MBS

PUBUCATiONS AlllQP

NBSIR 88-3750

Alaskan Marine Mammal Tissue

Archival Project: A Project

Description Including Coilection

Protocols

Paul R. Becker

Alaska Office, Ocean Assessments Division

National Ocean Service

National Oceanic and Atmospheric Administration

Anchorage, Alaska 99513

and

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

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Center for Analytical Chemistry

Gaithersburg, Maryland 20899

March 1988

-QC

100

.056

#88-3750

1988

C.2

idy was funded by the Mineral’s Management Service, Department of the

, through an Interagency Agreement with the National Oceanic and

iheric Administration, Department of Commerce, as part of the Alaska Outer

mtal Shelf Environmental Assessment Program.

Stimulating America's Progress 1913-1988

Research Information Center

iNalional Bureau of Standards

Gaithersburg, Maryland 20899

ALASKAN MARINE MAMMAL TISSUE

ARCHIVAL PROJECT: A PROJECT

DESCRIPTION INCLUDING COLLECTION

PROTOCOLS

NBSIR 88-3750

Paul R. Becker

Alaska Office, Ocean Assessments Division

National Ocean Service

National Oceanic and Atmospheric Administration

Anchorage, Alaska 99513

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

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

Center for Analytical Chemistry

Gaithersburg, Maryland 20899

March 1988

This study was funded by the Mineral’s Management Service, Department of the

Interior, through an Interagency Agreement with the National Oceanic and

Atmospheric Administration, Department of Commerce, as part of the Alaska Outer

Continental Shelf Environmental Assessment Program.

and

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

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Director
PREFACE

The Alaskan Marine Manunal Tissue Archival Project was initiated in 1987 with

financial support from the Minerals Management Service, Outer Continental Shelf

(OCS) Studies Program. Although the emphasis is on the collection of tissues

for analysis of contaminants that may be associated with the petroleum industry, it is also recognized that the development of an archive of marine

mammal tissues collected and stored using carefully controlled procedures

provides a resource that goes beyond the immediate needs of the OCS Program.

Such an archive developed over several years provides a resource of materials

for future investigators addressing questions concerning the transport of

elements and compounds (contaminants and non- contaminants) throughout the polar

ecosystem, regardless of source. It is hoped that this resource will gain wide

support from the many agencies involved in marine mammal research and management, environmental assessment and management, as well as organizations and

individuals with interests in the polar ecosystem, as a whole.

This document provides the basic information on Project objectives and management, justification for the species, tissues, and contaminants of interest, and

specific instructions for collecting, handling, and storing samples. At this

time, the protocols have been employed only in the collection of northern fur

seal tissues, therefore, the details are somewhat biased toward this species.

As these procedures are applied to the sampling of other marine mammals, the

protocols will probably have to be modified. Therefore, this document represents the first in a series of reports providing the most recent protocols used

by the Project. Specific comments on the protocols and other contents of this

and future reports which can be used to improve the project are welcome.

Paul R. Becker

Ocean Assessments Division

National Ocean Service, NOAA

701 C Street, Box 56

Anchorage, Alaska 99513

ii
ACKNOWLEDGEMENTS

Numerous individuals and organizations have provided advice and encouragement

during the development of this project, including suggestions for species

selections with justifications and practical advice for design of the sampling

protocols. Prominent among these have been:

Steve Treacy, Minerals Management Service; Kathy Frost and Lloyd Lowry, Alaska

Department of Fish and Game; Howard Metsker, U.S. Fish and Wildlife Service;

Drs. Robert DeLong, Charles (Chuck) Fowler, and Linda Jones, National Marine

Mammal Laboratory, National Marine Fisheries Service; Benjamin P. Nageak, Dr.

Thomas Albert, Charles Brower, and the staff at the Department of Wildlife

Management, North Slope Borough; the Alaska Eskimo Whaling Commission and the

Eskimo Walrus Commission.

The protocols described in this report were tested and refined during the July,

1987, sampling of northern fur seals on St. Paul Island. This work could not

have been accomplished without the expert advice and aid provided by Dr. Steve

Zimmerman, Chief, Alaska Office of Marine Mammals and Endangered Species,

National Marine Fisheries Service. Dr. Zimmerman set the stage for logistic

support and local cooperation that allowed us to sample the St. Paul Island fur

seals. Staff from the National Marine Mammal Laboratory provided numerous pointers on the practicality of sampling this species in the field.

A special note of appreciation is extended to Dr. Terry Spraker, College of

Veterinary Medicine, Colorado State University. Dr. Spraker provided expert

consultation on the sampling of this species, removed the tissues from the

animals in the field, and produced a necropsy report on each of the individual

animals sampled. Additional support was provided by Dr. Pat Kozloff, St. Paul,

and Sara Pumphrey, College of Veterinary Medicine, Texas A6cM University.

The successful testing of protocols at St. Paul could not have been conducted

without the cooperation and help provided us by the residents of St. Paul and

the Tanadgusix (TDX) Corporation. Key individuals were Pat Kozloff, Darleen

Melovidov, John R. Merculief, and Gregory Fratis, seal harvest foreman, TDX

Corporation.

This study was funded by the Mineral's Management Service, Department of the

Interior, through an Interagency Agreement with the National Oceanic and

Atmospheric Administration, Department of Commerce, as part of the Alaska Outer

Continental Shelf Environmental Assessment Program.

iii
CONTENTS

Page

PREFACE ii

ACKNOWLEDGEMENTS iii

LIST OF FIGURES v

LIST OF TABLES v

INTRODUCTION 1

Goals and Objectives ; . . 2 Management System 2

BACKGROUND 3

Species of Interest 4

Selection Criteria 7 Species Evaluation 8

Contaminants 11

Uptake, Transport, and Compartmentalization 14

Excretion 16

Accumulation 17

Choice of Tissues for Archival 18

Variability in Contaminant Measurements 20

METHODS 22

Collection Protocols 22

Materials 22

General Organization 23

Stage I, Tissue Removal 25

Stage II, Tissue Processing 28

Stage III, Tissue Packaging and Shipping 32

Sample Archival 33

Monitoring of Sample Stability During Storage 33

Quality Assurance 34

REFERENCES CITED 35

APPENDICES 39

Data Recording Forms: Instructions 39

Special Instructions 44

Contamination Sources 44

Procedures for Cleaning Titanium Knife 45

Procedures for Preparing Pinniped Teeth 45

iv
O

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CO

FIGURES

Page

1. Absorption, Distribution and Excretion

of Xenobiotics 15

2. Generalized Collection Protocol 24

TABLES

l.a. Marine mammals of principal interest 4

l.b. Marine mammals of principal interest 5 Marine mammals of principal interest 6

Trace organic contaminants of interest 12

Trace elements of interest listed in descending priority

by groups 13

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INTRODUCTION

The concept of archiving biological and environmental samples for retrospective

analysis is recognized as a major component of systematic environmental monitoring. The long-term storage of carefully selected, representative samples in an environmental specimen bank is an important complement to the real-time

monitoring of the environment. These archived samples permit:

1 . The use of new and innovative analytical technology that was not

available at the time the samples were archived, for clear state-ofart identification and quantification of analytes of interest, and

2. The identification and quantification of analytes that are of

Interest at the present but that were not of interest at the time the

samples were archived.

The retrospective analysis of archived samples allows the comparison of present

and past analytical techniques and values, thus providing continued credibility

of past analytical values, and allowing flexibility in environmental monitoring

programs

.

Marine mammals are considered top predators in the marine environment.

Chemical analysis of their tissues can be particularly useful in determining

whether bioaccumulation of contaminants (and potential biological effects)

associated with human industrial activities, including offshore petroleum and

mineral extraction, is occurring in marine food chains. The collection of

marine mammal tissues over a period of several years will provide an archive of

samples that can be used to determine baseline contaminant levels against which

future contaminant measures can be compared.

Collections of marine mammal tissues began in 1987. Protocols for collecting,

handling, shipping, and storing the tissues were developed and are being tested

through cooperative efforts by Minerals Management Service (MMS) , National

Oceanic and Atmospheric Administration (NOAA) , and National Bureau of Standards

(NBS) and through coordination with National Marine Fisheries Service (NMFS),

U.S. Fish and Wildlife Service (USFWS) and Alaska Department of Fish and Game

(ADF&G)

.

The Alaskan Marine Mammal Tissue Archive is maintained by NBS in the National

Biomonitoring Specimen Bank (NBSB) , Gaithersburg, Maryland. This facility,

designed for long-term storage, is the result of 10 years development Involving

cooperative efforts between NBS and EPA, and several years of comparative

studies with specimen archiving programs of West Germany, Japan, Sweden, and

Canada. Other agencies using the NBSB include EPA, U.S Department of Agriculture (USDA) , Food and Drug Administration (FDA) , the National Cancer Institute

(NCI), and the National Status and Trends Program of NOAA.

1
Goals and Objectives

The goal of the Alaskan Marine Mammal Tissue Archival Project is to archive a representative collection of Alaskan marine mammal tissues for future

contaminant analyses and documentation of long-term trends in environmental

quality. The Project has three objectives for the first two years:

1 . Collect Alaskan marine mammal tissues that are suitable for

determining levels of organic and inorganic contaminants.

Collections of tissues for archival are being limited to freshly killed

animals taken by researchers or taken in subsistence hunts. When a sample

archived by this project is analyzed, the researcher must have confidence

that the sample was collected as prescribed in acceptable protocols (refer

to Methods Section) . No dead and stranded animals nor old specimens

archived from past programs will normally be accepted by this project. As an additional task, however, tissue collections held by other individuals

and organizations are being cataloged and their suitability for future

contaminant analysis by this project evaluated.

2. Transport, catalog, and curate the tissues in a condition suitable

for long-term storage and eventual contaminant analyses.

After collection, samples are packaged, transported, cataloged, and

archived according to protocols consistent with those employed by the

National Biomonitoring Specimen Bank. Storage is under liquid nitrogen

vapor at -150 “C, which is the best condition available for minimizing

sample degradation. Samples will be selected by OCSEAP/MMS for future

contaminant analysis. Emphasis will be on those contaminants associated

with offshore mineral extraction. Requests by other researchers and

agencies for archived samples will be considered on a case-by-case basis.

3. Determine the most appropriate collection protocols for long-term

specimen banking of marine mammal tissues.

Field collection protocols were tested in July 1987 during sampling of the

northern fur seals on St. Paul Island. The protocols were evaluated as to

their practicality and suitability for obtaining uncontaminated samples of

four tissue types (liver, kidney, blubber and muscle) and were revised as warranted. These revised protocols are found in the "Methods" section of

this report; they will provide the guidelines for future tissue sampling.

Protocol evaluation will continue throughout the life of the Project as more species are sampled. Selected tissue samples may also be analyzed to determine

the suitability of each tissue with respect to levels of inorganic and organic

contaminants

.

Management System

The collection, packaging, and shipment of samples are being conducted by

OCSEAP/MMS researchers under existing separate programs, and through cooperative efforts with other management, survey and research programs of agencies

such as NMFS, USFWS , and ADF&G.

2
Cataloging and archiving of samples is done by the National Biomonitoring

Specimen Bank (NBSB) , National Bureau of Standards at Gaithersburg, Maryland.

Procedures are consistent with those employed by the NBSB in support of the

National Status and Trends Program.

Requests for any tissues for analyses or other uses will be considered.

Release of the tissues to outside investigators will be contingent upon the

approval of the sponsor of this project, MMS , after consultation with OCSEAP,

NBS , and the organization originally providing the sample. Release of these

tissues will depend on a determination that a surplus of requested tissues

exists beyond anticipated sampling or analytical needs and that such analyses

or uses will be performed cooperatively by MMS and the requesting organization.

Requests for samples will require a written proposal that includes the

following information:

1. Individual/organization making the request,

2. Purpose of the proposed study,

3 . Researcher/laboratory conducting the analyses

,

4. Kind of analyses to be performed/purpose of analyses,

5. Procedure/instrumentation/detection level,

6. Accuracy/precision to be expected,

7. Quality control procedures to be used, and

8. Agreement to provide OCSEAP/MMS, NBSB, and the individual or organization originally providing the samples with the data resulting

from the analyses

.

The Project will publish Annual Tissue Inventory Reports. These reports will

provide the current inventory of tissues maintained in the Archive and the

results of any chemical analyses.

Opportunities for cooperative efforts and exchange of information with the

Canadian Wildlife Service and the Canadian Department of Fisheries and Oceans

are being pursued. An important source of contaminant data on species common

to both Alaska and Canada is the Canadian Wildlife Service's National Registry

of Toxic Chemical Residues and National Specimen Bank (Elliott 1987)

.

3
BACKGROUND

Species of Interest

The marine mammals of principal interest include: polar bears, bowhead and

belukha whales, Dali's porpoise, walrus, Steller sea lions, northern fur

seals, bearded seals, ringed seals, spotted seals, harbor seals, and sea otters. These animals represent a range of sizes, habitat use, and subsistence

values. Table 1 presents a matrix of these animals and the factors most

prominent in determining which species will be sampled, and when and where the

sampling will probably take place. These factors are:

1 . the geographic range of the species

,

2. geographic range of a single population (whether local or migratory),

3. mode of potential contamination through the food chain (bottom or pelagic feeder)

.

4. whether it is a subsistence species.

Table la. Marine Mammals of Principal Interest

Factors PLBR BWHD BLKA DLPR

Geographic Range Broad Broad Broad Broad

Local vs Migratory

Migr . (ice) Migr. Migr. Migr.

Feeding Mode Top Pred. Pelag. (Zoopl .

)

Pelag. (Fish) Pelag. (Squid, Fish)

Subsistence Mode Major Major Major None

Biological

Information

Good Adequate Adequate Adequate

Ease of

Collecting

No Yes No Yes

Ongoing Program Yes(FWS) Yes(MMS) No Yes(NMFS)

Collection Period

(months)

? 4-8 5-7 5-8

Collection Area ? Barrow

Wainwright

Point Hope

Kivalina

S. Bering (Gill

Net Fishery)

Contaminant

Information Yes Yes Yes ?

PLBR,

BWHD,

BLKA,

DLPR,

Polar Bear

Bowhead Whale

Belukha Whale

Dali Porpoise

4
Table lb. Marine Mammals of Principal Interest

Factors WLRS STSL HBSL FRSL

Geographic Range Broad Broad Broad Broad

Local vs Migratory Migr . (ice) Migr. Local Migr.

Feeding Mode Benthic (Clams) Pelag. (Fish) Pelag. (Fish) Pelag. (Fish)

Subsistence Use Major Minor Minor Major

Biological

Information Good Good Good Good

Ease of Collecting Yes Yes No Yes

Ongoing Program Yes(FWS) Yes(NMFS) No Yes(NMFS)

Collection Period

(Months)

5-8 ? ? 7-8

Collection Area St. Lawrence I. Little Diomede

Nome

Point Hope Barrow

? Icy Bay Tugidak I

.

Glacier Bay

Izembek Lagoon

Pribilofs

Contamination

Information Yes Yes Yes Yes

WLRS,

STSL,

HBSL,

FRSL,

Walrus

Steller Sea Lion

Harbor Seal

Fur Seal

5
Table Ic. Marine Mammals of Principal Interest

Factors BDSL RGSL SPSL SEOT

Geographic Range Broad Broad Restricted Broad

Local vs. Migratory — rngiatoty ^ xce ) — Local

Feeding Mode Benthic Pelag. (Fish,

Zoopl. Crustaceans)

Pelag. (Fish) Benthic

Subsistence Use Major Major Major No

Biological

Information Adequate Good Adequate Good

Ease of Collecting No Yes Yes? No

Ongoing Program No No No Yes(FWS)

Collection Period 5-8 5-8 5-8 ? (Months)

Collection Area Nome Prince William

Kotzebue Sound, Kodiak

Wainwright

Barrow

Shishmaref

Contaminant

Information Yes Yes Yes 7

BDSL, Bearded Seal

RGSL, Ringed Seal

SPSL, Spotted Seal

SEOT, Sea Otter

6
5. availability of baseline biological information,

6. ease of collecting fresh samples where source is predictable and

protocol can be followed,

7. whether agency programs exist that can provide collections,

8. periods when collections are feasible,

9. geographical areas of potential collection, and

10.

availability of contaminant information.

Selection Criteria

Five of these 10 factors function as principal criteria for the selection of

the species to be sampled. Each of these criteria are discussed below.

The Geographic Range of the Species . A species with a broad geographic range

would be of more value for monitoring purposes and tissue archival than one of

a restricted range since such a species would provide some measure of com- parability on an international basis. Samples from a single species from a variety of locations throughout the Arctic or within the U.S. on a north-south

or east-west gradient could provide the comparability which could be used in

interpretation of contaminant levels found in that species. For example, a species such as the harbor seal, which occurs in both the Atlantic and Pacific,

and from Alaska to Baja California would provide more comparative opportunities

than the ribbon seal which occurs only in the Bering and Chukchi seas

.

Mode of Potential Contamination through the Food Chain (Bottom or Pelagic

Feeder") . Selection of species to sample should be representative of the range

of feeding modes which occurs in marine mammals. Rather than restricting the

sampling to only pelagic fish eating mammals, species representing plankton

feeders, benthic feeders, and top predators (e.g., feed on seals and walrus)

will also be sampled.

Subsistence Use . A substantial portion of the diet of coastal residents of the

Beaufort, Chukchi and Bering seas is marine mammals. Selection of marine

mammals which are consumed by humans, or which utilize the same species as commercial fisheries, would not only provide information on environmental

contaminant levels, but also data with possible implications regarding human

health. Areas such as the Bering Sea export fish all over the world, yet there

is no nearby human population to easily test for contaminants derived from

these fish. Marine mammals are readily available indicators of unusual levels

of cont^lminants that could cause human health concerns

.

Availability of Baseline Biological Information . Interpretation of contaminant

information requires biological knowledge regarding the species of interest.

This knowledge includes life cycle information, behavior, distribution and

movements of populations, age and reproductive status, feeding behavior, and

basic physiology. Such information would be necessary in order to determine

where and how contaminants might be accumulated, to explain the physiological

response of the animal , and to determine how internal regulation of con- taminants and biochemical byproducts might affect the health of individual

animals and populations

.

7
Ease of Collecting Fresh Samples where Source is Predictable and Protocol can

be Followed . The archival of tissue samples and its contribution to the

monitoring of contaminants require that samples be reliably and economically

available on a long-term basis. The species chosen should not be so rare that

future collection might be jeopardized by restrictive permit requirements. If

samples are to be obtained from hunters, the hunt should occur predictably at a location which is near a commercial airport and where samples can be adequately

processed and preserved. Meeting the standards of sampling protocols might be

difficult at any collection location. Hunting often occurs at remote camps

with no electricity, under less-than-sterile conditions, and under extreme

weather conditions that hampers normal human activities

.

Species Evaluation

Presented below are discussions of each of the marine mammal species of

interest as related to the selection criteria for the tissue archival. With

the exception of Dali's porpoise, these discussions are based on information

provided by Kathy Frost (personal communication 1987) .

Polar Bear (Ursus maritimus) . This mammal is widely distributed throughout the

Arctic. Its management is subject to international treaty, and it has been a species of interest relative to contaminant monitoring and archival programs in

Canada. The polar bear is a top predator; contaminants present in its tissues

may be reflective of those present in lower trophic forms (fish, seals, etc.)

and passed up through the "food chain". Basic biological data are available

for this mammal. USFWS research on this species is ongoing and involves

monitoring the subsistence harvest and radio tracking.

Polar bears are eaten by humans, but to a lesser extent than seals or walruses.

Tissue sampling would require development of a network among hunters, and

coordination with the agency managing this species. If regulations are implemented that require the sealing of polar bear hides, it might be possible

to build a sampling scheme to obtain necessary archival material. Sampling

protocol would have to accommodate field collection of specimen material, most

likely by standard hunting implements. Hunting does not occur within a predictable time frame but is spread over many months. Thus, in order to be

economically feasible, sampling would have to be coordinated by on-site agency

personnel rather than by a special sampling team.

Bowhead Whale (Balaena mvsticetus) . The bowhead whale occurs in the western

Arctic of Alaska and Canada, and in the eastern Arctic. It feeds on zooplankt- on (primarily copepods and euphausiids) , and in this regard has its closest

trophic connection to the ringed seal which feeds primarily on euphausiids and

arctic cod. Baseline biological information on the bowhead is more limited

than for some other Arctic marine mammals, but the basics of seasonal

migrations and food habits are known. At present, there is considerable

controversy regarding age determination, but research is ongoing.

The bowhead whale is one of the most important marine mammals used by coastal

residents from St. Lawrence Island to Kaktovik. Although the bowhead harvest

is small, biologists are present (in Barrow) to obtain and care for samples;

additional personnel should not be required. Because of high international

visibility, funding and personnel will probably be available in future years to ensure a successful tissue archival program. There would probably be

8
considerable local interest. In the past, the North Slope Borough Department

of Wildlife Management in cooperation with the Alaska Eskimo Whaling Commission

have collected and analyzed tissues from this species.

Belukha whale (Delph inapterus leucus) . This animal is widely distributed

throughout the Arctic and Subarctic; the western Arctic population is shared by

Canada and Alaska. The belukha eats primarily fish, cephalopods, and some crustaceans, and are trophically similar to spotted seals. They consrune many

of the same species taken by commercial fisheries (herring, salmon, pollock,

etc) . Although more data are needed, baseline information is available on distribution, reproduction, and food habits. This odontocete cetacean (toothed

whale) presents a different taxonomic group than seals and walruses, which are pinnipeds, and may metabolize and store contaminants somewhat differently.

The belukha whale is a major subsistence species in Alaska and Canada. The

logistics for obtaining tissue samples might be complicated. The largest hunts

in Alaska occur at Point Hope or Kivalina in the spring, and in Kotzebue Sound

during early summer. Since imposition of the bowhead quota, hunters at Point

Hope spend less time on the ice and consequently take fewer belugas. The

timing of this hunt is variable depending on weather, ice conditions, and other

hunting activities . There are no ADF&G or USFWS offices in these communities

,

nor do agency personnel monitor the harvest. The Kotzebue Sound belukha hunts

have traditionally been the largest in the state. However, they are unpredictable; during the past three years, the harvest has been near zero. The main

belukha hunting camp (Elephant Point) can be accessed only by charter aircraft

or small boat. Running water and electricity are not available, so it would be

difficult to process and store tissue samples.

Pali's Porpoise (Phocoenoides dalli) . Dali's porpoise occurs in the North

Pacific within Japanese and Russian waters eastward to the North American

coast. This species is especially abundant in the Sea of Okhotsk and the

southern Bering Sea, ranging at least as far north in the summer as the

Pribilof Islands. Although separation of the different stocks and details of

migrations are poorly known, genetic evidence indicates that the animals of the

southern Bering Sea are of single stock (Linda Jones, personal communication).

This species feeds primarily on squid, pelagic fish and crustaceans.

Background information is adequate

.

Although Dali's porpoise is not a subsistence species, tissue samples could

probably be obtained from the NMFS Cetacean Research Program. NMFS collects

whole animals caught by the Japanese high seas gillnet fishery in the southern

Bering Sea. These animals are quick frozen on board the Japanese vessels and

then returned to the National Marine Mammal Laboratory for study. If proper

arrangements are made ahead of time, application of appropriate protocols for

obtaining tissues for archival from the specimens is quite feasible.

Pacific Walrus (Odobenus rosmarus divereens) . The Pacific walrus does not

occur throughout the Arctic. There is, however, an Atlantic subspecies which

could be used in comparisons. The walrus is a benthic feeder, eating primarily

bivalve molluscs which are known to bioaccumulate some environmental con- taminants. It is a major subsistence resource to coastal residents of Alaska;

the Atlantic walrus is eaten by the Canadian Inuit. Baseline biological data

regarding distribution, movements, reproduction, and food habits are available,

and studies are ongoing.

9
Hunting for walrus occurs every year in locations which provide access to air

freight services and reasonable laboratory and freezer facilities. ADF&G

maintains field offices in several walrus hunting villages. USFWS hires people

each year who might assist in sample collection. Hunter participation could

possibly be coordinated through the Eskimo Walrus Commission. There is already

considerable interest in contaminants in walrus because of the recent attention

drawn to the high cadmium levels in this species.

Steller Sea Lion (Eumetopias iubatus) . The Steller sea lion occurs from the

Bering Sea south to California. Its diet is similar to that of the harbor seal

and includes commercial species such as salmon, herring, and pollock. Baseline

information on sea lions is relatively good.

There has been a substantial decline in numbers of this species since the

1970' s for unknown reasons and it might be declared depleted, which could make

sampling difficult. However, it may be desirable to obtain archival material

from a declining population. Tissues would probably have to be obtained

through directed sampling efforts and in conjunction with other research by

NMFS or ADF&G.

Northern Fur Seal (Callorhinus ursinus) . The northern fur seal summers in

Alaskan waters and winters south to California. It also occurs in Japanese and

Soviet waters. This species eats primarily fish and squid, and utilizes

species which are commercially harvested, particularly pollock. Extensive

baseline data exists.

Fur seals are harvested annually (July-August) on the Pribilof Islands under

very controlled conditions. There has been a steady 5-7% per annum decrease

in the numbers of Pribilof animals in recent years. Causes for this decline

are unknown. Efforts are underway to declare fur seals depleted. The current

harvest should allow ready access to samples, but it includes only subadult

(3-4 year old) males. Unless special additional sampling were done in conjunction with other research activities, there would be little opportunity to

archive samples from females or reproductive -age animals. NMFS personnel

currently conduct research on the Pribilof Islands and could aid in the sample

collection.

Harbor Seal (Phoca vitulina) . This species occurs from Bristol Bay south to

Baja California, along eastern and western Atlantic coasts, and in Japan.

Populations are localized and do not migrate to any extent. This would allow

comparative sampling in different parts of its range. Harbor seals eat

primarily fish and lesser amounts of invertebrates such as shrimp and octopus.

Their diet includes many species which are also commercially fished, including

salmon, herring, pollock, and smelt. They may compete quite extensively with

fisheries. The data base on this animal is extensive.

Harbor seal populations in parts of Alaska are currently declining, for unknown

reasons. This species is not a primary subsistence species; therefore, tissue

samples would have to be obtained through collections conducted as part of

other research, or through collections directed solely at obtaining archival

material

.

Bearded Seal (Erignathus barbatus) . The bearded seal is circumarctic in

distribution, but is less abundant than the ringed seal. It is similar to the

10
walrus in being a bottom feeder, but the bearded seal's diet is generally more

diverse. It is a preferred human food species because of its relatively large

size (up to 250 kg). Baseline biological information is available.

Bearded seals are hunted primarily in the spring, beginning not long after

pupping. Because of their large size, almost all bearded seals are butchered

on the ice during the hunt. Only the meat or other desired parts are returned

to the beach. This would make it more difficult to ensure that S£impling was accomplished according to prescribed procedures.

Ringed Seal (Phoca hispida) . The ringed seal is circumarctic and the most

abundant of the northern pinnipeds. It feeds pelagically on mostly non- commercial species, such as arctic and saffron cod. It is the mainstay in the

diet of the polar bear and an important source of food for the northern Eskimo.

Baseline biological data on this species are quite extensive.

Ringed seals are harvested throughout northern Alaska, including the larger

population centers of Nome, Kotzebue, and Barrow, and obtaining samples should

be straightforward. Local customs determine that whole seals are brought back

to be butchered by the women, which should facilitate sampling of tissues.

Seals are hunted primarily in the spring just after pupping and breeding.

Spotted SeaKPhoca largha) . Although closely related, this species is not as widespread as the harbor seal. It occurs in the Bering and Chukchi seas and,

in much smaller numbers, in the Beaufort Sea. Its diet is similar to that of

the harbor seal and beluga whale. The biological data base is less complete

than for the harbor seal but is probably adequate for selecting and characterizing archived specimen material. Spotted seals are hunted by coastal

Eskimos in the spring and autumn but the largest and most predictable hunts

occur in small remote villages.

Sea Otter (Enhvdra lutris) . The sea otter is abundant and is increasing in

Alaska. It is widely distributed from the Bering sea south to California. It

is a bottom feeder, often eating commercial species such as Dungeness crab and

abalone. This species is not a significant food item for humans, nor for any

other large carnivore. It would be difficult at present to obtain specimen

material except through a directed collection program. Existing and proposed

research plans do not include sacrifice of animals.

Contaminants

The contaminants of interest (Tables 2 and 3) are essentially those being

addressed by NOAA's National Status and Trends Program (Lauenstein 1986).

These include organic contaminants, consisting of aromatic hydrocarbons,

chlorinated compounds, and polychlorinated biphenyls (PCBs) , and trace elements, consisting primarily of heavy metals. Other contaminants will probably

be added to these lists as the project develops. Candidates for inclusion are:

additional cyclodienes such as toxaphene and endosulphan, polychlorinated

dibenzodioxins and furans , nitrosamines and other nitrogen containing com- pounds, and organometallic compounds of elements such as arsenic, mercury,

phosphorous, selenium, and tin.

The trace elements of Table 3 are listed in descending priority by groups,

based on the classification of Coyer (1986). The high priority elements are

11
Table 2. Trace Organic Contaminants of Interest. These are Consistent with

Those Being Analyzed by NOAA's National Status and Trends Program

(Lauenstein and Young, 1986). Alternate names as listed in "The

Merck Index"

.

Aromatic and Heterocvclic Alternate Name Hydrocarbons

Acenaphthene 1 , 2 -Dihydroacenaphthylene

Anthracene Paranaphthalene

Benz [a] anthracene 1 , 2 -Benzanthracene

Benzo [ a ] pyrene 3 ,4- Benzpyrene

Benzo [e]pyrene 1,2 -Benzpyrene

Biphenyl Dephenyl ; phenylbenzene

Chrysene 1,2- Benzphenanthrene

Dibenz [a,h] anthracene - - - Dibenzo thiophene - - - Fluoranthene l,2-(l,8-naphthylene) benzene

Fluorene o-Biphenyleneme thane

Naphthalene - - - Perylene Dibenz [ de , kl ] anthracene

Phenanthrene - - - 1-Methylphenanthrene - - - Pyrene Benzo [ de f ] phenanthrene

Chlorinated Comnounds

Aldrin 1,2,3,4,10, 10-Hexachloro-l ,4.4a, 5 , 8 , 8a-hexahydro1,4:5 , 8-dimethanonaphthalene

alpha-Chlordane 1,2, 4, 5, 6, 7, 8, 8-Octachloro-2 , 3 , 3a ,4 , 7 , 7a- hexahydro - 4 , 7 - me thano - IH - indene

o ,p' -ODD l-Chloro-2- [2 , 2-dichloro-l- (4-chloro

phenyl) ethyl Jbenzene

p,p' -DDD 1, l-Dichloro-2 ,2-bis- (p-chlorophenyl)ethane

0 , p ' -DDE l-Chloro-2- [dichloro-1- (chlorophenyl)

e theny1 ] b enzene

p , p ' -DDE 1,1'- (Dichloroethenylidene) -bis (4-chlorobenzene)

0 ,p' -DDT 1- (4-Chlorophenyl)ethyl]benzene

p,p'-DDT 1 , 1'

- (2 , 2 , 2-Trichloroethylidene)bls [4-

chlorobenzene

]

Dieldrin 3 , 4 , 5 , 6 , 9 , 9-Hexachloro- la, 2, 2a, 3, 6, 6a, 7,7aoctahydro-2 , 7 : 3 , 6-dime thanonaphth[ 2 , 3-b ]

oxirene

Endrin 1, 2 , 3 ,4, 10 , 10-Hexachloro-6,7-epoxy1,4, 4a, 5, 6, 7, 8, 8a-octahydro-endo,endo1,4:5, 8-dlmethanonaphthalene

Heptachlor 1 , 4, 5 , 6 , 7 , 8 , 8-Heptachloro-3a ,4 , 7 , 7a-tetra

hydro -4, 7 -methano-lH- Indene

Heptachlor epoxide 1 , 4 , 5 , 6 , 7 , 8 , 8-Heptachloro-2 , 3 -epoxy- 3a ,4,7,7atetrahydro-4 , 7-methanoindan

Hexachlorobenzene - - - Lindane ( gamma -BHC) Gamma -Hexachlorocyclohexane

Mirex 1 , la , 2 , 2 , 3 , 3a,4,5 , 5a, 5b , 6-Dodecachlorooctahydrol , 3 ,4-metheno-lH-cyclobuta[c ,d]pentalene

trans -Nonachlor 1,2, 3, 4, 5, 6, 7, 8 , 8-Nonachloro-2 , 3 , 3a , 4 , 7 , 7a- hexahydro - 4 , 7 - me thano - IH - indene

PCB' s

Dichlorobiphenyls - . . Trichlorobiphenyls - - - Tetrachlorobiphenyls - - - Pentachlorobiphenyls - - - Hexachlorobiphenyls - - - Heptachlorobiphenyls - - - Octachlorobiphenyls - - - Nonachlorobiphenyls - - -

12
Table 3. Trace elements of interest listed in descending priority by groups.

This list is a modification of those being analyzed by NOAA's

National Status and Trends Program (Lauenstein and Young 1986)

.

Classification is based on that of Coyer (1986).

High priority elements :

A. Toxic metals; can result in multiple effects:

As Arsenic

Cd Cadmium

Hg Mercury

Pb Lead

B. Essential metals with potential for toxicity and/or related to

toxicity of Group A, above:

Cu Copper

Cr Chromium

Se Selenium

Zn Zinc

Medium priority elements :

A. Essential metals with potential for toxicity:

Co Cobalt

Mo Mo lybdenum

B. Elements related to metal metabolism:

Ca Calcium (also Vitamins D and E)

Fe Iron

C. Minor toxic metals significant to oil industry:

Ni Nickel

V Vanadium

Ba Barium

Low priority elements :

A. Minor toxic metals:

Ag Silver

A1 Aluminum

B Boron

Be Beryllium

Mn Manganese

Sb Antimony

Sn Tin

Te Tellurium

T1 Thallium

13
those most commonly associated with environmental contaminant problems. These

consist of highly toxic, non-essential metals that can produce multiple

effects, metals essential to mammals but with potential for toxic reactions at

elevated levels, and metals that interact with other more toxic metals to

modify their toxicity.

The medium priority elements consist of essential metals that have moderate

potential for toxicity, elements related to metals metabolism and important to

the interpretation of trace element concentrations, and minor toxic metals

significant due to their elevated levels in crude oils.

The low priority elements consist of elements of low to moderate toxicity.

These will probably not be considered in routine tissue analysis. Although

beryllium is considered to be a highly toxic metal, it is included in the low

priority group since the principal mode of uptake is via the respiratory system

and is associated with heavy levels of fossil fuel combustion.

The contaminant load of an organism is the result of many factors associated

with the contaminant, the organism, and the environment. Among these factors

are the physiochemical characteristics of the contaminant, the relative

quantities of the contaminant released into the ecosystem, the transport rate

within the environment, the detoxification/degradation processes operating in

the environment, and the uptake, accumulation, and internal regulation pro- cesses of the organism, itself. The availability and toxicity of a contaminant

is related to its organic/inorganic form, valent state, reaction and

competition with other elements or compounds, and lipid solubility.

Uptake. Transport, and Compartmentalization

The basic mammalian routes of absorption, distribution and excretion of

xenobiotics are conceptualized in Figure 1. In this model, a whole animal is

visualized as consisting of peripheral components (organs) which are linked to

a central component (the circulatory system), but not to each other. The

circulatory system, consisting of blood, lymph, and extracellular fluid, is the

component that receives the contaminant from and returns it to the exterior of

the organism and distributes the contaminant to all of the organs.

Of the three major routes of contaminant uptake (ingestion, inhalation, and

dermal absorption) , the one of most importance to marine mammals is probably

ingestion. For example, food contributes over 90% of the organochlorine intake

by mammals (Campbell et al. 1965).

Regardless of method of uptake, substances have to move across biological

membranes in order to enter the circulatory system and in order to be transported from the extracellular fluid into the individual tissue cells. There

are many absorption mechanisms including: filtration, active transport,

facilitated diffusion by association with carrier molecules, pinocytosis

(formation of lysosomes), and passive diffusion. For the majority of compounds

that might be of significance from an environmental standpoint, however, the

primary mechanism of absorption is passive diffusion (Tinsley 1979; Klaassen

1986) .

Although small water-soluble molecules can diffuse through aqueous channels in

the biological membrane, for the majority of xenobiotics which have relatively

14
Ingestion

1

GI Tract

Inhalation Dermal Absorption

Lung

L.

Blood, Lymph, and ~

Extracellular Fluid

Organs

Soft Hard

Tissues Tissues

Muscle Bone

Skin Teeth

Spleen Nails

Brain Claws

etc

.

Feces Urine Expired

Air

Secretions

Excretion Uptake

Target organ of accumulation

Principal site of metabolism

Figure 1. Absorption, Distribution, and Excretion of

Xenobiotics. Modified from Klaassen (1986)

15
large molecular structure (the organic compounds) , the rate of absorption is

controlled by the partition coefficient of that substance. This is usually

expressed as the octanol/water partition coefficient. The larger this coefficient, the more hydrophobic (and less polar) the compound, and the greater the

tendency for the compound to pass through biological membranes. The lipid

fraction determines the membrane permeability to hydrophobic, lipid soluble

(lipophilic) substances. The tendency of organic acids and bases to be

absorbed is also influenced by the PKa and pH of the environment in which

absorption takes place (Tinsley 1979). Some compounds that have structures

similar to normal metabolites may move by other pathways and thus their

absorption characteristics can be different from those absorbed via passive

diffusion.

Transport and distribution of an absorbed substance usually occurs rapidly, and

the rate of distribution to the tissues of each organ is determined by the

blood flow through the organ and the ease with which the chemical crosses the

capillary bed and penetrates the cells of the particular tissue (Klaassen

1986). After entering the blood, xenobiotics may be associated with the blood

cell proteins or with the plasma. Several plasma proteins can bind foreign

compounds, the most important being serum albumin. While bound, the serum albumin/xenobiotic complex can not cross capillary walls into tissues; however,

the binding is a reversible process and contaminants can be released at target

tissues

.

The natural environment contains many types of toxic substances that can be

absorbed by the tissues of an organism. The survival of an organism depends on

its ability to mediate this stress by: (1) excretion of the toxic substance,

and (2) storage of the xenobiotic in a non- toxic form (in a storage depot) away

from the site of toxic action (target organ)

.

Excretion

Excretion of a lipophilic xenobiotic depends on conversion of the compound to a water soluble form and elimination in urine, feces and other excretory fluids.

Lipophilics in excretory fluids are usually reabsorbed by diffusion into

cellular membranes and, therefore, are not easily eliminated. An exception to

this is the lipophilic secretions produced by the scent glands of mammals

(Larsson and Lindegren 1987) . Considering the relatively small volume of these

secretions, however, little elimination probably occurs via this route.

One route of excretion which occurs through secretory structures in mammals and

which has an important bearing on differences in contaminant levels between

mature males and females is lactation. The consistently higher burdens of

organochlorines (as well as many heavy metals) in males as compared to female

mammals has been linked to the loss of contaminants through lactation and

parturition in the females.

The transfer of lipophilic xenobiotics from maternal fat stores to the

offspring through secretion of lipid-rich milk has been suggested by Addison

and Brodie (1987) to be particular important in marine mammals. At least for

the grey seal (Halichoerus grypus) , the transfer of organochlorines from the

female to fetus has been shown to occur to a greater extent via lactation than across the placenta (Addison and Brodie 1977); and Honda, et al. (1986) showed

16
the importance of lactation in excretion of lead, nickel, and selenitun in

striped dolphin (Stenella coeruleoalba)

.

Organisms have many biotransformation processes (mostly involving enzymatic

reactions) for converting lipophilic xenobiotics to hydrophilic metabolites.

Of the organs involved in biotransformation of xenobiotics (liver, lung,

kidney, intestinal gut flora, skin, and gonads), the liver is the most

important (Sipes and Gandolfi 1986)

.

Unfortunately, the metabolites resulting from the biotransformation or intermediate compounds leading to the metabolites are more toxic in many cases than the original xenobiotics. This is particularly tme for some chemical

carcinogens, organophosphates and a number of compounds that cause cell

necrosis of lung, liver and kidney (Sipes and Gandolfi 1986) .

Accumulation

As is suggested in Figure 1 , contaminants may accumulate in any tissue types or organ: liver, kidney, lung, fat, bone, teeth, nails/claws, hair, muscle, skin,

spleen, brain, etc. The concentration potential for any single contaminant,

depends on the chemical properties of that contaminant and the physical and

chemical properties of the tissue or organ in question.

Because certain organs appear to store large amounts of specific contaminants

in non-toxic form, these have been termed "storage depots". The major storage

depots in mammals are fat, bone, kidney, liver, and plasma. Although not

commonly thought of as a storage depot, the plasma proteins bind certain

xenobiotics preventing them from crossing capillary walls. This is usually a temporary and reversible reaction, depending upon the equilibrium established

between the xenobiotic/protein complex and the free xenobiotic in the plasma,

and also depending on competition from other chemicals for binding sites on the

proteins

.

Fat and bone are relatively long-term storage depots. Accumulation in fat is

primarily determined by the lipophilic nature of the xenobiotic. Bone,

however, is a relatively complex tissue and accumulation here can be quite

complicated. Bone is an important storage depot for heavy metals, such as lead

and zinc. Accumulation in bone can involve incorporation within the lipid

component, association with collagen (copper), incorporation within the red

marrow (iron and cadmium), or deposition with calcium (lead, manganese, zinc)

(Yamamoto, et al 1987).

The liver and kidney are the principal organs involved in the metabolism,

detoxification and excretion of contaminants. As such, they are also important

storage depots for trace elements and organic contaminants. The latter is

probably directly related to the fat content of these organs. Metal accumulation is related to the existence of metal-binding proteins, especially the

thioneins, which are low molecular weight proteins containing a sulfhydryl

group. The thiol group of these molecules has two outer orbitals with unpaired

electrons, easily shared with a Ilb group metal cation with vacant external

orbitals, such as cadmium, mercury, and zinc (Roesijadi 1981). The result is

the formation of metallothioneins which bind and store many metals in a non- toxic form in the kidney and liver.

17
One would expect the storage depot to be independent of the site of toxicity

(target organ). In many cases this is true. The principal site of toxic

response for lead and for organic pesticides is the nervous system; however,

the major storage depot for lead is bone and for organic pesticides it is fat.

This is not so straight forward in the case of the kidney. Although the

formation of metallothioneins allows for the storage of many metals in a non- toxic form, it also appears that at certain elevated metal levels, this

system may be overwhelmed and irreversible damage to renal tubules and nephrons can occur (Goyer 1986) .

Choice of Tissues for Archival

Ideally the tissue to be sampled in any monitoring program should be a main

target of the contaminant, preferably a target organ or tissue which is

indicative of the major toxic effect of the contaminant. Levels detected in

this way would correspond directly to the effect caused. However, as is

pointed out by Aguilar (1983), some target tissues may be more difficult to

collect than others and may deteriorate more quickly (example, nerve tissue).

It may be more appropriate for practical reasons to collect tissue which is not

the main target, but gives a representative picture of residue levels present

in the rest of the body (total body burden). This has been successfully done

for some birds (Dindal 1970; Capen and Leiker 1979; Friend et al. 1979; Larsson

and Lindegren 1987) and significant research on this approach for marine

mammals has been conducted by Aguilar (1985, 1987), Bergman et al. (1981),

Honda et al. (1986), Reijnders (1984, 1986), and Tanabe et al. (1981). As

Moriarty (1985) points out, most animals are exposed to fluctuating levels of

contaminants. Since the levels in the various compartments of an organism are controlled by the dynamic equilibrium established between the circulatory and

peripheral compartments, strong correlation between contaminant levels in the

various tissues of accumulation is difficult without sustained constant

exposure

.

The accumulation of organic contaminants (organochlorines , organometallic

compounds, aromatic hydrocarbons, etc.) is directly related to the lipid

solubility of the compound. Tissues containing large amounts of lipids are,

therefore, candidates for organic contaminant analysis. Such tissues include

blubber, visceral fat, liver, muscle, brain, and bone.

Figure 1 is loosely based on the compartmental models commonly used in animal

physiology and pharmacology (Moriarty 1983). However, the compartments of such

models are not necessarily defined by organ or organ systems, but by the

chemistry of the contaminant. A compartment is a mass of a contaminant that

has uniform kinetics of transformation and transport, and whose kinetics are different from those of all other compartments (Moriarty 1983) . Based on this

definition, a compartment would be best represented by a cellular component

(such as lipid) rather than an organ (such as liver) or even a tissue t3q>e

(such as adipose)

.

Defining a compartment based on the chemistry of the contaminant is significant

relative to analysis and data Interpretation. A good example of this is the

analysis and interpretation of contaminants associated with blubber. The

blubber in some marine mammals has been found to have consistently higher

18
levels of organic contaminants as compared to other organs, but with relatively

large variations between individual animals and between replicated samples.

Much of this variation is dependant upon the body condition of the animal.

Since most of the pollutant is incorporated into the body of mammals through

food, a change in the relationship between food intake and fat deposition may

account for changes in the concentrations of a given chemical in the body

tissues. Metabolism of reserved fatty tissue during periods of little feeding

may result in fat loss and an increase in the concentration of organic con- taminants in the remaining blubber. Dilution of residue concentration due to

the rapid expansion of the fat compartment during growth has been found in

neonates and juveniles of man and other mammals (Aguilar 1985)

.

More significant than the total volume of fat tissue may the amount of stored

lipids. The blubber does not increase or decrease from cellular gain or loss,

but by reduction of adipocite vacuoles which means that there may exist a minimum thickness from which the blubber can continue freeing lipids without an appreciable reduction in its thickness (Aguilar 1985) . Several investigators

have found that, except for brain tissue, there is no marked difference between

the organochlorine concentration in the different tissues of an individual when

the pollutant concentrations are expressed on a lipid weight basis (Jensen et

al., 1969; Linko et al., 1974; Perttila et al., 1986). As will be discussed

below, the exception of brain tissue may be related to the molecular structure

of the brain lipids, as compared to the storage lipids of other organs.

The normalization of organochlorine pollutants in different tissues of

cetaceans through reporting on the basis of lipid content was discussed by

Aguilar (1985) relative to compartmentation of the pollutant and reliability of

tissue sampling procedures. Most past studies of organochlorines in marine

mammal tissues have compared concentrations on wet weight basis. Concentrations have been found to be highest in the blubber, but with also the greatest

variability in the blubber. Since the apolar organochlorines are lipophilic

they would tend to concentrate in the lipid portion of the tissue. Reporting

of the concentrations based on lipid content should reduce this variability.

Lipids are also found in liver, kidney, muscle and brain, and, therefore,

higher concentrations of lipophilic organic contaminants should also be found

in the lipid portion of these tissues. Aguilar (1985) supposes that the organochlorines are shared among different organs and tissues in proportion to their

fat content (fat compartment). In his review of organochlorines in whales,

Aguilar (1985) also points out that lipid material consists of many different

compounds with varying degrees of apolarity. The less polar triglycerides and

non- esterified fatty acids (NEFA) should favor accumulation of organochlorine

compounds over more polar lipids, such as phospholipids (Kawai and Fukushima

1981; Aguilar 1985). Therefore reporting concentrations based on triglycerides

plus NEFA might reduce the variability of the measurements even further.

In comparison to blubber levels, the brain concentrations of xenobiotics have

been found to be lower by an order of 10:1 than in other tissues, such as lung,

liver, kidney, blubber, and muscle (Hayes 1975) with lesser variation between

individuals and between replicated samples (Delong et al. 1973; Robert DeLong,

personal communication, 1987). This has been the case even when concentrations

are expressed on a lipid basis. This could be due to the blood-brain barrier

to passage of materials between the circulatory system and brain. This would

be particularly effective relative to the transport of ionized compounds;

19
however, this barrier might not be as efficient relative to liposolubles

(Aguilar, 1985). In fact, Walker (1975) indicates that the barrier is very

permeable to organochlorines . The basic constituents of the cerebral lipids

are the phospholipids, which are less conducive for accumulations of liposoluble compounds and, therefore, these compounds may explain the lower levels of

organochlorines found in brain tissue. The brain levels of these contaminants

probably remain relatively independent of body conditions since phospholipids

are maintained even during starvation.

Statistically, the brain might be better for analysis than blubber; however,

there are still questions regarding the ability of different contaminant

compounds to cross the blood-brain transport barrier (Kurtz, nd.). There is

also the problem of obtaining brain tissue without contaminating the sample,

since one would either have to cut through the skull under field conditions

without disturbing the brain or return the entire head to the lab for

dissection.

The liver is a principal candidate for tissue sampling since it is the major

detoxification site for the contaminants of interest, and for some marine

mammals it is an item of human consumption. The liver generally has sufficient

lipid content that it is suitable as an accumulator of organic as well as inorganic compounds and may also represent a higher proportion of metabolites

than remaining tissues.

Tissues commonly sampled for trace elements include liver, heart, kidney,

muscle, and bone. Although all of these organs appear to accumulate different

trace elements to different degrees, both the liver and kidney appear to be

principal organs of concentration for most elements. For example, Goldblatt

and Anthony (1983) reported that, in the northern fur seal, the largest concen- trations of most metals have been found in the liver and kidney while elevated

levels of lead and nickel have commonly been found in bone as well as liver.

Considering all of the factors discussed above relative to the advantages and

disadvantages of certain tissue types for contaminant analysis, six rather

simple but practical criteria were followed for initial selection of tissues to

be included in the Archive. These criteria are:

1. The minimum two 150 g samples can be obtained from the tissue (see

Methods Section)

.

2. The tissue will provide a homogeneous sample.

3. The sample is conducive to precise anatomical description.

4. The tissue is accessible to sampling techniques.

5 . The tissue has a potential for concentrating both inorganic and

organic contaminants

.

6. The tissue is commonly consumed by humans.

Based on these criteria, tissues will be routinely collected from liver,

kidney, blubber, and skeletal muscle. Other tissues will be considered on a special case bases and as the sampling protocols are developed.

Variability in Contaminant Measurements

The preceding section of this report discusses several factors contributing to

the variability in contaminant data in marine mammals. However, emphasis has

been on normalization of the data through selection of appropriate units for

20
expressing concentrations (wet weight, lipids, triglycerides, etc.) Other

factors are also involved in the variations among individuals of the same species and among individuals of the same populations that have been found in

past studies of contaminants in marine mammals.

An examination of the variation in organochlorine levels in marine mammals by

Aguilar (1987) resulted in his listing of eight major sources of variability.

These sources, which probably apply to contaminants in general are:

1. Nutritional state of the animals.

2 . Sex and age

.

3. Differential metabolism and excretion.

4. Food chain level (varies depending on age and sex).

5. Distance from contaminant source (variation in exposure).

6. Variation in tissue sampling.

7. Selection of appropriate units for expressing concentrations (wet

weight, dry weight, etc.).

8. Tissue preservation and analytical procedures.

Aguilar (1987) points out that, ideally, any comparison of pollutant

concentrations between different marine m2unmal populations should use only

individuals of the same age, sex, reproductive category and fattening state.

He also suggests that sampling within a given size range of only one sex (preferably males because of the usual higher tissue loads) on a limited time

period would help to overcome many of the variability problems. Within a program such as the Archival Project, the ability to direct the sampling such

that the above restrictions are applied will be difficult. The Archival

Project will be relying upon samples collected in cooperation with ongoing

research and management programs of other agencies and such control may not be

possible. However, for some pinniped species sex and age may be controllable.

For example, northern fur seal samples obtained during the subsistence harvest

will come from 2-3 year old males, since these are the only animals taken

during the harvest. Sex and age may also be controllable sampling factors for

walrus , and perhaps other species

.

All sources of variation can not be controlled during sampling. However, as samples are collected and archived, knowledge of variability factors can be

used to establish guidelines on what comparisons can be made and what

interpretations can be derived from these comparisons.

Collection, storage, and analytical conditions should be exactly the same for

all samples in order for comparisons between samples to be valid. Samples

preserved under different conditions are not directly comparable unless the

possibility of differential pollutant derivation or loss can be reasonably

excluded (Aguilar 1987). The Archival Project can control some of the factors

contributing to measurement variations, such as the variation in tissue sampling, the selection of appropriate units for expressing concentrations, and

tissue preservation and analytical procedures. The collection protocols

presented in the Methods Section of this report are intended to provide this

control over sampling and preservation procedures. Analytical procedures and

the selection of concentration units for variability reduction will continue to

be investigated by the Project.

21
METHODS

Sample collections will be conducted in cooperation with research and/or

resource management programs of federal, state and local agencies and organizations. Samples to be archived, including those obtained incidentally, will be

collected, documented and handled in accordance with the standard protocols

developed for the project in 1987, and which are consistent with those employed

by the NBSB, NBS . Sample storage and inventory procedures will be in accor- dance with those routinely performed at the NBSB. This will include specimen

storage under liquid nitrogen vapor at -150 ®C.

Collection Protocols

The collection protocols for this project are modifications of those used by

the NBSB for the collection of human liver S 2imples (Harrison, et al. 1981;

Zeisler, et al. 1983a), and protocols used by NOAA's Status and Trends program

for the collection of fish, bivalves, and sediment samples for the purpose of

specimen banking and environmental contaminant analysis (Lauenstein 1986;

Lauenstein and Young 1986).

The quality of logistic and support facilities vary within Alaska. The

necessity for collecting materials in remote areas under relatively primitive

conditions, will probably require the development of more than one protocol.

Within the limitations imposed by the conditions under which collections are made, the intent of each of the collection protocols is to obtain fresh

well-defined tissue samples uncontaminated by extraneous sources of trace

elements and organic compounds, and to package and transport these samples as quickly as possible under conditions which eliminate or minimize chemical

changes within the tissues prior to storage.

Materials

The following materials are used for collecting marine mammal tissue samples:

Container with blue ice for transporting bagged samples from collection

site to processing site.

Teflon sheeting with adhesive backing for covering processing surfaces

(usually lab benches)

,

1 Dry shipper (LN2 cooled) with shipping container,

LN2 in container for freezing samples on site,

Dewar and lid,

Lab coats (disposable)

,

Insulated gloves, face shield, and tongs for handling the LN2 and frozen

samples

,

Balance for weighing samples (triple-beam balance is suitable),

Surgical scissors, forceps and screwcap vials with buffered formalin for

histological samples.

Labels for exterior of sample jars.

Tape for securing exterior jar labels.

Shipping labels.

Data recording forms,

5' X 10' polyethylene sheet, and

Garbage bags for the collection of used materials in the field for later

disposal

.

22
In addition, the collection of two replicates of a single sample requires:

4 pairs non- talced vinyl gloves,

2 Teflon FEP (Fluorinated ethylene propylene) bags to be used as sheets

for clean working surfaces,

2 Teflon FEP bags for transporting samples from the field to the

processing facilities,

2 Teflon jars (180 mL, 49 mm diameter, 120 mm length) with lid labels,

2 500-mL Teflon bottles containing high purity distilled or best available

water for rinsing samples,

1 Titanium blade/Teflon TFE (Tetrafluoroethylene) handle knife, and

High grade ethanol for rinsing knife (1 L)

.

For the collection of associated biological/environmental data, the following

equipment will also be required:

Thermometer for air temperature.

Measuring tape (metric)

,

Heavy duty scales for weighing animal (where practical)

,

Short metric ruler for measuring blubber thickness.

Camera for recording field procedures (optional)

General

The collection protocol consists of three stages: tissue removal from the

individual animal, tissue processing to obtain representative samples, and

packaging/shipping of the samples to the archive (Figure 2) . The division of

the protocol into stages is used as an aid in organizing and simplifying the

collection procedures.

Stage I . tissue removal, will occur out-of-doors in the field under

conditions of limited control. Procedures for tissue removal will vary

depending upon the group of mammals being sampled (e.g., pinnipeds vs cetaceans), but should be the same for individuals of the same species.

Stage II . tissue processing, will occur indoors in the majority of cases and under laboratory conditions where possible; this includes shipboard

laboratories

.

Stage III , sample packaging and shipping, should be relatively standard

for all tissues and should not vary, while the processing of tissues might

vary depending on the availability of laboratory facilities near the

collection site and the tissue type to be sampled.

Standard forms for recording information pertinent to the sample collections

are presented in Appendix A.

23
Stage I

Figure 2. Generalized Collection Protocol

24
Stage I. Tissue Removal

1. The size of the tissue sample removed from an animal should be sufficient

to provide two subsamples of 150-200 g each for archival. This amount of

material will be required in order to maintain long-term archived samples

plus aliquots for periodic analysis.

2. The anatomical location of tissue removal is specified in order to

maintain consistency and comparability between the same tissue types.

3. Sterile, cleaned, non- talced vinyl gloves will be used by all personnel

involved in sample removal and handling. Precaution must be taken

throughout the procedures to reduce the risk of chemical contamination of

the sample. Contamination may originate from the individual performing

the work (do not smoke during the procedure), the atmosphere, the skin of

the animal, the instruments used in the dissection, and any chemicals that

happen to be in the area where the work is being performed, (refer to

"Contamination Sources", Appendix B)

4. No animal will be considered a candidate for sampling, if tissues cannot

be collected within 10 hours after death (air temperatures <40 °F) and if

handling of the carcass between time of death and tissue removal can not

be documented.

5. Procedures for Pinnipeds and Sea Otters (from Fay, et al. 1979):

a. Record the weather information and individual animal data on the data

recording forms

.

b. For carcasses that can be moved, place carcass ventral side up on 5'

X 10' polyethylene sheet; straighten the spine (e.g., by grasping the

head and pulling) . Measure the length (tip of snout to tip of tail

flesh, in a straight line) and axillary girth (cm). For animals that can not be moved, place the polyethylene sheet on the left side of

the carcass to provide a working surface beside the animal.

c. Make an incision 4-5 cm long over the sternum, midway between the

axillae, cutting through the skin and blubber. Measure the blubber

thickness (mm) at this point.

d. Remove the ventral body wall from chin to anus, cutting through the

costal cartilages, and lay it out, skin side down, to one side as a work area. Confirm the gender.

Note! In the case of the northern fur seals, the pelt is traditionally

removed from the animal before the body cavity is opened. For this

species the animal is placed on its back on the polyethylene sheet and

incisions are made around the neck and flippers, and through the skin from

the chin to anus. The animal is then turned over on its ventral side

(still on the polyethylene sheet) , the head is held in place by a forked

steel bar, and the skin of the back of the head is grasped and pulled

posterior to remove the entire pelt. The body cavity is then opened and

the internal organs removed as described below.

25
6. Procedures for Cetaceans (from Fay, et al. 1979):

a. Record the weather information and individual animal data on the data

recording forms

.

b. For animals that can be moved, place carcass left side up on 5' x 10'

polyethylene sheet. For animals that can not be moved, place the

polyethylene sheet to the side of the carcass to provide a working

surface beside the animal. Measure the length (tip of snout to fork

of tail, in a straight line) and axillary girth (cm).

c. Make an incision through the skin and blubber about midway along the

side and measure the blubber thickness (mm). The incision should be

small enough to prevent distortion, but its size will depend on the

size of the animal and thickness of its blubber.

d. With the animal lying on its side, remove the skin and blubber from

the left lateral body wall and lay it out, skin side down, to one side as a work area. Confirm the gender.

7. The tissue samples are removed as soon as possible after opening the body

cavity. Opening of the body cavity and initial cutting of the skin to

expose muscle and adipose tissue may be performed with high quality

stainless steel dissection tools previously rinsed in the high purity

water

.

a. Liver. Note the general appearance of the liver before removal,

including any unusual coloration, texture, shape, etc.

(1) For the smaller species (northern fur, ringed, spotted, and

harbor seals and sea otter: Remove the entire liver from the

animal and place in a clean Teflon bag for immediate transport

to the processing area, or place it on a Teflon sheet on a wooden board for dissection in the field (see Stage II. 4 for the

dissection instructions) . Wetting the wooden surface enhances

the adherence of the Teflon sheet. Ligaments of the liver may

be cut using surgical scissors which have been previously

rinsed. (If liver membrane is ruptured or stabbed, the sample is

not acceptable)

.

(2) For the larger species (walrus, bearded seal, and cetaceans):

Using the titanium knife, remove a 300-400 g section from the

liver and place in a clean Teflon bag for immediate transport to

the processing area. For animals with multi- lobed livers

(pinnipeds) , this section is taken from the posterior portion of

the left anterior lobe. For animals with liver divided into two

lobes by a shallow indentation in the posterior edge (cetaceans) , this section is taken from the posterior portion of the

left lobe.

b. Kidney . Note the general appearance of the kidney before removal,

including any unusual coloration, texture, shape, etc.

26
( 1 ) For the smaller species (northern fur, ringed, spotted, and

harbor seals and sea otter: Remove both kidneys from the animal

and place in a clean Teflon bag for immediate transport to the

processing area. Both kidneys are required in order to provide

a total sample size of 300 g. (Note which is the left and which

is the right kidney) . Attachments of the kidney may be cut

using surgical scissors which have been previously rinsed (If

the kidney membrane is ruptured, the sample is not acceptable).

(2) For the larger species (walrus, bearded seal, and cetaceans):

Remove the left kidney from the animal and place in a clean

Teflon bag for immediate transport to the processing area. Attachments of the kidney may be cut using surgical scissors

which have been previously rinsed (If the kidney membrane is

ruptured, the sample is not acceptable)

.

If the entire kidney is too large to be transported back to the

processing area, remove a 300-400 g. section from the posterior

end of the left kidney using the titanium knife and place in a clean Teflon bag for transport.

c. Adipose Tissue (Blubber") . The anatomical site of blubber removal

will depend on the distribution of fat layers on the animal and the

butchering procedures used during subsistence harvests; it will,

therefore, be rather species specific.

(la) For animals with relatively thin blubber layers, such as northern fur seals and sea otters: Using the titanium knife, remove sections of blubber from along the backbone until the

required 300 g are obtained. Any small portions of muscle

tissue will be removed at the processing site. Avoid collecting

blubber from near the areas where the initial cuts through the

skin were made since these areas are contaminated with loose

hair

.

(lb) For animals with relatively thick blubber layers: In the region

of the sternum (or to the left of the point where the blubber

thickness measurement was made) remove a rectangular section

(300 g) of blubber using the titanium knife previously rinsed in

high purity water. This section should be a vertical section

from just below the skin to the surface of the muscle.

(2) Place the blubber sfimple in a clean Teflon bag for immediate

transport to the processing area.

d. Muscle .

(1) Using the titanium knife, remove strips of muscle tissue from

along the backbone until approximately 300 g are obtained. Avoid

collecting muscle tissue from near the areas where the initial

cuts through the skin were made since these areas are contaminated with loose hair.

27
(2)

Place the muscle sample in a clean Teflon bag for immediate

transport to the processing area.

8. For most of the pinnipeds the upper canine teeth are used to determine

age. The procedures for extracting and preparing the teeth for

examination are given in Appendix B.

Stage II. Tissue Processing

1. Tissue processing will take place indoors within a laboratory facility and

under a clean air hood, if possible. At a minimum, the tissue processing

will take place in a covered and enclosed area free of obvious sources of

contamination such as cigarette smoke, fuel oil fumes and smoke, laboratory formaldehyde, etc. The processing area of the laboratory should be

cleaned to remove dust and the working surfaces covered with Teflon

sheeting.

2. Only titanium knives are to be used to cut samples during Stage II. Two

knife cleaning procedures are presented in Appendix B. Cleaning Procedure

I can be performed at the processing site. Procedure II will be performed

only at the NBS facilities, or at another suitable laboratory.

3. If tissue sections are taken for histological work, this information is to

be recorded in the appropriate space on the data reporting forms. The

results of the histological analysis should be reported to the NBSB. Any

change in the physical location of the histological slides should be

reported to the NBSB. Investigators who intend to dispose of histological

slides after analysis are encouraged to transfer these materials to the

NBSB, where they will be cataloged, cross-referenced to archived tissue

samples, and maintained in the NBS archives.

4 . Liver

a. If the whole liver is transported to the processing facility :

(1) Weigh the Teflon bag containing the liver specimen. Use an empty bag as a tare. Record the weight on the Data Recording

Form.

(2) Cut open the Teflon bag containing the specimen so that it

provides a flat sheet for rinsing the liver.

(3) Rinse the surface of the specimen with water from the Teflon

bottle. Pour approximately 100 mL or more of the water from the

Teflon bottle over the specimen to wash off blood and other

fluid. Rub the specimen with gloved hand, if necessary, to remove blood, etc. Pick up the specimen and, while held in the

air, rinse the Teflon surface with the water. Place the

specimen back on the Teflon sheet, unwashed side facing up.

Rinse this side with another 100 mL of water. Allow the

specimen to drain for several minutes.

(4) The seal liver consists of four to eight long finger-like lobes.

Using the titanium knife, remove the two anterior lobes to give

28
two samples (Sample A and Sample B) of 150-200 g each. Each

sample must fit in a Teflon jar with a volume of 180 mL (49 mm

diameter and 120 mm length)

.

(5) Rinse the titanium knife with the water and rub with the gloved

fingers to remove all blood and fluids from the knife before

they have time to become dried on. Rinse the knife with ethanol

and air dry.

(6) Continue to Stage III, Tissue Packaging and Shipping.

a. If a sample of the liver (300-400 g) has been taken in the field and

transported to the processing facility:

(1) Cut open the Teflon bag containing the specimen so that it

provides a flat sheet for rinsing the liver.

(2) Rinse the surface of the specimen with water from the Teflon

bottle. Pour approximately 100 mL or more of the water from the

Teflon bottle over the specimen to wash off blood and other

fluid. Rub the specimen with gloved hand, if necessary, to remove blood, etc. Pick up the specimen and, while held in the

air, rinse the Teflon surface with the water. Place the

specimen back on the Teflon sheet, unwashed side facing up.

Rinse this side with another 100 mL of water. Allow the

specimen to drain for several minutes.

(3) Using the titanium knife divide the specimen into two equal

samples (Sample A and Sample B) of 150-200 g each. Each sample

must fit in a Teflon jar with a volume of 180 mL (49 mm diameter

and 120 mm length)

.

(4) Rinse the titanixim knife with the water and rub with the gloved

fingers to remove all blood and fluids from the knife before

they have time to become dried on. Rinse the knife with ethanol

and air dry.

(5) Continue to Stage III, Tissue Packaging and Shipping.

5 . Kidney

a. If both kidneys were collected from the animal:

(1) Weigh the Teflon bag containing the kidneys. Use an empty bag

as a tare. Record the weight on the Data Recording Form.

(2) Cut open the Teflon bag containing the specimens so that it

provides a flat sheet for rinsing each kidney.

(3) Rinse the surface of each kidney with water from the Teflon

bottle. Pour approximately 100 ml or more of the water from the

Teflon bottle over each kidney to wash off blood and other

fluid. Rub the specimen with gloved hand, if necessary, to remove blood, etc. Pick up the specimens and, while held in the

29
air, rinse the Teflon surface with the water. Place the

specimens back on the Teflon sheet, unwashed side facing up.

Rinse this side with another 100 mL of water. Allow the kidnejrs

to drain for several minutes

.

(4) In some cases each kidney will approach the weight of the

required specimen size for each subsample (150 g) and no subsampling will be necessary. The right kidney will provide

Sample A and the left kidney will provide Sample B. If subsampling is required (or if cutting of the sample is necessary to

fit the sample into the jar), use the titanium knife to remove a 150 g section from the kidney. Each sample must fit in a Teflon

jar with a voliome of 180 mL (49 mm diameter and 120 mm length).

b. If the left whole kidney was collected from the animal:

(1) Weigh the Teflon bag containing the kidney. Use an empty bag as a tare. Record the weight on the Data Recording Form.

(2) Cut open the Teflon bag containing the specimen so that it

provides a flat sheet for rinsing the kidney.

(3) Rinse the surface of the kidney with water from the Teflon

bottle. Pour approximately 100 mL or more of the water from the

Teflon bottle over the kidney to wash off blood and other fluid.

Rub the specimen with gloved hand, if necessary, to remove

blood, etc. Pick up the specimen and, while held in the air,

rinse the Teflon surface with the water. Place the specimen

back on the Teflon sheet, unwashed side facing up. Rinse this

side with another 100 mL of water. Allow the kidney to drain

for several minutes

.

(4) In some cases the entire kidney may approach the weight of the

required specimen size and no subsampling will be necessary. If

subsampling is required (or if cutting of the sample is neces- sary to fit the sample into the jar), use the titanium knife to remove a 150 g section from the kidney. Each subsample must fit

in a Teflon jar with a volume of 180 mL (49 mm diameter and 120 mm length)

.

c. If samples of kidney tissue were collected from the animal:

(1) Cut open the Teflon bag containing the specimen so that it

provides a flat sheet for rinsing the kidney sample.

(2) Rinse the surface of the specimen with water from the Teflon

bottle. Pour approximately 100 mL or more of the water from the

Teflon bottle over the specimen to wash off blood and other

fluid. Rub the specimen with gloved hand, if necessary, to remove blood, etc. Pick up the specimen and, while held in the

air, rinse the Teflon surface with the water. Place the

specimen back on the Teflon sheet, unwashed side facing up.

Rinse this side with another 100 mL of water. Allow the sample

to drain for several minutes.

30
(3) Using the titanium knife, remove two subsamples of 150 g each

from the kidney sample. Each subsample must fit in a Teflon jar

with a volume of 180 mL (49 mm diameter and 120 mm length)

.

d. Rinse the titanium knife with the water and rub with the gloved

fingers to remove all blood and fluids from the knife before they

have time to become dried on. Rinse the knife with ethanol and air

dry.

e. Continue to Stage III, Tissue Packaging and Shipping.

6 . Blubber

a. Cut open the Teflon bag containing the specimen so that it provides a flat sheet for rinsing the blubber.

b. Pour approximately 100 mL or more of the water from the Teflon bottle

over the specimen to wash off blood and other fluid. Allow the

specimen to drain for several minutes.

c. Use the titanium knife to remove any remaining portions of muscle

attached to the blubber. Excise a 300-400 g section from the

specimen.

d. Using the titanium knife divide the specimen into two equal samples

(Sample A and Sample B) of 150-200 g each. Each sample must fit in a Teflon jar with a volume of 180 mL (49 mm diameter and 120 mm

length) . Rinse the titanium knife with the water and rub with the

gloved fingers to remove all blood and fluids from the knife before

they have time to become dried on. Rinse the knife with ethanol and

air dry.

e. Continue to Stage III, Tissue Packaging and Shipping.

7 . Muscle

a. Cut open the Teflon bag containing the specimen so that it provides a flat sheet for rinsing the muscle.

a. Pour approximately 100 mL or more of the water from the Teflon bottle

over the specimen to wash off blood and other fluid. Allow the

specimen to drain for several minutes.

b. Use the titanium knife to remove any portions of blubber attached to

the muscle tissue. Excise a 300-400 g section from the specimen.

c. Using the titanium knife divide the specimen into two equal samples

(Sample A and Sample B) of 150-200 g each. Each sample must fit in a Teflon jar with a volume of 180 mL (49 mm diameter and 120 mm

length) . Rinse the titanium knife with the water and rub with the

gloved fingers to remove all blood and fluids from the knife before

they have time to become dried on. Rinse the knife with ethanol and

air dry.

31
d. Continue to Stage III, Tissue Packaging and Shipping.

Stage III. Tissue Packaging and Shinning

1. Place each sample in individual pre-cleaned Teflon jars. Tare the weight

of the jar and weigh each individual sample. Record the weights on the

Data Recording Form and sample labels. Affix the sample labels to the

jars with and place the lid labels in the jar lids.

2. Freeze each sample by immersing in the LN2 10 minutes.

Liquid nitrogen should not be stored in sealed containers. Personnel

handling LN2 cautioned to wear boots, cuffless trousers, non-absorbent

aprons, loose insulating gloves, and face shields.

3. The LN2 shipper should be filled with liquid nitrogen for at least 6 hours

to fully prepare it for shipping. This is required to fully saturate the

absorbent inside the shipper. Pour off the excess LN2 and place the

frozen samples in the shipper (10-12 sample boxes per shipper).

4. Once full transport the shippers to the NBS; do not store in intermediate

freezers

.

5. Double check the Data Recording Forms for completeness and accuracy. Any

deviations or modifications of the protocol -must be noted on the sampling

form.

6. Place a copy of the completed forms in the shipper; another copy is

retained by the collector for project records.

7. Samples should be shipped within 48 hours or as soon as possible after

sample collection using 24 hour express package service to:

National Bureau of Standards

Route 270 and Quince Orchard Road

Building 235, Room B118

Gaithersburg, Maryland 20899

Attn: Barbara Koster (301) 975-6291

In most cases, samples will have to be shipped commercial-air to Anchorage

and then 24 hour express package service to the Archive. The shippers

must not contain LN2 when shipped. Maximum holding time for the shippers

is 10-12 days. Shipping expenses will be borne by OCSEAP. Do not ship

late in the week, i.e., Thursday, or Friday, or before holidays, unless

special arrangements have been made with the shipping service, OCSEAP, and

NBS.

8. The Specimen Bank personnel should be notified by telephone as soon as possible after the specimens are shipped:

Barbara Koster (301) 975-6291 (FTS, 879-6291), or Rolf Zeisler (301) 975-6290 (FTS, 879-6290), or Steve Wise (301) 975-3112 (FTS, 879-3112)

32
Sample Archival

Samples are received at NBS and transported to the Specimen Bank facility. The

shippers are unpacked and samples are inspected for any packaging problems and

for unsuitable temperature. Sample data forms and samples are compared to

insure that they correspond and that all information has been included. These

samples are stored in a temporary storage LN2 freezer and are logged into the

temporary storage log book. They remain in temporary storage until assigned an NBS number and permanent LN2 freezer space.

When the samples are moved into the permanent freezer location, a storage form

is completed and the information is entered into the inventory form on the

IBM-AT computer. Depending on the geometry of the samples, various types of

containers are used to hold them in the LN2 freezer. The samples may be placed

in long cylindrical tubes containing several samples or small square freezer

boxes in metal racks. These s 2UDaples will remain stored in the LN2 freezer and

the conditions monitored until they are requested for analysis.

Monitoring of Sample Stability During Storage

In order to evaluate the stability of the archived tissues, NBS monitors the

concentrations of selected contaminants in 20% of the tissue specimens.

Aliquots of those specimens selected for monitoring are initially analyzed to

establish the baseline levels. Repeated analyses of aliquots of these tissues on a regular basis (every one or two years) a provides measure of any change

from the initial baseline concentrations. Besides providing a baseline to

evaluate sample storage stability, these analyses also serve two other

purposes

:

1. They provide some real-time measure of contaminant concentrations for

monitoring purposes

.

2. They provide a baseline for comparing contaminant levels using

present analytical techniques with those measured iii the future by

other laboratories using different methodologies.

In selecting the contaminants to be monitored, emphasis has been placed on

those elements and compounds which may be linked to the oil and gas industry in

Alaska. These are the heavier molecular weight aromatic hydrocarbons and trace

elements, such as Ba, Cr, Ni, and V (Tables 2 and 3). Other contaminants which

are known to occur in elevated levels in the tissues of marine mammals (Cd, Hg,

Pb, Zn, and selected chlorinated hydrocarbons) will also be monitored on a routine basis.

Of the two subsamples (Samples A and B) of each tissue which are archived,

sample "A" is maintained in long-term storage in the LN2 freezers while sample

"B" is used for the initial baseline analyses and storage stability evaluation.

Samples to be analyzed are homogenized using a cryogenic homogenization

procedure designed to reduce the likelihood of changes in sample composition

due to thawing and refreezing (Zeisler et al. 1983b). The sample homogenate is

then aliquoted into small Teflon jars (7-10 g) for analysis and for storage as the homogenate

.

33
The analytical approach for the baseline determinations of the trace elements

focuses on the use of a multi-element analytical technique [neutron activation

analysis (NAA) ] to provide data on a large number of trace elements using only

a limited amount of sample. Additional analytical techniques (voltammetry arid

atomic absorption) are being used to provide data on elements of high priority

that are not routinely measured by NAA (e.g., Pb and Ni) and to provide quality

control data for selected elements by comparing data from two different

analytical techniques.

Capillary gas chromatography with electon capture detection is used to measure selected chlorinated hydrocarbons and high performance liquid chromatography

with fluorescence detection is used to measure aromatic hydrocarbons.

During the initial year of analyses, all four tissue types from the first year

collection of northern fur seals will be analyzed to determine the suitability

of each tissue for monitoring of contaminant levels and analytical problems

associated with each tissue.

Quality Assurance

NBS is an active participant in the OCSEAP's Quality-Assurance Program for

Trace Petroleum Component Analysis, which is coordinated with the

Quality-Assurance Program of NOAA's National Status and Trends Program.

34
REFERENCES

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Addison, R. F. and P. F. Brodie (1987), Transfer of organochlorine residues

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35
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.

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Hayes, W. J. (1975), Toxicology of Pesticides, Williams and Wilkins,

Baltimore, Maryland.

Honda, K. , Y. Fujise, R. Tatsukawa, K. Itano and N. Miyazaki (1986), Agerelated accumulation of heavy metals in bone of the striped dolphin,

Stenella coeruleoalba . Mar. Environ. Res. 20:143-160.

Jensen, S., A. Johnels, M. Olsson and G. Otterlind (1969), DDT and PCB in

marine animals from Swedish waters. Nature 224:247-250.

Jones, L. (1987), Personal communication. National Marine Mammals Laboratory,

NMFS, Seattle, Washington.

Kawai, S. and M. Fukushima (1981), Relation between lipid composition and

the concentrations of organochlorine compounds in the various organs of

striped dolphins Stenella coeruleoalba . In: Studies on the Levels of

Organochlorine Compounds and Heavy Metals in the Marine Organlsns, T.

Fujiyama, Ed., University of Ryukyus, Ryukyus.

Klaassen, C. D. (1986), Distribution, excretion, and absorption of toxicants.

In: Gasarett and Doull's Toxicology: The Basic Science of Poisons, 3rd

Ed., C. D. Klaasen, M. 0. Amdur and J. Doull, Eds., Macmillan Publishing

Co., New York, pp. 33-63.

Kurtz, D. A. (nd) , Analysis of Northern Fur Seals from the Years 1975-1981

for DDT Analogs and PCB Mixtures, Unpublished paper. Department of

Entomology, Penn State Univ. , University Park, PA, 15 pages plus figures

and tables.

Larsson, P. and A. Lindegren (1987), Animals need not be killed to reveal

their bodyburdens of chlorinated hydrocarbons. Environ. Poll. 45:73-78.

36
Lauenstein, G, G. (1986), National Status and Trends Program for Environmental

Quality Benthic Surveillance Project: Specimen Banking Project, Ocean

Assessments Division, Office of Oceanography and Marine Assessment,

National Ocean Service, NOAA, Rockville, Maryland, 34 pp.

Lauenstein, G. G. and D. R. Young (1986), National Status and Trends Program

for Environmental Quality Benthic Surveillance Project; Cycle III Field

Manual, NOAA Technical Memorandum NOS OMA 28, Rockville, Maryland, 26 pp.

Linko, R. J., Kaitaranta, P. , Rantamaki and L. Eronen (1974), Occurrence of

DDT and PCB compounds in Baltic herring and pike from the Turku

archipelago. Environ. Poll. 7:193-207.

Moriarty, F. (1983), Ecotoxicology : The Study of Pollutants in Ecosystems,

Academic Press, London, 233 p.

Perttilia, M. , 0. Stenman, H. Pyysalo and K. Wickstrom (1986), Heavy metals

and organochlorine compounds in seals in the Gulf of Finland, Mar.

Environ. Res. 18:43-59.

Reijnders, P. J. H. (1984), Man- induced environmental factors in relation to

fertility changes in pinnipeds. Environmental Conservation 11(1): 61 -65.

Reijnders, P. J. H. (1986), Reproductive failure in common seals feeding on

fish from polluted coastal waters. Nature 324:456-457.

Roesijadi, G. (1981), The significance of low-molecular weight metallothioneinlike proteins in marine invertebrates: Current status. Mar. Environ. Res.

4:167-179.

Sipes, I. G. and A. J. Gandolfi (1986), Biotransformation of toxicants. In:

Gasarett and Doull's Toxicology: The Basic Science of Poisons, 3rd Ed., C.

D. Klaasen, M. 0. Amdur and J. Doull, Eds., Macmillan Publishing Co,, New

York, pp. 64-98.

Tanabe, S,, R. Tatsukawa, H. Tanaka, K. Maruyama, N. Miyaraki and T. Fujiyama

(1981), Distribution and total burdens of chlorinated hydrocarbons in

bodies of striped dolphins ( Stenella coeruleoalbal . Agric. Biol. Chem.

45:2569-2578.

Tinsley, I. J. (1979), Chemical Concepts in Pollutant Behavior, John Wiley &

Sons, New York, 265 pp.

Walker, C. H. (1975), Variations in the intake and elimination of pollutants,

In: Organochlorine Insecticides: Persistent Organic Pollutants, F. Moriarty, Ed., Academic Press, London.

Yamamoto, Y. , K. Honda, H. Hidaka and R. Tatsukawa (1987), Tissue

distribution of heavy metals in Weddell seals, (Leptonvchotes weddellii ')

.

Mar. Poll. Bull. 18(4) : 164-169

.

37
Zeisler, R. , S. H. Harrison and S. A. Wise, Eds. (1983a), The Pilot

National Environmental Specimen Bank-Analysis of Human Liver Specimens,

National Bureau of Standards, Special Publication 656, Gaithersburg,

Maryland, 128 p.

Zeisler, R. , J. K, Langland and S. H. Harrison (1983b), Cryogenic

homogenization procedure for biological tissues. Anal. Chem. 55:2431-2434.

38
APPENDIX A

DATA RECORDING FORMS: INSTRUCTIONS

Examples of the data recording forms are provided on pages 42 and 43. Standard

biological/environmental information is recorded on the first page (see page

42). The boxes at the top of this page provide space for the NBS identification codes. These are assigned to the samples when they arrive at the NSBS.

Other information recorded on this page are:

1. OCSEAP ID Number . This is a 10 digit alphanumeric code assigned by the

OCSEAP office before collections are made. The first three numbers (692)

refer to the OCSEAP Research Unit Number. The next four letters are abbreviated common names for the species sampled (FRSL = Fur Seal) . The

last three numbers are ascending numbers that identify the individual

animal sampled.

2. Species . Genus/species name.

3. Sample Source . Information pertinent to identifying the agency,

person(s) , research or management program providing or aiding in providing

the samples is entered here.

4. Site ID . This the most common name of the location where the animal is

sampled (killed). This should be as specific as is practical.

5. Lat. Long . The latitude and longitude of the location where the animal is

sampled. This should be to the nearest tenth of a minute, if possible.

6. Time of Death . Recorded as the day, month, year and hour. The month

should not be numbered, but should be written in abbreviated form (Jan,

Feb, May, Apr, May, Jun, Jul, Aug, Sep, Oct, Nov, Dec) and the hour should

be on a 24-hour basis (example, 6:00 pm is reported as 1800).

7. Method of Collection . The method by which the animal is sacrificed.

8. Intermediate Storage . Method of storage of animal carcass before removal

of tissues. If tissues are taken immediately after death, no entry is

necessary.

9. Weather Conditions . Space is provided for the notation of weather

conditions occurring during the sampling procedures (wind, temperature,

precipitation) , particularly anything that would be pertinent relative to

contamination sources

.

10. Sex. Age, and Method of Age Estimation . Blocks are provided for

indicating the sex of the animal and the age (in years). Method of age

estimation and the name of the individual making that determination should

also be recorded here.

11. Weight. Estimated or measured weight of the animal sampled should be

recorded in the space provided.

39
12. Length . This is measured as snout to tail tip length along a straight

line for pinnipeds, sea otters, and polar bears, and as snout to fluke

notch length along a straight line for cetaceans.

13 . Axillary Girth .

14. Sternal Blubber Thickness .

15. For Females . Blocks are provided to note if the animal sampled is

lactating or pregnant.

16. General Appearance of Individual . Any comments which describe the healthy

or unhealthy appearance of the animal, external parasites, evidence of

trauma, or body condition of the animal.

17. General Appearance of Organs . Any unusual appearances of any of the

internal organs, particularly those to be sampled. If they all appear

normal, note this also.

18. Stomach Contents . Food items if present are identified and some impression of degree of fullness recorded. If internal parasites are present, they are also noted.

19. Additional Samples . Samples collected from the same animal for other

research purposes are recorded in this space. This information includes

the kind of sample(s), for what purpose, and the name and location of the

individual receiving the samples.

The second page of the data recording form (see page 43) provides for the entry

of data and information specific to the tissue samples themselves.

20. OCSEAP ID Number . This is the same number as found on the first page

(Item #1). It is entered here in case the two pages are accidentally

separated.

21. Sample Type . The type(s) of tissue samples collected from the individual

animal are indicated by circling the appropriate category or by writing

the name in the space provided.

22. Time of Collection . Entered as in Item #6 on the first page of the data

recording form. This is the time at which the tissues are removed from

the animal in the field.

23. Collected by . Name of the individual actually removing tissue samples

from the animal in the field.

24. Intermediate Storage . Space is provided to indicate how tissue samples

collected in the field are stored during transport to the processing site

(method of cooling, transport containers, etc.).

25. Time of Preparation . Entered as in Item #6 on the first page of the data

recording form. This is the beginning time at which tissues are processed into subsamples and placed in containers in preparation for LN2 freezing.

40
26 . Time of LNo Freezing . Entered as in Item #6 on the first page of the data

recording form. This is the time at which the samples are frozen in LN2 . It is basically concurrent with the time at which preparation of the

subsamples are completed.

27. Time Shipped From Site . Entered as in Item #6 on the first page of the

data recording form. This is the time at which the samples leave the

sample processing site enroute to the NSBS, Gaithersburg, Maryland.

28. Time Received at Archive . Entered as in Item #6 on the first page of the

data recording form. This is the time at which the samples are received

at the NSBS, Gaithersburg, Maryland.

29. Processor . The name of the individual(s) processing the samples for LN2 freezing and shipment to the NSBS.

30. Shipper . The name of the person responsible for shipping the samples to

the NSBS.

31. Receiver . The name of the individual receiving the samples at NSBS.

32. Protocol . If the standard or modified protocol was used to collect or process the samples, this is noted here. If a modified protocol, the

nature of the modification is recorded.

33. Remarks . Space is provided for any additional remarks pertinent to the

collection, processing, or archiving of the tissue samples.

34. Histological Samples . If samples are collected for histological slides,

these are recorded here. In all cases, histological samples are to be

taken from B subsamples only. The Individual/Organization making the

slides, the Final Destination of the slides, the kind of Tissues Sampled,

and the method of sample preseirvation (if appropriate) are recorded here.

35. Sample Weight . The weights of subsamples are recorded here. Note, that

in some cases the collection of whole organs might be necessary in order

to provide enough tissue for a subsample. If that is the case, and if

these tissues are paired, the location of the specific organ should be

noted by the subsample space (In the example, the entire right kidney is

subsample A and the left kidney is subsample B)

.

36. Prepared bv . The name of the individual filling out the data recording

form.

All of the appropriate information for express shipping the samples are provided in the lower right hand comer of the second page. This includes the

street, building, and room address of the NSBS, as well as the name and

telephone number of the individual responsible for receiving the samples.

41
NATIONAL BiOMONITORING SPECIMEN BANK

Sampling Data - OCSEAP/MMS

Alaskan Marine Mammal Tissue Archival Project

OCSEAP ID Number Species

Sample Source

Site ID Lat. Long.

Time of Death:

day mo yr Intermediate Storage (Temp/remarks)hr

Method of Collection

Weather Conditions (wind, temp., precip.)

Sex: Male Female

Weight: Estimated

Length: Snout-Tail Tip

Snout-Fluke Notch

Axillary Girth:

Sternal Blubber Thickness

Age

Weighed

Method of Age Estimation

kg. For Females:

cm.

cm.

cm.

cm.

Lactating

Pregnant

General Appearance of Individual:

General Appearance of Organs:

Stomach Contents:

Additional Samples:

42
OCSEAP ID Number

Sample Type: Liver Kidney Muscle Blubber Other

Time of Collection: Collected by

day mo yr ir

Intermediate Storage (Temp/remarks)

Time of Preparation:

Time of LNa Freezing:

Time Shipped From Site:

Time Received at Archive:

hr

day mo yr hr

Processor

Shipper

Receiver

Protocol: Standard Modified (Please note modification below)

Remarks:

Histological Samples:

Individual/Organization

Final Destination

Tissues Sampled

Sample Weight: A B

Liver gKidney gMuscle ggggPrepared by:

Name (print)

Blubber

Other

A B

ggggSignature

43
APPENDIX B

SPECIAL INSTRUCTIONS

Contamination Sources

The following is taken from a discussion of contamination sources one might

encounter during the sampling of human liver tissue in a hospital autopsy room

(Harrison, et al. 1981). Many of the sources are also pertinent to the Alaskan

Marine Mammal Archival Project.

The control of contamination requires diligent attention to detail. A lack of

contamination control has made existing banks of samples useless for general

trace constituent analyses. Since it is impossible to point out every source of contamination that may be encountered, some of the most obvious sources will

be described to provide a general awareness of the problem. The following

example will illustrate the effect of contamination. A 1 /*g flake of stainless

steel contains approximately 100 ng of nickel. The natural occurrence of

nickel in human liver is at levels of 1 - 2 ng/g of sample. As we generally

use about 1 g of sample for analysis, a tiny flake of stainless steel in our sample could produce an analytical result which was 100 times higher than the

true value

.

In an autopsy room, the air, the implements, counter tops, and working

personnel are sources of contamination. The air may contain trace vapors of

formalin, xylene, and hair spray. It may also contain particles dust, cigarette ash, and wear particles from motors. Implements and working surfaces may

be contaminated with chemicals used for cleaning and salt and oils from human

contact (via hands, etc.). A common contaminant introduced by human hands is

gold from jewelry. Cigarette ash contains relatively high amounts of cadmium

and organic compounds. Cigarette smoke contains trace quantities of hundreds

of organic compounds

.

The dust- free gloves should be used liberally as they are easily contaminated.

Picking up a pen to record a weight contaminates them, grasping the Teflon

bottle to wash the liver contaminates them, adjusting eyeglasses, touching your

face, touching the outside of the bag which contains the clean Teflon sheets or bags contaminates the gloves

.

Recent research has pointed out other sources of contamination. One trace

organic study found some interesting and unusual compounds in human tissue.

They were later found to have been introduced when the sample was cut on a styrofoam board. In another case, the calcium content of a liver was found to

be unusually high. It was traced back to contamination from the contents of

the stomach. Liquid nitrogen contains oil and dust. The two sealed bags are provided to protect the sample as it is frozen in the LN2

.

The examples above will give the reader an appreciation of the potential

chemical contamination problem.

44
Since marine mammal tissues will be collected under conditions of less control

than the sampling referred to above, contamination sources may be an even greater problem in the Alaska Marine Mammal Archival Project. Sources would

include wind-blown dust, rainfall, boat, aircraft and vehicle fuel and exhaust

fumes, fuel oil and gas fumes from indoor heating units, and tools used in

native subsistence harvest, to name just a few.

Procedures for Cleaning Titanium Knife

There are two cleaning procedures for the titanium knives and other reusable

implements: the first is to be completed after finishing a sample and the

second is used after knife sharpening or after excessive contamination. The

titanium knives should be sharpened only with the silicon carbide stone

provided and only these knives should be sharpened with this stone.

Cleaning Procedure I

After placing tissue sections in bags and before leaving the sample preparation

area, the knife should be rinsed using high-purity water. While rinsing, and

with gloved hands, run fingers over the blade and handle of the knife to help

remove any adhering blood or tissue. This is best done before any fluid or tissue has a chance to dry on the knife. In the laboratory, the knife should

be rinsed again, as above, with water and then with ethanol, the knife is then

placed on a clean surface (do not touch the blade) and allowed to air dry,

preferably in a laminar flow hood. The knife should then be place in a Teflon

bag, made from the Teflon sheets, for storage and transported to the next

sampling site. The implements should at no time be touched with ungloved

hands

.

Cleaning Procedure II

This procedure should be applied after excessive contamination of the

implements and always after a knife is sharpened. Rinse the implement as described in cleaning procedure I. The knife may be disassembled to clean if

necessary. In the laboratory, the implement is placed in a clean container and

covered with 99.5% ethyl alcohol for one to two hours. The implement is then

covered with high purity water overnight. The implement is covered with dilute

hydrochloric acid (one part hydrochloric acid and ten parts high-purity water)

for half an hour. The implement is then removed from the acid, rinsed with

high-purity water, and covered with dilute nitric acid (one part nitric acid

and ten parts high-purity water) for half an hour. The implement is again

removed from the acid and rinsed with high-purity water. The implement is

removed from the washing container and placed on a clean surface to air dry,

preferably in a laminar flow hood. Only the knife handle should touch the

drying surface. The clean, dry implement should be stored in Teflon bags made

from Teflon sheets.

45
Procedures for Preparing Pinniped Teeth for Aging

Using bone saw, bolt cutters, or other appropriate tool remove the upper

maxilla of the animal containing the upper canine teeth. Boil the upper

maxilla for 20 minutes to soften the bone. Remove the two upper canines and

extract the pulp from each tooth. Boil the teeth for 10-15 minutes in a solution of one tablespoon sodium triphosphate per one liter of water. Dry and

label each tooth with following information:

OCSEAP ID Number

Species

Sex

Collection Date

46
NBS»n4A (REV. 2 -80 ) U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See instructions)

1. PUBLICATION OR

REPORT NO.

NBSIR 80-3750 j

1 2. Performing Organ. Report NoJ 3. Publication Date

‘ * APRIL 1988

4. TITLE AND SUBTITLE

Alaskan Marine Mammal Tissue Archival Project: A Project Description Including

Collection Protocols

5. AUTHOR(S)

wise, S.A., Koster, B. J., Zeisler, R.

6. PERFORMING ORGANIZATION (If joint or other than NBS, see instructions)

NATIONAL BUREAU OF STANDARDS

U.S. DEPARTMENT OF COMMERCE

GAITHERSBURG, MD 20899

7. Contract/Grant No.

8. Type of Report & Period Covered

9. SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street, City. State. ZIP)

10.

SUPPLEMENTARY NOTES

I I Document describes a computer program; SF-185, FIPS Software Summary, is attached.

11.

ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here) The Alaskan Marine Mammal Tissue Archival Project was initiated in 1987 with

financial support from the minerals Management service. Outer Continental Shelf (OCS)

studies Program. Although the emphasis is on the collection of tissues for analysis of

contaminants that may be associated. with the petroleum industry, it is also recognized

that the development of an archive of marine mammal tissues collected and stored using

carefully controlled procedures provides a resource that goes beyond the immediate

needs of the OCS Program. Such an archive developed over several years provides a resource of materials for future incestigators addressing questions concerning the

transport of elements and vompounds (contaminants and non-contaminants) throughout the

polar ecosystem, regardless of source. This document provides the basic information on Project objectives and management,

justification for the species, tissues, and contaminants of interest, and specific

instructions for collecting, handling, and storing samples. At this time, the protocols

have been employed only in the collection of northern fur seal tissues, therefore, the

details are somewhat biased toward this species. As these procedures are applied to the

sampling of other marine mammals, the protocols will probably have to be modified.

Therefore, this document represents the first in a series of reports providing the most

recent protocols used by the Project.

12.

KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolons)

Specimen Banking, collection protocols, marine mammals, pollution, trace elements,

organic contaminants.

13.

AVAILABILITY

K 1 Unlimited

I I For Official Distribution. Do Not Release to NTIS

I I Order From Superintendent of Documents, U.S. Government Printing Office, Washington, D.C.

20402.

Order From National Technical Information Service (NTIS), Springfield, VA. 22161

14. NO. OF

PRINTED PAGES

53

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