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NBS

PUBLICATIONS

NBS SPECIAL PUBLICATION 656

THE PILOT NATIONAL
NATIONAL BUREAU OF STANDARDS

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The Pilot National Environmental

Analysis of Human Liver Specimens m

Specimen Bank — 'V^IUv^

RATIONAL BUREAU OF STANDARDS

LIBRARY AUG 2 5 1983,

no

Edited by:

Rolf Zeisler, Sally H. Harrison, and Stephen A. Wise

Center for Analytical Chemistry

National Measurement Laboratory

National Bureau of Standards

Washington, DC 20234

Research supported in part by:

Inhalation Toxicology Division

Health Effects Research Laboratory

Office of Research and Development

Ui. Environmental Protection Agency

Research Triangle Park, NC 27711

Project Officer: George M. Goldstein

Ait

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director

Issued August 1983
Library of Congress Catalog Card Number: 83-600561

National Bureau of Standards Special Publication 656

Natl. Bur. Stand. (U.S.), Spec. Publ. 656, 135 pages (Aug. 1983)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE WASHINGTON: 1983

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402

Price $5.50

(Add 25 percent for other than U.S. mailing)
Foreword

In 1979, the Environmental Protection Agency and the National Bureau

of Standards completed construction on a Pilot National Environmental

Specimen Bank at NBS. This pilot program was initiated to (1) develop and

evaluate protocols for contamination-free sampling of environmental specimens; (2) develop analytical methods for the trace analysis of inorganic

and organic pollutants; (3) develop and evaluate storage conditions which

would permit the long-term storage of samples without change in pollutant

concentrations; and (4) evaluate a specimen bank as a means of storing

samples for pollutant trend monitoring over time and permitting retrospective analyses as concerns for new pollutants arise and as new analytical

techniques are developed.

In 1975, an international agreement to share knowledge and samples

between the United States and the Federal Republic of Germany was instituted

and in 1981, a pilot environmental specimen bank was initiated in Germany.

This international collaboration has led to a fruitful exchange of ideas

and analytical intercomparisons which have demonstrated the comparability

of data on an international scale.

The report which follows summarizes several years of careful research

and lays the groundwork for evaluation of specimen banking as a viable

long-term mechanism for evaluating environmental pollutants and monitoring

pollution in our biosphere. In addition to the obvious benefits which

arise from meeting the goals outlined above, there are several additional

and noteworthy benefits which have accrued from the pilot program. It is

this sum total of knowledge gained that should be evaluated in judging the

concept of specimen banking. The sampling, storage, and analysis protocols

developed when coupled with the statistical treatment of the data, have

given added insight into quality assurance mechanisms for environmental

analyses. It is our opinion that improved data quality through careful

research is of itself a major benefit to the complex field of pollutant

analyses. The protocols developed for sample collection and storage in a contamination-free mode yield benefits that go well beyond the sampling of

human livers for the pilot specimen bank; the lessons learned are equally

applicable to any occurrence which would require contamination-free

analysis for impact/hazard assessment. Furthermore, experience gained

through this pilot program is a valuable lesson in the development of a reference laboratory capable of performing high priority, problem-solving

analyses when a calamity has occurred and capable of serving as an advisory laboratory for field analyses performed on a continuing basis.

i i i
Finally, with the pilot experience at its current state of maturity,

one can envision the broad applicability of the specimen banking concept

to real-time and trend monitoring. Beyond the concerns of environmental

pollution the concept is equally valid to monitoring nutritional status,

occupational exposure and key chemicals related to health state of a populace. It is with all these aims and benefits in mind that the concept

of a full-scale specimen bank needs to be evaluated by the American

scientific community.

Harry S. Hertz

Acting Director

Center for Analytical Chemistry

iv
ABSTRACT

This work was performed under a joint EPA/NBS research program to develop

state-of-the-art protocols for the sampling, storage, and analysis of biological and environmental -type matrices as part of a Pilot National Environmental

Specimen Bank program. The purpose of this report is to summarize the

experience and results obtained for the first sample type, human liver. The

sample collection protocol was evaluated with respect to costs and suitability

of donor selection criteria. An analytical protocol was implemented for the

determination of trace elements in the liver samples using the techniques of

atomic absorption spectrometry, isotope dilution mass spectrometry, neutron

activation analysis, and voltammetry. Individual sections of this report

describe in detail the procedures used and the results obtained for each of

these analytical techniques. The analytical results for the determination of

31 trace elements in 36 liver specimens are presented and discussed. Analytical methodology for the measurement of organochlorine compounds is also

described.

This report is submitted in partial fulfillment of EPA Interagency

Agreement EPA-81 -D-X0105 by the National Bureau of Standards.

Key words: atomic absorption spectrometry; cryogenic homogenization ; gas

chromatography; human liver; inorganic analysis; isotope dilution

mass spectrometry; neutron activation analysis; organic analysis;

organochlorine pesticides; specimen banking; trace elements;

voltammetry.

DISCLAIMER

Certain commercial equipment, instruments, or materials are identified

in this Report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau

of Standards, nor does it imply that the materials or equipment identified

are necessarily the best available for the purpose.

V
TABLE OF CONTENTS

PAGE

FOREWORD iii

ABSTRACT v DISCLAIMER V

SECTIONS

1. The National Environmental Specimen Bank — Background and History. . 1 S. A. Wise

2. Evaluation of the Sampling and Storage Protocol for Human Liver

Samples 5

Section 2 Appendix A 19

K. A. Fitzpatrick, S. H. Harrison, R. Zeisler, and S. A. Wise

3. Cryogenic Homogenization of Biological Tissues 21

J. K. Langland, S. H. Harrison, B. Kratochvil, and R. Zeisler

4. Development and Verification of a Protocol for the Analysis of

Inorganic Constituents in Human Livers 35

R. Zeisler

5. Determination of Selected Trace Elements in Human Liver Samples by

Atomic Absorption Spectrometry 39

T. C. Rains, T. A. Rush, and T. A. Butler

6. The Determination of Lead, Thallium, Copper, and Nickel in Human

Liver Samples by Isotope Dilution Mass Spectrometry 47

J. W. Gramlich, L. J. Powell, K. A. Brletic, and T. J. Murphy

7. Instrumental Neutron Activation Analysis for the Determination of

Mineral and Trace Elements in Human Liver Samples 55

J. Bailey, K. A. Fitzpatrick, S. H. Harrison, and R. Zeisler

8. The Determination of Nickel, Copper, Lead, Cadmium, and Zinc in

Human Liver Samples by Voltammetry 77

E. J. Maienthal and W. F. Koch

9. Results of the Inorganic Analysis of the First Year Human Liver

Collection 81

R. Zeisler

vi
Table of Contents Continued

PAGE

10. Development of Analytical Methodology for the Determination of

Organochlorine Compounds in Human Liver Samples 91

W. F. Kline, C. F. Allen, S. N. Chesler, L. R. Hilpert, and

S. A. Wise

APPENDICES

I Development of a Radiochemical Separation Procedure for Tin 99

R. R. Greenberg

II Ultratrace Determination of Platinum in Biological Materials via

Neutron Activation and Radiochemical Separation 103

R. Zeisler and R. R. Greenberg

III Determination of Organomercury Cations in Tissue Samples Using

Liquid Chromatography with Amperometric Detection 115

William A. MacCrehan

IV Modification of Centrifugal Filtration Device for Elimination of

Sorption Losses 121

W. A. MacCrehan

V Neutron Capture Prompt Gamma-Ray Activation Analysis of Human

Liver Samples 123

D. F. Anderson

VI Publications of the U.S. EPA/NBS National Environmental

Specimen Bank 127

Vii
LIST OF FIGURES

NUMBER PAGE

Section 2:

1. Age and sex distribution of donors for the liver samples collected

during 1980-81 in the Pilot National Environmental Specimen Bank

Program 11

2. Age distribution of the U.S. population (1980) compared to the

donors of the first 378 liver samples in the Pilot National

Environmental Specimen Bank Program 12

3. Sampling and storage scheme for human liver samples during the

first year 15

4. Storage of a liver specimen in the storage facility of the Pilot

National Environmental Specimen Bank at the National Bureau of

Standards 16

Section 3:

la. Ball mill B: (1) milling body, (2) grinding ball 24

lb. Teflon® ball mill, Teflon® scoop, and Teflon® jar used in the

homogenization and storage of human liver specimens 24

2a. Disk mill #2: (1) milling body, (2) grinding disk and ring, and

(3) aluminum holder 25

2b. Teflon® disk mill and aluminum adapter plate 25

3. Details of the construction of the Teflon® disk mill 26

4. Teflon® mortar and plunger used to prefracture liver specimens ... 27

5. Cold dry box 28

24

6. Sampling diagram of Na in human liver homogenate 29

7. Particle size distribution of frozen tissue after homogenization

in Teflon® mills 30

viii
List of Figures Continued

NUMBER PAGE

Section 9:

1. Elemental concentrations in 36 individual liver specimens and

concentration ranges reported in the literature 82

2. Imprecision of the analytical techniques under the constraints

of the analytical protocol as observed for the determination

of cadmium in human liver 84

3. Concentrations of six essential trace elements in human liver. ... 86

4. Concentrations of six pollutant trace elements in human liver. ... 87

Section 10:

1. Gas chromatograms from the analysis of human liver samples 94

Appendix II

1. Platinum detection limits in dependency of Pt to Au ratio and_

irradiation time at a neutron flux of 5 • 10^^ • n • cm'^ • s~^. . . 107

2. Interfering activity from i^^Au(n,Y)^^^Au(n,Y)i^^Au 108

Appendix III

1. Effect of the alkyl chain length of the n-alkyl thiol on the

retention of ethylmercury and methylmercury. . 118

2. Reversed-phase LC separation of mercury species 119

Appendix IV:

1. Glass-modified centrifugal filtration apparatus 122

2. Organomercury determination 122

1x
LIST OF TABLES

NUMBER PAGE

Section 1

1. History of the Environmental Specimen Bank Program in the U.S. ... 2

Section 2:

1. Sample Collection Costs 7

2. Evaluation of Case Selection Criteria 9

3. Percentage of Human Liver Samples from Various Sources During

1980-1981 10

4. Donor and Sample Information for Human Specimens Analyzed During

the First Year of Sample Collection (1980-81) 13

5. Sample Processing/Storage Costs 17

Appendix A - Human Liver Sampling Protocol 19

Section 3:

1. Cryogenic Homogenization Units Used in the Pilot NESB Program. ... 23

2. Homogeneity of Two Cryogenically Homogenized Livers 33

3. Concentration of Sodium and Chlorine in Mussel Sieve Fractions

(mg/g Dry Weight) 34

Section 4:

1. Analytical Protocol for Human Liver Specimens 36

Section 5:

1. Instrumental Parameters for Atomic Absorption Spectrometry 40

2. Trace Element Concentrations in Human Liver Samples as Determined

by Atomic Absorption Spectrometry 44

X
List of Tables Continued

NUMBER PAGE

Section 6:

1. Trace Element Concentrations in Human Liver Samples as Determined

by Isotope Dilution Mass Spectrometry 51

2. Concentrations (Dry Weight) of Lead and Thallium and Their

Relative Ratios 52

Section 7:

1. Nuclear Parameters and INAA Conditions for the Analysis of Human

Liver Samples 56

2. Trace Element Concentrations in Human Livers as Determined by

Instrumental Neutron Activation Analysis 60

3. Analysis of Bovine Liver (SRM 1577) by INAA 72

Section 8:

1. Trace Element Concentrations in Human Liver Samples as Determined

by Voltammetry 80

Section 9:

1. Trace Element Levels in 36 Human Liver Specimens 83

Section 10:

1. Determination of p^p'-DDE in Human Livers by GC-MS 93

2. Concentrations of Selected Organochlorine Pesticide Residues in

Human Liver Samples 95

3. Concentrations of Selected Organochlorine Pesticides in Human

Liver Homogenate from the German Pilot Environmental

Specimen Bank 96

Appendix I:

1. Tin Concentrations in Standard Reference Materials 100

2. Concentration of Tin in Human Liver Specimens 100

xi
List of Tables Continued

NUMBER PAGE

Appendix II:

1. Nuclear Data for Platinum and Gold Analysis and for Major

Interferences 105

2. Determination of ^^^Au to ^^^ku Ratio 110

3. Platinum and Gold in Orchard Leaves (SRM 1571) Ill

4. Platinum and Gold in Bovine Liver (SRM 1577) and Citrus Leaves

(SRM 1572) Ill

5. Platinum and Gold and Human Liver Specimens 112

6. Concentrations of Platinum in Human Liver Samples 114

Appendix III:

1. Outline of Methods for the Determination of Methylmercury in

Tissues 116

Appendix V:

1. Prompt Gamma-Ray Activation Analysis (PGAA) Results for Human

Liver Samples 124

xii
SECTION 1

THE NATIONAL ENVIRONMENTAL SPECIMEN BANK - BACKGROUND AND HISTORY

by

S. A. Wise

The concept of environmental specimen banking for archiving and retrospective analysis of biological and environmental samples has been recognized

recently as an important component of systematic environmental monitoring.

Participants at three international workshops, held in 1977, 1978, and 1982

issued recommendations for the establishment of environmental monitoring and

specimen banking programs (1-3). The historical development during the

1970's of the concept of a National Environmental Specimen Bank (NESB) in the

United States has been reviewed previously by Goldstein (4,5). The history

of the Environmental Specimen Bank in the U.S. is summarized in Table 1.

Several workshops held in the early 1970's identified the need for a systematic collection, storage, and analysis of environmental specimens and finally,

in 1973, the U.S. Environmental Protection Agency (EPA) proposed the

establishment of a NESB system. The purpose of such a national system would

be [1] to detect changes in the environment on a real-time basis using

bioaccumulators as indicators, (i.e., monitoring) to assess the effectiveness

of environmental control measures, [2] to distinguish such changes from

natural inputs, and [3] to provide a "bank" of well preserved and documented

environmental samples for retrospective analyses in future years as analytical

techniques improve or as new pollutants are identified.

Since 1975 the National Bureau of Standards (NBS), in conjunction with

EPA, has been involved in research relating to the establishment of a National

Environmental Specimen Bank. The initial plans and preliminary results of the

evaluation of methodology for the NESB have been described previously (6-11).

At an EPA/NBS Workshop on "Recommendations and Conclusions on the National

Environmental Specimen Bank" held in 1976 (6), a pilot NESB program was proposed. This pilot program was designed to evaluate the feasibility of a national program by providing actual working experience in all aspects of

specimen banking, i.e., specimen collection, processing, storage, analysis,

and data management. The major goals of this pilot study were: [1] to

develop analytical protocols for sampling, processing, and storage of four

types of environmental accumulators; [2] to evaluate and improve analytical

methods for the determination of both trace element and organic pollutants in

biological matrices; [3] to establish baseline data on selected environmental

specimens; [4] to evaluate the feasibility of long-term storage at various

conditions; and [5] to provide a "bank" of samples for retrospective analyses

1
TABLE 1. HISTORY OF THE ENVIRONMENTAL SPECIMEN BANK PROGRAM IN THE U.S.

1972 NAS/NRC^ Workshop - Identified Shortcomings of Uncoordinated Sampling,

Storage, and Analysis of Environmental Specimens

1973 GERHD/NAS/NRC^ - Identified the need for National Environmental

Specimen Bank (NESB)

EPA Workshop - Proposed NESB Concept

1974 EPA/NBS - Interagency Agreement for Evaluation of Research

Methodology for NESB

1975 Established Bilateral Agreement with Federal Republic of Germany

for Collaborative Research in Specimen Banking

1976 EPA/NBS Workshop - Recommendations for the Design of a Pilot Program

to Study the Feasibility of NESB

1977 International Workshop on "The Use of Biological Specimens for the

Assessment of Human Exposure to Environmental Pollutants" held in

Luxembourg

1978 International Workshop on "Monitoring Environmental Materials and

Specimen Banking" held in Berlin, Germany

1979 U.S. Pilot Environmental Specimen Bank Facility at NBS Completed

1980 Collection and Analysis of Human Liver Samples Initiated

1982 Collection and Analysis of Mussel Samples Initiated

International Workshop on "Environmental Specimen Banking and

Monitoring as Related to Banking" held in Saarbrucken, Germany

^National Academy of Science/National Research Council (NAS/NRC), Geochemical

Environment in Relation to Health and Disease (6ERHD).

in future years. The experience gained during the pilot study will be

necessary to evaluate the feasibility of establishing a National (or International) Environmental Specimen Bank.

Four types of environmental specimens, which represent environmental

accumulators, were selected for inclusion in the NBS pilot program as a result

of the EPA/NBS Workshop on "Recommendations and Conclusions on the National

Environmental Specimen Bank". These sample types and specific samples

representative of the sample type are: [1] human soft tissue - liver,

[2] aquatic accumulator - marine mussels, [3] food accumulator - grain or

milk, [4] air pollutant accumulator - moss, lichen, or air particulate filters.

Human liver was selected as the first sample type for inclusion in the pilot

2
specimen banking program. As each new sample type is incorporated into the

program, collection, storage, and analysis of the previous sample type(s) will

continue. The number of sample types and samples was minimized so that the

pilot program would not be overwhelmed with samples or analyses, but could

focus on the protocol development and research aspects necessary to provide

reliable analytical data. In November, 1979 a special "clean" laboratory/

storage facility (12) was completed at NBS to initiate the Pilot National

Environmental Specimen Bank program. Since 1980, NBS has been involved in the

sampling, sample processing, storage, and analysis of the first two sample

types, human liver and mussels. In this report the experience gained by NBS

during this pilot program relating to sample collection, processing, and

storage of human liver samples is described.

At the "International Workshop on Monitoring Environmental Materials and

Specimen Banking" held in 1978 in Berlin, a major conclusion and recommendation was that "Pilot specimen banks should be established as soon as possible

in order to study the problems of organizing long term storage in banking

programs" (Ref. 2, p. 1). The joint U.S. EPA/NBS Pilot National Environmental

Specimen Bank is an example of the implementation of such recommendations.

Since 1975, EPA/NBS and the Environmental Agency of the Federal Republic

of Germany (FRG) have been pursuing collaborative research in the area of

environmental specimen banking as part of a bilateral agreement on the

environment. In 1981 the Pilot Environmental Specimen Bank Facility for the

FRG was completed. The activities of the U.S. EPA/NBS pilot program are being

coordinated closely with the efforts of the German Pilot Environmental

Specimen Bank Program (13-15). Participants of both programs meet annually

to exchange information and experience for the benefit of both programs.

Samples have also been exchanged between the two programs for interlaboratory

comparison purposes.

Through the EPA/NBS pilot program, the U.S. is looked upon as a leader

in environmental specimen banking research. In recent years interest in

specimen banking has increased and several other nations (e.g., Japan and

Canada) have initiated specimen banking programs.

REFERENCES

1. Berlin, A., A. H. Wolff, and Y. Hasegawa. The Use of Biological Speci- mens for the Assessment of Human Exposure to Environmental Pollutants.

Martinus Nijhoff Publishers, The Hague, The Netherlands, 1979. 368 pp.

2. Luepke, N.-P. Monitoring Environmental Materials and Specimen Banking.

Martinus Nijhoff Publishers, The Hague, The Netherlands, 1979. 591 pp.

3. Lewis, R. A. Environmental Specimen Banking and Monitoring as Related

to Banking, Martinus Nijhoff Publishers, The Hague, in press.

4. Goldstein, G. M. Plan for a National Environmental Specimen Bank.

EPA-600/1 -78-022. U.S. Environmental Protection Agency, 1978. 27 pp.

3
5. Goldstein, G. M. The National Environmental Specimen Bank — Its Concepts,

Its History and Its Objectives. In: The Use of Biological Specimens for

the Assessment of Human Exposure to Environmental Pollutants. A. Berlin,

A. H. Wolff, and Y. Hasegawa, eds. Martinus Nijhoff Publishers, The

Hague, The Netherlands, 1979. p. 165.

6. Rook, H. L., and G. M. Goldstein. The National Environmental Specimen

Bank. NBS Special Publication 501. U.S. Government Printing Office,

Washington, DC, 1978. 54 pp.

7. Rook, H. L. , and P. D. Lafleur. Establishing and Operating a Specimen

Bank for Environmental Monitoring. In: The Use of Biological Specimens

for the Assessment of Human Exposure to Environmental Pollutants.

A. Berlin, A. H. Wolff, and Y. Hasegawa, eds. Martinus Nijhoff

Publishers, The Hague, The Netherlands, 1979. p. 309.

8. Gills, T. E., and H. L. Rook. Specimen Bank Research at the National

Bureau of Standards to Insure Proper Scientific Protocols for Sampling,

Storage, and Analysis of Environmental Materials. In: Monitoring Environmental Materials and Specimen Banking. N.-P. Luepke, ed. Martinus

Nijhoff Publishers, The Hague, The Netherlands, 1979. p. 263.

9. Maienthal, E. J., and D. A. Becker. A Survey of Current Literature on Sampling, Sample Handling, and Long-Term Storage for Environmental

Materials. NBS Tech. Note 929, U.S. Government Printing Office,

Washington, DC, 1976. 34 pp.

10. Becker, D. A., and E. J. Maienthal. Evaluation of the National Environmental Specimen Bank Survey. EPA-600/1 -77-015. U.S. Environmental

Protection Agency, 1977. 269 pp.

11. Gills, T. E., H. L. Rook, and R. A. Durst. The National Environmental

Specimen Bank Research Program for Sampling, Storage, and Analysis.

EPA-600/1 -79-01 7. U.S. Environmental Protection Agency, 1979. 59 pp.

12. Moody, J. R. Design Principles and Construction of the Pilot Specimen

Bank Facility. In: Pilot Program for the National Environmental Specimen

Bank - Phase I. S. H. Harrison, R. Zeisler, and S. A. Wise, eds.

EPA-600/1 -81 -025. U.S. Environmental Protection Agency, 1981. p. 8.

13. Stoeppler, M., H. W. Durbeck, and H. W. Niirnberg. Pilot-Umweltprobenbank.

In: Jahresbericht 1979/80 der Kernforschungsanlage Jtilich GmbH, 1980.

p. 55.

14. Umweltprobenbank — Ergebnisse der Vorstudien und Projektierung der

Pilotphase. Vol. I (Part 1 and 2). Berlin, Umwel tbundesamt , 1981.

461 pp.

15. Kayser, D. , U. R. Boehringer, and F. Schmidt-Bleek, The Environemntal

Specimen Banking Project of the Federal Republic of Germany. Environ.

Monitoring and Assessment, 1_ 1982, p. 241.

4
SECTION 2

EVALUATION OF THE SAMPLING AND STORAGE PROTOCOL

FOR HUMAN LIVER SAMPLES

by

K. A. Fitzpatrick, S. H. Harrison, R. Zeisler, and S. A. Wise

A major goal of the Pilot National Environmental Specimen Bank (NESB)

Program is the development of protocols for the sampling, processing, and

storage of four types of environmental samples. During the past three years

(1980-1982) the pilot NESB program has been involved in the collection,

processing, storage, and analysis of the first sample type, human liver. In

this section the experience gained during this program relating to the collection and storage of the human liver samples will be described. This experience

will provide the basis for evaluating the feasibility of establishing a National Environmental Specimen Bank.

SAMPLE COLLECTION

Sampling Protocol

Due to the extremely low levels of trace element and organic pollutants

found in most environmental samples, caution must be exercised during sample

collection and sample processing to avoid contamination. A detailed protocol,

designed to provide samples suitable for both trace element and organic

analysis, has been developed and implemented for the collection of human liver

samples. A summary of the liver sampling protocol for the pilot program is

given in Appendix A [for the complete detailed protocol see Harrison et al .

(1,2)]. This liver sampling protocol was developed in conjunction with those

individuals performing the autopsies, and the implementation of the protocol

required periods of education and close cooperation to achieve a suitable

protocol within the bounds of practicality. Different disciplinary views and

the need for careful communication were evident from perceived meanings of

such terms as "clean", i.e., interpreted as "sterile" by autopsy personnel vs. "non-contaminated chemically" by analytical chemists.

The sampling protocol was designed to avoid possible contamination of the

sample by either inorganic or organic constituents. Teflon®* materials (e.g.,

sheets, bags, and storage jars) were selected as the most suitable material

for non-contamination of the sample with respect to both inorganic and organic

*Three types of Teflon® material were used: perfluoralkoxy (PFA), tetrafluoroethylene (TFE), and fluorinated ethylene propylene (FEP).

5
constituents and for low diffusion rates of water (3-5). The protocol

specifies the use of such non-contaminating items as dust-free non-talced

vinyl gloves, pre-cleaned dust-free Teflon® FEP sheets and bags, high-purity

water, and a titanium/Teflon® TFE knife. These items are provided by NBS to

each collection site to achieve uniformity in sampling materials. A special

titanium-bladed knife with a Teflon® TFE handle was designed and constructed

for use during cutting of the specimen. This special knife is used to avoid

trace element contamination by various constituents associated with a regular

stainless-steel scalpel/knife (e.g., Ni and Cr) and to limit the possible

contamination to a single element which is currently of no environmental or biological interest, i.e., titanium. The use of a titanium knife for removal

of human tissues was a recommendation of previous workshops on specimen

banking (6,7). The liver samples are sealed in Teflon® FEP bags, frozen in

liquid nitrogen (LN2), and shipped to the pilot NESB facility at NBS in a special biological shipper at LN2 vapor temperature.

To eliminate potentially infectious liver samples from the specimen bank,

a blood sample to be used for hepatitis B screening is removed from the donor

at the time of autopsy. In addition, a liver section from the right lobe is

removed for preparation of histological slides. These slides are examined by

a pathologist for evidence of abnormality and/or infectious diseases and then

stored in the specimen bank facility. The liver samples are placed in

"temporary" storage in a LN2 vapor freezer at the bank until the results of

the hepatitis screening are available. Any samples found to be infectious

are returned to the pathologist for incineration. These precautions are followed to protect the analysts involved in sample processing and analysis

and to provide biologically normal livers. Even after this screening, the

human liver samples are regarded and handled as potentially infectious.

Processing of the liver samples (i.e., sample transfers during homogenization

and subsampling) is performed in a biological safety hood.

A data form, provided with each liver sample, contains information about

the donor and specimen, e.g., date of birth, sex, residence, ethnic group,

height, weight, smoking history, occupation (if known), date and time of

death and autopsy, diagnosis of autopsy, and liver specimen weight (see

Appendix A and Refs. 1 and 2).

The sampling protocol for human livers developed for the pilot specimen

bank program was recently adopted as a model for collection of human tissue

at a workshop entitled "Protocols - Mineral /Elemental Analysis of Human

Tissues" held in St. Louis, MO (8). The goal of this workshop was to prepare

specific protocols for sample selection, collection, preservation, and

analysis of human tissues. These protocols were to be published as guidelines

for those involved in research on human body burdens. The St. Louis workshop

was organized as a follow-up meeting to the "Workshop on Research Needed to

Improve Data on Mineral Content of Human Tissue" (9).

Sampling Protocol Evaluation

During the first two years of the pilot specimen bank, 350 liver samples

from three geographical locations (Baltimore, MD; Minneapolis, MN; and

Seattle, WA) were collected using this protocol. During the remaining years

6
of the pilot study, liver samples will be received at a rate of approximately

100 per year and predominately from one location (Seattle). At the completion

of the first year of human liver collection, the sample collection protocol

was evaluated with respect to such items as: initial set-up costs for each

site, sample procurement costs, transport time, time required for receipt of

hepatitis results and histological slides, and suitability of the donor

selection criteria.

Sampling Costs

The costs associated with the sample collection are summarized in Table 1.

After the initial set-up cost of about $3000 per collection site, the liver

samples were obtained at an approximate cost of $250 each during the first

year of collection (300 samples total). The major portion of the sampling

cost was the sample procurement fee which included such items as: part-time

salary for a technician to work with autopsy personnel to obtain samples, fees

for hepatitis testing, and miscellaneous supplies (e.g., liquid nitrogen,

solvents for cleaning the knife, etc.). The second largest expense was the

shipping cost of the biological shipper to and from the collection site.

During the second year, the cost of materials and sample procurement increased

by about 10 percent; however shipping costs were lower due to the more efficient use of the large shippers (5 samples) rather than the small shippers

(1 sample).

TABLE 1. SAMPLE COLLECTION COSTS ($U.S.)

Initial Set-up Costs Per Site

Item Cost/Site

Biological shipper

Large (5 samples) or 1125

Small (1 sample) (290)

Shipping container (large) 375

Knife-titanium blade/Teflon® TFE handle 150

Heat sealing unit 1380

$3030 Sampling costs (non-reusable items ) Item Average Cost/Liver Sample

Non-talced vinyl gloves (4 pair) 2.,00

Teflon® FEP sheets (3) 6.,00

Teflon® FEP bags (4) 8.,00

High purity water 3.,00

Shipping (to NBS and return.

express air service) 46.,00

Sample procurement contracts (year 1) 180.,00

(i.e., technician salary, liquid

nitrogen, hepatitis testing, and

miscellaneous expenses)

Miscellaneous items 2.00

(e.g., labels, cardboard containers, etc.)

$247.00

7
Donor Selection Criteria--

The donor selection criteria were designed to provide samples which are representative of a "normal" liver immediately prior to death (see Appendix A

for the selection criteria). Livers were excluded which were physically

injured, diseased, infected, or chemically abnormal (e.g., drug overdose).

Medical examiners' offices rather than hospitals are more likely to have

sufficient cases which satisfy these criteria. In all three sampling sites

most of the samples were obtained through the medical examiners' office. In

Baltimore and Seattle the samples were obtained almost exclusively from

medical examiners' offices (100 percent and 96 percent, respectively). At the

Minneapolis site, samples were obtained from four different locations including medical examiners' offices and hospital autopsy rooms. The majority of

the samples in Minneapolis (60 percent), however, were provided by a research

clinic autopsy room.

Regarding donor selection criteria, the sampling experience at one site

(Seattle), i.e., total cases available for sampling, types of cases, number of

cases rejected, and reasons for rejections, is summarized in Table 2. At this

site the major factor for case rejection ('\^20 percent of the cases autopsied)

was the time requirement that the autopsy be performed within 24 hours after

death. An additional 10-20 percent of the cases available were lost because

the part-time technician was not available for sampling on afternoons and

Saturdays. In Table 3 the percentages of samples obtained from various sources (i.e., cause of death) at each location are summarized. At the

Baltimore and Seattle sites where the samples were obtained predominately

from medical examiners' cases, the primary sources were accidents, suicides,

and homicides. However, at the Minneapolis site the samples provided by the

research clinic were primarily from deaths due to natural causes. Legal

constraints in the State of Washington prevented the use of any tissue from a probable homicide case (i.e., the body is considered to be evidence). However, at the Baltimore site, where such restrictions do not exist, homicide

cases provided a major source of samples (34 percent). Of the 350 samples

collected through 1981, 2 percent were rejected because of positive hepatitis

results and 4 percent were rejected based on the results of the histological

examination. Additional samples (4 percent) were rejected due to lost

hepatitis results or no hepatitis test possible. The samples which were

rejected because of possible hepatitis contamination were returned to the

pathologist for incineration.

8
TABLE 2. EVALUATION OF CASE SELECTION CRITERIA (SEATTLE)

Total cases in first year = 1290

Samples needed = 100

Samples obtained during first year = 88 {^^7% of total cases available)

Technical reasons for sample rejection

Case Type

Total

Cases

1 . View only

(no autopsy)

2. Homicides

3. Natural death

4. Traffic

accidents

5. Suicides

6. Accidents

Cause for Rejection

77o Used for evidence

17% Puncture wounds

(medic assistance)

18% Torn, hemorrhage,

contaminated

97o Not autopsied

21% Drugs, fire,

alcohol ics

Laceration by pathologists/technicians

Autopsy performed

more than 24 hour

after death

Technician unavailable

during autopsy

Estimated

Percent

Rejected

28%

7%

7%

1%

30-40%

9
TABLE 3. PERCENTAGE OF HUMAN LIVER SAMPLES FROM

VARIOUS SOURCES DURING 1980-1981

Baltimore Seattle Minneapol is Total

Suicides 13 20 3 13

Homicides 34 0 0 10

Traffic accidents 36 36 12 29

Other accidents

(e.g. , falls, burns

drownings, etc.)

5 15 9 11

Natural causes

(e.g., diseases, heart

problems, etc.)

3 26 73 33

Miscel laneous 9 3 3 4

The age and sex distribution of the donors for the 350 samples collected

during 1980-81 are illustrated in Figure 1. As shown in Figure 1, the age

and sex distributions of the donors for the Baltimore and Seattle sites are predominately young and male because these samples were obtained from medical

examiner's cases; whereas for the Minneapolis samples which were predominately

from a research clinic (60 percent), the majority of the donors were older

(>60 years old). For all sites the majority of the donors were male {'^1^

percent). Thus, the selection criteria and the major source of samples

(i.e., medical examiners' offices) tend to provide "abnormal" age/sex distributions of donors when compared to the Census Bureau data on the age/sex

distributions of the U.S. population (see Figure 2). Regarding racial

information, 83 percent of the total 350 donors were Caucasian, 14 percent were Black, and 3 percent were other (e.g., Hispanic, Oriental, and American

Indian). The majority of the non-Caucasian donors were included in the 100

donors from the Baltimore site (54 percent Caucasian and 45 percent black).

Both the Seattle and Minneapolis sites provided mainly Caucasian donors (93

and 97 percent, respectively). The collection of a set of samples with

age/sex/race distributions representative of the normal population was not

the primary goal of the pilot program. However, the sample collection process

could be more selective to provide a representative sample set for a NESB.

Of the first 350 liver specimens collected, approximately 10 percent were

analyzed. Donor information for the 36 samples analyzed is summarized in

Table 4. As additional samples are analyzed during the pilot program

to provide baseline data (^^SO/year) , a more normal age/sex/race donor

profile will be selected.

10
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TABLE 4. DONOR AND SAMPLE INFORMATION FOR HUMAN SPECIMENS ANALYZED DURING

THE FIRST YEAR OF SAMPLE COLLECTION (1980-81)

Sample Hours Between Concentration

Identification . Ethnic Death and . Factor (dry

No. Sex Age Group Autopsy Smoker weight/wet weight)

L1B0002 M 61 4 14.5 2 0.279

L1B0005 M 27 1 8 2 0.302

L1S0018 M 43 4 12.5 2 0.287

L1M0023 F 22 4 8.5 2 0.240

L1S0027 M 52 4 11.5 2 0.226

L1S0037 M 82 4 15.5 0 0.235

L2B0041 M 45 4 13.3 2 0.256

L2B0042 M 17 4 19 2 0.248

L 1 DUUtO r 91 7 7 oc U . c / o L2M0048 M 17 4 12.0 2 0.230

L2S0049 M 24 4 5.8 2 0.240

L1S0053 M 60 4 11.5 2 0.278

L2M0062 F 84 4 14.3 2 0.221

L1B0074 M 16 1 14.5 2 0.281

L1S0080 M 32 4 15 2 0.230

L1M0083 M 29 4 10 2 0.300

L1S0086 M 19 4 12 2 0.299

L 1 ouuyu M 0 / o 1

1 nu c L1S0091 M 38 4 14.8 1 0.305

L1S0092 M 36 4 21 .3 1 0.262

LIBOIOO M 66 4 21 .5 2 0.302

L1M0105 M 74 4 4.5 0 0.269

LI SOI 07 M 33 2 21 .5 1 0.287

L1S0110 M 75 4 22.8 2 0.280

L1M0118 M 33 1 9 2- 0.293

L1M0119 M 49 4 17 2 0.240

L1B0121 M 26 1 12 2 0.257

L1S0123 M 27 4 13.5 2 0.280

L1M0126 M 70 4 11.8 2 0.297

L1M0127 M 86 4 11.8 2 0.217

LI SOI 28 M 54 4 19 2 0.265

LI SOI 29 M 69 4 14.5 2 0.279

L1M0130 M 23 4 5 2 0.259

L1M0131 M 56 4 5 2 0.270

LI MO 140 M 27 4 20.5 2 0.285

L1M0141 M 69 4 17.5 2 0.206

^The third letter/number in the identification no. indicates the geographical

.location, i.e., B = Baltimore, MD; M = Minneapolis, MN; and S = Seattle, WA.

M = male; F = female.

j1 = Black; 2 = Hispanic; 3 = Oriental; 4 = Caucasian; and 5 = unknown.

1 = yes; 0 = no; and 2 = unknown.

Concentration factor for conversion of results from dry weight to wet weight

basis.

13
A major conclusion from the experience of the first year of collection

was the need for a technician whose primary responsibility was to work closely

with autopsy personnel to select, remove, and prepare the liver samples.

Since these technicians were responsible for implementing the sampling protocol, NBS personnel worked closely with them to stress the importance of

following the protocol precisely. When a sample stored in the specimen bank

is analyzed, the researcher must have confidence that the sample was collected

exactly as prescribed in the protocol.

STORAGE SCHEME

The storage scheme for the liver samples will be described in detail to

illustrate the approach used in the pilot NESB program. This scheme is

designed to evaluate the question of appropriate temperature for storage of

biological samples and to provide a "bank" of wel 1 -characterized reference

samples. The liver samples are received at the specimen bank facility as duplicate sections of the left lobe, identified as Sections "A" and "B" (see

Figure 3). All of the "A" sections are placed in long-term storage at LN2

vapor temperature, and the "B" sections are used for the storage evaluation.

Approximately 30 of the "B" sections per year are homogenized using a special

cryogenic homogenization technique (see Section 3) to provide about 20

aliquots per sample of 6-8 g each. The sample remains frozen during homogenization and the sample aliquots are transferred to the Teflon® PFA jars

inside a cold nitrogen atmosphere dry box to minimize water condensation on the frozen samples. To investigate the question of the appropriate temperature for long-term storage, these aliquots are stored in Teflon® jars under

four different conditions: frozen at -25 °C, at -80 °C, and in liquid

nitrogen vapor (-120 °C to -190 °C) and room temperature after freeze-drying.

These aliquots will be reanalyzed at one-year intervals during the pilot

study and the results compared to the data found in the "real-time" analysis

(i.e., analyses performed soon after homogenization) to determine if changes

in the concentration of trace elements or trace organics (e.g., organochlorine

pesticide residues) have occurred. The "A" sections are reference samples

which may be used to re-evaluate results obtained for a particular "B"

section. At the end of the pilot program, the "A" sections will represent a valuable bank of wel 1 -characterized, documented samples available to the

scientific community. In addition, a large quantity of analytical data from

the analyses of the "B" sections will be available for many of these liver

samples

The storage area of the NBS pilot facility (see Figure 4), contains

three LN2 vapor freezers (500-L capacity each), four compressor type biological storage freezers (382-L capacity each), and a shelf cabinet for storage at room temperature. The Teflon® packaged liver specimens are received at NBS

in cylindrical cardboard tubes (57 mm O.D. x 117 mm) which provide a standard

geometry to facilitate easier sample storage. These samples are stored in

the LN2 vapor freezers in larger cylindrical tubes (6.35 cm O.D. x 64.8 cm)

with five specimens (sections) in each tube. Each LN2 vapor freezer will

hold a maximum of 170 tubes or 850 sections. In the compressor freezers

whole liver sections are stored in trays containing 16 samples each, three

trays per shelf, six shelves per freezer for a total of 288 samples per

14
Donors 100/Y

Baltimore,

Maryland

Section B

Storage Evaluation

Donors Donors Donors

100 / Y

Minnesota

Donors 100/Y

Seattle. Washington

Section A

Long Term Storage

-120 °C to -196 °C

30/

Cryogenic

l^otnogenlzatlon

Transfer to

;^20 Teflon PFA Jars^ 6-ag/Jar

UJars^ UJarsj (4 Jars]

Real Time

Analysis

Store LN2 freezer

Store

-80 »C

Store

-25 "C

Freeze dry

store Room temp,

Figure 3. Sampling and storage scheme for human liver samples

during the first year.

15
Figure 4. Storage of a liver specimen in the storage facility of the Pilot

National Environmental Specimen Bank at the National Bureau of

Standards.

16
freezer. Sample storage location is identified by freezer, shelf, tray, and

position (or tube identification for a LN2 vapor freezer). The homogenized

liver subsamples are stored in 15-mL Teflon® PFA jars. The outside surface

of the jar lids contains a removable Nomex® Aramid (a synthetic paper)

identification label. These jars are stored in square cardboard boxes (140 mm X 140 mm X 50 mm) which are labeled with a three-digit number embossed on the side. These boxes (eight jars/box) are stored in racks in both the LN2

vapor and compressor freezers. When a sample is placed in the bank, a storage

file (presently on hard-copy) is created which includes the following information: sample identification number, storage identification number (describing

storage location in the freezers), sample weights, and storage dates (in and

out) [see Harrison et al., (1,2)]. Currently, three compressor-type freezers

are used for -80 °C storage and one freezer is used for -25 °C storage.

Freeze-dried samples are also stored in boxes in an enclosed, dust-free

cabinet at room temperature. A cost summary of the major items used in the

NBS pilot storage facility and for the sample processing is provided in

Table 5.

TABLE 5. SAMPLE PROCESSING/STORAGE COSTS

Sample Processing Costs ($U.S.)

Cold dry box 2750

Disk mill instrument 5300

Teflon® TFE mill head 1200-1500

Teflon® PFA jars (03.65 ea.) 365/100 jars

Teflon® TFE scoops (10) 200

Storage Facility Costs

Liquid nitrogen vapor freezer (4) @ 6320 25,280

Compressor type freezers (4) @ 3853 15,412

with LN2 backup, remote alarm

Storage shelves, racks, containers 792

Automatic dialing alarm system 2100

Biological safety hood 4100

Histological slide cabinet 495

Dust free cabinet 213

Cardboard tubes 114/100 tubes

Liquid nitrogen per freezer 0 $0.1 0/L 360/year

Oxygen monitor 990

Fuel cells 110/year

LN2 storage containers (210L) 6 @ 1300 7800

17
As a safety precaution to protect the stored specimens, the operating

temperatures and power supply of the compressor freezers, LN2 vapor freezers,

and storage facility are monitored by an automatic dialing alarm system. This

alarm system will dial selected telephone numbers to report a fault when the

temperature exceeds a pre-selected limit or the power fails. The compressor

freezers are also equipped with a LN2 back-up system to cool them in the event

of an extended power failure.

REFERENCES

1. Harrison, S. H., T. E. Gills, E. J. Maienthal, H. L. Rook, S. A. Wise,

and R. Zeisler. The National Environmental Specimen Bank Pilot Program.

In: Trace Substances in Environmental Health-XIV, D. D. Hemphill, ed.

University of Missouri, Columbia, MO, 1980. p. 329.

2. Harrison, S. H. , R. Zeisler, and S. A. Wise. Pilot Program for the

National Environmental Specimen Bank - Phase I. EPA-600/1 -81 -025.

U.S. Environmental Protection Agency, 1981. 54 pp.

3. Gills, T. E. , and H. L. Rook. Specimen Bank Research at the National

Bureau of Standards to Insure Proper Scientific Protocols for Sampling,

Storage, and Analysis of Environmental Materials. In: Monitoring

Environmental Materials and Specimen Banking, N.-P. Luepke, ed.

Marti nus Nijhoff Publishers, The Hague, The Netherlands, 1979. p. 263.

4. Moody, J. R. , and R. M. Lindstrom. Selection and Cleaning of Plastic

Containers for Storage of Trace Element Samples. Anal. Chem., 49:2264,

1977.

5. Durst, R. A. Container Materials for the Preservation of Trace

Substances in Environmental Specimens. In: Monitoring Environmental

Materials and Specimen Banking, N.-P. Luepke, ed. Martinus Nijhoff

Publishers, The Hague, The Netherlands, 1979. p. 198.

6. Berlin, A., A. H. Wolff, and Y. Hasegawa. The Use of Biological Specimens for the Assessment of Human Exposure to Environmental Pollutants.

Martinus Nijhoff Publishers, The Hague, The Netherlands, 1979. 368 pp.

7. Luepke, N.-P. Monitoring Environmental Materials and Specimen Banking.

Martinus Nijhoff Publishers, The Hague, The Netherlands, 1979. 591 pp.

8. Hopps, H. C, and B. L. O'Dell. Proceedings of Workshop on Trace

Elements in Human Tissue, held October 15-17, 1981, St. Louis, MO.

9. Hopps, H. C, and B. L. O'Dell. Research Needed to Improve Data on Mineral Content of Human Tissues. Federation Proc, 40:2111 , 1981.

18
SECTION 2

APPENDIX A

Human Liver Sampling Protocol

A. Donor Case Selection Criteria

1. Liver expected to be "normal" and uninjured (lacerated or punctured).

2. Donor has no known history of alcoholism, hepatitis, drug abuse,

gross sepsis, long-term disease treatment, tuberculosis, cirrhosis,

liver carcinoma, chronic circulatory failure, or congestion.

3. Death was not due to chemical or drug overdose/exposure.

4. Autopsy performed less than 24 hours after death.

5. Donor has not been embalmed or frozen.

6. Liver has a total weight greater than one kilogram.

B. Donor/Sample Information Requested:

1. Autopsy identification number;

2. Date and time of death and autopsy;

3. Place of autopsy and attending pathologist;

4. Date of birth, sex, and ethnic group (e.g., Caucasian, Black,

Oriental , etc. )

5. Height and weight of donor;

6. Occupation, diseases, smoking history (if known);

7. Diagnosis of autopsy;

8. Comments regarding any deviations from the prescribed protocol or anything special about the donor/sample.

19
C, Sample Collection

1. Personnel involved in liver removal /handl ing should wear the dustfree non-talced vinyl gloves provided by NBS; no smoking is allowed

in autopsy room during sample collection.

2. The liver is removed as soon as possible after opening the body and

is placed on a clean Teflon® FEP sheet (If liver membrane is

ruptured, sample is not acceptable for this pilot program).

3. Liver is rinsed slightly with high purity water to remove blood and

surface contamination, allowed to drain, and transferred to a clean

Teflon® FEP sheet on the autopsy room scales for weighing.

4. Liver is placed on a Teflon® FEP sheet on a flat surface for

dissection. Using the titanium knife, the left and right lobes are separated to obtain a left lobe sample of <400 g.

5. Using the titanium knife, the left lobe sample is divided into

anterior and posterior sections (designated as A and B) of

approximately equal size.

6. Each section is placed in a dust-free Teflon® FEP bag, the air is

squeezed out, and the bag is then sealed with a heat sealing unit.

The sample is weighed and a label containing the autopsy identification number and the sample weight is placed on the bag; the sample

is then placed in a second bag and sealed again.

7. The double-bagged samples are placed in the cylindrical cardboard

containers provided and immersed in liquid nitrogen for 10 minutes.

8. The liver specimens should be stored in the cryogenic biological

shipper for no longer than 10 days before shipping to the pilot

facility using 24 hour express package service.

9. A copy of the completed donor information form is sent with the

sample.

10. During the autopsy a sample of blood (generally from within the

heart) is collected for hepatitis B antigen screening and a liver

section is removed from the right lobe for preparation of

histological slides.

11. The hepatitis results are reported to NBS as soon as available

and the histological slides are sent to NBS with results of the

histological examination. Another copy of the donor information

form is sent to NBS containing any additional information from

the autopsy report.

20
SECTION 3

CRYOGENIC HOMOGENIZATION OF BIOLOGICAL TISSUES

by

J. K. Langland, S. H. Harrison, B. Kratochvil, and R. Zeisler

INTRODUCTION

A difficult task in the Pilot NESB program is the chemical characterization of a bulk sample, the human liver. Despite the macroscopic homogeneous

appearance of human liver, large inhomogeneities have been reported within a single liver when one gram test portions were used (1). The concentrations

of essential trace elements may vary by a factor of four, while other trace

elements may vary by as much as a factor of 1000 (2). Most analytical techniques use relatively small test portions for the measurements and therefore,

the analyst is confronted with this inhomogeneity problem when a precise and

accurate characterization of the bulk sample is desired. To obtain trace

data on the bulk sample and to make results from different test portions of

the same liver equivalent for the purpose of storage and storage evaluation

in the pilot program (see reference 3 for a discussion of the storage

evaluation scheme), homogenization of the bulk sample is required.

The reduction of the bulk sample to a laboratory sample suitable for the

analytical techniques used is a critical step in an analytical measurement.

Heterogeneity and contamination of the analytical sample may produce poor

results which do not reflect the status of the bulk sample. If no precautions

are taken, the uncertainties due to the sample inhomogeneity and the sampling

process may become the limiting factor, thus reducing the usefulness of highly

precise and accurate analytical techniques. Thus, a major research effort of

the Pilot NESB Program has been directed towards the development and evaluation of a system for contamination-free homogenization of soft tissues.

METHODS FOR HOMOGENIZATION OF TISSUE

A variety of devices are in use for particle size reduction and homogenization of tissue, e.g., Waring-type blenders, ball mills, glass tube and

pestle grinders, and hydraulic or cutting shear homogenizers. The size

reduction step requires mechanical interaction (e.g., cutting, chopping, and

grinding) of an implement with the biological matrix. These implements are commonly made from metals (e.g., stainless steel) or from plastic laminates

or glass. These implements provide sharp edges and/or high resistance which

are necessary to reduce the size of tissue subdivisions. These interactions

21
can cause severe contamination of the biological material; therefore blenders

and similar apparatus have been excluded from use in the Pilot NESB Program.

In addition, some types of blenders generate excessive heat which can lead to

losses of trace constituents or changes in organic compound structures.

Several years ago Iyengar and Kasperek (4) evaluated the technique of

cryogenic homogenization (Brittle Fracture Technique) for relatively small

biological samples (5 to 15 g each). Cryogenic homogenization utilizes the

principles of milling and grinding of hard materials after the tissues are frozen at temperatures below -140 with liquid nitrogen. At this temperature the hardness of all tissues is increased to a point where impact or pressure causes fracture and reduction to smaller pieces. The mill is

constructed of Teflon® which has several essential advantages for the

homogenization procedure. [1] Teflon® is relatively pure, and therefore, the

danger of contamination of the sample with trace elements (other than carbon

and fluorine) and organic compounds by abrasion and leaching is minimal (5).

[2] Parts made from Teflon® are chemically inert and withstand thorough

cleaning procedures with acids and organic solvents. These cleaning

procedures also reduce the rish of contamination greatly. [3] Teflon®

provides the specific heaticapacity (1.05 kJ/kg»K) and insulation (heat

conductivity = 0.07 W/nrK) to maintain mill and sample at cryogenic temperatures throughout the homogenization process, thereby preserving the physical

and chemical state of the sample. [4] Teflon® has the density required for

the grinding balls and disks and, at near liquid nitrogen temperature, the

hardness and strength (without becoming brittle) to efficiently grind frozen

biological tissues.

DESIGN AND CONSTRUCTION OF CRYOGENIC HOMOGENIZATION APPARATUS

The capacity of the "Mikro-Dismembrator" described by Iyengar (4) was

too small for the Pilot NESB Program. Initially, sample sizes of up to 50 g were considered for the human liver banking program. During the final

implementation of the storage and analysis plan (3), the sample size was increased to 150 — 200 g; for future specimens (e.g., mussels), a sample size

of up to 1000 g is anticipated. These requirements led to a series of

prototype developments summarized in Table 1. For the initial experiments

the ball mills were patterned after that of Iyengar (4) (Figures la and lb);

however, mechanical limitations and the performance evaluation (see below)

made the search for another type of mill necessary. The Teflon® disk mills

developed during this study fulfill all the necessary requirements; therefore,

the description of the construction details will be limited to the disk mills.

22
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(UE r- 3 fO I— +J O O

CO ro E E o o o o CVI cn oro ro o

to Euo

LO

CVJ

CVJ

(O

roo

lO

LO

ro

CQ CO • >)c

laE

(U o

«l-ou

•r—3

a.

0) a:

<a

s- o

U- c

(U 3 o Od 3

4-* (U E

s: •r-(U o :3 c s:

t—

#1 to

0) E

z. ^ to •r— 3 C

-oc o

1—

cu

+->

X

cu (U Q. •1—

t/1 00

23
Figure la. Ball mill B (see Table 1): (1) milling body, (2) grinding ball.

Figure lb. Teflon® ball mill.

Teflon® scoop, and

Teflon® jar used in

the homogenization and

storage of human liver

specimens.

A "Shatterbox" disk mill made by Spex Industries, Inc. (Metuchen, NJ) was available. A Teflon® mill head was constructed (disk mill #1) to fit

into the holding clamps of the "Shatterbox" (Figure 2). This disk mill head was patterned exactly after the commercial steel heads with only a slight

increase in volume in spite of the much larger outer dimensions due to the

increased wall thickness.

24
H —p

Figure 2a. Disk mill #2 (see Table 1): (1) milling body, (2) grinding

disk and ring, and (3) aluminum holder.

Figure 2b. Teflon® disk mill

and aluminum adapter

plate.

After a short time of preliminary use (which gave excellent performance,

but provided insufficient capacity), a stress problem created a fracture along

the bottom inside corner of the mill head (Figure 3 upper diagram). This

crack was probably caused by rapid changes in temperature. The sharp edge of

the bottom of the chamber provided a starting point for the fracture. Later

designs (disk mills #2 and #3) have a smooth curve between the bottom and the

wall (Figure 3 lower diagram). As an additional precaution, the heads are cooled and warmed slowly to avoid rapid temperature changes.

25
The larger heads (#2 and #3) were adapted to fit a modified Siebtechnik

G.M.B.H. (Mulheim (Ruhr), Federal Republic of Germany) "Swinging Mill". The

large heads undergo significant changes in size with changes in temperature.

The linear expansion coefficient of Teflon® was determined to be about

1.2 X lO"'^ °C~i . With a change of about 40 "C during operation, the diameter

of the head increases by 1.2 mm. Therefore, the bottom of the mill is

beveled and fits into a beveled aluminum holder on the Siebtechnik machine

(Figure 3). With this arrangement a firm support of the mill head is ensured

throughout its operation.

All mill heads, disks, and rings were machined from solid Teflon® PTFE

stock. Rods with the desired diameters were commercially available. However,

working with Teflon® pieces the size of the mill heads is not easy. Teflon®

(TFE) is an unstable material, i.e., it will cold flow, "move", warp, etc.

During machining, close tolerances are difficult to maintain. The additional

problem of possible different contraction of the various parts during cooling

down also had to be considered. For example, seals are flat surfaces on the

top of the heads pressed together by the clamping holder of the Siebtechnik

machine.

26
AUXILIARY EQUIPMENT FOR CRYOGENIC HOMOGENIZATION

The liver samples used in the Pilot NESB program are frozen at liquid

nitrogen temperature. They are in a cylindrical shape about 10 cm long and

5 cm diameter. These tissue blocks do not fit into the disk mill and must be

broken into smaller pieces. This operation is accomplished by direct impact

in a Teflon® mortar with a Teflon® plunger (Figure 4). The liver is placed

into the cooled chamber (LN2 vapor temperature) and the plunger is inserted.

An 18 kg weight is dropped from a height of about one meter onto the plunger.

After several hits, the liver is fractured into small pieces with a maximum

size of about 2 cm^.

18 Kg Weight

one metre fall.

Teflon Plunger

Teflon

Mortar

2ZZZZZZZZ

Liver Sample

Figure 4. Teflon® mortar and plunger used to prefracture liver specimens

27
The samoles are maintained at cryogenic temperatures (below -60 °C) to

insure the chemical stability of the samples. These temperatures are maintained -From the time of removal of the specimen from storage through sample

nrenaration. After the prefracture, the sample pieces are inserted into the

cooled disk mill and after two minutes the homogenization is complete resulting

in a fine, frozen Dowder (particle size <40 mesh). The powder is subsampled

into Teflon® containers in a cold box (Figure 5) at temperatures below -60 °C.

A variety of small sample handling tools (e.g., scoops, scrapers, brushes, etc)

have been constructed of Teflon® to orevent contamination of the samples.

Figure 5. Cold dry box.

PERFORMANCE EVALUATION OF CRYOGENIC HOMOGENIZATION TECHNIQUE

In the frozen state the liver homogenate has a particulate physical

nature. The sampling properties of particulate materials have been studied

extensively (6,7), and Ingamells (8) has developed a very practical model to assess sampling. Ingamells' proposed sampling constant, K^, can be used for

direct comparison of the performance of the different cryogenic homogenization

systems. To determine experimentally, a particulate homogenate is subsampled at different sample sizes and analyzed. If the analytical error is

sufficiently small, the sampling constant can be determined using the

equation = wR^, where R is the observed relative standard deviation and w

is the mass of the respective subsamples. The sampling constant corresponds

to the weight of the sample required to limit the sampling uncertainty to 1 percent with 68 percent confidence. A sampling diagram derived from this

relation can be used to predict analytical errors and their dependence on sample size.

28
To evaluate the performance of the homogenization procedure, a radiotracer experiment was performed using ^^Na. In this experiment a 5-g subsample

was irradiated and then homogenized with the bulk material. Subsamples of

different weights were then removed from the homogenate and the amount of ^^Na

determined. From these experiments the sampling diagrams shown in Figure 6

were generated. The comparison of ball mill B with disk mill #1 (100-g

capacity) is shown in Figure 6. For the ball mill, a sampling constant of

= 32 g was obtained indicating that the material was not sufficiently

ground and mixed to allow accurate sampling using a typical 1-g analytical

subsample. The inadequate performance of the ball mill led to the design of

a disk mill which yielded a = 0.95 g. Using the disk mill, the precision

of the analytical data from a 1-g sample will be determined by the precision

of the analytical techniques rather than by sampling uncertainty.

(0

CM

<

o

0)Q. v>

•a

0)

E

Figure 6,

2.9

2.7

2.5

2.3

2.1

1.9

1.7

0.01

24

Sampling Diagrams For Na

I

1 ^ —Human Liver in Teflon Ball Mill • — Mytilus Edulis in Teflon Disk Mill

KgCX)

J_ 0.1 1.0 10 100

Sample Weight (g)

Sampling diagram of ^^Na in human liver homogenate,

To evaluate the performance of the mills with respect to particle size

reduction, efforts were undertaken to measure the actual particle size distribution of the homogenate obtained from the two different milling procedures.

In general, sieving is an easy way to determine particle sizes; however,

cryogenic sieving of frozen samples is much more difficult. After some procedure development, the sieving experiments were performed in a large LN2

freezer which provided the necessary cold and dry atmosphere to minimize

melting and particle agglomeration due to moisture condensation. A standard

set of metal sieves with the following mesh sizes, 40 (top), 60, 80, 100, 140,

and 200 (bottom) was used to determine the particle size distribution of the

homogenized tissues.

29
The performance of the mills was evaluated using three different types

of tissue and the results of these experiments are summarized in Figure 7. A

direct comparision of the results obtained with the ball mill and the disk

mill reveals that the disk mill is much more effective in producing a small

particle homogenate from beef liver. This matrix was selected to simulate

the behavior of human liver tissue. After homogenization with the disk mill,

virtually all of the material passes through the 40 mesh sieve, i.e., the

particles are less than 0.46 mm in diameter. The results shown in Figure 7 are reproducible within ±10 percent (Is) for constant weight, temperature,

homogenization time, and sieving time. Therefore, only the disk mills were considered for further evaluation and development.

I

I

1 100 140

( mesh)

LIVER

LIVER, disk mill

T

200

ADIPOSE

MUSCLE

Figure 7. Particle size distribution of frozen tissue after homogenization

in Teflon® mills. Approximately 150 g of tissue were homogenized

with a grinding time of 4 min; liver homogenized in ball mill B and liver, adipose, and muscle tissue homogenized in disk mill #2.

30
Two additional tissue types were selected to test the performance of the

disk mills: pork fat (adiposis abdominalis) to simulate human adipose tissue

(which is used frequently for monitoring of organic pollutants) and beef muscle

to simulate a fibrous tissue. The adipose tissue was the most difficult tissue

for this homogenization technique. In all experiments with the pork fat, a fraction of up to 16 percent remained on top of the 40 mesh sieve; many times

this fraction still contained several 1 cm^ chunks which were not broken up

during the homogenization process. These larger pieces may have hindered the

action of the mill. It was necessary to reduce the weight of the sample to

about 120 g for appropriate functioning of the mill since the volume of the

fat tissue homogenate is considerably larger than a corresponding mass of

liver. In addition, the mill had to be maintained as close as possible to LN2

temperature to maintain the fat in a brittle state. Therefore, the aluminum

support plates were also precooled in LN2. Although the mill still did not

perform as well with the fat tissue as with the liver tissue, the properties

of the homogenate appeared to be suitable for analysis. The fibrous muscle

tissue was also not completely homogenized during the selected 4 min cycle.

About 14 percent of the material remained on the 40 mesh sieve. This fraction

consisted mostly of fibers about 1 cm long and 1 mm thick. Although a considerable number of smaller fibers were found in the 40 to 60 mesh fraction,

these fibers could be broken up by further milling. After an additional 4 min

homogenization, only 2 percent of the material was left in the course fraction

above 40 mesh. For the particles passing through the 60 mesh sieve, the

particle size distribution for the fiberous tissue and the adipose tissue was smaller compared to the liver sample.

Using the information about particle size distribution and some assumptions as to the chemical nature of the liver, the weight of sample required to

hold the sampling standard deviation to a preselected level can be theoretically calculated starting with the computation of N, the number of particles.

The value of N may be calculated from the relation

where di and 62 are the densities of the two kinds of particles, d is the

density of the sample. Pi and P2 are the percentage compositions of the

component of interest in the two kinds of particles, P is the overall average

composition in percent of the component of interest in the sample, R is the

percent relative standard deviation (sampling error) of the sampling operation, and p and 1-p are the fractions of the two kinds of particles in the

bulk material (7,9). For a two component particle mix made up of cells and

fluids and for 40 mesh particles, the element Na requires a one-gram sample

to reduce the sampling error to one percent. This is very consistent with

the sampling constant of 0,95 g found as described above.

In addition to demonstrating the reduction in particle size, data supporting the homogeneity of the material was obtained by elemental analysis of

the homogenate. Two differently prepared human liver homogenates were each

subsampled into 20 jars with each jar containing about 7.5 g (fresh weight).

N

= 100(P^ - P2)

R P p(l - P)

31
The subsamples were lyophilized and randomly selected test portions of 250 mg

lyophilized material (corresponding to about 1 g fresh weight) were analyzed

for 26 elements using neutron activation analysis. The analytical procedures

are described in Section 7. The results which include the observed standard

deviation R from the set of 10 samples and the uncertainty a due to counting

statistics of the individual measurements are summarized in Table 2. If the

analytical uncertainty (in this case o) is small or insignificant relative to

R, then R is an estimate of the inhomogeneity in the material. None of the

elements determined showed any inhomogeneous distributions exceeding the

analytical uncertainty when either the ball mill or the disk mill was used.

The sample standard deviation R depends mainly on the dominant analytical

uncertainty a which is due to the counting statistics. Therefore, no significant difference can be observed between ball mill and disk mill. However,

the disk mill provides a finer particulate homogenate as shown in the sieving

experiments and, because of mechanical requirements, can homogenize larger

samples. For the majority of elements, R is smaller than 5 percent and less

than 2 percent for several essential trace elements, for which large differences among 1-g test portions were observed previously (1). This suggests

that subsampling errors due to inhomogeneity can be confined to less than

2 percent.

In another experiment, mussel {Mytilus edulis) samples were homogenized

in a disk mill and then sieved. The sieved fractions were then analyzed by

instrumental neutron activation analysis (INAA) for determination of selected

trace elements. Representative percentages of the amount of material in each

sieve fraction were obtained. Some interesting results from this experiment

are shown in Table 3. The sodium and chlorine concentrations increase with

decreasing particle size. These results may indicate that a fraction con- taining NaCl , probably due to ice crystals from the salt water contained

inside the mussel shell at the time of shucking, grinds to a finer particle

size than the organic material.

The results obtained during the performance evaluations of the various

mills provide important knowledge on the relation of the analytical test

portion to the bulk material. This information significantly increases the

quality and the usefulness of the analysis. The favorable comparison of the

theoretical calculations of sampling parameters with experimental results

validates the assumption that frozen tissue homogenate has a particulate

physical nature.

Thouqh handling of the parts of the mill and of the materials at low

temperatures is inconvenient, the Teflon® disk mill is recommended as an effective, contamination-free device for particle size reduction and homogenization of biological tissue. Homogenization at cryogenic temperatures

reduces the loss of volatile components and changes in composition during the

size reduction step. Samples as large as 1000 g can be homogenized using

this technique. The procedure uses a commercially available drive unit which

is fast and simple to operate. The quality and quantity of the samples

produced should make this technique useful for sample preparation of

biological tissues for analysis.

32
TABLE 2. HOMOGENEITY OF TWO CRYOGENICALLY HOMOGENIZED LIVERS, MEAN CONCENTRATIONS BASED ON WET WEIGHT.^

Ball Mill B, Liver ID: L1B0014B Disk Mill #2, Liver ID: L1M0344B

Element Method _N X R (%) a {%) _N X R (%) a (%)

Major and Minor Components

(mg/q) (mg/g)

c PGAA o 143 5.0 3.0

N PGAA 8 29.5 4.1 2.0 _

H PGAA 8 20.2 2.6 0.3 _

P PGAA 8 3.01 7.0 16

K PGAA 8 2.95 3.1 1.0

K INAA 1

2. 92 3.6 2.3 11 2.8 6.0 9

S PGAA 8 2 13

CI PGAA 8 1.08 3.0 1 .5

CI INAA 10 1.00 3.3 1.0 11 1.38 2.2 1 .0 Na INAA 10 0.84 1.4 0.2 n 1.10 1.2 1 .2

Trace Components

(yg/g) (pg/g)

Fe INAA 10 196 0.85 0.7 10 430 1 .4 0.4

Mg INAA 10 150 20 10 11 150 n' 9* Zn INAA 10 41.9 0.87 0.2 10 59.5 1 .2 0.3

Rb INAA 10 9.52 2.2 1.3 10 (1)^ 1.2 0.7

Cu INAA 10 5.88 16 10 Br INAA 10 1 ^ u . o in 1 9 1 .

Cd PGAA 8 1.31 3.4 1.5

Cd INAA 10 1.34 7.0 6.0 10 0.98 4.9 6.5

Mn INAA 10 1.23 5.0 3.5 11 1.31 3.2 2.3

Ul tratrace Components

(ng/g) (ng/g)

Se INAA 10 518 1.5 0.7 10 512 1.9 1.0

B PGAA 8 486 3.4 6.0

Mo INAA 10 180 7.0 7.0 10 528 1.2 10

Hg INAA 10 140 6.0 2.5 10 107 11 5.0

Co INAA 8 47.1 4.1 1.0 10 46.5 1.8 0.9

Cr INAA 8 25 30 25 10 26 17 15

La INAA 10 21 h 12 13 10 20 25 25

Cs INAA 10 (1)^ 7.0 5.0 10 14.9 5.4 3.8

Sb INAA 10 16.7 5.0 4.0 10 6.1 4.6 6.6

Sc INAA 10 0.17 12 13 10 0.40 14 10

The analysis was carried out on freeze-dried samples. Dry weight factors of the

individual samples (f=0.2626 to 0.2703) were used for conversion to wet weight.

Relative values of specific activity only (no standards used).

NOTE: N = number of subsamples analyzed. J. = mean concentration. R = relative sample

standard deviation, a = Relative uncertainty of individual determination due to

counting statistics. PGAA = neutron capture prompt gamma activation analysis.

INAA = instrumental neutron activation analysis.

33
TABLE 3. CONCENTRATION OF SODIUM AND CHLORINE IN MUSSEL SIEVE FRACTIONS

(mg/g DRY WEIGHT).

Sieve Fraction Unsieved >40 40-60 60-80 80-100 100-400 140-200 <200

Mesh Range

Weight Percent 2 25 18 14 126 12 3

Na 61.0 54.0 53.8 57.2 61.9 65.5 70.7 71.8

CI 111 79.8 96.7 102 no 116 121 128

REFERENCES

1. Lievens, P., J. Versieck, R. Cornelis, and J. Hoste. The Distribution

of Trace Elements on Normal Human Liver Determined by Semi automated

Radiochemical Neutron Activation Analysis. J. Radioanal. Chem., 37:483,

1977.

2. Schicha, H. , K. Kasperek, V. Riedel, L. E. Feinendegen, K. Vyska, and

W. Miiller. Trace Elements in Normal Mammalian Tissue and Corresponding

Malignant Tumors. In: Nuclear Activation Techniques in the Life

Sciences, International Atomic Energy Agency, Vienna, 1972. p. 451.

3. Harrison, S. H. , R. Zeisler, and S. A. Wise. Pilot Program for the

National Environmental Specimen Bank - Phase I. EPA-600/1 -81 -025.

U.S. Environmental Protection Agency. 54 pp.

4. Iyengar, G. V., and K. Kasperek. Application of the Brittle Fracture

Technique to Homogenize Biological Samples and Some Observations Regarding the Distribution Behavior of the Trace Elements in Different

Concentration Levels in a Biological Matrix. J. Radioanal. Chem., 39:

301, 1977.

5. Moody, J. R., and R. M. Lindstrom. Selection and Cleaning of Plastic

Containers for Storage of Trace Element Samples. Anal. Chem., 49:2264,

1977.

6. Visman, J. A General Sampling Theory. Mat. Res. Stds., 9:8, 1969.

7. Kratochvil, B., and J. K. Taylor. Sampling for Chemical Analysis.

Anal. Chem., 53:924A.

8. Ingamells, C. 0., and P. Switzer. A Proposed Sampling Constant for Use

in Geochemical Analysis. Talanta, 20:547, 1973.

9. Benedetti-Pichler, A. In: Physical Methods of Chemical Analysis,

W. M. Berl, Ed., Academic Press, New York, Vol. 3, 183, 1956.

34
SECTION 4

DEVELOPMENT AND VERIFICATION OF A PROTOCOL FOR THE ANALYSIS

OF INORGANIC CONSTITUENTS IN HUMAN LIVERS

by

R. Zeisler

INTRODUCTION

General criteria for the selection of analytical techniques for specimen

banking activities have been discussed previously (1). These selection criteria are: sensitivity, accuracy, reliability, availability, cost, precision,

speed, applicability to specific elements and species, minimal sample pretreatment, and multielement capabilities. Although a large number of trace

element analytical techniques which fulfill all or most of the above criteria

can be mentioned, each with their own advantages and limitations, only a few

can be selected for a routine monitoring program. From the well-established

analytical techniques available at NBS, the following were considered and

evaluated for the human liver analyses: atomic absorption spectrometry (AAS),

inductively coupled plasma emission spectrometry (ICP), isotope dilution mass spectrometry (IDMS), neutron activation analysis (NAA), voltammetry (VOL), and

x-ray fluorescence analysis (XRF).

COVERAGE OF PRIORITY ELEMENTS

Although all stable elements (and some radioactive elements) may occur in

biological tissues, only a small number are known for their biological functions: the major constituents of the organic matrix, the mineral elements,

and the essential trace elements. Equally limited is the number of elements

which have known toxic effects on biological systems at trace levels. The

first priority in the development of the specimen bank analytical protocol has

been the determination of as many elements of interest from the above groups

as possible. The elements of interest are those of environmental concern

[pollutant elements as defined by the 1978 workshop on specimen banking (1)

and EPA (2)] and minerals and trace elements of biological interest which may

be important for the assessment of nutrition and health related aspects of the

individuals (Table 1). With the measurement of as many elements as possible

from these groups, a set of data will be created which has the same analytical

basis (i.e., sampling protocol, processing, and measurement procedures),

thereby possibly providing new information about interelemental relationships,

the relationships of elements to health and nutrition, and the impact of

pollutant elements on the biological system. Some additional elements are

35
TABLE 1 . ANALYTICAL PROTOCOL FOR HUMAN LIVER SPECIMENS

Priority^ Method Detection Limit (yg/g)'^

RpDC 11 r\r\0 n nnofi

Na 3 NAA 1 .0

Mg 3 NAA 2.0

Al 2 AAS/NAA 0.1/0.5

CI 3 NAA 20

v •3O IN MM 1 nn

Sc 4 NAA 0.0001

V 1 NAA 0.02

Cr 1 NAA 0.02

Mn 1 AAS/NAA 0.2/0.05

Fe 3 NAA 1.0

Co 1 NAA 0.0005

Ni 1 AAS 0.01

Cu 2 AAS/IDMS 0.2/0.1

NAA/VOL 1.0/0.2

7n 1

1 NAA/VOL 0 1/10

As 1 AAS/NAA 0.001/0.02

Se 1 AAS/NAA 0.05/0.01

Br 3 NAA 0.1

Rh NAA n 1 Mo 1 NAA 0.2

Ag 2 NAA 0.02

Cd 1 AAS/NAA/VOL 0.01/0.1/0.05

Sn 1 r NAA 0.005

Cs

/I4

M A A NAA U. U 1 La 4 NAA 0.05

Ce 4 NAA 0.05

Pt 1 NAA^ 0.000002

Au 4 NAA 0.0001

Hg 1 NAA 0.005

Tl 1 IDMS 0.0001

Pb 1 IDMS/VOL 0.01/0.01

^1 = First priority elements from Ref. 1.

2 = Additional pollutant elements from Ref. 2.

3 = Biological mineral and trace elements.

4 = Elements possibly suitable for monitoring contaminations.

'^For human liver samples with the applied analytical protocol using 'vl g

(wet weight).

^Newly developed radiochemical NAA procedures, not yet routinely applied.

36
determined since they are detectable by a multielement technique thereby

providing additional information at relatively little additional cost.

The techniques considered for this program are limited in their sensitivities. These limitations are made greater by the small sample size available

for analysis in the Pilot NESB Program. The attractive multielement techniques

ICP and XRF could not be readily applied to the NESB liver samples bcause of

the limited amount of sample available for analysis and, therefore, the lack

of sensitivity for many of the elements of interest. Using the remaining four

techniques (i.e., AAS, IDMS, NAA, and VOL), nearly complete coverage of the

elements of interest (31 elements) has been achieved. The implemented analytical techniques are summarized in Table 1. Table 1 also includes the

experimental detection limits which were achieved during the analysis of 36

liver specimens collected during the first year of the Pilot NESB Program.

During the first year not all of the first priority elements (Ref. 1 and

Table 1) were determined in the complete set of 36 human liver samples.

Fluorine was excluded from the protocol because of the use of Teflon® materials

for homogenization and storage. Four elements (Be, Cr, V, and Ni) were at or below the detection limits of the applied analytical procedures. A modification in the AAS procedure allowed the determination of nickel in some of the

samples. The NAA procedure was not sensitive enough for the low concentrations of chromium in most of the samples. However, improvements in the NAA

instrumentation will provide improved sensitivity, thereby allowing for

determination of chromium at these low levels in the second-year liver analyses.

The analytical protocol for the second year will also be modified to include

the determination of chromium by AAS in addition to NAA. Vanadium is also

expected to be determined in the future by NAA using improved counting

techniques. To determine the low levels of beryllium successfully, research

has to be directed towards a more sensitive procedure, unless much larger

samples are made available for the AAS procedure to allow a preconcentration

step before the analytical measurement.

The experimental detection limits reported in this work (see Table 1) are somewhat higher than literature data which are generally reported for optimized

conditions. Since these techniques are obviously more sensitive if the experimental parameters are optimized, there is significant potential for improved

procedures which would result in greater sensitivity and more complete

elemental coverage in the future. In addition, recently developed analytical

methodologies (see Appendices I, II, and V) will be incorporated. In spite of

the above mentioned limitations of the applied analytical protocol, the data

set for the first year liver samples (31 elements in 36 liver specimens) represents the most complete data set available using four analytical techniques.

QUALITY ASSURANCE

The second criterion in the development of the analytical protocol was the implementation of two procedures to assure the quality of chemical

analysis: (1) the simultaneous analysis of standards (Standard Reference

Materials) with the same or a similar matrix as the analyte and (2) the

determination of constituents by two or more independent analytical methods.

37
All of the methods which were considered for use in this protocol have

been used for the certification of biological and environmental Standard

Reference Materials (SRM's). During the planning of the analytical phase of

the Pilot NESB Program, the capabilities of various analytical methods were evaluated on the basis of results from the ongoing certification analyses of

SRM 1577a and b, bovine liver, (renewals of the widely used SRM) . The similarity of the SRM matrix made the analytical procedures immediately available

for the human liver analyses. During the course of the first-year human liver

analyses, several of the analytical procedures were validated by analyzing the

SRM 1577 parallel to the NESB specimens (see Sections 6 and 7 and Appendix I).

The parallel analyses of SRM's provided an essential method of validation

for the acceptability of the results during actual routine implementation.

However, occasional blunders, contamination, and/or analytical errors still

may occur and provide incorrect data particularly when only one sample is

available for analysis. Good analytical practice calls for replicate and

independent determination of the constituents in a sample. Due to the limited

amount of analytical test portions of each liver sample in the pilot program,

a three-fold approach was incorporated into the analytical protocol to attain

a second means of continuous quality assurance. First, NAA was selected to

analyze the homogenate of each specimen in duplicate because of the small

sample size required for this technique. Second, as many elements as economically possible were determined using different techniques on additional

sample aliquots (Table 1). The use of two different techniques serves in

place of replicate analysis by one technique to eliminate the occasional

analytical blunder and also to protect against systematic errors of a single

technique. Third, sufficient sample homogenate is banked (as well as the

complete second section of each liver sample) to repeat an analysis if a result appears to be questionable.

CONCLUSION

The outlined philosophy for the selection of analytical methodology for

the analysis of human liver samples in the Pilot NESB Program has resulted in

a working analytical protocol for the determination of a large number of

elements in each of the selected specimens. Using this analytical protocol,

in conjunction with the previously described sampling protocol, should provide

high quality data for most elements of environmental and biological interest.

This protocol is a flexible analytical plan which is open for improvements in

analytical methodology as they may occur as well as for the addition of new procedures which provide for the inclusion of additional priority elements or

for better quality control.

REFERENCES

1. Luepke, N.-P. Monitoring Environmental Materials and Specimen Banking.

Marti nus Nijhoff Publishers, The Hague, The Netherlands, 1979. 591 pp.

2. U.S. Environmental Protection Agency. Quality Criteria for Water,

Environmental Protection Agency, Washington, DC, 1976.

38
SECTION 5

DETERMINATION OF SELECTED TRACE ELEMENTS IN HUMAN LIVER SAMPLES

BY ATOMIC ABSORPTION SPECTROMETRY

by

T. C. Rains, T. A. Rush, and T. A. Butler

INTRODUCTION

Atomic absorption spectrometry (AAS) is a unique analytical technique

for the determination of metallic elements. The absorption of radiant energy

by ground state atoms in the gaseous state is the basis of AAS. When radiation of proper wavelength passes through a vapor containing ground state

atoms, some of this radiation will be absorbed by excitation of particular

atoms (1). The magnitude of the absorption signal is directly related to the

number of these ground state atoms found in the optical path. Ground state

atoms necessary for the measurements are produced from the sample material by

vaporization of the material introduced into an appropriate excitation source followed by dissociation of the molecular species into neutral atoms.

Normally these steps are carried out by a nebulizer associated with a flame or electrochemical device (1-3). The basic components of an AAS instrument

are described in detail in the literature (2).

Since AAS is a relative technique, a quantitative determination can only

be carried out by comparison to a reference solution. If the behavior of the

sample is different than the behavior of the reference solution, it is implied

that an interferent is present. Depending upon the cause, the interferent can

be classified as chemical, physical, background, or spectral. While background and spectral interferences are based on measurement of non-specific

signals, chemical and physical interferences have an influence (positive or negative) on the total number of atoms formed per unit volume of the absorption cell. Of the interferences encountered in biological tissue, such as human liver, the chemical form is most prevalent. Also, chemical interferences

are most likely to prevail in electrothermal atomization than in flame

excitation devices. For those elements being determined in human liver and

described in this report, (i.e., Cu, Mn, Al , Be, Ni , Cd, As, and Se) , AAS

with electrothermal atomization was used because the concentration of the

analytes were below the detection limits by flame excitation devices.

The first furnace or electrothermal atomization device proposed for AAS

was described by L'vov (4). In his early work, a solution was placed on the

end of an electrode and evaporated to dryness. The sample was then vaporized

39
by a dc arc into a carbon furnace. This device produced impressive detection

limits but was limited because of power requirements and poor precision.

Massmann (5) constructed a somewhat simple graphite furnace which is

basically being used by all manufacturers today.

In this report procedures are described for the determination of selected

trace elements in human liver by AAS using electrothermal atomization or hydride generation.

EXPERIMENTAL

Reagents

High purity acids were prepared using subboiling distillation (6). All

reagents used in sample preparation or matrix modification were prepared and

stored in clean Teflon® FEP bottles unless otherwise stated.

Instrumentation

The instrumental systems used in this study consist of a Perkin-Elmer

Model 603 and Perkin-Elmer Model 5000 atomic absorption spectrometers with

HGA-2100 and HGA-500 graphite furnaces, respectively. A 20-yL aliquot of

sample was introduced into the furnace using an autosampler. The instrumental

parameters are given in Table 1.

TABLE 1 . INSTRUMENTAL PARAMETERS FOR ATOMIC ABSORPTION SPECTROMETRY

Element

Wavelength

nm

SBW'^ nm Scale

Expansion

Drying Charring

T-s

Atomization

T-s

Al^ 309.3 0.7 3 125-40 800-40 2700-6

Be 234.7 0.7 5 125-40 1000-40 2800-6

Cd^ 228.8 0.7 1 125-40 800-40 2300-6

Cu 324.7 0.7 1 125-40 700-40 2700-6

Mn^ 279.5 0.7 1 125-40 800-40 2700-6

Ni^ 223.0 0.2 5 125-40 800-400 2700-6

As 193.7 0.7 3 Hydride gener^tti on/heated

quartz cell

Se 196.0 0.7 3 Hydride generation/heated

quartz cell

^L'vov platform used within the graphite cell. The internal gas was argon.

^SBW = Source bandwidth.

^T-s = Temperature ("C) and seconds.

40
L'vov Platform

Interferences encountered with electrothermal atomization devices are more pronounced than in most flame systems, and the analyst has to rely upon

the standard addition technique or close matching of standards with unknowns

to correct for the interferences. Recent improvements in electrothermal

atomization-AAS have greatly reduced analytical interferences. Graphite used

in the absorption cell is a porous material which is easily penetrated by

liquids and gases. Atomic vapor can freely pass through a 1-mm thick wall of

hot graphite. Coating the graphite tubes with a thin layer of pyrolytic

graphite has been found to greatly reduce the effects of the porosity of the

graphite and increase the sensitivity for some elements. By inserting a L'vov platform in the graphite absorption cell, it is possible to atomize the

sample at nearly constant temperature conditions (8). This reduces analytical

interferences by volatilizing the sample into a gas which is hotter than the

surface from which the sample is volatilized.

While these platforms can be obtained commercially, they can be prepared

in the analyst's laboratory with a minimum of cost. For the system used in

this work, the platforms are constructed by cutting the two ends of a graphite

tube into six (three from each end) 7-mm x 5-mm grooved, curved sections.

These cuts are made using a small stainless steel saw. After the sections

are cut, the sides of each section are filed so that the platform will fit

the inside contour of the graphite tube (8).

The graphite tube is positioned in the furnace head. The right window

is temporarily removed and the platform inserted. The platform is then

centered directly beneath the sample port using a metal rod. Adjustments of

the automatic injector tip are made to insure that it does not come into

contact with the platform surface.

Interferences

Sublimation of the analyte during the processing step is virtually

unknown in flame AAS; however, in AAS with electrothermal atomization it is

of major importance. Since the atomization of a sample in a graphite tube

furnace can only take place from the graphite surface, it is essential that

the sample remains in good contact with the hot graphite surface for as long

as possible to become completely atomized. In contrast to flame AAS, the

formation of sulfate, phosphate or oxides in the graphite tube is often

advantageous.

Standardization

Working standards were prepared by serial dilution of aqueous stock

solutions, as described by Dean and Rains (2). These were acidified with

HNO3 to one percent to match the acid concentration in the sample solution.

Standard addition procedures were used to check for the presence of matrix

suppression or enhancement and to correct for these interferences when they

were present (7).

41
Sample Preparation

The freeze-dried samples were dried for 24 hours at ambient temperature

in a vacuum oven equipped with a liquid nitrogen trap. From a dried sample, a 0.5 to 1 g test portion was transferred to a Teflon® beaker. The sample was digested overnight after the addition of 10 mL of high purity HNO3. Then 5 mL

of HClOit and 1 mL of H2S0it were added and the sample evaporated to near

dryness. Then 1 mL of HNO3 was added and the sides of the beaker washed down.

The solution was warmed to redissolve the solids. Then, the solution was transfereed to a lO-mL volumetric flask and diluted to the calibrated volume.

The sample solution was transferred into a clean plastic (common polyethylene)

bottle for storage. The elements Al , Be, Cd, Cu, Mn, and Ni were determined

using the instrumental conditions as described in Table 1. The validity of

each analytical result was checked by the single addition method (7).

The samples for As and Se determination were prepared as described

below. A 0.5-1 g test portion of the previously dried sample was transferred

to a digestion flask and 10 mL of HNO3 and 3 mL of H2S0tt were added. A water

condenser was attached and the solution heated gently for one hour. The

water from the condenser was drained and 5 mL of HC10i+ added, the sample was then heated to sulfuric acid fumes. The flask was allowed to cool and 10 mL

of HCl was added slowly, the solution was reheated for 10-15 minutes and then

transferred to a 50-mL volumetric flask. The sample was then diluted to the

calibrated volume with water. Arsenic and selenium were then determined by

AAS using hydride generation.

The influence of some acids upon the analyte in the graphite furnace can

be attributed to the formation of easily volatile compounds which sublime as the molecule. Losses owing to deflagration of the salt at increased temperature play a part, especially when using perchloric acid and occasionally with

nitric acid. Since perchloric acid is necessary to completely oxidize the

human liver sample, a method had to be devised to remove the perchl orate

before atomization. Different matrix modifiers were found not to be effective

when perchl orate was present. To alleviate the interferences of perchl orate

the digestion procedure was modified to include the addition of sulfuric acid

and heating the sample to sulfuric acid fumes. While the sulfate ion was found in previous studies to suppress many analytes by AAS with electrothermal

atomization, with the L'vov platform the sulfate suppression was largely

removed.

Even with the L'vov platform, calcium caused a severe suppression of the

absorption signal for beryllium. In order to compensate for the interference,

calcium was added to all standards to match the calcium concentration in the

liver sample.

Cadmium compounds are known to sublime at relatively low temperatures;

therefore, it is difficult to volatilize the matrix during the charring step

without loss of cadmium. To alleviate this difficulty, a one percent solution of {NHi+)2HP0tt was added to sample and standards. With the addition of

phosphate the interference of the matrix on the cadmium absorption signal was largely removed.

42
Hydride Generation

The determination of arsenic and selenium is considerably influenced by

the fact that their resonance lines are in the vacuum UV spectrum range.

Arsenic can only be determined satisfactorily in the best AAS instrumentation

because its strongest resonance line is at 193.7 nm. Very few chemical

interferences were observed for arsenic in an air-acetylene flame; however,

the disadvantage of the flame is that 60 percent of the light energy emitted

by the radiation source (hollow cathode lamp or electrode! ess discharge lamp) was absorbed (9). The argon (entrained air) — hydrogen flame produced the same sensitivity with only 10 percent absorption of the light energy. A

typical detection limit for arsenic and selenium with the argon (entrained

air) — hydrogen flame is 0.02 yg. A further increase in sensitivity for

these elements can be obtained with the hydride system. With NaBH^ as the

reducing agent and an electrodeless discharge lamp, a detection limit of

0.0005 yg was attained. These elements are relatively interference free

provided the arsenic is pre reduced from As^ to As^^^ with potassium iodide

and Se^^ to Se^^ with HCl

RESULTS AND DISCUSSION

The results of the determinations of Be, Al , Mn, Ni , Cu, As, Se, and Cd

for 36 human liver samples are summarized in Table 2.

43
TABLE 2. TRACE ELEMENT CONCENTRATIONS IN HUMAN LIVER SAMPLES AS DETERMINED BY

ATOMIC ABSORPTION SPECTROMETRY. ------ Element Concentration (ug/g wet weight) -----

Sample

Identification Be Al Mn Ni Cu As Se Cd

L1B0002 <5xl0"'' 0.79 1 .8 <p.oi 4.96 0.011 0.44 3.71

L1B0005 <5xl0"^ 0.61 1.6 2.1 0.013 0.34 0.98

L1S0018 iSxlO""^ 0.90 2.0 2.8 0.017 0.49 2.97

L1M0023 <5xlO"'' 0.63 0.47 5.36 0.0023 0.68 2.1

L1S0027 <5xl0"'' 1.5 1.8 <0.01 4.44 < 0.0009 0.54 0.94

L1S0037 iSxlO"'^ 0.24 2.46 2.98 0.0078 0.41 2.0

L2B0041 <5xl0"'' 0.2 1.1 3.24 0.0042 0.50 0.84

L2B0042 <5xl0"'^ 0.51 2.0 5.02 0.0053 0.46 0.66

L1B0045 iSxlO"'' 0.34 1.6 2.0 0.0075 0.49 1.4

L2M0048 <5xlO"'' 0.70 0.83 <p.oi 6.84 0.0052 0.65 0.79

L2S0049 <5xlO''* 0.81 1.5 2.2 0.0091 0.48 0.45

L1S0053 iSxlO""^ 0.30 0.95 <0.01 5.80 0.027 0.65 2.4

L2M0062 <5xl0''* 2.2 1 .8 <0.02 5.92 0.0031 0.47 3.54

L1B0074 <5xl0"'^ 0.68 1.6 <0.01 5.87 0.0092 0.46 1.7

L1S0080 £5x10"^ 0.2 1 .3 0.01 4.97 0.0095 0.50 0.54

L1M0083 <5xl0"'^ 0.99 0.64 <0.01 5.25 0.011 0.50 0.82

L1S0086 <5xl0"'^ 0.45 1.4 <0.01 6.38 0.023 0.59 0.59

L1S0090 <5xl0"'' 0.93 1.1 <0.01 8.23 0.0085 0.53 2.2

L1S0091 <5xl0"'' 0.56 1 .2 0.02 5.58 0.0082 0.45 1 .4

L1S0092 <5xl0"'* 0.64 1.2 <0.01 10.8 0.0037 0.64 1.9

LIBOIOO £5x10"'' 1.6 1.6 <0.02 11.1 0.0069 0.54 2.4

L1M0105 <5xl0"'* 0.55 1.2 0.04 8.81 <0.0011 0.55 1.1

L1S0107 <5xl0"'' 1.1 1.5 0.037 10.1 0.011 0.63 2.6

LlSOllO <5xlO"'' 0.53 1.0 0.036 4.36 0.0064 0.61 2.1

L1M0118 <5xlO"'' 0.56 2.0 0.09 4.57 0.0070 0.59 2.5

L1M0119 <5xlO"'' 31 .6 1 .8 0.10 4.93 2.0 0.48 1.8

L1B0121 <.5xl0"'' 0.67 1.1 0.013 7.49 £0.0010 0.49 0.39

LI SOI 23 <5xl0"'' 0.42 1.2 0.47 8.41 0.012 0.53 1.8

L1M0126 <5xl0"'' 11 2.0 0.062 7.39 0.097 0.56 4.84

L1M0127 <5xl0"'' 2.1 1.1 0.076 4.26 0.0094 0.44 0.63

LI SOI 28 <5xl0"'' 0.34 0.61 0.02 8.98 0.022 0.56 1.2

LI SOI 29 < 5x1

0*^ 1.2 1.3 0.02 12.5 0.013 0.62 2.5

L1M0130 £5x1

0"5

0.2 1.2 0.029 6.6 0.012 0.52 0.45

L1M0131 <5xl0"'' 0.92 0.62 0.02 4.97 0.0087 0.46 0.76

L1M0140 <5xlO"'' 0.60 1.4 0.02 6.89 0.010 0.60 1.1

L1M0141 <5xl0"'' 0.79 0.95 0.014 4.45 0.014 0.41 0.25
REFERENCES

1. Dean, J. A., and T. C. Rains, eds. Flame Emission and Atomic Absorption

Spectrometry. Vol. 1. Theory. Marcel Dekker, New York, 1969.

2. Dean, J. A., and T. C. Rains, eds. Flame Emission and Atomic Absorption

Spectrometry. Vol. 2. Components and Techniques. Marcel Dekker,

New York, 1971.

3. Christian, G. D. , and F. J. Feldman. Atomic Absorption Spectroscopy.

In: Application in Agriculture, Biology, and Medicine. WileyInterscience, New York, 1970.

4. L'vov, B. V. Atomic Absorption. In: Spectrochemical Analysis. Adam

Hilger, London, 1970.

5. Massmann, H. Spectrochimica Acta 236:215-226, 1968.

6. Keuhner, E. C, R. Alvarez, P. S. Paulsen, and T. J. Murphy. Anal. Chem.

44:2050, 1972.

7. Dean, J. A., and T. C. Rains, eds. Flame Emission and Atomic Absorption

Spectrometry. Vol 3. Elements and Matrices. Marcel Dekker, New York,

1975.

8. Slavin, W. , and D. C. Manning. Anal. Chem. 51:261-265.

9. Menis, 0., and T. C. Rains. Anal. Chem. 41:952-954, 1969.

45

SECTION 6

THE DETERMINATION OF LEAD, THALLIUM, COPPER, AND NICKEL IN

HUMAN LIVER SAMPLES BY ISOTOPE DILUTION MASS SPECTROMETRY

by

J. W. Gramlich, L. J. Powell, K. A. Brletic, and T. J. Murphy

INTRODUCTION

Isotope dilution mass spectrometry (IDMS) has been used extensively for

the certification of elemental concentrations in Standard Reference Materials

(1,2) and is regarded as a "definitive method", i.e., a method of proven

accuracy. In IDMS, the quantity of an element present in a material is

determined from the change produced in its isotopic composition by the

addition of a known amount of a stable isotope of the element. After

equilibration and separation of the element, the altered isotopic composition is measured by thermal ionization mass spectrometry and the concentration

of the natural element is calculated.

To achieve high accuracy, careful attention must be given to the

elimination of possible systematic errors due to errors in the calibration

of the spike isotope, isobaric interferences, non-equilibration of spike and

natural isotopes, and contamination or blank. In a carefully designed

analysis these errors are negligible and the major sources of error are the

random errors present in the isotope ratio measurements for the spike calibration and the analysis. These errors are reflected in the precision of the

isotopic measurements and are on the order of ±0.05 to 0.2 percent for many

elements. Therefore, an absolute accuracy of ±0.1 to 0.4 percent is possible

even for a very low concentration in a complex matrix.

Because of the high accuracy attainable with IDMS, this technique was used to determine lead, thallium, copper, and nickel in a number of human

liver samples while restricting sample size to 0.5 g or less.

Due to the low concentrations expected for thallium and nickel in human

liver, it was necessary to develop procedures for these elements. This method

development was accomplished using SRM 1577a, Bovine Liver. This SRM material

is certified for lead and copper but not for thallium or nickel. Based on the results using SRM 1577a, the procedures described in this report were

adopted for the determination of these elements in the human liver samples.

47
EXPERIMENTAL

The liver samples had been previously freeze-dried; however, since they

may have absorbed water on standing, they were redried by overnight vacuum

drying at room temperature. Approximately 0.5-g samples of each liver were

spiked with ^^^Pb, ^^^Tl, ^^Cu, and ^^^i . The samples were then wet-ashed by

the addition of 10 g HNO3 and 1 g HCIO1+ and heating (covered) on a hot plate.

The covers were removed and the samples were heated until fumes of perchloric

acid were no longer visible. The sides of each beaker were washed down with

water and the heating repeated. The residues were taken up in 20 mL of H2O,

covered with Parafilm and two-50 mm platinum wires were inserted through the

film. One wire was connected to the anode of a d.c. -power supply and the

other wire was connected to the cathode. The solution was electrolyzed over- night at 2.0 V applied while stirring magnetically. (Lead and thallium

deposit on the anode as Pb02 and T1203 and copper deposits on the cathode as

Cu metal.) The electrolyte was reserved for the determination of nickel.

Determination of Lead and Thallium

The mixed Pb02 — TlzOs deposits were stripped from the electrodes with

dilute HNO3 — H2O2. The resulting solution was evaporated to dryness and the

residue was taken up in two percent (w/w) HNO3 so that the lead concentration

was about 100 yg/mL. The concentration of thallium is taken at whatever

level is present in the solution, since past experience has shown that the

results for thallium are relatively insensitive to the amount of material

under the analysis conditions employed.

A single filament rhenium source was used for the sample mounting and

analysis. Prior to sample loading, the filament was degassed in a vacuum and

under a potential field for 30 minutes at a current through the filaments

of 3 A.

A 5 uL drop of a collodial silica gel solution was dried on the filament

with a current of 1 A for 5 minutes. A 5 yL drop of the sample solution

(0.5 yg Pb) was then dried on the silica gel layer under the same conditions.

A 5 yL drop of 0.75 N H3PO1+ was then dried on the filament at a current of

1.3 A for 5 minutes followed by 1.5 A for 5 minutes. The filament was then

heated at a current of 2 A until the excess H3P0tt was seen to fume off. After

fuming had stopped, the filament temperature was increased to approximately

800 °C for 2 seconds. The filament was then immediately loaded into the mass spectrometer.

The analysis procedure was as follows: the filament temperature was increased to produce a Tl"*" ion current of approximately 3 x 10'^^ A at the

collector. The temperature of the filament was about 700 °C. A steady signal

intensity was obtained and 20 minutes of data were collected starting at 15

minutes into the analysis. After measurement of the thallium isotopic ratios,

the filament temperature was increased to 1100 °C. After 5 min and 10 min the

filament temperature was increased to 1150 °C and 1200 °C, respectively. Lead

isotopic measurements were obtained between 30 min and 55 min after the

filament temperature was increased to 1100 °C.

48
The thallium blanks for this procedure averaged 0.02 ng Tl or about

1 percent of the thallium in the samples. The lead blanks averaged 2.3 ng or about 0.2 percent of the lowest lead concentration. Due to limited samples,

natural lead isotopic compositions were measured for only two samples and one composite from several samples. The average isotopic composition was used for

calculation of concentration.

Determination of Copper

The cathode from the electrolysis was used for the determination of Cu in

the sample. The electroplated copper was dissolved with a few mL of 25

percent (w/w) HNO3 and the resulting solution was evaporated to dryness. The

residue was taken up in a drop of 2 percent (w/w) HNO3 for mass spectrographic

analysis. The analyses were performed using a triple filament thermal ionization mass spectrometric procedure. In this procedure, approximately 20 yg of

copper, dissolved in 2 percent (w/w) HNO3 solution was electroplated onto

each of two zone-refined rhenium sample filaments. After the plating was completed, the filaments were flushed with a stream of high purity water to remove residual acid and dried under a heat lamp. The filaments were mounted

into a triple filament configuration, loaded_into the mass spectrometer, and

the system was evacuated to less than 5 x 10"^ torr.

The mass spectrometric procedure is designed to yield a stable ion

current of 4-7 x 10'^^ A for the ratio measurement. This is accomplished by an initial adjustment of the ionizing filament to 2120 °C and maintaining

this as a constant temperature throughout the analysis. The sample filament

temperature is increased in a series of discrete increments, at precise time

intervals, to yield predetermined signal intensities. At 30 minutes into the

heating pattern, a series of three sets of the ^^Cu/^^Cu ratio measurements

are obtained.

Determination of Nickel

The samples from the determination of Pb, Tl , and Cu were again electrolyzed at 2.0 V applied using Pt wire anodes and cathodes. If Cu was noted on the cathode, the procedure was repeated until copper was no longer detected.

The solution was then made ammoniacal with 0.5 mL of concentrated ammonium

hydroxide and Ni was electrodeposi ted on the cathode by electrolyzing over- night at 2.4 V applied. The electrodeposited nickel was stripped from the

electrode with a few mL of 25 percent (w/w) HNO3 and the resulting solution

was evaporated to dryness.

The altered nickel isotope ratios were then measured by thermal ionization mass spectrometry using a silica-gel procedure to enhance ionization. A

platinum filament was coated with a layer of silica-gel. The sample residue

was taken up in 2 percent (w/w) HNO3 and a drop equivalent to about 5 yg of

Ni was added to the silica gel. A drop of 0.75 N phosphoric acid was added

and dried. The filament was transferred to a nitrogen atmosphere and dried

at 900 °C for 15 s (temperature was measured using a pyrometer). The

filament was then loaded into the source of the mass spectrometer and the

analysis was started when the pressure reached 1 x 10"^ torr. At t = 0, the

49
temperature was increased to 1150 °C, at t = 5 min to 1200 °C and at t = 10

min to 1250 °C. Ratio data were taken from 20 to 40 min.

RESULTS AND DISCUSSION

To monitor the effect of the increasing influx of anthropogenic sources of trace elements on human health, it is imperative to collect accurate baseline data regarding the concentration of environmentally important substances

in the present population so that future changes can be detected. Lead and

thallium are two elements that are known to be toxic at trace levels. To

achieve the required levels of sensitivity and accuracy, an IDMS method was developed and applied to human tissue samples. Since the available samples were small (0.5 g dry weight) and the expected concentrations low (1-5 ppb

Tl), blanks for the procedure would have to be extremely low. A method that

accomplished these objectives was developed using SRM 1577a, Bovine Liver, as

a control sample.

The results of the determinations of Pb on 0.5-g samples of SRM 1577a,

Bovine Liver, yielded an average value of 0.136 ± 0.012 yg Pb/g compared to

0.134 ± 0.08 yg Pb/g for one gram samples using a more extensive separation

technique (Pb only). The thallium concentration was found to be 3.1 ± 0.1 ng

Tl/g. Lead blanks for the procedure averaged 2.0 ng Pb and the thallium

blanks averaged 0.03 ng Tl

In addition, three samples of SRM 1577a were analyzed for copper with

this method using 0.5-g samples. The samples averaged 151.7 yg/g Cu.

SRM 1577a has not yet been certified for copper, but determinations by spark source IDMS and atomic absorption on large samples yield concentrations of

152 to 164 ppm Cu.

The results of the determination of these elements in 13 human liver

specimens are summarized in Table 1. The trace element concentrations were determined on a dry weight basis and then converted to wet weight concentrations using the concentration factors (dry weight/wet weight) for each

specimen (see Table 4, Section 2 for concentration factors). The thallium

concentrations are estimated to be accurate to ±1 percent mainly due to

blank; the lead concentrations are estimated to be accurate to ±1 percent

mainly due to uncertainty in isotopic composition. The copper blanks

amounted to about 0.5 percent of the copper present in the samples. The

total uncertainty in the copper measurement is estimated to be ±1 percent.

50
TABLE 1. TRACE ELEMENT CONCENTRATIONS IN HUMAN LIVER SAMPLES

AS DETERMINED BY ISOTOPE DILUTION MASS SPECTROMETRY

Element Concentration (yg/g wet weight)

Sample Tl ^ Identification Ni Cu (xlO""^) Pb

L1B0002 <0.08 5.09 0.335 1.15

L1B0005 <0.03 4.45 0.370 0.39

L1S0018 <0.03 5.30 0.292 0.811

L1M0023 N.D. 4.98 1.67 0.17

L1S0027 <p.l 8.56 0.39 0.992

L1S0037 <_Q.2 5.25 0.148 0.263

L2B0041 N.D. 6.08 1.38 0.665

L2B0042 <^0.05 9.01 0.518 0.788

L1B0045 N.D. 3.52 0.363 0.25

L2M0048 <^D.07 6.05 0.300 0.776

L2S0049 <0.60 3.79 0.441 0.513

L1S0053 N.D. 5.42 1.28 1.66

L2M0062 <0.07 5.08 0.477 0.254

The results of the lead and thallium determinations and the relative

ratios of these two elements are shown in Table 2. Results in Table 2 are reported on a dry weight basis for comparison to the bovine liver SRM.

51
TABLE 2. CONCENTRATIONS (DRY WEIGHT) OF LEAD AND THALLIUM AND THEIR

RELATIVE RATIOS

Sample

Identification

Concentrations

uq Pb/q

(dry weight)

ng Tl/g

Ratios

Pb/Tl

L1B0002 4.16 1.21 3436

L1B0005 1.31 1.24 1056

L1S0018 2.83 1.02 2774

L1M0023 0.71 7.07 100

L1S0027 4.38 1.70 2576

L1S0037 1 .11 0.62 1778

L2B0041 2.56 5.31 482

L1B0042 3.21 2.11 1521

L1B0045 0.90 1.31 687

L2M0048 3.41 1.32 2583

L2S0049 2.15 1.85 1162

L1S0053 6.05 4.66 1298

L2M0062 1.15 2.16 532

SRM 1577, Bovine Liver 0.134 3.1 43

Earth's Crust^ 20

Granite^ 33

Shale^ 20

Basalt^ 40

^Source - R. D, Reeve and R. R. Brooks, "Trace Element Analysis of

Geological Material," J. Wiley & Sons, N. Y., NY, 1979, pp. 80-81.

These results show that thallium concentrations in human liver are in the same range as in bovine liver (1-7 ng Tl/g compared to 3 ng Tl/g in the bovine

liver), whereas lead in human liver is considerably elevated over bovine liver

(1-6 yg Pb/g compared to 0.1 yg Pb/g). This increase becomes more evident

when one compares the ratio of lead to thallium found in various types of

samples. For most of the human liver samples, this ratio exceeds 1000/1

while the ratio for bovine liver is near that of the earth's crust and various

rocks, 20-40/1. These results could be due to the fact that cattle are less

exposed to major anthropogenic sources of either lead or thallium. In contrast, humans are exposed to anthropogenic sources of lead but not of

thallium. Thus, monitoring the trend of Pb/Tl ratios could be an indicator

of the degree of lifetime exposure to anthropogenic lead sources.

52
The determination of nickel in the liver samples was attempted on a research basis. The expected concentrations, <1 yg/g, were lower than any

previous nickel determinations by IDMS. In addition, the dimethyl glyoximeion exchange separation method which was used previously provided blanks which

were too high for these determinations. The silica gel mass spectrographic

method was developed which employed much smaller amounts of Ni for isotope

ratio determinations. To reduce the blank, Ni separations were performed by

electrodeposition from ammoniacal solution. Preliminary experiments showed

good nickel recoveries from synthetic solutions. The recoveries of Ni from

the SRM Bovine Liver samples were promising; however, the analytical results

showed the SRM material to be inhomogeneous for nickel.

Although these initial experiments for the determination of nickel were

promising, difficulties were encountered during the actual analyses of the

human liver samples. These difficulties were: [1] low signal for some samples during the MS analysis indicating poor recovery, and [2] a few high

blanks. Because of these problems, only upper limits are reported for those

samples that gave good intensity during the isotopic analysis. Results for

samples with low intensities are not reported. The results for nickel are summarized in Table 1. Even though these analyses were not completely

successful, further development of this method may yield a procedure that can

be used to determine Ni accurately in human liver specimens at the low ppm

level on 0.5 g samples.

REFERENCES

1. Moore, L. J., J. W. Gramlich, and L. A. Machlan. Application of Isotope

Dilution Mass Spectrometry to the High Accuracy Trace Analysis of

Environmental and Health Standards. In: Trace Substances in Environmental Health-IX. University of Missouri, Columbia, MO, 1975, p. 311.

2. Machlan, L. A., J. W. Gramlich, T. J. Murphy, and. I. L. Barnes. The

Accurate Determination of Lead in Biological and Environmental Samples

by Isotope Dilution Mass Spectrometry. In: Accuracy in Trace Analysis:

Sampling, Sample Handling, and Analysis. P. D. LaFleur, ed. NBS Spec.

Publ. 422, 929, 1976.

53
54
SECTION 7

INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS FOR THE DETERMINATION OF

MINERAL AND TRACE ELEMENTS IN HUMAN LIVER SAMPLES

by

J. Bailey, K. A. Fitzpatrick, S. H. Harrison, and R. Zeisler

INTRODUCTION

Instrumental Neutron Activation Analysis (INAA) is a well-established

multielement technique which has been applied to the chemical analysis of a wide variety of matrices and has been used frequently to analyze human tissues

(1-3). Using INAA, inorganic constituents can be measured in a broad dynamic

range from virtually 100 percent to 10'^^ g/g gpcl^ -jp special cases, even

lower. For most practical applications, INAA is matrix independent and free

of chemical blanks since most materials are analyzed without chemical sample

preparation. When applied to biological materials, INAA is sufficiently

sensitive for the determination of most of the important pollutant trace

elements and essential trace elements since the major constituents (H, C, N,

0) do not form measurable interferences. Even these matrix elements can be

determined using the Prompt Gamma Activation Analysis (PGAA) method which is

currently under evaluation (see Appendix V).

The goal of the INAA protocol for the analysis of human livers is the

determination of as many priority elements as possible with the limited amount

of sample available in the Pilot NESB Program. The "chemical separation" of

the elements is achieved by selection of appropriate irradiation and counting

conditions according to the nuclear parameters of the elements to be determined. The nuclear parameters and the conditions for the determination of

the 22 elements included in this protocol are summarized in Table 1.

PROCEDURE

Samples of 36 human liver specimens were obtained for instrumental

neutron activation analysis. Each sample consisted of one Teflon® jar con- taining 1-1.5 g of freeze-dried liver homogenate. Duplicate samples from

each jar were prepared for analysis by pressing 0.3 g of the material into

pellets using a stainless steel pellet press. Transfer of the material and

preparation of the pellets were performed in a normal fume hood to avoid

potential biohazard exposure of the persons handling the material. The pellets were packaged for irradiation in precleaned polyethylene irradiation bags

under clean room conditions.

55
TABLE 1 . NUCLEAR PARAMETERS AND INAA CONDITIONS FOR THE ANALYSIS OF HUMAN

LIVER SAMPLES

7~~Zs T~a Irradiation Time at a Neutron Flux of

^^°^°P^ \ 5 X 10-i3n cm-2s-i

^^Na IS.OOh 60s

^^Mg 9.48min 60s

^^Al 2.31min 60s

^^Cl 37.3min 60s

^^K 12.36h 60s

^^Sc 83. 9d 4h

^^V 3.75min 60s

^^Cr 27. 8d 4h

^^Mn 2.576h 60s

^^Fe 44. 6d 4h

^°Co 5.263y 4h

^^Cu 12.8h 60s

^^Cu 5.10min 60s

^^Zn 243. 8d 4h

^^As 26. 3h 4h

^^Se 120. Od 4h

^^Br 35. 4h 4h

^^Rb 18. 6d 4h

99Moi^99m^c 66.2h-v6.02h 4h

250. 4d 4h

115^^115mj^ 53.5H_>4.5h 4h

^^^CS 2.046y 4h

^^°La 40.27h 4h

^^^Ce 32.38d 4h

^^^Sm 46. 8h 4h

^^^Au 2.697d 4h

^^^Hg 46.59d 4h

G. Erdtmann and W. Soyka, Die x-Linien der Radionuklide, Band 1, Berichte

.der Kernforschungsanlage Jiilich-Nr. 1003, April 1974.

Value of 1078.8 keV given in the above reference is incorrect.

56
Before Counting

^^'"^^ ^'^ ^'^^ (^eV)

3h 5min 1368.6, 2754.1

2.5min 5min 843.8, 1014.4

2.5min 5min 1778.8

2.5min 5min 1642.4, 2167.5

3h 5min 1524.7

40-70d 24h 889.3, 1120.5

2.5min 5min 1434.2

40-70d 24h 320.1

3h 5min 846.6. 1811.2

40-70d 24h 1099.3, 1291.6

40-70d 24h 1173.2,1332.5

3h 5min 511.0

2.5min 5min 1039.0

40-70d 24h 1115.5

6-8d 4h 559.1, 657.1

40-70d 24h 135.9, 264.5, 279.5, 400.7

6-8d 4h 554.3, 776.5, 1044.0

40-70d 24h 1076.8^

6-8d 4h 140.5

40-70d 24h 657.7, 884.6

6-8d 4h 336.2

40-70d 24h 604.7, 795.8

6-8d 4h 1596.2

40-70d 24h 145.4

6-8d 4h 103.2

6-8d 4h 411.8

40-70d 24h 279.2

57
Primary standard solutions were prepared by dissolving ultrapure metals

or compounds in acids. The acids and water were prepared using a sub-boiling

distillation technique (5). The concentrations of these primary standards were 10"^ to IC^ g/g. From these primary standards, multielement standard

solutions were prepared which have concentrations suitable for the irradiation

and counting methods used. The elements are mixed together in groups which

provide chemical stability of the mixtures as well as groups which avoid

interferences in the gamma spectra. 0.1 -mL aliquots of the standards were

pipetted onto Whatman 41 5-cm diameter filter papers. After air-drying, the

filter papers were folded to 1 cm^ area or pressed into pellets to match the

geometry of the samples. The standards were also sealed in precleaned

polyethylene bags.

The samples were irradiated twice to obtain data on all the elements of

interest. The first, short irradiation with two subsequent counts provides

data on Na, Mg, Al , CI, K, Ca, V, Mn, and Cu. The second, long irradiation

with two subsequent counts provides data on Sc, Cr, Fe, Co, Zn, As, Se, Br,

Rb, Mo, Ag, Cd, Cs, La, Ce, Au, and Hg. For the short irradiation one sample

and standard_were irradiated together for 1 min at a nominal flux of

5 X lO^^n'cm'^s"^ . The samples and standards were transfered to clean,

unirradiated, polyethylene bags and counted.

After measurement of the short lived isotopes, the pellets were repackaged in clean polyethylene bags and irradiated for 4 hours. Ten samples and

two of each primary standard used were packaged together in one irradiation

capsule. The samples were irradiated for a 2 x 2 hour flipped irradiation

(which results in uniform neutron dose to all samples and standards) at

5 x 10^ ^n^cm'^s"^ . After 6 days decay the samples and standards were repackaged in clean polyethylene bags and counted for 4 hours each at a distance of

10 cm from the detector. After a 40 to 70 day decay the samples and standards were counted a second time, this time 5 cm from the detector. The second

count was for 24 hours for the samples and 4 to 18 hours for the standards.

In addition to the human liver samples, samples of NBS SRM 1577 Bovine Liver were analyzed periodically to check accuracy.

The following equipment was used: The counting of the short-lived

nuclides was carried out on a Ge(Li) detector (1.88 keV FWHM at 1332, 3.33%

efficiency) connected to a 4096 channel Nuclear Data (ND) 100 analyzer. The

counting intervals were preset at 5 min real time. Livetime and pileup

corrections were accomplished by accumulating a 60 Hz pulser peak at a non- interfering energy in the spectrum. The data were transmitted to the NBS

Nuclear Data 6620 system and stored on hard disk. The long-lived nuclides were counted on "Gamma-X" detectors (1.69 keV FWHM, 2.49%, and 1.65 keV FWHM,

2.50%) and spectral data were accumulated directly in 8192 channels of the

ND 6620 system and stored on disk. The ND activation analysis software was used for the data reduction. The spectral data were then transferred to

magnetic tape for permanent storage.

58
RESULTS AND DISCUSSION

The results of the INAA determinations in the human liver samples are reported in Table 2. Results are reported for replicate pellets from each

sample. For a given liver sample, the first row of data are for the "A"

pellets, the second row for "B" pellets. The uncertainty for each sample is

±la due to counting statistics alone. The results on the SRM Bovine Liver

check samples are summarized in Table 3 according to the analysis date. The

data for Al are somewhat erratic, both for replicate comparisons in the first

nine samples and in the detection limits in later samples. At the <_1 ppm

level which occurs in most human liver samples, the determination of Al by

INAA is marginal primarily due to counting statistics and the high background

from Na and CI activity. The possibility of some Al contamination from the

sample processing of the first few samples exists and this may explain the

poor agreement between replicates for these samples (e.g., see samples

L1B0005, L2B0041 , and L1B0045). Due to counting equipment problems, most of

the "A" pellets of the later samples had very high detection limits (i.e.,

<2-4) because they were counted after much longer decay times than normal

which makes replicate comparisons impossible. However, for samples where

comparison with AAS data is possible, agreement was generally good. The

concentrations of V, Cr, and As were equal to or below the detection limits

obtained by the irradiation and counting procedures employed. These detection

limits vary somewhat from sample to sample due to the decay time and the

background from other elements in the sample.

An incomplete set of data is reported for the elements Cs, La, Ce, Sm,

and Au. These elements were not on the environmental or biological priority

lists (see Section 4, Table 1); however, when it was found that they could be

measured in some or all of the samples, primary standards for these elements were added to the set making quantitative data possible.

Samples of SRM 1577 Bovine Liver were analyzed (Table 3) as periodic

check standards for quality control. It is important to note that over the

one year time interval during which these analyses were performed, several

different sets of primary standards, different analysts, and some different

irradiation and counting conditions were used. The use of SRM's provides a good check of the accuracy of the technique. A few aberrant values were

observed which require some comment. In the 10/27/81 short analysis, the

following elements Na, K, Mn, and Cu (which were determined in the same count) were between 5 and 10 percent high. As this trend is not observed in the

samples analyzed at the same time (L1S0090-B, LIBOIOO-B, LlSOllO-B, L1M0118-B,

L1B0121-B, L1M0126-B, L1M0127-B, L1M0131-B, and L1M0140-B) when compared to

the 'A' pellets of these samples, the high values were probably due to a counting error in this one count. The Hg values in the SRM 1577 analyses are

all very high compared to the certified value. Mercury was apparently

volatilized from the standards during irradiation. This problem, which

certainly varies in degree from irradiation to irradiation, may account for

the poor replicate agreement for Hg in the human liver samples.

59
TABLE 2. TRACE ELEMENT CONCENTRATIONS IN HUMAN LIVERS AS DETERMINED BY

INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS

Sample Element Concentration (yg/g wet weight) - - - - Identification Na Mg Al CI K

LI B0002 855±25 170+17 <0 6 1 180+40

854± 6 210±30 <p. 6 1330±20 2770180

L1B0005 609± 7 1 36+ 10 49+0 08 869+ 9 ?9i n+7n

630±10 145±14 0. 92±0. 08 869± 9 3160170

L1S0018 587± 9 1 39± 1

0 94+0 08 722+ 8 2850+80

581± 9 182±15 <p. 3 794± 8 2840170

L1M0023 1190+35 139+26 <3

1240±13 130±30 <P. 5 1450120 2190170

L1S0027 Urn i ^\J\J ^ 1 1232+13 142+19 <0 1 275+n ?540+6n

1211±13 91±19 <p. 4 1336±11 2740160

L1S0037 1073+1

141+16 n\J m 45+0 1 1 1 214+1

??4n+fin t.t.'TU— U(J 1122±16 108±18 0. 44±0. 10 1278114 2490170

L2B0041 810+ 9 150+20 n 49+0 08 1112+10 2960+70

81 0± 9 203±19 1. 07±0. 09 1244110 3010170

L2B0042 932+14 ^ \J L. — 1 ~ 128+19 0 43+0 08 1016+12 2200+60

938±14 103±14 0. 38±0. 09 1035112 2240160

L1B0045 740+1

122+16 71+0 10 1061+12680+70

645±10 82±16 0. 39±0. 08 983111 2410160

L2M0048 1900+60 1 20+20 <0 Q 1 900+60 1 O / U— uu 1880±10 150±40 <P. 9 1880+30 1390170

L2S0049 1191+13 182+19 <0 1 366+1

2450+60 C^'-T sJ\J — \J\J 1259±13 117±18 <P. 4 1376112 2600170

L1S0053 960+ 3 140+20 <1 1060+30 2510+70

970± 3 130±30 <0. 6 1180120 2440180

1 PMDflfi? 980+30 290+^^0 N D. 1 lOO+'^O

lOlOtlO 100±30 2. 3*±0. 2 1090120 2120160

LI B0074 T 1 OA 1 '^A 1120±30 122±17 <0. 6

1 IT OA 1 r A 1580150 O O 00_i_ "70 2290170

1134± 7 160±40 <p. 7 1730120 2520180

L1S0080 1570±50 <82 <2 2020160 1890180

1604±16 <110 <p. 8 1980120 2130170

L1M0083 720±20 140±30 N. D. 1040130 3040190

720± 7 150±30 <0. 8 1010120 2800170

LI S0086 930±30 110±18 0. 8 ±0. 2 1230140 2970190

960±10 150±30 <0. 5 1220120 2840170

L1S0090 1540±50 190±30 <4 1620150 2230190

1498± 5 140±20 0. 77±0. 14 1540120 2240170

60
Sc

Element Concentration (yg/g wet weight)

V Cr Mn Fe

\. y/-u . Uo;X 1<u . Uo ^n<U 1 oyi4-n no 1 . o4xU. Uo coo occ.

-1- c ± D (3.8 ±0.6)xl0"'^ <0 .04 <0 .03 1 .15±0.02 522 ± 4

<U. £X 1<U ^n<U no . Uo 1 on 4-n nc 1 .o1±U.Ud TOO 1 00 J. A ± 4

N.D. <0 02 0 .039±0. 009 1 27+0 05 130 + 4

<U <U no 1 cn-)-n nc 1 . bU±U. Ub 0"7C o/b J- c ± 0

<lxlO"'^ <0 02 <0 02 1 56+0 05 418 + 3

u/ n Ko+n no U. oyxu.Uo 1 on 1 ^U ± y

<lxlO"'* <0 04 <0 02 0.61 ±0.04 121 + 6

^tX 1 U

<rn <u^n<U no 1 7/1+n nc 1 . /4±U. Uo OOQ -1- 0

<0. 02 <0 03 1 .82+0.06 213 ± 2

M n IN . u

<U

no ^n<U. no 1 70+n nc 1 . / c±\j . Ub 1 yo

-1- o (7.8 ±0.5)xl0"^ <0. 02 <0 02 1 .91±0.05 199 ± 2

^•n <unovc. n QQ+n nc U . oy±U . Jb

(2.0 ±0 3)xl0''* <0. 03 <0. 02 0 87+0 05 218 ± 2 M n N.D. <u. no ^ n<U noU^ 1 /iO-i.n nc 1 .4o±U.Ub A 1 4 1 . nj. n U± U

0.07±0 02 <0. 02 <0 02 1 57+0 05 46. 4± 0

< 1 X 1 U <U. no ^n<U. noUo ^ 1 /i4.n n/i

1 . 1 4±U . U4 y/ ± 1 N.D. <0. 02 <0. 02 11 .03+0.04 • W W — W • W 1 87 ± 8

<0X 1<u

n/i n <UnKUo 1 nn+n n/i

1 . UU±U . U'f 1 on 1 yu

4- O ± d

<lxlO"'* <0. 06 <0. 01 0.93+0.04 192. 5± 1 M n IN . U

<U

n"? uo ^n<U

mU 1 1 cc-i-n nc 1 . bD.±U . Uo ocob. O J. 1 o± 1 (2±1 )xlO~'* <0. 03 <0 02 1 48+0 06 94. 2± 1

^ 1 . D ±U. o ^ X lU <U. ncUo ^n<U. ncUo 1 00+n 1

1 . o^±U . 1 c

cn/i bU4 4- C ± b

(3.4 ±0 4)xl0"^ <0. 04 <0. 02 1 07+0 03 521 ± 4

(4.8 ±0.4}xl0 <U. n "7 0/ U. UbD±U. nno UUo 1 c 1 J. n n c

I . b l±U.Ub one^:9d ± 0

(4 6 +0 2)xl0''^ <0. 04 0. 089±0. 008 1 32+0 04 298 ±10

<0XlU <U. ncUo <u.

n/i U4 1 /I cxn n/i

1 . 4b±U. U4 oUy J- /I ± 4

<2xl0"'* <0. 05 <0. 02 1 .41±0.04 316 ± 3

<lxlO ^ <0. 07 <0. 04 0.96±0.04 f\ r\ r\ 209 ± 3

llxlO"'* <0. 05 <0. 02 0.94±0.04 215 ±10

<2xlO"'* <0. 1 <0. 03 1 .47±0.05 274 ± 3

^^SxlO'** <0. 04 <0. 04 1.33±0.04 266 ±15

(3.6 ±0.3)xl0"'^ <0. 03 <0. 03 1 .69±0.05 265 ± 3

llxlO"*^ <0. 04 lO- 03 1.67±0.05 268 ±10

(2.0 ±0.4)xl0"^ <0. 1 <0. 02 1.28±0.04 226 ± 3

(3.7 ±0.3)xl0"'^ <0. 03 <p. 04 1.19±0.03 222 ± 2

61
TABLE 2 CONTINUED

Sample

- - - - Element Concentration (yg/g wet weight) - - - - Identification Co Cu Zn As

L1B0002 0.0605±0.0008 5.9±0.8 57.7 ±0.2 <0. 04

0.0589±0.0007 5.8±0.8 57.82±0.14 <0. 06

L1B0005 0.039 ±0.001 3.0±0.5 43.3 ±0.9 <0. 03

0.026 ±0.001 4.6±0.6 44.5 ±0.9 <0. 02

L1S0018 0.0424±0.0006 4.6±0.5 39.74±0.08 <0. 03

0.0498±0.0006 4.6±0.5 41.9 ±0.4 <0. 06

L1M0023 0.0312±0.001 6.1±1 .6 39.4 ±0.9 <0. 02

0.0310±0.001 3.7±0.8 37.9 ±1.5 <0. 03

L1S0027 0.0342±0.0007 7.0±0.7 97.2 ±0.9 <0. 05

0.0319±0.0005 7.8±0.8 94.97±0.16 <0. 05

L1S0037 0.0286±0.0002 5.3±0.7 64.5 ±0.1 <0. 05

0.0319±0.0005 3.6±0.7 65.5 ±0.6 <0. 05

L2B0041 5.U0.6

0. 031 4±0. 0005 5.3±0.5 49.5 ±0.5 <0. 05

L2B0042 0.0437±0.0005 8.U0.5 51 .8 ±0.1 <0. 05

0.0476±0.0005 10.U0.8 54.2 ±0.5 <0. 05

L1B0045 0.0316±0.0006 5.0±0.7 28.6 ±0.3 <0 06

0.0288±0.0003 4.2±0.6 27.04±0.06 <P 03

L2M0048 0.046U0.0007 6.1±1 63.5U0.17 <0 05

0.0449±0.0003 6.3±1.0 62.78±0.11 <0 05

L2S0049 0.033U0.0002 5.1±0.7 37.64±0.07 <0 05

0.0372±0.0005 <2.4 39.4 ±0.4 <0 05

L1S0053 0.0485±0.0007 <3.6 54.37±0.16 <0 04

0.0484^0.0004 4.7±0.8 55.25±0.12 <0 03

L2M0062 0.0514±0.0006 <21 70.30±0.17 <0 03

0.0496±0.0020 4.4±0.7 69.2 ±0.8 <0 02

1 1 Rnnvzi L 1 DuU /

U . U'+O'fit u . uuuo 0 . 03: 1 . 1 DU . / 1 iU .

o <u , UD

0.0445±0.0006 4.7±0.9 52.86±0.13 <0 03

L1S0080 0.0372±0.0006 <5 52.8 ±0.2 <0 11 0.0358±0.0015 7.8±1.0 55.2 ±1.5 <0 08

L1M0083 0.0500±0.0006 <7 62.9 ±0.2 <0 06

0.046 ±0.002 5.5±0.8 61.4 ±1.0 <0 02

L1S0086 0.0525±0.0006 6.9±0.9 58.40±0.15 <0 04

0.0523±0.0015 7.0±0.9 58.9 ±1.5 <0 03

L1S0090 0.0472±0.0005 <6.6 56.0 ±0.2 <0 05

0.0483±0.0005 7.3±0.5 55.52±0.11 <0 04

62
Se

Element Concentration (yg/g wet weight)

Br Rb Mo Ag

0.426±0.003

0.456±0.004

2.70±0.03

1.64±0.05

13.7 ±0.9

13.2 ±0.5

0.47

0.57

±0.05

±0.04

N.D.

0.011 ±0 .002

0.34 ±0.02

0.38 ±0.02

1.84±0.03

1.42±0.02

6.0 ±0.2

5.8 ±0.2

0.25

0.23

±0.04

±0.04

<0.01

<p.oi

0.430±0.006

0.424±0.006

1.89±0.17

2.54±0.04

10.3 ±0.08

11.6 ±0.08

0.34

0.36

±0.04

±0.04

<0.01

<0.008

0.37 ±0.04

0.49 ±0.02

1.68±0.02

1.76±0.03

3.6 ±0.4

5.6 ±0.4

0.28

0.26

±0.03

±0.03

N.D.

0.012 ±0 .001

0.546±0.005

0.566±0.005

2.48±0.04

1.84±0.18

6.34±0.07

5.73±0.07

0.63

0.67

±0.04

±0.04

<0.009

<p.oi

0.381±0.005

0.37U0.002

4.20±0.02

5.50±0.09

3.67±0.05

4.6U0.05

0.63

0.58

±0.04

±0.04

<0.009

<0.007

0.434±0.003 3.18±0.09 8.1U0.05 0.60 ±0.04 <0.008

0.373±0.005

0.370±0.002

3.29±0.02

4.30±0.10

5.06±0.05

5.50±0.05

0.53

0.59

±0.04

±0.04

<0.02

<0.007

0.402±0.006

0.4C4±0.003

2.84±0.05

2.12±0.02

4.76±0.06

4.2U0.03

<0.14

0.30 ±0.04

<0.01

<0.006

0.634±0.005

0.643±0.003

8.33±0.07

9.17±0.09

4.0 ±0.4

3.47±0.15

0.52

0.38

±0.05

±0.04

<0.008

10.004

0.534±0.005

0.555±0.005

3.02±0.02

4.0U0.12

5.05±0.05

5.9U0.05

0.43

0.44

±0.04

±0.04

<0.007

<0.009

0.613±0.005

0.645±0.003

2.1U0.02

2.28±0.03

9.2 ±0.8

8.3 ±0.3

0.36

0.32

±0.04

±0.04

o.on7±o,

0.0126±0,

.0021

.0014

0.409±0.004

0.45 ±0.02

1.90±0.02

2.37±0.04

5.1 ±0.4

4.3 ±0.3

0.76

0.69

±0.04

±0.04

<0.008

<0.006

0.424±0.005

0.473±0.005

1.78±0.11

1.98±0.03

4.2 ±0.4

4.5 ±0.2

0.58

0.55

±0.05

±0.04

<0.008

0.0063±0,.0018

0.466±0.005

0.50 ±0.02

41.2 ±2.5

47.8 ±0.7

3.9 ±0.4

4.5 ±0.3

0.62

0.55

±0.06

±0.06

<0.008

0.0096±0,.0011

0.509±0.003

0.543±0.0020

1.79±0.02

1.6U0.02

9.9 ±0.6

9.5 ±0.6

0.29

0.35

±0.04

±0.03

<0.008

<0.007

0.564±0.004

0.626±0.015

1.77±0.02

2.17±0.03

8.6 ±0.6

8.3 ±0.4

0.60

0.83

±0.03

±0.04

<0.007

£0.007

0.544±0.004

0.496±0.004

2.33±0.14

2.69±0.03

6.3 ±0.5

5.5 ±0.3

0.98

1.02

±0.04

±0.05

0.0058±0,

<0.005

,0013

63
Sample

Identification

- - Element Concentration (yg/g wet weight) -

Cd Cs La

L 1 DUUU^ J . of± u . uo u . u 1 jy± u . uuuo \/ . lX\J . o )X\\J

1.1 ±0.1 N.D. N.D.

Lduulo (J . O l± U . UD IN . uIN . u. 0.84±0.06 N.D. N.D.

L 1 OUU 1

^ . O l± U . UD M n IN . u

M n In . u

2.74±0.07 N.D. N.D.

1L1

1 Mnn?^ nVyJC'j U . 0D± U . UD M n IN . u. IN . u. 0.62t0.06 0.0049± 0.0003 0.009 ±0.002

1 1 Qnn97 L 1 jUUt9 91-t.n 07 In . u. nU.U^^ 099 +n±U.UU.3 nm

2.07±0.07 N.D. N.D.

1 1 <^nn'57 L 1 oUUJ/ 1 . Oo± U . U/ M n IN . u. N n IN . u

1 .75±0.07 N.D. <0.009

M n In . U

In . U. In . U. 0.83±0.06 N.D. 0.078 ±0.003

u . / U± U • UD In . u. In . u. 0.66±0.06 N.D. 0.0079± 0.0021

L 1 DV'JHO 11 . A9+n tt±U . 07u/ M n IN . u

<U.UUo

1 .31±0.06 N.D. N.D.

1 ?Mnn4R (/L

^ H . fU-f) Q£ U . 7Win"3 / y A 1 U iN . U . ~0.36±0.08 (5.4±0.4)xl0'3 <p.oi

<0.

~o!43±0.06 N.D. <p.oio

^ • utiu . uo U . U 1 HOZ U . uuuo U . KJl. I Z U • UUJ

2.19±0.08 0.01 54±0. 0005 0.032 ±0.003

1 PMnnfi? <J • / Ux U . U7 (R^O./xU./yAlU 7+n 7Win"3 NIN . nU. 4.19±0.08 (9.1±0.6)xl0"3 <0.012

1L iRnn7d 1 DVJVJ / t 11.71Ql+nlU 99

\ o . Dxu . oy A 1

(1 7+1 4.Win"3

1 41+0 08 f? 8+0 7^xl0'3 <0 009

L1S0080 0.52±0.12 (3.4±0.7)xl0"3 0.139 ±0.006

0.56±0.12 (5.8±l.l)xl0"3 0.132 ±0.004

L1M0083 0.79±0.07 (8.6±0.7)xl0"3 N.D.

0.66±0.06 (8.8±0.7)xl0"3 0.026 ±0.003

L1S0086 0.44±0.05 0.013 ±0.001 N.D.

0.42±0.06 0.008 ±0.001 0.066 ±0.003

L1S0090 2.15±0.09 0.0068±0.0005 (5.8±0.3)xl0'2

1.85±0.08 0.0066±0.0006 (5.7±0.4)xl0"2

64
Ce

- - - Element Concentration (ug/g wet weight) - - -

Eu Sm Au Hg

0.123±0.004 N.D. <0.001 N.D. 0.101±0.003

0.n0±0.004 <.1.7xl0'3 <^0.03 (2.7±0.5)xl0"^ 0.n0±0.002

N.D. N.D. N.D. N.D. 0.117±0.003

N.D. N.D. N.D. N.D. 0.098± 0.003

N.D. N.D. N.D. N.D. 0.02910.003

N.D. N.D. N.D. N.D. 0.06310.003

N.D. N.D. N.D. <2xl0"'+ 0.12710.002

0.022±0.002 N.D. N.D. N.D. 0.08110.004

N.D. N.D. N.D. N.D. 0.05410.005

N.D. N.D. N.D. N.D. 0.05010.002

N.D. N.D. N.D. N.D. 0.13010.002

N.D. N.D. N.D. N.D. 0.13110.002

N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. 0.04910.003

N.D. N.D. N.D. N.D. 0.05210.002

N.D. N.D. N.D. N.D. 0.07110.002

N.D. N.D. N.D. N.D. 0.06610.003

N.D. N.D. N.D. N.D. 0.06110.002

<0.02 N.D. <0.001 <2xlO"'* 0.03210.003

<0.006 N.D. <0.001 N.D. 0.10610.001

N.D. N.D. N.D. N.D. 0.02710.002

N.D. N.D. N.D. N.D. 0.04810.002

0.028±0.005 N.D. <0.001 (8.8±0.5)''^ 0.12010.003

0.030±0.002 N.D. <0.0009 N.D. 0.13310.001

<o.on N.D. <0.001 <2xlO"'* 0.02610.002

<0.009 N.D. N.D. N.D. 0.03710.002

<0.02 N.D. <0.0014 <2xlO"'* 0.08110.003

<p.oi N.D. N.D. N.D. 0.08210.002

0.11410.005 N.D. (3.9±0.4)xlO"S <4xl0'^ 0.11310.003

0.119±0.004 N.D. N.D. N.D. 0.10910.004

0.013±0.003 <1.6xlO"'* (8.6±0.7)xl0"3 <2xlO"'^ 0.10810.003

<0.02 N.D. (8.8±0.7)xl0'3 N.D. 0.06910.005

0.086±0.004 <1.7xlO"^ <9xl0"'^ <l.lxlO''^ 0.09710.003

0.078±0.004 N.D. N.D. N.D. 0.08710.003

0.062±0.003 N.D. <lxl0"3 <1.7xl0"'^ 0.08110.002

0.053±0.005 N.D. N.D. N.D. 0.11810.003

65
TABLE 2 CONTINUED

Sample

Identi fi cation Na

Element Concentration tyg/g wet weight)

Mg Al CI K

LI S0091 a60±30 140±20 <1 11 90±40 2620±90

963±7 1 fU+JU <u

7/ 1 oUU+^U A n A A r\f\ 2500±80

LI S0092 1410±40 110±30 <3. 3 1500±40 2330±70

141 0±10 1 1 0-1-90 1 1 U+tU n 1 3 1 fT 1 + 1 U

O O *5 A C A 2230±50

LI BOlOO 800±20 140±20 <1 1190±40 2870±90

790±10 1 OU+^U 1 . o j+U

1 7 11 11 70-1-1 / U+ 1 0u 2750±50

L1M0105 1270±40 1 50±30 <4 1260±40 2710±110

1280±10 1 ^U+fU <u

711 AQO-i-90 2780±90

LI SOI 07 888±26 130±30 N. D. 1205+9 2800±90

933±10 210±30 1

2 ±0. 2 1246±15 O AAA n A 2900±70

LI SOI 10 878±28 182±20 <1 1300±40 2890±110

890±10 185±22 1 . A yi .A 04±0. 1 /I 14 1 O A A 1 A 1200±1

2900±50

LiMons 1010+30 152±30 <10 1100±30 2530±80

1010±10 1 20±20 0. ^ A . A 69±0. 1

1 OdO±1 0 2430±50

L1M0119 1160±40 107 ±24 29 ±2 1400±40 3090±120

1166±7 1 80±40 33. \ ±0. b 1 D/b± 1

2970±80

L1B0121 1360±40 1 02±20 <2 1 750±bO 2290±80

1 370±10 1 1 n+?n 171 n+1

ooon . cn il^yUibU

LI SOI 23 1100±30 149±25 <3 T O AA yi A

1 390±40 2320±70

1138±12 1 DU+OU o 2bD0±/0

L1M0126 760±20 <270 N. D. 840±30 3070±90

750±10 1 yu+tu \ <J . u +u

c 7Q7-I-Q 1 A A A . C A 3000±50

LI M0127 1660±11 N.D. N. 1580±17 2165±60

1 680±10 1 \ J±C\j 1

/ D+U

1 A 1 (;aoj.i 0 2230±b0

LI SOI 28 1290±40 260±80 N. D. 1470±50 2220±70

1 330±10 <\j. QO

1 Q90-).90 A 1 AA OA

21 90±80

L1S0129 1100±30 1 50±20 <1 1 500±50 2720±80

1111±7 oon-i-An ttU+ T'U 1 > •JO+U . 9? O ^ ^ A T AA 2630±100

L1M0130 1280±40 160±30 <2 1560+50 2220±80

1317±15 120±30 <0. 6 1540±20 Z2b0±/U

LI MOT 31 1200+40 N.D. <1 1780+60 2610±100

1240±10 126±25 0. 89±0. 16 1760±10 2600±50

L1M0140 1220±40 95±30 <4 1640+50 2600+100

1230±10 130±20 0. 61±0. 14 1590±20 2470±50

L1M0141 1580±50 150±20 <2 1610±50 2040±80

1613±16 130±30 <0.,6 1580±20 2120±70

6&
Sc

Element Concentration (yg/g wet weight)

V Cr Mn Fe

<lxlO''+ <0.06 £0.05 1.03±0.03 209 ± 3

<2xl0 ^ <0 .05 £0. 02 1 AO A AO1 . 02±0. 03 Ol A

21

± 2 ^ZxlO''^ <Q.08 £0.07 0.71±0.03 437 ± 5

<3xl0 ^ <0.03 £0. 05 0. 72±0. 02 >l o o 432 ± 4

(9 ±l)xlO"^_ <0.06 £0.02 1.49±0.05 235 ± 3

{9.3±0.8)xl0 £0.03 N.D. 1 .45±0.02 235 ± 2

(2.0±0.2)xl0"'+ <0.08 £0.04 1.24±0.13 287 ± 3

{3.0±0.5)xl0 £0.05 £0.02 1 . 08±0.03 295 ± 2 (4.3±1 .4)xl0'^ <0.2 0.083±0.011 1.34±0.04 188 ± 3

(6.9±0.4)xl0"^ £0.04 £0.03 1.44±0.04 188 ±10

(2.4+0.3)xl0''+ <0.06 <0.07 1.20±0.03 427 + 5 (2.1±0.4)xl0"'+ £0.03 £0.03 1.17±0.02 421 ± 3

<4xl0"'+ <0.2 <0.03 2.07±0.04 57. 8± 1.0

T4.1±0.2)xl0"'+ £0.Q2 £0.02 2.00±0.03 60. 1± 0.9

{2.0+0.5)xlO"'+ <0.10 <0.08 1.39±0.03 68. 4+ 1.4

<2xlO"'* £0.06 £0.02 1.27±0.04 124. 9+ 1.2

(2.4±0.7)xl0 ^ N.D. <0. 03 N. D. 126 ± 6

<8xl0"5 £0.05 0.234±0.010 0.95±0.04 139 ± 2

(1 .23±0.18)xl0 ^ <0.03 <0. 04 A f\A A AO 0. 94±0. 02 141 ± 1.4

<9xl0"5 £0.08 £0.03 1.06±0.03 304 + 3

<lxlO ^ <0. 04 <0. 03 1 1 A . A A>l

1 . 1 0+0. 04 318 ±12

(1.2±0.1)xl0'3 £2 £0.05 1 .83±0.03 88 ± 2

(1 .4±0 .1 )xl 0

•= <0 .03 N.D. 1

d6±0. 02 N.D.

(4.0±0.7)xl0;;'+ N.D. £0.04 0.81±0.22 148 + 2 (3.3±0.2)xl0 £0.03 <0. 02 A OA A AO

0. 84±0. 03 147. 7± 1.2

<2xl0"^ £2 £0.02 0.75±0.03 273 + 3

£2x1 0'*+ £0.05 A r\ o £0.02 0.62±0.03 272 ± 2 <2xl0"^ £0.06 £0.03 1.15±0.03 125 + 2 £3x10"^ £0.05 £0. 03 1 1 O A AO

1 . 12±0. 03 122. 1± 1.3

<3xl0"^ <0.07 <0.03 0.96±0.04 341 + 4

£lxlO"^ £0.04 £0.03 1.06±0.04 362 ±15

<2xl0'^ <0.05 <0.02 0.70+0.03 270 + 3

"Tl .4±0.3)xl0"4 £0.04 £0.03 0.66±0.02 275 ± 2 <2xl0"^ <0.09 <0.02 1.27+0.03 260 + 3

Tl .4±0.3)xl0"^ £0.03 £0.04 1.19+0.03 260 ± 2

<lxlO"'+ <0.06 <0.02 0.84+0.03 428 + 4

<2xl0"^ <0.04 <0.03 0.78±0.05 440 ±20

67
TABLE 2 CONTINUED

gg^plg

- _ - . Element Concentration (yg/g wet weight) - - - - Identification Co Cu Zn As

LI S0091 0.0376±0.0006

0.0353±0.0005

<3

4.6±0. 8

30.66+0.11

30.35±0.09

<0.03

<0.04

L1S0092 0.0435+0.0006

0. 041 2±0. 0010

13 +2

10.8±0. 5 59.06±0.16

55.3 ±0.2

<0.04

<0.05

LIBOIOO 0.0564±0.0006

0.0584±0.0006

12 ±2

9.7±0. 4

54.7 +0.2

55.79±0.13

<0.05

<0.04

L1M0105 0.0503±0.0005

0.0499±0.0006

<6.4

9.8±1. 1 63.7 +0.2

65.02±0.14

<0.04

<0.05

Li SO 1 0/ 0.075 iO.OOa

0.055 ±0.001

< 1 1 7.7±0. 7

c/i 0 .no D4.£ +U.^

62.4 ±0.6

<u . uo <0.03

LI SOI 1

0 . 0b48±0 . 000/

0.0526±0.0006

C C.I b.D±l

5.0±0. 4

/IT 01 . n 1/1 4 1 . o 1 +U. 14 41.25±0.10

<U. Uo

70.04

1 1 Mm 1 o

L 1 MU 1 1 o u . Uby^±u . UUU/

0.0601 ±0.0007 4.5±0. 4

44 . /U+U .6

45.62±0.n

<U

<0.04

1 1 \iir\ inn LlMOl 1 9 U . 0b08±U . 000/

0.0484±0.0005

0.052 ±0.002

<0

5.9±0.

N.D.

8

70 Ql . n 1

Q

/^ . o 1 +u. 1

y

71.88+0.15

72.4 ±1.0

<U . UD

70.04

£0.03

LI B0121 0.0339±0.0005

0.0335±0.0004

6.9±1

8.2±0. 5 42.64±0.12

42.97±0.009

<0.05

<0.04

LI SOI 23 0.0458±0.0005

0.046 ±0.002

9. 0±2

1Q.2±0. 8

56.14+0.14

57.0 ±2.0

<0.03

<0.03

L1M0126 0.135 +0.001

0.131 ±0.001

<64

6.9±0. 4

65.49+0.18

63.65±0.14

<0.06

<0.04

L1M0127 0.0446±0.0006

0.0452±0.0005

N.D.

3.5±0. 5

48.4 +0.2

48.25±0.09

<0.05

<0.04

LI SOI 28 0. 041 0±0. 0005

0.0398±0.0005

<53

12.2±1. 0

40.2 +0.1

40.19±0.10

<0.05

<0.03

L1S0129 0.0524±0.0007

0 0521+0 0006

8.4±1

15.1+0. 9. 66.9 ±0.2

65 59+0.15

<0.05

<0.04

LlMOl 30 0.0357±0.0006

0.0381±0.0010

<4.9

5.9±0. 8

48.9 +0.2

52.0 ±2.0

<0.04

<0.040

LlMOl 31 0.0366±0.0005

0.0359±0.0006

<4.1

3.9±0. 4

37.9 +0.1

38.92±0.10

<0.05

<0.04

LlMOl 40 0.0458±0.0006

0.0448±0.0006

<6

9.1±0. 5 46.8 +0.1

46.85±0.012

<0.06

<0.04

L1M0141 0.0275±0.0002

0.0263±0.0010

<4.2

4.6±0. 9

52.66±0.1

53.0 ±2.0

<0.06

<0.07

68
Element Concentration (yg/g wet weight)

Se Br Rb Mo Ag

0.494±0 005 2^ • 26C—\J + 0 .02 8.0 ±0.7 0.56±0.04 <0 006

0.520±0 005 2.45 ±0 03 6.3 ±0.3 0.52±0.04 lO 006

0.661±0 .005 4 58 + 0 03 10.2 ±0.9 0.55±0.04 0 018 ±0. 002

0.613±0 008 5.19 ±0 05 7.1 ±0.3 0.48±0.05 0 016 ±0. 004

0.599±0 004 2.43 + 0 15 6,1 ±0.6 1.2U0.06 <0 006

0.51 5±0 004 2!76 ±0 03 5.8 ±0.3 1.26±0.05 1.0 007

0.45 ±0 01 1 67 +n 02 9.8 ±0.6 0.38±0.04 0 009 ±0. 002

0.492±0 .005 1.76 ±0 02 9.3 ±0.4 0.53±0.05 lO 008

0.640±0 006 1 .42 ±0 09 7.9 ±0.6 0.99±0.05 <0 009

0.652±0 08 1.61 ±0 02 8.9 ±0.5 0.96±0.04 <_0 007

0 643+0 006 2.12 ±0 02 8.2 ±0.8 0 21+0 04 <0 008

0.621±0 004 2.45 ±0 03 6.'7 ±0^3 0.20±0.04 lO 007

0 566+0 005 1.18 ±0 01 9 4+07 0 44+0 05 <0 005

0.554±0 005 1.37 ±0 02 7.31±0.18 0.40±0.05 006

0.473±0 005 1.08 ±0 02 5.5 ±0.4 0 70+0 04 <0 009

0.493±0 005 1.12 ±0. 02 4.9 ±o!2 0.97±0.05 <Q 007

0.520±0. 020 1.127±0. 019 5.4 ±0.3 0.69±0.04 006

0.486±0 004 3.29 ±0, 03 4.9 ±0.4 1.00±0.05 <0 006

0.472±0 004 3.71 ±0. 04 3.36±0.18 1.0U0.05 lO 005

0.494±0 004 1.23 ±0 01 9.6 ±0.7 0.80±0.04 0 0055±0. 0016

0.546±0 020 1.44 ±0 02 8.4 ±0.5 0.82±0.04 lO. 006

0.574±0 005 1.35 ±0 02 12.8 ±1.0 0.88±0.06 0 014 ±0. 002

0.539±0 005 1.46 ±0. 02 9.5 ±0.4 0.97±0.06 (8H:2)xlO"3

0.504±0 005 2.10 ±0 18 5.2 ±0.4 0.39±0.03 <0. 006

0.462±0 002 2.71 ±0. 03 5.44±0.14 0.25±0.04 lO 004

0.494±0 005 1.99 ±0. 03 7.4 ±0.5 0.44±0.3 <0 008

0.535±0 .005 2.11 ±0 02 7.4 ±0.3 0.48±0.04 . 1.0 006

0.674±0 006 2.40 ±0 15 8.8 ±0.6 1.07±0.05 <0 008

0.687±0 005 2.77 ±0 03 9.6 ±0.4 1.08±0.05 <0. 007

0.496±0 005 2.03 ±0 12 6.4 ±0.6 0.72±0.04 <0 007

0.553±0 .020 2.35 ±0 04 7.7 ±0.4 0.88±0.05 <0 005

0.442±0 .003 2.76 ±0 .03 6.4 ±0.4 0.40±0.04 <0 008

0.439±0 .004 3.09 ±0 03 5.50±0.16 0.35±0.04 ±0 007

0.568±0 .003 2.71 ±0 .04 6.6 ±0.4 0.4U0.04 <0 007

0.548±0 .004 2.67 ±0 03 6.2 ±0.3 1.18±0.05 IP 007

0.375±0 .002 35.6 ±0 .4 3.5 ±0.2 N.D. 0 0087±0. 0014

0.420±0 .020 36.7 ±0 .4 3.8 ±0.2 0.43±0.04 0 008 ±0. 002

69
TABLE 2 CONTINUED

^^i^plg

_ - _ _ Element Concentration (yg/g wet weight) - - - - Identification Cd Cs La

L1S0091 1.23±0.07 (7.0 ±0.6)xl0' N.D.

1 .38±0.07 (5.7 ±0.6)xl0"3 (8±2)xl0"3

L1S0092 1 .28±0.08 (1.19±0.08)xl0' N.D.

1 .47±0.08 (8.9 ±1 .3)xl0"3 (5.7 ±0.3)xl0"2

LIBOIOO 2.39±0.10 (7.0 ±0.6)xl0"3 (6.2 ±0.4)xl0"2

2,31±0.08 (6.4 ±0.6)xl0"3 (1 .ll±0.04)xl0'^

L1M0105 0.54±0.06 (1.23±0.08)xl0"2 N.D.

1.02±0.07 (1 .26±0.07)xl0"2 0.024±0.003

L1S0107 2.53±0.10 (6.8 ±0.8)xl0"3 0.208± 0.006

2.66±0.08 (6.4 ±0.6)xlO"3 0.204±0.004

LlSOllO 2.19±0.08 0.013 ±0.001 N.D.

2.33±0.08 0.013 ±0.001 0.049±0.003

L1M0118 2.9 ±0.1 (1.18±0.07)xl0"2 N.D.

2.90±0.08 (1 .31±0.07)xl0"2 0.039±0.003

L1M0119 1 .62±0.07 (1.29±0.05)xl0'2 N.D.

1.61±0.07 (1 .15±0.07)xl0"2 0.126±0.004

1 .37±0.07 (1 .23±0.05)xl0"2 0.037±0.004

L1B0121 <0.2 (2.0 ±0.5)xl0"3 N.D.

<^0.2 (3.2 ±0.5)xl0"3 <0.01

L1S0123 1.93±0.07 (9.3 ±0.6)xl0"3 N.D.

2.01±0.07 <^0.007 0.058±0.003

L1M0126 5.20±0.12 (1 .06±0.08)xl0'2 0.160±0.004

4.95±0.09 (1 .10±0.08)xlO"2 0.160±0.004

L1M0127 0.63±0.06 (1.05±0.06)xl0"2 <7xl0'3

0.50±0.06 (1.20±0.05)xl0"2 <0.012

LI SOI 28 1.33±0.06 (9.8 ±0.7)xlO"3 N.D.

1 .05±0.08 (1 .07±0.07)xl0"2 0.025±0.003

L1S0129 2.87±0.11 (2.08±0.09)xl0'2 (2.8 ±0.3)xl0'2

2.74±0.08 (1 .98±0.08)xl0"2 (3.6 ±0.3)xl0 ^

L1M0130 0.57±0.07 (8.1 ±0.7)xlO"3 0.021

U

HctU . UD ^ / . U lU . H ^ X 1 u L1M0131 0.73±0.07 (1.13±0.06)xl0"2 N.D.

0.76±0.07 (9.9 ±0.7)xlO"3 (4.2 ±0.4)xl0"2

L1M0140 3.95±0.1 (6.9 ±0.6)xl0"3 N.D.

1.00±0.07 (8.1 ±0.6)xl0'3 (4.2 ±0.3)xl0"2

L1M0141 <0.4 0.0122±0.0005 N.D.

<0.4 0.013 ±0.001 <0.012

70
Ce

- - Element Concentration (yg/g wet weight) - - -

Eu Sm Au Hg

<0.015 N.D. <9xl0~'+ 0.0003±0.0001 0.060 ±0.002

<0.01 N.D. <;ixl0"3 N.D. 0.058 ±0.002

(7.03±0.07)xl0"2 N.D. <lxl0'3 <2xl0"'* 0 109 +0 003

(9.7 ±0.9 )xlO"2 N.D. "N.D. ~N.D. 0.200 ±0.005

0 058 +0 004 N.D. (1 .20+0.04)xl0"3 <2xl0''* 0.0565+0 0019

0.115 ±0 .006 N.D. N.D. ~N.D. 0.182 ±0.004

(1 7 +0 4' IX10~2 <7xl0"5 N.D. <1 .8x10"^ 0.070 ±0.003

(1.6 ±0.3 )X10'2 N.D. <0.001 N.D. 0.0456+0 0019

0.176 ±0 .006 IN . U. \j . / ±U.U4jXIU \ L . D±U . 0 ;X lU U.Uyb ±U.UUJ

o!l70 ±0 !oio N.D. N.D. N.D. 0.104 +0 005

0 066 +0 006 M n IN . u

<^XIU r\ r\oo -i.n nrrs U.U<iy ±U.UUo

0.065 ±0 005 N.D <0.0009 N.D. 0.082 +0.003

0 060 +0 005 M n IN . u

<u . uu

^if./±U.D;XIU r> 11/1 4-n nr>o U . II 4 ±U . UU^i

0.052 ±0 004 N.D. N.D. 0.178 ±0.003

0 048 +0 004 < i . oX 1

IN. U. \ c. . 0±U . / jX 1 U (J.yJcO ±U.UU/

0.053 ±0 004 N.D. N.D. N.D. 0 0461+0 0018

0.052 ±0 004 N.D. <0.001 N.D. 0.038 ±0.005

<0.02 N.D. N.D. <2xl0~'* 0.078 +0 002

<0.014 N.D. <0.002 "N.D. 0.146 ±0.002

0.075 ±0 004 N D N D <2xl0''* 0 059 +0 002

0.076 ±0 004 N.D. <0.001 "N.D. 0.075 ±0.002

0.223 ±0. 007 . N D 0 0051+0 0004 0 047 +0 001 0 116 +0 003

0.234 ±0 007 N.D. N.D. N.D. 0.203 ±0.007

<0.014 N.D. <0.0012 <1 5x10"'^ 0 038 +0 002

ip.oii N.D. "N.D. "N.D. 0.0522±0.0018

(9 4 +n V|ylO"2 <0.0002 N.D. <2xl0'^ 0.039 ±0.002

(1.9 ±0.3]|X10"2 "N.D. <0.001 N.D. 0.0253±0.0017

(3.4 ±0.4])X10"2 N.D. <0.0014 <7xlO"'^ 0.056 +0.003

(4.5 ±0.4])X10"2 N.D. 70.001 "N.D. 0.051 ±0.002

<0.012 N.D. <0.0011 <2xlO"'^ 0.053 ±0.002

(2.5 ±0.3;)X10"2 N,D. N.D. N.D. 0.051 ±0.002

(4.3 ±0.3]|X10"2 <1.4xl0"'^ N.D. <2xl0"'^ 0.099 ±0.002

(4.2 ±0.4^)X10'2 N.D. N.D. N.D. 0.083 ±0.002

(4.1 ±0.4;)X10"2 <1 .5x10"'+ N.D. <2xl0''^ 0.071 ±0.002

0.043U0 .0005 N.D. N.D. N.D. 1.300 ±0.003

<0.008 <lxlO"'^ N.D. <4xl0"3 0.521 ±0.006

<0.01 N.D. N.D. N.D. 0.42 ±0.02

71
TABLE 3. ANALYSIS OF BOVINE LIVER (SRM 1577) BY INAA

Date Na Mq Al CI K

9/80A 2470±20 570±60 2.7±0.5 99201220

9/80B 2470±20 580±50 3.2±0. 4 9560+250

3/14/81 2290±20 600±160 N.D. 2750130 99701210

3/26/81 2300±20 550±110 N.D. 2810±30 97401200

9/15/81 2400±30 710±100 <1.8 2990±40 102001300

10/27/81 2680±20 520±70 3.6±0. 5 2800±30 113001200

9/ 5/80

6/15/81

9/23/81

11/14/81

Certified Values 2430±130 604±9 (2700) 97001600

Date Sc V Cr Mn

9/80A <0.07 9.6410.11

9/80B ^0.06 10.1 10.1

3/14/81 <.1.6 10.9 10.6

3/26/81 <0.7 10.1 10.4

9/15/81 <0.12 10.0 10.2

10/27/81 <0.09 10.8 10.5

9/ 5/80 N.D. N. D. 6/15/81 <2xlO"'* 0. 126±0.020

9/23/81 <0. 10

11/14/81 (2.3±0.7)xlO"'^ <0. 15

Certified Values 0. 08810.012 10.3 11.

72
TABLE 3 CONTINUED

Ua ue re UO Pi I 7i->

9/80A

9/80B 189±3

3/14/81 203±22

3/26/81 177±13

9/15/81 198±4

10/27/81 215±2

9/ 5/80 249±3 0.260 ±0.003 133. 1± 0.4

6/15/81 247±4 0.2412±0.0021 125. 6± 0.4

9/23/81 254±3 0.250 ±0.003 132. 0± 0.4

11/14/81 254±4 0.253 ±0.002 135. 0± 0.3

Certified Values 268±8 (0.18) 193±10 130 ±13

Date As Se Br Rb

9/80A

9/80B

3/14/81

3/26/81

9/15/81

10/27/81

9/ 5/80 <0.15 0.99 ±0.03 15.08±0.11 20.4±0.2

6/15/81 <p.2 1.028±0.010 9.3 ±0.5 20.2±2.0

9/23/81 N.D. 1.12 ±0.14 10.64±0.11 21.8±1.0

11/14/81 <0.11 1.078±0.013 11.26±0.12 19.6±0.8

Certified Values 0.055±0.005 1.1 ±0.1 18.3±1.0

73
TABLE 3 CONTINUED

Date Mo Ag Cd Cs

9/80A

9/80B

3/14/81

3/26/81

9/15/81

10/27/81

9/ 5/80 4.6 ±0.2 0.080±0.015 <_1 .0 N.D.

6/15/81 2.7 ±0.2 0.058±0.004 <0.9 (9.1±1 .2)xl0'3

9/23/81 3.12±0.17 0.049±0.006 <0.8 (1 .22±0.19)xl0"2

11/14/81 3.28±0.18 0.062±0.006 <0.9 (8.8±1 .8)xl0"3

Certified Values (3.4) (0.06) 0.27±0.04

Date La Ce Au Hg

9/80A

9/80B

3/14/81

3/26/81

9/15/81

10/27/81

9/ 5/80 N.D. N.D. N.D. 0.122±0.013

6/15/81 <p.02 <p.04 <6xl0"'* 0.375±0.006

9/23/81 <0.04 <0.05 N.D. 0.426±0.008

11/14/81 <0.03 <0.06 N.D. 0.376±0.011

Certified Values 0.016±0.002

NOTE: N.D. in Tables 2 and 3 indicates "not determined".

74
REFERENCES

1. Lievens, P., J. Versieck, R. Cornells, and J. Hoste. The distribution of

trace elements in normal human liver determined by semi automated radiochemical neutron activation analysis. J. Radioanal. Chem., 37:483, 1977.

2. Schicha, H. , K. Kasperek, V. Riedel, L. E, Feinendegen, K. Vyska, and

W. Muller. Trace elements in normal mammalian tissue and corresponding

malignant tumors. In: Nuclear Activation Techniques in the Life

Sciences, IAEA, Vienna, 1972. p. 451.

3. Brune, D. , G. Nordberg, and P. 0. Wester. Distribution of 23 elements

in the kidney, liver and lungs of workers from a smeltery and refinery

in north Sweden exposed to a number of elements and of a control group.

Sci. Total Environ. 16:13, 1980.

4. Yukawa, M. , K. Amano, M. Suzuki -Yasumoto, and M. Terai . Distribution of

trace elements in the human body determined by neutron activation

analysis. Arch. Environ. Health., 35:36, 1980.

5. Kuehner, E. C. , R. Alvarez, P. J. Paulsen, and T. J. Murphy. Production

and analysis of special high-purity acids purified by sub-boiling

distillation. Anal. Chem., 44:2050, 1972.

75

SECTION 8

THE DETERMINATION OF NICKEL, COPPER, LEAD, CADMIUM, AND ZINC IN HUMAN

LIVER SAMPLES BY VOLTAMMETRY

by

E. J. Maienthal and W. F. Koch

INTRODUCTION

Vol tammetry, in particular cathode-ray polarography (1), has been used

extensively for the analysis of a variety of materials, including biological

samples, for trace metal content (2,3). This electrochemical method is based

on the current-voltage relationship of reducible metal ions at a mercury-drop

electrode when diffusion to the electrode is the rate-determining step. The

potential of the mercury electrode is scanned from about 0.0 to -2.5 volts

relative to a saturated calomel electrode. Species can be identified by the

potential at which electrochemical reduction occurs. Species can be quantitated by the degree of reduction that occurs as indicated by the diffusion

limited current. Concentration is directly proportional to this current and

can be calculated through use of a theoretically-derived equation. However,

due to the complexity of the equation, standard solutions and calibration

curves are customarily used for quantitation. Standard solutions have a further advantage in that matrix effects can be compensated for by simulating

the sample matrix in the standard solution. Standard addition techniques can also be employed.

EXPERIMENTAL

Several samples of the cryogenically ground liver specimens have been

analyzed by the voltammetric technique. Great care was taken throughout the

procedure to avoid contamination. Teflon® jars (which had been cleaned by

soaking in 1:1 HCl , then 1:1 HNO3, followed by a mixture of hot dilute HCIO1+,

H2SO1+, and HNO3) were used to transfer the samples from the cold storage

facility to the chemical analysis laboratory. Samples were weighed directly

into these tared jars. Empty jars were taken through the procedure to serve

as blanks. All sample dissolution and preparation were performed in laminar

flow hoods. All reagents used were of the highest purity; acids were purified

by sub-boiling distillation.

77
DISSOLUTION PROCEDURE

The lids and interior sides of the Teflon® jars containing the samples were rinsed with about 10 mL of nitric acid into the jars. After standing

for 12 hours at room temperature, the samples in the Teflon® jars were heated

to about 90 °C on a hot plate until clear yellow solutions were obtained. The

samples were then quantitatively transferred to 100-mL Teflon© beakers,

rinsing with nitric acid. After addition of 5 mL of perchloric acid, the

samples were fumed, washed down again with nitric acid, and fumed to dryness

to completely oxidize the organic matrix. Solutions for the establishment of

calibration curves were started through the procedure at this point. The

walls of the beakers were rinsed with a few mL of hydrochloric acid and the

samples again taken to dryness at 90 "C. The residues were dissolved in 1 mL

of 10 mol/L hydrochloric acid and diluted to about 25 mL with distilled water

in a separatory funnel. Iron, which will interfere with the voltammetric

measurement, was extracted into 20 mL of isobutyl acetate and discarded. The

aqueous layer from each separatory funnel was transferred back to Teflon®

beakers. The organic material remaining from the extraction was destroyed by

fuming to dryness with 10 mL of nitric acid and 2 mL of perchloric acid. The

samples were redissolved in 0.5 mL of hydrochloric acid and 10 mL of water.

A few grains of ascorbic acid were added to each to reduce any residual iron.

NICKEL EXTRACTION AND DETERMINATION

The samples, free of organic material and virtually free of iron were transferred to separatory funnels. One mL of 1 percent dimethyl glyoxime was added to each.

The nickel complex was separated from the other metals with three

extractions (15, 10, and 10 mL portions respectively) into chloroform. The

three extracts were combined in a Teflon® beaker, evaporated to dryness at

90 °C and treated with nitric acid and perchloric acid to destroy the organic

material. After rinsing the walls of the beakers with hydrochloric acid, the

samples were evaporated to dryness. The nickel was redissolved in 1.0 mL of

1:40 sulfuric acid:water and 0.25 mL of pyridine. The resulting solution was transferred to a polarographic cell and deaerated with argon for 10 minutes.

The nickel reduction peak was measured at -1.1 volt relative to a mercurypool anode with a differential cathode ray polarograph. The second cell of

the polarograph contained the blank solution.

COPPER, LEAD, CADMIUM, AND ZINC DETERMINATION

After the extraction of the nickel complex into chloroform, 10 mL of

ammonium hydroxide and 7 mL of 0.1% sodium diethyl dithiocarbamate (precleaned

by chloroform extraction) was added to the aqueous portion in each separatory

funnel. The copper, lead, cadmium, and zinc complexes were then extracted

with 15, 10, and 10 mL portions of chloroform. The three chloroform extracts

of each sample were combined in a Teflon beaker, evaporated to dryness, and

treated with nitric and perchloric acids to destroy the organic material.

After rinsing the interior sides of the beaker with hydrochloric acid, each

sample was evaporated to dryness. The residue was redissolved in 2.0 mL of

78
ammonium acetate-acetic acid solution (pH = 4.7). After deaerating with

argon, the reduction peaks were measured with a differential cathode ray

polarograph using the second cell to contain the blank solution. The peaks

occurred at -0.35 V, -0.7 V, -0.9 V, and -1.3 V relative to a mercury-pool

anode for copper, lead, cadmium, and zinc, respectively.

RESULTS

The results of the determination of nickel, copper, zinc, cadmium, and

lead in 24 human liver samples are summarized in Table 1.

The values for nickel are reported as "less than" values and are in the

proximity of the minimum detection limits of the procedure. It is anticipated

that additional research in the areas of background correction and reagent

purification would lead to lower detection limits and more quantifiable values

for nickel

REFERENCES

1. Davis, H. M. , and J. E. Seaborn. A differential cathode ray polarograph.

In: Advances in Polarography. I. S. Longmuir, ed. Pergammon Press,

London, 1960, p. 239.

2. Maienthal, E. J. Analysis of botanical standard reference materials by

cathode ray polarography. J. Off. Anal. Chem., 55:1109, 1972.

3. Maienthal, E. J. The application of linear sweep voltammetry to the

determination of trace elements in biological and environmental

materials. In: Trace Substances in Environmental Health-VIII.

D. D. Hemphill, ed. University of Missouri, Columbia, 1974, p. 243.

79
TABLE 1. TRACE ELEMENT CONCENTRATIONS IN HUMAN LIVER SAMPLES AS

DETERMINED BY VOLTAMMETRY

- - Element Concentration (yg/g wet weight) - - Sample

Identification Ni Cu Zn Cd Pb

L2M0062 <0. 04 4. 73 71. 7 2 .76 0. 25

1 4. 48 70. 9 3 .31 0. 28

L1B0074 6. 50 89. 5 1 .59 0. 627

6. 44 67. 0 1 .66 0. 670

L1S0080 <o. 02 3. 75 817 0 .275 0. 352

<.0- 02 4. 26 71. 7 0 .565 0. 474

L1M0083 <o. 05 5. 58 71. 4 0 .25 0. 74

<_0. 05 5. 21 69. 5 0 .28 0. 72

L1S0086 <0. 05 7. 85 40. 6 0 .15 1. 2

<.0- 05 6. 94 33. 1 0 .21 1. 1 L1S0090 <o. 1 7. 14 29. 7 1 .0 0. 28

<.0- 1 11. 8 40. 6 0 .84 0. 79

L1S0091 <o. 03 4. 72 32. 7 0 .76 0. 20

<.0- 03 4. 94 31. 2 0 .88 0. 27

L1S0092 <o. 1 2. 88 16. 1 0 .55 0. 17

<_0. 1 9. 88 26. 0 0 .85 0. 79

LIBOIOO <0. 06 7. 39 43. 1 2 .06 0. 440

<0. 06 6. 71 55. 3 1 .95 0. 514

L1M0105 <o 05 4. 86 59. 6 0 .949 0. 721 <u

4. 65 58. 9 0 .758 0. 716

L1S0107 <o 05 6. 23 59. 0 1 .42 0. 58

n 1

1 c0 . QQ 00 . oJ

1

. 0/ nu

DO

L 1 0\J I 1 u

n UD AH . ZL7 •37 . A 1

1 . f 1 DO

<.o 1 3. 66 38. 6 1 .67 0. 70

L1M0118 <o 06 3. 00 30. 0 2 .64 0. 40

<.o 06 2. 87 33. 5 3 .00 0. 38

LlM0n9 <o 05 1. 69 71. 0 1 .10 0. 12

L1B0121 <o 1 7. 10 34. 0 0 .18 0. 23

n <y1

1 8. 25 23. 0 0 .15 0. 28

LI SOI 23 <P 06 5. 47 55. 3 1 .4 1. 03

L1M0126 <o 06 5. 28 63. 0 2 .68 0. 357

L1M0123 <.o 04 2. 62 46. 6 0 .455 0. 14

LI SOI 28 <o .05 6. 75 39. 2 0 .953 0. 434

L1S0129 <o .06 6. 98 46. 1 1 .27 0. 15

L1M0130 <o .05 5. 73 48. 8 0 .345 0. 429

L1M0131 <o .05 3. 63 37. 8 0 .65 0. 22 <o .1 4. 06 35 7 0 .73 0. 35

L1M0140 <o .06 6. 21 49. 4 1 .02 0. 413

L1M0141 <o .04 3. 12 57 5 0 .358 0. 399
SECTION 9

RESULTS OF THE INORGANIC ANALYSIS OF THE FIRST YEAR HUMAN LIVER COLLECTION

by

R. Zeisler

SUMMARY OF RESULTS

The distribution of 31 trace elements in 36 human liver specimens is

summarized in Figure 1. This figure is a plot of the more than 1,000 data

points obtained for the analysis of human liver samples during the first year

and also includes the concentration ranges reported for human livers by

Iyengar et al., (1). These results are also summarized in Table 1 (e.g., mean

value, median value, and range). Although many samples are prepared for

analysis and storage by freeze drying, the pilot NESB program is currently

reporting analytical results on the basis of fresh weight. An advantage of

fresh weight is its direct relation to reality and it appears to be a basis

which can be reliably obtained. By using the adopted sampling protocol which

requires immediate sealing of the samples in Teflon® bags and deep freezing,

the loss of moisture after sampling is minimized. Teflon® serves as an effective barrier for water (2), and the cryogenic conditions reduce the

partial pressure of volatile species.

Accuracy and Precision of the Analytical Data

The accuracy of the applied methods has been demonstrated and the

analytical imprecision can be minimized when the analytical procedures are optimized for the determination of a single element. For example, IDMS

provides data of high precision and accuracy on thallium and lead with an estimated measurement error of <1 percent. AAS, NAA, and VOL have repeatedly

demonstrated their capabilities in determining many of the priority elements

with uncertainties ranging from 1 to 5 percent. However, a quantitation of

the accuracy and precision of an individual result is virtually impossible

unless it consists of many observations and statistics can be applied. The

implemented analytical protocol provides only single observations for many

elemental data points. However, conclusions about the accuracy of the

measurements may be possible from a careful evaluation of those data points

which have been obtained by multiple observations. Cadmium has been selected

for this purpose because three analytical techniques were used and the

elemental concentrations in the data set vary by a factor of 25 with the

lowest values close to the detection limit obtained by the applied procedures.

81
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82
TABLE 1. TRACE ELEMENT LEVELS IN 36 HUMAN LIVER SPECIMENS

(CONCENTRATION IN yg/g WET WEIGHT)

:iellci 1 C Number of

OaMI|J 1 t:b rlcail Standard

UcV 1 cl L 1 Uil ricu 1 an r\a 1 1 y c

Be 0 < 0.0006

Na 36 1091 343 1105 185-1890

Mg 35 146 26 140 100-195

Al 36 2 5 0.7 0.20 -31.1

CI 36 1370 320 1370 760-2100

K 36 2520 350 2520 1380-3040

Sc 14 5.7x10"1+ 6.1x10"'+ — < 1. 0x1 0''^ -0.0024

V 0 — — <0.02

Cr 3 0.11 0.11 — <0.01 -0.234

Mn 36 1.2 0.4 1.2 0.60 -2.03

Fe 36 230 120 223 43-510

Co 36 0.042 0.010 0.045 0.0135-0.0605

Ni 18 0.037 0.027 0.022 <0.010 -0.100

Cu 36 6.3 2.0 5.7 3.50 -10.8

Zn 36 53 13 54 27.8 -96.1

As 34 0.012 0.016 8.9x10"

"3 <0.001 -0.097

Se 36 0.50 0.080 0.50 0.350 -0.650

Br 36 4.7 8.9 2.2 1.11 -44.5

Rb 36 7.0 2.6 6.3 3.54 -13.5

Mo 36 0.57 0.25 0.54 0.21 -1.23

Ag 10 9.8x10"

"3 4.0xl0"3 — <0,005 -0.018

Cd 36 1.6 1.1 1.4 0.20 -4.96

Sn 9 0.35 0.22 0.22 0.135 -0.712

Cs 27 9.5x10"

"3 4.1xl0"3 9.8x10"

"3 2.0xl0"3-0.020

La 24 0.045 0.036 0.026 7.0xlO"'+-0.160

Ce 18 0.066 0.49 0.041 <1.4xl0"3-0.221

Pt 11 1.8x10"

"5 1.5xl0"5 1.5x10"

"5 5.0xl0"6-5.7xl0"5

Au 20 0.003 0.010 2.0x10' 8.0xl0"6-0.047

Hg 35 0.086 0.074 0.070 0.026 -0.47

Tl 13 6.1x10"

"4 4.9x10"'+ 3.9x10'

"1+

1 .48xl0"'+-16.7xl0"'+

Pb 36 0.55 0.34 0.46 0.120 -1.660

83
The precision of the single determinations is a function of the concen- tration for all three techniques. Figure 2 was derived from the actual

measurements of cadmium in the 36 human liver specimens. Clearly, concentrations above 1 yg/g can be determined reliably with less than 7 percent

imprecision using any one of the methods. NAA and VOL significantly increase

their precision with higher concentrations when compared to AAS. Above 1 yg/g

the observed standard deviation of the data obtained by all three techniques

is in the range from 0.5 to 10 percent. A point by point evaluation did not

reveal a bias of any of the techniques to lower or higher values, therefore

the data can be regarded as accurate within the limits of the precision. In

the concentration range below 1 yg/g, the results have to be weighted by the

much more favorable precision of AAS. The other two techniques provide data

with deviations from the AAS value of up to 30 percent; thus the usefulness of

these techniques is reduced to the possible discovery of gross errors. The

cadmium determinations illustrate a problem encountered in environmental trace

element measurements where large concentration ranges exist. In such cases a combination of different techniques is most likely to provide the most

reliable data.

30 1-

0.5 1 Concentration (fig/g)

Figure 2. Imprecision of the analytical techniques under the

constraints of the analytical protocol as observed

for the determination of cadmium in human liver.

A similar evaluation was performed for the other elements where observations by different techniques were available. The respective ranges of

elemental concentrations and corresponding relative errors of the techniques

is summarized in Table 2. For each of these elements at least one method can provide measurements of most concentrations at a level of precision suitable

for the storage evaluation i.e., imprecision <10 percent (see reference 2 for

discussion of the storage evaluation study). For NAA the imprecision for most

priority elements is between 1 and 7 percent for the observed concentrations,

while AAS and VOL are providing data with 2 to 20 percent imprecisions

depending on the concentration found. Only in the case of aluminum did the

84
currently applied techniques, (the combination of AAS and NAA) provide results

with a 10 to 15 percent imprecision. However, good agreement was found

between the methods especially for the few very high values of aluminum where

the imprecision was less than 2 percent. In general, the limited quantity of

material for multielement determination on individual samples did not

excessively add to the uncertainties of the various techniques.

TABLE 2. COMPARISON OF DYNAMIC IMPRECISION OF FOUR ANALYTICAL TECHNIQUES

Element

rnnppnl" K*;^ "H nn nnp

(vig/g wet weight) AAS

Percent Imprecision -

IDMS NAA VOL

Al 0.3 - 2(31) 22- 5(1.6) >30 -9(1.5)

Mn 0.6 - 2 25- 5 4.5-1.5

Cu 3.5 -11 7- 2 0.5 20 -6 6-1

Zn 28 -96 0.4-0.2 25-2

Se 0.4 -0.65 18-14 1.2-0.9

Cd 0.3 - 5 8- 6 23 -2 >30-1.5

Pb 0.12- 1.7 0.2 15-3

DISCUSSION OF SOME FINDINGS

Conclusions on environmental impacts, health related statements, etc. are not readily available from the small number of specimens analyzed,

especially since the specimens in this first year of analyses were not

selected to represent the population or parts of the population (see Table 4,

Section 2 for information on the donors). However, some interesting findings

can be pointed out to illustrate the potential utility of a specimen banking

program in relation to monitoring environmental trends.

A significant result of this study is the very narrow range of concentration data for many essential trace elements (figure 3) as compared to the wide

scatter of pollutant element concentrations (figure 4). For example, the

concentrations of Se vary by only a factor of 1.8 and other essential elements

(Mg, Co, Cu, and Zn) vary by only a factor of 2-3.5, whereas several pollutant

elements (Al , As, Cd, Hg, Pb, and Tl ) vary by as much as a factor of 100.

Since the biological variability of the individual specimens appears to be

reasonably small, these broad ranges for the pollutant elements in the data

may point to sources of pollution to which the individuals were exposed.

The range of the selenium data is even lower if individuals from the same geographical area are studied. Evaluation of the data by the NBS

Statistical Engineering Division revealed a statistically significant differ- ence in average Se values for samples from Baltimore (0.44 yg/g) compared to

Minneapolis (0.51 ug/g) and Seattle (0.56 yg/g). Similar evaluations

indicated that possible geographic differences may exist for several other

85
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•r— M4-> O

E

dJ O)

10 CD

to E

O) fO

s> X

•r- (U

CO 4->

rt3 ^- O

o '1— T3

</) E

c

o

(/) +-> O)

»a E

S- •r— -ME

<U T3 O •r- E

o o o

86
I II III nil II

III tiiiii I

I iiiiiiiiiiiiii

I I iiiiiiiiiiiii ill I I

I III I mini nil I

I I Hill llllllllllll I

I I I I 1 I I I I I llllllllllll

jQ

Q.

0)

I

O

n

<

cu>

p—

CI * fO QJ

+^

C.

</) +-> C

(U CI

«

(I)

cu c

Z3

u) o

O ^—

tr^ i\J S- fo +^ +-' (O

+-> T3

c

fO M—

-M O

(L) cr Os- X

•P" O) CO +-> ro M- a O a V) c

c

o

4-> QJ (O C

s~ •p4-> c

(U D

o •r— c

o "o o

C7>

(M (M CO

6/6ry •(NOIlVdlN30NOO)o^o|

87
elements. The trace element data were also evaluated with respect to possible

correlations of variables such as age and sex with elemental concentration and

correlations of different elements with each other. Because of the relatively

small data set, the results were only partially successful in suggesting some possible correlations. However, as real-time analyses provide additional data

in each year of the pilot program, the potential for substantiating these

possible correlations will increase significantly. Because of concern about

possible analytical error, some data points which exceeded the previously

reported ranges were also investigated. The highest iron value, 522 yg/g,

was from a 40-year steel worker, while the highest zinc value, 96.1 yg/g, was found in the liver of an individual who worked in a galvanizing plant.

Many pollutant trace elements were found at concentration levels which

are on the low side or below previously reported data (1). Specifically,

levels of aluminum, arsenic, thallium, and lead are significantly lower than

the concentrations reported in the literature (see figure 3). One possible

interpretation of these differences is that the observed trends are the

consequence of the careful contamination control in the Pilot NESB Program.

The precautions taken to preserve the integrity of the samples, especially

the minimization of possible sources of contamination during sampling and

sample preparation, would naturally result in lower concentrations.

The selection of the specimens anfl possible low exposure of most of the

donors to pollutant sources may offer another explanation for the low

pollutant trace element concentrations. Recent significant and general

improvements in the environmental concentrations and therefore reduced human

exposure to those elements may have already resulted in lower human body

burden. In particular for the case of lead, this interpretation of the

results is supported by the significant decrease of lead levels in the air

since about 1974 (compare for example Ref. 3 and 4) and the latest findings

of lower blood lead levels in children (5,6).

Also a direct comparison of 1973 data from Baltimore residents (7) with

the data in this study shows this trend within a specified geographical

location. In 1973 the mean lead concentration of 22 livers from residents

of Baltimore was 2.5 yg/g with a range of 1.0-6.3 yg/g. In 1981 the mean

lead concentration of 8 livers was 0.58 yg/g with a range of 0.25-1.15 yg/g.

However, there is a significant element of uncertainty in this comparison,

since the applied analytical approaches cannot be compared. This uncertainty

can only be overcome with a formal and documented monitoring and banking

program.

88
REFERENCES

1. Iyengar, 6. V., W. E. Kollmer, and H. J. M. Bowen. The Elemental

Composition of Human Tissue and Body Fluids. Verlag Chemie, Weinheim,

New York, 1978.

2. Harrison, S. H., R. Zeisler, and S. A. Wise. Pilot Program for the

National Environmental Specimen Bank - Phase I. EPA-600/1 -81 -025.

U.S. Environmental Protection Agency, 1981, p. 19.

3. Kowalczyk, G. E., C. E. Choquette, and G. E. Gordon. Chemical Element

Balances and Identification of Air Pollution Sources in Washington,

D.C. Atmospheric Environment, 12:1143, 1977.

4. Kowalczyk, G. C. , G. E. Gordon, and S. W. Rheingrover. Identification

of Atmospheric Particulate Sources in Washington, DC Using Chemical

Element Balances. Environ, Sci . Technol., 16:79, 1982.

5. Billik, H. I., A. S. Curran, and D. R. Shier. Analysis of Pediatric

Blood Lead Levels in New York City for 1970-1976. Environ. Health

Presp., 31:183, 1979.

6. Marshall, E. EPA May Allow More Lead in Gasoline. Science, 215:1375,

1982.

7. Poklis, A. and C. Freimuth. Lead Distribution in Soft Tissues of

Baltimore Residents, 1973. Environ. Contam. Toxicology, 15:311,

1976.

89

SECTION 10

DEVELOPMENT OF ANALYTICAL METHODOLOGY FOR THE DETERMINATION OF

ORGANOCHLORINE COMPOUNDS IN HUMAN LIVER SAMPLES

by

W. F. Kline, C. F. Allen, S. N. Chesler, L. R. Hilpert, and S. A. Wise

INTRODUCTION

In the Pilot National Environmental Specimen Bank Program (NESB),

environmental samples will be analyzed for both inorganic and organic constituents. During the first year of sample collection in this program, the human

liver samples were analyzed only for trace elements, and analytical methods

for selected organic pollutants were evaluated for implementation for the

second year samples.

In contrast to trace element analyses, screening for large numbers of

organic compounds is difficult because of the requirements of specific extraction, digestion, and/or isolation procedures for various compound classes.

As a result, two classes of organic compounds, organochlorine compounds and

polycyclic aromatic hydrocarbons (PAH), have been selected for inclusion in

the current analytical protocol for the pilot NESB program. Additional

classes of compounds will be added to the program as resources increase and

analytical expertise for these groups is developed. For the human liver

samples the major focus of the organic analyses is the determination of

organochlorine compounds (i.e., organochlorine pesticide residues) due to

their widespread use and their abundance in human tissues. Because of the

extremely low levels of PAH expected in human liver samples (possibly due

only to sample contamination), PAH will be measured only in selected liver

samples. However, for the analyses of the second specimen type, mussels, the

determination of PAH will be a major concern in addition to the organochlorine

compounds.

EXPERIMENTAL

Sample Preparation

The human liver samples are analyzed for organochlorine compounds following the general scheme of solvent extraction, liquid chromatographic cleanup,

and capillary gas chromatographic analysis with selective and sensitive

electron capture detection. An extraction/cleanup procedure used for liver

analysis by investigators in the Pilot Environmental Specimen Bank Program of

91
the Federal Republic of Germany was adopted for use in the NBS pilot program

(1). A 2 g sample of the liver homogenate (prepared by cryogenic homogenization as described in Section 3) was thoroughly mixed at room temperature with

approximately 50 g of anhydrous sodium sulfate in an agate mortar and pestle.

The resulting mixture was a dry powder. The liver/sodium sulfate mixture was placed in a 22 mm x 350 mm glass column with about 1 cm of sodium sulfate on the top and bottom of the sample. Solutions containing the internal standards

(pentachlorobenzene, aldrin, and/or decachlorobi phenyl) were added at the top

of the sample column. The liver/sodium sulfate mixture was then extracted by

eluting 200 mL of a 2:1 mixture of hexaneracetone (all solvents were commer- cially available "distilled in glass" or "pesticide analysis" grade) through

the column. The extract was concentrated to about 50 mL using a rotary

evaporator. An aliquot (1 mL) was removed, evaporated to dryness using a rotary evaporator, and the residue weighed to determine the percent extractable fat (lipid). The bulk of the extract was transferred to the top of a 22 mm x 350 mm glass column packed with 25 g of 60-80 mesh Florisil (activated

by heating at 400 °C overnight, then deactivated by adding 3 percent by weight

of water). The chlorinated hydrocarbon pesticide residues were eluted from

the column in the initial fraction of 150 mL of 20 percent methylene chloride

in hexane. This fraction was concentrated in a rotary evaporator and then

analyzed by gas chromatography (GC) with electron capture detection or by gas

chromatography-mass spectrometry (GC-MS).

Quantitation of Organochlorine Pesticide Residues by Gas Chromatography (GC)

For the GC analyses with electron capture detection, the chromatographic

separations were performed on a fused silica SE-52 capillary column (30 m x 0.25 mm i.d.). The samples were injected at an initial temperature of 200 °C

and the temperature was then programmed at 3 °C/min to 290 °C. Standards of

the compounds of interest were run under identical conditions to determine

retention times and electron capture detector response. Quantitation was based on comparison to internal standards of pentachlorobenzene, aldrin, and

decachl orobi phenyl

Quantitation of p,p'-DDE in Human Liver Extracts by GC-MS

For the GC-MS determinations the GC separations were performed on a 30 m X 0.25 mm i.d. fused silica SE-30 wall -coated, capillary column which

was interfaced directly to the ion source of a quadrupole mass spectrometer.

The column was temperature programmed from 150 "C to 275 °C at a rate of

4 "C/minute. The injection port and GC-MS interface were maintained at

275 °C. Under these conditions the aldrin (internal standard added during

sample preparation) and p^p'-DDE (1 ,2-bis-(p-chlorophenyl )-l ,1 -dichloroethylene) were eluted as symmetrical peaks at approximately 13 and 18 minutes,

respectively.

Selected ion monitoring detection was used to enhance sensitivity and

insure selectivity by minimizing any co-el uting interferences. Two characteristic, intense ions were monitored for both the p^p'-DDE and the aldrin

internal standard. In each case the ions monitored contain chlorine atoms

which yield characteristic isotope patterns. Ions at m/z 246 and 248 were monitored for the p,p'-DDE and ions at m/z 263 and 265 were monitored for

92
aldrin. Ion intensity ratios for each analyte in the samples were compared

to ion intensity ratios for standards of the pure compounds to insure that

there were no co-el uting interferences. A mass spectrometer dwell time of

100 ms was used to insure at least 20 data points across each chromatographic

peak.

A solution of pure aldrin and p^p'-DDE was run under identical conditions

as the sample extracts to determine relative response factors in the selected

ion monitoring mode. Determinations of the p^p'-DDE concentration in the

liver extracts were made based on the selected ion record peak areas, the

aldrin (internal standard) spike concentration, and the experimentally

determined relative response factors.

RESULTS AND DISCUSSION

The analytical methodology for the determination of organochlorine

compounds in human liver has been developed and applied to several samples

from the Pilot National Environmental Specimen Bank Program. The gas chromatogram of one of the samples is shown in Figure lA. The major chlorinated

compound in the samples analyzed was identified by GC and MS as p,p'-DDE

which is a major metabolite of p^p' -DDT (1 ,1 -bis(p-chlorophenyl )-2 ,2,2-

trichloroethane) . Quantitative results for the determination of p^p' -DDE in

three samples are summarized in Table 1. Table 1 also includes the results of

an interlaboratory sample split with the U.S. EPA Health Effects Research

Laboratory (2). Trace quantities (5-30 ng/g wet weight) of the following

organochlorine species were also present: hexachlorobenzene (HCB), B-isomer

of hexachlorocyclohexane (e-BHC), trans-nonachlor, dieldrin, p^p'-DDD

(2,2-bis(p-chlorophenyl)-l ,1-dichloroethane) , and p^p'-DDT. These compounds

will be measured in 'v30 samples/year during the pilot specimen banking program.

Heptachlor epoxide and oxychlordane are also present but coelute as one peak

under these GC conditions. Trace quantities of polychlorinated biphenyl (PCB)

isomers were found in the sample shown in Figure lA. Total concentration of

PCB isomers will also be determined on selected samples.

TABLE 1. DETERMINATION OF p,p'-DDE IN HUMAN LIVERS BY GC-MS

Sample I.D. GC^

(yg/g wet weight)

GC/MS GC (EPA)^

M0136

B0025 0.72 ± 0.04

0.23 ± 0.01

0.75 ± 0.04

0.77^

0.81^

0.23^ 0.26

SOI 29 0.35 ± 0.01

Uncertainty based on four determinations, reported as one .standard deviation from the mean. Single determinations.

^Mean of two determinations, range of ±7 percent.

Single determination.

93
Human Liver Samples

A

a

a

"o. d.

'Major PCB Peaks (Arochlor 1260)

a. A

&

O

-V—

-r

10

-1

30

I 15 Time(min)

I 20

1^

25

Figure 1. Gas chromatograms from the analysis of human liver samples,

(A) Liver sample from NBS/EPA Pilot Program, (B) Liver

homogenate from German Pilot Environmental Specimen Bank

Program.

94
Several studies have reported data for selected pesticide residues

(primarily HCB, DDE, and dieldrin) in human liver samples (3-6). Kraul and

Karlog (3) reported ranges of DDT and metabolites (predominately DDE) at

0.62-22 yg/g extractable fat (mean of 6.0) and dieldrin at 0-1.07 yg/g

extractable fat (mean of 0.29) in human livers for 77 samples from Denmark.

Bj0rseth et al . , (4) found mean levels of HCB and DDE of 0.15 and 1.87 yg/g

extractable fat for 10 liver samples from Norway. The results of the analyses

samples from the Pilot NESB program will add considerable information to

the existing data base for pesticides in human livers.

A sample of a human liver homogenate from the German Pilot Environmental

Specimen Bank was analyzed as part of an interlaboratory comparison of

methods. The gas chromatogram from the analysis of the German liver homogenate

is shown in figure IB and the quantitative results are summarized in Table 2.

The quantitative results from the analysis of a liver sample from the NBS pilot

program are also given in Table 2 for comparison. Of particular interest in

comparing the two samples is the large quantity of hexachlorobenzene present

in the German Sample as compared to the U.S. sample. In addition, when

quantitation is on an extractable fat basis, the U.S. sample is higher in

concentrations of several compounds. Analyses of several liver samples from

the U.S. pilot program indicate that the levels of HCB are approximately a factor of 10 lower than the German homogenate. Acker and Schulte (7) reported

higher levels of HCB in human fat from Germany when compared to fat samples

from the Norwegian study (4), 6.4 vs. 0.11 yg/g extractable fat.

TABLE 2. CONCENTRATIONS OF SELECTED ORGANOCHLORINE PESTICIDE RESIDUES IN

HUMAN LIVER SAMPLES^

German Homogenate^ U.S. Sample^

Compound

ng/g ng/g

(wet weight) extractable fat

, ng/g

(wet weight)

ng/g

extractable fat

Hexachlorobenzene 413 + 20 3110 + 260 8 + 3 190 + 66

a-BHC 0.9 + 0.1 23 + 2

3-BHC 88 + 9 670 + 120 44 + 12 1100 + 330

trans-nonachlor 8 + 1 60 + 15 27 + 6 670 + 170

p^p'-DDE 305 + 9 2310 + 315 195 + 59 4840 + 1590

Dieldrin 22 + 4 170 + 40 83 + 38 2030 + 960

p^p'-DDD 34 + 5 260 + 60

p^p'-DDT 25 + 5 190 + 40 25 + 6 606 + 163

^Measurements based on three samples, uncertainty is ±la calculated standard

deviation based on 2 degrees of freedom.

^13% average extractable fat.

^4% average extractable fat.

95
The gas chromatog rams shown in figure 1 contain aldrin, pentachlorobenzene, and/or decachlorobi phenyl as internal standards for quantitation.

Recent results indicate that quantitation based on aldrin provides more reproducible results than pentachlorobenzene or decachlorobi phenyl (see Table

3). In addition, aldrin is rarely found in biological samples since it is

readily converted to dieldrin.

TABLE 3. CONCENTRATIONS OF SELECTED ORGANOCHLORINE PESTICIDES IN

HUMAN LIVER HOMOGENATE FROM THE GERMAN PILOT ENVIRONMENTAL SPECIMEN BANK^

Internal Standard Internal Standard

Aldrin Decachlorobi phenyl

ng/g ng/g

Compound (wet weight) (wet weight)

Hexachlorobenzene 413 ± 20 427 ± 60

B-BHC 88 ± 9 90 ± 13

t2'ans-nonachlor 8 ± 1 8 ± 2

p,p'-DDE 305 ± 9 316 ± 31

Dieldrin 22 ± 4 23 ± 5

p^p'-DDD 34 ± 5 35 ± 7

p^p'-DDT 25 ± 5 26 ± 4

^Measurements based on three samples, uncertainty is ±la calculated

deviation based on two degrees of freedom.

96
REFERENCES

1. Gebeftigi , I. Institute fUr Okologishe Chemie der Gesellschaft fiir

Strahlen-und Umwel tforschung mbH, Munich, Germany, personal

communication.

2. Bristol, D. Health Effects Research Laboratory, U.S Environmental

Protection Agency, Research Triangle Park, North Carolina, personal

communication.

3. Kraul , I., and 0. Karlog. Persistent Organochlorinated Compounds in

Human Organs Collected in Denmark 1972-73. Acta Pharmacol. Toxicol.,

38:38, 1976.

4. Bj0rseth, A., G. Lunde, and E. Dybing. Residues of Persistent

Chlorinated Hydrocarbons in Human Tissues as Studied by Neutron

Activation Analysis and Gas Chromatography. Bull. Environ. Contam.

Toxicol., 18:581 , 1977.

5. Hattula, M. L. , J. Ikkala, M. Isomaki, K. Maatta, and A. U. Arstila.

Chlorinated Hydrocarbon Residues (PCS AND DDT) in Human Liver, Adipose

Tissue, and Brain in Finlan d Acta Pharmacol. Toxicol., 39:545, 1976.

6. Morgan, D. P., and C. C. Roan. Chlorinated Hydrocarbon Pesticide

Residues in Human Tissue. Arch. Environ. Health, 20:452, 1970.

7. Acker, L. , and E. Schulte. Die Naturwissenschaften, 61:32, 1976.

97

APPENDIX I

DEVELOPMENT OF A RADIOCHEMICAL SEPARATION PROCEDURE FOR TIN

R. R. Greenberg

Although tin has been shown to be essential for mammalian nutrition (1),

it can be highly toxic in some forms, i.e., the alkyl tin species (2).

Unfortunately, tin cannot be readily determined in biological materials

because of the naturally occurring low levels. For example, very few biological reference materials with known tin concentrations are available.

Relatively little reliable data exist concerning the tin concentrations in

human and animal tissues and fluids.

Neutron activation analysis can be used to determine tin in most biological materials at the naturally occurring levels if a radiochemical procedure

is used to separate tin from interfering elements and if the background level

of radiation (noise) is reduced. Both the ^^^Sn and ^^"^nisn isotopes can be

used to quantify tin if the interfering ^^^Jb (for ^^^Sn) and '^'^Sc (for

ii^msn) isotopes are removed. Removal of these interferences was accomplished

with a separation procedure based on the extraction of Snli+ into toluene.

The sample is dissolved with nitric, sulfuric, and hydrofluoric acids and

heated to fumes of sulfuric acid. The sample is cooled and hydrofluoric acid

is again added. The sample is again heated to sulfuric acid fumes and a large fraction of the selenium present is driven off as. volatile fluorides.

This hydrofluoric acid treatment is repeated and the sample allowed to cool.

A few drops of hydrogen peroxide are added to insure that all tin is in the

upper, +4, oxidation state and excess peroxide is destroyed by the addition

of water and boiling. If necessary, the solution is filtered to remove insoluble oxides and sulfates. The solution is transferred to a separatory

funnel and potassium iodide is added. The tin is then extracted into toluene

as the iodide (Snli+). The aqueous fraction is discarded and the toluene

fraction is washed twice with a potassium iodide-sul furic acid solution. Tin

is then back-extracted into a basic, aqueous solution of Na2EDTA. The

sample is counted and the tin concentration is determined using either the

^^''''^Sn isotope or the iismjp daughter of ^^^Sn. Tin recovery for this

procedure is highly quantitative (>99.5 percent).

This procedure has been evaluated by analyzing several NBS SRM's. The

results obtained are compared with literature values in Table 1. Analysis of

human liver samples is currently in progress and the results of the first 10

samples are summarized in Table 2. Due to the long halflives of the radionuclides used for the tin determination, this radiochemical procedure can be

applied after the instrumental multielement neutron activation analysis.

99
Thus, no additional samples are needed in the NESB program and the data are obtained from the very same subsample.

TABLE 1. TIN CONCENTRATIONS IN STANDARD REFERENCE MATERIALS (nq/q)^

SRM Found Li tpratu rp^

Orchard Leaves (1 571290 ± 17^ 294 ± 16

290 ± 20

4100

Citrus Leaves (1572) 235 ± 12

Bovine Liver (1577) 20 ± 3 21 ± 3

10

^Weight basis — as received (not dried).

^Reference 3.

^Uncertainties are ±ls sample standard deviation, >5 degrees of

freedom.

TABLE 2. CONCENTRATION OF TIN IN HUMAN LIVER

SPECIMENS (nq/g)

Sample

Identification Wet-Weight Basis

L1S0090 301 ± 18^

L1S0092 164 ± 11 LIBOIOO 224 ± 14

LI SOI 10 211 ± 15

L1M0118 135 ± 8

L1B0121 501 ± 12

L1M0126 670 ± 18

L1M0127 712 ± 21

L1M0131 213 ± 8

L1M0140 221 ± 12

^Uncertainties are ±ls based on counting

statistics of a single determination.

100
The implemented radiochemical procedure is an exceptionally clean

separation. The resulting reduction in background radiation is better than a factor of 1000, and no scandium or terbium can be detected using other, more

intense, y-rays, indicating decontamination factors of >20,000 for scandium

and >1000 for terbium. The only element observed to accompany tin in the

procedure is selenium for which decontamination factors of up to 10^ are observed.

REFERENCES

1. Schwarz, K. , D. B. Milne, and E. Vinyard. Biochem. Biophys. Res.

Commun. 40:22, 1970.

2. Schroeder, H. A. Environment 13:18, 1971.

3. Gladney, E. S. Compilation of Elemental Concentration Data for NBS

Biological and Environmental Standard Reference Materials. Los Alamos

Informal Report LA-8438-MS, 1980.

101

APPENDIX II

Journal ofRadioanalytical Chemistry, Vol. 75, Nos 1-2 (1982) 27-37

ULTRATRACE DETERMINATION OF PLATINUM

IN BIOLOGICAL MATERIALS VIA NEUTRON

ACTIVATION AND RADIOCHEMICAL SEPARATION

R. ZEISLER, R. R. GREENBERG

Center for Analytical Chemistry, National Bureau of Standards, Washington, DC 20234 (USA)

(Received November 2, 1981)

A neutron activation analysis scheme based upon a radiochemical separation of the

activation products has been developed. The method utilizes the inherent sensitivity of the

activation reaction "'PtCn, 7)"'Pt and counting of the daughter nuclide "'Au. This

nuclide is radiochemically separated from interfering activities tiy homogeneous precipita- tion as elemental gold. The remaining interference of the secondary reaction *"Au(n,7)"*Au(n,7)"* Au from gold in the samples is quantitatively assessed and corrected. During this process accurate gold concentrations in the samples are obtained at ultratrace levels. The analysis scheme is applied to gold and platinum determinations in biological Standard Refraence Materials and human liver specimens. Gold and platinum are determined at concentrations of 5 10 g/g, and at higher levels.

Introduction

Concern continues about the possible impact on the biosphere from increasing use of platinum in industrial and medical applications, and interest is increasing

in the development and application of analytical methods to determine platinum

in enviroimiental and biological materials. Platinum has not yet been shown to accumulate in the environment, and adverse effects have not been established despite the intense interaction of the element and its compounds with biological sys- tems. However, it has been assumed that platinum will be detected in measurable

quantities in the environment with the end of the last decade, as platinum may

accumulate to higher concentrations, and more sensitive methods are developed.

Two possible major sources contributing to environmental hazards and human

body burden are the noble metal catalysts employed to reduce automobile emission and the administration of platinum-containing drugs in cancer chemotherapy.

Although only a small quantity of the metal is used in an automobile catalytic converter, the annual consumption by the ntomotive industry is estimated to be

about 18 700 kg (600 000 troy ounces) of platinum.' The therapeutic utilization

/. Radioaml. Chem. 75 (1982) 27

103
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

of platinum complexes is still in its beginning stages. However, increasing medication and pharmacological research with therapeutic doses of the complexes up to 80 mg/kg body weight introduces the element into the environment possibly in form of highly bioactive compounds.

Procedures have been developed for many analytical techniques, including neutron activation analysis (NAA), to determine platinum, but originally most

have been concerned with the analysis of geological and industrial materials containing trace amounts of the element, or the evaluation of reagents and techni- ques applied in industrial processes. In a few instances the methods have been

expanded to applications in the biological and enviromnental fields,^ and elev- ated platinum levels have been determined successfully in enviroiunental and biol- ogical matrices.^ NAA has been applied to determine the element in tissues and

blood after administration of antitumor drugs containing platinum coordination

compounds of the cis-Pt(L)2Cl2 type (L = N-coordinated amino or peptide ligands).^''

However, previous attempts to measure baseline concentrations have failed in many

investigated matrices because the reported sensitivities of the respective techniques were several ng/g, while the actual levels were at least one order of magnitude

lower.

Quantitative analysis of elemental concentrations below 1 ng/g almost always

requires rigorous minimization of analytical blanks, or at least the rigid control of the blank. The application of NAA techniques provides a blank-free analytical

step leaving only pre-irradiation handling as a controllable source of analytical blanks. To take full advantage of the capabihties of NAA and the method's intrinsic sensitivity for the determintion of platinum at levels below 1 ng/g, a newlydeveloped NAA scheme with radiochemical separation of the activation products

is investigated in this study. The technique is applied to the determination of

naturally occurring concentrations of platinum in biological Standard Reference

Materials (SRM's) and in human liver tissue.

NAA method

The different platinum isotopes undergo a multitude of nuclear reactions with

reactor neutrons. For analytical purposes, the most useful reactions are: "*Pt(n,7y"Pt and "*Pt(n,7)'"Pt03r)»"Au. The nuclear data for the

respective reactions and the common interferences for the analysis via the use of these reactions are summarized in Table 1. As demonstrated in Table 1, the use of either one of the reactions requires

radiochemical separation from the interferences to achieve accurate determinations

at low levels. In addition, with the expected extremely low levels of platinum in

28 J. Radiomud. Chem. 75 (1982)

104
R. ZEISLER. R. R. GREENBERG: ULTRATRACE DETERMINATION

Table 1 Nuclear data for platinum and gold analysis and for major interferences

Element Isotope Half-Ufe Gamma-energy,

keV

Activity*

Analytical interest

Pt

Au

"''Pt

•"Au

1 y 8 Au

Potential interferences Hg

Pt K(3'2 X-ray

Au Kp'l X-ray

Bi Kai X-ray Ca "'Sc

Sn •""Sn

Lu •"Lu

Au •"Au

18.3 h

3.15 d

2.697 d

64.7 h

3.41 d

14.0 d

6.74 d

3.15 d

77.35

191.31 158.37 208.20 411.80

77.35

77.8 77.9

77.1 159.40

158.40 208.34

158.37 208.20

1.6 - 10"'

3.9- 10"'

14.7

3.2 101.2

6.7 - 10"'

fluorescence

fluorescence background

2.1 • lO-'

6.2 - 10-*

2.2 - I0-' 15**

3.3**

*Gamma emission of respective energy per nanogram of the element and

per second, after one hour irradiation time at a thermal neutron flux of 5

' n $"• • cm"', and three days decay time. "Experimental value for irradiation facility RT-3 at the NBS Reactor.

10^

biological materials, radiochemical efforts should be aimed at "background and

interference-free" counting of the respective nucUde to achieve best possible limits of detection, i.e., the separation should be selective for the product nuclide. The

use of the isotope '"Pt imposes problems which cannot be easily controlled. The addition of platinum carrier during the radiochemical separation increases the contribution of the interference by the Pt K0'2-emission. The numerical correction

of this effect is severely hampered by the difficult determination of the respective Pt-X-ray peaks in the spectrum. The Pt—X-rays interfere with the Au—X-rays

produced in the contacts of the detector and other background radiation. Limits

of detectioii for radiochemical NAA via '"Pt are reported in the range of several ng/g.'

The determination of platinum via the daughter nuclide *"Au has the greatest

inherent sensitivity. The specific activity produced during irradiation and subse- quent decay of "'Pt is considerably higher than for *"Pt, and the higher rela-

/. Radioanal. Chem. 75 (1982) 29

105
R. ZEISLER, R. R. GREENBERG; ULTRATRACE DETERMINATION

tive emission rate of the main gamma line provides additional sensitivity (Table 1). The longer half-life allows a more rigorous radiochemical procedure with several steps to provide interference-free counting vdth low background. The weU known

interference by *'Sc at the 158.35 keV line can be controlled by a careful radio- chemical separation. Any possibly remaining *'Sc activity can be detected by the use of a high-resolution detector. The 159.40 keV gamma energy would distort the platinum gamma line and show up as a doublet or shoulder in the spectrum. The

quality of the separation can be further ascertained by the search for *'Sc activities. A more serious problem can occur when the 208.2 keV line of *"Au is used. The interfering "'Lu is often disregarded but can lead to serious over- estimation of platinum concentrations. Other elements likely to follow gold in the radiochemical separation are noble metals, rare earth elements, and hafnium,

which might cause increased background in the respective energy range. When complete separation is achieved, the only remaining interference to be

considered is the second order *"Au(n, 'y)"*Au(n, 'y)*"Au reaction from the

naturally occurring gold in the sample matrix. Second order interferences have

been discussed in the literature' and the magnitude of these interferences have

been tabulated for specific irradiation condition conditions.® The second order

reaction is a function of t* for t <^ T of the respective nuchde (4> = neutron

flux, t = irradiation time, T = half life) and can become a severe interference for the determination of platinum with relatively hig|h gold levels, a situation that

substantially raises the lower limit of detection for platinum. In biological materials where the concentrations of gold may not be significantly higher than that of platinum, this interference can be quantitatively assessed and corrected. However, this correction depends on an experimental determination with limited ac- curacy and precision. The enor in the determination of the "*Au to *"Au

ratio, both nuclides are formed from the naturally occurring "'Au, may become

the governing factor in the determination of platinum. Considering the t^ dependence of the interfering reaction, the detection limit for platinum may not be lowered due to better counting statistics. I>ue to the inaccuracy in determining

the *'*Au to *"Au ratio optimum irradiation times can be estimated to achieve

the lowest relative statistical error for given platinum to gold ratios in the analytical sample [Eq. (1)].

Vmptt + mAut* mAut^E . '^rel = K I + — 1 (1)

mpf t mp^ t

30 /. Radtoaml. Chem. 75 (1982)

106
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

where a,ei — relative statistical error for the 159.35 keV peak;

K — constant containing nuclear parameter from activation equatE

tion, and specific neutron flux conditions; — mass of platinum and gold in the sample; — irradiation time; and — non-random error for the "^Au to "'Au ratio.

The non-random error in the determination of the ''*Au to '^'Au ratio is caused

by several effects as discussed below. A realistic estimate of E= 15% is used in Eq. (1) to generate the optimization diagram (Fig. 1) for the relative sensitivity

of the platinum determination as a function of the statistical error of the cor- rected 158.35 keV peak and as a function of gold concentration and irradiation time.The inaccuracy and imprecision of the ''®Au to *''Au ratio determination

are mainly caused by two effects. The variations in the neutron flux energy spectrum and intensity over the sample volume have larger than usual effects on the activation rate because of the considerable resonance activation integrals

and the dependence of the interfering reaction. The second effect is the dis- tortion of the gamma spectrum at the 158.35 keV line by the ''®Au backscatter "peak". This distortion is shown in Fig. 2. The peak area determinations are very

difficult and even with 100 000 counts in the 158.35 keV ganuna line the uncertainty of this measurement is at least 2 to 3%.

Fig. 1. Platinum detection limits in dependency of Pt to Au ratio and irradiation time at a neutron flux of 5 • 10" n • cm (RT-3 position of NBS Reactor). The minimum

for Pt = Au = 1 corresponds to a detection limit of about 20 10'" g/g in a biological matrix

Irradiation time^ h

/. Radioanal. Chem. 75 (1982) 31

107
R. ZEISLER. R. R. GREENBERG: ULTRATRACE DETERMINATION

158.35

5s,V3 10'^

lh,4 9 10'

ncm"

n cm'

160 180 Energy ,keV

Fig. 2. Interfering activity from ' "Au(n. -y)"* Au(n. 7)' " Au. Spectra obtained from pure

gold standards

Sample preparation

All samples have been homogenized and freeze-dried. Approximately 1.5 g of

each material is pressed into a pellet of about 1 cm' volume. A blank consisting

of cellulose powder is treated the same way. To assure representative sampling,

up to three pellets (5 g total weight) are used for the determinations in Standard

Reference Materials. Standards are prepared by dissolving the pure metals and

pipetting calibrated solutions on filter paper (Whatman 41), drying and pelletizing.

Samples and standards are then sealed in precleaned bags made from linear polyethylene and packaged in fixed geometry in the irradiation rabbit. The sample

preparation is carried out under clean room conditions.

Irradiation and counting

Irradiations have been carried out in the pneumatic tube facilities of the NBS

Research Reactor at a flux of 5 • 10* ' n • s"' • cm"* for time intervals of 15 min

to 1 h. After 24 h decay the samples and standards are unpacked and processed.

They are ready for counting 3 to 4 days after irradiation. The samples and stand- ards are counted in close geometry on high-resolution Ge(Li) detectors (18% relative efficiency, FWHM 1.9 keV at 1332 keV). The spectral data are collected with

32 J- Radioanal. Chem. 75 (1982)

Experimental

108
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

the Nuclear Data* 6600 multichannel analyzer system. Special effort, including

hand-integration, has been used for the determination of small peak areas.

Radiochemical procedure

The principle of this newly-developed radiochemical separation scheme is to

dissolve the sample matrix completely, to equilibrate the produced *"Au and

*"Au activities with the gold carrier, and to separate the gold from the other matrix activities in as pure a form as possible. Fifty ml of a 2 : 1 mixture of

cone, nitric acid/sulfuric acid containing 80 mg of gold carrier and 10 Mg scan- dium is heated gently to dissolve the tissue samples. The plant materials require

initial addition of hydrofluoric acid to dissolve silicate components. After eight to fifteen hours at elevated temperatures (~90 °C), hydrochloric acid is added

with additional nitric acid if necessary to form aqua regia and to equihbrate the

gold completely. The temperature of the solutions is then raised to boil off nitric and hydrochloric acids. Blackening of the solution indicates remaining organic material which is subsequently destroyed by adding portions of nitric acid until all organic material has vanished. Aqua regia is used again to ensure equiUbration.

The temperature of the solution is then slowly brought up to over 250 °C, boiling off the more volatile acids. At temperatures above 250 °C, gold compounds, especially AuCla and AujOs, decompose and an auto-reduction to elemental gold takes

place. This process is sufficiently slow to allow the gold to homogeneously precipitate in the form of fme particles or leaflets which agglomerate into nugget or

leaflike precipitates without the inclusion of matrix activities. This process takes

several hours. After its completion, the gold precipitate is thoroughly washed with

HNO3, H2O, HCl and H2O in that order. The precipitate is dried on a hot plate

and weighed for yield determination. The chemical yield for this separation is generally 95—98%. The precipitate is sealed in a polyethylene bag and rolled out

to a thin foil of about 1 cm^ area for counting.

The plant materials yield some insoluble white precipitates with gold precipitate. These precipitates seem to be metal oxides. They contain high rare earth •and hafnium activities. The contaminating activities caimot be separated by washing. Consequently, the gold precipitate is dissolved in aqua regia. The solution is filtered, sulfuric acid added, and the gold is precipitated again. With another

2—5% loss of yield, a clean gold sample is obtained for counting.

'Certain commercial equipment, instruments, 01 materials are identified in this paper to

specify adequately the experimental procedure. Such identiHcation does not imply recom- mendation by the National Bureau of Standards, nor does it imply that the materials or equipment identifled are necessarily the best available for the purpose.

/. Radioanal. Chem. 75 (1982) 33

3

109
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

Table 2 Determination of " ' Au to ' "Au ratio (Concentration in standards 10"' g/g)

Detector Apparent Pt '»'Au Au in standard "»Au Ratio

1 h irradiation rx

6690

95

5.90 ± 0.07*

6.26 ±0.17

6.63 ± 0.20

6.00 ± 0.10

5.958 5.918

5.782

6.122

0.99 ± 0.15**

1.06 ±0.18

1.14 ±0.16

0.98 ±0.15

15 min irradiation 6690

0.92 ± 0.06

1.03 ±0.10

4.299

4.390

0.21 ± 0.03

0.23 ± 0.04

*Error due to counting statistics (I a). •Experimental error (1 a), five degrees of freedom for 1 h

irradiations and three degres of freedom for 1 S min irradiations.

Results and discussion *"j4m to *"y4« ratio determination

Each sample irradiation has been conducted with a set of pure gold standards. The data from those irradiations for the *"Au to *"Au ratio determination are compiled in Table 2. The measured standard deviation of the pooled data express

the difficulties encountered in the precise determination of this ratio. However,

this ratio needs to be determined as accurately and precisely as possible, and improvement can be expected with the planned setup of a Compton suppressor sys- tem. The 411.80 keV peak of *'^Au is already determined with high accuracy and precision. The analytical scheme thus provides the possibility for a highly

accurate and precise determination of gold at ultratrace levels. The detection limit of gold in the investigated materials has been found to be less than 10"'* g/g.

Biological Standard Reference Materials

Three different materials have been analyzed: Orchard Leaves (SRM 1571),

Citrus Leaves (SRM 1572), and Bovine Liver (SRM 1577). The platinum and gold

concentrations determined in this study are given in Tables 3 and 4 and compared with data found in the Uterature.^*' The wide scatter of gold data in Bovine Liver can be explained by inhomogeneity in the material. This inhomogeneity

has been observed in this study, and much more consistent results can be obtained

with increased sample sizes (up to 5 g).

34 /. Radioanal. Chem. 75 (1982)

110
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

Table 3 Platinum and gold in Orchard Leaves (SRM 1571)

(Concentration 10"" g/g)

Sample No.

Irradiation time

Interference* Au Pt

1 1 h 1780 ±250** 1440 ±200** 1344 ± 1** (340 ±320)**

2 1 h 1505 ±100 1500 ±250 1496 ± 1 (0 ±270)

3 15 m 660 ±330 310 ±. 40 1404 ± 3 (350 ±320) 4 15 m 636 ±185 300 ± 40 1387 ± 6 310 ±190

5 15 m 428 ± 75 280 ± 40 1530 ± 1 150 ± 80 X± Is 1430 ± 80 -200

Literature 970 ± 90 1800 ±1000

89 200 ±15 200^ 89 200 - 1.2 • 10* »

'Calculated as platinum concentrations. **Errors are random errors or sum of random and noiuandom errors [Eq. (1)] for a single determination.

Table 4

Platinum and gold in Bovine Liver (SRM 1577) and Gtrus Leaves (SRM 1572)

Bovine Liver (Concentration 10"'* g/g)

Sample No. Pt Au

1 28 ± 22* 68.9 ± 0.3* 2 99± 23 53.2 ± 0.3 3 94 ±23 41.9 ±0.1

4 58 ± 18 69.1 ± 0.1 Literature ND 200-30 000

Qtrus Leaves

(Concentration 10"" g/g)

Sample No. Pt Au

1 100 ± 30* 105.8 ± 0.2* 2 40 ± 30 105.7 ± 0.2 3 50 ±40 119.0 ± 0.5

'Errors are random errors or sum of random and nonrandom errors [Eq. (1)] for a single determination.

/. Radioanal. Chem. 75 (1982) 35

3* m
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

The comparison of the data for platinum in Orchard Leaves demonstrates the

difficulties encountered in the determinations of this element. The data in Table 3 demonstrate the effect of shortened irradiation time to lower the detection limit for platinum in the presence of relatively high gold concentrations. Despite this optimization we could only obtain a positive identification for platinum in Or- chard Leaves at about 200 • 10"'^

g/g taking into account the counting statistics of the 158.35 keV peak and the uncertainty for calculating the interference by

gold. However, this level appears to be more correct than previously reported

concentrations, which were orders of magnitude higher. The radiochemical sepa- ration of interferences is crucial, e.g., disregarding the rare interference from lute- tium would have given a false platinum concentration of about 40-10"' g/g. The

accuracy of gold determinations by activation analysis has been demonstrated pre- viously.'" The good agreement of the gold data with previous determinations by

activation analysis' suggest also good accuracy for the platinum data, since both

isotopes *"Au and *"Au have the same chemical behavior during the radiochemical separation and are counted simultaneously. The new SRM Citrus Leaves has

a much lower gold level, therefore we have been able to determine platinum at the ultratrace level (Table 4). It would be of interest to analyze a similar material

collected recently to compare with these SRM's which were collected more than

ten years ago. Possibly an increase of platinum in the environment could be detected. During collection and preparation of these materials, extreme caution has to be exercised to avoid contamination with gold. The SRM Orchard Leaves was

probably contaminated during its production, fortunately homogeneously, since

Table 5 Platinum and gold in human liver specimens

(Concentration 10"" g/g) NESB ID Pt Au

m9s

B9014

B9041-A

B9041-B B0942

80949

54 ± 13* 114 ±23

127 ± 60

240 ± 70 ND

ND 2652 ±1*

64 ± 1 71.9± 0.2 143.U0.3

29'8 ±1

496 ± 2 ND: not detectable. 'Errors are random errors or sum of random

and nonrandom errors [Eq. (1)] for a single determination.

36 /. Radioanal. Chem. 75 (1982)

112
R. ZEISLER, R. R. GREENBERG: ULTRATRACE DETERMINATION

the observed gold level is abnormally high compared to its crustal abundance and

the other trace elements in the material. The Bovine Liver data suggest that gold

might be an inhomogeneously distributed contaminant. The platinum determination

is occasionally impossible because of unexpected high interference from gold. The

platinum data have too large enors to judge on the distribution of this element.

Human liver specimens

The results for five selected specimens out of the National Environmental Specimen Bank program are presented in Table 5. The gold data fall well in the range of reported results,' ' no data for platinum have been reported previously. These

first results on the liver specimens are an indication for the current baseline level.

If ratios of platinum to gold in the Hver are compared with its crustal abundance

ratio (Pt : Au = 2 : 1), no elevanted platinum levels can be detected in this Umited

number of specimens. Further analysis of randomly selected samples from the National Environmental Specimen Bank will provide a first baseline for platinum in human liver.

References

1. Metal Statistics 1979, Fairchild Publications, New York, 1979.

2. P. SCHUTYSER, A, GOVAERTS, R. DAMS, J. HOSTE. J. RadioanaL Chem., 37 (1977) 651.

3. R. A. NADKARNI, G. M. MORRISON, J. RadioanaL Chem., 38 (1977) 435.

4. A. F. LeROY, M. L WEHLING, H. L. SPONSELLER, W. S. FRIAUF, R. E. SOLOMON. R, L. DEDRICK, C. L. LITTERST, T. E. GRAM, A. M. GUAMINO, D. A. BECKER, Biochemical

Medicine, 18 (1977) 184.

5. R. ZEISLER, F. LUX, H, SEIDENBERGER, H. SCHOENENBERGER, W. BECK, Nuclear

Activation Techniques in the Life Sciences 1978, IAEA Vienna, 1979, p. 467.

6. B. W. WILKINSON, J. TOTH-ALLEN, NucL Techn., 13 (1972) 103.

7. V. K. YURTCHENKO, E. N. GILBERT, G. V, VERIOVKIN, V. A. MIKHAILOV, J. Radio- anaL Chem., 41 (1977) 137.

8. J. P. OP DE BEEK, J. RadioanaL Chem., 3 (1969) 431, and J. RadioanaL Chem., 4 (1970)

137.

9. E. S. GLADNEY, AnaL Chim. Acta, 118 (1980) 385, and Informal Report LA-8438-MS,

Los Alamos, 1980.

10. R. M. LINDSTROM, S. H. HARRISON, J. M. HARRIS, J. AppL Phys., 49 (1978) 5903.

11. G. V. IYENGAR, W. E. KOLLMER, R J. M. BOWEN, The Elemental Composition of Human

Tissues and Body Fluids, Verlag Chemie, Weinheim/New York, 1978.

/. RadioanaL Chem. 75 (1982) 37

113
ADDENDUM TO APPENDIX II

TABLE 6. CONCENTRATIONS OF PLATINUM IN HUMAN LIVER SAMPLES

Concentration

Sample Identification (pg/g wet weight

LI BOOT 4 15

L2B0041 30

L2B0042 57

L1M0083 17

L1S0086 5

L1S0092 18

L1B0121 8

L1M0127 20

LI SOI 28 16

L1M0130 10

L1M0131 11

114
APPENDIX III

DETERMINATION OF ORGANOMERCURY CATIONS IN TISSUE SAMPLES

USING LIQUID CHROMATOGRAPHY WITH AMPEROMETRIC DETECTION

by

William A. MacCrehan

INTRODUCTION

Mercury has long been recognized as an important environmental toxin.

Of the various forms of mercury, the alkylmercury cations have been found to

be the most toxic (1). Thus, the determination of the levels of various

organomercury species in biological tissues is important in addition to the

measurement of total mercury concentration. Other workers have reported the

presence of methylmercury in aquatic samples (2,3) and in human tissues (4)

and ethylmercury in plants (5). It would be particularly valuable to measure

the different mercury species in available reference materials and to

establish baseline values in different tissue samples.

ORGANOMERCURY DETERMINATION

The current method for organomercury determination in tissue samples

involves a laborious digestion, extraction, derivatization, and sample

clean-up, followed by gas chromatography with electron capture detection (4,6).

An outline of this method is shown in Table lA.

As an alternative to this gas chromatographic method, a simplified

procedure based on liquid chromatography with electrochemical detection (LCEC)

has been developed. The new sample preparation is outlined in Table IB.

Organomercury compounds in tissues are thought to be bound to protein

sulfhydryl groups (7). Acidification with strong acid liberates the organomercury cations from the weak-base complexes:

R-Hg-S-protein + H"*" + Br" »- RHg-Br + H-S-protein.

The reagent under investigation consists of 1 moL/L HCl , 1 mol/L KBr and 0.05

percent v/v of an n-alkyl thiol (C^SH). A concentration of HCl was selected

which was strong enough to protonate the weak-base protein thiol but not so strong as to cause the acid hydrolysis of the organomercury compounds. The

Br" is added to help competitively complex the mercury cations. The addition

of the thiol to the extraction reagent was required as an antioxidant to

prevent the formation of Br2 from the reaction of Br' with atmospheric O2.

115
otherwise, the Br2 formed rapidly oxidizes the organomercury compounds to

inorganic mercury (II).

TABLE 1 . OUTLINE OF METHODS FOR THE DETERMINATION OF METHYLMERCURY IN TISSUES

A B

weigh 0.5 to 2.0 g sample

add 45% NaOH, 1% Cysteine

heat at 100 °C for 0.5 hour

add 8 mol/L urea, 3 mol/L HCl

0.5 mol/L CuSO^

shake 5 minutes

extract with benzene twice with

centrifugation, transfer

add 0.01 mol/L Na2S202 shake,

followed by centrifugation,

transfer to a new tube

add benzene, extract, transfer

add Na^SO^-Florisil , transfer

analyze by GC-EC

weigh 250 mg lyophilized sample

add 750 yL 1 mol/L HCl , 1 mol/L KBr,

0.05% C SH

n heat at 40 °C and sonicate for 0.5

hour

filter centrifugal ly

add 50 yL concentrated NH^OH

purge with Ar and analyze by LCEC

Total time 0.75 hour

Total time 3.5 hours

An important aspect of the sample preparation is the digestion and filtration of the lyophilized tissues. For the new method a commercially available

centrifugal filtration apparatus was employed. This apparatus permits the

digestion and filtration to be performed without transfer. It was necessary

however, by replacing the original plastic sample and

borosilicate glass (see Appendix IV for details).

to modify this assembly

receiver containers with

RESULTS AND DISCUSSION

A new separation of organomercury species has been developed for this

work. Previously, neutral complexes of the organomercury cations were formed

with 2-mercaptoethanol (2-ME) for LC separations (8,9). However, for measure- ments on tissue samples, the additional selectivity of differential pulse

electrochemical detection was required (9,10). In the new method, the

selectivity of the chromatographic separation was increased so that simple

amperometric detection could be employed. This was accomplished by the use

of n-alkyl thiols as complexing agents for the organomercury cations. These

reagents form even less polar, neutral complexes with the cationic mercury

species than the 2-ME thus providing greater chromatographic selectivity. In

116
the reversed-phase separation, the complexes are retained much longer than

the polar matrix constituents which are co-extracted by the aqueous sample

reagent. The complexation reaction takes place "in-situ" during the chromatographic separation:

RHg-Br + H-S-(CH2)^CH3 ^ > RHg-S-(CH2)^CH3 + H"^ + Br".

The effect of the chain length of the n-alkyl thiols on the retention of

ethyl- and methylmercury in a reversed-phase separation is shown in Figure 1.

As expected the longer alkyl chains increase retention.

The LC separation of methyl-, ethyl-, and inorganic Hg(II) is shown in

Figure 2A. This separation is achieved using an octadecyl modified silica

(5 ym) column and an 85 percent methanol /water eluent containing the Cy thiol.

The detector is a thin layer electrochemical cell with a 1.2 mm gold amalgam

working electrode and an applied potential of -825 mV.

Preliminary results of the new method are shown in Figure 2B. Samples

of NBS RM 50 Albacore tuna were prepared by the acid extraction procedure and

analyzed by LCEC. Preliminary quantitative results for methylmercury in the

two samples agreed with that found by the previously reported method (9).

Future work will focus on expanding the sample preparation to include the

recovery of inorganic mercury(II) as well as the alkyl species which will allow

a simultaneous determination of all the expected forms of mercury in tissues.

LITERATURE CITED

1. Thayer, J. S., Jr. J. Organometal . Chem., 76:265, 1974.

2. Westbb, G. Science, 181:567, 1973.

3. Bache, C. A., W. H. Gutemann, and D. J. Lisk. Science, 172:951, 1971.

4. Cappon, C. J., and J. C. Smith. J. Anal. Tox., 5:90, 1981.

5. Fortmann, L. C, D. D. Gay, and K. 0. Wirtz. EPA Technical Publication

EPA-600/3-78-037.

6. Cappon, C. J., and J. C. Smith. Anal. Chem., 49:365, 1977.

7. Zepp. R. C. , N. Baughman, and D. M. Cline. Envir. Lett., 6:117, 1974.

8. MacCrehan, W. , and R. A. Durst. NBS Technical Note EPA-600/7-79-211

9. MacCrehan, W. , and R. A. Durst. Anal. Chem., 50:2108, 1978.

10. MacCrehan, W. Anal. Chem., 53:74, 1981.

117
1 1 1 r~—

l_l I I III

3 4 5 6 7

Carbon Number

of Mercaptan

Figure 1. Effect of the alkyl chain length of the n-alkyl thiol on the

retention of ethyl mercury and methyl mercury. Conditions: column

- octadecyl modified silica, 5 ym particles, solvent - 72%

MeOH/H20 with 0.05% v/v of each thiol.

118
Hg(ii)

Inject

"1—I

\—I—r4 8 12 16 20

Minutes

T

1 nA

i

^vJ[MeHg+ —I—

4 8

Inject

Minutes

(A) (B)

Figure 2. Reversed-phase LC separation of mercury species. Conditions:

col umn-Spheri sorb ODS 5 ym, 0.4 x 25 cm; eluent-85% methanol/

water, 0.05 moL/L NH^OAc, 0.05% n-CyHisSH at 1.0 mL/min;

detector-amperometry at a gold amalgam electrode (1.2 mm diameter button) at -825 mV.

A. Sample amounts 1.0 x 10^ mol/L of each (about 45 ng).

B. Detection of methylmercury in tissue sample (NBS RM-50).

119

APPENDIX IV

Modification of Centrifugal Filtration Device for Elimination of Sorption Losses

William A. MacCrehan

Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards. Washington, D.C. 20234

The preparation of many liquid samples for analysis fre- quently requires nitration to remove suspended solid matter. This is particularly true for samples prepared for liquid

chromatography, where particles in the micrometer range can

clog protective column frits. Several devices are commercially

available for the filtration of liquid samples in the sub-milliliter range. Two approaches that we have used are sjrringe-membrtme filters and centrifugation with withdrawal of the su- pernatant liquid. Although both approaches are effective for removing particles, each has disadvantages. For example,

syringe-membrane filters are cumbersome to use for routine

work, and considerable sample volume may be lost as the filter becomes clogged. Withdrawal of the clear supernatant liquid after centrifugation is difficult, and some liquid is always

This article not subject to U.S. Copyright. Published 1982 by the American Chemical Society

121
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 • 839

Figure 1. Glass-modified centrifugal filtration apparatus: (1) txvosicate

glass sample compartment, (2) 10/18 Inner Joint cut to a letngth of 9.75 mm, (3) silicone washer, (4) filter membrane, (5) filter support, (6) original plastic filter holder, (7) 10/18 outer Joint cut to a length of 17.25 mm, (8) borosillcate glass receiving container.

retained in the interstitial volume of the "plug".

A new approach to the problem of small volume filtration

is to combine the action of membrane filtration and centri- fugation into one system. One commercially available microfilter device (Bioanalytical Systems MF-1 Microfilter) consists of a capped, upper sample compartment, a filter membrane and support, and a lower receiving tube. The

sample to be filtered is placed in the sample compartment,

the appropriate membrane-filter size and material are chosen, the apparatus is assembled, and the sample is centrifuged at high speed. The force generated drives the liquid through the

filter and the filtrate is collected in the receiving tube. The

microfilter is designed to be useful for the filtration of volumes

in the range of 50-1000 tiL. The material from which this particular assembly is constructed is a polyethylene-polypropylene copolymer.

The particular application for which filtration was required was the extraction of organomercury cations from lyophilized

tissue. The extraction involves digestion and ultrasonic ag- itation at 50 for 0.5 h using a reagent that consists of 1 mol/L HCl, 1 mol/L KBr and 0.1% v/v 3-mercaptopropanol

(3-MP). The sample is digested, filtered, and sampled in the

microfilter apparatus, without any transfers. The results of the new method wiU be described in a subsequent publication.

While the microfilter efficiently filters out the solid material with high recovery of the liquid, significant loss of organo- mercury cations was observed. Methyl- and ethyhnercury were found to be retained on the plastic container material,

probably as the neutral bromo or mercapto complexes.

Glass-Modined Microniter. Since no loss of organo- mercury complexes was observed to occur on borosilicate glass in this concentration range, new sample and receiver compartments were constructed from this material. The modified

microfilter assembly is pictured in Figure 1. Standard borosilicate inner and outer 10/18 joints were cut on a glass saw

to fit the filter support compartment. The ends of the tubes were closed, and a small bulge was blown on the end of the sample compartment to increase the volume to about 2 mL.

The glass was carefully annealed to avoid stress cracking upon

centrifugation.

The original commercial filter support was stiU used even though it was fabricated from the same plastic as the other

J

MeHg'*^ T

2 nA 1

EtHg*

~T—I—I

1 2 4 6 8 Minutes

"1—

2 4 6 8 Minutes Minutes

B

Figure 2. Organomercury determination. Conditions: column, conv

merdaBy available C-18 bonded phase silica (5 tun spherical particles) 0.4 X 25 cm; solvent, 80% MeOH/HjO, 0.1 mol/L NH^OAc pH 5, 0.02% 3-mercaptopropane; flow rate, 1.0 mL/mIn; detector electrode, Hg/Au 1.2 mm diameter; potential, -0.75 V; current offset, 20 nA; (A) glass-modmed microfilter; (B) original plastic microfilter; (C) methanol

leach of middle chromatogram fitter after water wash. Starting concentration of metiiylmercury (MeHg'*') and ethylmercury (EtI-ig'*') were

5 X 10-« mol/L.

compartments. This was possible because of the short contact time and small plfistic surface exposed to the sample on this part of the apparatus.

Recovery Study. The performance of the glass-modified

filtration apparatus was compared to the original plastic

containers by using the filters for the digestion^ ultrasonic

agitation, and filtration of a standard solution of methyl- and

ethylmercury in the micromolar concentration range. The first chromatogram in Figure 2 shows the results obtained after

filtration through the glass-modified filter. The center chromatogram is from an identical solution prepared by use

of the original plastic filter. The third chromatogram shows

a methanol solution obtained by rinsing the plastic filter with

distilled water and then leaching the plastic (used for the

center chromatogram) with 200 /xL of solvent. The partial recovery of sorbed organomercury compounds can be seen in

this last chromatogram. The recoveries obtained for the

glass-modified filter, expressed as a mean plus or minus the

relative standeird deviation in percent for five samples was

98 ± 3% for methylmercury and 100 ±4% for ethylmercury

compared to 75 ± 5% (methylmercury) and 56 ± 21%

(ethylmercury) for all-plastic filters. Cleaning. The plastic parts of the filter holder can be

cleaned for reuse by so£iking in 30% H2O2 overnight. Any

remaining tissue and the organomercury compounds are re- moved by this treatment. No deterioration of the polyethylene/polypropylene copolymer was noted after 10 such

cleanings. The glass sample compartment and receiving tube can be cleaned by soaking in concentrated nitric acid overnight.

Received for review September 10, 1981. Accepted December

18, 1981. To describe adequately materials and experimental

procedures, it is occasionally necessary to identify commercial

products by manufacturer's name or label In no instance does such identification imply endorsement by the National Bureau

of Standards nor does it imply that the particular products

or equipment is necessarily the best available for that purpose.

122
APPENDIX V

NEUTRON CAPTURE PROMPT GAMMA-RAY ACTIVATION ANALYSIS OF HUMAN LIVER SAMPLES

by

David F. Anderson

A series of human liver samples were analyzed by instrumental neutron

capture prompt Y->^ay activation analysis (PGAA) at the University of MarylandNBS reactor facility. The lyophilized samples (in powder form) were formed

into 1.25 cm diameter pellets, packaged in thin Mylar film and irradiated in

the thermal neutron beam (thermal flux = 2 x lO^n/cm^'S, Cd/Au ratio 55/1).

Full descriptions of the system and its capabilities can be found in

References (1-3).

Eight liver samples from specimen L1B0014, one from L2S0035, and one sample from NBS SRM 1577 Bovine Liver were evaluated and the results are presented in Table 1. For L1B0014, the individual samples' maximum statistical errors (not shown in Table 1) for y-ray counting and elemental sensitivity was

<1 percent for H and K, 1.5-3 percent for C, N, S, CI and Cd, and >3 percent

for B, Na and P. Using the mean and a values for H and K, the homogeneity of

the eight samples is then 2.7 ± 0.1 percent.

The elemental concentrations in SRM 1577 found for H, N, Na, P, S, K,

and Cd were in agreement with NBS certified and literature values. The

carbon determination appears to be slightly low ('^4 percent), while the CI is

'^^2 percent high, compared to several literature values (2,4,5), but these

values have significant error (up to 11 percent) as reported. The PGAA data

are included in the paper presented at the Conference on Modern Trends in

Activation Analysis, Toronto, Canada, June, 1981 (6).

123
TABLE 1 . PROMPT GAMMA-RAY ACTIVATION ANALYSIS (PGAA) RESULTS FOR HUMAN LIVER

SAMPLES

Element 1 4 5

L1B0014 Subsamples

6 10 12 14 17

H {%) 7.42 7.44 7.48 7.08 7.31 7.38 7.00 7.50

B (yg/g) 1 .78 1.70 1.82 1.81 1.69

__a __a __a

c {%) 53.6 51.3 51.0 48.3 48.4 54.1 52.4 56.3

N {%) 10.73 10.52 10.45 10.32 10.43 11.02 10.60 11.67

Na {%) 0.25 0.26 0.30 0.31 0.26 0.27 0.25 0.29

P {%) 0.97 1.13 1.12 1.15 1.09 1.21 1.03 1.03

s (%) 0.782 0.787 0.775 0.716 0.756 0.789 0.764 0.795

CI (%) 0.388 0.395 0.395 0.378 0.389 0.405 0.382 0.407

K {%) 1 .069 1.098 1.088 1.034 1.053 0.103 1.028 1.118

Cd (ug/g) 4.78 4.84 4.83 4.49 4.73 4.84 4.52 4.90

Sample

Mass (g)

0.92014 0.97486 0.97244 0.95312 0.96260 0.87620 0.99784 0.90496

^Different system setup, B background too high for reliable determination.

124
Meania L2S0035 SRM 1577

7.33 ±0.19 9.85±0.11 7.01 ±0.01

1.76 ±0.06 a ___a

51.9 ±2.8 66.2 ±3.0 48.7 ±0.4

10.72 ±0.44 13.51±0.09 10.39 ±0.12

0.27 ±0.02 0.94±0.02 0.25 ±0.04

1.09 ±0.08 a M B 1.0 ±0.3

0.77 ±0.03 1.00±0.01 0.76 ±0.04

0.392±0.010 0.85±0.01 0.29U0.001

1 m j.n AO

1 .07 ±0.03 11d±0.02 no.ybnc j.n ±U.UIm

4.74 ±0.16 9.6U0.16 0.22 ±0.04

0.3039 1.33886

125
REFERENCES

1. Failey. M. P. PhD Thesis. Department of Chemistry, University of

Maryland, 1979.

2. Failey, M. P., D. L. Anderson, W. H. Zoller, G. E. Gordon, and R. M.

Lindstrom. Anal. Chem., 51:2209, 1979.

3. Anderson, D. L. , M. P. Failey, W. H. Zoller, W. B. Walters, G. E. Gordon,

and R. M. Lindstrom. J. Radioanal. Chem., 63:97, 1981.

4. Gladney, E. S., D. B. Curtis, and E. T. Jurney. Anal. Chim. Acta, 110:

339, 1979.

5. Zikovsky, L. , and E. A. Schrweikert. J. Radioanal. Chem., 37:571, 1977.

6. Harrison, S. H., R. Zeisler, and D. Anderson. Conference on Modern

Trends in Activation Analysis. Toronto, Canada, June, 1981.

126
APPENDIX VI

PUBLICATIONS OF THE U.S. EPA/NBS NATIONAL ENVIRONMENTAL SPECIMEN BANK

1. Becker, D. A. Environmental Sample Banking - Research and Methodology.

In: Trace Substances in Environmental Health-X, D. D. Hemphill, ed.

University of Missouri, Columbia, 1976. p. 353-359.

2. Maienthal, E. J., and D. A. Becker. A Survey of Current Literature on Sampling, Sample Handling, and Long-Term Storage for Environmental

Materials. National Bureau of Standards Technical Note 929. U.S.

Government Printing Office, Washington, DC, 1976. 34 pp.

3. Maienthal, E. J., and D. A. Becker. A Survey of Current Literature on Sampling, Sample Handling, and Long-Term Storage for Environmental

Materials. Interface, 5(4):49-62, 1977.

4. Rook, H. L., and G. M. Goldstein. Recommendations of the EPA/NBS Workshop on the National Environmental Specimen Bank. U.S. EPA-600/1 -77-020,

1977. 55 pp.

5. Becker, D. A., and E. J. Maienthal. Evaluation of the National Environmental Specimen Bank Survey. U.S. EPA-600/1 -77-01 5, U.S. Environmental

Protection Agency, 1977. 269 pp.

6. Goldstein, G. M. Plan for a National Environmental Specimen Bank. U.S.

EPA 600/1-78-022, U.S. Environmental Protection Agency, 1978. 27 pp.

7. Rook, H. L., and G. M. Goldstein. The National Environmental Specimen

Bank. NBS Special Publication 501. U.S. Government Printing Office,

Washington, DC, 1978. 59 pp.

8. Gills, T. E., H. L. Rook, and P. D. LaFleur. Evaluation and Research of

Methodology for the National Environmental Specimen Bank. EPA-600/1 -78-

015, U.S. Environmental Protection Agency, 1978. 66 pp.

9. Goldstein, G. M. The National Environmental Specimen Bank; Its Concepts,

History and Objectives. In: The Use of Biological Specimens for the

Assessment of Human Exposure to Environmental Pollutants, A. Berlin,

A. H. Wolff, and Y. Hasegawa, eds. Martinus Nijhoff Publishers, The

Hague/Boston/London, 1979. p. 165-176.

127
10. Rook, H. L. , and P. D. LaFleur. Establishing and Operating a Specimen

Bank for Environmental Monitoring. In: The Use of Biological Specimens

for the Assessment of Human Exposure to Environmental Pollutants,

A. Berlin, A. H. Wolff, and Y. Hasegawa, eds. Martinus Nijhoff

Publishers, The Hague/Boston/London, 1979. p. 309-312.

11. Gills, T. E., and H. L. Rook. Specimen Bank Research at the National

Bureau of Standards to Insure Proper Scientific Protocols for Sampling,

Storage, and Analysis of Environmental Materials. In: Monitoring

Environmental Materials and Specimen Banking, N.-P. Luepke, ed. Martinus

Nijhoff Publishers, The Hague/Boston/London, 1979. p. 263-270.

12. Durst, R. A. Container Materials for the Preservation of Trace Substances

in Environmental Specimens. In: Monitoring Environmental Materials and

Specimen Banking, N.-P. Luepke, ed. Martinus Nijhoff Publishers, The

Hague/Boston/London, 1979. p. 198-202.

13. Gills, T. E. , H. L. Rook, and R. A. Durst. The National Environmental

Specimen Bank Research Program. EPA-600/1-79-017, U.S. Environmental

Protection Agency, 1979. 59 pp.

14. Harrison, S. H. , T. E. Gills, E. J. Maienthal, H. L. Rook, S. A. Wise,

and R. Zeisler. The National Environmental Specimen Bank Pilot Program.

In: Trace Substances in Environmental Health-XIV, D. D. Hemphill, ed.

University of Missouri, Columbia, MO, 1980. p. 329-340.

15. Harrison, S. H., R. Zeisler, and S. A. Wise. Pilot Program for the

National Environmental Specimen Bank - Phase I. EPA/600/1-81-025,

U.S. Environmental Protection Agency, 1981. 54 pp.

16. Zeisler, R. , S. H. Harrison, and S. A. Wise. Analysis of Human Liver

Specimens in the U.S. Pilot National Environmental Specimen Bank Program.

In: Proceedings of the International Workshop on Specimen Banking and

Monitoring as Related to Banking. R. A. Lewis, ed. Martinus Nijhoff

Publishers, The Hague/Boston/London, in press.

17. Wise, S. A., K. A. Fitzpatrick, S. H. Harrison, and R. Zeisler. Operation of the U.S. Pilot National Environmental Specimen Bank Program.

In: Proceedings of the International Workshop on Environmental Specimen

Banking and Monitoring as Related to Banking. R. A. Lewis, ed. Martinus

Nijhoff Publishers, The Hague/Boston/London, in press.

18. Zeisler, R. , S. H. Harrison, and S. A. Wise. Trace Elements in Human

Livers Using Quality Control in the Complete Analytical Process.

Biological Trace Element Research, Biol. Trace Element Res., in press.

19. Zeisler, R. , J. K. Langland, and S. H. Harrison. Cryogenic Homogenization Procedure for Biological Tissues. Anal. Chem., in press.

128

*n.S. GOVERMMENT PRINTING OITICE : 1983 0-580-997/5153
NBS'n4A iREv. 2-8C

II C n\ r O T ^ C ^ ^\ ^A\.A U^S* D t T . Or Q,t^MM, 1 PlIRI l^"ATinw OR 2* Performing Organ. Report No. 3. Publication Date BIBLIOGRAPHIC DATA

REPORT NO.

August 1983 SHEET (See instroctidns; NBS SP 656

4. TITLE AND SUBTITLE

The Pilot National Environmental Specimen Bank - Analysis of Human Liver Specimens

5. AUTHOR(S)

R. Zeisler, S. H. Harrison, and S. A. Wise

6. PERFORMING ORGANIZATION {\\ joint or other thon NBS, see instructions) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE S. Type of Report & Period Covered

WASHINGTON, D.C. 20234

Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City, Stote, ZIP)

Health Effects Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

10. SUPPLEMENTARY NOTES

Library of Congress Catalog Card I^Iuinber: 83-600561

I I Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here)

This work was performed under a joint EPA/NBS research program to develop

state-of-the-art protocols for the sampling, storage, and analysis of biological and environmental -type matrices as part of a Pilot National Environmental

Specimen Bank program. The purpose of this report is to summarize the

experience and results obtained for the first sample type, human liver. The

samole collection protocol was evaluated with respect to costs and suitability

of donor selection criteria. An analytical protocol was implemented for the

determination of trace elements in the liver samples using the techniques of

atomic absorption spectrometry, isotope dilution mass spectrometry, neutron

activation analysis, and voltammetry. Individual sections of this report

describe in detail the procedures used and the results obtained for each of

these analytical techniques. The analytical results for the determination of

31 trace elements in 36 liver specimens are presented and discussed. Analytical methodology for the measurement of organochlorine compounds is also

described.

This report is submitted in partial fulfillment of EPA Interagency

Agreement EPA-81 -D-X0105 by the National Bureau of Standards.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

atomic absorption spectrometry; cryogenic homogenization ; gas chromatography; human

liver; inorganic analysis; isotope dilution mass spectrometry; neutron activation

analysis; organic analysis; organochlorine pesticides; specimen banking; trace

13. AVAILABILITY elements; valtammetry

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