Postmortem and toxicological evaluations of animals that die in rehabilitation centers provide valuable information for determining the primary and contributing causes of death. The results can be used by veterinarians to improve the care of animals during an oil spill. Furthermore, the evaluation of data following the event provides the basis for improving the overall care of animals in future spills. To differentiate between the various causes of mortality, it is important to include data from the clinical history, necropsy results histopathology, and toxicology of each animal. Therefore, organized record keeping and tissue labeling are critical.
The following procedures were used for the postmortem examination of sea otters during the EVOS and are recommended for future spills. We list the tests in order of importance. Ranking is based on the probability that the procedure will contribute to understanding the cause of mortality in oiled sea otters.
General
Hematology, serum chemistry, necropsy results, and histopathology of select tissues should be evaluated for each sea otter that dies in a rehabilitation center. Because of the expense, toxicological analyses will probably be limited to a representative sample of animals. The degree of external oiling, reproductive status, age, and sex of the animal should be recorded, as these factors may affect the response to contamination. External oil exposure should be categorized as heavy (> 60% body coverage), moderate (30-60% body coverage), light ( 30% coverage or light sheen on fur) or unoiled (no visual or olfactory detection of oil). (See Chapter 4 for further discussion.)
Complete necropsies should be performed on all sea otters that die during rehabilitation and on fresh carcasses found in the wild. Necropsies should be performed within two hours of the animal’s death. If this is not possible, the carcass should be refrigerated until postmortem examination. Standard veterinary procedures for the necropsy of small mammals are recommended. Geraci and Lounsbury (1993) provide an excellent guide for conducting necropsies and collecting tissues from marine mammals. A pathology team consisting of a veterinary pathologist assisted by a veterinary clinician or a laboratory technician should be established. All necropsy records should be summarized and entered on standardized forms (Appendix 2, Forms A, B; Haebler et al., 1990 Download PDF) and into a computer data base. Photographic or videotaped documentation should be included and annotated.
The carcasses are first examined externally. After opening the abdomen and thorax, the presence or absence of abnormalities should be noted for each organ or tissue. All major organs should be weighed during necropsy. However, special care must be taken to avoid contaminating tissues that will be collected for residue analysis for petroleum hydrocarbons. To avoid cross-contamination, stainless steel or titanium dissecting instruments should be cleaned with dichloromethane. Samples of liver, lung, kidney, brain, muscle, and fat (bile, urine, stomach contents, intestinal contents, and placenta when possible) should be collected for complete petroleum hydrocarbon analyses. Samples from each tissue should also be obtained for histopathological examination. Additional samples will be required for microbiology, parasitology, and virology.
Tissue samples may be requested by the state and federal trustees (i.e. USFWS or the Department of Fish and Game). These agencies will provide the investigators with the permits required for collecting, handling, and analyzing tissues from marine mammals.
Histopathology
Pathologic changes in tissues provide important information for determining the cause of death. Tissue samples for histopathology should be collected from major organ systems at the time of necropsy, and should include normal appearing tissues as well as abnormal tissues (See list in following paragraph). Samples for histology should be less than 1 cm thick and preserved in 10% neutral buffered formalin. Several parallel slits in larger samples will facilitate penetration by the preservative. After fixation, the tissues should be embedded in paraffin and sectioned at four microns Junqueira et al., 1986). Mounted sections are usually stained with hematoxylin and eosin, and then examined microscopically.
Following is a list of sea otter tissues that should be collected during necropsy for hostopathological evaluation.
Thyroid
Pancreas
Cross section trachea and esophagus
Adrenals
Thymus
Kidney
Heart
Urinary Bladder
Lung
Skeletal Muscle
Liver
Bone Marrow
Diaphragm
Eyes
Gallbladder
Brain w/Optic nerves
Stomach
Pituitary
Small Intestines
Skin
Large Intestines
Lymph Nodes
Spleen
Gonads
Residue Analyses for Petroleum Hydrocarbons Tissue samples (> 50 gm) for residue analysis should be trimmed with a clean titanium knife and frozen individually in acid washed (I-Chern TM) jars with teflon lid inserts. Fluids such as urine and bile should be placed in amber-colored jars to prevent photodegradation. The samples should be kept frozen at -70 C. Warmer temperatures during storage could cause a breakdown of organic contaminants (Geraci and Lounsbury, 1993).
The evaluation of petroleum hydrocarbons in tissues is expensive and provides limited information. Unlike many of the chlorinated hydrocarbon pesticides, petroleum derived hydrocarbons are metabolized and do not bioaccumulate to significant levels in many tissues. Without detailed information about the duration of oil exposure and the previous health of the animal, it is difficult to interpret the results of tissue residue analyses. Oiled sea otters may succumb to the lethal effects of hypothermia or stress before there is a significant toxicological effect of petroleum hydrocarbons in the tissues (Mulcahy and Ballachey, 1993, 1994). In view of this, we recommend limiting residue analyses to selected tissues from animals with the most complete medical records and history of oil exposure.
Following the EVOS, we examined four tissues (liver, lung, brain, and kidney) from twenty-two sea otters that died in rehabilitation centers. The study groups included heavily (n = 7), moderately (n = 5 and lightly (n = 6) oiled animals, as well as unoiled sea otters (n = 4). Because the high lipid content of the brain samples interfered with the standard analyses used for petroleum hydrocarbons, the results were considered unreliable. Future attempts to assess petroleum hydrocarbon levels in samples of brain and blubber must consider such difficulties associated with analyzing lipid-rich tissues.
Standard protocols for measuring petroleum hydrocarbons in tissues lave been established and are used by many analytical laboratories. Approximately five grams of tissue are homogenized, extracted, and concentrated. The extracts are analyzed for individual petroleum hydrocarbons by gas chromatography and mass spectrometry.
Selection of a commercial laboratory for petroleum hydrocarbon analyses should be based in part on experience with protocols specific for biological tissue samples. Many laboratories that analyze nonbiological samples may not be suitable for tissue analysis. Problems routinely encountered during tissue residue analyses include interference from lipids and other naturally occurring hydrocarbons, and focal areas of contamination within tissues. The analysis should include a quantitative discrimination of individual polycyclic aroma hydrocarbons (PAHs) and saturated petroleum hydrocarbons (PHCs). The laboratories that were used for tissue residue analysis during the EVOS are listed here:
Clinical laboratories for the analysis of petroleum hydrocarbons in biological tissues:
Biological Tissues
Battelle Ocean Sciences
397 Washington Street
Duxbury, Mass 02332
(617) 934-0571
Blood
National Medical Services, Inc.
2300 Stratford Ave., PO Box 433A
Willow Grove, PA 19090
(215) 657-4900
Biological Tissues and Blood
Geochemical and Environmental Group
Texas A%26M University
833 Graham Road
College Station, TX 77845
(405) 690-0095
