Alaskan Marine Mammal Tissue Archival Project

PAUL R. BECKER, *'1 BARBARA J. KOSTER, 2 S T E P H E N A. WISE, 2 AND ROLF ZEISLER 2

1Ocean Assessments Division, National Oceanic and Atmospheric Administration, Anchorage, Alaska 99513; and 2Center for Analytical

Chemistry, National Institute of Standards and Technology, Gaithersburg, MD 20899

Received June 5, 1989; Accepted October 23, 1989

ABSTRACT

A project to establish an archive of Alaskan marine mammal tissues was conceived in 1987 to be a part of the National Biomonitor- ing Specimen Bank (NBSB). Protocols and field collection of marine mammals, long-term storage, and analysis are summarized in this paper. Instrumental neutron activation analysis has been used for an initial evaluation of trace element content in samples of northern fur seal (Callorhinus ursinus) from the Pribilof Islands. The findings agree with previously observed trace element levels in northern fur seals. The archived specimens can be used in future studies when compari- sons of past and present pollution levels are needed.

Index Entries: Specimen bank; long-term storage; marine mam- mals; Alaska and marine mammals; trace element analysis and ma- rine mammals; neutron activation analysis; northern fur seal (Cal- lorhinus ursinus); ringed seal (Phoca hispida).

*Author to whom all correspondence and reprint requests should be addressed.

Certain commercial equipment, instruments, or materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for this purpose.

Biological Trace Element Research Editor: G. N. Schrauzer �9 1990 by The Humana Press Inc.

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330 Becker et al.

INTRODGCI]ON

Within the last decade, Alaska has experienced a tremendous in- crease in industrial activities, such as mineral extraction and offshore oil exploration. Concerns have been raised as to what impact these indus- tries have, and will have, on the Alaskan environment. To address some of these concerns, the Minerals Management Service (MMS) and the National Oceanic and Atmospheric Administration (NOAA) initiated the /idaskan Marine Mammal Tissue Archival Project (AMMTAP) in 1987 (1).

Most marine mammals are considered top predators in the marine environment. Chemical analysis of their tissues can be particularly useful in determining whether bioaccurnulation of contaminants is occurring in marine food chains. Not only is the impact on the marine food chain important, but of equal importance are the human health implications. Marine mammals are a prime food source for many of the native Alaskan people. Additionally, fish taken from areas such as the Bering Sea are expdrted all over the world, yet there is no nearby human population to easily test for contaminants derived from these fish. Hence, the Alaska marine mammal tissues can provide information that may be of world- wide interest. The collection of marine mammal tissues, over a period of several years, will provide an archive of samples for the determination of baseline contaminant levels and against which future contaminant mea- sures can be compared.

Only in the past decade has the concept of an environmental sped- men bank for the archiving of biological and environmental samples for retrospective analysis been recognized as an important component to systematic environmental monitoring. For 10 years, the National Insti- tute of Standards and Technology (NIST) has been involved in environ- mental specimen banking activities. These activities resulted in the devel- opment of collection, storage, processing, and analysis procedures for long-term archiving of a variety of environmental specimens, including human liver, fish muscle and liver, marine bivalves, sediment, and marine mammal tissues. Although the total scientific value of the banked samples is not fully known at this time, the current uses of the banked samples, and the implementation of the concept, have already contrib- uted to major monitoring programs in the US and abroad. The National Biomonitoring Specimen Bank (NBSB) can serve as a valuable resource for the assessment of long-term trends of pollutants affecting human and environmental health, in particular for those pollutants that have been unnoticed thus far, or could not be measured in the past (2).

Several species of marine mammals have been selected for study in the Alaskan Marine Mammal Tissue Archival Project. The species are selected on the basis of criteria, such as availability, migration habits, feeding habits, reference information, and animal and organ size. The animals are collected with the cooperation of NIST, the National Marine Fisheries Service (NMFS), the US Fish and Wildlife Service (USFWS), the

Alaskan Marine Mammal Tissue 331

Alaska Department of Fish and Game (ADF&G), and the North Slope Borough Department of Wildlife Management, as well as many individu- al Alaskan native hunters and hunting organizations. Sample collections have been taken during the Pribilof Island subsistence hunt of the north- ern .fur seals (Callorhinus ursinus) and at Barrow, Alaska during the summer ringed seal (Phoca hispida) hunt. Animals considered for future sampling include: the harbor seal (Phoca vitulina), the bearded seal (Erig- nathus barbatus), the Pacific walrus (Odobenus rosmarus divergens), belukha whale (Delphinapterus leucus), and polar bear (Ursus maritimus).

MATERIALS AND METHODS

One of the primary concerns of the AMMTAP is to avoid contam- inating the tissues during the collection and handling process, thereby assuring the integrity of the sample. Therefore, detailed sampling proto- cols have been established for handling the animal from the time it is sacrificed until the time the tissue sample is permanently archived in the NBSB. The principles of such procedures have been evaluated previously with the human liver project (3). These protocols cover: the use and cleaning of specific equipment, such as the titanium instruments and Teflon bags (Clean Room Products, Ronkonkoma, NY) or Teflon jars (Savillex, New Prague, MN), the cleanliness of the physical area-where the sample is packaged, sample handling procedures, and the procedure for quick freezing and shipment of the samples to the NBSB. Addi- tionally, sample information forms are filled out for each animal from which samples are taken. The forms include information such as, loca- tion and time of sampling, size and sex of animal, weight of the tissues processed, weather conditions, handling times, and personnel responsi- ble. Tissue histology and age determination are added to the form later. This information is stored at NBSB in hardcopy and on computer.

Because laboratory facilities are not always available at the sampling site, a completely self-contained sampling kit was designed for use in field situations. The kit included all supplies and implements necessary for sampling one animal. The kit has been evaluated twice during field collection and was found to be a viable solution for sampling in the field.

Four types of tissue were sampled from each animal during the Pribilof hunt: liver, kidney, muscle, and blubber. For future collections, the muscle tissue has been eliminated from the process owing to homog- enization difficulties and low levels of analytes. Three hundred grams of each tissue are considered essential for archiving. Each 300 g tissue sample is divided into two replicate samples: one section (A) is placed in the specimen bank at liquid nitrogen temperatures for long4erm storage, and the second section (B) is homogenized and part of the tissue is analyzed to provide baseline data.

332 Becket et a].

To evaluate the significance of the various tissues for environmental studies, three tissue types were selected for analysis. Initial determina- tions of both inorganic and organic constituents were performed on cryogenically-prepared homogenates (4) from two northern fur seals from the 1987 Pribilof Islands hunt. The contaminants of interest includ- ed both organic compounds (polycyclic aromatic hydrocarbons, chlori- nated pesticides, and polychlorinated biphenyls) and trace elements.

The determination of 23 trace elements was performed on three tissue types from two northern fur seal samples using instrumental neutron activation analysis (INAA), and the results are reported in this paper. The procedure followed the approach developed for analyzing human tissue from the NBSB (5). The tissue homogenates were freeze- dried, pressed into 200 mg pellets, and irradiated. The blubber tissue could not be transformed into a sample suitable for the INAA procedure and, therefore, was not analyzed. The INAA procedure consisted of a

17 2 1 120-s irradiation at a fluence rate of 2"10 neu t ron /m/s , followed by two couffts on a high resolution gamma spectrometer, and a second 16-h irradiation followed by two counts after appropriate decay times. Ele- ment standards for quantitation by the comparator method and certified reference materials (NIST SRM 1577 Bovine Liver, NIST SRM 1572 Citrus Leaves, and IAEA MA-A-2/TM Fish Flesh) for quality assurance were included in the irradiation sets. A special high-count-rate gamma spec- trometer, combined with loss-free counting techniques (6), was used to lower the instrumental detection limit for vanadium.

RESULTS AND DISCUSSION

The results of the initial INAA studies are listed in Table 1. Replicate samples from the various tissues did not deviate more than 5% relative if the analytical uncertainty, owing to counting statistics, was suffidently small. This result is in agreement with earlier findings on human liver tissue in the NBSB (7). The analysis of the certified reference materials did not reveal any bias. The following elements were also included in the quantitative evaluation, but were below their detection limit in all investi- gated tissues: Sr, La, Ce, Sm, Eu, Tb, Hf, Ta, Au, and U.

Both the human and seal liver tissues have comparable element concentrations that allow for a similar suite of elements to be detected with the established procedure. The seal kidney and muscle show signifi- cantly different trace element concentration profiles for some elements. For example, arsenic is highly enriched in muscle tissue, whereas other elements, such as silver and cadmium, were below our detection limit. Therefore, as in human monitoring, the liver tissue may be the most suitable tissue for the determination of both inorganic and organic con- stituents. These limited results are comparable to results reported by

Alaskan Marine Mammal Tissue 333

Table 1 Trace Element Concentrations in Northern Fur Seal Tissues"

LIV|I UDIiI 105CLI Seal 1 Seal 2 Seal I Seal 2 Seal 1 Seal 2

h 760.4fl.0 S$|,3t5.5 1305.0110 1332.3*10.2 $05.4t3.7 47S.4t3.6

I l l 134t4.5 213'4.4 180.5.4 160.0,4.2 231'3.$ 212/4.5

IL 0.533t0.001 0.322t0.042 1,599t0.085 0 ,780.0 .053 ~,31310.041 0.712i9,049

C1 1116113 811112 1578A18 1611111 531,9t8,1 L?l.~t, I

[ )316150 3155,5| 2610109 2464133 3150131 3615136

Ca 38.3*4.4 30.6*3.8 38.1*4.4 53.1,4.0 31.5t3.7 22.1t2.5

Sc ~0.60112 ~0.0||13 ~0.00311 ~O.JO|l| O.OOilt)tO.OOlOO7 0.00008710.000104

V 0.014310.0062 0.1088*1.0044 ~O.IL3 $O.|fl ~0.0073 !|.OiT3

Ct | .0251t0.0044 0.0551,0.0050 0.0300, | .0036 0.0100*0.0032 0.061220.0030 0.031620.0030

h 4.17320.045 3 .18520.036 1 .15710.022 1 .26010 .015 1.1630,0.0158 0.1857,0.1643

fe 171.8,1.0 110.8t0.3 10.OJiO.0J 51.70,1.37 75.12,3.08 71.50*0.45

|.OISI0*O.O001| 0.1162410.00016 0.02501i0.00117 0.01733*0.0|114 O.J1501011.OiOlS! 0.|03641*0.0n057

56.34t0.82 17.0311.49 103t|.52 8.71i0.35 2.2|*|.45 2.07*0.30

|a 38.53tl.43 37.83t0.43 63.28t0.47 311810.28 35.00,1.36 33.43*|.25

is ~1.092 |.18710.020 0 .164 ,1 .021 0 . 5 i 3 1 0 . 0 2 3 0 .171 t3 .312 i . | lLti . iL|

Se 6.285i0,052 5.450*0.045 3 .156 i0 ,032 3.06610.026 3.LQ$10,026 2.147i0.018

ib 1.874t0.047 1 .755 ,0 .045 1 .36110.033 1o038t0 .030 1 .518 ,0 .028 1.418,0.028

Ha 0.38t0o10 0.05,9.11 ~0.40 ~0.~ ~0.23 ~0.28

if 0.1721*0.3123 1.3815,0.1137 ~|.0144 0.0129,0.1011 ~0.0030 ~0.0121

Cd 10.51,0.3| 8.01t0.13 0|.0|10.79 29.0210.31 $0.Z6 90.39

S| 1.|9105,9.00127 0.0037519.00033 ~0.10169 ~0.90954 Si .OI048 0.0190020.01016

1 0.44729.071 1.7419.12 ~0.32 ~9.10 ~0.23 ~|.21

Cs 0.01691,0.00048 0.32880*0.09053 0.02474t0.90050 0.0231219.00044 0.04045,9.90a46 0.04321t0.00045

�9 INAA results (mg/kg), errors represent uncertainty of a single determination and are comprised of counting statistics and estimated uncertainty owing to standards and irradiation and counting geometries.

others for trace e lement levels in this species (8,9). More detailed baseline studies, as well as values for other trace e lements , can be obtained from those samples in the same collection, which have not yet been analyzed. The long- term stability of samples being s tored in the NBSB has been recently conf i rmed (10).

Al though the emphas is is on the collection of tissues for the deter- ruinat ion of contaminants that may be associated with industrial activ- ities, it is also recognized that the d e v e l o p m e n t of an archive of marine m a m m a l tissues, which have been collected and stored using carefully

334 Becker et ai.

controlled procedures, provides a resource that goes beyond the immedi- ate needs of the program. Such an archive, developed over several years, provides a resource of materials for future investigators addressing ques- tions concerning the transport of elements and compounds 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 and environmental assessment and management, as well as organizations and individuals with interests in the polar ecosystem.

ACKNOWLEDGMENT

This work was supported by the Minerals Management Service, Department of the Interior and the National Oceanic and Atmospheric Administration. Special acknowledgment is extended to the seal harvest crews of St. Paul, Pribilof Islands, Alaska, TDX Corporation, NMFS, and the North Slope Borough Department of Wildlife Management, Barrow, Alaska, for working with us to obtain samples.

REFERENCES

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2. S. A. Wise, B. J. Koster, R. M. Parris, M. M. Schantz, S. F. Stone, and R. Zeisler, Intern. J. Environ. Anal. Chem., 37, 9-106 (1989).

3. S. A. Wise and R. Zeisler, Environ. Sci. Technol. 18, 302A-307A (1984). 4. R. Zeisler, J. K. Langland, and S. H. Harrison, Anal. Chem. 55, 2431-2434

(1983). 5. R. Zeisler, R. R. Greenberg, and S. F. Stone, J. Radioanal. NucI. Chem. 124,

47-63 (1988). 6. G. P. Westphal, J. Radioanal. Chem. 70, 387-410 (1982). 7. R. Zeisler, S. H. Harrison, S. A. Wise, Biol. Trace Elem. Res. 6, 31-49 (1984). 8. C. J. Goldblatt and R. G. Anthony, J. Environ. Quality, 12(4), 478482 (1983). 9. D. R. Smith, Report to NMFS, Juneau, Alaska by Creative Educational

Consultants, Monterey, California, 1986, 5 pp. plus 4 pp. appendix. 10. R. Zeisler, R. R. Greenberg, S. F. Stone, and T. M. Sullivan, Fresenius Z.

Anal. Chem. 332, 612-615 (1988).