🚀 go-pugleaf

To follow is the technical summary of a study I completed not long ago for the
Department of Interior of the United States.  The full report is available
from:

   U.S. Department of the Interior.
   949 E. 36th Ave, Rm 603, Anchorage,
   Alaska 99508-4302 USA

I hope you will find this information useful.  Those especially interested in
the new sampling technology developed by ETT should contact me directly.
However, I will be out of contact (in Europe) during the 1 June - 1 August
period.

   Richard H. Lambertsen, Ph.D., V.M.D.
   Ecosystems Technology Transfer, Inc.
   P.O. Box 6788
   Titusville, FL 32782-6788  U.S.A

____________________________TECHNICAL SUMMARY_______________________________

STUDY TITLE:  A Baseline Method to Evaluate Chemico-biological Interactions
Involving Cetacea

REPORT TITLE:  A Baseline Method to Evaluate Chemico-biological Interactions
Involving Cetacea, with Description of an Improved Whale Biopsy System Designed
for Multidisciplinary Research.

CONTRACT NUMBER:  14-35-0001-30553.

SPONSORING OCS REGION:  Alaska.

APPLICABLE PLANNING AREAS:  All.

FISCAL YEAR(S) OF PROJECT FUNDING:  1991; 1993.

COMPLETION DATE OF REPORT:  June l993.

COST(S):  FY 1991: $56,590;  FY 1993: $435;  Cumulative Project Cost: $57,025.

PROJECT MANAGERS:       J. Montague, J. Geiselman.

AFFILIATION:    U.S. Department of the Interior.

ADDRESS:        949 E. 36th Ave, Rm 603, Anchorage, Alaska 99508-4302.

PRINCIPAL INVESTIGATOR*: R.H. Lambertsen.

KEY WORDS:  Gulf of Maine; Massachusetts Bay; large cetaceans; endangered
species; humpback whale; toxicology; sampling methods; organochlorines; PCB's;
molecular biology; sex determination; P450 cytochrome oxygenase; biological
effects; histochemistry; tissue culture; cryogenics; photoidentification;
ecosystem integration; ecosystems health.

BACKGROUND:  The likely roles of large whales in concentrating man-made
chemicals from contaminated coastal zones and transporting these en masse to
pelagic and benthic habitats indicate possible health risks to endangered
species, other biota, and man.  In U.S. coastal waters, increases in the number
of deaths and apparent mortality rates of marine mammals, including humpback
whales, since the l970's have led to speculation that toxic pollution of the
coastal zone is impairing the resistance of these animals to disease.  In
parallel it has been suggested that these gigantic mammals serve as useful
sentinels of environmental degradation, yielding insight into the health of
large marine ecosystems because of their environmental integrating capability.
  To address these issues, and to better interpret existing data on tissue
contaminants found in stranded whales, toxicological baselines representing
free-ranging whale populations are needed.  Without baseline information it is
difficult if not impossible to judge the pathological significance of tissue
contamination found in dead, moribund, or beached whales. Traditional methods
to obtain baseline toxicological data, however, have relied principally on
postmortem study of animals taken directly from the wild.  Taking a different
approach, the present study developed a supra-vital method to obtain
toxicological and other types of information from free-living large cetaceans. 
This involved modification of an older, standard whale biopsy instrument to
enable acquisition of high quality tissue samples of sufficient volume to allow
high sensitivity toxicological analyses of tissue contamination in living
whales.  The promising possibility of cohort analyses, in which known
individuals might be studied again at a later date, thereby was achieved.  An
initial field test was conducted on humpback whales in the southern Gulf of
Maine, during the feeding phase of the annual migration of this species.

OBJECTIVE:  To develop and demonstrate a supravital method needed a) to
evaluate pathways of ecotoxicological transport by large whales of
anthropogenic pollutants and b) to establish baseline measures of tissue
contamination in  free-ranging populations of endangered whales.

DESCRIPTION:  A new whale biopsy system was designed to obtain efficiently a
larger quantity of tissue for biologic, genetic, toxicologic, and veterinary
research.  All parts of the sampling device to make contact with the sample
were made of titanium or Teflon, materials which meet the protocols of the
National Institute of Standards and Technology for archival quality sampling of
marine mammal tissues.  After successful land-based testing of the prototype on
pieces of whale blubber, 24 units were produced and then field tested in
Massachusetts Bay on living humpback whales (Megaptera novaeangliae). 
Immediate behavioral responses were noted in 18 cases.  In 10 animals, a
statistical comparison was made of respiratory and other behavioral variables
recorded for 30 minutes before and 30 minutes after the samples were taken.
  The tissue biopsies, subdivided within one hour of their acquisition at sea,
were preserved in liquid nitrogen and by other means for subsequent laboratory
studies conducted by a collaborating group of specialists. Laboratory study
determined 1) sample viability (by means of in vitro  cell culture); 2) sex of
the animals sampled (by application of the human pDP1007 y-chromosome specific
molecular probe); 3) cytochrome P4501A oxygenase activity in endothelial cells
which line the blood vessels of the skin (by means of immunohistochemical
staining); 4) lipid concentration at different depths across the blubber (by
microgravimetry after solvent extraction of tissue lipid); 5) concentrations of
the toxic man-made products transnonachlor, hexachlorobenzene, ppDDE, dieldrin,
a-chlordane, heptachlor epoxide, and polychlorinated biphenyls (PCB's) in the
blubber (by gas chromatography, in some cases followed by mass spectroscopy); 
and 6) mathematical profiles describing the concentration of selected toxic
contaminants across the blubber (by computer analysis of contaminant data vs
subsample depth in four animals).  On the basis of the data obtained, and
available information on the sighting histories of the whales sampled, the
possible best use of this sampling strategy for ecotoxicology was considered.

SIGNIFICANT CONCLUSIONS:  The results supported the conclusion that a more
information-effective, cost-effective, and humane whale biopsy system has been
successfully developed and field-proven.  On this basis, the instrument
developed can be recommended for future research and monitoring applications
that are carried out selectively by specially trained personnel.  The combined
advantages likely will enable a) nondestructive tissue sampling of all large
Cetacea, limited to the skin and blubber, b) long term cohort studies of
involving known individuals.
  Measurement of contaminant profiles across the blubber in particular
suggested a way to detect feeding-related exposure of endangered large whales
to lipophilic toxicants on a local geographic scale.  In combination with
sighting histories, the contaminant profiles determined were consistent with
the possibility of elevated rates of food-web transfer of organochlorine
pesticides and PCB's in Massachusetts Bay, in the Southern Gulf of Maine. 
Previously published data on natural variation in lipid content across the
blubber of fin whales, Balaenoptera physalus, also suggested a way to measure
feeding + anabolic success in this and possibly other large baleen whale
species.  As a physiologic measure of body condition, this could significantly
enhance sighting surveys seeking to evaluate the status or health of sentinel
whale populations.

STUDY RESULTS:  Powered by a pneumatic projection mechanism, 21 of 24 (88%)
experimental darts hit the study animals targeted.  The majority of these
experimental darts struck within a small target area at or near the base of the
dorsal fin.  Nineteen out of 20 (95%) fired in the intended design
configuration obtained a useful sample.  One trial dart fired with the biopsy
instrument still contained in its sterile wrap did not function. The biopsies
obtained were of sufficient size for distribution of 0.5-1.0 cc sample portions
to 6-8 different research groups studying population structure, sex
distributions, inbreeding coefficients, aging methods, histopathology,
genotoxicity, ecotoxicology, and physiology.  Seven out of nine (78%) samples
plated in vitro  were viable and showed active cell proliferation, without
bacterial contamination, 8 to 20 days after the initiation of cell cultures.
  Molecular biological study enabled the sex of study animals to be determined,
with results from application of the pDP1007 probe serving effectively as a
quality control over an earlier, visual method of sex determination in the
humpback whale.  The immunohistochemical findings, by comparison, were
negative, and accordingly suggested both the absence of induced cytochrome P450
1A oxygenase activity in endothelial cells of the skin of the animals sampled,
and the lack of an in vivo detoxification response.  However, further study
which employs other immunohistochemical staining methods than used here are
needed to examine more thoroughly the possibility of an in vivo  detoxification
response
-- a biological effect of exposure to toxicants that would be suggested by
P4501A cytochrome oxygenase activity in the skin.
  Measurable concentrations of the transnonachlor, hexachlorobenzene, ppDDE,
dieldrin, a-chlordane, heptachlor epoxide, and polychlorinated biphenyls
(PCB's) were found in the blubber of all the animals sampled.  This indicated
unequivocally that endangered humpback whales are exposed to these toxic
anthropogenic products, possibly while feeding in U.S. coastal waters. 
Contrary to expectation, however, detailed analysis of the concentrations per g
of lipid of hexachlorobenzene, transnonachlor, ppDDE, and total PCB's,
conducted in four animals, generally revealed no significant downward gradient
in these contaminants across the blubber. Furthermore, two of these four
animals were well known to utilize heavily Massachussets Bay, in the Southern
Gulf of Maine, as a feeding area.  The only exception was one individual, which
had been previously sighted in the Gulf of Maine only once, and which
apparently did not use this region intensively as a feeding habitat.  Only the
sample from this animal showed a significant downward gradient in contaminant
concentration  per g lipid from the outer to the inner blubber layer, in both
hexachlorobenzene and total PCB's.  Thus, in light of published evidence that
the inner blubber layer of large baleen whales is an especially dynamic tissue,
and more closely reflects recent feeding-related assimilation of anthropogenic,
as well as natural compounds, the pattern of tissue contamination observed was
consistent with the possibility of an elevated rate of exposure to toxic
organochlorines, including PCB's, in Massachussetts Bay.
  Results of the analysis of behavioral responses to the sampling technology
developed were compared with published data describing the behavioral response
to the older, "standard" whale biopsy system, which is powered by a crossbow. 
On balance, this comparison suggested that the potential for adverse biological
effect is less with the newly developed instrument. With both systems,
respiratory variables and estimated swimming speeds did not change
significantly between the 30 minute pre- and 30 minute post biopsy periods. 
Also, quantitative distributions of immediate behavioral responses, ranked by
intensity, appeared essentially similar.  However, the improved trajectory,
range, control, and power of the new unit substantially reduced the number of
failed biopsy shots and decreased disturbance of study animals caused by close
vessel approach and repeated biopsy attempts.  The need for repeated sampling
of individual whales to supply different investigative purposes was likewise
reduced because archival quality samples suitable for multiple research uses
were obtained.

STUDY PRODUCTS: 1) Improved whale biopsy technology, 2) Lambertsen, R.H.,
Baker, C.S., Weinrich, M.T., Modi, W.S.  A Baseline Method to Evaluate
Chemico-biological Interactions Involving Cetacea, with Description of an
Improved Whale Biopsy System Designed for Multidisciplinary Research.  A final
report by Ecosystems Technology Transfer, Inc., University City Science Center,
3624 Market Street, Philadelphia, PA 19104  to the Minerals Management Service,
949 E. 36th Ave, Rm 603, Anchorage, Alaska 99508-4302. Contract
14-35-0001-30553.  59 pp., with technical drawings and 18 pp appendix giving
photographs of study animals.

*P.I.'s affiliation may be different than that listed for Project Managers.