022 Molybdenum in Drinking Water.Pdf

i . _... . -- ABSTRACT . . .. . Molybdenum is a trace element whicii occurs widely in nature and plays an important role in industrial society. The primary industrial use of nolybdenun is as an alloy in steels. Major releases to the environment have been associated with several industries including molybdcnum mining and milling, uranium minina and milling, and oil refining. ._ Molybdenun. also plays an important biological role as a micronutrient for plarits and animals. At :ngh levels it can be toxic to animals. While concentrations in surface waters are generally less than 5 VgMo/L, concentrations as high as 500 pgMo/L have been reported in some drinking waters. Concentra- tions in water greater than 20 ;rgMo/L are almost certainly anthropogenic. ConveGtional wastewater and water treatment technologies are ineffective in the removal of molybdenum.

The average human intake via food for the Ui!ited States is 170 iigMo/day while the average intake via drinking water is less than 5 pgMo./day. While no adverse healtt effects have been reported in the Unite6 States, there are reports in the Russian and Tndinn literature of both biochemical and clinical effects in humans at intakes ranging from 1 to 10 mqFlo/day. Rnpid urinary excretion appears to provide considerable protection at inzakes less than 1 mgMo/day. This report reviews the data on rnolyhdenum as it relatrs to the effects of its occurrence iil drinking water.

The report also reviews the results of an interdisciplinary study carried out by the authors. The authors recommend a guideline of 50 pg;lo/L for the maximum concentration in drinking water.

This report was submitted in fulfillment of Grant No. R-SO3645 by the Environmental Trace Substances Research Program, University of Colorado under the sponsorship of the I1.S. Environmental Frotection Agency. This report covers the period Erom April 7, 1975 to September 30, 1978, and work was completed as of September 30. 1978. 1 .. Cok'respondence should he addressed to: R. .. Willard Chappell i . Director, Cnvirorunental Trace Substances Research Proqram . : Campus Box 215 University cf Colorado Bouider, Colorado 60309 ...... ! CONTENTS p . 1 ...... Fo&',iord ;...... ,. .iii. 1 astract ...... iv i. . Figures ...... vii 2 : Tables ...... iX 'Acknow1edqmen:s ...... xi .. 1:: Introduction ...... LI...... 1 .. Cment on Concentration Units ...... 2 2 . Conclusions and Recommendations 3 Conclusions ...... 3. Recommendations ...... 4 3 ... Chemical properties ...... 6 General Chemistry ...... 6 .. Bioinorganic Chemistry ...... 7 4 .. Measurement Tachnic;ue:; ...... 10 SappLing ...... 10 Analyses ...... L ...... 12 Colorimetric Methods ...... 12 ... P:to!nic i\bsorption Spectrophotometry ...... 13 Other Xethri;?:; ...... 13 5 . production and ...... IIst? production 15 .. Industrial Use 'of Elolybdenum and Its Conpollndo ...... 17 6 . Environmental Fate ...... 19 Rocks and Soils ...... 19 AirIndustrial ...... Exposure - ?-;:iinq and Milling ...... 20

Indust-rial 'Exposure - . ;h:i.Lting ...... 22 water ...... 24 .. plarlts ...... 27 f ~ood...... 28 Industrial Souryes . i ...... 30 ...... Coal Comhztiorl ...... 30 Molytd"nvm Mixling and Milling ...... 30 Molykwrencun Snelting ...... 31 tirhn.ium Mining and i4illing ...... - ...... 31 Steel z:id Capper >!illi.ng, Oil Refining, and Claypit Mining ...... 32 pernova1 Tfc'nnology...... 32 7 . piochcrn.istry arid pletabolism ...... 34 Biochemical Function ...... 34 Work of the Colorado M(:)lybdenm Project ...... 35 Giscussion ...... 37 .. .. , '. .

' i .: ..!.. Conclusions ...... 38 ,..- , ._-r. Metabolism ...... 39. ..I Tissue Distribution in Animals ...... 44 47 .. 8.. Biological Effects ...... ,...... ,,ti Toxicity''-. Animals .. 47 > ... Intrrduction ...;...... :...... '...... -...... ?7 I. 47 ! i. Livestock ...... I' i Laboratory. AnFmls - Acute Toxicicy ...... 49 t?'...... < -: ' .. . Laboratory Anim-ds - Chronic Toxicity ...... -. ;...... 52 1 ,i.!i Summary ...... 60 ;i!! .. I. -Human Dietary Irrtakes and Nutritional Requirements ...... 61 i -j .. ... Biolcgical. Effects of Molybdenum in Humans ...... 65 ..

. Deficiency ...... 65 .'' I '; Toxicity ...... 65 i-ji11 -i Human Health Effects and Present Study ...... 67 .. I .j References ...... 78. . i' j -. .i Appendices .. . . ' i.1 A. 92 ij Supplementary Calculation of Guideline ...... !! ., .. B. Analysis for Molybdenum, Spectrophotometric Method ...... 9'; C. Analysis for Mdybdenum, Atomic Absorption spectrophotometry . 98. i.I., ,:1 ._ .. .. q ,. .. 1- j .. .. ,I .. ".. '1.'.I .. .. . '< . ., ...... ;j ' .I .. .. :i .. ' it ' :i .. I. ! .. ., .. .. .' i ",j .. :i .. ... '. i: . .I, .*

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I r/ k FIGURES

Number . --Page - i! - .- i 1 Eh-pH diagram of areas of predominance of aqueous species of i molybdenum ...... 8 ! t 1 2 Atmospheric release in metric tons/year molybdenum ...... 16 i 3 R-value versus serum uric acid concentration ( r = -0.84) ...... I i 4 R-value versus log (serum molybdenum) [r = -0.84) ...... 39 I i 5 Percent of molybdenUm dose remaining in the gastro-intestinal t tract versus time, and percent of dose in accumulated urine 1 versus time after dosing ...... f ! 40 i 6 IJercent of molybdenum-99 dose accumulated per gram of tissue (wet weight) for kidney and blood versus time after dosing ...... 42 I 7 Percent(wet weight) of molybdenum-99 for liver anddose adrenals accumulated versus per time gram after of tissue dosing ..... 43

8 Weightid average nolybdenum concentration of ten tissues (dry weight basis) from male rats receiving indicated molybdenum concentrations in their drinking water ...... 44

9 Hypothetical dose-response curve for molybdenm ...... 48

10 Oxygen consumption in sleeping rats versus molybdenum concentration 54 in drinking water ......

11 Unstfessed animal activity as measured by nmbcr of squares entered 56 per minute in an open field arena ...... I I 12 Effdctdenum of and cold copper stress in ox1a defined serum ceruloplasfin diet at the indicated in rats given levels molyb- ...... 57 i3 Calculated daily intake of molybdenum, according to sex and age groups for the whole United States ...... 62 ...

14 Calculatea daily intake of molybdenum per kilogram body mass, Ii accordrng to sex and age groups for the whole United States ...... 63 i{. :-\I-11 .-- --

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15 Contribution of major food rategorics to daily molybdenum intake of male subjects, according to age ...... 64

16 r Plasma molybdenun concentrations of control subjects and uf workcrs in a molybdenum smelter in Denver ...... 69

17 Urinary concentrations of 14 workers in a molysdenum s-nflter ...... c 70 18 Histogram of plasma molybdenum concentrations in 42 noma1 subjects from the Denver area ...... 73

19 Comparison of the daily urinary molybdenum excretion in male subjects with different daily intakes from food and-water ...... 77

A-1 Molybdenum concentration of water required to produce a 50% increase in daily intake through water-based beverages ...... 33

I ..

TABLES

.. Number Page. 1 Some Physical Properties of Molybaenum ...... 7

2 Consumption of Molybdenum by End Use (1974) ...... --17

3 Some Use Applications for Molybdenum and Its CGciPounds ...... 17

4 Concentrations of Molybdenum in Various Rock Types ...... 19

5 Soils with Anomalous Molybdenum Concentrations ...... 20

6 Comparisoh of Dust Survey Letrels in a Col.orado Molybdenum Mine ..... 21

7 Molybdenum Content of Respirable Dust in.- a Colorado Molybden*-.m f Mine ...... 23 i ! 8 Moiybdenum Levels in Respirable Dust and Total Dust ...... 24 i f 9 Molytdenum Content of Waters and Stream Sediments ConLyninated f1. by Various Industrial Activities ...... 26 f 10 Molybdecum Content of Foonstuffs ...... 29 t 11 Urir Acld Production of Lysed Human Eryt!:rocytes Using Thin t Layer Chromatography ...... 36

I 12 Results of C14 ATP Formation ...... 37 I i 13 Molybdenum Concentrations (ppm dry weight basis) in Tissues of t Rats Given Different Levels of Molybdenum as Na2Mo04 in their f Drinking Water ...... 45 I- ..i -i 14 Effect of Molybdenum on Litter Size in white Rats ...... 50

! 15 Representative Neights for Xats on Various 3ieLary Regimes ...... 51 t a. 16 Summary of Test Results ...... 59

17 Serum Ceruloplasmin and Uric r.cid Concentrations in Molybdenum Factory Workers and in Control Males ...... 68

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18 Cuzparison of Ser*xa Ccruloplasxin and Uric Acid Levels and of the Urinary I3cretior.s of .ulolybdenum and Copper in 1.975 and 1977 in the Golden Area ...... 73

19 Canpariscr. of Scrm Ceruloplasmin and Cric Acid kvelc m..l of the Urinary Excrctions of Holybdcqun an3 Coppr in 1Lo Gro~ps r $ of Workers at Xater Treatment Plafits ...... 74

20 Cunparison of Serm Ceruloplasmir. and Uric Acid Levels and of the Urinary .%lyidcnum and Copper Excretions in Desidents of Summit County; Hales Only ...... 75

21 Conparison of Serum Ceruloplasmin an? Uric Acid-Levels and of

tho Urinary :!olybdcnum and Copper Excretions in Residents of I Summit County; Females Only ...... 75

22 Caparison of Biochemical Assays in Male Subjects frm the > .DeEv'3r, Breckenridgs, and Frisco Areas ...... 75

A-1 Variability of Daily YolybdenBa Intake ...... 93

I' t c ACDKlWLZDGMENTS

The authors grazefully acknowledge the work of many graduate students, i I undergrzduate students, and technicians. We also appreciate the assistance __A- of several official:; of local citicr and municipalities. Without their CG- operation the community tap water surveys and hunan sampling pregram would-- have been impossible. Special thanks are given to Ms. Terry Tedeschi for her expert handlino of the administrative details encountered durilg this i' study. The preparation of this rt.port was simplified and its ccntents were I. I rjreatly improved b\p the editorial assistance and many helpful suggestions of Ms. Kathy K. Petersen.

1 .. The following is a list of the authors and their affiliations: -.___+ , ,/' / Willard €2. Ch'tppell, Ph.D. Director, Environmental Trace Substances Research Program University of Colorado, Boulder, Co'n-ado Professor, Uepaytment of Physics University of Colorado, Denver, Colorado Professor, Depertment of Preventive Medicin" University or Colorado Medical Center, Denver, Colorado

Robert R. Megle.1, Fh.l). !,1, Director, Arialytical Laboratory

Envi ronmental Trace Sibstarices Research Program IC University of Colorado, Boulder, Colorado <.

I' kafael Moure-Eraso, M.Sc. I 'T Health and Safety Office '. Oil Chemical and Atomic Workers International Union (1 i Denver, Colorado

Cliva C. Solomons, Ph.D. Professor and Director of Orthopedic llesearch University of Co].orado Medical Center , Wnver, Colorado 8, i Theodora A. Tsongas, Ph.D. Clicical Instructor, Department of Preventive dedicine .=nd

Comprehensive Health Care 4 University of Colorado McdicaL Center, Denver, Colorado ! .I i f. Philip A. Walravens, M.D. I 1 Assistant Clinical Professor, Department cf Pediatrics and ! repartment of Preventive ?:ec?icine i University of Colorasiz Medical Center, Denver, Colorado

I_ ;i; J -, - -

SECTICN 1

I "wo-thirds of the elements occurring in the cartn'r; crust are mare abun- ! i dant than molybdenum, yet it is among the fifteen trace elements that are essential to plants and animals. Molybdenum is also economiczlly important as 1I I a component of metal alloys, fertilizers, catalysts, and anti-corrosive agents. i Its rapidly growing production and use represents a potential for increased I release and distribution in the envxrom:ent. The purpose of this report is to t describe the human health effects that water-borne molybdenum may havc. 1 i

** In spite of its relatlvely low natural abundance the anount of soil mo- 1 L lybdenum available for plant uptake appears to be sufficient in most areas. Geochemical anomalies leading to molybdcnum deficiencies in plants have beeq l -! i* described; hovever, nutritional deficiencies in himans have nct bccn docu- I i I mented. Therefore, the human nutritlonal requirement 1s probhbly low, or moc!?rn food distribution practices tenG to meliorate any deficiencies in I i I t local!y produced ioods. i i i I ! ! Molybdenum is similar to otter essential trace elements in that it exhibits a detrimental biochemical cffcct whcn thc animal 's intake exceeds the ! 1 optimum arnsunt. The syrrptoms, of molybdenum toxicity and the dic.tary coxfen- I tration at which symptoms oczur varies with spccier;. Pliich has bccrr published i _/-- the effects of cxcessivc nolybdcncm consumption by ca:tlc, about and the 1 mulybdenum-lnduccd disease in runinants, mo! ybdenosis, !)as hccn well docu- i 1 merited. liowevcr, little is known about the human hcalth cffccts. The fol- 1 ! lowing sections rcview the evist-ing 11teraturc rclcvarlt to thc asscssmcnt oE the hman health effects of ex~osurcto ,inornalouLily high cvncentratlons of 1 1 molybdenum. This rc*port also ircludcs the results ubtalncd by the authors '1 in a stud:, of hman cxposurcs to molybdenum in water, food, and air. Yost surface and qround waters contain about 1 ~3l.10,'L. Stream vaters draining undisturbed molybdcnuy mineral dcposi ts are also low. Therefore, waters contaiiiing nore tiban 10 to 20 pgNo/L arc uslially associatcd with human activities such as mining, upgiadinq, or ather iridustr ial proccssinq. Several municipal water supplies in Colorado cxhihit anomalously hiqh molybdenum concentrations. This report describes tk.. neasurenent of scvfsral biochenical parmeters on selected groups of individuals who ~e~e~vt?tneir drlnking water from these sources. The stud-{ also includes a11 extensive food sampling pro-

I gram to determine the moiybdenun intake derived from food consu?ption.

con- .. supplemented with relevant animal studies. The conclusions pre=ented in Section 2 are therefore based upon the best available data from the lite-atcre, <.:id tlid authors’ own work on both hurnans and animals.

Brief d6:scriptions of the chemistry of molybdenum, its production, use, c and enviro;mental fate precede the discussion of the biochemistry and biological effects. The referet:ce list does not cite all of the published work on the shject. However, it represents the bulrc of the relevant work upon which , i the conclusions and recommendations are based. , - !I ‘,Y).YAENT or+ CONCENTFSTION UNITS I: il Concentrations of trace constituents in solid samples are rzported in parts per n?.l:ion (ppn). This unit is equivalent to micrograms of analyte per gram of smple. Concentrations of an analyte in liquid samples are reported in micrograms analyte per liter of solution. This convention is convenient I since most natural waters contain less tt.an 20 ugMo/L. Concentratiorls of ten to one thousand times tihe “natural” levels are only observed as a result of anthropogenic activities. In order to emphasize the order of magnitude dif- :’ ferences between natural and contaminatec waters large concentrations have not been converted to the more comon multiples such as mg/L. , ii

SECTION 2

-. I CONCLUSIONS AXD RECOMMENDATIONS ."

CONCLUSIONS __ .- While conclusive evidence that molybdenum is required by huxans is lack- -- ing, there is general agreement that it should be considered as one of the Ii.> essential trace elements. The absence of any documented deficiencies in humans indicates that the reqtiired level is much less than the average intake of 180 grgMo/dny in the United States.

Except for industrial workers, the intake of molybdenum is a.lmost entire- tJ :? ly via fod. and beverages, including water. 4bnomally high food intakes, as high as 10 to 15 mgM.to/day, have been documented in India and the U.S.S.R. and I are suspected in Turkey. Because of the interregional distribution systeio in the United States, it unlikely that molybdenum toxicity will be encountered I ' is I in humans due to food intake. There could be exceptions where sigl;ll"icant !, parts of the diet come from one particular geographical area or for indivi- :: I i'I , duals on a limited diet. ! !. I i Natural molybdenum concentrations in grcund and surface waters are rarely I i 'Y I. more than 10 to 20 UgMo/L. Conccntratlons significantly higher than these %2 I levels are almost certainly due to indllstrial contanlrlation. Since conven- ! tional wastewater and water treatment facilities remove very little (0 to 'i 20%) molybdenum, drinking water concentratiorls will be close to those of the untreated source.

I. field studies indicate that molybdenum is a biological antagonist to copper. 'i'hus, the usual mechanism of molybdenum toxicity i:; the induction of copper deficiency. Symptoms in cattle and other animals c'dn often be reversed by the addition of sw2plemental copper to the diet. -. . .. While there are people in the Unfted states with water supplies containing greatly elevated concentraticiis of rnolybdenum, the corresponding daily 4 intakes are still at least ari order of magnitude less than those people in the U.S.S.R. who were described as exhibiting a gout-likz disease. Industrial workers in the United States are exposed to intakes that can be as high as 50 mgMo/day without violat'-q OSHA standards.

. Molybdenum in the dict and in water is readily absorbed and rapidly excreted. The rapid excretion, primarily iri urine, provides an efEective mechanism for regulating molybdenum concentrations in blood and presumably other tissues. In our stuay of humans having crater supplies containing as much as 200 \IgMo/L, we found that while urinary cmccntrations were increased, sermi molybdenum concentrations remained normal. No changes in copper metabolism were observed. However, Deosthale and Gopalen (1) have observed an increase in daily urinary copper excretion in human subjects receiving 500 to 1,000 pgMo/day in their diets.

In summary, no biochemical or clinical effects were observed in humans whose water supplies coritained' up to 50 ugMo/L. Increased urinary excretion of molybdenum wes observed in humans whose water supplies contained 50 to 200 pgMo/L. Deosthale and Gopalan (1) observed increased copper excretion in humans having daily intakes of 500 to 1,500 pgMo/day, but they did not ob- serve any changes in uric acid excretion. In a study of molybdenum workers exposed to a minimum daily intake oE 10 mgMo we found greatly increased blood and urine levels of molybdenum. We also found significant increases in uric acid excretion, but these levels were still within a normal rang& for humans. We also found a significant increase in serum ceruloplasmin compared to nor- mals, consistent vrith the results of Deosthale and Gopalan (1). In addition, we found an increased xanthine oxidase activity. However, these levels were still within a normal range for humans. Kovalskii and others (2), studied a human population receiving 10 to 15 mgMo/day. Thcy found greatly increased uric Gcid levels, decreased copper excretion, and a high incidence of a gout- like disease. They postulated that the increased uric acid excretion and gout-like disease were due to increased xanthine oxidase (a molybdenum- .. containing enzyme) which was in turn due to the abncrmsl molybdenum intake.

.. ..RECOMMENDATIONS . ,- In view of the absence of any documented human cases of molybdenum deficiency, it seems likely that the average daily inttke of 180 pq Mo is well &eve the minimum daily requirement. All micronutrients are characterized by a range of deficiency, a range of sufficiency, arid beyond these, toxicify. Since the exact size of the range of sufficiency is unknown, it would seem prudent to avoid large increases beyond the average dietary irtake of 180

\igMojlay:---On-the--other-hand, clinical effects have been reported at 2 d,OOO - - ... .~ ' . ..- .. ,_ . to 15,000 pgMo/day and biochemical effects in the range of 500 to ~O,LJOUgMo/ ...... , . r. .. , ' '1 ... .. -i ...... !' f I *. I day. The "no-effect'' level cannot be pinpointed with certainty, but we feel ! i it is probably not less t\an 5QO uqYo/day. A concentration of 50 pgMo/L in drinking water would contribute about 100 CgMo/day to the total diet which

I would make the average total daily intake 280 pgMo/day. Even for 15 to 17 year old males, the group having the largest average dietary intake at 250 -1 \ UgMo/day, the total would be 350 pgMo/day. Since no differences were seen be-

1 tween humans on 1 pgMo/L and on 50 ugMo/L, it appears that 50 pgP.lo/L repre- I (A supplementary method for calculat- '. .i sent:; a "safe" levelin drinking water. ing this level is found in Appendix A.) I rs Thus, we believe 50 pgMo/L represcnts a useful guideline for drinking

water. Our data indicate that somewhat higher levels can probably be taler-- 1. +- Where levcls above 50 l.IgMo/L occux ated, at least for short periods of time. 1 it is almost. certain to be as the result of industrial contamination. While such concentrations are not often encoankered, recent studies by ourselves and others show that some communities in different zegions of the United States I do have finished water supplies containinq more than 50 lJgFlo/L. other such 1 communities will t>e encountered in the future.

It is not likely :hat a drinking water standard for molybdenum is required. But we Selirve that 50 ygblo/L represents a prudent guieeline.

;

i 5 1 '. CHEMICAL PROPERTIES i *. .. GENERAL CHEMISTRY I?. __ . -_ The chemistry of molybdenum is so extensive that it is necessary to limit ij the discussion to chemical and physical properties that are important under :?, conditions which occur naturally in the environment or in biological systems. :: I1 Molybdenum, atomic number 42, is a transition element with the outermost ji:i electronic structure 4d55s'.. It exhibits oxidation states from 2- KO 6+. '1 1. However, the oxidation states below 2+ are generally found only in organ+ :f metallic compounds. Of the remaining oxidation states, only 3+, 4+, 5+, and '1 6+ are important in aqueous solution. Oxides and sulfides of these oxidation states are the principal solid inarganic species which occur naturally. Crys- talline oxides and sulfides are well characterized and are widely used induc- trially. Molybdenum also forms the tetrahedral poly atcmic anion molybdate, MOO^^-, and isopolyanions such as M07024~-. These anions form salts with a variety of cations and these compounds are used in industrial applications. Some of the important naturally occurring compounds are the minerals: molybdenite. powellite, wulfenite, ferrimolybdite, xlsenannite. Table 1 shows the chemical formulas for these compounds. .Most of these minerals are only !i t' slightly soluble at natural pHs and oxidation-reduction conditions. li -- (I 1: The relative stabilities for naturally occurring minerals in equilibrium r; with water and dissolved spe-ies can be illustrated by using theoretical Eh- I' pH diagrams. The species oxidation potential, relative to the hydrogen elec- !! trode, is plotted as a function of pH. Such a diagram shows the regions of stability for the various species under consideration. Figure 1 shows an Eh- pH diagram for the predominant aqueous molybdenum species (3). The shaded area represents the range of natural Eh-pH characteristics which have been measured for shallow ground waters and fresh waters (4). From this diagram it can be seen that most natural waters exhibit conditions under which I MoOq7- would be the principal stable species. At pHs below 5 and cnder more oxidizing conditions the hydrogen molybdate anion (HI.1004-) is expected to be -- the predominant species. Such Eh-pH conditions are likely to be found in some acid mine drainages or industrial effluents. Similarly, at very low pHs and under slightly less oxidizing conditions, the cationic Mo02+ is the principal species present.

When there are significant concentrttions of other ionic species such as ..--, I -_ - I iron, calcium, sulfate, etc., these species can also be included in the Eh-pH -* __ 1 diagram. The presence of sone of these species can significantly alter the i aqueous molybdenum chemistry. However, the following generalization remains: I

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Atomic number ----.-- 42 Atomic mass ------95.94 Atomic radius 7 - ---- 1.403 Density ------10.28 c3/cm3 (20OC) i L Melting point ------2620 f 10°C - i Boilincj point ------4825OC i1 Formulas of common minerals: I ; !, / / Molybdenite ------MOB 2 ___ powellite ------CaMo04 wulfenite ------PbMoOq

Ferrimolybdite ----- FeZ03- 3.. 52Mo03. lo. 4H20 IbI 1 11semz.nnite ------M0308 -- . -I_

the principal dissolved molytden~lmspecios in the natural environaen: is molybdate. For s. more detailed description of molybclenum mineral solubllity . and aqueous geochemistry the reader is directed to the work of D. Kaback (3).

BIOINORGANIC CHEMISTRY I i.t.

In order to understand t!ie biochemistry of the molybdenum-bearing enzymes !' . . 1 i! it is helpful to exa..ine the chemical interactions between molybdenum and bio- i.. logically iinportant molecules. !Jhile the exact nature of the molybdenum- I. protein interactions is irlcompletely understoodl severai generalizations regarding these interactions can be nade. The 3+, 4+, 5t1 and G+ oxidation states of molybdenum are the most important in biological systems (5). A protein can bind to a transition element through oxygen, sulfurl or nitrogen. In general, hi$-.?r oxidation states lead to oxygen binding while lower oxidation states favor sulfur or nitrogen bindinq. Oxygen and sulfur are favored over .- .. nitrogen as Ligands. This is partly due to the ability of oxygen and Fulfur to partlally neutralize thc positive charge of the metal atom (6). The neutral nitrogen in most ligands does not afford this aCded stabilization. .. .. ' The coordination number of molybdenum with most organic ligands is five or .- six. This is in contrast to other biologically important transition elements such as iron, copper, and zinc which are four coordinate (6).

Ipho.-p i 9 ro Theie is considerable evidence that the molybdenum in xanthine oxidasedase ! andana otherVCIK~ enzymes=l.u ,...--- is__ bound. through the m2rcaptc yroup of cysteine Ir;JAuueresidue (7). Bot?iBot'i Mo (V) and Mo (VI) are kncwn to form complexes with histidine.hlstlalne. The molybdenm!lybdenum enzyme:; are catalysts in oxidation-reduction reactlons. Molybdenw

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SECTION 4

MEASIIIWENT TECHNIQVES

Waters, geological solids, plants, animal tissues, and biological fluids all have complex chemical characteristics which must be considered b-fore an adequate sampling protocol can be devised. The choice of a sampling procedure depends upon field sainpling conditions, the chemical characzeristics of the material being sampled, and the purpose to which the analyses will be put. Since the purpose of this report is tc preser.t data on the human health effects of molybdenum in drinking water, we shall limit GUT discwssion to sampling methods whic'l have a direct bearing on this study. Therefore, water sampling ahd analysis will be discussed in detail.

No single method of collection and treatsent is suitable for all types of water samples. Each chemical constituent of water may affect the stability of other constituents. Preservat5on methods ,ghich ensure quantitative recovery of molybdenum may not effectlvely preserve other trace elements. Therefore, it is essential that the adequacy of the sample treatment be confirmed for each element of interest before extensive sampliric, is undertaken. The following section describes the results Tf a study of sampling factors which affect the validity of molybdFnum analyses. . Adsorption of trace elements on the surface of sample \-slls is potentially the most serious source of error in water smpling (8). The extr.nt of molybdenum adsorption, at equillbrium, depends upon the container materlal, ionic strength, pii, and molybdenum concentration (9). The maximum adsorptlon of molybdenum on polyethylene occurs at about pH 4. At this p!I about 25% is adsorbed from an aqueous solution of 5 ]1gt.lo/L. About 3% adsorption occurs at 100 ~.igMo/L. Adsorption is variable and greater when iron is present in solution at 2 ing/L. Coprecipitation or coadsorption with hydrated ferric oxides can severely reduce the recovery of molybdenum from improperly treated samples. Glass, linear, and cross-linked polyethylene containers are suitable for collection and storage of water samples when the samples are acidified to less than pH 2. Samples stored at pH 2 in these bottles for three years showed quantitative recovelies. The light weight and unbreakable nature of polyethylene containers makes them cmvenient for field collection and ship- *- ping. Bottles may be reused ii they are thoroughly cleaned with a detergent wash followed by a dilute nitric and/or hydrochloric acid wash. While no molybdenum 'contamination was found in unwashed new bottles, several other trace elements present serious contamlnation problems. Acid washing of new bottles is recommended when other trace elements are to be determined (10). used. When human or animal intake is to be determined, filtration may not be appropriate since molybdenum present in suspended colloidal m+??.rial is consumed along with dissolved molybdenum. The tradition of f.ilt&inj samples through 0.45 micron membrane filters was based upon the desire to remove biological materials; however, this practice complicates the interpretation of the existing literature.' This poorly justified filtration practice has become the arbitrary criterion for distinguishing between "dissolved" and "suspended" species. Experienced workers are aware of the tendency of membrane filters to retain species smaller than the nominal filter pore size. The effective pore size of these filters is reduced as the menhane becomes clogged. In addition, membranes heavily loaded with colloidal ixon hydroxides can strongly adsorb some dissolved species. These limitations should be considered when - __ ~ interprets data obtained on filtered samples.

Analyses obtained on uqfiltered natural water samples can also complicate pretation. This is particularly true of sam?les which contain sedissnt r large amounts of suspende3 material. These materials cm serve as . sinks €or adsorption and/or sources of elemental contamination through desorp- tion or dissolution. Smples ubtained from rapidly flowing streams often carry large amounts of material which settle' out under less turbulent'flou conditions. Sampling these wa'iers requires great care. Adequzte molybdemm analyses may be octainad by allowing these waters to settle in the sample bottle. Aliquots of the supernatant may be used for analysis. Such sanples should not be acidified since the added acid may dissolve s3me of the solid material. Samples should be analyzed as soon a; possible after collection.

s Most drinking and natural waters can be analyzed without filtration since most of the molybdenum present remains in solution as the molybdate or hydrogen molybdate species. Extremes of pH or high concentrations of other species such as iron are not likely to be found in most drinking waters. Figure I, the Eh-pH diagram shown in Section 3 of tP.is report, illustrates the stability fields of the principal aqueous species. Furtheryore, most of the naturally occurring molybdenum-bearing minerals which hay occur in sec?inients are not y soluble under the chemical condi5ions cF natural waters.

biological growth in stored samples can also chanqe the trace el*?ment content of stored sample$. Since most housekli tap waters are chlcr.nated no pretreatment is necesssry. However, untreztecl water? niay be trepted with bacteriostatic agents to retard biological activity. One mL of chloroform per liter of sample is sufficient to Frevent growth and does not inti-rfere with the analysis.

Freezing of samples is frequently recomnended for preservatim rJf tra:e elements in waters. Wintei collection of natural waters often precludes pro- per pretreatment of samples in the fjeld be'.'ore thiy freeze. Since freezifig . can irreversibly alter tha stability of somt! trace clencnts (11), a study to determine the effects 0: freezing of rno1;adenum in waters was performed. This study showed that freezing does nr,t affect thz malytical concentration of molybdenum. Treatment in the fie1.i is always che preferred method of sample preservation; however, samples which remain frozen uritil analysis can later be treated with acid in the laboratory to rcvcrsibly recover molybdenum. 11 when ircn is preserit at co:ic-t'ritrdtions qre~tcrt'!idn 1 mqF-/L poor iror! recov- ezy is obtdirird vnicfi iR ?:ur:i Affects L: c molyud!r7nllm recovery from unacidrfred iron rich water sanples. it oinpreknsive burvc-y of tk.e current literacur2 on .xmple hand1i;:g is avdrlaul I iii J:I !:bs 'rec!l:l:cJ1 !:?re (12).

A??ALYs €5

, . -- Quantitative analysis of, rolybdciiurn as a :.ra,ce co!il;ti>uerit has been . atteiaptcd by a wide variety of techniques. Xhilc! emission !jcectroscopy, x-ray fluorescence, and neutron activation havc been silcmssfully. used in the analy-. sis of aqueous sa.~ple.~,the cost arid Availability of ?!-IC; instrunentation required. by these methods prccludcs their use by the majority of analytical l&- oratories responsible for vatcr quality moriitorii:y. Fierefare, cclorimetric . and atonic absorption spectrop!ioto&try' arc the most-_xidely $sed analytical detection techniques today.

Colorimetric Methods

- .. ,a ~... , . ..-. -. ,...... I...

-..

..,. ... , . -e " -I-- ---.,., ---+-*"r-.- Yl-*u.- ---....,--*...-"- .,.. -3.". *, ,..- Tw....*-#- .."* -",.-.--*-- ."- E i $ 4 i i i ? i exchange resins have also been used to preconcentrate the sample' and to remove i interferents (17,18). Modifications and various improvements a-:e described i t in severai references (14, 19-23). \, i -Atomic ;\bsorpt:on Spectrophotomctry

I .) For most metal elements atomic' absorption spectrophotmietry (A.A.S.) is ! thz preferred method of analysis because of its speed and simplicity. How- i ever, accurate molybdenum analy.i,s by A.A.S. can be difficult and the consum- 6- - ing. Molybdenum is one of several metals which forms refractory oxides in flames. This tendency can be minhized by the use of a nitrous oxide-acetylene I reducirig Elaxe. However, even in reducing flames, the pro2ensity to renain in I an oxidized form is enhanced by the presence of other interfering elements.- I One study of mlybdenum absorption in flames has shown that 46 added ionic I species affect the signal intensity (24). The magnitude of the interference depends upon the concentration of the interferent. Therefore, complete separ- ation of molybdenum from potential interferents is necessary to insure accurate analyses. Adequate separations can be accomplished by ion exchange separ- ation or by complexation-extraction techniques (14,16,25,26). The comp1S:xcltion-extraction procedures used in the colorimetric metbods have been used for analysis of molybdenum by flame A.A.S. In these procedures the organic phase bearing the molybdenum complex is aspirated into the flame for i detection and quar.tification (25). These techniques tend to be more time I1 i consuming thar. the colorimetric procedures. L

Anothcr difficulty cncounterel with the R.A.S. determination oE molybde- nuii is its low practical sensitivity of about 500 )~q?to/L. Even with large Eircconccntration factors the dctecticn limit of about 10 UgMo/L is insuffi- cient to; m.iny waters. Dircct analysis of natural waters without preconcen- cration 1s riot pok;siblc since the detection limit of the zethod is between 59 arid 190 \J~/L. >lost. nalural arid clrinkin.~waters must be conceylrated by a factor of at least tcn before adequate irec cis ion can be cchieved.

2'lamc.;Icss A.A. techniques have recently become more widely used. In this tcchnicp~catomizati2n is accomplished by introducir..; a small aliquot (usually 10-50 \!L) of the aqueous sample into a graphite furnace or cup. The i

graphite furnace or cup is resistance heated to about 3,UOO0C by passing an ...' . electric currctit through it. Tt:c electrothermally indcccd atomization process .. replaces tnc flame ~nduccdatomization af conventional A.A.S. This method is much marc :.enFji tiw than flame A.A. techniques. Detection limits of 1 UJ/L nay be actij cvad withgut preconcentrating the sample. However, this method is also susceptible to I ritcrfercnces. Several ions severely suppress the absorption siqnal and high dissolved salt coccentrations make the detection :init is,. poorer. However, since most tap waters are low in dis+?l.ied solids, they may .* be analyzed directly by this method. With some modifications the method'is 1t I- applicable to the dctcnnination o€ molybdenum in bi-logi cal materials when 10s; concentratlons and only sm2l!. samples are available (27).

Other !4cthods--

Emission rpec:rooraphy (E.S.) has been used for the analysis of molybde- riui.71 in solid samples. However , water analysis by E.S. requires precipitation

...... ,.. of molybdenum as a complex which can be dry mounted in the instrument elec- I. . trodes. This method is tedious and time consuming (13). Inductively coupled plasma spectrometz-~,(I.C.P.S.) is now being applied to a variety of trace element analyses. This newiy developed technique is sufficiently sensitive to permit the direct analysis of waters without preconcentration. The ionic emission spectra generated in the high temperature argon plasma substantially .. .. decrease the interelement interferences observed ir tile lower temperature .. 1 .. flanes of A.A.S. Detection limits for molybdenum jn the lo4 W/L region are , .. being reported DY several instrument manufacturers. This instrumentation is . _..... -, ... -.- ...... - ... . . very expensive; however, the advantages of high sensitivity, low detection ... limits, wide dyn'amic range, and multi-element capability will make this technique very valuable for triLce elerrent studies. In Appendices B and C we describe the methods we have used .for colcrimetric and AAS analyses. PRGD'JCTION I

The prinicpal mineral from which molybdenum is cbtained is molybdenite (Nos2) (28). The product obtained from the milling of crude or'd containing molyl-denum, molybdenite concentrate: generally contains 90% or :.lore Nosi- Almost all molybdenite concentrate is then converted to technical-grade mofyb- dic oxide 34~0~)which is the base material for productioh of various chemical compounds, f errcmolybdenun, and purified molybdenum. Soae lubricant-grade MoS2 is prepared directly from the concentzate by additional grinding and 'flotation.

Yechriical grade molyUic oxide is usually produced by roasting in a mul- tipla hearth furnace at temperatures up to 600°C (28). Pure molybdic oxide is obtained by sublimation or selective recrystallization of technical-grade mclykxlic oxide {90-95% NoOj) at about l,OOO°C to l,lOC°C. It is used as a base ruterial for metallLC molybdenum and for sodium and ammonium molybdztes. Technical grade molybdic t:xide is added as a charge material or directly to cast iron and to a larae proportion of steels and other alloys.

In i976 produztion by the United States was 50 million kg (29). The total world productlon was ?6 mill.ion kg. Approximately 35% is produced as a byproduct from ores of copper, tuiiqsten, 3r.d uranLuIn; the remaining 65% is racovered fran ores prcccsscd for molybdenum. Approxirnately 60% of the U.S. production is presell~ly&+-a ired from the Climax and iienderson mines in Colo- rado vhich aie operated b:. the Climax Ilolybdenum Corporation, 3 division of AMAX I 1r.c. Other prodl1scrs includc the Molybdenum Corporation of America, Duval Sierri tal and Kcnr.ccott Copper Ccrporation. T.w primary axid four byproduct producers account for over 95% of the domestic output (30). .. The consumption of mclybdenum in 1974 is summarized in Table 2. Table 1 3 lists some common molybdenum compounds 2nd their use. i

iI! - 'TABLE 2. CONSUYPTIOh' Or MOLYB2ENUM RY END USE (1974) (23) f

4 .... - Alloy steel 44% I! Stainless steel 21z I* Tool steel . 11% 1: Chemicdls an8 lubricants 8% - --I3 ------Cast iron ani steel-mill 6% rolls # I Special and super alloys 5% Molybdenum metal 4% Miscellaneous 1% ------

TABLE -3. SOi4C USE A2PLICATIONS FOR MOLYBDFXUM AND IT'S COi.lPE!DS - I10 1y bdenum iron-base s.1 loys; cracking catalysts; iri fertilizers Molyhdic oxide production of ammonia fron hydrogen and nitrogen Cobalt molybhte desulfurization of gasolines f I!olybder.urr drsulf ide lubricant

Molybdenum pentachlorldc Tricdel Crafts chloricaticn of aromatics and alkylations; vapor phase depositjot1 I of molybdenum ccatirigs l b10 1y bd e n um ne x a c a r bo ri y 1 vapor phase deposition of rr..Jlybdenm i coatings t! M*>lySdcnuni acetylacctonate catalyst for polymerization of poly- i I urethane foam Molybd enum oxalate in photochemical systems Molybdenbl dithiocarbcunate lubricant additlvc Ammonium molybdate laboratory rcdyent for determination of phosphorus, bri>mates, cholesterol; i.

arsenic in fccds ai!- . -7- --_ FIolybdcnum tannate coloring of leather \

i~olyijd,itc~s(e.g., zinc piginencs for priritinq inks, lacquers, I --- mo lyhda t e) - paints; vitreous enamek i ....- . .~.. .. . - . .. - .. ,=__ ~,.~,-.,. _.,; - ....

.. .: ._. . . .- . . . . _.-..--.&-I.------?---' ---.

One particular use which may increase dramatically over the next few decades is in :he cacallrtic hydrocracking of coal to liquids. If this proves to be feasib1E an 1 commercial scale, then to silpply 9,490 quadrillion joules (9 auadrillion BTLs) of enerqy by coal conversion in 1985 we will need to con- vert almost. 353 million metric tons of coal (32). Conversion will require ahost 1-8 million kg of molyi>denum. Whlle this is a small percentage of the current U.S. production of over 45 millron kg/year, one estimate of coal conversion requirements in the year 2000 would iiT,ply the need for 11 mililon kg of molybdenun, for this purpose (32). Ot.her energy related uses include the use of molybdenum in elevated temperatuze ste~lswhlc!i are widely; used in steam and gas turbines, and in the steam generating sections of fossil or nuclear-fueled power plants. These and other applications are discu:;sed in ! reference 29. I ! 1, __

.. . .i,

: ’;... ,

SECTION 6

ENVIRONMENTAL FATE

ROCKS AND SOILS

The average abundance of molybdenum in the earth’s crust is or one part per million (ppm) (33). Table 3 summarizes information on the molybdenun contents of some rock types. ..

- TABLE 4. CONCENTRATIGNS OF MOLYBDENUM IN VARIOUS ROCK TYPES (33) ‘ Rock type Range - Average - -____----_---ppm ------

Basaltic (igneous) 0.9-7 1.5 Granitic (iqneous) 1- 6 1.6 i *... Shale 1- 3 2 . , !: Black shale 1-300 10 i f‘ Limestone --- 0.4 i Phosphorite 5-100 30 i 1 i f i Economic molybdenum deposits contain 200 ppm Mo or more, with the lower concentrat~onsgenerally mined as a byproduct of copper mining. Oil shale from the Green River formation in Colorado contains approximately 30 ppm Mo

The total molybdenum concentration in soils can vary quite widely, but most soils contain between 0.6 and 3.5 ppm 140 (35). The average content of most soils has been reported to be 1 to 2 ppm No (36).

.. i I

I quite high. Table 5 summarizes data on some SOLJS containing naturally high i* 1 i levels of molybdenm and others which are high be-ause of contamination. i

TABLE 5. SOILS WITH ANOMALOUS MOLYDPENUX CONCENTPATIONS - I * -1 SourLe Range Mean

I __ ------ppp, ------___ fl Natural sources: Soils covering a mineralized area (38) 27-190 i 76

1. Soil derived from a marine'black 2-85 - 12 I shale (39) _. i i Alluvial soils - eastern footslopes 5-32 17.4 1 of Sierra Nevada (37) I I Soils formed frcm volcanic ash - 13.5-18.6 14.9 Kauai, Hawaii (37)

Industrial sources: Soils downstream from a molybdenum mine 1.3-4,250 59 and mill - Colorado (38) Soil irrigated with water contaminated 49-72 61 t by a uranium mill (40) Wo miles from molybdenum smelter - Pennsylvania (41) 10-7 2 29 - --

elevatedClearly, molybdenum both naturalconcentrations and industrial in soils. source<; Whcrecan concentrations give rise to extremelyexcc-t-d 3 '/

to 5 ppm, a geological anomaly or industrial contaalnation is the likely BX- / planation. Soils adjacent to mineralized areas generally show elevated concentrations of molybdenurr. and offer a rcliable guide to mineralization.

CpncentratiorlsI of molyb3enum in ambient air are quite low ranging from below detection limits to 0.03 mgMo/m3 (42) . Thus, under ordinary conditions there is a negligible contribution from air to human and animal daily intake i of molybdenum. I .. i Industrial Exposure - Minincj and I4illlng IL : ! lybdenum.In the Various industrial environmental setting, therelevels can haw be beena considerable rzported in exposure mining opera-to mo- --.-- __ tions. In Table 6 levels of dust are reported that were measured during two - extensive surveys of a molybdenum mining, crushing, and milling operation. ih- -

., 20

I I ' ... :: .._. .I

TABLE 6. C(JP1PARISON OF DUST SURVEY LE?'L'IS IN A COLORADO MOLYBDENUM MINE !43, 44, and Personal Corrmunication, Wenster Area National Laboratory for Occupational Safety and HealLh, July 1975) -,-_I_--- -- ...------_- - - -.--- 1953 PHS study totGust irpinger method - 19'75 OCAW study Operation No. of Range of dy-19t Avg . dust concen- No. of Respirable Avg . respir- samples* conccntratlon MFFCF;' tration NPPCF samples!! dust conccn- able dust tration concentration mg/m mgjn' - -- - Miri i ng 439 71.6 - 0.3 %Si02 14.4 45 1.230-0.104 0.471

Crushi ng 61 98.3 - 0.4 36-39 7.5 , 11 6.079-0.275 1.318 I ' ?.I u Xili ing 33 3.9 - 0.3 1.9 '6 0.210-0.046 0.142 ! 1 Sclrface shops 35 74.1 - 0.3 4.5

Open pit ------14 0.682-0.129 0.318 I -- i I. * I Ten minutes stationary samples 1 I ! The milling operation is remarkably less dusty t:ian the others due to the high percentage of water used in the process. However, the proce.-ses of drying, packing and loading are potential areas of exposure to dust containing high percentages of molybdenite (90% bloS2).

The data of the 1959 Public Health Service are difficult to use to estimate Fxposure to molybdenite because the sampling techniques used counted the particles without determining their mass. Therefore, it is not possible to determined the gravimetric concentration of molybdenite in the airborne dust. -._. In our study stationary 10L samplcs were collected over 10 minute periods. The Oil Chemical and Atomic Workers (OCAW) 1975 study deternined the respirable mass of dust at the breathing zone of the worker.* Each sample was collected for 480 minutes (a full shift) and represents about 820 L of sample / .air. The concentration of molybdenum in the respirable dust collected in the I i OCAW 1'375 study are shown in Table 7. It is possible to estimate the exposure to molybdenum in the mining and processing of molybdenite by assruning that a "sthndard man" inhales 10 m3/day during an eight hour workday. This estimation is also found in Table 7. .!

Industrial Exposure - Smelting

In 1975, the environmental levels of molybdenum were measured in a smelting (roasting) operation (45). Settled dust, impinger samples and high volume, respirable size, filter samples were collected. Conce2tratior.s of molybdenum, as Moo3, in settled dust samples ranged from 57% to G1%. The quantitative analyticai method was colorimetry. The molybdenum species were confirmed by x-ray diifraction. Three impinger samples encompasslng the ! roasting operation gave concentrations of Moo3, which ranged from 3 mg/m3 to 33 mg/m . About 180 L of air were collected and passcd through the same Sam* pling trains consisting cf two impingers in series. Based on a time-motion study of the exposed workers, thc time-weiqhted average was found to be 9.5 mgMo/m 3 .

In orcler to estimate respirable fraction, lhree hiqh volume samples were also collected in the same areas whcre impingers were placed. X-ray diffrac- tion was again used to determine the molybdenum species. Moo3 was the idsn- tified compourld and its concentratio;. in the collected dust varied from 52.4% to 77.9% 1400j. The concentr,;tions of molybdic oxide were then determined by spectrophotometry. Values ranged from 4.6 to 1.1 r,:q?lo/m3. For an estimation of body burden, see Table 8.

No reports of levels of exposures in industrial operations using molybdenu- in the Unit& States were found in the literature. : ! A few U.S.S.R. studies (46,47) report environm*?ntal concentrations of \2.? Moo3. Fcrty samples were taken above a crucible and in the breathing zone of

I t

* As defined in Interim Guidc for Respirable Mass Sampli?g AIliR Journal, Vol. 31, p.-133, 1970. . ___ .. 22

_- I &+- 4.. 4.. 1.1--.r--- ..c--.l(lr

i . 'I

I 6. . I . . ..

;. ;. . . ..* ,. . . . . , ...... -. _*___.______A .-.. .- . . .-.. .. . ____ ~ ...... I ,. ..,

TABLE 8. MOLYBDENUM LEVELS It1 FESPIRABLE DUST AND TOTAL DUST (45) -- Base of roaster First tier Second tier -- Respirable dust:

Dust concentration (mg/m 3 1 1.33 3.01 6.25 Percent molybdenum in dust 77.8 52.1 71.0 c Molybdenum, content of dust (mg/m3 ) 1.32 - 1.56 4.39

Total dust: ,

rngMo/m3 of air 3.04 9.11 33.28 - __ Hours-exposure/worker* 4 1.5 1.5

8-hour TWA = (3.04 x 4) +-(9.11 x 1.5) + (33.28 x 1.5) = 9.47 mgMo/m 3 -8 -- ____ * Does not total 8 hours since there is virtually zero exp(xiiu';p during 0.5 hour lunch break and two 0.25 t.our breaks.

workers during a smelting operation involving ~1003. The conce11tratlcns of Moo3 in the breathing zone of the :corkers involved in tne process averaged 0.22 mgMa/m3. Hsghest values reported WC:~ C.4 to 0.5 mgMo/m3. The area above the crucible was also sampled showing con-entrations from 1.4 to 5.4 mgMo/m3 depending on the molybdenrlm coritent of the ore. In another factory ,rroducing high 1;qJrity molybdenum the air conccntratlon rarigcd from 6.4 to 10 rngMo/m3 of Man.13. The analyzical method was culorimet.ry but no description of the methods of' sample collecti.on 1s reported (46).

Another U.S.S.K. studv reported sir corm?ntracion in two chei.iica1 plants producing molySdenun salts. The range of concentrations found in the first plant was 0.5 to 200 ?gt.lo/m3 of mO3. 111 I-hc ,zcond plant the cmcentrations were 0.2 to 30 n,qMo/m'. No analytical metl1~3sor sample sizes are giver? (47;.

Clearly industrial exposures can lead to a greatly increased daily int.ike of mclybdenum. Indeed, the prcsent OSHA standard of 5 mg/m3 soluble nolybdenum would, over an eight-hour prrlod, lead to an average intake of 50 mg which is more than 200 timer, the normal himan daily intake.

WATER

It is estimated that 3.G x 10" grams of mclybdenum are released per year into the surface waters of the world by natural proce$ses (48). Conce!ltra- tions in most waters are less than 20 \Jmb\o/L. Average rnolybdenum -*-

-. I. I 1: )j 1- I concentrations in sea water rarge between 4 and 12 vgMo/L (49,501. In 1963, , Durum and Haffty (51) estimated the median inolybderlum content of major North I =American rivers to be 0.35 @Mo/L. In an extensive survey of CJlnrado surface L f E waters, Vogeli and King (52) found that 87% of 299 samples from 197 stations coiitained less than 10 \JC$dO/L and concluded that concentrations of more than j 5 UgMo/L in the surface waters of Colorado were probably due to molybdenum k mineralization and/or molybdenum pining an6 nilling. i* 1 *;P Vinogradov (53) found a normal mo1ybde;ium background concentration of 3 pgMo/L in the groundwaters'of the U.S.S.R. A recent study of groundwaters 1. - -I:t in Colorado by the U.S. Geological Survey (personal communication) found that i .E 98 out of 156 samples cental-.-d less than 1 pgbio/L--only 10 simples contained more than 10 pgYo/L and :he highest concentration found was 28 vgMo/L. - ._- E Runnells and Kaback (38) compared waters driining highly mineralized t areas with waters draining areas containing normal concentrationa of molybde- 1. nun in rocks and soils. They found that molybdenum mineralization did not I contribute significantly to molybdenum concentxations in surface waters. (Most ore bodies contain molybdenum as MoS2). Water from streams drajniiq highly mineralized areas rarely contained more than 1 to 2 \igt.lo/L. dith the b exception of surface waters that are very aciciic and have a high content ,I -3f ! particulate xz-rric iron, molybdenum occurs principally in the form of a truly I dissolved, filterable species (54). Norma1 concentrations in stream sediments, as measured in the -80 mesh i fraction, are reported to be in the range of 1 to 5 ppm No. Concentrations f ranging froin 1CI to 200 :pm Mo have been found in sediments fron strearrs that drain relatively undistLrbed nataral deposits of molybd.-num in the Uaited . States (38). Sediments derived from black marine shales in England may con- i tain as mu.=h as 300 ppm 410 (39). The concentration of molybdenum in stream . sediments has been shown to increase with decreasing grain size (38). Thus, stream sedimen-ts, as opposed to water, do reflect mineralization.

1 Contamination of surfirce and ground waters has been documented in several i studies. Kopp an3 Kroner {SS) corducted an extensive sampliKy of water fro= I rivers and lakes of the United States from 101'2 to l'.,.i7. Water smples were I taic.>n from 100 sampling stations in the vicinity of 1. .ghly populated areas, I i industrial areas, recreatianal use areas, state and iidtional boundaries, and t ' otiier potential problem areas. Of the 100 stations sampled, 38 had ma-rimum I concentrations of molybdenum in water greater than 100 iJgMo/L and 26 stations nad mean molybdenum concentracions in water samples greater than 50 lJg140,'L. i The detection llmit for molybdepcm in this study was 4:) JlgMo/L. t .* There are many industries involve3 in the production and use of molybde- 1 num compounds. These i;.dustries may be sources of n;olybdenum contamination l of the environment. The only ones that have been studied in detail are molybdenun mizing, milling, and smelting; but siqnificant contributions of mo- l lybdenum to t!w environment can result from uranium and copper mining and t dlling (56), shale or1 production (34), and coal-fired power plants (57). The concentratioris of molybdenum in tha. aqueous effluents from these zources i can be as high ac 850 rngMo/L (a uranium mill) (58), and the release rates can I I be as high as l00,GOO kg of molybdenum per year (a molybdenrun mill) (58). I I __- - _-I 25 j

._ - -

-.-._ _-- - - I - .*-- ,u...- L...... -PI_ - _., 1

1 , .. .. .,~...... ,~.,.. ... , ...... , _.. -.. ._..~ ... - ....

of this seccion. I:!dustriil Sources.

.. Ground water dowr. qcai'c lent so. ooo ' .._ from uranium mill (Colo.1 r, I%

i water supply fron a stx5axn draining J molybdenum mine and nil1 site, a!id i' during 1971, tap water samples contained an dveraqc of 4-10 !gCIdL. During + -. f late 1974, molyWcnlun con:entratrons wert. still averdyinq 440 \Jq$lO/L, when I the mill ceased operation. klowever, by .Jar.uary of 1975, the tap water molyb- denunr concentrations had decreased to about 159 ;!qUo/L. In June 1975 the con- centratLon was found to be GI) pg.'lo/L. The decrease was probably due to the sprtng rumff. The concentration of molyldr?num in tap water samples from . i '\ Goiden during 1377 had a mean of 30 pgHc/L. I , .. ..4 . Another suburban Denver community receives watez from the same source, but stores tne water ir. a large reservoir prior to fir1:shirtq 2nd distribution. I , Tt.e molybdenum concentration of this community's domestir watcr has decreased- i at d slower rate than that of ;olden, to approximateiy 83 pgrclo/L by October ., 1977.

Frisco. Colorado, a mountain ccmnunity, derives Its domestic water from Ten Hile Creek, which drains another large rnolylxlenua mining area. Silver- thorne, ColrJrado receives its watfr from the Blue River just below DillGn Reservoir. Tap wztcr molybdenum concentratiorls in rhese two cmsnities have varied between 100 and 400 Ug:.!o/L fron 1974 to 1977. I

LXnver, Colorado dravs water for domestic use from D~llo:iReservoir at .' .. . , certain times of 'tie year. The concentration of.molybdenum in Denver's tap 1 water varies considerably, dcpcndlrlg upon t.dp locJ.'iion and time of year. Since 1'375 thc molybdenum conccntrations in tap watcrs in Deiivcr have varied Sctvccn less than 1 ~igMo/L and RO :iijNo/L. Barrictt and others !64) reWrtr>d nolyldcnm concentrations in somc knver tdp eiatcrs as nign as 1.13 \iqt-io/L in 1369. Grcatl~oi~se.>ad othcrs (62) report a mdximum value for tap waters sim- pled irr the Uilitcd States of 276 !!glo/L from a Dcilvcr tal3 in 1975.

.. It can be coricluded that caivzentrations of more than 20 )13Mo/L in surface, grciuiid, or tap watt?rs are very likely bc anthropoqenlc. 1. . to . ,'.i

PIASTS

Molybdenum plays an important role as a micronutricnt for plallts. 331 i942 molybdcnum was discovercd to be a 1imit:ny factor to clover ;xoduction in some Australian pastures (65). Microorgafilsms require molybdenum for ni- trorjw fixatlon and for the eiizymcs which catalyze the reduction of nitrate to nitrite. Ilolybdancm is riow a comon component of fertilizers in many .* parts of the Vnitcci States. Several scil parameters can influence the availability of molybdenum to plants. These include soil pll, soil moisture, sulfate, aFd phosphate. Mo1ybdc:num availability is generally higher at hiqh pH,

.. 1 ?ow sulf3tc, high moisture lcvels, and hign ijhorphatc (37,GS).

Khile normal concentrations of molybdenum in plants arc 1 to 2 ppm, a rar?ge of tenths of a ppm to hundreds- of ppm have been obazrved (65). LegUmCs appear to take up more molybdenum than other pia-its. Til 1338 Ferguson and co-xorkers (66) reported that a sever? discas,: 111 cattle was cause by abnor- Ii i mally hiqh concentrat-ions of molybdcnum in forage. When the mo:yLc'crlm in for- - age excccds t can-advursciy affcct the health of Trazinq c~ttlc(3'9,671. i -- ~

27 3 i i I. L --- 2.4. d I( . , ', '. '\. I -1 i / 1 The additions of amounts of molybdenum ranging from a few hundredths of a kilogram per acre per year to several kilograms per acre per year (dekending on various soil parameters, climate, etc.) can significantly increase the molybdenum content of plants (68). Irrigation with water containing anomalous concentrations of molybdenum can lead to vlant Uptakes that could be deleter- 8 ious to animal or human health if these constituted an important part of the diet (69). The Water Quality Criteria Committee (70) recommended a maximum concentration of 10 PgMo/L in irrigation water for ccntinuous use on all soi1.s.

Vlek (71) and Lindsay develoned a model for molybdenum uptake that pre- dicts molybdenum concentrations in plants as'a functior. of molybdenum in irrigation water arid other parameters. For a common Colorado soil they found -I _...... that the use of irrigation 3:rater containing 100 ug!-!o/L would lea? to plant . i -..j concentrations toxic to cattle in apprcximately 15 years. I ! ! .-., j _. FOOD i 1 The molybdenum content'of food varies with the type of fmd and the geochemical region in which it is grown. IegUmes, cereal grains, leafy vege:a- : bles, liver, and kidney beans are among the foods which usually contain higher . ..A' concentrations of molybdenum than fruits, root and stem vegetables, muscle meats, and dairy products (72).

Deosthale and Gopalan (1) found widely dj Cfering molybdenum contents (0.21 and 1.39 ugMo/gm) in two varieties of Sorqhwt vulgare Pers. grown and used as an important dietary staple in same regions of India. Indian rice was reported to contain about half as much molybdcnm as the sorghE-i (i3).

Other irivestigators have found. great variability in the molybdenum con- teCt of foods (74,75). The variation in content was as grcat ,in Sdm!JleS of the smie food from within a region as it was between samples of the same type of food grown in different areas of the world (75).

rnAsongas tnd co-workers (76) in our study detelmined the tnoiybdcnum content of foods collected in a market basket sampling program which smpled foods from six major supermarke.t chain stores in the Denver, Colorado metropolitan area. There was very little variati.cn found in the molybdenum content of particular food iterras from store to store, and so seasorial trands in the molybden9 content were apparent in sdmples collected over one-and-a-half years. i I .I .-. In our study (77). 10 food itens makirg up the greatest bulk in the 'typical American' diet were saxpled most extensively. Of these, 'white' p. enriched bread and eggs contained the highest concentrations of niolybdenum or1 a wet weight basis. Ground beef, iccburg lettuce, and apples were lowest in molybdenum. Analysis of supplemental samples provideJ data on the molybdenum concentration.oi foods in each of 24 U.S.D.A. food categories (78) (see Thle 10). -. .. .. Data on the average molybdenum concentration of milk samples (whole, ., - . . . . skim, and 2% butterfat).collected during this study aqc consi;tent with those \> ./ __. - j

I 2

TABLE 10. MOLYBDENUM CONTENT OF FOODSTUFFS - Mean Mo pg/gm wet wt. - I1 hlilk, milk drinks 0.06 cream, ice cream 0.06 i Cheese 0.11 i i Eggs 0.086 i Beef 0.04 1t

Pork 0.029 . 1

Meat mixtures 0.039' ' Poultry: turkey, chicken 0.047 Fish -< 0.01 Legumes: dry beans. pea?, lentils, 1.63 mixtures rhts, nut butter .. 0.02 Fats, oils < 0.01 Breads 0.21

Bakery 0.27 Cereals: cooked and rezdy-to-eat 0.55 Mixtures: pastas, spaghetti, macaroni 0.41 Toma toes 0.039 i Citrus fruits -< 0.01 i- Other fruits, fruit mixtures 0.036 Earb green vegetables 0.03 Dee? yellw veqetables 0.24 Potatoes 0.065 Other vegetables , mixtures 0.15 r- Sugar, sweets -< 0.01 --

- previously reported from Colorado (63,7R), California (79), the United Kingdov (80), Germany (81), and other areas (82). Average valuss for milk sampled in ail of these area; variad between 20 and 60 pgMo/L. Several investigators .* b3ve found that changes in the dietary molybdenum intake of cows do affect the molybdenum content nf milk (82,83), and the concentrations can vary quite considerably on a local Level. liowevcr, when compared with other foodstuffs, milk does not usually contain high concentrations of molybdenum. Lt is im- prtant to note. however, that the greatest portion of the dietary molybdenum

39 . .-. - I ~ _.__-_.-~ .._____ ~ -._.-__ _r* ."-**-. e-- ---,-. .-.------.

-I intake among children in the United States is provided by milk (76). Milk is consumed in greatly varying amounts by different segments of the population of r thc United States. 11- is therefore important to consider the trace element I. content of foods relative to their contribution to the total dietary intake i when examining the impact of trace elements in the diet. Further information on the daily intake of molybdenum can be found in Section 8 under Biological Effects of Molybdenum in Humans.

.. i., INDUSTRIAL SGURCZS

Coal Corhustion

Coal combustion is probably the largest source of molybdenum to the at- mosphere. It has been estimated that 550 metric tons of molybdenum were emit- ted due to coal combustion in the United States in 1970, as compared to 900 metric tons of molybdenum emicted frcm all air pollution sources (80). The molybderium conceiitration in coal varies widcly with the concentrations in western coal ranging from 1 to 15 ppm (57). Mass balance studies indlcate a successively increasing enrichment in the outlet ashes. A ;ingle 1,600 mega- watt power plant may emit 909 rr.etric tons of molybdenum pe'~year (57).

Kaakinen (57) investigated the molybdenum concentrations and bioavailability in bottom ash and fly ash. He found concentrations ranging from 3 ppm in the bottom as11 to 37 ppm in the fly ash collected by the electrostatic pre- cipitator. Approximately 15% of the molybdenum in the bcttom and fly ash is available for upt,-ke by plants. Dreesen and others (84) found that approximately 60% of the .nolybdenun in the coal ash from one plant in New Plexics could be extracted from the ash obtained from the ash pond et this plant.

Thus, as the use of coal increases, especially with the impetus to change to coal from other fuels, the emission, both aqueous and atmospheric, of molybdenum from this source will become increasingly important. Since the release into tha surface waters of the C.7ited States averages about 1,80C metric tans per year (85), the atmospiwcic emissions alone due to coal combustion were approximatcly one-third tho total background value in 1970.

Other energy sources swh as oil shale (34) and uranium (58) afe also sources of molybdenum contamination. Some spwific instances involvinq uranium mining and milling are discussed in the part or1 Uranium Mining and P!illing.

-Molybdenum Mining and Milling

Three molybdenum mining and xnilling operationc have been invzstigated in detail: the Climax and Urad operations in Colorado and the Questa operation in New Mexico (38,581. While these facilities dealt with the mining and concent ration of nalfbdenum as MoS2, wlricirl is very insoluble, sufficient solubil- ization occurred during the operations to lead to concentrations on the order of 1,000 to 10,000 ligMo/L in the waters that are decanted from the tailicgs. The quantities of water released to surface streams depended on the size of the operations, on the ilmount of water which was recycled, and on the quaiitity . of precipitation-and. run-off- into the tailinqs pond?. 30

.... I. .. .c

The larg?st releases which were seen were thos;c from the Climax mine in .- Colcrado. Becituse of large quantities of water from snow melt, the operation had to release 3.7 to 6.2 billion liters 2er year (3,000 to 5,C100 acre feet ' per year) of water from the tailiiigs pofids into the receiving strcm. Th? total molybdenum release was about 100,000 kg in 1972 The result was a ? (86). sianificant increase in thc mol}cdenum l.*vels in surface waters downstream. L 11, 1972, Dillon RcSer-Jair, which 15 about 13 miles downstr.-arn from Climax, had an average molytvlenum concentration of about 300 Jcj>io/L (86). Climax is now taking steps to reduce i:s release to about 6,800 kg l'er year by diverting rur<- ,. off water around t?.e Lailin

The Urad cperation (whicli ceased production in 1974) and the Questa operation a;e much smaller than the Cliaa:.. opr-'tion and have much smaller release ..- -. rates. Concentratio!ls of molybdenum in r eccivinq waters for these 0psratior.s ' vary from 560 ;!q?:o/L to 1,500 mrj>lo/L. Since the receiving stre&- for the New Nexico opcratiorx flows into Lhe Rio Grande and is greatly diluted before any significant hunan use occursI the only concern with this effluent is that of potential impac'.s on li*rest-ock through irrigation. The release from the Urad mine bid lead tc very Iirqh sc.ncentrations (-IOG to 500 IigMo/L) in the tap Thc concentration of xolybdenum in these water .- water comnunitic?; Cowt1strei.m. 8- supplies has decreaseti sub-tantidlly sincc the operation ceased.

-Molybdenum Sne 1ti 11% The most thorwyilly documented study 3f environnient+l contamination d~e '. to molyixlenurn smcltincj was reiwrtL*3ily iiornick ailcl others (41). They rnves- tigated a.1 drc<-isuri-ouridl:?q A plailt- located in we:;t.cri1 Pcrinsylvania. Stack emissions, wiiicn se:~1cport.er1 .st 25 tcj 100 kcj p~rad:!, hid led tcj increased molyldenun; concc,rit:xatJons I II soil:; and plai.t$ in ::le area surrouridir.g the _I plant. 5011 co::ci":if.rittiofii'; in nc.ar!;y farms were hlg!~as 72 ppNo and mo- 1ybder.osi.i WJS rc2ortcd in some cdttlc. 11: bdditio'i, one sample of water from -- a nearhy stream was found to corc-nin 1,000 ;~qt.lo/L. Sear a r.olybd&num smelt~nq plaiit in the I4etiierlaricIs fol-acjc? wa? found to cO:it-i\iII m31ybdenura concentrations as high (is %@ i'pn (87).

Cranium Nin; :1'7 arid ::illi:irj ---- ,

Molyhdenuni is freclucnt1:i found in liirjh concentrations in uranium ores. major cont'?mir,ant in the effluents from uranium , As a rcstllt it is frquently bpcratiorlr;. Three c;ices of ~ubs~~ntidlmolyldcrium contanination of the cnvi- These ronmcnt asso'ci'ttcd witli ur.dnIzn niniiiq anti mi 1Li:iy 11a7:2 ken reported. involvcd a draniurn nil1 iri ?:orth Dakota (ad), a uranium open pit mine in Texas /# (89) and a uranium rnill in Colorado (-58). _-

The North L)akr,:a okcrcition involvcd A plant where a Jranlfcrous ligrri tP coal W~Sasiicd to upgra2e the uralium cont-c:it. I'1y ash relcased from the plant gave rise tr inc:'eascd soil and forayr. levcls of molybdenum in a tiearb./ farm and a scbere problem of molybdenosis in catCle. --__ . -. -_ The Texxq c3se involved open pit ~LI:.s wh1ch intcrceFted shallow aquifers. Elevated mo1;rb;cnLn CCI'LCCII~rations in surface w,iters, soils, and forage were I eporreci and nolybd!cnc>sis 1 n livestock was documented. 31

I I ./ . ..4

- - ______lll_ ----..--..--.,.- __I I, I 1 i1' / .i ----;-- - ._ I 1 /'- I ...... ,...... I .

...... -...... ,...,...... _- - .. ~ -.--...... _-_- I ..

The Colorado mill beyan operation in 1953. In 1965 farmers located down hydroloqic gradient: from tic mill reported a decerioratron in the health of their cattle. A study of tbe mill and the surrounding area indicated that leakaqc: from thc tailings pmds containing 85P,OOO ug!!o/L contaminated the underlying aquifer. The water from the farms' wells used for irrigation and stock water conta~ncdas much as 50,OOG liqMo/~. Forayc taken from the farms was shokn to contain as mucii as 300 F~TMa.

Aqueous effluent from ziome uranium operations in Hew Mexico are reported to contain as much as 1,000 ~i$-!o/L, (personal comuAiicatlon). Uranium mining . and milling, therefore, can contribute significant amounts of molybdenum into the environment, particularly as an aqueous eff lwnt wh1,cn can affect drinking water quality. .1 Steel and Copper Millinq, Oi? Refining, and Claypit :-lining ._ ! While the largest use of molybdenum products is in steel, very little is known about emissions from steel plants. One study reports highly elevated concentrations of molybdenum in forage accompariied by molybdenosis in cattle downwind from a steel plant (90). . -_ In some copper milling opzrations, molybdcnum is the major contaminant in the acpeous eff1uer.t. Concentrations in the effluent havc been measured to be as high as 30,000 $g>lo/L (91).

I Si ce mo1ybdenum-conr;lirling catalysts are used in the refining of OL~, oil refineries represent anotlicr pssi source of molybdenum into the environment. Ore such incident involved a fincry in England which was reported to time lost 0.45 to 3.6 mctric tons of molyblc~iiunper month during i9GO and 1961 (32,33). Elevated molyhdt-rrun concentratin7:rs in forage, due to these emissions, resulted in nolybdcnosis of cattle.

In Missouri a claypiti was fsund to be thc source of molybdcnurn which caused severe problcns in a nearby herd of cdttle. Some plants in the area were fo~mdta contain as much as 750 ppm Mo (33).

IU3IOVkL TECiilJOLOGY

The nost thc -*igh study of: the removal of molybdenum by conventional water dnd wastcwatcr treatment was rtportcd by Zemclnsky (34;. In this study tw2:ve wntcr treatment plants, ten wastewater treatmenc plants, .:r.3. two wastewater t-ertiary treatment I ilot plants were sampled at vario>is tixs over a one year period. The samples here analyzed for 19 trace metals including molybde- "?'e removal of molybdenum in conventional plants was very low, averagirig nun. ~-/ only 15%. The plants studied included the Alvdrado Plaiit in California which supplit%spart of th? water for Sar. Diego as we11 as treatment plants in Boulder, Denver, Golden, a:id other- communities in Colorado. The plant vhlch had tile highest nnolybdenum concenkrations was the Golden, Colorado plant. At that time the a':crage concentration during the year was 360 ~g!.lo/L in the in- fluent. The avc-rage removal tor the plant was 14%.

32 _" r I_I ___-I_ 1_-_-- . . __ . --I_____- I___.. -*.. _-_.- <--.----. - i 1: I !? I I i I f Zemansky concluded that. t reason for the consistently low removals of I I molybdenum was t!ie fact that molybdenum is present primarily as the molybdate I I anion. Since many colloids present in water are negatively cha-ged, sorption i f and removal of molybdenum does not occur, except perhaps where an excess of i alum is added (3 situ;tion which is generally avoided). i t _I Zemansky also found th-lt most wastewater treatment plants have a very low i percentage removal of molybdenum. For most plants only 4% to 16% removal was i i observed. Carbon adsorption was observed to be moderately effective (about i I 50% removal) at the Lake Tahoe plant. j-i I. .: I i Runnells and others (95) showed that significant quantities of molybdenum ! ! were removed from solution below an outfall of acjd mine drainage. They con- !.I, I cludzd that the presence of ferric iron and the low pH led to the insolubili- zation of molybdenum. Zander (96) demonstrated that this technique could be 1 used to remove molybdenum from industrial wsste streams. The process used

I involved the addition of ferric iron and subsequent dissolved-air flotation. -I Renoval efficiencies of hetter than 99% we-e obtained. Typical molybdenum I concentration in a treated effluent which initially contained 15,000 pgMo/L I i : WdS 110 ugMo/L.

I One molybdenum rrining and milling operation is using ion exchange to

treat their effluent. They report removal efficiencies of approximately 98% I I in treating water containing 6,000 ugl"lo/L (persona! cammunication) . I i Ii t 1 ! 5

.. 1 ...... -. I. I ?.

...... -. SECTION 7 1 I. I-? BI021JEhlISTRY AND METABOLISM ._ - -- -. - -!-: BIOCHEMICAL FilNCTION !:

.I The biology of molybdenum is a vast topic which is described i.n the sci- . entific literature in several languages. No attempt. will be made here to give an exhaustive treat!.ent of the subject-, especially as several excellent recent reviews are available (50,82,97). Utilizing these sources a brief outline of the biological function of molybdenum is 0resen:ed with special reference to our.approach to molybdenum-related effzcts in toae, gut, arid bhod cells. , . .. Molybdenum is. 1-apidly absorbed ;:.:om r0.e-i rlno water by gut and placenta (98) when present iAs the moblydate ci' i:ricxide., but not as .the disulfide, and it is rapidly excreted via the uri.i& in inan (-73). Exogenous or endogenous inorganic sulfate spJcifically ir.crcctse.s molyl'3+?iil.:m excretion and reduces -its retention in tissues (100). MolybdenLm alao affects the utilization of copper--increased molybdenum intake. resc1tir;y in copper depletion and vice 'versa (101). The protective action of cupper 2nd sulfate on molybdenum toxicity has Seen utilized in the thersp )]tic: ximiriisti:at.icn of copper sulfate to

livestock' suff&ring from molybdcnosis, ' '; 32). Ti?riqstat.~is' a rnolybdenun anfag- onist and can fnduce a functional mol\ ~d;:iwn 2efici.ency (103) Molybdenun may .' 1:: . I1 mediat2 the release of iron .from intecti n~~:.:or-;.?(104) hut the importance 'I ,... 1 .. . .. of this effect is being questioned. Moiybd'tntim is wilely distributed in the :: .i tissues of the body ranging' from approximately 3 pprri ih ir: liver to approxi- I mately 0.15 ppm I40 in lung, brain, arid muscle ('82). . The skelet?ri ccntains ;I...... over 50% of the total body molybdenum, Fres~Jmiblydue to the large surface : '7 .. area of the miners% phase of bone and the possibi.lity of phosphate-molybdate .. ! exchange reactions in hydroxyap&tite crystals. Hone, tendon I. and cartilage i. abnormalities, as well as osteoporosis (105). have bee-n Zeen in animals with molybdenosis. Toot-h enamel 'h.rls appreciable quantities of molybdenum of the .. ..:. ,.. order of 5 ppm' arid xas thought:. tr3 confer a degree OF' czries-resistance, but this has &en disp::ted, ancl the cffccts seen are ,thouqht 1.0 be due to an ef- . fect of flLoride alone (i.0~). II .- Molybdenum in blood. is present in the plasrncr and IS also firmly bound to re3 cells (107), values of lcss than 5 to 1s ppb being conmon 111 the general population. The homcostatic control of molyI>dcnum is not as efficient as that Eor sodium and potassium since moly?x?cnum levels several tims the normal range are oDscrved in ind1:strlal workers ex?osed to nolybd-nun (IOC).

The essential biochemical fitnction of molybdenuiin is related to tne activ- a-- -- ities o'l the enzymes xznthine oxidase (109), dldehyde oxidase, sulfite oxi- - -_ d;se, nitrogenase, and riitrate reductase (82). Ma1ybAi.num participates in the >

34 ; I

-i I i , 1, ?' ./

I reaction of xanthine oxidase with cytochrome C and facilitates the reduction of cytochrome C by aldehyde oxidase (110). In milk, xanthine oxidase has two inolecules of FAD, 29. atoms molybdenum, and 69. atoms Fe/molccule of protein. Three isozynes of xanthine oxidase are present in milk. One of tkese enzymes (K02) contained only molybdenum whereas the other two (KO4a and KG4S) contain- .. .. ed copper (ill). Adaptation to feedin9 molybde,ium or ccppcr occurred by .. chmging the metal content of these isozymes. Xanthine oxidase ir the body

converts hypoxanthine to xanthine and xanthine to uric acid. High intakes of ?'

molybdenum in man (10 to 15 mgMo/day) have been associsted with an increased 7 incidence of uric acid gout. Lower intakes, up to 1.5 mg?lo/day, had no observ- able effect on uric acid excretion but increased copper excretion (108,111).

In order to study the basic chemistry of molybdenum, nonenzymic mdels for the nitrogenase system have been proposed utilizing complexes of molybdate thioglycerol or cystine and Na BV4 acting on substrates of-nitrogenase including nitrogen (112). The rates of substrate reduction in the mqdel system were lower but parallel to those of nitrogenase. Nitrogends;i- rcductions, althouqh exothermic, were accelerated by ATP to a greater extent than by ADP and FLYP. To explain this effect it was suqgested that nucleotides form proionated complexes with MooX which catalyzes the hydrolysis of the nucleotide phosphate. MooX is converted to Xored throuqh a dehydroxylated derivative of 'loox. Thus, molybdenum in the complex is activated by ATP for it: role in nicroqenase- like behavior. ThP efficizncy of this model complex could be enhanced by the addition of iron in the fcrm of ferredoxin model compounds.

Work of the Colorado Molybdenum Project

The investigators of tile present piojeCt were faced with t:ie difficult problem of measuring molybdenum effects at low levels of exposure. Conse- quently, in orcler to detect the biochemical effects oE ntc,lybdenum, very scnsi- tive subclinical terts were devised ill the area of bone cctabolism, xanthine . , oxidase in red blood cells, and adenosine ti2,)hosphdte (Xis) metabolism in I erythrocytes and platelets.

Bone Calciup Transport--

t, This model system employs the excised ulna of the rat with periosteum intact and measures the *vitro transport of calcium 45 from bone to a phys-

-? ioloqical bathing solution pumped over the bone (113). The rate constants cDmputed for the mathematical model of this three compartmental system indi- 1 cated a dose response for rats supplemented with molybdenum at the levels of - 1C mgllo/L and 100 ngMo/L in their drinking water (114). 'Ine most pronoilnccd effect on calcium transport related to molybdenum exposure was a signiEicantly increase<: "-ss of exchangeable calcium from the bone at a rate approximatelv three tires the normal rate at a concentration of 100 mqMn/S ii~ciie water. Bone molybdenum content increased a!n%st eiyitt-fold from 0.71 ppm to 5.3 ppm _- 5 of ash. ::3 ;icjiriL'icant changes werc seen in hoqe calcium and phosphate contents.

The model enables one to study the effccts nf molybi.cnum ingestion in animals at relatively non-toxic levels in water or food but would not b@ suitablz for _lower concei trations--lower than 1 mrjM~/L. App1ic:ation to humans would also be difficult to standardize and use due to L!ie necessity for a bone -- _-__ - 35 1 i

..i _. -= j. -~ ._- -,. ..L . ------.*- I....,.. ____ - - -___-.I-- .-- -. "..- -.. -- ':

~14ATP Met ab01 ism-- Table 12 shows the results of C14 ATP formation and related substances from C14 adenine precursor by red cells and platelet-rich-plasma (PRP). P I

_.- TABU 12. RESULTS OF C14 ATP FORMATION

14 ATP+ADP Location C R-- Cells Serum Mo: vgMo/L NIP

Denver Mo plant* R RBC 18-363 1.5-7.8 PRP 18-363 Summit County 2-40 RBC . ___ 4-34 PRP 5-33 __

.Marsden Not viable RBC 5-13 - Not viable PRP Denver suburb 2.5-11 RBC -. 4.9-14 . PRP 5-25 Golden 6.8-12- 2 RBC 2.0-17 PRP 5-43

* Only 31% of this group had R values in the normal range, whersas over 90% of 8, r all the other groups had normal R value-, i.e., 4.5 or higher. In this '?..< group alone a negative co:-relation of i? xi?h serum uric acid concentration <1 of r = -0.e-l was seen (Fig. 3). A plot of R -Jersus log (serum Mo) is snown in Figure 4. The correlation coefficient here is also -0.84. W.en sodium molybdate was added e vitro to platelets or red cells at a concentration of 0 to 100 ppa no significan: change in ATP production or pool size was soen.

--Discussion

finding methods of suitable sensitivity and capabie of adaptation to I' field studies on humans was difficult. I:evertheless, by the end of the perio6 14 of support considerable skill 1iad been developed in analyzing blood from vol- a.-~ unteer:; in bacchec of 20 per day. Xanthifie oxidase activity in hemolyzed red cells is low corpared to other tissues suc? as liver or intestine, but is measurable using,: the isotopic technique. One subject with clinical gout had i- greatly increased levels of red cell uric acid praduction. However, no signi- ,, ficant correlation of xanthine oxidase with serum molybdenum was found.

I With regard +-o p3atelet ATP metabolism, the molybdenum smelter workers ;D i4 had disturbances which were signiflcantly correlated with both serum molybde- _i I num concentration arld uric lcid Concentration which was elevated in this i group. None of the other ?roup? had signlficant deviations of platelet metabolism. Whethzr these abnormalities vere solely due to the effect of molybdenu! or whether Gther factors in the korkinrj environment contributed to the !

SERUM URIC ACiC rng% ,\,

results could not be detcimincd. Ilowever, if plans to reduce the exposure to molybdenum-containing dust arr- carried out it should Le possible to re-study these individuals after tlic:ir, blood levels of molybdcl-,,&! have decreased to normal.

Conclusions

Sensitive metirods for detecting potential environmental toxic; ty have been devise?.

Significant changes in platelet adenine metabolism were seen in industrial wQrkers processing MoO3, but not in any of the non-industrial populations on various rnolybtlenum. intakes.

No changes in red c~llxanthine oxidase actlvity were seen in an]' of the subjects which caiild be related to exposure to mo1ybdenu;n.

...... , ... .- ..... z _____.-____--L ...._ ..-...... , .. .." . .. ..

I- i i ..

.: C' ......

Figure 4. K value versus log (serum Mo) (r = -0.84).

METABOLISM

For trace elements, little is kriown about the bioavai3abilityI zhso-qtion, transpcrt, rlimination, or movement in and out of tisrues (116). Dat.a on these aspects of the ph:rsiology of molybdenum are mostly limited to the salu- ble inorganic forms such as sodium molybdate (Na2MoO4) (82) and molybdic tri- oxi6e (MoO3: (117,118). Balance and tracer studies have show^: that these form are readily absorbed in the qut of non-ruminant animals such as rat!; (118-122) and swine (98,101,123-125) an2 that most irrtake i5, eliminated in the urine within 74 hocrs (Vi<;. 5). In ruminants, eliminatiop eDf molybdate is much slower. Six LO seven 23ys are rzquircd for climiIiation of most of a dose and only 10% to 15%of the dcse appears in the urirle. The remaining fraction appears in the feces (116\. While most researchers hale :,tudied relatively soluble mdlybdenum compounds, Fairhall and others (117) also cxposed guinea pigs to dusts of Kolybdenite (MoSz), a highly insoluble cmpound. The tissue concentrations, with the exception of the lungs, of the exposed ariimals did not dj ffer signiflcently from the controls; whereas expor-.ures to dusts containing similar concentrations of molybdic trioxide ar d calcium molySdate led to grcatly in-reased molybdenum concentrations in ihe liver , kidnzy, and other

.. .a.. -' 39' ; .- *' .. -... '. . ._ ...... f'

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0.1 I 3 ,6 9 12 15 18 21 24 Tlmr !n hours

Fiqurc 5.

t [ tissL2s. Since ttx E mlylxlatc (scc Sect t.. cies are usciul for

Pharmacokinctic sC~~ciiCs-ofsodiicn molybtI,i:.c (tiapood) wcrc performed in this iaborakory (119, 116, l.:!'lj nsi:lg ritfin-cd rats. T!ic animals wcrc force-fed doses of. mo]y?x:e::um-39 .is t!ic mol:;i-.c!atc SJ1.t. Eirj?it.\r-t;hrc? i?crcci?t. of the dosc: was absorbed in thz Stonach, 13% iri thc ii;qxr small iritcstine,. arid 38 i.n the .. lower sinal1 intes'line. ti0 absorpticti occurrctl iri ttic larcjc i.ntestinc. These resuYts agrc!c with the less cixtcn:'i.ve work of nibr arid !.etier (120), and Nie- lands and coxorkcrs (.11.9). ;\ri absorption half-tir:c? of 0.88 lioufs was measlitred .. for both the stomach and ttic 1 JCT ';?ortion of tiic. small i.ntcstinc; 'rhc simi- .. . larity of absorp?:ion rates rxiy i:Irli.c.ttc that thc abs!)r!ztio:i mecharii:;m Is the sine in both p?rts of 'tile ?ut. Uj'takc throt:qh t!ic srornach wall was ais0 ub- . served by others (120). Our cl,ita on thc stomach .arc c:snsistent. with passive, movcrnent through interire1lulsr port s:iacr.s. .--- .. it nay tc, hnwcvcr, that abmrption in tht. intestine ran proceed by a dif.'crcnt mechanism. ~ic!ic~t aric? 'icsterfcld (ilf3) ~uqq~rtr>d that: xanthinc oxidase might he a carrlc*r fur the transport of' molvb?-.hurn. Ttiey ba:.cd thcrr

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f .. I' ' . :'.i., .! .....!: * : .. i!

1. TIP16 In hour6

Figure G. Percent oi r.iolybdenum-99 dose accmulated per grm of tissue (wet weight) for kid- iiey 3nd blood versus time after dosinq. .. . .- of these results is not clear kuc they agree with those from other laborator- ies (132) which showed relatively high lew-rs of molybdenum in bot!? these glands. Such acxmufation, whebl dietary illtake of 1fIol~;Jde:QIIis elevated, niqht have detrincnt21 effects on the stress respanse in which the secretions of both these glanZs play an irlportant role. Results of experF:nen:s which in- : .. dicate such eEf'ect.:; are discussed in Section 8. .'! .:

EliminaLion of nolywenun ir. the urine vas very rapid. The kirietics are I bipharic and they ~~loselyfollow the chanqcs ln the blood (Fig. 5). Seventy I c percent OE a dose is eliminated in the first three to six hours. The rate of I b elimindtron then decreases iLnd 87% of the dose has been clim~llatedafter 24 I hour:;. These pcrcentages arc conslstcnt with the results of Blbr and Lener I ', (120), but th,ey <:re much h:.cjher than thosc of other- researchers (e.g., 118). I It can be see," tiiat rolybdmm is elimindted rapidly in the urine after it has been rapidly ahorbed. 1'.i I .. I ..I !' To test the possihi iity of adaptation to high intake levels, inolybdenilm- i 39 was also cjiqen to rats which had been on molybdenum in their drinkirlg water /- i' 0, 10, 100, and 1,003 ng'lo/L since birth (126,133). 'rnosc o:i the higher ,I at ~ /- lel-els (100 and 1,000 :ig!!o/13) eliminatcd the molybdenum-93 faster than those 0 10 mg:,lo;L. -- --I on and lnls is of interest in view of results shown in Figure 8 - -1 L -_

i __

42 1

I I i ... ,..' .. . -,4 . ._ !; I \ I _-- Figure 7. Percent of molybdenum-99 dose accumulated i per gray of tissue (wet weight) for liver f and adrcnals versus time after dosing.

where accumulation is higher t 10 mgMo/L than at 100 mgMo/L indicating that there is an adaptation to the high intake.

.i Little is known of the transport of molybdenum in the blood and its stor- , acre in tissues. Bibr and Lencr (134) tested two of the oxidation states of ! molybdenum expected to be found in biological systems. (Moly'udenuni-V exists in the molybdate-cysteine complex while Mo-VI is the oxidation state found in the molybdats ion.) They showed that, at most levels, No-V binds to many of ! the plasma globulins while No-VI binds to almost none. This indicates that there may Le a differince in the way that molybdenum from food (possibly Mo-V) and froni water (1.lo-VI) are carried in the blood. If this is true, then uptake, 'I residence time, and elimination of molybdenum by cells may be different for t 1 the two forms. Furthermore, all of the absorption siudies cited above were - ,i performed with molybdate, the inorganic form, but nothing is known about the .: bioavailability and absorption of organically bound molybdenum. It is possi- I ble tl-.at much of the organlcally bound molySdenum exists as the nolybdate- ; cysteine complex, the form in which molybdenum is found in all the known mo- .* , lybdenum bearing enzymes (135).

.. Figure 8. Weighted average molybdenum concentration of ten tissues (dry wcight basis) from rsale rats recei-riny. indicated molybdenun concentrations in their drinking water.

conclusions regarding potential detrimental effects of excess molybdenum, additional studies of various chemical forms of molybdenum should be performed.

The only facts known about the storaqe of molybdenum is that apparently large amounts of active and inactive xanthine and sulfite oxidases are stored in the liver (131). Jchnson and Xajagopolan (131) depleted rats of molybdenilm by I’eeding then tungstate over several weeks. During this time liver xankhine oxidase and sulfite oxidase activities decreased more rapidly than did the total molyhdenum in the liver. This discovery led to the suggestion that there js an unknown non-proteih form in which molybdenum is stored.

the ! In summary, inorganic molybdate which exists in drinking water is I -_ rapidly and nearly completely absorbed. It moves into and out of most tissues I very rapidly, and It is excreted in the urine at nearly the same high rat- .is it is absorbed. Animals receiving elevated levels of molybdate rapidly attain , . balance and there is no progressive accumulation of this substance. Much still remains to be learned of the physiology 0; molybdenum ir: humans and other- non-ruminants.

’I TISSUE DISTRIBUTION IN ANIMALS !. I: i concentration oE tissues obtained from adult rats. Figure 8 shows the average tissue concentration plctted against intake levels. It can be seen that ani- t mals on 0 and 100 mgMo/L in their water exhibit lower tissue concentrations than animals on 10 and 1,000 rngMo/L. The expected dose-response effect was I1 therefore not observed. Adaptation to the higher levels has been suggested as it an explanation for this phenomenon (133). It should be noted that the average values given here do not represent whole-body burdens but are biased toward i 2 high concentrations because we sampled all tissues which were previously found to be high in molybdenum. Only two low-concentration tissues, muscle and 1 -_ - brain, were included. Whole body analyses perform in this laboratory show -. .a .a smaller differences between the dose Groups. Howe * the sme basic pattern I---- is found. The molybdenum concentrations found in the tissues of the rats re- l ceiving drinking water containing 10 mgMo/L are similar to those found by i - /- Fairhall and others (111) in guinea pigs which had been given 50 mg of Mo as

. ~ Moo3 ny oral administration with a syringe. I- 1 The tissue Concentrations in the controls were very similar to those seen i by other workers. In general, rats on a normal dietary intake of molybdenum have 3 hisher molybdenum concentration in their livers than in most other tis- i’ sues (kidney, spleen, brain, muscle, etc.) (132,136). Similar results have been found for human tissues. At greatly increased molyhdenum intakes the I highest molytdenum concentrations are found in the kidneys of the exposed animals rather than the livers. This result has also been ohtained for guinea pigs (117) and goats (50). In addition, Table 13 also shows that there is considerable upcake by the testes. This result is potentially significant in

TABLE 13. MOLYBDENUM CONCENTRATIONS (PPM DRY WEIGHT BASlS) IEi TISSUZS OF RATS GIVEP! DIFFERCIJT LEVELS OF MOLYBDENUM AS Na2Mo04 IN THEIR DRINKING WATER

Mo in water 0 10 100 1,000 ti mgMo/L

Ad7 Iilals 0.5720.09 19t12.9 4.320.41 4025.7 (i3) (14 1 (10) (10) Brain 0.2f0.06 2.420.31 0.7920.1 5.450 -6 (13) (15) (10) (10) Muscle , 0.12f0.6 2.420.8 1.120. L 7.2f0.35 i (20) (16) (12) (10) Kidney \ 1.750.3 54k9.6 18k-l. 3 7729.2 I (21) (15) (12) (10) i

.. .. . -

. .,. .

.. .- .. a' .. ._ .. . Miile conclusive e-r:ds:ice that molybdenum is essential to animals has not ,. been re?ortled , Sierc is i..idcspread agreement among researchers that this ele- rent is indced essentitl (67). b'or esseritial clement.; there are three ranges of biclogical ef fec~r--3eSisiency, adequacy, and toxicity. While these ranges have not been we1 1-def ined for molybd~n~mor any other essential trace' ele- rents, IQrtz (141) has !J~O~XIS~~d tentative dose-response curve which is shown in Figure 5. Bccausc of the typ2 of beli3vior exhibited in this curve, it is very difficult to study the toxlco1oq ~calprcperties of an essential trace elenent such as molybi-',enim, zinc, coppcr, cobal t, and nickel. Moreover, en- vjroiirnentdl cxposurcs to thew elemciits arc rarely at the extreme ends of the curves, alcliouah dcutr? yoxicity nay occur from occupational exposures. Be- cause food sources contalri SrariaLlcl siiioti,its oE essential trace elements, and bcca.asc th3 conceniratioi?s i :I pl aiits ,JL-C ctetcriniiicd by t'arious geocheinlcal para-nctcrs such as pii, it is zea;onublc Lo assuiiit: tlmt animals can adapt to mo3araLc changes in d,il ly ii:take.

Thus, the pr

FriScrg and otlicrs (50) rzcently reviewed the research 011 the toxicity of rr.ol>bdenum. One ch.iracteris'iic of the toxicological properties of molybdenum is the yLcat var-ability froni spcies to species both in terms of the toxic doses and the effect. Cattle are by far the most susccptible (671, while sheep are sont".di;it lesa tolerant; horscs md pigs are the most to1erar.t of farm anrmals. uts, guintLL 2~75,and rabbits are intermediate between siieep and horses.

Livestock

The most commn c!inical disoiricr in livestock due to hiqh molybdenum intake is known as nolylxl.?nosis (or izu t, or peat scwrs in som? areas). This disease is characterized by didii-!icd (scouring), discoloration of hair, loss .. _.- -. - 4 7

tration was lethal to rats (155). llortality was also produced in our laboratory by force-feeding rats a daily cquivdent of 2,500 ppm !lo (about 50 mgMo/ te of 20 inL k1-Q r3r 23 q food). :.loi'tality was 100% in s anemia W~Sthe ninst ol~viouc;effect. L'ver and kid- , normally dzrk rcd, were almost white in ,111 caws.

Fats exhibit relatively mi13 cffecLs at 1or.er intdkes (ll&,133) as lcng for. Hats glvcn molvbdenum as molybdate at 250 or lifctinic in their drinkinq water did not exhibit any it). simptons. Rat piips €1 om tlams that >ad been on l,OG@ myMo/L most of their lives tiere mdintained cc ntinually on the sxie intake. This ex- ! g, rough hair, sterile males, and some hyperactivlty, the lif- spar1 noticeably. Fentales produced pups which re reduced in sizc (40% to 50% of normal) ; however, the Timber of pups was from untreated clans (Table 14). The maternal perform- as similar to rdh on much lower levels. The number of born, and resorbed fetuses was within norms1 limits, ations of increased incidence of tu:iors cr teratogenic ffects. Other workers (118) have found similar 1-esults for rats on 500 and 800 p:m Mo in thoir fcod. At intermediate dose levels (50, 100, 250 ppm Mol g~owthrates were significantly reduced bun not- as dramatrcally as at 1,000 ngMo/L (Table 15) and litters were somcNliat smaller than in controls. Other- wise therc were no obvious clinical symptoms of toxicity.

TABLE 14. CFrCCT OF I~IOLYRDEIJdN----- ON LITCER SIZE I14 WIIITE RATS -.I_-- -_L. ---

~ 110 in watrr (mg/L) 0 10 1ou 1,000 17 25 16 6 I.

_-______I 11. ?to. 5 12.41-0.3 . U.GC3.9 10. OkO. 56 8 - 15 6 - 16 2 - 14 9 - 12 !.

---- ~ -- - !. ..

The nost consiste:lc effect OF hjgh itiolybrlvnm intake in rats is the de- qrowth or weight loss. Since this is usually 'Accompanied by anor- . exia, it has been srigqested that thc ceiqht loss is due to reduced food intake rather than some Other metabolic proc-ss. Sciw others suggest that the rats develop an ability to recognize t!;e presence of molybder1 'TP i!: t!:c 3.a~dli6 voluntarily relect the food (155). This hypot-tiesis ('as been dlsputed by Arringtoi and others who reported pal r-feedl ng experiments which demonstrated that reduced food intake vas not the primary cause of stur.ting (155). v ." Y It was mentioned previously that male raLs whose mothers were on 100 to ' 1,000 mgMo/L in their <:a1 -7r and <:ha theinselvr': 7.z.e on Lh? same level showed a high incidence of sterility. Tlie effcct of excess molyoderium on male fer- tility has been reported earlicr in both calves a~drats. In a study reported by Jeter a?< Davis (138) I,onq-Wans rats i-ecei*~cdvaryiiig amounts of sodium molykiiate irt their diets. ~cvcnty-fiveperccriL oi Lhc niales which were fed ___ 50

i _. - __.-

i i i REPRESENTATIVE hTIGHTS~ FOR-HATS ON VARIOUS DIETARY RJGIMES I” -- TABLE 15. Fully Exposed From weaning !, i Defined diet -- 61 days [* Mo ppm - 56 days

\ 0.5 32e12.9 (8) 245+14* (8) 10 267215.1 (8) 261+13* (8) -- - __ - -. rt Mo in water i mgMo/L 60 days , 0 251t9.6 (15) 250515.4 (8) 4 [. I - -

10 ~ 202?12* (30) 245f10.4 (8) !Li -- 100 210+14.1* (21) 242k14.5 (8) I’ 1,000 108*7.9* (15) I j1 Mo in water 1.: mgMo/L -76 days 0 329+6.5* (8) j 5 316*5.3* (8! -1i

10 312!5.4* (8) i; - 1. * i Different from 0 mgMo/L group at p = <.OS. Values without * are not statis- I t ticaliy different. from controls. i

from weaning ,>n a diet containing 80 ppm Mo and 140 ppm Mo were found to be sterile. The limited histolocical exmirations wk.lch were performed showed varying degrees of seminiferous tubule degeneration jri the rats receiving high doses of molybdenum. Yhoxas and Moss (139) found severe degeneration of the seminiferous tubules in male calves which were given a diet containing 300 ppm Mo to 400 ppm Mo (4 to 8 ngMo/kg body weight) over 130 days. The.calves were also reported to show 3 marked decrease in lihiics. In a pdrt of Secticn 7 (Tissue Distributi.ix? is L-Limais) data were presented showing that molybdenum is readily accuxrulated in the testes of rats. I Some of pe effects of high molybdenum intake; (equivalent to about 190 ppm Mo in the’diet or more) that have been rcportes are summarized below: L. -Fats Rabbits -r.Y Loss of appetite Loss of appetite Loss of weight Loss of weight Rough coat Alopecia Growth retardation Dermatosis i ---- Anemia Anenia 1 - -- Mendibular exostose; Skeletal deformities I __ . __ - 51 I I i I ! i i i Ii -- -’+I - __ ?fez - SrA..*&d i I/ ’I ;I ..

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-Rats Rabbits !" Bone defornities JoiFt deformities Histopathology of liver and Decreased thyroxin kidney disease Increased liver copper -. s Increased xanthine oxidase Increased uric acid IrnpaireC alkaline phosphatase -.- and sulfide oxidase activity Male sterility

-Laboratory Anim.,ls - Chronic Toxicity .. While considerable research has been done on the effects of very high levels (>lo0 ppm) of molybdenum in the diet, considerably less work has bzen performed on long-term, low levcl exposxe. As noted in Section 5, the concentration of molybdenum in water rarely exceeds 1 llgMo/L and the highest level observed outside of an industrial water was 50 mgMo/L. Thus, it is important to study the effects of levels of.molybdenum corresporlding to perhaps . 5 to 10 ppm blo in the diet.

Schroeder (140,157) has studied reproduction effects in r.ice receiving 10 mgMo/L in their water and 0.25 ppm Mo in their food. He found significantly increased rates of young deaths and dead litters iri this group after three generations. The controls received the same food dith 1 mgNo/L in the .dater. His findings suqgested the follcwing order of toxicity with respect to reproduction:

Hg > C3 > Pb > Sc > No > Ti > Ni > As.

Suttle (358) fez animals a copper deficient diet, thereby depleting

^I on some animal functions. KO additional anilnal treatments were required to \ produce these effects. However, effects on other anirnal functions were only observed when the animals were subjected to stress. The applicatjon of stress to experimental animals was chosen to amplify the effects of subclinical toxic -1 doses. The working hypothesis is that the potential toxicant will alter the animals' ability to adapt to an externaily applied stress, or that stress will -_ intensify the otherwise subclinical effect of the toxic substance and clinical ______- 52 - ...

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-- < .-.- --. /, ' \\ , / --. __ - I ...... r...... \. . , '...... ' ...... , .. ..

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symptoms will be expressed at normally subclinical doses. In addition, stress produces a measurable response in several pLysiologica1 activities. Objective physical peasurements simplify the quantification of tbe respwse ?o experimental variables. Examination of the stress response is also useful ill re- lating the laboratory animal experiments to humans since humans are subjected to a wide rar.ge of stresses. .. .?n exyrimental protocol was developed to test the rets. Several differ- i ent s5te;ses m.-e applied ar.d their effects on various phrsiologicai and be- j hz-*-iorai activities were measured (122). Male, Sprague-Dawley rats were i! . .. *l either (a) raised from ddms maintained on identical test diets (fully exposed) or (b) bought as weanlings (3 weeks old) and started immediately on the test intake of molybdenur,i. Physiological and behavioral tests were begun at 7 wecks and continued until sacrifice at 17 weeks. Cxygen consumption was mea- - sured on sleeping rats oncz each day for four days. After this, behavic-r and activity were tested in an open Field arena (159) once a day for four moie days. Each animal was the:. subjected to a non-dzstructive psychological stress (fear) and tha immediite response was measured. The entire sequence was repeated separately for oxygen consunption measurements and for Open Field testing. The determination of oxyqen consumption proved to be a useful mea- . sure of garera1 body metabolism and health. The behavioral tests were ex- , pected to be sensitive in?icators of impaired neural function. , At 15 weeks tne rats were subjected to 4 days at 4OC to S°C, and at 17 weeks they were kept at 37OC for 16 hours and then sacrificed. Tests carried out and data gathered will be discussod below. By using several different approaches it was possible to identify some of t!:e subtle effects of excess i molybdenum. 1, i Growth-- 1" The effect:, of possible toxicants on growth is a ccmon measure of toxi- I' city (160). A siqnificarit reduction in growth was observed for rats receiving ! i 5 mqMo/L to 10 myMo/L (in the form of sodium molybdate) in their drinking I< water (Table 15). However, these concentraticns of malybdste in the animals' 6,

food prcduccd no effect. This difference can be explaine3 by the difierence I in tntal molybdenum ingested. (A rat receiving 10 ppm Mo in food ingests about 252 jlgMo/day while a rat receiving 16 ngMo/L in water ingested 350 to 400 ugMo/day from water and 25 pgMo/day from its food.) \iei.ght losses dimin- rc ished as the rats' weights apprcached 50n to 600 grams. For these larger rats . doses of 1,000 mgMo/L were required Eor significant weight differences.

Tests on Unstressed Rats--

e= OxycJen Consumption (V-O2L--Tfie sleeping and awake unstressed metabolic rates (moascred by oxygen ccnsumptton) oi animals receiving 10 mgMo/L in water were significantly highe; tk.an colitrol animals (no added molybd=num-- c see Fiq. IO). Oxygen consumption WAS also elevated in animals receiving 50 mgMo/L and 100 mgMc/L. However, no cledr dose-response effect was observed since the oxyc,en consumptions were only siightly higher than that for the 1 10 mgMo/L animals. This rcsult is similar to Suttle'r- results in this phenomenon. However, we helievc that the toxicity of molybdenum is not simply a function of the copper to molybdenum ratio As proposed by Miltinore and Mason i 1 __ i ? d3 I i ',, __ - -. .- - - t I f

(142). but that it also depends on the dietary copper intake in the sense that there is a threshold or “adcquate” value. kfi.en copper intake falls below that xralue tile animal is much Fore susceptible to molybdenum toxicity *.ban when the cepper Intake is riore than adeqmte. Since the rats which received the higher levels of molybderrum (50 to 100 mq:.’o/L) all rccciwd adequate coppcr (7 to 10 ppm Cu), the cffect of the a3ditional molyhdenum exposurc was reduced. The largest elevation above the controls was obtained with a group of i rats on fooC low in coprxr and elevated in molybdenuni (2.5 and 10 ppm, respec- 1 tively). in these animals, +-he sleeping V-O2 was nearly double that of the i .C control group receiving 10 ppm Cu and 0.5 ppm ‘10 (161). In a11 attenpt to ex- f- plain this result we examiced the fine structure qf the adreml cortex of ani- i ! mals receiving 0, 10, and 100 mgMo/L in their drinking water. The animals receiving the highest level showed statistically higher numbers of mitochondria- i in their cells when compared to controls. Animals receiving 10 mgMo/L also hac? higher mitochondria counts ’>ut the numbers were riot statistically signif i- cant (162). Thus, the larger number of mitochondria could result in higher oxyge.9 consumption.

open Field-Activity levels (number of squares entered) in the Open Field / were highci for unstressed rats receiving 10 mg.’lo/L than control animals (see Fig. 11). KO differencc was observ..ci for animals receiving 5 mgEfo/L. Rats on 10 rng?lo/L increased their activity considcrahl y during the last three days of the five day clrlals. This was not true for the control rats (163). In Open i Field testidq it rs commonly obscrvcd that rats will settle down to consistent activity in trials two through five, bit molybdenurn ‘appcars to have modified i 1 this rec.iilt. This bcliavior may the result of t.hc generally metahol- I be higher ? ic r.3tc i 1 -- $ - -P1 isma Gl~icosc--Clucose is mobilized uritlcr stress Ly epinephrine and cor- i ticosterords. Copi-’cr-concaii~rnyei::ymec, are inportant in the synthctic path- I’ ways of both of tliese hormones (ICJ). ‘r:ierc:fvrc plama glucosc was measured E as a possible iiidicator of molyklcnum LoxLcit)’. The plasma qlucose levels for i unstressed ex;xi?rjmcrital ‘inimals and controls WCLC similar (average 155 m%). riowcver, stressed nnin.31~rcccivinq elevated mcjlybdenim sflowed significantly 1, lowcr plasma glucose levels comparcd t.o sti-csscd c:ontrol.: on normal levels of molybcicnum. The largest rcductions were obscrvcd with animals receiving the 1 lowest coppcr lc-rels (2.5 ~ym). I‘

---~erulo!,Iasnin--Cerulojiiasm~n -- is a coi,pcr-containing eiiz~mewhich is syn- thesi7cd in tti? irver. ‘he enzyme is found in plasma, axd uridcr stress it can be mobilized from 1 iv?r storcs. BCCJUSC of the known interactions between molybdenum and coi)per (ICE;), the activit)‘ of this was measured as a *- enzync potential indicator of toxicity.

I Molybdenum at 5, 10, and 50 mg!’-!o/K. in wat.cr had no effect on the cerulo- ... plasmin activity in rats eatinq I.& Clioir. This food contains 7 to 10 ppm CU and t!iis conc’.ntrat.ion appears to be adquatc (164). Animals receivirig a de- i EineJ diet lox in copper (2.5 ;>pm) and cxccs.s molybdenum showed siqnificantly i . lower ccru1opl.isinin activity (p

- -_- __ - _-- 5 5 t

_..- ~ -- ,‘ / ’. -- /- <- I

1 75 ." . '. i . .. , ..

.....

,!,' ... ..' ~ . ,. .. I. i' , . , .'. ;' , ..

I. I

.. ,.. C 1 1 CONTROL COLD

.. EFFECT OF 24-HOUR COLD STRESS ..

Figure 12. Effect of cold stress on serum ceruloplasmin in rats . .- ...

.Y I given molybdentm and copper in a defined diet zit the .... indiceted levels. Vertical bars show one standard error. There were eight anbals in each test group.

-- . ;. . 1. 57 , ...... -......

. .-.. . .- .. ..., . - ...... ,...... activities (p=O.OS). These results suggest that copper must be below "adequate" in order for increased molybdenum to measurably reduce ceruloplasmin acti.vity.

HernatolrJz--Tl-.e appearance o E achromotrichous anemia iri rats receivi:ig .. ..! .. elevated mdyb=ler.um levels suggested t.hat some hematological factors might be .. affected. However, 5, 10, 50, 100, or 1,000 ppn Mo in food or water produced .. .. no sig-iifica. t. effects on hematocrit, hemoglobin, or blood cell nllmbers. >. .. While one s;ud,. (165) did report anemia in rats receiving 400 ppm Plo in their I. . diet (as sodium molybdate.) other studias (116) have .failed to reproduce this, result.

/--- On the other hand, there is a rather consistent picture of hematological complications in rabbits on high doses of molybdenum. Several authors have reported clecreased hernoglobin and hematocrit in rabbits on rations cmtaining 1,000 to 4,000 pp~r'M*I (150,165,166).

Tests on Stressed Rats-- I 1 Drop StreE--Tne drop stress is a s!icrt-term Don-destructive fear stress , which was expected to amplify the effects of molybdenum on symg~thoricnervous ! system activity via altered secretion of catecholamines. After reZeated free;fall in a cage, the metabolic respo:.sL, cc~risistsof a rapid decline in axygen consumption followed by a rapid ri:,e to more than double the rate ob- , served for th? sleeping animal. After the pcak consurption rate is attained +&e rate decreases for 15 to 20 minutes until the animal reaches its'awake resting rate. Rats (bred from dams on the same level) exposed to 10 mgMo/L froin conceptiqn showed shorter total respocse times than contrcl s (p/O.OS) . No other measured parameters showed significant effects (161).

sen Field Behsv&--kats were subjected to a similar drop stress and I+ - immediately placed In an ope11 field aiea for a four minute test. The open field test was reFeated (the stress has not repeated) 24 hours later and four I- days later to determine sny pcrsistenc effects of the original stress. The .. immediate response to the drop stress for all animal groups was a large reduction ir, actlvity (squares ent-ered) . The acLivity in all groups decreased t-o about the same level and no significant ef€ects of molybdenum were found on any aspdcts 05 behavior. There was no indication of memory impairmcct when the tsts wert rel?eated. Other tests of memory, using a different pro- tocai, als.3 showed tha: *-xcessr.iolybdenum had no effect.

Some leletcrious effects of molybdenum were found at 5 and 10 ppin Mo. The aninials shoked reduced growth, increased metabolism as measured by oxygen \! consumption, and increased activitv as measured by dpen field tests. Some of the deleterious effects were brought out by applicqtion of stress. In all t cases the excess nio! ybdel.um produced a reduction in the animals' stress response. Lo simple relationship between dose and responsz was observed. Evi- dence exists for an adaptation at higher molybdenum doses (near 100 ppm).

Some of the deleterious c'ffects appeared only when the a:iimal was receiv- ixg a diet which was low in copper. Results obtained in these experiments ..

indicate that the copper:molybdentun ratio is not sufficient for predicting the l response to excess molybdenum. There exists a minimum adequate copper concentration below which relativcly small increases in molybdenum intake can in- Iis duce adverse effects. Excess molybdenum produced no effect on memory or henla- i, tological measures such as blood cell counts, hematocrit, or hemoglobi.1. ! * The results of the tests performe'! nn the rats are .;hewn in Table 16. i The designations low and high dietary molybdenum correspond to 1 ppm MO or less and 5 ppm Mo and 10 ppm Mo, respectively. A plus sign (+3 indicates an d increase in the measured parameter and a minus sign (-) indicates a decrease. ... - . .. A zero (0) indicates no significant difference from control. The effect of molybdenum on ceruloplasmin activity and plas.!;a glucose concentrations showed

,. a dependence on dietary copper intake. These variables are indicated where relevant. (Low copper is 2.5 ppm; higher copper is 7 to 10 ppm.)

! la SABLE 16. SUMMARY OF TEST RESULTS I. ! - I h, ! Open Field Test i' i I (Activity)

I Unstressed Drop stressed I i 1 High - I* Eo + I' - .I I Low Mo Control t - I 1 t I - TABLE 16. (continued) Ceruloplasmin ! (Activity of enzyme) Unstressed Drop stressed

*> High Cu Low Cu High Cu Low Cu High Mo ------

- Low Mo Control -- - -- r* 1

-1 summary ! :

The toxicological properties of molylslenum are-very complex. No doubt i! this complexity is caused in part by the role of rwlybdenum as an essential trace element, thus leading to a more complicated picture than is the case for l a substance which does not play such a role.

me most susceptible species are gcnerally rL-minants and in particular, cattle and sheep. Since the mid-1930's when molybdenum was shown to be the cause of a lang-recognized diserce in cattle known as "peat scours" or "teart," many cases of molybdenosis in livrstock have been reported. In most cases the sources of the excess molyhdenw, were natural. But in some cases, industrial activity was the cause. In most cases tne symptoms--which are normally diarrhea, emaclation, arid male sterllity--c&n often be reversed by the addition of cop^ ,i to the ,animal's diet.

t The interaction among molybdenum, copper, and sulfate has been studied in several species. One hypothesi s has been that a Cu-140-S compound is formed which renders coppcx unavailable to the animal (167,168) . I Rats, rabbits, and quinea pigs are much less susceptible to nolybdenum toxicity than cattle and sheep. Whereas clinical effects can be induced in I cattle grazing forage containtng 10 to 20 pp-n tilo (nonnal plants contain 1 to 2 ppm Mo), 100 to 400 ppm 110 in the diet is required to induce clinical mani- 1 festations in rats. The spptons vary from species to species and include: I i weight loss, growth reduction, skeletcl deformitlos, and sterility in males.

Subclinical effects have been reported iii rats, mice, and guinea pigs - ! receiving dieLary levels that are 5 to 10 times normal (the equivalent of :* :* i 5 to 10 ppm Mo in food). These consist of reproducrlve effects, effects on

3 copper netabolism, growth retardation , and a ger,crally lowered respollse to I stress. * 1

I These results indicate that for several species the upper end of the range of sufficiency or the safe range is about 5 to 10 times the "normal" level. Since no diets have yet teen devised which are so low in molybdenum as to irid*lce a deficiency, the lower end of the safe range (that is, the minimum dail?, requirement) is as yet unknown. _- .. ,. - ...... ' . GO , .--. - - I ...... 1 i

HUMAN DIETARY INTAKES AND NUTRITIONAL ZEQUIRENENTS

It is estimated on the basis of balaqce studies (169-171) that 30% to 80% ! of the molybdenum consumed is absorbed. Eased upon urinary excretion of subjects in our studies, we estimate the minimum absorption of molybdenum from food and water be between 25% and 30% of the intake. It is not known whe- to I ther molybdenum in drinking water is absorbed more rapidly than that contained in food. Little information is available on the relative amounts of molybde- i num absorbed by humans under varylng conditions or the influence upon molybde- ! num absorption of other components of the diet, particularly protein and other tu trace elements. The discussion on Tissue Distribution in Animals in Section 7 ' I '. presents information on the absorption of molybdenum which has been derived from rat and other animal studies. - .-- Water usually considered tobe a minor factor in the intake of molyb- is ,- .. denum and many other trace elements (G0,72,82). If we assume a molybdenum intake from food at about 180 \lgMo/day (from our work), persons consuming one to two 1itf:rs of water per day which contains less than 10 UgMo/L will not de- rive a signifi\:ant portion of their dail,? molybdencm intake from their drinking water. If a person consumes water containing 40 to 50 ugMo/L, such as in the Denver metropolitan area, then the water contributes one-fourth to one-half of the daily molybdenum intake. If we assume a food intar

Based upox: the work of a number of investigators, Friberg and others (50; state that the usual intake of molybdenum via the diet can vary between 100 and 590 pgMo/day.

Investigators in the U.S.S.R. hpve estimated the daily intake of molybdenum from rhe diet at 329 to 376 ugMo/day for adults (172), 156 to 161 pgMo/day for children (173), and 200 to 500 pSMo/day for children and adults (174).

On the basis of total diet samples from different areas of the United Kingdom, Hzmilton and Minski (175) reported a mean daily intake of 12b UgMo/ day.

Schroeder and others (132) estimeted that the average diet in the United . States contained 335 I.lgMo/day, with a range between 210 and 460 ugMo/day, based on analyses of samples of camplete hospital diets.

k- Wester (176) examined duplicate samples of the hospital diets of two patients, dcriny six periods of five days each. Average daily intakes for each Ff2riOd varied between 250 and 1,000 pgMo/day. " Tipton and Stewart (177) found daily intakes averagjrig 110, 120, and 460 PCJ Mo for three subjects in a balance study. Wester (170) studied four healthy subjects in a 10 day metabolic study. The daily intake of molybdenum _.- varied between 115 and 245 pg Mo. Robinson and others (171) found a daily I i i I- 6 1. - i i - .. - 1 I i .- c_i

\. .

I I intake in New Zealand of 46 to 96 lJg MO on meatloaf.

Our study (76) has found the average daily intake OF molybdenum in the United States to vary betweep 120 and 240 pg!lo/day, depending upon age, sex, and family inccme. We estimated the average da'ly intake of molybdenum via 1' tLe diet in t!ie United States to be 180 pgMo/da.j, based on a market basket sampling program, and a method of estimation using U.S.D.A. published esti- mates of food consumption (77). Figures 13 and 14 show our calculated daily molybdenum intake for each age groLp and the daily nolybdenum intake for each age group per kg oody mass. The intake by males was gererally Treater than -1i-. - that by females. Persons with a lower annual family income generally had a ! higher intake of molybdenum per day, which was assumed to be due to the :I i greater proportion of legumes and cereals making up their diets. i !; .

...... 1; .. 1...... f; ,. 1; . . 1,

1 I I r

*cn I 8 - -- -I1 I In - -- CUmm i .Age Group

l Figure 13. Calculaked daily intake of molybdenum according to sex I and age groups for the whole United States. Open blocks: males; shaded blocks: females; cross-hatched areas: male ..I and female children less than nine years old. i'

62 . ,. . ..

__ ..

xx 6th *. Age Group Nrc) ! i t Figure 14. Calculated daily intake of molybdenlm per kiloc~ramof body mass, according to sex and ?:?e groups for the whole United States. Bl~cl, legend^ 2'; ;.n Yiyure 13. L ?

:he proportional contrlbucxoll of molybdcnum to the total dietary intake varies witk sqe because of L!X rtitferl-nces in the makc-L:p of the diet for these age groul:s (see "Food" zn Scctlon 6 on Che molybdenum content of foO2S.) For example, milk. makes up til,: larqcst contr~b~~iion of molybden-m to chc diets < of children in the IJnited Staks (approximately 30%), whereas gralns and le- I gmes provide mast of the molvbdeilclm 3n adult r,iets in the United States. Figure 15 illcstrates ho,r the cont ributiotl of molybdenum to the total dietary intake from foods in 24 caterjories ci~anqcswT-th age.

- blolybdenun's esselltldllty is related :o its role in the molybden1.m c'3n- - taining metal loenzymes xantiiine osil?ase, SUI f ite oxidase, and aldehyde oxidase. me tissue concentrat~onof aldehyde o~ic?acchas heen shown to be related to the molybjenum intake 111 dn~mals,dnd i~lcllrr-cteVidCnCe polnts to this rela- .. tionship with the other cn?,pes (55). Minimum dietary rcquii,:.,lcnts for molybc!: nun compatible wlth satisfactory growth aRd health cannot bc qiven for any animal species including the human species. t.lolybd.enurn deficiency has not been observed under natural conditions with any animal specizs (82j. I\lt!lccgh il defjclency of molybdenum in plants

I I f

i AGE - milk cheese ice cream beef pork -~ pou!try fish meat mixtures ems beans peanuts, nut s _--- citrus fruit _-- - tomatoes potatoes cereals

'I pastas treads bakery fats c23cr veg e toble s-dk qr een ,~e~etotdes-yellow vegstable mixtures fruits

Figiire 15. Contribution of major fool1 categcries to daily moly1)denum intake of male subjects, accordi11q to age. The verticzl width of each bar is proporLiona1 to the fractional portion which the f~o3type contributzs to the daily total intake. Exaxples: >!ilk contributes 29% of the daily irhtake of young cliil ire? and only 10% among 75 yc ar dd adillts. - . The contribution from breads incredses irom 5% to 13% as ': the individual's age increases. The age scale shown is nonlinear but follcvs the groups shown in th? previous figure ;. There is a sljght decline in bean consumption among 113 to 19 year old males.

has been implicated in the etiology of human illness in South Africa (1781, human requirements have not been determined. The information available from balance studies is difficult to assess due t3 varyrng amounts of molyGbenL. in the diets, and inconsistent results with respect to positive and negative balance in subjects with differing dietary intakes of molybdenum (169-171).

Results cf ssn;e dpidamiolojicdl driG aliirndl studies k.ai-e indicated that - r,-olybdenum exerts sorile effect in reducing the incidence of dental caries (179- 103). In some of the epidcmiologic studies, the association with a high molybdenum water supply was not clear: food sources may have been mcre important contributors of molybdenum. Two separate epidemiologic studies in the Unlted States in California (79) and in Calorado (63) were not able to find evidence

-. ~ 64

_A< -J i

J I i .?

1 supportinq the hypothesis that molybdenum exerts an anti-caries effect. In i i the California study, dental caries rates were compared in children living in areas with arid wichout molybdenosis problems in cattle. In the Colorado study, ! dietary intakes of molybdenum among children examined were considered to be 1 comparable due to similar soiirces of food scpply. No consistent association ! could be found between the use cf a high molybdeimm content water supply and

,I a reduced incidence of dental caries. 'I

BIOLOGICAL EFFECTS OF MOLYBDENUI IN IIUYANS - .. - - - - -_. - - - - $7 -* Deficiency

While molybdenum is considered an essential nutrient, the experimental 1. induction of deficiency states in animals has been difficult and qenerally required the addition of tungsten to the diets. Only recently have molybdenum responsive syndromes been described in chic::s (184) and in goats (185). In humans, cutritional deficiency of molybdeniun, per se, has not been described. There has bezn, however, a report of deficient function of sulfite oxidase, a molybdenum-metallo enz-me, in an infant with neurolcgical abnormalities who died at nine months of age in a decoricate state (186). There were excessive and _I mounts of S-sulfo-L-cysteine, sulfite, thiosulfate in the patient's urire,

I concomitant with decreased amounts of i:iorganic sulfate. Such a pattcrvi could i- be explained by a block in the conversion of sulfite to sulfate. Since three I of the patient's siblings had died in infancy ti-h neurological disorders, the 1 authors postulated that this disorder was a? i7herited metabolic disease that they called "sulfite oxidase deficiency. "

I .' ! Toxic i t y ..., It ! Because of the known toxic effects of molybdenum in animals, there has been concern over possible deleterious health effects of molybdenum exposure or ingestion in hlunans. Many of the studies on irdustrial mo1ybder.w~exposure 1 were performed in the U.S.S.R., thz results of which have been summarized in ! an exce1;ent critical review by Friberg and others (50).

Early signs of pneumoconiosis werz foun5 by Mogilevskaja (cited In ief. 501 in the chest x-rays of a 44 year old woman exposed for five pari to molybdenum arid 14003 in dusts at concentrations ranging from l to 3 mgMo/m3, and in a 44 year old man exposed for four years to concentrations varying between 6 to 19 $gMo/m3. Fully developed radiological finaings of pneumoconiosis were noted inla 34 year old man after seven years of exposure to the latter concentration,\ where most of the dust particles were below five microns in diameter. I *-e three subjects, who were anong 19 workers in a molybdenum reducing shop, had variable rcspiratory complaints.

Kolybdenum-induced hyperuricemia was reporte3 by Kavalskii and others in 1961 among inhabitants of a molybdenum-rich province in Armenia (2). In this large and comF?.ex study, 278 of the adult population of two settlements com- l plained of joint pains, particularly of the knees, the interphalapgeal joints, I and the metatarso-phalangeal joints of the feet. AI-ticular deformities, i erythema, and edema of the joint areas were noted; hepatomegaly was present

! i . . .- ......

and' renal and gastro-i.ntcstina1. disorlars were' also reported but no specific aetai3.s giyen. Hyperuricemia- and hyperuricosuria were demonstrated in 17 subjects, in some "healthy" subjects from the same region,. and iri five controls ' from a molybdenum-poor provincc. Xlevat.io!is of blood molybdenum levels were found f-n the sick subjects, accomp;lIi.icri by decrcases i!i blood copper 'concentratio; 5. Urina:ry excretion of moly1;denum c1i.d not clif fer arnong the "sick" and ' . "healthy" subjects.fro3 the Armenian province , but bcessive urinary excretion ' 4 of copper was noted in the sick subjects. The reportc:d control levRls of blood and urina-7 copper raise some d.cubt as.to thc accuracy of the measurements in this study, and the controi blood mo1ybderlu:n concentrations also s,eem '.-

high. Serum xanthine oxidase a,ctivity was approxinatell. doLLbh?d in the sick ', ... persons, and was noted to Le proportional to increments in blood, molybdenum . levels. . Analysis oi the dietary iiltak'es of, the in!-.abi.tants of the, molybdenum- rich province showed daily consumptions of molyddenum t3 qverage 19 to 15 'mg No and that of copper to approximate 5 to 10 mq Cu. Outsihe'the molybdenum- rich pzovince, daily intakes of 1 to 2 mq KO and 1.0 to 1.5 mg Cu were found. While -this study lacks adequate control groups and has trace element valaes which axe difficult to interpret, the data presented seems to show increased uric acid levels in blood and urine of the subjects livlng in. the molybdenum- rich province, which may have caused a -gouty-type syV,drome. .. .., Hyperuricemia is also mentioned in two other studies in the U.S.S.R. ac-. . companied by arthrolgias, and, elevations of serum bilirubin, cholestrol. and globulin leveis among .workers in coppcr-:nolybdenum plants . No precise laboratory values were presented, hence tlie validity of these reports is difficult .' to *assess (SO). Ecolajan, iii i!365, exami.ncd, 500 workers from a molybdenum and copper'ninc and cornpa-ed these workers to a control group of equal'size fror.1 +.he. general populatioii in t;;c area. The dust levels in the mines were report- . ed..to exceed the xaximu;i pennissiblc concentratj.ai1 (in U.S.S.R. , MPC = 6 mgXo/ .. 3 L: ) sonic 10 to 100 -fold: Eany workers corilpl.a~~~nedof lion-specific symptoms . such as weakness, fatigue, h-adaches, .iri-itabE.li.ty; poor appetite, stomach . pains, weight loss, irritated skin, c?i:.ziness, and tremor (187). TI?.? author . ccncluded that molybcicnum exposurc was 3ccoinpanj.ed hy impairment of central ' '. nervous systen: functions. X?iile.the stiiiics in the U.S.S.3. may b-. difficult to interprct in a scie-ti.fi.2' maniie:-, they did st.<.r,iul.ate further research 3n the, effects of molybdenurn~onhuman iit?3l.t\1, with cpcciflc emphasis on uric acid and copper metabolj.srn. -. .. .. MolyLAenurn er,riciir:ient [~t'sor

They then supplemented the sorghua with 1,GO0 :tg Flo as anunoniun molybdate to provide 'a &i].y 'intake of 1,540 ]~qXo,/day, which resulted in a further'increase i,n rnean urinary corpcr exretior? to 77 pgcu./?.-1 hours. Urinary uric acid excretion remained uncitanqed at the three lev:?l.s of mQIybr!enwrr .intake, whereas the mean i SI> pla..;na copper Leiicl.:; increas&I froin 8O.s ? 1.1 ].lgCu/dl on ,.the low molybdenum diet to 113 2 .0.5 I!cj&~,/dl at tlir .high intake. During the ., . .

I . .. I ......

balance studies, daily copper intakes remained constant at 2.4 mgCu/day, as did the fecal copper elimination, vhich averaged 1.83 mgCu/day. The authors concluded that increases in molybdenum irgestion did not impede copper absorption, but that molybdenum either 'prevented cellular copper uptakc, or mobilized tissue c6pper storesI or a combination of both rnezhanisins. The result .would be an increase in plasma levels of circulating copper with subsequent increments in urinary copper excreticn. They also suggested that changes in uric acid metabolism would occur only at higher intakes of molybdenum.

Human Health Effects and Present Study

Molybdenum contamination of drinking water supplies occurs in some por- .< tions of Colorado as 3. result of seepage from tailing ponds, at molybdenum mines, into downstream creeks and rivers (58). In our study levels of molyb- denpm as high 2s 400 werc found in the vater supply of city of pgMo/L tht- .... Golden water source which received effluent from the tailings ~OS%of the ! Urad mine. Such high levels were cause of concern and the U.S. Environmen- I_ a I! tal Protection Aqency sponsored an interdisciplinary study by this group of the biologicdl effects of molybdenum in humans, with €he particular aim ?f recommending m acceptable lzvel of molybdenum for drinking wate: in the United State:;. TQ accomplish this purpose, it was necessary to first determine normal concentrations of molybdenum in plasma and urine, and biological samples were collected from suhjects in the Denver area, where the water content of mol-rbdonum does not generally exceed 50 pgMo/L. Since nolytdenum was known to affect copper and uric acid metabolism, the blood samples were also assayed €or ceruloplasmin (a cuproprotein that normally contains '30% of circulating plasma copper) and/or coFper content, and serum uric acid levels were determined. The urinary levels of copper and creatinine were also measured, the latter being useful to judge the completeness of a tLmed urinary collection, and Cor analysis of uric acid to creatinine and molybdenum to creaknine ratios. ?%e levels of serum glumatic-oxalecetic ticnsiiminase (SGOT) were measured as an indicator of hepatic function and serum creatinine and/or blood urea njtrogen (BUN) were also assayed as indicatorn of renal func- ,.,. tion. Medical histories were obtained at the 'time of sanple collections, primarily on male subjects. The known effect of female estrogenic hormones on .. ,' plasma copper and ceruloplasmin levels could mask any molybdenum-induce3 .* changes. The resulix of the biochemical assays peiformed on these subjects were then compared to those of subjects with high mclybdenum exposure. Such exposure arose from the presence of the metal in drrnking water or in industrial settings ,< /. One study of industrial exposure was performed r7t a rozsting plant in Denver where molybdenum sulfide is converted to molybdenum oxides (45). .- Twenty-five workers at the plan: responded to a general medical questionnaire and provided a blood sample. Among these workers seven had no complaints; six had incurrec! tm upper respiratory infection in the preceding two weeks; six complained cf tqth joint paS;,s and backaches; another four complained of joint I' * pains; and anotner four of b2ckaches alone. Diarrhea was rneritioned by five workers, headaches by four, 2nd non-specific bair or skin changes by eight of the workers. In general, the cc,tipplaints were not very specific and there were no complaints of gout or renal stones among the workers. PSr1mo;iary function tests (forced vital capacity and €arced expiratory volume/second) were normal

67 ! : - ... ! _--

in 22 of the 25 workers, but evidcnce for ,uld obstructivi! lung disease was present in three subjects aged 32, 52, and 59 ycars. .. Total dust samples were co?lected in me roaster area and an eight-hour time weishted a\.orage ('IWA) of exposure to mclybdenum was ci?lculated to be 9.47 rncj!.!ojm3. X-ray diffractiai studies of the dust. showed it to be mainly composed of molybdic oxide (PloG3) and other soluble oxides Ji molybdenum. Respirable dust samples were collected with a system that limited cnti-y to particles equal or smaller than 10 microns in diameter. The molybdenm concentration of respirable du:;t at ';he base of ::le master was 1.02 ngMo/m3 of air. Sirce a worker breathes an average 10 m3 of air 2er eight-hour shift and t!!e particle size was sufficiently small to allow peatetration into at least the upper airways, the minimum daily body burden of molybdenum was calculated to be 10.2 mq NO.

The biochemical assays performed on thc workers werc compared to levels obtained from a cor.tro1 group, which at that time,-consisted of research per- sonnsl at the University of Colorado Medical Center. The complEte blood counts vere normal but the industrial workers demoastrated significant increases In serum ceruloplasmin and milder increases in serum uric acid levels (Table 17).

TABLE 17. SERUM CERULOTLASMIN AND SRTC ACID CONCENTRATIONS IN MOLYBDENUM FACTORY WOIKERS AN3 IN CONTROL MALES (XEAN 2 p-4 SE) ' .- - I Ceru Loplasmin Uric acid E'-.mbe r (mrj/d 1) (mg/dl) -. Workers 125) 50.47 5 1.38 5.90 F 0.24 Cantrols I, 24) 33.50 i 1.33 5.01 i 0.25 p-value -- ~0.005 <0.025 -

Fourteen of thc 24 control subjects hzd pldsrn,l molybdenum cc,ncentratioris less than 5 uqXo/L wbich was the lower limit UE detection. TI? thr? 10 remaining control subjects the plasma molybdenun levels ranged from 5 to 34 liqz.lo/L. In contrast, mong thi? factory workers, plasma nolybdcnum conccntrations ranqed from 3 to 365 DO%/L, anu thcre wcrc no samplcs tclow 5 pc;!-lo/i. Only 10 of the 25 sarrples were within tlic range of the control; levels of plasina nolyhde- nun from 35 to 100 Uq!.:o/T, wcre present in seven subjects, from 101 to 299 pg Mofi in fice and greater 2nan 300 \iqNo/L in three*. These drrfcrences were highly sicjnificant (p

Fourteen of the industrial wDrkers providel urine collections which un- fortunately were incomplctc, as determined by calc1:iat:or of the daily urinary creatinirx excretions. Clrlnary mo1ybdenl:m zontcnt was hcncc cypressed in ug/L and for cortrol subjects the range was frm 20 to 230 :jgMa/L. In +-he samples from the workers only two were within the normal range. Levels from 450 to 1,000 pgHo/L b: re present 111 ncvcr, subjects, fron 1,COO to 3,009 LIg>lo/L in

68 (r .. i' -. -t. . 1. . . ..

...... , .. 'z : 1 .A ' ' . ... , ...... I. J ' . %<.. . : .. \. i...... i ... ., . . , . ,. .. .

1-1 1-1 Control 1-1 1-1 Factory Workers

_I- ...... -. .. . -. .. .., .

Plasma Molybdenum ().g/l)

Figure 16. Plasma moly'ndenm concentratiops of control subjects and of workers . in a molybdenum smelter in Denver.

four, and 11,000 pqXo/L in one (see Pig. 171. The urinary copper excretion was normal (less than 50 uqCu/L) i.1 13 of tile 14 subjects, but hypercupruria (347 ugCu/L) was present in the last samplc. The urznary uric ac;d/creatinine ratios were normal (i.e., less than 0.75) in the 14 workers. / /' This pilot investlgation denonstrated absorpl ion of :,Qlybdenum froin dust particles with sub:ie.joerrt excretion by the kidneys. It could not be deter- .- L mined wiiethcr molyhdenuT was absorbed through thi: lungs or by the gastro- i7testinal tr6:: :F+er sk*ailowirlg of sccretions. The wide range of plasma molybcienim levels in the workers may be explained by the time of day when the >anples were taken. For some worhers, the samples were taken prior to the eight-houL shift., others in the middle of the work day, and som at the ed of the work day. _-

The increments i!i mean sexum uric acid levels of the Denver factory workers were not a5 marked 3s those described L-)I Kovalskii and others (2) and hy- percricosurla was not r'ouxl in our study. TP~increases in mean scm ceruloplasmin levels among Denver factory wOrkr7rs were paralleled bg proportions1 changes in plasma copper content, and hence in discordance with the L are RuTsian aiitlior's fin:?ivgs of decreased levels of plasma copper. E1evatio:l of . serwq cerulcplasmin xu?d lx expected, however, according to mosthale and Gopalan's hypothesis (188), if molybdenum exposure leads to mobilization of ' f tissue coppcr storcs within thc hspatocyte, with subsequent synthesis of cZ- 1 rulopLasmm to pzevent intracellular copper toxicity (164).

*.is pilot investigation in factory workers raised the question of whe- t trier sc-rm ceruioplasn,ln levels could be used as a:i indicator of excessive mo- I _- lybdenunr exposure. mile the mean uric acid levels were alsc significantly -1 1

- ^___ __ 69 t3 I 1 1 1 I

/'* -- - - / .,,- \'\. /' I - 0 i *I 800- -- -,(i _- - - -- a L 8, :i t 600- e I a L d) 3 400-

200 - 0 Q 1 ! with the urinary copper levels varying between 5 to 12 :igCu/L. Calculatiuns of the diurnal creatinine excretion shoded low values in four of the samples. In the remaining two, which appeared sdequate, the 24-hOUr uricsry molybdenum excretion was 41 pg and 48 pg, respectively. ,Urine uric acid to creatinine ratios were normal.

alood samples were obtained from 18 miners at the Questa mine in New alsa > Mexico. The mean 2 SE serum ceruloplasmin for 18 Qursta miners wss 40.94 f 1.66 ug/dl and the mean serum uric ac~dwas 6.03 5 0.27 rrg/dl for 15 subjects. I The uric acid level; of three miners were not included in the calculation because one had long-standing gout (serum uric aci3: 10.1 ngidl) and two others

were taking antihypertensive medications (serum uric acid: 8.8 and 10.2 mgjdl). 1 Plasma molybdenum levels were below 5 pqXo/L in 12 of the 18 simples, and ranged from 6 to 18 ;Igb:o/'L in the remaining six samples. Because of technic?: difficulties it was not possible to collect timed urine speciaens in the Questa miners, Dut aliquots of urine were provided by 11 miner?,. The uric acid to creatinine ratios were normal. The urinary c~olybdenumconcentrations varied from 10 to 74 pgXo/L arid the molybdenun to creatinine ratios (crinary -'. molybdenum in pg/L/urinary creatinine in mg/L) ran3ed from 0.02 to 0.05.

The results obtained in the mincrs.diffcre2 from those of the factory workers in. v:xious sspects. The plasma and ur!.nary concentrations of molybdenum were uniformly low in the miners, which might be eicpecterf, since the subjects were mainly exposed to molybdenite*(MoS2), 3 compound known to be rela-. tively insoluble. In contrast, the se~muric acid levels were highei among the miners, but this hyperuricemia cou?d be a consequcnce of pvlycythemia secondary to altitude. ScPdvjlle, where the Climax miners resicic, is situated at lQ,000 fcec above sea level, and wn~lethe altitude of the Qucsta village approximates 7,000 feet , the actual mine 1ocatrc.n is around 9,000 feet.

The mean :jcrup\ ceruloplasmin levc?l.; in the miaers were high.?r than in the Depver co-itrol subjects, hdt dLd no+ attain the levels demonstrate3 by the ' workers at thz molybdenum roasting piant. Sincc no evidence for excessive molybrienwn exposure was present, the cjucstic I of whether altitude might also I _- affect serum ceruloplscnin levels was critcrtairicc! 2nd a dccision was made EO \ collect samples from subjects liviny at hiqh altituc-c and not involved in molybdenum nuning opera: ions. Thc necessity for cactior) in the interpretation of serum ceru1opla;mic and iric acid levcls, as indicators of molybdt?num ex- 1 posure, became evijent with these studies, while at tnc same time, the relra- I I bility of plasma and urinary molybdenu. values increaseti.

In aiditi.cn to these stucijes of industrial extxxxre, molyhdenum is pre- - sent, as previously mentioncd, in scme drinking water supplies of cmunities .,C" in Colorado. T!ie highf:;t quantlties of molybdenum used to be found in the . wGtc-r of the city of Goldcn whcre levels of 300 to 4CO ligt*lo/L had, on czca-

8 sions, bien mcasured. t!ow3-rerI cessation of the operations at the Urad molyb- ,- c de?-- mine resulted in decreased contamination of the Clcar Creek river, and the levels of nolybderrum in Golden water supplies have been decreasing since 1974. To dctermine whether molybdenum in watcr was accompanied by any health

ef fccts or changes in bioc-hcmical lnrilcatorsI ke studjed var~ouspopulations wi;ose water supplies diffcrcd in rnolybdenuq contcnt. hs controls a group of subjects were selected from the @pi-,ver area, where the molySdenum in hater

.. 71 , .. . -..--. , .. , .:

I fluctuates from 1 to 53 UgMo/L. ' Blood samples were collected on 42 male sub- ! jects whose sge rangec'c from 19 to 46 years. The mean f SE serum ceruloplasmin level for the group was 30.41 ? 1.03 pg/dl and the mean ser1.m uric acid was 5.34 f 0.20 pg/dl, both levels being in accordance with published normal values. Plasma molybdenum concentrations varied from less than 5 pgMo/L in 18 subjects to 34 pgMo/L. Fourteen subjects had levels from 5 to 9 ?IgfFo/L; levels from 10 to 19 pglrlo/L were found in seven persons, from 20 to 29 pgkIo/L in I one, and the two remaining subjects had plasma molybdenum concentrations of ! 33 and 34 pgMo/L, respectively (see Fig. 18). Red blood cell molybdenum con- I tent varied bztween 1ess.than 5 to 38 pgMo/L. Twenty-four hour urine col1ec.- tions were perfomec on 14 controls, fcr which complete results are available ! on 12 subjects. The urinary uric acid to creatinine ratios were normal in i all subjects. The molybdenum concentration varied from 120 to 230 pgMo/L, with the 11.~;in24-hour excretion of molybdenum being 87.25 f 18.02 pg (SEI. Ii Urirary copper concentrations varied from 4 to 25 pgCu/L, the mean daily cop- ! per excretion being 12.70 i 1.50 \1gCu/24 hours. Urinary molybdenum to creati- i nine ratios rangad from 0.01 to 0.11. i< ! Since the levels of molybdenum in the water of the city of Golden were I decreasing, a sanple collection was organized in March 1975, at which time the i I. water levels of nolybGenum approximated 200 pgr.lo/L. Blood samples were drawn 1 on 13 univzrsity students at the School of !.lines and the mean 5 SE serum I i cerulopLa.jmin wa; 40.31 5 2.55 mg/dl, a level significantly higher (p<0.005) than for the control ~rcup. The mean serum- uric acid was 4.35 f 0.4.6 mg/dl, 1 signifisan;ly lower than for the control group. The mean daily moly9denum I excretian in four 24-hour urines wab -35 f 34 114, also significantly higher than for the control group (p

Another sample collectio~~was organized in the Golden area in September 1977, by which time the water content of Ino1ybder.m had dropped to approximately 40 pgHo/L. Blnoc' samples were collecteci again in 13 students and cornpar<-scn of the serun ceruloplasmin and uric acid levels and of the urinary copper &ad molybcienurn excretions is s!iown in Table 18.

The decrease. in molybdenum water content in t!ie ?*;olden area was accompanied by normalization of ths s-?rum cerulopiasmin levels and hy it decrcase in the mean daily urinary molybdenum excretion. The incxease in mem serum uric acid levels cipanonstrated by the students in 1977 has to be interpreted with caution, since at the time of collection, it was evident that many of the students ,lad been drinking beer, and alcohol is known to increase serum uric acid levels through inhibition of renal secretory mechanisms. The students were . \.,- asked to cstimate their daily intake of all beverages and tt,e estimated quantities varied between two to three liters per d-iy, of which approximately one- half were water or beverages made from water. Eence, the daily contribution of molybdenum water in 1975 would have approximated 300 pg. Calculdtions of the molybdenum intake from foods shod that ior 20 year old male subjects the daily ingestion approximates 210 pg, ;o the total molybdenum ingesteri in -food and beverages in 1975 would have reachec! 500 pg daily. At such a level of ii.take, the serum uric acid levels were not affected, serm cerulop1asr.h concentrations increased, an3 plasma molyblenuti levels were within the normal .

.. .

I i. ,. .. .. , .. .. . '. '. i .. /. ., i . .. 'I 'I :i ., ..,.

C5 5-9 10-19 20-29 30-40 Plasma .Molybdenum (pq/l)

Figure 18. Hist.ograms of plaljma mo1ybdcnu:n concentrations in 42 normal subjects from the Denver area.

TABLE 18. COHPiRLSON 01' SERUM CERULC.PLASMIN AND URIC PCID LEXdLS AND OF T~I,~JRTMRY EXCRETIONS or MOLYBDENUM AND COEPER IN 1375 AND IC77 IN VIE- GL;LDEN AREA (MEAN 2 SEI (N) * 1375 (N) 1977 p-value --- Serum f;eruloplasmin (13) 40.31 C 2.55 (13) 34.15 2 i.74

Urinary copper ( 4) ll.G3 k 4.02 ( 8) 14.20 ? 1.62 N.S. i-5 (Yg/24 hrs) - i * i: Number of subjects i .* .- --

i i __ __ 73 1

.,, .. ! .. range. 'helve of the 13 subjects in 1975 had plasma molybdenum concentrations less than 5 bgWo/L. The uriiiary copper excretion did not seem to be affected by the elevated levels of molybdenum in the water. In contrast, the urinary excretion of nalybc'enum was higher, thus indicating renal clearance of the excess molybdenum ingested.

Another occasion to compare two populations with different water concentrations of molybdenm occurred in the rcetropolitan Denver area. The molybdenum concentrations in water of a Denver suburb fluctuated in 1977 between 80 to 100 ugPlo/L while the levels in the city of Denver were generally less than 40 pgXo/L. Blood and urine samplcs were collectad from 13 workers at the . suburkn municipal water treatment plant an3 comparison was performed with the results obtained from samples collected from 16 workers at a Eenver area water treatment plant (T'ble 19). ?he plasma molybdenum :wels were within the normal range in the workers from both area treatment plants. Serum ceruloplasmin . levels were sirni1z.r in both groups, and there were no significant differences in the daily urineiry excretion of copper, which was higher in the workers from the suburban treatment plant.

TABLE 19. COlYPARISCN OF SERUM-CERULOPLASMIN AND URIC ACID LEVELS AND OF THE URINARY EXCFETIONS Or MOLYBDENUPI AND COPPER IN 'IWO GROUPS OF WOiNERS AT WATER TREATNEP~T PIANTS (MLAN 2 SE) - --- (I)) Drnvcr (N) Suburban are& P-value I --- -.-_ Serum cerulopiasmin (16) 3tr.63 2 1.59 (13) 38.46 t 0.32 N.S. (mg/d 1) Serum uric acid (i3! 6.25 t 0.32 N.S. (mg/dl) Urinary molybdenum ( 9) 88 It 19 ( 3) 97 2 10 N.S. (9g/24 hrs) Urinary copper ( 8) 12.63 2 1.65 !lo) 22.91 ' 1.90 .0.0',,5 (pcj/24 hrs) .- --

Ar. occa..;iori to coqarc amther ?roup of subjects with different concentrations of mlybdenum 1r1 kater arose from a unique situation existinq in neighboring coi.munities in the Summit County area of the Rxky Plountains. Tn the water supp,ies of Sreckcnridge and 5illon, only traces of molybdenum have ' ken found whereas in Frisco and Silverthurne, levels betwecn 100 tCJ 400 pgMo/ L havz beon repeatecliy measured by this group. Furthermorc, these communities are situated at. an alcitude of 9,O.X fwt and hence it is possible to deter- ---* mine whether altitude causi:c? the chaircjts in serum uric acid and/or ceruloplas- nin levels found in the rninc:s.

Blod and mine samples were ColiPcted From if3 mqle and female inhabitants who had resided in the area for at least two years. Nine male and eight female subjects were fron the high-molybdenum areas, and five males and six females served as controls from the low-molybdenum areas of Ereckenridge and Dillon. The results of bicchemiaal assays performed on the male subjects are shown in Tale 20; no significant differences were found. The j-lasma molybdenum lecels in subjects from both sexes and from both drinking water areas were within the normal range. Corresponding results of biochemical tests in the females are shown in Table 21, where, interestingly, the female residents from the high-molybdenum area excreted significantly more rcolybdenum in the urine. a

I COMPARISON OF SERUM CERULOPLASMIN AND URIC ACID LEWIS i 1- 1- AND OF *rm UJUNARY MOLYBDENUM AND CCPPER EXCRETIONS IN ! RESIDENTS OF SUNYIT COUNTY {bKAt? ? SE); MALES ONLY 1. Low molybdenum High molybdenum - _- !

Serum uric acid (5) 6.58 f 0.32 (9) 6.68 f 0.33 N.S. 8

(mrj/dl) 15 I: Urinary molybdenum (4) 72 2 14 (6) 112 +- 32 N.S. t :; (pg/2 4 hrs 1 (6) 23.96 2 6.30 N.S.. ’?i* Urinary copper (4) 14.95 f 1.59

TABLE 21. COYPARISON OF SERUM CERULOPLASMIN AIli) “;;IC ACID LEVELS !1: AND OF THP URINARY MOLYBDENUM AND COPPER EXCFSTIONS IN ! * RESIDENTS OF SUMMIT COUNTY (MEAN’ SEj; FEMALES ONLY - ’. I,

Low molybdenum High molybdenum in water in water - (N) (10 W/L) (N) (140 pg/L) p-value Serum ceruloplasmin (6) 49.33 ? 4.03 (7) 49.57 2 3.41 N.S. (mg/d 11 I Serum uric acid (6) 5.10 +- 0.17 4.63 5 0.28 N.E. (8) 1 (mg/dl) 1I1

c Urinary molybdenum 57 2 9 (8) 100 5 0.025 L (5) 14 I (l~g/24hrs) I Uninary coFper ‘ (5) 8.71 2 1.28 (8) 22.61 2 7.24 N.S. i F (~g/24hrs) /. , ,= An estrogen efcect on serum cerulnplzsmln concentrations is possiSle, as is . the protective effect of female hormones on uric acid levels. Also of interest was the comparison between the serum levels of ceruloplasmin and uric acid obtained in Summit County to those obtained in the Denver area control groups. jz Significant elevations of serum ceruloplasmin (p<0.005) and of uric acid (p

TABLE 22. COMPARISON OF BIOCHEMICP-. =SAYS IN MALE SUBJECTS FROM THE DFNVER, BRECWNRIDGE, AND FRISCO AREAS (MEAN SE)

i' Water rolybdenum Denver Breckenridge Frisco I\ content (N) 0-50 ugXO/L (N) 20 PgMo/L (N) 150-200 pgMo/L I ii

Serum ceruloplasmin- (42) 30.41?.'1.03 (3) 41.00'0.71* IS) 37.22'2.58* I

I. !J I. .... (mg/dl) :: ', .. Serum uric acid (42) , 5.3410.20 (5) 6.5850.32* (9) 6.68?0.33* -. I

(mg/dl) I' .. $1 .. i. Urinlry nolybdenum (12) 87218 (4) 72214 (6) ii2~ :2 (ug/24 hrs) 'i ii Urinary ccpper (12) 12.70t1.50 (4) 14.9551.53 (6) 23 .96+6.36*

. .. .. (pg/24 hrs) '.

* ' 9 .< Significantly higner th;..n for the Denver control values (p<0.025 or less) I

I' ,I The results of these different investigations point out the difficulties assessins molybdenum cxposure with indirect indicators, cerulo- of i.e., senun I plasmin an& uric acid levels. 'While serum ceruloplitsmin levels seemed to be

affected by mclybdenum exposure in the molybdenum factory workers and in the I 1975 Golden erea st.udent5, ceruloplasmin levels were also affected by gender, altitude, and occupation. Similarly, the potentiai effects of molybdenum on serum uric acid values cxld be masked by age, gender, alcohol Fqtake, and altitude. With so many variables, it became evident that the best indicators of molybdenum exposure were the plasma and urinary lewis of the metal. It was

.l encouraging tc find levels of plasma molybdenun within the normal racge (5 to i. 34 ugMo/L) among subjects who were consuming water with 200 L!gMo/L. Increased ingestion was generally accompanied by increased urinary excretion of molybde- . num (see Fig. 19), a finding noted in the Golden area stLdents, the resiuents b".. of Summit County, and the mdybdcnm factory workers. In the la'L:er, workers - where molybdenm exposure was far greater than those in any of the other situ- ations, the plasma levels and the urinary molybdexlum to creatinine ratios were .. elevated. Reual clearance of excess molybdentun thus seems to function as a _-_- , - protective homeostatic mechanism in humans as it does in animals. Excessive - I_

......

. ..

'. . ,

.. ...1; n .. c E I 3'

h , :' 8: ...... I'

.. -5 +.

I I I e250 200-300 300-400 400300 >600 pg Molybdenum Injested Daily

Figure 19. Comparison of the daily urinary molybdenum excretion (mean ? SE) in male subjects with different doily intakes from food and water. summic? County I --- Breckenr idge Summit County I1 -- Frisco Golden 1975 ------Students in 1975

urinary copper excretion wak not demonstrated in the present studies, even when significaqt differences in copper excretion were shown. Nor was increased urinary uric acid excretion found in the various samplings performed. Since no bioche iical changes were found in subjects consuming water containing up to 50 pgl.lo/L, it can be assumed that such a level does not cause any adverse biochemical or health effects. Biochemical changes were seen in some subjects at 100 UgMo/L. The argument is further corroborated by the observa- tions in the Golden area students where the changes in molybdenm water concentrations were accompmied by normaljzation of serum ceruioplasmin levels and of urinary molybdenum excretion.

.. .. 77 ......

~ "- ...... - , ...... , .. .. .I ... I...... -.'- ...... ,. - ...... - ...... ,...... ,. ' ...... ,

...... -. :. , 1. Deosthale, Y.G, and C. Gopalan. The Effect of Molybdenum Levels in Sor- t g!iun (Sorghum vulgare Pers.) on Uric Acid and Copper Execretion in Man: r-. British J. Nutr., 31:351-354, 1974. i 2. Kovalskii, V.V., G.E. ‘farovaya, and D.M. Shmanvonyan. Changes in Purilie Metabolism in Humans and Animals Living in Biogeochemical Areas with High Molybdenum Concentrations. 2. Obsc. Biol., 22:179, 1961. I.! i.” 3. Kaback, D.S. The Geochemistry of Molybdenum in Stream Waters and Sedi- ments, Central Colorado. Ph.D. Thesis, University of Colorado, Boulder, Colorado, 1977. s. 4. Baas-Becking,+L.G.M., I.R. Kaplan, and D. Moore. Limits of the Natural Envixonment in Terms of pH and Oxidation-Reduction Potentials. J. Geology, 68:243-284, 196C..

5. Rollinson, C.L. The Chemistry of Chromium, Molybdenum, and Tungsrren. Pergarnon Press, New York, New York, 1973. pp. 700-742.

6. Williams, R.J.P., and R.A.D. Wentworth. Molybdenum in Enzymes. In: Pro-

. ceedinq~First International Conference on the Chemistry and Uses of Molybdenum, P.C.H. Mitchell, ed. Climax Molybdenum Co., London, England, $ 1974.

I 7. Bray, R.C., and J.C. Swan. Molybdenum Containing Enzymes. Structure an3 Bonding. 11:107, 1972.

8. Subrumanian, K.S., et. al. PreserTation of Some Trace Metals in Samples of Natural Waters. - Anal. Chem., 50:444-448, 1978.

9. Zurclova, J., J. Prasilova, and P. Benes. The State of Adsorption Be- havior of Traces of Molybdenum (VI) in Aqueous So1utior.s. J. Inorg. Nucl. Chem., 35:909-919, 1973. I--

I 12. Maienthal, E.J., and D.A. Becker. A Survey of Current Literature on I Sampling, Sarnplz Handling and Long Term Storage for Enviromtental Materials. NBS Technical Note No. 929, 3,s. Department of Commerce..

13. Crouthamel, C.E., and C.E. Johnson. Thiocyanate Spectrophotometric Determination of Plolybdenum and Tungsten. Anal. Chem., 26:1284-1291, 1954.

__ 14. Fishman, E.I.J., and E.C. I4allory. Determination of Molybdenum in Fresh A J. Water Pollution Control Fed., 40 r. Waters - Comparison OK Methods. (Srppl) :R67-R7lI 1968.

15. Ward, F.N. Determination of Molybdenum in Sojl and Rocks. Anal. Chem., - 23:7e8, 1951. --,

. 16. Ward, F.M., H.M. Nakagawa, and C.B. Hunt. Geochemical Investigation of Molybdenum at Nevares Spring in Death Valley, California. U.S. Geolog- ical SLrvey Professional Paper No. 207, 1969. pp. B454-456.

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153. Suttle, N.F. The Nutritional Significance of tke Cu:Mo Interrelationship to Ruminants and No>-runinants. In: Trace Wbstances in Environmental Health - VII, D.D. Hemphill, ed. University of Miss.,uri, Cclumbia, Missouri, 1914. i f 159. McReynolds, W.E., N.W. Wier, and J.C. DeFries. Open Field Sehavlor in Mice: Effect of Test Illumnatlon. Fsychon. Sci., 9:277-2:0, 1967.

160. LaVia, M.F. and R.B. Hill. Principles of Fathobiology. Oxford Univer- sity Press, New York, New York, 1971.

161. Heppe, M.S. The Influence of Ingested Molybienum in the Standard and Stressed Metabolism of the White Rat. M.A. Thesis, ‘Jniversity of Colorado, Boulder, Colorado, 1976.

162. Buchanan, G.M. The Effect of Eigh Dietary Molybdenum on Rat Adrenal Cortex. M.A. Thesis, University of Colorado, Boulder, Colorado, 1973.

163. Hurlinan, M., and P.W. Winston. Effect of Excess Molybdenum on Open t’ield Behavior in Rats. .I. Co10.-Wyo. Acad. Sci., 1O:irr press, 1978. (abstract) -1

164. Evans, G.W. Crspper Homeostasis in the Mammalian System. Physiol. Rev., 53:535-592, 1973

165. Ostrom, C.A., R. VanRc-er., and C.W. Miller. Changes in the Connective Tisscle of Rats Fed Toxic Diets Containing Molybder,um Salts. J. Dent. .L Res., 40:520-528, 1961. I -.- t 89 !.,

z 1

/ / --___I_ -. ------1-

f 1 3- .I i t

156. McCarter, A., R.R. Riddall, and G.A. Robinson. Molybdenosis Induced in Laboratory Rabbits. Can. J. Brochem., 40:1415-1425, 1362. I 167. Huisingh, J., and G. Ilatrone. Interactions of Molybdenum in Animal Nutrition. In: Ilolybdenum in the Environment, Vc? 1, W.R. Chappell aird K.K. Petersen, eds. hrcel Dekker, Inc., New '-ork, New York, 1977. pp. 125-148.

168. Mills, C.F., et al. Mechanisms of t..e hhlybdenun Sulfur Antagonism of - . Copper Utilization by Rdnants. in: Trace Elcyent Metabolism in Man -* and Aminals, - 3, M. Kirchjcssner, ed. Freising-Wiehanstephan, West Germany, 1978. .i

169. TiTton, I.H., P.L. Stedart, and J. Dickson. Patterns of Element--1 Excre- ,' tion in Lon.3 'i'erm Balance Studies. Health Physics., 16:455-462, 1969. I

170. Wester, PA. Trace Element Balances in Relation to Variation in Cakium Intake. Atheroschlerosis, 20:?07-212, 1974. t i1 i 171. Robinson, M.F., et ai. Metabolic Balance of Zinc, Copper, Cadmium, Iron, i i Molybdenum, and Selenium in Young New Zealand Women. British J. Nutr., 20:195-205, 1973. I. i -i 172. Gabovich, R.D. and O.A. Kulsyaya. Voprosi Pitanya, 23:65, 1964. (cited ! i , i by Friberg, L., et. al., 1975, ref. 50) 38 i i: i 1-13. Vorbjeva, A.I., and E.V. Osmolovskaya. On Molybdenum and Cobalt Balance I in Preschool Children. Gig. Sscit., 11:108-109, 1936. (in itussian) I i (cited by Fribery, L., et sl., 1975, ref. 50) I .I 174. Smolyar, V.I. Nicroclement Requirements of Children and Adults. Gig. f Sanit., 86-89, 1372. (in Russian) (cited ky rriberg, L., et al., 1975, f ref. 50) i 175. Hamilton, E.I., and M.J. Minski. Ahundance of the Chemical Elements in Man's Diet and Possible Relations with Environmental Factors. Science of the Total Env., 1:375-394, 1972/73.

176. Wester, P.O. Trace Element Balances in Two Cases of Pancreatic Inauf- ficiency. Acta. Ned. Scand., 190:155-161, 1971.

177. Ti-ton,i, I.H., and R.L. Stewart. Patterns of Elemental Excretion in Long- Tdrm Balance Studies. 11. In: Internal Dosimetry. Annual Progress Report. Ilealth Physics Division, University of Termessee, 1970. pp. 303-304.

I, i 178. Eurrell, R.J.W., W.A. Roach, and A. Shadwell. Escphngeal Cancer in the i i Eantu of the Traiiskei Associated with Mineral Deficiency in Garden ?lants. J. Natl. Cancer Inst., 36(2):201-209, 1966. - -_ 179. Adler, C., and J. Straub. Water-borne Caries Protection Agent Other than Fluoride. Acta. Med. Acad. Sci. Hung., 4:221, 1953. .- ......

4 APPENDIX A /* . SUPPLEWNTARY CALCULATION OF GUIDELINE

The guideline of 50 ugMo/L for human driqking water is based upon anhd and human studies described in the preceding sectio-is. In order to "check" this guideline, we have perfomed the following calcilatiolls based upoq c?ie- tary intakes. While the validity of the initial assumptions necessary for Lhe calculations may be argued, we believe that the calculations provide a useful order of magnitude check on the guizeline.

Early humans probably uid not have a "balanced" diet since availability of game and vegetable stuffs was variable. The hunter-gatherers probably ex- isted on a vegetarian diet for short periods of time an3 on a ;neat 2iet at other times. These short-term fluctuations of food j:itske rrobably resulted in short-term deficiencies and excesses of trace elements (see Table 10, Sec- , tion 6). Some level of ndapt-&lity to variable intakes probably still exists in humans today. Plodern food distribution pi-accices nnd dietary habits tend tc minimize the fluctuations of trace element. intakes. However, sone differences in food consumption exist between the sexes and among tbe various .%ge .'3coups. Socioeconomic factors and dietary prefsrences also lead to different. ,a intakes. Since there is no evidence that severe deficiencies OT exccsses occur in the United States' population, we :my assume that the biol~gizaland sxioeconomic differences are within the Adaptability of humans. \ Table A-1 shows the varizbility of dietary intake from foods for several ,-'' biological and socioeconomic: qroups. These results were obtained by calculaf- :ng the maximum difference in daiiy molybGen*m intake for the various grOGFd. (Percentages are based on the a-..ernge intake or the two grol;ps !3sirig comp,;red. See Figcres 13 and 14, Sectio:; 8.) These calculations show that the ma::imum in :akc difference obsezved between the sexes or with aye is skut 50%/, ' Socia- .. 6.c.momic variables produce differ enccs less than or equal to biologl :a1 -/aria- bles. If we assume that the dxitrvcd biologically-based intake v?,i'iat'Lon of .. 50% is the maximum perturbation tnat may be tolerated Withcut o9;erVin.j ef- .. fects, it is posslble to calculate the concentratlw of m;,lyb.ieIiLm *: then LR water which would produce an equivalc-rit increasc. I. Using the calculated dietary intakes (Figure 13, Scc$ori a), arid the .', -" 5. availability data on consumption of water-based %?veraq-'s (coffee, tea, caft .. drinks from reference 77) one czn calculate 'thhf. molybY-enUn concentratic.1 in water which would produce a 5C% increase in dictary,.lntake.

The results of these calculaticns Eor the ?-,er;icc individual in each age and sex categcry are shown in Figure IrL. "he, 'rtp~drentlyhigh rancentrations I i i i . - /.- i ! / I TAR1.E P.-1. VAFI.ABILITY OF DP.ILY k!!OLYBDEMZX INTAKE I: i -- .- -- _--- I Dif f zrences .%ximan 's variation t t -- --r I t ! Biolog< ccll '.ge (males) 47 i (adults only) Age (female.-:) 47 r:*; Sex (males* vs females) 22 dI Age (ug,'day/'Ag body weight) 47 i _- __ ._.-t-a I . Sociceconon Ic Incorte (low* VF hirjh) 52 f .. .I -- Urbanization (ursan vs rural*) .! Geographic (North U.S. vs South U.S.') 156 [- 8. I ! Othe'. Meat vs vegetarian*? 58 I s. i - --- . - _. ----. _____p-___.____--

i .I . Group with higher mciybcienum ir.taLe. .. I i! 'Vegncarian 6iet estimated by s&stituting a prFL>ortionateincrease of all !i ncn-neat compor.aits for meats (estimate only) j ,' I,

i ! i

I ! 1

5. 1;

I ! j!t' I.

i ! 4 -ma111 Age Group -.

Figure A-1. Molybdenum concentration of water requirld -- / - to prcduce a 50% increase in daily intake - -- through water-based beverages.

_. - I 93

1 ! 'r li - i I ,I 1 %I 1' .-5 I I,'I i i

of nolybdenua in water permissible among youngei individuals is mainly due to the small anoU;lt of water-based hewragas consumed by this group. tiosever, rurrong adults the predicted m1ybdcr:um cottcentration which I-ould produce a 50% increase in Zaily intake approaches lOC! ugP.o/C. If we assume that water In- take is abol;t equal to the water-Lased beverage intake', then the "permissible" water concentration would k deut 50 ug.C(o/L. This estimate is the sane as the guic'eeline based upon the studies described in previous sections. The aqreemsnt between the calculated estimate and the guideline may be fortbi-.ucw buc it serves as a useful order of magnitude check.

-1 .r - _-

I 1 t t > 'i

- .--_I_ 1. t i. The maxi;um water-based beverage intake of 0.05 liters-occurs among males i: aqed 35 to 54. Cabling this ~nou:~';leads to J value close to the 2.0 llters ! typicaliy used by thc U.S. Env,rwmental Plotection Agency. .._ - t. _. i 94 i -_-____ -____- __ :I ';

I' i;

i ! i

I /I APDENDIX B I

.- ANALYSIS FOR MCLYBL)ENUM, SPECTROPHOTOMETRIC METHOD . - ! __- ! '. -_ fiiocyanate has been used extensively in the colorimetric analysis cf ! . _-..- molybdenum (13-16). The conventional analytical method has been modifie:l ! slightly to suit our needs. Water samples can be routinely analyzed with pre- c cision limits of abaut pg/L (without prior coccentration steps). 25 This pro- - # ._ cedure is outlined below.

ANALYSIS OF WATERS FOR MOLYBDENUM

1. Pipet 50 mL of the unki-own or standards inCo a 250 ml. separatory funnel. (A blank shou,d also be carrled through this procedure. I - __ The standards whicb *;euse usually range Frd 50 to 500 Vg/L. I I' Wically wc run four: 50, 100, 300, 500 ;Ig/L.) ! . I' I -I 2. Add 2 mL of concentrated HC1 co the :;amples. The acid is required I I to prepare *he solution for die formation of the thiocyanate com- i1. plexatlon reactlon. Thls reaction requircs that the final solution I ! be about 1 I! in acid. t 'i

3. Add a9proxinately 0.,2 CJ of sodium tartrate. This "ties up" any tungstate which may Ks present. It wo,lld interfere with the analysis for moLybder.um since tungsten also forms a colored com- ': plex (yellox) with thiocyanate. i 'i I 4. Add 0.5 mL of 1%ferrous ammonium sulfilte. I Prepare this solution by weLghing out 1 g of the reagent grade Fe(NHqS04)2. Dissolve .I this salt in 100 mL of deionized water and 1 m5 of concentrated j sulfuric acid.

5. Add 3.0 mL of 10% KCNS. Prepare this solution fresh daily by .- . weiqhing 5.0 g of the salt arid dissolving it in 45 mL of deionized-1 water. i I ? I Allow the solutior. to stand for about 15 minutes. This step is very imiortant. I'ull color will not develop if time is not given for the complexarion reaction to be ccvpleted. The solution will be pink, orange, or red.

7. Add 9.0 mL oE 10% stannous chloride solution. Prepare this by I mI, weighing out 100 g of the salt an6 dissolving it in 125 of con- i,-- centrated HCl. Heat the mixture until all of the salt dissolves. -. I- -i

95 i _<_I.

i I 1>

. __ ~ . ?.. --- I i I I'/

~ I I AI / 1 -- ! ..* < 1 1 The solution should be clear. Slowly add this solution to 900 mL of deionized nator. Store this solution in a bottle which contains a few pieces of purz t:'n metal.

8. Allow the solution to stand for at least 15 minutes. The pink or red color should disappear. Only a pale amber color should remain. This is the thiocyanate-molybdenum complex. t 9. A& 10.0 mL of isc?-amyl alcohol to the separatory funnel. Shake che funnel vigorously until the color leaves the aqueous,phase and enters the organic phase. Thirty to sixty seconds should be suf- f icient.

10. Allow the pkases to separate (about 15 minutes). Drain off the aqueous layer and discard it. Be sure to get all of the water out. If necessaq, drain off a little of the organic layer to "flush" - any water oat of the stopcock bore. ---,--- e4 11. Drain the colored organic layer into a test tube. Add a spatula full of granular anhydrous scdiun sulfate to the solution and allow it to stand about 30 minutes. This drying step helps to . remove any turbidity xhich could interfere with the spectrophotometric readings.

12. Read the absorbance at 465 nm versus the blank. Prepare a Beer's Law plot of absorbance versus concentration usiny the standards. Use the plot to determiroe the concent-:ations of the unknowns.

%. In this :ab we usually do i*. linear least sqiares regression analysis on the standard poin.ts. From the equaticn of the line we can calculate the concentration of the unknowns.

The conventional thiocyanate procec?are has been shortened to permit more rapid analysis of samples. '( The increased speed of the analysis has been gained at the expense of some precision. The standard error of estimate is typically +15 I.ig/L. This method is referred to as the semi-quantitative ~ro- cedure. Most natural waters and aqueotis smples can be analyzed by this procedure. i

/--- I Kore Rapid Procedure I This procedure is csed for the rapid processing of large numbers cf sam- - ples. Up to fifty samples may be analyzed at a time. If the samples are ' 's higher than 1 mg/L thz solution should be diluted to fall in the range of the #' /' standards. This procedure uses 30 mL test tubes. All of the steps and wait- ing times are the same dS in the method der-ribed above. A standard deviation .. of about 15 ug/L can be expected when this method is used.

The following are the chwiges in reagent quantities to be used:

.. 1. 10 mL samples and standards. .. , ...... , .... . , r_ . _.._-,.,,. .... I . .-.*, ...... " -: .. .. _......

, 2. 0.5 mL concentrated HC1.

3. 0.05 g tartrate (exact amount not critical).

4. 0.1 mL of 1% ferrous ammonium sulfate solution.

6. 2.0 rnL of stannous chloride sclution...... -. =I 7. Extract into 4.0 mL of iso-amyl alcohol. . ; .T' -? ...._. 8. Centrifuge the test tubes and contents at 2,003 rpm for about I __ I five minutes. iI

9. Draw off the colored organic phaseby using an 8" Pasteur pipet. .I .I :i 10. Diy the extract over anhydrous sodium sulfate. (omit if AA is used on the organic extract!)

11. Allow the extracts to stand for about me-half hour. Read the absorbance at 465 nm. Use 1 cm absorbtion cells.

. .. i APPENDIX C

. _..' , .... ANALYSIS FOR MOLYBDENUM, ATOMIC ABSORPTION SPECTROPHOTOMETRY (MS) . . . ., -...... - .. . I...... Molybdenum analysis by flame AAS is susceptible to many interferences. Direct aspiration of aqueous samples is not recommended because of these inter- Cerences. In addition, the method generally affords a detection limit (signal to noise ratio of 2.0) of about 50 pgMo/L. Therefore the method has insuffi- -1 -. .. - . cient sensitivity and precision to adequately determine the "contamination" of most waters.

Acceptable results can be-obtained using f1axr.e AhS by preconcentrating the samples prior to analysis. The analyte should also be separated from po- $ tential interferants. Both of these requirements can be accomplished by using a complexation-solvent .:xtraction procedure. Several publications describing such procsdures are referenced In Section 4 of the text. -;? . In this laboratory we have successfully used the following procedure. It consists of the complexation imd solvent extraction technique used for the spectrophotometric determination of molybdenum dzscribed in the preceding pages. However, in this modification molybdennm detection is acconplished by aspirating the organic solvent containing the analyte into a nitrous oxide- acetylene flame. Lower detection limits and higher sensitivity are achieved ,-_- ,by using greater preconcentration factors (sample volume to solvent volume ratios). In addition, the sensitivity is improved when organic solvents are used in flame analysis of refractory metals such as molybdenum.

.I Sample aliquots of 10 to 15 mL are taken for analysis. (This sample volume permits a detection limit of about 5 pgMo/L.) The following method is analogous to the solvent extraction technique described in the preceding pages. r

1. Add 0.5 g sodium tartrate and ctir.

2. AddI 0.1 mL 1%ferrous aradnium sulfate solution. n I I 3. Add 0.5 mL 10% potassium thiocyanate solution; stir; wait 15 minutes. molybaenm using flame a t0rni.c I absorption. ! t! AA ~iaraneters--Perkin-Elm~r360

Lamp wevelength--313.3 rm i Lzmp current--30 na Flame f120+cety:ene--fue1 rich, 5 cm single slot nitrous oxide head -'II Scale expaasion x 25 - Aspiration rate--the nebulizer system is slightly adjusted for ." I the alcohol to aspirate moderately slow 1 The standards arc 50, 100, 200, an2 300 \igHo/L. An is0;myl alcohol I i blank is used. ?%e common practico in chis laboratory is to include tuo to three of each standard, depending on the size of the run, to insure that there nitor the calibratioa of the instrument.._ . ion is determined by using several six second time averages r each sample and standard. A Beckmar 10" strip chart recorder i:: used to splay thc time integrals. !4fter anaiysis the peak !>eights are measured and are calculated.

or electrothermal atomizatian techriiqurs whish uce a heated graphite furndce or cup my also be uscd for nc-lybdcnun analysis. l'he graphite furnacc technique permits, molybdenum actermin~tlonat tho ng levei with snlall s~~plevoluvcs. *.is tech uquc is, Ixwcver, prone to matrix interfrsi- ences when used for molybderi~.m riilalysLs Tfit? boll129 point of molybdenum metal is abut 4,8OO0C. This is about 2,OUO0C akcwc the vlilx~muntemperature obtained in chc graphite furn.\cc. 1%~sindicates that molybdenum atomization can only take place by a nechanlsm which inclutlcs the formatlor of a compound L which is volatile at 2,d3O02. This com;~ouiid tlicri can dissociate in the vapor phase to release molybdcrt*xn dtoms. Any ions or compounds which affbct the complicated atomization *ncchanism w: li iiltcr the scnsitlvity of the method. . This crucial fact is the prinury reason for thc cxtrcme matrix sensitivjty of t!!e method.

ipal intcr€erencc cncountcrcd in this study rcsults from the simultarcous ciresence of two icnic species, an alkali mctal ion such as potassium or SodilJ~and the s-llfate ion. !:either the cation or anion produce sjg- \ riificant interference whcn they are expcrimentally added wlth other counter ions. A sample containi?g 50 :~u:-!o/L will a1)pear to contain 30 llgMo/L then sulfate (in thc presencc of sodium) is prcscnt at 1,000 mg.;L. A ten percent ssion results whcn sulfate is present at 200 ng/L.

In this study all atomic absorption measurcnents were nade with a Ferkin- Elmer Model 360 Atomic Absorption Spcctroph-Itoi,.-ter cqurppcd with ;i Perkin- Elmer HGA 210C Zraphitc Furnacr. The spcctrophotomcter conditi0r.s were: Silt--Alt. 0.7 rm; Mode--TCl; I:rip--lSX. A Pcrkin-Elmcr molytx'.enum hollow cathode lamp was operated at 3C ma. The nonochrorimttor was set to pass the 313.3 nm resonifice linc of molyldcnum. The graphite furnace conditions were: -1 Dry--50 sec at 120%: Char--4O sec at 1,EOOoC; Atomize--8 scc at :!,ROOOC. The i argon purge gas was set at con+inous flow (normal) at "20" units on tne fled . I __ - 93

.. - -