I. Introduction

The Marine Animal Resource Center (MARC) was formed in 1976 by concerned scientists and citizens in Seattle, Washington. MARC was formed with the stated purpose of facilitating research and fostering the well being of marine mammals. Over the past sixteen years, MARC has set up an extensive sit-ending network and telephone network, and is in the process of fat mining a comprehensive analytical laboratory.

The phone system is the life blood of MARC. Private citizens, State Patrols and government officials have access to the phone number twenty four hours a day, 365 days a year. The types of calls received range from sightings to reports of strandings or deaths. The sightings ate usually "sailing" sea lions, resting harbor seals and the occasional Grey whale or floating log. The stranding calls consist of harbor seal pups on the beach or log boom, and grey whales beaching during low tide. The deaths cover the whole range of marine mammals (see Chart I).

Over the past sixteen years, MARC has collected tissue samples, blood chemistries, pathological and toxicological information from animals situated in the previously mentioned circumstances. We have also expanded into water and sediment chemistry studies. We are now developing a data base to format this information in order to provide fat scientists a comprehensive picture of what is occurring with marine mammals in the Puget Sound. As of yet, the organizations responsible for marine mammal work in the Puget Sound do not have the means to compile an overall system for analysis of marine mammal deaths in the Puget Sound.

This lack of "response and responsibility" has resulted in confusion, lack of cohesion and a poverty of behavioral data "elating to marine mammals in the Puget Sound. Are any of these mortalities related? Are harbor seals "stranding" in large numbers? How does the composition of the Puget Sound's sediment affect they whale feeding? These questions and many more are in need of answers. Various agencies claim pollution does not affect marine mammals in Puget Sound and others say it plays an important role. Who is right? As this point and many others are argued, it is the animals that continue to provide the answers. In this paper I will show how MARC is trying to listen.

II. Sightings

A. Pinnipeds

Over the past ten years, MARC has received 786 sight calls on pinnipeds (see Chart I). The majority of these calls are of "sailing" California sea lions. When the general public observes this behavior, they are concerned that the sea lion may be sick or dying. The "sailing" behavior is explained to them and they are requested to report any other sightings. A MARC response team member confirms the observation and logs it into response files. We also receive copies of boat logs on sightings of sea lions and seals, which include observations on buoys, log booms and marine piers.

We also receive "false alarms;" calls which usually turn out to be floating logs that, at a distance, look like marine mammals.

B. Cetacea

A total of 214 cetacean sightings have been recorded by MARC in this ten year period; the majority of these have been grey whales feeding or swimming in the various bays in Puget Sound. A local Seattle T.V. station, KOMO, has been of great help in getting film footage of these whales from their helicopter. This has aided MARC in responding to these calls quickly and obtaining an accurate record of the animal's behavior. Over the past two years we have filmed over twenty grey whales feeding and swimming in Puget Sound. MARC has also received observations from the Coast Guard and The Whale Museum. These sightings aid us in keeping track of how many grey whales enter Puget Sound.

III. Strandings and Mortalities

A. Pinnipeds

Many of the pinniped strandings are baby harbor seals picked up off the beach. MARC has an educational program in effect, but we still receive over five to ten pups per season, brought in by private citizens. After blood is drawn and the health of the animal is determined "releasable," it is tagged and released into Puget Sound. Over the past sixteen years, MARC has received 106 pups, of which 92 have been rehabilitated to the point of release. The main cause of mortalities is not yet pin-pointed; theories range from hooks and pollution to "natural" diseases. Recently the, Armed Forced Institute of Pathology has discovered a herpes virus in a yearling harbor seal that MARC received from Port Gamble, Washington (Pletcher, 1990).

B. Cetacea

As with the pinnipeds, the precise cause of cetacea mortalities in Puget Sound has yet to be determined. Factors such as pesticides, heavy metals, bacterial diseases and "natural" causes have all been discussed as causative agents. Histopathological and toxo logical studies have been performed on a number of Grey whale tissues, but none have been conclusive. Over the past ten years a steady increase in grey whales appearing in Puget Sound has brought greyer whale deaths. Many agencies have discussed how samples should be collected, tested, and analyzed, but these have yet to be any solid reason for the increase in these deaths. I will elaborate on this problem in the discussion to follow.

IV. Chemical Analysis

When an injured or sick marine mammal is brought into MARC, our staff veterinarian examines for specific problems, draws a blood sample and administers the proper medication if deemed necessary. Once the blood work has been examined and the animal's health is in recovery, we are able to get it into out rehabilitation pool to increase its strength through exercise and feeding. Once this is completed the animal is tagged and released.

V. Analytical Research

A. Overview

Many marine mammal researchers collect and record data based on the physical condition of the animal in the field. Such information may include but may not be limited to weight, length, girth, sex, and age. Field necropsies may provide more immediate information, but usually organs and specific tissues must be prepped and sent out for more specific analysis. The majority of these tissues are sent out for histopathological examination.

In 1984, an increase in the observation of Grey whales in the Northern Puget Sound region was reported. MARC researchers as well as toxicologists from National Marine Fisheries cooperated in obtaining information from these animals. In his 1984 report, Malins a blood and brain tissue aluminum level of 2.2 ppm. Other contaminants include lindane, chlordane and a variety of other toxic materials. This issue has become one of controversy among different factions of scientists who are willing to "shrug off" and disregard available data without more intensive and in depth analytical testing.

The goal of the Marine Animal Resource Center is to approach each animal as an unknown entity. We simply don't know the history of a particular animal, such as where exactly did it come from, what is its home range, what were the main constituents of its diet prior to death? In treating each animal as an unknown, we may elect to run a battery of chemical tests to determine whether or not any industrial pollutants may be present Such an approach will allow us to establish population and species specific baselines that we should expect to encounter in the future.

Some marine mammals, Grey whales in particular, feed in the sediments of Puget Sound; this fact makes them ideal bio accumulators and good subjects for chemical analysis. The industrial nature of some Puget Sound locales (i.e. Bellingham, Ferndale, Seattle and Tacoma) may lead one to test for a variety of organic and metallic pollutants.

B. Sample Collection

One of the most important aspects of any analytical procedure is sample collection methodologies and chain-of-custody documentation. Tissue and organ samples should be removed from the body by a clean, contaminant free stainless (surgical) steel implement. Experience has shown that sample collection in the field does not always follow ideal conditions. This is also the case with decontamination of sampling equipment. Calambokidis et. al. (1990) recommended equipment decontamination with methylene chloride. We have felt that, for our purposes, methylene chloride decontamination procedures may cross contaminate field samples thereby skewing laboratory data. MARC staff employs a DI water, Alcanox DI water cleaning procedure. Utilizing a good laboratory detergent and adequate rinse should make the instrument suitable for resampling. Clean DI rinse water will then be poured over sampling equipment into containers for testing along with other samples collected. This will provide us with quality assurance data regarding sampling techniques. Samples will be placed into EPA cleaned glass containers with Teflon lined lids. Sample containers are then sealed with chain-of-custody seal and placed into a cooler with blue ice for transportation to the lab. Full chain-of-custody documents will accompany the samples from their point of generation to the laboratory.

C. Metals Analysis

There is some consideration being given to sample preparation before analysis. La Cognata et. al. (1987) of John Carroll University has examined three alternate methods of sample digestion prior to analysis by Atomic Absorption Spectra-photometry. These include hydrofluoric, perchloric, and hydrochloric acid digestion. All of these methods give significantly different values for the metals tested for. We have selected a strong acid digestion utilizing hydrofluoric acid (acid digestion of sediments, sludges, and soils, EPA method 3050). This method is mostly for soils and sediments in the area from which the animal was found. This method most closely resembles the processes found in mammalian pathways for digestion in the gut. We are currently trying to modify this procedure to actually duplicate the process in the mammalian stomach. Other procedures utilized for metals analysis may include EPA method 3040, dissolution procedures for oil, greases, or waxes. Table I indicates the metals tested for and their respective testing procedures.

Table 1. Methods for Determination of Metals

As we accrue more information on metals present in tissue and sediment samples we may modify and expand current methods being employed.

D. Organic Analysis

Samples being analyzed for organic contaminants should be stored properly, transported to a laboratory, and analyzed within four days of collection. There has been some discussion among researchers regarding suitability of testing for organic pollutants in decaying flesh of animals that have been stranded for an in determinant amount of time. Work by the Michigan Rudabon Society on nesting shore bit ds has indicated the presence of DDT and DDE analogs many years after the use and production of these materials have been banned by federal regulators It is out thought that persistent contaminants are worth looking for as they may aid us in determining the genes-al disposition of these compounds in Puget Sound. Persistent materials that are heavier than water are of specific importance as they are more likely to be consumed by bottom feeding organisms when they fit st enter the food chain.

Table 2. Determination of Organic Analytes by Gas Chromatographic Methods (40 CFR Part 261)

In addition to the stated analytical methods, a screen utilizing various GC/MS methods may be employed to the areas of testing. All samples will be stored, transported, and analyzed in accordance with EPA and Puget Sound Estuary Protocols.

VI. Discussion

From the data presented in this paper, there is not sufficient evidence to deter mine a specific cause of death of most marine mammals in Puget Sound. A few die of natural causes, such as old age, myocardial infarctions or "disease." Some die of unnatural causes such as bullet holes or net entanglements, but the majority of deaths fall into the "unknown" category. Past literature suggests a wide range of theories, altogether fairly scientific in their conclusions. Polychlorinated biphenyls (PCB) were found in forty one stranded Pilot whales along Newfoundland and Canada (Muir 1988). Fourteen humpback whales dies along Cape Cod after feeding on Atlantic mackerel containing Suxetoxin a dinoflagellate neurotoxin responsible for paralytic shellfish poisoning in humans (Geraci 1989). Here in Puget Sound we have found traces of aluminum oxide and various pesticides in Grey whale tissue samples (Gornall, Pers. Comm.) and a herpes virus in harbor seals (Pletcher, 1991).

The one common factor that is hindering the correct and future research of marine mammal deaths in Puget Sound is funding. One other organization performs research on marine mammal deaths and receives funding from governmental grants. Much of this research is concentrated on harbor seals and little toxicology work is performed. In order to carry out this type of work thoroughly and accurately, a great deal of time and money is required. A specific protocol for analyzing for toxins in marine mammal tissues needs to be formatted. LaGognata (1987) discusses three varying methods of toxicological analysis of Northern fur seals. Are these adaptable to Grey whales or to Harbor seals? A complete histopathological workup should also be performed. Presently, the Armed Forces Institute of Pathology is working either MARC to analyze the marine tissue samples we collect.

Also, we must assure that properly trained individuals are performing the necropsies. A pathologist and a veterinarian familiar with pinniped and cetacea anatomy should be on hand during the necropsy. Time and time again, we encounter individuals making generalized statements about cause of death who have no qualification or proper testing procedures in place to do so.

Figure 1A.

Figure 1B.

References

1.  Calamlbokidis, J., G. Steiger. Recommended Guidelines for Sampling Marine, Mammal Tissue Samples for Chemical Analyses in Puget Sound and Adjacent Waters. Prepared for U.S. Environmental Agency. December, 1990 draft.

2.  Geraci, J.R., D.M. Anderson, et al. Humpback whales (Megaptera novaeangliae) fatally poisoned by clin flagellate toxin. Am. J. Fish. Auguat. Sci. 46 (11) 1895-18987 1989.

3.  Locognata, S., Edwin J. Skoch. A comparison of Techniques for the Extraction of Heavy Metals of Tissues. Proceedings from the 18th Annual IAAAM Conference 85­101, 1987.

4.  Malins, D.C. Letter to Washington State Department of Ecology. September 26, 1984.

5.  Muir, D.C.G., Wageman, R., et al. Organochlorine Chemical and Heavy Metal Contaminants in White Beaked Dolphins (Lagenorhynchus albirostris) and Pilot Whales (Globicephala melaena) from the Coast of Newfoundland, Canada. 1988.

6.  Pletcher, J.M., D.V.M. Letter to Marine Animal Resource Center. February 1, 1991.