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\\ 0I r* j h 4 AIRBORNE LEAD IN PERSPECTIVE ADVANCE COPY A Report Prepared by the Committee on Biologic Effects of Atmospheric Pollutants of the Division of Medical Sciences it National Research Council National Academy of Sciences #' Washington, D, C. 1971 DUP050055272 Prepared under Environmental Protection Agency contract no. CPA 70-42, a * DUP050055273 PREFACE The United States Congress passed the Clean Air Act in 1963 and has since strengthened it through amendments. Responsibility for issuing air quality criteria guidelines to assist the states and cities in developing standards for air quality was assigned to the Environmental Protection Agency (.EPA), The scope of the guidelines, as stated in the Clean Air Amendments of 1970, includes "effects on soils, water, crops, vegetation, man-made materials, animals, wildlife, weather, visibility, and climate, damage to and deterioration of property, and hazards to transportation, as well as effects on economic values and on personal comfort and well-being." The National Academy of Sciences (NAS) was asked to prepare evaluative reports of current knowledge of selected atmospheric pollutants to serve as background for preparation of criteria documents and EPA decisions, and to make recommendations for research needed where sound info mat ion is lacking. In the Division of Medical Sciences of the National Research Council (NRC), the Committee on Biologic Effects of Atmospheric Pollutants was fomed to coordinate and supervise the activities of ad hoc panels of experts brought together to pool their knowledge of specific pollutants. Because most of the members of both the Committee and the Panel on Lead were biologists, this report treats the strictly biologic aspects thoroughly, whereas the material on nonbiologic effects of lead, which came from con sultants, is brief. The report reviews the scientific knowledge of the effects of lead on human health and welfare, enumerates the factors that alter these effects, and points out areas where data are lacking; it is an attempt to place in i DUP050055274 perspective the role of airborne lead in the biosphere. In its early planning, the Panel decided to consider biologic effects of lead not necessarily attributable directly to atmospheric sources and not neces sarily at levels of exposure as low or as prolonged as those related to general ambient air. Such consideration was necessary because lead attributable to emission and dispersion into general ambient air has no known harmful effects. It was only by considering the circumstances in which lead is manifestly harmful that some perspective could be gained as to the significance of contributions from general atmospheric sources . The Panel was not asked to comment on air pollution standards and has not done so. At the Panel fs first meeting, in July 1970 * a tentative timetable and outline were developed that seemed reasonable within the constraints of EPAfs needs. Sections were then assigned to panelists and consultants, who prepared drafts that were revised by the full Panel and circulated to reviewers from the Committee, to outside specialists, to appropriate I5IRC divisions, and to the WAS Report Review Committee. The bibliography is not intended to be exhaustive, hut it contains the most important references to the primary literature sources on lead through 1970 and early 1971- Translations of most of the foreignlanguage reports are available from the National Translation Center. Throughout the report, the units are consistent with common usage of workers in the area of the discussion. The term f,lead particles" will be recognized as meaning "lead-containing particles." * ii DUP050055275 Only by the cooperation of many persons was preparation of the report within 9 months made possible. They responded generously, enthusiastically, and competently, The roster lists the names of contributors and consultants, to all of whom the Panel expresses great appreciation. In addition to the generous involvement of panelists, the response of EPA staff to requests for help was indispensable. The Panel's needs for information services were met in part by the NRC Advisory Center on Toxicology, the NAS Library, the National Library of Medicine, and the Air Pollution Technical Informal tion Center, Members of the Committee Who had specific responsibilities to the Panel were Paul B, Hammond (Chainnan, Panel on Lead), Bertram D. Dinman, Victor G. Laties, and Gordon j. Stopps, The indexed report will be published by the National Academy of Sciences in its series "Biologic Effects of Atmospheric Pollutants.11 iii DUP050055276 COMMITTEE ON BIOLOGIC EFFECTS OF ATMOSPHERIC POLLUTANTS Division of Medical Sciences, National Research Cornell Roster from November 1970 to April 1971 DuBois, Arthur B., School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 191Q4 - Chairman Bacon, Vinton W* , College of Applied Science and Engineering, University of Wisconsin, Milwaukee, Wisconsin 53201 Baetjer, Anna M., School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Cooper, W, Clark, School of Public Health, University of California, Berkeley, California 94720 Corn, Morton, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Dinman, Bertram D., School of Public Health, University of Michigan, Ann Arbor, Michigan 48104 Golberg, Leon, Institute of Experimental Pathology and Toxicology, Albany Medical College, Albany, New York 12208 Hammond, Paul B., College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55101 Hicks, Samuel P. , University of Michigan Medical Center, Ann Arbor, Michigan 48104 Laties, Victor G., University of Rochester Medical Center, Rochester, New York 14620 Idlienfeld, Abraham M., School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Meier, Paul, Biomedical Computation Facilities, University of Chicago, Chicago, Illinois 60637 iv * p 9 DUP050055277 COMMITTEE ON BIOLOGIC EFFECTS OF ATMOSPHERIC POLLUTANTS - Continued Pittis, James N., Jr., Statewide Air Pollution Control Center, University of California, Riverside, California 92502 Stopps, Gordon J., Haskell Laboratory , E.I. duPont de Nemours'and Company, Newark, Delaware 19898 Taylor, 0. Clifton, Statewide Air Pollution Control Center, University of California, Riverside, California 92502 Vostal, Jaroslav J., University of Rochester Medical Center, Rochester, New York 14620 Boaz, T. D. , Jr. , Division of Medical Sciences, National Research Council, Washington, D* C., Executive Director PANEL ON LEAD Hammond, Paul B, , University of Minnesota College of Veterinary Medicine, St. Paul, Minnesota 55101 - Chairman Aronson, Arthur L., New York State Veterinary College, Cornell University, Ithaca, New York 14850 Chisolm, J. Julian, Jr., Baltimore City Hospitals and Johns Hopkins University Medical School, Baltimore, Maryland 21224 Falk, John L., Rutgers University, New Brunswick, New Jersey 08903 Keenan, Robert G., George D. Clayton & Associates, lire. , Southfield, Michigan 48075 Sandstead, Harold H. , Vanderbilt University School of Medicine , Nashville, Tennessee 37203 Marshall., Louise H., Division of Medical Sciences, National Research Council, Washington, D. C., Staff Officer V DUP050055278 CONSULTANTS AND CONTRIBUTORS Albert, Roy E., Institute of Environmental Medicine, New York University Medical Center, New York, New York 10016 Blodgett , Frederick M., Marquette School of Medicine, Milwaukee, Wisconsin 53233 Brown, Thomas, Food and Drug Administration, Washington, D. C. 20204 Bryson, Reid Av* University of Wisconsin, Madison, Wisconsin 53706 Chow, T. J,, Scripps Institution of Oceanography, University of California, La Jolla, California 92037 Cohen, Norman, institute of Environmental Medicine, New York University Medical Center, New York, New York 10016 Daines, Robert H., College of Agriculture and Environmental Science, Rutgers University, New Brunswick, New Jersey 08903 Dews, Peter B., Harvard Medical School, Boston, Massachusetts 02115 Doe, Bruce R., U. S. Department of the Interior, Branch of Isotope Geology, Denver, Colorado 80225 Eisenbud, Merril, Institute of Environmental Medicine, New York University Medical Center, New York, New York 10016 Goyer, Robert A., University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 Habibi, Kamran, Petroleum Laboratory, E. I. duPont de Nemours and Company, Wilmington, Delaware 18898 Hardy, Harriet L., Occupational Medical Clinic, Massachusetts General Hospital, Boston, Massachusetts 02115 Kehoe, Robert A., Kettering Laboratory, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229 Langway, Chester Cr, Jr., U. S. Army Cold Regions Research and Engineering Laboratory, Hanover, New Hampshire 03755 Lin^Fu, jane, Maternal and Child Health Services, Health Services and Mental Health Administration, Rockville, Maryland 20852 vi * & ) DUP050055279 Consultants and Contributors - Continued Locke, L. N., Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, Maryland 20810 Lodge , James P., Laboratory of Atmospheric Sciences, National Center for Atmospheric Research, Boulder, Colorado 80302 Machta, Lester, Air Resources Laboratory, National Oceanic and Atmospheric Administration, Silver Spring, Maryland 20910 McNesby, James R., National Bureau of Standards, Washington, D. C. 20234 Miller, Donald F-, Food and Drug Administration, Washington, D. C. 20204 Montgomery, John M., National Paint* Varnish and Lacquer Association, Inc., Washington, D. C. 20005 Morgan, Jean M., Veterans Administration Hospital, Birmingham, Alabama 35233 Pierrard, John M., E. I. duPont de Nemours and Company, Newark, Delaware 19898 Rizzo, Frank j,, textile Research and Engineering Division, U* $ Army Natick Laboratories, Natick, Massachusetts 01760 Schaefer, Vincent J., Atmospheric Sciences Research Center, State University of New York, Albany, New York 12201 Shemin, David, Northwestern University, Evanston, Illinois 60201 Six, Kathryn M., University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 Tepper, Lloyd B., Kettering Laboratory, University Of Cincinnati, Cincinnati, Ohio 45219 Ter Haar, Gary, Ethyl Corporation Research Laboratories, Ferndale, Michigan 48220 Tschudy, Donald P., National Cancer Institute, Bethesda, Maryland 20014 Tyler, Germund, Department of Plant Ecology, University of Lund, Sweden Ulmer, David D., Biophysics Research Laboratory, Peter Bent Brigham Hospital, Boston, Massachusetts 02115 vii DUP050055280 Consultants and Contributors - Continued Viechnicki, Dennis J., Ceramics Division, Army Materials and Mechanics Research Center, Watertown, Massachusetts 02172 Wood, John M., School of Chemical Sciences, University of Illinois, Urb an a , Illinoi s 6l801 v r viii r DUP050055281 CONTENTS introduction Chapter 1 Chapter 2 Chapter 3 Chapter k Chapter 5 Chapter 6 Chapter 7 Chapter.8 Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G References Glossary PaSe. LEAD IN THE ECOSYSTEM LEAD AND PLANTS INPUT AND DISPOSITION OF LEAD IN MAN BIOLOGIC EFFECTS OF LEAD IN MAN BIOLOGIC EFFECTS OF LEAD IN DOMESTIC AND WILD ANIMALS AIRBORNE LEAD ALKYL COMPOUNDS NONBIOLOGIC EFFECTS OF LEAD CONCLUSIONS AND RECOMMENDATIONS FOR RESEARCH PARTICLE SIZE SAMPLING AND ANALYTIC METHODS FOR LEAD DATA AND CALCULATIONS FOR FIG. 3-3 COLLECTION AND STORAGE OF BIOLOGIC SAMPLES SUMMARY OF TOXICOLOGIC DATA TREATMENT AND COSTS Of1 LEAD POISONING IN MAN TREATMENT AND COSTS OF LEAD POISONING INCATTLE 1 5 51 6j 105 277 295 303 312 A-l B-l C-l D-l E-l P-1 G-l DUP050055282 Introduction In the midst of currently intensified concern over the quality of the environment, it is both logical and proper to select lead for indepth scrutiny, particularly from-the standpoint of the consequences of atmospheric emissions. The amount of lead vented into the atmosphere over the United States as a result of combustion of lead-containing automobile fuel additives and from miscellaneous other sources is measured in hundreds of tons per day and is increasing in proportion to the ever^expaiiding use of the automobile. Because the uses of lead for purposes other than as a fuel additive are many and varied, it is important to. develop a sense of perspective wherein the conse quences of lead input into the environment fran all major sources are evaluated. Although this document is concerned with the hazards associated with the dispersion of lead into the environment, the consequences of dis persion should also be viewed from the standpoint of depletion of a natural resource. The earth's lead resources are being mined at the rate of 2.2 x 10^ kg/year to meet worldwide needs The net loss relative to the earth's total lead resources, as with other minerals consumed by man, should be a factor in any decisions that will increase the rate of consumption of lead. "Only by supplementing our fairly definite knowledge of measured and indicated reserves--or working inventory--with crude guesses and analogies tending toward -L :- : DUP050055283 the optimistic, can we at this time see sufficient reserves to QOQ equal lead demand during the balance of this century." The extensive mining and processing of lead dates back to preChristian times... Its low melting point ( 327 C )., malleability , and ductility, favored its early use for the manufacture of metal products. It- also tarnishes in air and forms a film of oxide, which accounts for its not being readily corroded. This property also made it highly desirable for many applications. In addition to numerous vises by civilized man, lead has contributed greatly to knowledge of geologic time. 11 Studies of lead isotopes in meteorites have been very fundamental to all of earth science," l(5l and have led to determination of the accepted age of the earth. h52 Lead has four radioactive isotopes (lead-210, -211, -212, and -214); lead-210, a natural 6 and r-emitter with a half-life of about 22 years, . 24 has been useful as an atmospheric tracer and in precipitation dating. Most of the salts of lead are highly insoluble and, in nature, lead exists mainly as the least soluble of the common salts, lead sulfide (galena). Lead carbonate (cerussite), lead sulfate (anglesite), and lead chlorophosphate (pyromorphite) also are common naturally oecurr^ ing forms. The interaction of lead with organic matter also results in the formation of stable complexes. Sulfhydryl, carboxyl, and amine coordination sites are all involved in the complexing of lead -2- O DUP050055284 with organic matter . The low solubility of lead in the aqueous phase of natural systems is manifested in many ways directly rel evant to the understanding of the behavior of lead in the environ ment. Some consequences of this fundamental characteristic are that lead is poorly transferred from soil to plants and that the concentration of lead in natural bodies of water is extremely low in proportion to the concentration in the beds of lakes and streams. In animal systems, the consequence is poor uptake and slow excre tion.. The net effect of these sluggish dynamics, however * is a high degree of accumulation with prolonged exposure. Animal tissues have excellent lead-binding characteristics. The persistence of lead added to segments of the environment to which man is exposed thus results in legitimate concerns over adverse cumulative effects. The concern today is mainly over the possible hazards to living organisms that might result from the widespread dissemination of lead by man into the general environment, and particularly over the insidious effects of long-term exposure. Because the mining, smelting, and commercial uses of lead have been so extensive for such a long time, a considerable body of knowledge already exists regarding the harmful effects of excessive exposure in man, animals, and plants. These effects generally have been observed only at levels of exposure higher than apply to the general populations of people , animals, and plants not near primary sources of man-made lead products. The primary goal of this report was to -3- V ; ...........' DUP050055285 assess the likelihood that man, animals, and plants would he brought into the range of harmful levels of exposure now or in the near future and the circumstances that might make that possible. This goal required that the Panel address itself to questions pertaining to the magnitude and distribution of lead in the environment, both in the context of present conditions and in the context of likely future conditions, assuming the continuation of current technologic trends. It was also neces^ sary to attempt to estimate minimal toxic exposure levels, draw ing from the vast literature pertaining to the toxic effects of lead, The frustrations engendered by the attempt to dp that found expression in the form of recommendations for future research. A rDUP050055286 CHAPTER 1 NATURAL OCCURRENCE OF LEAD LEAD IN THE ECOSYSTEM "Pollution" implies the introduction of an undesirable substance. Any; amount of a purely man-made substance in the environment is present obviously because man put it there. Other substances considered to be pollutants may occur naturally as well; that is the case with lead. Description of the source, magnitude, and distribution of man-generated lead pollution can hardly be undertaken without some knowledge of the natural background levels. Considering that man has mined and used lead for many centuries, accurate estimation of contemporary natural background of lead is difficult. Calculations of natural contributions have been made on the basis of geochemical information. Natural sources apparently contribute only insignificantly to present-day concen trations of lead in the atmosphere. o 451 be about 0.0005 Mg/m air, Natural concentrations have been estimated to and result from airborne dust contain ing on the average 10-15 ppm lead12 and from gases diffusing from the earth*s crust. ^ The latter source results in the presence of lead-210 in the atmosphere ranging from 7-1 x 10"^ dpm/kg air at ground level to about 70 x 10~3 dpm/kg air in the lower stratosphere* 79 Detectable quantities of lead-210 reach the earth's surface in precipitation and on dust particles. 190 ' 2 5 7 * 326 Perhaps the most useful records on national concentrations of lead are to be found in chronologic layers of snow strata, available in quiescent ice sheets in perpetually frozen polar regions. Annual layers of snow from Greenland and the Antarctic have ^Reliable age data of annual layers have been obtained from radioactive (natural and artificial) and stable isotopic investigations of deep ice core samples: namely, total beta activity (primarily Sr-90 and K-40, useful for the last 10-20 years); Pb-210 (for the last 100-150 years); Si-32 (for the last 1000 years); and 0-l3 (combined with physical models, for the last 150,000 years). -5- DUP050055287 4l8 been analyzed. Annual ice layers from the interior of northern Greenland show that lead concentrations increase from less than 0.0005 yg/kg of ice at 800 B.C. to more than 0.2 yg/kg in 1965 (Fig. 1-1). The ice layer corresponding to 1750 A.D. represents the beginning of the Industrial Revolution, and the lead concentration at that date is 25 times greater than natural levels. During the second half of the 18th century, lead concentrations tripled, and from 1935 to 1965* they abruptly tripled again. The sharpest rise occurred after 19 40. Today, lead concentra tions in Greenland snows are about 400 times the natural levels. These data dramatically document the rise of lead in some components of the environment, but they must not be taken as representative of worldwide circumstances. For example, levels of lead in Antarctic continental ice sheets fail to show similar rises. Concentrations of lead in ice before 1940 were below 0.001 yg/kg and rose to only 0.02 yg/kg after 1940. The difference between lead concentrations in northern and southern polar snows is ascribed to barriers to northsouth tropospheric mixing, which hinder the migration of aerosol pollutants from the northern hemisphere, where most industrial emissions occur, to the Antarctic. Few chronologic records of lead concentrations are as complete as that described for polar regions, but there are data that reflect increasing lead concentrations in some other ecosystems. For example, it is esti mated that the preindustrial lead content in marine water was about 0.02-0.04 yg/kg. 123 Today, surface waters in some areas of the Mediterranean and Pacific contain as much as 0.20 and 0.35 yg/kg. -6- DUP050055288 respectively , and only deep waters, below 1,000 m, appear uncontaminated (Fig. 1-2). The lead content of fresh waters also has increased in recent times. The available data suggest that the mean global natural lead con- 365 tent of lakes and rivers is 1-10 ng/liter. The soil in rural areas of the United States has lead concentrations usually similar to the average jpfl content in the earth1 s crust, 10-15 ug/g. But in many cities, the concentration of lead in soil is much higher, suggesting substantial but relatively localized pollution. There are few examples of long-term trends in lead concentrations of biologic materials. Ruhling and Tyler1*77 demonstrated chronologic in creases in lead concentrations in Swedish mosses from i860 to 1968 (Fig. 1-3). The principal increases were believed to correspond to increased combustion of coal in 1875-1900 and of leaded gasoline in 1950-1968. Other evidence of man *s large contribution to total environmental lead is found in the geographic pattern of atmospheric lead concentrations: high over major urban centers, lower by a factor of 10 or more over rural areas remote from human habitation, and lower than that by a factor of 100 over the itdd-Pacific.1^^,!^ A useful tool for distinguishing between industrially produced lead and lead that occurs naturally in contemporary soils and water has *2 g been analysis of its isotopic composition. ' Many lead ores have a characteristic composition fixed during mineral genesis, and the isotopic eonposition of commercially important ores has been surveyed. 73 126 In addition, Chow and Earl have determined the isotopic composition of several lead ores, gasolines, aerosols, waters, and marine sedi- ments. The average isotopic composition of lead in sediments from the Pacific and Atlantic Oceans is characteristic of lead from the -7- DUP050055289 Quaternary Period, deposited thousands of years ago. But lead in coastal surface seawater is characteristically from the Tertiary Period and is probably no longer the product solely of natural weathering. The isotopic compositions of lead in gasoline additives and in atmos pheric aerosols from cities where the fuel was obtained are similar to each other and represent isotopically distinct lead ores of the 1 Tertiary Period or earlier. Chow and Earl4* infer that the aerosols could not be derived from weathering of natural surface material and instead come primarily from automotive exhausts. However, to ascribe this lead specifically to manufactured tetraethyl lead, one would have to know whether the isotopic composition of tetraethyl lead differs from that in all the other products of industry that contain lead and also enter the ecosystem by weathering, burning, etc. MAN-MADE SOURCES OF LEAD Patterns of Lead Consumption The consumption of lead in the United States has not increased sub stantially in the last 20 years (Table 1-1) . Msre than one-third of the lead consumed is recycled from previous consumption. This recycled fraction also has been fairly stable in recent years. There has been, however, a considerable shift in the pattern of lead consumption. Thus, while the use of lead for the manufacture of paint pigments has been decreasing, the use for manufacture of storage batteries and anti-knock gasoline additives has risen dramatically (Table 1-2). As can be seen in Table 1-3, the uses of lead are diverse and numerous . > -8- DUP050055290 The electric-storage-battery industry has been the largest single user of lead in the United States for many years. In 1968, it accounted for 38.7# of all lead consumed in this country. There is an almost even distribution of lead between the metallic form used to make the grids (which contain 4-8# antimony) and lugs and the lead oxides (litharge, PbO; red lead, FbgOjj; and black oxide, FbgO) that constitute the active material pasted on the plates. The second largest consumer of lead is the petroleum industry. In 1968, this industry used almost 262,000 tons of primary forms of lead for gasoline additives (lead alkyls), or 19.7# of the total lead consumption* In addition, this industry uses lead in the form of pipe and sheets for the fabrication of corrosion-resistant equip ment. Lead is used in heavy-duty greases--for example, in gear com pounds for drilling machinery, and for use under water, where its lubricating properties are effective after exhaustion of the grease* About 1000-2000 tons per year are consumed in this use, which does not apply to automotive greases. The paint industry uses white lead [basic lead carbonate, 2PbC03*Pb(0H)2] and red lead in paints intended only for outdoor use, for which their weathering characteristics make them valuable. About 50# of the white exterior house paints on today's market are oil-based and contain amounts of white lead that vary with the manufacturers' formulas. Lead chromates provided some Ik ,000 tons (as Pb) of yellow, green, and red pigment colors in 1968. These find many uses, for example, in traffic marking paints and printing inks.-^5 The paint industry uses several other -9- DUP050055291 lead compounds, including white basic lead sulfate and blue basic lead sulfate* The lead associated with "lead pencils" is lead chromate in the yellow paint pigment. Lead has found numerous applications in the ceramics industry. Glazes for china and structural clay products may contain lead oxides or lead silicates. Examples of the latter include glazed bride, lightweight aggregate, and porcelain enamels for aluminum and steel. During firing at high temperature, the interaction between lead and silica forms inert substances. Miscellaneous uses of lead, on a tonnage basis, include the use of lead arsenate as an insecticide (decreasing since 1946) and the use of lead in electric cable insulation, hose, pipe, sheet, and floor coverings and as a stabilizer in vinyl plastics. Emission of Lead to the Atmosphere Amounts. A national inventory of the sources of lead emission in the United States for 1968 by the Air Pollution Control Office has provided a data bank (summarized in Table 1-4) that shows that the inorganic emission from the combustion of leaded gasoline constitutes approximately 98$ of the total emission of lead from the listed sources. It should be pointed out that this inventory does not include attrition of the innumerable lead-containing items that are subject to weathering and often are burned or otherwise disposed of after thay have outlived their usefulness. The dispersal of lead compounds has many other sources: exhausting of workroom atmospheres; the abrasive action of >> -10- 'tr ...; DUP050055292 automotive traffic on lead-painted lane dividers on streets and highways; resuspension of lead by high-speed cars; burning of lead-painted surfaces of houses, bridges, and other structures before repainting; welding of lead-painted structural or other steels; weathering of painted surfaces, with the resulting flaking and distribution into the atmosphere of a portion of the lead-bearing dust; incineration of leaded plastics and other materials whose usefulness has ended; recovery of lead from old battery cases, lead pipe, lead-sheathed cable, and sheet lead in secondary smelters ; and, on a small scale, the welding and soldering operations conducted in plumbing and electrical repair shops. The magnitude of these additional sources of emission could he considerable and is unknown.* In comparison with the burning of gasoline, the burning of coal has not been a serious source of atmospheric lead. In 1968, the production of coal in the United States amounted to 50h million tons. Analysis of 827 samples of domestic coal provided a weighted average of 7 parts of lead per million of coal This concentration would produce 3*528 tons of lead, of which 7UJS either remained in the clinker or was collected with the fly-ash, (in modern furnaces burning pulverized coal, about 95~99% of the fly-ash is removed by electrostatic precipitators, and only a small portion of the lead is emitted as aerosols. )op Probably no more than 920 tons escape to the atmosphere each year. Significant amounts of lead used to be introduced into the atmosphere klQ by smelting lead ores. At the beginning of the Industrial Revolution, in 1750* smelting technology resulted in the loss of much lead fume. Unpublished data (J. Wagman, personal communication) indicate that about 10% of the total particulate matter in municipal incinerator effluent may be lead. -11- DUP050055293 During the 19th century, smelting procedures were improved; and by the early 20th century, it became economically feasible to recover lead smelter fume, which resulted in reducing the fraction of fume loss by a factor of k. During the last four decades, the loss fraction has been further reduced by a factor of 8 (Table 1-5). Thus, the emission of lead into the atmosphere in the Northern Hemisphere from smelting currently is small, in comparison with the emission from burning gasoline. In 1968, for example, (.Table 1-lf) the comparative values were 985 tons of lead emissions from smelting and 181,000 tons from burning leaded fuels. Physical Characteristics . Probably equal in importance to the absolute amounts of lead emitted to the atmosphere are its physical characteristics, in that they determine in large part its subsequent disposition. For example, the largest lead-bearing or pure lead particles will fall to the earth near the emission source and enter the ecosystem there, whereas the smallest particles will remain air borne and enter the ecologic cycle far from the emission source. The small particles (less than 2 ym in mean equivalent diameter) become especially important when it is considered that they tend to be retained by the lungs and accessory airways when inhaled, to be absorbed or coughed up and swallowed later . Aerosol lead may be emitted to the atmosphere in the form of dust, fume, mist, or vapor, depending on the source material and the method of generation. -12- DUP050055294 Pusts are disperse systems of particles or aggregates of particles 1-150 \m in diameter that are thrown into the air by mechanical activi~ ties^^a during blasting, crushing, drilling, grinding, and other industrial, constructional, or agricultural processes* Smaller particles tend to remain airborne longer, and particles less than about 10 pm in diameter can be inhaled into the respiratory tract. Examples include the oxides and salts of lead that become airborne during handling or packaging. Fumes are formed in chemical reactions, including combustion, and in such physical processes as distillation, sublimation, calcination, and condensation. Particle size is 0.2-1 pm* Examples are the fumes of metallic lead and lead oxide generated as vapors in smelters or foundries during melting operations and then condensed from the vapor state. Mists are formed by the condensation of water vapor on submicroscopic 271 particles of dust or gaseous ions or by the atomization of liquids. Sprayed lead arsenate insecticide is probably the only example of a mist of a lead compound that is important in air pollution studies. Vapors are formed from liquids by an increase in temperature. Like gases, they produce true solutions with the atmosphere. However, as soon as they regain their liquid state, from a decrease in temperature, they form mists and fumes. Evaporated lead alkyls are the best examples of vaporous forms of lead. -13- DUP050055295 Particle size is the major determinant of the residence time of lead emitted into the atmosphere and of the degree of dispersion from the point of emission. Particle size also determines the degree to which lead is deposited and retained in the airways in man. Consideration of particle size distribution in ambient air therefore is of major importance. (A discussion of the measurement of particles appears in Appendix A). The particle size distribution of urban lead aerosols has been determined by a number of investigators. Robinson and Ludwig**^ report a mass median equivalent diameter (MMED) of 0.25 ym with 25# of the particles smaller than 0.l6 ym and 25$ larger than 0.43 vmi. Their data are from sizedistribution analyses of samples collected from 59 urban sites with a modified Goetz Aerosol spectrometer in Los Angeles, San Francisco Ray area, Cincinnati, Chicago, and Philadelphia. There was little variation among these widely scattered places in MMED of the particles, and the results are representative of lead aerosols from a variety of urban sources, inasmuch as there was a fairly uniform division of samples between residential, commercial, and industrial areas in each of the five cities* In another study, the average MMED was 0.18 ym in Cincinnati and 0.42 ym in a nearby suburbIn Cincinnati, 75# of the aerosol mass consisted of particles less than 1 ym in MMED, and in the suburb, 65# were smaller than 1 ym. Other studies conducted in California,*1^indicate that 50-90# of the lead particles in urban air are smaller than 1 ym. * -14- DUP050055296 A comparison of estimated emissions of lead from several sources in the Cincinnati area indicates that the principal, contributor is the coinbustion of leaded gasoline , 379each gallon of which contains about' 2 . 4 ` g of lead as antiknock additive. In 1962, Mueller and his associates at the California Department of Public Health determined the size of particles in the exhaust emissions 3^4 of three popular makes of automobiles operated in cruise conditions. They reported that 62-80# by weight of the particles were smaller than 2 pm in MMED, and of these, more than 68# were smaller than 0.3 ym. These results for cruising automobiles are in substantial agreement with 46 7 those reported by Robinson and Ludwig for general ambient air without specification as to source of lead. 228 In I97O, Habibi used a new experimental system designed to study the wide range of particle sizes emitted in automobile exhaust in cyclic driving conditions .* He reported data obtained with an Andersen and This consists of a programed chassis dynamometer and a 40-ft tunnel in which the exhaust gases are diluted with filtered air in a. known ratio, and proportional sampling is conducted under isokinetic condi tions. In addition, a total exhaust filter was prepared by packing an l8-in.-diameter, 24-in.-long drum , with a high-efficiency fiberglass medium. The filter was claimed to be capable of direct mounting on the tailpipe and could be used in direct road operation to determine exhaust emission rates as affected by road vibrations and thermal fluctuations associated with normal (start-stop) driving patterns. -15- DUP050055297 a Monsanto impactor showing an MMED of 0.6 ym for lead particles during steady-state operation at '4'5 &ph. This is in reasonably good agreement 344 with the results of Mueller et al. in cruise conditions with autoinobiles manufactured and tested in 1962. However, of greater interest to re searchers in the field of air pollution is Habibi's finding of heavy deposits of large 11 gritty" particles ranging from 300 to 3,000 pm in diameter. These deposits began to form along a l6- to 20-ft section on the floor of the dilution and sampling tunnel at the point where the exhaust gases expanded to the wall of the tunnel; the deposits appeared to be due to gravitational settling. More than 95% by weight of the lead so deposited was present as coarse particles, and the total quantity of this deposit amounted to 10% of the lead originally contained in the fuel consumed by the automobile. Chemical Forms. Definitive data on the inorganic forms of lead in the atmosphere are lacking, except for the identification of several lead compounds emitted in the exhausts of automobiles using leaded gasoline as fuel. The chief lead emission products are (in particles of equivalent diameter between 2 and 10 ym) lead bromochloride (PbCl*Br) and (in particles smaller than 1 ym) the alpha and beta forms of ammonium chloride and lead bromochloride (NH^Cl 2PbCl Br, 2NH]+C1rPbCl Br), minor quantities of lead sulfate (PbS04) 5 and the mixed oxide and halide (FbO PbCl*Br^20) . The chemical and physical characteristics of these unstable compounds are largely unknown and constitute a very difficult area of investigation that may be more important than is generally appreciated. It has been noted that * h -16- DUP050055298 little 2PbBrCl *NH^C1 was found in hot exhaust gas collected at the tail pipe. It was, however, found after cooling and mixing of the exhaust with the ambient air, and it has been suggested that this compound Is 229 less easily photolyzed than PbCl'Br* If phosphorus compounds have also been added to the fuel, up to 20% of the exhausted lead may be in the form of a phosphate-halide compound [SPb^CPO^^vPbCl-Br]. New data (j. M. Pierrard, personal communication) have been made avail able on samples collected near heavy traffic (at the Delaware Memorial Bridge tollgate) and in locales where the lead aerosols were assumed to have aged after emission. They apparently showed different surface compositions, which would be consistent with a conversion mechanism expected to release the less reactive hydrogen halide, rather than the more reactive molecular halogen, from the particles. The literature contains only meager characterization of the inorganic forms of lead from other emission sources. Many of the oxides and salts of lead probably are dispersed into the atmosphere during their chemical preparation, use, and disposal and during the burning of lead-containing fuels , such as coal. Very small amounts of tetraethyl lead (TEL) and tetramethyl lead (TML) in gasoline blends may escape to the atmosphere through vents on the carburetor and fuel system during evaporation of the fuel, as found in the three-city study, 539described in the next section* It was pointed out in that report, however, that, because these organic lead compounds are less volatile than gasoline, they tend to remain behind in the unevaporate d portion. Furthermore, TEL and TML are -17- DUP050055299 light-sensitive and undergo photochemical de composition when they 539 reach the atmosphere. The presence of TEL and TML in ambient air is discussed in greater detail in Chapter 6. ^v Nothing is known about the existence of organic forms of lead in the environment from biotransformation. The methylation of lead by bacteria has been studied in the laboratory using lead acetate (Fb the principal valence form of lead in exhaust emissions) as a substrate No evidence was found of [^C ]tetramethyl lead formation from [^C]methylcobalamin. (j. M. Wood, personal communication) . The quantitative methods for determination of lead are discussed in Appendix 3 * Lead in the Atmosphere Concentration Gradients. The concentration of lead in ambient air is closely correlated with the density of vehicular traffic, at least in the United States, It is highest in what might be termed Vehicular microclimatesin large cities, decreases with averaging of place-to-* . place variation within total city climates, then tapers off as one progresses into the suburbs or smaller towns and finally into rural areas. These gradients with reference to locale are also subjected to diurnal and.seasonal cycles. The character of these patterns has been sampled only here and there , and the overall picture is still incomplete. Also, it is difficult to make reliable comparisons because methods of sampling and techniques of analysis have not been standardized. Thus, it is impossible to compare lead concentrations -1&. of air taken close to traffic with that of air sampled from the roof of a tall building. In the areas of highest concentration, the concentrations are consider ably higher than would be encountered if values from various points in city air were averaged. Atkins, reporting the distribution of lead in the Palo Alto, California, environment^ for very short sampling times (5 min), found up to 19 ug/m^ a few feet from moving traffic. He also found close correlation between time of day and atmospheric lead concentrations; the concentrations were highest in the heavy traffic hours around 8:00 a.m. and 4:00 to 5:00 p.m. In Los Angeles (Table 1-6), concentrations were highest in the morning rush hour and for some reason never returned to the morning level during the after noon rush hour. Regardless of the reasons, these microclimate concen trations in the vicinity of heavy traffic are of potential significance to the health of people who spend the working day in them. It should be noted that the mean concentrations of atmospheric lead did not fluctuate much during the day; at least this would appear to be the case in the Los Angeles samples cited. The dilution of atmospheric lead from these points of high concentra tion is considerable. Even in Los Angeles, where vehicular traffic is unusually heavy, overall concentrations of lead are considerably lower in the ambient air than in the microclimates near vehicular traffic. An extensive study to determine the variation in the annual, seasonal, monthly, and diurnal average distributions of lead and total particulate -19- DUP050055301 matter in the atmosphere of three U.S. cities was conducted by the Public Health Service in collaboration with the American Petroleum Institute, the Automobile Manufacturers Association, the California State Department of Public Health, E. I. duPont de Nemours and Company, Ethyl Corporation, and the Kettering- Laboratory, University of Cincinnati. Sampling sites were selected to represent four geographic and land-use Glassifications: rural, residential, commercial, However, the samples were, taken at various above-ground levels and therefore, should no and industrial. / Atmospheric sampling was performed^ continuously from 3une l5ol com_ through May 1962 A summary of the results of the lead found during this pared period for Cincinnati , Los Angeles, and Philadelphia is presented in Table 1-7, which shows that the general urban concentrations ranged from about 1 to 3 yg/m^, depending on the area sampled. The average concentration of lead for all samples collected during the year was 1.4 yg/nr in Cincinnati, 2.5 in Los Angeles, and 1.6 in Philadelphia. The highest average concentration for all samples collected . during a single month at any single sampling site was 3.1 yg/m in Cincinnati, 6.4 in Los Angeles, and 4.4 in Philadelphia. The highest individual concentra 's tions were 6.4 yg/nr in Cincinnati, 11.4 In Los Angeles, and 7*6 in Philadelphia. A uniform sampling procedure was used in each of the cities, and the samples analyzed by the same technique, those from Cincinnati by the Kettering Laboratory, those from Los Angeles by the California Health Department Laboratory, and those from Philadelphia by the U.S. Public Health Service Laboratory. To ensure com- . parability of results, frequent comparative analyses were made . Preliminary data on samples taken in 1968-69 from the same sites indicate that air lead concentrations at some individual sites are higher than in 1961-62.^5 When monitored concentrations of airborne lead are averaged, it appears that higher mean concentrations of lead are correlated positively with size of city. This relation is illustrated in Fig. 1-4, showing averages of composite samples collected quarterly and analyzed with a low-temperature ashing procedure and emission spectroscopy. -20- DUP050055302 There is evidence of a decrease in lead concentrations as one moves outward from the core area of the larger cities. The National Air Surveillance Networks (NASN) of the Environmental Protection Agency is continually sampling the atmosphere for lead and other pollutants in many parts of the United States. In most cities, the average concentration of lead in the atmosphere in 1953-66 ranged from 1-3 Mg/m-, hut suburban and nonurban stations averaged 0.1-0.5 Mg/m^ and many concentrations at some of the veiy rural stations, were less than 0.05 pg/m^. 3 5 4 55 ,65 7 * 8 Data from the NASN for 217 urban stations during 1966 and 1967 showed an annual average lead concentration of 1.1 ug/m^. Samples averaged ,3 quarterly at individual sites ranged from 0.02 to 19 Mg/m * The non urban stations showed an average of 0.21 pg/m^ near the city, 0,096 at q Qc an intermediate distance from the city, and 0.022 in remote areas. p Yearly Trends. The consumption of lead alkyl fuel additives Is in creasing substantially every year. It cannot be assumed, however, that the concentration of lead In urban air is increasing in parallel. Indeed, most of the available data suggest that the concentration of lead in air, even over the largest cities, is increasing only very slowly if at all. The most impressive data on this point come from analysis of the .atmosphere In Cincinnati, Ohio:, initiated by the Kettering Laboratory in 1938 and continuing to the present.11 In 1941, a lead monitoring program was inaugurated and sampling was conducted s intermittently and randomly at 1-50 stations with electrostatic pre cipitators. In 1955, continuous sampling was begun at three repre sentative sites with high-volume samplers fitted with tared glass -21- DUP050055303 fiber filters; sampling took place over 24-hr periods and particulate matter was collected from 2,000-m^ air samples. During the 1961^-1962 period, samplers with 3-pm membrane filters were used to collect samples continuously at four stations representative of. v industrial, commercial, central residential, and peripheral residential sites. These sampling periods ranged from 2 to 3 days. A mobile station was used intermittently to investigate special conditions. The 1961-1962 samples averaged 325 m^ and 480 of air for the 2- and 3-day sampling periods, respectively. The results of the studyIl8with the same spectrographi c and dithizone methods over the 20 years of the study showed a gradual, continual downward trend in the mean and median concentrations of lead in the atmosphere of Cincinnati. The mean and median concentrations were 5.1 and 3.4 jjg/m^, respectively, in 1941 and 1.42 and 1.27 yg/m^ in 1962. It is important to mention that there is some bias in the results obtained during the earlier period of this 20-year study: a greater proportion of industrial and other specially selected sites were represented in the samples collected at that time. However, the investigator believed that there was little doubt about the down ward trend when results obtained during the different periods and the sampling conditions were examined. Although the volume of traffic and the consumptican of leaded gasoline increased greatly during the period of study, there was a simultaneous decrease in the quantity of .coal consumed for heating purposes and in the mass of dustfall from all sources, resulting in less lead in the urban Cincinnati atmosphere. r t v -22-- DUP050055304 Furthermore, an increasingly effective enforcement of the smoke abate ment ordinance, improved collection and disposal of particulate*matter from industrial operations, and changes in traffic flow and land use in the metropolitan area of Greater Cincinnati each contributed to the decrease in atmospheric lead concentration during the 1946-1962 period. 539 329 Landau et al. of the Air Pollution Control Office discussed the average concentration of lead in air in 31 cities of the NASN sampling network for which data had been obtained in at least one year of each of three periods--1957-1961, 1962-1965, and 1966-1967 They reported that the average lead concentration had decreased slightly from the first to the second period. Lead concentrations for the third period were about double those of the second period. However, the data for the third period were obtained by an analytic procedure different from that used during the first two periods, and the authors assume that the increase in lead concentration between the second and third periods is due primarily to the change in analytic method. The NASN data cited earlier for 1953 through 1966 are from samples taken at many different sites and exact information is not available when sites were changed. It is difficult, therefore, to tell whether the atmospheric concentration of lead increased or decreased during that period, but the data seem to indicate that the concentrations remained fairly constant. Nelson,428 reported concentrations of lead in air measured in suburban Salt Lake City during the period 1944-1965. Average lead concentra tions for 5-year periods beginning in 1944 were l.l4, 0.75, 0.70, and 23- DUP050055305 0.51 ug/m For 1964 and 1965, the average was 0,30 ug/m^, The reason for the decrease may be improvement in control of particulate matter emitted into the atmosphere from industrial sources, The relation of some average airborne lead concentrations at selected urban and non urban stations to lead consumed as gasoline additives is shown on Fig. 1-5. Chow and Earl1--suggested that the lead concentration in San Diego is increasing at the rate of 5% per year. This estimate is based on weekly sampling at a single site and on yearly averages of MSN for 1959 and 1966 at this site of 1.12 and 1.84 yg/m^, respectively. However, it is speculative to interpret citywide trends of lead in air from measurements taken at a single site, in that many investi gations have shown that lead concentration decreases logarithmically with increasing distance from the source. In view of the disparate results from different cities , it is not possible to make any generality about trends. But it is possible to say that, if there are any upward trends, they are not very substantial, CYCLING OF LEAD IN THE BIOSPHERE The fate of lead emitted into the atmosphere is poorly characterized* Figure 1-6 presents an ecologic flow chart that summarizes most of the major compartments and pathways taking part in the cycling of lead in the biosphere, a biogeochemical process. Because of the lack of* significance attached to lead in natural systems in past years, there has been no comprehensive treatment of its movements within living > -2k DUP050055306 systems. There is not sufficient information on the chemical forms, amounts, and rates of transfer of lead from one compartment of the environment to another to permit treatment of the subject in terms of systems analysis. However, fragmentary data afford some insight into the movement of lead in the biosphere. These data come primarily from studies of lead-210, a radionuclide important in tracing the pathways 79 and determining flux rates of lead, as stressed by Burton and Steward* This section documents what is known about the general move ment of lead between compartments of the biosphere, details of which are treated in subsequent chapters. Lead in Precipitation Lead is removed from the air by aggregation and by precipitation. From analyses of lead-210 in rainwater, its residence time in the atmosphere was calculated to be 7-30 days, depending on environmental 79 ,190 conditions. Whether the residence times are valid for atmospheric lead aerosols resulting from gasoline combustion is not known, but 549 Ter Haar et al. found that stable lead and lead-210 were well mixed in air. If the residence time of atmospheric lead aerosols from automotive exhaust is 1-4 weeks, the turnover rate of these materials may be such that a worldwide steady state of lead already has been reached in the atmosphere. Lazrus and .co-workers ^42 nave sampled atmospheric precipitation of lead at 32 U.S. stations and found a correlation between the number of gallons of gasoline used and the concentration of lead in rainfall in each area. The average lead concentration was 34 pg/liter in precipi -25- DUP050055307 tation, with the median somewhat lower, about 10 jig/liter$ the highest concentration found was about 300 jig/liter. The authors pointed out that, on the average, at least twice as much lead is found in atmospheric precipitation as in water supplies, which implies to them the existence of a process by which lead is depleted after precipita tion reaches the earth's surface. This conclusion is supported by 316 recent Russian studies that indicate the tendency of lead to be present in suspended matter, to be insoluble in surface waters, and to be removed by natural sedimentation or filtration. 5^9 Ter Haar et al. found, in a study carried out in a semi rural area, that an average of 1 yg of lead per square centimeter per year is pre cipitated out of the atmosphere. Although they had sparse data, there seemed to be a correlation between the type of rainfall and its lead concentration: showers had lower concentrations than slow, even rainfall. Weather preceding the rainfall may affect the lead content, in view of their finding of high concentrations after thermal inversions It seems likely that today the global mean natural lead content of lakes and rivers lies between 1 and 10 yg/liter. The average lead content of 33 water samples of major rivers in North America was 6.6 yg/liter; and of 440 lake and river water samples in Maine was 2.3 yg/liter (range, 0.03-115 yg/liter) Goldberg^^ observed a rapid depletion of lead-210 in un filtered samples along the paths from origins to outfalls of the Sacramento and Colorado Rivers . Assuming that stable lead species and lead-210 have similar -26- ' DUP050055308 V r\ chemistry, he concluded that some inorganic or biochemical processes removed lead from solution. In marine waters, Goldberg also found a rapid turnover and transfer of lead to deeper waters , which he credits to some process of biologic transfer. Lead in Soils The natural concentration of lead in soils is primarily a function of the 23k geologic source of the parent material. The usual range is about 2-200 ppm, exclusive of areas near deposits of lead ores.265 * If15 599 Specific areas of increased concentration due to man-made sources (e.g. , mining and fallout from industrial operations and motor vehicles) have been discussed earlier. In addition to these sources, soils receive on the average 1 pg/cm^- year from precipitation (discussed previously), and 0.2 pg/cm2~ year may be deposited in dustfall. 549 Using these averages , Ter Haar et al. estimated the contribution of stable and radioactive atmospheric lead to soil at about 0.2# of the existing natural burdens of soil lead per year* This amounts to about 0.04-4 ug/gper year and might explain the failure of most investigators to detect a significant accumulation of lead in soils remote from heavy traffic and industrial areas. There appears to be a natural mechanism by which lead tends to be moved upward in the earth*s crust. Many workers have shown that a sharp lead profile , higher at the surface than at underlying depths, is frequently found. Swaine and Mitchell,54l in a study of lead -2T- DUP050055309 profiles of eight soils in Scotland, found that the lead content was approximately halved in going from the surface to a depth of 50 in* 599 Wright, Levick, and Atkinson reported a similar result for virgin soils of four great soil groups of Canada. Soils in both studies were from uncultivated areas and far from industrial contamination. Goldschmidtzok and Hibbard255 also reported higher concentrations of lead and other elements in the upper horizon. Swaine and Mitchell, in their study of Scottish soils, found a 30-fold decrease in lead with depth in some cases. They stated: The surface accumulation of Pb is the outstanding effect observed in the total trace-element contents of the pro files examined., . .Ten-fold increase calculated on the basis of the air-dry material in the surface horizon, is not uncommon. The effect is greatest in organicrich uncultivated surface horizons, and is even more pronounced when expressed on a mineral matter or ash basis....These soils are generally remote from industrial areas and from high-density motor traffic; being -un cultivated, contamination from tractors is excluded. The obvious explanation would appear to be an accumulation of Pb through plants, much more pronounced than for other elements. Presumably an insoluble complex which holds the Pb in the surface horizon--whether organic or inorganic is not yet established--is being formed on the decay of the plant material. iv * -28- DUP050055310 Numerous investigators have studied the concentration of lead in depth profiles of soils along highways. All reported decreasing lead content of these soils with distance from the highway and with depth beneath the surface. The data of Lagerwerff and 327 Specht (Table 1-8) typify these findings. There was about 65-75# reduction in the lead content of the top 5 cm of soil as the sampling site was moved from 8 m away to 32 m away from a high way with traffic densities ranging from 7*500 to 48,000 cars per day. Further examination shows that, at a depth greater than 5 cm, four soil samples taken l6-32 m from a highway with a traffic density of 23,000 cars per day provided analytic values of lead that are lower than the frequently cited l6 ppm (l6 ug/g of dry soil). Although comparable samplings gave higher values than these, it is apparent that the penetration of lead into the soil drops rather uniformly as interpreted from the data in Table 1-8. The concentration of lead in street dust and surface soil of large cities is extremely high. In a study of 77 midwestern cities ranging 266 in population from 100,000 to 1,000,000, the average concentration of lead in dust collected from residential and commercial sites was 614 1,636 pg/g and 2,413 yg/g, respectively. The concentration of lead in surface soils in city parks is also very high: Balboa Park, San Diego, 194 pg/g; Golden Gate Park, San Francisco, 560 pg/g and MacArthur Park, Los Angeles, 3,357 pg/g. -29- "liead in Biota Studies of lead-210 also have provided knowledge of the movement of fc 595 lead into plants. For example, Wilson and Cline concluded that' soil lead is largely unavailable for uptake by plants, only 0.003-0.005# of the total amount of lead in soil being available for such uptake. Several investigators have concluded that the primary 189 257 39k source of the lead taken up by plants is rainfall, and not soil. 3 3 561 However, Tso et al. believe that a significant amount of soil lead is available to plants, 398 most plants. in any event, soil lead is not absorbed by An interesting situation exists with regard to the concentrations of lead-210 in animals of the Arctic, where relatively high concentrations have been found in the biota. This occurs because the lead nuclei tend to accumulate on the sedges and lichen, which attain high concentra tions because of their slow growth. This vegetation forms a large part of the diet of the caribou and reindeer, which in turn constitute a substantial fraction of the diet of predators in these regions. 260a The concentrations of lead-210 found by Holtzman in the reindeer and the caribou were 7 and 11 pi cocuries/g of bone ash, respectively. The flesh of the reindeer contained 0.05 picocurie/g wet, and that of the caribou, 0.08 picocurie/g. However, the wolf, which preys on these animals, was found to have only 1 picocurie/g of bone ash and 0.02 picocurie/g of flesh. The reason for the lower concentrations of lead-210 in the wolf than in the animals it eats is that lead*-210 is a bone-seeker; thus, the concentrations tend to be higher in bone -30- DUP050055312 than in flesh. Because the predators are flesh-heaters, the lead concen tration in the predator tends to decrease as one follows the food chain. Accumulation of lead in hones of vertebrates may be a general rule, inasmuch as this also was observed in fish, with lead-210 in bone exceed- 26l ing that in muscle by a factor of over 50. SUMMARY The relative inertness and malleability of lead enhance its usefulness to man; it occurs in nature chiefly as a sulfide. Lead radionuclides are useful in geologic dating and in documenting the cycling of the metal in ecosystems . Current world production is about 2.5 million tons per year , about b0% in the United States , Yearly U. S. consumption of primary and secondary (recycled) lead is about 1.3 million tons and has almost doubled in the last 30 years. The largest consumer is the electric-storage-battery industry (39$)> followed by the petroleum industry, which uses 20$ of the total for gasoline additives. Lead consumption for these two uses has increased with the production of antiknock gasoline and batteries at the same time that the manufacture of lead-containing paints and insecticides has decreased, but the magnitudes of the absolute amounts differ widely. Patterns of lead emission to the atmosphere have changed in modern times. The initial increase due to smelting and coal-burning that started with the industrial revolution has been checked hy improved industrial controls, but the decrease in those emissions has been more than offset by emissions from automotive exhaust fumes, Today , -31- DUP050055313 about 98# of the airborne lead that can be traced to its source comes from combustion of gasoline. Geographically, there is a logarithmic increase in atmospheric lead concentration from mid-ocean, to remote high mountains, to seashore, to suburban and urban environments. In spite of the rapid increase in the consumption of lead alkyls used in automotive fuels, however, the concentration of lead in urban air is, in general, rising only slowly, presumably because of dispersal. The disposition of lead emitted to the atmosphere (and indirectly to other ecologic compartments) depends on its physicochemical form and on meteorologic factors that help to dissipate it. About half the lead-containing particulate matter from automotive exhausts is removed from the air by gravity within a few hundred feet of roadways. The remaining lead consists of aerosols that are largely airborne until removed by precipitation. The mean residence time of lead in the atmosphere, calculated at 7-30 days, reflects the efficiency with which the aerosols are removed by precipitation. Much of the lead entering aquatic systems via precipitation and runoff is not water-soluble and apparently is removed from water by sedimentation. The low solubility of lead in water also is an important factor in terrestrial systems, because it affects the ability of plants to assimilate lead. Precipita tion tends gradually to increase the lead content of soils, but little of this lead enters food chains because of its limited availability to primary producers. The role of micro-organisms in the biotransfer of lead and almost all aspects of transfer rates between compartments* of the ecosystem are two areas in which there is little or no information. V -32- DUP050055314 TABLE 1-1 Lead Consumption in United States (Excluding Alaska, Hawaiiand Puerto Rico) 1940-19688, Lead consumption % thousands of tons Smelter, primary c lead lpUo m2. i?60 1965 166 1967 533 508 1*79 382 1*18 44l 380 Recovery of second ary lead 260 1*82 502 1*70 576 573 574 Imports^ 282 5l*2 ,. 1+62 360 350 435 499 Stocks at primary smelters and refineries on 31 Dec. HA HA HA 250 83 HA 125 Consumption of metal. primary and secondary 782 1238 1213 1021 1241 1324 1261 1968 467 551 426 90 1318 a >o*t Adapted from U.S. Bureau of the Census. ^Preliminary. cFrom domestic and foreign ores and bullion. d Includes lead imported for immediate consumption plus material entering country under bond; includes lead in pigs, bars, bullion, ores, and matte. -33- DUP050055315 TABLE 1-2 Lead Consumption for Selected Uses in United States^ Year 1935 19 ^0 1945 1950 1955 I960 1965 1968 Lead consumption, thousands of tons As white lead 80 66 In gasoline additives 37b 5013 In storage batteries 175 220 36 ?6 60 36 114 398 18 165 380 8 164 353 8 225 6 262 555 513 a poo Adapted from Minerals Yearbooks, .. _ 193o-19o8. ^Calculated from gasoline consumption x 2.0-4 g/gal. -34- DUP050055316 TABLE 1-3 Lead Consumption in United States, 1968, by Products9, Product Metal products Ammunition Bearing metals Brass and bronze Cable covering Caulking lead Casting metals Collapsible tubes Foil Pipes, traps, and bends Sheet lead Solder Storage batteries: Battery grids, posts. etc. Battery oxides Terne metal Type metal Pigments White lead Red lead and litharge Pigment colors Other Lead Lead consumption tons Product Chemicals consumption tons 82,193 Gasoline antiknock 18,441 21,021 53,456 additives Mis cellaneous chemicals 261,897 629 Subtotal 262,526 49,718 Miscellaneous uses 8,693 Annealing 4,194 9,310 6,114 21,098 Galvanizing Lead plating Weights and ballast 1,755 389 16,768 28,271 74,074 Other, unclassified uses Subtotal 23,106 17,924 250,129 263,574 1,427 27,981 Subtotal 915,500 5,857 86,1*60 14 ,163 3,234 Subtotal 109,734 -35- Total 1,328,790 O DUP050055317 TABLE 1-3 Footnotes Adapted from Minerals Yearbook,^3 1969s P 61+7. Vi ^Includes lead content of leaded zinc oxide and other pigments. ^Includes lead that went directly from scrap to fabricated products. -36- DUP050055318 TABIJ2 1-4 Lead Emission i n United States, 1968a Emission source Gasoline combustion Coal combustion Fuel oil combustion Lead alkyl manufacturing Primary lead smelting Secondary lead smelting Brass manufacturing Lead oxide manufacturing Gasoline transfer Total Lead emitted* tons /year 181,000 920 24 8l0 174 8ll 521 20 36 184,316 aData from National Inventory of Air Pollutant Emissions and Controls, a loose-leaf data bank on file at the Bureau of Air Pollution Sciences, Environmental Protection Agency, Research Triangle Park, North Carolina, 27709. -37- DUP050055319 TABLE 1-5 Lead Aerosol Production in Northern Hemisphere8, Lead smelted, thousands Year of tons 1753 100 1815 1933 1966 200 1600 3100 Lead converted to aerosols, * 2 2 0.5 0.06 Lead Lead aerosols burned as from smelters, alkyl, thousands thousands of tons of tons 2- h8 10 <2 300 Lead converted to aerosols, % hO ho Lead aerosols frpm alkyls thousands of tons h 120 4l8 Adapted from Muzorumi et al -38- DUP050055320 TABLE 1-6 Atmospheric Concentrations of Lead in Traffic--Los Angeles Area (in Micrograms Per Cubic Meter)8, Lead concentration, Route Parked along freeway*> Parked along freeway Parked along freeway Parked along freeway Sampling time 0600-0900 0900-1600 I6OO-I8OO Days Weekdays Weekdays Weekdays Saturday HS/g---------------- ------No. Description- Mean Range samples Morning rush 38.0 26,9-5!*.3 7 Midday 2k .1 16.6-31.1 7 Afternoon rush l8.lt 8.7-25. It 7 - 19.9 17.7-22.2 h Freeway traffic Freeway traffic Freeway traffic Freeway traffic 0600-0900 0900-1600 1600-1800 - Weekdays Weekdays Weekdays Saturday Morning rush 29.3 Midday 21.5 Afternoon rush 22.2 - 25.3 10,9-1*1.3 1*.5-39.2 10.5-1*3.1 10.0-71.3 35 39 34 32 Los Angeles downtown traffic Lcs Angeles downtown traffic Los Angeles downtown traffic Los Angeles downtown traffic O6OO-O9OO Weekdays Morning rush 4 23.6 19.1-29.9 0900-1600 Weekdays Midday 10.5 8.1*-12.2 I6OO-I8OO Weekdays Afternoon rush 15.3 12.1*-i 8.6 - Saturday - 9.4 8.6-10,3 6 k h 4 a 539 Adapted from Public Health Service Publication No. 999-AF-12 Parked on shoulders of major freeway in downtown Los Angeles -39- DUP050055321 TABLE 1-6 - Continued Route Pasadena downtown traffic Pasadena downtown traffic Pasadena downtown traffic Pasadena downtown traffic Sampling time Pays Description Lead concentration, e s Zs ! No. Mean Range samples 0600-0900 Weekdays Morning rush . 0900-1600 Weekdays Midday 11.9 8.6-1 It. 6 1600-1800 Weekdays Afternoon rush _ _ 6 w- Saturday 12. i. 12.3-12.lt 2 Total 191 V * -1*0- DUP050055322 TABLE 1-7 Concentration of Lead in Atmosphere8, Annual average values: Downtown Outlying area All stations Atmospheric lead concentration, ug/m^ Cincinnati Los Angeles Philadelphia 23 12 l,k 2.5 3 1 1.6 Seasonal distributions: (all stations) Summer Fall Winter Spring 1.3 1.7 1-3 1.3 1.9 2.8 3.1 2.1 1.4 1.9 1.9 . 1.4 Diurnal distributions (annual -all stations stated as a fraction of annual mean): 2300-0300 0300-0700 0700-1100 1100-1500 1500-1900 1900-2300 1.3 1.1 1.4 0.8 0.9 1.0 1.0 1.1 1.2 0.7 0.8 1.1 0.9 0.8 1.4 0.8 1.1 1.1 539 aAdapted from Public Health Service Publication No. 999*-AP-12. -4l- DUP050055323 TABLE 1-8 Lead Content in Roadside Soil and Grass as a Function of Distance from Traffic and Grass Depth in Profile^ Site __ I vest of U.S, 1, near Plant Industry Station , Beltsvine, Md. Distance from road, m Lead content yg/g dry vt Soil profile layer, cm Grass. 0-5 5-10 1Q*-L5 8 68.2 522 460 4l6 16 47.5 378 260 104 32 26.3 164 108 69 II vest of southbound lanes , Washington-Baltimore Parkvay, Bladensburg, Md. 8 16 32 51.3 30.0 18.5 540 300 202 105 l40 60. 98 60 38 III vest of Interstate 29, Platte City, Mo. 8 21.3 242 112 95 16 12.5 140 io4 66 32 7.5 6l 55 60 327 Adapted from Lagerverff and Specht. -42 DUP050055324 TABLE 1-8 - Continued Site IV north of Seymour Road, Cincinnati, Ohio Distance from road, m Lead content, Vig/g dry Wt Soil profile layer, cm Grass 0-5 i=i2. 10-15 8 l6 32 ' 31.3 150 29 26.0 101 lU 7.6 55 10 11 8.2 6.1 -U3- DUP050055325 FIGURE 1-1. Increase of lead in snow at Camp Century, Greenland since 4l8 800 B. C. Reprinted with permission from Murozumi et al* FIGURE 1-2. Read profiles in the major oceans . Reprinted with permission from Chow.12fAtlantic waters were sampled at sites relatively free of continental influence.) FIGURE 1-3, Lead concentrations in samples of Hylocomium splendens , Pleurozium schreberi and Hypnum cupressiforme collected in Skane, Sweden from i860 to 1968. The line indicates 15-year averages. 477 Reprinted with permission from Ruhling & Tyler. FIGURE 1-4. Mean lead concentration versus population for 87 urban stations having more than 30,000 persons in I960 of the Rational Air Sampling Network, 1966 and 1967. The line is drawn through points determined by grouping the data geometrically by population intervals and finding the median concentration at the median population, (Data from R". I. Larsen, personal communication) . 568 FIGURE 1-5. Amounts of lead consumed as gasoline additives and mean lead concentration in air sampled at selected sites for I960 to 1968, from analyses made with the new procedure* 14-18 sites averaged for each point, (Air data from R. I, Larsen, personal communication). FIGURE 1-6. Ecologic flow chart for lead showing possible cycling pathways and compartments. -44- DUP050055326 FIGURE 1-1. Increase of lead in snow at Camp Century, Greenland, since 418 800 B. C. Reprinted with permission from Murozumi ejt al. f via O .1$ r-O--i--i 800 BC O .08 -.06 -.04 -.02 ------ 1--i --r --t 1750 AO 1800 1850 Age of comples r* r -T t ~t -t --f*] *r--1Tf" 1800 1850 45 DUP050055327 FIGURE 1-2. Lead profiles in the major oceans. Reprinted with 123 permission from Chow. (Atlantic waters were sampled at sites relatively free of continental influence.) -46- DUP050055328 FIGURE 1-3. Lead concentrattons in samples of Hylocomium splendens. Pleurozium schreberi. and Hvnnum cupressiforme collected in Skene, Sweden, 1860-1968. Line indicates averages of each 15-year period. 477 from Ruhling & Tyler. Reprinted with permission -47- DUP050055329 MEAN LEAD CONCENTRATION (1966-7) VS. POPULATION (1960) Go a3 ao. jje euu/Grf #u o j j .bj j .u 0d u o q pB0~| uea|/\| --U8-- DUP050055330 FIGURE 1-5 ............ ..: ..i- ' ... ' --r'... -49- DUP050055331 Ficuro i-(>. Ec.o logic flow chart .for lead .showing possible cycling pathways find compirtmontis, -50- DUP050055332 CHAPTER 2 LEAD AND PLANTS Lead in plants could arise from several sources, including (l) lead present in the soil, either naturally or added artificially (e.g., by spraying) or as a result of industrial activity; (2) lead present in rainwater and ground water; and (3.) lead present in the air. Each of these sources may in turn affect the roots, stems and leaves of plants in a different manner. The Effect of Lead in Soil Lead is a natural, but minor, constituent of agricultural soils and plants. Swaine^0 gave a range of lead in agricultural soils of 2-200 yg/g. Most investigators report soil lead concentrations within these limits; however, a few have found a higher lead content in some soils. The average concentration of lead in soil is thought to be about 16 yg/g.^^ The lead content of the soil is not constant with depth. It is normal for the lead content of undisturbed soil to decrease with depth. This and the lead gradients in soils near busy highways are discussed in detail in Chapter 1. The amounts of lead reported in plant material have varied widely. The lead content of the leaves of different species growing in three wooded areas in New Jersey ranged from less than 0,3 to 30 yg/g.^ In several qrr O crop plants , it averaged less than 10 yg/g. -51- DUP050055333 In discussing plant uptake of lead from soil, it is probably more appropriate to consider soluble lead than total lead. Brewer*1 gives a range of 0.05-5 pg/g of soluble lead in soils. Difficulty arises, however, when one tries to decide what should be used as the extracting agent that simulates the root of the plant. At best, a correlation can be made between the extractability of lead and the ease with which the soil releases its lead to the plant. The relation of extractabiliy of lead chloride added to the soil, its organic matter content, and pH was demonstrated by Mac Lean et al. 375 using oats and alfalfa. This work showed that low humus content and high acidity favor lead uptake by plants from soil. It also showed that When large amounts of lead (LOGO pg/g) were added to the soil, the availability of the lead to the extracting agent, IN ammonim acetate, could be correlated roughly with the availability of lead to the plant. The typical effects of adding lead to soil are summarized in the work of Baumhardt He added lead to soils in field plots in amounts varying from about 20 to 1000 pg/g of soil. Corn (Zea Mays X.) was then grown on this soil for 2 years. He concluded that there was no reduction in germination on any plot treated with lead, and it had no effect on plant height, date of silking, or grain yield. The lead content of the leaves was significantly greater when the amount of lead added was about 250 pg/g, but there was no detectable increase in the lead content of the kernel at any lead concentration studies. The results showed that the length of time following the addition of lead has an influence on its biologic availability. Thus, the lead content -52- DUP050055334 of the plant portion without the grain grown in soils receiving more than 250 ug/g was about half as great in the second year of harvest as in the first year* Extraction with IN NH^OAc confirmed that the lead 291 was less available in the second year, Keaton also noted the de crease in soluble lead with time. The importance of soluble lead versus total lead is further shown by a greenhouse experiment conducted by Marten and Hammond,^ in which an eightfold increase in the total lead content of a soil did not result in a significant increase in the lead content of bromegrass grown in it. The trace amounts of lead absorbed by plants from soils can be increased by increasing the lead content of the soil or by decreasing the binding lj, 15 capacity of the soil for lead. Motto et^aU reported on the absorption of lead by plants from solutions. In a greenhouse experiment, five crop plants--carrots (a root crop), potatoes (a tuber crop), tomatoes (a fruit), corn (a grain), and lettuce (a leafy vegetable)--were grown in acid-washed sand to which complete nutrient solutions were added daily until the plants were well established. When the plants were established in the sand culture, the sand was washed by leaching with distilled deionized water. Each cult Tire was then treated with nutrient solutions that contained no phosphorus and either no lead or 1, 2, or k pg/g of lead as lead nitrate. These solutions were added to the growing plants daily for a week; then the sand was washed and complete nutrient solution was added daily for a week. This was repeated until the crops were ready for harvest. All harvested plant parts were washed in distilled deionized water to which detergent had been added. The results of this experiment are recorded in Table 2-1, The roots of each crop absorbed soluble lead -53- DUP050055335 and translocated some of it to aboveground parts, except the leaves of potatoes, fruits of tomatoes, and the grain of corn, which showed little or no increase in lead. In lettuce, potatoes, and tomatoes, the small feeder roots were analyzed, and their lead content was very high; in corn and carrots, larger roots were analyzed, and their lead content was much lower, indicating poor translocation even in the roots. In these and other experiments using solution culture, 358 the concen trations of lead in the nutrient solution were Very high, compared with the concentrations of lead that might he expected in solutions in soil, hut the over-all amount of lead in the plants was not extremely high* This further confirms the poor translocation of lead in plants. Studies by Motto et al. in the field indicate similar results. They found that lead was absorbed through the root system with some translocation to other parts of the plant. The fruiting and flowering parts of the plant contain the smallest amount of lead and show little effect of changes in the amount of lead supplied. Several studies have been made of the relative importance of air, 15'U water, and soil as sources of lead in plants. Dedolph et al. studied the effects of lead in air, water, and soil on the concen tration of lead in perennial rye grass and radishes. Filtered air was used for half the plots. Both grass and radishes were found to derive about 2-3 ug of lead per gram of dry matter from the soil when -5h- DUP050055336 the concentration of lead in air and in water was zero, They concluded that there are substantial amounts of soil-derived lead within plants and that soil has long been and remains an important source of lead in plants. Ter Haar,*^ in a similar study, investigated the effects of lead in air and soil on the lead concentration of the edible and inedible portions of several important types of food crops. These were studied by growing crops in greenhouses supplied with filtered and un filtered ambient air and in plots planted in long rows perpendicular to a busy highway. The lead content of the soil varied from 65 yg/g near the road to 25 yg/g remote from the road. On the basis of these crops, the conclusion was that lead occurring naturally in the soil is the main source of lead in the edible portion but that, in the concentration range studied, the variation of lead content of the soil did not affect the lead content of the crops (Tables 2-2, 2-3, 2-4). These studies further showed that fresh unprocessed foods grown in filtered air contained quantities of lead similar to those in foods in the marketplace. Thus, it seems that food as purchased probably is not significantly contaminated with lead unless processing is done in a careless manner, Schuek and Locke496 studied five crops--cauliflower, tomatoes, cabbage, strawberries, and oranges. In spite of their being grown near heavily -55 traveled, highways with up to 50,000 cars per day , the amount of lead found in the five crops in an untreated state was never greater than 1 ug/g fresh weight , and the average lead concentration for the entire crop area studied was only one-tenth or even one hundredth of that.. Although their conclusion that crops are not inclined to absorb lead through the root system disagrees with some of the authors cited earlier, it may be that the pH of their soil, or some other physical or chemical characteristic of the soil, led to this conclusion. Little systematic work has been done on the uptake of lead from soil of different types, in which the effects of pH and other physicochemical variables may account for the differences found. The Effect of Lead in Water The effects of lead in soil discussed in the preceding paragraphs are undoubtedly actually the effects of trace amounts of lead in "soil solution". The factors of soil type and probably pH, which strongly affect the uptake of lead, influence the amount of lead in solution in the ground water. In nearly all cases, this concentration will be very low because of the strong coordination of lead to soil. The effect of lead in rainwater on lead concentration in plants 154 was studied in work mentioned earlier in which the lead-in-rainwater variable was separated from the lead-in-soil and lead-in-air variables. -56- DUP050055338 Thera was no detectable change in the lead concentrations in any part of grass and radishes sprayed with 40 pg/liter of lead chloride. The Effect of Lead in Air Many authors substantiate the conclusion that lead in air increases the concentration of lead in the leafy parts of plants near highways , hut does not affect the compact portion of the plant- Kloke and Riebartseh 313 found higher concentrations of lead in grass grown 88 near busy highways . Cannon and Bowles found higher concentrations of lead in vegetation grown near a highway than in that grown some distance 578 away. Warren and Delavault correlated lead content in plants with highway traffic. They determined lead in tree stems collected from an area remote from highway traffic and in stems of the same species collected in an area of heavy traffic. The lead values for the remote area ranged from 0.4 to 2.0 yg/g dry weight, and for the heavy traffic from 2 to 52 pg/g 178 dry weight. Everett et j l U measured the lead content of unwashed privet leaves collected from sites along main highways and remote from highways in England. They found an average of 86 pg/g dry weight in the leaves near the highway and 45 pg/g dry weight in the sites away from the highway. Ruhiing and Tyler*4^ have studied the concentrations of lead in mosses. They observed that, because the mosses obtain their minerals chiefly from -57- DUP050055339 precipitation and settled dust, the moss could be used as an index for surveying deposition of airborne heavy metals. They found in southern Sweden that the concentration of lead in mosses has risen from about 25 yg/g in the 19th century to the present average of about 100 yg/g. The interpretation of these findings is discussed in Chapter 1. 154 The experiments already cited of Dedolph et, al. using filtered and unfiltered air showed no effect of lead in air on radishes, but the eontentration of lead in grass was about doubled when the lead concen tration was raised from zero to about 1 yg/m^. Studies near a busy highway confirmed these results. Grass grown at increasing distances from the road contained concentrations of lead that could be correlated with the concentration of lead in the air. The study by Ter Haar548 cited earlier concluded that airborne lead contributed about 0.5 - 1.5# of the lead content of the U.S. diet. Of . the ten crops studied--wheat, potatoes, tomatoes, sweet com, carrots, cabbage, oats, rice, leaf lettuce, and snap beans--eight were not affected by the concentration of lead in air. In both field and greenhouse . tests, the inedible portions of the plants (bean leaves, com husks, soy bean husks, and oat, wheat, and rice chaff) showed a 2- to 3-fold increase in lead concentration when grown near the road or in the greenhouse with unfiltered air (Tables 2-2, 2-3, 2-4). 58- Lelr349 compared rye and potato plants growing within 15 ft of traffic with similar plants growing more than 300 ft from the highway. The lead contents of chaff and green tops from near the highway were higher by a factor of about two5 whereas those of kernels and tubers were un affected. Motto et al. 41v5 grew five commercial crops /(lettuce, tomatoes, com, potatoes, and carrots) along highways supporting 12,500, 47,100 and 49,000 cars every 24 hr. These crops were grown 30, 100, and 250 ft from each of the three highways. The soil in all locations was a sassa fras loam. All the plants except the com were grown to usable maturity; the corn was harvested after the kernels became hard. The data (Table 2-5) demonstrate the tendency for lead from air to accumulate on leaves and other exposed aboveground plant structures. These washed samples indicate that the lead from the air or the soil did not increase in the portions of plants consumed by man, except for lettuce leaves, which exhibited a highly significant increase in lead in the samples gathered near the highways supporting the most traffic. All investigators reach the same conclusion. In a narrow band near the highway, the concentration of lead on the surface of foliage is proportional to the concentration of lead in the air. On the protected portions of the plants (e.g., seeds and roots) , which in almost all cases are the edible portions, little or no effect is noted. -59- DUP050055341 Seasonal Variations The importance of the growing state of the plant in the plant 's lead content is noted by several investigators. Daines at al. iho report an increase in atmospheric lead during the winter months t increasing the opportunity for lead to be deposited from the air. During the summer months, plants are normally growing, thus diluting the lead deposited on their leaves with new tissue. The lead deposit on leaves would be expected to increase with increasing exposure time. Chadwick and Chamberlin^00 also found that less lead remained if it was applied during the summer to grass than if it was applied during the winter after the same weathering period. The lead content in privet foliage was greater during September through April than during May through Aa ugus4-t. 178 Mitchell and Reith1*03 found that there is an increase in lead in the aboveground portion of the plant when active growth stops. In autumn, the lead content of the aboveground portion of pasture herbage begins to rise from about 1 ug/g dry weight to 10 ug/g dry weight. It may reach 3Q-40 ug/g in the winter, 'The authors believe the increase in lead content of the aboveground portion when the plant is dormant may indicate translocation of lead from roots to tops during the winter months or loss of organic matter through respiration, rather than uptake from the soil. They ruled out the possibility of surface contamination from lead in air and soil contamination. -6O- DUP050055342 This study indicates that caution is necessary when comparing lead concentrations in materials. If they are harvested in different seasons, the results may not he comparable.' More work is needed'to learn the influence of seasonal variation and the effect of aging stress on lead uptake* One further point should he made. Even in the absence of lead in air, the leafy portions of plants are often higher in lead than the 5h8 rest of the plant. Ter Haar observed this in his greenhouse study. 204 Goldschmidt observed it as early as 1933. He stated that the min eral solution enters the plants through the roots and concentrates at the point of greatest evaporation--namely, the leaves. The concentration of lead in grass near a highway is of special interest, because animals may consume it. Chow6l0 found up to 60 yg/g dry weight in grass cut within about 40 ft of U.S. Highway 1 in Maryland and the Baltimore-Washington Parkway. With an increase in traffic from 11,000 to 32,000 cars per 12 hr, Kloke and Riebartsch313 found an increase in roadside grass from 16 to 6.0 yg/g dry weight. Leh^ found 180 yg/g dry weight on the median strip, 120 yg/g dry weight 6 ft from the edge, and 35 yg/g dry weight about 20 ft from the edge pf a road reported to carry 30,000 cars in 12 hr. Dedolph et al. 154 found that for a road carrying 30,000 cars per day the concentration of lead in grass was correlated with the concentration -6l DUP050055343 of lead in air* Forty ft from the road, the lead in air was 2.3 Rg/m^, and the grass contained 15 Rg/g dry weight. At 120 ft from the road, the lead in air was 1,7 Rg/m3, and the grass contained Q.k ng/g diy weight. 1^15 Motto et al. analy zed grass clippings gathered at seven diet emcee (0-225 ft ) from highways supporting 12,800-5^,700 cars daily (Table 2-6), All samples were divided: one portion was washed in four changes of distilled deionized water to which a small amount of detergent was added for the first washing; the second portion was analyzed without washing. The average lead content of unwashed orchard grass clippings gathered at the highway edge (255 Rg/g dry weight) was more than five times that of grass gathered 225 ft from the highway. It was of interest that the decrement in lead content was steepest in the first 75 ft from the highway. If the average lead content of the grass samples reported by Motto et. al. were charted against distance from the highway, 1 ip the curve would be very similar to that of Baines et al:. for atmos pheric lead versus distance from a highway* Paines et al. reported a decrease in atmospheric particulate lead of over 30% between 10 and 150 ft from the highway. bl3 Motto et. al. further report that washing the grass removed lead from all samples studied; however, those gathered in the first 75 ft lost the greatest amount of lead (nearly 30%) in washing. The fact that -62- DUP050055344 lead can be removed from leaves by washing and that lead in grass is correlated with lead in air demonstrates that all or much of the lead on leaves that comes from the air is as stated by Schuck and Locke present "as a topical dust coating *" Effects Although a considerable volume of literature is devoted to studies of lead in soils and plants, there seems to be no reliable evidence that lead injures plants in nature. In fact, extensive investigations summarized by Bradshaw et al. TO show that some species of plants have adapted to habitats near mining operations that contain lead, zinc, and copper in amounts that are toxic to nonadapted populations of the same species. Jowetx2 Si showed that a high tolerance to lead had de veloped in many populations of Agrostis tenuis (colonial bent grass) growing on disused lead mines in mid-Wales . The tolerance mechanism is specific for the metals separately, and it is known that there is no significant difference between tolerant and nontolerant plants in the amount of toxic metal absorbed. However, tolerant plants can be distinguished from nontolerant by both calcium and phosphate response. 2 Wilkins'^ reported that lead tolerance in Festuca ovina (sheep fescue) seemed to depend on a single gene with large effect. Toxic effects on plants of experimentally administered lead have been manifested by a variety of signs.* There are reports of delay in germination of ^During the period when lead arsenate was used as an insecticide on fruit trees, injury to apple and peach foliage was common. However, this phytotoxicity was produced by the arsenical and not the lead part of the compound. -63- DUP050055345 seeds of cress and mustard after treatment -with concentrations of lead ion above 0.01%, and subsequent growth; ? of delay in root growth and leaf and flower production in hyacinth bulbs exposed to lead ion;^ 1L.7 and of contributory effects to frenching of tobacco. At the cellular level, toxic effects on plants have been found experi mentally affecting cell walls, nuclei, and mitochondria. From studies using Allium Cepa (onions), Zea Mays (corn), and Viola Faba (broad bean), po/ Hammett reported the concentration of lead within the nuclei and cell walls of the growing portions of roots He found that lead retarded cell proliferation while allowing cells to increase in size. In further studies, he noted that the mitotic (dividing) nuclei of the growing roots "have a special avidity for lead," and that a reaction between lead and an organic sulfhydryl occurred in the area of normal rapid elongation. Of interest is the comment that "the sulfhydryl group stimulates cell proliferation in mammals and in lower organisms," Lead nitrate-induced mitosis has been found indistinguishable from that induced by colchicine, with spindle disturbances and chromatid formation in root tips of Allium Cepa, 353 In a small number of experi ments, barley seeds soaked for J hr in 0.2M solutions of lead nitrate seemed to be more susceptible than controls to x-radiation. 535 Koeppe and Miller, 315' using corn mitochondria, found that lead chloride (50-62 uM/liter) in either potassium chloride or sucrose-containing -64- DUP050055346 media stimulated oxidation of exogenous reduced nicotinamide adenine dinucleotide. This increased oxidation was not affected by the presence of phosphate. These studies further indicated that lead chloride . (12,5 uM/liter) inhibited oxidation of succinate in the absence of phos^ phate. inasmuch as phosphate is essential for plant growth, the effect on succinate oxidation would seldom be encountered in nature . The effects of high concentrations of lead on animal cell mitochondria are described in Chapter 4. In all the experiments discussed here, the concentrations of lead bore no resemblance to the concentrations present in soil solutions* In all cases, effects were noted only at concentrations several orders of magni tude higher than would be present in the soil solution, even in a soil highly polluted with lead. Summary 1. Plants can absorb soluble lead through their roots and translocate some of it to their aboveground portions. There is a natural concentra tion of lead in plants that comes from the natural lead content of the soil. 2. Widely varying concentrations of lead in soil have little effect on the lead content of the plant. The lead naturally occurring in soils is largely 'unavailable to plants, as is the airborne lead in soils along heavily traveled highways. 3. Lead in air does not measurably increase the lead content of the edible portions of most plants. 4. Leafy portions of plants within about 75 ft of busy highways contain higher concentrations of lead than do plants growing further away. This is true for a narrow band on both sides of the highway. There is little 65- DUP050055347 or no evidence that this lead can be translocated downward to the under ground portions of the plant * 5 . Because about 50# of the lead on leaves of plants growing near busy highways can be removed by a water wash, it must be concluded that much of the lead is present as a surface deposit. Although the lead particles in the air are small enough to enter the open stomata of plants (unless prevented by electrostatic forces), there is no evidence that lead par ticles from air enter the leaf. 6. Even in the absence of lead in air, leafy portions of plants generally contain more lead than the other parts. 7. Rainwater does not appear to be a significant source of lead in crops. 8. Stresses on a plant, such as senescence, may increase the concentra tion of lead in it. 9. Evidence that lead, as it occurs in nature, is toxic to vegetation is lacking. However, in studies using roots of some plants and very high concentrations of lead, lead has been reported to concentrate in cell walls and nuclei in mitosis and to inhibit cell proliferation but to allow the continued increase of cell size. In addition, on the basis of corn mitochondria, it has been reported that lead chloride stimulated the oxi dation of exogenous reduced nicotinamide adenine dinucleotide and decreased the oxidation of succinate in the absence of phosphate. Because phosphate is essential for plant life, the importance of the effect of lead chloride on the oxidation of succinate in plant cells is uncertain. 66-. - DUP050055348 (CABLE 2-1 Concentration of Lead in Plants Grown in Acid-Washed Sand in a Greenhouse in 1968, (jjg/g dry weight), Motto et al.(Ul5) Treatment ppm Pb Carrot 0 i 2 4. - Tops Root s . 8,7 3.1 . 16 7.9 19 18 27 21 Corn Tassel Leaves Stalk Husk Outer Inner Roots Kernel Cob 7,8 11 0,6 2.9 3.9 3.7 1.0 2.4 8.1 19 11 7.9 7.3 12 1,7 2.7 7.4 39 28 15 13 22 2,8 4.3 9,2 88 44 23 16 35 3.3 10 Lettuce Leaves Roots Potato Leaves Stems Roots Tuber Tomato Leaves Stem Root Fruit 5.7 7.6 11 7.6 30 1.0 8.1 3.6 17 3,6 12 16 108 182 7.8 29 200 0.6 12 55 451 2.6 8.1 24 418 2,5 15 89 690 2.3 37 332 12 123 764 1.2 16 87 739 2.1 -66 a- DUP050055349 TABUS 2-2 Lead in Crops--Greenhouse Studies3, Lead Content, ug/g (dry) Ail* Soil Leaf lettuce Cabbage bead Cabbage-^un harvested leaves Tomatoes Beans Bean leaves Sweet corn Kernel Cob Husk Carrots Potatoes Wheat Unfiltered air 1.45 Mg/m3 17.1 6,6C 1.0 4.5 0.59 1.4 20.9e 0.22 0.43 6.9 1.7 0.30 0.l8 Filtered air 0.09 ug/m^ 17.1 3.2 1.1 5.8 0.72 1.2 7.9 0.27 0.69 1.8 2.1 0.33 0.l6 b LSD 1.3 0.35 1.6 0.26 0.52 8.8 0.22 0.24 1.5 1-9 0.12 0.06 aNbdified from Ter Haar.^ to Least significant difference, different from otber numbers in row at 95# level of confidence. -66b- DUP050055350 TABLE 2-3 Lead in Crops ^-Highway Studies 9* Lead Content, ug/g (dry) Feet frcm road Air Soil 30 2.3C Mg/m3 65c 120 1.7d Mg/m3 40d 520 *0 1,1 Mg/m 25 ISP Leaf lettuce Cabbage bead 6.5 0,5.6 5-0 0.86 4.8 0.83 3,1 0.44 Cabbage-- unharvested leaves 6.4c Tomatoes 1.3 Beans 1,9 8.9d 1,2 1.2 4.0 1.6 0.90 1.3 1,3 0.47 Potatoes 0.48 0.64 0.40 0.27 Sweet corn Kernel Cob 0.39 0.74 0.21 0.55 0.83c 0.68 0.31 0.21 Husk 12.6e 6.6 5.7 3,6. Soybeans Beans 0.28 . 0,12 0.10 0.10 00 p . Husk 15.9C 5.3 0.22 Oats Kernel 0.47 . * 0.53 0.37 Chaff Carrots 31.4a 1.6 15.5 12.8 1,5 15.5 0.61 Wheat Kernel Chaff 0.62 17.8 0.42 9-8d 0.48 6.2 0.17 1.6 -66 c- DUP050055351 TABLE 2-3 - continued aModified from Ter Haar.^ *u Least significant difference ** cSingle letter shows this value different from others in row at 95# level of confidence. %*wo letters in same row show these values different from others at 95# level of confidence. -66d~ 0 DUP050055352 TABLE 2~U Lead in Rice8, Lead Content, ug/g (dry) Road U. S. 90 Sample Grain Hulls Straw Feet from road 30 0.17 1220.18 LSD13 0.0 4 3.9C 1.9 0.98 k,l 2.5 1.2 1-10 Grain Hulls Straw Feet from road. 30 700 LSD13 0.23 0.24 0.04 4.9 2.9 1.6 5 83c 2.13 0.35 ^Modified from Ter Haar.^ ^ Least .significant difference. c Different from other numbers in row at 95% level of confidence. -66 e- DUP050055353 in COO CM CM CM M Qft) CO . O CO (MC0O U O Oo CM * 5$ CO co on CM CM X ft! o cd CO VpO\ rH ir\ r0H l CO CM 9 H CQ 1 r<HHD J-J <U X! K Mt CM a0) CO u cd o oo CM to CM CM o Q # o M* CO o m rH CM rH - rH CO o CO co CO Ml- co CO co 00 CO Mtol- o VO in $ <o CM CO CM CM CO CO CQ Ml- m VO ov CM r>. vo CO CM CO CM 5 CO in o vO in co CQ d\ Mi- CO rH CO r--1 in rH CO CO CO 00 r>- mirH ON o *4 cn rH co CO 00 CM o CM m in m 1 rH3 O t> MUHH MH CtJ U H oB 4uH Qi aO 4C-d QCHO M4HJ CO & CO r* SL >> n cd CM *CHO ur1 X <3 gCaL * AXt #\ rH H CoQ # 0H 1 4J cd A +J 4<0J0 O0 a PM m 0 BCL a 4J oIMH od CO H0a* w 4QOJ Pd o*0 H CCaQO> Hcd t<ou c>ftd) rH C40-Q CO W0 uo3u O IH <u M00 -66f- / DUP050055354 TABLE 2-5 c o n tin u e d 0.5 9.0 1 ^Adapted from Hot to efc a l u X3 <1CM M<U D CO cuoo CM CO (u0 :O0SA ft SWO fr#* .* SWOO iSHO St r* sp c m 3 os oo CM CO U") H <M rH CM t-A VO oo os co oo c m co CM MS' OrHs o o <rHt CoOs O * OS <t 60 CO CM rH in 00 O rH O CM St CO M0 co CM .* O# Oo TC--M1 rC>M u cod O os co St r>* . CM liO m CM O0 CM c0 oo O sf vO vP ps o st *3- m co c m .* vo r^r^<r sr rM CM CM 00 rH . 00 f^CMCM U ftSi r- vj D XI WO co CM St CM ft f*t 00 o iH oo H CM rH Os sO v rH rH CM U O A tuo Osft sO CM CO C0 CO co U0 H rH St W0 pH 00 co r~i CO CM 60 tn CM ON HWO oft co ctf o o O in CM rH O ooft O SO CO CO CM VO rH pH SCOO CiHM CCMM 60 SO 00 rH CM *o 4-1 hcudi e 0 u CO V 4J Q c u i 0 o r--^ <u 0 u 0) X 0 t; <U o 0 4-1 4-1 <u CO 0) CO O > c<dD CO 4J O f 0 4-1 cd 4J d> co > cd CBD a) 4J X co CO 4-> Q fS <U XD H <u 4-1 O 4J > cd a 6 4<-u1 IQ 4-1 O r4 D o Cl . H -66g- DUP050055355 CHAPTER 3 INPUT AND DISPOSITION OF DEAD IN MAN Lead has been detected in all tissues- of man and animals , even in environmental settings remote from man-made sources of lead. In assessing the potential hazards of environmental lead, the foremost consideration is the margin of safety between ordinary rates of input and rates that are demonstrably toxic. Subsidiary considerations are the relative contributions of air, food, water, and other sources and the relative importance of the several portals of entry into the body. This chapter emphasizes the input and disposition of lead in man under ordinary conditions in contrast to extraordinary exposure to lead, which is the subject of Chapter 4. Some animal data are cited, but mainly as evidence of what is thought to occur in man. SOURCES AND ROUTES OF INPUT Food, water, and other beverages are the major sources of lead input in man and probably in most animals. There have been numerous studies of the concentration of lead in various foods and beverages. Diet Exposure to lead from drinking water is influenced by the water's source, treatment, and distribution system. These facts have been known for many years, as evidenced in a letter by Benjamin Franklin"^ on the "bad effects of lead taken inwardly." One of the sources of lead that Franklin cited was rainwater collected from lead roofs overhung with trees that shed acid-producing leaves. Modern work has now documented Franklin's observations. -67- DUP050055356 The lead content in public water supplies of the 100 largest cities in the United States in 1962 ranged from traces to 62 yg/liter; the higher concentrations reflect'sizable additions of lead from pollution in some localities. Continuous monitoring of the water supplies of the United States since 1962 has demonstrated that their lead content has in general not exceeded the U. S. Public Health Service fs prescribed standard of 50 yg/liter.^"*' Kehoe2^ found values of 3-^Q yg/liter for water from 35 U. S. towns and cities and pointed out that the lead content of drinking water may be high if the piping or joint luting used contains lead or if the water stands undisturbed in lead pipes or luting of a new building for a weekend. In such conditions, concentrations as high as 920 yg/liter have been obtained. Aside from those special circumstances, lead con centrations in drinking water have been observed to be little different from those in the oceans, in rural ponds and streams, and in well water. Etfinger points out that samples from surface-water stations in heavily populated and industrialized areas where waste pollution is high contain only small amounts of lead in solution, probably because of rapid precipitation and coordination of insoluble lead from waters con taining silt, which acts as excellent coordination sites for. precipitating lead. Organic species in the water also coordinate lead and aid in 378 removing it from water. A recent report of 2,595 distribution samples found that 25# contained no measurable lead and about 73# contained less than the mandatory 50 yg/liter; 41 samples contained more than this limit. -68- hJ DUP050055357 From all these findings it is evident that drinking water contributes on the average a small amount of lead to the body burden. On the basis of the data cited, it seems unlikely that many people drink water containing more than 50 yg/liter with any consistency. The average intake of lead from water and water-based beverages cannot be stated with any pre cision. But, assuming 20 yg/liter and a daily consumption of 1 liter, the lead consumed from water would be 20 yg/day. This estimate would apply to an adult; for an infant or young child, it would be proportionally greater (i.e., on the basis of body weight). The amount of lead input in man from food depends on the natural lead content of foods, factors that may increase it, and the amount of food ingested. Of prime interest is the effect of exposure to automobile exhaust on the lead content of comestibles. Also to be considered are factors associated with the storage and preparation of foods. 306 Kehoe et al. found lead in every item of food obtained from the field and from dwellings of the inhabitants of a primitive region 451 far from industrial and mining activities. Patterson estimated that the natural lead content of food should be 0.01 yg/g wet weight (0.01 ppm) and concluded that most of the lead present today in food is from industrial sources. The lead content of food today is con- siderably greater than 0.01 yg/g. Schroeder and co-workers4q 4 have made extensive examinations of lead in food. On a fresh-weight basis, they found about 1.2 (range, 0-1.5) yg/g in condiments, 0.5 (range, 0,2-2.5) yg/g in fish and seafood, 0.2 (range, 0-0.37) yg/g in meats and eggs, 0.4 (range, 0-1.39) Ug/g in grains, 0.2 (range, 0-1.3) yg/g -69- DUP050055358 in vegetables, and no lead detectable by his analyses in fresh whole milk. 408 `svft Monier-WiHiams and Warren and DelavaulV 1 u estimated about 0.2 pg/g of food. jhe contribution of atmospheric lead to the amount in vegetables and fruits is V not clearly known. If significant, it is probably not due tp^rrect uptake by plants from the air or water.^5,548 recent studies^^^E^have define the extent of the contribution of lead dispersed into the atmosphere along high ways to the lead content of vegetation. Both investigations (which are discussed in detail in Chapter 2) showed that the amount of lead found on soils and plants in such areas tends to decrease with distance from the highway and that one-half to two-thirds is removable by washing* except in the case of rough-surfaced foods, such as strawberries. In the case of unwashed animal feed, lead may find its way from aerially contaminated grasses and other vegetation to meats consumed by man. There is no evidence that this possible source of food contamination has measurably altered the concentrations of lead in animal food products, such as meat and milk, over the last 30 years. Because milk is a major source of nutrition for infants and young children, there is special concern for the contribution of this food to total dietary lead intake. The concentration of lead in milk was reported in connection with an outbreak of bovine lead poisoning.The con centration of lead in milk was linearly related to the coexistent concentration of lead in the blood of 9 cows having varying degrees of lead expos we. In this same study, concentrations of lead in the milk of 8 cows not known to be exposed to an unusual amount of lead averaged 0.009 mg/1 iter. This average is slightly lower than the values reported by another group in 1940.303 The total dietary intake of lead is generally estimated to be about the same in recent years as it was in 1943, if the estimates of Cholak and Bambach*^0 356 -70- DUP050055359 Assuming that a person consumes about 2,000 g of food and drink per day, Schroe&er and co-workers>95 estimated that lead intake averages 120 100-500 yg/day, depending on the foods eaten. Cholak and Bambach estimated the intake of lead in food to be about 300 yg/day; Kehoe,299 whose analytical work was performed by associates in the Kettering Laboratory, estimated a similar amount on the basis of a large number of duplicate daily samples of food and beverages. Harle|^^ Conducted an especially useful study in New York City. He has determined the lead concentration in various foods and estimated the yearly intake of lead on the basis of the Department of Agriculture consumption statistics for food for an adult man at 103 mg or about 285 yg/day, a figure which is consistent with other investigations, iyewigK- points out that no food or group of foods is either a large or a constant contributor to lead in man, because man's diet is com posed of a wide variety of individual items and various foods con tribute various amounts of lead. Lewis estimated the average daily dietary intake of lead at about 300 yg, with a range for most people of 100-2,000 yg. The range can vary markedly from person to person and city to city, on the basis of choice and opportunity and specific habits. The daily dietary intake of lead of persons 1-3 years old without unusual nondietary exposure has been estimated at 130 yg, 38 * 113 on the basis of the daily fecal excretion of lead (with urinaiy losses not accounted for). The greater lead intake of infants, compared -71- DUP050055360  with adults, on a unit-tody-weight basis, probably is related to infants' higher caloric and water requirements. It is not Known whether the apparent greater intake is accompanied by a correspondingly greater total output; the critical balance studies have not been conducted, as they have in adults. Respiration Inhalation of air is a smaller potential source of input than ingestion of food and water. In view of the great variations of lead in air with season and locale, the potential intake by this route is as variable as the potential intake from food and water. For example, data cited in Chapter 1 indicate that the median concentration of lead in the air of Q Los Angeles in 1962 was 2.5 pg/m , although some stations in the city 3 recorded concentrations as high as l6.9 pg/m . In most suburban areas, by contrast, the concentration probably seldom exceeds 0.5 pg/m^, and for rural areas, 0.1 pg/m^. Thus, there is at least a fivefold varia tion in atmospheric lead exposure, depending on whether one is a resident of downtown Los Angeles or of a typical suburb (2.5 Pg/in v$. 0.5 Pg/m^). The variability of potential lead intake by inhalation is further increased by the variability of total air inhaled, which depends for example, on the degree of physical activity engaged in. A man doing light work for 8 hr a day would inhale more than twice (22.8 vs. 10.8) as much air as a mail at rest all day (Table 3-1). If one superimposes on this range a twenty-five fold difference in atmospheric lead concentration between rural and urban air, the potential daily intake of lead could vary from 1.1 yg for a rural *0 + * -72~ DUP050055361 man at rest all day to 57*0 pg for a man engaged in light work in downtown Los Angeles and residing there continually while not at work (Table 3-1) The comparable amount of lead potentially inhaled by a suburbanite who comnutes to the city for light work is 30.6 pg. As in the case of dietary intake, potential respiratory intake in infants is greater than that in adults on a unit -body-weight basis when exposed to comparable concentrations of airborne lead. A 1-year-old child inhales 6 m air per day, compared with the 23 mr per day in the case of an adult. Again, the significance of this proportionally greater inhalation of lead per day is obscure because of lack of studies of the metabolism of lead in infants. The proport i onally greater intake might be accompanied by a correspondingly greater rate of excretion. ABSORPTION AND EXCRETION Although food and water are generally a greater potential source of lead in man than is the air, there are still many 'uncertainties as to their relative contributions, mainly because of the imperfect state of knowledge concerning the fate of the lead that is inhaled. The con tributions of food and water to the total daily assimilation of lead have been studied extensively. Much was learned from detailed, pro longed balance studies conducted in man at the Kettering Laboratory, University of Cincinnati, under the leadership of R. A. Kehoe. By analysis of samples of all the food, water, and incidental beverages ingested and of the total urine and feces excreted, the net balance of intake vs. output in a number of men was determined over a period of many months * This was done in conditions of both normal and -73- DUP050055362 abnormally bigh intake . The results of these studies have been reported over a span of many years and were summarized in 196I They show that at normal intake the amount of lead excreted generally slightly exceeds the amount ingested in food, water, and incidental beverages. The failure of lead intake to match lead excretion was thought to be due to the un measured contribution to the intake made by lead inhaled in air. A typical set of data is presented in Table 3-2. It is apparent from these data for the first seven periods of 8 weeks each that any one period may yield either a slight increase or a slight decrease in the total lead content of the body without any apparent relation to the small fluctuations in the level of intake. In the case of this particular sub ject, a change in residence and in diet at the end of the seventh period resulted in approximately a doubling of the rate of lead intake, with a pronounced accumulation of lead. In conditions approximating a steady state, in which oral input roughly parallels urinary and fecal output, the urinary excretion of lead is about 10% of the oral input. In this respect, the data in Table 3-2 are typical of KehoeVs findings in human subjects, and it is inferred that net absorption of lead from the gastrointestinal tract is approximately 10%, The contribution to total excretion of lead assimilated through the airways was not determined. Inclusion of this additional factor would have resulted In a reduction of the estimated proportion of gastrointestinal absorption. More complete studies involving normal ambient air lead and normal dietary lead have not been reported. -74- DUP050055363 In additional studies by the same author, a change from slight decrease to increase in body lead (i.e., lead accumulation) was demonstrated with 299 addition of small daily doses of a lead salt to the diet, continually for as long as h years . Although the, urinary excretion of lead increased roughly in proportion to the daily intake of lead, new levels of equi librium were not reached within the period wherein urinary and fecal excretion consistently equaled or exceeded oral intake* inasmuch as increasing the level of oral intake of lead resulted In an increase In urinary excretion, it can be concluded that a small fraction of the lead ingested is indeed absorbed. The time required for approximate balance between Input and outgo to be achieved when lead is added to the diet is unknown, but is more than 2 years. It is not known how long it takes to achieve a new steady state when intake is substantially increased, or whether indeed a steady state ever is achieved. For that matter, the rate at which approximate equilibration of intake and outgo is achieved under usual dietary conditions is not known. Virtual equilibration certainly does not occur during fetal life. The concentration of lead Increases rather rapidly throughout fetal life in the bones, whole body, and, to a lesser 262b degree, liver. Some confirmation of Kehoe's work is provided in two Japanese studies as to the approximate balance of dietary intake with outgo and as to the persistence of accumulation of lead In the body during lohg periods of abnormally high oral intake /^ a^^9`Further consideration is given to the long-term accumulation of lead in man in a later section. -75- DUP050055364 The influence of dietary factors on the absorption of lead In man has been 299 studied to some degree by Kehoe. Increases and decreases in dietary calcium and phosphorus , alone and in combination, had no significant influence on the retention by the body of small daily doses of lead (l mg). Studies in rats indicate that high dietary levels of both 527 calcium and phosphorus decrease the amount of dietary lead retained, whereas vitamin D has the opposite effect. 528 The relative contributions of the gastrointestinal and urinary routes to total excretion of lead under a variety of conditions is not clearly known. The data presented in Table 3-2 indicate that about 9Q$ of the dietary lead is eliminated in the feces. Some of this lead has probably been absorbed and re-excreted into the gastrointestinal tract by biliary secretion and by secretion and epithelial exfoliation elsewhere in the gastrointestinal tract. After the intravenous administration of lead-212 to four men, the average urinary excretion in the first 24 hr was 4.6$ of the dose and practically ended in that period. During the first 48 hr after administration, only 0.l8$ of the dose appeared in the feces. Owing to the slow appearance in the feces of substances excreted in the upper bowel, it is not certain that these results reflect 63 the true relative contributions of urine and feces . Booker et al. conducted similar studies in two human subjects. They found no lead-212 in the feces during the first 24 hr after injection and 4.42$ in the urine. During the second 24 hr, however, they found 1.42$ in the urine and 1.5$ in the feces. This suggests that the fecal route of excretion may well contribute as much as the urinary route to total excretion. Long-term studies of lead excretion in the baboon indicate that the -76- DUP050055365 ratio of urinary to fecal excretion of lead is approximately 2:1. Similar studies in rats^T and in sheep^ indicate that in these species the fecal excretion of lead is even greater than that in urine; in these two studies, biliary excretion was found to he much greater than excretion elsewhere in the gastrointestinal tract. Knowledge concerning the contribution of respired air to the total amount of lead entering the body is insufficient to allow anything better than general approximations. A reasonably sound estimate can be provided for the fraction of the inhaled lead that is deposited in the airways, but very little is known concerning its fate once de posited. Lead particles deposited in the nasopharyngeal region may be swallowed or ejected by nose-blowing or expectoration. Particles deposited in the trachea and bronchi may migrate up to the pharynx by ciliary-mucus transport to be swallowed or expectorated later, or they may enter the systemic circulation. Even particles deposited in the alveolar bed may be phagocytized by migratory macrophages and conveyed back up the airways to be swallowed and passed through the gastro intestinal tract largely unabsorbed. The degree to which inhaled lead is initially deposited in the respiratory system has been studied by a number of investigators . Mehani^'' reported an average deposition of 37$ for nonindus t rially exposed men and 39$ and 47$ for two groups of men industrially exposed. Unfortunately, he did not report the particle size distribution for the inhaled lead. -77- DUP050055366 in recognition of the fact that particle size is an important determinant of the deposition and retention of inhaled particles, some investigators have characterized the particles under study in this regard. The informa tion available concerning the size distribution of lead particles in the environment (see Chapter l) seems to indicate that the major concern in respiration should be the fate of respired lead particles with mass median equivalent diameters (MMED's) in the range of 0.1-1.0 ym and that the average MMED in the general atmospheric environment is about 0.25 ym. There are only two reported studies in which the deposition of lead in the lungs was measured and particle size was recorded. The first of these studies was by Kehoe, ^9 The lead was in the form of lead sesquioxide generated by burning tetraethyl lead. Particles of two size groups were used: an average diameter of 0.05 ym with 90# from 0,02 to 0.09 m; and a median diameter of 0.9 ym with 90$ less than 2 ym. Diameters were determined by electron microscopy. A diameter of 0.05 ym for lead sesqui oxide as seen in the electron microscope represents an MMED of approximately 0.25 ym, which is identical with the MMED for lead reported for the general atmospheric environment 1+67 by Robinson and Ludwig* Particles of lead sesquioxide having a diameter of 0.9 ym as viewed in the electron microscope would, by contrast, have an MMED of 2.9 ym, which is considerably higher than is encountered in the general environment. (See appendix A for particle-size conversion.) In both cases, the concentration of lead o in the air was 150 yg/m which is much higher than is encountered in the general environment. The deposition in the respiratory tract associated with exposure to the smaller particles was 36$. -78- DUP050055367 That is almost identical with the 37% reported by Mehani for nOn- QGC industrially exposed men. ' Deposition of the larger particles was 46$. 436 The other major study of lead deposition was reported by Nozaki. Many particle sizes were compared, and an effort was made to distinguish between pulmonary and tracheobronchial deposition. The concentration 3 of lead was 10 mg/m , which is extremely high, even for an industrial situation. The source of lead was fumes generated in a high-frequency 262 induction furnace. Particle size, which was closely controlled, varied from 0.05 to 1 pm and, as in Kehoe* s studies, was determined visually with an electron microscope. Deposition was observed in conditions of both rapid shallow respiration and slow, deep respiration. For particles having a diameter of 0.05 pm (by electron microscopy), the total deposition in the respiratory tract for slow, deep respiration was similar to that reported by Kehoe, but was appreciably lower for rapid, shallow respiration (Table 3-3). 395 299 430 The observations by Mehani, " Kehoe , and Kozaki suggest that the deposition in the respiratory tract of lead from the ambient air is approximately 37$ All these studies were deficient in one respect or another, and more extensive studies are needed in which particle size and rate and depth of respiration are controlled and varied in a systematic fashion, preferably using lead concentrations in the general range to which the public is currently being exposed. -79- ......0 '* '* * " ' :* DUP050055368 Very little is know about the clearance of lead from the respiratory tract. Only one investigator has provided any useful information on the retention characteristics of lead in a form similar to that in general ambient air, hut it was at a concentration much higher than would he ordinarily encountered/ In the respiratory experiments cited earlier, Kehoe determined the excretion of lead in urine and feces before, during, and after termination of inhalation of small and larger particles of lead oxide ( average MMED, 0.25 pm; median MMED, 2.9 pm). During the long periods of inhalation, the excretion of lead of both size groups in urine increased. Only during inhalation of the larger particles, however, was the excretion of lead in feces increased. The increase was attributed to passage of inhaled load into the gastrointestinal tract by swallowing. It was estimated that k0% of the lead deposited in the airways was transferred to the gastro intestinal tract. In this case, 50% of the lead particles were greater than 2.9 pm in MMED. It was probably the particles in this range of diameter that were transferred to the gastrointestinal tract. Even assuming total retention the usual amount of input of lead via the airways is probably less than that via food and water. Given 37% deposition and even total retention, a lead concentration of 2.5 pg/m^ of air, and inhalation of 23 m- of air per day, a person would absorb 21 pg of lead per day originating in the air, compared with roughly 30 pg/day originating in food and water. In the specific case of the -80- DUP050055369 data cited in Table 3-2, the estimated concentration of atmospheric lead at that time and place was 2,0 ug/m . Adding the amount of lead absorbed from inhaled air would convert a net decrease of 1.35 mg observed during the first seven 8-week periods to an increase of 5*3 mg. Work is in progress oh a study that is mentioned because it may have important implications regarding the retention of lead inhaled by man at low concentrations. These studies should be viewed with the caution due preliminary findings (T. B. Griffin, j. C. Russell, L. GoXberg* F. Coulston, and J. Bradley j personal communication). Bats and rhesus monkeys (M, mulatta) have been exposed to continuous inhalation of lead sesquioxide particles at atmospheric concentration of lead 21.5 Mg/oT* for 1 year. Also, more than 20 human volunteers have been exposed 23 hr daily to inhalation of 10.9 + 3*1 (S.D.) Mg/m3 for up to 4 months. These studies may provide much needed information concerning the retention of lead inhaled by man at relatively low con centrations for a prolonged period. -81- DUP050055370 ACCUMULATION The extensive balance studies performed by Kehoe led him to conclude that in normal environmental circumstances man achieves fairly early in life and maintains a steady state with regard to lead wherein the rate of output approximately equals the rate of input .297 * 300 9 303 In support of this conclusion, he cited a limited study of the concentration of lead in the bones of people of various ages in which no age-related increase was apparent. More extensive studies have been conducted regarding the concentration of lead in human tissues. Some investigators report no marked variation with age; 1+37,55^ others report 262b, 49 5 substantial increases. ' In these studies, the concentration of lead in bone (expressed in terms of ash) increased up to the age of 40 and perhaps beyond. A similar age-related increase was noted in U95 the wall of the aorta; the possibility that the increase might be due to disease (e.g., calcification) was not ruled out. The rising concentrations of lead in bone and aorta were found in both U.S. acr4 foreign populations. Age-related increases were also found in some other organs of U.S. subjects but not of foreigners. There are two possible explanations for these increases. The first is that the older people were formerly exposed to higher concentrations of lead than they are today and have retained this lead in their dense -82- DUP050055371 bone. This is probably not true with regard to oral intake of lead in food, but it may be true of lead in drinking water. One can only speculate as to changes in respiratory exposure, because air sampling programs are too new to allow comparison of today 's air with the air of 30 years ago. The other possibility is that lead is sufficiently accumulative to result in increasing concentrations through middle life even at a constant rate of intake. Studies of the rate of loss from the body of single doses 59,372 of radioactive lead in rats, 51*3 rabbits, 1*2 and dogs have been reported. In all cases, the rate of loss conformed to power- function kinetics. That is, the fraction of the dose remaining in the body was less and less readily excreted with the passage of time, perhaps as the lead slowly moved into the dense bone and became metabolieally inaccessible. These observations were made on animals and were of relatively short duration, compared with the lifespan of man, but they are nonetheless compatible with the data that suggest some accumulation over man's lifespan. Accumulation of lead in the body is not necessarily paralleled by an increase in hazard. The concept of biologic availability must be con sidered. Elements whose accumulation is accentuated by progressively decreasing availability for excretion are bone-seekers. They are poorly excreted because they become relatively isolated from the blood as they move into dense bone matrix, which is a compartment having a very low turnover rate. In the case of radioactive bone-*seekers, such as strontium-90, burial In the matrix of bone does not result In biologic -83- DUP050055372 inactivation, because the emitted radiations influence biologic processes that occur well beyond the irmnediate atomic environment. In the case of stable elements, such as the species of lead (lead-207) that is the major source of environmental contamination, isolation in dense bone is equated with biologic inactivation. The accumulation of lead in the body with age may also involve some soft tissues, such as the wall of the aorta* The biologic availability of lead in soft tissues may similarly decrease with age. In evaluating the relative contributions of various environmental sources of lead to total input in the general population , dermal contact cannot be totally ignored. Some finite amount of lead is continuously coming into contact with the skin from air, water, and clothing. These contacts 89,5^7 are not considered significant, although critical experi ments of the type conducted to evaluate the significance of normal dietary intake have not been reported. Because the output of lead in urine and feces is so nearly accounted for by oral and respiratory input, dermal absorption is surely insignificant in usual conditions of lead exposure. Fat-soluble lead alkyl compounds, however, are readily absorbed from the skin and constitute an occupational hazard (see Chapter 6). Lead is lost in sweat, falling hair, and discarded and desquamated skin to some variable, imprecisely known degree* The concentration of lead in sweat approaches that in urine. 9 It has been observed that, during long-term constant intake of lead added to the diet, accumulation of lead in the body decreased in the summer months, but this was accounted for by increased losses in urine and,, perhaps also. * ^ -84- DUP050055373 301,302 feces. Thus, in the temperate zone at least, the foie of sweat in total lead balance seems insignificant. The influence of ambient temperature on lead metabolism has been studied to some degree because of the known seasonal variation in the incidence of lead poisoning in infants, the incidence being appreciably higher in 27,225 the summer months than during the rest of the year. The mechanism 28,29,81 has been investigated in rats and mice. The en hancement of toxicity by high ambient temperature was accompanied by considerable reductions in both fecal and urinary lead excretion and was favored by dehydration. The effect was most pronounced when exposure to high temperature was delayed until after the administration of lead. The finding in experimental animals that lead excretion decreased when ambient temperature was high does not agree with the observations in man mentioned earlier. The apparent difference between man and animals in this respect is not understood. DISTRIBUTION IN THE BODY In ordinary conditions (steady state or nearly steady state of lead retained by the general population), over 90# of the total amount of 495 lead in the body is in the skeleton. The concentration found in the tissues is variable, being highest in bone, intermediate in liver, kidney, and aorta, and low in muscle and brain. The concentration in most other tissues lies between those in kidney and muscle. 495 The data reported by Schroeder and Tipton indicate that the concentration of lead is greater in the tissues of U.S. citizens than -85- DUP050055374 in those of some foreign populations, particularly Africans and Swiss (Table .3-U'):. This, combined with the fact that the concentration of lead in most tissues appears from the limited data to increase with age among U.S. citizens but not among foreigners, was interpreted to mean that North Americans have reached a level of lead intake that exceeds their bodies1 capacity to maintain a steady state of input and output, whereas foreigners have notThat is an interesting speculation, but whether it ultimately proves correct or incorrect, it fails to consider the biologic or toxicologic significance of the data, a fact that the authors recognized. What is known about the relation of the concentration of lead in the body to its toxicity has been learned mainly by study of the relation of symptomatology and exposure to the concentration in biologic fluids, principally the blood, especially under the conditions of occupational exposure at known levels. Lead in human (and animal) blood is associated predominantly with the erythrocytes. In one recent analysis of the distribution of lead in blood, the concentration in erythrocytes gl was approximately 16 times greater than the concentration in serum. Analytic data are usually reported as micro grams per 100 ml whole blood or micrograms per 100 g whole blood.* Obviously, some serious errors of * Throughout this document blood lead content is expressed as per 100 g of whole blood, because it is a more accurate measure than by volume, which would be about 5$ higher. ,86- interpretation can result from comparing whole-blood concentrations in people with low hematocrit values to those in people with normal hema tocrit values (see Chapter 4), The manner in which lead is associated with erythrocytes is not well understood. There have been numerous studies involving the addition of lead in vitro. The validity of such studies is questionable, because lead added in vitro can be removed by dialysis with EDTA (ethylenediaminetetraacetic acid) for 24 hr, whereas lead originally in the cells is not affected. 132 This study and others indicate that lead normally associated with erythrocytes is only slowly exchangeable with lead in plasma. It is reasonable to suppose that the concentration of lead reflects the contemporaneous exchangeable material in the tissues perfused by blood. A similar rationale applies to the lead concentration in urine. Indeed, during periods of change in the rate of dietary lead intake, the changes in concentration of lead in blood and urine occur more or less proportionally, with blood exhibiting a somewhat lesser fluctuation than uri,ne 299 The concentration of lead in blood is considered more useful than that in urine, because blood is notsubjectto ^ content, as is urine; nor to the influence of changes in renal excretory capacity. However, it should be pointed out that no data exist concerning the correlation between the concentration of lead in blood and that in other tissues of the same persons. The difficulties of making such a study are compounded by the distribution of the body burden of lead in exchangeable and nonexchangeable pools. -87- DUP050055376 The concentration of lead in the blood of the general population has been studied extensively and found fairly stable* When the analyses are performed competently, the 90# range found in various population groups such as post office employees or city health department employees is of the order of two-fold. 539 The constancy of blood lead content is further evident in that it does not change significantly with age, ... 539 either among North Americans 262 c or among foreigners . Similarly, there is no increase in the concentration of lead in serum with age in 8l U.S. citizens. Although it is valuable (in terms of degrees of exposure) to know how much lower lead concentrations are in the blood of the general population than in the blood of persons who experience overt signs of lead toxicity, what one really wishes to know is the difference in lead intake between healthy and lead-poisoned people. That is what makes the relation between lead concentration in blood and lead exposure important. The relation between lead concentration in the blood of the general population and total lead input into the body per unit time is not known, and cannot be in the individual case because of the great variety of opportunities for exposure to lead in the modern environment. Although a few studies have been reported in individual experimental subjects in which the balance between dietary lead intake and excretion was determined, in no case was a determination also made of the actual concentration of lead being inhaled throughout the entire 2k hr of every day of the experimental period. Ideally > one would wish to know the amount of lead absorbed per unit time by persons whose blood lead -88- DUP050055377 concentrations are also known. Even a knowledge of the amount of lead inhaled and ingested and the contemporaneous concentration of lead in the blood would be useful as an index of lead absorption* ,/TTfai king what information is currently available! an analysis has been reported by Goldsmith and Kexter^^ of the relation between blood lead and estimated exposure to atmospheric lead. In the analysis,air sampling data and blood lead determinations that were not necessarily taken at the same time and place had to be used, A dose-response relation was suggested by a plot of lead concentration in blood against concentration in air. This relation was elaborated by the inclusion of data from experimental subjects exposed 206 to high, known concentrations of lead sesquioxide. The regression line for the data was calculated solely on the basis of the epidemiologic data, and the point was made that the experimental data only reinforce the validity of the relation (Fig. 3-2), The blood data used were those from men only, because the concentration of lead is known to be lower in women than in men living in similar environmental circumstances. 539 The validity of calculating a regression line from these data has been challenged on the basis of the variability of the absorption of lead from larger sources (e.g., food), but not the general proposition that the atmosphere is a 536 source of lead that can be absorbed, A signlficant regression, in any case, could not be said to exist for the data below the value of approxi- mately 2 iig/nr . Thus, the regression line cannot be applied with confi^ dence to the exposure conditions affecting the general population; this is so even for the general population of most large urban centers, inasmuch as average ambient air concentrations even in these centers do not generally o exceed 2 ug/m . -89- DUP050055378 The most extensive study of the relation between exposure to atmospheric lead and lead concentration in blood was conducted under the auspices of the U.S. Public Health Service from 1961 through 1963Several thousand atmospheric samples were collected and analyzed for lead, as well as samples of blood and urine from over 500 persons. The study was con ducted in the metropolitan areas of L&s Angeles> Philade1 phia, and Cincinnati, which were selected because their atmospheric lead concen trations were known from prior investigations. A general trend was noted toward an increase in the concentration of lead in the blood of the different groups as they varied from rural or suburban areas to the central cities in their place of residence or work (Table 3-5). It was inferred that atmospheric lead contributes to total blood lead, although differences in dietary lead intake among the groups might have contributed to the differences in blood concentrations. In a preliminary examination of data from a more recent study involving 1441 women, no relation could be detected between exposure to lead in the ambient air (0.17-3.39 Mg/m^) and blood lead (L. B. Tepper> personal communication). In the studies cited above, cigarette smokers have slightly higher blood lead concentrations than nonsmokers of similar residence and place of work. This can be attributed to the inhalation of additional lead from cigarettes, because each cigarette generates about 1-2 Mg of airborne lead 136 when burned. However, although the majority of workers have found consistently increased blood lead content among cigarette smokers. 351 others have failed to confirm this finding (M. McLaughlin and G. J. Stopps, personal communication). -90- DUP050055379 The purpose of the Goldsmith-Hexter regression line above (Fig, 3-2) was to adduce evidence concerning the contribution of atmospheric lead to the body burden of lead in the general population. The same approach has been used by Danielson in an effort to describe a dose-response relation between total daily lead assimilation and lead concentration in bloolvii- The author used the epidemiologic data of Goldsmith and Hexter for blood lead and for presumed atmospheric lead. He then assumed that all these groups were getting the same amount of lead, 350 ug/day, in their diets, and were inhaling 15 w? of air per day. To get from potential input to actual input, he assumed 10$ retention of dietary lead. Data for two experimental subjects receiving 1 and 2 mg of lead per day added to their diet were included. A regression line was then plotted, giving a dose-response relation. Although this approach seems sound, the assumptions are somewhat at variance with available information. The retention of lead in the lungs probably is considerably lower than the 50$ assumed in this particular report. Even if one assumes total retention of lead deposited in the respiratory tract, the data available suggest that lung deposition should be about 37$ for particles of the size usually encountered in ambient air, and not 50$. Another assumption that seems to have been in error was that an average man inhales 15 m^ of air per day. Current information suggests that 23 would be a better estimate for "reference man" engaged in light working activity (Table 3-1). Using epidemiologic data for lead concentrations in the blood of various groups of men whose exposure to atmospheric lead differed?**4 and data l -91- DUP050055380 for two experimental subjects whose dietary intake of lead was known and who were exposed to atmospheric lead estimated at 2 Mg/m3 (Subjects 299 E. B. and M, R., ) the relation between blood lead and daily lead intake can be estimated (Fig. 3-3). This is a revision of the dose-response relation discussed above, using the same sources of data on lead in inhaled air and blood, and assuming complete retention of the 30% or 31% deposited, from inhalation of 23 of air per day, and oral intake of 300 Mg of lead per day with 10Jif absorption. Some may find it difficult to accept the assumption that all the lead deposited in the airways is retained and absorbed. The only evidence available on this point was provided by Kehoe.^9 His study of the inhalation of lead sesquioxide suggested that all the particles deposited in the airways are retained if the particles are small (0.25 Mm MMED). On inhalation of such an aerosol for 22 weeks, 8 hr/day, the urinary excretion of lead doubled within 2 weeks and persisted at that level for the duration of the period of exposure. Essentially total freedom from the swallowing of lead moved upward from the lungs and their passages and total retention of the lead deposited in the respiratory tract is inferred from the absence of a corresponding increase in fecal excretion of lead. Retrograde movement of lead from the airways back into the nasopharynx therefore seems not to have occurred. By contrast, Kehoe's experiment with the larger lead particles (50% >2.9 um MMED), suggest that only particles exceeding 2.9 ym in MMED are subject to such retrograde movement. This is inferred from the estimate that approximately kO% of the total lead, by weight, in this experiment was swallowed after lung deposition. The rest was probably retained -92- DUP050055381 in the airways and eventually absorbed into the body. The tendency for particles to be deposited in the nasopharynx, as opposed to the lower airways, increases with particle size. Thus, if 50$ of the particles exceeded 2.9 ym in MMED and only 40$ of the lead, by weight, was trans ferred to the gastrointestinal tract, almost all the particles below 2.9 ym in MMED were probably retained in the lungs. Inasmuch as 90$ 469 of the lead particles in ambient air are smaller than 1.6 ym in MMED , it would seem from KehoeVs data that retention of deposited lead particles of the size found in ambient air is essentially complete. Even if one were to assume only 30% retention of the inhaled lead, the calculated total intake of lead over the range of 20-80 yg/100 g of blood would differ by less than 10$ from the amount calculated on the assumption of 37$ retention (Fig. 3-3). It is not the purpose of this chapter to discuss the biologic effects of lead. However, it will be established in Chapter 4 that biologic effects, including clinical lead poisoning, can be related to blood lead concentrations, if only in a general way* SUMMARY The diet is the major source of lead In man and probably In most animals. The lead content of most water supplies in the United States does not exceed 50 yg/liter, and general distribution samples have the same content. The daily intake of lead from this source for an adult is probably about 20 ug. -93- DUP050055382 The natural lead content of food has been estimated at 0.01 ppm, but some analyses show higher levels--up to 2.5 ppm, depending on the type of food. On the basis of the accepted average of 0.2 ppm, the dally adult intake, depending on factors determining the food eaten, is 100-500 )jg. The daily average oral intake of lead in this country is thus about 300 yg, and limited data indicate that this figure has not changed significantly during the last 30 years. Infants on a mixed diet are estimated to take in 130 yg/day, proportionally more than adults on a body-weight basis but not on the basis of metabolic rate or caloric requirement, The atmospheric contribution to the lead content of foods is probably not significant; near heavy sources, such as highways, lead contained in particulate matter is largely washed from plant parts consumed by man* Fallout probably contributes significantly to the intake of animals only under very unusual circumstances. The net absorption of lead from the gastrointestinal tract is 5-10# if the intake is not substantially increased. Under usual dietary con ditions, therefore, about 30 yg of lead are absorbed daily by man from food and beverages. Calcium and phosphorus intake seem not to alter absorption substantially; nothing is known of other dietary influences in man. Data on normal fecal and urinary excretion and on excretion after administration of lead-212 in man and experimental animals suggest that the fecal route is at least as important in lead loss as the urinary route. -94^ DUP050055383 The contribution of inhaled lead to the total daily assimilation of lead in man is not clearly known. Speculation as to its contribution is based on limited studies of lung deposition and on data concerning atmospheric lead concentrations in the general environment, rather than in the specific environment of persons determined continuously for periods of weeks or even days, Liang deposit ion is estimated to be approximately 37$ in man . The lung clearance of inhaled lead is largely unknown. What little information there is suggests that, ip. th$ ppi^icle size range found in ambient air, lead deposited in the respiratory tract undergoes very little if any oral transfer to the gastrointestinal tr:j|.g:t. For all practical purposes, all the lead deposited in the lung seems to be retained. On the basis of all the above considerations, it is to be expected that the atmospheric contribution of lead to the total daily assimilation by a standard man engaged in light activity would be from 0.8 pg to 6.3 pg, depending on where he lives and works. Epidemiologic data gathered to date have not provided convincing evidence that inhaled lead contributes to the concentration of lead o in the blood at atmospheric concentrations below 2-3 pg/nr, There is some evidence of a contribution when the atmospheric concentration " of lead is higher. The exposure of people in the general population to lead results in some accumulation in the body up to and perhaps beyond the age of 40 , as determined by analysis of tissues. There is no apparent concurrent increase in lead circulating in the blood. The biologic significance and reason for the increase are not known. -95- DUP050055384 Activity a level TABLE 3-1 Potential Dally Intake of Lead by inhalation Air 3 Inhaled> m Potential lead intake* ue @ 0.1 ug/m^ @ 0.5 @ 2.5 tig/ra^ 8 hr working ("light activity11') 9,6 8 hr nonoccupational activity 9.6 8 hr resting 3.6 Total 22.8 0.96 0,96 0,36 2.28 4.8 4.8 1.8 11.4 24.0 24,0 9.0 57.0 j <* - q Of a "standard man" weighing TO kg, between 20-30 years of age, 175 em tall and 1.8 sq. m. surface area* A -96- DUP050055385 TABLE 3-2 Lead Ingestion and Excretion of a Normal Human Subject 8-week periods 1st 2nd 3rd 4th 5 th 6th 7th Subtotal 8 th 9 th Subtotal Total Lead ingested, mg Lead excreted, mg total In feces In urine Net change in body lead 13.59 13.31 13.16 11.51 9.30 9.24 12.75 82.86 22.21 18.17 40.38 123.24 11.95 10.45 15.13 13.63 13.82 12.45 11.59 10.41 8.93 7.88 9.05 8.16 13.74 12.86 84.21 75.84 17.61 15.85 15.05 13.76 32.66 29,61 116.87 105.45 1.50 1.50 1.37 1.18 1.05 0.89 0.88 8.37 1.76 1.29 3.05 11.42 +1.64 -1.82 -0.66 -0.08 +0.37 +0.19 -0.99 -1.35 +4.60 +3.12 +7.72 +6.37 a Adapted from Kehoe 299 -97- DUP050055386 TABLE 3-3 g Deposition of Lead Inhaled by Man Respirations/min Tidal air* cc Particle diameter* til Particle. deposition* % i 0,6 0,4 10 1350 0,2 0.1 0.08 0,05 63.2 59.0 50.9 48.1 39.3 40.0 42.5 1 0.6 0.4 30 450 0.2 0,1 0.08 0.05 35.5 33.5 33.0 29,9 27,9 26.5 21.0 1+36 aAdapted from Nozaki M * -98- DUP050055387 Lead in A d u lt Human T is s u e s A c c o rd in g to G e o g ra p h ic A rea o f sam ples in w hich le a d was fo u n d ; i f no fig u r e is g iv e n , le a d was found in a l l sam ples. 2| O CM CM I S| *1 o l\ 1 00 ON csi CM o CO I o o V a4 vp O Sf C<NM in & NO 0o x o 00 to x> o *o a> co 14 4J to o 4J W cO JO 4J <0 0) e <u p V) <0 u t>N 0) CD M C0 cn CD CD r-t 4J 0> C O 60 u U H 0> CD *0 PU .O cO 6 tJ : O cO H MQ) 4- cO 4CJO m h . i o t>-4 Tr3t ctf to (D cfl cl) to H u . O o <! 53 PM P W H C t-3 pm H 33 PQ CO PQ H co 43 O T3 -99- J DUP050055388 TABLE 3-5 Lead Concentration in Blood of Selected Groups of Males d Mean lead concentration in blood, No. me/100 eb subiects GrouD 0.011 9 Suburban nonsmokers, Philadelphia 0.012 16 Residents of rural California county 0.013 10 Commuter nonsmokers, Philadelphia 0.015 14 Suburban smokers, Philadelphia 0.019 88 City employees, Pasadena 0.021 33 Commuter smokers, Philadelphia 0.021 36 City health department employees, Cincinnati 0.021 155 Policemen, Los Angeles 0.022 11 Live and work downtown, nonsmokers, Philadelphia 0.023 0.024 140 Postoffice employees, Cincinnati / 30 Policemen, nonsmokers, Philadelphia^ 0.025 191 Firemen, Cincinnati 0.025 123 Policemen, Cincinnati 0.025 55 Live and work downtown smokers, Philadelphia 0.026 83 Policemen, smokers, Philadelphia ^ 0.027 86 Refinery handlers of gasoline, Cincinnati (1956) 0.028 130 Service station attendants, Cincinnati (1956) 0.030 40 Traffic polie emen, Cincinna ti 0.030 60 Tunnel employees, Boston 0.031 17 Traffic policemen, Cincinnati (1956) 0.031 14 Drivers of cars, Cincinnati -100- DUP050055389 TABLE 3-5 (continued) Mean lead concentration in blood, mg/100 gb 0.033 0.034 No. subjects 45 48 1434 Group_____________ ., . Drivers of cars* Cincinnati (1956) Parking lot attendants* Cincinnati (1956) Total 1Adapted from Three Cities Study. 539 ^Values are those determined for the 1965 study, except where otherwise indicated. -101- DUP050055390 FIGURE 3~1. Retention of lead in four human subjects at daily doses 299 of 0.3, 1, 2, and 3 mg Pb. Modified from Kehoe. -102- DUP050055391 MEAN BLOOD LEAD (jttg/IOOjg) FIGURE 3-2, Mean blood lead concentration for epidemiologic and experimental respiratory exposures with regression from epidemiologic data only . Redrawn from Goldsmith.20^ -103- DUP050055392 30 50 100 200 tig Pb assimilated/day 500 FIGURE 3-3. Relation between total absorption of lead and concentration 3 of lead in blood. Calculations based on assumptions of 23 m air inhaled per day, absorption of 10# of 300 yg Pb/day oral intake, and retention of 37# (solid line) or 30# (broken line) of inhaled lead, See Appendix C for original data (solid line) and calculations. -104- DUP050055393 3 V ~ / wpttVv.v CHAPTER k BIOLOGIC EFFECTS OF LEAD IN MAN Knowledge of the 'biologic effects of lead in man is based largely on the 3tudy of persons with overt clinical lead poisoning and on experience in the medical supervision of workers industrially exposed to lead. Exposure to lead in these groups is considerably greater than exposure likely to be encountered by the general population , whose intake of lead is derived from normal food sources and the ambient air. Similarly, most of the available experimental data on intact animals are based on studies in which high doses of lead have been administered. Although the effects of high doses of lead in man and experimental animals do not necessarily permit precise estimates of possible biologic effects of lower doses, they do point to areas of metabolism and function in man in which further study of the doseresponse relationships at the subclinical level is appropriate. Types of exposure associated with clinical lead poisoning are also important, because they represent clear and present risks to some segments of the population. Because the total amount of lead ab sorbed by a person may be derived from several sources, airborne lead must be considered in relation to other sources to which various population groups may be exposed. ETIOLOGY OF LEAD POISONING IN MAH Uncontrolled industrial exposure to airborne lead, the eating of leadpigment residential paints by children with pica, and the drinking of illicitly distilled lead-contaminated whiskey are responsible for most -105- DUP050055394 of the cases of overt clinical illness due to lead poisoning today* What information is available concerning dose-response relationships in man is derived largely from the study of groups poisoned in those ways. The burning of discarded battery casings in the home and con tamination of food and drink by improperly lead-glazed earthenware vessels have also been responsible for severe illness from time to time. Industrial Exposure to Lead and its Compounds A brief perusal of Table 1-3 should indicate which population in the United States stands at greatest risk of suffering untoward effects engendered by lead and its compounds* Review of those data indicates that metal products and miscellaneous and unclassified uses account for *J2% of the lead consumed in the United States. Consideration of each of the products under these rubrics in terms of the biologic availability of the lead therein makes it apparent that as finished products they do not, in normal use, pose threats to human health. However, because fabrication may include heating, grinding, dissolu tion and volatilization, spraying, or other manipulations productive of biologically available forms of lead, workers engaged in each type of manufacture shown in Table 1-3 represent industrial populations that are at risk of lead absorption. By contrast, normal use of the products poses essentially no risk to the general population of users. The ubiquity of lead--in contrast with other non ferrous metals--in industry is indicated by a U* S. Public Health Service list of 113 potential occupational exposures^^attended by a risk of lead poisoning. '4 -106- DUP050055395 A reliable definition of the extent of the risk of occupational lead exposure is unavailable. In contrast with numerous other technologically advanced nations, the United States has no data on the prevalence of occupational ,lead poisoning. Except for a few of the states--whose reporting systems are of questionable adequacy-no data of this nature are available. It is hoped that the.Occupa tional Safety and Health Act of 1970 will lead to the accumulation of such data. Estimates of the magnitude of the industrial health problem posed by lead exposure are fragmentary. H. E. Stokinger (personal communi cation) in a canvas of the persons in public agencies largely responsi ble for control of industrial lead exposure, attempted to collate their estimates of the trends in reported cases. Evidence in this survey based on lead measurements in air and urine indicated that "lead exposures had decreased by a factor of several magnitudes in ... 7 or 8 major categories representing the major uses of lead since 193^." On the basis of hitherto unassembled data, it seems that this decrease had "continued but at a slower rate since 19*+5 * t! Such impressions require some amplification. These reports repre sented mostly workers employed in relatively large corporations; large employers are more accessible to regulatory agencies than small shops. Furthermore, large corporations often provide lead-exposure control and medical surveillance programs, because they are economically better able to afford such environmental and medical controls . The potential magnitude of the problem of occupational lead poiosning is -107- DUP050055396 not clarified if one considers that approximately two-thirds of all workers in the United States work for employers of 100 persons or fewer. There are also large groups of peripatetic workers * for. example, in construction, for whom few data are available. Stokinger, in 86 surveys of 26 different operations, found indicap tions of excessive exposure to lead, as judged hy blood analysis , over a 4-year period in 10-35$ of exposed workers (Table 4-1), It should be emphasized that these data do not represent systematic sampling; often, they represent only the blood samples representative of persons thought to be suffering from lead poisoning, and many workers possibly suffering from lead absorption were not sampled. The manpower resources of the few effective official agencies are insufficient to permit representative sampling, and blood samples were sought from employers (usually involving large corporations) who were knowledgeable as to the risks of lead exposure. To place occupational exposures in their proper perspective as regards the general population risk, it is appropriate to consider the atmos pheric concentrations associated with clinical occupationally induced lead poisoning. Data generated over many years indicate that cases of overt lead poisoning usually are associated with air concentrations of over 0.5 mg/ur^ A blood concentration of 80 yg/100 g of blood may produce symptoms and signs of lead poisoning in an exposed worker and a concentration of 100 yg/100 g of blood will usually produce clinical manifestations: However , some investigators200 5oi* have found 50-80 yg/100 g of whole blood lead content associated with mild lead poisoning symptoms. -108- DUP050055397 The magnitude of the differences between workers 1 exposure to atmos pheric lead (and their blood lead concentrations) and community ex posures (and the general population's blood lead concentrations) should place into proper perspective the risks of the occupationally exposed. These differences, coupled with a large potential working population at risk, should indicate the relative gravity of the occupational lead problem, compared with the lead problem of any other population group. It is reasonable to assume that any ill-defined potential health risks associated with long-term lead exposure that the general population may carry are also present among the large group occupationally exposed to lead. Thus, the occupationally exposed carry both the risk of overexposure that is usually minimal in the general population and a relatively higher risk of whatever leadrelated harm may befall the general population as a result of community exposure. Lead-Based Paints on Housing Surfaces The clear association between lead poisoning in children with pica and old deteriorating and dilapidated urban housing in the United 192,225,270,272,36^,407,530,592 States, England, and Australia is well-documented. The source of the lead is lead-pigment paints in one or more of the layers of paint applied to the woodwork and plastered and papered walls of dwellings before World War II, when lead pigments were common constituents of both interior and exterior house paint. In such paint, oji.O lead may constitute 5-1+0$ of the final dried solids. ""These layers persist in flaking multilayer paint chips, crumbling plaster, and cracking wall paper in old substandard housing that is still in use. -109- DUP050055398 Within the dwellings , window sills and frames are apparently the most common locations"^^nibbled by young children. According to the I960 U. S. Housing Census, 30.6 million dwelling units occupied in.i960 363 were built before 19^0. Of these, 5.6 million were classified in i960 as deteriorating and 1,8 million as dilapidated. (Comparable data from the 1970 U. S. Census will not be available until after January 1972. ) In Baltimore, 90% of the reported cases of lead poisoning are in children who live in multiple-dwelling, rented housing units. Chisolm and Harrison^*^reported that old paint containing more than 1% lead (by weight) could be found on at least one of the surfaces of the home accessible to a young child in almost every case of poisoning. In 102 of 105 cases, at least one source con taining more than 5% lead was found at the child's residence. In a survey of 100 randomly selected blocks of dwellings in Baltimore, lead in excess of 1% in paint was found on interior surfaces in 70$ of 667 dwelling units, Similar surveys of old housing in Phi lade lphia*' and London'3 have likewise revealed that 70-80# of interior painted surfaces contained more than 1% lead. In these three surveys, only a few samples from each dwelling unit were analyzed. A limited survey in Baltimore''^indicated a 98# probability of houses with positive findings for lead if a greater number of samples per dwelling unit had been analyzed. In the investigation of actual cases, the analysis of 20-25 different samples is usually required to identify all the interior surfaces that are both positive for lead (more than 1# lead) and accessible to young children. ` Geographic * -110- DUP050055399 spot maps have been kept by the Baltimore City Health Department during the last 30 years. They indicate that, as old Inner-city dwellings are replaced by urban renewal and other modern constructionthe loca tion of reported cases moves outward from the center of the city and that cases are now found in older housing in nearly all parts of the city (E. W. Dahle, Jr. , personal communication). In 1955, the American Standards Association developed a standard specifying that, paints for toys, furniture, and the interior of dwellings should not con tain "harmful quantities" of lead. The standard, now known as ANSI Standard Z66.1T limits the lead content to less than 1% lead, in the final dried solids of fresh paint. This excludes lead pigments (usually white lead or lead carbonate and lead sulfate in oil-based paints), but it does not necessarily eliminate other lead additives in the total paint formulation. MSI Standard Z66.1 also limits the concentrations of antimony, arsenic, cadmium, mercury, selenium, and barium in paints conforming to the standard. This standard was developed on the basis of a number of factors, including the observa tion that clinical cases of poisoning were associated with sources containing more than 1% lead in paint and the use of semi quantitative gravimetric methods of analysis at the time for testing paint samples.287 Newer methods of detection based on the principle of x-ray fluorescence may call for revision of this 1% w/w standard, inasmuch as the result obtained by x-ray fluorescence analysis is expressed as weight of lead per unit of exposed wall surface (mg/cm^).^^ Several prototype portable nondestructive detectors for in situ detection of lead in housing surfaces are now available. Their use can simplify and -111- DUP050055400 greatly speed detection. Because the detectors measure the amount of lead in 10 or more layers of paint, they provide a more useful value in terms of the dose of lead contained in multilayered flakes of paint of various thicknesses. For example, 10 layers of paint containing 1# lead would contain 10 times as much lead per unit area as one layer containing 1% lead, although both the 10-layer and the one-layer paint flake would give a concentration of 1# by traditional gravimetric analysis. One report111^showed four paint fragments with a surface A of approximately 5 cnr weighing 2.68 g and containing 9 5# lead, or 254 mg of lead; had these fragments contained 0.95# lead, their removal would not have been required under the 1% gravimetric standard, although they would have contained 25 mg of lead, (The 1# gravimetric standard is the basis of all currently applicable municipal ordinances.) The amounts of lead found in the feces of children with lead poisoning11^(range, 0.37-225 mg/24 hr) correspond to the amounts of lead found in multilayer paint chips with an exposed surface area of 1-5 cm2 . Before 1940, lead pigments were widely used in both interior and exterior paints. Beginning about 1940, titanium dioxide, a less expensive white pigment, began to replace lead pigments, especially white lead, in interior paints. Even so, in Baltimore, for example, lead-pigment paints could still be bought for interior use until 1958, when a municipal ordinance designed to prohibit their sale was 407 passed. " , As of 1970, approximately 50# of exterior oil-based paints contain some lead in their pigments; the other 50# are latex -112- paints and do not contain lead, except for small amounts that might be used for tinting purposes, according to information supplied by the National Paint, Varnish and Lacquer Association. The laws and ordinances pertaining to the labeling, use, and removal of lead-based paints extant in October 1970 are summarized in Table 4-2. At that' time, four states had laws pertaining to one or two of these aspects of the problem, and 10 municipalities had ordinances prohibiting the use of paints containing more than 1% lead in interior paints and requiring the removal by scraping or other means or covering of interior surfaces painted with high-lead paints accessible to children in dwelling units occupied by children with blood lead levels greater than 60 ug/100 g of whole blood. In some cities, enforcement has been difficult and far from satisfactory, especially where enforcement has been under criminal codes.3 63 In Baltimore and Philadelphia, enforcement is carried out as a civil proceeding under the Hygiene of Housing Code, and the fine for noncompliance is cumulative for each successive day of noncompliance after a grace period of approximately 4-6 weeks. Also, surfaces must be inspected after removal of the old paint before repainting is allowed. In New York City, under the 1970 ordinance, the city will itself remove old lead-based paint and recover the cost through a lien on the property, if the owner fails to do so after proper notification. A number of the municipal ordinances (Table 4-2) require removal only from the residence in which the child with high blood lead concentration lives and from public access passages; other apartments and areas in multiple unit buildings are not affected.* *R. A. Roland, letter dated October l6, 1970. -113- DUP050055402 The Lead-Based Paint Poisoning Prevention Act (Public Law 91^695), signed into law on 14 January 1971 was to assist local governments in coping with the problem of lead poisoning in old, poorly maintained egg* houses. The paint Industry has complied with labeling provisions under the Federal Hazardous Substances (Labeling) Act (i960) and even earlier , under ANSI Standard Z66.1. The hazard presented by lead- based paint Is related both to the lead content of the paint and to poor maintenance of painted surfaces It is easy for a child to pick and eat loose flakes from chipped and deteriorating painted surfaces. The lead content of such paint flakes is far In excess of the amount of lead likely to be inhaled, even in congested areas, so it seems clear that the direct ingestion of lead-containing paint chips is the principal environmental cause in clinical cases of lead poisoning in children. The question of the lead content of dusts as they may contribute to children's total oral intake of lead is considered later in this chapter. Improperly Lead-Glazed Earthenware Vessels Acidic foods and beverages^--including tomatoes, tomato Juice, most fruits and fruit juices, cola drinks, such alcoholic beverages as wine and cider, and pickles and relishes stored in vinegar or cidercan dissolve the lead in improperly lead-glazed earthenware (pottery) containers. Foods and beverages so contaminated have been responsible 31) 2kC for fatal and nonfatal cases of human lead poisoning. The many factors that influence the amount of lead that can be leached into food or beverage include the ratio of lead oxide * Congress has appropriated $7.5 million for FY 1972. 114 DUP050055403 to silica in the formulation of the frit, the presence of pigments and other constituents in the frit, the temperature at which the glaze is fired, the acidity and temperature of food or beverage stored in the vessel, and the' length of time the acidic food or 256,310 beverage remains in contact with the improperly glazed vessel. Extreme care is required to formulate and fuse a safe glaze on earthenware vessels . Of particular importance is the low temperature used for firing (less than 1150 C) earthenware pottery glazes . Safe conditions are not easily ensured by amateur potters. But stoneware is fired at 1200-1260 C and presents no hazard because the residual lead, after firing, is fused completely as an insoluble lead silicate glaze. In one case, a man was found to be consuming 3.2 mg of lead per day in a chilled cola drink, which he drank nightly from a home-handicrafted 246 earthenware mug. In another case, apple Juice stored in an earthen ware pitcher was found to contain 157 mg/liter if stored in the pitcher , 310 310 for 3 hr and 1300 mg/liter if stored for 3 days. Klein et_ al. tested 117 earthenware vessels purchased in Montreal, Canada, over the counter through retail outlets. Approximately 50% of the items tested had a "lead release" considered unsafe by current standards. These and other data are summarized in Table 4-3. The Canadian Department of Consumer and Corporate Affairs has confirmed these findings, especially in earthenware produced by studios and hobbyists and in imported items. Re-evaluation of current regulations, practices, and control measures are now under consideration in Canada and the -115- DUP050055404 United States to reduce this potential hazard to the general public. It is highly unlikely that hobbyists will ever be able to produce earthenware items that are consistently safe for use as food and beverage containers . Visual inspection of an item does not permit a purchaser to distinguish between glazed earthenware (which may not be safe) and stoneware (which is safe). ^ piscarded Battery Casings Discarded automobile battery casings made of wood^^and vulcanite20"1* have been used as fuel in wood-burning cook stoves and fireplaces during times of extreme economic distress among the poor. Fatal and nonfatal eases of lead poisoning, chiefly among young children, have been traced to this source. Such outbreaks have been controlled by halting the distribution of the casings to the poor and by institu- *,, 593,201 tion of measures for the safe disposal of the casings. Whether lead poisoning in these outbreaks resulted from inhalation of lead fume or dust or from ingestion of lead dust is not entirely clear; 201 however, Gillet thought that ingestion was the major route of absorption. He noted that the maximal temperature obtainable in domestic cookstoves in use in Rotherham (1,000 C) was not sufficient to volatilize lead and that almost all the cases of poisoning occurred in children of preschool age, the age group in which pica is most prevalent. Inhalation of lead dust by young children or contamination of food in these outbreaks might, however, result in a higher dose of lead per unit of body weight, compared with that to which adults -116- DUP050055405 were subjected, "because of children's higher metabolism per unit body weight. * The problem of the actual route of entry into the body (lung or gastrointestinal tract) remains unsolved in these battery^ casing outbreaks; but in either case, the dose to the child would be higher. The burning of battery casings is the only nonindustrial type of potential respiratory exposure to lead that has been associated with actual human cases of lead poisoning. Illicitly Pistilled Whiskey Lead is a common contaminant of the illicitly distilled whiskey (moonshine) manufactured in the United States. The reason is the method of construction of the stills. Automobile radiators are frequently used as condensers, with the other components connected by lead soldering. Thus, hot vapors and the liquor come into con tact with metallic lead. Although the forms in which lead is present in the liquor have not been identified, it has been suggested that acetic acid in the distillate may react with lead to form lead Studies in respiratory physiology^^ indicate that oxygen consumption requires an intake of 5-6 m3 of air per 1000 cal metabolized. A 1-year-old child weighing 10 kg would metabolize 1000 cal per day on .3 the average and inhale 5-6 m of air . In conditions of comparable environmental exposure, a child would inhale two to three times as much of a given pollutant as would an adult per unit of body weight. The younger the infant, the higher the metabolic rate and the greater the difference in dose between adult and child on the basis of body weight. -117- DUP050055406 acetate. 32 In any case, the concentration of lead in moonshine whiskey may exceed 1 mg/liter in 30% of samples (Table 4-4). In contrast to moonshine, concentrations of lead in 20 brands of alcoholic beverages assayed by the Laboratory Services Branch of EPA at Cincinnati, Ohio, were found not to exceed 0.4 mg/liter; 12 of the brands contained less than 0.2 mg/liter. y 7G 1 The use of moonshine whiskey is widespread. The map (Fig. 4-1) indicates the major areas of manufacture and distribution. Although the amounts produced are unknown, it was estimated in 1968 that more than 36^ million gallons were produced. 409 The incidence of clinical poisoning by lead from moonshine is unknown; as is the case with many unusual illnesses, the frequency of diagnosis is directly related to physician awareness of the problem. Miscellaneous Sources Sporadic cases of classic human lead poisoning have been traced to lead-painted children*s toys and furniture, lead toys and baubles eaten by children, lead nipple shields, home battery manufacture, artists* paint pigments (used In hand mixing), lead dust in shooting galleries (with the attendant at risk), soluble lead compounds con veyed in lead pipes, ashes and fumes of painted wood, certain patented ''medications," jewelers* wastes, and lead type in schools for the blind. Lead has been used In cosmetic products through the years; there is no information with respect to whether it presents a significant hazard to health. -118- DUP050055407 CLINICAL EFFECTS Lead poisoning can give rise to several well-known but nonspecific clinical syndromes' of illness in man, including anemia and the* syndromes of acute abdominal-colic, acute encephalopathy, chronic encephalopathy, peripheral neuropathy, and chronic or late lead nephropathy with or without secondary gout. Under conditions of prolonged or recurrent uncontrolled excessive exposure, the clinical picture in a given person is of recurrent episodes of acute lead poisoning on a background of slowly progressive renal insufficiency 23,84,174,4o4,542 and cerebral incompetence. Each of the syndromes of acute lead poisoning may abate spontaneously on cessation of the hazardous exposure. A single episode of acute encephalopathy in a young child may be followed by brain damage varying in severity from subtle learning deficits to profound mental deficiency. Clinical descriptions of lead poisoning may be found in the writings of Tanquerel and Aub et_ al.,3 in modern texts ,no * 4o4 and in various reviews 15 84,174 It has long been thought that young children might be more susceptible than adults to the toxic effects of lead* Certainly, the clinical manifestations of a variety of diseases are more devastating in a very young child than in a mature adult* The observed differences in responses between children and adults may be attributed to children1s rapid growth, which limits their capacity to respond to adverse agents. Factors associated with rapid growth may well limit a child's capacity to compensate for the absorption of increased amounts -119- DUP050055408 of lead, but this hypothesis has not been documented at the clinical level. The early symptoms of lead poisoning are subtle, subjective, and nonspecific and therefore not so readily recognized in children. Less severe signs and symptoms, of lead poisoning, such as anemia and acute abdominal colic, are the clinical manifestations most often recognized in adults. In children, such mild symptoms are often either overlooked or attributed to other disease states, so that poisoning due to lead is more likely to be recognized first at a late or severe stage on the basis of nervous-system involvement (acute encephalopathy). Most adult lead poisoning is associated with occupational exposure, which is usually less severe than the types of exposure associated with childhood lead poisoning. The imbibers of lead-contaminated moonshine whiskey, who may be subject to very high doses, as are children, are usually first recognized clinically as having acute encephalopathy or late lead nephropathy. When the factor of dose is taken into account, the clinical response-- particularly at high doses--appears to be comparable in children and adults. Clinical responses to moderate increases in soft-tissue lead levels are ill-defined, particularly in very young children. Never theless, a rapidly growing child's response to moderately increased levels of lead may well differ from that of the adult, despite the current inability to perceive the response. Anemia Lead poisoning gives rise to a mild hypochromic and sometimes microcytic anemia. The anemia is also associated with shortened red-cell life span, -120- DUP050055409 reticulocytosis, and the presence of basophilic stippled cells in the peri.p,heranl blood,, . 45,46,224,253,2769352,5?5,580 _The symptoms of this anemia include pallor, waxy sallow complexion, easy fatigability, irritability, and headache. In young children unable to describe such symptoms, anemia may give rise to decreased play activity,, and irritability may masquerade as a "behavior disturbance." Anemia due to lead poisoning has many morphologic features in common with the anemias of iron deficiency and thalassemia; hence, these three con ditions are not always easily distinguished. Basophilic stipple cell counts may be influenced by the method of preparation of the smear46 and by the presence or absence of anticoagulants. Bone marrow preparations in lead-poisoning anemia consistently show greatly increased numbers of sideroblasts > which distinguish the anemia due to lead poisoning from that due to iron deficiency states. In the absence of iron deficiency, the iron content in serum and bone marrow may be increased in lead poisoning. The clinical symptoms of anemia due to lead are indistinguishable from those of chronic anemias with a variety of other causes. Anemia due to lead is often seen in associac tion with acute abdominal colic. Acute Abdominal Colic In adults, acute abdominal colic due to lead poisoning is often preceded by headache and may be associated with generalized muscle aches. These are followed by constipation and, within a few days , attacks of crampy diffuse abdominal pain. When pain and constipation to the point of obstipation are severe, there may be vomiting, anorexia -121- DUP050055410 with associated weight loss and easy fatigability, and a complaint of a bad taste in the mouth. This sequence of events usually develops over a period of 1-2 weeks. 277 In patients without obvious exposure to lead, this syndrome may resTilt in inappropriate laporotomy. In very young persons, acute abdominal colic due to lead poisoning finds its clinical expression in the form of anorexia, apathy, irritability, refusal to play, pugnaciousness, episodic vomiting, and constipation. These symptoms may abate spontaneously on cessa tion of the abnormal exposure to lead. Acute Encephalopathy Encephalopathy is the most severe acute clinical foim of lead poisoning. It may arise precipitously with the onset of intractable seizures, lapse into coma, and cardiorespiratory arrest. Prodromal manifesta tions may or may not occur. In fatal cases, death usually occurs within 48 hr of the initial seizure, unless life is supported by artificial cardiopulmonary devices. This brief catastrophic sequence may also occur on a background of anemia and mild colic in patients who are tinder observation but are not thought to be very ill until the initial seizure. The fulminant form of encephalopathy usually develops within a week. Vomiting, which may have been sporadic previously, becomes increasingly frequent, persistent, and forceful. Apathy progresses to drowsiness and stupor, interspersed with lucidity or hyperirritability. In severe eases, these alterations in the state -122- DUP050055411 of consciousness progress rapidly to coma and convulsions during the final 48 hr. 15 > 110 About 20% of patients have a history of recent onset of clumsiness and show frank ataxia on examination. Other neurologic manifestations * such as paralysis and weakness, are usually found post!ctally. The prodroma of acute encephalopathy due to lead poisoning also include subtle changes in mental attitude, sluggishness, poor memory , inability to concentrate , restlessness, and hyperirrita-^ bility. Adults may report mental depression, persistent headache, 23 vertigo, and tremor shortly before the onset of convulsions . Both the onset and the clinical course of acute encephalopathy are unpredictable. Symptoms may abate at any point in the sequence if the patient is removed from gross exposure to lead. 106,111 Two reports indicate that one-third to one-half of children with acute encephalopathy due to lead poisoning may also manifest acute renal injury in the form of the Fanconi syndrome (generalized renal hyperaminoaciduria, glycosuria, and hyperphosphaturia in the presence of hypophosphatemia). In addition, patients with acute encephalopathy may also be oliguric. Pathologically, the most extensive neuronal injury is in the cerebellum, althou^i there is diffuse injury to nerve cells throughout the brain. Vascular injury is the basic lesion responsible for diffuse severe cerebral edema and the pressure effects of acute encephalopathy. Factors that increase the risk of death include diagnostic lumbar puncture and diagnostic and therapeutic neurosurgical procedures during the acute phase. These and other im portant aspects of clinical diagnosis and management are discussed elsewhere.1^'111 -123- DUP050055412 The division of encephalopathy into "severe" and "mild" groups is mainly of prognostic significance in terms of survival and the severity of neurologic sequelae. Patients whose condition is .classi fied clinically as "severe" are those with either intractable seizures or coma or both for 2b hr or longer. Patients whose condition is classified clinically as "mild" encephalopathy include all those with the above features lasting less than 24 hr and those with less severe impairment of consciousness. Ataxic patients without impaired consciousness are sometimes included in groups classified as having 4s6 "mild" encephalopathy and sometimes grouped separately. Acute lead encephalopathy today is associated with the following types of exposure : Ingestion of lead-pigment paint chips by children with pica, burning of discarded battery casings in homes for fuel, con tamination of beverages by improperly lead-glazed earthenware vessels, and lead-containing illicitly distilled whiskey. Chronic Encephalopathy Exposure to lead occasionally produces clear-cut progressive mental deterioration in children. They are usually over 3 years old. Their clinical history indicates normal development during the first 12-18 months of life or longer, followed by a steady loss of motor skills and loss of speech to the point of mutism. These children may also have severe hyperkinetic and aggressive behavior disorders and a poorly controlled convulsive disorder. There is often evidence of current excessive absorption of lead. Blood lead concentrations are in excess of 60 pg/lQO g of whole blood, and x-rays may show heavy multiple bands -12 k- DUP050055413 of increased density at the metaphyses of the growing long bones. These findings in older preschool children indicate prolonged increased absorption of lead. This clinical entity has been termed "chronic encephalopathyIts etiology is not at all clear. It represents the final clinical expression- of diffuse cerebral injury incurred during infancy and early childhood and may be due to a number of traumatic9 toxic, viral, and bacterial agents. Such children also often have persistent pica, so that the evidence of current increased absorption of lead may be the result of pica superimposed on prior cerebral injury of a different cause. The diagnosis of acute lead encephalopathy can easily be missed repeatedly, and so this syndrome may also represent the sequel of prior episodes of acute lead encephalopathy and much higher blood lead concentrations in the past. The picture is also indistinguishable from the pattern seen after recurrent documented episodes of acute lead poisoning with or without acute encephalopathy, as described by Byers and Lord0 and Perlstein and Attala. 1*56 The older medical literature2*3 9 koh contains references to a similar picture of progressive premature dementia in adults in association with prolonged, grossly excessive occupational, exposure to lead and a history of recurrent episodes of acute symptomatic lead poisoning. Fortunately, this is rare today, although it may conceivably occur In imbibers of lead-containing whiskey. Pe ripheral Neuropathy The distinguishing clinical feature of the peripheral neuropathy of lead poisoning is predominance of motor involvement with minimal or -125- DUP050055414 absent sensory abnormalities . There is a tendency for the extensor muscles of the hands and feet to be involved. Three clinical forms are noted. In the firsts patients with acute abdominal colic may also complain of very severe pain and tenderness in the trunk muscles , as well as pain in the muscles of the extremity. As .the pain and tenderness subside , weakness may emerge, with very slow recovery over the ensuing several months. No epidemiologic data based on careful long-term follow-up are available to assess the frequency of this syndrome as a component of classic lead colic. In the other, more common form of peripheral neuropathy due to lead poisoning, the neuropathy is described as a painless peripheral extensor weakness occurring either after termination of excessive expos tire or after long, lib moderately increased exposure, Eramerson has described patients with pes cavus deformities resulting from old peripheral neuropathy. This suggests that neuropathy of sufficient severity may cause irreversible impairment of peripheral nerve function. The recent report of Catton et al con*cerning subclinical peripheral neuropathy in the absence of obvious clinical manifestations of lead poisoning suggests that the entire question of peripheral neuropathy needs re-evaluation. In the third fora, neuropathic and myopathic features are almost indistinguishable. When muscle fascieola tions and proximal girdle atrophy are present the findings resemble those 518 of amyotrophic lateral sclerosis. * Late or Chronic Lead Nephropathy The nephropathy seen in patients with a history of one or more episodes of acute lead intoxication is characterized by progressive and apparently irreversible renal insufficiency. Under the light microscope, renal biopsies and postmortem specimens show nonspecific interstitial fibrosis, tubular degeneration, and glomerular and vascular changes -126- DUP050055415 in small arteries and arterioles. 411,465 Under the electron microscope, characteristic tubular lesions similar to those in chronic experimental pi c 107 465 466 lead poisoning ? may be found in humans. > Functionally and clinically , chronic lead nephropathy is characterized by progressive azotemia. In some patients hyperuricemia with or without manifest gout may be associated with renal insufficiency. 33 The clinical differentiation 1?4 between primary gout and lead gout is well described by Emmerson. This clinical entity has, in recent years , been called "chronic lead nephropathy11 by Riehet et aJ.,197,1*65'^6 ln France, by Lilis et al.360 in Roumania (in adults with chronic uncontrolled occupational expos lire to lead), by Danilovie^^ in Yugoslavia (in older children and adults consuming leadcontaminated flour), and by Morgan et al.^11 in the southeastern United States (in long-term imbibers of lead-contaminated illicitly distilled whiskey). Wye, in Queensland, Australia, was the first to note an association between chronic lead poisoning in childhood and delayed-onset chronic nephropathy . Because of the interval between the apparent termination of abnormal lead intake and the onset of clinical renal insufficiency and gout, the term "late" is sometimes applied to this lead nephropathy. As discussed by Lilis ert al.,there is considerable controversy in the literature as to the causative role of lead in progressive renal insufficiency with or without hypertension. Although Henderson*^ found a very high frequency of chronic nephritis among survivors of -127- DUP050055416 childhood lead poisoning in Queensland, Australia, Tepper was un able to confirm this finding in Boston in young adults with a documented history of lead poisoning during early childhood. The apparent- differ ence between the group studied by Tepper and the other groups is in the length of exposure. In the subjects studied by Tepper, acute lead poisoning occurred during the preschool years, whereas the other groups were subjected to prolonged uncontrolled exposure to lead for a period estimated at 10 years or longer. At the time that the several groups of patients with chronic nephropathy underwent renal function studies, spontaneous urinary lead excretion was normal; but the response to the CaEDTA mobilization test for lead was abnormal every time it was measured.-^3*17^*465 jsuujje^on^^ found this the most suitable measurement for differentiating between nephropathy apparently due to lead and other forms of chronic nephritis. His patients with lead nephropathy could be distinguished on the basis of bonebiopsy lead content, but not on the basis of spontaneous daily urinary lead output. Emmerson did not measure blood lead content; however, Richet^00 reported minimally increased blood lead content in associa tion with abnormal EDTA mobilization of lead in patients with renal injury attributed to the metal. Although the bulk of clinical evidence apparently points to prolonged and excessive absorption of lead as the central causative agent for this form of nephropathy, the possibility that other unrecognized factors play additive, synergistic, or modifying roles has not been adequately investigated and so cannot yet be excluded. For example, none of the reports includes measurements of other heavy metals This topic is discussed in detail later in this .chapter. -128- DUP050055417 Relation Between Symptoms of Acute Lead Poisoning and Lead Content of Blood It is rarely possible in clinical situations to determine the amount of lead absorbed before the onset of symptoms of lead poisoning. In one case .,2 46 a man was estimated to have ingested approximately 3.2 mg/day for a period of 2 years before the onset of symptoms; however, no blood lead determination was made before treatment. The studies of Kehoe^99 in human adult volunteers who were fed known amounts of lead indicate that blood lead content may serve as an index of the degree of current and recent absorption of lead. Figure 4-2 shows the distribution of blood lead concentrations before treatment in relation to symptoms in 195 previously reported cases of childhood lead poisoning. Also shown are the concentrations from 98 fatal cases. The mean concentrations in fatal cases and in mild and severe cases of nonfatal acute encephalopathy were equivalent (approximately 330 yg/100 g of whole blood) :. Allvbut one of 98 cases of fatal encephalopathy were associated with blood lead concentrations of 150 yg/100 g of whole blood or greater. Like-* wise, symptoms compatible with acute lead poisoning were, with few exceptions, found in children with concentrations greater than 100 yg/100 g of whole blood. Symptomatic lead poisoning has also been seen in persons with severe anemia in whom blood lead levels were less than 80 yg/100 g of whole blood* It cannot be stated un equivocally that the subjects classified in Fig. 4-2 as asymptomatic were entirely asymptomatic; some had mild symptoms compatible with both mild lead poisoning and other disease states that were present -129- DUP050055418 at the time of examination. On the basis of clinical Judgment, the symptoms were attributed to the acute infections found in the children, rather than to lead. Others- ^ have reported comparable ranges of blood lead content for children with and without symptoms of lead poisoning. The total urinary output of lead during 10 days of chelation therapy has been measured in 9 children with acute encephalopathy. The data have been recalculated from a previous report111 and are summarized in Table h-6. The "chelatable lead" as determined by urinary output of lead does not represent the total body lead burden. Nevertheless, the values for chelatable lead shown in Table h-6 exceed by a factor of 20 the estimates of total body lead burden based on postmortem analysis of tissues for lead by Barry QO and Mossman. For normal children of comparable age and body weight, they estimated total body lead burden at 0.23 mg/kg of body weight (range 0.09-0.38 mg). Such comparisons suggest that acute encephalopathy is associated with 20-fold or greater increments in body lead burden. In adults, Kehoe reports that'clear-cut symptoms of acute lead poisoning^9 are associated with a blood content greater than 80 yg/100 g of whole blood, and most other clinical reports are in agreement. This clinical impression is based almost entirely on observations in occupationally exposed men who are otherwise healthy. Reports of some other investigators, however, have noted that symptoms and signs consistent with mild lead poisoning may be associated with blood lead concentrations of 50-80 yg/100 g of whole blood.^0 >504 reports -130- DUP050055419 are difficult to evaluate: in some instances the affected adult has been separated from his occupational exposure for up to several weeks prior to the blood lead measurement; in other cases the methods of analysis may not give results comparable to those obtained with the "tlSPHS" or comparable dithizone method. To these factors must be added the uncertainties in the clinical evaluation of. the nonspecific symptoms which are compatible with mild lead poisoning. Adequate medical supervision of men occupationally exposed to lead requires not only serial measurements of blood lead concentration * urinary ALA concentration, and hemogram but also repeated, careful, clinical evaluation by a physician familiar with each worker, his exposure, and his work habits. Such comprehensive medical supervision is essential in order to determine what component of any illness, is attributable to the individual's occupational exposure to lead. This problem is discussed by 503 50! Selander and Cramer. Selander et_ ai. noted evidence of hepatocellular injury in some patients with symptoms of lead poisoning and blood lead content in the lower range. As hepatocellular injury is not usually observed in industrial lead poisoning, they suggested that the use of alcohol might modify a person's susceptibility to lead poisoning from the clinical viewpoint. It should be noted also that the hepatocellular injury may have been due to alcohol alone. As acute lead poisoning and acute alcoholism induce many similar symptoms, the differential diagnosis may be exceedingly difficult and uncertain. Little is known of the influence of coexisting diseases that may modify the subject's response to lead. Permanent Neurologic Sequelae At least one-fourth of young children who survive an attack of acute <1 encephalopathy due to lead poisoning sustain severe permanent neuro %J logic sequelae113,526 and Harrison11^ reported and Byers has confirmed the observation that the return of a child following a single -131- DUP050055420 known attack of acute encephalopathy to the same hazardous home environment increases his risk of severe permanent brain damage to almost 100$ Byers and Lord0 and Byers0,3 have delineated the nature of the central nervous system injury that follows symptomatic lead poisoning in early childhood. The injury does not differ qualitatively from that which follows any diffuse cerebral injury incurred during early childhood (such as severe head trauma or viral or bacterial encephalitis or meningitis). In its most severe form, acute lead encephalopathy may be followed by cortical atrophy, hydrocephalus ex vacuoa severe convulsive disorder, idiocy, and blindness. Such a result is becoming increasingly rare, and subtle neurologic deficits and mental impairment are the more common outcomes . These include lack of sensory perception and perseverance despite I.Q, scores of 80-100, or better, on verbally oriented intelligence tests. Form and proportion, are distorted. Motor incoordination and lack of sensory perception severely impair learning ability. Often the handicap is not recognized until after the child enters school. Such children also have short attention spans and easy distractibility. With respect to behavioral aberration , it is difficult to determine how much is due to organic brain damage and how much represents the response of the affected child to the many facets of his total environmental deprivation, nevertheless, many children with documented prior attacks of symptomatic lead poisoning develop hostile, aggressive, and destructive behavior patterns, which, in turn, may precipitate their exclusion from school and a demand for institutionalization. Although seizure disorder and behavioral abnormalities tend to abate during adolescence, mental incompetence is permanent (j. j* Chisolm, Jr., unpublished data, and Bmmerson^^). Perl stein and Attala^^ studied b-25 children 6 months to 10 years after the initial diagnosis of symptomatic lead poisoning or asymptomatic increased lead absorption was made. A total of 39$ & -132- DUP050055421 of the group showed one or more of the following: profound mental retardation, seizure disorder, paresis, or blindness; the severity of the sequelae appeared to be related to the severity of the .acute episode. The findings are summarized in Table 4-5. Among the 59 children in the group who had had acute encephalopathy, Ql% had one or more of the severe sequelae listed in the table. Of 1*3 who presented with seizures, 6j% had one or more of the sequelae. Among the 232 who presented with acute abdominal colic, 31$ had one or more of the sequelae. Among the 351 patients who presented with symptomatic lead poisoning but no fever, 24.2$ were found at follow-up to have developed seizure disorder, which strongly suggests a causal relation between the seizure disorder and symptomatic acute lead poisoning. The mental re tardation found in asymptomatic children is difficult to evaluate. Whether children without encephalopathy have long-continued excessive intake of lead with repeated but undiagnosed episodes of acute poisoning is not known, but is probable, inasmuch as no definitive steps were taken to terminate the exposure and the initial age range at the time of diagnosis was 9 months to 8 years. Recurrent episodes of poisoning are mentioned in some patients. Similarly, Byers and Lord84 noted permanent mental subnormality in 19 of 20 children who did not have clear-cut encephalopathy but who sustained recurrent bouts of acute poisoning during the preschool years. Because the true incidence of lead poisoning in young children is not known, the incidence of significant permanent injury to the central nervous system is also not known. Furthermore, all the studies just reviewed are retrospective. -133- DUP050055422 Whether asymptomatic increased lead absorption can cause subtle but permanent impairment of nervous system function in young children is not now known, , 1*07 Moncrieff et^ al, 1 reported minimally increased blood lead concen tration (40-80 ug/100 g of whole blood) in almost half of a group of mentally retarded children. It is not possible on the basis of their data to determine whether there is any causal relation between the mental retardation and the observed increase in blood lead content. Many mentally subnormal children have persistent pica. Consequently, mental impairment with other causes might well have preceded the increased intake of lead; the final clinical picture would then repre sent the interaction of several etiologic factors. The older medical literature refers to similar mental impairment, 2.3 >404. encephalopathy , and seizures associated with uncontrolled occupa tional exposure to lead in adults. Lead poisoning due to ingestion of lead-contaminated moonshine whiskey may give rise to a range of symptoms that spans the full clinical spectrum from the mild abdominal colic seen in industrially exposed workers to the acute encephalopathy seen in young children. This wide spectrum of clinical disease is apparently related to the wide range of lead content in various batches of moonshine. The following clinical observations are qualitative. Patients of all economic and social strata have been found. Examination of the military service records of some patients with lead poisoning from moonshine suggests that -134- DUP050055423 they formerly had jobs in military service that required personal initiative and reliability. At the time they were examined for lead poisoning they were incompetent * As the intellectual function of these patients has not been studied in detail and no epidemiologic studies have been performed to document these impressions > nor to separate the effects of lead from those of alcohol on nervous system function, it is suggested that this is an area for future research. Clinical Diagnosis of Lead Poisoning In the absence of a positive history of abnormal exposure to lead, the clinical diagnosis of lead poisoning is easily missed (e.g., in children with pica and imbibers of moonshine whiskey) . The character istic sequence of clinical events, with a positive history of known exposure, may alert the physician to the possibility. However, because the signs and symptoms of the syndromes of acute lead poisoning are shared by other diseases--particularly acute alcoholism and acute intermittent hepatic porphyria^*^ "diagnosis may be over looked. Likewise, physical examination rarely reveals findings specific for lead poisoning. The classic "lead line" on the gums requires the presence of poor dental hygiene and even then is seen only in persons with severe chronic exposure , as occurs in moonshine drinkers. The signs of acute encephalopathy are those of increased intracranial pressure from whatever cause. The signs of chronic encephalopathy may resemble the signs of a variety of cerebral degenerative diseases. Physical examination does not distinguish -135- DUP050055424 lead colic from a variety of acute surgical and nonsurgical conditions of the abdomen, especially peptic ulcer, pancreatitis, and acute intermittent porphyria. Lead neuropathy does show a predeliction for motor weakness involving the most frequently used extremities , which may help to distinguish this form of peripheral neuropathy from other forms with more obvious sensory abnormalities. Lead myelopathy is difficult to distinguish from anyotropie lateral sclerosis. Microscopic examination of the peripheral blood may reveal only the features of iron-deficiency anemia. On routine examination the urine may be normal or show the presence of minimal albuminuria, glycosuria, and nonspecific abnormalities. Basophilic stippling of red blood cells is an inconstant feature of gross lead poisoning and requires special techniques for consistent results. However, microscopic examination of aspirated bone marrow samples almost always reveals stippled sideroblasts in classic lead poisoning; Because of the nonspecificity of the clinical features, diagnosis requires a high index of suspicion, knowledge of the epidemiology of lead poisoning, and some specialized diagnostic tests, which are discussed later in this chapter. The principal pitfalls of clinical diagnosis are failure to obtain and establish an exposure history and failure to appreciate the rapidity with which classic plumbism may progress. The clinical subtlety of lead poisoning is exemplified by the measures that may be necessary to establish the diagnosis when it is due to 410 a moonshine drinking. If the patient has ingested lead-contaminated whiskey shortly before hospitalization, his blood and urinary concen trations of lead are usually increased. But if ingestion has been -136- DUP050055425 more remote , blood and urine levels may not be increased, iiiOa The severe anemia sometimes found in these patients must be taken into account in interpreting their blood lead concentrations,* The diagnosis of increased body burden of lead is sometimes possible only after administration of a chelating agent, such as CaEDTA* and measurement of the urinary excretion of lead. Observations on the 10 patients with overt lead poisoning shown in Table h-7 illustrate this fact. Symptomatic lead poisoning has been seen in persons with severe anemia in whom blood lead concentrations were less than 80 pg %. -137- DUP050055426 MEffiABOUC AND FUNCTIONAL EFFECTS IN MAN Lead is not known to be an essential trace element. No studies pertinent to this question have been reported* However, various segments of the population dp carry concentrations of lead in blood and urine at which minimal changes in pyrrole, metabolisHi can be detected. The observed minimal metabolic changes are of the adverse type. This section takes up the known adverse metabolic and func tional effects of increasing concentrations of lead on heme synthesis, the kidney, the nervous system, and other organ systems in man. Observations in experimental models that complement, confirm, and amplify observations in man with respect to the effects of lead on heme synthesis, the kidney, and the nervous system are included. The effects of lead that have been examined in experimental bio chemical systems, but not in intact man, are described later in this Chapter. * ~ Biosynthesis of Heme Current knowledge of the biosynthesis of heme is based largely on experimental studies in avian erythrocytes and embryos , mammalian erythrocytes and liver, chloroplasts, and Rhodopseudomonas spheroides, a facultative anaerobic bacterium. These findings , which have been reviewed recently 5indicate that the metabolic pathway shown in Pig. 4-3 is the common pathway in mammals and photosynthetic bacteria and plants for the formation of heme or chlorophyll. The necessary enzymes have been found widely distributed in mammalian tissues^!? ,210 ,219, 220 ,336393 so it is highly probable that each cell synthesizes its own heme -138- DUP050055427 for the formation of its particular hemoproteins (hemoglobin, myoglobin, cytochromes, and catalases). A modified form of heme, the corrin moiety, is a major component of vitamin B12. The intracellular organization of the pathway in animal tissues is apparently as follows: The initial step (formation of ALA*) and the final two steps (COPROGEN 111 PHOTO 9 and PHOTO 9 + Fe heme) are mediated by intramitochondrial enzymes, whereas the interned!ate steps are mediated by soluble enzymes in the cytoplasm. Regulation is by negative feedback control through repression of ALAS formation and activity21^ ^3-6 by heme. There is some evidence that heme and PROTO 9 may also inhibit the activity of ALAD, the second enzyme in the pathway. This is an Important consideration with respect to lead in that PROTO 9 accumulates in lead poisoning. In liver and in chick embryos , some sex steroid metabolites and drugs in physiologic concentrations exert important modifying effects on the 218,288 de novo production of ALAS. Thus, some flexibility in response to the needs of the organism is apparent. Also, there is some evidence that globin synthesis is synchronized with heme synthesis.39 \6,109 Lead clearly inhibits the formation of heme at several points ,^o/,po^^7|hovn in Fig> 1*_3. Classic lead poisoning in both humans and experimental animals is characterized by accumulation of nonheme iron and PROTO 9 in red blood cells , accumulation of ALA in serum,and the increased excretion in urine of ALA and C0PR0 III (the oxidation product of C0PR0GEN III). ALA and PROTO 9 are the substrates of SH-dependent enzymes, 109,111,199,220,336 ^buAt COPROGEN III apparen_tly i.s not. *See Glossary for abbreviations. -139- DUP050055428 Increased excretion of PBG and URO in human lead poisoning has been reported only in more severely affected cases ,200,231 The transfer of iron from transferrin into human reticulocytes is only partially inhibited (20# inhibition at a lead concentration of 10"*^ M) ;^^ however, electron microscopic studies reveal the accumulation of nonheme iron in developing red blood cells in the form of ffferruginous micelles," which are responsible for the ringed sideroblasts character istic of lead poisoning and dense aggregations of ferritin in damaged mitochondria. ^*276.,487 These accumulations of altered cytoplasmic and nuclear remnants (RNA and altered microsomal and mitochondrial fragments) are responsible for the basophilic stippling of red blood cells characteristic of human and experimental lead poisoning. ^ ,48 f In vitro studies show that in man lead is tightly bound to red blood cells and not readily desorbed,3597 and electron microscopy shows lead deposited on the outer surface and within the membrane of red blood cells . ^ The localization of lead in red blood cells in vivo is not known. That the accumula tion of porphyrins and metalloporphyrins within mitochondria may have physiologic significance is suggested by the experimental observation in mouse liver by Silverstein-^ that metalloporphyrins in concen trations of 1CT5 M and 10M may inhibit the activities of other intramitochondrial enzymes . Waldron^^has summarized the experimental studies on the overall effects of lead on heme and protoporphyrin formation in avian red blood cells when glycine is used as substrate--minimal inhibition at -l40- DUP050055429 a lead concentration of 10""^ M, partial inhibition at 10 ^ M, and complete inhibition at .10and 10^ M. In vitro observations show that inhibition by lead can be partially reversed by the addition of such SH reagents as glutathione (GSH), cysteine , BAL, and d-penicillainine,199,220,357 Many studies indicate inhibition at some stage before the formation of ALA. For example . Morrow al.^1^ report that lead inhibits ALAS in in vitro preparations (12% inhibition at a lead concentration of 10 M, and hb% inhibition at 10 ^ M). Although the intracellular distribution and concentrations of lead are largely unknown, the enzymatic inhibition demonstrable at 10~^ M in vitro is generally con*- sidered to be physiologically significant in the intact animal. The increased FEP and FEC in red blood cells, increased COFRO III in urine, and increased ALA in serum and urine indicate that these are the steps in heme biosynthesis most susceptible to inhibition by lead in man. The inhibitory effect of lead on heme biosynthesis in organ systems other than hematopoietic tissue has received relatively little study .109 That some of the ALA found in human urine may arise from tissues other than the hematopoietic system is suggested by the finding of measurable amounts of ALA in the absence of demonstrable erythropoiesis i.n urine in 507 humans . Gibson i*.*9 e^ al. showed that lead . (10 J Mv) partially inhibited ALAD activity in beef liver. Recently, Millar 400 et al. reported that ALAD activity is inhibited in the blood, brain , and liver of lead-poisoned suckling rats . The inhibition in liver was 60% of that observed in blood, whereas the inhibition in -l4l- DUP050055430 brain was 80-90# of that in blood. However, in one experiment in which the lead intake in six suckling rats was sufficient to produce a mean blood lead concentration of 30 yg/100 g of whole blood, the degree of ALAD activity in brain was not significantly reduced in comparison with the controls Further studies of heme synthesis . in tissues other than erythrocytes (especially brain and kidney) are needed, because the quantitative aspects of the rate and regulation of heme synthesis in these tissues may well differ from those in the hematopoietic system. The potential for significant interactions between lead and other heavy metals has received little attention. Passow, Rothstein, and Clarkson^^O point out the biologic complexities introduced by differ ences in membrane and intracellular distributions of various metals, the variety of ligands with which metals can interact, and the com petition among metals for various types of binding sites. For example, in Rhodopseudomonas spheroides lead,manganese, and cobalt increase the requirement for iron for the conversion of C0PR0GEN III Sok i+ll to PROTO 9. Similarly , Morrow et al. have reported that ferrous iron in low concentration has a mild stimulatory effect on the activity of ALAS, whereas lead has an inhibitory effect in the same system. In beef liver, for example, copper, mercury, and silver are more effective inhibitors of ALAD in vitro than is lead,^9 *but are not known to be significant inhibitors of ALAD in vivo in man. This enjoins one to be cautious in the transfer of in vitro observa tions to in vivo situations.. In occupationally exposed lead Workers , Rubino et al.^^ reported a statistically significant positive correlation between rising concentrations of copper and PROTO 9 in red blood cells in the peripheral circulation. These preliminary experimental and .c.linical data strongly suggest that some of the important metabolic effects of lead may be modulated by competition, between lead and other metals at various stages in the biosynthesis of heme. Clearly, potentially competitive metal-metal interactions involving lead , such as those found in Rhodopseudomonas spherpides, merit considerable study. In particular, inasmuch as lead can cause a functional impairment in the utilization of iron, persons with iron-deficiency states and persons with disorders characterized by functional impairment in iron utilization (such as copper deficiency, vitamin Bg deficiency, and thalassemia) deserve study with respect to their susceptibility to given levels of lead absorption. Only in some of the inborn errors of porphyrin metabolism and some toxic porphyrias are increases in pyrroles and ALA in the tissues and excreta comparable with those in classic lead poisoning in humans ,108*231,^92, The pattern of pyrrole excretion found in lead poisoning differs from that found in acute intermittent porphyria and other hepatocellular disorders, as shown in Table k-8. Erythropoietic protoporphyria is associated with very high concentrations of PROTO 9 in red blood cells. Deficiency and impaired utilization of iron are also associated with moderate increases in PEP of the same order of magnitude as those found in asymptomatic increased lead absorption.231,357,^87>580 Only classic lead poisoning is associated with FEP concentrations -ll*3- DUP050055432 comparable with those in erythropoietic protoporphyria, an uncommon genetically determined disorder. The adverse metabolic effect most specifically associated with lead is the inhibition of ALAD. When ALAD is inhibited, its substrate, ALA, accumulates; the accumulation is reflected by an increase of ALA in serum and urine. The increased excretion of ALA in urine in some porphyrias is the result of increased production of ALA and does not result from inhibition of ALAD.163'210'218'288'1*22*1*^, 560 Several authors^29152,251,357>^00s^23 using different in vitro techniques have found that ALAD activity is inhibited in circulating red blood cells in humans and that the degree of inhibition increases with the concentration of lead in the blood. An approxi mately fourfold change in the level of ALAD, as measured in vitro, is found in hemolysates of peripheral blood from persons whose blood lead content is within the normal range (i.e., 5-40 yg/100 g of whole x 15^,251,400 blood;. In humans, this effect is apparently specific for lead; ALAD activity is not impaired in human iron-deficiency anemia/^ Hernberg et al report no relationship between ALAD activity in hemolysates of blood and the concentration of mercury in blood and urine in man. Nakao et^ al.^3 report normal ALAD activity in blood from cases of arsenic poisoning, carbon monoxide poisoning, acute intermittent porphyria, several types of anemia, four cases of leukemia, and 46 cases of 21 different neurologic disorders. Only in acute alcoholic intoxication has a transient decrease in ALAD activity in blood been reported--in adult human volunteers who drank 300 ml of whiskey in 1 hr,33 Although a number of disease states r * -Ikk- DUP050055433 remain to be evaluated, no other exceptions have been reported to date. Haeger-Arons en31 found that urinary* ALA excretion was normal in human adults with a variety of hepatic, hematologic, `and neoplastic diseases. The available evidence indicates, therefore, that the combination of inhibition ofALAD act increasing excretion of ALA in urine is jassocia.ted. in, increasing concentrations of lead; the only known exception Is the transitory effect of acute drunkene ss. Hember^lj252 has suggested that impaired ALAD activity in red blood cells is the earliest evidence of an adverse metabolic effect of environmental exposure to increasing levels of lead. The logarithm of the activity of ALAD in hemolysates of red blood cells has an inverse linear relationship with the concentration of lead in blood when concentration is between 5 and 95 yg/100 g of whole blood (Fig. The interpretation of these in vitro findings is. highly pertinent to the question of airborne lead. However, the conditions under which ALAD activity is measured must be considered.. In all the reported procedures, known amounts of ALA are added in vitro to crude hemolysates of whole human blood, the hemolysate is incubated, and after a suitable time, the amount of PBG formed is measured. "Ghosts" or membrane fragments are not removed. Such cofactors as GSH, which are present in blood in varying amounts and which partially reverse inhibition due to lead in samples of blood from lead-poisoned subjects ^ are not added in optimal amounts in the reported procedures. Because the chemical aspects of DUP050055434 this in vitro assay are not completely understood, any conclusions concerning its in vivo significance should still be considered tentative. 4It .is, however, clear that decreasing ALAD activity, as measured in vitro in human blood, is associated with Increasing urinary excre tion of ALA and increasing concentration of lead in blood. Nakao ejt al. 423 report a negative linear-correlation between the logarithm of ALAD activity in blood and the excretion of ALA in urine as blood lead content rises from kO to l65 yg/100 g of whole blood. Selander and Cramer 503 find a correlation between blood lead content and urinary ALA, when ALA in samples of urine collected for approximately hr in the morning is measured by the method of 392 Mauzerall and Graniek. This method measures aminoacetone, a substance unrelated to lead, as well as ALA; although this does not affect the significance of the findings, it may obscure the location of the apparent threshold to some extent. Selander and CramerVs data (Fig. U-5)^^ indicate that there is an exponential relation between blood lead concentration and urinary ALA excretion. As blood lead concentration rises above Lo yg/100 g of whole blood, urinary ALA excretion rises at a progressively increasing rate. Considerable Variation is seen in workers with blood lead concentra tions between ho and 59 yg/100 g of whole blood, 29 of the 33 workers with concentrations of 60 yg/100 g or higher have urinary ALA values ' that exceed the upper limit of normal (5 + 2 S.D. ) for the method of analysis used. 231 In a group of young children with blood-lead 146- DUP050055435 conceatrations betveen 25 and 75 yg/100 g of whole blood* an apparently similar curyilinear relation can be seen between the eluantit a^ dally output of ALA in urine -and bipod lead concentration (Fig. 4-6)/ However, in a group of adolescents who-had lead poisoning at least 10 years previously and normal blood lead content (8-40 yg/100 g of whole blood) at the time of study, the excretion of ALA (mg/24 hr per m^) showed no significant correlation with blood lead content, (Fig. 4-7). None of these studies is entirely satisfactory by itself from the point of view of methodology, completeness, or groups studied. Never- theless, all are consistent with the hypothesis that the exponential increase in ALA excretion associated with, blood lead content abpve approximately 40 yg/100 g of whole blood signifies inhibition^ of ALAD that is physiologically significant in vivo. The curvilinear nature of the relation between ALAD in blood, ALA in urine, and "chelatable11 lead with rising blood lead concentration further indicates that the inhibition of ALAD by lead accelerates as blood lead rises ufrom 40 to 80 yg/100 g of whole blood and higher. ^ At blood lead con- cen traWpM.^^1 35 Ug/100 g of whole blppdn stimulatory nor .inhibitory effeptS:: oflead h^ in vivo in relation to AL^ activity. At these "normal" levels of lead in blood, hemoglobin syn^ red blood cell formation* ^ increase and ALA excretion is apparent unaffected. JBms, the higher activities of ALAD activity in hemolysates of peripheral blood in persons with very low blood lead content may reflect an in vitro measure of "reserve enzyme capacity11 that is not essential. Studies -1U7- * DUP050055436 using more specific methods for the determination of ALA in urine, as well as studies of the factors that may influence ALAP activity in vivo, may in the future delineate more precisely the concentrations of lead in blood and soft tissues at which compensatory biologic mechanisms must be invoked. Functional Effects of Lead on Red Blood Cells in Man Hasan and Hernberg,2^*3 Waldron ,575 and Griggs22** have summarized the in vivo effects of lead, on red blood cells in man as follows: accumulation of lead, increase in osmotic resistance, increase in mechanical fragility, increased glucose consumption, increased loss of potassium at 37 C in a 2-hr incubation, decrease in sodium and potassium ATPase activity in membrane fragments , , decreased life span, and increased proportion of immature red cells in the circulation (increased numbers of reticulocytes and baso philic stipple cells). For the most part, these represent effects on membrane functions. Hemberg ert al, ,2^ using tritiated di-isopropyl fluorophosphonate reported nonrandom shortening of human red blood cell life span, which they interpreted as indicating a true shortening of life span. Significant reduction (to less than 105 days) was observed at blood lead concentrations greater than 80 ug/lOO g of whole blood. In a patient with clinical lead poisoning, non- 1+5 random destruction was also observed, suggesting intravascular hemolysis which is consistent with the jaundice sometimes seen in very severe acute lead poisoning. In Finnish adults occupationally exposed to lead, glucose consumption was increased, but three metabolic -lL8~ DUP050055437 functions of red blood cells were not altered---lactate production, glucose-6-dehydrogenase (G^PDH) activity, and lactic dehydrogenase activity. However, in Italywhere a much higher incidence of G6PDH deficiency may be anticipated' on a genetic basis, red blood cell preparations from men who were occupationally exposed to lead and had both clinical and subclinical lead poisoning showed statistically significant lowering of glutathione (GSH) content, glutathione stability, and Ct 6PDH content compared with normal controls g 6PDH deficiency is a rather widely distributed genetic abnormality in persons of Mediterranean origin.h1i This enzyme lies in the pathway that is essential for the maintenance of * normal GSH content in the tissues. The genetic defect renders affected persons peculiarly susceptible to hemolytic crises on exposure to primaquine, naphthalene, sulfonamides j and other substances * In view of the rather large reserve of GSH in red blood cells and the effectiveness of GSH in reversing the inhibitory effect of lead and other heavy metals on AMD activity in vitro, the question of susceptibility of otherwise healthy primaquine-sensitive persons to increased lead content is, at present, unanswered. The anemia so frequently seen in clinical lead poisoning is complex. Under conditions of severe acute exposure, hemolysis predominates whereas prolonged and increased exposure of the sort usually associ ated with clinically evident lead poisoning leads ultimately to erythroid hypoplasia and marked morphologic changes in the bone marrow. The usual picture is of a mild to moderately compensated 575 -1^9- DUP050055438 hemolytic anemia, with hemoglobin concentrations rarely less than 10 g per 100 g blood, The hemolytic component is small in most circumstances. Impaired heme synthesis is the major cause of the anemia. The degree of reduced red cell.life span correlates most closely vith the reticulocyte count. ^ As the lead content in the hematopoietic tissues increases, the compensatory mechanism becomes inadequate and anemia follows, Frank anemia is usually not apparent until sustained blood lead concentrations exceed about 80 yg/lOO g of whole blood. Even in the absence of frank anemia, various somewhat less serious degrees of lead exposure may be associated with abnormalities in membrane functions, maturation, metabolism of red blood cells, and biosynthesis of heme .2^b Kidneys At the histologic level, the unique intranuclear inclusion bodies have long been considered pathognomonic of severe lead poisoning in man. They are readily reproduced in experimental lead poisoning in rats, and there is some evidence that they represent an inert lead-protein complex,212,213,214,2l6 GQyer^^ has proposed that inclusion bodies represent a storage mechanism by which renal cells can maintain lower cytoplasmic concentrations of lead. This provocative hypothesis remains to be proved. Experimental evidence and human autopsy and biopsy specimens of Goyer Galle and Morel-Maroger^-97 and Richet et_ al.indicate that lead has adverse metabolic effects on enzymes in the mitochondria and other cell organelles. , -150- DUP050055439 At the functional level, renal glycosuria has long teen recognized as a concomitant of clinical lead poisoning. More recently, hyperamino > aciduria (of the generalized renal type) has been reported in asympto- matic men occupationally exposed to inorganic lead ^ and in children with acute symptomatic lead poisoning.10^ ,596 clinical investi gators have used rather insensitive semiquantitative techniques for the most part. Clearer delineation of any threshold for hyperamino aciduria would require the use of clearance techniques and ion-exchange chromatography. Experience in ether disease states affecting the kidney indicates that aminoaciduria of the generalized renal type is a most sensitive, nonspecific index of functional injury to proximal renal tubular cells, and that the biologic significance of this type of aminoaciduria is not nutritional (i.e., the loss of nutrients In the form of glucose and amino acids is inconsequential).. In children, it is associated with clinical illness and blood lead concentrations greater than 80 jjg/100 g of whole blood and it is reversible on termination of exposure.Studies in rats with high . doses of lead demonstrate that hyperaminoaciduria is correlated with alterations in mitochondrial structure and function. In addition, fructosuria and citraturia have been reported in children with acute load poisoning, but the significance of these observations remains entirely unexplained.^^ In children with acute encephalopathy and blood lead concentrations greater than 150 pg/100 g of whole blood, the full Faiieoni triad *) -151- DUP050055440 (hyperaminoaciduria, glycosuria, and hypophosphatemia in the presence of hyperphosphaturia) has been reported and is estimated to occur in approximately one-third of patients with acute encephalopathy caused by exposure to lead. The significance of the hypophosphatemia in this triad is that it in turn can result in demineralization of bone.^5 The Fanconi triad is evidence of severe injury of proximal renal tubular cells and can be caused by many agents , including lead and such other heavy metals as cadmium and uranium. In children with severe lead poisoning, this injury to the renal cells is also reversible after treatment and cessation of gross lead intake, it may be tenta tively concluded that the full Fanconi triad is associated only with severe clinical lead poisoning. It may also be tentatively concluded that hyperaminoaciduria alone is a manifestation of severe over exposure to lead; it has been reported in severe occupational exposure, and it is highly unlikely, that exposure f to lead in the ambient air alone can cause hyperaminoaciduria. This belief is buttressed by observa tions in children with clear-cut symptomatic lead poisoning who do not exhibit hyperaminoaciduria as measured hy semi quantitative paper chromatographic techniques. That even the acute Fanconi triad is reversible is not surprising, in view of the regenerative capacity of renal tubular cells. Similarly, clinical investigative experience indicates that profound injury to proximal tubular cells is the prerequisite of hypophosphatemia and demineralization of bone and is associated only with severe forms of lead poisoning. -152- DUP050055441 Observations in men who have drunk lead-contaminated whiskey suggest that high levels of lead intake or long-continued exposure to lead from this source may be associated with impairment of other renal tubular transport mechanisms. - Renal tubular acidification may be impaired, as, apparently, may the ability of patients to conserve sodium when they are fed a diet low AQQ in sodium, The latter suggestion is based on observation of men who had drunk illicitly distilled whiskey. Similar observations have not been reported in patients with less complicated conditions or in experimental animals. Therefore, an effect of lead on renal sodium conservation must be considered tentative. Another function of the kidney that may be impaired by lead is the secretion of renin by the juxtaglomerular complex in response to sodium deprivation. ' ^ The physiologic variables that stimulate this complex to secrete renin are not entirely understood. Inasmuch as it is composed of cells of the distal convoluted tubule (macula densa) and of juxta glomerular cells, which are epithelioid cells in the wall of the afferent glomerular arterioles, it seems possible for lead to affect either component of the complex. However, because renal tubular cells appear particularly susceptible to the effects of lead, for the present it is reasonable to assume that the effect of lead on the juxtaglomerular complex is mediated primarily through the cells of the macula densa. In any case, the effect has been found reversible following treatment of patients with CaEDTA. -153- DUP050055442 An interesting consequence of the chronic renal injury due to lead is the syndrome of saturnine gout. 174 Lead interferes with the secre- 33 tion of urates by the renal tubules. Hence, although uric acid is not overproduced (as in other forms of gout), the body pool of urates increases, and crystals are deposited in the joint spaces, resulting in the classic gouty diathesis. In contrast with other forms of gout, the frequency of disease is the same in both men and women. In addition, according to the experience of Eramerson 174 and 411 Morgan^ saturnine gout is almost always associated with im*- paired renal function. Nervous System Information on the adverse effects of lead on the function of the nervous system in man is limited almost entirely to clinical and 454' postmortem observations. The pathologic studies of Pentchew and Pentchew and Garro 455 on the brains of children dying from acute encephalopathy and observations in experimental animals indicate that the most severe tissue injury is found in the cerebellum and in capillary endothelial cells. Recent experimental studies, particularly those of Lampert and 328 194,195 Schochet and Fullerton, have shed some new light on the peripheral neuropathy of lead poisoning. Their findings indicate that chronic feeding of relatively high doses of lead in the diet are necesary to produce the lesions. Electron microscopic studies show that segmental demyelination was associated with injury to the Schwann cell (including injury to its mitochondria), that regeneration can -154- DUP050055443 occur, and that eventual "onion "bulb" formation and scarring of the nerve fiber result from chronic poisoning. These observations are V consistent with observations in humans that peripheral neuropathy associated with lead is associated with high-level chronic and uncontrolled exposure (usually occupational). However, Catton et al, 99 report finding minimal electrophysiologic abnormalities in peripheral nerve conduction in a few occupationally exposed workmen whose blood lead concentrations were 70-80 pg/100 g of whole blood and who were considered otherwise clinically asymptomatic. This is the only report of clinical investigation concerning the relation between peripheral nerve conduction and low exposure to lead. Similarly, the interesting j? .*t o report of Simpson et_ al. concerning an acetylene burner (for 20 years) with a complex neuropathy resembling progressive muscular atrophy (amyotrophic lateral sclerosis) points to chronic highlevel exposure to lead as the etiologic agent. Although the avail able but scanty evidence indicates that neuropathy in humans is associated with chronic high-level exposure, the cases Just cited suggest that the question of peripheral nerve function, especially as it may be related to the occupational type of exposure, needs re-examination with the newer e le etrophysio logic techniques. It is most unlikely that lead in ambient air alone can cause sufficient absorption of lead to produce this kind of injury. The effects of lead on behavior and performance in man and experimental animals are discussed later in this chapter. '> -155- DUP050055444 Other Organ Systems In addition to the well-documented effects on the hematopoietic system, kidneys, and nervous system, some reports suggest that patients .with clinical lead poisoning, and often some complicating intercurrent disease, may also exhibit adverse effects in other organ systems. The following observations are presented here in the hope that they will stimulate research to establish more clearly whether these effects are concomitants of classic lead poisoning and, if so, to establish the soft-tissue levels of lead at which they become evident. The effect of lead on the endocrine and reproductive systems has received relatively little attention. Its impairment of thyroid function both in vitro52.2 and in vivo in rats482> 604 and in man406>485 has been reported. In vitro observations on thyroid slices522 have shown that lead, like other heavy metals , impairs uptake of iodine by the gland. In the Intact lead-poisoned rat*482,604 uptake of iodine-131 and conversion of iodine to protein-bound iodine are retarded, females being more severely affected than males. In lead-poisoned man,406,485 the 24-hr uptake of iodine-131 is sometimes decreased. Administration of thyroid-stimulating hormone will usually overcome the effect. The various studies indicate that lead may injure the pituitary-thyroid axis, at the level of the thyroid, the anterior pituitary, and possibly the hypothalamus. A second effect of lead on the hypothalamus and/or anterior pituitary is a decrease in secretion of pituitary gonadotropic hormones observed in some patients. 463>484 ^ appggrs that lead may also impair function of * r* -156- DUP050055445 the pituitary-adrenal axis . Some patients with nonfulminant or severe lead poisoning due to ingestion of illicitly distilled whiskey have teen shown to have a decreased responsiveness to an inhibitor (metapyrone) of 11-beta hydroxylation in the synthesis of cortisol. Urinary secretion of 17-hydroxycorticosteroids, and plasma concentrations of cortisol and immunreactive ACTH have been found decreased in response to this test . Some patients also do not have an appropriate increase in plasma cortisol when made hypoglycemic with insulin. Most of those tested with exogenous ACTH responded normally. At present, it is suggested that lead, and not some other constituent of illicitly distilled whiskey, is the toxicant responsi ble for the decreased functional reserve of the pituitary and possibly the adrenal gland of these patients. Support for this opinion comes from two studies^^k 5 456a carried out with methods less definitive than desirable. In one involving 25 persons with chronic tetraethyl lead poisoning, many were found to have a decreased pituitary reserve, but they were normally responsive to exogenous ACTH. In the second, workers occupationally exposed to organic lead also revealed evidence of impaired pituitary-adrenal function. Concerning the human adrenal cortex, according to Makotchenko, the responsiveness of the zona fasciculata to exogenous ACTH may sometimes be suppressed in lead-poisoned persons---a finding also observed in a few men with lead poisoning due to illicitly distilled whiskey.^84 Function of the zona glomerulosa (aldosterone secretion) has also been found suppressed in some men with lead poisoning due to illicitly distilled whiskey.483 -157- DUP050055446 The effect of lead on human reproduction has not been studied in de tail in recent times. It was recognized 75 years ago that lead poisoning in women industrial workers resulted in decreased fertility ooo and an increased abortion rate, Exposure of women to concentrations of lead similar to those prevalent in industry at the turn of the century no longer occurs and the effect of lead on human reproduction is primarily of historical importance. However, lead is known to cross the placenta^ and it is conceivable that ingestion of lead- contaminated whiskey during the first trimester of pregnancy occa- 447 sionally causes fetal injury. Excessive exposure to lead during pregnancy has resulted in neurologic disorders in children. In child.ren 193,516 and adult. s -421,464 w.ith acute lead. poisoning, cardiac arrhythmias have been found by electrocardiograph. Altera tions in cardiac conduction have been noted consistent with a diagnosis of myocarditis and were responsive to treatment with CaEDTA. In two adult cases, the abnormalities recurred on renewal of excessive exposure but were again responsive to treatment with CaEDTA. The mechanism of this effect is unknown, but the clinical circumstances strongly suggest an association with acute lead poisoning. In view of the myopathy that has been reported in skeletal musculature,^** these findings cannot be overlooked; on the contrary, they call for research. -158- DUP050055447 EVALUATION OF BODY BURDEN Clarification of the relations between lead and human health requires that human subjects be evaluated by measurements of both the. degree of lead absorption and its storage and of some adverse metabolic and functional effects attributable to increased concentrations of lead in soft tissues. The evaluation should be made with regard for the recent level and duration of absorption of lead and possible exposure to high levels of lead in the remote past. Types of Measurement Blood lead concentration and, in the presence of normal renal function, endogenous urinary lead excretion are the most widely used indices of recent and current absorption of lead. In "healthy" adults208,299,539 and children^0 without undue exposure to lead (such as industrial exposure or paint ingestion by children), blood lead concentration ranges between approximately 10 and 40 jjg/100 g of whole blood. In acute lead poisoning, blood lead concentration is usually grossly increased (greater than 100 yg/100 g of whole blood) in association with current excessive absorption (see ELg. 4-2). The following factors must be taken into account in interpreting a given blood lead concentration: proper collection of whole blood sample and reliability of laboratory performing the analysis (see Appendixes B and D), hematocrit value, current or recent administration of chelating agents that temporarily decrease blood lead content, presence of hemolytic anemia, and period since termination of undue exposure.1071 Long-term continuous administration of a chelating -159- DUP050055448 agent such as d-penicillamine suppresses blood lead content to the normal range during the diuresis of lead, 111 9 202 and intermittent chelation therapy is associated with fluctuating blood lead content. Marked changes in the mass of circulating red blood cells may influence whole-blood lead content, in that 90# or more of the lead in blood is attached to the red cells ,1^8>^70 perS0ns with moderate to severe anemia, clinical evaluation of the significance of a given blood lead content may be facilitated by correcting the observed concentration to the approximate value that would be expected if the patient1 s packed red cell volume (hematocrit) were within the normal range.# The relevance of such corrections must, however, be assessed further through the use of other indices of lead absorption and adverse effects (e.g., response to chelating agents and ALA excretion). Subjects whose high-level exposure has terminated several months to several years previously may still have evidence of increased body lead burden, as measured by the CaEDTA mobilization test and blood lead concentration that are minimally to moderately elevated.1115360,460,1+65 Endogenous (spontaneous) urinary lead excretion may be defined as the excretion of lead that occurs normally in the absence of chelating # This correction may be made according to the following simple calculation: expected blood lead = & -Lea<* x hematocrit normal for age and sex. observed hematocrit This correction should be made in subjects with moderate or severe anemia due to lead or other causes, but probably is not warranted in persons with minor deviations from normal hematocrit. f -l60- DUP050055449 agents. It is best estimated by quantitative measurements of the daily 2^-hr output of lead to minimize diurnal variations'^ in the excretion of lead, as well as variations in the overall concentra tions of solutes in urine. However, most of the available data in adults are based on measurements of the concentration of lead (in micrograms per liter) in morning or random samples of urine. In adults without undue lead exposure, the concentration of lead in such samples is generally less than 80 yg/liter.208,299,539 In young children, the concentration of lead in random samples of urine is of little diagnostic use; quantitative 2k-hr collections are re quired and normal concentrations (less than 55 yg/2^ hr) may be found in severe acute lead poisoningIn men occupationally exposed to lead who appear to be in good health, the concentration of lead in urine (in micrograms per liter) is increased. However, in chronic lead nephropathy and in persons with other forms of renal Insufficiency , endogenous urinary lead excretion (in micrograms per 2k hr) may be within the normal range in the presence of increased body lead burden.^33,173,360,kll,465 jn subjects remotely occupationally exposed to lead and in moonshine drinkers, endogenous urinary lead / . a 460.466 excretion may also be normal (see table 4--7). Although the measurement of endogenous urinary lead excretion in timed collections of urine from healthy adults may be suitable for epidemiologic purposes, it is clearly inappropriate in patients with renal impairment from what ever cause. Body lead burden may be estimated by direct measurement of lead con tent in bone biopsies, but that has rarely been done in the living -l6l- DUP050055450 Due account should he taken of differences between flat bone (rib, skull) and dense tubular bone (femur, humerus)39 if results are expressed as micrograms per. gram of fresh tissue. No difference is apparent (except for teeth)' if results are expressed as micro- grams per gram of ash. The lead content of shed deciduous teeth may provide an index of past gross lead absorption.1^ Such measurements in bony tissues do not, however, give an estimate of the fraction of "exchangeable11 lead in bone, which can be rapidly mobilized. Emmerson1^ showed that increased bone lead concentration is positively correlated with the increased output of lead in urine that is provoked by the administration of calcium EDTA. (This tech nique is commonly known as the CaEDTA mobilization test for lead.) This finding is consistent with experimental observations that a large fraction of the lead mobilized by CaEDTA is derived from bone. Various investigators111 >173,4ll,U60,465,466 have shown that an abnormal response to the CaEDTA mobilization test is the most reliable way (short of bone biopsy) to demonstrate an increased body lead burden when high-level exposure took place much earlier and particularly in subjects with renal insufficiency. Heavy reliance on this test is also attributable, in large measure, to the widespread unavailability of accurate blood and endogenous urine lead analyses. According to Emmerson,1^ the CaEDTA mobilization test may differentiate between nephropathy due to lead and renal insufficiency due to other causes (Fig. *1-6). In subjects with renal insufficiency, urine must be collected quantitatively for 4 days after a single intravenous infusion of 1 g of CaEDTA.1^ The technique of the test may affect the result. -162- DUP050055451 According to Emmerson*s technique, the upper limit in healthy adult subjects is less than 600 yg of lead excreted during k days. Teisinger and Srbova^ use a different technique; their results -are shown in Table U-9. If renal function is normal, a 24-hr urine collection is adequate.. In conditions of current and recent exposure, the CaEDTA or d-penici 1 lamine mobilization test is correlated with the excretion of heme precursors and hence provides an index of the "met abolically activeM lead, which is thought to represent the fraction of the totalbody lead that is responsible for adverse metabolic effects.111?H^138 Thus, the CaEDTA mobilization test is currently the most widely used procedure for estimating both current and remote absorption of in creased amounts of lead. Although this test serves as one of the important current criteria for the diagnosis of nephropathy due to lead in adults, it cannot be used to make a positive retrospective diagnosis of acute lead encephalopathy in subjects with permanent brain damage. Children who have sustained permanent brain damage as a result of acute encephalopathy may not have a persistently increased residual "mobile" fraction of the body lead burden, as measured by the CaEDTA mobilization test. These and other data relative to the metabolism of bone in children and adults suggest that the CaEDTA mobilization test has as yet undefined limits as an index of high levels of lead absorption and storage in earlier years. Because of its usefulness in diagnosis and in studies of dose-response relations, there is urgent need for the standardization of procedures for administering the CaEDTA mobilization test. Standardization -163- DUP050055452 would greatly facilitate both comparisons and interpretation of results from different laboratories. Experimental observations show un-^ equivocally that oral administration of CaEDTA can increase thfc absorption of lead present in the gut ; consequently , oral CaEDTA tests may give misleading estimates of the "mobile" fraction of the body lead burden, in that the amount of lead measured in the urine, may include that absorbed from the gut owing to oral administration of the drug. Untoward reac tions to CaEDTA are apparently related to both dose and rate of parenteral administration. The lower dose (l g) and the lower rate of infusion (l hr) used by Emmerson1^ provide both an adequate margin of safety and meaningful data. No untoward reactions have been reported by Emmerson, even in adults with renal insufficiency , and a comparable technique has also been used in children without untoward reaction (25 mg/kg of body weight administered intramuscu larly to a maximal dose of 1 g). (For intramuscular injection, procaine is added.) Urine should be collected quantitatively for 24 hr (4 days in persons with renal insufficiency). This test should not be used in symptomatic patients or those with blood lead content greater than 100 pg/100 g of whole blood, because the dosage may be insufficient to meet their therapeutic needs. Clinical judgment is clearly required in the use of this test in persons with high blood lead content, but no significant reactions have been reported in persons with normal or slightly increased blood lead content. Selander has used d-penicillamine in a standard routine way with other drug support. With impaired renal function, the use of CaEDTA or d-penicillamine should be approached with caution, particularly when they are used therapeutically. -l64- DUP050055453 The measurement of heme precursors (PROTO, COPRO, ALA) provides the most sensitive index of current and recent absorption of inorganic lead salts . Normal values in "healthy" subjects and those with overt clinical lead poisoning are listed in Table 4-10. Increases in urinary ALA and COPRO precede the appearance of clinical symptoms, and the values decrease when abnormal absorption is terminated. Measurement of urinary COPRO apparently is relatively insensitive to increments in occupational exposure to airborne lead. Various workers agree that > among urine tests , urinary ALA provides the best index of the level of absorption of leadl^S,153,231,504 (see Fig. 4^-5). In persons with blood lead content over 40 yg/100 g of whole blood, arithmetic increases in blood, lead concentration are apparently associated with exponential increases in urinary ALA and COPRO excretion, as well as exponential increases in the "mobile" or "metabolically active" lead in the tissues, as measured by the CaEDTA mobilization test. Most of the analytic procedures currently in use for measuring ALA in urine are based on the method of Mauzerall and Granick .392 Haeger-Aronser30 recently reported that the method of Grabecki ;et al,,217 which does not require chromatography, gives comparable results. These methods, as noted by Mauzerall and Granick, also measure aminoacetone, an aminoketone that apparently fluctuates with food intake.lo5 ' 390 Aminoacetone excretion is not influenced by lead,'*"1*"' but such methods may still be suitable for the control of exposure of industrial workers. However, more specific methods?^, 5^5 ...... ' -w -165-- ........ '-& DUP050055454 that exclude aminoacetone are indicated in studies designed to measure normal and minimally increased urinary excretion of ALA. Measurement of ALA in random urine samples in children is not suited for the early detection of increased lead absorption.52 Owing to the wide variation in the concentration of ALA in random samples of urine from young children, large proportions of both false-negative and false-positive results have been encountered in attempts to adapt this test for mass screening. Because of this variation, the test seems relatively in sensitive in children with blood lead concentrations from less than 80 to 90 yg/100 g of whole blood. This is in contrast to experience in the medical supervision of industrially exposed workers, in whom serial measurements may be made under rather standardized conditions. In such persons, it is the trend of frequently repeated measurements that is most useful to the industrial physician. In industry> urinary ALA is one of several indices of the level of absorption that may be used serially in evaluating individual workers. This is quite different from the situation among children, in whom the attempt has been made to use a single determination of the ALA concentration in random samples of urine as the sole diagnostic test. ALA is stable in acid urine, hut not in neutral or alkaline urine.Hi>231 Hydrochloric acid, refrigeration, and protection from light may be used for preservation until analysis. Critical factors in the analysis of FEP and FEC in blood and COPRO in urine are discussed by Schwartz et al.^8>^99 Wramne.^ Among the important factors are use of dilute iodine as the oxidant in the A -166- DUP050055455 analysis ,avoidance of ether, (it may contain peroxides ) stabilization of COPRO in urine by addition of alkali (sodium carbonate)4^ and sulfanilamide, and protection from light . The use of ethyl acetate in place of ethyl ether is safer and minimizes interference due to peroxides. Although the quantitative determina tion of porphyrins in blood is limited largely to research, a simple rapid technique for estimating the proportion of fluorescent erythro cytes (due to PHOTO) by fluorescence microscopy is reported to cor relate well with CaEDTA mobilization test results in the case of highlevel current exposure. 427 Diagnostic Criteria The various types and degrees of adverse metabolic, functional, and clinical effects attributable to increasing concentrations of lead in the tissues may be related to the concentration of lead in blood and, to a limited extent, to endogenous urinary lead excretion. This is shown schematically in Table 4-11, in which much of the information presented earlier in this chapter is summarized. The table is based on the concept that the rates of absorption, excretion, and storage of lead are interdependent. When the rate of absorption exceeds the rates of excretion and storage, the concentration of lead in the soft tissues rises. As soft-tissue concentration rises, adverse metabolic effects begin to appear, and these are followed by adverse functional and clinical effects. Borne of the functional and clinical effects are reversible. The concentrations of lead in blood shown in the table have not been corrected for deviations in hematocrit -167- DUP050055456 from normal. The overlapping values reflect, therefore, the effects of anemia and the uncertainty of clinical diagnosis, particularly in persons with mild symptoms and other disease states associated with symptoms similar to those of lead poisoning. In persons with blood lead concentrations lower than approximately 1*0 yg/100 g of whole blood, no adverse effects related to current exposure have been demonstrated in vivo, although changes in the level of ALAD activity in hemolysates of human blood have been demonstrated in vitro. These may be classified diagnostically as normal or "healthy" persons . Epidemiologic data indicate that most of the general population is in this category .20Blood lead concentrations in this range may be found in persons whose excessive exposure(s) have been remote in time. Level XI in Table 4-11 designates persons with a minimal increase in urinary ALA excretion only and is associated with blood lead concentrac tions between approximately 40 and approximately 60 yg/100 g of whole blood. Epidemiologic data 9 79 7 indicate that some groups residing or working in congested urban areas might be expected to show a minimal increase in urinary ALA if it were measured. Mean blood lead concen trations between 4.0 and 6.0 yg/100 g of whole blood have been reported in two studies of urban children. with respect to ambient airborne lead, all population groups so far reported are in Levels I and II, as defined in Table 4<-ll. The term "asymptomatic increased lead absorption" is used to designate persons with evidence of increased lead absorption and storage and increased concentrations of ALA and pyrroles in blood and urine, but * -I68- DUP050055457 without evidence of renal impairment, anemia due to lead, or other symptoms compatible with acute lead intoxication. The hematologic disturbance is compensated by increased red blood cell production. Alterations in the properties" of red blood cells may be demonstrable in vitro in this group. It also appears that some may have abnormali-^ ties in peripheral nerve conduction. It is rare to find subjects with blood lead concentrations greater than 60 pg/100 g of whole blood who have not had some exposure to lead, in addition to that found in normal diet and ambient air in urban areas. With respect to children, the term n.asymptomatic increased lead absorption" has been defined in a few cities for legal purposes as a confirmed blood lead concentration greater than 60 pg/100 g of whole blood. It has been recommended recently that this limit be revised downward.lOT Table 1-11 indicates considerable overlap in blood lead concentrations between Level III (compensatory biologic mechanisms invoked) and Level IV (acute lead poisoning). For example (Fig. ^-5)? there may be much variation in urinary ALA, as well as in the excretion of other heme precursors at blood lead concentrations between hO and 80 pg/100 g of whole blood. Sustained blood lead concentrations above approximately 80 or 90 pg/100 g of whole blood are uniformly associated with deranged heme synthesis, as well as other clinical manifestations of lead poisoning. The diagnosis of acute lead poisoning is based on a full consideration of metabolic, functional, and clinical factors. Classic acute lead poisoning has been defined as the presence of lead-related anemia. -I69- DU P050055458 symptoms exclusive of central nervous system manifestations, or both. In the presence of anemia due to other causes , severe anemia due to lead poisoning, or other diseases (such as renal insufficiency 'and sickle cell disease) that may modify a subject's response to increased lead content in the tissues, symptomatic lead poisoning may be associated with blood lead concentration as low as approximately 50 or 60 yg/100 g of whole blood, If inorganic lead contributes to the symptom complex in such patients, either heme precursors (e.g., ALA' in urine) will be greatly increased, the response to the CaEDTA mobilization test will be abnormal, or both. In view of the nonspecificity of mild symptoms of acute lead poisoning and the ease with which they may be confused with acute alcoholism, acute intermittent porphyria, and various surgical and nqnsurgical abdominal conditions, a thorough laboratory evaluation with respect to lead is essential, and even then, differential diagnosis may be difficult. Acute lead encephalopathy is almost always associated with blood lead concentrations greater than 120 yg/100 g of whole blood (Fig, 4-2) 9 marked diuresis of lead in response to chelation therapy, and the clinical findings described earlier in this chapter. According to present criteria, the diagnosis of chronic lead nephropathy requires the demonstration of an increased body lead burden by the CaEDTA mobilization test (or bone biopsy) and the presence of functional renal impairment as previously described. Permanent residual effects of acute central nervous system injury after acute lead encephalopathy is not necessarily associated with the persistence of an increased body lead burden as measured by the CaEDTA mobilization test. ::K -170- DUP050055459 All reported instances of permanent injury to the nervous system and kidneys in man that have "been attributed to lead have been associated * with a history of single episodes of acute lead encephalopathy, recurrent episodes of symptomatic plumbism, or prolonged absorption of greatly increased amounts of lead. The exposure to lead of persons with permanent renal or neurologic injury caused by lead must have been, at some time, greatly in excess of that which can be attributed to lead in the ambient air. Whether persons who meet the above criteria for asymptomatic increased lead absorption may also manifest subtle impairment in renal and nervous system function * or for that matter endoerinologic function, as they do in heme synthesis and various other hematologic indices, is not known; as indicated in Table 4-11, no data are available from which such estimates can be made. Summary Blood lead concentration, spontaneous urinary lead excretion, and the CaEDTA mobilization test may be Used to measure current and recent levels of expos\ire to lead. Spontaneous urinary lead output , as determined either quantitatively or qualitatively may not be increased in persons who have an increase in the mobile fraction of body lead burden but who also have renal insufficiency. In the presence of renal insufficiency, urinary lead output should be measured quanti*tatively for 4 days after the parenteral administration of' CaEDTA. The available data indicate that the CaEDTA mobilization test pro vides an estimate of the mobile fraction of the total body lead burden and -171- DUP050055460 experiments in animals suggest that most of the mobile fraction is derived from bone. However* it appears that only a fraction of the lead in bone is mobilized by chelating agents. Autopsy analyses indicate that bone biopsy is required for estimation of total body lead burden* but this has rarely been done in the living. However* it is the mobile frac tion of the total body lead burden that is apparently responsible for the adverse immediate effects of lead. The CaEDTA mobilization test may not necessarily provide an index of irreversible tissue injury that has resulted in tissue scarring in the very remote past. The concentrations of heme precursors In body fluids and urine pro vide the best understood metabolic indices of the adverse effects of lead. Of these * ALAD activity in hemolysates of blood and ALA In urine have been most extensively studied with respect to various degrees of absorption of lead. FEP has been studied in normal popula tions and in persons with overt lead poisoning* but not at inter mediate subclinical levels of increased lead absorption. Studies of UCP at intermediate levels of increased leacl absorption are limited. Nevertheless, the pattern of increase for all these heme precursors appears to be similar. Arithmetic increases In the lead content in blood above 40 yg/100 g of whole blood are associated with exponential increases in urinary ALA output* urinary CQPRO output* and urinary output of lead after administration of CaEDTA (or d-penici 11 amine). Significant correla-. tions are also found between the chelatable lead and the excretion of heme precursors and suggest that urinary ALA and UCP provide an A -172- DUP050055461 index of the "metabolically active11 or toxic fraction of the total body lead burden. For the clinician, the curvilinear relation between blood lead content and ALAD, ALA, UCP, and chelatable lead indicates that arithmetic increases in blood lead concentration are associated with an exponentially decreasing margin of safety. For example, an in crease in blood lead content from 10 to 60 pg/100 g of whole blood may be associated with relatively little change in health status, whereas an increase from 60 to 80 yg/100 g of whole blood markedly increases the risk of symptomatic illness. The diagnostic evaluation of human subjects may be based on the concept of exponentially in creasing risk that attends arithmetic increments in blood lead con tent. It is possible that increments in spontaneous urinary lead output may follow a similar pattern, although the available data do not show this clearly. -173- DUP050055462 EPIDEMIOLOGY Children Today in the United States, lead poisoning in children is believed to be due almost entirely to the repetitive eating of leaded house paint. Most of the available data are based on retrospective analyses of small groups of cases from cities in which childhood lead poisoning is a reportable disease and in which the municipal health departments have active programs--namely, New York City, Chicago, Philadelphia, and Baltimore . Although the nationwide incidence of the disease in children is unknown, some epidemiologic features are well established: prevalence of pica in preschool children, seasonal variation, age distribution of cases, association with dilapidated and deteriorating housing, race, and sex. The term "pica" has been defined as the repetitive ingestion of things that are not food (e.g., string, dirt, paper, putty, paint, clay, and cigarette butts). It is important in a variety of accidental poisonings in preschool children. 5^9 The behavioral, and biologic factors responsible for this age-related activity are not understood. 370 401 Lourie and his associates 5 have stressed disturbed parent-child relations as a factor that can intensify pica in the child, whereas I29 Neumann has suggested that it may be a vestigial instinct. Lourie and associates^*and Sobel'^ found that the habit begins at the age of about 12 months ., that it may persist until the age of 3-5 years, and that it occurs in at least 50# of children in both middle-class and poverty groups. Thus, pica is apparently a -174- DUP050055463 rather common behavioral activity in normal preschool children, and it may become intensified in response to stress. The well-recognized association between childhood lead poisoning and 53,130,225,270,272,364,497,592 old housing has been. discussed earlier in this chapter. No differences related to sex have been found. The vast majority of reported cases are found in Negro and Puerto Bican children. This concentration of cases is apparently related to the high concentration of such children in deteriorating urban housing and not to any known genetic, racial, or ethnic factors. The seasonal distribution of the fatal and nonfatal cases for which blood lead concentrations are given in Fig. 4-2 are shown in Fig. 4-9. This is characteristic of the seasonal distribution reported through- out the United States.53,130,27,272,364,592 Between 85 and 90% of all fatal and nonfatal symptomatic cases are recognized during the months of May through October. Blanksma et_ al.^3 re ported data that suggest that seasonal factors are also operative, even in "normal" circumstances. In a group of 746 control children under 6 years old without undue exposure to lead, the mean blood lead content for the entire year was 23.5 .7*8 pg/lOO g of whole blood. However, within this group, the mean peak blood lead content by month of testing was found in June (mean, approximately 36 pg/100 g of whole blood), with a secondary peak in February . This observation requires confirmation. -175- DUP050055464 Within the preschool age range, 80-85$ of all reported fatal and non- 1 P70 P7P c q a c t q p fatal cases occur in children 12-35 months old. 5 9 ? (Fig. 4-10). After a survey of 103 children from the metropolitan Philadelphia area who did not have anemia, a history of pica, or * VfO signs suggestive of poisoning, Robinson et_ al^. report that blood lead content is constant between the ages of 6 months and .13 years (median, 27 yg/100 g of whole blood; middle 90$ range, 15-40 pg/lOO g of whole blood). This may be contrasted with the findings in 1953-1954 in a metropolitan pediatric clinic serving children who lived exclusively in dilapidated housing. The findings in 333 children distributed by age and blood lead content are shown in Table 4-12 > Among 219 children 10-36 months old, 106 (48.4$) had blood lead concentrations of 50 pg/100 g of whole blood or higher and eight (3.7$) had blood lead concentrations of at least 100 yg/100 g of whole blood, the point above which the risk of encephalopathy be comes significant. Localized concentrations of cases in the very worst housing areas have been noted in other cities in 1- and 2-year,- olds j270,530 Although age-related data are not given, Blanksma al.^ noted differences between various geographic areas within Chicago in the incidence of blood lead concentrations over 50 yg/100 g of whole blood (Table 4-13). However, there are no published data on blood lead content in healthy children who live in modem safe housing. Estimates of the incidence of lead-paint poisoning and the prevalence of blood lead concentrations that may be associated with adverse health -176- DUP050055465 v*| 'w* * '..) effects can be made only on the most tenuous grounds. With the exception of a few of the larger cities, it is not a reportable disease. For the most part, recognition is based on the case finding approach, which in turn depends on the level of awareness in the local medical community and the availability of diagnostic laboratory facilities. Only in Chicago (since October 1966) and in New York City (since January 1970) are prospective mass-screening programs currently in operation for young children. The Chicago program is concentrated in nine areas in the city and includes the operation of an ambulatory treatment center. About 68,800 children under 5 years old in these areas (47$ of that segment of the population) were tested in 1967 and 1968 (Table 4-13). Of these, 3*935 (5.7%) had blood lead concentrations of 50 yg/l00 g of whole blood or higher. 53 An additional 48,000 children were tested in 1969 The numbers and percentages of children found each year to have blood lead concentra tions of 50 yg/100 g of whole blood or higher were 2,379 (8.5$) in 1967, 1,556 (3.8$) in 1968, and 1,172 (2.4$) in I969. During 1967, and 1968, 1,15^ (l.68$) of the children tested were treated as cases of poisoning, and in 1969, 456 (0.95$) were treated as cases of poisoning. This decrease after the first year (1967) in the numbers of reported cases may be attributed to the backlog of previously un^ recognized cases, first identified at the inception of the screening program. A "steady state" may be anticipated when a backlog of older children is finally reduced and new cases are confined to the 1- to 2-year-old group representing the annual input of new births. A con tinued input of new cases at a steady rate may be anticipated so long -177- DUP050055466 as the hazard in old housing exists and the number of deteriorating dwellings containing lead-based paints remains unchanged* In New York City, during the first. 7 months of 1970, 64,644 blood specimens were analyzed for lead by the Health Department; 2,070 children with blood lead concentrations of 60 yg/lOO g of whole blood or greater were found, and two deaths attributable to lead 530 poisoning were reported. The distribution of blood lead concentrations indicates a mean of approximately 20 yg/100 g of whole blood with 11$ greater than 50 yg/100 g and 0.2$ 110 yg/100 g or greater in children 1-6/* years old. 530 The epidemiologic data cited above are, in general, limited in that only children with blood lead concentrations greater than 50 or 60 yg/100 g of whole blood are identified. The percentage of children in these groups with concentrations greater than 40 yg/100 g of whole blood is clearly much higher. Elsewhere in this chapter, evidence is presented that urinary ALA excretion increases as blood lead content rises above 40 yg/100 g of whole blood and that increasing urinary ALA excretion represents metabolic interference due to lead in the biosynthetic pathway for the synthesis of heme. ' Monthly dustfall samples were collected in 77 midvestern cities in 266,6i4 1968. The mean lead concentration in dust from various sectors of each city was calculated after averaging the results for all the cities as follows: residential> 1,636 jig/g of dust; commercial, 2 >413 M-g/g of dust; and industrial l>512 Ug/g of dust. The swallowing of as much as 1 g of such dust could result in the oral intake of an 178- DUP050055467 amount of lead that exceeds by a factor of 10 or more the estimated mean daily intake of lead from normal food and drink in nonexposed young children (130 pg/day excreted in feces). 5 Although the available data on the distribution of blood lead concentrations and other epidemiologic data clearly indicate that this alone does not account for clinical lead poisoning in children, the swallowing of lead-contaminated dusts may well account, in large part, for the higher mean blood lead content in urban children^^^5 and the rather large fraction whose blood lead content falls in the range of 40-60 pg/100 g of whole blood, thereby bringing them into the range in which increased urinary excretion of ALA may be observed. For a child with pica for paint, the combination of the ingestion of a few chips of paint and an increased intake of lead from con taminated dusts would provide a total lead intake sufficient to cause symptomatic illness. In summary, adequate epidemiologic data based on proven methods of lead analysis (dithizone technique) on the distribution of blood lead concentrations in young children without undue exposure to lead are not available . The problem is complicated by the prevalence of pica in young children. The few data that are available are based largely on samples of children in metropolitan areas. In these groups , mean blood lead concentrations are somewhat higher than those found in adults, but in one report the range did not exceed 15-^0 pg/lOO g of whole blood. One unpublished report showed that rural and suburban children have lower blood lead content than -179- DUP050055468 adults in similar environments. However, large-scale screening pro grams in urban areas suggest that perhaps 5-10$ of young children who live in deteriorating old housing have concentrations of lead in their blood that may be associated with adverse metabolic effects at the very least ; and 1-2$ may have evidence of lead poisoning. Where active screening programs are in operation, the numbers of reported cases increases while the numbers of reported deaths decrease to well under 1% of reported cases. Season, age, pica, and housing are the identified epidemiologic determinants of childhood lead poisoning in the United States today. The extent to which airborne lead in congested urban areas contributes to increased lead absorption and lead poisoning in children is not clearly defined. With respect to young children, it is not a matter exclusively of inhalation and particle size, inasmuch as young children mouth and actually eat things that are not food rather indiscriminately. Airborne lead wastes from such sources as automotive emissions and the weathering and demolition of old build ings can be expected to have a significant additive effect on the total intake * This would be sufficient to evoke compensatory metabolic responses that are now considered sub clinical (such as increased urinary ALA) at the very least . It may be estimated that dustfall from airborne lead, if swallowed, can make a significant contribution to a small child's total lead intake and thereby contribute to the occurrence of lead poisoning, especially in urban areas. Even so, the direct ingestion of lead-pigment paints is clearly the principal environmental source in cases of severe acute lead poisoning in young children. -180- ; * DUP050055469 Adults In studying diseases due to a single environmental agent, such as lead, three main stages in man^ state of knowledge may he distinguished. The first is the recognition of the diseased state, without apprecia tion of the nature of the causative agent. It is at this stage of man's knowledge that Paul of Aegina and Avicenna described patients suffering from "colicky affections11 accompanied by paralysis which may well represent lead poisoning, but by the second century B.C, some men had already entered the second stage of man's knowledge of the biologic effects of lead, with the recording of unequivocal evidence linking the disease to the causative agent, lead. At that time , Nikander wrote his "Alexipharmaca" describing both colic and paralysis as the result of ingesting litharge (lead oxide). The third stage of knowledge of the biologic action of an agent, such as lead, comes with the demonstration that the severity of the effect produced is directly related to the amount of the agent . This third stage is often delayed, because only severe disease produced by massive doses is recognized at first and more subtle effects are missed. Such situations are illustrated by the sporadic and usually accidental lead exposures reported after 1500, at about which time the development of printing made the dis semination of such reports easier and their preservation more certain. In 1572, an epidemic of lead poisoning occurred in the French province of Poitou, owing to drinking wine adulterated with litharge; from then on, repeated epidemics of lead poisoning due to intentional -l8l- DUP050055470 or accidental adulteration of cider,rum/^* or foocP^aor to drinking water conyeyed in lead pipes'3 ' were reported. More recently, there have he eh outbreaks of lead poisoning presumably due to airborne lead particles'from the burning of old automobile batteries to provide domestic heating.222 * 593 it is with the beginning of the Industrial Revolution, with the splitting of the manufacturing process into discrete operations performed by separate groups of workers with different degrees of lead exposure, that it becomes possible to study sickness rates and relate them to the amount of lead in the workers1 environment. One of the first studies that clearly showed a relation between degree of lead exposure and amount and severity of the sickness 166 produced was that of Puckering in 1908. As a re soilt of his measurements of the amount of lead in the air breathed by workmen in a number of British factories and his records of the number of men with signs of lead intoxication, Legge and Goadby suggested in 1912 that, "if the amount of lead present in the air breathed con tains less than 5 milligrammes per 10 cubic metres of air, cases of encephalopathy and paralysis would never, and cases of colic very rarely occur. And this figure is a quite practical one in any process amenable to locally-applied exhaust ventilation. Somewhere about 2 milligrammes, or 0.00.2 gramme, of lead we regard as the lowest daily dose which, inhaled as fume or dust in the air, may^ in the course of years, set up chronic plumb ism." 34 8 * -182- DUP050055471 With the demonstration that exposure to a specific amount of lead caused a specific biologic response and that the clinical response became less as the dose of absorbed lead diminished, the prevention of the disease depended increasingly on the ability of engineers to control the amount of lead in workmen * s environment. This ability to control lead poisoning by a combined medical and engineering approach backed by government regulations is illustrated by the ' figures for notified cases of lead poisoning in Great Britain, which ' q 1l 7 decreased from 1,058 in 1900 to 239 in 1928. Similar studies in the United States l64 5 222 confirmed that the number of cases of plumb ism was positively correlated with the amount of lead in the air, as shown in Table 4<-l4.* Because the major route of entry for lead in an industrial worker is his lungs, information from the study of industrial populations commonly relates the clinical or biochemical findings to the con centration of lead in the air. But similar relations between the amount of lead absorbed and the biologic effect produced have been found for ingested lead299 and for lead injected intravenously as a therapeutic agent. 43` It has also been found that the degree of exposure to lead is reflected in the concentrations of lead in the blood and urine of exposed persons. Such relations may vary considerably when the values from different persons are compared, but the variation is much less when the average or median For reasons given in the discussion of Table 4-15, data such as those in Table 4-l4 can be used to show qualitative relations only, and no conclusions as to allowable limits for the concentration of lead in the air should be drawn from this information alone> -183- DUP050055472 values for groups of persons are studied. The values in Table 4-15 should be used only to show a general tendency for increasing ex posure to be reflected in increased blood lead concentrations, ' inasmuch as the exposure to lead is hot completely described in terms of particle size, which is known to play an important part in the deposition and absorption of lead in lungs. Another factor that tends to reduce the degree of correlation between the lead concentration in blood and that in the air in the study summarized in Table 4-15 is the medical control exerted by the physicians in charge of the workers. This control removed many of the men in the high-exposure group and placed them in areas of lower exposure; they appear in Table 4-15 under the heading "Exposure changed." 594 A more recent study- that illustrates the relation between lead exposure and urine and blood lead concentrations yielded the data in Table 4-l6. In this study, by Williams , although the number of men observed (39) was not large , the use of personal air samplers and the number of observations made make the results more valuable than usual. From these data, Williams calculated correlation co efficients (y) and regressions of the biochemical value (Y) with the mean 8-hr lead-in-air concentration (X). The results are shown below; 7Y X Blood lead 0.90 30.1 + 201 X - 0.0975 + 0.00399 Y Urine lead 0.82 45.5 + 486 X - 0.0277 + 0.00138 Y A m -181+- DUP050055473 Experimental exposures of human beings to known concentrations of airborne or ingested lead have provided graphic data describing the relation between degree of exposure and blood and urine lead concen trations , ^ All the information so far discussed deals with occupational or experimental exposure, but a similar correlation between lead con centrations in air and blood has been found at air lead concentra- . 3 206 tions above 2 yg/m in the urban environment. There have been no cases of characteristic lead poisoning due to lead as a community air pollutant. Therefore, from this point on, attention will be centered on studies of health effects of lead other than classic lead poisoning. Three main epidemiologic approaches have been used in seeking such health effects: to compare the health of occupational groups with documented lead exposures with the health of groups not so exposed, to study persons with a given disease and compare their tissue concentrations of lead with those of persons who do not suffer from the disease but are otherwise similar, and to study the geographic distribution of a given disease and compare it with the geographic distribution of a type of lead exposure. Health Effects. The first method, if properly carried out , probably offers the chance of the greatest precision, b.ut it suffers from some limitations inherent in the nature of an employed population, such as restrictions in age range and initial state of health. In the case of the lead industries, it is also likely to be pre dominantly a male group. -185 DUP050055474 There have been very few epidemiologic studies of groups occupationally exposed to lead in which a large number of factors were documented and the results in different exposure groups conqpared. One of the earliest of such studies to use modern epidemiologic methods was of persons exposed to lead arsenate as a result of its use as an insecticide. As in most epidemiologic studies,. which may be looked upon as naturally occurring experiments in which the investigator has hS6 little or no control over the experimental design , this study/of the orchardists in the Wenatchee area of Washington State was complicated (as far as this discussion is concerned), in that it dealt with ex posure to lead arsenate. This raises the question of the relative importance of the lead, compared with the arsenical, portion of the lead arsenate molecule in the overall toxicity of the compound. An attempt to answer this was made by special studies -in animals . It was found that the greater toxicity of the molecule is to be attributed to the lead radical, rather than to the arsenic; no synergistic action of lead and arsenic was found. 179 The lead concentrations , shown in Table U-17, are not as high as those sometimes seen in other industrial exposures, but that makes them particularly important:, because the urine lead concentrations range from slightly higher than commonly found in urban communities to the same as in moderate lead exposures in industry . Among the factors studied in assessing the health of the orchardists were weight, blood pressure, diseases of the cardiovascular system, skin dis orders, eye irritation, chronic nervous diseases, blood dyscrasias. : * -186- DUP050055475 kidney disease, pulmonary tuberculosis, visual acuity, syphilis, neoplastic disease, and fertility. Each was studied to find out whether it had been modified by the lead arsenate exposure. Insofar as comparative data for other populations were available , no evidence was found that any of these factors was altered by the exposure. Special.attention was given to medical examination of children, because in the Wenatchee area, where orchards surrounded the communities or the houses in which they lived, there were unusual opportunities for children to be exposed to lead arsenate Insecticide sprays and spray residues On branches, leaves, and grass, in addition to residues Ingested on apples. In only one respect did these children differ from children in other districts : their urinary lead and arsenic concentrations were nearly twice as high as those of a group of 18 children measured at the same time in Washington, D.C. (who had a mean urine lead content of 0.026 mg/liter; SD, 0.0128). There was no indication of adverse effects of lead arsenate exposure on the health of the Wenatchee children. Although the study of persons exposed to lead arsenate has many deficiencies as a source of information on the possible biologic effects of lead as an urban air pollutant, it is particularly important for two reasons: it is one of the few modern studies that include a substantial number of women of childbearing age and it is one of the few studies that include children exposed to lead in a form other than paint. The study (previously referred to)l6h of storage-battery workers whose blood lead concentrations were shown in Table ^-15 also looked for effects -187- DUP050055476 of exposure to lead on the incidence of diseases other than plumbism. Of the 766 men studied, 75$ had been employed in electric^storagebattery plants for more than five years, and about 12$ for 20 years or more. In this study, the incidence of disease in the highexposure group (exposed to 0.15 mg/m or more) was compared with the incidence in the low-exposure group (exposed to less than 3 0.15 mg/m ), The prevalence of arteriosclerotic-hypertensive heart disease did not increase with increasing atmospheric lead concentra tion. In the age range 45-54, which covers the period when de generative cardiovascular disease is making its appearance, 53 workers were exposed to atmospheric lead concentrations below 0.15 mg/m^, and 32$ were found to have arteriosclerotichypertensive heart disease as defined in the study. Of the 66 workers in the same age range exposed to concentrations in excess o of 0.15 mg/m , 32$ had this type of heart disease. Unfortunately* there was no cohort study. Another statistical treatment, shown in detail in Table 4-l8, is to use the population exposed to less than 0.15 mg/m^ as a basis for predicting the number of cases that would be expected if the same incidence prevailed in all age groups of the more heavily exposed populations* If this assumption were correct, one would expect to find 35 cases of heart diseases of the arteriosclerotichypertensive group in 263 workers 25-64 years old exposed to atmospheric lead concentrations in excess of 0.15 mg/m . Making allowance for the differences in age distribution, the 37 cases found agree closely with the expected number. The agreement is even closer -l88- DUP050055477 for the 102 workers who changed jobs within 5 years of the time of study, the expected 13.7 checking the observed number of cases, l4. Thus , it may be concluded that an increase in lead exposure was- not accompanied by any increase in the prevalence of this group of diseases of the cardiovascular system. When lead-affected workers are compared in the same way as before with workers exposed to less than 0.15 mg/m^ of air, one would expect to find 27-3 cases of arteriosclerotic-hypertensive heart disease in the l69 lead-affected workers 25-61+ years old. Actually, there were 34 cases, but, considering the standard deviation (4.78) attached to such an estimate, the difference is not statistically significant. Application of a chi-square test confirms this con clusion. Response to a standard exercise test was as good in men exposed to lead concentrations of 0.15 mg/or more as in men of comparable age exposed to lower concentrations. Men who had changed jobs during the preceding 5 years also, as a class, had equally satis factory returns to pre-exercise pulse rates and systolic and dia stolic blood pressure. Blood pressures, measured with a mercury sphygmomanometer, agree closely with the average values for 6,667 industrial workers in nine other industries studied by the Public 72 Health Service. As an additional check, a tabulation was made of men with pre-exercise blood pressures in excess of 150 mm Hg. The number and percentage with higher systolic pressures, for 72 storage-battery workers compared with other industrial workers . -I89- DUP050055478 agree closely , shoving that hypertension, of this degree is no more prevalent in the storage-b attery industry than in other industries. Information on the effect of exposure to lead on the cardiovascular system has been given in some detail because many earlier writers thought this system was affected by lead. However, after reviewing those authors, Aub concluded that, nalthough the general clinical opinion holds that arteriosclerosis is a result of chronic plumbism, this idea is based entirely on the occurrence of this condition in many post mortem examinations. That arteriosclerosis is much more frequent in those exposed to lead than in other individuals of the same age, however, needs much more statistical proof before it can lt23, P-121 be accepted. Some 20 years later, Cantarow and Trunrper, in their book89^ on lead poisoning, claimed that lead at such high concentrations that lead poisoning has occurred or is likely to occur may be associated with degenerative vascular disease. Support for the view that lead at high concentrations may be linked to degenerative vascular disease is found in a British study of battery workers,1^0 in which three exposure grades were defined as: Grade A, no exposure; Grade B, negligible exposure; and Grade C, exposure represented for the last 20 years by a mean urine lead content of 0.100-0.250 mg/liter (in the past, these have frequently exceeded 0.250 mg/liter). The mean exposure periods for the pensioners in Grades B and C were 35*3 and 32.3 years, respectively; for men who died while employed, the mean exposure periods for Grades B and C were 26.8 and 21.8 years, respectively. There was found to be a * -190- DUP050055479 significant excess of deaths from all causes among the pensioners in Grade C, hut not in either of the other grades. When the deaths attributed to vascular lesions are studied in this series, a highly significant excess of deaths due to cerebrovascular catastrophes was found among Grade C pensioners. As can be seen in Table 4-19, although the deaths of pensioners before 1951 showed an excess of observed over expected in both Grades B and C, after 1950 only Grade C shows an excess of deaths. This suggests that improvements in hygienic standards, with a consequent decrease in lead exposure, may have resulted in the observed improvement in mortality experience. It is unfortunate that this study, initiated to learn the incidence of cancer among battery workers, did not yield more information on the physiologic status of the men; for instance, the incidence of hypertension in the various exposure groups would have been most interesting. The apparent difference between the experiences of European and North American investigators studying the effect of lead on the blood vessels is thought by Lane to reflect the working conditions on the two sides of the Atlantic, although he points out that the study of Dree sen et^ al.^P^ included only 12% of men with more than 20 years of exposure and that there has been no adequate study of U.S. workers exposed to high concentrations of lead for one or two decades . The study of Cramer 137 supports the view that lead workers in carefully supervised plants do not, even after long exposure, have a higher risk of hypertension than nonlead workers. He studied 364 workers, of whom -191- DUP050055480 82 had been employed for more than 20 years and 265 for more than 10 years. The incidence of hypertension in various age and exposure groups was studied and compared within groups and with data gathered in an extensive Norwegian study of men not exposed to lead. In none of these comparisons was there any evidence of an effect of lead exposure, in terms of either duration or severity, on the incidence of hypertension. The same confusion of claims and counterclaims regarding the effects of lead surround the subject of kidney disease and hypertension. Views on these conditions as on arteriosclerosis, appear to depend on the characteristics of the lead exposure of the workers studied. The earlier observers saw many more cases of heavy prolonged exposure to lead than is possible today and were convinced that chronic kidney disease and hypertension were direct, although delayed, consequences of exposure to lead. The extensive literature on this matter has been reviewed by Radosevic. h'-o2 Browning7' h points out that no actual undue rise in blood pressure of workers in the eleetric-storage- 4l battery industry was demonstrated by Belknap, Tgh Dreesen, or Lane, 332 that the absence of significant change shows that the ex posure had not reached a dangerous level, and that the relative absence of chronic renal disease in lead workers is probably due to the improve ment of conditions in modern lead industries. It appears that modern lead workers in well-supervised plants , although still absorbing more lead than other workers, do not run a risk of arteriosclerosis, hypertension, or kidney disease due to exposure ?* r * -192- DUP050055481 to lead. This conclusion is supported by the Wenatchee study, with its generally moderate level of lead exposure, and the studies of oc Baader c: *7p and Vigltani. The epidemiologic experience with groups of persons exposed to lead and other materials through their consumption of illicitly distilled whiskey is dealt with elsewhere in this publication, but in general it may be said that the kidney kll disease seen in such groups is associated with high lead absorption. Susceptibility Differences. In concluding this review of information from epidemiologic studies of lead workers, it is important to raise the question of whether some groups of workers are more susceptible than others to the effect of lead. As in every question of biologic effects of lead, the degree of exposure must be eon side red. Differences in susceptibility might show up in conditions of severe lead exposure that would not be common in the general population; it is also possible that some persons are unusually susceptible to "normal1* degrees of exposure to lead. On balance, the available evidence favors the former rather than the latter situation. Two groups have been the main subjects of concern, controversy, and legislation with regard to exposure to lead: women and children. The controversy over whether they are more susceptible than men to lead poisoning involves many of the problems that were touched on in discussing the relation of lead to kidney disease. An additional factor in the case of women and children that makes resolution of the controversy difficult is the banning of both groups from the lead trades in most western countries for the last 50-70 years. Thus, the -193- DUP050055482 only available occupational data pertain to a period when severe and prolonged exposure to lead was common and the criterion of a lead effect was clinical lead poisoning, Oliver liliO said in 1902: So fa r as occupational exposure to lead is concerned, my opinion is (l) that women are more susceptible than men; (2) that while female liability is greatest between the ages of 18 and 23 years, that of men is later; and (3) that while females rapidly break down in health under the influence of lead, men can work a longer time in the factory without suffering, their resistance apparently being greater." However* German authors at about the same time felt that the apparent suscepti bility of women to lead poisoning was explained> not by their sex> but by the fact that they were usually more poverty-stricken than the men, undernourished, and obliged to do work for their families in addition to their factory work. Also, women's skirts and hair collected the lead nr dust, so that they carried it home with them after work. Support for this view is presented by Hamilton for the U.'S* battery industry around the beginning of the century . In areas where men belonged to strong unions, they enjoyed good pay and living conditions, while the nonunionized women were underpaid, poorly housed, poorly fed, and subject to the worry and strain of supporting dependents on low wages. In these conditions, a much larger proportion of women than of men suffered from lead poisoning. In the non-unionized pottery industry, in which both men and women were making low wages and were subject to poor conditions, the incidence of lead poisoning was slightly greater among men . * * -194- DUP050055483 Information supporting the greater susceptibility of women to lead is 332 given by Lane, who quotes a British departmental committee report on the dangers attending the use of lead in the manufacture of earthen ware and china. The report makes it possible to compare the incidence of lead poisoning in men and women engaged in the same work--ware cleaners, dippers, and dipping assistants. The incidence of lead poisoning in females was shown to be 2-3 times that in males. Of course, such factors as work habits, labor turnover, and general health might influence these statistics, but the reporting committee was sufficiently impressed with the data to express its belief that women are more susceptible to lead poisoning than men. If the statistics on relative susceptibility of men and women leave much to be desired, there is even less information on whether lead poisoning produces significantly different clinical pictures in men and women. In one of the few comparisons undertaken, there is a suggestion of such a difference. Prendergastlr>8 found the following incidences of various expressions of lead poisoning among 6k0 cases of plumbism in the Staffordshire potteries: CO 11C paralysis convulsions blindness (total) blindness (partial) incidence, % men women 77.6 69.8 57.0 30.0 15.0 3k.9 2.3 7.7 3.5 10.2 -1.95- DUP050055484 These admittedly sketchy data suggest that, when there has been sufficient exposure, to lead to cause clinical poisoning, women are more likely to have central nervous system manifestations and men , peripheral nervous system manifestations . This finding might be related to the greater likelihood that men will be engaged in heavy work, and the amount and type of physical effort have been thought to affect the incidence and location of muscular paralysis. In the study of the earthenware and china workers previously referred to, the committee found that the incidence of miscarriage per 1.00 women was almost 3 times as great among those who continued to work with lead after marriage than among those not occupationally exposed to lead. 3T3 Lund In a review of the effect of lead on reproductive capacity, concluded, on the basis of studies conducted before 1920, that, "even when the mortality of that time is taken into account, the number of productive pregnancies is abnormally small" (in mothers occupationally exposed to lead). The earlier writers--Hamilton, Oliver., Legge, Cantarow and Trumper--were unanimous in their view that lead in high concentrations affected the reproductive capacity of women and that, in the light of the poor hygienic conditions in the lead trades at that time, women should not work in these trades. As a result of * these views, legislation was enacted in many countries barring women from occupations in which they would be exposed to lead. Therefore, there are almost no data on the effect of the generally much lower concentrations of lead now prevailing in these industries on reproductive 426 capacity. The Wenatchee study remains as the only source of -196- ** ,, v O DUP050055485 evidence, and that study found no effect on fertility. In summarizing their study of the fertility of men and women in the Wenatchee study, the authors concluded that 11 The instances reported in the literature of an effect of lead on human fertility appear to be limited to men and women who were far more heavily and much more regularly exposed to lead than the residents of Wenatchee. It would appear that a clinical state approaching that of frank lead poisoning is necessary before the fertility of men or women is affected." Most of the early writers on the health of lead workers have remarked on the individual variability shown by persons exposed to high levels of lead--some men become poisoned within a few weeks, whereas others can work for years in the same surroundings without apparent harm. Undoubtedly, some of this variation in response was due to differences in working habits , and even today similar variations can be found, although now it is more likely to be confined to a difference in the amount of lead absorbed, rather than to variations in overt toxicity. For instance, there is a good correlation between blood and air lead concentrations with relatively little variation from man to man when the major portion of the lead exposure is from the air; but when the work involves such jobs as hand pasting of battery plates, with a much greater chance of "local" lead contamination, the variability of lead absorption between men is as much as fourfold. SO h Therefore, it is probable that at least some of the apparent individual resistance to lead poisoning seen by the earlier writers reflects variations in working habits, and therefore in lead absorption. There remains, however, a -197- DUP050055486 likelihood that different persons respond differently to a given dose of lead, it seems unlikely that this difference would he of any practical importance at the degrees of lead exposure now found in the general population,20 hut.this merits further study. Such studies should first consider persons who are known to have biochemical defects in systems of the body that are recognized as influenced by lead, such as sickle cell anemia, glucose-6-phosphate dehydrogenase deficiency, the porphyrias, thalassemia, and gout. Relation of Tissue Concentrations of Lead to Disease. Although studying the tissue levels of lead in various diseases and looking for a con gruence between disease distribution and lead exposure are attractive concepts, when the results are positive, the investigator is faced with a formidable task in dealing with the question: Did lead cause the disease under study? This problem of deciding whether two variable^^T are merely associated or causally related is dealt with by Bradford Hill. Among the aspects to be considered in deciding for or against a causal relation between two variables, Hill suggests: strength of association, consistency, specificity, temporal relation of association, dose-response curve, biologic plausibility, coherence with known facts, experimental evidence , and judgment by analogy . Tissue lead concentrations have been studied in two diseases other than classic lead poisoning: renal disease in Queensland, Australia, and multiple sclerosis in England. The Queensland study of bone lead content 250 in renal disease , showed 160 eases of Bright's disease (chronic renal disease) in 866 autopsies. Of those 20-49 years old at the time 1* * ^ -198- DUP050055487 of death, 6*5% had no known cause for their renal disease and the re mainder suffered from diseases recognized as involving the kidneys. The hone lead concentrations of the persons with renal disease of known causation were significantly lower than those of the persons with renal disease of unknown causation; this lends support to the belief that longstanding absorption of lead in sufficient quantity can cause chronic renal disease. The source of the lead in the Australian cases was childhood ingestion of exterior house paint. The authors of the study suggest that surveys of the lead content of tissues taken from persons who die of renal disease of unknown cause might be useful in providing diagnostic clues to the cause. Their conclusions have been supported by Emmerson*173 who Was able to show, in a group of Australian patients with renal disease of undetermined origin, that the mobilization of lead from bone by a standardized infusion of calcium EDTA gave evidence of a significant past exposure to high concentrations of lead in 12 of l6 cases, which suggested that lead played a part in the causation of the renal disease. 87 Campbell reported that teeth from patients with multiple sclerosis contained* on the average, significantly higher concentra tions of lead than teeth from normal, healthy persons. This observation was followed up by Butler,80 who observed that physiologic changes accompanying chronic diseases may promote the deposition of metals in the skeleton, as illustrated by the increased deposition of zinc in teeth in tuberculosis. Butler analyzed urine, blood, cerebrospinal fluid, and bone in 31 patients with multiple sclerosis and in 54 persons -199- DUP050055488 with other neurologic disease. Lead concentrations in the two series did not differ significantly from commonly accepted normal values. Examination of necropsy tissue, including brain and spinal cord, from patients in whom the diagnosis' of multiple sclerosis was confirmed histologically did not show significantly increased lead content. Butler concluded that his study did not support the view that lead plays a part in the etiology of multiple sclerosis. Geographic Distrlbution. The final epidemiologic relation to be ex plored is that of geographic distribution of a disease to some index of exposure to lead. Two diseases have been considered in this light: 577 multiple sclerosis and cancer. Warren has drawn attention to the great variation in the incidence of multiple sclerosis in different geographic areas of Norway, Scotland, Sweden, England, and Canada; some of those areas with a high incidence of the disease have lead-bearing rocks. There are many reasons for not accepting this as a causal relation. It is difficult to obtain adequate statistics on the true incidence of a disease whose prevalence is normally between 30 and 60 per 100,000 of population; if deaths are the basis of the reporting, mortality rates of the disease may vary from zero to 4 per 100,000. In the areas reported as having a high incidence, such as the Orkney and Shetland Islands of Scotland, with a total population of around 36,000, the addition of a single case of a disease that is particularly difficult to diagnose with certainty can increase the overall prevalence by as much as 25$. Additional impediments to accepting a causai relation 325 are the strong North^South gradient in prevalence; the lack of 4* V -200-- DUP050055489 recognition of multiple sclerosis as a sequela of industrial exposure to lead; the failure to show increased lead content in the brain, blood, 80 or urine of multiple sclerosis patients; ` and the evidence that multiple sclerosis is probably an autoimmune disease or possibly related to an earlier viral infection. 515 The epidemiologic evidence that lead can cause cancer in man is mentioned more because of the seriousness of the disease than because of the strength of the evidence. Warren577 points out that, if variations in cancer mortality rates are plotted on a geographic basis, striking 12 differences appear among some circumscribed areas. Alien-Price, on the basis of a survey in Devon, England, claims that the incidence of cancer is higher in persons living on the highly mineralized Devonian geologic formation than in persons living on the adjoining carboniferous and granite formations. Warren states that in some cases rocks containing more than 1000 ppm of lead can be correlated with some of the high-cancer areas encountered by Allen-Price, but this was not a consistent finding. Warren mentions two other rather tenuous examples and suggests that further studies be carried out. Apart from the difficulties, mentioned earlier, of obtaining reliable mortality statistics for small geographic areas, the even larger problem of the reliability of the information on which the cause of death is based is a well-recognized epidemiologic problem. Although it is known that lead at concentrations of 1$ or more in the diet causes renal cancer in rats (see earlier in this chapter for a dis cussion of these experiments) , there is no evidence that this happens -201 DUP050055490 in other species, including man Lane's study of lead workers was undertaken specifically to examine the incidence of cancer in this occupational group; no Increase in the incidence was found. Although many human diseases have been alleged to be caused by lead, cancer is not one of them; even in the days of heavy and prolonged exposure to lead sufficient to cause renal disease, lead was not thought by clinicians to be linked to cancer. &.. ! -202 vv o DUP050055491 BEHAVIORAL EFFECTS Behavioral Effects in Man Overt and Acute Behavioral Changes. Lead poisoning is characterized by a range of overt symptoms already described in detail in previous sections of this chapter. Among the changes in man associated with higher nervous system function are severe headaches, depression, insomnia* and irritability. Sensory effects have also been commonly described. These are usually abnormal sensations and excessive sensitivity. Especially prominent have been visual disturbances ranging from mild, transitory changes to partial or complete blindness. Motor effects include paralysis, which usually develops slowly and varies in severity; muscular twitching and tremors; and epileptiform seizures. Gasoline-sniffing by adolescents has been reported, as well as one extreme case in an adult in which lead encephalopathy developed *31+1 Hallucinations, excitability , impairment of recent memory and a slightly ataxic gait were associated with increased blood lead content. Ordinarily, gasoline-sniffing would not produce lead poisoning, owing to the comparatively low volatility of tetra ethyl lead, as discussed more extensively in Chapter 6. Insidious Behavioral Changes. Biochemical changes occur at blood lead concentrations well below those defining industrial toxicity and are perhaps the correlates of insidious changes. For example, interference with heme biosynthesis is the earliest evidence detected -203- DUP050055492 as the blood lead content rises above 40 ug/100 g of blood, Lane*^* has pointed out that only the lead worker undergoing some toxic episode comes to medical attention. The worker who has become . ' slowly and insidiously poisoned, .who is tfbelow par," but without acute manifestations, appears to be well because he presents no overt health problems . However, he may be subject later to chronic 331 nephritis and cerebral hemorrhage * 24*3 As Hardy points out, 1*norispecificity of sign and symptom, delayed diagnosable damage because of the body's incredible margin of safety, and more than one insult acting like lead or with lead require sophisticated attention to the potential effect of low doses of lead*--in much the same manner as low levels of ionizing radiation have been studied since the use of atomic energy for military purposes in 1945.11 If the notion of "insidious poisoning" is valid, one might expect that workers exposed to lead concentrations below those which produce overt symptoms of toxicity would also undergo behavioral changes similar to the sensory, motor, and other alterations characteristic of frank lead poisoning, but to a lesser degree. However, no investigations of this have been reported. Nonetheless, a responsible company physician In sufficient contact with his workers is in a position to evaluate the early behavioral changes resulting from low-level poisoning. Given a familiarity with the baseline behavior of a worker, the physician can be alerted by the frequency with which changes In certain symptom categories occur that are otherwise difficult to Interpret--irritability, lassitude, constipation, headaches. Insomnia, -204- DUP050055493 abdominal cramps, and other diffuse complaints --as well as any increase in accident rates. In experimental .administration of lead to human subjects for long. periods, blood concentrations rose and then remained fairly constant* 295 298 However, while the blood remained in a steady state with regard to lead content, the body lead burden slowly increased as a small difference between lead intake and lead output persisted. This picture of positive lead balance should be viewed against the clinical background of lead poisoning in making a decision about the possible effects of low levels of exposure to lead: f,The symptoms of lead poisoning are, initially at least, rather vague; irritability and other mood changes predominate in the early stages, frank psychosis and encephalopathy later. The long biologic half-life results in so slow a buildup of toxic levels in the body that no connection may seem evident between the beginning of exposure to a chronically noxious environment and the development and progression of the symptoms of lead poisoning."207 -205- DUP050055494 Late Behavioral Effects of Early Exposure. At least 23% of those who survive acute encephalopathy from lead poisoning in early childhood have permanent central nervous system injury. ^6 The nature of the residual 'behavioral deficit cannot he easily characterized as "mental retardation,11 inasmuch as mOst studies report the majority of the intelligence-test scores as in or above the "dull normal1* level. For example, Thurston ert al. ^553 a 5- to 10-year study of 11 cases of lead poisoning in young children, found no Sianford-Binet intelligence-test scores lower than dull normal. However, more specialized tests of visual-motor function (Graham and Kendall test, Bender Gestalt test) yielded results very similar to those seen in children with train damage due to birth injuries or cerebral anoxia. Although the initial hyperactivity syndrome disappeared by the time the later tests were administered, only three of the children were doing satisfactory schoolwork. 8k Similar results have been reported by Byers and Lord, Bradley and Baumgartner,^ Mellins and Jenkins , and Jenkins and Mellins. 271 5^ * The damaging effects of lead poisoning associated with frank encephalopathy seem well substantiated, but an intriguing question -206- * O DUP050055495 remains: -whether excessive lead ingestion in young children produces permanent central nervous system and behavioral deficits if the magnitude of ingestion is not sufficient to result in demonstrable encephalopathy. Habitual ingestion of lead paint can increase the body lead burden and result in a chronic hyperkinetic-aggressive behavior disorder. Whether this condition. Which lacks the features of acute encephalopathy9 nonetheless produces neurologic damage via chronic derangements of metabolism is important to determine , because 117 pica for lead paint often implies protracted, recurrent episodes. 84 Byers and Lord studied 20 schoolchildren who had been hospitalized for lead poisoning in early childhood. None had shown evidence of encephalopathy and they were judged, at the time of hospitalization, to have made complete recoveries. Only one made satisfactory progress in school, the others having various intellectual and emotional diffi culties. However, by current standards, many of these cases would be judged as mild encephalopathy. In a recent review of this area, 590 Wiener concluded that, although most studies report behavioral impairment due to lead poisoning, none satisfies the demands of statistical control. There are also problems with respect to sampling bias and the application of various diagnostic procedures and definitions. The evidence with respect to the special effects of early exposure to a relatively small increment in lead concentration, particularly in its implications for later behavior, remains -unclear. However, existing studies, if not definitive, nevertheless afford some presumptive evidence of central nervous system dysfunction. -207- DUP050055496 Behavioral Effects in Experimental Animals Toxic Behavioral Effects. Although the nascent sub discipline of he^ havioral pharmacology has developed rapidly over the last 10-15 years ^56 >307>551 behavioral toxicology is much younger in this country,** Consequently* only a few experimental studies are available that deal with the behaviorally toxic properties of lead. Using a water T-maze, in which an animal must swim into the correct arm of the T to escape from the situation, Bulloch et. al J found that administration of tetraethyl lead (TEL) had little effect on the performance measured. Rats were given TEL intraperitpneally for 8 days to a total dose of 15 mg/kg of body weight. Four days after the last dose, maze training was begun. Escape times differed only slightly between control and experimental groups, and swimming times not at all. Other animals were given TEL injections after training had started. There were no performance differences between experi mental and control groups. All TEL-treated rats showed tremor, ataxia, fighting, and after a few days, convulsions and death. The water T-maze is apparently a rather insensitive instrument for evaluating TEL toxicity; nor has it proved suitable for evaluating other pharmacologic agents. Using a classically conditioned motor response in rats, Gusev reported finding behavioral impairment when animals were exposed to high and low atmospheric concentrations of lead oxide for 6 hr daily for 6 months. The actual number of exposure days, excepting -208- DUP050055497 the off-days, was 148-150 days* Using force and latency-of-^response measures, no impairment was seen at average air concentration of 1.13 yg/m3 of lead. At the higher dose level (11 yg/m3), disturbed reflexes began to occur about 1.5-2 months after the start of exposure and increased in severity over the exposure period; baseline conditionedreflex activity was re-established 10-23 days after lead exposure was discontinued. As exposure time increased, differential reactions to strong (bell) and weak (light) conditioned stimuli were often dis*rupted, and positive reactions to a negative conditioned stimulus (buzzer) also occurred. No changes in the formed elements of the blood were seen in any of the experimental animals. Histopathologic changes in the central nervous system were noted in rats and rabbits 3 exposed to about 11 yg/m of lead oxide, and the rat bone lead con tent was 10 times as high as that in either control animals or those exposed to the lower dose. The author did not discuss the probable contribution to the total lead intake produced by grooming of the fur. In an experiment using the same methods as Gusev, 508 Shalamberidze 3 found that a lead sulfide concentration of 48.3 yg/m (calculated as metallic lead) produced disturbed conditioned reflexes in rats exposed to ore.dust inhalation 6 hr daily for 6 months. Novakova using similar classic conditioning techniques, re ported that combined chronic doses of arsenic (0.0025 mg/kg) and lead (0,005 mg/kg) were additive in their effects and disrupted the acquisition of conditioned reflexes. These behavioral tests were administered between the fourth and eighth months of chronic dosing. -209- DUP050055498 Only two studies in this country of the behavioral effects of low- level exposure to lead, were found. Goldfish were given shock avoidance training and then exposed to specific concentrations of lead nitrate for 48 hr. Tests after 24 and 48 hr of exposure , with different groups being exposed to different lead concentrations * yielded significant behavioral impairment at concentrations as low as 0.07 ppm. This is only 1/857 of the concentration that is lethal to 1% of the animals, and it approximates the concentration in potable water. Mercury was more, and arsenic and selenium .less toxic than lead. Impairments were found at 0.003, 0.1, and 0.25 ppm, respectively In the light of Russian experiments on the behavioral toxicity of atmospheric lead, it is interesting to note that these results show impairment at a similar order of magnitude, Using a spatial escape response in fish, Jones 279 showed that the three-spined stickleback (Gasterosteus) would escape from lead nitrate solutions as dilute as 1 ppm, whereas the threshold for minnows (Phoxinus phoxinus) was 0.4 ppm. All the drugs commonly associated with the production of physical dependence (amphetamines, barbiturates, morphine, alcohol) also suppress the paradoxic, or rapid-eye-movement (REM), phase of sleep. They also produce a long rebound of this REM phase on withdrawal of 442 the drug. In the light of this sensitivity of the REM phase of sleep to drugs, it is interesting that the chronic absorption of lead also affects REM sleep Rats given lead acetate (lv5 mg/ml) in their drinking water showed * -210- DUP050055499 altered REM-phase patterning, Ibis could fee related to the fact that an early sign of plumbism is insomnia.89 Developmental Effects of Early Exposure. If the developmental processes of the fetus or immature organism are subject to pathologic alteration by exposure to lead, such pathologic changes will necessarily have behavioral implications. If rats nursing their young are fed diets containing either 1 or h% lead carbonate, lead appears in the milk 455 473 and affects the neonates. 9 The young show evidence of lead poisoning, faulty growth, and various neurologic changes', including paraplegia, changes in the cerebellum and striatum, and blood-brain barrier dysfunction* Although various lead salts administered (at 50 mg/kg) to hamsters on day 7, 8, or 9 of gestation produced skeletal malformations in sacral and tail vertebrae recent evidence reveals a low degree of teratogenic effects in rats 300 and mice. Little radioactive lead was found to cross the placenta. However, Barltrop^ cites evidence of placental penetration by lead in rats and presents evidence suggesting maternofetal blood-lead equilibrium in women. 211- Suggested Lines of Behavioral Investigation Perhaps the most controversial and also the most pressing aspect of the lead-exposure problem is the effect of chronic exposure to low concen trations of lead in the environment. the extent to which such chronic exposure influences behavior needs to be evaluated with reference to well-understood behavioral baselines. Long-term studies, similar to those undertaken by the Russian investigators, but using operant conditioning as well as other methods, need to be instituted. There is no substitute for the chronic exposure of organisms to various dosages and the consequent delineation of dose-effect functions. Demonstrations that some dosage "has an effect" on behavior provide an insufficient base from which to proceed toward the study of be havioral mechanisms of action. The lessons afforded by behavioral pharmacology in this regard are well worth noting. Some areas of investigation could be used to illuminate the particular effects lead may be suspected of having at low dosages, inasmuch as lead poisoning produces peripheral neuropathy*^ ^5^5 and muscular changes8Q^ and lead ions produce preganglionic transmission blockthe effects of chronic exposure to lead on motor-control tasfcsxo might yield early indications of the behavioral toxicity of lead. Lead poisoning also produces various visual disturbances. These might be evaluated not Only by acuity and flicker-fusion determinations, but by the use of the evoked-response technique. Promising investiga tions along this line have been instituted by Xintaras et^ al/^^ r -212- DUP050055501 In. evaluating the effect of lead on sleep patterns, Xintaras et al. have opened the area of complex, natural behavioral sequences to behavioral toxioology. The study of complex learned-behavior * iQk . schedules is likely to prove indispens able, but the effects of lead exposure on the patterning of HEM. phases, thresholds of aggression , 269 and food*-fluid intake patterns should also receive attention. And ethologic studies of courtship and parental sequences should not be neglected, because they may provide early indications of wildlife problems not likely to be discovered in laboratory studies* In studying the toxicology of lead, it is imperative for investigators to use well-known agents in the research program as reference standards to validate their experimental arrangements. Thus, Xintaras et al.^^ used the well-known effects of pentobarbital on the evoked response to validate their evoked-response preparation before studying the lesser-known effects of carbon monoxide and ozone. Lead is not the only toxic substance to which an organism might be subjected at a particular time, so investigators should also consider the effects of lead combined with other pollutants (such as carbon monoxide) or probable vehicles (such as ethanol). The possible synergistic effects of various pollutants are largely unknown; 557 but the not uncommon combination of alcoholism and lead poisoning is known to be a medical problem. 102 9 24h -213- DUP050055502 Finally, the relative sensitivity of the fetus, neonate, child, and adult to chronic, low-level exposure to lead needs investigation, as do the reversibility of toxic effects and the evaluation of residual effects. The possibility of specific susceptibility of the developing brain at various fetal and Juvenile stages should be evaluated most carefully. Some of these studies could be done on lower organisms, particularly primates, but, in light of the serious suspicions that lead may produce some unknown fraction of the mental retardation and perceptual disorders found in children exposed to this element in their environment epidemiologic end other correlational and clinical studies of this problem should he encouraged. -214- DUP050055503 OBSERVATIONS IN EXPERIMENTAL ANIMAL SYSTEMS Biochemistry Lead has a strong affinity for some biochemical ligands, including the epsilonamino group of lysine, the carboxyl group of glutamic and aspartic acids, the sulfhydryl groups of cysteine, imidazole residues, and the phenoxy group of tyrosine. Consequently, it may displace metals from enzymes, modify tertiary structure of enzymes, and block enzyme substrate interactions. When lead is so concentrated as to exceed the ability of the numerous reactive sites in and on cells to receive it, metabolic consequences ensue that are manifest by lead poisoning. However, as in deficiency disease, poisoning may be recognized clinically somewhat later, because of the insensitivity of usual clinical methods of diagnosis. An understanding of the evolution of poisoning at the cellular level is essential to an understanding of clinical lead poisoning and the possible VT4 effects of ambient lead. Rothstein/ has suggested that enzymes associated with the cell envelope may be among the most sensitive to lead. He pointed out that the envelope is the first point of contact between the cell and lead in the interstitial fluid. Some of the apparent effects of lead on membrane transport in the erythrocyte and renal tubule support his hypo thesis. Rothstein believes that the internal milieu of the cell may dilute low amounts of lead that pass the external membrane. Some organelles within the cell seem particularly susceptible to lead, including raito- , , . 213 , , 96,216, . 96 chondrxa, , nuclei, and microsomes. Their sensitivity to lead and apparent accumulation of the metal, as demonstrated by lead-210 96 Vfi tracer studies, extend Rothstein's hyppthesisi that cell membranes -215- DUP050055504 are particularly susceptible to lead, to include intracellular membranous structures and their enzymes, inasmuch as the cell envelope is not a static barrier between the interior of the cell and the external environ ment, but rather is a membrane across which many metabolites travel, both actively and passively, entry of lead into the cell, perhaps in place of calcium, and its subsequent attachment to ligands on the membranes of organelles is not in conflict with the Bothstein hypothesis. Effects of lead bound to mitochondria have been evaluated in the renal 212,216 tubule of the rat. Isolated mitochondria demonstrate im paired oxidative phosphorylation, in addition to defective structure of their membranes. Ultrastructural transformation is inhibited, and 2lb those which do transform have labile membranes and decreased ability to phosphorylate ADP in pyruvate-malate substrate. Those which do not transform from the condensed to the orthodox form in the presence of pyruvate-malate substrate are thought to have impaired electron transport. Of interest in terms of the effects of lead on mitochondrial enzymes is its inhibition of 1ntramitochondrial aspects of heme synthesis. The observations of Goyer and co-workers on mitochondrial respiration and phosphorylation are of particular interest in view of the recent 562 in vitro studies by Ulmer and Vallee on the dithiol enzyme, lipoamide dehydrogenase. This enzyme is part of the macromolecular complex of enzymes that synthesize acetyl coenzyme A and succinyl coenzyme A from pyruvate and a-ketoglutarate, respectively. Lead, at concentra tions of 6.$ x 10*"^ M will inhibit its action. Studies on a second -216- * DUP050055505 dithiol molecule in the enzyme-lipoi-c*-acid complex, similar to those done on lipoamide dehydrogenase, have apparently not been reported* f* it is of interest j however , that dl-q-lipoic acid did not protect* mice 534 from a lethal dose of lead. . Inhibition of lipoamide dehydrogenase by lead is probably representative of the effects of lead on many dithiol enzymes. Because the studies were in vitro their direct application to man is not possible. They simply lend support to the concept that very small concentrations of lead can inhibit critical enzyme systems* Evidence that lead may also suppress protein synthesis through alteration of the tertiary structure of MA or inhibition of reac tions in which it participates has come from the finding that RNA from lead-intoxicated experimental animals contains considerable lead'* and from the in vivo concentration of lead-210 by liver microsomes. 96 According to Sroezynski, 533 lead poisoning in rabbits does impair protein synthesis, Phosphorus-32 tracer studies suggest that RNA synthesis may also be inhibited. 417 At concentra- -5 562 tions of 10 M, lead causes hydrolysis of Escherichia coli RNA. Whether this occurs in vivo at lower concentrations is unknown. If transfer RNA were hydrolyzed by lead, incorporation of amino acids into peptides would be suppressed. Lead at 10 -4 M suppresses the l4 activity of enzymes involved in the incorporation of C-leucine into transfer RNA of coli. It is thought that this is due to inhibition . . -4 of the sulfhydryl enzyme aminoacyl synthetase. Because 10 M is a greater concentration of lead than usually found in intact mammalian systems , it is most improbable that these observations would have any bearing in man, -217- DUP050055506 Lead is also concentrated by cell nuclei, as documented in liver and 96,216 kidney and implied by the chromosomal abnormalities present in 417 leukocytes from lead-intoxicated mice and lymphocytes from lead- , 350 intoxicated men and by the bizarre mitotic figures sometimes found in bone marrow smears taken from lead-intoxicated men. The intranuclear inclusions in liver and kidney are currently thought to be inert lead-protein complexes and they may well represent a protective storage mechanism. 215' The mechanism of lead's effect on chromosomes is unknown. However, because the abnormalities produced are of the "gap-break" type and are not paired between chromatids, it is thought that they occur after DNA replication. Considering that activation of DNase from liposomes will apparently damage chromosomes 13 and that DNase activity is markedly 323 increased in urine from lead-poisoned rats, In 7 Muro and Gayer have speculated that increased DNase activity in lead-poisoned cells may produce these chromosomal effects. The significance of these observations for human health is unknown. It seems clear that the concentrations of lead necessary to produce these abnormalities are much greater than those found in the environment. For the present* these studies may be viewed as interesting laboratory phenomena with no known genetic implications. Of the enzymes associated with the cell envelope, those involved in sodium-potassium transport appear susceptible to lead at concentre*- tions found in lead-intoxicated man. Hasan et al. 247 have reported -218- DUP050055507 that erythrocyte sodium-potassium ATPase activity is lower in lead- intoxicated men than in controls (4.42 + 1.23 x ICT^ vs, 6.34 + 1.29 -13 x 2S4 ~~ x 10 ), Their finding is consistent with Joyce et al. fs earlier * finding and their own studies, which indicate that potassium leaks from and sodium enters lead-poisoned erythrocytes incubated in vitro. 290 Again, the implications of these findings are unknown. Some workers are of the opinion that the sodium-potassium ATPase enzyme system may be of major importance in maintaining the correct internal milieu in the cell. Whether significant alterations in the internal milieu occur in vivo as a consequence of lead seems not to have been reported. Of interest in this regard is the apparently decreased renal sodium- retaining ability in some men with lead intoxication due to ingestion 483 of illicitly distilled whiskey . This has not been investigated in experimental animals in controlled conditions; therefore, the minimal dose of lead necessary to produce the effect has not been determined. In addition, the effect seems to have been related to long, heavy exposure; therefore, it seems unlikely to be of significance with regard to ambient lead. Physiology Absorption of lead from the gastrointestinal tract appears to be regulated by some of the same physiologic mechanisms as control the absorption of calcium and phosphorus. Increased dietary calcium and phosphorus will, in fact, decrease the absorption of lead, and decreased dietary intake of calcium appears to increase lead absorption. 5H It has been shown in rats given lead in their drinking water (at -219- DUP050055508 200 jjg/ml) that low dietary calcium will greatly increase the severity of anemia and biochemical indices of lead poisoning Bone lead content is higher and bone calcium content is lower than those in rats given the same amount of lead in their drinking water and a normal calcium diet intake. 521 It appears that vitamin D will also enhance the ab sorption of lead from the gastrointestinal tract.528 mechanism of action is as yet undefined. The composition of the diet ingested apparently may influence the occurrence of lead poisoning. Baernstein and Gran<^ reported that a 20% casein diet would protect rats from lead chloride (lh%) in the diet to a greater extent than a 6 or 13% casein diet. Addition of methionine or cysteine to the 6% casein diet decreased the mortality and improved the weight gain of both lead-fed and control rats. These studies are supported by the work of Gontzea jrt al. Recently, it has been shown that cysteine in vitro will protect activity of 6ALA dehydrase from lead in rabbit liverThe apparent protective effects of some diets against poisoning by lead shot in ducks are discussed in Chapter 5. It has been suggested that synthesis of nicotinic acid from tryptophan is impaired in experimental lead poisoningExperimentally, it appears that nicotinic acid may decrease porphyrinuria in lead-poisoned I4.I4. if80 rabbits 5 p but this finding has not been confirmed in rats. In support of the suggestion that lead produces abnormalities in tryptophan metabolism, xanthurenic acid excretion in the urine has been found to increase. However, when Tenconi and Acocella 5^.5 A -220- DUP050055509 carried out tryptophan loading tests in rats, they did not find an increase in xanthurenic acid excretion. At present, there is no evidence that nicotinic acid has therapeutic value in lead poisoning* It appears that chromium may have a protective effect on survival of 1*95 rats given low doses of lead for long periods. The data suggest that chromium deficiency makes rats more susceptible to lead. Implica-* tions of these observations for man are as yet unclear. Other Effects Because many factors are suspected of influencing both the severity and the clinical manifestations of lead toxicity, it would seem useful to be able to recognize common denominators . The common mode of action of a number of the factors discussed is the effect of mobilizing lead into a transportable or diffusible form. Little Is known presently about the biochemical nature of diffusible lead. Likewise, the toxicology of lead at the cellular or molecular level is ubiquitous and probably entails a number of mechanisms , depending on the physiologic or biochemical process involved. What is known about these reactions is discussed in detail elsewhere and only briefly mentioned here. A summary of the toxicologic indices of lead poisoning from the published literature is presented in Appendix E. Lead is known to interact with some enzymes; those concerned with heme synthesis, particularly d-aminolevulinic acid dehydrase, are the best studied and have been discussed, in detail earlier. Lead ions also 2lU impair the oxidative and phospho ry1a11ve functions of mitochondria. -221- DUP050055510 and it is also suggested that it interferes with transmission of impulses at preganglionic nerve endings by reducing the output of acetylcholine. 319 In vitro studies have shown that lead may impair protein synthesis by In spite of the sophistication of these studies , the present level of knowledge of these effects of lead is not complete enough to identify a common physicochemical property of the lead ion* Understanding of synergistic and antagonistic factors in lead toxicity will be greatly improved when more basic knowledge of the metabolism of lead is available * The possibility that lead at physiologically significant concentrations affects mutation in plant or animal cells has no firm experimental evidence. The effect of high concentrations of lead on plant cells has been discussed in Chapter 2. Novick and Roth^*^ have reported in Staphylococcus aureus a mutational transfer of resistance to lead (as well as to penicillin, cadmium, and mercury); these results are not yet confirmed. The chromosomal damage found in experimental lead poisoning by Muro and Goyer^^ has also been mentioned earlier. In all investigations cited, lead was introduced at higher concentrations than the organism would normally encounter or, in fact, could survive for long. It is not known whether low concentrations of lead may have an effect on somatic mutations that very likely contribute to the aging process. I -222- ) DUP050055511 Although epidemiologic studies have not shown any relation between lead exposure and the incidence of cancer, several investigators have been able to show that lead experimentally administered in very high doses is carcinogenic. The first observation of neoplasia induced by lead was reported by Zollingerwho injected lead phosphate subcutaneously into rats, yielding renal adenomas and adenocarcinomas. Similar results were obtained by other workers with oral or parenteral lead phosphate, 65,218,387,^71,558,571 lead acetate, or basic lead acetate in rats and in mice. 570 Tumors of testis, adrenal, thyroid, pituitary, prostate, and lungs have arisen in the course of long-term dietary administration 603 of lead acetate to rats. In a study of cerebral gliomas induced in 14.1+5 rats by various agents, Oyasu obtained the highest yield with lead subacetate; coadministration of 2-acetylarninofluorene did not increase the incidence of gliomas. Beginning with the classic account by Weller`S of the "blastophoric" effect of chronic lead poisoning in man and animals, the evidence has accumulated of a teratogenic action of high concentrations of lead 82 98 2 89 salts. Results with the chick embryo 9 9 have supplemente?dd aa n h& earlier study of intravenously administered lead in the rabbit. 183 Perm and Carpenter have reported developmental malformations in the golden hamster, resulting from intravenous administration of a lead salt at a dosage of 50 mg/kg on day 7, 8, or 9 of pregnancy. Despite the accumulated evidence ir. laboratory animals on very high dosage, teratogenic effects of lead have not been seen in cattle or 273,513 sheep. -223 DUP050055512 SUMMARY Body Lead Burden Postmortem analysis of tissues for lead indicates that approximately 95# of the total body lead burden in man is stored in bone. Although the concentration of lead in bone appears to increase during life until at least 1*0 years of age, the concentrations of lead in the various*soft tissues, excluding aorta, normally remain low and relatively stable throughout adult life. Limited age-related data in adults without Known industrial or other unusual exposure to lead indicate that blood lead content also remains constant and within the "normal" range throughout life. These observations are consistent with the hypothe sis that the total body lead burden may be divided into two major pools: a small "mobile" fraction found primarily in the soft tissues and a much larger, but relatively "nondiffusible" fraction, which is apparently tightly bound in matrix of bone. In man, increments in the "mobile" .fraction of the total body lead burden are associated with the Known acute toxic effects of lead. Blood lead content and the "chelatable" lead* provide an index of this small "mobile" fraction of the total body lead burden. In persons with normal renal function, spontaneous urinary lead excretion (which has long been used as a measure of current and recent absorption of lead) may provide similar information, but this point has * I.e., the quantity of lead excreted in urine in response to a standard ized dose of a chelating agent, such as CaEDTA or d-penicillamine. -224- DUP050055513 I not been clearly documented. Current evidence concerning the contribu tion of the nondiffusible fraction to the soft tissue pool isincomplete. In general, it is sequestered in bone and is therefore not likelyto be associated with adverse health effects. Concentrations of lead in bone are usuallly greater in persons from industrialized countries than in those from the less developed countries. Effects of Lead on Biosynthesis of Heme Studies in experimental models and in severe acute lead poisoning in man indicate that lead can inhibit the biosynthesis of heme and the utilization of iron. Acute lead poisoning in man is characterized by a decrease in ALAD activity in vitro in blood, an increasein ALA in serum and urine, an increase in UCP, and an increase in FEP.* * UCP and FEP levels have been studied in relation to toxic concentrations of lead in man, but not extensively in relation to minimal increases in tissue lead content and in subclinical lead poisoning. UCP is affected also by hepatic injury, and increased FEP content is associated with both deficient supply and impaired utilization of iron. In man, inhibition of ALAD activity in hemolysates of peripheral blood and increased ALA in serum and urine apparently represent a specific response to rising lead content in soft tissues. (The only reported exception is transient impairment of ALAD activity in the blood of men with acute alcoholic intoxication.) -225- DUP050055514 Significant relations have been found in man between blood lead content and (1) the in vitro assay of ALAD activity in hemolysates of peripheral blood, (2) urinary excretion of ALA, and (3) "chelatable" lead as measured by the CaEDTA mobilization test. There is an inverse linear relation between the logarithm of ALAD activity in vitro in hemolysates of blood and the concentration of lead in blood when blood lead content is between 5 and 95 Pg/100 g of whole blood (or more). Throughout the "normal" range of blood lead concentration (5-40 pg/100 g of whole blood), ALAD activity in blood, as measured in vitro, decreases by about 75%. Some studies, however, indicate that the excretion of ALA (the substrate of ALAD) in urine remains constant and low in adults and adolescents with blood lead content less than approximately 40 pg/100 g of whole blood. (This observation has not been confirmed in young children; no children with blood lead content less than 25 pg/100 g of whole blood have been studied for this.) But arithmetic increases in blood lead content above approximately 40 pg/100 g of whole blood are correlated with a continuing exponential decrease in ALAD activity in hemolysates of peripheral blood, an exponential increase in urinary ALA excretion, and an exponential increase in "chelatable" lead. When blood lead content exceeds 60 pg/100 g of whole blood, it appears that most persons may be expected to shown an increase in urinary ALA excretion. The panel reached this conclusion on the basis of evaluation of several studies, none of which by itself could be considered adequate to support the conclusion. When all the available data are considered together, they are consistent with the hypothesis that the inhibition of ALAD activity in vivo in intact man becomes physiologically significant as blood lead content rises above -226- DUP050055515 approximately 40 pg/100 g of whole blood and that the partial inhibition observed is reflected by an increasing rate of excretion of its substrate (ALA) in urine* As sustained blood lead content exceeds approximately 80 pg/100 g of whole blood, impairment in the biosynthesis of heme in the hematopoietic tissue can no longer be compensated by an increased rate of production* Failure of this compensatory mechanism is, in turn, followed by frank anemia. The biosynthesis of heme is probably also impaired in other tissues; however, the level at which significant impairment in the biosynthesis of heme may occur in other tissues has not been defined. The biologic significance of declining ALAD activity in vitro in red blood cells in persons with blood lead concentrations less than 40 pg/100 g of whole blood is not presently understood. It may be tentatively suggested that, in such persons, this in vitro phenomenon represents a nonessential "reserve enzyme capacity,11 insofar as red blood cell formation is con cerned* Asymptomatic persons with blood lead concentrations between 40 and 80 ug/100 g of whole blood are apparently able to compensate by increasing their rate of red cell production* However, lead poisoning is associated also with impaired utilization of iron, so that persons with either iron-deficiency states, disorders associated with impaired utilization of iron, hemolytic anemias, chronic infectious diseases, severe liver disease, or renal failure may not be able to compensate as well for soft tissue or "mobile" lead concentrations associated with blood lead concentrations in the range of 40-80 pg/100 g of whole blood. Such population groups have not been adequately examined in this regard. -227- DUP050055516 Clinical Effects and Epidemiologic Considerations Studies of the clinical effects of increased tissue lead content are based almost entirely on the clinical evaluation of industrial workers in the lead trades, children in urban areas, and imbibers of lead-con taminated illicitly distilled whiskey. These sources of exp.osure to inorganic lead salts constitute the major health hazards associated with lead in the United States today. The other potential environmental sources of lead listed early in this chapter have been responsible for sporadic cases and minor outbreaks of clinical lead poisoning. Occupa tional health supervision and the institution of measures to minimize exposure of workers have greatly reduced the occurrence and incidence of severe lead poisoning in the lead trades since the early 1900*s. Today, estimates of the magnitude of the industrial health problem posed by exposure to lead are fragmentary, because adequate incidence data are , not available. The principal occupational problem probably lies in small shops, whose owners do not provide adequate medical surveillance and leadexposure control programs, as are usually maintained by large corporations. (Hazards due to exposure to lead alkyls are limited to a very small number of closely supervised workers employed in the manufacture of tetraethyl and tetramethyl lead.) As with occupational exposure, epidemiologic data concerning the incidence of lead poisoning in children are also inadequate. The available data do suggest, however, that 5-10% of preschool children residing in deteriorating urban housing consume sufficient lead to have "increased lead absorption," as judged by blood lead concentrations, and that approximately 1% may be found to have symptoms compatible with the diagnosis of clinical lead poisoning. The extent to which lead-bearing -228- DUP050055517 1 dust in urban areas may contribute to the problem is not known. Also unknown is the magnitude of the problem in moonshine-whiskey drinkers but it is suspected that it may be significant} especially in the southeastern part of the United States, Today, it is generally true that clinically evident lead poisoning most frequently result^ from the absorption of lead through the gastrointestinal, rather than the respira tory, tract. To place the question of lead in the ambient air in proper perspective, it may be stated that clinically evident disease due to inhalation of inorganic lead salts does not usually occur unless the 3 concentration in the air exceeds 0.5 mg/m --roughly 100 times the maximal concentration reported in the ambient air in urban areas and 1000 times that reported in rural ambient air. Clinical studies indicate that clear-cut clinical signs and symptoms of acute lead poisoning are related to the degree of current and recent absorption of lead and, in the absence of severe anemia, are almost always associated with blood lead concentrations greater than 80 ug/100 g of whole blood. At higher concentrations, the severity of acute clinical manifestations is not closely associated with and cannot be precisely predicted from blood lead concentration. It can be stated only that derangement of heme synthesis is always evident and that the risk of symptomatic illness increases markedly as blood lead content rises above 80 ug/100 g of whole blood. The factors that may precipitate acute lead poisoning are not clearly understood. Whether symptoms compatible with acute lead poisoning may, in fact, be attributed to lead in persons with blood lead content between 50 and 80 ug/100 g of whole blood is contro- -229- DUP050055518 versial. In part, the controversy arises because of the subtle and non specific nature of the early symptoms of lead intoxication* Not only is clinical diagnosis difficult in lead-intoxicated persons, but the general unavailability of adequate laboratory diagnostic facilities limits their clinical evaluation. At present, it is unlikely that the biologic significance of blood lead concentrations between 50 and 80 yg/100 g of whole blood can be resolved on the basis of routine clinical evalu ation alone. The clinical interpretation of observed blood lead content can be greatly facilitated in the presence of moderate to severe anemia if it is corrected for low hematocrit values. This correction is not indicated for mild deviations from expected hematocrit. Although there is no scientific basis for correction of hematocrit, available clinical reports indicate that patients with moderately severe anemia, clear-cut symptoms of poisoning, and blood lead content of less than 80 yg/100 g of whole blood will usually be found to have a corrected blood lead content of over 80 yg/100 g if the correction is made. Clinical evaluation of difficult cases may be facilitated by use of the CaEDTA mobilization test. The acute toxic effects of lead on the hematopoietic system, kidneys, and nervous system are summarized in Table 4-11. Whereas the toxic effects of lead on hematopoiesis (anemia) and the kidneys are apparently reversible, injury to the central nervous system might not be. Indeed, at least 25% of the survivors of acute lead encephalopathy are said to sustain permanent brain damage. Chronic lead nephropathy and nervous system sequelae (even in the absence of known encephalopathy), as recog nized clinically today, are apparently associated with long-continued abnormal exposure to lead and recurrent symptomatic episodes of poisonine. -230- DUP050055519 The occurrence of central nervous system sequelae in at least 25% of children after acute encephalopathy appears a reasonable estimate. However, the frequency of residual central nervous system deficits attributable to less severe lead poisoning in children cannot be accu rately estimated from the published clinical material. Epidemiologic studies in adults occupationally exposed to lead have revealed no evidence of an association between lead and cancer or multiple sclerosis and no evidence of increased mortality due to degenerative cardiovascular disease. Workers under today1s careful medical supervision in plants with adequate exposure control programs have not been found to have an increased incidence of hypertension. Past occurrences of hypertension, renal insufficiency, and fetal wastage in occupationally exposed adults were associated with uncontrolled protracted exposure. Biochemical and Functional Considerations There is no evidence that lead is an essential trace element, although the hypothesis that minute amounts may serve some essential function in metabolism has not been examined. On the contrary, all experimental data obtained both in vitro and in vivo show that the metabolic effects of lead in concentrations as low as 10'^M are of the inhibitory or adverse type. At the enzymatic level, the inhibitory effect of lead on sulfhydryldependent enzymes is well documented in vitro: sulfhydryl-dependent enzymes clearly inhibited by lead include two in the biosynthetic pathway for heme formation (ALAD and heme synthetase) and lipoamide dehydrogenase, an essential component in the pyruvate and a-ketoglutarate decarboxylase -231- DUP050055520 systems. It seems clear that lead forms ligands with groups other than disulfide and sulfhydryl in biologic systems, but this area has received only cursory attention. At the cellular level, the formation of respira tory pigments (heme and cytochrome), energy production, and some membrane functions appear to be the aspects of metabolism most susceptible to the adverse effects of lead. Although the effects of lead on the biosynthesis of heme in red blood cells are reasonably well delineated, gaps in knowledge remain and little is known of its effect on heme synthesis in other tissues. Evidence obtained from red blood cells and rat kidney indicates that the structure and function of the mitochondria are altered by lead. In the kidney, the most sensitive consequence of this impairment of mitochondrial function appears to be aminoaciduria. Aminoaciduria apparently represents a response to the high soft tissue concentrations of lead associated with severe acute poisoning in man; the question has not been examined subclinically. With regard to membrane functions, the significance of the finding of decreased sodium-potassium ATPase activity in vitro in red cell fragments in men with increased lead exposure is unknown. Whether signifi cant alterations occur in vivo in man as a consequence of this particular in vitro effect of lead is not known. It is, however, possible that the apparently decreased renal sodium-retaining ability of some men with lead poisoning due to ingestion of illicitly distilled whiskey may result from a comparable impairment in membrane transport in the kidney. This question has not been examined in experimental animals in controlled conditions, so the minimal dose of lead necessary to produce the renal effect has not been determined. Inasmuch as the effect appears to be related to long and heavy exposure to lead in illicitly distilled lead-contamined whiskey, -232- DUP050055521 it is unlikely to be important with regard to lead in ambient air and a normal diet. From the limited experimental data, it can only be surmised that inter actions between lead and other metals--such as iron> manganese, and cadmium--, may be important. Similarly, factors that apparently affect absorption, storage and excretion of lead in experimental conditions--such as heat, dehydration, vitamin D deficiency, and dietary calcium and phosphorus imbalances--have not been examined in sufficient detail to estimate their significance for intact man. The possible effects of increased lead con tent in the soft tissues on integrated nervous system function (behavior and performance) have received scant attention. It is suspected that these effects, when studied in intact experimental animals, might be best approached in nonhuman primates. The alterations in endocrine function observed in men with lead poisoning due to the ingestion of illicitly distilled whiskey are incompletely understood. The adverse effect on thyroid function appears clearly due to lead; similar effects have been produced by feeding lead to rats. Because the other endocrine abnormalities described have not been repro duced in animals or described in patients with lead poisoning due to industrial exposure (with the exception of adverse effects on renin secretion), the presence of other contributing factors in moonshine has not been ruled out. The significance of intranuclear inclusions in proximal renal tubules of man and animals with lead intoxication is incompletely understood. Goyer's suggestion that they represent inert lead-protein complexes and that they -233- DUP050055522 may serve as a protective storage mechanism in liver and kidney is provoca tive* It is noteworthy that these inclusions have not been described in other organs. There is scant knowledge with respect to the biologic binding and intracellular localization of lead: it appears to bind to mitochondria, microsomes, or nuclei. The physiologic implication of this binding is a subject for further investigation. The significance of the many experimental studies for intact man is unknown particularly because most of the studies have been based on high dosages of lead without attention to dose-response relations. Subclinical Effects The uncertainties concerning the threshold at which the biologic effects of lead become clinically manifest are reflected in Table 4-11. This table includes the concept that physiologic mechanisms may be able to compensate--at least temporarily--for increases in the '`mobile11 fraction of the total body lead burden. In the case of the hematopoietic system, biologic compensation is apparently accomplished by increasing the rate of red blood cell production when blood lead content is 50-80 ^g/100 g of whole blood. For renal and nervous system function, the thres holds for clinical correlation with the biochemical effects of lead are not known. Present knowledge suggests that significant uncompensated impairment of renal and nervous system function is unlikely in persons whose blood lead content does not exceed about 50 or 60 }ig/100 g of whole blood. In patients with sustained higher blood lead content, it appears likely that thresholds for adverse functional effects in these organs will be exceeded. The long-term consequences of sustained small increases in -234- DUP050055523 soft tissue lead content are not known* Available epidemiologic information suggests that clinically obvious and severe adverse effects do not occur in such conditions. The degree of subclinical effect is an area in which research is urgently needed; reliance should be placed on clues provided by experimental biochemical studies in approaching this problem in man* -235- DUP050055524 TABLE 4-1 Percentage of Workers Tested Having Blood a Lead Concentrations Above Allowable Limits Year No. blood specimens 1966 1967 1968 1969 Total 214 114 136 10 4 568 Blood samples indicating lead poisoning No. %_ 74 35 17 15 13 10 14 13 118 Estimated no. exposed13 430 215 483 276 l,4o4 ^ram H. E. Stokinger, personal communication. Allowable limit * 100 jig/100 g of whole blood, ^Estimates might be incorrect by a factor 9f 2 or 3. -236- DUP050055525 State: Connecticut Illinois Kansas New York TABLE 4-2 State and Local Laws and Ordinances Pertaining to Use of Lead in Interior House Paints (October 1970)a Labeling Use prohibited Removal reauiredc 1967 1958 1958 no 1967d no no 1970e no . . no no no Municipality: Baltimore, Md. Cincinnati, Ohio Cleveland, Ohio Jersey City, N. J. New Haven, Conn. New York, N. Y. Philadelphia, Pa. St. Louis, Mo. Washington, D. C. Wilmington, Del. 1958 1960 1970 1962 -- 1959 1966 1970 -1963 1958 1960 1970 1962 1968f 1970 1966 1970S 1970 1963 1951 1960 1970 1962 1968f 1970 1966 1970& 1970 1963 aAccording to information supplied by National Paint, Varn: and Lacquer Association, Inc., Washington, D . C. (legislation thought to be pending in Minneapolis-St. Paul, Minn., and Newark, N. J., as of October 1970). -237- DUP050055526 bLabel on paint can conforms to ANSI Standard Z66. 1, which identifies paints containing <1% lead as safe for interior use. Label must state whether paint conforms to this standard. Ordinances invoked in dwelling units of children found to have blood lead levels >60 Mg/100 g of whole blood in most cities listed. .. ^Prohibits use of paints not conforming to ANSI Standard Z66. 1 on interior of dwellings owned or planned by municipalities or other housing agencies. Does not apply to privately owned dwellings. eAlso prohibits use of paints containing >1% lead on porches. ^Prohibits use of paints containing >1% lead on both interior and exterior surfaces accessible to children in all dwellings, whether publicly or privately owned. Requires proper maintenance of surfaces, ^Prohibits use of paints containing >1% of lead on interiors. "Exterior" requirement limited to flaking or peeling surfaces. -238- DUP050055527 K arthenvare Glaze S urfaces Report o f Lead E x tra c tip n s from o0 ooH1 \ la ON P 1 p e oc H P o cd U P X <D cd 0) P o la oo1 H oO r--1 A Oo H | O C\1 O CM 1 t-- 1 11 q H CO LA vql p Hi CM 1 VO LA vo| P H 1 CM H VO P P| P CM | i--1 on r-- i LA LA t-- p| VO vo o I f--1 on voia ] O v| ON C-- rH l VO h I on on 0} CoD cd P 01 <D N cd H m ON CM 00 P o| c*- t- P (--i P | H rH <D bcd > r--1 P cd Cm H t>> O cd u U2 o CD P i3 P O } o 2 03 cd rH Pop cd T* o a P0 CD H H Eh p o22 hO O <$ tO MPE P 03 06) fi P 03 og<oD PQ <D rH P &2 pj C/1 O02 O22POH O rH on i--11 cdl P ol 2 H i--CD1 t*! E02 P TC! P P cd <T3 3J -239- CD 59 o rH 8 P 2 O Cm CD Cm Cd 03 CD u o *2 H 03 co o o cd CD --f Cmo E P P c-- V > P CD 03 2 P 03 CD P T* H a cd u H P CD a cd CD b 2 p 2 2 CD &c CD P g O o 2 T23 pcd 3c p C/3 P CO 2 0 p CO p G OE e cd P o TJ 'd c cd CO oP P 03 P P cd 2 o H d q cd P E O <M 2 CD P 2 2 O o (D P P 2 CD > O pe 2 H cd p PO 03 <D rH P oCO 03 2 O H *P ' oCO & E O a CD N 2 H W) P 2 a <D P P r-| Tl On P P P H > Td a p cd CD 2 p 2 O H P H 03 O Pw 0 o >? cd rH a t* 2 <d (D H to i--1 cd o M P V2O H P o 03 *--<Di nd1&f20cf DUP050055528 00 O 00 a S 00 -5f o CM CM T oi--i A & bC OHI CM LA ON CM O oo vo CM o CM A -4- 8& CM CM 8 rO vo CM LA CM o H Ot- 00 O CM Lead Content o f Moonshine Whiskey' Data from A lc o h o l, Tobacco and Firearm s D iv is io n L a b o ra to ry, In te rn a l Revenue S ervice -o^or on CM -C=Mr ON On CM LA -cr LA 00 8 oon CM vo CM CM CM ON CM CM vt vo VO 01 0 H no *0 0 cd CO N 0 01 . o a HOoO o CM oA a cB CM H ccJ 0 Vct,f Cc co H oc H 0 w bo Eh 0 0 cO 0 -p p rH m to H CO 0 H 0 & * 42 43 P p CO vAo aaS 23 0 CO o3 CO co -=r H o too i--i H eu 0, flj H o5 to H CO bQ H CO O (0 4 AA 00 VO CM rH rH O Pu CtJ J oo 0O CO 42 p o S5 42 P 3 C CoGCO $ H tO-n H Po -2fc0- DUP050055529 CM O CM CM co uo c\ tr> ON 00 j*| O st VO O oo m II m cm vO c m I sh rH I ON O X4J 6Q30 XX 3 x 3 X3I C0 Cd ol wo o p <g 0 1 1 ON o I rH X 3 > I 3 O co Q 04 SI rH | ON co o 3 --1 r-l i a H M B oX O x CO X a) o 4-i e 1 CO o a > ol St CO H CO SI UO CirO> 1I 0AO0 ON r-l 00 I co CO I so rH I rH rH CO OS VO CO r> ouos II CCMO CM 4-1 rl 0 30 H >X 04 ri Vi 3 0. rl 3 X rl 3 0 H 3 X 3 3 r-4 00 03 03 t4 3 03 03 CO Vl ri CJ 0 0< 0 rl M ode.of O nset in 425 P a tie n ts 33 40 1 ON U0 VO vO 3 CM co rl .*81 X co 1 4J o UO vO r--1 rH < Si ON uO p rrl 03 o 3 rl 3X r-4 l MH Q 0I 3P H to X3 CO 1 <u Vi e^| 3 rl I 00) P o st Oo CO SI 1--1 fH cr 3 CO cd ts d1 ON CO st uo st H r> xX 0a, x co o a ol 00 co CM 00 sf W rH si CM CO vO CO CO o 3 CO 3 3 4J 60 3 0 60 VO 3 3 P u\ H 3. 3 ON St I CO x- 4-1 rl CM fM St # 3 .3 MM cd cu 0 rH t-i 0 3 cd 3 X 3 .4-1 I 0t 3 orH 00 o X < O CO 0 i--) u 3 *3 0 f>> a rl 3 rH 3 > rH 3 ON -a 3 .3 UO 0 0 X 0 *H 3 3 cu O 4J 0 4-1 WH 0 CO 0 0 rHd 0 H 4-1 0cu 0 0 > 3 3 o 03 PI r-l 3 x 0 r3-l V0 n-l 04 3 "0 3 0 O 3 HoW CO CO Po T) 03 Ps VI r-l X cd cd a. 4J 04 0 0x X rH I0Q u rrl cd X 4-1 Cd cd X 3 M X0 O H 00 H 0 X0 4J a CO a o O XX 4J co rl > MM oX 0 0 0 cu 0 X XH 0 0 c30r coo 3> Vi T3 xo 1--1 0 XtJ o X rH 0 3 H X P Vl o P 3 CO X Q m P HXo 0 0 x MH 03 0> 4-1 04 3 *3 < 3 3 O* 3 CO 3 r-l 04 rl 44 r-4 3 X 0 3 X 0 3 rl X 3 O04 3 3 X X 3 X X O X o MM -2ia- DUP050055530 TABLE 4-6 Excretion of Lead by Children with Acute Lead Encephalopathy Under the Influence of Chelating Agentsa Patient R. S. M.S. W.K. M.G. A.P. C.R. A.W. K.LeCR.N. Age, months 18 18 22 24 25 26 26 27 34 Lead excretion, mg/10 davs 23.38 40.24 55.83 51.04c 30.82 45.05 70.37 42.33c 70.99 Body weight, M, 8.77 9.0 8.94 12.8 9.0 11.0 14.5 13.5 12.9 Lead excretion> mg/kg body weight. 2.67 4,47 6.25 3.99 3.42 4.1 4,85 3.13 5.5 Mean Median 11.2 11.0 4,26 4.1 aThese data have not previously been published. Additional data on these patients were published by Chisolm.1^1 Chelation therapy was given in two 5-day courses separated by an interval of 2-5 weeks. cAn estimated loss of 3 tng occurred during the second course of therapy. Loss excluded from recorded totals. A -242- DUP050055531 TABLE 4-7 Effects of Lead Poisoning Due to Chronic Ingestion of Illicitly Distilled Whiskey on Selected Measurements for Assessing Lead Poisoning*8, * * * Patient Blood lead content , jjg/lOOg13 Hematocrit value Baseline urine lead content^ Vr /liter- PostCaEDTA ur ine lead, yg/liter * Qualitative urinary doproporDhvrin 1 80 21 2 120 25 3 130 35 4 110 . 25 5 80 24 6 50 30 7 60 29 8 70 28 9 50 28 10 80 27 < 40 d *0rO* V 180 2,200 positive 140 9,200 positive 1,300 positive 40 2,200 positive 180 2,700 positive 40 930 negative 30 2,100 positive 90 8,500 positive 50 2,000 not done 3,300 positive < 450e a Unpublished data from H, H. Sandstead. ^Lead analysis performed by the Toxicology Laboratory, State of Tennessee, Department of Public Health. Urine collections were for 2U hours (8 A.M. to 8 A.M.) c2 g CaEDTA was given intravenously over 6 to 8 hours starting at 8 A,M:. ^From Goldwater and Hoover.*^ From Teisinger and Srbovd, -2 4 3- DUP050055532 TABLE 4-8 Patterns of Increased Pyrrole Excretion in Urine o Acutely Symptomatic Pa tlent sa _____ Pyrrolesb_____ Disease ALA PBGC URO COPRO Lead poisoning Acute intermittent porphyria +++ 1 1 11` 0 -K-H- + Tt -- I Tf T-i ri +4+ +-44+ Acute hepatitis (toxic and infectious types) 000 4--H+ Acute alcoholism 00+ +-44+ aAdapted from Chisolm*'^ ^0 = Normal; H--H4+ degree of increase; ALA 5-aMnolevulinic acid; PBG - porphobilinogen; URO - uroporphyrin; C0PR0 * coproporphyrin. ^Qualitative Watson-Schwartz test for PBG. V -244- DUP050055533 CaEDTA M o b iliz a tio n T e s t ^ Technique o f T e is in g e r and Sirboyi* fed a 8Q *s 4rJljD<J* u xs= Uy g. q *rp st CM aH yc m J25 c H 8<3 a <RW iR oR < O R R<D y3 x a> 0R R< TyyJ r--1 yO 5Rf *ar-} W *H CN > cd Ry c 0) p 8 3 H m R vr P PQ p CO CM ON CM m n CM H rH on sO CM p O CO .v | .v 1 |x V 1 |X V 1 CO 00 CM sO St r-i m vrRo H '# PO on ON CM 00 <* ON 00 p P CM M Po ,1vv* | .V 1 |x v 1 I f-H VO CM on r--( SO rR * o* o* oop y CyO 33 r-l i-- <d y >> 0y0 y yy8 Ry R W y> X w y yCO 3 H y 3 r--1 y >> 0y0 y 8yy Ry R r y> X w m *r Qo CO CO R R g R H R R 8 R rR yy Uy CyJ CyJ X> *R m x> ?rl m CJ p y 0y3 8^ ON P ON P wy yCO y oa Jz; un cJ 31 pi r| OJ i St 8 R P 5 mI- Ty3 CO 0 a, K w X* H 8 y3 cn H ,>y*N iR P y y3 R 3 cq ROR 3 CO yy .09 ' Xn 3 0O *H R XJ CO >> 3 XJ O y0 3y i y> tR0 R 4J 00 3 R 00 y CM rR v 00 *3R CO y Ry y Ry y 3y yy >R 00 Ry R CO y y3 R 8 Ry 3y R Xy> Ry 3y R a< i--i XJ Os XI XJ yyy R y CM R O LA m R * Vy > g n XPJ CO ryR o w R > JO R CO 3 T> g H Ry MR 3 R no P R R y3 Ry y 3 O r-i y R y R R 3 533 cr* op y R 3 R R R P Xy) CO H y CO R 3 3 P R Ry y EM rl R rR 8 3 rH P8 R T> rR y D CO Oy <R m CO oy 0Xy) R y04 > "O CM p XI y y 3 R H TJ ) iR R <s S O 3 y X) -21*5- DUP050055534 Heme P re c u rs o rs in B lood and U rin e in "H e a lth y " and Lead-Poisoned Humans &$ 00 00 uON\ Os it s 00 00 5\ 5> 00 fc- ctor C-- oo A* gO GO OV tf\ PO Al - boo a* CO CQ to CQ CQ 4J 4J 4J P a) rH rH rH rH CQ rH 33 33 0> ed 13 TJ *o p 0) rH a J3 B Q) 01 PH <s P <u *0 <3 T) P <U *3 P4 03 4J M U nj 0) CQ CO rHp <3 <3 03 03 CU <U CQ CO cu & rH H Jp <J rH rH rH rH o H 3 3 r( O. a T3 <*3 6 w o rH a rl w a c^ 0 1a o a *uoauoah ,x/- ca cd O CM vO vp 1 P cb OP O rH iH O P uII PO-l CCOO /N r* oo *Ca3>d H H CnM II a BV v-v rH vO oo AA M/ VH so 00 rH vO M0 O CJjl 1H : 6 1 CM r> M0 M0 CM uII UII so CM O II 1-1 CO . so M0 11 oo II W uII A OO ov CM CM JL w o r-H II vp-;/ JL VaH cn CQ Si* CM CM rH & mo CT> M *s CM rH s-^ SO MO rH 00 rH uo s +l* <o a o co n fl >s $ Wowldto uII 00 CM |L B CM iH 1 II .H* *k uIIft <o O r^. CM CO oo II P li B IL B rH 1 SO 00 M0 CM rH o o IL E IL IL S uII o . rH IL B co - o IL B rO J-C n * o to a rH s o o rH 9Wi toO PH 3 y p no rid 0 ed 3 rH B o o r-H a w toO Ph 3- -2l*6- o g s jH aB cd o Bo CO rH to H too PM 3 Q O rH OB cd B oo CQ iH CO rH toO a3 DUP050055535 03 40-1 0 O0 0*43 >> 3 H CO a, 34 cd T5 CO <i> aw CO <d fart 00 HD 3 03 <u 0 *H cd 0 u 3 1-1 Q 3 a Q CO TJ HD h rl a) 0 u 0 CO cd a PM <d 0 y HD 0 u 0 cd cd o $ o i--i i <u h4 ,g sj- IS p CO 0 p-H cfl co &rCJ 4OJ +1 U M fl) 0 O t h *n cd *>n cd PC* 4C.O33 NSj~)*' H0D 00 dLTv\ 54 <M CM CO i--1 00 Oi--! CO H44 3 HD <J HCo<DUO M <u hd rH CM H 6 rW* 0 U HD rH rl 6 / * /-s sCOr VO r--1 It < r\ 00 r>. CM JL i 55 a \mS t--i CSI II v/ O < r>- 34 rH cn 1 A r> O co CM (1 II B 34 r-i O O LA O O cNj .Hfr -3* rHj 0 03 U co --44 HD 44 1 r4 i--1 3 r4 3 HO d HD < <J in X A --00 HD s-/ i i m O in o /~s 9 oo rH 1 rH vO o B m 43 i o II X st O 34 P VO rH co + /HCM N ,0 <t 60 PM o rH CM 3- J >*A HD CD >3 43 P4 O 0 rH A < O *1*-->>1, <J HD 43 IH 60 O oO 34 < 3\/ O rH 0 o 43 rH 43 44 rH CM H. ro CM $ CM co to 44 44 rH rH 33 HD HD <3 < id 0y CO CO 09 !* CM W oo p 00 o vO aI oo mp io o in II II 34 34 o*o*N ; r-i mP /%Ci^zIHOIs; &II ro--s1 i1r+--n--111. V+O1 m 1BL S!L 3 i to- 304 CO 304 rHS CO oro--1 0\ 360 43y34 cCC>dOOs >% rB-l >> H4>4 rl 4JHH4> 4y4 < 4y4 4O4 <C Q 4C4O <*3| rl a <* 3 r\ /Hi p in CM wJ3l 'JJw2L*' cs<mionioot uii vrinHO O1s SrHt II 34 P O SCMi* mrpHt II 34 4-j C6O rH CM aJL 4o3 HP 34 o rH 34 43 ja i . B 43 34 Opq CM S 0 >o 43 <u 0 3H4 P oP rH ^ 60 3. st *C*M** 630- -247- DUP050055536 R e fe re n c e s Lead P oisoning and Increased lead a b so rp tio n Type o f (Mean a n d /o r Range)aS u b je c ts LoCn\ CO ,0 H (U 43 44 -a o -d P w r-< r4 43 & 00 m rP un o i 60 o c0 <u H or> O rH (0 a 43 C a) M u rH |M rG H o i--f a rH 43 a) rH p rH . rl rH <M Pm P *H 6 0 MM s 1 60 43 a J5 cHn oHn GO cn cn _q- : q> VO VO h: Ol CVI .nt- CVI CM rH 43 CO H u 43 rH rH 3 rv *P O rH h? VO 1 I rH T3 PM <0 P to rH 4J CO 44 rH 0 rH (0 CO P Or 3 >* P X w *P < & r-s cans as cn m si* i o vh O r*H 00 II V O VD 00 r-4 6*5 B1L !L 6 m as JO 4J >r P 0 X 60 4* v. X hi n 40 X sf <t CM CN Ss 60 60 a3 <r*N 00 It s5-5/ cooOn o CS rs. CM i rH iOon 1 m so rH ll II In M vO & VH CM scin* 1 in ; Sf : ll w /V A CM rH n, rH CM r A . 1 O O JB. wS3 k N-' V/ rH OV co O O 00 JL V/ o m r> rH cCnS rH o1 rH . <7% <s P +1 CS 6 o CO VO 00 Pv H m \0 u u IBL ti u B1. !BL o u 43 vi es Vs 60 3. Oft u <u S3 H B oo rH H 43 Mi es u 43 si cs u 43 Scsi i-4 N. `V. 60 60 60 60 P 3- 3 3- 3 -248- u 43 scsi v. 60 3 xoc cd a GO % OtaHo. o uo fit 8 S ! <u t>- Pu -C=Or SI c f4 co xW40>4O tJ o 43 44 Spoo4 1 <u 4? s <u 42 hJ 43>v * H ealthy S u b je c ts (Mean jk 1 S*D. and/or Range) DUP050055537 ( perrtianerit) I 3 T> OOP (ft PM i U V* PP j s a-s 6C c H e 0 (ft H 0 CM p 1C > (0(ft P 1 -r4 pOC 0 P r (ft X Vi P p OO P 43 X M-4 V. p *H 3 (ft U 0 LM *3 0 c (0p a X >% O <9 p p P p e (ft V. p P u> P c t 4 3 Vi r< (ft 43 O P U 0 & P V P Vi 3 (ft O a X P OO--( p 1--1 +1 43 V> *r( (ft p Vi H P 6> PP c pV 3O XL c aCO ga: u *o. e. u oJC >uc(oft {c<*0 u *3P glycosiiria( f) (re ve rsib le ) 000 TJ r- P -40H3 >% ^o>e ( 0fa (0 MMM 4ftl-ffjl **E(-ftt (wuU. (X, s- 4<> a 5 fj -rl 0 or-* 6 r-4 , > C J3 PJ 2 3W 4p3 uft) U(Vfti wMP-ii HOB P> *~4 p *H 0 *3 >. *-3 KO *Ch bO u PCGL Q 4= C JC* >> 3 c g 5 3 403 -2U9- tubular function) DUP050055538 DUP050055539 Level I No d e m o n s tra b le in vivo e ffe c t Level I I M inim al stib clin ica l m etabolic e ffe c t, Level I I I Compensatory b io lo g ic mechanisms invoked Level IV Acute lead poisoning ^ 3 T> O U 03 Q> QJ <n U >-* Q> > 4MQ--1)l T*U QJ QJ >X <J C O X XO3 03 XC *0cH4?. -CO0x) oQJ 0# X o> o xa P 3E 0) x3 o c O QJ d5 d H QJ <0 X X X QJ dX o CJ *g a X B 0) QJ TABLE 4 -1 1 c o n tin u e d a Ha &3 CO 8 -P d qj 4g o >s & .J < S 60 HOo-i XToQ3J X >d >v*H X O 03 WX Pq>QJjJ UOa2 x0uE) $ O OU Mr* HQj *-dHH r--OHH H g r-4 X QJ *H O 03 > 6X x XQJ -5HS 3 QEJ Xu Xfl 4-1 4*-Q-H1J M 0OC} QJ O E> r-J Ql *rl. QJ T5 > > QJ O C tJ 55 *H o st O PO V o sr O --H 01 > QJ r--* tx o X >1a QJ o H d M C r-4 QJ o XX X> 3 U U-l C 03 O X 0 OS o X QJ 60 X rH Xa O d x T3 o T3 QJ o O r-4 O Cl (0 o "V. O QJ X .X .60 X OS a X <P> r--1 VO d QJ o QJ X H 03 QJ XX QJ r-4 H C .3 r-4 H T) X *0 60 Pa a Xq j -odp <o a I E a<u d s s pw qj p c Oh PT -at r--1 A QJ QJ CO * Id -251- DUP050055540 A D is tr ib u tio n o f C h ild re n A cco rd in g to Bipod Lead Content and Age O IVO 00 CO <1* rM 0<!0 Q CM VO CO CO CM vOI O CO CO 00 CO CO O CM p rH o U0 r-f O OCMMfOf^O COCMO CO wuCCfi o c m r-i r*. vo r>. rH O 0) <00 O <t o\ p vo T*> rH 00 P <t O P U0 CM CM O -H CM r-i 00 00 iO O O P O O *rc<H0tUf 'O rPooHQ 40HJ cpud aoaoaO) MM o 60 oP 1--oo1 P r-<H0 00 o tdo) JH 0 P r-H p CM 1 o CM P C1O CO Os I O <r p UO | o in p p | o VO P 1 o P 00 i o oo p p i o p U0) JS 0H0 MO OP VtOil *1 i? d 8 H 8 <0 P ft rS cd* -252- DUP050055541 D ata o f S urvey A reas fo r C hicago B lood Lead S c re e n in g P rogram , 1967-1968 Figures given to nearest hundred. 50 M.g/100 g o f w hole b lo o d o r h ig h e r 5 4-1 O 00 is SO CO rH is sO Sf sf rs Oosoon co co 00 is m sf is in tH in G<u CsO TO 42 .00 o r-l CN| o\ cm 00 CM Oiss -< Sf |s 00 m r^. sf vO 00 m 00 sf rs oo m CM co Os 6 40rl u o mh o so n CO CO 00 CM CM st vO sO 00 n rH sf in m sf is sf O ep O o O ooo O o oO so * in> ft rs . O CO ft sf co V) CO in rH Os sf r-l roH oo OO o oo CM oo so &*$ g<p no rfi W) 6 H 42 1 is rH m OS rH CO in sf Os SO CM CO H is. m fO m 00 o CO cino so in Vso0 rs CM CM sf rH is is CM so is m CM co CM Os vO m in 00 sO os <J I rH CM Sf in SO rH 3 o CO rH rH CM CO CO sf OO 00 CO CM O Ooo 00 m rH ft n rH co m CO OO Os is #% sf rs O oO rH ft IS sp O O IS 00 rH sf pP TGVuO 40 4000. o 40 CO CO rH 00 is IS Os so sf CO Os rs SO CM os rH rH CO m SO 00 on : Osis CM OS sO CO CM q o OS o>in rs SO rH CO rH rH is in coftCM CO CO Sf CM CM IS SO OS <D 1 u ^ <31 m rH OS CO CM rH CM o rs CO CM rs sf rH rH is. Os rH CO LA G 0> U O O o --*1 cd| -o rH rQ *rl O o m S CM * ft ft o rH SO * ft O Sf CM ft ft OO CO fl u\ Q)| 43 o CM rH CM m sf VO CO CM 00 PQ O 53 CM Tp G rH rO 4) in 0 /-s U co T3 u co O P rH 41 u SO ih H T3 o .43 c 121 O p ctf Os 4) 1--i >s V G CD O G ctf CO 43 Q> UU CO O o O o o o o ? O <D rH m co 0% CM CM is 0% in co sf in CO 0% o' m in Os sf 0% IS rH rH CM CM rH CM CM rH Sf rH CO rH 10 4J O rH CM CO sf in so rs op OS 1 H co X G CO rH PQ g 0 Pi 4H TJ <U 44 a t0 T3 < 0 -253- DUP050055542 TABLE 4-14 Cases of Plumblsm Among Battery Workers in Areas Having Different Lead Exposures8 Atmospheric lead concentration. mg/m^ of air No, men exposed Men with early plumbism No. % 0 - 0.074 0.075-0.14 0.15 -0.29 i 0.3 97 4 4.1 84 6 7.1 168 50 29,8 125 67 53.6 164 aAdapted from Dreesen si. ai A -254J DUP050055543 Average and Median Blood Lead C ontent (D lth iz o n e Method) in o CM CvOO O *t in oo o St in CM o mm Qo oo 9 3 Oom 3O <3 3 o vO OSt vO -< o <o <o oo co in 33 St CM ipn*- co oo co oO Sot st o 33 oo oSt o CvOOO H o COM o 1o--l or-o. co CM 00 OO o CO 00 CM o> CHO o oo st St CO ON CO oCM CM o oo ocu g A <0 3 oo svt> Km VO St St oo Q CM CM St st c- CO co Oo Oo vO O CM CM st mm oo oo u 3 m O XO. W 1 <U U O d <y r--) rcQd rdM *M vO CM CM in co st CoM o 9} 60 d <u jQ 6 <Vd4 <0 *pl (0 T3 5 s t> c * o CO mCM CO o <u u 60 a <u ,Bo cd cd n rl Q) 'O :o < vO CO O a) M 60 3 0) cd cd jQ6 M <u H *XJ 23 4 i st r>. CO CM oo a) w 60 d *o<0 *gS cd *4 <u tHd S3 4 * *1 l Qu3 -255- + m &4 *<09 RJ DUP050055544 TABLE 4-l6 Departmental Means and Standard Errors of 8-hr Lead-in-Air. Blood, and Urine Lead Concentrations of Workers in Ble ctric-Storage-Battery Plant St Air lead Blood lead* Urine lead concentration, concentration, concentration, Job m. workers mg/m3 ._____ ,,____ yg/l00 g blQQd yg/liter___ Mean S.E. Mean S.E. Mean S.E, Machine pasting 6 0.218 0,025 74.2 4.7 163.8 21.2 Hand pasting 8 0.150 0.029 63,2 9.2 111.3 14.1 Forming 9 0.134 0.013 63.O 2.7 ll4,0 7.2 Casting 6 0.052 0.003 - - 87.9 6,8 Plastics dept. A 5 0.012 0.0008 27,. 2 1.4 34.5 3.2 Plastics dept. B 5 0.009 0.0008 29.1 1.6 34.8 2.0 a Adapted from M. K. Williams et al 594 -256- o DUP050055545 TABLE 4-17 Urine and Blood Lead Content of Persons 1b Wenatchee Study; by Severity of Exposure8, Group Low exposure: Men Women Urine lead content, Blood lead content Ho. Average, SD, Ho. Average, SD, analyses yg/liter yg/liter analyses Hg/100 g pg/100 146 35 123 28 21 l48 26 19 '124 26 11 10 Intermediate exposure-: Men 102 43 30 108 30 11 Women 25 27 15 27 22 10 High exposure: Men 386 88 6o 329 44 16 Women 6,1 46 25 58 34 13 Children under 15: Boys 81 53 39 17 37 15 Girls 65 54 4o l4 36 10 a Adapted -from ,426 A. Weal et tal. -257- DUP050055546 C om putation Sheets, Showing Number o f Gases o f A rtfe rio s c le ro tic -H v p e rte n s iv e a ffe c te d p Tp>MO X0PW> w P cp0o T3 eH| Bj3 CO CO < *0 CpO co MP OU XOi Mp3 CO 4aaPpj. pX cpCyPoO Eoaxd nCpoO A .IS 0 4J TP3 cOOPo o J?5 p0aX CQO wX -po wXfi r ->r4J xH>uu-:1 JP!>4 CO XO) CpO CO p a > X! WC0O3 r>P-i pJ~>4 0) >Mri >H r-P4 4J rC-4O pPc ; PUo B JOh 0 PI < TUPyp? a* pX cpo CO py PP4 pH P S TP3 MBOJcPMP4o PCO P CPBO CO o in wxP4 1o-1 CO Oa o )25 Xp CH XpI npo u0 4UPJ po fi W rOpwe CO p yc CO H CpO p CO Q< on B 6BC in uu P o 0 W tj CpO 0p4 WX cc<ooD O J2 CpO CO py TJ p CO O $3 p oXP n CO 5P0 PP cH Q *4- in m Xi 50 X o * CM O n H CM CO T3 r-4 00 CM r*4 00 CO CO CM t-4 co m r-4 00 m; CM p; X O CM CM o co <t* r-4 vp T3 CM P CM 4* 00 \> vp CM t-4 c m CM t-4 m vp co n CM H P co CO r-4 co n- SP y r4 r--1 r-4 CO CM rQ co in m t-4 <t 4* st* <t co m VP iii1 m in n m P CM co si* m st* H l>. co r-4 1 CO t-4 in co t-4 P 4J P -258- .(* DUP050055547 co 43 Pnj CO TJ <0 pc0o O 0O' S53 4c2o O vor41nt W1-4 <u XMP(PQg0! ill r-C4O 4pC2O Vuco g 4oP-1 4PWm c4s P41rC1ltC00aC0<--44J4O4OO40)l r-4 4a<p1<<20400 Vr-D4 Oi-14N SvoOst r-4 a s t<0> r-o4 W0B 0H TJ wX 44 0 (0 tOcpjo PQ Tpc<Jo0 O5 4PC3O TC<JO0 * 5O3 4CdO PCdO> T> Jo25 TPCCOXQ<JOOU TJ CPc>COoO 4O2 T0J) p Uco 0 cXo M co 3 <5 T! a <0 CO CCO 4m P TJ TCJO m>PCO CO *rl wO srHo ON T--1 1 VO 4O2 4g2 0 CM Or-N4 TC<JO0 1O23 CO 4>cOP(D0o <5 tC0jO; 4p3 H r CO CO 4P3 _ TJ TJ --i pCO CD CO c PoCO 0 X w 4pPCC2OO u o CHO P0(40 cO 0> Q) o O UOXC0O 53 CO 44 O 4m PP CO CO O CO > T> 00 LO 00 CO CO CM CM o co O N0 N. CM o r-4 UO vO Or-N4 rp-N4 1 NCOM r-14 IT) Or-N4 OTmNI CO P CO a 0 or4 0 CO Oa P CO a CP CIMI <1* CM on VO ON LO VO CO r-4 co CO vO CO ON CM CO r-4 r-4 S0 vD Or-N4 Or-N4 1| vCOM vO <t rO-N4 Or-N4 TJ <0 >% r-o4 04 6 W -259- * roH O: O V oi CO DUP050055548 FIGURE 4-1. Production and Distribution of Illicitly Distilled Whiskey in the United States, 1968. Reprinted with permission ling from 5it6ensed Beverage Industries, Inc. ^ -260- * DUP050055549 FIGURE k-1. Production and Distribution of Illicitly Distilled wnissey m the United States, 1968. Reprinted with permission from Licensed Beverage Industries, Ine. 1+09 MOONSHINE PRODUCTION AND TRANSPORTATION -26i- DUP050055550 FIGURE 2. Relation between concentration of lead in blood and clinical symptoms in children with acute lead poisoning. The criteria for each of the diagnostic groups are as stated in Chisolm and Harrison, J except that spinal fluid examinations were not performed after 1956; after 1956, the minimal clinical criterion for the diagnosis of acute encephalopathy was the presence of drowsiness or stupor. The data presented in this figure have not been previously published. They are taken from the records of patients previously reported in Chisolm^^9^- and Chisolm and Harrison.^3,115 Records of blood lead content before treatment were not available for 15 patients included in these reports, most of whom had asymptomatic increased lead absorption . Patients included in these reports who died are included in the death group. The data on deaths caused by lead-paint poisoning were obtained from the Baltimore City Health Department records. Records of blood lead content were available for 98 (1936-1970) of 132 reported deaths (1931-1970). Where blood lead content was not obtained during life, diagnosis is based on postmortem studies. A breakdown of deaths into 5**year periods (1936-1970) shows no significant variation in either distribution or mean of blood lead content in fatal cases. All blood lead analyses included in this figure were performed by the Baltimore City Health Department (dithizone method). The values shown are not corrected for deviation from normal hematocrit. Some of the children included in the group designated as "asymptomatic increased lead absorption" * -262- DUP050055551 had mild symptoms that at the time of examination were' attributed to other disease processes rather than to lead. In such instances, the nonspecificity of mild clinical symptoms made differential diagnosis difficult. Of considerable interest is the spread of values under each clinical category and the overlap of laboratory values from category to category. The relatively low concentrations of blood lead found in some of the children may in part be due to anemia, but they also suggest that a blood lead concentration may not always exclude lead as the cause of illness if it is not as high as usually observed in illness. Blood lead concentrations >60 pg/100 grams in children are for all practical purposes compatible with the diagnosis of disease due to lead if there is clear evidence of impaired heme synthesis and the other clinical findings support the diagnosis. -263- DUP050055552 DUP050055553 -26 k- ASYMPTOMATIC INCREASED LEAD ABSORPTION ACUTE LEAD POISONING D AGN0S1S ACUTE LEAD ENCEPHALOPATHY u FIGURE 4-3. Lead interferes with the biosynthesis of heme at several enzymatic steps, with the utilization of iron, and, in erythrocytes, with globin synthesis. Inhibition of ALAD and heme synthetase (Steps 2 and 6), which are SH-dependent enzymes, is well documented and accumulation of the substrates of these enzymes (ALA and PROTO 9) is characteristic of human lead poisoning. Inhibition of ALAS (Step l) is based on experimental evidence only. Whether there is enzymatic inhibition or other factors operate at Step 5 is not clear; neverthe less, increased urinary excretion of COPRO is prominent in human lead poisoning. Minor increases in PBG and URO in urine are occasionally reported in severe lead poisoning. The utilization of iron is impaired; although the in vivo mechanisms are unclear , nonheme iron (ferritin and iron micelles) accumulates in red blood cells together with damaged mitochondria and other fragments not found in normal mature erythrocytes Serum iron may be increased in humans with lead poisoning, but without iron deficiency states. Heme synthesis is reduced and, in red blood cells, globin synthesis is apparently impaired, although the mechanisms responsible for reduced globin synthesis remain unknown. -265- ENZYMATIC STEPS INHIBITED BY LEAD FIGURE 4-3 s t e p s in b io s y n t h e s is o f h e me in h ib it e d b y l e a d NORMAL PATHWAYS PORPHYRIN FORMATION IRON UTILIZATION METABOLITES AND ABNORMAL PRODUCTS ACCUMULATED INHUMAN LEAD POISONING :V * -266- 1 DUP050055555 FIGURE Correlation between ALAD activity and blood lead con centrati on of 158 persons representing different degrees of natural and occupational exposure to lead. Note logarithmic scale on ordinate. Solid circle, medical students; open circle, workers in printing shops; solid square, automobile repair workers; open square, 3! lead smelters and shipscrappers. Redrawn from Hernberg et_ al. A LAD A ctivity in Units/ml Red Blood Cells -267- DUP050055556 FIGURE 4-5. Values for ALA in urine plotted against those for lead in blood. The broken lines mark the upper normal limits. Redrawn from Selander and Cramer. ALA mg / 100 ml urine -268- DUP050055557 FIGURE 4-6. Relation between blood lead concentration and daily excretion of ALA in urine in a group of young children without clinically evident lead poisoning. The group consisted of 51 children between 1 and 5 years old, drawn from an inner-city clinic population in Baltimore, Maryland. The range of blood lead concentration was 25^-75 pg/lOO g of whole blood. Whole blood samples for lead determination were drawn on admission to the hospital and analyzed by the laboratory of the Baltimore City Health Department by a dithizone technique. ALA output was measured in quantitatively collected 24-hr urine specimens by a method that excludes aminoacetone. Statistical analysis of the data indicates that ALA output is related to age and height but that it is best related to surface area in young children, in that the slope and regression between daily ALA output and surface area intersect at the origin. Therefore, expression of ALA output as mg/nn per 24 hr completely compensates for differences among the children due to age and body mass. A highly significant curvilinear relationship is best fitted by the following quadratic equation: mg/m2 per 2h hr = 2.4135 - (0.07500 x Blood Pb) + 0.001265 (Blood Fb) . For this fitted quadratic equation, <0.01 and the standard error of the fitted line is + 0.6. A highly significant linear relation is also found in this group between blood lead con centration and the log of ALA excretion (j d <0.001). Within this narrow range of blood lead concentration (25-75 yg/100 g of whole blood), the data suggest that ALA excretion apparently increases only as blood lead concentration rises above approximately 40 yg/100 g of whole blood; however, the absence of a threshold for this relation cannot be excluded in these young children. -269- Statistical analyses were performed by Dr, David E. Mellits, Department of Pediatries, Johns Hopkins Medical School. These studies have not been previously reported (J. J. Chisolm, Jr.). They were supported by DHEW grant EC-R01-002D1 from HIH, HSMHA study project 464, MCHS grant MC-R-240012-01-0, institutional research grant FR-53-78 to the Johns Hopkins Medical School, and a grant from the Thomas Wilson Fund, Baltimore , Maryland, The studies were carried out on the Pediatric Clinical Research Unit of the Johns Hopkins Medical School, which is supported by DHEW grant R-M01-00052. * * FIGURE 4-7. Relation between blood lead concentration and daily ALA excretion in urine in a group of adolescents with no known abnormal exposure to lead for 10 years or longer. The group consisted of 55 adolescents who had had acute lead poisoning as young children but no * known unusual exposure to lead for at least 10 years before the studies shown here . Blood lead concentration was determined by a dithizone technique, and ALA was determined on quantitative 24-hr urine collections co by a method that excludes aminoacetone. ^ ALA output is expressed as mg/m per 24 hr to facilitate comparison with the data in Figure 4-6. Within the range of blood lead concentration in this group (8~4o yg/100 g of whole blood), no correlation was found between blood lead concentra tion and ALA excretion. This suggests that ALA excretion is low, constant , and independent of blood lead concentration when blood lead concentration is less than approximately 40 yg/100 g of whole blood in adolescents. These studies were supported by DHEW grant EC-R01^-00201 from National Institutes of Health. They were carried out in the Clinical Research Center of the Department of Medicine, Johns Hopkins ^ University School of Medicine, which is supported by DHEW grant 5-M01-RR-00032. -270- DUP050055559 FIGURE 4 -6 . Blood Lead Concentration /(jug Pb TOOg of whole blood) -271- DUP050055560  2O no> 8 o " CT I C .2 io CM 1 O FIGURE 4 -7 * O -272- I -'ll DUP050055561 FIGURE 4^-8. The values for the CaEDTA lead excretion in relation to previous lead exposure and disease. Method of administration of CaEDTA: 1 g of CaEDTA in 250 ml of a 5% aqueous solution of glucose was administered by intravenous infusion over the course of 1 hr. Urine was then quantitatively collected for 4 days. Be drawn 173 from Ifaffiierson. PATIENTS WITH NORMAL RENAL FUNCTION || PATIENTS WITH RENAL INSUFFICIENCY MINIMAL LEAD EXPOSURE INDUSTRIAL 1I DUE TO LEAD LEAD EXPOSURE ' NOT DUE TO LEAD INDUSTRIAL LEAD EXPOSURE CONTROLS >3.0mg - 'NORMALS' 2.5 oX 1.5 -< ** 1.2 UJ m <0 g t0.6 m m --m *.. 0.3 . .* ... S m *T | 99 9 -273- ..... ...............*................................... Jf v v-,v,v ..... DUP050055562 FIGURE 4-9. Seasonal distribution by* month, of report of diagnosis of fatal and nonfatal cases of lead poisoning and increased lead absorption. (Same cases shown in Jig, 4-2). FIGURE 4-10, Numbers of cases for each diagnostic classification grouped by month of age at time of diagnosis, (Same cases shown in Fig. 4-2). -274- DUP050055563 o CO 0M0 <<N0 !N CM CM CON C*=O Ct~O 5- Vc m- O ^ 00 CO CM r .r t ....r T~T ri--r o>z 88aggsg^^ FIGURE l*-9 . MONTHS 0 1 483 2 ffi TO $ 34-* r*3i aa oo <2 -j C x : aa f8 c a> a> Ion|# -<JDP <*3o <~a3 Qtac3>o -L.,. L. 1 8 CO CM 8 CM CM CM O 00 CD CM r- o- J___ L J N -D 00 CD sioarans do saaaiAinN gg aQ;. CD E2* mES DUP050055564 FIGURE U-10 AGE A T TIME OF DIAGNOSIS (GROUPED BY MONTHS) S 3 t CS CO a Si a CD i--i--i--i--i--r , oC ................ I i ?i 5o >E. >" 1 II- O JC JC g> o 3 S *E I! 11 -- fin s f |<1_i <3 <3 o* P to on si O) oir>> s C E 1 11 ia^x>oo<>op^ CM I1I J.... 1 S3svo do syaawnN -276- E I 00 <fr O DUP050055565 CHAPTER 5 BIOLOGIC EFFECTS OF LEAD IN DOMESTIC AND WILD ANIMALS DOMESTIC ANIMALS It generally is recognized that lead is the most common cause of acci dental poisoning in domestic animals. The condition is diagnosed most frequently in cattle and dogs * It should be kept in mind that a dis*cussion of lead poisoning in domestic animals must differ from the approach taken for man* Whereas subtle, subclinical effects of lead are highly relevant and important for man, similar considerations regarding animals are not practical. Lead poisoning in animals usually is recognized only when overt clinical signs of poisoning are apparent. The natural curiosity and licking habits of cattle make any available lead-containing material a potential source of poisoning. Some of the sources incriminated are lead-base paint (either from discarded paint cans or from paint peeling from walls), used motor oil, discarded oil filters, storage batteries, some types of greases and putty, and linoleum. v These sources can be found in the vicinity of farm buildings and in dumps located in pastures. It is noteworthy that these sources are rarely incriminated in lead poisoning of horses * Horses are much more selective in their eating habits than cattle. They usually do not lick old paint cans, storage batteries, or peeling paint, nor do they find the taste of motor oil attractive. Histories of exposure in dogs commonly include chewing on objects painted with lead-base paints (e.g. , when home remodeling entails -277- .. f r. . .... ......................... ' Vy-. " > :................................r .................................................................... y. ........ DUP050055566 scraping of plaster and old paint), eating linoleum, or ingesting lead materials , such as lead slugs and curtain weights Dogs less than 6 months old are affected more commonly than older do gs, hut this may he related to the almost completely indiscriminate eating habits of younger dogs. Several outbreaks of lead poisoning in domestic animals have been recorded in which the apparent source of metal was contamination of pasture or crops by industrial lead operations. These outbreaks differ from the more common cases of lead poisoning described previously, in that several animals may be involved. Some of the areas involved in Forth America are St. Paul, Minnesota;2^ ^20 oocr Belleville, Pennsylvania; Trail, British Columbia, ^and Benecia, California. bkh Deaths of horses attributable to lead poisoning have been noted in the Benecia area since the early 1900fs. It is reported that the presence of lead and arsenic has made it impossible to raise oil horses near smelters in East Helena, Montana, for several decades. Pastures and crops are contaminated by fumes and dusts that are emitted by lead industries and settle on the surrounding countryside. Animals that eat this vegetation can accumulate sufficient lead to produce clinical signs of lead poisoning, A number of studies have been made to determine whether the lead found in vegetation is of direct airborne origin or due to translocation from soil. These studies have been reviewed recently by Mueller and Stanley ^,416 a who conclude that translocation from soil does not contribute more than 15 jig of lead per gram (dry weight)of forage, even when plants are grown in soil containing about -278- & p J DUP050055567 700-3000 ug/g. Thus , amounts of lead in excess of 15 ug/g most likely are due to direct aerial fallout. The extent to which contamination can occur is illustrated by concentrations of 3200 pg/g (dry weight) found in corn leaves 75 yards from a lead smelter.23 It has been estimated that 6-7 mg/kg-day constitutes a minimal cumulative fatal dosage of lead for cattle. 238 This intake represents a concentration of about 200 ppm in the total diet. The cattle studied were approximately 2 miles from a smelter but were fed lead-contaminated hay and corn silage grown in fields adjacent to the smelter. A fatal case of lead poisoning occurred after approximately 2 months on this diet, and an intake of approxi mately half this dosage had no observed effect on cows at another farm in the previous winter. In this eonnnection, it is of interest that dosages of 5-6 mg/kg-day were fed to cattle for a period of 2 years with no observable 9 10 clinical effects, but that longer intake at this rate may be fatal. There is some evidence that horses are more susceptible than cattle to the chronic ingestion of lead. Whereas horses contacted lead poisoning on pastures adjacent to a lead smelter in the Trail area, cattle grazing in the same area 335appeared healthy. At one farm adjacent to the St. Paul smelter, horses succumbed to lead poisoning in March after a winter lead intake in their hay estimated at 2.k mg/kg-day. D It was not possible to determine the lead intake from past lire grazing in the previous summer. However, inasmuch as cows and horses had similar pasture that summer and the winter ration for the horses contained appreciably less lead than that for the cows, it would seem that cumulative toxicity occurred somewhat more readily in horses. It is of interest to consider that pasture grass containing over 80 pg of lead per gram (dry weight) was toxic to horses in the Benecia area.^1^8, A horse will eat about 21 g dry matter/kg body weight/day. Thus, a minimal toxic dosage could be estimated at 1.7 mg/kg-day--a figure close to the previous estimate. -279- DUP050055568 Although the evidence above suggests that horses might be more sensitive to lead than cattle are, a consideration of the grazing habits of horses precludes eny firm conclusions. Horses occasionally pull forage out by the roots and eat the roots and attendant soil with the forage Cattle rarely, if ever, do that, probably because their jaw structure makes it impossible. The soil near smelters usually contains far greater amounts of lead than does the forage. It is apparent that a horse showing a marked tendency toward this habit could ingest far greater quantities of lead than would be estimated from the analysis of forage alone. Several workers have documented high concentrations of lead in grass along high ways. Motto et al^1^ found in grass samples immediately adjacent to the roadside an average concentration of lead (dry weight) of 255 mg/kg; the concentration decreased with increasing distance from the highway (see Table 2-6) . It is appropriate to consider whether concentrations in grass of this magnitude con stitute a hazard to animals such as cattle and horses. Even if the entire diet of cattle were obtained from forage immediately adjacent to a major highway, overt clinical poisoning probably would not occur. Cattle eat approximately 22,5 g of dry matter per kilogram of body weight per day. Thus, the ingestion of grass immediately adjacent to the highway (255 mg/kg dry weight) would provide a daily intake of approximately 5*7 mg/kg. Horses, however, appear to succumb to lead poisoning at a lower daily intake than cattle. If their diet consisted exclusively of forage obtained 75 ft from a major highway (99 mg/kg dry weight, Table 2-6), overt lead poisoning conceivably could occur. A horse eats approximately 21 g of dry matter per kilogram of body weight per day, and the ingestion of forage containing 99 mg/kg dry weight would provide a daily intake of approximately 2.1 mg/kg. Exclusive consumption of forage 125 ft from the highway would provide a daily intake of approximately 1,U mg/kg, somewhat below that associated with clinical lead poisoning in the horse. -280- DUP050055569 It is emphasized, however, that it would he highly unlikely for animals to obtain their entire diet, the year around, from grazing within 125 ft of a major highway. Even in the absence of fences, animals usually prefer to graze in areas farther from major highways. It is only natural for persons residing close to smelters near which animals are dying of lead poisoning to be concerned about their own health. In many cases, such persons eat produce from home gardens. Analyses of blood and urine of these persons by local public health officials have not revealed evidence of increased lead absorption. It is emphasized that horses and cattle are vegetarians and, if raised in the vicinity of a lead industrial operation, may subsist entirely on contaminated vegetation. Probably only a small fraction of the total diet of human beings would consist of food grown in the vicinity of a lead operation.' Furthermore, it is customary for people to wash garden produce (or to husk corn) before its consumption. This practice undoubtedly removes appreciable quantities of surface lead deposits. Inasmuch as the animal and human populations breathed the same air and human residents in the area did not show evidence of increased lead absorption, it may be concluded that the animals received nearly all their lead burden through oral ingestion. All domestic species with lead poisoning exhibit various degrees of derangement of the central nervous system, gastrointestinal tract, muscular system, and hematopoietic system. Differences occur clinically, however, in the relative severity of signs referable to those organs and tissues. The most striking syndrome is presented by young calves . The calf may suddenly begin to bellow and stagger about with rolling eyes and frothing mouth and often blindly crashes into objects. This phase may last up to 2 hr before sudden collapse. In less severe cases, depression, anorexia, and colic may be observed, and the animals may be blind, grind their teeth, move in a circle, push against objects, and be ataxic. The less severe signs are more frequent in adult cattle, although the syndrome of maniacal excitement is not uncommon. In sheep, the syndrome consists mainly of depression, anorexia, abdominal pain, and (usually) diarrhea. Excitatory phases have never been reported for sheep. Anemia is common during chronic ingestion of lead. -281- DUP050055570 In horses, the syndrome consists mainly of depression, stupor, knuckling at the fetlocks, and laryngeal paralysis that produces an obstruction in the air passage and causes the horse to "roar," Anemia is conmonly .associated with lead poisoning in horses.13 1 Gastrointestinal or central nervous system signs are seen with equal frequency in lead poisoning in dogs. At some time during the course of poisoning, approximately $7% of dogs show GI signs consisting of emesis, colic, diarrhea, and anorexia. Approximately 76% of dogs show CNS signs consisting of hysteria and convulsions . Anemia and basophilic stippling are commonly associated with lead poisoning in dogs and are considered to he of diagnostic significance Abortions have been reported in ewes that graze in lead-mining areas. A high rate of abortions and of failures to conceive were noted in ewes fed finely divided metallic lead at a rate sufficient to induce signs 76 of poisoning. The lethal dose of lead appears to be considerably lower in pregnant than in nonpregnant ewes. Cattle and horses have given birth to normal offspring after excessive lead exposure ,171*512 but the small number of animals reported (five) makes it impossible to state that lead has no effect on the fetus in these species. A high concentration of lead in blood or tissues, associated with clinical signs, is considered to be the best criterion for a diagnosis of lead poisoning. The presence of basophilic stippling of red blood cells, immature (especially nucleated) red blood cells, and acid-fast inclusion bodies in the cells of the kidneys has been reported to be -282- y y DUP050055571 commonly associated with lead poisoning.^*A discussioh of the treatment and costs of lead poisoning in cattle is found in Appendix G. BIRDS Lead poisoning has been recognized in waterfowl and upland game bird species for at least 100 years. Lead poisoning was considered to be a serious problem in ducks and other waterfowl in the United States by 1919.^^ Recently it was estimated that a million ducks, geese, and swans die of lead poisoning each year in the United States.^ This figure becomes even more impressive when it is considered that most of the birds die after the hunting season and thus represent a loss of breeding stock. Although many birds die from lead poisoning from minor wounds, the principal, and probably the only significant, source of lead is spent lead shot that is ingested in the birdsf search for gravel. Approximately 6,000 tons of lead shot are deposited on waterfowl habitats each year.171 Cases of lead poisoning occur in upland game birds, as well as waterfowl.^1 As in the case of waterfowl, the primary source of lead appears to be spent lead shot, as evidenced by the recovery of the material from the gizzards of affected birds. Efforts to develop a safer material than lead for shot began as early as 1936.221 Copper and hard iron are safer materials, but ballistic considerations and damage to gun barrels and Chokes make the materials unsuitable. The Sporting Arms and Ammunition Manufacturing Institute (SAAMl) recently supported a $100,000 study by the Illinois Institute of Technology to find a material more suitable than lead. Soft iron pellets were found to be the best substitute. Although soft iron pellets -283- DUP050055572 compared satisfactorily with lead for killing ducks in flight ^ the cost of manufacturing them is at present prohibitive Another problem with soft iron shot is that the material hardens with age. Shot made 2 years ago has hardened by 25#, and that will cause a scoring of gun barrels and chokes As with mammals , the presence of lead in birds is a constant finding. A study including between one and 37 representatives of 28 different bird species with no known ,,excessive,, lead exposure revealed concen trations in fresh liver ranging from 0.3 to 7*0 ppm.^ Lead, as well as several other inorganic ions , was measured in pheasant populations in Illinois. Concentrations (wet weight) ranging from 0.22 to 0.27 yg/g of blood, 0.09 to 0.84 yg/g of liver, and 0.11 to 0.27 yg/g of kidney were measured in apparently healthy pheasants A lethal amount of lead for duck can be absorbed from one #6 lead shot. Variable mortality occurs after the ingestion of up to six #6 shot, which is nearly always lethalA daily dose of 6 mg/kg-day (as lead nitrate) for 137 days did not produce any observable ill effects, but when the dose was increased to 8 and 12 mg/kg-day, the survival periods averaged 28 and 25 days, respectivelyThe deposition of lead in soft tissues was almost proportional to dosage, but no appreciable differences were measured for bone. When the dosage of lead for the two birds that received 6 mg/kg-day for 137 days was increased to 12 mg/kg-day, death occurred at about the time expected for the new dosage. The long period on the lower dosage seemed to have little effect on the rate at which clinical signs developed or on the deposition -284- DUP050055573 of lead in soft tissues. When excretion was expressed as' a percentage of total dose, an average of only 3% was excreted in the first week, hut the average increased to 6j% and 63% for the second and third weeks, respectively. Concentrations of lead in muscle are very low in mammals, hut it has heen reported that breast muscle and liver from a pheasant dying of lead poisoning contained 42 and 169 ppm, respectivelyA total of 29 shot were recovered from the gizzard. However, concentrations of lead in muscle of mallards dying of lead poisoning are considerably lower than 42 ppm (L. N. Locke, personal communication). The usual picture of lead poisoning in waterfowl consists of lethargy, weakness, flaccid paralysis, emaciation, anemia, greenish diarrhea, impaction of the proventricuius, and distention of the gall bladder. The large pectoral muscles waste away, and in some cases the sternum is covered merely with a thin layer of fascia* muscle, and skin---the so-called "razor-keel" or "hatched-breast1,.1^^>^,^^2^^ Acid-fast intranuclear inclusion bodies of the renal tubular cells are commonly but not always found in cases of lead poisoning. A number of workers have stated that basophilic stippling of red blood cells does not occur. It has been suggested that lead may be important in producing sterility among many birds that in other respects appear to have recovered eompletely from clinical lead poisoning.567 This contention was not sup*ported by a study designed to evaluate breeding performance of mallard -285- DUP050055574 drakes. 103 The data indicated that drakes recovering from lead poisoning did not exhibit a significant loss of fertility; similar studies with females, however, were not carried out. It has been reported that some green feeds , such as coontail (Ceratophyllum demersum) will prevent lead poisoning in mallards fed levels of lead shot that will kill birds maintained only on cracked or whole corn.^0aTn another study, ducks maintained on corn and on a diet of grain and duck pellets developed acid-fast intranuclear inclusions in renal tubular cells when given one or three #6 lead shot orally. No lesions developed in ducks maintained on a duck pellet ration and fed the same number of shot. It is not known whether these dietary factors affect the absorption or the biologic effects of the lead, ZOO ANIMALS Chewing on cage bars painted with lead-base paint has been incriminated as a source of lead poisoning in bats^^ and in nonhuman -primates. AQUATIC ORGANISMS Contamination of natural waters by effluent from lead mines has long been recognized in England, A report of the River Pollution Commission of 167^ (cited by Jones^S ) described the disappearance of fish from streams fouled by effluent from lead mines and deaths of waterfowl, horses, and cattle in the vicinity of streams. Lead also can be present in effluent associated with a number of industrial and manufacturing operations. 286- DUP050055575 Probably the first definitive experiments on lead poisoning in fish go q t go were carried out by Carpenter. An explanation was sought for the continued absence of fish in rivers passing through old mining ' areas. Minnows placed in a river in cages remained normal until heavy rains occurred. The concentration of lead in the river suddenly in creased from an immeasurable value to 0.3-0.4 mg/liter, and the minnows died. It was reasoned that the rain dissolved surface lead deposits and carried them into the river. The effect of lead on lower forms of life is not well documented, but it appears to be less toxic than in higher forms. For example, insects rir/ were shown to be less sensitive than fish to the effects of lead. Concentrations of lead in excess of 0.1 mg/liter appear to inhibit the 286,602 self-purification of water by biologic means in water-storage reservoirs. Concentrations of lead in excess of 0.5 mg/liter have been reported to retard the growth of protozoa and to retard nitrification in sewage 286 purification procedures. Acute Toxicity of Lead to Freshwater Fish For purposes of this review, "acute toxicity1' will be defined as in*eluding effects that occur within 2 weeks of exposure to lead. Available evidence suggests that the principal biochemical lesion occurs outside the body. When fish were placed in a solution con taining insufficient lead to cause death, a film of coagulated mucus formed over their bodies, with concomitant respiratory distress.^3 Recovery occurred when the film was shed. Analysis of the film for -287^ DUP050055576 lead accounted for almost all the lead in the original solution. No lead could he found in the bodies of the fish, hut it should he pointed out that the exposure was brief (hours ) and that the method of lead analysis23was not as sensitive and accurate as methods in use today* Observation of these effects led to formulation of the "coagulation 177 278 SR6 film anoxia" theory, 99 The film of coagulated mucus that appears over the entire body is particularly prominent over the gills; the insoluble material interferes with the respiratory function of the gills, resulting in acute respiratory distress and death by suffocation. This effect is not peculiar to lead; it can be produced by toxic concentrations of other heavy metal ions , including zinc, iron, copper, cadmium, mercury, manganese, cobalt, nickel, silver, gold, and aluminum 9^,162,172,278 The effecib hag been demonstrated for many species of fresh-water fish, but apparently not for salt-water fish. Lead may precipitate in sea water before toxic concentrations are attained. Thomas^^could not poison killiefish, which can live in both fresh and sea water, with lead in sea water, because the material precipitated out of solution; in fresh water, lead nitrate (3 mg/liter) was fatal in 12 hr. Some species of fish are considerably more susceptible than others to the toxic effects of inorganic lead. Carpenter93 observed that the action of lead on goldfish was the same as on trout, sticklebacks, and minnows, but goldfish were more resistant, Jones^^stated that goldfish appeared able to tolerate indefinitely 1 mg of lead per liter of soft tap water, whereas 0.1-0.2 mg/liter proved fatal to sticklebacks. The amount and nature of gill secretions may explain variations in species -a88~ ^ j DUP050055577 susceptibility to lead. 2 Goldfish produce a copious gill secretion. When exposed to 10 mg of lead per liter, goldfish produced so much precipitated mucus that the solution he came milky and sediment collected on the bottom of the vessel. Ellis112suggested that, if the concentra tion of a pollutant, such as lead, were low enough or if the source were limited so that it acted on the fish for only a short time, the secretion of additional mucus might wash away the precipitated material before serious toxicity to the fish occurred. It is difficult to define acutely toxic concentration of lead for fish; experimental results from different laboratories vary considerably. Such variables as dissolved oxygen concentration, solution pH, volume and number of exchanges of experimental solution, and duration of exposure are not always controlled.12 Water temperature is an im portant factor. A 10 C rise in temperature reduces survival time by 50%.Probably one of the most important factors is the degree of water hardness. Lead is readily precipitated as the carbonate or hydroxide; thus, hard waters tend to decrease the effective concentra tion of lead. As water hardness (expressed as calcium carbonate) increased from ik to 53 mg/liter, the concentration of lead in solution decreased from 8 to 1.6 mg/liter.21 Another important point regarding the presence of calcium is that it appears to decrease the toxic effects of lead.20 For example, in solutions containing 1 mg of lead (as lead nitrate) per liter with 0, 5> 10, 20, and 50 mg of calcium (as calcium nitrate or chloride) per liter, survival times averaged 1, 3, 6, 7, and 10.5 days, respectively. In this case, precipitation of lead could not -289- account for the decreasing toxicity with increasing concentrations of calcium. It was concluded that calcium somehow prevents the precipi^ tation of mucus by lead. Calcium was shown to have a similar pro-^ teetive effect on the toxicity of other metals. 91 Carpenter * 93 and 280 Jones have reported some of the lowest demonstrable concentrations of lead (0.1-0.4 mg/liter) toxic to fish. They used either distilled or soft tap water in their studies, which might be expected to provide optimal conditions for the toxic effects of lead. However, it has been pointed out1^2 that other species of fish probably are more sensitive to lead than those studied by Carpenter and by Jones, and concentrations of 0.1 mg/liter therefore probably do not represent minimal concentrations of lead toxic to sensitive fish in the conditions most conducive to poisoning. The behavioral effects of acute exposure to very low concentrations of lead in goldfish are discussed in Chapter 4. Chronic Toxicity of Lead to Fresh and Salt Water Fish and Other Aquatic Organisms Few studies are available concerning the chronic toxicity of lead to fish. Anemia has been reported in catfish exposed to solutions of 50 mg/liter for periods of 16-I83 days Somewhat similar findings were reported in guppies exposed to 1.24 and 3.12 mg/liter (total water hardness, 80 mg/liter) for periods of up to 129 days.139 In addition to blood changes, histologic studies revealed renal changes consisting of expanded tubular lumens and a lack of lymphoid tissue, a lack of mesenteric fat, cellular elements in the myocardium suggestive of -290- 4 * DUP050055579 degenerative changes, and retarded gonadal development. There was no demonstrable consistent alteration of respiratory epithelium or evidence of an accumulation of coagulated mucus, but the possibility of some damage to the respiratory system was suggested by the frequent finding of granular debris in the branchial blood vessels . The histopathology, as well as growth inhibition and retardation of sexual maturity, sug gested that the secondary effects of inanition or stress were the most prominent features of chronic lead poisoning in fish. Growth inhibition also was observed in salt-water plaice when exposed to solutions of 4 mg of lead per liter.159 There have apparently been no studies with fresh-water fish in which concentrations of lead have been measured simultaneously in fish and water. Such information would be essential for evaluating the ability of fish to concentrate lead from a surrounding medium, Kehoe, Thamann 306a 24$ . ,,and Cholak and Harley have reported lead concentrations of 0.24 and 588 0.16 ppm in fresh-water fish. `Wetterberg reported concentrations as high as 12 ppm in liver, 5.7 ppm in gills, and 1.4 ppm in muscle of fish taken from a lake near a rich lead mine. No analysis of the lake water for lead was reported. Analyses of over 1500 samples from natural water sources near watertreatment plants over a $-year period throughout the United States revealed measurable quantities of lead in fewer than 20% of the samples analyzed, and 27 samples were over the U, S. acceptable limit for >318 drinking water of 0.05 mg/liter. The highest value recorded was 0.l4 mg/liter in the Ohio River near Evansville, Indiana, a heavily -291- DUP050055580 industrialized area. The authors pointed out that the total concentra tion of lead in a body of water would be higher than that in flowing water because the presence of carbonate and hydroxyl ions in natural still waters tends to induce precipitation of lead as insoluble lead salts. This was borne out in a study measuring the concentration of lead in particles larger and smaller than 0.45 \xm at various depths hoo in Lake Hamilton, Arkansas. The concentration of larger lead particles increased with increasing depth. Information is not available to state with any degree of certainty whether the occurrence of lead in natural waters of the United States constitutes a serious threat to fish or to humans who eat the fish. There seem to be no reports of fish kills due to pollution of natural waters in the United States by lead. In addition to areas close to water-treatment plants, it would appear highly desirable to analyze both water and fish for lead downstream from industrial operations likely to emit lead. An evaluation of lead concentrations in the water and fish of lakes used heavily by motorboats would also be desirable. The amount of lead emitted into the water from an outboard motor burning leaded gasoline (0.7 g of lead per liter) appears to be related to the size of the motor and the speed of operation.17 S A 10-horsepower engine operated at one-half to three-fourths throttle was shown to emit into the water 0.229 g of lead per liter of fuel consumed, whereas a 56-hp engine operated at full throttle emitted 0.121 g/liter. -292- DUP050055581 There is no question hut that some marine organisms can concentrate the lead present in sea water. The normal concentration of lead in M sea water is stated to he around 0.-00003 mg/liter Although concen trations of lead reported to occur in seafood are relatively low, they \ do indicate considerable concentration from the surrounding medium, kok For example, Schroeder et al, ^ reported a range of 0,17-2,5 ppm in seafood, with an average of 0.5 ppm and only one sample exceeding 0.87 ppm. Harley245reported a concentration of 0,31 ppm in shellfish. 459 Pringle et al. reported average wet-weight concentrations of 0.47, 0.70, and 0.52 ppm in eastern oysters, soft-shell clams, and northern quahpgs, respectively. The remarkable ability of the eastern oyster to concentrate lead was demonstrated by exposing oysters to flowing sea water containing lead concentrations of 0.025, 0.05, 0.1, and 0.2 mg/liter. After 49 days, the total accumulations of lead amounted to 17, 35, 75, and 200 ppm (wet weight). The highest concentrations of lead in the oyster occurred in liver, and the lowest concentrations, in muscle tissue. Oysters exposed to the two lower experimental con centrations of lead appeared normal. Oysters exposed to the higher experimental lead concentrations (0.1 and 0.2 mg/liter), however, grossly showed considerable atrophy and diffusion of the gonadal tissue, edema, and less distinction of hepatopancreas and mantle edge. In view of the insolubility of lead in sea water in usual conditions, it would be very interesting to know whether conditions could arise that would permit lead concentrations of 0.2 mg/liter to exist in sea water in a chemical state such that it could be absorbed by marine organisms to the extent shown in this experimental study. -293- DUP050055582 SUMMARY Lead poisoning is the most frequently diagnosed poisoning of domestic animals. The sources most commonly incriminated include lead-base paints, storage batteries, and used motor oil, Airborne lead from smelting and other lead-using industries has caused poisoning of cattle and horses in localized areas, by contaminating hay and pasture vegetation. Airborne lead from vehicular exhaust emissions has not been shown to cause poisoning of domestic animals . Lead poisoning is an important health problem in waterfowl. The only source of lead ever reported to cause poisoning is spent lead shot from hunting. There is no evidence that lead constitutes a health problem to fish in the United States. But there is very little information on which to base any firm conclusion. Very few analytic data have been reported on concentrations of lead in fish in natural or experimental conditions. What data are available suggest that soluble lead is not present in natural waters of the United States in concentrations likely to be toxic to fish. There is no published evidence of any trend toward increased concentrations of soluble lead in natural waters. Much of the mandispersed lead that is eventually washed into natural waters is probably precipitated owing to the presence of carbonates, hydroxides, and organic ligands in the water and settles to the bottom. There is no evidence that lead precipitated on the bottom of natural waterways is harmful to fish. -294- % * DUP050055583 CHAPTER 6 AIRBORNE LEAD ALKYL COMPOUNDS The atmosphere may contain gaseous lead alkyls, in addition to particu late inorganic lead. Lead alkyl vapors result from the production of lead antiknock compounds and their subsequent handling and use as gasoline additives* Precise information on the concentration of lead alkyl vapor in the ambient air is scanty, because until a few years ago the only available methods of analysis required the sampling of large volumes of air and their precision and specificity were not as good as could be wished. The analytic methods in use before 1962 were developed mainly to monitor the air in lead alkyl manufacturing plants .^^^^'^^ecause the threshold limit value for tetraethyl lead (TEL) was 75 yg of lead per cubic meter of air, the analytic methods were designed to measure TEL concentrations close to this figure. That even at this comparatively high lead alkyl concentration in the air some of the analytic methods were inadequate is shown by the recovery of only 30%*-iQ% of the TEL 36l in the air by the aqueous iodine method. By 1967* a new method was developed using an activated charcoal scrubber, that achieved an o acceptable level of accuracy (+ 0.008 yg/nr of air) in the hands of well-trained personnel, and an effort was made to measure the much smaller quantities of lead alkyl compounds in suburban air.52k However, the large volumes of air (100-200 m^) that had to be passed through the scrubber made the method suitable only for measuring average concentrations over periods of several days. Snyder carried out some -295- DUP050055584 limited sampling in Los Angeles with this method; at a time when the average concentration of inorganic lead in the air was 3.56 ug/m^ of air over a 6-week period, the organic lead concentration averaged ' O.O78 yg/rn^ of air, with a range of 0.0^7^0.106 yg/m^. The method of analysis used during the survey of atmospheric lead in three urban communities^^was not accurate enough to allow more than the general comment that !&t most the alkyl lead concentrations did not reach 10 percent of the inorganic, lead values and probably were considerably less." In 1970, A. Laveskog published a method that combined the use of gas chromatograph material to trap alkyl lead compounds, desorption, separation, and detection (carried out in a mass spectrometer). The method is sensitive to within 10 ng of tetramethyl lead (TML) or TEL per cubic meter of urban air. 34q This method made it possible, although by no means simple, to measure the TEL and TML in the urban air accurately with relatively short (15 min) periods of sampling. LEAD ALKYLS IN THE AIB Because lead alkyls are readily broken down by light and heat, their presence in the atmosphere is transient. In some conditions, however, sizable short-lived peak concentrations can be attained. For example, the combustion process within the engine coupled with the passage of the exhaust gases through the hot exhaust manifold and muffler system normally results in no more than 100 yg of lead alkyls per cubic meter of exhaust gas leaving the tailpipe. This gas is rapidly diluted by o the surrounding air to give concentrations of 0,02-2 yg/m- of air. o -296- DUP050055585 However, if measurements are made at the tailpipe of a poorly tuned car starting fully-choked from cold, transient peak lead alkyl concentra tions up to 5000 ug of lead per cubic meter of air may occasionally be reached; such peaks last for a matter of seconds and fall below 1000 yg/m^ within 2 min and to 4-30 yg/m^ in 10 min. It must be remembered that these measurements are made in the exhaust gas stream before dilution with ambient air. If such a cold-started fully-choked car were to pass a sampling station in the street where the average lead alkyl level might be 0,02-0.Oh yg/m^ of air, a peak of about 0.5 yg/ir3r might be measured as the car passed by. 340 Other sources of lead alkyl vapor in the ambient air due to operation of a car are gasoline evaporated from the fuel system, particularly from the carburetor after the hot engine is shut off, and from crank case nblow-by.,! The crankcase emissions contain a high percentage of unbumed air-fuel mixture. Both these sources of lead alkyls have been or are being reduced or removed by antipollution devices built into new cars now being sold in the United States. The devices include the recycling of the blow-by gases and control systems designed to contain, and recycle evaporative losses from the carburetor and fuel tank. Sources that are more difficult to control involve the distribution and dispensing of gasoline containing lead alkyl and include displaced fuel tank vapors, entrained fuel droplets in the displaced vapors, and liquid gasoline spillage. -297- DUP050055586 Because of the much higher vapor pressure of gasoline than of TEL or TML and the small amount of lead alkyl in a gallon of gasoline, lead alkyl vapors are not present to a significant degree above liquid ' gasoline until almost all the gasoline has evaporated. When a gasoline spill occurs and the gasoline is allowed to evaporate completely, as may happen in filling a gasoline tank from a pump, a transient high concentration of lead alkyl in the ambient air can be found. Filling station attendants more than most persons are exposed to the lead alkyls in the air around service stations, as well as to sources of inorganic lead, e.g., from cylinder head deposits, crankcase oil, and dust from mufflers and other parts of the exhaust system. In addition, some service station attendants engage in the grinding of body solder. Such attendants have been "studied and their urinary lead concentrations found within the range in the general public, although, as might be expected from the possibility of absorbing lead from more than one source, concentrations for individual attendants may be toward the high end of the normal range (up to 30 ug/liter).305 CLINICAL RAMIFICATIONS it 37i TEL was discovered by Lowig in 1852, but the first cases of occupa tional poisoning were reported in 192^4, shortly after TEL began to be manufactured in the United States as a gasoline additive. The main site of action in lead alkyl poisoning is the central nervous system. In severe cases in man, a toxic psychosis develops, with hallucinations, delusions, and excitement, which may end in delirium and death. If the mental symptoms disappear, recovery is very likely * X -298- DUP050055587 but may take 2-6 months* In contrast with poisoning with inorganic lead, lead alkyl intoxication seldom if ever is accompanied by abdominal colic, abnormalities of the red cells , or peripheral neuropathy. The earliest symptom of lead alkyl poisoning in man is insomnia; in cases of more severe exposure, that is followed by lack of appetite, nausea, vomiting, and diarrhea. Continuing exposure commonly leads to com plaints of irritability, restlessness, nervousness, and anxiety, and these may be followed by the more serious mental symptoms mentioned earlier. As with toxic effects of all chemicals, the relation of the amount of the chemical absorbed to the type and severity of the symptoms is very important in establishing whether a clinical picture is due to a particular chemical. /Podds^and Kehoe' ^agree that, if the urinary excretion of lead is less than 0.1 mg/liter of urine at a time when such symptoms as those described are present, the cause is unlikely to be the absorption of TEL. Mild symptoms may occur with a concen tration of 0.15 mg/liter of urine, but TEL poisoning is generally associated with concentrations of about 0.3 mg/liter or greater. ei.fil.537 Stoppe/ found no adverse effect of TEL exposure on health (as measured by history of important chronic diseases, height and weight, blood pressure, hematology, ECO abnormalities, or other findings on physical examination) when 348 TEL workers were compared with 348 matched controls not working in TEL production. Lead excretion in the TEL group was 0.064 +_ 0.033 mg/liter of urine, and in the control group, 0.03 + 0.016 mg/liter of urine. The health of those who had -299- DUP050055588 previously worked in TEL production but were no longer so engaged was also studied and compared with the health of the other two groups. No significant differences in health were found between any of the groups. Although the acute toxicity of organic and inorganic lead differs considerably, it is possible that the chronic toxicity depends more heavily on the lead ion than on the chemical form of lead at the time of absorption. That is suggested by the fact that, if the lead con centrations in the urine of workers exposed to organic or inorganic lead are kept below 150 yg/liter and the concentrations in the blood are kept below TO mg/100 g of whole blood, the Workers 1 health is protected."1 86,3|| has not been found necessary to set lower biologic safety limits for exposure to organic lead than for exposure to inorganic lead; this suggests that the safety of long exposures to either inorganic or organic lead depends more heavily on the average amount of lead ion present in the soft tissues and less heavily on whether an inorganic or an organic form of lead enters the body. Because TML is more volatile than TEL {TML boils at 110 C, TEL at 199 C), some concern was felt by manufacturing companies and public health authorities when TML was introduced as a component of gasoline antiknock mix around 1959 DeTreville et_ al., however, reported in 196Z^%hat in their experience there was less hazard associated with the manufacture of TML than of TEL* The smaller hazard associated with TML is explained partly by a slightly lower absorption of TML, as shown by the somewhat lower urinary lead concentration of workers in f S' -300- DUP050055589 TML areas , than of workers in TEL areas , at the same concentrations of 362 TEL and TML vapor (measured as lead) . EXPERIMENTAL STUDIES In addition to observations in man, toxicity differences between TEL kgi and TML have been found experimentallyf Schepers found that the oral approximate lethal dose of TEL in rats was 11 mg of lead per kilogram of body weight and that of TML was 83 mg/kg--at least a sevenfold difference in acute toxicity. Similar differences have been found 1 kg ___ when the compounds were given by inhalation. ^The metabolism of TEL suggests that it is a compound of very low toxicity, but that it is broken down in the body to triethyl lead, a water-soluble compound that causes the clinical picture typically associated with lead alkyl +tox.i c.it. y.iko In experiments on liver and brain homogenates , Cremer^9tlso showed that the enzyme system that converts TEL into triethyl lead is found only in the liver. Bolanowska^ carried these studies a step further with rats and showed that 2k hr after the administration of TEL, $0% of the total lead in the soft organs was in the form of triethyl lead. The concentration of triethyl lead remained steady for several days. The other metabolite of TEL was inorganic lead, which was produced within the first 2k hr after a single exposure to TEL but had been removed from the soft tissues within a week. SUMMARY Practical and precise methods of measurement of the con centrat ion of lead alkyls In the ambient air have only recently become available. Preliminary data based on these newer methods suggest that the broad -301.. DUP050055590 generalization contained in the three-city survey, that "at most the alkyl lead concentrations did not reach 10 percent of the inorganic lead values and probably were considerably less ,11 remains true * It is now possible to demonstrate peak concentrations lasting from a fraction of a minute to several minutes during which the lead alkyl concentration may reach 50% of the inorganic lead concentration when the sampling point is close to a cold-started automobile or a spill of gasoline containing lead antiknock compounds. . Offsetting this difference in ambient concentrations to some extent is the relatively greater acute toxicity of lead alkyls than of inorganic lead compounds. Judging from the few clinical cases , these differences do not appear in practice to apply to long human exposures extending over a working lifetime. -302- DUP050055591 CHAPTER 7 NONBIOLOGIC EFFECTS OF AIRBORNE LEAD NONBIOLOGIC EFFECTS ON MATERIALS The Panel has not looked exhaustively at the nonbiologlc effects of. airborne lead, but it has inquired specifically about possible effects on two important categories of material: natural and man-made textile fibers and glass. In addition, it offers some brief comments on auto motive exhaust catalysts and microelectronic devices. The possible role of lead aerosols in affecting atmospheric visibility and in serv ing as nuclei for water vapor condensation is also discussed. Organic fibrous materials used in clothing and in a large number of household and utilitarian items fall into two basic categories: the natural fibers, among which wool and cotton are economically the most significant, and man-made or synthetic fibers. Normal values for lead content apply both to the cotton plant and to the cotton fiber grown in normal-lead-content soils, and for plant ash c are between 0.008% and 0.18% (80-1800 ppm). Ikk The lead content of wool has been reported to be Ir3 mg/100 g. 233 Data for human hair reported by Kraut and Weber is consonant with the overall lead content of 1.7 mg/100 g from normal persons not work ing in activities involving lead products. Data obtained during assess ment of damage to wool fibers induced by the use of plumbite solutions in chemical treatments and photochemical degradation showed that the uptake of lead by wool is related to the formation of lead-sulfur com plexes with sulfhydryl groups generated by the splitting of disulfide 552 linkages in the wool structure (F. J. Rizzo, unpublished data). Thorsen -303- DUP050055592 has more recently shown that staining of wool by lead occurs strongly in the paracortex of the fiber, and progressive levels of staining in this component may be taken as an indicator of increased damage to the fiber. By extrapolation from these facts, one may thus assume that the lead content of wool fibers is associated with free sulfhydryl groups. The distribution of lead in the tissues of animals to which lead salts are deliberately injected favors the bone structure, 486 which suggests that the concentration in the wool fiber will be limited even when feeds containing high concentrations of lead are ingested by the sheep. In the modern fiber producing facilities of the industry, lead as a structural element has been supplanted by other materials that greatly reduce the lead and other nonfibrous components that tend to interfere with the color of the fiber and often its processing and chemical reac tivity characteristics. The lead content of man-made fibers to which lead compounds have not been deliberately added is generally below that of natural fibers. By far the most significant source of lead in textiles is its deliberate addition to achieve specific functional properties not inherent in the basic fiber of the fabric. In some instances, it is accidental--for example, in yarns that have been delustered by the use of titanium diox ide pigments in which lead is a natural impurity. Organolead compounds and lead compounds formed in situ with many organic acidic compounds of high molecular weight have been applied to textile materials to impart waterproofing or water-repellence, fungusproofing, and occasionally delustering characteristics to textile materials. 135 Other compo sitions involving compounds with more favorable toxicologic and der-* matologic characteristics are available today and are therefore more -304- DUP050055593 generally used both for clothing materials and for textiles that go into industrial and other nonclothing items. Where it is desirable to use lead compounds in processing clothing materials* it is obvious that preference is for nonabsorbable (not fat soluble) forms that, cannot enter the body through the skin (see Chapter 9) a The synthetic fiber industry introduces small amounts of metal compounds into the polymer before its extrusion in fiber form to provide antioxidative properties,-^ heat stability, and photochemical stability and to depress some degradative tendencies. The degree and nature of chemi cal bonding of lead and other additives is not always clear, but they obviously are sufficiently bound to the polymer substrate by either physical or chemical processes to remain essentially undiminished during the active life of the textile materials. The protective quali ties of lead against gamma and x rays are well known, Textile fabrics 413,506 impregnated with lead salts and fabric composites with lead sheets are items of commerce for both laboratory and medical shielding purposes. Deposits of airborne contaminants (including lead) are known to have deteriorative properties on textile materials, but it is not known how much, if any, of the deterioration may be due to lead itself. A signifi cant factor that limits the impact of contaminants on clothing materials is laundering or dry cleaning, which reduces deposits to levels that should be close to the levels in the fiber when it was produced. Airborne lead has no effect on glass at low temperatures (25 C) and con centrations, Lead will react with glass at temperatures above about 443 400 C, which precludes using glass where such high temperatures exist with lead. -305- The catalytic surface destruction by lead in automotive exhaust control devices appears to be of major importance, Such effects are not strictly from ambient atmospheric lead, but rather from lead that would otherwise have been distributed in the atmosphere, There appear to be some problems with respect to the effects of particulate matter in general on micro electronic devices, but it is not possible to assign any of these effects specifically to lead. (McNesby, J. R., personal communication), NQNBIOLOGIC EFFECTS OH ATMOSPHERIC VISIBILITY Char Ison and Pierrard"^^ estimated that automotive lead emissions could be responsible for one-fourth or more of the observed visibility degrada tion noted at times near freeways. The magnitude of this estimate depends heavily on assumptions made because of lack of data on two important prop erties of the lead aerosol. These assumptions were: that the lead aerosol always has a size distribution similar to that of the aged urban atmos pheric aerosol, and that short-term lead aerosol particle-mass concentra tions in such areas exhibit large excursions about their longer-term mean values, Since the appearance of that paper, further data have been published that suggest that the contribution of lead emissions to visibility reduction i ]ip should be reconsidered. Paines et al. 'reported a decrease of 50% in airborne lead mass concentration between 10 and 150 ft from a highway, with a similar reduction between 30 and 250 ft from the highway. The particle-size distributions showed little change in the fraction of col lected lead at the third impactor stage {mass median equivalent diameter (MMED) - 3.5 pm for/^=5.85 g/cnf|] as a function of distance from the highway. but they showed a decrease for larger particles and an increase for smaller 306- DUP050055595 4l6 ones. These observations support the approach of Mueller, who esti mated the size distribution of atmospheric suspendable lead (by assuming 22$ settling of all particles larger than 10 ym MMED) from Habibirs 229 size distributions of automotive emitted lead Habibi et al. haVe since reported that 57% of the mass of the lead emitted by the average car on the road is in particles larger than 9 ym MMED. However, atmos- 468,469 374 pheric measurements by Robinson et, al. and Limdgren show MMED of 0.1-0.5 ym for lead-bearing particles. These observations indicate that a rapid and significant change takes place in the size distribution of the lead aerosol in the time between its emission and its arrival at a point even, say, 30 or 40 ft from the source. It appears that a suitable first approximation is that the air-suspendable fraction of the emitted aerosol (MMED more than about 10 ym) is conserved. The magnitude of the reduction in lead mass concentrations and the implicit change in size distribution over short distances suggest that an estimate of visibility reduction by lead particles cannot be soundly based on the assumption that the lead aero sol has, at the same time, a size distribution comparable with that of the atmospheric aerosol and a high mass concentration corresponding to proximity to the source. In an effort to clarify the role of exhaust lead particles in visibility reduction, a sampling program was conducted in September and October 1970, at a site adjacent to the heavily^traveled San Bernardino Freeway at El Monte, California, 15 miles west of downtown Los Angeles. Continu ous records were made of (1) light scattering coefficient, which is inversely proportional to visibility, (2) carbon monoxide concentration, -307- DUP050055596 (3) wind speed and direction, and (4) temperature and relative humidity. Although analysis of the data from this study is not yet complete, some useful points have already emerged (J. M, Pierrard, personal communication), Visibilities encountered during the observing period varied over more than an order of magnitude. No deliberate bias was introduced in choos*ing periods for lead sampling runs* except that no samples were taken be tween midnight and 6 a.m. Ten-minute average values were formed from the records of the continuously recorded variables listed above, and these data analyzed statistically. Correlation coefficients between lead and carbon monoxide concentrations were slightly smaller than those (0.79-0.81) found at Los Angeles area sites during the three-city survey.539 For visibility of less than 5 miles, the correlation coefficient between 10min lead and carbon monoxide concentrations is 0.66 (N * 156); for visi bility of greater than 5 miles, r - 0.75 (N = 335), The statistical associations provide no information on the absolute magnitude of the contribution of lead particles to visibility reduction. The failure to find a significant association between visibility and the con centration of lead suggests that the effect of lead particles in visi bility reduction is outweighed by other factors in the case of low visi bility. The probable dominance of other elements, such as hygroscopic particles, is also suggested by the improved performance of complicated quadratic models in accounting for light scatter. However, lead particles in the air must make some contribution to total light scatter. The size-fractionated aerosol collections mentioned earlier were made to provide estimates of this contribution. Unfortunately, analy sis of these samples is incomplete, so no estimate is yet possible. It does appear that the large, easily settleable particles were already greatly depleted in the lead aerosol sampled at the site, in that the average of 18 lead size distributions analyzed to date yields an MMED ^ t 468,469 of approximately 0.2 pm, in agreement with the results of others for the dispersed urban lead aerosol. The ratios of peak 10-min average lead concentrations to the averages over 1 and 2 hr were 1,20 and 1.28, respectively. Although peaksmean ratios of lead concentration may be larger than this closer to the source, large particles that would con~ tribute strongly to mass concentration near the source would not make significant contributions to light scatter. It therefore appears un-* much as likely that lead particles could contribute as/25% of visibility reduc tion. WEATHER MODIFICATION BY AIRBORNE LEAD The formation of large masses of ice crystals either increases or decreases precipitation and may be related to airborne lead insofar as 48< lead contributes to the possibility of the formation of nuclei. Schaefer observed the production of abundant freezing nuclei when iodine vapor was allowed to react with automotive exhaust; he interpreted the reaction as 44-9 a lead '-halide interaction. In a recent study in the Boulder-Denver area, iodine vapor production of freezing nuclei was more than 1 order of magnitude above the background level , yielding concentra tions of 10-300 nuclei per liter during both surface and airborne sampling. This concentration range is considered adequate for effective weather modi fication. Furthermore, the interaction with iodine vapor generated plenti ful freezing nuclei even at 9,000 ft above sea level, which is frequently the level of summertime cumulus cloud bases in the Denver area. Sampling was conducted as high as 14,000 ft, where high concentrations of latent -309- DUP050055598 ice nuclei were found when conditions were favorable to turbulent mixing. During periods of inversion, very low concentrations were observed at the higher levels. A study by Hogan^^ of io dine -1reat e d automotive exhaust indicated that 60% of the detected lead particles had a radius of 0.2-1.0 pm. The mini mal detectable size was 0.2 urn, and therefore at least 60% of these par ticles will have low terminal fall velocities and long expected residence times in the atmosphere. Supportive evidence that lead contributes to nuclei formation is the fact that concentrations of lead and other particles 5kg are known to be effectively reduced by rainfall. It should be noted that there is still little more than circumstantial evidence that lead particles, in the absence of artificial treatment, actually become ice nuclei, but the possibility of widespread effects should be kept in mind.^0 SUMMARY The reportedly low lead content in natural fibers is related to the rela tively low solubility of most lead compounds found in the soil, the low natural concentration of lead in normal soils, and, in the case of wool, the metabolic balance in the tissues. Man-made textile fibers are produced today with very low lead content, except those to which lead is deliberately added for specific protective qualities. The low concentra tions of lead in fibers encourage the conclusion that such concentrations may be within the tolerance limits of humans for lead compounds, even if the concentrations are labile to the point of complete transfer from the textile material to human skin. Given the knowledge that fiber producers -310- DUP050055599 subject their products to toxicologic and dermatologic testing for reasons of self-protection, the role of lead when it is deliberately added would not seem to achieve significance in terms of potential hazard to man except where , skin damage exists and lead is in a fat-soluble form- The secondary factor in this respect is the low level of addition that is effective in achieving the desired results. Lead concentrations in the air are too small and usually not at a high enough temperature to affect glass. Lead-containing particles in auto motive exhaust are destructive to metallic surfaces only when they are no longer airborne. It is not possible to assign a destructive role to lead in contamination of microelectronic devices. Re-examination of the statistical association of concentrations of lead to carbon monoxide concentration, light scattering coefficient, wind speed and direction, and temperature and relative humidity provided no information on the extent of lead's contribution to light scatter, especially at low visibility, where other ambient factors apparently come into play. There are few capable people working in these compli cated problems and there is much more work to be done before they can be treated adequately. The modification of weather by interaction of lead with iodine vapor needs much more field research before conclusions can be drawn as to its usefulness. -311- DUP050055600 CHAPTER 8 CONCLUSIONS AND RECOMMENDATIONS FOR RESEARCH EXTENT OF ENVIRONMENTAL POLLUTION WITH AIRBORNE LEAD The extensive information assembled in this document leaves little doaibt as to whether man has substantially contaminated some parts of his en* vironment with lead. It is also clear that one major source of general environmental contamination is the combustion and dispersal of lead alkyl compounds used as automotive fuel additives. The lead alkyls also are by far the most readily and completely dispersed of all the various substances generated by the lead-using industries, owing to their aerosol form. The air over the largest American cities has a concen tration of lead 20 times greater than the air over sparsely populated areas of the country and 2,000 times greater than the air over the mid-Pacific Ocean. Although there is evidence that this concentration profile is due largely to gasoline-additive lead, the high degree of dispersal associated with the venting of burned lead alkyls into the air has minimized the effect of the rapid rise in consumption of leaded gasoline on the atmospheric concentration of lead, with the result that the average lead content of the air over most major cities apparently has not changed greatly over the last 15 years. The net result is that correspondingly little change in the character and magnitude of the effects of atmospheric lead on biologic systems will likely occur for some years to come. We are, in short, not dealing with a rapidly shifting scene in this respect. However, more recent information is not in complete agreement with this conclusion and might slightly modify it . A deliberative evaluation of the data would be possible only if the Panel were reconvened. -312- DUP050055601 From the point of view of contamination of the total environment with lead, the lead-using industries have contributed more tlian h.as the single source of combusted leaded fuel. Two to three times as much lead is added to the total environment in the form of paint pigments and'metallic products as in the form of lead alkyls. Some undefined fraction of this non re cycled lead finds its way into the ecosystem by surface weathering and leaching, dumping, and burning. The environmental fate of this lead is ill-defined, but it is probably chiefly returned to the soil without emission to the air. Any proposal for the removal of lead from auto motive fuels to rid the environment of lead pollution must take into consideration the fact that the fate of other lead products , such as paints and manufactured items, is largely unknown. The Panel can do no more than call attention to the lack of information on this point. Recommendation 1: Expanded study of lead chemistry in nature. The Panel encourages the study of the chemistry of lead in the environment--in emitted particulate matter, in soil, and in water. The physics of particles and aerosols in general is comparatively well known, but the chemical forms of lead need elucidation before its effect on man can be fully understood. Knowledge of the complex chemistry of biotrans formation of lead by lower organisms is of considerable im portance. A prerequisite to research on the chemistry of lead in the environment is the training of atmospheric chemists competent to undertake it or the reorienting of trained chemists in this direction. *** -313- DUP050055602 The transfer of lead from the atmosphere to the soils can be demon strated by several examples. Within a narrow band along major roadways, the concentration of lead bears a direct relation to the density of automobile traffic. The surface soil of parks and the street dust in large cities also is heavily contaminated with lead. However, rural soils where crops are grown show little evidence of lead contamination attributable to burned lead alkyls. Similarly, the waters of streams and lakes have about the same concentration of lead today as in 19^0 , at which time the consumption of lead alkyls was considerably less than it is today. There is no evidence that the amount of lead in the diets of people has changed substantially since 19^0. This is consistent with the observed trends concerning lead in soil and water. Furthermore, the concentration of lead in edible plants is little affected by even large increases of lead in soil above rural concentrations. Surface deposition of lead on plants from the atmosphere occurs but seems to be small, except in the immediate vicinity of major roadways or of other sources of lead emission. The concentration of lead in animal food products also seems to have changed imperceptibly if at all over the last 30 years. Recommendation 2: Monitoring food and drink for lead content. It is recommended that an extensive monitoring program of lead in common items of food, drinking water, and miscellaneous beverages be instituted now, to provide reliable benchmarks for the future. The protocol for securing this information should be standardized and should be detailed as to description -314- * * DUP050055603 of sampling technique, analytic procedure, specification of water content , and geographic and other environmental character istics of the sampling site. The lack of film evidence that concentrations of lead in food and drinking water are increasing is not very reassuring. Past studies of lead in foods have teen so poorly described and so deficient in sample size as to make . the data of very questionable value as benchmarks for measuring changes over the years.* In assessing the dietary sources of lead, the possible contamination during preparation and storage of food and drink must be taken into account. Periodic publi cation of updated figures in a useful form would enhance the value of such studies to nutritionists and public health officials. *** The general picture that emerges from consideration of lead in the en vironment is of a steep gradient of pollution emanating from the cities, in proportion to their size. The absence of significant upward trends in the concentration of lead in rural soils and water and in the food sources of man in the last 30 years indicates that the great increase in the combustion of lead alkyls has not had a large impact on the intake of lead by nonurban people and animals , except perhaps by direct inhalation. Air has not been monitored long enough or extensively enough to allow any firm conclusions on this point. However, the monitoring activities in urban centers since i960 have not yet suggested any substantial upward trend. -315- DUP050055604 Recommendation 3; Improved aerometric data gathering and sampling near populations at risk. Currently, after 26 years of being concerned with air pollution, our meteorologic broadcasts are limited to announcements of the , local air pollution index in a relatively few cities of this country . Much effort should be expended in the field of meteorology, coupled with the use of tracer techniques, to use this important branch of science for a more complete understanding of the factors that bear on dissipation and nondissipation of air pollutants* Specifically, most meteorologic data are now collected at airports near the fringes of urban centers and do not necessarily describe the conditions in the city, where the industrial, municipal, traffic, and other emission sources are, or in the surround ing rural environs. Also, some polluting elements (such as lead) are difficult to measure in a built-up area. A net work of aerometric stations should be established in each urban area to obtain meteorologic and pollutant data that are representative of the area and can he used to provide overall values for the city as a Whole. Furthermore, this network should be extended to cover the natural air move ments from one district or region to others, to form a national meteorologic program that is air-pollutionoriented. The continued and orderly development of the Storage and Retrieval of Aerometric Data (SAROAD) system by the Environmental Protection Agency.^2 is a step in this direction, whose usefulness will be determined by the quality of the data that enter the system. -316- 4 DUP050055605 For individual air sampling sites, the Panel believes it im portant to point out that the site should he related to the community at risk. If air samples are for analysis of pollutants potentially hazardous to man* for example, they should be taken where the population is greatest--near the street, rather than on the roof of a tall building. If young children constitute the population at risk, then the sampling intake should be closer to the surface than If adults are the only concern. This principle also applies to testing of natural waters subject to industrial contamination. In this case , it would be desirable to collect representative samples from sites removed from water treatment facilities to obtain complete data on the concentrations of lead (and other trace elements) resulting from discharge by lead-using or lead^producing industries along the waterways . The importance of these measurements is apparent when it is realized that the lead may be con centrated by different species of fish or other aquatic organisms. *** EFFECTS OF AIRBORNE LEAD ON MAN Only in the urban setting is man possibly exposed to hazardous circumstances relative to lead pollution* occupational exposures in the lead-fusing industries excepted. The exposures are mainly the consequences of atmospheric emissions. The high concentrations of lead in urban air and on the surfaces of parks and streets constitute a source of intake additional to the usual dietary sources and. in special circumstances may be a substantial source. -317- DUP050055606 The contribution of atmospheric sources of lead to the total body burden of city dwellers varies considerably, and depends on the particular' urban complex, and on the place of residence and of work within that complex. An extensive survey of men in three large urban complexes has suggested but not shown with definitive evidence a strong association between the concentration of lead in the blood and time spent in areas of high automobile traffic density. Although this evidence of the impact of lead inhalation is inferential, limited experimental data are consistent with the conclusion that the amount of inhaled lead is about one-half (and in special circumstances twice) the amount that comes from diet , depending on the particular urban microclimates encountered in the course of daily activities. However, it is not possible, on the basis of available epidemiologic evidence, to attribute any increase in blood lead concentration to exposure to ambient air below a mean lead concentration of about 2 or 3 yg/m^; only special small groups of people in large cities are exposed to higher mean atmospheric concentrations. Autopsy data strongly suggest that the body burden of lead in the general population increases with age. Mpst of the increase is attributed to an increase in the amount of lead in bone , but it seems likely that some other tissues also are involved. The concentration of lead in the blood does not increase with age. This suggests that the readily exchangeable pool of lead in the body generally reaches a steady state early in life and that the increases in body burden with age probably involve a relatively inert, slowly exchangeable pool of lead* the toxicologic significance of which has not been evaluated. -318- y DUP050055607 Recommendation 4: Research on body burden and distribution of lead as related to age and its biologic significance. More age-related studies are needed to evaluate further the concept that the "mobile" fraction of the total body lead burden remains constant throughout life; this hypothesis is presently based on very limited data. Age-related studies should also include some measure of biologic response to increasing concentrations of lead in soft tissues (e .g. , ALAD in blood and excretion of ALA in urine). The concept that the nondiffusible fraction is tightly bound in bone and therefore biologically inert requires more detailed examination. In mammalian systems, the relations between parathyroid hormone, thyrocalcitonin, bone , and kidney are intimate and important in the metabo lism of bone mineral. Research concerning the effects of these relations on lead metabolism and clearance from the body should therefore be carried out. Data obtained from such studies will provide a better understanding of the significance of the "nondiffusible" fraction of body lead to health. *** Poisoning clearly attributable to airborne lead exposure was at one time a serious and frequently encountered disease among workers in lead smelters and in the lead-using industries. Today, industrial lead poisoning is much less commonly encountered and is seldom seen in its more severe forms, because industrial hygiene programs and better diagnostic procedures have substantially reduced the. hazard. However, in small shops with relatively little supervision, potentially dangerous conditions exist, the extent of which is at present unknown. -319- DUP050055608 In addition to airborne lead in smelters and in lead-using industries, other sources of lead poisoning have been identified. The most serious and crippling current form of lead poisoning is encountered in infants and young children living in deteriorating housing of the cities; usually, the source of poisoning is leaded paint. This type of paint is no longer used for interior surfaces, but the pre valence of poorly maintained pre-1945 housing constitutes a special hazard to those who live in it. Other miscellaneous sources of lead poisoning of both children and adults include illicitly distilled whiskey, improperly lead-glazed earthenware, old battery casings used as fuel, and an assortment of manufactured items, such as leadcontaining toys. The entity of severe clinical lead poisoning is manifested as a constellation of effects on the central nervous system, the gastrointestinal system, the hematopoietic system, and the kidneys. Other organs (such as the thyroid gland and the heart) may be involved to varying degrees. In the most severe form of poisoning, profound disturbances of the central nervous system are prominent and permanent damage to the brain may occur. Damage to the kidneys also is prominent and may be permanent. The life span of erythrocytes is shortened, with or without coexistent anemia. This form of lead poisoning is encountered today mainly among infants and heavy drinkers of illicitly distilled whiskey. Recommendation 5: Establishment of comprehensive medical and environmental control programs to eliminate clinical lead poisoning in special groups. Occupationally exposed workers and young children are in urgent need of sustained medical supervision and effective environmental control programs. Prospective epidemiologic programs are needed to evaluate the extent of the risk to these groups and to assess the effectiveness of measures designed to minimize such risks. In such programs, consideration should be given to total exposure derived from all environmental sources. It is hoped that implementation of the Occupational Safety and Health Act of -320- DUP050055609 19TO and the Lead-Based Paint Poisoning Prevention,Act of 1970 will provide the needed epidemiologic data and facili*** tate the realization of these goals, Analysis of confiscated samples of illicitly distilled whiskey indicates that this may also he a significant cause of lead poisoning. Sampling of populations known to he at risk is needed to evaluate the extent of this problem. Potential sources of hazard, such as improperly lead-glazed earthen ware, should he controlled hy systematic sampling before distribution to the general public. There is no substitute for astute clinical observation and case-finding. These techniques provide clues concerning incompletely understood aspects of lead poisoning, which should be studied experimentally. The obvious adverse effects of excessive lead on the nervous system, kidneys, and hematopoiesis are well recognized at the clinical level, but they are not adequately understood at the subclinical level. Furthermore, dose-response relations need to be established with regard to these effects. Case studies suggest that neuropathy and myopathy may he significant but inadequately recognized aspects of lead poisoning both clinically and sub clinically* These possibilities are in need of careful study. Even less well understood are the apparent effects of lead on various endocrine organs. Experimental and clinical investigative efforts are appropriate to elucidate the. clinical significance and mechanism of these effects* Wider availability of adequate laboratory diagnostic services is essential if progress is to be made toward these recoKH mended clinical and epidemiologic goals. Analytic problems inherent in the determination of lead in biologic samples suggest that reliable data may be obtainable only in larger laboratories with adequate quality-control procedures and experience in lead determinations, In addition to estimates of the level of exposure and of the mobile fraction of the total body lead burden, adequate medical supervision also requires measurement of some metabolic response, such as ALAD or ALA. k k * In its mildest forms , clinical lead poisoning is usually seen where close medical supervision (e.g., in industry) can facilitate early identification of toxic signs and symptoms. In such circumstances , mild anemia with shortened red-cell life span is noted, often with headaches and generalized muscle aches. Constipation and diffuse abdominal pain generally follow. These effects are readily re versible by removing the worker from the source of lead. The first clearly defined clinical signs of lead poisoning usually do not occur at blood lead concentrations lower than SO jig/100 g of whole blood, except in cases where anemia prevails. In such cases, lower concentrations may be associated with signs or symptoms of poisoning, v * -322- DUP050055611 because the analysis of lead per unit volume of blood is performed on an abnormally low number of red blood cells , to which more than 90# of lead in blood is specifically associated. At blood lead concentrations of l0-80 yg/100 g of whole blood, the excretion of the heme precursor ALA in urine is increased because of inhibition of the conversion of ALA to porphobilinogen (PBG) by the enzyme ALAD. With respect to hematopoiesis, this loss of heme pre cursor may be compensated completely, for there is no evidence at present of interference with the maintenance of normal hemoglobin levels in the blood of adults at this level of exposure. Implications for the synthesis of other heme pigments has not been adequately explored. Nonetheless, this action must be viewed as undesirable, in that it does represent an interference with the availability of an essential metabolite required for normal body function, which in some circum stances might prove deleterious. r--N At concentrations of blood lead below about 40 yg/100 g of whole blood J an additional biochemical effect is noted. Inhibition of ALAD in circulating red blood cells in proportion to the concentration of lead found in the cells can be demonstrated in vitro. Ibis relation seems to apply even at the lowest concentrations of lead detectable in vivo in the peripheral blood of man, but its biologic significance is dubious, because it is unaccompanied by any detectable biologic effects in intact man. -323- DUP050055612 Recommendation 6: Additional information on dose-response relations of lead. A fuller understanding of the biologic significance of the in vitro inhibition of ALAD in blood is needed. Because it is likely that herae synthesis will be inhibited in other tissues, as well as n blood, it seems likely that the assay in blood may be a model of the effects of increasing lead content in other organs. Dose-response relations with respect to heme synthesis need studying in other tissues and particularly in the central nervous system. Relations between ALAD, ALA, and the "mobile" or "chelatable" fraction of the total body lead burden require further careful clinical study, especially in the range of 5-80 ug/100 g of whole blood, to determine more accurately the apparent "threshold" for this effect. All age groups, as well as subgroups of the population that may be less able to compensate for impairment in heme synthesis, should be studied. Such studies not only should have sound epidemiologic design, but should use timed, quanti tative urine collections, standardized mobilization tech niques , and more specific methods for measuring ALA than have been used in the past. Dose-response relations that can be worked out in intact experimental animals should be evaluated in correlative studies in man, in whom eareful controi of dosage is not altogether possible. A clear understanding of the dose-response relations with respect to lead in this area of metabolism can serve as a basis V * is -321+- DUP050055613 of reference in the study of other, less well understood adverse metabolic and functional effects that have been attributed to lead. More precise studies are needed of the relation between atmospheric lead exposure in the urban environment and the concentration of lead in the blood, perhaps by the use of personal monitors . Only by balance studies of individual subjects can the proportion of body burden due to atmospheric intake be precisely determined. Dosages can only be approximated in human populations* There fore , carefully controlled studies in intact animals are needed to buttress apparent dose-response relations that are now estimated for man. Particular attention should be given to central and peripheral nervous function and to renal function at dosages that produce subclinical effects. Assays of endocrinologic functions may also offer a readily available avenue of approach, as may studies of rates of catabolism of various drugs, both in intact animals and in isolated systems. Investigations of interactions between lead and other metals may also be productive , and a thorough understanding of the binding and transport of lead in the tissues is urgently needed. There is need to carry out animal studies to determine the threshold at which metabolic abnormalities begin to occur in such fundamental processes as oxidative phosphorylation and electron transport , as well as the threshold for effects on dithiol enzymes* Studies should be done in a large popu lation of animals (e.g. , rats) raised on a hydroponic ally -325- DUP050055614 grown diet ultra-low in lead, measuring the length of ex^* posure, the concentrations of lead in blood and other tissues, and effects on heme synthesis. Comparison of these results should be made with animals on a '`normal11 diet and a diet with a moderate lead content--e.g., 100 ug/g dry weight . With suitable guidelines from studies in animals, prospective correlative investigations in man may be undertaken. Some of the questions obviously in need of answers are: (l) At what low hut chronic degree of overexposure are behavior and per formance compromised? (2) Does aging, particularly, as it may affect renal and vascular function, modify man's response to minimal increases in soft tissue lead content? (3) Are fetuses, infants, and young children more sensitive to lead than mature adults? Answers to these questions are needed most to perait proper medical supervision of children and industrial workers , in whom subclinical adverse effects are most likely to be found. More lead balance studies of the type pioneered by Kehoe are needed, but they should be designed to include atmos pheric lead intake along with intake from food and beverages. Fortunately, this type of study is underway and is expected to yield significant information. Comparable studies pertinent to the metabolism of lead in human fetuses, infants, and children should also be done. Owing to ethical considerations, much of this type of experimentation -326 V * DUP050055615 will have to be carried out in appropriate experimental models. Nonhuman primates may be particularly valuable in the study of effects related to nervous system function. if if * * The suspicion has long been entertained that lead exposure of long duration at low concent rations might have some subtle effects on the health and behavior of people apart from the classic syndrome of lead poisoning. Retrospective studies have been made of some health characteristics of populations that were exposed to abnormally high concentrations of lead but did not show classic lead poisoning. In no case has it been possible to unequivocally link the incidence of human diseases other than lead poisoning to lead exposure. Admittedly, the list of diseases to which such studies might be addressed is virtually inexhaustible, but the Information gathered to date does not encourage the belief that lead at concentrations encountered in the general population increases the susceptibility of people to disease. However, people with iron-deficiency states and hemolytic anemia may be more susceptible than others. The subtle effects on behavior of low lead exposure of long duration without prior acute exposure may be manifest in two types of.disorders: the dulling of mentation and chronic hyperkinesis . No information is available regarding the possibility of cause and effect in what may be an extremely important problem. -327- Recoimsndation 7: Further search for possible subtle effects of prolonged low-level exposure to lead. There is need for further study of the possible relations between disease and lead exposure. The particular population studied should be relatively stable, and whole family groups should be studied. The study should include several health indices and other aspects of health besides those related to environmental pollut1on, The importance of determining whether insidious poisoning due to low-level expos tire to lead occurs is apparent. Current information does not afford an adequate basis for the evaluation of this critical area of concern. The only studies available on the effects of long-term inhalation of lead on animal behavior are those of the Russian investigators. It is important for research in this area to confirm or deny the development of behavioral toxicity at comparably low, chronic exposures to lead. Although frank encephalopathy produced by early exposure to lead seems to result in mental retardation, the evidence on the late effects of less severe early poisoning is still unclear. *** Two special categories of people are currently exposed to lead of general atmospheric origin to a degree that seems undesirable. In both, the problem is limited largely to exposures in the inner areas of some large cities. The first category consists of men who are more or less continuously exposed to unusually high concentrations of lead in ambient air in the course of their work (e.g., garage workers, traffic policemen. * % -328- DUP050055617 workers in the lead trades), which may res nit in blood lead concentra^ tions in excess of 40 yg/100 g of whole blood. Even within this small population, this level of blood lead concentration is probably attained by only a relatively small proportion of those exposed. To reach the very high blood lead concentration compatible with clinical lead poisoning would probably require in these special groups approximately a fivefold increase beyond current levels in lead assimilation for a long period. The other special category of inner-city inhabitants for whom the level of lead in ambient air poses a significant threat consists of infants and young children. Recent surveys of large-city children indicate that many have blood lead concentrations in the range of 40-*60 yg/100 g of whole blood. These high blood lead concentrations cannot be ascribed specifically to the inhalation of lead, although that is a possibility. It is also possible that these infants and children eat leaded paint in quantities too small to produce acute poisoning and that at least some of their lead burden comes from the ingestion of lead-bearing street dust and soil, which often attains lead concentrations in excess of 2,000 yg/g. Assuming the validity of the relation between daily lead intake and blood lead content (see Fig. 3-3), the daily ingestion by a child weighing 10 kg of O.kl g of street dust with a lead content of 2,000 yg/g would result ultimately in a blood lead 329- concentration compatible with clinical lead poisoning , even without allowing for additional lead acquired by inhalation, from normal dietary sources, or from coincident ingestion of leaded paint. Likewise, approximately 44 mg of street dust daily would suffice to increase the daily lead assimilation from the level associated with a blood lead content of 20 yg/100 g of whole blood to that associated with Vo yg/100 g of whole blood. This is an undesirable level of exposure. These estimates of the amount of street dust or soil which ultimately would elevate the concentration of lead in the blood to dangerous levels may be too low because they assume continued intake for prolonged periods. Unfortunately the amount of street dust or city soil swallowed or inhaled by young children is totally unknown. Recommendation 8: Study of the ingestion patterns of children. In addition to the applicability of recommendations 3 and 4 to the health of inner-city inhabitants, the Panel recommends that research be intensified on the broad subject of pica. This behavioral activity has implications for social problems far beyond those associated with lead poisoning in children. *** The most effective way of controlling lead poisoning in man and animals is removal from the source of lead. In acute toxic episodes chelating agents are valuable and sometimes life-saving adjuncts in the treatment of lead poisoning. Hecommendation 9: Standardization of diagnostic tests and study of therapy* In relation to estimates of the mobile fraction of the hocfyburden of lead, there is a need to develop a standardized CaEDTA mobilization test for use in asymptomatic adults and children. The dose of CaEDTA is related to the size of the person (preferably estimated by height or surface area), and CaEDTA is given parenterally over a defined period. Urine should also be collected over a carefully defined interval. Modification of the test for Use in renal disease needs exposition. Also, data should be accumulated on the efficacy and safety of repetitive chelation therapy with CaEDTA and d-penicillamine. *** SIGNIFICANCE TO OTHER FORMS OF LIFE Most livestock and wildlife are far removed from the high ambient air concentrations of lead that have been shown to warrant some concern with respect to the health of man. It is conceivable that the grasses along heavily traveled roadways (more than 50,000 vehicles per day) might in some cases acquire enough surface deposition of lead to constitute a hazard to grazing animals. However, for a hazard to exist animals would have to confine their grazing to a band of 125 ft or less on either side of the heavily traveled roadways, which is extremely unlikely . The actual atmospheric hazard is to livestock grazing in the vicinity of lead smelters when stack emissions are not well controlled. There is no evidence that clinical lead poisoning of household pets can be attributed to atmospheric sources of lead. The etiology of lead poisoning in pets is similar to the etiology in infants, in that it is most common in very young animals, which -331- DUP050055620 share with human infants the tendency to eat or chew on foreign objects. including many that contain lead. It is not known, however, 'Whether household pets eat lead-contaminated street dust and soil any more than infants. Lead does not appear to be a hazard to aquatic animals, such as fish. This is no doubt related to the fact that only very low concentrations exist in solution, most of the lead in natural waters being insoluble and apparently nonavailable. Lead poisoning is an important health problem in waterfowl. The only source of lead reported to cause poisoning is spent lead shot from hunting. As in the case of animals, there is no evidence that atmospheric sources of lead are injurious to plants. Although some plants may be susceptible to lead in the natural environment , in soil and water lead is generally in a form that is largely nonavailable to them. Recnm endation 10; Additional study of susceptibility of animals and plants to lead. Long-tern studies axe needed to define more precisely the level of lead intake that can be tolerated by the major species of domestic animals likely to be exposed to en vironmental lead hazards . The newer methods for the assessment of the body burden of lead and its biologic effects in man should also be investigated and applied to these animals. -332- DUP050055621 The assimilation and susceptibility to lead of a wide range of indigenous plant species should be studied, especially those that are consumed by man and domestic animals. These studies are important to determine the possibility that some plants may have an unusual capacity to assimilate lead from the biosphere or a high susceptibility. Plant studies should also be made on a wide range of exposures to lead, but especially at the lower concentrations that the plant might be expected to encounter> rather than at very high experimental concentrations. The importance of such studies lies in acquiring information on and detecting a possible threshold for mutagenesis. k kk -333- * ...... ........ ...... -tr- DUP050055622 APPENDIX A 'PARTICLE SIZE Many disciplines are involved in the study of particles. Each appears to have devised its own system of nomenclature to classify particles 425 with respect to size, physical state, origin, etc. In terms of particle size, the main reason for this confusion is that an irregularly shaped particle has no unique dimension. Its size can be expressed only in terms of the diameter of a sphere that is equivalent, to the particle with regard to some stated property,^^uch "equivalent" spheres are those which have the same volume as the particle, the same surface area as the particle, the same free-falling velocity in a fluid as the particle, and the same projected area as the particle when viewed in a direction perpendicular to the plane of greatest stability. It is evident that different methods of particle-size determination measure different equivalent diameters. For example, microscopes measure equivalent diameter based on projected area, whereas sedi mentation techniques measure equivalent diameter based on free-falling velocity. It is also evident that the numerical values for the various equivalent diameters can be the same only for spherical particles and the divergence of the values will increase with particle irregularity. In dealing with atmospheric particles, size is usually presented as the diameter of a sphere of unit density that has the same terminal fall velocity in still air as the particle in question. For spherical particles larger than approximately 1 pm, the terminal fall velocity in still air can be calculated by Stokes's law: A-l ..............: v' " * '...................- V" ; ............i" .......................................................r*..... DUP050055623 V= l8n in which v ~ terminal velocity, cm/sec, g - acceleration of gravity, cm/sec2 d = particle diameter, cm. o = density of particle , g/cm , 3 = density of air, g/cm , and n - viscosity of air, poises* The expression has an upper limit of applicability, owing to the fact that, when a certain terminal fall velocity is reached, the particle generates a significant "wake." A lower limit of applicability is reached when the particles become small enough that air resistance is no longer continuous but is the result of individual collisions with air molecules. For most purposes, the terminal velocity of particles smaller than about 1 pm can be determined with the Stokes-Cunningham equation: r * * in which A is a constant close to unity (0.9 is often used) and X is the mean free path of the fluid. When comparing the geometric and equivalent diameters of a particle with a density greater than 1, it is evident from Stokes's law that the geometric diameter will be smaller. Consider, for example, a spherical particle of lead sesquioxide with a geometric diameter of 3 0.9 pm and an assumed density of 10 g/cm . The equivalent diameter A-2 DUP050055624 d, in micrometers, as calculated from Stokes*s law, is: . d2 (1 - p2)= (0.9)2 (10 - p ) .- Assuming that the density of air is negligible, compared with the lead particle, a2 = (0.9)2 x 10 - 8,1 d = 2,84 pm. Similarly , a lead sesquioxide spherical particle of 0,05 pm with an assumed density of 10 g/cm^ would have an equivalent diameter, as calculated from the Stokes-Cunningham equation, of: ,2 n + 2&L) = (0*95.)^ 10 (l + x 104) d (1 + - flcF) iu u + 0#Q5 Taking a value of 0.9 for constant A and 1 x 10 ^ cm for the mean free path of air, the equivalent diameter is: d - 2.6l x 1(T5 cm d = 0.26 pm In the case of poly dispersed aerosols, the size distribution of the particles can be presented in a number of ways. Because atmospheric particles usually follow a log-normal distribution, the accepted method is to plot a cumulative distribution on special graph paper with logprobability scales. Thus, the equivalent diameter is plotted on a log scale on the Y axis , and the total fraction of particles that are larger than that diameter is plotted on a probability scale on the X axis. The equivalent diameter is determined by size-measuring instruments, such as impactors calibrated in terms of spherical particles A-3 of unit density. The particulate fractions are conveniently calculated as the emulative weight percent oversize. For a truly log-normal distribution, this type of size-distribution plot will provide a straight line, The equivalent diameter corresponding to the 50# oversize value is known as the "mass median equivalent diameter" (MMEP) of the aerosol. A number of workers have. determined the MMEDs for lead in the atmosphere 3I7 467 and report values in the range of 0.15-0.3 urn, * A-4 DUP050055626 APPENDIX B SAMPLING AND ANALYTIC METHODS FOR LEAD The methods presented in this appendix are reliable, sensitive, and essentially free from interference. LEAD IN THE ATMOSPHERE Dithizone Method The Tentative Method of Test for Lead in'the Atmosphere, ASM Designation D 2681-68T,# is recommended for the sampling and analysis of particulate 1 and nonparticulate (organic) lead. 1. Scope 1.1 This method covers the determi nation of lead in the atmosphere. It involves separate measurements of par ticulate lead and nonparticulate lead. For the purpose of this method, nonparticulate lead is that which will pass a 0.45-/xm membrane filter and includes the organic lead. This procedure is designed to measure ambient lead con centrations above 0.2 /xg of lead/m3. The method employs a special short-term sampling procedure using an iodine absorber for the nonparticulate lead, and a membrane filter to collect particulate lead. It is satisfactory for measuring lead with an accuracy of 0.2 /xg of lead/m3, 2. Summary of Method 2.1 Sample--The sample of air is drawn through a sampling train consist ing of the 0.45-/xm pore membrane filter 1 Under the standardisation procedure of the Society, this method is under the jurisdiction of the ASTM Committee P-22 on Methods of Atmospheric Sampling and Analysis. A list of members may he found la the ASTM Year Book. Accepted I'eb. 14, 19G8. and then through a special sampling tube containing crystalline iodine (1,2,3,$),2 A sample of 2 m3 is collected, 2.2 Particulate Lead--The. membrane filter is digested with nitric, sulfuric, and perchloric acids to remove all organic matter and to dissolve the lead. The lead is determined by the colorimetric dithi zone procedure (4). 2.3 Nonparticulate Lead--The iodine crystals are dissolved in acidified potas sium iodide solution and the excess iodine is reduced with sodium sulfite. The lead is determined by the colori metric dithizone procedure. 2.4 Dithizone Method--The dilute acid solution containing the lead is treated with citric acid and is made alkaline with an ammoniacal buffer solution. The lead is extracted with successive por tions of a chloroform solution of dithi zone. The lead is then transferred into the aqueous phase by extraction with an acidic buffer. This acidic buffer is treated with an ammoniacal buffer solution and the lead is re-extracted into chloroform as lead dithizonate. The lead dithizonate 2 The boldface numbers in parentheses refer to the list of references appended to this method Reproduced with permission of the American Society for Testing and Materials, 191-6 Race Street, Philadelphia, Pennsylvania 19103. B-1 "*VT.. ................ H" DUP050055627 2 Tes t f q h Le a d in t h e At mo s p h e r e (D 2681) is measured spectrophotomctricaliy at 5)0 m/i (4). 3. Significance 3.1 This method is sensitive and rela tively free from interferences. While it is intended primarily for measuring ambi ent atmospheric lead concentrations above 0.2 /ig of lcad/m* for atmospheric survey, careful attention to details and effective control of contamination will allow lower concentrations to be deter mined. 3.2 The procedure is based on the removal of particulate lead by a mem brane filter and the reaction of nonparticulatc lead with crystalline iodine. 4. Definitions 4.1 particulate lead--the lead collected on a membrane filter with a nominal pore size of 0.45 pm. 4.2 nonparticulate lead--that lead which passes an 0.45-pm membrane filler and is collected on crystallized iodine jn accordance with the described sampling procedure. 4.3 For definitions of other terms used in this method, refer to ASTM Defini tions I) 1356, Terms Relating to At mospheric Sampling and Analysis.3 5. Interferences 5.1 Dithizonc gives colored complexes with bismuth, cadmium, cobalt, copper, gold, lead, mercury, nickel, palladium, platinum, silver, stannous, thalious, and zinc ions. Stannous and thalious ions are oxidized in the wet-ashing technique to prevent interference. Aluminum and iron are complexcd with citric acid. Cadmium and zinc are complexed with cyanide in ammoniacal solutions. Bis muth is removed in the double extraction procedure which leaves the lead in solu tion free from interference from other metals. 3 Appears in this publication. 6. Precautions 6.1 Dithizonc is insoluble in water, but is soluble in ammonium hydroxide, and many organic solvents. Mild oxidizing agents convert it to diphenylcarbadiazonc which is yellow and docs not react with metals. The presence of hydroxylamine hydrochloride in the solution prevents the oxidation. If the reagent contains some yellow oxidation product, it must be removed by rccrystallization. 6.2 The determination of lead in small quantities by this method requires meticulous attention to technique. Good precision is not usually obtained without some experience with a dithizonc pro cedure. Precision may be improved by knowledge of, and close adherence to, the suggestions that follow. 6.2.1 All glassware used in the method must be borosilicate glass. It is also imperative that contamination be pre vented by rinsing with nitric acid (1+1) and completing the cleaning with several distilled water rinses. 6.2.2 For the dithizonc extraction, .always use separatory funnels that have not been used in the other parts of the procedure. After use for the dithizone extraction, the separatory funnel is leadfree if rinsed only once with distilled water. Washing with a mixture of dithi zone solution and buffer solution is one pf the most effective ways of deleading and of testing for the absence of lead. 6.2.3 Use the same reagents and solu tions in the same quantities for a group of determinations and the corresponding blank. When a new reagent is prepared or a new stock of membrane filters is taken or new stock of chemicals is re quired, a new blank must be prepared. 7. Apparatus 7.1 Absorption Cells, matched, chem ically strengthened aluminosilicate glass, covered or stoppered, 50 mm long. 17-30 App* B P. 2 B-2 DUP050055628 App. B Te s t f o r Le a d in t iie At mo s p h e r e (D 2681) P. 3 7.2 Iodine Crystal Scrubber--See Fig. 1, 7.3 Carbon Trap-*-Pack n glass tube 18 in. long by 2.5 in. in diameter with activated carbon 10 to 30 mesh with 3 in. of glass wool between the activated carbon and the one-hole rubber stopper located at each end of the tube. 7.8 Membrane Filter? of 0.45 pm pore size. Blanks must be performed for each lot of filters (see Fig. 1). 7.0 Separatory Funnels, Squibb-type, 125-ml and 250-ml capacity. 7.10 Spectrophotometer,fl 7.11 Vacuum Pump, capable of draw- mg 10 liters of air/min. t 4pr - TO CHARCOAL TRAP, GAS METER ond PUMP l?io. 1--SamplinR Train. 7.4 Horosilicale Glassware shall be used in this method. 7.5 Erlcnmeyer Flaskt wide-mouth, 500-ml capacity. 7.6 Filter Holder/ for a membrane filter with a diameter of 2 in. or 47 mm (see Fig. 1). 7.7 Gas Meter--An integrating gas meter capable of measuring from 10 liters of air/min. 4 Gelmnn No. 1200A Open Filter Holder 2 in., or No. 4273 Dispoz-It 2 in. with filter; Millipore Aerosol Open-type Filter Holder (47 mm) XX50 047 10 or MAWP 0 37 AO Aerosol Gravimetric Analysis Monitor with Filter, have been found satisfactory. 8. Reagents and Materials 8.1 Purity of /?eageH/j--Reagcnt grade chemicals shall be used in all tests. Un less otherwise indicated, it is intended that all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such spccifications are available.7 Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without decreas ing the accuracy of the determination. 8.2 Purity of Water--Unless otherwise indicated, references to water shall be understood to mean distilled water or referee reagent water conforming to ASTM Specification D 1193, for Reagent Water.3 8.3 Ammonium Hydroxide (sp gr 0.90) --Concentrated ammonium hydroxide (NH/)H). No t e 1-Low-lead ammonium hydroxide and hydrochloric acid manufactured lor the electronics industry are available and are satis factory. 8.4 Bujfer Solution--Dissolve 400 g 6 Millipore membrane filter Tyi>e HA, 0.45pm pore size; Gelmun AM-6, 0.45-^m pore size; or Schleicher and Schuell, Type B. 0.45-pin |X>re size may be acceptable. * Beckman Model DU or equivalent, equipped with 50-mm cells, has been found satisfactory. 7 "Reagent Chemicals. American Chemical Society Specifications," Am. Chemical Sew., Washington, D. C. For suggestions on the test ing of reagents not listed by the American Chemical Society, see "Reagent Chemicals and Standards," by Joseph Rosin, D. Van Nostrand Co., Inc., New York. N, Y,, and the "United States Pharmacopeia." tMO B-3 Tk .s t f o r Le a d in t h e At mo s p h e r e (D 2681) of citric acid in water, arid 6 drops of phenol red indicator solution, then add XI MW until the solution turns pink, then add 50 g of potassium cyanide. To another beaker add 13.5 g of hydroxylamine hydrochloride and sufficient water to dissolve the salt. Add 6 drops of .phenol red indicator solution, and add NH4OH until the solution turns pink. Mix the two solutions and dilute to 1570 ml with water. 8.5 Clark and hubs Buffer Solution p/l J.'l Dissolve 17.0 g of potassium arid phthalntc in water. Add 1.4 ml of lid and dilute to 1000 ml with water. 8.6 Chloroform--Place 1000 ml of chloroform in a 2000-ml separatory fun nel. Dissolve approximately 10.0 g of hydroxylam ine hydrochloride in 50.0 ml of water and make the solution am monia ral to phenol red indicator solution with NH4OH. Adel this solution to the chloroform in the separatory funnel. Shake well. Allow the aqueous layer to separate and filter the chloroform through a fluted filter. To stabilize, add 10 ml of absolute alcohol to the filtered chloroform. 8.7 Oiihizone--An especially pure grade is required.8 8.8 Dilhizonc Solution--Dissolve 40 mg of dilhizonc in 1000 ml of chloroform. This solution is stable for only 4 weeks and should be kept in a refrigerator. Allow the solution to warm to room tem perature before use. 8.9 Hydrochloric Acid (sp gr Concentrated hydrochloric acid (HCl) (Note 1). 8.10 Iodine Crystals-*-Sieve crystals through a 16-mesh stainless steel sieve, and use only the portion that passes the sieve. Store crystals in the dark in lead-free brown glass bottle (Note 2). Purify the sieved crystals by washing with dilute nitric acid (Note 3). 8 Kastman Kodak white liiliel requires nopurifi cation and has been found satisfactory. No-rfc 2 -Iodine crystals tend to segregate in a storage bottle. When weighing 20.6 g fur an analysis, invert the l>oUle ami mix the fine particles with some of the larger ones to obtain a representaliyc jx>rtion of the sieved particles. About 30 percent of }>ariiclc8 finer than 30 mesh arc necessary for complete lead recovery at high sampling rates. No t e 3--Dclcad the surface of the iodine crystals by adding 30 to 50 ml of HNOi (1+1) to lHe iodine scrubber. Rinse with 25 to 30 ml of water. Add 25 mi of methanol solution and with a rubber bulb force all extraneous alcoholic solution from the scrubber. Care must be exer cised to prevent -the solution of the fine particles in the alcoholic water wash solution. 8.11 Indicator Solutions'--Vhenol red and methyl orange. Dissolve 0.1 g of the indicator in water and dilute to 100 ml. 8.12 Methanol Solution--Add 600 ml of methanol to 300 ml of water. 8.13 Nitric Acid (/+/)---Prepare by adding 1 part of nitric acid (HN03, sp gr 1.42) to 1 part of water. 8.14 Nitric Acid (/+4)--Prepare by adding 1 part of HNO# (sp gr 1.42) to 4 parts of water. 8.15 Potassium Cyanide Solution-- Dissolve 15 g of potassium cyanide (KCN) in 1000 ml of NH4OH (sp gr 0.90). Caution, see Note 4. No t e 4: Caution--Handle potassium cya nide with rubber gloves in a vented hood and wash hands and gloves frequently. Drain excess and waste solutions in a hooded sink that is not used for the disposal of acids, and flush with water. 8.16 Perchloric Acid [72 percent). 8.17 Potassium Iodide, Acidified-- Prepare just before each analysis by mixing 5 parts of potassium iodide solu tion with 1 part of HN03 (1+4). 8.18 Potassium Iodide Solution--Dis solve 500 g of potassium iodide (KI) in water and dilute to 1000 ml with water. 8.19 Sodium Sulfite Solution--Dis solve 200 g of sodium sulfite in 1000 ml of water. 8.20 Standard Solution I--Dissolve ir-n App. P. 4 B B-4 / - -:v v- ......... DUP050055630 App. B Tk s t f o r Le a d in t h e At mo s p h e r e (D 2681) P- 5 0.1600 g of lend nil rale in 200 ml of walor in a 10()0 m l volumetric.Husk. Add 8 ml of IiXOa (sp gr 1.42) and dilute to 1000 ml with water (lead concentration - 0.10 mg/ml), 8.21 Standard Solution //--Add 10 ml of Standard Solution I to a 1000-ml volumetric flask and dilute to 1000 ml with Clark and Lubs pH 3.4 buffer (8.5) (lead concentration 1 ftg/ml). 8.22 Sulfuric Acid (sp gr I.S4)--Con centrated sulfuric acid (HaSO<). 9, Sampling 9.1 -Draw air at the rate of at least 10.0 liters/min through the sampling train (Fig. 1). The components of the sampling train arc: (/) 0.45-gm mem brane tiller to collect the particulate matter and particulate lead, (2) a tube containing 20 g of crystalline iodine to collect the nonparticulute lead. (Note 3), (.?) an activated carbon trap to protect the pump from volatilized iodine, (4) a gas meter to measure the air, and (5) a pump to draw the air sample. 9.2 Collect approximately 2 In3 of air (P) per sample. Do not take an air sample so large that it would reduce the iodine below 10 g, the minimum amount that should remain after each test (see Note 6), 9.3- Tap the sides of the iodine scrub ber every 5 to 10 min with a pencil or rod to ensure an even bed (cross section) of iodine crystals. 9.4 Treat the membrane filter for particulate lead as described in 11.1. Treat the iodine scrubber for nonparticu late lead as described 11.2. 9.5 BlanksPrepare a reagent blank of all reagents. Use the same portions of each reagent used in the analysis. Treat the reagent blank similarly as the sample in all steps; (7) for the membrane filter as described in H.l and 11.3; and (2) for the iodine scrubber as described in 11.2 and 11.3. Read the micrograms of lead from the calibration curve and sub tract the appropriate blank from the amount of lead found in the sample. 9.5.1 A blank is not required for each determination once an average blank has been established. A new blank must be prepared when the supply of any reagent including distilled water or of membrane fdters is changed. 10. Calibration Curve 10.1 Lead from Particulate Pilfer-- Pipet 0.0, 2.0, 5.0, 10.0, 15.0, and 20.0 ml of Standard Solution II respectively into each of six Squibb-type 125-ml separatory funnels and dilute each solu tion to 50 ml with Clark and Lubs buffer. Add 2 drops of methyl orange indicator solution and treat the solutions as de scribed in 11.1.2. Subtract the absorb ance of the 0.0-ml sample from the absorbances of the other solutions. 10.1.1 Construct a calibration curve by plotting the absorbances of the solu tions against micrograms of lead, 10.2 Lead from Absorption on Iodine Crystals--The amount of iodine in the scrubber at the end of the sampling procedure may influence the zero reading of the curve. Separate calibration curves must, therefore, be prepared for 10 and 20 g of iodine remaining in the scrubber. No t e 5 --The influence of iodine on the re sults is in dispute. The calibration of lead with varying amounts of iodine present is offered so that a check can be made. If the amount of residual iodine has no effect on the results, the curve prepared in 10.1 will suffice. If the iodine has an effect on the results, the calibration curves prepared as described in 10.2.1 will be required. 10.2.1 Pipet 0.0, 2.0, 5,0, 103), 15.0, and 20.0 ml of Standard Solution II respectively into each of six beakers containing' 10 g of iodine crystals. Pre pare a similar series of samples using 20 g of iodine in each beaker. Treat the sam ples as described in 11.2.1, 11.2.2, and it-ji B-5 ............................V.........' ' ' ' -,-v ' ..............* ..... . ' ........' ' DUP050055631 6 Te s t f o r Le a d in t h e At mo s i'h f .r k (P 2681) 11.2.3. Subtract 1 he absorbance of the O.O-ml sample from lhe alworbiihccs of (be .other solutions. 10.2.2 Const met a calibration curve by plotting the absorbances of the solu tions against niicrograms of lead. ' 11. Procedure 'M.| Particulate Lead: 11.1.1 Remove the membrane filler from the holder. Place it in a JOO-ml beaker and add 5 ml of water, 5 ml of UX();i (sp gr 1.42), 3 ml of HsSO* (sp gr 1.84), and 0.5 ml of perchloric acid (72 percent). Digest on the hot plate until the solution is white or pale yellow . Finally,-remove the last of the carbonaceous material by heating the sample until dense white fumes appear. Allow the solution to cool. Add 10 ml of HNOa (14-4) and boil the solution to dissolve any lead sulfate. Dilute the mixture to 25 ml`and filter into a 250ml Squibb-type separatory funnel. Use another 25 ml of water to wash and transfer the sample. 11.1.2 Add phenol red indicator solu tion. Add NH<OH (sp gr 0.90) until the solution turns yellow. Add 25 ml of buffer solution and 10.0 ml of the ditbizone solution, shake for 1 min, allow to separate, and run the dithuonechloroform layer into a 125-nil Squibbtype separatory funnel. Repeat the treatment with 5-ml portions of dithizone solution until the last addition retains its original green color. Discard the aqueous layer. 11.1.3 Treat the combined chloroform-dithizone layers as described in UJ. 11.2 Sonparticulate Lead: 11.2.1 Set the iodine scrubber in a beaker of suitable size and dissolve the iodine crystals in the scrubber by adding acidified KI in small quantities until all of the crystals are dissolved. Rinse the scrubber with water and collect all washings in the beaker. 11.2.2 Add sodium sulfite solution until the iodine color disappears (2.6 ml of suliitc solution is equivalent to 1.0 g of iodine). Use 3*ml portions, .adding each portion with stirring until all the iodine color is gone (Note 6). Transfer the mixture to a 250-ml -Squibb-type separatory funnel. Add phenol red indi cator solution. Add NIDOH (sp gr 0.90) until the solution is ammoniacat. Add 25 ml of buffer solution and 10.0 mi of the dithizonc solution, shake for 1 min, allow to separate, and run the dithizoncchloroform lay er into a second 125-ml Squibb-tv|ic separatory funnel. Repeat the treatment with 5.0-ml portions of dithizonc solution until the last addition retains its original green color. Discard the aqueous layer. No t k 6 The volume of reducing solution required Is a measure of ihc amount of iodine that has remained in the tube following the period of sampling. At least 10 g of iodine should remain in the tube after sampling. If less than 10g remain, discard the test. Practice has shown that the air scrubbing for nonpurl iculatc lead becomes inefficient and erratic if the iodine level drops below lO g. 11.2.3 Treat the . combined chloroform-dithizonc extracts as described in 11.3. 11.3 l.CAid Determination-Dithizonc Ex tract: M.3.1 Add to the chloroform-dithh zone extracts (obtained in M.1.3 and It.2,3) 50.0 ml of the Hark and Lubs pH 3.4 buffer, 2 drops of methyl orange indicator solution, and shake. Adjust the pH of this solution to 3.4 bv matching the color against 50 ml of pH 3,4 buffer solution to which 2 drops of methyl orange indicator solution have been added. Use HC1 or NH/IH to make the match. Shake for 1 min. Compare colors. Match again if necessary. Shake for 1 min and let stand to allow the layers to separate. Discard the chloroform layer. Add'5 ml of chloroform to the solution in the funnel. Shake, let stand to sepa- IT-II App, B P. 6 B-6 > DUP050055632 Te s t f o r Le a d in t h e At mo s p h e r e (D 2681) 7 rate layers, and discard the chloroform layer. If the lead concentration is 10 idg or less per sample, treat the aqueous layer as described 11.3.3. For concen trations above 10 jug per sample, treat the aqueous solution as described in 11.3.2 and 11.3.3. Usually the particu late lead level will be above 10 jig and 11.3.2 and 11.3.3 should be followed. For the. nonparticulate lead sample, proceed as in 11.3.3, 11.3.2 Drain the aqueous layer into a 100-ml volumetric flask. Dilute to vol ume with Clark .and Lubs buffer solu tion. Place a 10-mi aliquot in a 125-unl Squibb-type separatory funnel and dilute to approximately 50 ml with Clark and Lubs buffer solution. 11.3.3 Add ammonium hydroxide to the aqueous solution until ammoniacal. Add 20 ml of the KCN solution. Add 20 ml (buret or pipet) of dithizone solution and shake for 1 min (Note 7). Drain and discard the first 2.0 ml of dithizone solution and then fill a 50-mm absorption cell with the chloroform-dithizone solu tion. Read- the absorbance compared to chloroform with the spectrophotometer set at 510 nyx. tion of the lead dithizonate at this step must be done exactly in the same manner each time to . reduce errors or variations in the results. 11.3.4 Determine the micrograms of lead by comparison with the previously prepared calibration curve and correct . for blank. 12. Calculation 12.1 Particulate Lead: 12.1.1 Calculate the concentration of particulate lead, Cp, in micrograms per cubic meter of air sample as follows: * Wp X 1000 X 10 c, -- where: Wp = micrograms of particulate lead from calibration curve, V ~ liters of sample volume, 1000 = factor to convert to cubic meters, and 10 = factor for aliquot (11.3.2). 12.2 Nonparticidate Lead: 12.2.1 Calculate the concentration of nonparticulate lead, Cnp, in micrograms per cubic meter of air sample as follows: ,, Wnp X 1000 Cnp - y No t e 7--Usually a 1-min shaking period is specified for shaking the solution with dithizone- where: chloroform to extract the lead. If shaking is Wnp micrograms of nonparticulate done by hand, care must be exercised to prevent warming the container by the hand since the solubility of lead dithizonate in aqueous phase or in the chloroform phase will vary depending lead from calibration curve, V = liters of sample volume, and 1000 = factor to convert to cubic , on the temperature of the mixture. The extrac meters. Re f e r e n c e s (1) Cholak, J., ``Analytical Methods for Deter mination of Lead," Archives Environ^ mental Health, AEHLA, Voi8,1964, p. 222. (2) Snyder, L. J. and Henderson, S, R., "A New Field Method for the Determination of Organic Lead Compounds in Air/' Analytical Chemislry, ANCHA, Vol 33,1961, pp. 1175-1180. (3) MacPhee, R. D,, Eye, M. G., and Parkin son, E. E., "A Method for Monitoring Organic Lead in the Atmosphere," Air Pollution Control District, County of Los Angeles, APCPA, September, 1962. (4) "Methods for Determining Lead in Air and Biological Material," American Public Health Association Inc., APHYA, 1790 Broadway, New York, N, Y., 1955. (5) Snyder, L. J., "Determination of Trace Amounts of Organic Lead in Air," Ana lytical Chemistry, ANCHA, Vol 39, 1967, pp. 591-595. Comment. The Millipore type HA filter, with a 0.45 pm pore size, is reported to have a 99.9% efficiency for collecting particles as small 532 as 0.03 pm. Blank filters from each new batch should be analyzed by the selected procedure, and the appropriate averaged correction value must be applied to the results of sample analyses. B-7 DUP050055633 Crystalline Iodine, used in this method to collect organic lead, is not lOOfo efficient for this purpose, according to L. J. Snyder and H, R, Henderson .^inyder has since used 30- to 50-mesh activated charcoal to sample organic lead in 100- to 200-m3 air samples near a Lo's Angeles freeway at a rate of 0,7 cfm. The lead was converted to inorganic, - 52'k lead and analyzed by the dithisone method' with a precision of +0.008 ug/itr based on laboratory experimentation with tetramethyl lead. .<* Alternate Method for the Determination of Lead by Atomic Absorption Spectr ophotome try During the last 6 years, atomic absorption spectrophotometry has emerged in the United States as a very rapid, specific, and sensitive method for the analysis of metallic elements, including lead, in any type of sample that can be put into solution. Using an air-acetylene flame to produce the "atomic cloud'* of sample constituents, a sensitivity of 15 pg/l00 g of whole blood is now being achieved routinely with dilute acid solutions of ashed blood samples. The accuracy and precision of this method are extremely high; results obtained by this method compare very favorably with those provided on duplicate samples with a spectrographic procedure applied in other laboratories. Thus, the reliability and rapidity of atomic absorption analyses for lead are making this technique attractive to many laboratories. The coefficient of variation of the atomic absorption method, as determined by repetitive measurements of a set of blood lead standard samples over a period of 6 months, was found to be 8#.. However, by the preparation of a separate standard curve for each group of samples, the true coefficient of variation can be reduced to 1.5# (H. I. Grander and R. G, -Keenan, unpublished data). Apparatus. All glassware must be borosilicate and must be lead-freed by rinsing with warm 1:1 nitric acid and distilled water before use. B-8 DUP050055634 1. Atomic absorption spectrophotometer. Numerous instruments, available commercially, are being used for the analysis of lead in sample solutions,. The Techtron models AA4, AA5, and AA120, or equivalent, have been found satisfactory. 2. Volumetric flasks, glass-stoppered, 50 ml. 3. Pipets, volumetric, assorted sizes. 4. Beakers, Phillips, conical, 125 and 250 ml. Reagents and Materials 1. Acid, hydrochloric (sp gr 1,19), A.C.S. Grade. Prepare dilute reagent as needed. 2. Acid, nitric (sp gr 1.42), A.C.S. Grade. 3. Acid, sulfuric (sp gr 1.84), A.C.S. Grade. 4. Distilled water, shown to be lead-free by atomic absorption spectropho tome trie analysis. Used throughout the method where water is mentioned. Procedure. To the cooled acid digest of the sample resulting from the treatment described in Sec. 11.1,1 of ASTM Method D 2681, add 10 ml of HNO^ (1:4) and boil the solution, as stipulated, to dissolve any lead sulfate. Transfer the solution to a 50-ml volumetric flask, dilute to volume with water, and mix. Analyze the solution, or a diluted aliquot if the lead content is high, by atomic absorption spectrophotometry. The following characteristics apply to the Techtron instruments: Current: Slit: Burner height setting: Support pressure (air) setting: Fuel flow (acetylene) setting: Analysis line: 6 ma 300 nm 3.0 15 3 0 2170 A . - ............................... ............... :* \ , ' '* \ " B-9 ...........* - "r'"- '''' ' '"t": ' --r . ' DUP050055635 Obtain the absorbance of the sample solution, Correct this value by subtracting from it the absorbance of an -unused membrane filter carried through the entire procedure. Calculations. Estimate the micrograms of lead per milliliter of sample solution from the standard curve prepared daily at the same time each group of unknown samples is analyzed* Multiply by 50 (and by any additional aliquot reciprocal if further dilution of the prepared sample solution was required for a high lead sample) to obtain the total number of micrograms of particulate lead in the total air sample. The concentration of lead per cubic meter of air is calculated as follows: o Ug/m iig/ml x 1000 x 50 x arly additional aliquot reciprocal liters of air sampled Comment. The atomic absorption method may be used for the analysis of lead in any prepared dilute acid solution (< 3N) of a sample follow ing the wet oxidation of organic material and the solubilization of lead with mineral acids. By standardization with a set of lead solutions of known concentrations with each group of samples, it is possible to minimize the effect of varied gas-flow regulator settings and to reduce the relative standard deviation to less than 1*5$ (& Grander R. G. Keenan, unpublished data). The acidity of the Sample solutions should be less than 3N to prevent corrosion of the burner. Modification of this method for continuously measuring lead is currently being field-tested by the Environmental Pro tec ti on Agency. B-10 DUP050055636 LEAD IN BIOLOGIC MATERIALS Lead in blood, urine, feces, and body tissues may be analyzed by, the ,fUSFHS,f Method for Determining Lead in Air and in Biological Materials2^ or by the APHA Methods for Determining Lead in Air and in Biological Materials^8, (the latter provides for the separation of bismuth dithizonate at pH 3.4), Methods and precautions for the collection, preservation, and storage of biologic samples are described in Appendix C. t-USPHSlt Method for Determining Lead in Air and in Biological Materials* `HE USPHS method for the determina- precision have been evaluated periodically by i- tion of lead is a double-extraction, mixed- die analysis of replicate samples of blood and color dithizone procedure which is especially urine. For these evaluations, pooled biologi Convenient for lead analysis in the absence of cal samples have been used. In each series bismuth. Although this method has evolved of test samples, one set of replicates contained from the early experimentation conducted lead only in its physiologic form whereas the with dithizone procedures in this Division remaining sets contained this lead plus known starting in 1937, it has never been published incremental quantities of an inorganic lead in complete detail but only in an abbreviated compound. Statistical analyses of the result form as part of a committee report.1 Many ing lead recovery data have been made. The chemists have suggested that the complete results of the analyses of the latest series of procedure along with its supporting data test samples of blood are reported in this should be published. paper. During the past 26 years, the method has been refined through the efforts of the chem Reagents ists who have used it in this Division. Con sequently, these improvements have been in corporated on a gradual basis in the separate series of mimeographed copies of the method distributed to those requesting them. Hence, it is appropriate now to publish the com plete details of the fully refined method along with the associated information on sample collecting, ashing and other preparative pro cedures. Of equal importance, however, is the need to report on the results of the repetitive test ing of the method of which the accuracy and Analytical grade reagents are used. Puri fication is essential when analyzing biological tissues and fluids because of the very low levels of lead in these materials: purification of reagents may not be required for air samples containing quantities of lead suffi ciently greater than that present in the re agent blank. A reagent blank sample, is car ried through the entire procedure with each set of unknown samples (air, biological, or other type) and its analyzed lead content is subtracted from each analytical result to cal culate the net quantity of lead in each unknown sample. Present address: U. S. Department of Health, Education, and Welfare, Public Health Service, Division of Air Pollu A boiling rod is used to prevent bumping tion, Cincinnati 26, Ohio. in the flasks when distilling reagents. This The use of trade names in this paper does not constitute an endorsement by the Public Health Service. is prepared by cutting 3 or 4 mm O.D. glass * Reproduced with permission of the / merican Industrial Hygiene Association, 25711 Southfield Road, Southfield, Michigan 48075 B-ll DUP050055637 Appendix B Page 12 tubing to a length which is one cm greater tlian the height of the flask. The tubing is sealed at a spot about one cm above the bottom end which is firepolished but left open. Before each use, the liquid is shaken out of the bottom section and the rod inserted in the flask. As the flask is heated a steady stream of ait and vapor bubbles issues from agent may be prepared more convenientlyfrom tank ammonia, using a small wash bottle to scrub the gas and a sintered glass delivery tube which extends to the bottom of the reagent bottle. The ammonia gas is absorbed in double-distilled water until the solution reaches the desired specific gravity, the open space, thus providing nuclei for C/i/oro/orm.-^Use a brand with a state- smooth boiling. ment on the label that the chloroform passes Double^distilled Water.--To distilled water in an all borosilicntc-.gla.ss still add a crystal each of potassium permanganate and barium hydroxide and redistill. Use for reagent and biological sample solutions unless tests indicate that single-distilled water is satisfactory ; single-distilled water is usually adequate for determinations on air samples. the American Chemical Society test for suit* ability for use in dithizone procedures. In addition, each batch of chloroform should be purchased in glass containers only and should be tested as follows in the laboratory to make sure that it is satisfactory for preparing the dithizone solutions: add a minute quantity of dithizone to a portion of the chloroform in a test tube, shake gently, then stopper with a cork. The faint green color Nitric Acid. Concentrated.--Redistill in an all borosilicatc-glass still the ACS reagent grade add. 69.0rr minimum, specific gravity 1.42. Use an electric heating jacket on the boiling flask to minimize danger of its breakage. and a boiling rod to prevent bumping, should be stable for one* day. Our experience has indicated that the procedures for reclaiming used .chloroform arc tedious, timeconsuming, sometimes unsuccessful, and no longer warranted in view of the commercial availability of acceptable reagent grades, which otherwise would be severe. Discard t],rx rf d::t:!!atc; this may be wm* bined with the add allowed to remain in the flask at the end of the distillation and used for washing glassware. The reagent is conveniently dispensed from a small automatic burette. Xo grease should be used on the Extraction Dithizone,--Dissolve lb mg of diphenyl thiocarhazone (dithizone), Eastman Kodak Co. Xo. 3092, or equivalent, in one liter of chloroform. Store in a brown bottle in the refrigerator, stopcock. Standard Dithizone.--Dissolve 8 mg of A Uric Acid, 1:99.--Dilute 10 ml of the redistilled, concentrated acid to one liter with double-distilled water. diphenylthiocarbazone in one liter of chloroform. Store in a brown bottle in the refrigerator but allow to warm to room temperaturc before using. Age for at least one day, then standardize as described in the proce- Ammonium Hydroxide, Concentrateddure. Restandardize every few months. Distill in an all borosilicatc-glass still 3 liters of the ACS reagent grade, 28.0^r minimum, specific gravity 0.8957 at 60~F. into 1.5 liters of double-distilled water, contained in a 2liter reagent bottle which is chilled in anice bath. Continue the distillation until the bottle is filled up to the previously marked 2-liter level. Submerge the condenser tube deeply in the water in the receiver, hut withdraw it before discontinuing the heat to avoid siphoning back of distillate. Thisre* Sodium Citrate.--Dissolve 125 gm of the 2 Xa,C,.H;,07ll H=0 salt in sufficient distilled water to provide a solution nearly 500 ml in volume. Adjust the pH to 9-10, using a very small quantity of phenol red indicator solution (strong red color! and fresh, pHydrion test paper to check the pH. Extract in a large separator)' funnel with a 100 mg per liter solution of dithizone and finally with the extraction dithizone reagent until a green B-12 * * * DUP050055638 Appendix B Page 13 Industrial Hygiene Journal extract is obtained with the latter reagent. Add a small volume of lead-free citric acid until an orange color (pH 7) appears. Ex tract the excess dithizone repeatedly with chloroform until a colorless extract is ob tained. Remove the last traces of chloroform. Hydroxylamine Hydrochloride.--D i $so 1 ve 20 gm of the salt in distilled water to provide a volume of 65 ml. Add a feu* drops of mcresol purple indicator, then add ammonia until the indicator turns yellow (pH 31. Add a sufficient quantity of a 4solution of sodium diethyldithiocarbamatc to combine with metallic impurities, then mix. After a few minutes extract repeatedly with chloro form until the excess carbamate reagent has been removed, as indicated by the absence of a yellow color in the final chloroform extract tested with a dilute copper solution. To the aqueous solution of the hv/lrovvr-Mwjrjn hydrochloride add redistilled. fi.Y hydrochlo ric acid until the indicator turns pink, and adjust the volume to 100 ml with doubledistilled water. Potassium Cyanide.-- (Danger! Highly poi sonous!!) To 50 cm of potassium cyanide in a beaker, acid sufficient distilled water to make a sludge. Transfer the sludge to a separator)' funnel previously marked to show 100-ml volume. Add a small amount of distilled water to the beaker and warm. (Potassium cyanide cools the solution as it dissolves, thus retarding the solution process.' Add tills warm water to the separatory funnel but do not permit contents to exceed the 100-ml mark. Shako, then let stand until the contents come to room temperature. A prac tically saturated -solution results. Extract the lead by shaking repeatedly with portions of the extraction dithi/one solution until the lend has been -removed. Part of the dithi/one dissolves in the aqueous -plias" but enough remains in the rhlorofi >rm to color it. A green extract indicates that ail the lead lias`been completely extracted. Most of the ditlii/one In the aqueous phase is then removed by repeated extrac tions with pure chloroform. Dilute the concentrated solution of potassium cyanide with double-distilled water to 500 ml. It should not be necessary to filter the solution, if the directions are followed precisely. Extraction is carried out before dilution because the higher pH of the dilute solution is less favorable. (No t e: A colorless solution usually results if above directions are followed. Occasionally aging results in a brown color or precipitate due to polymerization of hydrogen cyanide. This does not interfere with use of the reagent if it is carefully decanted. Old potassium cyanide reagent may lose enough strength to cause insufficient complexing of large amounts of zinc.) Anwionia^cyanide Mixture.--Mix 200 ml of the purified I0r< potassium cyanide re agent with 150 ml of distilled ammonium hydroxide (specific gravity 0.9, corresponding to 2&4fr XH,' and dilute to one liter with double-distilled water. If the measured spe cific gravity of the ammonia is not 0.9, use tire equivalent volume as calculated from a table of specific grav ity vs. percentage am monia. Standard Lead Solution.--Dissolve 1.5984 gm of pure lead nitrate in one liter of l :99 nitric acid to provide a strong stock solution containing one mg Pb per ml. Pipet exactly 20 ml into a 500-ml volumetric flask and make to mark with 1:99 nitric acid to give a dilute stock solution containing 40 Pb per ml. (A standard lead solution. 10 >ig Pb/ml, was stable in 1:99 nitric acid for three years.) Prepare a working solution, contain ing 2 /*g Pb per nil. just before it is needed by pipetting 5 ml of the dilute stock solution into a 100-ml volumetric flask and making to mark with 1:99 nitric acid. Phenol Red.--O.l^r aqueous solution. Ashing Aid Acid.--Dissolve 25 gm potas sium sulfate in sufficient redistilled concen trated nitric acid to make 100 ml. White Petrolatum,- Supplied in a glass jar, for greasing stopcocks. To du ck on the purity, put a pinch of this petrolatum in a beaker, add a few milliliters of the -standard dithi/one and swirl. If the dithizone is no B-13 DUP050055639 Appendix g Page 14 .longer green after a few minutes, the mate rial is unsatisfactory for greasing .stopcocks. Apparatus A Beckman Model DU Spectrophotometer has been used in this laboratory since this instrument became available in the 1940s. However, the Beckman Model B and the Bausch and Lomb Spectronic 20 have been shown to give comparable results for blood lead determinations, provided that appropri ate standardizations are conducted with each instrument. Other laboratories, whose results are reported in this paper, have presumably used a diversity of available photometers and spectrophotometers. In our laboratory, 22 x 175 mm matched test tubes are used in most spectrophotometric procedures employing the Model DU. w hich is fitted with a tube holder which docs not interfere with the use of the instrument with regular ccllsT* These same tubes are used in the Model B fitted with a test tube adaptor. A 34-inch tube, supplied by the manufacturer, is used in the Bausch and Lomb Spectronic 20. Borosilicato glassware is used throughout the procedures (except for vacutainers used for blood sampling . Ashing is performed in 125- or 250-ml Phillips beakers. Automatic burettes arc used for the addition of most reagents. The extractions arc conducted in Scjuibb-type. 125-ml separatory funnels sup ported in electrically operated shakers pro vided with timer switches. The stopcocks of the separatory funnels are greased with while petrolatum .'purchased in a glass jar rather than ill a metal can or tube- unless Teflon stopcocks, which require no grease, are used. All glassware should be reserved for trace analysis only, to avoid possible gross contam ination. Soak all ashing beakers in a detergent so lution (Alconox or Duponol is .suitable') im mediately after each usage to "prevent any material from drying on the surfaces. Rinse 8-10 times with hot wa ter and store in a dustproof drawer or cabinet until needed. Use the following acid cleaning, lead-freeing techniques immediately before the next use of the glassware: Rinse the ashing beakers with a saturated solution of sodium dichro- matc in concentrated sulfuric acid. Leave a 1-2 ml portion in the beaker or flask (pro portionally less in a small volumetric flask!). Add about 5-10 ml of warm tap water and allow the hot solution to flow pv.cr all inner surfaces to remove the last traces of grease. Rinse with three or four portions of cold tap water. Rinse with one portion of either con centrated or 1:J nitric acid, as preferred. (This wash nitric acid may be used repeat* edlv until it loses its strength.) Then rinse successively with three or four portions each of tap water, distilled and double-distilled water. Set the beakers upright on the bench and cover with a clean dust-case or a large piece of filter paper (or otherwise protect from dust). Under no circumstances is glass ware turned upside down to drain on a towel or cheesecloth placed on a laboratory bench Use an oven operating at 105C if dry glass ware is required. Separatory funnels are rinsed with tap water immediately after use. If a high lead sample was present or if a visible precipitate remains on the inside, it is rinsed with a small portion of 1:1 wash nitric add (which is dis carded L followed by tap water. The stop* pored funnels are stored in double-deck racks. Immediately before use. stopcocks are re* frrnpc^H jf nececsarv Then the -funnel* an? rinsed with wash acid, four times with tap water, and four times with distilled water. Each rinse is accomplished by shaking with the stopper, then draining through the stop cock with two or three turns. Spectrophotometer tubes are rinsed four times each with tap and distilled water im mediately after use. They are placed upright in a large beaker and dried in art oven at 105'C. then stored under a dust-cover. Occasional!y they are cleaned with dichro ma te-sulfuric acid and nitric acid as described above. (.Vo t e : With this method of cleaning glassware we have never encountered cross-contamination from chromium, lead, or from any other trace element being determined routinely in this labora tory.) Analytical Procedure 1. Warm the sample ash (prepared as de scribed in tlvc following sections) with 2 ml B-14 A ry DUP050055640 Appendix B page 15 Industrial Hygiene journal of concentrated nitric acid for a few minutes, then add 25 mi of distilled water, heating on the hotplate until a clear solution is obtained. 2. Cool to room temperaure. Add to the solution in the beaker one ml of hydroxylamine hydrochloride. -I ml of sodium citrate (10 ml is required for a urine sample), one drop of phenol red indicator, and titrate to a strong red color with concentrated am monia reagent. Add a few drops excess of ammonia to make sure that the pH is be tween 9 and 10. using fresh pHydrion test paper to check the pH. (No t e : Phenol red has a weak orange-red color in strong acid, yellow in weak acid, and a red color in alkaline solution. Do not mistake the first color for that produced in alkaline medium!) 3. Transfer the sample quantitatively with double-distilled water rinsings to a 125-ml Squibb separatory funnel containing 5 ml of the potassium cyanide reagent. 4. Add 5 ml of the extraction dithizone and shake two minutes, after releasing the initial pressure by momentarily opening the stopcock o.t the inverted separatory funnel. Allow -the chloroform layer to settle. 5. Draw off most of the extraction dithizonc into a second funnel containing exactly 30 ml of 1:99 nitric acid. 6. Add a second 5^ml portion of extrac tion dithizone to the first funnel and shake as before. Allow the layers to separate and combine the extracts in the second funnel. Continue this process with fresh portions of extraction dithizone until the reagent remains green. A rough estimate of the lead present in the sample may be made on the basis of 20 fig for each cherry-red 5-tnl extract portion. 7. Shake the second funnel for two min utes to transfer the lead to the 1:99 nitric acid layer. Allow the layers to separate. Dis card the chloroform layer. 8. Shake the nitric acid solution with approximately 5 ml of reagent chloroform and lot settle. Drain the settled chloroform through the stopcock bore as completely as possible without loss of the aqueous layer. Evaporate the last drop of chloroform cling ing to die upper surface of the liquid. (No t e 1: Start a zero lead standard at the be ginning of this step by placing 30 ml of 1:99 nitric acid in a separatory funnel. This zero lead stand ard will be used to set the spectrophotometer at zero absorbance for each scries of samples being analyzed.) (No t e 2: If the quantity f load estimated for any sample exceeds the 25 range of the color imetric determination, pipet an appropriate aliquot of the nitric acid solution at the end of step 7 into a clean separatory funnel containing 5 ini 1:99 nitric acid to minimize errors caused by possible leakage of the stopcock, add sufficient additional 1:99 nitric acid to make 30 ml total volume, and continue with step 8.) (No t e 3: Start lead standards at this point if required. Add 5-ml portions of 1:99 nitric acid to each of four separatory funnels, then 2.5. 5.0, 7.5, and 12.5 ml of dilute standard lead solution (2 fig Pb/ml) from a burette, respectively to the separa tory funnels, finally add the proper quantity of 1:99 nitric acid to make total volume 30 ml in each. Continue with step 8.) 9. Add 6.0 ml of the ammonia-cyanide mixture, exactly 15.0 ml of the standard dithizone. and shake two minutes. Allow the layers to separate. Drain the chloroform layer containing the lead dithizonatc into a clean, dry test tube, and cork the tube immediately. 10. Decant this solution carefully into a dry photometer tube leaving the water be hind. If any water spots are visible in the optical light path, transfer again to another photometer tube. 11. Set the spectrophotometer at a wave length of 510 m/t. 12. Set the instrument at zero absorbance using the zero lead standard solution. 13. Read the absorbances of the samples and of the reagent blank. 14. Calculate the lead content of each by multiplying its absorbance by the standardi zation factor (which is the slope of the stanardi/ation plot in micrograms of lead per unit of absorbance.) Subtract the blank value from the gross lead content of each sample to obtain the net amount of lead expressed in micrograms. Special Materials for Blood Sampling 1. Vacutainers, Becton-Dickinson, No. 3208. 20-ml or 10-ntl. complete with stoppers arc used for blood sampling. Tlvc vacutainers B-15 ...... .....'- .......... ..... *...... : ............. DUP050055641 Appendix B Page 16 are used repeatedly and are lead-freed by .the Collecting and Ashing Blood Samples technique described previously. Blood is re moved from the vacutainers and the stoppers, after each vise, bv soak inn' in eold tap water. When no further visible trace' of blood re mains on these items, they are soaked over night in the detergent solution. They are then rinsed repeatedly with hot tap water to Collect a 10-inl sample of whole blood using a lead-free vacutaincr and a sterilized, stainless steel needle. In the laboratory, transfer the sample to a weighed, lead-free, 125-ml borosi]irate Phillips beaker. No aliquoting of the blood is permissible, as most remove alkaline materials. The vacutainers of the lead is present in the clot. Determine are then subjected to the .chromic and nitric the weight of the blood sample to the nearest acid cleaning procedures. The stoppers are 0,01 gram, weighing rapidly to minimize soaked for 20- to 30-minute periods, three evaporation. Add 2 ml of ashing aid acid times, with single distilled water and finally reagent. Add 7 ml of concentrated nitric three times with double distilled water. The acid. (This ashing system permits the analyst lead-freed vacutainers arc dried at 105 C, fitted with clean stoppers, and stored in a drawer reserved for them. Layers of cheese cloth are placed between the separate layers of -vacutainers and the drawer is sealed with masking tape to prevent the admittance of any dust. They are evacuated just before shipment to the field, A vacuum tester is used both in the laboratory and field to test for loss of vacuum, which usually will not occur until stoppers have been used several times. to handle a large number of samples at a time as die blood clot breaks up readily and smoothly without bumping and without re quiring the constant attention of the analyst.) Place the samples on a hotplate operating about 130CC and evaporate just to dryness. After the water is driven off in the initial evaporation to dryness, keep the beaker cov ered with a lead-free watchglass to increase the reflux action of the concentrated acid. This server, to wach solids dcv.ii from the 2. Vacuum Tester, High Frequency, Fisher Cat. No. 1-179. or equivalent. 3. Needles. Becton-Dickinspn. Gauge 20. one and one-half inches in length, stainless steel, B-D Xo. 3200 X. As these needles are used repeatedly, check the tips for burrs by drawing them across the thumb nail. When sides to the hotter zone at the bottom, and also reduces the amount of acid needed. Cool the beaker briefly and then add succes sive portions of the nitric acid ranging from 2 ml down to 0.5 ml as the ashing proceeds. Do not remove the watchglass at any time but merely slide it back sufficiently to facili burrs develop either discard the needles or tate each new' addition of the acid. Each file off the burrs. After filing, they must be time, as soon as the residue becomes light recleaned. Yacutaincr needles are soaked in colored, heat on a 400CC hotplate just long a dilute detergent solution. A Bccton-Dick- enough to blacken the residue, then remove inson Needle Cleaner. No. 3200 G, is used to and cool the sample. Throughout the re force detergent solution and subsequent rinse mainder of the ashing procedure, alternately water through the needles. Needles are sub jected to thorough rinsing with distilled water. They are then placed in stcri tubes and either autoclaved or heated for two hours in a drying oven operating at 180C. The stcritubes arc then fitted with rubber caps. heat the sample with a few drops of nitric acid on the 130C hotplate and bake the residue for the few minutes required to dark en it on the 400C hotplate. Finally, the residue will remain pale yellow or light brown (due to iron content) after heating for 5- 4. Stcritubcs, Bccton-Dickinson, No. 3200 10 minutes at the high temperature. Avoid D. with rubber caps. excess baking at this stage as the ash will 5. Stil le ts for No. 3200 N needles, 20 become decomposed to a difficultly soluble Gauge, two and seven-eighths inches long. form. It is now ready for solution and analy (These BD items are available from the Bccton- sis. Report results as milligrams of lead per Dic'kimon Company, Rutherford, New Jersey.) 100 grams of whole blood. B-16 A V DUP050055642 Appendix B Page 17 Industrial Hygiene Journal Collecting and Ashing Urine Samples Use lead-free, narrow-mouthed, reagenttype, borosilicate, 250-ml bottles provided with standard taper glass stoppers to collect grab samples of urine. Add 2.U ml of a 37^ formalin solution as a .preservative, shaking the bottle 10-12 times after the contribution of the urine to mix the specimen with the formalin thoroughly. Alternatively, urine specimens may be col lected in 125-ml polyethylene bottles contain ing as a preservative 100-200 mg of EDTA (acid form) per bottleP This is convenient and economical for shipping samples consid erable distances. If the urine sample is clear and only one or two days old. measure a 50 ml portion into a graduated cylinder. However, if the sample is older, much of the lead may be in a sediment or on the walls of the bottle and must be dissolved before aliquoting. Transfer the entire specimen to a glass-sloppcrcd grad- iintrr'l rvllnrlor tK/ volume, ririSC the sample bottle with three small portions of concentrated nitric acid and add these rins ings to the cylinder. Mix thoroughly (Cau tion! Old samples may foam over.) Note the total volume and remove an aliquot equiva lent to 50 ml of urine for analysis. Transfer the aliquot portion to a lead-free, 250-nil borosilicate Phillips beaker and add 5 ml of redistilled concentrated nitric acid. Evaporate just to dryness on a hotplate operating at about 130C. Cool, add sufficient nitric acid to moisten the residue and cover the beaker with a lead-free watchglass. Heat on the 130C hotplate and then alternately bake for a few minutes and digest with minimal amounts of nitric acid (as described in the ashing method for blood) until a white residue remains after the final heating for 5-10 minutes at the high temperature. The sample is now ready for solution and analysis. Report results as milligrams of lead per liter of urine. Procedure for Air Samples It is convenient to wash out samples in electrostatic precipitator tubes with redistilled ethanol, using a special policeman made with a rubber disc cut to fit the tube like a piston, and transferring the sample through a short stem funnel into a 250-ml Phillips beaker; gently evaporate just to dryness, (Ethanol is helpful in removing greasy deposits on the walls of the precipitator tube. Some chemists may prefer hot 1 to oc/c nitric acid to transfer the sample.) Transfer impinger samples or membrane filter samples to Phillips beakers. If little ash is expected (usually for impinger or membrane filter samples), add 2 ml of ashing aid acid reagent. (The presence of this salt -will prevent loss of lead by glazing onto the surface of the beaker during ash ing.) Otherwise add 1-2 ml nitric acid. Evaporate to dryness. Continue ashing with nitric acid at a moderate heat until organics arc destroyed. Dissolve the ash in 2 ml of concentrated nitric acid and distilled water and then trans fer quantitatively to a 100-inl volumetric flask and make to mark. Pipet a suitable aliquot into a separately funnel, containing about 5 ml of double-distilled water, add sufficient additional double-distilled water to make the total volume about 25 ml, and apply the Analytical Procedure, starting with step 2. In step 3, as the sample is already in a sep aratory funnel, merely add the cyanide. The amount of lead present in the aliquot may be estimated as described in step 6. If it is less than a few micrograms, an additional aliquot may be added to the same funnel, and the pH readjusted with ammonia. The extraction is then continued, and extracts combined with those collected previously in the second funnel. If the estimated amount of lead exceeds the range of the method (25 ininograms) . take an aliquot as described in Note 2, step 8, When calculating the results, make allow ance for the total number of aliquots. If convenient, aliquot the reagent blank in the same manner so that the correction represents the same amounts of ashing and extraction reagents as are present in the sample. How ever, the blank correction is usually small for air samples. Report results as milligrams of lead per cubic meter of air. B-17 DUP050055643 Appendix $ Page 18 Lead in Paint on Sheet Metal lost on the rubber stoppers. In thp latter case, Cut a small disc of known area from the sheet without dislodging the paint. A 7/32inch shearing type metal punch is convenient. AVeigh the disc to .the nearest 0.01 milligram. Transfer the disc to a 125-ml Phillips beaker shipment must Jx made using sealed glass ampoules. Experimentally it was shown that inorganic lead, in the form of either the chloride or nitrate, was not lost when samples wen* stored so that there was no contact be and add 3.0 ml of methylene chloride. Swirl tween the blood and the rubber stoppers. the beaker for several minutes. If necessary, Experimental studies also were made of use a fresh, clean wooden slick to help dis lodge the paint completely from the metal surface. Remove the* stripped metal disc with dean forceps, blot with filter paper, and airdry. Rewcigh. Calculate the loss in weight as the amount of paint film removed by the losses of lead during the ashing of blood samples. If the samples were overheated de liberately so that carbonized material flashed into flames, up to one-third of the lead was lost. By adding 0.5 gram of potassium sulfate methylene chloride treatment. (in the ashing aid acid) good results were Transfer the beaker to a steam bath and evaporate off the methylene chloride. Add 3.0 ml of concentrated nitric acid and heat until a clear solution results. Apply the pro cedure for air samples, beginning with the second paragraph. Results obtained with this method may be expressed both as milligrams of lead per square inch of surface and as percentage lead in the paint film. obtained: even in the absence of flashing, 5% more recovery was obtained for inorganic lead added to blood samples. The final color of the ashed residue depends upon the degree of heating but docs not affect the recovery of lead. Overheated samples have a brown*ish residue due to presence of iron oxides and may require prolonged heating before a clear solution of the ash is obtained at the start of the analysis. No loss of lead was Comments on Analysis of Biological Materials This iiia!uiu .iiM\ be i egai ded as essentially found to occur upon standing of the dissolved sample for 20 hours prior to completion of analysis. specific for lead in biological materials. Any tin which may be present is oxidized during Evaluation of Method for Lead in Blood the ashing procedure to the stannic state, A supply of citratcd human blood from which is not extracted by the dithizone. Al expired stock in a blood bank was mixed in though bismuth is an interference with the a lead-free, borosilicate glass carboy. Approx procedure it is very-rarely found in biological imately 2.000*gram portions of this pooled samples. The bismuth present in oral medi specimen were transferred to each of four cations generally appears in the feces and previously weighed, lead-free, glass-stoppered, does not enter the blood or urine unless taken borosilicate glass bottles. The weight of the over a prolonged period!4 The intravenous blood in each bottle was then determined to injection of bismuth medication for treat the nearest gram. Appropriate quantities of ment of such diseases as syphilis is now lead, as a solution containing one mg Pb per seldom practiced as it has been supplanted ml. were added from a burette to provide by penicillin. Thallium would be measured inorganic lead concentrations of 0, 0.050, as lead but. as it is rarely encountered in 0.123, and 0.271 mg Pb per 100 gm of whole biological materials, it does not constitute an blood in the hollies, which were labelled P, interference from the practical viewpoint. S, T, and AV. respectively. These concentra Studies were made of the losses of lead in tions of added lead, plus the mean normal blood samples shipped in vacmainers fitted value of 0.020 mg Pb per 100 gm blood with rubber stoppers. There was no loss of determined by analysis of 12 replicate por lead present in its .normal physiologic form, tions of sample using the described although lead in the inorganic form added method, provided total lead concentrations of to whole blood was shown by analysis to be 0.020, 0.070, 0.145, and 0.291 mg Pb per 1-00 B-18 Appendix B Page 19 Industrial Hygiene Journal Ta b l e I Replicate Analyses by USJPHS Laboratory (All values expressed as mg Pb per 100 gm whole blood or per 100 ml of 1% HNFOs solution) Sample Gated. Lead Content Replicate Analyses Sum Mean Percent Recovery of Total Pb . Standard Deviation from Gated. Value Coefficient of Variation, % P 0.020 0.0186 0.0237 0.0212 0.0195 0.0179 0.0204 0.0200 0.0192 0.0203 0.0236 0.0187 0.0182 0.2413 0.0201 95.7d 0.0019 9.5 Sb 0.070 0.0693 0.0700 0.0714 0.0700 0.0708 0.0689 0.0697 0.0680 0.0670 0.0620 0.6871 0.0687 98.1 0.0030 4.3 Tb 0.145 0.1469 0.1424 0.1434 0.1433 0.1406 0.1376 0.1349 0.1339 0.1590 0.1360 1.4180 0.1418 97.8 0.0081 6.6 Wb 0.291 0.2876 0.2819 0.28Q9 0.2791 0.2916 0.2721 0.2741 0.2853 0.2890 0.2780 2.8196 0.2820 96.9 0.0115 4.0 Xc 0.075 0.0717 0.0753 0.0722 0.0753 0.0703 0.0734 0.0706 0.0737 0.0731 0.0740 0:7296 0.0730 97.3 0.0028 3.7 yc 0.160 0.1485 0.1510 0.1456 0.1524 0.1426 0.1451 6.1448 0,1511 0.1543 0.1451 1.4805 0.1481 98.7 0.0045 3.0 (a) Unspiked blood sample containing 0.020 mg Pb per 100 grams of blond, as determined from analyses reported in table. (b) Blood Sample containing calculated 0.020 mg Pb pcr 100 grams spiked with 0.050, 0.125, or 0.271 mg inorganic Pb' 100 grama blood. .(c) 1% nitric acid solutions spiked with indicated amounts of inorganic lead, d) From intercept of line in Figure 1. gm of whole blood for the series of four samples. The blood was kept under refrig eration except during laboratory handling prior to shipment to the collaborating labo ratories. Replicate sets of samples, of approximately lO-ml volume, were removed periodically from the bottles during the initial 10-day period, weighed, and analyzed. This proce dure was repeated for five sets of samples to make sure that the calculated lead content of each sample was correct. Upon obtaining this assurance, sets of lead-free vacutainer tubes were loaded with approximately 10 ml of the blood samples and the tubes were flame-sealed. During the next two months five sets of these vacutainer samples which had been retained in our laboratory under refrigeration were analyzed. These latter analyses were spread out over this period while the remainder of the samples were being analyzed by the collaborating labora tories* The results of all of the analyses con ducted in this laboratory on the blood sam ples arc presented in Table I. In addition to the blood lead values, this tabic also lists the results of our analyses of standard samples X and Y which contained respectively 0.075 and 0.150 mg Pb per 100 ml in 1# nitric add. The data presented in Yable I (excluding sample P) showing the mean lead recoveries ranging from 96.9 to 98.7fc, demonstrate the accuracy to be expected of the method. The lower portion of Table I shows the standard deviations and the coefficients of variation. The coefficients of variations in the 3% to 5fc region indicate the highly satisfactory precision possible with this method. The same data arc reproduced graphically in Figure 1. The quantity of lead added is plotted as the abscissa and the total amount of lead found is plotted as the ordinate. Extrapolation of the linear curve to zero ordinate has provided a value of 0.021 mg Pb per 100 grams of whole blood as the probable true initial concentration of lead in the supply of pooled blood. The slope of this curve is 0.971, which represents a mean re covery of 97.1**.. Furthermore, the 0.0201 nig Pb per 100 gm of blood obtained as the mean of the 12 replicate analyses of the pooled \lood supply is in close agreement with the 0.0201 analytical value from the graphical computation (0.021 x 0,971). B-19 DUP050055645 Appendix P Page 20 Ta b u II Replicate Analyses by Ten Collaborating Laboratories (All v aIu ps are thi' mran of twn analyses timi art* i,xprwiMi(l aa mi Pb per 100 Rm ot whole blood or per 100 ml of 1% HNCj solution} Sample Laboratory Caled. Value of total Pb A B C D E F G H K L Sum Mean Percent Recovery of Total Pb Standard Deviation from Caled. Value Coefficient of Variation. % Ps 0.020 0.032 0.022 (Q.00A)d 0.020 0.028 0.030 0.023 0.028 0.025 0.024 0.238 0.0264 125,7 0.0082 41.0 Sb 0.070 0.078 0.007 0.060 0,067 0.088 0.070 0.066 0.071 0.068 0.065 0.700 0,0700 100.0 0.0078 ll.l Tb 0.145 0.150 0.134 0.150 0.130 0.137 0.135 0.128 0.142 0.145 0.130 1.381 0.1381 95.2 0.0110 7.6 Wb 0.291 0.270 0.277 0.260 0,255 0.290 0.280 0.256 0.303 0.307 0.288 2.786 0.2786 95.7 0.0227 7.8 Uic 0.075 0.068 0.081 0.080 0.068 6.080 0.073 0.078 0.071 0.080 0.679 0,0754 100.5 0.0055 7.3 0.160 0.149 0.148 0.160 0...1.3.S 0.160 0.147 0.158 0.140 0.160 1,355 0.1505 100:3 0.0098 6J5 <ai I'nspikcd blood sample containing 0.020 mg Pb per 100 grams of blood, as determined from analyses reported in table, (bl Blood sample containing calculated 0.020 mg Pb per 100 grams spiked with 0.050, 0.125, or 0.2.71 mg inorganic Pb/100 gm. (ci T'f nitric arid solutions spiked with indicated amounts of inorganic lead. (d) Discarded. m- INORGANIC Pb ADDED / 100 G* 0LOOO Fig u r e 1. Evaluation of the method for lead in blood. The analytical data reported for this same series of samples by the 10 collaborating laboratories of State and local occupational health agencies using the USPHS method, are presented in Table II and plotted in Figure I. These data show satisfactory lead recoveries except for sample P which yielded mostly high results. It should be noted that each value is the mean of two analyses in contradistinction to the individual analyses reported in Table I. With the diversity of analysts, reagents, and instrumentation em ployed in this collaborative effort, one might expect larger coefficients of variation than those obtained by a single analyst. These coefficients for the collaborators' results are about double those obtained by one chemist in our laboratory for the corresponding sam ples. However, it is pertinent to mention that the majority of these collaborating labora tories do not perform blood lead determina tions on the routine basis that has been our practice for many years. Therefore, \ve believe that these data also demonstrate a remarkably good performance of this method for blood lead analyses. Summary The complete details of the USPHS method for the determination of lead in air and in biological materials have been pre sented. Also included is a rapid procedure for the removal of a known area of paint film from a sheet metal surface and the deter mination of its lead content in terms of per centage or of milligrams of lead per square inch of surface. Complete information has been provided on the successful associated procedures for the cleaning of glassware, the collection of blood and urine samples, the transfer of atmospheric particulate samples for the ash ing treatment after collection by the common sampling techniques, the wet ashing proce dure using nitric acid, and the purification of reagents. The method has been tested periodically with resulting minor procedural changes dur ing the past quarter-century. The results of the most recent evaluation of the final method in this laboratory provided a mean lead recovery of 97.1% for lead in whole blood samples. The coefficient of variation was 9.5% for twelve replicate analyses of the pooled blood supply and it ranged from 4.0% to 5.6% for ten replicate sets of the other three blood samples which contained total lead ranging up to 0.291 milligram of lead per 100 grams of whole blood. References 1. Co mmit t e e o n Ch e mic a l Pr o c ed u r e s or t h e Oc c u p a t io n a l He a l t h Se c t io n , Ame r ic a n Pu b l ic He a l t h As s o c iat io n , Inc.: Methods for Determining Lead in Air and in Biological Materials. American Public Health Association, Inc., 1790 Broadway, New York 19, N. Y. (1955). 2. Sal t zman , B. E.: Matched Test Tubes in the Beckman DU Spectrophotometer. AnaL them. 27: 1207 (1955), 3. Ne l s o n , Ke n n et h W.: Personal communication. 4. El k in s , H. B., and B. P. W. Ru o t o l o : Notes on De termination o Lead by Dithizone Method. Part II. Interference from Bismuth and Tin. Amer. Ind. Hyg. Assoc. Quart, 14: 111 (1953). Comment. The "USPHS" method is free from interference except bismuth, thallium, and stannous tin. Tin, however, is oxidized to the noninterfering stannic form during ashing. Thallium and bismuth are currently seldom encountered in biologic samples. If bismuth is expected to be present, the APHA dithizone method (see ref. 1 above) or the atomic absorption or spectrographic methods described previously should be used. LEAD IN FOODS The Standard Methods of the Association of Analytical Chemists (formerly the Association of Official Agricultural Chemists), 19^5 or 1970 Edition, 439 should be consulted for the analysis of lead in foods. Procedures are given for the separation of lead from some metallic interference, notably, from tin present in canned foods, alkaline earth phosphates, bismuth, and iron in some food products before analysis by the dithizone method. B-21 DUP050055647 Maay food products contain substances that precipitate in the ammoniaeal solution of the sample from which lead must he extracted as the dithizonate. These precipitates include the phosphates of calcium and magnesium, iron and aluminum hydroxides, and silicates. Lead is occluded in the precipi tates and thus may be lost during the analysis. Substances containing more of these substances than can be kept in alkaline solution with the citric acid reagent must be subjected to a removal of lead as the sulfide after ashing. ' Dissolve the ashed sample in hydrochloric acid. Add 20 ml of a 50% solution of lead-free citric acid, and adjust the pH to 3*0-3.4 with ammonium hydroxide, using bromophenol-blue indicator. If the iron content colors the solution strongly, a spot plate may be used to assist in adjusting the pH. If the anticipated lead content of the sample is small (less than 0.1 mg), 5-10 mg of lead-free copper sulfate (CuSO^ . 5HgO) must be added to serve as a eoprecipitant and "collector11 of lead sulfide. Precipitate the sulfides by passing hydrogen sulfide into the solution for 3-5 min to achieve saturation. Then filter the solution immediately through a fine fritted filter disk, using suction. Dissolve the sulfides (without previous washing) with 5 ml of hot nitric acid, drawing solution through the filter disk into the original flask in which the samples had been gassed. Wash the filter with hot distilled water, Stopper the flask and shake to dissolve any sulfides adhering to the walls. Boil to expel residual hydrogen sulfide. The sample is now ready for analysis by the dithizone or other analytic procedure. B-22 y DUP050055648 LEAD IN DUST1^8, AND SOILS Lead in street or factory dusts should he dissolved hy digesting and leaching with nitric acid {l:k) and hot 20% ammonium acetate, the extracts are filtered and analyzed, after appropriate dilution with distilled water, using one of the recommended dithizone, atomic absorption, or spectrographic methods. The selected dithizone method must he applied after proper provisions for the elimination of inter fering substances.- The atomic absorption and spectrograph! c methods are highly specific and may be used successfully. Care must always be taken to avoid molecular absorption errors with atomic absorption (the salt effect); this is accomplished by lowering the sample concen tration in the solution as required for lead (or other elements) being analyzed. The emission spectrograph, with its unparalleled degree of specificity, has been used to great advantage since 1933 for the determination of lead in biologic and environmental samples. 119 5 122 This method offers the advantage of detecting and measuring simultaneously other elements that are in the sample and constitute possible interference with the dithizone method. Although its precision is not as great as that of the dithizone method, its error of measurement of lead may be reduced to about +_ 10% by the use of wisely selected internal standard and spectroscopic buffer conditions plus the analysis of biologic B-23 DUP050055649 samples in triplicate. Industrial laboratories are using directreading spectrographs for routine, rapid analysis of many thousands of biologic samples of blood each year. 321+ Under rigorously con trolled conditions, this method has proved to be extremely reliable for the periodic blood lead analyses required for monitoring workers1 exposures--an excellent example of a biologic monitoring technique. However, its expense puts the spectrograph beyond the budget of many hospitals and health departments. Other methods that have been popular for lead analyses include electrolysis and polarography. However, these methods were capable of handling relatively few samples per day and still required ex tensive preliminary treatment of the samples to avoid spurious results. GENERAL COMMENTS OH METHODS FOR LEAD The several published dithizone methods for lead will provide reliable data if the analyst is alert to the requirements of a high degree of purity of reagents, lead-free sample containers and laboratory glass ware, lead-free Vacutainers and needles without a soldered joint, a thoroughly clean laboratory, and the isolation of glassware likely to have become contaminated with a "high lead11 sample until it has been deleaded. The last requirement is frequently overlooked and can cause spurious lead values in samples analyzed later in the same glassware. Extreme care must be taken to ensure the complete removal of traces of lead from such glassware, using hot nitric acid and rinsing; this B-24 glassware must be tested analytically to confirm that it has been com pletely freed of lead before permitting its reuse for any types of samples. * In wet-ashing biologic materials, care must be taken to avoid the > Hflashing11 of samples, which always results in a loss of lead. Molecular membrane filters are recommended, whenever feasible, for collecting particulate lead from the atmosphere. They are easily mounted for microscopic analysis and they are readily digested with oxidizing acids and thus permit rapid preparation of a sample for analysis . Airborne lead should be sampled at a height of 5-10 ft above ground level to keep the sampler above most of the coarser particles stirred up at ground level. In some studies conducted close to highways , it is necessary to use an elutriator to remove the coarse particles (greater than 10 \m) before they reach the filter. Several other methods for the quantitative determination of lead in biologic samples are being developed or are now in use. Further evaluation of these methods may be necessary to assess their value as analytic-diagnostic tools, but it is appropriate to mention them here. The use of an ion-selective electrode consisting of a lead sulfide- silver sulfide crystal membrane has been used to determine lead in urine samples; a sensitivity down to the parts -pe r-b1 Hion level is 430 claimed. The method requires no sample preparation and is B-25 DUP050055651 relatively rapid and inexpensive. The ion-selective electrode can measure only free ions, and therefore the method does not readily lend itself to the analysis of lead in blood, In blood, lead is usually bound to red cell constituents to a significant degree* Other blood proteins also may interfere with the electrodes. The method may prove applicable to hydrolyzed blood samples, A method that requires relatively small amounts of sample, as well as very little sample preparation, is anodic stripping voltammetry (ASV). This method, as described by Matson ,391 relies on the concentration of an electroactive ion, such as lead, on a negative electrode during a relative long plating time (5-60 min). Alter plating, the polarity of the electrode is reversed and increased over a relatively short period (2-30 sec), resulting in a sharp current peak proportional to concentration. Sample treatment involves digestion of the sample in perchloric acid and dilution with water before ASV. Because different forms of the metals, as well as different organic complexes, will affect the plating kinetics of ASV, this method in conjunction, with other qualitative or quanti tative methods may lend itself to the study of the nature and kinetics of metallo-organic complexes in biologic and nonbiologic systems, 235 Hammer et al. have recently used atomic absorption spectroscopy to analyze hair lead content and demonstrated a correlation of hair lead content with environmental exposure to lead. Although the study did not attempt to relate hair lead content to clinical or subclinical illness, the use of hair samples to estimate subacute or chronic exposure to heavy metals is clearly feasible. B-26 V DUP050055652 Personal samplers are available from several manufacturers. These samplers are battery-operated; are complete with a rotameter, which should be calibrated with the sampling device attached in-line; and are used in monitoring individual worker exposure to dusts and fumes in mines and factories * The collected particles are separated into respirable and nonrespirable portions, the respirable portion being collected on a preweighed membrane filter. The sampling device is usually worn on the lapel in the approximate breathing zone ; the pump is clipped to the belt. The use of this sampler permits the evaluation of a person*3 inhalation exposure to a dust or fume. B-27 DUP050055653 APPENDIX C DAtfA AND CALCULATIONS FOR FIG. 3-3 Air lead Blood lead content, content, yg/lOO g yg/m^ Total daily lead assimilation,a ug whole "blood 12 l6 13 21 19 19 24 25 30 31 21 31 30 48b 63d air 0.12 0.5 1.0 1.0 1.9 2.2 2.4 2.2 3.8 4.2 5.2 5.5 6.3 2.0 2.0 at 30$ lung retention at 37$ lung retention 30.83 31.02 33.45 34.26 36.90 38.51 36.90 38.51 43.11 46.17 45.18 48.72 1)6.56 50.42 45,18 48.72 56.22 62.34 58.98 65.74 65.88 74.25 67.95 76.81 73.47 83.61 148.80 152.02 221.30 224.52c lung retention 30% 37$ m = 54.7605 54.2467 b =-69.2052 -69.9312 y = yg Fb/100 g blood x = log yg Pb assimilated daily' C-l DUP050055654  V ^Assumptions: 23 M^/day inhaled air; 30 yg/day lead assimilated from food and water. ^Subject M. R.2" cDaily oral lead intake ;M. R., 1.350 mg; E. B., 2.075 mg;2^ 1055 absorption assumed. ^Subject E, other data from*^^ C-2 DUP050055655 APPENDIX D COLLECTION AND STORAGE OF BIOLOGIC SAMPLES Valid analytic data require not only reproducible and accurate analytic techniques, but also great care to prevent either contamination or losses of lead and metabolites during collection, transport, and storage of biologic samples. A number of reports refer to the collect tion of blood and urine in "plastic'1 containers and the use of -"plastic" syringes and "disposable" needles. With the introduction of newer micro techniques of analysis, specimen collection will require even greater care than in the past. Polypropylene and Teflon are the preferred plastic materials for trace metals analysis, so blood and urine are best collected with polypro pylene syringes and sample containers. Similarly, needles should be of stainless steel with a polypropylene hub. Vacuum tubes of silica glass designated as "lead-free" are also in wide use and are suitable. Anticoagulants (such as heparin or citrate) added to bloodi-collection outfits and acids or other preservatives added to urine must be shown on analysis to be "lead-free" (i.e ., maximal permissible quantity of lead should be insignificant in relation to the quantity of lead in the sample). In general, collection equipment must be washed in mineral acids and then rinsed in copious amounts of water that has been "deionized" by passage through mixed ion-exchange resin beds; such water (resistance, 2-3 x 106 ohms) may contain up to 200 ppb k51 of lead. In general, specimen collectors should be obtained either from an experienced laboratory performing the analyses or with . D-l DUP050055656 their advice regarding the use of commercially available special leadfree equipment. In general, for urine containing CaEDTA (from patients receiving the drug), addition of hydrochloric acid to the collection bottle permits analysis for both lead and ALA and storage of urine for up to several -weeks before analysis. The stability of ALA^*^ and ,^99coproporphyrin^^l n urine has been studied and reported. The data may be summarized as follows: Samples are preferably collected and stored in the dark at 4 C. Repeated thawing and freezing can result in serious losses, so freezing should be avoided. ALA is stable in acid urine (pH, 1-5), so an acid preservative, such as hydrochloric or possibly glacial acetic acid, is needed, unless the analysis is carried out within a few hours. For 24-hr collections, enough acid must be added to yield a final concent 111 tration of 0.001-0.1 N, The ALA content of acidified and refrigerated urine specimens remains constant for several months. Schwartz et noted that unpreserved urine samples should be analyzed for coproporphyrin within 30 min because of the instability of this substance at the usual acid pH of urine. Samples collected and preserved with sufficient sodium carbonate to yield a final pH of 6,5-8.5 are suitable for analysis for up to 10 days if kept refrigerated in dark bottles. For 24^-hr collections, sulfanilamide may be added to sodium carbonate in the collection bottle to suppress bacterial growth (J. J. Chisolm, Jr., unpublished). :Y D-2 DUP050055657 The most widely used method of determining ALA in serum'is that of Haeger-Aronsenanalyzed her samples within 5 hr. Hernberg et al. 251 found that ALAD activity, as measured by the technique of Bon signor et al . was stable up to 5 hr at 3, .25 and 30 C, but all analyses in their laboratory were done within 3 hr of sampling. Uoo EL liar et al., who used a somewhat different technique for measuring ALAD activity in red bipod cells, carried out the analysis within 1 hr of sampling. The available reports do not include data on the stability of protoporphyrin and coproporphyrin in blood; in view of the known difficulties in handling these substances in the laboratory, it is suggested that protoporphyrin and coproporphyrin determinations in blood be carried out as soon as possible. Measurement of lead in blood and urine provides at once indices of exposure and absorption. Lead in these fluids may be measured by spectral analysis, the colorimetric dithizone technique, atomic absorption spectrophotometry (AAS), polarography, and anodic stripping voltammetry (ASV); these methods are described in Appendix B. Each technique requires suitable preparation of the sample before final determination. Spectral analysis is considered the most accurate , but its use is limited by economic considerations. Polarography is widely used in Europe but not in the United States. In the United States, the dithizone technique and AAS find the widest use for lead determinations in clinical laboratories. Long experience with and refinement of the dithizone technique make it the standard of reference. A minimum of 0.5 (preferably 1 jjg) of lead in the final sample volume is needed for accurate results. For blood, this requires a sample of P-3 ................ ...,,...... v . -.- - DUP050055658 5-15 ml. In recent years, because of the time-con sianing and exacting requirements of the dithizone method, MS has been explored in the hope of speeding and simplifying the analytic procedure and reducing the sample volume needed for accurate analysis in blood and urine. Methods have sometimes been hastily introduced without reference to comparability with older methods . Selander and Cramer have published an AAS method for measuring lead in urinethat shows excellent agreement with a dithizone technique . Their MS methods^^ por lead in blood give results that even at low concentrations, are comparable with the results of spectral analysis. These and other reports make it clear that important interference does exist and must be corrected in AAS techniques; (l) In sample-preparation techniques that omit the ashing or wet-digestion step, blood and urine must be totally free of clots and particulate matter, to avoid low recoveries. (2) With single-beam MS instruments, the sample must be read not only at an absorbing lead line (2170 or 2833 X), but also at an adjacent nonabsorbing lead line, to correct for nonspecific light-scattering and -absorbing effects. (3) Double-beam AAS instruments with provisions for background correction presumably accomplish the same correction, but the published data are inadequate to evaluate this point. (4) An ultraviolet-sensitive photo- o multiplier is essential when the 2170 A line is used for analysis. Micro-AAS techniques 15^a and ASV techniques 391 have the potentiality for measuring lead in nanogram and subnanogram quantities and hence may reduce the sample requirement for blood to less than 0.2 ml, which is readily obtainable by capillary sampling; these techniques are currently being developed. d -4 DUP050055659 Regardless of the method used, it is clear that the analyzing laboratory should be experienced in the technique, have experienced personnel, be performing lead analyses regularly, and incorporate adequate quality and specificity control measures into its procedures. iese factors are especially important when low concentrations of lead in blood and urine are being measured. It is unlikely that a laboratory that per forms such analysis only occasionally will produce consistent and OA D reliable results. A recent report0 showing wide discrepancy between different laboratories points up the further need for interlaboratory studies when new techniques are introduced and at other times to ensure maintenance of adequate standards of quality with established techniques. D-5 DUP050055660 SUMMARY OF TOXICOLOGIC DATA OF EXPERIMENTAL LEAD POISONING anim als 150 APPENDIX E E-l P oo pP I dr & 2 0 .3 4J 0 S * 44 1 43 O CO P H 0 0 :P H 44 rl 44 3E O 13 0 0 *4 -U a e ft a a a CO uCCPOOi CM Hc U Q0H 00 *CCM0S0HOO> T0O 10>>r3l *>r4 A . CH3O r0H <a0 0a HrH < to Pi 13 co pH 13 1s03 1Cw0>>3rO3l 1I03V rCHO 0SPBP HH < **00) 5 M X* L/O . Cu0>O) w0 CM PH u >0H *3 o O X1 00 (3 oo M o 00 A6 00 M 00 o 00 00 b rH B B o o o CM CO m rH rH op 00 r* At op 00 6S oo CM 1^. rH CM 00 AL op B o CO CO a a o CO aa o CO <u 44 0 00 <M- 1 0 0 0 13 4J 4-1 330 > 0 13 r--1 U 0 0 /N 0 0 0 *4 4-1 0 0 rl 44 H CL. 0 13 0 JG 44 Pi Pi 0 2 4-1 44 w0 O rl rH co 0 u u to P 0 0 o44 X 44 4-1 0 44 O 00uM Br-4 0 P u U i--1 U 0 H *rl 44 Pi 4J 44 0 O O u 0 3 *H o xs 43 0 U H H PH 0 S o U CO H to 0 44 4J &4 Q 0 U rH CM A 0 13 13 13 13 13 0 CO 13 13 13 13 Xi 13 13 4-1 0 0 0 0 0 4- V 0 0 0 0 u 0 0 is 0 0 0 0 0 0 AA AA A0 0 0 -3 0 A 00 A h3 0 4-1 0 U 4J H P 13 0 0 A 0 I! 10W3 3 MOM 0 1>3 a3 EM 404 0 4J 03 <$ R e f. 00 0 VO 0 75 tv* Cr* C~ 0000 44 4J 4J 44 a3 O3 O3 O3 < <1 < <U c~* o- 00 4-1 4-1 33 03 << c-- 0 4-J O3 DUP050055661 L iv e d 8 days 5.0 mg/kg CO tH 2 C0 a T3 r4 <D oPW S3 <0 P > <D q> -o 4- fiu p P >> 4-1 O B CO ** cu 4J P 0 *4-1 0) PS o PS LA t-- P O O - e- in g a m <u 0) Q) U 4-1 P PP O PP <5 <J <D .4-4 P O < aa DUP050055662 4040 4040 00 N "Ss f00 B 0B0 o CM o o . w so r> 00 d H tit mm i 40 o CO d rH O <u 3u po4 idH roB to 4rJf to UM NH 4040 N 4040 V* 4040 \ 4040 4040 X 4040 4040 V '**>* 4040 S tH 4o3 00 S B a* F f F f F O y rH o O .* o O o O o O to os o o CO sfr PN. oo d rH rH H 1--1 H pH CM rH ft ft ft ft ft ft -ft ft 4040 4040 4040 X FFF o O CO CO sf ft .ft ft d *3 (U to > <u H rH W <0 rH *0 rH fts rH to 43 IH *r4 44 O u cy rH <u d 43 4J H O 11 H n3 to <D rH <0 rH *3 0s rH O. u oo ua* rH 43 H y M H Iw d tnoa 0<1u td 3 S PH 00 MM o > <0 3 a3 >s U H <0 y 44 3 y < * -P 91 mm <d J ir\ OI d o 04 dt o ) a S fo ix, P* ;s* a ^ X X Pt DUP050055663 Lived 2 h r 7 .0 ing/kg T--i<30 60 60 60 60 60 60 60 **>* >> A* r* 0 S* f f IP I1 IP 0 y O p O P o o oO to m in IT) VO 00 r*-4 1--4 e i--4 t-4 rH VH 0 P<3QMdD 1 CO rl eH JrcoO .jO oa rl 4tuoJ 4-1 60 r*i f6KS0 *3 6B0 60 60 s. IP f &60 60 60 >>* fff oOOo o oo # cSi CM CO cn on VD 1--CO1 o 25 2BH a<0 toX0C>O): 3 o 0a) E6Hs XVC>3O)) mcu oS Oli| PI *> iisn- ;-*P a 0a TC<JUO u6s JPHQ x0ui-)i rol JO2 HVI s s Ft, 00 a> 4J <OP X to S -Jd P* JSJ X ...........: v . v* \ ' V;.w.i,w.v:-v: -:-:v... DUP050055664 S u rv iv e d 42.0 mg/kg 1--1 ~i H 0 bC bO bO b0 b0 bO bo bO bo bO O bO bO bO bO bO *! Q) J4 <D M& > tM ff S IF I1 ff f f 45 ff ff fff r-4 t-s 0 Q O o O O O O O O O Q o o o o * o d i-m St in oo CM st vO r^. o vO d CO vO CM st UO *rl i--i i--1 rM rH CM CM CM CM co sf `rM CM CM co CO CO <s tw i .D Q to d H o V d H 4- 4J P B td o TD u p4 td 4-J a a ft a .a ft eu 6 & .a a a a a aa TD c<dD pd td u <U 4J u j S fM<d I TODJ Od S2J td o r-t fe 4M 0 > (U TD du P 4-> H CO 4) 4J P O < D TD t<dD $u <u B cud H 1*4 Pm P4 <D o3M < DUP050055665 S u rv iv e d 90.0 mg/kg vH U cd 0oo 60 60 60 60 60 60 60 60 60 60 60 60 60 60 44 44 44 44 44 44 44 44 44 44 44 3 44 44 44 44 Ss Ns N X *>Sw 60 \ f IP060 60 60 es 600 600 ji5 600 IP 5>S 0 600 600 o o Q o O O o o O O O CM O P 0> . CM St vO <t H in CM in cn in M3 in m cn r>. t-j p> CM O' H o # G O vO O 00 00 oo aH O4-4 <u 3 ( & & a ft & & & & & & & & ft ft G<U > CO r"*"t 0 0 d T3 rl <0 G CO cd 3 04-4 > 0a) T3 ,3 in u H CO 4<-10 0 64 G -p rx - L^T\ G8 P c<du in su 4M0J) H 4-1 ^ a fa a 2 a fa fa a a 3 DUP050055666 o CO r4 U -0M 2 CO r--f 0 e cox> T-l 0 o 6 CO co d 4-1 0 > <u H3 pu d >> 4-> H CO # 4-C 0Q a *Tc0d3 >* t<Md3U M <U H S coos 4-1in Pm Pm Pm Fm 0M ud DUP050055667 R esult CO P d Po to 0 91 13 <U 43 p d P 91 d P a H CO CO >% <9 rH 0 13 os P > H * MM H <0 13 *H rH r* rM cBd A P co 9) H y 'd m O c0 "O a P d 91 13 13 p p y 01 <0 <a0) o 4H 2B ctf rH CM p P 91 g O rH PP <0 O CO > MH H MH oi CO MH P 4H 91 P 13 P <U 0 (0 H o p P0 POh >P, 0 o to i3 O rH to rM 0) Vd 9) CO O rl W 9) d rJ c0 d a) P GJ O rl 91 13 P 9) O MH oin d <0 2 rP CO p I i 13 P o<9 91 rH o PrH to to rO P CM W P a 0) s H cd rl 13 13 (0 r0H 9) u P H H P CO 9; p d H M 13 (0 13 d CO 91 p p *H 13 rl P d rl P O V H ' d. 13 91 00 P CO rH d rl CO rH CO {> 2 d P u 91 rl H 13 cd p 13 91 P CO 4 P O 8 & <0 PH 00 00 dd 60 op BS 00 00 \ wa1 00 BB co a m cn in CM CM 00 r-H 00 00 00 AS w CO PO s d 13 w w B sB 00 r-> CO CO . l CO co co O rH rH mcBr CO co 1d3 cd r* 9H1 13 o >2 d00 00 B CM rH Ml o f p ro te in catabolism E-8 O Pm OA o PH Ao. a 2B oPM Pm O PM M a te ria l g iv e n _______ t r a t io n Dose ai p dtpc9oo1 CO P91 dcp(00d) cd P.cPta9C9odO11 P91 co :p<U u to 13 >t94o1 Tpt0o))I 13 .t94o1 1C934O1 used 00c- p 91 91 PP PP uU <3 < CQ c* Po o cd CO 13 >> O cd u CO rH 13 H as d O P co do i CM P !>- du d study 4H co 91 A! Od rt>o* OO st 91 91 PP cd Cd pai p 91 yO cd d 13 13 cd cd 941 941 CM H to d 91 U 91 rl d 0 >fr p CM du w 00 rH r> o rH d Q p G\ IT) CO (8 days) DUP050055668 B m . 0 sfr CO & MM 1 X . o CO d *p4 o 0) d r+ 4J H 4-> 35 Qd CO IM PM 0 4J d CO d rl H CO v d n O 3 0d v4 o o M4 r4 rl a CO 4J d <D X! rl t>0 60 M Oh CO i u 0 U M B >> d rl CO 0 0 & CO d CO CO 0 CO 0) d o d > 0 d 0 60 U CO d <0 r-l d CO 4J Oh r-l *d o r-4 X y 1-1 >> CO co 4- M d3 r--4 0 CD a; CD w 0'. 0 H 4J 0 CO u H d rH CD H > dd >. r--1 y l"l 3 *d CO H r-4 d 1-4 od cM IV 0 dd O TO r\ 0) d 0 CO y co rl CO d CO M > 0 4J CD :M d r4 4J td <0 MM in CO O r-4 r4 *H CO CD > H rl oH CL fl) CO CO M-l H o st > o A d St CO 0) d a d d n0 W<D d op "D CO H r--1 CO a CO H 0 :4J u <0 $ P r--4 0 r--1 CD 0 CO o > H d rl Q U O d 0 rrl X! 00 r-l r-l 0 CD rd 1 M 0) a a o H r-l rl d y 4-1 sd Od S3 < O rl $5 r* 4-> 4J 00 *H *M dd mm MM C0 o Od fr* 00 H a o rH o i> >> r-l r--1 rl rl <0 0 dd 5* 5^ in in MM mm 0o yy yy CM CM O&4 H EH tro p h y > fa tty in filtr a tio n E-9 0 t> 0 H 4J w0 U rH 0 0y rl: 0 u 0d w 0 m0 a Si 0 u 0 4J 0 0 0 d 0 0 0 4J 0 4J 0 y 0 d 0 0 S\ CO 122 0 d Q CO 0 01 d d CO 00 03 St /-\ CM w X! /N w <s rd rd in 4-1 /-s rs 4J 4-1 0 r> d 0d >M 0 m y Ps y y 0 y 0 J>> 4J 0 rl 0 H a rl a rl d H 4-1 > d d d d d 0 r4 0d o o o> 0 < 0 o > a eu 3 d u CM M CM m00 6 6:> 4-i X3 w H <0 O V v m & CinO n t-t fm** m 0 4J 0 4J 0 y 0 d 0 0 S <N y in /-s rl vO CO d CO oi 0 u crs d 6 n o 0 4-1 c<uou o 0 iJ0 yH aoM CCMIO CO o days) DUP050055669 E-10 CO 00 0 OM 0) 0 73 73 w CO CO 00 X0 44 O 4-1 o0)o d0 >N 73 ,*4 0& 0 0 oo O 0o 00 a !> 0 73 0 0 73 00 :3 4O3 4J CSO 73 e H Pi 0 d X) (4 00 w u 0 73 i-4 0 *08 73 0 t >> 44 > rl rl CO d 0 0 O a rH <44 0 <0 4*H3 o O u 0du d 0 r4 > 44 0 CO 0 00 0CO 0 M u d 73t j S03 U H X d rl 4J D CO 00 CO CP d 0 M CD H .d 00 d I d03 73 43 O 44 43 r4 4J 0 H d vCwO 0 73 44 (0 0 Pi Pn O 0 44 r-4 u 00 0d Pi 44 1-4 V du o 0 3 g 44 73 0 0 A d r4 0 43 44 0 M p* >N 73 M 0 0 Pn 0 44 d 43 d Q *rl H 0 H 0 M 44 d H r-^ < M 73 00 Q >7 A 43 0 M 0 M 2i 0d > 44 0 >> P% 0 0d 73 rl 0 0 S 0 d rl O U 0 (3 2 0i w U 0 44 0 d 0 pi 44 r-4 2 p Pi 0 44 2 44 0 H4 D 44 0 O U CO I 44 d a5 d 0 Pi 73 44 O 0 73 d d 00 o r-4 43 0 0 00 0 <44 p > 0 73 m i--i 0 a <44 1 jQ o 0d rl 0 0 d H 4J rl 44 3 80 Q 73 U o2 0 44 e? rl 4*J 0rl 73 O d H {xT as CM o P*4 4J >N 0 0 rl 73 73 00 P4 &O O ON r-4 P* > 00 73 73 00 1 S CM vp r--I oo B d< CM > 0 > rl oc r-l 0 H Pi 0 44 a 0 1--1 ' 0 0 B 73 rl 0 d0 0d <44 0 > 0 73 CP d >1 44 B0 4-4 0 & S] 0 44 404 0 O 0 73 0 3 > 43 4JI uI rl d o 6 ON JHsf 0 00 44 44 44 0 44 0 4J 0 44 000 UUU 000 73 73 73 0 00 a00 .-3 rd 20 <N* /-N 0 y-N y > 0 u pi 0 o Pi 0 r4 oa 73 o *r-l d 0 0 rl Ps a o 0 Pn Pi 43 Q sCOy pi 4a3 r--l N/ pi 43 U r--l V cn S! C-- VO 3 t DUP050055670 p JWp3) dco o PP d p 4J C0O) CO 8 > P P d hHd P 5 *o p CO p CO pp dP pP HO rl HP3 P3 CO H P H tK P IsS *ri d p pd-i 00 Q CO dP r-l O O P5 CO p P 4H P0 O $3 o d TJ p HD CO P CO P P Q Pd P Pd HD 0 50 H P 4J CO rl r3-l P 00 i3 dP QP rH CO p HD l"H CO o S ;rP--l P rC-Ol M3 co dH P3 Pa 4$30 rl P Op CL X P j CL & HD p P J3 P O CL . P P CL O p CL P rH O CL CP p 3 Cfl CO 0 p > dd 2 HD p H 5P a P CO P P co h d o 0 P p o P Pp P u X P CO rH d M s d H P d CO <D p n p 1CaO g V p HD P f CL e< HD O P f. <0 P O rH O CO rH JD P H rH HD HD P cd P d P CO d rl CO dPP 'H P d W rH P o H aP * *iH 3 o J25 s HD O O o Q if to1 o a <> H 5 IT**) B'S oO 5-$ 50 Ea a to CO p SCOo p {> CO CO p 8 HD HD >> CO O co st v> rH 1--4 p > p CM d d> d H H P r-l > rH 50 50 50 50 50 r. 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' v-,-,-r- ........V, .................................'V': ' v ^x'""""> DUP050055671 E-12 O 00 P >> OJ b C0 CU * cd C4 CU CO cu d 00 PS u CU cd <u o M Mi PS d cd <u Mi cu o r-->>1 H CM 44 50 d 2a J 44 p 0 >^ o a rl Ml P cu CU 0 TJ rl CO cd 44 CU p 44 4J >ri cd P d S> >4 M-i CO 4* P Cd d cu CP H SP 0) o 4c3o rl P P CO 4 A Ml s rl M > cu 44 !* > CU r-l P cu <U 5 <U 43 44 44 rrl cd CU 4c4u o cd 43 SHP I 8 d <u P CO cu 440340 CO P d 43 P c<ou | <0 d 44 O 43 44 p rl CU ? 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Type o f a n im a ls DUP050055672 polychrom atophi1ia , re tic u iu--4 CaO 0 (4 it--c0oOi *Q4> 0 CoO H-4 0 tTr0o>4 OCM TO0O Jc3o ir----CO44 n 400HJ CUHO 4o0J r4 n 0 uCO rH CO TCOO O' 000 1 o oC0HO b0, st* tr<--ju4 t--4 r-4 r4--M00J)4 4--CM<JO0i i u o -B00 a 0 t--4 VN CO TCPOOX oo> l <fr -ri----0OH)44 fc4 TCC:OOOx OON 1 O tT--0O4 r HH rt CO TCPOOX rCtOf* 440V<-J4u4 TO 0 tQ-4 P CO oC0oHO N<0 rO-4 c0O0 Xi u 4r1C--JO44 H4) 400-4 M>0c00b *> ti----44 <0 CO i-o(00r404 LB0 Mo 4COS00O M r-04 t-4 CrB-O4 JX Tr0O-4 r* 4o-1 J0Ca00O>) trt----o-444 <<0p0 S cino 0H J4H0J TJ 0rl TO 0O0 4-i 0 JO r-4 rH <3 P HM SS3 a) r0oP u i--04 * CO T0^ON * 1r0H--44 -f<Co000-0Ul4L T0OO TOH0O PM CO 0 r-4 o 0 *r4 r4 4J s0 TO U 0 4-J PX 0 TO X00 00 00 M 00 M CM X V- V 00 00 00 B B B 00 CoO o o CM o CoM I**- Px T0O 00 sCM-/ N T0J o 00 Sx >T0-Os om 60 00 o CM 6 mrH iH Om i--4 o&4 O04 O04 O0| O 04 00 t> r4 6* i-4 0 r4 U 0 U CO t-4 00 B TO iH 0 0 CO 00 *W O > 0 TJ CL D Jx 4-J H CO M-4 1 0 & 2R1 0 40 0 0 <0 0 TO 4J 4J 0 rH 0 0 OX0 U 40 0 0 0 H0 0 o 0 0 0 2B U 0 TO rQ TO TO 0 000 3- 3 3 3 0 4J 0 0 O JO M 0 P TJ 0 co -s Jx CO Oo p0 u JX p m 0 Hr4 r-4 TJ H 0 r4 co fx ca 0 0 TO 0 i 0 o0 0 rJP* O O rlf* TO uLi L< CO U u CO 6JO tP w v-/ xi s-/ u u Uu 09 si* NO cb co <CTM) (30 days) DUP050055673 1 00 H XJ 0 0 44 0 0 0 0) 0 *rl H iH 4J w >> O 00 43 a X X o0 XJ 0 44 44 0 o O 0 0g 000 0 rf rH 00 W X! a0 w 0 JO 0 u iH VO tW XJ 00 o 0 J0 M 0 4J 0 44 w H O 0 OO O0 0 H H 44 CO 0 44 A r^S rrf O 0 oB B 0 W (0 0 W --1 0 00 44 iH 0 >> 0 0 43 43 0 H MH w 0 44 w AW 00 44 U4 XJ 00 0X 0 *0H *W 0 *H XJ B 0 H 44 XJ r4 0 00 a0 .0 0 X? 0 0 XJ *H 0 44 0 0 X> X> iH 0 0X 0 *r4 0 60 0 rH 0 a0 u XI w w 0 GS iH 0 44 > 0 ad 00 a H >4 0 0XJ H m *% XJ 0 60 0B w 44 #1 00 |H O iH wB 0 XJ 0 W 0 *H 0 U 0 0 >V 0 *r4 0 W 0 60 XJ XJ O 0 a ao 0 a XJ oo O *r4 r4 j p 0 iH M o 0 43 00 40 44 a 0 0 0 O 44 0 XJ ** 0 0 43 0 0 0 M a CM H > r-4 W 00 a B AW 0 0O 0 I 4-1 0 oM **' a 0o O rl r4 0 V *W B A 1 M S2W 44 0 >> W 0 w 0 W 44 H . .a 0u K a> *r4 o 44 0 XJ 44 V00 XJ 0 60 0 00 0 r-4 *trl W 0H sB 0 44 >> a 3 0 XJ O $N d 0 > H 60 o 0 0 C H U 3B ir--P1 O W 44 0 f-i &0 H 0 o *W 3 0 0 TO XJ E-14 >0> xj oCO 0a) p3 a f oW VM co 440a3J B IS okD1o 00 M B AoW o CM <w i 43 0 (0 a w o 0 H 44 w 44 30 2o U oi 0 44 n to 1% h 00 a> 3 m 0 > 0 XJ a3 > 44 Eh 0 mJ 0 oa| o J0uuO xcjd $ MTO u0 H 0 d 0 w 00 43 V4 U 0o0 <u T0 Houri 4o3 x0> ,3 o H d 00 0t w vO 43 \4 P 0 < VD to DUP050055674 a Q 43 M pP pa TP3 ba)o do p p to M4 *D 0 S pP p tWo to *t) H mh o X P d 43 P U o H 43 PPH u 60 4b rP>H (30 J s 4OH a<u ob <3 H w 00 4J o p /-> 60 o CO 60 1--1 Q aO *3 O X3 60 p o rH O a p rH vO 43 J* CO 60 CM i 0O VPx <* 6 in CD i> CO rH 1 m . 4o4 oCH 00 o i 4b co d rl o d *rl rH 44 s CO X) P CO 44 Cco4 Poh aP pP > CO rl 43 3. CO rH o CO 43 rH A. M CD X 44 CO s ha3) <0 P a x> rl p ! c CO /-N CO 3 c* CO 43 4J VM y O rl >d P XJ A. 3 u0 4J 43 d 0 a sd rH H CO O 4H J<D & o VO O d| VO dB H co 1 44 XJ 60 d 44 <0 d P H 0P p rl rH P i XI Pi PP rl rH p XJ CO p rH P CO p rH 3 p <g P3p d 44 rH 60 H I p> 43 p aP rH ao d 0 M 43 O -c3or E-15 P II w H $* 4J 3 P 4b 3 60 H 4ao* P A. a d rH P CO CM P 3 co P d 60 d P rl 43 3 CO O rH *r-) 4^ t-H P 0a), o PM x) <o 3 to 3 O d <u O. >o CO P P CL. P d 44 P a) co P P 4o3 CO P 3o Opo Pp tdo CO H u P CO 3 rH P rH S S CO tu 43 o CO 43 P X> p CO d *d po d rH 43 po Opu Pi-t Pcu P p wp o Q 0 to a CCOO A. 4b <0 CO o P y P P V rH P .5 4^ co P CO bco IH ,, w P CO d p CO w CO II J P o 43 +J O 4U3 rH 4? 4b 4*J P rH P y <2 ft A <5 3 <q 3 C 3 < y X3 P MH 60 43 Route o f DUP050055675 APPENDIX F TREATMENT AND COSTS OF LEAD POISONING IN MAN TREATMENT OF LEAD POISONING The management of asymptomatic increased lead absorption (arbitrarily defined as a blood lead concentration greater than 50 pg/100 g of whole blood in the presence of a normal hematocrit) and symptomatic acute lead poisoning in man consists of two interdependent components: separation from noneccupational sources of exposure to lead and selective use of chelating agents as an im portant adjunct for the rapid safe reduction of high lead content in the soft tissues. Control of environmental exposure is the sine qua non of any treat ment program. The adjunctive use of chelating agents should be instituted at the asymptomatic stage of illness. The need for this approach is supported by the evidence that single episodes of acute encephalopathy and recurrent episodes of less severe acute lead poisoning may be associated with irreversible injury to the nervous system and kidneys. *113,173>17**>360 >411,456 , 465 Chelating agents have been used almost exclusively for the treatment of acute lead in toxication; brief courses of chelation therapy have not been shown to mobilize the entire body lead burden, as estimated by the CaEDTA mobilization test In the case of occupational exposures, separation from the source is readily achieved by a variety of means known to industrial physicians and plant sanitarians. Selander and Cramr^^ have presented evidence that rotation of workers may, in some conditions, minimize exposure. In cases of lead poisoning associated with the burning of battery casings, Improperly lead-glazed culinary ware, and other unusual sources of lead, the patient can be quickly and permanently F-l DUP050055676 separated from the source once it is identified. If undue exposure can be quickly controlled, asymptomatic patients with blood lead concentrations less than about TO or 80 Mg/100 g of whole blood may be treated on an ambulatory basis, and patients with higher blood . lead content, with or without symptoms, may require only brief periods of hospitalization for parenteral injection of chelating agents and supportive care. However, when the environmental exposure and other social and behavioral factors cannot be controlled quickly , prolonged hospitalization is necessary if the patient is to be separated effectively from such environmental exposures as leaded paint in deteriorating housing or lead-contaminated whiskey. The medical literature contains one report of the treatment of children with pica for lead paint on an 613 ambulatory basis, including some cases of encephalopathy; the authors deemed the ambulatory-treatment program a success in the sense that there were no fatalities, but the adequacy of the environmental control measures cannot be judged from this report and no evaluation of residual #effects is given. The treatment of children with blood lead concentrations greater than 80 Mg/100 g of whole blood on an ambulatory basis may involve considerable risk to the patients in view of the very high levels of absorption associated with this type of exposure (Fig, 4-2 and Table 4-6). There is considerable clinical experience with three chelating agents that are effective in lead poisoning: calcium disodium ethylenediamine^ tetraacetic acid (CaEDTA), 2,3-dime rcaptopropanol (BAL), and ^-penicillamine. Diethylenetri aminepentaaceti c acid (DTPA) and dimercaptosuccini e acid (DMS) have received brief clinical investigative trials only. CaEDTA and BAL are effective by injection only. 111 Both parenteral and oral F-2 DUP050055677 preparations of d-penicillamine are available in Europe. Few comparative studies have been reported, but one report indicated that depended 11amine is almost as effective as CaEDTA when given parenterally, but less effective 504 when given orally. In the United States, parenteral preparations of ^-penicillamine are not available, and the single oral preparation (250-mg capsules) for adults is currently classified as an investigational drug by the Food and Drug Administration (FDA) when used for the treatment of lead poisoning, (its use for the treatment of Wilson's disease and cystinuria is not restricted.) Chisolm^11 has proposed that the choice in dosage of chelating agents should be based on the physician's estimate of the mobile fraction of the body lead burden in patients with current and recent increased lead absorption. Thus, the highest doses consistent with safety would be indicated in the patients estimated to have the highest lead content in the soft tissues. Limited clinical experience indicates that the simul taneous administration of BAL and CaEDTA is more effective in terms of survival, reversal of metabolic toxicity, and rate of reduction of soft tissue lead content than is CaEDTA alone in patients with acute symptomatic lead poisoning and in patients with blood lead concentrations greater than 80 ug/100 g of whole blood without symptoms but with known gross exposure. Although they are more efficacious in certain situations, the intramuscular use of CaEDTA and the higher doses of BAL used in this regimen are not cur rently approved by the FDA. In patients with lower lead content in blood, HI 202 504 either parenteral CaEDTA or oral dependcillamine provides adequate therapy. * * The rationale for this approach, critical aspects of supportive therapy. F-3 DUP050055678 and adverse drug reactions are reviewed elsewhere *Ill * 202 Limited clinical data suggest that the use of d-penicil1amine in patients with renal insufficiency must be approached with great caution; further experience may prove that it is contraindicated in such patients. The incidence of adverse drug reactions cannot be estimated from the available clinical data, although experimental studies indicate that adverse reactions are dose-related. x It has been reported that the excretion of heme precursors in urine as quantitatively measured in 24-hr samples provides the best index of the quantity of lead that will be excreted under the influence of chelating agents and a better index of the "chelatable lead11 in subjects with current and recent abnormal exposure to lead than blood and urine lead content. This is particularly useful in asymptomatic subjects with blood lead content of 50-80 ug/lOD g of whole blood and acute or intermittent abnormal exposure to lead. In such patients, there may be considerable variation in the adverse metabolic responses to lead and the need for chelation therapy. Alternatively, the response to chelating agents can be measured directly by determining urinary lead output during therapy. Therapy should be terminated when the diuresis of lead diminishes toward normal. However, only oral d-penicillamine can be given practically and safely on a continuous basis for more than a few days.' the above statements are based entirely on the limited clinical reports of a few authors, it should be noted that chelation therapy without careful clinical supervision may be dangerous. Facilities for F-J* y DUP050055679 determination of lead in urine or blood and for repeated evaluation of a patient are essential for the proper use of chelating agents. The lack of wider clinical experience and a more systematic approach to chelation therapy can be traced largely to the general unavailability of the necessary laboratory techniques in the general community (especially blood and urine lead analyses). Even accurate analyses are of little clinical use in the day-to-day management of patients if the results are not immediately available. At present, the use of chelating agents varies widely from clinic to clinic and is based largely on clinical evaluation and whatever laboratory techniques happen to be available in a particular clinic. In patients with asymptomatic increased lead absorption, chelation therapy promptly suppresses the adverse metabolic effects of lead on heme synthesis. Because the subclinical effects of lead on the nervous system are not well defined, no evaluation can be made in this regard. Limited data with respect to kidney function indicate that the Fanconi syndrome and the altered renin-aldosterone response to sodium deprivation are reversible and respond quickly to chelation therapy In patients with chronic nephropathy, CaEDTA has been used largely for diagnostic purposes only .^3*17^ >*H1 ,^56 The scarring and other permanent tissue damage reported in such patients make it unlikely that they would be responsive to chelation therapy . The spectrum of renal injury suggests, however, that subclinical functional renal injury may be reversible at some point before the late and a 377 F-5 DUP050055680 apparently irreversible form in which it is now recognized clinically. So evaluation of the efficacy of chelation therapy in this type of patient has been found in the literature, With respect to encephalopathy , it is clear that at least 25% of childhood survivors sustain permanent and often profound brain damage regardless of the form of therapy used, The efficacy of chelation therapy as it may relate to minimal cerebral injury is unknown. In summary, the available but limited data suggest that chelation therapy should be instituted in patients with increased absorption and storage of lead. Soft tissue concentrations of lead definitely should be maintained well below those associated with clear-cut clinical symptoms and functional injury. Chelation therapy is effective for this purpose, provided that subsequent exposure is controlled. There are insufficient data to judge the extent to which an excessive body burden of lead, once accumulated, can be safely and effectively mobilized. Control of hazardous exposure offers the most effective means of preventing the accumulation of an increased body lead burden. Chelation therapy is of limited efficacy in persons with acute encephalopathy, in terms of the occurrence of central nervous system sequelae in survivors, despite therapy. DIRECT MEDICAL COSTS OF LEAD POISONING No comprehensive estimate of total medical and related expenses attributable to lead poisoning can be made at this time because of the many medical and environmental factors involved. In general, the type of exposure, the age of the patient, and the severity, recurrence, and sequelae of F-6 * y, A i- v DUP050055681 the illness are the important variables that influence total direct medical costs in cases of human lead poisoning. In cases associated with circum scribed types of exposure (e.g., occupational exposures) or such sources as unsafe earthenware culinary items, direct medical costs may be limited to the costs of brief hospitalization and professional care. Provided that treatment is instituted before the occurrence of irreversible tissue injury and that hazardous exposure is promptly terminated, no further direct medical expenses would be anticipated. In cases associated with late diagnosis, "high-dose" types of exposure, and the initiation of medical treatment after the occurrence of irreversible tissue injury, even a single episode of acute encephalopathy without re-expo sure will entail long-teim medical expense for the sequelae of severe acute lead poisoning. In cases associated with uncontrolled types of environmental exposure, the likelihood of recurrence of acute illness, recurrent hospitalization, and expenses related to permanent injury is increased. Recurrent exposure and illness axe major considerations in children with pica, in unsupervised small-shop occupational situations, and in the use of "moonshine" whiskey. In young children, separation from exposure is the essential component of therapy; therefore, hospitalization is frequently indicated. Out patient treatment with daily injections of chelating agents in children with persistent pica and continued intense environmental exposure is neither effective nor humane. Out-patient treatment may reduce public medical expense, but it increases costs to the parents of the affected children in the form of transportation costs, loss of time from Work, and neglect of other members of the family. The epidemiology of child hood lead poisoning is such that these costs fall most heavily and directly F-7 DUP050055682 on idle poor , who are least able to pay . Treatment costs may be divided into six general categories: l) direct medical costs for acute and con valescent care, 2) after-care and excess school costs for the partially brain-damaged, 3) custodial care for the permanently and severely injured, k) correction of hazards in housing, 5) preventive health supervision and 6) supporting municipal and state health department activities. No broad generalizations concerning the economic impact of childhood lead poisoning can be made at this time. Diagnostic and treatment facilities vary widely among communities, few of which have programs which can be considered either comprehensive or wholly adequate. However, limited experience in one community with regard to direct medical expenses in a group of ^5 children may be illuminating. These children were treated under the general medical policy that no child, when found to have increased lead absorption, with or without symptoms, is returned to a "leaded" home. Under this medical policy, the child is first treated in a general hospital for a brief period of time and then placed in a convalescent facility until a safe dwelling* is found for the family. (At present, Baltimore, Maryland is the only U. S. city known to have such an "extended pediatric care" or convalescent facility for young children.) Table F-l lists actual direct medical costs in field operating conditions incurred in 1965-1970 by a group of k5 children with increased lead ab sorption. The group of included 10 survivors of acute encephalopathy. The average total time of acute and convalescent hospitalization was 100 * "Safe dwelling" in this sense is defined as modem public housing or adequately repaired old housing. p-8 DUP050055683 days, and the average direct hospital cost for 3** patients was $2,71*9. Almost all these children were hospitalized at public expense in hospitals that at the time of hospitalization had a blanket basic charge and did not charge for such extras as special nursing and intensive care. Not represented in Table P-1 are the two highest hospital bills! which were over $8,000 each. In one instance, the high cost resulted from repeated hospitalization for complications of encephalopathy, and in the other, from an excessively long wait for admission to public housing. This experience is not unique, and the cost would be greater in other cities where children are retained in general hospitals for periods of 1-3 months while informal quests for safe housing go on. In view of the 1 rapid rise in hospital costs during the last 5 years, it can be estimated that direct hospital costs for the ^5 children shown in Table F-l would average $5,1*98 in 1970 (Table F-2). Chelation therapy with BAL and CaEDTA followed by oral administration of d-penicillamine requires an average of only 10 days of hospitalization. Asymptomatic children do not require treatment in a general hospital; they can be handled in a convalescent facility at a lower cost. Under these circumstances, hospital costs beyond 10 days are attributable directly to delays in the completion of necessary housing repairs. Baltimore allows up to 6 weeks for comple tion of housing repairs before legal action is taken. New York and Philadelphia now limit the time allowed for compliance, repair an apartment at municipal expense , and take out a lien on the property to recover the costs. Sustained medical followup for children with pica who live in substandard old housing is indicated for preventive purposes throughout the years of pica or for an average period of 3 years. The current costs for this are F-9 DUP050055684 estimated at $120, on the basis of a fee of $15 per out-patient clinic visit. For those with lead poisoning, the follow-up period ranges from 2 to 10 years, or $225-675 per patient. The salary of a medical social worker or comparable paramedical personnel at one worker per 50 cases should be added to this figure. For children with moderately severe permanent brain damage who require special schooling* excess school costs related to transportation costs and smaller classes are currently estimated at $1200 per pupil per year, or $l4,40Q for 12 . years of school through high school per damaged child. For children who require institutionalization for custodial care, current costs at the Rosewood State Hospital in Maryland are approximately $4000 per year per patient . Data for other institutions in Maryland where such patients have been hospitalized in the past are not available, and comparable data from other parts of the country are not immediately available to the Panel. During the last 13 years, 19 children have been admitted to the Rosewood State Hospital as a result of lead encephalopathy during early childhood (J. J. Chisolm, Jr., personal communication); one has died and two have recently been given leave of absence, leaving l6 children in residence at a current cost of $64,000 per annum. These patients have been hospitalized for a total of l48 patient-years, or 7*7 years per patient. None of the l6 remaining in residence is considered suitable for discharge. If they survive to the age of 65, the expenditure for 60 years of institutional care at $4,000 per annum would be about $240,000 per severely damaged child. The total direct medical cost can therefore be roughly estimated according to the final clinical outcome as follows: (l) asymptomatic increased lead absorption without obvious residual permanent injury, $1500-2000 rJ ^ * A A F-10 DUP050055685 per patient ; (2) moderate permanent brain damage (special schooling re quired) , $18,0Q0 per patient; and (3) severe permanent brain damage (institutional care required), $2^5,000 per patient at current medical costs, These direct treatment costs may be contrasted with the cost of the repairs to substandard housing to eliminate the paint hazard. In Baltimore, estimated costs range from $150 to $1200 per apartment, depending on the extent of repairs needed. New York City estimates costs at $1263 per apartment Because New York City repairs many apartments itself through the municipal emergency repair program, actual figures may be available soon; the program was instituted in April 197O. Adequate housing repair is therefore comparable in dollars with the direct medical costs of treatment of a single asymptomatic child with increased body lead burden. Also, repairs to substandard housing can prevent lead poisoning in all the children who may live in a given house during the remainder of the house's useful existence. If houses were inspected and repaired before the onset of pica, direct medical costs could be totally eliminated. Preventive health care costs for children cannot be precisely estimated. With the exception of that in New York City, no comprehensive U* S. program is in existence. New York City estimates such costs at $12.50 per child for initial case finding and $6.51 per child for each follow-up visit, including blood lead tests. Current research is aimed at reducing the cost of screening both children.and housing through improved tech niques with a potential for automation and portability for field testing. F-ll DUP050055686 TABLE F-l Total Days of Hospitalization and Estimated Direct Medical Costs for 45 Children with Initial Blood Lead Content Greater than 80 Mg/100 g of Whole Blood (Including 10 Children with Acute Encephalopathy) who are not Discharged to Home until Removal of Old Lead-Pigment Paints from Home Environment - Baltimore , Maryland% 1965-1970 Duration of hospitalization days Acute care Convalescent care (general hospital) (Happy Hills Hospital) Total Total 1108 3402 1+510 Mean per patient Median per 24.7 75.7 100.lt patient 21 50 71 Range (80# of patients) 4 - 70 20 - 203 25 - 253 Total Mean per patient Median per patient Range (90$ of patients) Actual costs for 34 patients, $a 93,377 2,746 2,175 1,068 - 4,8l4 Basie hospital rates roughly doubled between 1965 and 1970* Most of the 34 children for whom complete hospital charges could be verified were hospitalized during 1965, 1966, and 196? at public expense. F-12 DUP050055687 TABLE F-2 Estimated Current Average Direct Medical Costs to 1+5 Children, Based on 1970 Basic Hospital Hates in Baltimore , Maryland v Unit Total (1+5 patients ) Mean per patient Estimated costs, $ Acute care Convalescent care 100/day 40/day 110,800 136,080 (1108 days) (3402 days) 2462 3024 (24.6 days) (75.6 days) Total 246,88O (1+510 days) 5,486 (100.2 days Note : Children were handled under the general, policy that no child is discharged until either home is "deleaded" or family moves into safe modern housing; length of hospitalization is detexmined by this factor, rather than by severity of acute illness. P-13 DUP050055688 APPENDIX G TREATMENT AND COSTS OF LEAD POISONING IN CATTLE Lead poisoning of domestic animals is generally considered to be a disease of low morbidity and high mortality. . In general, it is felt by clinicians that, if an animals lives, the prognosis for recovery is good. For example, many calves are blind during acute manifesta tions of the disease. If a calf lives, sight usually returns within a week, at least enough for the animal to move about with no difficulty, The chelating agent calcium disodium ethylenediaminetetraacetate (CaEDTA), used as a successful treatment for lead poisoning in man, has also been used successfully as an antidote for lead poisoning in ^ 2 260,35^,355,556 cattle. The dosage of administration of CaEDTA to lead-poisoned cattle in the reports cited was based largely on toxicity and metabolic studies in 1 D rr laboratory animals. To establish a more definitive basis for the use of CaEDTA in the treatment of lead poisoning, a study was made in which CaEDTA was administered at various dosages to calves previously given lead. 21 Previous studies had established that the amount of lead mobilized by a single rapid intravenous injection into calves of 110 mg of CaEDTA per kilogram of body weight was proportional to the concentration of lead in their erythrocytes at the time of treatmentThis dosage probably produced the maximal amount of lead that could be mobilized G-l DUP050055689 in calves by a single rapid intravenous injection; a range of doses of II-I65 mg/kg did not produce significantly greater urinary excretion. This relation served as the standard base of reference for evaluating other dosages of CaJEDTA administration. The study indicated that optimal conditions of lead mobilization were provided by concentrations of approximately 135 um of CaEDTA per liter of plasma and higher, maintained for 10-12 hr. 21 These concentrations could be achieved by the constant intravenous infusion of CaEDTA at 110-220 mg/kg over 12 hr or approximated by two rapid intravenous injections of 6 mg/kg 6 hr apart, More than twice as much lead could be mobilized by these pro cedures as with a comparable dose given as a single rapid intravenous injection. The administration of CaEDTA as a 12-hr infusion for 3 consecutive days proved to be no more effective in mobilizing lead than a single 12-hr infusion. Because these experiments were carried out with calves that did not have clinical signs of lead poisoning, it might be argued that the conclusions would not necessarily apply to clinical cases of bovine lead poisoning. It cannot be stated unequivocally that higher concen trations of lead in the body would not require higher concentrations of CaEDTA for maximal lead mobilization. However, the concentrations of lead measured in liver (2,3-3*2 ug/g wet wt.), kidney cortex (2.8-15.0 yg/g wet wt.), and blood (0.27-0.58 ug/ml) of untreated animals given lead were about half the concentrations commonly encountered in clinical cases of lead poisoning. pl*2 Furthermore, there A -G-2 DUP050055690 was no significant difference in lead mobilisation when CaEDTA was in fused for 12 hr with a fourfold variation in the rate of administration (110-440 mg/kg). # This suggests a ready capacity to cope with the greater amounts of lead that would be involved in actual poisoning. With regard to the lack of additional benefit. attained by repeating 12-hr infusions beyond the first infusion, a clinical situation might yield different results, because there might be a much larger reservoir of unabsorbed lead in the gastrointestinal tract, which would provide a greater continuing absorption of metal by the circulation. However, multiple daily infusions at 220 mg/kg over 12 hr would be hazardous because of the toxicity of CaEDTA. It would therefore seem that, even in the presence of tissue concentrations of lead greater than those produced experimentally, 12-hr infusions of CaEDTA should not exceed 220 mg/kg and probably should not be instituted more than once every other day* Probably no more than three treatment^ should be given. A rational basis for an intermittent schedule of therapy also is provided by evidence that CaEDTA acts to remove some lead from . bone and that additional time is required for redistribution of lead 240 from soft tissues to the CaEDTA-sensitive sites in bone, A very crude estimate of what the economic cost of lead poisoning might be in cattle affected with clinical lead poisoning in the United States is based on an extrapolation of the estimated incidence of lead poisoning in Tompkins County, New York, an area served by the Ambulatory Clinic of the New York State Veterinary College. G-3 DUP050055691 Wo estimate is being made for the cost of lead poisoning 4n other species of animals or for the cost of a past-ore or crops contaminated by lead fallout from a smelting or mining operation. This probably conservative estimate indicates the difficulty of trying to assess the cost of a disease in domestic animals for which no reporting system exists. A survey of a knackery^^ (an establishment that receives and processes animal carcasses) in Northern Ireland estimated that lead poisoning accounted for 1.7# of the deaths of adult cattle and 4.5# of the deaths of young calves. This incidence was considerably greater than the number of clinical diagnoses and suggested that cases of lead poisoning were not being diagnosed, for a variety of reasons. The cost estimate of lead poisoning in cattle is based on the following facts, estimates, and assumptions. The total cattle population of the United States in 1969 was 123,784,000 head.^^ In Tompkins County, > , 565 New York, the cattle population was 24,410. From this county, the Ambulatory Clinic of the N.Y.S. Veterinary College treats approximately four cases of lead poisoning per year, or approximately one case of lead poisoning per 6000 cattle per year. If this rate holds through- out the United States, it is estimated that 20,000 cases of lead poisoning occur in the United States per year. At an assumed cost of treatment per animal (based on two calls plus drugs) of $25, an assumed mortality of 50# (probably higher), and an assumed average value of an animal of $150, the cost of treatment is $500,000 and the loss is $1,500,000^-or a total cost per year of $2,000,000 for lead poisoning in cattle. G-4 JL DUP050055692 REFERENCES The following 6 references have been deleted from the text and should be deleted from the list of references: 69, 282 , 292 , 293, .31U, k02. Seven references (nos. 609-615) were added to the text too late to he included in the alphabetized list. 1. Abernethy, R. G., M. J. Peterson, and F . H, Gibson. Spectrochemical Analyses of Coal Ash for Trace Elements. Bureau of Mines Report R 1^-728l, Washington, D. C.: U. S. Department of Interior, 1969. 30 pp. 2. Acocella, G. 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Durch chronische Bleivergiftung erzeugte Nierenadenome und -carcinome bei Ratten und ihre Beziehungen zu den entsprechenden Neubildungen des Menschen. Virchows Arch. (Path. Anat.) 323:69^-7101953* 607. Zook, B. C., J. L, Carpenter, an d E. B, Leeds. Lead poisoning in dogs. J. Amer. Vet. Med. Assoc. 155 1329-1342, 1969. 608. Zook, B. C., R. M. Sauer, and F. M. Garner. Lead poisoning in Australian fruit bats (Pteropus poliocephalus). J. Amer. Vet. Med. Assoc, 157:691-69^, 1970. % * i DUP050055772 SUPPLEMOTTAL REFERENCES Baumhardt, G. R, Absorption by and Growth of Corn (Zea Mays L.) with Soil Applied Lead. Master's thesis, University of Illinois*, 1970, 51 pp. Chow, T. J. Lead accumulation in roadside soil and grass. Nature 225:295-296, 1970, Foreman, H. Toxic side effects of ethylenediaminetetraaeetic acid, J. Chronic Dis. 16:319-323, 1963. Nehls, G. J., D. H. Fair, and J. B. Clements. National air databank open for business. Environ. Sei, Tech, k:902-905, 1970, Sachs, H. K., L, A. Blanksma, E, F. Murray, and J. J. O'Connell. Ambulatory treatment of lead poisoning: Report of 1,155 cases. Pediatrics k6:389- 396, 1970 Shy, C. M., D. 1* Hammer, V. A. Newill, and W. C. Nelson. Health Hazards of Environmental Lead* In House Technical Report, Community Research Branch, Bureau of Air Pollution Sciences*, Environmental Protection Agency, 1971* (to be published) Tepper, L, B, Seven-City Study of Air and Population Lead Levels. An Interim Report. Department of Environmental Health i College of Medicine, University of Cincinnati, 1971* 11 PP* DUP050055773 GLOSSARY To aid readers unfamiliar with terms outside their field of expertise, definitions are provided to promote comprehension without the irritation of going elsewhere to 11 look it up." The glossary consists of terms not explained in the text, and they are defined in the sense in which they are used. Specialists and panel members in the several disciplines have been consulted, in addition to the usual dictionary sources. AAS: Atomic absorption spectrophotometry. Abdominal colic: See Colic. Absorption (of lead) : Transfer of lead into the organism via intestinal wall, alveolar surface, or skin, with a demonstrable effect. Accumulation (of lead): The net positive difference between intake and output of lead over an extended period. Acid-fast: Describes a cell or bacterium that retains a dye that has a negatively charged molecule. ACTH: Adrenocorticotropic hormone , a hormone secreted by the pituitary gland that stimulates the adrenal cortex. Acuity: Sharpness of perception with respect to ability to resolve detail. Aerosol: A system in which the dispersion medium is a gas , and the dispersed phase is not large enough to settle out under the influence of gravity. Aerosol particles: Solid particles from 0.1 pm to 1 x 10~12 pm in diameter dispersed in a gas. -1- DUP050055774 0 ALA: 6 -aminolevulinic acid, C00H^CH2-QH2^H--CH2--H2, formed from succinyl coenzyme A and glycine (see Fig. 4-3). ALAJD: 6-aminolevulinic acid dehydrase {syn. 6-aminolevulinic acid dehydratase, 6-aminolevulinic acid dehydronase): The enzyme found in cells that catalyzes formation of porphobilinogen from <5-aminolevulinic acid (See Jig. 4-3). ALAS: See 6-aminolevulinic acid synthetase. Albuminuria: Presence in urine of albumin, a protein that is a normal constituent of blood. Ambient air: The surrounding, mixed air; ''ambient community air" refers to air in residential urban areas; "ambient urban air" refers to air in downtown areas with relatively heavy traffic. 6-aminolevulinic acid synthetase: The enzyme that converts succinyl coenzyme A and glycine to ALA (See Fig. 4-3). Amyotropic lateral sclerosis: A disease characterized by a hardening of the lateral columns of the spinal cord with muscular atrophy. Anorexia: Loss of appetite. Anoxia: Relative lack of oxygen; may be due to lack of blood carrying normal amounts of oxygen or to normal perfusion of blood carrying reduced amounts of oxygen. Anterior pituitary (syn. anterior hypophysis): Part of the gland of internal secretion at the base of the brain, producing hormones that act on adrenal cortex, thyroid gland, gonads, and skelton. 4 Glossary 2 DUP050055775 ASV: Anodic stripping voltammetry: An electrochemical method of analysis. Ataxia: Failure of muscular coordination* Atomization: Reduction to fine particles, usually in a spray, BAL: 2,3-dimercapto-l-propanol (British Anti-lewisite). Balance experiments; Experiments on man or other animals that involve quantitative measurements of intake (via respiration and ingestion) and loss (via exhalation and excretion) of a specific element or substance. A positive balance means that more is taken in than is lost over a specified time. Basophilic stippling: The characteristic appearance of some erythrocytes in the blood that in certain conditions contain cytoplasmic material that stains deeply with basic dyes. Bender-Gestalt test: A performance test requiring reproduction of the configuration in line drawings, * BIOSPHERE: The part of the earthfs crust, waters, and atmosphere where living organisms can subsist. Biota: The animal and plant life, collectively. Biotransfer: The process by which living organisms , such as bacteria, can convert a chemical compound into another. Blood^brain barrier: The barrier created by semipermeable cell walls and membranes to passage of some molecules from the blood to the cells of the central nervous system. Glossary 3 DUP050055776 Body Burden: The total amount of a specific substance (for example* lead) in an organism, including the amount stored and the amount absorbed. CaEDTA: Abbreviation for edathamil calcium disodium, which is the calcium disodium salt of ethylene diaminetetraacet ate, a chelating agent. CaEDTA is used in the study, diagnosis, and treatment of poisoning by various heavy metals, including lead. CaEDTA mobilization test: A test in which a known quantity of CaEDTA is injected parenterally and the amount of lead excreted in urine during a known period beginning immediately thereafter is measured. This procedure is used both clinically and experimentally> and is thought to provide an index of the mobile fraction of the total body lead burden. Cerebellum: A large dors ally projecting part of the brain having the special function of muscle coordination and maintenance of equilibrium* Cerebral anoxia: Relative lack of oxygen in the brain. Chronic nephritis: Chronic inflammation of the kidneys. Citraturia: The presence in the urine of citric acid. The term usually implies the presence of increased quantities of citric acid in the urine. Colic: A paroxysmal pain in the abdomen, due to spasm, distention, or obstruction of any one of the hollow viscera. Community exposure : Exposure of any community or group of persons to contaminant(s). * 4 Glossary k DUP050055777 Complexing: In chemistry, the process of incorporation into other com pounds, such as through hydration, oxygenation, halogenation9 and chelation. Contamination: Contact with an admixture of an unnatural agent , with the implication that the amount is measurable* o lx Conyers ion factors: For air, 1 yg/nr = 8.5 x 10~ ppm by weight; for water, 1 yg/liter " 1 ppb; for soil, food, forage, etc., 1 yg/g (dry, ashed, or wet weight) - 1 ppm or 1 yg/kg = 1 ppb. Coordination site: Chemical configuration of a mole exile where inter action between it and another molecule occurs. COPED (syn. CP.): Coproporphyrin, the oxidized form of coproporphyrinogen. It is a byproduct of intermediary heme biosynthesis found in tissue fluid and the excreta. COPROGEN III: Coproporphyrinogen III, an intermediary metabolite in heme biosynthesis . It is a natural precursor of heme . COPROGENASE: Copropo rphyrinogen as e , the enzyme that converts coproporphy rinogen III to protoporphyrin IX. Coproporphyrinogen (syn. coprogen): A fully reduced colorless tetracarboxylic tetrapyrrole. Isomers I and III are found in biologic systems. Corpus striatum: A subcortical mass of grey and white substance in each cerebral hemisphere , containing the caudate nucleus and the. lentiform nucleus * Glossary 5 DUP050055778 Cortical atrophy : Wasting away of the outer layer(s), e .g., of the brain or kidney. Deionized water: Water that has been specially distilled or treated to remove inorganic ions and salts. Organic substances may be removed by special processes or prior distillation. Demyelinatlon: Destruction of the myelin, a fatlike substance forming a sheath around certain nerve fibers.. Deposition: Refers to particles inhaled but not exhaled. Dithizone methods: Colorimetric methods of analysis for lead that involve the reaction of lead with diphenylthio carbazone to foim lead dithizonate, which is measured spectrophotametrically at 510 mu* dpm: Disintegrations per minute. Earthenware: Vessels or other utensils made of fired clay. ECP: Erythrocyte coproporphyrin. Edema: Abnormal accumulation of tissue fluid in the connective tissue or serous cavities. EDTA: Ethylenediaminetetraacetic acid, used in the form of calcium, disodium salt as a chelating agent to complex with lead and other metals and remove them from the body by urinary excretion. Effluent: The gaseous or liquid discharge of waste products, which may or may not contain environmental pollutants. I ^ Glossary 6 DUP050055779 Electrostatic precipitator: A device for removing small particles of smoke, dust, oil, mist, etc., from air by passing the air first through an electrically charged screen, which gives a charge to the particles, then between two charged plates, where the particles are attracted to one surface. Elutriator: A machine for separating heavy and light mineral particles by washing and straining or decanting. Emesis: Vomiting. Encephalitis: inflammation of the brain. Endogenous lead: head that has already entered the body. Endogenous urinary lead: The urinary excretion of lead that occurs normally in the absence of chelating agents. Epithelioid cells: Cells resembling epithelium. Erythrpid hypoplasia: Decreased formation of erythroid elements of the blood (i .e., red blood cells). Erythropoiesis: Formation of red blood cells. Evoke d-response technique: A technique widely used in electrophysiology whereby a stimulus (e.g., electric shock, light flash, click, etc.) is applied peripherally to the electrode used to detect the response. Exposure level: The concentration of the contaminant to which the population in question is exposed. Glossary 7 DUP050055780 Extensor muscles: The muscles under voluntary control that, when con tracted, extend the limbs. Fanconi syndrome: There are several Fanconi syndromes. As used in this document, refers to the triad of glycosuria, hyperaminoaciduria, and hypophosphatemia in the presence of hyperphosphaturia. This triad is associated with injury to proximal renal tubular cells. FEC: Free erythrocyte coproporphyrin. VFEP: Free erythrocyte protoporphyrin. Flicker-fusion: The fusion of intermittent flashes of light into a sensation of continuous brightness * Fructosuria; The presence in the urine of fructose, a monosaccharide formed from the breakdown of more complex sugars and normally converted ultimately (during metabolism) to C02 and H2O. Glycosuria: The presence in the urine of glucose, a monosaccharide formed from more complex sugars and normally retained in the body as a source of energy, Gouty diathesis: Predisposition to gout. G6PDH: Glueose-6-phosphate dehydrogenase, an enzyme important in the maintenance of adequate concentrations of reduced glutathione in red blood cells. Deficiency of this enzyme is inherited as a sex-linked t rait. ^ j \ Glossary 8 DUP050055781 GSH: Reduced glutathione. Hematopoietic system: The system of cells in the bone marrow, spleen, and lymph nodes concerned with formation of the cellular elements of the blood. Hemogram: A clinical term used to encompass several hematologic indices, including hematocrit, hemoglobin, and red blood cell count. Hepatic porphyria: An inborn error of metabolism characterized by increased formation and accumulation of pyrroles in the liver. Hepatocellular injury: Injury to the cells of the liver. Histopathology: Abnormal structure of plant and animal cells at the microscopic level. Hydrocephalus "ex vacuo": increased volume of cerebral spinal fluid within the cranial vault, associated with decreased volume of cortical tissue, as in severe cortical atrophy. Hyperaminoaciduria: Presence in the urine of above-normal amounts of amino acids. hyperkinetic: Abnormally Increased muscular movement. Hyperkinetic-aggressive behavior disorder: A disorder characterized by overactivity, restlessness, distractlbility, and short attention span. Hyperphosphaturia: Above normal amounts of phosphate compounds in the urine. Glossary 9 DUP050055782 Hyperuricemia: Abnoimal amounts of uric acid in the blood. Hypochromic anemia: A condition characterized by a disproportionate reduction of red cell hemoglobin, compared with the volume of packed red cells. Hypophosphatemia: Abnormally low amount of phosphate compounds in the blood. Hypothalamus : The posterior portion of the forebrain that includes the nuclei of nerve cells that exert control over visceral activities, water balance, temperature, sleep, etc. Illicitly distilled whiskey: Whiskey that is distilled and sold without payment of federal excise taxes. Interstitial fibrosis: A progressive formation of fibrous tissue in the interstices In any structure; in the lungs, it reduces aeration of the blood. Intranuclear inclusion bodies: Round, oval, or irregularly shaped bodies occurring in the nuclei of cells. Iron deficiency: A deficiency of iron-containing foods in the diet such that not enough iron is available for incorporation into newly formed hemoglobin. Knuckling: Involuntary flexing of the fetlock joint* Laparotomy: Surgical incision through the abdominal wall. & ^ v y v Glossary 10 DUP050055783 Latency of response: The time between the application of a stimulus and the beginning of the response to that stimulus. LDH: Lactic acid dehydrogenase* Leached: Subjected to the action of percolating water or other liquid that removes the soluble parts. Lead intoxication - see lead poisoning. Lead poisoning (syn. plumbism, saturnism): A disease condition reflecting the adverse effect of the absorption of lead into the system. Lead sesqui oxide (syn. lead trioxide): FbgO^ Lead subacetate (syn. lead monosub acetate, monobasic lead acetate): Pb(C2H3Q2)2-2Pb(0H)2. Ligand: A molecule, ion, or atom that is attached to the central atom of a coordination compound, a chelate, or other complex. Luting: A substance for packing a joint to make it impervious to gas or liquid. Medical surveillance: Kepeated medical examination, including physical examination and laboratory examination of blood and urine specimens, to discover any change from baseline ("normal") conditions. Meningitis: Any disease producing inflammation of the meninges, or membranes that envelop the brain and spinal cord. Glossary 11 DUP050055784 Metabolites: End products of metabolic processes that transfora one compound into another in living cells. Metalloporphyrins: A combination of a metal with porphyrin, e. g., iron and heme . Metaphyses (singular metaphysis): The wider part at the end of the shaft of a long bone, which during development contains the growth zone and consists of spongy bone. Microcytic anemia: A condition in which the majority of the red cells are smaller than normal. Microsome: One of the finer granular elements of protoplasm. Mitochondria: Small granules or rod-shaped structures seen by differential staining in the cytoplasm of cells. MMED (Syn. MMD): Mass median equivalent diameter. See Appendix A. Mobile fraction of the body burden; The fraction of the total lead content of the body that can be removed by chelating agents* Moonshine: See illicitly distilled whiskey. jybtpr skills: Skilled movements that depend on the integrity of the nervous system for control. Myelopathy: Pathology of the muscle fiber. Myocarditis: Inflammation of the heart muscle. FASN: National Air Surveillance Networks. tV i r *) Glossary 12 DUP050055785 Nerve conduction: Passage of a nerve impulse manifested by an electrical impulse that travels along the nerve . Nitrification: Oxidative process that converts ammonium salts to nitrites and nitrites to nitrates. Nondestructive detectors: Instruments that can measure a variable without sampling or otherwise causing destruction of the material in which the variable occurs. Normal blood lead: Range of 2-^0 yg/g of whole blood. Nutrient solution: A solution in which tissue cultures are grown containing the necessary ingredients to nurture growth. Obs tipation: Ext reme constipation. Oliguria; Deficiency in the formation and excretion of urine. Operant conditioning (syn. Instrumental conditioning): The experimental procedure of presenting an animal with a reinforcing stimulus immediately following the occurrence of a certain response. Organelle: A specific particle of organized living substance in most cells. Organic brain damage: Structural impairment or change in the brain. Paraplegia: Paralysis of both lower limbs due to spinal disease or injury. Parental sequences: Sequential behaviors associated with rearing of the young of a species. Glossary 13 DUP050055786 Parent material: The original rock from which the soil in question was made. * Paresis: Incomplete paralysis. PBG: Po rphobilinogen Pes cavus deformities: Exaggerated height of the longitudinal arch of the foot due to disturbed balance of the muscles. pH: A symbol denoting the negative logarithm of the hydrogen ion concen tration in gram-atoms per liter. Phosphorylation: introduction of the phosphoryl group into an organic compound. Pica: Ingestion of nonfood items. Pituitary-adrenal axis: The interrelation of the anterior pituitary and adrenal glands whereby the activity of one is stimulated or inhibited by a hormone of the other, e.g., regulation of ACTH secretion by the blood level of adrenal corticoids. Pituitary gonadotropic hormones: Hormones secreted in the anterior pituitary that control the gonads. Pituitary-thyroid axis: The interrelated activities of the anterior pituitary and thyroid gland. Plutybism - See lead poisoning. Pollution: Admixture of an agent with the implication that the effect is measurable. Glossary l4 i- DUP050055787 Porphyria: A disturbance of porphyrin metabolism characterized by marked increase in formation and excretion of porphyrins or their precursors.. Porphyrins : Any one of a group of iron- or magnesium-free pyrrole derivatives that occur universally in protoplasm and form the basis of the respiratory pigments of animals and plants. Postictaliy: Following a stroke or seizure, such as an acute epileptic attack. Preganglionic transmission block: Blocking of nerve impulse transmission at the synapse before the nerve enters the ganglion. Premature dementia (syn. dementia praecox): a large group of psychoses of psychogenic origin, often recognized shortly after adolescence. Primary gout: A condition characterized by abnoimal purine metabolism producing an excess of uric acid in the blood, chalky deposits (chiefly urates) in the joints, and attacks of acute arthritis. Primary producers: Green plants that are able to fix carbon dioxide and give out oxygen. Prodromal manifestations: Premonitory signs, indicating the approach of a disease or other morbid state. PROTO: See protoporphyrin PROTO 9: Protoporphyrin 9, an isomer of protoporphyrin. Glossary 15 DUP050055788 Protoporphyrin: The most important natural porphyrin, whose iron complex, united with protein, occurs as hemoglobin, myoglobin, catalase, and certain respiratory pigments, . - Pyrrole: A ring compound consisting of h carbon atoms and 1 nitrogen atom with 5 hydrogen atoms, that is a component of chlorophyll, hemin, and many other important, naturally occurring substances. Reactive site: A chemical configuration on a molecule with which a bond is made by other specific molecules. Renal insufficiency: A state in which the kidneys are unable to remove a sufficient proportion of the effete matter of the blood. Rotameter: A device that measures the flow of a gas based on the height in a calibrated cylinder to which it pushes a rotating bobble. Sacral vertebrae: The 5, normally fused, vertebrae at the posterior end of the spinal column that form the sacrum. SC: Sickle cell-hemoglobin C disease, characterized by sickle cell anemia and the presence of C hemoglobin. Schwann cell: One of the large nucleated masses of protoplasm lining the inner surface of the neurilemma, a membrane wrapping the nerve fiber. Sequela: Any lesion or affection following or caused by an attack of disease. SH: Free sulfhydryl group consisting of sulfur and hydrogen atoms. * / Glossary 16 DUP050055789 Side roblasts.: Early cells in the red blood cell series that contain granules of free iron as detected by the prussian blue reaction. Signs: Conditions that are evident to the examining physician but are not obvious to the patient, as are symptoms. Spindle: The fusiform figure of achromatin in the cell nucleus during %. mitosis, consisting of fine threads connecting the centrosomes, which have separated to opposite ends of the spindle. SS: Sickle cell anemia, a hereditary, genetically determined hemolytic anemia accompanied by the presence of S hemoglobin. Stanford-Binet intelligence-test: A revised version of the Binet-Simon test for determining relative intellectual development; consists of a series of questions and tasks graded with reference to the ability of the "normal" child to deal with them at successive age levels. Stoneware:: A hard, opaque, vitrified ceramic ware. Striatum: See Corpus Striatum. Sub clinical lead poisoning: Toxic effects of lead that do not produce clinically discernible signs. Surface horizon: The earth1 s surface. Synergistic effects: Joint effects of two or more agents, as drugs, etc., which, when taken together, increase each others effectiveness Tared: Preweighed before use in the sampling procedure. TEL: Tet rae thy1 lead. Glossary IT DUP050055790 Thalassemia! A hereditary, genetically determined hemolytic anemia with familial and racial incidencej divided into a number of categories based on clinical severity and the type(s) of hemoglobin contained in the red blood cells, TML: Tetramsthyl lead. r Translocation: The transfer of metabolites, nutritive material, or other substances from one part of a plant to another. Tubular lesions: Lesions of the tublies of the kidney causing impairment of its re absorptive capacity. UCP: Urinary coproperphyrin Upper horizon: The ipper 6 in. of earth, immediately beneath the surface, Urban atmosphere: The atmosphere over the center (downtown) of a city. UBO: Uroporphyrin, C^QH^gO^gU^, a porphyrin eeewring in the urine. Vaeutainers (trademark): Sealed ampules, maintained under a slight vacuum and containing an anticoagulant, into which blood samples may be draw directly. Vertigo; Dizziness. /Glossary 18