EPA-600/1-75-004
November 1975
Environmental Health Effects Research Series
MOLYBDENUM - A TOXICOLOGICAL APPRAISAL
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
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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
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH
EFFECTS RESEARCH series. This series describes projects and
studies relating to the tolerances of man for unhealthful
substances or conditions. This work is generally assessed
from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical
specialities, study areas include biomedical instrumentation
and health research techniques utilizing animals - but always
with intended application to human health measures.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
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EPA-600/1-75-004
November 1975
MOLYBDENUM - A TOXICOLOGICAL APPRAISAL
by
Lars Friberg, Pamela Boston, Gunnar Nordberg,
Magnus Piscator, and Karl-Henrik Robert
Department of Environmental Hygiene
Karolinska Institute
and
National Environment Protection Board
S-104 01, Stockholm 60, Sweden
Contract No. 68-02-1210
Project Officer
Robert J. M. Horton
Special Studies Staff
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
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. Environ-
mental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation
for use.
11
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TABLE OF CONTENTS
PAGE
CHAPTER 1 INTRODUCTION l
CHAPTER 2 ANALYTICAL METHODS 3
2.1 COLORIMETRIC METHODS 3
2.2 EMISSION SPECTROGRAPHY 5
2.3 NEUTRON ACTIVATION 6
2.4 ATOMIC ABSORPTION 6
2.5 QUALITY CONTROL AND INTRA-LABORATORY COMPARISONS 7
TABLE 2:1 8
CHAPTER 3 PRODUCTION, USES AND OCCURRENCE 9
3.1 PRODUCTION 10
3.2 USES 10
3.3 OCCURRENCE H
3.3.1 In ambient air and fuel H
3.3.2 In work environment 12
3.3.3 In water and marine organisms 13
3.3.4 In soils 15
3.3.5 In food 17
3.3.6 In tobacco 18
3.3.7 Daily intake 18
3.3.8 Conclusions 19
TABLES 3:1-3:4 21-24
CHAPTER 4 METABOLISM 26
4.1 UPTAKE AND ABSORPTION 26
4.1.1 Absorption following inhalation 26
4.1.1.1 In animals 26
4.1.1.2 In human beings 27
4.1.1.3 Conclusions 27
4.1.2 Gastrointestinal absorption 27
111
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PAGE
4.1.2.1 In animals 27
4.1.2.2 In human beings 29
4.1.2.3 Conclusions 31
4.2 EXCRETION 31
4.2.1 In animals 3^
4.2.1.1 Urine and alimentary tract 31
4.2.1.2 Milk 32
4.2.1.3 Bile 32
4.2.1.4 Hair 33
4.2.2 In human beings 33
4.2.3 Conclusions 34
34
4.3 TISSUE DISTRIBUTION
34
4.3.1 In animals 34
4.3.1.1 Single exposure
4.3.1.1.1 Oral route 34
4.3.1.1.2 Intravenous route 3 5
4.3.1.2 Repeated exposure 35
4.3.1.2.1 Oral route 35
4.3.1.2.2 Inhalation 37
4.3.1.3 Distribution within organs 37
4.3.2 In human beings, "normal exposure" 38
4.3.3 Conclusions 41
TABLES 4:1-4:7 42-46
FIGURES 4:1-4:3 47-49
CHAPTER 5 MOLYBDENUM AS AN ESSENTIAL ELEMENT IN
ANIMAL NUTRITION 50
5.1 MOLYBDENUM DEFICIENCY AND WOLFRAMATE INHIBITION
STUDIES 50
5.2 THEORETICAL BACKGROUND TO THE QUESTION OF
MOLYBDENUM ESSENTIALITY - ENZYME STUDIES 53
5.2.1 Xanthine oxidase 53
5.2.2 Other molybdoflavoproteins 55
5.2.2.1 Sulfite oxidase 55
5.2.2.2 Aldehyde oxidase 56
IV
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PAGE
5.3 MOLYBDENUM AND DENTAL CARIES 56
5.3.1 In animals 56
5.3.2 In human beings 5?
5.4 CONCLUSIONS 58
TABLES 5:1-5:2 59-60
CHAPTER 6 TOXICITY 61
6.1 "TEART" DISEASE - MOLYBDENUM POISONING IN RUMINANTS 61
6.2 IN HUMAN BEINGS 63
6.2.1 Effects on lungs 63
6.2.2 Hyperuricemia 64
6.2.3 Other effects 66
6.3 IN ANIMAL EXPERIMENTS 66
6.3.1 Single exposure 66
6.3.1.1 Injection 66
6.3.1.2 Inhalation 67
6.3.3 Repeated exposure 69
6.3.3.1 Oral administration, general aspects 69
6.3.3.1.1 Experiments with common laboratory
animals 6 9
6.3.3.1.2 Experiments with cattle, trials to
produce "artificial teartness" 74
6.3.3.2 Repeated exposure through inhalation,
common symptoms and effects on lungs 75
6.3.3.3 Effects on blood 78
6.3.3.4 Effects on bone and connective tissue 81
6.3.3.5 Effects on liver 83
6.3.3.6 Effects on kidney 84
6.3.3.7 Myocardial effects 8 5
6.3.3.8 Effects on the thyroid gland 85
6.4 CONCLUSIONS 86
TABLES 6:1-6:10 88-97
FIGURE 6:1 98
v
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PAGE
CHAPTER 7 INTERACTIONS OF MOLYBDENUM WITH COPPER,
SULFATE, PURINES AND PROTEINS 99
7.1 INTERACTIONS WITH COPPER AND SULFATE 99
7.2 INTERFERENCE WITH PURINE METABOLISM 105
7.3 INTERFERENCE WITH PROTEIN METABOLISM, STUDIES ON
ENZYME IMPAIRMENT 106
7.4 SUMMARY 107
CHAPTER 8 CONCLUSIONS AND DISCUSSION CONCERNING
POSSIBLE HEALTH EFFECTS OF MOLYBDENUM
ON HUMAN BEINGS 111
REFERENCES 114
VI
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CHAPTER 1 INTRODUCTION
In 1972, a preliminary review on molybdenum (by Karl-Henrik
Robert, M.D. and Pamela Boston, B.A.) was submitted to the
U.S. Environmental Protection Agency according to a contract
between that agency and the Department of Environmental
Hygiene of the Karolinska Institute. Based on this report,
it was decided to make a more extensive and detailed review.
The present document is a result of that decision and has
been carried out under contract 68-02-1210 between the
same two organizations. The project officer for both the
preliminary and the final report has been Robert J.M.
Horton, M.D. of the U.S. Environmental Protection Agency.
The focus of "Molybdenum - A Toxicological Appraisal"
is upon an evaluation of the metabolism and toxic effects
of molybdenum which can be of relevance for human beings.
Some data have been gathered and assessed in connection
with analytical methods, production, uses and occurrence.
The document has been prepared by a group on molybdenum.
The names of the members are listed on the title page.
Individual chapters were drafted by Magnus Piscator, M.D.
(chapters 2-3) and Karl-Henrik Robert, M.D. (chapters
4-7).
In addition, Velimir Vouk,x* Ph.D. of the World Health
Organization kindly contributed by drafting all sections
dealing with the U.S.S.R. literature available only in
the original language and also gave valuable criticism
on the work as a whole. A collaboration with the WHO
has further been under way through the participation
v)
Address: Velimir B. Vouk, Ph.D., Chief, Control of
Environmental Pollution and Hazards, Environmental Pollution
Division of Environmental Health, World Health Organization,
1211 Geneva 27 Switzerland.
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of Dr. Piscator in two meetings on molybdenum organized
by that body.
At the same time that this document was submitted to the
U.S. Environmental Protection Agency, a conference on
molybdenum in the environment was held in Denver, Colorado,
Papers presented during that conference have not been
possible to take into consideration in the present report.
For further details the reader may get in touch with The
x)
Molybdenum Project .
xAddress: The Molybdenum Project, Duane F-1033, University
of Colorado, Boulder, Colorado 80302 U.S.A.
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CHAPTER 2 ANALYTICAL METHODS
Molybdenum is a transition element in group 6B of the peri-
odic table. It can occur in 6 valence states: 0, +2, +3,
+4, +5 and +6, making its chemistry very complicated and
hence creating difficulties in analysis. More than 50
inorganic forms are known (Handbook of Chemistry and Physics,
1974/1975) . With its great complexing power,- molybdenum
easily forms chelates, a property which has been used for
analytical purposes. Colorimetric methods have been used for
decades for determination of molybdenum, but during later
years neutron activation, atomic absorption and X-ray fluores-
cence have also come into the picture. For more detailed
discussions on the determination of molybdenum reference is
made to recent reviews by Meglen and Glaze, 1973, 1974.
2.1 COLORIMETRIC METHODS
More than 100 years ago it was discovered that reduced
molybdenum in an acid solution formed a colored complex with
thiocyanate, which could be extracted into an organic
solvent. This formed the principle for the most common
method for determination of molybdenum - the thiocyanate
colorimetric method. Numerous articles have been published
on this method and many modifications have been proposed
(e.g. Dick and Bingley, 1947, 1951, Karlsson, 1961, Fishman
and Mallory, 1968, Kim and Zeitlin, 1968, Duval, 1971,
Khosla and Rao, 1971, Meglen and Glaze, 1973, Savariar,
Arunachalam and Hariharan, 1974, Yatirajam and Ram, 1974).
The basic procedure has been to take up the sample in di-
luted hydro-chloric acid after wet or dry ashing. Thio-
cyanate, usually the potassium salt, and tin (II) chloride
are added together with an iron salt. Hexavalent molybdenum
is reduced to pentavalent molybdenum, but reduction to other
valence states may occur simultaneously to some degree. The
colored complex formed is extracted into an organic solvent,
which may be ethylether, isoamylalcohol, butylacetate,
isopropyl ether, methyl isobutyl ketone or N-benzylaniline
-------
in chloroform or a mixture of chloroform and isoamyl alco-
hol. The color is usually read in a spectrophotometer at a
wavelength varying from 465 to 500 nm depending on the
method used.
The thiocyanate method is very complicated, as indicated by
the many different modifications proposed. One important
point seems to be that tin chloride causes reduction not
only to pentavalent molybdenum, but also to other valence
states. This has been claimed to be surmountable by using
copper and thiourea as reducing agents, and extracting with
N-benzylaniline (Khosla and Rao, 1971) or by using hydrazine
as a reducing agent and tribenzylamine in chloroform as an
extracting agent (Yatirajam and Ram, 1974). Another factor
is that small amounts of iron must be present (Dick and
Bingley, 1947) . Many compounds, especially tungsten, may
interfere with the determination of molybdenum by the
thiocyanate method. Interference from other compounds seems
to depend to a large extent on the procedure chosen. No
consistent pattern has appeared, but titanium, platinum,
silver, silica, chromates and nitrates have been shown to
interfere in some procedures.
Some media, e.g. water, have generally low molybdenum
content which may make pre-concentration necessary. Ion
exchange (Fishman and Mallory, 1968) or precipitation with
oxine and tannic acid (Kim and Zeitlin, 1968) have been
used. The thiocyanate method seems to allow the determi-
nation of about 0.01-0.1 ug molybdemun/g sample. Precision
has been reported to be about 10% in vegetables with a molyb-
denum content of 0.1-0.2 ug/g and 1-2% at concentrations
above 1 ug/g. In one study (Fishman and Mallory, 1968) the
thiocyanate method was compared with three other methods.
As shown in Table 2:1 it was proven to be accurate when
determining molybdenum in water. In plant material (clover)
the thiocyanate method showed a mean of 0.96 ug/g (3 deter-
minations) and neutron activation 0.94 ug/g (9 determi-
-------
nations) (Van Zanten, Decat and Leliaert, 1962).
Another colorimetric method is the dithiol method, according
to which hexavalent molybdenum is combined with toluene-3,4-
dithiol to form a colored compound, which can then be ex-
tracted into an organic solvent (Stanton and Hardwick, 1967,
1968, Ssekaalo, 1971, Cardenas and Mortenson, 1974, Quin and
Brooks, 1975). As seen in Table 2:1 this method gives
similar results as the thiocyanate method for determination
of molybdenum in water.
The dithiol method has had many applications and has been
subjected to many modifications. For more details and
references see e.g. Quin and Brooks, 1975.
Another method which involves complexing of hexavalent
molybdenum uses 2-amino - 4-chlorobenzenethiol hydrochlo-
ride, also forming a colored complex, which can be extracted
with chloroform (Ssekaalo and Johnson, 1969). This method
was tested on barley and milk, the latter material requiring
a separation of molybdenum from phosphate prior to final
analysis.
2.2 EMISSION SPECTROGRAPHY
Spectrographic methods have been used to determine molybdenum
in plants and water, and in animal and human tissues (e.g.
Yip, Shaw and Nace, 1961, Meltzer et al., 1962, D' Alonzo
and Pell, 1963, Tipton et al., 1963, Niedermeier and Griggs,
1971). Molybdenum has been precipitated with e.g. 8-hydro-
xyquinoline (Heggen and Strock, 1953, Fishman and Mallory,
1968) as a pre-concentration step. In Table 2:1 such a
method is seen to give similar results as other methods for
determination of molybdenum in water. By concentrating and
extracting from large samples concentrations of a few ng/g
can be measured. Concentration of molybdenum in the sample
has also been accomplished by complexing with tert-carbate
(N-pyrrolidinodithiocarbamic acid-sodium salt) and extraction
-------
into chloroform (Voth, 1963).
2.3 NEUTRON ACTIVATION
Neutron activation analysis has been employed in several
investigations (Bowen, 1959, Van Zanten, Decat and Leliaert,
1962, Samsahl and Brune, 1965, Livingston and Smith, 1967,
Kjellin, 1968, Lunde, 1968, Pillay and Thomas, 1971, Morgan
and Holmes, 1972, Plantin, 1973). After irradiation molybdenum
is separated by a complexing agent or ion-exchange. Both
99 101
Mo and Mo may be used. The detection limit is reported
to be 0.1 ug for Mo (Van Zanten, Decat and Leliaert,
QQ
1962) and 0.2 ng for Mo (Morgan and Holmes, 1972). Good
agreement was found when the Mo method was compared to a
colorimetric method (see section 2.1).
2.4 ATOMIC ABSORPTION
Molybdenum is not well suited for analysis by atomic absorption
in flame (Johnson, West and Dagnall, 1973, Meglen and Glaze,
1973). A claimed sensitivity of around 0.01 ug/ml (Roussos
and Morrow, 1968) has not been supported by other authors.
The sensitivity is generally relatively low. Molybdenum is
not easily atomized, forming refractory oxides in the flame.
After extraction into organic solvents concentrations of 1-
3 ug/g in fertilizers could be determined (Hoover and
Duren, 1967.- Koirtyohann and Hamilton, 1971) . In fresh
water molybdenum has been determined at concentrations of
from 230 to 3200 ug/1, in which range the agreement with
other methods is good (see Table 2:1). In this case the
analysis was carried out on a dithiol-MIBK extract (Fishman
and Mallory, 1968).
Flameless methods have been recommended, but data are not
available as to how they compare to other methods. A
sensitivity of 0.035 ug/g has been reported (Johnson, West
and Dagnall, 1973) . Muzzarelli and Rocchetti, 1973, determined
-------
molybdenum in seawater concentration by ion-exchange with a
graphite atomizer after separation and from their data it
can be calculated that about 0.03 ug/ml can be detected in
the concentrated solution.
2.5 QUALITY CONTROL AND INTRA-LABORATORY COMPARISONS
A considerable number of methods have been used to determine
molybdenum in different media. In many studies, the method
proposed has only been tested on artificial solutions or on
a very limited number of samples, and generally not been
compared with other methods. Comparisons among different
methods inside the laboratory have been carried out, as in
the study by Fishman and Mallory, 1968 (see Table 2:1). In
other studies a few standard samples have been tested. Very
few data on inter-laboratory comparisons have emerged.
Meglen and Glaze, 1974, performed intra-laboratory checks in
which they compared their own colorimetric (thiocyanate)
method with an atomic absorption method, and they found a
good agreement for water samples with a very high content of
molybdenum, i.e. in the mg/1 range. In an inter-laboratory
check, they compared their own results on the thiocyanate
method for water samples with two other laboratories, one
using a colorimetric method and the other both colorimetric
and atomic absorption methods. All water samples contained
between 250 and 450 ug/1, and there was a good agreement
between laboratories.
The U.S. Geological Survey distributed 6 mineral or soil
samples to 85 laboratories in the U.S. for the deter-
mination of molybdenum and 14 other elements. The values
submitted on the molybdenum samples varied widely, e.g.
in one mineral sample from 1 to 500 mgAg (Allcott and
Lakin, 1974).
No data on the accuracy of methods used for the determina-
tion of molybenum in animal tissues and body fluids are
available.
7
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TABLE 2:1
MOLYBDENUM IN FRESH WATER (ug/1) DETERMINED
BY FOUR DIFFERENT METHODS. (Modified from
Fishman and Mallory, 1968).
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Thiocyanate
920
6
3000
400
8
290
330
3800
260
19
1800
260
550
2
Spectrographic
900
2
2800
300
7
270
280
3500
220
14
1600
230
500
3
Dithiol
960
6
3200
390
7
320
330
3900
250
21
1900
300
550
1
Atomic
absorption
970
3200
230
280
530
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CHAPTER 3 PRODUCTION, USES AND OCCURRENCE
A vast literature has accumulated on the aspects of molyb-
denum treated in this chapter. A complete review of the en-
vironmental behavior of molybdenum has not been possible
to give here. The distribution and transport of molybdenum
in environmental compartments present a very complex pattern,
intimately connected with the basic properties of the earth's
surface.
For details on production technology, supply-demand rela-
tionships, consumption patterns and the like, see reviews
by Morning, 1969, and Sheridan, 1970.
Recently an extensive project on transport, distribution
and effects of molybdenum has been undertaken in Colorado,
where the largest molybdenum deposit in the world is
situated and mined. Two comprehensive progress reports
(Transport and the Biological Effects of Molybdenum in
the Environment, Progress Report, January 1, 1973, and
Progress Report, January 1, 1974, University of Colorado,
Boulder, Colorado and Colorado State University, Fort
Collins, Colorado) have already emerged from the University
of Colorado and Colorado State University. Readers inter-
ested in the flow of molybdenum in the environment are
x\
referred to that project for more extensive information '.
Some data on the occurrence of molybdenum in soil, plants
and food will also be found in Chapter 6 in connection
with effects on livestock and human beings.
x)
'The leader of this project is Dr. Willard R. Chappell;
His address: The Molybdenum Project, Duane F-1033,
University of Colorado, Boulder, Colorado 80302.
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3.1 PRODUCTIQN
Molybdenum does not occur in the native state, but is
obtained from minerals, such as molybdenite (MoS2), wul-
fenite (PbMo4>, ferrimolybdate (FeMoO3 x H2O) and jordi-
site (amorphous MoS2). Many other minerals contain molyb-
denum, but these four are the ones used for commercial
exploitation.
Molybdenite is the greatest source of molybdenum, the
largest deposit being the one in Climax, Colorado. 75%
of the world's known reserves of molybdenum are in the
western parts of North and South America. Large deposits
are also known to exist in the U.S.S.R., Canada and
Chile. In the U.S. the main part of the produced molyb-
denum comes as a primary product from the processing
of molybdenite, but a substantial fraction is obtained
in connection with processing of copper, tungsten or
uranium ores. Molybdenum is also obtained by recycling
of scrap materials. in Table 3:1 the total known produc-
tion of molybdenum, excluding U.S.S.R. and some other
countries in the eastern hemisphere, is shown.
3.2 USES
In Table 3:2 the consumption of different, forms of molyb-
denum in the U.S.A. during a one-year period is depicted.
Molybdenum's ability to harden steel has made it a metal
of growing importance in this century. About 85% of the
produced molybdenum is used in the manufacturing of al-
loys, such as alloy steels, tool and high speed steels,
stainless steel, alloy cast iron and alloys with non-
ferrous metals. Molybdenum may be used in steel alone
or in combination with other alloy materials, such as
chromium, manganese, nickel and tungsten. Especially
the weapon and aircraft industries have found great use
for molybdenum alloys. Molybdenum has a low coefficient
10
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of expansion and high tensile strength, making it very
suitable for the latter industry.
Other products which may contain molybdenum are spark
plugs, certain parts of X-ray tubes and electrodes in
electrically heated glass furnaces. In the chemical
industry molybdenum compounds are used as catalysts
and as chemical reagents, e.g. for the determination
of phosphorus. There are also many pigments that contain
molybdenum compounds. Molybdenum is an essential element
in plant and animal nutrition, and molybdenum compounds
are added to soils or waters for increasing plant and fish
production respectively.
3.3 OCCURRENCE
3.3.1 In ambient air and fuel
In the U.S. molybdenum concentrations in ambient air
have been reported to be 10-30 ng/m in urban areas and
0.1-3.2 ng/m in non-urban areas (Air Quality Data, 1966).
The concentrations of molybdenum in air are thus low. It
has been suggested that its high boiling point (melting
point 2617°C, boiling point 4612°C) allows molybdenum to
remain to a large extent in the ash after combustion of
e.g. coal.
Analyses of fly ash from 18 power stations in the U.K.
showed molybdenum concentrations ranging from 10-40 ug
per gram, with one sample showing 180 ug/g (Smith, 1958) .
Molybdenum concentrations in coal from different parts
of the U.S. varied from 0.28-15 ug/g (Kaakinen and Jorden,
1974). Andersson and Grennfelt (1973) reported that molyb-
denum concentrations in light oils were below 0.1 ug/g
and in heavier oils up to 0.52 ug/g. It was estimated
that the total emission from burning of oils in Sweden
was about 2.5 tons/year. Lindau and Sundberg, 1974, cal-
11
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culated that the total yearly emission of molybdenum in
air in Sweden amounted to about 70 tons, 45 tons coming from
the iron-alloying industries *
Deposition of molybdenum from air emissions has been meas-
ured in Sweden by determining molybdenum in moss. The nor-
mal molybdenum concentration in moss (Hypnum cypressiforme)
was found to be around 1 ug/g. The maximum value in moss
in Stockholm of 7.6 ug/g was found near a waste disposal
plant, where the molybdenum came from incinerated material.
In a Swedish city with large metal processing industries,
levels of up to 400 ug/g were found in moss. Near a large
steel works a maximum level of 560 ug/g was found (Lindau
and Sundberg, 1974).
3.3.2 In work environment
The only data on industrial levels available to us have
been those reported from the U.S.S.R. by Mogilevskaya,
1963, and Golyakova, 1971, each author reporting from
two different plants. For effects associated with these
levels, see Chapter 6.
In 40 samples taken above a crucible during a smelting
process involving MoO- (analysis by thiocyanate method,
sampling time not stated), Mogilevskaya found an average
value of 1.39 mg/m when the molybdenum content of the
alloy was 4% and an average of 5.4 mg/m when it was 17%.
The area above the crucible was distinguished from the
breathing zone of the workers, where the mean concentra-
3 3
tion was 0.22 mg/m and sometimes reached 0.4-0.5 mg/m .
In another factory, this one producing molybdenum wire,
she stated that the air concentration of MoO- fluctuated
3
between 6.4 and 19 mg/m .
Golyakova, 1971, reported some values found during the
processing of the final products in the hydrometallurgy
of tungsten and molybdenum salts and oxides. In air
12
-------
samples from two plants (sampling time not stated)
she recorded a range of 0.5-200 and 0.2-30 mg Mo/m ,
respectively, in the main working places.
3.3.3 In water and marine organisms
Harvey, 1945, summarized earlier data which indicated lev-
els of molybdenum in sea water below 1 ug/1. More recent
data indicate that the concentration is around 10 ug/1
(Black and Mitchell, 1952, Sugawara et al., 1962, Head
and Burton, 1970, Sournia and Citeau, 1972, Muzzarelli
and Rocchetti, 1973). Fresh water generally contains less
than 10 ug/1. Durfor and Becker, 1964, in a study of the
water supplies of the 100 largest cities in U.S., found
that the median concentration was 1.4 ug/1. Only in one
supply was a high value of 68 ug/1 found. Runnells et al. ,
1974, found average concentrations from 1.2-4.1 ug/1 in
4 rivers of the American West, whereas in the Climax area
in Colorado where mining is extensive, molybdenum concen-
trations were as high as several mg/1 of water. In the same
study molybdenum concentrations in the sediments varied in
the 4 rivers between 17 and 57 mg/kg dry weight, whereas
in the Climax area an average of 530 mg/kg dry weight was
found, the highest value being 1760 mg/kg. These data
agree well with earlier reported data by Turekian and
Scott, 1967, who found that molybdenum concentrations
in sediments of rivers in the eastern parts of the U.S.
ranged from 5-35 mg/kg.
In 170 California lakes concentrations of molybdenum
from < 0.3 to 100 ug/1 (median value 0.4) were found
(Bradford, Bair and Hunsaker, 1968). Soluble molyb-
denum concentrations in one California lake have
been shown to have large seasonal variations, from
< 1 to 15 ug/1 during one year (Dumont, 1972).
Runnells et al., 1974, also reported no difference be*
tween molybdenum concentrations in unfiltered and fil-
13
-------
tered water indicating that molybdenum was not bound to
particles in the water phase.
Bostrom and Wester, 1967, determined molybdenum in water
in three Swedish cities. Samples were taken from the raw
water, the water works, a water reservoir and from the
tap. As seen in Table 3:3 treatment and distribution do
not cause any changes in molybdenum levels. These results
indicate a low removal of molybdenum in water treatment
plants, support to which is given by Zemansky and Jorden,
1973, who found that only between 11-15% of the molybdenum
was removed during treatment.
Molybdenum in tap water was determined in 17 Swiss cities.
In 16 the concentrations were around 1 ug/1 or less, whereas
in one city a concentration of 29 ug/1 was found (Wenger
and Hogl, 1968) . In the Tomsk region in U.S.S.R. concentra-
tions between 0.11 and 0.15 ug/1 were found in summer, whereas
in winter between 0.03 and 0.06 ug/1 were found (Osmolovskaja,
1967). High concentrations of molybdenum in tap water were
found in Denver, Colorado. The source is the Dillon reser-
voir, which drains the mining area in Climax. In the reser-
voir molybdenum concentrations vary between 200 and 400 ug/1.
Values above 50 ug/1 are considered to be due to mining
activities (Chappell, 1974). High molybdenum concentrations
(25,000 ug/1) in ground water have been found in mining
areas in Colorado (Chappell, 1974) .
The fate of molybdenum released into water from molybdenum
mines was discussed by Asmangulyan, 1965. Molybdenum
sulfide, which is the compound released, is only slightly
soluble in water, but is oxidized to more soluble molyb-
dates and gradually converted to a molybdenum sulfate
complex.
In the marine environment molybdenum has been determined
in algae. Black and Mitchell, 1952, found that molybdenum
14
-------
concentrations in algae were relatively low, the concentra-
tions being between 30 and 240 ugAg wet weight, 100-1320
ug/kg dry weight. Lunde, 1970, determined molybdenum in
seaweed from the Norwegian coast and recorded concentra-
tions from 300 to 5,800 ug/kg. It is not stated in the
article whether the values are on dry or wet weight basis.
Young and Langille, 1958, found concentrations of molyb-
denum from 230 to more than 1,.000 ug/kg on a dry weight
basis in algae from the Atlantic coast of Canada.
3.3.4 In soils
Ever since molybdenum was shown to be essential to plants
and excess molybdenum in soil was shown to cause high con-
centrations in herbage, in turn causing the so-called "teart"
disease in cattle (Ferguson, Lewis and Watson, 1943), many
reports from different parts of the world have been devoted
to the occurrence and properties of molybdenum in soil.
In this section no attempt is made to discuss different
types of soil with regard to geochemical properties. By
going to the original references, the reader in many cases
may find such details.
A general question has been that of molybdenum availa-
bility to plants, since this depends not primarily upon
the total molybdenum content of the soil, but upon such
factors as whether the soil is acid or alkaline, organic
matter and levels of phosphate and sulfate. Whereas most
other metals are more available in acid soils, molybdenum
is more available in neutral or alkaline soils.
Data from some earlier studies in different areas of the
world have been compiled by Iyer and Satyanaryan, 1958,
and Reddy, 1964, the concentrations reported varying from
0.1-10 mg/kg of total molybdenum. In mining areas and near
molybdenum-emitting industries considerably higher values
have been reported (Chappell, 1974).
15
-------
Concentrations of available molybdenum between 0.2 and 0.7
mg/kg have been regarded as "normal", whereas concentrations
below or above are regarded as "deficient" or "excessive"
(Pines, 1963). One common method for the determination of
available molybdenum is extraction with ammonium oxalate at
pH 3.3 according to Grigg, 1953. Extraction with oxalate of
pH 3.0 (Gupta and McKay, 1966) with ammonium acetate, disodium
EDTA or water (Williams and Thornton, 1973) has also been
used.
Pines, 1963, found that in soils in Israel with pH from 6.7
to 8 total molybdenum varied from 1.6-8.4 mg/kg, whereas
available molybdenum varied from 0.2-4.4 mg/kg. Gupta and
McKay, 1966, found in soils from eastern Canada, that avail-
able molybdenum varied from less than 0.01-0.1 mg/kg. The pM
of these soils varied from 4.5-7.8 and the available molyb-
denum was significantly correlated to the pH of the soil.
In Ireland, Brogan, Fleming and Byrne, 1973, determined
molybdenum in a large number of samples. The available molyb-
denum varied from 0.05-6.5 mg/kg. In this study the highest
levels of available molybdenum were found in acid soils.
In India Iyer and Satyanarian, 1958, found available molyb-
denum ranging from 0.01-0.22 mg/kg, with the lowest values
found in soils with low pH.
Reddy, 1964, analyzed both total and available molybdenum
in a large number of soils in a region in India. The former
.aried from 0.5-4.1 mg/kg and the latter from 0.12-2.26 mg/kg.
On an average the total molybdenum concentration was 26 times
the available concentration. Up to pH 7.9 there was an in-
crease in available molybdenum with increasing pH.
Grigg, 1960, 1961, has presented exhaustive data on total
and available molybdenum in soils of different types on
the northern and southern islands of New Zealand. Total
16
-------
molybdenum on the northern island varied between 0.8 and 4.6
mg/kg and available molybdenum from 0.03-1.11 mg/kg. On the
southern island available molybdenum concentrations ranged
from 0.05-0.45 mg/kg.
Molybdenum has been determined in sewage sludge by Berrow
and Webber, 1972. They found that molybdenum concentrations
varied between 2 and 30 mg/kg dry weight, with 35 of 42
samples below 10 mg/kg. Andersson and Nilsson, 1972, found
that adding sewage sludge containing 0.68 mg/kg dry weight
of molybdenum to soil with a total molybdenum content of
0.53 mg/kg dry weight caused an increase with about 50%
in the molybdenum concentrations in the plants. Molybdenum
in fertilizers was determined by Hoover and Duren, 1967,
who found levels from 2.6-5.9 mg/kg in four samples. In
five samples Koirtyohann and Hamilton, 1971, found from
< 2 to 5.2 mg/kg.
3.3.5 In food
A number of reports on the molybdenum content of various
food items have appeared, showing variations both among and
within different classes of foodstuffs. An extensive re-
view on molybdenum concentrations in foodstuffs has been
given by Schlettwein-Gsell and Mommsen-Straub, 1973, who
compiled data from 26 original papers.
In Table 3:4 data from several reports have been assembled
on molybdenum in some common foodstuffs. The highest amount
of molybdenum has been found in the parts of the vegetables
above ground, whereas root parts generally have considerably
lower contents. Especially legumes and cauliflower can have
very high concentrations of molybdenum. Meat and other animal
products generally have low concentrations of molybdenum. The
molybdenum content of milk has attracted interest since it
may reflect variations in molybdenum content of animal feed.
17
-------
Data on molybdenum in food will also be found in Table
6:3 in connection with a description of human effects
in a molybdenum-rich area.
Archibald, 1951, reported normal cow milk to average 73
ug Mo/kg, with a range of 18-120. Feeding the same cows
500 mg Mo as ammonium molybdate daily raised the concen-
tration in milk to 371 ug/kg. Molybdenum in the milk of
cows is bound to the enzyme xanthine oxidase, meaning that
the activity of the enzyme in such milk is proportional to
the content of molybdenum (Hart/ Owen and Proudfoot, 1967),
Whether the differences between plant products depend en-
tirely on uptake of molybdenum from soil or somewhat on
the contribution of analytical differences is hard to
ev-.luate. The good agreement between different analyses
of milk gives some assurance that the different methods
give similar results.
3.3.6 In tobacco
In tobacco Voss and Nicol, I960, found molybdenum con-
centrations of 0.3-1.76 ug/g, mean 0.87. Reddy, 1964,
f oi-incl 0 .36 ug/g in Indian tobacco.
3.3.7 Daily intake
Schroeder, 1970, estimated that in the U.S. the average
diet contained 335 ug molybdenum, range 210-460. In the
U.S.S.R. estimates of intake in children were 156-161
ug/day (Vorobjeva and Osmolovskaya, 1970) and in adults
329-376 ug/day (Gabovich, 1964). According to Smolyar,
1972, diets of both children and adults in the U.S.S.R.
contain between 200 and 500 ug of molybdenum. Hamilton
and Minski, 1972/1973, determined molybdenum in total diet
samples collected from different regions of the U.K. They
found an average of 128 ug/day (S.D.+ 34). Tipton and
Stewart, 1970, followed three subjects for long periods
in a balance study. The daily intakes were on an average
18
-------
110, 210 and 460 ug. Wester, 1971, determined the daily
intake of molybdenum in two hospital patients by neutron
activation analysis of duplicate samples of the hospital
diet. Average amounts in 6 periods of 5 days each varied
from 250 to 1,000 ug. Wester, 1974a,studied also 4 healthy
subjects in a metabolic study. The daily intake of molyb-
denum varied between 115 and 245 ug (average for 10 days).
The above mentioned data show quite a good mutual agreement.
From the discussions in the section on molybdenum in food-
stuffs, it is obvious that if a diet is mainly based on
leafy vegetables and legumes, the intake may be consid-
erably higher. This is illustrated by the metabolic study
by Robinson et al., 1973, in which the diet was based on
meatloaf, and included no other vegetables, resulting in
a low daily intake, less than 100 ug molybdenum. The in-
take from water and air will be small compared to the in-
take from food, except in some areas where mining activities
may cause increases in molybdenum concentrations in drink-
ing water. It is not known if any molybdenum is released
from cigarettes during smoking.
3.3.8 Conclusions
The natural variations of molybdenum concentrations in
the environment are large, depending on geological factors.
Concentrations in water and soil may vary with a factor
of more than 10 causing both deficient and excessive con-
centrations for plants and ruminants in some parts of the
world. In areas where molybdenum ore is processed, con-
centrations in soil and water may increase considerably.
In soil the available molybdenum is of greater importance
than the total amount of molybdenum for plant nutrition.
The availability is dependent on pH and other factors in
the soil, being greater in alkaline soils. Molybdenum
concentrations in air are generally low. The variations
in foodstuffs, especially plants, are very greatly depen-
dent both on species and soil characteristics. In general,
19
-------
high concentrations are found in leafy vegetables and
legumes, whereas edible roots have a lower content.
Animal products are generally low in molybdenum. Daily
intake of molybdenum can be estimated to be between 100
and 500 ug. In areas where molybdenum ore is mined con-
siderable contamination may occur, which can cause high
concentrations in drinking water and thus daily intakes
of more than 1,000 ug.
20
-------
TABLE 3:1 WORLD PRODUCTION OF MOLYBDENUM, EXCLUDING
U.S.S.R. AND SOME OTHER COUNTRIES IN THE
EASTERN HEMISPHERE, 1956-1969. (From
Morning, 1969).
Year
Total (in thousand pounds):
1956-60
1961
1962
1963
1964
1965
1966
1967
1968
1969
63,300
74,000
59,300
75,000
78,000
98,531
124,988
126,273
125,735
142,802
"1,000 pounds =0.45 tons
21
-------
TABLE 3:2 CONSUMPTION OF MOLYBDENUM (in thousand pounds contained molybdenum) BY END USES
IN THE UNITED STATES IN 1969. (From Morning, 1969).
to
10
End Uses
Steel:
Carbon
Stainless and
heatresisting
Alloys
Tools
Cast irons
Super alloys
Alloys for cutting
and wear-resistant
materials
Alloys for welding;
for hard- facing rods
and materials
Magnetic alloys
Other alloys
Mill products made
from metal powder
Chemical and Ceramic
Pigments
Catalysts
Other
Misc.
Totals
(indep . roundings )
Molybdic
dxides
3,068
4,259
18,768
2,045
1,050
338
W
W
412
Uses:
731
1,514
35
2,128
34,349
Ferro- . Ammonium and
molybdenum Sodium
Molybdenum
468
1,883
2,309
1,204
3,140
597
W
383
W
87
W
371
W
W W
1,065 418
11,135 789
Other Mo ,
Materials**'
W
117
28
237
84
1,581
3
29
W
109
1,899
W
785
478
5,348
Totals
3,536
6,259
21,105
3,486
4,274
2,516
3
r
412
W
196
1,311
1,102
1,514
820
4,089
51,622
x) Incl. calcium molybdate
xx) Incl. purified Mo disulfide, molybdenite concentrate, molybdenite concentrate added directly to
steel, Mo metal powder, Mo metal pellets
W = witheld, confidential
-------
TABLE 3:3 MOLYBDENUM CONCENTRATIONS IN WATER (ug/1)IN THREE
SWEDISH CITIES. (From BostrSm and Wester, 1967).
Raw water Water works Water reservoir Tap water
Stockholm 43 4 3
Gothenburg 22 1 3
Malmo 996 9
23
-------
TABLE 3:4 MOLYBDENUM CONCENTRATIONS (ugAg wet weight) IN DIFFERENT
FOODSTUFFS.
Average con-
centration
Reference
Country
Milk
73
29
36
25
200
17
38-48
xxx)
Archibald, 1951
Kirchgessner, 1957
Voth, 1963
Kiermeier and Capellari, 1958
Stanton and Hardwick, 1968
Schroeder, Balassa and Tipton, 1970
Lupea and Vranceanu, 1972
Ward, 1974
U.S.A.
Germany
U.S.A.
Germany
U.K.
U.S.A.
Roumania
Colorado, U.S.A.
Potatoes
780
1004')
20-180
160
30
100
55
<20
80
Reddy, 1964
Le Riche, 1968
Warren, Delavault and Fletcher, 1971
Warren, Delavault and Fletcher, 1971
Schroeder, Balassa and Tipton, 1970
Lupea and Vranceanu, 1972
Lupea and Vranceanu, 1972
MacLean and Langille, 1973
MacLean and Langille, 1973
India
U.K.
Canada "Indust-
rial areas"
Canada "Normal
area"
U.S.A.
Roumania "Area 1"
Roumania "Area 2"
Canada "Light
farming"
Canada "Inten-
sive farming"
Cabbage
460
20
340
570
Reddy, 1964
Schroeder, Balassa and Tipton, 1970
MacLean and Langille, 1973
MacLean and Langille, 1973
India
U.S.A.
Canada "Light
farming"
Canada "Inten-
sive farming"
x) From cattle grazing on pastures with acid soils.
xx) From cattle grazing on pastures with alkaline soils.
xxx) Averages for 5 sampling periods during one year.
*) Calculated from dry weight values in the article, assuming a ratio
of 1:5 of dry weight to wet weight.
**) Calculated from ash values in the article.
Air-dry basis.
24
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TABLE 3:4 CONTINUED
Average con
centration
Reference
Country
Cauliflower
1,920
260-2,200
Reddy, 1974
Bovay and Rod, 1964
India
Switzerland
Legumes
1,000
(peas)
750-2040
6,030
(peas)
1,099
(peas)
310-4,800
180
(peas)
Wheat
230-400
490
300
190-780
199
640-5,870
179-222
Schall and Schall, 1962
Reddy, 1964
Los, Piatnizkaja and Samsonova, 1966
Lupea, Vranceanu and Waltraut, 1969
Schroeder, Balassa and Tipton, 1970
MacLean and Langille, 1973
Schall and Schall, 1962
Reddy, 1964
Kirchgessner and Friesecke, 1969
Basargin and Peregudova, 1969
Lupea, Vranceanu and Waltraut, 1969
Schroeder, Balassa and Tipton, 1970
Lupea and Vranceanu, 1972
Germany
India
U.S.S.R.
Roumania
U.S.A.
Canada " Inten-
sive farming"
Germany
India
Germany
U.S.S.R.
Roumania
U.S.A.
Roumania
25
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CHAPTER 4 METABOLISM
4.1 UPTAKE AND ABSORPTION
Data presented in the immediately following sections do
not cover interactions of molybdenum with other compounds
such as sulfur and copper. Such studies are treated separa-
tely in Chapter 7. For general aspects of accumulation of
toxic metals, their absorption, excretion and biological
half-times, the reader is referred to reports by the Task
Group on Metal Accumulation, 1973, and the Task Group on
Metal Toxicity, in press.
For understanding the metabolism and toxicity of molyb-
denum, the solubility of the different compounds must be
taken into consideration. With regard to solubility in
biological fluids there are only data available from
Mogilevskaja, 1963, who stated that the solubilities of
molybdenum metal and molybdenum trioxide were 3.8 and
125 mg/100 ml serum and 4.6 and 50 mg/100 ml gastric
juice respectively. The solubility was higher in alka-
line media (Na.CO.,) than in acid media (0.3% HC1) . Ammonium
paramolybdate was easily dissolved in water, serum and
gastric juice. Calcium molybdate is practically insoluble
in water, whereas sodium molybdate has a high solubility
(Handbook of Chemistry and Physics, 1974/1975).
4.1.1 Absorption following inhalation
4.1.1.1 in_a*!im.a:Ls_
Fairhall et al., 1945, exposed groups of guinea pigs to the
dusts (particle size not given) of molybdenite (average
concentration 285.9 mg Mo/m ) , calcium molybdate (158.9 mg
3 3
Mo/m ), molybdenum trioxide (204.7 mg Mo/m ) and a fume of
molybdenum trioxide (53.0 mg Mo/m ), one hour daily, five
times per week, for five weeks. From the resultant distri-
bution (see Table 4:1) in organs, it is obvious that hex-
avalent compounds (calcium molybdate, molybdenum trioxide)
26
-------
are absorbed, even though no quantitative evaluations can
be made. Tissue concentrations, with the exception of the
lungs, after exposure to molybdenite were not higher than
among controls. The trioxide fume gave unexpectedly lower
concentrations in the tissues than did the trioxide dust.
Confining themselves to distribution among different organs,
the authors did not discuss how large the total body reten-
tion was, nor what proportion of the molybdenum retention
was actually gastrointestinally absorbed following mucocil-
iary lung clearance.
4.1.1.2 2Ln_human_beings.
No data are available.
4.1.1.3 Cone ].us_ions_
One study on animals shows that different molybdenum com-
pounds are absorbed, but a quantitative evaluation cannot
be made from the data on hand. No human data on absorp-
tion after inhalation are available.
4.1.2 Gastrointestinal absorption
4.1.2.1 .In_anima_ls_
Fairhall et al., 1945, gave 50 mg of Mo as Mo03 by oral
administration with a syringe to three pairs of guinea
pigs weighing between 350 and 450 grams. The distribution
among organs at intervals of 4 hours, 16 hours, and 48
hours after administration is given in Table 4:2. The
cumulative amount found in the feces up to 48 hours
was 5.8 mg, the remaining portion in the gastrointestin-
al tract being 1.29. If all of this is counted as a not-
absorbed amount, the total absorption would be around
85%. Fairhall et al. further gave evidence of a rapid
absorption of molybdenum when they measured the blood
values of molybdenum after the administration of 100
27
-------
mg of MoO3 to two rabbits weighing 1,880 and 1,620 g
by means of a stomach tube. The blood values increased
from 2 ug/g blood before exposure to more than 10 times
the initial value 400 min. after administration (15 and
20 ug/g blood respectively). Considerable doubt must be
expressed as to the validity of the blood values. One
reason for this is the large variation seen in Table
4:2; another reason is the very large difference seen
between Fairhall et al.'s "normal" values in animals
and "normal" values in humans (section 4.3.2). The latter
are 100-1,000 times lower than the values given by Fair-
hall et al., 1945.
In an experiment by Neilands, Strong and Elvehjem, 1948,
13.34 mg of Mo given as radioactive MoO, (hexavalent) was
introduced into the back of the throat of 12 rats that had
been starved for one day before the experiment. The study
is not suitable for an exact assessment but shows that at
least 10-20% of the molybdenum had been absorbed as early
as 2.5 hours after administration. At that time the liver
contained 28 ug and the kidneys 60 ug of molybdenum. In
the stomach there was 1,537 ug and in the intestines 215
ug. The authors did not state whether these figures meant
tissue alone or tissue plus contents. The carcass had a
concentration of approximately 825 ug. After 2 days at
least 35% of the given dose must have been absorbed since
during day two, 4,692 ug was excreted with the urine and
molybdenum was still present in internal organs.
Van Campen and Mitchell, 1965, studied absorption of (NH J ,
99
MoO. from ligated segments of the female rat gastrointes-
tinal tract. The abdominal cavity was opened, the segment
ligated, and molybdenum thereafter injected into it. The
isotope was readily absorbed from the stomach as well as
from the intestines at all levels down to the colon (absorp-
28
-------
tion from the last was not examined). The uptake was counted
as the total uptake by the blood, heart, kidneys and liver
in percentage of given dose two hours after administration
(2.7 uMol Mo). The means for uptake from the different seg-
ments fell in the following order: the duodenum (9.46%)
> ileum (8.28%) > midsection (6.75%)> stomach (3.6%).
Each figure is the mean of 5 animals (5 animals in each
"segment group").
Anke et al., 1971, studied the absorption of molybdenum in
four lactating goats following oral ingestion. The animals
were fed a diet to which was added 1.5 mg Mo as MoO- per day
99
and animal for two weeks. Subsequently, 3 mCi of MoO- was
99
injected via a stomach tube and levels of Mo in the feces,
urine and different organs were determined for a period of
96 hours. It was calculated that on an average at least
99
34.7% of the given amount of Mo was absorbed from the
gastrointestinal tract (based on molybdenum found in tis-
sues, urine and milk). The total percentage found in the
feces, 51%, was counted as the not-absorbed amount.
Miller et al., 1972, compared absorption in calves and
99
pigs after doses of Mo. Pigs were given the isotope in
feed, whereas calves were given the isotope in milk by
nipple pails (abomasal dose) or by gelatin capsules (rumen
dose). It was found that if the rumen was by-passed, ab-
sorption in calves was similar to that in pigs whereas
rumen doses were absorbed to a lesser extent. It was pos-
tulated that molybdenum became less available by some
process in the rumen.
4.1.2.2 ]Cn_human_be ings
Tipton, Stewart and Dickson, 1969, analyzed 22 elements
in the complete daily diets (self-chosen), and in the to-
tal daily urinary and fecal excreta of three men for a
29
-------
period of 50 weeks. Analysis was made by arc emission
spectrography. As can be seen from Table 4:3, all three
subjects had a positive balance of molybdenum. Subjects
A and B had more than 50% of the total urinary-fecal con-
tents in their urine. Considering the dietary/fecal ratio
of these two subjects it seems likely that the absorption
was well over 50%. In subject C the absorption was probab-
ly less than 50%.
A three-day balance study was conducted on 11 healthy
schoolchildren by Alexander, Clayton and Delves, 1974.
The analytical method employed was atomic absorption.
They concluded that 77% of the ingested molybdenum was
absorbed.
In an 18-day balance study on 4 women, Robinson et al.,
1973, found that between 38 and 72% of the daily in-
take was in feces, indicating that from 28 to 62% had
been absorbed. Molybdenum was determined by a dithiol
method.
Bostrom and Wester, 1968, studied one healthy man and
two patients with osteomalacia for two 5-day balance
periods. From their data it can be seen that from 30
to 76% of the molybdenum might have been absorbed by
the patients. The values for the healthy man were 38
and 76% in the two periods. Molybdenum was determined
by neutron activation. Wester, 1974a, studied 4 healthy
men during two 5-day periods, the one period involving
low and the other high calcium intake. From his data it
can be estimated that absorption was 52% (44-62) and 47%
(29-65) during the two periods.
30
-------
4.1.2.3 Conclusions^
Hexavalent molybdenum is readily absorbed from the gastro-
intestinal tract, 40%-85%, in animals. Naturally occurring
molybdenum in human food is absorbed to a high degree,
probably 25-75%. The data on humans are based on a very
limited number of observations.
4.2 EXCRETION
4.2.1 In animals
4.2.1.1 Uri.ne_ eind a_limentary_tra£t
Fairhall et al., 1945, gave guinea pigs molybdenum as MoO3
per os in doses of about 20 mg daily for ten days. After
two days, the daily total urinary and fecal contents were
equalizing the administered doses. The urine contained
about three times the amount that appeared in the feces.
The excretion of the absorbed molybdenum seemed almost
complete already three days after the ten-day exposure
period.
Neilands, Strong and Elvehjem, 1948, in their experiment
on rats, referred to in section 4.1.2.1, found after single
oral introduction of 13.34 mg of Mo (given as radioactive
MoO.,) 4,692 ug Mo in the urine during the second day after
exposure, compared to only 697 ug in feces.
In the study by Anke et al., 1971, on lactating goats
(section 4.1.2.1), the cumulative urinary excretion of
99
Mo during 4 days following a single peroral dose of
99
MoO3 was 25.4% out of the ingested amount while 51% was
in the stools.
Robinson et al., 1964, reported on the excretion of intra-
99
venously administered Mo as sodium molybdate in water
to two groups of six cows each. The cows in one of the
31
-------
groups had been on a deficient diet and had symptoms of
reduced growth, low milk production and low serum copper
99
levels. The excretion of Mo in urine followed an ex-
ponential pattern during the observation period (80 hours)
with a half-time of 19.9 ± 1.4 (S.E.) hours in the normal
cows and 18.7 ± 1.2 in the nutritionally deficient cows.
The biological half-time for the body as a whole cannot
be evaluated as excretion via feces and milk was only
followed for a short period.
In two experiments, Amon, Scheler and Peters, 1967, stud-
ied the excretion of Mo in rats following two s.c. doses
99
of 2 mg of Mo03 given on day 1 and 4 of the experiments.
Within 14 days 43.5% and 79.4% respectively had been ex-
creted in the urine, and 2.6% and 9.7% in the feces.
4.2.1.2 Mi l.k_
In Anke's experiment referred to in 4.1.2.1 (Anke et al.,
1971), a cumulative amount of 2.4% of ingested molybdenum
was found in the milk of lactating goats four days after
administration.
4.2.1.3 Bile_
Caujolle and Roche, 1935, studied the biliary excretion
of molybdenum in two dogs with bile fistulas following
s.c. injections of molybdenum salts. One dog weighing
25 kg received 5 g of sodium molybdate and the other
weighing 15 kg 0.2 g of ammonium molybdate. Six hours
after the injection, the first dog had excreted 33.5
mg of molybdenum in the bile and the second dog 2.2 mg.
For comparison the corresponding urinary amount was 91.9
mg and 16 mg respectively.
32
-------
Fairhall et al., 1945, at autopsy of their experimental
guinea pigs, also found a considerable amount of molyb-
denum in bile (see sections 4.3.1.1 and 4.3.1.2).
4.2.1.4 Ha ir_
The total amount of molybdenum retained in the body after
96 hours in the experiment by Anke et al., 1971, was 6.9%.
Out of the retained amount 0.2% appeared in the hair. The
authors pointed at a steady rise of molybdenum concentra-
tion in hair as long as 96 hours after the administration.
The activity in hair at this point was 6 times higher than
the activity 24 hours after administration.
4.2.2 In human beings
Excretion in man was studied by Rosoff and Spencer, 1964.
99
Carrier-free Mo in ammonium hydroxide was injected in-
travenously (50-100 uCi) and the subsequent urinary and
fecal excretion was determined. The cumulative urinary
99
Mo excretion in 10 days was 24% in one subject and 29%
in another, while the corresponding fecal excretion was
6.8% and less than 1%, respectively (Figure 4:1). The re-
tention of the metal seems higher in man than in animals
from this study. What part the considerably smaller doses
could have played is not known.
The total excretion of molybdenum under "normal" steady
state conditions is not known. Data from Tipton, Stewart
and Dickson, 1969, tend to show that 25-50% of the ingested
amount is excreted via urine.
In Figure 4:2, data have been compiled on urinary excre-
tion of molybdenum in relation to dietary intake in in-
dividuals studied by various investigators. It is obvious
that a relationship exists.
33
-------
Data on daily urinary excretion of molybdenum in "normal"
human subjects have also been given by Meltzer et al.,
1962 (spectrography) and Wester, 1973, 1974b. On the average,
the excretion in the groups varied between 49 and 81 ug
per day. In New Guinea Adkins, Barraes and Schamschula,
1974, found daily averages ranging from 4 to 20 ug of
molybdenum (method not stated) in 10 groups of "normal"
subjects.
4.2.3 Conclusions
Studies referred to have dealt with hexavalent molybdenum
compounds. Limited animal data indicate a rapid excretion.
A more or less complete excretion of molybdenum took place
during the first two weeks after a single s.c. exposure.
In one study on humans the retention 10 days after an
intravenous injection of molybdenum was higher, only
30-40% of the dose being excreted.
The main channel of excretion is through the kidneys.
Excretion through bile is likely to be the main channel
of fecal excretion. There are indications of a small
excretion through milk, sweat and hair.
4.3 TISSUE DISTRIBUTION
4.3.1 In animals
4.3.1.1 Single_exposure
4.3.1.1.1 Oral_route_
In Fairhall et al.'s study (1945) three pairs of guinea
pigs were given 50 mg of molybdenum as molybdenum trioxide
by oral administration with a syringe, and were killed at
4 hours, 16 hours and 48 hours afterwards. Molybdenum was
analyzed with the thiocyanate method. The molybdenum con-
centration in kidneys decreased from 46 ug/g wet weight
after 4 hours to 7 ug/g after 48 hours. In the liver there
was a decrease from 20 to 3 ug/g and in the spleen from 26
34
-------
to 18 ug/g. The concentration in the bile was between
20-30 ug/g during the whole study (Table 4:2).
A similar study on 12 rats divided into 6 groups of two
rats each was made by Neilands, Strong and Elvehjem, 1948
The rats were given 13.34 mg of molybdenum trioxide la-
99
belled with Mo in the back of the throat via a syringe.
The rats in three of the groups were also given immedi-
ately thereafter 0.5 ml of a solution of copper sulfate
corresponding to 5 mg of copper. Two rats from both the
non-copper and the copper groups were killed after 2
hours, 26 hours and 51 hours.
The molybdenum concentrations in different organs and
biological material are given in Table 4:4; The distri-
bution was rather similar to the one found by Fairhall
et al., 1945. It is further seen that bones contained
appreciable amounts of molybdenum. The excretion from
the organs was rapid in these experiments.
4.3.1.1.2 _lntrayenous_ route_
Durbin, Scott and Hamilton, 1957, administered 3 uCi of
99
Mo as Na2MoO. intravenously to three rats. After 4
hours 1/3 was excreted via the urine, 1/3 was in the
liver, and the rest was distributed among the gastro-
intestinal tract, blood, soft tissues and skeleton.
Liver, kidney and pancreas contained the highest con-
99
centrations of Mo.
4.3.1.2 Re£eated_exposure
4.3.1.2.1 _Ora^ route
Fairhall et al., 1945, performed a long-term experiment
on rats -fed different molybdenum compounds which varied
as to concentration (10-500 mg molybdenum per rat and
day) and as to period of time (10-500 days). In rats giv-
en molybdenite there was no mortality and concentrations
35
-------
of molybdenum in liver, kidney and bone at the end of the
experiment were low (2-6 ug/g wet weight) and the same re-
gardless of whether the rats were given 10 or 500 mg molyb-
denum per day during a 44-day period. Control animals had
3-6 ug molybdenum per gram tissue, the studies thus in-
dicating that there could only be a very low absorption
of molybdenite.
Molybdenum trioxide, calcium molybdate and ammonium molyb-
date were absorbed and the mortality was between 25-100%.
In some cases the data were based on surviving animals
while in other cases deceased (time of death not given).
This makes it impossible to evaluate accumulation ten-
dencies and dose-related differences. The thiocyanate meth-
od was used for analysis.
In four lactating cows, Huber, Price and Engel, 1971,
found concentrations of molybdenum in kidney and spleen
to be several times higher than those in the liver, 42.3
and 95.0 versus 10.4 ug/g dry weight following a six
month's intake of a basal ration of 53 mg Mo/kgdiet giv-
en as sodium molybdate. Blood, brain and muscles con-
tained considerably less metal. Bones were not analyzed.
Four other animals were fed 173 mg Mo/kg diet. In the
spleen there was no increase in molybdenum compared
to the 53 mg/kg group, while in blood, kidneys and liv-
er the concentration increased with a factor of 2-3.
In the study by Anke et al., 1971, four goats were each
fed 1.5 mg molybdenum as molybdenum trioxide daily for
99
two weeks. Hereafter 3 mCi of MoO3 were administered
through a stomach tube. 96 hours after this the animals
99
were sacrificed and their tissues analyzed for Mo. The
results are presented in Tables 4:5 and 4:6. 6.9% of the
administered dose was found in the body, excluding the
gastrointestinal tract, and the figures in the tables are
36
-------
given in percent of this amount. The bones contained the
highest amount, 27.4%, followed by ti j liver, 19.9%, and
the kidneys, only 3.4%. The concentration (radioactivity
per gram dry weight) was highest in the kidneys, being
followed by the liver.
4.3.1.2.2 Inhalation_
Groups of guinea pigs were exposed to the fumes and dusts
of different molybdenum compounds one hour daily, five
times per week for five weeks (Fairhall et al., 1945).
In Table 4:1 the different tissue concentrations in ani-
mals dying during the test, sacrificed at the end of
the test, and sacrificed two weeks after the test, are
given. As expected, molybdenite after inhalation also
gave very low concentrations in the tissues, apart from
the lungs. For more details see 4.1.1.1.
At the end of the exposure the concentrations of molyb-
denum were higher in animals exposed to molybdenum tri-
oxide dust than in those exposed to calcium molybdate
or molybdenum trioxide fume. The low concentration after
exposure to molybdenum trioxide fume is remarkable as
is the fact that exposure to fumes gave the lowest mor-
tality. The possibility of methodological errors must be
borne in mind but the authors offered some explanation in
differences in solubility of the two compounds.
The drop in the molybdenum content of most tissues two
weeks after the end of exposure indicates low retention
of the metal. No estimations or calculations on the total
retained amount of molybdenum are given.
4.3.1.3 Di£tribution_within organs
In a study by Brinkman, Miller and Engel, 1961, groups of
rats were fed between 200 and 500 mg Mo/kg diet for six
37
-------
weeks. Hereafter the livers of the animals were removed,
homogenized and centrifuged. Molybdenum was usually found
to be concentrated in the supernatant (>60%) while nuclei
and debris contained <15%, mitochondria 15% and microsomes
10%.
4.3.2 In human beings, "normal exposure"
It has not been possible to find any studies dealing with
distribution in man following experimental administration
or industrial exposure to molybdenum.
Butt et al., 1964, spectrographically determined molyb-
denum concentrations in blood in three groups of individuals
in the Los Angeles area, totally 266 subjects in two dif-
ferent hospitals (two patient groups and one personnel
group) and found values between non-detectable to 34 ng
per ml. Mertz et al., 1968, also using spectrography, found
an average concentration of 6 ng/ml serum, but described
a wide scatter (0.1-30 ng/ml) among individuals (69 sub-
jects in Germany).
By neutron activation analysis Morgan and Holmes, 1972,
found average concentrations in whole blood of 0.8 and
1.2 ng/ml in 8 normal men and 5 normal women respectively
in the U.K. Brune et al., 1966, found 3.3 ng/ml in whole
blood and Kjellin, 1968, detected <1 ng/ml in cerebro-
spinal fluid and 15 ng/ml in brain tissue. Wester, 1973,
found an average concentration in serum of 5.6 ng/ml in
8 healthy subjects. The three latter studies involved
subjects in Sweden.
Allaway et al., 1968, studied the influence of the geo-
graphical area on the blood molybdenum content. The study
encompassed 229 subjects from 19 different areas in the
United States. 75% of the samples examined by the thio-
cyanate method showed a concentration of molybdenum of
38
-------
less than 5 ng/ml of whole blood. Still, they found two
collection sites where more than 70% contained more than
this amount, and in one of these areas the maximum detected
value was 410 ng/ml.
Polonskaja, 1968, determined molybdenum in the blood of
47 pregnant women and 12 non-pregnant controls in the
U.S.S.R. The controls had 166 ng/ml of blood? in the
pregnant women, the blood level of molybdenum first de-
creased and then increased again. From the 12th to the
19th week of pregnancy, the concentration was 129 ng/ml;
from the 28th to the 38th week: 152 ng/ml; from the 39th
to the 40th week: 234 ng/ml. After parturition the con-
centration remained high for 5-6 days (213 ng/ml). The
reason that these blood values were high compared to the
other reports is not known.
Tipton and Cook, 1963, reported levels of 27 different
trace elements in 29 spectrographically analyzed tissues
of 150 adult subjects from the United States. As for molyb-
denum, this metal was observed in every sample of liver
(147/147), in almost every sample of kidney (140/144),
and adrenals (10/13), and only occasionally in other
tissues. The median values were, respectively, 1 ug/g,
0.3 ug/g and 0.7 ug/g wet weight.
Plantin, 1973, determined molybdenum in kidney cortex,
liver and heart by neutron activation in 8 autopsy cases
in Sweden, 51-63 years of age. The average concentrations
in the respective organs were 0.2, 0.88 and 0.022 ug/g
wet weight. Samsahl, Brune and Wester, 1965, also in
Sweden, found 0.41 and 0.50 ug/g wet weight in two livers
by neutron activation.
Schroeder, Balassa and Tipton, 1970, gave the changes
with age in the main concentrations of hepatic and renal
39
-------
molybdenum by decade of life in the United States and
in other countries as seen in Figure 4:3. The method of
analyses was emission spectroscopy. Here the concentra-
tions are given in ug/g ash. According to Tipton and
Cook, 1963, the ash percents of wet weight for liver and
kidney are 1.3% and 1.1% respectively. There were no sig-
nificant differences between the United States and the
non-U.s. subjects. Molybdenum was relatively low in the
newborn, rising to a peak (about 1.1 ug/g in livers and
0.4 ug/g in kidneys) in the second and third decade of
life, and declining thereafter, particularly in the liver.
In this material total body content of the United States
subjects was less than 9 mg.
Pribluda, 1964f reported the molybdenum concentrations
in liver and kidney of 80 subjects aged 17 to 77 years
in the U.S.S.R. The subjects died accidentally and had
no signs of organic disease. The thiocyanate method was
used for determining molybdenum. The results were evalu-
ated statistically and are shown in Table 4:7. The con-
centration of molybdenum in the human liver was about
2-3 times higher than in the kidneys.
In another report from the U.S.S.R., Gurskaja, 1966,
determined molybdenum by the thiocyanate method in the
ash of the amnion of 60 fetuses and the (villous) chorion
of 90 fetuses. The arithmetic mean concentration of molyb-
denum in the amnion was 3.5 - 0.27 (S.E.) ug/g ash; in
the (villous) chorion 0.6 - 0.02 ug/g ash. The ash con-
tent of the tissue was 0.47 and 1.05% respectively.
Fetuses from women in the age group 21-25 had more molyb-
denum in the amnion (4 ug/g ash) than from those in the
31-35 year group (2.3 ug/g ash). The difference was stat-
istically significant (P< 0.02). There was no age-related
difference with regard to the molybdenum concentration in
the (villous) chorion.
40
-------
4.3.3 Conclusions
There is a fairly good uniformity in results between the
different animal studies dealing with the tissue distri-
bution of molybdenum. Thus, it seems clear that the metal
is concentrated to a higher degree in kidneys, liver, and
bone tissue, the highest values invariably being in the
kidneys. On the other hand, the retention of the metal
is low, the major part of administered doses in animal
experiments being excreted within hours or days, meaning
a biological half-time of an approximately similar value.
Human studies have only been concerned with "normal ex-
posure", that is, no studies dealing with distribution
following experimental or industrial exposure have been
published. Estimations of blood levels in different in-
dividuals show a wide scatter, but most data support a
normal value of only a few ng/ral whole blood. The geo-
graphical area may be of considerable importance. There
are indications of an increasing molybdenum level in the
human liver and probably kidneys to the second and third
decades of life, and a slight decline thereafter. The
liver values are higher than the kidney values, the liver
reaching on the average a concentration of 0.5-1 ug/g
wet weight and the kidney about 0.25 ug/g wet weight.
There are no studies implying an accumulation of the metal
throughout life.
41
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TABLR 4:1. DISTRIBUTION OF NOLTBDEJUN (ug Ho/g wet weight) IN GUINEA PIOS FOLLOWING INHALATION OF VARIOUS MOLYBDBNUM COMPOUNDS (Modified fro»
Fairhall et al., 1945).
Compound
Molybdenum
sulfide
Molybdenum
trioxide
dust
Calcium
molybdate
Molybdenum
trioxide
Molybdenum
trioxide
Controls
Total Average
number cone en-
exposure tration
24 286
24 205
26 159
?S 191
25 53
Peroent Animals died during test Animals
mortality
Liver Kidney Long Spleen Bone Liver
4.2 2
51.0 12 21 17 16 521 10
20.8 4 6 126 8 20 3
8.3 5 69 13 2
0 2
1
killed
Kidney
1
27
11
4
4
1
at end of test
Lung Spleen Bone
387 1 2
20 17 24
179 6 21
86 6
3 4 6
1 S 3
Animals killed two weeks after test
Liver Kidney Lung Spleen Bone
23 57 11
36 132 4 12
12 38 5
? 1 24 5
1Include! weak animals killed after 10 exposures.
-------
TABLE 4:2
DISTRIBUTION** OF MOLYBDENUM IN TISSUES OP GUINEA PIGS
AT DIFFERENT INTERVALS FOLLOWING ORAL ADMINISTRATION
OF 50 mg OF MOLYBDENUM AS MOLYBDENUM TRIOXIDE.
(Modified from Fairhall et al., 1945).
Interval after dosage
Kidneys
Spleen
Bloodxx)
Bile
Liver
Lungs
Muscle
Feces (total Mo)
Urine (total Mo)
Gastrointestinal tract
(total Mo)
4 hours
46
26
26
28
20
31
10
2,200
19,900
16 hours
20
12
5
20
8
10
2
990
11,100
5,080
48 hours
7
18
50
30
3
9
1
5,800
13,200
1,290
x)
'Values for tissues given as ug Mo/g wet weight and for feces,
urine and gastrointestinal tract as total cumulative molybdenum
in ug.
xx'Special comments, see text.
TABLE 4:3 MOLYBDENUM IN DIET, URINE AND FECES (ug/day, mean ± S.E.)
IN THREE HUMAN SUBJECTS. (Modified from Tipton, Stewart
and Dickson, 1969).
Subject
A
B
C
Diet
210 +
460 +
110 +
Feces
20
80
10
88 ±
99 +
71 ±
17
15
3
Urine
110
130
26
± 10
± 10
± 6
43
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TABLE 4:4 DISTRIBUTION5^ OF RADIOACTIVE MOLYBDENUM IN TISSUES OF
99
RATS GIVEN A SINGLE ORAL DOSE OF Mo03 LABELLED WITH Mo
ALONE AND WITH COPPER. (Modified from Neilands, Strong
and Elvehjem, 1948).
Group No.
Average weight, g
Time interval between
dose and death, hours
Mo dose , mg
Cu dose, mg
1
132.6
2.1
13.34
2
150.3
26.1
13.34
3
142.9
51.0
13.34
4
155.8
2.5
13.34
5.00
5
96.2
26.4
13.34
5.00
6
71.4
51.4
13.34
5.00
Molybdenum in tissues
(ug/g wet weight)
Blood
Stomach
Kidneys
Liver
Intestine
Bone
Heart
Lungs
Carcass
Feces
28.8
176.4
18.5
9.1
13.9
10.9
9.0
15.8
8.5
2.3
5.8
7.7
1.2
25.4
4.8
1.5
1.6
3.2
1-.8
2.9
7.1
0.8
6.2
3.0
1.0
1.2
0.6
290.9
13.2
9.3
14.3
5.4
9.0
10.3
10.0
12.4
8.0
3.0
19.3
9.7
1.7
25.5
6.4
1.9
3.4
3.9
2.3
4.6
2.8
1.6
14.4
3.9
1.6
2.2
1.5
597.4
x) Each column represents the average from two rats.
44
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TABLE 4:5 RELATIVE PERCENTAGE DISTRIBUTION (MEAN AND S.D.)
QQ
OF RETAINED "Mo IN TISSUES OF FOUR GOATS 96
HOURS AFTER ORAL ADMINISTRATION.
(From Anke et al., 1971).
Skeleton Liver
Mean
S.D.
27.4
9.2
19
4
.9
.7
"Rest"
14.4
7.2
Head
14.3
13.6
Muscle
11.6
12.7
Blood
8.7
9.3
Kidneys Hair
3.4
1.5
0
0
.2
.18
Ovaries
0.1
0.004
TABLE 4:6 RELATIVE CONCENTRATION3^ (MEAN AND S.D.) OF RETAINED "MO IN
TISSUES OF FOUR GOATS 96 HOURS AFTER ORAL ADMINISTRATION.
(From Anke et al., 1971).
Mean
S.D.
Kidney
100
16
Liver
60
22
Ovaries
28
10
Blood
18
2
Head
12
10
Skeleton
12
6
"Rest"
7
2
Hair
3
4
Muscle
3
2
Given as radioactivity/g dry weight. Highest activity = 100.
45
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TABLE 4:7 MOLYBDENUM IN LIVER AND KIDNEYS (ug/g wet weight)
OF "NORMAL" HUMANS OF DIFFERENT AGES.
(Modified from Pribluda, 1964).
Age, years
Liver
Kidneys
17-25 26-44 45-59 60-77
17-25 26-44 45-69 60-77
Number
Arithmetic
mean
Standard
deviation
23
27
20
10
0.64 0.61 0.54 0.57
0.21 0.18 0.12 0.15
23
27
20
10
0.22 0.24 0.24 0.24
0.06 0.06 0.09 0.06
46
-------
Cumulative excretion
dose percent
30 ,
FIGURE 4:1
99,
Cumulative excretion of Mo in man after an
intravenous injection of carrier-free Mo in
ammonium hydroxide.
Subject lj
Subject 2. (From Rosoff and Spencer,
1964) .
47
-------
Urinary
molybdenum D = Alexander.Clayton and Delves. 1974
Jjg/24hrs 0_ Bostrom and Wester, 1968
250 1 O= Robinson et al., 1973
X = Wester, 1974
200-
150 -
100 -
50-
cP x
D
-I 1 1 1 1 1
50 100 150 200 250 300 Dietary molybdenum
FIGURE 4:2 Relationship between daily intake and urinary
excretion of molybdenum. All data are from
balance studies on healthy individuals. The
data from Bostrom and Wester, 1968, Robinson
et al., 1973, and Wester, 1974a, represent
individual values. The data from Alexander,
Clayton and Delves, 1974, represent the mean
values from eleven healthy schoolchildren.
These values were originally given as ug/kg
body weight and have been converted to 20
kg body weight.
48
-------
Mo
/ug/g ash
140
120
100
80
60
20
.x'^v--1
Molybdenum
/ .#•'
•
o*
•
o
. •.
•o.
0-10 1-9 10-19 20-29 30-39 40-49 50-59 60-69 70-84
Months Age, years
FIGURE 4:3
Mean concentrations of molybdenum, according
to age, in livers (upper curves) and kidneys
(lower curves) of United States and non-U.S.
subjects, ug/g ash. There were 226 livers
(solid dots, dash-dot line) and 217 kidneys
(solid dots, dashed line) from U.S. subjects
and 179 livers (open circles, dotted line)
and 181 kidneys (open circles, dash and 3 dots)
from non-U.S. subjects. Note the similarity
of the curves. The first 3 points for U.S.
values were based on 18, 33 and 3 samples
respectively, the remainder on 10-45 samples
in each decade. The first non-U.S. point was
based on only 2 samples, the remainder on
7-37 samples in each decade. (From Schroeder,
Balassa and Tipton, 1970).
49
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CHAPTER 5 MOLYBDENUM AS AN ESSENTIAL ELEMENT IN
ANIMAL NUTRITION
5.1 MOLYBDENUM DEFICIENCY AND WOLFRAMATE INHIBITION STUDIES
One way of studying the essentiality of an element is to
exclude it from the diet of experimental animals. This
can be technically difficult since complete absence of
the element is often required before anything definite
can be said about its role in metabolism. Tracer doses of
some essential elements are sufficient to maintain a nor-
mal metabolism.
In an experiment by Higgins, Richert and Westerfeld, 1956,
female weanling rats were raised and maintained on a puri-
fied diet poor in molybdenum, approximately 20 ug Mo/kg,
for three generations. No difference arose in the develop-
ment of these animals compared with a group that received
an additional 200 ug molybdenum per kg given as NaMoO..
The animals grew at a normal rate, reproduced, and appeared
normal in all respects. The livers of the third generations
of animals contained 1.89 ± 0.15 and 2.24 ± 0.19 ug/g dry
weight of molybdenum respectively. In order to decrease
the tissue levels of molybdenum further, six animals were
given an inhibitory metal, wolframate, 4.5 mg W/kg diet
in the form of Na^WO^ as a supplement to the molybdenum-low
diet. This increased the urinary excretion of molybdenum
from less than 0.07 ug/day to about 0.6 ug/day and de-
creased the liver molybdenum to 0.81 - 0.18 ug/g dry
weight. Still, no symptoms or effects on the animals could
be found.
A similar experiment was carried out by the same authors
on chicks (see Tables 5:1 and 5:2). Feeding a synthetic
diet containing approximately 20 ug/kg of molybdenum to
day-old chicks for five weeks (group 2 in Tables 5:1 and
5:2) failed to produce a molybdenum deficiency syndrome in
50
-------
this species. At the simultaneous administration of tung-
sten, however, something that might have been molybdenum
deficiency developed. 45 mg/kg of tungsten (as Na2WO4)
given to 29 chicks (group 4) significantly reduced the
growth of the animals compared to a control group given
an "ordinary" chick diet. The mortality after five weeks
was 24% compared to zero in the control group. The
liver molybdenum was 0.22 ug/g dry weight compared to
3.56 ug/g in the control group (group 1) and other tissues
of group 4 such as proventriculus, intestines, kidneys,
brain and muscles contained from non-detectable to 0.08
ug/g dry weight. The adverse effects were largely overcome
by adding more molybdenum to the diet. 22 chicks were given
the same amount of tungsten (45 mg/kg) plus 2 mg/kg of
molybdenum. These animals grew at a normal rate, showed
normal values of molybdenum in their tissues, but still
showed mortality, 9% after five weeks. To draw any def-
inite conclusions from this experiment is impossible,
since another element was given in high amounts.
The findings of Higgins, Richert and Westerfeld, 1956,
are partly confirmed by Teekell and Watts, 1959. They
studied the growth response of 1,790 chicks to the diet-
ary supplementation of different levels of molybdenum.
One group of chicks came from dams that received a reg-
ular diet, and another group from dams that received
tungsten in their diet. The latter group showed a stat-
istically significant positive growth response to molyb-
denum supplementation. The result is difficult to evalu-
ate for several reasons, first because of the presence
of another element, tungsten. Secondly, the tungsten
supplement to dams did not, according to a table in this
survey, decrease the tissue level of molybdenum in the
chicks.
51
-------
The tungsten in itself was transferred from the dams
to the chicks. This would indicate that the "negative"
growth response in chicks from tungsten-supplemented
dams is a direct effect of the tungsten rather than an
indirect effect of a decrease of molybdenum.
Tungsten was used by Cohen et al., 1973/ and Johnson,
Rajagopalan and Cohen, 1974, to produce molybdenum defi-
ciency in rats. The activities of the enzymes sulfite
oxidase and xanthine oxidase in liver were reduced to
about 10 percent of the control activity when 100 mg
tungsten per kg of diet was given, but no toxic signs
appeared in the rats. Activity was rapidly restored by
giving molybdenum (Cohen et al., 1973). Johnson, Rajago-
palan and Cohen, 1974, could show that rats with de-
creased activities of sulfite oxidase were less resistant
to bisulfite, the LD5Q being considerably less in molyb-
denum-deficient rats. In such rats, survival times after
inhalation of sulfur dioxide at concentrations of 925
and 2,350 ppm (2,313 and 5,815 mg/m ) were much shorter
than in controls while there was no difference when the
3
exposure was 590 ppm (1,475 mg/m ). It was concluded
that at the higher exposures systemic effects of the
bisulfite derived from the sulfur dioxide occurred, but
that at the lower exposure the effect was a direct effect
on the lungs. In that case, the sulfite oxidase activity
in the lungs did not influence the outcome. Neither bi-
sulfite nor sulfur dioxide induced sulfite oxidase activ-
ity. There was no difference in the 2-week mortality after
4 hours of exposure to sulfur dioxide at concentrations
ranging from 224 to 1,319 ppm between controls and
tungsten-treated animals.
Sheriha, Sirny and Tillman, 1962, fed a synthetic diet
that contained approximately 10 ug/kg of molybdenum to
52
-------
six lambs for 46 days. Three of those animals received
an addition of 2 mg Mo/kg diet supplied as Na^MoO., but
this supplement did not result in higher weight gains
compared to the other three animals. The authors con-
cluded that under the experimental conditions of this
trial, the molybdenum requirement of sheep was less
than 0.01 mg Mo/kg diet.
5.2 THEORETICAL BACKGROUND TO THE QUESTION OF MOLYBDENUM
ESSENTIALITY - ENZYME STUDIES
5.2.1 Xanthine oxidase
Metalloflavoproteins, playing a fundamental role in ani-
mal metabolism through the "electron transport chain",
are a group of enzymes containing both metal and flavin
firmly bound to protein. Molybdenum was found to be a
constituent of the enzyme xanthine oxidase by Westerfeld
and Richert, 1953, and DeRenzo et al., 1953. Earlier
studies by Westerfeld and Richert had revealed evidence
that the levels of liver and intestinal xanthine oxidase
in rats were dependent on some unidentified dietary factor.
It could be supplied by adding liver residue or soy flour
to a purified diet and it was not destroyed by ashing.
In 1953 this factor was isolated and identified as molyb-
denum. Xanthine oxidase from different animal tissues
has later been purified and analyzed, and has been a
subject of several studies (see Bray and Swann, 1972,
for review of work on this enzyme and other molybdenum
enzymes up to 1972).
In vitro studies have revealed a wide range of oxida-
tions that are catalyzed by xanthine oxidase. Its main
biological function is considered to be the oxidation
of two purines, xanthine and hypoxanthine, to uric acid.
In the experiment by Higgins, Richert and Westerfeld,
1956, commented on in 5.1, the molybdenum depletion
53
-------
of rat and chick tissues caused by molybdenum-poor diets
and tungsten supplement was accompanied by a decrease in
xanthine oxidase activity in all tissues analyzed. Com-
parison between group 1 ("ordinary" chick diet) and
group 4 (20 ug Mo/kg and 45 mg WAg) revealed a signifi-
cant, sharp decrease in xanthine oxidase activity and
molybdenum content in the tissues of the latter group
(see Tables 5:1 and 5:2). Further, this depletion was
accompanied by changes in the uric acid excretion: about
one half of the uric acid normally excreted by chicks was
replaced by a mixture of xanthine and hypoxanthine. In a
study by Leach et al., 1962, on chicks, the decrease
of xanthine oxidase activity parallel to growth inhibition
during tungsten-induced molybdenum deficiency was con-
firmed. Added dietary molybdenum reversed the tungsten
effect on xanthine oxidase activity but growth inhibition
was only partially reversed (mortality was not discussed).
Hence the authors suggested that the growth inhibition
was not caused by interference with the activity of this
enzyme. It is difficult to compare this study with the
study by Higgins, Richert and Westerfeld as the dietary
level of molybdenum and the tungsten supplement were
much higher (1.72 mg molybdenum per kg and 500-2,000
mg W per kg).
In vitro studies by Green and Mazur, 1957, demonstrated
how xanthine oxidase, reduced by the oxidation of hypo-
xanthine or xanthine to uric acid, could release iron
from hepatic ferritin. In 1958 the biological role of
this mechanism had been confirmed in in vivo studies
on guinea pigs, rabbits and dogs (Mazur et al., 1958).
The administration of xanthine oxidase substrates
(xanthine and hypoxanthine) to the animal led to an
increase of plasma iron. Further, a reduction of blood
pressure through hemorrhage led to a sharp increase in
54
-------
plasma iron and uric acid in blood samples from the
hepatic vein. According to the authors this is indicat-
ive of an increase in activity of this mechanism, medi-
ated by the physiologic demand for iron at tissue hypoxia.
The ferritin - xanthine oxidase system should be a part
of the homeostatic mechanism for the regulation of iron
in the circulation. For more details in this matter, see
a review by Seelig, 1972.
5.2.2 Other molybdoflavoproteins
5.2.2.1 £3ulfite oxi.dase
The occurrence of molybdenum in this enzyme was first
established as a result of EPR work (electron paramag-
netic resonance) by Cohen, Fridovich and Rajagopalan,
1971. This enzyme catalyzes the oxidation of sulfite to
sulfate which is necessary in the mammalian metabolism
of the sulfur amino acids and related sulfur-containing
compounds. Irreverere et al., 1967, described a case of
possible inherited disease: sulfite oxidase deficiency.
The patient was born with neurological abnormalities and
deteriorated to a decorticate state in nine months. The
patient's urine contained abnormally increased amounts
of S-sulfo L-cysteine, sulfite and thiosulfate, and mar-
kedly reduced amounts of inorganic sulfate. This pattern
would be explained by the presence of a block in the
conversion of sulfite to sulfate, a postulation that was
confirmed by post mortem studies of the patient s tissues.
The authors suggested that the pathologic changes could
have been caused by the toxic effects of accumulation of
sulfite or other metabolites (or deficiency of inorganic
sulfate). Three of the patient's siblings had died in
infancy with neurological disorders, leading the authors
to suspect the disorder of being a hereditary metabolic
defect, "sulfite oxidase deficiency".
55
-------
5.2.2.2 Aldehyde_oxidase_
This enzyme is third of the mammalian molybdoenzymes and
has many properties in common with xanthine oxidase (see
review by Mahler and Green, 1954, Bray and Swann, 1972) .
It is known to catalyze the oxidation specifically of
aldehydes and various nitrogen-containing aromatic hetero-
cyclic compounds (Knox, 1946). In 1964 Rajagopalan and
Handler showed that it is also capable of oxidizing
hypoxanthine but not xanthine. Neither the need for this
enzyme nor its correlation with molybdenum have been
subjected to any studies so far.
5.3 MOLYBDENUM AND DENTAL CARIES
In 1953 Adler and Straub discovered that in certain
communities in Hungary caries incidence was lower than
would have been expected from the fluoride content of
water in these areas. Later studies by Nagy and Polyik,
1955, showed that the drinking water contained unusually
high concentrations of molybdenum, 100 ug/1, suggesting
that this factor caused the reduction in caries. Up to
the year 1967 three epidemiological studies of caries
frequency in man had revealed evidence of a caries
protective effect of molybdenum in the diet (review by
Jenkins, 1967) , in turn confirmed by several animal
studies.
5.3.1 In animals
Adler, 1957, supplemented the drinking water of two
generations of rats with 100 ug Mo/1 given as ammonium
molybdate and found a significantly reduced caries fre-
quency in the second generation (4 out of 21 compared
to 11 out of 22 in a control group). This result was
confirmed by Adler and Porcsalmy, 1961. Van Keen, Ostrom
and Berzinskas, 1967, found an approximately 10% reduction
of caries frequency by the supplementation of 24 mg Mo/kg diet
56
-------
Mo/1 given as sodium molybdate or ammonium molybdate to
rats, i.e., a much weaker effect of a much higher concen-
tration of molybdenum. This experiment, however, was
undertaken with only one generation of rats (21 days old,
duration of experiments 5 weeks) and it is possible that
the positive effect of the molybdenum is mainly active
during the neonatal period and early development of the
teeth.
In 1973, Bowen found no caries preventive effect of molyb-
denum when monkeys were given 2 mg sodium molybdate/1 water
in a 5-year experiment beginning at 11-17 months of age.
The mechanism behind the caries preventive effect of
molybdenum is a subject of controversy, stookey and
Muhler, 1959, 1964, and Stookey, Roberts and Muhler, 1962,
have provided evidence of a synergistic effect of
molybdenum and fluoride in rats and hamsters. Measurements
of the fluoride retention in the carcass and skeleton
of experimental animals showed higher values of fluoride
in the molybdenum-exposed animals, indicating that molybdenum
may act metabolically to increase the "availability"
of the fluoride ion (a substance that has a documented
anti-caries effect).
5.3.2 In human beings
Anderson, 1969, found evidence for the caries preventive
effect in humans that had been postulated by early investi-
gators (see Jenkins, 1967). 682 twelve-year-old children
from a "molybdenum" area (or "teart11 area; see Chapter
6) in Great Britain and from "control" areas were examined
for the presence of dental caries. Further, drinking
water, herbage, milk, urine and extracted teeth were
analyzed for their molybdenum contents. Samples from
the molybdenum area were found to hold slightly but sig-
57
-------
nificantly higher levels of molybdenum compared to the
control areas. Pooled urine samples from the molybdenum
area contained 57 ug/1 compared to 33 ug/1 in the con-
trol area. The dental caries prevalence was said to be
significantly lower in the molybdenum area than in the
control area.
5.4 CONCLUSIONS
Data from enzymatic animal studies provide backing for
the concept of molybdenum's essentiality. The metal is
a constituent of three mammalian metalloflavoproteins,
xanthine oxidase, aldehyde oxidase and sulfite oxidase.
A report of congenital sulfite oxidase deficiency and
its postulated correlation to a deadly syndrome is
indicative of an absolute need for at least this enzyme
(and consequently molybdenum). Animal experiments indicate
that reduction in the activity of sulfite oxidase in-
creases the susceptibility to bisulfite.
Trials to produce a "molybdenum deficiency syndrome"
by feeding animals a diet low in molybdenum have failed
in all instances so far. Assuming an absolute need for
the metal, this is indicative of an extremely low
requirement as it has evidently not been possible to
reduce the dietary molybdenum level to a sufficient
degree. Thus, it has been necessary to introduce an
element with a known property of inhibiting molybdenum
in the trials, tungsten. With this technique it has been
possible to produce an assumed molybdenum deficiency
syndrome in chicks consisting of reduced weight gain and
even death. In rats, no toxic effects were noted, however.
There is evidence of a caries preventive effect of
molybdenum both in animals and human beings.
58
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TABLE 5:1 THE EFFECT ON GROWTH AND TISSUE XANTHINE DEHYDROGENASE ACTIVITIES OF CHICKS FED
DIETS CONTAINING VARYING AMOUNTS OF TUNGSTEN (as Na2W04) AND MOLYBDENUM (as
(From Higgins, Richert and Westerfeld, 1956).
01
vo
2)
Diet , . Additions to Body weight ' (g) Xanthine dehydrogenase activities
group ; diet (ragAg) At 3 wks At 5 wks (ram-3 02/20 min/flask3) )
1
2
3
4
5
6
1
8
9
W
45
45
45
45
94
94
Mo
218
217
0.2 203
169
2 196
20 203
60 186
149
60 177
Intestine Liver
343
311
339
311
339
332
322
274
324
8
8
5
1
7
7
6
1
5
32
32
27
4
34
35
29
3
29
Kidney Pancreas
11
12
11
1
11
17
14
2
12
6
4
5
1
4
5
7
1
5
'Group 1 was fed Park and Pollard starter mash; group 2 was fed the synthetic diet to which no molybdenum
was added, but which contained approximately 0.02 mg/kg Mo.
'Starting weights averaged 40 to 44 g for all groups. Twenty-nine chicks per group for groups 4 and 8;
22 chicks per group for all others.
''Xanthine dehydrogenase values were determined on 8 chicks from each group after 6 weeks on the diets,
using 28.3 mg of liver or kidney and 84.9 nig of intestine or pancreas per flask.
-------
TABLE 5:2 MOLYBDENUM CONTENT (ug Mo/g dry weight) OF CHICKEN TISSUES AFTER SIX WEEKS ON
DIETS CONTAINING VARYING AMOUNTS OF TUNGSTEN AND MOLYBDENUM. (Same diet groups
as in Table 5:1). (Modified from Higgins, Richert and Westerfeld, 1956).
Diet group
Prevent riculus
Intestine and colon
Kidney
Brain
Skeletal muscle
Liver
1
0.
0.
4.
0.
3.
21
51
44
14
56
2
0.
0.
3.
2.
15
24
09
52
3
0.18
0.35
3.48
0.17
0.10
3.35
4
0
0.05
0
0
0.08
0.22
5
0.19
0.76
1 .65
0.05
0.10
2.73
6
0.83
1.11
6.85
0.12
0.44
3.55
7
1.51
1.82
>8.80
0.59
1.07
4.43
8 9
6
>5
14
0
0
0.20 4
.79
.75
.45
.82
.77
.11
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CHAPTER 6 TOXICITY
6.1 "TEART" DISEASE - MOLYBDENUM POISONING IN RUMINANTS
Since the middle of the 19th century a bovine disease
named "teart" has been known. The disease affects only
ruminants of special pastures and is mainly characterized
by diarrhea and concomitant emaciation. If left too long
on teart land, the cattle may even die, but a very rapid
recovery upon removal to non-teart pastures is typical.
The illness is described as follows: diarrhea may start
within 24 hours of putting the cattle in the pasture.
The dung becomes extremely loose and watery. The animals
lose condition rapidly, their coats becoming rough and
lustreless and changing to a more greyish tone.
Sheep are affected to a certain degree, but horses and
swine are resistant. The illness found its etiology
in 1937 through spectrographic analyses of "teart" herbage
from Somerset in England (Ferguson, Lewis and Watson,
1938) , this herbage containing considerably higher
amounts of molybdenum than herbage from non-teart fields
close-by. The same authors presented a detailed study
on the "teart" pastures of Somerset in 1943, and the
data are reviewed by Ferguson, 1944.
The areas in question are in central Somerset, north
Somerset, Gloucester and Warwick. The degree of teartness
varies from field to field, from season to season and
is proportional to the molybdenum content of herbage.
From Table 6:1 it can be stated that a diffuse "borderline"
should lie somewhere around 12 mg Mo/kg in dry matter.
The molybdenum contents vary widely from 4 mgAg to
59 mg/kg in different fields, at the same time of the
year, and the seasonal variation at the same farm is
also marked. Table 6:2 shows that "teart" is most pronounced
61
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in autumn, after which the "teartness" decreases, the
winter hay containing the lowest levels of molybdenum.
Though containing not insignificant concentrations of
molybdenum, winter hay rarely produces "teartness".
This, according to the authors, stems from a lower pro-
portion of water soluble molybdenum in hay.
The authors also tested the postulation that molybdenum is
the etiologic factor by feeding molybdic salts to some
animals, thus producing the same symptomatic picture (see
6.3) .
In 1946 "teart" was also reported from Kern County in
California by Britton and Goss. These authors described
the same symptoms as those from Somerset, but in addition,
a hypochromic microcytic anemia. The average morbidity
in the cattle was about 80%. Analyses of alfalfa from
the area in question revealed a molybdenum content of
10.3 mg/kg, i.e. only at Ferguson, Lewis and Watson's
"borderline". Ferguson, Lewis and Watson compared the"teart"
of Somerset with copper deficiency in cattle reported by
Brouwer et al., 1938. The resemblance between the two
illnesses made Ferguson, Lewis and Watson test copper as
an antidote to "teart". This was quite successful, "teart"
being completely cured by the addition of copper salts
(1-2 g of copper sulfate/animal) to the diet. Thus, the
etiology of the chronic molybdenum poisoning in cattle
could be said to be more a matter of the balance in the
dietary molybdenum-copper composition than a matter of
the dietary molybdenum level per se. Accordingly, the
relatively lower molybdenum content found in the Britton
and Goss study might be explained by a low copper content
in the herbage.
Hogan et al., 1971, reported on sheep from New Zealand.
The animals were grazed from April to October on pastures
62
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that contained about 5-7 mg/kg dry natter of copper, and
5-8 mg Mo per kg in the beginning of the trial period.
During August the molybdenum content rose rapidly to
approximately 20 mg/kg. (The authors supplemented the
diet of certain groups with copper and selenium and com-
pared these groups with "controls". Selenium plays a
role in copper metabolism, but did not give any remarkable
effects in this study). The animals demonstrated symptoms
that were not described in cattle in the foregoing stud-
ies . Besides diarrhea and anemia, some animals were suf-
fering from lameness. Out of 9 animals grazing on past-
ures with high levels of molybdenum, 4 were affected
(this was noted in September) and were killed and autop-
sied. They showed lesions concerning the skeleton-muscle
system: rib, humerus, and femur fractures, muscle inser-
tions having been stripped off the bone together with
periost, and hemorrhages of periost and muscle (see
6.3.3.4).
6.2 IN HUMAN BEINGS
6.2.1 Effects on lungs
Mogilevskaja, 1963, reported 3 cases of pneumoconiosis
out of 19 workers exposed to metallic molybdenum and its
oxide. A 44-year-old woman working in a molybdenum re-
ducing shop, exposed for 5 years at concentrations in
the range of 1-3 mg/m and a 44-year-old man exposed
for 4 years at concentrations fluctuating between 6-19
mg/m , showed early signs of pneumoconiosis on X-ray
examination. The woman complained over difficulties in
breathing, and general weakness, the man over dry cough.
A 34-year-old man exposed for 7 years to concentrations
between 6 and 19 mg/m showed fully developed pneumoconi-
osis (multilayer shadows of a nodular nature and slight
emphysema). This man complained over difficulties in
breathing, pain in the chest and expectoration, parti-
cularly in the morning. The dust contained different
63
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proportions of the metal and its oxide. 90% of the par-
ticles were below 5 u.
6.2.2 Hyperuricemia
Akopajan, 1964a, examined 73 workers in a copper-molybdenum
plant and 10 control subjects. The highest levels of uric
acid in blood were observed in miners who were the most
exposed group. The increased blood uric acid was found in
34 out of 37 workers who complained of arthralgia. The
results are difficult to evaluate because the author does
not report the values of uric acid in blood; the exposure
is not defined either. Avakajan, 1966a, also described
hyperuricemia while studying 85 workers in a copper-molyb-
denum plant in Kadzaran. He further noted an increase in
the concentrations of bilirubin, globulins and cholesterol
in blood but no data were given in this study.
x)
Kovalskii, Yarovaya and Shmavonyan, 1961/and Yarovaya,
1964, reported on a high incidence of gout in an area
of Armenia having 77 mg MoAg and 39 mg Cu/kg in the soil.
Kovalskii, Yarovaya and Shmavonyan, 1961, on the basis of
molybdenum levels in different food products, calculated
the total molybdenum and copper intake for an adult man
in this area compared to a man in a control area (10-15
mg molybdenum and 5-10 mg copper, versus 1-2 mg molybdenum
and 10-15 mg copper respectively). The concentrations
of molybdenum and copper in food products and in drinking
water in the molybdenum-rich area were compared with
those in the control area. In this region of the U.S.S.R.,
more than 50% of the diet is based on locally grown prod-
ucts. Selected values for molybdenum and copper concen-
trations in food are given in Table 6:3. As is seen through-
out the table, the values from the molybdenum-rich area
as regard molybdenum are considerably higher. It is not
known however why the values in the control area are
_ _
The treatment of this report is based upon our access
to a complete translation made by GSran Persbagen, M.B.
at the Department of Environmental Hygiene of the Karolinska
Institute.
64
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higher than usually seen in "normal" areas (see 3.3.7).
A medical survey of 400 subjects from two villages in the
molybdenum-rich area of which 262 persons were 18 years
or older revealed a prevalence of symptoms similar to
gout in 31% from one of the villages and 18% from the
other, these percentages pertaining to the adults only
and making a total of 71 persons. The authors claimed
that similar symptoms normally occurred in 1-4% of the
population of the U.S.S.R. The symptoms were characterized
as arthralgia in the knee-joints, hands and feet. Joint
deformities were also reported. Symptoms from the gastro-
intestinal tract, liver and kidney were said to be common-
ly observed. The description of the symptoms is not given
in such a way as to allow a judgement as to whether they
are actually identical with symptoms of gout. Concerning
the symptoms from the gastrointestinal tract, liver and
kidney, no details whatsoever are furnished.
A more detailed study was performed on 52 subjects in
the molybdenum-rich province and on 5 subjects from a
control area. In Table 6:4 the uric acid values in blood
and urine from the 52 examined subjects, some with and
some without symptoms of gout, are presented, along with
the values of the 5 control subjects.
In the paper there are no data whatsoever regarding
how the exposed and control subjects were chosen nor
were any detailed data on age distribution given. Another
point of difficulty is that only 5 controls were included,
but it should be noted that their values agree with what
is considered "normal" (Documenta Geigy, 1970). In spite
of the difficulties in interpreting any bias in connection
with the selection of the study groups, the data presented
seem to show an increased uric acid level in blood and
urine in the subjects living in the molybdenum-rich area.
65
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Concerning concentrations of molybdenum in blood and
urine, data from Table 6:5 reflect the excessive expo-
sure to molybdenum inasmuch as the levels of molybdenum
are considerably higher in the molybdenum-rich province
than in the control area. The same reservations apply
concerning the selection of subjects as were discussed
in connection with Table 6:4.
6.2.3 Other effects
Eolajan, 1965, examined 500 workers in a molybdenum mine
and plant and a control group of equal size (general popu-
lation of the same area). The majority of workers were
miners in copper-molybdenum mines (both underground and
surface). According to the information of a previous study,
dust levels exceeded, on the average, the MPCXJ value 10-
100 times (MFC for metallic Mo and insoluble Mo compounds
is 6 mg/m3 in the U.S.S.R.). (Note: the composition of dust
is not given). A large percentage of workers had various
unspecific symptoms and signs including general weakness,
fatigue, headache, irritability, lack of appetite, epi-
gastric pain, pains in joints and muscles, increasing
loss of weight, red and moist skin, tremor of the hands,
sweating and dizziness. On the basis of a neurological
analysis of these symptoms and signs, the author con-
cluded that a long-term exposure at the levels mentioned
results in the impairment of the central nervous system.
6.3 IN ANIMAL EXPERIMENTS
6.3.1 Single exposure
6.3.1.1 Injection
Maresh, Lustok and Cohen, 1940, studied the acute toxicity
of intraperitoneal injections of sodium molybdate. The
authors gave 20 rats, weighing from 114 to 260 grams, from
45 to 350 mg Mo/kg body weight. Ten rats receiving 114 mg
_
;MPC = Maximum Permissible Concentration
66
-------
Mo/kg and below showed a few transitory symptoms and re-
mained well. The 10 rats receiving from 117 mg Mo/kg and
above died within a few hours .
Fairhall et al., 1945, studied the acute toxicity in guinea
pigs following intraperitoneal injections of molybdenum
compounds. They followed the animals 4 months after the
injection and noted the mortality rate (see Table 6:6) .
In 12 animals receiving 800 mg ammonium molybdate per
kg, mortality was 100% (probably within a few hours)
whereas those receiving 80 mg/kg underwent no mortality
at all. 75%, receiving 400 mg molybdenum trioxide/kg,
died within 4 days, and the majority of those within 2
hours . The injection of calcium molybdate and molybdenite
resulted in a much lower mortality.
In experiments on cats, Cilingarajan, 1965, found that a
single i.v. injection of 25 mg/kg of sodium molybdate
(Na2MoO. • 2H2O) produced a moderate increase in arterial
blood pressure (measured in the carotid artery) , and a
stable prolonged increase in the tonus of the nictitating
membrane. The pressor effect of noradrenaline was poten-
tiated. Introduction of acetylcholine was followed by
hypotension. A dose of 50 mg/kg reduced the pressor effect of
noradrenaline significantly; the nictitating membrane
contracted on stimulation of the preganglionic fibers
of the cervical nerve.
6.3.1.2 _
Mogilevskaja, 1963, exposed 45 rats for one hour to the
dusts of metallic molybdenum (25-30 g/m ) , molybdenum
trioxide (12-15 g/m3) , molybdenum dioxide (10-12 g/m )
and ammonium paramolybdate (3-5 g/m ) . The distribution
and particle size are seen in Table 6:7. With the exception
of some irritation of the upper respiratory passages, the
67
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author could find no adverse effects upon the appearance
of the animals, which were followed 4 weeks after admin-
istration.
In another experiment rats were exposed for 2 hours to an
aerosol of molybdenum trioxide (concentrations of the dust
being below 0.064 g/m3). The animals showed no signs of
poisoning, and increased in weight parallel to controls
for 2 weeks after administration. Microscopical examina-
tion, however, revealed evidence of "dystrophic processes"
in the heart, liver and kidneys, and "necrotic processes"
and "signs of regeneration" in the liver.
In a third series of experiments, rats were given a single
intratracheal dose of a 50-mg suspension of the dust of
metallic molybdenum (17 rats) or molybdenum trioxide (8
rats). 85-90% of the particles had a diameter of less than
2 u. In the case of animals exposed to molybdenum dust
a progressive interstitial process and a formation of dust
granulomata were seen after two months. There were collagen
fibers in the thickened intraalveolar septa as well as in
the granulomata. The changes progressed and gave rise to
peribronchial and perivascular fibrosis. At the sites of
accumulation of dust, "whole fields of collagen fibers
and emphysema" were found. Molybdenum trioxide also gave
rise to interstitial fibrosis, but here the most marked
changes were observed in the regional lymph nodes (pro-
liferative processes and marked fibrosis),.
Dzukaev, 1970, studied the effects of the intratracheal
administration of powdered (metallic) molybdenum on rabbits.
32 animals were given 1.5-2.0 ml of molybdenum powder sus-
pended in a physiological solution (70-80 mg/kg body weight;
particle size: 36% <2 urn; 47% <5 umj 17% >5 urn). Two
animals died from pneumonia within 10 days. The remaining
68
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30 animals were killed after 3, 6 and 9 months respectively.
The author stated, "In 9 months, histological examinations
showed slight diffuse pneumoconiosis with interstitial
pneumonia." The author does not give a more detailed account
of what number of animals was used in each group or what
the prevalence of pathological findings was.
6.3.3 Repeated exposure
6.3.3.1 Oral_admi.nistratipn/_general_a£pects_
6.3.3.1.1 Ex£eriments with_c£mmon 3:aboratory_animal.s_
Fairhall et al.f 1945, studied the toxicity of molybdenite,
molybdenum trioxide, calcium molybdate and ammonium molyb-
date on white rats by feeding the animals from 10-500
mg/animal/day for a varying number of days up to 232 days.
No signs of poisoning appeared in any of the groups in-
gesting molybdenite while all groups receiving hexavalent
molybdenum,were affected: loss of appetite, loss of weight,
"they became quiet and listless and their furs harsh and
rough". Daily doses in excess of 100 mg were uniformly
fatal. Moreover, the 10 mg groups showed a mortality vary-
ing from 25-50% during the test period (see Table 6:8) .
The approximate LD5Q of daily repeated doses in mg of
molybdenum per kg per day for molybdenum trioxide was
given as 125, for calcium molybdate 100 and ammonium molyb-
date 333. These figures only serve as a comparative toxi-
city between the three compounds, as the "LD50" in re-
peated exposure is a vague term.
Neilands, Strong and Elvehjem, 1948, placed 21-day-old rats
in groups of 4 on a diet containing 500, 1,000 or 5,000
mg MoAg as sodium molybdate. All four rats receiving 5,000
mg Mo/kg died shortly after the first week. All groups
showed growth retardation (see Figure 6:1) and emaciation,
but the authors negated any other effects such as diarrhea
(see 6.1) or changes in blood picture (see 6.3.3.3).
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Growth retardation was also reported by Gray and Daniel,
1954, who fed 5 rats 800 mg Mo/kg in the diet given as
sodium molybdate, this level being insufficient to cause any
mortality within an experimental period of 6 weeks. Mean
increase of weight for the five rats within this period was
94 g compared to 152 g for a control group.
Williams and Van Reen, 1956, fed groups of 6 rats 800 mg
Mo/kg diet, 1,200 mg/kg and 1,400 mgAg/ respectively,
given as sodium molybdate. After 5 weeks a control group
had an average weight of 218 g, whereas the experimental
groups, in order according to dose, weighed 171, 121 and
87 g. Many of the rats developed an intermittent diarrhea,
but there was no mortality. The authors related the poor
weight gain to reduced food intake, but excluded the pos-
sibility of palatability as a cause of the depressed intake
as 5 rats given sodium molybdate by stomach tube also showed
poor food intake.
Ostrom, Van Reen and Miller, 1961, fed rats 400 mg Mo/kg
diet as sodium molybdate for 5 weeks. The authors con-
firmed the growth depression described by earlier investi-
gators, but also found anemia and mandibular exostoses.
These symptoms were highly correlated with the growth
depression, i.e. all three symptoms tended to appear in
those rats that were most affected. Thus, out of 20 rats,
10 rats showed the toxic signs, whereas 10 rats were
relatively unaffected. The hemoglobin values (g/100 ml)
of 7 rats with exostoses on an average were depressed
from 13.8 to 8.4 while 8 rats without exostoses showed
normal hemoglobin values, 13.2 on the average.
In order to find out whether the growth depression upon
molybdenum intake is proportionally correlated to a
reduced food intake or if some other metabolic process
is causing the disorder, Arrington, Ammerman and Moore,
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1965, pair-fed rats for 4 weeks. Each pair consisted
of 1 rat receiving 500 or 1,000 mg Mo/kg diet given as
sodium molybdate, and 1 control. The intake of the pair-
control was limited to the reduced intake by the pair-
mate. Still, the weight gains of the "molybdenum rats"
were significantly lower than their pair-mates in both
the 500 mg/kg and the 1,000 mgAg groups. This effect
was also studied by measuring the food intake of ad
libitum fed rats for 6 weeks using the term "feed per
unit gain". The corresponding figures for controls,
500 mg/kg and 1,000 mg/kg were 4.7, 5.1 and 8.6 respect-
ively, the latter experimental group value being signi-
ficantly different from controls. The matter of reduced
feed efficiency is confirmed on rats by Gray and Daniel,
1954, already referred to in this chapter.
Greseva and Iliev, 1973, fed 4 groups of 36 white rats
(body weight 100 - 10 g) for 17 weeks with diets con-
taining naturally occurring molybdenum bound in peas,
or added molybdenum as Mo03. The total dose received
was 2,365 ug (controls, normal concentration of Mo in
peas); 24,487 ug (peas + Mo03); 11,550 ug (peas with
naturally high content of Mo - grown on Mo-rich soil);
and 24,497 ug (normal peas + MoO.J . The mortality in-
creased from Group I to Group IV as follows: 14.2%,
13.4%, 34.6%, 57.2%.
Three groups of 8 guinea pigs in each were given 25,
100 and 200 mg molybdenum per animal per day of either
molybdenum trioxide or calcium molybdate (Fairhall et
al., 1945). The compounds were administered in a 10%
gum arabic solution into the back of the animals'
throats by means of a syringe. The oxide proved most
toxic, 25 mg molybdenum per day leading to a 75% mor-
tality, and both the 100 mg and 200 mg doses leading
to 100% mortality. The corresponding numbers of days
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on test were 99 days, 27 days and 6 days respectively.
Calcium molybdate gave a 12.5% mortality at 25 mg molyb-
denum within 95 days and 25% at both 100 mg and 200 mg
molybdenum per animal per day, also within 95 days. Tox-
ic ity symptoms were not described.
Arrington and Davis> 1953, fed sodium molybdate to 31
(22 weanlings and 9 adults) rabbits. Molybdenum was
added to a commercial ration (containing 2.7 mg Mo/kg
and 16.4 mg Mo/kg of copper) providing 140, 500, 1,000,
2,000 and 4,000 mg Mo/kg diet. Growth data were collected
from weekly weights during 12 weeks of 22 weanling rabbits
placed on the experiment at 6 weeks of age. Other symp-
toms as anemia and skeletal deformities were also stud-
ied, and the results are given in Table 6:9.
Rabbits fed 1,000 mg/kg diet or more developed a toxic
syndrome, which was characterized by anorexia, loss of
weight, alopecia, slight dermatosis, anemia and death.
In some young rabbits an abnormality of the .front legs
developed. The syndrome that started to develop within
4 weeks for younger rabbits, and took a "somewhat longer
time for mature animals", had a tendency to become worse
until the animal died or was given copper therapy. Growth
was retarded only in those rabbits which developed other
symptoms of toxicity. At the two higher doses the animals
lost weight instead of gaining. Hemoglobin values were
depressed to between 3.2 g/100 ml and 8.0 g/100 ml com-
pared to controls between 12.0 g/100 ml and 15.0 g/100
ml and the anemia also appeared together with the other
symptoms.
McCarter, Riddell and Robinson, 1962, induced molyb-
denosis in 52 rabbits by feeding an oats-alfalfa diet,
containing approximately 2,000 mg of sodium molybdate
per kg diet. All animals were affected; by the 5th week
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mortality was 65%. Epiphyseal line fractures appeared in
some animals after only 12 days. Hemoglobin was decreased
to approximately 65% and of the 34 animals that died, 33
had lost 4-41% of their body weight. These authors also
noted diarrhea in some test animals.
Arrington, Ammerman and Moore, 1965, also studied feed
efficiency in rabbits, and used the same method as
already described for rats. The rabbits consumed less
feed and gained less than controls, but in this species
feed efficiency was not affected. 1,000 mg Mo/kg diet
did not significantly reduce intake or gain in this study.
Rats consume proportionally larger amounts of food than
rabbits, and the authors suggested this fact to be at
least a part of the explanation for the species differ-
ences. (Already 500 mg Mo/kg diet resulted in decreased
feed efficiency in rats).
Probst, 1971, negated noxious effects in broilers at
the supply of up to 6.95 mg Mo/kg feed given as ammonium
molybdate. The author is actually recommending an addition
of 5-5.5 mg molybdenum/kg feed to broilers as this level
improves weight gain and feed efficiency in the animals.
Davies et al., 1960, tested from 100 to 8,000 mg Mo/kg
given as sodium molybdate in the diet of chicks. Each
group contained from 18 to 20 birds. A slightly decreased
weight gain started to appear first at 500 mg/kg diet,
but became more severe at higher doses and at 8,000 mg/kg,
the 4 week weight was only 1:6 of that of the controls.
Mortality was inconsistent at levels over 500 mg/kg (some
of the groups had a slight mortality that was not correlated
to the dose-level), but 6,000 and 8,000 mg/kg gave rise
to a mortality of 33 and 61% respectively- Hemoglobin was
slightly increased at levels up to 2,000 mg/kg, but still
higher levels produced anemia.
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Lepore and Miller, 1965, tested the egg production and
embryonic viability in hens after the supplement of 0,
500, 1,000 or 2,000 mg Mo/kg diet added as sodium molyb-
date. Each group contained 6 animals. The animals laid
15% fewer eggs than controls when fed a diet containing
500 mg Mo/kg. The decreases at 1,000 and 2,000 mg MoAg
were 50% and 80% respectively. The feeding of 500 mg
Mo/kg diet resulted in an egg concentration of 16-20 mg
Mo/kg, this concentration leading to the death of all
embryos.
6.3.3.1.2 Ex£eriment£ with_ca11le,_tria 1 s_ to_produce
^artific^ial^ teartness_"
As already commented upon in 6.1, Ferguson, Lewis and
Watson, 1943, tested whether molybdenum could be
a cause of "teart" by feeding molybdic salts to exper-
imental animals. Serious diarrhea in three out of four
cows arose in 5-10 days on non-teart pastures, by feeding
the animals 1,710 mg of sodium molybdate daily. This dose
contained 680 mg of molybdenum, according to the authors
corresponding to 50 mg Mo/kg in the dry matter of a normal
daily ration on pasture. These cows were brought up on
"teart-land" which, according to the authors, makes ani-
mals weaker and more susceptible to molybdenum poisoning.
Cattle on winter rations, and cattle brought up on sound
land required higher doses (approximately 200 mg/kg dry
matter) and some animals did not react even to these
doses.
Britton and Goss, 1946, referred to in 6.1, gave 5,000
mg sodium molybdate daily to a heifer calf (6 months old)
for 7 months. In 6 weeks the animal was thin, rough-
coated and grey in color. The animal also became in-
creasingly emaciated. At the end of the trial the weight
was 355 pounds, compared to a control calf weighing 490
pounds. In spite of the demonstrated toxic signs, the
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authors did not note any diarrhea or anemia. A month after
the trial, the test calf was recovering rapidly.
Cook et al., 1966, treated 24 yearling steers with 0
mg (control), 1.5 mg (low level) and 3.0 mg (high level)
of molybdenum per kg body weight daily. The weight of
the animals was approximately 264 kg, and molybdenum
was administered as sodium molybdate in gelatine cap-
sules . The total daily amount of molybdenum would be,
according to these figures, approximately 400 and 800
mg respectively, i.e. close to the amounts tested by
Ferguson, Lewis and Watson, 1943. The experiment was
planned for 150 days, but after 100 days three animals
receiving the high level of molybdenum had died, making
the authors reduce the daily intake to 1.5 mg per kg body
weight for the remaining test animals for the final 50
days. Profuse diarrhea occurred within 2 weeks in most
animals and was most pronounced in the high level animals.
At 100 days the control animals had gained an average of
81 kg, whereas the low and high level groups lost 24 and
69 kg respectively. These authors also noted anemia in
several animals.
Huber, Price and Engel, 1971, studied molybdenum toxicity
in lactating dairy cows fed a basal ration containing
6 mg Cu/kg. Overt symptoms of molybdenum toxicity (diar-
rhea, "inanition" etc) were observed in three cows con-
suming a diet with 173-200 mg Mo/kg given as sodium molyb-
date. A diet containing 53-100 mg Mo/kg (trial period 6
months) failed to produce any symptoms in this study.
6.3.3.2 Re£eated_exposure through inha^aj^ion^ common
syrap_toms_and_ef_fects_on lung£
Groups of guinea pigs were exposed to the dusts (particle
size not given) of molybdenite, calcium molybdate and
molybdenum trioxide respectively one hour per day 5 times
75
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per week (Fairhall et al., 1945, as referred to in 6.3.1
and 6.3.3.1) . Twenty-five guinea pigs were exposed to molyb-
denite dust (average molybdenum content 286 mg/m ). With
the exception of one animal, dying after only 3 days, the
appearance of these animals was normal (tissues levels
were extremely low, see section 4.3). Fifty-one animals
were exposed to an average of 205 mg Mo/m as molybdenum
trioxide. This exposure led to loss of appetite, loss of
weight, diarrhea, muscular incoordination, loss of hair
and a mortality rate of 50% during the experimental period.
Upon histopathological examination the lungs showed a small
to a moderate amount of alveolar and bronchial exsudate,
but no other significant findings. The calcium molybdate
dust was administered to 24 animals in an average concentra-
tion of 160 mg Mo/m and led to a 20% mortality but "no
other toxic signs" were evident. The same model of admin-
istration was used in another experment, exposing 12
guinea pigs to the fume of molybdenum trioxide 5 days/week
for 5 weeks. Two levels of concentrations were used, 191
and 53 mg Mo/m . Only one animal given the high concentra-
tion died, and no further evidence of toxicity was apparent.
As the fume generally is more toxic than the dust a more
pronounced toxic effect had been anticipated.
Mogilevskaja, 1963, exposed rats to dusts of metallic
3 3
molybdenum, 12-15 g/m , molybdenum dioxide 8-10 g/m ,
molybdenum trioxide 8-10 g/m and ammonium paramolybdate
0.5-2.5 g/m one hour daily for 30 days. With the exception
of one death and slight growth depression the rats exposed
to metallic molybdenum and molybdenum dioxide appeared
normal during the exposure period. In lungs there was a
considerable deposit of dust with thickening of the intra-
alveolar septa, which contained connective tissue fibers.
The toxic effects of molybdenum trioxide were, however,
more pronounced. Weight gain in this group was only 8.6
g on the average, compared to 31 g in a control group. The
76
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lungs showed macroscopical hemorrhages, marked perivas-
cular edema, and large areas of hemorrhages into the
alveolar spaces. Mortality rate was not mentioned. The
exposure to ammonium paramolybdate showed the most marked
toxic effects, a 100% mortality and more serious effects
of the same type as described for molybdenum dioxide.
In another experiment by Mogilevskaja, 1963, rats were
exposed to a condensation aerosol of molybdenum trioxide
for 2 hours "on alternate days" for 2 months at a concen-
tration of 3-10 mg/m . 90% of the particles were smaller
than 1 u. During this entire period, no changes in the
condition of the animals were noted. The lungs showed
thickening of the alveolar walls, interstitial pneumonia
and areas of collapse and emphysema.
Dzukaev, 1970, demonstrated a "slight diffuse pneumoconiosis"
in rabbits 9 months after a single intratracheal administra-
tion of powdered molybdenum at 70-80 rag/kg. When 40
mg of molybdenum were administered a second time 6 months
later, note was made of an intensified inflammatory process
in the interstitial tissue resulting in a "vague pneumoscler-
osis and symptoms of focal pneumonia without the formation
of cellular-fibros foci".The changes were not visible by
radiography in vivo but could be demonstrated well by radio-
graphy on isolated lungs.
Lukasev, Siskova and Knys, 1971, exposed rabbits and rats
(the number is not stated) for 3.5 months, 4 hours daily, to
molybdenum trioxide dust by inhalation. The concentration
changed cyclically from 210 to 10 mg/m within 25 minutes.
The animals were divided into 3 groups (the size of groups
is not given): 1) controls; 2) exposed to molybdenum trioxide
and 3) exposed to molybdenum trioxide and also given an oral
dose of sodium sulfate solution (50 mg of sulfate per rabbit
and 5 mg per rat). On autopsy, the animals exposed only to
77
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molybdenum trioxide showed point hemorrhages in the lungs
and in some cases polyemia. There were no changes in other
organs. Microscopic examination revealed extensive incid-
ence of hemorrhages, destruction of alveoli, emphysema,
interstitial pneumonia and bronchitis. Rabbits which were
treated with sodium sulfate showed interstitial pneumonia
and bronchitis but no hemorrhages. In rats exposed only
to molybdenum trioxide, there were no hemorrhages but
interstitial pneumonia and bronchitis were well developed.
Interstitial pneumonia and bronchitis were less well dev-
eloped in rats treated with sulfate. The authors did not
give any information on mortality or the prevalence of
pathological changes. Collagen fibers were not found
in either rabbits or rats. No changes were observed in
the content of mucopolysacharides as compared with con-
trol animals.
6.3.3.3 Effects^ on_blood_
As is already described in 6.1, 6.3.3.1 and 6.3.3.2,
anemia is a common constituent of the molybdenum toxicity
syndrome in many species (cattle, sheep, rabbits, guinea
pigs, rats, chicks).
Britton and Goss, 1946, referred to in 6.1 and 6.3.3.1,
characterized the anemia appearing with "teart" disease in
cattle as hypochromic and microcytic with a high lymphocyte
and platelet count. Analysis of alfalfa from the area in
question revealed a molybdenum content of 10 mg/kg.
Cunningham and Hogan, 1959, did not find hematological
effects in sheep grazing on pastures providing 8 mg Mo/kg
diet and 7 mg Cu/kg diet. Hogan et al., 1971, referred to
in 6.1, in a study on sheep on pastures high in molybdenum
(up to 20 mg/kg diet) reported, however, "significantly lower
hemoglobin and hematocrit levels in those animals not receiving
78
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copper as an antidote" but prevalence and actual values
were not given.
Arrington and Davis, 1953, produced toxic symptoms in rabbits
by feeding the animals sodium molybdate in doses from 1,000
mg Mo/kg diet and higher (see Table 6:10 which presents some
"representative animals"). The first changes in hemoglobin
and the number of red blood cells were observed at the same
time as other evidence of toxicity appeared.
Anemia in rabbits has also been reported by McCarter, Riddel1
and Robinson, 1962. They fed the animals 2,000 or 2,340 mg
sodium molybdate/kg feed for 5 weeks Hemoglobin and heraatocrit
was decreased to approximately 65 and 60% respectively. The
anemia was characterized by the authors as a microcytic,
hypochromic anemia, but detailed values are not given.
Molybdenosis was produced in 12 young rabbits by giving
them 4,000 mg/kg as sodium molybdate in the diet for 4
weeks (Valli et al., 1969). Decreases in hemoglobin, hema-
tocrit, but not in hemoglobin saturation were apparent af-
ter 11 days and were marked at the 18th day. The authors
further described a marked erythroid hyperplasia of bone
marrow (while the myeloid series was not so affected)
developing in all test rabbits by the llth day. In this
experiment the prevalence of anemia was 100%, but neither
hemoglobin nor hematocrit values were given.
Ostrom, Van Reen and Miller, 1961, referred to in 6.3.3.1,
reported on anemia in 21-day-old rats fed 400 mg Mo/kg
diet as sodium molybdate. Hemoglobin and hematocrit values
were on an average significantly and markedly reduced in
those rats that were also affected by exostoses (8.4 com-
pared to 13.8 for controls, and 29.2 compared to 43.7
as mean values respectively). MCHC (mean corpuscular
hemoglobin concentration in percent) was slightly de-
79
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pressed, 29.5 compared to 31.5. White blood cells were not
affected.
In an experiment by Gray and Daniel, 1954, hemoglobin
values were only slightly depressed by the feeding of
800 ing Mo/kg diet as sodium molybdate to 21-day-old
rats. The contemporaneous weight gain was less in the
exposed group than in the control group. Thus, in 6 weeks
the mean (5 animals) weight gain was 94 g and the mean
hemoglobin value 14.28 g/100 ml while the corresponding
values for a control group were 152 g and 14.49 g/100 ml.
As the authors used the same molybdic salt and the same
age of animals as in Ostrom, Van Reen and Miller's study,
the difference in results is somewhat surprising. The
difference was only affecting the blood picture as weight
depression and mandibular exostoses were reported by both
author groups. In Ostrom, Van Reen and Miller's study,
albino rats of the NNRI-B strain were fed a "purified
diet" while in the Gray and Daniel study rats of the
Sprague-Dawley strain were fed a "basal diet" supplemented
with copper. Either difference in strains or in dietary
levels of copper "or other dietary constituents" should be
responsible for this difference in result.
The existence of molybdenosis in rats without the contem-
poraneous existence of anemia is also described by Neilands,
Strong and Elvehjem, 1948, in Sprague-Dawley rats on a
^urified ration which received 500, 1,000 or 5,000 mg Mo/kg
.iet as sodium molybdate. At all levels, depressed growth
and emaciation were encountered but the blood picture was
normal (hemoglobin, hematocrit, red blood cells and white
blood cells). Sprague-Dawley rats also failed to develop
anemia in an experiment by Johnson, Little and Bickley, 1969
The animals were fed up to 1,200 mg Mo/kg diet as sodium
molybdate. The authors concluded that the hematological
system of this rat strain is likely to be resistent to
molybdenum intoxication.
80
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The study by Davies et al., I960, on chicks indicates a
strong resistance of this species to high dietary levels
of molybdenum. First at 4,000-6,000 mg Mo/kg diet were
changes in blood pictures apparent.
6.3.3.4 E ffec_ts_ on_bone and £onn£ctive_ti:S£ue
Osteolathyrism is a disease caused by ingestion of seeds
from the sweet pea (Lathyrus odoratus). The disease can
easily be experimentally reproduced by feeding the sweet
pea or its toxic compound 0-aminopropionitrile (BAPN),
to animals. Its histological features are recaptured by
Gardner, Dasler and Weinmann, 1958, and it is evident
that the disease has many characteristics in common with
the bone disorders in molybdenosis (disorders of epiphysis
of long bones, periostal new bone formation at the sites
of muscle attachments, loosening and detachment of tendonous
insertions, tearing of the periostem etc.). It also, to
some extent, resembles certain disorders in the bone tissue
of man (osteogenesis imperfecta, osteopetrosis, Pagets
disease, Perthes disease, Osgopd-Schlatter disease).
Hogan et al., 1971, reported on connective tissue lesions
in 4 out of 9 sheep on high molybdenum pastures (up to
20 mg/kg dry matter, corresponding copper values were
5-7 mg/kg ppm dry matter) . One animal showed rib fractures
and a transverse fracture of the humerus. Another one dem-
onstrated "stripping" of 3 muscle insertions together with
periost of the right humerus. A third demonstrated the
same type of lesions on both humeri plus a fracture of the
left femur. The fourth of the affected animals exhibited
"stripping" at the same site as the secondly mentioned
sheep.
Ostrom, Van Keen and Miller, 1961, demonstrated mandibular
exostoses in 10 rats out of 20 fed 400 mg Mo/kg diet per
day as sodium molybdate. At the start of the experiment,
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the animals were 31 days of age, and the duration of the
experiment was 5 weeks. The exostoses were situated at
insertion sites of muscles and were according to the
authors histologically resembling "osteolathyrism".
Lalich, Groupner and Jolin, 1965, fed rats 1,000,
1,500 and 2,000 mg of sodium molybdate per kg feed. The
authors used young animals, 39-45 g, and checked the body
weight every other day. If the rats stopped eating and
lost weight, the diet was supplemented with 0.5 mg of
copper sulfate per day for 1-3 days, but the experiment was
continued for 6 weeks whereafter the animals were killed
and autopsied. Twenty-seven of 33 rats developed deformi-
ties of the long bones, and femur, tibia and humerus were
equally affected. The changes consisted of shortening of
bones and increase of shaft diameter, enlargement of fe-
moral condyles, and the tibial head, and gross deformities
of knee joints. Several rats demonstrated palpable mandi-
bular exostoses. At microscopical examination it was found
that the greatest deformities occurred at the ends of the
long bones, particularly the epiphyseal plates (widening
of the plates and distortion of the chondroblastic alignment)
In contrast to the alterations observed in the epiphyseal
plates, only relatively minor changes were encountered in
the trabeculae, and cortical bone. Some rats were given
copper sulfate or copper carbonate from the start of the
experiment, and none of these animals developed any bone
disorders.
In the experiments on rabbits performed by Arrington
and Davis, 1953, McCarter, Riddell and Robinson, 1962,
and Valli et al., 1969, commented upon in Chapter
6.3.3.1 and 6.3.3.3, bone disorders appeared together
with the other symptoms. In the first study "front leg
abnormalities", i.e. deformities in joints and in one
case twisting of humerus, developed in 7 weanling animals
82
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out of 22 (1,000 mg Mo/kg diet and more were required to
produce the symptoms). Five of 7 test animals that were
X-rayed demonstrated epiphyseal line fractures and epi-
physeal plate widening in the study by McCarter, Riddell
and Robinson, 1962, and this finding was confirmed by
Valli et al., 1969. In this study 6 out of 12 animals
fed 4,000 mg Mo/kg diet as sodium molybdate for 4 weeks
demonstrated intra-cartilaginous epiphyseal fractures in
the humeri. The femoral epiphyseal plates were increased
because of widening of the zone of chondrycytes. The
latter authors compared the pathomorphology with "osteo-
lathyrism", suggesting an interference with the same
"metabolic pathway" in the two disorders.
6.3.3.5 Effect^ on_l_iver_
Fatty changes in the liver are a common result in histo-
pathological studies on experimental molybdenosis. They
have been reported by Fairhall et al., 1945 (25 mg and
more per day of molybdenum dioxide orally to guinea pigs
for 14 days); Asmangulyan, 1965 (50 mg/kg body weight
per day, of ammonium molybdate orally to rabbits for 6
months); Rokicka, 1969 (120 mg molybdenum/kg/day as ammonium
molybdate intraperitoneally to rats for 30 days); Mogil-
evskaja, 1963 (inhalation by rats of an aerosol of molyb-
denum trioxide in a concentration of 3-10 mg/m on alternate
days for 2 hours/day for 2 months)j Valjcuk and Sromko,
1973 (5 mg/kg body weight/day as ammonium molybdate orally
to rabbits for one year). Both Fairhall et al., 1945,
and Mogilevskaja/ 1963, also noted necrotic foci in the
liver.
Grigorajan and Brutajan, 1968, reported significantly re-
duced cholesterol levels and increased bilirubin levels
in sheep given large doses ( >1.5 g) which generally pro-
duced molybdenosis.
83
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Liver function was also studied by Avakajan, 1966b, on
6 dogs with 4 controls; 4 dogs were fed from 500 mg (20
mg/kg body weight) to 4,700 mg ammonium molybdate in
their diets for 5.5 months; 2 dogs received the same
doses of sodium molybdate for 3 months. The increase of
bilirubin in blood serum of dogs exposed to ammonium
molybdate was highly significant. The mean ± S.E. was
for all dogs prior to the experiment 3 - 0.28 ug/ml.
At the end of the experiment, the exposed group showed
values of 5.4 - 0.29 ug/ml and the control group 3.1
- 0.12 ug/ml. The 6ame effect was seen on dogs exposed
to sodium molybdate. At high doses (1,500 to 4,700 mg) ,
serum cholesterol was significantly increased (220 mg
per 100 ml as compared to 114 mg/100 ml in the controls),
and esterified cholesterol reduced to 51%. Total serum
proteins were somewhat increased and the albumin/globulin
ratio significantly decreased (0.92 to 0.71).
6.3.3.6 E f.f ects^ on_kidney_ _
Histopathological changes in kidney in experimental molyb-
denosis, mainly described as only "dystrophic changes"
or "swelling" of cells are described by the following:
Fairhall et al., 1945 (25-200 mg/animal per day of molyb-
denum trioxide orally to guinea pigs for 14 days);
Mogilevskaja, 1963 (inhalation by rats of an aerosol of
molybdenum trioxide in a concentration of 3-10 mg/m 2
hours/day on alternate days for 2 months); Asmangulyan,
1965 (50 mg/kg body weight/day of ammonium molybdate orally
to rabbits for 6 months); Valjcuk and Sromko, 1973 (5
mg/kg body weight/day as ammonium molybdate orally to
rabbits for one year); Lukasev, Siskova and Knys, 1971
(inhalation by rabbits and rats of molybdenum trioxide
4 hours/day for 3.5 months. Concentration of dust was
changed cyclically between 210 and 10 mg/m ).In the latter
study the findings were described as "atrophy" of renal
tubules. The nui her of animals and prevalence of patho-
morphological changes were not given.
84
-------
Grigorajan and Tatevosajan-Makarajan, 1970, exposed 4
groups of 10 rats to sodium molybdate by stomach tube
(0.025 mg Mo/kg? 1 mg Mo/kg? 20 mg Mo/kg and 100 mg
Mo/kg body weight). The first 3 groups received molybden-
um each day for 150 days? the fourth group only 30 days
as the large dose killed the animals rapidly. Animals were
kept in metabolic cages to obtain 24 hours urine. The
authors examined some physicochemical properties of urine/
the presence of proteins, blood and sugar? and changes
in weight and micromorphology of kidneys. Small doses
of molybdenum (0.025 and 1 mg/kg) had no effect on renal
function. High doses (20 and 100 mg/kg) impaired renal
function.
6.3.3.7 Myocardial_effects_
Fairhall et al., 1945, fed up to 200 mg/day of molybdenum
trioxide to guinea pigs for 14 days and did not find any
significant histopathological changes in the heart. A sim-
ilar opinion was given by Birjukova, 1971, injecting 70-100
ug/kg per day subcutaneously to 15 rats for 20 days. Valli
et.al., 1969, and Lukasev, Siskova and Knys, 1971, on the
other hand, did observe histopathological changes in the
myocardium in connection with other signs of toxicity.
Andreasajan, 1968, did electrocardiographic recordings on
6 dogs fed rising doses of molybdenum starting from 500
mg (20 mg/kg body weight) and reaching 4,700 mg daily for
3-5.5 months. Four dogs were given ammonium molybdate and
two sodium molybdate. The author described a series of EGG
abnormalities that were noted at the beginning of the first
month.
6.3.3.8 Eff:ec:ts on__the_thyroid_gl.and_
Widjajakusuma, Basrur and Robinson, 1973, studied the ef-
fect on the thyroid gland of rabbits fed a diet containing
3,000 mg Mo/kg given as sodium molybdate. The animals were
examined daily for gross signs of molybdenum toxicity, and
85
-------
hemoglobin and body weight were checked weekly. When molyb-
denosis was evident, plasma thyroxin (T.) concentrations
were significantly decreased (mean for 14 molybdenum-fed
rabbits being 2.31 compared to 4.40 for 19 controls).
6.4 CONCLUSIONS
There are few reports on molybdenum toxicity in man. In
industry a few cases of pneumoconiosis and hyperuricemia
attributed to exposure to molybdenum have been reported.
Although the data have not been possible to analyze in
strict detail due to lack of information on the epidemic-
logical methodology in the report, one study has indicated
that molybdenum might be a causal factor behind hyperuricemia
and an increased prevalence of gout-like symptoms in a gen-
eral population in two villages in Armenia, the molybdenum
content in the soil in this area being very high. Concen-
trations in several vegetables were considerably higher in
this area than in the control area (e.g. potatoes 11 mg/kg
vrs 3.3 mg/kg and beans 82 mg/kg vrs 5.1 mg/kg).
A disease termed "teart" disease has been reported in
ruminants grazing on pastures with high levels of molyb-
denum, the "risk zones" seeming to lie somewhere between
10 and 20 mg/kg dry matter in the herbage. This disease
afflicts only cattle and sheep and is not seen in horses
and swine. "Teart" is characterized mainly by diarrhea,
anemia and emaciation that can be so pronounced as to
lead to death. The animal typically recovers rapidly
once it is removed to "non-teart" pastures low in molyb-
denum. The disease can also be cured by the addition of
higher levels of copper to the diet.
Experimentally produced molybdenosis is reported in
many species (cattle, sheep, rabbit, guinea pigs, rats,
hens etc.) and is in repeated exposure experiments, mainly
86
-------
characterized by reduced weight gain and emaciation,
an anemia resembling iron deficiency anemia, and de-
fects in the skeletal system and connective tissues re-
sembling "osteolathyrism". The dietary levels used in
long-term experiments for rats are generally over 400
mg Mo/kg diet, this level in one experiment being report-
ed to produce molybdenosis in 50% of the exposed animals.
A 75% mortality resulted among guinea pigs fed 25 mg molyb-
denum per day and animal, given as molybdenum trioxide
while calcium molybdate brought a 25% mortality among the
same species when they were fed 200 mg molybdenum per
animal and day, indicating different toxicity levels for
different compounds. The same study gave evidence that only
the hexavalent molybdenum compounds (soluble) are toxic
while molybdenite (molybdenum disulfide) is harmless.
The metal seems to be toxic to about the same "magnitude"
in the different species, but its toxic manifestations
seem to differ somewhat among different animals. Thus,
skeletal deformities seem to be more a constituent in the
molybdenum syndrome of rabbits and rats than in the other
species. Diarrhea, perhaps the most typical manifestation
of molybdenosis in cattle, is only rarely described in other
species.
The mode of administration in the different experiments
is generally through ingestion or administration via a stomach
tube. A few reports on inhalation or intratracheal admin-
istration have been published. In some instances it has
been possible to produce a pneumoconiosis-like picture,
especially at intratracheal administrations of high levels
of metallic molybdenum or molybdenum trioxide.
87
-------
TABLE 6:1 -AVERAGE MOLYBDENUM CONTENTS (n\g/kg dry weight) OF
HERBAGE SAMPLES3^ IN RELATION TO TEART DISEASE.
(From Ferguson, Lewis and Watson, 1943).
Year Farm
1937 W. Hambridge
Go. Hambridge
Gr. West Camel
Co. West Camel
D. West Camel
K. Butleigh
Cd. Pi 1 ton
Wh. Kings don
M. Kings don
P. Babcary
H. Pylle
Lu. Kingsdon
1938 W. Hambridge
Co. West Camel
K. Butleigh
Wh. Kingsdon
Arithmetic mean, 1937
Arithmetic mean, 1938
Teart
51(5)
14(2)
25(3)
20(6)
52(2)
40(1)
30(1)
30(1)
59(4)
49(2)
18(2)
27(1)
33
38
Mildly Non-
Teart Teart
4 (2)
5 (1)
14(4)
12 (2)
12(2)
4 (2)
4 (1)
6 (1)
3 (2)
24(1)
13(1)
4(12)
4 (4)
5(12)
4 (3)
16 5
4
x)
'The figures in parentheses refer to the number of samples
analyzed.
88
-------
TABLE 6:2 SEASONAL VARIATION IN MOLYBDENUM CONTENT (mgAg
weight) OF HERBAGE AT FARM W. (From Ferguson, Lewis
and Watson, 1943).
Apr May June July Aug Sept Oct Dec
1937
1938
1939 a
1939 b
1940
32
49
45
74
34
42
62
66
26
49
62
48
53
87
66
93
108
68
80
79
66
102
29
89
-------
\o
o
TABLE 6:3 URIC ACID IN BLOOD AND URINE IN SELECTED PERSONSX) WITH AND WITHOUT A GOUT-LIKE
DISEASE IN A MOLYBDENUM AREA AND IN A CONTROL AREA. (Modified from Kovalskii,
Yarovaya and Shmavonyan, 1961) .
No. of
Area Status of subjects subjects Statistic
Molybdenum 111
province
Molybdenum Healthy
province
Molybdenum 111 +
province Healthy
Control area Controls
17 M,
Sl
ml
35 M2
S2
m2
t (1-2)
52 M,
j
S3
m3
5 M4
S4
m4
t (2-4)
t (3-4)
Blood No . of
Urine
mg/100 ml subjects mg/day
8
1
0
5
2
0
5
6
2
0
3
1
0
2
4
.1 17
.8
.4
.3 34
.1
.4
.1
.2 51
.3
.3
.8 5
.0
.5
.6
.4
824
285
67
649
234
40
2.2
707
247
42
432
89
40
3.9
4.7
Note: M = arithmetic mean, m = standard error, s = standard deviation, t = t-test
x) Not all subjects with the gout-like disease are included in the table. It is not known on
what basis the ones who are included were selected from the total group of 71 with clinically
overt gout.
-------
TABLE 6:4
CONCENTRATIONS OF MOLYBDENUM AND COPPER IN BLOOD AND URINE IN SELECTED*^ PERSONS WITH
AND WITHOUT A GOUT-LIKE DISEASE IN A MOLYBDENUM AREA AND IN A CONTROL AREA.
(Modified from Kovalskii, Yarovaya and Shmavonyan, 1961).
Blood
Status of
Area subjects
Molybdenum 111
province
Molybdenum Healthy
province
Molybdenum 111 +
province healthy
Control Controls
area
Statistic
M
Sl
ml
M2
S2
m2
t(l-2)
M3
S3
m3
M4
S4
m4
t(2-4)
t(3-4)
No. of Mo
subjects ug/ml
16 0.31
0.08
0.02
31 0.17
0.08
0.01
6.4
47 0.22
0.10
0.01
5 0.06
0.02
0.01
7.1
11.4
Cu
ug/ml
1.13
0.25
0.06
1.38
0.26
0.05
3.1
1.29
0.30
0.04
1.83
0.20
0.09
4.5
5.5
Urine
No . Of Mo
subjects ug/1
6 290
20
10
11 280
40
10
0.7
17 280
30
10
4 160
30
20
5.5
5.5
Cu
ug/1
3,040
140
60
1,840
300
90
12.0
2,260
320
80
1,200
240
120
4.6
7.6
Note: M = arithmetic mean, m = standard error, s = standard deviation, t = t-test
x) Not all subjects with the gout-like disease are included in the table. It is not known on what
basis the ones who are included were selected from the total group of 71 with clinically overt
gout.
-------
TABLE 6:5
CONCENTRATIONS OF MOLYBDENUM AND COPPER IN SOME FOOD
PRODUCTS IN A MOLYBDENUM-RICH AREA AND IN A NORMAL
AREA. (Modified from Kovalskii, Yarovaya and
Shmavonyan, 1961).
Food
x)
products '
potatoes
cabbage
coma toes
beans
bread / brown
bread, white
cow's milk
beef liver
beef
mutton
eggs
Molybdenum-rich area
ug Mo/kg
11,000
5,200
8,500
82,000
280
510
280
340
59
34
8
drinking water 10
ug Cu/kg
3,200
5,900
29,000
17,000
740
2,100
190
2,100
400
510
490
270
Normal
ug Mo/kg
3,300
40
2,700
5,100
410
390
47
98
12
10
3
5
area
ug Cu/kg
7,000
22,000
17,000
14,000
5.900
2,800
290
11,000
820
680
910
110
x)
' vegetable products in dry weight
animal products in wet weight
92
-------
TABLE 6:6 PERCENT MORTALITY FOLLOWING A SINGLE INTRAPERITONEAL INJECTION OF A MOLYBDENUM
COMPOUND IN GUINEA PIGS. (Modified from Fairhall et al., 1945).
vo
U)
Molybdenum
compound
Molybdenite
Molybdenum trioxide
Ammonium molybdate
Ammonium molybdate
Calcium molybdate
Number of
animals
12
8
8
12
6
Amount injected
(mg/kg)
800
400
80
800
400
4 days
17
75
0
100
0
Mortality i
4 weeks
17
75
0
0
%\
/
4 months
25
75
0
17
-------
TABLE 6:7
PARTICLE SIZE DISTRIBUTION OF THE DUST OF METALLIC MOLYBDENUM AND OF
MOLYBDENUM COMPOUNDS USED FOR INHALATION EXPERIMENTS ON RATS.
(From Mogilevskaya, 1963) .
vo
Substance
Metallic molybdenum
Brown molybdenum dioxide
Molybdenum trioxide
Ammonium paramolybdate
Percentage of Particles of Different Sizes
less than
2.5 u
55
2.5-5 u
35
26 25
24 : 25
33 ; 31
5-7.5 u
6
22
30
18
7.5-10 u
3
12
14
8
more than
10 u
1
15
7
10
-------
TABLE 6:8 PERCENT MORTALITY AND DISTRIBUTION OF MOLYBDENUM IN TISSUES OF RATS AFTER PROLONGED INGESTION
OF A MOLYBDENUM COMPOUND. (Modified from Fairhall et al., 1945).
vo
en
Compound
Molybdenite
Molybdenite
Molybdenite
Molybdenum trioxide
Molybdenum trioxide
Molybdenum trioxide
Molybdenum trioxide
Molybdenum trioxide
Calcium molybdate
Calcium molybdate
Calcium molybdate
Calcium molybdate
Calcium molybdate
Ammonium molybdate
Ammonium molybdate
Ammonium molybdate
Control (average)
No. of
animals
8
8
8
8
10
10
8
8
10
10
10
10
10
8
8
8
48
Estimated
daily con-
sumption
(mg Mo)
10
100
500
10
25
50
100
500
9
21
43
86
430
10
100
500
0
No. of
days on
test
44
44
44
120
137
137
14
8
137
128
137
57
17
232
13
9
137
Estimated
total molyb-
denum intake
(ug)
400
4,400
22,000
900
1,800
1,700
1,100
3,100
900
1,300
3,200
3,600
4,700
2,300
1,000
3,200
0
Percent
mortality
0
0
0
50
80
90
100
100
50
100
60
100
100
25
100
100
15
Concentration of molybdenum
(ug/g wet weight)
Liver
2
2
1
12
8
36
24
49
4
5
9
8
18
9
16
16
3
Kidney
4
6
4
11
19
33
14
39
12
21
17
17
35
36
16
8
6
Bone
6
5
6
23
25
40
25
28
34
79
37
41
5
-------
TABLE 6:9 SYMPTOMS IN RABBITS FED DIETS CONTAINING DIFFERENT CONGENTRATIONS
OF MOLYBDENUM AS SODIUM MOLYBDATE. (Modified from Arrington and Davis, 1953)
vo
Concentration of
Molybdenum in diet
(mg/kg)
No. of
animals
Deaths
Average sur- .
vival timc Body
Initial, g
weight
Final, g
Anemia
Alopecia
and der-
ma tosis
Front leg
abnormality
Weanling rabbits
4,000
2,000
1,000
500
140
Control
4,000
2,000
1,000
140
Control
2
5
5
5
5
5
2
3
2
2
4
2
4
_2>
0
0
0
2
2
0
0
0
30 967
44 887
716
700
671
738
Mature rabbits
51
54
803
739
1521
1948
1917
1844
2
5
4
0
0
0
2
3
1
0
0
o11
4
4
0
0
0
0
2
1
0
0
1
4
2
0
0
0
0
0
0
0
0
1)
2)
Deaths occurred early and no alopecia observed.
Severe toxic symptoms developed in three rabbits and death impended; copper therapy
initiated to prevent death.
-------
TABLE 6:10 HEMOGLOBIN AND RED BLOOD CELL COUNTS IN RABBITS
FED DIETS CONTAINING DIFFERENT CONCENTRATIONS OF
MOLYBDENUM AS SODIUM MOLYBDATE.
(Modified from Arrington and Davis, 1953).
Concentration of
Molybdenum in diet
(mg/kg)
Control
Control
Control
Control
1,000
1,000
1,000
1,000
2,000
2,000
2,000
2,000
Week of
experiment
12
17
17
17
13
7
5
3
4
17
13
9
Hemoglobin
(g/100 ml blood)
13.2
15.0
14.5
12.0
3.5
6.3
6.3
8.0
5.5
7.3
3.2
4.5
Erythrocytes 3
(million cells/mm
6.16
6.42
5.79
5.08
1.55
3.39
3.16
3.46
1.89
3.50
1.20
1.53
97
-------
Mo
grams
180-
140
lOOl
60
4 weeks
FIGURE 6:1
Growth retardation in rats fed a purified diet
containing molybdenum (Na2Mo04•2H20).
A, diet without Mo(control)j a, diet contained
500 mg Mo/kg; 0, diet contained 1,000 mg Mo/kg
X, diet contained 5,000 mg Mo/kg. (From
Neilands, Strong and Elvehjem, 1948).
98
-------
CHAPTER 7 INTERACTIONS OF MOLYBDENUM WITH COPPER,
SULFATE, PURINES AND PROTEINS
7.1 INTERACTIONS WITH COPPER AND SULFATE
The toxicity of molybdenum in animals is dependent to a
large extent upon the supply of other compounds. Most
attention has been devoted to the influence of copper
and sulfur compounds, as witnessed by the large number
of reports on this subject. For penetration of certain
aspects of this relationship, the reader is referred to
Underwood, 1971. In most studies large amounts of molyb-
denum and other compounds have been given and the relev-
ance of the findings for long-term exposure to excessive
amounts of molybdenum in human beings is not known.
That the administration of copper sulfate could cure
"teart" disease was shown by Ferguson, Lewis and Watson,
1943. They tried this compound as an antidote since Brouwer
et al., 1938, had described a "teart-like" syndrome in
copper-deficient cattle, which could be cured by copper
sulfate.
Other copper compounds have also had a beneficial effect
upon cattle diseases caused by excessive molybdenum, e.g.
copper glycinate (Cunningham, 1957, Cook et al., 1966).
Copper sulfate has been shown to be more effective than
copper glycinate in curing molybdenotic heifers (Clawson
et al., 1972).
Subsequent to the early experiments on cattle diseases,
copper sulfate has been shown to counteract effects caused
by excessive molybdenum in other species such as rats
(Neilands, Strong and Elvehjem, 1948), rabbits (Arring-
ton and Davis, 1953) and guinea pigs (Arthur, 1965).
In 1953 Dick showed that the dietary intake of sulfates
other than copper sulfate was of importance for the met-
99
-------
abolism and effects of molybdenum. Sheep ingesting 10 mg
molybdenum per day excreted more molybdenum and had lower
blood levels of molybdenum when they received hay with a
high content of sulfates. Reduction in tissue levels of
molybdenum was found in a later study (Dick, 1956). Dick,
1956, summarizing his studies on sheep, reported that when
the intake of sulfate was low, daily doses of 0.3-100 mg
for long periods had no effect on blood copper levels. In
contrast, when intakes of sulfate were high, rapid rises
in blood copper took place upon the administration of 60
or 90 mg of molybdenum. Even 15 mg of molybdenum had an
effect on blood copper when more than 5 grams of sulfate
were given. It was also shown that the rise in blood cop-
per was wholly attributable to a rise in plasma copper.
When sulfate was given in doses from 1,000 to 6,000 mg/day
and molybdenum from 2.5 to 25 mg/day, it was found that
decreases in levels of copper became greater with increasing
sulfate intake. Thus 15 mg of molybdenum and 1,400 mg sulfate
had the same effect as 5 mg molybdenum and 2,500 mg sul-
fate. These studies showed conclusively that effects of
molybdenum on copper metabolism in sheep were intimately
related to the intake of sulfates.
Throughout the subsequent years, the literature on the
interrelationships of molybdenum, copper and sulfate has
greatly expanded. Wynne and McCiymont, 1955, reported
that copper levels in liver of sheep on a low copper
and molybdenum intake were not affected by the dose of
400 mg sulfate/kg diet, while 4,000 mg sulfate/kg caused
lowered liver levels of copper and symptoms of copper
deficiency. Similar results were obtained by Kline, Hays
and Cromwell, 1971, when giving 2,200 mg sulfate/kg diet
for 88 days to sheep receiving 11 mg of molybdenum and
15-33 mg of copper/kg diet. In pigs, however, the same
authors did not find any changes in liver levels of copper
even after prolonged exposure to high dietary levels of
molybdenum (50-100 mg/kg) and sulfate (1,000-5,000 mg/kg).
100
-------
Goodrich and Tillman, 1966, found that sheep given large
doses of sulfate (4,000 mg/kg diet) for two months under-
went a reduced weight gain when the molybdenum intake was
low (2 mg/kg diet), but not when the molybdenum intake
was higher (8 mg/kg diet). Marcilese et al., 1969, did
not find that sulfate (4,000 mg/kg diet) had any impact
upon copper metabolism in sheep on a low molybdenum in-
take, while 50 mg Mo/kg diet together with sulfate did
produce changes in copper metabolism.
In cattle on a low intake of copper and sulfate for 300
days, liver concentrations of copper decreased when from
5 to 50 mg molybdenum/kg diet was given, but no toxic signs
arose (Vanderveen and Keener, 1964). When sulfate (3,000
mg/kg diet) was given, 50 mg Mo/kg caused hair changes,
which were reversed by copper. Higher molybdenum levels
(100-200 mg/kg diet) together with sulfate resulted in
signs of molybdenosis but liver levels of copper were
not decreased. In calves, Cox et al., I960, did not find
any significant changes in liver concentrations of copper
when molybdenum concentrations in the diet were 250 to
800 mg/kg.
In sheep sulfate increased the urinary excretion of molyb-
denum (Dick, 1953), whereas milk molybdenum levels fell
when high doses of sulfate were given (Hogan and Hutchin-
son, 1965).
Miller, Price and Engel, 1956, gave rats molybdenum and
sulfate for six weeks, the levels in food being 75-300 and
400-3,300 mg/kg respectively. Molybdenum alone caused in-
creases in liver copper and reduced weight gains, but 800-
2,200 mg/kg of sulfate alleviated both the weight reduc-
tion and the increase in liver copper at the lowest molyb-
denum exposures (75-100 mg/kg diet).
101
-------
Johnson and Miller, 1961, found that the growth depression
brought about in rats by 600 mg Mo/kg diet was partially
ameliorated by 500 mg sulfate/kg diet, whereas copper had
no effect.
In copper-deficient rats, Gray and Daniel, 1964, showed
that small amounts of molybdenum in the 'diet (10 mg/kg)
caused toxic symptoms, intensified when sulfate was also
given. In copper-supplemented rats, larger doses of molyb-
denum (800 mg/kg) were needed to produce symptoms, in
which cases sulfate prevented the effects of molybdenum.
It was also found that methionine (1.2% in diet) prevented
the copper accumulation and alleviated the symptoms caused
by molybdenum, but to a lesser extent than sulfate.
Miller and Denton, 1959, showed that sulfate reduced molyb-
denum levels in liver of molybdenum-treated chicks. When
copper was given as well, further reductions in molybdenum
levels occurred. Copper supplementation did not improve
growth in molybdenum-treated chicks. Molybdenum alone
or together with sulfate slightly increased the liver
levels of copper.
Davies et al., I960, compared sodium sulfate to ammonium
sulfate in chicks, finding that the latter compound was
more toxic. Sodium sulfate promoted growth in molybdenum-
treated chicks, but did not prevent accumulation of molyb-
denum. Methionine had no effect on the toxicity of molyb-
denum.
In guinea pigs, copper supplements prevented some of the
drop in weight gain caused by molybdenum. Dietary levels of
200 mg Mo/kg diet caused a slight increase in liver levels
of copper after 8 weeks, but higher molybdenum intake
(1,000 and 2,000 mg/kg) caused reductions in liver copper.
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Lukasev and Siskova, 1969, noted that ammonium paramolyb-
date administered orally to rabbits (5 mg Mo/kg body weight)
for 5 days reduced the urinary excretion of free sulfates
but increased the excretion of bound sulfates (calculated
as the difference between total sulfates and free sulfates).
When 50 mg/day of sulfate was given simultaneously with
molybdenum, the urinary excretion of sulfates was normal.
There was no effect of molybdenum on the urinary excretion
of indican and thiocyanate. Lukasev and Siskova, 1971b,
measured the distribution of molybdenum and copper in rab-
bits after inhalation exposure to molybdenum trioxide.
Simultaneous exposure of rabbits to molybdenum (inhalation
of Mo03) and oral application of sodium sulfate (50 mg/day
per animal) did not significantly affect the urinary excre-
tion of molybdenum. The excretion of copper was increased
following exposure to molybdenum; this effect was signi-
ficantly reduced by sulfates (P< 0.02).
Several attempts have been made to explain the mechanisms
behind the complex interrelationships of molybdenum, copper
and sulfate.
To study the influence of molybdenum upon copper metabolism/
64
Compdre et-al., 1965, injected Cu as the chloride in
molybdenum-treated animals and controls. Higher plasma
levels of Cu and a lowered uptake of Cu in the liver
were found in the molybdenum-treated animals compared to
controls. In another group of molybdenum-treated rats,
which were supplemented with copper chloride in the diet,
the uptake of Cu in the liver was higher than in controls.
These results were interpreted as showing the blockage by
molybdenum of copper metabolism in the liver.
Marcilese et al., 1969, gave molybdenum and sulfate in
feed at concentrations of 50 and 4,000 mg/kg respectively
for 120 days to a group of sheep. This group was compared
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to two other groups, one a control group, and the other a
group receiving 4,000 mg/kg diet of sulfate. The plasma
clearance and distribution of Cu were studied after in-
jection of sheep's plasma incubated with the isotope. Where-
as sulfate alone had no influence on either the metabolism
of the radioisotope or stable copper, molybdenum together
with sulfate caused considerable decreases in the uptake
of Cu in the liver and decreases in the contents of
64
stable copper in the liver. The disappearance of Cu
from plasma was about twice as rapid in the control group
as in the molybdenum-treated animals. It could also be
shown that in control animals considerably more of the
Cu was in the ceruloplasmin fraction than in animals
given molybdenum.
Molybdenum exposure has been shown to decrease the activity
of the enzyme sulfide oxidase in the liver (Mills et al.,
1958). Halverson, Phifer and Monty.- I960, found that ex-
cessive dietary cystine given rats on a high molybdate -
low copper diet worsened symptoms caused by molybdenum.
The toxic effects of cystine were prevented or reversed
by copper. It was postulated that a decrease in sulfide
oxidase activity could cause excessive amounts of sulfide
in the tissues, which could interfere with copper metab-
olism.
Another factor could be the formation in vivo of a copper-
molybdenum complex, as postulated by Dowdy and Matrone,
1968a, 1968b, and Dowdy, Kunz and Sauberlich, 1969- The
copper in this complex would have low availability for the
organism.
Regarding the role of sulfate, the data by Dick, 1953,
1956, suggested that sulfate mobilized stored molybdenum
by interference with molybdenum transport across membranes.
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7.2 INTERFERENCE WITH PURINE METABOLISM
Yarovaya, 1964, briefly reported changes of purine metab-
olism in humans under the conditions of high molybdenum
concentrations in food products. It was established that
"2-10 times higher than normal" content of molybdenum
(and "relatively low copper concentrations") in daily
food rations increased the concentration of molybdenum
in blood approximately 4 times. This led to an increase
of xanthine oxidase activity by 100%, which in turn was
conjectured to result in the high uric acid content and
high incidence of gout registered in these areas high in
molybdenum.(Kovalskii, Yarovaya and Shmavonyan, 1961?
see section 6.2).
Increased xanthine oxidase activity when molybdenum is
given as a supplement in the food is confirmed by Gusev,
1969, The author studied effects of molybdenum on the
purine metabolism in a chronic experiment on 50 rats.
Rats were fed standard diet which included the essential
elements. One group (10 animals) served as the control;
the second group received the standard diet from which
molybdenum and copper were excluded as far as possible.
Three other groups of 10 animals received by stomach
tube each day a solution of ammonium molybdate (5, 50
and 500 mg Mo/kg^'respectively). After six months the
animals were killed and the following measurements made:
xanthine oxidase in liver; uric acid in blood and urine?
and ceruloplasmin in serum. The xanthine oxidase activity
increased with increasing dose of molybdenum. The con-
centration of the uric acid in the control group was
0.95 mg/100 ml in blood and 0.71 mg/day in urine. The
corresponding values in animals receiving 5, 50 and 500
ug Mo/day ^ere 1.26, 2.29 and 2.27 mg/100 ml in blood,
and 0.88 mg, 1.04 mg and 0.87 mg/day in urine.
There is some discrepancy here due to the uncertainty as
to how to interpret the U.S.S.R. units expressed as MKG.
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Kovalsky and Vorotnitskaya, 1970/ found two peaks of xan-
thine oxidase activity, and consequently of uric acid
levels, in liver and kidney of rats at different "Cu/Mo-ratios"
in the diet. One peak appeared at a Cu/Mo ratio of 1.6
(Cu/Mo =80 ug/50 ug per day, copper given as copper sul-
fate and molybdenum as ammonium molybdate) and one at a
Cu/Mo ratio of 16 (Cu/Mo = 320 ug/20 ug). In both instances
xanthine oxidase activity was approximately doubled compared
to controls (Cu/Mo = 4) but the ratio of 1.6 (relatively
high molybdenum) produced a higher peak of uric acid than
the ratio of 16 (relatively high copper). The two peaks
made the authors suggest two different xanthine oxidase
enzymes, one containing molybdenum and one containing
copper. The reason for the higher uric acid response in
the high molybdenum case is not clear but xanthine oxidase
induction is suggested as the reason for the higher prev-
alence of gout in a molybdenum-endemic area (see section
6.2) .
7.3 INTERFERENCE WITH PROTEIN METABOLISM, STUDIES ON
ENZYME IMPAIRMENT
Mills et al., 1958, studied some enzyme systems in rats
suffering from molybdenosis induced by feeding the animals
800 mg/kg diet of Mo and/or 0.29% sulfate, these elements
being supplied as sodium molybdate and sodium sulfate,
respectively. The molybdenum supplement resulted in a
36% depression of growth in a five week period and the
inclusion of sulfate largely prevented this effect on
growth. After this period the rats were sacrificed and
their enzyme activities in liver and kidney were measured
and compared to controls. It was found that the enzyme
sulfide oxidase was depressed to about 56% of normal val-
ues in livers. Further, alkaline phosphatase was elev-
ated in liver and depressed in kidney. The latter changes
were prevented by the sulfate supplement. No effect was
reported on the levels of activity of cysteine desulfhy-
106
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drase and aryl sulfatase or on the rate of oxidation of
cysteine sulfinic acid in tissue homogenates. Thus the
authors failed to detect a dysfunction of sulfur metab-
olism which could account for the protective effect of
sulfate against molybdenum toxicity. Further the signi-
ficance of the determined effects of sulfide oxidase and
alkaline phosphatase was discussed in terms of a possible
impairment of protein metabolism. Indications of metab-
olic protein derangement were found in measurement of
protein/DNA and protein/RNA ratios in liver and kidneys,
but the possible linkage between the anticipated protein
derangement and the changes in enzymatic impairment re-
mains to be further explored.
Johnson and Miller, 1961, 1963, confirmed the concept of
impairment by molybdenum of protein metabolism and alka-
line phosphatase activity in rats. These authors found a
depression of femur alkaline phosphatase activity and
also reported an alleviation of the molybdenum-induced
enzyme depression by added dietary sulfate. Through
controlled feed intake studies it was shown that feed
restrictions leading to a growth depression of the same
magnitude as the molybdenum supplementation to "ad libi-
tum" fed animals led to an equivalent depression of al-
kaline phosphatase activity. Thus, the authors concluded
that the decrease in enzyme activity induced by molyb-
denum feeding was the result of growth depression and
not of the ingestion of molybdenum per se. (Basal ration
restriction to about 62% of the rations of "ad libitum"
fed animals resulted in a growth depression equivalent
to the one achieved by molybdenum supplement in the diet
of 600 mg/kg given as sodium molybdate). Impairment of
protein metabolism was established through an increased
excretion in urine of nitrogen (47.9% of consumed nitrogen
compared to 29.5% for controls) and amino acids by rats
fed 400 mg/kg diet of molybdenum given as sodium molybdate,
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A complicated derangement of the amino acid levels in
blood of rabbits fed 25-1000 ug Mo as ammonium molyb-
date was reported by Val'chuk and and Koval'skaya,
1970.
Lukasev and Siskova, 1971a, found that an oral daily dose
of 5 mg Mo/kg body weight (as ammonium molybdate) for seven
months increased significantly the total amino acids
(paper chromatography) in the liver and the kidneys. The
comparison was made with a control group fed only the
standard diet and another experimental group receiving
the same dose of molybdenum and 50 mg/kg per day of
sulfate. The addition of sulfate had no significant ef-
fect. When the molybdenum dose was reduced to 0.5 mg/kg
daily and given for 4.5 months/ the urinary excretion
of amino acids first decreased and then increased again.
There was no change in the amino acid content in serum
and organs. The effect of sulfate (50 mg/kg) showed up
only at the end of the experiment (increased urinary
excretion of amino acids as compared to the control and
molybdenum group). The distribution of individual amino
acid content in the serum, liver and kidneys was changed
in both groups receiving molybdenum (0.5 and 5 mg/kg).
Sulfates had small influence.
Avakajan, 1966b, gave dogs (4 experimental and 2 control
animals) ammonium molybdate daily in increasing oral doses
from 500 mg (20 mg/kg body weight) to 4.7 g for 5.5 months,
and measured cholinesterase activity in serumj thiol groups
in serum, liver, spleen, kidney, lungs, heart and brain
tissue; and molybdenum and copper in blood, liver, heart,
spleen, brain, kidneys and lungs. Molybdenum in doses from
500 mg to 1.5 g in 1.5 months increased the activity of
cholinesterase and the concentration of thiol groups in
serum. Higher doses given for 4.5 months resulted in a
decrease of both the cholinesterase activity and the con-
108
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centration of thiol groups in serum and in organ homo-
genates.
In order to study the mechanism behind bone disorders of
animals fed a high molydenum intake, Feaster and Davies,
1959, fed 2,000 mg/kg of molybdenum as sodium molybdate
to rabbits and studied the resultant effect on the Ca-P
metabolism of the animals. After having been fed the diet
for five weeks (the animals showed the symptoms described
in Chapter 6 including deformity of the joints, anemia and
diarrhea) they received a single oral dose containing 3 uCi
45 32
each of Ca and P and were killed at different intervals
after 6 hours to 14 days. Neither soft tissue levels nor
urinary or fecal excretion of total or radioactive Ca or P
indicated any impairment in the absorption of these ions
from the intestine. Further, there were no indications
of any derangement of bone metabolism of Ca and P. The
bones of the experimental animals did weigh significantly
less than those of the controls, indicating a decrease of
"organic matter" rather than "demineralization" as a causal
factor behind the molybdenum-bone disorders observed in
the animals.
7.4 SUMMARY
There is a complex relationship between molybdenum, copper,
sulfate and probably also some other sulfur compounds.
Species differences exist, e.g. sheep are more susceptible
to imbalances between the elements than pigs. Copper gen-
erally has a beneficial effect on the symptoms caused by
excessive molybdenum but the action of sulfur compounds,
especially sulfate, is not so clearly understood, both
positive and negative effects having been reported,
depending upon the copper status. The association between
molybdenum exposure, hyperuricemia and gout-like symptoms
reported in Chapter 6 could find an explanation in terms
of an impaired purine metabolism through an enhancement
109
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of xanthine oxidase activity by molybdenum. Moreover, there
are indications of an interference with other enzymes as
sulfide oxidase and alkaline phosphatase.
110
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CHAPTER 8 CONCLUSIONS AND DISCUSSION CONCERNING
POSSIBLE HEALTH EFFECTS OF MOLYBDENUM ON
HUMAN BEINGS
Molybdenum is considered to be an essential element. It
is a constituent of some enzymes including xanthine
oxidase which oxidizes xanthine or hypoxanthine to uric
acid. Although there are no minimum requirement data for
human beings, information from other mammals tends to
show that the necessary amount of molybdenum is very
small. Therefore, the concentration of molybdenum in
a normal diet with all probability will be sufficient
for maintaining physiological levels of the metal in
humans.
Despite the fact that human beings are exposed to relat-
ively high amounts of molybdenum (100-500 ug/day), pri-
marily via food, there is no substantial age-related
accumulation of molybdenum in the body. Concentrations
in those organs in which molybdenum is mainly found,
i.e. liver, kidney and bones, are relatively low, in the
order of below 1 ug/g wet weight. Molybdenum occurring
naturally in the diet is absorbed to a high degree from
the gastrointestinal tract (probably 25-75%) but is
eliminated rapidly, primarily via the urine. The bio-
logical half-time is not well established in humans but
the major part of the absorbed molybdenum is eliminated
within days or, at a maximum, a few weeks.
Animal data show that the metabolism of molybdenum is
closely related to the metabolism of copper and sulfur
compounds.
Most observations on the toxicity of molybdenum are
based on data from animals. It is clearly shown that
an excessive exposure to molybdenum via food can give
111
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rise to a severe disease in ruminants which involves
diarrhea, anemia and emaciation and may progress to
death. The disease may be prevented or cured by the
administration of copper compounds or removal of animals
from the areas high in molybdenum. The disease is termed
teart disease and has been possible to reproduce exper-
imentally. It has also been possible to show toxic effects
resembling teart disease in several of the common labor-
atory animals. In addition, several forms of bone defor-
mities, including exostoses, have been induced. The doses
required are excessive and much higher than those to
which human beings eating a normal diet may be expected
to be exposed, even in molybdenum-rich areas. In several
studies 200-400 mg of soluble hexavalent molybdenum com-
pounds per kg diet were required over prolonged periods.
Information into possible toxic effects in human beings
is scarce. There are data from the U.S.S.R. literature
pointing towards the possibility that exposure to some
molybdenum dusts may give rise to pulmonary disorders
in the form of pneumoconiosis. Similar effects have
been reproduced in animals. No conclusions concerning
the necessary exposures to give rise to this condition
in humans can be drawn at this time.
There have also been reports on effects of molybdenum on
purine metabolism. It has been shown in studies from the
U.S.S.R. that people exposed to molybdenum (concentration
not known) in industry have increased levels of uric
acid in urine and in blood. Moreover, a report from the
U.S.S.R. from 1961 has indicated that people living in
an area of that country with high dietary intake of molyb-
denum (by the authors calculated to 10-15 mg/day as com-
pared to 1-2 mg in the control area) show an increased
prevalenceof joint-disorders claimed to be gout-like as
well as an increase in blood levels of uric acid and an
112
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increase in the urinary excretion of uric acid. The reason
for the high daily intake value in the control area is not
known. The results are very difficult to evaluate since no
details are given concerning the study population and the
selection of subjects; furthermore, the control group con-
sisted of only five subjects. Nevertheless, the data fully
motivate further studies, not only in the U.S.S.R. but also
in regions of other countries where the exposure to molyb-
denum via diet is high, one reason being that from the
theoretical standpoint, effects like those reported might
well appear. Thus, molybdenum exposure could give rise to
an increase in xanthine oxidase activity which in turn
should give rise to an increase in uric acid formation.
The reason that effects have not been reported from other
parts of the world may be a lack of studies focusing on
these effects. Target groups of special importance are
1) persons living in areas with a high molybdenum content
in food, particularly in such areas in which the inhabitants
are dependent upon locally produced food; 2) persons from
non-contamined areas with consumption habits favoring a
high molybdenum intake and an imbalance in relation to
other dietary constituents of importance, e.g. copper.
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141
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-75-004
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
MOLYBDENUM - A TOXICOLOGICAL APPRAISAL
5. REPORT DATE
November 1975 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Lars Friberg, Pamela Boston, Gunnar Nordberg,
Magnus Piscator, and Karl-Henrik Robert
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Environmental Hygiene
Karolinska Institute and
National Environmental Protection Board
S-104 01, Stockholm 60, Sweden
10. PROGRAM ELEMENT NO.
1AA001
11. CONTRACT05J5JEKT NO.
68-02-1210
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
13. TYPE OF REPORT AND PERIOD COVERED
Final - 18 months
27711
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This review presents and evaluates the available material on
the metabolism and toxicology of molybdenum with emphasis on
those aspects of relevance to human beings. Agricultural and
environmental aspects of molybdenum are less completely
covered. Special efforts have been made to include recent
publications in Russian, and to coordinate this work with a
similar effort under way in' the Environmental Health Criteria
Programme of the World Health Organization.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Molybdenum
Toxicology
Metabolism
6T
6P
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
148
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
142
4USGPO: 1975 — 657-695/5343 Region 5-11
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