AlllDE 7fiTlDS

NBS SPECIAL PUBLICATION 740

i NBS

PUBLICATIONS

NATL INST OF STANDARDS & TECH R.I.C.

A1 11 027891 05

/Progress In environmental specimen bank QC100 .U57 NO.740 1988 VI 988 C.2 NBS-PUB

PROGRESS IN ENVIRONMENTAL

SPECIMEN BANKING

STEPHEN A. WISE

ROLF ZEISLER

GEORGE M. GOLDSTEIN

EDITORS

f

QC

100

U57

1988

c.2

1

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS
Tm he National Bureau of Standards' was established by an act of Congress on March 3, 1901. The Bureau's overall goal is to strengthen and advance the nation's science and technology and facilitate their effective application for public benefit. To this end, the Bureau conducts research to assure international competitiveness and leadership of U.S.

industry, science arid technology. NBS work involves development and transfer of measurements, standards and related science and technology, in support of continually improving U.S. productivity, product quality and reliability, innovation

and underlying science and engineering. The Bureau's technical work is performed by the National Measurement

Laboratory, the National Engineering Laboratory, the Institute for Computer Sciences and Technology, and the Institute for Materials Science and Engineering.

The National Measurement Laboratory

Provides the national system of physical and chemical measurement;

coordinates the system with measurement systems of other nations and

furnishes essential services leading to accurate and uniform physical and

chemical measurement throughout the Nation's scientific community,

industry, and commerce; provides advisory and research services to other Government agencies; conducts physical and chemical research; develops,

produces, and distributes Standard Reference Materials; provides

calibration services; and manages the National Standard Reference Data

System. The Laboratory consists of the following centers:

• Basic Standards^

• Radiation Research

• Chemical Physics

• Analytical Chemistry

The National Engineering Laboratory

Provides technology and technical services to the public and private sectors

to address national needs and to solve national problems; conducts research

in engineering and applied science in support of these efforts; builds and

maintains competence in the necessary disciplines required to carry out this research and technical service; develops engineering data and measurement

capabilities; provides engineering measurement traceability services; develops test methods and proposes engineering standards and code

changes; develops and proposes new engineering practices; and develops

and improves mechanisms to transfer results of its research to the ultimate

user. The Laboratory consists of the following centers:

Applied Mathematics

Electronics and Electrical Engineering^

Manufacturing Engineering

Building Technology

Fire Research

Chemical Engineering^

The Institute for Computer Sciences and Technology

Conducts research and provides scientific and technical services to aid Federal agencies in the selection, acquisition, application, and use of

computer technology to improve effectiveness and economy in Government

operations in accordance with Public Law 89-306 (40 U.S.C. 759),

relevant Executive Orders, and other directives; carries out this mission by

managing the Federal Information Processing Standards Program,

developing Federal ADP standards guidelines, and managing Federal

participation in ADP voluntary standardization activities; provides scientific and technological advisory services and assistance to Federal agencies; and

provides the technical foundation for computer-related policies of the Federal Government. The Institute consists of the following divisions:

Information Systems Engineering

Systems and Software

Technology

Computer Security

Systems and Network

Architecture

Advanced Computer Systems

The Institute for Materials Science and Engineering

Conducts research and provides measurements, data, standards, reference

materials, quantitative understanding and other technical information

fundamental to the processing, structure, properties and performance of

materials; addresses the scientific basis for new advanced materials technologies; plans research around cross-cutting scientific themes such as nondestructive evaluation and phase diagram development; oversees Bureau-wide technical programs in nuclear reactor radiation research and

nondestructive evaluation; and broadly disseminates generic technical information resulting from its programs. The Institute consists of the following Divisions:

• Ceramics

• Fracture and Deformation^

• Polymers

• Metallurgy

• Reactor Radiation

'Headquarters and Laboratories at Gaithersburg, MD, unless otherwise noted; mailing address Gaithersburg, MD 20899. ^Some divisions within the center are located at Boulder, CO 80303.

'Located at Boulder, CO, with some elements at Gaithersburg, MD
Itesearch Informatiaa Center

National Bureau of Standards

Gaithersburg, Marylaaid 2.0899

Progress in Environmental

Specimen Banlcing

Stephen A. Wise\ Rolf Zeisler\ and George M. Goldstein^, Editors

^Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, MD 20899

^Health Effects Research Laboratory

U.S. Environmental Protection Agency

Research Triangle Park, NC 21771

Research Supported in part by.

Office of Health Research

Office of Research and Development

U.S. Environmental Protection Agency

Washington, DC 20460

April 1988

U.S. DEPARTMENT OF COMMERCE, C. William Verity, Secretary

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director
Library of Congress Catalog Card Number: 88-600530

National Bureau of Standards Special Publication 740

Natl. Bur. Stand. (U.S.), Spec. Publ. 740, 217 pages (Apr. 1988)

CODEN: XNBSAV

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 1988

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402-9325
FOREWORD

The American Specimen Bank Project has gone successfully through both

feasibility and pilot phases, and the potential usefulness of such a specimen

banking system is clearly evident. It is easy to imagine how stored samples

can be used to show trends of various existing environmental chemicals, and how

we can go back and analyze stored samples should additional chemicals of

interest to our program offices appear. At this point in time, it is critical

that we consider the overall objective and an organizational management scheme

for the Specimen Bank Project on a national scale, since other U.S. government

agencies have recently joined with the National Bureau of Standards (NBS) and

Environmental Protection Agency (EPA) in the banking of environmental and

nutritional specimens. Additional agencies may also join the ef fort- - several

having expressed interest in what a National Specimen Bank can contribute.

Consequently it has become important to consider formulation of a "charter" for

a National Environmental Specimen Bank . This formal charter would indicate

such things as the types of samples to be collected, stored, analyzed, and who

might have access to these stored samples. This charter would also set up a policy board of members from the various agencies to guide the Specimen Bank

efforts and oversee its management.

The effort of developing a National Environmental Specimen Bank must be

coordinated with the various environmental monitoring networks presently in

existence. The desired coordination between the Specimen Bank and the monitoring programs will also assist us in better assessing exposure and doses at the

cellular sites where the toxic actions occur. The U.S. EPA is currently

planning extensive research initiatives dealing with Human Exposure and

Advanced Methods of Extrapolation. Both programs would benefit from Specimen

Bank results

.

It would also be relevant to assess the results from analyses of specimen

bank tissues and the corresponding benefit to our regulatory programs. There

is already a good indication that this may be possible as evidenced by the

reduction in the level of lead in human blood and livers since EPA required a reduction in the lead content of gasoline.

The key to a successful Environmental Specimen Bank, and the most

challenging effort, will be to establish a National Specimen Bank Program, to answer specific and hard scientific questions, questions such as: What is the

trend for specific chemicals in the environment and in the human? What

remedial actions need to be taken if the chemicals are on an upward trend? Are

new chemicals appearing which we need to be concerned about? Have remedial

actions taken been effective to reduce this environmental and health risk? The

results of these questions will assist our policy makers and regulatory staffs

to make better founded decisions in their effort to provide a better and

healthier environment for all of us. In other words, we need to determine what

we want and what we expect to obtain from such a specimen bank.

On an international scale, the Germans have developed a full National

Environmental Specimen Bank, and several other countries such as Japan, Canada,

and Sweden have seen the usefulness of Specimen Banking. Other countries may

follow the lead established by the German-American cooperation. There is,

iii
already one international environinental monitoring effort underway (HEAL) and

it is possible in this effort to do additional sample collection, storage and

analyses on a global scale. Such efforts must be nurtured. We all live on

this small planet and we breathe the same air. We have seen how events in our country will affect all of us. We need to discuss, in the near future, how and

by what mechanisms we can bring about such international cooperation. Through

an international, well-coordinated Environmental Specimen Bank Network, we should be able to ascertain the chemical trends in the environment which effect

all of us, from acid rain to nuclear fall-out.

Erode Ulvedal , Ph.D.

Senior Health Scientist

Office of Health Research

U.S. Environmental Protection Agency

iv
ABSTRACT

In the past decade, interest in the concept of specimen banking for the

archiving of biological and environmental samples for future analysis has

increased significantly, and specimen banking is now recognized as an integral

part of systematic environmental monitoring. Since the establishment of pilot

Environmental Specimen Banking Programs in the U. S. and the Federal Republic

of Germany (FRG) in the late 1970' s, formal meetings have been held annually

between representatives of these two programs to discuss results and future

needs related to specimen banking. In recent years representatives of similar

programs in Japan, Canada, and Sweden have joined in these meetings to expand

the exchange of information.

In October, 1986, the 10th U.S. -German Seminar of State and Planning on Environmental Specimen Banking was held at the Virginia Institute of Marine

Sciences in Gloucester Point, Virginia. At this meeting the current status of

specimen banking activities in the U. S., FRG, Canada, and Japan was presented

and discussed. This publication contains the proceedings of that meeting with

contributions describing various activities related to banking and analysis of

samples from aquatic, atmospheric, terrestrial, and human monitoring programs.

DISCLAIMER

Certain commercial equipment, instruments, or materials are identified in

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

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

V
TABLE OF CONTENTS

PAGE

List of Figures viii

List of Tables xiii

SECTIONS:

1. Ongoing Activities of the Environmental Specimen Banking

Program in the Federal Republic of Germany - Ulrich Boehringer 1

2. Status of Specimen Banking Activities at National Bureau of

Standards - Stephen A. Wise, Barbara J. Koster, and

Rolf Zeisler 10

3. Specimen Banking: A Key Element of the National Oceanic

and Atmospheric Administration's National Status and

Trends Program - Gunnar G. Lauenstein and John A. Calder 19

4. Influence of Environmental Chemicals in the Aquatic

Environment - Istvan Gebefiigi, Jan-Peter Lay, and

Friedhelm Korte 31

5. Analytical Methods for the Determination of Organic Contaminants

in Marine Sediments and Tissues - Michele M. Schantz

Stephen N. Chesler, Barbara J. Koster, and Stephen A Wise 40

6. Comparative Investigations on Trace Metal Levels in Brown Algae

and Common (Blue) Mussels at the Same Location in the Baltic Sea

and the North Sea - M. Stoeppler, F. Backhaus , M. Burow,

K. May, and C. Mohl 53

7. Inorganic Methods and Results for Marine Bivalves and

Sediments - Susan F. Stone, Donald A. Becker, Barbara J. Koster,

Peter A. Pella, Gerald Slater, Manoranjani, P. M. Tillekeratne

Rolf Zeisler, and Ronald W. Sanders 62

8. Improved Analytical Methodology for the Determination of PCBs

and Pesticides in Human Liver Specimens - Reenie M. Parris,

Stephen N. Chesler, and Stephen A. Wise 74

9. The Determination of Trace Elements in Human Livers - Rolf Zeisler,

Robert R. Greenberg, and Susan F. Stone 82

10. National Ambient Air Monitoring Program National Particulate

Network - Stanley F. Sleva and Dennis Shipman 91

11. Chemicals in Indoor Air - Istvan Gebefiigi and Friedhelm Korte 101

vi
TABLE OF CONTENTS (Cont.)

PAGE

12. Banking of Atmospheric Particulate Matter Samples for Long-Term

Monitoring of Atmospheric Pollution and Related Reference

Material at the National Institute for Environmental

Studies - Yoshinari Ambe , Hitoshi Mukai , and Kensaku Okamoto 108

13. Analysis of PAH in Air Samples Obtained by Long- and Short-Term

Procedures - J. Jacob, G. Grimmer, K.-W. Naujack, and

G. Dettbarn 122

14. Trends and Effects of Environmental Contaminants Determined from

Analysis of Archived Wildlife Samples - J. E. Elliott,

R. J. Norstrom, S. W. Kennedy, and G. A. Fox 131

15. Wildlife as an Indicator for Environmental Contaminants in

Monitoring Systems - Jasper Holm 143

16. Regional Monitoring of Pollutants with Honey Bees - Jerry J.

Bromenshenk 156

17. Quality Assurance as Applied to Long-Term Monitoring of Chemical

Residues and Specimen Banking - R. Turle, R. J. Norstrom,

and T. Won 171

18. Preparation of Sample Material for Environmental Specimen

Banking Purposes - Milling and Homogenization at Cryogenic

Temperatures - J. D. Schladot and F. W. Backhaus 184

19. The Potential Role of Environmental Specimen Banking in Biosphere

Reserves - William P. Gregg, Jr 194

APPENDICES:

A. List of Meeting Participants 200

vii
LIST OF FIGURES

Section 1

Figure 1. Organization of the environmental specimen bank of the Federal

Republic of Germany

Figure 2. Landscapes of the Federal Republic of Germany (published in:

Hydrologischer Atlas der Bundesrepublik Deutschland)

Section 2 :

Figure 1. Development of specimen banking activities at the National Bureau

of Standards

Section 3 :

Figure 1

Figure 2

Specimen bank sampling sites- -Benthic Surveillance Project

Specimen bank sampling sites- -Mussel Watch Project

Section 4 :

Figure 1. Disappearance of hexachlorobenzene , pentachloronitrobenzene , and

4-chloroaniline from the water of experimental ponds

Figure 2. HCB-derived residues in an experimentally polluted small pond.

A; Notonecta glauca, B; Libellula quadrimaculata, C; Lemna minor,

D; Ranunculus aquatilus, and E; sediment

Figure 3. PCNB-derived residues in an experimentally polluted small pond.

A; Notonecta glauca, B; Libellula quadrimaculata, C; Lemna minor,

D; Ranunculus aquatilis, and E; sediment

Figure 4. Disappearance of pentachloro- and 2 , 4 , 6 - trichlorophenol from the

water of the treated compartments

Figure 5. Dynamics of daphnia concentrations in the treated and untreated

compartments

Figure 6. Concentration dynamics of three plankton species in the TCPtreated compartments

Figure 7. Dynamics of concentration of three plankton species in the

PCP- treated compartment

Figure 8. Dynamics of concentrations of Chroococcus limneticus in the

control compartments

viii
Figure 9. Dynamics of diatom concentrations (Nitzschia acicularis) in the

treated and untreated compartments

Figure 10. Concentration dynamics of heterotrophic microorganisms in the

water of the treated and untreated compartments

Section 5 :

Figure 1

Figure 2.

Figure 3

Figure 4.

Figure 5

Figure 6

Flow diagram for the analysis of sediment samples

Flow diagram for the analysis of bivalve tissue samples

Chromatograms from the GC analysis of PAH fractions from a calibration solution (2) sediment extract, and (3) mussel extract

Chromatograms from the GC analysis of PCB fractions from a (1)

calibration solution and (2) mussel extract

Chromatograms from the GC analysis of pesticide fractions from a

(1) calibration solution, (2) sediment extract, and (3) mussel

extract

Flow diagram of the procedure used by NOAA laboratories for

analysis of the intercallbration sediment sample

Section 6

Figure 1 Sampling locations for the comparative study of trace metal

uptake and accumulation in brown algae (Fucus vesiculosus) and

mussels (Mytilus edilus)

Section 7 :

Figure 1. Sequential analysis procedure

Section 8 :

Figure 1

Figure 2.

Figure 3

Flow diagram of the analytical procedure

Gas chromatogram from the analysis of aminosilane Fraction I (electron capture detection)

Gas chromatogram from the analysis of aminosilane Fraction II

(electron capture detection)

ix
Section 9 :

Figure 1

Figure 2

Section 10 :

Figure 1

Figure 2

Section 11

Figure 1

Figure 2

Section 12

Figure 1

Figure 2

Figure 3

Section 13

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Flow chart for the irradiation, decay and counting cycles of the

activation analysis procedures

Comparison of As results in the 1984 human liver set using instrumental and radiochemical neutron activation analysis (INAA

and RNAA) with recent literature data

Particulate sampling network - 1953

Particulate sampling network - 1986

Correlation between air humidity and PCP-concentrations in a model experiment

Lindane and pentachlorophenol accumulation on cotton surface

Banking program of atmospheric particulate matter samples

Sampling locations

Change of benzo [ a] pyrene in atmospheric particles during storage

Schematic representation of the PAH-profile analysis of air

suspended matter

Time -dependence of PAH-concentration during 24 hours

(benzo [ ghi ] perylene , benzo [ajpyrene , dibenz [ a , h ] anthracene

)

Time-dependence of PAH-concentration during 24 hours

(benzo [ b ] naphtho [2,l-d]thiophene, benz[a] anthracene , benzo [ e ] pyrene)

Correlation between yield and boiling points of various PAH

Dependence of the yield on the air flow velocity

X
Section 14 :

Figure 1

.

Figure 2.

Figure 3

.

Section 15 :

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Section 17

Figure 1

Figure 2

Figure 3

Figure 4

PCB , DDE, oxychlordane and 2378-TeCDD concentrations in pools of

Herring Gull eggs (8 to 10 eggs per pool) from Scotch Bonnet

Island, Lake Ontario and Big Sister Island, Lake Michigan 1971-

1982, as determined from re-analysis of samples stored in a specimen bank

High performance liquid chromatograms which illustrate the

differences in liver porphyrin patterns in Herring Gull chicks

from Port Colborne, 1974 (a) and Scotch Bonnet Island, 1974 (b)

Organochlorine levels (wet weight) in Northern Gannet eggs from

Bonaventure Island, Quebec, 1969-1984, as determined from

re-analysis of samples stored in a specimen bank

Regions for monitoring activities with wildlife in the east of

Lower Saxony

Percentage frequency distribution of lead content in liver of

various wildlife

Percentage frequency distribution of cadmium content in liver of

various wildlife

Sum frequency function of random sample values for DDT in various

wildlife

Sijim frequency function of random sample values for PCB's in

various wildlife

Toxic metal content in liver and kidney of roe deer in different

areas of Lower Saxony (mean value in mg/kg wet weight)

Standard of Aroclor 1254, EC detection, chromatographed on a

6' X 1/8" OD, 6% QF-1 and 4% SE30 on Chromosorb W (AW) column at

190°, N2 flow of 40 mL/min

A 1:1 mixture of Aroclors 1254 and 1260, chromatographed on a 60m

DB-5 fused silica capillary column, EC detection, initial

temperature 100° for 2 min. , then 10°/min. to 150°, then 3°/min

to 300°, the carrier velocity 25 cm/sec

DDE in Gannet eggs, Bonaventure Island, Quebec, 1969-76

PCBs in Gannet eggs from Bonaventure Island 1969-76

xi
Section 18 :

Figure 1. Flow sheet showing the tasks of the environmental specimen bank

Julich

Figure 2. Flow sheet of Sample Preparation Procedure (SPP) for

environmental species

Figure 3. Determination of grain size in fish material-dab (limanda

limanda)

Figure 4. Homogeneity study of fish material -dab (limanda limanda) by

determination of Pb and Cd in 6 random subsamples out of 950

Figure 5. Homogeneity study of fish material-dab (limanda limanda) by

determination of As in six random subsamples out of 950

Figure 6. Homogeneity study of fish material-dab (limanda limanda) by

determination of Se in six random subsamples out of 960

I

xii
LIST OF TABLES

Section 1

Table 1

Table 2

Section 2

Table 1

Table 2

Section 3

Table 1

Table 2

Table 3

Section 5

Table 1

Table 2

Table 3

Section 6

Table 1

Table 2

History of the Animals According to the Complete System of Nature

Developed by the Knight Carl von Linne Designed by C.F.W. Roth, ca. 1783 Weimar, Garden-House of J.W. Goethe

Preliminary List of Specimen Types in the Environmental Specimen

Bank

Existing and Proposed Projects in the Biomonitoring Specimen

Banking Program

Inventory of Specimens in the Biomonitoring Specimen Bank

Trace Organic Contaminants to be Determined by the NS&T Program's

Specimen Banking Project

Major and Trace Elements Determined for Archived Samples

National Status and Trends Specimen Bank Inventory

Mean Concentrations of Selected Organic Contaminants in NOAA

Intercalibration Sediment (ng/g dry sediment)

Concentrations of Selected Organic Contaminants in Six 1985 Benthic

Surveillance Sediments (ng/g dry sediment)

Concentrations of Selected Organic Contaminants in Six 1985 Mussel

Watch Bivalve Tissue Samples (ng/g dry bivalve tissue)

Contents of Trace Metals in Bimonthly Collected Samples of Algae

(Fucus Vesiculosus) and Mussels (Mytilus Edulis) at Eckwarderhoerne

(North Sea)

Comparative Trace Metal Determinations in Algae and Mussels from

the Same Collection Site, Summarizing Overview for Arsenic,

Mercury, Lead, Cadmium, and Nickel

xiii
Section 7:

Table 1.

Table 2.

Table 3.

Table 4.

Section 8 :

Table 1.

Section 9 :

Table 1.

Table 2.

Section 10

Table 1.

Table 2.

Section 11

Table 1.

Section 12

Table 1.

Table 2.

Table 3.

Elemental Concentrations for Selected Reference Materials

(concentrations based on dry weight)

Elemental Concentrations for Certified Reference Material IAEA

Sediment SD-N-1/2 (concentrations in mg/kg dry weight)

Elemental Concentrations for Selected Bivalve Samples

(concentrations based on dry weight)

Elemental Concentrations for NS&T 1985 Benthic Surveillance

Sediment Samples

Range of Concentrations of Selected PCBs and Pesticides in 24 Human

Liver Samples

Ranges of Trace Element Concentrations in Selected Human Liver

Specimens

Limits of Detection (/zg/kg) for Seven Critical Elements

Comparison by Pollutant of National Air Monitoring Stations 1977

vs. 1986

National Summary of SLAMS Monitors

Pentachlorophenol , Lindane, Chlorinated Dibenzodioxin and

Dibenzofuran in Samples of Wood, Dust, and Textiles

Comparison of the Concentration of Benzo [ a] pyrene in the

Simultaneously Sampled Four Filters

Change of B[a]P in Atmospheric Particles after 4 Years Storage

Analytical Values for NIES Certified Reference Material No. 8 "Vehicle Exhaust Particulates"

xiv
Section 13 :

Table 1.

Table 2.

Table 3.

Table 4.

Section 15

Table 1.

Table 2.

Table 3.

Table 4.

Section 17

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Section 18

Table 1.

PAH Profiles on Air Particulate Filters (ng PAH/m'^ )

PAH-Profiles Collected on a Glass Fiber Filter and a Subsequent

Porapak PS Filter (ng PAH/m^

)

PAH-Profiles on Glass Fiber Filters (ng PAH/m^

)

PAH-Profiles Recorded at a Vehicle- Exhaust Polluted Location

Different Contaminations to be Expected in 5 Regions in the East of

Lower Saxony

Criteria for Bioindicators in Monitoring Studies

Chlorinated Hydrocarbons in Organs of One Year Old Roebucks from

the Forest Near Braunschweig

Limits of Detection for Elements Determined by ICP

Pesticides Determined in Canadian Wildlife in 1968

Changes in CWS Analytical Methodology for PCBs 1968-1986

Analysis of Gannet Eggs from Bonaventure Island 1969

Re-analysis of 1969 Gannet Eggs, Bonaventure Island

Interlaboratory Comparison of CWS 1979 Reference Material

Application of Acceptable Criteria

Homogenization Study of Fish Material by Determination of

Chlorinated Hydrocarbons

XV

SECTION 1

ONGOING ACTIVITIES OF THE ENVIRONMENTAL SPECIMEN BANKING PROGRAM

IN THE FEDERAL REPUBLIC OF GERMANY

Ulrich R. Boehringer

Umwe 1 tbundes am

Federal Environmental Agency

Bismarckplatz 1 D-1000 Berlin, 33

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

At the 8th Seminar of State and Planning on Environmental Specimen Banking

in Gaithersburg , I announced the intention of the German Federal Government to

establish a permanent German Environmental Specimen Bank in 1985. The results

obtained up to that time indicated that the pilot project on Environmental

Specimen Banking would be concluded successfully. As you know, this plan of

the Federal Government was realized. The first steps of this activity were presented at the 9th seminar in Reisensburg last year.

At that seminar, I gave a survey of the areas for sampling and of the

proposed specimens that were under discussion at that time. The progress made

since then will be presented by my German colleagues in other sections of this

publication. I will give a survey of the organization of the permanent bank

and explain the list of the specimen types proposed.

II. ORGANIZATION

Under the supervision of the newly established Federal Ministry for the

Environment, Nature Conservation and Nuclear Safety, the Federal Environmental

Agency coordinates the permanent German bank. The ministry has to harmonize

the program with the following three ministries (Figure 1): (1) the Federal

Ministry for Research and Development, (2) the Federal Ministry for Youth,

Family, Women and Health, and (3) the Federal Ministry for Food, Agriculture

and Forestry.

The Umwe 1 tbundes amt (Federal Environmental Agency) guides the bank

scientifically and administratively, harmonizes the project with other national

and international activities, and initiates Research and Development projects.

The database of the bank will be connected with the environmental information

and documentation system of the agency. The Umwe 1 tbunde s amt will also be the

secretariat of the scientific advisory committee.

The work that needs to be done to run the bank is too specialized to be

carried out by just one institution. Therefore, five institutes are at present

involved in this program.

1
First of all, the two banking facilities are in Julich at the Nuclear

Research Center (KFA) and in Munster at the Institute for Pharmacology and

Toxicology of the Westphalian Wilhelm-University . The bank in Julich is

responsible for:

September tg86

E.")* I ronmentd I tcec:mef Sank of tfte federal RpmudIic or Germany

Federal Minister 'or the

coorainduofi witri Fed. Minister for ftesearcn and Tecnnology (Ahft). Fed. Mm. for Touti, Faniily, Women and Healtri (BHJFFG). Fed. Hin. for Aqricultu-^ (9HL) Envirorment . hatu^-e Conservation

and Nuclear Safety (BHU) coordination *ith federal states (Lander)

governmental suoervision

international agreements. EEC

Federal E TV I ronmen t Agency (UBA)

Sc lent i f

Ad V 1 scry

- scienltflc and administrative supervision initiation and suoervision of R + 0 projects Comn 1 1 tee

and steering secretariat of the scientific advisory co-nnittee [not yet es aDiished)

- further development r environmental Information and documentation system ~|

- Harmonization with national and

| L

(umpl:s)

I Internationa! activities

^

Nuclear Research Center (rPA)

JOl ich bank for biological specimen

- acQuisiton of marine sanoles

- analyses for inorganic substances

- logistics and transport I

Munster Un 1 rs I ty

bank for human organ samples

- acQuisition of nuinan organ samples

_ -

- _delect

n Ajj

ro.iirS_

data ______ Drocessing fc" the environmental specirien bank

university of the Saarland samp ling

Geseilschaft fur Strahlen- und

Urmieltforscnung. Neuheroerg (q^^ analyses for ^roamc SbDstarces

Biccnerical Instit'jte for

Environfliental Carcinogens,

Figure 1. Organization of the environmental specimen bank of the

Federal Republic of Germany

1. storage of the biological specimens, other than the human organ

samples

2. collecting the marine samples,

3. inorganic analyses, and

4. transportation of the samples under cryogenic conditions.

The university in Munster is responsible for all activities in the human

sector: sampling, characterization, analysis, and storage. Additionally, the

database for the German Environmental Specimen Bank will be set up in Munster.

The University of the Saarland, the Chair of Biogeography of this

University, to be exact, is responsible for collecting the biological samples

and for their characterization. The two other institutes, the Institute for

2
Ecological Chemistry at the Gesellschaft fur Strahlen - und Umweltforschung in

Neuherberg near Munich and the Biochemical Institute for Environmental Carcinogens in Grosshansdorf near Hamburg, analyze the samples for halogenated

organic ingredients and polycyclic aromatic hydrocarbons (PAH) , respectively.

In addition, several Research and Development projects are on-going or planned.

These activities are described in detail in the papers in other sections of

these proceedings (see Sections 4, 6, 11, 13, 15 and 18).

III. SPECIMEN SELECTION AND AREA SELECTION

After we had solved the technical questions involved in the operation of an environmental specimen bank, we had to select representative areas for

sampling and the representative species that live in these areas. As you know,

the plant and animal species were classified by the Knight Carl v. Linne . His

compilation of zoological species, called the history of the animals, is shown

in Table 1

Carl V. Linne also founded the Museum of Natural History in Sweden at

which an Environmental Specimen Bank is also located [1].

As you can see, Linne divided the animals up into mammals, birds, fishes,

insects, worms, and amphibians. You can imagine how difficult it is to select

representative and suitable species for an environmental specimen bank. For

example, look at the mammals in Table 1. For man alone two species exist, the

day species and the night species.

In following this compilation we had three workshops, where this problem

of the selection of species was discussed extensively.

1. 1977 in Luxembourg:

The use of Biological Specimens for the Assessment of Human

Exposures to Environmental Pollutants [2];

2. 1978 in Berlin:

Monitoring Environmental Materials and Specimen Banking [3]; and

3. 1982 in Saarbrucken:

Environmental Specimen Bajiking and Monitoring as Related to

Banking [4]

It was agreed at these workshops that the three spheres, the human, the

aquatic, and the terrestrial, should be represented in an environmental

specimen bank.

In 1985, we had two meetings of experts for the selection of species for

the German Environmental Specimen Bank. The following list shows the species

proposed by the experts (Table 2). Some of these species have already proved

suitable during the pilot project, e.g., grass, poplar leaves, zebra mussels,

3
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and aigae. I wiil give some comments on other specimens investigated for

inclusion in the permanent Specimen Bank.

1. Not enough sample materials can be obtained from the earthworm.

Therefore, a research project is under way to investigate how the

sampling method can be optimized. The other problem is how to remove the soil from the intestines. Three methods exist: to

feed the earthworms cellulose, to dissect the earthworm and wash

the soil out of the earthworm or to massage them.

2. A research project is in progress to investigate what tissue of

the roe deer is best suited for the specimen bank.

3. Since we cannot obtain enough eggs from the black bird, we will

have to choose another species. The pigeon is being discussed as

a substitute species.

4. The Herring Gull (Larus argentatus) of the Great Lakes is not

identical with the European Herring Gull. Furthermore, the

European Herring Gull gets its food from waste dumps among other

sources; it is a scavenger or a so-called food opportunist.

Therefore, this species is not very representative for the

environment

5. For the marine sphere, we are presently discussing whether a tern

species, the Sandwich Tern (Sterna sanvicensis) , is suitable for

the purposes of the bank.

6. The specimen "air-suspended particulates" has been added to the

list since our last meeting.

The other main question has been the selection of areas. The Federal Republic

of Germany has different types of landscapes, from the sea in the north to the

Alpes in the south (Figure 2). Therefore, you find very different ecosystems

and the question arises which of these are the most representative ones. Furthermore, the sampling areas should also be used for environmental monitoring and ecosystem research. The following lecture on the Selection of National

Ecological Assessment Parks in the Federal Republic of Germany will give you

information on this subject.

IV. INFORMATION SYSTEM

Background information on the samples collected is essential for making a comprehensive trend analysis. We will start in the near future a research and

development project to establish an information system for the database. Not

only do we have to collect the information directly concerning the sample,

e.g., sampling site, sampling date, characterization and storage, but it is

also necessary to describe the system out of which the sample is collected,

e.g., data on geography, meteorology, soil, land use, etc. The systems are dynamic; therefore, the data has to be updated regularly. Together with this

information, the specimens can be classified and assessed much better. It will

6
7
take some time and effort until we know the minimum set of information needed

for a sampling site.

V. EXAMPLE FOR THE VALUE OF STORED SAMPLES

In conclusion, I will present a recent result from the 6th International

Symposium on Chlorinated Dioxins , held in Fukuoka, Japan, September 16-19,

1986. It is a classic example for the value of stored samples. The

chlorinated dioxins, highly toxic chemicals in multiple animal species, are known to be the synthetic contaminants of certain synthetic chemicals.

Relatively recently, it has been proposed that the chlorinated dioxins may be

formed in trace amounts, at least, by the combustion of wood not treated with

chemicals: the trace chemistry theory of the origin of dioxins.

If this is the case, chlorinated dioxins would have existed prior to the

synthesis of chemicals known to contain trace amounts as contaminants, essen- tially at the beginning of this century. Thus, animals and man might have

evolved in the presence of these compounds.

More recently, Kites and coworkers found, in sediment cores from the

bottom of certain lakes, dioxins to have been present only relatively recently,

probably related to chemical manufacture and possibly incineration.

Schecter and coworkers [5] (State University of New York) found in non- industrialized countries low levels of dioxin in human tissues and higher

levels (1,200 ppt) in adipose tissue of persons in industrial countries. This

suggests an anthropogenic origin of most polychlorinated dibenzodioxins

To provide further data to test this hypothesis as to the anthropogenic

origin of most dioxins, Schecter and coworkers analyzed human liver and lung

tissue from two female Eskimos, frozen over a hundred years ago, and meat found

in their home, as well as charred wood from an ancient California redwood tree.

Since the lungs were found on histologic evaluation to have pneumoconiosis from

burning of fuel combined with poor ventilation, lungs as well as liver were of

interest. They found non detectable levels of dioxin. Therefore, the results

are consistent with an anthropogenic origin for most dioxin and not with the

above mentioned theory.

VI . ACKNOWLEDGMENT

Let me conclude my remarks with thanks to Stephen A. Wise and his

coworkers from National Bureau of Standards for organizing this seminar and to

EPA for supporting it. We welcome the fact that other government agencies in

the USA have joined the Environmental Specimen Banking Program. It

demonstrates the increasing importance of this task for the future.

8
VII . REFERENCES

Olsson, M. (1985) , The Environmental Specimen Bank at the Swedish Museum

of Natural History, International Review of Environmental Specimen

Banking, S. A. Wise and R. Zeisler, Eds., NBS Spec. Publ. 706, U.S.

Government Printing Office, Washington, DC, pp. 22-33.

Berlin, A., Wolff, A. H., and Hasegawa, Y. , Eds. (1979), The Use of

Biological Specimens for the Assessment of Human Exposure to Environmental Pollutants, Martinus Nijhoff Publishers, The Hague, 368 pp.

Luepke , N.-P., Ed. (1979), Monitoring Environmental Materials and

Specimen Banking, Martinus Nijhoff Publishers, The Hague, 591 pp.

Lewis, R. A., Stein, N. , and Lewis, C. W. , Eds. (1984), Environmental

Specimen Banking and Monitoring as Related to Banking, Martinus Nijhoff

Publishers, Boston, MA, 358 pp.

Schecter, A., Gross, M. , and Dekin, A. (1986), Human Tissue Levels of

PCDD and PCDF in Over One Hundred Year Old Frozen Eskimo Tissue, Meat,

and California Redwood Charcoal and Their Relation to the Trace Chemistry

Theory of Dioxin Origin, Abstracts of Dioxin '86, 6th Int. Symp. on Chlorinated Dioxins and Related Compounds, Fukuoka, Japan, Sept. 16-19,

1986.
SECTION 2

STATUS OF SPECIMEN BANKING ACTIVITIES AT THE NATIONAL BUREAU OF STANDARDS

Stephen A. Wise, Barbara J. Koster, and Rolf Zeisler

Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, Maryland 20899

USA

I . INTRODUCTION

Since 1975 the National Bureau of Standards (NBS) and the U.S.

Environmental Protection Agency (EPA) have been involved in the development of

methods and procedures for environmental specimen banking. In 1975 the Federal

Republic of Germany (FRG) and the U.S. EPA agreed to cooperate in the study of

environmental specimen banking activities and proposed the establishment of

"pilot phase" specimen banks in both countries. The purpose of the EPA/NBS

Environmental Specimen Bank Program was to determine the feasibility of longterm storage of environmental and biological specimens for the determination of

inorganic and organic constituents. In 1980 a "pilot" environmental specimen

bank was established at NBS to evaluate the feasibility of a large-scale

National Environmental Specimen Bank. The pilot Environmental Specimen Bank

program was to provide working experience in all aspects of specimen banking,

e.g., in specimen collection, processing, storage, and analysis.

The approach of the EPA/NBS pilot

experience in the collection, storage,

mental accumulators: (1) human tissue,

air. The goals of the pilot phase were:

Environmental Specimen Bank was to gain

and analysis of four types of environ-

(2) marine tissue, (3) food, and (4)

1. To develop procedures for the sampling, processing, storage, and

analysis of four types of environmental accumulators;

2. To evaluate and improve analytical methods for the determination

of trace element and organic pollutants in biological matrices;

3. To evaluate the feasibility of long-term storage of environmental

samples under various conditions;

4. To establish baseline data on selected environmental samples, and;

5. To evaluate the potential application of specimen banking for

trend monitoring and for providing samples for retrospective

analyses as concerns about new pollutants arise and as new

analytical techniques are developed.

Since 1980 the EPA/NBS Pilot Environmental Specimen Bank has been involved in

the collection, storage, and analysis of the first sample type, human liver

tissue. However, the remaining three sample types (i.e, marine, food, and air

10
accumulators) were not incorporated fully into the EPA pilot banking program as originally proposed.

During the past year the specimen banking concept at NBS has been modified

to reflect the interests of other agencies in specimen banking activities. A

proposed concept for the development of specimen banking activities at NBS is

outlined in Figure 1. The development of the specimen banking concept consists

of three phases: the pilot phase, project phase, and network phase. The pilot

phase is near completion and has been described briefly above and in other

publications [1-3]. The project phase and proposed network phase are described

in the following sections.

II. PROJECT PHASE

In 1986 the program moved towards a "project phase" of the Biomonitoring

Specimen Bank Research Project. This title reflects the change from solely

environmental aspects to the inclusion of specimens of nutritional and medical

importance. At present there are five existing specimen banking activities

within the NBS specimen banking program, and several other projects are proposed for incorporation into the program in the coming years (see Table 1).

Table 1. Existing and Proposed Projects in the Biomonitoring

Specimen Banking Program

EXISTING PROJECTS:

1. EPA Environmental Specimen Bank Program (human livers)

2. NOAA NS&T Marine Monitoring Program

3. NCI Chemoprevention Program

4. lAEAAfSDA/FDA Trace Nutrients in Human Diet Project

5. NOAA Alaska Marine Mammal Archive

PROPOSED PROJECTS:

1. EPA Human Adipose Tissue Monitoring Program

2. National Health and Nutrition Examination Survey (NHANES-III)

A. Existing Projects

EPA Specimen Bank Program

The EPA-NBS Specimen Bank Program will continue to serve as the focal

point for specimen banking-related activities during the project phase. The

pilot phase of the EPA specimen banking activities resulted in a number of

outputs including the following:

1. Development of protocols for the sampling, processing, and storage

of environmental/biological specimens (e.g., human liver and

11
SPECIMEN BANK CONCEPT

1980 - 1985

PILOT PHASE

NATIONAL ENVIRONMENTAL

SPECIMEN BANK

1986 - 1990

PROJECT PHASE

BIOMONITORING SPECIMEN BANK

RESEARCH PROJECT

COORDINATING COMMITTEE

Participants' Meeting

1990 +

NETWORK PHASE

NATIONAL BIOMONITORING SPECIMEN

BANK SYSTEM

NBS SPECIMEN BANKING

RESEARCH AND OA

LABORATORY

XT I ^—I I

'

1 I

'

1

1

1

I HUMAN . . MARINE . . NUTRITION. . ADIPOSE . LIVER SPECIMEN SPECIMEN TISSUE

BANK '

' BANK '' BANK ' BANK '

I I I I I I I I

Figure 1. Development of specimen banking activities at the National

Bureau of Standards

12
marine bivalves) ; development of cryogenic homogenization

procedures for biological specimens;

2. Improved analytical methods for the determination of inorganic and

organic constituents in environmental/biological samples;

3. Analytical baseline data for inorganic constituents on 96 liver

specimens from three collection periods (1980, 1982, and 1984);

analytical data for organic constituents (PCBs and pesticides) on

60 liver specimens from two collection periods (1982 and 1984);

4. An archive of over 500 human liver specimens and a large

collection of mussels which are available for future

investigations

The expertise developed during the EPA pilot project has been implemented in

other specimen bank projects at NBS as well as by researchers in other programs. For example, sampling and homogenization procedures for human liver and

mussels developed in the pilot project were used as the starting point for the

development of the protocols used in the National Status and Trends Program

sponsored by the National Oceanic and Atmospheric Administration (NCAA) (see

discussion below) . The existing analytical data base on inorganic and organic

constituents in human livers collected from 1980 to the present and the over

400 unanalyzed specimens in the bank represent a substantial resource and a unique opportunity for EPA to investigate pollution trends. During the next

year the activities of the EPA-NBS project will be directed towards the

evaluation of the analytical data base for possible trends and the evaluation

of the compositional stability of the huiman liver specimens stored under

different conditions.

NCAA National Status and Trends Program

In 1985 a specimen banking project was initiated at NBS with the National

Oceanic and Atmospheric Administration (NCAA) as part of their National Status

and Trends (NS&T) Program to monitor the U.S. marine environment [4]. The NS&T

program consists of two monitoring programs: (1) the Benthic Surveillance

Project, in which sediment and fish tissue (muscle and liver) are collected

annually from approximately 50 coastal sites and (2) the Mussel Watch Project,

in which bivalves (mussels and oysters) and sediment are collected annually

from approximately 150 coastal sites. As part of the NCAA NS&T program, NBS

provides the following specimen banking activities:

1. Develop sampling, homogenization, and storage protocols for

sediment, bivalve, fish muscle, and fish liver samples;

2. Archive specimens of sediment and tissue from 12 sites of the

Benthic Surveillance Project and mussels/oysters and sediment from

30 sites in the Mussel Watch Project each year; a complete set of

samples from all sites will be archived after five years.

13
3. Analyze selected samples of sediment, bivalves, and fish livers

for the determination of selected trace elements (15 elements) and

organic compounds (organochlorine pesticides, PCBs , and PAH);

4. Analyze sediment and tissue intercalibration materials as quality

assurance exercises.

In addition to the specimen banking activities for the NOAA NS&T program,

NBS is involved in the NOAA NS&T program as the laboratory responsible for

organic analysis quality assurance activities. These activities include the

preparation and distribution of control and/or intercalibration materials and

calibration solutions to the various laboratories involved in making analytical

measurements for the NS&T program and organizing annual quality assurance

workshops to discuss the quality of the analytical data generated within the

program. NOAA is also supporting NBS in the preparation of a marine sediment

to be issued as a Standard Reference Material for the determination of organic

pollutants. Both of these activities complement and support the efforts

related to specimen banking of marine specimens.

lAEA/FDA/USDA/NBS Nutrients in Human Diet Program

NBS, the Food and Drug Administration (FDA), and the Department of

Agriculture (USDA) are collaborating in a coordinated research program

sponsored by the International Atomic Energy Agency (IAEA) to obtain

comparative data on the dietary intakes of 23 nutritionally important minor and

trace elements in a number of developing and developed countries [5]. In the

U. S. effort, which combines the expertise of the three participating government agencies, four aspects are addressed: (1) benchmark total diet samples

from the FDA Total Diet Study (TDS) are prepared and analyzed to provide data

for the IAEA project; (2) representative portions of these benchmark samples

are stored in the NBS specimen bank facility; (3) research is carried out to

develop analytical techniques for the determination of a number of constituents

in total diet samples and to develop a total diet reference material; (4) the

immediate and long-term stability of such total diet samples with respect to

organic and inorganic nutrients during handling and storage are investigated.

Several tasks related to the total diet study are included in the

expansion of specimen banking activities to this field:

1. Bank aliquots of mixed total diet samples based on the FDA market

basket collections;

2. Methods development, analyses, and quality assurance;

3. Intercomparison of analytical methods for trace elements in

existing dietary reference materials;

4. Preparation of U.S. mixed diet research material from the 200

different food groups in the FDA Total Diet Study (10 kg); and

5. Determination of long-term stability of trace constituents in

the total diet composite.

14
Future collections of this total diet sample will include the variations in

season as well as the four major areas of FDA's TDS . Within several years, a complete inventory of the FDA market basket survey will exist in the proposed

Biomonitoring Specimen Bank.

National Cancer Institute (NCI) Chemoprevention Program

As part of a quality assurance program of the National Cancer Institute

Chemoprevention Program, NBS is serving as the reference laboratory for the NCI

contract laboratories involved in the determination of nutrients in human

serxjm. The following analytes are currently included in the study: zinc,

selenium, beta-carotene, retinol, alpha- tocopherol , and ascorbic acid. As part

of this program NBS prepares and distributes intercalibration samples to the

various laboratories. Since the long-term stability of these nutrients in serum (frozen and lyophilized) is unknown, selected intercalibration serum

samples and calibration solutions have been archived under various storage

conditions. These samples are periodically removed from the bank and analyzed

to determine the stability of these nutrients in the specimens. These stability investigations will provide additional information on the behavior of

various matrices with respect to long-term specimen banking.

NOAA Alaska Marine Mammals Tissue Archive

NOAA will be banking selected tissues from marine mammals for the future

determination of contaminants associated with offshore oil and gas development.

A pilot program has been initiated to develop and implement sample collection

protocols for liver, muscle, and adipose tissue from marine mammals. Fur

bearing seals will be the first specimen type included in this study. Additional species of Alaska marine mammals from will be incorporated into the

archive in future years

B. Proposed Projects

Several other projects have been discussed as possible additions to the

Biomonitoring Specimen Bank Program to broaden the scope of specimen banking

activities within the program.

EPA Human Adipose Tissue Monitoring Program

Since the mid-1960' s, EPA has conducted a monitoring program to measure

organochlorine pesticides and PCB's in human adipose tissue. At present

adipose tissue samples are collected from approximately 1000 sites across the

U.S. Portions of the tissue specimens have been stored when excess material

was available after analyses. Recently, this tissue monitoring program has

expanded their analytical procedures to include volatile organics as well as the semi-volatile compounds measured in the past. EPA has expressed interest

in including NBS in this program to serve in a quality assurance and specimen

banking role. NBS could store a small, representative number of specimens from

the program under more rigid conditions to serve as reference samples and to

investigate storage stability.

15
National Health and Nutrition Examination Survey (NHANES-III)

As the National Center for Health Statistics (NCHS) plans the third

National Health and Nutrition Examination Study (NHANES-III), with participation of the EPA, the Centers for Disease Control (CDC) , and other interested

agencies, the opportunity exists to incorporate the concept of specimen banking

into the program to enhance the usefulness of the data gathered. As part of

NHANES-III, aliquots of the serum and urine specimens analyzed in the various

assays (or a selected fraction of the total specimens analyzed) should be

banked for future analysis. Discussions are in progress concerning how

specimen banking activities can best be utilized in NHANES-III.

III. NETWORK PHASE

The project phase will provide experience in the sampling, processing,

storage, and analysis of a variety of biological matrices as summarized in

Table 2. The existing projects in the "project phase" now include three of the

four environmental accumulators originally intended for the EPA/NBS pilot

Environmental Specimen Bank Program (i.e., human, marine, and food). The

project phase will also bring together the different government agencies

responsible for contaminants, nutrients, and biologically important

constituents in these various matrices to coordinate their efforts in specimen

banking. This cooperation will form the basis for the establishment of a National Biomonitor ing Specimen Bank System consisting of a network of satellite banks directed towards specific projects as illustrated in Figure 1. EPA/NBS Specimen Bank would serve as a resource facility to conduct research

related to the needs of specimen banking and as a quality assurance laboratory

for the analytical measurements required in the various satellite banking

programs

IV. REFERENCES

[1] Zeisler, R. , Harrison, S. H., and Wise, S. A. (1983), The Pilot National

Environmental Specimen Bank - Analysis of Human Liver Specimens, NBS

Spec. Publ. 656, U.S. Government Printing Office, Washington, DC, 135 pp.

[2] Zeisler, R. and Wise, S. A. (1985), Quality Assurance and Protocols in

Sample Preparation of Biological Samples, In: Biological Reference

Materials: Availability, Uses, and Need for Validation of Nutrient

Measurements, W.R. Wolf, Ed., John Wiley & Sons, Inc., New York, NY, pp

257-279.

[3] Wise, S. A. and Zeisler, R. (1984), The Pilot Environmental Specimen Bank

Program, Environ. Sci. Technol. 18, 302A-307A.

[4] Lauenstein, G. G. and Calder, J. A. (1988), Specimen Banking: A Key

Element of the National Oceanic and Atmospheric Adminstration' s National

Status and Trends Program, In: These Proceedings.

16
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5] Co-ordinated Research Programme on Human Daily Dietary Intakes of

Nutritionally Important Trace Elements as Measured by Nuclear and Other

Techniques, International Atomic Energy, Vienna, Austria. Report of the

First Research Coordination Meeting Gaithersburg , Beltsville and

Washington, DC, March 19-22, 1985.

18
SECTION 3

SPECIMEN BANKING: A KEY ELEMENT OF THE NATIONAL OCEANIC AND ATMOSPHERIC

ADMINISTRATION'S NATIONAL STATUS AND TRENDS PROGRAM

Gunnar G. Lauenstein and John A. Calder

National Oceanic and Atmospheric Administration

Rockville, Maryland 20852

USA

SUMMARY

The National Oceanic and Atmospheric Administration has initiated a Specimen Bank for estuarine and coastal samples, as part of its National Status

and Trends Program. During the first year, sample collection protocols were developed for the collection of benthic fish, bivalve molluscs, and associated

sediments. Specimens from over 40 sites nationwide have now been submitted for

inclusion in the Specimen Bank, which is housed at the National Bureau of

Standards in Gaithersburg , Maryland. Because specimens are preserved at liquid

nitrogen vapor temperature, degradation is expected to be minimal for decades.

The specimens will provide the opportunity to derive retrospective baseline

values for environmental contaminants of future concern and to reevaluate the

past when new and improved analytical procedures become available in the

future. Analyses of selected samples collected during the first year are presently under way for organic and trace elemental contaminants to provide a basis for assessing stability of the specimens while in storage.

I. INTRODUCTION

The concept of retrospective analysis prompted experiments that compared

chemical constituents in environmental materials preserved for other purposes

with chemical constituents in freshly collected samples. In the 1960's, for

example, pollution was studied in Sweden by researchers who compared chemical

constituents of recent samples of bird tissues with those of bird skins

preserved in academic and museum collections dating from 1840 [1]. In the

1960's, the kepone content in shellfish and sediments from the James River

(Virginia) was similarly determined and compared with archived tissue samples

to establish the earliest date of this chemical's occurrence in the river [2].

In the United States, a pilot Environmental Specimen Bank Program was established in 1980 at the National Bureau of Standards (NBS) , sponsored in

part by the Environmental Protection Agency. Other Federal agencies, including

the Food and Drug Administration, Department of Agriculture, National Cancer

Institute, and the National Oceanic and Atmospheric Administration (NOAA) , of

which the Ocean Assessments Division is a part, have joined in specimen banking

activities at NBS.

19
II. BACKGROUND

In 1984, NOAA's Ocean Assessments Division (OAD) initiated a new program

cailed the National Status and Trends (NS&T) Program, within which a series of

activities are being undertaken to quantify the current status and long-term

temporal and spatial trends of key contaminant concentrations and biological

indicators of contaminant effects in United States coastal and estuarine

environments [3]. The program's purposes are to provide highly reliable data

on concentrations of toxic chemicals in marine fish, shellfish, and sediments;

to measure biologic parameters that accurately reflect anthropogenic stress;

and to assess marine environmental quality and recommend Federal actions needed

to maintain or to improve it. Key questions the program intends to answer are

1) what are the current environmental quality conditions of the nation's

coastal zone, and 2) are these conditions getting better or worse?

Sampling and analysis for the NS&T Program are performed with well

understood methods and adherence to statistical requirements so that a known

confidence level can be assigned to all data. Sampling at all sites is con- ducted in a uniform manner, using the same techniques. Analytical procedures

adhere to quality assurance protocols that have been established for all

laboratories participating in the NS&T Program. The activities include a series of interlaboratory comparison exercises using appropriate reference

material and the convening of specialized technical education and training

workshops [ 3 ]

The NS&T Program has two monitoring efforts, the Benthic Surveillance

Project and the Mussel Watch Project. Under the Benthic Surveillance Project,

samples are collected annually from over 50 sites around the United States

coastline, including Alaska. Both benthic fish and associated sediments are sampled. Sediments were selected because they are known reservoirs of contaminants; bottom fish were selected because they are reliable indicators of local

pollution effects and because their exposure to toxic chemicals is linked

primarily to bottom sediments.

Under the Mussels Watch Project, samples are collected annually from over

150 sites around the United States coastlines, including Alaska and Hawaii. As

in the Benthic Surveillance Project, not only are organisms collected but

associated sediments are collected as well.

Bivalve molluscs are a logical choice as sentinel organisms since they

have the ability to concentrate environmental contaminants, they are cosmopolitan, they have the ability to tolerate contaminated environments, they are sessile, and they appear to have a minimal ability to metabolize organic

contaminants, including petroleum hydrocarbons, PCBs , and DDT [4-6].

One element of the NS&T Program is the archiving of samples from both the

Benthic Surveillance and Mussel Watch projects. The methods of collection,

preparation, and storage of samples for a specimen banking program are critical

to maintaining the integrity of the sample and its value for future analytical

comparisons. The methods used in the specimen banking component of the NOAA

NS&T Program are described in this paper.

20
III. BENTHIC SURVEILLANCE SPECIMEN BANKING PROTOCOLS

For the Benthic Surveillance Project, two duplicate aliquots of 150 g each

are collected for both benthic fish liver and muscle tissue. Duplicate 150 g

samples of sediments are taken from depositional sites near to the fish

collection sites. Approximately 10 sites, of the 50 NS&T Benthic Surveillance

sites, are added to the Specimen Bank each sampling season (Figure 1).

Special precautions are taken with the dissection tools both during a dissection and after the tools are cleaned. Before initiating a set of

dissections and after sharpening the titanium knives, the tools are wiped of

extraneous materials, scrubbed in a detergent solution, rinsed with tap water

followed by a distilled water rinse and finally transferred to a fume exhaust

hood where they are carefully rinsed with methylene chloride.

To ensure that tissues are not contaminated, fish are necropsied in

positive-pressure hoods using only specially cleaned materials and tools. For

the dissection, two tool groups are used, one for the removal of the liver

samples and the other for fish muscle collection. Each tool group in turn has

two sub-sets. The first set of tools in each group is used to enter the

sampling field, i.e., to cut through the body wall to access the body cavity

(for liver) or to cut through the potentially contaminated epidermis to sample

fish muscle [7]. The actual removal of the liver or muscle tissue occurs with

the use of titanium knives . These knives are used to avoid the problem of iron

and/or chromium contamination of samples.

Dissected tissue replicates are placed into pre-cleaned Teflon bags, which

had been packaged under Class- 100 clean room conditions, and then the bags are heat sealed and placed in a second bag that is also heat sealed.

For the Benthic Surveillance Project, two duplicate sediment aliquots of

150 g each are also taken at each site. Samples are collected with the use of

a box corer. Two 50 g subsamples, (replicates A&B) are taken using Teflon

cylindrical corers from each of three sampled stations per site. These are then composited in Teflon bags to yield two duplicate 150 g composites per

site. To ensure that only a minimum of sample degradation occurs following

collection, samples of fish tissue and sediments are frozen in liquid nitrogen

in the field.

IV. MUSSEL WATCH SPECIMEN BANKING PROTOCOLS

The NS6cT Program's Mussel Watch Project also supplied duplicates of

bivalves and sediment samples from 30 sites nationwide (Figure 2). Each site

consisted of three stations; each station provided two batches of approximately

16-18 mussels (mytilus edulis/mytilus californianus) or two batches of 10

oysters (crassostrea virginical) for a total of approximately 100 mussels or 60

oysters per site. After collection, the bivalves are sorted for size on a noncontaminating tray and then rinsed with seawater and scrubbed with a noncontaminating brush. Bivalves are divided into two batches and then sealed

in Teflon bags for storage. Sediment samples for the Mussel Watch Project are handled in the same manner as for the Benthic Surveillance work. Due to

21
Cl.

22
23 i
logistical difficulties, the large sample volume and the possibility that

bivalves may explode if immersed fresh in liquid nitrogen, samples from the

Mussel Watch Project are stored and shipped on dry ice before the final storage

in liquid nitrogen freezers at the NBS

V. NS&T Cryogenic Storage Facility

Because of its long-term experience with cryogenic specimen storage and

related specimen banking activities, the NBS was selected for the NS&T Specimen

Bank. The facilities available at NBS for storage of NS&T materials include a Class- 100 clean-air laboratory designed to minimize sample contamination from

the laboratory environment during sample handling, processing, and storage.

The laboratory has two sections, one for trace element analysis and the other

for trace organic analysis. An additional room has been designed for long-term

storage of environmental and biological samples and contains the NS&T liquid

nitrogen vapor freezer whose volumetric capacity of 500 liters can be

maintained at temperatures ranging from -120 to -150 °C.

VI . Analyses

Using a Teflon disk mill developed by the NBS, a portion of the tissue

samples sent to the Specimen Bank are homogenized at cryogenic temperatures.

Cryogenic homogenization reduces loss of volatile components and reduces the

likelihood of changes in sample composition due to thawing and refreezing.

Experiments with mussel tissue indicate that after homogenization, virtually

all the frozen material passes through a 0.46 mm mesh sieve [8].

Environmental contaminants of concern to the NS&T Program include aromatic

hydrocarbons, chlorinated organics including polychlorinated biphenyls, and

trace elements (Tables 1 and 2). Using the following methods, the NBS will

supply the NS&T Program with data for some samples included in the Specimen

Banking portion of the program.

For the determination of organic contaminants, the sample homogenate is

first mixed with precleaned anhydrous sodium sulfate and internal standards are added. The sodium sulfate mixture is then Soxhlet extracted using methylene

chloride. The fractions containing the aromatic hydrocarbons, polychlorinated

biphenyls, and chlorinated organics are isolated using a silica microf ractionation column, gel permeation column, and finally an aminosilane liquid

chromatographic column. The concentrated fractions are analyzed by gas

chromatography using flame ionization detection or liquid chromatography using

fluorescence detection for the aromatic hydrocarbons and by gas chromatography

using election capture detection for the polychlorinated biphenyls and

chlorinated organics [9].

Selected trace elements are determined in the sediment and tissue samples

using a combination of instrumental neutron activation analysis and x-ray

fluorescence analysis. The combination of these two techniques has been

demonstrated to provide data for up to 44 elements in bivalve samples [10].

24
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26
Table 2. Major and Trace Elements Determined for Archived Samples

(in order of atomic number)

Chemical Abstracts

Registry Number

Al Aluminum 7429-90-5

Si Silicon 7440-21-3

Cr Chromium 7440-27-3

Mn Manganese 7439-96-5

Fe - Iron 7439-89-6

Ni Nickel 7440-02-0

Cu Copper 7440-50-8

Zn Zinc 7440-66-6

As Arsenic 7440-38-2

Se Selenium 7782-49-2

Sn Tin 7440-31-5

Sb Antimony 7440-36-0

Ag Silver 7440-22-4

Cd Cadmium 7440-43-9

Hg Mercury 7439-97-6

Pb Lead 7439-92-1

VII. CONCLUSIONS

Sample collection protocols developed and used for the collection of

benthic fish, bivalve molluscs, and associated sediments have provided speci- mens from over 40 sites nationwide (Table 3) , which have now been submitted for

inclusion in the Specimen Bank housed at the National Bureau of Standards in

Gaithersburg , Maryland. Specimens are preserved at liquid nitrogen vapor

temperature to inhibit degradation for decades. Initial analysis of archived

samples provides data that is used for Quality Assurance purposes since the

data can be compared to data generated by other NS&T projects. In addition,

this primary data set will allow a quantification of archived sample degradation, if any. Future analysis of specimens will allow the derivation of

baseline values for newly defined environmental contaminants and a reassessment

of the past when new and improved analytical procedures became available.

VIII. REFERENCES

[1] Olsson, M. (1985), The Environmental Specimen Bank at the Swedish Museum

of Natural History, In: International Review of Environmental Specimen

Banking, S. A. Wise and R. Zeisler, Eds., NBS Spec. Publ. 706, U.S.

Government Printing Office, Washington, DC, pp 26-32.

[2] Huggett, R. J., Nichols, M. M. , and Bender, M. E., (1980), Kepone

Contamination of the James River Estuary, In: Contaminants and Sediments, Vol. I, R. A. Baker, Ed., Ann Arbor Science, Ann Arbor, MI,

pp 33-52.

27
Table 3. National Status and Trends Specimen Bank Inventory

Archive Collection N W

ID# Date Location Site Species Latitude Longitude

BS1S001 19Jul85 Dana Point 33 26.8 11741.5 BS1F002 19Jul85 Dana Point White croaker 33 26.8 11741.5 BS1L003 29Jul85 Dana Point White croaker 33 26.8 11741.5 BS1F005 18Jul85 Dana Point White croaker 33 26.8 11741.5 BS1L006 18Jul85 Dana Point White croaker 33 26.8 11741,5 BS1S007 21Jul85 San Pedro Bay 33 44.5 116 10.5 BS1F008 20Jul85 San Pedro Bay White croaker 33 44.5 116 10.5 BS1L009 20Jul85 San Pedro Bay White croaker 33 44.5 116 10.5 BS1S010 12Aug85 Elliott Bay 4741.0 122 21.0 BS1F011 12Aug85 Elliott Bay English sole 4741.0 122 21.0 BS1L012 12Aug85 Elliott Bay English sole 4741.0 12221.0 BS1S013 14Aug85 Nisqually Reach 4741.0 122 44.5 BS1F014 13Aug85 Nisqually Reach English sole 4741.0 122 44,5 BS1L015 13Aug85 Nisqually Reach English sole 4741.0 122 44,5 BS1S016 05Sep85 Round Island, MS 30 19.0 88 36,0 BS1F017 04Sep85 Round Island, MS Atlantic croaker 30 19.0 88 36.0 BS1L018 04Sep85 Round Island, MS Atlantic croaker 30 19.0 88 36.0 BS1S019 09SGp85 Miss. R. Delta 29 05.0 89 04.0 BS1F020 08Sep85 Miss. R. Delta Atlantic croaker 29 05.0 89 04.0 BS1L021 08SGp85 Miss. R. Delta Atlantic croaker 29 05.0 89 04.0 BS1S022 23Sep85 Corpus Christi 27 50.0 97 17.0 BS1F023 20Sep85 Corpus Christi Atlantic croaker 27 50.0 97 17.0 BS1L024 20Sep85 Corpus Christi Atlantic croaker 27 50.0 97 17.0 BS1S025 140ct85 Charlotte Harbor 26 51.0 82 07.0 BS1F026 140ct85 Charlotte Harbor Spot 26 51.0 82 07.0 BS1L027 140ct85 Charlotte Harbor Spot 26 51.0 82 07.0 BS1S028 17Jul85 Chesapeai^e Bay Spot 38 55.7 76 25.0 BS1F079 27Mar86 Great Bay Winter Flounder 39 31.0 74 23.0 BS1S080 25Mar86 Great Bay 39 31.0 74 23.0 BS1L083 08Apr86 Boston Harbor Winter Flounder 42 20.0 71 00.0 BS1F084 08Apr86 Boston Harbor Winter Flounder 42 20.0 71 00.0 BS1S085 08Apr86 Boston Harbor 42 20.0 71 00.0 BS1L086 07apr86 Buzzards Bay Winter Flounder 41 35.0 70 45.0 BS1F087 07Apr86 Buzzards Bay Winter Flounder 41 35.0 70 45.0 BS1S088 08Apr86 Buzzards Bay 41 35.0 70 45,0 BS1F097 29Jun86 Bodge Bay Starry Flounder 38 18.0 123 02.0 BS1L098 29Jun86 Bodge Bay Starry Flounder 38 18.0 123 02.0 BS1S099 30Jun86 Bodge Bay 38 18.0 123 02.0 BS1F100 08Jul86 Southampton Shoal Starry Flounder 37 54.0 122 25.0 BS1L101 08Jul86 Southampton Shoal Starry Flounder 37 54.0 122 25.0 BS1S102 08Jul86 Southampton Shoal 37 54.0 122 25.0 BS1F103 16Jul86 Santa Monica Bay Hornyhead Turbot 33 53.0 118 26.0 BS1L104 16Jul86 Santa Monica bay Hornyhead Turtx)t 33 53.0 118 26.0 BS1S105 16Jul86 Santa Monica Bay 33 53 0 118 26.0 BS1F106 19Jul86 Dana Point Hornyhead Turbot 33 27.0 117 42.0 BS1L107 19Jul86 Dana Point Hornyhead Turbot 33 27.0 11742.0 BS1S108 19Jul86 Dana Point 33 27.0 117 42.0 BS1S109 28Jul86 Elizabeth River 36 50.0 76 22.0 BS1S110 26Jul86 Lower Chesapeake 37 10.0 76 10.0 BS1S111 18Aug86 St. John's River 30 24.0 81 33.0 BS1L112 19Aug86 St. John's River Atlantic Croaker 30 24.0 81 33.0 BS1F113 29Aug86 St. John's River Atlantic Croaker 30 24.0 81 33.0 BS1S114 15Aug86 Sapelo Sound 31 33,0 81 14.0 BS1L115 13Aug86 Sapelo Sound Atlantic Croaker 31 33.0 81 14.0 BS1F116 14Aug86 Sapelo Sound Atlantic Croaker 31 33.0 81 14.0 MW1M029 11Jan86 San Diego Imperial Beach M. californianus 32 40.8 11708.2 MW1Y030 11Jan86 Galveston Bay Todd's Dump C. virginica 29 30.1 94 54.0 MW1S031 10Jan86 Galveston Bay Todd's Dump 21 30.1 94 54.0 MW1S032 20Jan86 San Diego Imperial Beach 32 36.0 117 10.0 MW1M033 27Jan86 Palos Verdes Royal Palms State Park M. californianus 33 43.5 118 19.6 MW1M034 27Jan86 Anaheim Bay West M. californianus 33 44,2 11807.9 MW1Y035 04Feb86 Savannah River Estuary Tybee Island C. virginica 33 44.2 80 50.6 MW1S036 05Feb86 Savannah River Estuary Tybee Island 31 588 80 50.6 MW1M037 06Feb86 Point Dume M. californianus 34 00,2 118 48.4 MW1S038 31Jan86 Matagorda Bay Gallinipper Point 28 35.0 96 34.0 MW1Y039 31Jan86 Matagorda Bay Gallinipper Point C. virginica 28 35.0 90 634 MW1S040 24Jan86 Lower Laguna Madre South Bay 26 02.6 97 10 5 MW1Y041 24Jan86 Lower Laguna Madre South Bay C. virginica 26 02.6 97 10.5 MW1S042 18Jan86 Calcasieu Lake St. John's Island 29 51.0 93 23.0 MW1Y043 18Jan86 Calcasieu Lake St. John's Island C. virginica 29 51.0 93 23.0 MW1 Y044 12Feb86 Charleston Harbor Fort Johnson C. virginica 32 43.7 79 51.9 MW1S045 1 1 Feb86 Charleston Harbor Fort Johnson 32 43.7 79 51.9

28
Table 3. (Continued)

MW1M046 23Jan86 Buzzards Bay Round Hill M. edulis 41 32.2 70 55.1 MW1S047 05FGb86 Buzzards Bay Round Hill 41 32.2 70 55.1 MW1M048 13Jan86 Boston Harbor Dorchester Bay M. edulis 42 18.1 71 02.0 MW1S049 16Jan86 Boston Harbor Dorchester Bay 42 18.1 71 02.0 MW1M050 03FGb86 NarragansGtt Bay Conanicut Island M. edu.lis 41 29.8 71 23.2 MW1S051 03Feb86 NarragansGtt Bay Conanicut Island 41 29.8 71 23.2 MW1 S052 12Feb86 AnahGim Bay West Jetty 33 44.0 118 06.0 MW1M053 12FGb86 Long Island Sound Housatonic River M. edulis 41 10.1 73 06.6 MW1S054 13FGb86 Long Island Sound Housatonic River 41 10.1 73 06.6 MW1M055 2lJan86 WhidbGy Island Possession Point M. edulis 47 53.2 122 25.3 MW1M056 04FGb86 Yaquina Head M. calilornianus 44 40.6 124 04.7 MW1M057 06Feb86 Coos Bay Russell Point M. edulis 47 53.2 124 13.0 MW1S058 29Jan86 Whidbey Island Possession Point 47 54.7 122 20.7 MW1S059 07Feb86 Coos Bay Russell Point 43 26.1 124 13.0 MW1 S060 08Feb86 Yaquina Head 44 37,0 124 00.4 MW1S061 01Mar86 Point DumG 33 59.9 118 46.9 MW1 Y062 04Mar86 ChGsapGakG Bay Smith Point C. virginica 37 47.6 76 17.1 MW1S063 04Mar86 ChGsapeake Bay Smith Point 37 47.6 76 17.1 MW1S064 18FGb86 Palos Verdes Royal Palms State Park 33 43.5 118 19,6 MW1S065 20FGb86 RookGry Bay Henderson Creek 26 01.8 81 43.8 MW1Y066 20Feb86 RookGry Bay Henderson Creek C. virginica 26 01.8 81 43.8 MW1S067 05FGb86 Mississippi Sound Pascagoula Bay 30 21.1 88 37.0 MW1Y068 05FGb86 Mississippi Sound Pascagoula Bay C. Virginica 30 21.1 88 37.0 MW1 S069 06FGb86 Mississippi Sound Pass Christian 30 19.8 89 19.6 MW1Y070 06FGb86 Mississippi Sound Pass Christian c. virginica 30 19.8 89 19.6 MW1S071 27FGb86 Cat Point Bar Apalachicola Bay 29 43.0 84 52.5 MW1Y072 27FGb86 Cat Point Bar Apalachicola Bay c. virginica 29 43.0 84 52.5 MW1Y073 12Mar86 Baltimore Harbor Mountain Point Bar c. virginica 39 04.4 76 24.7 MW1S074 12Mar86 BaltimorG Harbor Mountain Point Bar 39 04.4 76 24.7 MW1M075 1 5Mar86 Hudson Raritan Estuary Lower Bay M. edulis 40 34.2 7403.4 MW1 S076 13Mar86 Hudson Raritan Estuary Lower Bay 40 34.2 74 03.4 MW1M077 25Mar86 Delaware Bay Arnolds Point Shoal C. virginica 39 23.1 75 25.9 MW1S078 25Mar86 Delaware Bay Arnolds Point Shoal 39 23.1 75 25.9 MW1M081 04Mar86 Columbia River Fort StGvens M. edulis 46 13.9 124 00.5 MW1S082 04Mar86 Columbia River Fort StGVGns 46 13.9 123 51.9 MW1Y089 26Mar86 BrGnton Sound Bay GardornG C. virginica 29 35.9 89 38.5 MW1 S090 26Mar86 BrGnton Sound Bay GardGrnG 29 35.9 89 38.5 MW1Y091 29Mar86 Caillou LakG C. virginica 29 1 5.3 90 55.5 MW1S092 29Mar86 Caillou LakG 29 1 5.3 90 55.5 MW1 S093 11Apr86 San Francisco Bay San Mateo Bridge 37 35.8 122 14.5 MW1M094 11Apr86 San Francisco Bay San Mateo Bridge M. edulis 37 35.8 122 14.5 MW1S095 21Apr86 Point St. GGorgG 41 43.3 121 13.9 MW1M096 29Apr86 Point SL GGorge M. calilornianus 41 44.9 124 12.4

Key for first column fourth letter F = Fish muscle sample

L = Liver sample

M = Mussel sample S = Sediment Sample Y = Oyster sample

Species key

Atlantic croaker

English sole Hornyhead Turbot Spot

Starry flounder White croaker

Winter floundor American oystor Blue mussel

California mussGl

Micropogon undulatus Parophrys vetulus Pleuronichthys verticalis Leiostomus xanthurus

Platichthys stellatus Genyonemus lineatus Pseudopleuronecles americanus

Crassoslrea virginica Mylilus edulis Mytilus calilornianus

29
[3] Ocean Assessments Division (1986), The National Status and Trends Program

for Marine Environmental Quality, FY86 Program Description, National

Oceanic and Atmospheric Administration, Ocean Assessments Division,

Rockville, MD , 35 pp.

[4] Tripp, B. W. and Farrington, J. W. (1984), Using Sentinel Organisms to

Monitor Chemical Changes in the Coastal Zone: Progress or Paralysis,

Submitted to the Coastal Society, 9th Annual Conference, Atlantic City,

NJ .

[5] Farrington, J. W. (1983), Bivalves as Sentinels of Coastal Chemical

Pollution: The Mussel (and Oyster) Watch, Oceanus 1983, Vol. 26, No. 2,

pp 18-29.

[6] Farrington, J. W., Albaiges , J., Burns, K. A., Dunn, B. P., Eaton, P.,

Laseter, J. L. , Parker, P. L. , and Wise , S. A. (1980), Fossil Fuels, In:

The International Mussel Watch, National Academy of Sciences, 70 pp.

[7] Patterson, C. C. and Settle, D. M. (1976), The Reduction of Orders of

Magnitude Errors in Lead Analyses of Biological Materials and Natural

Waters by Evaluating and Controlling the Extent and Sources of Industrial

Lead Contamination Introduced During Sample Collecting, Handling, and

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

Analysis, P. D. LaFleur, Ed., NBS Special Publication 442, U.S.

Government Printing Office, Washington, DC, pp 321-351.

[8] Zeisler, R. , Langland, J. K. , and Harrison, S. H. (1983), Cryogenic

Homogenization of Biological Tissues, Anal. Chem. 55, 2431-2434.

[9] Schantz, M. M. , Chesler, S. N. , Koster, B. J., and Wise, S. A., (1988),

Analytical Methods for the Determination of Organic Contaminants in

Marine Sediments and Tissues, In: These Proceedings.

[10] Stone, S. F., Zeisler, R. , and Sanders, R. W. (1987), Sequential

Determination of 44 Trace Elements in Marine Bivalves, Anal. Chem., In

press

30
SECTION 4

INFLUENCE OF ENVIRONMENTAL CHEMICALS IN THE AQUATIC ENVIRONMENT

Istvan Gebefugi, Jan-Peter Lay, and Friedhelm Korte

Gesellschaft fur Strahlen-und Umweltforschung, mbH, Munchen

Institut fur Okologische Chemie

D-8042 Neuherberg

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

Since 1976, a research group at the institute has been carrying out field

experiments with xenobiotics in small natural ponds. The aim of these investigations is to study the fate of environmental chemicals and their effect on population dynamics of several species. Further information for interpreting

laboratory results from chemical testing experiments and verification by longterm experiments is needed. Our knowledge about the occurrence of environmental chemicals in aquatic systems is limited; neither the corresponding

residues of the chemical nor the biologicals have been thoroughly investigated.

We expected data about effects by single doses in short-term as well as long-term experiments with continuously low dosages of environmental chemicals.

The ecotoxicological behavior was studied by the elucidation of the influence

of the chemicals on population dynamics of phyto- and zooplankton species and

by observation of the fate of chemicals by measuring the concentrations in

water, sediment, and biota (accumulation studies) [1-3].

As a first example of our investigations, the results of a long-term field

study will be shown. The object of this work was the elucidation of the longterm behavior, dispersion, accumulation, and metabolism of hexachlorobenzene

pentachloronitrobenzene , and 4-chloroaniline (Parachloroaniline) in small

experimental ponds. The concentrations of the chemicals during the application

phase were chosen to be environmentally relevant, but still enabling residue

studies in parts of the systems over several vegetation periods. Aromatic

organochlorine compounds are known to be persistent in the aquatic environment.

Out of this group of contaminants, RGB and pentachloronitrobenzene (PCNB) have

been used as examples for highly chlorinated chemicals, and 4-chloroaniline

(4-CA) as a lower chlorinated compound.

II. EXPERIMENTAL SECTION

For the implementation of the program, six small ponds were dug in a marsh-meadow near a larger pond. After 12 months, a high diversity of plants

and animals could be observed in all ponds. One experimental pond was treated

with each of the following chemicals: RGB, PCNB, 4-CA, the others served as controls. The average size of the ponds was 2-3 m in diameter and about 0.8m

in depth. During the investigation period, the biotopes had no continuously

measurable influx or drain of water.

31
The concentration of each chemical in the water was maintained at about 50

Mg/L (50 ppb) over a period of 4 to 6 weeks. The totai amounts of the chemicals applied during the dosing period, calculated as concentrations, were 250

Mg/L for HCB and PCNB , and 480 /ig/L for 4-CA. The chemicals were applied with

traces of the respective '• ^ C- labelled materials to allow low level counting

analys is

III. RESULTS AND DISCUSSION

In Figure 1 , the decrease in concentrations of HCB and PNCB in the water

is shown, they have a comparable course.

-,— , 1

\—-—1 1 T

1 1 1 1 1

1 r 4 8 12 16 20 32 44 56

days

after application

Figure 1. Disappearance of hexachlorobenzene , pentachloronitrobenzene

and 4-chloroaniline from the water of experimental ponds

It was found that after 1.3 days (for HCB) and 1.8 days (for PNCB) after

the last application, 50% of the initial concentrations still could be detected

in the water phase. The rapid decrease of both chemicals in the water may be

caused by evaporation into the atmosphere and mainly by adsorption to sediment

or absorption by biota. As residue analyses demonstrate, the biocides in the

water were quickly transported to the bottom sediment which then acted as pool

for further dispersion (via water) into the biota.

Figure 2 shows the HCB-derived residues in an experimentally polluted

pond. In the sediment of the HCB- treated biotope, there is a buildup phase of

residues over several weeks, followed by a very slow declining phase. A

similar disappearance of residues can be observed in all fauna species. In the

PNCB-biotope (Figure 3), comparable residual concentrations for all analyzed

samples were measured. The residual behavior of 4-CA, chemically quite

different from the other model substances, follows a similar but not equal

residual pattern, with a high initial buildup of concentrations in the biota

and slow decline in the sediment. The concentrations of 4-CA in gold fish

32
mg/kg

7

weeks

after application

I nov march sept march sept marc

I 1976 1977 1978 1979

last application

Figure 2. HCB-derived residues in an experimentally polluted small pond.

A; Notonecta glauca, B; Libellula quadrimaculata, C; Lemna minor,

D; Ranunculus aquatilus, and E; sediment

Figure 3. PCNB-derived residues in an experimentally polluted small pond.

A; Notonecta glauca, B; Libellula quadrimaculata, C; Lemna minor,

D; Ranunculus aquatilis, and E; sediment

33
(Carassius auratus) were determined in the gastrointestinal tract, muscles,

fat, and whole body.

The highest initial levels were found in the gastrointestinal tract (about

8 mg/kg) and the highest level after 10 weeks in the fat (about 7 mg/kg) . In

the same time, the concentration in the whole body was reduced, maybe the most

important result with respect to "Environmental Sample Banking."

In a short-term experiment, the effects of 2 , 4 , 6 - trichlorophenol (TCP) and

pentachlorophenol (PCP) on the population dynamics of several planktonic

species were studied. For this investigation, a natural pond was divided into

six compartments with tubes of 50 cm diameter resulting in a volume of about

200 L each. TCP and PCP were applied into the water with single doses of 5 mg/L and 1 mg/L, respectively. These are equivalent to about three times the

EC5g for TCP and two times the EC50 for PCP as determined in laboratory

toxicity tests with Daphnia magna.

A. Disappearance of TCP and PCP From Water

As shown in Figure 4, the concentration of both chemicals declined to

about 10% of their initial values 24 days after treatment. This decrease may

be due to evaporation from water, to adsorption to seston and absorption by

biota. It is evident that significant residues of the two chemicals remained

in the aqueous layer over the whole experimental period of 24 days, and thus were available for bioaccumulation and adverse affects to planktonic organisms.

Figure 4. Disappearance of pentachloro- and 2 , 4 , 6- trichlorophenol

from the water of the treated compartments

34
B. Effects Of TCP and PCP and BIOTA

As expected from the applied concentrations, both chemicals were highly

lethal to Daphnia pulex (Figure 5) . Three (PCP) and nine days (TCP) after the

applications, the total daphnia populations declined to zero and did not

recover during the 24 days of the experiment. Considering the important

intermediate role of daphnia in the food chain as consumers of algae and

detritus, it can be assumed that their disappearance affected the stability of

the phytoplankton and planktonic predator communities. Therefore, changes in

the community structure of the phytoplankton could have been caused either

directly by the chemicals or indirectly by changes in daphnia densities.

tinM (days)

<ft*f application Figure 5. Dynamics of daphnia concentrations in the treated and

untreated compartments

Phytoplankton

In Figures 6, 7 and 8, the population dynamics of three selected

phytoplankton species are presented. Chroococcus limneticus (blue-algae)

Trachelomonas hispida (flagellate) and Euglena acus (flagellate) were selected

as representatives of blue-green/mixotrophic species because of their natural

abundance and characteristic response to the chemical perturbation.

In the TCP as well as in the PCP treated compartments, the population

densities of these autotrophic and mixotrophic plankton showed distinctive

trends in response to chemical exposure. As compared to their control population, C. limnetus decreased significantly demonstrating direct sensitivity to

the chemical pollutant (primary effect) , whereas the mixotrophic species showed

a significant increase after chemical treatment. This increase may mainly be a consequence of the reduced grazing pressure by daphnia (secondary effect) or

35
(individual* / lOnm)

CHROOCacuS = TRACHEL0M0N4S

o----o= EUGLEN*

» W 12 14 1« n JO 71

aa**nlk>n (doyij Figure 6 Concentration dynamics of three plankton species in the

TCP- treated compartments

(individu*l»/ lOml

BOOC' «XtD

30000

2000O

sooo 4000

3000

200O

500 too

200

100

= CHROOCOCCUS Uulotfopfiic puncton)

= TRACh€LC>40NAS ^'nu>tmft^ fXincion)

= EUOLENA (innolropfiK: pUnclon)

10 12 li 16 18 20 22 24 tim€ (<M)r»

Figure 7. Dynamics of concentration of three plankton species in

the PCP- treated compartment

36
( individuals/ I0nr>|)

5 00(H

4 000

-r10T—I—I—I—I—

]L 16 18 20 22 24

days

after application

Figure 8 . Dynamics of concentrations

the control compartments , and Euglena spec, were not

compartments

of Chroococcus limneticus in

Note: Trachelomonas spec,

presented in the control

interspecific competition and probably not a direct consequence of chemical

exposure

As shown in Figure 9, the presence of PCP and TCP in the water results in

the complete elimination of diatoms (Nitzschia acicularis) 11 and 17 days,

respectively, after chemical application. The high sensitivity of diatoms to

both chemicals is in agreement with the classification of these species as representatives of clean water associations according to water quality standard

II [4]. In the control compartments, a species equilibrium appears after a slight decrease in numbers at the beginning of experiment due to effects of

compartmentalization. The increase of heterotrophic microorganisms (bacteria)

in all treated compartments, following the reduction of the autotrophic

organisms, exemplifies the drastic alteration of the structure in the

contaminated biotopes (Figure 10). Watanabe [5] found a comparable increase

six weeks after single application of PCP by field soil.

37
datom*** / 10 mi noo

18 20 22 24

time (d«vs)

Figure 9. Dynamics of diatom concentrations (Nitzschia acicularis) in the

treated and untreated compartments

260 260 2iO

220 200

180

160

1A0 120 100

806040

20

microorganisms/ lOmI

.10'

. r

= controls

/

• .7 — • = PCP

» * =TCP

tim* aftfr apptkatlon (days)

Figure 10. Concentration dynamics of heterotrophic microorganisms in

the water of the treated and untreated compartments

38
IV. CONCLUSIONS FOR SPECIMEN BANKING

For the purpose of sampling design strategies for Environmental Specimen

Banking, the following items have to be pointed out:

1. sediments can lose/decrease their chemical contents (concentrations).

It is reasonable (suggestive) to collect and store "fresh" or new sediments (e.g., sediments following one vegetation period) for

surveying and comparing the concentrations of organic chemicals.

2. Data of residue kinetics for organochlorine chemicals suggest that fat

tissue (adipose tissue) from fish samples should be stored in addition

to liver and muscle tissue.

3. Knowledge about population figures (density, dynamics) in the water

collecting sites are necessary information to characterize the sampling

sites for aquatic ESB-samples.

V. REFERENCES

[1] Lay, J. P., Schauerte, W. , and Klein, W. (1984), Effects of

Trichloroethylene on the Population Dynamics of Phyto- and Zooplankton in

Compartments of a Natural Pond, Environ. Pollut. 33, 75-91.

[2] Schauerte, W. , Lay, J. P., Klein, W. , and Korte, F. (1982), Long-Term

Fate of Organochlorine Xenobiotics in Aquatic Ecosystems, Ecotoxlcology

and Environmental Safety 6, 560-569.

[3] Schauerte, W. , Lay, J. P., Klein, W. , and Korte, F. (1982), Influence of

2 , 4 , 6 -Trichlorophenol and Pentachlorophenol on the Biota of Aquatic

Systems, Chemosphere 11, 71-79.

[4] Streble, H. and Krauter, D. (1974), Mikroflora and Mikrofauna des

Susswassers, Kosmos , Stuttgart, 36-37.

[5] Watanabe , I. (1977), Pentachlorophenol-Decomposing and PGP-Tolerant

Bacteria in Field Soil Treated with PGP, Soil Biol. Biochem. 7, 99-103.

39
SECTION 5

ANALYTICAL METHODS FOR THE DETERMINATION OF ORGANIC CONTAMINANTS

IN MARINE SEDIMENTS AND TISSUES

Michele M. Schantz , Stephen N. Chesler, Barbara J. Koster,

and Stephen A. Wise

Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, Maryland 20899

USA

I . INTRODUCTION

Samples representative of the marine environment were one of the four

types of specimens originally proposed for storage in the Pilot Environmental

Specimen Bank established at the National Bureau of Standards (NBS) in conjunction with the Environmental Protection Agency. In 1985, NBS began archiving

marine sediment, fish tissue, and bivalve molluscs as part of the National

Oceanic and Atmospheric Administration's (NCAA) National Status and Trends

(NS&T) Program. The purpose of the NS&T program is to quantify the current

status, as well as long-term trends, of contaminant concentrations and biological indicators of contaminant effects in the United States' coastal and

estuarine environments [1]. The NBS Environmental Specimen Bank receives

samples from two NS&T activities, the Benthic Surveillance Project and the

Mussel Watch Project. In the Benthic Surveillance Project, benthic fish

samples (muscle and liver tissue) and sediment samples are archived from 12

sites per year of the 50 sampling sites within the project. In the Mussel

Watch project, bivalve molluscs and sediments are archived from 30 sites per

year of the 150 sampling sites within the project. The collection of specimens

for the NS6tT specimen banking activity are described in more detail elsewhere

[2] .

For each sample to be archived, NBS receives duplicate samples (designated

as A and B) of approximately 150 g each, in the case of the sediment, fish

liver, and fish muscle, or, in the case of the bivalve molluscs, either 30

oysters or 50 mussels per sample. Sediments from six of the 1985 Benthic

Surveillance Project sites and bivalve molluscs from six of the Mussel Watch

Project sites were selected for inorganic and organic analysis at NBS. For

these samples, one of the duplicates was shucked (bivalve molluscs only),

homogenized, and subsampled for analysis. The analyses of the twelve samples

and an intercalibration sediment sample provided by NCAA for the determination

of selected polycyclic aromatic hydrocarbons (PAH), chlorinated pesticides, and

polychlorinated biphenyls (PCBs) are discussed in this paper.

II. EXPERIMENTAL SECTION

Subsamples from the "B" portions of six sediment samples received from the

1985 Benthic Surveillance collection were analyzed for organic contaminants.

Samples from the following Benthic Surveillance sites were selected for

40
analysis: Chesapeake Bay, MD ; Mississippi River Delta, LA; Corpus Christi, TX;

Dana Point, CA; Nisqually Reach, WA; and Elliott Bay, WA. Subsamples of the "B"

portions of six samples received from the 1985 Mussel Watch collection were

also analyzed. Samples from the following Mussel Watch sites were selected for

analysis: Pascagoula Bay, FL (oysters, Crassostrea virginica) ; San Diego, CA

(mussels, Mytilus califomianus) ; Fort Johnson, SC (oysters, Crassostrea

virginica); Galveston Bay, TX (oysters, Crassostrea virginica); Baltimore

Harbor, MD (oysters, Crassostrea virginica); and Dorchester Bay, MA (mussels,

Mytilus edulis) . These same samples were analyzed for the determination of

inorganic contaminants as described elsewhere [3].

Flow diagrams of the analytical procedures used for the analyses of the

sediment and bivalve tissue samples are shown in Figures 1 and 2, respectively.

An aliquot of wet sediment or bivalve tissue was first mixed with precleaned

sodium sulfate. Known amounts of appropriate internal standards were added to

the tissue/sodium sulfate mixture. The following internal standards were used:

1 -butyl pyrene for the determination of PAH, deuterated 4,4' DDT for the

determination of chlorinated pesticides, and PCB #198 (2, 2', 3, 3', 4, 5, 5',

octachlorobiphenyl ) for the determination of PCBs . This mixture was then

Soxhlet extracted using methylene chloride, and the extracts were evaporatively

concentrated to approximately 300 /iL.

For the sediment extracts (Figure 1), the concentrate was placed onto a small precleaned silica column (Sep Pak, Waters Associates, Milford, MA) where

the more polar components of the sediment extract were retained. For the

bivalve tissue samples (Figure 2), the concentrated extracts were injected onto

a liquid chromatographic preparative scale size exclusion column (10 /^m

polystyrene/divinylbenzene gel column, 100 A, 25 mm i.d. x 60 cm. Polymer

Laboratories, Inc., Amherst, MA). The void volume of the system plus a lipid

fraction were discarded before collection of the fraction of interest (i.e.,

the fraction containing the PAH, chlorinated pesticides, and PCBs). For both

the sediment and bivalve samples, the fractions collected were concentrated to

approximately 400 /iL for the final liquid chromatographic fractionation on an aminosilane column (LC-NH2).

At this point, the samples were divided in half (approximately 200 /xL

each). One aliquot was used for the LC-NH2 isolation of the PAH fraction, and

the other aliquot was used for the LC-NH2 isolation of the chlorinated pesticide and PCB fractions. In both cases, the column used to isolate the fractions was a semi-preparative aminosilane column (7.9 mm i.d. x 30 cm, /xBondaPak

NH2 , Waters Associates, Milford, MA). The PAH fractionation procedure has been

described previously [4-8]. For the fractionation of the chlorinated pesticides and PCBs, the mobile phase was 100% hexane for the PCBs and lower

polarity pesticides. The pesticides of interest in this fraction were hexachlorobenzene , heptachlor, aldrin, 2,4' DDE, 4,4' DDE, and mirex. At this

point, the mobile phase was changed to 5% methylene chloride in hexane for

elution of the more polar pesticides. The pesticides of interest included in

this fraction were lindane, heptachlor epoxide, a-chlordane, trans -nonachlor

,

dieldrin, 2,4' DDD, 4,4' DDD , 2,4' DDT, and 4,4' DDT. Further details of this

fractionation procedure will be published elsewhere [9]. At least two

calibration solutions containing the compounds of interest and the same

41
SEDIMENT SAMPLE

SOXHLET EXTRACTION

SILICA SEP PAK

I

LC- NH2

PAH FRACTION

1

LC-NH2

GC-FID

1

LC-FLUORESCENCE

PCBs AND

LOWER POLARITY

PESTICIDES

GC- ECD

MORE

POLAR

PESTICIDES

GC- ECD

Figure 1. Flow diagram for the analysis of sediment samples

BIVALVE TISSUE SAMPLE

SOXHLET EXTRACTION

SIZE EXCLUSION

CHROMATOGRAPHY

LC- NH2

1

PAH FRACTION

1

LC-NH2

1

GC-FID LC-FLUORESCENCE

PCBs AND MORE

LOWER POLARITY POLAR

PESTICIDES PESTICIDES

GC- ECD GC-ECD

Figure 2. Flow diagram for the analysis of bivalve tissue sample

42
internal standards were taken through identical Soxhlet extraction and

fractionation steps

.

The three fractions from the LC-NH2 step were evaporatively concentrated

to 200 nh for gas chromatographic (GC) analysis. The column used was an immobilized nonpolar fused silica capillary (30 m x 0.25 mm i.d., 0.25 nm coating thickness) . For GC analysis of the PAH fraction, a flame ionization

detector (FID) was used, and 2/iL of the sample was injected in a splitless

mode using hydrogen as the carrier gas. For the GC analysis of the other two

fractions, an electron capture detector (ECD) was used. As above, 2 /iL of the

sample was injected but in a split mode using helium as the carrier gas. For

the analysis of the intercalibration sediment sample, a reversed-phase LC

method with fluorescence detection was also used to determine the PAH [8,9].

This LC- fluorescence method provides a second analytical technique for com- parison with the GC analyses, and this method will be used to determine PAH in

sediment and bivalve samples in which the PAH concentrations are too low for

measurement by the GC procedure.

III. RESULTS AND DISCUSSION

In Figure 3, chromatograms are shown for the GC analysis of the PAH

fraction from (1) a calibration solution (2) a sediment extract, and (3) a mussel extract. Chromatograms from the GC analyses of the PCB and pesticide

fractions from mussel and sediment extracts are shown are shown in Figures 4

and 5 .

An intercalibration sediment sample distributed by NOAA was analyzed using

the method shown in Figure 1 and by three laboratories utilizing the method

shown in Figure 6 [10]. The method shown in Figure 6 uses two gravity-fed

chromatographic columns, a silica/alumina column which separates the saturated

hydrocarbons from the aromatic hydrocarbons and a Sephadex LH-20 column which

removes the lipid and biogenic material. Using the method shown in Figure 6, one fraction containing the PAH, PCBs , and chlorinated pesticides is obtained.

The results ( in ng/g dry weight) from the analysis of the intercalibration

sediment sample are compared in Table 1 . There was good agreement among the

laboratories for the determination of PAH concentrations. The agreement among

all the laboratories for the PCB and pesticide concentrations, however, was not

as good.

Sediment samples from six sites in the Benthic Surveillance project were analyzed for the determination of PAH, PCBs, and pesticides. The concentrations (given in terms of ng/g dry sediment) of selected compounds in these

sediment samples are summarized in Table 2. The concentrations for individual

PAH range from less than the detectable limit to 5280 ng/g dry weight of

chrysene/triphenylene in the Corpus Christ! , TX sediment. The majority of the

pesticide concentrations are less than the detectable limit. The highest PCB

congener concentration (58 ng/g dry weight of PCB #153) was also found in the

Corpus Christi, TX sediment.

Subsamples of bivalve specimens from six sites in the Mussel Watch Project

were also analyzed. The concentrations (given in terms of ng/g dry tissue) of

43
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46
SEDIMENT

TUMBLER

T

SILICA / ALUMINA

COLUMN

SEPHADEX

LH-20 COLUMN

AROMATIC AND

CHLORINATED

HYDROCARBONS

GC-FID

AND GC-ECD

Figure 6. Flow diagram of the procedure used by NOAA laboratories

for analysis of the intercalibration sediment sample

47
Tabie 1 . Mean Concentrations of Selected Organic Contaminants in NOAA

Intercalibration Sediment (ng/g dry sediment)

Compound Lab 1^ Lab 2^ Lab 3^ NBS-GC*' NBS-LC^ '

Phenanthrene 2300 2200 3200 2420(220) 2280(170)

Anthracene 510 650 730 710 (60) 490 (40)

Fluoranthene 3900 3700 5600 3660(430) 3420(190)

Pyrene 4100 3900 5800 3930(360) 3640(260)

Benz [ a ] anthracene 1500 1400 2100 1840(200) 1390(110)

Chrysene + Tripheny lene 2600 2100 3600 2700( 190)

Benzo [ e ] pyrene 1600 1400 2000 1860(100)

Benzo [ a ] pyrene 1800 1700 2700 2540(170) 2140(140)

Pery lene 510 460 710 770 (40) 570 (30)

Hexachlorobenzene 0 .

0 .32 <0 . 5 Heptachlor epoxide <0. 3 <0.8 <2 0.6(0. 1)

Q-Chlordane 0.9 2 2 <0.2

Trans -nonachlor 0.4 0.9 <1 0.9(0. 1)

4,4' -DDE 7 10 9 1 .9(0. 1)

2,4' -DDT 4 5 <2 1 .4(0. 1)

4,4' -DDT <0.5 10 <2 2.4(0. 1)

PCB #47 8 9 14 9.5(0.4)

PCB #101 70 64 63 117 (5)

PCB #153 110 77 72 127 (8)

PCB #194 8 17 9 10.0(0.4)

^Reference [10]

''Numbers in parentheses are standard deviations of a single measurement and

< numbers indicate minimum detectable limit for the compound in the sample.

^ LC- fluorescence ana lysis of PAH fraction

48
Table 2. Concentrations of Selected Organic Contaminants in Six 1985 Benthic

Surveillance Sediments (ng/g dry sediment)^

Compound

Phenanthrene 2040(230) 50 (2) 490 (70) 170(10) 120(20) 400(10)

Anthracene 930 (30) 50 (4) <50 140(10) 30 (1) 180(10)

Fluoranthene 1060 (50) 20 (3) 540 (10) 230(20) 100(10) 640(30)

Pyrene 1040 (70) 260(30) 1800(140) 300(20) 120 (4) 250(20)

Benz [ a ] anthracene <50 <20 <50 40 (4) 50 (3) 260(30)

Chrysene + Triphenylene 1650 (10) 50 (2) 5280(220) 70(10) 90 (2) 640(40)

Benzo [ e ] pyrene 870 (80) 100(10) 760 (30) 140 (4) 60(10) 330(30)

Benzo [ a ] pyrene 1130 (50) 160(10) 2130(270) 630(20) 130(20) 500(20)

Perylene <50 <20 <50 120(10) <30 <50

Hexachlorobenzene <23 3 10.3(0.7) <13.4 6.5(0. 3) <1.7 <5.2

Heptachlor epoxide <3 5 <0.3 <2.7 5.2(0. 1) <0.2 <0.8

a-Chlordane <1 7 <0.2 <1 .2 <0.4 <0.1 <0.4

Trans -nonachlor <2 6 <0.2 <1.8 <0.6 <0.2 <0.5

4,4' -DDE 3 9(0. 2) <0.2 <3.1 <0.5 <0.2 1.6(0.1)

2,4' -DDT <0 5 <0.1 <0.4 <0.

1

<0.

1

<0.

1

4,4' -DDT <0 7 <0.1 <0.5 <0.2 <0.

1

<0.

1

PCB #47 <9 4 <0.8 <14.8 <1 .7 <0.7 <2.

1

PCB #101 15 5(0. 6) 9.3(0.2) 51.9(3.5) 18.0(0. 9) <0.4 24. 1(0.4)

PCB #153 <2 5 6.6(0.1) 58.0(3. 1) 13.7(0. 4) 5.2(0.2) 33.3(0.4)

PCB #194 18 9(1. 0) 4.3(0.1) 19.9(1 .0) 9.5(0. 3) 0.3(0.1) 34.9(0.5)

^Average of 3 extracts each analyzed 3 times. Numbers in parentheses are the

standard deviation of a single measurement. The < values indicate the minimum

detectable limit of the instrument for this compound in this sample.

Chesapeake Bay, MD

•^Mississippi River Delta, LA

"^Corpus Christi, TX

^Dana Point, CA

^Nisqually Reach, WA

8 Elliott Bay, WA

49
selected compounds are suiiunarized in Table 3. The PAH concentrations are, in

general, lower in the bivalve tissue samples (Table 3) than in the sediment

samples (Table 2) with values ranging from below the minimum detectable limit

to 270 ng/g dry weight of phenanthrene in the Dorchester Bay, MA mussel tissue.

The 4,4' -DDE concentrations are higher in the bivalve tissue samples than in

the sediment samples ranging from 7 ng/g dry tissue in the Galveston Bay, TX

oysters to 163 ng/g dry tissue in the San Diego, CA mussels. The highest PCB

congener concentration in the bivalve tissue samples was 93 ng/g PCB#101

(Dorchester Bay, MA).

I¥. CONCLUSIONS

The procedures described in this paper were used to analyze an intercalibration sediment, as well as six sediment and six bivalve samples

collected as part of the NOAA NS&T Specimen Banking program. The liquid

chromatographic fractionation steps provide relatively clean fractions containing the analytes of interest. These methods will be used to provide baseline

values for organic contaminants in representative marine specimens banked for

the NS&T program.

V . ACKNOWLEDGEMENT

The authors acknowledge the assistance of Gunnar G. Lauenstein in the

coordination of the Specimen Banking activities for the NS&T Program of NOAA.

We acknowledge the cooperation and assistance of the following individuals in

the collection of samples: Sandy Freitas (Battelle New England Marine Laboratory) , Andy Lissner (Pacific Northwest Laboratory) , Gary Wolff (Texas A & M

University). Robert C. Clark, Jr., (National Marine Fisheries Northwest and

Alaska Fisheries Center) , Peter Hanson (National Marine Fisheries Southeast

Fisheries Center) , and Vincent Zdanowicz (National Marine Fisheries Northeast

Fisheries Laboratory) . We acknowledge the assistance of Kevin Diamond in the

preparation of the bivalve specimens from the NS&T Program. This work was supported in part by the Ocean Assessments Division, NOAA.

REFERENCES

[1] Ocean Assessments Division (1986), The National Status and Trends Program

for Marine Environmental Quality, FY86 Program Description, National

Oceanic and Atmospheric Administration, Ocean Assessments Division:

Rockville, MD , 35 pp.

[2] Lauenstein, G. G. and Calder, J. A. (1988), Specimen Banking: A Key

Element of the National Oceanic and Atmospheric Administration's National

Status and Trends Program, In: These Proceedings.

[3] Stone, S. A., Becker, D. A., Koster, B. J., Pella, P. A., Sleater, G.,

Tillekeratne , M. P. M. , and Zeisler, R. (1988), Inorganic Analytical

Methods and Results for Marine Bivalves and Sediments, In: These

Proceedings

50
Table 3. Concentrations of Selected Organic Contaminants in Six 1985 Mussel Watch

Bivalve Tissue Samples (ng/g dry bivalve tissue)^

Compound PB^ SD^ GB^ BH^ DB8

Phenanthrene <20 <30 <50 <40 90(10) 270(20)

Anthracene 80(10) <30 <40 50(10) 30(10) 30(10)

Fluoranthene 100(10) <20 <170 260(10) <30 <100

Pyrene 90(10) <30 170(10) 200(10) 40(10) 130(10)

Benz [ a ] anthracene <80 <40 50(10) 70(10) <20 70(10)

Chrysene + Triphenylene 210(20) 50(10) 110(10) <50 50(10) 210(10)

Benzo [ e ] pyrene 40(10) 50(10) 70(10) <40 20(10) 80(10)

Benzo [ a ] pyrene <90 50(10) <50 <30 <30 60(10)

Perylene <40 <50 <60 <30 <40 <40

Hexachiorobenzene <0.2 0.6(0.1) 1.0(0.1) 3.1(0.3) 15.2(0. 1) 1.5(0.1

Heptachlor epoxide <0.2 <0.2 <0.7 <0.2 <0.

1

5.5(0.2

Q-Chlordane <0.

1

0.8(0. 1) <0.4 <0.2 3.5(0. 1) 12.4(0.2

Trans -nonachlor <0.2 <0.2 <1 .6 2.2(0.1) 2.5(0.1) 6.1(0.2

4,4' -DDE 10.8(0.6) 163.4(5.9) 8.4(0.8) 7.3(0.7) 14.6(0.3) 28. 1(1 .0

2,4' -DDT <0.6 76.0(2.2) <0.3 <0.3 13.6(1 .0) 0.6(0.1

4,4' -DDT <0.8 14.7(1.3) <2.4 <1.3 <0.5 0.7(0.1

PCB#47 <0.9 3.0(0.1) 4.0(0.2) <1.8 3.0(0. 1) 49.8(3.7

PCB#101 11.3(0.9) 13.7(0.8) 5.7(0.3) 10.2(0.3) 10.8(0.9) 93.0(4.4

PCB#153 16.9(0.9) 30.0(0.6) 9.2(0.3) 11.9(0.3) 13.0(0.2) 79.7(2.5

PCB#194 <0.4 0.4(0. 1) 0.3(0.1) <0.4 <0.3 0.2(0.1

^Average of two extracts each analyzed in triplicate. Numbers in parentheses are the

standard deviation of a single measurement. The < values indicate the minimum

detectable limit of the instrument for this compound in this sample.

^Pascagoula Bay, FL - oysters

'^San Diego, CA - mussels

'^Fort Johnson, SC - oysters

^Galveston Bay, TX - oysters

^Baltimore Harbor, MD - oysters

^Dorchester Bay, MA - mussels

51
[4] Wise, S. A., Chesler, S. N., Hertz, H. S., Hiipert, L. R. , and May, W. E. (1977), A Chemically Bonded Aminosilane Stationary Phase for the HighPerformance Liquid Chromatographic Separation of Polynuclear Aromatic

Compounds, Anal. Cham. 49, 2306-2310.

[5] Hertz, H. S., Brown, J. M. , Chesler, S. N., Guenther, F. R. , Hiipert,

L. R. , May, W. E., Parris , R. M. , and Wise, S. A. (1980), Determination

of Individual Organic Compounds in Shale Oil, Anal. Chem. 52, 1650-1657.

[6] Wise, S. A., Benner, B. A., Chesler, S. N., Hiipert, L. R. , Vogt, C. R.

and May, W. E. (1986), Characterization of the Polycyclic Aromatic

Hydrocarbons from Two Standard Reference Material Air Particulate

Samples, Anal. Chem. 58, 3067-3077.

[7] May, W. E. and Wise, S. A. (1984), Liquid Chromatographic Determination

of Polycyclic Aromatic Hydrocarbons in Air Particulate Extracts, Anal.

Chem. 56, 225-232.

[8] Kline, W. F., Wise, S. A., and May, W. E. (1985), The Application of

Perdeuterated Polycyclic Aromatic Hydrocarbons (PAH) as Internal

Standards for the Liquid Chromatographic Determination of PAH in a Petroleum Crude Oil and Other Complex Mixtures, J. Liquid Chromatogr . 8,

223-237.

[9] Parris, R. M. , Wise, S. A., and Chesler, S. N., Unpublished Manuscript.

[10] MacLeod, W. D. , Jr (1986), Trace Analysis and Quality Assurance for

Extractable Organic Chemicals, In: Summary Report of NOAA-NBS Quality

Assurance Workshop, December 1985,1986; J. K. Taylor, Ed., NBSIR 86-3446,

National Bureau of Standards, Gaithersburg , MD, pp. 4-11.

52
SECTION 6

COMPARATIVE INVESTIGATIONS ON TRACE METAL LEVELS IN BROWN ALGAE AND COMMON

(BLUE) MUSSELS AT THE SAME LOCATION IN THE BALTIC SEA AND THE NORTH SEA

M. Stoeppler, F. Backhaus, M. Burow, K. May, and C. Mohl

Institute of Applied Physical Chemistry

Nuclear Research Center (KFA) Julich

D-5170 Julich

FEDERAL REPUBLIC OF GERMANY

SUMMARY

In order to obtain basic data for trace metal contents in a marine algae

(Fucus vesiculosus) in comparison to a marine mussel (Mytilus edulis) , arsenic,

cadmium, lead, total mercury, methylmercury , and selenium have been determined

in specimens found close together at several locations in the North Sea and the

Baltic Sea. The results indicate that the levels of cadmium, total mercury,

methylmercury, selenium and possibly lead are somewhat to significantly higher

in mussels, whereas higher arsenic and nickel levels are found in algae. From

these data it appears that the simultaneous use of algae and mussels, at least

for a distinct investigation period, may have advantages for the Environmental

Specimen Bank in the Federal Republic of Germany.

I . INTRODUCTION

During preliminary research of the Pilot Environmental Specimen Bank (ESB)

Program of the Federal Republic of Germany performed from 1976 to 1984 and from

similar investigations at the National Bureau of Standards (NBS) in the United

States of America, it has been shown that specimen banking from the present

state-of-the-art is now feasible [1-8]. Thus, the Federal Government decided

in 1984 to establish a permanent ESB in the Federal Republic of Germany

beginning in January 1985. Since the pilot phase was predominantly aimed at

technical, analytical and organizational studies, the first part of the

permanent operation was a transition program. This phase aims mainly at

identification and selection of representative sampling areas and appropriate

species for environmental monitoring and assessment in addition to current

collection and storage of already identified specimen types. From the beginning, our institute was responsible for tasks in the marine environment. Thus,

prior to and within the transition phase of the ESB program, we performed some investigations with marine species. The first species selected, as a result of

collaboration with the Institute of Marine Research of the University at Kiel,

F.R.G., was the brown algae (seaweed, Fucus vesiculosus) as a community forming

dominant species in coastal areas of the Baltic as well as of the North Sea.

In order to obtain reliable data for variations of trace metal contents with

location and season, we started a long-term study of sampling and analysis in

bimonthly intervals in 1982 including sea water analysis for baseline levels.

The results achieved so far indicate some seasonal dependence of metal levels

at nonpolluted sites in the Baltic Sea with the highest values observed in the

53
winter and the lowest values occuring in the summer, as best documented for

arsenic [ 9 ]

While it is supposed that algae incorporate trace elements predominantly

from the dissolved state, bivalves as filter feeders are able to scavenge

metals and organometallic compounds from particulate matter [10,11]. By

including the Common (or Blue) mussel (Mytilus edulls) in our marine research

and collection program, we would be able to make use of already existing

international mussel watch programs [12,13] and of the experience of the U.S.

Environmental Specimen Bank with this specimen [4,8]. In this context, it is

of importance to compare values obtained for both species collected at exactly

the same site and at the same time. The goal was to find out if those values

could be used at least partly to distinguish between dissolved and total metal

levels or if other important differences, e.g., due to physiological reasons,

could be seen and used for monitoring purposes. In this preliminary evaluation, the data found for mussels and algae collected from selected locations in

1984, 1985, and 1986 are reported, some facts discussed, and the preliminary

conclusions drawn.

II. SAMPLING SITES

Mussels as well as algae have been collected at the same site and at

different times at the following locations: Eckwarderhoerne , Nordstrand and

List/Sylt (North Sea), Glucksburg, Kronsgaard, Langholz, Kiel Strande, and

Dahme (Baltic Sea) as shown in Figure 1. At the Eckwarderhoerne site. Jade

Bay, we performed a detailed, study which is still ongoing, with bimonthly

collections starting in February 1985 that will include the analysis of trace

metals in water.

The above mentioned areas, except Nordstrand serve as current collection

sites for biannual collection and long-term storage of algae and mussels for

permanent banking. Within this concept, however, all sampling sites in the

western Baltic Sea contribute to a composite sample for the ESB since this area can be considered as an area that has only minimal metal pollution thus

providing baseline levels for the marine species now under study.

III. EXPERIMENTAL SECTION

A. Sampling and Sample Preparation

Fucus vesiculosus occurs in large communities in the littoral region of

the collection area and thus can be easily collected manually at low tide

wearing precleaned polyethylene gloves. Usually a sample amount of approximately 800 g from each community (6-7 randomly taken individual specimens) is

collected at the sampling site. The algae are then further prepared as follows. Adhering material (small animals, plants, inorganic parts) were separated first, e.g., with plastic forceps. The sample is then rinsed three

times in a stainless steel sieve with clear seawater from the sampling site.

Excess water is removed by rotating the sieve in the air. Next, the algae are transferred into weighed stainless steel vessels and immediately placed in

54
00
cryogenic transport containers where a temperature below -150°C is maintained

over liquid nitrogen (gas phase storage)

Mytilus edulis also occur in rather large communities at the investigated

sites providing enough material for the preparation of representative samples.

At low tide approximately 200 single mussels, shell length from 2 to 6 cm (equivalent to approximately 1000 g soft tissue) have been collected, rinsed,

and stored as described above for algae. The mussels were not allowed to

depurate in our experiments. Usually all sampling sites have been treated in

the same manner during a sampling campaign and the deep frozen samples

transported to the Environmental Specimen Bank in Jiilich in special vans.

Preparation of the samples for metal analysis is performed in the

following manner. The samples are thawed overnight in clean benches (class 100

environment) until the material is just below 0 °C. Algae can be passed

directly through a mincer made from an aluminum alloy, while the mussels have

to be dissected (separation of soft parts from the shells) prior to

homogenization in a household mixer.

A subsample of approximately 100 g (fresh weight) is taken from each

homogeneous material and stored at -20 °C for a few days until subsequent

analysis of total mercury and methylmercury . The remaining material is then

subjected to a freeze drying procedure on PTFE coated steel dishes and followed

by a moisture content determination. Subsequently, the whole materials are milled with agate ball mills until the particle size is < 200 fim. The latter

procedure is controlled by sieving. The material is then transferred into

250-mL polyethylene bottles with wide necks and stored in the dark at 15°C

until analysis.

B. Analytical Procedure

Total mercury analysis is performed on fresh material after wet digestion

with a mixture of nitric and perchloric acid in closed quartz vessels [14]

using instrumentation designed and optimized in our laboratory that allows the

routine determination of total mercury at the low /xg/kg-level [2,7]. Analysis

of methylmercury starts with a simple hydrochloric acid cleanup, ion exchange

separation of CHgHgCl from ionic mercury, UV decomposition of methylmercury and

subsequent determination as ionic mercury [15]. Quality assessment of the

latter procedure has been done via gas chromatography with Electron Capture

Detection (ECD)

Total and inorganic arsenic as well as total selenium is determined in all

materials as described elsewhere in detail using the hydride technique after

different sample treatment procedures with electrothermal atomization by use of

a PE MHS-20-system [9]. The remaining elements cadmium, lead, and nickel are determined after pressurized decomposition of 0.2-0.3 g subsamples (dry weight)

with ultrapure or purified (e.g., by low boiling distillation) analytical grade

nitric acid in PTFE vessels [2,7] by the application of improved GFAAS

(Graphite Furnace AAS) procedures [7,16]. Evaluation is performed, wherever

possible, against Environmental Specimen Bank Specific Reference Materials in

order to maintain an optimal accuracy and precision for routine measurements

[17] .

56
IV. RESULTS AND DISCUSSION

Table 1 summarizes all the data obtained for total arsenic, selenium,

mercury, lead, cadmium, and nickel in bimonthly collected samples of algae and

mussels starting with February 1985 at Eckwarderhoerne site. Jade Bay, North

Sea (see Figure 1). At this site, we have also started water analysis which

will be discussed elsewhere since the bimonthly collection and comparative

investigation will be continued and expanded to other elements, since at the

moment no clear seasonal variations can be seen.

In Table 2 the results are summarized for comparative measurements of the

above mentioned elements, except selenium, in algae and mussels at various

collection sites from spring 1984. This includes Nordstrand and List/Sylt from

the North Sea, as well as Strande, Kronsgaard, Dahme , Langholz, and Gliicksburg

from the Baltic Sea.

From Tables 1 and 2, it is evident that there can be seen some obvious

differences in elemental contents between both specimen types which at least,

will stimulate further detailed comparative studies for the benefit of the ESB

program in the FRG . Due to the fact that the mussels were not allowed to

depurate during this series of experiments and that it appears from more recent

studies with algae that uptake may occur also from particulate matter [18], the

differences found have to be evaluated further. Nevertheless, these data

clearly show that, irrespective of uptake mechanisms, arsenic and nickel values

certainly are higher in algae, while selenium, mercury, cadmium, and possibly

lead are higher in mussels. It is also interesting to note that mussels

contain higher methylmercury levels. From the ratio total mercury to methylmercury in algae , one could speculate that the aqueous phase has a methylmercury level that is well below 10% of total mercury. An excess appears

to be due to uptake from particulate matter or biological enrichment.

V. ACKNOWLEDGMENTS

The authors express their gratitude to the following Federal Ministries

that financially supported the whole ESB Program coordinated by the Federal

Environmental Agency (Unweltbundesamt) : Until December 1984, Bundesminis terium

fur Forschung und Technologie, until June 1986, Burndesminis terium des Inneren

and recently Bundesminis terium fur Umwelt, Naturschutz und Reaktorsicherheit

,

Bonn, FRG, to Mrs. M. Roth and Mr. N. Commerscheidt for their valuable

technical assistance.

VI . REFERENCES

[1] Boehringer, U. R. , Ed. (1981), Umweltprobenbank, Ergebnisse der

Vorstudien und Proj ektierung der Pilotphase, Umweltbundesamt , Berlin.

[2] Kayser, D., Boehringer, U. R. , and Schmidt-Bleek, F. (1982), The

Environmental Specimen Banking Project of the Federal Republic of

Germany, Pilot Phase, Environmental Monitoring and Assessment 1, 241-255.

57
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59
[3] Stoeppier, M. , Durbeck, H.W., and Nurnberg, H.W. (1982), Environmental

Specimen Banking: A Challenge in Trace Analysis, Talanta 29, 963-972.

[4] Lewis, R. A., Stein, N., Lewis, C. W., Eds. (1984), Environmental

Specimen Banking and Monitoring as Related to Banking, Martinus Nijhoff

Publishers, Boston, MA, 358 pp.

[5] Stoeppier, M. , Backhaus , F. , Schladot, J. D., and Nurnberg, H. W. (1984),

Concept and Operational Experiences of the Pilot Environmental Specimen

Bank Project in the Federal Republic of Germany, In: Environmental

Specimen Banking and Monitoring as Related to Banking, R. A. Lewis,

N. Stein, and C. W. Lewis, Eds., Martinus Nijhoff Publishers, Boston, MA,

pp 95-107.

[6] Nurnberg, H. W. (1984), Realization of Specimen Banking, Summary and

Conclusions, In: Environmental Specimen Banking and Monitoring as Related to Banking, Lewis, R. A., Stein, N., Lewis, C. W. , Eds., Martinus

Nijhoff Publishers, Boston, MA, pp 23-66.

[7] Stoeppier, M. (1985), Trace Metal Analysis for the German Environmental

Specimen Bank, In: Biological Reference Materials: Availability, Uses

and Need for Validation of Nutrient Measurements, Wolf, W. R. , Ed., John

Wiley, Inc., Chichester, 281-297.

[8] Wise, S. A. and Zeisler, R. , Eds. (1985), International Review of

Environmental Specimen Banking, NBS Spec. Publ. 706, 54 pp.

[9] Stoeppier, M. , Burrow, M. , Backhaus, F. , Schramm, W. , and Nurnberg, H.W.

(1986), Arsenic in Seawater and Brown Algae of the Baltic and the North

Sea, Marine Chemistry 18, 321-334.

[10] Philipps, D. J. H. (1977), The Use of Biological Indicator Organisms to

Monitor Trace Metal Pollution in Marine and Estuarine Environments - A

Review, Environ. Pollut. 13, 281-317.

[11] Stoeppier, M. (1979), Choice of Species, Sampling and Sample Pretreatment

for Subsequent Analysis and Banking of Marine Organisms Useful for

Mercury, Lead and Cadmium Monitoring, In: Monitoring Environmental

Materials and Specimen Banking, Luepke , N. P., Ed., Martinus Nijhoff

Publishers, Boston, MA, pp 555-572.

[12] Goldberg, E. D., Ed. (1980), The International Mussel Watch, National

Academy of Sciences, Washington, DC, 248 pp.

[13] Popham, I.D. (1985), Musseling in On Heavy Metal Pollution in Estuarine

Environments Using Principal Components, Can. Tech. Rep. Fish, Aquat.

Sci. 1268, 81-90.

[14] May, K. and Stoeppier, M. (1984), Pretreatment Studies with Biological

and Environmental Materials, IV. Complete Wet Digestion in Partly and

Completely Closed Quartz Vessels for Subsequent Trace and Ultratrace

Mercury Determinations, Fresenius. Z. Anal. Chem. 317, 248-251.

60
[15] May, K. , Stoeppler, M. , and Relsinger, K. (1987), Studies in the Ratio

Total Mercury/Me thy Imercury in the Aquatic Food Chain, Toxicological and

Environmental Chemistry 13, 153-157.

[16] Stoeppler, M. , Mohl, C, Novak, L. , and Gardiner, P.E. (1986),

Application of the STPF Concept to the Determination of Aluminum,

Cadmium, and Lead in Biological and Environmental Materials, In: Welz,

B. , Herausg, Fortschritte in der atomspektroskopischen Spurenanalytik,

Band 2, VCH-Verlagsges . mbH, Weinheim, 419 pp.

[17] Stoeppler, M. , Backhaus , F. , and Schladot, H.D., and Commerscheidt , N. (1987), Environmental Specimen Bank Specific Reference Materials,

Fresenius. Z. Anal. Chem. 326, 707-711.

[18] Luoma, S.N., Bryan, G.W., and Langston, W.J. (1982), Scavenging of Heavy

Metals from Particulates by Brown Seaweed, Marine Poll. Bull. 11,

394-396.

61
SECTION 7

INORGANIC ANALYTICAL METHODS AND RESULTS FOR MARINE BIVALVES AND SEDIMENTS

Susan F. Stone, Donald A. Becker, Barbara J. Koster, Peter A. Pella,

Gerald Sleater, Manoranjani P. M. Tlllekeratne^ , and Rolf Zeisler

Center for Analytical Chemistry,

National Bureau of Standards

Gaithersburg, Maryland 20899

USA

and

Ronald W. Sanders,

Pacific Northwest Laboratory,

Richland, Washington 99352

USA

I . INTRODUCTION

When studying the elemental composition of biological and environmental

samples, analytical chemists are frequently confronted with the limitations of

a particular analytical technique when applied to unique or small samples.

Generally, any analytical technique or even several techniques can only

determine a fraction of the elements contained in the samples. Since it can be

assumed that most of the elements in the periodic table occur at various levels

in every biological or environmental matrix and may be important for various

reasons, an analytical technique or combination of procedures that covers all

elements may be desirable.

An ideal combination of analytical methods would fulfill many of the

following requirements: 1) each individual technique can in principle assay all

elements; 2) the elemental quantitation is feasible from major components to

ultratrace constituents; 3) the techniques are non-destructive; 4) the procedures provide for internal quality control, e.g., via independent determinations of selected elements; and 5) only validated techniques are used which are suitable for routine analysis.

A unique sequence of instrumental analytical methods has been investigated

that included non-destructive analyses steps by X-ray fluorescence (XRF)

prompt gamma activation analysis (PGAA) , instrumental neutron activation

analysis (INAA) , and final destructive steps utilizing NAA with radiochemical

separations (RNAA) for additional sensitivity [1]. This approach has been

applied to twelve bivalve samples and six sediment samples from the National

Oceanic and Atmospheric Administration (NCAA) National Status and Trends (NS&T)

program [2], as well as to a collection of mussels from the National Bureau of

Standards (NBS) / Environmental Protection Agency (EPA) Specimen Bank Program

[3].

Current address: Sri Lanka Standards Institution, Colombo, Sri Lanka
II. EXPERIMENTAL SECTION

A. Sample Preparation - Bivalves

Marine bivalves were collected from Narragansett Bay, R.I. (more than 5000

organisms) and recently from 30 collection sites (60 to 100 organisms from

each) as part of the NOAA National Status and Trends Program. The species

involved were Mytilus edulis, Mytilus califomlanus , and Grasses trea virginica.

The organisms were scrubbed, rinsed, cooled at dry ice temperatures, shipped to

NBS and then stored in liquid nitrogen vapor (-150°C) in the NBS/EPA Specimen

Bank. The organisms had been separated into batches and sealed in Teflon bags.

Each batch consisted of 50 to 60 mussels or 30 oysters. One batch from the

Narragansett collection and two batches of mussels and four batches of oysters

from the 1985 NS&T collection were selected for analysis. After a batch was selected for analysis, the bivalves were shucked at -5°C. A shucking implement

with a titanium blade and Teflon handle was employed. After the shell was

opened, the contents were scooped out with another titanium instrument and

placed in clean Teflon bags for storage in liquid nitrogen until the tissues

were homogenized.

A cryogenic homogenization procedure [4] was used for the next step of

sample preparation. The vessels used for pre- fracturing and grinding were constructed of Teflon, to minimize contamination from trace metals. Disk mills

of two different capacities (0.4 and 1 kg) were used to prepare the mussel

homogenate . The resulting fine powder of fresh frozen tissue has been shown to

have adequate homogeneity so that 1 g subsamples are representative of the bulk

sample [ 5 ]

.

Following homogenization, portions of the powder were subsampled into

Teflon jars and stored at liquid nitrogen vapor temperatures. Just prior to

analysis, a subsample was removed from storage and freeze-dried . This dry

material was then pressed into pellets with a KBr pellet press (#186-0025

Perkin Elmer Corp., Norwalk, CT) to prepare it for the analytical techniques.

B. Sample Preparation - Sediments

Duplicate samples of bottom sediments from Benthic Surveillance sites were collected and banked as part of the NS&T program. Each sample consisted of a combination of aliquots, approximately 50 g each, from three stations per site.

Each aliquot was obtained from a box core or grab sampler using a cylindrical

Teflon corer. After a sample was taken, sediment was pushed out of the top of

the container and the top 3 cm of the core (approximately 50 g) was sliced off

and scooped into a clean Teflon bag using a titanium knife. Two additional

station aliquots were combined into the same Teflon bag to obtain a total site

sample of about 150 g. The bags were then sealed and stored in liquid

nitrogen.

Six sediment samples were selected from the 1985 NS&T collection and

removed from cryogenic storage. They were then partially thawed and thoroughly

mixed in the Teflon bags. Approximately 50% of the wet material was used for

organic analysis [6]. The remaining portion was freeze-dried. The dried

samples were transferred to Teflon jars, frozen at LN2 temperature, and

63
ground/mixed with Teflon balls at cryogenic temperatures with a Spex Mixer/Mill

(Model 8000, Spex Industries, Edison, N.J.)- Pellets from the dry material were then prepared as described above for neutron activation analysis.

Additional sample preparation was necessary for the XRF procedure [7].

The sample aliquots used for XRF analysis were prepared as fused glass disks,

in the ratio of 1 part sample to 12 parts lithium tetraborate (LTB) flux.

Because each sample had a different loss on ignition (LOI) during fusion, this

ratio did not hold when the samples were fused directly. To maintain the

constant ratio, the samples, as received, were first ignited at 1050 °C (the

fusion temperature) and the LOI of each was determined. The ignited sample

material was then fused. The elemental concentrations determined for the fused

aliquots were converted to sample concentrations by use of the LOI.

C. Sequential Analysis Scheme For Bivalves

The sequence of analytical steps followed in this procedure has been

described elsewhere [1] and is illustrated in Figure 1. Only a single 250 mg

aliquot of freeze-dried tissue is needed to obtain quantitative results on the

listed elements. This sample is usually pelletized and four assays are first

performed using the XRF-BFP technique. Then, the same sample aliquot is repelletized and PGAA is applied to it. After the collection of the prompt gamma

spectra, the pellet is submitted to a short irradiation followed by two decay

and counting cycles for the INAA assay. After sufficient decay for safe

handling, the pellet is submitted to a long irradiation followed by two decay

and counting cycles. All the information that can be obtained non-destructively has been recorded at this point and the sample is then submitted to a radiochemical separation.

D. Analysis Procedure For Sediments

Separate aliquots of freeze-dried material were used for each procedure

because of the delicate nature of the pellets, which easily disintegrated

during handling. An INAA procedure similar to the above mentioned procedure

was applied to the sediment samples to obtain results for 32 elements. The

applications of XRF and PGAA were investigated to increase the information that can be obtained from a single aliquot. However, XRF could only be done via the

fusion technique [7], and also PGAA had to use different aliquots. Therefore,

these methods have been used only to study their feasibility and results from

these techniques are only included from the initial evaluation analyzing

control materials.

Silicon measurements via an epithermal neutron activation analysis (ENAA)

procedure [8] were added to the analytical techniques applied to the sediments.

This procedure is non- destructive and could be incorporated into a sequential

analysis scheme. However, it presently lacks the sensitivity required for the

determination of silicon in biological tissues, and so could not be used for

the biological tissue analyses.

64
Arir - RPP Drr

PGAA

Si, P, S, CI, K, Ca, Mn, Fe,

Ni, Cu, Zn, As, Se, Br,

Rb, Sr, Zr, Mo, I, Ba, Pb

H, B, C, N, Na, P,

S, CI, K, Cd

INAA - SHORT

IRRADIATION

INAA - LONG

IRRADIATION

RADIOCHEMICAL

SEPARATION

Na, Mg, Al, Ci, K,

Ca, V, Mn, I

Sc, Cr, Fe, Co, Zn, As,

Se, Br, Rb, Sr, Mo, Ag,

Cd, Sb, Cs, Ba, La, Ce,

Sm, Eu, Hf, Au, Hg, Th

Sn

Figure 1. Sequential analysis procedure. A single 250 mg aliquot of

freeze-dried tissue is carried through the individual

analytical methods in sequence to obtain quantitative

results for 45 elements. Titanium is not reported in this

work because implements containing this element are used.

65
E. Quality Assurance

The sequential analysis procedure provides for internal quality assessment

on each individual sample through determinations of several of the elements by

more than one technique. However, to obtain additional information and since no sequential procedure was carried out on the sediments, Standard Reference

Materials (SRMs) from the National Bureau of Standards and other Certified

Reference Materials (CRMs) were used during the initial evaluation of the new

sediment procedure. The following control materials were used in these

analyses: SRM 1566 Oyster Tissue [9], the National Institute of Environmental

Sciences (NIES) CRM No. 6 "Mussel Tissue Powder" [10], and International Atomic

Energy Agency (IAEA) CFM Sediment SD-N-1/2 [11]. During sample analysis, at

least one sample of a reference material was used with each set of samples (a

set being samples of similar type analyzed under the same conditions) . These

reference materials do not undergo any sample preparation except drying and

pelletizing

III. RESULTS AND DISCUSSION

Tables 1 and 2 summarize the results obtained in the initial evaluation of

the analytical procedures. The performance of the sequential analysis scheme

for the bivalve samples was tested via the analyses of the NIES CRM No. 6 and

SRM 1566. The results of these analyses are summarized in Table 1. For the

sediment samples, the CRM SD-N-1/2 was used to evaluate the performance of the

analysis procedures. These results are included in Table 2. These data

indicate the elemental coverage by the individual techniques, the betweentechnique agreement, as well as comparability with the corresponding certified

values

The sequential analysis scheme was used for the characterization of the

seven selected bivalve samples, and includes results for 45 elements. The

results for each batch are summarized in Table 3. This table contains pertinent mean concentrations and uncertainties. The elemental concentrations

vary greatly among the species as well as among the collection sites, as expected

In Table 4, the INAA results for the sediment samples are given. The

sediment samples presented different analytical problems because of spectral

interferences, which were not serious in the analyses of the bivalve samples.

The following significant interferences made a determination of the respective

elements almost always impossible. After correcting for the terbium interference at the 392 keV line, tin was no longer instrumentally detectable. A

radiochemical separation is necessary for its determination. Both silver and

selenium were at the detection limit for INAA in most of the sediments. The

two main silver lines had interferences; europium at the 657 keV line and

scandium at 889 keV, which greatly overshadowed the silver's 884 keV line.

Selenium, at 264 keV, had a tantalum interference.

Copper was not detectable by INAA due to the high levels of sodium and

chlorine (this was also true of the bivalve samples) . The XRF analyses were
Table 1. - Elemental Concentrations for Selected Reference Materials (concentrations based on dry weight)

NIES CRM - Mussel SRM 1566 - Oyster

XRF PGAA INAA CERTIFEDa XRF PGAA INAA CERTIFIED^

H (mg/kg) / olAH/r3UUU l\j /VA/Xj\A/ B (mg/kg) 13.0±1.4 _ _ 9.210.3 C (mg/kg) - 540000130000 - - - 490000170000 - - N (mg/kg) J juuuxzuuu Na (mg/kg) 920011300 96001200 1 r\fV\f\-l.'i AA 10000+3 UU C A AAXOAA j4UU+zUU < 1 AAO-T AA

Me (me/ke) 16001400 21001100 1100+300 1280190

Al (mg/kg) - - 24516 (220) - 26916 - Si (mg/kg) 540±220 JOUll JO P (mg/kg) 7770±30 890011100 (7700) 75001300 (8100) S (mg/kg) 1250O±10O 122001500 90001300 73001100 (7600)

CI (mg/kg) 175001100 179001200 17000+400 94001300 104001200 (10000) K (mg/kg) 5400±1400 5900+100 57001600 54001200 97001300 - 86001500 9690150 Ca (mg/kg) 16001100 1 1 AA_1,T AA 13001300 1 jUOllOU 1 rfCAA-t-£AA 1 /^AA-t'J AA 1 <AA4-OAA

So 43.011.3 72.310.6 V (mg/kg) 5 0.60*^ S4.6 /. /XU.Z T 1 4-A 1

Cr (mg/kg) A CA_1.A AC 0.5910.05 A <C1 J.A AT 0.6310.0 / A TiCXA Al 0. /O+U.Oj O.oV+U.z / Mn (mg/kg) 17.0±0.2 15.610.2 16.311.2 16.2±1.0 _ 15.010.4 17.511.2 Fe (mg/kg) 160±1 - 16215 15818 191±7 - 20912 195134 Co (tig/kg) 32813 (370) 31213 (400) Ni (mg/kg) 1.24 ±0.03 A Al J.A A/C 0.9310.UO A mo-A A A 1 CkI 4-A 1 Q

Cu (mg/kg) 4.7±0.2 4.910.3 jV .4±1 .6 Oj.UXj.j Zn (mg/kg) 10912 10811 10616 860130 _ 85417 852114 As (mgAg) 9.910.1 - 9.310.3 9.210.5 13.610.5 - 13.210.1 13.411.9 Se (mg/kg) 1.5110.10 1 11 ^A A A 1.33x0.04 (1.5) O OXA 1 O O 1 /CXA A'l z. loiU.Uz O 1 4-A C at (mg/kg) lUo. jxU.O CO O+O 1 ZJo.oXZ. 1

Kb (mg/kg) Z.o 1±U.Uj 1 Cy4 J.A f\fi 4 /lA+A ylT 't.'tUXU.^Z 't.UZXU. iz Sr (mg/kg) 16.710.4 _ 20.912.6 (17) 10.310.6 S6 10.3610.56 Zr (mg/kg) - - 2.010.3 - Mo (mg/kg) (.SU.Z> Ag (Mg/l^g) O70X 14 ftonxcin ovuxyu

Cd 0 75+0 08 0.7610.05 0.8210.03 3.310.4 2.8+0.3 3.510.4 Sn (mg/kg) - 4.62+0.09 - Sb Oig/kg) 11.512.2 6.511.5

I (mg/kg) 1213 (2.8) Cs (jxg/kg) 27±2 _ 16+3

Ba fme/kc) <, 6 S 10 ^16

La (Mg/kg) _ 260120 _ _ 320110 - Ce (mg/kg) 0.3210.02 0.4310.02 Sm (Mg/kg) 37.411.3 65.711.3 Eu (Mg/kg) 6.610.4 15.410.4 20^

Hf (Mg/kg) 8.511.1 Au (Mg/kg) 3.510.5 Hg (Mg/kg) 5818 (50) 5818 57115 Pb (mg/kg) 1.810.7 0.9110.04 ^1.4 0.4810.04 Th (Mg/kg) 38.711.4 4012 (100)

'Certified concentrations and their uncertainties, as well as information values ( ), are given as defined in the Certificates of Analysis (9,10)

''Uncertainties are Is sample standard devialiwi

'-Concentrations are literature values (13)

•^Limits of detection are given as defined in Ref. 12

67
Table 2. Elemental Concenlrations for Certified Reference Material IAEA Sediment SD-N-1/2

(Concentrations in mg/kg dry weight)

XRF PGAA INAA CERTIFIED

H 218101180* B 80.3±1.0 - - C 55 0000± 140000 - - N <20000 - - Na - 107501180 (10300-10700) Mg 1.05+0.21 (0.74-0.79) Al 39090±90 366001200 (35800-38500)

Si 2901001500 28160012500 (261000-292300) S 360011300 - - CI - 9080+910 (9000-9600) K 18110160 17000+1000 (14600-16600) Ca 55430120 5200012400 (51200-56000) Sc - 7.09810.068 7.10 (6.90-7.21) V - 77.713.2 77.7 (65.0-80.7) Cr - 149.41+3.5 149(125-161) Mn 86517 809.919.2 777 (728-801) Fe 39000+600 3692013400 (35300-37800) Co - 11.7310.04 12.1 (11.2-12.7) Ni 30.2+1.8 - 31.0(27.0-34.0) Cu 88116 - 72.2 (68.1-75.2)

Zn 492123 438.4+3.6 439 (423-452) As - 65.010.9 50.0 (2.4-60.0) Se - 2.24510.037 (2.30-3.78) Rb 70.5+5.8 66.412.1 74.2 (68.6-77.0) Sr 293.011.7 279.014.4 291 (277-303) Mo - 2.3010.60 (1.30-2.60) Ag - 2.04310.041 (1.30-2.60) Cd 10.4710.56 9.111.5 11.0(10.0-12.0) Sn - ^18 Sb - (3.222+0.050)xlO-3 3.62 (3.20-3.95)xlO-3

Cs 4.593+0.028 4.9 (4.1-5.4) Ba - 248121 289 (276-310) La - 35.410.6 31.9 (30.0-35.5) Ce - 53.4710.80 60.3 (58.2-65.0) Sm - 6.2410.09 5.58 (5.40-6.35)

Eu 1.24610.068 1.16(1.12-1.30) Au 0.036110.0019 (0.03-0.04) Hg 1.44810.074 (0.06-1.72) Pb 140110 120(112-132) Th 7.09010.075 7.04 (6.85-7.20)

^Uncertainties are la counting statistics of samples and standards

68
Table 3. - Elemental Concentrations for Selected Bivalve Samples (concentrations based on dry weight)

Species

Site

Mvtilus edulis Mvtilus califomianus Crassostrea vjreinica M2N0086a MW1M048 MW1M029 MW1Y030 MW1Y044 MW1Y068 MW1Y073

Element n=6'' n=2 n=2 n=2 n=2 n=2 n=2 H (mg/kg) 6420011000^ 6700012000 676001200 708001400 64200+200 644001400 720001400 B (mg/kg) 38.5±0.9 41.510.8 40.410.7 21.610.3 39.811.8 34.911.9 18.510.8 C (mg/kg) ^60000+10000 430000140000 4700001100000 4900001120000 500000130000 3500001130000 N (mg/kg) / **tf\ft / J\ n n 1 86000+4000 10100014000 7000014000 5700014000 8400015000 108000+12000 Na (mg/kg) 466001200 42000+200 222001100 5020012400 486001200 222001100 Mg (mg/kg) 7300±1100 67801210 64201190 3800+120 70001450 75901450 40701120

Al (mg/kg) 905±140 64817 95619 29114 64917 1588114 16813

Si (mg/kg) 24001300 21001300 13001400 8001400 24001500 70001400 <700 P (mg/kg) 92001400 8900+400 126001500 72001300 75001500 63001300 76001400

S (mg/kg) 21800+2000 206001800 217001800 124001500 18300+1000 128001500 1 16001600

CI (mg/kg) 10370013600 845001500 748001400 403001200 876001500 881001500 388001200 K (mg/kg) 122001200 1100011000 1370011000 10300+600 1530011000 1180011000 96001600 Ca (mg/kg) 54001400 6600+500 10300+500 71001400 140001800 81001800 164001600

Sc (jtgAg) 150110 115.610.5 246.011.0 52.910.2 145.810.6 297.410.1 76.3+0.4 V (mg/kg) 2.010.5 1.910.2 2.610.2 1.710.1 9.3+0.3 4.210.3 <0.6

Cr (mg/kg) 1.7410.07 4.4810.03 1.8010.02 0.81+0.03 1.6010.03 2.5210.03 1.39+0.03 Mn (mg/kg) 26.111.1 13.810.9 17.611.0 11.510.6 15.811.0 54.211.2 23.410.6 Fe (mg/kg) 557120 557+3 620+3 209+1 49213 106015 23012 Co (Mg/kg) 538+1

1

36912 64112 32911 46513 85614 76113

Ni (mg/kg) 3.210.1 ^1.5 <,\A 2.610.5 S1.9 2.710.7 9.411.2

Cu (mg/kg) 7.010.6 15.810.9 9.410.7 10414 9715 7513 317+16 Zn (mg/kg) 131+1 117+1 143+1 144919 1462+9 2354118 5766135 As (mgAg) 11.010.6 8.110.2 8.0+0.3 6.310.2 34.7+0.5 16.610.4 4.610.2 c-oC (mg/kg) 2.54+0.05 2.8210.16 2.7210.02 2.5810.02 2.8610.02 2.6210.40 3.6810.02

Br (mg/kg) 46718 420114 398110 209+7 392120 340112 190110 Rb (mg/kg) 6.9510.15 6.4110.08 5.3710.10 4.2610.08 A n C.1.A 1 A 4.y3lU. 14 3.9810. 16 2.8310.15

Sr (mg/kg) 6513 4812 83+4 41110 8616 5613 5914

Zr (mg/kg) 2.211.0 <2 <3 <2 2.010.9 <3 <2 Mo 1.610.4 <2.4 <3 <1.4 <2 3.810.5 <2 Ag (Mg/kg) 80110 1710120 1250120 3780140 3380140 2840140 5850160

ca (mgAg) 1.910.4<1 0.510.4 0.5+0.5 2.710.4 0.510.4 1.810.4 7.8+0.4 Sn (mgAg) 0.1710.03 <7 1.4610.07 0.6010.06 0.45+0.07 <0.44 0.6810.10 Sb (Mg/kg) 3713 5012 2312 11+2 18+3 22+3 <20

I (mgAg) 1313 <10 <10 <5 £10 <12 <6 Cs (Mg/kg) 6915 5412 35+2 3213 3214 9214 <0.03

Ba (mgAg) 8.412.1 4.110.9 8.010.8 5.710.8 <10 10.612.0 10.012.0 La (Mg/kg) 520180 3821100 1040150 190110 360120 1360130 180+10 Ce (mgAg) 0 9?+0 07 0 790+0 019 1.5910.020 0.28510.008 0.88010.014 2.51410.019 0.314+0.013 Sm (Mg/kg) 5512 8112 19613 41412 9313 28814 5112 Eu (Mg/kg) 20.311.1 16.110.4 30.710.5 10.6+0.1 19.910.5 62.31.1 8.810.7

Hf (Mg/kg) 8017 Au (Mg/kg) 313123 66.816.5 4.910.9 16.510.6 5.411.4 3.9+0.8 29.0+0.7 Hg (Mg/kg) 158+24 225.016.6 8416 9816 81+8 14918 9611 Pb (mgAg) 4.610.4 13.111.0 2.111.0 <1.8 <2 <2 <2 Th (Mg/kg) 12219 12812 22112 44+2 23913 333+3 34+2

* Site Identification - M2N0086 - Narragansett Bay, RI MW1M048 - Dorchester Bay, MA

MW1M029 - San Diego Harbor, CA

MW1Y030- Galveston HarbOT. TX

MW1Y044 - Fort Johnson, SC

MV/1Y068 - Pascagoula Bay, MI MW1Y073 - Baltimore Harbor, MD

'Undesignates the number of replicate samples analyzed

Uncertainties are la sample standard deviation

^Error includes additional uncwtainty due to the uncertainty of the subtracted blank

69
Table 4. Elemental Concentrations for NS&T 1985 Benthic Surveillance Sediment Samples'

Sample DC 1 CAfllb DO 1 OUl J DO lOUZZ DORC 1 C(Y>R iOU/5

Element Na 26610±330 292201380 303501340 1 15601190 394401490 197201340 Mg 10200±630 192601920 208101920 7590+490 2107011000 121701830

Al 66200±0.035 77320+410 7476+360 417201350 683701520 62110+370

Si 307000±4200 205500+4900 26430014200 35670014700 228900+5200 25000016600

CI 6100±140 95001200 3860+180 2320190 56220+600 23720+550 K 22500±1100 <380O ^4000 204001900 2670011500 2550011300 Ca 23400±1600 2670011800 3590012200 1020011000 3150012100 4430011000

Sc 9.546±0.091 16.4710.16 11.7410.11 5.33510.051 11.80+0.11 17.7010.17 V 58.3±3.0 108.013.8 92.013.5 44.612.5 95.214.0 112.613.4 Cr 35.7±1.0 82.311.9 36.510.9 41.511.0 56.511.3 114.312.7 Mn 365.6±5.0 631.417.9 662.218.4 276.614.2 551.217.0 1070115 Fe 18970±180 455101420 355401330 167401160 37530+350 546301510 Co 4.98±0.02 16.3510.06 12.8110.05 7.9210.03 11.3410.04 36.6410.13 Zn 49.72±0.42 163.611.4 76.3010.65 55.5410.47 155.311.3 365.013.0 As 4.24±0.28 16.77+0.48 2.6610.37 6.84+0.38 10.4410.46 20.81+0.71

Se <0.07 0.31210.028 <0.10 <0.07 <0.10 1.5610.05 Rb 62.5±1.1 41.712.0 50.212.0 63.311.4 108.6+2.3 134.613.6

Sr 284.6±4.4 413.115.1 420.215.2 214.413.2 173.114.3 184.716.3 Mo 3.45±0.43 4.8610.59 4.4410.79 2.06+0.31 4.5810.56 5.46+0.68 Ag <0.05 0.40110.024 50.08 <0.04 SO. 10 0.20810.040

Cd <6 <9 <8 <5 <9 <12 Sn <9 <13 <15 <9 <15 <24 Sb 0.47610.0.025 2.70810.048 0.143+0.025 0.51510.019 0.635+0.029 1.39710.051 Cs 2.481±0.015 2.54 110.016 2.63210.017 2.45710.015 8.331+0.048 8.337+0.049 Ba 463125 425+30 428123 667+17 662+27 453136

La 29.510.5 22.0+0.4 23.310.4 34.710.6 39.210.7 57.311.0 Ce 48.12+0.73 37.5010.57 38.24+0.58 51.2810.77 63.7110.96 91.011.4 Sm 5.9010.08 4.7910.07 4.4810.06 5.5810.08 6.8110.09 10.6810.15 Eu 1.34410.073 1.39710.076 1.27310.070 1.15110.063 1.42510.078 2.1510.12 Au 0.00510.0007 0.024610.0019 sO.006 0.009610.0009 0.0114+0.0016 0.028510.002:

Hg <0.10 0.82410.029 <0.11 0.172+0.019 0.272+0.013 0.60810.051 Th 10.3710.12 5.2310.06 6.3910.07 7.6410.08 12.3710.13 13.5410.15

^Concentrations in mg/kg dry weight; uncertainities are lo counting statistics of samples and standards

''Site Identification BSlSOOl - Dana Point, CA

BSlSOlO- Elliott Bay, WA

BS1S013 - Nisqually Reach, WA

BS1S019 - Mississippi River Delta, MI

BS1S022 - Corpus Christi, TX

BS1S028- Chesapeake Bay, MD

70
also unable to obtain copper results, since copper alloys with the platinum

fusion crucible leading to a high systematic error (±45%)

.

Lead results were not reported by XRF due to the interference with

arsenic; the arsenic line with the lead line. A direct overlap

correction could not be made due to the possible loss of arsenic during fusion.

IV. CONCLUSIONS

The sequential analysis procedure which was evaluated and applied in this

work to bivalve samples is an efficient approach towards the accurate determination of approximately half of the elements in the periodic table, and

included many of the elements important for environmental and marine studies.

Of course, this list of elements is very dependent on their concentrations and

may not be feasible in all samples of this matrix. However, only slight

modifications should be needed to optimize the determinations in these marine

tissues. Undoubtedly, with more effort, in particular epithermal activation,

reirradiations and added radiochemical steps, the list of elements could be

further increased to include elements of concern such as silicon, palladium,

platinum, and bismuth.

Considerable additional work is needed to design a similar sequential

analysis procedures for the sediment samples. In particular, improvements in

sample preparation methods prior to the XRF analysis need to be made to avoid

the problems observed in this work. However, the application of XRF to the

instrumental analysis of these complex samples may still be limited by matrix

problems such as micro- inhomogeneities

.

Nonetheless, the analysis scheme described above has provided much useful

information on a great number of elements in the marine tissues as well as in

the sediments. The developing database on the samples from U.S. coastal and

eustuarine waters in conjunction with ongoing Specimen Banking programs will

provide us with a measurement yardstick for reference in future years.

V. ACKNOWLEDGEMENT

The authors acknowledge the assistance of Gunnar G. Lauenstein in the

coordination of the Specimen Banking activities for the NS&T Program of NOAA.

We acknowledge the cooperation and assistance of the following individuals in

the collection of the samples: Donald Phelps (U.S. EPA Narragansett) , Sandy

Freitas (Battelle New England Marine Laboratory) , Andy Lissner (Pacific

Northwest Laboratory), Gary Wolff (Texas A & M University) Robert C. Clark, Jr.

(National Marine Fisheries Northwest and Alaska Fisheries Center) , Peter Hanson

(National Marine Fisheries Southeast Fisheries Center) , and Vincent Zdanowicz

(National Marine Fisheries Northeast Fisheries Laboratory) . We acknowledge the

assistance of Kevin Diamond in the homogenization and preparation of the

bivalve specimens from the NS&T program and Lavern Baker (Battelle-PNL) for

assistance in the XRF-BFP analyses. This work was supported in part by the

Ocean Assessments Division, NOAA, the Office of Health Research, Office of

71
Research and Development, U.S. Environmental Protection Agency, and by the U.S.

Department of Energy under contract DE-AC06 - 76RLO 1830.

VI . REFERENCES

[I] Zeisler, R. , Stone, S. F. , and Sanders, R. W. (1988), Sequential Analysis

of 44 Trace Elements in Marine Bivalves, submitted to Anal. Chem.

[2] Lauenstein, G. G., Schantz , M. M., Wise, S. A., Zeisler, R. (1986),

Specimen Banking in the National Status and Trends Program: Development

of Protocols and First Year Results, Oceans '86 Conference Record, IEEE,

New York, NY, 586-589.

[3] Wise, S. A. and Zeisler, R. (1984), The Pilot Environmental Specimen Bank

Program, Environ. Sci. Technol. 18, 302A-307A.

[4] Zeisler, R. , Harrison, S. H., and Langland, J. K. (1983), Cryogenic

Homogenization of Biological Tissues, Anal. Chem. 55, 2431-2434.

[5] Stone, S. F. and Zeisler, R. (1984), Evaluation of Biological Samples for

Specimen Banking and Biomonitoring by Nuclear Methods, Proc . 5th Conf. on Nuclear Procedures in Environment and Energy Technology, J.R. Vogt, Ed.,

NTIS U.S. Dept. Energy, Springfield, VA, CONF-840408, 801-812.

[6] Schantz, M. M. , Chesler, S. N., Koster, B. J., and Wise, S. A., (1988),

Analytical Methods for the Determination of Organic Contaminants in

Marine Sediments and Tissues, In: These Proceedings.

[7] Sleater, G. A. (1986), XRF Analysis of Marine Sediments (NOAA/NBS

Specimen Bank) by Automated Borate Fusion, In: Technical Activities 1986,

Center for Analytical Chemistry, NBSIR 86-3486, National Bureau of

Standards, pp 150-151.

[8] Becker, D. A. (1986), Determination of Silicon by Fast Neutron Activation

Analysis, In: Technical Activities 1986, Center for Analytical Chemistry,

NBSIR 86-3486, National Bureau of Standards, pp 41-42.

[9] Certificate of Analysis, Standard Reference Material 1566, Oyster

Tissue, (1979), National Bureau of Standards, Gaithersburg , MD 20899,

USA.

[10] Okamoto, K. and Fuwa, K. (1984), Certificate for NIES Certified Reference

Material No. 6, Mussel, National Institute for Environmental Studies

(NIES), Yatabe-machi , Tsukuba, Ibaraki , 305, Japan.

[II] International Atomic Energy Agency (IAEA) Certified Reference Material

SD-N-1/2, (1985), Musee Oceanographique , 98000 Monaco.

[12] Currie, L. A. (1968), Limits for Qualitative Detection and Quantitative

Determination, Anal. Chem. 40, 586-593.

72
[13] Gladney, E.S., Burns, C.E., Perrin, D.R., Roelandts, I., Gills, T.E.

(1984), 1982 Compilation of Elemental Concentration Data for NBS

Biological, Geological, and Environmental Standard Reference Materials,

NBS Spec. Publ. 260-88, U.S. Government Printing Office, Washington, DC,

231 pp.

73
SECTION 8

IMPROVED ANALYTICAL METHODOLOGY FOR THE DETERMINATION

OF PCBs AND PESTICIDES IN HUMAN LIVER SPECIMENS

Reenie M. Parris, Stephen N. Chesler, and Stephen A. Wise

Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, MD 20899

SUMMARY

An analytical method is described which was developed for the

determination of low ng/g levels of selected individual polychlorinated

biphenyl (PCB) congeners and chlorinated organic pesticides in human liver

specimens from the Environmental Specimen Bank at the National Bureau of

Standards (NBS) . Two liquid chromatographic fractionations are utilized to remove analytical interferences and to separate the analytes of interest into

two fractions prior to quantification by high resolution gas chromatography

with electron capture detection.

I . INTRODUCTION

As part of the Environmental Specimen Banking program sponsored in part by

U. S. Environmental Protection Agency, NBS has been involved with the sampling,

processing, storage and analysis of human liver samples since 1980. This

project is part of a continuing effort to archive and characterize several

types of environmental samples for future retrospective analyses for both

inorganic and organic components. Detailed protocols on the collection and

storage of these specimens have been described previously [1].

Prior to the development of the analytical methodology described in this

paper, organic analyses at NBS of these human liver specimens focused on the

determination of selected chlorinated pesticides using classical column

chromatography on Florisil as a pre-separation step [2]. The methods used

previously have been modified to include individual PCB congeners as analytes,

to utilize more efficient and reproducible liquid chromatographic preseparation techniques, and to separate the species of interest into two

fractions for the analytical measurements. The five individual PCB congeners

selected as analytes are among those currently used by many researchers as marker compounds for the identification and quantification of PCB contamination. The liver specimens were also analyzed for the determination of seven chlorinated pesticides. The analytical scheme consists of extraction of the

sample, two liquid chromatographic fractionation steps, and quantification by

capillary gas chromatography with electron capture detection.

74
II. EXPERIMENTAL SECTION

A. Sample Collection and Preparation

The samples analyzed were collected in 1984 in Seattle, Washington. The

liver specimens were frozen within one hour of autopsy and shipped to and

stored in the NBS specimen bank at liquid nitrogen vapor temperatures

(-150 °C) . Prior to analysis, the liver specimens (150 g) were cryogenically

homogenized in a Teflon disk mill [3]. Aliquots of this frozen liver particulate homogenate were transferred to Teflon storage jars and were stored at

liquid nitrogen vapor temperature [1]. A flow diagram is shown in Figure 1 for

the analytical procedure used for the analysis of the human liver specimens for

the determination of selected PCB and chlorinated pesticide analytes

EXTRACTION

CLEANUP

QUANTITATION [

GRIND LIVER SAMPLE WITH SODIUM SULFATE

+ IS

ELUTION WITH PENTANE/ACETONE

% EXTRACTABLE FAT DETERMINATION

SEC FRACTIONATION

I NP-LC FRACTIONATION

I CAPILLARY GC-ECD

Figure 1 . Flow diagram of the analytical procedure

B. Sample Extraction

Each weighed sample (2.5 g) was ground with precleaned anhydrous sodium

sulfate (100 g) at room temperature in a mortar with pestle. This mixture was transferred to a glass column (20 mm i.d. x 360 mm) equipped with a Teflon

stopcock at the lower end. An aliquot containing known masses of the internal

standards (IS) was pipetted onto the top of the column. The internal standards

used were PCB 103 [4], PCB 198, and deuterated-4 , 4 ' -DDT (dg). These IS's were not significantly present in the samples and had appropriate LC and GC elution

zones. This mixture was then eluted with 200 mL of 2:1 (v:v) pentane/acetone

The rate of flow was adjusted so that about 6 h were required for the extraction. The extract was concentrated to approximately 20 mL in a rotary evaporator, transferred to a centrifuge tube, and reduced to 1 mL with a stream of

precleaned nitrogen gas. Methylene chloride (5 mL) was then added to the

extract. A typical set of samples consisted of six liver samples, two aliquots

of liver samples that had been analyzed in the previous set, two aliquots of

gravimetrically prepared calibration solutions, and one aliquot of hexane

Each sample was spiked with the internal standards, extracted, fractionated,

and analyzed as shown in Figure 1.

75
C. Percent-Extractable Components Determination

The w/w percent of the liver extracted as defined by the following formula

was determined for each liver extract. This is often called "percent extractable fat" . A 90 /iL aliquot of known mass of the extract was transferred to a tared foil pan. The methylene chloride was allowed to evaporate at room temperature (1 h) and the mass of the remaining residue was determined.

100 x M2 X E

% extractable = — ; Ml X L

components

where: Ml = initial mass of aliquot

M2 = mass of aliquot residue after solvent evaporation

L = mass of liver extracted

E = mass of total extract

D. Size Exclusion LC Fractionation

The remainder of the extract was filtered with a precleaned 0.5 /xm glass

microfilter (disposable unit) , and evaporatively concentrated to 600 ^L. To remove most lipid and other large molecular weight components from this complex

liver extract matrix, the extract was fractionated on a preparative-scale size

exclusion column (2.5 cm i.d. x 60 cm). The column material was a divinylbenzene-styrene copolymer with pore size of 100 A and particle size of 10 /zm. With methylene chloride as the mobile phase, the fraction containing the PCBs

and chlorinated pesticides was collected according to previously calibrated

retention zones. The volume of the fraction was reduced to 400 fih after the

solvent was changed to hexane

E. Aminosilane LC Fractionation

The entire fraction was injected onto a normal phase aminosilane semipreparative column (7.9 mm i.d. x 300 mm). Two fractions were collected

according to previously determined retention zones. Initially, the mobile

phase was 100% hexane and Fraction I which contains the PCBs and two pesticides

(4,4' -DDE and hexachlorobenzene) was collected. The mobile phase was changed

to 1:20 (v:v) methylene chloride/hexane and Fraction II was collected which

contains the remaining pesticides of interest (4,4' -DDT, 2,4' -DDT, heptachlor

epoxide, transnonachlor , 4,4'-DDD and dieldrin) . The voltjme of each fraction

was reduced to 150 /^L prior to GC analysis.

F. Gas Chromatographic Analyses

For the gas chromatographic analyses, 1.5 /xL aliquots of each fraction

were injected into a gas chromatograph equipped with an electron capture

detector and an immobilized nonpolar fused silica column (30 m x 0.25 mm x 0.25

/xm film thickness) . A split mode of injection was used with helium as the

carrier gas.

76
III. RESULTS AND DISCUSSION

Typical chromatograms of Fraction I and II from the analysis of a human

liver sample are shown in Figures 2 and 3. The PCBs are identified according

to the Ballschmiter-Zell numbering scheme [4] . A range of results from 24

samples for each analyte is listed in Table 1. The concentrations of the PCBs

ranged from 0.5 to 30 ng/g of wet liver tissue and concentrations of

chlorinated pesticides ranged from 1 to 400 ng/g wet liver tissue.

- IV. CONCLUSIONS

By including these liquid chromatographic fractionation steps in our procedure, low ng/g levels of selected PCB congeners and chlorinated pesticides

in human liver tissue can be determined because 1) many matrix components that

could interfere with the quantitative analysis and/or foul the analytical

system are removed, 2) analytes can be separated into several fractions which

precludes coelution of some components during the analytical measurement step

and 3) sample contamination and exposure during the analysis is minimized.

These LC procedures are also less labor intensive than most classical column

sample pre- treatments and are automatable.

V. ACKNOWLEDGEMENTS

The authors acknowledge the cooperation and assistance of Dr. V. F. Garry,

University of Minnesota, Minneapolis, Dr. N. K. Mottet, University of Washington, Seattle, and Mrs. D. Kenny, Harborview Medical Center, Seattle, for the

careful collection of the liver samples. We acknowledge the assistance of

W. F. Kline, K. A. Fitzpatrick, and G. Harrington in the homogenizatlon and

preparation of the liver samples.

VI . REFERENCES

[1] Zeisler, R. , Harrison, S. H. , and Wise, S. A., Eds., (1983), The Pilot

National Environmental Specimen Bank - Analysis of Human Liver Specimens,

NBS Spec. Publ. 656, U. S. Government Printing Office, Washington, DC,

135 pp.

[2] Kline, W. F., Allen, C. F. , Chesler, S. N., Hilpert, L. R. , and Wise , S. A., (1983), Development of Analytical Methodology for the Determination of

Organochlorine Compounds in Human Liver Samples, In: The Pilot National

Environmental Specimen Bank - Analysis of Human Liver Specimens, R. Zeisler, S. H. Harrison, and S. A. Wise, Eds., NBS Spec. Publ. 656, U. S. Government Printing Office, Washington, DC, pp 91-97.

[3] Zeisler, R. , Langland, J. K. , and Harrison, S. H. (1983), Cryogenic

Homogenizatlon of Biological Tissues, Anal. Chem. 55, 2431-2434.

77
_ o

OJ

961; 93d (SI) c

•f-i s

LU o

•H

•UO

0) 4J

0) -o

0)U

:3 u

a,oco

•Uo

OJco

•r-l uo

0)c

4-1O

V)C

0)

4Jo

1-1 (4-1 e

bOOu

<«eou

j:ow

n3 O

78
3SN0dS3ti 033

79
Table 1. Range of Concentrations of Selected PCBs and Pesticides in

24 Human Liver Samples

Analyte Concentration

Compound ng/g liver tissue (wet)

PCB 10 11.1-8.3

(2,2' ,4,5,5' -pentachlorobiphenyl)

PCB 138 3.4 - 29

(2, 2', 3, 4, 4' , 5' -hexachlorobiphenyl)

PCB 153 4.6 - 29

(2,2' ,4,4' ,5,5' -hexachlorobiphenyl)

PCB 170 0.9 - 11

(2,2' ,3,3' ,4,4' , 5-heptachlorobiphenyl)

PCB 180 2.4 - 20

(2,2' ,3,4,4' ,5,5' -heptachlorobiphenyl)

Hexachlorobenzene 1-30

4,4'-DDE 32 - 400

Heptachlor epoxide/oxychlordane^ 6.7 - 42

Transnonachlor 2.0 - 14

Dieldrin 3.6 - 17

4,4'-DDD 1.8 - 130

4,4'-DDT 1.4 - 250

% Extractable Components 2.4 - 6.4%

In heptachlor epoxide equivalents

80
Ballschmiter , K. and Zeil, M. , (1980) Analysis of Poiychlorinated

Biphenyls (PCB) by Glass Capillary Gas Chromatography, Fresenius . Z. Anal.

Chem. 302, 20-31.
SECTION 9

THE DETERMINATION OF TRACE ELEMENTS IN HUMAN LIVERS

Rolf Zeisler, Robert R. Greenberg, and Susan F. Stone

Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, MD 20899

USA

I . INTRODUCTION

Since 1980, the pilot National Environmental Specimen Bank (NESB) program

at the National Bureau of Standards (NBS) has examined the collection, processing, storage, and analysis of human livers [1]. Sampling protocols, handling

procedures and analytical methods have been developed and implemented taking

into consideration the requirements for valid analytical results. Sampling and

handling included the use of clean-room technology, specific clean implements

and packaging materials made from titanium and Teflon, and flash-freezing and

preservation of the tissues at liquid nitrogen temperature. More than 500

human liver specimens have been collected and banked from several locations in

the U.S. since the pilot program began, representing all age groups above age

fourteen. A representative portion of the samples included in the bank has

been analyzed to provide data for real time monitoring, determination of long

term stability, and evaluation of today's analytical capabilities.

Initially, a combination of four techniques provided a comprehensive data

set for 36 human livers. However, several key trace elements were not detectable in all samples or insufficient data was obtained from a particular

analytical technique. Frequently, analyses had to be repeated to detect low

levels or to confirm results. This expensive approach needed improvement to

reduce the amount of sample required and to limit the nximber of techniques and

analytical steps required. In addition, the naturally occurring levels of

several pollutant trace elements were approximately one order of magnitude

lower than previously reported, thus requiring more sensitive analytical

procedures. Since NAA had been used for the determination of most of the above

elements, on individual 1 g (fresh weight) samples, further expansion of NAA

and lowering of its detection limits could effectively achieve this goal.

Therefore, we modified the instrumental NAA procedures and added radiochemical

separation procedures that would provide the additional sensitivity and

precision required.

II. EXPERIMENTAL SECTION

A. Sample Preparation

Human liver specimens were selected from the 1982 and 1984 collections of

the pilot NESB program. The samples were obtained from the NESB storage in the

form of aliquots of cryogenically homogenized liver specimens [2]. These

homogenates , stored at liquid nitrogen temperature in Teflon jars, consisted of

82
5 to 7 g of fresh material in the form of a finely divided powder. Before

analysis, the selected aliquots were freeze-dried at -5 °C and 1 pascal until

stable weight was reached. Water loss ranged between 70 and 80%, and the

individually determined results were used to correct analytical values to a fresh weight basis. Portions of the freeze-dried material, weighing typically

250 mg (equivalent to 1 g fresh weight) , were then pressed into 13 mm diameter

pellets with a Perkin-Elmer KBr pellet press. Control materials (NBS SRM

1577a, Bovine Liver) and primary standards (multi-element solutions on Whatman

42 filter paper) were pressed into similar pellets. The pellets were individually sealed in acid cleaned linear polyethylene film for irradiation. The

pellets were re-packaged in a similar manner for counting and re-irradiation.

Pelletizing greatly facilitates the handling of the samples, and to date, no contamination from the pellet press has been observed in numerous analyses of

biological materials with even lower trace element concentrations, e.g., milk

powder, and rice and wheat flour.

B. Neutron Activation Analysis

The instrumental NAA procedures followed the general principles that were outlined previously [3]. A sequential analysis scheme, consisting of several

irradiation, decay and counting cycles for each sample, provides optimum

information from each individual sample. All irradiations were performed in

the pneumatic tube facilities RT3 and RT4 of the NBS research reactor. For the

analyses done with modification B (see below) , we utilized the higher fluence

rates available at the upgraded 20 MW research reactor of the NBS [4].

All counting was done on high resolution, high efficiency gamma spectrometers (typically 1.9 keV FWHM for 1332 keV and 25% relative efficiency). The

gamma spectra contained 8192 data channels with 0.40 keV/channel (1st and 2nd

counts) or 0.25 keV/channel (3rd and 4th counts) resolution. The spectra were accumulated with Nuclear Data 6000 Series MCA's and the data were stored on disk for automatic processing. Pileup and live time corrections in the 1st and

2nd counts were done from a 100 Hz pulser peak stored with the spectral data;

for the other counts, ADC live times were taken and pileup corrections were calculated from a calibration curve.

To improve the detection limits in INAA, a Compton suppression system was used for some counts. It consists of a central Ge (Li) -detector surrounded by a sodium iodide scintillator with internal sample position, and a Harshaw NC-26R

Compton suppressor analyzer [5]. This system is particularly effective with

these samples during the 3rd count where a more than 20 fold reduction in the

background under the 559 keV line of arsenic can be achieved. We have dedicated two ADCs for the simultaneous acquisition of the Compton suppressed

spectra along with the normal data. Random coincidence suppression of the As,

Cd, and Cr counts were estimated by comparison of the intensities of single

line emitting nuclides, such as Zn-65, in both spectra. The necessary correction, between two and ten percent, was determined individually for each sample

spectrum

The analysis scheme that was applied to the first set of human liver

samples [1], and the revised scheme that was used during this investigation are illustrated in Figure 1. Modification A was applied to the 1982 set of human

83
Procedure A Procedure B

1st irradiation

1st count: 5 min

2nd count: 10 min

2nd irradiation

3rd count

A: 4 hours

B: 6 - 12 hoiurs

Compton Suppressed

6 - 12 hours

A: selected samples

B: all samples

4th count : 12 hours

1 min, RT-3

2 min, RT-4

I

Al, V. Mg. CI, Cu

Na. K, Mn

4 hours, RT-3

As, Cd, La, Mo

Sm, Br

Cd, As

Cr, Co, Fe, Zn,

Se, Rb, Ag, Hg,

Th

90 sec, RT-4

decay - 2 min

decay - 2.5-3 hours

decay - 20-40 days

I

Al, V, Mg, CI, Cu

Na. K, Mn

4 hours, RT-3

decay - 5-7 days I

Fe, Co, Zn, Se,

Rb, Mo, Ag, Sb, La,

Sm, Au, Hg, Th

Cr, As, Cd

decay - 30-50 days

Radiochemistry

Cr, Sn Ag, Cr, As, Mo,

Sb, Se, Sn

Figure 1. Flow chart for the irradiation, decay and counting cycles of the

activation analysis procedures (Modification A for the 1982 set of

human livers, Modification B for the 1984 set)

84
livers, modification B to the 1984 set. In modification A, no changes were made to the Irradiation cycles. The Improvements were focused on lowering the

detection limits during count 3 by Implementing Compton suppression, and

evaluating a radiochemical step for long-lived nuclides to be Implemented after

count 4. The simultaneous Irradiation of at least six samples In each rabbit

resulted In greatly varying sensitivity for arsenic because of the relatively

short half life (T,^ = 26.3 h) of Its radionuclide. This drawback was eliminated in modification B, where only two to three samples were Irradiated

at a time, thus providing practically equal sensitivity for the determination

of arsenic in each sample after a 6 to 7 day decay. The radiochemical separation in modification A provided results for chromium and tin. However, silver

and antimony were not considered. To Include these elements, and further lower

the detection limit for arsenic, a different separation was used in

modification B.

C. Radiochemical Procedures

Following the completion of the instrumental assays, radiochemical

procedures were added to obtain better sensitivity for several low level trace

elements. In modification A, efforts were focused on combining existing

procedures for the determination of chromium [6] and tin [7]. In modification

B, more elements were Included in an adapted group separation [10] for counting

after 9 to 10 days of decay.

For the radiochemical separation, the samples were digested after the

addition of appropriate carriers, using a stepwise addition of an appropriate

combination of acids in several steps. Evaporating the solutions to dryness

was avoided to prevent loss of selenium and chromium. In modification A, a liquid/liquid extraction of the Cr(VI) trlbenzy lamlne (TBA) complex In chloroform was used for the specific separation of this element [6]. Following the

separation, the chloroform was evaporated and the chromium taken up in 15 mL of

concentrated HNO3 and H2S0^ (1:1) for counting.

The aqueous phase was then used for the separation of tin by liquid/liquid

extraction of Snl^ in toluene. Before this could be accomplished, all chloride

was eliminated by adding concentrated HNO3 and gently heating for 48 hours.

The residual organic material (TBA) was digested with H2SO^/HN03 and H2O2 by

heating In a Teflon beaker. The solution was then adjusted to approximately

20% H2S0^. A mixture of 10 mL of 5 mol/L KI and 20 mL toluene were added to

extract the Snl^ . The tin was back-extracted from the organic phase with 20 mL

of 5% Na2EDTA solution. The tin-containing solution was counted after 12

hours, allowing for the buildup of In- 11 3m, and the 392 keV gamma line was used

for quantitation.

In modification B, the 3rd count was a compromise, achieving some additional sensitivity with the Compton suppression; however, this action gives up

the usual advantage for nuclides with half-lives greater than 70 hours, which

commonly are counted after longer decay periods . This was done to obtain

sufficient sensitivity for arsenic in the radiochemical separation step that

follows the instrumental assay. Samples were digested as described above and

then submitted to a previously described group separation on an Inorganic Ion

exchange column prepared from hydrated manganese dioxide (HMD, Carlo Erba) [7].

85
The HMD had been preconditioned with phosphoric acid to minimize the retention

of radioactive phosphorus which causes excessive bremss trahlung background. In

addition to the previously investigated elements, Cr, As, Se, Mo, Ag, and Sb

it was found that the retention of Sn is quantitative from 1 mol/L HNO3 . After

washing with 1 mol/L HNO3/O.OO25 mol/L H3 PO^ , the HMD column was counted

immediately on a gamma spectrometer and in some cases a second time after

additional decay.

III. RESULTS AND DISCUSSION

A major part of this work was to determine up to 30 elements for baseline

studies in human livers collected during 1982 and 1984. The ranges of elemental concentrations found in the sixty selected specimens are summarized in

Table 1. As discussed above, improved INAA and RNAA procedures were implemented to lower detection limits and to include additional elements. The

significant improvements in detection limits achieved for the low level trace

elements Cr , As, Ag, Sn, and Sb are summarized in Table 2. These detection

limits represent the element concentrations at which, with 95% probability, a sample with the given concentration would generate a detectable signal [8].

The lower detection limits permitted the determination of the aforementioned

elements in all 1984 specimens.

In Figure 2, the data obtained for arsenic in the 1984 human liver

specimens are summarized. The plotted data are ranked according to increasing

values and are compared to "best" regional values [9]. The figure illustrates

the improvements in the analytical procedures. The Compton suppression lowers

the detection limits to the naturally occurring levels, but this is still

insufficient for about 50% of the actual concentrations. The value of RNAA is

clearly established at these levels. The comparison of RNAA with INAA at the

higher levels shows good agreement between both procedures, re-affirming the

quantitativeness of the separation procedure. It should be noted that the

recent literature data [9] are an order of magnitude lower than previously

reported values [10].

When this procedure is applied to specimen bank samples, only one additional technique is needed. Voltammetry complements the activation analysis

measurements in an ideal way by providing results for Ni , Cu, Cd, and Pb . Most

of the values for these elements in Table 1 were obtained by this technique

[11]. This combination of the two techniques significantly reduces the amount

of sample needed as well as analytical effort spent. The second technique

would also serve to estimate the quality of the results. Since INAA results

are not affected by the matrix and interferences are known and correctable, the

comparison of Cu and Cd results would allow a reasonable assessment of the

quality of the results. If there is good agreement between the two techniques

for those two elements, it can be assumed that the quality of the rest of the

results is good. This highly efficient approach will be useful in the future

by providing more results on human livers, without sacrificing the quality of

the analytical data.

86
Table 1 . Ranges of Trace Element Concentrations in Selected Human Liver

Specimens

1982 Collection 1984 Collection

Element Low High Low High

Na 720 2265 750 2140

Me 81 . 5 171 104 246

Al 0. 525 2 . 22 0. 29 7 . 31

CI 1019 2418 984 1980

K 1645 3942 1330 3090

Sc 0.000096 0.00093 0.00028 0.00051

V <0.016 <1 .05 0.011 0.026

Cr <0.01 0.18 0.01 0. 12

Mn 0.608 1 . 964 0. 742 2 . 152

Fe 73 . 22 441.3 38 .

6

915

Co 0.026 0.059 <0 . 017 0 . 074

Ni 0.0027 0 . 037 0.0029 0 . 036

Cu 3 . 62 17 .

2

2 . 60 23 . 8 Zn 29 .

3

103 36 . 97 99 . 87

As <0 . 001 0.0268 0 . 0022 0.0203

Se 0. 327 0.631 0. 348 0.838

Rb 1 . 74 8.07 2 . 701 8 . 930

Mo 0.241 1 .41 0. 332 1 .41

Ae <0 . 003 0 . 0347 0 00086 0.0341

Cd 0.28 6.05 0

.

440 3.268

Sn 0.050 2.420

Sb <0.0008 0.0122 0.0013 0.0772

Cs 0.0012 0.019 0.0079 0.0146

La <0.008 0.417 0.0017 0.0191

Ce <0.01 0.52 <0.022 0.086

Sm <0.0009 0.0154 <0.0003 0.0033

Au <0. 00008 0.06164

Hg 0.0423 0.329

Pb 0.138 0.926 0.23 1 .03

Th <0.002 0.0047

87
Table 2. Limits of Detection [8](^g/kg) for Seven Critical Elements

* MODIFICATION A MODIFICATION B

•

Element INAA INAA RNAA INAA INAA RNAA

Normal Suppressed Normal Suppressed

Or 10 4 0.5 40 15 1.0

As 30 5 - 10 5 1.0

Se 6 - 35 0.5

Mo 25 20 - 45 30 40^

Ag 4 - 40 -^0.7

Sn 300 200 10 3000 2000 30

Sb C^^Sh) - 2 0.7 0.06

Sb (I24sb) 3 0.8

^Due to coincident gamma lines, Ag and Se are not included.

^The 739 keV line of ^^Mo was used to avoid an additional decay period

required for the ^^Mo - ^^"Tc equilibrium to be established.

88

t
5-1

C 0) o

.—I ^ U)

•U -P -P C C S O 01

!-i

U

4-) cnC 4-1

•t-l -H C ^3 Z

4-) 0) a; T3 :3 u>ce

00

CO

4-1 0) n3 0) > 5 H 4->

4-1 0)

4-1 CC

4-1 c3 C

CO o

0) >-l

!-l 4J W 0) OJ < C

U-l rH •!-( O Cfl Q O o e • M dJ 

•H ^ ON

S-l

O

—

tfl

O e T3 4J o nj cc

•1-1

89
IV . ACKNOWLEDGEMENTS

The authors acknowledge the cooperation and assistance of Dr. V. F. Garry,

University of Minnesota, Minneapolis, Dr. N. K. Mottet, University of Washington, Seattle, and Mrs. D. Kenny, Harborview Medical Center, Seattle, for the

careful collection of the liver samples. We acknowledge the assistance of

W. F. Kline, K. A. Fitzpatrick, and G. Harrington in the horaogenization and

preparation of the liver samples.

V. REFERENCES

[I] Wise, S. A., and Zeisler, R. (1984), The Pilot Environmental Specimen

Bank Program, Environ. Sci. Tech. 18, 302A-307A.

[2] Zeisler, R. , Langland, J. K. , and Harrison, S. H., (1983), Cryogenic

Homogenization of Biological Tissues. Anal. Chem. 55, 2432-2434.

[3] Greenberg, R. R. , Fleming, R. F. , and Zeisler, R. (1984), High

Sensitivity Neutron Activation Analysis of Environmental and Biological

Standard Reference Materials. Environ. Intern. 10, 129-136.

[4] Becker, D. A., and Williamson, T. (1985), Characterization of the NBSR at

20 MW, Nat. Bur. Stand. (U.S.) Tech. Note 1217, 120-129.

[5] J. R. Vogt and R. Zeisler (1983), A Compton Suppression System for

Background Reduction and Improved Sensitivity, Nat. Bur. Stand. (U.S.)

Tech. Note 1178, 154-159.

[6] Greenberg, R. R. and Zeisler, R. , A Radiochemical Procedure for

Ultratrace Determination of Chromium in Biological Materials, J.

Radioanal. Nucl. Chem., in press.

[7] Greenberg, R. R. (1986), Elemental Characterization of the National

Bureau of Standards Milk Powder Standard Reference Material, Anal. Chem.

58, 2511-2516.

[8] L. A. Currie (1968), Limits for Qualitative Detection and Quantitative

Determination. Anal Chem. 40, 586-593.

[9] Iyengar, G. V. (1985), Concentrations of 15 Trace Elements in Some

Selected Adult Human Tissues and Body Fluids of Clinical Interest from

Several Countries: Result from a Pilot Study for the Establishment of

Reference Values, Berichte der KFA Julich - Nr. 1974, 156 pp.

[10] Iyengar, G. V., Kollmer, W. E., Bowen, H. J. M. (1978), The Elemental

Composition of Human Tissues and Body Fluids, Verlag Chemie , Weinheim/New

York, 151 pp.

[II] Stoeppler, M. and Ostapczuk, P. (1986), Report of Analysis, Nuclear

Research Center (KFA) Julich, 1986.

90
SECTION 10

NATIONAL AIR MONITORING PROGRAM NATIONAL PARTICULATE NETWORK

Stanley F. Sleva and Dennis Shipman

Monitoring and Data Analysis Division

Office of Air Quality Planning and Standards

U.S. Environmental Protection Agency

Research Triangle Park, North Carolina 21771

USA

I. INTRODUCTION

Section 110 of the Clean Air Act [1] (CAA) requires ambient air quality

monitoring and data reporting for purposes of State Implementation Plans

(SIPs) . The Environmental Protection Agency (EPA) promulgated regulations in

August 1971, for the preparation, adoption and submittal of SIPs under Section

110 of the Act [2]. Included in these regulations were requirements for States

to establish and operate ambient air monitoring networks and to report the data

to EPA. These data were to be used for such purposes as evaluation of national

control efforts, tracking attainment and maintenance of the National Ambient

Air Quality Standards (NAAQS) , reevaluation of the NAAQS , development of

national control policies and strategies, and the preparation of national

trends in ambient air quality.

In the mid-70's, several critical reviews [3-6] of environmental

monitoring programs identified major deficiencies in several aspects of the

existing monitoring systems. A Standing Air Monitoring Work Group (SAMWG) was formed to review and provide recommendations to overcome these deficiencies.

The group was composed of representatives of EPA, State and local agencies.

After an extensive and comprehensive review of the monitoring program, SAMWG

issued an air strategy report which called for the revision of the 1971

monitoring regulations [5]. The suggested revisions would, in effect, correct

the major shortcomings of the existing air monitoring programs and would also

respond to requirements of Section 319 of the 1977 CAA.

The Environmental Protection Agency in May 1979 promulgated air monitoring

and reporting regulations which significantly revised the previous regulations

as recommended by SAMWG [7]

.

II. HIGHLIGHTS OF THE 1979 REGULATIONS

Briefly, the major elements of the revised regulations are the following

[8]:

1. Provides for fixed and movable monitoring sites.

2. Establishes uniform monitoring criteria.

_ 3. Requires that reference or equivalent methods be used.

91
4. Imposes an annual network review.

5. Requires public reporting of an air quality index daily.

6. Provides for the quarterly reporting of all NAMS data and the

annual sununary reporting of SLAMS data.

]

III. TYPES OF MONITORING STATIONS ) Three types of stations are provided for in the regulations. Each is

designed to serve different needs and must be established according to

different reporting, siting, and instrumentation requirements.

State and Local Air Monitoring Stations (SLAMS)

SLAMS consist of a carefully planned network of fixed monitoring stations

whose size and distribution is largely determined by the needs of State and

local air pollution con-trol agencies to meet their respective SIP

requirements. These stations are the backbone of the ambient monitoring

program relative to State Implementation Plan (SIP) activities. To ensure quality data, the SLAMS are required to meet the following:

a. Each site must meet network design criteria and criteria for

instrument exposure and sample inlet design.

b. Each SLAMS network must include a quality assurance program.

c. All sampling methods and equipment must meet EPA reference or equivalent requirements

d. Acceptable data validation and recordkeeping procedures must be

followed.

In addition, data from the SLAMS must be summarized and reported annually

to EPA.

National Air Monitoring Stations (NAMS)

The NAMS are a subset of the SLAMS network with emphasis being given to

urban and multi-source areas. In effect, they are the key SLAMS with emphasis

on areas of maximum concentration and high population density. NAMS, like

SLAMS, must conform to EPA siting criteria and operate according to quality

assurance procedures that equal or exceed EPA's minimum specifications. The

NAMS differ from the SLAMS in that:

a. NAMS must use continuous automated instruments for gaseous

pollutants

b. A minimum number of NAMS must be established in specified

urbanized areas

92
c. NAMS data must be reported to EPA on a quarterly basis.

d. Adherence to quality assurance, siting and probe criteria must be

achieved two years earlier than for SLAMS.

Special Purpose Monitoring (SPM)

Special Purpose Monitoring stations provide for special studies needed by

the State and local agencies to support SIPs and other air program activities.

SPMs are not permanently established and thus, can be adjusted easily to

accommodate to changing needs and priorities. SPMs can be used to supplement

the fixed monitoring network as circumstances require and resources permit.

IV. UNIFORM MONITORING CRITERIA

The monitoring stations of the SLAMS and NAMS must adhere to the uniform

monitoring criteria described in the regulation. These criteria cover quality

assurance, monitoring methods, network design, and probe siting.

The quality assurance program consists of two functions. One function is

the assessment of the quality of the data through precision and accuracy estimates. This function must be adhered to explicitly. The second function is

the control and improvement of the quality of the data and is described in

general terms. Each state is allowed to develop a quality control system

appropriate for its situation.

Criteria were established for the monitoring methods that must be used for

all SLAMS and NAMS. Each station must use reference or equivalent methods

which are designated by EPA. Exceptions to the requirements may be allowed for

unique circumstances where it can be proven that the method to be used will

essentially provide the same results as an approved reference or equivalent

method. It is further required that except for TSP, only continuous

instruments be operated at the NAMS.

Procedures for the design of the monitoring networks to meet specified

monitoring objectives are provided. Spatial scales of representativeness are included as a means of providing a useful system for classifying stations as well as to serve as a physical basis of defining sample station characteristics. The spatial scales of representativeness that are most important are microscale, middle, neighborhood, urban, and regional scale.

After selecting the general location of a monitoring station, a decision

must also be made about the specific siting of the instrument probe of sampler

inlet. In order to ensure consistency in this decision making process, probe

siting criteria that must be followed were incorporated into the regulations.

Pollutant specific criteria for the horizontal and vertical probe placement of

the sampling probe, its spacing from obstructions and roads, as well as probe

material and pollutant sample residence time are provided. Recognizing that some stations may not meet the probe siting requirements while still producing

acceptable data, a waiver provision for such stations may be obtained. For

93
NAMS , the requests are to be forwarded to the Administrator, for SLAMS to the

Regional Administrator.

V. OTHER FEATURES

Three other items of major importance in the regulations are the annual

network review, the reporting of the Pollutant Standard Index (PSI) daily, and

air monitoring data reporting requirements

In order to ensure optimal use of monitoring resources as well as meet

changing data needs, the regulations require that the SIP monitoring plan

provide for an annual review of the state network. Furthermore, if the annual

review indicates that a change in the monitoring system is called for, the

state must develop and implement a schedule for modifying the system.

In response to Section 319 of the Act, the regulations also contain a requirement that states report a uniform air quality index to the general

public daily. By January 1, 1981, urban areas greater than 500,000 population

were required to begin reporting, and by January 1, 1983, all urban areas exceeding 200,000 individuals. As of September 1986, over 100 urban areas were using the PSI.

Air quality data reporting for NAMS must be done on a quarterly basis.

Under the requirements of the regulations, validated data must be submitted to

EPA within 120 days of the end of the calendar quarter.

Data collected from SLAMS must be summarized annually, certified by the

state senior air pollution control officer, and submitted to EPA by July 1.

In line with congressional requirements, EPA reviews most of its

environmental regulations at five year intervals. Therefore, in 1984 EPA,

state, and local agencies started a review of the Part 58 air monitoring

regulations. In March 1986, revisions to the regulations were promulgated.

The revisions were relatively minor resulting in the adoption of the 1980

census as a basis for determination of the NAMS network size, minor modifications to the number of required total suspended particulate (TSP) samplers and

sulfur dioxide (SO2) monitors according to population size and pollution

levels. In addition, some changes in the quality assurance program were made.

Table 1 illustrates the changes in the number of state and local agency

air monitors which have taken place between 1977 and 1986. Overall, the number

of monitors has decreased from 9459 in 1977 to 4645 in 1986 for a total

reduction of 51 percent. Nitrogen dioxide (NO2 ) and SO2 bubblers and TSP

samplers have decreased while continuous monitoring of Ozone (O3 )

, Carbon

Monoxide (CO), NO2 and SO2 remained very stable, 1846 continuous monitors in

1977 versus 1842 monitors in 1986. Table 2 shows the yearly change in the

total of SLAMS monitors. The largest changes occurred in 1979 and 1980, a drop

of 1530 between 1978 versus 1979 and 1616 between 1979 and 1980. Since 1981,

the network has stabilized and the number of monitors is expected to remain

close to these levels for the next several years.

94
Table 1. Comparison by Pollutant of National Air Monitoring Stations

1977 vs. 1986

1986

Pollutant 1977 SLAMS* NAMS

Total Suspended Particulate Matter 3987 2396 638

Sulfur Dioxide 2382 547 218

Continuous 600 541 218

Bubbler '

1782 6

Carbon Monoxide 470 447 113

Nitrogen Dioxide 1588 252 57

Continuous 234 238 57

Bubbler 1324 14

Ozone 542 616 214

Lead 490 416 105

Total 9459 4674 1345

^Include NAMS

Table 2. National Summary of SLAMS Monitors

Year NAMS SLAMS TOTAL

1977 9459

1978 8800

1979 7350

1980 5734

1981 1277 3655 4932

1982 1354 3478 4832

1983 1351 3412 4763

1984 1347 3377 4724

1985 1344 3330 4674

1986 1345 3300 4645

VI. NATIONAL PARTICUIATE NETWORK

Currently, at the national level the closest approach to any type of air

quality monitoring specimen banking program would be the National Particular

Network (NPN) . The network is based on the hi-volume sampling (hi-vol) method

for particulate matter in the atmosphere. The current network can be traced

back to a 1953 cooperative program between the U.S. Public Health Service and

95
17 state and local health departments to sample for the protein content of

airborne particulate matter [10]. Figure 1 shows the approximate location of

the original network. These particulate samples were sufficiently large to

allow additional chemical analyses to be added to the original scope of the

program. The additional analyses included determinations for acetone or benzene soluble organics , radioactivity, chlorides, fluorides, nitrates,

sulfates and 23 metals. Sampling equipment and central laboratory services

were provided by the U.S. Public Health Service's Sanitary Engineering Center

in Cincinnati, Ohio while state and local organizations provided the personnel

for particulate matter sample collection and site operation. As the years

passed, additional communities elected to participate in the program expanding

it by 66 sampling sites by 1956. Some of these additional sites operated on an every other year or every third year basis. During the early years of

operation, the principal objectives for the network were as follows:

1. Determine the extent and nature of air pollution.

2. Study trends in the levels of various atmospheric contaminants.

3. Investigate the relationships between air pollution and

socioeconomic, geographic, topographic, meteorological and other

factors

On January 1, 1957, this program was officially named the National Air

Sampling Network. Between 1957 and 1961, the network was expanded to 250

stations. Approximately 140 were scheduled to sample every other year leaving

110 operating annually; therefore, about 180 stations were operating during a given year [11]. These operating sites were to collect samples on a randomized

biweekly preset schedule. In 1967, the network was renamed the National Air

Surveillance Network (NASN) . By 1968, the network had grown to about 350

sites, about half of which also operated every year [12]. In December of 1970,

the U.S. Environmental Protection Agency (EPA) was formed from several other

Federal Programs. The NASN was taken over and has been administered by EPA

since that date. During the early 1970' s, the sampling frequency for the NASN

stations was changed to one sample every 12th day instead of one biweekly

sample and the NASN size remained stable. However, during the 70' s, the number

of TSP hi-vol samplers operated by state and local agencies grew to approximately 3000. The next principal change to the NASN occurred in 1981, when the

National Filter Analysis Network was reevaluated and reduced in size to 167.

The network was renamed the National Particulate Network (NPN) in 1984. The

NPN consisted of 167 sites, 16 of which were rural or background sites, 17

located in vicinity of coking operations to measure benzo [ a] pyrene (BaP) and

the remaining 134 sites located in urban settings which generally had a population of 150,000 or greater. Since that time, approximately 30 sites have

been added to the network for a special study. Some of these sites will be

discontinued at the conclusion of the study in 1987 and the resulting network

will be approximately 175 sites. Figure 2 shows the approximate locations of

the current network.

96
97

Over the years the pollutants measured have changed, as have analytical

methods. Currently, the filters are reanalyzed for arsenic (As), chromium

(Cr) , cobalt (Co) , barium (Ba) , beryllium (Be) , cadmium (Cd) , copper (Cu) , iron

(Fe) , manganese (Mn) , molybdenum (Mo) , nickel (Ni) , lead (Pb) , vanadium (V) , and zinc (Zn) . The filters from 60 of the sites are also analyzed for benzo-

(a)pyrene (BaP) . The differences in pollutants measured were influenced by

varying data needs as well as problems with analytical methods, emerging

technology, etc.

From the beginning of the NASN program in 1953 until 1982, the unused

portion of filters from all stations participating in the program were stored

(banked) by the Federal Agency to provide for future retrospective analyses.

However, because of storage space availability and funding levels, beginning in

1982, filters are banked for a period of 10 years and then removed from the

bank

.

It is believed that metal constituents of TSP collected on these filters

will remain stable for relatively long periods of time; however, organics and

many inorganic compounds are known to decompose over time; therefore, no retrospective analysis for substances other than metals is contemplated. While EPA

banks filters only for a period of 10 years, each state and local government

agency has its own policy and there may be some of which have filters banked

from specific monitoring locations for periods of 20 to 30 years.

VII . REFERENCES

[1] Clean Air Act as Amended August 1977, Section 110(a)(2)(c), 42 U.S.C.

7410(a)(2)(c).

[2] Requirements for Preparation, Adoption, and Submittal of Implementation

Plans," Federal Register 36 (158), 15486 (August 14, 1971).

[3] Environmental Monitoring, (1977), A Report to the U.S. Environmental

Protection Agency from the Study Group on Environmental Monitoring,

National Academy of Sciences, Washington, DC.

[4] A Report to the President and the Congress, National Advisory Committee on Oceans and Atmosphere, Sixth Annual Report, Washington, DC, June 30,

1977.

[5] Air Monitoring Strategy for State Implementation Plans, EPA-450/2 - 77 -010

,

U.S. Environmental Protection Agency, Research Triangle Park, N.C.,

27711, June 1977.

[6] Environmental Quality, The Eight Annual Report of the Council on Environmental Quality, September 1976.

[7] Air Programs: Ambient Air Quality Monitoring, Data Reporting, and

Surveillance Provisions, Federal Register 44 (92), 27558 (May 10, 1979).

99
[8] Hoffman, A. J., Sleva, S. F. , and Cox, W. M. (1979), "Revised Air Quality

Surveillance Requirements for State Implementation Plans, J. Air Poll.

Control Assoc. 29, 7, 704-707.

[9] Air Programs: Ambient Air Quality Monitoring, Data Reporting, and

Surveillance Provisions," Federal Register 51 (53): 9582 (March 19, 1986).

[10] Air Pollution Measurements of the National Air Sampling Network, U.S.

Department of Health, Education, and Welfare, Public Health Service,

Cincinnati, Ohio, 1958.

[11] Air Pollution Measurements of the National Air Sampling Network, Analyses

of Suspended Particulates, 1957-1961, U.S. Department of Health,

Education and Welfare, Public Health Service, Cincinnati, Ohio, 1962.

[12] Air Quality Data for 1968 from the National Air Surveillance Networks and

Contributing State and Local Networks, U.S. Environmental Protection

Agency, Research Triangle Park, N.C., 1972.

[13] Air Quality Data for Metals 1970 through 1974 from the National Air

Surveillance Networks and Contributing State and Local Networks, U.S.

Environmental Protection Agency, Research Triangle Park, N.C., 1972.

100
SECTION 11

CHEMICALS IN INDOOR AIR

Istvan Gebefugi and Friedhelm Korte

Gesellschaft fur Strahlen- und Uraweltforschung mbH

Institut fur Okologische Chemie

D-8042 Neuherberg

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

The hygienic importance of air contamination in workplaces has been

commonly known for decades. For the protection of workers, maximum concentration limits have been fixed for chemicals whose impact can be damaging to one's

health. The so-called maximum workplace concentration values ( "MAK" -values

)

were established. They are valid for the surrounding air at industrial

workplaces. Although there is a total body exposure, additional detailed

precautions, such as the wearing of gloves and protective clothing or instructions not to eat in the workplace, provide for the highest possible

safety

.

But there are also burdens in other confined spaces where no handling of

chemicals takes place. The substances responsible for this are summed up by

the expression "indoor chemicals" and defined as non-occupationally introduced

chemicals in confined spaces. In contrast to industrial chemical burdens which

are restricted by the above mentioned maximum workplace concentration values,

indoor chemicals are up to now not subject to limit regulations. The importance of this lack of regulations is outlined by the fact that people in well

industrialized countries spend more than 80% of their time in confined spaces.

The formaldehyde burden in a chipboard factory for instance, is regulated for

the workers in the production units by maximum workplace concentration values,

but not so for the secretaries in the offices. In schools and homes, where the

chipboards can emit formaldehyde, the maximum workplace concentration regulations are not valid and not reasonable. (To complete the picture, it should be

mentioned that there exists a recommendation by the German federal health

agency saying that in indoor spaces and recreation rooms the formaldehyde

concentration in the air should not be above 0.1 ppm, approximately a tenth of

the maximiom workplace concentration value.)

Chemicals can come into indoor air from many different sources. Passive

exposure can take place by intake from outside, contamination of the housing

ground, building materials with residues, but also household furnishings.

Active contamination, e.g., caused by man himself can arise by habits such as smoking as well as the use of household chemicals, sprays, moth deterrents,

cleansing, and polishing agents, etc.

101
II. INITIAL INDOOR POLLUTION RESEARCH

The Institute for Ecological Chemistry of the GSF was asked in 1974 to

conduct measurements of halogenated phenols in a home, since it was suspected

that these substances, ingredients in some wood protection products, caused

symptoms of illness in one of the inhabitants. In the investigated room, the

proportion of wood to the remaining surface was 1:3. The wood, as the

proprietor stated, had been treated according to instructions with a wood

protection agent. This product contains, like many others, about 6%

pentachlorophenol (PCP) [1].

First of all, PCP concentrations in the air as well as in nontreated

walls, carpets, furniture, etc. were determined. The resulting air concentration of PCP varied between 50 and 100 /ig/m'^ . The air analyses were repeated

half a year and one year later. The PCP values detected then, were lower and

varied between 0.6 and 1.7 ;ug/m'^ . For these concentration differences, no explanation could be found at first. Incorrect measurements with deviation

values higher than a power of ten could be at least considered as improbable,

since multiple measurements had been made. In the house concerned, about 20

serial investigations had been made, which agreed with the above mentioned

concentration range. In the same house, a flat with a separate entry, which

had no wooden parts, by simultaneous measurements showed no PCP content. When

the medical symptoms indicating that the illness possibly correlated with the

PCP content in the house grew stronger, it was deemed necessary to make

comparative measurements in another building. This, we thought, might lead to an understanding of the great variations of PCP content in the air.

As a model room for this study, a closed hall (swimming baths, 6 x 12 m) was selected. The walls and the ceiling were wood panelled yielding a treated

wooden surface around 210 m^ . The swimming baths were furnished with a heat

pump so that no outdoor air could get in. The condensed water of the heat pump

was available for analytical investigations.

The wood in the hall had been treated according to instructions with

another wood protection product that has about the same PCP content as the one used in the above mentioned house. Inside the room, air temperature, humidity

and air exchange could be held constant for a certain time. Two investigations

of four weeks each were carried out. The results can be interpreted to

indicate that at least humidity is an important factor affecting the PCP

concentration in the indoor air.

III. SUMMARY OF THE FIELD AND MODEL STUDIES

Detectable PCP and TCP values in non- treated samples lead to the

conclusion that after treatment with wood protection products containing

pentachlorophenol, a contamination of all objects within a confined space is

inevitable. The values measured in plaster, wood and in other objects vary

between 0.2-26.0 mg/kg for (PCP) and 0.4-5.5 mg/kg for 2 , 3 , 4 , 6- tetrachlorophenol (TCP). Consequently, they are contamination sources by themselves. In

addition, it is sure that PCP, TCP and other chlorinated attendant substances

in wood protection products are transported in the air. The model studies

102
proved a certain correlation between PCP air contamination and humidity.

However, by knowing the room temperature and the humidity content, the con- centration values cannot be predicted (see Figure 1).

1 2 3 4(A) 5 6{A) 7 8(A) 9 10 n{A) 12 13(A) 14 15(A) 16

Figure 1 . Correlation between air humidity and PCP-concentrations

in a model experiment

The humidity can be considered as a type of "solvent" for PCP, so that the

variations in PCP values can be explained as the result of varying extraction

yields. It can be stated as a fact, however, that the vapor emission and

uptake in the system influences these variations. Increasing the temperature

causes an increase in the diffusion velocity of pentachlorophenol

.

For a better ranking of the data from the field and model studies, case studies were carried out on selected objects. The aim was to determine clearly

the contamination and to detect by close analysis as well as by model

experiments the paths of non- industrial exposition in confined spaces.

IV. CASE STUDIES

For the case studies, private households were selected where a massive use

of material protection agents containing pentachlorophenol and lindane had

taken place. In the most part, information was available about ways, amount,

and time of application. The two chemicals mentioned above served as model

substances for these studies because of their wide spread use and their

sensitive analytical characteristics. The well-known and highly toxic

103
impurities of technicai pentachlorophenol , i.e., polychlorinated dibenzodioxins

and dibenzofurans , were also intended to be included in the study.

A. Case A

Family A lives in a wood house which the family has been renovating mostly

in a 'do-it-yourself style since 1975. In the course of those efforts, about

200 L of wood protection products containing pentachlorophenol and lindane had

been applied not only to the beams, walls (outside and inside), and doors, but \ also to a great part of the furniture, too. Shortly after application of the

chemical agents, all four members of the family complained of being ill. Skin

rashes, headaches, concentration weakness, and hair loss were the symptoms.

These complaints have been growing stronger until the present time, especially

with one person.

Since regular wood protection products contain about 6% pentachlorophenol

and about 1.5% lindane, nearly 12 kg pentachlorophenol and 3 kg lindane had

been used within this house. All inhabitants claimed that the symptoms men- tioned disappeared during longer absence from the house, e.g., on holidays.

In spite of the wood samples' high content of pentachlorophenol and

lindane (beams of the atelier and living room) , only small concentrations were detected in the air. If the risk assessment in this flat would have been made

only according to the air concentration values, one must have had to qualify

the concentrations as harmless.

The textiles out of this flat, however, were all highly contaminated. i Since these textiles consisted mainly of clothing and bed linen, i.e.,

substances worn close to the skin, it was confirmed that uptake of the chemical

does not only take place via inhalation but via direct skin contact, especially

after a longer time following application. The blood samples taken for

comparison showed relatively low values.

B. Case B

In this case, the application of the wood protection product had taken

place in 1976. No detailed information about the amount or type of the product

could be provided. The blood investigation had been made at a time when none

of the six members of the family lived any longer in the contaminated flat. An

air measurement was not carried out.

The wood samples in this case showed high contents of pentachlorophenol

and lindane too. A strong contamination of the whole home had occurred. House

dust and textile samples showed high pentachlorophenol values. In this case

also, the conclusion was that exposure to the chemical was due to direct skin

contact. Accordingly, high pentachlorophenol values could be detected in the

blood

104
C. Cases C, D, AND E

These three cases also showed the same findings. The air samples were very low contamination; often the chemicals were not even detectable in the room air.

The wood samples as the indoor sources of the chemicals often showed high

concentrations of pentachlorophenol and lindane even after ten years. A

spreading around in the whole flat had taken place. Apparently, the biocidal

active agents accumulate strongly on wood and cotton (case E)

.

In contrast, the concentrations on mixed tissues and pure synthetics are about a factor of 10 lower. A risk assessment with the help of blood samples

should not be recommended based on our investigations since broad variations

did not provide a relevant correlation to the concentrations in indoor spaces.

V. MODEL EXPERIMENTS FOR INVESTIGATING THE BEHAVIOR OF PENTACHLOROPHENOL

AND LINDANE IN CONFINED SPACES

To verify the observed high contamination of textiles, model experiments

were performed [2,3]. Boxes the size of 50x50x50 cm served as model rooms. They were made of untreated pinewood, the bottom covered by a 2 mm aluminum

sheet and the top consisting of a 0.50 cm thick glass plate the size of 50x50

cm.

Into the model room six cotton strips were inserted. Onto the side walls

the active agents PCP and lindane had been applied under reproducible conditions. An air exchange rate of about 1.6 was arranged. The temperature was

20 °C, the relative humidity 45%. These circumstances are the standard for a not specifically heat insulated flat. As can be seen from Figure 2, pentachlorophenol and lindane accumulate on the cotton. For pentachlorophenol, an increase of up to 24.8 Mg/g cotton can be observed after 225 hours. After that

time an equilibrium seems to have been established. This equilibrium con- centration of pentachlorophenol on cotton amounts to about 20 Mg/g- For

lindane, saturation is established after only 70 hours with an equilibrium

concentration of about 6 Mg/g cotton.

The concentration profile of both substances in the air escaping from the

box is contrary. The PCP concentration is rising for about 50 hours to a value

of 430 ng/m'^ , and then decreases to below 100 ng/m^ . The lindane concentration

in the air escaping the box is higher than that of PCP; although, the

concentration on the cotton is lower.

VI. ANALYSIS OF THE TOXIC IMPURITIES IN SAMPLES OF

WOOD, HOUSE DUST, AND TEXTILES

In four cases, samples of wood, house dust, and textiles were analyzed for

chlorinated dibenzodioxins and dibenzofurans . In all cases, highly chlorinated

dibenzodioxins and dibenzofurans could be identified definitely in the wood.

The concentrations are at the low ppb level. In three cases, these compounds

105
Figure 2. Lindane and pentachlorophenol accumulation

on cotton surface

could also be found in the house dust. These compounds also seem to be spread

via air in the treated homes [4] . In the textiles analyzed, the concentrations

were below the detection level. The results of these analysis are listed in

Table 1.

VII . CONCLUSIONS

As a result of the case and model studies, it can be concluded that in

indoor spaces, chemicals which are less volatile than 10"^ mbar , accumulate on surfaces, especially textiles. The contamination of persons staying indoors

takes place not only directly via air (inhalation). Skin contact with con- taminated surfaces can lead to a significant additional burden. The so-called

total body intake of the chemicals in indoor spaces is possibly more relevant

than inhalation. The results of the case studies seem to confirm this. By

analyzing PGP impurities, a contamination of house dust with octachlorodibenzodioxin could be detected. Therefore, a contamination of inhabitants in rooms treated with wood protection products with toxic impurities of PGP could

be classified as probable.

106
Table 1. Pentachlorophenol , Lindane, Chlorinated Dibenzodioxin and

Dibenzofuran in Samples of Wood, Dust, and Textiles

OCDD OCDF PCP Lindane

(ng/g) (ng/g) (/^g/g) (Mg/g)

WOOU i

.

J Z . 0 826. 0

Dust 1: 1

.

46

Textile 1: -- -- 1

.

20 --

Wood z

:

++ ++ 376. 0 i Z . D Dust 1

:

4.2 8.7 16. 2 2.4

i exui j.e o . z

.

2. 3

Wood 3: 71 .0 8. 4 3.62

Dust 3: ++ ++ 7 . 0

Textile 3: 3. 26 1 .

1

Wood 4: 27.8 45.0 15.3

Dust 4: 8.4

Textile 4: 2.6

VIII. REFERENCES

Gebefugi, I., Parlar, H., and Korte, F. (1979), Occurrence of

Pentachlorophenol in Enclosed Environments, Ecotox. Environ. Safety 3,

269-300.

Gebefugi, I. and Korte, F. (1984), Indoor Contamination of Household

Articles Through Pentachlorophenol and Lindane, Indoor Air 4, 317-322.

Ruh, C, Gebefugi, I., and Korte, F. (1984), The Indoor Biocide

Pollution, Occurrence of Pentachlorophenol and Lindane in Homes, Indoor

Air 4, 309-315.

Gebefugi, I. (1986), Chemikalienbelastung in Innenraumen, Areas Review

for Architecture and Science 4, 172-176.

107
SECTION 12

BANKING OF ATMOSPHERIC PARTICULATE MATTER SAMPLES FOR LONG-TERM MONITORING

OF ATMOSPHERIC POLLUTION AND RELATED REFERENCE MATERIAL AT THE

NATIONAL INSTITUTE FOR ENVIRONMENTAL STUDIES

Yoshinari Anbe, Hltoshi Mukal, and Kensaku Okanoto

Division of Chemistry and Physics

National Institute for Environmental Studies (NIES)

Yatabe-Machi, Tsiikuba, Ibaraki 305

JAPAN

I . INTRODUCTION

The idea of environmentaL specimen banking can be applied to many kinds of

environmental samples including atmospheric samples [1,2]. The distinguishing

characteristics of atmospheric samples compared to other environmental samples

are: (1) high variability, both spatial and temporal, which presents a problem

for representability of the sample, (2) the amount of matrix is high and the

concentration of pollutant is low, therefore, large volume sampling and highly

sensitive analytical methods are needed, and (3) reactions with light,

oxidants, container walls, etc., which lead to sample handling problems.

Of the three phases of atmospheric samples gas, rain, and particulate

matter , the storage of gaseous samples is most difficult from a practical

standpoint because it not only requires a large space for storage, but there

are also many problems with the sample stability, e.g., reactions with other

components, adsorption on the container wall, etc.

We selected atmospheric particulate matter samples collected on filters as

a most promising sample for specimen banking related to atmospheric pollution.

Using these samples, we started a small pilot specimen banking program a few

years ago to systematically collect samples and preserve them for future retrospective analysis. In addition to particulate matter, rain and snow samples were collected although not so systematically, and are also stored in the bank.

Parallel to the banking program, a storage study on the stability of

benzo [ a] pyrene in the atmospheric particulate matter on the filter is in

progress. It was revealed that a part of the benzo [ a ] pyrene in the samples

disappeared during storage. In connection with the above program, we prepared

an environmental reference material for the analysis of particulate matter

which is representative of vehicle exhaust particles. The outline of the

preparation of this reference material is presented in this paper.

II. PILOT PROGRAM FOR BANKING ATMOSPHERIC PARTICULATE MATTER SAMPLES AT NIES

The atmospheric particulate matter samples stored in the bank for a long

time can be used for the following purposes:

108
1 To monitor the occurrence of new pollutants especially new man-made

chemicals in the atmosphere.

2. To analyze old samples by future improved analytical techniques.

3. To record a more precise trend of long-term air pollution by

simultaneously analyzing a series of samples.

4. To repeat analysis of past samples when any problems occurs in the

future

.

5. To analyze retrospectively presently unnoticed atmospheric

pollutants

.

The following are the conditions needed for the stored samples to be

available for the above mentioned uses [1,2].

1 . The relevant constituents in the samples at the time of sampling

should be preserved both qualitatively and quantitatively for several

decades

.

2. The samples to be stored should be meaningful as indicators of

change in the environment.

3. The origin and the history of the samples should be well recorded.

In order to realize an effective and appropriate banking program for

atmospheric particulate matter samples, the following items should be studied.

1 . Relating to Sample Collection

a. Sampling sites (distribution, height)

b. Sampling time and duration

c. Sampling method

i. Sampler (high volume sampler, low volume sampler, Andersen

sampler)

ii. Filter (membrane, glass fiber, quartz fiber, metal, etc.)

d. Sample amounts

e. Sample transportation

2 . Relating to Sample Storage

a. Sample size

b. Container (size, form, material)

c. Light condition

d. Humidity

e. Temperature

f. Atmosphere (air, other gases)

g. Pre treatment

109
h. Storage room

1. Storage duration

3 . Relating to Storage Management

a. Registration system

b. Storage room capacity

I. Amounts of samples

II. Replacement of samples

c. Countermeasures for accidents (stoppage of electricity, fire,

earthquake, and other natural disasters)

After having considered the problems mentioned above and the preliminary

results of the storage experiment described In the next chapter, a small pilot

program for long-term storage of atmospheric particulate matter samples was Initiated at the National Institute for Environmental Studies (NIES) In Japan

In 1983. The purpose of this program was to examine the availability and

usefulness of stored samples for monitoring long-term changes of atmospheric

pollution by retrospective analysis of stored sample materials.

For organic substances In the atmospheric particulate matter other than

polycycllc aromatic hydrocarbons (PAH) , the Information on the long-term

stability Is very scanty. Some estimates, however, on the stability may be

possible from considerable other Information, such as chemical characteristics,

chemical structure, stability in short-term storage, etc. Though storage at

-20 °C is not always the perfect condition to preserve organic substances, this

condition of storage was selected mainly for practical reasons. As the concen- trations of heavy metals and other elements, other than volatile ones like Hg, are considered to be stable for a long time, the stored filter samples can be

used to monitor the pollution by such substances. The outline of this program

is as follows (Figure 1).

Tokyo

(polluted)

Tsukuba

(suburban)

Monthly Samples

NIES

Okinoshima Is.

(remote)

Storage

( -20°C, in Ar )

Analysis

( PAH, Heavy metals )

over 20 years

Figure 1. Banking program of atmospheric particulate matter samples

110
A. Sample Collection

Atmospheric particulate matter is collected using a high volume air

sampler on a quartz fiber filter on the first day of each month at Tsukuba, a suburban area which is located 50 km northeast of Tokyo.

At Okinoshima Island, which is a rural and remote area lying in the Japan

Sea as shown in Figure 2, samples are collected at the top of a mountain (300 m

high) for a month on a filter with low volume air samplers. Sampled filters

are sent by mail to Tsukuba where storage facilities exist.

N

I I I—I

I

Sampling Locations

Figure 2. Sampling locations

Samples are also collected in a highly polluted urban area in Tokyo using

high volume air samplers. This sampling program is planned to continue for 20

years and the number of sampling sites is expected to increase in the near future

.

B. Storage of Collected Samples

Prior to storage, the collected sample filters are registered and a part

of the sample is used to determine PAH, heavy metals, and other pollutants for

real-time monitoring of atmospheric pollution. The major part of the sample is

placed on a stainless steel plate (20 cm x 25 cm) and stored in a airtight

stainless steel plate box (30 cm x 30 cm x 50 cm) filled with argon gas to

111
prevent oxidation. The container box is placed in the -20 °C storage room to

store for a long period as an environmental sample bank.

C. Sample Registration And Management

All the necessary information on each sample such as the characteristics

of the sample, sampling conditions, and storage conditions are recorded on a card and stored in a personal computer.

D. Storage Of Rainwater And Snow Samples >

In addition to atmospheric particulate matter, rain water, and snow

samples, which are occasionally collected from various regions, are also stored

at -20 °C condition. Polypropylene bottles and glass bottles are used as containers for these samples.

III. STABILITY OF BENZO[A]PYRENE IN THE STORED ATMOSPHERIC

PARTICULATE MATTER SAMPLES

Information on the stability of pollutants in the atmospheric samples

during long-term storage over months is scanty. However, as to the stability

of PAH (polycyclic aromatic hydrocarbons) in atmospheric particulate matter on

the filters during sampling, several studies have indicated that some degradation of PAH occurs on the filters presumably affected by UV-radiation, NO3 , O3

and other oxidants in the atmosphere [3-8]. Tomingas [3] reported that nearly

50% of benzo [ a] pyrene collected on glass fiber filters disappeared after one year's storage at room temperature.

These results suggest that protection from such factors is necessary for

long-term storage of PAH in atmospheric particulate matter. In this study, an experiment was conducted on the long-term stability of benzo [ a ] pyrene , as a representative PAH in the atmospheric particulate matter on the sampling

filter, under several storage conditions to examine the suitability of the

storage method.

A. Experimental Section

Samples

Atmospheric particulate matter was sampled at the roadside near Shijuku

Gyoen Park, Tokyo in June, 1982. The road had heavy vehicular traffic and a high content of particulate matter and PAH derived from vehicle exhaust gas was expected in the samples. Samples were collected on four sheets of quartz glass

fiber filter (Palliflex 2500 QAOT) and the air was sampled for 70 h using four

sets of high volume air samplers simultaneously which were set side by side.

Sampling was repeated twice to prepare two series of sample filters having

different contents of particulate matter and benzo [ a ] pyrene

From one sheet of sampled filter (20 cm x 25 cm), 12 pieces of small discs

(5 cm diameter) were cut out as analytical samples using a stainless steel

punch

112
Analytical Procedure

The analytical sample filter disc was folded and dissected into 5 nun wide

strips with stainless steel scissors and put into a 10-mL centrifuge tube.

After adding 10 mL of acetonitrile and extracting ultrasonically for 15

minutes, the solvent was centrifuged for 7 min (300 rpm) . The extracted

benzo [ a] pyrene in the supernatant solvent was determined by HPLC coupled with a fluorescence detector under the following conditions [9-12]:

Instrument: HPLC (Waters 700A)

Column: )u-Bondapak C^^g (i.d. 1/8 in. x 1 ft.)

Solvent: acetonitrile/water 80/20

Flow Rate: 2.0 mL/min

Detector: fluorescent detector (Hitachi lOLC) (excitation wavelength

366 nm, fluorescence emission wavelength 403 run)

The analytical result was expressed as benzo [a] pyrene weight per weight

(ppm) of atmospheric particulate matter. The weight was made constant in the room with 50% relative humidity and a temperature of 20 °C.

Storage Experiment

The experiment was designed to check the effects of temperature and oxygen

on the stability of benzo [ a] pyrene in the samples during storage under the

following four conditions:

(1) +20 °C in air

(2) +20 °C in argon gas

(3) -20 °C in air

(4) -20 °C in argon gas

The sample filters were placed on a stainless steel plate (20 cm x 25 cm)

and placed in airtight stainless steel boxes (30 cm x 30 cm x 50 cm) filled

with air or argon gas and stored in the 20 °C or the -20 °C storage room. Twelve pieces of analytical sample filter discs were stored under each of the

four conditions and two discs each were programmed to be taken out from each

container to measure benzo [a] pyrene after 6 months, 1 year, 2 years and 4 years

from the start of the experiment. The determination was repeated twice for

each analytical sample disc and two series of samples with different benzo[a]-

pyrene content, which were sampled at different times, were used in this

experiment.

Since the similarity of the stored samples at the start of the experiment

is essential for the comparison of the analytical results at a later date to

determine whether a change in the samples has occurred, the homogeneity of the

distribution of benzo [a] pyrene in the sample filters was examined prior to the

storage experiment.

113
B. Results And Discussions

Recovery of Analysis and Blank Test

The recovery of benzo [ a] pyrene in this analytical method was examined by

adding a small amount of benzo [ a] pyrene solution on the filters and analyzing

by the same procedure. The percent of recovery was above 92% for each run. The blank value of the filter for benzo [ a ] pyrene was 0.0 ppm.

Distribution of Particulate Matter and Benzo [ a 1 pyrene Concentration on the

Sampled Filter

The coefficient of variance of the weights of particulate matter of 12

analytical sample discs cut from one sheet of filter was 1.4% and that of

benzo [ a] pyrene was 4.5%, both showing nearly homogeneous distribution on the

filter. The concentrations of benzo [ a] pyrene on four sheets of filters were approximately equal as shown in Table 1. This also gave a reliable homogeneity

in the samples at the time of the start of the experiment.

Table 1. Comparison of the Concentration of Benzo [ a] pyrene in

the Simultaneously Sampled Four Filters. Sampling

was Repeated Twice (1st and 2nd run)

Sampler No. 1st run 2nd run

1 5.2 ppm 8.4 ppm

2 5.2 8.6

3 5.4 8.2

4 5.2 8.7

Mean 5.3 ppm 8.5 ppm

C . V. 2.9?h 2.2^i

Storage Experiment

Table 2 summarizes the analytical results of benzo [ a] pyrene concentration

in the atmospheric particulate matter at the time of the start of the experiment, after 6 months, 1 year, 2 years, and 4 years, for each series of samples

stored under each of four storage conditions. The duplicate analysis for each

disc agreed within ± 2%. The trends of changes expressed as percentage of the

initial values (100%) are illustrated in Figure 3. The two series of samples

tested gave nearly similar results, both decreasing in their benzo [ a] pyrene

concentrations according to the storage conditions.

The average rate of decrease of benzo [ a] pyrene during the test period (4

years) was higher for the samples stored at 20 °C decreasing to about 35% of

the initial value. The samples in -20 °C, on the other hand, were both reduced

on the average by 12% of the initial values. Thus, the effect of the

difference of temperature was significant. The difference of the atmosphere in

114
o

o

<:

CNI CO O O

U ea

aU

>^ •

CM

e

ON a^

r-i in <1- r~-

S-i 6^ ON

u

CQ in

E

aB

CM r~- LO 00

U

0)

4-1

CO 5-1 <:

•r-l

a

e

0) o

CM o

CM
0)

O)Soc3o

0)

0)o

(5aoa

(/) oE

(0

0)as

0)c

116
the storage box did not show any remarkable effect in this experiment; but, a problem still remains as to whether the substitution of air by argon gas was complete or not; further studies are needed.

The reasons for the decrease of benzo [ a] pyrene in this experiment cannot

be explained adequately. The box was closed and dark inside, no effect of

light can be considered to have occurred. From these differences in the

behavior of benzo [ a] pyrene in the different temperature conditions, it is

proposed that some bacteriological or chemical reactions may have occurred

other than the decomposition by the reaction with oxygen or oxidants, assuming

that none of the benzo [ a] pyrene could escape by evaporation.

A rapid decrease in the initial stage is remarkable for the storage both

at 20 °C and -20 °C. The difference between the rate of decrease during the

first 6 months and that of the following periods is remarkable as shown in

Figure 3, which shows that in the first 6 months' storage the rate of decrease

was approximately twice that in the next 6 months and during the following 1 year the rate of decrease became almost zero under the -20 °C condition,

whereas under the 20 °C condition the concentration still decreased by several

percent. These results suggest that benzo [ a ] pyrene in atmospheric particulate

matter consists of an easily changeable part and stable part at -20 °C.

The experiment is still going on and the final results will be obtained

after one or two additional years. From the results obtained so far, it was concluded that the storage method applied in our storage program does not

preserve the sample completely, but as the rate of reduction of benzo [ a] pyrene

after 6 months is quite low, at least 80% of the initial amount is expected to

be preserved after 20 years storage and the samples can be utilized for a retrospective analysis.

As to other organic substances in the atmospheric particulate material,

the information on the long-term preservability is very limited, and similar

experimental studies will be needed to increase the usefulness of the stored

samples

IV. PREPARATION OF ENVIRONMENTAL REFERENCE MATERIAL

VEHICLE EXHAUST PARTICULATES"

The National Institute for Environmental Studies (NIES) has prepared

several kinds of environmental reference materials. One of the newest ones is

"NIES No. 8 Vehicle Exhaust Particulates," which will be of use as a reference

for the analysis of elemental composition of atmospheric particulate matter

samples. In spite of increasing demand for various types of reference materials of atmospheric particulate matter in many areas of environmental

sciences, the availability of such reference materials has been limited to only

the three Standard Reference Materials issued by the U.S. National Bureau of

Standards (SRM 1648, "Urban Particulate Matter" for trace element; SRM 1649,

"Urban Dust Organics ; " and SRM 1650, "Diesel Particulate Matter" for

organics) , probably because of the difficulty in the collection of sufficient

amounts of material. Under these circumstances, the preparation of a new type

of atmospheric particulate reference material, which is closely related to

117
automobiles pollution problems, has been undertaken at NIES in cooperation with

Keio University.

A. Sample Collection and Preparation

The material used for this reference material was collected from

electrostatic precipitators in huge ventilators connected to a highway tunnel.

The electrostatic precipitators were placed behind moving cloth filters and the

contribution from pavement material and soil was small in the collected

samples. About 7 kg of the material was used for the preparation of this

reference material.

During preparation procedures, special care was taken when handling such a large amount of potentially hazardous material. Because this material readily

produced a dust cloud and had an irritating smell of gasoline, it was treated

under wet conditions whenever possible, for safe operation with respect to

occupational health and danger of explosion.

After examining three methods to prepare a homogeneous and easy- to -handle

material safely, a "paste-granule" method was adopted making a paste with

ethanol. About 300 g of the material and 1 liter of 35% ethanol were mixed

well in a 10-L polyethylene container. After repeating this mixing procedure

for the remaining samples, all mixtures were combined together in a large

polyethylene tray, mixed again, and air-dried for 2 weeks.

The material was transferred to aluminum trays, dried in an air-oven at

60 °C for about 5 days and crushed into fine powder in polyethylene bags with a wooden hammer. After passing though a 2 mm nylon screen, the powder was mixed

again in a 30-L polyethylene bottle by rolling it on a ball-mill apparatus.

The mixed powder was packaged into small gas bottles (7 g each)

B. Homogeneity Assessment

A homogeneity test of the final product was performed by acid-dissolution

followed by inductively coupled plasma emission and atomic absorption analyses.

Six bottles were randomly selected from the lot of 1000 bottles and 5 aliquots

(about 300 mg each) were taken from each bottle (total 30 samples)

For the elements Ca, Al , Na, K, Zn, Mg, P, Pb , Sr, Cu, Cr, Ni , V, Co and

Cd, variations between bottles were not significant. For Fe , Ti, and Mn on the

other hand, between-bottle variations were significant, though the reason for

this inhomogeneity is not clear.

C. Sample Dissolution

Certified values for this reference material are issued based on analyses

of the entire samples. Decomposition procedures, therefore, should be designed

to achieve complete dissolution of the material.

118
The dissolution procedure used at NIES was as follows:

Weigh 300 mg of the sample into a Teflon PTFE 100-mL beaker. Add 5 mL of

concentrated nitric acid, cover with a Teflon PTFE watch glass and heat at

160 °C for 3 h on a hot-plate. After cooling, add 5 mL of perchloric acid and

heat at 180 °C for 8 h with the cover on. After washing the inside of the

beaker with distilled water, add another 5 mL of perchloric acid and continue

to heat at 200 °C for 8 h with the cover on, until the contents become a yellow

clear solution. Remove the Teflon cover and add 5 mL of hydrofluoric acid to

dissolve the small amount of remaining residues. Heat the contents at 200 °C

to near dryness. After cooling, add a small volume of warm distilled water and

0.5 mL of nitric acid, transfer the contents quantitatively to a 50-mL polypropylene bottle and dilute it 100-fold with distilled water. Thus, 300 mg of

sample is diluted to 30 g of solution.

D. Certified Values for Vehicle Exhaust Particulates Reference Material

The initial stage of this certification process requires analytical data

for the various elements to be obtained by independent and established analytical techniques. At NIES, the analyses of the vehicle exhaust particulate

reference material were carried out by atomic absorption spectrometry, flame

emission spectrometry, inductively coupled plasma emission spectrometry and

spectrophotometry. Collaborative studies on the elemental analysis of the

Vehicle Exhaust Particulate sample have been performed by 20 participating

laboratories. The analytical data obtained by isotope dilution mass spectrometry, neutron activation analysis and spectrofluorimetry and by the above

mentioned techniques were provided by the collaborating laboratories.

Table 3 shows the certified and reference values for NIES Vehicle Exhaust

Particulates reference material. The certified values are based on results of

determinations by at least three independent analytical techniques. Technical

considerations and statistical outlier tests were first applied and the

certified value is the mean of the acceptable values. Certified values are provided for Al , As, Ca, Cd, Co, Cr, Cu, K, Mg, Na, Ni , Pb , Sb , Sr, V and Zn,

while reference values are reported for Ag, Br, Ce , Cs , Eu, La, Lu, Mo, P, Rb

Sc, Se, Sm and Th. The elemental composition of NIES Vehicle Exhaust

Particulates CRM is considered typical of automobile emission particulates.

The NIES Vehicle Exhaust Particulates CRM is available in bottles containing

7 g on request from NIES [13].

119
Table 3. Analytical Values for NIES Certified Reference

Material No. 8 "Vehicle Exhaust Particulates"

Certified values

Minor constituents Trace constituents

Content (wt .%)* Content (/^g/g) *

, c , e 0.53±0 02 pl^a , c , d 217±9

p^-^a , c , e , g 0.33±0 02 Sj-a , c , e 89±3

Na^ . b , c , e 0. 192±0 008 Cu^ . c , d . e 67±3

j^a , b , c , e 0.115±0 008 Qj-a , c , e 25.5±1. 5 2^a.c .d.e 0.104±0 005 j;j£a , c , d , e 18.5±1

5

I4ga . c , e O.lOliO 005 ya . c , e , f 17±2

Sb^ ^ ^ 6 . 0±0

4

3 . 3±03 2 . 6±0

2 Cd^ . c , d , e 1 . 1±0. 1

Reference values (fig/g)*

p 510 Se 1 3 Ag 0 20

Br 56 La 1 2 Sm 0 20

Mo 6 4 Sc 0 55 Eu 0 05

Rb 4 6 Th 0 35 Lu 0 02

Ce 3 1 Cs 0 24

*0n an "as received" basis.

Analytical techniques used:

® atomic absorption spectrometry;

Aflame emission spectrometry;

'^inductively coupled plasma emission spectrometry;

'^isotope dilution mass spectrometry;

^neutron activation analysis;

^ spectrophotometry

^ spec trofluorime try

V. REFERENCES

Luepke, N. P., Ed. (1979), Monitoring Environmental Materials and

Specimen Banking, Martinus Nijhoff Publishers, The Hague, 591 pp.

Lewis, R. A., Stein, N., and Lewis, C. W. , Ed. (1984), Environmental

Specimen Banking and Monitoring as Related to Banking, Martinus Nijhoff

Publishers, Boston, MA, 358 pp.

120
[3] Tomingas , R. (1979), Remarks on the Sampling Procedures for Polycyclic

Aromatic Hydrocarbons from the Atmosphere, Fresenius. Z. Anal. Chem. 297,

97-101

[4] Peters, J. and Siefert, B. (1980), Losses of Benzo [ a] pyrene Under the

Conditions of High Volume Sampling, Atmos . Environ. 14, 117-119.

[5] Grosjean, D., Fung, K. , and Harrison, J. (1983), Interactions of

Polycyclic Aromatic Hydrocarbons with Atmospheric Pollutants, Environ.

Sci. Tech. 17, 673-679.

[6] Cimberle, M. R. , Bottino, P., and Valerio, F. (1983), Decomposition of

Benzo [ a] pyrene Deposited on Glass Fiber Filters and Exposed to Sunlight,

Chemosphere 12, 317-324.

[7] Nikolaou, K. , Masclet, P., and Mouvier, G. (1984), Sources and Chemical

Reactivity of Polynuclear Aromatic Hydrocarbons in the Atmosphere, Sci.

Total Environ. 32, 103-132.

[8] Galasyn, J. F. . , Horing, J. F., and Soderberg, R. H. (1984), The Loss of

PAH from Quartz Fiber High Volume Filters, J. Air Pollut. Control Assoc.

34, 57-59.

[9] Fox, M. A. and Staley, S. W. (1976), Determination of Polycyclic Aromatic

Hydrocarbons in Atmospheric Particulate Matter by High Pressure Liquid

Chromatography with Fluorescence Technique, Anal. Chem. 48, 992-998.

[10] Matsushita, H. , Arashiya, K. , and Handa, T. (1975), Simple Microanalysis

of Benzo [a] pyrene in Air Suspended Particulates Using Ultrasonic

Extraction, Bunseki Kagaku 25, 264-268.

[11] Das, B. S. and Thomas, G. H. (1978), Fluorescence Detection in High

Performance Liquid Chromatographic Determination of Polycyclic Aromatic

Hydrocarbons, Anal. Chem. 50, 967-973.

[12] May, W. E., Chelser, S. N., Hertz, H. S., and Wise, S. A. (1982),

Analytical Standards and Methods for the Determination of Polynuclear

Aromatic Hydrocarbons in Environmental Samples, Inter. J. Environ. Anal.

Chem. 12, 259-275.

[13] Okamoto, K. (1987), A New Certified Reference Material, Vehicle Exhaust

Particles, Anal. Sci. 3, 191-192.

121
SECTION 13

ANALYSIS OF PAH IN AIR SAMPLES OBTAINED BY LONG- AND SHORT-TERM PROCEDURES

J. Jacob, G. Grimmer, K.-W., Naujack, and G. Dettbam

Institute for Environmental Carcinogens

D-2070 Grosshansdorf

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

When the pilot phase of the German Environmental Specimen Bank was started, the terrestrial and aquatic environment were represented by various

kinds of specimens. However, the matrix air was not addressed mainly for

technical reasons, since no adequate and reliable sampling procedures seemed to

be available at that time. It was expected that broad basic studies had to be

carried out before air samples could become a regular matrix for the bank.

One of the goals of specimen banking is to correlate human diseases with

pollution, and one of the best known examples for this is human lung cancer. According to a study of the Department of Epidemiology and Statistics of the

American Cancer Society about 83% of the lung cancers in the United States may

be attributed to tobacco smoking, and there are some strong indications that

air pollution also plays an important role as a risk factor for this disease.

It can be expected that emissions, at least from coal combustion and from

vehicles, contribute considerably to the carcinogenesis in the human lung.

There are various classes of compounds that might be responsible for this such

as polycyclic aromatic hydrocarbons (PAH), nitroaromatics , thiaarenes,

asbestos, etc. As a result of these emissions, the importance of air as a matrix for an environmental specimen bank is obvious.

At least as far as PAH are concerned, there are various problems

associated with air sampling related to the volatility of the lower-boiling

compounds, which may result in re-evaporation of already collected compounds

from the filter, and to the sensitivity of some PAH against NG^;. , ozone, sulfur

dioxide, and other photooxidants resulting in irreversible destruction. These

reactions form compounds which are possibly even more biologically active than

the original PAH, such as nitro-PAH, ketones, aldehydes, peroxides and

quinones , about which we know very little at the moment - analytically as well

as biologically.

In this paper some preliminary data are presented on the different PAHconcentrations as found after long-term and short-term sampling.

II. EXPERIMENTAL SECTION

Air sampling was performed with commercially available instruments. For

sample sizes of 15 m^/h, a LIB- instrument was used equipped with glass fiber

filters of 113 cm^ and 500 cm^ surface possessing a separation degree of

99.998% for particles with a diameter of 0.3-0.5 fj.m. For sample sizes of 2-2.3

122
m'^/h a small filter instrument Derenda GS/050/3 with a 12.5 cm^ filter was

used. To check the volatility of PAH, a subsequent Porapak PS filter was used.

The clean up is schematically represented in Figure 1

.

Filter, exfracfed with toluene + internal standard _ ^ toluene

filtration on silica gel / cyclohexane

. — ^

cyclotiexane

chromatography on Sephadex LH20 / isopropanol

isopropanol

1 dissolved in toluene.

Glass-capillary gas- chromatography

Figure 1. Schematic representation of the PAH-profile analysis of air

suspended matter

III. RESULTS AND DISCUSSION

A first set of results was obtained from an urban area predominantly

polluted by domestic and industrial heating, a coke plant and to a lesser

extent by vehicle exhaust. Two samples were collected in parallel: the first was obtained after a 24 h collecting period using one filter for a total of 326

m^ air equal to 13.6 m"^/h. The second sample consisted of 24 subsamples each

obtained after one hour collecting during the same period. In this experiment,

between 11 and 13 /h air passed through each of the filters. In Figures 2 and 3, the time dependence of various PAH concentrations are shown. All

compounds follow the same trend despite the different chemical reactivities

which can be expected for the various PAH. (Note: No specifically low values

for benz [ a] anthracene or dibenz [ a ,h] anthracene were found, although both are sensitive anthracenoid structures.)

A first maximum appears between 6 and 7 o'clock a.m., followed by a second

one at 9 a.m.; these maxima may be attributed to the morning rush hours. A

huge maximum then is observed at 5 p.m. and there is also a maximum at about 1-

2 a.m.

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Of greater interest is the comparison of the sum of the 24 single one- hour samples and the long-term sample (24 h) . On the basis of a 100%

separation for the short-term (one hour) collection, which is not completely

realistic for the low-boiling compounds, a significant decrease of the

recoveries for the lower-boiling compounds was observed, as shown in Table 1.

Table 1. PAH Profiles on Air Particulate Filters (ng PAH/m^ )

1 2 Sample 24xlh lx24h Yield 2 Volume (m'^ ) b.p. (°C) 277. 16 326 39 Rel. to 1

Fluoranthene 384 1

31 0 50 38.2%

Pyrene 394 112 0 33 29.5%

Benzo [b ] naphtho [ d , 2 -

1

] thiophene 430 0. 52 0 15 28.8%

Benz [ a ] anthracene 438 0. 85 0 31 36.5%

Chrysene + Thiphenylene 441/439 2. 69 0 86 31 .9%

Benzof luoranthenes [b+j+k] 480 3. 55 2 83 79.7%

Benzo [ e ] pyrene 493 163 1 17 71.8%

Benzo [ a] pyrene 496 0. 91 0 67 73.6%

Indeno [1,2, 3 -cd] pyrene 140 1 09 77.9%

Dibenz [ a , h] anthracene 0. 21 0 22 104.8%

Benzo [ ghi ] pery lene 500 173 1 60 92.5%

Anthanthrene <0. 16 0 16 100.0%

Coronene 525 0. 75 0 .74 98.7%

When the recoveries of the various PAH are plotted against their boiling

points a linear curve is obtained which, however, seems to depend on the total

amount of PAH collected on the filter (Figure 4)

From the data, it may be predicted that compounds with boiling points above

480 °C can be collected quantitatively and compounds boiling below about 340 °C

such as dibenzothiophene , phenanthrene , and anthracene cannot be collected at

all by this commonly used filter system. The curves do not indicate any

chemical conversion of the PAH investigated, if they do not already decompose

within the first hour. The re-evaporation can also be demonstrated by adding a Porapak PS filter following to the glass fiber filter as demonstrated in

Table 2.

It was demonstrated that the air flow velocity onto the filter

significantly influences the PAH concentration separated by a particle filter

system. Although only two measurements have been made, one may conclude with

caution that the lower-boiling PAH are more affected than the higher-boiling

ones. This is shown in Figure 5, in which two air flow velocities have been

used, 7.6 cm/s and 52.6 cm/s . Both samples have been taken at identical times

and at identical locations.

126
127
Table 2. PAH-Profiles Collected on a Glass Fiber Filter and a Subsequent

Porapak PS Filter (ng PAH/m^

23 hr (GF)* 23 hr (PS)*

Sample Volume (m^ ) 57 . 89

Fluoranthene 0. 55 = 2. 1% 25. 80

Pyrene 0. 54

A 2. 7% 19. 51

Benzo [b ] naphtho [ 2 , 1 -d] thiophene 0 20

A 36. 4% 0 35

Benzo [ c ] phenanthrene + BghiF 0 60

A 23. 1% 2 00

Benzo [ a ] anthracene 0 69

A 53. 8% 0 60

Chrysene + Triphenylene 1 78

A 48. 0% 1 93

Benzofluoranthenes [b+j+k] 4 64

A 90. 0% <0 40

Benzo [ e ] pyrene 1 85

A 90. 0% <0 20

Benzo [ a ] pyrene 1 48

A 90. 0% <0 20

Indeno [ 1 , 2 , 3 - cd ] pyrene 1 24

A >90% <0 10

Benzo [ ghi ] perylene 2 37

A >90% <0 10

Anthanthrene 0 18

A 100%

Coronene 1 33

A 100%

"GF: Glass fiber filter (12.5 cm^ 2.5 /h)

*PS: Porapak PS (2.0 g)

ng PAH/m3

o •CHR

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1

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1 1 I 10 50 v(air flow velocity) i Figure 5. Dependence of the yield on the air flow velocity

128
Slopes of the curves are comparable for compounds with similar boiling

points. The slopes approach zero for PAH with boiling points of about 450 °C.

Unfortunately, results were less consistent in samples linked predominantly to

vehicle traffic.

Very sensitive compounds such as cyclopenta [ cd] pyrene and anthanthrene are significantly reduced in concentration when compared to more stable compounds

such as benzo[e] pyrene and coronene . The ratio 24 x lh/1 x 24h is abnormally

high for cyclopenta [ cd] pyrene and anthanthrene when compared to PAH having the same boiling points, as shown in Table 3 which compares long- and short-term

sampling at a car parking area. At this location, which is predominantly or almost entirely polluted by vehicle exhaust, a comparative experiment with

sampling times of 24 x Ih and 1 x 24h has been repeated three times, and the

results were very disappointing (Table 4)

Table 3. PAH-Profiles on Glass Fiber Filters (ng PAH/m^

)

1 2 Yield 2 Sample 24xlh lx24h Rel. to

Volume (m^

)

328 17 329 05 1

Fluoranthene 43 32 7 39 17.0%

Pyrene 36 19 6 44 17.8%

Benzo [b ] naphtho [ 2 , 1 -d] thiophene 1 87 1 20 64.2%

Cyclopenta [ cd] pyrene 6 72 0 40 6.0%

Benz [ a ] anthracene 3 60 2 29 63.6%

Chrysene + Thiphenylene 8 08 4 58 56.2%

Benzofluoranthenes [b+j+k] 9 58 5 73 59.8%

Benzo [ e ] pyrene 4 55 2 47 54.3%

Benzo [a]pyrene 3 45 1 76 51 .0%

Indeno [1,2, 3 -cd] pyrene 2 80 1 69 60.4%

Benzo [ ghi ] perylene 5 53 3 17 57.3%

Anthanthrene 0 87 0 25 28 . 7%

Coronene 3 22 1 58 49.1%

Air flow velocity: 7.5 cm/s

Although, the recoveries of the lower boiling compounds are significantly

reduced, the higher-boiling compounds generally were not found to be in the

100% range but rather in the 50%-range. Table 4 also gives the absolute

amounts of benzo [ a] pyrene . The atmospheric conditions were different in all

three experiments and they might be responsible for the great variations,

especially for the low concentrations of benzo [ a] pyrene and benz [ a] anthracene

in the second experiment. On the other hand, no correlation between NO^^ , SO2 , and ozone levels, which were measured simultaneously (not shown in Table 4),

and the PAH-profile could be observed. Until further systematic experiments

have been carried out, these data cannot be interpreted satisfactory. However,

from the information available, one may question whether the concentrations of

benzo [a] pyrene and other PAH measured in air, as reported in the literature,

129
Table 4. PAH-Profiles Recorded at a Vehicle-Exhaust Polluted Location

Three Independent Experiments During Different Days) . Yields

Calculated from 1 x 24h Sampling

1* 2* 3**

Fluoranthene 16 22 17

Pyrene 13 19 18

Benzo [b ] naphtho [ 2 , 1 -d] thiophene 11 30 64

Benzo [ a ] anthracene 11 28 64

Benzofluoranthenes [b+j+k] 20 51 60

Benzo [ e ] pyrene 23 45 54

Benzo [ a] pyrene 29 (18) 54

Indeno[ 1,2, 3-cd]pyrene 50 65 60

Benzo [ ghi ] pery lene 47 41 57

Coronene 67 42 49

Absolute Benzo [ a] pyrene (ng/m^

1.7 4.2 4.6

*Filter surface 113 cm'' ; **filter surface 500 cm^

are a reliable basis for any meaningful conclusions. It could well be that

most of these data are underestimating the actual PAH-concentrations in the

matrix air.

III. CONCLUSIONS

1. During long-term sampling, there is a considerable re -evaporation of the

lower boiling PAH collected on glass fiber filters after 24 h and even

earlier. This problem can be overcome by using a subsequent Porapak PS

filter. The effect depends on:

the absolute amount of PAH collected on the filter, and

the air flow velocity.

The effect can be neglected for PAH boiling above 480 °C

(benzofluoranthenes ) , but data for pyrene and fluoranthene have to be

regarded with great caution. They might not be very reliable. Hence, in

general, short-term and low-volume sampling should be preferred and the

air flow velocity onto the filter should be less than 8 cm/s

2. In places which are predominantly polluted by vehicle exhaust emission,

hitherto unidentified effects influence the recoveries and the profiles of

PAH collected on glass fiber filters. Various reasons may be responsible

for this among which sunlight-catalyzed reactions are the most probable

ones. Further basic and systematic investigations are required to understand these detrimental effects. Long-term sampling, however, cannot be

recommended for the active collection of the matrix air.

130
SECTION 14

TRENDS AND EFFECTS OF ENVIRONMENTAL CONTAMINANTS DETERMINED

FROM ANALYSIS OF ARCHIVED WILDLIFE SAMPLES

J. E. Elliott, R. J. Norstrom, S. W. Kennedy and G. A. Fox

Canadian Wildlife Service

National Wildlife Research Centre

Ottawa, KIA 0H3

CANADA

I. INTRODUCTION

Most developed countries introduced measures during the past 20 years to

control the release of persistent toxic chemicals to the environment. However,

chemical inputs continue from manufacturing, waste dumpsites, transport

accidents, ocean dumping, incineration and open uses especially in developing

countries. Substantial amounts of chemicals such as the polychlorinated

biphenyls (PCBs) , persisting mainly from previous usage, continue to cycle

through the environment [ 1 ]

Agencies in some countries have established programs to monitor persistent

contaminants in the environment. A number of cooperative international

monitoring projects have also been established over the past two decades [2-4].

The storage of environmental samples for future use, or specimen banking, is a natural complement to environmental monitoring [5]. Environmental agencies in

a number of countries now operate specimen banks [6,7].

This paper will briefly discuss current specimen banking activities by

Canadian federal agencies working in the Great Lakes. We will then discuss the use of samples from the National Specimen Bank of the Canadian Wildlife Service

(CWS) to determine both levels and effects of environmental contaminants in the

Great Lakes and the Gulf of St. Lawrence.

II. SPECIMEN BANKING BY CANADIAN AGENCIES IN THE GREAT LAKES

The three Canadian agencies involved in monitoring environmental

contaminant levels in the North American Great Lakes also operate specimen

banks. The Canadian Wildlife Service maintains the most comprehensive bank

largely in support of its monitoring programs [8,9].

The Canadian Department of Fisheries and Oceans monitors contaminant

levels in larger predatory fish such as Lake Trout (Salvelinus namaycush) and

Coho Salmon (Oncorhynchus kisutch) in the Great Lakes [10]. Homogenized

aliquots of whole fish, invertebrates and plankton are archived at -80 °C [11].

Other fish samples are stored in chemical fixatives. Information on samples in

the bank is on computer.

The National Water Research Institute of Environment Canada maintains a bank of Great Lakes sediment samples [12]. Surficial sediments were collected

131
from locations throughout the Great Lakes, homogenized, freeze-dried,

aliquotted and stored at room temperature.

The specimen banks operated by Canadian agencies in the Great Lakes are not formally coordinated as in the Federal Republic of Germany's Environmental

Specimen Banking project [13]. However, coordination of effort is maintained

by direct contact among the scientists involved, by the federal government

Great Lakes Water Quality Program Working Group, and via committee structures

of the International Joint Commission (IJC) which oversees the monitoring of

contaminants in the Great Lakes [4]

III. MONITORING CANADIAN WILDLIFE

Our monitoring of environmental contaminants in Canadian wildlife samples

is often designed to meet two complementary objectives: 1) determination of

levels and trends in the environment using wildlife as indicator organisms and

2) assessment of the effects of chemicals on wildlife health. Here we discuss

the use of archived Herring Gull (Larus argentatus) eggs to determine trends of

organic contaminants in the North American Great Lakes. The second objective

is pursued at both the population and subcellular levels and examples are presented using banked samples.

A. Monitoring Trends in Contaminant Residues

Biological and chemical surveillance of Great Lakes Herring Gulls has been

part of the IJC Great Lakes Surveillance Program since 1974. Two geographically separated colonies from each lake and colonies on connecting channels are monitored annually [14]. The Herring Gull is a useful indicator of persistent,

lipophilic contaminant levels in the Great Lakes. It is a colonial species

which feeds mainly on aquatic food chains. Upon reaching breeding age, the

Herring Gull is a year-round resident in the Great Lakes. Seasonal movements

between lakes and changes in diet can confound interpretation of Herring Gull

residue data. A bioenergetics based model has been developed to aid data

interpretation [15,16].

Until 1986, we collected and analyzed ten individual eggs from each

monitor colony. Statistical analysis of ten years' accumulated data showed

that a strategy of pooling samples would reduce analytical costs and still

provide a reliable estimate of mean residue levels and trends [17,18]. We

continue to collect, process and bank individual Herring Gull eggs. In this

way, we shall always have the option for individual analyses should the trends

in pooled sample means require greater statistical treatment. We have established a schedule of annual pooled analyses for most colonies with each lake

and connecting channel receiving individual analyses on a rotating schedule,

i.e., every five years.

Since the Herring Gull program began in 1974, the number of chemicals

reported on a routine basis has increased considerably. Also, methodological

changes and improvements have rendered some of the earlier data of questionable

value. We decided in 1982 to do a comprehensive retrospective analysis of

Herring Gull eggs from the specimen bank in order to determine temporal trends

132
for a variety of chemicals. We searched our specimen bank records to locate

colonies where adequate numbers of Herring Gull eggs had been collected and

archived regularly since the early 1970s. We selected two locations, Scotch

Bonnet Island in Lake Ontario and Big Sister Island in Lake Michigan. We

prepared 50 gram pools of Herring Gull egg homogenate for each year and

analyzed the pools for a variety of contaminants. The stability of

organochlorine residues in stored Herring Gull eggs has been demonstrated

[19,9].

Figure 1 shows contaminant trends for three of the major chlorinated

organics (PCBs, DDE, oxychlordane) found in Herring Gull eggs from Lake Ontario

and Lake Michigan. Declining trends are evident for PCBs and DDE. Oxychlordane remained relatively constant throughout the study period. The higher

levels of chlordane derived compounds in Lake Michigan reflect the more agricultural nature of the drainage basin. Both lakes are also surrounded by

large urban populations and extensive industrial developments, accounting for

high PCB concentrations

The controls imposed in Canada and the U.S. in the 1970s on use and

disposal of PCBs and many organochlorine pesticides presxjimably account for the

declining concentrations of PCBs and DDE in Herring Gull eggs. Oxychlordane is

the major metabolite of cis and trans - chlordane , the main components of

technical chlordane. Chlordane was used extensively in North America after the

restrictions on DDT, and controls were only imposed in the late 1970s. Ongoing

use of chlordane as a termiticide and atmospheric deposition from long range

transport from areas of broader use may also contribute to the relatively

constant levels of this chemical.

Figure 1 also shows trends for a tetrachloro dibenzo-dioxin (TeCDD)

congener, 2378-TeCDD, also from analysis of the archived samples from Scotch

Bonnet and Big Sister Islands. Among the dioxin congeners, only 2378-TeCDD

levels in Lake Ontario Herring Gull eggs from the early 1970s were significantly elevated over levels from Lake Michigan [20]. This indicated that the

dioxin source to Lake Ontario contained predominantly 2378-TeCDD. 2,4,5-T was manufactured in the Niagara Falls area for many years and is probably the

origin of most of the 2378-TeCDD in Lake Ontario.

B. Monitoring Contaminant Effects on Biochemical State

Impaired reproduction observed in a variety of Great Lakes colonial water

birds during the 1960s and 1970s has been related to chemical contaminants

[14]. With the exception of some species in the Green Bay area of Lake

Michigan [21,22], productivity of colonial water birds is now thought to be

normal throughout the Great Lakes [23,24]. Although contaminant concentrations

have decreased at least in Herring Gulls, levels are still much higher than

found elsewhere in Canada [25]. The focus is now on possible sublethal effects

on Great Lakes wildlife.

Chronic exposure to the polyhalogenated aromatic (PHA) compounds including

the biphenyls (PCBs) , dioxins (PCDDs) and furans (PCDFs) produces a suite of

toxic effects in most species studied [26]. Among those effects, induction of

hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity has been

133
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investigated in wild species and proposed for monitoring of birds [27,28] and

fish (Spies et al

1982) . Many PHA congeners have been shown to disrupt the

heme biosynthetic pathway [30]. Disruption of the pathway by PHAs causes a change in the normal "porphyrin pattern" resulting In elevated levels of highly

carboxylated porphyrins (HCPs) in tissues and excreta. In a recent paper, Koss

et al. [31] showed that livers of pike, Esox lucius , from the Rhine River

contained much higher HCP levels than livers of pike from the less polluted

River Lahn. Livers of adult Herring Gulls collected in 1985 from the Great

Lakes had much higher HCP levels than in Herring Gulls or other avian species

collected from more pristine sites [32]. In an earlier study, Herring Gull

chicks collected from the Great Lakes in 1974 had total porphyrin levels which

correlated with exposure to PHAs [33].

Livers from prefledged Herring Gull chicks, collected in 1974 from two

Great Lakes colonies, were re-analyzed in 1985 for porphyrins. HCPs in some Herring Gull chick samples from Scotch Bonnet Island, Lake Ontario were ten

times higher than any of the samples from Port Colborne, Lake Erie (Figure 2).

A six month study of porphyrin stability in rat liver homogenate frozen at

-20 °C showed no apparent degradation of HCPs [34,35].

Methods of sample collection and preservation for study of biochemical or histological effects of contaminants may differ from those required for

measuring contaminant levels. Collection protocols can be designed to meet

different objectives; however, many samples, including the bulk of our CWS

National Specimen Bank have been collected for chemical analyses. Such samples

can still be used for measuring biochemical effects in the form of levels and

patterns of stable metabolites such as porphyrins.

C. Correlating Contaminant Residue Trends With Population State

Northern Gannet (Sula bassanus) populations in the Gulf of St. Lawrence

declined during the late 1960s and early 1970s [36]. Adult and juvenile birds

and eggs were collected on a number of occasions from 1968 to 1976 at Bonaventure Island (48 30'N 64 09 'W) and stored in the CWS specimen bank. Analysis of

those samples showed high levels of DDE and other organochlorine contaminants.

In 1984, we collected more eggs at the colony and retrieved a subset of the

eggs from the specimen bank. We analyzed those eggs for organochlorines using

current analytical methodology.

One objective of the study was to reassess the role of chemicals in the

gannet reproductive decline using a more extensive organochlorine database.

The data strongly suggests that high levels of DDE and thin eggshells were associated with the period of reduced reproductive success [37,38].

We also saw an opportunity to determine organochlorine trends in the Gulf

of St. Lawrence ecosystem using the gannet as an indicator seabird species

[17]. The St. Lawrence River drains a highly industrialized, heavily populated

region of North America. The Gulf of St. Lawrence also receives drainage water

from four surrounding maritime provinces. Long range atmospheric transport

from the North American mainland is a further source of airborne chemical

contaminants to the gulf.

135
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Figure 3 shows trends of DDE, PCBs , a-HCH and oxychlordane in gannet eggs

from Bonaventure Island. Only random samples of the eggs laid were used for

trend assessment. Eggs that failed to hatch were also re-analyzed from some years in order to determine difference in contaminant levels between those eggs

and samples of eggs laid random from the same year [37].

DDE levels declined significantly throughout the study period. Aerial

application of DDT to control forest insects around the Gulf of St. Lawrence

stopped in 1969. Legislative controls on DDT use in Canada and the U.S. came

into effect the following year. Declining DDE levels in gannet eggs from the

gulf are probably the result of reduced DDT input from both regional and wider

North American sources.

PCB levels also decreased in gannet eggs during the course of the study

but at a slower rate than DDE. Re-analysis of PCBs from specimen bank samples

has been particularly useful: Analytical methods used in 1969 seriously

under-estimated PCB levels which resulted in the appearance of a flat or increasing trend for PCBs in gannet eggs from 1969 to 1976 [39].

With the exception of heptachlor epoxide, chlordane-related isomers were not measured routinely in wildlife samples prior to 1976. The re-analysis of

archived samples provides a trend picture for this group of compounds.

Oxychlordane concentrations in gannet eggs were constant throughout the period

1969 to 1976, paralleling the situation in Great Lakes Herring Gulls. This

does not reflect increasing Canadian usage during that time frame. However,

the decrease in oxychlordane by 1984 would appear to follow 1978 restrictions

on use in Canada.

HCH residues were not routinely measured in our wildlife samples until

1978. Although not statistically significant (p < 0.05, regression test)

levels of a-HCH increased with time as indicated in archived gannet eggs. We

could find no published data on HCH trends with which to compare our results.

Increasing levels in the Atlantic coastal environment are possible as a result

of long range transport from areas of continued use [40]

IV. SUMMARY

A specimen bank can be a valuable addition to an environmental monitoring

program. Canadian agencies involved in monitoring wildlife, fish and water in

the Great Lakes ecosystem currently maintain specimen banks.

The Canadian Wildlife Service has been monitoring environmental

contaminant levels in Great Lakes Herring Gulls since the early 1970s. Remains

of these and other samples are routinely archived. Series of Herring Gull eggs

collected from 1971 to 1982 at colonies in Lakes Ontario and Michigan were analyzed for a broad suite of contaminants. PCB and DDE levels in Herring Gull

eggs declined substantially at both colonies. Oxychlordane levels remained

constant in Herring Gull eggs at both colonies throughout the study period.

2378-TeCDD levels declined at both locations.

137
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Increased liver levels of highly carboxylated porphyrins have been

measured in wildlife from environments heavily contaminated with polyhalogenated aromatic hydrocarbons (PAHs) . Measurement of tissue porphyrin levels

is a promising tool for monitoring biochemical effects of PHAs

.

Analysis of Northern Gannet eggs from the Canadian Wildlife Service

specimen bank showed trends in organochlorine residues from 1969 to 1984 in the

marine ecosystem of the Gulf of St. Lawrence. Levels of DDE and PCBs declined

throughout the study period. Oxychlordane levels remained quite constant while

Q-HCH levels increased throughout the study.

V. ACKNOWLEDGEMENTS

We gratefully acknowledge the late, Y. Ouellette, for his careful

reorganization of the specimen bank samples and records. H. Won, M. Simon and

M. Mulvilhill performed the chemical analysis.

VI . REFERENCES

[1] Eisenreich, S. J., Looney, B. B., and Thornton, J. D. (1981), Airborne

Organic Contaminants in the Great Lakes Ecosystem, Environ. Sol. Technol.

15, 30-38.

[2] International Council for the Exploration of the Sea (1977), The ICES

Coordinated Monitoring Programmes, 1975 and 1976, Cooperative Research

Report No. 72, Charlottenlund , Denmark, 27 pp.

[3] Organization for Economic Cooperation and Development (1980), Chemical

Trends in Wildlife, Paris, 249 pp.

[4] International Joint Commission (1985), 1985 Report on Great Lakes Water

Quality, Great Lakes Water Quality Board Report to the International

Joint Commission, Windsor, Ontario.

[5] Luepke , N. -P. (1979), Monitoring Environmental Materials and Specimen

Banking, Martinus Nijhoff Publishers, The Hague, 591 pp.

[6] Lewis, R. A., Stein, N., and Lewis, C. W. (1984), Environmental Specimen

Banking and Monitoring as Related to Banking, Martinus Nijhoff

Publishers, The Hague, 358 pp.

[7] Wise, S. A. and Zeisler, R. , Eds., (1985), International Review of

Environmental Specimen Banking, NBS Spec. Publ. 706, U.S. Government

Printing Office, Washington, DC, 54 pp.

[8] Elliott, J. E. (1984), Collecting and Archiving Wildlife Specimens in

Canada, In: Environmental Specimen Banking and Monitoring as Related to

Banking, R. A. Lewis, N. Stein, and C. W. Lewis, Eds., Martinus Nijhoff

Publishers, Boston, MA, pp 45-66.

139
[9] Eliiott, J. E. (1985), Specimen Banking in Support of Monitoring for

Toxic Contaminants in Canadian Wildlife, In: International Review of

Environmental Specimen Banking, S. A. Wise and R. Zeisler, Eds., NBS

Spec. Publ. 706, U. S. Government Printing Office, Washington, DC,

pp 4-12.

[10] Shear, H. (1984), Contaminants Research and Surveillance - A Biological

Approach, In: Toxic Contaminants in the Great Lakes, J. 0. Nriagu and

M. S. Simmons, Eds. , John Wiley and Sons, Inc. , New York, NY.

[11] Hyatt, W. H., Fitzsimons, J. D., Keir, M. J., and Whittle, D. M. (1984),

Great Lakes Biological Tissue Archive Studies, Canadian Technical Report

of Fisheries and Aquatic Sciences, No. 1497, Burlington, Ontario, 58 pp.

[12] Bourbonniere , R. A., Van Sickle, B. L. , and Mayer, T. (1986), The Great

Lakes Sediment Bank, I. National Water Research Institute, Environment

Canada, Burlington, Ontario.

[13] Boehringer, U. R. (1985), The Environmental Specimen Banking Project of

the Federal Republic of Germany, In: International Review of

Environmental Specimen Banking, S. A. Wise and R. Zeisler, Eds., Nat.

Bur. Stand. Spec. Publ. 706, U.S. Government Printing Office, Washington,

DC, pp 13-21.

[14] Mineau, P., Fox, G. A., Norstrom, R. J., Weseloh, D. V., Hallett, D. J.,

and Ellenton, J. A. (1984), Using the Herring Gull to Monitor Levels and

Effects of Organochlorine Contamination in the Canadian Great Lakes, In:

Toxic Contaminants in the Great Lakes, J. 0. Nriagu and M. S. Simmons,

Eds., John Wiley and Sons, Inc., New York, NY, pp 425-452.

[15] Norstrom, R. J., Clark, T. P., Jeffrey, D. A., Won, H. T., and Gilman,

A. P., (1986a), Dynamics of Organochlorine Compounds in Herring Gulls

(Larus argentatus) : I. Distribution and Clearence of '^ ^ C DDE in Freeliving Gulls, J. Environ. Toxicol. Chem. 5, 41-48.

[16] Norstrom, R. J., Clark, T. P., Kearney, J. P., and Gilman, A. P. (1986b),

Herring Gull Energy Requirements and Body Constituents in the Great

Lakes, Ardea 72, 11-32.

[17] Gilbertson, M. , Elliott, J. E. , and Peakall, D. B. (1987), Seabirds as Indicators of Marine Pollution, In: The Uses of Birds, A. W. Diamond

and F. Filion, Eds., ICBP Technical Publication, Cambridge.

[18] Struger, J. and Collins, B. T. , A Statistical Evaluation of the Great

Lakes Herring Gull Monitoring Program, Canadian Wildlife Service, Ottawa,

(manuscript in preparation)

[19] Norstrom, R. J. and Won, H. T. (1985), Long-term Preservation of Egg and

Tissue Homogenates for Retrospective Determination of Organochlorine

Compounds: Freezing Versus Freeze Drying, J. Assoc. Off. Anal. Chem.

68(1), 129-135.

140
[20] Norstrom, R. J. (1986), Long-Term Trends of PCDD and PCDF Contamination

in the Great Lakes, Presented at the 6th Internat. Symp . on Dioxins and

Related Compounds, Fukuoka, Japan, Sept. 16-19, 1986.

[21] Hoffman, D. J., Rattner, B. A., Sileo, L. , Docherty, D., and Kubiak,

T. J. (1987), Embryotoxicity , Teratogenicity, and Aryl Hydrocarbon

Hydroxylase Activity in Forster's Terns on Green Bay, Lake Michigan,

Environ. Res. 42, 176-184.

[22] Kubiak, T. J., Harris, H. J., Smith, L. M. , Stalling, D. L. , Schwartz,

T. R. , Trick, J. A., Sileo, L. , Docherty, D. E., and Erdman, T. C,

Microcontaminants and Reproductive Impairement of the Forster's Tern on Green Bay, Lake Michigan - 1983, U.S. Fish and Wildlife Service, East

Lansing, Michigan, manuscript in preparation.

[23] Weseloh, D. V., personal communication.

[24] Weseloh, D. V., Mineau, P., Teeple, S. M. , Blokpoel, H. , and Ratcliff, B. (1986), Colonial Waterbirds Nesting in Canadian Lake Huron in 1980,

Canadian Wildlife Service, Progress Notes, No. 165, 28 pp.

[25] Canadian Wildlife Service, unpublished data.

[26] Poland, A. and Knutson, J. C. (1982), 2 , 3 , 7 , 8-Tetrachlorodibenzo-p-Dioxin

and Related Halogenated Aromatic Hydrocarbons: Examination of the

Mechanism of Toxicity, Ann. Rev. Pharmacol. Toxicol. 22, 517.

[27] Ellenton, J. A., Brownlee , L. J., and Hollebone, B. R. (1985), Aryl

Hydrocarbon Hydroxylase Levels in Herring Gull Embryos from Different

Locations on the Great Lakes, Environ. Toxicol. Chem. 4, 615-22.

[28] Peakall, D. B., Norstrom, R. J., Rahimtula, A. D., and Butler, R. D. (1986), Characterization of Mixed- function Oxidase Systems of the

Nestling Herring Gull and Its Implications for Bioeffects Monitoring,

Environ. Toxicol. Chem. 5, 379-85.

[29] Spies, R. B., Felton, J. S., and Dillard, L. (1982), Hepatic MixedFunction Oxidases in California Flatfishes are Increased in Contaminated

Environments and by Oil and PCB Ingestion, Mar. Biol. 70, 117-127.

[30] Marks, G. S. (1985), Exposure to Toxic Agents: The Heme Biosynthetic

Pathway and Hemoproteins as Indicator, ORG Grit. Rev. Toxicol. 15(2),

151-179.

[31] Koss, G., Schuler, E., Arndt , B., Seidel, J., Seubert, S., and

Seubert, A. (1986), A Comparative Toxicological Study on Pike (Esox

lucius L.) from the River Rhine and the River Lahn, Aquat. Toxicol. 8,

1-9.

141
[32] Kennedy, S. W. , Fox, G. A., Norstrom, R. J., and Wigfield, D. C,

Porphyria in Herring Gulls: A Biochemical Response to Chemical

Contamination of Great Lakes Food Chains, Canadian Wildlife Service,

Ottawa, (manuscript in preparation)

[33] Gilbertson, M. and Fox, G. A. (1977), Pollutant-associated Embryonic

Mortality of Great Lakes Herring Gulls, Environ. Pollut. 12, 211-16.

[34] Kennedy, S. W. (1986), Porphyria in Great Lakes Herring Gull: A

Biochemical Indicator of Polyhalogenated Aromatic Hydrocarbon Exposure,

Canadian Wildlife Service Report, Ottawa.

[35] Kennedy, S. W. , Wigfield, D. C, and Fox, G. A. (1986), Tissue Porphyrin

Pattern Determination by High-Speed High- Performance Liquid

Chromatography, Anal. Biochem. 157, 1-7.

[36] Nettleship, D. N. (1976), Gannets in North America: Present Numbers and

Recent Population Changes, Wilson Bull. 88(2), 300-13.

[37] Elliott, J. E. , Norstrom, R. J., and Keith, J. A., Organochlorines and

Eggshell Thinning in Northern Gannets (Sula bassanus) from Eastern

Canada, 1968-1984, Environ. Pollut., in press.

[38] Chapdelaine, G., Laporte, P., and Nettleship, D. N. , Population,

Productivity and DDT Contamination Trends of Northern Gannets (Sula

bassanus) at Bonaventure Island, Quebec, 1967-1984, Can. J. Zoo., in

press

[39] Turle, R. , Norstrom, R. J., and Won, W. T. (1986), Quality Assurance as Applied to Long Term Monitoring and Specimen Banking, In: These

Proceedings

[40] Tanabe, S., Tatsukawa, R. , Kawano , M. , and Hidaka, H. (1982), Global

Distribution and Atmospheric Transport of Chlorinated Hydrocarbons HCH

(BHC) Isomers and DDT Compounds in Western North Pacific, Easter Indian

and Antarctic Oceans, J. Oceanograph. Soc . Japan. 38, 137-148.

142
SECTION 15

WILDLIFE AS AN INDICATOR FOR ENVIRONMENTAL CONTAMINANTS

IN MONITORING SYSTEMS

Jasper Holm

Staatliches Veterin^^runtersuchungsamt

D-3300 Braunschweig

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

The government Veterinary Research Institute in Braunschweig is

responsible for the hygienic control in foods of animal origin. The controlled

area, in the east of Lower Saxony, comprises extensive agriculture, integrated

heavy industry as well as the mining of toxic metals, and includes various

power stations. Thus, the environmental chemicals such as the toxic metals and

organochlorine residues in the human food chain are of great importance.

Within the last 10 years we have established an efficient cause-oriented

system for monitoring the contaminants, using wildlife to indicate the residues, in the terrestrial ecosystems where also the production areas for human

consumption are to be found. The advantages of this indication method are as follows

1. Wildlife are directly confronted in their natural habitat with single

or multiple pollutants all the year round. Therefore, the accumulation of different pollutants may follow the emission levels of their

home ranges.

2. Wildlife seem to have a higher degree of contamination than farm

animals [1-3]. Regional differences, therefore, can be observed more

easily. The food chain of wildlife and the ruminants may have

parallels thus giving us some information about the contamination

degree in cattle and the human food chain.

II. ACTIVITIES OF THE FIRST RESEARCH PROGRAM

As a first step a research program was carried out, sponsored by the

Ministry of Public Health in Bonn, where the levels of contamination in game

had to be determined. Five differently structured regions, each with an area

of 1500 km^ , were selected in the east of Lower Saxony where different levels

of contamination could be expected. The regional classification was programmed

on the basis of previous data obtained from other indicators (fish and cattle).

In Table 1 the influence of contamination to be expected is outlined. These

areas are outlined on the map shown in Figure 1.

The selection of the indicators (roe deer, red deer, wild boar, hare,

nearly 1500 animals) used in this study was made in accordance with different

criteria (distribution, living area, biotop, nutrition) as summarized in

143
Table 1 . Different Contaminations^ to be Expected in 5 Regions in the East of Lower Saxony

REGION CONTAMINATION EMISSIONS AGRICULTURE

1 . Luchow-Dannenberg Low No Industries

Low Traffic

Grain and Cattle

Production

Forestry

2. Braunschweig

3. Gottingen

4. Helmstedt - Wolfsburg

Middle Industry and

Traffic Emissions

Grain and Cattle

Production

Forestry

5. Goslar - Osterode High Mining- Indus try

Emissions

Forestry

Cattle

Heavy Metals Pb and Cd

Table 2. Questionnaires were sent out to the hunters asking for details of

animals killed (species, sex, date killed, age, and weight) and for information

Table 2. Criteria for Bioindicators in Monitoring Studies

CRITERIA ROE DEER RED DEER WILD BOAR HARE

Capreolus

Capreolus L.

Cervus

Elaphus L.

Sus

Sorofa L.

Lepus

Europaeus P

West

Distribution Europe

Near East

Iran

West, South

East

Europe, Asia

North Africa

Eurasia West

Europe To

Ural

Living

Area

9 25 - 30 HA

O 25 - 40 HA

5 Km and

more

To 250 HA 9 4 - 10 HA

30 - 38 HA

FOREST FOREST FOREST AGRICULTURAL

Biotop Agricultural

Area

Agricultural

Area

Area

Forest

Nutrition Herbivore

Selecting

Concentrates

Herbivore

Prefering

Roughages

Omnivore Herbivore

Selecting

Green

Grasses

According to Ellenberg (1977), Fradrich und Fradrich (1973),

Hofmann (1978), Krautforst (1975), Meynhardt (1978),

Muller (1980), Raesfeld und Vorreyer (1978),

Schneider (1978)

144 I

I,
Figure 1. Regions for monitoring activities with wildlife in the

east of Lower Saxony

145
on the vegetation and other parameters of the region of origin. The analysis

data (20,000 for lead, cadmium, mercury, and arsenic from liver, kidney,

muscle, and hair) and 8,000 for the chlorinated hydrocarbons (body fat) were stored with the questionnaire data in an IBM 4341-2 computer with an SPSS

statistical packet (Rel 9) [4] . The results of this study have been reported

previously [5,6] and are described briefly below.

1. The lead content increases from roe deer, red deer, wild boar to hare.

Regional differences can be seen from 1 : 3 for roe and 1 : 12 for

hare in low, middle, and high contaminated areas. The percentage

frequency distribution of the lead content in liver confirms the

results as shown in Figure 2.

2. The cadmium content in the kidney is 57 times higher than in the

liver. The lowest cadmium content can be found in red deer (see

Figure 3). The hare, especially, demonstrates very high differences

of the regional cadmium contamination. Mercury contamination is

fairly high in wild boar and hare. Regional contaminations can be

observed in areas of high agricultural production.

3. The regional differences of chlorinated hydrocarbons are fairly

restricted (see Figures 4 and 5). Wild boar, in particular, have a somewhat higher DDT content (sum frequency function of random sample

values for DDT) , while hare have a lower PCB content in their body fat

(Sum frequency function of random sample values for PCB)

Kleminger [7] defined the bioindicators in more detail in this project.

He suggested selecting roe deer and hare for bioindication . Hares showed an excellent accumulation especially for toxic metals, but unfortunately they have

disappeared in many ecosystems in Europe. Therefore, we decided to give roe deer the preference within the activities of the Environmental Specimen Bank

[3]. The advantages are their widespread distribution, their high population

densities, their easy availability, the relatively small home ranges they live

in, and finally roe deer serve as man's food. During the hunting season of

1983/84 nearly 700,000 roe deer were killed in Germany.

When we calculated the residue contents of different wildlife species from

the point of view of their influencing parameters (age, area of origin, etc.), we observed certain difficulties in the data set having a too high range of

contamination. An example is shown in Figure 6 which shows the toxic metal

contents in liver and kidney of roe deer in different areas of Lower Saxony.

Figure 6 summarizes the lead and cadmium content of liver and kidney in

different age groups in areas with low, middle and high contamination.

Although we know that animals from areas with high contamination should also

have the highest cadmium contents, the diagram indicates otherwise. One reason

is the different numbers within the age groups, so that the age parameter

overlaps the region of origin parameter.

146
groups With

low and middle contaminati on

groups with

high contamination

50 , iO

30

20

.

10

0 J

PL Toe deer

<d 0,2 O.U 0,6 0,8 I0ii ^9

50-,

1.0

30

20\

10

0

m'/o 50 ,

^ 0

30

20 .

10

0 J

red deer n= 31

<d 0.2 0,i 0.6 0,8 /,0>/

Wild boar

<d 0.2 O.i, O.b Q8 7,0>? ^

9 0.

8 0

30.

20.

10

0 .

hare

n-3£,3

<d 02 O.U 06 08 1,0>1

n= Hi,

-< 4=1

' ' I—1—I—I—(—1

\—' I'^gy <d 0,2 O.U 0,6 0.8 1,0 >1 ^9

n-- 187

Cd 0,2 O.U 0,6 0,8 10>1 kg

-I*—1—1—S—I—

r

0= 26

1^9/, <d 0.2 0^ 0.6 0,8 ',0>1 ^9

'n^ 17

<d 0,2 0,U 0,6 0,8 W>1 ^9

d - detect I 0 n limit

mean value m mny wet weight

kg Figure 2. Percentage frequency distribution of lead content in

liver of various wildlife

147
groups with

low and middle contamination

groups with

high contammat ion

in°/o 50.

i.0-

solo -

70 J

0

I n%

50 T 40.

30.

20 .

10 .

0 .

I r 50

iO .

30 .

20.

W

0 .

roe deer

n = 59d

<d

r—l ""9/

0.2 OA 05 0 8 W>1 ^9

n= 3 1 red deer

-fi^T—I

i—I

I—I—t—I—I—I—I ^9/

<d 0.2 0,L 0.6 0.8 10>1 l<g

wild boar

150

-^Z

d 0.2 0,U 0,6 0,8 10>1 ^

I n %

50-

LO -

30 A

20

10

0 J

hare

n= 3£. 3

9/, <d 0,2 0^ 0,6 0.8 / 0 > ' ^5

n = 1 lit

I I I I I » I I t t rngy <d 0.2 0,U OS 0.8 1,0 >l l<g

n 1

I I I ! I 1 ( I P aI I d 0,2 O.i, 0.6 08 i,0>l >'9

n= 26

1—I—I—I—I—i f"^/, d 0.2 Q,i, 0,6 0,8 1.0>!

n-- 17

<d 2 i> 6 8 w>W i<g

d = detec f I 0 n limit

mean ^lue m rng, wet weight

kg

Figure 3. Percentage frequency distribution of cadmium content in

liver of various wildlife

148
Figure 4. Sum frequency function of random sample values for

DDT in various wildlife

149
700.

7 0,

0,001

I 1

.

I I

1 I 0,00 5 0,0 7

I

' 0,0 5 ni—

0, /

roe c/eer

red deer

wild boar

hare

0, 5 7,0

T r _, , mgy fat

' Ug

5.0

Figure 5. Svun frequency function of random sample values for

PCB's in various wildlife

150
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151
III. WORKING MODEL FOR THE BIOINDICATOR ROE IN THE ENVIRONMENTAL SPECIMEN BANK

A. Sampling

The reasons for taking roe into the Specimen Bank have been outlined.

Since the areas we took the samples from within the first research program were relatively large, we now decided to limit the areas to three forestries having

a size of only 8,000 hectare each. In addition, the age was limited by

choosing yearlings which represent the largest group within a hunting season.

These decisions were supposed to limit the influence of the parameters "region"

and "age" so as to enable us to observe the contamination range of the indicator more exactly. Thus, we hope to stabilize the indicator for trend analyses

in the Specimen Bank.

One main problem to be solved is the correct sampling of organs for the

Specimen Bank. Since the chlorinated hydrocarbons were taken on a priority

list of studies for the Specimen Bank, the question of organotropy should be

solved for these contaminants. Of further interest are also the toxic metals

and other relevant elements. In order to establish the best accumulation, we chose the liver, kidney and brain, which, along with the body fat, will be

checked for a period of two years in the three forestries involved.

The hunters were trained to collect liver and kidney samples not

contaminated by shot, vegetation, or intestinal injury. The samples are collected at the forestry stations in freeze packs and the fresh material is

sent immediately to the Veterinary Institute in Braunschweig. After the organs

have been inspected and prepared they are frozen to a temperature of -80 °C.

The liver is subdivided for pesticide and metal analyses, and the necessary

amounts are reserved for the Specimen Bank in Jililich. The right kidney is

reserved totally for Jtilich, where already more than 130 organs have been

collected and deep frozen.

The analysis work is carried out after homogenization. A sophisticated

clean up of the samples is used prior to analysis by capillary gas chromatography. The determination of 15 elements is performed by inductively coupled

plasma (ICP) spectroscopy after wet digestion.

B. Chlorinated Hydrocarbons

The mean values for chlorinated hydrocarbons in 19 yearlings from the

forestry in Braunschweig are listed in Table 3. We observed a significantly

higher contamination with chlorinated hydrocarbons in the liver than in the

kidney. Furthermore, the range of contamination seems to be rather narrow. The main CH components to be found in the liver are a-BHC, ^-BHC, r-BHC and

4,4' -DDE. The FCB's are mentioned in Table 3 that now are controlled in the

human food chain. We observed significantly higher contents for PCB 52, 138,

153 compared with the other components.

152
Table 3. Chlorinated Hydrocarbons® in Organs of One Year Old

Roebucks from the Forest Near Braunschweig

Liver Kidney

n n 1 nvi'nj^t'AH

M© 3.11 nean O L.LL . L/C V .

a-BHC 0 . 304 0 24 V_/ . *J Z. _J 0 01

0 . 245 0.18 0 . 130 0 . 05

7-BHC 0 . 086 0 . 04 0 . 100 0 09

(7-BHC 0 . 024 0 . 02 ND

HCB 0 . 054 0 . 04 0 . 033 0 . 02

4,4' -DDE 0 . 154 0.17 0 . 038 0 03

4,4' -DDD 0 . 061 0 . 03 ND

4,4' -DDT 0 . 014 0 . 01 ND

Hep tachlor ND ND

IN U ViiJ ocs 0.009 0.02 ND

PCB 28 0.018 0.02 0.022 0.02

PCB 52 0.035 0.03 0.041 0.02

PCB 101 0.013 0.01 ND

PCB 138 0.073 0.04 0.024 0.01

PCB 153 0.076 0.04 0.025 0.01

PCB 180 0.030 0.02 0.017 0.01

®Mean values in mg/kg fat, N = 19

ND = Not Detectable

C . Trace Elements

The trace element determination by ICP created difficulties since the

detection limits of some elements are high. Therefore, the ICP method for

element determination in mammals is also criticized, because the contents are partly too low. In the last two years, we have developed a digestion method

with high tissue concentration and a suitable aspirator.

In Table 4 the elements and their limits of detection are summarized.

From Ca to Zn the concentration in liver and kidney in roebucks is well over

the detection limit; however, from Al to V the determination may become

difficult, and Pb , Sb , Tl are not detectable by this method. Liver and kidney

seem to have different abilities for accumulating the elements.

IV. CONCLUSION

The results demonstrate that the liver might be the organ from roe deer to

be sampled and stored in the Specimen Bank. The organ offers enough material

for multiple analysis of different contaminants. The distribution of the

elements and the chlorinated hydrocarbons is very homogeneous, so that

subsampling of this organ is possible. The accumulation of the contaminants

seems to be higher than in the kidney.

153
Table 4. Limits of Detection for Elements Determined by TCP

Element Limit of Detection mg/L Content in Liver^ Content in Kidney^

Ca 0.,050 39.2 56.,5

Cd 0.,005 0. 181 0,,895

Cu 0.,050 11.6 8..94

0 090 1 20 • -J Mg 0.,010 163 143

Mn 0,,002 4.66 1

.73

Zn 0,,050 29.5 36..7

Al 0..200 1.32 1

.92

Or 0,.010 0.332 0..251

Mo 0..020 0.559 0,. 126

Ni 0,.020 0.034 0 .063

V 0 .010 0.063 0 .046

Pb 0 .030 ND+ + ND

Sb 0 .050 ND ND

Tl 0 .030 ND ND

^Mean values in mg/kg freshweight of one year old roebucks (N = 19)

^ND = Not Detectable

V. REFERENCES

[1] Hecht, H. (1986), Unterschiede im Schwermetallgehalt bei Haus- und

Wildschweinen und ihre Ursachen, Mitteilungsblatt der Bimdesanstalt fur

Fleischforschung Nr. 92, 6962-6970.

[2] Holm, J. (1985), Uberwachungsmodelle fur Umweltkontaminanten in Form von

Monitoringsystemen, Fleischwirtsch. 65, 1405-1407.

[3] Mtiller, P. (1985), Cadmium-Konzentrationen bei Rehpopulationen (Capreolus

capreolus) und deren Futterpflanzen, Zeitschrift fur Jagdwissenschaft 31,

146-153.

[4] Holm, J. (1983), Aufbau eines ursachenorientierten Monitoring-Systems fiir Schadstoffbelastungen beim Wild, Problemstellung und Aufbau des MonitoringSystems, Fleischwirtsch. 63, 1764-1766.

[5] Holm, J. (1984), Belastungen von Wild mit Schwermetallen aus unterschiedlich strukturierten Herkunftsgebieten, Fleischwirtsch. 64,

613-619.

[6] Holm, J. (1984), Belastungen von Wild mit chlorierten Kohlenwasserstoffen

aus unterschiedlich strukturierten Herkunftsregionen, Fleischwirtsch. 64,

970-973.

154
Kleiminger J. (1983), Untersuchungen uber die Eignung von freilebenden

Wildarten als Bioindikatoren zur Erfassung von f lachenhaften Schwermetallkontaminationen in Niedersachsen , Hannover vet. med. Diss.

Kleiminger J. and Holm, J. (1985), Aufbau eines ursachenor ientierten

Monitoring-Systems fiir Schadstoffbelastungen beim Wild, Wahl eines

geeigneten Bioindikators fur die Anzeige von Schadstoffen , Fleischwirtsch.

65, 394-399.

155
SECTION 16

REGIONAL MONITORING OF POLLUTANTS WITH HONEY BEES

Jerry J . Bromenshenk

Department of Zoology

University of Montana

Missoula, Montana 59812

USA

I . INTRODUCTION

Identification and mitigation of problems associated with hazardous

chemicals is a worldwide concern. Vast quantities of toxic chemicals are produced, transported, and used throughout the world. There are opportunities for

entry of these materials into the environment at each stage in their existence.

Anthropogenic materials are continually moved across natural and political

boundaries by man, or by physical and biological processes [1]. Contaminants

end up in air, soil, water, and eventually in living organisms. Monitoring

programs are needed to detect serious environmental contamination before

critical or irreversible damage has been sustained [2].

Effective monitoring of this myriad of substances is confounded by our lack of knowledge about the potential hazards. Often, we are unable to

predicate the consequences of chronic exposure to mixtures of chemicals

understand mechanisms of action, identify indirect as well as direct effects,

or determine the assimilative capacity of ecosystems [3].

We can detect incredibly small quantities of many substances, but their

hazard to man, other organisms, and ecosystems is largely unknown. Improved

measurement procedures do not necessarily increase our ability to extrapolate

to biological effects. Environmental protection is a biologically oriented

goal: the protection of life. Attainment of that objective can only be

determined by assessing biological parameters [4] . Although considerable

research has been conducted to develop biologically oriented monitoring

capability, only a few examples of dedicated ongoing biomonitoring programs

exist, and few have any legal status.

II. BIOMONITORING AND SPECIMEN BANKING

We define biomonitoring as any biologically based measurement of

environmental quality. Bioindicators like the miner's canary, some lichens,

and certain aquatic invertebrates, generally display rapid, unambiguous

responses such as presence or absence, avoidance, or death. Usually, indicator

organisms are particularly sensitive to changes in environmental conditions.

They often are not well suited for long-term monitoring. For example, the

death of the miner's canary provides an immediate warning of toxic gases but

yields no further information.

156
Ideally, a monitor organism, unlike an indicator, should be relatively

hardy so that responses can be assessed over extended periods of time. In

addition, a biomonitor should possess one or more readily quantified parameters

that can be related to pollution exposure levels and any environmental effects.

Specimen banking provides extended monitoring capability over time.

Deferred analysis provides a means of retrospectively examining environmental

problems as we become aware of them or as new techniques are made available for

investigation [5]. Specimen banking often emphasizes sampling and analyses of

environmental scavengers, organisms that accumulate or concentrate chemicals.

This perspective turns our attention to biological receptors and essential

issues such as bioavailability and bioeffects, rather than merely quantifying

amounts of specific contaminants in air, soil, or water.

As specimen banking moves from pilot phases to established programs,

fiscal commitments must be made, since most specimen banks rely upon cold

storage facilities. Loss of adequate funding would jeopardize current

activities and all preceding efforts. Maintenance of cold storage places a high priority on long-term undertakings.

Real-time biomonitoring programs in the United States, and presumably

other countries, are frequently subjected to unpredictable fluctuations in

funding, justification being that sampling efforts can be temporarily reduced

or suspended. This on-off approach has seriously hampered development of

biomonitoring techniques, establishment of long-term monitoring programs, and

our ability to follow environmental trends.

Protection of human health and the environment requires not only a commitment to monitoring and banking programs , but also requires further

chemical, toxicological and ecological research, development of control

technologies, hazard assessments, improved data management, and international

cooperation [1-3]. Unfortunately, the cost of these activities precludes

testing and monitoring ail potentially toxic chemicals [1].

This underscores the importance of specimen selection and the need to link

specimen banking and real-time monitoring, especially if we bank materials to assess potential environmental problems that may transpire in the future. Our

ability to store samples is limited. Materials selected for banking should be

indicative of properties valued by humans, especially those pertaining to

welfare and health. Samples must be ecologically and chemically meaningful as well as representative of environmental conditions [1-3,5].

These issues received considerable attention at the 1978 and 1982

international workshops on banking and environmental monitoring. A variety of

organisms were recommended for environmental monitoring. The actual species or set of species selected is dependent on monitoring objectives, the

aforementioned issues, and site-specific considerations [2,3].

157
III. HONEY BEES AND REAL-TIME MONITORS

Following the 1978 international workshop, the U.S. Environmental

Protection Agency (EPA) sponsored a pilot investigation of the feasibility of

using honey bees (Apis mellifera) for monitoring and banking. The pilot study

built upon work initiated in 1974 at the University of Montana (UM) . In 1982,

investigators from the quantitative ecology and statistics divisions of Pacific

Northwest Laboratories (PNL) joined the UM and the EPA in a cooperative

research effort. By 1985, we were using honey bees in application modes to

determine exposure levels, identify sources, and map the distribution of trace

elements and radionuclides.

Beekeepers have long associated bee mortalities with exposure to

industrial effluents. One of the earliest damage awards resulted from a 1912

legal suit filed by a beekeeper against a lead and silver work located on the

Rhein [6]. In 1972, Debackere [7] noted that honey bees could serve as pollution indicators in a review of air pollution, bee mortalities, toxicity

data, and transfer of various industrial chemicals to pollen, nectar, water,

and bees. Shortly thereafter, studies appeared that focused on developing the

biomonitoring potential of bees [8-48].

A bee colony contains thousands of forager bees. They forage relatively

large areas (7 km^ or more, assuming they fly 1.5 km from the hive). During

each flight, a bee may sample up to 350 flowers and may undertake more than 100

trips per day [49]. Thus, it is not surprising that bees provide spatially

integrated and averaged samples of their forage area [11,31,41,46,47].

Honey bees return to their hives with nectar, honeydew, pollen, resin, and

water. They sometimes 'mistake' dusts and powders for pollen [10] and caulking

materials for resin [50]. Their bodies may be contaminated by air pollutants

while flying or by contacting dusty surfaces. Electrostatic charges on the

surface of the bee may play a role in the gathering of small particulates [47].

Pollutant uptake into body tissues may occur from ingestion, inhalation, or absorption through the exoskeleton [7]. Contamination of nectar and pollen may occur via direct contact with chemicals in the ambient air, from wind resus- pended soils, or by transfer through plant tissues from soil or water. Control

of hive temperature and humidity by fanning to promote air circulation and the

spreading and evaporation of water constitutes other potential mechanisms for

the introduction of contaminants into bee systems [7,10,31,41,46,47]. Bees

encounter contaminants in or on air, water, soil, or plants, effectively

providing multi-media monitoring [46,47].

IV. MONITORING POLLUTANTS BY RESIDUES IN BEES AND BEE PRODUCTS

Extensive studies have been made on pesticides and their effects on bees.

A complete review of the pesticide literature is beyond the scope of this

paper. The Agricultural Act of 1970 authorized the U.S. federal government to

indemnify bee losses due to the use of registered and approved pesticides.

Documentation of losses required proof of pesticide exposure, usually based on chemical analysis of dead and dying bees [49] . Although this program no longer

exists, it generated well-developed procedures for determining pesticide

158
residues in bee tissues as well as other indicators of poisoning such as reduced brain cholinesterase levels [51].

A three year Italian study combined bee mortality data analysis of

pesticide residues to identify pesticide pollution in the province of Forli.

In orchards where bee mortalities were highest, bees, larvae, and honey

contained 17.0, 1.64, and 1.68 ppm of dithiocarbamates , respectively, as well

as residues of DDT/DDE and organophosphates [33].

Pesticides can be monitored with bees even when dosages are below toxic

levels. In eastern Montana, we found trace levels of the DDT/DDD/DDE

chlorinated hydrocarbon complex in forager bees collected live at all 14

apiaries sampled in 1975 and 7 of 11 apiaries sampled in 1976 [18]. These

apiaries were located on rangeland near alfalfa fields where DDT application

had been banned ten years earlier.

In addition to pesticides, radioactive and trace elements, especially

heavy metals, have been monitored using bees, honey, or bee-collected pollen.

Investigators at Cornell University, USA [10] examined 47 elements in honey

bought from groceries and collected from bee colonies near underground zinc

mines, a major highway, and an industrial area in Niagara County, New York.

They found elevated levels of several chemicals from automobile exhausts and

industries. Honey processed in a galvanized metal extractor had elevated

levels of zinc, while high levels of tin were thought to have come from

processing or storage in metal containers. In another study, Cornell

researchers examined 16 elements in honeys from around the globe. Concentrations of iron varied from 5.8 to 183 ppm, zinc from 2.0 to 172 ppm, while

chromium and nickel levels paralleled the concentration of iron in some samples. Variations in elemental concentrations were thought to be a result

of leaching from metal containers, industrial pollution, or naturally occurring

variations in soil geochemistry [24]. In another study, they found that pollen

collected by bees foraging on asters growing on a New York fly ash dump

contained high concentrations of selenivim [14]. A post-closure investigation

of a nuclear reprocessing plant did not discern any measurable amounts of beta

or gamma emitting elements in honey, pollen, propolis, or water [19]. Nor

could any significant increase in gamma- radiation be detected in honey collected within 16 km of the Three-Mile Island nuclear power plant after an accidental release of radioactive materials in March 1979 [25].

For many years, honey bees have been utilized as a constant surveillance

monitoring system of radioactive waste disposal sites at Los Alamos National

Laboratory in New Mexico, USA. Hakonson and Bostick successfully used bees to

determine the bioavailability of radioactive tritium, 137-cesium, and plutonium. Concentrations of these radionuclides in bees closely followed fluctuations of radionuclides in other components of the surrounding environment.

Tritiated water was most readily transferred to hive components, including

honey, but there appeared to be discrimination against transfer of 137-cesium

to honey [12]. This research led to the establishment of a monitoring network

of bee colonies, which is maintained at potentially contaminated areas at the

Laboratory. As an example, honey samples from 1980 contained low levels of

7-beryllium, 137-cesium, tritium, and 22-sodium, and undetectable levels of

159
mercury, 238-plutoniuin, and 239-plutoniuin. One sample from a hive near a waste

stream outfall had detectable levels of uranium [31].

In a cage feeding experiment, uranium uptake by bees was investigated

using syrup spiked with an uranium tracer. Highest uranium concentrations were found in the bees themselves with lower levels in comb, larvae, and honey [26].

Another caged bee study examined tritium kinetics in honey bee colonies. Bees

were fed tritium and measurements made of rates of uptake and depuration. An

attempt was made to model the results and apply them to field situations. The

model was not satisfactory, probably because accurate predictions of stack

releases were impossible due to variable wind patterns in complex terrain and

time -dependent variations in tritium releases [39].

At Los Alamos, the highest concentrations of tritium, cesium, and uranium

occurred in the bees themselves. Investigators at the Oak Ridge National

Laboratory, Tennessee, USA, also placed bees near waste disposal sites. Bees

foraging for pollen or nectar showed little evidence of radioactive

contamination, but water brought back to the hive for evaporative cooling

provided a mechanism for radionuclide transfer to colony components [43].

Our ongoing research includes deploying bees at other USA facilities near radioactive waste disposal sites (in ponds or buried) , nuclear reprocessing

plants, and nuclear power plants. Our initial results indicate that bees can

be used to monitor an array of radionuclides, including 137-cesium, tritium,

60-cobalt, 51 -chromium, and 65-zinc. Significantly elevated levels of gamma

emitting radiation was observed in adult bees, but not necessarily pollen or brood. At one site, 137-cesium found in pollen was transported to hives by

pollen foraging bees, but not by nectar or water gatherers [51].

In Pennsylvania, USA, lead levels in honey bees were compared to those of

wildflowers frequented by the bees. Flowers from a site located along a highway contained an acreage of 13.6 ppm lead; flowers from the control site

0.2 ppm. Lead concentrations in bees were 28.1 ppm near the highway and 1.4

for the control site. Analysis of bee legs revealed concentrations of lead in excess of 50 ppm, which suggest pollen as a more likely route of accumulation

than nectar [30].

European researchers also have used bees to monitor inorganic elements.

Honey was obtained from 18 locations near Stolberg/Rhe inland, FRG , where the

air contains high concentrations of lead and cadmium. Honey samples had 0.016-

0.8 ppm lead and 0.009-0.125 ppm cadmium. A sample of pollen, one of beeswax

and two samples of propolis had considerably higher levels of lead and cadmium

than honey: 2.1 ppm lead, 0.322 ppm cadmium; 6.2 ppm lead, 0.103 ppm cadmium;

and 41 ppm lead, 0.327 ppm cadmium, respectively. A correlation was found

between lead and cadmium in honey and in dust (r=0.76, rsp. 0.50) [45]. In

West Berlin, a study of 2600 urban colonies was initiated to monitor heavy

metals. Lead concentrations in honey varied from 0.02 - 1.8 ppm [37]. A Dutch

investigation found wide variations in the lead and cadmium contents of honeys

attributable to species of plant foraged, geographic region, and whether honey

was derived from nectar or honeydew. Average lead and cadmium levels were somewhat higher for honeydew honey than floral honey: 0.89 ppm lead and 0.018

160
ppm cadmium versus 0.31 ppm lead and 0.014 ppm. A sample of honey from

California contained 0.76 ppm lead and 0.016 ppm cadmium [36].

In a Bulgarian study, pollen and honey collected near two copper mills

contained higher than normal levels of copper, zinc, and phosphorus. Hive

samples from various regions, including agricultural areas, also displayed

widely varying levels of manganese, cobalt, iron, sodium, calcium, and

potassium. Trace element content of pollen was 2 to 3 times higher than that

of honey [9]. In a Great Britain study of nineteenth century copper mining

region, bee-collected pollen contained elevated levels of manganese, zinc,

copper, and lead and reflected those of stream sediments. Using the published

data, we computed coefficients of variation (CV's) for within site variation

due to colony specific differences in metal concentrations. For duplicate

samples of pollen from six colonies per site, CV's ranged from 4.3 to 81.2%.

The authors concluded that differences in trace element uptake by each species

of plant would account for much of the variability [34].

A study conducted during summer and winter in an industrial region near Saarbrucken, FRO, examined the bees themselves, rather than pollen or honey.

Bees displayed levels of cadmium ranging from 0.4 to 3.1 ppm and lead from 2.6

to 69.1 ppm [35]

These findings are consistent with our own studies near smelters in

western Montana, coal-fired power plants in eastern Montana, in rural, urban,

and industrial zones of the Puget Sound region of Washington, and in agricultural and semi-arid desert regions of eastern Washington and central Idaho

[8,16-18,20,22,23,28,29,32,40,42,46-48]. We mapped widespread distribution

(over thousands of square kms) of arsenic, cadmium, and fluoride based on concentrations of contaminants in bees. We also detected widely varying levels

of lead, copper, zinc, nickel, titanium, strontium, bromine, iron, rhodium,

aluminum, and manganese. Levels of most of these chemicals in pollen approximated those in bees, except for fluoride which was much higher in bees.

Contaminant levels in pollen displayed greater temporal and colony specific

variability than concentrations in bees. CV's for arsenic and cadmium in bees

averaged about 20% and were dependent on time of year, proximity to source, and

other factors [47].

Several authors have suggested that bees concentrate or biomagnify certain

elements. This is clearly evident for air- or water-borne fluoride. Fluoride

levels in bees usually exceeded those of air, water, or pollen by one or more orders of magnitude [17-18,22,23,28,42,47]. In a study of an aluminum reduction facility, fluoride content of bees provided a means of statistically

distinguishing more differences in exposure levels among sites than did calcium

formate plates (static air monitors) . In addition, decreasing fluoride levels

in bees over a period of several years provided evidence that curtailment of

emissions as evidenced by improved air quality was mirrored by bees. Partial

correlation analysis suggested that the bees accumulated substantial amounts of

fluoride from the air, while pollen and plant blossoms (sources of nectar and

pollen) contributed considerably less fluoride than air [42].

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V. CHOICE OF COLONY COMPONENT FOR MONITORING

During our 12 years of research with honey bees, we have examined a wide

array of trace and radioactive elements in forager bees, nurse bees, pupae,

pollen, honey, and wax. We have chosen the forager bee as the primary sample

unit. Contaminant concentrations in forager bees tend to be equal to or greater than levels in nurse bees, pollen, or wax and usually are higher than

levels in honey. Forager bees are the oldest bees in a colony (longest

exposure time) , are easy to obtain, and provide a temporally and spatially

integrated sample of air, soil, water, and vegetation [47]. Pollutant concen- trations in bees display less within colony, between colony, and temporal

variability than levels in pollen or wax.

Other investigators have chosen honey for their primary sample unit,

probably due to the potential of ingestion of toxic chemicals by humans.

However, concentrations of toxic pollutants generally are lower in honey than

in bees or pollen and, to the best of our knowledge, reported values usually

are not thought to constitute a hazard to consumers.

Pollen constitutes another potential means of transfer of toxic chemicals

to human. In recent years, pollen has been marketed as a 'health food'. We

found that concentrations of toxic elements such as arsenic, cadmium, and lead

in pollen collected near smelters often exceeds safety standards for other food

products (unpublished data). However, most pollen sold in US stores is

collected from rural areas, few people eat pollen, and those who do usually

ingest small quantities. It seems unlikely that human exposures to toxic

substances in pollen would exceed acceptable health safety guidelines.

We have found bee-collected pollen useful for providing information about

levels of hazardous chemicals in or on plants. Combining this knowledge with

microscopic identification of plant species yields information about the

specific areas and plants foraged and helps pinpoint the locality of the

pollutant source.

Beeswax offers a possibility of providing a historic record of pollutant

exposure, since the comb in a hive may be many years old. We found that

concentrations of heavy metals varied widely in wax sampled from different

places within the same hive. The amounts appeared to be dependent on the age

of the wax, i.e., new wax contained lower levels than old. We are unaware of

any practical way of accurately determining the age (days-years) of wax in

existing hives

Propolis has seldom been analyzed for environmental contaminants. Altmann

[45] found 20 times more lead in propolis than in pollen and a 50-fold increase over honey, cadmium valves in propolis approximated those for pollen, but were higher than those for honey. Bees use propolis to seal and waterproof their

nest cavities or hives. It is made from saps and gums exuded by trees.

Propolis may have value for averaging samples from woody plants, but it is a difficult substance to collect and handle because of its adhesive nature.

Also, it is composed of an indeterminate number of substances and has no specific chemical formula [50].

162
VI. EXPOSURE AND EFFECTS MONITORING WITH HONEY BEES

Examining chemical residues in a biological receptor such as a honey bee

in order to detect and follow spatial and temporal trends in contaminant levels

is often referred to as exposure or concentration monitoring. Addressing the

consequences of exposures to these substances requires a different approach,

which we term effects monitoring. Ideally, effects monitoring can be linked

with exposure monitoring and correlated by dose response information. With

honey bees it is possible to combine these functions. The individual bee is

susceptible to hard such as poisoning and can be a sensitive indicator, while

the colony functions as a monitor, since it is remarkably adaptive and capable

of surviving all but the most severe pollution insults [46]. Effects monitoring can provide an early warning of pollutant induced perturbations and provides information about the nature and severity of the impacts to bees, to the

products they provide, and most importantly to the pollination of croplands and

natural ecosystems.

It is hoped that exposure and effects monitoring with bees can be used to

reveal the presence of chemicals detrimental to human health. In 1982, we were

the first to identify such an area. A commercial beekeeper reported severe bees losses near an inoperable copper smelter. The bees and pollen contained

extremely high levels of arsenic and other toxic metals. Four years later, a study by the EPA and the Center for Disease Control (CDC) found unacceptably

high levels of arsenic in the hair and urine of children living in the area foraged by the bees. Relocation of families to reduce health risk is now being

considered [46].

We have developed specialized procedures for exposure and effects

monitoring. A particularly promising technique is the use of mini-hives and

colonies. Each consists of a small box containing a queen and usually less

than 0.5 kg (4-5 thousand) bees. Mini-hives are easy to manage and transport

and are inexpensive. They are useful for effects monitoring because we are able to quickly and accurately make measurements that normally are time

consuming and difficult using full-size hives and colonies. For exposure

monitoring, mini-hives allow us to inexpensively fill voids in sampling grids,

provide additional replication, and place bees where risk of loss is high.

VII. EXPOSURE MONITORING PROTOCOLS

Well documented and standardized monitoring protocols are essential to

data quality and comparability. Slight changes in procedures may significantly

affect results. For example, we found that fluoride levels in bees vacuijimed

from frames inside the hive were approximately one-half those of bees collected

at the hive entrance [18]. What constitutes a representative sample varies

with the chemicals of interest and component examined. As few as 25 bees may

be adequate to provide a representative sample for heavy metals in a colony.

On the other hand, a study conducted near a nuclear power plant indicated that

only a few in several hundred pollen pellets contained detectable amounts of

cesium. The non-uniform distribution of cesium was thought to reflect individual bee foraging patterns relative to localized sources [personal

communication, L. Cadwell, Pacific Northwest Laboratories].

163
Avoiding inadvertent contamination of field collected samples requires

specialized procedures. We clean the front of the hive, block the entrance

with a polycarbonate shield or fiberglass screen, and aspirate incoming bees

directly into sample storage bags. Pollen is collected by entrance mounted

traps, made of relatively inert materials such as polycarbonate plastic, teflon

or stainless steel. Traps are precleaned, attached to hives, and left in place

for 4-6 hours. Pollen is poured from the traps directly into storage bags.

Bee and pollen samples are quick frozen in the field. All sampling equipment

is acid washed and kept in dust-proof containers until use. Sample storage

bags are presealed with a perforated closure that is torn from the bag at the

time of sampling. After sampling, the bags are immediately resealed.

Chain of custody is documented by a permanent identification number on each bag, a sample label card which is completed when the sample is collected,

and field and laboratory chain-of -custody logs containing serially numbered,

tracking sheets.

In the laboratory, samples are freeze or oven-dried (45 °C) and ground or cryogenically fractionated into a fine powder. Preparation for analysis varies

according to chemicals of interest. For heavy metal analysis, we utilized

sealed-vessel, high pressure digestions in high purity nitric acid.

Accuracy, precision, and completeness of field and laboratory parameters

are addressed by statistical evaluation of a matrix of collated, quantifiable

parameters. Quality assurance is provided by adherence to written protocols

and corrective action as needed. Data quality criteria include replicate

samples, co-location of bee hives with conventional instrumentation such as high volume air samplers, field blanks, standardized siting, sampling, and

analysis, performance and systems audits, interfield and laboratory comparison

studies, internal and external quality control checks, control charts,

traceability of samples, standards, and instruments, regularly scheduled

preventative maintenance of facilities and equipment, certified reference

materials, standard additions, our own standard bee tissues, spiked samples,

consistency of reporting units (e.g., ppm dry weight), standardized data

formats, data analysis, validation, and reporting. Our procedures and results

from statistical assessments of data quality have been published [46-48].

Detailed protocols for field sampling, laboratory methods, and quality

assurance are available from the UM.

VIII. BEES AND SPATIAL MONITORS

To the best of our knowledge, every reported investigation of honey bees

as environmental monitors has concluded that bee colonies are well suited to

this task. Bees are particularly valuable as self-sustaining, multi-media

samplers of large geographical areas. We know of no other way of costeffectively acquiring such spatially integrated environmental samplers.

To fully realize this potential, we need to do more the merely report

concentrations of specific chemicals in bees or bee collected materials. We

have found a relatively new statistical approach called kriging [47] to be

useful for mapping pollutant distributions. Kriging uses a weighted moving

164
average technique in which point estimates or block averages are calculated for

a specified grid. Factors such as proximity of observations to points or areas

of interest, the "structure" of observations, and systematic drift or trends

are taken into account. Two and three-dimensional contour maps can be derived

from grid estimates, while confidence bands for individual isopleths can be

obtained from the collection confidence intervals for kriging estimates [47].

Using this technique, we were able to map widespread distributions over thousands of km^ for many elements. Our kriging maps generally show dispersion

patterns similar to isopleth maps derived from modeling of soil or air concen- trations, but reveal discernible patterns over larger areas, implying longerrange transport. We also have been able to identify pollution "hot spots" and can partition chemicals to different point sources.

Combining kriging with preliminary observations can yield exact estimates

of the needed sampling intensity for low or high resolution monitoring for

spatial pattern. Factors needed are block size estimates, grid spacing, and

the size of the multiplicative standard deviation (because data is log transformed) . Interrelating these factors with the variagram model enables us to

predict the outcome of various proposed sampling scenarios and to recommend

site specific sampling strategies (high spacing and numbers)

Sampling intensity (space between hive locations) is dependent on the

purpose of the monitoring and the desired degree of definition of spatial

patterns. Sampling intensity also depends on expected values for contaminants.

Greatest accuracy is usually desired in high impact areas where high and

rapidly changing concentrations of pollutants engender regulatory, health, and

environmental concern. In contrast, for routine screening of areas where

relatively low contaminant values are expected, low resolution and relatively

high percentage errors data still provides informative maps. Hive spacing can

be far apart where low resolution data with a relatively high percentage of error is acceptable. For highest resolution, we recommend at least one hive in

each unit of foraging range, approximately 7 km^ throughout the area of

interest

IX. COMMENTS AND CONCLUSIONS

Honey bees of the same species. Apis mellifera, thrive as managed colonies

or as wild colonies throughout the world. Beekeeping is practiced over a greater portion of the earth's surface than any other branch agriculture, and

other types of agriculture depend on bees. Bees are not only kept in rural and

agricultural areas, but also in cities. Highest honey yields are obtained from

Australia, North America, and parts of South America; whereas 83% of the hives

and 90% of world's beekeepers are found in Europe, Asia, and Africa. Worldwide, approximately 52 million colonies of bees are kept in nearly all areas where people reside [49-50]. This provides an in-place, potential global

monitoring network.

Moreover, our experience indicates that this form of monitoring is less

expensive than most other approaches. From a relatively few sample locations,

it is possible to use bees to search for and map environmental contaminants

165
over large geographical areas with a reliable degree of resolution. Since bees

gather materials over a range of several kilometers, hive placement is less

critical than, for example, a high volume air sampler, which requires electrical power and should be a specified distance above the ground and away from

buildings or trees. Furthermore, sampling can be successfully performed by

beekeepers. Thus, we believe that this monitoring approach could be employed

even in countries or regions lacking electrical power or access to

sophisticated monitoring instruments.

To determine how bees can most effectively contribute to monitoring needs,

particularly in terms of integrating information obtained with decision-making

and regulatory processes will require continued investigation. However, it

should be apparent that the function of bees as self-sustaining, multi-media

spatial monitors ideally complements the temporal advantages of specimen

banking. Combining a powerful, real-time, spatial analysis monitor with longterm temporal analysis capability should maximize information return. Thus, we believe honey bees should be seriously considered for incorporation into

specimen banking programs.

X. ACKNOWLEDGEMENTS

Our successful development of honey bees as environmental monitors has

been a cooperative research effort, funded primarily by the U.S. EPA, the U.S.

DOE, and the UM. Dr. John M. Thomas, PNL, was responsible for the statistical

design and evaluation of field and laboratory investigations. Dr. Eric M.

Preston, Project Officer, U.S. EPA, is credited with recognizing the significance of such a tool and laying the groundwork for this work. Janet L. Gudatis, Stan R. Carlson, and Mark L. Dewart were principal contributors to the

field and laboratory research at UM. Jeanne C. Simpson and Mary Ann Simmons,

PNL, conducted the kriging, which added a critical component of the sophistication of this approach. Finally, much of the work was performed with the

assistance of several hundred of the beekeepers of the Pacific Northwest

states

It is hoped that the work that has been carried out in many nations of the

world can be coordinated and intensified in the future.

The opinions expressed in this paper reflect the views of the author and

are not necessarily those of any sponsoring agency.

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166
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[6] Steche, W. (1975), Industrial Development and Its Effects on Beekeeping,

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[7] Debackere, M. (1972), Industrial Air Pollution and Apiculture, Vlamm,

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[8] Bromenshenk, J. J. and Carlson, C. E. (1975), Impact on Insect

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[9] Shabanov, M. and Ibrishimov, N. (1975), Assessment of Trace Elements in

the Environment on the Basis of Honeybee Foraging Activity, Acta

Microbiologica, Virologica, and Immunologica 21, 105-108.

[10] Tong, S. C, Morse, R. A., Bache , C. A., and Lisk, D. J. (1975), Elemental Analysis of Honey as an Indicator of Pollution, Arch. Environ. Health

30, 329-332.

[11] Toshkov, A. S., Shabanov, N. M. , and Ibrishimov, N. I. (1975), Attempts

to Use Bees to Prove Impurities in the Environment, C.R. Acad. Bulg. Sci.

27, 677-702.

[12] Hakonson, T. E. and Bostick, K. W. (1976), Availability of Environmental

Radioactivity to Honeybee Colonies at Los Alamos, J. Environ. Qual. 5(3),

307-310.

[13] Bortitz, S. and Reuter, F. (1977), Untersuchungen uber den Fluorgehalt

von Bluten in Bebieten mit bienengefahrdenden Immissionen, Archiv fur

Gartenbau 25(5) , 247-255.

[14] De Jong, D., Morse, R. A., Gutenmann, W. H., and Lisk, D. J. (1977),

Selenium in Pollen Gathered by Bees Foraging on Fly Ash-Grown Plants,

Bull, of Envrion. Contamin. Toxicol. 18(4), 442-444.

[15] Kirkham, M. E. and Corey, J. C. (1977), Pollen as Indicator of

Radionuclide Pollution, J. Nuclear Agricult. Biol. 6(3), 71-74.

167
[16] Bromenshenk, J. J. (1978), Yet Another Job for Busy Bees, The Sciences

18(6), 12-15.

[17] Bromenshenk, J. J. and Gordon, C. C. (1978), Terrestrial Insects Sense

Air Pollution, In: Proceedings of the Fourth Joint Conference on Sensing

of Environmental Pollutants, November 6-11, 1977, New Orleans, Louisiana,

American Chemical Society, Washington, DC , 5 pp

[18] Bromenshenk, J. J. (1978), Investigation of the Impact of Coal-Fired

Power Plant Emissions Upon Insects, Entomological Studies in the Vicinity

of Co Is trip, Montana, In: The Bioenvironmental Impact of a Coal -Fired

Power Plant, Third Interim Report, Colstrip, Montana, December, 1978,

U.S. Environmental Protection Agency, Corvallis Environmental Research

Laboratory, Corvallis, Oregon, EPA-600/3-78-021 : 140-212.

[19] Gilbert, M. D. and Lisk, D. J. (1978), Honey as an Environmental

Indicator of Radionuclide Contamination, Bull. Environ. Contamin.

Toxicol. 19(11), 32-34.

[20] Bromenshenk, J. J. (1979), Monitoring Environmental Materials and

Specimen Banking Using Terrestrial Insects with Particular Reference to

Inorganic Substances and Pesticides, In: Monitoring Environmental

materials and Specimen Bailing, N.-P. Luepke , Ed., Martinus Nijhoff

Publishers, The Hague, pp 132-155.

[21] Canteneur, R. (1978), L'Abeille et son Utilitie dans la Detection Des

Pollutions, La Sante de L'Abeille, December, 159-163.

[22] Bromenshenk, J. J. (1979), Honey Bees and Other Insects as Indicators of

Pollution Impact from the Colstrip Power Plants, In: The Bioenvironmental Impact of a Coal-Fired Power Plant, Fourth Interim Report,

Colstrip, Montana, December 1978, E.M. Preston and T.L. Gullet, Eds.,

U.S. Environmental Protection Agency, Corvallis Environmental Research

Laboratory, Corvallis, Oregon, EPA-600/3-79-044 : 215-239.

[23] Bromenshenk, J. J. (1980), Accumulation and Transfer of Fluoride and

Other Trace Elements in Honey Bees Near the Colstrip Power Plants, In: The Bioenvironmental Impact of a Coal-Fired Power Plant, Fifth Interim

Report, Colstrip, Montana, April 1980, E.M. Preston and D.W. O'Guinn,

Eds., U.S. Environmental Protection Agency, Corvallis Environmental

Research Laboratory, Corvallis, Oregon, EPA- 600/3 - 80-052 : 72-95.

[24] Morse, R. A. and Lisk, D. J. (1980), Elemental Analysis of Honeys from

Several Nations, American Bee Journal, 522-523.

[25] Morse, R. A., Van Campen, D. R. , Gutenmann, W. H., Lisk, W. H., and

Collision, C. (1980), Analysis of Radioactivity in Honeys Produced Near

Three-Mile Island Nuclear Power Plant, Nutrition Reports International

22, 319-321.

168
[26] Gladney, M. D., Curtis, D. B. , Perrin, D. R. , Owens, J. W. , and Goode , W.

E. (1980), Nuclear Techniques for the Chemical Analysis of Environmental

Materials, LA-8192-MS Informal Report, Los Alamos National Laboratory.

[27] Romanow, L. R. (1980), Effects of an Air Pollutant, Solid Rocket Fuel

Exhaust on the Honey Bee Apis mellifera and Other Insects, MS Thesis,

North Carolina State University, Raleigh, North Carolina, 78 pp.

[28] Bromenshenk, J. J. (1981), Fluoride and Arsenic Concentrations in Honey

Bees Near Colstrip, In: The Bioenvironmental Impact of a Coal-Fired Power

Plant, Sixth Interim Report, Colstrip, Montana, August 1980, E.M. Preston

and D.W. O'Guinn, Eds., U.S. Environmental Protection Agency, Corvallis

Environmental Research Laboratory, Corvallis, Oregon, EPA-600/3-81-007

210-227.

[29] Bromenshenk, J. J. and Rice, P. M. (1982), Air Pollutants and Plant

Fertilization, Proceedings of the Tenth Pollination Conference,

Carbondale, Illinois, 10, 95-101.

[30] Pratt, C. R. and Sikorski, R. S. (1982), Lead Content of Wild Flowers and

Honeybees Apis mellifera Along a Roadway, Possible Contamination of a Simple Food Chain, Proceedings of the Pennsylvania State Academy of

Sciences 56(2), 151-152.

[31] Wallwork-Barber, M. K. , Ferenbaugh, R. W. , and Gladney, E. S. (1982), The

Use of Honey Bees as Monitors of Environmental Pollution, American Bee

Journal 12, 770-772.

[32] Bromenshenk, J. J., Dewart, M. L. , and Thomas, J. M. (1983), Pollution

Monitoring Using Networks of Honey Bees, VI Congress Mondial Pour La

Quality de L'Air, May 16-20, Paris, Presentation des Posters 6, 155-158.

[33] Celli, G. (1983), L'apa Come Insetto Test Delia Salute di un Territorio,

L'apicoltore Modern© 75, 133-140.

[34] Free, J. B., Williams, I. H. , Pinsent, R. J. F. H., Townshed, A., Basi,

M. S., and Graham, C.L. (1983), Using Foraging Honeybees to Sample an Area for Trace Metals, Environ. Intern. 9, 9-12.

[35] Hoffel, I. and Muller, P. (1983), Schwermetallruckstande in Honigbienen

(Apis mellifica L.) in Einem Okosystem (Saarbrucken) , Forxun StatdteHygiene 34, 191-193. *

[36] Kerkvliett, J. D. (1983), Lood-en Cadmiumgehaltes van Honig, Maandschr.

Bijent. 85 (10) , 251-253.

[37] Kulike, H. and Voget, M. (1983), Bienenhonig als biologischer Indikator

fur die Blei- und Cadmium- Immission aus der Luft , Allg. dt. Imkerzty

17(10), 323-324.

[38] Lilley, W. (1983), Bee Miners Join British Columbia Gold Hunt, American

Bee Journal 123(9), 635-637.

169
White, G. C, Hakonson, T. E., and Bostick, K. W. (1983), Fitting a Model

of Tritium Uptake by Honey Bees to Data, Ecological Modeling 18, 241.

Bromenshenk, J. J., Thomas, J. M. , and Simpson, J. C. (1984),

Biomonitoring Using Honeybees, Battelle Pacific Northwest Laboratories,

Annual Report for 1983 on Interagency and Other Research, p. 20-21.

Crane, E. (1984), Bees, Honey and Pollen as Indicators of Metals in the

Environment, Bee World 65(1), 47-49.

Dewart, M. L. (1984), Pollution Monitoring in the Flathead Valley with

Honey Bees, M. S. Thesis, University of Montana, Missoula, MT, 22 pp.

Eldridge, J. S., Stegnar, P., Cakes, T. W. , and Teasley, N. A., Jr.

(1984) , The Use of Bioindicator Organisms and Specialized Analysis

Techniques in a Program of Environmental Surveillance, Health Physics

47(1), 118-119.

Terzic, L. , Terzic, V., Krunic , M. , and Brajokovic, M. (1984), Honey Bee

Poisoning Caused by Arsenic from Copper Smelter Smoke, Acta Veterinaria,

Beograd 34(1) , 57-62.

Altmann, G. (1985), Beiastung von Blutenhonig mit Schwermetallen und Ihre

Herkunft, Apidologie 16(3), 197-198.

Bromenshenk, J. J. (1985), Monitoring Air Pollution, More Work for

Honeybees, Western Wildlands 11(3), 27-32.

Bromenshenk, J. J., Carlson, S. R. , Simpson, J. C, and Thomas, J. M.

(1985) , Pollution Monitoring in Puget Sound with Honey Bees, Science

227(4687), 632-634.

Bromenshenk, J. J. and Preston, E. M. (1986), Public Participation in

Environmental Monitoring, Environ. Monit. Assess. 6, 35-37.

Dadant and Sons (1975), The Hive and the Honey Bee, Revised Edition,

Journal Printing Company, Carthage, IL, 740 pp.

Morse, R. A. and Hooper, T. (1985), The Illustrated Encyclopedia of

Beekeeping, E. P. Dutton, Inc., New York, NY, 432 pp.

Barker, R. J., Lehner, Y. , and Kunzmann, M. R. (1978), The Constancy of

Cholinesterase Activity in Adult Honeybees, Journal of Apicultural

Research 17(4) , 173-175.

Cronn, R. C. and Bromenshenk, J. J. (1986), Monitoring Contaminants with

Honey Bees at the INEL, FY- 1986 Annual Report to the Idaho DOE Operations

Office, U.S. Department of Energy, Idaho Falls, Idaho, 7 pp.

170
SECTION 17

QUALITY ASSURANCE AS APPLIED TO LONG-TERM MONITORING OF CHEMICAL

RESIDUES AND SPECIMEN BANKING

R. Turle, R. J. Norstrom, and H. T. Won

Wildlife Toxicology and Suirveys Branch

Canadian Wildlife Service

Environment Canada

Ottawa, KIA 0E7

CANADA

I . INTRODUCTION

The main purpose of long-term monitoring of toxic chemical residues is to

establish changes in concentration with respect to time. The analytical

chemist may not be concerned with identifying the physical sources of the con- taminants or to interpret the trends in terms of biological effects , populations, etc., but the chemist is charged with producing analytical results that

are accurate, precise and comparable to previous results, that is, results

without bias with respect to the true value, however obtained. The reasons for

this are obvious; compounds may be mis- identified and trends in the levels of

contaminants may be mis-interpreted. Also, it may not be possible to see the

effects of regulatory action on chemicals entering the environment, if

imprecision masks changes in actual levels.

Because of this need to produce accurate data, the chemist is very

concerned with Quality Assurance (QA) and all that it entails. Two successful

aspects of the QA program of the Canadian Wildlife Service (CWS) long-term

monitoring studies have been the ability to retrieve samples from a specimen

bank and the use of reference materials. The retrieval of samples ensures that

the analyst can determine levels of compounds that he was unable to determine

in the past and, perhaps as importantly, ensures that changes in methods are not obscuring long-term trends. The establishment of reference materials

enables a direct comparison between methods, analysts and laboratories over a long time frame.

In this paper we discuss the changes in methodology for organochlorine

insecticides (DCs) and polychlorinated biphenyls (PCBs) over a 15 year period

and compare the results of the original analysis and the re-analysis of samples

retrieved from the CWS National Specimen Bank. The National Specimen Bank has

been described previously [1], and in another paper in this volume [2]. The

criteria used to assess results based on reference materials developed by CWS

will also be discussed.

II. METHODOLOGY CHANGES IN OC AND PCB ANALYSIS

In the 1960's, there was an increasing concern with the effects of

organochlorine insecticides in the environment and especially with their

effects on avian reproduction [3]. At that time, projects were in effect in

171
the Canadian Wildlife Service to measure the levels of the then conunonly used

organochlorine insecticides given in Table 1

Table 1. Pesticides Determined in Canadian Wildlife in 1968

lindane

heptachlor epoxide

kel thane

dieldrin

o,p' -DDT

me thoxychlor

tedion

heptachlor

aldrin

DDE

DDD

p,p' -DDT

endrin

To place the situation in its analytical context, we must examine the

methods in use at that time. The analytical technique of choice was the gas

chromatograph equipped with packed columns and electron capture detectors,

which were then exceedingly troublesome. In about late 1968, a concern was being expressed about the industrial chemicals known as PCBs . As a consequence

it was decided that some effort should be made to determine the levels of PCBs

in Canadian wildlife tissues [4] . Analysts of that era realized that these

mixtures could interfere with the analysis of DDT and its metabolites. Thus,

methods were developed to separate PCBs from the organochlorine pesticides.

In the first procedure used by CWS contractors [5], it is likely that any

PCBs would have interfered with the DDT determination, because the method

lacked a column clean-up and separation step. It was regarded as a major

innovation when it became physically possible to separate PCBs from DCs using a Florisil column [6]. This was achieved using hexane as an eluant to remove

PCBs and then followed by 20% ether :hexane to remove most of the OC pesticiderelated residues. Only DDE proved difficult to separate from PCBs. During the

following years, interest in PCBs remained high and resulted in considerable

improvement in analytical methodology as shown in Table 2. Also during this

period of time new analytes such as technical BHC (hexachlorocyclohexane

isomers) , mirex and chlordane-related compounds were added into the method.

Perhaps in looking back to 1969 we should realize how difficult it was to

quantitate PCBs from a chromatogram given the packed column technology of that

era. It was extremely difficult to decide which peaks to choose for quantitation and what they represented. Indeed, at that time little was known about

the composition of the individual Aroclors [4] . It is only recently that all

of the PCB congeners have been synthesized and the relative response factors on an electron capture detector determined [7]. Quantitation was then based on averaging peaks 8 and 10 of either Aroclor 1254, as shown in Figure 1, or Aroclor 1260 [8]. For most wildlife samples Aroclor 1260 was used for compari- son as this appeared to be the best visual match of peaks. This was found to

be as accurate as averaging all major peaks. Peaks 8 and 10 turned out to be

good choices, since they were composed mainly of 2 , 4 , 5 , 2

'

, 4

'

, 5 ' -hexachlorobiphenyl and 2 , 3 , 4 , 2

'

, 4

'

, 5 ' -hexachlorobiphenyl respectively [9]. These two

isomers (lUPAC No. 153 and 138 [10]), are major constituents of both Aroclor

172
Table 2. Changes in CWS Analytical Methodology for PCBs 1968-1986

1968 Chemical separation from OCs based on removal of

aromatics by nitration. Quantitation based on packed

column chromatograms

.

1969 Florisil column separation introduced.

1970 Carbon-celite filtering at -70 °C to improve clean-up.

1972 Alumina-celite filtering.

1974 2% water deactivation of Florisil.

1976 Quantitation based on 1:1 mixture of Aroclors 1254

and 1260.

1979 Introduction of glass capillary GC columns. Quantitation

based on specific peaks for Aroclor 1254, Aroclor 1260

and 1:1 mixture of the two.

1981 Fused silica capillary GC columns.

1982 Mini-column Florisil clean-up.

1986 Specific PCB congener analysis introduced.

1254 and 1260, and the PCB levels obtained were therefore not particularly

sensitive to the proportion of these Aroclors in environmental samples. —j- --4--*

er

~r

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.

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i

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——

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9 10.5 12 13.5 15 IN MINUTES

16.5 :

-

i- [_ Figure 1. Standard of Aroclor 1254, EC detection, chromatographed on a

6' X 1/8" OD, 6% QF-1 and 4% SE30 on Chromosorb W (AW)

column at 190°, N2 flow of 40 mL/min. Reproduced with

permission of L.M. Reynolds from Ontario Research Foundation

Report 68-18

From about 1976 a 1:1 mixture of Aroclor 1254 and 1260 was used for

calibration. On a 60 m DB-5 fused silica column (Figure 2), congener 138 was used for calculation of total PCB content because it corresponded to peak 10 in

the packed column analysis and the results obtained were therefore comparable

173
Figure 2. A 1:1 mixture of Aroclors 1254 and 1260, chromato graphed on a 60m

DB-5 fused silica capillary column, EC detection, initial

temperature 100° for 2 min. , then 10°/min. to 150°, then 3°/min

to 300°, the carrier velocity 25 cm/sec

to those obtained prior to the introduction of capillary columns. Congeners

118 and 180 can be used to estimate the Aroclor 1254 and Aroclor 1260 levels,

respectively, because these congeners are unique to each Aroclor [11].

III. CASE STUDY: ANALYSIS OF GANNET EGGS 1969 - 1984

Results obtained over the period 1969-1976, were compared with analyses on

the samples retrieved from the specimen bank and re-analyzed in our own labs in

1984 [12]. The condition of storage of these samples varied from -10° to40°, depending on the period. The results obtained from ten Gannet (Sula

bassanus) eggs collected and analyzed in 1969 are given in Table 3. The

results of the re-analysis of six 1969 egg samples are shown in Table 4. As

has been noted the number of analytes has greatly increased. The original and

re-analysis of the samples for the determination of DDE and PCB are compared in

Figures 3 and 4 for the years 1969 to 1976.

174
Table 3. Analysis of Gannet Eggs from Bonaventure Island 1969 [12]

(10 analyses of 10 eggs)

Residue Result (mg/kg wet weight)

Mean _ 95% CI Range

DDE 14.5 12.3-17.1 10.0-20.6

dieldrin 0.500 0.415-0.603 0.365-0.818

heptachlor epoxide 0.058 0.044-0.076 0.029-0.099

Aroclor 1260 ^ 5.11 3.81-6.87 1.81-8.57

%fat ' 4.54

%water 84.2

Table 4. Re-analysis of 1969 Gannet Eggs, Bonaventure Island [12]

(6 analyses of 6 eggs)

Residue Result (mg/kg wet weight)

Mean 95% CI Range

DDE 18.5 10.6 -32 .3 9.5- 35 .8

DDT 1 .67 0 .703 -3. 95 0 .687 -3 .93

DDD 0. 811 0 .524 -1

26 0 .416 -1 .48

dieldrin 0. 641 0 .452 -0. 911 0 .467 -1 .04

heptachlor epoxide 0. 029 0 .019 -0. 043 0. 019- 0. 053

Mirex 0. 030 0. 030- 0. 030

Q-HCH 0. 021 0 .013 -0. 035 0. 013- 0. 037

)3-HCH 0. 006 0 .003 -0. 010 0. 004- 0. 013

7-HCH 0. 003 0 .002 -0. 005 0. 002- 0. 005

oxych1 o r dane 0. 093 0 .061 -0. 143 0. 052- 0. 154

trans - chlordane 0. 021 0 .014 -0. 031 0. 015- 0. 039

cis-chlordane 0. 240 0 .163 -0. 353 0. 158- 0. 385

cis-nonachlor 0. 173 0 .130 -0. 230 0. 125- 0. 250

HCB 0. 075 0 .045 -0. 125 0. 044- 0. 128

Aroclor 1254:1260 23. 9 14.3 -39 .9 13.1- 43 .4

%fat 4.44

%water 83.0

175
Original

analyses

1984

analyses

/ / / / y / / / / /

00

CO>

CO

'cu

CDCD

CTi

20

15

10

0

1969 1973 1974 1976

year

Figure 3 DDE in Gannet eggs, Bonaventure Island, Quebec, 1969-76

Original 1984

analyses analyses

p / / / / ^ y y y y y

10

>

rocu

'cu

cu

25

20

15

10

5

0

/ /

/ /

/ / /

/ /

/ /

/ /

/ /

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/ /

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y /

^ / /

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' / /

/ /

'' / /

'*' / /

/ /

*' x'' /

•* / /

*' / /

•' y /

'' /

1969 1973 1974 1976

years

Figure 4. PCBs in Gannet eggs from Bonaventure Island 1969-76

176
The DDE results are comparable as can be seen from examining the

confidence intervals and ranges in the two data sets, Tables 3 and 4. The re- analysis suggests that the original determinations in 1969 significantlyunderestimated PCB levels (Figure 4). However, given that these results are not necessarily obtained from the same samples, the results from 1973 onwards

are in excellent agreement. PCB levels appeared to have increased from 1969 to

1976 based on the original data, but this was obviously incorrect. Most probably, much of the data for PCBs from the late 60' s and early 70' s is questionable and should be interpreted with caution, unless the accuracy of the

analysis can be verified by analysis of archived samples. These results show

the distinct advantage in being able to retrieve samples from a specimen bank

for re-analyses to identify compounds not originally determined, as well as to

correct or confirm the original analyses

In the case of PCBs the difference in results was largely a matter of

quantitation, that is, the nature of the "analyte" changed with time. For

example, Aroclor 1260 results from earlier analyses are not equivalent to 1:1

Aroclor 1254:1260 results, which explains most of the difference in the results

of the two analyses of the 1969 egg samples. In 1969, quality assurance was not the concern it is today. Setting up a control or reference material to use over the long haul may have flagged the problems in the earlier analyses.

The lessons from this exercise are several. The first is that the effects

of changes in analytical methods with time are not fully recognized by many of

those who interpret residue data, and may lead to erroneous conclusions. The

classic example is the alleged decline in the levels of silica in Lake Michigan, which is given in many texts [13]. What actually happened is that

chemists did their job better and used more accurate methods. Obviously there was a failure in communication between the chemists and the interpreters

regarding the bias in individual methods. In preparing this paper, it was quite a challenge to document all the various changes over the period in

question. All chemical residue data banks should have a method dictionary and

every change should be documented. Also, every time a method is changed there

should be a statistical comparison between the old and new methods to ensure

they are equivalent and are not biased between one another. This should be

part of the quality assurance procedures during the development of new methods.

Ideally, there should be a reasonable period when both methods are in use. If

a new method is shown to be more accurate, then re-analysis of stored samples

should be carried out to verify that the overall long-term trend is correct.

If the new method shows a substantial difference between original and retrieved

samples, then correction factors for past analysis may possibly be applied to

correct long-term trends. It must, of course, be shown that the difference is

not due to slow degradation of the compounds in question. The only alternative

to method changes is to freeze methodology, but such a course would not allow

analysts to take advantage of new instrumentation and techniques which provide

better specificity, accuracy and productivity. There is actually a long-term

study where this approach has been taken [14].

177
IV. ASSESSMENT OF ANALYTICAL RESULTS

How do we prevent data quality problems in the future? Quality control is

especially difficult when the majority of analyses are done by a laboratory

outside of the organization, which has been the case for a number of years for

CWS . We may demand proper quality assurance protocols, but there is no certainty that they will be followed. Ultimately, we must assess the

analytical results to see if they are acceptable in terms of prescribed

requirements for accuracy and precision. This has required the incorporation

of blind-numbered reference materials into a set of samples. While there are a number of good SRMs (Standard Reference Materials) available from the National

Bureau of Standards and elsewhere for use in analysis for metals, there are no comparable SRMs for organic constituents. Because of this, several reference

materials have been developed by CWS specifically for the assessment of

analytical results.

A. CWS Reference Materials

The first of these materials was developed in 1979, primarily for use with

the Herring gull egg surveillance program for the International Joint Commission (IJC) [15]. A second reference material was developed in 1980, mainly

because the first had high levels of PCBs . Another reference material was developed in 1986, primarily for use in dioxin analysis. All of these materials are whole egg homogenates . They were initially analyzed in the CWS

laboratories and have been used mainly to assess the analytical performance of

contractors, and to monitor the in-house laboratory performance. These materials have been re -analyzed over a period of time and have been found to be

stable [1,16].

The 1979 reference material has been used to compare CWS results with

those of the Ontario Research Foundation and the U.S. Fish and Wildlife Service

in Patuxent, Maryland. It has also been used within the Canadian Federal

Interdepartmental Committee on Pesticides check sample program. Thus, we have

been able to establish good values for levels of a number of chemical

constituents for this material (Table 5) . A need has been expressed for

developing liver and breast muscle reference materials in a similar manner in

response to concerns about the edibility of wild foods.

B. Assessment Criteria

In using reference materials for the assessment of results from a contract

laboratory, criteria must be established prior to the awarding of a contract.

The three criteria which were originally proposed for the acceptance or rejection of results are based on the concepts of systematic error (Eg), total error

(E^), and acceptable ranges based on multiples of standard deviation (SD) , and

maximum coefficient of variation (CV^j^^^) [17].

The Eg criteria (or percent relative bias) is defined as

Eg = (X - x,3f)/(x^,f) • 100 (%)

where x - mean of replicate SRM analyses.

Xj.

g f = reference value

178
Table 5. Interlaboratory Comparison of CWS 1979 Reference Material

Residue CWS ORF FICP USFWS

HCB 0.192

± 10 (21)

0.153

± 13 (15) n . a

n. a

p,p' -DDE

Aroclor

1254

Aroclor 1254

+1260 1:1

Aroclor

1260

4.83

± 16 (21)

28.47

± 18 (10)

83. 12

± 21 (11)

33.99

± 13 (21)

3.903

± 10 (13)

n . a

72.94

± 10 (13)

n . a

4.64

± 13 (5)

n . a

88.6

± 3 (4)

n . a

5. 38

± 7 (5)

n . a

n . a

34.4

± 7 (5)

Mirex

oxychlordane

els- chlordane

0.122

± 11 (21)

0.194

± 4 (21)

0.0287

± 10 (15)

0.102

± 25 (13)

0.192

± 15 (13)

0.0215

± 13 (17)

0.139

± 14 (3)

0.202

± 15 (2)

n. a.

n . a

0.184

± 6 (5)

n . a

cis-nonachlor

p,p' -DDD

heptachlor

epoxide

dieldrin

p,p' -DDT

0.0609

± 30 (15)

0.0581

± 15 (15)

0.1373

± 12 (21)

0.2779

± 9 (21)

0.0475

± 41 (10)

n . a

0.0350

± 53 (13)

0.110

± 22 (13)

0.257

± 14 (13)

n.d.

n. a

0.069

± 5 (4)

0.129

± 25 (8)

0.28

± 36 (5)

0.065

± 9 (5)

n.d.

n.d.

0.123

± 21 (4)

0.258

± 21 (4)

n.d.

All results expressed in mg/kg wet weight, ±CV (%) , number of determinations

in parentheses; ORF, Ontario Research Foundation; FICP, Federal

Interdepartmental Committee on Pesticides check sample program (6-7 labs

in duplicate); USFWS, U.S. Fish and Wildlife Service, Patuxent , Maryland.

Note: n.a. = not analyzed, n.d. = not detected.

179
Normally, the Eg should be less than 50%, based on the observation of Elgar

[18] that, in interlaboratory studies with pesticides at low levels, rarely is

there agreement below a Eg = 25%. Thus, Eg = 50% is a suitable limit at very

low levels when the absolute bias is acceptable but the relative percentage

bias can be quite high such as near the detection limit.

The total error, E^ , provides a value of the effects of both SE and

precision

Et = (Eg + 2 SD)/(x^3f) • 100 (%)

This concept proposed by McFarren [19] sets an arbitrary value of 50% as the

maximum acceptable total error. The total error criterion would consider

acceptable a set of analyses of an SRM with a CV < 15% and Eg < 20%.

The third criterion of acceptable ranges based on multiples of standard

deviations is very useful when there is a history of analyses. We have chose

± 2 SD for a set of 5 SRM analyses in a batch of samples. Experience has shown

that this can be obtained for residue analysis by a single laboratory. It is

analogous to the control chart used in many laboratories.

The CVj^gjj criterion is based on

Briefly, it has been shown that, as increases. From this they developed

value curves"

the work of Frehse and Timme [20].

analyte concentration declines the CV

a concept of "first category measured

This can be extended to the general use

CV„,, = CV, (x,/x)^°^ f (%)

where CV^^^^^ = maximum permissible CV

CVq = CV at the detection limit

X = mean of replicate analysis of blind sample

Xq = detection limit

f = the factor by which CV^^^^ diminishes per order of magnitude.

For example, if f = 2 and CV, = 100, the equation reduces to

CV.ax - (Xo/x)°-3 • 100 %

For pesticide residue analysis, the form of the equation

CV,,, = 13.7 x-°-^3i (%)

based on x„ = 0.01 mg/kg, CV„ = 100% and CV,,, = 15% at 1 mg/kg. This is

equivalent to f = 2.7. As an example, for a mean of 0.0895 mg/kg, CV^^^^ is

38.8%.

These equations allow CV^^^^^ to increase as concentrations decline in a systematic manner. A practical lower limit of CV,,, is 15%. Normal practice

has been to determine the minimum expected value of CV^^^ in our own labs by

use of replicate analysis.

180
These criteria have been grouped so that the data manipulation can be

performed by computer, and the data sets failing to meet the criteria can be

identified. Thus, acceptability can be based on:

a) Et < 50 %

b) Mj.gf < M ± 2 SD where M^.^^ is mean of reference material and M is prior

mean of reference material

c) E3 < 50%, CV < CV„3,.

Ideally, an acceptable result would pass all three criteria, but this often

does not occur for various reasons. When a result fails to pass all criteria,

it is compared to previous data sets and judged to see if there is consistent

bias, and if previous contracts had similar problems in the past. An example

is given in Table 6.

Table 6. Application of Acceptable Criteria

DDE DDD DDT Oxychlordane

Contractor Mean 4 828833 0 04 0 048333 0 21667

SD 0 612185 0 01 0 024832 0 25819 n 6 6 6 6 CV 12 679 25 51 3782 11 9168

Reference Mean 5 61 0 0367 0 153 0 232

SD 0 28 0 004 0 024 0 010 n 15 12 12 12

CV 5 0 11 3 16 0 4 4

Systematic Error E3 -13 93 8 99 -68 41 -6 61

Total Error 35 76 63 48 100 87 28 86

Acceptable data?

a) Et < 50% YES NO NO YES

b) M^ -2SD<M<M^+2SD NO YES NO YES

c) E3<50%,CV<CVmax YES YES NO YES

In this example, the relatively high CV for DDE has caused the result to

fail criterion b) , but the accuracy is good. Similarly, the high CV for DDD

has resulted in failure to pass criterion a) , even though the CV is not

unreasonable for a value so near to the detection limit. For these residues,

criterion c) seems to give a better assessment of data quality than a) or b) . The DDT result clearly fails all criteria because of a large negative bias

(E3 = -68%), whereas oxychlordane passes all criteria. Thus, only the last

181
criterion is written into the contract but the other, criteria are used in the

assessment

V. CONCLUSIONS

Accurate analytical determinations are a prerequisite for reliable

determination of long term trend monitoring data. Changes in methodology, if

not properly documented, may lead to erroneous interpretations. Appropriate

uses of specimen banking, reference materials and rigorous quality assurance procedures can be devised to prevent or minimize such errors

VI . ACKNOWLEDGEMENTS

The authors wish to thank J. A. Keith, W.K. Marshall, and J. E. Elliott

for their interest and comments.

VII. References

[1] Elliott, J. E. (1985), Specimen Banking in Support of Monitoring for

Toxic Contaminants in Canadian Wildlife, In: International Review of

Environmental Specimen Banking, S. A. Wise and R. Zeisler, Eds., NBS

Spec. Publ. 706, 4-12.

[2] Elliott, J. E. and Norstrom, R. J. (1987), Trends and Effects of

Environmental Contaminants Determined from Archived Wildlife Samples, In:

These Proceedings

[3] Ratcliffe, D. A. (1967), Decrease in Eggshell Weight in Certain Birds of

Prey, Nature 215, 200-210.

[4] Peakall, D. B. (1972), Polychlorinated Biphenyls : Occurrence and

Biological Effects, Residue Reviews 44, 1-21.

[5] McCully, K. A. and McKinley, W.P. (1964), Determination of Chlorinated

Pesticides in Fat by Electron Capture Gas Chromatography, J. Assoc. Off.

Anal. Chem. 47, 652-660.

[6] Reynolds, L. M. (1971), Pesticide Residue Analysis in the Presence of

Polychlorinated Biphenyls (PCBs) , Residue Reviews 34, 27-57.

[7] Mullin, M. D., Pochini , C. M. , McCrindle, S., Romkes, M. , Safe, S. M. , and Safe, L. M. (1984), High-Resolution PCB Analysis: Synthesis and

Chromatographic Properties of All 209 PCB Congeners, Environ. Sci.

Technol. 18, 468-476.

[8] Reynolds, L. M. , Ontario Research Foundation CWS Contract Report 68-18.

182
[9] DeFreitas, A. S. and Norstrom, R. J. (1974), Turnover and Metabolism of

PCBs in Relation to Their Chemical Structure and the Movement of Lipids

in the Pigeon, Can. J. Physiol. Pharmacol. 52, 1080-1094.

[10] Ballschmiter , K. and Zell, M. (1980), Analysis of Polychlorinated

Biphenyls (PCB) by Glass Capillary Gas Chromatography, Fresenius . Z. Anal. Chem. 302, 20-31.

[11] Norstrom, R. J., Patterns and Trends of PCB Contamination in Canadian

Wildlife, IEEE MONTECH'86 Conference on PCBs and Replacement Fluids,

Montreal, 1986, 1-9.

[12] Elliott, J. E. , Norstrom, R. J., and Keith, J. A., Organochlor ine

Insecticides and Eggshell Thinning in Gannets from Eastern Canada,

Environ. Pollut. , in press.

[13] Shapiro, J. and Swain, E. B. (1983), Lessons from the Silica Decline in

Lake Michigan, Science 221, 457-459.

[14] Addison, R. F. , Brodie , P. F. (1984), DDT has Declined More Than PCBs in

Eastern Canadian Seals during the 1970s, Environ. Sci. Technol. 18,

935-937.

[15] Won, H. T. and Norstrom, R. J. (1980), Analytical Reference Materials:

Organochlorine Residues in CWS-79-1, a Herring Gull Egg Pool from Lake

Erie, Canadian Wildlife Service; Manuscript Report No. 41.

[16] Norstrom, R. J. and Won, H. T. (1976), Long-Term Preservation of Egg and

Tissue Homogenates for Determination of Organochlorine Compounds:

Freezing versus Freeze-Drying , J. Assoc. Off. Anal. Chem. 68, 129-135.

[17] Turle, R. and Norstrom, R. J., Guideline to Practical Quality Assurance

for Contract Analysis, Canadian Wildlife Service Technical Report (in

press), 1987.

[18] Elgar, K. E. (1979), The Variability of Residue Results, with Particular

Reference to the CODEX study on Organochlorines in Butterfat, Advances in

Pesticide Science 3, 668-672, Pergammon, Oxford.

[19] McFarren, E. F., Liska, R. J., and Parker, J. M. (1970), Criterion for

Judging the Acceptability of Analytical Methods, Anal. Chem. 42, 358-361.

[20] Frehse, H. and Timme , G. (1980), Quantitative Residue Analytical

Reliability: Beatitude through Application of Latitude, Residue Reviews

73, 27-47.

183
SECTION 18

PREPARATION OF SAMPLE MATERIAL FOR ENVIRONMENTAL SPECIMEN BANKING

PURPOSES - MILLING AND HOMOGENIZATION AT CRYOGENIC TEMPERATURES

J. D. Schladot and F. W. Backhaus

Institute of Applied Physical Chemistry

Nuclear Research Center (KFA) Julich

D-5170 Julich

FEDERAL REPUBLIC OF GERMANY

I . INTRODUCTION

One of the most important tasks of the environmental specimen bank (ESB)

Julich is the preparation of sample material to be stored for future investigations. The collected material is directly deep frozen in the gaseous phase over liquid nitrogen (see Figure 1). Further sample treatment is carried out

only at cryogenic temperatures to avoid any deterioration and losses of organic

compounds. For this reason, we implemented a sample preparation procedure

(SPP) by which the sample material is treated at very low temperatures

(<-140 °C).

The data sheets for sample collection with all the important data

(anamnesis of sample, weather conditions, possible visible emission sources,

etc.) are complemented by the data of the sample procedure.

The sample preparation procedure is divided into different single steps

(see Figure 2). The temporarily stored sample material will be pre-crushed

first with a pre-crusher type ZMl , Mercodor, Frankfurt, FRO, with 2 drive

shafts with 12 rotating knives each. The 24 knives are all coated with TiN to

avoid contamination. By pre-crushing , the sample material is pre-mixed while

some of the pre-crushed material is recycled [1]. After this pre-crushing

step, the material is temporarily stored in stainless steel containers until

the material is milled and homogenized. All data from this first preparation

step are gathered in particular data sheets and utilized later on the computer

together with all other preparation data.

The next step is milling and horaogenization of the sample material at

cryogenic temperatures. For this preparation step, we have set up a grinding

device, type CRYO PALLA'^ , KHD Klockner Humboldt Wedag, Koln, FRO. This

grinding device is cooled with liquid nitrogen for hours until the cooling

capacity of the mill will keep the cold material at cryogenic temperatures.

During the milling process, no liquid nitrogen is fed into the device. The

pre-crushed sample material is fed to the grinding cylinder by a metering

trough. By limiting the speed of the metering trough, the grain size of the

sample material can be adjusted. The pre-crushed sample material is milled in

the grinding cylinder by stainless steel rods or in some cases by PTFE grinding

rods, depending on the strength of the material. The rods have a diameter of

20 mm. About 65-70% of the volume of the grinding cylinder is filled with the

grinding media. By this, the material is milled to a fine powder with grain

size <200 /xm. The cold, fine powdery sample material is collected in stainless

steel vessels.

184
185
M 7Z z: • 1-4 m i-i

ith3

DO ti

o Dated o

nS p; O M a* u

Q-i H

o 5c; > z:

O

m $= ^ M

M o

W

OJ tn'—

!= E

-1-1

ITS != M .1-4 tt, M

= 1

o

!=

CD

M

1 M

o ITi

nj o

nS

GO 1 O in

!=

-i-H m

!=O

ITS =! I—

OS m

E lU o

nS CO

186
The collected data from this preparation step contain:

1. Amount of material fed into the grinding device.

2. Amount of collected fine powdery sample material.

3. Temperature of material before milling in the grinding cylinder

and temperature of homogenized sample material.

4. Feeding speed of the metering trough, etc.

Because of the limited space of the pilot environmental specimen bank building,

it is not possible to carry out the sample preparation procedure (SSP) as a one-step process at present.

To test this sample preparation procedure for environmental specimen

samples, we have treated comparable material, e.g., fish [dab (limanda limanda)

from the Baltic Sea and cod (gadus morhua) from the North Sea] , - kale

[ (brassica oleracea) from the region surrounding the Bornhoved Lakes], and

rhubarb [ (rheum rhabarbarum) from the Jililich Region] .

In the following, an example of the sample preparation procedure is given

for fish material [dab (limanda limanda)]. Sixty-five individuals of dab,

weighing about 7.5 kg, were tested according to the sample preparation procedure for environmental specimen bank samples. The material was pre-mixed by

crushing it with the Mercodor ZMl several times. The device was cooled with

ice, then cooled to low temperature in liquid nitrogen. The pre-crushed

material was collected in stainless steel containers (5.5 L volume) and cooled

in the gaseous phase over liquid nitrogen. After a short temporary storage in

the gaseous phase of liquid nitrogen, the pre-crushed material was fed to the

cold milling device by a metering trough.

The temperature of the material prior to crushing was -186 °C. By cooling

the pre-crusher by deep cold ice the temperature of the material after precrushing was: -172 °C. For temporary storage the pre-crushed material was cooled again in the gaseous phase over liquid nitrogen.

The grinding device CRYO PALLA^ was cooled with liquid nitrogen within a few hours to a temperature of -190 °C. During the milling process no more liquid nitrogen was fed into the milling device. The temperature of the precrushed sample material was recorded by a measuring system in the metering

trough. Another temperature sensing unit is located in the grinding cylinder.

The temperature of the grinding cylinder was controlled during the milling

process, and the temperature usually increased to -160 °C.

The powdery sample material (grain size <200 ^m) was then collected in

stainless steel containers (volume 5.5 L) , and cooled over liquid nitrogen.

The temperature of the fine powdery material decrease to -180 °C.

II. EXPERIMENTAL SECTION

187
The fine powdery sample material (dab), weighing about 7.2 kg was subsampled into cold glass containers (Wheaton scintillation flasks) with

quartz spoons under a laminar flow box. The glass flasks were cooled in the

gaseous phase over liquid nitrogen. The weight of each subsample was measured

and recorded. Generally, the weight of a subsample with dab material is about

7 g (cold fresh weight). While subsampling, the material in the stainless

steel containers and the glass flasks was cooled by liquid nitrogen and the

surrounding atmosphere under the laminar flow box was filled with cold nitrogen

vapor

Fifty random subsamples of dab (limanda limanda) out of 950 subsamples

were analyzed by our colleagues of the Institut fiir okologische Chemie , GSF

Neuherberg. They determined different chlorinated hydrocarbons. The obtained

values show the homogeneity of the subsampled material (see Table 1). A

discussion of these values is presented elsewhere [2]. Other random subsamples

were used for the determination of the grain size of the milled material.

Various methods were used: e.g., sieving analysis, microscopic measurement,

and scanning photo- sedimentographic analysis (see Figure 3). Furthermore, in some random subsamples the distribution of Pb , Cd, As, and Se was analyzed. Pb

and Cd were analyzed using solid sampling Zeeman AAS (see Figure 4) [3]. As

and Se were determined using sodium-borohydride - AAS after wet chemical

digestion (see Figures 5 and 6) . All these methods show that the homogeneity

of the subsamples is satisfactory for most elements.

III. CONCLUSIONS

The tests proved that we are now able to homogenize the ESB sample

material according to a sample preparation procedure (SPP) . If all single

steps of a "uniform" sample preparation procedure are strictly adhered to and

if all resulting data within these steps are recorded, it should be possible to

compare samples of the same matrices prepared in different institutions, e.g.,

common mussel (mytilus edulis) from U.S. coastal waters prepared at the ESB of

the National Bureau of Standards (NBS) and common mussel from German coastal

waters (North Sea and Baltic Sea) prepared at the ESB in Julich, or hioman liver

samples prepared at the ESB of the NBS and human liver samples prepared at the

ESB for human organs at the University of Miinster.

IV. ACKNOWLEDGEMENT

The authors wish to thank the Ministry of the Interior, and from June

1986, the Ministry of the Environmental, Protection of Nature and Reactor

Safety, Bonn, for financial support. We wish to thank DI . M. Burow for the

determination of As and Se in homogenized fish samples; N. Commerscheidt for

his helpful assistance in sample preparation; Dipl. Biol. K.-H. Grobecker, Griin

Analysengerate , Wetzlar; and DI . C. Mohl for the determination of Pb and Cd

with Zeeman Solid Sampling AAS. Furthermore, we wish to thank our colleagues

from the Institut fur okologische Chemie, Dir. Prof. Dr. F. Korte , GSF,

Neuherberg, Dr. I. Gebefugi and Dr. K. Oxynos , for the determination of

chlorinated hydrocarbons in homogenized fish material, and our colleagues from

the Institut fur Meereskunde, University Kiel, Dr , K. Rumohr and Dr. M. Weigelt

188
Table 1. Homogenization Study of Fish Material by Determination of

Chlorinated Hydrocarbons^

Pos . HCB a-HCH 7-HCH ^-HCH DDE Die Idr in A 60-1

1 mean x 1 . series 1 1 5.3 1L . q QO _> 47

2 mean x 2. series 1 4 4 7 4.6 1X . 1. 1 0 eO 46

3 mean x 3. series 1 2 4 8 5.2 1 9 1 1 X X . s

b 4 mean x 4. series 1 4 5 4.2 0 Q 7 7 53

5 mean x 5 . series 1 1 6 1 5.4 b 13. 6 4 4 50

6 mean x (pos. 1-5) 1 2 5 4.9 1. 2 10. 6 3 4 49

7 std. dev. S (pos. 1-5) 0 15 0 6 0.5 0. 15 2. 2 0 6 3 8 -1.96 s (S 95%) 0 8 3 8 3.9 0. 8 6. 3 2 2 43 =

9 +1.96 s (S 95%) 1 5 6 2 5.9 1

.

5 14. 9 6 3 55

10 median (pos. 1-5) 1 1 4 8 5.2 1

.

2 10. 5 3 3 49

11 mean x (total) 1 2 4 9 4.9 1. 1 10. 5 3 4 49

12 std. dev. (total) 0 25 1 1 0. 4 2. 3 0 85 7 13 P min (95%) 0 9 3 8 3.8 0. 7 8. 2 2 6 42^

14 P max (95%) 1 4 6 6 1

.

5 12. 9 4 3 56

15 median (total) 1 4 8 5 1 10 3 50

^Data provided by K. Oxynos , I. Gebefugi, GSF Neuherberg, Inst, of Ecological Chemistry

^Missing value

'^95% range of normal distribution, within the range are 95% of all (900) values.

^Forecast interval for the mean value x of 5 random samples of the total amount (900).

189
SCANNING PHOTO SEDIMENT06RAPHIC ANALYSIS

Figure 3. Determination of grain size in fish material -dab (limanda limanda)

190
Pb /ug/g

>16l

f >10

8 initial weight; 0.0^ - 0.^ mg

f

'

t

'—I—J 1 1 1 1 , , 5. 6. 3. 2. l.j 1. 1 2. 3. ^- 5. 6. sample aliquot

l.day 2. day 3. day

sn - 20

>400 vr^n

, 207 >270, » ^2^0

CD NG/G iSOf

J20

80 -

initial weight: 0.1 - 1.5 mg

• -v. V--. •.. •

•

•

1. 2. 3. 4. 5. 5. jl.2. 3, 4. 5.: 5. 6. ; sample aliquot

1 day ' 2. day ' 3. day \ SM - 20 5030Z

Figure 4. Homogeneity study of fish material-dab (limanda limanda) by

determination of Pb and Cd in 6 random subsamples out of

950. Determination by solid sampling atomic absorption

carried out at different times

191
As 2-r mg/kg

1.2

!•

0.&

0.&

0.4

0.2

a

+ X + std

- X - std

< mean

1 2 3 4 5 6

Figure 5. Homogeneity study of fish material-dab (limanda limanda) by

determination of As in six random subsamples out of 950

Se

mg/kg

0.4t

0.3--

0.2--

0.1--

+ X + std

- X - std

»« mean

-+-

Figure 6

2 3 4 subsamples

Homogeneity study of fish material-dab (limanda limanda) by

determination of Se in six random subsamples out of 960

192
for their helpful assistance in collecting the fish material (dab - limanda

limanda)

IV. REFERENCES

[1] Protokoll der Status Seminar Sitzung, November 1982, Reisensburg, FRG

[2] Schladot, J. D., Backhaus , F., and Reuter, U., Beitrage zur Umweltprobenbank , 1. Studie zur Errichtung einer Kaltmahlapparatur , Jill

Spez 330, 1985. .

[3] Mohl, C, Grobecker, K. -H., and Stoeppler, M. (1987), Homogeneity Studies

in Reference Materials with Zeeman Solid Sampling GFAAS , Fresenixis Z. Anal. Chem. 328, 413-418.

193
SECTION 19

THE POTENTIAL ROLE OF ENVIRONMENTAL SPECIMEN BANKING IN BIOSPHERE RESERVES

William P. Gregg, Jr.

U.S. Department of the Interior

National Park Service

Washington, DC 20013-7127

USA

I . INTRODUCTION

When the Man and the Biosphere Program (MAB) was officially launched by

the United Nations Educational, Scientific, and Cultural Organization (UNESCO)

in 1971, one of its thirteen project areas was "The Conservation of Natural

Areas and the Genetic Material They Contain." The central focus of this

project was the establishment of a global network of protected sites called

biosphere reserves, to conserve representative examples of each of the world's

biomes as benchmarks of environmental quality and centers of basic and applied

ecological research. In 1972, the idea became a key recommendation of the

United Nations Conference on the Environment, or Stockholm Conference. In

1973, UNESCO issued criteria for biosphere reserves [1]. In 1975, the

International Union for the Conservation of Nature and Natural Resources (lUCN)

published a global biogeographic classification as a framework for selection

[2]. And in 1976, the UNESCO's International Coordinating Council for MAB

designated the first 29 biosphere reserves.

Since that time, the network has grown rapidly to include 266 units in 70

countries (November 1987 data). About two-thirds of these units are in the so- called developed countries. About 100 of the 193 biogeographical provinces in

the UNESCO classification are represented. Of the world's major biomes,

temperate broad- leaf forests and mixed mountain systems are particularly well

represented. Significant gaps remain elsewhere, particularly in major lake

systems, coastal and marine areas, and most tropical biomes [3].

Biosphere reserves are selected on the basis of their potential to carry

out and integrate conservation, information/logistic, and development function

[4].

II. CONSERVATION FUNCTION

Biosphere reserves are of exceptional importance in conserving ecosystems

and genetic resources. They should be large units of landscape -- the majority

of existing units exceed 20,000 hectares in size, and about a fourth exceed

100,000 hectares. These landscapes are planned and managed for purposes

ranging from strict protection to intensive, yet sustainable production.

Throughout, the objective is to sustain natural processes and the evolutionary

potential of indigenous biota. However, at the heart of each biosphere reserve

are one or more securely protected core areas, which conserve representative

examples of natural or minimally disturbed ecosystems where human uses are

194
strictly controlled. In the United States, legislatively established

wilderness areas within national parks comprise most of the core areas. A few

countries --for the most part those lacking well-established systems for land

management and environmental regulation- -are establishing biosphere reserves by

law. However, in most countries, biosphere reserves represent a voluntary

approach to conservation, using existing legally protected areas as building

blocks

One promising approach adopted in some countries, including the U.S., is

to use the biosphere reserve as a framework for linking ecologically and

functionally complementary protected areas in a particular biogeographical

region. In this way, the biosphere reserve becomes a symbolic and practical

basis for cooperation among governmental and private administrators of important conservation real estate. During the last few years, some significant

assemblages of protected areas have been established as biosphere reserves. For example, a recently designated biosphere reserve in the warm deserts of

California links five units under four different administrators totalling 1.3

million hectares. It is worth noting that the global network already contains

many of the world's outstanding conservation areas, such as the Manu (Peru),

the Bavarian Forest (Germany), and Yellowstone (U.S.) National Park. Biosphere

reserve status encourages functional linkages between these areas and surrounding lands, and can provide a framework for coordinating efforts to conserve regional ecosystems and provide for their intelligent use and management.

III. INFORMATION/LOGISTIC FUNCTION

The International Network of Biosphere Reserves is the only permanent

global association of protected areas dedicated to generating, sharing, and

applying ecological information for human well-being. Each reserve should be a focus for long-term monitoring, comparative and interdisciplinary research, and

related demonstration and training. The core areas should be centers for care- fully designed monitoring for early warning of natural and man-caused changes,

with special emphasis on chemical contamination. In the surrounding areas,

experimental manipulations should be carried out, monitored, and compared with

the core to develop and assess ecosystem use and rehabilitation strategies.

Throughout, the biosphere reserves are centers for the testing and adapting of

new methodologies for monitoring, research, assessment, and prediction. The

actual accomplishments and potential of the site in these fields thus are essential considerations in selection. The administrator's commitment to

facilitate research is another, as is willingness to participate in MAB's

unique international framework for scientific cooperation on environmental

problems. Given these considerations, it is not surprising that the network

includes many leading field stations for developing the theory and practice of

ecology. For example, the biosphere reserves in the U.S. include ten former

biome research sites in the International Biological Program and nine of the 15

sites currently supported by the National Science Foundation's long-term

ecological research program (November 1987 data)

195
IV. THE DEVELOPMENT FUNCTION

Information and skills developed in the biosphere reserve should provide

the basis for ecologically sustainable development. One objective is develop

strategies for ecosystem use and management on site which can be transferred

from the reserve to the surrounding region. The strategies must be culturally

appropriate, economically feasible, and capable of implementation by participating institutions. This process requires cooperation between natural and

social scientists in carrying out the necessary research, and, in many

countries, continuing involvement of local populations to assure that they

benefit from the research and development programs. Another objective is to

obtain information on site which can influence national environmental and

development policies. For example, many, if not most, biosphere reserves in

the U.S. and other developed countries have monitoring program.s to detect and

assess atmospheric pollutants in natural ecosystems (Gregg and Giogel, 1981).

In the U.S., the results of these activities have been important factors in

decisions affecting the siting and design of major developments where

atmospheric pollution has been an issue.

V. THE ROLE OF ENVIRONMENTAL SPECIMEN BANKING

Systematic banking of environmental specimens as part of an integrated

program of ecological monitoring is consistent with the mission of biosphere

reserves as bellweathers of ecological health (and, in some developing coun- tries, perhaps also of human health). By providing a basis for retrospective

assessment of chemical contaminants in the environment, environmental specimen

banking could serve all major functions of biosphere reserves. In conservation, it could improve the protected area manager's capability to detect trends

potentially affecting the evolutionary potential of indigenous biota. In

logistics, it could provide a new and exciting area for interdisciplinary and

international cooperation, both in the conceptualization and implementation of

effective banking programs, and in developing and testing of research hypotheses based on analysis of banked specimens. In development, it could provide

information useful in policy decisions on technology and development, and help

ensure that these decisions on technology and development, and help ensure that

these decisions minimize or avoid significant effects from environmental

toxicity

For their part, many biosphere reserves afford particular advantages in

developing and coordinated specimen banking program. Some of the more important

• they are representative of larger biogeographic regions, hence results

are likely to be widely applicable

• they have an establish mission in ecological research, improving

likelihood of logistical and financial support

• many have a good history of scientific activity, required for

establishing concepts and goals for banking

196
• size of the reserve and distribution of ecosystems are generally

adequate for repeated sampling

• secure protection of minimally distributed core areas helps assure continuity in baseline monitoring

• land use practices in designated buffer areas adjoining the core areas

are normally defined and predictable, reducing possibility of

unexpected human influences

• most are in remote locations; and are generally not subject to acute

chemical episodes

• each site is part of an established network suitable for assessing the

distribution and effects of chemicals

• the MAB framework for international cooperation facilitates sharing

perspective and results, and technology transfer

In a 1984 action plan, UNESCO underscored the role of biosphere reserves

for long-term monitoring of ecological processes, global biogeochemical cycles,

and effects of human uses on the biosphere [6]. The plan is a unique document

for a unique network. It calls for 35 actions, which are being implemented by

MAB National Committees and the various United Nations agencies and nongovernmental organizations which have endorsed the plan. It specifically calls on United Nations Environment Programme (UNEP) , in collaboration with other United

Nations agencies, to seek support for monitoring, among other things, background levels of pollutants and biological indicators of environmental change.

In this context, the plan provides ready justification for incorporating

specimen banking as an adjunct of monitoring in biosphere reserves.

Notwithstanding these considerations, environmental specimen banking

remains to be tested and implemented in biosphere reserves. In discussions

with biosphere reserve managers and scientists, I find general acceptance of

the need for baseline monitoring of pollutants (e.g., a methodology developed

for UNEP's Global Environmental Monitoring Program is now being used in five

U.S. reserves [7]), but considerable uncertainty over the requirements and

potential benefits of specimen banking.

First, the technology is unfamiliar. Recent conceptual and technical

advances in Germany and elsewhere have yet to be effectively communicated

widely in the United States. To help address this problem, the U.S. and German

MAB National Committees, in cooperation with domestic agencies and institutions

in both countries, have recently published an English adaptation of the

guidelines prepared for the German National Environmental Specimen Bank [8,9].

In addition, Lewis [10] has prepared a chapter on environmental specimen

banking for the National Park Service's Museum Handbook, which is now being

revised. These publications will be generally distributed and should raise

awareness of the technology, and the many considerations in implementing it

effectively.

197
Second, there is concern about what to include in a bank. Although, many

reserves monitor a range of abiotic and biotic parameters, only a few could

reasonably be considered for banking. Even if clear goals and objectives are established, it remains no easy task to select the ecosystem components most

likely to be burdened with introduced chemicals, or which govern the transformations and fate of these chemicals in the ecosystem . . . much less operate an effective program after these decisions have been made. To my knowledge, no biosphere reserve has undertaken the interdisciplinary planning needed to

develop an effective specimen banking program.

Finally, there is concern over the cost of facilities and operations.

Relative to the total cost of the integrated monitoring programs in many

biosphere reserves, the cost of a banking component is likely to be modest.

Nevertheless, the concern is likely to remain until the cost of alternative

administrative arrangements can be demonstrated on the basis of practical

experience

In conclusion, there seems little question that biosphere reserves can play an important role in developing and applying specimen banking

technologies, in concert with monitoring, in early warning and predictive

assessment of environmental contaminants. We now have MAB guidelines for

planning environmental specimen banks based on the German experience. Preparation of a prototype plan for a biosphere reserve would seem to be an important

first step in fostering understanding and support within the biosphere reserve

network. It would certainty seem worthwhile at this time to determine the

interest of international (e.g., UNESCO, UNEP) and domestic (e.g., national MAB

committees) agencies and institutions in supporting such an effort.

VI . REFERENCES

[1] UNESCO 1973, MAB Expert Panel on Project 8: Conservation of Natural Areas

and the Genetic Material They Contain, Merges, Switzerland, 25-27

September 1983, MAB Report Series No. 22, UNESCO, Paris, 66 pp.

[2] Udvardy, M. D. F. (1975), A Classification of the Biogeographical

Provinces of the World, I. U.C.N. Occasional Paper No. 18, I.U.C.N.,

Morges, Switzerland.

[3] I. U.C.N. 1983, MAB Information System: Biosphere Reserves, Complication

3, September 1983, I. U.C.N. Conservation Monitoring Center, Available from

UNESCO MAB Secretariat, Paris, 61 pp.

[4] UNESCO 1986, Scientific Advisory Panel on Biosphere Reserve, Report of

Second Meeting, La Paz, Bolivia, 18-24 August 1984, Unpublished Manuscript

(draft) , 29 pp.

[5] Gregg, W. P. and Goigel, M. M. (1983), Putting the Biosphere Reserve

Concept into Practice: The United States Experience, In: Ecology in

Practice, Part I: Ecosystem Management, F. diCastri, et al., Eds.,

Tycooly International Publishing, Limited, Dublin, Ireland, pp 460-484.

198
[6] UNESCO 1984, Action Plan for Biosphere Reserves, Nature and Resources

20(4), 1-12.

[7] Wiersma, G. B., Brown, K. R. , and Crockett, A. B. (1978), Development of

Pollutant Monitoring System for Biosphere Reserves, Publication No. EPA600.4-78-052, Environmental Protection Agency, Environmental Monitoring

and Support Laboratory, Las Vegas, Nevada.

[8] Lewis, R. A. (1985), Richtlingen fur den Einsatz einer Umweltprobenbank in

der Bundesrepublik Deutschland auf Okologischer Grundlage, Universitat des

Saarlandes, Saarbrucken, Federal Republic of Germany, 190 pp , Plus

Appendices

.

[9] Lewis, R. A. (1987), Guidelines for Environmental Specimen Banking with

Special Reference to the Federal Republic of Germany, U.S. MAB Report No.

12, U.S. Dept. of the Interior, National Park Service, 182 pp.

[10] Lewis, R. A. (1986), Guidelines for Environmental Specimen Banking in the

Federal Republic of Germany: Ecological and Managerial Considerations,

The Environmental Professional 8, 138-148.

199
APPENDIX A

LIST OF MEETING PARTICIPANTS

Dr. Yoshinari Ambe

Division of Chemistry and Physics

National Institute for Environmental

Studies

Yatabe -machi , Tsukuba, Ibaraki 305

JAPAN

Dr. Richard Batiuk

Chesapeake Bay Program

U.S. Environmental Protection Agency

410 Severn Avenue

Annapolis, MD 21403

Dr. Ulrich Boehringer

Unweltbundesamt

Bismarckplatz 1 1000 Berlin 33

FEDERAL REPUBLIC OF GERMANY

Dr. Jerry J. Bromenshenk

Department of Botany

University of Montana

Missoula, MT 59812

Dr. Stephen N. Chesler

Organic Analytical Research Division

A113 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr. Maria Donner

U.S. Environmental Protection Agency

MD-56

Environmental Monitoring System

Laboratory

Research Triangle Park, NC 27711

Dr. John Elliott

National Wildlife Research Center

Environment Canada

Ottawa, Ontario KlA 0E7

CANADA

Dr. Istvan Gebefugi

Institut fur Okologische Chemie der

Gesellschaft fvir Strahlen-und

Umweltforschung-Munchen

Ingolstadter Landstr. 1 Post Obserschleissheim

D-8042 Neuherberg

FEDERAL REPUBLIC OF GERMANY

Dr. Wilfred Goerke

Ministerium fur Umwelt, Naturschutz

und Reaktorsicherheit

Postfach 12 06 29

D-5300 Bonn 1 FEDERAL REPUBLIC OF GERMANY

Dr. George M. Goldstein

U.S. Environmental Protection Agency

University of North Carolina

Medical Research Building C224-H

Chapel Hill, NC 27514

Dr. Robert R. Greenberg

Inorganic Analytical Research

Division

B108 Reactor Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr. William P. Gregg

Office of Science and Technology

U.S. Department of Interior

National Park Service (484)

Washington, DC 20240

Dr. Jasper Holm

Staatliches Veterinaruntersuchungsamt

Dresdenstrasse 6

D-3300 Braunschweig

FEDERAL REPUBLIC OF GERMANY

Dr. Kenneth Hood

Office of Research and Development

(RD-682)

U.S. Environmental Protection Agency

401 M Street, S.W.

Washington, DC 20460

200
Dr. Robert J. Huggett

Virginia Institute of Marine Science

School of William and Mary

Gloucester Point, VA 23062

Prof. Dr. Jurgen Jacob

Biochemisches Institut ftir

Umweltcarcinogene

Lurup 4

D-2070 Grosshansdorf

FEDERAL REPUBLIC OF GERMANY

Prof. Dr. Fritz H. Kemper

Institute for Pharmacology and

Toxicology

University of Munster

Domagkstrasse 12

D-4400 Munster

FEDERAL REPUBLIC OF GERMANY

Ms. Barbara Koster

Organic Analytical Research Division

A133 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr. Reinhard Kruse

Staatliches Veterinaruntersuchungsamt

ftir Fische and Fischwaren

D-2190 Cuxhaven - F

FEDERAL REPUBLIC OF GERMANY

Mr. Gunnar G. Lauenstein

National Oceanic and Atmospheric

Administration

NOAA/NOS/OOMA

N/OMA32

Rockville, MD 20852

Dr. Robert A. Lewis

Lehrstuhl fur Biogeographie

University of Saarland

D-6600 Saarbrucken

FEDERAL REPUBLIC OF GERMANY

Dr. David Mage

Senior Science Advisor

U.S. Environmental Protection Agency

Environmental Monitoring Systems

Laboratory (MD-56)

Research Triangle Park, NC 27711

Dr. Willie E. May

Chief, Organic Analytical Research

Division

A113 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr . Hugh McKinnon

U.S. Environmental Protection Agency

401 M Street, S.W.

Washington, DC 20460

Dr. Ray Merrill

U.S. Environmental Protection Agency

Industrial Environmental Research

Laboratory (MD-62)

Research Triangle Park, NC 27711

Ms. Reenie M. Parris

Organic Analytical Research Division

A113 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr. Curt Reimann

Deputy Director for Resources and

Operations

National Measurement Laboratory

A363 Physics Building

National Bureau of Standards

Gaithersburg, MD 20899

Mr. Phil Robinson

Design and Development Branch

Office of Toxic Substances

U.S. Environmental Protection Agency

401 M Street, S.W.

Washington, DC 20460

Mr. Michele Schantz

Organic Analytical Research Division

A113 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

Dr. Johan Schledot

Kernforschungsanlage Julich

Institut 4

Angewandte Physikalische Chemie

P.O. Box 1913

D-5170 Julich

FEDERAL REPUBLIC OF GERMANY

201
Dr . George Southworth

Environmental Science Division

Oak Ridge National Laboratory

Oak Ridge, Tennessee 37830

Dr. Wayne R. Wolf

Nutrient Composition Laborator

Human Nutrition Center

U.S. Department of Agriculture

Beltsville, MD 20705

Dr. Markus Stoeppler

Kernforschungsanlage Jtilich

Institut 4

Angewandte Physikalische Chemie

Dr. Rolf Zeisler

Inorganic Analytical Research

Division

B108 Reactor Building

National Bureau of Standards

Gaithersburg, MD 20899

P.O. Box 1913

D-5170 Julich

FEDERAL REPUBLIC OF GERMANY

Dr. Stanley F. Sleva

U.S. Environmental Protection Agency

Monitoring and Data Analysis

Division (MD-14)

Research Triangle Park, NC 27711

Ms. Susan Stone

Inorganic Analytical Research

Division

B108 Reactor Building

National Bureau of Standards

Gaithersburg, MD 20899

Mr. Richard Turle

National Wildlife Research Centre

Canadian Wildlife Service

Environmental Canada

Ottawa KIA 0E7

CANADA

Dr. Erode Ulvedal

Office of Health Research (RD683)

U.S. Environmental Protection Agency

401 M Street, N.W.

Washington, DC 20460

Dr. Gerhard Wagner

Lehrstuhl fur Biogeographie

University of Saarland

D-6600 Saarbriicken

FEDERAL REPUBLIC OF GERMANY

Dr. Stephen A. Wise

Organic Analytical Research Division

A113 Chemistry Building

National Bureau of Standards

Gaithersburg, MD 20899

202
NBS'n4A iREv. 2-ec) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See instructions)

1. PUBLICATION OR

REPORT NO.

NBS/SP-740

2. Performing Organ. Report No. 3. Publication Date April 1988

4. TITLE AND SUBTITLE

PROGRESS IN ENVIRONMENTAL SPECIMEN BANKING

5. AUTHOR(S)

Stephen A. Wise, Rolf Zeisler, and George M. Goldstein

6. PERFORMING ORGANIZATION (If joint or other than N6S. see instructions) 7. Contract/Grant No. NATIONAL BUREAU OF STANDARDS

8. Type of Report & Period Covered

U.S. DEPARTMENT OF COMMERCE

GAITHERSBURG, MD 20899 Final

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street. City. State. ZIP)

10. SUPPLEMENTARY NOTES

Library of Congress Catalog Card Number 88-600530

I I Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11. ABSTRACT (A 200-worcl or less factual summary of most significant information. If document includes a si gnificant

bibliography or literature survey, mention it here)

In the past decade, interest in the conLept of specimen banking for the

archiving of biological and environmental samples for future analysis has

increased significantly, and specimen banking is now recognized as an integral part of systematic environmental monitoring. Since the establishment

of pilot Environmental Specimen Banking Programs in the U. S. and the

Federal Rebuplic of Germany (FRG) in the late 1970' s, formal meetings have been

held annually between representatives of these two programs to discuss results

and future needs related to specimen banking. In recent years representatives

of similar programs in Japan, Canada, and Sweden have joined in these meetings to

expand the exchange of information.

In October, 1986, the 10th U.S. -German Seminar of State and Planning on Environmental Specimen Banking was held at the Virginia Institute of Marine

Sciences in Gloucester Point, Virginia. At this meeting the current status of

specimen banking activities in the U. S., FRG, Canada, and Japan was presented

and discussed. Ibis publication contains the proceedings of that meeting with

contributions describing various activities related to banking and analysis of

samples from aquatic, atmospheric, terrestrial, and human monitoring programs.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

atmospheric pollution; gas chromatography; human tissue analysis; inorganic analysis;

marine pollution; neutron activation analysis; organic analysis; pollutaants;

specimen banking; trace element analysis

13. AVAILABILITY

^pC] Unlimited

I I For Official Distribution, Do Not Release to NTIS

2CX1 Order From Superintendent of Documents, U.S. Government Printing Office, Washington, D.C.

20402. Q Order From National Technical Information Service (NTIS), Springfield, VA. 22161

14. NO. OF

PRINTED PAGES

217

15. Price

USCOMM-DC 6043-P80


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