United States Health Effects Research EPA-600/1 -79-01

Environmental Protection Laboratory May 1979

Agency Research Triangle Park NC 27711

Research and Development

The National

^

Environmental

Specimen Bank

Research Program

for Sampling,

Storage, and

Analysis
RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental

Protection Agency, have been grouped into nine series. These nine broad cate- gories were established to facilitate further development and application of en- vironmental technology. Elimination of traditional grouping was consciously

planned to foster technology transfer and a maximum interface in related fields. The nine series are:

1. Environmental Health Effects Research

2. Environmental Protection Technology

3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

6. Scientific and Technical Assessment Reports (STAR)

7. Interagency Energy-Environment Research and Development

8 “Special” Reports

9 Miscellaneous Reports

This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RESEARCH series. This series describes projects and studies relating to the toler- ances of man for unhealthful substances or conditions. This work is generally

assessed from a medical viewpoint, including physiological or psychological

studies. In addition to toxicology and other medical specialities, study areas in- clude biomedical instrumentation and health research techniques utilizing animals — but always with intended application to human health measures.

This document is available to the pubfic through the National Technical Information Service, Springfield, Virginia 22161.
EPA-600/1 -79-017

May 1979

THE NATIONAL ENVIRONMENTAL SPECIMEN BANK RESEARCH PROGRAM

by

T. E. Gills, H. L. Rook, R. A. Durst, Editors

Center for Analytical Chemistry

National Bureau of Standards

Washington, D. C. 20234

Contract No. EPA-78-D-X0105-1

Project Officer

G. M. Goldstein

Clinical Studies Division

Health Effects Research Laboratory

Research Triangle Park, North Carolina 27711

HEALTH EFFECTS RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U. S. ENVIRONMENTAL PROTECTION AGENCY

RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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.
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.

This report documents one aspect of an International effort, supported

by EPA, to provide a comprehensive environmental monitoring program to assess the relative risk of environmental hazard to the health and well-being

of our population and to aid in the improvement of our environmental quality.

This program, the National Environmental Specimen Bank, will serve as an environmental warning system by providing real time chemical analysis of

collected specimens. In addition, this system would permit the use of

tomorrow's more sensitive and more specific methods of chemical analysis on stored samples. The advantages of such a program will permit us to assess

the effectiveness of our 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.

F. Gordon Hueter, Ph.D.

Director

Health Effects Research Laboratory

iii
ABSTRACT

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

state-of-the-art protocols for sampling, storage, and analysis of biological

and environmental- type matrices. This report is a compilation of research

papers and/or efforts describing developed or adopted procedures for retrospective analysis of biological and environmental samples. Preliminary protocols

for sampling, sample handling, and sample storage are given for human liver

autopsy tissue in addition to methods for the accurate measurement of selected

toxic elements in biological and environmental materials. Analytical methods

employed were neutron activation analysis (NAA) , polarography , and isotope

dilution spark source mass spectrometry (IDSSMS)

.

This report is submitted in partial fulfillment of EPA Interagency agreement EPA-78-D-X0105 by the National Bureau of Standards for the 1978 contract

year

.

iv
CONTENTS

Page

Disclaimer ii

Forward iii

Abstract iv

List of Figures vi

List of Tables vii

Acknowledgments viii

Section 1 - Development of a Preliminary Protocol for Sampling,

Sample Handling, and Long-Term Storage of Human

Liver 1

Section 2 - Research on Freezer Storage ............... 14

Section 3 - Container Materials for the Preservation of Trace

Substances in Environmental Materials 16

Section 4 - Design and Construction of the Pilot Bank Facility . . 22

Section 5 - Organic Mercury in Tissues 25

Section 6 - The Determination of Trace Elements in New Food Grain

SRM's Using Neutron Activation Analysis 27

Section 7 - Cadmium Analysis by Radiochemical Neutron Activation

Analysis 32

Appendices

I. Simultaneous Determination of Arsenic, Antimony, Cadmium,

Chromium, Copper, and Selenium in Environmental Material by

Radiochemical Neutron Activation Analysis 37

II. The Quantitative Determination of Volatile Trace Elements in

NBS Biological Standard Reference Material 1569, Brewers

Yeast 40

III. Chemical Preparation of Biological Materials for Accurate

Chromium Determination by Isotope Dilution Mass Spectrometry . 50

IV. The Determination of Zinc, Cadmium and Lead in Biological

and Environmental Materials by Isotope Dilution Mass

Spectrometry ..... 54

v
List of Figures

Section 4:

Figure 1. Specimen Bank Laboratory/Storage Facility
List of Tables

Page

Section 2:

Table 1. Analyses of a Stored Bovine Liver, Storage and

Analysis Protocol Tests . . 15

Section 3:

Table 1. Annual Rate of Water Loss from Plastic Containers .... 17

Table 2. Trace Elements in Plastics Determined by Neutron

Activation Analysis 18

? Table 3. Impurities Leached from Plastic Containers (ng/cm“) . ... 19

Section 6:

Table 1. Irradiation Conditions and Data Used for Instrumental

Analysis 28

Table 2. Irradiation Conditions and Data Used for Radiochemical

Activation Analysis . 29

Table 3. The Determination of Trace Metals in Rice and Wheat

Flour in pg/gm 30

Section 7

:

Table 1. Cadmium in Various SRMs 34

Table 2. Copper in Various SRMs 35

Table 3. Sub-Bituminous Coal SRM 1635 35

Table 4. Bovine Liver SRM 1577 36

Table 5. Orchard Leaves SRM 1571 36

vii
ACKNOWLEDGMENTS

The authors of this report wish to acknowledge their appreciation to the

Environmental Protection Agency for its funding. We especially want to thank

the Project Officer, Dr. George M. Goldstein, Chief of the Clinical Pathology

Branch for his consultations and program guidance.

viii
SECTION 1

DEVELOPMENT OF A PRELIMINARY PROTOCOL FOR SAMPLING,

SAMPLE HANDLING, AND LONG-TERM STORAGE OF HUMAN LIVER

by

E. June Maienthal

INTRODUCTION

In response to the increasing concern with environmental pollution, the

National Bureau of Standards (NBS) in collaboration with the Environmental

Protection Agency (EPA) has developed a preliminary protocol for sampling,

sample handling and long-term storage of human livers, which are to be monitored for trace element and trace organic compositions for the Pilot National

Environmental Specimen Bank which will be located at NBS. The criteria for

arriving at these guidelines will be discussed, and the preliminary guidelines

up to the stage of subsampling will be given.

Considerations

From an earlier literature survey (1) , it was obvious that much of the

work concerning environmental sampling, sample handling, and long-term storage

is of questionable value owing partially to sources of contamination from

various steps in the sampling and sample processing or to losses of some of

the trace constituents through adsorption, volatility, etc. It was also

obvious from a survey of existing environmental collections that few of the

collections surveyed would be of sufficient value for environmental trace

inorganic and organic determinations (2) . It was necessary, therefore,

prior to the establishment of the Pilot National Environmental Specimen

Bank, to set up a valid working protocol.

The rationale and plans for the National Environmental Specimen Bank

(NESB) have been described by Goldstein, where he states "... regardless of

the outcome of the NESB, the methodology protocol development would provide

the scientific community with state-of-the-art standardized protocols for sam- ple collections in a variety of ecologically important materials. The cost

benefit of this alone is incalculable." (3).

The protocol to be described was arrived at as the result of work con- ducted at NBS, a past literature survey (1), and additional literature investigated and consultations with other workers in the field, including chemists,

biochemists, pathologists, cryobiologists , physicians, and equipment suppliers

The necessity of obtaining and storing a sample valid for both trace organic

and inorganic constituents requires very rigid control of the sampling materials and methods, shipping methods, storage containers and storage conditions.

1
Although shipping and storage are the last items in the protocol, they

will be discussed first in order to explain the reasons for certain other steps

of the procedure. It was generally concluded that it would be necessary to

freeze the specimen as soon as possible after sampling for long-term banking.

Frozen storage is usually done at about -20 °C (approximate home freezer temperature) , -80 °C (approximate dry ice temperature) , or at -196 °C (liquid

nitrogen temperature). At -20 °C, however, samples often may not be frozen,

and at -80 °C, molecular or enzymatic activity may still occur (4,5). Although this might not affect the stability of the inorganic constituents, some speciation and organic changes could occur. Some chemical changes are reported to occur as low as -130 °C (5) , and some physical changes such as devitrification are reported to take place at temperatures substantially lower

than -130 °C (6) . Meryman had earlier reported that no biochemical activity

should take place below -130 °C (7), but states that on the basis of Dowell

and Renfret's work (6), further study of this activity at low temperatures

should be investigated (7a). Mazur has reported that at -196 °C, no aqueous

reaction occurs and that a tissue sample should be preserved, unchanged

indefinitely at that temperature (8) . Cravalho also states that to arrest

biochemical and physical processes, the lower the storage temperature the

better, and that there is no lower temperature limit for storage of biomaterials (8a) . It would appear therefore, that liquid nitrogen would be an ideal

storage as well as shipping medium, having the additional advantage of being

relatively inexpensive and readily available. Laessig, et al. describe some

of the earlier uses of liquid nitrogen for storing and shipping biological

samples in tanks by air freight with no difficulty (9) . These tanks were used

by Laessig, et al. for epidemiological serum studies in remote field areas and were found adaptable under the most severe circumstances. The tanks are commercially available from several manufacturers and are being used routinely

for tissue shipping and storage by groups such as the Tissue Bank of the Naval

Medical Research Institute. The use of dry ice has not been as satisfactory

for shipping, in part because of frequent failures and thawing of samples, and

also because of possible pH changes of the sample owing to diffusion of carbon

dioxide into the container (4). Because of cell rupture and tissue leakage

during thawing, a sample cannot be salvaged for many analytical purposes by

refreezing once it has been thawed. Omang and Vellar have described the

concentration gradients occurring in biological samples during freezing and

thawing (10)

.

The choice of donor specimen must also be under some restrictions. For

the pilot study and to check the preliminary protocol, normal livers which show

no erratic concentrations of the constituents to be determined are desired.

For this reason, certain donors are to be excluded, such as those: having

liver weights less than about 1000 grams; stored at temperatures above 4 °C;

previously frozen; deceased more than 24 hours; a history of alcoholism, gross

sepsis, viral hepatitis, tuberculosis, cirrhosis, liver carcinoma, chronic

circulatory failure or congestion, chemical or drug overdose or exposure; or previously embalmed

.

Ideally the sample should be taken immediately after donor death since

significant enzymatic changes occur with time (11) , but at present this would

not be generally possible; so on a practical basis an upper limit of 24 hours

has been set.

2
Ideally, the autopsy should be performed in a laminar-flow clean room

atmosphere, because the chances of contamination from the typical autopsy room environment are very great. Again, this is not at present a practicality;

hence, as few manipulations as possible are to be done prior to sealing the

sample in the cleaned container.

Most autopsies must be performed in a sterile atmosphere requiring the use of sterile surgeon’s gloves which are typically latex, powdered both

inside and out. This type of glove would be prone to introduce trace element

contaminations, such as zinc from the latex itself, and other trace elements

from the powder. A producer of sterile, clean-room packaged, talc-dust free,

polyvinyl chloride gloves has been located, and these gloves have been found

acceptable for use at the autopsy facilities so far contacted. It must be

remembered, however, that there may be a chance of trace organic contaminations of the liver surface from the plastic or plasticizers used in the PVC,

as will be discussed later. This organic contamination could occur with latex

gloves also. For the first samples taken, relatively standard surgical instruments are to be used — unrusted, cleaned steel for the liver removal, and an unrusted, cleaned, hardened carbon steel knife for slicing the left lobe into

two equal sections. It must be remembered that possible surface contamination

from the knives may occur, as discussed previously (1), particularly as shown

by Versieck, Speecke and coworkers (12,13), and by Maletskos, et al. (13a).

A number of other types of less-contaminating implement materials are being

considered and will be discussed later.

The liver section should be rinsed with pure distilled water to remove

gross external contamination, put into its container, sealed, and immediately

frozen in liquid nitrogen.

One problem arising from freezing a section of liver complete with its

blood and extracellular fluid is that the amount of fluid contained can vary

greatly in a normal human being, depending on time and a variety of physiological factors, thus causing an apparent difference in compositions from

sample to sample because of varying amounts of fluid present at time of death.

The fluid can differ markedly in composition from that of the tissue. This

has been shown by LeBaron and coworkers by analysis of human kidney tissues

and fluids (13b) . Iyengar and Kasperek have used a very promising approach

which will be investigated here and possibly incorporated into the protocol

(13c). They rinse the liver sections three times, squeezing the sections

gently between clean polyethylene sheets (Teflon would probably be used here)

in between rinsing processes, thereby eliminating most of the blood. The sections were then frozen in liquid nitrogen and gently pressed between two

Perspex plates. This results in the fracture into small pieces which could be

picked out with plastic forceps, separating the remaining blood vessels and

homogenizing the rest by the brittle fracture technique using a special Teflon

vessel and a Teflon ball with a metal core (14).

A very important item of concern is the choice of container and this is

discussed in more detail in another section of this report. This also was discussed at length previously (1) and in the Proceedings of the 7th Materials

Research Symposium (14a) . It would be desirable to have one type of sample

container suitable for both organic and inorganic constituents. Although some plastics can be cleaned sufficiently for trace element analysis (1,15), the

contamination for organic constituents and migration of plasticizers has been

3
often shown (1,16,17,18). Also, most of the plastics will not withstand

liquid nitrogen temperatures without breaking. One lot of polypropylene cryogenic tubes also showed the presence of sodium, aluminum, chloride, and titanium (18a). Properly cleaned Pyrex containers are suitable for organic con- stituents, but are notoriously bad for trace elements through contamination

and/or adsorption (1,19). The latter reference also found significant zinc

contamination from polypropylene containers (19)

.

The polyf luorinated hydrocarbons such as Teflon, FEP or PFA, after appropriate cleaning appear to be the most suitable for both organic and inorganic

constituents. They will also withstand liquid nitrogen temperatures if not

subjected to too severe physical stresses.

A preliminary experiment was done at NBS freezing pork liver in a heatsealed 5-mil Teflon FEP bag in liquid nitrogen with promising results (19a).

The bags are evacuated immediately prior to sealing to increase the sample

stability (20,21). The packaged sample should be put in a forming cylinder

prior to freezing in liquid nitrogen in order to obtain a more compact, easily

handled package for shipping and storing.

The liver sections are weighed separately in order that weight loss or gain can be checked after they are received at NBS and periodically during

storage. One problem with Teflon is that it is porous to gases, and the

samples may be subject to moisture loss or gain. If this should prove to be a problem, a vapor barrier such as aluminized Mylar or glass may be required in

the protocol.

The liquid nitrogen shipping containers can also be used as storage con- tainers prior to shipping the livers to NBS. These containers have an absorbent around the inner walls, so that during shipping only vapor-phase nitrogen

need be present. The type which will probably be used for the first experiments has a static holding time of 11 days after the absorbent is saturated.

The two halves of the left liver lobe are to be sent back to NBS in

order that different subsampling and storage techniques may be investigated.

One section will be stored untouched in the event that the subsampling procedures (as yet unfinalized) should in some way alter or contaminate the other

section. It must also be emphasized that the sampling protocol is preliminary

and will necessarily change as unforeseen problems are encountered and

improved methods arise. It is now thought that the subsampled section will be

stored in approximately 8 mL PFA bottles (which may also require an outside

vapor barrier) . It is possible that glass might be used for the samples for

the organic determinations.

A major problem remaining is how to subsample and homogenize the liver

section without contaminating it. Various methods have been suggested, including Waring blenders, meat grinders, ultrasonic tissue homogenizers , food

processors, cryogenic grinding and brittle fracture. Some preliminary studies

have been made at NBS on subsampling, homogenization, analysis by neutron

activation, freezing, storing, and reanalysis of beef liver (21a, 21b). A

number of types of sampling tools have been used which seem to offer much less

chance of contamination than the steel knives mentioned earlier. Iyengar and

Kasperek used a specially prepared Suprasil-quality-quartz knife for slicing

4
the liver, and used the brittle fracture technique, as mentioned earlier, at

liquid nitrogen temperatures utilizing a specially prepared Teflon container

and Teflon ball with a metal core (13c). Lievens, et al. also used an ultraclean spec-pure quartz tube to obtain liver subsamples (22) . Thompson and

Bankston studied contamination arising from grinding and sieving devices made

from different types of materials (23). They found that a boron carbide

mortar introduced little or no contamination (except for boron) and that

stainless steel or brass sieves introduced appreciable levels of cobalt,

copper, iron, manganese, nickel, lead, tin, and zinc. Maletskos and coworkers

used a stainless steel knife to cut a frozen segment of tissue, then a special

high purity boron nitride knife to shave the surfaces which had been in contact

with the stainless steel (13a) . Folsom developed several different types of

plastic sampling tools for frozen tissue sampling (24) . These, however, might

not be suitable for trace organic samples. Another interesting sampling

possibility involves the use of the laser. Hislop and Parker used a carbon

dioxide laser for cutting bone with no trace element contamination (25)

.

Auth, Doty, and coworkers have developed a "laser blade" or "laser scalpel"

using high power argon laser radiation transported via a low-loss optical

fiber into a sharp transparent quartz knife (26) . This device has proved very

useful for operations in which massive bleeding is a problem. This device has

the effect of cauterizing the incision and sealing the blood vessels. Of course for the trace constituent analysis the several millimeters of cauterized tissue would need to be removed by an implement which would be noncontaminating for the constituents of interest for the particular determination.

A number of additional references concerned with problems involved in

trace analysis of tissue or biological matrices are listed (27-44) .

It must be emphasized again that the attached protocol is preliminary and

will change as improved methods are developed.

5
LIVER SAMPLING PROTOCOL

I. DEMOGRAPHIC DATA

a. Record autopsy identification number and NBS number on label provided and attach to sample bag (Note 1).

b. Collect and include donor information with shipment and send a copy under separate cover. Donor information should contain as a minimum: the autopsy identification number; the NBS number; date and

time of death; date and time of autopsy; weight of whole liver and

separate left lobe sections; donor height; weight; age; sex; and

ethnic group (Note 2)

.

II. SAMPLE HANDLING

a. Sterile, cleaned PVC gloves will be provided and should be used for

liver removal and handling. Extreme precaution must be taken

throughout the autopsy procedure to reduce the risk of contaminating

the liver sample. Contamination may result during the autopsy from

the donor, the individual (s) performing the autopsy, the atmosphere

and/or the surgical instruments.

III. SAMPLING

a. The liver is identified and excised with instruments supplied by NBS.

The liver should be removed as early as is feasible after death (but

not later than 24 hours) and as close to the beginning of the autopsy

as normal routine permits (Note 3)

.

The liver will not at any time during the autopsy procedure be placed

on any surface other than cleaned Teflon sheets provided by NBS.

b. The liver is placed on an inclined surface (to provide rapid drainage) covered with a Teflon sheet. Hashing is accomplished by pouring

approximately 250 mL of distilled water (Note 4) over the liver surface. The liver is then turned over and the washing procedure

repeated

.

c. Excess water is allowed to drain off the liver prior to weighing on another Teflon sheet.

d. Left lobe is dissected with the knife provided by NBS.

e. Left lobe is cut into equal halves through an axis of symmetry with

the knife provided.

6
f. A section is taken from a cut surface of the left lobe (with the

knife supplied) for the preparation of a histological slide. A

slide and copy of the pathological findings is sent under separate

cover as per lb above.

g. Each liver section is immediately placed in separate Teflon bags

provided by NBS and weighed separately on Teflon sheet as in IIIc.

h. Double heat seal each bag (with the twin heat sealer supplied by

NBS) to within about 1 inch of one side. Insert a precleaned

Teflon tube, attached to a hand vacuum pump (both supplied by NBS)

,

into the unsealed corner of the bag. Evacuate each bag and double

heat seal across the remaining open corner while maintaining the vacuum.

i. The sealed Teflon bags are then placed in the Teflon cylinders, provided by NBS, and frozen by immersion for at least 15 minutes in

liquid nitrogen contained in a plastic Dewar supplied by NBS

(Note 5)

.

j . Remove the Teflon bag from the cylinder and inspect the bag for

obvious damage. If loss of sample integrity is encountered, both

liver sections should be discarded.

k. Prefill the shipping container with liquid nitrogen so that there

is at least 4 inches of standing liquid. This may be estimated by

using a dipstick. The liquid nitrogen level must be maintained

(Note 6)

.

l. ( Transfer the Teflon-packaged frozen liver sections to the shipping

container. Check and maintain the liquid nitrogen level as directed

in Illk above until ready for shipment.

m. Store liver specimens no longer than 10 days or until 4 lobes

(8 sections) have been collected.

n. Draw off excess liquid nitrogen, cap securely, and ship to the

National Bureau of Standards by a carrier (to be designated later)

.

Include with the shipment a copy of the information listed in la and

lb, as well as the Restricted Article Statement required by the CAB.

Copies of information from la and lb, along with the histological

slide should also be sent under separate cover (mentioned in lb,

Illf above) (Note 7)

.

7
NOTES FOR PROTOCOL

1. Polyester tape, labels, and dispenser are to be supplied by NBS . Labels

are to be affixed to the sample bag by wrapping tape completely around

bag and overlapping ends one-half inch.

2. At a later date in this protocol, additional donor information will be

sought for a specified number of samples. This will include: smoking

and drinking habits, socioeconomic status, residential environment, and

employment history.

3. Liver samples are to be excluded if donors have: a total liver weight of

less than 1000 grams (35 oz) ; livers which have been ruptured or punctured; been stored at temperatures above 4 °C, or have been previously

frozen; had a history of alcoholism, drug addiction, or long-term drug

therapy; had gross sepsis, viral hepatitis, tuberculosis, cirrhosis,

liver carcinoma; had a history of chronic circulatory failure or congestion; died due to chemical overdose or exposure; or embalmed donors.

4. Water used in washing the liver will be provided by NBS or obtained from

a local source that has been previously analyzed by NBS and shown to be

of acceptable quality.

5. Precautions should be observed when working with liquid nitrogen. Liquid

nitrogen should not be stored in sealed containers. When transporting by

elevator, personnel should not accompany Dewar because of the possibility

of elevator and/or Dewar failure. Personnel handling liquid nitrogen are cautioned to wear boots, cuff less trousers, non-absorbent apron, loose,

insulating gloves and face shields.

6. The cryogenic shipping container weighs 27 kg (59 lbs) empty. With the

absorbent filled with liquid nitrogen, the weight increases to 38 kg (84

lbs) . The container requires approximately 14 liters to fill the absorbent. The static evaporation rate is approximately 1.2 liters/day.

7. A Government Bill of Lading (GBL) will be supplied to cover shipping

expenses. Forms for the Restricted Article Statements will be provided by

NBS. These forms must be included with each shipment.

8
REFERENCES

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

Materials. NBS Tech. Note 929, Oct. 1976.

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

Protection Agency, 1977.

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

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

4. Strong, D. M. Tissue Bank Division, Naval Medical Research Institute,

Bethesda, MD 20014. Private communication.

5. Peterson, Jr,, W, D. , and C. S. Stulberg. Freeze Preservation of

Cultured Animal Cells. Cryobiol. , 1:80, 1964.

6. Dowell, L. G. , and A. P. Renfret. Low Temperature Forms of Ice as Studied by X-Ray Diffraction. Nature, 188:1144, 1960.

7. Meryman, H. T. Mechanics of Freezing in Living Cells and Tissues.

Science, 124:515, 1956.

7a. Meryman, H. T. Review of Biological Freezing, In: Cryobiology,

H. T. Meryman, ed. Academic Press, London and New York, 1966. pp . 72.

8. Mazur, P. Preservation of Embryos and Organs. Talk presented at the 2nd

Annual Meeting of the American Association of Tissue Banks, May 15-18,

1978, Boston, Mass.

8a. Cravalho, E. G. The Cryopreservation of Living Cells. Technology Review,

Oct. /Nov., 1975. pp . 31.

9. Laessig, R. H. , F. P. Pauls, T. A. Schwartz. Long-Term Preservation of

Serum Specimens Collected in the Field for Epidemiological Studies of

Biochemical Parameters. Health Lab. Sci., 9:16, 1972.

10. Omang, S. H. , 0. D. Vellar. Concentration Gradients in Biological Samples

during Storage, Freezing, and Thawing. Anal. Chem. , 269:177, 1974.

11. Barker, E. D. Post-Mortem Loss of Hepatic Microsmal Mixed Function

Oxygenases. Res. Comm, in Chem. Path, and Pharm. , 7:321, 1974.

12. Versieck, J. , A. Speecke, J. Hoste, and F. Barbier. Trace Contaminations

in Biopsies of the Liver. Clin. Chem., 19:472, 1973.

9
13. Speecke, A., J. Hoste, and J„ Versieck. Sampling of Biological Material,

In: Accuracy in Trace Analysis: Sampling, Sample Handling, Analysis,

Vol. I, P. D. LaFleur, ed. Proc. 7th Materials Res. Symp., NBS Special

Publication 422, August 1976. pp. 299.

13a. Maletskos, C. J., M. D. Albertson, J. C. Fitzsimmons, M. R. Masureaker,

and Chung-Wai Tong. Sampling and Sample Handling of Human Tissue for

Activation Analysis. In: Trace Substances in Environmental Health — IV,

pp. 367. D. D. Hemphill, ed. Proc. of U. of Mo. 4th Annual Conf., 1970.

13b. LeBaron, G. J., W. H. Cherry, and W. F. Forbes. Studies of Trace-Metal

Levels in Human Tissues — IV. The Investigation of Cadmium Levels in

Kidney Samples from 61 Canadian Residents, p. 44. In: Trace Substances in

Environmental Health —XI. D. D. Hemphill, ed. Proc. of U. of Mo. 11th

Annual Conf. 1977.

13c. Iyengar, G. V., and K. Kasperek. Application of the Brittle Fracture

Technique (BFT) to Homogenize Biological Samples and Some Observations

Regarding the Distribution Behavior of the Trace Elements at Different

Concentration Levels in a Biological Matrix. J. Radioanal. Chem.

,

39:301, 1977.

14. Iyengar, G. V. Homogenized Sampling of Bone and other Biological Materials. Radiochem. Radioanal. Letters, 24:35, 1976.

14a. LaFleur, P. D., ed. Accuracy in Trace Analysis: Sampling, Sample Handling, Analysis. Proc. 7th Materials Res. Symp., Vol. I, II. NBS Special

Publication 422, August 1976.

15. Moody, J. R., and R. M. Lindstrom. The Cleaning, Analysis, and Selection

of Containers for Trace Element Samples. In: The National Environmental

Specimen Bank, pp. 19. H. L. Rook and G. M. Goldstein, eds. 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, Md . , Aug. 19-20, 1976. NBS Special Publication

501, February 1978.

16. Missen, A. W. , and S. J. Dickson. Contamination of Blood Samples by

Plasticizer in Evacuated Tubes. Clin. Chem., 20:1247, 1974.

17. Daun, H., and S. G. Gilbert. Migration of Plasticizers from Polyvinylchloride Packaging Films to Meat. J. Food Sci. , 42:561, 1977.

18. Jaeger, R. J., and R. J. Rubin. Migration of a Phthalate Ester Plasticizer from Polyvinylchloride Blood Bags into Stored Human Blood and Its

Localization in Human Tissues. New Engl. J. Med., 287:1114, 1972.

18a. Lindstrom, R. M. , National Bureau of Standards, Private Communication.

19. Subramanian, K. S., C. L. Chakrabarti, J. E. Sueiras, and I. S. Maines

.

Preservation of Some Trace Metals in Samples of Natural Waters. Anal.

Chem., 50:444, 1978.

19a. Harrison, S. H., National Bureau of Standards, Private Communication.

10
20. Duncan, W. Clean Room Products, Bay Shore, NY. Private Communication.

21. Seidman, S. C., et al. Effect of Degree of Vacuum and Length of Storage

on the Physical Characteristics of Vacuum Packaged Beef Wholesale Cuts.

J. Food Sci., 41:732, 1976.

21a. Gills, T. E. , S. H. Harrison, and H. L. Rook. Research on Freezer

Storage. In: Section 2, this publication.

21b. Harrison, S. H. , and T. E. Gills. Investigation of Sampling and Storage

Techniques for Environmental Specimen Banking Using Neutron Activation

Analysis. Talk presented at the 5th Symposium on the Recent Developments

in Neutron Activation Analysis, July 1978, Oxford, England.

22. Lievens, P. , J. Versieck, R. Cornells, and J. Hoste. The Distribution of

Trace Elements in Normal Human Liver Determined by Semi -Automated Radiochemical Neutron Activation Analysis. J. Radioanal. Chem., 37:483, 1977.

23. Thompson, G. , and D. C. Bankston. Sample Contamination from Grinding and

Sieving Determined by Emission Spectrometry. Applied Spectroscopy,

24:210, 1970.

24. Folsom, T. R. Nonmetallic Tools for Sampling, Frozen Tissues for Trace

Metal Analyses. USAEC Health and Safety Laboratory Report, HASL-249,

1-19-24, April 1, 1972.

25. Hislop, J. S., and A. Parker. The Use of a Laser for Cutting Bone Samples

Prior to Chemical Analysis. Analyst, 98:694, 1973.

26. Auth, D. C. , J. L. Doty, D. Neal, D. Heimbach, R. Wentworth, J. Colocousis,

and P. W. Curreri. The Laser Blade: A New Laser Scalpel. Proc. 2nd

International Surgical Laser Symp., Oct. 23-26, 1977, Dallas, Texas.

27. Koch, Jr., H. J., E. R. Smith, N. F. Shimp , and J. Connor. Analysis of

Trace Elements in Human Tissues. Cancer, 9:499, 1956.

28. Iyengar, G. V., K. Kasperek, and L. E. Feinendegen. Determination of

Certain Selected Bulk and Trace Elements on the Bovine Liver Matrix using

Neutron Activation Analysis. Phys. Med. Biol., 23:66, 1978.

29. Persigehl, M. , H. Schicha, K. Kasperek, and L. E. Feinendegen. Behavior

of Trace Element Concentration in Human Organs in Dependence of Age and

Environment. J. Radioanal. Chem., 37:611, 1977.

29a. Persigehl, M. , H. Schicha, K. Kasperek, and H. J. Klein. Trace Element

Concentration in Human Organs in Dependence of Age. Beitr. Path., 161:209,

1977.

30. Bernhard, U., K. Kasperek, A. Hock, K. Vyska, Chr . Freundlieb, and L. E.

Feinendegen. Changes in Trace Element Concentrations in Human Serum

after Parenteral Application of Amino Acids. J. Radioanal. Chem.,

37:383, 1977.

11
31 . Kollmer, W. E., G. V. Iyengar. Normal Concentrations of Various Elements

in Organs and Body Fluids. Compilation of Data Published by Various

Authors. Gesellschaft fuer Strahlen - und Umweltf orschung m.b.H.,

Neuherberg/Muenchen (F.R. Germany) Abt. fuer Nuklearbiologie , Dec. 1972.

32. Schicha, H., K. Kasperek, L. E. Feinendegen, V. Siller, and H. J. Klein.

Inhomogeneous but Partly Parallel Distribution for Cobalt, Iron, Selenium, Zinc, and Antimony in Different Regions of Liver, Lung, Kidney,

Heart and Aorta, Measured by Activation Analysis. Beitr. Path. Bd.,

146:55, 1972.

33. Schicha, H. , H. J. Klein, K. Kasperek, and F. Ritzl. Activation Analytical Estimation of Some Elements in Several Organs and in Cancerous Tissue.

Beitr. Path. Anat., 138:245, 1969.

34. Sumino, K., K. Hayakawa, T. Shibata, S. Kitamura. Heavy Metals in Normal

Japanese Tissues. Arch. Environ. Health, 30:487, 1975.

35. Velandia, J. A., and A. K. Perkons. Survey of 43 Constituent Elements in

Kidney Samples by Instrumental Activation Analysis. J. Radioanal. Chem.,

20:715, 1974.

36. Hock, A., U. Demmel, H. Schicha, K. Kasperek, and L. E. Feinendegen.

Trace Element Concentration in Human Brain. Brain, 98:49, 1975.

37. Khera, A. K. , and D. G. Wibberly. Some Analytical Problems Concerning

Trace Metal Analysis in Human Placentae. Proc. Analyt. Div. Chem. Soc.,

34D, 1976.

38. Sharma, R. P., and J. L. Shupe. Lead, Cadmium, and Arsenic Residues in

Animal Tissues in Relation to Those in Their Surrounding Habitat.

Science of the Total Environment, 7:53, 1977.

39. Langmyhr, F. J., and J. Aamodt. Atomic Absorption Spectrometric Determination of Some Trace Elements in Fish Meal and Bovine Liver by the

Solid Sampling Technique. Anal. Chim. Acta, 87:483, 1976.

40. Borjeson, H., and E. Fellenius. Towards a Valid Technique of Sampling

Fish Muscle to Determine Redox Substrates. Acta physiol, scand., 96:202,

1976.

41. Cornells, R., A. Speecke, and J. Hoste. Neutron Activation Analysis for

Bulk and Trace Elements in Urine. Anal. Chim. Acta, 78:317, 1975.

42. Brune , D. Interference-Free Trace Element Analysis of Water, Biological

Specimens and Food Sources by Means of Nuclear Techniques. Science of

the Total Environment, 2:111, 1975.

43. Johnson, D. E., R. J. Prevost, J. B. Tillery, and R. E. Thomas. The

Distribution of Cadmium and Other Metals (Lead, Zinc, and Mercury) in

Human Tissue. EPA-600/1-78-035 , U. S. Environmental Protection Agency,

May , 1978.

12
Lt iPods'

Health Physics, 9:89, 1963

c v.,

'

-
SECTION 2

RESEARCH ON FREEZER STORAGE

by

T. E. Gills, S. Harrison, and H. L. Rook

An important part of the NESB research program has been a study and evaluation of freezer storage for long-term storage of tissues and/or other biolog

ically active samples. The effects of microbiological action on trace constit

uents concentrations and distributions are well documented. Freezing has long

been applied as a technique for storage, however no study has been previously

made to document the reliability with respect to inorganic ions of this method

of storage for more than a short period of time. Recently, the analysis and

subsequent reanalysis of a selected animal tissue (bovine liver) that had been

stored frozen at -80 °C for a period of one year gave important information on

the effects of freezer storage for select elements, namely iron, copper, zinc,

molybdenum, selenium, cobalt, and arsenic. The original organ had been subset

sampled using stainless steel scalpel blades and the remaining portion homogenized using a commercial food blender. The homogenate was poured into six

precleaned polypropylene ice cube trays which were sealed in 1 mil thick

polyethylene film and stored in a chest-type -80 °C freezer.' Analyses were

performed on the subsets and homogenates to provide initial trace elemental

concentrations and to verify contamination-free homogenization of the sample.

For the one year reanalysis, one tissue cube was removed from each of the six

trays and the trays resealed for future studies. The selected tissue cubes were freeze-dried and analyzed using the same analytical procedures utilized

in the initial analysis. A comparison of the results of the reanalysis to

those originally obtained is given in Table I. For the elements determined,

the capability to procure a tissue and store it frozen for one year for retrospective analysis has been demonstrated. In addition, results indicate that

subset sampling can be a viable sampling strategy when applied to relatively

homogenous tissues such as liver, etc. Important elements such as chromium,

cadmium, iodine, lead, etc., though not analyzed in this set of samples, are currently being evaluated for their stability in tissues during freezer

storage

.

The sampling implements used in this work are not suggested for sampling

or homogenization but were utilized to provide a base for evaluating contamination with respect to elements such as chromium. Work is currently being done

to assess contamination from sampling implements and their effects upon analyt

ical uncertainty of real data.
Table I

Analyses

ofa

Stored

Bovine

Liver

Storage

and

Analysis

Protocol

Tests

(Concentrations

Reported

on

Dry

Wt.

Basis)

Pg/g

G3

CU

cn 2

cn

r-4cn0C

cu

5-1 P

TJ

cuooG

Ph 3 3 3 3 3 « 60

CO \ 1—

CN CN

w) +1 + 1 + 1 C Nf m m

lO

m cn o o o o o o • • o o O o O

CJ +1 +1 +1cn

n- e'- O o en • • o

LO LO cn O o o

<D • • • CO +1 +1 +i o o CNl co cn

CM

m i—1 o o o « • a +1 +1 +i OA o 00 00 e • • m CM CN G +i +1 +1 NS n O n

cn Nt cn P oi—

m cn c +i -h +i oo n n m in in

<r MO

0) +l +1 +1 Pm \D On ii

CNOoo

+1LO

n-O

CN0

+101

CNII + 1 a>

cnli

<r

+im

+1LO

CNOoo

+1mOo

+1

CTl (N

N-O

+1CN00+ 1 cn

cn

+icn

in

CN

+1m

+1

cnooo

+1mOmo

+iCN

00o

+1CNoo

+1oo

cn

CN

+1O

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cnC

cd +1

cu <n

iwo

cn 4H CU cn o <—

G

PH 00 Nmm

m o in • B 1—

cn •H o cd 4-4 2 cn cd /

. •H K > r> ^—

,

s CU •K +, n i— ro cn g

”1" as <U + G •H ^

<U '—

u 4-1 cd cn PH cd cd T3 i—

tn COGG cn

G T—

H 4—1 0) cu cu 4-1 <u cd cd

g

CD \—

oo on cu i—

4J

C cu

CU co a- o o cn a, in Cd 4-) rH rQ £ 6 e rP e CU 14-4 a o o p cd pj pJ cd £ CO CO 2 in in O • •

<fi

G

CO • • • * O • M i—

i—

> pj

G

M

i—i—

M > G

M

w < PJ

H

cuP

cn

cn •HHC

CUNO

5-i pH

5-1 Om

n

cn cu

cu cu

5-1 Sn pH pH + *

15

-dried
SECTION 3

CONTAINER MATERIALS FOR THE PRESERVATION OF

TRACE SUBSTANCES IN ENVIRONMENTAL MATERIALS

by

Richard A. Durst

INTRODUCTION

The success of the National Environmental Specimen Bank will be determined

in large part by the ability to preserve the integrity of the trace substances

in samples during long-term storage. It is widely recognized that the storage

of environmental materials is subject to a variety of uncertainties when one

is considering the identity and levels of trace elements and organic compounds.

Changes in the forms and concentrations of the numerous environmentally

important substances in specimens stored for extended periods may occur in

several ways. Processes such as surface adsorption and sample degradation may

reduce the concentrations of various components and/or produce species which

may not have been present in the original sample. On the other hand, contamination from the container and evaporation of specimen fluids could lead to

apparent increases in trace substance concentrations or losses in the case of

volatile trace components. In addition, superimposed on these processes are

the factors which will affect their rates, such as, container material, contact

time and area, storage temperature, pH, and initial species concentration. All

of these factors are important considerations in evaluating the suitability of

various containers for long-term storage.

There have been numerous studies performed in recent years directed toward

the identification of suitable container materials and optimum storage conditions, but much of this work is contradictory (1). In many cases, the analytical data were invalidated because of problems not associated with the storage

but with other links in the analytical chain such as sampling and procedural

contamination

.

Container Materials

While certain container materials can be eliminated a priori, it is

usually necessary to consider the container composition vis-a-vis the type of

sample and/or the components of interest in the sample. Accordingly, it is

unlikely that samples intended for trace organic analysis would be stored in

plastic containers or acidified water samples in glass containers if trace

elements were to be determined. While these are obvious examples of

16
incompatible sample/container combinations, even these may be acceptable

under certain storage conditions and for special requirements.

The National Environmental Specimen Bank is somewhat unique in that both

trace elements and trace organics are of environmental concern and a choice

must be made as to storing the samples in a single container material suitable

for both types of components or in two different container materials optimized

for the components of interest. In the former case, the choices are much more

limited and compromises must be made, whereas in the latter, the increased

complexity of separate sampling, sample handling and storage procedures may be

excessive. In the EPA/NBS Environmental Pilot Bank, this is one of the questions to be answered by careful evaluation of a variety of container materials

under different cleaning procedures and storage conditions.

At NBS , most of the research to date has been concerned with trace element contamination and losses, while trace organic problems have largely been

avoided by the use of carefully cleaned glass containers and immediate freezing of the samples. In a recent study (2), twelve different plastics were

examined by gravimetry, isotope dilution mass spectrometry, and neutron activation analysis in order to evaluate the rate of water loss, the levels of

impurities present in the plastics, and the quantities of trace elements

leached from the plastics during acid cleaning.

The annual rate of water loss, based on weighings at 17 and 66 days, are given in Table 1. The polypropylene. Teflon, and conventional polyethylene

have water loss rates compatible with long-term storage of aqueous samples,

whereas the other plastics may be adequate for shorter storage periods (2)

.

TABLE 1. ANNUAL RATE OF WATER LOSS FROM PLASTIC CONTAINERS

Material % Loss/year*

Polypropylene 0.04

Teflon 0.05

Conventional polyethylene 0.1

Polyvinyl chloride 0.5

Polymethylpentane 1 Polycarbonate 2

Average values based on weight loss measured at 17 and 66 days.

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

water loss, these values should not be construed as permeability data but

merely typical losses from these types of containers. These losses could be

reduced in practice by sealing the container in a vapor barrier or storage at an ambient relative humidity comparable to that of the aqueous samples.

The concentrations of trace elements contained in the plastics were determined by neutron activation analysis (2)

.

The ten plastics studied showed

17
striking differences in trace element composition as illustrated in Table 2 for several of the elements determined.

Table 2. Trace elements in plastics determined by Neutron

Activation Analysis (Concentrations given in ng/g)

Element CPE LPE PP PMP PS PC TFE FEP

Na lxlO 3 15xl03

5xl03 200 2x 10

3

3x10

3 160 400

A1 500 3xl0

4

6xl0

4 6xl 0

3 500 3xl0 3 230 200

Ti 5xl03

6xl0

4 5xl0 3 lxlO 3

Mn 10 20 10 20 60

Co 40 6 Zn 5xl05 4 3x10

4 Br >20 800 >5 >2 >1 3x10 >2

Sb 5 200 600

Au 0.1 0.6 0.04 0.03 0.4

CPE = conventional polyethylene; LPE = linear polyethylene;

PP = polypropylene; PMP = polymethylpentane; PS = polystyrene;

PC = polycarbonate; TFE and FEP = types of Teflon.

In agreement with the results of other workers, the purest materials appear to

be Teflon, PS, and CPE. Comparison of these data with those obtained in leaching experiments indicate that the bulk of most trace element impurities present

is distributed throughout the matrix, rather than concentrated at the surface.

The acid-leaching experiments were performed on four types of plastic

using two acids prepared from ultra-pure reagents. In this study (2), the

plastic bottles were first rinsed with distilled water to remove any surface

contamination and then filled with a 1+1 mixture of ultra-pure HNO^ or ultrapure HC1 and ultra-pure water (3) . The leaching process was allowed to proceed

for one week at room temperature (80 °C for the FEP bottle) before the analyses

were performed. The results obtained by isotope dilution mass spectrometry are given in Table 3 for a selected group of the impurity elements measured.

The results of these experiments indicate that HNO 3 and HC1 leach various

elements with different efficiencies, and it is recommended that both acids, in

sequence, be used for cleaning these containers (2). After cleaning, the

Teflon and CPE bottles have been found to be the least contaminating. Based on experience at NBS and reports of other studies, a recommended method for cleaning plastic containers has been proposed ( 2 )

1. Fill container with 1+1 analytical reagent grade HC1.

2. Allow to stand for one week at room temperature (80 °C

for Teflon)

18
3. Empty and rinse with distilled water.

4. Fill with 1+1 analytical reagent grade HN0 3

.

5. Repeat steps 2 and 3.

6. Fill with the purest available distilled water.

7. Allow to stand several weeks or until needed, changing water

periodically to ensure continued cleaning.

8. Rinse with purest water and allow to dry in a particle- and

fume-free environment.

2 Table 3. Impurities Leached from Plastic Containers (ng/cm )

Element FEP

hno 3 HC1

LPE

HN0 3 HC1

CPE

HN0 3 HC1

PC

hno 3 HC1

Pb 2 2 2 0.6 0.7 18 0.3 10

A1 6 4 1 4 1 10 5 3

Na 6 2 10 6 8 42 3 8

Sn 1 1 1 <1 <0.8 <0.8 0.2 13

Cd 0.4 0.6 0.2 0. 2 0.2 0.2 0.3 <8

Se 0.2 0.8 0.4 0.4 3 <0.3 0.5 <0.5

Zn 4 4 8 9 2 1 0.8 -

Cu 2 6 0.4 1 2 0.7 0.8 <6

Ni 2 0.8 1.6 0.8 0.5 0.3 0.7 0.3

Fe 20 16 3 1 3 1 3 <49

Cr 0.8 4 0.2 0.8 0.8 0.3 0.3 <5

Mg 8 1 0.6 0.4 0.7 0.7 2 0.8

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

.

For the storage of biological tissues and fluids, rapid freeze drying

immediately after sampling has been recommended but suffers from the disadvantage that some volatile components may be lost (1) . A more viable approach

would appear to be the immediate freezing of the sample or sub-samples to the

lowest conveniently attainable temperature. This approach serves two purposes:

1) it reduces or stops both chemical and biological processes which could

result in sample changes, and 2) it reduces the mobility of sample and con- tainer components thereby lessening the possibility of contamination and/or

losses due to adsorption or volatility. However, because of concentration

gradients which may be produced during freeze-thaw cycles and physical changes,

e.g., cell destruction, caused by freezing, it is highly recommended that all

sub-sampling sites usually precludes this procedure because of the probability

19
of sample contamination during the sub-sampling. Instead, the sample, which

has been frozen as rapidly as possible to avoid component fractionation, is

sub-sampled while still in the frozen state at a location which has a clean

facility.

When the sample is frozen, especially at cryogenic temperatures, the

reduced interaction between the sample and container permits greater flexibility in the choice of container material. However, even under these conditions, it is inadvisable to store samples intended for trace organic analysis

in any type of plastic container which incorporates additives such as plasticizers, organometallic or other stabilizer antioxidants, colorants, or any

other components which may be leached or volatilized from the plastic. Teflon

is the only plastic which has been found suitable for trace organic storage at

cryogenic temperatures, and which, after proper cleaning, is also suitable for

the storage of samples intended for trace element analysis.

Glass (quartz or Pyrex) , with Teflon (or aluminum foil) lined caps, is

also suitable for trace organic samples and may be satisfactory for frozen

trace element samples. At the present time, the key to long-term storage

appears to be storage at cryogenic temperatures to reduce reactions and interactions to a minimum. Under these conditions, both glass and Teflon appear to

be suitable storage container materials.

20
REFERENCES

1. Maienthal, E. J. and D. A. Becker, "A survey of current Literature on Sampling, Sample Handling, and Long-Term Storage for Environmental Materials," NBS Tech. Note 929, U. S. Government Printing Office, Washington,

D.C. 20402, 1976.

2. Moody, J. R. and R. M. Lindstrom Anal. Chem. 49:2264, 1977.

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

44:2050, 1972.

21
SECTION 4

DESIGN AND CONSTRUCTION OF THE PILOT BANK FACILITY

by

H. L. Rook, T. E. Gills, J. Koskoris, R. A. Durst

A final proposal and design for the construction of the Pilot Bank lowtemperature storage facility has been made. The Pilot Bank facility will have

the capacity to house 20 replicate samples for each of the approximately 4000

input specimens planned for the five-year storage period. These will be

derived from four classes: human tissue, aquatic tissue, food grain, and air

accumulator. Nominal sample size will be five-to-ten grams per replicate

sample. Samples will he stored at approximately -196 °C liquid nitrogen temperature, -80 °C, -25 °C, and freeze dried material stored at room temperature. The multiple storage conditions for the same sample will allow direct

evaluation of the candidate storage procedures. Protocols for sampling and

sample handling as discussed earlier in this report will be followed.

The pilot facility will be made up of an existing NBS clean room which

will be upgraded to a Class-100 clean lab, and a new Class-100 storage area which will be constructed adjacent to the existing clean room (see figure 1).

The existing clean room will be modified to provide ultra-clean preparation

laboratory conditions equivalent to a trace constituent analytical laboratory.

Samples will be protected with ultra-clean air (99.97% efficient on all particles 0.3 micrometer and larger). Personnel will be protected by performing

all operations with potentially pathogenic samples (liver) in a biological

clean work station.

The air flow pattern and work function will be designed to eliminate or minimize contamination caused by work functions. The preparation laboratory

will be divided into two principal work areas by means of a central dual air

return wall. A conventional fume hood and clean air module providing vertical-flow, Class-100 air will be located in the inorganic analysis section.

A biohazard enclosure and clean air module providing Class-100, charcoalfiltered air wj.11 be located in the organic analysis section of the laboratory. False walls will also be located at the ends of this laboratory to

provide additional air return to the clean air modules containing HEPA

filters

.

The Class-100 storage area will consist of a room approximately 3.7 m by

7.3 m (12' x 24') connected to the existing clean lab via an air lock. The

cleanliness of this storage area will have a particle count of fewer than 100

22
particles 0.3 micrometer and larger per cubic foot. Freezers will be located

in this area for the storage of specimens in liquid nitrogen freezers (approximately -196 °C) and compressor-type freezers at -25 and -80 °C. Racks will

also be provided for freeze-dried samples. In order to avoid the possibility

of build up of potentially harmful levels of nitrogen which could lead to

oxygen starvation, provision will be made to vent and replace sufficient

storage room air to ensure safe oxygen levels. In addition, the freezer compressors will be hermetically sealed in order to minimize potential contamination from wear metals and/or organic vapors.

It is anticipated that this laboratory/storage facility will be completed

in mid-summer of 1979. Human liver specimens will be acquired immediately

upon completion of this facility.

23
32’

-11

Figure 1. Specimen Bank Laboratory/Storage Facility

Emergency Exit

-
SECTION 5

ORGANIC MERCURY IN TISSUES

by

R. L. Zeisler, T. E. Gills

Increased interest of ecologists and analysts has been focused on the

ratio of organic mercury to total mercury content of biological and environmental samples. The determination of the total mercury content of these

matrices using Neutron Activation Analysis (NAA) procedures has been extensively investigated in the past years, and some Radiochemical Neutron Activation Analysis (RNAA) methods are considered fully reliable for this purpose.

So far, the determination of organic mercury has been carried out by use of

other methods, e.g. gas-chromatography. Thus, two samples have to be analyzed

by different methods to yield the desired information on the ratio of organic

mercury to total mercury content.

Well developed extraction and volatilization techniques are currently

being investigated for the isolation of methylmercury from biological tissues.

Since the possible radiolytic damage to organic mercury compounds during irradiation cannot be fully assessed, we are developing and applying a modified

extraction technique to be used in a pre-irradiation and "normal" RNAA scheme.

This will yield information about the changes of the chemical form of organic

mercury during irradiation.

The separation of methylmercury is based on the volatilization of methylmercury cyanide formed in the reaction of methylmercury in a sample with

hydrocyanic acid released by the interaction of a cyanoferrate with sulfuric

acid at elevated temperatures. The methylmercury cyanide released is captured

on cysteine paper in a microdiffusion cell. The paper is then placed in a flux of neutrons and the mercury quantitatively determined by neutron activation analysis.

The feasibility of this method is currently being tested. SzilardChalmers Reactions can change the chemical status of the mercury, and possible

losses of mercury from the sample have to be assessed. Test analyses have

proven that irradiation has to be performed in sealed quartz containers.

Samples can then be counted either "instrumentally" in the irradiation con- tainer or after using the combustion methods. Both require comparable samples

and standard configurations. The use of mercury deposits on filter paper (comparable to the proposed cysteine paper) is being evaluated.

25
In addition, the volatilization method for mercury determination after

the reduction of the inorganic mercury by SnCl 2 will give the possibility of

cross-checking of the mercury contents. The reduction method is claimed to be

selective for the inorganic mercury (only 0.5% of the organomercury is

released). This is, however, critically dependent on the chemical treatment

of the samples. This has to be carefully investigated.

The procedure utilizes the volatility of mercury and its compounds, more importantly the inherent sensitivity of neutron activation analysis along

with minimum use of reagents and solvents should provide increased accuracy

over currently used methods.

26
SECTION 6

The Determination of Trace Elements in New Food Grain SRM’s

Using Neutron Activation Analysis

by

Thomas Earl Gills, Mario Gallorini, Harry L. Rook

INTRODUCTION

Increasing concern with possible toxic elements in the food chain plus

growing recognition of the importance of a number of trace elements in nutrition make the accurate determination of trace elements essential (1) . The

National Bureau of Standards, in an interagency agreement with the Food and

Drug Administration, is involved in developing and certifying selected elements

in food grain as a part of the NBS Standard Reference Materials Program. The

food grains chosen, rice flour and wheat flour, are currently being analyzed

to determine trace element concentrations. Many of the elements considered to

be toxic to human health are present at the ppb level, and their analyses

require selective radiochemical separations. This paper presents results

obtained using radiochemical and instrumental neutron activation analysis to

determine thirteen trace elements whose effect on human systems ranges from

toxic to essential. The following elements were determined in the food grain

SRM's: Fe, Zn, Mn, Co, Na, K, Br, As, Sb, Se, Cu, Rb, and Hg.

EXPERIMENTAL

Samples and Standards :

Homogenized samples of Wheat and rice flours were vacuum dried at a pressure of 0.55 mm Hg for 48 hours in order to obtain a moisture content correction factor. For elemental standards, primary solutions were made by the dissolution of high purity salts or metals in NBS high purity acids (2)

.

Irradiations

:

All samples and standards were sealed in Suprasil quartz vials and irradiated in the high flux pneumatic irradiation facility of the NBS Research

Reactor. In this position the nominal neutron flux is 5xlO I0 n*cm *sec

with a Cu/Cd ratio of ^80. Within the irradiation container the radial flux

variation is <_ 2 percent (3) .

27
Reagents for Radiochemical Separations :

Chromatographic reagents used for radiochemical separations consisted of

inorganic ion exchangers developed by Girardi, et al. (4,5). Tin Dioxide (TDO)

,

Cuprous Chloride (CUC) and Hydrated Manganese Dioxide (HMD) were obtained from

Carlo Erba, Italy.

Counting :

All samples and standards were counted on a 66 cm

3 volume Ge(Li) detector

coupled to a 4096 channel pulse height analyzer. For multiple analyses an automatic sample changer system was employed. After each count the accumulated data were transferred to a magnetic tape and were processed by the quantitative analysis program QLN (6)

INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS (INAA)

The following elements were determined by INAA: 3r, Co, Fe , K, Mn, Rb,

Zn, Na , and Se. The irradiation conditions and data used for the instrumental

analysis are shown in Table 1. Different irradiation times were used to obtain the proper conditions for maximum sensitivity and accuracy. Approximately 300 mg of each food grain SRM, along with appropriate standards, were encapsulated in Suprasil quartz and irradiated in the high flux pneumatic tube

facility of the NBS Reactor. After allowing the samples to decay for the

specified time, samples and standards were post-weighed into fabricated polyethylene bags and counted. The resultant spectra were reduced to individual

photopeaks associated with specific isotopes of interest and peak areas com- puted both by the total peak area method and by a peak fitting method. Elemental concentrations were determined by the direct comparator method.

Table 1. Irradiation Conditions and Data Used for Instrumental Analysis

Element

Det 'd

Isotope

Measured

Half-Life

(1%)

Irradiation Time

Decay

Time

Counting

Time (sec)

Peaks Used

Evaluation

for

(MeV)

Fe

^ y Fe 44.6 d 8 hr 30 d 5 X 10 1.099, 1.292

Co

60

Co 5.27 y 8 hr 30 d 5 X io

4 1.173, 1.332

Zn

65

Zn 244 d 8 hr 30 d 5 X io

4 1.115

Zn

69M

Zn 13.9 h 8 hr 30 d 5 X io

4 .439

Se

75

Se 120 d 8 hr 30 d 5 X io

4 .136 .265

Rb •J3 DO 18.7 d 8 hr 30 d 5 X io

4 1.077

Mn

56MMn 2.58 h 3 min 2 hr 4 X io3 .847, 1.811

Na

24vNa 15.03 h 1 hr 30 hr 4 X io

3 1.369, 2.754

Br

82

Br 35.34 hr 1 hr 30 hr 4 X io

3

. 776, .619

K

42k 12.36 hr 1 hr 30 hr 4 X 103 1.525

28
RADIOCHEMICAL SEPARATIONS (RNAA)

The concentrations of arsenic, antimony, copper, selenium, and mercury

were determined by RNAA. The irradiation conditions and data used for the

radiochemical analysis are shown in Table 2. Inorganic ion exchangers were

used in the determination of As, Sb, Se, and Cu while a combustion distillation

procedure was used in the determination of Se and Hg . Complete descriptions

and details of these procedures are given elsewhere (7-9).

Table 2. Irradiation Conditions and Data Used for Radiochemical Activation

Analysis

Element

Det ’d

Isotope

Measured

Half-life

(TJs)

Irradiation Time

(hr)

Decay

Time

(hr)

Counting

Time

(sec )

Peaks used for

Evaluation

(MeV)

As

76

As 26.4 hr 4 36 5 x 10

4 .559, .657

Cu

64

Cu 12.7 hr 4 36 5 x 10

4 .511, 1.345

Se

75

Se 120. d 4 36 5 x 10

4 .136, .265

Hg

197

Hg 64.1 hr 4 36 5 x 10

4 .077

Sb

122

Sb 2.7 d 4 36 5 x 10

4 .564

Sb

iZ4

Sb 60.3 d 4 36 5 x 10

4 .603, 1.691

RESULTS AND DISCUSSION

The results of the instrumental analysis are shown in Table 3. The experimental design of the conditions for irradiation and counting was optimized to

obtain the best statistical parameters. Under optimized conditions, the precision obtained was relatively good. The results of the analysis of NBS SRM

Orchard Leaves 1571 also indicates relatively good accuracy.

The results obtained using radiochemical separation procedures are also

summarized in Table 3. The values represent the averages obtained from the

different radiochemical separation procedures.

The results shown constitute only one independent method of analysis

(NAA) and are not considered NBS certified values. However, these results

give a preliminary trace elemental profile of typical food grains (rice and

wheat flour)

.

The procedures used in this work have been evaluated with respect to the sources of error that can affect the accuracy and precision of the results

obtained

.

29
INSTRUMENTAL

ACTIVATION

ANALYSIS

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d3 +i +1 +1 +i +i +i +1 +i +1 +1 +1 +1 +1 +1

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30

in

parentheses

indicate

information

values

only.
REFERENCES

1. Tanner, J. T. and M. H. Friedman. Neutron Activation Analysis for Trace

Elements in Foods. Proceedings 1976 International Conference Modern

Trends in Activation Analysis, 1, p 244.

2. Kuehner, E. C., R. Alvarez, P. J. Paulson and T. J. Murphy. Production

and Analysis of Special High Purity Acids Purified by Sub-Boiling Distillation Anal. Chem. 44:2050, 1972.

3.

Becker, D. A. and P. D. LaFleur. Characterization of a Nuclear Reactor

for Neutron Activation Analysis J. Radioanal. Chem. 19:149-157, 1974.

4. Girardi,

1969.

F., R. Pietra and E. Sabbioni

Euratom Techn. Rep. EVR 4287,

5. Girardi

,

F., R. Pietra and E. Sabbioni

Radiochemical Separations by

Retention on Inorganic Precipitates. 1968 International Conference on Modern Trends in Activation Analysis. NBS Special Publication 372,

Vol. 1, pp 639.

6. Yule, H. P. (NUS), J. P. F. Lambert and S. K. Gerauson (EPA-NC) . Automatic Activation Analysis Code for Component Identification and Quantitative Result Computation Transactions of the Amer. Nucl. Soc. 17:128-129,

1973.

7. Gills, T. E., M. Gallorini and R. R. Greenberg. Determination of

Selected Toxic Elements in Biological Matrices Using Radiochemical Activation Analysis. Proceedings of the International Conference Nuclear

Methods in Environmental and Energy Research, 11 October 1977, University

of Missouri-Columbia.

8. Rook, H. L., T. E. Gills and P. D. LaFleur. Method for Determination of

Mercury in Biological Materials by Neutron Activation Analysis .Anal.

Chem. 44 , No. 7, June 1972.

9. Rook, H. L. Rapid, Quantitative Separation for the Determination of

Selenium using Neutron Activation Analysis Anal. Chem. 4A, No. 7, June

1972.

Certain commercial equipment, instruments, or materials are identified in

this paper in order to adequately specify the experimental procedure. In no case does identification imply recommendation or endorsement by the National

Bureau of Standards, nor does it imply that the materials or equipment identified is necessarily the best available for the purpose.

31
SECTION 7

Cadmium Analysis by Radiochemical Neutron Activation Analysis

by

Robert R, Greenberg, Mario Gallorini, Thomas E. Gills

Cadmium has been suspected of causing detrimental health effects in

humans even at very low levels. Since Cd is commonly found at the trace or ultra-trace level, many analytical techniques do not possess the sensitivity

to accurately determine the concentration of this element in many environmentally important materials. In addition, there are some instances in which the

total amount of material available for analysis is very small, such as for

certain types of atmospheric particulate samples, or hair samples. Analytical

techniques capable of measuring very small amounts of Cd are therefore

required

.

Radiochemical neutron activation analysis (RNAA) was used at the National

Bureau of Standards to analyze Cd in a variety of matrices. RNAA offers the

advantages of high sensitivity, excellent selectivity, and no chemical

blank. Furthermore, the ability to add carriers during the chemical dissolution and separation enables quantitative recovery of the element.

The concentrations of many elements can be determined instrumental ly —

that is, without separating them from the activated matrix. Other elements,

however, such as Cd, usually have to be determined radiochemically , or isolated from other neutron-activated products. Many different types of radiochemical separations can be used for RNAA, such as: solvent extraction,

distillation, precipitation, ion-exchange chromatography, and electrodeposition. The method we use to isolate Cd is solvent extraction. Zinc diethyldithiocarbamate [Zn(DDC) 2 ] in chloroform will quantitatively extract Cd from

an aqueous solution over a pH range of from 1 to 12.

In addition to Cd, Zn(DDC)2 also extracts Cu. In many matrices, Cu can interfere with the determination of Cd by producing a high background level of

radiation. This problem can be eliminated by first extracting with bismuth

32
diethyldithiocarbamate [Bi(DDC) 3 J in chloroform which removes Cu but not Cd.

Cu can then also be analyzed if desired.

Preparation of Metal DDC Compounds

The Zn(DDC )2 and Bi(DDC )3 compounds are prepared by mixing aqueous solutions of NaDDC and either Zn ( 1103)2 or Bi ( 1103 ) 3

. The M(DDC) compound formed

is insoluble in water and precipitated. The precipitate is filtered, washed

with water, and dissolved in chloroform. An equal volume of ethanol is added

to the solution which is then set aside to allow the chloroform to evaporate

at room temperature. After the chloroform evaporates, the M(DDC) X compound

crystallizes in the remaining ethanol. The crystals are filtered and allowed

to dry at room temperature.

Procedure

Prior to irradiation, the samples and primary standards were encapsulated

in cleaned quartz vials. The standards consisted of a solution prepared from

high purity metals dissolved in high purity HNO 3

. The samples and standards

were then irradiated together in the NBS reactor at a thermal neutron flux of

5 x 10 13 n*cm 2 *sec 1 for times ranging from one to four hours.

After irradiation, the radioactive samples were allowed to decay for

three days after which they were transferred to dissolution vessels. Three

types of vessels were used depending on the matrix: Erlenmeyer flasks. Teflon

wet-ashing vessels and a Teflon-lined bomb. One hundred pg of Cu and Cd

carriers were added to each vessel and the samples were dissolved. Various

acid mixtures were used including HNO 3 alone, KNO 3 — H 2 SO 4 and HNO 3 — HClOg

(not in the bomb). If silica was present, some HF was also added.

-f" o After dissolution, 100 mg of Zn z hold-back carrier was added to minimize

exchange between the radioactive Zn of the samples, and the organic Zn of the

Zn (DDC )

2

solution used to extract the Cd . The pH of each solution was adjusted to 1.5 with NH 3 and the volume brought to about 60 mL with deionized

water. Each solution was then transferred to a 125 mb separatory funnel and

shaken for 30 minutes with 20 mL of 0.003 mol/L Bi(DDC )3 in chloroform using

a shaking machine. The organic fraction containing Cu was drained into a 120 mL polyethylene bottle and the aqueous phase was washed for 15 seconds

with an additional 10 mL of the Bi (DDC) 3 /CHCI 3 solution. The wash was then

combined with the first Bi(DDC )3 fraction and retained for counting.

Twenty mL of 0.005 mol/L Zn(DDC ) 2 in chloroform was then added to the

aqueous phase still in the separatory funnel, which was then shaken for five

minutes. The organic fraction was drained into a second 120 mL polyethylene

bottle, and the aqueous phase washed for 15 seconds with an additional 10 mL

of the Zn(DDC) 2 /CHCI 3 solution. The wash was combined with the first

Zn(DDC )2 fraction and was retained for counting.

Two different procedures were followed for the standards. One standard

was prepared by pipetting a known amount of irradiated solution into a dissolution vessel with carriers and some unirradiated sample material. This

material was dissolved in the same manner as were the samples used for analysis. A second standard was pipetted directly into 1 mol/L HNO 3 along with

carriers. 3 oth standards were then subjected to the same separation procedure used for the samples.

33
Counting

The samples and standards were counted on Ge(Li) detectors with active

volumes of from 60 to 75 cm 3 coupled to 4096 channel pulse height analyzer

systems. The Bi(DDC )3 solutions containing Cu were counted immediately after

separation. The 511 keV peak produced from the annihilation of positrons

emitted by ^ 4 Cu was used for analysis.

The Zn(DDC )2 solutions containing Cd were allowed to decay for at least

24 hours to establish the equilibrium between * 1

5

Cd and its daughter 115m

ln.

The 336 keV line from 115m

in, and the 527 keV line from 1 1

5

Cd were both used

for analysis. Computer code QLN1 was used for data reduction supplemented by

hand integrations of poorly defined peaks.

Results and Discussion

A large number of National Bureau of Standards Standard Reference Materials (SRMs) were analyzed by the above procedure, indicating the applicability of this method to a wide variety of matrices. The Cd and Cu results

obtained are compared with the certified values in Tables 1 and 2. Very good

agreement is observed demonstrating the accuracy of this technique.

Table 1. Cadmium in Various SRMs

Concentration-yg/g

This Work Certified

Orchard Leaves (SRM 1571) 0.116 + 0.008 0.11 + 0.01

Pine Needles (SRM 1575) 0.194 ± 0.009 <0.5

Bovine Liver (SRM 1577) 0.295 ± 0.015 0.27 + 0.04

Rice Flour (SRM 1568) 0.029 ± 0.005 0.029 + 0.004

Wheat Flour (SRM 1567) 0.030 ± 0.005 0.032 + 0.007

Sub-Bituminous Coal (SRM 1635) 0.030 ± 0.002 0.03 + 0.01

River Sediment (SRM 1645) 10.2 ± 0.4 10.2 + 1.5

Urban Particulate (SRM 1648) 71.2 ± 3.7 75 + 7

This two-extraction radiochemical separation scheme is very versatile and can be used as a part of a larger multi-element analysis scheme. One such

scheme used at NBS involves the use of an ion-exchange resin, HMD (Hydrated

Manganese Dioxide) prior to the extractions. HMD will quantitatively retain

As, Sb, Se, and Cr from a 1 mol/L HN0 3 solution. To perform this separation,

we normally dissolve the sample in HN0 3 + HCIO 4 with some HF if silica is

present. The samples are heated to incipient dryness and then brought to a volume of 20 mL with 1 mol/L HNO 3

. The solutions are passed through plastic

chromatographic columns, each containing a bed of HMD 7 mm by 30 mm. The

columns are washed twice with 1 mol/L HNO 3 and dismantled for counting. Under

34
Table 2. Copper in Various SRMs

Concentration-yg/g

This Work Certified

Orchard Leaves (SRM 1571) 11.6 + 0.4 12 + 1 Pine Needles (SRM 1575) 3.04 + 0.16 3.0 + 0.3

Bovine Liver (SRM 1577) 185 + 7 193 + 10

Rice Flour (SRM 1568) 2.12 + 0.09 2.2 + 0.3

Wheat Flour (SRM 1567) 2.21 + 0.10 2.0 + 0.3

Sub-Bituminous Coal (SRM 1635) 3.56 + 0.18 3.6 + 0.3

these conditions Cu and Cd are completely eluted. The eluted fraction is then

subjected to the two-extraction procedure described above. Various SRMs were

analyzed using this procedure and the results obtained are compared with the

certified values in Tables 3-5. Very good agreement is observed.

Table 3. Sub-3ituminous Coal SRM 1635

Concentration-yg/g

This Work Certified

As 0. 44 ± 0.,05 0.,42 1 0.15

Sb 0. 12 0.,01 (0 .14)*

Se 0.,82 + 0.,04 0..9 0.3

Cr 2. 48 + 0.,08 2..5 + 0.3

Cd 0..030 + 0.,002 0..03 + 0.01

Cu 3., 56 + 0,.18 3..6 + 0.3

Values in parentheses are MBS information only values.

35
Table 4. Bovine Liver SRM 1577

Concentration-yg/g

This Work Certified

As 0 ..054 + 0 ..004 (0. 055)

Sb 0 ..010 + 0 . 002 -—

Se 1 .,06 + 0 .,06 1 ., 1 + 0 .,1

Cr 0 .,085 + 0 ., 009 0 . 088 + 0 ,,012

Cd 0 .,295 + 0 .,015 0 .,27 + 0 ,.04

Cu 185 + 7 193 + 10

*Values in parentheses are NBS information only values.

Table 5. Orchard Leaves SRM 1571

Concentration-yg /g

This Work Certified

As 9. 7 + 0.4 10 + 2 Sb 2.8 + 0.1 2.9 + 0.3

Se 0.09 + 0.01 0.08 4- 0.01

Cr 2.67 + 0.15 2.6 + 0.3

Cd 0.116 + 0.008 0.11 + 0.01

Cu 11.6 + 0.4 12 1

Conclusions

The two-extraction radiochemical separation scheme for Cu and Cd described in this paper is both highly selective and extremely sensitive. The

simplicity of the method allows a quantitative recovery of both elements,

thus avoiding a calculation of chemical yield. Furthermore, a relatively

short time is required to carry out the complete procedure. One person can separate about 15 samples a day.

36
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 • 1479

Appendix I

Simultaneous Determination of Arsenic, Antimony, Cadmium,

Chromium, Copper, and Selenium in Environmental Material by

Radiochemical Neutron Activation Analysis

SVL Gaflorini , 1 R. R. Greenberg, and T. E, GUIs*

Center for Analytical Chemistry, National Bureau of Standards, Washington, D C. 20234

A multielement radioartaiytical procedure for the simultaneous

determination of As, Cr, Se, Sb, Cd, and Cu has been de- veloped. An inorganic ion exchanger coupled to a solvent

extraction system was used for the selective separation of these elements from neutron activated matrices. The method

has been used for centrificafion of environmentally related NBS

SRMs.

In studies of environmental pollution, elements such as

arsenic, antimony, chromium, cadmium, copper, and selenium

are among the most interesting because of their toxic nature. These elements are usually found at very low concentration

in most samples of interest. The accurate analysis of these elements can be accomplished only by techniques that are very

sensitive and selective. Neutron activation analysis usually meets these requirements; however, in many cases the ele- ments to be determined must be separated selectively from

neutron activated matrices. Utilizing radiochemical neutron

activation analysis (RNAA), one can obtain maximum sensitivity, accuracy, and selectivity ( 1 ). Usually, the choice of the radiochemical procedure to be

followed is dependent upon the elements to be determined,

the nature of the materials to be analyzed, and the simplicity

of the chemistry. Several radiochemical procedures such as solvent extraction, distillation, ion-exchange chromatography,

precipitation and electrodeposition can be used to obtain

satisfactory results ( 2

-6). However, when used alone, most

of these procedures are not suitable for simultaneous multielement analysis. Furthermore, when several chemical steps

are necessary, quantitative separation becomes extremely

1 Guest worker from Laboratorio di Radiochimica e Analisi per

Attivazione del C.N.R., Pavia, Italy.

difficult; often the determination of chemical yields, which

can contribute to the analytical error, is required. In this work, a multielement radiochemical separation

procedure has been tested and optimized to determine six elements simultaneously in different NBS environmental

Standard Reference Materials (SRM’s). Hydrated manganese

dioxide (HMD), an inorganic ion exchanger developed by

Girardi et al. (7, 8), was used for the retention and subsequent

determination of As, Cr, Sb, and Se, while a solvent extraction system using diethyldithiocarbamate compounds (9, 1

0

) was

used to determine Cd and Cu.

In an attempt to demonstrate applicability to matrices

having different chemical compositions, the following NBS

materials were analyzed: Orchard Leaves (SRM 1571), Bovine

Liver (SRM 1577), and Subbituminous Coal (SRM 1635).

EXPERIMENTAL

Reagents and Apparatus. The HMD inorganic exchanger was obtained from Carlo Erba, Milan, Italy. Bismuth and zinc diethyldithiocarbamates (DDC) were pre- pared by mixing aqueous solutions of Na (DDC) with their re- spective nitrate salts (9). The MIDDCL compounds formed

precipitated from water. The precipitates were filtered, washed

with water, and dissolved in CHC13

. An equal volume of ethanol was added to the CHC13 solution which was then set aside to allow the CHC13 to evaporate at room temperature. After evaporation, the MiDDClj compound crystallized in the remaining ethanol. The crystals were filtered and allowed to dry at room temperature.

Ion-exchange columns were made of polyethylene with an

internal diameter of 7 mm and packed with 3 cm of hydrated

manganese dioxide. A Teflon-lined digestion bomb and Teflon wet-ashing vessels were used for sample dissolutions. Standards and Carriers. A multielement primary standard was prepared from solutions of high purity metals dissolved in NBS high purity HN03 and/or H2 S04 (11). A multielement

carrier solution was prepared by mixing solutions of the salts or

Published 1978 by the American Chemical Society 37
Table I. Determination of As, Sb, Se, Cr, Cd, and Cu in NBS SRM Orchard Leaves (1571 ) and SRM Bovine Liver (1577)°

rna trice NBS Orchard Leaves 1571 NBS SRM Bovine Liver 1577

element certified values this work certified values this work

As 10 ± 2 9.7 ± 0.4 (0.055) 0.054 ± 0.004

Sb 2.9 ± 0.3 2.8 ± 0.1 (

) 0.010 ± 0.002

Se 0.08 ± 0.01 0.09 ± 0.01 1.1 ± 0.1 1.06 ± 0.06 Cr 2.60 ± 0.3 2.67 ± 0.15 0.090 ± 0.015 0.085 ± 0.009

Cd 0.11 * 0.01 0.116 ± 0.008 0.27 ± 0.04 0.30 ± 0.02

Cu 12 ± 1 11.6 ± 0.4 193 ± 10 185 ± 7

a Concentration in Mg/g- The reported value for each element consisted of ten determinations. Values in parentheses are NBS information values.

metals of interest dissolved in distilled water, HN03 , and/or

H2S04. The concentration of each element in the multielement

carrier solution was ~1 mg/mL.

The use of HC1 or chloride salts was avoided to prevent possible

losses of volatile chloride compounds during sample dissolution. Sample Preparation. Special attention was given to sample

handling prior to irradiation in order to avoid any possible contamination. Teflon-coated spatulas and forceps were used to transfer the samples into cleaned silica vials. Sample sizes ranged

from 200-300 mg. Each irradiation capsule contained six vials, one liquid multielement standard, and five samples of the materials to be analyzed.

Irradiation. The standards and samples were irradiated in the RT-3 pneumatic tube facility of the NBS Research Reactor.

In this position, the nominal neutron flux is 5 X 1013 n-cnT2 s”

1

. The radial flux variation within the irradiation container is <2%

(12).Sample Dissolution. After irradiation and two days cooling

time, the quartz vials were washed with coned HN03 and distilled water, cooled in liquid nitrogen to reduce the internal pressure, and opened. The contents were weighed and transferred to chemical dissolution containers along with 500 ^<L of the multielement carrier. Two types of dissolutions were performed to

test for possible losses of volatile compounds. These were; (1) Dissolution in glass Erlenmeyer flasks or open Teflon wet-ashing

vessels using 10 mL of coned HN03 , HC104 , and HF in a 10:3:1

ratio. (2) Dissolution in Teflon-lined bomb using 10 mL of fuming

HN03 and HF in 20:1 ratio. These acid mixtures were tested and found satisfactory for the subsequent radiochemical procedures.

Hydrated Manganese Dioxide (HMD) Separation. The

solutions obtained from the Erlenmeyer flasks or Teflon vessels were evaporated to a final volume of approximately 1 mL, cooled, and brought to a 20-mL final volume with 1 M HN03

. The

solutions obtained from the bomb were evaporated in Teflon

vessels to a volume of about 2 mL. One mL of coned HC104 was

then added and the samples were heated to fumes of HC104 , cooled, and brought to a final volume of 20 mL with 1 M HN03

. The 1 M HN03 solutions were then passed through HMD

columns (7-mm diameter and 3-cm height) which had been

preconditioned with 1 M HN03. The flow rate was adjusted to

0.5 mL/min by varying the packing density of the HMD. Sel- enium, chromium, arsenic, and antimony were retained on the HMD bed. Sodium-24, a major mterference in both the biological

and environmental matrices, passed through in the eluate. The

columns were washed with 20 mL of 1 M HN03 , dismantled, and

the HMD was transferred to polyethylene counting vials while

the eluted fraction was saved for the subsequent Cu and Cd

separations.

Metal Diethyldithiocarbamate Extraction. The pH of the

eluted fractions was adjusted to 1.5 with NH4 OH, and 100 mg

of Zn holdback carrier was added. The solutions were then placed

in separatory funnels and 20 mL of 0.003 M bismuth diethyldithiocarbamate [Bi(DDC)3 ] in chloroform was added in order

to extract the copper. The solutions w’ere then shaken for 30 min,

utilizing a shaking machine. The organic fractions containing the copper were placed in polyethylene bottles for counting. The

cadmium remained in the aqueous fractions and was extracted with 20 mL of 0.005 M zinc diethyldithiocarbamate [Zn(DDC) 2 ] in chloroform. Five minutes of shaking time was found sufficient

Table II. Determination of As, Sb, Se, Cr, Cd, and Cu in NBS SRM Subbituminous Coal (1635)a

Matrice

NBS SRM Subbituminous Coal (1635)

Element certified values this work

As 0.42 ± 0.15 0.44 ± 0.05

Sb (0.14) 0.12 ± 0.01 Se 0.9 ± 0.3 0.82 ± 0.04 Cr 2.5 ± 0.3 2.48 ± 0.08

Cd 0.03 ± 0.01 0.029 ± 0.003

Cu 3.6 ± 0.3 3.56 ± 0.18

a Concentration in pg/g. The reported value for each

element consisted of ten determinations. Values in parentheses are NBS information values.

for a complete extraction of the cadmium complex into the organic

phase. The resultant extract was also placed in polyethylene vials for counting.

The Zn holdback carrier was added to minimize exchange

between activated Zn from the sample and the organic Zn used

for the Cd extraction. Radioactive Zn can interfere with the detemination of Cd by producing a high background level of

radiation. Standards. Standards for analysis were prepared in two ways. The first was prepared using the same procedure as used for the sample dissolution. HMD separation, and M(DDCL extraction. The second standard was prepared by pipetting a known amount

of irradiated standard solution directly onto HMD in a polyethylene counting vial for the determination of As, Sb, Se, and

Cr. Copper and Cd standards were pipetted into 1 M HN03 acid and extracted in the same manner as the samples.

Counting. The samples and standards were counted on Ge(Li) detectors with active volumes of 63 to 75 cm3 coupled to 4096-

channel pulse height analyzers. The HMD fractions were counted

twice; first, 4 days after irradiation for measuring short-lived radioisotopes of arsenic and antimony, and then 3 weeks after

irradiation for measuring the long-lived radioisotopes of selenium and chromium. Because of the very low concentration of chromium in Bovine Liver, and the high background coming from

32 P .3” bremsstrahlung, counting times of from 5 to 10 h were

necessary. The organic fractions containing the Cu were counted immediately after separation. The organic fractions containing

the Cd were allowed to stand for 24 h to establish the equilibrium between U5Cd- U5lEIn. -y rays from the decay of both these isotopes were used for Cd analysis. Data reduction was accomplished by a quantitative analysis

computer code (13) and hand integration of poorly defined peaks.

RESULTS AND DISCUSSION

The results are shown in Tables I and II. The results indicate very good agreement between certified and found

values, demonstrating that the procedure is accurate. During the dissolution of the samples in the Teflon bombs

and in open Teflon containers, no appreciable losses of As,

Cr, Se, Cu, and Cd were found. Large losses of antimony (up

to 80%) were observed in some cases during the dissolution 38
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 • 1481

in the open Erlenmeyer flasks due to the adsorption of Sb onto

the glass surface of the flasks. The quantitative recovery of

this element was impossible even with repetitive washing with

1 M HN03

. No losses of antimony were observed, however,

when the samples were dissolved in open Teflon vessels or

in the Teflon-lined bomb.

A previous study by L. T. McClendon ( 14 ) cited apparent

losses of organometallic chromium compounds during initial acid digestion. Because these volatile compounds were not

characterized, all Bovine Liver samples analyzed in this work

were digested in a Teflon-lined bomb. However, when the

digested samples from the Teflon-lined bomb were boiled with

HCIO4 -HNO3 , no appreciable losses were observed. These

observations give support to the theory that the first digestion

in the Teflon bomb converts the organochromium compounds

to inorganic chromium, which is not volatile in nonchloride

systems. Furthermore, standards for analysis that were di- gested in HC104 -HN03 , and those pipetted directly were

always in total agreement.

The retention of arsenic, selenium, chromium, and antimony on HMD in 1 M HN03 is highly selective and quantitative,

while the elution of Cd and Cu is complete. The decontamination of the elements of interest from sodium-24 and other

radioactive matrix interferences can be estimated to be a factor of 10

a

.

CONCLUSION

The simplicity of the method permits a rapid isolation of the radioisotopes of interest without many chemical steps. The quantitative recovery avoids the calculation of the chemical yield. The sensitivity and accuracy obtained in this work demonstrate the validity and the precision of this ra- diochemical procedure.

LITERATURE CITED

(1) T. E. Gills, M. Gallorini, and R. R. Greenberg, Proceedings of Third

International Conference on Methods in Environmental and Energy

Research, University of Missouri-Columbia, Mo., October 1977.

(2) K. Heydorn and E. Damsgaad, Talanta, 20, 1 (1973).

(3) A. Gaudry. B. Maziere, and D. Comar, J. Radioanal. Chem., 29, 77 (1976).

(4) H. M. N. H. Irving, J. Radioanal Chem., 33, 287 (1976).

(5) K. Kudo, T. Shigematsu, and K. Kobayashi, J. Radioanal. Chem., 36 , 65 (1977).

(6) M. Gallorini, M. DiCasa, R. Stella, N. Genova, and E. Orvini, J. Radioanal. Chem., 32, 17 (1977).

(7) F. Girardi, R. Pietra, and E. Sabbioni, J. Radioanal. Chem., 5, 141 (1970).

(8) J. Cuypers, F. Girardi, and F Monsty, J. Radioanal. Chem., 17, 115 (1973).

(9) A. Wyttenbach and S. Bajo, Anal. Chem., 47 , 1813 (1975).

(10) S. Bajo and A. Wyttenbach, Anal. Chem., 48, 902 (1976).

(1 1) E. C. Kuchner, R. Alvarez, P. J. Paulson, and T. J. Murphy, Anal. Chem.,

44 , 2050 (1972).

(12) D. A. Becker and P. D. LaFleur, J. Radioanal. Chem., 19, 149 (1974).

(13) H. P. Yule and H. L. Rook, J . Radioanal. Chem., 39, 255-261 (1977).

(14) EPA Technical Report EPA-600/1-77-020, April 1977, p 44.

Rlcdiysg fcr April IS, 1978. Accepted June 13, 1978.
Appendix II

Tiie Quantitative Determination of Volatile Trace

Elements in NBS Biological Standard Reference

Material 1569, Brewers Yeast

Harry L. Rook

Analytical Chemistry Division

National Bureau of Standards

Washington, D. C. Wayne Wo 1

Nutrition Institute

U.S. Department of Agriculture

Beltsville, Maryland

ABSTRACT

In the past few years, a large body of analytical data has been reported on trace levels of chromium in biological samples. From data on materials such as NBS Standard Reference Material

1577, Bovine Liver, and IAEA standard materials, it is now apparent that much of the reported Cr data are in error.

It has been suggested that some of the analytical problems

may be due to the presence of a volatile organic complex of Cr

in many biological matrices.

In an effort to resolve the question of Cr volatility, a series of experiments have been conducted on a new NBS Standard

Reference Materia 1 --Brewers Yeast SRM 1569, which has been certified for Cr content. The experimental design allowed for

the quantitative collection of volatile species in a thermallyheated vacuum distillation system over a temperature range of 150-325°C. A small fraction of the total Cu (<1%), about 251 of the total Hg and about 50% of the total Se were trapped and determined quantitatively. Arsenic, Ag and Au were also

observed in the trapped fraction.

INTRODUCTION

The accurate determination of Cr in biological samples has histori-

• I y been difficult. Analytical methods such as atomic absorption

pectroscopy and neutron activation have the required sensitivity to determine ng levels of Cr but reported analytical data on biological

materials, such as NBS Standard Reference Material (SRM) 1577 Bovine Liver,

have been widely divergent (5). More recent data have indicated that the current status of Cr analysis has not greatly improved (6). It has been suggested that part of the analytical error may be due to the presence of

a volatile organic species which may be lost in certain sample-preparat ion procedu res (7,8).

In general, analytical verification of volatile trace elemental

species in biological systems has been limited. Elements such as Hg and

Pb have been shown to form elemental or simple molecular species which

vaporize upon mild thermal treatment. The awareness and confirmation of

possible vaporization losses of other metals which are not normally

considered volatile is becoming of increasing importance. Two recent studies have reported no Cr losses during ashing and acid digestion of

Reprinted from Trace Substances In Environmental Heulth-XI. 1977. A symposium. D. D. Hemphill, Ed., (c) University of

Missouri. Columbia.

40
biological samples to which radiotracers of C

i— 51 were added (5,6). A more recent study has reported as much as 30% volatilization loss of Cr from neutron activated brewers yeast samples during dissolution with

concentrated acids (4 )

.

In an effort to resolve the question of Cr volatility, a vacuum distillation experiment has been designed and conducted on a new NBS Standard Reference Material, SRM 1569 Brewers Yeast, which is certified

f-or total Cr content. The experimental design enabled volatile species

to be collected from samples heated in the range of 150-325°C, and for the quantitative determination of those elemental species amenable to neutron

activation. In addition to Cr volatility losses were found and quantified

for mercury, Se and As. Other elements such as Br and I were observed to volatilize but were not quantified due to experimental conditions of the NA procedure.

MATERIALS AND METHODS

The basic experimental system is pictured in Figure 1. The system

consists of a tube furnace (Lindbera-, Type JJ035 Hevi-Duty, 3 0 0 W , Watertown, Wl*) used to heat a 19 mm i.d. quartz combustion tube containing the sample and a thermometer One end of the Quartz tube was closed with a standard taper ground glass removable stopper. The other end was fitted

with a second standard taper joint, to which was sealed a 30 -cm piece of

8 mm i.d. quartz tube. This extension was passed straight through a liquid nitrogen ( LN 2 ) trap and was used to collect the distillate. The

trap was constructed of styrofoam with holes bored through the sides to seal to the collection tube. The collection tube was connected to a vacuum pump with rubber vacuum tubing. A second liquid N trap was placed

between the pump and the collection tube to prevent back streaming of pump

oil vapors and other contaminants.

COMBUSTION

TUBE

FIGURE 1- VACUUM AND COLLECTION SYSTEM.

In conducting each experiment, the quartz collection tube was first

rinsed several times with ultrapure HC1 and HNO 3 to remove trace element

I n order to adequately describe materials and experimental procedures, it wa s occas i on a 1

1

y neces sa ry to identify commercial products by manufacturer's

name or label. In no instance does such identification imply endorsement

by the National Bureau of Standards nor does it imply that the particular

products or equipment is necessarily the best available for that purpose.

41
contamination from the inner surface (3)- The combustion tube was also

cleaned and purged by heating to 800°C with a low flow of oxygen through

it. The collection tube was then sealed to the sample tube and the system

again purged. Following the precleaning procedure, approximately one g of MBS SRM 1569 Brewers Yeast was weighed into an aluminum foil sample boat. The sample was placed into the combustion tube along with a thermometer

whose bulb rested against the sample boat. The collection tube was then connected to the vacuum system, liquid N was added to the collection trap, and the system was evacuated.

The furnace was preheated to the desired temp and placed around the sample tube. The entire system was then heated to the desired equilibration temp and maintained at that temp (+ 5°C) for 15 min. During this 15" min period the entire system, up to the LN 2 condenser, was heated gently

with the oxygen-gas torch to prevent condensation of the vapors outside

of the collection trap. Care was taken to prevent decomposition of the vapors due to excessive heat in the tranfer line. The volatilized

material from the sample was collected in the liquid N trap portion of the collection tube. For several of the higher temp runs, the trap had to be periodically moved several mm closer to the sample tube to prevent a solid

plug of condensed material from forming inside the collection tube. All of the condensed material, however, remained at liquid N temp throughout

the run. At the end of the 15 - min collection period, the furnace was removed

and the combustion tube allowed to cool. The collection tube, while still

under vacuum, was sealed off between the combustion tube and the liquid N collection trap. The other end of the collection tube was then sealed

with the sample still frozen and under vacuum. The sealed samples were

then set aside for irradiation and analysis.

The analytical portion of the experiment was conducted by non- destructive neutron activation. For analysis, A of the sealed quartz

tubes containing the condensed volatile material were irradiated together

with one quartz tube containing 300 pi of a mixed trace element water

standard. The A samples were placed in a single irradiation container so that one sample from each of the temperature levels, ranging from 150-300°

C, were irradiated together. The samples and standard were irradiated for

A hr at a thermal neutron flux of "v 6xl0 13 n-cm" 2 sec" 1

. After irradiation,

the samples were allowed to decay for 2 days to eliminate short halflived radioactivity and counted on a large volume Ge(Li) detector coupled

to a A096 channel pulse height analyzer. The samples were then allowed

to decay for 2 wk and were recounted to determine those elements with

half lives greater than a few days. The resultant gamma-ray spectra were

recorded on magnetic tape and processed off-line by computer. The individual photopeaks were quantified by the total peak area method. Quantitative results were obtained by direct comparison of the specific activity

•f the samples and standard.

RESULTS Detectable amounts of 6 trace elements were observed in trapped

fractions distilled from the brewers yeast. The amount of each element

trapped was dependent upon the maximum temp to which the sample was heated.

The quantities of those trace elements observed were normalized to the weight of yeast used in individual experiments and are given in Table I. The amount of each element in the trapped fraction increased with

increasing temp in all cases except Ag. However, the quantity of the element volatilized and the dependence on temp varied among the individual

elements, as shown graphically in Figures 2-5. The data in Table II reflect elemental concentrations in the yeast

residue relative to those in the original unheated yeast sample. These

data are corrected for total wt loss of material from the sample due to

42
vacuum distillation. The elemental data are given relative to initial

yeast concentration because the absolute trace element content of the SRM Yeast has been determined only for Cr. Approximately 50% of the total Se

about 25% of the total Hg and less than 1% of the total Cr content were

lost at the 300°C temp. The total weight loss of material was between 30 and A0% at that temp. For Cr these data are consistent with the observed

volatility data. Although a volatile Cr component was observed, the quantity, "v.003-. 005 pg/g, was within measurement error of the total certified

Cr content in the yeast. Scandium, Fe and Co were not detected in the trapped fraction and no differences were observed in any residue samples

compared to initial samples.

TABLE I. TRACE ELEMENTAL LOSS FROM BREWERS YEAST

BY VACUUM DISTILLATION

pg trapped/g sample

Temp °C

E 1 emen t 150 190 205 250 295 305

Hg 0.001 A 0.006 ... 0.017 0. 10 0.055

Se 0.0013 0.0027 0.0018 0.036 0. 16 0.23

C r 0.0028 0.0021 0.0021 0.0050 0.0038 0.0052

As 0.06A 0.060 0.050 0.10 0.22 0.32

Ag 0.00A5 0.0005 0.0028 0.0027 0.0010 0.00A2

Au 0.00010 ... 0. 00006 0.000A0 ... ...

TABLE II. RELATIVE ELEMENTAL COMPOSITION OF YEAST RESIDUE

Concentrations Relative to Temp °C Unheated Yeast

E 1 emen t 0 0 200 265 290 305

Sc 0.996 1.01 0.992 1.00 1 .00 1.02

Fe 1 .00 0.983 1.00 1 .00 1 .01 0.983

Co 1 .00 1.00 0.905 1.01 0.910 0.933

C r 1.01 0.990 1 .06 0.920 1.02 1 .06 Hg 0.990 1.01 XXX 1.01 1 .06 0.7A

Se 1 . 0A 0.960 1.12 0.6A 0.AA 0.57

% wt loss 0 0 7% 22% 39% 32%

DISCUSSION

The appearance of detectable amounts of several trace elements in the vacuum distillate of a sample of brewers yeast suggests the presence of volatile elemental species in biological materials. For some metals such

as Hg , these species may volatilize at temps lower than 150°C, while for the other elements observed in this work the volatilization began between

200 and 250°C. This is also the temp at which visible charring of the brewers yeast occurred (Table ll). Thus, the volatility and loss of these elements appears in general to be more of a thermal degradation of the matrix with concomitant release or decomposition of the naturally

occurring metallic species, resulting in a volatile product. The

A3
analytical significance of these losses, especially in methods which rely upon thermal decomposition of the organic matrix prior to solubilization

and analysis, is obvious. For example, the data from this study indicate

that up to 50% of the total Se content can be volatilized upon moderate

heat ing.

TEMPERATURE »C

FIGURE 2- COLLECTED VOLATILE MATERIAL VS. TEMPERATURE-MERCURY.

Figures 2-5 show some very interesting trends. If one assumes that

the ideal pattern for volatilized material would follow a curve as depicted in Figure 6, there is a temp. A, below which no loss occurs. At temp A, losses begin due to natural volatility or thermal degradation. At any temp above A, the amount of volatilized trace element will be determined by the rate of formation, vapor pressure of the volatilized species

and he length of time at that temp. In this experiment the length of

time at the maximum temp was held constant; therefore, the amount of

material volatilized was dependent upon the rate of formation of the volatile species, up to the limiting total amount of that species or its precursor. At point B in Figure 6, the limiting condition has been reached and no more material is volatilized.

44
TEMPERATURE *C

FIGURE 3- COLLECTED VOLATILE MATERIAL VS. TEMPERATURE-SELENIUM.

If we examine the experimental data for the different elements in terms of this idealized curve, a consistent trend is apparent. For Hg

(Fig. 2) we see a logarithmic increase throughout the temp range studied,

indicating that we are between transition points A and B for this element.

The initial volatilization temp for Hg is below 150°C. At 300°C, a significant portion of the total volatile Hg was not vaporized in the 15* min distillation period; thus point B had not been reached. This interpretation is consistent with the Hg loss data in Table II. The data for Se (Fig. 3) show the entire curve as idealized. The initial volatilization point is around 175°C and we see that the amount

of trapped Se appears to be reaching a maximum at 300°C. Thus, a significant portion of the total volatile species has been vaporized in 15 min

at 300°C. Again, the loss data in Table II are consistent in that approximately 50& of the total Se in the sample was lost. This implies that there are at least 2 different fractions or species of Se in the brewers

yeast and that they are approximately equal in amount. With the knowledge

that different chemical species or forms may have widely differing effects

45
or functions in biological systems, this confirmed multiplicity of Se species is of great significance.

TEMPERATURE «C

FIGURE 4— COLLECTED VOLATILE MATERIALS VS. TEMPERATURE-ARSENIC.

For As (Fig. 4) we see the beginning of the idealized curve with

transition point A at about 200°C . At the maximum temp of 300°C we have

not yet volatilized a significant portion of the total As species as the curve is still increasing logarithmically.

The amounts of trapped Cr (Fig. 5) are significantly higher for the samples collected above 200°C than for those collected below that temp

although the absolute amounts are very low. These data show that about

5*10 ng/g of Cr is volatilized by thermal degradation between 150°C and

300°C. Although this is a very small fraction of the total content, it signifies a fraction of Cr in this sample which is chemically different

from tne bulk. It has been shown that determination of Cr in biological

materials by thermal heating and atomization in graphite furnaces is very susceptible to chemical form and matrix in which it occurs (7,8). This

existence of a fraction of Cr that can be directly distilled supports

those earlier findings. In the studies which reported no losses of Cr during ashing and acid digestion with added radiotracer (1,2) there was no

46
assurance that the Cr-51 tracer was present in, or had exchanged with,

the organic forms of Cr in the samples. Also, the experiments were

designed to determine the amount of Cr retained in the sample after treat- ment, but were not specifically designed to determine small Cr losses via

volatilization. In the recent study reporting loss of up to 39% of the Cr during dissolution with concentrated HCIO 4 and HNO 3 , mixtures were collected and the losses due to volatility were quantitatively verified

(4). Due to the prior neutron irradiation and the severe conditions of

acid dissolution, it is highly probable that the Cr species originally

present in the sample was changed during the procedure. In our studies,

the sample was subjected to only a thermal stress and no chemical treatment was carried out. These studies do reemphasize the importance of initial chemical speciation and matrix upon the potential for formation of

volatile species during thermal or chemical reactions.

TEMPERATURE *C

FIGURE 5- COLLECTED VOLATILE MATERIAL VS. TEMPERATURE-CHROMIUM.

In summary, we have shown that chemical species of several trace elements exist in brewers yeast which can be volatilized by thermal

heating under vacuum distillation, and that the temperature dependence of

the quantitative distillation varies among the different trace elements.

47
We have also shown that in a given biological material, some trace elements exist in more than one chemi ca 1 state. The potential exists for

use of this type of differential pyrolysis or fractional distillation in studies of chemical speciation of these trace elements.

TEMPERATURE *C

FIGURE 6- IDEALIZED VACUUM DISTILLATION CURVE.

LITERATURE CITED

Jones, G. 8., R. A. Buckley and C. S. Chandler. 1975. The volat i 1 -

i ty of chromium from brewers yeast

80:389-392.

during assay. Anal. Chem. Acta

2. Koirtyohann, S. R. and C. A. Hopkins. 1976. Losses of trace metals

during the ashing of biological materials. Analyst 101:870-875-

3. Kuehner, E. C., R. Alvarez, P. J. Paulsen and T. J. Murphy. 1972. Production and analysis of special high purity acids purified by

sub-boiling distillation. Anal. Chew. kh: 2050-2056.

A. McClendon, L. T. Determination of chromium in biological matrices

using NAA application of SRM's. Radio Anal. Chew. 8: (Accepted for pub 1 i cat i on )

.

5. McClendon, L. T. 197A. Selective determination of chromium in biological and environmental matrices. In: Trace Substances in

48
Environmental Health - VIII, D. D. Hemphill, Ed., University of

Missouri, Columbia, pp. 2 5

5 ~ 2 5 7

6. Parr, R. 1976- Problems of chromium analysis in biological

materials: An international perspective with special reference to results for analytical quality control samples. In: Proc. Int. Conf. Modem Trends in Activation Analysis (to be published).

7. Wolf, W. , W. Mertz and R. Masironi. 1974. Determination of chromium

in refined and unrefined sugar by oxygen plasma ashing by flameless

atomic absorption. J. Ag. Food Chem. 22:1037-

8. Wolf, W. 1976. Preparation of biological materials for chromium

analysis. In: Proc. 7th Materials Research Syrrrp. , NBS Special

Publication 422, 605"610, U.S. Government Printing Office, Washington.

DISCUSSION

Inquirer: W. R. Faulkner, Vanderbilt University, Nashville, TN

Q. Is it likely that vol Cr might be appreciable at temps greater than 305°C and less than charring temps?

A. Not likely because Cr cone is beginning to plateau at temps less than

305°C. There was difficulty because of clogging of trap at temps

greater than 305°C. Inquirer: Richard A. Peabody, V. A. Hospital, Albany, NY

Q. Has chromyl chloride been observed directly in your analytical system

for volatile components, presumably formed by interaction of HC1 with

Cr in digests?

A. No, not in our system. The procedure used in this study was vacuum distillation of volatile materials directly from the brewers yeast.

No acid digestion, using HC1 or any other acids, was used. Also, the neutron activation technique used to quantify the amounts of trace elements volatilized would not detect chemical compounds, such as chromyl chloride; it is a total elemental technique.

49
Appendix III

Chemical Preparation of Biological Materials

for Accurate Chromium Determination by

Isotope Dilution Mass Spectrometry

Lura P. Dunstan and Ernest L. Garner

National Bureau of Standards

Washington, D. C.

ABSTRACT

The current interest in trace elements in biological

materials has created a need for accurate methods of analysis.

The source of discrepancies and variations in chromium concentration determinations is often traceable to inadequate methods

of sample preparation. Any method of Cr analysis that requires

acid digestion of a biological matrix must take into consideration the existence or formation of a volatile Cr component. In addition, because Cr is often present at concentrat ions less than 1 pg/g, the analytical blank becomes a potential source of

error.

Chemical procedures have been developed for the digestion

of the biological matrix and the separation of Cr without either

large analytical blanks or significant losses by vol at i 1 i za t i on

These procedures have been used for the analysis of NBS Standard

Reference Material (SRM) 1569 Brewers Yeast; SRM 1577 Bovine

Liver; SRM 1570 Spinach and other biological materials including

human hair and nails. At this time, samples containing 1 pg of

Cr can be determined with an estimated accuracy of 2 %.

INTRODUCTION

The accurate determination of chromium in biological materials

presents a considerable challenge to the analytical chemist. Because the concentration of Cr is often less than 1 pg/g, it is imperative that the analytical method be sensitive and non-contaminating. Isotope dilution

mass spectrometry (iDMS) is capable of precise and accurate concentration

determinations on submicrogram amounts of an element, and as a result, a major limitation of this method is the magnitude and variability of the analytical blank. Through the use of clean room facilities, pure reagents

and apparatus fabricated from quartz and Teflon, the variability of the

Cr analytical blank can be maintained at a level of less than 10 ng. Control of the analytical blank at this level permits the determination of

1 pg of Cr with an estimated accuracy of +2%. For biological samples, accuracies of this magnitude are not only

dependent on controlling the analytical blank, but on preventing loss of

the volatile Cr compounds during the wet oxidation of the matrix. The presence of volatile Cr compounds in various biological materials has been discussed (3, A) and it is suspected that many of these compounds may volatilize at low temperatures. In addition, the formation of volatile

Cr compounds during the wet oxidation process must be avoided. For this reason, perchloric acid should not be used since the formation of chromyl

chloride is likely to produce losses of Cr. The loss of other volatile

components can be controlled by performing the wet oxidation procedure in a quartz reflux system.

Reprinted from Trace Substances In Ens ironmental Healih-XI 1977 A symposium. D D. Hemphill. Ed.. © University of

Missouri. Columbia.

50
Once the digestion of the matrix is complete and the spike and natural Cr have equilibrated, it is no longer necessary to maintain

complete recovery. However, recoveries greater than 80% are essential for

the accurate determination of ng amounts of Cr, since a few ng of blank

are sufficient to significantly bias the concentration measurement. Thus,

understanding and controlling the effects of the analytical blank and the

loss of volatile Cr compounds are the major factors in the accurate determination of Cr in biological materials by IDMS.

MATERIAL AND METHODS

The dissolution, separation and purification of all samples were

performed in a Class 100 clean air environment to minimize particulate

contamination. With the exception of the ceric sulfate solution, all reagents used for these analyses were purified by sub-boiling distillation

(2). The ceric sulfate was prepared from high purity cerium (IV) oxide by several evaporations with sulfuric acid.

The 50 Cr spike, a 961 enriched isotope, was calibrated by comparison

with 2 different solutions of high purity natural Cr. Weighed aliquots

of the spike and natural solutions were mixed and equilibrated and the concentration of the spike was determined by IDMS. The quartz reflux system used for the wet oxidation of the organic

matrix consisted of a 125 ml Erlenmeyer flask fitted with a quartz condenser. The refluxing action was aided by filling the condenser with quartz

chips and by running chilled water through the condenser jacket.

The chemical separation procedure is similar for all biological

materials discussed in this paper. The sample is placed in a quartz

Erlenmeyer flask and 50 Cr spike is added to alter the natural 50 Cr/ 52 Cr ratio. Sample wts ranging from 1 g for spinach and brewers yeast up to 5

g for blood serum were used. The flask is then fitted with a condenser

and chilled water is run through the jacket for approximately 1/2 hr. Ten

g of concentrated ( 70% ) nitric acid are added through the top of the con- denser and the temp is increased gradually to avoid excessive foaming and frothing of the sample. After any initial reactions have ceased, 9 g of

concentrated ( 38%) sulfuric acid are added. This mixture is digested for approximately 5 hr while gradually increasing the temp to *v250°C. The condenser jackets are then drained and the refluxing action is continued

overnight. In cases where silica is present in the matrix (e.g. brewers

yeast and spinach), the contents of the flask are transferred to Teflon

beakers and treated with 1 g of concentrated (361) hydrofluoric acid.

The Cr is then oxidized with ceric sulfate, separated by extraction

with methyl isobutyl ketone and back extracted into water. The resulting

solution is evaporated to dryness and the purified Cr is analyzed by

surface ionization mass spectrometry.

All isotopic ratio measurements were made using a 15 - cm radius of curvature, solid sample, single focusing mass spectrometer. The details

of the tungsten filament surface ionization procedure for Cr will be published (1). All isotopic ratio data were corrected to the absolute

isotopic composition of a Cr isotopic reference standard (SRM 979) which

was analyzed under the same conditions as the samples.

RESULTS AND DISCUSSION

The results of Cr determinations on various biological materials are shown in Tables 1-1 II. The effect of the blank varies with the concentration level, and as the amount of Cr determined approaches the submicrogram range, the accuracy of the method becomes primarily dependent upon

the magnitude and variability of the blank. The uncertainty for the determination of Cr in Brewers Yeast SRM 1569 (Table I) is the estimated

accuracy and includes allowances for the precision of the ratio measure51
ment, the calibration of the 50 Cr spike, the variability of the Cr analytical blank and sample inhomogeneity.

TABLE I. CHROMIUM CONCENTRATION IN SRM 1569 BREWER'S YEAST

Bottle No. yg Cr/g

5 2.07

127 2.10

1 28A 2.09

15A 2.08

3A0 2.23 253A (1) 2.13 253A (2) 2.12 Average 2.12 + 0.13'

a The uncertainty (ts) is the estimated accuracy and includes allowances for the precision of the ratio measurement, the calibration

of the 50 Cr spike, the variability

of the analytical blank and sample

i nhomogene i ty

.

TABLE II. CHROMIUM CONCENTRATION IN SRM 1577 BOVINE LIVER

No. of

Bottle No. Determi nat ions ng Cr/g

1 A 95.6

2 3 85.3

3 2 89-3

A A 97-8

Average 92.

0

a

Analytical blanks averaged 1

3

7 ng

TABLE III. CHROMIUM CONCENTRATION IN OTHER BIOLOGICAL MATERIALS

Matrix yg Cr/g SRM 1570, Spinach A. 67

Ha i r 2.65 Toena i 1

s

2.93 Blood serum 0.008

52
Since both SRH 1569 Brewers Yeast and SRM 1577 Bovine Liver were

suspected of containing volatile Cr compounds, an attempt was made to check for possible losses. This process involved taking additional

samples of each SRH, which were spiked after the wet oxidation of the matrix had been completed. Any losses of Cr up to this point in the analysis should produce a measured ratio depleted in natural Cr and thus yield lower concentration data. In both cases discussed above, the Cr concentrations determined by spiking before and after dissolution were

within the estimated uncertainty of the analysis and as a result it would

appear that if a volatile constituent is being lost, the amount is probably ve ry sma 1 1

.

The bovine liver data in Table II exemplify the analytical problems

encountered when analyzing ng levels of Cr. These measurements were part

of a preliminary experiment to evaluate the IDMS technique for Cr in the bovine liver matrix and to test for sample inhomogeneity. Two-g samples

of the liver were analyzed so that the total amount of Cr was approximately 200 ng. However, because the variability of the analytical blank

was +7 ng , the accuracy was estimated to be + 4% for each determination.

At this concentration level, the effect of even relatively small changes

in the blank made it difficult to ascertain the cause of sample variations.

Table III contains a summary of concentration determinations on other

biological materials. The blood serum represents the lowest Cr concentration determined to date. Although 5

_ g aliquots of serum were taken for analysis the magnitude of the analytical blank was 25% of the total Cr in the sample.

CONCLUSION

By employing techniques that minimize the magnitude and variability

of the analytical blank as well as losses of volatile Cr compounds, the concentration of Cr in biological materials can be accurately determined.

Although the magnitude and variability of the analytical blank becomes

increasingly significant as the amount of Cr approaches the low ng range, uncertainties of 2% or less are possible for the determination of at least one pg of C r

.

ACKNOWLEDGMENTS

We wish to express our appreciation to Joy J. Shoemaker and Karen A. Brletic for manuscript preparation, to William A. Bowman, III for instru- ment maintenance support and to our colleagues of the Analytical Spectrometry Section for their helpful discussions.

LITERATURE CITED

1. Dunstan, L. P. and E. L. Garner. 1977- The determination of

chromium by surface ionization mass spectrometry. (in preparation).

2. Kuehner, E. C., R. Alvarez, P. J. Paulsen and T. J. Murphy. 1972. Production and analysis of special high-purity acids purified by sub- boiling distillation. Anal. Chem. 44 : 2050-2056

.

3- Wolfe, W. R. and F. E. Greene. 1976. Preparation of biological

materials for chromium analysis. In: Accuracy in Trace Analysis

:

Sanpltng, Sample Handling, .Analysis. P. 0. LaFleur, Ed., NBS Spec.

Publ. 422, Vol. I, U.S. Government Printing Office, Washington,

pp. 605-610.

k. Wolfe, W. R., W. Mertz and R. Masironi. 1974. Determination of chromiun in refined and unrefined sugars by oxygen plasma ashing

flameless atomic absorption. J. Ag. Feed Chem. 22:1037-1042.

53
Appendix IV

The Determination of Zinc, Cadmium and

Lead in Biological and Environmental Materials

by Isotope Dilution Mass Spectrometry

J. W. Gramlich, L. A. Machlan

T. J. Murphy and L. J. Moore

National Bureau of Standards

Washington, 0. C.

ABSTRACT Techniques have been developed for the accurate and pre- cise determination of the concentration of zinc, cadmium and

lead using thermal ionization mass spectrometry. These tech- niques have been applied to the analysis of a variety of biotogical materials such as blood, hair and nails, and to environmentally related materials such as water, air particulates and

foss i 1 fue 1 s

Uncertainty in the accuracy of the isotope dilution method

is less than 0.5% for all 3 elements, and is nearly independent

of element concentration from the % level down to the ng/g (ppb) range where the analytical blank becomes a significant contribution to the uncertainty.

The mass spect romet r i c methodology and chemical separation

procedures are presented along with examples showing the effect

of sample impurities on the quality of the analytical data.

INTRODUCTION

The toxicity of cadmium and lead, and the associated hazards resulting from increased pollution of the environment by these elements, has received considerable attention during the past decade. The serious patho

logical effects of Cd (3,8) and the hematological disturbances associated

with lead poisoning (2) have been extensively reviewed in the literature.

Zinc is an essential element for growth and maintenance of body functions;

it is a constituent of several enzymes and a cofactor for certain enzy- matic reactions. There is ample evidence to suggest that the metabolisms

of Cd and Zn are closely related and that Cd has the ability to exchange

with Zn and thus produce changes in enzymatic activity (3)- The close

biochemical and geochemical association of Zn and Cd make their collective

study in biological and environmental systems highly desirable.

As the concentration of these trace elements approaches the ng/g

(ppb) range, it becomes increasingly difficult to maintain accuracy and

precision in routine analytical measurements. Well characterized trace element standard materials are essential to ensure quality control and to develop or verify analytical methods. Although not suited for routine

analysis because of the time and expense involved, isotope dilution mass spectrometry provides an extremely valuable tool for trace element analysis of standard materials, for refereeing discrepancies between other

analytical methods and for determining small sample inhomogeneities.

Samples to be analyzed are spiked with a known amount of a separated

isotope of the element of interest. After dissolution of the sample and

equilibration of sample and spike, quantitative recovery is no longer necessary because only the altered isotopic ratio is of interest. Herein

Reprinted trom Trace Substances In Environmental Health-XI. 1977. A symposium. D. D. Hemphill, Ed.. © University of

Missouri. Columbia.

54
lies a major advantage of the isotope dilution technique over other

analytical methods. Because only a representative portion of the sample

is needed for analysis, rigorous chemical purification can be employed to obtain a high purity, matrix-free sample that can be compared directly

with pure standards of known absolute or relative isotopic abundances.

The high degree of sensitivity afforded by the mass spect romet r i

method allows the concentrations of Zn, Cd and Pb, as well as many other

elements, to be determined with both high accuracy and precision at con- centrations below one mg/g. The practical lower limit of concentration measurements is governed by the magnitude and variability of the analytical b 1 ank

MATERIALS AMD METHODS Accurate mass spect romet r i c measurements require a high purity sample

for analysis. Trace amounts of impurities may result in reduced signal

intensity and alteration of the filament fractionation pattern (A). In addition, isobaric interferences from Ge , Ni, Pd, Sn and In may affect the determination of Cd and Zn. Although the exact chemical separation and

purification procedures may vary somewhat, depending on the particular

mdti ia, trie loiiowiiiy (jiocedure is generally applicable. A known amount

of sample is spiked with weighed aliquots of 67 Zn, J11 Cd, and 206 Pb solutions. The sample is then digested with a mixture of hydrochloric,

nitric and perchloric acids. After dissolution and equilibration of the sample and spike, the 3 elements are separated from each other and the matrix by anion exchange chromatography. Further purification of Pb and

Cd is accomplished by electrodeposition: Pb by anodic deposition as Pb0 2 (1) and Cd by cathodic deposition from an ammoniacal solution. Further

purification of Zn is frequently necessary to remove traces of Fe which

interfere with the mass spect romet r i c analysis. This is accomplished by

additional anion exchange chromatography.

Since the limiting factor for the accuracy of trace element analysis

is often the magnitude and variability of the analytical blank, all chemical preparation is conducted in a clean room facility (7) using acids and water prepared in this laboratory by sub-boiling distillation (5). A class 100 clean air environment is also utilized to dry the samples on the mass spectrometer filaments.

All analyses reported here were performed on 30 cm radius, 90° mag- netic sector mass spectrometers utilizing thin lens z-focusing sources and

Faraday cage collectors equipped with 50? transmission grids shadowing a series of suppression grids (10). The suppression grids are designed to provide cubic suppression of secondary particles between the transmission

grid and the collector-defining slit. The remainder of the measurement

circuits consisted of a vibrating reed electrometer, voltage to frequency

converter, scaler and programmable calculator. Timing, magnetic field

switching and data acquisition were controlled by the programmable calculator.Zinc, Cd and Pb were analyzed from separate loadings onto single

filament rhenium sources employing the silica ge 1 -phosphor i c acid tech- nique for ionization enhancement (1,9)-

RESULTS AMD DISCUSSION

The concentration of Pb at or below the ug/g level has been routinely

determined in this laboratory for several years. Samples relevant to this

discussion include porcine, bovine and human blood (0.03-1 ug/g), human

hair and nails (28 and 1.6 -g/g respectively), botanical materials (1-10

Mg/g) , gaso 1 i ne (1 2-700 _g/g) and wa te r (0 . 02- 1 ug/g) . The determination

of Zn and Cd concentrations by isotope dilution has been recently undertaken, based on results which indicated that sufficient signal intensities

could be generated using the silica ge 1 -phosphor i c acid technique (6,9)

55
Cadmium produces signal intensities and stabilities comparable to those obtained with Pb; however, the ionization appears to be easily

poisoned by trace impurities, especially Zn. Electrodeposition as a final purification step has been found necessary to obtain maximum signal intensity. The analytical blank for Cd is less than 1 ng, which is substantially better than the typical 1-5 ng blanks for Pb. Measurement precision at present appears to be in the range of 0.11-0.2% (see Table 111).

Zinc produces a signal intensity at least an order of magnitude

lower than Cd or Pb. This fact, combined with relatively high and variable blanks, restricts the practical concentration limits to samples con- taining more than 100 ng of Zn. Fortunately, Zn is abundant in nature and samples below the u/g level are not prevalent. Further research is expected to identify and reduce the sources of the blank. The results obtained by isotope dilution mass spectrometry on 2 new candidate NBS Standard Reference Materials are reported in Tables I and II. These SRMs should be of particular interest to those involved in environmental monitoring.

TABLE I. ZINC, CADMIUM AND LEAD IN URBAN PARTICULATES (SRM 1648)

Bottle No. Percent Zn ug Cd/g Percent Pb AO 0. A769 76.06 0.6597

1A8 0.478A 75.65 0.6590

162 0. A730 77-98 0.6595

757 0. 4768 75-95 0.6598

808 0. A7'tl 76. 1

A

0.6579

1636 0.

A

76 I 76.05 0.661

1

Average O.A759 76.31 0.6595

Std. Dev. 0.0019 0.8A 0.0010

Range 0.005A 2.33 0.0032

TABLE II. ZINC, CADMIUM AND LEAD IN COAL (Cadidate NBS SRM 1632a)

Bottle No. pg Zn/g ug Cd/g ug Pb/g

1 26. 9A 0. 1688 12.21

2 26.88 0. 1698 12.35

16 27-09 0. 1692 12.10

93 27-82 0. 1 7A6 12.33

Average CO 04 0.1706 12.25

Std. Dev. 0. A3 0.0027 0.12 Range 0.9A 0.0058 0.25

56
TABLE III. REPETITIVE MASS CADMIUM AND LEAD

(Candi date

SPECTROMETRIC ANALYSES OF

IN COAL - BOTTLE NO. 1 NBS SRM 1632a)

ZINC,

pg Zn/g pg Cd/g pg Pb/g

26.932 0. 16895 12.206 26.938 0.16871 12.203 26.973 0.16896 12.205 26.919 0. 16869 12.206 26.914 0. 16893 12.204

Average 26.935 0. 16885 12.205

Std. Dev. 0.023 0.00014 0.001 Range 0.059 0.00027 0.003

As mentioned previously, tne precision of the isotope dilution method

allows the identification of sample inhomogeneities which are often masked

by the imprecision of other analytical techniques. The data in Table III represent 15 separate mass spect romet r i c analyses on a one g sample taken from bottle No. I of SRM 1632a. These data are typical of the precision

expected from the mass spect romet r i c measurements. Comparison of the data

in Table III with those in Table II indicates that the variation between

bottles is the result of sample inhomogeneity rather than measurement

imprecision. Statements on the accuracy of the concentration measurements

must include not only the measurement precision and blank uncertainty con- tribution, but also components to cover possible sources of systematic

errors such as the uncertainty in the concentration of the separated iso- tope and the isotopic composition of the separated isotope and sample.

Thus, although the mass spect romet r i c precision for Pb analyses is generally less than 0.1%, an accuracy statement of 0.2% is generally assigned

to the data. At present the accuracy of the Zn and Cd analyses is estimated to be + 0.5%. This value is expected to decrease as experience is gained and sources of possible systematic errors are further investigated.

CONCLUSIONS

Isotope dilution mass spectrometry is an accurate and precise analytical technique capable of determining Zn, Cd and Pb concentrations from

the % level into the ng/g concentration range where the analytical blank

becomes a limiting' factor in the accuracy. The technique is particularly

useful for the standardization of reference materials and for measuring

small sample inhomogeneities.

LITERATURE CITED

1. Barnes, I. L., T. J. Murphy, J. W. Gramlich and W. R. Shields. 1973- Lead separation by anodic deposition and isotope ratio mass spectrometry of microgram and smaller samples. Anal. Cherr. 45 : 1 88 1 - 1 884 . 2. de Bruin, A. 1971- Certain biological effects of lead upon the animal organism. Arch. Env. Elth. 23:2*9~261.

3. Friberg, L. T., M. Piscator, G. F. Nordberg and T. Kjellstrdm. 197**. Cadniurr. in the Environment, 2nd ed. CRC Press, Cleveland.

4. Gramlich, J. V. 1976. The effect of sample purity on mass spectrometric isotope ratio measurements. In: Pros. 24th Ann. Conf. Mass Scectronetry and Allied Tories, San Diego, pp. 498-499-

5. Kuehner, E. C., R. Alvarez, P. J. Paulsen and T. J. Murphy. 1972.

57
6 .

The production and analysis of acids purified by sub-boiling distillation. Anal. Chem. 44 : 2050-2056

.

Moore, L. J., J. W. Gramlich and L. A. Machlan. 1975- Application

of isotope dilution to the high accuracy trace analysis of environmental and health standards. In: Trace Substances in Environmental

Health - IX, D. D. Hemphill, Ed., University of Missouri, Columbia,

pp. 311-316.

7. Murphy, T. J. 1976. The role of the analytical blank in accurate

trace analysis. In: Accuracy in Trace Analysis: Sampling, Sample

Handling and Analysis, P. D. LaFleur, Ed., NBS Spec. Publ . 422, U.S. Government Printing Office, Washington, pp. 509-539-

8. Nilsson, R. 1969. Aspects on the Toxicity of Cadmium and its Compounds. Swedish Nat. Sci. Research Council, Ecological Research

Comm. Bull. 7, Stockholm.

9. Rosman , K. J. R. and J. R. DeLaeter . 1974. The abundance of cadmium

and zinc in meteorites. Geochim. Cosmochim. Acta 38:1665-1677-

10.

Shields, W. R., Ed. 1966. Analytical Mass Spectrometry Section:

Instrumentation and Procedures for Isotopic Analysis, NBS Tech. Note

277. U.S. Government Printing Office, Washington, pp. 1-99-

58
TECHNICAL REPORT DATA

(Phase read Instructions on the reverse before completing)

1, REPORT NO. 2. EPA-600/1-79-017

3. RECIPIENT'S ACCESSION NO.

4. TITLE AND SUBTITLE

THE NATIONAL ENVIRONMENTAL SPECIMEN BANK

RESEARCH PROGRAM FOR SAMPLING, STORAGE , AND ANALYSIS

5. REPORT DATE

May 1979

6. PERFORMING ORGANIZATION COOE

7. AUTHOR(S)

T.E. Gillis , H.L. Rook, R.A. Durst, Center for Ana.

Chemistry, and National Bureau of Standards Washington , DC

8. PERFORMING ORGANIZATION REPORT NO.

9. PERFORMING ORGANIZATION NAME AND ADDRESS

Clinical Studies Division

Health Effects Research Laboratory

Research Triangle Park, N.C. 27711

10. PROGRAM ELEMENT NO.

1HE775

11. CONTRACT/GRANT NO. EPA-IAG-D5-0568

12. SPONSORING AGENCY NAME AND ADDRESS

Health Effects Research Laboratory HERL , RTP

Office of Research and Development

U.S. Environmental Protection Agency

Research Triangle Park, N.C. 27711

13. TYPE OF REPORT AND PERIOD COVERED

14. SPONSORING AGENCY CODE

EPA 600/11

15. SUPPLEMENTARY NOTES

16. ABSTRACT

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

state-of-the-art protocols for sampling, storage, and analysis of biological

and environmental- type matrices. This report is a compilation of research

papers and/or efforts describing developed or adopted procedures for retrospective analysis of biological and environmental samples. Preliminary protocols

for sampling, sample handling, and sample storage are given for human liver

autopsy tissue iri addition to methods for the accurate measurement of selected

toxic elements in biological and environmental materials. Analytical methods

employed were neutron activation analysis (NAA) , polarography , and isotope

dilution spark source mass spectrometry (IDSSMS)

This report is submitted in partial fulfillment of EPA Interagency agreement EPA-78-D-X0105 by the National Bureau of Standards for the 1978 contract

year.

7. KEY WORDS AND DOCUMENT ANALYSIS

DESCRIPTORS b. IDENTIFIERS/OPEN ENOED TERMS c. COSATl Field/Group

toxic elements

polaro graphy

environmental samples

lung tissue

I1

06 ,c ,e ,f ,t

07 ,c

|I8. DISTRIBUTION STATEMENT

RELEASE TO PUBLIC

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19. SECURITY CLASS , This Report)

UNCLASSIFIED

21 . NO. OF PAGES

63

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