyanide-containing compounds
to experimental animals 142
6.9 Plasma thiocyanate and vitamin B12 levels in neuropathy
patients and in controls with miscellaneous diseases. . . . 143
6.10 Embryolethal and teratologic effects of BAPN in pregnant
rats 148
6.11 Effect of BAPN and AAN on fetal development in baboons. . . . 150
6.12 Comparison of effect of antidotes on cyanide poisoning
in dogs 150
7.1 Primary uses of some major cyanide compounds 166
7.2 Typical electroplating wastes containing cyanides 168
7.3 Inorganic cyanide wastes 169
7.4 Tolerances for cyanide residues on foods 172
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FOREWORD
A vast amount of published material is accumulating as numerous
research investigations are conducted to develop a data base on the
adverse effects of environmental pollution. As this information is
amassed, it becomes continually more critical to focus on pertinent,
well-designed studies. Research data must be summarized and interpreted
in order to adequately evaluate the potential hazards of these substances
to ecosystems and ultimately to public health. The Reviews of the Environ-
mental Effects of Pollutants (REEPs) series represents an extensive com-
pilation of relevant research and forms an up-to-date compendium of the
environmental effect data on selected pollutants.
Reviews of the Environmental Effects of Pollutants: V. Cyanide
includes information on chemical and physical properties; pertinent
analytical techniques; transport processes to the environment and sub-
sequent distribution and deposition; impact on microorganisms, plants,
and wildlife; toxicologic data in experimental animals including metabo-
lism, toxicity, mutagenicity, teratogenicity, and carcinogenicity; and an
assessment of its health effects in man. The large volume of factual
information presented in this document is summarized and interpreted in
the final chapter, "Environmental Assessment,", which presents an overall
evaluation of the potential hazard resulting from present concentrations
of cyanide in the environment. This final chapter represents a major
contribution by James L. Way from Washington State University.
The REEPs are intended to serve various technical and administrative
personnel within the Agency in the decision-making processes, i.e., in
the development of criteria documents and environmental standards, and
for other regulatory actions. The breadth of these documents makes them
a useful resource for public health personnel, environmental specialists,
and control officers. Upon request these documents will be made available
to any interested individuals or firms, both in and out of the government.
Depending on the supply, the document can be obtained directly by writing
to:
Dr. Jerry F. Stara
U.S. Environmental Protection Agency
Health Effects Research Laboratory
26 W. St. Glair Street
Cincinnati, Ohio 45268
R. J. Garner
Director
Health Effects Research Laboratory
XI
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ACKNOWLEDGMENTS
The authors are particularly grateful to R. F. Kimball, Oak Ridge
National Laboratory (ORNL), for reviewing preliminary drafts of this
report and for offering helpful comments and suggestions. The advice
and support of Gerald U. Ulrikson, Manager, Information Center Complex,
and Jerry F. Stara, EPA Project Officer, and the cooperation of the
Toxicology Information Response Center, the Environmental Mutagen Infor-
mation Center, and the Environmental Resource Center of the Information
Center Complex, Information Division, ORNL, are gratefully acknowledged.
The authors also thank Carol Brumley McGlothin and Maureen Hafford,
editors, and Donna Stokes and Patricia Hartman, typists, for preparing
the manuscript for publication.
Appreciation is also expressed to Bonita M. Smith, Karen L. Blackburn,
and Donna J. Sivulka for EPA in-house reviews and editing and for coordinat-
ing contractual arrangements. The efforts of Allan Susten and Rosa Raskin
in coordinating early processing of the reviews were important in laying
the groundwork for document preparation. The advice of Walter E. Grube
was valuable in preparation of manuscript drafts. The support of R. John
Garner, Director of Health Effects Research Laboratory, is much appreciated.
Thanks are also expressed to Carol A. Haynes and Peggy J. Bowman for typing
correspondence and corrected reviews.
Xlll
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ABSTRACT
This study is a comprehensive, multidisciplinary review of the
health and environmental effects of cyanide and specific cyanide deriv-
atives. Over 500 references are cited.
Cyanide production in the United States was about 700 million
pounds in 1975, most of which was used for acrylonitrile production.
The most important applications of inorganic cyanides are electroplating
and metal treatments. Improper storage, handling, and disposal account
for isolated instances of cyanide release to the environment. Tobacco
smoke is probably one of the major sources of cyanide exposure to the
general public.
Cyanide is a general respiratory poison acting by inhibition of
cytochrome oxidase. Uptake occurs through inhalation, ingestion, or
skin absorption. Because of the known dangers of cyanide, accidental
acute poisonings are uncommon. Evidence of exposure to low levels of
cyanide over prolonged periods is not well recognized. There is little
data concerning the carcinogenic, teratogenic, and mutagenic properties
of cyanide or the distribution and transformation of cyanides in air,
land, or water.
This report was submitted in partial fulfillment of Interagency
Agreement No. D5-0403 between the Department of Energy and the U.S.
Environmental Protection Agency. The draft report was submitted for
review in December 1976. The final report was completed in November 1977,
xv
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SECTION 1
SUMMARY
1.1 PROPERTIES AND ANALYSIS
Cyanides are defined as organic or inorganic compounds which contain
the -C=N grouping. Hydrogen cyanide (HCN) is a colorless liquid which
boils at 25.7°C and freezes at -13.2°C; the gas is lighter than air and
diffuses rapidly (Section 2.2.1). Free HCN is very reactive and occurs
only rarely in nature; it is usually prepared commercially from NH3 and
CHi, at elevated temperatures with a platinum catalyst. In aqueous solu-
tion, HCN is a weak acid with the ratio of HCN to CN being about 100 at
pH 7.2, 10 at pH 8.2, and 1 at pH 9.2. Both HCN and cyanide salts form
complexes with metals; this property is responsible for the industrial
utility of these compounds. Cyanides from industrial activity are released
mainly to water and, to a lesser extent, to the atmosphere. The degrada-
tion of cyanides through waste management procedures usually produces car-
bonates and nitrogen gas (Section 2.2.1.4).
Cyanide complexes a variety of metals, especially those of the tran-
sition series (Section 2.2.8). Ferricyanides and ferrocyanides have a
variety of industrial uses but do not release free cyanide unless exposed
to ultraviolet light. Thus, sunlight can lead to the formation of cyanide
in wastes containing ferricyanides and ferrocyanides.
Cyanogen [(CN)2] is an extremely poisonous, flammable gas which has
a vapor pressure of about 5 atm at 20°C. It is prepared industrially from
HCN and oxygen at 300 to 600°C with a silver catalyst and is used chiefly
in the chemical industry as a high-energy fuel and for organic syntheses.
Cyanogen reacts slowly with water to form HCN, cyanic acid, and other com-
pounds. It is extremely reactive and is probably rapidly degraded in the
environment.
Cyanates contain the -OCN radical. Inorganic cyanates, which are
formed by oxidation of cyanide salts, are reactive and hydrolyze in water
to form NH3 and bicarbonate. Alkyl cyanates trimerize readily to form
cyanurates. Alkyl isocyanates contain the -NCO radical and are formed
from cyanates. They, too, are reactive; however, they are insoluble in
water.
Thiocyanates (-SCN) are formed from cyanides and sulfur-containing
materials and are more stable than cyanates (Section 2.2.4). Solutions
of thiocyanates form HCN in acidic media; degradation of thiocyanate
wastes is accomplished by procedures similar to those used for cyanide
wastes.
Nitriles are defined as organic cyanides (RCN). They are easily
prepared and exhibit a marked tendency to polymerize (Section 2.2.5).
Most nitriles are fairly insoluble in water but are soluble in organic
solvents. Acrylonitrile is an important raw material for the textile
and rubber industries.
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Cyanohydrins, R2C(OH)CN, are toxic compounds which can decompose to
HCN or CN~ under environmental conditions (Section 2.2.6). Calcium cyan-
amide (CaNCN) is commonly used as a fertilizer that reacts in the soil
to yield urea.
Suitable analytical procedures are important for the detection of
cyanide within the environment. Preparation of aqueous samples usually
involves alkalizing the sample, removing interfering sulfides by precipi-
tating and filtering lead sulfide, and removing interfering fatty acids
by extracting the acidified aqueous phase with isooctane, hexane, or
chloroform (Section 2.3.1). Additional steps may be necessary to remove
other interfering substances. The sample can then be analyzed by one of
several procedures (Section 2.3.2). Absorption spectrophotometry is prob-
ably the most widely used technique for determining cyanide concentrations
of 1 mg/liter or less; modifications allow detection of cyanide down to
5 yg/liter. Speedy analyses and little sample preparation are attributes
of the cyanide ion-selective electrode; the silver iodide membrane elec-
trode is useful in the 10"3 to 10"5 M range. Indirect atomic absorption
spectrometry is adequate for cyanide detection but is not extensively
used. Fluorometry has a low detection limit but is presently limited in
application. Gas chromatography is a sensitive procedure for detecting
cyanide, usually by converting it to a cyanogen halide.
1.2 ENVIRONMENTAL OCCURRENCE
Cyanide production in the United States was about 700 million pounds
for 1975; about 52% was used for acrylonitrile production, 18% for methyl
methacrylate production, 14% for adiponitrile production, 7% for sodium
cyanide production, and 9% for a variety of uses (Section 7.2). Inorganic
cyanides have many uses; the two most important applications are electro-
plating and metal treatments (Table 7.1). Hydrogen cyanide is used as a
rodenticide and an insecticide. Organic cyanides, such as acrylonitrile,
are used for the production of acrylic and modacrylic fibers, nitrite
elastomers, and plastics.
Industries concerned with the production and use of cyanide compounds
generate wastes which contain large amounts of cyanide; for example, elec-
troplating wastes contain 0.5% to 20% cyanide (Section 7.3). Paint manu-
facture and use, the steel industry, and mining operations all produce
wastes with a high cyanide content. Fortunately, the toxicity of cyanides
is well-known. Waste management procedures are standard practice and
remove most of the cyanide. Three standard procedures for cyanide destruc-
tion are alkaline chlorination (most common), electrolytic decomposition,
and ozone oxidation (Sections 2.2.1.4 and 7.5). Cyanide can also be con-
verted to less toxic compounds such as cyanate and ferricyanide. Improper
storage, handling, and disposal account for isolated instances of cyanide
release to the environment.
Data on the distribution of various cyanide compounds within the
environment and on their transformations are sparse. Cyanides are not
adsorbed or retained within soils (Section 7.4.1). Microbial metabolism
apparently can rapidly degrade cyanide to C02 and NH3 and thus eliminate
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any soil accumulation problem. Under anaerobic conditions, cyanides are
converted by microbes to gaseous nitrogen compounds which escape to the
atmosphere.
Cyanides are uncommon in U.S. water supplies. Small quantities of
hydrocyanic acid, cyanide salts, or complexed cyanides sometimes occur
(Section 7.4.2); however, they usually do not exceed the recommended max-
imum limit of 10 ppb. Volatile cyanides are not usually detected in the
atmosphere (Section 7.4.3).
1.3 BIOLOGICAL ASPECTS IN MICROORGANISMS
A wide variety of microorganisms are able to metabolize cyanide.
These organisms may play a role in the treatment of cyanide wastes. If
a mixed population in a sludge process has not been exposed to cyanide,
small cyanide concentrations (e.g., 0.3 ppm cyanide) can be toxic (Sec-
tion 3.2.2.2.1). The population can become acclimated to cyanide, after
which they are able to degrade wastes with higher cyanide concentrations.
The presence of cyanide will affect the population structure in mixed
population systems.
Several species of fungi are able to take up and metabolize cyanide
(Section 3.2.2.2.2). The products of cyanide metabolism vary. Fusarium
solan-i degrades cyanide to C02 and NH3, whereas Khi-zoGtoni-a solani converts
cyanide to a-aminobutyronitrile. The nitrile group of a-aminobutyroni-
trile is then hydrolyzed to give a a-aminobutyric acid. An unidentified
basidiomycete can convert HCN to 4-amino-4-cyanobutyric acid, which is
then hydrolyzed to glutamate. Thus, cyanide nitrogen is eventually re-
leased to the environment as NH3 and cyanide carbon is released as C02.
Bacterial species which metabolize cyanide have also been isolated,
but often the details of their metabolism have not been described (Section
3.2.2.2.3). ChTomo'baotei^Lwn v-Lolacewn can apparently produce $-cyanoala-
nine and Y~cyano~a~amin°butyric acid from cyanide. Subsequent hydrolysis
of the nitrile groups in these products returns the cyanide nitrogen atom
to the environment as NH3. Several species of bacteria form thiocyanate
from cyanides and thiosulfates.
Microbes are also able to synthesize cyanide (Section 3.2.2.1). The
biochemistry of cyanide production is not completely known, however. In
some fungi, cyanide production is associated with autolysis, whereas in
others it is formed throughout the growth cycle. Various bacteria produce
cyanides. Chr>omobaoteT-iwn violaceum can synthesize HCN from glycine.
Again, some reports have suggested that cyanide can be synthesized by
growing cultures, while others report that cyanide is synthesized from
nonproliferating cells.
Cyanides are toxic to a wide range of microorganisms, but quantita-
tive data are sparse (Sections 3.3.1 and 3.3.2). Miororegma heterostoma
(protozoa) and Seenedesmus quadincauda. Calga) are very sensitive, having
toxicity thresholds of 0.04 and 0.16 ppm CN~, respectively (Section
3.3.1.1). Some bacteria are killed by exposure to 1 ppm cyanide; however,
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some other microorganisms have much higher tolerances. One gram-negative
bacterium can tolerate up to 50 ppm cyanide; the fungi AspePgHlus sp.
and Rhizopus ni.gpica.ns can tolerate up to 100 and 200 ppm cyanide, respec-
tively. Besides decreased growth, the effects of exposure to cyanide
have been reported to include altered cell morphology, decreased motility,
and mutagenicity. Some microorganisms have become essentially resistant
to cyanide.
Inhibition of respiration is an extensively studied effect of cya-
nide. In addition to the cyanide-sensitive electron transport pathway,
many microbes have a cyanide-insensitive pathway. Examples are more com-
mon and better studied in the bacteria and fungi but are also known in
algae and protozoa.
Cyanide inhibits DNA replication in Esoher^ichla ooli. and perhaps
DNA-repair processes in Chtcmydomonas ve-Lnhafdl. Amino acid transport
in bacteria is also inhibited by cyanide. The above inhibitions were
observed with 0.5 to 17 mM KCN. Cyanide also inhibits various microbial
enzymes such as B-hemolysin, nitrite reductase, and nitrate reductase.
In summary, data exist concerning the cyanide inhibition of growth,
respiration, and other physiological processes in isolated microbial sys-
tems, but there are virtually no data on the effects of cyanide exposure
on natural microbial communities in soil and water. An exception is the
mixed microbial populations in sewage sludge. Cyanide exposure most
likely occurs sporadically and in low concentrations. Many microbes
metabolize cyanide and many others synthesize it; however, the integra-
tion of these processes into the ecology of a given community is unknown.
1.4 BIOLOGICAL ASPECTS IN PLANTS
Free cyanide is not found in higher plants. Although the supporting
data are sparse, plants can actively release HCN to the environment (Sec-
tion 4.2.2). The ecological implications of such a release are unknown.
Many plants, if not all, have the ability to metabolize administered cya-
nide (Section 4.2.1). A major pathway is the reaction of cyanide with
cysteine to form g-cyanoalanine, which is then hydrolyzed to asparagine.
Other conversions of g-cyanoalanine occur in some species. Some
lathyrogenic compounds, found in the genera Lathyrus and Viaia, are formed
from g-cyanoalanine (Section 4.2.5). The lathyrogens are products of nor-
mal plant metabolism and do not indicate cyanide exposure. The origin of
the endogenous cyanide radical in the lathyrogenic plants is unknown.
The cyanide radical is found in a variety of naturally occurring
plant compounds. These compounds include cyanogenic glycosides, glyco-
sides, lathyrogenic compounds, indoleacetonitrile, and cyanopyridine
alkaloids.
Plants that contain cyanogenic glycosides are potentially poisonous
because bruising or incomplete cooking can lead to hydrolysis of the gly-
coside and release of HCN (Section 4.2.3). There are about 20 major
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cyanogenic glycosides, of which only one or two occur in a given species.
They are synthesized from amino acids and sugars and are found in a vari-
ety of economically important plants including sorghum, flax, lima bean,
cassava, and many of the stone fruit. Their functions and further metab-
olism within the plant are unknown. Glucosinolates (thioglucosides) are
also common secondary plant products and are formed in members of the Cru-
ciferae (Section 4.2.6). Upon injury to these plants, the glucosinolates
can be hydrolyzed to organic nitriles, isothiocyanates, or thiocyanates,
depending on the pH. These compounds can produce human maladies (Section
6.3.3). The remaining natural products which contain cyanide groupings
are of lesser importance and are restricted to a few taxa.
Nitrile herbicides are used in a variety of situations; all appar-
ently act as metabolic inhibitors or uncouplers of electron flow (Section
4.3.4.5). They are used either as a preemergent herbicide (dichlobenil)
or as a postemergent, contact herbicide (ioxynil and bromoxynil). Their
metabolism is not well characterized, but they do not release cyanide
(Section 4.2.9).
The major effect of cyanide on plants is the inhibition of respira-
tion, which occurs through the inhibition of cytochrome oxidase (Section
4.3.1). Inhibition is not always complete, however, and the remaining
activity is referred to as cyanide-insensitive respiration. Insensitive
respiration varies with physiological status and pretreatment and is
apparently due to electron flow through an alternate pathway which is
not inhibited by cyanide. Inhibition of enzymes other than cytochrome
oxidase is usually a result of (1) complexing of a metal cofactor, (2)
reaction with carbonyl groups, or (3) reaction with disulfide bonds
(Section 4.3.3).
Without respiration and the resulting ATP, a variety of other phys-
iological processes, including ion transport and translocation, fail.
Interestingly, the early stages of germination are stimulated by cyanide
exposure, presumably due to the more extensive use of the pentose phos-
phate pathway during this period (Section 4.3.4.1). Cyanide produces
chromosomal aberrations in some plants, but the mechanism of this action
is unknown (Section 4.3.4.4).
Hydrogen cyanide has been used in greenhouses, in the field, and in
commercial fumatoria as a fumigant to control insect populations (Section
4.3.4.6). This use can lead to cyanide residues in treated foods. The
chemical forms of these residues are not known, but maximum residue limits
are established for various foods.
1.5 BIOLOGICAL ASPECTS IN HUMANS AND EXPERIMENTAL ANIMALS
1.5.1 Metabolism
1.5.1.1 Uptake — Intake of cyanide can be by inhalation, ingestion, or
absorption through the skin (Section 6.2.1). Whatever the route, cyanide
is readily absorbed into the bloodstream and carried throughout the body.
Death soon after exposure to lethal concentrations of cyanide is evidence
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of rapid absorption. Cyanide rapidly appears in the. blood after inhala-
tion of both HCN and cyanide salt dusts. Cyanogen, a gas at room temper-
ature, releases hydrogen cyanide upon hydrolysis and is, therefore, toxic;
no absorption values were available. When inhaled, the toxicity of halo-
genated cyanogens is comparable with that of HCN, but this should not infer
toxicity is due to the cyano moiety. Some nitriles may also represent an
inhalation hazard.
Ingestion of cyanides also causes the rapid appearance of cyanide
in blood. Cyanogenic glycosides found in some plant foodstuffs release
hydrogen cyanide during digestion, but release is slow and appearance of
symptoms is delayed. Food in the stomach tends to delay absorption of cya-
nides . Hydrogen cyanide in liquid or vapor form can be absorbed through
the skin; absorption is more rapid if the skin is cut, abraded, or moist.
1.5.1.2 Transport and Distribution — Once cyanide is in the bloodstream,
it is distributed to other body tissues. Some cyanide binds to methemo-
globin in the blood (Section 6.2.2.1). This binding is reversible and
cyanide is released to plasma when the free cyanide concentration in the
blood drops. After the initial exposure to cyanide, levels of cyanide in
tissues other than blood increase considerably.
Cyanate reacts irreversibly with free sulfhydryl and amino groups
of blood proteins to form carbamyl derivatives. Sodium nitroprusside is
reported to react nonenzymatically with hemoglobin in red blood cells to
form cyanide. Cyanogen chloride (CNC1) is also rapidly converted to HCN
in the presence of red blood cells.
1.5.1.3 Detoxification — Most cyanide reactions, including those respon-
sible for toxicity and for detoxification, occur within cells. Inhibition
of cytochrome oxidase is probably the major reaction causing cyanide tox-
icity. The major detoxification pathway is the reaction of cyanide with
thiosulfate in the presence of the enzyme, rhodanese, to produce thiocya-
nate (Section 6.2.3.1). Most tissues contain rhodanese; liver, kidney,
brain, and muscle have higher levels than other tissues. Rhodanese is
found within mitochondria. The content of the other reactant, thiosul-
fate, is low in the body; added thiosulfate can increase the LD50 for
cyanide; thus, sulfur donors may be the limiting substrate for cyanide
detoxification.
Minor detoxification pathways also exist. Cyanide combines with
cystine to produce 2-aminothiazoline-4-carboxylic acid or 2-iminothiazo-
lidine-4-carboxylic acid. This last compound is inert and is excreted.
Cyanide also is believed to be metabolized through formic acid to form
C02. In addition, cyanocobalamine (CN-B12), a cyanide-containing form of
vitamin Bi2, is formed after cyanide exposure. However, the amounts of
hydroxocobalamin (Bj.2) in the liver are sufficient to detoxify large doses
of cyanide. Injection experiments in humans suggest that CN-Bj.2 is not
metabolized; however, contradictory evidence also exists. Vitamin Bj.2
given to mice shortly before or after cyanide reduces the toxicity of
cyanide. The binding of cyanide by methemoglobin in blood also reduces
free cyanide concentrations.
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1.5.1.4 Excretion — Studies with mice and guinea pigs show that 1% to
2% of injected HCN can be eliminated by exhalation prior to death. How-
ever, most cyanide is metabolized and excreted in the urine as thiocya-
nate (Section 6.2.4). Tobacco smoke contains cyanide; the urine and body
fluids of smokers contain higher concentrations of thiocyanates than those
of nonsmokers. Levels of thiocyanate in urine increased from 3.7 mg/liter
for people smoking 10 cigarettes per day to 20.0 mg/liter for people smok-
ing 40 cigarettes per day. Cyanocobalamin is also excreted in the urine.
1.5.2 Effects
1.5.2.1 Mechanism of Action — Cyanide inhibits enzymatic activity by
binding to the metallic cofactor in metalloenzymes (Section 6.3.1). Cyto-
chrome oxidase, the most sensitive enzyme known, is completely inhibited
by 10~s moles/cm3 of cyanide. Oxygen cannot then be utilized and cyto-
toxic anoxia occurs. Death results from depression of the central nervous
system (CNS), the tissue most sensitive to anoxia. Cyanide in higher
concentrations also inhibits other heme enzymes (catalase and peroxidase)
as well as nonheme metalloenzymes (tyrosinase, ascorbic acid oxidase, and
phosphatase).
1.5.2.2 Acute Effects — The effects of cyanide depend on the degree and
rate of production of histotoxic hypoxia (Section 6.3.2). Fatal doses
produce a brief stage of CNS stimulation and then depression followed by
hypoxic convulsions and death. At 2000 ppm HCN in air, the first breath
brings immediate deep, rapid breathing with collapse, convulsions, and
cessation of breathing occurring within a minute. The total amount of
HCN absorbed in rapid death may be as low as 7 mg/kg body weight. For
HCN absorbed through the skin the LD50 is about 100 mg/kg body weight.
Ingestion of KCN or NaCN produces similar effects but over a longer time
(5 to 20 min after ingestion). About 3.5 mg HCN per kilogram body weight
is absorbed when death occurs after NaCN or KCN ingestion.
Nitriles may first exert a pharmacologic action due to the entire
molecule, but if cyanide is released from the nitrile faster than the
cyanide can be detoxified, cyanide toxicity symptoms may occur.
1.5.2.3 Chronic Effects — Because of the known dangers of cyanide, acci-
dental acute poisonings are uncommon. Exposure to low levels of cyanide
over prolonged periods produce symptoms that differ from acute exposure
and are not well recognized (Section 6.3.3).
Recently, chronic cyanide uptake has been correlated with diseases
such as tobacco amblyopia, retrobulbar neuritis in pernicious anemia,
Leber's optic atrophy, and Nigerian nutritional neuropathy. Defects in
cyanide detoxification processes may play contributing roles. The neu-
ropathies may result from demyelination of nerves in the CNS caused by
cyanide-induced anoxia. Cyanide exposure can lead to pathology of the
CNS in experimental animals. Tropical neuropathies in humans are charac-
terized by optic atrophy, nerve deafness, and sensory spinal ataxia (Sec-
tion 6.3.3.1). Chronic cyanide intoxication has been implicated by some
reports in these diseases as well as in tropical amblyopia. Intake may
result from the use of cassava, a cyanogenic plant, as a staple in the
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diet. Patients with these neuropathies have elevated plasma levels of
thiocyanate and vitamin B12. Other factors may contribute to the symp-
toms. For instance, a deficiency of sulfur in the diet may decrease the
cyanide-to-thiocyanate detoxification process and, thus, increase the
severity of the chronic symptoms.
Cyanide and thiocyanate may also be involved in endemic goiter and
cretinism (Section 6.3.3.2). Again, the relationship occurs in tropical
areas where cassava is a food staple. Thiocyanate, produced from detox-
ification of cyanide, has antithyroid activity and could be a factor,
along with iodine deficiency, in goiter.
1.5.2.4 Carcinogenesis, Teratogenesis, and Mutagenesis — There is a pau-
city of information concerning the carcinogenic, teratogenic, and mutagenic
properties of cyanide, and the information that is available warrants
further investigation.
g-Aminopropionitrile (BAPN) is teratogenic, but this behavior is
apparently a function of the whole molecule. Cleft palate was produced
in 98% of the offspring of rats fed 270 mg BAPN on day 15 of the gesta-
tion period.
1.6 BIOLOGICAL ASPECTS IN WILD AND DOMESTIC ANIMALS
Since cyanide is a general respiratory poison acting by inhibition
of cytochrome oxidase, acute exposure of aerobic organisms to cyanide may
result in a histotoxic anoxia. Uptake, absorption, transport, distribu-
tion, and acute toxic effects in mammals are similar to that described
for human and experimental animals in Section 1.5. Acute poisoning may
occur when animals graze on cyanogenic plants such as sorghum and Sudan
grass. Chronic cyanide poisoning also has been reported in grazing ani-
mals. Grazing on cyanogenic plants has been reported to induce sulfur
deficiency in sheep, presumably because the sulfur is used to detoxify
the released cyanide. In the rainy season, sheep feeding on the cyano-
genic, low iodine plant Cynodon pleetostaohyum exhibit hypothyroidism.
In this latter case, the hypothyroidism may be due to low dietary iodine,
release of cyanide, or a combination of both factors.
Fish are the other major group of animals for which some cyanide
data are .available. Data on uptake, absorption, and excretion were few.
Absorption probably occurs through gills and the gastrointestinal tract.
Toxicity data suggest that absorption is not a limiting factor. In one
field study, cyanide concentrations of 0.05 to 0.1 ppm were lethal to most
game fish. Toxicity varies with species, physiological conditions, and
with water pH, temperature, and content of oxygen and other solutes. Tox-
icity of cyanide is reduced by complexation with some cations such as iron
and nickel. Generally, lowered oxygen concentrations in water or an in-
crease in temperature increases the toxicity of a given concentration of
cyanide. There is surprisingly little information on the interactions of
cyanide with birds.
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1.7 CONCLUSIONS
1. The major cyanide compounds of environmental interest are hydrogen
cyanide, cyanide salts, and nitriles.
2. Cyanide forms stable complexes with most of the transition metals;
however, cyanide can be released from some iron complexes with ultra-
violet light.
3. Absorption spectrophotometry and volumetric titrimetry are two use-
ful techniques for cyanide detection.
4. No exact numbers are available for current cyanide production in
the United States, but production for 1976 was estimated to be about
700 million pounds. Most of this amount (52%) was used for acrylo-
nitrile production.
5. Wastes generated from the metal plating and finishing industry con-
tain high concentrations of cyanide; however, cyanide removal from
these wastes is accomplished mainly by alkaline chlorination, elec-
trolytic decomposition, or ozone oxidation.
6. Improper storage, handling, and disposal account for most instances
of environmental cyanide release.
7. Tobacco smoke is probably one of the major sources of cyanide expo-
sure to the general public.
8. Little is known about the distribution and transformation of cya-
nides in air, land, or water. Cyanides are not usually found in
air and are not retained in soils. The recommended maximum concen-
tration of cyanide in water (10 ppb) is seldom found to be exceeded.
9. Cyanide is a relatively reactive compound that is not found to accu-
mulate in the environment. Various microbes are able to degrade
cyanide to carbon dioxide and ammonia. Most other organisms can
also metabolize or detoxify cyanide. Cyanide probably does not
bioaccumulate nor biomagnify in food chains.
10. Although some bacteria, fungi, and a few higher plants are able to
synthesize cyanide, the impact of this release on the environment
is not known.
11. Cyanogenic glycosides, which occur in a variety of food and crop
plants, may release cyanide only during destruction of the plant
(i.e., crushing, bruising, or ingestion). This release is not known
to be a source of lethal quantities of cyanide to humans, but in
tropical areas where these plants form a major dietary staple,
chronic cyanide exposure has been proposed.
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10
12. Inhibition of aerobic respiration is the best-studied effect of
cyanide on aerobic organisms. The lethal dose varies with species.
In humans, death results from depression of the central nervous
system, the tissue most sensitive to anoxia.
13. Uptake of cyanide by animals occurs through inhalation, ingestion,
or skin absorption. Absorption readily occurs and cyanide is
rapidly distributed by the blood system to other body tissues.
14. Some cyanide reacts with methemoglobin to form cyanmethemoglobin.
The major mammalian detoxification mechanism is attributed to the
rhodanese-catalyzed reaction of cyanide with a sulfur donor to pro-
duce thiocyanate, which is then excreted in the urine. Some alter-
native detoxification pathways also exist.
15. Accidental acute cyanide poisoning occurs but is uncommon in humans.
16. Chronic human effects are not well recognized and they have been
implicated in the tropics. Diseases such as amblyopia, retrobulbar
neuritis, Leber's optic atrophy, and various neuropathies occur in
tropical areas where cyanogenic plants (e.g., cassava) form a large
portion of the diet. Endemic goiter and cretinism may be partially
related, in some cases, to chronic cyanide exposure.
17. 3-Aminopropionitrile apparently is teratogenic in some mammals.
18. Little information exists on the metabolism and effects of cyanide
on other groups of organisms (birds, amphibians, reptiles, and
invertebrates).
19. Fish are sensitive to cyanide; sensitivity varies with species,
physiological condition, pH, temperature, oxygen level, and occur-
rence of other compounds in the water. Cyanide concentrations
greater than 0.1 ppm are lethal to many species.
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SECTION 2
CHEMICAL AND PHYSICAL PROPERTIES AND ANALYSIS
2.1 SUMMARY
Cyanides comprise a group of organic and inorganic compounds con-
taining the CN radical. Many of these compounds are extremely useful in
industry. The widespread usefulness of cyanides stems from the tendency
of these compounds to form strong complexes with most metals — particu-
larly those of the transition series. This characteristic accounts for
the use of cyanides in metallurgy, electroplating, and metal-cleaning
operations.
The complex-forming tendency of cyanides is also responsible for
their toxicity; cyanide ions form stable complexes with various metals
in enzyme systems, interfering with their normal operation. In particu-
lar, the cyanide ion reacts with the trivalent iron present in mitochon-
drial ferricytochrome oxidase, preventing further oxidation and reduction
reactions and causing a histotoxic anoxia. Detoxification of cyanide-
complexed ferricytochrome oxidase requires either (1) reversal of the
complexing reaction by the introduction of a competing chemical species,
such as methemoglobin which contains trivalent iron, or (2) introduction
of a compound which reacts with cyanide to form a less toxic product. An
exogenous sulfur donor, sodium thiosulfate, is usually used to convert
cyanide to a less poisonous species. The resulting thiocyanate is con-
siderably less toxic than the cyanide ion. Toxicity of the various organic
cyanides sometimes depends on whether cyanide ions are formed during
their metabolism. In general, the lower aliphatic nitriles are more toxic
than the purely aromatic nitriles. Solutions of ferrocyanides and ferri-
cyanides have relatively low toxicity unless they are exposed to sunlight
or ultraviolet radiation, in which case hydrogen cyanide and cyanide ions
are formed. The nitroferricyanides are believed to dissociate in vivo
to form cyanide ions and therefore may be toxic. Cyanogenic glycosides
are natural compounds produced in a variety of plants. Although presumed
to be relatively harmless in the pure state, these substances are capable
of producing quantities of hydrogen cyanide when hydrolyzed.
Many chemical treatments for removal of cyanides from industrial
wastewaters have been suggested; however, only three processes are suffi-
ciently versatile and economical for widespread use. Alkaline chlorina-
tion, probably the most frequently used method, is adaptable to both large-
and small-scale use. Electrolytic decomposition is effective for wastes
containing high concentrations of cyanide but is not useful for processing
dilute solutions. Ozone oxidation may be an economical alternative to
alkaline chlorination for selected cyanide wastes, but it is not effec-
tive for all cyanide complexes.
A variety of good analytical methods is available for determining
cyanide in environmental samples. Absorption spectrophotometry and vol-
umetric titrimetry are the most widely used techniques, mainly because of
11
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12
their inherent methodical and technical simplicity and low cost. Ion-
selective electrodes allow direct and sensitive measurements of cyanide
in selected samples. Indirect atomic absorption spectrophotometric meth-
ods are useful for samples containing cyanide in the parts per million
range. Fluorometry and gas chromatography can be sensitive methods for
analyzing cyanide. The latter technique also can distinguish speciated
cyanides.
2.2 PHYSICAL AND CHEMICAL PROPERTIES
Cyanides are compounds containing a characteristic group, the cya-
nide radical (CN). Many of these compounds have separate names which
designate distinctive features of the cyanide group. The most important
classifications are hydrogen cyanide, cyanogen, cyanate, isocyanate, thi-
ocyanate, nitrile, isocyanides, cyanohydrin, cyanogenic compounds, and
complex cyanide compounds. Pertinent physical and chemical properties
of these groups are discussed in the following sections.
2.2.1 Hydrogen Cyanide and Salts
Hydrogen cyanide, a widely used industrial chemical known also as
hydrocyanic acid, prussic acid, and HCN, was first prepared by Scheele
in 1782.
2.2.1.1 Physical Properties — Hydrogen cyanide is a colorless, flamma-
ble liquid or gas which boils at 25.7°C and freezes at -13.2°C. It is
miscible with water and alcohol but is only slightly soluble in ether
(Stecher, 1968). The penetrating odor of hydrogen cyanide is reminiscent
of bitter almonds. The gas is lighter than air (0.947 at 31°C, air = 1)
and therefore rises and diffuses rapidly. Hydrogen cyanide polymerizes
spontaneously and sometimes violently when it is not absolutely pure
(Faith, Keyes, and Clark, 1965, p. 456). A small quantity (0.1% to 0.5%)
of sulfuric or phosphoric acid is usually added to hydrogen cyanide to
inhibit this reaction (Montgomery, 1965, p. 583). Other physical prop-
erties of hydrogen cyanide are listed in Table 2.1. The chief physical
characteristics of sodium cyanide and potassium cyanide, the two most
important salts of hydrogen cyanide, are given in Tables 2.2 and 2.3,
respectively.
2.2.1.2 Chemical Properties — Hydrogen cyanide is a weak acid; in solu-
tions at ordinary pH values, the molecular form, HCN, predominates. At
25°C the pH of HCN is 9.2 (Montgomery and Stiff, 1971). Thus, hydrogen
cyanide is readily liberated from solutions of its salts when the solu-
tions are treated with strong mineral acids. This behavior sometimes
causes inadvertent gassings in chemical laboratories when waste cyanide
and acid solutions are flushed through a common drain (Poison and
Tattersall, 1969, p. 130). Hydrogen cyanide is effectively absorbed by
bases such as alkali hydroxides and soda lime.
The widespread usefulness of hydrogen cyanide is related to the
strong tendency of this compound and its salts to form complexes with
many metals. For example, sodium cyanide is used in metallurgy for the
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13
TABLE 2.1. PHYSICAL PROPERTIES OF HYDROGEN CYANIDE
Melting point
Boiling point
-13.24°C
25.70°C
0°C
10 °C
20 °C
1 ft
Specific gravity, aqueous solution, d18
10.04% HCN
20.29% HCN
60.23% HCN
Vapor pressure
-29.5°C
0°C
27.2°C
Vapor density at 31 °C (air = 1)
Surface tension at 20 °C
Viscosity at 20.2°C
Specific heat
-33.1°C
16.9°C
Heat of fusion at -14 °C
Heat of formation
gas
liquid at 18 °C, 1 a tin
Heat of combustion
Critical temperature
Critical density
Critical pressure
Dielectric constant
0°C
20°C
Dipole moment, gas, at 3 to 15 °C
Dissociation constant, K\Q
Conductivity at 0°C
Heat of vaporization
Heat of polymerization
0.7150
0.7017
0.6884
0.9838
0.9578
0.829
50.24 mm Hg
264.39 mm Hg
807.23 mm Hg
0.947
19.68 dynes /cm
0.2014 cP
13.95 cal/mole
16.94 cal/mole
1.72 kcal/mole
-30.7 kcal/mole
-24.0 kcal/mole
159.4 kcal/mole
183. 5 °C
0.195 g/ml
55 kg/cm2
158.1
114.9
2.1 x 10~8 esu
7.2 x 10-10
3.3 x 10-6 JT1 cm"1
6027 cal/mole
10.2 kcal/mole
Source: Adapted from Montgomery, 1965, Table 1, p. 575.
Reprinted by permission of the publisher.
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14
TABLE 2.2. PHYSICAL PROPERTIES OF SODIUM CYANIDE
Melting point
100%
98%
Boiling point (extrapolated)
Density
cubic
orthorhombic
molten, at 700°C
Vapor pressure
800 °C
900 °C
1000°C
1100°C
1200°C
1300°C
1360°C
Heat capacity, 25 to 72°C
Heat of fusion
Heat of vaporization
Heat of formation, AH^, NaCN (c)
Heat of solution, AH - in 200 g-mole
Hydrolysis constant, K. , 25°C
563.7 (+1)°C
560 °C
1500°C
1.60 g/cm3
1.62 to 1.624 g/cm3
1.22 g/cm3 (approx)
0.76 mm Hg
3.34 mm Hg
12.4 mm Hg
36 mm Hg
90 mm Hg
204 mm Hg
314 mm Hg
0.33 cal/(g)(C)
75.0 cal/g
761 cal/g
-21.46 kcal/g-mole
+0.36 kcal/g-mole
2.51 x 10~5
Source: Adapted from Mooney and Quin, 1965, Table 1, p. 586.
Reprinted by permission of the publisher.
extraction of gold from ores and in electroplating baths because it forms
stable soluble complexes of the type Au(CN)2~:
SNaCN + 4Au + 02 + 2H20 = 4NaAu(CN)2 + 4NaOH.
Similar behavior makes alkali cyanide solutions excellent for cleaning
silverware and other precious metals and is responsible for their general
use in industry as metal cleaners (Arena, 1974, p. 210). The complexing
nature of cyanides is also utilized in chemical synthesis and photography.
Other aspects of the chemistry of cyanides have been reviewed in detail
by Williams (1948).
2.2.1.3 Synthesis and Occurrence — Large quantities of hydrogen cyanide
are synthesized in the United States (Section 7.2); Faith, Keyes, and Clark
(1965, p. 455) estimated the production in 1963 at 136 million kilograms
(300 million pounds). Although it can be manufactured from a variety of
starting materials (e.g., coke-oven gas, calcium cyanide, or formamide),
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15
TABLE 2.3. PHYSICAL PROPERTIES OF
POTASSIUM CYANIDE
Melting point
100%
96.05%
634. 5°C
622 °C
Density
cubic at 20 °C 1.553 g/cm3
cubic at 25 °C 1.56 g/cm3
orthorhombic at -60 °C 1.62 g/cm3
Specific heat, 25 to 72 °C 0.24 cal/(g)(C)
Heat of fusion 3.5 kcal/g-mole
Heat of formation, AHi -26.90 kcal/g-mole
Heat of solution, AH +2.8 kcal/g-mole
Hydrolysis constant, 25 °C 2.54 10~5
Solubility in water at 25 °C 71.6 g per 100 g H20
Source: Adapted from Mooney and Quin, 1965,
Table 3, p. 597. Reprinted by permission of the
publisher.
in most modern plants hydrogen cyanide is synthesized by passing ammonia,
air, and natural gas over a platinum catalyst at an elevated temperature:
2NH3 + 302 + 2 CH* -> 2HCN + 6H20.
The product is then concentrated by distillation, stabilized to prevent
polymerization, and stored in cylinders (Faith, Keyes, and Clark, 1965,
p. 454).
Free hydrogen cyanide occurs only rarely in nature because of the
high reactivity of the molecule. However, the gas may sometimes be found
in the atmosphere as a result of manufacturing operations, the incomplete
combustion of nitrogen-containing materials (Poison and Tattersall, 1969,
p. 130), or more often, the fumigation of ships, warehouses, or agricul-
tural areas. Usually, the concentration of cyanide used for fumigation
is <1%. Cylinders of liquid hydrogen cyanide are the most economical
sources of gas for such work; however, for convenience, various absorp-
tion preparations are sometimes used [e.g., Zyklon (liquid hydrogen cya-
nide absorbed in fuller's earth), HCN discoids (liquid hydrogen cyanide
absorbed in wood fibre discs), and Saftifume briquets (a mixture which
produces hydrogen cyanide and cyanogen chloride from sodium cyanide and
sodium chlorate mixed with sand)] (Jacobs, 1967, p. 723).
Hydrogen cyanide and its salts enter industrial waste streams from
ore extracting and mining processes, synthetics manufacturing, coal-
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16
coking furnaces, photographic processing, and operations involving the
electroplating of zinc, cadmium, and precious metals. The electroplat-
ing industry is considered a major source of cyanide waste (Watson, 1973).
The cyanide content of natural waters, however, is usually low because
of the reactivity of cyanide and the waste treatment procedures (Section
7.4.2).
2.2.1.4 Chemical Treatment of Cyanide Wastes — Many chemical treatments
for removal of cyanide from industrial wastewaters have been suggested;
however, only a few of these processes are sufficiently versatile and
economical for widespread use. The current, most-used methods appear to
be alkaline chlorination, electrolytic decomposition, and ozone oxidation
(Watson, 1973, p. 147) (Section 7.5).
Alkaline chlorination involves the treatment of the waste cyanide
solution with alkali and chlorine gas at room temperature. In most in-
stances, the cyanide radical is completely disrupted and the carbon frag-
ment is converted to carbonate and the nitrogen to nitrogen gas:
2NaCN + 5C12 + 12NaOH -> N2 + 2Na2C03 + lONaCl + 6H20.
Special equipment is required for the safe addition of chlorine, and
some form of agitation is needed to obtain adequate mixing and reaction
rates. The overall reaction is slow, usually requiring hours for comple-
tion, especially if the solution contains appreciable quantities of heavy
metals. The process is better suited to the large-scale rather than the
small-scale user because of the need for instrumentation to control the
addition of reagents and the quality of the effluent.
Small-scale users can eliminate the problems of handling and meter-
ing' chlorine gas by using solid hypochlorites such as sodium hypochlorite
(NaOCl), calcium hypochlorite [Ca(OCl)2], or bleaching powder (CaOCl2).
These compounds destroy cyanide without the addition of alkali:
2NaCN + SNaOCl +H20->N2+ NaHC03 + SNaCl,
4NaCN + 5Ca(OCl)2 + 2H20 •* 2N2 + 2Ca(HC03)2 + 3CaCl2 + 4NaCl,
2NaCN + 5CaOCl2 + H20 -> N2 + Ca(HC03)2 + 4CaCl2 + 2NaCl.
The process is relatively simple; although agitation is still required,
it is only necessary to add the hypochlorite as a solution or as a solid
to the wastewater. The reaction is more rapid with hypochlorites than
with chlorine gas. However, the cost of the hypochlorite process is
about twice that of the chlorine treatment (Watson, 1973, p. 158).
Electrolytic decomposition is frequently used to process waste con-
taining high concentrations of cyanide. Usually, the waste solution is
electrolyzed for 10 to 20 days at about 94°C. At first, cyanide is com-
pletely converted to carbon dioxide and ammonia; later, as the solution
becomes more dilute, the reaction fails to go to completion and cyanate
accumulates in the electrolyte. The cyanate is usually decomposed by an
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17
alternate process. The cyanide concentration of the waste solution after
electrolysis usually varies from 0.1 to 0.4 ing/liter (Watson, 1973, p.
160). Obviously, this method is unsuitable for use with wastes contain-
ing low concentrations of cyanide.
Ozone may be used to replace chlorine in the treatment of some cya-
nide wastes. Solutions containing zinc, nickel, and copper cyanide com-
plexes are readily processed by this reagent. The cobalt complex, however,
is resistant to this treatment (Kandzas and Mokina, 1969). Ozonation was
used at the Boeing Aircraft metal-working plant in Wichita, Kansas, to
detoxify a 500-gpm stream containing 25 mg/liter cyanide. Complete oxi-
dation of cyanide to cyanate was achieved with partial conversion of
cyanate to final end products (Anonymous, 1958). Ozone also has been
recommended for use in recovering or destroying complex cyanides present
in commercial photofinishing waste solutions (Hendrickson and Daignault,
1973).
2.2.1.5 Chemical Basis for Toxicity — Cyanides are among the most rapid-
acting poisons known (Goodman and Gilman, 1970, p. 934). The toxicity
of these compounds is due to their dissociation into cyanide which com-
plexes with the metals present in various enzymes, inhibiting their cata-
lytic activity (Fassett, 1963, p. 1993) (Section 6.3.1). The specific
reactions depend on unspecified physiological conditions and are not well
characterized; however, the tendency of cyanide ions to form complex ani-
ons with transition metals, as shown by the following equations, is well
established (Durrant and Durrant, 1962, p. 582). The indicated reactions
may generally be considered simplified examples of more complex cellular
chemistry:
NaCN -v Na+ + CN~,
6CN" + Fe2 + ->- [Fe(CN)6]A-,
6CN" + Fe3 + •* [F6(CN)6]3~,
6CN" + Mn3+ + [Mn(CN)6]3",
6CN~ + Co3+ -> [Co(CN)6]3~.
The reaction which is most sensitive to cyanide and which is believed to
be predominantly responsible for toxicity involves the complexing of tri-
valent iron contained in mitochondrial cytochrome oxidase. This complex-
ing prevents further oxidation and reduction reactions in the normal
electron transport system and causes a histotoxic anoxia. Death from
cyanide exposure is not due to inhibition of oxygen transport but to the
inhibition of cytochrome oxidase and therefore, the failure of tissue
utilization of oxygen, especially at the central nervous system (Section
6.3.1) (Arena, 1974, pp. 135-211).
2.2.1.6 Chemistry of Detoxification — In principle, it should be possi-
ble to reduce the toxicity of ingested cyanide by two different tactics:
(1) supplying an alternate source of trivalent iron to compete with the
-------
18
ferri cytochrome oxidase for the available cyanide ion or (2) introducing
a reagent which converts the cyanide ion to a less toxic form. In prac-
tice, both techniques are usually used. Obviously, implementation must
be limited to rapid reactions which do not drastically upset the equilib-
rium of the patient. For example, sodium thiosulfate is the reagent usu-
ally chosen to implement the second technique. With the aid of the
mitochondrial enzyme sulfurtransferase (rhodanese), this reagent reacts
with the cyanide ion to form the relatively harmless thiocyanate ion,
which is readily excreted in the urine (Goldstein, Aronow, and Kalman,
1974, p. 266):
CN~ + S2032~ -> CNS" + S032~.
This enzymatic reaction occurs in vivo since sulfurtransferase is widely
distributed in the tissues. This reaction is very rapid, but is incapa-
ble of handling massive doses of cyanide primarily due to substrate limi-
tation of the sulfur donors (Gleason et al., 1969, p. 75). Additional
discussion of cyanide detoxification in humans is found in Section 6.2.3.
2.2.2 Cyanogen and Cyanogen Halides
Cyanogen, the simplest compound containing the cyanide group, has
the formula, (CN)2 (Table 2.4). It is a colorless, flammable gas which
freezes at -27.9°C and boils at -21.2°C. Its vapor pressure at 20° is
about 5 atm. Cyanogen is soluble in and slowly reacts with water to pro-
TABLE 2.4. PHYSICAL PROPERTIES OF CYANOGEN
Boiling point -21.17°C
Melting point -27.9°C
Critical pressure 59.6 atm
Critical temperature 128.3°C
Density of gas 2.321 g/liter
Density of liquid at boiling point 0.9537 g/ml
Dipole moment 0.38 x 10~18 esu
Heat of combustion 261.7-261.9 kcal/mole
Heat of dissociation 120-130 kcal/mole
Heat of formation (298.1°C, gas) 69.1-73.8 kcal/mole
Heat of vaporization at boiling point 0.5778 kcal/mole
Surface tension at boiling point 21.98 dynes/cm
Trouton's constant 22.94
Source: Adapted from Brotherton and Lynn, 1959, Table 1,
p. 843. Reprinted by permission of the publisher.
-------
19
duce hydrogen cyanide, cyanic acid (HOCN), and other compounds (Kleinberg,
Argersinger, and Griswold, 1960, p. 356). The gas has a latent energy
comparable to acetylene; mixtures of cyanogen and oxygen are explosive.
It is used in the chemical industry as a high-energy fuel (Brotherton
and Lynn, 1959). Cyanogen and its halide derivatives are also used in
organic synthesis, as fumigants and pesticides, and in gold-extraction
processes. It is also encountered as a rocket or missile propellant, in
processes which involve heating of nitrogen-containing organic compounds,
and in the exhaust gases of blast furnaces (Fishbein, 1973).
Cyanogen is extremely reactive; it does not long remain unchanged in
the environment after introduction. Cyanogen and its halide derivatives
are extremely poisonous; their toxicity is comparable to hydrogen cyanide,
probably because of their conversion of the latter in the body (Fishbein,
1973, p. 361):
(CN)2 + H20 = HCN + HOCN,
CNC1 + H20 = HCN + HOC1.
Cyanogen is most conveniently prepared in the laboratory by the addi-
tion of potassium cyanide to copper sulfate:
2CuSOz, + 4KCN -> Cu2(CN)2 + 2^30,, + (CN) 2.
Commercial production is based on a more efficient reaction in which
hydrogen cyanide is oxidized at 300°C to 600°C with air and a silver
catalyst:
4HCN + 02 (air) -> 2(CN)2 + 2H20.
Cyanogen halides are formed when an aqueous solution of hydrogen
cyanide or mercuric cyanide is treated with the free halogen. They are
also obtained by the action of a mixture of bleaching powder and a halide
acid on sodium cyanide (Williams, 1948, p. 6). Cyanogen chloride is a
colorless, volatile liquid which boils at 13.8°C and freezes at -6°C. It
is soluble in water, alcohol, and ether (Stecher, 1968). The vapor is
highly irritating; its toxicity is like that of hydrogen cyanide (Section
6.2.2.1). The properties of the bromide are similar.
The chemistry of cyanogen and its derivatives has been reviewed
extensively by Brotherton and Lynn (1959) and by Williams (1948).
2.2.3 Cyanates and Isocyanates
Inorganic cyanates are compounds containing the radical -OCN; they
are formed when cyanides are treated with mild oxidizing agents (Norbury,
1975):
KCN + 02 (air) -> KOCN.
Crystalline alkali cyanates are colorless or white stable solids, but
aqueous solutions of these compounds readily hydrolyze to ammonia and the
-------
20
corresponding bicarbonate. Some physical properties of these salts are
shown in Table 2.5. Sodium and potassium cyanates, when compared to cya-
nide are relatively nontoxic to humans and animals (Arena, 1974, p. 164).
However, the use of cyanate in the treatment of sickle cell anemia (Cerami,
1974) did induce various toxic lesions such as cataracts (Nicholson et
al., 1976) and polyneuropathy (Peterson et al., 1974). Details of metab-
olism are unknown, but it is presumed that the toxic effect is caused by
the cyanate ion per se and not by breakdown products (Fassett, 1963, p.
2034). Because of their relative instability and low toxicity, inorganic
cyanates probably pose few environmental problems. Alkyl cyanates can be
prepared by the action of sodium alkoxide on a cyanogen halide, but these
esters usually trimerize immediately to form the cyanurate (Sidgwick,
1966, p. 462). Cyanates are encountered chiefly in manufacturing opera-
tions, especially the preparation of organic compounds (Zuzik, 1972).
TABLE 2.5. PHYSICAL PROPERTIES OF SOME CYANATES
r, ^ i Melting point
Cyanate Formula .„ .
( w
Ammonium NH^OCN Decomposes, 60
Lead PbOCN Decomposes
Potassium KOCN 315
Silver AgOCN Decomposes
Sodium NaOCN 550
Specific
grav±ty Water
1.34 Very
soluble
Slightly
soluble
2.0 Decomposes
(hot)
4.00 Soluble
(hot)
1.94 Soluble
Solubility
Ether Benzene
Slightly
soluble
Soluble
Slightly Slightly
soluble soluble
Source: Adapted from Zuzik, 1972, pp. 937-940.
Potassium cyanate reacts with dialkyl sulfate to form alkyl isocya-
nates which have the general formula RNCO. These volatile liquids are
very reactive, have a pungent odor, are insoluble in water, but are sol-
uble in acetone, ethyl acetate, toluene, and kerosene. Alkyl isocyanates
are widely used in the production of polyurethane plastics, foams, fibers,
and surface coatings. Two of these compounds, toluene diisocyanate and
diisocyanatodiphenyl methane, are known to cause asthmatic reactions in
sensitized subjects (Morgan and Seaton, 1975). The mechanism of inter-
action is unclear, but isocyanates are known to react with free amino
groups in proteins; they should thus be capable of forming antigens
(Fassett, 1963, p. 2033).
2.2.4 Thiocyanates
Compounds containing the radical SCN are known as thiocyanates; they
are formed by treating cyanides with sulfur or sulfur-containing reagents
(Latimer and Hildebrand, 1951, p. 298):
KCN + S •* KSCN.
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21
Thiocyanates are frequently encountered in the environment; they are
widely used in printing and dyeing textiles, photography, analytical
chemistry, insecticides, and in the manufacture of artificial mustard
oil (Stecher, 1968).
2.2.4.1 Physical and Chemical Properties — Thiocyanates are more stable
than cyanates; they form complexes with many elements, usually coordinat-
ing through the nitrogen atom to the first transition series metals and
through the sulfur atom to the metals of the second and third transition
series (Cotton and Wilkinson, 1962, p. 467). The alkali and alkaline
earth thiocyanates are colorless, deliquescent salts which dissolve eas-
ily in water, alcohol, and acetone. Other physical properties of potas-
sium thiocyanate are given in Table 2.6. Dry thiocyanate salts are
decomposed on heating with concentrated sulfuric acid:
2KSCN + eHaSOi, -> K2SO<, + (NHz,)2S04 + 2CO + 2H20 + 6S02,
but hydrogen cyanide is usually formed when acids are added to thiocyanate
solutions, especially under oxidizing conditions (Williams, 1948, p. 258).
Thiocyanate dissociates into cyanide and sulfate when electrolyzed:
KSCN + 30 + 2KOH -+ KCN + KaSO,, + H20.
Potassium thiocyanate reacts with alkyl halides or alkyl hydrogen
sulfate to form liquid esters which have a garliclike odor and varying
degrees of toxicity. Several of these esters are used as insecticides
(Dreisbach, 1971, p. 117).
2.2.4.2 Toxicity of Thiocyanates — Although small quantities of thiocya-
nates are normally present in human cells, presumably through the detox-
ification of dietary cyanides by the enzyme sulfurtransferase (Section
2.2.1.6), larger quantities cause chronic and acute poisoning (Arena,
1974, p. 600). The poisoning mechanism appears to involve the reaction
of thiocyanate with oxyhemoglobin to yield sulfate and cyanide ions by
the lacto-peroxidase-hydrogen peroxide system (Chung and Wood, 1971) and
thiocyanate with sulfite to yield thiosulfate and cyanide ions (Goldstein
and Rieder, 1951). The latter reaction is not the reverse of the sulfur-
transferase (rhodanese) reaction described in Section 2.2.1.6 as it is
attributed to the action of an enzyme, thiocyanate oxidase (Williams,
1959, p. 392).
2.2.4.3 Detoxification of Thiocyanate Wastes — The ready rupture of the
sulfur-carbon bond occurring in reactions described in Section 2.2.4.1
indicates that methods normally used in detoxifying cyanide wastes can
also be used effectively for wastes containing thiocyanate.
2.2.5 Nitriles and Isocyanides
Nitriles are organic cyanides with the general formula RCN. They
can be prepared by many different chemical reactions such as the treat-
ment of (1) potassium cyanide with alkyl sulfates or alkyl halides, (2)
amides with a dehydrating agent, or (3) aldoximes with acetic anhydride.
-------
TABLE 2.6. PHYSICAL PROPERTIES OF SOME IMPORTANT CYANIDES
Compound
Potassium
thiocyanate
Acetonitrile
Ethyl isc cyanide
Lactonitrile
Amygdalin
Potassium
ferricyanide
Potassium
ferrocyanide
Sodium
nitroferricyanide
Color Formula
Colorless KSCN
Colorless CH3CN
C2H5OCN
Colorless CH3CH(OH)CN
C2QH27NOH
Red K3Fe(CN)6
Yellow KitFe(CN)6'3H20
Red Na2(NO)Fe(CN)5-2H20
Formula Melting .,. . Density or
. B Boiling point .
wt point (°c) specific
(g) (°c) gravity
97 173.2 Decomposes 500 1.886 (14°C)
41 -42 81.6 0.783 (25°C)
71 Decomposes 162 0.89 (20 °C)
71 -40 182 0.9877 (20°C)
457 223
329 Decomposes 1.85 (25 °C)
422 -3H20, 70 Decomposes 1.85 (17 °C)
1.72 (20°C)
Solubility
Water
Soluble
Miscible
Insoluble
Miscible
Very soluble
(hot)
Soluble
Soluble
Soluble
Alcohol
Soluble
Miscible
Miscible
Miscible
Slightly
soluble
Insoluble
Insoluble
Soluble
Ether
Miscible
Miscible
Soluble
Insoluble
Insoluble
Source: Compiled from various sources.
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23
One of the most important industrial compounds, acrylonitrile (H2C =
CHCN), is produced by the catalytic addition of hydrogen cyanide to acet-
ylene and by the reaction of ammonia with propylene (National Academy of
Sciences, 1975, p. 214). The simple aliphatic nitriles are liquids; higher
members of this series are crystalline solids. Acrylonitrile, acetoni-
trile, propionitrile, and n-butyronitrile boil at 77.3, 81.6, 97, and
118°C, respectively. Nitriles have limited water solubility but are gen-
erally miscible in all proportions with most of the common organic sol-
vents such as acetone, benzene, methanol, petroleum ether, and toluene.
Other physical properties of important nitriles are given in Tables 2.6
and 2.7- There is a marked tendency for nitriles to polymerize; acrylo-
nitrile polymerizes explosively in the presence of concentrated alkali.
The tendency to polymerize is the basis for the use of acrylonitrile and
adiponitrile in the manufacture of acrylic or nylon fibers and synthetic
rubber. Nearly 550,000 tons of acrylonitrile were manufactured in seven
plants in the United States during 1972 (National Academy of Sciences,
1975, p. 213). The occurrence of synthetic nitriles in the environment
is largely due to losses associated with the production, transportation,
and consumption of acrylonitrile and related compounds. In the United
States, the annual loss of acrylonitrile is estimated to be about 7000
tons (National Academy of Sciences, 1975, p. 222).
TABLE 2.7. PHYSICAL PROPERTIES OF ACRYLONITRILE
Boiling point
Freezing point
Specific gravity at 20°C
Vapor density, air = 1
Viscosity at 24°C
Surface tension at 24°C
Flash point, open cup
Latent heat of vaporization at 25°C
Latent heat of fusion at 25"C
Refractive index at 25°C
Water solubility at 20°C
Solubility of water in
acrylonitrile at 20°C
Solubility in most organic solvents
77.3°C
-83.55°C
0.8060
1.83
0.34 cP
27.3 dynes/cm
0°C
7.8 kcal/mole
36.20 kcal/mole
1.3888
5.35%
3.1%
Miscible
Source: Adapted from National Academy of Sciences,
1975, taken from outline on p. 209. Reprinted by permis-
sion of the publisher.
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24
Isocyanides (formerly called isonitriles or carbylamines ) are organic
compounds which have the general formula RNC (Cahn, 1974, p. 102). The
alkyl isocyanides are colorless liquids which have low molecular weight
and distill without decomposition. The methyl, ethyl, and phenyl deriv-
atives boil at 59, 79, and 166°C, respectively (Durrant and Durrant,
1962, p. 583). They are slightly soluble in water and readily soluble
in alcohol and ether. Isocyanides possess a very disagreeable odor and
are generally considered hazardous, although little research has been
performed with them. Early investigators reported methylisocyanide to
be more toxic than hydrogen cyanide and ethylisocyanide to be eightfold
less so. It is uncertain if the usual antidotes for hydrogen cyanide are
effective in treating poisoning by isonitriles (Fassett, 1963, p. 2031).
Although known since 1868, isocyanides are little used outside the labor-
atory; they are not widely distributed in the environment.
2.2.6 Cyanohydrins
Cyanohydrins are organic compounds having a cyanide and a hydroxyl
radical attached to a common carbon atom; they comprise a special class
of nitriles whose general formula is R2C(OH)CN. Cyanohydrins are pre-
pared by treating carbonyl compounds with hydrogen cyanide or chlorohy-
drins with sodium cyanide (Fieser and Fieser, 1961, p. 418):
R2CO + HCN -> R2C(OH)CN,
R2C(OH)C1 + NaCN -> R2C(OH)CN + NaCl.
The most common Cyanohydrins are lactonitrile, CH3CH(OH)CN; glycoloni-
trile, HOCH2CH; and 2-methyl-lactonitrile, (CH3) 2C(OH)CN. They are all
water-white, highly reactive liquids which are very soluble in water,
acetone, alcohol, and ether. The physical properties of lactonitrile are
given in Table 2.6. Cyanohydrins are used chiefly as chemical intermedi-
ates in the production of pharmaceuticals and synthetic resin; under
environmental conditions they can decompose to hydrogen cyanide or the
cyanide ion. Cyanohydrins can be extremely toxic by ingestion, skin
absorption, and eye contact (Fassett, 1963, p. 2020).
2.2.7 Cyanogenic Glycosides
Many plants are capable of releasing hydrogen cyanide (Conn, 1969) .
These plants do not contain hydrogen cyanide as such but rather contain
cyanogenic glycosides, which have the general formula:
CN
Ri — C — OR3
where Rj = an alkyl or aryl group,
R2 = a hydrogen atom or methyl group,
R3 = usually, D-glucose.
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25
Amygdalin, the best-known representative of this group, is a g-glycoside
of mandelonitrile (Table 2.6). It is found in the seeds and leaves of
many members of the group Rosaceae (Williams, 1959, p. 402). Other im-
portant cyanogenic glycosides are prunasin, many stone fruits, other
members of the Rosaceae group, sambunigrin (black elder leaves), dhurrin
(sorghum), and linamarin and lotaustralin (white clover, lima beans, and
cassava). Many common vegetables also contain cyanogenic glycosides:
maize, millet, field bean, kidney bean, sweet potato, and lettuce (Oke,
1969).
In general, the pure glycoside is harmless; it becomes toxic only
when in vivo conditions permit hydrolysis of the compound to liberate
hydrogen cyanide. Formation of hydrogen cyanide frequently begins when
the plant is crushed and proceeds rapidly to completion in the rumen or
intestines of most animals (Oke, 1969). Further discussion of the cyano-
genic glycosides is found in Section 4.2.3.
2.2.8 Complex Cyanide Compounds
Cyanide forms many complex compounds, especially with the transi-
tion metals, but only those few that are relevant to this discussion are
described below.
2.2.8.1 Ferricyanides — Potassium ferricyanide, K3Fe(CN)6, is a ruby
red, crystalline solid which is slowly soluble in water but only slightly
soluble in alcohol. The aqueous solution is unstable and decomposes
slowly on standing. Other physical properties are listed in Table 2.6.
Potassium ferricyanide is widely used in making blueprints, staining
wood, photography, dyeing wool, tempering iron and steel, electroplating,
and in analytical chemistry (Stecher, 1968); it is frequently present in
waste streams from such operations. The sodium salt, Na3Fe(CN)6, is less
soluble in water but has properties similar to the potassium salt. Ferri-
cyanides have relatively low toxicity because they do not normally liber-
ate cyanide when acidified nor are they believed to be metabolized to
cyanide in vivo (Arena, 1974, p. 211).
2.2.8.2 Ferrocyanides — Potassium ferrocyanide, Ki,Fe(CN)6, is a yellow
efflorescent, crystalline solid which is soluble in water and insoluble
in alcohol (Table 2.6). The aqueous solution decomposes slowly on stand-
ing. It is used in dyeing wool and silk, tempering steel, process engrav-
ing, photography, and in analytical chemistry. The sodium salt has similar
properties and is used in blueprint paper, photography, pigments, dyes,
and metallurgy (Stecher, 1968). Ferrocyanides are often present in waste
streams from the various operations mentioned above. Like ferricyanides,
the ferrocyanides have relatively low toxicity (Arena, 1974, p. 211).
Although neither ferrocyanides nor ferricyanides normally produce
hydrogen cyanide or the cyanide ion, these toxic substances are readily
formed when solutions of ferrocyanides or ferricyanides are treated with
ultraviolet radiation, as from sunlight (Hendrickson and Daignault, 1973,
-------
26
p. 6). The mechanism by which this result occurs was proposed by Lur'e
and Panova (1964, cited in Hendrickson and Daignault, 1973):
4Fe(CN)6it" + 02 + 2H20 -> 4Fe(CN)63~ + 40H",
4Fe(CN)63- + 12H20 ^ 4Fe(OH)3 + 12HCN + 12CKT.
These authors reported that in the presence of sunlight, 75% of the orig-
inal ferrocyanide concentration in a solution was oxidized in five days
and that the ferrocyanide disappeared completely in 10 to 12 days. This
reaction has considerable toxicological significance. In an experiment
performed by the U.S. Air Force Environmental Health Laboratory, Kelly Air
Force Base, a photographic solution containing ferrocyanide killed no fish
in 96 hr without sunlight present but generated over 220 times the LCSO of
free cyanide in 6 hr when it was exposed to sunlight (Hendrickson and
Daignault, 1973, p. 6). Clearly, ferrocyanide and ferricyanide wastes
should not be discharged to streams where exposure to sunlight can occur.
2.2.8.3 Nitroferricyanides — Sodium nitroferricyanide, also known as sodium
nitroprusside or sodium nitroprussiate, has the formula Na2(NO)Fe(CN)5»2H20.
It is a ruby red crystalline solid which is soluble in water but only
slightly soluble in alcohol (Table 2.6). Aqueous solutions slowly decom-
pose on standing. Sodium nitroferricyanide is used chiefly in analytical
chemistry for the detection of organic compounds, S02, and alkali sulfides
(Stecher, 1968). The potassium salt has similar properties.
Nitroferricyanides are used in the treatment of hypertensive disease
(Page et al., 1955) and are believed to decompose in vivo, liberating
cyanide (Section 6.2.2.1).
2.3 ANALYSIS FOR CYANIDES
2.3.1 Sampling and Sample Handling
Cyanides and cyanophoric substances may occur in the environment,
and the principal requirements for handling each of these sample classes
are discussed in the following sections.
2.3.1.1 Cyanides in Air — Hydrogen cyanide and other volatile cyanide-
containing compounds are not normal constituents of air. They occasion-
ally occur, however, as emissions from electrolytic plating plants, fumi-
gation of buildings or ships with hydrogen cyanide, incomplete combustion
of nitrogen-containing substances, or from chemical processing operations
(Katz, 1968, p. 102). There is increasing concern with cyanide in the
air due to its production by catalytic converters (as a pollution con-
trolling device in automobiles) and by home fires due to the increased
plastic content in homes. Samples are usually collected by drawing the
polluted air through a liquid-filled bubbler or impinger-type collector.
Countercurrent scrubbers and spray columns can, also be used. These col-
lectors are normally charged with an alkaline solution of sodium or potas-
sium hydroxide. This solution is then processed as described in Section
2.3.1.2 prior to analysis. Hendrickson (1968) discussed sampling devices
and procedures in detail.
-------
27
2.3.1.2 Cyanides in Water — Cyanides do not normally occur in domestic
wastewater; however, they are frequently important compounds of trade
and industrial waste effluents (Leithe, 1973, p. 102). Cyanides may be
present as free hydrocyanic acid, simple cyanide salts (e.g., sodium or
potassium cyanide), varying decomposable cyanide complexes (e.g., potas-
sium zinc salt), sparingly decomposable potassium ferricyanide, or one
of various organic compounds (Section 2.2).
To minimize loss of the volatile hydrocyanic acid during storage,
aqueous samples should be adjusted to pH 11 or greater by adding sodium
hydroxide. After this pH adjustment, aqueous samples are frequently
treated with a small quantity of lead carbonate and filtered to remove
hydrogen sulfide which may interfere with the subsequent analysis. Vol-
atile fatty acids, if present, are removed by acidifying the sample with
acetic acid from pH 6 to 7 and extracting the aqueous phase with a small
quantity of isooctane, hexane, or chloroform. Multiple extractions and
long contact time should be avoided to minimize the loss of hydrogen cya-
nide (American Public Health Association, American Water Works Associa-
tion, and Water Pollution Control Federation, 1971, p. 400). If oxidative
impurities, such as free chlorine, are present, sufficient ascorbic acid
is added to the sample to reduce them. The cyanide can be separated from
any remaining impurities by treating the sample with sulfuric acid and
distilling the resulting hydrogen cyanide into a receiver containing so-
dium hydroxide (Leithe, 1973, p. 104). Additives such as ethylenediamine-
tetraacetic acid, potassium iodide, or salts of copper(I), mercury, or
magnesium may be used during the distillation to liberate cyanide. Anal-
ysis of the purified sample can now be performed by the analytical method
of choice.
2.3.1.3 Cyanides in Biological Media — Cyanides may be recovered, with
varying degrees of success, from most biologic materials by procedures
similar to that described in Section 2.3.1.2; typically, the fluid or
macerated tissue is warmed with water, lead, and trichloroacetic acid.
The last reagent precipitates proteins and acidifies the sample, releas-
ing hydrogen cyanide. The hydrogen cyanide is collected in a sodium
hydroxide solution for subsequent analysis (Shanahan, 1973).
2.3.2 Methods of Analysis
Cyanide in environmental samples can be determined by a variety of
procedures. Procedures which are currently important or which show prom-
ise of future usefulness are described in this section. The performance
and limitations of each method are emphasized rather than minute opera-
tional details and are summarized in Table 2.8. Because of sensitivity,
precision and accuracy obviously vary not only among different methods but
also among various models of equipment and different operators (Karasek,
1975) . Therefore, the tabulated data should be considered representative
rather than definitive. Performance data cited by developmental labora-
tories usually are obtained under optimized conditions and procedures;
interlaboratory comparisons, when they exist, offer more realistic com-
parisons of these characteristics.
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TABLE 2.8. METHODS FOR DETERMINING CYANIDE
Analytical method
Absorption
spectrophotometry
Volumetric
titrimetry
Ion-selective
electrodes
Indirect atomic
absorption
spectrophotometry
Fluorometry
Gas chromatography
Sources :
a. . , -. .
Important application
Natural and treated
waters, trade and
industrial efflu-
ents , biologic
materials
Natural and treated
waters and trade
and indus trial
effluents where
concentrations
exceed 1 mg/
liter cyanide
Natural and treated
waters , industrial
wastewaters
Industrial effluents
polluted waste-
water
Natural and treated
waters , processed
industrial efflu-
ents , biological
materials
Natural and treated
waters , industrial
effluents , biologic
fluids and solids
, ,
Limit of
detection
0.5 ug in a 15-
ml solution*3
1 to 5 yg/liter^
0.02 mg/literc
0.1 mg/liter^
25 pg/liter^
60 ppbe (iron
complex)
30 ppbe (silver
cyanide)
1 ppb-f
0.2 yg/ml?
25 tig/ml?1
•
Precision ,
, _ . Accuracy
(relative , n
(relative
standard ,
, . . \ error)
deviation)
8.3% (0.06 2% to 7% (1.5
mg/liter)C pg/liter)^
15.1% (0.62 2% to 15% (0.28
mg/liter)c to 0.62 mg/
1.2% (40 liter)c
yg/literr
2% (>1
mg/liter
cyanide)
0% to 5% (0.2 0% to 5% (0.2
ppm cyanide) ppm cyanide)"
2.2% (3 ppm)e
1.5% (2 ppm)e
11% (2.6 ppb)f
2.5% (10 yg/ml/( 2% (7 yg/ml)^
• i-
Interf ering substances
Sulf ides , thiocyanates ,
and fatty acids inter-
fere but are removed
by the sample prepa-
ration procedure.
These are believed to be
removed in the sample
preparation step .
Strongly complexing
cations , sulf ide
None reported
Sulf ide, per sulf ate,
f erricyanide , mercury
(II) , and iron(II)
interfere.
When treated with
chloramine-T, thiocya-
nate yields a peak
coincidental with
cyanogen chloride
which is about 2% or
3% of that from an
equal concentration of
cyanide .
-p
Selectivity
Substances yielding
cyanide when
digested with sul-
furic acid will be
determined .
All cyanide-yielding
substances will be
determined .
All substances which
yield cyanide ions
will be determined.
Both techniques have
good specificity
for cyanide.
Only cyanide ion is
determined.
Cyanide is determined .
00
^Goulden, Afghan, and Brooksbank, 1972
°U.S. Environmental Protection Agency, 1974.
Frant, Ross, and Riseman, 1972.
eDanchik and Boltz, 1970.
JRyan and Holzbecher, 1971.
9Sass et al., 1971.
^Valentour, Aggarwal, and Sunshine, 1974.
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29
2.3.2.1 Absorption Spectrophotometry — This analytical method is based
on the formation of a colored molecular species of cyanide. The amount
of absorbed light is compared with a previously determined calibration
plot and is related to the cyanide concentration in the sample by the
calibration data. Various absorption compounds are used by different
analysts (Humphrey and Hinze, 1971; Leithe, 1973, p. 107; Nomura, 1968;
Scoggins, 1972). Probably, the most frequently used compound is the
blue dye formed by treating cyanide first with chloramine-T, then with
an aqueous pyridine solution of bispyrazolone and 3-methyl-l-phenyl-5-
pyrazolone. The detection limit, effective range, and relative standard
deviation of this method are 0.5 yg in a 15-ml aqueous solution, 1 to 5
yg in a 25-ml aqueous solution, and approximately 2%, respectively. If
the blue dye is concentrated by extraction into a small volume of organic
solvent, greater sensitivity is obtained. When 10 ml of n-butyl alcohol
is used, the sensitivity, effective range, and relative standard devia-
tion is 0.1 yg, 0.2 to 2 yg, and 3.9%, respectively (American Public
Health Association, American Water Works Association, and Water Pollution
Control Federation, 1971, p. 406). A modification of the pyridine-
pyrazolone method by Goulden, Afghan, and Brooksbank (1972) has a detec-
tion limit of 5 yg/liter cyanide. Sulfides, heavy metal ions, fatty
acids, substances that hydrolyze to give cyanide ions, and oxidizing
agents which are likely to destroy cyanide during the distillation step
interfere with the determination of cyanide by this method, but they can
be eliminated or minimized by the treatments described in Section 2.3.1.2.
Absorption spectrophotometric methods are widely used to determine
cyanide in a variety of environmental samples, including natural and
treated waters, trade and industrial effluents, and biological materials
(Goulden, Afghan, and Brooksbank, 1972; Leithe, 1973, p. 107; Shanahan,
1973).
2.3.2.2 Volumetric Titrimetry — If the cyanide content of the alkaline
distillate described in Section 2.3.1.2 is sufficiently large — 1 mg/
liter or more (U.S. Environmental Protection Agency, 1974, p. 40), it can
be determined by volumetric titration with an appropriate reagent. For
example, silver nitrate combines with cyanide according to the equation:
2CN- + AgN03 = [Ag(CN)2]~ + N03 .
One milliliter of 0.01 N silver nitrate is thus equivalent to 0.52 mg
of cyanide ion. The indicator frequently used for this reaction is p-
dimethyl-aminobenzalrhodanine. When the end point of the titration is
reached, excess silver ions react with the indicator to produce a char-
acteristic color change. A blank reagent value is subtracted from the
result (Leithe, 1973, p. 106).
Nickel salts are also suitable titrants:
4CN- + Ni2+ = Ni(CN)42~.
One milliliter of 0.01 M nickel sulfate corresponds to 1.04 mg cyanide.
Murexide, which is usually used as the indicator, changes from blue vio-
let to orange yellow at the end point of the reaction.
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30
Para-dimethylaminobenzalrhodanine, the indicator commonly used for
the silver nitrate titration, is sensitive to about 0.1 mg/liter silver
or to about 0.05 mg cyanide. The minimum detectable concentration of
cyanide by the titration technique thus approaches 0.1 mg/liter if a
500-ml sample is used. The relative standard deviation is about 2% for
distilled samples that contain at least 1 mg/liter cyanide (American
Public Health Association, American Water Works Association, and Water
Pollution Control Federation, 1971, p. 403). Volumetric titrimetry is
commonly used as a supplement to the molecular absorption spectrophoto-
metric method for analysis of cyanide in drinking, surface, and saline
waters and in domestic and industrial wastes when the concentrations in
these samples exceed 1 mg/liter cyanide.
2.3.2.3 Ion-selective Electrodes — Cyanide can be determined potentio-
metrically in certain industrial wastewaters by a specially designed
electrode which contains a membrane of silver sulfide and silver iodide.
When the electrode is immersed in a sample, iodide is released at the
membrane surface in an amount proportional to the cyanide in the sample:
2CN- + Agl -> Ag(CN)2" + I~.
The liberated iodide is sensed by the electrode and determines the elec-
trode potential. Consequently, the electrode response follows the
relationship,
E = E + S log [CN-],
X
where [CN~] = the concentration of the cyanide ion,
S = slope of the electrode response curve.
Only free cyanide ions are detected; molecular hydrogen cyanide and other
forms of complexed cyanide do not activate the electrode. Accordingly,
samples must be adjusted to pH >11 before measurement to ensure the pres-
ence of cyanide as cyanide ion. In addition, strong complexing cations,
such as nickel or copper, must be sequestered with ethylenediaminetetra-
acetic acid to free complexed cyanide prior to measurement. Sample color
and turbidity do not interfere with the use of the cyanide-specific elec-
trode and time-consuming sample distillations are eliminated. This method
is therefore convenient for direct measurements in the field as well as
for automated use in the laboratory. Although the silver sulfide-silver
iodide membrane electrode can detect levels of cyanide as low as 50 ppb
(Riseman, 1972), practical applications are currently limited to about
0.3 ppm (Frant, Ross, and Riseman, 1972).
Lower concentrations of cyanide can be measured by the electrode-
indicator technique. In this method, a small volume of indicator solu-
tion, Ag(CN)2~, is added to a sample in which a silver sulfide membrane
electrode is immersed. Three to five successive additions of a standard
cyanide solution are then made to the sample, with observations of the
electrode potential before and after each addition. A plot of these val-
ues on Gran's plot paper results in a straight line which extrapolates
to the original cyanide concentration of the sample. The practical detec-
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31
tion limit of the method is about 25 ppb. An upper limit, imposed by the
formation of higher silver cyano complexes, is about 260 ppm (Frant, Ross,
and Riseman, 1972). Metal ions which form stable cyanide complexes inter-
fere but may be controlled by the addition of appropriate amounts of di-
sodium ethylenediaminetetraacetate. Sulfide interferes and should be
removed by the addition of a slight excess of lead(II). Other anions
normally present in wastewater do not interfere.
The accuracy of the electrode-indicator technique is excellent; the
relative error for synthetic samples containing 200 to 2000 ppb cyanide
in the presence of several complexing cations was 0% to 5%. (Frant, Ross,
and Riseman, 1972). The method appears attractive for the analysis of
natural waters and industrial wastes (Blaedel et al., 1971). Ion-selective
electrode techniques have been discussed at length by Durst (1969).
2.3.2.4 Indirect Atomic Absorption Spectrometry — Cyanide cannot be
determined directly by atomic absorption spectrometry; however, there
are two general indirect techniques which can be'used. In one technique,
an insoluble metal cyanide compound is formed, and the metal in the pre-
cipitate or the excess metal in the supernatant solution is determined
by atomic absorption spectrometry (Danchik and Boltz, 1970). The cyanide
concentration is then computed from the measured concentration of the
metal and the stoichiometry of the precipitation reaction. In the other
general indirect technique, a stable metal-cyanide complex is formed,
the complex is extracted, and the metal content of the extract is deter-
mined by atomic absorption spectrometry (Danchik and Boltz, 1970; Jungreis,
1969; Manahan and Kunkel, 1973). As before, the concentration of cyanide
is computed from the measured concentration of the metal and the stoi-
chiometry of the reaction.
Danchik and Boltz (1970) used both techniques in developing proce-
dures for determining cyanide. In one instance, they converted the cyanide
initially present in the sample to the dicyano-bis(l,10-phenanthroline)-
iron(II) complex which was extracted in chloroform, dissolved in ethanol,
and aspirated into the air-acetylene flame of an atomic absorption spec-
trophotometer. The absorption of iron is measured at 248.3 ran. The sen-
sitivity of the method is about 60 ppb cyanide and the calibration is
linear up to 5 ppm when ethanol is the solvent. The precision of the
method is good; samples containing 2.85 ppm cyanide are determined with
a relative standard deviation of 2.2%.
An alternative method of Danchik and Boltz (1970) is based on the
precipitation of silver cyanide and the determination of the excess sil-
ver in the supernatant solution. The absorbance is measured in a luminous
acetylene-air flame at 32.81 nm. The sensitivity of the method is good —
about 30 ppb cyanide. The useful range is from 0.3 to 2.5 ppm cyanide.
Samples containing 1.4 ppm cyanide are analyzed with a relative standard
deviation of 1.5%.
Indirect atomic absorption spectrophotometric methods have suffi-
cient sensitivity and precision for use in determining cyanide in many
industrial effluents and other polluted wastewaters. However, they are
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32
not widely used, probably because of the general convenience and adequacy
of competing methods.
2.3.2.5 Fluorometry — Fluorescein fluoresces when exposed to ultravio-
let light, but its reduced form, the leucobase of fluorescein, is inactive
until oxidized. Oxidation of the latter occurs readily in the presence
of cupric and cyanide ions but not with cupric ions alone. These rela-
tionships form the basis of a very sensitive method for determining cya-
nide at the parts per billion level (Ryan and Holzbecher, 1971).
The analytical procedure consists of adding copper chloride, the
leuco-base, and borate buffer to the sample to be analyzed. After the
sample solution is mixed, it is irradiated with light of 488-nm wave-
length; the resulting fluoresence is measured at 514 nm with a spectro-
fluorometer using 1-cm quartz cells. The intensity of the emitted light
is related to the cyanide concentration of the sample by a previously
prepared calibration curve. The detection limit of the method is excel-
lent — less than 1 ppb cyanide; the relative standard deviation for sam-
ples containing 2.6 ppb cyanide is about 11%. Bromide, chloride, chlorate,
fluoride, iodide, nitrate, acetate, thiocyanate, and sulfate ions do not
interfere when present in concentrations up to 500 times the cyanide con-
centration. However, sulfide does interfere and must be removed. Per-
sulfate and ferricyanide interfere strongly at ion to cyanide molar ratios
of 1:1. Cobalt(II), nickel(II), and cadmium do not interfere in 500-fold
excess. A 50-fold excess of zinc or aluminum, 25-fold excess of manga-
nese, and a 5-fold excess of iron(III) also are without interference.
Equivalent amounts of mercury(II) and iron(II) interfere and must be
removed (Ryan and Holzbecher, 1971).
This laboratory-tested procedure has not received extensive testing
under field conditions. However, it appears attractive for determining
low concentrations of cyanide in natural and treated waters, processed
industrial effluents, and biological materials. Other fluorometric meth-
ods for determining cyanide have also been published (Guilbault and Kramer,
1965; Guilbault, Kramer, and Hackley, 1967; McKinney, Lau, and Lott, 1972).
Disadvantages of the fluorometric method reported to Guilbault are the
lack of sensitivity and the lack of linearity in the response.
2.3.2.6 Gas Chromatography — Unlike analytical methods previously de-
scribed, the gas chromatographic technique readily distinguishes various
species of cyanide compounds. In general, hydrogen cyanide and other
cyanide-containing compounds have different characteristic retention times
in the chromatographic column and can be identified by this criterion.
For example, Lo and Hill (1972) used this method to determine cyano com-
pounds and goitrin in rapeseed meal. Sass et al. (1971) measured mix-
tures of various nitrites used by military and law enforcement agencies.
Claeys and Freund (1968) used this technique to determine undissociated
hydrogen cyanide in an aqueous cyanide solution. In their procedure,
molecular hydrogen cyanide was removed from the sample by air sparging,
concentrated in a cold trap, and analyzed in two serially connected col-
umns containing 15% 1,2,3-tris(2-cyanoethyoxy)-propane on 60- to 80-mesh
Chromosorb W/DMCS and 50- to 60-mesh Porapak Q, respectively. The car-
rier gas was nitrogen; a flame ionization detector was used. Samples
-------
33
containing 1 yg/liter hydrogen cyanide are readily analyzed. The cali-
bration curve is linear up to 2000 yg/liter.
Valentour, Aggarwal, and Sunshine (1974) separated cyanide from
blood, urine, gastric contents, and aqueous solutions by microdiffusion,
treated the separated cyanide with chloramine-T, and extracted the result-
ing cyanogen chloride with hexane. The latter is measured with a 1.8-m
(6-ft) long, 0.635-cm (1/4-in.) diameter, stainless steel chromatographic
column packed with 7% Halcomid M-18 on 90- to 110-mesh Anakrom ABS and
equipped with an electron capture detector. The sensitivity and preci-
sion of the method are good; as little as 0.25 yg/ml can be distinguished
from the impurities in the blank and the relative standard deviation for
samples containing 10 yg/ml cyanide is 2.5%.
The gas chromatographic technique is not widely used for determin-
ing cyanide in environmental samples because other methods are more con-
venient and less time consuming for routine samples. However, the inherent
sensitivity and selectivity of the method ensure its application to spe-
cialized samples, especially those requiring differentiation of cyanide
species.
2.3.3 Comparison of Analytical Procedures
At the present time, absorption spectrometry is probably the most
widely used technique for determining cyanide in concentrations of 1 mg/
liter or less (American Public Health Association, American Water Works
Association, and Water Pollution Control Federation, 1971). Of the many
variations of this technique, the Konig reaction, which involves the
pyridine-pyrazolone reagent, is the most extensively used (Boltz, 1973,
p. 218). This well-seasoned method is sensitive to about 0.5 yg cyanide
in a 15-ml sample in its usual form and to 5 yg/liter with recent modifi-
cations (Goulden, Afghan, and Brooksbank, 1972). Its accuracy is adequate
for analysis of cyanide in natural waters as well as treated industrial
effluents. It is also amenable to automation.
Volumetric titration methods are "standard" and widely used in ana-
lyzing water samples and industrial effluents when the cyanide concentra-
tion is greater than 1 mg/liter (American Public Health Association,
American Water Works Association, and Water Pollution Control Federation,
1971). The need for simple equipment and conventional laboratory proce-
dure make this approach attractive in small laboratories.
The relatively new ion-selective electrode determination of cyanide
is less versatile than the previously mentioned techniques, but it is
attractive for selected application because sample preparation is usually
eliminated and analyses are obtained speedily. The silver iodide membrane
electrode is useful in the 10~3 to 10~s M concentration range; above 10~3
M cyanide, the electrode life is shortened by the formation of soluble
silver cyanide complexes (Riseman, 1972). Greater sensitivity can be
achieved with the silver sulfide membrane electrode and the use of KAg(CN)2
electrode indicator solution (Frant, Ross, and Riseman, 1972). Both tech-
niques are amenable to automation, but strongly complexing cations must
be controlled by adequate sample pretreatment.
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34
Indirect atomic absorption spectrophotometric determinations of cya-
nide are based on well-established atomic absorption techniques for meas-
uring such cations as iron(II) and silver (Danchik and Boltz, 1970). The
sensitivity and precision of these methods are adequate for many treated
industrial effluents and other polluted wastewaters. However, indirect
atomic absorption methods are not yet widely used — probably because of
the general convenience and adequacy of the absorption spectrophotometry
and titrimetric techniques.
The determination of cyanide by fluorometry is relatively new and
presently limited in application. Fluorometry is very sensitive — gen-
erally two orders of magnitude more sensitive than colorimetric proce-
dures; analyses at or below 10 ppb are feasible, even in the presence of
foreign ions (McKinney, Lau, and Lott, 1972). Accuracy at this concen-
tration level is good (Ryan and Holzbecher, 1971). Few ions interfere,
but mercury and sulfide are exceptions and must be removed. Nevertheless,
fluorometry appears attractive for the analysis of cyanide in most nat-
ural and treated waters, as well as processed industrial effluents and
wastewater.
The gas chromatographic method of determining cyanide is also sensi-
tive and precise, particularly when it is converted to cyanogen chloride;
in addition, it readily distinguishes speciated forms of cyanide-containing
molecules. These characteristics commend its potential use for the anal-
ysis of cyanide from biological materials, gaseous and liquid effluents,
and wastewaters, but it is little used at present.
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35
SECTION 2
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Determination. Int. J. Environ. Anal. Chem. (Great Britain) 1:159-168.
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39
57. Sass, S., T. L. Fisher, M. J. Jascot, and J. Herban. 1971. Gas-
Liquid Chromatography of Some Irritants at Various Concentrations.
Anal. Chem. 43(3) :462-464.
58. Scoggins, M. W. 1972. Ultraviolet Spectrophotometric Determination
of Cyanide Ion. Anal. Chem. 44(7):1294-1296.
59. Shanahan, R. 1973. The Determination of Sub-microgram Quantities of
Cyanide in Biological Materials. J. Forensic Sci. 18:25-30.
60. Sidgwick, N. V. 1966. The Organic Chemistry of Nitrogen, 3rd ed.
Clarendon Press, Oxford, England. 909 pp.
61. Stecher, P. G. (ed.). 1968. The Merck Index, 8th ed. Merck and
Co., Inc., Rahway, N.J. 1713 pp.
62. U.S. Environmental Protection Agency. 1974. Methods for Chemical
Analysis of Water and Wastes. Report No. EPA-625-/6-74-003,
Washington, D.C. 298 pp.
63. Valentour, J. C., V. Aggarwal, and I. Sunshine. 1974. Sensitive Gas
Chromatographic Determination of Cyanide. Anal. Chem. 46(7):924-925.
64. Watson, M. R. 1973. Pollution Control in Metal Finishing. Noyes
Data Corp., Park Ridge, N.J. pp. 147.
65. Williams, H. E. 1948. Cyanogen Compounds, 2nd ed. Edward Arnold
and Co., London. 443 pp.
66. Williams, R. T. 1959. Detoxication Mechanisms, 2nd ed. John Wiley
and Sons, Inc., New York. 796 pp.
67. Zuzik, J. B. 1972. Nitriles, Cyanates. In: Encyclopedia of Occupa-
tional Health and Safety, Vol. 2, L. Parmeggiani, ed. McGraw-Hill,
New York. pp. 937-940.
-------
SECTION 3
BIOLOGICAL ASPECTS IN MICROORGANISMS
3.1 SUMMARY
Microorganisms play an important role in the environmental conver-
sion and cycling of many organic and inorganic compounds; they also serve
as models for studying other biological systems. Although the details of
the fate of cyanide compounds in the environment are not completely elu-
cidated, microorganisms can degrade cyanide and, thus, may play an impor-
tant role in detoxification of this substance.
Several species of bacteria and fungi can synthesize cyanide. An
even wider variety of bacteria and fungi can degrade cyanide compounds
introduced into the environment by industries or by cyanogenic plants.
No data were found on the metabolism of cyanide by protozoa or yeast, ex-
cept in conjunction with mixed populations. The production of cyanide by
microorganisms has been associated with active growth and/or with autol-
ysis. The importance of microbially produced cyanide in the ecosystem
cannot be assessed at the present time. Unacclimated microbial popula-
tions may be very sensitive to cyanide (0.3 ppm cyanide is toxic to bac-
teria in sludge); however, acclimated populations in activated sludge can
often completely oxidize nitriles to ammonia. Degradation of concentra-
tions as high as 60 ppm CN~ has been reported.
The toxic effects of cyanide include those effects which are observ-
able in whole cells or in populations: (1) alteration of respiration,
(2) morphological changes such as swelling or filamentation, (3) increased
lag periods for growth, (4) reduced biological oxygen demands, and (5)
one case of induced mutation. Of the reported cyanide-induced effects on
physiology, most studies are concerned with respiratory resistance and
sensitivity. Inhibition of amino acid transport, alterations in nucleic
acid metabolism, and inhibition of nitrogen metabolism are biochemical
effects resulting from cyanide intake. The effects of cyanide are quite
diverse among different microbial species. For further discussion of the
effects of cyanide on microorganisms, the reader should consult Knowles'
(1976) review.
3.2 METABOLISM
3.2.1 Uptake
Uptake of cyanide has been observed in several species of bacteria,
fungi, and algae. No data were available on uptake of cyanide by proto-
zoa or yeast. Evidence for uptake usually consists of demonstrating that
cyanide can be metabolized by the microorganism. The carbon and nitro-
gen atoms of cyanide are transferred into a variety of metabolic end-
products, depending on the species (Section 3.2.2.2). Whether cyanide
is altered or converted before uptake into the cell is unknown. For ex-
ample, Skowronski and Strobel (1969) reported that a strain of Bao-illus
40
-------
41
pwnilus, isolated from Fargo clay, decreased the concentration of cyanide
in Trypticase soy yeast broth culture containing 0.1 M KCN (Figure 3.1).
Growth was not observed in this medium. Cyanide was found to be actively
metabolized; 14C02 was produced by the bacteria in the presence of KlitCN.
Disappearance of cyanide coincided with the growth of Bacillus megatevium
in broth culture containing a much lower KCN concentration (1 mM) (Gastric
and Strobel, 1969). Growth in the control medium (minus KCN) appeared at
3 to 4 hr, whereas growth in the test medium was not apparent until 10 to
12 hr. In fungi and an alga, addition of [ li(C] cyanide has led to the pro-
duction of labeled organic compounds, again suggesting that cyanide uptake
occurs (Section 3.2.2.2).
ORNL-DWG 76-15519
< 2
o
.^STERILIZED CONTROL
'"""""
LIVE
VBACTERIA
12
18 24
TIME (hr)
30
36
Figure 3.1. Amount of cyanide remaining in a medium containing
10"x M KCN per milligram of a strain of Bacillus pwnilus as a function
of time. Source: Adapted from Skowronski and Strobel, Figure 2.
Reproduced by permission of the National Research Council of Canada
from the Canadian Journal of Microbiology, Vol. 15, pp. 93-98, 1969.
Knowles (1976) reviewed several other reports indicating microbial
utilization of cyanide for growth. He suggested that cyanide might be
assimilated by conversion to formic acid via formamide, after the action
of cyanide hydratase and formamide hydrolyase. Another pathway could be
similar to that described for cyanogenic organisms:
unit
glycine
HCN
V
ATP(?)
(3-cyanoalanine
asparagine
aspartic acid + NH3
Other possible pathways of cyanide incorporation have been discussed by
Howe (cited in Knowles, 1976).
-------
42
3.2.2 Biotransformation
This discussion of biotransformation is concerned with the production
and degradation of cyanide by microorganisms. The following reactions are
discussed: carbon + nitrogen ^ cyanogenic compound ^ cyanide.
3.2.2.1 Production of Cyanide
3.2.2.1.1 Production by fungi — The basidiomycetes are the best-studied
group of cyanogenic fungi. Losecke (1871) was the first to describe a
cyanogenic fungus — Marasmi-us OTeades. Robbins, Rolnick, and Kavanagh
(1950) detected HCN production by an unidentified fungus (isolated from
white cedar) grown on liquid corn steep medium and associated the HCN pro-
duction with autolysis. Lebeau and Dickson (1953) found that an uniden-
tified psychrophilic basidiomycete which caused snow mold on alfalfa and
other forage plants produced HCN during active growth on the mycelium as
well as during autolysis. Mycelial growth was correlated with the quan-
tity of HCN produced when the organism was grown on synthetic (Richard's
solution plus pectin as the carbon source with various nitrogen sources)
and plant media. The greatest amount of HCN was produced when the fungus
was grown on soybean meal and alfalfa crown tissue (2500 and 700 ppm HCN,
respectively, of the 2-g oven-dried samples).
Table 3.1 lists 31 known cyanide-producing species of fungi. Phot-i-
ota awcea has been studied in detail. Young, fresh, fruiting bodies of
cultured P. aurea did not form HCN unless damaged (Bach, 1956) . Older
mycelia seemed to produce more HCN than young mycelia, and HCN production
was greatest under relatively poor growth conditions. Dead cells of fruit-
ing bodies and of mycelia did not exhibit HCN production. Thus, HCN pro-
duction seemed to be dependent on labile enzyme systems and occurred only
when the cells were grown under adverse conditions. Oxygen seemed to be
required for HCN production. The optimum temperature for the reaction was
about 20°C and the optimum pH for the basal solution was 5.9. Also, urea
production increased as HCN increased. It was not determined whether urea
was a precursor of HCN or was merely produced simultaneously. Bach found
that P. aurea did not differ in its nutritional requirements from those of
noncyanogenic fungi.
Ward and Lebeau (1962) correlated HCN production by an unidentified
pathogenic basidiomycete (snow mold) with autolysis rather than with growth
or with specific substrates. The particular isolate (type B) produced HCN
in infected hosts (grasses and legumes) and in complex and synthetic media.
Lebeau and Hawn (1963) found that the mycelial stage of Marasmius oreades,
the fairy ring fungus, produced HCN when grown on malt-yeast-glucose medium
and when found in lawns. Fruiting bodies also produced HCN. The produc-
tion of cyanide may be important in the pathogenesis of M. oreades toward
its grass host.
Ward (1964) reported that an unidentified sterile basidiomycete grown
on malt extract, yeast extract, and glucose accumulated an unstable cyano-
genic compound in the mycelium which yielded free HCN upon autolysis. Fig-
ure 3.2 shows that a bound form of HCN can be detected much earlier in the
-------
43
growth cycle than free HCN. Tapper and MacDonald (1974) subsequently estab-
lished that the major cyanogen in snow mold extracts were glyoxylic acid
cyanohydrin. Ward and Thorn (1965) have also extracted an unstable cyano-
genic compound from M, oreades; its properties are similar to those of
glyoxylic acid cyanohydrin.
TABLE 3.1. CYANOGENIC FUNGI
Mucoraceae
MUCOT oyanogenes Guyot
PolypoTaceae
Polyporus frondosus Dicks, ex Fries
Polyporus g-iganteus Pers. ex Fries
PolypoTus put-flans Pers. ex Fries
Trametes amygdalea Maire
Agai"Lcaceae
LeuoospOT-L
Cantharellus oarbonarius A. & S. ex Fries
Cl-itocybe alexandri, (Gill.)
Clitocybe stavipes (Pers. ex Fries)
Clitocybe cyathifoxmis (Bull, ex Fries)
Clitocybe geotropa (Bull, ex Fries)
Cl'Ltocybe gigantea (Sow. ex Fries)
Cl-itocybe -infundi-bul-iform-is (Schaeff. ex Fries)
Cli-tocybe nebularis (Batsch ex Fries)
Clitooybe obbata (Fries)
Clitocybe parilis (Fries)
Marasmius alliaceus (Jacq. ex Fries)
Marasmius globulax"Ls Fries
Marasmius hca"io1oTum DC. ex Fries
Marasmius oreades Bolt, ex Fries
Marasm-ius pevfoTans Hoffm. ex Fries
Max>asnrius peTonatus Bolt. ex Fries
Marasmius ramealis Bull, ex Fries
Marasmius votula L. ex Fries
Collyb'ia arborescens Henn.
OmphaliM. gr-iseopallida Desm. ex Fries
Pleurotus porrigens (Pers. ex Fries)
Trioholoma oognatwn (Fries)
Tr-Lcholoma goni-ospeTmwn Bres.
TT-icholoma nudum (Bull, ex Fries)
Ochrospon
Phol-Lota aurea (Matt, ex Fries)
Pholiota aaperata (Pers. ex Fries)
Source: Adapted from Bach, 1956, Table 14,
p. 70. Data collected from several sources.
Reprinted by permission of the publisher.
-------
44
ORNL-DWG 77-(5520R
500
400
300 —
>- 200
(00 —
4 6 7 8 9 10 \\ 12 (3 14
TIME (days)
Figure 3.2. Production of HCN during growth of a snow mold fungus
in shake culture. Free HCN collected by aeration, total HCN by steam
distillation. Source: Adapted from Ward, Figure 1. Reproduced by per-
mission of the National Research Council of Canada from the Canadian
Journal of Botany, Vol. 42, pp. 319-327, 1964.
The mechanism of cyanide production- in these organisms is not known.
Stevens and Strobel (1968) reported that the snow mold converted valine and
isoleucine to HCN via linamarin and lotaustralin (cyanogenic glycosides)
(Section 4.2.3) as intermediates. However, Ward and Thorn (1966) and Ward,
Thorn, and Starratt (1971) have clearly demonstrated that carbon-2 of gly-
cine is the precursor of HCN in this organism. Ward, Thorn, and Starratt
(1971) also provided strong evidence that the HCN supposedly produced from
valine in Stevens' and Strobel's experiments was erroneously identified.
Finally, Tapper and MacDonald (1974) were unable to detect linamarium or
lotaustralin in the fungus as reported by Stevens and Strobel.
3.2.2.1.2 Production by bacteria — The production of cyanide by bacteria
is well known. In early studies, Clawson and Young (1913) reported that
several strains of Bacillus pyocyaneus (now classified as Pseudomonas
aeruginosa) produced HCN when grown aerobically on gelatin, egg, broth,
milk, agar, Dunham's peptone solution, and cotton-seed meal. Baaillus
violaceus (Chromobacterium lividwn) also produced HCN when grown on gela-
tin and egg. Patty (1921) found that oxygen was required for HCN produc-
tion by B. pyocyaneus (P. aeruginosa); that HCN was not produced by a
filtrable extracellular enzyme; that pigment production, gelatin liquefac-
tion, and HCN production were independent but apparently related functions;
that whole egg medium and a synthetic medium (as used in the methyl red
test) supported the best HCN production; that pH optimum for HCN produc-
tion was 5.4 to 5.8; and that the amount of HCN produced varied among
strains. P. aeruginosa can produce cyanide when grown on a synthetic
medium with glycine as the only nitrogen source (Lorck, 1948). After
small amounts of nutrient broth or yeast extract were added to the syn-
thetic glycine medium, HCN production increased. Hydrocyanic acid yield
-------
45
at 26°C was three times greater than at 37°C, and the maximum HCN concen-
tration occurred at 25 hr and declined at 48 hr.
More recent work on cyanide production by Pseudomonas species has
been done on a Pseudomonas isolate by Wissing (1968, 1974, 1975), on P.
aevug-inosa by Gastric (1975), and on P. fluoresaens by Freeman et al.
(1975). A Pseudomonas strain isolated from a water reservoir produced
the greatest amount of cyanide when grown on succinate plus glycine methyl
ester or on glucose plus glycine and D,L-methionine (Wissing, 1968). The
greatest amount of cyanide was produced just before the stationary phase,
after which production rapidly decreased. If an additional carbon source
was added to the cultures, HCN production again increased. The largest
quantities of HCN were produced in Tris/HCl buffer at pH 8.3. From his
studies, Wissing (1974) proposed that glycine (H2N—CH2-COOH) is oxidized
in two steps, first to an imino acid (HN=CH-COOH) and then to HCN and C02.
A linear relationship existed between the amount of glycine added to a cul-
ture and the amount of HCN produced (Figure 3.3). The strain used, desig-
nated as strain C, contained no a type cytochromes but contained b, c3 and
o types. Inhibitor studies suggested that the oxidative enzymes involved
were flavoproteins. When Pseudomonas was treated with the oxidizing agent
phenazine methosulfate before sonication, 8% of the HCN-producing activity
of untreated intact cells was recovered in a cell-free preparation (Wissing,
1975). With the addition of flavinadenine dinucleotide (FAD), 16% of the
activity was recovered. Gradient centrifugation and subsequent electron
microscopy of the three fractions revealed that the main enzyme activity
was associated with cytoplasmic membranes. The effect of FAD on HCN pro-
duction indicates that flavoproteins may be involved in the bacterial con-
version of glycine to HCN.
1000
O
c
Q
LL)
o
o
UJ
cr
o
x
500
ORNL-DWG 76-15523
O
/
O
0 500 1000
GLYCINE ADDED (nmoles)
Figure 3.3. Regression line showing the molar ratio between added
glycine and cyanide recovered when whole bacteria were fed varying
amounts of glycine. Similar dots refer to experiments made with the
same bacterial preparation. Source: Wissing, 1974, Figure 6, p. 1292.
Reprinted by permission of the publisher.
-------
46
Gastric (1975) suggested that HCN is a secondary metabolite of P.
aeruginosa. Strain 9-D2 (isolated from a septic human burn) grown in 2%
peptone medium did not produce cyanide until the growth rate began to de-
cline. Figure 3.4 shows three phases of growth and cyanogenesis: "(1)
lag phase and active growth, no cyanide production, 0-6 h; (2). decelera-
tion of growth, active cyanide production, 6-10 h; (3) limited growth and
cyanogenesis, past 10 h." No further cyanide production was noted with
incubation from 14 to 48 hr. The production of cyanide seemed to depend
on protein synthesis, as shown by an 85% reduction in cyanide production
with the addition of chloramphenicol (25 yg/ml, final concentration) to
a 6-hr culture. Other factors affecting cyanogenesis were temperature,
oxygen, and iron. Maximum cyanide production occurred at temperatures
from 34°C to 37°C. Iron had a stimulatory effect at concentrations greater
than 1 uM. Anaerobic growth was inhibitory. Growth of strain 9-D2 in a
synthetic medium lacking glycine decreased cyanide production by 80% (un-
published data by Gastric, cited in Gastric, 1975). This result is similar
to that found with Chromobacterium violaceum (discussed below). The author
suggested that cyanide may act as a regulator of glycine levels. Of the
other species of Pseudomonas tested, only P. fluoresoens and P. polyoolor
were cyanogenic. Several other bacteria (e.g. , Escherichia col-i, Strep-
tococcus faecal-is, and Bacillus megateri-um') did not produce cyanide under
test conditions.
10.0
ORNL-DWG 76-15524
- 0.300
CYANIDE
PRODUCTION
6 10
TIME (hr)
- 0.200
- 0.100
0.000
o
o
£
o
X
Figure 3.4. Time course of cyanide production in response to growth
of Pseudomonas aeruginosa strain 9-D2 in 2% peptone at 37°C. Source:
Adapted from Gastric, Figure 1. Reproduced by permission of the National
Research Council of Canada from the Canadian Journal of Microbiology,
Vol. 21, pp. 613-618, 1975.
-------
47
Freeman et al. (1975) used a very sensitive gas chromatography—mass
spectrometry method to detect HCN production by P. fluorescens. They
detected HCN production from two isolates grown on Trypticase soy agar
plus 0.5% yeast extract or on sterile chicken medium. A closely related
taxon, P. putida, did not exhibit cyanide biosynthesis, nor did Flavo-
bacterium, Cytophaga, Moraxella, or Aci.netoba.cter isolates.
Growing cultures and nonproliferating cells of Chromobacterium
violaceum can synthesize HCN from the carbon-2 of glycine (Brysck, Corpe,
and Hankes, 1969; Brysk, Lauinger- and Ressler, 1969; Michaels and Corpe,
1965; Michaels, Hankes, and Corpe, 1965). Cells grown in 1% (wt/vol) pep-
tone medium produced the largest amount of HCN during the lag phase of
growth (Figure 3.5) (Michaels and Corpe, 1965). A synergistic effect of
glycine and methionine on cyanide formation by C. violaceum was found
(Michaels and Corpe, 1965; Michaels, Hankes, and Corpe, 1965). The amount
of cyanide produced depended on the concentrations of glycine and methio-
nine in a glutamate-salt medium. A possible mechanism for the formation
of C02 from the carboxyl group of glycine and of cyanide from the a-carbon
is shown in Figure 3.6. However, neither the intermediates nor the enzymes
proposed were demonstrated.
Thus, of the 34 bacterial species surveyed in this document, only
seven species, representing two genera, were shown to be cyanogenic (Table
3.2): P. aerugi.nosa, P. fluorescens., P. chlorovap'his, P. aureofaciens,
C. l-ivi-dim, and C. violacewn.
ORNL-DWG 76-15525
— 9.5
CYANIDE/VIABLE CELL (X10~11)
6 8 10
TIME (hr)
14
16
Figure 3.5. Time-course production of cyanide by Chromobacteriim
violaceum (in 1% peptone medium) in relation to growth. Source:
Adapted from Michaels and Corpe, 1965, Figure 1, p. 109. Reprinted by
permission of the publisher.
-------
48
H
I
H-C-COOH
I
NH,
[o]
-H2O
O
II
» C 1MH
c=o
1
OH
-H20
OH
HCN+
HO-C
I
N
C-OH
II
N
ORNL-DWG 76-15526
COOH
N JST
II I
HOOC XC-COOH
^N'"
Sym-Triazine-
Tricarboxylic
Acid
HCN + CO2
OH
Cyanuric
Acid
Oxalic
Amide
Nitrile
Figure 3.6. Hypothetical pathway for cyanide formation from gly
cine. Source: Michaels, Hankes, and Corpe, 1965, Figure 1, p. 124.
Reprinted by permission of the publisher.
3.2.2.2 Degradation of Cyanide — Microorganisms are essential in the
treatment of cyanide-containing wastes and in the natural purification of
aquatic environments polluted with cyanide or related compounds. For
example, Stolbunov (1971) found that the occurrence of thiocyanate-decom-
posing microorganisms seemed to be associated with points of industrial
and household wastes discharge.
Few data were found on the metabolism of cyanide by algae. The green
alga CKloTella pyrenoidosa can assimilate cyanide (Fowden and Bell, 1965).
Thirty minutes after [ li*C] cyanide was added to the culture, large amounts
of radioactivity occurred in g-cyanoalanine and later (from 120 to 300 min)
in Y~glutamyl-3-cyanoalanine. Cells grown anaerobically assimilated only
negligible amounts of [ lilC] cyanide.
Erickson, Maloney, and Gentile (1970) found that four species of phy-
toplankton, Cyolotella nana, Amph-Ldiniim carteri, Skeletonema eostatum,
and Isoohrysis gaZbana, could not directly metabolize nitrilotriacetic
acid, a chelating agent used as a builder in detergent formulations. Cya-
nide was not significantly toxic to these algae.
Data on the utilization of cyanide by protozoa and yeast were found
only in conjunction with mixed populations.
-------
49
TABLE 3.2. OCCURRENCE OF CYANOGENESIS IN BACTERIA
Organism
HCN
production
Reference
Pseudomonas spp.
P. aeruginosa
P. aloaligenes
P. awreofaciens
P. cepacia
P. cichorii
P. chloroaphis
P. denitrificans
P. dimunata
P. fluorescens
Pseudomonas isolate
P. marginalis
P. ma't tophilia
P. poly color
P. pseudoalcaligenes
P. put-Ida
P. putrifaciens
P. stutzeri
Chromobacterium spp.
C. lividum
C. violaceum
Acine tobacter
Aloaligenes oderans
Alcaligenes sp.
Bacillus megateriurn
Bacillus subtilis
Bordetella bronchiseptica
Cytophaga
Escherichia coli
Flavobacterium sp.
Herellea sp.
Mima sp.
Moraxella sp.
Serratia rnarcescens
Staphylococcus aureus
Streptococcus faecalis
+ Clawson and Young, 1913
+ Patty, 1921
+ Lorck, 1948
+ Castric, 1975
- Michaels and Corpe, 1965
Castric, 1975
+ Michaels and Corpe, 1965
Castric, 1975
Castric, 1975
+ Michaels and Corpe, 1965
Castric, 1975
Castric, 1975
+ Castric, 1975
+ Freeman et al., 1975
Michaels and Corpe, 1965
+ Wissing, 1968, 1974, 1975
Castric, 1975
Castric, 1975
+ Castric, 1975
Castric, 1975
Castric, 1975
Freeman et al., 1975
Castric, 1975
Castric, 1975
+ Clawson and Young, 1913
Michaels and Corpe, 1965
+ Michaels and Corpe, 1965
+ Michaels, Hankes, and Corpe, 1965
+ Brysk, Corpe, and Hankes, 1969
+ Brysk, Lauinger, and Ressler, 1969
+ Ressler et al., 1973
Freeman et al., 1975
Castric, 1975
Castric, 1975
Castric, 1975
Michaels and Corpe, 1965
Castric, 1975
Freeman et al., 1975
Michaels and Corpe, 1965
Castric, 1975
Michaels and Corpe, 1965
Castric, 1975
Freeman et al., 1975
Castric, 1975
Castric, 1975
Castric, 1975
Freeman et al., 1975
Michaels and Corpe, 1965
Michaels and Corpe, 1965
Castric, 1975
-------
50
3.2.2.2.1 Degradation by mixed populations — Mixed microbial populations,
such as those found in activated sludge, may either be extremely sensitive
to cyanide or may be able to degrade cyanide compounds in industrial wastes
and in sewage. As little as 0.3 ppm cyanide can be toxic to bacteria in
activated sludge (Berry, Osgood, and St. John, 1974). The disruption of a
treatment process with cyanide may have more serious effects than the toxic
effect of cyanide alone (Murphy and Nesbitt, 1964). All treatment processes
are disturbed if shock loads of cyanide are added to unacclimated cultures.
Unacclimated aerobic systems will produce poor quality effluents if more
than 1 ppm cyanide is added (Corburn, 1949, and Lockett and Griffiths, 1948,
both cited in Murphy and Nesbitt, 1964). Acclimation to cyanide apparently
occurs when cultures are exposed to low cyanide concentrations. Rheinheimer
(1974) stated that cyanides may kill organisms involved in the reminerali-
zation process in aquatic environments.
Several different additives can decrease the minimum retention time
required for complete oxidation of thiocyanate and cyanide in a continuous
activated sludge process for treating coke-oven wastes (Catchpole and Cooper,
1972). Alanine and p-aminobenzoic acid gave good results but proved uneco-
nomical. Glucose was effective and economical. Pyruvic acid metabolism
was thought to be an important factor in the improved treatment process.
The acclimated complete-mixing activated sludge process (utilizing a feed
of domestic sewage) was capable of degrading cyanide loads as high as 5 mg
cyanide per gram mixed-liquor-volatile solids per hour with 99% efficiency
(Murphy and Nesbitt, 1964). The rate of degradation was adequate for use
with industrial effluents. Under equilibrium conditions of the system,
one-third of the cyanide carbon was used by the sludge microorganisms for
respiration and two-thirds was converted to cellular material. The occur-
rence of ammonia and nitrite in the effluent indicated some biological fail-
ure; the cyanide nitrogen is completely converted to nitrate in a properly
functioning system. The above study was a pilot plant operation of a com-
plete-mixing activated sludge process which proved to be a very successful,
efficient, and economical means of degrading cyanide wastes. Some organisms
isolated from a batch-fed system (600 ppm cyanide) which could rapidly de-
crease cyanide levels included: (1) a gram-positive coccus which grew
slowly and destroyed cyanide in a few days, (2) a gram-positive sporulated
bacterium which grew slightly faster, and (3) a mobile gram-negative Pseu-
domonas strain which grew rapidly (Rayand and Bizzini, 1959, cited in Murphy
and Nesbitt, 1964).
Organic cyanides can also affect bacterial population structure and
function. Ludzack et al. (1958), using Ohio River water from the intake of
the Cincinnati Water Works, tested the effects of six different nitriles on
the oxidation by and ecology of microbial populations. From qualitative
observations (plate counts and stained slides made at irregular intervals),
the variety of organisms was less in the nitrile-treated water than in the
untreated river water, but often the total plate counts were higher in the
treated water. Organisms found in systems treated with different organic
nitriles are shown in Table 3.3. A greater variety of organisms was usu-
ally present in the lactonitrile- and oxydipropionitrile-fed systems. Spe-
cific organisms normally found in the river water were not given. Of the
nitriles studied, oxydipropionitrile was the most resistant to microbial
-------
Nitrile
51
TABLE 3.3. OCCURRENCE OF MICROORGANISMS IN NITRILE-FED OHIO RIVER WATER
TEST OXIDATION SYSTEMS0
Bacteria
Molds
Gram-negative
cocci and
bacilli
Pseudomonas
aerug'inosa
Several
different hyphae
and spores
Protozoa
Trichoderma
Acrylonitrile
Acetonitrile
Adiponitrile
Benzonitrile
Lactonitrile
Oxydipropionitrile
x indicates common occurrence.
Source: Adapted from Ludzack et al., 1958, p. 307.
oxidation, but after acclimation of the organisms it was rapidly consumed.
Other authors have reported that acrylonitrile can be utilized by micro-
organisms. The Dow Chemical Company (cited in National Academy of Sciences,
1975) found that acrylonitrile could be 50% oxidized in ten days and com-
pletely oxidized in 20 days to NH3 by an activated sludge seed. Ryckman,
Rao, and Buzzell (1966) listed acrylonitrile among the compounds which
could be removed from aquatic environments by acclimated microorganisms.
3.2.2.2.2 Degradation by fungi — The fungi Fusarium solani and Stemphyliwn
loti may well be used to eliminate cyanide from industrial waste. F. solani
can degrade cyanide to ammonia and C02 (Shimizu, Taguchi, and Teramoto,
1968; Shimizu and Taguchi, 1969; Shimizu et al., 1970). Shimizu, Taguchi,
and Teramoto (1968) found that the overall cyanide degradation activity was
20 mg cyanide per milligram dry cell weight. The degradation rate decreased
with increased age of the culture and with increased loading of the system.
In further experimentation, the optimum cyanide concentration for maximum
degradation was found to be less tha.n 100 ppm cyanide (Shimizu, Fuketa, and
Taguchi, 1969).
S. loti, a fungal pathogen of the cyanogenic plant bird's-foot trefoil
(Lotus cornieulatus L.)> has a tolerance for HCN (Fry and Millar, 1972).
Tolerant spores (adaptation affected by incubation in 0.1 mM KCN for 2 hr)
or enzyme preparations from these spores convert hydrogen cyanide to form-
amide. The enzyme responsible for this conversion, formamide hydrolyase,
has an optimum pH range of 7 to 9.
can
Pathways of cyanide metabolism have been proposed for the fungi which
utilize and detoxify cyanide. Table 3.4 lists the amounts of radio-
activity incorporated into amino acids by various fungi (Allen and Strobel,
1966). Rhisopus nigfioans, Marasmius oreades, and all species of Pholiota
contained radioactive alanine. Other amino acids were also found to be
radioactive, but to a lesser extent. Fusariwn nivale contained labeled
asparagine. Of the fungi tested, Pholiota auvivella appeared to assimilate
the most HCN.
-------
52
TABLE 3.4. COMPARATIVE ASSIMILATION OF H1LtCN BY VARIOUS FUNGI
Organism
Photiota adi-posa
Pholiota aurivella
Pholiota praecox
Clitocybe illudens
Marasmi,us ofeades
Khizoctonia solani
Fusariwm nivale
Fusarium solani
AspeTgillus flavus
Phoma betae
Rhizopus nigricans
Medium
PD
PD
PD
PD
PD
Syn
Syn
Syn
Syn
Syn
PD
Days of
growth
34
28
12
25
28
10
14
7
8
15
4
Dry wt
(mg)
42
36
70
149
50
95
90
130
93
142
73
Radioactive
amino acid
detected
Alan in e
Alanine
Alanine
None
Alanine
None
Asparagine
None
None
None
Alanine
Specific
activity
(myCi/ymole)
1.76
6.54
5.86
0.00
4.14
0.00
0.43
0.00
0.00
0.00
1.58
Syn — synthetic; PD — potato dextrose.
Source: Adapted from Allen and Strobel, 1966, Table 1. Reproduced by per-
mission of the National Research Council of Canada from the Canadian Journal of
Microbiology, Vol. 12, pp. 414-416.
Rh-izoctonia solani, can take up and metabolize HCN. Mundy, Liu, and
Strobel (1973) proposed a pathway by which R. solani. can condense H1Z*CN
with ammonia and propionaldehyde to form -a-aminobutyronitrile:
H — C'
GIL
HCN
NrL
-c/-
H
ira.
H2N
— C — H
propio-
naldehyde
a-aminobutyro-
nitrile
a-aminobutyric
acid
They detected labeled a-aminobutyronitrile and a-aminobutyric acid after
administering K14CN to drained cultures. The concentration of KlilCN used
was not stated but was said to be nontoxic. When labeled aminobutyric acic
was administered to cultures, labeled C02 was detected in sufficient quan-
tities to indicate its rapid metabolism. Precursor-product relationships
suggested that a-aminobutyronitrile is probably a precursor of a-amino-
butyric acid; enzymatic studies further substantiated this. An unidenti-
fied psychrophilic basidiomycete can combine KCN, succinic semialdehyde,
and ammonia to form 4-amino-4-cyanobutyric acid, which can then be con-
verted to glutamate (Strobel, 1967). Figure 3.7 illustrates how cyanide
could be converted to C02 in a cyclical process.
3.2.2.2.3 Degradation by bacteria — Apparently, a wider variety of bac-
teria can degrade cyanide than can synthesize it. Such degradative orga-
nisms discussed in this section are Corynebacterium, Arthrobaater, Bacillus,
Thiobacillus, Esaheriahia coli, and an Actinomyces isolate.
-------
53
ORNL-DWG 76-15528A
SUCCINIC SEMIALDEHYDE
x--
HCN
4-AMINO-4-CYANOBUTYRIC V-AMINOBUTYRIC ACID
ACID
C02
2H 0 x^
2 y^ GLUTAMIC ACID
NH4
Figure 3.7. A cyclical process for converting HCN to C02 which
involves glutamate, y-aminobutyric acid, succinic semialdehyde, and
4-amino-4-cyanobutyric acid. Source: Adapted from Strobel, 1967,
Figure 6, p. 3268. Reprinted by permission of the publisher.
Ware and Painter (1955) isolated a bacterium from sewage which could
grow on silica gel medium with potassium cyanide as its sole source of
carbon and nitrogen. The organism, which was "provisionally classed" among
the Actinomycetaceae, had gram-positive branching filaments, was aerobic
and autotropic, had aerial hyphae and conidia, and was inhibited in culture
by agar or peptone. It utilized cyanide at concentrations up to 15 ppm,
but the best growth occurred on cyanide concentrations of approximately
4 ppm. Ammonia was produced from the breakdown of cyanide, but the fate
of the cyanide carbon was unknown.
An Arthrobaoter' strain was isolated by Aaslestad (1961, cited in
Murphy and Nesbitt, 1964) from the same pilot plant used by Murphy and
Nesbitt. Cultures of this strain could degrade concentrations of cyanide
up to 60 ppm. Aaslestad hypothesized that cyanide could act as a sole
nitrogen source but not as a sole carbon source for the organism. Ammonia
inhibited a growing culture. The strain could also convert cyanide to
thiocyanate in the presence of sulfur; however, thiocyanate was not thought
to be an intermediate in the normal degradation process.
An unclassified cyanide-resistant bacterial strain from soil polluted
with wastewater from an electroplating plant exhibited three peaks of
cyanide-degrading activity at pH 5.3, 7.0, and 10.0 (Figure 3.8) (Furuki
et al. 1972). At pH 10.0, cyanide degradation was highest in the expo-
nential growth phase; the organism grew well in alkaline media containing
400 to 500 ppm cyanide.
The major pathway for cyanide assimilation in higher plants is the
reaction with cysteine to form $-cyanoalanine (Section 4.2.1). A variant
of this pathway involving serine instead of cysteine is apparently found
-------
54
LJ
O
O
100 r-
90 -
80
70
60
O 50
O
t 40
en
o
Q_
O 30
o
UJ
Q
20
10
0
ORNL-DWG 76-15529
.1
_L
/6 hr
3 hr
_L
3.0 4.0 5.0
6.0 7.0
pH
8.0 9.0 10.0
Figure 3.8. Effect of pH on the degradation of cyanide by a
cyanide-resistant bacterium. Source: Adapted from Furuki et al., 1972,
Figure 11, p. 302. Reprinted by permission of the publisher.
in some bacteria (Knowles, 1976). Brysk, Corpe, and Hankes (1969) and
Brysk, Lauinger, and Ressler (1969) reported that nonproliferating Chromo-
'bacterium wiolaoeum cells incubated with glycine, methionine, and succi-
nate accumulated 3-cyanoalanine (Figure 3.9); these workers postulated
serine as the acceptor of HCN. Young cultures of C. wiolaoewn also accu-
mulated Y~cyano~a~L-aminobutyric acid (Brysk and Ressler, 1970). The
formation of y-cyano-a-L-aminobutyric acid by the organism was greatest
at the end of the lag phase and decreased with increasing age. Ressler
et al. (1973) have described the reactions involved and have purified the
enzyme involved. The only g-cyanoalanine synthetic activity in cell-free
extracts of Bac-illus megater'Lwn was associated with 0-acetylserine sulf-
hydrase (Gastric and Conn, 1971). This enzyme converts Nalz*CN and either
0-acetyl-L-serine or L-cysteine to g-cyanoalanine-^C. L-Serine and cya-
nide produced much less 3-cyanoalanine. Since the authors detected no
cysteine-linked 3-cyanoalanine synthetase in extracts, they suggested that
this enzyme either was lacking in this strain or was labile and easily
inactivated.
Bacteria which are fairly resistant to cyanide are sometimes found
in the environment of cyanogenic plants. A strain of EaQ'illus pwnilus
isolated from Fargo clay cropped in flax (a cyanogenic plant) for 73 years
-------
55
GLYCINE
ORNL-DWG 77-15530A
GLYOXYLATE
I
C., UNIT (FORMALDEHYDE)
N5, N10-METHYLENE
TETRAHYDROFOLATE
HCN
SERINE
r
iS-CYANOALANINE
ASPARAGINE
ASPARTIC ACID IN CELLS
Figure 3.9. Proposed pathway for the incorporation of cyanide by
Chromobacterium violaceum. Source: Adapted from Brysk, Corpe, and
Hankes, 1969, Figure 2, p. 326. Reprinted by permission of the
publisher.
could survive in saturated KCN solutions (Skowronski and Strobel, 1969).
Labeled ^C02 and
15NH,+
were produced from the metabolism of K14C15N by
the organism. Bacteria obtained from the standard medium with 10~ M KCN
were incubated in a modified Dulbecco's medium with 10"1 M KCN and 11 yCi
of K^CN. In the nitrogen experiment, all conditions were the same except
that 2 mg of K15CN instead of Kli
-------
56
ORNL-DWG 76-15531
16-
14-
_ 12-
c
1
>io-
•o
* 8-
0 °
o" 6-
O
+•
J
4-
2-
x "-
/^
TOTAL ag /
' O
14C AS 14C02 /
l
/
/
/
0 I
1
1
/
1
o' TOTAL LLQ
+ I
/ 15N AS 15NH.
o/ ^
o-"**""
-8
-7
-6
-5
-4
-3
-2
-1
CP
18 36
TIME (hr)
54
72
Figure 3.10. Production of
li*
15
*C02 and 15NH3
as a function of time in cells exposed to K14C15N.
by a strain of
Source:
Adapted from Skowronski and Strobel, Figure 3. Reproduced by permission
of the National Research Council of Canada from the Canadian Journal of
Microbiology, Vol. 15, pp. 93-98, 1969.
For rhodanese activity, the optimal ratio of thiosulfate to cyanide was
1.5-2.5:1 for intact cells and 1:1 for purified rhodanese in vitro.
Atkinson has also developed a mutant strain of B. steapothermophi'lus
which is resistant to 10~3 M NaCN and contains five to six times the rho-
danese activity of normal cells. Atkinson, Evans, and Yeo (1975) found
that rhodanese production seemed to be independent of medium composition
and was higher in continuous culture than in batch cultures.
CoTynebacter-ium n-itr-iloph-ilus can assimilate organic nitriles such
as acetonitrile, adiponitrile, butyronitrile, propionitrile, and succino-
nitrile (Mimura, Kawano, and Yamaga, 1970). The organism has been proposed
as a useful addition to activated sludge for treatment of nitrile wastes.
-------
57
3.3 EFFECTS
3.3.1 Growth Effects
The effects of cyanide on the morphology and growth of microorga-
nisms include increased lag times for growth, altered cell morphology,
decreased biological oxygen demand (BOD), and decreased motility. The
alga Saenedesmus quadricauda was very sensitive to cyanide, having a
toxicity threshold of 0.16 ppm cyanide, whereas 20 ppm was toxic to the
alga Mioroeystis aeruginosa (Table 3.5). As previously stated (Section
3.2.2.2.1), sewage organisms can be quite sensitive to low cyanide con-
centrations (0.3 to 1 ppm cyanide). Fifty percent of the BOD of sewage
organisms was inhibited by 15 ppm cyanide (Table 3.5).
3.3.1.1 Protozoa — Table 3.5 shows the effects of cyanide, ferrocyanide,
and thiocyanate on a variety of microorganisms. The protozoan Micvoregma
heterostoma was the most sensitive to cyanide with a toxicity threshold
of 0.04 ppm CN-.
Sodium cyanide is also lethal to several other protozoa. Willard
and Kodras (1967) reported that 50 ppm sodium cyanide caused death or
disintegration of all bovine rumen protozoa in 24 hr (protozoa with no
internal or external cilia movement were considered dead). In the con-
trol sample, approximately 50% of the protozoa were dead at 24 hr. A
diverse population of rumen protozoa was observed: oligotrichs found
were En~tod.i-ni.wn, Ophvyoscolex, Polyplastron, Metadinium, and others;
holotrichs found were Isotrichia and Dasytriaha.
3.3.1.2 Bacteria — Few reports presenting cyanide toxicity data for bac-
teria were found. Eschevichia coli had a toxicity threshold of 0.4 to
0.8 ppm cyanide (Table 3.5). Some bacteria can develop substantial res-
piration in the presence of cyanide (Section 3.3.2.1) (Henry and Nyns,
1975); however, effects on the growth of these cultures are not usually
given. Examples of extreme resistance have been reported.
Two gram-negative rod-shaped bacteria, isolated from the rhizosphere
of the cyanogenic cassava or tapioca plant (Manihot utilissima), could
tolerate up to 50 ppm KCN in soil extract agar (Sadasivam, 1974). Potas-
sium cyanide had little effect on the mean generation time of E. ooli
when D-xylose was used as the carbon source (Ashcroft and Haddock, 1975) .
The generation time was 1.5 hr without cyanide and 2.3 hr in the presence
of 1 mAf KCN. When sodium succinate was used as the carbon source, how-
ever, no growth was detected with concentrations greater than 0.25 mA?
KCN. With 0.15 mAf KCN, the mean generation time increased to 7 hr from
the control value of 2.4 hr.
Using phase-contract microscopy, Ingram, Thurston, and Van Baalen
(1972) observed no morphological changes in cells of the blue-green bac-
terium Agmenellum quadruplicatim exposed to KCN (concentration not given) .
Chloramphenicol and penicillin G induced temporary filamentous forms. A
strain of Bacillus pwnilus grown in 0.1 M KCN did form long filaments after
60 min (Skowronski and Strobel, 1969). These filaments did not revert
back to normal morphology when they were put back into medium with KCN.
The organism could survive in concentrations of 2.5 M KCN.
-------
TABLE 3.5. TOXICITY OF CYANURATES AND CYANIDES TO MICROORGANISMS
Chemical compound
Test organism
Test conditions
a Concentration
(ppm)
Remarks
Ammonium thiocyanate
Potassium cyanide
Potassium ferrocyanide
Sewage organisms FW, LS
MioTooystis aeruginosa FW, LS
(algae)
Sewage organisms FW, LS
Soenedesmus quadricauda SB, FW, LS
(algae)
Escherichia coli SB, FW, LS
(bacteria)
Mieroregma heterostoma SB, FW, LS
(protozoa)
Saenedesmus quadricauda SB, FW, LS
(algae)
Escherichia coli. SB, FW, LS
(bacteria)
Synthetic sewage FW, LS
>5000
50% inhibition of
20 °C
20 Lethal; lab study on algae
culture (90% kill)
15.0 50% inhibition of BOD, 20 °C
0.16 Toxicity threshold, four
days at 24 °C
0.4-0.8 Toxicity threshold, one to
two days at 27 "C
0.04 Toxic threshold, 28 hr ,
27 °C
0.15 Toxic threshold, four
days at 24 °C
1000 No adverse effect, 27 °C
0.75 50% reduction BOD
t-n
00
, SB — static bioassay; FW — freshwater; LS — lab study.
BOD — biological oxygen demand.
Source: Adapted from Becker and Thatcher, 1973, Table J, pp. J.2-J.10. Data collected from several sources.
-------
59
Bowdre and Krieg (1974) proposed that cessation of motility of the
bacterium Spirillim volutans be used as an indicator of toxicants in
industrial effluents. However, the only nitrile compound tested in this
laboratory study, nitrilotriacetic acid (90 ppm, neutralized with KOH),
did not affect the motility of the cells even after 75 min.
Blum, Nolte, and Robertson (1975) tested the effects of cyanoacry-
late compounds on possible resident and/or pathogenic microorganisms of
the body. These compounds have been used as tissue adhesive and hemo-
static agents in medicine and dentistry and may possibly be used in peri-
dontal surgery. Pseudomonas aeruginosa and the fungus Candida albi-cans
were resistant to isobutyl and trifluoro cyanoacrylate compounds, as
determined by disc sensitivity tests. Staphylococcus aureus was the most
sensitive organism tested. Lactobacillus casei was slightly resistant.
The authors suggested that the growth inhibition observed in their plat-
ing assays was due to vapors rather than diffusion of the cyanoacrylates.
3.3.1.3 Fungi — Similarly, few data are available on the effects of cya-
nide on the growth of morphology of fungi. An Aspergillus sp. and Rhizo-
pus nigriaans grown on potato dextrose agar tolerated 100 ppm and 200 ppm
KCN respectively (Sadasivam, 1974). If such organisms could metabolize
cyanide from the root region of cyanogenic plants, toxicity to other neigh-
boring organisms might be nullified. Cyanide as an alleopathic agent has
not been well documented, however.
A 50% reduction in growth of Saccharomyaes ceTevisiae with no effect
on average cell size resulted from additions of n-butyronitrile (4000 ppm),
3-cyanopyridine (1000 ppm), propionitrile (4000 ppm), and m-valeronitrile
(4000 ppm) (Loveless, Spoerl, and Weisman, 1954). Approximately 50% reduc-
tion in growth of E. coli cells with no effect on cell size resulted from
additions of 1000 ppm acrylonitrile, 1000 ppm 3~chloropropionitrile, and
3 ppm sodium cyanide (Loveless, Spoerl, and Weisman, 1954). Compounds
tested by Loveless, Spoerl, and Weisman which inhibited both growth and
division of S. cerevisiae are listed in Table 3.6.
3.3.2 Metabolic Effects
3.3.2.1 Respiration — Data on the metabolic effects of cyanide on micro-
organisms deal primarily either with respiratory sensitivity or with en-
hancement. Examples of both cyanide-insensitive and cyanide-sensitive
respiratory chains are found in fungi, algae, protozoa, and bacteria
(Henry and Nyns, 1975).
3.3.2.1.1 Protozoa — Effects of cyanide on protozoal respiration include
enhancement, resistance, and sensitivity. Various protozoa exhibit a
degree of cyanide-insensitive respiration at some stage during their life
cycles (Henry and Nyns, 1975). Bloodstream forms are apparently insensi-
tive to cyanide. They lack mitochondria, a functional Krebs cycle, and an
electron transport chain. A pleomorphic strain of Trypanosoma lacked
cytochromes and a fully active Krebs cycle, and its respiration was insen-
sitive to cyanide (Hanas, Linden, and Stuart, 1975). Ray and Cross (1972)
proposed a branched electron transport chain in Trypanosoma mega in which
-------
6Q
TABLE 3.6. COMPOUNDS INHIBITING BOTH GROWTH AND
DIVISION OF SACCHAROMYCES CEREVISIAE
Compound
Acrylonitrile
Cyanamide
Cyanoacetic acid
Malononitrile
Sodium cyanide
Concentration in
culture medium
(ppm)
1000
300
4000
40
125
Percent of
control
weight
52
37
45
62
38
Percent of
control
size
170
160
220
150
150
Source: Adapted from Loveless, Spoerl, and Weisman, 1954,
Table l,_p. 640. Reprinted by permission of the publisher.
one branch contained cytochromes c and a~a3 and was cyanide sensitive.
The other branch contained a cyanide-insensitive oxidase identical to
cytochrome o. The cyanide-sensitive oxidase was inhibited by low concen-
trations of carbon monoxide, and the cyanide-insensitive oxidase was inhib-
ited only at high carbon monoxide concentrations.
Calvayrac and Butow (1971) found that increased respiration via a
cyanide-resistant pathway occurred when antimycin A was added to Euglena
graailis, strain Z, growing on lactate. Giant mitochondria were observed
several hours after the addition of antimycin. Electron micrographs showed
altered structure with many cristae in the matrix. Succinic oxidase from
Crithidia fasoiculata mitochondria was inhibited 50% at concentrations of
5 \iM KCN and 99% at 50 yAf KCN (Kusel and Storey, 1973). The organism
appeared to have cytochrome a3 as its sole terminal oxidase. No cyto-
chrome o was detected.
3.3.2.1.2 Bacteria — Bacterial respiratory systems are inhibited by low
concentrations of cyanide (Slater, 1967). Gel'man, Lukoyanova, and
Ostrovskii (1967) have listed the effects of cyanide on many bacterial
respiratory enzymes (Table 3.7). Recent studies on cyanide-resistant
respiratory chains deal with the following bacteria: Azotobacter vine-
landii (Jones and Redfearn, 1967; Jones, 1973), Beneckea natriegens
(Weston, Collins, and Knowles, 1974), Escherichia coli (Ashcroft and
Haddock, 1975; Pudek and Bragg, 1974, 1975), Photolaateriwn phosphoreum
(Yoshikawa and Oishi, 1971), Chromatium strain D (Takamiya and Nishimura,
1974), Rhodopseudomonas capsulata (Melandri, Zannoni, and Melandri, 1973;
Zannoni et al., 1974), Pseudomonas saecharophila (Donawa, Ishaque, and
Aleem, 1971), Bacillus aereus (McFeters, Wilson, and Strobel, 1970), and
Aohr-omobacter strain D (Arima and Oka, 1965).
-------
TABLE 3.7. EFFECT OF CYANIDE ON BACTERIAL RESPIRATORY CHAINS
Bacterium
Concentration
(AO
Enzyme system
Inhibition
Pseudomonas sp.
Pseudomonas aeruginosa
Aerobaoter
aerogenes
Azotobaster vinelandii
Pasteurella tularensis
Staphylooooaus aureus
Myaobaeterium phle-i
Aoetobaoter xylinum
Saraina lutea
Esoherichia eoli
Corynebaoterium
diphtherias PW8SP
Myxoooaaus xanthus cells
Myxococeus xanthus
microcysts
Miaroaoaaus denitrifiaans
Aoetobaater peroxydans
Xanthomonas phaseoli
Proteus vulgaris
10"3 Malic oxidase 50-80
3 x 10"4 Cytochrome 96
c551:nitrite:02
oxidoreductase
10'k Cytochrome c:02 96
oxidoreductase
10"3 Succinic oxidase 95
10~3 Pyruvic oxidase 95
10 Pyruvic oxidase 50
10~3 NADH oxidase 85
2 x lO"4 NADH oxidase 48
10"2 Succinic oxidase 80
10"3 NADH:nitrate reductase 70-90
ID"3 NADH oxidase 100
Succinic oxidase 100
6 x ID'2 NADH oxidase 80
10'3 NADH oxidase 60
3 x 10"3 Succinic oxidase 95
NADH oxidase 55
1Q-3 Malic oxidase 100
10"3 Succinic oxidase 96
7 x 10"3 Succinic oxidase 100
Malic oxidase 100
2.5 x 10"3 Succinic oxidase 50-60
10"3 NADH oxidase 78
10-3 NADH oxidase 29
lO"4 Cytochrome c oxidase 100
10"3 Lactic oxidase 90
5 x 10"3 Succinic oxidase 87
3.3 x 10"3 NADH oxidase 84
Source: Adapted from Gel'man, Lukoyanova, and Ostrovskii, 1967, Table 19,
p. 142. Data collected from several sources. Reprinted by permission of the
publisher.
3.3.2.1.3 Fungi ~ Some of the fungal genera containing cyanide-resistant
respiratory systems are Monilie'l'la, Candida, Neurospora, and Saocharomyces.
Moniliella tomentosa has a branched respiratory chain; one branch is cya-
nide sensitive and contains cytochromes a, b, and o, while the other branch
is cyanide insensitive (Hanssens, D'Hondt, and Verachtert, 1974). A variant
strain of Candida utilis is less copper dependent than the wild type because
of a terminal oxidase which bypasses cytochrome oxidase and cytochrome c.
This alternate pathway is insensitive to cyanide (Downie and Garland, 1973).
A comparison of the respiratory chains of wild-type and poky Neurospora
arassa strains was made by Lambowitz and Slayman (1971). Wild-type cells
were sensitive to cyanide, having a cytochrome chain similar to that of
higher organisms. However, poky cells possessed two alternative oxidase
systems — one similar to that of the wild type and thus cyanide sensitive
and the other insensitive to cyanide concentrations which maximally inhibit
-------
62
the cytochrome chain. Schwab (1973) reported that 02 uptake of mitochon-
dria from copper-depleted N. arassa cells was only slightly sensitive to
1 mAf KCN (02 uptake was 94% of control mitochondria). The insensitivity
was due to a branched respiratory chain; one branch contained a cyanide-
insensitive oxidase perhaps similar to the oxidase system in N. cvassa
described by Lambowitz and Slayman (1971). Resting cells of M. tomentosa
exhibited approximately 60% inhibition of 02 uptake by 10"3 M cyanide,
whereas more aged cells (48-hr-old culture) were even more resistant to
cyanide — 30% inhibition with 10"3 M cyanide (Hanssens, D'Hondt, and
Verachtert, 1974).
Bergquist et al. (1974) described the pleiotropic effect of mutations
occurring at loci affecting isoleucine and valine biosynthesis in N. crassa.
The cytochromes of the mitochondria from mutants were altered, as shown by
reduced 02 uptake, different cyanide sensitivities, different ratios of
cytochrome & to a, and generally lower cytochrome concentrations. Oxygen
uptake of the wild-type strain was inhibited by concentrations of KCN as
low as 0.01 mM, but the wild type did contain a component which was rela-
tively cyanide resistant. Three of the mutant strains tested were more
resistant to cyanide than was the wild type.
Concentrations of 10"5 to 10"3 M KCN inhibited oxygen consumption by
11% to 25% in cultures of the fungus Fusariwn 1-ini (Weiss-Berg and Tamm,
1971). However, if the steroids deoxycorticosterone and digitoxigenin
are added with KCN, oxygen consumption is increased above controls. Pre-
sumably, this increase in 02 uptake is due to hydroxylation of the steroids,
and the function of cyanide is to inhibit cytochrome oxidase.
Von Jagow and Klingenberg (1970) used ferricyanide as an electron
acceptor for localizing two dehydrogenases in Sacchcccomyoes carlsbergens-Ls.
Both enzymes were connected to the cytochrome chain via the ubiquinone pool.
Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and KCN were used as un-
couplers in studying respiration of wild-type and petite strains of S.
oerevisiae. Cyanide (3 mAf) and CCCP (10~6 M) caused degradation of mito-
chondria in yeast (S. aerevisi-ae, wild type) cells.
3.3.2.1.4 Algae — The respiration of most algae apparently is sensitive
to cyanide. Henry and Nyns (1975) listed only two species, Euglena gra-
ailis (also classified as a protozoan) and Nitetla clavata, which are
able to develop a distinct mitochondrial insensitive respiration.
One of the terminal oxidases of the green alga Chlopella vulgaris
was not inhibited by 1 mAf cyanide or 0.1 mM thiocyanate (Sargent and
Taylor, 1972). This enzyme differed from the typical cytochrome oxidase
in that it had about one-fourth the capacity for oxygen uptake and was
resistant to cyanide.
Although CCCP (Section 3.3.2.1.3) apparently acts as an uncoupler,
recent data with Chlorella vulgaris suggest that degradation of CCCP also
can lead to a significant production of HCN (Pistorius et al., 1975).
Additional information is necessary to determine if the uncoupling prop-
erties of CCCP are due in all cases to cyanide production.
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63
3.3.2.2 Nucleic Acid Metabolism — In E. ooli. cyanide blocks the propaga-
tion of the replicating fork of the circular chromosome (Olivera and
Lundquist, 1971). The extremely rapid inhibition is noticeable in less
than one-thousandth of the generation time (Cairns and Denhardt, 1968).
These KCN-inhibited bacteria could initiate DNA synthesis after being
infected with bacteriophage X174. Nazar and Wong (1969) reported that
25 \iM cyanide produced immediate inhibition of RNA synthesis in E. ooli-
strain 1ST" but did not affect DNA synthesis. With a higher concentra-
tion, Olivera and Lundquist (1971) found that 0.5 mM KCN caused an imme-
diate inhibition of thymine incorporation into the DNA of exponentially
growing E. soli- strain 15T~. After a 1-hr lag period, however, incor-
poration resumed and eventually growth occurred (Figure 3.11). Weigel
(1974) found that KCN decreased ATP and deoxynucleoside triphosphate
(dNTPs) concentrations within a few seconds in conjunction with the rapid
decrease in DNA replication. This decrease in ATP and dNTPs is presum-
ably responsible for the decrease in replication (Weigel and Englund, 1975).
The site of cyanide action is apparently complex because both aerobically
and anaerobically grown cells are inhibited. Thus, inhibition is not due
only to inhibition of electron transport but is "due to the ability of this
highly reactive compound to react non-specifically with many proteins and
ORNL-DWG 76-15532
TIME (hr)
Figure 3.11. Inhibition of DNA synthesis by cyanide.
Adapted from Olivera and Lundquist, 1971, Figure 1, p. 266.
by permission of the publisher.
Source:
Reprinted
-------
64
other molecules." In contrast to the data of Olivera and Lundquist (1971),
Weigel and Englund (1975) did not observe a recovery from KCN inhibition.
CTilcmydomonas reirihardi cells treated with the DNA-methylating agent
methyl methanesulfonate (MMS) exhibited a rapid drop in survival when
exposed to 10"3 to 10'2 M KCN (Loppes, 1967). The survival of untreated
cells did not decrease when exposed to these same cyanide concentrations.
If the MMS-treated cells were incubated overnight in buffer prior to cya-
nide addition, the cyanide sensitivity was almost completely lost. The
cyanide posttreatment was thought to inhibit some respiratory processes
involved in repair of DNA damage by MMS so that repair could not proceed.
The only data available for cyanide effects on viruses were concerned
with the formation of an HCN-photoproduct upon ultraviolet irradiation of
tobacco mosaic virus RNA in the presence of HCN (Borazan, 1973). The photo-
product chromatographed similarly to a known uridine-HCN photoproduct. HCN
was weakly bonded to the RNA with or without irradiation and most HCN could
be removed by washing. However, a residual amount always remained complexed.
The author suggested that the bond might involve a trace metal within the
RNA.
Wagner et al. (1950) used KCN as a mutagen to produce biochemical
mutants of Neurospora crassa (conidia treatment). Mutation rates were
less than those obtained by direct ultraviolet irradiation.
Radioprotective effects against x-irradiation in E. coli B have been
produced by malononitrile, methyl malononitrile, benzal malononitrile,
the 4-chloro- and 4-oxy- derivatives of benzal malononitrile, acetonitrile,
propionitrile, butyronitrile, ethyl malononitrile, cyanoacetamide, and
l,l,6,6-tetracyano-2,5-dimethyl-l,5-hexadien (Hernadi et al., 1968). Of
the organic sulfocyanide derivatives tested, none were effective radio-
protectors.
3.3.2.3 Amino Acid Transport — Amino acid transport can be inhibited by
cyanide. Examples of such inhibition were found in bacterial systems.
Glycine and L-serine transport by membrane vesicles of Thioba.c'il'Lus
neapolitanus was completely inhibited by 10 mA? cyanide (Matin et al.,
1974). In Pseudomonas putida, L-lysine transport was inhibited 95% by
17 TaM KCN (Miller and Rodwell, 1971). In membrane vesicles of E. coli,
ML308-225, 2 mAf NaCN inhibited D-lactate-driven transport of proline by
50% and internally generated NADH-driven transport by 25% (Futai, 1974).
A concentration of 10 mM NaCN inhibited these processes by 88% and 87%
respectively. Ferricyanide (0.5 to 4.0 mM) inhibited transport stimulated
by additions of NADH to the medium but did not inhibit transport stimulated
by internally generated NADH.
3.3.2.4 Enzymatic Activities — Cyanide inhibits many enzymatic reactions
involved in processes such as hemolysis and nitrogen cycling. In 12 strains
of E. aoli- isolated from intestines of pigs with edema disease, 40 uM KCN
inhibited g-hemolysis 80% to 90% (Short and Kurtz, 1971). Complete inhi-
bition was obtained with 400 yAf KCN. Cells were tested in early, middle,
and late exponential growth periods. Cyanide did not inhibit the 6-hemolysis
of cell-free supernatants.
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65
Cyanide can also affect nitrogen metabolism. For example, Schloemer
and Garrett (1974) found that KCN (1 mM) decreased nitrite transport in
Neupospora ovassa by inhibiting nitrite reductase by 95%; the inhibition
appeared to be reversible. The enzyme NADH—nitrate oxidoreductase of
ChloTella vulgari,s is converted in the presence of NADH and cyanide to an
inactive form which can be reactivated by ferricyanide (Lorimer et al.,
1974; Solomonson, 1974; Solomonson and Vennesland, 1972). The cyanide
binding was directly proportional to the amount of enzyme inactivation
(0.066 nmole of cyanide per unit of enzyme inactivated) (Lcrimer et al.,
1974). Pistorius et al. (1975) reported that C. vulgavis produced HCN
at high light intensities, high 02 tension, and low C02 tension. Although
there is no direct evidence, Lorimer et al. (1974) stated that the cyanide
inactivation of nitrate reductase in C. vu1gavis may be a natural control
mechanism to inactivate that enzyme when ammonium is the nitrogen source.
Vennesland and Jetschmann (1976) suggested that the production and excre-
tion of glycolate by illuminated cultures of (7. VulgaT-is at high 02 and
low C02 tensions may be regulated by internal HCN production. Externally
added cyanide, hydroxylamine, hydrazine, or semicarbazide were able to
stimulate glycolate synthesis. Nitrate reductase A from Aer-obaoter aevo-
genes and M-ioTooooQus den-itr-if-loans is strongly inhibited by cyanide
(Pichinoty, 1969). This inhibition is not completely reversible. Approx-
imately 50% of the enzyme activity could be restored when nitrate and
chlorate were used as substrates. In M. denitvifioans and P. aeruginosa,
p-phenylene-diamine-N02~ reductase was also inhibited by cyanide (Pichinoty,
Bigliardi-Rouvier, and Rimassa, 1969). Nitrate reductase from Nitrobacter
agtl-is inactivated by NADH could be reactivated by 0.5 mM ferricyanide
(Herrera and Nicholas, 1974).
Various pesticides were found to stimulate the growth of Azotobaoter
vlnelandi-i. in culture (Peeters et al., 1975). Calcium cyanide, however,
decreased growth in culture (Table 3.8). Most of these pesticides de-
creased the amount of nitrogenase extracted from these cells. Data on
the inhibition of the isolated enzyme by a few of these pesticides are
given in Table 3.9 (data for calcium cyanide was not reported).
Other microbial enzymes are inhibited by cyanide. For example, 1 mM
NaCN inhibited purified 6-aminolaevulate dehydratase from photosynthet-
ically grown Rhodopseudomonas spheroides (van Heyningen and Shemin, 1971).
The enzyme C02 reductase from Chlostridium pasteurianwn ATCC 6013 was
inactivated by lO"*1 M cyanide (Thauer et al., 1973).
Cyanogen bromide is often used to characterize the active site of
an enzyme and apparently cleaves a polypeptide chain by reacting with
methionine (Lehninger, 1970). Examples include analyses of trypsin from
StTeptomyoes (Olafson et al., 1975) and of invertase from Saocharomyces
eerevisiae. This compound, however, is apparently not of environmental
concern.
-------
TABLE 3.8. INFLUENCE OF VARIOUS PESTICIDES ON THE YIELD OF BACTERIAL MASS OF
AZOTOBACTER VINELANDII AND THE SYNTHESIS OF NITROGENASE ENZYME COMPLEX
Compound
None
Ammonium thiosulfate
Calcium cyanide
Potassium cyanate
Cyanuric acid
Nirit Supra (2 ,4-dinitrophenyl thiocyanate)
Applied dose
(g/10 liters)
1.2
0.2
1.2
Saturated
1.3
Yield of wet bacteria
(g/10 liters of medium)
165
474
74
174
200
394
Specific
. . . a
activity
5
0
0
0.04
0.26
0.5
Specific activity of nitrogenase enzyme complex in nanomoles of C2HI+ per minute per milligram
of protein.
^Soluble in culture medium.
CPoorly soluble in culture medium.
Source: Adapted from Peeters et al., 1975, Table II, p. 405. Reprinted by permission of the
publisher.
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TABLE 3.9. EXTENT OF INHIBITION OF SPECIFIC ACTIVITY OF NITROGENASE ENZYME COMPLEX
OF AZOTOBACTER VINELANDII BY SEVERAL PESTICIDES
Compound
Ammonium thiocyanate
Potassium cyanate
. ,b
Cyanuric acid
Nirit Supra (2 , 4-dinitrophenyl thiocyanate)
Use
Herbicide
Herbicide
Insecticide
Fungicide
Concentration
(mM)
3.5
3.5
0.1-1
0.7
Inhibitiona
30
30
0
50
Percent inhibition is indicated at the concentration given in the concentration
column. When no inhibition is observed, the range of concentration tested is given.
^Soluble in water.
Q
Soluble in dimethyl sulfoxide.
Source: Adapted from Peeters et al., 1975, Table IV, p. 406. Reprinted by permission
of the publisher.
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68
SECTION 3
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SECTION 4
BIOLOGICAL ASPECTS IN PLANTS
4.1 SUMMARY
Free cyanide is not found in intact plant cells. Many plant species,
such as cassava, sorghum, flax, cherries, almonds, and lima beans, contain
cyanogenic glycosides which release hydrocyanic acid (HCN) when they are
hydrolyzed. It should be pointed out that cyanogenic compounds are far more
widely distributed than any of the other nitrile compounds discussed in this
section. These glycosides are biosynthesized from amino acids and sugars.
The amounts formed depend on the physiological status of the plant; younger
plants and tissues often have a higher glycoside content than mature parts.
Glycosidase, the enzyme responsible for glycoside hydrolysis, is usually
compartmentalized within the cell and contacts the substrate only when the
plant is bruised or crushed. Free HCN may be released by living roots of
sorghum, but other cyanogenic plants are not known to possess this ability.
The production and release of cyanide to the environment through normal
processes of death and decomposition have not been studied.
Other plant species neither contain cyanide nor release it upon injury.
The Cruciferae contain glucosinolates (mustard oil glycosides) which hydro-
lyze when the cell is injured and release isothiocyanates, thiocyanates, or
organic nitriles. These glucosinolates are synthesized from amino acids
and sugars. Within the Cruciferae, indoleacetonitrile may be a natural
metabolite involved in synthesis of indoleacetic acid and/or glucobrassicin
(a glucosinolate).
Although no definitive evidence on whether HCN fixation is ubiquitous
in all plants has been shown, some plants possess the ability to metabolize
externally added HCN. The major route appears to be condensation of cyanide
with cysteine to yield 3-cyanoalanine, which is subsequently hydrolyzed to
asparagine. The synthesis of asparagine by this pathway is probably of
minor importance in total asparagine biosynthesis. In common vetch,
g-cyanoalanine condenses with glutamic acid to produce a lathyrogenic com-
pound, Y~glutamyl~B-cyanoalanine. In Lathyrus, g-cyanoalanine is converted
to g-aminopropionitrile and then is condensed with glutamic acid.
Cyanopyridine alkaloids, natural compounds of limited distribution in
the plant kingdom, contain the cyanide group but do not release cyanide
during either synthesis or degradation. Pseudocyanogenic glycosides, natural
compounds which are limited to the Cycadaceae, release HCN only under the
condition of alkaline hydrolysis.
The major effect of cyanide on metabolism is through the inhibition of
respiration by complexation of iron in cytochrome oxidase. In some species,
cyanide inhibition of respiration is not complete and the proportion that is
insensitive varies with plant organ, ageing, and physiological status of the
tissue. Details of the electron transport chain for cyanide-insensitive
respiration are unknown, but this chain apparently involves only one site
for adenosine triphosphate (ATP) synthesis.
77
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78
In vitro cyanide can inhibit a variety of enzymes by complexation of
metal cofactors, by combination of undissociated HCN with carbonyl groups,
or by combination of the cyanide ion with disulfide bonds. The significance
of these reactions in vivo is unknown.
Because cyanide inhibits respiration, and hence, ATP production, it
also inhibits a variety of processes directly or indirectly dependent on ATP
(e.g., ion uptake and translocation in phloem). However, in some species
cyanide can stimulate germination — perhaps by increasing the flow of carbon
through the pentose phosphate pathway. Cyanide in the presence of oxygen
has been observed to increase chromosomal aberrations in broad bean roots.
Nitrile herbicides act as electron flow inhibitors or uncouplers in
respiration and/or photosynthesis. No release of cyanide from these com-
pounds has been observed. Dichlobenil is a preemergent herbicide; ioxynil
and bromoxynil are postemergent contact herbicides.
4.2 METABOLISM
The cyanide group is present in a variety of natural organic compounds.
The major group of these compounds is the cyanogenic glycosides, which are
rather widely distributed within the plant kingdom (Conn, 1969). The metab-
olism of these glycosides is considered in some detail in this section;
damage (bruising, crushing, etc.) to cells of plants containing these com-
pounds causes enzymatic release of hydrocyanic acid (HCN).
The metabolism of nitrile herbicides is briefly discussed. Herbicidal
activity apparently is not related to the formation of free HCN and, where
studied, metabolism of these herbicides does not involve HCN release.
4.2.1 Hydrocyanic Acid Incorporation
Although little or no free HCN occurs in plants (Robinson, 1975), plants
are able to metabolize externally added HCN. Blumenthal-Goldschmidt, Butler,
and Conn (1963) observed that the cyanogenic plants sorghum, flax, and white
clover converted H14CN to the amide-carbon atom of asparagine. However, the
ability to fix HCN was not related to the cyanogenic nature of these plants.
Barley, pea, and red clover, none of which contain cyanogenic glycosides,
also convert HCN to asparagine.
Blumenthal-Goldschmidt, Butler, and Conn (1963) initially proposed that
g-cyanoalanine was an intermediate in the conversion of HCN to asparagine
and that it was formed enzymatically by the reaction of HCN with serine. In
a preliminary enzyme study, however, cysteine was a better substrate than
serine for asparagine production (Floss, Hadwiger, and Conn, 1965). Sub-
sequently, the enzyme 0-cyanoalanine synthetase, which catalyzes the reaction
cysteine + HCN -»• g-cyano-L-alanine + H2S ,
was isolated (Hendrickson and Conn, 1969). This enzyme is responsible for
the production of 3-cyano-L-alanine in common vetch, lupine, and sorghum
(Blumenthal-Goldschmidt et al., 1968), in several other vetches — Lathyrus
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odovatus, Eoball-ium elater-iwn, and ChloTella pyrenoidosa (Fowden and Bell,
1965), in EscheT-iah-ia ooli (Dunnill and Fowden, 1965), as well as in
numerous other plants that convert HCN to asparagine (Conn and Butler, 1969).
In common vetch (Vic{,a sativa) most HCN was converted into an unknown
compound instead of asparagine (Blumenthal-Goldschmidt, Butler, and Conn,
1963); this unknown compound was later shown by Ressler, Giza, and Nigam
(1963) to be y-L-glutamyl-3-cyano-L-alanine. Fowden and Bell (1965) then
demonstrated that common vetch and C. pyrenoi-dosa contained a glutamyl
transferase that added glutamic acid to g-cyano-L-alanine and that this
transferase was absent in other Via-ia species. Since a majority of the
species which metabolize HCN by the g-cyano-L-alanine pathway produce
asparagine instead of the glutamyl peptide, they must lack the transferase
and contain instead an enzyme that converts 3-cyanoalanine to asparagine.
Gastric, Farnden, and Conn (1972) have partially purified an enzyme from
blue lupine which catalyzes this reaction. The plant enzyme is distinct
from the asparaginase of E. soli, which does catalyze the conversion of
3-cyanoalanine to asparagine (Jackson and Handschumacher, 1970).
3-Cyanoalanine synthetase occurs in the mitochondrial fraction of
plant seedlings (Floss, Hadwiger, and Conn, 1965; Hendrickson and Conn,
1969) and reaches a maximal concentration in five-day-old seedlings of
Lup-inus angustifolius (Lever and Butler, 1971). Since the maximum enzyme
activity occurred prior to maximal accumulation of asparagine, the latter
authors concluded that the 3-cyanoalanine pathway was not a major route
for asparagine biosynthesis in the blue lupine. Other authors (Gastric,
Farnden, and Conn, 1972; Oaks and Johnson, 1972) who have examined the
3-cyanoalanine pathway in other plants have also reached the same conclusion.
The physiological significance of the 3-cyanoalanine pathway for HCN
metabolism is not clear. Abrol and Conn (1966) and Abrol, Conn, and Stoker
(1966) presented evidence that endogenous turnover of cyanogenic glycosides
in lotus and in Nandina domestica Thunb. releases HCN, which is then incor-
porated into asparagine. In those plants that do not produce cyanogenic
glycosides, the situation is more puzzling. The enzyme system for metabo-
lizing HCN is present, but the endogenous source of HCN is unknown. Conn
and Butler (1969) have suggested that the 3-cyanoalanine pathway is a meta-
bolic activity acquired early in evolution and retained by species that no
longer have a need for such a process.
A few plants apparently release HCN to the environment through normal
metabolism as opposed to release during destructive actions such as bruis-
ing or crushing. Sorghum roots released free HCN — about 0.005 mg per
plant per 24 hr for one variety and about 0.02 mg per plant per 24 hr for
another variety (Rangaswami and Balasubramanian, 1953). The occurrence of
this ability in the plant kingdom and the ecological significance of HCN
release have not been well studied.
Cyanides and organic nitriles can react with nitrogenase, the enzyme
complex responsible for nitrogen fixation (Burns and Hardy, 1975). The
reaction involves the cleavage of the C=N bond and produces ammonia and the
corresponding hydrocarbon. Cyanide probably does not undergo a reaction
with nitrogenase within the cell because of its high affinity for metallo-
proteins. Organic nitriles have varying reactivities with nitrogenase,
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80
depending upon the nature of the hydrocarbon position. There is little
information on the in vivo significance of such reduction.
4.2.2 Cyanide Release from Plants
The effects of the natural release of HCN from sorghum roots on the
neighboring biota have not been studied. Rangaswami and Balasubramanian
(1963) reported that sorghum roots released HCN and suggested that this
release may be responsible for the sparsity of serious fungal or bacterial
root diseases in sorghum. The isolation of an HCN-resistant Aspevg-illus
ni-gev strain from the sorghum rhizosphere, not from the soil itself, could
indicate that HCN is rapidly metabolized in the rhizosphere and, thus, is
not available for toxic action.
The relationships among HCN, cyanogenic glycosides, and disease resist-
ance in plants have been the subject of several studies. Reynolds (cited
in Timonin, 1941) correlated the resistance of flax to Fusariwn lini with
the HCN "recovered from plant tissues." Timonin (1941) presented evidence
that HCN was produced and excreted by the roots of a wilt-resistant flax
variety but not by a susceptible variety. Presumably, the HCN was released
by the hydrolysis of the cyanogenic glycoside linamarin. Millar and Higgins
(1970) showed that HCN was released from the cyanogenic glycosides linamarin
and lotaustralin during the infection of bird's-foot trefoil with Stemphyliim
loti. This fungus was more resistant to HCN than were other fungi which were
not parasitic to bird's-foot trefoil. S. loti also produced a 3-glucosidase
which could hydrolyze the cyanogenic glycosides from a trefoil strain that
did not possess an endogenous 3-glucosidase.
Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and its derivatives
are effective uncouplers of oxidative and photosynthetic phosphorylation
and are used in biochemical analysis (Pistorius et al., 1975). When CCCP
was added to spinach grana or CKLoTella cultures, cyanide was released;
the amount released was stimulated by light and oxygen. Presumably, the
cyanide was derived from the cyano group of CCCP- Further work is necessary
to determine if this cyanide generation is responsible for the uncoupling
activity of CCCP.
4.2.3 Cyanogenic Glycosides
4.2.3.1 Catabolism and Anabolism of Cyanogenic Glycosides — In higher
plants, the major group of compounds that contains the cyanide group is the
cyanogenic glycosides (Robinson, 1975). These compounds have been identified
in approximately 1000 plant species, comprising 90 families and 250 genera
(Conn, 1969). No specific comments on the evolutionary relationships among
these families were found in the literature, although Alston and Turner
(1963) stated that "no clear cut systematic implications are evident."
These compounds are quite common in certain families. For example, cyano-
genic glycosides occur in 150 species of Rosaceae, 100 species of the
Leguminosae, 100 species of the Gramineae, 50 species of the Araceae, 50
species of the Compositae, and lesser numbers of the Euphorbiaceae, Passi-
floraceae, Ranunculaceae, and Saxifragaceae (Alston and Turner, 1963).
Within these families, cyanogenic glycosides can be used to determine
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81
systematic relationships. Alston and Turner (1963) cited the work of Gibbs
and Hegnauer to demonstrate that "cyanogenesis" (defined as the release of
cyanide from plants; whether the cyanide is actually released from cyano-
genic glycosides is usually not determined) is common in the Rosaceae
subfamilies Pomoideae and Prunoideae but not in the Rosideae or Spiraeoideae,
Characterization of the particular cyanogenic glycoside in each species is
essential for further systematic studies.
Because of the occurrence of cyanogenic glycosides in a variety of
foods consumed by humans and the subsequent release of HCN during cell
destruction, care must be taken in the preparation of these foods to avoid
cyanide poisoning (Conn, 1973a; Montgomery, 1969). This problem is more
critical in parts of the world where cyanogenic plants (e.g., cassava)
constitute a major portion of the diet. Some food plants containing cyano-
genic glycosides and thus having a cyanide-releasing potential are almonds,
cassava, lima beans, macadamia nuts, bamboo shoots, numerous stone fruits,
sorghum, and corn.
Poisoning of livestock by cyanogenic plants is also a problem which
has occurred in various countries (Kingsbury, 1964). In the United States,
cattle have been poisoned by eating various species of Sorghum (grain sor-
ghum, Sudan grass, Johnson grass). Under dry growing conditions, arrow-
grass (TT-igloch-in marit-ima and Tv-Lglooh-Ln palustna) can develop concen-
trations of cyanogenic glycosides which release enough cyanide to poison
sheep and cattle (Radeleff, 1970).
Approximately 20 different cyanogenic glycosides are known and all
have the basic structure:
0 )S-GLYCOSYL
— CN
where R and RJ refer to hydrogen, alkyl, or aryl groups.
Table 4.1 lists the major cyanogenic glycosides and representative
species in which they occur. The particular cyanogenic glycoside in many
plant species has not been identified and many possibilities for substitu-
tion within the general formula exist; therefore, additional glycosides
may be discovered and characterized. For example, Seigler et al. (1975)
reported the isolation of dihydroacacipetalin, a new cyanogenic glycoside
from Acacia sieberiana var. woodii, and Tantisewie, Ruijgrok, and Hegnauer
(1969) described deidamin, a newly characterized cyanogenic glycoside from
Deidamia clematoides (Passifloraceae).
The cyanogenic glycosides themselves are not highly toxic to animals.
Hydrolysis of the cyanogenic glycoside releases HCN, which is the toxic
agent in cyanophoric plants. Hydrolysis occurs when the plant cells are
injured, allowing previously compartmentalized hydrolytic enzymes to
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82
TABLE 4.1. NATURALLY OCCURRING GLYCOSIDES
Name
Formula
Source
Acacipetalin
Derivatives of valine, isoleucine, and leucine
Acacia stolonifera Burch.
CHgOH
-0 0
Cardiospermin
CH-OH
I ' / "-(-LJ--
-q p—/ CH'
CN
H
OH
Sapindaceae
Linamarin
Lotaustralin
CH2OH Me
-0 p—(- Me
CN
OH
,OH C2H5
0 0—f--CN
.OH Y Me
Manihot utilissima Pohl
(cassava), Liniian
usitatissiimm L. (flax),
Phaseolus lunatus L.
(lima bean), Hevea
brasilisnsis Muell.,
Trifoliwn repens L.
Same as Linamarin
(R)-Amygdalin
Derivatives of phenylalanine
CH2OH
0 0— CH
«
HO^I - / H ^J'
Prunus amygdalus Stokes,
many other Rosaceous
species
(R)-Lucumin
-CH, CN
J-0 «JLH
,— 0 0 —C
frX ^°V°^H
H0^ H (°H A
OH H0\—_/ H
OH
Sapotaceae
(R)-Prunasin
CH-OH fN
/
0 O — fr — H
O
^=^
Primus padus L., many
other species from a
variety of families
(S)-Sambunigrin
Sambucus nigra L.,
Acacia cunninghamii.
(R)-Vicianin
HO, — o o— C
OH HO - f H
OH
©
Vicia angustifolia Roth L.
(continued)
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83
TABLE 4.1 (continued)
Name
Formula
Source
Probable derivatives of phenylalanine
(R)-Holocalin
CH,OH f"
I 2 /
>—0 0—t-H
jtX the
Leguminosea, Caprifoliacea
(S)-Zierin
CH,OH CN
Zievia laevigata Sm.
Derivatives of tyrosine
(S)-Dhurrin
Sorghum vulgare Pers . ,
Fhyllanthus gasstroemi
Muell.
p-Glucosyloxymandelonitrile
CH2OH
(OH
OH
Berberidaceae,
Ranunculaceae, Goodia
latifolia Salib.
(R)-Proteacin
HO H
HO
H | 'OH
HO
Proteaceae, Ranunulaceae
(R)-Taxiphyllin
HO OH
Taxus baccata
Compounds with cyclopentene ring structure
Barterin (tetraphyllin B)
CH2OH NC
-0 0
HO*—f H
HO
May occur in
Flacourtiaceae and
Passifloraceae
Deidaclin
CH,OH NC
2
o
HO^ ( H
HO
May occur in
Flacourtiaceae and
Passifloraceae
Gynocardin
CH,OHNC, /=\
>-o o
fcX
10 1 H
Gynooardia odorata R. Br.
Pangium edule Reinw.
Source: Modified from Seigler, 1975 and Conn, 1969.
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84
degrade the glycosides. Hydrolysis usually occurs in two steps. For
instance, linamarin,
0—GLUCOSE
CH3 C CN.
CH3
is first hydrolyzed by a g-glucosidase, linamarase, to yield glucose and
acetone cyanohydrin,
OH
CH3 C CN
CH3
The cyanohydrin is then cleaved by an oxynitrilase to yield acetone and
HCN (Conn, 1969, 1973a). Some plants containing cyanogenic glycosides do
not possess the necessary degradative enzymes (Robinson, 1975), but this
is the exception rather than the rule.
The glucosidases isolated from a given species have a certain amount
of substrate specificity. For example, flax glucosidase hydrolyzes lina-
marin, which is found in flax, and the chemically related lotaustralin,
found in Lotus sp. and flax, but not amygdalin found in Prunus sp. The
oxynitrilases from different species also exhibit varying degrees of sub-
strate specificity (Conn, 1969).
Biosynthetic pathways for cyanogenic glycosides involve the conversion
of specific protein amino acids to the corresponding aldoximes and their
subsequent conversion to the nitrile. The nitrile is hydroxylated and the
sugar moiety is then attached (Figure 4.1). Thus, L-tyrosine is ultimately
converted to dhurrin and toxiphyllin; L-phenylalanine to prunasin, amygdalin,
and vicianin; L-valine to linamarin; and L-isoleucine to lotaustralin (Conn,
1973i>). Feeding of radioactively labeled amino acids to plants (e.g., valine
and isoleucine to flax) or of labeled hydroxynitriles (acetone cyanohydrin
or butanone cyanohydrin fed to flax) results in the formation of labeled
cyanogenic glycosides (linamarin and lotaustralin in flax) (Hahlbrock and
Conn, 1971). In the previous example, both glycosides are apparently formed
in vivo by the same glycosyltransferase.
The physiological role of cyanogenic glycosides in plants is not known.
However, some workers have postulated their involvement in the coevolution
of plants and insects (Jones, 1973). They are apparently actively metabo-
lized (Abrol and Conn, 1966) and do not represent metabolic end products.
4.2.3.2 Content of Cyanogenic Glycosides in Various Plants — The relation-
ship between the potential for producing hydrocyanic acid (HCN-p) and
various factors has been well studied in the diverse genus Sorghum because
of its economic importance as a crop and the threat of livestock poisoning.
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85
ORNL-DWG 76-15533R
R,
H
H
COOH
H
CH
R2 NH2
AMINO ACID
R,
OH
•CN
R2 NOH
ALDOXIME
I
H
CN
R.
a-HYDROXYNITRILE
(CYANOHYDRIN)
0 - SUGAR
CN
R2
NITRILE
R2
CYANOGEN 1C GLYCOSIDE
Figure 4.1. Generalized biosynthetic pathway for cyanogenic glyco-
sides. Source: Adapted from Conn, 1969, Figure 11, p. 525. Reprinted
by permission of the publisher.
Sorghum genotypes all contain dhurrin as a precursor of HCN. Harrington
(1966) reviewed earlier work and concluded that "(1) HCN-p varies with
species and cultivar, (2) HCN-p decreases as plant height and age increase,
(3) HCN-p varies with location and climate, (4) moisture stress results in
higher HCN-p and (5) HCN-p is higher in first growth than in aftermath
growth."
An easy screening procedure is necessary to select cultivars with low
HCN-p. Whole plants of Sorghum variety Piper had an average content of
about 12 to 17 ppm HCN-p (fresh weight basis), while the variety Suhi-1
averaged about 70 to 80 ppm HCN-p (Benson, Gray, and Fribourg, 1969). An
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86
analysis of HCN-p of leaf samples from these plants showed that leaf samples
are not a suitable measure of whole plant HCN-p content. Prediction of
HCN-p of sorghum varieties was not possible from measurements of various
morphological features although some weak correlations were observed (James
and Gray, 1975).
The concentration of HCN-p in various organs of Greenleaf Sudan grass
(sorghum) are shown in Figure 4.2 (Loyd and Gray, 1970). Dry seeds contain
little HCN-p; young plants contain the highest concentrations. Correlations
between total HCN-p and dry weights of individual aboveground parts were
positive but not significant for any cultivar. In a sorghum—Sudan grass
hybrid the HCN-p content of the whole plant was 372, 254, and 204 ppm for
plants of 50-, 120-, and 155-cm height (Wolf and Washko, 1967). At all
three heights the leaf blade had the highest concentration of the parts
studied.
Sorghum subjected to mild frost showed increased HCN-p content within
one to six days after the frost; however, severe freezing led to death of
the plant and a concommittant loss in HCN-p of the plant (Wattenbarger et
al., 1968). Presumably, this rapid depletion of HCN-p was due to the enzy-
matic release of HCN from the cyanogenic glycoside.
Examples of the HCN-p content of some plants are given in Table 4.2.
Contents of different varieties (e.g., cassava) can vary considerably.
DeBruijn (1971) presented a thorough study of factors affecting the cyanide
content in cassava plants. The cyanogenic glycoside content of leaves de-
creased with age and the concentration in tubers was higher in the bark than
ORNL-DWG 76-15534
1 5 1
DAYS
4 5 6 7 8 9 10 11
WEEKS AFTER EMERGENCE
12 13 14 15
Figure 4.2. Concentration of hydrocyanic acid potential in plant
parts of Greenleaf Sudan grass (sorghum). Source: Adapted from Loyd
and Gray, 1970, Figure 2, p. 395. Reprinted by permission of the
publisher.
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87
TABLE 4.2. CYANIDE CONTENT OF SELECTED PLANTS
Plant material
Cyanide content
Reference
Bitter almond
Spicy almond
Sweet almond
Peach leaves
var. Alexander
var. Mariska
var. Ford
Cassava roots
var. Palmeiras
var. CEPEC 62
var. IAC 780
var. Itapecuru
var. Engole boi
var. CEPEC
Bitter cassava
dried root cortex
stem
whole root
Sorghum, young plant
Bamboo
tip
stem
Lima bean
Java
Puerto Rico
Burma
Arizona
America
European contoneaster fruit
Hedge contoneaster fruit
Miniature crab apple fruit
American mountain ash fruit
European mountain ash fruit
280 ppm
250 mg/100 g
86-98 ppm
22-54 ppm
39 ppm
74 ppm
120 ppm
378 ppm
313 ppm
200 ppm
68 ppm
41 ppm
27 ppm
245 mg/100 g
113 mg/100 g
55 mg/100 g
250 mg/100 g
800 mg/100 g
300 mg/100 g
312 mg/100 g
300 mg/100 g
210 mg/100 g
17 mg/100 g
10 mg/100 g
1.08 mg/100 g frozen wt
2.75 mg/100 g frozen wt
1.70 mg/100 g frozen wt
1.80 mg/100 g frozen wt
6.47 mg/100 g frozen wt
Gyorgy et al., 1969
Montgomery, 1969
GyBrgy et al., 1969
Gyorgy et al., 1969
Gyorgy et al., 1969
Gyorgy et al., 1969
Gyorgy et al., 1969
Esquivel and Maravalhas, 1973
Esquivel and Maravalhas, 1973
Esquivel and Maravalhas, 1973
Esquivel and Maravalhas, 1973
Esquivel and Maravalhas, 1973
Esquivel and Maravalhas, 1973
Montgomery, 1969
Montgomery, 1969
Montgomery, 1969
Montgomery, 1969
Montgomery, 1969
'Montgomery, 1969
Montgomery,
Montgomery,
Montgomery,
Montgomery,
Montgomery,
Jeffrey and
Jeffrey and
Jeffrey and
Jeffrey and
Jeffrey and
1969
1969
1969
1969
1969
Wiebe, 1971
Wiebe, 1971
Wiebe, 1971
Wiebe, 1971
Wiebe, 1971
in the inner sections. Concentrations varied considerably among tubers on
the same plant, but no correlation with tuber size was evident. Nitrogen
additions to soil increased the glycoside content in leaves and roots,
whereas additions of potassium and "farmyard manure" decreased the content.
In pot experiments, drought increased the glycoside content of young plants.
In field experiments, however, a dry season in three areas did not increase
the glycoside content in roots. These and other observations indicate that
the final glycoside content at a given time or developmental stage is the
result of various interacting physiological processes.
4.2.4 Pseudocyanogenic Glycosides
Pseudocyanogenic glycosides, a series of toxic compounds found primarily
in members of the Cycadaceae, have the formula CH3—N=N^-CH2—0—(sugar) (Miller,
1973). Although acid or enzymatic hydrolysis of these compounds does not
produce free HCN, treatment with cold alkali will release HCN; for example:
macrozamin
acid
N2 + CH3OH + HCHO + primeverose
macrozamin
cold NaOH
N2 + HCN + HCOOH + primeverose
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88
Table 4.3 lists the common pseudocyanogenic glycosides (Seigler, 1975).
These compounds have the same nitrogenous moiety but differ in the constituent
sugar.
4.2.5 Lathyrogenic Compounds
Certain compounds that produce distinct maladies in animals ingesting
them are called lathyrogenic compounds because they were initially found in
the genera Lathyrus and Vi-G-ia. For example, y-glutamyl-3-aminoproprio-
nitrile is produced in Lathyvus and Y~Slutamyl-3-cyanoalanine in Vic-La
sat'Lva. In Lathyrus, $-cyanoalanine (formed by HCN reacting with cysteine)
is converted to 3-aminopropionitrile, which then condenses with glutamic
acid (Ferris, 1970). 3-Cyanoalanine is not converted to asparagine in
V. sat'lva because of the lack of the hydrolase enzyme (Gastric, Farnden,
TABLE 4.3. PSEUDOCYANOGENIC GLYCOSIDES
Name
Formula
Source
Cycasin
O. O — CH~N = N-
HO^ f H
HO
Cycas revoluta Thunb.
Macrozamin
0 —CH,—N = N —Me
Macrozamia sp-iralis
Miq . , M. reisdle-i C.
Cl. Gardner
Neocycasin A
CH2OH
CHZOH
(OH A
ON - H
0 0—CH,—N= N-Me
HON f H HO
HO
Cycas revoluta
Neocycasin B
CH2OH
0 0—CH
— CH,—N = N-Me
Cycas r-evoluta
Neocycasin E
0 0—CH2-N-N-Me
HO
Cycas revoluta seeds
The position of the oxygen in the azoxy group is not
established.
Source: Modified from Seigler, 1975 and Miller, 1973.
-------
and Conn, 1972) and is therefore available for other conversions such as
the addition of the glutamyl group. Another neurolathyrogenic factor,
a,Y-diaminobutyric acid, is produced in V. sat-iva and L. odoratus pre-
sumably by y-reduction of g-cyanoalanine. These compounds are apparently
formed by normal metabolism and not in response to exposure to cyanide in
the environment.
4.2.6 Glucosinolates (Thioglucosides)
Glucosinolates have the general formula
S — GLUCOSE
and upon enzymatic hydrolysis during crushing of the plant, isothiocyanates
or thiocyanates are rapidly formed (VanEtten, Daxenbichler , and Wolff,
1969) . The proposed mechanism is
S — GLUCOSE H20 _ | S —
R— C, - »-Sof~+ GLUCOSE +R—CX
THIOGLUCOSIDASE *
S4- R-C=
ORGANIC NITRILE .
ISOMERASE
R—N=C=S R—S—C=N
ISOTHIOCYANATE THIOCYANATE
The products formed by enzymatic hydrolysis depend on pH. At pH 3 to 4
glucobrassicin (3-indolylmethyl glucosinolate), isolated from BTassica
species, is hydrolyzed to produce indolylacetonitrile (an organic nitrile) ,
sulfur, sulfate, and glucose. At pH 7 the products of hydrolysis are
3-indolylmethyl isothiocyanate, sulfate, and glucose (Ahmed et al., 1972).
The 3-indolylmethyl isothiocyanate is unstable and breaks down to 3-hydroxy-
methylindole and the thiocyanate ion.
Glucosinolates are mainly found in members of the Cruciferae family
(in all 300 species examined) and consist, so far, of some 50 compounds of
which only one or two predominate in any given species (Table 4.4). Highest
concentrations of glucosinolates are found in seeds, but roots and leaves
also contain measurable amounts. Concentrations in plant tissues vary with
environmental factors and species variety. Measurement of thiocyanate pro-
duction in the crucifers usually involves disruption of the cells and incuba-
tion, during which time the glucosinolates are hydrolyzed to thiocyanates.
This amount is then referred to as the thiocyanate content of the tissue.
For example, the thiocyanate content of Burpee White radish roots increased
linearly with sulfate levels in plants grown in 0.5-strength Hoagland's
solution, but the content did not increase in roots of the French Breakfast
variety (Bible and Chong, 1975a). Neither variety showed an increase in
thiocyanate content with sulfate when grown in double-strength Hoagland's
solution. Thiocyanate content of Burpee White radish roots was greater on
organic soil than on loam soil and increased with cooler conditions in both
soils (Bible and Chong, 19752?). Spring- and fall-grown cruciferous vegeta-
bles often have a higher than average thiocyanate content. Thiocyanate
content of Burpee White and Champion radish roots decreased as development
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90
TABLE 4.4. GLUCOSINOLATES IN DOMESTICATED
CRUCIFER PLANTS
Plant
Glucosinolate(s) present
For food
Brassiaa oleraaeae
cabbages, kale, brussel
sprouts, cauliflower,
broccoli, kohlrabi
Brassica aampestris
turnips
Brassica napus
rutabaga
Lepidium sativwn
garden crest
Raphanus sativus
radish
Sinigrin
Glucobrassicin
Progoitrin
Gluconapin
Neoglucobrassicin
Progoitrin
Gluconasturtiin
C??)-2-Hydroxy-4-pentenyl-
glucosinolate
Progoitrin
Glucobrassicin
Neoglucobrassicin
Glucotropaeolin
4-Methylthio-3-butenyl-
glucosinolate
Glucobrassicin
For condiments
Amoracia lapathifolia
A. TUStiaana
horseradish
Brassica aarinata
Ethiopian rapeseed
B. juncea
Indian or brown mustard
B. n-igra
black mustard
Sinapis alba
white mustard
Sinapis arvensis
charlock
Sinigrin
Gluconasturtiin
Sinigrin
Sinigrin
Sinigrin
Sinigrin
Sinigrin
(continued)
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91
TABLE 4.4 (continued)
Plant Glucosinolate(s) present
For feed as processed seed meal
Brassiaa campestris Gluconapin
rape, turnip rape, Polish Progoitrin
rape, rubsen, naverte Glucobrassicanapin
Glucoalyssin
Glucoraphanin
Brass-lea napus Progoitrin
rape, Argentine rape, Gluconapin
winter rape Glucobrassicanapin
Gluconasturtiin
Glucoiberin
Sinalbin
Crambe abyssiniea ept-Progoitrin
crambe, Abyssinian kale Sinigrin
Gluconapin
Gluconasturtiin
Organic radicals in the glucosinolates given are as
follows: sinigrin, allyl-; glucobrassicin, 3-indolymethyl-;
progoitrin, (.ff)-2-hydroxy-3-butenyl-; gluconapin, 3-butenyl-;
neoglucobrassicin, ]l/-methoxy-3-indolymethyl-; gluconasturtiin,
2-phenylethyl-; glucotropaeolin, benzyl-; sinalbin,
p-hydroxybenzyl-; glucobrassicanapin, 4-pentenyl-; glucoalyssin,
4-methylsulfinylbutyl-; glucoraphanin, 5-methylsulfinylpentyl-;
glucoiberin, 3-methylsulfinylpropyl-; epi-progoitrin, (5)-2-
hydroxy-3-butenyl--
Source: Adapted from VanEtten, Daxenbichler, and Wolff,
1969, Table 1, p. 484. Data collected from several sources.
Reprinted by permission of the publisher.
from seeding to the rosette stage occurred and remained at a low level
during the reproductive stage (Chong and Bible, 1974). Thiocyanate content
of foliage decreased during the rosette stage but increased considerably
during the reproductive stage, especially the early bolting stage.
The goitrogenic effect of some members of the cabbage family (Brassi-
caceae) is apparently due to isothiocyanates (mustard oils) , L-5-vinyl-
2-thiooxazolidone (goitrin), and thiocyanate (Michajlovskij, Sedlak, and
Kosterkova, 1970). These compounds are released from glucosinolates when
the plant material is crushed and the previously compartmentalized enzyme
thioglucosidase (sometimes called by the trivial name myrosinase), which
perhaps occurs in specific cells, is then able to hydrolyze the glucosino-
lates. The amount of these compounds varies considerably in different
species. Raw cabbage and kale contained the equivalent of 10 to 30 mg
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92
isothiocyanate per 100 g tissue, kohlrabi contained 7 mg per 100 g, and
cauliflower contained almost no isothiocyanates. Boiling vegetables alters
the concentrations of the various constituents. Heating inactivates the
enzymatic processes, but at higher temperatures thermal hydrolysis of glu-
cosinolates and disintegration of goitrogens increases. In addition, the
more volatile components are lost. Selection of vegetable varieties with
lower glucosinolate concentrations is possible. For instance, rapeseed,
cultivar Bronowski, has about one-twentieth the amount of unsaturated
nitriles as the normal cultivar (Bvassioa aampestris) and about one-tenth
the amount of goitrin (Lo and Hill, 1972). In seeds of B. oampestris var.
Yellow Sarson, cyano-3,3-epithiobutane is the major product of glucosino-
late hydrolysis, while in seeds of B. campestris var. Echo the major hy-
drolysis products are butenyl- and pentenylisothiocyanate (Kirk and
MacDonald, 1974).
Glucosinolates are apparently biosynthesized from an amino acid; the
nitrogen atom is retained in subsequent transformations (Underbill, Wetter,
and Chisholm, 1973). Labeling experiments have demonstrated that L-methio-
nine and D,L-cysteine are probable sources of the sulfur atom. Again, the
function of these glucosinolates and their further metabolism within the
cell are unknown. Conjecture is that glucobrassicin (3-indolylmethyl
glucosinolate) may serve as an auxin reserve; however, conversions to auxin
have not been unequivocally demonstrated (Miller, 1973). Although the
formation of toxic thiocyanates and isothiocyanates from glucosinolates is
difficult to experimentally test, it may serve as a protection mechanism
for plants against foragers (Whittaker and Feeny, 1971).
4.2.7 Indoleacetonitrile
Indoleacetonitrile (IAN), a substance which can be isolated from some
plants, is structurally similar to indoleacetic acid, a naturally occurring
auxin. Glucobrassicin, a glucosinolate found in many crucifers, can be
converted to IAN by myrosinase (Section 4.2.6). Since this enzyme is
normally separated from its substrate in the intact plant, IAN may be an
artifact of the extraction methods used (Mahadevan and Stowe, 1972). This
idea was further supported by the fact that IAN could be found only in
plants containing glucobrassicin. However, some IAN was found in these
plants even when precautions were taken to inactivate myrosinase. The
limitations upon detection of minute quantities of metabolites must be
considered before it is concluded that a given compound does not naturally
occur.
Indoleacetaldoxime (IAOX) can be converted to IAN and then to indole-
acetic acid (IAA) in a variety of plants (Mahadevan and Stowe, 1972). The
conversions of IAOX to IAN, to glucobrassicin, and to IAA in crucif ers
suggest that IAN may be a natural intermediate. The significance and
relative importance of such a pathway for IAA formation in vivo is unknown.
Although there is agreement that tryptophan is the precursor of IAA, the
major pathway from tryptophan to IAA is uncertain. Schneider and Wightman
(1974) discussed the evidence for severaal possible pathways of IAA bio-
synthesis.
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93
4.2.8 Cyanopyridine Alkaloids
At present, the only cyanopyrldine alkaloids which have been isolated
from plant materials are ricinine,
OCH
isolated from E-ioi-nus oommuni-s L. (castor bean), and nudiflorine,
NC
isolated from Trewia nudiflora Linn. (Ferris, 1970). Biosynthesis of
ricinine probably involves the incorporation of the nicotinamide skeleton
into ricinine with subsequent formation of the nitrile group. If Hli*CN is
fed to R. aommunis, the cyano group has a high specific activity. Since
the amide group of asparagine also has a high activity, ricinine biosynthesis
may proceed through either asparagine or 3-cyanoalanine. The only data
available for the catabolism of ricinine are for the bacterium Pseudomonas,
which hydrolyzes ricinine to produce ammonia and an organic acid.
4.2.9 Nitrile Herbicides
The nitrile herbicides, which have been developed rather recently,
presently consist of three major compounds: 2,6-dichlorobenzonitrile
(dichlobenil), 4-hydroxy-3,5-diiodobenzonitrile (ioxynil), and 3,5-dibromo-
4-hydroxybenzonitrile (bromoxynil) (Ashton and Crafts, 1973). Each com-
pound is only sparingly soluble in water. Salts of ioxynil and bromoxynil
are usually applied as sprays to foliage. Dichlobenil is often applied to
soils to inhibit germination of seeds and growth of actively dividing
meristems. It is volatile and is quickly lost from soils.
Metabolic studies of dichlobenil, usually with mature plants, have
suggested the pathway outlined in Figure 4.3 (Verloop and Nimmo, 1969).
The three- and four-position conjugated derivatives are as toxic as dichlo-
benil, while 2,6-dichlorobenzoic acid shows growth-regulating action.
Since dichlobenil is generally used as a preemergence herbicide, metabolic
studies with older plants may not directly relate to herbicidal properties
in seedlings or embryos. Metabolism of ioxynil apparently leads to benzoic
acid and iodide ions and "presumably bromoxynil would undergo similar
reactions" (Ashton and Crafts, 1973). No data suggest that free HCN is
formed by the metabolism of any of these nitrile herbicides. Dichlobenil
-------
ORNL-DWG 76-15527
Hydroxylotion
Cl
Cl
0-2%
Figure 4.3. Scheme showing the metabolites of dichlobenil in bean leaves after a
five-day uptake of a 12 ppm solution via the roots (R = biopolymer, e.g., polysaccharides)
Source: Verloop and Nimmo, 1969, Figure 2, p. 368. Reprinted by permission of the
publisher.
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95
can be absorbed by all plant organs, but movement through the plant is
slow. One reason Is that dichlobenll adsorbs to llgnin, which is present
in xylem walls. Little evidence exists for phloem transport. Some move-
ment may occur by vapor diffusion through the intercellular air spaces in
the plant.
loxynil can be translocated as evidenced by chlorosis on untreated
leaves and by autoradiography after [14C]ioxynil treatment (Ashton and
Crafts, 1973). Although movement was slow, radioactive label was found in
young leaves at the apex. Treatment of roots with [14C]ioxynil resulted in
the transport of small amounts to the foliage. Selective toxicity between
grasses and broadleaf species and among different broadleaf species may
partially be due to differential spray retention. Other factors must also
operate, however, since selectivity could not be abolished by the use of
wetting agents.
4.3 EFFECTS
4.3.1 Cyanide and Respiration
4.3.1.1 Inhibition of Respiration — The toxic effect of cyanide is due to
its ability to inhibit various enzymes. Cytochrome oxidase, the terminal
electron acceptor of the electron transport chain, is probably the most
sensitive (Hewitt and Nicholas, 1963). Cyanide is known as a potent in-
hibitor of respiration and its action can ultimately lead to death of the
organism. Although inhibition of cytochrome oxidase and other enzymes is
usually due to complexing by cyanide of the metal ion from metalloenzymes,
other inhibitory actions of cyanide are known (Section 4.3.3). Both un-
dissociated cyanide (HCN) and cyanide ions (CN~) inhibit cytochrome oxidase.
It is uncertain whether cyanide binds with the Fe3+ or the Fe2+ form of
cytochrome oxidase.
4.3.1.2 Cyanide-Insensitive Respiration — Although cyanide is a potent in-
hibitor of cytochrome oxidase, respiration, as measured by oxygen uptake,
is not completely inhibited by cyanide in many plant species. The propor-
tion of respiration that is insensitive to cyanide varies with the particular
species and plant organ and with the physiological state of the tissue or
organ (Henry and Nyns, 1975). Table 4.5 lists species that have cyanide-
insensitive respiration (CIR) and the extent to which cyanide either inhibits
or, in some cases, stimulates respiration.
Mitochondria appear to be the organelle in which the insensitive
respiration occurs. Use of inhibitors and various substrates have estab-
lished that a CIR path coexists with the main respiratory electron transport
chain (ETC). Whether the CIR path branches from the main electron transport
chain or whether it is an independent chain utilizing similar substrates is
uncertain. Hydroxamic acids apparently can inhibit the CIR pathway but not
the normal ETC pathway.
Some adenosine triphosphate (ATP) production does occur during opera-
tion of the CIR pathway. In most systems exhibiting CIR, the adenosine
diphosphate (ADP) to oxygen ratio in the presence of cyanide is about 1 com-
pared to about 3 for the ETC. Most of the CIR is also insensitive to anti-
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96
TABLE 4.5. RELATIVE CYANIDE-INSENSITIVE RESPIRATION OF HIGHER PLANTS
Relative respiration rate (%)
Species Whole Cell-free Isolated Submitochondrial
tissue extract mitochondria particles
Monocotyledoneae
Arales
Araceae
Aerissima amurense 102
Amorphophallus rivieri 124
Arum creticum 96
Arum -Italicum 100 100 95
Anon maaulatum 150 100
100 73
175 80
94
45
162
Biarum tenuifolium 95
Philodendron grandifoliwn 159
Sauromatum guttatum 50 100
Symplocarpus foetidus 123 82
62
75-100
50
124
97
86
61
62
Liliaceae
Alliim oepa 87
Graminales
Gramineae
Hordeum sp. 81
36
Hordeum vulgar'e 70
Oriza sp. 75
Oriza sativa 50
Tritiaum sp. 145
115
91
Triticwn aestivum 35
Zea mays 147
Dicotylodoneae
Laurales
Lauraceae
Persea americana 109
Persea gratissima 132
Leguminosales
Leguminosae-Papilionoidea
Lathyms odorata 100
Phaseolus auveus 20 20
36
65
25
Phaseolus vulgar-i-s 20
34
Phaseolus sp. 125
Pisum sativim 15
Vigna sinensis 20
Fagales
Fagaceae
Fagus sylvatica 145
115
(continued)
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97
TABLE 4.5 (continued)
Relative respiration rate (%)
Species
Whole Cell-free Isolated Submitochondrial
tissue extract mitochondria particles
Sapindales
Aceraceae
Acer pseudoplatanus 62
Coniferales
Cupressaceae
Cedms atlant-ica 102
Cedrus 1-tbani 96
Cruciferales
Cruciferae
Brassiaa napus 123
Brass-ica oleracea 10
Sinapsis alba 120
Chenopodiales
Chenopodiaceae
Beta vulgaris
red beet 111
131
sugar beet 105
Polemoniales
Convolvulaceae
Ipomea batatas 121 50
68
38
61
33
49
Solanales
Solanaceae
Lycopersicon esculentum 58
N-ieotan-ia tdbaaum 64
Salomon tuberosum 110 30
75 30
94 58
89
58
84
Ombellales
Umbelliferae
Daucus carota 106
151
128
30
Asterales
Compositae
EeUanthus tuberosus 35
100 x the ratio of the respiration rate measured after the addition of cyanide to
the respiration rate measured in the absence of cyanide. Specific cyanide concentrations
were not included.
Source: Adapted from Henry and Nyns, 1975, Table 1, pp. 4-5. Data collected from
several sources. Reprinted by permission of the publisher.
mycin, an inhibitor acting on the cytochrome ~b level. Since ATP production
at sites II and III occurs after cytochrome b in the ETC, ATP is probably
not produced at these sites during cyanide inhibition. Synthesis of ATP
may occur at site I before cytochrome b if the cyanide-insensitive pathway
branches off the ETC, or alternatively, the CIR chain may contain just one
site of ATP synthesis.
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98
An important facet of the CIR pathway is that it is inducible in cer-
tain organisms and is often related to ageing or changes in physiological
state (Henry and Nyns, 1975). For example, fresh potato tuber discs had 70%
of their respiration inhibited by 0.4 mM NaCN, but after ageing in water for
20 hr the entire endogenous respiration was insensitive to 0.4 mM NaCN.
Infection of Soorzonera discs by Agrobacterium tumifaciens decreased the
cyanide inhibition (using 1 mM NaCN) from 60% in uninfected discs to 25% in
infected discs. Injury can also increase the percentage of respiration in-
sensitive to cyanide. Higher concentrations of HCN (1 mM) decreased the
difference between uninfected and infected tissue to 75% and 65% inhibi-
tion, respectively. Protein synthesis is apparently necessary for induc-
tion of the CIR chain although the exact roles of cytoplasmic and mitochon-
drial protein biosynthesis are not yet defined.
Flow of electrons through either the ETC or the CIR chain is in some
way modulated in the cell. The physiological role of such a pathway is not
completely understood. The high CIR in portions of the inflorescence in
some araceous plants (e.g., skunk cabbage) produces an increase in tempera-
ture and may be related to flowering at the end of winter. The occurrence
of the CIR pathway in injured tissues may serve to remove toxic or unwanted
substances, especially if ATP synthesis "is not required or is impossible,
as is the case in tightly coupled mitochondria that have depleted ADP"
(Henry and Nyns, 1975).
4.3.2 Inhibition of Photosynthesis
Photosystem I is inhibited by KCN, presumably at the site of plasto-
cyanin, a component of the photosystem electron transport chain. With
isolated chloroplasts and purified spinach plastocyanin, Berg and Krogmann
(1975) found evidence that KCN removes copper from plastocyanin. The
apoprotein produced, apoplastocyanin, remains in the chloroplast membrane
and is unable to transfer electrons to photosystem I.
4.3.3 Inhibition of Enzymes
Besides the inhibition of cytochrome oxidase (Section 4.3.1.1), cyanide
also inhibits a variety of other enzymes. Inhibition can occur (1) by the
complexing of the cyanide ion with metals in metalloenzymes, (2) by the
combination of undissociated HCN with carbonyl groups of aldehydes or
ketones to form cyanohydrins, or (3) by the irreversible reaction of sodium
cyanide with disulfide bonds (Hewif't and Nicholas, 1963).
Inhibition by reaction of cyanide with metals in enzymes has been
demonstrated in several cases, including uricase, nitrate reductase, tyros-
inase, carbonic anhydrase, and carboxypeptidase (Hewitt and Nicholas,
1963). Many complexities exist in the inhibition patterns observed for
certain enzymes. In spinach, the NADH-dependent nitrate reductase is in-
hibited by cyanide only when the enzyme complex is in the reduced state
(Relimpio et al., 1971). Preincubation of the enzyme with nitrate reduces
or abolishes the cyanide inhibition. The authors suggested that molybdenum,
an essential cofactor of the enzyme, in a reduced state is irreversibly
complexed by cyanide. In many cases of enzyme inhibition by cyanide, the
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99
exact mechanism of inactivation is unknown. The mere presence of a metal
within the functional enzyme does not mean that the observed inhibition by
cyanide is through metal chelation. Studies with the use of other inhibitors
which react with carbonyl and disulfide groups are necessary to determine
the inhibitory mechanism.
Yeast phenylalanine ammonia-lyase is inactivated by NaCN, presumably
by reaction with carbonyl groups (Hodgins, 1971). The active center of the
enzyme contains a carbonyl group and a linear relationship was observed be-
tween enzyme inactivation and incorporation of Na14CN into the enzyme,
suggesting that the labeling and inactivation events are the same. Hewitt
and Nicholas (1963) also reported that cyanide might inhibit by reacting
with a carbonyl-containing substrate for a given enzyme and suggested that
this may be the manner of inhibition for amino acid decarboxylases which
use pyridoxal phosphate as a coenzyme.
An example of the third inhibition mechanism is the inactivation of
xanthine oxidase by cyanide (Massey and Edmondson, 1970). The addition of
cyanide to disulfide bonds leads to the production of the thiocyanate group,
possibly by the following reaction:
/s /s-
PROTEIN I + CN~ »• PROTEIN
S SCN
Additional data suggest that persulfide formation may also be involved with
the following scheme at the active site of the enzyme:
PROTEIN— S—S~ 4- CN~ »• PROTEIN — S" + CNS"
4.3.4 Physiological Effects
When cyanide is added, the main effect first observed is inhibition of
respiration. This inhibition can ultimately result in death of the cell,
tissue, organ, or organism, presumably because no ATP is formed to serve
the many processes which require a continuous supply. Lack of ATP leads to
breakdown of many cellular functions and ultimately leads to death.
Because the major site of inhibition is in electron transport, cyanide
has been a useful tool for determining if various processes directly depend
on aerobic respiration. For example, cyanide causes a rapid depolarization
of the electrical potential of plant cell membranes (Anderson, Hendrix, and
Higinbotham, 1974; Higinbotham, Graves, and Davis, 1970). Depolarization
results from inhibition of an electrogenic pump (membrane-bound) apparently
caused by a decreased ATP concentration in the cell. The ATP decrease and
electropotential decay have similar half-times (Slayman, Lu, and Shane, 1970).
However, care must be used in interpreting data obtained with the use of
inhibitors. In this case, cyanide, an effective metal chelator, may also be
acting on a metalloprotein at the site of the pump (Anderson, Hendrix, and
Higinbotham, 1974).
There are few studies on the effects of low cyanide concentrations —
concentrations less than that which inhibits respiration. This lack of
reported data is probably due to the sensitivity of the cytochrome oxidase
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100
enzyme system in the plant and the effective metabolism of cyanide to
asparagine in a variety of plants (Section 4.2.1). However, some interest-
ing observations have been reported and are discussed in the following
sections.
4.3.4.1 Germination — Several reports have demonstrated that cyanide ex-
posure can increase or stimulate germination in several species. For
example, Mullick and Chatterji (1967) found that seeds of two legumes,
Clitoria ter-natea Linn, and Rhynaosia minima B.C., showed increased inhibi-
tion, germination, and early seedling growth when exposed to cyanide solu-
tions. The maximum effect was observed when the seeds were soaked for 24 hr
in 100 ppm NaCN at 35°C. Roberts (1969) summarized the data supporting oxi-
dation hypotheses which explain dormancy and the release from dormancy in
cereals. Presumably, most respiratory metabolism during early germination
is through the pentose phosphate pathway (PP). The terminal oxidase involved
is cyanide insensitive and, hence, germination would not be inhibited by
cyanide. A small amount of respiratory metabolism occurs by the Embden-
Meyerhof-Parnas glycolysis pathway and, thus, by the citric acid cycle and
the ETC; therefore, cyanide stimulation of germination could be explained
by an increased inhibition of this pathway, with shunting of metabolites
into other pathways, perhaps the PP pathway. The relative importance of the
PP pathway decreases during the later stages of germination (to different
extents in different species), and degrees of cyanide inhibition can be
observed at this time. The terminal oxidase, which is cyanide insensitive
and operates during the early stages of germination, has not been identified.
Hendricks and Taylorson (1972) found that potassium cyanide in concen-
trations from 0.003 to 3.0 mM (maximum at 0.1 mM) promoted seed germination
in lettuce (Lactuaa sativa L.) and pigweed (Amaranthus albus L.). They
suggested that blocking of the ETC promoted germination and that germina-
tion, therefore, may involve cyanide-insensitive respiration. They sup-
ported this hypothesis with data showing that aryl hydroxamates and thio-
cyanates, both of which inhibited cyanide-insensitive respiration in some
systems, also inhibited germination in Amavanthus and Laotuoa. The rela-
tion of these observations to operation of the PP pathway was not explored.
Whatever mechanism exists, the crucial observation is that cyanide inhibi-
tion of the ETC allows for an altered metabolism which, in an unknown
manner, stimulates seed germination in different species.
4.3.4.2 Anomalous Growth Response in Presence of Iron — Israelstam (1968)
reported that cyanide retarded growth of Phaseolus vulgar-is more severely
in the presence than in the absence of iron. Subsequent data showed that
a similar phenomenon occurred in wheat seedlings (Israelstam, 1970), al-
though in both wheat and bean, growth in the absence of cyanide was greater
than in its presence. In nutrient solution experiments, iron did not pre-
vent the uptake of cyanide (Kli4CN) (Alam and Israelstam, 1975). The pres-
ence of cyanide in the nutrient solution slightly decreased the amount of
chlorophyll in the leaves. Photosynthesis and respiration were inhibited
by cyanide to about the same extent as height growth. Cyanide plus iron
inhibited photosynthesis and respiration 63% and 70%, respectively, of
iron-treated controls. Cyanide without iron inhibited photosynthesis and
respiration 34% and 50%, respectively, of untreated controls. Similar
kinetics for the appearance of 1AC in leaves were observed in the presence
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101
and absence of iron; the chemical form of 14C in leaves was not determined.
Thus, the extent of metabolism of 1ACN could not be estimated. While these
observations are interesting, considerable work is necessary to determine
their importance.
4.3.4.3 Cyanide and the Translocation Process — Cyanide has been used to
examine the translocation of photosynthate by phloem. Like other physio-
logical processes inhibited by cyanide, the mode of transport inhibition
is probably by inhibition of ATP production (i.e., inhibition of cytochrome
oxidase). Pertinent questions concern whether cyanide exerts its effect
locally in the sieve tubes or is carried in the transpiration stream and
exerts its effect at the loading (source) site and whether the inhibitory
effect is reversible. Application of cyanide (gaseous and aqueous forms)
to stolons of Saxifraga sarmentosa L. inhibited transport of 137Cs and
natural li*C assimilates, but cyanide did not move to any great extent
toward the daughter plant (Qureshi and Spanner, 1973). The effect appar-
ently occurred locally within the sieve tube, not at the source or sink,
and was completely reversible.
4.3.4.4 Chromosomal Aberrations — The effects of cyanide on the occurrence
of chromosomal aberrations have been examined in conjunction with work
defining the mechanism of chromosomal mutation caused by radiation. D'amato
and Gustafsson (1947) observed that pretreatment of barley with 1 x lO"*4
and 1 x 10" 3 M KCN prior to x-irradiation increased the mutation frequency
in barley. However, pretreatments of 1 x 10~2 M KCN produced a lower
mutation frequency than in the water presoaked controls or dry seed series.
Lilly and Thoday (1956) found that cyanide and oxygen together produced an
inhibition of mitosis and a higher percentage of chromosomal aberration
in V-io-la fdba roots than that produced by the absence of oxygen. Treatments
of cyanide without oxygen and oxygen without cyanide produced no observable
increase in aberration frequency. Mikaelsen (1954) reported that 10"3 to
5 x 10"u M NaCN decreased the frequency of production of chromosome frag-
ments and chromosome bridges during exposure to gamma irradiation (11% to
25% protection). Kihlman (1957) and Kihlman, Merz, and Swanson (1957) con-
firmed Lilly and Thoday's results but also showed that Visia faba exposed
to x rays and KCN had a higher frequency of chromosome aberrations when
oxygen was absent. A threshold concentration of 5 x 10"= M KCN produced
this effect, while for aberrations produced by KCN plus oxygen in the ab-
sence of x rays about 10"A M was necessary. The stimulus for this work was
the old hypothesis that H202 was the agent responsible for chromosomal
aberrations produced by ionizing radiation. The justification for cyanide
use was its ability to inhibit cytochrome oxidase, peroxidase, and catalase
and thus to allow the buildup of peroxides in the cell.
4.3.4.5 Effects of Nitrile Herbicides — Many organic nitriles apparently
have herbicidal properties. The relationships between the cyano group and
herbicidal activity and among breakdown products of the herbicide are not
understood (Barnsley and Yates, 1962). Although the cyano group may be
important for herbicidal activity, HCN is not released from these compounds
and, thus, activity is due to the particular structural characteristics of
the whole molecule. All three herbicides apparently act as inhibitors of
electron flow or uncouplers in respiration and/or photosynthesis (Ashton
and Crafts, 1973).
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102
4.3.4.5.1 Dichlobenil — Dichlobenil (2,6-dichlorobenzonitrile) Is an
Inhibitor of germination and growth of plant parts which contain actively
dividing meristems (i.e., embryos, buds, and shoot and root meristerns).
Both monocotyledons and dicotyledons are affected. Dichlobenil is often
used as a preemergence herbicide for annual weeds in orchards and vineyards
(Ashton and Crafts, 1973). Because it produces little contact damage and
does not migrate to any depth in soil, trees and established crops with
deeper root systems are not affected.
Dichlobenil inhibits active transport and cell division and, thus,
growth. At lethal concentrations it produces a blackened appearance of
apical meristems (Ashton and Crafts, 1973). Although it has limited move-
ment in the plant, root treatment with dichlobenil decreases movement of
assimilates from leaves to buds, stems, and roots. Dichlobenil has been
observed to uncouple oxidative phosphorylation in mitochondria.
4.3.4.5.2 loxynil and bromoxynil — loxynil is a postemergence contact
herbicide used to control broadleaf plants that are frequent weeds in
cereal and grass crops and are resistant to 2,4-D (Ashton and Crafts, 1973).
Bromoxynil is used to control broadleaf plants that tolerate phenoxy her-
bicides. Both bromoxynil and ioxynil are applied when weeds are in their
early growth stages. Necrosis is the first visible symptom after bromoxynil
or ioxynil treatment. Eventually, chlorosis occurs in the surrounding areas
and in untreated leaves.
loxynil inhibits both respiration and photosynthesis. Stimulation of
02 evolution was found at concentrations of 5 x 10~8 to 5 x 10~7 M, while
inhibition of 02 evolution was found at 5 x 10~6 to 5 x 10"3 M (Paton and
Smith, 1967). Thus, uncoupling may occur at the lower concentrations and
blockage of electron flow may occur at higher concentrations. Low concen-
trations of ioxynil can also inhibit electron transport in photosynthesis,
perhaps at the level of plastoquinone. Herbicidal activity could be ex-
plained by either or both modes of action.
4.3.4.6 HCN as a Fundgant — Hydrogen cyanide released from Ca(CN)2 is used
as a fumigant for insect control in stored grain, tobacco and vegetable
plant beds, greenhouses, and soils. It is registered for use on almonds,
dried beans, citrus, cocoa beans, grains, nuts, dried peanuts, and spices
(Thomson, 1974). Details of the fumigation process are described in Lindgren
and Vincent, 1962.
The use of HCN is presently restricted and tolerance limits have been
set for a variety of vegetables (Section 7.7, Table 7.4). Waldron, Robb,
and Sleesman (1970) showed that greenhouse tomato fruits did not build up
residues of cyanide after fumigation and airing. Various factors, however,
affect the penetration of the fumigant into the fruit or vegetable and its
retention within the food (Sinclair and Lindgren, 1958).
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103
Little useful information was found on the chemical form of cyanide in
fumigated foods or on metabolic conversions in these foods. Lopez-Roman,
Barkley, and Gunther (1971) reported that the water solubility of HCN limits
its use on many vegetables and most fruits other than citrus because in
contact with moist commodities it usually causes burning, discoloration,
wilting, and flavor changes, which render the commodities unmarketable.
These authors demonstrated that citrus fruits can sorb considerable amounts
of HCN, as measured by the decrease in HCN concentration in the fumatorium.
These results were compared to an empty fumatorium control to determine
sorption onto the fumatorium walls. No determinations of the chemical form
of cyanide in the fruit were made. Desorbtion of cyanide was small over a
90-min period. Lindgren and Vincent (1962) reported that much of the sorbed
HCN will be released from food due to its high vapor pressure. However, at
pH 7 and above, some conversion to metal salts will occur and these salts
will be stable within the food.
Page and Lubatti (1948) demonstrated that fumigation of dried fruit
with HCN converted fructose into the corresponding cyanohydrin. The pro-
portion of cyanohydrin formed increased dramatically with moisture content
of the dried fruit. Even 50 days after fumigation, the total cyanide content
was quite high.
Cyanide used as a preservative for raw vegetables may inhibit some
enzymes that affect flavor and quality. Lipoxygenase catalyzes peroxida-
tion of lipids and is thought to be responsible for quality deterioration
in vegetables (Flick, St. Angelo, and Ory, 1975). Lipoxygenase was inhibited
by cyanide to a greater extent in homogenates of purple eggplants than in
homogenates of white or green eggplants.
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SECTION 4
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Illuminated Grana or Algae. FEES Lett. (Amsterdam) 59(2):162-166.
-------
109
67. Qureshi, F. A., and D. C. Spanner. 1973. Cyanide Inhibition of Phloem
Transport along the Stolon of Saxi-fvaga sarmentosa L. J. Exp. Bot.
24(81):751-762.
68. Radeleff, R. D. 1970. Cyanides and Cyanogenetic Plants. In:
Veterinary Toxicology, 2nd ed. Lea and Febiger, Philadelphia, Pa.
pp. 50-58.
69. Rangaswami, G., and A. Balasubramanian. 1963. Release of Hydrocyanic
Acid by Sorghum Roots and Its Influence on the Rhizosphere Microflora
and Plant Pathogenic Fungi. Indian J. Exp. Biol. (India) 1:215-217.
70. Relimpio, A. M., P. J. Aparicio, A. Paneque, and M. Losada. 1971.
Specific Protection against Inhibitors of the NADH-Nitrate Reductase
Complex from Spinach. FEES Lett. (Amsterdam) 17(2):226-230.
71. Ressler, C., Y. H. Giza, and S. N. Nigam. 1963. Biosynthesis and
Metabolism in Species of Vetch and Lathyrus of y-Glutamyl-g-cyano-
alanine: Relation to the Biosynthesis of Asparagine. J. Am. Chem.
Soc. 85:2874-2875.
72. Roberts, E. H. 1969. Seed Dormancy and Oxidation Processes. In:
Dormancy and Survival, Symposia of the Society for Experimental
Biology, No. 23. Academic Press, New York. pp. 161-192.
73. Robinson, T. 1975. The Organic Constituents of Higher Plants, 3rd ed.
Cordus Press, North Amherst, Mass. pp. 320-323.
74. Schneider, E. A., and F. Wightman. 1974. Metabolism of Auxin in
Higher Plants. Annu. Rev. Plant Physiol. 25:487-513.
75. Seigler, D. S. 1975. Isolation and Characterization of Naturally
Occurring Cyanogenic Compounds. Phytochemistry (Great Britain) 14:9-29.
76. Seigler, D. S., C. S. Butterfield, J. E. Dunn, and E. E. Conn. 1975.
Dihydroacacipetalin — A New Cyanogenic Glucoside from Aoac-ia s'iebeT'iana
var. woodii. Phytochemistry (Great Britain) 14:1419-1420.
77. Sinclair, W. B., and D. L. Lindgren. 1958. Factors Affecting the
Fumigation of Food Commodities for Insect Control. J. Econ. Entomol.
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Membrane Potential and ATP Concentrations in Neurospor>a. Nature
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-------
110
79. Tantisewie, B., H.W.L. Ruijgrok, and R. Hegnauer. 1969. Die Verbrei-
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Verbindungen bei den Parletales und bei Einigen Weiteren Sippen
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-------
SECTION 5
BIOLOGICAL ASPECTS IN WILD AND DOMESTIC ANIMALS
5.1 SUMMARY
As a general respiratory poison, cyanide would be expected to be
toxic to many organisms that depend on aerobic respiration for life. It
is rapidly absorbed from the gastrointestinal and respiratory tracts and
has behaved similarly in all organisms tested. However, threshold levels
do vary widely with different species.
The major threat of cyanide poisoning to livestock is through the
ingestion of plants containing cyanogenic glycosides. The effects can
vary from acute to chronic intoxication.
Cyanide generally is toxic to fish at 0.1 ppm. Species variations,
of course, do occur with regard to cyanide sensitivity. Although cyanide
is toxic to birds, there is little recent information on the effects of
cyanide in birds. Invertebrates are also poisoned by cyanide; however,
the toxicity levels, in most cases, are not well defined.
5.2 MAMMALS
5.2.1 Metabolism
Metabolism of hydrogen cyanide and its various salts by mammals is
discussed in Section 6.2. Absorption readily occurs through inhalation,
ingestion, or skin contact. The greatest danger to wild or domestic ani-
mals is through either ingestion of plants with a high cyanogenic glyco-
side content or deliberate poisoning by man.
5.2.2 Toxic Effects
This section discusses cyanide poisoning in animals that have eaten
forage with a high cyanogenic glycoside content. Microbes in the ruminal
fluid of sheep can hydrolyze cyanogenic glycosides to release free hydro-
gen cyanide (HCN) (Coop and Blakely, 1949). The hydrolytic enzyme pres-
ent in the plant will also release some cyanide during digestion, but this
amount is small compared to that released by the rumen microbes. Free
HCN is released when ruminal fluid from the cow is incubated with glyco-
sides or trefoil plant leaves containing glycosides (Bansal and Seaney,
1970). HCN is released in ruminant and nonruminant animals' stomachs.
As a result animals grazing on sorghums, Sudan grasses, corn, and other
cyanogenic plants may be poisoned. These animals manifest initial excit-
ability with generalized muscle tremor; polypnea and dyspnea follow. They
may also salivate, lacrimate, defecate, and urinate. Ultimately, there
is loss of righting reflex, pupillary dilatation, gasping, and convul-
sions. Death can occur quickly (Burrows and Way, 1977), depending on
the dose administered.
Ill
-------
112
In acute cyanide poisoning, the effects are usually obvious. The
animal is in severe distress and either dies or rapidly recovers. Chronic
poisoning, however, is more difficult to diagnose. The often subtle ef-
fects may result secondarily from complications which develop from long-
term cyanide ingestion. Horses grazing on Sudan grass and hybrid sorghums
in the southwestern United States developed a syndrome with clinical signs
including posterior ataxia, urinary incontinence, cystitis, and myeloma-
lacia of the lower spinal cord (Van Kampen, 1970). Offspring of mares
that had eaten Sudan grass during early pregnancy developed musculoskel-
etal deformities. Sudan grass and hybrid sorghums are known to be cyano-
genic plants, but the disease symptoms were similar to lathyrism, which
occurs from the ingestion of lupine or related plants. Van Kampen (1970)
suggested that sorghum may contain lathyrogenic compounds since it pos-
sesses the metabolic precursors (Section 4.2.5).
A secondary effect from ingesting cyanogenic glycosides in sorghum
and Sudan grass may be induced sulfur deficiency, which results when a
significant proportion of the sulfur ingested by an animal is used to
detoxify the cyanide released from these plants. Wheeler, Hedges, and
Till (1975) found that sheep grazing on sorghum and Sudan grass forage
gained significantly more weight when they were given supplemental sul-
fur. Sheep with access to salt licks containing 8.5% sulfur (average
daily sulfur intake 0.82 g/sheep) gained up to 88% more live-weight than
control sheep with access to licks containing 0.1% sulfur (average daily
sulfur intake 0.01 g/sheep). The authors suggested that a sulfur defi-
ciency might be suspected if animals fail to gain weight when grazing on
young sorghum species with high HCN content.
A thyroid dysfunction occurring in sheep feeding on star grass
(Cynodon pleatostaahyum), a plant with a high cyanogenic glycoside and
low iodine content, is similar to a condition reported in some human pop-
ulations (Section 6.3.3.2). In this case, the sheep developed enlarged
thyroid glands and gave birth to lambs which were either stillborn or died
shortly after birth (Herrington, Elliott, and Brown, 1971). Star grass
only produces hypothyroidism during the rainy season when the cyanogenic
glycoside content is high (180 ppm as HCN). According to Herrington,
Elliott, and Brown (1971), an iodine and mineral supplement can prevent
the effect of the glycosides.
5.3 FISH
5.3.1 Metabolism
Few data were found on the metabolism of cyanide in fish. Uptake
and absorption of cyanide are apparently rapid since lethal quantities of
cyanide cause death within minutes. Detoxification mechanisms are not
well described. Doudoroff (1976) cited data showing that thiosulfate
administered in the water with cyanide reduced the toxicity of cyanide.
Presumably, the thiosulfate would increase the detoxification rate of
cyanide to thiocyanate.
-------
113
5.3.2 Effects
Cyanide release to waters is responsible for several fish kills each
year (U.S. Federal Water Pollution Control Administration, 1964-1969).
Most cyanide kills are usually through accidental release of cyanides to
the environment rather than inadequate treatment procedures (Section 7.5).
For example, a newspaper reported a fish kill in Melton Hill Lake near
Oak Ridge, Tennessee, on August 22, 1975, which was attributed to cyanide
from a metal-plating industry (Anonymous, 1975). In addition to pollu-
tion from human activities, decomposing plant materials which naturally
occur in surface waters may contribute to the cyanide content of the water.
The extent of this contribution is not known, however. Because industrial
effluents containing cyanide may find their way into waterways, much in-
formation has been collected on toxicity of various cyanides to aquatic
organisms. Table 5.1 lists a wide range of toxic levels for different
fish. Concentrations greater than 0.1 ppm are toxic in most cases. Fig-
ure 5.1 demonstrates that the median lethal concentration of sodium cya-
nide for five species of freshwater fish decreased only slightly with
increasing exposure time (Cardwell et al., 1976). Goldfish were found
to be less sensitive than the other four species.
ORNL-DWG 76-15521
A FATHEAD MINNOW
• BLUEGILL
• BROOK TROUT
A CHANNEL CATFISH
O GOLDFISH
10 20 50
EXPOSURE TIME (hr)
100
200
500
Figure 5.1. Relationship between median lethal concentration (LC50)
of sodium cyanide as CN" and exposure time for five species of freshwater
fish. Source: Adapted from Cardwell et al., 1976.
The toxicity of cyanides varies with temperature, dissolved oxygen,
mineral concentration, and pH of the water (Doudoroff, 1976; National
Academy of Sciences and National Academy of Engineering, 1972). The
exact response differs among various species of fish.
-------
114
TABLE 5.1. TOXICITY OF CYANURATES AND CYANIDES TO FISH
Chemical
compound
2- (2-Butoxyethoxy)
ethyl thiocyanate
l,3,3-Trimethyl-2-
norbornyl thio-
cyanate and
isobornyl thio-
cyanate
Octyl thiocyanate
2 , 4-Dinitrophenyl
thiocyanate
Methanediol
thiocyanate
p-Dimethyl-
aminophenyl
thiocyanate
Phenyl p-dimethyl-
aminiso
thiocyanate
Allyl iso-
thiocyanate
Ammonium
thiocyanate
Cyanide
Test
organism
Cyprinus carpio
C. carpio
C. carpio
(7. carpio
C. carpio
C. carpio
C. carpio
C. carpio
Kuhlia sandvicensis
K. sandvicensis
K. sandvicensis
Carassius auratus
Gambusia af finis
G. affinis
G. affinis
K. sandvicensis
Lepomis sp .
Tinea tinea
Lepom-is auritus
Lepomis macrocnirus
K. sandvicensis
L. macrochirus
L. macrochirus
L. macrochirus
Lebistes reticulatus
L. reticulatus
L. reticulatus
L. reticulatus
Notemigonus
crysoleucas
Micropterus
salmoides
M-icropterus dolomieu
Pomoxis annularis
Pimephales promelas
Salvelinus
fontinalis
Trout
Trout
Trout
Fish^
Fish
Fish°
Fish
Test
conditions
FW, LS
FW, LS
FW, LS
FW, LS
FW, LS
FW, LS
FW, LS
FW, LS
SW, LS
SW, LS
SW, LS
FW
SB, FW, LS
SB, FW, LS
SB, FW, LS
SW, LS
FW
FW
CB, SB, FW, LS
FW
CB, SB, FW, LS
SB, FW, LS
FW
CB, FW, LS
CB, FW, LS
CB, FW, LS
CB, FW, LS
CB, SB, FW, LS
CB, SB, FW, LS
CB, SB, F¥, LS
CB, SB, FW, LS
CB, SB, FW, LS
FW
FW
FW
FW, FS, LS
(river)
FW, FS, LS
FW, FS, LS
FW, FS (river)
FW, FS (river)
FW, FS (river)
FW
Concentration
(ppm)
79, 93
112
118, 131, 135
101, 104, 143
140, 310
74, 96, 104
164, 174, 178
129, 187, 202
0.05
0.1
1 . 0
1600
910
420
114
20.0
280-300
1600
0.06^
0.12-0.18
0.06"
0.18
0.15
0.42
0.28
0.26
0.24
0.06C'
0.066'
0.06°
0.06°
0.06°
0.1
0.2
0.08
0.05
0.02
1.0
>0.1
0.05-0.1
0.03
0.53-0.65
Remarks
No effect, 67 hr
Death <60 hr*
No effect, 43 hr*
No effect, 48 hr*
No effect, 91 hr
No effect, 44 hrfc
No effect, 46 hr*
No effect, 45 hr*
Slight irritant response
24-hr lethal limit
24-hr TLm, acute; high
turbid water, 16-23 °C
48-hr !!,„, turbid water
96-hr Tiro, turbid water
No observable response
(2-min exposure)
Killed in 1 hr
24-hr lethal limit
96-hr !!,„, acute; hard and
soft water
96-hr TL,,,, acute
96-hr TLm
50% kill in 20 hr; pH 7.25;
24-24. 5 °C
50% kill in 30 hr; pH 7.25;
24-24. 5 °C
50% kill in 43 hr; pH 7.25;
24-24. 5 °C
<407. kill in 80 hr; pH 7.25;
24-24. 5 °C
Overturned in one day
Overturned in 50 min
Causes immediate signs of
distress
Will kill all fish in 130-
136 hr
No kill in 27 days
Will kill in 20 min
Lethal to all fish
Lethal to most game fish
Lethal to some fish
Toxic; toxicity increased
over temperature range
of 1.2 to 25.4°C
(continued)
-------
115
TABLE 5.1 (continued)
Chemical
compound
Cyanide (CN~)
Cyanide (a)
zinc (b)
(Zn++)
Cyanides
(metal complexes)
n-Butyl carbitol
thiocyanate
Hydrocyanic acid
Hydrogen cyanide
Methyl iso-
thiocyanate
p-Chlorophenol
isocyanate
Potassium
cuprocyanide
Potassium cyanate
Potassium cyanide
Test
organism
L, macrochirus
L. macrochirus
L. macrochirus
L. macrochirus
L . macrochirus
L. macrochirus
L. macrochirus
L. macro chirus
K. sandvicensis
La.god.on rhomboides
L. rhomboides
Freshwater fish
(cyprinids)
L. rhomboides
L. rhomboides
Salmo gai.Td.neTi,
K. sandvicensis
K. sandvicensis
K. sandvicensis
Rhinichthys
atratulus
/?. atratulus
P. atratulus
B. atratulus
R. atratulus
R. atratulus
K. sandvicensis
Brachydanio rerio
B. rerio
C. auratus
G. af finis
K. sandvicensis
K. sandvicensis
K. sandvicensi s
L. macrochirus
L. macrochirus
L. macrochirus
Test
conditions'3
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SW,
SB,
SB,
SB,
SB,
SB,
SB,
SW
SW,
SW,
SW,
CB,
CB,
CB,
CB,
CB,
CB,
SW,
SB,
SB,
SB,
SB,
SW,
SW,
SW,
CB,
CB,
CB,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
SW,
SW,
FW,
SW,
SW,
FW,
LS
LS
LS
LS
FW,
FW,
FW,
FW,
FW,
FW,
LS
FW,
FW,
FW,
FW,
LS
LS
LS
FW,
FW,
FW,
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
Concentration ^ ,
, , Remarks
(ppm)
0.18
0.07
0.24
0.26 (a)
3.90 (b)
0.16 (a)
2.43 (b)
20.0
0.069
0.10
0.069
0.05
0.07
20 0
0.1
1.0
20.0
0.38
0.47
0.71
0.53
0.69
1.10
20.0
0.49
=11.7
0.1-0.3
1.6
0.1
1.0
10.0
0.55
0.45
0.57
96-hr Tim, acute
No kill in 96 hr
Total kill in 96 hr
96-hr TL^, acute
No kill in 96 hr
Toxicity is generally a
function of molecular
HCN level.
Violent reaction, 2-min
exposure
24-hr Tim, acute
All fish died
Lowest lethal molar concen-
tration was 7.7 x 10~6-
24-hr TL^, acute
Maximum dose, no kill
48-hr TI^, acute; toxicity
related to molecular HCN
exposure
Slight irritant response,
2-min exposure
Violent irritant response,
2-min exposure
No observable response,
2-min exposure
24-hr TI^, acute; =20°C,
pH 7.8-8.0; CN to Cu ratio
of 4.0
24-hr TL,,,, acute; =20°C,
pH 7.8-8.0; CN to Cu ratio
of 3.7
24-hr T^, acute; =20°C,
pH 7.8-8.0; CN to Cu ratio
of 3.0
4-hr Tim, acute; CN to Cu
ratio of 4.0
4-hr TLm, acute; CN to Cu
ratio of 3.7
4-hr TI^, acute; CN to Cu
ratio of 3.0
No observable response,
2-min exposure
48-hr Tim, acute; adults,
24°C, soft water
48-hr TL^,; eggs, 24°C,
soft water
Killed in three to four
days , hard water
48-hr T^, acute; high
turbid water
Slight irritant response,
2-min exposure
Moderate irritant response,
2-min exposure
Violent irritant response,
2-min exposure
96-hr TL^, acute; small fish
96-hr Tim, acute; medium
fish
96-hr TLm, acute; large fish
(continued)
-------
116
TABLE 5.1 (continued)
Chemical Test
compound organism
S. trutta
S. trutta
S. toutta
S. fontinalis
S. fontinalis
S. fontinalis
Trout
Trout
7±shd
Fishrf
Potassium K. sandvicensis
thiocyanate
K. sandvicensis
Sodium cyanide Anguilla ctnguilla
C. auratus
C, auratus
C. carpio
GasteTosteus
aculeatus
Ictalurus melas
Ictalurus natalis
K. sandvicensis
K. sandvicensis
Lepisosteus osseus
Lepomis cyanellus
L. cyanellus
L. cyanellus
L. cyanellus
L. cyanellus
L. cyanellus
L. macrochirus
M. salmoid&s
Minnows
Minnows
Minnows
N. ci>ysoleucas
Phoxinus phoxinus
P. promelas
P. promelas
P. promelas
S. trutta
Sodium cyanide L. macro chirus
Total CN (a) L. macrochirus
Molecular HCN (b) L. macrochirus
L. macrochirus
L. macrochirus
L. macrochirus
Test a
conditions
CB,
CB,
CB,
SB,
SB,
SB,
FW
FW
FW,
FW,
SW,
SW,
CB,
CB,
SB,
SB,
CB,
SB,
SB,
SW,
SW,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
FW,
FW,
FW,
SB,
CB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
FW,
FW,
FW,
FW,
FW,
FW,
LS
IS
LS
LS
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
LS
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
LS
LS
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
FS, LS
FS, LS
FS, LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
Concentration Remarks
(ppm)
0.1
0.5
1.0
0.009
=0.09
0.05-0.08
0.11
0.11
0.25-0.35
0.6-1.0
10.0
20.0
0.49
4.9
1.0
1.0
0.49
0.25
1.0
2.0
1.0
1.0
0.25
1.0
0.5
5.0
1.0
<0.5
0.15
1.0
0.3
0.5-0.7
0.8
0.25
0.49
0.35
0.23
0.24
0.49
0.54 (a)
0.50 (b)
0.96 (a)
0.90 (b)
0.72 (a)
0.62 (b)
Threshold dose, 45 min;
15.6 Ch
Threshold dose, 13 min;
15.6 Cft
Threshold dose, 8.5 min;
15.6 Ch
Reduced ability to swim by
507.; 8-10°C
48-hr TL^,, acute; 8-10°C
Minimum lethal concentra-
tion; 8-10 °C
Loss of equilibrium in 2
hr; 7-9°C, dissolved
oxygen 11 ppm
Loss of equilibrium in 10
min; 7-9 °C, dissolved
oxygen 3 ppm
Minimum lethal dose; hard
water, 20°C
Minimum lethal dose;
distilled water, 20°C
No observable response,
2-min exposure
Slight reaction; 2-min
exposure
Killed in 12 hr; 17-18 °C
Killed in 12 hr; 17-18 °C
100% kill in 5-48 hr; 26 °C,
PH 7.2-8.7
100% kill in 5-14 hr; 26 °C,
pH 7.3-7.5
Killed in 8 hr; 17-18 °C
Not lethal in 72 hr; 24.4°C
100% kill in 5-10 hr; 24-26°
C, pH 7.0-8.2
Violent reaction, 2-min
exposure
Slight reaction, 2-min
exposure
100% kill in 3.4-4.2 hr;
23°C, pH 7.3-7.8
Lethal to most in 72 hr;
24.4°C
100% kill in 1.0-2.8 hr;
pH 5.5-9.0
100% kill in 4.1-6.0 hr,
24-26°C, pH 7.6-8.0
Moderately effective as
repellant (lake study)
Avoidance response
No response
96-hr TL^, acute; hard water
100% kill in 0.7-1.4 hr;
23-27°C, pH 7.0-8.9
No effect in 24 hr
25% mortality in 24 hr
100% mortality in 24 hr
Not lethal in 72 hr; 24.4°C
Killed in 6 hr; 17-18°C
96-hr TLm, acute; hard
water, 25 °C
96-hr Tl^, acute; soft
water, 25 °C
48-hr TI^,, acute
Killed in 2 hr; 17-18 °C
129 min, median resistance
time
50 min, median resistance
time
91 min, median resistance
time
(continued)
-------
117
TABLE 5.1 (continued)
Chemical
compound
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
M.
M.
M.
Test
organism
macrochirus
macrochirus
macrochirus
macroahirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
macrochirus
dolomieui
dolomieui
dolomieui
Rasbora heteromorpna
R.
K.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
Sa
S.
S.
S.
S.
S.
S.
atratuius
atratuius
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
gairdneri
Imo trutta
trutta
trutta
trutta
trutta
trutta
trutta
Test
... a
conditions
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
CB,
CB,
CB,
SB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
SB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
CB,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
Concentration
(ppm)
0.
5.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
0.
0.
0.
0.
0.
2.
0.
0.
0.
0.
5.
10
0.
16
0
28
42
35
45
33
47
33
44
39
45
45
12
1
2
0
074
22
26
15
16
0
3
2
1
105-0.155
0
.0
31
0.14
0.
0.
1.
2.
0.
0.
1.
2
5
0
0
1
5
0
Remarks
48-hr TL^, acute, adults;
24°C, soft water
Lethal in 1 hr
24- and 48-hr TL^,, acute;
20°C
96-hr TL^; hard water, 18°C
96-hr TL,,,; hard water, 30°C
96-hr I!,,,; soft water, 18°C
96-hr TL,,,; soft water, 30°C
48-hr TL^,; soft water, 18°C
48-hr TLm! soft water, 30°C
48-hr TLm; hard water, 18°C
48-hr HOT; hard water, 30°C
96-hr T!OT, acute; toxicity
is dependent on temperature
dissolved oxygen, and
hardness of water
96-hr TLm, acute; "normal"
dissolved oxygen
96-hr TLm, acute; "low"
dissolved oxygen
Threshold dose, 200 min;
21.1°Ce
Threshold dose, 30 min;
21.1°Ce
Threshold dose, 14 min;
21.1°Ce
20% kill in seven days
24-hr TI^, acute; =20°C,
pH 7.7-8.0
4-hr TI^
28. 8-min mean survival time
when acclimatized 24 hr in
test tank; 50. 8-min mean
survival time when accli-
matized 191 hr in test
tank
39.0-min mean survival time
of smallest fish (5.5-6.25
cm); 16.0-min mean survi-
val time of largest fish
(16.5-17.25 cm)
2.7-min mean survival time;
17-18°C, pH 7.4-8.0
8. 8-min mean survival time;
17-18°C, pH 7.4-8.0
12.1-min mean survival time;
17-18°C, pH 7.4-8.0
2523-min mean survival time;
17-18°C, pH 7.4-8.0
Lethal; one-half died in 10
hr at <5 ppm; 17°C, pH
7.8-8.2; survival time
increased at higher
dissolved oxygen levels
Lethal in 1 hr
Total kill <3 min, acute;
17.5°C
Total kill in <10 min, acute
Total kill in 165 min, acute
62 min, mean death time;
15. 6° C/
16 min, mean death time;
15. 6° C/
8.5 min, mean death time;
15 . 6° C/
5 min, mean death time;
15 . 6° C/
Threshold dose, 300 min;
Threshold dose, 30 min;
15.6°C2
Threshold dose, 12 min;
15.6°C9
(continued)
-------
118
TABLE 5.1 (continued)
Chemical
compound
Isobornyl
thiocyanate
Isobornyl thio-
cyanoacetate
Thiocyanic acid
(various esters)
Test
organism
L. macrochirus
L. maeroch-irus
K. sandv-icensis
K. sandvicensis
I. punctatus
I. melas
K. sandvicsnsis
X. sandvicensis
I. cyanellus
L. macFochirus
N. crysol&uaas
S. gairdner-i
Ptyc'hoche-i'Lus
oregonens-is
Oncorhynchus kisutch
0. tshauytscha
S. gairdneri
Test
conditions
SB,
SB,
SW,
SW,
SB,
SB,
SW,
SW,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
FW,
FW,
LS
LS
FW,
FW,
LS
LS
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
Concentration ., .
Remarks
(ppm)
0.17 (a)
0.155 (b)
1.0
0.2
1.5
0.1
1.0
0.6
0.4
1.5
<0.7
10.0
10.0
10.0
10.0
700 min, median resistance
time
Violent reaction, 2-min
exposure
Slight reaction, 2-min
exposure
24-hr TLm, acute
24-hr TLm, acute
No irritant response;
2-min exposure
Violent irritant response;
2-min exposure
24-hr TLy,, acute
24-hr TL^, acute; data at
20-23 C, no increase in
kill after 24 hr
24-hr TL^, acute
24-hr TI^, acute; 11 °C
Acute death in 24 hr
Acute death in 24 hr
Acute death in 24 hr
Acute death in 24 hr
aSB - static bioassay; CB constant-flow bioassay; FW = fresh water; SW = sea (salt) water; LS lab
study- FS = field study.
^Concentration is dose, milligrams per kilogram, force-fed; 18.3°C.
GNo species survived more than 10 hr at doses over 0.06 ppm.
"Species not given.
eAll data from graph; dissolved oxygen near saturation, =8.8 to 9.8 ppm.
/Data from graph; alkalinity and pH had little effect over ranges studied; dissolved oxygen 8.1 to
9.0 ppm.
i?Data from graph; dissolved oxygen near saturation, ^9.0 to 10.0 ppm.
^Data from graph; dissolved oxygen near 5.0 ppm.
Source: Adapted from Becker and Thatcher, 1973, Table J, pp. J.2-J.10. Data collected from several
sources.
In a review of cyanide effects on fish, Doudoroff (1976) concluded
that fish are more susceptible to cyanide at reduced oxygen concentrations.
This susceptibility is more noticeable with lower levels of cyanide (about
0.1 ppm). If the dissolved oxygen concentration of the water is reduced
from 10 ppm to 4 ppm, the toxicity of cyanide increases by a factor of
about 1.4, possibly because the fish must pump more water through their
gills to obtain enough oxygen (Skidmore, 1974). Morgan and Kiihn (1974)
measured the breathing rate of largemouth bass and found a rapid rate
increase in response to a sublethal cyanide concentration.
Generally, an increase in water temperature increases the metabolic
rate of fish, which, in turn, increases the toxicity of a respiratory
poison such as cyanide (Doudoroff, 1976); however, there are some excep-
tions. Toxicity also varies with fish species (Table 5.1), age, stage in
life cycle, activity, metabolism, and acclimatization (Doudoroff, 1976;
Skidmore, 1974). Doudoroff has summarized the relationships among water
temperature during exposure, acclimation temperature, and toxicity. Ex-
cept for the above generality, no further simple relationships are
apparent.
No general relationship between body size and resistance of a given
fish species to cyanide is apparent (Doudoroff, 1976). For example,
Anderson and Weber (1973) reported an excellent linear correlation be-
-------
119
tween the lethal response to cyanide and the cyanide concentration in
comparisons of groups of male guppies of similar weight. The linear cor-
relation between cyanide concentration and response in fish of different
weight classes was poor. Manipulation of the data, however, did produce
a linear correlation between concentration and the logarithm of the cya-
nide concentration divided by a fish weight factor. Doudoroff cited other
examples in which resistance to a given cyanide concentration was not
related to fish size.
The response to a given concentration of cyanide can be modified by
previous exposure (Doudoroff, 1976). Neil (1957, cited in Leduc, 1966)
found that brook trout exposed to sublethal cyanide concentrations of
0.01, 0.03, or 0.05 mg/liter as CN~ survived longer than controls when
they were subsequently exposed to KCN at 0.4 to 0.5 mg/liter as CN~. When
challenged at 0.3 mg/liter as CN~, however, brook trout previously exposed
to 0.03 or 0.05 mg/liter died sooner than controls. Trout previously ex-
posed to 0.01 mg/liter lived longer than controls. Brockway (1966, cited
in Leduc, 1966) obtained results with cichlids which were also difficult
to interpret. Juvenile cichlids exposed to 0.02, 0.06, or 0.10 mg/liter
cyanide as HCN were more resistant than controls to challenge with rela-
tively low concentrations of cyanide. Those previously exposed to 0.1
ppm cyanide as HCN were less resistant than controls when challenged with
relatively high lethal levels.
Natural waters which receive industrial wastes contain a mixture of
pollutants and the contribution of each component to total toxicity is
difficult to evaluate. Zinc and cyanide are reported to be antagonistic
(Cairns and Scheier, 1968; Chen and Selleck, 1969). Marking and Dawson
(1975) presented a method in an attempt to describe additive toxicity of
chemicals in water and to assign significance to the additive toxicity
index. Using the data of Cairns and Scheier (1968) on zinc and cyanide
toxicity to fathead minnows, Marking and Dawson (1975) calculated an
additive index of -1.37, which indicated considerable antagonism. This
should be expected as the zinc ion forms a stable complex with cyanide.
Brown (1968) described another method to attempt to estimate the
toxic potential of a mixture of poisons by dividing the concentration of
each poison in a mixture by its individual 48-hr LC50 and summing these
proportions. If the sum is less than one, less than half the fish would
die in 48 hr and if greater than one, more than half would die in 48 hr.
The values in Table 5.2 were determined for rainbow trout. In spite of
obvious problems with this method such as unequal changes in toxicity of
various components of the mixture with changing water quality (hardness,
pH, alkalinity-, dissolved oxygen, etc.), it is felt that it may serve as
a guide but does not substitute for actual tests.
Doudoroff (1956, 1976) and Doudoroff, Leduc, and Schneider (1966),
recognizing that industrial effluents often contain cyanide in associa-
tion with heavy metals (e.g., electroplating and metal-finishing plant
wastes), examined the effect of cyanide-heavy metal complex formation on
toxicity to fish. The toxicity of cyanide complexes, of course, varies
considerably. In most cases, the toxicity is less than that of a compar-
-------
120
TABLE 5.2. ESTIMATION OF THE TOXIC PROPORTION OF EACH POISON
AND THE TOTAL TOXICITY OF THE MIXTURE FOR RAINBOW TROUT
Poison
Ammonia
Phenol
Zinc
Copper
Cyanide
Sum of the proportions
Concentration
in water
(mg/liter)
6.3
1.39
0.62
0.12
0.01
48-hr LC50
(mg/liter)
31.13
4.6
2.8
0.4
0.07
Proportion
of
48-hr LC50
0.20
0.30
0.22
0.30
0.17
1.19
Source: Adapted from Brown, 1968, Table 2, p.
by permission of the publisher.
731. Reprinted
able amount of free cyanide. It has been suggested that HCN alone often
determines the toxicity of solutions of complex cyanides (Doudoroff,
Leduc, and Schneider, 1966; Doudoroff, 1976). Because of the dissocia-
tion of the metallocyanide complex, free HCN would always be present in
equilibrium with the complex. It would be difficult to dissociate the
toxicity due to the metallocyanide complex itself from cyanide.
Formation of the nickelocyanide complex markedly reduces the toxic-
ity of both cyanide and nickel. This complex is reasonably stable and is
not decomposed by direct sunlight; however, dissociation occurs in dilute
solution at an acid pH. For example, the toxicity to fish of the nickel-
ocyanide complex (NaCN combined with NiSOA) increased tenfold when the
pH of the test solution was reduced from 7.8 to 7.5 (Doudoroff, 1956). A
pH change from 8.0 to 6.5 increased the toxicity more than a thousandfold.
The TL50 value was 0.75 mg complex per liter at pH 6.5 and 1300 mg com-
plex per liter at pH 8. The basis for this observation is that a lower-
ing of the pH value enhances the dissociation of the metallocyanide
complex to form hydrogen cyanide.
Copper, which is fatal to fish at 1 ppm in soft water, can be detox-
ified by complexing with cyanide, as can the very toxic synergistic mix-
ture of copper and zinc. In extremely soft water with thorough aeration,
copper ions can be released from the cyanide complex and the solution
becomes toxic.
One additional factor affecting the toxicity of a cyanide compound
to fish is decomposition of wastes to HCN. Burdick and Lipschuetz (1970)
reported a fish kill in a New York stream resulting from ferrocyanide and
ferricyanide in concentrations well below the lethal concentration [up to
8732 ppm potassium ferrocyanide was nontoxic to trout in a 1-hr exposure
(Ellis, 1937, cited in Burdick and Lipscheutz, 1970)]. This enhanced
toxicity is attributed to the photodecomposition of these compounds lib-
erating HCN (Section 2.2.8.2). Concentrations of potassium ferrocyanide
-------
121
or ferricyanide as low as 2 ppm were rapidly toxic to fish (Rhi-ni-chthys
atvatulus atratulus, Semot-ilus atromaoulatus atromaculatus, and Hybo-
gnathus regi-us).
The literature on the toxicity of nitriles to fish has been reviewed
by Doudoroff (1976). The toxicity of lactonitrile is similar to that of
cyanide because it undergoes rapid hydrolysis in water to free cyanide.
Median tolerance limits for acrylonitrile are in the 10 to 50 ppm range
for several species of fish. The lethal effects of acrylonitrile may be
attributed to factors other than the liberation of cyanide. Toxicity
of other nitriles appears to be due predominantly to the organic nitrile
itself rather than to cyanide.
5.4 BIRDS
5.4.1 Metabolism
There is a paucity of information on the uptake, absorption, and
distribution of cyanide in birds. Also, there is little information con-
cerning the excretion of cyanide or its metabolites by birds.
5.4.2 Effects
There is surprisingly little information on acute and chronic toxic
effects of cyanide on birds. A bird kill, possibly due to ingestion of
cyanogenic plant material, was reported in British Columbia (Cameron,
1972).
5.5 INVERTEBRATES
5.5.1 Metabolism
The details of cyanide uptake, absorption, transport, and biotrans-
formation within invertebrates are not well known. Detoxification of
cyanide in some groups of invertebrates may be similar to that found in
mammals (National Academy of Sciences and National Academy of Engineering,
1972). Thiocyanates are formed in mollusks, parasitic helminthes, and
insect larvae of Gasterophilus equi (Khan and Bederka, 1973). Rhodanese,
the enzyme converting cyanide to thiocyanate, is also found in blowflies.
There are not enough data, however, to generalize that all members of a
given invertebrate taxa possess a similar detoxification mechanism.
Bond (1961a) has summarized information on relative susceptibility
of different insects to HCN fumigation. The data suggested that more
tolerant insects were able to exclude HCN from their bodies. Studies
with the granary weevil, SitophHus granari-us (L.), showed uptake to be
linear with time if the fumigant concentration remained constant (Bond,
1961a). Neither rhodanese activity (thiocyanate production) nor detoxi-
fication by combination with cysteine to form 2-imino-4-thiazolidine
carboxylic acid was found. Similar results were obtained with the desert
locust, Soh-istooeToa gTegavia Forsk. Later studies showed that HCN does
inhibit respiration in insects, but complete inhibition was only observed
-------
122
in S. granar-ius (Bond, 19612?). The response of S. granar"ius was quickly
inhibited (15 min) long before the lethal dose (4-hr LDSO is 8 ing/liter)
was absorbed (4 hr). Isotopic tracer studies suggested that a major por-
tion of the absorbed HCN is excreted as amino acids in the feces of S.
granarius (Bond, 1961c). Some cyanide may also have been metabolized to
cyanocobalamin and C02.
5.5.2 Effects
The effects of cyanide on invertebrates are not too well documented.
"Paralysis" occurs in insects which have been fumigated with HCN; recov-
ery occured in some organisms (e.g., S. granai"Lus) which were exposed to
LDSO concentrations of 8 mg/liter for 4 hr even though respiration was
completely inhibited within 15 min (Bond 1961a, 1961&).
Defensive secretions of cyanide have been found in millipedes of
the order Polydesmida (Eisner and Meinwald, 1966). These millipedes,
when placed in HCN killing jars, also seem to be more tolerant of HCN.
In the millipede Aphelori-a, cyanide is generated in a two-compartmented
organ by hydrolysis of mandelonitrile. Cyanide generation occurs outside
the gland when the components of the two compartments are mixed during
ejection.
Table 5.3 lists the toxicity of cyanide to various aquatic inverte-
brates. The data are insufficient to determine sensitive or resistant
groups of invertebrates. The National Academy of Sciences and National
Academy of Engineering (1972) suggested that levels greater than or equal
to 0.01 ppm cyanide are hazardous in the marine environment, whereas
levels less than 0.005 ppm present minimal risk. They also suggested
that a level of 0.005 ppm cyanide not be exceeded in any waters with
aquatic life.
-------
123
TABLE 5.3. TOXICITY OF CYANIDES AND THIOCYANATES TO AQUATIC INVERTEBRATES
Chemical
compound
Ammonium
thiocyanate
Cyanides
Potassium
cyanide
Sodium cyanide
Organism
Asetlus aquaticus
(amphipod)
Carinogammams
(amphipod)
Midge larvae
Cvicotopus bicinctus
(midge fly)
Physa heteroalita
(snail)
Bivalve larvae
Daphnia magna
(cladocera)
D. magna
D. magna
Hydropsyche sp . , larvae
(caddis fly)
Lyrmacea sp. , eggs
(snail)
Lymnacea sp, , eggs
Lyrmacea sp . , eggs
Physa heterostropha
P. heterostropha
Stenonema rubmm
(mayfly)
D. magna
(cladocera)
Garmarus pulex
(amphipod)
G. pulex
Polycelis nigra
(planaria)
Test
, . . a
conditions
FW
FW
FW
FW,
SB,
SW
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
SB,
CB,
CB,
SB,
FS
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
FW,
(river)
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
LS
Concentration
100 ppm
100 ppm
50 ppm
<3. 2 ppm
O.A32 ppm
0.014 ppm
2.0 ppm
0 . 7 ppm
0 . 4 ppm
2 . 0 ppm
796.0 ppm
147.0 ppm
130.0 ppm
1.08 ppm
0.48 ppm
0.5 ppm
<3.4 ppm
0.005 N
0.0001 N
0.0006 M
Remarks
No noticeable. harm
No noticeable harm
Killed
Survived and matured
96-hr TL^, acute
Lethal
24-hr and 48-hr TL^,, acute
72-hr TLm, acute
96-hr TLm, acute
48-hr TL^, acute; soft
water, 20-22. 2 °C
24-hr and 48-hr TI^,
72-hr Tiro
96-hr Tim
96-hr Tim, acute; ''normal"
dissolved oxygen
96-hr TI^, acute; "low"
dissolved oxygen
48-hr TL^,, acute
Concentration to nearly
immobilize; Lake Erie
water, 25 °C
Survive =1.5 hr; 13-14 °C
Survive =3 hr; 13-14 "C;
temperature and pH
affect survival
Toxic threshold, survives
48 hr; pH 6.8, 14-18 °C
SB = static bioassay; CB constant-flow bioassay; FW fresh water; SW sea (salt) water;
Data collected from several
LS lab study; FS field study.
Source: Adapted from Becker and Thatcher, 1973, Table J, pp. J.2-J.10.
sources.
-------
124
SECTION 5
REFERENCES
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ship Between Body Size and Lethal Response of a Teleost Exposed to
Environmental Toxicants. Proc. West. Pharmacol. Soc. 16:139-140.
2. Anonymous. 1975. State Says Cyanide Caused Lake Fishkill. The Oak
Ridger, Oak Ridge, Tenn., September 5, 1975. p. 1.
3. Bansal, R. D., and R. R. Seaney. 1970. Action of the Cow Ruminal
Fluid on C^
40(4):413.
Bansal, R. D., and R. R. Seaney. 1970. Action of the Cow Ruminal
Fluid on Cyanogenetic Glucoside — A Note. Indian J. Anim. Sci. (India)
/, ru/, ~\ • /, i •}
4. Becker, C. D., and T. 0. Thatcher. 1973. Cyanurates and Cyanides.
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WASH-1249, Battelle Pacific Northwest Laboratories, Richland, Wash.
pp. J.1-J.13.
5. Bond, E. J. 1961a. The Action of Fumigants on Insects: I. The
Uptake of Hydrogen Cyanide by Sitophilus gr>anca"ius (L.) During Fumiga-
tion. Can. J. Zool. (Canada) 39:427-436.
6. Bond, E. J. 196L&. The Action of Fumigants on Insects: II. The
Effect of Hydrogen Cyanide on the Activity and Respiration of Certain
Insects. Can. J. Zool. (Canada) 39:437-444.
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Fate of Hydrogen Cyanide in Sitophilus granarius (L.). Can. J. Bio-
chem. Physiol. (Canada) 39:1793-1802.
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9. Burdick, G. E., and M. Lipschuetz. 1970. Toxicity of Ferro- and
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11. Cairns, J., Jr., and A. Scheier. 1968. A Comparison of the Toxicity
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Combined. Prog. Fish Cult. 30(1):3-8.
12. Cameron, J. F. 1972. Natural Substances Suspected of Killing Birds
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125
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No. EPA-600/3-76-008, U.S. Environmental Protection Agency, Duluth,
Minn. pp. 22-26, 97-101.
14. Chen, C. W., and R. E. Selleck. 1969. A Kinetic Model of Fish
Toxicity Threshold. J. Water Pollut. Control Fed. 41(8):R294-R308.
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of Cyanides and Cyanogenetic Glucosides in Sheep: I. Activity in
the Rumen. N. Z. J. Sci. Technol. Sect. A (New Zealand) 30:277-291.
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17. Doudoroff, P. 1976. Toxicity to Fish of Cyanides and Related Com-
pounds. Report No. EPA-600/3-76-038, U.S. Environmental Protection
Agency, Duluth, Minn. 155 pp.
18. Doudoroff, P., G. Leduc, and C. R. Schneider. 1966. Acute Toxicity
to Fish of Solutions Containing Complex Metal Cyanides, in Relation
to Concentrations of Molecular Hydrocyanic Acid. Trans. Am. Fish.
Soc. 95:6-22.
19. Eisner, T., and J. Meinwald. 1966. Defensive Secretions of Arthro-
pods. Science 153:1341-1350.
20. Herrington, M. D., R. C. Elliott, and J. E. Brown. 1971. Diagnosis
and Treatment of Thyroid Dysfunction Occurring in Sheep Fed on
Cynodon pleotostaohyus. Rhod. J. Agric. Res. (Rhodesia) 9:87-93.
21. Khan, M.A.Q., and J. P- Bederka, Jr. (eds.). 1973. Survival in
Toxic Environments. Academic Press, Inc., New York. pp. 195-201.
22. Leduc, G. 1966. Some Physiological and Biochemical Responses of
Fish to Chronic Poisoning by Cyanide. Ph.D. Thesis. Oregon State
University, Corvallis, Ore. 146 pp.
23. Marking, L. L., and V. K. Dawson. 1975. Investigations in Fish
Control: 67. Method for Assessment of Toxicity or Efficacy of
Mixtures of Chemicals. U.S. Department of the Interior, Washington,
D.C. 8 pp.
24. Morgan, W.S.G., and P. C. Kiihn. 1974. A Method to Monitor the
Effects of Toxicants Upon Breathing Rate of Largemouth Bass (Mi,OTO-
ptevus salmoides Lacepede). Water Res. (Great Britain) 8(1):67-77.
25. National Academy of Sciences and National Academy of Engineering.
1972. Water Quality Criteria 1972. U.S. Environmental Protection
Agency, Washington, D.C. 533 pp.
-------
126
26. Skidmore, J. F. 1974. Factors Affecting the Toxicity of Pollutants
to Fish. Vet. Record. 94:456-458.
27. U.S. Federal Water Pollution Control Administration. 1964-1969.
Fish Kills Caused by Pollution, 1964-1969. U.S. Government Printing
Office, Washington, D.C. 110 pp.
28. Van Kampen, K. R. 1970. Sudan Grass and Sorghum Poisoning of
Horses: A Possible Lathyrogenic Disease. J. Am. Vet. Med. Assoc.
156(5) :629-630.
29. Wheeler, J. L., D. A. Hedges, and A. R. Till. 1975. A Possible
Effect of Cyanogenic Glucoside in Sorghum on Animal Requirements
for Sulphur. J. Agric. Sci. (Great Britain) 84:377-379.
-------
SECTION 6
BIOLOGICAL ASPECTS IN HUMANS
6.1 SUMMARY
Cyanide, an extremely toxic substance, can quickly cause death after
inhalation, ingestion, or cutaneous absorption. For example, a hydrogen
cyanide concentration of 0.3 mg/liter (270 ppm) in air is immediately fatal
to humans. Fortunately, recovery is common if treatment is instituted as
quickly as possible. Humans may be exposed to cyanide as hydrogen cyanide,
a cyanide salt, cyanogen, or other cyanide-containing compounds such as
nitriles. Cyanogenic glycosides from some plants release cyanide upon
hydrolysis, but symptoms are delayed following ingestion because hydrol-
ysis is relatively slow.
A detectable amount of cyanide is generally found in all biological
materials; however, cyanide usually is not cumulative. Therefore, plasma
cyanide levels may not be significantly higher in normal persons than in
individuals with greater than average exposure to cyanide (e.g., cigarette
smokers). However, thiocyanate, the major detoxification product of cya-
nide, may be a better index of cyanide exposure, as it is present in sig-
nificantly higher amounts in the plasma of cigarette smokers than in the
plasma of nonsmokers.
Cyanide may be metabolized via several pathways. The major pathway
is an enzymatic reaction (rhodanese) with thiosulfate to produce thiocya-
nate, which is excreted in the urine. Cyanide also may react with cystine
to produce nontoxic 2-aminothiazoline-4-carboxylic acid, or it is believed
to enter one-carbon metabolism through formic acid and be oxidized and
exhaled as carbon dioxide. Reaction of cyanide to cyanate with hydroxo-
cobalamin to form cyanobalamin also has been described. Cyanide is
excreted in the urine predominantly as thiocyanate.
Cyanide is believed to produce its toxic effects predominately by
combining with metal ions in enzymes. In acute poisoning, inhibition of
cytochrome oxidase prevents body cells from utilizing oxygen resulting
in a histotoxic anoxia.
Treatment of acute cyanide poisoning is based on both binding and
detoxifying cyanide. Sodium nitrite is employed to convert hemoglobin
to methemoglobin which binds cyanide as cyanmethemoglobin. Thiosulfate
is given as substrate to convert cyanide to the less toxic thiocyanate
in the presence of the enzyme rhodanese. Some cases of chronic cyanide
exposure have been treated with vitamin Bi2a (hydroxocobalamin).
Acute effects caused by cyanide depend on how fast and to what extent
histotoxic hypoxia is produced. The quicker cyanide levels build up in
tissues, the more severe the response. The first breath of cyanide at
2000 ppm causes immediate hyperpnea with collapse, convulsions, and ces-
sation of breathing within one minute. When death occurs this rapidly,
127
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128
the total absorbed dose may be 7 mg/kg. Ingested cyanide salts are rela-
tively more slowly absorbed and symptoms may not appear for 5 to 30 min.
Up to 3.5 mg/kg body weight may be absorbed when death occurs.
Some nitriles release cyanide slowly and, initially, the entire mol-
ecule may exert a pharmacological action. With chronic intake of organic
nitriles, such as the cyanogenic glycosides in many plant foodstuffs, the
slow release of cyanide may result in chronic cyanide poisoning. Various
tropical neuropathies, Leber's optic atrophy, and tobacco amblyopia are
correlated with chronic cyanide uptake. Demyelination of nerves in the
central nervous system as a result of cyanide-induced anoxia may be re-
sponsible for the neuropathies.
Experimental studies have shown that cyanide compounds can e^ert an
anticancer effect. Laetrile therapy has been used experimentally
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129
6.2.1.2 Inhalation — Hydrogen cyanide vapor is absorbed rapidly through
the lungs (Gettler and St. George, 1934; Poison and Tattersall, 1969).
Because HCN has a pKa of 9.2 and exists primarily as the acid under bio-
logical conditions, absorption across the alveolar membrane should be
rapid (Fingl and Woodbury, 1970; Wolfsie and Shaffer, 1959). Human inha-
lation of 270 ppm HCN vapor brings death immediately, while 135 ppm is
fatal after 30 min (Fassett, 1963).
Cyanide absorption following inhalation of very low concentrations
is indicated by the observation that smokers have higher thiocyanate lev-
els in plasma and other biological fluids than do nonsmokers (Maliszewski
and Bass, 1955; Wilson and Matthews, 1966). Cyanide levels usually are
not significantly different (Pettigrew and Fell, 1973; Wilson and Matthews,
1966), as these levels probably reflect rapid conversion of cyanide ab-
sorbed from inhaled tobacco smoke to thiocyanate (Johnstone and Plimmer,
1959; Osborne, Adamek, and Hobbs, 1956; Pettigrew and Fell, 1973). Inha-
lation of cyanide salt dusts is also dangerous because the cyanide will
dissolve on contact with moist mucous membranes and be absorbed into the
bloodstream (Davison, 1969; Knowles and Bain, 1968).
Inhalation exposure to cyanogen [(CN2)] is likely because it is a
gas at room temperature. Absorption values are not available, but the
lethal dose for rats is greater than that of hydrogen cyanide (McNerney
and Schrenk, 1960). Besides releasing hydrogen cyanide and cyanate upon
hydrolysis, cyanogen is also an irritant causing eye and nasal inflamma-
tion at concentrations as low as 16 ppm.
Halogenated cyanogens such as cyanogen chloride or cyanogen bromide
are comparable in toxicity to hydrogen cyanide when inhaled and cause
marked irritation of the respiratory system with hemorrhage and pulmonary
edema (Fassett, 1963; Prentiss, 1937). Quantitative absorption data are
not available.
The organic nitriles are another class of potential cyanide-releasing
compounds that may present an inhalation hazard. It is not possible to
generalize on whether the primary toxicity of these compounds resides with
the cyanide moiety, the organic nitrile molecule itself, or its metabo-
lite^). For example, the 2-cyanopyridinium ion is metabolized to liberate
cyanide (Way and Way, 1968), whereas the 4-cyanopyridinium ion liberates
no cyanide and is believed to be metabolized to a carboxamide. Although
organic nitriles may have the same toxicity as HCN, this should not infer
that the primary toxicity can be attributed to cyanide liberation.
6.2.1.3 Percutaneous Absorption — Hydrogen cyanide in either liquid or
vapor form is absorbed through the skin (Drinker, 1932; Potter, 1950; Tovo,
1955; Walton and Witherspoon, 1926). Absorption is probably increased if
the skin is cut, abraded, or moist. Many accidents involving skin con-
tamination also involve inhalation exposure; the contribution due to skin
absorption in these cases is difficult to assess. Potter (1950) described
a case in which liquid HCN ran over the bare hand of a worker wearing a
fresh air respirator. Cyanide inhalation was prevented, but the worker
collapsed into deep unconsciousness within 5 min, suggesting significant
percutaneous absorption.
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130
Cyanogen vapor is apparently not absorbed percutaneously to a sig-
nificant degree. Rabbits exposed to 10,000 ppm cyanogen for 8 hr under
conditions preventing inhalation exposure exhibited no toxic effects
(McNerney and Schrenk, 1960). Some organic nitriles are also absorbed
through and irritate the skin (Dudley and Neal, 1942; Fassett, 1963;
Graham, 1965).
6.2.2 Transport and Distribution
6.2.2.1 In Blood — Regardless of the route of uptake and absorption,
cyanide enters the bloodstream and is carried to other body tissues.
Cyanide readily forms complexes with metal ions such as iron or copper.
This property is probably responsible for most of its acute toxic effects,
particularly when cytochrome oxidase is involved (Sections 6.3.1 and
6.3.2). Within the bloodstream, cyanide can bind with methemoglobin to
form nontoxic cyanmethemoglobin (Chen and Rose, 1952; Williams, 1959).
As the plasma cyanide concentration decreases, cyanide continues to dis-
sociate from cyanmethemoglobin and eventually is metabolized or excreted
(Albaum, Tepperman, and Bodansky, 1946; Smith and Gosselin, 1966).
The site of major toxic effects, cytochrome oxidase, and the major
detoxification system, rhodanese, are found almost exclusively intracel-
lularly (de Duve et al., 1955). A minor conversion of thiocyanate back
to cyanide can occur in red blood cells (Goldstein and Rieders, 1951,
1953).
Tobacco smoke is probably the most important daily source of cyanide
exposure for the general population (1600 ppm HCN in cigarette smoke, re-
ported in U.S. Department of Health, Education, and Welfare, 1964), but
due to the rapid excretion and detoxification to thiocyanate, cyanide
levels are not significantly higher in plasma of smokers than in plasma
of nonsmokers (Wilson and Matthews, 1966). Plasma thiocyanate levels,
however, are significantly higher in smokers than nonsmokers (Lawton,
Sweeton, and Dudley, 1943; Maliszewski and Bass, 1955; Trasoff and
Schneeberg, 1944; Wilson and Matthews, 1966) due to its slow excretion.
When sodium nitroprusside [Na2Fe(CN)5NO] is incubated with some bio-
logical materials, it releases cyanide. The most active biological mate-
rial is red blood cells. Red cells are more active in rats and mice than
in humans, but this difference disappears upon hemolysis of the cells,
indicating a species difference in red cell permeability to nitroprusside
(Hill, 1942; Page et al., 1955; Smith and Kruszyna, 1974). Cyanide has
been reported to be released from nitroprusside via two nonenzymatic
reactions: (1) a slow, nonspecific reaction with free sulfhydryl groups
and (2) a fast reaction with hemoglobin. In animals, the free cyanide
produced from nitroprusside has been reported to be from the reaction
with hemoglobin (Smith and Kruszyna, 1974).
From a chemical viewpoint, cyanogen chloride (CNC1) should be rela-
tively stable in blood since it decomposes slowly in aqueous solution up
to pH 8 (blood pH is around 7.4); however, in reality CNC1 is rapidly
broken down in blood (Aldridge and Evans, 1946). Thirty percent or more
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131
of CNC1 was converted to HCN in the presence of red blood cells; no HCN
was produced with serum alone although the CNC1 was still rapidly broken
down. Aldridge and Evans (1946) concluded that the lethal effects of
CNC1 were due to the formation of HCN and that CNC1 was eventually con-
verted to thiocyanate, the major cyanide metabolite in the body (Section
6.2.3.1).
6.2.2.2 In Organs — When sufficiently sensitive detection methods were
used, cyanide was found in trace amounts in all biological materials test-
ed (Boxer and Rickards, 1951). The destruction of cyanide in blood and
organs of female rabbits receiving HCN or KCN was reported by Ballantyne
et al. (1972). Equal doses of the cyanide radical were administered (8
mg/kg in these studies). A comparison was made between cyanide levels
in tissues containing blood and in those from animals perfused with saline
to remove the blood (Table 6.1). Cyanide levels in whole blood and serum
were higher after injection of HCN than after injection of KCN, reflecting
TABLE 6.1. COMPARISON OF CYANIDE CONCENTRATIONS IN TISSUES
FROM RABBITS KILLED BY HCN WITH CONCENTRATIONS IN TISSUES FROM
RABBITS KILLED WITH KCN
Tissue
Cyanide concentration
mean + standard error
a
HCN
KCN
Containing blood
Skeletal muscle
Kidney
Liver
Spinal cord
Brain
Whole blood
Serum
35.0 + 5.2
74.7 + 10.3
148.7 + 32.3
48.5 + 4.9
145.3 + 37.2
685.0 + 83.0
275.0 + 18.0
29.6 + 2.4
52.0 + 11.0
82.0 + 8.0
36.8 + 3.5
106.5 + 12.4
453.0 + 34.0
161.0 + 21.0
<0.5
<0.05
<0.005
Perfused with saline
Skeletal muscle
Kidney
Liver
Spinal cord
Brain
Whole blood
Serum
9.3 + 2.7
11.0 + 4.3
43.7 + 13.5
49.8 + 14.7
289.0 + 67.7
761.0 + 129.0
261.0 + 48.0
7.8 + 2.4
2.3 + 1.1
6.5 + 0.8
22.5 + 3.8
98.0 + 5.0
438.0 + 8.0
134.0 + 8.0
<0.7
<0.1
<0.025
<0.2
<0.02
<0.05
<0.05
Concentrations expressed in micrograms CN per 100 g wet
tissue and micrograms CN per 100 ml blood or serum. Blood was
removed from the left ventricle of perfused animals before saline
was injected.
^Significance of difference in cyanide concentrations between
animals killed with HCN and those killed with KCN.
Source: Adapted from Ballantyne et al., 1972, Table IV,
p. 216. Reprinted by permission of the publisher.
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132
a more rapid absorption of the weakly ionized HCN. The authors stated:
"There was generally no significant difference in the concentration of
cyanide in blood-containing tissues or saline-perfused tissues removed
from animals killed with HCN compared with the concentration in corre-
sponding tissues from rabbits killed with KCN; the only exception was a
somewhat lower concentration of cyanide in perfused livers from the rab-
bits killed with KCN compared with perfused livers from the animals killed
with HCN." However, it seems that the cyanide level in saline-perfused
brain tissue is also significantly lower in KCN-killed animals than in
HCN-killed animals.
Cyanide determinations of autopsy samples from three human cyanide
poisonings were reported by Finck (1969) (Table 6.2). Two deaths resulted
from ingestion of unknown amounts of a cyanide salt (which salt was not
reported) and one death resulted from inhalation of cyanide gas.
TABLE 6.2. CYANIDE LEVELS IN HUMAN TISSUES AND FLUIDS
AFTER FATAL CYANIDE POISONING
Cyanide content
. (mg/100 g or mg/100 cc)
Sample
Gastric contents
Lung
Blood
Liver
Kidney
Muscle
Brain
Urine
Fat
Case la
0.03
0.50
0.03
0.11
0.07
0.20
Case 2
15
0.09
0.75
0.40
0.35
0.30
0.25
0.20
0.20
Case 3
20
0.70
0.80
0.50
0.40
0.06
, Death from inhalation of cyanide gas.
Death from ingestion of cyanide salt.
Source: Adapted from Finck, 1969, Table 1, p. 357.
Reprinted by permission of the publisher.
6.2.2.3 Placenta! Transfer - The relatively high pKa value of HCN and
its ready absorption through the respiratory tract, gastrointestinal tract,
and skin indicate that it also should cross the placenta; however, no
direct data confirming placental transfer were found in the literature.
Also, no fetal abnormalities were reported in women suffering from var-
ious neuropathies believed to be partly caused by chronic cyanide poison-
ing (Section 6.3.3.1). Andrews (1973) compared thiocyanate levels in
maternal and cord blood samples from 50 consecutive deliveries and found
a direct correlation (r = 0.914).
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133
6.2.3 Detoxification
Although cyanide interacts with some substances in the bloodstream
(Section 6.2.2.1), most reactions occur intracellularly within organs.
These include detoxification reactions as well as biochemical reactions
which have been attributed to producing the predominant toxic effects
(Sections 6.3.2 and 6.3.3). Some of the possible metabolic pathways for
cyanide are shown in Figure 6.1.
CN-
Major path
CNS- -
minor
path
2-imino-thiazolidine-
4-carboxylic acid
HCN
in expired air
_CN- pool
HCNO
CO,
ORNL-DW6 76-15522
Excretion
_+ cyanocobalamin
HCOOH -> metabolism of
one-carbon
compounds
some excreted
in urine
Figure 6.1. Fate of cyanide ion in the body. Source:
1959, p. 393. Reprinted by permission of the publisher.
Williams,
6.2.3.1 Detoxification by Thiocyanate Production — The conversion of cya-
nide to thiocyanate (SCN~) is the major detoxification pathway, requiring
a sulfur donor such as thiosulfate and the enzyme, rhodanese (Lang, 1933a,
1933&). Rhodanese (sulfurtransferase) is found to be widely distributed
in animal tissues, especially in the liver. Cosby and Sumner (1945) par-
tially purified this enzyme and it was subsequently crystallized by Sorbo
(1953). Himwich and Saunders (1948) compared rhodanese activity in var-
ious tissues of dogs, rhesus monkeys, rabbits, and rats. They found that
the rhodanese activity in liver, kidney; muscle, and adrenals was quite
variable among different species, whereas brain rhodanese activity was
similar among these species (Table 6.3). The dog differed from other
species examined by having the highest enzyme activity in the adrenals
rather than in the liver. Detoxification via rhodanese also occurs in
other tissues; however the rhodanese contents usually are lower than the
liver.
-------
TABLE 6.3.
RHODANESE ACTIVITY IN TISSUES OF THE DOG, RHESUS MONKEY, RABBIT, AND RAT
(mg CN converted to CNS per gram of tissue)
Tissue
Suprarenals
whole
cortex
medulla
Liver
Brain
cortex
caudate nucleus
midbrain
cerebellum
medulla
Spinal cord
cervical
lumbar
sacral
Heart
Kidney
Testes
Epidydymis
Ovaries
Lung
Spleen
Muscle
Intestine
duodenum
j e j unum
Eye
Optic nerve
Salivary gland, parotid
Lymph node
Pancreas
Thyroid
Anterior pituitary
Whole blood
Erythrocytes
Plasma
Dog
Rhesus monkey
Number
Range of
observations
2
(5
2
0
0
(4
0
0
0
0
0
0.
0,
0,
0,
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
<0.
.14-3.60
.46, 4.50)
.86-5.62
.27-1.12
.78-1.46
.91, 6.28)
.34-0.92
.27-1.06
.52-1.35
.21-1.22
.38-1.52
.15-1.08
.12-0.84
.16-1.41
.11-0.14
,42-0.74
.32-0.41
,29
,42
.16-0.17
10-0.14
03-0.19
05-0.11
,04
02
35
05-0.36
08-0.13
14-0.28
05-0.94
26
01-0.02
01-0.02
01
6
2
2
7
7
7
6
7
7
7
4
4
6
6
5
1
1
3
2
6
3
1
1
1
3
2
4
3
1
2
2
1
0
10
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
Rangea
.14-1.
.98-15
(5.98)
.27
.34-0.
.22-0.
.33
.49-0.
.56-0.
.20-0.
.23-0.
.48-0.
.46-3.
.38-0.
.11-0.
.12-0.
.23-0.
.99
Rabbit Rat
Number Number Number
of Range of Range of
observations observations observations
35
.16
50
80
85
57
42
28
82
58
46
21
34
57
.12-0.44
3
4
1
2
2
1
2
2
2
2
3
4
3
2
2
3
I
2
1.
7.
1.
0.
1.
0,
0.
0.
0,
0,
6.
0,
0
0
0
0
24-3.94
,98-18.92
41-1.44
13-0.18
,17-1.39
,63-1.24
.91
.89-0.90
,35-1.74
.59-1.10
.20-7.69
.32-0.36
.30
.40
.20
.18
2 0.27-0.41
9 14.24-28.38
2 0.70-0.72
2
2 0.73-1.13
2
1
2 0.16-0.18
2 0.23-0.27
3 0.56-0.74
3 10.44-11.08
2 1.24-1.61
1
1
1
1
2
9
2
2
2
2
2
2
2
OJ
-p-
Figures in parentheses are single observations falling outside the normal range.
Source: Adapted from Himwich and Saunders, 1948, Table 1, p. 351. Reprinted by permission of the publisher.
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135
Sodium thiosulfate was found to be an effective sulfur donor for
rhodanese (Table 6.4) (Chen and Rose, 1952; Himwich and Saunders, 1948).
The substrate specificity of the sulfur donor for rhodanese was investi-
gated by So'rbo (1953). These studies indicated that the substrate spec-
ificity for rhodanese requires that a sulfur atom be adjacent to another
sulfur atom and that one of the sulfur atoms be free. Under optimal in
vitro conditions, the rhodanese content in dog liver is sufficient to
detoxify over 4000 g cyanide in 15 min. The comparable value for total
skeletal muscle is 1763 g. These composite values are over 1000 times
the lethal dose for dogs (Section 6.3.2). This infers that rhodanese
itself, because of its high tissue content and turnover number, is not
the limiting factor in cyanide detoxification. The rate limiting reac-
tion probably can be attributed to the content and physiologic disposi-
tion of the sulfur donor. The thiosulfate content of the body is low and
exogenously added thiosulfate can substantially increase the LD50 of cya-
nide. For example, Sheehy and Way (1968) increased the LDSO of potassium
cyanide in mice by a factor of 4- to 6-fold by an intraperitoneal injec-
tion of sodium thiosulfate after signs of cyanide poisoning became appar-
ent. Similar results were reported by Chen, Rose, and Clowes (1934),
Frankenberg and Sorbo (1975), and others. Auriga and Koj (1975) found
a protective effect of rhodanese and/or thiosulfate on the respiration
of cyanide-poisoned, isolated mitochondria from rat liver and muscle and
from beef liver and heart.
Rhodanese and cytochrome oxidase are localized in the mitochondria
(de Duve et al., 1955); therefore, the rhodanese-catalyzed reaction must
TABLE 6.4. REPLACEMENT OF SODIUM THIOSULFATE
BY OTHER SULFUR-CONTAINING COMPOUNDS
Percent
Compound activity of
standard system
Sodium thiosulfate 100
Sodium sulfide 4
Sodium tetrathionate Spontaneous conversion
Thiourea 4.5
a-Naphthylthiourea 4.6
Thiouracil 1
Dithiobiuret 1
Methionine 1
Cystine 1
Cysteine 1
Thiodiglycol 0
Diphenylsulfide 0
Diphenyldisulfide 0
Source: Himwich and Saunders, 1948, Table 3,
p. 352. Reprinted by permission of the publisher.
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136
occur intracellularly. The exogenous sodium thiosulfate has limited mem-
brane permeability, especially when compared to cyanide (Crompton et al.,
1974, cited in Auriga and Koj, 1975; Himwich and Saunders, 1948). Thio-
cyanate can be converted, to a limited extent, to cyanide; however, this
dose not occur via rhodanese. There also is a minor conversion of organic
thiocyanates to cyanide which is catalyzed by glutathione S-transferases
(Habig, Keen, and Jakoby, 1975). These enzymes cleave organic thiocya-
nates to form cyanide and the respective asymmetric disulfide of
gluthathione.
6.2.3.2 Minor Detoxification Pathways — Theoretically, any of the meta-
bolic processes that convert cyanide into a less toxic compound or any
substances that bind cyanide, such as methemoglobin, should reduce the
toxicity of cyanide. Vitamin B12a (hydroxocobalamin) occurs naturally
in the body in small amounts (Drouet et al., 1951; Mollin and Ross, 1953);
however, even if all the vitamin B12 in the liver was vitamin B12a, it
would combine with only 25 ug cyanide (Wokes and Picard, 1955). Hydroxo-
cobalamin has a hydroxyl group bound to the cobalt atom and when cyanide
is added to hydroxocobalamin, the cyanide is incorporated into vitamin
Bi2 (cyanocobalamin) (Brink, Kuehl, and Folkers, 1950; Kaczka et al.,
1950).
Formation of cyanocobalamin (CN-Bi2), a cyanide-containing form of
vitamin Bi2, is important for two reasons: it is a minor cyanide detoxi-
fication pathway and it may minimize chronic cyanide intoxication (Section
6.3.3.1). Little or no cyanocobalamin is found in the plasma of normal
human subjects (Linnell, MacKenzie, and Matthews, 1969). The major plasma
Bi2 components are methylcobalamin (coenzyme B12) and a mixture of hydrox-
ocobalamin (OH-Bi2) and deoxyadenosylcobalamin. Mushett et al. (1952)
administered potassium cyanide to mice and successfully antagonized the
toxic effect of cyanide with OH-B12. The largest portion of cyanide in
the urine appeared as CN-Bi2 with a smaller portion as thiocyanate. There
was no CN-Bia detected in the urine of controls. The formation of CN-B12
from other vitamin Bi2 chemical species may serve as a detoxification
function by scavenging some cyanide which might otherwise react with cyto-
chrome oxidase. Formation and excretion of CN-B12 could deplete the body's
store of active vitamin Bi2 during chronic cyanide exposure. The role of
vitamin B12 in metabolic processes has been discussed by Brown (1973),
Stadtman (1971), and Wokes and Picard (1955).
Vitamin B12 (cyanocobalamin) administered intraperitoneally or intra-
venously into mice produced no toxic effects, indicating that the cyano
group is tightly bound (Winter and Mushett, 1950), as this dose of vita-
min B12 possessed a cyanide content of 32 mg/kg. In humans, half of an
intramuscular or subcutaneous injection of vitamin B12 was excreted with-
in 3 to 5 hr and almost all was excreted within 24 hr (Boxer and Rickards,
1951). The cyanide apparently was not released from the injected vita-
min B12 in their experiments. Reizenstein (1967) reported that a thera-
peutic dose of cyanocobalamin in humans was excreted as cyanocobalamin in
12 hr in the urine. Guinea pig liver could convert cyanocobalamin to
hydroxocobalamin in vivo and in vitro at a rate of 0.1 to 0.4 myg/g of
liver per day. Cima, Levorato, and Mantovan (1967) reported that cyanor
-------
137
cobalamin was decyanated to hydroxocobalamin in vitro by rat liver and
kidney. The decyanation was due to an enzyme system which they called
"cyanocobalamin-decyanase." Release of cyanide from vitamin Bi2 in humans
is supported by the observation that some patients treated for Leber's
disease (Section 6.3.3.1) with vitamin Bi2 developed unusually severe
optic atrophy, a condition associated with chronic cyanide poisoning
(Foulds et al., 1968).
Hydroxocobalamin (100 to 250 mg/kg) is an effective cyanide antagonist
Hydroxocobalamin, but not cyanocobalamin, was effective in antagonizing
cyanide intoxication and lethality in mice (Mushett et al., 1952). The
studies on hydroxocobalamin have been confirmed by Friedberg, Grutzmacher,
and Lendle (1965). Treatment of chronic cyanide poisoning in humans with
hydroxocobalamin is discussed in Section 6.3.3.1. Additional information
on the chemistry of vitamin B12 can be found in Brown (1973).
Although cyanide blocks the tissue utilization of oxygen (Section
6.3.1), therapy with oxygen nevertheless enhances the protective effects
of other conventional cyanide antidotes (Burrows, Liu, and Way, 1973;
Isom and Way, 1974; Sheehy and Way, 1968; Way, Gibbon, and Sheehy, 1966a,
I966b; Way et al., 1972).
Another minor detoxification reaction of cyanide occurring in vivo
is the spontaneous reaction with cystine, yielding 3-thiocyanoalanine
which tautomerizes to 2-aminothiazoline-4-carboxylic acid or the equiva-
lent isomer, 2-iminothiazolidine-4-carboxylic acid (Schoberl and Hamm,
1948). These products apparently are metabolically inert. Rats given
subcutaneous injections of NaCN excreted 15% of the dose as 2-iminothiazo-
lidine-4-carboxylic acid (Wood and Cooley; 1956).
The carbon atom of cyanide apparently enters the one-carbon metabo-
lism through formic acid (HCOOH) and also is further metabolized and ex-
creted as respiratory carbon dioxide (Boxer and Rickards, 1952&) . These
studies, using rats and dogs, were conducted with isotopically labeled
Na14CN or thiocyanate (NaS^CN). About 30% of the injected thiocyanate
was oxidized to carbon dioxide in the rat during the nine-day experiment.
6.2.4 Excretion
A small amount of cyanide is eliminated unchanged through the lungs.
Friedberg and Schwarzkopf (1969) found 1% to 2% of HCN given intraven-
ously to guinea pigs was eliminated by the lungs before respiration ceased;
this value was increased three- to fourfold when artificial respiration
was applied. Hydrogen cyanide is found in respired air of normal humans
and rats (Boxer and Rickards, 1952a). Most of the cyanide, under normal
conditions, is metabolized to other compounds and excreted in the urine.
The major metabolic product is thiocyanate, as discussed in Section 6.2.3.1
and shown in Figure 6.1. Thiocyanate is normally found in body fluids
because cyanide and thiocyanate are related compounds and are regularly
ingested in the diet (Section 6.2.1.1) and inhaled from tobacco smoke.
Because tobacco smoke is a significant source of cyanide, thiocyanate
levels in urine and other body fluids are higher in smokers than in non-
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138
smokers (Lawton, Sweeney, and Dudley, 1943; Maliszewski and Bass, 1955;
Pick, 1910; Wokes and Moore, 1958). Djuric, Raicevic, and Konstantinovic
(1962) reported that thiocyanate appeared only sporadically and in trace
amounts in the urine of nonsmokers. Thiocyanate levels in urine were
directly proportional to the number of cigarettes smoked, varying from an
average of 3.7 mg/liter with ten cigarettes per day to an average of 17.5
mg/liter with 40 cigarettes per day. Boxer and Rickards (1952a) found
thiocyanate levels ranging from 8.3 to 20.0 mg/liter in urine from "normal
subjects"; however, smoking histories were not given.
Wood and Cooley (1956) reported that in rats receiving NaCN, 80% of
the cyanide was excreted in the urine as thiocyanate. Cyanide is also
found in the urine but in much lower concentrations than thiocyanate
(Boxer and Rickards, 1951). As mentioned in Section 6.2.3.2, cyanide is
also excreted in the urine as cyanocobalamin.
6.3 EFFECTS
6.3.1 Mechanism of Action
Cyanide has a high affinity for certain metal ions. As a result,
enzyme systems which require metal ions may be susceptible to cyanide
inhibition. Oxidative enzymes and coenzymes in which iron is sometimes
in the ferric (Fe3+) state are especially sensitive. Cytochrome oxidase,
the terminal enzyme in the mitochondrial electron transport chain is espe-
cially sensitive to cyanide and is completely inhibited by 3.3 x 10~s
moles/ml of cyanide, producing a cytotoxic anoxia which leads to the symp-
toms seen in acute cyanide poisoning (Chen and Rose, 1952; DiPalma, 1971;
Way, Gibbon, and Sheehy, 1966a, 1966&). One molecule of cyanide combines
with the iron in one molecule of cytochrome oxidase to form an inactive
complex (Yoshikawa and Orii, 1972). Cyanide produces a histotoxic anoxia,
that is, it prevents the tissue utilization of oxygen. Death from cya-
nide poisoning is probably due to a cerebral anoxia (DiPalma, 1971).
Cyanide combines with other heme-containing enzymes such as catalase
and peroxidase as well as some nonheme enzymes such as tyrosinase, ascorbic
acid oxidase, and phosphatase. However, these enzymes are much less sen-
sitive to cyanide than cytochrome oxidase (DiPalma, 1971).
6.3.2 Acute Effects
In general, the effects of cyanide poisoning depend on the severity
and rate of production of the histotoxic hypoxia. The quicker critical
cyanide concentrations are attained in tissues, the more severe the effects
and the smaller the dose required for a given effect. Inhalation of HCN
leads to the most rapid absorption in tissues. Cyanide salts and other
cyanide-containing compounds, such as organic nitriles and cyanogenic gly-
cosides, release cyanide at different rates and in different amounts;
therefore, the doses required for a given effect vary greatly. In addi-
tion to those effects due to cyanide, some cyanide-containing compounds
have pharmacological effects not related to cyanide.
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139
The acute effects produced by cyanide have been known for hundreds
of years and many case studies of cyanide intoxication have been published.
The course of events following exposure to cyanide has been described in
numerous publications (DiPalma, 1971; Dreisbach, 1971; Fairhall, 1969;
Gleason et al., 1969; Grant, 1974; McAdam and Schaeffer, 1965; Montgomery,
1965; Mooney and Quinn, 1965; Morgan and Seaton, 1975; Oke, 1969; Poison
and Tattersall, 1969; Rentoul and Smith, 1973; Sollmann, 1957; Swinyard,
1970; Way and Way, 1968). The following general symptoms were compiled
from these references.
Normally, a fatal cyanide exposure produces a brief stage of central
nervous system stimulation followed by depression, hypoxic convulsions,
and death. A concentration of 2000 ppm HCN in air (2.4 mg HCN per liter
of air) gives a very brief sensation of dryness and burning in the throat,
a feeling of warmth, and shortness of breath. The first breath produces
immediate hyperpnea (deep, rapid breathing) and sometimes an outcry. Col-
lapse, convulsions, and apnea (cessation of breathing) occur in less than
a minute. The heart may continue to beat for several minutes after breath-
ing stops. The dose causing rapid death may be less than 7 mg/kg body
weight, while the LD50 for HCN absorbed through the skin (which takes
much longer) is about 100 mg/kg body weight.
Similar effects follow ingestion of KCN or NaCN, but they may be
slower due to the slower absorption from the gastrointestinal tract.
Within 5 min hyperpnea occurs from chemoreceptor stimulation and vomiting
results from irritation of gastric mucosa and central stimulation. With-
in 5 to 20 min after ingestion, a variety of symptoms are seen including
unconsciousness; convulsions; flushed, hot, dry skin; full, rapid, irreg-
ular pulse; high systolic with low diastolic blood pressure; trismus of
jaw muscles; and gasping. Hypoxic dilation of the pupils, vascular col-
lapse, and cyanosis then occur.
Certain organic nitriles cause a sequence of events, such as CNS
depression that may be due to the pharmacologic action of the entire mol-
ecule rather than cyanide per se. These effects may or may not be fol-
lowed by cyanide poisoning, depending on whether cyanide is released. The
slow increase in cyanide levels over a prolonged time may reveal symptoms
which are due to inhibition of enzymes other than cytochrome oxidase.
Tables 6.5, 6.6, 6.7, and 6.8 present quantitative data on the effects
of various compounds which contain the cyanide moiety in humans and
experimental animals.
6.3.3 Chronic Effects
The acute effects of cyanide on the body are well described and well
recognized. However, the possible dangers of long-term exposure to low
cyanide levels that in single doses do not produce clinical signs of poi-
soning are not well understood. Only recently have possible correlations
been implicated between chronic cyanide uptake and specific diseases such
as tobacco amblyopia, retrobulbar neuritis in pernicious anemia, Leber's
optic atrophy, and Nigerian nutritional neuropathy.
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140
TABLE 6.5. HUMAN RESPONSE TO INHALED CYANIDE AND CYANIDE-CONTAINING COMPOUNDS
Compound
Cyanogen bromide
Cyanide concentration
(mg/liter)
(ppm)
Response
0.40
0.035
0.006
Reference
Hydrogen cyanide
Cyanogen
Cyanogen chloride
0.3
0.2
0.15
0.12-0.15
0.40
0.120
0.005
0.0025
270
181
135
110-135
16
159
48
2
I
Immediately fatal
Fatal after 10-min exposure
Fatal after 30-min exposure
Fatal after 1/2 to 1 hr or
later, or dangerous to life
Nasal and eye irritation after
6 to 8 min
Fatal after 10-min exposure
Fatal after 30-min exposure
Intolerable concentration,
10-min exposure
Lowest irritant concentration,
10-min exposure
Prentiss, 1937
Prentiss, 1937
Prentiss, 1937
Fassett, 1963
McNerney and
Schrenk, 1960
Prentiss, 1937
Fassett, 1963
Fassett, 1963
Fassett, 1963
92 Fatal after 10-min exposure Prentiss, 1937
8 Intolerable concentration Prentiss, 1937
1.4 Greatly irritating to Prentiss, 1937
conjunctiva and the mucous
membranes of the respiratory
system
The various neuropathies probably result from demyelination of nerves
in the central nervous system brought about by cyanide-induced anoxia.
Ferraro (1933) gave repeated doses of cyanide to cats and monkeys (amount
not reported) and found diffuse demyelination in the white matter local-
ized in the corpus callosum. Hurst (1940) attempted to duplicate these
results in monkeys by giving cyanide at near lethal doses daily or at
longer intervals but did not obtain consistent results. If a nonlethal
cyanide level was maintained in the rat for 20 to 30 min by HCN inhala-
tion or by slow intravenous injection of KCN, the white matter was always
damaged (Levine, 1967). Distended vacuoles appeared at one point on the
axons that crossed the corpus callosum (Hirano, Levine, and Zimmerman,
1967). Lesions first appeared and were more severe in an area of white
matter. Smith et al. (1963, cited in Smith, 1964) showed that compara-
tively small doses of cyanide given at long intervals produced histolog-
ical changes in the central nervous system of the rat.
From experiments on mice, Isom, Liu, and Way (1975) concluded that
cyanide exposure could induce a marked alteration in normal carbohydrate
metabolism and that this alteration might be associated with pathological
conditions related to cyanide exposure.
6-3.3.1 Tropical Neuropathies and Amblyopias - The fully developed syn-
drome of tropical neuropathies is characterized by optic atrophy, nerve
deafness, and sensory spinal ataxia (Money, 1958). Various fragmentary
forms of the fully developed syndrome may occur. Tropical amblyopia was
first studied in Nigeria by Moore (1930, 1932, 1934a, 1937, cited in
Osuntokun et al., 1968). Cyanide was suggested as a possible contributing
factor by Clark (1935), Monekosso and Annan (1964), Monekosso and Wilson
(1966), and Osuntokun (1968). The diet of many people in tropical areas
-------
TABLE 6.6. ANIMAL RESPONSE TO INHALED CYANIDE AND CYANIDE-CONTAINING COMPOUNDS
Compound
Animal
Cyanide
concentration
(rag/liter)
Hydrogen cyanide
Cyanogen
Cyanogen chloride
Cyanogen bromide
Aery lonit rile
Acetonitrile
Isobutyronitrile
Propionitrile
Rat
Guinea pig
Rabbit
Cat
Dog
Mouse
Mouse
Rat
Rat
Rat
Rabbit
Cat
Mouse
Rat
Rat
Rabbit
Cat
Dog
Dog
Dog
Goat
Mouse
Cat
Rat
Rat
Rat
Rat
Guinea pig
Guinea pig
Rabbit
Rabbit
Cat
Cat
Dog
Dog
Dog
Dog
Dog
Dog
Rhesus monkey
Monkey
Rat, male
Dog
Rat
Rat
0.
0.
0.
0.
0.
0.
4.
0.
0,
8,
0,
0.
1.
I.
2.
3.
0,
0,
0.
1.
2.
1
1
0,
1.
0,
1,
0,
0.
0,
0.
0,
0.
0.
0.
12
35
35
35
125
.5
.26
.851
.851
,508
.84
42
.0
,40
.80
.0
.3
,12
,8
.0
.2
.28
,38
.58
.25
,29
.56
.33
.60
,213
.24
,24
.33
(ppm)
110
315
315
315
115
235
2000
400
400
4000
400
200
400
1200
120
48
320
400
230
230
75
100
130
635
265
575
135
260
153
275
50
75
100
100
110
110
75
153
7500
8000
5500
9500
Response
Fatal in 1.5 hra
Fatal0
Fatala
Respiratory paralysis, 2 min;
death, 5-10 mina
Fatala
Recovered, 15-min exposure
Fatal, 13-min exposure
No deaths, 45-min exposure
Fatal, 60-min exposure
Fatal, 15-min exposure
Fatal, 1.8-hr exposure
Fatal, 1/2-hr exposure
Fatal to some, 3— min exposure
Fatal, 10-min exposure
Fatal, 5-min exposure
Fatal, 2-rain exposure
Fatal, 3. 5-min exposure
Fatal, 8-hr exposure
Fatal, 7. 5-min exposure
Fatal,
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142
TABLE 6.7. LD50 OF COMPOUNDS CONTAINING CYANIDE MOIETY AFTER SKIN
ABSORPTION BY RABBITS AND GUINEA PIGS
Compound
Animal
Dose
Skin response
Reference
Cyanogen Rabbit 10,000 ppm No effect, 8-hr exposure3 McNerney and Schrenk, 1960
Acetonitrile Rabbit 1.25 ml/kg Fassett, 1963
Isobutyronitrile Guinea pig <5 ml/kg Slight irritation Fassett, 1963
Propionitrile Guinea pig <5 ml/kg Slight irritation Fassett, 1963
ainhalation exposure was prevented.
TABLE 6.8. ACUTE TOXICITY OF CYANIDE AND CYANIDE-CONTAINING COMPOUNDS TO EXPERIMENTAL ANIMALS
Compound
Hydrogen cyanide
Potassium cyanide
Sodium cyanide
Calcium cyanide
Cyanogen chloride
Cyanogen iodide
Acetonitrile
Propionitrile
Isobutyronitrile
Animal
Rabbit, male
Rabbit, female
Mouse
Rat , male
Rabbit, male
Rabbit, female
Rat
Dog
Rat
Mouse
Rabbit
Pigeon
Frog
Mouse
Rat
Rabbit
Rabbit
Rabbit
Cat
Cat
Dog
Rat
Guinea pig
Rat
Rat
Guinea pig
Guinea pig
Mouse
Rat
Dosage Route of a Effect
(mg/kg) administration
1.50 (1.27-1.80)
0.95 (0.81-1.11)
6.02 + 3.3
8.7-11.5
3.06 (2.61-3.63)
3.27 (2.70-4.08)
15 (11-21)
5.36 + 0.28
39 (30-51)
39.07
20.038
43.53
111-143
27-36
44
23.5
19-40
15
18
23
19-30
1.7-8.5
0.18
50-100
25-50
25-50
10-25
5-10
50-100
IM
IM
OR
OR
IM
IM
OR
SC
OR
SC
SC
SC
SC
SC
SC
OR
SC
IV
OR
SC
SC
OR
OR
OR
IP
OR
IP
OR
OR
LD50
LD50
LD50
LD
LD50
LE50
LD50
LD50
LD50
LD
LD
LD
LD
LD
LD
LD
LD
LD
LD
LD
LD
LD50
LD50
LD50
U>50
LD50
LD50
LD50
LD50
Reference
Ballantyne et al. ,
Ballantyne et al.,
Streicher, 1951
Gaines, 1969
Ballantyne et al . ,
Ballantyne et al.,
Smyth et al. , 1969
Chen and Rose, 1952
Smyth et al. , 1969
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Hunt, 1923
Fassett, 1963
Fassett, 1963
Fassett, 1963
Fassett, 1963
Fassett, 1963
Fassett, 1963
Fassett, 1963
Fassett, 1963
1971
1971
1971
1971
IM — intramuscular; OR — oral; SC — subcutaneous; IP — intraperitoneal.
includes cassava, sometimes as a major component of the diet. Cassava
has a high cyanogenic glycoside (linamarin) content (Section 4.2.3),
which releases HCN on enzymatic or acid hydrolysis. Osuntokun, Monekosso,
and Wilson (1969) compared the prevalence of neurological disorders in
two Nigerian villages that differed in amount of cassava eaten (64.3%
cassava meals in one village vs 10.8% in the other) but were similar with
respect to the population's mean age, weight, height, and prevalence of
sickling. A degenerative neuropathy occurred with a relatively high fre-
quency in the village with high cassava consumption. It should be pointed
out that factors other than cyanide such as protein deficiency, riboflavin
deficiency, abnormal vitamin Bi2 metabolism, and infections may be in-
volved. These other factors still may be related to cyanide. For example,
-------
143
lack of substrate for normal cyanide detoxification can occur because of
the deficiency of sulfur-containing amino acids in the diet (Osuntokun
et al., 1968). Most etiological studies have focused on chronic cyanide
intoxication (Monekosso and Wilson, 1966; Osuntokun, 1968; Osuntokun et
al., 1968; Osuntokun, Monekosso, and Wilson, 1969; Osuntokun, Aladetoyinbo,
and Adeuja, 1970). From studies on 320 patients with Nigerian ataxic
neuropathy, Osuntokun (1972) reported that protein-calorie deficiency and
deficiency of water-soluble vitamins were not important in the etiology
of the disease but contributed to the clinical picture. These patients
had demyelination of peripheral nerves and resultant decreased conduction
velocity of motor nerves.
Makene and Wilson (1972) concluded, from data on thiocyanate and
vitamin B12 levels in plasma of Tanzanian patients with ataxic tropical
neuropathy, that the condition may be attributed to chronic cyanide in-
take from cassava. Data in Table 6.9 show that thiocyanate levels were
significantly higher in eight patients with ataxic neuropathy than in nine
controls at the P < 0.1% level. Vitamin B12 levels, both with and with-
out cyanide added to the extraction mixture, were significantly higher
at the 5% level in ataxic neuropathy patients.
Other neuropathies such as West Indian amblyopia, tobacco amblyopia,
and Leber's optic atrophy are all characterized by visual field defects and
may be attributed to cyanide as a possible etiological factor. MacKenzie
and Phillips (1968) examined the visual field of ten West Indian ambly-
opia patients. There is no direct evidence that cyanide was involved,
however, the above authors felt that it was a possible factor. The ques-
tion was raised that Leber's optic atrophy may be an inborn error of cya-
nide metabolism which becomes apparent when the body is confronted with
a source of cyanide such as tobacco smoke, cassava, or certain infections
(Wilson, 1956). It is of interest to note that in tobacco amblyopia,
vitamin Bi2, particularly as hydroxocobalamin, is an effective treatment
(Chisholm, Bronte-Stewart, and Foulds, 1967). Chisholm, Bronte-Stewart,
and Foulds (1967) administered to patients 1.0 mg of cyanocobalamin or
hydroxocobalamin parenterally daily for two weeks, followed by 1.0 mg
twice weekly for four weeks, and then 1.0 mg at monthly intervals. Fig-
ure 6.2 shows the improvement in vision with time of treatment, especially
with hydroxocobalamin.
TABLE 6.9. PLASMA THIOCYANATE AND VITAMIN B12 LEVELS IN NEUROPATHY PATIENTS
AND IN CONTROLS WITH MISCELLANEOUS DISEASES
Patients with Patients with
ataxic neuropathy miscellaneous diseases
Thiocyanate 9.3 + 1.9 ymoles/100 ml plasma 2.8 + 0.82 ymoles/lOO ml plasma
812 + CN 700 + 118 pg/ml plasma 407 + 60 pg/ml plasma
612 - CN 380 + 64 pg/ml plasma 227 + 34.2 pg/ml plasma
Source: Adapted from Makene and Wilson, 1972, Table 1 and Table 2, p. 32.
Reprinted by permission of the publisher.
-------
144
g
in
LU >>
Ld o
"^> O
O -
or o
CL 77,
LU
ORNL-DWG 76-15518
HYDROXOCOBALAMIN
O/
/ o
o/
CYANOCOBALAMIN
01 2 34567
DURATION OF TREATMENT (months )
Figure 6.2. Rate of visual improvemeat per month in patients with
tobacco amblyopia treated with parenteral hydroxocobalamin or cyanoco-
balamin. Source: Adapted from Chisholm, Bronte-Stewart, and Foulds,
1967, Figure 1, p. 451. Reprinted by permission of the publisher.
Earlier, Heaton, McCormick, and Freeman (1958) achieved some success
in treating tobacco amblyopia with cyanocobalamin (100 mg) parenterally.
The treatment was discontinued after six months if the symptoms were gone.
Small amounts of hydroxocobalamin are present in commercial preparations
of cyanocobalamin and may actually have been the effective substance
(Smith and Duckett, 1965). Bronte-Stewart, Chisholm, and Lewis (1968)
reported a case of tobacco amblyopia that did not respond to cyanocobal-
amin treatment but did improve when hydroxocobalamin was administered.
Foulds et al. (1968) reported a similar situation in one, and possibly
two, cases of Leber's hereditary optic atrophy that did not respond to
cyanocobalamin but did improve with hydroxocobalamin. Because cyanoco-
balamin may actually accelerate development of optic atrophy, hydroxo-
cobalamin has been specifically recommended (Foulds et al., 1970).
Cyanide has been implicated as a possible etiological agent in var-
ious ^ human neuropathies. Lessell (1971), however, suggested caution in
attributing the etiology of human nerve disorders to cyanide. Studies
with rats (Lessell, 1971; Lessell and Kuwabara, 1974) indicate that nerve
damage from cyanide in experimental cyanide lesions and human disorders
were similar. However, the cyanide dose necessary to produce experimental
-------
145
nerve damage was in the lethal range. Also, in the rat the corpus cal-
losum is much more sensitive to cyanide than is the optic nerve, whereas
in human disorders ocular involvement is often the predominent or only
indication of brain involvement.
6.3.3.2 Goitrogenic Action — Cassava may contribute to endemic goiter
and cretinism. A high incidence of mental deficiency in many regions
where endemic goiter occurs suggests a possible common etiological factor
(Ermans et al., 1972). Iodine deficiency is generally considered to be
involved in endemic goiter; however, in some cases it may not be the only
factor (Delange, Thilly, and Ermans, 1968). A study of many communities
throughout Idjwi Island in Lake Kivu (Republic of Zaire) showed a severe
and uniform iodine deficiency in the whole population, but endemic goiter
occurred in only one region. Cassava was eaten in especially large amounts
in this region (Delange and Ermans, 1971). Thiocyanate, resulting from
detoxification of cyanide released from the cassava, may be exerting an
antithyroid action, as this is a well known effect of thiocyanate (Barker,
1936; Barker, Lindberg, and Wald, 1941; Ermans et al., 1972). Thiocya-
nate, a metabolite of the cyanogenic glycoside linamarin in cassava, can
affect thyroid function, producing an endemic cretinism (Ermans et al.,
1972) . Although there is no direct evidence indicating that cassava con-
sumption is responsible for endemic goiter and cretinism in this region,
experimental data in rats are consistent with data from subjects in the
goitrous area (Ermans et al., 1973). Ermans et al. (1973) presented the
following observations:
1) The investigations first show that in rats, continu-
ous intake of cassava is capable of causing changes in iodine
and thiocyanate metabolism which are similar to those obtained
by prolonged administration of thiocyanate.
2) Ingestion of thiocyanate or cassava entails marked
depletion of iodine stores: depletion is fairly moderate in
iodine-supplemented rats. This depletion is very severe in
iodine-deficient rats and is associated with major changes in
intrathyroidal metabolism which iodine deficiency alone is
incapable of causing.
3) Chronic ingestion of thiocyanate does not necessarily
cause blocking of the thyroidal iodide pump; iodine uptake by
the gland seems, on the contrary, to be increased, probably
due to thyrotropic stimulation triggered by iodine depletion.
This does not preclude transitory inhibition during the phase
of thiocyanate absorption.
4) Administration of thiocyanate or its precursors even
in increasing doses does not necessarily entail a very marked
rise of SCN concentration in the blood. Evidence of increased
ingestion is only obtained by measurement of urinary excretion
or estimation of plasma turnover of SCN.
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146
5) The iodine depletion seems to be mainly due to an
increased loss of iodine in urine related to a blockage of the
tubular reabsorption of this ion by an excess of SCN.
Other naturally occurring goitrogenic agents are present in some
foods (Greer, 1962). Endemic goiter has existed for a long time in Slo-
vakia in Czechoslovakia and 70% or more of the adult women in endemic
areas in this region have goiters (Podoba, Michajlovskij, and Stukovsky,
1973). An inverse correlation exists between iodine in the drinking water
in the various communities and the incidence of goiter. Also, vegetables
of the Brassica family, which have a goitrogenic potential, are consumed
in large amounts and have a high sulfur and thiocyanate level.
6.3.3.3 Carcinogenesis, Teratogenesis, and Mutagenesis — There is a
paucity of information on the carcinogenic, teratogenic, and mutagenic
properties of cyanide and the available evidence warrants further exami-
nation. Cyanide has been employed as an antitumor agent in experimental
animals and in humans (Brown, Wood, and Smith, 1960; Perry, 1935; Stone,
Wood, and Smith, 1959). It appears to have a selective inhibitory effect
on certain tumor tissues such as the Ehrlich Ascites tumor and Sarcoma-
180 in mice. The regional perfusion studies of pelvic tumor with cyanide
in humans apparently can be done without evidence of toxicity. In these
latter studies there was no evidence of a decrease in either tumor size
or number of metastasis, but the pathologic cellular changes of the
tumors were encouraging.
A long-standing claim has been made for an anticancer effect by the
cyanogenic glycosides (Krebs, 1970; Morrone, 1962; Navarro, 1959); how-
ever, these claims have been vigorously refuted by various laboratories
(Lewis, 1977). The rationale of cancer treatment with the cyanogenic
glycoside, amygdalin (also called laetrile) is a postulated selective
hydrolysis of amygdalin by a 3-glucosidase, to free cyanide, benzalde-
hyde, and sugar at the cancer site. The cyanide was then proposed to
selectively attack the cancer cell which presumably is deficient in rho-
danese. Normal cells, which contained rhodanese were assumed to possess
adequate concentration of the sulfur donor to be able to detoxify cyanide
and therefore would suffer no permanent damage.
The opponents of this rather simplistic theory have pointed out
(Greenberg, 1975; Lewis, 1977) that many tumor tissues are not selectively
enriched in 3-glucosidase nor are they low in rhodanese. Moreover, there
are numerous studies citing a lack of antitumor activity of amygdalin in
model tumor systems (Hill et al., 1976; Laster and Schnabel, 1975; Lev!
et al., 1965; Wodinsky and Swiniarski, 1975). It is noted, however, that
no large scale carefully documented study of the effectiveness of amyg-
dalin as an antitumor agent has been carried out on human patients.
Chronic suppression of prolactin secretion in C3H/HeJ female mice
by 6-methyl-8-g-ergoline-acetonitrile significantly inhibited development
of mammary hyperplastic alveolar nodules and greatly reduced mammary
tumor incidence (Welsch, Gribler, and Clemens, 1974).
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147
3-Aminopropionitrile (BAPN) was teratogenic when tested in pregnant
rats (Abramovich and Devoto, 1968; Barrow and Steffek, 1974; Herd and
Orbison, 1966; Wilk et al., 1972) and in pregnant baboons (Steffek and
Hendrickx, 1972) . Wilk et al. (1972) studied the metabolism and distri-
bution of BAPN and cyanoacetic acid (CAA), a major metabolite, in rats.
The teratogenic agent was BAPN rather than CAA; the incidence of cleft
palate was correlated with maternal and fetal levels of BAPN. Day 15 of
gestation is the critical time for cleft palate induction and administra-
tion of BAPN on day 14 or 16 produced few or no cleft palates. Oral
administration of 270 mg BAPN on day 15 led to 98% incidence of cleft
palate in offspring. Six hours after BAPN administration to pregnant
rats on day 15, an embryonic level of 106, 42, and 16 yg BAPN per gram
of tissue corresponded to an incidence of cleft palate of 98%, 28%, and
0%, respectively. Oral CAA did not produce cleft palate although high
embryonic CAA levels resulted. Rabbits, which metabolize BAPN very effi-
ciently, had a low incidence of cleft palate in these studies, supporting
BAPN as the active agent.
Barrow and Steffek (1974) also obtained teratologic and other embryo-
toxic effects with BAPN in rats. Day 15 of gestation was an important
time for embryotoxic effects with no gross malformations occurring if BAPN
was administered before day 14 although resorptions did occur. Generally,
higher doses (e.g., 5000 mg/kg body weight) caused fetal resorptions.
Lower doses at certain times produced various fetal abnormalities (e.g.,
2500 mg/kg on days 14 to 15) such as ecotcardia, gastroschisis, and cleft
palate. Complete data are shown in Table 6.10.
Pregnant baboons were given BAPN to extend the earlier studies
(Steffek and Hendrickx, 1972). Only three animals were used in this pre-
liminary study and each was given a different dose size via a different
route at a different time during gestation; therefore, the teratogenic
potential could not be determined. The data are presented in Table 6.11.
Palate formation and closure occurs during days 40 to 50 of gestation in
baboons. The effects of aminoacetonitrile (AAN) were also tested in this
study. The results are difficult to interpret. The appearance of cleft
palate in each twin of a dizygous set was taken as an indication of a
common environmental etiology (i.e., AAN administration).
6.3.4 Treatment for Cyanide Poisoning
Because cytochrome oxidase is the most sensitive enzyme to cyanide
(Section 6.3.2), any substance that can effectively compete with cyto-
chrome oxidase for the cyanide ion could be an effective antagonist.
Methemoglobin is an effective competitor (Albaum, Tepperman, and Bodansky,
1946) and the body can tolerate blood concentrations of 30% with no symp-
toms and up to 70% before lethal levels are reached (Bodansky, 1951).
The accepted treatment for acute cyanide poisoning (Chen, Rose, and Clowes,
1933, 1934; Hug, cited in Chen and Rose, 1952) is an intravenous injec-
tion of sodium nitrite (0.3 g in 10 ml water) to convert a portion of the
hemoglobin to methemoglobin, followed immediately by an intravenous injec-
tion of thiosulfate (12.5 g in 50 ml water) to supply a sulfur donor, the
substrate, for rhodanese. Usually amyl nitrite is inhaled by the patient
-------
TABLE 6.10. EMBRYOLETHAL AND TERATOLOGIC EFFECTS OF BAPN IN PREGNANT RATS
Days
treated
0 (controls)
1-7
8-13
9
10
11
11-12
11-16
11-17
11-17
11-18
11-18
11-20
11-20
12
12
13
13
13-14
13-14
13-15
13-16
13-17
13-17
13-18
Dose
(mg/kg)
1000
1000
3500,
4250,
5000
3000,
3500,
4250
3500,
5000
4250
1000
1000
4000
1000
2000
250
500
3000,
3500,
4250
5000
2500,
3000,
3500,
4250
5000
3000
3500,
4000
2500
1000
1000
4000
1000
Number
of
litters
111
10
15
9
15
12
4
19
4
5
6
3
3
5
15
5
24
6
8
9
12
7
5
5
8
Resorp-
tions
No.
79
3
74
12
12
34
27
78
14
50
40
31
9
31
29
43
42
35
42
76
63
8
4
21
50
%
7
3
54
16
9
43
100
47
34
100
69
100
29
55
25
81
23
61
47
100
47
14
8
100
66
Number
of
living Ecto-
fetuses cardia
1124 0
83
64
63
116
45
87
27
18
22
25
89
10
139
22
48 2
70 5
51
43
26
Abnormalities
Cleft Osteolathyritic
Gastro- B d T°tal palate effects
schisis T a,
M0' '° No. 7. No. % Degree
0 00 00000
24 27 87 100 Mild to
moderate
1 4 27 100 Mild
14 78 18 100 Mild
22 100 Very mild
25 100 Mild
10 100 Very mild
6 27 Very mild
1 3 6 1 2 11 23 Mild
3 1 9 11 7 10 3 4 Very mild
19 37 51 100 Mild
22 51 43 100 Moderate
23 88 26 100 Moderate
00
to severe
(continued)
-------
TABLE 6.10 (continued)
Days
treated
13-20
14
14
14
14
14
14
14
14-15
14-15
14-15
14-15
15
15
15
15
15
15-16
15-17
16
16
16
16
16
16
16-17
16-17
17
17
17
18
19
19
21
Dose
(mg/kg)
1000
2500
3000
3500
4000
4500
5000
5500,
6000
2500
3000
3500
4000
2500
3500
4000
4500
5000
3000
2000
1000
2500
3000
3500
4000
4500
1000
2000
1000
2500
3500
2500
2500
2500
2500
Total
Number
of
litters
8
6
5
6
5
4
9
7
30
11
12
9
13
6
15
8
4
3
3
3
24
8
8
6
3
10
4
4
7
3
4
11
5
2
480°
Resorp-
tions
No.
78
8
3
60
14
20
54
73
188
87
91
89
4
16
95
66
36
27
13
3
112
40
52
36
20
8
22
1
37
17
28
45
0
0
%
96
12
6
85
34
53
47
95
55
81
93
100
4
31
68
72
100
96
43
9
42
43
65
66
50
8
55
3
50
68
72
47
0
0
Number
of
living Ecto-
fetuses cardia
3
56
44
11
27
18
60
4
154 11
21 4
7
96
36 1
44 1
26 1
1
17
27
152
54
28
18
20
91
18
30
37
8
11
51
39
14
Abnormalities
Total C*
GaStr° Both • Pal
schism %
No.
3
3
1 31
1 27
3 11 25 16 4
4 19
14 3
1 35
12 5 12
1 44
1
144
54
28
17
20
16
18
eft Osteolathyritic
ate effects
%
100
7
3
12
4
43
14
27
16
100
95
100
100
94
100
18
100
No.
3
15
8
11
9
8
16
6
9
10
26
1
17
130
54
28
18
20
80
18
30
35
8
11
51
%
100
27
18
100
33
44
27
29
100
10
72
100
100
86
100
100
100
100
100
100
100
95
100
100
100
Degree
Severe
Very mild
Very mild
Very mild
Very mild
Mild
Mild
Mild
Mild
Mild
Mild
Mild
Mild
50 severe,
80 mild
44 severe,
10 mild
Severe
Severe
Severe
42 mild, 38
severe
Severe
Mild
25 severe,
10 mild
Severe
Mild
16 severe,
35 mild
Does not include controls.
Source: Adapted from Barrow and Steffek, 1974, Table 1, pp. 168-169. Reprinted by permission of the publisher.
-------
150
as an emergency means of inducing methemoglobinemia because it can be
given immediately while sodium nitrite and thiosulfate injections are
being prepared. Data from Chen and Rose (1952) showed the comparative
effects of these compounds alone and in combination on cyanide poisoning
in dogs (Table 6.12).
TABLE 6.11. EFFECT OF BAPN AND AAN ON FETAL DEVELOPMENT IN BABOONS
Drug
BAPN
AAN
Days of
treatment
during
gestation
38-50
37-48
43-48
38-50
38-50
38-50
38-41
38-48
40-48
43-48
43-48
45-48
46-48
Dose
(mg kg"1 day"1)
200
300
500
20
20
40
70
60
60
75
75
130
130
Route
of
administration
IM
OR
IV
IM
IM
IM
IM
OR
OR
OR
OR
OR
OR
Results
Fetal resorption
Fetal resorption
Fetal maceration, spina
bifida
Abortion
Normal
Normal
Abortion
Fetal resorption
Normal
Abnormal flexure of digits on
right foot
Twins, both with cleft palate
and abnormal curvature of
arms and legs
Normal
Normal
IM — intramuscular; OR — oral; IV — intravenous.
Source: Adapted from Steffek and Hendrickx, 1972, Table 1, p. 172. Reprinted by
permission of the publisher.
TABLE 6.12. COMPARISON OF EFFECT OF ANTIDOTES ON CYANIDE
POISONING IN DOGS
Antidote
Number
of
dogs
LD5g of sodium
cyanide + standard
error
(mg/kg)
Ratio of LD50's
of sodium cyanide
in treated and
untreated dogs
None 16
Sodium thiosulfate 11
Amyl nitrite 13
Sodium nitrite 13
Amyl nitrite and
sodium thiosulfate 17
Sodium nitrite and
sodium thiosulfate 21
5.36 + 0.28
18.4 + 0.9
24.5 + 1.2
27.1 + 3.1
60.9 + 3.0
96.7 + 23.6
1
3
5
5
11
18
Source: Adapted from Chen and Rose, J. Am. Med. Assoc., May,
Vol. 149, Table 1, p. 114, copyright 1952, American Medical Association.
-------
151
It would seem reasonable to employ oxygen with the classic nitrite-
thiosulfate therapy, as oxygen appears to enhance the antidotal effect
of this combination (Sheehy and Way, 1968; Way, Gibbon, and Sheehy, 1966a,
1966£>; Way et al., 1972). Also various cobalt containing compounds are
employed as a cyanide antagonist (Burrows and Way, 1977; Evans, 1964;
Friedberg, Grutzmacher, and Lendle, 1965; Isom and Way, 1973; Mushett et
al., 1952; Paulet, 1958; Rose et al., 1965).
-------
152
SECTION 6
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SECTION 7
ENVIRONMENTAL DISTRIBUTION AND TRANSFORMATION
7.1 SUMMARY
Production figures for cyanide are over 700 million pounds per year
and are steadily increasing. Figures indicating increased yearly produc-
tion are available for dicyandiamide, acrylonitrile, and other organic
nitriles. The primary users of cyanide compounds include the electro-
plating, steel, plastics, and various chemical industries.
Cyanide wastes are produced by various industrial processes. Some
attempts have been made to control waste cyanide but an appreciable amount
is still being discharged into the environment. Sources of cyanide that
enter the environment include the steel and electroplating industries,
mining operations, catalytic converters on automobiles, home fires, and
hospital laboratories.
Data concerning cyanide movement in soils are fragmentary. Cyanide
is believed to not be strongly adsorbed or retained by soils. Since most
cyanide salts are soluble, they move through soils and are converted to
other compounds or are fixed by trace metals.
Cyanides are believed to be relatively uncommon in most U.S. water
supplies. When they do occur, they are usually less than the U.S. Public
Health Service limit. Cyanides in the atmosphere probably are increasing
as a result of pollution. However, data concerning the distribution and
transformation of cyanides in air are lacking.
Of the many processes which have been proposed for cyanide removal
from industrial wastewater, only a few appear to be economically feasible
and are commonly used (e.g., alkaline chlorination, electrolytic decom-
position, and ozonation). Improper management of cyanide wastes has led
to various incidents of environmental damage.
7.2 PRODUCTION AND USAGE
Information concerning the level of cyanide production has been
regarded as confidential since 1939 (Fairhall, 1969). Only fragmentary
production figures are available. The estimated U.S. production capacity
for hydrogen cyanide varies from different sources. It was 202 million
kilograms (445 million pounds) as of early 1964 (Montgomery, 1965) and
700 million pounds in 1976. Of this amount, 52% was used in acryloni-
trile production, 18% in methyl methacrylate production, 14% in adi-
ponitrile production, and 7% in sodium cyanide production; the remaining
9% was used for various purposes.
The world production capacity (excluding the USSR and its sat-
ellites) for sodium cyanide is greater than 90,800 metric tons (100,000
tons) per year (Mooney and Quin, 1965). Actual usage in 1963 was about
164
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165
64,000 to 73,000 metric tons (70,000 to 80,000 tons). World production
of potassium cyanide was about 4500 metric tons (5000 tons). Free world
production of dicyandiamide was 90,800 metric tons (100,000 tons) in 1962
(McAdam and Schaefer, 1965). Melamine production consumed 80% to 90% of
this output. Recent U.S. production of acrylonitriles has been about
5 x 10s metric tons and has grown at about 12% per year (National Academy
of Sciences, 1975). Exports in 1972 were 2470 tons, about 0.5% of the
total production. Imports amounted to 1.0 metric ton (1.1 tons).
The primary uses of some major cyanide compounds are given in Table
7.1. A large percentage of cyanide usage is by the electroplating, steel,
and chemical industries.
7.3 SOURCES
Cyanide wastes are produced by various industries, and only a rela-
tively small fraction of cyanide is believed to escape into the environment.
The largest amount of cyanide wastes is generated by the electroplating
industry (Table 7.2). Because cyanides are such good complexing agents,
they are used in plating baths. The concentration of cyanides in liquid
wastes of the National Association of Metal Finishers member plants ranges
from 9 to 115 ppm, with an average of 72 ppm (Reed et al., 1971). Waste
streams of the electroplating industry may contain 0.5% to 20% cyanide
before treatment. Approximately 9,660,000 kg (21,300,000 Ib) of cyanide
wastes is discharged each year by 2600 U.S. electroplating plants (Table
7.3).
Paint manufacturing generates cyanide wastes. About 20,000 kg (45,000
Ib) of cyanide is lost each year through 16.8 million kilograms (37 million
pounds) of solvent-based waste paint sludges (Ottinger et al., 1973a).
Additionally, residues in used paint containers provide an estimated
141,000 kg (310,000 Ib) of cyanide each year to the environment.
The steel industry also provides cyanide input. Weak ammonia liquor
from the Bethlehem Steel Corporation results from coking 4350 metric tons
(4800 tons) of coal per day and contains 20 to 80 ppm cyanide as CN~ and
700 to 1300 ppm as thiocyanate (Cousins and Mindler, 1972). Typical coke
oven liquor contains 6 ppm cyanide, with a range of 0 to 8 ppm (Pearce
and Punt, 1975). A liter of coke oven gas normally contains about 2.3
mg of HCN (0.5 to 1 grain per standard cubic foot) (Mitachi, 1973).
Cyanide enters the environment as a result of mining operations.
Raw wastewaters from copper mine and concentrator operations contain 0.06
ppm cyanide (Hallowell et al., 1973). The Tjikotok mineral processing
plant in West Java discharges 1.25 x 10s kg of waste (containing about
800 mg sodium cyanide per kilogram) per year to the river Tjimadur (Bowen,
1971).
Laboratory wastes provide a minor cyanide emission. Hospital labs
in Buffalo, New York, have discharged 5600 g of cyanide per year to the
environment CPragay, 1974). It is estimated that a laboratory in a 1000-
bed hospital in the Buffalo area would discharge 930 g of cyanide per
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TABLE 7.1. PRIMARY USES OF SOME MAJOR CYANIDE COMPOUNDS
Compound
Uses
Reference
Acrylonitrile, C2HN
Cadmium cyanide, Cd(CN)2
Calcium cyanide, Ca(CN)2
Calcium cyanamide, CaCN2
Cuprous cyanide, CuCN
Cyanogen,
Cyanogen bromide, CNBr
Dicyandiamide,
Hydrogen cyanide, HCN
Lead cyanide, Pb(CN)2
Melamlne, C3N3(NH2)3
Nickel cyanide, Ni(CN)2'4H20
Potassium cyanate, KOCN
Potassium cyanide, KCN
Potassium f erricyanide, K3Fe(CN)5
Production of acrylic and modacrylic fibers,
nitrite elastomers, plastics
Electroplating
Ore cyanidation, froth flotation, fumigation,
HCN production, ferrocyanide production,
rodenticide, case hardening of steel, cement
stabilizer
Fertilizer, defoliant, weed killer, production
of melamine, steel production
Electroplating, medicine, insecticide, oxygen
removal from molten metals, underwater paint,
organic nitrile separation
Fumigant
Organic syntheses, fumigant, pesticide, gold
extraction, cellulose technology
Melamine manufacture, vinyl resin stabilizer,
curing agents for epoxy resins, textile-
drying assistant, starch fluidifying agent,
guanidine salt production
Rodenticide, insecticide, electroplating, ethyl
lactate, acrylonitrile synthesis, ferrocyanide
manufacture, lactic acid, chelating agents,
optical laundry bleaches, Pharmaceuticals
Insecticide, electroplating
Melamine-formaldehyde resins, textile fire
retardants, bactericide, tarnish inhibitor
Electroplating
Weed killer, chemical intermediate
Electroplating, steel hardening, extraction of
metals from ores, nitrile manufacture, fumi-
gation, photography, silver polish
Photography, blueprints, metal tempering,
electroplating, pigments
National Academy of Sciences, 1975
Ottinger et al., 1973&
National Academy of Sciences, 1975
McAdam and Schaefer, 1965
Ottinger et al., 1973i>
Hardy and Boylen, 1971
Mooney and Quin, 1965
McAdam and Schaefer, 1965
Hardy and Boylen, 1971;
Montgomery, 1965
Ottinger et al., 1973i
McAdam and Schaefer, 1965
Ottinger et si., 1973b
Zuzik, 1974
Hardy and Boylen, 1971;
Hamilton and Hardy, 1974
Hardy and Boylen, 1971
h-1
ON
(continued)
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TABLE 7.1 (continued)
Compound
Uses
Reference
Potassium ferrocyanide, KitFe(CN)g • 3H20
Silver cyanide, AgCN
Sodium cyanate, NaOCN
Sodium cyanide, NaCN
Zinc cyanide, Zn(CN)2
Tempering of steel, process engraving, pigment
manufacture, dyes
Electroplating
Organic syntheses, heat treatment of steel,
Pharmaceuticals
Metal treatment, electroplating, synthesis of
organic intermediates, ore extraction, organic
chemical synthesis, photography, silver polish
Medicine, electroplating
Hardy and Boylen, 1971
Ottinger et al., 1973fc
Hardy and Boylen, 1971
Hamilton and Hardy, 1974;
Ottinger et al., 1973&
Ottinger et al. , 1973*
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168
TABLE 7.2. TYPICAL ELECTROPLATING WASTES CONTAINING CYANIDES
Waste description
Liquid waste containing 15% sodium cyanide, a 10% mixture of sodium ferro-
cyanide and sodium ferricyanide, and traces of nickel and zinc
Stripping solution containing 13% sodium cyanide, sodium hydroxide, and
600 ppm copper
0.8% cyanide, 3300 ppm zinc, 165 ppm nickel, and trace of silver in a 1%
sodium hydroxide solution
Slurry containing 20% sodium ferrocyanide, 2% zinc and insoluble material,
and 50% water
Slurry containing 2.5% zinc ferrocyanide, 2% calcium fluoride, 3% chromic
hydroxide, and 80% water
1% potassium ferrocyanide and <50 ppm lead, nickel, chromium, and copper
combined in an aqueous 10% sodium hydroxide solution
3% to 5% sodium cyanide and 1% to 3% nickel, cadmium, copper, and zinc in
an aqueous 10% sodium hydroxide solution
Source: Modified from Ottinger et al., 1973e, Table 3, p. 23.
year. A similar hospital in the Rochester area would discharge 3500 g.
The cyanide is generated mostly during hemoglobin and uric acid
determinations.
Less than 908 kg (2000 Ib) of cyanide compounds is awaiting disposal
in Department of Defense storage facilities. These compounds include
sodium, calcium, copper, potassium, and silver cyanides, and potassium
ferrocyanide, and potassium ferricyanide. All except calcium cyanide
were acquired for plating purposes.
7.4 DISTRIBUTION AND TRANSFORMATION IN THE ENVIRONMENT
7.4.1 Distribution and Transformation in Soils
Data concerning the distribution and movement of cyanides in soil
are limited and fragmentary. Because the toxicity of cyanide is well
known, treatment of wastes is a standard practice. High concentrations
in the soil are unusual and are nearly always the result of improper waste
disposal (Section 7.5). Although many herbicides contain a nitrile group
and are directly applied to soils, the cyanide group is not believed to
be released from these compounds during breakdown and, thus, is not re-
sponsible for herbicidal activity. Since herbicidal activity is appar-
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TABLE 7.3. INORGANIC CYANIDE WASTES
Source and
material
Cyanides from
electroplating
Paint sludge
cyanides
sludge
Paint residue
cyanides
old paint
Sodium cyanide
Calcium cyanide
Copper cyanide
Potassium cyanide
Silver cyanide
Potassium
ferri cyanide
Potassium
f errocyanide
Bureau of the Census regions
I II III IV V VI VII VIII IX
Annual waste production (Ib/year)
2.78 x 106 6.07 x 106 6.86 x 106 0.96 x 106 1.04 x 106 0.49 x 106 0.77 x 106 0.15 x 106 2.20 x 106
1,100 9,900 13,800 2,900 3,850 2,150 3,350 550 7,300
0.92 x 106 8.12 x 106 11.32 x 106 2.40 x 106 3.16 x 106 1.76 x 106 2.74 x 106 0.44 x 106 5.97 x 106
0.18 x 105 0.57 x 105 0.62 x 105 0.23 x 105 0.47 x 10s 0.20 x 105 0.30 x 105 0.13 x 105 0.41 x 105
13 x 106 41 x 106 44 x 106 16 x 106 34 x 106 14 x 106 21 x 106 9 x 106 29 x 106
Stored wastes (Ib)
1,400 16
180 25
100 32
2
16 10
4
12
Total
21.32 x 106
44,900
36.83 x 106
3.11 x 105
221 x 106
1,416
205
132
2
26
4
12
Source: Ottinger et al., 1973c, Table 1, p. 135.
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170
ently related to the integrity of the whole molecule, these herbicides
are not herein discussed.
Cyanide ions are not strongly adsorbed or retained by soils (Murrmann
and Koutz, 1972). The cyanide salts of most cations are soluble (except
AgCN) but move only a short distance through soil before being biologic-
ally converted under aerobic conditions to nitrates (microbial degrada-
tion to NH3, then conversion to N03~; Section 3.2.2.2) or fixed by trace
metals through complex formation. Under anaerobic conditions, cyanides
denitrify to gaseous nitrogen compounds which enter the atmosphere. The
cyanide ion is not involved in oxidation-reduction reactions (Murrmann
and Koutz, 1972).
The carbon and nitrogen of cyanide are converted to carbonate and
ammonia, respectively, in nonsterile soils (Strobel, 1967). Doubly la-
beled cyanide (li*ClsN) has shown that retention of the cyanide nitrogen
by soil is greater than retention of the cyanide carbon. Compounds such
as cyanamid, thiourea, dicyandiamide, guanidine nitrate, guanylurea, and
uramon are rapidly converted to ammonium and ultimately nitrate when they
are applied to soils as fertilizers at rates up to 100 ppm (Fuller, Caster,
and McGeorge, 1950).
7.4.2 Distribution and Transformation in Water
Cyanides occur in water as (1) free hydrocyanic acid (HCN), (2) sim-
ple cyanides (alkali and alkaline earth cyanides), (3) easily decompos-
able complex cyanides such as Zn(CN)2, and (4) sparingly decomposable
complex cyanides such as [Fe3+(CN)6]3~, [Fe2+(CN) 6] *~, and Co(CN)il. Com-
plex nickel and copper cyanides assume an intermediate position between
the easily decomposable and sparingly decomposable compounds (Leithe,
1973). The recommended limit for cyanide in U.S. waters is 10 ppb; con-
centrations of 200 ppb and above constitute grounds for rejection of the
water supply (U.S. Department of Health, Education, and Welfare, 1962).
Concentrations of cyanide exceeding the mandatory limit are usually
a result of improper waste management (Section 7.5). A survey of 969 U.S.
public water supply systems revealed no cyanide concentrations above the
mandatory limit (McCabe et al., 1970). In 2595 water samples, the high-
est cyanide concentration found was 8 ppb and the average concentration
was 0.09 ppb. All but one sample of United Kingdom waters contained less
than 50 ppb cyanide (Reed and Tolley, 1971). The exception contained
0.1 ppm.
7.4.3 Distribution and Transformation in Air
Volatile cyanides (e.g., hydrogen cyanide) are not normal atmospheric
contaminants. These compounds occur in air only occasionally via emission
from plating plants, fumigation, or other special operations (Stern, 1968).
As a result, no data were found concerning the distribution and transfor-
mation of cyanides in the atmosphere.
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171
7.5 WASTE MANAGEMENT
Due to the great toxicity of most cyanide compounds, the elimination
of cyanide from wastewaters is standard practice. The three main categor-
ies of removal techniques are (1) complete destruction of the cyanide ion,
(2) conversion of the cyanide ion to the cyanate ion, and (3) conversion
of the cyanide ion to some other less toxic form such as ferrocyanide
(Reed et al., 1971).
The most frequently used method of cyanide destruction is alkaline
chlorination. Wastewaters are treated with chlorine gas in an alkaline
solution to oxidize cyanide usually to carbon dioxide (carbonate ion) and
nitrogen (Section 2.2.1.4). If desired, the reaction may be controlled
to oxidize cyanide only to cyanate (Lawes, 1972; Watson, 1973).
Hypochlorites may also be used to destroy cyanides (Section 2.2.1.4).
This method involves essentially the same reactions as alkaline chlori-
nation. The active ingredient may be supplied as sodium hypochlorite,
calcium hypochlorite, or bleaching powder (Green and Smith, 1972; Watson,
1973) . Other possible methods of destruction include acidification, reac-
tion with aldehydes, electrolytic decomposition, ionizing radiation, and
heating (Lawes, 1972; Ottinger et al., 1973£>; Watson, 1973).
The acute toxicity of cyanate ion is about a thousand times less
toxic than the cyanide ion, and hence, may be discharged to the environ-
ment in low concentrations in some areas. The conversion uses chlorine
gas in a reaction similar to alkaline chlorination. Hypochlorites are
also used. Other oxidants proposed for the conversion of cyanide to cya-
nate include ozone, kastone (peroxygen), and permanganate (Green and
Smith, 1972; Lawes, 1972; Ottinger et al., 1973&; Watson, 1973).
Another method for converting cyanide to other less toxic forms is
the use of iron salts. The salt, usually ferrous sulfate, complexes with
free cyanide in aqueous solution and causes it to precipitate. This
method is commonly used in Europe but not in the United States (Ottinger
et al., 1973&; Watson, 1973).
Other methods described in the literature for cyanide waste treat-
ment include complexation by polysulfides or nickel salts, ion exchange,
evaporation, incineration, dilution, lagooning, and biological destruc-
tion (Avery and Fries, 1975; Cousins and Mindler, 1972; Green and Smith,
1972; Lutin, 1970; Murphy and Nesbitt, 1964; Muzzarelli and Spalla, 1972;
Ottinger et al., 19732?; Reed et al., 1971).
Improper management of cyanide wastes can result in damage to plant
and animal life. Contamination may occur as a result of improper storage,
handling, or disposal of cyanides. A landfill near Denver, Colorado, has
leaked cyanides to the surrounding area. Tests of surface drainage have
indicated the presence of cyanide in ponded water downstream from the site.
According to the site operator, significant amounts of cyanide were dis-
charged into pits at the disposal site (U.S. Environmental Protection
Agency, 1974).
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172
Cyanide, unknowingly released from a sewage plant in Oak Ridge, Ten-
nessee, was responsible for the death of 4800 fish in Melton Hill Lake
near the sewage outfall. The source of the cyanide was not known, but
it was believed to be from a metal-plating industry (Anonymous, 1975).
Disposal of cyanide wastes in a gravel pit near Cologne, Germany,
resulted in groundwater contamination by hexacyanoferrate of potassium.
Fortunately, tests revealed that this salt was relatively innocuous, so
the water supply was not shut down (Effenberger, 1964).
About 1500 55- and 30-gal drums containing cyanides disposed of near
Byron, Illinois, resulted in long-range environmental damage and live-
stock death. Surface water runoff from the area contained up to 365 ppm
cyanide. Measures have been taken to prevent further contamination (U.S.
Environmental Protection Agency, 1975).
7.6 BIOMAGNIFICATION AND CYCLING
There is no report of cyanide biomagnification within the food chain.
Cyanide is not likely to accumulate in food webs because low doses are
rapidly detoxified by most species and large doses result in death (Sec-
tion 5). Data concerning the cycling and transformation of cyanide in
the environment were not found.
7.7 CYANIDE IN FOODS
United States tolerances for HCN residues on foods are given in
Table 7.4. No data for cyanide residues on processed foods were noted.
A discussion of the cyanide compounds which occur naturally in edible
plants can be found in Section 4.2.
TABLE 7.4. TOLERANCES FOR CYANIDE RESIDUES ON FOODS
Food
Tolerance
(ppm)
Reference
Beans (dried)
Cocoa beans
Nuts
Citrus fruits
Grains
Spices
25 U.S. Environmental Protection Agency, 1969
25 U.S. Environmental Protection Agency, 1969
25 U.S. Environmental Protection Agency, 1969
50 Rules and Regulations, 1971a
75 Rules and Regulations, 197Ib
250 U.S. Environmental Protection Agency, 1969
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173
SECTION 7
REFERENCES
1. Anonymous. 1975. State Says Cyanide Caused Lake Fishkill. The Oak
Ridger, Oak Ridge, Tenn., September 5, 1975. p. 1.
2. Avery, N. L., and W. Fries. 1975. Selective Removal of Cyanide from
Industrial Waste Effluents with Ion-Exchange Resins. Ind. Eng. Chem.
Prod. Res. Dev. 14(2):102-104.
3. Bowen, H.J.M. 1971. Testing the Recovery of Silver and Gold from
Liquid Mineral Wastes. Radiochem. Radioanal. Lett. (Switzerland-
Hungary) 7:71-73.
4. Cousins, W. G., and A. B. Mindler. 1972. Tertiary Treatment of Weak
Ammonia Liquor. J. Water Pollut. Control Fed. 44(4):607-618.
5. Effenberger, V. E. 1964. Verunreinigung eines Grundwassers durch
Cyanide (Contamination of a Groundwater Through Cyanide). Arch. Hyg.
Bakteriol. (Munich) 148:271-287.
6. Fairhall, L. T. 1969. Hydrogen Cyanide. In: Industrial Toxicology.
Hafner Publishing Co., New York. pp. 272-274.
7. Fuller, W. H., A. B. Caster, and W. T. McGeorge. 1950. Behavior of
Nitrogenous Fertilizers in Alkaline Calcareous Soils: I. Nitrifying
Characteristics of Some Organic Compounds Under Controlled Conditions.
Technical Bulletin No. 120. University of Arizona, Tucson, Ariz.
pp. 451-467.
8. Green, J., and D. H. Smith. 1972. Processes for the Detoxification
of Waste Cyanides. Met. Finish. J. (London) 18:229-235.
9. Hallowell, J. B., J. F. Shea, G. R. Smithson, Jr., A. B. Tripler, and
B. W. Gonser. 1973. Water-Pollution Control in the Primary Nonferrous-
Metals Industry, Vol. 1. Report No. EPA-R2-73-247a, U.S. Government
Printing Office, Washington, B.C. 168 pp.
10. Hamilton, A., and H. L. Hardy. 1974. Industrial Toxicology-, 3rd ed.
Publishing Sciences Group, Inc., Acton, Mass. pp. 221-228.
11. Hardy, H. L., and G. W. Boylen, Jr. 1971. Cyanogen, Hydrocyanic Acid,
Cyanides. In: Encyclopedia of Occupational Health and Safety, Vol. 1.
McGraw-Hill Book Co., New York. pp. 352-354.
12. Lawes, B. C. 1972. Control of Cyanides in Plating Shop Effluents:
What's on the Shelf Now. Plating 59:394-400.
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174
13. Leithe, W. 1973. Cyanides. In: The Analysis of Organic Pollutants
in Water and Waste Water. Ann Arbor Science Publishers, Ann Arbor,
Mich. pp. 102-105.
14. Lutin, P. A. 1970. Removal of Organic Nitriles from Wastewater Sys-
tems. J. Water Pollut. Control Fed. 42:1632-1642.
15. McAdam, J. R., and F. C. Schaefer. 1965. Cyanamides. In: Kirk-Othmer
Encyclopedia of Chemical Technology, 2nd ed., Vol. 6, R. E. Kirk and
D. F. Othmer, eds. John Wiley and Sons, Inc., New York. pp. 553-573.
16. McCabe, L. J., J. M. Symons, R. D. Lee, and G. G. Robeck. 1970. Survey
of Community Water Supply Systems. J. Am. Water Works Assoc. 62:670-687
17- Hitachi, K. 1973. Cleaning Na Absorbents in Tailgas Recovery Circuits.
Chem. Eng. 80:78-79.
18. Montgomery, P. D. 1965. Hydrogen Cyanide. In: Kirk-Othmer Encyclo-
pedia of Chemical Technology, 2nd ed., Vol. 6, R. E. Kirk and
D. F. Othmer, eds. John Wiley and Sons, Inc., New York. pp. 574-585.
19. Mooney, R. B., and J. P. Quin. 1965. Alkali Metal Cyanides. In:
Kirk-Othmer Encyclopedia of Chemical Technology, 2nd ed., Vol. 6,
R. E. Kirk and D. F. Othmer, eds. John Wiley and Sons, Inc., New York.
pp. 585-601.
20. Murphy, R. S., and J. B. Nesbitt. 1964. Biological Treatment of
Cyanide Wastes. Engineering Research Bulletin B-88. The Pennsylvania
State University, University Park, Pa. 66 pp.
21. Murrmann, R. P., and F. R. Koutz. 1972. Role of Soil Chemical Pro-
cesses in Reclamation of Wastewater Applied to Land. In: Wastewater
Management by Disposal on the Land. Special Report 171, U.S. Army Cold
Regions Research and Engineering Laboratory, Hanover, N.H. pp. 48-76.
22. Muzzarelli, R.A.A., and B. Spalla. 1972. Removal of Cyanide and
Phosphate Traces from Brines and Sea-water on Metal Ion Derivatives of
Chitosan. J. Radioanal. Chem. (Switzerland-Hungary) 10:27-33.
23. National Academy of Sciences. 1975. Acrylonitrile. In: Assessing
Potential Ocean Pollutants. Washington, D.C. pp. 209-227.
24. Ottinger, R. S., J. L. Blumenthal, D. F. Dal Porto, G. I. Gruber,
M. J. Santy, and C. C. Shih. 1973a. Recommended Methods for Reduc-
tion, Neutralization, Recovery, or Disposal of Hazardous Waste,
Vol. 1. Report No. EPA-670/2-73-053-a, U.S. Government Printing
Office, Washington, D.C. p. 85.
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175
25. Ottinger, R. S., J. L. Blumenthal, D. F. Dal Porto, G. I. Gruber,
M. J. Santy, and C. C. Shlh. 1973&. Recommended Methods of Reduc-
tion, Neutralization, Recovery, or Disposal of Hazardous Waste, Vol. 5.
Report No. EPA-670/2-73-053-e, U.S. Government Printing Office,
Washington, D.C. pp. 115-143.
26. Ottinger, R. S., J. L. Blumenthal, D. F. Dal Porto, G. I. Gruber,
M. J. Santy, and C. C. Shih. 1973c. Recommended Methods for Reduc-
tion, Neutralization, Recovery, or Disposal of Hazardous Waste, Vol. 14,
Report No. EPA-670/2-73-053-n, U.S. Government Printing Office,
Washington, D.C. p. 23.
27. Pearce, A. S., and S. E. Punt. 1975. Biological Treatment of Liquid
Toxic Wastes. Effluent Water Treat. J. 15(l):32-39.
28. Pragay, D. A. 1974. Pollution Control and Suggested Disposal Guide-
line for Clinical Chemistry Laboratories. Am. Lab. 6:9-23.
29. Reed, A. K. , J. F. Shea, T. L. Tewksbury, R. H. Cherry-, Jr., and
G. R. Smithson, Jr. 1971. An Investigation of Techniques for Removal
of Cyanide from Electroplating Wastes. Report No. 12010 EIE 11/71,
U.S. Government Printing Office, Washington, D.C. 87 pp.
30. Reed, C. D., and J. A. Tolley. 1971. Hazards from the Kitchen Tap.
J. R. Coll. Gen. Practic. (Great Britain) 21:289-295.
31. Rules and Regulations. 1971a. Fed. Regist. 36(132):12901.
32. Rules and Regulations. 1971&. Fed. Regist. 36(172) :17646.
33. Stern, A. C. (ed.). 1968. Air Pollution, Vol. 2, 2nd ed. Academic
Press, New York. p. 102.
34. Strobel, G. A. 1967. Cyanide Utilization in Soil. Soil Sci.
103(4) :299-302.
35. U.S. Department of Health, Education, and Welfare. 1962. Public
Health Service Drinking Water Standards. U.S. Government Printing
Office, Washington, D.C. pp. 39-40.
36. U.S. Environmental Protection Agency. 1969. EPA Compendium of
Registered Pesticides III, May. p. H-211.
37. U.S. Environmental Protection Agency. 1974. Disposal of Hazardous
Wastes. Publication No. SW-115, U.S. Government Printing Office,
Washington, D.C. 110 pp.
38. U.S. Environmental Protection Agency. 1975. Hazardous Waste Disposal
Damage Reports. Report No. EPA/530/SW-151, U.S. Government Printing
Office, Washington, D.C. pp. 3-5.
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176
39. Watson, M. R. 1973. Cyanide Removal from Water. In: Pollution
Control in Metal Finishing. Noyes Data Corp., Park Ridge, N.J.
pp. 147-179.
40. Zuzik, J. B. 1974. Nitriles, Cyanates. In: Encyclopedia of
Occupational Health and Safety, Vol. 2. McGraw-Hill Book Co.,
New York. pp. 937-940.
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SECTION 8
ENVIRONMENTAL ASSESSMENT OF CYANIDE
James L. Way
Washington State University
Pullman, Washington
8.1 PRODUCTION, USES, TRANSPORTATION, AND POTENTIAL ENVIRONMENTAL
CONTAMINATION
8.1.1 Production
The estimate for the U.S. production of hydrogen cyanide was approxi-
mately 700 million pounds in 1976, and industrial production has increased
annually in the past decade. Most of the inorganic cyanides, such as the
sodium and potassium salts, are prepared from hydrogen cyanide.
8.1.2 Preparations and Transportation
Hydrogen cyanide is sold as a gas or as a technical grade prepara-
tion, containing 2%, 4%, 10%, or 96% to 99.5% hydrogen cyanide. A sta-
bilizer, such as phosphoric acid, is usually present to minimize its
spontaneous polymerization and associated explosive consequences. This
material is normally transported in tanks, 75-lb cylinders, steel drums,
or 5-lb bottles. Hydrogen cyanide is transported predominantly by rail
and to a lesser extent by trucks and barges. The Interstate Commerce
Commission considers the liquid and the solid forms of cyanide to be
class B poisons.
8.1.3 Uses
The predominant users of cyanide are the steel, electroplating,
mining, and chemical industries. It is used as an intermediate for the
manufacturing of various plastics, synthetic fibers, inorganic salts,
and nitrites. Cyanide is also used in photographic development, in the
fumigation of various vehicles and buildings (for rodent control), and
in agriculture. The predominant plastic synthesized from hydrogen cya-
nide is acrylonitrile.
8.1.4 Potential Environmental Contamination
8.1.4.1 Natural Causes — The liberation of hydrogen cyanide into our
environment is not totally due to human activity. There are various
natural sources which could contribute hydrogen cyanide to our environ-
ment. The extent of these contributions is difficult to assess. There
is a'possibility that long before humans inhabited the earth, hydrogen
cyanide in our atmosphere may have been higher than at the present time.
Cyanide has been observed in the atmospheres of the sun and other rela-
tively cool stars, particularly the carbon stars. Hydrogen cyanide is
177
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178
the second polyatomic organic molecule detected historically in inter-
stellar space by the use of microwave absorption spectroscopy. It is
considered to be one of the more important precursors for the abiotic
formation of amino acids, purines, and pyrimidines. Chemical emission
studies have suggested that the original heteropolypeptides on earth may
have been synthesized spontaneously from hydrogen cyanide and water with-
out the intervening formation of amino acids. The formation of cyanide
in the atmosphere is not unreasonable when one realizes that some of the
present methods for the production of hydrogen cyanide are the inter-
action of ammonia with methane and the interaction of nitric oxide with
various hydrocarbons. Hydrogen cyanide can be produced and excreted
from various plants, fungi, and bacteria. Also, some plants release
cyanide when they are crushed or macerated.
8.1.4.2 Human Related
8.1.4.2.1 Water — Regarding the discharge of cyanide waste into our
waterways, the Federal Water Pollution Act (PL 92-500) includes cyanide
in the effluent quality standards. Cyanide wastes are produced by a
variety of industries; however, only a small fraction of the cyanide
will escape into the environment if adequate waste treatment procedures
are instituted. The electroplating industry uses a considerable amount
of cyanide, and it is estimated that over 20 million pounds of cyanide
waste ultimately is discharged each year. Prior to treatment of this
waste, the cyanide content is approximately 0.5% to 20%. The steel
industry also is a potentially high contamination source. It is of
significance to note that the Environmental Protection Agency (EPA)
filed a refuse act suit against the ARMCO Steel Company for discharging
pollutants containing cyanide into Houston, Texas, shipping channels.
This discharge was in the form of a continuous-process generated efflu-
ent stream. The case of the United States versus ARMCO Steel Company
(C.A. 70-H-1335) was prosecuted successfully by the EPA. Other sources
of cyanide entering the environment would be from various mining opera-
tions, paint manufacturing processes, electroplating, and to a lesser
extent, photographic laboratories.
8.1.4.2.2 Air — There are various potential sources for the release of
hydrogen cyanide in air. One source would be the atmospheric emissions
of hydrogen cyanide from the petrochemical industries. Another recent
source would be from cars equipped with malfunctioning catalytic con-
verters, as Bell Laboratory has reported that a mixture of nitric oxide,
carbon monoxide, and hydrogen can produce varying amounts of hydrogen
cyanide. However, due to the sulfur content of gasoline and the water
content of automobile exhaust, the amount of hydrogen cyanide formed is
believed to be relatively low. In a closed environment, the maximum
emission level of hydrogen cyanide from raw exhaust is approximately
10 ppm. In an open environment under extremely adverse conditions, the
vehicle equipped with a three-way catalyst should not raise the hydrogen
cyanide level over 1.1 ppm.
Another source of hydrogen cyanide in air which does not receive
much attention is from home fires. With the increased use of plastics
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179
in homes, there is a potential for the combustion of various plastic
materials which may liberate hydrogen cyanide. It is generally recog-
nized that certain plastics, such as polyurethane, will liberate hydrogen
cyanide upon pyrolysis. The amount of hydrogen cyanide formed is de-
pendent upon the conditions under which pyrolysis occurs. Lastly, prob-
ably one of the major sources of atmospheric hydrogen cyanide affecting
man is tobacco smoke. It should be emphasized that the use of low tar,
low nicotine, or filter cigarettes does not necessarily reduce the hydro-
gen cyanide concentration in cigarette smoke.
8.1.4.2.3 Foods — Another potential source of hydrogen cyanide exposure
affecting man and animals results from the ingestion of substances which
either contain or liberate cyanide. The fumigation of various foods may
result in a cyanide residue that can persist for an extensive time period.
The United States lists tolerances for hydrogen cyanide residue on vari-
ous food products. These tolerances vary from 25 ppm in beans to 250 ppm
in spices. There are various edible plant products containing naturally
occurring substances which can release cyanide. Many of these plants
contain cyanogenic glycosides and therefore can potentially liberate
cyanide. This is of particular concern because some of these plants
comprise a major dietary constituent in various countries. Toxicity of
these cyanogenic plants is also a problem for various range animals and
wildlife. Poisoning of these herbivores is more prevalent under drought
conditions when these animals become less selective in their source of
forage. Also, dry growing conditions have been reported to enhance the
development of higher concentrations of cyanogenic glycosides in certain
plants. The effect of ingestion of low levels of cyanide on a long-term
basis is an area of study warranting further investigation.
8.2 ENVIRONMENTAL PERSISTENCE
8.2.1 Biomagnification and Cycling
Cyanide is a nucleophilic agent which is readily metabolized. When
one considers the high toxicity of cyanide in combination with its
chemical reactivity and rapid biotransformation, it is not surprising
that there have been no reports of biomagnification or cycling of cyanide.
8.2.2 Persistence in Foods
When various food products are fumigated with cyanide, a cyanide
residue may persist. Feeding these fumigated foods to laboratory animals
results in an increased excretion of urinary thiocyanate (a major
metabolite of cyanide).
8.2.3 Persistence in Soils
There are very few studies on the persistence of cyanide in soil.
Usually the movement of cyanide in soil is quite limited because it is
either complexed by trace metals or metabolized by various microorganisms.
If a high concentration of cyanide is found in the soil, it usually can
be attributed to improper industrial waste management procedures.
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180
8.2.4 Persistence in Water
The U.S. Public Health Service recommends a limit for cyanide in
U.S. water of 10 ppb (and EPA indicates a limit of 5 yg/liter). Unless
the cyanide is complexed with metals, most of it will exist as free
hydrogen cyanide, and an appreciable amount may be either volatilized
into the atmosphere or converted into other compounds by various orga-
nisms. Because of these various factors, concentration of cyanide ex-
ceeding the mandatory limits in water usually can be attributed to
improper waste disposal.
A recent survey of U.S. public water supplies revealing no concentra-
tion of cyanide above the mandatory limits is not reassuring in view of
the factors previously discussed, as high pulses of cyanide from various
industries into our waterways can occur without detection unless a con-
tinuous monitoring system is used.
8.2.5 Persistence in Air — Under normal circumstances, hydrogen cyanide
is present in the atmosphere in sufficiently low concentrations to not
be detected by standard procedures. The emission of hydrogen cyanide
into the atmosphere usually can be attributed to electroplating or fumi-
gation operations or to the pyrolysis of various plastics. There was
some concern that in addition to the gas-phase hazard, the airborne
water droplets generated by the combustion processes could trap enough
hydrogen cyanide to exert deleterious effects. An example would be the
combustion of polyvinyl chloride where the hydrogen chloride gas re-
leased can be trapped in water droplets in sufficient concentration to
be of concern. However, in the atmosphere around fire, the hazards of
respirable water droplets exposed to hydrogen cyanide is relatively
minimal when compared to the toxicity of the gas-phase hydrogen cyanide.
8.2.6 Waste Management
The disposal of cyanide in wastewater is still a significant problem
for various industries with respect to the effluent quality standards in
the Federal Water Pollution Act (PL 92-500). The most common method
used in management of cyanide wastewater in this country is alkaline
chlorination where the cyanide is oxidized to carbon dioxide and nitro-
gen. In some European countries, the cyanide is complexed to iron salts
as an alternative method. The mere fact that a variety of methods are
being described in the current literature for cyanide waste treatment
suggests that cyanide disposal still is a significant problem to some
industries. Other methods employed in cyanide waste treatment include
complexation with various metals, biologic transformation by cyanide-
resistant microorganisms, ionizing radiation, ion exchange processes,
ozonation, charcoal adsorption, and reverse osmosis.
8.3 EFFECTS OF AQUATIC AND TERRESTRIAL ORGANISMS
8.3.1 Aquatic Organisms
The National Academy of Sciences and the National Academy of
Engineering in 1972 indicated that 0.005 ppm of cyanide should represent
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181
a maximal safe concentration in water containing aquatic life. Also,
these academies indicated that 0.01 ppm or higher is hazardous to the
marine environment. This is of interest because the recommended limit
for cyanide in U.S. waters is 0.01 ppm and concentrations exceeding
0.2 ppm constitute grounds for rejection of the water supply (U.S.
Department of Health, Education, and Welfare, 1962). Because cyanide
not infrequently is found in waterways due to industrial effluents, some
studies have been conducted on the effects of cyanide on fish. The
toxicity of cyanide to fish varies with temperature, oxygen, mineral
content, and the pH values of the water. It should be pointed out that
most of the investigations influencing the determination of our standards
have been acute studies. There are very few studies concerned with the
long-term effects of cyanide on aquatic life. Since cyanide concentra-
tions in the range of 0.03 ppm have been reported to be lethal to fish,
it is reasonable to expect that lower concentrations of cyanide probably
would elicit some toxic manifestations in fish. It should be mentioned
that in studies on aquatic invertebrates, 0.014 ppm is lethal to bivalve
larvae. Also, the survival time of G. pulex is greatly shortened by
0.003 ppm of sodium cyanide. These data should generate some concern
about the paucity of information available on effects of cyanide on
aquatic life both in freshwater and seawater. The information which is
available should be further evaluated. It is also important to recog-
nize that chronic studies are essential to determine the long-term
effects of cyanide on the life cycles of these various aquatic inverte-
brates and vertebrates. It should be emphasized that most of the acute
studies focus on rapid lethal effects as the end point of cyanide
toxicity. If one looks at more subtle cyanide effects, such as behav-
ioral effects on fish, then the sensitivity of organisms to cyanide
becomes more apparent. It has been reported that 10 yg/liter of cyanide
(0.01 ppm) will impair the swimming performance of salmonid fish. In
chronic studies performed on brook trout the following parameters were
monitored: growth and survival of adults and spawning characteristics
(i.e., number of eggs per female and egg viability, growth of embryos
and juveniles, and embryo and juvenile survival). In these studies
the maximum acceptable toxic concentration (MAT) of hydrogen cyanide was
estimated to be between 6.0 and 11 yg/liter (0.006 to 0.011 ppm). Further
studies conducted using the fathead minnow gave similar results. It would
seem that chronic studies would provide a more valid rationale for setting
standards for concentrations of various toxicants, such as cyanide in our
waterways.
8.3.2 Terrestrial Organisms
The group of animals exposed most frequently to the threat of cya-
nide poisoning by the ingestion of cyanogenic plants is range animals,
particularly cattle and sheep which graze on forage with a high cyano-
genic glycoside content. In ruminants, the microorganisms in the rumen
would hydrolyze the cyanogenic glycosides, liberating hydrogen cyanide.
As pointed out earlier, this is most apt to occur during periods of
drought, as the selectivity of forage by range animals becomes less
discriminating. It is also believed that the cyanogenic glycoside
content increases in some plants under dry climatic conditions. Under
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these conditions one would expect that livestock would be most apt to
develop chronic as well as acute toxicity symptoms. There have been
reports of possible chronic cyanide toxicity in horses; however, attempts
to relate a well-defined toxin to symptoms of poisoning observed under
field conditions are rather tenuous, as the effects can be ascribed to
other factors present in the forage in addition to cyanide. For example,
the syndrome described in the horse could be ascribed to lathyrogenic
factors present in the forage rather than to cyanide itself. Another
deleterious effect on livestock that results from grazing on forage
which has a high cyanogenic glycoside content is thyroid dysfunction.
This is attributed more to the formation of the metabolite of cyanide,
thiocyanate, rather than to cyanide itself. It is of interest to note
that various wild herbivores, such as deer and elk, have been observed
to graze not infrequently on forage which contains a high content of the
cyanogenic glycosides; yet, cyanide poisoning in wildlife has not been
reported to any appreciable extent.
There have been some studies on the effect of cyanide on birds;
however, they have been quite limited. Early studies on sparrows and
pigeons have led to the general inference that birds are more suscepti-
ble to cyanide than mammals. Whether this impression is warranted or
not is difficult to assess due to the limited studies conducted. The
effects of cyanide on a variety of mammals have been studied in great
detail. There is not uniform consensus that the dog may be more sensi-
tive to cyanide than other species. Some investigators have noted that
dogs have a lower rhodanese content than other animals and have ascribed
this greater sensitivity to cyanide to a depressed detoxification mech-
anism. One should interpret the cyanide toxicity data in mammals with
some degree of caution because the variation in susceptibility to cya-
nide from one species to another depends on many factors such as the
route of administration and ambient temperature.
8.4 EFFECTS ON HUMAN HEALTH
8.4.1 Toxic Effects
The acute toxic effects of cyanide are well recognized. Cyanide
intoxication can occur by exposure to the gas, the liquid, or inorganic
salts. Hydrogen cyanide can be easily absorbed by inhalation as well
as by topical and oral routes of administration. The rapidity with
which cyanide exerts its lethal effect is well recognized by the general
population.
Suspected chronic cyanide poisoning has been described and is
alleged to be a rather serious and debilitating intoxication. Low-level
chronic cyanide poisoning in man is difficult to document clearly in
many cases since a variety of other causes can contribute to the signs
and symptoms observed. Chronic poisoning may be occurring more fre-
quently than is presently realized. Under industrial conditions where
the cyanide is usually inhaled, a variety of signs and symptoms have
been grouped to form what can be presently described as a "cyanide
syndrome." These toxic effects involve the gastrointestinal tract,
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thyroid, and central nervous system. Other purported cases of chronic
cyanide intoxication from the ingestion of cyanogenic foods have been
reported. The syndrome of this tropical neuropathy is characterized by
affliction of the nervous system producing optic atrophy, nerve deafness,
and various types of ataxia. Demyelinization of fiber tracts in the
central nervous system and peripheral nerves with resultant decreased
conduction velocity has been described. In many of these studies, it is
difficult to unequivocally ascribe the human nerve disorder specifically
to cyanide; however, in some cases of tobacco amblyopia, hydroxocobalamin
is effective in improving vision, providing rather convincing evidence
that the lesion is more apt to be ascribed to cyanide exposure.
It should be noted that low chronic doses of cyanide have been
reported to produce neuropathic lesions in man; however, attempts to
experimentally produce neuropathic lesions in laboratory animals still
require rather high doses of cyanide.
8.4.2 Teratogenic, Mutagenic, and Carcinogenic Effects
There have been no detailed studies which implicate cyanide as a
teratogenic, mutagenic, or carcinogenic agent. There are also no well-
documented studies employing controlled low-level chronic exposure of
cyanide. Further studies along these lines are warranted, as cyanide is
a very reactive nucleophile which should distribute rather widely through
body compartments, should be permeable to cell membrane, and should attain
reasonable concentrations in the fetus. It is of interest to note that
cyanide has been reported to have antineoplastic properties and that it
has been employed in clinical trials on humans. Epidemiological compar-
isons attempting to link cyanide with decreases in the incidence of tumors
in the human population have not been very convincing.
8.4.3 Treatment
The time-honored basis for the treatment of cyanide poisoning pro-
posed by Dr. K. K. Chen over 40 years ago involves a combination of drugs
to bind and to detoxify cyanide. This therapeutic combination employed
sodium nitrite to form methemoglobin, which would combine with cyanide
to form cyanmethemoglobin, and sodium thiosulfate, which serves as a sub-
strate for the enzyme, rhodanese, to convert cyanide to thiocyanate. More
recently, oxygen was proposed as an integral part rather than as an ad-
junct therapy to this antidotal combination, as it strikingly enhances the
protection against cyanide poisoning. This therapeutic regimen has pro-
tected against 20 LDSO doses of cyanide. Recently, some have advocated
that intensive supportive medical care with special attention to pulmo-
nary function may be of importance in treating cyanide poisoning. It
would be of interest to determine whether such concepts as ventilated
control and use of diuretic agents would contribute significantly to the
treatment of cyanide poisoning.
Although the antidotal combination of sodium nitrite and sodium
thiosulfate is used rather extensively in the United States, the use of
cobalt EDTA is more prevalent in Europe, particularly in England and the
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Scandinavian countries. The rationale for the use of cobalt is that it
will form a stable metal complex with cyanide, thereby preventing its
toxic effect. It seems more reasonable that the older conceptual approach
to treat cyanide intoxication would be more efficacious (i.e., to bind
cyanide as well as to detoxify it). If cobalt EDTA is to be employed,
then it would seem that a more efficacious antidotal combustion would be
to use cobalt EDTA in combination with sodium thiosulfate. More recently,
new effective cyanide antidotes are being reported, and surprisingly,
some of these antidotes neither combine nor detoxify cyanide.
8.5 POTENTIAL HEALTH HAZARDS
8.5.1 Occupational
Since cyanide is widely used in large amounts in various industries,
this will remain a potential health hazard to workers, particularly because
of the volatility of hydrogen cyanide and its ability to be absorbed by
the inhalation and cutaneous routes. The U.S. standards proposed are to
establish concentrations where no employee will suffer impaired health,
functional capacity, or diminished life expectancy as a result of the work
experience. The proposed standards developed by the National Institute
of Occupational Safety and Health (NIOSH) apply to the processing, manu-
facturing, and use of hydrogen cyanide and its salt or their release as
intermediates, by-products, or impurities as practicable under the Occu-
pational Safety and Health Act of 1970. These standards were developed
for general occupational exposure and should not be extrapolated to the
general population. Both the federal standard and the American Confer-
ence of Governmental Industrial Hygenists (ACGIH) adopted threshold limit
values (TLV) for hydrogen cyanide and alkali cyanide which greatly exceed
the TLV instituted in some countries. It should be pointed out that the
past recommendations by NIOSH are 8-hr time-weighted averages. The pres-
ent federal standards are probably too high to provide adequate protection
from the systemic effects of hydrogen cyanide and prevent the erosional
effects on the nasal septum produced by the alkalinity of the cyanide
salts.
8.5.2 General Population
There are potential health hazards to the general public from expo-
sure to hydrogen cyanide and its salts. In countries where cassava is
eaten as the major staple food, the high cyanogenic glycoside content pre-
sents a potential chronic health hazard to the general population. Because
of the increased use of plastics in homes, liberation of hydrogen cyanide
in home fires upon combustion of the plastics is coming under closer scru-
tiny in some countries. With regard to other sources of exposure to hydro-
gen cyanide on a long-term low-level basis, malfunctioning catalytic
converters in automobiles do emit hydrogen cyanide; however, even under
the most adverse conditions, projected on freeways, the general consensus
is that this source alone would not be considered to be "hazardous" to the
general public. The more likely concern to the general population would
be restricted to those who reside near the electroplating, steel, mining,
and paint manufacturing industries. Although cyanides can be released
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into the atmosphere, they are more likely to be released into the water-
ways. A recent incident of massive continuous outpouring of industrial
effluent containing cyanide into waterways (Section 8.1.4.2.1) is of con-
siderable concern. A more reliable system to continuously monitor indus-
trial effluents containing cyanide needs to be instituted.
8.6 POTENTIAL ENVIRONMENTAL HAZARDS
Cyanide has been shown to be potentially toxic to microorganisms,
aquatic invertebrates and vertebrates, and terrestrial animals. Suffi-
ciently high concentrations of cyanide have provided a disruptive influ-
ence to the environment. Although hydrogen cyanide is volatile and birds
have been reported to be extremely sensitive to cyanide, there is little
information on avian poisoning as a result of the release of cyanide into
the atmosphere.
Probably the greatest controllable sources of cyanide entry into
the environment are industrial effluents in waterways. This can be a
disruptive influence on the aquatic environment. Most studies to project
recommended levels are based on acute lethal effects; however, investiga-
tions of the effects of long-term low-level dosages on aquatic organisms
are essential. For example, the present allowable cyanide levels in U.S.
waters could exert toxic effects on the life cycles of various fish. There
is a paucity of information on the effects of chronic low levels of cya-
nide on the life cycle not only of fish but other aquatic organisms and
microorganisms. The monitoring of the U.S. waterways indicating levels
of less than 10 ppb does not necessarily imply that the problem of cyanide
contamination is relatively minimal. Severe damage may result from the
high pulsing of cyanide into waterways, which would not be detected by
the present monitoring system. The factors which probably minimize the
environmental hazard of cyanide are its chemical properties of volatility
and reactivity. The pKa of cyanide indicates that it would be present
predominately in the form of hydrogen cyanide, and therefore, its vola-
tility upon agitation of the water would minimize its environmental per-
sistence. Also, the biologic reactivity of cyanide should contribute to
its rapid removal from aquatic systems.
Another environmental hazard, which can be indirectly attributed to
cyanide is the growth of various forage plants which have a high content
of cyanogenic glycosides. The toxicity of this forage to livestock
already has been discussed.
8.7 REGULATIONS AND STANDARDS
The American Conference of Governmental Industrial Hygenists (ACGIH)
adopted a threshold limit value (TLV) for hydrogen cyanide on a time-
weighted average of 10 ppm (11 mg/m3). In 1971, the ACGIH TLV was indi-
cated to present a twofold safety margin against mild symptoms and a
sevenfold or eightfold margin against the lethal effects of cyanide. The
present federal standard for hydrogen cyanide is also 10 ppm on a time-
weighted average (29 CFR 1919.1,000 published in 39 FR 23541 on June 23,
1974) and is based on the 1962 ACGIH TLV. There are 13 foreign countries
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and 6 states in the United States that have set standards for hydrogen
cyanide. It is difficult to compare these values as some countries use
ceiling values, whereas other countries use time-weighted averages. It
is of interest to note that five countries have set 0.3 mg/m3 (0.27 ppm)
as their standard, whereas the standards of most of the other countries
are comparable with those of the United States. In the U.S. Department
of Health, Education, and Welfare document (1976) on cyanide, it is recom-
mended that employee exposure to hydrogen cyanide not exceed 5 mg/m ,
which would be determined at a ceiling concentration on a 10-min sampling
period.
The ACGIH TLV for alkali cyanide is 5 mg/m3 on a time-weighted aver-
age and the federal standard is identical (29 CFR 1919.1,000 published
in 39 FR 23541 on June 27, 1974, as amended) and is based on the 1968
ACGIH TLV. It is of interest to note that there is a 17-fold difference
in the standards between some foreign countries and the United States
and that the United States has the higher TLV for alkali cyanide.
The federal standard and ACGIH TLV would protect against the acute
toxicity from cyanide. However, with the cyanide salts the current fed-
eral standard of 5 mg/m3 of cyanide on an 8-hr time-weighted average was
recently suggested for revision by NIOSH, as it appeared to be too high
and allowed for substantial excesses above that concentration for short
time intervals. The U.S. Department of Health, Education, and Welfare
document (1976) recommends that the same value of 5 mg/m3 of cyanide be
retained, but that the time base be changed from an 8-hr time-weighted
average to a ceiling concentration on a 10-min sampling period. This was
suggested to protect the workers from the systemic effects of cyanide and
also to minimize the occurrence of nasal septum erosion produced by the
alkalinity of the inorganic cyanide salts. These federal standards and
threshold limit values may not have taken into account the low-level con-
centrations of cyanide which have been reported to produce chronic tox-
icity. It should be pointed out that 4.2 to 12.4 ppm has been reported
to produce various signs and symptoms of chronic cyanide toxicity.
The recommended limit for cyanide in U.S. water is 10 ppb. Concen-
tration in excess to 200 ppb constitutes ground for rejection of the water
supply (U.S. Department of Health, Education, and Welfare, 1962). The
U.S. Environmental Protection Agency recommended limit is 5 yg/liter.
With regard to the effects of these limits on aquatic microorganisms and
fish, it should be pointed out that, because cyanide lacks environmental
persistence, the survey of U.S. public water supply systems revealing no
cyanide concentrations above the mandatory limit is not reassuring, as
high pulses of cyanide into waterways can cause profound changes in aquatic
organisms and microorganisms without being detected by the methods pres-
ently employed.
In attempting to assess the effects of long-term exposure from the
few studies conducted on occupational workers, the question always arises
whether the signs and symptoms observed from purported chronic intoxica-
tion from cyanide may be attributed to other factors. However, the
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frequency of reported incidents of toxic manifestations resulting from
exposure to low concentrations of cyanide has probably been sufficient
to describe a "cyanide syndrome." The NIOSH recommendation of a lower
exposure limit to hydrogen cyanide appears to be justified, particularly
since these measurements were recommended to be conducted on a 10-min
sampling period rather than an 8-hr time-weighted average. It should be
pointed out that the USSR, Romania, Hungary, and Bulgaria recommend
what appears to be ceiling concentrations of 0.3 mg/m3 and Czechoslovakia
recommends a standard of 3 mg/m3 on a time-weighted average. Also in the
United States, Hawaii recommends a standard of 20 ppm (approximately 22
mg/m3) as a ceiling value. Pennsylvania also recommends this same stand-
ard as a 30-min ceiling value.
8.8 CYANIDE ANALYSIS
8.8.1 Biologic Sample
Analysis of cyanide is usually conducted using samples of whole
blood. These samples should be obtained as soon as possible after cya-
nide exposure and analyzed immediately. Storage of blood samples can
result in erroneous analytical values. It should be pointed out that
variability in the temperature at which the samples are stored will con-
tribute to variability in the final readings. Frozen blood samples may
give lower values than those not frozen. Although most assays for cya-
nide are conducted using whole blood, there are some investigators who
object to the use of whole blood and recommend the use of serum or plasma.
However, in many cases these investigators may be unaware that the correla-
tion among serum, plasma, and whole blood cyanide levels have been quite
good at the higher cyanide concentrations.
8.8.2 Analytical Methods
The colorimetric method employing the Kb'nig reaction with pyridine-
pyrazolone reagent is one of the most widely used procedures for cyanide
analysis. It is a sensitive and accurate method; however, it is sub-
jected to interference by some anions. Many of the studies showing the
efficacy of sodium thiosulfate in detoxifying cyanide by converting it to
thiocyanate based upon this method of analyses may be erroneous. Sodium
thiosulfate when acidified forms polythionic acids which volatilize, pre-
sumably as sulfur dioxide, and are trapped in the alkaline media as sul-
fite anion. It is this latter anion which will interfere with the colori-
metric determination of cyanide. Therefore, the rapidly falling cyanide
levels attributed to sodium thiosulfate administration in cases of cya-
nide poisoning may be erroneously low due to the anion interference with
the colorimetric method rather than the presumed enhanced conversion of
cyanide to thiocyanate. The more recent method for determining cyanide
which employs ion-selective electrodes is quite sensitive. It is a much
more convenient and rapid method for analyzing cyanide. Automation has
been achieved employing these electrodes and commercial models are
available.
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Various fluorometric methods are available for the determination
of cyanide. In general, the fluorometric methods are sensitive, but the
results obtained are not as consistent as with the colorimetric method.
One of the fluorometric methods has the advantage that it is not subjected
to anionic interferences as with the pyridine-pyrazolone procedure. How-
ever, this specific fluorometric method does not give a linear response.
Automation has also been obtained using a fluorometric method.
Gas chromatographic methods of measuring cyanide are usually based
on its conversion to cyanogen chloride. This conversion is needed because
most gas chromatographic detectors are not very sensitive to cyanide it-
self. The measurement of cyanide as cyanogen chloride is quite sensitive
and it is surprising that this method is not more widely used.
8.9 SUMMARY OF OPINION AND PROJECTED RESEARCH NEEDS
The concentration of cyanide allowed in waterways should be reeval-
uated with some consideration directed toward the long-term effects of
cyanide on life cycles, growth, and survival as well as biochemical, phys-
iological, and behavioral effects. The present limits have been primarily
based on acute toxicity studies where lethality is the end point. Inves-
tigations using fish clearly delineate the limitations of this type of
data. Not only should chronic studies be conducted, but these studies
should be conducted in a variety of aquatic organisms and microorganisms
in addition to fish.
In an area where potential for entrance of cyanide into waterways
exists, analysis should be conducted employing a continuous monitoring
system rather than intermittent sampling. The environmental persistence
of cyanide is probably quite low; therefore, periodic entrance of hydro-
gen cyanide into our waterways can promote considerable damage to aquatic
life without being detected. Because of the rapid action, high toxicity,
and low environmental persistence of cyanide, periodic monitoring of cya-
nide in waterways does not produce sufficient assurrance that an ecologi-
cal hazard does not exist.
The potential formation of hydrogen cyanide in home fires warrants
further studies. Pyrolysis of various plastics involved in home construc-
tion should be studied and the interaction of the various gases liberated
should be investigated from a toxicological viewpoint.
Intensive low-level long-term studies on cyanide intoxication in
mammals should be undertaken. These studies should include measurement
of concentrations of cyanide, its metabolites, thyroid function, EEC,
blood chemistry, and in the case of humans, medical history, occupation,
smoking habits, history of potential exposure, and air samples in living
and occupational areas. Controlled laboratory studies on the effects of
chronic low-level administration of cyanide should be conducted. Substan-
tial studies of effects of long-term low-level cyanide exposure by the
oral and inhalation routes should be undertaken with particular attention
focused on the neuropathic lesions which may be produced. These types
of studies may provide a more valid rationale in establishing federal
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standards and threshold limit values for hydrogen cyanide and inorganic
cyanide.
With respect to livestock, more studies are needed on the various
factors, such as drought, which may increase the concentration of cyano-
genic glycosides in plants.
There is a paucity of information on the teratogenic, mutagenic,
and carcinogenic effects of cyanide at the acute, subacute, and chronic
levels, and the information which is available should be further
investigated.
Although the antidotal therapy of cyanide intoxication is quite
efficacious, additional studies are warranted because of the high toxicity
and rapid action of cyanide. These studies should focus on supportive
medical therapy, better antidotes, and different antidotal combinations
and should be conducted not only on laboratory rodents but on other
species including range animals.
Epidemiological studies may be warranted in an attempt to correlate
low-level long-term exposures of cyanide to various toxic manifestations.
For example, in occupationally exposed workers and in the general popula-
tion in vicinities of high cyanide concentrations, cyanide levels may be
correlated with the incidence of skin dermatitis, nasal septum lesions,
thyroid dysfunction, and urinary thiocyanate levels.
Continuous automatic monitoring equipment to measure cyanide in air
and water should be encouraged and these devices should include automatic
alarm systems when predetermined levels are exceeded.
Studies on the production of hydrogen cyanide under various condi-
tions in malfunctioning catalytic converters should be further investi-
gated. These studies should focus on those factors which can promote
maximal formation of hydrogen cyanide and on its toxicity alone and in
combination with other automobile emission gases in regard to possible
potentiation of toxicity.
The effect of cyanide on wildlife and range animals, particularly
those which could graze on foliage with a high cyanogenic glycoside con-
tent, warrants further studies.
The mechanism of uptake, metabolism, and biosynthesis of cyanide
in soils, plants, and various fungi merits further investigation. At
the present time there is little information on the mechanism of cyanide
liberation by plants and microorganisms, and almost no information on
their contribution to total cyanide in the environment.
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SECTION 8
REFERENCES
1. U.S. Department of Health, Education, and Welfare. 1962. Public
Health Service Drinking Water Standards. U.S. Government Printing
Office, Washington, D.C. pp. 39-40.
2. U.S. Department of Health, Education, and Welfare. 1976. Criteria
for a Recommended Standard . . . Occupational Exposure to Hydrogen
Cyanide and Cyanide Salts. Washington, D.C.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
k TITLE AND SUBTITLE
5. REPORT DATE
Reviews of the Environmental Effects of Pollutants:
V. Cyanide
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Information Center Complex, Information Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
10. PROGRAM ELEMENT NO.
1HA616
11. CONTRACT/GRANT NO.
IAG D5-0403
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory Cin-OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45219
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This is a review of the scientific literature on the biological and environ-
mental effects of cyanide. Included in the review are a general summary and a
comprehensive discussion of the following topics as related to cyanide and
specific cyanide compounds: physical and chemical properties; occurrence;
synthesis and use; analytical methodology; biological aspects in microorganisms,
plants, wild and domestic animals, and humans; distribution, mobility, and
persistence in the environment; assessment of present and potential health and
environmental hazards; and review of standards and governmental regulations.
More than 500 references are cited.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDEDTERMS
c. COSATI Field/Group
^Pollutants
Toxicology
Cyanide
Health Effects
06F
06T
3. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
!1. NO. OF PAGES
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
ir U.S.GOVERNMENT PRINTING OFFICE: 1978-748-189/318
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