ENVIRONMENTAL
CHEMICALS
HUMAN & ANIMAL HEALTH
2nd Annual Conference
1973
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ENVIRONMENTAL CHEMICALS
HUMAN AND ANIMAL HEALTH
Proceedings of 2nd Annual Conference
Sponsored by
Colorado State University
College of Veterinary Medicine
and Biomedical Sciences
Institute of Rural Environmental Health
and'
Division of Pesticides Programs
Environmental Protection Agency
Edited by Eldon P. Savage
Held at
Colorado State University
Fort Collins, Colorado
July 25-29, 1974
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CONTENTS
Page
Contents i
Preface ill
Conference Participants v
Current Aspects of Occupational Chemical Carcinogens
Donald V. Lassiter 1
The Absorption, Biotransfonnation and Excretion of
Environmental Chemicals
Frederick W. Oehrae 7
PASS—Its Function and Application Potential for Chemical
Accidents
Henry C. Schroeder 21
Nitrates as Human and Animal Health Hazards
Frederick W. Oehme 25
Correlation of Nitrate Levels with Human Health
Status in a Watershed
Janet G. Osteryoung 39
Industrial Hygiene Significance of Isocyanate Exposures
B. Gunter 45
Laboratory Identification of Petroleum Pollution Problems
William S. Dunn 47
Controlling Emissions from New and Used Cars and
Alternate Motor Vehicle Power Sources
Lane W. Kirkpatrick 53
PCB's in Ambient Air
John Tessari, Eldon P. Savage, Joseph W. Malberg,
and H. William Wheeler 61
Correlation of Environmental and Human Pesticide Levels
on a Geographic Basis
Anne Yobs 75
Antibiotic Resistance and Animal Feed Additives
Leslie P. Williams, Jr. and Carey L. Quarles 81
Health Hazards Associated with the Sale of Pesticides
in Food Outlets
Eldon P. Savage and John D. Tessari 99
Survey of Pesticide Morbidity in Kentucky
E. Edsel Moore 103
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Page
Epidemiology and Prevention of Pesticide Poisoning
John I. Freeman 115
Effects on Reproduction in Quail from Low Level Pesticides
Carey L. Quarles, Eldon P. Savage, G. R. J. Law and
John D. Tessari 123
Trace Elements in Unprocessed Plant Foodstuffs
Hansford T. Shacklette, James A. Erdman, and
John R. Keith 129
The Total Diet Program: The Tenth Year
Dennis Manske 145
Differentiation of Incidents of Infection in Chemical
Etiologies
Keith R. Long 151
Sampling Technology
Bill Stevenson 157
Epidemiology of Carbon Monoxide Poisoning at High Altitudes
Eldon P. Savage, Joseph W. Malberg, John Tessari
and H. William Wheeler 167
Panel: Current Status of Disposal of Toxic Chemicals
Robert Harding, Fred M. Applehans, H. William Wheeler
and Gary Gingery 173
Hazardous Contaminants: Chlorinated Dibenzodioxins and
Chlorinated Dibenzofurans
James Edward Huff and John S. Wassom 175
Mycotoxins
D. W. Lawellin 199
National Pesticide Monitoring Program in the Environmental
Protection Agency
G. Bruce Wiersma 201
Effects of Lead on Micrococcus Luteus and Subcellular
Components
Thomas G. Tornabene 205
Acute and Chronic Exposures to Pesticides
David L. Mick 227
Anhydrous Ammonia Accidents
L. W. Knapp, Jr 229
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Preface
The Second Annual Conference on Environmental Chemicals: Human
and Animal Health was held on the campus of Colorado State University
during the week of July 25-29, 1973.
The purpose of this Conference is to explore the environmental,
ecological, human and animal health effects of environmental chemicals.
Over 100 people representing 29 states and Canada attended the 1973
Conference.
As co-chairman of the Conference with Dr. Anne Yobs of the Environ-
mental Protection Agency, I wish to express our sincere appreciation to
fellow staff members of Colorado State University and the Environmental
Protection Agency for assistance in the program.
E. P. Savage
Editor
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iv
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CONFERENCE PARTICIPANTS
PROGRAM MEMBERS
Applehans, Fred M., B.S., M.S. Institute of Rural Environmental Health,
Department of Microbiology, Colorado State University, Fort Collins,
Colorado.
Baca, Joseph R., United States Food and Drug Administration - Analytical
Chemist, 240 Hennepin Avenue, Minneapolis, Minnesota.
Bagby, John R., Jr., M.S., Ph.D., Director, Institute of Rural Environ-
mental Health, Professor of Microbiology, Department of Microbiology,
Colorado State University, Fort Collins, Colorado.
Baird, Justus, Epidemiologist - Houston Health Department, 1115N Mac-
Gregor, Houston, Texas.
Barefoot, Howard, Georgia Department of Natural Resources - Solid Waste
Management, 535 Highland Avenue, S.W., Atlanta, Georgia.
Bell, LTC Raymond E., Chief, Radiological and Chemical Protection Branch,
Health and Environment Division, Academy of Health Science, United
States Army, Fort Sam Houston, Texas.
Bindeman, John, Montgomery County Health District, Supervisor, Bureau of
General Services, 451 West Third Street, County Government Plaza,
Dayton, Ohio.
Bishop, Vincil C., D.V.M., Arkansas State Department of Health, District
Supervisor, Meat Inspection Division, Arkansas SHD, 4815 West Mark-
ham Street, Little Rock, Arkansas.
Burnett, William C., D.V.M., Food and Drug Administration (Veterinary
Medical Officer) 4126 Cedar Lane, Kansas City, Missouri.
Caplan, Paul E., United States Department of HEW - NIOSH, Deputy Director,
Division of Technical Services, DHEW - PHS - NIOSH, United States
Post Office Building, Room 508, Cincinnati, Ohio.
Case, A. A., M.S., D.V.M., School of Veterinary Medicine, University of
Missouri, Department of Veterinary Clinics, Columbia, Missouri.
Cholas, Gus, D.V.M., M.P.H., Assoicate Professor of Microbiology, Depart-
ment of Microbiology, Colorado State University, Fort Collins,
Colorado.
Christopherson, A. Reese, Department of the Navy, WESTNAVFACENGCOM -
Entomologist, Post Office Box 727, Code 10A, San Bruno, California.
Collier, John R., D.V.M., M.S., Ph.D., Professor of Microbiology, Depart-
ment of Microbiology, Colorado State University, Fort Collins, Colo-
rado.
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Creswell, John L., Mason and Hanger - Silas Mason Company, Inc., Natural
Resources Manager, Post Office Box 561, Burlington, Iowa.
Davis, William, Jr., State Pesticide Project Coordinator, Memphis and
Shelby County Health Department, 814 Jefferson Avenue, Memphis,
Tennessee.
Dominick, Harvey J., Illinois Department of Public Health, Entomologist,
535 West Jefferson, Springfield, Illinois.
Dunn, William S., M.S., Chief Chemist, Division of Laboratories, Colorado
State Department of Health, Denver, Colorado.
Elder, James B., Bureau of Sport Fisheries and Wildlife, Division of
Ecological Services, Chief, Ecological Monitoring and Pesticides
Branch, Federal Building, Fort Snelling, Twin Cities, Minnesota.
Elliott, John, Specialist in Pesticide Education, Cooperative Extension
Service, Auburn University, Cooperative Extension Service, Extension
Cottage, Auburn, Alabama.
Faulkner, C. E., Regional Environmental Coordinator, Bureau of Sport
Fisheries and Wildlife, Twin Cities, Minnesota.
Faulkner, Lloyd C., D.V.M., Ph.D., Professor and Chairman, Department
of Physiology and Biophysics, Colorado State University, Fort
Collins, Colorado.
Flake, Harold W., Jr., USDA, Forest Service - Entomologist, Section
Head, Insect Detection and Evaluation Section, United States Forest
Service, Timber Management, 517 Gold Avenue, S.W., Albuquerque,
New Mexico.
Fitzwater, William D., Environmental Protection Agency, Biologist, 1501
South Edgewood, Apartment. 563, Arlington, Virginia.
Frederickson, Luther E., D.V.M., Tennessee Department of Public Health,
Director of Veterinary Medicine, Suite 101 Capitol Towers, Nashville,
Tennessee.
Freeman, John I., D.V.M., M.P.H., Chief, Veterinary Public Health Section,
North Carolina State Board of Health, Raleigh, North Carolina.
Gibson, John W..Department of Agriculture, Agricultural Environmental
Division, Room 122, State Capital Building, Oklahoma City, Oklahoma.
Gingery, Gary, M.P.H., Administrator, Pesticide Division, Montana State
Department of Agriculture, Helena, Montana.
Gunter, Bobby J., Ph.D., National Institute of Occupational Safety and
Health, Region VIII, Denver, Colorado.
Harding, Robert, Ph.D., Pesticide and Hazardous Waste Disposal Section,
Chief, Environmental Protection Agency, 1860 Lincoln Street, Suite
900, Denver, Colorado.
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Heath, James E., Veterinary Diagnostic Laboratory, Tennessee Department
of Agriculture, Box 40627 Melrose Station, Nashville, Tennessee.
Hill, John E., Dow Chemical, U.S.A., Research Industrial Hygienist, Post
Office Box 888, Golden, Colorado.
Hoffmann, Buzz L., Ph.D., Department of Health, Education and Welfare,
Food and Drug Administration, Office of the Assoicate Commission
for Science, Environmental Health Scientist, 5600 Fishers Lane, Room
7-79, Rockville, Maryland.
Hueneberg, Carl, Superintendent, Sanitation Section, 824 Civil Engineering,
Box 25, 279, APO San Francisco, California.
Huff, James E., Ph.D., Assoicate Director, Toxicology, Information Systems
Office, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
Humphrey, Harold E. B., Michigan Department of Public Health, Environmental
Epidemiology, 3500 North Logan, Lansing, Michigan.
Jagger, Herbert F., R.P.S., Director, Environmental Health, Northeast
Colorado Health Department, 822 Columbine, Sterling, Colorado.
Johnsen, Richard E., Associate Professor of Entomology, Department of
Zoology and Entomology, Colorado State University, Fort Collins,
Colorado.
Johnson, Michael, Environmental Health Services, Department of Microbiology,
Colorado State University, Fort Collins, Colorado.
Kirkpatrick, Lane, Ph.D., Technical Secretary, Colorado Air Pollution
Control Commission, Colorado State Department of Health, Denver,
Colorado.
Knapp, L. W. , Jr., Ph..D., Director, Institute of Agcultural Medicine,
Department of Preventive Medicine and Environmental Health, The
University of Iowa, Oakdale, Iowa.
Kokoski, Charles, Ph.D., Assistant Director of Petitions Review of the
Division of Toxicology, Bureau of Foods, Food and Drug Administration,
Washington, D.C.
Lassiter, Donald V., Ph.D., Environmental Health Scientist, Office of
Research and Standards Development, National Institute for Occupational
Safety and Health, Rockville, Maryland.
Law, Jack, Ph.D., Department of Animal Scineces, Colorado State University,
Fort Collins, Colorado.
Lawellin, David, B.S., Ph.D. candidate, Department of Microbiology,
Colorado State University, Fort Collins, Colorado.
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Levy, Michael John, 551 Brookline, Brookline, Massachusetts.
Link, James B., Major, United States Air Force (DEM), Director of Opera-
tions and Maintenance, Robins Air Force Base, Warner Robins, Georgia.
Linn, Lloyd G., Jr., Assistant to the Director of the Environmental
Laboratory, State of Alabama Health Department, Environmental Health
Laboratory, 716 West Shawnee Drive, Montgomery, Alabama.
Long, Keith R., Ph.D., Professor and Vice-Chairman, College of Medicine,
Department of Community Health, The University of Iowa, Iowa City,
Iowa.
Lovry, Stanton, Chemist, Toxicology Division, United States Army Environ-
mental Hygiene Agency, Commander United States Army Environmental
Hygiene Agency, Attention: Mr. Stanton Lovry, Aberdeen Proving
Grounds, Maryland.
Lund, John L., Sr., CMS, USAF, School of Aerospace Medicine, EDE, Building
150, Room 212A, Brooks Air Force Base, San Antonio, Texas.
Malberg, Joseph W., Institute of Rural Environmental Health, Department of
Microbiology, Colorado State University, Fort Collins, Colorado.
Mandel, Robert M., Environmental Protection Agency, Pesticides Accident
Investigator, 100 California Street, San Francisco, California.
Manske, Dennis D., M.S., Supervisory Chemist, Food and Drug Administration,
Kansas City Field Office, Kansas City, Missouri.
Mason, Fred D., M.S., Kansas City Missouri Health Department, Chief Environ-
mentalist, Section 110, 10th Floor, City Hall, Kansas City, Missouri.
May, Jon R., Ph.D., United States Public Health Service, NIOSH, Environ-
mental Toxicologist, Parklawn Building, 5600 Fishers Lane, Room
10-28, Rockville, Maryland.
Mathis, Bob, Ph.D., Epidemiologist—Veterinarian, Veterinary Services
Division, United States Department of Agriculture, Animal Plant
Health Inspection Service, Federal Center Building Number One,
Room 738, Hyattsville, Maryland.
McCarron, James E., Supervisory Microbiologist, Food and Drug Administra-
tion, 500 United States Customhouse, Denver, Colorado.
Meyers, Howard, Ph.D., Bureau of Veterinary Medicine, Food and Drug
Administration, 5600 Fishers Lane, Rockville, Maryland.
Mick, David L., Ph.D., Project Director, Iowa Community Pesticides Study,
Institute of Agricultural Medicine, The University of Iowa, Oakdale,
Iowa.
Middendorf, William B., Director, Bureau of Community Environmental Control,
Department of Environmental Resources, Post Office Box 2351, Fulton
Building, Harrisburg, Pennsylvania.
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Moore, E. Edsel, B.S., Director, Pesticides Program, Division of Environ-
mental Services, Kentucky Department of Health, Frankfort, Kentucky.
Morrison, Sumner M., Ph.D., Director of Environmental Health Services,
Professor of Microbiology, Department of Microbiology, Colorado
State University, Fort Collins, Colorado.
Morton, William E., M.D., Environmental Medicine Division, University
of Oregon Medical School, Portland, Oregon.
Muchnick, Carl, M.D., Epidemiologist, 702 Josephine Street, Austin, Texas.
Muller, Harry D., Ph.D., Extension Poultry Scientist, Cooperative Extension
Service, University of Georgia, Extension Poultry Science Department,
Athens, Georgia.
Norvell, Michael J., Ph.D., DNS, BVM, FDA, 5600 Fisher Lane, Rockville,
Maryland.
Oehme, Frederick W., D.V.M., Ph.D., Professor of Toxicology and Medicine,
Director, Comparative Toxicology Laboratory, Department of Surgery
and Medicine, Kansas State University, Manhattan, Kansas.
Ogg, James E., Ph.D., Professor and Head, Department of Microbiology,
Colorado State University, Fort Collins, Colorado.
Osmun, John V., Ph.D., Director, Operations Division, Office of Pesticide
Programs, United States Environmental Protection Agency, Washington,
D.C.
Osteryoung, Janet G., Ph.D., Institute of Rural Environmental Health,
Assistant Professor, Department of Microbiology, Colorado State
University, Fort Collins, Colorado.
Quarles, Carey L., Ph.D., Associate Professor, Department of Animal.
Sciences, Colorado State University, Fort Collins, Colorado.
Ramos, Henry, National Institute for Occupational Safety and Health,
Industrial Hygienist, 5259 Waltella Place, Cincinnati, Ohio.
Rosenberg, Myron C., D.V.M., Chief, Antiparasitic Drugs Branch, BVM,
Food and Drug Administration, VM330, Room 6B16, 5600 Fishers Lane,
Rockville, Maryland.
Saiiabia, James, Department of Health, Chief Chemist, Institute of Health
Laboratories, Post Office Box 1730, Hato Rey Station, Hato Rey,
Puerto Rico.
Savage, Eldon P., M.P.H., Ph.D., Chief, Chemical Epidemiology Section,
Institute of Rural Environmental Health, Associate Professor, De-
partment of Microbiology, Colorado State University, Fort Collins,
Colorado.
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Schmid, Robert J., Chemist, Food and Drug Administration, 500 Customhouse,
Denver, Colorado.
Schroeder, Henry C., Ph.D., Pesticide Accident Officer, United States
Environmental Protection Agency, Rocky Mountain-Prairie Region,
Denver, Colorado.
Shoultz, Phillip, Indian Public Health Service, United States Public
Health Service, Window Rock, Arizona.
Shacklette, Hansford T., Ph.D., Research Botanist, United States Depart-
ment of the Interior Geological Survey, Denver, Colorado.
Smart, Phil, Malaria Project Manager, USAID-PHS Manila, APO San Francisco,
California.
Sitorius, Marvin, Nebraska Department of Agriculture, Chief, Bureau of
Plant Industry, Post Office Box 94756, Lincoln, Nebraska.
Steinberg, Marshall, LTC, Director Laobratory Services, United States
Army Health Service Command, United States Army Environmental Hygiene
Agency, Building 2100, APG, Maryland.
Stevenson, Bill, Ph.D., United States Forest Service, State and Private
Forestry, Room 1205 B, Rosslyn Plaza, 1621 North Kent Street, Arling-
ton, Virginia.
Tarrant, Webb A., Chemist, Food and Drug Administration, 500 United States
Customhouse, Denver, Colorado.
Tessari, John D., B.A., Chemist, Institute of Rural Environmental Health,
Department of Microbiology, Colorado State University, Fort Collins,
Colorado.
Tornabene, Thomas G., Ph.D., Associate Professor, Department of Microbiology,
Colorado State University, Fort Collins, Colorado.
Watson, Kayle N., Jr., Fort Wayne and Allen County Building of Health,
Director Rodent Control, City County Building, One East Main Street,
Fort Wayne, Indiana.
Walker, Terry, State Division of Health, Advisory Sanitarian, Post Office
Box 1788, Mail Stop 4-1, Olympia, Washington.
Weedon, James R., D.V.M., M.P.H., Texas State Health Department, Veterinary
Public Health Division, 1100 West 49th Street, Austin, Texas.
Wheeler, H. William, B.S., Research Associate, Institute of Rural Environ-
mental Health, Department of Microbiology, Colorado State University,
Fort Collins, Colorado.
Whitcomb, Dr. Donald, Arizona State Department of Health, Chief Chemist,
1716 West Adams Street, Phoenix, Arizona.
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White, James, New Mexico Environmental Improvement Agency, Post Office
Box 2348, Santa Fe, New Mexico.
Wiemann, 0. J., M.P.H., Chief, Milk, Food and Drug Section, Colorado
Department of Health, Denver, Colorado.
Wiant, Chris, Administrator, Illinois Department of Public Health, Product
Safety Program, 535 West Jefferson, Springfield, Illinois.
Wiersma, Bruce, Ph.D., Ecological Monitoring Branch, United States Environ-
mental Protection Agency, Washington, D.C.
Williams, L. P., D.V.M., M.P.H., Dr.P.H., Chief, Epidemiology and Zoonoses
Section, Institute of Rural Environmental Health, Associate Professor,
Department of Microbiology, Colorado State University, Fort Collins,
Colorado.
Wyrick, Brenda, Chemist, Department of Health Education and Welfare,
Communicable Disease Center, Center for Disease Control Laboratory,
Fort Collins, Colorado.
Yobs, Anne R., M.D., Chief, Training and Education Branch, Operations
Division, Office of Pesticide Programs, United States Environmental
Protection Agency, Chamblee, Georgia.
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CURRENT ASPECTS OF OCCUPATIONAL CHEMICAL CARCINOGENESIS
Donald V. Lassiter, Ph.D.
National Institute for Occupational Safety and Health
The Occupational Safety and Health Act of 1970 established two
new organizations: The National Institute for Occupational Safety and
Health (NIOSH) within the Department of Health, Education and Welfare
and the Occupational Safety and Health Administration (OSHA) within the
Department of Labor. In very general terms, NIOSH was mandated the
responsibility for occupational safety and health research leading to
the development of recommended occupational safety and health standards
for the consideration of OSHA, which was mandated the responsibility
for promulgation and enforcement of such standards. NIOSH fulfills its
statutory obligation to provide recommended standards to OSHA through
the documentation of criteria upon which the NIOSH recommendations are
based. These recommendations and the accompanying documentation are
included in Criteria Documents which are transmitted to OSHA and are
published for the use of the general public. These documents, then,
form the basis of the standards promulgated by OSHA.
The other route available for standard setting by OSHA is through
the promulgation of an emergency temporary standard by its publication
in the Federal Register. This route is more expedient and is reserved
for emergency situations. It was via this emergency temporary route
that the standard concerned with control of occupational exposure to
14 chemical carcinogens was recently promulgated.
It has been estimated that well over 50% of all human cancer is
caused by chemicals present in the environment. Boyland has further
estimated that 90% of all human tumors result from the action of
chemicals of environmental or endogenous origin with the remaining
10% due to viruses and radiation. Many of the chemicals present in
our environment have been tested for carcinogenicity and the National
Cancer Institute (NCI) has published a series of four monographs which
summarize the experimental evidence related to the carcinogenic
potential of approximately 1,000 of these chemical substances. The
NCI list is included in a larger listing published by NIOSH entitled
the Toxic Substances List, which is published annually as required
by the Occupational Safety and Health Act of 1970. This latter list
identifies chemicals which have been documented to be toxic, carcinogenic
or otherwise neoplastic. Several other Federal agencies including the
Department of the Army, the Food and Drug Administration (FDA), and the
Environmental Protection Agency (EPA) are also known to possess similar
lists of chemicals which have a demonstrated toxic or carcinogenic
effect.
The legal mandates of several Federal agencies related to the
control of human exposure to carcinogenic substances are well known.
The Delaney amendment to the Federal Food, Drug, and Cosmetics Act has
provided the FDA with a mandate to exclude those food additives which
have a demonstrated carcinogenic potential for humans or animals. The
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Occupational Safety and Health Act of 1970, while not addressing the
question of occupational exposure to carcinogens directly, does provide
that, insofar as practicable, no worker shall suffer diminished health,
loss of functional capacity, or decreased life span as a result of his
work experience. It is clearly evident that occupationally-induced
cancer touches on all three of these areas. The entry of OSHA into the
area of mandatory occupational safety and health standards for the
control of carcinogens in the workplace has forced the reconsideration
of questions regarding carcinogenicity which hithertofore had remained
largely open-ended and in the exclusive domain of the oncologists and
other biomedical scientists concerned with mechanisms of carcinogenicity.
Although various Federal agencies including the FDA, NCI, Department of
Agriculture (USDA) and the National Center for Disease Control (NCDC)
have been active in the area of cancer control, the promulgation by the
Department of Labor on May 3, 1973, of an emergency temporary standard
for the control of 14 carcinogens marked the first Federal attempt to
control exposure to chemical carcinogens in the workplace. Prior to
that time only the State of Pennsylvania had enacted legislation to
effectively control worker exposure to chemical carcinogens. For the
most part the 14 chemical carcinogens included in the emergency standard
had been considered to be potentially carcinogenic for humans by
industrial hygienists, including the American Conference for Governmental
Industrial Hygienists (ACGIH). This non-governmental organization had
included, in a separate appendix, a growing list of chemical carcinogens
in its yearly listing of threshold limit values (TLV). The recommended
control was that no exposure to these substances should occur by any
route. In the 1972 TLV list the carcinogens were arbitrarily divided
into two groups according to whether they were considered to be primarily
animal or human carcinogens, and in the 1973 list this same classification
scheme was altered to include exposure values for several of the
substances.
The 1972 ACGIH TLV list served as the principal guideline for the
NIOSH recommendation to OSHA that occupational exposure to 15 carcino-
genic substances should be controlled. These substances were:
1. 4-Aminodiphenyl
2. 4-Nitrobiphenyl
3. bis(Chloromethy1)ether
4. Chloromethyl methyl ether
5. 4-Dimethylaminoazobenzene
6. N-Nitrosodimethylamine
7. 2-Acetylaminofluorene
8. S-Naphthylamine
9. a-Naphthylamine
10. Benzidine and its salts
11. 3,3'-Dichlorobenzidine and its salts
12. Ethyleneimine
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13. 4,4'-Methylene-bis(2-chloroaniline) - commonly
called MOCA
14. $-Propiolactone
15. Dimethyl sulfate
Dimethyl sulfate was later deleted from the list because of
insufficient evidence of its carcinogenic hazard. It should be mentioned
that a-naphthylamine was never included as a carcinogen by the ACGIH and
in a recent letter to the Department of Labor the chairman of the TLV
committee has stated that he does not consider a-naphthylamine to be
carcinogenic for humans.
The emergency temporary standard promulgated by OSHA is primarily a
work practice standard containing control procedures for the isolation
of processes involving potential exposure to any of the 14 chemicals.
An advisory committee appointed by the Assistant Secretary of Labor for
Occupational Safety and Health is currently meeting to recommend a
permanent standard which, by law, must replace the existing emergency
temporary standard within six months following the date of its
promulgation. It is expected that the permanent standard will contain
provisions for the eventual control of the 14 substances by the issuance
of permits for operation by the Department of Labor. The change from a
predominantly work practice situation to a use permit situation will
require the careful evaluation of permit applications and may require
on-site inspections by the Department of Labor to ensure that control
measures are implemented and maintained.
One feature of the current emergency temporary standard is the
requirement that employees be apprised of the potential cancer hazard
involved with exposure to any of the 14 substances. This particular
requirement has been greeted with great concern by the chemical industry,
with no small amount of controversy concerning the actual carcinogenicity
of certain of the chemical substances included in the standard. From
the NIOSH point of view, however, there is adequate reason to consider
that occupational exposure to each of the 14 substances should be
controlled.
From the historical viewpoint the term occupational cancer has been
intimately associated with the dyestuffs industry and with the older
term, "aniline cancer." With the introduction of the "aniline" dyes,
workers in this industry began to acquire exposure to a number of
previously unknown synthetic aromatic amines derived from benzene and
naphthol. Rehn is credited with the first published account of
occupational bladder cancer associated with exposure to the chemicals
in this industry in 1895.
In the 1930's several important papers were published by Hueper,
Bonser and Berenblum concerning the etiology of "aniline cancer." The
exposure of an individual to a number of chemicals in this industry,
however, did little to assist in the search for etiologic agents. In
his 1934 review Hueper mentioned the beginning of what was to become a
growing controversy concerning the etiology of occupational bladder
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cancer. He stated that aniline, benzidine, and naphthylamine were the
principal etiologic candidates but emphasized that in 1931 Hamilton had
recognized major epidemiologic "pitfalls" including:
1. Worker exposure to more than one suspect compound,
further complicated by shifting of workers
between departments;
2. Different degrees of exposure hazard between
processes;
3. Unsuspected impurities in trace amounts possibly
more harmful than the parent compound;
4. Composition of dyes and production methodology
in different factories complicating statistical
comparison.
To a very great extent these same epidemiologic "pitfalls" are valid
to the present day. This is not the case for several of the more potent
carcinogenic aromatic amines, however. The capacity of g-naphthylamine,
benzidine, 4-aminodiphenyl, or 4-nitrodiphenyl to induce bladder cancer
in humans represents one of the more well documented cause and effect
relationships in occupational medicine. Mixed exposure to other aromatic
amines, including 3,3'-dichlorobenzidine and a-naphthylamine has
precluded the direct assertation that these are, likewise, carcinogenic
for man, although both have induced cancer in animals.
Herein lies one of the more important considerations concerning the
documentation of carcinogenic hazard and the extrapolation to man of
positive tests for carcinogenicity in animals. Which animals, exposed
by which routes to the substance in question, represent the best models?
Although no hard and fast rules are available as guidelines, it is
generally recognized that evidence documenting the induction of tumors
in at least two animal species by routes comparable with possible human
exposure should pre-exist prior to the consideration that the substances
are potentially carcinogenic for humans. Such general considerations
must be evaluated within the legislative mandate stated earlier; that
all employees are to be protected insofar as practicable. The dilemma
created by these circumstances requires that all available information
and data concerning the hazard of occupational exposure to a given
substance be reviewed and evaluated. Each experimental investigation
must be evaluated on its own merits before any extrapolation to an
occupational environment can be attempted. In this regard, experimental
evidence which is published in the open literature must be accorded
greater consideration than unpublished studies, or than the unsupported
statements of employers or employees.
The problem of occupational carcinogenesis as concerns a specific
chemical substance is largely a question of hazard evaluation. In some
instances an Increased incidence of tumor production in a defined work
force has thrown suspicion on a chemical substance previously considered
either innocuous or, at most, hazardous from aspects other than
carcinogenic potential. In other instances the results of animal
experimentation have demonstrated a carcinogenic potential for a specific
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chemical substance. The mechanisms of carcinogenesis are far from
being completely understood, and the variables surrounding occupational
carcinogenesis do little to clarify the situation even if a specific
agent is suspect. The more important aspect of occupational cancer must
be the recognition of cause and effect, at least from the standpoint of
control procedures. The hazard associated with occupational exposure
to a specific chemical substance must be considered not only from the
aspects of its acute or chronic toxicity, but also from its potential
to induce tumors. There can be no clear distinction between classic
toxicity and tumorigenesis until the mechanisms of both are completely
understood for a given chemical substance. The literature is replete
with instances of tumor induction in animals exposed by one or more of
several routes to a large variety of chemical substances. Although even
one such finding should immediately alert industry and governmental
agencies alike to a possible problem, certainly in depth review and
critical evaluation is necessary prior to establishment of mandatory
control standards by such agencies. Such in depth review requires that
all available information and data bearing on the problem be considered
and evaluated on its merits, and with the limited resources available
to NIOSH this is the direction which has been taken. The Institute has
produced a hazard review document for each of the 14 chemical substances
named in the emergency temporary standard and has submitted them to OSHA
for consideration by the DOL advisory committee on carcinogens. NIOSH
intends to make the 14 hazard reviews available to the public as an
Institute publication in the near future.
Some have criticized the promulgation of standards for the control
of chemical substances based solely on their potential to induce cancer
as being premature and not based on sound scientific evidence that all
of the substances in question were proven carcinogens for man. Certainly
degrees of carcinogenicity do exist and it is entirely possible that
thresholds for tumor induction may exist. If this latter hypothesis can
be proven, then control procedures based upon ceiling concentrations may
be feasible. Until such time, however, only the most stringent of
control procedures will suffice to assure that employees are adequately
protected from chemical substances considered to be potential human
carcinogens. The assessment of carcinogenic potential for a specific
chemical substance must include the consideration of published informa-
tion, monitoring and control data from affected industry, and the in
depth, epidemiologic experience of affected employees. Negative
experience in industry regarding incidence of cases of human cancer
must be considered not only in terms of pure versus mixed exposures,
but also in the light of the proven experience in the dyestuffs industry
that the average latency period for development of bladder cancer is
approximately 20 years.
Surely the knowledge that a chemical substance has a demonstrated
potential to induce cancer in animals must be evaluated by industry in
terms of establishing minimum controls, apprisal of employees, and
accurate record-keeping procedures on environmental levels of employee
exposure and health experience. In view of the latency period documented
in the dyestuffs industry for induction of bladder cancer, nothing short
of follow-up until the death of the employee will permit the accurate
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assessment of epLderaiologic studies for employee exposure to many of the
chemicals presently in use today by industry. Neither NIOSR nor OSHA
has either the manpower or the funding to undertake such a task; it will
await to be seen whether industry will rise to the challenge to bridge
this gap.
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THE ABSORPTION, BIOTRANSFORMATION, AND
EXCRETION OF ENVIRONMENTAL CHEMICALS
Frederick W. Oehme
Kansas State. University
The environment of man and animals is composed of chemicals.
Most of these chemicals are static and even though placed in direct
contact with the biological system are not absorbed and therefore,
produce no effect. The soil, our shelter from the elements, and
our clothes are examples of this static environment. Other portions
of the environment (water, food, air) are intended for absorption
and have a direct and usually desirable effect upon the organism.
Because the latter environments do enter mammalian systems, foreign
chemicals contained therein may produce undesirable effects. For that
matter, foreign chemicals in any portion of the environment may pro-
duce toxic syndromes if they come in contact with and enter the biological
system. The ultimate outcome of such exposure to environmental
chemicals is then dependent upon the ability of the System to
detoxify and excrete the offending material.
The number of foreign chemicals in the environment of man and
animals is constantly increasing. Fortunately, mammalian systems
have several barriers to prevent absorption of foreign chemicals and
additionally numerous physiological and biochemical mechanisms to
detoxify, biotransform, and hasten excretion of any such chemicals
that are absorbed into the system. Further, these various mechanisms
are capable of adaptation and response to the presence of a variety
of compounds. Thus, the biological system may be viewed as a constant-
ly adapting unit that is sensitive to some chemicals, but able to
respond uniquely in the presence of a wide variety of foreign materials.
It is fortunate for man and animals that this is the case; for were
it not so, the continually increasing chemical environmental burden
could have long since reduced animal life to a handful of debilitated
and degenerated specimens.
A variety of factors determine the specific effect that a foreign
chemical in the environment ultimately has upon individuals living
within that contaminated environment (Table 1). Prominent among these
factors are the degree to which absorption of the foreign chemical
occurs, the various biotransformation processes available to the
chemical, and the rapidity and degree to which excretion of the foreign
compound occurs. It is these three phases which ultimately decide
the fate of the foreign chemical and the influence it might have upon
the contacting organism.
Exposure and Absorption
Since environmental chemicals are found in all phases of the
environment, exposure may occur through a variety of routes. Most
commonly, however, exposure occurs by inhalation of contaminated air,
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application to the skin, or by ingestion with water or foods. Thus
the initial contact of the foreign chemical with the body frequently
is with the surface of the respiratory tract, the skin, or the diges-
tive tract. Allthe surfaces are faced with epithelial cells and these
cells form the initial barrier to any penetration of foreign chemicals.
The chemical properties of epithelial cells are those of a
protein-lipid-protein "sandwich" (1,6,8,9). Hence chemicals that
have similar properties would be able to "mix" with the chemical
components of the "sandwich" and thereby move through the cell bar-
rier into other portions of the biological system. The single most
important property which permits this "mixing" and thereby penetra-
tion, is the lipid or fatty character of the foreign compound. Chem-
icals that are unionized under the conditions of contact with the
epithelial cell are capable of rapid diffusion and movement through
the cell barrier (4,8). Hence the pH at the cell surface, the pKa
of the foreign chemical, and the degree of ionization of that chemical,
as well as the degree of solubility in the surface fluids to increase
surface area, all are important in determining whether the foreign
chemical will move across the initial cell barrier (2,4,5,6,9).
In the respiratory system, the foreign material is usually
dispersed in the inhaled air and is in contact with a moist surface
that is specifically designed for penetration by gaseous chemicals.
This is an ideal situation for absorption by foreign chemicals of
small molecular size and in a vapor or unionized (organic, non-polar,
fat-soluble) state. Hence the absorption of foreign chemicals from
inhaled air is usually rapid and relatively complete. The filtering
mechanisms of the respiratory tract are capable of holding out larger
particles of a diameter less than 1 micron. Unfortuantely, except
for those chemicals adhered to air-borne particulate matter, most
foreign compounds are uniformly dispersed in the inhaled air and reach
the deeper portions of the respiratory tract (alveolar sacs) in sig-
nificant concentration.
Absorption of foreign chemicals following application to the
exposed skin is variable and depends heavily upon the physical state
(surface area) and chemical state (degree of fat solubility or non-
ionization) of the environmental compound. Materials such as insect-
icides and petroleum products are rapidly absorbed and penetrate the
body tissues. Other compounds, such as heavy metals, are essentially
nonabsorbed if applied to the skin in aqueous solutions or attached
to particulate matter. Only if such compounds are in an organic
matrix, so that lipid compatibility is approached, does any signif-
icant (usually less than 10%) absorption take place. Even some of the
more potent toxins and venoms are innocuous if applied to the unbroken
skin since their chemical form is largely nonlipid.
Most intoxications occur via the oral route of exposure. En-
vironmental chemicals occur commonly in water supplies and, because
of agricultural use of food-chain contamination, may appear in animal
feeds or human foods. While such materials are usually present in
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only small concentrations, occasional large quantities are found when
accidents in food processing or handling occur. Continual ingestion
of small quantities of potentially toxic environmental chemicals may
accumulate in biological tissues and eventually produce problems;
however, the most spectacular problems from environmental chemicals
usually result from large scale contamination of feeds or foods and
resulting poisoning. The digestive tract is expertly designed for
the absorption of a large number of chemicals. Here also, however,
the importance of lipid solubility in promoting passage of chemicals
across the intestinal tract mucosa is of paramount importance. The
pH of the stomach permits acid compounds to remain in an unionized
form and diffuse rapidly into the blood stream (1,6,9). Further down
the digestive tract more basic chemicals are favored by the intestinal
pH to become unionized and be rapidly absorbed. The large surface
area of the intestinal tract, the enzymes of the mucous membranes and
glandular systems, and the large quantities of secretions produced
in response to digestion all favor the absorption and systemic ac-
cumulation of ingested materials. Of significance in digestive ab-
sorption is the particle size of the gross material ingested. If
it is capable of dissolving in the digestive tract fluids, absorption
is favored. Large particle sizes of materials result in great hinder-
ance of solubility and diffusion across the digestive tract membranes.
Environmental chemicals that are not absorbed from biological
surfaces are usually discarded by physical forces or by biological
processes. Unabsorbed particulate matter in the respiratory tract
is coughed up following the secretion of mucus by the lung and dis-
charged by that route. Smaller particles that are unable to rapidly
pass the lung surfaces may be trapped by adjacent lymph glands and
removed by that process. Workers in environments heavily contaminated .
with fine particulate matter are frequently found to have enlarged
and heavily-loaded pulmonary lymph structures. Environmental con-
taminants resting on skin surfaces are usually removed by mechanical
brushing or bathing. Unabsorbed materials in the digestive tract
have only 12-18 hours to penetrate the intestinal mucous membranes.
Chemicals unable to achieve the lipid-like state during that time are
expelled in the feces to contribute to the environmental contamination
produced by organic wastes. ,
Regardless of the route of exposure and subsequent absorption,
following penetration of a series of biological membranes from the
skin surface through subcutaneous tissues and eventually blood cap-
illary walls, the absorbed environmental chemicals reach the circulat-
ing blood and are dissolved in the biological fluid which circulates
throughout the body and eventually reaches all functioning tissues
and organs. The organism is then faced with the task of dealing
with the foreign chemical and hastening its elimination from the system.
This is accomplished via detoxication ;or biotransformation (metabolism)
followed by excretion.
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Biological Detoxication (7)
All Individuals have mechanisms to detoxify and metabolize foreign
materials. These are normally existing systems present to various
degrees in all animals to modify the effect of an absorbed chemical.
They are outlined in Table 2.
Foreign chemicals may be detoxified physiologically by the animal
having increased digestive tract secretions, by vomiting, or by developing
diarrhea following ingestion of the chemical. These responses serve
to dilute the chemical and clean the digestive tract of the toxic
material. Following absorption of the chemical, it may be bound to
protein in the blood or tissues. The foreign chemical may be stored
in inert and inactive forms in body fat or bone. Physiological ex-
cretion of the material from the body may be by elimination via the
breath, body secretions, bile, or urine. Excretion by the kidney and
urine is the most common means by which the body rids itself of a
foreign chemical.
The second major mechanism for detoxifying chemicals is via
enzymes found throughout the body (2-5,8,10). The purpose of enzymatic
attack is to increase excretion and thereby decrease body levels of
the chemical. The liver, kidney, and intestinal tract mucosa are especially
high in detoxifying enzymes, but all tissues in the body are capable
of metabolising foreign chemicals to varying degrees (8). The goals of bio-
chemical detoxification are to modify the chemical so that it may be
more easily further detoxified, to make it water-soluble so that it
may be excreted more easily in the urine, or to split or destroy the
active form of the chemical so that it is no longer capable of producing
its toxic effect (10). While the vast majority of detoxification
steps decrease toxicity or cause inactivation of the foreign chemical,
enzymatic attack occasionally results in increased toxicity through
the production of metabolites that are more toxic than the parent
compound. Adaptation of the system to foreign chemicals is possible
through receptor tolerance and enzyme induction (2,4,5,8).
Biotransformation
The biological system's numerous attempts to attack, alter,
or modify foreign compounds has been the subject of extensive investi-
gation the past two decades (2-5,8,10). Even so, the products of
such studies have resulted in the development of general concepts
and specific information on only relatively few of the numerous potentially
hazardous foreign chemicals.
Although, all tissues in the body are capable of attacking foreign
chemicals, the bulk of the activity is accomplished in the selected
tissues mentioned earlier. Hence chemicals absorbed from the diges-
tive tract are ferried directly to the liver which has initial access
to the compounds. This protective device from oral intoxication al-
lows a significant portion of the detoxication process to occur before
the potentially toxic materials are distributed to the total body. The
processes in chemical alteration are accomplished by enzymes located
10
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largely in the microsomes of the cell. Three major processes may
occur there: Oxidation, reduction, and/or conjugation. These first
two reactions are subject to wide variation depending upon the chemical
groups under attack and the related chemical structures. Since enzymes
are vitally necessary, deficiencies in specific proteins, co-factors,
or genetic deficiencies in enzymatic capability play important roles
in the rate of these processes. Oxidation and reduction have been
called "Step I" reactions since they are frequently only the initial
accomplishment of adding or removing oxygen or hydrogen. This is
commonly followed by the "Step II" process of conjugation—the adding
of a commonly found biological moelcule (glucose, sulfate, or an amino
acid) to the foreign chemical that initially was slightly altered
through oxidation or reduction. It has been descriptively stated that
the "Step I" reactions are designed to "put handles" on the chemicals
undergoing biotransformation so that they would be more easily dealt
with and attacked by the "Step II" reactions.
A fourth enzymatic process that alters foreign chemicals is
hydrolysis. This procedure is somewhat more limited than the previous
three and occurs largely by circulating enzymes in the biological
fluids rather than within cells. The result of hydrolysis is the splitting
of the foreign chemical and the adding of hydrogen to one produce while the
hydroxyl grouping is added to the other.
As the processes of biotransformation are continually active,
at any one time the body fluids contain a mixture of the absorbed
unaltered chemical, varieties of the partially metabolized compounds
(oxidized, reduced, and/or hydrolyzed), and numerous species of the
conjugated end products of the total procedure. Since the effect of
biotransformation is to make the previously unionized chemical into
a more water-soluble (ionized, polar) derivative, each one of these
biochemical offspring have different affinities and properties while
circulating throughout the body. The unchanged, largely fat-soluble
chemical is likely to be attached to plasma protein during its intra-
vascular travels. During its voyage it will be frequently released
from its protein-binding and may cross a variety of biological mem-
branes to penetrate various organs, tissues, or fluid compartments.
This particular species will frequently become sequestered in body
tissues high in fat, since it is to these materials that it would
have the greatest likeness. Hence body fat reserves and brain may
accumulate and store such chemicals. Other compounds of the unionized
form may bind to tissue protein, such as muscle or liver or kidney
tissue, and remain there until again released and redistributed to
other portions of the system. Occasionally, some chemicals have
specific affinity for particular matrices and are attracted to bone,
teeth, or even hair follicles. The effect of such distribution and
storage is to have accumulation of certain environmental chemicals
in specific biological tissues. Hence previous exposure to environ-
mental chemicals may be determined by assay of selected organs and
tissues.
The partially modified chemical structures (those having under-
gone oxidation or reduction, for example) are in a state of flux and
are subject to further enzymatic action. While awaiting this, the
11
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c hem.I CM In Qiul themsolvort IOHM I at.-noliibl.i,-. ami more .In tin- l.oul/w.1
or polar form. They thus tend I.CHH to attach to biological tlscmes
and tend more to be carried freely in the body fluids. In this state
they are subject to filtration and excretion processes much as any
foreign, relatively polar (water-soluble) compound is.
The chemicals having already undergone the "Step II" reactions
are extremely water-soluble with a very short biological half-life
in the circulating fluid. Such chemicals are heavily polar and are
rapidly filtered by the kidney and excreted via the watery urine.
Once conjugation is completed, the foreign chemical is essentially
inactive and will be excreted in a matter of minutes.
Because of the variety of environmental compounds and the number
of byproducts that each produces through the biotransformation process,
it is easily seen that a significant number of foreign chemical species
may be circulating within the biological system at any one time. If
one further introduces the deliberate absorption of chemicals by med-
ications, dietary supplements, and drugs taken in response to the
pressures of our civilization, it can readily be seen that the circula-
tion may be literally filled with a large variety of foreign chemicals.
All of these are competing for enzymes, metabolism, and excretion routes.
Some of their actions are additive on particular organs or body functions,
Other chemicals may react with one another to cancel their effects,
some of which may be those for which medications are therapeutically
being taken. In other instances the foreign chemicals may produce
alterations in the enzyme pathways and the bio-chemical systems by
which they are normally handled. This general competition-synergisia-
induction has been loosely termed "chemical interaction" and has
recently begun to attract significant medical interest (7). As our
understanding of biotransformation processes and the reactions that
may occur between various chemical species and various biological re-
ceptors and tissues increases, this particular field will grow in
importance and health significance.
Excretion
Excretion of foreign chemicals occurs largely when they have
been reduced to the water-soluble (polar) state, and they can be
eliminated through the large volume excretion of urine. Since polar
compounds are not capable of passing biological membranes, essentially
all the polar chemical filtered through the kidney appears in urine.
The non-protein-bound unaltered (unionized) foreign chemical and the
"Step I"-processed metabolites circulating in the plasma are also
filtered by the kidney, but because of their lipid-soluble state, most
of the unchanged chemical and some of the oxidized or reduced compounds
are reabsorbed by the kidney tubules and reenter the circulation.
Occasional active "carrier" systems may be involved in this reabsorption,
but it is largely a diffusion process. Concurrently, some secretion
of circulating chemicals occur from the plasma bathing the kidney
tubules and results in a small amount of unaltered compounds in the urine.
The most efficient excretory process, however, is the filtration and
retention of water-soluble biotransformation products in urine.
12
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A second significant excretion mechanism for polar (water-
soluble, conjugated) chemicals is through the liver and biliary ex-
cretion. This process appears to be one of active secretion from
the hepatocyte and is primarily associated with highly polar conju-
gates (such as glucuronides) and those that have a molecular weight
greater than 300 and have some binding affinity to plasma and hepatic-
ce.1.1 protein (8). Such compounds are excreted via the bile and appear
promptly in the digestive tract. Here they may be eliminated with
the feces or may undergo an enterohepatic circulation in which reab-
sorption of the parent chemical results. This occurs due to bacterial
action on the conjugated chemical in the digestive tract causing a
splitting of the conjugate (for example, into a glucuronide moiety
and an aglycone), with resulting reabsorption of the now fat-soluble
aglycone. By this process biliary excretion may be incomplete and
can result in the recycling of the same chemical structure several
times. By-in-large, excretion of foreign chemicals in the bile accounts
for a significant but not extremely large portion of the excretory
process.
Other excretory processes also exist, but are of relatively
less significance and require that the excreted chemical be in a
relatively unionized (fat-soluble) state (2,5,8). Many volatile
compounds are excreted unchanged in the expired air by merely dif-
fusing back across the alveolar membrane into the air sacs. Excretion
may occur through secretion of fluid by glands in the digestive tract,
particularly the stomach and intestine. To be a successful process,
such activity requires that the pKa of the secreted chemical be such
that when in the digestive tract the pH of the contents converts the
chemical to the polar, ionized, and hence nonreabsorbable form. To
a minor extent, excretion of foreign compounds through other body
secretions also occurs. Such processes occur by passive transport
of the unionized molecule and are seen in sweat, saliva, milk, and
the genital secretions of various animal species.
Excretion of the foreign chemical is vitally important to the
health of the affected individual, since only in this way can it
rid the body of the environmental chemicals to which it is continually
being exposed. Were it not for excretory systems, the body burden of
such materials would rapidly saturate available storage sites and
circulating levels would of necessity increase. The excretion mechanisms
provide a process for discharging these undesirable chemicals from
the biological system. Interestingly, the excretion rates can be
increased in instances of heavy biological contamination or intoxica-
tion, providing another illustration of nature's adaptive ability
to its surroundings. In clinical difficulties, therapy of poisoned
individuals is largely aimed at increasing the excretory ability of
the various routes of elimination.
Overview of the Biological Process
A schematic presentation of the biodynamics involved in the
absorption, metabolism and biotransformation, and exretion of foreign
chemicals in the biological system is given in Fig. 1. Although only
oral intake is illustrated, other routes of exposure can also be included,
13
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The digestive tract absorption is depicted as leading to levels of
the foreign compound in the circulating blood which is in equilibrium
with other biological fluids, tissues, organs, and receptors. The
processes of distribution by passage through biological membranes can
result in accumulation of chemicals in various tissues and systems.
Flow of the chemical to liver tissue results in metabolism and exre-
tion in bile or return to the circulating blood. Excretion is illus-
trated as a 2-way process to and from the intestinal tract and kidney,
while elimination of chemicals from the lung and glandular tissue is
not reversible.
The dynamics of such a system are largely a balance between
the chemicals being taken in through various routes and the elimination
of the compounds and/or their metabolites through several different
organ systems. A balance between intake and out-flow is thus apparent,
with enzymatic processes of biotransformation occurring as an effort
to modify the foreign chemicals to less-toxic materials that are more
easily excreted. As long as the intake is compensated for by the out-
flow, the system is in a "healthy balance". In instances of overwhelm-
ing absorption, however, the circulating level of foreign chemical(s)
rises rapidly to produce a state of intoxication. The system is then
in a "toxic balance" since the excretion mechanisms are insufficient
to handle the large amount of toxic chemical. Although adaptation
may occur if sufficient time is allowed, most poisonings are acute
and death frequently results before biological compensation and adapta-
tion can occur. Further, biological compensation is limited in its
extent and heavily overwhelming amounts of foreign chemicals may far
exceed the limited capacity for adaptation.
It is into this area that therapy for poisonings is attempted.
The use of antidotes are intended to rapidly "capture" and hasten
elimination of the circulating toxin. Support of fluid therapy is
largely intended to maintain and aid kidney function and urine excretion.
Digestive tract lavages and laxatives are utilized to remove unab-
sorbed material rapidly from the digestive tract and limit the potential
for reabsorption. The maintenance of cardiac and respiratory function
is vital to prolonged biological functions so that respiratory elimina-
tion of the chemical^)may continue and biotransformation is permitted
to inactivate the offending compound(s). The therapy of poisonings
is thus largely to assist the normal processes of biotransformation
and allow them to respond to the toxic insult.
There seems little doubt that the future years will see con-
tinuing development of new and more biologically-potent chemicals,
and with it the hazard for animal and human exposure will increase.
It is at least comforting to know that the biological system has
several mechanisms for coping with accidental or intentional exposure
to such materials. An understanding of the absorption, biotransforma-
tion, and excretion of foreign chemicals by organisms is important
to the study of potentially toxic environmental effects—and an intimate
knowledge of the specifics of such processes is vital to the success-
ful therapy of clinical instances of intoxications.
14
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Summary
Foreign chemicals are continually present in the environment
of man and animals. Mammalian systems are in a constant state of
balance—the intake compensated for by the out-flow. The intake is
largely determined by the route of exposure and the chemical charac-
teristics of the environmental compound. Under normal conditions of
exposure to small or moderate amounts of environmental chemicals,
the system is capable of biotransforming and detoxifying such materials
into compounds more easily handled by the mammalian system. These
are largely converted to more water-soluble materials and excreted
in the urine, bile, and less commonly through other excretory routes.
In situations of massive exposure to foreign materials, or when re-
peated exposure to moderate amounts of chemicals result in accumulation
in body systems, toxicoses may result. These are essentially an over-
whelming of the biological mechanisms for detoxifying and excreting
such materials. The hazard associated with environmental chemicals
is greatly increased if pre-existing disease modifies the normal
biological detoxification processes. Therapy to assist intoxicated
individuals is largely aimed at increasing excretory processes and
maintaining or restoring the physiological balance between the amount
of environmental chemical absorbed and the level capable of being
excreted.
15
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References
1. Ari'ens, E. J.: A General Introduction to the Field of Drug
Design. In Drug Design, Vol. I. E. J. Ari'ens, Editor. Academic
Press, N. Y. 1971.
2. Brodie, B. B. and Gillette, J. R., Editors: Concepts in Bio-
chemical Pharmacology, Part 1 and 2. Handbook of Experimental
Pharmacology, Volume XXVIII. Springer-Verlang, N. Y. 1971.
3. Fishman, W. H.: Chemistry of Drug Metabolism. Charles C. Thomas,
Springfield, 111. 1961.
4. Goldstein, A., Aronow, L., and Kalman, S. M: Principles of Drug
Action. Harper & Row, N. Y. 1968.
5. La Du, B. N.., Mandel, H. G., and Way, E. L., Editors: Fundament-
als of Drug Metabolism and Drug Disposition. Williams & Wilkins,
Baltimore. 1971.
6. Notari, R. E.: Biopharmaceutics and Pharmacokinetics. Marcel
Dekker, N. Y. 1971.
7. Oehme, F. W.: Chemical Interactions. In Environmental Chemicals:
Human and Animal Health. A. R. Yobs and E. P. Savage, Editors.
Colorado State University, Fort Collins. 1972.
8. Parke, D. V.: The Biochemistry of Foreign Compounds. Pergamon
Press, N. Y. 1968.
9. Swarbrick, J.: Biopharmaceutics. Lea & Febiger, Philadelphia.
1970.
10. Williams, R. T.: Detoxication Mechanisms, 2nd Ed. John Wiley &
Sons, N. Y. 1959.
16
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Table 1 — Factors Affecting Toxicity of Environmental Chemicals
1. Degree of chemical exposure
2. Physical and chemical properties of compound
3. Route of exposure to chemical
A. Absorption of chemical
5. Biotransfonnation of chemical
a. Distribution
b. Metabolism
c. Accumulation
d. Elimination
6. Species of animal involved
7. Size, age and sex of exposed individual
8. Health of exposed individual
9. Individual biochemical variations
17
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Table 2— Biological Detoxication Mechanisms
1. Physiological detoxication
a. Secretions
b. Vomiting, diarrhea
c. Binding to protein and tissues
d. Excretion in breath, secretions, bile, urine
2. Biochemical detoxication
a. Enzymatic attack
b. Liver, kidney, intestinal mucosa
c. Make easier to detoxify further, make more
water-soluble, split or destroy
d. Most detoxication decrease toxicity or
inactivate
e. Adaptation, enzyme induction
18
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I«4T/\KE
HVER
bi'o-
INTESTIME
I
Pl-l/ID
CMS
•if
reo«.phJ<"
hindin
Irxnrf (Art
•**•
a fllNOIA(6
•tf-
^f
44r
LUMG
GLANDS
PKcooirrs
r e. o. .V.r isl- ioi
F6CES
Figure 1 — Biodynamics of a Schematic Biological System
19
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20
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THE PESTICIDE ACCIDENT SURVEILLANCE SYSTEM
Henry C. Schroeder, Ph.D.
U.S. Environmental Protection Agency
The Environmental Protection Agency (EPA) is responsible under a
series bf Federal statutes including the Federal Insecticide, Fungicide,
and Rodenticide Act (FIFRA) and, more recently, the Federal Environmental
Pesticide Control Act (FEPCA), for preserving the quality of the environ-
ment for the well-being of mankind. Pesticide chemicals are one of a
variety of potentially hazardous materials the agency seeks to regulate
through legislated authority. In order to facilitate the regulation of
pesticides, the EPA has established a comprehensive nationwide reporting
system which includes both a capability for basic accident reporting and
for investigating a representative sample of pesticide accidents. This
reporting system is known as the Pesticide Accident Surveillance System
(PASS). It is a regionally orientated computer information system
designed to handle pesticide episode data. By episode, we mean pesticide
accidents or incidents; an accident being defined as an undesirable
effect (such as injury, illness, or death) to humans, animals, plants
or the environment, resulting from the use or misuse of a pesticide; and
an incident as an alleged happening involving a pesticide which has or
has not been verified as having had an undesirable effect on humans,
animals, plants or the environment. An incident may or may not be worthy
of an investigation, depending upon the circumstances.
The system has a capability for both basic accident reporting and
for investigating a representative sample of pesticide mishaps. Priori-
ties have been established for investigation of pesticide episodes but
we have been encouraging the reporting of all accidents or incidents
where a pesticide is believed to have been involved, regardless of their
extent or severity.
In the last year, the accident program was reorganized within EPA's
Office of Pesticide Programs (OPP) and given higher priority. Under the
reorganization, the Accident Investigation Branch (AIB) of OPP's Opera-
tions Division has been designated as the operational unit responsible
for coordinating all pesticide accident activities. The AIB serves as
the focal point for EPA's regional pesticide accident reporting activi-
ties, as well as for the accident reporting systems of other federal
agencies. This includes the collection, analysis, interpretation, and
dissemination of data.
The basic forms and procedures for implementing PASS have been (and
still are being) developed by Chase, Rosen, and Wallace, a management
consulting firm under contract to EPA. These forms and procedures are
being employed on a trial basis at this stage, and thus are subject to
later modification. EPA wishes to encourage State agencies to submit
recommendations to the Regions for improvement of the system. At the
start, there will inevitably be problems to be solved.
21
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The first, step in Implementing the system was to contact each State
and Federal agency (e.g., health, agriculture, wildlife, etc.) that had
knowledge of pesticide accidents and to solicit the agency's cooperation
in collecting accident or Incident data. Most agencies have agreed, and
one of their officials has been identified as the focal point for
organizing and collecting reports within that agency. The regions are
providing the necessary orientation and training to insure uniform
collection and recording of data.
When a source in the reporting system learns of a pesticide episode,
he completes a pesticide report form (PERF) (Figure 1), and forwards it
to the EPA Regional Office as soon as practicable. Certain episodes
require immediate attention and in such cases, the source notifies his
regional contact by telephone as soon as possible. Situations where a
hazard still exists or a human fatality has recently occurred call for
immediate notification of the region. Once a report is received, it is
the region's responsibility to screen it for any apparent discrepancies
and decide whether to conduct a follow-up investigation. This decision
is based upon the report itself, the priorities for investigation,
manpower resources, requests from AIB, etc.
The major objectives of the accident investigation program are
essentially two-fold. First, we hope to develop reliable estimates of
the extent and nature of the consequences of pesticide uses oh man and
his environment. Our second objective is to provide specific cause and
effect data to modify or develop programs, or to take regulatory actions,
e.g., label revision or suspension and/or cancellation of product
registrations, in order to reduce adverse consequences of pesticide
usage.
As corollary objectives, in terms of benefits to State and Federal
agencies, the program will also provide:
1. Special reports or data to all cooperating State
or Federal agencies
2. Material for education and safety programs
3. Accurate and up-to-date information on a timely
basis to the news media or other interested parties
4. A Statewide, regionwide, and nationwide episode
early warning system
5. Assistance in conducting accident investigations,
responding to and helping with emergencies, and
laboratory assistance where necessary.
PASS has been designated for use by professionals who have some
knowledge of pesticides so that we can get reliable, sound data without
the wide range of discrepancies we have had in the past.
22
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Figure 1. Pesticide episode reporting form.
PESTICIDE EPISODE REPORTING FORM
Shaded Areas To Be Completed By EPA Region.
Kl« No- . i i i t j
Reporting Agency codi \_, >
u
EPA Region No.
u
Type of action af^ n(J ^
" A foi deletion of episode ' i
D 8 'or n« episode
' C tor corrections or additions to report
FIPS location code ;
" . State County City ': :
\ . » . . 1 . . . V .
;.-..» i'
GENERAL Mo Da Yr
Date of Episode 1 • I .1.1 Location of Episode i . i • i i i i i i i i i i i i ii i i i i ,
u v » (include state, county, city) . »
Mo Da Yr
Date of Report I . I . I . I Reporting Agency.
Reported by: . . . . i . . . i . . . . . . i Telephone no. . . . ./. . . .-. . . . . (..,., ,1
ii a 30area / eit. •»
NATURE OF EPISODE (Check all that apply)
l.D Accidental 2. D Intentional 3. D Self induced 4. D Homicidal . 5. D Undetermined
n ii it ir u
Was pesticide stored in original container? l.O^Yes 2. DwNo 3. D,,Unknown 4. D,,Not Applicable
Did usei follow label instructions? l.DSJYes 2.D,,No 3. D^Unknown 4. D«Not Applicable
Could the user comprehend label? l.Ds,Yes 2. Dj,No 3. DwUnknown 4. DMNot Applicable
Was pesticide container failure probable? l.D,,Yes 2.D,,No 3. DsjUnknown 4.D,,Not Applicable
Was pesticide applied aerially? l.DlsYes 2.O«No 3.0,,Unknown 4. D(,Not Applicable
CIRCUMSTANCES OF EPISODE (Check all that apply)
l.D Transportation 2. D Disaster (Fire, 3. D Disposal of 4. D Agricultural 5. D Industrial 6. D Home 7. D Other
"' OF Pesticide « Flood, etc.) » Pesticide " " " "
PESTICIDE INVOLVED (For multiple pesticide products use additional forms)
Doeslabelbearfederalreg.no.? l.D Yes 2..D No 3. D Unknown Federal Registration No. i i i i i i i i i i i
IS -II II II II
Type of Pesticide: l.D Insecticide 2. D! Herbicide '3. D Fungicide 4.D Rodenticide 5.D Disinfectant
(Check all that apply) 6. D Other (specify)
code
Product Nime i_i—i_i—i i i—i—1_i_i—1_i_i—i—i—i_j_i—i i i i j—. i . . • .—.. .....,., i,.,..
n »
Active Ingredient
A. I. Code • ' i i ' ' ' C. C. Code *-+->
Active Ingredient ^ , Co;!, .i;. , , , ,", c. C. Code uxlj
Active Ingredient
A. I. Cnde i •- • • • • • C. C. Code L.
it is it
. i . Total No. Affected Lj^J No. of Fatalities ' <' ' ' ""• HosP'tal'2e^ „ „ No. Not Hospitalized.
Received Medical Attention
Check age groups of humans affected: l.D Under 5 2.D 5 to 16 3. D 17 to 65 4. D Over 65 5.D Unknown
• » II II 11 !l
Was episode job-related? l.D Yes 2. D No 3. D Unknown
ANIMAL
',.' i v Total No. Affected • • • • • N" "»"< l.D Livestock 2.D Wildlife 3.D Birds 4. D Fish 5.D Pets 6.D Bees
31 .'i }f j; 31
[jTj Specify breed/species ', • „' jSpeciescode.
PLANT LIFE
. i . . No. Acres Attccled or i—i—L-J-I No. Plants Affected l.D Ciops 2. D Trees 3D Shrubs 4. D Grasses
" " " ii ii n re
5. D Flowers 6. D Ornamentals
Specify variety /species
Species code \ , ,
CONTAMINATION (Check all that apply)
l.D Soil 2.D Air 3.D Water 4.D Food 5.D Vehicle 6.D Building 7. D Other
11 u M 11
INVESTIGATION (Check all that apply)
Were samples taken for laboratory analysis? l.D Yes 2. D No 3. D Unknown
it i; il >
l.D Investigation planned by reporting agency 2. D Not planned by leporting agency 3. D Other
II 10 II
REMARKS (Additional space on reverse sic'e): ,
1 D Inv^Uts'lc.ipUneit'jytPAi^icn 2. H Mot idjrir-i:! i;> f.fK'nkn 3. U Oih?i ________
'i'-mafion by: •_..• ,1 ..,-.- ..•*_ . ___ L_I ,L_J.-._I..J._I HM Ccn!ir-ic-.f
;V
FORMACC I DLC72
23
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NITRATES AS HUMAN AND ANIMAL HEALTH HAZARDS
Frederick W. Oehme
Kansas State University
The public awareness of foreign chemicals in our environment has promoted
the current high interest in pollution. As a result, the applied health
fields (medicine, veterinary medicine, dentistry, nursing, public health,
and others including social and psychological health) have been stimulated
to increase their concern for the environmental burden of foreign chemicals
(36,40).
When considering environmental pollution, we must recognize three basic
surroundings—air, water, and soil. Each of these is a dynamic system
of sensitive and balanced ecological systems. In many instances, the
biotic and abiotic entities of one biological system are interconnected,
and the entities within: the eco-systems of each biological system are cyclic
and interrelated. The most studied relationship is that involved in the
phases of the food chain cycle within an ecological system.
A disturbance in any one phase of food chain results in an upset within
this balanced system. Once stability is disrupted, the order of organisms
within the system change and the system itself becomes less diversified.
Destruction of the system may even occur. In nature, stability results as
a function of time. All living things can alter the precise balance within
these biological systems. However, man is the only living creature capable
of developing stability through technology.
Nitrogen is widespread in nature and is found in all three of the basic
ecological systems. Nitrate is an inorganic anion formed by oxidizing
elemental nitrogen. The anion is a significant component of the nitrogen
cycle of soil and water; it is found in the atmosphere at a much smaller
concentration (14,24). Nitrate is an essential nutrient for plants and
it is ultimately metabolized by plants to plant protein. Whenever one of
these biological eco-systems is disturbed, nitrates may accumulate in soil,
water, or plants. The accumulation of nitrates in our environment con-
stitutes a potential public health and environmental hazard.
The levels of nitrates found in plants and surface waters have been steadily
increasing (7,44). In many rural areas of north-central United States,
excessive levels are reported in ground waters (2,39,41,49). In Kansas
groundwater supplies, nitrates range from 0.0 ppm to over 1000 ppm (39,41).
Results obtained by the Environmental Health Services of the Kansas State
Department of Health show that over 25% of the groundwater samples tested
from January, 1967 to December 31, 1971 exceeded 50 ppm; 6% of the ground-
waters tested were in the toxic range (39,41). The Public Health Service
Drinking Water Standards recommend no more than 45 ppm nitrate in rural
wells (38). The Kansas State Department of Health found 37 municipal water
supplies above the 45 ppm nitrate tolerance limit (23). Three municipal
water supplies exceeded the Kansas recommended limit of 90 ppm nitrate (23).
Surface waters (ponds, lakes, streams, and reservoirs) had nitrate levels
Presented at the Second Conference on Environmental Chemicals in Human and
Animal Health, July 23-27, 1973, Colorado State University, Fort Collins,
Colorado.
25
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ranging from 0.1 ppm to 69 ppm (39,41). Low nitrate levels in surface
waters are expected because of denitrification and since water biota
rapidly utilize nitrates as a nutrient source (25). However, if a small
pond receives the runoff of a feedlot or a heavy fertilized cultivated
field, it is apt to have a high nitrate content.
Nitrites are infrequently found in groundwaters. Only 1-4% of 5,000
samples tested in an extensive Missouri study had detectable nitrites
(27). The concentration of nitrites varied from summer to winter. The
limited finding of nitrites in groundwater results from the instability
of the ion and the infrequent testing for nitrites in groundwater (46).
Problems Associated with High Concentrations of Nitrates
The results of research studies of nitrate poisoning are inconsistent but
numerous clinical experiences have been reported. These contradictions
may be caused by: (1) variations in research design; (2) biological
variations in plants; (3) differences in nutrition, housing, and care of
experimental animals; (4) varying chemical and physical properties of the
toxicant; (5) the route, method, and dosage of the toxicant; and (6)
clinical inability to diagnose nitrate intoxication accurately (12).
Nitrate toxicity in animals results primarily from the ingestion of
nitrates in feed and water. Mayo, in Kansas in 1895, reported the first
case of nitrate toxicity in animals (51). He diagnosed the disease as
"cornstalk poisoning" since the cattle poisoned had ingested corn stalks.
Since then many cases of poisoning from the ingestion of plants and water
high in nitrates have been reported and the etiological agent has been
recognized.
Nitrate poisoning in humans was first documented by Comly (6) in Iowa.
In Minnesota, 139 cases of infant methemoglobinemia were reported from
the ingestion of well waters high in nitrate (2); the mortality rate was
10%. In Kansas, 13 cases of infant methemoglobinemia, with three deaths,
were reported during a period from the early 1940's to 1950 (49). Other
cases of acute nitrate poisoning no doubt have also occurred, but since
these are not reportable they probably have not been documented.
The earliest cases of nitrate poisoning in animals were due to acute
toxicity. The clinical signs generally were dyspnea, increased heart
rate and respiration, excessive salivation, muscular tremors, incoordina-
tion, cyanotic mucous membranes, coma, and death. The blood had a
characteristic chocolate color, but this color was not pathognomonic
since chlorate herbicides can cause similar signs and chocolate-colored
blood. In humans, primarily infants, the brownish-blue discoloration
was first noticeable around the lips, then spread to the fingers and toes
and over the face, and eventually covered the entire body (49). Other
signs included drowsiness, increased rate of respiration, and death.
The clinical signs of acute nitrate toxicity are due to anoxia and
appear when methemoglobin levels reach 30-40%. Death occurs when
methemoglobin levels reach 70-80%. The nitrate ion is reduced to
26
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nitrite in the digestive tract, which upon absorption oxidizes the iron
of the hemoglobin molecule from the ferrous (hemoglobin) to the ferric
(methemoglobin) form. The resultant methemoglobin (ferrihemoglobin,
ferriprotoporphyrin IX-globin) is incapable of reversibly binding molecular
oxygen (2). The vasodilatory effect of nitrites on the cardiovascular
system further induces tissue anoxia due to a lowered circulatory volume
and pressure.
Oxidation of hemoglobin to methemoglobin by the nitrite ion occurs at a
rate which is characteristically different for each animal species, but
there is little difference between individuals of the same species (46).
Similarly, the reduction of methemoglobin is at a rate characteristic
for each animal species. These two physiological processes appear re-
lated, even though there is large variation in the rate of methemoglobin
formation and its subsequent reduction. An imbalance of these two
physiological processes may explain differences in species susceptability
(and variations in signs) seen with nitrate poisoning.
Reports of chronic nitrate toxicity due to long-term, low-level nitrate
intake in animals are found, but documentation is inconsistent. The
controversial signs are abortions, lowered milk production, reduced rate
of gain, avitaminosis A, and a thyroid-interference syndrome. Other
signs, such as diarrhea, diuresis, arthritis in swine, and hydrocephalus
in rabbits, have also been observed with the consumption of sublethal
levels of nitrates.
Ingestion of well waters high in nitrates is the primary cause of infant
methemoglobinemia due to nitrate toxicity. However, Phillips (37) cites
cases of infant methemoglobinemia due to ingestion of vegetables high in
nitrate. Vegetables may develop excessive levels of nitrites due to
various processing and storage techniques (37,44). Although no cases of
infant methemoglobinemia due to such foods have been reported in the
United States, fatal cases have occurred in other countries (35). Orgeron
(35) reported poisoning from the ingestion of wieners containing over
5,000 ppm nitrite.
The use of nitrates and nitrites as curing ingredients in meat processing
is being challenged by various consumer groups (10) . Nitrosamines have
been found in many processed meats (15,31,32). These substances, formed
by the reaction between nitrites and secondary amines, under acid conditions,
are carcinogenic (28,46). The present tolerance limits for processed
meats are 500 ppm nitrate and 200 ppm nitrite (13).
"Silo Fillers' Disease", long recognized as caused by silo gases (nitrogen
oxides), is a result of ensiling plants high in nitrates. Cases of
pulmonary adenomatosis have been observed in cattle ingesting feed and
water high in nitrates (43). Research conducted with cattle using nitrogen
dioxide caused severe methemoglobinemia and lung lesions (43).
Vasodilation, a pharmacological action of nitrites upon the cardiovascular
system, is the cause of nitrite syncope and occurs in man and animals (44).
Cases of this type have been observed experimentally and clinically in
cattle and swine.
27
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Ingested nitrates are metabolized in the digestive tract by microflora.
Depending upon the organisms present, the pH, and the available nutrients
(trace elements and carbohydrates), the products formed from nitrate
reduction are: N03 ^
(Nitrate)
N02 ^ N205; N02; NO NH2OH ^ NH3.
(Nitrite) (Nitrogen Oxides tHydroxylamine (Ammonia)
Once formed, these products are absorbed and may be toxic if allowed to
accumulate. Hydroxylamine has produced experimental hemolytic anemia in
cattle and sheep, and is involved in methemoglobin formation (43,49).
Eutrophication of lakes, ponds, and streams is an increasing problem (19).
Nitrogen and phosphorus are thought to be the major limiting nutrients to
aquatic phytoplankton and plants. Excessive nitrate in waters results in
plant and animal life changes which usually interfere with the multiple
uses of water, reducing its esthetic and economic values and threatening
the destruction of our water resources (19). Premature death of a body
of water is a serious consequence.
Physiological Effect in Animals
Ruminants. Ruminant animals, due to their physiology, are more susceptible
to nitrate poisoning than monogastric animals (46,51). Cattle are more
susceptible to nitrate poisoning than sheep (45). The effect of nitrate in
ruminants, as in all animals, is variable and depends upon several factors
(9). The total nitrate intake, rather than the individual content in water
or feed, is critical. Previous adaptation to nitrate feed, nutritional
status, feeding practices, and proper rumen function will influence animal
susceptibility. Hungry animals are less tolerant to nitrate since they
consume feed rapidly. The quality of the ration is important; rations high
in vitamin A reduce the toxic effects of nitrate, and a good quality
carbohydrate ration encourages microbial action and reduced nitrate toxicity.
Nitrate toxicity occurs in ruminants in an acute and chronic form. The
acute toxicity is the result of nitrates being reduced to nitrites, which
then oxidize hemoglobin to methemoglobin, causing anoxia and death.
Methemoglobin formation occurs most rapidly in ruminant animals (46).
However, of all the ruminants, cattle have the slowest rate of methemoglobin
reduction. This might explain why cattle are more susceptible to nitrate
poisoning. Tillman ejt al^. (48) showed that nitrate reduction in the
rumen was dependent upon pH and the copper, molybdenum, and iron content
of the rumen.
Acute nitrate toxicity results from ingesting water containing 500 ppm or
more nitrate or feed containing in excess of 5,000 ppm (0.5%) nitrate.
Ingested nitrate is reduced by the rumen microflora to nitrite, which is
6-10 times more toxic than nitrate. However, high levels of nitrates
alone may cause acute gastroenteritis, diarrhea, diuresis, and petechial
hemorrhages on the pericardium.
Chronic nitrate toxicity may result from sublethal levels of nitrate. The
signs reported are primarily those of physiological interference and include
28
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vitamin A and E deficiencies, thyroid dysfunction, abortions, fetal
resorptions, poor conception rates, decreased milk production, and
lowered rates of gain. While these interference effects have been
reported in other animal species, their occurrence in the ruminant is
very controversial in view of experimental findings. Field cases of
chronic toxicity may seem quite conclusive, but it has been difficult
to prove that all other variables capable of producing the observed
signs have been excluded. Two separate field cases are perhaps pertinent
(39,41); the only controlled variable, water high in nitrates, resulted
in abatement of the clinical signs when it was eliminated.
Olson e_t^ al_. (34) surveyed 25 livestock operations. Twenty percent of
the surveyed farms had excessive nitrates present in feed and water
sources; in 15% of the operations, herd health problems could be related
to excessive nitrate intake. Work recently completed on farmsteads
with excessive nitrates in drinking water provides further evidence for
a chronic nitrate syndrome. The herd health problems observed were
suggestive of vitamin A and E deficiencies and of thyroid, interference.
Cases of excessive nitrates in feed and water associated with animal
abortions have also been reported (5,18,43).
Many borderline nitrate cases exist, but signs are apparently being
suppressed by high-level feeding of vitamins A, D, and E, supplemental
iodized or trace-mineralized salt, or high carbohydrate diet (4). The
existence of the acute nitrate condition is also probably more prevalent
than recognized.
Monogastric Animal. Monogastric animals are more resistant to excessive
levels of dietary nitrate than ruminants. Dogs have been fed up to 2%
(20,000 ppm) nitrate without any adverse health effects (34,38). Rats
have received 1% (10,000 ppm) nitrate in feed for a life-time without
adverse effects (38) . Healthy human adults are reported to be able to
consume large quantities of nitrate in drinking water with relatively
little, if any, ill effects (2,35).
However, physiological effects due to ingestion of nitrates in food or
water are present and variable. Variability and response may be explained
by metabolic and physiological differences between animals. Toxicity
usually results when nitrate in feed or food is reduced to nitrite prior
to ingestion. Under certain conditions, this reduction can occur in the
stomach. Some animal tissues are able to reduce nitrate to nitrite.
In swine, large doses of nitrites are required to produce significant
methemoglobin levels and acute toxicity (11). The pig has the slowest
rates of methemoglobin formation and methemoglobin reduction (46). A
lack of response to methylene blue treatment in pigs has also been noted.
Swine are said to only be susceptible to nitrite toxicity when the nitrite
is performed (1). This may occur when feed is mixed with water prior to
feeding and bacteria in the ration reduce the nitrate to nitrite. Waters
high in nitrate, left standing in metal troughs and waterers, may also be
sources of excessive nitrites (17). Reduction may occur by bacteria or
by chemical reaction of the nitrate with the metal in the troughs. The
toxic oral dose of nitrites for pigs is given as 88 mg./kg. (1).
29
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The clinical signs of chronic nitrate toxicity in swine are vitamin A
deficiency, thyroid dysfunction, decreased rates of gain, arthritis-like
conditions, and abortions. Lymphocytic leukocytosis and erythrocytosis
have also been reported (11). Perhaps some instances of iron-deficiency
anemia are a result of chronic nitrate consumption.
When given orally to dogs, nitrate was reduced to nitrite, but the amount
reduced varied from essentially none to that sufficient to produce
methemoglobinemia (44). When given orally or intravenously, the nitrate
ion caused excessive renal excretion of chloride, resulting in hypochloremia,
alkalosis, and digestive disturbances (16). Dehydration occurred due to
the diuretic effect of nitrates (16). Hydrocephalus has been noted in
puppies from dogs with chronic nitrate ingestion (33).
The physiological effects of nitrate in humans are largely unknown. Acute
nitrate toxicity is usually seen in infants, and results from ingestion
of well waters and vegetables high in nitrates. Increased susceptibility
of infants to nitrate toxicity due to age, diet, and other factors has
been speculated. The amount of nitrate content of food or water ingested
daily, the duration of exposure to food or formula, the presence of
nitrate-reducing bacteria in the upper gastrointestinal tract, and the
condition of the digestive mucosa are all factors effecting toxicity (49).
Comly deduced that infants were prone to upset stomachs and achlorhydria
(6). As a result, the stomach pH increased in alkalinity, allowing nitrate-
reducing organism to enter. A gastric pH above 4 supports the growth of
nitrate-reducing organisms (2,8). Digestive disorders producing injury to
the gastrointestinal mucosa and thereby increased absorption of nitrite
was evident in several cases of infant methemoglobinemia in Minnesota (2).
Immature enzyme systems are also of importance (6). Methemoglobin formation
rates in human adults are close to those observed in cattle. However, the
rate of methemoglobin reduction in man was the most rapid of all species
studied (46). Approximately 1% of the adult hemoglobin is present as methemo-
globin (22). This relatively constant level exists because of the reversible
balance between hemoglobin oxidation and methemoglobin reduction. Fetal
hemoglobin (hemoglobin F) is oxidized by nitrite to methemoglobin twice as
rapidly as in adult hemoglobin (hemoglobin A). Furthermore, the enzymatic
capacity of the erythrocytes of newborn infants to reduce methemoglobin to
hemoglobin is less than that of adults (22). This is probably due to a
developmental deficiency in the activity of DPNH-methemoglobin reductase
(diphosphopyridine nucleoxide) (22). Compared to adults, several clinical,
physiological, and metabolical factors predispose infants to methemoglobinemia
and acute nitrate poisoning.
Sources of Nitrates
The sources of nitrates contaminating ground and surface waters are numerous.
Some of these are: atmospheric nitrogen (14); soils and other geological
sources, such as limestone rock, calicites, caves, and playa deposits
(14,24); concentrated organic animal wastes from pigpens, privies, cesspools,
feedlots, and primary-treated sewage; decayed plant matter and silo drainage;
inorganic fertilizers; and industrial wastes.
30
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Much controversy exists regarding the sources of nitrate in waters. Commoner
(7) feels that improper and excessive use of chemical fertilizers is the
largest source. Kenney (25) found that fertilizers are important contributers,
but apparently are less involved than Commoner believes. Increased fertilizer
use has probably contributed to the nitrate levels in some of our lakes,
rivers, and streams.
Extensive research in Missouri revealed that excessive nitrates in ground-
waters frequently resulted from organic animal wastes (26). In Minnesota,
83 of 129 wells containing high-nitrate water were improperly located in
relation to barnyards, pigpens, privies, cesspools, and other sources of
animal organic wastes (2). Twelve of 22 states reporting to the Engineering
Section of the Committee on Water Supply, American Public Health Association,
reported similar findings (49).
Wide variations in nitrate sources also exist in Kansas. Metzler (30) re-
ported that nitrogenous materials of animal origin, especially sewage and
manure, were principle sources of nitrates. The Rush County Extension
Service (42) reported that barnyard runoff, septic tanks, cesspools, privies,
sewer outlets, and silage seepage were prominent sources of nitrates in
750 wells surveyed. The primary sources of nitrates in other studies (39,41)
appeared to be organic animal waste materials. Sixty-one of 88 wells
surveyed were improperly located near barnyards, cesspools, privies, and
septic tanks. Only two of the 88 wells were found to be properly constructed.
The accumulation of nitrates in forages, feeds, and vegetables has been
significantly related to the amount of chemical fertilizers applied to the
land (24,25,26,37). There was also a significant relationship between the
amount of fertilizer used and the leaching and/or runoff of nitrates into
waters (24,25). Crop yield and the application of chemical fertilizers
are directly related until a critical level of application is reached; beyond
that, they are indirectly related. In areas of excessive nitrate application,
plants tend to hold nitrates without metabolizing them, and crop yield
decreases (24) . Although the use of fertilizers by farmers may not always
meet prescribed application procedures, a recent survey indicates that
fertilizers are being administered properly (39). There was no statistical
relationship between fertilizer application and nitrate concentrations in
well waters on 47 farmsteads.
Detection of Nitrates
The analysis of nitrates in waters and feedstuffs are conducted either by
colorimetric measurements or by use of ion-specific electrodes.
The phenol-disulfonic acid method and the brucine method are colorimetric
procedures most frequently used in water nitrate analysis (29). In the
former test, a color reaction proportionate to the amount of nitrate present
is quantitated spectrophotometrically. In the latter procedure the color
intensity changes with time, and it is necessary to develop standards and
samples simultaneously (29). Both tests are quantitative, but interference
by nitrites or chlorides may occur.
31
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Several other tests have been reported for testing nitrates in natural
and waste waters (29). Nitrate analysis by reduction to ammonia, which
is detected by Nesslerization, or to nitrites which are detected by the
Griess-Ilsovay method, may be used. Ultraviolet and infared techniques
have also been suggested. Polarographic analysis for nitrate offers the
advantage of being adaptable to continuous monitoring.
The use of ion-specific electrodes is a quick and reliable method (29).
Although the electrode responds to anions other than nitrate, it does not
respond to cations. It is more selective for chlorate, periodide, and
chloride ions than for nitrate; thus, interferences may be expected. A
standard curve is prepared with known nitrate solutions, the sample is
then assayed, and results are obtained by extrapolation from the curve.
The electrode can be used for testing for nitrates in water or feedstuffs:
Nitrites may be determined in waters, serum, plasma, blood, and feedstuffs
by diazotization, using sulfonilic acid and alpha-naphthylamine hydro-
chloride to produce a reddish-purple color or by using sulfanilamide
and 1-naphthylethylene-diamine dihydrochloride to form a red color. These
color reactions are evaluated visually or with a spectrophotometer.
Several screening tests (spot-plate tests) are also available for nitrate
or nitrite. These tests are qualitative and only reveal the relative
amounts of the respective ions present. The diphenylamine procedure has
been very useful, and adaptation has been made to test for nitrates in
body fluids (20). The disulfanilic acid test is probably used most widely
for testing feedstuffs and water.
Treatment of Nitrate-Nitrite Toxicitv
The administration of a 1% solution of methylene blue is the treatment for
acute nitrate toxicity in all animals, including man. It is postulated
that methylene blue opens a new methemoglobin-reducing pathway requiring
TPHN and coenzyme factor II (22). However, the administration of excessive
methylene blue may cause additional methemoglobin formation (49). Recovery
in acute cases, when diagnosed early and treated promptly, usually occurs
within one-half hour of treatment. Removal from the nitrate-containing
water has resulted in spontaneous recovery in infants (2,49).
Treatment of chronic nitrate toxicity and signs of physiological inter-
ference consists of replacement therapy. High levels of vitamins A, D,
and E in the ration and added iodized salt and trace minerals are recommended,
Vitamin-A supplement alone has reversed signs of chronic nitrate toxicity
in cattle and swine (4). A good quality ration high in carbohydrates also
assists in treating chronic nitrate poisoning. Removal from dietary source
of nitrates is necessary for successful recovery.
Current Status
It is known that nitrates can and do accumulate in feeds, foods, and water,
and that this occurs largely as a result of man's own actions. Ingestion
of these chemicals can be hazardous in many ways. The accumulation of
nitrates in surface waters may also lead to biological destruction of these
32
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waters. Numerous regulations concerning sewage treatment, pollution
control, and use of nitrates in foods are currently in effect. Regulations
are also pending to place pollution taxes on large industries and feedlots.
Intense concern is currently directed at restricting the use of nitrates
in processed foods.
Although much is known about nitrates in our environment, more is still
required. What are the biochemical lesions occurring from the chronic
consumption of nitrates? Once nitrates are ingested, the effect and
excretion of various biological products require clarification. These
compounds may be precursors of several common disorders, such as anemia,
neoplasia, collogen disease, spontaneous abortions, and other idiopathic
conditions.
Specific research is needed to determine undetected nitrate sources. Soil-
borne sources are now being studied. Since nitrates are excreted in the
milk of cattle and women, some workers suggest that sufficient amounts
may occur in milk to cause infant toxicity. Ammonia fumes from feedlots
may be absorbed by nearby lakes and lead to pollution and eutrophication
(21).
Information is needed about the levels of nitrates that constitute hazards
to the human body. It is known that infants under 3 months of age and
pregnant mothers are most susceptible to nitrate toxicity. What about
persons with chronic digestive or respiratory problems? Nitrate poisoning
has developed from excessive nitrates in dialyzing solutions and from silver
nitrate applied to the skin of burn patients.
The occurrence of nitrosamines in processed meats and their role in produc-
ing cancer in rats has stimulated research. The United States Department
of Agriculture and the Food and Drug Administration are under increased
pressure to reevaluate guidelines for the addition of nitrates and nitrites
to foods.
The removal of nitrates from water is presently economically unfeasible.
New methods of water purification are needed, since nitrate concentrations
appear to be steadily increasing. Practical solutions to the problem of
farmsteads with high nitrate levels in well water are urgently required.
Merely finding a new low-nitrate water source is usually a difficult task.
Even when costs and attitudes are favorable for seeking another source,
water frequently cannot be found.
Only after these basic:problems are solved can further energies be directed
to controlling nitrate contamination of our feed, food, and water through
public education and governmental policies. However, a comfortable
understanding between environmental and agricultural agencies is necessary
for rational policies to result.
1 Summary •
Nitrates are an important part of natural ecosystems and may become
environmental problems if allowed to accumulate in waters and food. Toxicity
33
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is a common problem in animals; it is less frequent but still a significant
hazard in humans. The demand for greater crop productivity has increased
the use of nitrogenous fertilizers on cultivated and pasture land. Eco-
nomic pressures have created problems in handling wastes from large numbers
of food-producing animals concentrated in small areas. Water samples,
frequently miles away from the pollution source, have become contaminated
and unfit for human and animal consumption. The effects of nitrates on
the environment and man and animals are still confused. Measures are need-
ed to detect and prevent the occurrence of nitrates in water supplies and
to study the chronic effects of long-term ingestion of nitrate-containing
waters and foods.
References
1. Blood, D. C., and Henderson, J. A.: Nitrate and Nitrite Poisoning.
Veterinary Medicine, 3rd Ed., Williams and Wilkins Company, Baltimore,
1968.
2. Bosch, H. M., Rosenfield, A. B., Huston, R., Shipman, H. R., and Wood-
ward, F. L.: Methemoglobinemia and Minnesota Well Supplies. J^. Am.
Water Assoc., 42, (February 1950): 161.
3. Buck, W. B.: Diagnosis of Feed-Related Toxicosis. J.A.V.M.A., 156,
(1970): 1434.
4. Case, A. A.: Some Aspects of Nitrate Intoxication in Livestock.
J.A.V.M.A.. 130, (1957): 323.
5. Case, A. A.: Abortion in Swine Due to Excessive Nitrates. J.A.V.M.A.,
131, (1957): 226.
6. Comly, H. H.: Cyanosis in Infants Caused by Nitrates in Well Water.
J.A.M.A.. 129, (1945): 112.
7. Commoner, B.: Threats to the Integrity of the Nitrogen Cycle: Nitrogen
Compounds in Soil, Water, Atmosphere and Precipitation. Symposium on
Global Effects of Environmental Pollution, Dordrecht Pub. Co., Dallas,
1968, p. 70.
8. Committee on Nutrition: Infant Methemoglobinemia, The Role of Dietary
Nitrate. Pediatrics, 46, (September, 1970): 475.
9. Crowley, J. W.: Ways to Prevent Possible Nitrate Problems in Cattle.
Hoards Dairyman. 115, (1970): 934.
10. Curing Ingredients. The National Provisioner, 166, (February 12, 1972):
13.
11. Curtin, T. M., and London, W. T.: Nitrate-Nitrite Intoxication in Swine.
Proceedings 70th Annual Mtg. United States Livestock Sanitary Association,
(October, 1966): 339. :
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12. Dollahite, J. W., and Holt, E. C.: Nitrate Poisoning. S. A. Mediese
Tydskrif, 44, (1970): 171.
13. Federal Register (1968). Title 21, Ch. 1, Nos. 121, 1063, 121 and
1064.
14. Feth, J. H.: Nitrogen Compounds in Natural Water, A Review. J. Water
Resources Res.. 2, (1966): 51.
15. Galea, V., and Preda, N.: The Possible Formation of Nitrosomines in
Food Preserves, Abstract No. 5604. Excerpta Medica - Public Health,
Social Medicine and Hygiene, Vol. 17, No. 11, (1971): 952.
16. Greene, I., and Hiatt, E. P.: Behavior of the Nitrate Ion in the Dog.
Am. J. Physiol.. 176, (1954): 463.
17. Hanway, J. J., Herrick, J. B., Willrich, T. L., Bennett, C. C., and
McCall, J. T.: The Nitrate Problem. Special Report No. 34,
Cooperative Extension Service in Agriculture and Home Economics,
Ames, Iowa, 1963.
18. Harris, D. J., and Rhodes, H. A.: Nitrate and Nitrite Poisoning in
Cattle in Victoria. Australian Vet. J., 45, (1969): 590.
19. Hasler, A.: Man-Induced Eutrophication of Lakes. Symposium on
Global Effects of Environmental Pollution, Dordrecht Pub. Co.,
Dallas, 1968, p. 110.
20. Householder, G. T., Dollahite, J. W., and Hulse, R.: Diphenylamine
for the Diagnosis of Nitrate Intoxication. J.A.V.M.A., 148, (March 15,
1966): 662.
21. Hutchinson, G. L. and Viets, F. G., Jr.: Nitrogen Enrichment of
Surface Water by Absorption of Ammonia Volatilized from Cattle Feedlots.
Science, 166, (1969): 514.
22. Jaffe, E. R., and Heller, P.: Methemoglobinemia in Man. In Progress
in Hematology, Edited by Carl V. Moore and Elmer B. Brown, Vol. IV,
Grume and Stratton, New York, 1964, p. 48.
23. Kansas State Department of Health, Environmental Health Services:
Chemical Quality of Public Water Supplies in Kansas. Bulletin No.
1-7, Topeka, June 1965.
24. Keeney, D. R., and Gardner, W. R.: The Dynamics of Nitrogen Transformat-
ions in the Soil. Symposium on Global Effects of Environmental Pollution,
Dordrecht Pub. Co., Dallas, 1968, p. 96.
25. Keeney, D. R.: Nitrates in Plants and Waters. .J. Milk Food Tech.,
33, (October 1970): 425.
26. Keeney, D. R., and Walsh, L. M.: The Pollution Problem - Nitrates in
Ground Water. Hoard's Dairyman, 115, (October 1970): 980.
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27. Keller, W. D. and Smith, G. E.: Groundwater Contamination by Dissolved
Nitrate. The Geological Society of America, Special Paper No. 90, 1967.
28. Lijinsky, W., and Epstein, S. S.: Nitrosomines as Environmental
Carcinogens. Nature. 225, (1970): 21.
29. Mancy, K. H.: Instrumental Analysis for Water Pollution Control. Ann
Arbor Science Publishers, Inc., Ann Arbor, 1971, pp. 254-258.
30. Metzler, D. F.: An Investigation of the Sources and Seasonal Variations
of Nitrates in Private and Public Water Supply Wells, Particularly
with Reference to the Occurrence of Infant Cyanosis. Kansas State
Public Health Department, Project No. RG 4775, 1958.
31. Nitrites, Nitrosomines, and Cancer. The Lancet, London, 1, (1968): 1071.
32. Nitrosomine Found at Low Levels in Some Processed Products. The
National Provisioner. 166, (February 19, 1972): 54.
33. Oehme, F. W.: Unpublished data, 1971.
34. Olson, J. R., Oehme, F. W., and Carnahan, D. L.: Nitrate Levels in
Water and Livestock Feeds. Vet. Med./Sm. An. Clinician, 67, (1972): 257,
35. Orgeron, J. D., and Martin, J. D., and Caraway, C. T.: Methemoglobinemia
from Eating Meat with High Nitrite Content. Public Health Reports,
72, (1957): 189.
36. Osteryoung, J.: Nitrates: Human and Animal Health. In Environmental
Chemicals: Human and Animal Health. A. R. Yobs and E. P. Savage,
Editors. Colorado State University, Fort Collins, 1972, p. 151.
37. Phillips, E. E.: Naturally Occurring Nitrate and Nitrite in Foods in
Relation to Infant Methemoglobinemia. Food Cosmetic Toxicology, 9,
(1971): 219.
38. Public Health Service Drinking Water Standards. Public Health Service
Publ. No. 956, U. S. Dept. Health, Education and Welfare, Washington,
D. C., 1962.
39. Ridder, W. E.: Relationship of Nitrates in Kansas Ground-Waters to
Animal and Human Health. Thesis, Kansas State University, Manhattan,
1972.
40. Ridder, W. E., and Oehme, F. W.: Nitrates as an Environmental, Animal,
and Human Hazard. Clinical Toxicology, in press.
41. Ridder, W. E., Oehme, F. W., and Kelley, D. C.: Nitrates in Kansas
Groundwaters Related to Animal and Human Health. Am. J^ Public Health,
submitted for publication.
42. Van Meter, E. L.: Progress Report of the Rush County Nitrate Study.
Quoted 'in Rush County Water and Sewer Development, White, Hamele, Hunsley
and Associates, Salina, Kansas, 1972, pp. 116-131.
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43. Simon, J., Sund, 3. M. , Wright, M. J., and Douglas, F. D.:
Prevention of Noninfectuous Abortion in Cattle by Weed Control and
Fertilization Practices of Lowland Abortion. J.A.V.M.A. , 129,
(1955): 315.
44. Singer, R. H. : Environmental Nitrates and Animal Health. Southwest
. , 21, (Fall 1968): 13.
45. Smith, G. S . :. Diagnosis and Causes of Nitrate Poisoning. J.A.V.M.A. ,
147, (1965): 365.
46. Smith, J. E., and Beutler, E.: Methemoglobin Formation and Reduction
in Man and Various Animal Species. Am. J. Physiol., 210, (1966): 347.
47. Stoltenberg, H. I.: Sanitary Engineering Laboratory, Kansas State
Department of Health, Environmental Health Service, Topeka. Personal
Communication, 1972.
48. Tillman, A. D., Sheriha, G. M., and Sirny, R. J.: Nitrate Reduction
Studies with Sheep. J. An. Sci., 24, (1965): 1140.
49. Walton, G.: Survey of Literature Relating to Infant Methemoglobinemia
Due to Nitrate-Contaminated Water. Am. J. Public Health, 41 (August
1951): 986.
50. Winter, A. J.: Studies on Nitrate Metabolism in Cattle. Am. J. Vet.
Res.., 23, (1962): 500.
51. Wright, A. J., and Davidson, K. L.: Nitrate Accumulation in Crops
and Nitrate Poisoning in Animals. Advances in Agronomy, 16, (1964):
197.
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38
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CORRELATION OF NITRATE LEVELS AND HUMAN
HEALTH STATUS IN A WATERSHED
Janet G. Osteryoung
Colorado State University
Several recent articles and reports have reviewed the problem
of the occurrence and health hazards of nitrate (1-3). Our objective
is to cover briefly the health effects of nitrates and then to discuss
the requirements for epidemiological studies designed to explore the
relations between nitrate exposure and health for those health problems
in which a mechanism is not established.
The best understood health effect of nitrates on humans is
methemoglobinemia, which arises from reduction of hemoglobin by nitrite
either ingested or produced from nitrate in vivo. The reduction product
of hemoblobin, methemoglobin, cannot carry oxygen, and the clinical
effects in methemoglobinemia include cyanosis, apnea, and other symptoms
of impaired oxygen supply. Normal (steady state) methemoglobin concentra-
tions in the blood of adults are approximately 2% of total hemoglobin.
Levels substantially raised above this through exposure to high nitrate
levels in water or high nitrate or nitrite levels in foods can lead
to cyanosis and ultimately death. The populations especially at risk
are infants, pregnant women, and perhaps individuals with vitamin C
deficiency. The risk factor depends on the ability of the body to
reduce nitrate, the usual form of exposure, to nitrite, the toxic
species, and upon the ability of the methemoglobin reductase system
to convert methemoglobin back to hemoglobin.
Two recent studies have attempted to define the health effects of
sub-clinical methemoglobinemia and to establish normal values for methem-
oglobin in the populations at highest risk. Shuval and Gruener (4) have
studied methemoglobin levels in infants in Israel in areas of high nitrate
water supplies. They found no significant difference between the study
and control group blood methemoglobin levels by age, vitamin C intake,
use of formula or whole milk, and incidence of diarrhea. However, all
of the usual associations appeared as statistically insignificant indica-
tions . That is, higher vitamin C intake was associated with lower methem-
oglobin levels, and younger, lighter infants and those with diarrhea tended
to have higher methemoglobin levels. The range of values found was 0.9 -
1.47% and the average value was 1.1%. It is significant that in the
populations studied, fresh citrus fruits form a large part of the diet,
and 94% of the infants were breast fed or fed whole cow's milk. These
two factors tend to minimize the production of nitrite from nitrate in
the diet and to reduce nitrate exposure. Auxilliary evidence from clinical
records in Israel supports an association between diarrhea and methemoglobinemia.
In addition to epidemiological studies, Shuval and Gruener carried out
experiments on the toxicity of nitrite to rats under various conditions.
Of particular importance were those which showed long term effects after
the administration of nitrite had been stopped, and the great susceptibility
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of both dams and fetuses to serious health effects from nitrite exposure.
The original paper should be consulted for the details of this important
work.
Shearer e£ al (5) have studied methemoglobin levels in infants in
an area of California with high nitrate levels in the drinking water sup-
plies. In their study group individuals were estimated to have an exposure
to nitrate of greater than 22 mg N03~/da or approximately 6 mg/kg/da. A
3.6 kg baby drinking 2 liters of water a day with the maximum nitrate con-
centration recommended by the U.S. Public Health Service (45 mg/liter) would
have an exposure of 25 mg/kg/da. High methemoglobin levels were defined
as those over 4%. It was found that the normalized ratio of high methemo-
globin levels (study/control) was 5 for infants with diarrhea or upper
respiratory illness and 3 for well infants. This study provides convincing
evidence for widespread subclinical methemoglobinemia as result of the
exposure of infants to nitrate through water supplies high in nitrates,
even though the nitrate levels are within recommended standards. The public
health significance of these data cannot yet be evaluated however, because
there is no solid information on the effects of subclinical methemoglobinemia
on infant health or the health of the general population.
A second problem associated with nitrate exposure which is currently
receiving much attention is the production of carcinogenic nitrosamines.
Nitrite formed by nitrate reduction can react with secondary amines and
with other amine compounds to form nitrosamines:
+ RR"NNO + OH~
The compounds RR"NNO, the N-nitrosamines, are for most choices of R
and R" potent carcinogens and teratogens whose no effect levels have not
been established. Of the approximately 100 N-nitrosamines which have been
tested, more than 80% are carcinogenic in more than one test species. They
are organotropic in general, and more than 25% of those tested are specific
for esophageal cancer. There are good general arguments for taking the
results of tests of carcinogenic behavior on animals seriously. First,
cellular structure and mechanisms in the various species are similar, and
cancer is certainly a cellular phenomenon. Second, there is no case of
an identified compound carcinogenic in humans which has not produced cancers
in some animal species. In addition, with the tJ-nitrosamines, a thorough
investigation has been carried out of the metabolism of dimethylnitrosamine,
Me2NNO, in vitro in human and rat liver; the results were similar. Also,
the acute toxicity of N-nitrosamines in humans and animals such as the rat
is the same.
At what levels are N^-nitrosamines carcinogenic and to what extent
is the general population or selected high risk populations exposed to
these compounds? A recent publication of the International Agency for
Research on Cancer contains material bearing on these questions (6).
First, what exposure levels should be considered important? In
experiments using rats with 20-30 animals in each test group the following
results have been obtained. Using dimethylnitrosamine, Me2NNO, a single
40
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dose of 20 mg/kg produced cancers in all of the test animals. Continuous
doses of 2 mg/kg/da produced cancer in one of 26 test animals at 60 weeks.
In another set of continuous feeding studies, doses of greater than 0.15
mg/kg/da produced tumors in 100% of the test animals, 0.15 mg/kg/da pro-
duced liver cancer in 27 of 30 animals, and 0.075 mg/kg/da produced malign
or benign tumors of the liver and esophagus in 11 of 20 animals. A dose of
0.075 mg/kg/da corresponds to 0.75 - 1.7 mg/kg in the diet. The "marginal
effect" level is estimated to be 0.5 - 0.75 mg/kg. If a safety factor of
100 is allowed, this corresponds to a concern about contamination of foods
at the 5-10 yg/kg level.
The question of the exposure of people to fl-nitrosamines is especially
complicated by the extremely low levels which might be important, the dif-
ficulty of developing adequate analytical procedures, the lability of N-
nitrosamines, and the ubiquitous presence of their precursors in foods and
the general environment. In the case of foods, the main problem is the
deliberate adulteration of processed foods, especially corned meat products,
with nitrates and nitrites. From laboratory studies of the rate of JN-
nitrosamine formation from nitrite and various amine compounds as a function
of pH and of temperature, it is reasonable to assume that products such as
hot dogs can contain substantial amounts of N-nitrosamines. The problem
of analyzing meat products for _N-nitrosamines has been pursued in this
country primarily by Wasserman and coworkers (7); a typical result by Sen
(8) shows 5 of 59 samples tested having dimethylnitrosamine concentrations
in the range 0.01 - 0.08 ppm, or 10 to 100 times the 5-10 yg/kg levels
mentioned above. There are several well documented cases of animals (mink
and sheep) which have developed hepatic disease and cancer after being fed
fish meal, which is naturally high in trimethylamine, and which is preserved
with nitrite (1).
A second possible route of _N-nitrosamine exposure is the in vivo
synthesis of IJ-nitrosamines from nitrite and various amines. A class of
amine precursors of especial interest are drugs, particularly those which
are usedover a long time span, which contain functional groups which can
be nitrosated. Two important examples are disulfuram (antabuse) and
tolazamide, a hypoglycemic agent. These are administered to patients on
a long term basis at levels of 10 - 20 mg/kg/da. Compare this with the
marginal effect dose of dimethylnitrosamine in rats, 0.075 mg/kg/da. Less
than 1% nitrosation of the drug would correspond to roughly the same dose
in humans.
Concern about possible in vivo synthesis of N-nitrosamines is based
upon more than calculations and purely chemical experiments (6). Rats fed
a diet 0.08% in NaNC>2 and 0.25% in N-methylbenzylamine have developed esoph-
ageal tumors. A diet of 0.5% NaN02 anc' 0-5% morpholine for 56 days produced
death within one year from liver tumors. For comparisons sake, the recom-
mended maximum concentration of NaNC>2 in the diet of cattle is 1.5% and in
the diet of humans is 0.02%. Another important set of experiments involves
the development of lung adenomas in rats as a result of feeding diets con-
taining an IJ-nitrosamine, or a combination of nitrite and the secondary
amine precursor. Assuming that in the second case the N-nitrosamine is
41
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produced in the stomach of the rat, one can calculate the rate of production
of the N-nitrosamine from the concentrations, pH, and temperature in the
stomach. In these experiments, it was found that the ratio of the percentage
of lung adenomas in the group fed the precursors to the percentage in the
group fed the N-nitrosamine was equal to the calculated rate of production
of the N-nitrosamine in vivo. This is impressive evidence that the predic-
tions concerning In vivo synthesis are at least semi-quantitatively correct.
A further point of interest concerns the catalytic effect of thiocyanate
on the nitrosation reaction. Independent of the substrate, thiocyanate
causes a 20- to 25-fold increase in the rate of nitrosamine production at
pH 2 (the gastric pH) at a concentration of 0.1 mM. The typical concentra-
tion in human saliva is 0.5 - 1.0 mM, and 5-10 fold dilution occurs in
the stomach, resulting in gastric thiocyanate concentrations in the range
0.05 - 0.2 mM. Since the rate of nitrosamine production is linear in the
catalyst concentration, these concentrations would cause a 10 - 50 fold rate
increase in nitrosamine production. Smoking increases the level of thiocyanate
in saliva to about 5 mM which would result in a rate enhancement of 100 -
250 times the uncatalyzed rate. It has been suggested (6) that this effect
may be related to the incidence of cancer of the upper alimentary tract in
smokers.
It is generally accepted that the N-nitrosamines are potent carcinogens,
that they are teratogenic and mutagenic, and that they are extremely toxic
and dangerous. Although analytical problems have tended to obscure the
evidence bearing on the occurrence of N-nitrosamines in the environment
and the extent of human exposure to these compounds, there can now be no
serious disagreement with the statement that these compounds are formed in
small amounts in the environment, and that one of the matrices particularly
favoring their formation is various nitrited food products. It is also
generally conceded that the majority, perhaps 80 - 90%, of cancers are caused
by some environmental insult which is usually chemical. However, there is
now no evidence, positive or negative, that exposure to IJ-nitrosamines con-
stitutes a significant cause of cancer in the United States or in other
parts of the world. The answer to this question could be an important
contribution to methods of cancer prevention; failure to pursue the question
would be foolhardy.
The International Agency for Research in Cancer is now conducting
epidemiological studies in two areas of the world, the Caspian littoral
of Iran and Brittany in France, in which there is an unusually high incidence
of esophageal cancer. The ratio of incidence of esophageal cancer in males
to females is 3:1 world-wide while in the Iranian study area it is 0.6:1
The incidence in the Iranian study area divided by the incidence in the world
as a whole is 6 for males and 32 for females. Nitrosamines are being in-
vestigated as causal factors which might account for this unusual distribu-
tion (6, 9-10). This large and well organized study might provide informa-
tion on the importance of N-nitrosamines as environmental carcinogens.
Nitrate or nitrite exposure has also been associated with incidence
of essential hypertension and ischemic heart disease (1). There are also
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many clinical reports involving nitrate or nitrite exposure and myocardial
infarction. A recent and typical example (11) involved a 67 year old male
with a history of hypertension and myocardial infarction who ate sausage .
for dinner which caused cyanosis and other symptoms of methemoglobinemia
accompmiled by myocardial infarction. In this example, the correspondant
comments that the risk to health of an overdose makes the. use of nitrites
in foods unjustified even though the incidence of acute problems is low.
The problem of estimating the effects of continued exposure to nitrates
through food or water on general health is a much more difficult one than
that of detecting clinical episodes of poisoning. In particular, the sug-
gestion that essential hypertension and ischemic heart disease are associated
with high nitrate exposure bears on the most important public health problem
in the United States today, and must be further investigated. The require-
ments of such a study are more demanding than the usual epidemiological
study, because of the many sources of exposure, the complex interaction
between nitrate exposure and nitrite production in vivo, the association
of essential hypertension and ischemic heart disease with a host of other
environmental and genetic variables, and the notably bad record of epi-
demiological studies of heart disease. With the exception of industrial
chemical carcinogens, retrospective studies have had little success in
solving this sort of problem, and prospective studies, such as the Framingham
Study, have failed to meet the expectations for results, despite their cost.
Studies designed to investigate the connection between nitrate exposure
and hypertension or ischemic heart disease can be simplified by choosing
populations which have a large, relatively constant exposure to nitrate
through their water supply and comparing them with similar interspersed
populations which are not exposed to nitrate through that source. A health
index survey of these populations not only establishes the necessary back-
ground information, such as estimates of individual vitamin C intake, but
also provides information, such as immunization records, of general use to
public health personnel. A non-intrusive measurement, determination of
blood pressure, provides data that can be correlated with nitrate exposure
and other environmental factors. This measurement can be done by people of
modest training and skill, and again provides useful information, not only
to public health personnel, but to the individual, for early detection and
control of hypertension reduces the later incidence of hypertensive heart
disease. The establishment of a clear connection between hypertension and
nitrate exposure through such an approach would not only lead to immediate
prophylactic measures, but could also provide some scientific clues to help
understand the puzzling etiplogy of essential hypertension.
It is our view, dispite the contrary opinion of the National Academy
of Sciences/National Research Council (3), that human exposure to nitrates
could prove ^p_ _be an important public health problem. In light of the
evidence briefly summarized here, it would be most surprising if their
effeicts were found to be insignificant. At the present state of our know-
ledge it is the responsibility of scientists who can contribute to further
understanding of this problem to do so, either through their own research,
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through assistance to others, or through general efforts to keep abreast
of the literature in this area and to serve an educational function. in
addition, it is rational and conservative to make every effort which does
not cause serious economic dislocations to reduce nitrate and nitrite exposure
from food and water. In particular, public health officials should, within
the limits of their resources, encourage the use of alternative water supplies
in areas where they are contaminated with nitrate, especially for infants
and pregnant women. Physicians should explore the advisability of removing
preserved meat products from the diets of patients with a history of ischemic
heart disease. An finally, through "illogical" groupings of scientists,
or through serendipity, people of many disciplines and professions must
work together to obtain the right questions about nitrates in the environ-
ment and to obtain the best possible answers to those questions.
References
1. Osteryoung, J. Nitrates and water quality. In Environmental Chemicals;
Human and Animal Health. A. R. Yobs and E. P. Savage, Eds. Colorado
State University, Ft. Collins, pp. 151-172, 1972.
2. Oehme, F. W. Nitrates as human and animal health hazards. These
proceedings. 1973.
3. National Academy of Sciences/National Research Council. Accumulation
of nitrates. 1973.
4. Shuval, H. I. and N. Gruener. Epidemiological and toxicological aspects
of nitrates and nitrites in the environment. Am. J. Public Health.
62:1045-1062, 1972
5. Shearer, L. A., J. R. Goldsmith, L. Young, 0. A. Kearns and B. R. Tamplin.
Methemoglobin levels in infants in an area with high nitrate water supply.
Am. J. Public Health. 62 (9):1174-1180, 1972.
6. Bogovski, P., R. Preussman, E. A. Walker and W. Davis, Eds. N-Nitroso
compounds. Analysis and formation. IARC Scientific Publications No. 3.
1972.
7. Wassennan, A. E., W. Fiddler, R. C. Doerr, S. F. Osman and C. J. Dooley.
Dimethlnitrosamine in frankfurters. Food Cosmet. Toxicol. 10(5):681-
684, 1972.
8. Sen, N. P. The evidence for the presence of dimethyInitrosamine in meat
products. Food Cosmet. Toxicol. 10(2):219-223, 1972.
9. Kmet, J. and Mahboubi, E. Esophageal cancer in the Caspian littoral of
Iran: Initial studies. Science. 75:846-853, 1972.
10. Leete, E., W. C. Ellerbrock and J. Kmet and E. Mahboubi. Esophageal
cancer. Science. 179:228, 1973.
11. Lacher, J. W. Acquired methemoglobinemia. Rocky Mountain Medical
Journal. 70:41-42, 1973.
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THE INDUSTRIAL HYGIENE SIGNIFICANCE OF ISOCYANATE EXPOSURES
B. Gunter
National Institute of Occupational Safety and Health
Denver, Colorado
The isocyanates are a group of neutral derivatives of primary
amines and are of considerable industrial importance. Industrial uses
of the isocyanates include the production of polyurethane elastomers
for the manufacture of a wide range of products: rigid foam resins,
synthetic rubber, plastic coating for wire, in paints, varnishes,
adhesives, etc.
The first week in May, NIOSH received a request from an employer
in Colorado who had accidentally exposed himself and all of his employees
to toxic concentrations of isocyanate. As a result, they all became
sensitized to isocyanate. The results of this investigation will be
discussed.
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46
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LABORATORY IDENTIFICATION OF PETROLEUM POLLUTION PROBLEMS
William S. Dunn
Colorado State Department of Health
The problem of underground contamination of soil and water by
petroleum products has plagued both Public Health and Oil Inspection
Officials for years. In 1971-72, those of us in Public Health noticed
a very sharp increase in the number of incidents involving pollution of
waters in Colorado by petroleum products. Whether this is due to
better case finding and reporting or whether it is caused by an actual
increase in leakage or spills is difficult to determine. In any event,
we are all seeing some devastating effects of petroleum pollution.
Outside of the pure economic loss to the retailer, there now
appears the specter of litigation to recover damages to property, i.e.,
the Telephone Company spill in Englewood where considerable drilling
had to be done and time spent by company personnel in isolating and
assessing the hazard of gasoline under a building. Another example is
a restaurant that spent considerable money to bring a public water
supply to its premises because gasoline from an unknown source had
entered their private well.
If the petroleum product reaches navigable waters, then the EPA,
replete with lawyers and other "experts," is anxious, willing and able
to prosecute the miscreants. Our own State Water Pollution Control
Division must legally take action after a period of conciliation,
negotiation, arbitration and persuasion.
For the most part, contamination is unintentional and usually
due to lack of attention to inventory records, poor housekeeping, or
ignorance of the effects of pollution but a number of incidents are, in
my opinion, "pre-planned accidents" by companies or people who simply
don't give a damn. The problems as you can see are far more numerous
than the solutions.
Gasoline (and most other petroleum products) is toxic when ingested-
-even in rather small amounts. Assuming that one can get the water and
gasoline mixture past the nose and into the stomach without gagging, the
combination of hydrocarbons and additives such as tetraethyl lead,
ethylene bromide, and tricresyl phosphate can cause severe problems.
Plants, too,react adversely to water-gasoline mixtures. Soil character-
istics are also changed. These effects just described are long-term
effects and few if any effective means are available to Public Health
officials to counterattack the problems created.
A thousand gallons of gasoline are, figuratively, a drop in the
bucket in terms of Denver's daily consumption; but a drop of gasoline in
a citizen's drinking or irrigation water is too much and promptly brings
howls :of protest to Public Health officials to "Do something—NOW!"
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Identification of petroleum products by laboratory methodology is
still in a trial and error, cut and fit stage. Our efforts in analysis
of gasoline, especially, have followed several avenues. They can be
summarized in the following categories:
1. Gas chromatography by flame ionization
2. Gas chromatography by flame photometric detection
3. Infra-red spectrophotometry
4. Ultra-violet spectrophotometry
5. Column chromatography and solvent stripping
6. Thin layer chromatography of column extracts
7. Thin layer chromatography of dye components.
Despite all these techniques, we have only been moderately success-
ful. Most retail products in the Rocky Mountain area have a common
crudestock source. Refining processes are very similar, thus the
gasolines are very similar in the amount and type of hydrocarbon
constituent. The gasolines, as you are aware, all contain sulfur
compounds of various types, such as mercaptans, organic sulfides and
other thio compounds. Another distinguishing characteristic of
gasolines are the additives. But the type, number and amount of these
additives are closely guarded corporate secrets and access to specific
information is essentially non-existent. Certain classes of chemicals
are listed in patents for anti-knock, octane number improvers, and
deposit prevention but we are not now in the position to determine them
or their degradation products since they number in the hundreds of
chemicals, often in very small amounts.
One of the magic "black boxes" employed by chemists is called a
gas chromatograph (GC). While a thoroughly complicated looking instru-
ment, it has a basic purpose. It must have a means of separating
components and then a means of identifying them. To separate components,
a tube is filled with diatomaceous earth coated with a Carbowax compound.
This column is placed in the GC and a carrier gas such as nitrogen is
constantly passed through it. A tiny sample of gasoline is injected and
swept onto the column. The hydrocarbon components dissolve in the
Carbowax coating for an instant. Some are held in the coating for a
fraction of a second longer than others before being swept to the next
coated particle. By the time it passes through billions of particles,
these differences of a fraction of a second are extended to several
seconds or minutes so that a separation of the various components is
made. Detection of the compounds as they emerge from the column is
accomplished by a hydrogen-oxygen flame that burns the compound. The
change in electrical current called ionization is measured and converted
to a graph.
To detect the various sulfur compounds in gasoline, another column,
this time coated with a methyl silicone gum, is placed in the GC. As in
the previous case, an injected gasoline sample is swept by the carrier
gas onto the column, separated as before by the difference in solubility
in the coating, and passed into a Flame Photometric Detector. The
48
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compounds are burned as before but this time a photometric cell detects
a certain wavelength of light when compounds of sulfur or phosphorus
are burned.
In the infra-red spectrophotometry technique, a sample of gasoline
is placed between a source of infra-red waves and a. photocell that
can detect changes in the amount of light in the infra-red region.
Depending upon the number, type, and configuration of the atoms in the
molecules, the amount of infra-red light absorbed by the compounds can
be measured and converted into a graph.
Ultra-violet spectrophotometry works in a somewhat similar way
except that the wavelength of the light is in the ultra-violet region
rather than the infra-red region. The fluorescence of compounds is such
that many kinds of fluorescence are detectable by eye.
Column chromatography has been applied to identification of heavier
petroleum products such as lubricating greases, asphalts, tars, gilsonite
and crude sludges. In this technique, a buret is filled with a special
material called Florisil, a highly absorptive magnesium alumino-silicate;
and a sample of material to be identified is passed onto and through
the column. Light hydrocarbons that are highly non-polar are poorly
absorbed and pass through readily, but other compounds that do conduct
small amounts of electricity because of their molecular make-up and
configuration are absorbed. To separate these compounds, then, a series
of solvents is passed through the column, beginning with a non-polar
pentane, followed by other solvents that have a higher dielectric
constant number, that is, solvents that would conduct electricity more
readily. The solvents used are pentane, cyclohexane, carbon tetra-
chloride, benzene, ethyl ether, chloroform, acetone and finally methyl
alcohol. Each solvent strips certain individual components from the
column. Many of them have different visible colors and also different
colors when viewed by a black light.
After separating the components into various fractions by column
chromatography, another technique called thin-layer chromatography is
used. As the name indicates, a thin layer of silica-gel or alumina is
spread on a plastic sheet or glass plate. A spot of the material to be
separated is placed near the bottom of the sheet and placed in a tank
or vessel so the bottom of the sheet contacts a solvent. By capillary
action, the solvent moves up the plate slowly. Certain components of
the original spot are carried up the sheet, too. Some compounds are
more tightly bound to the silica-gel than other compounds. Those less
tightly bound move up the sheet further, and thus a separation is made.
The secret of thin-layer chromatography (TLC) is to know what solvents
or combination of solvents to use to effect a separation of various
compounds. Many of the column extracts are colorless or yellow-brown
to the naked eye but when viewed with a short-wave ultra-violet light,
many fluorescing compounds of blues, greens, yellows and browns are
seen. This serves as a means of comparison.
As you know, gasolines are colored by dyes—orange or bronze for
regular, red for premium, and blue for non-lead. Our most successful
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identification of petroleum pollution has been in this area. For
example, the dyes from Conoco Refinery, Refinery Corporation, and Denver
Pipeline products all appear to be identical to the observer; but when
separated by the TLC technique just described, a magic separation into
several individual dyes occurs. To accomplish this, a sample of
gasoline (about 15 ml) is passed through the Florisil column, washed
with carbon tetrachloride to remove excess gasoline and interferences
while leaving the dye absorbed. Addition of methyl alcohol is used to
push the dye off the column where it is recovered, evaporated and made
ready for TLC analysis. In the case of gasoline dyes, the different
colors are readily visible to the eye. In other TLC techniques,
identification must be made by spraying with other chemicals to produce
colors or spots. In this case, we have a built-in ready-made color.
Now that I have described the various techniques used by the
laboratory, let me tell you what successes and failures we have had.
In the case of GC by flame ionization, the gasolines are identical or
so nearly so that no identification can be made, whether they come from
Conoco, Tenneco, or Pipeline Products. Furthermore, extended contact
with ground and water changes them. The light volatile fractions soon
disappear; water dissolves some components more readily than others.
Thus, this technique is not of much use for identification. We can,
however, tell you whether the sample is gasoline, kerosene, light
diesel, mineral spirits, Stoddard Solvent, or lubricating oil.
Gas chromatography by flame photometric detection suffers from the
same problems. There is a lot of natural variation from day to day in
the refined product and contact with ground changes these compounds.
Furthermore, other sulfur compounds are leached from the soil and water
by the gasoline. No useful information can be achieved by this technique.
Infra-red spectrophotometry shows only that the gasoline is
primarily carbon and hydrogen atoms hooked together and cannot detect
minor differences in configuration. No useful information can be
achieved. Ultra-violet spectrophotometry shows no basic differences
in gasolines but can be somewhat useful in basic identification of
asphalts and tars.
Column chromatography and solvent stripping are useful in
determining asphalts, tars, lubricating oils, etc., but not for specific
identification of individual brands of products.
TLC of column extracts has been fairly successful in comparing
asphalts, tars, lubricating oils, fats, etc., when viewed by a short-wave
ultra-violet light, but lacks specific identification characteristics
due to close similarity of asphalts from various sources.
Our best success so far has been with dye identification of TLC
plates. This tells us which refinery the gasoline came from. But it
cannot tell the brand because many different companies purchase gasoline
from the same refinery. It is a simple rapid technique that is helpful
if about 10-15 ml of gasoline are available. If the dye is degraded by
soil components or water, the identification fails. If only gasoline-
saturated water is available, it fails since the dye is not water soluble.
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In conclusion, there are far more problems than solutions.
Despite all of our sophisticated laboratory instrumentation and chemical
techniques, no absolute identification can be made in most instances.
It comes right back to the most important thing—prevention of pollution
by petroleum. This means a lot of work by field personnel as they are
still the most important factor in the investigative process.
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52
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CONTROLLING EMISSIONS FROM NEW AND USED CARS
AND ALTERNATIVE MOTOR VEHICLE POWER SOURCES
Lane W. Kirkpatrick
Colorado Air Pollution Control Commission
America's cities are hurting to a large extent because of the auto-
mobile. Its exhaust is asphyxiating them, its demands are bankrupting
them, and its proliferation is, like the camel in the tent, forcing out
the trolleys, trains, buses, and people that give the cities life! (1)
Though I judge that urban air pollution problems will only be solved
using a pluralistic combination of control strategies, including vehicle
inspection and maintenance, vehicle modifications, and transportation
and land use planning and controls, today I will limit my presentation
to a very limited aspect of the automobile emissions control scene.
I have prepared this presentation because most people, including
environmental health workers, are interested in how air pollution control
systems on new and used cars will work. They are also interested in
how alternate power sources for motor vehicles may prove to be less
polluting than the internal combustion piston engine.
Control Systems on Pre-1974 Automobiles
Many air pollution control retrofit systems for older cars are
adaptations of the new car systems. So, in describing the following
systems, I do not differentiate between the new and used cars.
The principal systems for the control of emissions on new and used
cars include:
1. Positive crankcase ventilation (PCV)
2. Air pump (air injection system)
3. Evaporative emission control system
4. Exhaust gas recirculation (EGR)
5. Retarded spark.
Major Sources of Vehicle Emissions
The above-mentioned systems control the major sources of vehicle
emissions: exhaust gases, fuel evaporation, and crankcase vapors.
Exhaust emissions are simply those emissions of the exhaust system
of the car, either from the tailpipe or from leaks in the exhaust system.
Fuel evaporation emissions include fuel tank evaporation vapors as
a result of a non-sealed fuel tank, fuel evaporation from the carburetor
area, or evaporation as a result of fuel line leaks.
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Crankcase vapors are primarily unburned fuel and lubricating oil
vapors that escape complete combustion in the combustion chamber and slip
past the piston and piston rings into the crankcase area. This area is
opposite the piston from the combustion chamber. In the uncontrolled
vehicle, these gases are vented to the atmosphere.
System Description and Checks for Proper Operation
All examples presented here are simplified, and certain manufacturers
have systems that deviate from the basic systems described.
Positive crankcase ventilation. The positive crankcase ventilation
(PCV) valve regulates the flow rate of unburned crankcase vapors being
fed back into the intake manifold (found directly below the carburetor)
for combustion by a return to the combustion chamber. The PCV valve
allows for greater crankcase ventilation (or unburned combustion gas
recirculation) when greater acceleration and speed is demanded of the
vehicle. In more detail, ambient air usually passes through the air
filter, then through a hose connecting the air cleaner to the oil filter
cap. This filter cap is sealed to prevent the entrance of unfiltered
atmospheric air. From the oil filter cap, the air flows into the rocker
arm chamber and into the crankcase where it picks up "blowby" gases
(unburned gases that slip past the piston) and other vapor contaminants.
It then flows through a spring-loaded positive crankcase ventilation
valve and by hose to the carburetor base, into the intake manifold, and
ultimately to the combustion chambers.
The way to check the effectiveness of the PCV valve is to disconnect
it, attach a vacuum gauge or flow meter while the engine is operating
and see if the valve meets flow specifications (drawing about 15 inches
of mercury or 1.5 c.f.m. at idle for larger engines). A crude check can
be made, after disconnection, by shaking the device—a rattling sound
usually indicates the valve is working and is not stuck. Also, when the
engine is running, a "hiss" may be heard if the valve is working, and
a strong vacuum pull should be felt when a finger is alternately placed
over the valve outlet and removed.
Air injection system air pump. The air injection system is used to
add a controlled amount of air (hence oxygen) to the exhaust ports of
the combustion chambers, thus burning an appreciable amount of carbon
monoxide and hydrocarbons in the exhaust stream. Carbon monoxide and
hydrocarbon pollutants are, by this method, converted to carbon dioxide
and water vapor—both harmless gases.
The air injection system is comprised of a belt-driven air pump with
its associated plumbing. The pump takes ambient air and forces it through
an air distribution manifold, then through air injection tubes to the
exhaust manifold adjacent to each exhaust port.
Inspection of this system includes checking that the air pump belt
drive is in place and that it has the proper belt tension, that air in-
takes on the pump are free of obstructions, that the plumbing is properly
connected, and that the auto backfire valve and pressure release valve
on this system are operating according to manufacturer's specifications.
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Evaporative emission control systems. The evaporative control
system is a charcoal canister system which collects fuel vapors from the
fuel tank and the carburetor in an activated charcoal filled canister
usually located under the hood. Carburetor fuel evaporation vapors are
collected in the canister during vehicle non-operation; then during
operation, a reverse process occurs wherein vapors are drawn from the
canister back into the intake system and burned in the engine. Evapora-
tion vapors from the fuel tank are also stored in this canister during
vehicle operation and non-operation. This is a closed system which does
not allow fuel evaporation vapors to be released to the atmosphere.
Exhaust gas recirculation (EGR). The above systems are primarily
for the control of unburned hydrocarbons and to some degree, carbon
monoxide pollutants. EGR is primarily to control nitrogen oxide
emissions, and, to a lesser degree, other pollutants. The basic theory
of EGR is that in this system recirculated exhaust gas acts as an inert
dilutaht in the combustion chamber and serves to minimize combustion
temperatures, thereby reducing nitrogen oxide emission levels. This
system is controlled by a metering valve between the exhaust gas takeoff
and the intake manifold. This valve varies the amount of exhaust gas
admitted into the intake manifold by responding to a vacuum signal from
the carburetor.
Visual inspection of this system is difficult and about all that
can be done is to check to see that the vacuum hose from the carburetor
to the EGR valve is intact. Also, the top of the valve may be removed
to inspect as to whether the valve operates freely.
Retarded spark. Retarded spark means delaying, for a fraction of a
second, the time at which the spark is applied to start the combustion
of gases in the combustion chamber. This is a major way in which vehicle
manufacturers have controlled all three major vehicle pollutants—carbon
monoxide, hydrocarbons, and nitrogen oxides. Because of spark retard,
engine coolant temperatures are raised, primarily because less of the
combustion heat is converted to engine power, and this heat must be
extracted from around the combustion chambers. Correspondingly, engine
performance and gasoline economy are adversely affected because power
is lost for the sake of emission control.
Nitrogen oxides are controlled as a result of spark retard because
a cooler burn occurs. Carbon monoxide pollutants and hydrocarbon
pollutants are controlled because the burning occurs over a longer time
period, resulting in more complete combustion.
Control Systems for 1975 and 1976 Automobiles
1975 Federal new car emission control standards require about a 90%
reduction in carbon monoxide and hydrocarbon emissions over the 1970
cars. The National Research Council reports that four types of engines
will be able to meet this Federal standard. They are:
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1. A modified conventional engine equipped with an
oxidation catalyst
2. The carbureted stratified charge engine
3. The Wankel engine equipped with a thermal reactor
4. The diesel engine.
Of these four engine types, only the Wankel is seen by the National
Research Council as having a fair chance of meeting the Federal 1976
emission standards for nitrogen oxides; however, this standard is one
which the Environmental Protection Agency is considering relaxing.
A Description and Analysis of Alternative Engine Types
for Future Automobiles
A Modified Conventional Engine Equipped with an Oxidation Catalyst
This alternative is the internal combustion engine with all kinds
of gadgets and equipment added, including all emission control systems
heretofore mentioned as used on the pre-1974 vehicles. In addition,
there must be the installation in the car's exhaust system of a platinum
catalystic device designed to further control carbon monoxide and hydro-
carbons, and a second catalystic device and/or lower compression ratios
to control nitrogen oxides to meet the 1976 standards. Cost estimates
for the dual catalystic system vary but may be about $270, which may be
a disadvantage compared to other systems. Other disadvantages are
thought to be fuel economy, maintainability, durability, and availability
of both non-leaded gasoline and platinum catalyst material.
Carbureted Stratified Charge Engine
Combustion in a stratified charge engine is much like cupping your
hands to light a cigarette in the wind. A pre-combustion chamber tightly
encloses all the elements necessary to start combustion: an extra rich
fuel mixture from the carburetor or from a small pocket of air with a
dose of fuel from an injector, and a spark from the nearby sparkplug.
The initial mixture is very rich and easy to ignite. As the charge burns,
it moves out into the piston cylinder and forces the piston down. Some
unburned fuel is also driven out of the small pocket when an injector is
used, and it mixes with additional air in the cylinder supplied by a
second intake port to form a lean air-fuel mixture. (Another design
supplies the leaner fuel-air mixture directly from a second carburetor.)
This lean fuel-air mixture is progressively leaner with increasing
distance from the pre-combustion chamber. Hence the charge is "strati-
fied" (varying in fuel-air ratio). All other aspects of the engine are
typical of current piston power plants.
The cost of this system is favorable, about $70, when adapted for
smaller vehicles. Other advantages are improved gasoline mileage and
less retooling requirements for the auto manufacturers. Some loss of
performance may occur.
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The Wankel Engine Equipped with a Thermal Reactor
Instead of pistons and valves, the Wankel engine has one or more
triangular rotors. They move through a combination of sliding and
rotating motions determined by both an eccentric shaft and a reaction
gear fixed to the chamber's end plate.
Four actions take place in one circular motion of the rotor: intake
of a fuel-air mixture, compression of that mixture, power by ignition,
and the exhaust of combustion gases. The rapidly expanding gases created
by combustion drive the rotor around the chamber while delivering power
to the eccentric shaft. The spent gases are then swept out the exhaust
port by the rotor. This process is continuous on all three sides of the
rotor simultaneously.
Once most of the delicate tip sealing problem was solved, and wi.th
tough air quality requirements, world attention focused intensely on the
Wankel with its impressive list of advantages. It represents about half
the weight, less than half the number of moving parts, and occupies
about one-third of the space of a piston engine. Carbon monoxide and
hydrocarbon emissions can be effectively controlled using a thermal
reactor (a sparkplug fired, metal afterburning device located in the
car's exhaust system). A big advantage of the Wankel is its ability to
control the most evasive of the pollutants: nitrogen oxides (NOX). The
Wankel's combustion process takes place at relatively cool temperatures
because of the large surface area within the combustion chamber. Cool
combustion temperatures are a key to NOX control.
Disadvantages are poor fuel economy, still some sealing problems,
and short sparkplug life.
The Diasel Engine
This is how the diesel engine works: envision a conventional spark
ignition piston engine with the sparkplugs replaced by fuel injectors.
The intake valve delivers only air and the upward moving piston compresses
it to such a high pressure and temperature that combustion is spontaneous
the instant the fuel is delivered by the injector. This combustion
drives the piston down in the normal manner to deliver power to a crank
shaft. The spent gases are then pushed out through a standard exhaust
valve.
A diesel engine produced by Mercedes will meet the 1975 emission
standards for carbon monoxide and hydrocarbons. (2) Because diesels use
an excess amount of air, they produce practically no carbon monoxide or
hydrocarbon emissions. The emission "hangup" comes with the Federal
nitrogen oxide limit for 1976, as evidenced by a Mercedes engine tested
that exceeded that limit by three times. Little hope of meeting that
limit is seen, especially because high operating temperatures are an
inherent part of the diesel's operation.
For a couple of reasons, the diesel is not considered to be a viable
alternative to the piston:
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1. The diesel engine must be of heavier construction
for the same power than a gasoline engine (takes
up about twice the space)
2. More weight and size mean more expense
3. Visible emissions and odor problems can arise,
especially if injector and engine maintenance
falls behind schedule or is inadequate.
Other Alternative Engine Types
Stirling Cycle
Fuel burns continuously to heat a sealed-in working gas, usually
helium. This engine is thermodynamically quite efficient because no vast
quantity of heat is conserved using heat exchangers. Pistons move up and
down as a result of a carefully designed process wherein the working gas,
by alternate cooling and heating (causing gas compression and expansion)
causes an alternating up and down motion.
An external combustion process is a cooler combustion than that of
the "internal" combustion engine; therefore, nitrogen oxide emissions are
minimal and carbon monoxide and hydrocarbon emissions may meet standards
by selecting the proper fuel (as kerosene or propane) or can be controlled
using thermal converters or other conventional systems. Other advantages
are only five major moving parts, no reciprocating imbalance, good fuel
economy and low noise levels. There have been problems with acceleration,
engine size and mechanical layout, but these may be eliminated with better
research and development.
Gas Turbine
Heated air mixes with fuel, then is ignited by a single sparkplug
device. The combustion chamber encircles the turbine's central shaft.
Combustion causes the gases to greatly expand, and the outgoing gases
(with temperatures near 1700 F) drive a power turbine.
Carbon monoxide and hydrocarbon emission control problems are similar
to those of the "archaic" piston engine. Unfortunately, this engine's
high combustion temperature causes high nitrogen oxide emission levels.
Disadvantages are engine weight and size (comparatively speaking),
leisurely acceleration, and a high-pitched whine.
Gas mileage is fair and there is no engine vibration with which to
conterid.
Steam (Rankin Cycle)
In steam systems, all combustion is external. A working fluid,
usually water, is boiled in the vapor generator via heat from an external
burner. This energy transferred to the fluid is used to drive a turbine
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wheel by the steam expansion. Some heat from the exhaust gas Is trans-
ferred back into the vapor generator. A heat exchanger condenses the
steam to a liquid state for reuse in the vapor generator.
High combustion temperatures cause high nitrogen oxide emissions.
Gas mileage is poor, water consumption can be high, and the engine is
heavy—all adding up to little prospect of a steam car ever gracing your
driveway.
Electric Powered
Lead-acid batteries serve as the power source recharged by simply
plugging into a normal 120 volt plug.
There is speculation that electric cars will eventually comprise a
small but significant segment of the automobile use, especially in the
cities. Lead-acid batteries limit the speed to about 50 miles per hour .
and the range to about 80 miles. Nickel-cadmium batteries with a higher
energy density are more expensive by a factor of 10, and insufficient
reserves of these materials are available.
Fuel costs with electric power are comparable to gasoline charges
(unless gasoline prices continue to increase); but in the end, the effect
of power plant emissions must be weighed against that of controlling
automobile emissions.
Conclusion
As you can see, no simple solution to the urban smog problem
exists; in fact, perhaps a massive effort in developing extensive public
transportation systems may be as easy to implement and at the same time
help solve the energy crisis. Only a major effort directed toward all
possible air pollution control alternatives will achieve clean city air
by the end of this decade.
References
1. NTRDA Bulletin, vol. 58, no. 7.
2. Has the auto industry taken the wrong road to emission controls?
Design Engineering News, p. 11, April 1973.
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60
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PCB'S IN AMBIENT AIR
j. D. Tessari, E. P. Savage, J. W. Malberg, and H. W. Wheeler
Colorado State University
Abstract
Since the appearance of polychlorinated biphenyls (PCB's) as an
environmental pollutant, there has been an increasing interest in their
analyses and the interference they cause in the analyses of other chlo-
rinated hydrocarbons. The varied uses of PCB's and their wide spread
distribution in the food chain led us to question if man was being ex-
posed to residues of PCB's in ambient air. Levels of PCB's in the am-
bient air of grocery stores were determined and the influence of seasons
and physical environmental factors were considered. All of these points
were considered for two primary reasons: 1) occupational and customer
exposures inside the stores, and 2) contamination of food products by
PCB's within the stores. Of the seven stores sampled, all were positive
for PCB's. The levels of PCB's ranged from 0.6 to 5.0 ygAjn2. All sam-
ples were analyzed by gas liquid chromatography and thin layer chroma-
tography. Thin layer chromatography provides a sound approach for the
semi-quantitation of the stated PCB's as the various compounds of the
series have similar Rf values and therefore, produce a single spot.
Introduction
Since polychlorinated biphenyls (PCB's) were identified in environ-
mental samples in 1966, there has been an increased interest in their dis-
tribution in the ecosystem (1). Polychlorinated biphenyls have been detect-
ed in water and sediment samples (2). The Food and Drug Administration has
confirmed their presence in fish, milk, eggs and cheese (3). PCB's are
resistant to degradation bv means of the usual detoxification mechanisms,
they are insoluable in water, and are stored in milk and adipose tissue
(4). PCB's have been reported in human milk in California (5). In our
own laboratory, we found PCB's in human milk in 8 of 39 samples collected
from women living in rural Colorado (6). Finklea, et^ al. reported PCB's
in 43 percent of 723 plasma samples collected in Charleston County, South
Carolina (7). Yobs found 31.1 percent of 637 human adipose tissues collect-
ed from various locations in the U. S. contained measurable amounts of PCB's
(8).
As in the case of most industrial chemicals, loss figures to the
environment for PCB's are not readily available. Possible routes of entry
into the environment include 1) leaks from partially sealed hydraulic
systems; 2) leaks from transformers and heat exchangers; 3) spills in their
manufacture; 4) vaporization or leaching from formulations containing PCB's;
5) disposal of PCB waste products; 6) over-heating of the ballasts or capaci-
tors in fluorescent light fixtures; and 7) vaporization during incineration
of PCB containing products. The total rate of loss of PCB's into the
atmosphere is estimated to be approximately 1.5 to 2.0 x 103 tons per year
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The estimated emission of 1500 to 2500 tons a year of PCB's mainly
by vaporisation and open burning is thought to be concentrated in urban
areas. It is expected that most of the airborne PCB's will be absorbed
on particles. Since roughly three-quarters of the total suspended parti-
culates in urban areas are of non-agricultural origin (.10), and the emis-
sion of. total suspended particulates within cities Is oT the order of.
2 x 10^ tons per year (11), this gives rise to a rough estimate of PCB
levels on urban particulates of 50 to 80 ppm. While conducting air
studies to determine levels of organochlorine pesticides in grocery stores,
polychlorinated biphenyls were recovered in some of the samples. There-
fore, we decided to determine the levels of PCB's in stores and attempt
to trace the source of this contamination.
Methods
Seven grocery stores were initially selected for the study. All
stores were located within a ten mile radius of Fort Collins, Colorado.
Physical characteristics of the buildings were similar in most respects.
The typical store building was of concrete, brick or stone outer wall con-
struction, concrete floor, steel trussed roof, wallboard interior, tiled
floor, lighted with fluorescent light fixtures, and equipped with a forced
air natural gas heating and cooling system. Most of the store buildings
were less than ten years old. The handling of food products and types
of food products were similar in all grocery stores. The level of sanita-
tion inside the larger stores was usually quite satisfactory. In the
smaller stores sanitation was usually not as good as in the higher volume
establishments.
After the first round of sampling, a decision was made to concentrate
the sampling effort in the newest stores with periodic sampling in the other
stores. This allowed sampling during all four seasons:, winter, spring,
summer, fall and at various locations inside the store selected for inten-
sive study. A store that did not handle food products was selected for a
control and sampled to determine if PCB's were in ambient air in non-grocery
stores. The physical characterisitcs of the control store was similar in
every detail to the main study grocery store. Air sampling inside the
grocery stores was conducted using nylon cloth screens as previously describ-
ed by Tessari and Spencer (12). Infrared, Coulson Electroytic Conductivity
Detector and mass spectroscopy were used to analyze the nylon cloth and
also to aid in further verification of the PCB residues. Standard Aroclor
reference spectra were obtained using a Dupont 21-110B high resolution
mass spectrometer and a Dupont 21-490 mass spectrometer. The standards
were introduced using a direct insertion probe with sample and source at
80°C. The standards used were Aroclor 1221, 1232, 1242, 1248 and 1254.
Cloth samples were cut from a bolt of material to the dimensions of 23
inches by 17 inches to give !gra2 cloth screens. The cloth screens were
then placed in a beaker and cleaned with three washings of chloroform
methanol (1:1). The three washings were decanted and the cloth was then
cleaned with three washings of n-hexane again decanting the solvent washings.
Prior to sampling, the screens were saturated with solutions of 10 percent
ethylene glycol in acetone and the solvent decanted. The screens were
suspended in a wooden frame in the stores for a period of 5 to 7 days.
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After collection the screens were extracted for 4 hours using the Soxhlet
method of extraction with acetone-hexane (1:1) as solvent. The solvent
flasks were transferred to a rotary evaporator and the solvent mixture
was removed under a water aspirator suction at 37°C until the volume was
reduced to approximately 2 ml. The sample was then transferred quantita-
tively to a Florisil column prepared by adding 1 inch Na2SOit, 4 inches
activated Florisil and 2 inches Na2SOi+ to the chromatograph column. The
column was pre-rinsed with 100 ml n-hexane, and the sample was transferred
quantitatively to the column, and eluted with 200 ml 6% ethyl ether in
hexane. The collection flask was changed and the column was eluted with
200 ml 15 percent ethyl ether in hexane. Both eluates were treated sep-
arately and evaporated to 1 to 2 ml on a rotary evaporator. The eluates
were transferred quantitatively to a graduated tube and diluted to 10 ml.
All samples were analyzed by gas liquid chromatography using electron
capture and coulson detectors and thin-layer chromatography. After anal-
ysis by electron capture and coulson gas chromatography the 6 percent
Florisil fraction was transferred quantitatively to a concentrator tube
fitted with a modified micro-snyder column and concentrated to 1 ml or
less in a 100°C water bath. The remainder of the volatile solvent was
removed under a stream of nitrogen at room temperature and 2 ml of alco-
holic KOH was added. The column was reattached and the tube was immersed
in a 100°C glycerin bath for 30 minutes. The tube was then allowed to
cool, to room temperature and 2 ml of distilled water and 5 ml of hexane
was added. The tube was stoppered and mixed vigorously on a Vortex mixer
for 30 seconds. After the layers separated the top hexane layer was trans-
ferred using a disposable pipet to a 25 ml evaporator concentrator tube.
After transferring, 5 ml portions of hexane were again added for two
additional extractions as previously described. A glass bead was added
to the 25 ml evaporator concentrator tube containing the hexane extract,
a modified micro-snyder column was attached and the extract was concentrated
to 1 ml or less in a hot water bath. The tube was allowed to cool and the
extract was evaporated to dryness at room temperature. At this time, 2 ml
of oxidizing solution was added and the tube was immersed in a 100°C glyc-
erin bath for 30 minutes. The tube was then removed and allowed to cool
to room temperature. Ten ml of distilled water and 3 ml hexane was added,
the tube was stoppered and mixed vigorously on a Vortex mixer for 30 seconds,
The layers were allowed to separate and the hexane layer was carefully
transferred to a 13 ml graduated test tube with a disposable pipet. The
hexane extract was then concentrated under a stream of nitrogen at room
temperature to dryness. Exactly 0.05 ml of hexane was added and the tube
was stoppered and mixed on a Vortex mixer for 30 seconds. The extract
was then spotted on thin-layer plates for quantitation.
A composite air sample of store number six consisting of a 6 percent
Florisil fraction from an MRI air sampler, and two 6 percent Florisil
fractions from Jjjn2 cloth screens were concentrated in glass capillary
tubes by evaporating 1 cc of the sample in the capillary over 24 hours.
The sample was analyzed by a direct insertion probe on the Dupont 21-490
mass spectrometer.
63
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Apparatus and Reagents
a) Gas chromatograph—Micro Tek 220 (MT-220). Equipped with
parallel plate, electron capture detectors, utilizing 14.5 me nickel
63 sources and Coulson Electrolytic Conductivity Detector used in the
reductive mode.
Columns; Liquid phases: SE-30 (methyl silicone, GC grade), QF-1 (Tri-
fluoro propyl methyl silicone), OV-210. Column packings consisted of
mixed phases of (1) 1.5% OV-17/1.95% QF-1 (2) 4% SE-30/6% OV-210 and
(3) 3% OV-1.
Support material; Gas-Chrom Q 100-120 mesh.
Temperatures; Electron capture: injector port 245°C, transfer line
240 C, detectors 275°C, columns 200°C. Coulson: injection port 245°C,
transfer line 240°C, pyrolysis furnace 820°C, inlet block 230°C and
column 200°C.
Flow rates; Nitrogen carrier gas 60 ml/min for OV-17/QF-1 column,
100 ml/min for the SE-30/OV-210 column, and 60-80 ml/min for OV-1
column. Furnace gases to coulson, hydrogen 20 ml/min, nitrogen 80
ml/min.
b) Soxhlet extraction apparatus: with reflux condensers
(Van Waters and Rogers, San Francisco, California, NO. 27613-226).
c) Nylon chiffon screen: Blossom Touch, 100% nylon chiffon,
non-static finish, approximately 90 mesh, I.K.O. Industries, 4500
Joliet Street, Denver, Colorado.
d) Chromatographic columns: Florisil, 25 mm id, with Teflon
stopcocks and coarse fritted disks, equipped with 300 ml reservoir
at top.
e) Florisil: 80-90 mesh, activated by heating at 130 C at least
48 hours prior to use. The Florisil used was obtained from J. F.
Thompson, Perrine, Florida 33157.
f) Solvents Acetone: reagent grade, redistilled; hexane (Phillips
high purity normal), glass-distilled over metallic sodium before use;
ethyl ether, reagent grade, redistilled; ethanol reagent grade, redistilled;
benzene, Nanograde, Mallinckrodt Chemical Works, Saint Louis, Missouri;
methanol, reagent grade, redistilled.
g) Chemicals: ethylene glycol, pesticide quality, Matheson,
Coleman and Bell, EX 567, U5087; acetic acid, glacial reagent grade;
chromium trioxide, cryst., reagent grade; potassium hydroxide, pellets,
reagent grade; silver nitrate, cryst. reagent grade; aluminum oxide G,
Merck Laboratory Chemicals, Merck and Company, Inc., Rahway, New Jersey.
h) Utility bath: Sero-utility, Theico, precision, model 82, with
sufficient control to hold bath at 100°C, ±2°C.
i) Vortex mixer, variable speed
j) Rotary flash evaporator: Buchler Instruments, Fort Lee,
New Jersey.
k) Heating mantles: Equipped with'adjustable auto transformers
to fit extraction flasks.
1) Evaporator concentrator tubes, 10 ml and 25 ml, size 1025,
2525, #570050, Kontes Glass Company, Vineland, New Jersey.
m) Modified micro-Snyder columns, 19/22, #569251, Kontes Glass
Company, Vineland, New Jersey.
n) Thin-Layer equipment: Glass plates, 8" x 8", double strength
window glass (Pittsburg Plate Glass); developing tank, Thomas-Mitchell,
64
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8" x 4" x 8" deep (Arthur H. Thomas, Co.); Desage/Brinkmann standard
model applicator; Desage/Brinkmann drying rack, #0410100-6; Desage/
Brinkmann mounting board, //0410000-0; Desage/Brinkmann stainless steel
desiccator, #04-11-500-7 Brinkmann Instruments, Inc., Cantiague Road,
Westbury, New York, Spotting pipettes, 1, 5, and 10 ml, Kontes, //763800;
Kontes Glass Co., Vineland, New Jersey; Ultra violet light source, germ-
icidal lamp, Sylvania, //G15T8.
o) Standards: Aroclor samples for standards were obtained from
the Monsanto Chemical, Co., St. Louis, Missouri. Standard iso-octane
solution or Aroclors were prepared for gas chromatography at the follow-
ing concentrations: Electron capture detector, 500 pg/yl of Aroclor;
Coulson detector 100 ng/pl of Aroclor. Standards for TLC analysis were
prepared at four working concentrations of 25, 50, 100 and 400 ng/yl,
using hexane as the diluent.
p) MRI air sampler: Sampling device developed by Mid West Research
Institute utilizing a vacuum pump which draws air through ethylene glycol
contained in glass impingers.
Results
Table 1 shows the number of air samples collected at each of the
seven stores. Four stores were sampled one time only. The other three
stores were sampled twice, six and nineteen times respectively. A total
of 31 air samples were collected. Table 2 depicts the levels of PCB's
found at the six sampling sites. The range of PCB residues found in
grocery stores using the nylon chiffon screen was from 0.6 to 5.0
The mean was 1.8 yg/^zjn2. Store number six was sampled once using the
MRI sampler. The total amount of air sampled during this 72-hour sampling
period was 75.82 m3. PCB's were present at a level of 65.8 ng/m3.
In an attempt to trace the source of PCB contamination, different
locations within the main study store site number six were sampled. The
level of PCB's sampled by store location is shown in Table 3. Four pri-
mary locations in the store were sampled. These included one site near
or across the aisle from the pesticide display, one site at the north
end of the receiving area, one at the south end of the receiving area
and one close to the produce section. The samples ranged from 0.6 to
5.0 ugAjn2. The average for samples near the pesticide display was
2.12, the produce area 1.0, receiving area south 1.42, and receiving area
north 2.2.
The level of PCB's in ambient air by seasons of the year is shown
in Table 4. Based on the limited number of samples we analyzed, there
appeared to be no significant difference in seasonal occurrence.
Figure 1 compares chromatograms of Aroclor 1232, 1242, 1248 and
1254 standards to a chromatogram of an ambient air sample collected in
grocery store number six. Samples collected at various locations inside
this store were analyzed and many peaks obtained on electron capture gas
chromatography had identical retention times. Many of these specific
retention times were identical to those given by Aroclor standards men-
tioned above. Cloth screens that were not exposed inside grocery stores
but were cleaned and extracted in the same manner were also analyzed us-
ing infrared and mass spectroscopy. This analysis was conducted to
make sure the cloth contained no PCB material of any kind. The infrared
65
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spectra of the nylon pyrolysis indicated nylon 6 which is a polymer of
caprolactam.
A solid probe outgas study on the Dupont 490 mass spectrometer
indicated very little outgas products. Extracting the cloth with n-
hexane and acetone (1:1) indicated a polyethylene glycolic ester using
both I.R. and mass spectroscopy. Polyethylene glycols are used as
anti-static agents. The mass spectral analysis of the cloth screen
indicated dioctyl phthalate as the major component remaining after
evaporation of the n-hexane solution. Polyoxyethylene and fatty acids
were detected in trace amounts. No polychlorinated biphenyl components
were detected in this sample.
Mass spectrometry was then used to analyze cloth screens that
had been exposed in store number six and spectra were recorded at 80,
120 and 200°C. The spectra indicated PCB's dioctyl phthalate, fatty
acids and a polyoxyethylene material. The fatty acids and the poly-
oxyethylene material were present in trace amounts. The polyoxyethylene
material was noted only in the higher temperature spectra. The poly-
chlorinated polyphenyl fraction exhibited a 100% peak at mass 292 with
correct isotopic abundance at 290, 294 and 296 to indicate four chlorine
atoms. This spectral data indicates a biphenyl structure substituted
with four chlorine atoms. Isotopic cluster at masses indicating sub-
stitution with 2, 3, 5, 6, 7, and 8 chlorine atoms were also identified.
Polychlorinated terphenyls substituted with 7, 8, 9, 10, and 11 chlorine
atoms were noted in trace amounts. The mass spectrum of the PCS in this
sample corresponds to the Aroclor 1248 reference spectrum. The poly-
chlorinated terphenyls were not detected in the reference spectrum.
Discussion
Quantitation of PCB's from electron capture chromatograms is
complicated because the EC detector responds differently to each PCS
isomer. Quantitation by direct comparison of an unknown EC chromatogram
with those of Aroclor standards is difficult because individual peaks
in environmental samples are sometimes obscurred by pesticide residues,
are completely missing, or have considerably different relative inten-
sities. An advantage the EC detector has in PCS analysis is its sen-
sitivity. The practical sensitivity of this method using the EC detector
to an Aroclor 1254 standard is approximately 1 yg/izjn2 . Because of its
high sensitivity but lack of differential response, this detector was
used for a screening device and not used for quantitation. The Coulson
detector was used for varification and not for quantitation primarily
because of its lack of sensitivity. This detector is capable of de-
tecting a standard of Aroclor 1254 at approximately 20 yg/^sm2.
Thin layer chromatography is a useful technique in pesticide
residue analysis for the qualitative confirmation of results obtained
by means of gas chromatography (13). When an often used TLC system
such as aluminum oxide/hexane is used, very little information about
PCB's is obtained. TLC can, however, be used for semi-quantitation
of PCB's in the presence of organochlorine insecticides after a pre-
liminary oxidation step to oxidize DDE to 4,4-dichlorobenophenone (14).
Although this system does not separate different members of the Aroclor
series, it is sensitive and specific to PCB's with a lower limit of
0.6 pg/lzjn2.
66
-------
References
1. Risebrough, R. W., B. de Lappe. Environmental Health Perspec.,
Experimental Issue No. 1, April 1972.
2. Duke, T. W. , J. I. Lowe and A. J. Wilson. Bull. Environ. Contain.
Toxicol. 5:171, 1970.
3. Kolbye, A. C. Environ. Health Perspec., Experimental Issue No. 1.
April 1972.
4. Biros, F. J. Bull. Environ. Contain. Toxicol. 5:317, 1970.
5. Risebrough, R. W., J. Davis, R. Anastasia, F. A. Beland and J. H.
Enderson. Publication submitted to New England Journal of Medicine.
6. Savage, E. P., J. D. Tessari, J. W. Malberg, H. W. Wheeler and
J. R. Bagby. Bull. Environ. Contain. Toxicol. 9:222, 1973.
7. Finklea, J. Amer. J. Publ. Hlth. 62:645, 1972.
8. Yobs, A. R. Environ. Health Perspec., Experimental Issue No. 1,
April 1972.
9. Nisbet, C. T., A. F. Sarofim. Environ. Health Perspec., Experimental
Issue No. 1, April 1972.
10. Air Quality Criteria for Particulate Matter. U. S. Public Health
Publication AP-49, 1969.
11. Council on Environmental Quality. Environmental Quality. First
Annual Report of C. E. Q. Washington, D. C. 1970.
12. Tessari, J. D. and D. L. Spencer. J.A.O.A.C. 54:1376, 1971.
13. Kovacs, M. F. J.A.O.A.C. 48:1018, 1965.
14. Manual of Analytical Methods, Pesticide Community Studies Laboratories
Primate Research Center. Perrine, Florida, 1971.
67
-------
TABLE 1. Number of ambient air samples collected.
Site
1
2
3
4
5
6
7
TABLE 2. PCB's in air samples
Site
1
2
3
4
5
6
7
* (ug/W) N_ 29
Median- 1.4
Mean- 1.8
Number
2
1
6
1
1
19
1
of Colorado 1972-1973
PCB's*
2.5
1.3
1.3
3.7
4.5
1.3
2.5
1.3
1.0
0.6
0.6
1.3
5.0
0.6
1.3
0.9
1.9
1.9
0.6
0.6
1.3
1.3
2.5
2.1
2.5
0.6
1.9
1.9
2.5
Std. Deviation- 1.1
Range- 0.6-5.0 pg/Jsm2
68
-------
TABLE 3. PCB levels of air sampling locations within site number six.
Location
Near pesticide display
Near produce
Receiving area' - south
Receiving area - north
Date
6/6/72
8/29/72
7/18/72
8/29/72
8/29/72
12/11/72
12/11/72
10/9/72
12/11/72
3/12/72
1/24/73
10/9/72
12/11/72
1/24/73
3/12/73
PCB's*
1.3
1.3
5.0
0.9
0.6
2.1
1.3
1.9
1.3
1.9
0.6
1.9
2.5
2.5
1.9
* (yg/W)
TABLE 4. Levels of PCB's in ambient air by seasons.
Season
Winter
Spring
Summer
Fall
No. Samples
4
1
4
6
Average PCB's*
1.72
1.30
1.70
1.85
*(ug/W)
69
-------
Figure 1
2 4
AROCLOR 1232
68 10 12 14
MINUTES
70
-------
Figure 1
AROCLOR
10
12 14
16
MINUTES
71
-------
Figure 1
AROCLOR 1248
4
10 8 6 4
MINUTES
72
-------
Figure 1
AROCLOR 1254
IZ
IO 8
MlMTES
73
-------
STORE NO. 6
AIR SAMPLE
t
MINUTES
-------
LEVELS OF POLYCHLORINATED BIPHENYLS IN ADIPOSE
TISSUE OF THE GENERAL POPULATION OF THE NATION
Anne R. Yobs, M.D.
Environmental Protection Agency
Polychlorinated biphenyls (PCBs) have been reported in many different
parts of the environment in this country (1-3). The purpose of this
report is to present preliminary results from a monitoring program in
which, these materials are routinely sought and quantitated.
The Human Monitoring Survey, established in 1967 by the Pesticides
Program, DHEW (now Division of Pesticide Community Studies, EPA), deter-
mines on a continuing basis the exposure to pesticides experienced by
the general population by measuring levels of pesticide residues present
in tissues or excreted in urine. Initially, technological limitations
and resources restrictions limited the Survey to the identification and
measurement of those chlorinated hydrocarbon residues which are stored
in measurable amounts in mammalian adipose tissue or which can be meas-
ured in blood, these residues being a reflection of past exposure. The
initial program plan has been, and continues to be, reviewed frequently
with reference to technological and/or research developments to identify
other groups of pesticides and other materials of interest which can be
incorporated with slight modification of the existing program.
Samples are collected through the direct cooperation of pathologlsts
who are in hospital or private practice or who are in public service as
city or county medical examiners. Samples are collected from both sexes
in all age and racial groups. Adipose samples are collected from tissues
removed for therapeutic surgery or from postmortem examinations, the
latter including people who have died accidentally (trauma) or during
relatively brief hospitalization. No samples are accepted from institu-
tions for long-term care of the chronically ill patient.
All samples are analyzed by laboratories established under contract
with State Health Departments or at community study projects in Michigan,
Florida, and Colorado. These laboratories use analytical methodologies
specified by the program on the advice of technical consultants and are
required to maintain acceptable levels of performance as demonstrated in
both inter- and intra-laboratory quality control programs. Technical con-
sultation for analytical aspects of the Survey is provided by the Chem-
istry Branch, Perrine Primate Laboratory, EPA, under the direction of
Henry F. Enos, Ph.D., Chief.
In 1969, with the increased interest in polychlorinated biphenyls,
it was recognized that PCBs probably occur in several segments of the
environment and that their presence would distort analytical values for
certain pesticides, expecially selected DDT residues. The monitoring
75
-------
laboratories were therefore asked to report all samples suspected of
containing PCBs by gas-liquid chromatography. A reporting system was
developed, based on rough quantitation, as follows: negative, present
in usual amounts, present in greater or lesser amounts than.usual.
Samples collected in late 1968 and 1969 showed that PCBs were indeed
present in a significant portion of the general population of the country
over a wide geographic distribution. Positive samples were reported
from 14 states, including Michigan, New York, Minnesota, California,
Massachusetts, Georgia, Kentucky, Illinois, North Carolina, South Dakota,
Ohio, Louisiana, Delaware, and Arkansas. To insure that proper identi-
fication was being made, two samples with higher levels were confirmed
by combined gas liquid chromatography-mass spectroscopy (4).
Based on these preliminary findings and certain as yet unpublished
information about the effect of PCBs on the accuracy of measurement of
residues of DDT (Enos, H. F., personal communication), high priority was
given by the Perrine Laboratory to the development and evaluation of an
analytical method which would separate PCBs from pesticides permitting
accurate quantitation of pesticides and confirmation of PCB levels. The
resulting methodology is a further modification of the Mills-Olney-Gaither
procedure in which adipose tissue is subjected to extraction by petroleum
ether, acetonitrile partitioning, and Florisil cleanup. A portion of the
resulting 6% ethyl ether/petroleum ether eluate, in concentrate form, is
treated with KOH to effectuate dehydrochlorination of DDT and ODD to
their olefins, thus eliminating the problem of separating these pest-
icides from the PCBs. Oxidative treatment is then applied to convert
any interfering DDE to p,p'-dichlorobenzophenone which has an Rf value
different from the PCBs. The PCBs are then determined by thin layer
chromatography (5).
On April 15, 1971, all monitoring laboratories in the Human Monitoring
Survey adopted this methodology for the analysis of all adipose samples
with the lowest limit of sensitivity being 1.0 ppm. Aliquots of all
samples in which measurable amounts of PCBs are found are saved for
future confirmation by mass spectroscopy.
Results and Discussion
Analytical results for 637 samples have been reported and are summariz-
ed in Table 1 (51 additional samples have been analyzed but have not as yet
been reported). Of the 637 samples reported, 198 (31.1%) contained measur-
able amounts of PCBs and 125 (19.6%) contained trace amounts, with the
balance being negative. These samples were collected from 40 pathologists
in 38 cities distributed over 18 states and the District of Columbia. The
states are Arkansas, Illinois, Louisiana, Oklahoma, South Dakota, Tennessee,
Oregon, Pennsylvania, Florida, Kansas, Maine, Georgia, North Carolina,
California, Michigan, New York, Ohio, and Kentucky. Positive samples came
from every hospital, city, and state sampled. It, therefore, appears
that these materials are widely found in the population. Details of dis-
tribution by sex and race, collection procedure, and diagnostic groups
are summarized in Table 2. The difference between figures for the
76
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positive and negative groups does not appear to be significant, although
no statistical evaluation has been attempted because of the preliminary
nature of this report. Since a statistical design has been established
and is being followed for sample collection for the Human Monitoring
Survey, in due time valid statistical analysis will be possible and will
then be performed.
Summary
Polychlorinated biphenyls have been found in measurable amounts in
31.1% of 637 samples of human adipose tissue collected from the general
population as a part of the Human Monitoring Survey. Sample collection
involved 18 states and the District of Columbia. Positive samples were
obtained from every state sampled.
References
1. Gustafson, C. G. PCBs—prevalent and persistent. Environ. Sci.
Technol. 4(10):814-819, 1970.
2. Risebrough, R. N., P. Rieche, D. P. Peakall, S. G. Herman and M. N.
Kirven. Polychlorinated biphenyls in the global ecosystem. Nature
220:1098-1102, 1968.
3. Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. A polychlorinated
biphenyl (Arochlor 1254) in the water, sediment, and biota of Escambia
Bay, Florida. Bull. Environ. Contain. Toxicol. 5(2):171-180, 1970.
4. Biros, F. J. Polychlorinated biphenyls in human adipose tissue.
Bull. Environ. Contain. Toxicol. 5(4) :317-323, 1970.
5. Thompson, J. F., Editor. Analysis of pesticide residues in human
and environmental samples: for use by Community Studies Projects
and Pesticide Monitoring Laboratories, Pesticides Program. Section
9. Perrine Primate Research Laboratories, Environmental Protection
Agency, Perrine, Florida, 1971.
77
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Table 1 - Incidence of polychlorinated hiphenyls in adipose of the
general population of the nation
Reported Analyzed
No. % No. %
Total 637 100.0 688 100.0
Negative 314 49.3 235 34.2
Trace - <1.0 ppm 125 19.6 229 33.3
1-2 ppm 165 25.9 188 27.3
>2 ppm 33 5.2 36 5.2
78
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Table 2 - Descriptive statistics of samples analyzed
Negative
Total
Number
Sex
Male
Female
Race
White
Nonwhite
Samples from
Therapeutic Surgery
Postmortem
Diagnostic Group
Neoplasms 140-209
Circulatory
System 390-450
• Gastrointestinal
System 530-565
Liver & gall
bladder 570-575
Pancreas 577
Urinary System 580-595
Congenital
Anomalies 740-759
Accidents &
Violence E numbers
All Other
and trace
No.
439
221
218
381
58
95
344
77
121
44
20
0
11
5
16
145
439
- < 1 . 0 ppm
7
100.0
50.3
49.7
86.8
13.2
21.6
78.4
17.5
27.6
10.0
4.6
0.0
2.5
1.1
3.6
33.0
99.9
>!.(
No.
198
127
71
151
47
29
169
39
61
16
6
2
7
1
7
59
198
) ppm
100.0
64.1
35.9
76.3
23.7
14.6
85.4
19.7
30.8
8.1
3.0
1.0
3.5
0.5
3.5
29.8
99.9
79
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ANTIBIOTIC RESISTANCE AND ANIMAL FEED ADDITIVES
Leslie P. Williams, Jr. and Carey L. Quarles
Colorado State University
During the past thirty years the state of man's health has been
greatly improved by the use of sulfonamides and antibiotics. These drugs
have helped save the wounded and maimed of recent wars, decreased the
maternal and infant mortality rates and helped increase the life span
of the aged. They have found widespread use in veterinary medicine.
They have also been used in industry to improve food shelf life, and
in animal agriculture to increase weight gains and improve feed con-
version.
Although their use has increased tremendously, these antimicrobial
agents have not proved to be the panacea that they were first thought to
be. For example, when sulfonamides were first used as a treatment for
gonorrhea in 1936 there was a 90 percent cure rate. Less than a decade
later, 75 to 85 percent of the cases treated were drug resistant. A
similar situation occurred with meningococci, especially in military
populations. The majority of meningococci causing clinical disease
have been sulfonamide resistant since 1962 (1). Japanese investigators
documented the shift of Shigella isolates from drug-sensitive to multiple-
drug-resistant in three major hospitals. The change in frequency of re-
covery of resistant strains was as follows: Tokyo 1960 - 21%, 1964 - 52%;
Nagoya 1960 - 17%, 1964 - 50%; and Osaka 1960 - 16%, 1964 - 65%. They
also made the observation that patients with multiply resistant Shigella
also harbored multiply resistant E. coli (2).
The changes above took on new significance in the light of recent
findings reviewed by Watanabe (2) and Smith (3) that enteric bacteria
exhibited infectious drug resistance and that this characteristic could
be passed from non-pathogenic to pathogenic bacteria in the gut. Donors
and recipients could be any members of the Enterobacteriaceae.
, The distribution of R factors and the occurrence of resistant
enteric organisms in man was reviewed recently by a Japanese worker.
The percent carrying R factors varied from 95 percent of Shigella and
Salmonella isolates tested in Greece to lows of 20 percent E._ coli and
Proteus in Germany and 18 percent of Salmonella in France and the Nether-
lands (4).
While resistance to S. typhimurium had been studied extensively
by others in the United Kingdom, Smith and Halls were concerned with
E. coli because these organisms formed the bulk of the enterobacterial
flora of the G.I. tract in man and animals. They sampled healthy calves,
pigs, and fowl on different farms, and humans in various households with
the results shown in the following table adapted from the original:
81
-------
Incidence of drug resistant E^ coli in fecal
swabs of man, calves, pigs, and fowl.
Man Calves Pigs Fowl TOTAL
Number examined 24 50 50 18 142
Number with
resistant strains 15 34 35 17 101
(71%)
Number with
multiple-drug
resistance 10 18 15 13 56
(55%)
The majority of the resistant isolates was shown to have infective
(transferrable) resistance. The authors noted that some of the ampicillin
resistant strains from man were from caretakers working in intensive calf-
rearing units where this antibiotic had been used in the treatment of calf
diarrhea (5).
Similar findings were made from swine and fowl in Japan. A total
of 151 and 108 of each on a research farm were tested. They had been fed
on a dairy product containing tetracycline. All of the swine and 50% of
the fowl were resistant to tetracycline, chloramphenicol, streptomycin,
sulfanilimide, or combinations of these. Forty percent of the resistant
isolates from swine and 22% from fowl carried R factors. Domestic animals
subjected to maximal amounts of antibiotic pressures and wildlife subjected
to minimal pressures were examined in Illinois. Approximately 90 percent
of E._ coli isolated from calves and swine were resistant to common anti-
biotics, whereas the percentage in wildlife was 8-11 percent of the isolates,
These authors concluded that, "it is probable that antimicrobial drugs
facilitated the development, selection and growth of antibiotic-resistant
Escherichia coli in domestic animals" (6). These authors also cited inter-
esting data by Mare. It is summarized in the table below:
Prevalence of antibiotic resistant gram
negative bacteria in human and animal
populations not exposed to antibiotics
Origin of Number of % Antibiotic
Specimens Specimens Resistant
Kalahari Bushmen 47 19*
Pohwe River Valley Animals 201 8*
Kruger National Park Animals 334 9**
TOTALS 582 10%
* Ampicillin Resistant Only (None with R Factors)
** Multiply Resistant (None with R Factors)
By contrast, of 207 humans from Pretoria, South Africa tested, 78%
yielded drug-resistant strains (6).
82
-------
Mercer and his coworkers studied 555 isolates of E. coli from feces
of animals on 5 farms in the U. S. Antibiotic use on these farms was
determined by survey. They demonstrated a relationship between continuous
feeding of antibiotics and multiple resistance to antibiotics, and found
that resistant strains from continuous fed herds had a 2% times higher
rate of R factors. Their results are shown on the table below (7):
Relationship between exposure to antimicrobials
and frequency of resistances of Escherichia coli
% of Strains
Exposed
Farm 1
Farm 2
Farm 3
Farm 4
Farm 5
Swine
Calf
Subtotal
Farm 5
Dairy cows
Total
Nature of
Exposure
Continuous
Intermittent
Continuous
Continuous
Continuous
Intermittent
(therapeutic
at birth)
Not exposed
Total
strains
Multi-
ply
sensi-
tive
Singly
resis-
tant
Multi
ply
resis
tant
99
138
131
77
25
21
491
64
555
0.0
44.9
10.7
4.9
8.0
76.2
24.1
78.1
31.8
4.1
16.7
22.2
0.0
16.0
4.8
10.6
6.2
9.9
95.9
38.4
67.2
95.1
76.0
19.0
65.3
15.7
58.2
Use of Antibiotics in Feeds
In a brief review of the subject of antibiotic use in feeds Jukes
reported on their use in 1946 and stated that "in the early 1950's the
practice of adding antibiotics to animal feeds spread throughout the world"
(8).
In 1955, approximately 225,000 kilograms of antibiotics were
produced for use in animal feeds in the United States (9). The reported
usage in 1969 had increased to 1.25 million kilograms. Hays made the
following statement, "The wide acceptance of antibiotics has been based
on their established benefits of increasing growth rate, improving feed
conversion, and reducing morbidity and mortality from clinical and subclin-
ical infections" (10). Studies have shown that the response of a group of
animals to antibiotics is greater in contaminated environments. However,
workers are quick to point out that the use of drugs is not a substitute
for good sanitation practices (10). While some have stated that the rer-
sponse to antibiotics has decreased over the past decade, Hays reviewed
the data from midwest researchers in several states that confirm that the
use of antibiotics is still giving improved performance, thereby justifying
its continued use. He stated that "species involved, age of the animal,
adequacy of diet, and environmental conditions are all important factors
83
-------
affecting the response to antibiotics." He also feels that because of
the intensification of livestock production (raising livestock in a con-
fined situation) it is even more imperative that antibiotics are used (.10).
However, the true cost-benefit ratio from using antibiotics has not been
analyzed sufficiently in most instances.
Public Health Significance of Antibiotic Use in Feeds
The potential hazards of antibiotic use in feeds to the health of
man seems to fall in three areas as follows: 1) the infection of man with
resistant organisms that would cause clinical disease and thus be difficult
to treat; 2) the transfer of resistance from non-pathogens to pathogens
within the gut of man; and 3) the allergic reactions of sensitive persons
to antibiotic residues that may be present in meat or meat products.
Salmonella typhimurium isolates were studied to detect resistance
patterns in Great Britain. The majority of the resistant strains were
found to be phage type 29 associated with confinement calf operations where
treatment failures were common and calf losses high. This phage type was
often isolated from people experiencing salmonellosis. Isolates showed
multiple-drug resistance patterns. These investigators believed that the
increase in resistant calf strains was due to the increase in intensive
(confinement) calf rearing and the treatment and additives required to
keep these animals alive. A direct correlation seemed to exist between
calf and human resistance patterns and the increased isolation of this
phage type during the period 1964-1966.
Resistance transfer from normally non-pathogenic genera may be an
important factor in human health. Because E. coli constitutes a major
part of the gut flora, it is conceivable that more of these organisms
could be spread by food contamination (12). It is likely that a high
percentage could be resistant and transfer this resistance in a new host.
Smith (13) feels that drug resistant bacteria and particularly those with
RTF constitute a major hazard to hospitalized patients, especially those
with compromised body defense mechanisms. Taylor has stated that feeding
small doses of antibiotic will affect the development of E^ coli with RTF
that may reach man and complicate treatment (14).
Regarding antibiotics incorporated in feeds some authorities have
stated "The significance of residues of antibiotic in meat and edible
viscera from animals fed antibiotics is not entirely clear" (15). Some
investigators have shown no evidence of harm and therefore antibiotic
residues in foods seems to be in possible hypersensitivity reactions (15,
16). The main concentration of drugs may be in kidney,, liver, cecum, and
bones (16). Other authors have stated that evidence indicates that ac-
cumulation of drugs in tissue is the exception rather than the rule, that
drugs differ in their stability in vivo making withdrawal periods effective,
that variation in human consumption of animal products plays a role, and
that food processing will destroy most residues in meat (17).
The question of antibiotic residues in market meat has been answered
in part by a survey conducted in Illinois. Antibiotic residues were found
84
-------
in chicken livers (12%), lamb livers (16%), and swine kidneys (7%). Residues
of penicillin, dihydrostreptomycin, tylosin, and tetracylines were identified.
These investigators cited European data stating that 77% of veal meat con-
tained antimicrobial residues as opposed to the 20% prevalence in the domes-
tic veal they tested (18).
Effect of R Factors on Drug Therapy in Animals
In discussion of the control of neonatal colibacillosis in swine
Barum stated that their finding that 90% of enteric bacterial strains possess
R factor showed the urgent need for a chemotherapeutic agent that would act
on E. coli without their developing resistance (19). Another approach to
this would be to control the use of antibiotics now available so resistant
organisms would not emerge. Walton has stated that the resistance problem
is a serious complicating factor in the treatment of young animals affected
with colibacillosis. He feels that because of this, the widespread use
of drugs should be drastically reduced (20). This problem was also the
topic of an editorial in a prominent veterinary journal in 1967. The author
stated that if transferable resistance proved to be more common in the
future the widespread use of antibiotics against diseases of non-specific
origin should be restudied by veterinarians and others cpncerned with animal
health (21). Judging from reports of the past five years, the time to re-
study the issue may be now.
The British were the first to act. In 1969 the Swann Committee
released its report. They decided that low level feeding of antibiotics
and resulting resistance problems were a direct threat to human and animal
health. Only those drugs not used in treatment of disease could be used
as low level feed additives (22).
Early in 1972, the FDA released the report of the Task Force of the
Use of Antibiotics in Animal Feeds. They recommended that additional re-
search be done to evaluate the practice on a benefit-risk basis. Three
primary areas of concern were identified: (1) Human health hazards; (2)
Animal Health Hazards; and (3) Drug effectiveness (23).
The commissioner of FDA has required that all drug sponsors submit
by April 1974 results of studies of.the effect of low level feeding of
tetracycline, streptomycin, dihydrostreptomycin, the sulfonamides and pen-
icillin on the salmonella reservoir.
By April 1975 conclusive evidence must be given that no human or
animal health hazards exists because of their use or such use must be
discontinued.
The FDA in the interim is sponsoring research to answer questions
and provide guidance where there are voids in available information. One
such project is being conducted by the Department of Animal Sciences and
the Department of Microbiology. It is concerned with development of a
drug testing protocol for industry using multiple drugs, and induced in-
fection with E. coli. Trials were conducted in a controlled environment
85
-------
house (figure 1). Four hundred and fifty, one day old chicks of known
genetic stock were placed in 18 pens, fed antibiotic free feed for two
weeks, fed specified antibiotics (Table 1) for one week (and these continued
throughout the remaining five weeks of the growing period), artificially
infected orally and by air sac injection with a laboratory marked strain of
E. coli, observed for three days for morbidity and mortality, treated with
various specified antibiotic for 4 days and then followed through the finish-
ing period to slaughter. During the feeding period weight gains and feed
efficiency were documented weekly as was the development of antimicrobial
resistance of the indigenous E. coli flora. At slaughter the number of
condemnations, breast blisters and grade A's were recorded for each group.
Intestinal samples were collected and resistance patterns determined for
enteric bacteria. Carcasses were swabbed to demonstrate possible movement
of resistant bacteria into the human food chain.
Feed samples were assayed by a commercial laboratory in Denver to
determine the quality of feed mixing and the levels of antibiotic that the
birds were actually exposed to (Table 2).
A graphical study was made of the data to evaluate the response over
time (growth curve) and to locate any inconsistencies in the data. No
mathematical analyses were done on this aspect.
Randomized block (2 way) analysis of variance were performed on the
data from replicates two and three of the 18 treatments for the data obtained
at the 4th and 9th weeks. In addition, a factorial analysis of variance
was performed on the 16 treatments making up the low level-high level anti-
biotic treatment portion of the study. The 18 means were ranked for reporting
and,.where appropriate, Tukey's HSD was applied for separating the means
into subgroups.
These analyses were performed on replications two and three only
because trial one was not comparable. Oral dosing only was used in trial
one. Morbidity was low and only 37 birds died. When we switched to oral
dosing and air sac innoculation with E. coli at 108-109 organisms, virtually
all infected birds became ill and approximately 100 birds died in trials
two and three.
Results — Growth, and Slaughter Measurement
The average body weight in gram at the the eighth week (mean of
replication two and three) varied from 1896 (pen 5) to 2229 grams (per 18).
There was a significant difference (p=.01) between pens 17 and 18, and pens
1 thru 16 due to induced infection in the latter group. The total mortality
for same period ranged from 4 to 33 percent and followed the same pattern
as one would expect. The greatest losses were in the infected groups and
there was a significant difference (p=.01) between them and pens 17 and 18.
There was no statistically significant difference between the means for
feed efficiency for the 18 groups. These results are summarized in tables
3, 4, and 5.
86
-------
Ninety-eight percent of the carcasses produced from pens 17 and 18
(not infected) graded A. The same parameter for the other 16 pens ranged
from 69 to 95 and the average was 86. There was a significant difference
(P=.01) between the means for pens 17 and 18, and those for 1 thru 16 due
to low level and treatment level antibiotic in the feed, the interaction
between the two, and due to infection. These means and their rank order
are shown in Table 6. There was no significant difference between the mean
percentage of carcasses condemned in the various pen groups.
Results - Microbiology
Fecal samples were taken for three days in order to establish a
baseline of sensitivity patterns of the indigenous E. coli. It was noted
that there was a low amount of resistance to all antibiotics tested with
the exception of dihydrostreptomycin in trial three and this antibiotic
and tetracycline in trial two. Approximately 50% of the population was
resistant to these drugs. Those few which showed resistance to tetracycline
were also R Factor positive with only few exceptions.
Weekly fecal samples were taken after the first three weeks. Sen-
sitivity patterns were determined for each of three isolates from each
pen. It was noticed that tetracycline resistance increased to 100.% in
those pens receiving a ration containing chlortetracycline within one week
after first receiving that ration. Most of these tetracycline resistant
organisms were also R Factor positive.
Resistance transfer studies were performed on all E. coli isolates
which were resistant to tetracycline or dihydrostreptomycin. The procedure
developed by FDA, Beltsville, Md. was used for these tests. A greater
number of RTF positive cultures were isolated from those pens receiving
the ration with chlortetracycline added than were isolated from any of the
other groups. This was especially noted in trial three. There were ninety-
seven RTF positive isolates from pens 5, 6, 7, and 8 as opposed to thirty
from pens receiving tylosin additive, and twenty-two each from the pens
receiving penicillin additive and no antibiotics. In trial two, there were
forty-one positives from pens 5, 6, 7, and 8 as opposed to twenty-three,
fifteen and nineteen, respectively. The higher occurrence in trial three
could be due to the fact that RTF tests were run concurrently with the
sampling and isolation of the organisms, while the tests for trial two
were run after the experiment was completed. It is known that bacteria
may lose the ability to transfer R Factors over a period of time.
Probably the most important parameter measured in the microbiology
laboratory was the acquisition of the antimicrobial resistance by indigenous
E._ coli in the gut of the chicken. This was monitored by weekly examination
of the resistance patterns of the E. coli isolated from pooled fecal specimens
(one pool per pen representing the 25 birds in that pen). Isolates were
tested against sensi-discs containing tetracycline, ampicillin, furoxone,
kanamycin, sulfamethoxypiridazine, neomycin, naladixic acid, dihydrostrep-
tomycin, and added in replication 3 was chloraphenicol. They were almost
universally sensitive to ampicillin, furoxone, naladixic acid, and chlo-
ramphenicol. The statistical analysis was done on the combined means of
replications two and three using the data from pens 1 to 16 (only where
infection was induced. See tables 7 thru 11 attached) during the last
five weeks of life for the birds. For tetracycline there was a significant
87
-------
difference (P=0.001) between the means due to low-level feeding. The
percentages for birds fed chlortetracycline at low-level varied from 91
to 100 percent whereas in other pens it varied from 18 to 62 percent.
Somewhat the same pattern was observed for dihydrostreptomycin and kanamy-
cin. There was a significant difference in streptomycin resistance (P=
0.007) between the means due to low-level feeding. Birds fed chlortetra-
cycline at sub-therapeutic levels yielded between 82 and 100 percent
resistant J^_ coli whereas in the other groups the mean percents varied
from 36 to 73. The means for kanamycin differed significantly (P=0.004)
due to low-level feeding, also. Again the high group (53 to 83 percent
vs. 14 to 53 percent) was in the chlortetracycline fed Birds. There was
not a significent difference between the mean percents of sulfa resistance.
There was a significant difference in neomycin resistance (P=0.1Q) related
to low-level feeding.
The patterns of resistance found in E. coli from intestinal samples
taken in the second replication did not vary greatly from those found in
the composite pen dropping samples. The highest percent resistance to
tetracycline and dihydrostreptomycin was in specimens from birds fed low-
level chlortetracycline. The highest percentage of organisms resistant
to kanamycin, sulfas, and neomycin was in the pens fed low-level penicillin
with bacitracin fed birds a close second. The results of intestine samp-
lings are shown in Table 12. It was interesting to look at the percent
of E. coli recovered from intestines resistant to tetracycline and dihydro-
streptomycin by pen, and group them according to any contact (sub-therapeutic
or therapeutic) with tetracycline (C-pens 3, 5, 6, 7, 8, 11 and 15) and no
contact with it (NC-pens 1, 2, 4, 9, 10, 12, 13, 14, 16, 17, and 18). The
average percent of E. coli resistant to tetracycline was markedly different—
C-pens 81 percent, NC-pens 41 percent. The results were much the same for
dihydrostreptomycin—C-pens 77, NC-pens 48 percent.
The results of examining E. coli from intestines for antibiotic
resistance in the last replication are shown in Table 13. They follow
the same pattern of the previous replication, however, fewer of the iso-
lates were resistant to antibiotics other than tetracycline and dihydro-
streptomycin, and the percentages for these were even slightly lower.
These data from the intestinal and dropping samples demonstrate
the usefullness of the isolation facility and the procedures used for
these experiments. If they were not adequate the percent of resistant
organisms would not vary from pen to pen and between feeds. One of the
most important features of the house may be the air flow of the heating-
ventilating system which travels from the back of the pens into the hall
and exits at the far end to be exhausted to the outside.
Ten carcass swabs were taken from each group of birds and the E_»_
coli isolated (those susceptible to nalidixic acid) were tested for re-
sistance in the second replication only (see Table 14). Swabs from low-
level chlortetracycline fed birds had a higher percent E^ coli resistant
to tetracycline (85%) than those fed other rations (40 to 58%). This
same pattern was observed for dihydrostreptomycin. The percent for chlo-
ratetracycline fed birds was 93 whereas the other groups ranged from 53
88
-------
to 75 percent. We were able to determine the variation of percent of
resistant organisms by pen group from these samples because the individual
carcasses were spray washed rather than sending them as a total group through
a rotating chill-icing tank—the usual practice in the poultry processing
industry.
The carcass swabs from replicate three were only examined for
nalidixic acid _£. coli as these could be readily identified on plates
containing this antibiotic. Escherichia coli Fl were isolated at a very
low rate from these carcass samples.
References
1. Gill, F. A. and E. W. Hook. Changing patterns of bacterial resistance
to antimicrobial drugs. Am. J. of Med. 39:780-792, 1965.
2. Watanabe, T. Infectious drug resistance in enteric bacteria. N. Eng.
J. of Med. 275:888-893, 1966.
3. Smith, D. H. Salmonella with transferable drug resistance. N. Eng.
J. of Med. 275:625-630, 1966.
4. Mitsuhashi, S. Transferable drug resistance Factor R. University
Park Press, Baltimore, Md., 25-31, 1971.
5. Smith, H. W. and S. Halls. Observations on infection drug resistance
in Britain. Brit. Med. J. i:266-269, Jan. 29, 1966.
6. Huber, W. G., D. Korica, T. P. Neal, P. R. Schnurrenberger and R. J.
Martin. Antibiotic sensitivity patterns and R Factors in domestic
and wild animals. Arch. Environ. Health. 22:561-567, May, 1971.
7. Mercer, H. D., D. Pocurull, S. Gaines, S. Wilson, J. V. Bennett.
Characterisitcs of antimicrobial resistance of Escherichia coli from
animals: Relationship to veterinary and management uses of antimicro-
bial agents. Appl. Micro. 22:700-705, Oct., 1971.
8. Jukes, T. H. Discussion- the use of drugs in animal feeds. Proceedings
of a symposium. National Acad. of Sci., Publ. No. 1679, Washington,
D. C., 56-62.
9. U, S. Tariff Commission. Synthetic Organic Chemicals: United States
Production and Sales - 1955. U. S. Govt. Print. Office, Pub. No. 206,
Washington, D. C., 1957.
10. Hays, V. W. Biological basis for the use of antibiotics in livestock
production. N. Eng. J. of Med., 263:11-30, 1969.
11. Anderson, E. S. Drug resistance in Salmonella typhimurium and its
implications. Brit. Med. J. 333-339, Aug. 10, 1968.
12. Kampelmacher, E. H. Foods of animal origin as a vehicle for transmission
of drug-resistant organisms to animals and man. N. Eng. J. of Med.
263:318-326, 1969.
89
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13. Smith, D. H. The influence of drug resistant bacteria on the health
of man. N. Eng. J. of Med., 263: 334-345, 1969.
14. Taylor, J. Salmonellosis: The present position in man and animals.
II—Public health aspects. Vet. Rec., 80:147-154, 1967.
15. Report: An evaluation of the Salmonella problem. Natl. Acad. of
Sciences, Washington, D. C., pp. 8, 23, 63, 197 and 198, 1969.
16. Ullberg, S. Distribution and fate of drugs used in feeds. N. Eng.
J. of Med. 263:225-243, 1969.
17. Morrison, A. B. and J. C. Munro. Appraisal of the significance to
man of drug residues in edible animal products. N. Eng. J. of Med.
263:255-269, 1969.
18. Huber, W. G. The public health hazards associated with the non-medical
and animal health usage of antimicrobial drugs. Pure and applied
Chem. 21:377-388, 1970.
19. Bernum, D. A. The control of neonatal colibacillosis of swine, in
neonatal enteric infections caused by Escherichia coli. Editor,
B. Tennant, Ann. of N. Y. Acad. of Sci., 176:385-400, Jan. 7, 1971.
20. Walton, J. R. Infectious drug resistance in Escherichia coli isolated
from health farm animals. Lancet. 1300-1302, Dec. 10, 1966.
21. Freeman, A. Transferable drug resistance (Editorial) J.A.V.M.A.
150:884-885, April 15, 1967.
22. Smith, H. W. The "cost" of Swann. Vet. Record. 133-134, Jan. 31,
1970.
23. Report: The use of antibiotics in animal feeds, Bureau of Vet. Med.
FDA, Rockville, Md., 20852, 1972.
90
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Table 1.
Pen No.
Design of an experiment to show the relationship of antibiotics
fed at sub-therapeutic levels of response to treatment with
various antibiotics at therapeutic levels in chicks artificially
infected with a pathogen.
Low Level
Antibiotic
Infection
Induced
Treatment
Antibiotic
No. Chicks
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Table 2.
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Yes None
Yes Penicillin
Yes Chlortetracycline
Yes Tylosin
Yes None
Yes Penicillin
Yes Chlortetracycline
Yes Tylosin
Yes None
Yes Penicillin
Yes Chlortetracycline
Yes Tylosin
Yes None
Yes Penicillin
Yes Chlortetracycline
Yes Tylosin
No None
No None
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
450
Results of antibiotic assays of poultry feeds for the experiments
on the relationship of low level feeding of antibiotics to sub-
sequent therapy following induced EA coli infection, Colorado
State University, 1973.
Feed
Number
1
2
3
4
5
6
7
8
Antibiotic
Added
None
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Bacitracin
Level Added
Grams /Ton
None
Low level -
Treatment -
Low level -
Treatment -
Low level -
Treatment -
Low level -
25 g
100 g
50 g
200 g
50 g
900 g
50 g
Assayed
1
None
27
110
56
232
54
780
46
Level of
2
None
27
110
58
214
58
910
49
Antibiotics
3
None
26
100
58
217
58
920
52
Assays made by:
Industrial Laboratories Company
Denver, Colorado
91
-------
Table 3. Replications 2 and 3.
Average Body Weight - grams
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
1978.5
1927.5
1964.0
1908.5
1896.0
1932.0
1967.5
1962.5
2070.5
2083.5
1979.0
1936.5
2060.0
1956.5
1950.0
2059.0
2123.0
2229.0
Rank of
the Mean
11
3
9
2
1
4
10
8
15
16
12
5
14
7
6
13
17
18
Table 4. Replications 2 and 3.
Total Mortality (percentage)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
.285
.325
.220
.320
.280
.260
.260
.240
.220
.180
.240
.250
.220
.200
.320
.200
.040
.040
Rank of
the Mean
15
18
7
16.5
14
12.5
12.5
9.5
7
3
9.5
11
7
4.5
16.5
4.5
1.5
1.5
92
-------
Table 5. Replications 2 and 3.
Feed Efficiency
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
2.000
1.840
1'.905
1.915
1.945
1.905
1.900
1.910
1.860
1.895
1.830
1.930
1.825
1.915
1.875
1.795
1.980
1.920
Rank of
the Mean
18
4
9.5
12.5
16
9.5
8
11
5
7
3
15
2
12.5
6
1
17
14
Table 6. Replications 2 and 3.
Grade A (percentage)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
88.5
78.0
89.5
85.5
94.0
83.0
86.0
89.5
89.5
92.0
87.0
95.0
69.0
80.0
79.5
82.5
98.0
98.0
Rank of
the Mean
10
2
12
7
15
6
8
12
12
14
9
16
1
4
3
5
17.5
17.5
93
-------
Table 7. Replications 2 and 3.
Percentage of indigenous E^ coli resistant to Tetracycline
(average of Week 3 thru Week 8)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlor tetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
.280
.450
.590
.385
.915
.970
.970
1.000
.610
.355
.535
.485
.180
.300
.620
.445
.565
.635
Rank of
the Mean
17
12
8
14
4
2.5
2.5
L.OOO
7
15
10
11
18
16
6
13
9
5
Table 8. Replications 2 and 3.
Percent of indigenous E_._ coli resistant to Dihydrostreptomycin
(average of Week 3 thru Week 8)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep. 2 & 3
.365
.645
.735
.410
.890
.825
1.000
.970
.695
.400
.565
.485
.385
.660
.615
.585
.625
.570
Rank of
the Mean
18
8
5
15
3
4
1
2
6
16
13
14
17
7
10
11
9
12
94
-------
Table 9. Replications 2 and 3.
Percent of indigenous !E._ coli resistant to Neomycin
(average of Week 3 thru Week 8)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep 2 & 3
.190
.215
.280
.125
.310
.535
.435
.355
.360
.030
.300
.215
.090
.135
.250
.115
.135
.335
Rank of
the Mean
12
10.5
8
15
6
1
2
4
3
18
7
10.5
17
13.5
9
16
13.5
5
Table 10. Replications 2 and 3.
Percent of indigenous E. coli resistant to Kanamycin
(average of Week 3 thru Week 8)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment
Antibiotic
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
Mean of
Rep 2 & 3
.250
.250
.310
.265
.670
.820
.735
.530
.530
.320
.505
.450
.210
.135
.355
.250
.370
.600
Rank of
the Mean
14.5
14.5
12
13
3
1
2
5.5
5.5
11
7
8
17
18
10
14.5
9
4
95
-------
Table 11. Replications 2 and 3.
Percent of indigenous JS._ coli resistant to Sulfas
(average of Week 3 thru Week 8)
Pen No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Low Level
Antibiotic
Penicillin
Penicillin
Penicillin
Penicillin
Chlortetracycline
Chlortetracycline
Chlortetracycline
Chlortetracycline
Tylosin
Tylosin
Tylosin
Tylosin
None
None
None
None
None
Bacitracin
Infection
Induced
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Treatment Mean of
Antibiotic Rep. 2 & 3
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
Penicillin
Chlortetracycline
Tylosin
None
None
.470
.355
.165
.125
.365
.395
.375
.265
.445
.185
.210
.375
.265
.190
.235
.085
.135
.435
Rank of
the Mean
1
8
15
17
7
4
5.5
9.5
2
14
12
5.5
9.5
13
11
18
16
3
Table 12. Average percent of indigenous E. coli from intestinal samples
resistant to various antibiotics in relation to ration consumed-
Replication II. _
Ration
Antibiotic 1 2 4 6 8
Tetracycline
Ampicillin
Furoxone
Kanamycin
Sulfa
Neomycin
Naladixic Acid
Dihydrostreptomycin
43 63 81 39 46
26000
28 13 47 10 0
23 57 36 33 46
34 49 17 31 46
21 51 22 25 46
0 0 3 0 23
49 69 83 39 46
Ration 1 No Antibiotic
Ration 2 Penicillin, low-level
Ration 4 Chlortetracycline, low-level
Ration 6 Tylosin, low-level
Ration 8 Bacitracin, low-level
96
-------
Table 13. Average percent of indigenous E. coli from intestinal samples
resistant to various antibiotics in relation to ration consumed-
Replication III.
Ration
Antibiotic 1 2 4 6 8
Tetracycline
Ampicillin
Furoxone
Kanamycin
Sulfas
Neomycin
Naladixic Acid
Dihydrostreptomycin
12 21 76 18 20
00000
72200
6 7 25 6 20
16 10 12 16 40
0 0 2 0 10
22220
43 75 61 44 30
Ration 1 No antibiotic
Ration 2 Penicillin, low-level
Ration 4 Chlortetracycline, low-level
Ration 6 Tylosin, low-level
Ration 8 Bacitracin, low-level
Table 14. Average percent of indigenous E. coli from carcass samples
resistant to various antibiotics in relation to ration consumed-
Replication II.
Ration
Antibiotic 12468
Tetracycline
Ampicillin
Furoxone
Kanamycin
Sulfas
Neomycin
Naladixic Acid
Dihydrostreptomycin
54 50 85 50 40
2 0 10 3 0
62 18 38 53 10
20 30 20 35 30
18 30 8 33 40
50 23 10 10 30
00000
68 58 93 53 10
Ration 1 No antibiotic
Ration 2 Penicillin, low-level
Ration 4 Chlortetracycline, low-level
Ration 6 Tylosin, low-level
Ration 8 Bacitracin, low-level
97
-------
Figure 1. Poultry isolation house - floor plan
Colorado State University
c
Barrier Room /-Lab Area
:•>/
£
!
2
, f
• L
15
16
3
4
5
6
7
8
xj \y x/
)» /*~si > r~^> " /"
.•K.. ' • .' « i \ •(,.• . n.i IM-J / 5 ^\
f IftWTiii.*-*. .%• i-.' i -»-, r.Y,-» • f ^
u
^v -^
/
O
17
1
k
(/
G
\
)
-f-
-Feed
Storage
<
^Barrier Room WiCDecontamination
. 1 Area /
,~ One -Way
Q Foot Bath Traffic
98
-------
PESTICIDES SOLD IN GROCERY STORES
ARE POTENTIAL HEALTH HAZARDS1
Eldon P. Savage, John D. Tessari and Laurier P. Couture
Colorado State University
Many public health workers have voiced concern about the potential
health hazards associated with the sale of pesticides in grocery stores,
primarily because of the possible contamination of foodstuffs. This
contamination may occur in the room where the pesticide is received,
while the product is on display, through sacking and transfer from the
store to the consumer's home, or by airborne transport of pesticides in
store dust. The potential health hazard "represented by" or "associated
with" exposure of customers (especially small children) to pesticides
displayed in areas accessible to them is also of concern.
During the past legislative session in Colorado, only limited data
were available to support an effort to restrict the sale of pesticides
in grocery stores. Convenience for the consumer is the reason usually
given, and in some sparsely populated areas the grocery store may be the
only easily available source from which to buy these items.
During the spring of 1971, the Institute of Rural Environmental
Health (IREH) of Colorado State University, in cooperation with the
Colorado State Department of Public Health, gave a series of training
courses on environmental toxicology at five locations in the State.
A number of questions were raised by public health workers attending
these courses about the continued practice of selling pesticides in
grocery stores. The common practice of displaying and selling pesticides
in grocery stores raised a question about the potential contamination
of fresh produce and other food products by airborne particles inside
the stores.
To learn more about the potential health hazard, the chemical
epidemiology section of the IREH conducted a study during July-August
1971 of 29 stores in five northern Colorado communities.
Methods
The 29 stores randomly selected for the study included 17 super-
markets and 12 privately owned and operated grocery stores. An epidemi-
ologist from the IREH contacted each store manager, stated the purpose
of the study, and completed a survey form. The form included information
on pesticides displayed, location of displays in relation to fresh fruits
and vegetables, statements on labels of pesticide containers, types of
pesticide containers, types of pesticides sold, place where pesticides
were, received in the store, procedures for sacking pesticides for trans-
port by customers, and data regarding the training of managers and employees
in the safe handling of the pesticides.
1 Published in Health Services Reports, 87(8):734-736, October 1972.
99
-------
Results
The distance of pesticide displays from displays of fruits, vegetables,
and baked goods in the store is shown in table 1. In approximately half
Table 1. Percentages of 29 grocery stores displaying pesticides at various
distances from counters bearing fruits, vegetables, and baked
goods, August 1971.
Distance (feet)
Less than 10
11-20
21-30
31-40
41-50
More than 50
Fruit
display
24.2
6.9
17.3
0
3.4
48.3
Vegetable
display
17.3
17.3
6.9
6.9
3.4
48.3
Baked goods
display
0
0
6.9
3.4
6.9
55.2
the stores, pesticides were located more than 50 feet from the fruit
and vegetable displays, and in 24 percent of the stores, pesticide displays
were within 10 feet of the fruit displays. In 17.3 percent, the displays
were located less than 10 feet from the vegetable counters.
Because many people have expressed concern about the accessibility
to children of pesticides in grocery stores, two survey questions focused
on this problem. One question concerned the height of the pesticide
display above the floor and the other concerned pesticide labeling. As
shown in table 2, practically all pesticides were displayed on shelves
Table 2. Number of stores displaying pesticides at various heights above
the floor, by types of containers, August 1971.
Metal Plastic Paper Glass
Distance (feet) containers containers containers containers
Less than 1
1
2
3
4
5
11
3
5
3
3
0
8
1
2
6
0
2
9
1
2
3
0
4
2
0
6
4
1
3
that were within easy reach of children. The height range of the pesticide
displays varied from less than 1 foot to 5 feet off the floor surface.
The pesticides were packaged in metal, plastic, paper, and glass containers.
Metal, plastic, and paper containers were frequently displayed on shelves
2 feet above the floor.
Since warning, caution, and poison classifications on the label are
realted to the degree of pesticide toxicity, the distance form the floor
of the shelf at which toxic pesticides were displayed was noted. Few
stores handled pesticides that were labeled poisons. In the two stores
100
-------
that did handle them, they were displayed on shelves 3 feet above the floor.
In 18 stores, 62 percent, some pesticides with the caution label were
displayed less than 1 foot from the floor. In eight stores, 28 percent,
pesticides with warning labels were displayed on shelves less than 1 foot
above the floor.
The types of pesticides sold in the grocery stores were also noted.
Insecticides were sold in all stores, and in addition more than 90 percent
of the stores sold herbicides and miticides. Approximately one store
in four sold rodenticides, and 62 percent of the stores sold fungicides.
Tabulations of the types of chemicals in the pesticides sold were made.
The survey revealed that botanicals were sold in two-thirds of the stores,
and chlorinated hydrocarbons were sold in Slightly less than two-thirds.
Organophosphates, carbamates, and heavy metals were sold in approximately
one of every three stores handling pesticides (table 3).
Table 3. Types of pesticides sold in grocery stores.
Pesticide
type
Organophosphates
Carbamates
Chlorinated hydrocarbons
Botanicals
Heavy metals
Number
10
9
17
19
10
Stores
Percent
34
31
58
65
34
In the past, DDVP (0, O-dimethyl-2, 2-dichlorovinyl phosphate) strips
have been used extensively in homes, restaurants, campers, and horse barns.
Because a change in labeling of DDVP strips had recently occurred, we
were interested in determining if any of the stores had the product under
both the old and new label in their stock. More than two-thirds of the
stores had DDVP strips and one-third of these handled strips sold under
both the old and the revised label. On one label was the statement that
the strips could be used in homes, restaurants, motels, milk rooms, and
animal shelters, and on the other label was the statement that the strips
should not be used in kitchens, restaurants, or areas where food was
prepared.
An opportunity exists for contamination of food products by pesticides
in the room where supplies are received and at the checkout counter. We
were interested in determining the number of stores that used a common
room for receiving pesticides and grocery products and in observing any
procedure being followed to keep pesticides separated from groceries at
the checkout counter. In more than 70 percent of the stores surveyed,
pesticides and food products were received in the same room. In 24 of
the 29 stores surveyed, pesticides and food products were packaged sep-
arately at the checkout counter.
The desirability of providing training for managers and employees
in the safe handling of pesticides has been suggested. Our study showed
that less than 7 percent of the employees and employers had received any
specialized training in the handling of pesticides.
101
-------
Discussion
Although the record for safety as related to acute accidental exposure
to pesticides in grocery stores in this country is excellent, the potential
for exposure through direct contact and contamination of foodstuffs exist.
The fact that any person can walk into a grocery store and purchase
pesticides and groceries concurrently is not a good public health practice.
Some health departments have attacked the problem by prohibiting the sale
of pesticides in grocery stores. If this measure is not feasible, store
managers should be trained to use isolated areas away from fresh food
products for the display and sales of pesticides.
Pesticides in ambient air inside the store pose another problem.
Because legal tolerances of contamination of food products are low, the
possibility of contamination of fresh food products by airborne particles
in stores where pesticides are sold should not be overlooked. It is
absurd for producers to be extremely careful to avoid contamination of
food products before marketing—only to have the produce contaminated in
the marketplace.
102
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SURVEY OF PESTICIDE MORBIDITY IN KENTUCKY
E. Edsel Moore
Kentucky Department of Health
Nonfatal poisoning by pesticides in Kentucky, as in most States, is
not a reportable or notifiable disease. Reports of incidence are usually
difficult to obtain, making it almost impossible to assess the true
magnitude of this phenomenon at the State or national level.
The pesticide mortality rate for the United States has been
estimated at one per million population. The most accurate estimate for
the ratio of nonfatal to fatal poisonings is 100:1. Figures, however,
for this ratio may vary widely depending upon the criteria of severity
of illness used for defining a case of poisoning (1).
During 1968 through 1971, Kentucky's seven poison centers reported
less than 50 pesticide episodes to the National Clearinghouse for Poison
Control Centers, despite a State population of over 3 million, 3.7
million acres of agricultural crops annually, and 16 to 18 million pounds
of actual pesticide materials applied for agricultural/non-agricultural
use for the four year period (2).
It was felt that because of the restrictions being placed on the
use of chlorinated hydrocarbons (particularly DDT in Kentucky) and the
resulting shift to the organophosphate and carbamate insecticides, a
survey of hospital records at this time would be desirable to determine
whether or not an increase in non-fatal exposure had occurred due to
these more acutely toxic compounds. The survey would also serve as a
reasonable assessment of pesticide morbidity in Kentucky.
For the study, 40 public hospitals were selected to represent a
cross section of the State. Criteria for selection included rural versus
urban location, institutions with 25-50 beds, 50-100 beds and over 100
beds. These hospitals represent approximately 33.97% of the State's
118 public institutions and 6,327 beds (47.6%) of the State's total of
13,274 (3). Excluded from the total were State operated mental and T.B.
hospitals or other State or private institutions providing specialized
health care services. Three hospitals with less than 25 beds originally
selected for the survey were excluded because of inadequate record
keeping systems.
Methods and Procedures
Upon selection of the 40 hospitals, hospital administrators were
contacted and apprised that morbidity data from poison control centers
and other sources was grossly incomplete. All authorized the screening
of records for human pesticide episodes. This was coordinated through
the hospital medical records librarian. Thirty-one of the 40 hospitals
surveyed are located primarily in urban communities (over 2,500 population)
at different points across the State in or near areas of considerable
103
-------
pesticide usage. The remaining nine institutions are in small rural
agricultural communities..
In-patient records were screened for the four year period of
1968-71, with the exception of one hospital which did not begin
operation until 1969. Emergency treatment room records were included
for 1970-71 only. These records are not coded, and the task of screening
either the individual record or log book, depending on hospital policy,
would have been impractical. In-rpatient records of pesticide Incidents
are generally coded in seven or fewer codes, depending on the ICDA
(International Classification of Diseases Adapted) or PAS (Professional
Activities Study) coding system used by the hospital, and are less
difficult to acquire. Only three hospitals employed the obsolete
standard nomenclature coding system. Four different coding systems
were encountered in conducting this study. Refer to Appendix 1 for
codes pertaining to pesticides for these hospital coding systems.
LOCATION OF HOSPITALS INCLUO IN TIE SUMY
25 - 50 BEDS
50 - 100 BOB
OVER 100 BOG
BOUNDARY - EASTERN KENTUCKY COAL FIELD
Figure 1. Location of hospitals included in the survey.
104
-------
Results
Review of the hospital records revealed 975,114 in-patient
admissions (excluding newborns) for the period of 1968-71 and 871,720
emergency treatment room cases for 1970-71. Table 1 summarizes records
screened.
TABLE 1. Suanary o£ records screened. Number of Incidents Involving pesticides in parentheses.
Emergency room
Hospital
Loiii-T/llle General
Children's
St. Luke's
Kind's Daughters'
Bowling Green
(Starred County)
Jepaia Stuart' -Mem.
Western Baptist
Owensboro-Daviess Co.
Somerset City
Good Sanaritad
St. Joseph
Central Baptist .
0. of Ky. Med. Ctr.
Logan County
Haysvood
Norton's Infiraary
Jewish
Fleming County
St. Clair Med. Ctr.
Cocsiunity
John Graves Ford!
Hardin Memorial
Comnunity Methodist
Ohio County
McLean County
T. J. Saason
Bentoa Municipal
Trigg County
Murray-Calloway Co.
CaldveJ.1 County Men.
Clinton-Hickoan
Clinton County Mem.
Caverna Memorial
Franklln-Slrmson
Owen County Mem.
Flaget Memorial . •
Kallory Taylor
Kinj's Daughters'
(Shelbyville)
Mary Chiles
Hopkins County
Totals
19/0
63,915 (4)
28.29^ (42)
13.711 (5)
25,215 (4)
34,000 (3)
8,339 (2)
12,299 (7)
23,786 (0)
10,421 (4)
12,500 (6)
17,000 U)
13,923 (5)
30,536 (8)
4,792 (0)
8,874 (0)
7,637 (0)
14,467 (1)
• 3,060 (0)
6,946 (0)
4,361 (3)
1,743 (0)
12,026 (4)
6,969 (3)
5,383 (3)
1,451 (1)
6,329 (8)
2,515 (2)
1,661 (0)
5,877 (I)
1,842 CO)
1,617 (1)
306 (0)
2,528 (1)
3,734 (1)
4,209 (1)
2,883 (4)
684 (1)
3.633 (1)
1,907 (1)
11,555 (7)
423,438 (135)
1971
6-7,123
28,768
11,692
26,026
33,493
8,887
13,779
26,670
11,954
14,250
19,330
14,261
31,218
6,666
8,231
7,790
15,494
2,317
7,435
5,418
2,623
13,088
6,751
5,546
1,691
7,029
2,815
1,704
8,339
2,153
1,654
389
2,402
3,601
4,644
3,163
504
3,846
3.142
11.896
448,282
(0)
(48)
(3)
(0)
(7)
(2)
(10)
(12)
(5)
(3)
(3) •
(0)
(2)
(4)
(0)'
(0)
(2)
(1)
(2)
(4)
(0)
(5)
(3)
(5)
(4)
(6)
(1)
(1)
(3)
(2)
(2)
(0)
(2)
(4)
(1)
(3)
(0)
(0)
(0)
0)
(146)
1968
12,000
6,954
8,922
13,316
10,287
6,439
9,109
15,031
7,170
l'0,980
14,461
12,499
10,975
3,413
4,652
10,875
13,210
1,066
2,504
2,739
1,830
6,610
6,049
2,037
1,015
5,473
1,740
1,014
4,551
2,753
1,221
1,228
1,654
1,923
1,503
804
2,098
2,560
9,677
232,3-'i2
Emergency rood
Adalss
Grjnd
ilons
total
(0)
(5)
(0)
(0)
(0)
(0)
(0)
(2)
(0)
ay .
(0)
(1)
(1)
(0)
(0)
(0)
• (0)
(0)
(0)
(0)
(0)
(0)
(2)
(0)
(0)
(4)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
-
(0)
(0)
(0)
(0)
(0)
(0).
(16)
871
97J5
1,846
Admission
1969
12,300
7,210
8,824
13,585
10.446
6., 711
9,528
15,401
7.902
11,000
15,227
12,278
11,819
3,646
4,638
11,021
12,709
1,097
2,471
2,755
1,856
6,869
6,703
2,376
81)
5,863
1,892
1,003
4,34f!
2,761
1,254
1,330
1,661
1,910
1,833
1.45C.
725
2.117
2,487
10.777
2A0.6U
,720
jJli
,834
(1)
(2)
(0)
(0)
(0)
(1)
(4)
(3)
(2)
(0)
(2)
(0)
(3)
(3)
(0)
(1)
(0)
(0)
(0)
(0)
(0)
(1)
(0)
(0)
(0)
(2)
(0)
(0)
W)
(1)
(0)
CO)
(0)
(3)
(0)
(0)
(0)
(0)
(0)
(0)
(29)
1970
12,600:
7,990
9,348
13,732
11,236
6,838
9,950
16,579
8,451
11,100
13,115
14,262
13,140
.3,740
4,579
11,070
12,506
1,080
2,496
3,118
1,731
7,635
7,008
2,633
999
6,215
1,693
954
4,236
2,663
1,315
1,550
1,827
1,895
1.905
1,430
745
2,135
2,683
11.T77
249,559
(4)
(1)
(0)
(0)
(0)
(1)
(0)
(2)
(4)
(0)
(3)
(0)
W
(0)
(1)
(0)
(0)
(0)
(0)
(0)
(1)
(1)
(1)
(1)
(0)
(0)
(0)
CO)
(1)
(1)
(0)
(0)
(1)
(1)
(0)
(0)
(0)
(0)
(0)
-CLL
(31)
1971
12,329
7,568
9,131
14,215
12,011
7,205
10,070
16.903
8,252
9,862
16,142
11,743
14,532
4,027
4,705
11,601
12,272
1,127
2,549
3,550
1,488
8,067
6,790
2,317
1,028
6,147
1,748
1,052
5,080
2,673
1,357
1.714
1.696
1,378
1,991
1,393
589
1,996
2,827
11.477
252,605
incidents
(1)
(4)
(0)
(0)
(1)
(2)
(0)
(3)
(2)
(0)
(1)
(0)
(1)
(0)
(1)
(0)
(1)
(0)
(0)
(0)
(2)
(1)
(1)
(0)
(0)
(2)
(0)
U)
(0)
(0)
(0)
(0)
(0)
(2)
(0)
(0)
(0)
(0)
(0)
(2)
(28)
10
95
8
4
11
8
21
22
17
10
10
6
19
7
2
. 1
4
1
2
7
3
12
10
9
5
22
3
2
5
4
3
0
4
11
2
7
1
1
1
17
387
105
-------
As noted in the summary, 387 incidents of exposure to pesticides
were found that were serious enough to require physician attention. Of
these, 281 (72.6%) were emergency room cases that involved treatment
such as lavage or irrigation of the stomach, a medication for dermatitis,
injections of atropine, vitamin K and others. Several victims not
admitted were under observation up to three hours before release.
One hundred and six (27.4%) incidents involved hospitalization of
the victim for routine or intensive care. Nine cases subsequently
resulted in death, six of which involved intentional ingestion of a
toxic pesticide. None of the deaths was associated with usage. These
nine deaths represented 50% of the total pesticide related deaths by
accidental, suicidal or homicidal means during 1968-71 that were recorded
by the Kentucky State Health Department's Division of Vital Statistics.
Of the 106 in-patient incidents, 16 occurred in 1968, 29 in 1969,
33 in 1970, and 28 in 1971. There was only one institution that did not
record an incident related to pesticide exposure during the four year
period. This institution was located in a rural farm community. Two
additional hospitals did not record an emergency treatment room case, and
14 (35%) did not record an in-patient case. Ten of these hospitals had
fewer than 100 beds.
Considering both emergency room and in-patient cases, there were 168
and 174 total pesticide incidents documented at the 40 hospitals during
1970 and 1971, respectively, which represents only a token increase.
Accidents accounted for 359 (92.8%) of all incidents, while 28 were
intentional. None of the incidents was considered to be homicidal or due
to malicious poisoning.
Regarding the time of occurrence of all incidents, 80 were in the
A.M. hours, while 307 (70.3%) were in the P.M. hours, with the majority
being after 5 P.M. Emergency room cases involving pre-school age
children usually occurred before mealtime.
Forty-eight (12.4%) of the total incidents were agriculturally
oriented while 332 (85.7%) were domestic, occurring in and about the
home. The remaining seven were of an occupational or industrial nature.
Virtually all of the on-the-farm incidents resulted from pesticide misuse
or failure of the user to employ the proper protective devices and
clothing while working with highly toxic compounds that included
parathion, dieldrin, demeton, as well as other less toxic insecticides
that were being applied to tobacco or alfalfa. Tobacco is Kentucky's
leading farm cash crop, representing one third of the total farm income.
Eighty-one (20.9%) of the incidents resulted from dermal exposure
or inhalation of vapors or both. Three hundred and six (79.1%)of the
incidents involved oral ingestion of a pesticide occurring predominantly
in the urban environment. Incidents were prominent in both the affluent
and lower socio-economic neighborhoods. Most of these incidents were
due to indiscriminate use inside the home and the ready availability of
improperly stored pesticides, usually in the kitchen.
106
-------
There were 243 (62.8%) males involved and 144 females. Three
hundred and thirty-eight (87.4%) of all cases involved Caucasians; the
remaining 49 cases were Negroes (approximately 93% of the State
population is Caucasian).
Table 2 categorizes all incidents by age group. Note that 263
(68.0%) of the incidents involved pre-school children.
TABLE 2. Incidence of pesticide poisoning by age group.
Age No. incidents
1 year and under 53
1-5 years 210
6-10 years 15
11-20 years 30
21-40 years 38
41-60 years 28
Over 60 years 11
Unknown 2
Total 387
Pesticide incidents involving pre-school age children were far less
common in the in-patient cases (44) than in emergency treatment room
cases, which numbered 219 (83.3%).
Careful review of the treatment records of the 387 cases of
pesticide exposure revealed that 136 (35.1%)demonstrated manifestations
of poisoning which were observed and recorded by the attending physician.
There were other cases in which by virtue of the treatment rendered the
victims probably demonstrated signs or symptoms, but these were excluded
because the physician failed to record them.
It can be postulated that many additional incidents would have
produced clinical manifestations of poisoning had the victim not
received prompt and proper medical attention. Only 16.7% (44) of the
pre-school age victims required hospitalization for treatment and careful
observation, compared to 52% (25) of the agricultural victims.
Thirteen of the 40 hospitals surveyed, not necessarily in rural
areas, had 50 or fewer beds; 49 incidents or 12.9% of the total number
were recorded at these institutions, and only ten (20.4%)of these were
agriculturally oriented. The remaining 39 agriculturally exposed victims
previously alluded to were treated in the larger urban hospitals. This
suggests that the larger institutions may be better equipped to treat
individuals poisoned by pesticides. This is further substantiated by the
107
-------
fact that there were only 27 episodes (.7%) of the 387 recorded by the
nine hospitals located in rural communities.
Incidents occurred in every month of the year with, the greatest
number, 226 (58.4%), occuring during the peak pest control season, May
through September. Emergency room cases were significantly more frequent
during April through September, as illustrated in Figures 2 and 3.
FREQUENCY OF IN-PATIENT INCIDENCE -MONTHLY
40
35
30
S3 25
2
o
fe 20
3
I 15
10
JAN. ret. MAR. APR.MAY JUNE JULYAUt". SEPl7
OCT. NOV. DEC.
Figure 2. Frequency of in-patient incidence—monthly.
FREQUENCY OF EMERGENCY ROOM INCIDKNCE - MONTHLY
JAN. fEB. MAR. APR. HAY JUNE JULY AUC. SEPT. OCT. NOV. DEC.
Figure 3. Frequency of emergency room incidence—monthly.
A total of 44 different pesticides were specified and implicated in
the 387 incidents (.see Table 3). In 28 of the incidents, a combination
of two or more pesticide compounds were named that included DDT-dieldrin,
malathion-captan, malathion-endosulfan, chlordane-diazinon, household
108
-------
aerosol preparations, pyrethrum-piperonyl butoxide, dichloryos-dieldrin,
etc. Insecticides were named in 242 (62.5%) of the cases, rodenticides
in 154 (39.8%), herbicides in 17 (0.4%), fungicides in one, and a
fumigant in one.
TABLE 3. Pesticides implicated in all incidents.
Type of pesticide
insecticides
Unspecified insecticides
(23 of which were desig-
nated as "tobacco sprays")
Arsenic (all forms)
Malathion
Pyre thrum
Piperonyl butoxide
Paradichlorobenzene
Chlordane
Dichlorvos
Dieldrin
Sodium fluoride
Diazinon
DDT
Lindane
Parathion
Boric acid (for roach
control)
Lead (in combination
with arsenic)
Endosulfan
Roiinel
Toxaphene
Demeton
Dimethyl phosphorate
(snail bait)
Carbaryl
Coal tar (sheep dip)
TDE
Kepone
Methoxychlor
Ciodrin
Benzyl benzoate
Dichlorcme thane
Nicotine sulphate
Endrin
Kel thane
Number of
incidents
242
47
34
22
16
16
12
9
9
8
8
7
.
7
6
5
4
*r
A
*T
4
3
3
3
7
f.
2
2
1
1
1
1
1
1
1
1
1
type of pesticide
Rodenticides
Warfarin
Unspecified rat poisons
Coumarin
Plval
Strychnine
Prolin
Phosphorus
Herbicides
2,4-D
Sodium arsenite
Maleic hydrazide
Atrazine
Meta borate
Dalapon
Fungicides
Copper sulphate
Fumigant
Calcium cyanide
GRAND TOTAL
Number of
incidents
154
97
41
6
6
2
1
1
• 7
5
2
1
1
1
I -
1
1
1
415
109
-------
Arsenic (all forms) was the most commonly implicated insecticide
compound. The majority of episodes involving arsenic compounds can be
attributed to products formulated for household purposes, usually for
control of ants and roaches. This may not have been the case had
physicians identified all unspecified insecticides. Chlorinated
hydrocarbon insecticides accounted for 41 incidents while organophosphate
insecticides were implicated in 52 incidents.
Evaluation of the data did not reveal a significant increase in
organophosphate exposure cases during the last two years. However,
organophosphate and chlorinated hydrocarbon compounds combined were
responsible for 71% of the agriculturally oriented incidents involving
insecticides.
Discussion
The review of records indicates that pesticides, especially in the
urban environment, present a significant hazard particularly to pre-
school age children because of parents' faulty judgment in using and
storing chemicals. Although pesticides formulated for the home, garden
and lawn market are usually of low concentration and rarely cause death,
they do cause parents considerable anxiety. Prompt medical attention
has probably prevented illness in several instances.
Virtually all of the incidents in this study, in both the urban
environment and the agricultural sector, are attributable to 1) improper
storage, 2) indiscrimate use, and 3) failure to observe the proper
precautions when associated with the more toxic pesticides.
Although other hazardous materials such as drugs, solvents, cleaning
compounds, etc., were not included in the review, it was evident that
they contributed to substantially more incidents than pesticides despite
the fact that probably 90% of the families in this country currently use
a pesticide of some description for the control of a pest problem
sometime during the year.
All of the agriculturally oriented exposure incidents occurred
during the usage season whereas the urban incidents, although more
predominant during the peak pest season, occurred the year around.
The survey did not reveal a substantial number of exposure cases of
agricultural users to the highly toxic organophosphate insecticides as
was anticipated, although such exposure was reported by poison control
centers and other sources, particularly during 1970. A poison control
center in central Kentucky reported ten calls of inquiry by physicians
about parathion and other phosphate compounds during August and September
that year. It can be concluded that these cases were treated in hospitals
other than those included in the study and low grade intoxications
occurring in the rural sector were probably treated by physicians at
their offices, in small clinics, which were not included in the survey.
110
-------
Available pesticide morbidity data do not reflect a true incidence
of nonfatal exposure. In a recent interview of 75 pesticide applicators
and farmers participating in a Health Department Blood Cholinesterase
Testing Program, virtually all responded that they had on one or more
occasions experienced headaches, dizziness, nausea, numbness of limbs
and other symptoms when associated with different compounds for several
hours or days. The compounds involved included parathion, azinphosmethyl,
disulfoton, dieldrin, endrin and others. None of the persons interviewed
consulted a physician for observation or treatment. Most responded
that they felt the chemicals were responsible for the described symptoms
and when they appeared, discontinued use until they subsided.
Many complained about "Paris Green" having caused severe dermatitis.
This compound, which is no longer used, was very popular with tobacco
farmers years ago.
This study points out that the incidence of nonfatal exposure to
pesticides is a significant problem of unassessed magnitude and that data
from poison control centers are not a true indicator of the problem,
especially in Kentucky, because most of the exposure cases reported in
the study did not involve a center. As indicated previously, annual
reports received from the National Clearinghouse for Poison Control
Centers revealed less than 50 cases were reported from Kentucky's seven
centers during the time period this study covered.
Correlation of pre-school age children incidents for 1970-71
indicated an increase from 114 to 123 or 7.3%. Incident data compiled
by the National Clearinghouse for Poison Control Centers indicated the
problem in the age group under five years had been increasing but
stabilized during the years 1967 through 1970 (4,5). This may be
attributable to inadequate reporting.
It is hypothesized that had all of the State's 118 hospitals been
surveyed and all records reviewed for the 1968-71 period, over 1,200
exposure cases requiring physician attention would have been documented.
This study points out the need for improved reporting procedures,
particularly in Kentucky, and creating an awareness of the importance
of storing and using pesticides properly.
Ill
-------
References
1. Hayes, W. J., Jr. Occurrences of poisonings by pesticides. Archives
of Environmental Health. 9:612-625, 1964.
2. Moore, E. E. Pesticide sales and usage in Kentucky-—1968. Pesticide
Monitoring Journal 6(.4):379-387, 1973; and Moore, E. E. Revisitation
pesticide sales and usage in Kentucky-—1971. Unpublished report.
3. Index—facilities licensed as hospitals by the Kentucky State Board
of Health., From the Division of Health Facilities, Kentucky State
Department of Health, Frankfort, Kentucky.
4. Personal communication: Grotty, John J., M.D., Chief, National
Clearinghouse for Poison Control Centers, Washington, D.C.
5. National Clearinghouse for Poison Control Centers Bulletin,
September, October, November, 1971, p. 6.
112
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Appendix I. Hospital Coding Systems Pertaining to Pesticides
1962 (December) Revised Edition of ICDA
(Public Health Service Publication No. 719)
Codes
961.0 Mercury and its compounds
961.2 Arsenic and its compounds
961.9 Other metals chiefly nonmedicinal as to source (includes
thallium)
962.1 Strychnine
962.2 Other insecticides and rodenticides
962.8 Other gas, fumes or vapor (includes methyl bromide)
8th Revision of ICDA (Public Health Service Publication No. 1963)
Codes
964.2 Anticoagulants
985.0 Mercury and its compounds
985.1 Arsenic and its compounds
985.9 Other
989.1 Strychnine
989.2 Chlorinated pesticides
989.3 Other pesticides
Professional Activities Study System
Codes
964.2 Warfarin, Coumarin and other anticoagulants
985.0 Mercury and its compounds
985.1 Arsenic and its compounds
985.9 Other—thallium, etc.
987.4 Includes methyl bromide, methyl chloride
989.0 Cyanide
989 ,.1 Strychnine
989..2 Chlorinated pesticides
989..3 Other pesticides
113
-------
Standard Nomenclature of Diseases and Operations, Fifth Edition
Etiologic classification
32: Metals, metalloids, and their compounds, inorganic and organic
321: Arsenic, selenium, tellurium, and their compounds
322-326: Nonradioactive metals and their compounds
3238: Lead and its compounds
3243: Mercury and its compounds
3257: Thallium and its compounds
33: Simple organic substances other than those specified in categories 34-39
331: Hydrocarbons and halogenated hydrocarbons
3315: Halogenated aliphatic hydrocarbons
33151: Methyl bromide
3316: Halogenated aromatic hydrocarbons
36: Economic poisons and chemical warfare agents
361-362: Insecticides and poisons against other invertebrate pests
o/- -I -I __
Halogenated hydrocarbon insecticides
Halogenated hydrocarbon insecticides containing
organic oxygen
„,-, Halogenated hydrocarbon insecticides (miticides)
containing organic sulfur (with or without oxygen)
3615—
_,-,, Organic phosphorus insecticides
_-., Insecticides from botanical sources (and related
synthetic substances)
3619: Miscellaneous insecticides
363: Fumigants for space, soil and grain
3631' Nitriles exclusive of acrylonitrile (33751) and
hydrogen cyanide (3153)
3635: Naphthalene and derivatives used as fumigants
3637: Miscellaneous fumigants
,,,.. Herbicides (weed killers, defoliants, desiccants, soil
sterilants) and plant growth regulators
366: Fungicides
367: Rodenticides and other vertebrate poisons:
36711: Warfarin
36712: Pivalyl indandione
3672: Inorganic poisons
3673: Poisons of botanical origin except rotenone
3674: Miscellaneous
114
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EPIDEMIOLOGY AND PREVENTION OF PESTICIDE POISONING
John I. Freeman
North Carolina Department of Human Resources
Pesticides form one of the major agents contributing to the occupational
disease record of agriculture and because agriculture is one of the major
industries in the South, pesticide epidemiology is truly representative
of a specific occupational health problem (1). In 1964, there was a
striking increase in south Texas in the number of cases, the total (70)
was approximately equal to the number of cases observed during the pre-
vious four years combined. This rise coincides with the introduction of
certain organic phosphate insecticides used to control crop pests, espe-
cially insects attacking cotton (2). In south Florida, pesticides have
been responsible for 49% of deaths due to poisoning among children,
making these chemicals the leading cause of pediatric poison mortality
(3). Epidemiologically, pesticide poisoning can be categorized into two
major groups, i.e., the pediatric group and the occupationally exposed
group, the latter group being inclusive of farmers as well as various
aspects of the agri-business complex.
Epidemiologic Aspects
The epidemiology of poisoning due to pesticides is exceedingly complex.
The epidemiologic model of agent-host-environment is as applicable to
morbidity due to chemicals as it is to infectious agents; however, in
chemical epidemiology there is seldom a single causative factor. More
often than not, a multiplicity of factors are interrelated and signifi-
cant,when the sequence of.events is reconstructed. From field investi-
gations of acute pesticide poisoning cases, the following are considered
important factors in the epidemiology of pesticide poisoning (4).
Organochlorine vs. Organophosphorus Compounds. It is perhaps unfortunate
that the organochlorine pesticides preceded the organophosphorus ones.
Generally, the organochlorine compounds are less toxic than the organo-
phosphorus pesticides, which due to their low toxicity allowed farmers to
develop rather reckless procedures for handling pesticides. During the
late 1940's and early 1950's, the organochlorine pesticides became read-
ily available to farmers and it was during this time that farmers devel-
oped such practices as mixing pesticides with their arms, application
without protective clothing or masks, and improper storage and disposal
of unused material and containers. Due to the low toxicity of most
organochlorine pesticides, such gross violations of recommended proced-
ures seldom resulted in illness or death to the applicator. The organo-
chlorine era from the late 1940's to the mid 1960's^has firmly affixed
many improper procedures of handling pesticides in the farmer's mind.
This is perhaps the most important contributing factor in pesticide
epidemiology today.
The usage of organochlorine pesticides began to decline in the late
1960's, and the compounds that replaced them were the more toxic organo-
phosphorus materials. In making the transition to the use of organo-
phosphorus pesticides, farmers continued to use the same techniques and
115
-------
procedures that had been practiced during previous years. The amount
and degree of individual exposure remained fairly constant, but toxicity
of the pesticidal material—the organophosphorus compounds—was sub-
stantially increased, resulting in increased morbidity and mortality.
Accessibility. For years, pesticides have been offered for sale as an
over-the-counter item throughout the country. Highly toxic compounds
such as parathion were sold to both the educated and the illiterate. The
availability of highly toxic pesticides plus improper on-the-farm pro-
cedures account for most of the acute cases of toxicosis in the working
age groups.
Children, particularly those less than 6 years old, are victims of their
environment. Pesticides are readily accessible to children on most farms.
A survey conducted in an eastern North Carolina county revealed that only
10% of the farmers stored pesticides under lock and key. Pesticides were
found to be stored in every conceivable location on the farm, including
all the places in buildings that children normally play.
Storage of farm commodities under lock and key is not a new concept to
farmers. The survey also revealed that, without exception, gasoline was
stored under lock and key. This raises the question of priorities, i.e.,
protecting the dollar value of gasoline or protecting the lives of
children.
Accessibility of toxic pesticides is a common factor in the acute poi-
soning of both adults and children but epidemiologic aspects pertaining
to these two groups are quite different. In the adult group, society
permits the sale of highly toxic pesticides, predicated on the fulfillment
of labeled requirements. The label, which is explicit, detailed, and
informative as to correct usage, is the only protection afforded the pur-
chaser. Little or no regard has been given to the purchaser's ability to
read or to comprehend the label message. Thus illness and deaths continue
to occur because "they didn't read the label."
The accessibility of pesticides to children is generally due to negligence
of their parents. Pesticides for farm use are purchased and brought into
close proximity to the home. After application, unused material is
returned to the home environment and 90% is stored in such a way as to be
accessible to children.
Morbidity. Pesticides are a major cause of illness in our society today.
Data on total poisonings from pesticides reported in 3 recent years are
given (Table 1).
In North Carolina, the organophosphorus insecticides are the leading
cause of pesticide morbidity. Reported cases in North Carolina, by type
of pesticide, are given (Table 2). The organophosphorus insecticides are
cholinesterase-depressing compounds and most persons acutely afflicted
require hospitalization. Exposures and illness due to pesticides other
than the organophosphorus compounds are often treated in the emergency
room or on an out-patient basis and seldom require hospitalization of the
patient.
116
-------
TABLE I
Reported Poisonings by Pesticides United States 1968 - 1970 (5)
Cases Deaths
Pesticide 1968 1969 1970 1968 1969 1970
Insecticides 2,919 2,929 2,913 11 14 13
Rodenticides 1,185 1,192 1,132 4 6 6
Fungicides 99 90 106 0 0 0
Herbicides 254 281 287 96 1
Mothballs 922 813 933 0 0 1
Miscellaneous 360 397 358 0 0 0
TOTAL 5,739 5,702 5,729 24 26 21
When reported cases (Table 2) are tabulated by month of onset, it is
evident that pesticide-induced illness is seasonal. As would be expected
in an agricultural population, the greatest number of cases occur May
through September (Table 3), correlating with pesticide demands on the
major field crops produced by North Carolina farmers.
TABLE 2
Reported Poisonings by Type Pesticide - North Carolina 1970 - 1972
TYPE 1970 1971 1972
Organophosphorus 55 32 47
Organochlorine 27 10 9
Carbamate 969
Herbicides 348
Rodenticides 15 17 15
Other Pesticides 23 44 42
TOTAL 132 113 130
117
-------
TABLE 3
Reported Poisoning by Month - North Carolina 1970 - 1972
YEAR
MONTH 1970 1971 1972
January 025
February 044
March 357
April 6 8 10
May 11 18 12
June 10 17 15
July 22 14 18
August 45 24 26
September 15 13 11
October 5 7 13
November 735
December 10 2 4
TOTAL 134 117 130
Future Trends. Pesticides probably will continue to be used in all seg-
ments of agricultural production. The use of organochlorine compounds,
because of their persistence in the environment, has been declining
during the past several years and recent decisions relating to use of DDT
will further limit the use of the organochlorine insecticides. The
organophosphorus insecticides are being used more and more as replacement
compounds for the persistent organochlorines as they are removed from the
market. Based on experience thus far with the organophosphorus insecti-
cides, an increased use of highly toxic materials by the farming popula-
tion will result in an increased rate of pesticide-induced illness.
Another important trend is the activity at both state and federal levels
in legislative control on the use and application of pesticides. Ef-
fective pesticide legislation must protect the farmer but make available
for production the necessary compounds and protect consumers and the
environment.
Pesticide legislation is difficult to formulate and implement. It is both
time-consuming and expensive, yet it now appears that society demands and
is willing to support financially such legislation. Legislative controls
will undoubtedly have a curbing effect on many of the pesticide problems;
however, they are not the ultimate answer. The many disciplines that have
a vested interest in the pesticide problem must strengthen their working
relationships toward the common goal of reducing illness and deaths attrib-
utable to pesticides.
118
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Prevention
Planning. The logical first step in preventing pesticide poisoning would
be in selecting the type pesticide material to be used on individual
crops. Planning at this stage of crop production would allow the selec-
tion of a material that would be effective as well as one with a lower
toxicity when there are several products registered for the same use. An
example of such an option would be the choice of either a granular or
emulsifiable concentrate form of a contact nematocide. While both products
contain the same active pesticidal material and each is equally effective
as a nematocide, there is a striking difference in the hazard to the per-
son applying this pesticide in their two forms. Pesticides in granular
forms are less hazardous than emulsifiable concentrates. Safety planning
must also take into account who will actually be available to apply the
pesticides. In North Carolina, we observed that the highest percent of
pesticide-induced morbidity in agriculture workers occurs in the 10-14 age
group with only a slight drop in the 15-19 age group followed by a marked
drop in successive age groups. From case investigations, it is quite
clear that an inexperienced operator-applicator is more likely to exper-
ience pesticide poisoning than a more experienced adult.
Pesticide safety planning at the farm level should also include storage
of materials as well as well as disposal of unused materials and contain-
ers. A well-planned safety program must begin before the pesticide is
brought to the farm and continued until all material has been properly
used or properly disposed.
Application. Application accidents and exposures are by far the most
common type pesticide case among farm workers. Most cases associated
with the application of pesticides can be related to inadequate protective
devices and clothing. Protective clothing is an all inclusive term used
in pesticide language which has become meaningless unless it is defined
for specific pesticide uses. In discussing and recommending specific pro-
tective clothing, one must take into account the feasibility of the appli-
cator following instructions. All too often those of us who make recom-
mendations for protective clothing have never.been in a position to field
test our recommendations.
Generally speaking, a pair of non-absorbent gloves and boots are essential,
particularly when handling concentrated materials. Body protection can
generally be achieved by tight-weave cotton trousers and long sleeve
shirts. However, the applicator must be aware that frequent changing of
clothes is necessary and a change of clothes should be carried on the
equipment for an emergency change should a spill or other accident occur.
In the southeast, there are a very few instances where a rubberized suit
would be practical or even tolerable during the primary pesticide use
season.
Respirators are particularly effective in reducing the total body expos-
ure to those pesticides that are absorbent through mucous membranes and
the respiratory tract. Respirators are not particularly uncomfortable to
wear yet a very large percent of farmers are reluctant to wear such
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devices. From many conversations with farmers, both those who have ex-
perienced illness and those who have not, I have come to the conclusion
that the reluctance to wear a respirator is due to the fear of peer crit-
icism. This attitude is particularly prevalent among small farmers who
apply pesticide themselves rather than delegate the task to employees.
It has been our experience that most cases associated with the application
of pesticides could have been prevented by the use of gloves during the
mixing and recharging of spray tanks along with the use of a respirator
and tight-weave cotton clothes while actually operating the spray rig.
Along with the above recommendations, an adequate supply of soap and water
should be available in the field for accidental spills that contaminate
clothes and skin.
Storage and Disposal. Improper storage and disposal of pesticides is a
significant factor in poisonings that occur among the pediatric group.
Young children, to say the least, are adventurous and notorious for ex-
ploring the small world within which they live, i.e., the home environ-
ment. Those cases associated with storage and disposal could be most
readily prevented. Improperly stored or disposed pesticides are acces-
sible to those persons in the immediate environment. From a practical
and functional point of view, the pediatric group must be considered
illiterate in terms of reading pesticide labels or comprehending hiero-
glyphics that appear on pesticide labels. Therefore, to effectively
reduce pesticide-induced illness in the pediatric group, the accessibility
of these materials to this group must be reduced.
To accomplish such a reduction in accessibility, it is not necessary to
re-educate the farmer or teach him new methods of security. Farmers have
long practiced the lock-and-key technique of security for stored gasoline,
other farm commodities, tools, and equipment. Such security techniques
are for protection against theft but also, in part, for protection against
one's own family, particularly those family members that fall into the
pediatric group. If a farmer routinely locks his gasoline storage tank to
prevent employees from stealing gasoline and if a farmer routinely locks
his tool box to prevent his children from losing his tools, the question
is how do you motivate a farmer to exercise the same degree of security to
prevent poisoning of his family members by pesticides. There are perhaps
three approaches to attack this aspect of the pesticide problem. First,
the direct economic approach, such as higher insurance premiums for those
who do not have adequate storage facilities and do not exercise proper
security measures, would probably be the most effective; however, such an
approach would be most difficult to monitor and enforce. Secondly, legis-
lation could be enacted to require all pesticides to be stored under lock-
and-key and all containers be buried. Again the problem of enforcement
would greatly dilute the effectiveness of such legislation. A third
approach might be to utilize the technique of the social sciences in an
attempt to re-align farmers' priorities and attitudes toward the presence
of highly toxic chemicals in the home environment. This approach would
certainly be slow but may well be the most rewarding in the long run. It
is tragic to realize that many farmers must visit the Emergency Room of a
community hospital before they realize the necessity of proper pesticide
storage and disposal.
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Legislation. Legislation and the subsequent enforcement of pesticide laws
obviously will have some effect on the incidence of pesticide-induced
illness. For years, pesticides have been available over the counter to
anyone without restrictions. This in itself has contributed to the pres-
ent attitude that farmers have toward pesticides. Those who sell pesti-
cides devote all their time to extolling the virtues of their product and
neglect to warn the farmer that their product may be equally effective in
killing him and his family as it is in killing the target pest.
Legislation can be effective in bridging the gap in several areas that we
know are important in the prevention of pesticide poisonings. Through
dealer testing and licensure, we can be assured that those who sell
certain toxic pesticides to the end users possess certain knowledge about
pesticides that could be passed on to the purchaser. Through legislation
and subsequent development of restricted-use pesticides, the time and place
of the use of highly toxic materials can be somewhat controlled. Also,
through this technique, the functionally illiterate can be prevented from
purchasing and using highly toxic materials.
It seems quite reasonable to require those who sell highly toxic pesti-
cides to have available for purchase certain protective devices, partic-
ularly non-absorbent gloves and respirators. Such items are not readily
available in many rural areas or small towns. The availability of
protective devices would certainly be an influencing factor on a farmer's
attitude toward the pruchase of such items.
Legislation currently requires certain warning and precautions to appear
on the pesticide label. Experience has taught us that this method of
communicating a message to the end user has greatly failed. Likewise,
other written communications such as leaflets and pamphlets prepared by
pesticide manufacturers and extension service personnel has also failed
to adequately warn the end user of the dangers associated with certain
pesticides. Since written communication has not adequately conveyed the
danger message, perhaps it would be wise to explore the possibility of
requiring pesticide manufacturers to devote part of their verbal advertis-
ing time to warning the users of pesticides that their products may cause
illness and death. Radio and television are successful tools of advertis-
ing arid are effective in selling a product. It, therefore, seems reason-
able to assume that verbal communication via radio and television would
also be effective in forming an association between a product name and its
danger to human health.
Conclusions
Pesticides are a major cause of occupational illness in agriculture.
Pesticides are essential in agricultural practices; however, in a nation
that is losing farms at a rate greater than 100 per day, it would seem
essential to protect the agricultural work force against the occupational
hazards of pesticides.
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The epidemiology of pesticide-induced illness can be summed up in two
major areas—accessibility of highly toxic chemicals, and failure to
follow recommended procedures. Pesticides as a cause of illness, unlike
infectious agents, cannot be eliminated or removed from the environment.
To be effective, pesticides must be applied to fields and crops; thus,
human beings will be exposed. Assuming that this exposure cannot be
eliminated but can be minimized, the rate of pesticide-induced illness
can be reduced. Current trends in pesticide legislation are to minimize
the exposure of the functionally illiterate population to highly toxic
pesticides.
Accessibility to toxic pesticides can be controlled to some degree by
legislation; however, legislative controls must be followed by an educa-
tional program at the farm level in the proper procedures of handling
highly toxic pesticides. Agricultural organizations and industries must
assume a leadership role in educating the farming population in the correct
use of agricultural chemicals.
References
1. Davies, J. E., J. 0. Welke, and J. L. Radomski. Epidemiological
aspects of the use of pesticides in the south. Journal of Occupa-
tional Medicine. 7:12, 612-617, 1965.
2. Riech, G. A., G. L. Gallaher, and J. S. Wiseman. Characteristics
of pesticide poisoning in south Texas. Texas Medicine. 64(7):
56-58, 1968.
3. Davies, J. H., et^ al. Occurrence, diagnosis, and treatment of organo-
phosphate pesticide poisoning in man. Read before the New York Academy
of Science, May, 1967.
4. Freeman, J. I. and M. P.. .Mines. Epidemiology of pesticide poisoning
in North Carolina. J.A.V.M.A., 161(11):1492-1494, 1972.
5. Lisella, F. S. Epidemiology of poisoning by chemicals. Journal
of Environmental Health. 34(6):602-612, 1972.
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REPRODUCTION IN QUAIL AFFECTED BY LOW LEVEL PESTICIDES
C. L. Quarles, E. P. Savage, G. R. J. Law, and J. D. Tessari
Colorado State University
Introduction
With increasing concern for human and animal health and with ever
expanding awareness of the interaction of man with his environment,
industrial chemicals, pesticides and herbicides have been the topic of
many publications. Of particular concern has been the effects of these
substances on populations of wildlife, including fish, mammals and birds.
Ingestion of toxic amounts of certain chemicals has resulted in reduced
wildlife populations either by the direct action of the agent causing
death of animals of breeding age or by the indirect action of reduced
reproductive efficiency through physiological alteration in the complex
process of reproduction. Although the pathway of action of various
compounds differs, the result is often the same—declining population,
increasing awareness of the environment, and man's involvement at many
levels of interest and concern.
Much of the information and publicity about this complex subject of
environment, population, health, reproduction and relationships to man
has centered around birds. As early as 1955, DeWitt (1) reported that
chemical agents for the control of unwanted plants necessarily involved
contamination of foods for wildlife. Reproduction of bobwhite quail,
the mallard duck and ring-necked pheasant was either reduced or inhibited.
Quantities of pesticides in diets ranged from 0.5 ppm for aldrin to 1250
ppm for derivatives of 2,4-D (2,4-dichlorophenoxyacetic acid). Further-
more, diets containing one of the highly chlorinated polycyclic insecti-
cides inhibited the appearance of secondary sex characteristics in these
same species of birds.
The effects of sublethal or subacute exposure of birds to pesticide
residues is only partially known as there are many kinds of chemical
agents, many different species, and many unknown factors in wild
populations. Fowler e~b at, (2) summarized some of these observations
on a wide variety of changes in behavior, liver function, testicular
development, delay in ovulation, metabolism of steroids and lack of
calcium in egg shells.
In a different study, Porter and Wiemeyer (3) compared low levels
of dieldrin and DDT in the diet to sublethal high levels. Even at low
levels, treated sparrow hawks showed reduced reproductive success.
There are additional reports in the literature concerning reproduc-
tion in birds. Muller and Lochman (4) observed reduced egg production,
fertility and hatchability in mallard ducks fed sublethal levels of
various pesticides. Similar reduction in nesting and number of young
in nests have been associated with the presence of subacute levels of
chemical residues (5). An aspect of this topic which has not received
much attention is that concerning the interaction of two or more of these
123
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chemicals. As an example the work of Peakall (6) is cited. When both
DDT and dieldrin were fed in combination to pigeons, a greater increase
in metabolites of testerone was observed than if each was fed
individually. Conceivably, natural populations could be exposed
to multi-environmental chemicals and this experiment suggests that
certain chemicals can have a synergistic effect.
Experimental Material
The quail used to study effects of low level pesticides at
Colorado State University are not an indigenous species to this country.
Coturnix cotupnix japonica, or the Japanese quail, is a very excellent
bird for avian research. This bird is small, relatively inexpensive,
has a short generation cycle, high capacity for egg production and is
amenable to intensive rearing in cages for experimentation. Many of the
procedures and physiological measurements used in experiments with the
domestic chicken can be applied almost directly to the Japanese quail.
Furthermore, much of the work from experimentation in the laboratory can
be applied to natural populations with appropriate modifications based
on food chains, population ecology and natural habitats.
Objective
The present experiment was designed as a preliminary trial concerned
with the effects on reproduction of multiple exposure to more than one
chemical agent considered to be a possible hazard in the environment.
Procedures
Eggs for this study were obtained from the coturnix colony of Dr.
Henry Marks, U.S.D.A., Athens, Georgia. Sufficient eggs were hatched by
standard procedures to provide chicks for five treatments consisting of
three replicated pens in each treatment. In all, 114 quail were used in
this study, 24 per treatment except controls which consisted of 18 chicks.
The treatment diets were fed from day old until termination of the
experiment and consisted of a basal quail ration with the following
chemical agents added at the low rate of 1 ppm:
Ration Description
A Control
B Mercury (as mercuric chloride)
C Polychlorinated biphenol (PCB)
D DDT
E Combination (M + PCB + DDT)
Rations were prepared by micro-mixing in 70 pound lots and stored
in paper bags.
Management procedures during the growing period and reproduction
phase were standard practices of ad libitum water and feed for each pen.
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No medication or therapeutic agent was administered during the course of
the experiment.
During the growing period, body weights were obtained at 4, 6, 8 and
10 weeks of age. Mortality records were kept throughout as well as
records of aberrant behavior, excitability, cannabilism and rate of
feathering. These characteristics can reflect abnormal physiologic
functions not seen by body weight changes alone. .
After the onset of egg production (about 42 days) records were kept
of rate of lay for 32 days. Two settings of eggs provided data on
fertility, hatchability and embryonic mortality. The latter records
were divided into early (first week of incubation), late embryonic
mortality (last week of incubation) and mortality occurring after the
chicks have pipped the shell (pips). Shell thickness was recorded on
dry bases after each hatch. Chicks produced in each hatch were reared
by treatment and fed their respective diets to observe any transmittable
effects of the treatments applied to the parents.
All quantitative data were analyzed statistically by procedures of
analysis of variance and the Sheffe modification for comparisons of means.
After the second hatch of chicks, five birds per treatment were
sacrificed and tissue samples from the brain, liver and gonads were
excised for residue analysis. Details concerning the procedures and
results of chemical determination will be the subject of subsequent
reports.
Results and Discussion
Body weights (Table 1) showed no statistically significant differ-
ences among treatments at any of the ages of 4, 6, 8 or 10 weeks. It
appears, therefore, that the chemical agents used did not interact with
normal metabolic processes associated with body growth and normal
development of the skeletal bones and integuments.
TABLE 1. Body weights (grams) of quail of parental generation
fed treated diets from day of hatch.
Treatment
A-Control
B-Mercury
C-PCB
D-DDT
E- Comb ina t i on
Average
4 week
76*
78
78
80
78
78
6 week
103
106
103
106
104
104
8 week
110
113
110
112
109
111
10 week
109
111
112
113
111
111
* All figures are mean of 3 pens—all individuals weighed.
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Throughout the growing period, mortality was very low with no appre-
ciable death beyond the normal expectations during the first few days.
As shown in Table 2, egg production on a pen basis did not differ
significantly nor did shell thickness show statistically significant
differences among the treatments. Apparently, these low levels of
chemical agents did not influence the complex physiologic processes of
shell deposition and oviposition under the conditions of this experiment.
TABLE 2. Egg production (32 day period) and average shell
thickness of birds on experimental diets from day of hatch.
Treatment
A-Control
B-Mercury
C-PCB
D-DDT
E-Combination
Average
Number of eggs
21
19
21
18
21
20
Thickness
0.18
0.18
0.17
0.16
0.17
0.17
(mm)
In Table 3 are shown data concerning fertility and hatchability for
two hatches of eggs. Fertility, through natural mating, appeared not to
be influenced by the dietary treatments and was considered to be within
normal ranges. However, hatchability for both hatches showed a signifi-
cant effect due to rations. In hatch 1, eggs from females on treatment
A (controls) hatched at a greater rate than eggs from females on treat-
ment E (combination). In the second hatch, pairwise analysis indicated
the same differences (A vs. E) and also treatment C (PCB) and D (DDT) gave
hatchability which was significantly lower that hatchability of eggs from
females on the control diet.
TABLE 3. Average fertility and hatchability of all eggs set
in two hatches of quail eggs—parental generation.
Eggs set Fertility (%) Hatched (%)
Treatment 1212 12
A-Control
B-Mercury
C-PCB
D-DDT
E-Combination
Average
63
104
90
90
91
88
89
100
85
107
112
99
81
82
89
82
89
85
87
90
89
85
93
89
75
58
66
60
55
62
75
61
55
59
53
60
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In further analysis of the complex trait of hatchability, it can
be seen in Table 4 that the control ration showed considerably fewer
early dead embryos. Pairwise, statistical analysis of these data for
hatch 1 showed that birds on the control diet (A) produced significantly
fewer early dead embryos than birds on either treatments D or E (DDT
and combination). In hatch 2, treatment A gave significantly different
results from both treatments B (mercury) and E.
TABLE 4. Early (1-7 days) and late (12-18 days) embryonic
mortality of two hatches of quail eggs from
females on experimental diets.
Early (%) Late (%) Pip (%)
Treatment 12 12 12
A-Control
B-Mercury
C-PCB
D-DDT
E-Combination
Average
3.2
15.4
10.0
17.8
20.9
14.2
6.7
17.0
11.8
12.1
22.3
14.4
1.6
5.8
8.9
3.3
6.6
5.5
2.2
9.0
14.1
2.8
11.6
7.9
0.0
2.9
2.2
1.1
2.2
1.8
2.2
1.0
2.4
7.5
0.9
2.8
In hatch 1, the percent dead embryos in the late embryonic period
showed no significant differences among treatments but the combined data
from both early and late periods (total embryonic mortality) did show
significant differences between the control diet and the diet containing
the combination of chemical agents (E).
Considering hatch 2, the percent dead embryos in the late period was
also analyzed by statistical pairwise comparisons. The analysis showed
ration A (control) produced significantly lower late mortality than both
rations C (PCB) and E (combination). Likewise, ration D (DDT) showed a
significantly lower percent late dead embryos than both rations C and E.
When all embryonic mortality in hatch 2 is considered (both early
and late), pairwise comparisons of means indicated ration A gave results
significantly lower than did rations B, C and E. Also, ration D produced
fewer dead embryos than did ration E.
Considering both hatches and both periods of embryonic mortality, the
consistently significant comparisons were between data from females on the
control ration and the combination ration. This was particularly evident
and consistent during the early stages of embryo development.
It appears from all measurement data that hatchability, particularly
as influenced by early embryonic mortality, was sensitive to modification
by the feeding of the combinations of mercury, PCB and DDT. This implies
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that developing embryos within eggs laid by females on diets containing
these residues have less "resilience" than hatched chicks on feed or
than growing birds. The embryo within the shell has little or no way
of altering metabolic pathways of eliminating antimetabolites produced
during embryogenesis. In contrast, the "free-living" chick or growing
bird can eliminate degraded chemical agents not bound in tissues and
make physiologic adjustments when fed rations containing chemical
residues at these sublethal levels.
Regarding the subjective observations on feathering, excitability
and cannabilism, the only deviation from normal was observed in chicks
hatched from females fed a diet containing 1 ppm of mercury. These
chicks showed considerably more excitability than other lots of chicks.
Also, three birds in this same lot of chicks (on mercury) showed a
non-specific type of spraddle-legs not usually seen in growing quail.
Further observations are necessary to confirm this possible interaction.
Conclusion
From this experiment using the Japanese quail, it appears that diets
containing as little as 1 ppm of mercury, DDT or PCB do not affect the
characteristics measured. However, when all three chemical agents are
combined in the same ration, hatchability of eggs produced by females
fed this "combination" diet was significantly reduced. This reduction
in hatchability reflected an increase in embryonic mortality during
the earlier phases of incubation. Other treatments also tended to have
increased early embryonic mortality but not at statistically significant
levels.
References
1. DeWitt, J. B. Effects of chlorinated hydrocarbon insecticides upon
quail and pheasants. J. Agr. Food Chem. 3:672-676, 1955.
2. Fowler, J. F., L. D. Newsom, J. B. Graves, F. L. Bonner and P. E.
Schilling. Effect of dieldrin on egg hatchability, chick survival
and eggshell thickness in purple and common gallinules. B. Env.
Con. Tox. 6:495-501, 1971.
3. Porter, R. D. and S. N. Wiemeyer. Dieldrin and DDT: Effects on
sparrow hawk eggshells and reproduction. Science 165:199-200, 1969.
4. Muller, H. D. and D. C. Lochman. Fecundity and progeny growth
following subacute insecticide ingestion by the mallard. Poultry
Sci. 51:239-241, 1972.
5. Seidenstricker, J. D. IV and H. V. Reynolds III. Nesting, reproduc-
tive performance and chlorinated hydrocarbon residues in red-tailed
hawk and great horned owl in South Central Montana. Wilson B. 83:
408-418, 1971.
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TRACE ELEMENTS IN UNPROCESSED PLANT FOODSTUFFS
Hansford T. Shacklette, James A. Erdman and John R. Keith
Introduction
The U. S. Geological Survey became interested in the concentrations
of trace elements in plants in the early 1950's as a method of exploration
for mineral deposits. New methods of trace element analysis and concepts
of data interpretation were developed in this country, as well as in Canada
and Europe, that were successfully used in discovering buried ore bodies (1) •
The principle was established that high concentrations of certain elements
in plant tissues indicated, in many instances, the presence of underlying
mineral concentrations. The extension of this principle led to the belief
that differences in the element content of plants invariably reflected differ-
ences in the elemental composition of the associated soils; therefore,
regional patterns of soil element concentrations could be related to regional
patterns of the prevalence of certain diseases of animals and humans who
consumed the local plants. Impressive confirmation of this concept as ap-
plied to grazing animals was provided by early studies of selenium toxicity
in parts of Western United States (2), of copper deficiency in areas of the
Atlantic Coastal Plain (3), of molybdenosis in areas of California (4),
and of other element problems in various countries.
The relationship of trace elements in soils and plants to human
health and disease has been less easy to demonstrate, and most studies
have rested heavily on correlation rather than causation (5). Allaway
(6) stated, "Unquestionably, the fact that domestic animals frequently
derive all of their diet from a specific environment, in contrast to the
multisource diets common to people, has been a major factor in the success
of soil-plant-livestock studies." In 1964, a U. S. Geological Survey
project undertook a geochemical investigation of two areas in Georgia,
each consisting of nine counties, that were determined by the U. S. Public
Health Service to have greatly contrasting rates of human mortality from
cardiovascular diseases (7). Native plants and uncultivated soils and
garden vegetables and garden soils were collected from the two areas and
the chemical element compositions compared. Although soils from the two
areas invariably were found to differ greatly in element content, this
difference was but very weakly, or not at all, reflected in the chemical
composition of the native plants or the vegetables. The explanation of
this apparent contradiction of the principles of biogeochemistry lies
in the relative magnitude of the differences' in trace element composition
of soil overlying "barren" versus "mineralized" deposits, and the differ-
ences between soils in the two areas of Georgia. That is, the concentra-
tions of trace elements in the Georgia soils, even in the area where most
pronounced, were below the response threshold as expressed by anomalous
element concentrations in the plants.
The recently increased concern with environmental quality has caused
emphasis to be placed on the geochemical component of the environment, with
particular attention being given to problems of chemical contamination and
129
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pollution. Knowledge of normal or background geochemistry of the natural
environment is a prerequisite to an evaluation of the magnitude of any
geochemical alterations produced by man's activities. Baseline geochemical
data covering a wide range of chemical elements over large areas and
considering many kinds of natural materials have been largely lacking
because most studies have dealt with abnormal environments such as those
of mineralized areas, or areas where industrial pollution has been ex-
tensive. Some recent studies conducted by the U. S. Geological Survey
have contributed to the establishment of "normal" or "baseline" geochemical
values for rocks, soils, waters, and plants; and these background guide-
lines already have been applied to investigation of pollution problems
(8, 9).
A program of sampling the B horizon of soils and associated native
plants at sites about 50 miles apart on routes of travel throughout the
conterminous United States has been completed, and maps showing the
concentrations of 38 elements in soils at more than 900 sites have been
published (10, 11, 12). These data provide a relatively unbiased estimate
of the natural compositional variability of subsurface soils of this
country. Plant analysis data have not yet been published.
In an effort to learn how to efficiently characterize the total
geochemistry of a large area, a U. S. Geological Survey project under-
took a four-year investigation of the element concentrations in rocks,
soils, waters, native vegetation, and some crop plants in Missouri (13).
This study was conducted with the cooperation of the Environmental Health
Surveillance Center of the University of Missouri. This center simul-
taneously carried out studies of animal and human epidemiology in a search
for patterns of health problems that might possibly relate to patterns
of geochemical distinctiveness. The geochemical study has been completed,
and the results have been released in seven semiannual open-file reports
(14). Some results of the vegetation studies will be presented later in
this report.
In order to further explore some of the factors that may relate to
differences in soil-plant chemical relationships that were found in earlier
studies, we initiated in 1972 a program of sampling a wide variety of
food plants and associated soils from areas of commercial production
throughout the United States. In addition to meeting this objective,
the study was designed to provide some baseline data on the concentrations
of about 40 trace and major elements in the food plants. Current interest
in these baseline data centers around the evaluation of pollution effects
on vegetables and fruits and on allowable concentrations of certain .trace
elements in processed food of plant origin. The Food and Drug Administra-
tion at present is conducting a total diet study (the Market Basket
Sampling Program) of the element concentrations in foods as sold at
retail outlets throughout the country (15). Research workers in this
organization acknowledge that data on the normal range of concentration
of elements in the fresh or unprocessed food material is fragmentary or
lacking, and that this deficiency is a serious handicap in evaluating the
extent of change in element content of foods that is introduced through
130
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processing. We have contributed data to this organization, and further
exchange of information is anticipated.
Our on-going study of these food plants and soils encompasses 11
centers of commercial fruit and vegetable production selected to represent
a wide variety of food plants, soils, and climatic factors. We sample
only the food plants that may be eaten as grown with little or no prepara-
tion or processing. Autumn harvested fruits and vegetables were sampled
in Michigan, New York, and New Jersey, and winter harvested produce,
including citrus fruits, were sampled in the southern parts of Florida,
Texas, Arizona, and California. We observed no obvious pollution in any
of the areas sampled so far; but if pollution was present, growth of the
plants was not noticeably affected by it. During the current year we
plain to sample autumn produce in Idaho, Washington, central California
and Utah.
We sample each kind of food plant and associated soils within an
area according to a nested sampling design, with sufficient replication
at each level of the design to obtain a reliable measure of sampling
and analytical error. The vegetables and fruits are prepared soon after
sampling as if for table use, in a manner (as Dr. Warren and his associates
so nicely put it) "followed by a prudent housewife" (15). The prepared
samples are weighed in the field in order to get an accurate measure of
their normal fresh weight. The weighed samples are frozen and held in
this condition until dried to constant weight in a low temperature oven
(45-50 degrees C). The water content of the fresh produce can then be
calculated. After all samples have been dried they are pulverized and
submitted to the analytical laboratory where appropriate aliquots are
burned to ash, and the percentage ash yield calculated. The samples are
then analyzed in a random sequence with respect to both kind of sample
and geographical origin; the randomized order is unknown to the analyst.
The concentrations of each measured element can then be expressed on a
fresh weight, dry weight, and ash weight basis, to suit the various uses
which this kind of data may serve. Soil samples are air dried in paper
envelopes, pulverized in a ceramic mortar to approximate -200-mesh size,
and submitted for analysis in a random sequence.
Although the results of this comprehensive food plant study are not
yet available, we feel able to identify some general characteristics of
element absorption by food plants, and some chemical relationships of
food plants to soils. These conclusions are based on earlier studies
that we have conducted, and also on reports in the literature. The
selected examples of data are offered with the realization that some
conclusions may be modified or elaborated from our on-going food plant
study. For brevity in presentation, only a limited number of analyses
were chosen from larger data sets as being representative of tendencies
that are believed to exist in chemical characterization.
Geometric means are used in this report to express estimates of
the "typical" or "characteristic" concentrations in the various sample
media included in the tables. A measure of variation is given by the
geometric deviation, which is the antilog of the standard deviation of
the logs. The use of these measures of central tendency and variation
131
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was discussed by Shacklette, Sauer, and Miesch (7). Ratio, as used
in the tables of this report, is the number of samples in which the
element was found in a measurable quantity to the total number of
samples analyzed. The vegetation types of Missouri that are referred
to are those described by Kuchler (17), with modifications (14).
Trace Element Characteristics of Food Plants
Differences among plant species
By examining large sets of data on trace element analyses of plants,
the greatest differences in mean concentrations generally are found to
be among different species rather than among samples of the same species.
That is, several species growing together in a particular chemical environ-
ment may absorb greatly different amount of trace elements. Some species
regularly contain concentrations near the extreme end of the high concen-
tration range for a particular element in plants; these species are
designated accumulator plants in Table 1. This table indicates the cobalt
Table 1. Cobalt and molybdenum concentrations (ppm) in ash of some
accumulator and nonaccumulator species.
Detection Geometric Geometric Observed range
Kind and source of sample ratio mean deviation min max
Cobalt
Blackgum leaves; Georgia
Persimmon leaves; Georgia
Cabbage leaves; Georgia
27:30
3:30
1:30
400
<7
<7
7.67 <7
<7
<7
10,000
2,000
10
Molybdenum
Corn grains; Floodplain Forest,
Missouri
Soybene seeds; Floodplain
Forest, Missouri
Blackgum leaves; Georgia
8:8
7:10
1:30
18
10
<5
1.66
3.77
— — '
7
<5
<5
30
70'
15
accumulating tendency of blackgum trees. These trees exhibit such strong
affinity for cobalt that analyses of their tissues have been used as an
indication of cobalt deficient areas; less than 5 ppm in dry weight of
the leaves indicates that the area where they grew is dificient in this
element for the nutritional requirements of cattle and sheep (18).
Another species, persimmon, that grew at the same sites as the blackgum
shown in Table 1 absorbed much less cobalt, as is generally true of other
tree species. Cabbage has the low concentration that is characteristic
of vegetables.
132
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If we turn to a second trace element, molybdenum, we find that
blackgum is not an accumulator, but that corn grains and soybean seeds
accumulate relatively high concentrations. Other food plants and native
plants commonly contain low concentrations of molybdenum.
Species of hickory are known to concentrate the rare earth elements
in their tissues, even if growing in soils where these elements are
below the common limit of analytical detection. Yttrium in ash of
hickory stems ranges from <20-150 ppm, but when occasionally reported
in snap beans, blackeyed peas, cabbage, and tomatoes it ranges from
20-100 ppm in ash. Yttrium is not commonly detected in food plants;
we have found it only in cabbage leaves and corn grains, but some native
woody plants (particularly buckbush) regularly contain about 1.5 ppm
irv stem ash. In all our food plant samples, we have detected cerium
and neodymium only in a sample of tomatoes.
Genetic control of trace element accumulation is known to extend
below the species level to the level of natural or cultivated plant
varieties. Hemphill (19) stated that grafting vanencia orange scions
on different varieties of roots affected the major and trace element
concentrations in the orange leaf. Many different genetic variants may
occur in a population of plants generally considered to be of one species.
The genetic influence of these variants may be partly responsible for
the wide range of element concentrations reported for samples of a species;
only carefully controlled laboratory experiments could determine the presence
and extent of this effect. It is this phenomenon that offers the pos-
sibility of selecting variants or "strains" of food plants that accumulate
more of the desirable trace elements, and less of the noxious ones.
As a general rule, our data show fewer species differences in con-
centrations of the major essential plant nutrients magnesium, phosphorus,
and potassium than in concentrations of the trace elements. This observa-
tion suggests that a homeostatic principle may operate in the absorption
of the major elements. The magnesium concentrations in a variety of
plants are given in Table 2.
Table 2. Magnesium concentrations (percent) in ash of selected plant species.
Kind and source of sample
Detection
ratio
Geometric
mean
Geometric
deviation
Observed range
min max
Cultivated
Asparagus stems; Wisconsin
Bean, lima, seeds; Georgia
Bean, snap, pods; Georgia
Beet roots; Wisconsin
Cabbage leaves; Georgia
Corn grains; Missouri
Cucumber fruits, Wisconsin
Onion bulbs; Wisconsin
Potato tubers; Wisconsin
Native species
Black cherry stems; Georgia
Black cherry leaves; Georgia
Buckbush stems; Missouri
5:5
30:30
30:30
3:3
30:30
10:10
4:4
7:7
10:10
5.7
3.4
3.6
2.6
2.2
6.3
2.7
2.7
2.2
30:30
24:30
47:47
4.0
6.7
3.5
1.41 3 7
1.53 1.5 7
1.42 1.5 7
1.26 2 3
1.68 .7 7
1.18 5 7
1.22 2 3
1.40 2 5
1.56 1 5
1.49 1.5 7
1.26 5 >10
1.56 1.5 7
(continued)
133
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Table 2. (continued)
Kind and source of sample
Cedar stems and leaves;
Missouri
Hickory stems; Missouri
Oak stems; Missouri
Pine stems and leaves;
Missouri
Sweetgum stems; Georgia
Detection
ratio
50:50
19:19
50:50
49:49
28:28
Geometric
mean
3.0
2.5
1.8
3.5
2.6
Geometric
deviation
1.81
2.11
1.86
1.40
1.46
Observed
rain
.7
.5
.5
1.5
1.5
range
max
7
10
7
5
7
Strontium, a trace element not thought to be nutritionally essential,
exhibits great variation in its concentration in different plants, as
shown in Table 3. No definite explanation is available for the wide
Table 3. Strontium concentrations (ppm) in ash of selected plant species.
Detection Geometric Geometric Observed range
Kind and source of sample
Cultivated
Asparagus stems; Wisconsin
Bean, lima, seeds; Georgia
Bean, snap, pods; Georgia
Beet root; Wisconsin
Cabbage leaves; Georgia
Corn grains ; Missouri
Cucumber fruits; Wisconsin
Onion bulbs; Wisconsin
Potato tubers; Wisconsin
Native species
Black cherry stems; Georgia
Black cherry leaves; Georgia
Buckbush stems; Missouri
Cedar stems and leaves;
Missouri
Hickory stems; Missouri
Pine stems and leaves;
Missouri
Sweetgum stems; Georgia
ratio
5:5
30:30
30:30
3:3
30:30
10:10
4:4
7:7
10:10
30:30
30:30
47:47
50:50
19:19
49:49
27:27
mean
586
88
210
421
880
15
341
366
75
1,900
1,200
1,800
340
3,200
570
1,500
deviation
2.38
1.63
1.80
1.34
1.89
1.55
1.29
1.72
2.53
1.68
1.74
1.43
1.63
2.03
1.85
2.03
min
300
30
50
300
200
7
300
200
30
700
500
700
200
500
200
300
max
2,000
300
700
500
3,000
30
500
700
700
7,000
5,000
5,000
1,000
7,000
2,000
7,000
variation in mean strontium concentrations among the different kinds of
plants shown in this table. Contamination with soil cannot account for
the high values, because the typical strontium content of soil (geometric
mean, 120 ppm for United States soils) is lower than all strontium values
in this table except those for lima beans, corn, and potatoes. Steward
(20) suggested that a single gene determines the requirement for a specific
element in a plant species, while another gene determines the requirement
for another element. Perhaps the data in Table 3 express this degree of
genetic control of the element concentrations in the different plants.
134
-------
But this control alone, if operative, is not very precise, as indicated
by the wide range in concentrations for a species. This range likely
is not due entirely to the range in soil strontium concentrations, for
the range in the plants is greater than the range in soils at the dif-
ferent sites.
Differences among plant organs
The differences in concentrations of some trace elements in the
several organs of food plants may be striking, but a general rule cover-
ing all trace elements cannot be made. Zinc, for example, seems to be
concentrated in seeds and grains, rather than in fruits or vegetative
parts (Table 4).
Table 4. Zinc concentrations (ppm) in ash of different organs of selected
food plants.
Detection Geometric Geometric Observed range
Kind and source of sample
ratio
mean
deviation min
max
Corn grains; Missouri
Soybean seeds; Missouri
Blackeyed pea seeds; Georgia
Bean, lima, seeds; Georgia
Beet root; Wisconsin
Cabbage leaves; Georgia
Asparagus stems; Wisconsin
Carrot root; Wisconsin
10:10
10:10
29:29
30:30
3:3
28:28
5:5
8:8
1,800
870
750
600
391
340
283
177
1.29
1.09
1.27
1.18
1.29
2.57
1.46
1.46
1,200
840
400
400
300
100
200
100
2,800
960
1,200
1,000
500
5,000
400
300
Titanium, in contrast to zinc, tends to be more concentrated in
vegetative parts (Table 5). There is, however, considerable variation
Table 5. Titanium concentrations (ppm) in ash of different organs of
selected food plants.
Detection Geometric Geometric Observed range
Kind and source of sample
Asparagus stems; Wisconsin
Cabbage leaves; Georgia
Beans, snap, pods; Georgia
Blackeyed pea seeds; Georgia
Carrot root; Wisconsin
Beet root; Wisconsin
Bean, lima, seeds; Georgia
Soybean seeds; Missouri
Corn grains; Missouri
ratio
5:5
28:28
30:30
29:29
8:8
3:3
26:30
6:10
2:10
mean
182
85
73
37
28
27
8.3
5
<5
deviation
1.16
2.37
2.69
2.52
2.67
2.65
3.93
—
— —
min
150
15
3
5
10
10
<2
<5
<5
max
200
700
500
200
200
70
200
200
10
in concentrations in a suite of samples. Some, but certainly not all,
titanium that is found in plant samples may be only surface contamination.
135
-------
The titanium found in samples of seeds is not largely from dust contami-
nation if the seeds have been removed from the pods or other structures
with reasonable care. Titanium as well as other elements occurring as
surface contamination in food plants will be ingested, however, if not
removed by the "prudent housewife" or the commercial processor, the same
as if it were in the tissues.
Differences between herbaceous and woody species
The data on food plants in this report consist only of those for
herbaceous species; we do not yet have analyses of our samples of fruit
produced on woody plants (trees and vines). We do have extensive analyses
of native trees and shrubs, and these data will be used in the comparisons
that follow. Probably the most appropriate comparisons that our present
data afford are those of cabbage leaves to the leaves of native trees, and
stems of asparagus to the stems of these trees. The differences, however,
that are shown in large data sets between the concentration of many elements
in food plant and woody plant tissue, if each set of data is considered as
a group, are striking for certain elements. An example is provided in
Table 6 by the concentrations of barium in species belonging to the two
groups.
Table 6. Barium concentrations (ppm) in ash of herbaceous and woody plants.
Detection Geometric Geometric Observed raftge
Kind and source of sample
Herbaceous plants
Asparagus stems; Wisconsin
Cabbage leaves; Georgia
Cabbage leaves; Wisconsin
Corn grains; Missouri
Soybean seeds; Missouri
Woody plants
Black cherry stems; Georgia
Black cherry leaves; Georgia
Blackgum stems; Georgia
Blackgum leaves; Georgia
Buckbush stems; Missouri
ratio
5:5
28:28
30:30
11:11
10:10
8:8
30:30
30:30
30:30
30:30
48:48
mean
288
320
450
155
16
290
3,600
3,400
7,200
9,800
2,500
deviation
1.86
2.95
1.67
1.69
1.92
2.18
2.93
2.50
2.18
1.67 1
1.48 1
min
100
50
30
50
5
70
300
50
200
,500
,000
max
700
300
300
300
50
700
10,000
7,000
10,000
20,000
7,000
The same degree of difference shown for barium in the two groups
generally applies also for aluminum, cadmium, calcium, chromium, lead,
manganese, strontium, and titanium, with the woody plants always having
the larger amount. A possible explanation for this difference in element
concentration in herbaceous and woody plants may be the difference in
longevity and the depth of root penetration of plants in the two groups.
The herbaceous plants discussed in this report are all (except asparagus)
short lived. They grow and are harvested usually within a period of five
months or less, and their roots are largely restricted to the plow zone
of soil. The woody plants, in contrast, may live for many years, and
their roots commonly penetrate the C horizon of soil or grow even into
the more consolidated soil parent material. They have, therefore, a
136
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better opportunity to accumulate elements from the geochemical environment
in which they grow.
Accumulation of hazardous elements in food plants.
For this discussion, only cadmium, lead, and selenium will be
considered in this category of elements. Mercury was not detected in
concentrations as great as 0.025 ppm in any food plant sample. Of these
plants, only corn and soybeans were analyzed for cadmium. Nine species
of woody plant stems were analyzed for this element, and their concentra-
tions of cadmium tended to be somewhat greater than those reported for
the food plants, as indicated by the analyses of buckbush stems in Table
7. We have included the apparently anomalous geometric mean cadmium
Table 7. Cadmium concentrations (ppm) in ash of plant organs.
Kind and source of sample
Corn grains; Missouri
Floodplain Forest
Glaciated Prairie
Soybean seeds; Missouri
Floodplain Forest
Glaciated Prairie
Buckbush stems; Missouri
Glaciated Prairie
Unglaciated Prairie
Detection
ratio
6:8
9:10
10:10
10:10
47:47
48:48
Geometric
mean
.37
.62
2.3
.60
13
14
Geometric
deviation
1.72
2.34
2.33
2.04
.1.91
2.08
Observed
min
<.02
<.02
.7
.3
2.8
3.2
range
max
4.4
8.2
7.0
1.6
50
60
concentration in soybeans in this table because we suspect it may represent
contamination, possibly from cadmium associated with phosphate fertilizers.
Detectable concentrations of cadmium were not found, however, in the soils
associated with these soybean samples. The cadmium concentrations in corn
grains listed in Table 7 are not substantially different from a value of
0.26 ppm reported in wheat grains (21). The greatest cadmium concentra-
tions that we have found in plants, 60 ppm in ash, were found in the
sample of buckbush stems listed in Table 7 and in a sample of sumac from
the Glaciated Prairie vegetation-type area in Missouri.
We have found the occurrence of lead in measurable concentrations
in the ash of food plants to be very erratic (see Table 8). We have
also found that the range of concentrations in a suite of plant samples
Table 8. Lead concentrations (ppm) in ash or herbaceous and woody plants.
Detection
Kind and source of sample ratio
Asparagus stems; Wisconsin
Tomato fruits; Georgia
Bean, snap, pods; Georgia
Cabbage leaves; Georgia
Blackeyed pea seeds; Georgia
Carrot roots; Wisconsin
Black cherry stems; Georgia
Black cherry leaves; Georgia
5:5
8:30
2:30
1:30
4:29
3:8
30:30
30:30
Geometric Geometric
mean deviation
87
5.6
<10
<10
<10
<10
210
33
2.79
6.46
—
—
—
—
2.71
4.65
Observed range
min
25
<10
<10
<10
<10
<10
30
<10
max
300
300
30
20
70
25
L,000
700
137
-------
often tends to be extreme. Both facts suggest that lead pollution has
contributed to the concentrations found in the plants. However, analyses
of stems and leaves of woody plants almost invariably show lead concentra-
tions in stems to be two or more times the concentrations reported in
leaves. If the source of pollution is assumed to be airborne lead, leaves
would be expected to contain greater concentrations than would stems be-
cause of their greater surface area for deposition of the pollutant. The
question of the source of lead in our plant samples remains unanswered.
Facilities for selenium analysis were not available during our
studies of Wisconsin and Georgia vegetables, therefore, we have selenium
values only for samples of the Missouri study. Table 9 gives these values
Table 9. Selenium concentrations (ppm) in dry plant tissues.
Detection Geometric Geometric Observed range
Kind and source of sample
Corn grains: Missouri
Floodplain Forest
Glaciated Prairie
Unglaciated Prairie
Oak-hickory Forest
Soybean seeds; Missouri
Floodplain Forest
Glaciated Prairie
Unglaciated Prairie
Oak-hickory Forest
Buckbush stems; Missouri
Cedar stems and leaves;
Missouri
Oak stems; Missouri
ratio
8:8
10:10
10:10
10:10
10:10
10:10
8:8
9:9
46:46
50:50
48:50
mean
0.062
.072
.047
.040
.17
.098
.097
.077
.038
.021
.018
deviation
2.41
2.61
1.88
2.96
2.68
1.83
2.28
1.94
1.49
1.36
1.43
min
.01
.02
.02
.02
.06
.04
.04
.04
.02
.01
<.01
max
.20
.40
.15
.50
1.25
.25
.35
.40
.08
.04
.04
for corn, soybeans, and a few representative species of woody plants.
Our limited data suggest that selenium is more concentrated in seeds than
in vegetative parts of trees and shrubs. Excessive amounts of selenium
in forage plants are toxic to grazing animals, but these levels usually
are reached only in selenium-accumulating native herbs, or in other
plants that have derived their selenium from the decomposition products
of the selenium accumulators. Selenium in trace amounts is essential
for grazing livestock. Underwood (5) stated that pasture herbage contain-
ing 0.03-0.04 ppm selenium (in dry material) meets the requirement for
sheep. Our values for selenium in plant materials are well below the level
of 4-5 ppm in dry material indicated by Underwood as potentially hazardous
to livestock. Our plant samples are from areas where selenosis has not
been recognized as a problem.
Soil and plant chemical relationships
The numerous factors that determine the amounts of an element that
a plant will remove from its surrounding soil interact in an extremely
138
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complex manner. These interactions have been extensively studied for many
years, but even an outline of the major interactive principles is outside
the scope of this report. We will give only some results of our studies,
leaving the question of causal realtionships largely unanswered. In our
studies, only total element concentrations in the soils were measured.
The determination of the chemical compounds in which the elements occur,
and attempts to measure availability of the different elements in the soil
are, for the most part, impractical in rapid geochemical surveys of
large areas. The true measure of the availability of a soil element
to a plant can be determined by chemical analysis of the plant, but the
causal factors can seldom be determined in their entirety.
The total amount of an element in a plant sample does not neces-
sarily come only from the soil in which the plant is rooted. The effects
of the addition of elements to the plant sample by aerial transport, whether
as dust particles, soluble materials in rain, or atmospheric gases, are
difficult to estimate, but it is generally conceded that the greatest
amounts of the elements in ordinary land plants come from the soil by
absorption through the roots, unless gross aerial contamination has
occurred.
It was mentioned earlier in this report that in our Georgia soil-
plant study the pronounced differences in the trace element content of
soils from'the two distinct areas were not strongly, if at all, reflected
in the concentration of these elements in the plants that were sampled.
In our Missouri soil-plant investigations, summaries of chemical character-
istics were prepared according the vegetation-type areas. Significant
differences in concentrations, expressed as geometric means, of many
elements in the soils were found among the six areas. These soil dif-
ferences, however, failed to appear in the elemental composition of sumac,
a shrub that occurs throughout the State.
Correlation coefficients were calculated for some of the elements
found in samples of sumac and associated soils. They are presented in
graphical form in Figure 6. These graphs show the general lack of
correlation in sumac analyses and soil analyses. There are no strongly
positive correlations shown in any vegetation-type area; most points
are near the zero correlation level, and some show negative correlations.
Similar lack of correspondence in elemental composition of plants and
soils from the same areas was generally found in all of our soil-plant
relationship studies of native species and uncultivated soils.
Results obtained from our study of the elemental composition of
corn grains, soybean seeds, and pasture grasses and their associated
soils were similar to those of the study of native plants and unculti-
vated soils. We found that, in Missouri, the regional compositional
differences in these soils generally parallelled those of uncultivated
soils, and that, as with sumac, these differences were not clearly re-
flected in the chemical composition of the cultivated crops. Hemphill's
statement, "In general, the elemental composition of the plant will
reflect the composition of the soil or other growing media" (19) must be
taken in its broadest sense with respect to the data just described.
139
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Floodplain
Forest
Cedar Glade
MANGANESE
NICKEL
PHOSPHORUS
POTASSIUM
SELENIUM
SODIUM
STRONTIUM
TITANIUM
ZINC
140
-------
Floodplain
Foreat
Cedar
Glade
ALUMINUM
BARIUM
BORON
CALCIUM
CHROMIUM
COPPER
IRON
LEAD
MAGNESIUM
141
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The Missouri study gave further support to results obtained from analyses
of vegetables and garden soils in Georgia, which may be summarized by
stating that these plants, if growing in soils with typically low concen-
trations of trace elements, do not faithfully reflect differences in soil
concentrations of these elements. The differences in element concentra-
tions in the plant samples must then be attributed to the effects of other
site factors or to differences in the genetic complement of the plants
from the several areas. In the final analysis, however, the causal
factors of these geochemical differences have as yet no satisfactory
explanation.
References
1. Lovering, T. S. Work of the Geochemical Exploration Section of the
U.S. Geological Survey. Mining Engineering p. 1-4, October 1955.
2. Robinson, W. 0. Determination of selenium in wheat and soils.
Jour. Ass. Off. Agr. Chemists 16:423-424, 1933.
3. Neal, W. M., R. B. Becker, A. L. Shealy. A natural copper deficiency
in cattle rations. Science 74(1921):418-419, 1931.
4. Barshad, Isaac- Molybdenum content of pasture plants in relation
to toxicity to cattle. Soil Sci. 66(3):187-195, 1948.
5. Underwood, E. J. Trace elements in human and animal nutrition, 3rded.
New York and London, Acad. Press, 543 p., 1971.
6. Allaway, W. H. An overview of distribution patterns of trace elements
in soils and plants. New York Acad. Sci. Annals 199:17-24, 1972.
7. Shacklette, H. T., H. I. Sauer and A. T. Miesch. Geochemical environ-
ments and cardiovascular mortality rates in Georgia. U. S. Geol.
Survey Prof. Paper 574-C, 39 p., 1970.
8. Connor, J. J., H. T. Shacklette and J. A. Erdman. Extraordinary
trace-element accumulations in roadside cedars near Centerville,
Missouri. In Geological Survey Research 1971: U.S. Geol. Survey Prof.
Paper 750-B, B151-B156, 1971.
9. Ebens, R. J., J. A. Erdman, G. L. Feder, A. A. Case and L. A. Selby.
Geochemical anomalies of a claypit area, Callaway County, Missouri,
and related metabolic imbalance in beef cattle. U. S. Geol. Survey
Prof. Paper 807, 24 p., 1973.
10. Shacklette, H. T., J. C. Hamilton, J. G. Boerngen and J. M. Bowles.
Elemental composition of surficial materials in the conterminous
United States. U.S. Geol. Survey Prof. Paper 574-D, 71 p., 1971.
11. Shacklette, H. T., J. G. Boerngen and R. L. Turner. Mercury in the
environment—Surficial materials of the conterminous United States.
U.S. Geol. Survey Cir. 644, 5 p., 1971.
142
-------
12. Shacklette, H. T., J. G. Boerngen, J. P. Cahill and R. L. Rahill.
Lithium in surficial materials of the conterminous United States
and partial data on cadmium. U.S. Geol. Survey Cir. 673, 8 p.,
1973.
13. Connor, J. J., J. A. Erdman, R. R. Tidball and G. L. Feder. Environ-
mental geochemistry in Missouri—A multidisciplinary approach. Ln
Earth science and the quality of life, Symposium 1. Internat. Geol.
Cong., Montreal, Canada, p. 7-14, 1972.
14. U.S. Geological Survey, 1972a, Geochemical survey of Missouri, plans
and progress for first six-month period (July--December, 196.9). U.S.
Geol. Survey open-file report, 49 p.
, 1972b, Geochemical survey of Missouri, plans
and progress for second six-month period (January-June, 1970). U.S.
Geol. Survey open-file report, 60 p.
, 1972c, Geochemical survey of Missouri, plans
and progress for third six-month period (July-December, 1970). U.S.
Geol. Survey open-file report, 33 p.
, 1972d, Geochemical survey of Missouri, plans
and progress for fourth six-month period (January-June, 1971). U.S.
Geol. Survey open-file report, 63 p.
, 1972e, Geochemical survey of Missouri, plans
and progress for fifth six-month period (July-December, 1971). U.S.
GeoL. Survey open-file report, 145 p.
, 1972f, Geochemical survey of Missouri, plans
and progress for sixth six-month period (January-June, 1972). U.S.
Geol. Survey open-file report, 86 p.
_, 1973, Geochemical survey of Missouri, plans
and progress for seventh six-month period (July-December 1972). U.S.
Geol. Survey open-file report, 59 p.
15. U.S. Food and Drug Administration. General programs—Foods, Chap.20
of Compliance program guidance manual. U.S. Bur. Foods, Div. Com-
pliance Programs, Program Devel. Br., BF-323, p. 1-9, 1972.
16. Warren, H. V., R. E. Delavault and K. W. Fletcher. Metal pollution—
A growing problem in industrial and urban areas. Canadian Mining
and Metall. Bull., p. 1-12, July 1971.
17. Kiichler, A. W. Potential natural vegetation of the conterminous
United States. Am. Geog. Soc. Spec. Pub. 36, 116 p. + map, 1964.
18, Kubota, J., V. A. Lazar and K. C. Beeson. The study of cobalt status
in soils in Arkansas and Louisiana using the black gum as the indicator
plant. Soil Sci. Soc. America Proc., 24:527-528, 1960.
143
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19. Hemphill, D. H. Availability of trace elements to plants with
respect to soil-plant interaction. New York Acad. Sci. Annals.
199:46-60, 1972.
20. Steward, F. C. Plant physiology, V. 3, Inorganic nutrition of plants.
New York, Acad. Press., 1963.
21. Schroeder, H. A., A. P. Nason, I. H. Tipton and J. J. Balassa.
Essential trace metals in man—zinc, relation to environmental cadmium.
Jour. Chronic Diseases 20, 179-210, 1967.
144
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FDA's TOTAL DIET PROGRAM--THE TENTH YEAR
Dennis D. Manske
Food and Drug Administration
The Food and Drug Administration, Department of Health, Education
and Welfare, monitors residues of pesticides, industrial chemicals and
metals in the nation's food supply through several programs. One of
these, the Total Diet Program, is one of FDA's oldest residue monitor-
ing programs, having been in operation since June, 1964. The purpose
of this discussion is to trace the development of this program over
the last nine years. Included will be a comparison of the present
program with earlier programs and comparison of analytical findings
since the start of the Total Diet survey.
Total Diet Program - 1964
The first year's program involved three FDA district offices with
each of the three districts collecting and analyzing six samples (one
every two months).
Each sample consisted of food items purchased from retail food
stores in the three regions of the United States. The shopping list
was prepared in cooperation with the Household Economics Research Divi-
sion, USDA. It was designed to represent a two-week supply of food
for a 16-19 year old male. Although the shopping list totaled 117
items,, not all items were collected from all parts of the country
because of regional dietary patterns.
Each food item was prepared for consumption just as it might be
in the average home. For example, oranges were peeled, meat was roast-
ed, baked or fried, and potatoes were split into three groups for
baking, frying, or boiling. The foods requiring processing were pre-
pared by dieticians in institutional kitchens.
After preparation, the foods were separated into twelve classes
of similar foods (e.g. dairy products, legumes, garden fruits). A
homogeneous composite was prepared for each food class. Each of the
twelve food class composites was analyzed for organochlorine and
organophosphorus pesticides, chlorophenoxy acid herbicides, carbaryl,
arsenic, amitrole, dithiocarbamates and bromides.
Total Diet Program - 1973
In general, the 1973 program is just an expanded version of the
1964 program. No significant change has occurred in type or amount
of food, food preparation, or separation of food into twelve different
composites. In fact, one of the most significant points of the Total
Diet Program has been Its apparent resistance to change.
Most "change" has been in the form of expanded coverage in sampling
or analyses: For example, in 1965, the program coverage was expanded
from three regions of the United States to five regions.. FDA district
145
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laboratories in these five regions each collected and analyzed six
samples per year for a total of thirty samples. In 1970, the program
was modified to include samples from all parts of the continental
United States.
The year 1970 was also the time of a major modification of the program.
In October of that year, all Total Diet analytical work was consolidated
in FDA's Kansas City District laboratory. Several benefits resulted from
this consolidation. First of all, analytical expertise was increased
because of the possibility for chemists to specialize in residue work.
Secondly, consolidation led to a greater degree of efficiency and uni-
formity of Total Diet analytical work.
The year 1970 also saw the first major change in analyses required
by the program. Although analysis for cadmium had been added in 1967,
the other analyses required by the first program were still being performed.
Beginning in October, 1970, analyses for amitrole, dithiocarbamates, and
bromides were dropped from the program and analyses for mercury, poly-
chlorinated biphenyls (PCB's) and orthophenylphenol were added. The
latest additions to the program occurred in 1972, when analyses for zinc,
lead, and selenium were added.
Note that with the addition of new analyses (such as zinc and
selenium), the Total Diet Program is no longer strictly a "pesticide"
monitoring program. Many of the newer compounds may be present in food
not as a result of pesticide usage. Rather, they are present as naturally
occurring materials or as incidental contaminan ts (sometimes resulting
from environmental contamination). Even though these are not pesticide
chemicals, their occurrence in our food supply is significant enough to
warrant their inclusion in the program. The list of materials presently
examined for in the program is, then: (1) organochlorine residues; (2)
organophosphorus residues; (3) PCB; (4) carbaryl; (5) orthophenyl phenol;
(7) chlorophenoxy acid herbicides; (8) arsenic; (9) cadmium; (10) mercury;
(11) zinc; (12) lead; (13) selenium.
Analysis
Each composite is analyzed for organochlorine and organophosphorus
residues, chlorophenoxy acid herbicides, carbaryl, ortho-phenyl phenol,
and the following six metals: arsenic, cadmium, lead, zinc, selenium, mercury
(The three fatty composites—dairy products, meats and shortening—are
not examined for carbaryl and orthophenyl phenol.)
Methods of analysis for organochlorine and organophosphorus and
chlorophenoxy acid herbicides are given in the FDA Pesticide Analytical
Manual, Vol. I and II. Other methods are as follows:
a) Carbaryl and ortho-phenol, JAOAC _52_, 177-181 (1969), 736-738
(1965), and j>4, 975 (1971)
b) Arsenic, AOAC, llth edition, 25.008 and 25.016
c) Cadmium, JAOAC, March 1973, Official Changes In Methods. 25.C07
through 25.C11
d) Mercury, JAOAC 54, 202 (1971)
e) Lead, AOAC, llth edition, 25.044, and JAOAC 55_, 426 (172)
f) Zinc, AOAC, llth edition, 25.084
g) Selenium, JAOAC 51, 1039 (1968)
146
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Results and Discussion -
As discussed earlier, the Total Diet Program has been somewhat
resistant to any change in its basic approach. This consistency over
the years allows us to compare analytical findings from year to year.
These comparisons are generally valid even though they must be tem-
pered with knowledge of changing analytical techniques and capabili-
ties. One example of changing capabilities is the change in screening
level for organophosphorus residues with improved detection systems
such as the thermionic or flame photometric detector. This changed
the screening level for parathion from 0.05 ppm to 0.01 ppm. The val-
idity of these comparisons is enhanced by three important program re-
quirements: (1) use of proven methodology; (2) recovery studies con-
ducted with each analysis; and (3) requirement for confirmation of
all organophosphate, organochlorine, chlorophenoxy acid, or carbaryl
residues.
Table 1 shows the average incidence and daily intake of 22
pesticide chemicals. The data shown covers a seven year period,
1965-1970. This table is reprinted from the Pesticide Monitoring
Journal, Vol. 5, No. 4, March 1972, p. 331, "Dietary Intake of Pest-
icide Chemicals in the United States (111)" by Duggan and Corneliussen.
The data for 1971 and 1972 are being prepared for publication.
The figures for DDT and its analogs DDE and TDK are particularly
interesting since they show an increase in frequency of occurrence
of these residues. However, the daily intake figures tell a more
important story, i.e. a decrease in daily intake. Simply stated,
DDT is showing up more frequently but at lower levels. This is further
shown by a listing of percentage of times DDT was found at only "trace"
values. These percentages are: 4.1% in 1967; 10.7% in 1968; 7.9%
in 1969; and 13.0% in 1970.
In contrast to noticeable trends in DDT residues, dieldrin
occurrence and daily intake appears to be constant over the six-year
period, and malathion appears to be on the increase. The increased
occurrence of malathion is further hinted by the data illustrated in
Figure 1, which shows distribution of total daily intake of residues
by chemical class (reprinted from Pesticide Monitoring Journal, Vol.
5, No. 4, March 1972). Note that organophosphorus residues are be-
coming a larger and larger percentage of the total residue burden.
Data such as given in Table 1 and Figure 1 are extremely useful in
reviewing trends in pesticide use. Carefully applied, the informa-
tion can also be used to guide other pesticide surveillance programs.
Some examples of this guidance are given below:
1. Since the frequency of phosphate residues appear to be
increasing, we must utilize analytical methodology capable
of detecting a wider range of compounds of this type.
2. Earlier Total Diet results indicated that the highest inci-
dence of chlorinated residues in the diet occurred in the
dairy and meat composites. Our surveillance efforts should
then be directed toward milk, milk products, and animal
feeds.
147
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In addition to providing guidance for surveillance programs,
Total Diet results may trigger special surveys or follow-up for legal
action. An example of the former is the survey for PCB's in paper
products after Total Diet results showed excessive PBC's in a break-
fast cereal. Findings of metal residues have also resulted in nation-
wide surveys of various products such as a survey of spinach for cad-
mium residues.
My discussion, thus far, has been confined to a review of past
results and past programs. Let me now discuss our present program
and the program under which we will be operating for the next year.
One major area of expansion has been the increased attention to metal
residues in our food. Although arsenic and cadmium were part of the
program for many years, only recently did our attention turn to mer-
cury, zinc, lead, and selenium. Many planners would like to see this
list of metals in the Total Diet program expanded considerably. As
manpower permits, I can foresee metal analysis becoming a major thrust
of our Total Diet Program. Perhaps the greatest hindrance to expansion
now is a good reliable multi-metal analytical procedure.
Expansion of metal analysis will be accompanied by increased
concentration on nutritional elements. In 1974, it is hoped that we
can add analyses for calcium, iron, phosphorus, and iodine. Analytical
methodology is a problem here but we hope to have the answer shortly.
Attempts are being made to determine within each composite, which
products, if any, are prime carriers of residues. Four samples of
last year's program were selected for individual commodity analysis.
In this program, individual commodities of the dairy composite and the
meat composite were analyzed for organochlorine and organophosphorus
residues. The data on this are still being evaluated and will be
published in the future. Individual commodity analyses of this type
will continue at least in the foreseeable future.
Program changes will occur but these will be the natural result
of expanding interest in residues or new residue problems arising.
In summary, as the Total Diet Program approaches its tenth
anniversary, let me say that the program is alive and well, growing
and continuing to supply residue information to many agencies and
individuals.
148
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FIGURE 1 -- Distribution of Residues by Chemical Class, 1967-1970
0.12
0.11
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
11.2%
3.4%
15.5%
69.8%
\
\
\
\
- \
fy?Q ^c\x^
V*^
^*G*
^V>
3.7% (
5.0% 1
5.5%
85.8%
V%^
^
3.6%
5.4.7%
19.8%
71.9%
\
N,
\
\
\
V
CHLOR-
INATED
ORGAN 1C S
HERBICIDES
9.8%
5.0%
26.9%
67.3%
1967
1968
1969
1970
149
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TABLE 1
Average Incidence and Daily Intake of 22 Pesticide Chemicals
[T=< o.ooi MG]
COMPOUNDS
DDT
DDE
TDE
Dieldrin
Lindane
Heptachlor
epoxide
BHC
Malathion
Carbaryl
Aldrin
2,4-D
Diazinon
Dicofol
(Kelthane)
PCP
End r in
Methoxychlor
Heptachlor
Toxaphene
Per thane
Parathion
Endosulf an
Ethion
1965*
A B
37.5 .031
31.5 .018
19.4 .013
18.5 .005
15.8 .004
13.4 .002
6.5 .002
7.4 .15
5.6 .001
4.2 .005
0.5 .003
1.4 T
2.8 T
1.9 T
0.5 T
— —
1966**
A B
37.3 .041
33.0 .028
25.7 .018
21.3 .007
12.3 .004
12.0 .003
6.0 .004
5.3 .009
2.7 .026
3.7 .002
3.0 .002
3.0 .001
3.7 .002
3.3 ..006
2.0 T
1.6 T
1.0 .002
1.3 .001
1.0 T
1.6 T
0.3 T
1967***
A B
38.6 .026
31.1 .017
28.9 .013
15.3 .004
10.6 .005
8.9 .001
8.9 .002
3.6 .010
1.1 .007
3.3 .001
1.7 .001
0.3 T
5.6 .012
2.2 .001
1.7 T
0.8 .001
0.3 T
1.4 .001
0.3 T
1.1 .002
1968***
A B
44.1 .019
37.5 .015
31.1 .011
15.6 .004
15.3 .003
13.1 .002
9.7 .003
1.9 .003
3.9 T
0.6 .001
0.3 T
4.7 .010
1.9 .001
1.1 .001
1.1 .001
0.3 T
1.1 .002
0.6 .001
0.6 T
0.8 T
1.7 .001
1969***
A B
48.9 .016
39.4 .011
28.1 .005
25.3 .005
13.3 .001
12.2 .002
10.6 .001
5.8 .012
0.8 .003
1.4 T
0.3 T
3.9 T
3.6 ,007
2.8 .002
3.3 ' T
0.3 T
1.7 T
3.6 .004
1.1 .004
3.3 T
4.2 .001
1.7 .003
1970***
A B
55.6 .015
50.6 .010
32.8 .004
31.3 .005
13.3 .001
11.1 .001
13.6 .001
11.1 .013
0.8 T
0.3 T
5.8 .001
4.4 .004
1.4 T
1.9 .001
0.3 T
1.1 .001
5.0 T
5.3 .001
4.4 .004
o
m
,— i
" * 216 composites examined.
** 312 composites examined.
*** 360 composites examined.
A = Percent positive composites
B = Daily intake mg
-------
DIFFERENTIATION OF INCIDENTS OF INFECTION AND CHEMICAL ETIOLOGIES
Keith R. Long, Ph.D.
University of Iowa
The differentiation between incidents of chemical poisoning and
other diseases can be one of the most frustrating problems confronting
an investigator. Case referrals more often than not are accompanied by
scanty information and nearly always require additional investigation.
In many instances the illness will be of unknown etiology and chemicals
or pesticides become suspect by default.
Health problems, particularly those of a chronic nature, associated
with environmental contaminants tend to have multi-causality. Thus, the
health behavior of an individual is the result of a combination of fac-
tors and is governed by the interactions of a number of characteristics
governed by time. These characteristics include occupation, social and
cultural behavior, family group, ethnic and religious beliefs and socio-
economic levels. These characteristics interacting with biological
factors of age, sex, race and physiological and biochemical behavior
coupled in time and place with an exposure to a chemical contaminant
point out the multi-causal relationships that must be interpreted.
A differentiation of incidents of chemically induced poisoning is
based upon employing the fundamental principles of epidemiology. Such
illnesses are either acute or chronic and may occur in all degrees of
severity. Acute illnesses induced by pesticides are fairly well
understood; however, a great deal needs to be learned about chronic
illnesses that may have a chemical etiology.
The history of each case is of the greatest importance whether it
be acute or chronic. First of all, one must establish the fact that
there was exposure to a toxicant. Then it is necessary to know what
poison or poisons along with any solvents* or adjuvants that may have been
involved. In addition, the concentration and the amount of contaminating
material constituting the exposure as well as the type, duration and
frequency must be ascertained. In addition, it is most important to know
the period from exposure to onset of symptoms. Frequently cases will be
single exposures but it is also important to know all preceding known
instances of contact with the toxicant in question. All of this
information can be developed by taking a good occupational history,
either from the individual or his close associates. In addition, it is
often helpful to have a past and present medical history developed,
particularly if one is dealing with a sub-acute or chronic situation.
A very carefully conducted neurologic examination is essential in
pesticide poisoning cases because many of the symptoms associated with
pesticide intoxication are predominantly neurologic.
In some cases of acute poisoning where a physician has suspected a
toxicant and begun treatment promptly, the response to the therapy may
help establish the diagnosis. However, proof of adequate exposure to a
toxicant should be established after the crisis if not before.
151
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For one to understand and learn more about poisoning and treatment,
it is desirable to collect comparable data from a number of subjects.
This is often time consuming and patterns cannot be established in some
instances until a quantity of data from a number of subjects has been
collected. In addition to the symptomatology experienced by the subject,
a clinical laboratory evaluation is often helpful. Most of the tests
that are conducted are nonspecific in regard to any particular toxicant
but may indicate the alteration of a physiologic response by a toxicant.
While many of the symptoms of pesticide intoxication are primarily
neurologic in nature, continued exposure can produce pathologic changes
in the kidney and liver. In the diagnosis of acute poisonings and the
differentiation from infectious disease, measurements of cholinesterase
are often helpful in cases where organophosphorus or carbamate intoxi-
cation is suspected. In differentiating the more chronic relationships
some of the liver enzyme changes, particularly alkaline phosphatase and
transaminase performance, may be helpful. One word of caution, however,
is that diseases such as chronic liver disease or infectious hepatitis
may confuse the picture. In some instances the pesticide residue content
of the blood or tissues may be of some aid. In most instances there
is no single measure that will indicate exposure to a toxicant. The
diagnosis of a toxicant must be established by utilizing the information
from the occupational history relative to exposure, the symptomatology
and the clinical laboratory findings, particularly with respect to
cholinesterase and liver enzyme activity. Occasionally, it may be
helpful to perform an electroencephalograph or electromyograph.
Identifying the agent and developing the occurrence of exposure in
time and place with the onset of illness may enable one to come to a
fairly positive conclusion about the diagnosis of acute illnesses. One
must not overlook infectious and organic diseases that may mask or be
the true cause of the illness in question whether it be acute or chronic.
A differentiation of this situation can only be achieved by developing
a good past and present medical history to establish the relationship of
the subject's physiological well being to chemical exposure.
In summary, the differentiation between incidence of infectious or
organic disease and chemical poisoning can only be established through
careful evaluation and obtaining occupational and exposure history and in
some instances past and present medical history to establish the fact
that a toxicant was involved. Knowledge of exposure, symptomatology and
laboratory findings can then be employed to establish the diagnosis
relative to a suspected toxicant.
The following cases serve as examples of the type of investigation
needed to differentiate between toxic responses to chemicals and other
etiologic agents.
Case No. D.F.; The case involves a veterinarian and his wife who had
recently built a new lake shore house. The family had lived in the house
approximately three weeks. The veterinarian commuted to his office each
day a distance of about 35 miles. The wife spent most of her time in the
house. The house was paneled throughout and all exposed wood surfaces
had been treated with Panel Lite which contained 4.1% pentachlorophenol
diluted 1:4 for application.
152
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Complaints: The family complained that all the house plants wilted
and died and that their two dogs were showing symptoms of illness. The
wife complained of headache, gastrointestinal disturbances, loss of
appetite, and fatigue.
Examination of premises: The house is a rectangular two-story
structure with a walkout basement. The basement area is totally devoted
to a swimming pool, roofed with a planking that serves as a deck leading
out from the living room, which is directly above the game room adjacent
to the swimming pool. The house is richly paneled in rough fir and pecky
cypress. Every room in the house is wood paneled and was treated with
pentachlorophenol. The house is divided into two general areas. The
living room area has a cathedral type ceiling made of planking and
exposed beams which were also treated with the pentachlorophenol solution.
The living room is adjacent to the deck and pool area, with the other
side of the house consisting of an open balcony leading to bedrooms on
the second floor, with access gained by an open stairway.
Medical history: The wife complained of headaches, a heavy feeling
in her legs, fatigue, and shortness of breath. She spends about 23 of
every 24 hours inside the house. She has a dip in the pool at least once
a day and spends her time doing housework and other work in the house.
When interviewed, subject indicated her skin had burned for two to three
days. The dogs, a Boxer and a West Highland White, had shown symptoms of
intoxication. Both showed neurological symptoms, gastrointestinal dis-
turbances and spitting up. Both showed redness of the foot pads.
Clinical laboratory findings: Clinical biochemical data obtained
for the housewife included total protein 6.1, albumin 4.2, globulin 2.4,
SCOT 24, thymol turbidity 2, bilirubin 0.4, alk phosphatase 10.4, WBC
7500, Hb 12.5, Hctr 38, neutrophils 54, bands 2, lymphocytes 33 and
sedimentation rate 31.
A diagnosis of pentachlorophenol intoxication was made.
I. Exposed plants Concentration PCP (ppm)
Tomato (by pool) 94
Tomato (in kitchen) 33
Pepper (by pool) 9.6
Pepper (in kitchen) 14
(Recovery from an unexposed tomato gave 98%)
II. Air sample results Concentration PCP (ppm)
Sample #1 3.5
Sample #2 0.723
(Recovery from blank glycol was 113%)
Sample Concentration PCP (ppb)
Urine (housewife) 37
Blood (housewife) 1190
Blood (Boxer dog) 270
Blood (West Highland White dog) 280
Plant 1733*
* No literature, methodology, or data available.
153
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Case No. R.E.; At 10:30 A.M. on 8 May 1971, this farmer used Atrex 80
(herbicide) for two hours. From 1:00 P.M. to 4:00 P.M. on the same day,
he used Bladex (herbicide). The nozzles on the sprayer clogged several
times when he was using the Atrex and he cleaned them out with his bare
hands. By 4:00 P.M. of that day he was having an intense amount of pain
in his hands and forearms which grew steadily worse until he was seen at
the hospital at 3:30 A.M. on 9 May 1971.
Past history is remarkable in that he had had much difficulty with
Ramrod (herbicide) one year earlier. He had pronounced skin blistering
and dermatitis of a generalized distribution (hands, forearms, legs,
penis, and ears). Late in April 1971 he used Ramrod again and noted
mild skin blistering so he quit using it immediately. At that time he
was seen by the Iowa Pesticide Study Unit and told he was in good health.
Past skin history is essentially negative except as mentioned above.
He has no known allergies except as mentioned above.
When first seen at the University Hospital at 3:30 A.M. on 9 May
1971, significant physical findings were limited to the hands and
forearms. There was much swelling and pain in these parts. The hands
were erythematous and there were hemorrhagic bullae between the fingers.
There were no pulmonary or systemic physical findings present.
Diagnosis of acute contact dermatitis was made and the patient was
treated with 60 mg of Prednisone by mouth. He was also treated for pain
with intra-muscular codeine and sent home with oral codeine for pain
relief.
At 12:00 noon on 9 May 1971, the patient was once more seen at the
University Hospital. The hands appeared much more ecchymotic and many
vesicles were present over the entire surface of the hands. He was once
again treated with Prednisone 60 mg by mouth and started on acetic acid
soaks. In the week of 10 May 1971 to 14 May 1971 he was treated with
a decreasing dosage of oral Prednisone. He continued the acetic acid
soaks and was also given a 10-day course of Tetracycline 250 mg qid as
prophylaxis for infection.
He was next seen on 1 June 1971, and the dermatitis was healing very
rapidly. Residual scaling and ecchymosis were present on the hands at
this time.
He was last seen at the University Hospital on 29 June 1971 at which
time his dermatitis of the forearms and hands was 90% cleared. He was
discharged from the clinic at this time and was told that he would be
contacted for a return appointment so that patch testing to the various
herbicides could be performed.
Case No. R.V.: Subject is a 23-year-old white male who was born and
raised on a farm. In addition to his own farm, he farms with his father
and brother on their farms.
First signs of illness were noted on June 5, 1969, when his tongue
felt numb and on June 7, 1969 when his hands felt numb when he touched
154
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something cold. On June 8, upon awakening, he could not.control lips,
mouth, hands or arms. Subject was hospitalized on June 8.
A preliminary diagnosis of atypical Guillan-Barre syndrome was made.
Neurologists were not happy with the diagnosis. Spinal fluid protein was
only slightly elevated. Findings included polyneuritis, slightly elevated
spinal fluid protein, difficulty in breathing and marked weakness in arms
and facial muscles, and the upper extremities and 7th, 9th, 10th and llth
cranial nerves were affected.
The subjects farm was visited oh July 15, 1969. Five empty one pound
cans of 34.7% heptachlor seed treater, a partially empty 5 gallon can of
23.4% Amiben, a one pound bottle of 44%.chlordane partially empty, and an
empty quart bottle of Coopertox were found.
The subject began planting corn on May 12, 1969 and disced ahead of
the planter. Thimet 15G was used with the planter to treat 250 acres of
corn. Fifteen hundred pounds of Thimet were applied over a 10-day period.
Following planting the subject treated and harrowed an additional 150
acres of corn. Between May 12 and May 26, subject had used an additional
300 pounds of Thimet and 1000 pounds of aldrin 20G. Two hundred acres of
soybeans were planted and treated with Amiben. Subject did not work with
Amiben. Subject wore overalls, shirt, and 8 inch high shoes. He did not
wear respirator or gloves. Subject indicated soil was very dry and he
was covered with dust during times of planting and harrowing. On May 8,
cattle were sprayed using 1% gallons of Korlan. Subject was observed
spraying and was covered with spray mist. Subject also used Coopertox
routinely for spraying for fly control. The product contains 14% toxa-
phene and 5% lindane. About June 1, subject rotary hoed about 18 acres
of corn on which Thimet had been applied. Subject got 5-6 hours sleep
during corn planting season.
During the patient's eight day hospitalization the differential
diagnosis of his peripheral neuropathy was between organophosphate
poisoning due to pesticide exposure and Guillain-Barre syndrome. Con-
sultation with the EMG service revealed a denervation pattern in the
muscles of the upper extremities: left and right triceps, left and right
biceps, and left deltoid muscles. EMG of the right anterior tribial,
left gastrocnemius and right quadriceps were within normal limits. A
Harvey Masland test showed no abnormal fatigue. Neurology consultation
was in concurrence with findings of a peripheral neuropathy involving
the lower cranial nerves and cervical areas. The most likely cause was
the neurotoxic effect of the organophosphates. In addition to their
anticholinesterase activity, these drugs may cause neurotoxicity and
axonal swelling and breakage. The delayed reaction to organophosphate
was consistent with the clinical history. A Guillain-Barre syndrome is
doubtful despite the preceding upper respiratory condition. The charac-
teristic findings of Guillain-Barre are that of an ascending paralysis
and the albumino-cytological dissociation were not present in this case.
Medical history: On June 5, 1969, subject noted numbness in mouth
and lips while drinking a cold drink. On the following day, he became
dysarthric and could not swallow. Later he had parathesis of his hands
which was followed by weakness starting from his hands and progressing
155
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up his arms. He had no fever, tachycardia or diaphoresis. On June 8,
1969, he was admitted to the hospital and put on a respirator. Three
lumbar punctures were done showing no abnormality. EMG revealed
conduction times which were slower than one would expect for the
patient's age. Plasma cholinesterase was 1.3 pM/ml/min and red cell
5.4 yM/ml/min. Review of pesticide exposure revealed recent use of the
following organophosphorus compounds: Atgard, Thimet, Korlan and Co-Ral.
Atgard was used one week prior to admission and exposure was extensive
because the subject mixed the compound into hog feed with his hands. He
was discharged from the hospital on July 18, 1969.
Subject was admitted to University Hospital at Iowa City on
September 15, 1969 for evaluation of his neuropathy. Since his discharge
from the hospital in July, his five-week course was characterized by a
slow gradual improvement in strength. Physical examination showed a
well nourished, white male in no acute distress with obvious atrophy of
the pectoral, scapular and deltoid muscles. Vital signs were within
normal limits. Positive physical findings were a tracheotomy scar
(performed during period of earlier hospitalization), cafe au lait spot
on left side of back, and motor weakness in the following muscles:
biceps, triceps, and serratus anterior especially on the left side.
There was no sensory impairment and deep tendon reflexes were equal and
normal.
Laboratory studies revealed a normal CBC, urinalysis, stool guaiac,
BUN, creatinine, electrolytes, calcium, phosphorus, thyroid activity,
pro time, two hour post prandial blood sugar and serum uric acid. Plasma
and red cholinesterase were somewhat abnormal with levels of 1.96 and
7.9 pM/ml/min respectively. Spinal fluid examination revealed a protein
of 46, sugar of 63 and a cell count of 1 red cell. Serum protein electro-
phoresis was within normal limits.
156
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SAMPLING TECHNOLOGY
Bill L. Stevenson
United States Forest Service
To promptly and accurately evaluate chemical pesticides in biological
and environmental media, suitable representative samples of these sys-
tems insure quality analytical performance.
The identification and evaluation of pesticides present in samples
submitted to a laboratory may serve as a basis for the documentation of
an accident or incidental contamination from normal use. Laboratory data
is a necessity to identify current and potential public health problems
and protect the environment from unanticipated contamination.
Data from a competent laboratory based upon improper sampling can lead to
false conclusions concerning the pesticide profile of the "sample even
though properly analyzed. For example, let us assume that a laboratory
making routine blood analysis of the general population in a given area
is consistently, but erroneously, finding higher pesticide levels than
are being reported by similar laboratories in other areas. Plotted on a
national scale, it would then appear that the people of this area are
being exposed to higher levels of pesticides than the rest of the national
population.
Another example can be cited which involved complaints filed by several
women employees making draperies for mobile homes that some irritant was
present in the atmosphere. An investigation was made and a survey of
the work area revealed that the entire operation, including the storage
of bolts of drapery cloth, was confined to a room 12' x 15' with a three-
fourths ton air conditioner providing the only ventilation.
Through a literature review, it was learned that formaldehyde is used to
make colors "fast" in the dyeing process of cloth. This information indi-
cated a possible source of the problem.
The sampling procedure followed (to determine exposure levels to atmos-
pheric formaldehyde in work areas) was recommended by an excellent and
authoritative source. However, one small but important detail was
omitted in the sampling instructions—that was to use an impinger with a
diffusion attachment to disperse the air stream into tiny bubbles for
greater absorption in the collection media. This omission resulted in a
false reading indicating a safe working environment when in fact, the
formaldehyde vapors exceeded established threshold limits. A lawsuit
was instituted and was an embarrassment to all concerned.
Interferences are encountered in most analytical methods; therefore, it
is rare that a method is free from all interferences regardless of the
concentration of chemicals in a sample. Nevertheless, most methods will
yield reliable results with little interference when followed as pre-
sented by authoritative and approved methods.
157
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Analytical procedures are usually designed either to give the total
amount of an element present in a sample or to give the amount of a
certain definite solute species. Interferences affect either type of
analytical procedure and the results are a serious potential source of
error against which the analyst must always be aware.
The following general rules for sampling can be utilized in most
investigations (1):
1. Never take a composite sample unless the subject area to be
sampled is known to be uniformly contaminated.
2. Take a number of samples from the subject area that would best
represent possible areas of contamination.
3. Take the samples immediately, when reason for concern is
apparent. It may be too late later.
4. Label the samples with all the information immediately. You
may forget later.
5. Preserve the samples with whatever means are necessary. This
may be refrigeration, freezing, formaldehyde or solvent.
6. Make sure the means of preservation are compatible with
subsequent analysis. Samples of crab eater seals and pen-
guins from the Antarctic were shipped back to this country
wrapped in plastic. As a result the reported levels of DDT
are questionable due to the plasticizer in the plastic.
Don't preserve samples to be used for chlorinated hydrocarbon
analysis with chloroform. Don't preserve samples for phos-
phate analysis with formaldehyde. If you don't know what
analysis will be required, refrigeration is your best bet.
7. Take a large enough sample. Tomorrow will be too late to get
more.
8. Be prepared to take samples. Have sampling tools needed, knives,
scissors, trowels, spoons, etc. Have suitable bottles for
samples.
9. Label all samples.
10. Write a description of the situation as the investigation sees
it at the time of sampling. It is not unusual for the cost of
analysis in particular investigations to run $10,000. This
expenditure should not be jeopardized because the sampler is
too lazy to write down the situation as he sees it. Provide a
case history. This is needed for the chemist to get a clue as
to what to analyze for. He cannot possibly screen for the
million or so compounds that presently contaminate our environ-
ment.
11. Get samples to laboratory now. Don't wait two weeks while you
talk the situation over.
158
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Procedures for taking samples. Although pathologists should have adequate
knowledge concerning the collection and submission of autopsy samples for
chemical analysis, occasionally samples are contaminated by using improper
containers or solutions that interfere with analytical methods.
Samples from suspected human poisonings:
1. Quantity:
a. Tissue - 1-5 grams
b. Vomitus - 2 ounces or more
c. Stomach content - 2 ounces or more
d. Stomach wall tissue - 5 grams or more
e. Kidney - 1-5 grams
f. Liver - 1-5 grams
g. Blood - 5-10 ml
h. Urine - 5-10 ml
i. Brain - 1-5 grams
2. Packaging: Each tissue sample should be isolated in a
separate chemically clean glass jar or bottle.
3. Shipment: It is preferable to have the samples frozen
and shipped on Monday to the appropriate laboratory.
When samples are frozen, care should be taken that
enough dry ice is included to insure arrival of samples
in a frozen condition-
Soils. The area should be searched carefully for oil spots or areas that
look different from the surroundings. These can be sampled by using a
sampling tool made from a tin can or similar container. Cut both top and
bottom out evenly. Wash well and repeatedly. Rinse in clear water.
Press this can in suspect area to depth of 1^-2 inches and remove from
soil. Punch core taken into new quart mason jar. Take several more
cores from same areas. Seal jar and send to the laboratory after careful
marking to indicate where sample was taken, when, by whom, and number and
depth of cores (1).
Water. Water samples should be submitted in chemically clean, one-gallon
glass bottles.
Most natural water bodies are not completely homogeneous and obtaining a
truly representative sample will depend on the sampling technique em-
ployed, as well as the size and number of samples collected (2). Proper
sampling of large bodies of water should include a surface sample, an
intermediate sample and a bottom sample. Always use chemically clean
glass bottles for collecting samples.
Interfering elements are leached from plastic; therefore, plastic sample
bottles should never be used to collect samples for pesticide analysis.
159
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Swab samples. Sampling to determine contamination on a wall or duct or
other surfaces may be collected through the use of sterile wool, some
medical alcohol or acetone and plastic throw away gloves. Wearing the
gloves, wash the suspect area with swabs of cotton and solvent using
care not to let solvent squeeze out of the swab. Place used swab in
pint mason jar. Repeat on adjacent area. When labeling bottle, record
where sample was taken, by whom, how it was sampled and how large an
area (1).
Air Sampling. Sampling of air requires sophisticated equipment and
methods. Consult the Environmental Protection Agency, Ecological Mon-
itoring Branch, Division of Technical Services, Washington, B.C., for
specific information.
Other Sampling
Many other types of sampling are occasionally required such as grain,
feed, flour, food or other types of samples representing human or animal
exposure. It is highly important in such cases to inspect carefully the
material to be sampled in order to detect possible areas of contamination.
Sometimes only one sack in a shipment will show an oily spot of contamin-
ation, or the colored grain denoting seed treatment. Samples should be
drawn both from suspect and non-suspect areas and so labeled. If in
doubt, call the appropriate laboratory for guidance.
Labeling and Shipping
Without proper labeling or shipping, all the care and trouble of taking
the samples is wasted. Be sure the label is explanatory and well
attached. Ship samples on a Monday so that they arrive during the week
and not over the weekend where they might sit in a hot airport terminal
and deteriorate. If the samples are worth taking, they are worth packing
well and with extra dry ice or other refrigerant.
The following tables provide information for proper sample collection;
i.e., size of sample, packing storage, preservatives, and other helpful
information (1).
Type Poison Examples Types of Samples Needed
Chlorinated Aldrin Stomach and its content,
hydrocarbons Dieldrin vomitus, kidney, liver
DDT and fat.
Endrin, etc.
Fluorinated 1080 Stomach and its content,
compounds Sodium fluoride or vomitus if present,
liver, kidney and heart.
Inorganic Zinc phosphide Stomach and its content,
phosphides vomitus, liver and spleen.
160
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Type
Poison Samples
Types of Samples Needed
Organic
phosphates
Phosphorus
Cyanide
Alkaloids
Thallium
Cardiac
glucosides
Lead
Anticoagulants
Miscellaneous poisons,
toxic .compound unknown
TEPP
Parathion, etc.
Elemental
phosphorus
Cyanogen
Sodium cyanide
Strychnine
Thallium sulfate
Red squill
Lead acetate
Lead arsenate
Warfarin
Pival
PMP
Diphacin
Stomach and its content,
vomitus, brain, heartj
blood (if not deteriorated).
Stomach and its content,
vomitus, liver and kidney.
Stomach and its content
vomitus, throat tissue,
heart, brain and lung
tissue.
Stomach and its content,
vomitus, liver and kidney.
Stomach and its content,
vomitus, liver, kidney,
leg muscle and bone.
Stomach and its content,
vomitus and a portion of
intestine.
Stomach and its content,
vomitus, liver, kidney, and
long skeletal bones (leg).
Stomach and its content,
vomitus, heart, liver and
blood.
Stomach and its content,
vomitus, heart, liver,
kidney, brain, blood and
bone.
161
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Sample
private well
municipal
pre filter
at filter
treated
river
lake
TABLE I
Environmental Sampling
Size of Sample Packing*
Storage
Preservative
1 gallon
1 gallon ea.
1 gallon
1 gallon
SAMPLING OF WATER FOLLOWING INCIDENT
glass ambient
glass
glass
glass
ambient
ambient
ambient
none
none
none
none
Other Information
description of well
date
note where and when
taken
detailed location of
sampling site and .date
detailed location of
sampling site and date
Accompanying each set of samples should be a complete description
of incident and all pertinent information to guide laboratory
director in making analysis.
SAMPLING OF TISSUE AT AUTOPSY
fat
liver
lung
brain
gonads
spleen
muscle, etc.
1 to 5 gms
1 to 5 gms
glass bottle
or aluminum
foil
frozen or 10% Formaldehyde
glass bottle frozen
or aluminum
foil
not
acceptable
name of subject
site of sample
sample size
date, cause of death
name of subject
name of tissue
sample size
date, cause of death
*Where glass containers are specified they should be fitted with teflon or aluminum lining.
-------
OJ
Sample
urine
for chlorinated
hydrocarbons
urine for
phosphates
blood for
chlorinated
hydrocarbon
blood for
phosphates
blood for
cholinesterase
- Size of Sample
**25 ml of
24 hr. sample
if possible
**25 ml of
24 hr. sample
if possible
1-5 ml plasma
1-5 ml whole
blood
1-5 ml plasma
1-5 ml red
cells
TABLE I
Environmental Sampling (Cont.)
Packing Storage - Preservative
SAMPLING OF BODY FLUIDS
glass ambient 1/10%
Formaldehyde
glass
glass tube
oxaloted
glass tube
oxaloted
glass tube
heparinized
glass tube
heparinized
frozen none
frozen none
frozen none
refrigerated none
refrigerated none
Other Information
name of subject
description
whether 24 hr. or not
name of subject
description
whether 24 hr. or not
name of subject
reason for analysis
date, physician
name of subject
reason for analysis
date, physician
name of subject
reason for analysis
date, physician
name of subject
reason for analysis
date, physician
** A complete case history should be provided with all human samples.
-------
Sample
soil
sediment
wildlife
birds
mammals
or reptiles
fish
TABLE I
Environmental Sampling (Cont.)
Size of Sample
1 Ib.
Packing Storage
OTHER SAMPLING
Preservative
1 Ib.
20 gms muscle
(20 gms fat
if possible)
20 gms
tissue
glass or
metal can
glass or
metal can
glass
ambient
ambient
frozen
none
none
none
glass
frozen
none
Other Information
description of site
amo unt sampled,
by whom and why
description of site
amount sampled
by whom and why
detailed description
of sampling site
name of animal
approx. age if known
by whom and why
detailed description
of sampling site
name of animal
approx. age if known
by whom and why
Analysis of one animal, fish or bird will generally tell but little. Enough animals should
be sampled to provide a crossection exposure picture of the incident but certainly never
less than 10 subjects.
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References
1. Barthel, W. F., Sampling Procedures for Chemicals in the Environment,
U.S. Department of Health, Education and Welfare/Public Health Service,
Center for Disease Control, Atlanta, Georgia.
2. Brown, Eugene, Skougstad, M. W. and Fishman, M. J., Methods for
Collection and Analysis of Water Samples for Dissolved Minerals and
Gases.
165
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166
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CARBON MONOXIDE POISONINGS IN THE ROCKY MOUNTAIN AREA
Eldon P. Savage, Joseph W. Malberg,
H. William Wheeler and John D. Tessari
Colorado State University
Background
Carbon monoxide poisoning is a preventable hazard that still
frequently occurs in the Rocky Mountain Area. Since carbon monoxide
poisoning is not a reportafale disease, accurate morbidity and mortality
data are not available. Lehr noted that, based on evidence available
to the Injury Control Program of the United States Public Health Service,
at least 10,000 persons suffer chronic ill effects from exposure to
sublethal but debilitating levels of carbon monoxide (1). Mortality
data for carbon monoxide poisoning are collected from death certificates
and reported annually by the National Center for Health Statistics.
An example of this mortality in the United States is provided by the
1966 statistics which accounted for 1500 deaths; of these, 900 were
in homes, 100 in industry, and less than 200 on streets or highways (1).
Morbidity from carbon monoxide is not well documented but it
is estimated that over 10,000 persons per year are overcome or
incapacitated due to exposure to hazardous levels of carbon monoxide.
An average of 1,400 people per year die from carbon monoxide poisoning
in the United States. Sixty percent of the mortality cases are
reported to occur in the home and less than fourteen percent of these
cases occur on streets or highways. But the morbidity and mortality
figures may be misleading, and some concerned physicians and scientists
believe that low level carbon monoxide exposures may be responsible
for judgement accidents that occur in the home and on the highway.
Human tolerance to carbon monoxide is significantly decreased
as the altitude increases. The carbon monoxide emission from vehicles
also increases with altitude (2).
Methods
this paper reports on carbon monoxide cases occurring in rural
areas of Colorado and Wyoming. These data were taken from hospital
records in Leadville, Canon City, Salida, Fort Collins, Durango,
Greeley, Grand Junction, Burlington, Lamar, LaJunta, Craig and
Glenwbod, Colorado; Cheyenne and Laramie, Wyoming.
The age, sex, race, occupation, exposure source, symptoms and
treatment were recorded when these data were available. A total of
115 cases were reviewed from Colorado and Wyoming hospitals. The
study was conducted during 1970-72.
Results
Table one depicts the exposure source of carbon monoxide. A
total of 44 cases were caused from vehicular carbon monoxide.
167
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Approximately three quarters of these occurred when the victims
were riding in cars. Others were due to exposure in trailers and
campers and exposures when working on cars in residential garages.
Exposures due to faulty furnaces accounted for the largest
numbers of carbon monoxide poisoning cases. There were 48 cases
that occurred form faulty heating systems either due to clogged
flues, improperly vented furnaces or malfunctioning furnaces.
Occupational exposures accounted for six cases. Mechanics
were treated on four occasions.
Table two depicts the age and sex of carbon monoxide cases in
rural Colorado and Wyoming. The victims were evenly divided between
males and females for all age groups and no significant differences
were noted. The 16-24 and 40-55 age groups had a total of 23 cases
in each age group respectively.
The largest number of cases occurred during the months of
December and January. The number of cases dropped gradually through
April and May, rised precipitously during June and July, and then
dropped to the lowest number during August and September.
Table three depicts the importance of altitude and its relationship
to carbon monoxide poisoning. The Wyoming and Colorado communities
were divided into communities over 6,000 feet and into communities
under 6,000 feet. The population of communities in the above 6,000
feet and in communities below 6,000 feet were then calculated and
morbidity rates per 100,000 were tabulated. The morbidity rate
was 102.0 per 100,000 at altitudes greater than 6,000 feet and 23.3
per 100,000 at altitudes less than 6,000 feet.
Discussion
The age of the patients treated for carbon monoxide poisoning
ranged from two to 75 years and were evenly distributed with no
significant age group indicated. Sex was also of little significance
in that 57 males and 59 females were exposed. Occupation was not
of major significance in exposure. The primary source of exposure
was the home environment, including both houses and attached garages.
Fifty-three of the cases were reported due to improperly vented
furnaces. Four cases occurred in trailers and were also due to
improper venting.
Improper venting of stoves and furnaces and ignorance of the
source of carbon monoxide are major reasons for exposure in the
cases studied. Persons installing furnaces, stoves or heaters
should be made aware of the proper ventilating techniques for such
heating units. Many of them are personally installed and the owners
are unaware of the fact that carbon monoxide is produced as an
incompletely oxidized by-product and proper ventilating is necessary
to carry these fumes to an outside environment.
168
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A carboxyhemoglobin (COHb) level of 13% produces objective
changes due to an increase in vascular permeability. The threshold
limit value should be below this level or 50 ppm (3). It was noted
in a study of Los Angeles County (3) that concentrations in the
order of 50 ppm may result in temporary impairment of judgement and
motor performance.
Many victims of carbon monoxide poisoning have been diagnosed
and treated for such ailments as food poisoning, influenza, sinus-
itis, pneumonia, or typhoid fever. When carbon monoxide poisoning
is not correctly diagnosed the patients will generally return to the
same environment, which could have fatal results. Carboxyhemoglobin
tests can easily confirm or deny any suspicions of carbon monoxide
poisoning. In a report from the British Medical Journal (4) it was
reported that only 32.5% of the accidental exposures to carbon
monoxide were given oxygen therapy and they state, "in view of
the risk of neuropsychiatric sequelae current patterns of manage-
ment should be revised." COHb should be eliminated as quickly as
possible because its presence alters the dissociation curve of the
remaining oxyhaemoglobin—the affinity of carbon monoxide for haemo-
globin is about 250 times that of oxygen—impeding the oxygen release
to the tissues.
Results of the current study show that 67 of 116 patients received
oxygen treatment. Only one case resulted in sequelae, but that
resulted in the possibility of mental problems in a two year old
boy. Oxygen treatment might have prevented this if the attending
physician had been more aware of the possibilities of carbon monoxide
poisoning and had administered oxygen.
The importance of altitude should not be overlooked. The rate
per 100,000 population at 6,000 feet was 102. This compared to a
rate of 23 per 100,000 at altitudes less than 6,000 feet. The
altitude-carbon monoxide relationship needs further investigation.
The interrelationship between non-clinical levels of carbon monoxide
and driver judgement accidents may be a more severe problem in the
Rocky Mountain States in high altitudes than originally expected.
The month of occurrence is also an important factor. The large
number of cases occurring in the winter months is due to faulty
furnaces and the peak during the summer months is due to the large
increase of cases due to exposures in automobiles. If the total
number of accidental exposures to carbon monoxide poisoning in the
United States is to be reduced, those responsible for public health
will have to play a more active role.
-------
Table 1. Carbon monoxide poisoning, rural Colorado-Wyomlng
Exposure
Number of cases
Vehicular
Riding in car
Car in garage
Trailer
Camper
Furnace
Home
Work
Motel
Occupational
Mechanic
Cook
Unclassified
Unknown
44
53
TOTAL
31
9
1
3
48
3
2
4
1
1
Table 2. Age and sex of carbon monoxide poisoning victims in rural
Colorado and Wyoming
Age
TOTAL
Male
56
Female
59
Total
6 -
16 -
25 -
40 -
56 -
<5
15
24
39
55
70
70 +
Unknown
2
10
11
9
13
9
0
2
5
8
12
9
10
7
3
5
7
18
23
18
23
16
3
7
115
1 unknown sex and age
Table 3. Carbon monoxide morbidity, Colorado-Wyoming
Altitudes higher
than 6,000 feet (6 cities)
Altitudes lower
than 6,000 feet (9 cities)
Population
Number of cases
Rate/100,000
87,263
89
102.0
111,504
26
23.3
170
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References
1. Lehr, E. L. Carbon monoxide poisoning: a preventable environmental
hazard, American Journal of Public Health, 60: 283-93, Feb. 1970.
2. Gent, C., P. Astrup, P. Chullen, Jr., and G. Gerhardsson. Threshold
limit values for carbon monoxide, Archives of Environmental Health.
21:542-44, Oct. 1970.
3. Hexter, A. C., J. R. Goldsmith. California State Department of
Public Health, Berkley. Science, 172:265-66, April 16, 1971.
4. Carbon monoxide poisoning, British Medical Journal, 3:180, 1970.
171
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172
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CURRENT TRENDS IN DISPOSAL OF TOXIC SUBSTANCES
Current Status of Disposal of Toxic Chemicals
Robert Harding
U. S. Environmental Protection Agency
The disposal of toxic chemicals is an ever growing problem. The
quantity of toxic chemicals being used is increasing, which increases
the quantity to be discarded. Secondly, and perhaps of greater impor-
tance, the quantity of toxic chemicals allowed into the air and water,
through ever stricter federal and state laws, is being reduced.
Unless alternative disposal methods are developed, toxic chemicals
will probably be deposited on the land. This magnifies the seriousness
of potential ground water pollution problems and concentrations of large
amounts of undegraded toxic chemicals which may some day be excavated for
land development purposes.
The federal government has placed emphasis on pesticide and pest-
icide container disposal. Recommendations include incineration, burial,
soil degradation, chemical neutralization, photo decomposition, and
storage. Factors affecting, disposal methods are whether the pesticide
is an organic or an inorganic, and the metals contained.
The Biodegradation of_ p,p'-DDT and Aroclor 1254 in Soil
Obtained From a_ Solid Waste Disposal Site
Fred M. Applehans
Colorado State University
The rate of microbial degradation of p,p'-DDT and Aroclor 1254
inoculated into alfalfa-amended and nonamended soil obtained from the
Fort Collins-Loveland Solid Waste Disposal Site was recorded over a
twenty-two week period. The inoculated soil samples were waterlogged
and incubated at 30 °C for the duration of the experiment.
The p,p'-DDT was moderately degraded in both the alfalfa-amended
and nonamended soils; however, there was no significant difference in
the rate of degradation between the amended and nonamended soil prepara-
tions. No significant degradation of the Aroclor 1254 occurred over the
twenty-two week period, and likewise there was no difference in the de-
gradation rate between the Aroclor 1254 amended and non amended soil.
All four soil preparations had microbial cell counts characteristic
of a fertile soil at the end of the twenty-two week period. The Aroclor
1254 soil preparations had significantly greater populations than the
p,p'-DDT soil preparations. There were no significant population dif-
ferences between the amended and nonamended soils for each of the chlo-
rinated hydrocarbons. Therefore, the alfalfa amendment did not induce
cell growth and proliferation in the soil. Seven different genera of
microorganisms were isolated from the soil and identified.
173
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Mercury Contamination at a_ Solid Waste Site
H. William Wheeler
Colorado State University
Disposal of mercury and mercury containing products has recently
become a concern. Incidents such as those involving fish contamination
in both fresh and salt water and incidents of contamination of animal
feed illustrate the need for a broad base of knowledge in areas of
transport, conversion, and degradation prior to establishing safe methods
of disposal.
A recent study conducted by the Institute of Rural Environmental
Health attempts to establish background mercury levels in the area of
the Larimer County Solid Waste Disposal Site and determine possible
transport routes of natural and introduced contamination through the
area. Included is an effort to identify possible indicator species of
plant and animals.
Pesticide Disposal - Montana
Gary Gingery
Montana State Department of Agriculture
Pesticide disposal in Montana has been divided into three major
areas: surplus pesticides (pesticides that cannot be utilized or which
are mislabeled), excess pesticides (remaining material from products
properly labeled) and container disposal. Projects are currently under-
way to regulate each of these areas in combination or separately and to
educate all users on proper disposal techniques.
Records of past experiences with improperly disposed pesticides
and their containers are extremely limited. Problems that have occurred
involve such items as arsenicals, cattle dippings, scattering of containers
throughout the State, and severe soil contamination in and around commer-,
cial applicator mixing points.
Since 1969 the three departments^Agriculture, Health, and Fish
and Game-^have had a program to collect, store, and ultimately dispose
of surplus pesticides. These products are now being stored in an am-
munition bunker at Glasgow Air Force Base under strict security and fire
protection. To date, 75,000 pounds of dry material and 4,000 gallons
of liquids have been collected. Ultimate disposal remains to be an acute
problem; however, protection of citizens and the environment has resulted.
The major disposal problem with these materials is the large volume of
inorganics collected.
174
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HAZARDOUS CONTAMINANTS: CHLORINATED DIBENZODIOXINS
AND CHLORINATED DIBENZOFURANS
James Edward Huff and John S. Wassom
Oak Ridge National Laboratory
Introduction
Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic chemical.
TCDD, referred to most often in this paper, rivals many of science's most
toxic substances. In fact, it has been considered second to botulinum
toxin as a potentially toxic hazard to man. TCDD has been labeled the
most toxic chlorinated hydrocarbon in the chemical armamentarium. The
other chlorinated dibenzo-p-dioxins and the chlorinated dibenzofurans
are generally less toxic than TCDD. Toxicity of the individual chlorin-
ated derivatives correlates to some degree with structure.
This presentation will not consider the herbicide 2,4,5-trichloro-
penoxyacetic acid (.2,4,5—T) per se except in direct relation to TCDD as
an unwanted contaminant. After attending the National Institutes of
Environmental Health Sciences (NIEHS) conference on the Chlorinated
Dibenzodioxins and Chlorinated Dibenzofurans, we began a project to
collect the available literature on these contaminants to make them more
readily accessible to the scientific community. The annotated literature
collection appeared in September 1973 (2); a brief summary has been pre-
pared (3) whereas an extensive treatise (4) is nearing completion.
Following a brief, but high-lighted historical chronology of the
salient events leading to the culpability linkage between these relatively
simple chemical molecules and the disasterous biologic implications, we
will categorize some results from our continuing literature collection
and survey.
Historical Considerations
TCDD is an impurity associated with 2,4,5-T whereas the chlorinated
dibenzofurans are contaminants found in some polychlorinated biphenyl
(PCS) compounds—Arochlor, Clophen, Phenoclor. Unfortunately, these un-
wanted—and until recently undetected and unrecognized—contaminants are
more toxic and represent a potentially greater environmental hazard than
the compounds they contaminate. Thus, while major attention has been
focused on the toxicity of a persistent pesticide, impurities which are
in fact more toxic may have gone unnoticed.
Following the tragic world-wide thalidomide horror, attention was
focused on the potential and real hazards of chemically-induced birth
defects for the human population. Chemicals hitherto regarded as safe
became highly suspect. In 1963, the President's Advisory Committee recom-
mended initiation of a large-scale safety screening program for pesticices
and other industrial chemicals. Shortly thereafter, in 1964, the National
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Cancer Institute (NCI) contracted with the independent and privately
operated Bionetics Research Laboratory to do carcinogenicity, mutagen-
ecity, and teratogenicity testing on a number of pesticides. Bionetics
subsequently reported to NCI in the late 1960's, that 2,4,5-T was ter-
atogenic to mice. This report was not widely distributed and it was some
time before the scientific community became aware of these results.
Shortly thereafter, it was discovered that the 2,4,5-T sample used in
that study contained about 30 ppm TCDD. This revelation was the point
of departure for the increased research activity on dioxins and also
the furans.
Chemistry
The basic dibenzo-p-dioxin two-dimensional structure as shown in
Figure 1 has eight possible points of chemical addition. From the mono-
chloro- to the octachlorodibenzo-p-dioxin, a variety of isomers are pos-
sible—approximately 60 chlorinated derivatives can be imagined. The
skeleton dibenzofuran structure is also shown in Figure 1, resembling
dibenzo-p-dioxin except having a central furan nucleus.
Despite the high sensitivity of most existing analytical method-
ology, a compound such as TCDD exhibits toxic effects in the 0.1 to 1
ppb concentration range which is below the normal detection limit for
TCDD—10 ppb. Recently however, Baughman and Meselson reported a more
sensitive analysis for dioxins (5). Homogenized samples are saponified
in alcoholic potassium hydroxide and extracted with hexane. The extract
is shaken with sulfuric acid and chromatographed on alumina. Elution
with carbon tetrachloride-hexane removes DDE and PCB's. Chlorinated
dioxins are then eluted with dichloromethane-hexane. After this initial
clean-up, the TCDD containing fraction is further purified by preparative
gas-liquid chromatography and analyzed by mass spectroscopy, using a
multichannel analyzer (CAT) to average successive scans. This procedure
is reported reliable for TCDD detection in animal tissues down to levels
approaching one ppt. The method separates TCDD from DDE, PCB's, and
other chlorinated hydrocarbon residues.
Resultantly, chlorinated dibenzo-p-dioxins and chlorinated diben-
zofurans can be determined in chlorinated phenols, chlorinated phenoxy
herbicides, ronnel, fat, and combustion products using such analytical
techniques as gas chromatography, liquid chromatography, thin-layer
chromatography, and gas chromatography-mass spectrometry (6).
Formation Reactions
TCDD is produced as an unwanted side product in the industrial
synthesis of 2,4,5-trichlorophenol, an intermediate in the manufacture
of 2,4,5-T. Figure 2 illustrates some of the possible reaction mechan-
isms that may lead to contaminant formation. Under the influences of
high temperature, high pressure, and alkalinity—tetrachlorobenzene forms
trichlorophenol which continues in the synthesis of 2,4,5-T—or 2 molecules
of trichlorophenol may react to form TCDD. In like manner, hexachlorobenzene
goes to pentachlorophenol; two molecules may react to form octachlorodibenzo-
p-dioxin; unreacted hexachlorobenzene may act with pentachlorophenol (PGP)
176
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Figure 1. Parent structure of dioxins and furans
10
DIBENZO-p-DIOXIN
DIBENZOFURAN
177
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Figure 2... DIOXINS AND FURANS FORMATION REACTIONS
TETRACHLOROBENZENE
oo
HEXACHLOROBENZENE
CHORINE + PHENOLS
high temperature
high pressure
alkalinity
high temperature
high pressure
alkalinity
high temperature
2,4,5-TRICHLOROPHENOL->+2,4,5-T
2,4,5-TRICHLOROPHENOL+TCDD
PENTACHLOROPHENOL
PENTACHLOROPHENOL-* TCDD
HEXACHLOROBENZENE-TURANS
PENTACHLOROPHENOL + DIOXIN CONTAMINANTS
Fe
-------
to form furans.
Pentachlorophenol enjoys widespread use as a wood preservative.
Commercial grades have been found to contain up to 2500 ppm chlorinated
dibenzo-p-dioxins (7). The predominant dioxin is octachlorodibenzo-p-
dioxin, one of the least toxic members. Evaluating pentachlorophenol
toxicity in animals revealed that some untoward effects (chloracne, chick
edema disease, and histopathologic alterations) were caused by chlorinated
dibenzo-p-dioxin content. Purified pentachlorophenol did not produce
these effects.
The fungicide PGP has been shown to be contaminated with high amounts
of octachlorodibenzo-p-dioxin. Previously undetected contaminants belong-
ing to the chlorinated hydroxydiphenyl ethers are formed in the first step
of the dioxin formation (8). One substance, a nonachloro-precursor of
dioxin, is called predioxin and is formed from 2 molecules of PCP. The
other substance is isomeric to predioxin, called iso-predioxin, and is
incapable of ring closure to form dioxins.
Chlorodioxins are formed in a two-step condensation reaction from
ortho-substituted halophenoxy radicals or anions (9). Reaction of chlorine
with pentachlorophenol at elevated temperature proceeds by radicals;
anionic condensation products result from strongly exothermic reactions
of alkali metal salts of chlorinated phenols above 300°C. Reaction product
distribution depends on the total number of halogen substituents, the
crystal lattice arrangement of the molecule, steric effects, and an
electronic effect. Dioxin formation was the major condensation product
only for sodium pentachlorophenate.
Logically however, all products originating from chlorinated benzenes
treated with alkali are possible sources of chlorinated dibenzo-p-dioxins (10)
Toxicologic Aspects
The polychlorinated dibenzo-p-dioxins are both chemically persistent
and biologically active. The lethal dose and toxic manifestations are
structure and species dependent.
TCDD is perhaps the most potent small molecule toxin and teratogen
presently known. As little as 0.6 yg TCDD/kg body weight is lethal to .
one-half the experimental guinea pig population; 10 yg/kg resulted in
death to rabbits, while 1 yg/kg caused liver damage and chloracne (11).
The higher dioxin homologues tend to be less toxic.
Chick Edema Disease - In 1957 millions of commercially raised chickens
died mysteriously. The disease was called chick edema disease because
the characteristic overt signs were hydropericardium and ascites in
chickens. Symptoms included droopy and ruffled feathers and labored
breathing.
A toxic agent was traced to the fat portion of the chicken feed;
for want of more information, the edema causative was termed toxic fat
and more specifically chick edema factor. Single crystal x-ray crystal-
lography was used to determine the toxic substance as 1,2,3,7,8,9-
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hexachlorodibenzo-p-dioxin (12). Adverse effects were exhibited also
in monkeys, dogs, guinea pigs, rats, and pigs.
Chloracne - During the same year that the chick edema disease was rampant
in the eastern and midwestern parts of the United States, research reports
published in Germany indicated that certain chlorinated hydrocarbons
caused chloracne in industrial workers and experimental animals. Chloracne
or acneform dermatitis is a common occupational dermatitis characterized
by inclusion cysts, comedones, and pustules.
The agent responsible for causing occupational chloracne in 31
employees of chlorophenol producing factories was found to be TCDD (13).
The toxic action in animals was due to the alkaline hydrolysis product of
1,2,4,5-tetrachlorobenzene. TCDD and tri- and tetrachlorodibenzofuran
were active skin irritants and were especially effective acne-inducing
agents on rabbit ears (14). TCDD proved most toxic; the characteristic
changes were achieved with concentrations of 0.01 to 0.002%. Higher con-
centrations caused liver necrosis and death. Single oral administration
to rabbits of 0.05 to 0.1 mg/kg body weight caused death in one to two
weeks. Autopsy revealed necrosis and fatty degeneration of the liver.
Pure 2,4,5-trichlorophenol and pentachlorophenol did not cause chloracne.
TCDD formation from sodium trichlorophenolate was established and separa-
tion from the byproducts was accomplished.
A study and review of employee health, in a plant producing 2,4,5-T
and 2,4-D revealed chloracne in 13/73 male workers believed caused by
chlorinated dioxins (15). These chloracne conditions were correlated in
severity with the presence of scarring, hyperpigmentation, hirsutism,
and complaints of eye irritation but were not correlated with distinct
occupations within plants manufacturing 2,4,5-T, duration of employment,
or coproporphyrin excretion (16).
-12
As little as 4.66 x 10 mole (1.5 ng) of TCDD per chicken egg
induced hepatic delta-aminolevulinic acid synthetase (ALAS) activity in
the chick embryo (17); 1.5 ng caused doubling of ALAS activity and 1.55
x 10 ^ moles/egg (0.5 yg) caused a 35-fold stimulation of enzyme activity
(18). Enzyme induction is dose-related and prolonged in time, 70 percent
of the maximum induced activity was present 5 days after a single 150 ng
dose. A single dose of TCDD stimulates hepatic aryl hydrocarbon hydroxlase
(AHH) and cytochrome P-450 for 35 days and more in the rat. AHH activity
was induced in chick embryo liver. TCDD was linked to an outbreak of
porphyria cutanea tarda among workers in plants were 2,4,5-T was synthesized.
The chemically inert parent compound was suggested as not being the toxic
moiety, but that a highly reactive intermediate causes cell damage. Further-
more, at least three of the 2,3,7 and 8 positions on the ring must be
occupied to induce ALAS.
Induction of ALAS synthetase by TCDD is the first specific bio-
chemical action identified for this toxic compound. The relationship
between enzyme induction and the delayed hepatic necrosis presumably
responsible for the lethality of TCDD is not known.
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To follow the biologic disposition, TCDD was administered to male
rats in single oral dose of 50 yg/kg (19). The primary excretion route
was via the feces and the highest tissue concentration was in the liver.
Gross and microscopic examinations were performed on rats, guinea
pigs, and mice treated with TCDD (20). A spectrum of dose ranges and
schedules was used. Lymphoid organs (thymus, spleen, and lymph nodes)
were affected consistently. Thymus atrophy (dose realted decrease in
weight) was found to be a sensitive index of TCDD exposure. The most
severe hepatic effects were seen in rats that received .a lethal dose
of TCDD. The magnitude of the degenerative and necrotic liver changes
were diminished in guinea pigs and mice.
' Albino rats were grouped and treated with single oral doses of
either 0, 5, 25, 50, or 100 yg/kg TCDD in an acetone/corn oil mixture
(21). Animals that eventaully died continued to lose weight until death
while survivors exhibited a depressed weight gain. Ruffled hair coat,
hunched posture, inactivity, and jaundice were the overt signs seen in
the high dose group. Daily oral administration of 10 yg/kg caused death
in 15/16 rats with a mean time of 21.8 days. Death resulted in 9/10 female
guinea pigs after receiving an oral dose of 3 yg/kg—mean equaled 18.1
days. A single oral dose of 1, 10, or 50 yg/kg to adult mice had no effect
on appearance or body weight.
Ultrastructure changes in rat liver microsomes and mitochondria
were examined at various intervals from one to thirty days following a
single TCDD injection of 0, 5, or 25 mg/kg (22). Observed changes included:
proliferation of smooth endoplasmic reticulum (SER), mild increase in rough
endoplasmic reticulum (RER), and moderate swelling of mitochondria; at
seven days, large aggregates of SER, massive amounts of RER, and small
numbers of moderately swollen mitochondria were seen from the 25 mg/kg
dosed rats.
The persistent toxic effect of TCDD in rats is evidenced by death
as long as 15 weeks after a single oral dose (23). Alterations in liver
constitution (microsomes and cytochrome P-450) and drug metabolism (zoxa-
zolamine and hexobarbital), however, occurred within 24 hours after dosing.
From these sketchy observations it is clear that these compounds are
potentially hazardous to human health and should be avoided. TCDD's
culpability as a potential and real health hazard to man and animals is
without question. Those of you working with these compounds or contemplat-
ing such research should follow rigid laboratory safety procedures.
Mutagenicity
TCDD is reported mutagenic in two bacterial assay systems—
Escherichia coli SD-4 reversion to streptomycin independency and
Salmonella typhimurium TA 1532 reversion to histidine prototrophy (24).
The concentration of TCDD was 2 yg/ml. TCDD was weak in its prophage-
inducing activity in E. coli K-39 (lambda) cells and did not induce
mutations above the spontaneous level in S. typhimurium TA 1530. These
results indicate that an acridine-like intercalation with DNA may have
caused these genetic effects.
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Inhibition of mitosis and development of cytological abnormalities
observed in dividing endosperm cells of the African blood lily were
believed caused by TCDD, a contaminant of 2,4,5-T rather than the herb-
icide itself (25). In contrast to the no effect of 2,4,5-T, dramatic
inhibition of mitosis was observed in cells subjected to 0.2 to 1.0 pg/1
dioxin, 0.2 yg/1 dioxin plus 10~4M 2,4,5-T or 10-% 2,4,5-T containing
dioxin as a contaminant. These preparations also induced formation of
dicentric bridges and chromatin fusion with formation of multinuclei or
a single large nucleus.
2,4,5-T containing dioxin contaminants less than 0.1 ppm was evaluated
in a wild-type Drosophilia population, Canton-S 109 (26). Adult flies were
exposed to 250 ppm 2,4,5-T in their food-!within or later than 24 hours of
eclosion. Results indicated that this 2,4,5-T formulation effected early
oogenesis and caused chromosome disturbances which could result in sterility.
Carcinogen!city
Evidence implicating the dioxins or furans as carcinomimetics or
carcinogenic agents is presently lacking. The long-term or chronic studies
necessary to prove or disprove the potential carcinogenic activity of these
structurally-related compounds have not as yet been accomplished.
TCDD administered to rats caused induction of zoxazolamine hydroxylase,
marked reduction in phenobarbital sedative effect, and a decrease in hepatic
arginase (27). Dioxin therefore, according to these authors, has pronounced
inhibitory effect on the enzymatic systems as do the carcinogens benzo(a)-
pyrene and p-dimethylaminoazobenzene.
Others, however, disagree with that correlation (28). Although such
effects as described on the carcinomimetic activity of TCDD (27), the
relationship is by no means exclusive. For example, stimulation of hy-
droxlating enzymes can be induced by many compounds, including BHT, which
is not carcinogenic in long-term feeding studies (28).
Long-term studies are actively being pursued, however, or are being
planned for the near future. For example, King and Shefner (29) fed a
diet containing 2,7-dichloro- and octachloro-dibenzo-p-dioxin at dietary
levels of 1% and 0.5% to mice for 90 weeks and rats for 110 weeks. Results
are not in yet on this study. These chlorinated dibenzodioxins were also
dissolved in acetone and applied to the backs of mice three times a week
to assess their activity as complete carcinogens and/or promoting agents.
Octachlorodioxin caused skin tumor formation in only one female mouse.
No other dioxin produced papillomas.
Tera t og eni c i t y
In the late 1960's the Bionetics Research Laboratory reported to
the National Cancer Institute that 2,4,5-T was teratogenic to mice.
These results stimulated much activity in the scientific and regulatory
communities; a short time later however, the 2,4,5-T sample used in that
study was found to contain about 30 ppm TCDD. It was this later revelation
that caused the multi-investigator plunge into the chlorinated dibenzo-
dioxin and chlorinated dibenzofuran research area—evidenced by the increased
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number of research investigations on chlorinated dibenzodioxins.
TCDD was then studied to discern if this 2,4,5~T impurity could
account for fetal abnormalities in test animals. Rats received 0, 0.03,
0.125, 0.5, 2.0, and 8.0 yg TCDD/kg body weight/day on days 6 to 15 of
gestation (30, 31). No effect was observed on the fetus or mother at the
0.03 y/kg dose level. However, at the 0.125 level and up fetal mortality,
early and late resorptions, and fetal intestinal hemorrhage occurred. The
effects became more pronounced with the increase in dose. Maternal toxicity
was noted beginning at the 0.5 level which also increased with the dose.
At the 0.5 level, the number of fetuses were reduced, and the number of
resorptions and fetal deaths was increased. Histological examination of
fetuses revealed brain and intestinal hemorrhages and edema of subcutaneous
tissue (32). It was suggested that the teratogenic effects of 2,4,5-T
observed in the other studies may have been due to this contaminant (30
ppm) (33).
Commercial-grade 2,4,5-T containing 0.5 ppm TCDD did not induce a
teratogenic response when administered orally to rats on days 6 to 15
of gestation at a dosage level of 50 mg/kg/day (34).
Results from teratogenic studies with rats treated with 2,4,5-T,
2,4-D, and several derivatives were either negative or inconclusive (35).
The TCDD levels in these preparations were not known in all cases but
most were suspected to be less than 0.5 ppm.
Treatment of rabbits and rats with 2,4,5-T containing 0.5 ppm TCDD
did not cause any teratogenic or embryotoxic effects (36). Rats received
1, 3, 6, 12, or 24 meg 2,4,5-T/kg on days 6 to 15 of pregnancy. Rabbits
received 0, 10, 20, or 40 mg 2,4,5-T/kg on days 6 to 18 of pregnancy.
2,4,5-T containing less than 0.5 mg/kg TCDD induced fetopathy and
skeletal anomalies in progeny from female rats treated with a single daily
oral dose of 100 to 150 mg/kg on gestation days 6 to 15 (37). Number of
conceptions and numbers of viable and dead fetuses per litter gave no
indication that in utero treatment of off-spring with up to 100 mg/kg
2,4,5-T had impaired fertility.
Conversely, 2,4,5-T having a TCDD content of less than 0.1 ppm
caused embryotoxic effects and a significant increase in the incidence
of cleft palate in mice (38). Concentrations used in the study ranged
from 35 to 130 mg/kg 2,4,5-T: dioxin which were given orally on days
6-15 of pregnancy. The teratogenic no effect level of 2,4,5-T: dioxin
was found to be 20 mg/kg.
Interspecies differences are evident for the mouse and rat studies.
Other species have been used to further elucidate the teratogenic nature
of these contaminants.
2,4,5-T commercial samples given on days 6 to 10 of organogenesis
were feticidal and teratogenic in the golden Syrian hamster (39). The
incidence of the observed effects increased with an increasing content of
TCDD. The dioxin impurity caused edema and hemorrhages in newborn animals.
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Embryonic deaths have been recorded in the laboratory among birds
treated with PCBs (40). These deaths have been attributed to chlorinated
dibenzofuran contaminants. Also, birth defects in wild populations of
birds and sea lions are believed caused by chlorinated dibenzodioxins.
High resolution mass spectrometry was used to examine samples prepared
from aborted sea lions and dead embryos of the herring gull and osprey.
Technical grade 2,4,5-T which contained less than 0.05 ppm TCDD
was administered orally to forty pregnant Rhesus monkeys daily from day
22 through day 38 of gestation (41). Dose levels used in the experiment
were 0.05, 1.0, and 10.0 mg/kg. Hematology, clinical chemistry, and
urinalysis data were recorded for all females before and at various times
following treatment until parturition; no toxicity was observed. Examina-
tion of live born infants revealed no terata.
From the data discussed there is no doubt that these contaminants
are highly toxic and teratogenic. But some investigators express doubts
as to how much of an actual hazard they represent to human health.
Tabular summaries of the adverse biologic effects of dioxin are
shown in Tables I and II.
Environmental Considerations
Because of TCDD's chemical stability and its lipophilic nature, the
possibility exists that TCDD released into the environment could accumulate
in food chains. Much more work needs to be done, however, to determine
the hazardous consequences of environmental contamination from the chlo-
rinated dibenzodioxins and chlorinated dibenzofurans—those established
toxic impurities that accompany other agents into the environs.
TCDD does not leach in soils, does not reside in the economic portion
of plants growing in contaminated soil, degrades to about 50 percent after
one year in soils, and does not result from microbial or chemical condensa-
tion of 2,4,5-trichlorophenol in soil (42).
Both the chlorinated dibenzodioxins and dibenzofurans are unstable
to light in the presence of organic substrates (43). Even if generated
under environmental conditions, light provides a mechanism for rapid
destruction.
TCDD was not detected (<1 ppm) in 3-foot soil core samples in a
sandy area where a total of 947 pounds of 2,4,5-T per acre was applied over
a 3-year period (44). No TCDD residues were found in bald eagle tissue
gathered from locations in 15 states—lower limit of detection was 0.05
ppm.
Most organisms capable of degrading other chlorinated hydrocarbons
showed no ability to metabolize TCDD; a few exhibited a limited degree
of TCDD-metabolizing activity (45). TCDD leached from sand to organic
soil much less than did DDT. Using pesticide coated sand in aquaria
containing various organisms, TCDD had the lowest biologic accumulation
and affinity.
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Table I. DIOXIN TOXICOLOGY
LETHAL DOSE
ACNEGENIC EMBRYOTOXIC TERATOGENIC CEF RANGE
DCDD
TCDD +
HCDD +
OCDD
-
-H-
+
+
g/kg
+ + yg/kg
+ + mg/kg
g/kg
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Table II.
LEVELS OF TCDD GIVING VARIOUS BIOLOGICAL EFFECTS
TCDD TO OBTAIN EFFECT
LETHALITY (PPT BODY WEIGHT)
FEMALE RAT, SINGLE ORAL DOSE LD50 45,000
(OBSERVATIONS TERMINATED AT 44 DAYS)
MALE RAT, SINGLE ORAL DOSE LD50 ..23,000
(OBSERVATIONS TERMINATED AT 44 DAYS)
MALE GUINEA PIG, SINGLE ORAL DOSE LD50 600
(OBSERVATIONS TERMINATED AT 50 DAYS)
MACACA MONKEY, CUMULATIVE DOSE GIVING 5/5 DEATHS (280,000)
(AVERAGE SURVIVAL TIME 445 DAYS)
TERATOGENICITY
CLEFT PALATE IN 50% NMRI MICE, DAILY ORAL DOSE, 5,000
DAYS 6-15
INTESTINAL HEMORRHAGE AND SUBCUTANEOUS EDEMA IN 125-
50% SPRAGUE-DAWLEY RATS, DAILY ORAL DOSE, DAYS 6-15 500
EDEMA AND DEATH IN CHICKEN EMBRYO, SINGLE INJECTION 20
ENZYME INDUCTION
DOUBLING OF S-AMINOLEVULINIC ACID SYNTHETASE IN 30
CHICKEN EMBRYO, SINGLE INJECTION
MITOTIC ARREST
LILY ENDOSPERM, AMBIENT CONCENTRATION <200
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Guppies and coho salmon fingerlings were exposed to dioxin con-
centrations ranging from 0.056 ppt to more than 0.2 ppb for 24, 48, and
96 hours (46). The initial concentration was found to be more important
in causing death than duration of exposure. The coho salmon fingerlings
threshold response level for all exposure periods was between 0.056 and
0.56 ppt dioxin. Mosquito larvae, oligochaete worms, and pulminator
snails were maintained in water initially dosed with 0.2 ppb dioxin.
These aquatic organisms were less sensitive than fish.
Various fish and shellfish collected in 1970 from the Dong Nai and
Saigon Rivers and along the Can Guo Coast contained dioxin; catfish had
the highest concentration (47). Dioxin concentrations ranged from 18 to
814 ppt; Dong Nai River carp averaged 540 ppt dioxin (48).
These surprisingly high levels of dioxin in fish caught in Vietnam
have placed doubt on earlier governmental considerations about dioxin
residues. It had been previously thought that the dioxin content of
2,4,5-T was so low that there was little opportunity of residues appearing.
In addition to 2,4,5-T and silvex, any compound using trichlorophenol in-
termediates may be suspect.
Juvenile Atlantic salmon were fed dry fish food contaminated with
a mixture of 2.7 Mg/g di-, 5,7 tri-, 2.8 tetra-, and 9.1 octa-chlorodi-
benzofuran (49). Median mortality was 120 ± 30 days. Only octa-chlo-
rodibenzofuran was found in tissues of dead fish (0.03 yg/g in muscle
and 0.2 in the gut). Fish surviving 140 days feeding contained correspond-
ing values of 0.01 and 0.02 Mg/g.
These preliminary data tend to implicate these chemicals as potential
health hazards. In fact, the General Accounting Office (GAO) urged the
Environmental Protection Agency (EPA) to implement full-scale recall
procedures for suspended pesticides and raised questions on the dioxin
content of trichlorophenol herbicides (50). GAO stated that because
silvex, ronnel, erbon, and hexachlorophene can contain the same level of
dioxin as 2,4,5-T and because a safe level of dioxin has not been determined,
we believe EPA should establish a standard for dioxin content and prohibit
the use of all pesticides containing dioxin in excess of the established
standard.
The U. S. Environmental Protection Agency's Committee on 2,4,5-T
recommended that the registration of 2,4,5-T be restored with the follow-
ing exceptions (51): (a) a permissible residue of not more than 0.1 ppm
2,4,5-T on edible parts of food products and in water for human consump-
tion and (b) a limit of 0.5 ppm of contamination with TCDD, except that
in all formulations to be used around the home and recreational areas,
TCDD contamination chould be limited to 0.1 ppm.
Meanwhile, the U.S. Air Force has a surplus stockpile of about
2.4 million gallons of Agent Orange (50 percent 2,4,5-T and 50 percent
2,4-D); some of these mixtures contain as much as 28 times the maximum
acceptable safety limit of dioxin (52). Presently, dioxin concentrations
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for new herbicides are 0.1 ppm and for stocks already manufactured 0.5
ppm.
For the chlorinated dibenzodioxins and chlorinated dibenzofurans—
as with many other substances—one should not be content to do acute and
chronic toxicology studies on the parent compounds. Toxicity testing
must not be confined to the test agent per se, but should be extended to
its chemical and metabolic derivatives, its pyrolytic and degradation
products, and its contaminants and reaction products, especially when
various derivatives or degradation products are of toxicologic or environ-
mental consequence (53).
Literature Survey,
Up to 1969 the literature on dioxins was sparse, evidenced by only
30 publications from the earliest in 1934 to 1969. The seventies present
a different picture— 25 references in 1970, 65 in 1971, 70 in 1972, and
60 in the first half of 1973 (see Table III). The September 1973 issue
of Environmental Health Perspectives contains the proceedings from the
April 2-3, 1973 conference on Chlorinated Dibenzodioxins and Chlorinated
Dibenzofurans. The 35 papers include methods of detection; chemical
processes involved in formation, bioaccumulation, and degradation; toxicity;
pharmacologic effects;, and distribution and fate in the environment. This
compilation gathers into one publication most of the scientific knowledge
on dioxins.
The earlier literature dealt primarily with synthesis, chemistry,
and use; whereas the recent reports center on analytical detection, en-
vironmental contamination and movement, and toxicology . Table III also
illustrates the number of reports categorized under various key terms.
Sources and time periods searched for this literature compilation
are summarized in Table IV; as can be seen from the length of this list,
one must examine many literature sources to adequately blanket all the
location possibilities.
Most of the nomenclature or terms searched are listed in Table V.
Each particular author, journal, secondary abstracting service, and news
copy unfortunately utilize separate and distinct terminology when reporting
on the chlorinated dibenzodioxins and dibenzofurans. As can be seen from
Table V, it is vitally important, therefore, to become thoroughly familiar
with the sources being utilized before mounting a massive effort to collate
all that is written or reported about a particular compound, series, or
class of, compounds, or subject. The magnitude of the search effort for
this report is self-evident when noting all the necessary terms used.
As with most scientific literature reporting, names change over the
years—note that the parent names for dibenzo-p-dioxin were diphenylene
dioxide and phendioxin whereas for dibenzofuran the name biphenylene oxide
was used.
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Table III.
DIOXINS AND FURANS
LITERATURE TRENDS AND CATEGORIES
1934-1969 1970 1971 1972 1973
30 25 65 70 65
TOXICOLOGY ANALYSIS TERATOGENICITY
115 106 57
ENVIRONMENT MUTAGENECITY CARCINOGENICITY
32 7 4
189
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Table IV.
Sources and Time Periods Searched
Multidisciplinary Information Resources
Bibliography of Agriculture
Biological Abstracts
Biological and Agricultural Index
Bioresearch Index
Chemical Abstracts
Chemical-Biological Activities
Food Chemical News
Health Aspects of Pesticides Abstract
Bulletin
Vol.
Vol.,
Vol.
Vol.
Vol.
1965
Vol.
Vol.
Health Effects of Environmental Pollutants Vol.
Index Medicus Vol.
Pesticide Chemical News Vol.
Science Citation Index Vol.
Teratology Lookout Vol.
Toxicology Bibliography Vol.
Specialized Information Centers and Libraries
Environmental Mutagen Information
Center (EMIC)
Environmental Information System Office
(EISO)
Oak Ridge National Laboratory (ORNL)
Toxicology Information Response Center (TIRC)
21:1957 to Vol. 37(1):1973
31:1957 to Vol. 55(8):1973
19:1964 to Vol. 24:1970
1:1965 to Vol. 9(4):1973
1:1907 to Vol. 78(20):1973
to 1971
13(42):1972 to Vol. 15(9):1973
1:1966 to Vol. 6(4):1973
1:1972 to Vol. 2(3):1973
60:1956 to Vol. 14(5):1973
1(1-24):1973
1:1961 to Vol. 6:1965; 1966 to
1972
3:1972
1:1968 to Vol. 6(1):1973
190
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Table IV (Continued)
On-Line Computer Data Bases
MEDLine
TOXLine
Journals
Ambio
Archives of Environmental Health
Bulletin of Environmental Contamination
and Toxicology
Clinical Toxicology
Environment
Federation Proceedings
Food and Cosmetic Toxicology
Journal of Agriculture and Food Chemistry
Journal of the Association of Official
Analytical Chemists
Journal of Chromatography
Mutation Research
Residue Reviews
Science
Teratology
Toxicology and Applied Pharmacology
Vol. 1:1972 to Vol. 2:1973
Vol. 7:1963 to Vol. 26(2):1973
Vol. 1:1966 to Vol. 7:1973
Vol. 1(1):1971 to Vol. 6(1):1973
Vol. 12:1970 to Vol. 15(3):1973
Vol. 16(1):1957 to Vol. 32(4):1973
Vol. 1:1969 to Vol. 10(6):1972
Vol. 1:1953 to Vol. 21(2):1973
Vol. 40:1957 to Vol. 56:1973
Vol. 1:1958 to Vol. 73:1973
Vol. 1:1964 to Vol. 10(6):1972
Vol. 1:1962 to Vol. 41:1972
Vol. 157:1967 to Vol. 179:1972
Vol. 1:1968 to Vol. 6(2):1972
Vol. 1:1959 to Vol. 24(3):1973
191
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TaBle. V,
TERMS USED FOR LITERATURE SEARCHES
DIBENZO-P-DIOXIN
(DIPHENYLENE DIOXIDE)
(PHENDIOXIN)
2,3-DICHLORO-
2,7-DICHLORO-
2,3,7-TRICHLORO-
1,2,3,4-TETRACHLORO-
1,3,6,8-TETRACHLORO-
2,3,6,7-TETRACHLORO-
2,3,7,8-TETRACHLORO-
PENTACHLORO-
HEXACHLORO-
1,2,3,7,8,9-HEXACHLORO-
HEPTACHLORO-
1,2,3,4,6,7,8,9-OCTACHLORO-
BENZO-
BIPHENYL-
CHLORINATED
CHLORINATED DIBENZODIOXIN(S)
CHLORINATED DIBENZO-P-DIOXIN(S)
DI BENZO-
DIBENZO-
CHLORODIBENZO DIOXINS(S)
CHLORODIBENZO-P-DIOXIN(S)
DIBENZODIOXIN(S)
DIBENZO-P-DIOXIN(S)
DIBENZO-P-DIOXIN, TETRACHLORO-
DIBENZO-P-DIOXIN, OTHER CHLORO-
DIOXIN
HALOGENATED DIBENZO DIOXIN(S)
HALOGENATED DIBENZO-P-DIOXIN(S)
OCDD
POLYCHLORINATED DIBENZOBIOXIN
POLYCHLORODIBENZOBIOXIN
TCDD
DIBENZOFURAN
(BIPHENYLENE OXIDE)
3-CHLORO-
2,4-DICHLORO-
1,2,4-TRICHLORO-
TETRACHLORO-
1,2,3,4-TETRACHLORO-
PENTACHLORO-
BENZO-
BIPHEYL-
CHLORINATED
CHLORINATED DIBENZOFURAN(S)
CHLORODIBENZO-
DI BENZO-
DIBENZO-
DIBENZOFURAN
FURAN
HALOGENATED DIBENZOFURAN(S)
POLYCHLORINATED DIBENZOFURAN(S)
192
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Summary
The chlorinated dibenzodioxins and chlorinated dlbenzofurans are
toxic and potentially hazardous.
These chlorinated compounds are toxicogenic, mutagenic, and ter-
atogenic. The evidence on carcinogenicity is incomplete.
The environmental hazards are real and should, be eliminated; for
the most part, however, this environmental insult has been reduced sub-
stantially.
The annotated and keyworded literature collection is an ongoing
project; updated results will be published periodically.
References
1. Romberger, J. A. Meristems, growth, and development in woody plants:
An analytical review of anatomical, physiological, and morphogenic
aspects. U.S. Government Printing Office Technical Bulletin No. 1293,
October 1963.
2. Huff, J. E. and J. S. Wassom. Chlorinated dibenzodioxins and dibenzo-
furans: An annotated literature collection. Environ. Health Perspect.
No. 5:283-312, September 1973.
3. Huff, J. E. and J. S. Wassom. Health hazards from chemical impurities:
Chlorinated dibenzodioxins and chlorinated dibenzofurans. In press,
Int. J. Environ. Studies.
4. Dinger, C. Y., J. E. Huff and J. S. Wassom. A treatise on chlorinated
dibenzo-p-dioxins and chlorinated dibenzofurans. In preparation.
5. Baughman, R. and M. Meselson. An analytical method for detecting TCDD
(Dioxin): Levels of TCDD in samples from Vietnam. Environ. Health
Perspect. No. 5:27-35, September 1973.
6. Crummett, W. B. and R. H. Stehl. Determination of chlorinated dibenzo-
p-dioxins and dibenzofurans in various materials. Environ. Health
Perspect. No. 5:15-25, September 1973.
7. Johnson, R. L., P. J. Gehring, R. J. Kociba and B. A. Schwetz.
Chlorinated dibenzodioxins and pentachlorophenol. Environ. Health
Perspect. No. 5:171-175, September 1973.
8. Jensen, S. and L. Renberg. Contaminants in pentachlorophenol:
chlorinated dioxins and predioxins (Chlorinated Hydroxy-diphenyl
ethers). Ambio 1(2):62-65, 1972.
9. Langer, H. G., T. P. Brady, and P. R. Briggs. Formation of dibenzo-
dioxins and other condensation products from chlorinated phenols and
derivatives. Environ. Health Perspect. No. 5:3-7, September 1973.
193
-------
10. Jensen, S. and L. Renberg. Various chlorinated dimers present in
several technical chlorophenols, used as fungicides. Environ.
Health Perspect. No. 5:37-39, September 1973.
11. Milnes, M. H. Formation of 2,3,7,8-Tetrachlorodibenzodioxin by
thermal degradation of Sodium 2,4,5-Trichlorophenate. Nature
(London) 232(5310):395-396, 1971.
12. Cantrell, J. -S., N. C. Webb and A. J. Mabis. Identification and
crystal structure of a hydropericardium-producing factor: 1,2,3,
7,8,9-Hexachlorodibenzo-p-dioxin. Acta Crystallogr. Sect. B. 25
(1):150-156, 1969.
13. Kimmig, J. and. K. H. Schulz. Occupational acne (so-called chlor-
acne) due to chlorinated aromatic cyclic ethers. Dermatologia
115 (4):540-546, 1957.
14. Kimmig, J. and K. H. Schulz. Chlorinated aromatic cyclic ethers
as a cause of the so-called chloracne. Naturwissenschaften 44:
337-338, 1957.
15. Poland, A. P., D. Smith, G. Metter and P. Possick. A health survey
of workers in a 2,4-D and 2,4,5-T plant. Arch. Environ. Health
23:316-327, 1971.
16. Anonymous. Working with 2,4,5-T. Food Cosmet. Toxicol. 9:908-909,
1971.
17. Poland, A. P. and E. Glover. 2,3,7,8-Tetrachlorodibenzo-p-dioxin:
A potent inducer of 6-Aminolevulinic Acid Synthetase. Science
179(4072):467-477, February 2, 1973.
18. Poland, A. P. and E. Glover. Studies on the mechanism of toxicity
of the chlorinated dibenzo-p-dioxins. Environ. Health Perspect.
No. 5:245-251, September 1973.
19. Piper, W. N. and J. Q. Rose. The excretion and tissue distribution
of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the rat. Abstract No. 88,
Division Pesticide Chemistry, 162nd National Meeting of the American
Chemical Society, Washington, D.C., September 12-17, 1971.
20. Gupta, B. N., J. G. Vos, J. A. Moore, J. G. Zinkl and B. C. Cullock
Pathologic effects of 2,3,7,8-Tetrachloro-p-dioxin in laboratory
animals. Environ. Health Perspect. No. 5:125-140, September 1973.
21. Harris, M. W., J. A. Moore, J. G. Vos, and B. N. Gupta. General
Biologic effects of TCDD in laboratory animals. Environ. Health
Perspect. No. 5:101-109, September 1971.
22. Fowler, B. A., G. W. Lucier, H. W. Brown and 0. S. McDaniel. Ultra-
structural changes in rat liver cells following a single oral dose
of TCDD. Environ. Health Perspect. No. 5:141-148, September.
194
-------
23. Greig, J. B. Biochemical toxicity of TCDD in rat liver. NIEHS
Conference on Chlorinated Dibenzodioxins and Chlorinated Dibenzo-
furans, National Institute of Environmental Health Sciences, Re-
search Triangle Park, NC, April 2-3, 1973.
24. Hussain, S., L. Ehrenberg, G. Lofroth and T. Gejvall. Mutagenic
effects of TCDD on bacterial systems. Ambio 1(1):32-33, 1972.
25. Jackson, W. T. Regulation of mitosis. III. Cytologic effects
of 2,4,5-Trichlorophenoxyacetic Acid and of Dioxin Contaminants
in 2,4,5-T formulations. J. Cell Sci. 10:15-25, 1972.
26. Davring, L. and M. Sunner. Cytogenie effects of 2,4,5-Trichloro-
phenoxyacetic acid on oogenesis and early embryogenesis in Drosophila
melanogaster. Hereditas 68(1):115-122, 1971.
27. Buu Hoi, N. P., D. P. Hien, G. Saint-Ruf and J. Servoin-Sidoine.
Canceromimetic properties of tetrachloro-2,3,7,8-Dibenzo-p-dioxin
(dioxin). C. R. Acad. Sci. Paris, Ser. D 272(10):1447-1450, 1971.
28. Anonymous. Tetrachlorodibenzodioxin—intimations of carcinogenicity?
Food Cosmet. Toxicol. 9:909, 1971.
29. King, M. E. and A. M. Shefner. Carcinogenesis bioassay of chlo-
rinated dibenzodioxins and related chemicals. NIEHS Conference
on chlorinated dibenzodioxins and chlorinated dibenzofurans, National
Institute of Environmental Health Sciences, Research Triangle Park,
NC, April 2-3, 1973.
30. Sparschu, G. L., F. L. Dunn and V. K. Rowe. Teratogenic study of
2,3,7,8-Tetrachlorodibenzo-p-dioxin in the rat. Toxicol. Appl.
Pharmacol. 17(1):317-318, 1970.
31. Sparschu, G. L., F. L. Dunn and V. K. Rowe. Study of the terato-
genicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the rat. Ter-
atology 4:247 (abstract), 1971.
32. Khera, K. S. and J. A. Ruddick. Perinatal effects of dibenzodioxins
in Wistar rats. Abstract No. 87, Division Pesticide Chemistry,
162nd National Meeting of the American Chemical Society, Washington,
D.C., Spetember 12-17, 1971.
33. Sparschu, G. L., F. L. Dunn and V. K. Rowe. Study of the terato-
genicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the rat. Food
Cosmet. Toxicol. 9(3):405-412, 1971.
34. Sparschu, G. L., F. L. Dunn, R. W. Lisowe and V. K. Rowe. Study of
the effects of high levels of 2,4,5-Trichlorophenoxyacetic acid on
foetal development in the rat. Food Cosmet. Toxicol. 9(4):527-530,
1971.
195
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35. Khera, K. S., B. L. Huston, and W. P. McKinley. Pre- and postnatal
studies on 2,4,5-T, 2,4-D, and derivatives in Wistar rats. Toxicol.
Appl. Pharmacol. 19:369-370, 1971.
36. Emerson, J. L., D. J. Thompson, R. J. Strebing, C. G. Gerbig and V.
G. Robinson. Teratogenic studies of 2,4,5-Trichlorophenoxyacetic
acid in the rat and rabbit. Food Cosmet. Toxicol• 9(3):395-404,
1971.
37. Khera, K. S. and W. P. McKinley. Pre- and postnatal studies on
2,4,5-Trichlorophenoxyacetic acid and their derivatives in rats.
Toxicol. Appl. Pharmacol. 22:14-28, 1972.
38. Roll, R. Teratogenic effect of 2,4,5-T (2,4,5-Trichlorophenoxyacetic
Acid) in mice. Food Cosmet. Toxicol. 9(5):671-676, 1971.
(
39. Collins, T. F. X., C. H. Williams, and G. C. Gray. Teratogenic
studies with 2,4,5-T and 2,4-D in the hamster. Bull. Environ.
Contam. Toxicol. 6(6):559-567, 1971.
40. Bowes, G. W., B. R. Simoneit, A. L. Burlingame, B. W. deLappe,
D. B. Peakall and R. W. Risebrough. The search for chlorinated
dibenzofurans and chlorinated dibenzodioxins in wildlife populations
showing elevated levels of embryonic death. Environ. Health Perspect.
No. 5:191-198, September 1973.
41. Dougherty, W. H., F. Coulston and L. Goldberg. Non-teratogenicity
of 2,4,5-Trichlorophenoxyacetic acid in monkeys (Macaca mulatta).
Abstract No. 9, Twelfth Annual Meeting of the Society of Toxicology,
New York, N.Y., March 18-22, 1973.
42. Kearney, P. C., E. A. Woolson, A. R. Isensee, and C. S. Helling.
Tetrachlorodibenzodioxin in the environment: Sources, fate, and
decontamination. Environ. Health Perspect. No. 5:273-277, September
1973.
43. Crosby, D. G., K. W. Moilaners and A. S. Wong. Environmental
Generation and degradation of dibenzodioxins and dibenzofurans.
Environ. Health Perspect. No. 5:259-266, September, 1973.
44. Anonymous. TCDD residue disappears. Down to Earth 28(4):18,
Spring 1973.
45. Matsumura, F. and H. J. Benezet. Studies on the Bioaccumulation
and microbial degradation of 2,3,7,8-Tetrachlorodibenxo-p-dioxin.
Environ. Health Perspect. No. 5:253-258, September 1973.
46. Miller, R. A., L. A. Norris and C. L. Hawkes. Acute and Chronic
toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (Dioxin) in aquatic
organisms. NIEHS Conference on chlorinated dibenzo-dioxins and
chlorinated dibenzofurans, National Institute of Environmental
Health Sciences, Research Triangle Park, NC, April 2-3, 1973.
196
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47. Meselson, S. Vietnam dioxin contamination. Center for Short-
lived Phenomena, Event 51-73, No. 1611, Smithsonian Institute,
Cambridge, Ma., April 19, 1973.
48. Meselson, S. Vietnam dioxin contamination. Center for Short-
lived Phenomena, Event 51-73, No. 1627, Smithsonian Institute,
Cambridge, Ma., May 8, 1973.
49. Zitko, V., D. J. Wildish, 0. Hutzlnger and P. M. K. Choi. Acute
and chronic oral toxicity of chlorinated dibenzofurans to salmonid
fishes. Environ. Health Perspect. No. 5:187-189, September, 1973.
50. Anonymous. Recall program, another look at Trichlorophenols urged
on EPA. Pest. Chem. News 1(23):3-5, May 9, 1973.
51. Anonymous, Correction, Pest. Chem. News 1(22):2, May 2, 1973.
52. Shapley, D. Herbicides: Agent Orange stockpile may go to the
South Americans. Science 180(4081):43-45, April 6, 1973.
53. Epstein, S. S. Environmental Pathology. A Review. Amer. J.
Pathol. 66(2):352-373, 1972.
197
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198
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MYCOTOXINS
D. W. Lawellin
Colorado State University
Mycotoxins have become an important consideration in both human
and veterinary medicine. Fungi, involved in the elaboration of the toxins,
normally inhabit or have access to stored grains, decaying pasture forage
and other food stuffs which are consumed by humans or domestic animals.
The conditions for toxin formation vary for each fungus and each
toxin formed may give a different clinical response for each species of
animal involved. The possibility of secondary intoxication from the
ingestion of intoxicated animals also exists.
Certain toxins produced by members of the Aspergillus spp., Chaetomium
spp., Fusarium spp., Penicillium spp., Pithanyces spp., Rhizoctonia spp.,
Stachbotrys spp., Trichoderma spp., and Trichothecium spp. will be dis-
cussed as to their involvement in human and domestic animal toxicoses.
Future condiserations for needed research in the areas of specific
mycotoxin identification, prevention and treatment will also be suggested.
199
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200
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NATIONAL PESTICIDE MONITORING PROGRAM
IN THE ENVIRONMENTAL PROTECTION AGENCY
G. Bruce Wiersma
\
Environmental Protection Agency
The Environmental Protection Agency, under FIFRA amended, is
responsible for implementing and designing the National Pesticide
Monitoring Plan. This Plan is currently in the developmental stage,
but an existing National Pesticide'Monitoring Network has been
operated for several years and coordinated by the Monitoring Panel
of the Federal Working Group on Pest Management. The Agencies in-
volved on this Panel are the Food and Drug Administration which is
responsible for monitoring food.and feed, the Department of Agri-
culture which is responsible for monitoring red meats, the Bureau
of Sport Fisheries and Wildlife which is responsible for monitoring
freshwater fish and various birds. All the rest of the environmental
components that are monitored are the responsibility of the Environ-
mental Protection Agency. These include air, fresh water, estuaries,
soil, crops associated with the soil, human tissue and a pilot monitor-
ing program in the oceans.
I would like to explain in a little detail each of the National
Pesticide Monitoring Systems for which the Environmental Protection
Agency is responsible.
All monitoring systems operate through three basic phases:
1. There is the field collection of the samples
2. The laboratory chemical analyses of these samples
3. The analysis and interpretation of the data
I will use the National Soils Monitoring Program to describe
each of these three phases and then only describe the field phase
for the remaining system since that is the only phase that substantially
changes.
The National Soils Monitoring Program
, The National Soils Monitoring Program collects soil samples in
cropland, noncropland and urban areas. To date, 45 states have been
sampled for cropland soil, 27 states have been sampled for noncropland
soil, and 30 standard metropolitan statistical areas have been sampled
in our urban soil monitoring program. Soil samples are collected
using a soil core 3 inches deep by 2 inches in diameter. A sampling
site is usually made up of 10 acres with 50 soil cores collected over
that 10 acres, on a 5 x 10 grid. In the urban areas, this is modified
to use a 50 x 50 foot plot and a 4 x 4 grid.
201
-------
After the soil samples have been collected, they are screened
three times through V mesh screening and a 2-qt. subsample is taken.
Equipment is carefully washed to insure that there is no possibility
of cross-contamination between sampling sites. For each site a map
is made to enable us to go back to that site four years after the
original sampling to take trend data. In addition to collecting a
soil sample, a crop sample is collected on each site, when available.
Also, the following information is obtained from the farmer.
1. The crops grown on the site in addition to those sampled
2. Irrigation used, if any
3. The number of inches of irrigati9n
4. Pesticides used
5. Crop the pesticide was used on
6. Formulation that was applied
7. Pounds of active ingredient applied
8. Method of application
After the sample has been collected in the field, it is sent to
our central receiving laboratory at the Mississippi Test Facility.
Here the samples are analyzed using gas liquid chromatography; however,
in addition to gas liquid chromatography, we have a wide range of
confirmatory insturments available for our use including atomic
absorption spectrophotometry, liquid chromatography and mass spectrometry.
After the chemical analyses have been completed, the data is returned to
the Headquarters in Washington where it Is statistically analyzed using
small mini computers such as the Hewlett Packard, 9100-B and minipulated
and handled by our large computer support programs.
The National Estuarine Monitoring Program
The National Estuarine Monitoring Program has as its objective to
determine pesticide levels in fish in estuaries. Two basic species
of fish are collected, herbivorous fish and a carnivorous fish. They
are sampled twice a year. Only young of the year are attempted to
be collected. Originally, this program sampled crustaceans and shell-
fish, but the design was modified because it was felt that while
the crustaceans were good indicators of the presence of pesticides,
they were not as mobile as fin fish and therefore, we hoped that
fin fish would be a better overall indicator and integrator of the
pesticide pollution in an estuary.
The National Human Tissue Monitoring Program
The National Human Tissue Monitoring Program collects samples
of human adipose tissue to determine the baseline levels of chlori-
nated hydrocarbons. Seventy-five standard metropolitan statistical
areas have been allocated across the United States according to the
distribution of the population of the United States. In each of these
cities, a pathologist is contracted. He is given a quota based upon
the sex, age and race distribution of his particular area and he fills
202
-------
this quota by taking small 10 or 15 gram samples of adipose tissue,
usually at autopsy, and shipping them back to a central receiving
point in Washington. Here the samples are logged and checked against
the design requirements. If they meet the design requirements, the
pathologist is then paid and the samples are frozen and shipped on
to our support laboratories.
The National Water Monitoring Program
In the June issue of the 1971 Pesticide Monitoring Journal, there
was published an approved National Monitoring Plan for monitoring pest-
icide residues in fresh water. This Plan1 was never implemented due
to the inability to collect the necessary resources. However, thanks
to contacts on the Monitoring Panel, a cooperative program was worked
out between the U.S. Geological Survey and the Environmental Protection
Agency. The Geological Survey would collect the water samples and
the Environmental Protection Agency would do the chemical analyses.
Water samples were collected at 162 sampling stations in 17 major
river basins. Originally the design called for four water samples
to be collected a year, and two sediment samples; however, because
of a lack of funds associated with this program, in the first year
the sampling is only being collected in the fall for water and sediment.
The National Air Monitoring Program
When one considers pollution in air, pesticides do not usually
come to mind as being the primary pollutant; however, in certain
situations such as during the height of the spray season in the Wenatchee
Valley, pesticidesin air can be a serious problem. In addition, the
National Pesticide Monitoring Program for Pesticides in Air which
was in existence from 1970 torough 1972, detected, albeit at low
levels, a large number of pesticide residues.
The sampling device which was used in that program was an impinger
system. Basically, an air stream was bubbled through an ethylene
glycol solution and the pesticides contained in that air stream were
partitioned into the ethylene glycol. Some question has been raised;
however, as to the applicability of using a high volume air sampler.
The advantage of the high volume air sampler over the impinger
sampler is that is is a simpler machine and easier to operate in
the field and it samples a larger volume of air; therefore, the
analytical sensitivity required to support this air sampling device
is much less. This question has not been resolved and the air program
is currently suspended until a satisfactory resolution of this problem
can be achieved.
Ocean Monitoring
The oceans are generally considered an important sink for many
persistent pollutants and to date there has been no systematic
203
-------
monitoring of the oceans for pesticide residues on a continuing
basis. The National Marine Fisheries Service approached us suggesting
that a cooperative program might be established between the National
Marine Fishery Service and the Environmental Protection Agency.
Basically, they would supply ship time, collect the samples at
sea, and the Environmental Protection Agency would do the chemical
analyses and supply other monitoring support. Knowing that the
Environmental Protection Agency could not afford to pay the cost
of ship time, we took advantage of this situation. Fish samples
will be collected on five or six ocean cruises on both coasts of the
United States. This program is a pilot program. The effectiveness
will be evaluated and decisions will be made as to whether it should
be continued.
This covers the on-going National Monitoring Programs for which
the Environmental Protection Agency is responsible. These programs
are the direct responsibility of the Ecological Monitoring Branch
of the Technical Services Division, Office of Pesticides Programs.
While these programs are currently in effect they are also continuously
in a state of flux, changing design as necessary to meet the regulatory
information requirements of the Agency. Therefore, if additonal
information is required a request could be sent to the attention of
the author, appropriate branch, division office, and agency located
at Waterside Mall, Washington, D.C. 20460.
204
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EFFECTS OF LEAD ON MICROCOCCUS LUTEUS CELLS
AND MEMBRANE LIPID COMPONENTS
Thomas G. Tornabene, Larry G. Bennett,
Steven L. Peterson and Harry W. Edwards
Colorado State University
Introduction
Previous work done in our laboratory (24) revealed that the via-
bility of bacterial cells exposed to a medium containing lead for one
complete growth cycle (48 h) was not seriously affected; that virtually
all of the lead taken up by the cells was immobilized in the cellular
envelope; and, that mlcrobial systems are capable of abstracting sub-
stantial quantities of lead from media containing insoluble inorganic
lead-salt precipitates. This paper deals with the effects of longer-
term exposures of the natural human skin bacterium, Micrococeus luteus,
to lead and with experiments to identify membrane lipid compositional
factors associated with lead retention by growing cells. In the latter
experiments, interactions between lead and specific membrane lipids and
lipid mixtures were examined by placing lipid-impregnated disks in contact
with lead-containing solutions. Lead retention by specific membrane lipid
fractions in the disk experiments is compared to lead retention by mem-
branes of whole cells. The results are discussed in terms of composi-
tional factors associated with lead retention in membranes of M. luteus
cells.
Materials and Methods
Culturing conditions
Miaroooccus luteus ATCC 533 (16,25,26) was cultivated at 27 C by
the shake culture method in flasks containing Trypticase Soy broth (BBL)
with and without lead salts. Lead bromide (300 mg) and lead nitrate
(300 mg) were introduced into respective flasks containing 500 ml of
culture medium either by the dialysis procedure previously described (1)
or by directly adding it to the medium. The different exposure methods
appeared to have no observable effects on the cultivations of these cells.
In both procedures the majority of lead salts in the growth medium remained
as insoluble precipitates either on the bottom of the flask or in the
dialysing tubes. Only about 20% of the total lead salt added remained
solubilized or dispersed in the growth medium. The ability of bacteria
to abstract additional lead from water insoluble precipitates has been
previously acknowledged (24). The cells were harvested from the broth
preparations by centrifugation when the cells reached their early station-
ary phase of growth (48 h). The cells were washed twice with a saline
solution previously described (24) and recentrifuged. A 1 ml suspension
of the harvested and washed control (no lead) or lead treated cells (O.D. =
1.0, 580 nm) was used as the inoculum for the respective next cultivation.
This procedure was repeated until significant variations between the control
and lead treated cells were observed. Relative cellular yields were deter-
mined turbidimetrically at 580 nm. Cells were dried by lyophilization and
specific cellular yields determined on a dry weight basis. Each experiment
205
-------
was repeated a minimum of five times.
Lipid extraction and column fractionation
Washed cells from each preparation were immediately suspended in
the saline solution and extracted by the method of Bligh and Dyer (2)
for the removal of total free lipids. Virtually all of the lipids of this
organism are membrane associated. To prevent loss of lipid-lead complexes
that may exist with modified lipid properties, total lipid extracts were
not passed through Sephadex G-25 columns to remove possible uncomplexed
lead salts. Total lipid extract was then transferred to a column con-
taining heat activated silicic acid (Unisil, 325 mesh, weight ratio of
silicic acid to sample 30:1). The lipids were fractionated with the
following eluting solvents: hexane, benzene, chloroform, acetone, and
methanol to remove non-polar lipids, glycolipids, and phospholipids, respec-
tively. About 99% of the phospholipid phosphorus was in the methanol
eluate (for descriptions of lipid compositions of M. luteus cells, see
references 9, 10, 16, 19, 25). Carotenoid pigments were present in all
eluates except hexane.
Analysis of lipid contents
The lipid contents of the control and lead treated cells were
routinely analyzed on silicic acid coated thin-layer plates in solvent
mixtures of (A) diethyl ether, benzene, ethanol, and acetic acid (40:
50:2:0.2, by vol) as the first solvent; hexane and benzene (9:1, by vol)
as the second solvent for the separation of non-polar lipids (5); and
(B) chloroform, acetone, methanol, acetic acid, and water (50:20:10:10:5,
by vol) for separation of polar lipids (13). Components were visualized
by exposure to iodine vapors. Isolated components were scraped from the
plates and the components eluted with a mixture of chloroform, methanol,
and water (10:5:1, by vol). Membrane hydrocarbons were Isolated from the
lipid extracts chromatographically in hexane and identified as described
elsewhere (16,25,26). The acid and alkaline hydrolysates of the lipid
fractions were characterized according to analytical procedures previously
described in detail (23,27).
Examination of cellular subtractions
The isolation of cellular membrane, digested cell wall, and cyto-
plasm of control and lead treated cells was accomplished by lysozyme
digestion according to previously described procedures (24). The mem-
brane preparations were examined by electron microscopy. Carbon-stabilized
collodion coated grids were prepared according to the method described by
Dowell (4). A drop of the membrane suspension in distilled water was
placed on a coated grid and allowed to stand for several minutes. The
liquid was drawn off with filter paper and residual liquid was allowed
to evaporate. A very thin coat of carbon, <50 A, was evaporated over
the unstained, unfixed membrane preparations. Electron microscopic
examinations were performed on a Zeiss EM9A microscope at 60 kv accelerat-
ing voltage at magnifications of 6,000 to 19,000 X, and on an Hitachi
HU-12 equipped for high contrast microscopy at 50 kv at magnifications
of 18,000 and 27,000 X.
206
-------
Preparation of lipid impregnated millipore disks
Total as well as fractionated and purified M. luteus lipids from
the control cultures were further studied for their chemical affinities
for lead ions. The major lipid components of M. luteus are acyclic
.hydrocarbons (16,25,26), pigments (10), aminolipids (9), and phospholipids
(9). The following commercially prepared lipids (Supelco, Inc., Supelco
Park, Pennsylvania) were also included in this study: phosphatidyl glycerol
phosphate, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl
ethanolamine, phoshatidyl serine, tripalmitate, trioleate, tetracosane,
and methylated as well as unmethylated saturated and unsaturated fatty
acids.
Between 100 and 150 mg of total, fractionated, or pure lipids were
applied in chloroform to 47 mm thick fibrous Millipore prefilter disks.
Disks not containing lipids and those containing tetracosane, an n-alkane,
were used as controls. The disks were prepared in duplicate for each
experiment. The disks were taken just to dryness under a stream of nitrogen
followed by drying in vacua over potassium hydroxide for 10-15 min. One
disk was submerged in small petri dish (50 mm) containing 10 ml of either
lead bromide, lead nitrate, or lead acetate (0.1-0.4 mg lead salt per ml
of degassed unbuffered distilled water at a pH between 6 and 7). The
second disk was then added directly on top of the first one, permitting
a liquid layer to remain between them. The disks in the solution were
allowed to stand overnight at room temperature. The pH of the solution
remained constant throughout the experiment. The disks were then removed,
placed on a sintered glass filter, and washed by filtration with 200 ml
of water (greater quantities of water were ineffective in removing addi-
tional lead). The disks were dried as before and inserted into a Millipore
filter holder and washed successively with 200 ml each of chloroform,
acetone, methanol, methanol-water (9:10, by vol) and water. The eluates
were examined for lead and changes in properties of lipid components.
Analytical procedures
Cellular preparations as well as the eluates from the membrane-lipid
containing Millipore disks were analyzed by spectrophotometric methods.
Samples were analyzed for lead by dissolution in concentrated nitric acid
followed by atomic absorption spectrometric analysis with a Varian Techtron
AA-5 spectrophotometer. Visible and ultraviolet absorption spectra were
recorded for each lipid fraction with a dual beam Perkin-Elmer 120 auto-
matic spectrophotometer according to previously described procedures
(17). Infrared spectra of isolated components were taken in thin films
in carbon tetrachloride with a 257 Perkin-Elmer infrared spectrophotometer.
Phosphorus determinations were made according to the colorimetric procedure
of Allen (1).
Results
Effects of lead on bacterial cells
Light and electron microscope examinations of the cellular preparations
revealed no significant changes in cellular morphology and surface structure
207
-------
of test cells throughout this study. In the first 8 days of growth no
significant variations in culture yields and pigmentations were observed.
After the fifth consecutively complete growth cycle (10 days), the continued
growth of cells in medium containing lead resulted in gradually lower
yields of cells and decreasing intensity of pigmentation. The pigmentation
in lead treated cells in the ninth harvest (18 days) was reduced to just
a visibly detectable yellowish tint in contrast to the rich, deeply yellow
pigmented control cells; the yield of cells was reduced to one half of that
obtained for the control preparations. Electron microscopic examination of
preparations of these lead treated cells showed evidence that intracytoplasmic
materials were leaking from the cells. Protoplast preparations could not
be readily isolated from these cells; the protoplasts often lysed after a
brief lysozyme treatment. These results were consistently obtained in all
the repetitive experiments. No irregularities were observed in the control
cells. These observations suggest an osmotically sensitive condition
of the lead cultured cells due to changes in the properties of the cyto-
plasmic membrane. Electron microscopic examinations of membrane prepara-
tions of control and lead treated cells are shown in Figs. LA-ID. The
membrane preparation from control cells (Fig. 1A) reveals the rupture of
the essentially intact cytoplasmic membrane, some membrane enfolding, and
granular material interpreted to be cytoplasmic debris. The micrographs
reveal electron dense inclusions in three different membrane preparations
of lead treated cells (Figs. IB-ID); these inclusions were interpreted
to contain lead on the basis of their absence from control preparations
in all of the repetitive experiments and on the basis that significant
quantities of lead were detected in these isolated membrane fractions
by atomic absorption spectroscopy. These amorphous appearing electron
dense areas are essentially identical to those identified as lead-containing
aggregates in thin sections of nuclei of moss leaf cell (22), rat liver
mitochondria (29) and mammalian cells (8). The minimal amounts of lead
required for the formation of an electron dense inclusion have not been
determined. An additional noticeable feature in most lead treated membrane
preparations is the dissolution of the membranes (Figs. 1C and ID).
• The visualization of lead containing aggregates in the membrane
fraction supports the previous report (24) of lead immobilized in membranes
of M. luteus cells.
Effects of lead on cellular lipid composition
Table 1 shows the lipid content of cells, on a dry weight basis,
for lead treated and control cells after their 9th consecutive growth
cycle (18 days). The lipid content of lead treated cells is 30-56%
lower than that of the control cells. It should be pointed out that the
difference between the total lipid contents of cells grown in a medium
with different lead salts is not significant; the uptake for PbBr2 is
considerably slower than Pb(N03)2. Cells grown longer in PbBr2 provided
the same reduced level of lipids as Pb (1^03)2.
The amounts of lead removed from the cells in the lipid extracts
varied considerably among preparations. In most lead treated cells 30-40%
of the lead was removed by the Bligh-Dyer extraction; however, less than
1% of the lead remained with the chloroform lipid fraction. Between 60%
and 70% of the lead taken up by the cells remained with the lipid extracted
208
-------
cell debris. Repetitive washing of the cell debris with various organic
solvents or tris (hydroxymethyl) aminomethyl (Tris)hydrochloride buffer
at pH 7 containing 5 mg sodium ethylene-diamine-tetraacetate (EDTA)/ml
was ineffective in eluting additonal lead and lipids.
Chromatographic analyses of the individual components of polar
lipid fractions from both the control cells and lead treated cells re-
vealed patterns that were essentially the same and typical of those
previously described (9). The most significant difference between
phospholipids of control and treated cells was in the total quantities
observed; 40-50% less phospholipids were extracted from the lead-treated
cells as compared with control cells. Interestingly, the relative amounts
of individual lipids were not significantly altered. Studies on the
metabolism of individual phospholipids in lead treated cells are in
progress and will be reported elsewhere. No components in addition to
those observed in the control extracts were detected in the chromatographic
systems employed, minimizing the possibility of significant quantities of
lipids in the polar free lipid extracts consisting of lipid-lead complexes
with different chromatographic mobilities.
The hydrocarbon and fatty acid patterns obtained from lead-treated
cells were the same as those of the control cells (for presentations of
representative patterns, see Refs. 16, 25, 26). Chromatographic separa-
tion of the total non-polar lipid fractions by thin-layer chromatography
in a solvent system A revealed that only the components comprising the
pigment contents of the lead treated cells were either absent or greatly
reduced in quantities. The quantitation of the total cellular pigment
content (Table 1) was calculated spectrophotometrically assuming an
Er^ of 3 x 103 at 440 nm for the carotenoids (21). The spectral scans
of total lipid fractions extracted from the 9th harvest (18 days) of the
control and lead treated cells are shown in Fig. 2. The differences in
their spectral properties correlate with the observed visual reduction
of carotenoid components. Components absorbing between 325 and 224 nm
(Fig. 3), characteristic of napthoquinone (vitamin K) structures (20),
were not affected by lead treatments. The extracted and analyzed lipids
were then suspended in a solution of methanol-chloroform-water containing
0.01% EDTA (10:5:4, by vol). All of the lipids were recovered in the
chloroform phase after adjusting the volume ratios to 10:10:9 (by(vol),
respectively; however, virtually all of the lead in the chloroform-lipid
extract was partitioned into the methanolic-water phase. Re-examination of
carotenoid pigments showed no measurable differences in their spectral
properties (Fig. 2). The small amount of lead that was detected in the
chloroform-lipid extract was an apparent artifact of the experimental
procedure.
The results presented support a proposal that there are no signif-
icant amounts of specific stable individual lead-lipid components recovered
from the cells by the standard lipid extraction procedure employed for
free lipids. However, the absence of approximately one-half of the free
lipid content in the lead-treated cells demonstrates a definitive effect
of lead on M. luteus cells. Preliminary evidence indicates, by the signifi-
cant increase in the quantity of bound fatty acids, that a portion of the
missing free lipid is complexed or bound in some manner in the cell mem-
branes, rendering the lipids insoluble in the extracting lipid solvents
(work in progress).
209
-------
Lead treated cells from the ninth consecutive harvest (18 days)
grown in lead free medium exhibited a rapid reversal of the biological
and chemical effects observed above. The unhealthy lead treated cells
were restored to apparent normal states after the 3rd complete growth
cycle (6 days). Restoration of the cells was determined by culture
yields, osmotic stability, total lipid content, spectral analyses of
carotenoid content and the absence of lead inclusions in the membrane
preparation.
Further studies on the interaction of lead in a lipid milieu
The technique of using lipid impregnated Millipore disks was chosen
for this study because of the experimental accessibility of the prepara-
tions and for ease of studying the interactions with relatively large amounts
of both lipids and lead solutions. There were no significant differences in
the results obtained in this phase of the study on the basis of different
lead compounds employed; therefore, only the data using lead bromide is
presented.
The results in Table 2 show that of the 1.2 x 106 ng of lead re-
covered from two control disks containing a total of 250 mg of tetracosane,
only 4.5 x 103 ng (0.4%) of lead was retained by the lipids after water
washes of the disks. Virtually all of the tetracosane was removed in
the chloroform washing of the disks; methanol, and in some cases methanol-
water washings removed most of the lead retained by the impregnated disks.
Comparable retention of lead and its elution from the disks was also
observed in the control disks containing no lipids. Of the 1.2 x 106 ng
of lead recovered from two disks containing a total of 250 mg of extracted
total membrane lipids, 1.1 x 105 ng (9.0%) of lead was retained by the
membrane lipids after water washes of the disks. Successive chloroform,
acetone, and methanol washes of these disks contained near aliquots of
lead (Table 2), even though the majority of the membrane lipids (82%)
were removed in the chloroform wash. The above data clearly indicate that
the total membrane lipids are much more effective (nearly 25 times as
effective) at retaining lead than the pure tetracosane. The recovered
membrane lipids were unaltered chromatographically and relatively complete
lipid compositions were detected in each of the chloroform, acetone and
methanol washes by thin layer chromatographic procedures.
With disks containing total membrane lipids that were not treated
with lead, about 90% of the lipids were eluted in chloroform wash and
all of the lipids were recovered from the disks after the methanol wash.
In contrast, 14.2% of the phospholipids of the membrane-lipid containing
disks treated with lead still remained on the disks after all washes
(Table 2). Small quantities of carotenoid pigments were also visibly
retained. Methanolic-HCl washings of these membrane lipid disks followed
by a water wash were required to elute this remaining lipid content.
Fatty acids as well as trace amounts of acyclic hydrocarbons (which
should have been eluted in the chloroform wash) were detected by gas
chromatography (2-4) in the methanolic-HCl wash, indicating the occur-
renceof some form of bound lipid complex on these lead treated disks.
210
-------
In pursuit of defining specific interactions between lead and
individual lipid components, membrane llpid subtractions were studied
in the same manner. The results obtained from the Milllpore disks im^
pregnated with either the total non-phospholipids or the phospholipids
obtained by elution from silicic acid columns containing total free
membrane lipids are given in Tables 3 and 4, respectively. The 6.5%
'(7.9 x lO1* ng) of lead retained by 300 mg of the non-phospholipids is
'compared to the 4.2% (5.5 x 101* ng) of lead retained by 300 mg of phos-
pholipids. The data clearly demonstrate that the subsequent elution
of the lipids and lead are not dependent on the lipid phosphorus content.
In a manner similar to the total lipid disk experiments, substantially
greater quantities of lead were retained by membrane mixed lipid treated
disks as compared to the control disks containing tetracosane. In all
repetitive experiments, the retention of lead by lipids was substantially
lower in fractional membrane lipid preparations than in the total membrane
lipid preparations (Tables 2,3, and 4). There is a moderate difference
between the effects of lipid subfractions and those of total mixed mem-
brane lipids on the amount of lead that is eluted with the various sol-
vents. Large quantities of lead were removed in the chloroform, methanol,
and methanol-water washes of the Millipore disks containing lipid sub-
fractions from isolated cell membranes (Tables 3 and 4). This is in
moderate contrast to the approximate aliquots of lead eluted in the chloro-
form, acetone and methanol washes from total membrane lipid impregnated
disks (Table 2). Reduction in volume of chloroform washes of disks
containing lipid subfractions (Tables 3 and 4) from 200 ml to approxi-
mately 50 ml by evaporation at 40 C under a stream of purified nitrogen
resulted in a heavy precipitation of materials. The chloroform washes
were taken to dryness in voouo. Solubilization studies on the residues
revealed that 18,000 ng and 40,000 ng of lead of the non-phospholipids
and phospholipids, respectively, were resolubilized in 30 ml chloroform.
The solid residue was soluble in water. Similar insolubility problems
were encountered during concentrations of other washes. The data demon-
strate a definite increase in the solubilization of lead in a solvent
containing lipid mixtures. Ultraviolet, visible and infrared spectro-
photometric analyses (as well as NMR analyses of a few preparations) of
these chloroform soluble preparations provided no interpretable data that
could support the formation of a coordination complex between lead and
lipid components. The spectra of the lipid components were the same with
and without lead treatments. Chromatographically the lipid components
were the same as those of the controls.
Membrane hydrocarbons, individual microbial lipids, and pure com-
mercial lipids were placed on separate Millipore disks and studied in the
same manner. In none of these experiments was there significant retention
of lead or increased solubilization of lead in organic solvents. The
results from these experiments were identical to the controls.
i
Discussion
The uniformity in the cell wall surface and cell wall thickness
of cells treated with lead was indistinguishable from those observed in
non-lead treated cells. The visualization of electron dense areas by
electron microscopy (Fig. 1) and the spectrophotometric detection of
lead in the membrane subfraction support a previous report of there
211
-------
being substantial amounts of lead immobilized in the membranes of M.
luteus (24). The occurrence of lead in the membranes of cells does not
have a readily noticeable biological effect on M. luteus until the cells
have been exposed to lead over an extended period. Lead impregnated
cells were affected to the extent that apparent osmotically sensitive
cellular conditions resulted, as indicated by cellular leakage and proto-
plast instabilities. A definitive effect of lead was established by the
substantial reduction in the cellular free lipids.
None of the individual free lipids extracted from intact cells were
found to be associated with lead. The results were supported by the
absence of detectable lipid-lead components in the lipid impregnated
Millipore disk experiments. These results, in part should not be unex-
pected since zwitterionic lipids (phosphatidyl choline, phosphatidyl en-
thanolamine, etc.) should not strongly interact with cationic metals.
Other phospholipids found in M. luteus extracts are only moderately anionic
lipids and apparently ineffective in producing detectable quantities of
a plumbated lipid component. On the other hand, studies with lipid im-
preganted disks in contact with lead solutions demonstrated an appreciable
tendency for lead to interact only with complex lipid mixtures. These
properties observed are apparently not uncommon for metals; a similar
relationship has been reported for calcium ions (6). The mechanisms
responsible for the results observed are somewhat obscure. An explana-
tion of the observed uptake of lead by mixed lipids and unsuccessful
attempts to establish functionally specific plumbation of individual
membrane lipids could perhaps be explained by a type of macromolecular
complexing phenomenon. A complex of mixed lipids and lead could result
in a type of "bound" lipid that is insoluble in the organic lipid solvents
as evidenced by the retention of lipids on the thoroughly washed lipid
impregnated disks. This possible explanation could perhaps also account
for part of the high retention (60-70%) of cellular lead in the lipid
extracted cell debris with the simultaneous loss of a well-balanced free
lipid content. The increased solubilization of lead in solvents contain-
ing mixed lipids (Table 3 and 4) fits the scheme on the basis of a com- •
plexing interaction, but one that is sufficiently unstable in a solvent
system to account for the preceipitation of massive amounts of its lead
on reduction of solvent volumes. All free lipids analyzed spectrophoto-
metrically and chromatographically supported the scheme by the absence •
of evidence for stable ionic or covalent bonds between lead and lipid
components. The nature of the bound or complexed lipid forms has not
yet been determined. However, the combination of electronic and steric
factors in a lipid mixture may provide an environment suitable for the
nucleation of lead containing aggregates that include the "bound" lipids
and perhaps the electron dense inclusions which were evident in the
electron micrographs of the isolated membranes,of lead treated cells.
Lipid analyses of the heavily lead treated cells showed that indeed
the free lipid content of the cells was substantially lower. The most
obvious compositional changes were observed in the reduction of the phos-
pholipid phosphorus (40-50%) and in the spectra of the carotenoids (Fig.
2). It must be assumed that such a loss in membrane free lipids will
effect membrane stability and be responsible for at least part of the
osmotic sensitivity observed. It had been proposed that carotenoids have
212
-------
a role in stabilizing bacterial membranes (12, 19^21); however, a recent
report provides some evidence for the contrary (15). Support for the
proposal that carotenoids are involved in membrane stabilization was de-
rived from studies on the effects of diphenylamine on M. luteus cells
(12,20,21). The results from those studies are comparable to the results
reported here.
The mechanism by which lead affects the free lipid content and is
responsible for its quantitative reduction has not yet been resolved.
Work is in progress to specify the compositional and chemical changes
that occur in both the bound and free lipids of lead treated cells.
Acknowledgment
This research was supported by the National Science Foundation through
the Research Applied to the National Needs Program on Environmental Systems
and Resources, Grant GI-34813X. The authors are indebted to Kris Foster
for technical assistance.
References
1. Allen, R. J. L. The estimation of phosphorus. Biochem. J. 34:858-
865, 1940.
2. Bligh, E. G., W. J. Dyer. A rapid method of total lipid extraction and
purification. Can. J. Biochem. Physiol. 37:911-917, 1959.
3. Carroll, K. G., F. R. Spinelli and R. A. Goyer. Electron probe micro-
analyser localization of lead in kidney tissues of poisoned rats.
Nature. 227:1056, 1970.
4. Dowell, W. C. T. Carbon-stabilized collodion substrates for electron
microscopy. J. Appl. Phys. 28:634^635, 1957.
5. Freeman, C. P. and D. West. Complete separation of lipid classes on
a single thin-layer plate. J. Lipid Res. 7:324-330, 1966.
6. Hanahan, D. J. Metal ions participation in lipid protein systems,
_In J. Maniloff, J. R. Coleman, and N. W. Miller (ed.), Effects of
metals on cells, subcellular elements and macromolecules. C. C.
Thomas, Springfield, 111., p. 191-206, 1970.
7. Hoogeveen, J. T. Thermoconductometric investigation of phosphatidyl-
choline in aqueous tertiary butanol solutions in the absence and
presence of metal ions, jn J. Maniloff, J. R. Coleman and N. W.
Miller (ed.), Effects of metals on cells, subcellular elements and
macromolecules. C. C. Thomas, Springfield, 111. p. 207-229, 1970.
8. Hsu, F. S., L. Krook, J. N. Shirley and J. R. Duncan. 1973. Lead
inclusion bodies in osteoclasts. Science. 181:447-448, 1973.
9. Huston, C. K., P. W. Albro and G. B. Grindey. Lipids of Saroina
lutea. II. Composition of the complex lipids. J. Bacteriol. 89:
768-775, 1965.
213
-------
10. Isler, 0. In Carotenoids. Birkhouser Verlag Basel und Stuttgart,
1971.
11. Jensen, S. L., G. Cohen-Bazire, T. 0. M. Nakayama and R. Y. Stanier.
The path of carotenoid synthesis in a photosynthetic bacterium.
Biochim. Biophys. Acta 29:477-498, 1958.
12. Krinsky, N. I. The protective function of carotenoid pigments. In
A. C. Giese (ed.), Photophysiology, Vol. 3, Academic Press, New York,
p. 123-195, 1968.
13. Lepage, M. Identification and composition of turnip root
lipids. Lipids. 2:244-250, 1967.
14. Massey, V., and C. Verger. Studies on the reaction mechanism of
lipoyl dehydrogenase. Biochim. Biophys. Acta 48:33-47, 1961.
15. Mathews-Roth, M. M. and N. I. Krinsky. Carotenoid pigments and the
stability of the cell membrane of Sarcina lutea. Biochim. Biophys.
Acta 203:357-359, 1970.
16. Morrison, S. J., T. G. Tornabene and W. E. Kloos. Neutral lipids
in the study of realtionships of members of the family Mi-ovoooocaoeae .
J. Bacteriol. 108:353-358, 1971.
17. Ray, P. H. , D. C. White and T. D. Brock. Effects of growth temperature
on the lipid composition of Thermus aquatieus . J. Bacteriol. 108:
227-235, 1971.
18. Rosenthal, A. S., H. L. Moses, D. L. Beaver and S. S. Schuffman.
Lead ion and phosphatase histochemistry. I. Nonenzymatic hydrolysis
of nucleoside phosphates by lead ion. J. Histochem. Cytochem. 14 :,
698-710, 1966.
19. Salton, M. R. J. and A. F. M. Ehtisham-ud-din. The localization of
cytochromes and carotenoids in isolated bacterial membranes and
envelopes. Aust. J. Exp. Biol. Med. Sci. 43:255-264, 1965.
20. Salton, M. R. J. and J. H. Freer. Composition of the membranes
I isolated from several Gram-positive bacteria. Biochim. Biophys.
Acta 107:531-538, 1965.
21. Salton, M. R. J. and M. D. Schmitt. Effects of diphenylamine on
carotenoids and menaquinones in bacterial membranes. Biochim. Biophys.
Acta 135:196-207, 1967.
o
22. Skaar, H. , 0. E. Ophus, and B. M. Gullvag. Lead accumulation within
nuclei of moss leaf cells. Nature. 24:215-216, 1973.
23. Tornabene, T. G. Lipid composition of selected strains of
pest-is and ¥ersinia pseudotuberaulosis . Biochim. Biophys. Acta 306:
173-185, 1973.
24. Tornabene, T. G. and H. W. Edwards. Microbial uptake of lead.
Science. 176:1334-1335, 1972.
214
-------
25. Tornabene, T. G. and S. P. Markey. Characterization of branched
monounsaturated hydrocarbons of Saroina lutea and Sordino, flava.
Lipids. 6:190-195, 1971.
26. Tornabene, T. G., S. J. Morrison and W. E. Kloos. Aliphatic hydro-
carbon contents of various members of the family Miarococcaoeae.
Lipids. 5:929-937, 1970.
27. Tornabene, T. G. and J. E. Ogg. Chromatographic studies of the
lipid components of Vibrio fetus. Biochim. Biophys. Acta 239:
133-141, 1971.
28. Vallee, B. L. and D. D. Ulmer. Biochemical effects of mercury,
cadmium and lead, p. 91-128. JEn E. E. Snell (ed.), Ann. Rev. Biochem.
Vol. 41, Ann. Rev., Inc., California, 1972.
29. Walton, J. R. Granules containing lead in isolated mitochondria.
Nature. 243:100-101, 1973.
Table 1. Percent lipid composition of lead treated and control
Miorocoocus luteus cells1
Sample
Total lipids
Carotenoids
Control
2.38
0.0072
Lead Nitrate
1.08
0.0006
Lead Bromide
1.80
0.002
*Lipid compositions were obtained from cells in their ninth
consecutive complete growth cycle by repetitive extraction by the
Bligh-Dyer method until no further materials were detected in ex-
tracts. Percent lipid compositions were calculated on dry cell
weight basis. Pigment contents were determined assuming an E
of 3 x 103 for carotenoids.
1%
1 cm
215
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Table 2. The distribution of lead and total lipid phosphorus in sequential eluates of Millipore disks
impregnated with total lipids of M. luteus and exposed to a lead bromide solution1
N>
M
ON
1.
2.
3.
4.
5.
6.
7.
Samples
Supernatant
Water
Chloroform
Acetone
Methanol
Methanol-water
Disk (extracted)
Lead Recovered 1,
ng Pb
693,000
495,000
1
462
2,508
726
798
192,495
Control
Total Cellular Lipid
% Pb eluted
% Pb after H20 wash %P.
58.1
41.5
<0.1 <0.1
<0.1 10.2
0.2 55.8
<0.1 16.2
<0.1 17.8
99.7
-0-
-0-
-0-
-0-
-0-
-0-
-0-
ng Pb
726,000
363,000
25,410
39,600
33,000
3 ,300
6,250
1,196,560
% Pb eluted
%Pb after H20 wash yg P.
60.7
30.3
2.1 23.6
3.3 36.8
2.8 30.7
0.3 3.1
0.5 5.8
100.
3
15
4,440
96
80
9
770
%Pi
0.1
0.3
82.0
1.8
1.5
0.2
14.2
1Control consisted of 250 mg tetracosane applied to two Millipore disks; 250 mg of the total cellular
lipid sample was prepared in the same manner. All experiments were prepared in multiples; the data pre-
sented are an average of five experiments. Samples consisted of the reaction mixture and 200 ml of each
solvent used to elute the lead and lipids from the disks. Pb was determined by atomic absorption spectros-
copy; phosphorus (P.) was determined colorimetrically according to procedure of Allen (21).
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.Table..3.... The. distribution of lead in different solvent eluates of Millipore disks impregnated with the
non-phospholipids of M. luteus and exposed to a lead bromide solution1
NJ
1.
2.
3.
4.
5.
6.
7.
Samples
Supernatant
Water
Chloroform
Acetone
Methanol
Methanol-water
Disk (extracted)
Lead Recovered 1
Control
ng Pb %Pb
725,000 61.4
450,000 38.1
15 <0.1
9 <0.1
325 <0.1
5,750 0.5
596 <0.1
,181,695 96.1
Total Non-Phospholipids
%Pb after
H20 wash
-
-
0.2
0.1
4.9
85.9
8.9
ng Pb
800,000
350,000
25,000 218,000 CHC13
7,500 H20
5,000
35,000
12,000
2 ,240
1,229,240
% Pb
65.1
28.5
2.0
0.4
2.9
1.0
0.2
99.9
%Pb after
H20 wash
-
-
32.0
6.3
43.9
15.1
2.8
1The control consisted of 300 mg of tetracosane applied to two Millipore disks; the total non-phospholipid
sample (300 mg) consisted of combined hexane, benzene, chloroform and acetone eluates of a silicic acid
column containing total cellular lipids applied on two disks. Samples represent the reaction mixtures and
200 ml of each solvent used to wash the disks. Pb was determined by atomic absorption spectroscopy. Data
presented are an average of three representative experiments.
2Values represent amounts of Pb that were resolubilized in 30 ml of chloroform or water, respectively,
after the cholorform wash was taken to dryness.
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Table 4. The distribution of lead in different solvent eluates of Millipore disks impregnated with the
phospholipids of M. luteus and exposed to a lead bromide solution1-
Samples
1. Supernatant
2 . Water
3. Chloroform
4. Acetone
NJ
oo 5. Methanol
6. Methanol-water
7. Disk (extracted)
Lead Recovered 1
ng Pb
725,000
450,000
15
9
325
5,750
596
,181,695
Control
%Pb
61.4
38.1
<0.1
<0.1
<0.1
0.5
<0.1
90.6
Phospholipids
%Pb after
H20 wash
-
-
0.2
0.1
4.9
85.9
8.9
ng Pb
825,000
425,000
41,800 2 40, 000 CHC13
1,800 H20
1,350
10,000
800
887
1,304,837
%Pb
63.2
32.6
3.2
0.1
0.8
0.1
0.1
100.
%Pb after
H20 wash
-
-
76.2
2.5
18.2
1.5
1.6
control consisted of 300 mg of tetracosane applied to two Millipore disks. The lipid subtraction
(300 mg) consisted of the lipids isolated in the methanol eluate collected from a silicic acid column con-
taining total lipids and prewashed with hexane, benzene, chloroform, and acetone. 99% of total cellular
lipid phosphorus was in this fraction. Samples represent the reaction mixtures and 200 ml of each solvent
used to wash the disk. Pb was determined by atomic absorption spectroscopy. Data presented are an average
of 4 representative experiments.
2Values represent amounts of Pb that were resolubilized in 30 ml of chloroform or water, respectively,
after the chloroform wash was taken to dryness.
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List of Figures
Figure 1. Electron micrographs of representative unstained and unfixed
membrane subfractions of control (A) and lead bromide treated
cells (B, C and D).
Figure 2. Visible spectra of total carotenoid content of control (2.6 mg
lipids/ml, solid line), lead nitrate treated (7.8 mg lipids/ml,
dotted line), and lead bromide treated (5.9 mg lipids/ml, dashed
line) cells in 15% acetone in petroleum ether. Extracts were from
cells harvested from the ninth consecutive complete growth cycle.
Figure 3. UV spectra of total lipid content of control (1.2 mg lipids/ml,
solid line), lead nitrate treated (1.4 mg lipids/ml, dotted line),
and lead bromide treated (1.5 mg lipids/ml, dashed line) cells
in ethanol. Extracts were from cells harvested from the ninth
consecutive complete growth cycle.
219
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226
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ACUTE AND CHRONIC EXPOSURE TO PESTICIDES
Dave Mick
The University of Iowa
Pesticides are poisons. Pesticides are designed to kill. Although these
statements are rather sensational, they are true. Furthermore, the most
toxic types of pesticides generally do not differentiate between forms of
life. This responsibility is delegated to the individuals using pesticides.
It is a generally accepted fact that pesticides play a significant role in
the efficient production of quality food and fiber in this country. And it
seems unlikely that alternative types of pest control will replace pesticides,
at least not in the foreseeable future. Therefore, proper use of these
chemicals is of utmost importance if the adverse effects are to be kept at
a minimum. The responsibility is again delegated to those of us who use
pesticides.
No records are available to document all cases involving adverse effects of
pesticides. If these records were available, the prevalence of adverse
effects would probably be minimal when examined in conjunction with denominator
data such as the quantity of pesticides used. However, the fact that
pesticides continue to be implicated in cases resulting in human injury and
illness, animal loss, crop damage, etc. warrants careful scrunity of these
cases to determine the causes. Using this information in an educational
program will, hopefully, alert others and thereby prevent the occurrence
of similar incidents.
In Iowa, and probably elsewhere, the causes of almost all of the incidents
involving adverse effects of pesticides fall into 2 broad categories:
1. Carelessness (including misuse and not following label instructions).
Examples of these cases are as follows:
a. Application of the wrong pesticides.
b. Application of pesticides using applicators contaminated with other
pesticides.
c. Back-siphoning pesticides into wells.
d. Using old chemicals that have become obsolete.
e. Inadequate personal protection.
f. Pesticide spillage in locations easily accessible to pets, animals
and children.
g. Drift that produces crop damage, illegal residues, etc.
h. Carelessness on the part of the implement and pesticide manufacturers
which forces the operator to climb on a metal maze, carrying a heavy
container of toxic chemical, and finally pouring it through a small
opening.
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2. Improper storage and disposal of pesticides and pesticide containers.
Most of the situations that fall in this category could be classified
as carelessness.
Examples of these situations are as follows:
a. Storage of pesticides in other than the original containers.
b. Storing pesticides in close proximity to feed or food.
c. Transporting pesticides and other commodities in the same vehicle.
d. Usage of so-called "empty" pesticide containers for other purposes.
e. Improper disposal of "empty" pesticide containers and unwanted
pesticides.
The following statements are simple and trite but they will alleviate most of
the problems encountered from improper use of pesticides
1. Read and heed the label
2. Be careful
3. Use common sense
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ANHYDROUS AMMONIA ACCIDENTS
L. W. Knapp, Jr.
The University of Iowa
Problem
Each year farmers use unprecedented quantities of fertilizers as they
attempt to increase production of food and fiber to meet the demands of
the U.S. and the world. Consequently, new methods are ever sought out
which will reduce the time and costs of application, for timeliness of
application and reduction of labor, which is in ever decreasing supply,
are critical management factors.
Agricultural census figures indicate that while there is a steady drop in
the agricultural labor force and a similar decline in number of farms,
that the productive acreage has changed little and the output per man
hour is continually increasing. It is obvious that among other factors
which have brought this about, improved management, genetic improvements
in plants and animals, mechanization, control of pests and better
fertilization have been important factors.
One of the major fertilizer needs of today's agriculture is nitrogen,
which is usually supplied either as a liquid, in dry form, or as a
solution. Each of these types, all using anhydrous ammonia as their
basic ingredient, have their particular advantages and disadvantages of
use depending upon crop and season. However, as the largest quantities
of nitrogen are usually applied pre-plant or at time of planting, then
the economics of cost and ease of application are important considerations.
Consequently, anhydrous ammonia, which is 82% nitrogen, compared to 21%
to 45% for dry materials, and 20% to 41% for solutions, and which can
be easily liquefied (concentrated) for storage and application, makes it
an ideal and desirable fertilizer for this purpose. The demand for its
use, in Iowa, for example, is demonstrated by its growth since 1952-53
when 897 tons were applied, to a present plateau in 1972, of approximately
500,000 tons, evidencing its widespread use.
This increased use of ammonia, particularly in the anhydrous form, has
brought to the attention of physicians another new farm injury associated
with our changing agricultural technology. While no reliable figures are
available to determine the extent of the problem, sufficient numbers of
caustic burns, freezes, and eye damage cases are being seen by physicians
and hospitals to warrant everyone's attention. Thus it is timely to
discuss the special problems of ammonia-produced chemical trauma.
Ammonia is a colorless gas at atmospheric temperature and pressure with
a characteristic pungent, irritating odor. It is easily liquefiable under
moderate pressure; thus one can understand that if 113 cu. ft. of vapor
can be condensed to 1 cu. ft. of liquid, then there is good reason for
its being stored and transported as a liquid. Further, being stored
under pressure (approximately 100 - 125 psi), and having a vaporization
temperature of -28°F, contact with a stream of this vaporizing liquid
will cause freezing of tissue.
229
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Perhaps ammonia's most significant property is its affinity for water.
One volume of water will dissolve 1300 volumes of the gas; thus it
readily dissolves in living tissue causing caustic burns. Obviously
water is a first aid measure.
Equipment
Special equipment is used in the handling of anhydrous ammonia all the
way from the production tank to the bulk storage facility to the field
applicators. Obviously, several transfer operations are necessary to
change it from tank to tank and it is during such operations that most
injuries occur.
Similarly, aqua ammonia, a solution of anhydrous ammonia and water,
which is also an important agricultural fertilizer, unlike anhydrous
ammonia, does not have sufficient vapor pressure to permit transfer by
decompression; thus it must be pumped. Consequently, it is also handled
under pressure, necessitating special couplings and hoses to prevent
similar "leaks" and "escapes" which can be injurous to the worker.
In a typical anhydrous ammonia transfer operations, there are usually
four or more valves to open and close in a precise sequence; thus,
opportunities for human error and negligence are many and varied. Also
with the present system of couplers in general use there must be a
release of gas in the immediate vicinity of the operator to vent the
coupling assembly after the valves are closed down in order to more
easily release the coupling fitting. There can also be an inadvertent
release of gas should the operator fail to secure the valves properly
in a closed position or upon returning it to its storage position, if
the valve wheel should be accidentally struck.
Injury
After substantial exposure to ammonia, symptoms and signs of varied
character and severity may appear. The cause in all exposures is
ammonia coming into contact with moisture on body surfaces, vis., the
mucous membranes of the upper respiratory tract, skin and eyes. The
severity of injury varies with the amount and duration of exposure.
Respiratory problems may be life-threatening, but in terms of pain and
discomfort, skin injuries also loom as important. Exposure to ammonia may
injure the skin in two principal ways: freezing and alkaline caustic
action. If the skin is struck by the pressurized jet of liquid ammonia,
it is subjected to a temperature of -28° F, causing thrombosis of surface
vessels followed by ischemia and necrosis. Another mechanism of tissue
injury is the "chemical burn" which occurs whenver a. significant amount
of ammonia reaches the skin and is not immediately and thoroughly washed
off. The "chemical burn" is much more significant than the "freeze", in
that it produces much wider destruction. When ammonia reaches the skin
it quickly dissolves in surface moisture, producing the dissociate ions
230
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of ammonia hydroxide (NH^ + OH) and raising the pH. Ammonia, because
of its peculiar water solubility and rapid attainment of high pH, is one
of the most damaging alkaline agents causing denaturation of tissue
proteins, forming gel like alkaline proteinates, and saponifying the
fat of cell membranes.
In treating an ammonia burn of the skin, washing with water is essential,
while cool, wet compresses may ease the pain after thorough irrigation
has been accomplished. Particularly crucial is the avoidance of any
salves or greasy ointments which seem to promote deeper penetration of
ammonium hydroxide.
While respiratory injuries may be lethal and skin burns disfiguring, eye
injuries constitute the most serious hazard in regards to permanent
disability from anhydrous ammonia.
Since the mechanism of injury is similar for all alkaline solutions, the
determining factor in speed and extent of penetration into the eye
becomes the water solubility of the chemical involved. Ammonia's
fantastically high solubility places it at the top of the list of hazardous
substances, and because of the rapid penetration of ammonia into living
tissue, even prompt irrigation cannot be expected to remove all of the
chemical. Although complete elimination of the chemical is practically
hopeless, the amount penetrating varies with the duration of the exposure;
thus rapid and copious lavage is essential in limiting damage.
The pathological picture of an ammonia eye injury begins when the chemical
strikes the eye and dissolves in the surface moisture of the cornea. The
epithelium is destroyed and will slough shortly. Biochemical changes in
the stroma and disruption of the endothelium follow. If the exposure is
severe, the ammonia may reach the iris and the lens to start the process
of destruction there. Edema of the corneal stroma and the ciliary
processes begin within a few minutes. Damage to the walls of the limbal
and episcleral vessels may produce hemorrhage with a resultant beefy
red appearance and later neovascularization of these areas. Alternatively
thrombosis without hemorrhage may occur. Corneal opacity may develop
insidiously from one week to several weeks after the accident. Opacity
results from infiltration of the cornea by inflammatory cells with fibrous
tissue1 scarring and neovascularization following. The presence of tiny
blood vessels reaching across the cornea together with the thrombosis
of the limbal vessels makes a poor bed for a corneal transplant, and such
transplants are notoriously unsuccessful in ammonia injuries.
Conclusion
The final treatment of a serious ammonia eye injury ought to be handled
by an opthalmologist, but emergency copious lavage with water for fifteen
minutes must be implemented as quickly as possible by anyone.
For irrigating there is nothing better than clean water, and any attempt
at chemical antidoting is useless. If water is not available, any bland
fluid taay be used. Time is more essential than the particular liquid for
irrigation. Lavage should continue for fifteen to thirty minutes as tissues
may still be releasing ammonia fifteen minutes after washing begins.
231
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The expectation that each and every worker will understand the hazards
of ammonia, will wear face goggles, and have water nearby when handling
or transferring the chemical, is fallacious. To think that he will always
follow a pre-planned sequence of events for connecting and disconnecting
lines and hoses is equally in error. Thus, educational endeavors in the
safe handling of anhydrous ammonia need to be increased.
The farmer has to develop a greater respect for human hazards of ammonia
use and the first aid precautions he must make. Goggles during transfer
are a must as well as nearby water whenever a person works with anhydrous
ammonia.
Anhydrous ammonia application can and must be made safer.
References
1. Helmers, S., F. H. Top, Sr., and L. W. Knapp, Jr. Ammonia Injuries
in Agriculture. Journal of Iowa Medical Society. 271-280, May, 1971.
2. Helmers, S., W. H. McConnell, Jr., L. W. Knapp, Jr. Anhydrous Ammonia
Mishaps in Agriculture. Paper, 1970 Annual Meeting, American Society
of Agricultural Engineers, Minneapolis, Minnesota. July 7-10, 1970.
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