U.S. DEPARTMENT OF COMMERCE / National Bureau of Standards

Environmental Specimen Bank
NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards 1 was established by an act of Congress March 3, 1901. The Bureau's overall goal is strengthen and advance the Nation's science and technology and facilitate their effective application for public benefit. To tt end, the Bureau conducts research and provides: (1) a basis for the Nation's physical measurement system, (2) scientific ai technological services for industry and government, (3) a technical basis for equity in trade, and (4) technical services to pr mote public safety. The Bureau consists of the Institute for Basic Standards, the Institute for Materials Research, the Institu for Applied Technology, the Institute for Computer Sciences and Technology, the Office for Information Programs, and tl Office of Experimental Technology Incentives Program.

THE INSTITUTE FOR BASIC STANDARDS provides the central basis within the United States of a complete and consii

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Applied Mathematics — Electricity — Mechanics — Heat — Optical Physics — Center for Radiation Research — La

oratory Astrophysics 2 — Cryogenics* — Electromagnetics 2 — Time and Frequency 5

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1 Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D.C. 20234.

2 Located at Boulder, Colorado 80302.
The National Environmental Specimen Bank

Proceedings of the Joint EPA/NBS Workshop on

Recommendations and Conclusions on the

National Environmental Specimen Bank

Held at the National Bureau of Standards,

Gaithersburg, Maryland, August 19-20, 1976

Edited by

Harry L. Rook

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

and

George M. Goldstein (Also Project Officer)

Health Effects Research Laboratory

U.S. Environmental Protection Agency

Research Triangle Park, N.C. 27711

Sponsored by the National Bureau of Standards

and the U.S. Environmental Protection Agency

Interagency Agreement No. I AG-D4-0568

U.S. DEPARTMENT OF COMMERCE, Juanita M. Kreps, Secretary

Dr. Sidney Harman, Under Secretary

Jordan J. Baruch, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Acting Director

Issued February 1978
Library of Congress Cataloging in Publication Data

Joint EPA/NBS Workshop on Recommendations and Conclusions on the National Environmental Specimen Bank, Gaithersburg, Md.,

1976. The national environmental specimen bank.

(National Bureau of Standards special publication ; 501)

"Interagency agreement no. IAG-D4-0568."

Supt.ofDocs.no.: C13.10:501

1. National Environmental Specimen Bank-Congresses. I. Rook,

Harry L. II. Goldstein, George M. III. United States. National Bureau

of Standards. IV. United States. Environmental Protection Agency.

V. Title. VI. Series: United States. National Bureau of Standards.

Special publication ; 501. QC100.U57 no. 501 [TD171] 602Ms [363.6] 78-682

National Bureau of Standards Special Publication 501

Nat. Bur. Stand. (U.S.), Spec. Publ. 501, 59 pages (Feb. 1978)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1978

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

Stock No. 003-003-01 890-2 Price $2.30

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

The many benefits of our modern, developing, industrial society are

accompanied by certain hazards. Careful assessment of the relative risk

of existing and new man-made environmental hazards is necessary for the

establishment of sound regulatory policy. These regulations serve to

enhance the quality of our environment in order to promote the public

health and welfare and the productive capacity of our Nation's population.

The Health Effects Research Laboratory, Research Triangle Park,

conducts a coordinated environmental health research program in toxicology,

epidemiology, and clinical studies using human volunteer subjects. These

studies address problems in air pollution, non-ionizing radiation,

environmental carcinogenesis and the toxicology of pesticides as well as

other chemical pollutants. The Laboratory develops and revises air quality

criteria documents on pollutants for which national ambient air quality

standards exist or are proposed, provides the data for registration of new

pesticides or proposed suspension of those already in use, conducts research

on hazardous and toxic materials, and is preparing the health basis for

non-ionizing radiation standards. Direct support to the regulatory function

of the Agency is provided in the form of expert testimony and preparation of

affidavits as well as expert advice to the Administrator to assure the

adequacy of health care and surveillance of persons having suffered imminent

and substantial endangerment of their health.

As part of the Health Effects Research Laboratory's efforts to provide

a coordinated environmental health research program, the U.S. Environmental

Protection Agency and the National Bureau of Standards co-sponsored a

Workshop to review current technical developments and to make recommendations

affecting the proposed National Environmental Specimen Bank (NESB) . The NESB

is part of an International effort to monitor the environment for hazardous

substances. The advantages of such a program will permit the Agency to

assess the effectiveness of its present environmental control techniques

by monitoring pollutant trends, as well as establishing environmental

baseline levels of new pollutants or pollutants of current concern not

previously investigated.

N*John H. Knelson, M.D.

Director,

Health Effects Research Laboratory

iii
ABSTRACT

On August 19 and 20th, 1976, the National Bureau of Standards and the

U.S. Environmental Protection Agency co-sponsored a Workshop to review

technical developments and to make recommendations on implementation of the

National Environmental Specimen Bank. The Workshop consisted of a review

session where past considerations were discussed; a technical session where

recent analytical research relevant to the sample bank was abstracted and

discussed; and a planning session where planning and design of a prototype

banking system was outlined.

This report is a summary of the presentations, discussion, and conclusions of the Workshop attendees. The attendees represented a wide cross

section of interested Federal and Non-Federal research groups as well as

International representation including the International Tissue Banking

Program (Sponsored by the World Health Organization, The Commission of

European Communities and the U.S. Environmental Protection Agency) and the

Federal Republic of Germany.

The workshop concluded that with the ever increasing influx of new

man-made substances into our ecosystem, that a formalized, systematic

approach is needed to assess the environmental impact of these substances on

a national as well as an international level. The technology to initiate

a pilot banking program is presently available and was formulated into a

five-year pilot bank program. This program will be evaluated at each stage

of development.

This report was submitted in partial fulfillment of EPA Interagency

Agreement IAG-D4-0568 by the National Bureau of Standards under the partial

sponsorship of the U.S. Environmental Protection Agency. This report covers

the period August 19 and 20, 1976, and the work was completed as of

January 31, 1977.

Key Words: Analytical; National Environmental Specimen Bank; prototype;

research; sample bank.

iv
TABLE OF CONTENTS Page

Foreword iii

Abstract iv

Figures v

Acknowledgment vi

1. Introduction 1

2. Background and History 3

3. Results of Current Research Programs 7

4. Discussions and Recommendations 10

5. Conclusions 14

Bibliography 18

Appendices 19

A. The Cleaning, Analysis and Selection of Containers for

Trace Element Samples 19

B. Evaluation by Activation Analysis of Elemental Retention

in Biological Samples after Low Temperature Ashing .... 33

C. Determination of Chromium in Biological Matrices by

Neutron Activation: Application to Standard Reference

Materials.. 43

FIGURES

Number Page

1 National Environmental Specimen Bank (NESB) Concepts 15

2 Pilot Bank samples 16

3 NESB Pilot Bank 17

v
ACKNOWLEDGEMENTS

The U.S. Environmental Protection Agency and the National Bureau of

Standards wish to acknowledge their appreciation to all the participants

of the National Environmental Specimen Bank Workshop; Progress and

Planning. Without their involvement and guidance at this most crucial

time in the development of the National Environmental Specimen Bank,

this proposed Pilot Bank Plan would never have emerged. We especially

want to thank Dr. Arthur Wolff, representing the International Bank

Conference Sponsored by the Commission of European Communities, The

World Health Organization, and the U.S. Environmental Protection Agency;

and Dr. F. Schmidt-Bleek of the Umweltbundesamt , Federal Republic of Germany,

for representing the International Communities in this most vital

program of world-wide concern.

DISCLAIMER

This report has been reviewed by the Health Effects Research

Laboratory, U.S. Environmental Protection Agency, and approved for

publication. Approval does not signify that the contents necessarily

reflect the views and policies of the U.S. Environmental Protection

Agency, nor does mention of trade names or commercial products

constitute endorsement or recommendation for use.

vi
SECTION 1

INTRODUCTION

On August 19 and 20th, the National Bureau of Standards (NBS) and the

Environmental Protection Agency (EPA) co-sponsored a Workshop to review

current technical developments and to make recommendations affecting the

proposed National Environmental Specimen Bank (NESB) . The attendees

represented a wide cross section of interested Federal and non-Federal

research groups as well as representatives from The Federal Republic of

Germany and the International Tissue Banking Program, sponsored by the World

Health Organization (WHO), U.S. E.P.A., and Commission of European

Communities (CEC)

.

The Workshop was conducted as an essential part of the program planning, preliminary to the establishment of the National Environmental

Specimen Bank. There has been a concerted effort on the part of EPA and

NBS to initiate the NESB system. This system will provide a dual output

useful to many environmental monitoring and assessment programs; that of

credible real time monitoring data for an environmental early warning

system and that of providing well preserved and documented environmental

samples for future retrospective analysis.

The Workshop was divided into three half-day sessions. The first

session was a review of the rationale and objectives of the NESB system.

The second session was a review of scientific research pertinent to NESB

objectives. The final day was devoted to a planning and design of a

prototype system for the banking and analysis of environmental samples.

During this planning session, the following considerations were specifically

addressed

:

1. A review of the issues that created the need for the

National Environmental Specimen Bank with specific emphasis on

current environmental problems which would be aided by the

existence of a sample banking system.

2. The development of specific objectives, identifying the functions

that the bank would perform, and how it would meet the needs as

specified in the rationale statement.

3. Identify the types of specimens that would be stored in the bank,

including demographic and technological data, based upon existing

and anticipated future needs and objectives.

1
4. Identify and evaluate the potential users of the bank and set

forth guidelines to govern those who could use the bank and what

could be drawn from it.

5. Determine if the technology is presently available to initiate a

pilot program.

With the successful completion of these tasks, a plan was generated

for the establishment of a pilot bank program. This pilot bank will utiliz

scaled-up laboratory procedures established at NBS as part of the NBS/EPA

cooperative effort to provide standardized sampling, storage and analytical

protocols for environmental samples, and identify additional areas of

research and development that would be needed before the full National

Environmental Specimen Bank is formalized.
SECTION 2

BACKGROUND AND HISTORY

The storage of human tissues and other environmental samples for

analytical measurements is a natural outgrowth of the EPA environmental

monitoring system. The need for environmental monitoring is well established and is currently a major EPA responsibility. A comprehensive

monitoring system is an integral part of the EPA mandate to assess the

total environmental impact of pollutants that may lead to deleterious

effects on human health.

The health research objectives of EPA can be stated as:

1. To minimize adverse human effects

- Prevent exposure to harmful new agents

- Reduce exposure to existing agents

- Predict relative effects of control options

2. To quantitate the benefits of environmental control.

3. To optimize the environment for man's health and well-being.

A preliminary program using human tissue samples as monitors for trace

element pollutant burdens was initiated in the EPA human pollutant burden

studies program.

From the very onset of the human pollutant burden studies, short-term

tissue banking became an integral part of this program. Human tissues were

viewed as environmental dose integrators of multiple proportions. The

integration of a pollutant level with time was used to estimate pollutant

trends. Analyses of these pollutant trends served as a resource for

standards setting, inputs for cost-benefit assessments of pollution abatement measures, and guides for determining research priorities.

The problem orientated approach of the early pollutant burden studies

did not fully utilize the capabilities of a tissue banking program. At

that time, a tissue or group of tissues was collected to test a specific

hypothesis. These tissues were stored until they could be analyzed, and

were then discarded. As the pollutant burden program expanded, the need

for a fully developed tissue banking system became apparent.

3
This banking system was envisioned as having a dual function. First,

representative portions of samples included in the bank would be analyzed

at the time of introduction to provide real time monitoring and evaluation

of pollutant trends. Evaluation of these trends would serve as early

warning sentinels so that proper control measures could be taken to halt

rising human body burdens before irreversible damage could occur. Second,

a specimen bank would enable the analyst to use tomorrow's more sensitive

and specific methods of chemical analysis on today's samples. The improved

measurement methodology would enable health scientists to determine accurate

levels for substances that would be either undetectable or poorly analyzed

by today's less sensitive methodology. The existence of a specimen bank

would provide the opportunity to determine what the body burden of newly

recognized toxic substances was in the past and to determine if their

levels had changed with time.

In EPA's continuing effort to establish a National Environmental

Specimen Bank, a two day working session was held in February 1973 at EPA's

National Environmental Research Center, Research Triangle Park, North

Carolina to discuss and propose plans for the establishment of a National

Environmental Specimen Bank System.

The broad objectives of this working session were:

1. Establish current trends in human pollutant burdens (shortterm banking)

2. Creating a specimen bank that would provide retrospective

analytical capability (long-term banking)

One major recommendation from the working session was that a sample

banking system should be established at the national level that would cross

agency lines and provide human tissues representative of that period in

time from which the sample was taken. The proper storage of these tissues

would permit retrospective analysis using improved methodologies that were

likely to be available.

In 1972, the National Academy of Sciences/National Research Council

(NAS/NRC) addressed tissue banking when they stressed the lack of coordination in the numerous programs by components of Federal and State

governments, private industry and academic institutions to collect, store

and analyze specimens of environmental interest.

In an effort to upgrade the availability and long-term protection of

environmental samples and to make the information gathered with each

collection readily available, the Subcommittee on the Geochemical Environment in relation to Health and Disease of the National Committee for

Geochemistry, National Academy of Sciences /National Research Council at

their Asilomar Workshop in California (1972) recommended that a group of

specialists be convened to study this problem at its Capon Springs Workshop

in May 1973.

4
The Capon Springs Workshop concluded that the U.S. Government should

establish, on a permanent basis, a National Environmental Specimen Bank.

Initial coordinating and funding of the multi agency system should be

considered by the National Science Foundation (NSF) . The Environmental

Protection Agency should be considered as the most logical organization to

establish the system.

Four tasks were proposed to begin development of the NESB.

Task I . Conduct an inventory and assess the value of existing

specimen collections as potential candidates for participation in the NESB.

Task II . Establish a steering committee composed of representatives

from a variety of concerned groups that are providing funds, participating

in specimen collection, and operating monitoring programs. This committee

would be responsible for:

1. Developing the organizational and managerial structure

of the NESB.

2. Identify the types of specimens and information to be stored

in the Banking System.

3. Develop interim protocols for sampling, sample handling, and

storage of specimens to be included in the bank.

4. Plan for a data handling, storage, and retrieval system.

Task III . Identify research needs as determined during the implementation of the NESB. Areas already identified are:

1. Sampling strategies

2. Sample processing and storage procedures

3. Measurement strategy

Task IV . Conduct meetings at the national and international level, of

user and research groups, to exchange current information that would be

relevant to the NESB.

During this time, the rapid growth of EPA's human pollutant burden

program dictated the need for a sophisticated system of standardized

protocols for sample collection, preparation, storage and analysis.

In December, 1973, this need was addressed by the EPA and the NSF in

a meeting to formulate plans for the development of a National Environmental

Specimen Bank. At this meeting, a four point proposal was generated, with

joint funding being provided by NSF, EPA and NBS. This proposal was designed

to set the ground work for establishing a specimen bank; a bank that would

meet the requirements of both Federal and State regulatory agencies and

that of the academic community.

5
The four point proposal contained the following elements:

1. A survey of existing specimen collections

2. An evaluation of existing specimen collections

3. Research and development of sampling, storage, and analysis

protocols

4. The development of a planning document for the organization and

management of the NESB

The specimen collection survey, conducted nationally, was used to

generate a broad data base on various aspects of specimen banking. This

survey was conducted by the Oak Ridge National Laboratory.

NBS then had the responsibility of critically evaluating the survey

results to determine their utility and applicability to the National

Environmental Specimen Bank program.

In general, the survey evaluation revealed that the collections,

sampled and stored for purposes other than retrospective analysis, provided

little information that was applicable to the analytical nature of the

specimen bank program. Their use, however, was primarily in the area of

taxonomy (1)

.

Concurrent with the survey, NBS began and is continuing research to

generate state-of-the-art methodology for sample collection, preparation,

storage and analysis. This task is envisioned as a continuing function

for the life of the bank. This approach would allow for a continued

updating of methodology to meet the needs of the bank.

Finally, the last task was the formulation of a plan for the development

and operation of the bank. The clarification of this plan with respect

to several key issues was to be the final result of the present Workshop.

Relevant scientific issues which were discussed were:

1. Review issues and need for the bank

2. Development of specific bank objectives

3. Identify and specify sample types

4. Specimen collection, preparation and storage requirements

5. Evaluate and formulate methods of analysis

6
SECTION 3

RESULTS OF CURRENT RESEARCH PROGRAMS

Since January 1975, the Analytical Chemistry Division of the National

Bureau of Standards has conducted a continuing research program to improve

methodologies for the collection, storage, and analysis of NESB samples.

The program currently underway is pursuing three avenues of investigation. The first is a complete survey of available literature concerning problems of sampling, transporting, and storage of biological and

environmental samples for analytical purposes. The second is an active

research program to improve methodologies for sampling, sample handling,

and storage of the above matrices. The third area concerns the evaluation

and improvement of analytical techniques to be used for the analysis of the

trace constituents of interest. The latter two portions of the NBS research

program are currently directed primarily towards trace elements, but future

research will be directed toward other substances of interest, such as

trace organic species.

Literature Survey

The survey of recent literature entailed the use of both manual searching and computer assisted bibliographical retrieval services. These included

Medline, Chemcon, Biosis, Cain, Defense Documentation Center, and others.

The specific components of interest that were researched included trace

elements, pesticides, other trace organics, radionuclides, and microbiological species. This survey, containing over 200 references, represents a

single base of scientific data for the development of guidelines for

sampling techniques, container cleaning methods and storage techniques (2)

.

In general, the information found in the literature survey was limited,

often contradictory, and usually pointed out problems rather than solutions.

Several useful points of information that were documented include references

on trace element contamination of biological tissues due to stainless steel

sampling implements, and the substantial problems associated with currently

accepted water sampling and storage techniques.

Sampling and Storage

A major portion of the current NBS research program has been the

experimental evaluation of contamination and losses of the trace constituents

of interest during sampling, sample handling, and long-term storage. One

of the initial projects in this program was the evaluation of twelve

7
polymeric materials for their trace element content, and for the possibility

of removing these trace elements when contacted by liquid samples. This

study was made using three complimentary trace analytical techniques,

neutron activation analysis, atomic absorption, and spark source mass

spectrometry. The utilization of a multidisciplinary analyical approach

gave an almost complete coverage of trace elements of interest.

The results of this study indicated that many materials were grossly

contaminated by trace elements from plasticizers, formulators, and other

process materials. However, conventional polyethylene and Teflon were

found to be reasonably clean and it was generally found that less than 10

percent of the bulk trace element content could be leached out, even with

conditions as severe as a 2-hour hot leach with 6N acid. The complete

details of this study are included in Appendix A.

A second, and equally important part of the current research program

has been a study and evaluation of long-term storage techniques which would

be adequate for tissue and other biologically active samples. The effects

of microbiological action on trace constituent concentrations and distributions

are well documented. However, the mechanism for complete long-term elimination of that micro-biological activity is not well documented. Freezing

has long been applied as a technique for analytical storage, however no

study has yet been performed to document the reliability of this method of

storage for more than a short period of time.

More recent studies into lyophilization have demonstrated minimal

losses and/or contamination of trace elements during the sample processing.

The NBS has now documented the viability of the freeze-drying technique to

stabilize trace element composition. Standard Reference Material Bovine

Liver (SRM 1577) has been shown to be unchanged for more than five years.

The bulk material for this SRM was freeze-dried, ground, blended, and

bottled. This material was analyzed and certified for trace element composition

in 1972. To the present time, no documented evidence of trace element loss

or alteration has occurred. The results of a complete study of losses on

freeze-drying of liquid samples were published in Analytical Chemistry (3)

Finally, the technique of low temperature ashing (LTA) has been

evaluated for long-term storage and found to have many advantages. A

recent study at NBS investigated the loss of trace elements during plasma

ashing using both radioactive tracers, and activation analysis of samples

before and after ashing. The results obtained indicate that over thirty

(30) trace elements are retained quantitatively during LTA. Five elements,

mercury, osmium, and the halogens (chlorine, bromine, iodine), are not

quantitatively retained. It was also determined from the above studies

that contamination of the sample was not a measurable problem during

ashing. An added advantage to the LTA technique is that resultant samples

are easily composited and homogenized. The complete results of this

research are detailed in Appendix B.

8
Trace Element Analytical Techniques

The third portion of the current research program has been to evaluate

the effectiveness, and improve where necessary, the major analytical

techniques for environmental samples. The elements specified to be of

primary interest were mercury, lead, arsenic, selenium, nickel, vanadium,

copper, manganese, beryllium, chromium, platinum, and palladium.

Since space does not permit a complete detailing of efforts in this

area, two examples will be selected for brief discussion.

First, a complete study of the determination of chromium by neutron

activation was undertaken. As has been reported recently, there is increas

ing analytical evidence which suggests the presence of a volatile organometallic chromium species in some types of biological samples. Recent work

on the certification analysis of a candidate NBS SRM of brewer's yeast has

indicated that neutron activation with radiochemical separation is able to

determine reproducibly both the stable and the volatile species of chromium

and excellent agreement was obtained with other analytical results using

closed system desolution. A complete description of this work is given in

Appendix C.

The second example is the work at NBS on the modification of published

procedures for the cold vapor atomic absorption determination of Mercury.

Results from the determination of mercury in biological and environmental

samples exhibited a disturbing variability with many results having a

significant errors. A complete investigation of analytical errors, including losses during sample dissolution resulted in a detailed analytical

procedure which now yields consistently reliable results down to the 50

parts per billion (ng/g) concentration level (4).

9
SECTION 4

DISCUSSIONS AND RECOMMENDATIONS

The second day of the Tissue Bank Workshop was devoted to a review

of scientific issues and the formulation of recommendations specific to

the implementation of the NESB. On review of the issues that created

the need for the NESB, there was unanimous agreement that not only was the

concept of sample banking still of vital importance to the assessment of

low-level environmental contamination but that many of the original issues

which mandated the implementation of the NESB were heightened due to recent

environmental pollutant episodes. The Kepone episode in the James River

and the Chesapeake Bay was pointed to as a prime example where existing

specimens of documented validity would have been extremely useful to assess

the change in the environment of that pollutant. There were not and are no

samples of aquatic life or shell fish available from the Chesapeake Bay or

James River which can be used for determination of Kepone levels before the

start-up of the Kepone production in that area. Limited samples were

available from the Virginia Institute of Marine Sciences dating back 3-4

years. These samples have proven invaluable in establishing the extent to

which Kepone has affected the marine life. Had earlier samples been available from a banking system, a far better assessment of Kepone baseline

levels prior to the dumping episodes would have been available to environmental officials. With the large increase of man-made chemicals now being

put into our environment, it was concluded that issues such as the Kepone

insult in Virginia are surely to be on the increase rather than to remain

as an isolated situation.

A second possible function of the NESB system was raised and found by

the participants to be of vital importance. It was noted that an NESB

system should also include a real-time monitoring function. This function

would provide trend analysis data for the real time estimate of our environmental quality in addition to assessing the effectiveness of our current

pollutant control programs. This could logically be done by subsetting

selected samples that were planned for inclusion in the bank and conducting

real-time analyses on them. This would create an initial environmental

monitoring program with vastly improved state-of-the-art analytical methodology. The storage of data produced by this system would also provide a

mechanism for evaluating analytical methods with time. The combination of

time dependent data with the availability of similar samples would allow a

far superior method of quality control.

The single most important point to come from the Workshop was that the

NESB can serve many important functions, not just that of long-term retrospective analysis. The results of sample banking would surely impact on

10
monitoring and health affects research of the EPA, and would be of great

assistance to ongoing programs within the Department of Agriculture, Food

and Drug Administration, and other agencies (Figure 1).

A set of specific objectives for the sample bank were identified

during the second day's discussions. Those objectives are summarized as

follows:

1. The collection, preservation, and storage of selected environmental

samples using methodologies that had been documented to minimize

or eliminate alteration of trace constituents.

2. The real-time analysis of selected trace constituents using

methods of documented validity to obtain mdhit6 ring" trend data.

3. Research in analytical methodology utilizing both the accumulated

long-term data base and samples that have been stored in the

NESB. This research will lead to a self -improving set, of

monitoring data.

4. The periodic review of the operation of the banking system

relative to its valid input of samples and output of analytical

data.

With the realization that the NESB is a viable concept, both by need and

availability of technology, it was proposed that the NESB be initiated in the

form of a five-year pilot program for banking environmental samples. This

proposal was founded on the realization that a. system such as NESB could

quickly become overwhelmed with samples. This would invariably lead to a

breakdown of the physical storage mechanisms, but more importantly, would

likely lead to short cuts and compromises in operating procedures and

analytical methodologies used for this real time monitoring phase of the

system.

This position of moderate growth with careful monitoring of the program

was unanimously recommended by the attendees of the Workshop. It was

further recommended that the pilot program be initiated as soon as was

feasible. During this time, limited numbers of samples would be collected,

analyzed, and stored in a central facility. Problems encountered in the

collection, transportation, analysis, and storage would be carefully

monitored. Any dif f iciencies found in the system would be reexamined and

new research would be initiated as necessary. With complete accord on the

pilot scale concept, exact guidelines for this system were discussed and

agreed upon.

The identification of specific sample types which should be included

in the pilot sample bank during initial start-up of the system was discussed.

The major focus of attention was on the absolute requirement to minimize

both the number of samples and sample types in the pilot program so that

the banking system did not become overwhelmed with either samples or analyses

during its first years of operation. Unreasonably large numbers of avoidable

errors would destroy its credibility before it was even in full operation.

11
Thus, all participants recommended the inclusion of a modest sample set per

year for the first five years of operation with the focus on validating

credible storage and analytical results.

A reasonable figure of approximately two thousand samples a year,

split into four matrix types, was recommended (Figure 2). Also, it was

recommended that the samples be obtained from no more than two different

locations in the beginning phases, to minimize problems with logistics,

sample collection, and transport.

Nine different sample types representing all major phases of environmental samples were considered for inclusion in the bank. These included

air particulates, sediment, water, botanical, biological, and human samples.

It was recommended that samples which represented environmental accumulators

or integrators be emphasized as initial candidates, both due to the ease of

analytical manipulation of those samples and because they represented time

integrators of major pollutants present in our environment.

The first sample that was recommended for inclusion unanimously was a soft tissue sample that had an accumulator function in the human body, most

likely liver or kidney. The second and equally important sample type was

an accumulator of aquatic origin. Much discussion centered around the

exact nature of the accumulator and suggestions ranged from plankton to

shark tissue. Agreement was reached that a shellfish bivalve such as

oyster which passes large quantities of water through its system every day

and which tended to mirror increased concentrations of many toxic pollutants

was a good choice. The third sample type was a food material representing

a major input into the human diet. Consensus was unanimous that a food

grain or composite of grains was the best choice. The fourth sample type

was a collector of atmospheric or airborne pollutant materials. It was

recommended that material such as lichen or moss was a good indicator of

long-term trends in atmospheric pollutants and should be included' in the

initial bank. These four sample types were chosen for their diversity and

their utility to environmental monitoring programs. However, a second and

even more important consideration was that there is now enough scientific

evidence to be reasonably assured that the storage and analysis of the

trace element components in these materials could now be carried out with

integrity.

One of the last items for consideration at the Workshop was the

evaluation and formulation of viable analytical methods for the real-time

and retrospective analysis of samples from the NESB system. Part of the NBS

research effort has been to publish a compilation of analytical methods

currently being used for SRM certification analysis. These methods will be

available in the near future and were discussed at the Workshop. As the

pilot bank program expands, additional analytical laboratories will become

involved in the program. It was the concensus of opinion that rather than

have a single specified analytical method be used on samples from the NESB, a better approach would be to rely on the judgment of high quality analytical

laboratories to use methodologies that they had demonstrated as viable in

their own laboratories. It is widely recognized that many analytical

methods are capable of producing excellent results in the hands of a

12
talented analyst. However, in the improper hands, standard methodologies

do not necessarily generate credible results. To the contrary, they usually

generate a false sense of security which often leads to monumental mistakes.

It was thus recommended by members of the Workshop that a compilation of

recommended or evaluated methods be included in the NESB system, but these

be included only for the information or aid to the analytical laboratories

actually doing the work. The final decision on methodology would be left

up to the capable laboratories or scientists working on samples from the

bank. The laboratory capabilities would be under a strict quality assurance

program with blind analyses of representative materials as a foundation.

As a summary to the recommendations and conclusions of the Workshop, a

schematic drawing of the overall NESB system with its functions and responsibilities was put together in a composite form. This schematic represents

most functions and outputs to be derived from the NESB system and is given

in Figure 3. It was concluded by the members of the Workshop that a system

such as this would give reliable data for the evaluation of trends in trace

element and trace organic constituents of environmental and human nutritional

importance over a long period of time.

13
SECTION 5

CONCLUSIONS

There is a real need for the evaluation of sampling, sample handling,

and long-term storage methodology for the establishment of an effective

environmental specimen banking system. This formalized, systematic approach

to defining our current environmental hazards has never before been attempted

on a national scale. Studies are continuing in a randomized fashion and in

many cases without the proper validation of specimen banking procedures.

It is this type of information, however, that is currently being used by

the scientific community as well as State and Federal regulatory agencies

to propose environmental quality standards and limits for control technology.

If these types of monitoring programs are to continue, as they must to

protect our environment as well as the health of our population, then the

type of research program described here is required to establish and define

the necessary basic scientific information required for such a specimen

banking system. The NESB, when operational, will provide future generations

with an important resource for evaluating their current environmental

influences

14
NESB CONCEPTS \

ECOLOGICAL INDICATOR SAMPLE BANKING

REAL TIME RETROSPECTIVE ANALYSIS

ANALYSIS NEW POLLUTANTS

IMPROVED METHODOLOGY

FIGURE 1. National Environmental Specimen Bank (NESB) Concepts.

15
PILOT BANK SAMPLES

TWO GEOGRAPHIC LOCATIONS

2000 SAMPLES PER YEAR

HUMAN TISSUE

ACCUMULATOR

ATMOSPHERIC

INTERGRATOR:

LICHEN, MOSS, / FILTERS

AQUATIC

ACCUMULATOR

SHELLFISH BIVALVE

\

FOOD

GRAIN

FIGURE 2. Pilot Bank Samples.

16
NESB PILOT BANK

RESEARCH IN

ANALYTICAL

METHODOLOGY

NESB ADMINISTRATION

REAL TIME

MONITORING

LONG TERM

STORAGE

z

RESULTS

TIME SERIAL DATA

FOR TREND ANALYSIS

X

RESEARCH IN

SAMPLE PRESERVATION

AND STORAGE

RESULTS

SAMPLES FOR

RETROSPECTIVE ANALYSIS

z

QUALITY ASSURANCE

TIME DIFFERENTIAL

COMPARISONS

'« *. *

FIGURE 3. NESB Pilot Bank.

17
BIBLIOGRAPHY

1. ) NBS Special Publication EPA/NBS/IGA-D5-0568, "Evaluation and

Research of Methodology for the National Environmental Specimen

Bank", D.A. Becker, E.J. Maienthal and G.M. Goldstein.

2. ) NBS Technical Note 929, E.J. Maienthal and D.A. Becker.

3. ) Anal. Chem. , 4_7, 1685, (1975), S.H. Harrison, P.D. LaFleur, and

W.H. Zoller.

4. ) J.O.A.C., 55 No. 6, 1339, (1972),. T.C. Rains and 0. Menis.

18
APPENDIX A

THE CLEANING, ANALYSIS AND SELECTION OF CONTAINERS FOR TRACE ELEMENT SAMPLES

by

J. R. Moody and R. M. Lindstrom

National Bureau of Standards

Institute for Materials Research

Analytical Chemistry Division

Washington, DC 20234

19
ABSTRACT

Since the sample container represents one of the earliest and potentially

one of the largest sources of sample contamination, much of the analytical

accuracy will depend upon the choice of container materials and the method of

cleaning containers. Twelve different plastics have been examined by gravimetry,

isotope dilution mass spectrometry, and neutron activation analysis. The

present study examines the impurities present in the various plastics as well

as the actual level of impurities leached from the plastics.

Contemporary problems in analytical chemistry have forced the analyst to

lower the threshold for determining trace elements to the parts per billion

level (ng/g) or lower. More is involved, however, than simply lowering the

detection limit for a method. Blanks, sampling, sample handling, and storage

conditions often become dominant factors in a trace elemental analysis scheme.

Maienthal and Becker (1) have recently reviewed the literature on sampling,

sample handling, and storage. Problems are very severe for water and other

liquid handling and storage. Generally, the usage of moderately concentrated

mineral acid is considered the best way to prevent adsorption of trace metals

from water by the container walls. While the best rule will continue to be to

analyze a sample as soon as possible after sampling, the influence of the

sample container on the analysis blank cannot be overlooked.

At these low levels (ng/g or less) the analytical blank in all of its

manifestations becomes a dominant factor in the accurate determination of

trace constituents in a sample. Patterson and Settle (2), for example, have

reported that the majority of published data for lead in plants, animal tissue,

and water is in error due to gross levels of contamination or failure to

control the analytical blank. Murphy (3) has reviewed the role of the analytical

blank in accurate trace analysis and defined its sources and methods of control.

Often, as contamination is eliminated and other sources of the analysis blank

are brought under control, the true concentration of a trace element is found

to be even lower than previously determined.

Considering only the sample storage container, two primary methods of

blank (contamination) control are available. These complementary techniques

are the proper selection of container materials and the proper choice of a

method of cleaning the container. Robertson (4) provided a good introduction

to the subject by analyzing a number of reagents, construction materials, and

container materials for various trace elements and then underscoring the

implications for contamination of a sea water sample. The use of plastics and

quartz has become common practice in most trace metal analysis laboratories.

20
Numerous authors have addressed the problem of containers and blanks, and

suggestions for the proper way to clean laboratory ware and containers are

almost as numerous.

Since so much of the literature concerning storage materials and cleaning

procedures was contradictory (1) , an effort was undertaken at the NBS to

determine the best possible materials for sample storage containers. The

immediate objective was to determine which container should be used for various

applications such as Standard Reference Materials (SRM's) or the National

Environmental Specimen Bank. Depending upon the application, considerations

other than contamination levels must be anticipated (e.g., cost, commercial

availability)

.

For liquid samples, an important consideration together with low contamina1 tion levels is a low rate of permeability to water vapor. Although such

permeability data is available, it is difficult to apply to a commercially

fabricated bottle. The wall thickness varies from plastic to plastic, from

manufacturer to manufacturer as well as from bottle to bottle. This situation

is also complicated by the relative efficiency of the closures provided.

Thus, the experimental determination of the actual rate of loss of water from

various containers was indicated.

Experimental

A number of plastic materials were studied by several methods. Among

those materials examined were bottles constructed of conventional polyethylene

(CPE), linear polyethylene (LPE) , polypropylene (PP) , polymethylpentane (PMP)

,

polycarbonate (PC), polyvinylchloride (PVC) and several types of Teflon (TFE,

FEP) . In addition specimens of ethylenetetrafluoroethylene copolymer (Tefzel

or ETFE), Teflon PFA, polystyrene (PS), and Teflon pipe sealing tape (TPT)

were examined. The studies of these materials may be divided into three types

of experiments: neutron activation analysis (NAA) of the bulk impurities

within the plastics both before and after cleaning, isotope dilution mass

spectrometric (IDMS) analysis of the impurities leached from a container

during a cleaning process, and a gravimetric study of the loss rate of water

from containers.

Gravimetric Studies . The loss of water from a plastic container by

permeation is not trivial over a period of one year or more. Two containers

each of CPE, PP, PVC, PMP, and PC were obtained from commercial sources. The

containers were of a similar physical size. All of the containers were weighed

on a high capacity analytical balance. Half of the containers were then

filled with similar amounts of distilled water and re-weighed. The second

container of each type was retained as a weighing tare to correct for the

influence of changes in the barometric pressure, relative humidity and temperature and loss of plasticizer. The samples were re-weighed after 17 days and

66 days to establish a rate of loss of moisture.

IDMS Studies . Solutions contained in one liter or 500 mL size bottles of

FEP, CPE, LPE, and PC were examined by spark source IDMS (multi-element) and

by thermal emission IDMS (for lead only). The general principles and techniques

21
used have been given elsewhere (5,6). The bottles were rinsed with distilled

water to remove any surface contamination and three sets of bottles were

filled with a (1+1) mixture of pure HC1 or pure HN0 3 and pure water (7) . The

bottles were allowed to stand full for one week, with the exception of the FEP

bottle which was heated to 80 °C for one week.

Aliquots of the contents of one set of bottles leached by (1+1) HC1 were

spiked with 206 Pb. Aliquots from two other sets of bottles, leached by (1+1)

HC1 and (1 + 1) HN0 3 ^respectively, were spiked with a 19 .element multi-spike (7)

for the spark source mass' spectrometer (SSMS) ^ The spiked solutions were then

evaporated to several drops under a Class 100 laminar flow hood. The sample

solutions were then analyzed in the SSMS (multi -element) or thermal source

mass spectrometer (for Pb only)

.

The first set of bottles were re-filled with a (1+1) mixture of pure HNO3

and pure water and the entire cleaning, sampling, spiking, analysis sequence

was repeated as before. Finally, the bottles were filled with high purity 0.5

percent HNO3, sampled after one week and again after two months, and then the

aliquots were spiked with 206 Pb and subsequently analyzed.

NAA Studies . Specimens of 0.2-2 grams (4-12 cm2

) were taken with a steel

paper cutter from new 0.5-1 1 plastic bottles (CPE, LPE, PP, PMP, PC, PVC, and

FEP) , sheet (TFE and PFA) , bottle caps (ETFE) , boxes (PS) or tape (TPT)

.

Precleaning consisted of a light rinse or wipe with ethanol, and then with

distilled water. Contamination from the steel paper cutter was eliminated

after irradiation.

All irradiations were performed at a thermal neutron flux of 1.3 x 10 13 n

cm 2 sec 1 and a gamma flux of 2 x 10 7 rad/hr (8) . Other positions in the

reactor offer higher neutron flux, but at the expense of a much higher gamma

flux per neutron. Gamma counting was performed on two large- volume Ge(Li)

detectors. Peaks used for quantitation contained greater than 50 net counts

and better than 25 percent (relative standard deviation) precision from

counting statistics. Signals from many elements were obscured by other

dominant activities, notably Na, Br, and CI.

NAA Experiment 1 : The plastics were each irradiated for two minutes.

After irradiation the edges were trimmed to remove edge contamination and the

samples were counted. Then, the samples were cleaned in (1+1) HC1 by heating

at 100 °C for two hours, rinsed with high purity water, dried, and irradiated

and counted again. After a similar two-hour leach in hot (1+1) HNO3 the

plastics were analyzed a third time.

NAA Experiment 2 : Fresh samples from the same plastics were used in

measurements of long-lived activation products. After irradiations (4-6 hrs)

and decay of short-lived activities the edges were trimmed and the plastics

were counted. Then the samples were leached in (1+1) HC1 as before, rinsed,

and leached in hot (1+1) HN0 3

. The leach solutions and cleaned plastics were

then counted.

22
Results and Discussion

Gravimetric Studies . The results obtained for the materials studied are

given in Table 1. In addition, data previously obtained by Moody, et al , (9)

indicated an annual rate of water loss of ^0.05 percent from one liter Teflon

bottles. PVC, PMP, and PC containers may be excluded from consideration as

container materials due to their high permeability to water. Both Teflon FEP

and PP containers have a rate of loss of less than 0.1 percent per year. CPE

containers lost slightly more than 0.1 percent. Such losses may be minimized

or. largely eliminated by , sealing the bottle in a ba"g made of a 'commercially

available vapor barrier material i.e. polyethylene coated aluminized mylar.

None of the observed loss rates should be construed as permeability data since

the closure may have been responsible for a significant portion of the total

water loss.

All containers used were bought from commercial sources and are believed

to be similar to those most often used in the laboratory. Three of the containers (PP, FEP, and CPE) have water loss rates which are compatible with the

long term storage of samples while the others (PVC, PMP, and PC) have significant

rates of water loss. The results obtained for CPE are similar to those observed

by Curtis, et_ al_, (10). Humidity conditions during the study averaged about

40 percent R.H. The practice of using a desiccator partially filled with water

to act as a humidity chamber to slow transpiration losses is recommended

although one must be careful that high humidity conditions do not cause a

weight gain (absorption of moisture).

IDMS Studies . The results obtained for the leaching of lead from FEP,

LPE, CPE, and PC containers are listed in Table 2. Lead was chosen as a model

element for this study, partly because of the high accuracy obtainable by

thermal source isotope dilution mass spectrometry. The sequence of using HC1

first and then HNO3 conforms to long standing practice in our laboratories (7)

.

Most of the cleaning of these containers is accomplished by only one week of

soaking in HC1. The additional week of soaking in (1+1) HN0 3 removed additional

lead only from FEP and PC.

Patterson (2) has suggested that 0.5 percent HNO3 is more efficient for

cleaning Teflon containers of Pb contamination than is (1+1) HNO3. This was

tried after leaching with (1+1) HNO3 no further leaching of lead was detected

except from the PC container. After one month of leaching with 0.5 percent

HNO3, no further lead was leached even from the PC container. The greater

quantity of various elements leached from FEP may be partially explained by

the higher temperature (80° vs room temperature) used to clean the Teflon FEP

container. One advantage of Teflon is that it is chemically inert. The

extent of chemical attack on other materials can be quite severe even at

temperatures only slightly above room temperature.

Data obtained by SSMS is compiled in Tables 3 and 4 for impurities

leached by (1+1) HC1 and (1+1) HN0 3 respectively. Again, the Teflon FEP

bottles were heated to 80 °C while the others were leached at room temperature. Generally, the (1+1) HC1 leached more than the (1+1) HN0 3 except for

the FEP containers. A single element, Ca, is responsible for most of the

difference between the (1+1) HC1 and (1+1) HN0 3 in leaching Teflon FEP.

23
Values which are below 2 ng/cm2 or which are prefixed by < are upper limit

numbers. That is, the concentration is below the optimunTconcentration range

for the amount of spike isotope used or the concentration is near the blank or

detection limit.

NAA Studies . The amount of impurities found in the uncleaned plastics as

determined in NAA experiments 1 and 2 are summarized in Table 5. A comparison

of the impurities found in CPE in this study shows agreement with that found

by other workers (4,12-16) to approximately an order of magnitude. It is

interesting to note that there has been no clear change in trace elemental

impurities in over 25 years of manufacture (13) . The ten materials studied

show striking differences in trace element composition. The purest materials

are TFE and CPE, in agreement with the experience of numerous workers. Polystyrene, cut from a box used to package NBS Standard Reference Materials, was

also very clean. Many materials showed easily detectable amounts of a few

elements. Linear polyethylene contained large amounts (>10 ppm) of Na, Al,

Ca, CI, and Zn; polypropylene-Al , CI, and Ti; polymethylpentene-Zn; polycarbonate-Cl and Br; polyvinylchloride-Na and Sn; Teflon FEP-K (and 1 ppm of

W) ; Teflon PFA-C1; Teflon pipe tape-Al, Zn, and large amounts of Ti; and ETFElarge amounts of CI. Some of these elements are residues of polymerization

catalysts (17).

The results of the acid leaching experiments are presented in Table 6.

They are compiled from the differences in trace element composition of the

plastic after leaching in the first and second NAA experiment, and from direct

measurement of leached tracer in the second. The two experiments are generally

concordant in the conclusion that less than half of most trace elements are

leachable, and quantitatively concordant in the results for* sodium,', the only

element common to the two NAA experiments. This point is important because

the long irradiation in the second experiment makes those measurements an

imperfect model of a reagent or analyte stored in a bottle.

Those atoms of trace elements leached and detected in the leachate in

the second experiment are precisely those not representative of undisturbed

trace elements in an undisturbed matrix. The radioactive atoms underwent

recoil on absorption of a neutron and thus may have been more labile than

their inactive neighbors. In addition, the matrix suffered radiation damage

from some 10 8 rads of gamma radiation, with the result that all materials were

visibly browned and their physical properties were greatly changed. The

halocarbons were damaged additionally by the absorption of nuclear recoil

energy and beta radiation induced in major constituents of the matrix. Teflon

TFE and especially pipe tape were perceptibly embrittled, and PVC became black

after the long irradiation.

With the exception of Na in pipe tape, (perhaps a special case because of

its high surface/volume ratio), and to a lesser extent Na in CPE, the trace

elements studied are in general not leached from the polymer matrix even with

the rather severe acid treatment used here (note that the rule of thumb of a

doubling of reaction rate for every 10 °C rise in temperature makes our conditions correspond to a few weeks' immersion at room temperature). It may be

inferred that generally the bulk of trace elements present are distributed

throughout the matrix, and not merely on the surface.

24
The hydrochloric acid leach in experiment 1 increased the chloride concentration in all the plastics, but not grossly so. The added chloride contamination corresponds to a 10 4 cm thickness of 1:1 HC1 in the surface of polypropylene, and less in the other materials. Neither this chloride nor other

elements were entirely removed by the subsequent HNO3 leach. This inward

diffusion of HC1 is in accord with the water loss measurements, both experiments

• leading . to a diffusion coefficient ' for aqueous solutions in polyolefin" of 1 D=10 12 cm2 /sec. If the removal of trace contaminants depends on diffusion of

water as a rate-limiting step, then the inertness of trace elements within the

matrix is explained. With this value of D, the mean square diffusion length

in one year x = /2 Dt = 0.1 mm.

The observation that a second leach removes much less contamination than

the first indicates that only surface contamination is easily removed. The

cleanest plastic materials available must be cleaned further for the lowest

blanks. Many methods have been suggested and several have been shown to be

sufficient for the purpose at hand (1,2,7). Since continued cleaning with an

array of purest solvents would continue to clean any container (1) , the ideal

cleaning method will be forever out of reach.

Conclusions

Several commercially available plastic containers have been examined for

rate of transpiration of water. Those which have been found to be suitable

include Teflon FEP, and PP. With a suitable moisture barrier, CPE containers

can also be held to water losses of less than 0.1 percent per year. It is

recognized that there can be considerable bottle-to-bottle variation in wall

thickness and other parameters of construction. Results of the trace element

studies are especially suspect in this regard since even one occlusion in the

container wall can considerably affect the trace element levels.

The containers used for these studies were carefully selected to be free

from visible occlusions and were average in wall thickness, weight, and other

characteristics. While the trace element concentrations found by NAA and IDMS

cannot be said to be statistically representative of all bottles of these

types, the large differences observed between various plastics are probably

valid. In summary, the best containers are believed to be those constructed

of CPE and the various Teflons. Differences were observed between NAA and

IDMS results for trace elements leached from the plastics. At least some of

these differences may be procedural since all the NAA samples were heated

whereas only the FEP samples were heated for the IDMS study.

Another possible difference between the two results may be in the way the

samples were leached. For IDMS work the leaching was carried out within

intact bottles and the results are believed to be representative of the

contamination to which a sample would be exposed in an uncleaned bottle. In

contrast, the NAA studies examined the trace elements leached from both sides

of a small sample of plastic immersed in acid. Since at least some containers

are known to be blow molded, the higher results obtained by NAA may simply

reflect a difference in contamination levels between inside and outside walls,

the NAA data probably including contamination from the mold.

25
With the exception of Teflon FEP, (1+1) HC1 has been found to be the

better cleaning agent. However, HC1 and HNO3 appear to leach various elements

with different efficiencies, thus, the use of both acids one after the other

is recommended. It should not be surprising that in sequential cleaning

studies (for Pb by IDMS, various elements by NAA) , most of the cleaning is

accomplished in a short period of time. From the present work, that of Karin

et al . , (16), and other work in this laboratory (3,7) a procedure may be

suggested that is optimum for most trace work: First, fill the containers

with reagent grade (1+1) HC1 and allow them to stand for one week. Empty,

rinse with distilled water, and re-fill the containers with (1+1) HNO3 and

allow to stand for another week. Finally, empty, rinse, fill with the purest

available distilled water, and allow to stand until needed. Preferably, the

distilled water should be changed periodically to assure continued cleaning.

Teflon bottles should be heated through the first two acid leaches.

Finally, it should be recognized that despite all efforts to clean these

bottles, the data would seem to indicate that only the surfaces of the container

walls have been cleaned. While very low blank levels may be achieved in a relatively short period of time (several weeks) , there is insufficient data to

suggest what levels of contamination might occur over a very long period of

storage. One possible solution to this problem, depending upon the sample

involved, would be to freeze the sample for long term storage.

Then there is the matter of the cost of the containers. Currently, there

is greater than one order of magnitude difference in cost between FEP and CPE.

Teflon is the preferred container but its cost often mitigates against its

use. Other plastic materials are available which might be suitable for some

applications. However, we have deliberately chosen to examine only those

materials which are commercially available in suitable containers. The

analyst should keep abreast of changes in materials and methods of fabrication

which would have an influence on our results.

Acknowl edgement

The authors wish to acknowledge the contributions of T. J. Murphy,

P. J. Paulsen and J. W. Gramlich for the IDMS analysis of solutions stored in

plastic containers.

26
LITERATURE CITED

E. J. Mainthal and D. A. Becker, "A Survey of Current Literature on Sampling,

Sample Handling and Long Term Storage for Environmental Materials, NBS

Tech Note 929 (1976).

C. C. Patterson and D. M. Settle, in "Accuracy in Trace Analysis: Sampling,

Sample Handling, and Analysis," Proc. of the 7th IMR Symp., P. D. LaFleur,

ed., NBS Spec. Pub. 422, 321 (1976).

T. J. Murphy, in "Accuracy in Trace Analysis: Sampling, Sample Handling, and

Analysis," Proc. of the 7th IMR Symp., P. D. LaFleur, ed. , NBS Spec. Pub. 422,

509 (1976).

D. E. Robertson, Anal. Chem. 4£, 1067 (1968).

P. J. Paulsen, R. Alvarez, and C. W. Mueller, Applied Spectroscopy 30, 42 (1976)

I. L. Barnes, T. J. Murphy, J. W. Gramlich, and W. R. Shields, Anal. Chem. 45_,

1881 (1973).

E. C. Kuehner, R. Alvarez, P. J. Paulsen, and T. J. Murphy, Anal. Chem. 4£,

2050 (1972).

H. E. Despain, private communication.

J. R. Moody, H. L. Rook, T. C. Rains, and P. J. Paulsen, unpublished data from

the preparation of SRM 1642.

G. J. Curtis, J. E. Rein, and S. S. Yamamura, Anal. Chem. 4_5, 996 (1973).

C. C. Patterson, private communication to E. J. Maienthal.

R. E. Thiers, "Methods of Biochemical Analysis," D. Glick, ed., 5_, 274-309,

Interscience, New York (1957).

J. W. Mitchell, Anal. Chem. 45_, 429A (1973).

S. H. Harrison, P. D. LaFleur, and W. H. Zoller, Anal. Chem. 47_, 1685 (1975).

H. Sorantin and P. Patek, Z. Anal. Chim. 211 , 99 (1965).

R. W. Karin, J. A. Buono, and J. L. Fasching, Anal. Chem. 47_, 2296 (1975).

E. C. Kuehner and D. H. Freeman, in "Purification of Inorganic and Organic

Materials," M. Zief, ed., Marcel Dekker, New York 1969, pp. 297-306.

27
Table 1. Annual Rate of Loss of Water from Container Materials.

* » * l . * •

Container After 17 Days After 66 Days

CPE 0.116% 0.109%

PP 0.034% 0.049%

PVC 0.429% 0.601%

PMP 0.988% 1.018%

PC 1.65% 2.00%

Table 2. Lead Leached from Containers (ng/cm2

).

Bottle (1+1) HC1 (1+1) HN0 3 0.5% HN0 3 0.5% HN0 3

1 week 1 week 1 week 2 months

FEP 0.41 0.014

LPE 0.20

CPE 0.18

PC 0.36 0.023 0.023

no significant amount over blank level

28
Table 3. Impurities Leached from Plastic Containers by

(1+1) HC1 (ng/cm2).

Elements Teflon FEP LPE CPE PC

Pb 2 0.6 18 10

Tl <1 £0.6 3 0.7

Ba 2 1 0.3 3

Te 2 — 0.7

Sn 1 <1 <0.8 13

Cd 0.6 0.2 0.2 48

Ag <6

Sr <1 0.2 0.2 0.3

Se 0.8 0.4 ^0.3 <0.5

Zn 4 9 1.0

Cu 6 1 0.7 46

Ni 0.8 0.8 0.3 0.3

Fe 16 1 1.0 449

Cr 4 0.8 0.3 <5

Ca 2 60 0.8 ^16

K 1.6 1 0.7 <5

Mg 1.0 0.4 0.7 0.8

Al 4 4 10 3

Na 2 6 42 8

E58 Z89 Z81 £130

29
Table 4. Impurities Leached from Plastic Containers by

(1+1) HN03 (ng/cm2 ).

Elements teflon FEP LPE CPE PC

Pb 2 2 0.7 0.3

Tl <1 4I 1 <0.8

Ba 4 <0.2 2 0.3

Te 0.6 0.2 <^0.5 0.3

Sn 1 1 <0.8 0.2

Cd 0.4 0.2 0.2 0.3

Ag <8 0.2

Sr 0.2 1 0.2 <0.2

Se 0.2 0.4 3 0.5

Zn 4 8 2 0.8

Cu 2 0.4 2 0.8

Ni 2 1.6 0.5 0.7

Fe 20 3 3 3

Cr 0.8 0.2 0.8 0.3

Ca 80 0.6 10 3

K 2 2 2 2

Mg 8 0.6 0.7 2

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Al 6

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32
APPENDIX B

EVALUATION BY ACTIVATION ANALYSIS OF ELEMENTAL RETENTION

IN BIOLOGICAL SAMPLES AFTER LOW TEMPERATURE ASHING

by

George J. Lutz, John S. Stemple and Harry L. Rook

Activation Analysis Section

Analytical Chemistry Division

National Bureau of Standards

Washington, D.C. 20234

33
INTRODUCTION

Low temperature ashing (LTA) is the low temperature O100 °C) decomposition of organic or biological samples with atomic oxygen produced by an

electrodeless radio-frequency (RF) discharge. There are a number of

advantages in using LTA as the first step in an analytical method whether

by nuclear activation or otherwise:

1. Since the oxidation temperature is low, volatility losses are

minimized during the destruction of large quantities of organic material.

2. There are no electrodes to contaminate the sample.

3. A sample can be more readily dissolved after LTA than by alternative

methods. Wet ashing techniques with hot oxidizing acids or strong alkalis

are potentially hazardous, sometimes incomplete, and usually susceptible

to contamination because of the large amounts of reagents required.

4. High temperature ashing in a muffle furnace introduces the

possibility of contamination from containers or the furnace walls as well

as loss of some elements by volatilization. The convenience of the LTA

method can be demonstrated by reference to a publication concerning the

spectrophotometric determination of boron extracted from ashed animal

tissues (1) . Four grams of freeze-dried tissue were ashed 40-50 hours at

100-150 watt forward RF power. After the LTA treatment, the sample could

be dissolved in 2 mL of IN HC1.

5. In the X-ray fluorescence method, which is strongly matrix

dependent, LTA reduces the sample to a silicate-carbonate-oxide form,

which minimizes errors due to that problem.

6. In spark-source mass and emission spectrometry the ashed residue,

when mixed with powdered graphite, gives a uniform consistency which also

reduces matrix error.

With specific respect to activation analysis, one can identify several

advantages of beginning the analysis of a biological sample with freezedrying followed by LTA.

1. A very high neutron flux can badly char a biological or organic

sample leavig it with an undesirable consistency. That the LTA method is

very efficient for removing organic carbon is demonstrated by an American

Society for Testing and Materials procedure (2) which specifies the technique

34
in removal and hence determination of organic carbon as opposed to carbonate

in sediment samples.

2. LTA results in a substantial reduction in the volume of the

sample. This can yield a corresponding reduction in electron accelerator

or neutron generator irradiation time in photon or 14-MeV neutron activation

analysis

.

3. Since lengthy wet digestions are eliminated, a sample after

irradiation can be dissolved more quickly and hence short-lived nuclides

can be more quickly separated from the sample for counting.

4. In the instrumental photon activation analysis of samples containing

large amounts of carbon, it is usually necessary to delay counting for a

few hours after irradiation to allow the -^C produced by the reaction

12 C(y, n) 1 ^

to decay. Some shorter-lived nuclides are therefore not

detected. Many of them could be detected by removal of the carbon prior

to irradiating.

The possibility exists in the LTA method of the loss of elements of

interest during the ashing process. Studies to evaluate elemental retention during ashing have been conducted with radioactive tracers (3) . This

is not an entirely satisfactory procedure since a number of elements may

not be present in a biological sample in the same chemical form as was

used in the tracer studies. This paper describes a study, using activation

analysis, of loss or contamination of trace elements during LTA. It is

hoped that this will be useful not only in analytical chemistry for the

reasons listed above, but also to evaluate LTA as a potential processing

method for the retention of biological samples by the .National Environmental

Sample Bank over several decades of time for purposes of baseline studies

to evaluate increases or decreases in pollution.

EXPERIMENTAL

A commercial model LTA apparatus capable of 100 watts forward power

was used in these experiments. A schematic of the apparatus is shown in

figure 1. The sample was placed in a borosilicate glass ashing chamber

which was briefly rotated by hand to induce the organic sample to spread

as evenly as possible on the inside surface. The cold trap was filled

with crushed dry ice (liquid nitrogen would condense explosive ozone)

.

The system was pumped down to 1.3-2.6 Pa (10-20 millitorr). The oxygen

flow rate was adjusted so as to maintain a pressure of 13-40 Pa (100-300

millitorr). Forward power was usually set to the maximum of 100 watts.

Tuning to optimum was accomplished by observing the maximum intensity in

discharge glow in the tube.

35
BOROSILICATE GLASS ASHING CHAMBER

VACUUM GAUGE

TO

VACUUM

t \

^_ DRY ICE

"COOLED TRAP

VALVE ^

1__ >

FLOW GAUGE

RF GENERATOR AND

TUNING CONTROLS

REGULATOR

OXYGEN CYLINDER

Figure 1. Block diagram of LTA apparatus.

Preliminary experiments involving addition of tracers to biological

materials and determining retention of each isotope after ashing verified

previously published data (3) and served to establish and perfect the

basic ashing technique. After these experiments, it was decided to study

three NBS Standard Reference Materials in detail; 1571-Orchard Leaves,

1577-Bovine Liver, and 1632-Trace Elements in Coal.

Samples of about one gram were ashed for a period of several hours.

It was impossible to remove all of the ashed material from the tube, thus

it was necessary to determine the exact amount of material removed. This

was first attempted by weighing of the tube which proved difficult, since

the only balance capable of accommodating the 200 g in weight, 30 cm in

length ashing tube was accurate to only ± 2 mg. A second and more successful

technique involved the use of a nonvolatile radioactive tracer that wouldn't

36
interfere with later activation analysis. The uniform distribution of the

tracer throughout the bulk sample was demonstrated by taking several small

samples from the bottle and measuring their specific activities. Subsequently,

it was found possible to use the activation product of an element demonstrated

to be nonvolatile as an internal standard for this purpose.

The relative amounts of the elements in ashed and unashed samples

were determined by nondestructive neutron activation analysis with the NBS

10 megawatt nuclear reactor and photon activation analysis with the NBS

electron linear accelerator.

For the reactor irradiation a paired experiment was carried out using

approximately one gram of unashed material compared to the ashed residue

of one gram of material. Three series of neutron irradiations were conducted:

1. Fifteen-second irradiation followed by three countings at few

minute intervals,

2. Twenty-minute irradiations followed by 2-3 countings over a

period of one hour to a few days,

3. A four-hour irradiation followed by 2-3 countings over a period

of several days to a few weeks after irradiation (depending on Na-24

levels)

.

The two shorter irradiations were conducted at a flux of 1 . 3X10 1

3

n« cm 2 -sec 1

, and the longer irradiation at a flux of 5.6xl0 13 n*cm 2 -sec 1

.

Two series of photon irradiations were conducted with approximately

three grams of unashed material and the ashed residue of three grams of

material with an electron energy of 35 MeV and a beam current of about 50

uA.'

1. Thirty to sixty minute irradiation followed by 2-3 countings

over a period of two hours to a few days after irradiation,

2. Two to six hour irradiation followed by 2-4 countings over a period of two days to a few weeks after irradiation.

Irradiated samples were counted with a 75 cc Ge(Li) detector and a 4096

channel analyzer using one-half of the memory. Molybdenum, iodine, and

zinc by photon activation analysis were also determined with a low energy

photon detector (LEPD) . Photopeak counting rates were corrected for times

of irradiation and decay and ratio of weights of unashed material and

ashed residue corrected for weight loss.

37
RESULTS AND DISCUSSION

The experimental study of the observation of ashing losses was carrie

out in three phases. The first consisted of spiking samples of interest

with radioactive tracers of known chemical states. This phase was simple f to conduct as the isotopes could be so added to avoid interference in

radioactive counting. This first phase severed to assist in development ri

of handling techniques for subsequent work. The results with radioactive

tracers showed the retention of zinc, cadmium, arsenic, palladium, rhenium

antimony, iron, platinum, iridium, gold, and silver. A previous paper (3$'

had reported the loss of gold and silver but this phenomenon was not

confirmed here. Mercury and osmium were lost after several hours of ashing

in every case.

The second phase consisted of irradiating portions of the three

samples, counting them, then ashing and recounting. These experiments

added to the retention list the following elements: sodium, potassium,

rare earths, and selenium.

The third phase, which was the major part of this study, involved the

simultaneous activation, either by thermal neutrons or high-energy photons

of an untreated portion of the samples and an ashed portion. This study

thus established loss data on elements in the chemical form they would

actually be found in three representative matrices. Table 1 gives typical

weight losses and carbon losses as measured by the photonuclear reaction

1:i C(Y,an) 7 Be for an ashing period of approximately 12 hours and power of

approximately 70 watts. In addition to carbon, the loss of chlorine,

bromine, and iodine was observed in all three matrices as well as the

expected loss of mercury. Table 2 gives experimental recoveries of

retained elements.

TABLE 1. TYPICAL WEIGHT AND CARBON LOSSES FOR ASHING PERIODS OF

15 HOURS AND RF POWER OF 70 WATTS

% Weight Lost % Carbon Lost

Coal 80 75

Beef Liver 90 98

Orchard Leaves 75 80

38
Figure 2 shows, in the formate of the periodic chart, the elements

detected and not lost during ashing in all experiments. The symbols

in the individual square of each element, OL, C, and BL stand for

orchard leaves, coal, and beef liver, respectively, in which the

element was determined. In addition, some elements reported retained

in the literature determined by neutron activation analysis (4)

,

atomic absorption spectrometry, (5) or spectrophotometry (1) are

included. The numbers refer to references.

H HE

LI BE B

1 NE

NA

c

OL

BLK

cOL

RB~

c

OL

BLcs

cOL

BLFR

MG

cOL

BLCA

cOL

BLSR

c

OL

BA

cOL

BLRA

SC

c

OL

BL

LA

cOL

BLAC

TlcOL

~ZR~ c

OL

HF

c

OL

V

C

NB

TA

C

CR

cOL

BLMO

OL

BLw

C

MN

C

OL

BLTC

RE

FE

c

OL

BLRU

OS

CO

c

OL

BLRH

IR

NlcOL

~P0

PT

CU

5

AG

4

AU

ZN

cOL

BLCD

5

HG

AL

cOL

BL GA

c

IN

TL

5

SI

GE

SN

PB

cOL

AS

C

OL

"SB"

cOL

BL

Bl

SE

cOL

BL TE

PO

CL

BR

AT

AR

KR

XE

RN

CE PR ND PM SM EU .. GD TB DY HO ER TM YB LU

c c OL OL

BL BL

TH

c PA U NP PU AM CM BK CF ES FM MD NO LW

OL

BL

1. J.W. Mair, Jr., and H G. Day, Anal. Chem. 44 (1972) 201 5. Matrix was animal tissue.

4. D.Behne and P A Matamba, Z. Anal. Chem. 274 (1 975) 1 95. Matrix was blood serum.

5. B.B. Stafford, Proceedings 2nd Conf. Trace Subs, in Environmental Health (1 968) Univ. of Mo.

Matrix was atmospheric particulate.

Figure 2. Elements retained during LTA.

Although some of the results have been previously reported, we include

data here on many additional elements, including those particularly

susceptible to loss such as arsenic, selenium, and chromium, since they

are known to form compounds which are volatile at the temperatures of LTA.

39
TABLE 2. PERCENT OF ELEMENT RETAINED (EXPERIMENTAL)

Coal Orchard Leaves Beef Liver

Sodium 98 100 104

Magnesium 98 101 95

Aluminum 105 - 112

Potassium 101 106 - Calcium 99 101 99

Scandium 100 101 - Titanium 105 99 - Vanadium 98 - - Chromium 99 106 94

Manganese 102 102 105

Iron 96 102 99

Cobalt 108 95 106

Nickel 99 99 - Zinc 107 102 100

Gallium 91 - - Arsenic 110 92 - Selenium 99 105 - Rubidium 98 98 92

Strontium 98 106 - Zirconium 104 108 - Molybdenum 93 94 - Antimony 104 108 - Cesium 102 104 - Barium 105 109 - Lanthanum 107 95 95

Cerium 99 98 100

Europium 98 - 94

Hafnium 96 98

Tantalum 109

Tungsten 97

Lead 106 97

Thorium 103 92

It is also significant to note that, equal in importance to loss of

trace elements, there was no pick-up of contamination during the ashing

process as would be evidenced by an increase in the amount of the element

after ashing. This is in spite of the fact that only very ordinary precautions

were taken to guard against contamination during cleaning of the borosilicate

glass ashing tube with ordinary laboratory detergent followed by rinsing

with water, hot dilute hydrochloric acid and finally deionized water and

drying in an oven at 100 °C.

40
An application of immediate interest is those elements which upon

thermal neutron or photon activation yield radioisotopes with half- lives

in the range of 2-30 minutes and are not detectable in the typical biological

sample without separation. Since after ashing, biological samples, especiall

animal tissue, are rapidly dissolved in dilute mineral acid, one may

consider radiochemical separations in the determination of magnesium,

aluminum, titanium, and vanadium in the case of thermal neutron activation

and potassium and iron in photon activation.

ACKNOWLEDGMENTS

The authors thank the operating staffs of the NBS research reactor

and electron LINAC for the fine services provided. This work was supported

by the Environmental Protection Agency.

REFERENCES

1. J.W. Mair, Jr., and H.G. Day, Anal. Chem. 44, (1972) 2015.

2. V. Janzer, Private Communication.

3. C.E. Gleit and W.D. Holland, Anal. Chem. 34, C1962) 1454.

4. D. Behne and P. A. Matamba, Z. Anal. Chem. 274, (1975) 195.

5. B.B. Stafford, Proceedings 2nd Conf. Trace Subs, in Environmental

Health (1968) Univ. of Missouri.

41

APPENDIX C

DETERMINATION OF CHROMIUM IN BIOLOGICAL MATRICES BY NEUTRON

ACTIVATION: APPLICATION TO STANDARD REFERENCE MATERIALS

by

L.T. McClendon

Analytical Chemistry Division

National Bureau of Standards

Washington, D.C. 20234

43
ABSTRACT

Chromium is recognized to be an essential trace element in several

biological systems. It exists in many biological materials in a variety

of chemical forms and very low concentration levels which cause problems

for many analytical techniques. Both instrumental and destructive neutron

activation analysis were used to determine the chromium concentrations in

Orchard Leaves, SRM 1571, Brewers Yeast, SRM 1569, and Bovine Liver, SRM

1577. Some of the problems inherent with determining chromium in certain

biological matrices and the data obtained here at the National Bureau of

Standards using this technique are discussed.

44
INTRODUCTION

Chromium has been recognized as an essential trace element (1) in

human nutrition for several years. At high concentrations or in different

chemical states it can also cause deleterious or toxic effects. There is a

need for reliable methods of analysis for chromium at trace levels in a

variety of matrices, especially biological matrices. A laboratory intercomparison on environmental materials (2) pointed up the sad state of

chromium determinations as performed by most of the analytical community.

Also, the author has observed a wide range of results reported for chromium

in the National Bureau of Standards (NBS) Bovine Liver Standard Reference

Material (SRM 1577) . As a result of these observations and communication

with several analytical laboratories involved in the determination of

chromium in biological matrices, persons at NBS became convinced that more

biological materials needed to be analyzed and certified for chromium

content, in addition to other elements.

Neutron activation analysis, both instrumental (INAA) and destructive

(DNAA) , has been used to determine the chromium concentration in a variety

of matrices. The results obtained for chromium in three biological Standard

Reference Materials (SRM' s) --Orchard Leaves, Bovine Liver, and Brewers

Yeast--are described in this paper. The chromium content has been certified

by NBS in two of these SRM's, Orchard Leaves and Brewers Yeast.

EXPERIMENTAL

Samples Analyzed

Two of the biological materials analyzed were NBS Standard Reference

Materials Orchard Leaves (SRM 1571) and Bovine Liver (SRM 1577) . The

third material, Brewers Yeast, was furnished to NBS by the Nutrition

Institute, U.S. Department of Agriculture, Beltsville, Maryland, for

preparation, certification, and issuance as a Standard Reference Material.

This material is certified for total chromium concentration only and is

being prepared for sale by the NBS Office of Standard Reference Materials

as SRM 1569.

Preparation of Standards and Carrier

Two standards were used in this work. One standard was prepared by

dissolving a weighed amount of chromium metal (99.99% pure) in high purity

HC1 and diluting to a specific volume with high purity H20 to obtain the

desired Cr concentration. The second standard was prepared by dissolving

45
a weighed amount of NBS-SRM 136C (l^C^Oy) in high purity H2O and diluting

to volume to obtain the desired Cr concentration. Chromium carrier solutions

were prepared from analytical reagent grade CrCl3 and l^C^Oy and normally

contained 3 mg Cr/mL.

All reagents used in the analysis were of analytical grade, unless

stated otherwise.

Irradiation Conditions

Chromium was determined in the three biological materials using both

INAA and DNAA. The samples (200-350 mg) along with Cr standards were

encapsulated in clean quartz vials and irradiated for periods of one to

six hours in the NBS Reactor at a thermal neutron flux of lxl0 13 n cm 2

* sec" 1

. The samples were allowed to decay 3-6 weeks for INAA work and ^36 hours

for DNAA work to reduce the matrix activity before processing.

Procedure for INAA

Following a three-week decay the sample vials were washed clean of

exterior contamination with 1:1 HNO3 and H2O', frozen in liquid nitrogen

opened, and the material was transferred to clean polyethylene counting

vials. The amount of sample transferred was determined by weight. The

samples and standards were counted on a 75 cm 3 Ge(Li) detector coupled to

a 4096 channel pulse height analyzer for measurement of 51 Cr produced by

the 50Cr(n,y) Cr reaction. The concentration of chromium was determined

by the direct-comparator method.

Several brewers yeast samples and standards were preweighed into

clean quartz vials, sealed, and irradiated as already described. After

allowing these samples to decay for ^6 weeks, the vials were washed clean

of exterior contamination and counted (material still contained in vial)

as already described.

Procedure for DNAA

Following a 36-hour decay, the sample and standard vials were cleaned,

frozen in liquid N 2 , opened, and transferred (by weight) to a 50 mL Erlenmeyer

flask designed for dissolving materials in a closed system (3) and trapping

volatile material in an external solution. Chromium carried (y5 mg of both

Cr III and Cr VI) and 5-10 mL of concentrated HC104 -HN0 3 mixture (1:3)

were added to the flask. The flask was heated on a Pyrex top hot plate

until all the sample had dissolved and all the vapor fumes visibly trapped

in a 1:1 HN0 3 solution. The flask was removed from the heat and 5 mL of

hot Ce(S0tt ) 2 -3M ti 2 S0k solution (10% w/v) was added; the flask was heated

an additional 5-10 minutes to assure oxidation of and maintain the chromium

as Cr VI. After cooling, the sample solution in the flask was transferred

to a 50-mL extraction tube with 15 mL of 1.5 M HC1. The sample was then

extracted with 10 mL of 1 percent (w/v) tribenzylamine-chloroform solution.

A 5-mL aliquot was taken from the organic phase and counted on a 25 cc

Ge(Li) detector coupled to a HP 4096 computer-analyzer. The trap solution

46
from the dissolution step was transferred to a suitable counting vessel

and counted on this system also. The 320 keV gamma ray peak of 51 Cr was

integrated and the chromium concentration was determined by the directcomparator method using the two chromium standards subjected to the same

procedure. All of the chromium from these standards remained in the

flask. The trap solution was checked for chromium but none was detected.

The procedure is the same for open-flask dissolution of samples except a

regular Erlenmeyer flask is used, eliminating the trap solution.

Results and Discussion

Recent interlaboratory comparison studies (4,5) of analytical methods

and their results for the analysis of chromium in biological and environmental

materials showed very large differences in analytical values. During the

spring of 1974, a workshop on chromium analysis was held at the University

of Missouri in Columbia where these differences were discussed and recommendations made to resolve them. There was consensus among the participants

that biological standard reference materials, certified for total chromium

content by NBS, would provide a starting point for the analytical community

to critically evaluate their methods.

Chromium has been determined and certified in several materials by

NBS. However, this manuscript describes the results of chromium analyses

in three biological materials—Orchard Leaves, Bovine Liver, Brewers

Yeast--using neutron activation analysis (NAA) . Studies here in our

laboratory and other laboratories (6,7) on some biological materials had

suggested possible losses of chromium content during sample manipulation

(e.g., dissolution, charring, ashing, etc.). Instrumental neutron activation

analysis (INAA) and destructive neutron activation analysis (DNAA) was

used to determine the chromium concentration in these biological materials.

The use of INAA to determine the total chromium concentration in these

biologicals provided an M absolute" check for the values obtained using

DNAA and also other techniques which required sample treatment, the

results obtained for chromium in the three biologicals mentioned above,

using NAA and the NBS Reactor, are given in Tables 1-4.

By employing two dissolution techniques —closed system and open

flask— for the irradiated biologicals, the loss of chromium in the dissolution

step could be evaluated. The results obtained for chromium in Orchard

Leaves (Table 1) were essentially the same using INAA and the two dissolution

techniques, indicating no chromium is lost from this material in the

dissolution procedures used. The slightly higher INAA result for chromium

is expected. A large amount of matrix radioactivity is produced when

these biological materials are irradiated. Much of this activity results

in long-lived beta radiation creating a bremsst rah lung effect in the gamma

spectra when the sample is counted. This effect is very dominant in low

energy range of the spectra where the chromium photopeak is located so

that large background corrections are necessary. Radiochemical separation

of Cr (DNAA) eliminates the background interference. The results obtained

from open-flask dissolution (DNAA) of yeast (Table 2) were consistenlty

lower (^25%) than those obtained instrumental ly (INAA). The results

47
obtained from dissolution of brewers yeast in a closed system as described

in the DNAA procedure are in good agreement with the INAA results. The

same phenomenon existed in the determination of chromium in bovine liver

(Table 3).

The results in Tables 1-3 show chromium can be lost during sample

dissolution of two biologicals, brewers yeast and bovine liver. However,

no loss of chromium was observed in our experimental design for orchard

leaves. The chromium loss observed in brewers yeast and bovine liver is

probably an organic component, since there was apparently no exchange with

the inorganic chromium added to the sample before dissolution. Further

studies to characterize this component are underway in this laboratory.

Brewers yeast was preweighed, packaged, irradiated, and counted in

the same container in order to establish that chromium was not lost in the

irradiation step. The results in Table 4 show the amount of chromium

obtained using a polyethylene irradiation container (INAA-P.E.) are in

good agreement with those obtained from a quartz irradiation container

(INAA- Quartz)

.

The radiochemical procedure described for chromium (DNAA) provides

the analyst with a simple, rapid, and selective technique for chromium

determination in a variety of matrices. The procedure is also adaptable

for use by other techniques. Results of chromium analysis (from a variety

of techniques) reported in the literature in recent years show very large

differences in concentration on the same materials. Thus, it is hoped

these two biological materials, certified for total chromium concentration,

will help those involved in chromium analysis to evaluate their methods

and improve chromium results reported in the literature and elsewhere for

analytical quality control samples.

48
REFERENCES

1. W. Mertz, Physiol. Rev., 49_ (1969) 163.

2. L. McClendon, Trace Subst. Environ. Health, £ (1974) 255.

3. D.A. Becker and G.W. Smith, Mod. Trends in Activ. Anal. Conf . , College

Station, Texas (1965) 230.

4. J. Pierce, et al, Symp. on the Development of Nucl. Based Tech. for

Meas., Detection and Control of Environ. Pollutants, Vienna, Mar.

1976.

5. R. Parr, Mod. Trends in Activ. Anal. Conf., Munich (1976) 1414.

6. V. Maxim, et al, symp. Nucl. Activation Tech. in the Life Sciences,

Bled, Yugoslavia, April, 1972.

7. W. Wolf, Interface, 2 (1973) 31.

49
TABLE 1. CONCENTRATION OF CHROMIUM IN ORCHARD LEAVES,

SRM 1571, CPg Cr/gram)

Spl # INAA DNAA

A

DNAA

1 2.590 2.514 2.472

2 2.574 2.489 2.447

3 2.563 2.499 2.484

4 2.567 2.495 2.433

5 2.569 2.501 2.463

6 2.582 2.472 2.481

X 2.574 ± 0.010 2.495 ± 0.014 2.463

B

* NBS Certified Value: 2.6 ± 0.2 yg/g

Note: A - Closed system dissolution.

B - Open flask dissolution.

* Based on results of two independent analytical methods

This work and isotope dilution mass spectrometry.

50
TABLE 2. CONCENTRATION OF CHROMIUM IN BREWERS YEAST,

SRM 1569 (ug Cr/gram)

Spl # INAA DNAA

A

DNAA

B

1 2.079 2.070 1.583

2 2. 075 2.068 1.543

3 2.090 2.062 1.548

4 2.077 2.068 1.588

5 2.067 2.088 1.551

6 2.104 2.090 1.565

X 2.082 ± 0.013 2.074 ± 0.012 1.558 ± 0.015

NBS Certified value: 2.12 ± 0.05 yg/g — Based on results of two

independent analytical methods —This work and

isotope dilution mass spectrometry.

Note: A - Closed system dissolution.

B - Open flask dissolution.

51
TABLE 3. CONCENTRATION OF CHROMIUM IN BOVINE LIVER

SRM 1577 (yig Cr/gram)

Spl # INAA DNAA

A

DNAA

B

1 0.228 0.212 0.157

2 0.170 0.209 0.166

3 0.229 0.210 0.153

4 0.242 0.207 0.156

5 0.177 0.214 0.166

6 0.213 0.209 0.160

X 0,210 ± 0.030 0.210 ± 0.002 0.160 ± 0.005

Note: A - Closed system dissolution.

B - Open flask dissolution.

52
TABLE 4. CONCENTRATION OF CHROMIUM IN BREWERS YEAST,

SRM 1569, Using INAA (jig Cr/gram)

Spl # INAA - P.E. INAA - Quartz

1 2.079 2.131

2 2.105 2.087

3 2.117 2.137

4 2.081 2.092

5 2.120 2.149

6 2.075 2.120

X 2.096 ± 0.020 2.119 ± 0.025

53
NBS-114A (REV. 1 1-77)

U.S. DEPT. OF COMM. 1. PUBLICATION OR REPORT NO. 2. Gov't Accession

BIBLIOGRAPHIC DATA No. SHEET NBS SP-501

3. Recipient's Accession No.

4. TITLE AND SUBTITLE

The National Environmental Specimen Bank

Proceedings of the Joint EPA/NBS Workshop on Recommendations

and Conclusions on the National Environmental Specimen Bank Held

at the NBS, Gaithersburg, MD. , August 19- 20, 1976

5. Publication Date

February 1978

6. Performing Organization Code

7. AUTHOR(S)

Harry L. Rook and George M. Goldstein, Editors

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

iu. project/ l ask/ work Unit No.

11. Contract/Grant No.

12. Sponsoring Organization Name and Complete Address (Street, City, State, ZIP)

National Bureau of Standards and

U.S. Environmental Protection Agency

13. Type of Report & Period

Covered

14. sponsoring Agency Code

15. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number: 78-682

16. ABSTRACT (A 200-word or less (actual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.)

On August 19 and 20th, 1976, the National Bureau of Standards and the

U.S. Environmental Protection Agency co-sponsored a Workshop to review technical developments and to make recommendations on implementation of the National Environmental Specimen Bank. The Workshop consisted of a review

session where past considerations were discussed; a technical session where

recent analytical research relevant to the sample bank was abstracted and discussed; and a planning session where planning and design of a prototype

banking system was outlined.

This report is a summary of the presentations, discussion, and con- clusions of the Workshop attendees. The attendees represented a wide cross section of interested Federal and Non-Federal research groups as well as International representation including the International Tissue Banking

Program (Sponsored by the World Health Organization, The Commission of European Communities and the U.S. Environmental Protection Agency) and the Federal Republic of Germany.

The workshop concluded that with the ever increasing influx of new man-made substances into our ecosystem, that a formalized, systematic

approach is needed to assess the environmental impact of these substances on

a national as well as an international level. The technology to initiate

a pilot banking program is presently available and was formulated into a five-year pilot bank program. This program will be evaluated at each stage of development.

This report was submitted in partial fulfillment of EPA Interagency

Agreement IAG-D4-0568 by the National Bureau of Standards under the partial

sponsorship of the U.S. Environmental Protection Agency. This report covers

the period August 19 and 20, 1976, and the work was completed as of

January 31, 1977.

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name; separated by semicolons)

Analytical; National Environmental Specimen Bank; Prototype; Research;

Sample Bank.

18. AVAILABILITY ? Unlimited

1 ! For Official Distribution. Do Not Release to NTIS

I X| Order From Sup. of Doc, U.S. Government Printing Office

Washineton. D.C. 20402. SD Stock No. SN003-003-

| | Order From National Technical Information Service (NTIS)

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19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

21. NO. OF PAGES,

59

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(22. Price $2. 30

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