EPA-R3-73-042
MAY 1Q71 Ecological Research Series
IVI n I I 3 / U
Interaction between
Marine Organisms and Oil Pollution
vS
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport* and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
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EPA-R3-73-042
May 1973
INTERACTION BETWEEN MARINE ORGANISMS AND
OIL POLLUTION
by
Dr. Max Blumer
Dr. John M. Hunt
Dr. Jelle Atema
Lauren Stein
Project #18050 and 18080 EBN
Project Officer
Dr. C. S. Hegre
National Marine Water Quality Laboratory
Environmental Protection Agency
P. 0. Box 277
West Kingston, Rhode Island 02892
Prepared for
OFFICE OF RESEARCH AND MONITORING
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price $1.26 domestic postpaid or $1 QPO Bookstore
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EPA Review Notice
This report has been reviewed by the Water
Quality Office, EPA, and approved for publication.
Approval does not signify that the contents
necessarily reflect the views and policies of
the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for
use.
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ABSTRACTS
PART I
INTERACTION BETWEEN MARINE ORGANISMS AND
OIL POLLUTION
The present work has established the following:
I. Hydrocarbons in uncontaminated living plants and animals differ
in structure and molecular weight distribution from the hydrocarbons
in fossil fuels. We have established criteria and methods that
permit the detection of hydrocarbons from fossil fuels in the presence *
of biogenic hydrocarbons and vice versa.
II. Hydrocarbons are remarkably stable in marine sediments and in the
lipids of marine organisms, even chemically reactive hydrocarbons can
move unaltered through several trophic levels in the marine food web.
Degradation and dispersal eventually proceeds by physical (evaporation,
dissolution), by chemical (oxidation, polymerization) and by bio-
chemical (metabolism) processes.
III. There is now ample evidence for the importance of chemical communi-
cation between marine organisms, both with inter- and intraspecific
message systems. Our work shows again that only very low concentrations
of organic stimuli are required for communication. Consequently, such
processes appear especially prone to interference by pollutants at low
concentration levels.
This report was submitted in fulfillment of project #18050 EBN under
the sponsorship of the Water Quality Office, Environmental Protection
Agency.
PART II
SUBLETHAL EFFECTS OF CRUDE OIL ON LOBSTER,
CHQMARUS AMERICANUS) BEHAVIOR
Small quantities of crude oil (0.9 milliliters in 100 liters of sea-
water) interfere with some specific, possibly chemosensory, behavior
of the lobster, Homarus americanus. Timing of their feeding behavior
showed that the delay period between noticing food and going after it
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doubles when oil was added. The water soluble fraction of this crude
oil alone (in the 50 ppb range) does not have a noticeable effect on
behavior and feeding times. Morphological changes in odor receptors
after oil exposure were not detected by light and electron microscopy.
The results indicate that small quantities of oil mixed into seawater
constitute a noxious, bad smell in the lobsters environment, depressing
his appetite and chemical excitability.
Chemical analyses showed that before the addition of oil a great quan-
tity of lipids was present in the test aquaria. When the water was
brought in contact with an oil slick, the lipid concentration dropped
considerably. The same effect was seen in the alkane and the alkene-
aromatic hydrocarbon fractions. The fate of oil in seawater followed
the usual degradation pattern.
This report was submitted in fulfillment of project #18080 EBN under
the sponsorship of the Water Quality Office, Environmental Protection
Agency.
iv
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CONTENTS
Section
I
II
III
IV
V
VI
VII
VIII
IX
X
Section
I
II
III
IV
V
VI
VII
VIII
IX
X
PART I Page
Conclusions 1
Recommendations 2
Introduction 3
Identification of Oil Pollution 5
Fate and Persistence of Oil Pollution 8
Marine Chemotaxis 10
Acknowledgements 14
References 15
Publications 16
Appendix - Species List 20
PART II Page
Conclusions 25
Recommendations 27
Introduction 29
Methods 31
Results 41
Discussion 65
Acknowledgements 69
References 70
Pending Publication 71
Appendices 72
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FIGURES
Number Page
1 Connection of oil slick tank and lobster aquaria 35
in solubles experiment.
2 Flow chart of chemical analyses. 38
3 Appearance of oil slick over five day period, 43
first series of solubles experiment.
4 Appearance of oil slick over five day period, 44
second series of solubles experiment.
5 Gas chromatograms of pentane extract from oil- 45
water mixture.
6 Ultraviolet spectra of lipid extracts of solubles 47
on days 5-10.
7 Ultraviolet spectra of alkene-aromatic fraction 48
of day 10 and of whole crude oil.
8 Gas chromatograms of alkane fraction of solubles. 52 & 53
9. Gas chromatograms of alkene-aromatic fraction of 54
solubles.
10 Antennule with aesthetasc hairs and guard hairs. 56
11 Antennule in vivo (150x, light microscopy). 57
12 Detail of tip of aesthetasc hair in vivo (1500x, 58
light microscopy).
13 Detail of base of aesthetasc hair in vivo 59
(1500x, light microscopy).
14 Aesthetasc hair details (scanning electron 60
microscopy).
a) Tip, head-on view
b) Distal 1/3 portion
c) Base, head on view, with pores in antennule
15 Cross section of middle portion of aesthetasc 61
hair (12,000x, electron microscopy).
16 Detail of cuticle of Figure 15 (45,000x) 62
electron microscopy).
17 Detail of dendrites inside aesthetasc hair 63
middle portion (33,000x, electron microscopy).
18 Detail of dendrites inside aesthetasc hair middle 64
portion (29,000x, electron microscopy).
vi
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TABLES
Number Page
1 Selected behavior units used in analysis. 33
2 Frequency of occurrence for the be- 34
havior unit - "Crude, Antenna Wave".
3 Lipid concentrations and hydrocarbon 49
concentrations in sea water.
4 Ultraviolet absorbance of lipid extract 50
of one liter of sea water.
5 Infrared absorbances, normalized to 55
2955 cm-1 band.
vii
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PART I
INTERACTION BETWEEN MARINE ORGANISMS AND
OIL POLLUTION
Project #18050 EBN
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SECTION 1
CONCLUSIONS
The present work has established the following:
1. Hydrocarbons in uncontaminated living plants and anihials differ
in structure and molecular weight distribution from the hydrocarbons
in fossil fuels. We have established criteria and methods that
permit the detection of hydrocarbons from fossil fuels in the pre-
sence of biogenic hydrocarbons and vice versa.
2. Hydrocarbons are remarkably stable in marine sediments and in
the lipids of marine organisms, even chemically reactive hydrocarbons
can move unaltered through several trophic levels in the marine food
web. Degradation and dispersal eventually proceeds by physical
(evaporation, dissolution), by chemical (oxidation, polymerization)
and by biochemical (metabolism) processes.
3. There is now ample evidence for the importance of chemical com-
munication between marine organisms, both with inter- and intraspecific
message systems. Our work shows again that only very low concentra-
tions of organic stimuli are required for communication. Consequently,
such processes appear especially prone to interference by pollutants
at low concentration levels.
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SECTION II
RECOMMENDATIONS
1. This work has established the possibility to distinguish in en-
vironmental samples between biogenic and petroleum-derived hydrocarbons.
Our baseline data come mostly from the coastal regions of the Western
North Atlantic and should be extended to other coastal regions.
2. There are strong suggestions that biogenic hydrocarbons play vital
roles in the life processes of marine organisms. Therefore, oil pol-
lution may interfere with such processes at very low concentration
levels. This may be a critical research area.
3. This work has shown that hydrocarbon pollutants enter the marine
food web. Earlier work, based on subjective (taste) tests appears
now irrelevant. The survey for oil derived hydrocarbons in fisheries
products must be extended to other species and should be performed
routinely on products that reach the market because of obvious public
health implications.
4. No guidelines or legal limits exist for the acceptability of oil
pollution in fisheries products. EPA in cooperation with public
health authorities should establish such limits for the protection
of the consumer.
5. The case study on the persistence of oil pollution reported here
deals with a fuel oil spill. Other investigations using similar ob-
jective analytical and biological methods should be carried out with
other oils and in other climatic regions.
6. Oil pollution may be more damaging through its long term and low
level effects than through the gross esthetic impact. Research in
the areas of low level toxicity, persistent damage, interference
with chemotaxis or with reproduction has been neglected in the past.
Immediate attention both to basic and applied research in these areas
is needed.
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SECTION III
INTRODUCTION
The goals of our study of marine pollution were summarized in our initial
proposal dated May 27, 1968.
Identification of Oil Pollution
Marine hydrocarbons derive in part from living organisms and in part
from pollution. We plan to establish sensitive,criteria to distin-
guish hydrocarbons from either source. This knowledge is essential
if we are to measure in the oceans the spread of the true pollutants
and not of the ubiquitous natural hydrocarbons.
Fate and Persistence of Oil Pollution
Many stable pollutants, especially hydrocarbons, pass through the
marine food chain with little alteration but often with a rise in con-
centration. We plan to study the persistence and accumulation by marine
organisms, especially human food, of objectionable and hazardous com-
pounds derived from pollution.
Effect of Pollutants on Marine Organisms
Natural compounds in the sea play an important ecological role as
stimuli for the attraction of the sexes, for Inter- and intraspecies
recognition, in the finding of food and in short and long distance
migration. Marine pollution is likely to interfere with many of
these processes. Because of the extreme sensitivity of the organisms
to these stimuli, such interference may be more subtle and still have
a more catastrophic long-term effect than the gross toxicity of bulk
pollution. We plan to study the extent to which marine pollution
interferes with these processes and the concentrations which are harmful.
This work is important for pollution control through the identification
of pollution and in establishing the types of compounds which have to
be controlled and the levels at which they have to be held.
I
During the duration of this grant we have defined the natural hydrocarbon
content of a wide variety of marine organisms (8, 11, 17, 19, 24) and
the fate of these hydrocarbons in the marine food chain (.6, 7, 8, 24).
Progress during the first year of the grant period has influenced our
research plan. A large coastal oil spill near Woods Hole has provided
a testing ground for our concepts (6, 7, 15, 16, 22). We ha\/e succeeded
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in distinguishing the spilled oil from the natural hydrocarbon back-
ground (6, 7, T$, 18, 22, 23, 24) and have shown that the spilled oil
is incorporated into oysters and scallops (6, 7, 24), Thus, the first
and second objectives of the initial proposal have been essentially
achieved and brought to a field test. During the reporting period we
have gained much new information on the environmental behavior and
fate of spilled petroleum. This is relevant to the problem of oil
spill identification.'
Also, during the granting period we have gained experience in our
research on the effect of pollutants on marine organisms. As a result,
the research aims have been redefined, and instead of proceeding as
initially planned for a third year, we are now planning to proceed
along two separate but related lines: characterization of environmental
oil samples (oil spill identification) and marine chemotaxis.
Progress in the individual areas will be discussed in the order outlined
in the introduction; much of the progress is documented in publica-
tions (see list, Section XI).
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SECTION IV
IDENTIFICATION OF OIL POLLUTION
Hydrocarbons of recent, biochemical origin are minor but not un-
important components of all marine organisms (8, 11, 17, 19, 21}.
We find a great structural variety between the hydrocarbons of
different organisms. In spite of their relative complexity, the
biogenic hydrocarbons are easily distinguished from those of fossil
fuels. We find great differences in the structural types, in the
molecular weight distribution and in the relative predominance of
individual compounds. This is of obvious relevance to chemotaxonomy,
to the study of chemical transormation in subsurface sediments and
to the differentiation between recent hydrocarbons and fossil fuels
in the environment.
We have paid special attention to:
Hydrocarbons of Marine Phytoplankton
The hydrocarbon contents of 23 species of algae (22 marine planktonic),
belonging to 9 algal classes, were analyzed (17). The highly un-
saturated 3,6,9,12,15,18-heneicosahexaene predominates in the
Bacillariophyceae, Dinophyceae, Cryptophyceae, Haptophyceae and
Euglenophyceae. Rhizosolem'a setigera contains n-heneicosane, pre-
sumably derived from the hexaolefin by hydrogenation. Two isomeric
heptadecenes have been isolated: the double bond is located in 5-
position in the blue-green algae Synechococcus bacillaris and in 7-
position in 2 green algae. In all cases, the algae contain relatively
few hydrocarbons, mostly straight chain alkanes and alkenes and some
monomethylalkanes. Pristane is present, but at a low concentration.
Zooplankton, on the other hand, contains the complex assemblage of
CIQ and C20 isoprenoid alkanes and alkenes which are derived from
phytol (21). The hydrocarbon composition of ancient sediments and
of petroleum is far more complex; many isomeric compounds belonging to
different homologous series are found, olefins are absent, and ali-
cyclic and aromatic compounds occur at much higher concentrations
than in living organisms. This work suggests that hydrocarbon analysis
may be a tool for the detection of algal species in mixed plankton
or in mixed algal lipids ingested by certain herbivores. Also,
detailed hydrocarbon analysis enables the distinction between hydro-
carbons of recent btogenic origin and hydrocarbon pollutants from
fossil fuels.
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Hydrocarbons of Benthic Algae
Saturated and olefinic hydrocarbons were determined in 24 species of
green, brown and red benthic marine algae from tKe Cape Cod area
(Massachusetts, USA) (19). Among the saturated hydrocarbons, n-penta-
decane predominates in the brown and n-heptadecane in the red algae.
A Ci7 aIkylcyclopropane has been identified tentatively in Ulva
lactuca and Enteromorpha cbrhpressa, two species of green algae. Mono-
and diolefinic Ci5 and C]7 hydrocarbons are common. The structures of
several new Ci7, CIQ and C2i mono- to hexaolefins have been elucidated
by gas chromatography, mass spectrometry and ozonolysis. In fruiting
Ascophyllum nodosum, the polyunsaturated hydrocarbons occur exclusively
in the reproductive structures. The rest of the plant contains n-
alkanes from Ci5 to C2i- A link between the reproductive chemistry of
benthic and planktonic algae and their olefin content is suggested.
Our analyses of the hydrocarbons in benthic marine algae from coastal
environments should aid studies of the coastal food web and should
enable us to distinguish between hydrocarbon pollutants and the
natural hydrocarbon background in inshore waters.
An intriguing speculation is based on Paffenhofer's 0970) obser-
vation that the sex ratio of laboratory reared Calanus helgolandicus
depends upon the species of algae fed to the nauplii. The percentage
of males produced correlates with out analyses of heneicosahexaene
in the algal food.
This and the recent discovery of an olefinic hydrocarbon acting as
male attracting substance in the female gametes of a brown algae by
Mueller and co-workers, suggests specific biochemical roles for hydro-
carbons in marine organisms. Aside from their scientific implications
these findings suggest the possibility that olefinic hydrocarbons
from cracked fossil fuels might interfere with sensitive biochemical
processes in the sea at concentration levels that have not before
been considered potentially harmful. Further work in this area seems
urgent.
Fossil Fuels, Methods
We participated in anFAO symposium on the Detection and Monitoring
of Pollutants in the Marine Environment and chaired the Panel on Petro-
leum. A review of existing methods and a recommendation for further
research has been compiled and published (21).
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Characterization of the Water Soluble Fraction of Petroleum
The immediate toxicity of crude oil and oil product arises to a signi-
ficant part from the water soluble fraction of the lower boiling hydro-
carbons. Characterization of this fast spreading fraction is necessary
and had not been carried out before. We have analyzed water extracts
of crude oils and of kerosene by gas chromatography and mass spectro-
metry; the aqueous extracts consist predominately of aromatic hydro-
carbons. Thus, in the 160-260 C boiling range substituted benzenes
and naphthalenes predominate (20).
This work may aid in the identification of crude oil derived hydro-
carbons in organisms and in the water column, especially at some dis-
tance from the spill and it provides the background for toxicity
studies on aqueous oil extracts.
Identification of Mixed Fossil and Biogenic Hydrocarbons
The marine sediments in Buzzards Bay at West Falmouth, Massachusetts
have been contaminated by a fuel oil spill in September 1969 (.6, 7, 15,
16, 22). The area provides an ideal testing ground for this subject
in view of the gradation between heavy contamination to clean sediment.
We have established that biogenic and fuel derived hydrocarbons can be
detected and determined in each others presence, even when material
from one or the other source predominates strongly (22).
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SECTION V
FATE AND PERSISTENCE OF OIL POLLUTION
The Environmental Ageing of Fossil Fuels
The natural alteration of biogenic and fossil hydrocarbon mixtures
proceeds by physical (dissolution, evaporation), by chemical^(oxidation,
polymerization) and by biochemical (metabolic) processes. Different
processes affect the hydrocarbon composition of e mixture in different
ways and their relative role can be inferred from a chemical analysis.
We are studying the alteration of fossil fuels derived from marine spills
in subtidal and intertidal sediments on the open ocean and in organisms.
In the case of the subtidal sediments (22) the degradation is slow,
and proceeds primarily through biochemical attack and-to a lesser degree
through dissolution. Oil in intertidal sediments and on the open ocean
is altered primarily by evaporation and dissolution. In the case of
substantial thickness (tar balls) this affects primarily the outside
layer. Bacterial degradation of tar balls is negligibly slow; in
beach sands it may proceed more rapidly if a sufficient supply of
oxygen and of nutrients is present.
Work on this topic has begun during the last year of the present grant
and will be pursued at greater intensity in the future project on
Oil Spills Identification.
Hydrocarbons in the Marine Food Web
Earlier work at this laboratory had already established the persistence
of biogenic hydrocarbons in the marine food web. This has been comple-
mented with further studies both on biogenic and petroleum derived
hydrocarbons.
We have now studied the occurrence of an algal derived hydrocarbon (all-
cis- 3,6,9,12,15,18-heneicosahexaene} in marine animals (8). This
olefin is accumulated nonselectively by Rhincalanus nasutus from its
algal food together with the triglyceride liptds. Other related copepods
and other zooplankton species contain little or none of this hydrocarbon,
even when grown in cultures of algae that provide R. nasutus with
that olefin. The presence of this hydrocarbon in marine vertebrates
shows that mechanisms for its injection into higher trophic levels,
either via R_. nasutus or other unidentified vectors, exist and that
even such a highly unsaturated and unstable hydrocarbon is stabilized
within animal lipids.
8
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The Oil Spill of blest Falrnouth, Massachusetts
This incident has been described in published and unpublished reports
C6, 7, 15, 16, 22). The essential conclusions regarding persistence,
spread and biological effect of this oil spill remain the same. Oil
is still chemically identifiable and the original animal populations
have not returned except at the most lightly polluted marginal loca-
tions.
Biochemical degradation of the oil has accelerated during summer 1970
and the straight chain hydrocarbons have almost completely disappeared
at some locations. Branched and cyclic hydrocarbons persist and
the original boiling point distribution of the oil has been preserved
remarkably well (22).
Of special interest are the analyses at an offshore, subtidal station
at a considerable distance from shore. At this station oil from the
spill did not appear in the sediments until at least seven months
after the accident. During the following months the oil content
of the sediment increased further and by September 1970, one year
after the spill, the contamination reached a level similar to that
in the originally more polluted stations closer to shore (22).
Because of the more rapid bacterial degradation of normal paraffins,
ratios of normal to branched paraffins of similar boiling point are .
a sensitive measure of the relative degree of biodegradation of an
oil. In the sediments at West Falmouth, this parameter demonstrates
the gradual environmental modification of the oil. Remarkably, at
all stations the degree of degradation is halted and reversed for one
to several months during spring to early summer of 1970. This, and
the late appearance of oil at the offshore station discussed above
strongly suggest the movement of a less degraded oil from the same
spill from shore into the sediments of the open Buzzards Bay, most
likely as a result of sediment movement.
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SECTION VI
MARINE CHEMOTAXIS
Some natural organic compounds are of great importance to marine
organisms serving as signals conveying information about the en-
vironment. Organisms are extremely sensitive to the presence of
such stimuli. Because of this sensitivity and the very low con-
centration level at which the natural stimuli occur in the sea, pol-
lution may interfere with the natural chemotaxis of organisms in a
subtle but potentially catastrophic way. Our research in this area
is multidisciplinary and integrates chemical studies with those in
animal behavior and neurophysiology.
The principal objectives of the program are:
1. Survey of marine animals to determine the extent of their dependence
on chemical communication.
2. Development of sensitive bioassays for testing chemicals involved
in animal communication and for the detection of the interference by
pollutants with natural communication.
3. Isolation and structure determination of natural communicants,
synthesis of model compounds and studies of the relationship between
structure and activity.
The eventual goal is an understanding of the response of marine animals
to chemical stimuli; this will enable us to predict the effect of a
pollutant of a given structure on chemical communication in the ocean.
In our initial proposal to FWPCA we requested funds to initiate a
program to investigate the effects of pollution on marine chemotaxis.
The effort is now well underway and our effectiveness has been strengthened
through the hiring - through independent funding, of a neurophysiologist
and an animal behavior specialist. They perform a detailed survey on
the importance of chemotaxis to marine fishes. Methods Include anatomical
studies of brain and sensory centers, behavior studies and neurophysio-
logical investigations of organisms in miniature ecosystems.
Two chemical communication systems are being investigated in detail.
The first involves the attraction of starfish to oyster extracts;
the second involves the attraction of a flatworm to its host, the
horseshoe crab. In both cases the chemicals are water soluble, have
low molecular weights and are active in the part/per billion range.
10
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Starfish Chemotaxts
This work, introduced in earlier summaries, has been pursued in two
directions.
The starfish Asterias vulgaris was observed in aquaria and in a flow
tank in the presence of oysters, oyster tissue, and extracts of four
shellfish species (10). Oysters were approached after varying delays,
opened, and consumed; oyster tissue was rapidly approached and eaten.
Dilute (ppb) shellfish extracts were appraoched in a flow tank.
High concentrations of extracts, elicit posturing and stomach eversion
responses. This phase of our investigation was aimed at an under-
standing of the behavior of starfish in the presence of a chemical
stimulus and in the hope of the eventual isolation and identification
of the components of the stimulus system.
In addition, we noted during our investigation erratically high responses
to certain blank test solutions. These were traced to the presence
of compounds attractive to starfish in the perspiration of the experi-
menter. Several chemicals known to occur in human perspiration have
now been demonstrated to elicit an approach and feeding response in
starfish.
This unexpected finding again emphasizes the need for great
caution in planning and performing chemotaxis studies; in addition,
it has given us insight into the range of compounds which are sensed
by Asterias vulgaris.
Chemotaxis in the Symbiosis of Limulus and Bdelloura
The host specific substances released by the horseshoe crab Limulus
polyphemus, attracting the symbiotic flatworm Bdelloura Candida have
been investigated. Water from horseshoe crab tanks contains metabolites
which are attractive to the flatworm, whereas water from other crusta-
ceans has no effect. We have investigated and isolated two active
compounds that act together as stimulus. One has been identified as
trimethylamine hydrochloride, the other is still unidentifed. The
response of Bdelloura to synthetic organic compounds has been investi-
gated. Ammonium acetate, zinc chloride, dieldrin and aqueous kerosene
extracts had no immediately apparent effects on the response of Bdelloura
to Limulus at 100 ppm. On the other hand, iron chloride, phosphates
(typical of those in household detergents), detergents and mercuric
chloride interfered with the response of Bdelloura to Limulus water
at 100 ppm but not at 1 ppm.
11
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Exploratory Studies, onChemotaxis in Lobster and Alewife
With, increasing experience tn work. with, lower and relatively unso-
phisticated animals CAsterias, Bdelloural we have proceeded to ex-
ploratory work on higher organisms that are also more important com-
mercially.
a). The effects of kerosene and kerosene fractions on the behavior
of lobster Horoarus-americahus have been investigated. Kerosene was
fractionated into three fractions; straight chain hydrocarbons,
branched/cyclic hydrocarbons and aromatic hydrocarbons (water soluble
part of kerosene}. Before the test the animals were observed until
their normal behavior was classified into 65 behavioral units, such.
as attack, grooming, tail flips, etc. Kerosene caused an increase
in stress behavior, a high degree of grooming, stimulation of feeding
and an increase in,aggression. The aromatic hydrocarbon fraction
caused the same responses as those observed in the kerosene test
except that feeding behavior was inhibited, although attraction to the
stimulus was still evident. The branched/cyclic hydrocarbon fraction
caused only weak attraction and stimulation of feeding. The
straight chain hydrocarbons had no observable effect on behavior.
b). The recognition of the homestream by alewives (Alosa pseudohafengus)
returning to their spawning ground has been demonstrated to involve
the sensing of chemical stimuli that are specific to the homestream
water. We find that the active components in this "chemical finger-
print" are heat stable, nonvolatile, polar and of a molecular weight
below 1,000. Tests on processed fractions from the horaestream
suggest that acids and bases are involved but no lipids and inorganic
anions and cations.
We hope that continuation of this work will aid in predicting the
danger of pollution with certain chemicals to the homestream recogni-
tion of commercially important fishes such as the alewife and especially
the salmon.
Conclusions from Chemotaxts Studies.
These and other investigations in this field demonstrate the extent
to which chemotaxis mediates many important life processes of marine
organisms. The recent investigations by Mueller on chemotaxis in
brown algae have shown that hydrocarbons, previously thought to be
relatively inert byproducts of plant and animal metabolism, are
directly responsible for the attraction of male gametes to the egg
cell. Similarly, our work on the hydrocarbon composition of marine
benthic and planktonic algae suggests that hydrocarbons, may be in-
12
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tiraately involved in cell division or reproduction.
Obviously, chernotactic processes occur at the molecular level and
conversely, they can he interfered with by chemicals at the molecular
level. We have carried out exploratory work on the attraction of th.e
mud snail Nassarius, obsoletus to oyster extracts. This response is de-
pressed by minute amounts of aqueous kerosene extracts. CA saturated
kerosene extract after a dilution by 10' still depresses the food
response by as much as 40%). This suggests that pollution may be
ecologically damaging at concentration levels far below those normally
considered harmful.
i
Our investigations directly related to chemotaxis have been largely
exploratory; however, we have learned much about the complexity of
the processes involved in stimulus recognition, especially in higher
animals and about the necessary precautions that have to be taken in
experimentation.
We feel that it is important to continue this phase of the investi-
gation because of its implications.
13
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SECTION VII
ACKNOWLEDGEMENTS
We wish, to tfianfc. Dr. C. S. tlegre, Project Officer, for Ms support,
advtce and concern.
14
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SECTION VIII
REFERENCES
1. Muller, D, Q., Jaenicke, L., Donlka, JU., Afcfntoht, .T., "Sex
Attractant in a Brow Alga: C&eratcal Structure11, SCIENCE, Vol. 171,
pp. 815-817 C1971L
2. Paffenhoffer, G. A., "Cultivation of Calahus belgolandlcus Under
Controlled Conditions," Eelgolander wlss. Meeresunters. 20,
346-359 (1970).
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SECTION IX
PUBLICATIONS
Listed are all publications completed with partial or full
support of Grant 18050 EBN.
1. Blumer, M., Marine Hydrocarbons from Organisms and Pollution.
Abstract, A.C.S. Meeting, September 1970, Chicago, 111.
WATR 50.
2. Blumer, M., Oil Contamination and the Living Resources of the
Sea. A Review - presented at the FAO Technical Conference
on Marine Pollution and its Effects on Living Resources
and Fishing. Rome, December 9-18, 1970.
3. Blumer, M., On the Ecological Risks Inherent in Expanded Off-
shore Drilling and Oil Transport. Testimony. Jji Hear-
ings before the Subcommittee on the Judiciary United States
Senate, Ninety-first Congress, Second Session, The Petro-
leum Industry Part 5, Aug. 11 and 13, 1970. p. 1968-1976.
4. Blumer, M., On the Impact of Oil Port and Refinery Operations ;
on the Coastal Ecology and Food Derived from the Sea.
Testimony. Jj]^ Hearings before the Subcommittee on Air
and Water Pollution of the Committee on Public Works,
United States Senate, Ninety-first Congress, Second Session,
Machiasport, Maine, Sept. 8 and 9, 1970. p. 33-42.
5. Blumer, M., On the Environmental Effects of Increased Oil Traf-
fic in the Potomac. Testimony before the Conservation
and Natural Resources Subcommittee. Washington, July 22,
1970.
6. Blumer, M;, G. Souza and J. Sass., Hydrocarbon Pollution of
Edible Shellfish by an Oil Spill. Marine Biology. (1970)
i, 195-202.
7. Blumer, M., G. Souza and J. Sass., Hydrocarbon Pollution of
Edible Shellfish by an Oil Spill. Report to the Board of
Selectmen, Town of Falmouth, Mass., Unpublished Manuscript,
Woods Hole Oceanographic Institution, Reference'#70-1,
(1970).
16
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8. Blumer, M., M.M. Mullin and R.R.L. Guillard., A Polyunsaturated
Hydrocarbon (3, 6, 9, 12, 15, 18-heneicosahexaene) in the
Marine Food Web, Marine Biology, (1970) 6., 226-235.
9. Hunt, J. and M. Blumer., Oil Pollution in the Marine Environ-
ment. Presented at the 15th General Assembly of the Intnl.
Assn. for the Physical Sciences of the Ocean, Tokyo, Septem-
ber 13-25, 1970.
10. Whittle, K.J. and M. Blumer., A Predatory-Prey Relationship.
Sea Stars-Bivalves. The Chemical Basis of the Response of
As ten'as vulgaris to Crass ostrea virginica. A Bioassay, its
Application and the Partial Purification of an Active Ex-
tract. Woods Hole Oceanographic Inst., Unpublished Manu-
script, Ref. No. 70-20, (1970).
11. Youngblood, W. and M. Blumer., Hydrocarbons of Benthic Algae.
Paper presented at 6th A.C.S. Western Regional Meeting,
San Francisco, October 6-9, 1970.
12. Blumer, M., Oil Pollution of the Ocean. In: Man's Impact on the
Environment, ed.: T.R. Detwyler, McGraw Hill, New York (1971)
295-301.
13. Blumer, M., Scientific Aspects of the Oil Spill Problem. |n_:
Coastal Water Pollution, Pollution of the Sea by Oil Spills,
Committee on the Challenges of Modern Society NATO, Brussels,
Number 1 (1971) pp. 9. 1-9.24.
14. Blumer, M., Scientific Aspects of the Oil Spill Problem. Env.
Affairs 1 (1971) 54-73.
15. Blumer, M., H.L. Sanders, J.F. Grassle and G.R. Hampson., A
Small Oil Spill. Environment (1971) March, 1_3, 2-12.
16. Blumer, M., Verunreinigung der Gewaesser durch Oel, Zum Problem
der persistenten Chemikalien in der Umwelt. EAWAG, Dueben-
dorf-Zuerich, Separatum Nr. 398, (1971), 21 pages.
17. Blumer, M., R.L. Guillard and T. Chase. Hydrocarbons of Marine
Phytoplankton. Marine Biology (1971) 3_. 183-189.
!
18. Ehrhardt, M., and M. Blumer., The Identification of Oil Spills
by Means of Gas Chromatographic and Spectrometric Data.
Abstracts. 161st ACS Meeting, Los Angeles, (1971) WATR, 15.
,17
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19. Youngblood, W., M. Blumer, R.L. Guillard and J. Fiore., Saturated
and Unsaturated Hydrocarbons in Marine Benthic Algae. Marine
Biology (1971) 8, 130-201.
20. Boylan, D.B. and B.W. Tripp., Determination of Hydrocarbons in
Seawater Extracts of Crude Oil and Crude Oil Fractions,
Nature, (1971), 230. 44-47.
21. Blumer, M., P.C. Blockker, E.B. Cowell and D.F. Duckworth. Pet-
roleum. In: Guide to Marine Pollution, E.C. Goldberg, ed.,
Gordon anOreech, (1972) p. 19-40.
22. Blumer, M. and J. Sass. The West Falmouth Oil Spill, Data Avail-
able in November, 1971. II. Chemistry. Woods Hole Oceano-
graphic Institution Technical Report No. 72-19 (1972).
23. Ehrhardt, M., and M. Blumer., The Source Identification of Marine
Hydrocarbons by Gas Chromatography and Spectrometry. Environ.
Poll. (1972) 3., 179-194.
24. Ehrhardt, M. Petroleum Hydrocarbons in Oysters from Galveston
Bay. Environmental Pollution. (1972) in press. 3_, 257-271.
25. Zafiriou, 0. Response of Asterias vulgaris to Chemical Stimuli,
Marine Biology, 17, 1972, p. 100-107.
Consultation, Testimony and Analyses for Government Agencies
We have advised Dr. T. Murphy, Federal Water Quality Administra-
tion, prior to a hearing at the Department of the Interior.
We have advised Mr. E.R. Baird, U.S. Army Engineers, Norfolk
District, on oil pollution, in connection with a law suit prepared
by the Corps of Engineers.
We have served on a FWPCA-API panel on the toxicity of disper-
sants and have also served on a U.S. Coast Guard - National Academy
of Sciences Panel on Pollution Monitoring, and on a FWQA Panel in
Bacterial Degradation of Oil Pollution.
Further, we have testified at Government request:
On the Ecological Risks Inherent in Expanded Offshore Drilling
and Oil Transport: to the Subcommittee on the Judiciary, U.S. Senate.
On the Impact of Oil Port and Refinery Operations on the Coastal
Ecology and Food Derived from the Sea: to the Subcommittee on Air
and Water Pollution of the Committee on Public Works, U.S. Senate.
18
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On the Environmental Effects of Increased Oil Traffic in the
Potomac: to the Conservation and Natural Resources Subcommittee.
We have served as a member of the U.S. Delegation to the NATO/
CCMS Ocean Oil Spills Conference, Brussels, November 1970.
We have presented testimony at the request of the Maine Environ-
mental Improvement Commission.
At the request of EPA, we have carried out analyses of shellfish
speciments for the possible presence of petroleum derived hydrocarbons,
We have carried out analyses regarding shellfish pollution and
beach pollution for the Town of Falmouth, Mass., for the Department
of Public Health, Commonwealth of Massachusetts and for the Martha's
Vineyard Conservation Society.
Dr. Blumer was a member of the Pollution Committee, Town of
Falmouth, Massachusetts, 1970-1971.
19
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Class
Order
SECTION X
APPENDIX
Benth-ic Algae
Species
Date Cl97Ql/Col lec-
tion Station3
Chlorophyceae
Phaeophyceae
Rhodophyceae
Ulotrichales
Cladophorales
Siphonales
Ectocarpales
Chordarlales
Punctariales
Lamtnariales
Fucales
Bangiales
Cryptonemiales
Gigartinales
Rhodymeniales
Ceramiales
Enteromorpha compressa
Diva lactuca
Spongomorpha arcta
Codfum fragile ssp.
Tomentosoides
Ectocarpus fasciculatus
Pilayella littoral is
Chordaria flagelliformis
Leathesia difformis
Punctaria latifolia
Scytosiphon lomentaria
Chorda filum
Chorda tomentosa
Laminaria agardhii
Laminaria digitata
Ascophyllum nodosum
Fucus distichus ssp.
Edentatus
Fucus spiralis
Fucus vesiculosus
Porphyra leucosticta
Dumontia incrassata
Chondrus crispus
Rhodymenia palmata
Ceramium rubrum
July/2
25 May/1
25 May/1
July/2
25 May/1
25 May/1
25 May/1
1 June/3
1
1 June/3
1 June/3
25 May/1
14 June/1
25 May/1
25 May/1
25 May/1
25 May/1
25 May/1
25 May /I
25 May/1
25 May/1
25 May/1
25 May/1
Stations: 1 Sandwich Jetty, Barnstable, Mass.; 2 Little River,
Waquoit Bay, Mass.; 3 West Falmouth Harbor Jetty, West Falmouth, Mass.
20
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Baclllariophyceae
Dinophyceae
Cryptophyceae
Haptophyceae
Euglenophyceae
Cyanophyceae
Rhodophyceae
Xanthophyceae
Chlorophyceae
Plahktonic Algae
Cytlotella nana (3H)
Ditylum-brtgfttwellit (D. Bright. 1
Lauderta boreal is [Clone 141
RFitzosolenia setfgera (Rhizo)
Steeletonema costatum (Slcel)
Thalassiosira fluvtattlis (Actin)
Tfialassiostra sp.
Gonyaulax polyedra (GP 60e)
Gymnodintura splendens CGym. s.)
Pertdintuni trochoideum (Pert)
Peridtnium trochoideum, old culture
Cryptoraonas (Rhodomonas?) sp. (3C)a
Cryptoraonas, (Rfiodomonas?) old culture
Coccolithus huxleyi C.BT-6)
Isochrysis galbana (Iso)
Pheaocystis poucheti (Pp)
Pheaocystts poucheti C677-3)
Eutrepiella sp. (W. H. Eut.)
Oscillatoria woronichinii (Sm 24}
Synechococcus bacillaris (Syn.)
Porphyridium sp. CPorph)
Triboneraa aequale CNo. 50)
Undetermined sp. CGSB Stiocho)
Dunaliella tertiolecta (Dun.)
Derbesia tenuisstma (LB 1260)
a Now believed to be a species of Chroomonas.
b A freshwater algae, studied because of its clear taxonomic position,
No certain marine xanthophytes are available.
c Systematic position uncertain. May belong to the new class
Eustigmatophyceae.
21
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Copepods
Rhincalanus na&utus.
Eucalanus b.ungtl callfornicus
Calanus helgolandtcus Cpactftcus]
Shellfish
Crassostrea virgtntca
Aequipecten irradians
Asterlas vulgarfs
Ltraulus polyphemus
Bdelloura candtda '
Homarus amerfcanus
Also pseudoharengus
Nassarius obsoletus
Other Animals
22
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PART II
SUBLETHAL EFFECTS OF CRUDE OIL ON LOBSTER,
(HOMARUS AMERICANUS) BEHAVIOR
Project 118080 EBN
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SECTION I
CONCLUSIONS
1. Effects on behavior and feeding times of the lobster Homarus americanus
were seen only in the first experiment when crude oil was directly
added to their aquaria (ration oil-sea water = 1:100,000). No effects
could be measured in the second experiment when only the soluble
fraction of this crude oil was added. In the latter case, oil was
added to the water in the same amount (1:100,000) and the soluble
fraction was recovered from the sea water in the range of 50 parts
per billion (ppb).
2. The effects on behavior are interpreted as changes in water chemistry
sensing movements. The change in feeding time was caused by a doubling
of the waiting phase, which defines the time period in which the
lobster has noticed the food but has not yet left his shelter to
search for it.
3. Light and electron microscopy showed no observable changes in
morphology of odor receptors. The results are therefore interpreted
to be caused by the depressing quality of crude oil: a "bad odor"
effect.
4. In the experiment on effects of solubles, some petroleum hydrocarbons
among a large amount of lipids were present in the aquaria before ,
oil was added. After addition of oil, both lipids and hydrocarbon
concentrations went down considerably. The degrading oil showed
a different composition each day in both experiments (crudes and
solubles), in general following the usual pattern.
-25-
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SECTION II
RECOMMENDATIONS
It can be seen from the results of this study that levels of oil in sea
water can be determined which could be allowable for lobsters and other
marine animals. Using our techniques, sublethal effects can be observed
and quantified.
1. In an expanded effort different (higher) levels of oil
should be tested.
2. Other animals should be tested in a similar fashion.
3. The experiments should be repeated under natural conditions.
With the addition of the 3 experiments recommended above,reasonable
water quality standards can be set regarding petroleum hydrocarbons
in the sea.
-27-
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SECTION III
INTRODUCTION
It is well known that oil can kill marine animals, affecting both inhabitants
of surface waters and bottom dwellers for long periods of time (Blumer, 1970).
It is also becoming increasingly clear that marine organisms depend on chemical
communication in one way or another for individual and species survival. For
example, catfish cannot locate food without their sense of taste (Atema, 1971)
and lobsters utilize a sex attractant for mating (Atema and Engstrom, 1971).
The possibility of interference with chemical communication by extremely low
levels of crude oil has been pointed out (Blumer, 1970). Oil is not only
present as a surface film or in bottom sediments; several fractions also exist
in solution and emulsion in considerable quantities (Boylan and Tripp, 1971).
With the increasing chance of coastal oil spills and the understandable
public confusion on ill effects of oil on lobster populations in mind,
we have tried to develop methods of determining sublethal effects of crude
oil on lobsters under carefully controlled conditions. The results of such
a study on a much larger scale could be used to determine what level of
oil pollution becomes intolerable for marine life and might also indicate
what physiological mechanisms are involved in the effects of oil on the
behavior of these animals.
Oil is a complex stimulus; its effects on behavior are difficult to measure
because many hydrocarbon fractions have very different properties. After
various pilot studies on effects of oil and kerosine fractions, we decided
to use whole crude oil and its water soluble fraction. We selected from
the lobsters' behavior the most reproducible pattern; feeding behavior,
as a standard in which to measure sublethal oil effects.
As a measure for physiological damage, we chose to examine the microscopical
and submicroscopical structures of the aesthetasc hairs on the antennules.
These appendages serve an olfactory function and are, together with the
gills, probably the most sensitive membranes to the outside environment.
-29-
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SECTION IV
METHODS
A- Keeping of Animals and Test Procedures
Sixteen mature lobsters (Homarus americanus] caught locally around Woods
Hole, Massachusetts, were used in the experiments. Carapace lengths
(measured from the rear of the eye socket to the beginning of the
abdomen) ranged from 6.5 cm to 10.1 cm with most lobsters measuring
around 9 cm. Animals that had molted one to three months prior to the
start of the experiment were chosen to exclude molting and premolt
effects during the experiment.
All the animals were housed in individual 100 liter fiberglass tanks
with one glass window for observation. Tanks were maintained at approxiv
mately 22°C in a closed system. A large airstone in each tank provided
aeration and circulation of the water. All animals were given at least
two weeks acclimation time, prior to the beginning of the experiments,
during which they were starved. Dim room light and a diurnal schedule
were maintained throughout.
During the experiment, each animal was observed individually in its
behavior just before and after the introduction of food. The lobsters
were observed each day between 0800 and 1200 hours for ten consecutive
days. The order of testing individuals was constant, so that each
lobster was tested at the same time each day. A single observation
session for each animal consisted of two parts: (1) a ten minute period,
during which all behavior units were recorded; at the end food was
introduced; (2) the timing of the lobster's feeding behavior.
Food, a small piece of fresh mussel, Mytilus edulis, was slowly lowered
on nylon string at the opposite side of the tank to where the lobster
was situated. In order to do this two pieces of food were always
available, one on the left and one on the right side of the tank. The
introduction method did not elicit any immediate behavioral responses
attributable to visual or mechanical stimuli. On this amount of food
the animals were always kept hungry (McLeese, 1972).
Three periods of the feeding behavior sequence were then timed: 1)
the alerting phase; from introduction of food to alert, 2) the waiting
phase; from alert to the beginning of search, 3) the searching phase;
from the beginning of search to hit. Alert was determined by an increase
in antennule and/or exopodite rate while a general body movement marked
the beginning of search. Hit was the moment of first physical contact
with the food by the feeding appendages.
Two series of experiments were done, one with whole crude oil and one
with the water soluble fraction of crude oil. In the whole oil experiment
eight lobsters, four males and four females were used. All eight were
selected and treated simultaneously. In the experiments on the effects of
the water soluble fraction four male lobsters were tested at one time,
followed by an identical series of four females. Oil introduction methods
are reported later.
-31-
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The crude oil tests were done in the summer of 1971, the tests with the water
soluble fraction were done in the winter of 1972, In both experiments one
animal showed aberrant behavior and was eliminated. The tests were done with
seven animals for crude oil and seven animals for water soluble fraction.
B. Behavior Recording and Analysis
1. Behavior recordings were made in shorthand using behavior description
units (see Appendix Table I}., which we developed in the course of our work
on lobster behavior. The shorthand code was transcribed on data code
forms for analysis of the frequency with which each unit appeared in
each animal during each daily observation period. Superficial judgment
of the frequency scores resulted in elimination of several units and
grouping of others. The units that did not occur frequently enough
to allow statistical treatment were eliminated. The units "Rake 1,
2, 3" were grouped as one: "Rake". The five groom units were grouped
as "Groom". Table I lists the resulting units which were analyzed.
Of these units, three required further treatment before statistical
analysis. Two of those, "Beat" and "Fan", describe appendage movement
rates in three categories: "Slow, Medium and Fast". The third
describes "Claw Position" in "1, 2 and 3". Since these units must
occur in one of the three categories all the time, only their relative
occurrence is of importance. With our recording procedure it is not
possible to measure duration of each behavior unit. For example, an
approximation was obtained by calculating the number of "Fast" rates
as a fraction (in percent) of the total number of "Beat". For
instance, if for one animal during one observation period "Beat"
was recorded 10 times, of which two were Fast, three Medium and five
Normal, the relative occurrence was 20%, 30% and 50% respectively.
These normalized numbers (20, 30 50) were then analyzed as all other
units (Appendix Table II, a and b).
Analysis of the duration of feeding behavior phases were done as
for behavior units. The time in seconds was listed for "Alert",
"Wait" and "Search" for each animal and each day (Appendix Table III).
2. Statistical analysis of these behavior data is made difficult
by great individual differences between such complex animals as
lobsters. To allow for individual differences a non-parametric i
technique was used to test the significance of changes in unit '
frequencies. Treatment of all units, including normalized units /
and duration times was identical. A sample treatment will be /
given for the unit "Crude, Antenna Wave"*.
Table II lists the 10 experimental days (horizontal) and the seven
individual animals (Vertical). The numbers in the table represent
the frequency of occurrence of the behavior unit "Crude, Antenna
Wave" over a ten minute period per animal per day. In other words,
each number means how many times one lobster waved his antennae
in the ten observation minutes of that particular day. A x test
was used to. determine whether or not the unit frequency changed
significantly from the first five days (before oil) to the next
five days (after oil). For this, the two highest scores and the
* The units are preceded by "Crude" or "Soluble" to denote the
experiments -32-
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TABLE I
SELECTED BEHAVIOR UNITS USED IN ANALYSIS
Name
1. Antenna wave
2. Antennule wave
3. Wipe
4. Rake
5. Scoop
6. Move
7. Climb
8. Groom
9. Antennule: Beat Slow
Beat Moderate
Beat Fast
10. Fan:
Slow
Moderate
Fast
11. Claw 1
Claw 2
Claw 3
-33-
Definition
slow sweep of an antenna
change in antennule positior
wiping of the antennule by
the third maxillipeds
back and forth movement
of one or more walking
legs (pereiopods) across
the substrate while body
is still
lifting of walking legs
from substrate to mouth
any Undirected movement
of the body
raising of body against
the side of the wall to
a vertical position
picking, rubbing, scratching
parts of the body with the
walking legs
beating of the antennules
at a slow or normal rate
(0-60 beats per minute)
beating at a moderate rate
(60-120 bpm)
beating at a fast rate
(x 120 bpm)
slow fanning of exopodite
of 3rd maxilliped (90 bpm)
moderately fast fanning
(90-180 bpm)
fast fanning (>_ 180 bpm)
claws closed
claws half open
claws wide open
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TABLE II
FREQUENCY OF OCCURRENCE OF BEHAVIOR UNIT "CRUDE, ANTENNA WAVE"
Animal Days pre-oil Days post-oil
Number 12345 6789 10
1 45225 14 14 4 16 10
2 16 14 5 3 19 8 28 29 29 7
3 8 9 3 1 22 26 22 36 32 29
4 5316 11 17 7993
5 14 9012 10 1501
6 28006 10 14 661
7 10 8 2 5 18 2 5 20 9 12
-34-
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two lowest scores were considered. In case of a tie all tied numbers
in that animal were taken, until at least two numbers of both extremes
were represented. In cases of frequent zero scores (for instance see
Appendix Table Ha, "Crude, Scoop") this procedure resulted in large
numbers of low scores.
C. Introduction of Crude Oil
A volume of 0.9 ml of La Rosa crude oil was introduced on to the surface
water of each test aquarium on day six at 9 a.m. This represents an oil-
water ratio of 1:100,000. A uniform thin brown slick soon covered almost
the entire surface of the aquaria. Vigorous airstone bubbling resulted
in some of the oil adhering to the wall of the aquaria near the airstone.
Emulsification undoubtedly took place from the beginning (Boylan and
Tripp, 1971) . Near the end of the five day period the surface film had
disappeared. Only small brown particles floated at the surface. Many
such particles were stuck to the walls.
After completion of the ten day experiment the antennules of the ex-
perimental animals were removed and prepared for electron microscopical
examination.
D. Introduction of Water Soluble Fraction of Crude Oil
In order to expose the test animals exclusively to the water soluble
fraction of crude oil a 100 liter all glass mixing tank was set up
separate from the test aquaria housing lobsters (see Figure 1). Water
was pumped from near the bottom of the glass mixing tank into four
test aquaria using glass tubing, teflon connectors and a peristaltic
pump (Manostat standard model). Pumping rate was one gallon per minute.
The four aquaria received equal amounts of water and fed the extra
volume back to the mixing tank by gravity through glass tubing and a
silicone rubber end piece to minimize vandalism by the lobsters. The
outflow of the feedback was near the bottom of the oil slick tank,
away from the intake to insure good mixing. In the first five days, no
oil was present in the system. On day six, 4.5 ml of La Rosa crude oil
was introduced on the surface of the mixing tank. This volume was
calculated to result in an oil-water ratio of 1:100,000, similar to
the ratio used in the whole oil experiment. A variable speed electric
mixer (Eastern model 5 VA) was used at a constant low speed (200 rpm)
to insure proper circulation of water under the oil film without
causing an emulsion (Boylan and Tripp, 1971).
This series was completed for four male lobsters and repeated with
four female lobsters, one of which was eliminated as mentioned earlier.
E. Photographic Record
ft
Each day after the biological observations, black and white photographs
were taken of the slick in the mixing tank to follow its visual appearance.
F. Determination of Hydrocarbons and Lipids in Oil-Sea Water Mixture
1. Sampling and Extraction
To collect samples of the whole oil-water mixture for chemical
analysis a separate aquarium was set up, identical with the others, in-
cluding airstones, lobster size and feeding. No observations were
recorded on this lobster's behavior. Crude oil (0.9 ml La Rosa crude)
-35-
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Pump
Stirrer
Oil Slick Tank
l>
4 Lobster Aquaria
Figure 1. Connection of oil slick tank and lobster aquaria in solubles
experiment.
-36-
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was introduced on the water surface at 0900 hr. At 12 noon of that day (1) and
on days two three and fxve a volume of five liters was siphoned out of the
center of the tank. Day four was not sampled to keep the tank volume sufficiently
high for the lobster. }
Each sample was extracted four times with 80 ml redistilled pentane The
resulting 320 ml pentane extract was evaporated down to about 2 ml for analysis
by gas chromatography.
2. Gas Chromatography
Samples of the pentane extract (1/50 of volume) were injected in
a column (8 ft. 1/16 inch, 0.2% Apiezon L on textured glass beads, 80-100
mesh), which was temperature programmed from 50-220°C at 8°/minute and
maintained isothermally at 220 C. In order to identify the major peaks
in the chromatograms a standard was run using 1 yl La Rosa crude dissolved
in 50 yl pentane, of which 1/50 aliquots were used.
G. Determination of Hydrocarbons and Lipids in Seawater
A flow chart of the chemical analyses of lipid extract is presented in
Figure 2.
1. Sampling and Extraction
Samples of lobster tank seawater before and after oil was introduced
in the mixing tank, were taken each day at 12 noon starting at day five
of the experiment. A volume of 1250 ml was siphoned out of the center
of each test aquarium. The resulting volume (5 liters) was extracted
four times with 80 ml redistilled pentane. The pentane extract was
evaporated down to 2 ml for chemical analysis. Aliquots of this frac-
tion were used for the following analyses.
2. Ultraviolet-Visible Spectra
Subsamples of the lipid extracts were dissolved in redistilled
isooctane and spectra obtained using 1 cm quartz cells mounted in a
Gary Model 14 spectrophotometer.
3. Infrared Spectra
Subsamples of the lipid extracts were analyzed as thin films
between a 1 x 4 mm NaCl crystal and a larger crystal mounted on a holder
with a 1 x 4 mm mask. Spectra were obtained using a Perkin Elmer 337
infrared spectrometer with a beam condenser.
4. Column Chromatography (Methods of Blumer, 1970)
The lipid extracts (minus subsamples analyzed above) were dissolved
in 0.2 ml of pentane and charged to a 1.0 ml column of silica. The silica
had been deactivated with 5% water and extensively eluted with pentane prior
to charging the sample to the column in 0.2 ml of pentane- The saturated
hydrocarbons (alkanes) were eluted with 1.5 ml of pentane after collecting
void volume. Alkenes and aromatic hydrocarbons were eluted with an additional
6.0 ml of pentane. The pentane was removed from each eluate fraction by
evaporation under reduced pressure. The residual was taken up in 25-100 yl
-37-
-------
FIGURE 2.
Flow Chart of Chemical Analyses
Pentane Extract of Water
Aliquot to j
L«w»».«
infrared spectrometryi
Aliquot to
Ultraviolet and visible
spectrometry
Aliquot weighed to determine
IjLpid concentration.
5% H20
Column Chromatography
deactivated SiO-, -
1.0ml column.
V
1.
0.8 ml. pentane
eluate
(alkanes)
2.
6.0 ml. pentane
eluate
(alkenes and aromatics)
Aliquot weighed-
V
Aliquot weighed
Analysis by gas
gas Chromatography on 7.5 ft. 1/8" o.d.
3% Apiezon L /Chromasorb W 80/100 mesh.
temperature programmed 80-290^. at 6°/min.
-------
of CS2 and 2.0-10.0 yl aliquots were weighed on a Cahn Electrobalance.
The remainder of the sample was analyzed by gas chromatography.
5. Gas Chromatography
Hydrocarbons isolated from column chromatography were analyzed on
a 7.5 ft. 1/8" 3% Apiezon L Chromasorb W 80/100 mesh column. The column
was temperature programmed from 80-290°C at 6°/minute and maintained
isothermally at 290 C. All solvents employed in this analysis were redistilled.
Analysis of a blank showed no contribution by solvents and other chemicals
to the reported concentrations of lipids and hydrocarbons. UV absorbance
values were corrected for absorbance contributions from the solvents and
chemicals.
H. Microscopy of Chemosensory Hairs
In order to reveal possible structural damage of exposure to oil the
chemosensory hairs on the antennules, the so-called aesthetasc hairs
were examined by light and electron microscopy. Immediately after completion
of the experiments both outer rami of the antennules of the experimental
animals were cut. One ramus of each animal was used for light microscopy,
the other one for electron microscopy.
1. Light Microscopy
Freshly cut outer rami with aesthetasc hairs were kept in seawater
and immediately viewed and photographed with high power light microscopy and
oil immersion at 1000 x magnification. A Wild microscope was used with plan
focal objectives and high power electron flash to reduce exposure time and
improve contrast. To further decrease exposure times (to 1 m sec.) pic-
tures were taken on Kodak Tri X film exposed and developed at 1600 ASA in
Acufine developer. This procedure resulted in satisfactory pictures with
good contrast, and relatively free of vibration "fuzz".
2. Electron Microscopy
Antennular outer rami were cut and fixed immediately in 6%
glutaraldehyde in seawater for four hours. Further fixation in 2% phos-
phate buffered osmium tetroxide was done for 16 hours, after which the
specimens were dehydrated in an ethanol series. At 70% ethanol, the aes-
thetasc hairs were carefully removed from the antennule under a dissecting
microscope. In some cases individual segments were cut off the ramus
leaving the two rows of hairs attached to each segment. Dehydration
was then completed, after which the hairs were embedded in araldite
going through the following mixutres of araldite and propylene oxide:
0:1 - 0:1 - 1:3 - 1:1 - 1:0. The plastic was polymerized during 72
hours of baking at 70°C. Control specimens were prepared from fresh lobsters
using identical procedures. All specimens were cut and viewed in an
Hitachi electron microscope.
-39-
-------
SECTION V
RESULTS
A. Analysis of Behavior
The behavior units listed in Table I were analyzed individually for changes
in their frequency of occurrence during the first five days versus the
last five days. A sample treatment was already given for the unit "Crude,
Antenna Wave". All units were treated in this fashion. The complete
set of results can be found in Appendix Table II.
Three behavior units were found to change significantly: A x2 test showed
significance at the 0.01 level for "Crude, Antenna Wave", Crude, Antennule
Wave" and "Crude, Fan Slow". All three units increased in frequency in
the last five day period compared with the first five day period. The
frequency change of the unit "Crude, Beat Fast" 0.05 level, but here
a lower frequency of occurrence appeared in the last period.
These units were only significant in the Crude Oil experiment, but not
in tests with the Soluble fraction. In these experiments, none of
the behavior units changed significantly.
B. Analysis of Feeding Times
The three time phases of feeding behavior were analyzed for changed in
duration in the first five days versus the last five days. Treatment was
identical to that of the frequency of behavior units. The time in
seconds is listed in Appendix Table III for the complete set of data.
A significant change (0.01 significance) in feeding time between the
first and the last five days was found only for the phase "Crude, Wait".
The average "Crude, Wait" time was about doubled in the last five
days and represent the greatest change in the lobsters' behavior.
The mean wait time in the first five days was 17.2 sec. versus 36.4 sec.,
in the last five days. No other significant changes occurred.
C. Oil Slick Photographs
A visual idea of the oil slick at days 6-10 can be formed from the
photographs that were taken each day (Figures 3 and 4). The coherent
slick of day six is broken up at day seven and finely dispersed at day
eight. In the first series a fine granular appearance of the slick is
evident on days nine and ten. In the second series the slick is probably-
mixed in with the water to some extent after day nine due to malfunctioning
of the stirrer. Mixing was obvious at day ten, when practically no oil
was visible at the water surface.
First series, oil in female tanks, March 10-14, 1972
la. One hour after oil introduction (4.5 ml La Rosa crude); oil covers
about 20% of the water surface in an almost solid black-brown
slick (Figure 3A).
lb. Seven hours; oil covers the entire surface, broken up in vacuoles
up to 4 cm in diameter slightly lighter color (Figure 3B).
2. Twenty-five hours; oil globules up to 1 cm in diameter cover entire
surface (Figure 3C). -41-
-------
3. Forty-eight hours; same as twenty-five hours (Figure 3D); more stirrer
action visible.
4. Seventy-three hours; some thin 1 cm globules still present, but
appearance has become granular and gray (Figure 3E); much oil has
disappeared from surface.
5. One hundred hours; as seventy-three hours (Figure 3F); some oil
has stuck to the walls (Figure 3G).
Second series, oil in male tanks, March 22-26, 1972
1. Ten minutes after oil introduction (4,5 ml La Rosa, crude); solid
black slick covers about 10% of water surface (Figure 4A).
2. Twenty-seven hours; an oil film with large (up to 3 cm diameter)
and small vacuoles covers about 85% of surface (Figure 4B).
3. Fifty-five hours; a thin film full of small vacuoles (1 cm diameter)
covers the entire surface (Figure 4C).
4. Seventy-four hours; very thin granulated cover of oil over whole
surface (Figure 4D); some is coating walls. Stirrer malfunction
suspected.
5. Ninety-seven hours; almost all oil has disappeared (Figure 4E)
some is coating walls. Stirrer malfunction evident, though not
directly observed.
D. Analysis of Hydrocarbons and Lipids in Oil-Sea Water Mixture
Gas chromatograms for pentane extracts of 1) La Rosa crude oil and 2)
oil-water mixtures at day six; 3) day sev«n; 4) day eight; and 5) day
ten are shown in Figure 5. Major peaks can be matched,.well and their
fate followed over five days. The greatest observable effect is the
decrease of low boiling compounds and the increase in the. unresolved
envelope. In general individual peaks disappear in favor of the un-
resolved fraction; see also below.
E. Analysis of Hydrocarbons and Lipids in Sea Water
1. Hydrocarbon Concentrations and Lipid Concentrations
The results of the analyses of lipid concentrations and hydro-
carbon concentrations are presented in Table III. The important
aspects of the data are as follows:
-42-
-------
^;:«M«
"'-"-. ."=-:-" ^Ssi:"'. "- ; '* J »/!">-
^^&j£&'^$4
' ' . ^ "V*-" ""''-'-.- ^-^" ''' '*'.-*'?'
B
D
, : ->.« ''" v' .'
^r - < - **' :
:*-, ^ ". i -
G
Figure 3. Appearance of oil slick over five day period, first series of
solubles experiment.
-43-
-------
D
Figure 4. Appearance of oil slick over five day period, second series of
solubles experiment.
-44-
-------
DAY 1
DAY 2
DAY 3
DAY 5
LA ROSA CRUDE OIL
... ,:.r-=-''.'.'^-.: ^-".'.'I-"'-''"> '
Figure 5. Gas chromatograms of pentane extract from oil-water mixture.
-45-
-------
a) Lipid concentration decreases markedly following connection of the
oil mixing tank into the water circulating system of the experimental
tanks and continues to decrease until day nine where the trend is re-
versed. The day ten lipid concentration is high as a result of the
mixing of the oil slick throughout the oil-mixing tank and into the
experimental tanks when the stirring motor malfunctioned.
b) Total hydrocarbon concentration decreases after the oil-slick
tank is connected into the water circulating system. The total
hydrocarbon concentration then fluctuates until day nine where
the concentration decreases and then increases again on day ten
due to the stirrer malfunction.
c) The alkane concentration essentially parallels that of the total
hydrocarbon concentration.
d) The total alkene + aromatic hydrocarbon fraction concentration
fluctuates at a value near the lower detection limit of the methods
employed until day nine where an increase is noted. On day ten
the concentration again increases as expected when the stirrer mal-
functioned.
e) Generally the ratios of hydrocarbons to lipids, alkanes to lipids,
and alkenes + aromatics to lipids all increased throughout the course
of the experiment with the exception of a slight decrease from day
seven to day eight. The ratio of the alkenes + aromatics to lipids
shows the greatest increase of the three ratios.
f) The ratios of alkanes/alkenes + aromatics shows a definite de-
crease after day six and fluctuates at lower values through day ten.
2. Ultraviolet-Visible Spectra
Comparison of the UV spectra given in Figure 8 with appropriate
reference to the dilution factors of the lipid extract showed a steady
increase in the UV absorbance during the experiment after the connection
of the oil slick tank. Four absorbance bands were clearly defined in the
spectra and the absorbance of each band calculated per unit volume of
sea-water and corrected for absorbance contributions of the solvents
and chemicals used in isolating the lipids from the water. Corrections
were less than 0.005 absorbance units and were small compared to the
absorbance measured for the samples except for day five where the
absorbance of the sample was due entirely to the absorbance of unknown
compounds contributed by the solvents and chemicals of the extraction
procedure. The absorbance values of the four selected bands are
given in Table IV.
UV spectra of the alkene-aromatic hydrocarbons isolated by column
chromatography from the day ten lipid extract and the UV spectra of
aromatic hydrocarbons isolated from the La Rosa crude oil used in the
experiment are presented in Figure 7. The spectra are essentially
the same. A comparison of the UV spectra of the alkene-aromatic
hydrocarbon fraction of the lipid extract of day ten shows a sub-
stantial difference. This difference could be due to UV absorption
of polynuclear aromatic hydrocarbons present in the lipid extract,
but not eluted from the column or the presence of non-hydrocarbon
compounds soluble in pentane and absorbing in the UV region.
-46-
-------
2200
3000 3500 3000 3500 2200
WAVE LENGTH, A
3500
Figure 6. Ultraviolet spectra of lipid extracts of solubles on days 5-10.
-47-
-------
1.5
1.0
.3
*
Q;
O
0)
.1
.05
' ' ' ' I ' ' ' ' I ' '
DAY 10
ALKENE AROMATIC
LA ROSA CRUDE AROMATICS
1
2500
3000
3500
2500
3000
3500
WAVE LENGTH, A
Figure 7. Ultraviolet spectra of alkene-aromatic fraction of day 10 and
of whole crude oil.
-48-
-------
TABLE III - LIPID CONCENTRATIONS AND
HYDROCARBON CONCENTRATIONS IN SEAWATER
Day
Lipids
(yg/liter)
Total Hydrocarbons
(pg/liter)
Alkanes
(ng/liter)
Alkene-Aromatic
Hydrocarbons
(yg/liter)
RATIOS
Total Hyd/lipid
Alkanes/lipid
Alkene-Arom/1ipid
Alkane/alkene-arom.
5
1076
43.0
37.8
5.16
0.040
0.035
0.005
7.32
6
232
17.2
15.1
*
<2.01
0.074
0.065
0.009
7.23
7
182
24.5
16.3
8.29
0.135
0.090
0.046
1.97
8
109
12, .4
8,9
3.51
0.114
0.082
0.032
.2.54
9
370
91.8
59.7
32.1
0.248
0.161
0.087
1.86
10
1786
541.0
290.0
251.0
0.303
0.162
0 . 140
1.16
* Concentration could not be more than 2.01, the lower detection limit and
could be much less
-49-
-------
1 TABLE IV - ULTRAVIOLET ABSORBANCE OF LIPID
EXTRACT OF ONE LITER OF SEAWATER
Day
Wavelength A 5 6 7 8 9 10
2280 -- -0.604 0.514 0.676 18,20 14.60
2580 -- 0.158 0.194 0.232 4.30 8.84
2750 0.104 0.125 0.166 3.66 6.16
3120 0.017 0.008 16.30 2.14
-50-
-------
There were no detectable absorbance bands in the visible wavelength region
except for days nine and ten where weak absorbance was noted at 4000 A.
3. Infrared Spectra
The absorbance values for the absorption bands noted in the IR spectra
of the lipid extracts are given in Table V. The values are absorbances
normalized to the absorbance at 2955 cm. There has been insufficient
time to fully analyze the data. However, it is clear that the composition
of the lipids in the water is changing as is evidenced by a decrease
in absorbance at some wavelengths and increases in absorbance at other
wavelengths.
4. Gas Chromatography
The gas chromatograms of alkanes for days 5, 6, 7 and 8 (Figure 8) showed
very similar patterns with resolved peaks assigned to a series of n-alkanes
and branched alkanes eluting over a signal due to an unresolved complex
mixture of hydrocarbons. The signal due to the unresolved complex mixture
increased markedly on day nine and increased again on day ten (Figure 8).
Comparison of the gas chromatogram of alkanes present on day ten with
the alkanes isolated from the La Rosa crude oil used to form the oil
slick showed a depletion of the lower boiling hydrocarbons and a depletion
of the n-alkanes and branched alkanes relative to the unresolved complex
mixture. This unresolved complex mixture contains many hundreds of
cyclic and branched cyclic hydrocarbons - usually designated by the
term naphthenes and are characteristic of gas chromatograms of alkanes
isolated from crude oils and fuel oils.
The gas chromatograms of the alkene-aromatic fractions (Figure 9) of
the hydrocarbons showed the same features on days 5, 6, 7 and 8 with
only three peaks at retention indices 2044, 2081 and 2107 on Apiezon L
present in the gas chromatograms. No detectable unresolved complex
mixture was present. On day nine and day ten there was an unresolved
complex mixture signal present in the gas chromatograms. The three
peaks were also present eluting over the complex mixture signal. The
unresolved complex mixture on the day nine and day ten chromatogram
was probably due to naphtheno-aromatic hydrocarbons.
F. Microscopy
A morphological description of the olfactory sense hairs of the lobster,
the aesthetasc hairs, is given in Figure 10. These hairs are primary
candidates for disruption by chemical pollutants because their internal
structures, the olfactory dendrites and cilia, are probably immediately
accessible to rather large molecules in the environment (Ghiradella,
1968). The other non-chitinous body covering is found in the gill
membranes, which were not investigated. The rest of the lobster is
covered with hard chitin, impregnated with calcium.
No consistent differences other than normal variations could be observed
either at the light microscopical external level in_ vivo or at the
electron microscopical internal level after EM preparation (Figures 11-18).
-51-
-------
J EXTRACTION AND ANALYSIS BLANK
(INCLUDES ALL SOLVENTS, REAGENTS)
INCREASING TIME, TEMPERATURE L
22
b
JO
^3)
Figure 8. Gas chromatograms of alkane fraction of solubles.
-52-
-------
DAY 10
SENSITIVITY
10"11X16
V-J-
-UNRESOLVEO COMPLEX
MIXTURE
PHYTr
Hj
24
'25
23
2O
PHYT
U
17
PRIS
16
UNRESOLVED COMPLEX
MIXTURE
£
--'WHOLE LA ROSA CRUDE OIL
SENSITIVITY
* Automatic attentuation of detector signal.
Pris- Pristane Phyt- Phytane. Numbers refer to carbon chain lengths
of n-alkanes. Identifications are tentative only and are based on
coinjection with even numbered n-alkanes and/or interpolation between
even-numbered n-alkanes to obtain the retention index g., 2044 peak.
Figure 8. Gas chromatograms of alkane fraction of solubles.
-53-
-------
DAY 5
(SIMILAR ON DAYS
6,7.8) 'i-j
SENSITIVITY
UNRESOLVED COMPLEX
MIXTURE
CO-INJECTED WITH EVEN NO.
n-olkanes n-c,4-n-c24
UNRESOLVED COMPLEX
MIXTURE
UNRESOLVECi COMPLEX
MIXTURE
* Automatic attentuation of detector signal.
Pris- Pristane, Phyt- Phytane. Numbers refer to carbon chain lengths
of n-alkanes. Identifications are tentative only and are based on
coinjection with even numbered n-alkanes and/or interpolation between
even-numbered n-alkanes to obtain the retention index e.g., 2044 peak.
Figure 9. Gas chromatograms of alkene-aromatic fraction of solubles,
-54-
-------
TABLE V - INFRARED ABSORBANCES NORMALIZED
TO 2955 Cm"1 ABSORBANCE BAND
cm~l
3000
2955
2915
2850
1735
1715
1626
1450
1410
1379
1265
1100
1029
870
810
760
5
*-
1.000
.19
.10
.05
.04
.02
.06
.10
a
4.69
4.31
4.43
0.81
5.75
0.81
6
.10
1.40
.75
.45
.35
.30
.30
1.10
.95
1.10
0.35
1.15
0.50
Day
7
.23
--
1.03
.80
.53
.43
.03
.42
.12
.35
.22
.27
.10
.15
0.13
8
.16
--
1.55
.74
.58
.37
--
.32
--
.18
.45
.34
.45
.08
.37
.18
9
.10
1.69
.77
.13
.31
.46
.46
.59
.59
.59
.46
1.0
0.05
1.59
.73
.05
--
.07
.30
.16
.07
.07
.09
--
.07
.11
a -- not detected.
-55-
-------
B
1 cm
1 mm
^
0.5 mm
Figure 10. Antennule with aesthetasc hairs and guard hairs,
-56-
-------
Figure 11. Antennule in vivo (150x, light microscopy)
-57-
-------
I 1
Figure 12. Detail of tip of aesthetasc hair vivo (1500x, light micro-
scopy).
-58-
-------
Figure 13. Detail of base of aesthetasc hair in vivo (1500x light micro-
scopy).
-59-
-------
Figure 14. Aesthetasc hair details (scanning electron microscopy)
a) Tip, head-on view
b) distal 1/2 portion
c) base, heart-on view, with pores in antennule
-60-
-------
**'
I
n
' IP **-,,«
>
» -
,*
V
Figure 15. Cross section of middle portion of aesthetasc hair (12,000x
electron microscopy).
-61-
-------
V if*
'A. S<-'.- :;
Figure 16. Detail of cuticle of Figure 15. (45,000x electron microscopy'
-62-
-------
P^^ .-
c + *
^r*--..*"*.,^^ ^V 'i -""
- . . * -
^. fc :ijte? f?
? '
;.
"}V2k7 /-
.w %;r*.L4rr/« * / °
Figure 17. Detail of dentrites inside aesthetasc hair middle portion
(33,000x electron microscopy).
-63-
-------
Figure 18. Detail of dendrites inside aesthetasc hair middle portion
(29,000x electron microscopy).
-64-
-------
SECTION VI
DISCUSSION
Fate of Oil in Seawater
Some of the results from the more extensive chemical analyses performed
on the water soluble fraction can be expected to be generally valid for
the crude oil-water mixture also. Water analysis on day five before oil
exposure should be similar in both cases. It may be expected that,
since oil emulsified in the first experiment, bacterial degradation
takes place faster. A comparison of the gas chromatography results of
both experiments shows ,' indeed , great similarities in overall similarities
with time.
The results of hydrocarbon analyses show that there were probably
some petroleum hydrocarbons present in the experimental tanks prior
to exposure to an oil-slick. This could have been the result of
contamination of the natural seawater in the experimental tanks, or
despite extensive cleaning and leaching procedures, due to contamination
of the tanks with oil from a previous experiment.
The decrease in lipid concentration from day five to day six and con-
tinuing through day eight could be due to adsorption of lipids onto
the walls of the oil-slick tank or into the oil slick itself. The
addition of water to the system would only dilute the lipid concentration
to a value of 800 ug/liter, well above that of the 232 yg/liter measured
on day six.
The data of Tables III, IV and V and Figures 6 and 7 all indicate that
the lipid composition, lipid concentration and hydrocarbon concentra-
tion are changing throughout the experiment. The exact processes
involved are not clear. Chromatograms in Figures 5 and 8 suggest
that bacteria and other microorganisms are degrading the oil.
The comparison of the gas chromatograms of alkanes of La Rosa crude
and day ten alkanes show that the oil present in the tanks after five
days has been biochemically oxidized to the extent that n-alkanes and
branched alkanes have been reduced in concentration relative to the
naphthenes (unresolved complex mixture). This is expected in view of
the preferential oxidation of n-alkanes and branched alkanes relative
to the naphthenes as demonstrated by the many investigations reviewed
by ZoBell (1969).
The comparison of the UV spectra of lipids extracted on day ten and
the UV spectra of the alkene-aromatic fraction of hydrocarbons isolated
from the day ten lipid extract suggests that some of the UV absorption
of the day ten lipid extract is due to other than aromatic hydrocarbons
or to higher molecular weight aromatics than those eluted from the column
by the procedures employed i.e. more complex polynuclear aromatics
than phenanthrene and anthracene.
The increase in the hydrocarbon concentrations on day nine may have
been due to a short term malfunction of the stirrer which was not noted
by visual observations. This is conceivable in view of the failure of
the stirrer on day ten (also not observed) .
-65-
-------
In conclusion, the data indicate that several processes may be operating
at the same time during the course of the experiment with solubles. Chemical
analyses presently available show that the behavior of the test organisms
could have been affected by (1) hydrocarbons from the oil slick, (2) changes
in the composition and concentration of the lipids other than the hydrocarbons,
(3) influx of products of the biochemical oxidation of oil by microorganisms.
However, analysis of lobster behavior showed no changes caused by this
experiment; only in the experiment with whole crude oil mixed with sea water
could behavior changes be measured.
B. Behavior Changes
In order of significance, the following behavior units changed after the
lobsters were exposed to whole crude oil:
2
Behavior Unit Direction of Change X Significance
1. Crude, Waiting Phase increase 12.6 .001
2. Crude, Antennule Wave increase 9.7 -005
3. Crude, Antenna Wave increase 8.5 .005
4. Crude, Fan Slow increase 8.5 .005
5, Crude, Beat Fast decrease 3.1 .100
Exposure to the water soluble fraction of crude oil did not affect the
lobsters' behavior in a way that could be measured by our methods.
The changes in behavior observed after exposure to whole crude oil may
be summarized as changes in, water chemistry sensing movements: 1) Slow
rates of gill, bailers increased, which means that less water was passed
over the gills and around the lobsters' anterior end; 2) Fast antennule
rates decreased slightly, which means that the lobster "sniffed" (to
use a higher vertebrate analog) less intensely. Both results indicate
that there is a tendency for an increase in slow fanning and beating
movements. Finally, 3) Antennae and antennules moved more, which may
be the equivalent of an increase in head movements in higher vertebrates,
or more "looking around". The increase in antenna and antennule waves
may compensate for the decrease in gill bailer and antennule beating
rates; or, in vertebrate terms, head movements took over from sniffing
in the presence of oil in the water.
All phases of feeding behavior may involve sensory perception. The
first or alerting phase, in which the animal becomes aware of a chemical
stimulus, is probably determined by threshold perception of the chemical
5<:,ises, most likely by antennular aesthetasc hairs of the sense of smell,
The third or searching phase, in which the food is localized, probably
involves chemical and tactile senses in the detection of odor gradients
and water currents. The second or waiting phase, however, may or may
not be under sensory control.
-66-
-------
One explanation for the doubling of the waiting phase is that the animal
is still capable of perceiving chemical stimuli in its environment, but
that the ability to interpret the stimuli as food is impaired by direct
damage to the sensory receptor system. However, since no major morphological
changes took place in odor receptors, the aesthetasc hairs of the antennules,
as evidenced by light and electron microscopy, sensory disruption probably
did not occur at this level of oil exposure.
Discounting sensory damage, the waiting phase can be described as a period
in which the animal must build up motivation (i.e., sufficient attracting
stimulation) to leave the sheltered corner position and go out in search
of food, the chemical presence of which is presumably known after alerting.
Highly motivated animals (i.e., very hungry ones that are under little
stress from ambient light, the presence of the observer, oil or any other
environmental factor) become alerted and soon run off to find the food.
In extreme cases, this phase is reduced to zero. Since all animals were
kept hungry, a time increase in this phase must be due to the one
environmental stress not held constant; oil. One might say that oil
produces a bad taste (or odor) in the water which competes negatively
with the attractive properties of the food odor. This competing effect
of sensory inputs may take place at the receptor sites or, probably, in
more central parts of the nervous system.
There also remains the possibility that oil interferes directly with
the molecular properties of the food stimulus, changing its odor
characteristics. This would result in difficulty in recognizing the
chemical stimulus as food,hence a longer waiting time.
All explanations are based on the premise that less food stimulation
reaches the receptors or that more food stimulation is necessary to
offset the ill effects ("bad taste") of oil stimulation, Either way,
the waiting time increases to allow for motivation (here, level of
positive chemotactic stimulation) to build up. It may be this same
"bad taste" effect that changes the water quality sensing movements
of the lobsters' behavior in general. The possibility must be pointed
out that the change in water quality sensing movements could directly
affect the length of the waiting phase in feeding behavior.
It has been suggested that the changing chemical composition of degrading
oil may have different levels of toxicity for marine life (Blumer, 1972).
The small number of tests in relation to the individuality of the
lobsters' behavior made it impossible to analyze, on a day by day basis,
the oil effect corresponding with the chemical analyses. However, if
a particular day and, therefore, a particular composition of the de-
grading oil had caused a uniform spectacular change in behavior, it
would have been apparent in the results. Thus, at this exposure level,
no major differences could be seen in the effects of oil degrading
over a five day period.
In summary, the results show that crude oil, when mixed in small
quantities (1 ml in 100 1) in sea water, has immediate, measurable
effects on the behavior of adult male and female lobsters over a
five day period. Changes in water quality sensing movements were observed
and the time required to find food, even when hungry, was increased.
Sensory damages by oil at these low levels seems unlikely from the
results of behavior and microscopy, but interference with chemical
communication nevertheless takes place when oil represents a negative
-67-
-------
stimulus reducint the attraction of food. Long term effects and recovery
were not studied. The experiments confirm that the low boiling fraction
of oil rather rapidly disappears from sea water and that the higher
boiling fraction increases with time in the water column. A correlation of
behavior effects with the changing composition of degrading oil could
not be made.
-86-
-------
SECTION VII
ACKNOWLEDGMENTS
We want to express our great appreciation to Dr, John Farrington and Bruce
Tripp of the Woods Hole Oceanographic Institute who did the chemical
analyses of the water soluble fraction and crude oil-water mixture,
respectively. Without their help this study would have lost much of its
value.
We also want to thank Dr. Melbourne Carriker of the Marine Biological
Laboratory at Woods Hole for the generous use of his excellent light
microscopy equipment and Dr. Susumu Honjo and Joanne Antanavage of
W.H.O.I, for their assistance in the electron microscopy.
The Environmental Protection Agency (Dr. C. S. Hegre, Project Officer)
provided financial support under Grant No. 18080 EBN to Dr. John M.
Hunt of W.H.O.I.
-69-
-------
SECTION VIII
REFERENCES
1. Blumer, M., (1971), Scientific Aspects of the Oil Spill Problem. Environ-
mental Affairs 1_: 54-73.
2. Atema, J., (1971), Structures and Functions of the Sense of Taste in the
Catfish, Ictalurus natalis. Brain, Behav., Evol. £: 273-294,
3. Atema, J., and D.G. Engstrom, (1971), Sex Pheromone in the Lobster,
Homarus americanus. Nature 232; 261-263
4. Blunter, M., (1969), Oil Pollution of the Ocean. In: Oil on the Sea,
Plenum Press, pp. 5-13.
5. Boylan, D. B., and B. W. Tripp, (1971), Determination of Hydrocarbons
in Sea Water Extracts of Crude Oil and Crude Oil Fractions. Nature
230: 44-47.
6. McLeese, D. W., (1972). Initial Experiments on Growth of the American
Lobster in Captivity, Fisheries Research Board of Canada Technical
Report No. 320.
7. Blumer, M., (1970). Dissolved Organic Compounds in Sea Water:
Saturated and Olefinic Hydrocarbons and Singly Branched Fatty
Acids. In: Organic Matter in Natural Waters, D. W. Hood, ed-^, Inst.
Mar. Sci., U. of Alaska Publ. No. 1, pp. 153-167.
8. Ghiradella, H., J. Cronshaw and J. Case, (1968), Fine Structure
of the Aesthetasc Hairs of Pagurus, hirsutiuscutus (Dana). Protoplasma
6£: 1-20.
9. ZoBell, C. E., (1969), Microbial Modification of Crude Oil in the Sea.
In: Proceedings Joint Conference on Prevention and Control of Oil
Spills . American Petroleum Institute, pp. 317-326.
10. Blumer, M., (1972), Oil Pollution: Persistence and Degradation of
Spilled Fuel Oil. Science 176: 1120-1122.
-70-
-------
SECTION IX
PENDING PUBLICATION
Atema, J. and L. Stein. Sublethal Effects of Crude Oil on Feeding Behavior
in the lobster, Homarus americanus. Marine Behavior and Physiology, sub-
mitted.
-71-
-------
SECTION X
APPENDICES
App. Table I: Complete list of behavior units (Homarus americanus)
App. Table Ha: Frequency of occurrence of selected behavior units
App. Table lib: Frequency of occurrence of the behavior units "Beat", "Fan1'
and "Claw" presented in raw scores and normalized scores
App. Table III: Duration of Phases in Feeding Behavior
-72*
-------
Name
Beat
Slow
Moderate
Fast
APPENDIX TABLE I
COMPLETE LIST OF BEHAVIOR UNITS (Homarus americanus)
Definition
Beating of antennules at
the following rates:
0-60 bpm
60-120 bpm
^ 120 bpm
Wipe
Antennule position 1-5
Antennule wave
Antennule point
Antenna wave
Antenna wipe
Antenna position
normal
up
folded
back
Antenna feel
Antenna touch
Antenna point
Fan
Slow
Moderate
Fast
Maxilliped movement
Maxilliped rub
Maxilliped extended
wiping of an antennule
with 3d maxillipeds
It;
54-
change in antennule position
movement of antennules in
direction of stimulus
slow sweep of an antenna
wiping of an antenna with
3d maxillipeds
in front
straight up
folded back
between up and folded
quick, successive move-
ments of antennae over object
antenna touching object one time
movement of antenna in direction
of stimulus
Fanning of exopodites of 3rd
maxillipeds; rates:
0-40 bpm
90-180 bpm
>_ 190 bpm
swaying of 3d maxillipeds
moving 3d maxillipeds
against each other
stretching of maxilliped
down toward substrate
-73-
Code
aaS
aaM
aaF
aw
aa ; etc.
aT
aP
aW
Aw
AAn
AAu
AAf
AAb
AT
AP
exo S
exo M
exo F
mm
Ru
Me
-------
Name
C,law position 1
Claw position 2
Claw position 3
Claws
Up
Level
Down
Claw spread
Stretch claws
Withdraw crusher
Withdraw seizer
Shield
Body position
low on legs
medium on legs
high on legs
Crouch
Tail position 1-5
Tail arch
Tail hump
Defensive posture
Move
Walk
Definition
claw closed
claw ^ open
claw full open
claw raised above eye level
claw raised but below eye level
claw on substrate
claws raised and spread
extend claws in front of body
bring crusher claw close to body
bring seizer claw close to body
Keep claws closed in front, passive
body close to substrate
body partially raised off sub-
strate
body raised high
sit very low on legs and body
fairly compact
1-; 2-s 3-i; 4~>; 5=>;
curling tail upward
raising abdomen while tail
is down
Tail position 5; body raised,
claws 2-3 and up
any undirected movement of the body
walk
Code
Sl or Ci
or
S , etc.
SI
Sl
CIS
St.C
WCr
WSe
Sd
lo legs
M legs
H legs
Cro
T^ etc.
Arch
Hump
Def
GBM
W
-74-
-------
Name
Slow walk
Stop walk
Run
Reverse
Stop reverse
Walk sideways
Approach
Climb
Descend
Reverse descend
Raise rear
Fall
Pounce
Roll
Enter hole forward
Enter hole reverse
Turn
Rake
I
II
III
Scoop
Definition
slow walk
stop walk
fast walk
walk backwards
stop walk backwards
forward and lateral
walk at the same time
forward movement directed
toward stimulus
raising of body against side of
rocks, walls,, etc. to a vertical
position
climb down
climb down backwards
tail up on wall, rock etc.
(describe)
free fall descend
sudden lowering of body
usually accompanied by
feeding behavior
lateral rotation of body
crawl into hole forward
(denote which hole)
crawl into hole tail first
change direction of body
back and forth movement of
one or more walking legs
across the substrate while
body is still
slow rake
moderate rake
fast rake
lifting of walking legs
from substrate into mouth
-75-
Code
Slo W
Stp Jf
Run
Rev
Stp Rev
WS
app
Cl
D
RD
RR
FD
Pounce
roll
HF
H rev.
T
R I
R II
R III
sc
-------
Name
o
Pinch
Poke
Dig
Bulldoze
Lunge
Snap
Swinuneret wave
Stop swinuneret wave
Groom
antenna
antennuies
swimmerets
carapace
rostrum
eye
claws
legs
tail
Stop groom
Definition
opening § closing dactyls
of walking legs
poke into substrate with legs
moving substrate with legs
push gravel with maxillipeds
and/or claws
fast extension of claws
quick opening § closing of
seizer claw
beating of swimmerets
stop beating of swimmerets
picking, rubbing, scratching
parts of body with the walking
legs
Code
pin
Po
Dig
BD
lunge
snap
sw
stp sw
GR
AA
aa
sw
car
ros
eye
claws
legs
tail
stp Gr
-76-
-------
Definition
Code
AGGRESSIVE
Being pulled
Being pushed
Claw lock
Engage
Face off
Grab
Jab"
Positioning
Pull
Push crusher
Push seizer
Rip
Low rip
Shake
Roll animal over
Swat
Release
On Guard
MATING
Dismount
Ejaculate
Limp
while in claw lock, being maneuvered
by other animal forwards
in encounter, being maneuvered by other
other animal backwards
handshake position of animals while
in face off - crusher - crusher only
maneuvering of claws to get into claw
lock
face to face confrontation of two
animals within o'ne body length
claw placed around part of other
animal and bear down, including
crusher-seizer hold
poking at other animal's body
or claws with own claws
first stages of maneuvering in
claw lock
while in claw lock, maneuvering
other animal
with crusher, push other animal.
If not in claw lock, push will
be with back of claw
same as above but with seizer
bearing down with claws and
quick jerking of bodyhigh intensity
low intensity Rip
while in claw lock, movement of
claw that is locked toward other
animal and back toward self
(slow moving)
while in claw lock, due to strength,
turning other animal on its side
swinging of seizer toward other animal
(like a right hook)
letting go of claw lock
one claw raised and extended and other
claw close to body and down
walking away from mount
thrust of abdomen of male while mating
describes submissive animal when being
matedno resistance
puld
pud
CL
En
FO
G
ja
Pn
pul
PuC
PuS
Ri
loRi
Sh
RAO
Swat
Rel
OG
DisM
ejac
Li
Mount
II
III
Resisting
Turn animal over
beginning stagesclaws and/or
maxillipeds on another animal
1/2 way on other animal
fully on other animal
opposite of Limp
after mounting, turning the
submissive animal over
M I
M II
M III
Res
TAO
-77-
-------
APPENDIX TABLE Ha
Frequency of occurrence of selected behavior units
Unit: Crude, Antenna Wave
Days pre-oil Days post-oil
Animal Number 12345 6 ,7 r 8 9 10
1 45225 14 14 4 16 10
2 16 14 5 3 19 8 28 29 29 7
3 8 9 3 1 22 26 22 36 32 29
4 5 3 1 6 11 17 7 9 9 3
5 14 9012 10 1501
6 28006 10 14 661
7 10 8 2 5 18 2 5 20 9 12
Unit: Crude, Antennule Wave
Days pre-oil Days post-oil
Animal Number 12345 6789 10
1
2
3
4
5
6
7
Unit: Crude, Wipe
Days pre-oil Days post-oil
Animal Number 12345 6 7 8 9 10
1 14 11 10 2 3 7 7 13 8 6
2 3 13 0 13 0 5 11 7 5 3
3 78336 1 12 14 44
4 8 38 152 16408
5 3 13 4 15 6 13302
6 370 10 21 81533
7 8 10 206 72745
-78-
0
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
1
0
2
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
2
1
0
0
0
0
3
9
0
1
0
0
0
0
3
0
0
0
6
0
0
2
1
1
0
1
0
1
3
1
-------
APPENDIX TABLE I la continued
Unit: Crude, Rake
. , ,_ Days Pre-oil Days post-oil
Animal Number 1 23 4 5 _ 6 7 8 9 10
1 14 8 5 0 5 0 1 12 11 8
2 00200 05220
3 0 15 000 00351
4 01000 11714
5 39831 309 12 8
6 2520 16 10000
7 11100 02707
Unit: Crude, Scoop
Days pre-oil Days post -oil
Animal Number 12345 6789 10
1
2
3
4
5
6
7
Unit: Crude, Move
Days pre-oil Days post-oil
Animal Number 12345 _ 6 _ 7 _ 8 _ 9 _ 10.
I 13016 66355
2 31035 23563
3 23004 44 10 37
4 14323 42333
5 34202 23201
6 14137 41137
7 31314 22313
14
0
0
0
2
0
0
10
0
53
0
18
0
0
4
0
0
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
0
0
0
8
13
1
0
0
0
0
7
1
4
1
3
0
2
10
0
0
0
0
0
0
12
0
3
0
0
0
4
-79-
-------
APPENDIX TABLE Ha comt,
Unit: Crude, Climb
Animal Number
1
2
3
4
5
6
7
Unit: Crude, Groom
Animal Number
1
2
i
3
4
5
6
7
Days pre-oil
Days post-oil
1
5
0
0
0
4
0
0
1
15
0
0
1
0
1
2
2
2
0
0
1
0
0
0
Days
2
20
2
0
0
3
0
3
3
7
2
3
0
0
1
0
r.4
0
0
0
0
0
0
0
pre-oil
3 4
4
0
0
2
1
1
0
0
3
0
0
0
0
0
5
2
0
0
3
0
1
1
5
2
0
0
0
0
8
0
6
0
0
0
0
2
0
0
6
0
0
0
0
6
1
0
7
0
4
0
2
0
0
0
Days
7
8
0
5
0
0
4
0
0
8
0 19
1
0
0
0
1
0
2
0
0
1
0
4
9
2
0
0
1
0
0
0
10
3
1
0
0
0
0
0
post-oil
9 10
8
0
4
8
0
0
0
10
0
0
0
0
0
0
-80-
-------
APPENDIX TABLE Ila cont.
Unit: Soluble, Ant^na Wave
Days pre-oil
Animal Number
1
2
3
4
5
6
7
Unit: Soluble,
Animal Number
1
2
3
4
5
6
7
1
15
30
33
10
15
20
33
Antennule
1
5
2
0
2
0
5
1
2
5
27
19
16
41
3
15
Wave
Days
2
4
3
6
2
5
3
2
3
6
31
15
23
4
17
6
4
2
22
14
15
20
17
13
pre-oil
3 4
1
4
4
0
15
6
6
4
6
8
2
0
3
0
5
3
12
21
7
7
3
20
5
0
0
4
1
0
3
2
6
11
25
24
5
7
18
15
6
2
3
3
2
0
6
0
j
7
3
43
24
14
10
28
17
Days
7
1
0
2
3
10
2
3
JT
8
11
8
6
14
6
18
13
9
5
13
3
30
16
24
16
post- oil
8 9
3
1
1
2
2
6
2
1
2
2
2
2
3
5
10
14
49
3
13
17
5
15
10
0
1
6
0
1
5
3
-81-
-------
APPENDIX TABLE Ila cont.
Unit: .Soluble, Wipe
Days pre-oil Days post-oil
Animal Number
1
2
3
4
5
6
7
Unit: Soluble, Rafc?.
Animal Number
1
2
3
4
5
6
7
1
2
6
3
2
2
6
2
1
0
0
5
1
0
1
1
2
3
3
7
9
3
0
23
Days
2
3
0
4
12
3
0
0
3
5
5
10
2
3
1
3
4
0
5
5
5
9
2
13
pre-oil
3 4
2
0
7
4
1
0
4
0
2
1
2
0
0
2
5
3
7
6
4
4
5
3
5
2
2
6
0
0
1
2
6
3
7
6
6
3
2
23
6
3
5
1
7
0
3
5
7
2
3
8
12
0
3
3
Days
7
3
0
0
4
0
5
3
8
4
4
5
7
3
4
11
9
5
0
5
2
1
4
5
post-oil
8 9
0
5
3
12
3
3
2
1
0
1
0
3
8
2
10
2
6
3
6
4
5
3
10
1
3
10
10
0
6
0
-82-
-------
APPENDIX TABLE Ha cont.
Unit: Soluble, Scoop
Animal Number Days pre-oil Days post-oil
1
2
3
4
5
6
7
Unit: Soluble, Move
Animal Number
1
2
3
4
5
6
7
Unit: Soluble, Climb
Animal Number
1
2
3
4
5
6
7
1
0
0
1
0
0
0
0
1
0
0
0
5
2
3
1
1
0
0
10
0
0
0
0
2
0
0
1
27
11
0
2
Days
2
4
1
1
4
0
0
3
Days
2
0
0
3
0
4
0
0
3
0
0
0
0
0
0
1
4
0
0
0
3
0
0
1
pre-oil
3 4
1
2
1
1
1
0
0
0
0
3
1
1
1
0
pre-oil
3 4
0
0
5
3
0
0
0
0
1
4
0
0
0
0
r
%/
4
0
0
0
0
2
1
5
3
1
1
5
1
0
3
5
0
1
8
0
0
0
0
6
0
6
1
2
0
0
3
6
2
2
3
2
1
2
5
6
0
0
2
0
0
1
0
7
5
0
0
8
0
0
0
0 13
0
0
0
Days
,7
1
0
1
3
0
2
2
Days
7
0
14
2
0
0
1
0
0
0
7
9
0
0
2
0
0
0
2
10
2
1
3
1
0
0
0
post-oil
8 9 10
3
2
2
2
1
0
3
3
1
3
2
2
4
2
1
1
0
4
1
1
1
post-oil
8 9 10
0
0
0
0
0
0
0
2
0
0
0
0
0
0
1
1
0
0
0
0
1
-83-
-------
APPENDIX TABLE Ha qont.
Unit: Soluble, Groom
Animal Number Days pre-oil Days post-oil
2
3
4
5
6
7
1
0
1
0
6
4
3
0
2
1
2
9
25
12
0
0
3
0
0
0
1
0
0
1
4
0
8
3
10
0
0
0
5
7
0
2
0
1
0
0
6
0
5
0
0
0
0
1
7
13
16
30
3
0
0
0
8
8
1
33
0
9
0
3
9
0
1
22
0
13
0
0
10
1
0
16
0
0
0
0
-84-
-------
APPENDIX TABLE lib
Unit: Crude, Beat
Days pre-oil Days post-oil
Animal Number 1 25 4 5 6 7 8 9 10
1 Slow 02252 42332
% 0 25 40 83 50 50 100 38 43 33
Moderate 15312 40433
% 12 63 60 17 50 50 0 50 43 50
Fast 71000 00111
% 88 12 0 0 0 0 0 12 14 17
2 Slow 52410 10354
% 50 15 80 20 0 12 0 23 38 40
Moderate 5 10 146 74886
% 50 77 20 80 67 88 57 62 62 60
Fast 01003 03200
% 0 8 0 0 33 0 43 15 0 0
3 Slow 63334 11134
% 67 100 43 50 66 10 20 14 43 44
Moderate 30121 84345
% 33 0 14 33 17 80 80 43 57 56
Fast 00311 10300
% 0 0 43 17 17 10 0 43 0 0
4 Slow 14535 45635
% 17 57 71 75 46 40 63 86 50 100
Moderate 52214 53130
% 83 29 29 25 36 50 37 14 50 0
Fast 01002 10000
% 0 14 0 0 18 10 0 0 0 0
5 Slow 24345 26332
% 22 50 75 100 62 40 67 34 75 100
Moderate 43003 22410
% 44 38 0 0 38 40 33 44 25 0
Fast 31100 10200
% 34 12 25 0 0 20 0 22 0 0
6 Slow 24145 37448
% 40 33 100 50 72 43 88 80 57 80
Moderate 28041 41132
% 40 67 0 50 14 57 12 20 43 20
-85-
-------
APPENDIX TABLE lib cont.
v
Unit: Crude, Beat
Days pre-oil Days post-oil
Animal Number 12545 6 7 8 9 lp
Fast 10001 00000
% 20 000 14 00000
7 Slow 43437 55625
% 50 75 67 38 70 83 100 86 67 83
Moderate 41252 lOlli
% 50 25 33 62 20 17 0 14 33 17
Fast 00001 00000
% 0000 10 00000
-86-
-------
APPENDIX TABLE lib
-------
APPENDIX TABLE lib cont.
Unit: Crude, Claw
Days pre-oil Days post-oil
Animal Number 12345 6 7 8 9 10
1. Position I
%
Position II
%
Position III
%
2. Position I
%
Position II
%
Position III
%
3. Position I
%
Position II
%
Position III
%
4. Position I
%
Position II
%
Position III
%
5. Position I
%
Position II
%
Position III
%
6. Position I
%
Position II
%
Position III
%
7. Position I
%
Position II
%
Position III
7
70
3
30
0
0
0
0
3
75
1
25
0
0
3
75
1
25
0
0
2
100
0
0
3
75
1
25
0
0
0
0
0
0
0
0
0
0
4
100
0
0
4
40
6
60
0
0
4
67
2
33
0
0
0
0
1
50
1
50
2
100
0
0
0
0
2
40
3
60
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
33
4
67
0
0
2
100
0
0
0
0
0
0
2
100
0
0
1
25
3
75
0
0
0
0
2
100
0
0
2
100
0
0
0
0
0
0
1
50
1
SO
0
0
2
100
0
0
0
0
4
100
0
0
0
0
0
0
0
0
0
0
2
100
0
0
0
0
3
75
1
25
0
0
2
100
0
0
0
0
2
100
0
0
0
0
4
100
0
o
0
0
4
100
0
0
0
0
4
67
2
33
0
0
4
100
0
0
0
0
2
100
0
0
2
50
2
50
0
0
2
50
2
50
0
0
0
0
2
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
100
0
0
2
33
4
67
0
0
0
0
0
0
0
0
0
0
2
100
0
0
0
0
4
100
0
0
4
50
4
50
0
0
0
0
3
75
1
25
4
67
2
33
0
0
0
0
2
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
100
0
0
1
6
15
94
0
0
0
0
4
67
2
33
0
0
4
100
0
0
2
33
4
67
0
0
0
0
0
0
0
0
1
25
3
75
0
0
2
13
13
87
0
0
0
0
6
100
0
0
1
50
1
50
0
0
1
25
2
SO
1
25
0
0
2
100
0
0
0
0
0
0
0
0
0
0
2
100
0
0
0
0
12
100
0
0
0
0
2
100
0
0
0
0
4
100
0
0
0
0
2
100
0
0
0
0
2
100
0
0
0
0
0
0
0
0
0
0
3
75
1
25
-------
APPENDIX TABLE lib cont.
Unit: Soluble, Beat
Animal Number navs m-e oi i n
uays pre-oii 0ayS post-oil
L 2 3 4 5 6 789 10
1 Slow 10 7 10 10 S 6 6 8 S 11
% 77 64 91 100 83 , 33 60 67 24 79
Moderate 24101 11 4 4 11 3
% 15 36 9 0 17 61 40 33 52 21
Fast 10000 10050
* 80000 600 24 0
2 Slow 46798 12 ,6 3 15 5
% 20 21 44 69 36 57 43 20 100 25
Moderate 11 13 8 3 10 8 6 12 0 12
% 55 45 50 23 45 38 43 80 0 60
Fast 5 10 1 1 4 1 2 0. 0. . 3
% 25 34 6 8 18 5 14 0 0 15
3 Slow 10 8 9 11 6 8 6 6 11 13
% 50 50 39 55 24 33 35 60 92 93
Moderate 5697 15 16 7411
% 25 38 39 35 60 67 41 40 8 7
Fast 52524 04000
% 25 13 22 10 16 . 0 24 0 0 0
4 Slow 5 10 5 11 11 ., 11 5 12 7 11
% 26 63 21 65 58 58 25 55 24 65
Moderate 14 6 18 6 8 8 15 10 21 6
% 74 37 75 35 42 42 75 45 72 35
Fast 00100 00 ,010
% 00400 00030
5 Slow 11 9 17 12 10 14 11 10 20 14
% 55 50 100 57 77, 100 100 100 95 82
Moderate 97093 00013
% 45 39 0 43 23 0 0 0 5 18
Fast 02000 00000
% 0 11 000 00000
6 Slow 11 10 12 9 9 11 9 13 10 6
% 42 100 57 38 50 55 43 100 53 50
Moderate 14 0 9 15 9 9 12 0 9 6
% 54 0 43 62 50 45 57 0 47 50
Fast 10000 00000
% 40000 00000
7 slow 16 12 16 10 11 4 4 10 14 9
% 72 80 84 77 92 . 25 20 100 88 53
Moderate 6 3 3 3 1 12 16 0 2 8
% 28 20 16 23 8 75 80 0 12 47
Fast 00000 00000
% 00000 00000
-89-
-------
APPENDIX TABLE lib cont.
Unit: Soluble, Fan
Days pre-oil Days post-oil
Animal Number 12545 _6 7 * 9 10
1 Slow 47 10 03 75829
% 100 100 100 0 50 100 83 100 50 100
Moderate 00003 01020
% 0 0 0 0 50 0 17 0 50 0
Fast 00000 00000
% 00000 00000
2 Slow 25531 64026
% 40 83 100 100 100 86 33 0 100 100
Moderate 31000 14100
% 60 17 0 0 0 14 33 100 0 0
Fast 00000 04000
% 00000 0 33 000
3 Slow 74653 459 10 8
% 41 100 55 83 43 100 100 69 100 100
Moderate 30513 00400
% 18 0 45 17 43 0 0 31 0 0
Fast 70001 00000
% 41 000 14 00000
4 Slow 73789 10 3 10 4 11
% 100 100 64 100 100 100 100 100 44 92
Moderate 0 0 3' 0 0 00051
% 00 27- 00 0 0 0 56 8
Fast 00100 00000
% 00900 00000
5 Slow 8 0 0 0 11 0 10 10 6 6
% 100 0 0 0 100 0 100 77 50 100
Moderate 06600 00360
% 0 50 100 0 0 0 0 23 50 0
Fast 06000 00000
% 0 50 000 00000
6 Slow 5 0 8 0 10 11 12 0 10 10
% 83 w lUO 0 100 100 80 0 91 100
Moderate 10050 03010
-90-
-------
APPENDIX TABLE lib ciont.
Unit: Soluble, Fan
Days pre-oil Days post-oil
Animal Number 12345 6 7 8 9 1C[.
% 17 0 0 100 0 0 20 0 9 0
Fast 00000 00000
% 00000 00000
7 Slow 7 8 8 11 8 5 8 10 12 6
% 100 89 67 69 100 38 47 100 80 43
Moderate 01450 8 11 037
% 0 11 33 31 0 62 53 0 20 50
Fast 00000 00001
% 00000 OOOOt
-91-
-------
APPENDIX TABLE III - DURATION OF PHASES
IN FEEDING BEHAVIOR
CRUDE, ALTERTING PHASE
Animal Number Days pre-oil Days post-oil
1 2345 678910
1
2
3
4
5
6
7
:25 :15
:17 :22
:10 :21
:17 :06
:20 :15
:28 :20
:40 :10
:ll
:20
:25
:15
:25
:15
:35
:18
:15
:12
:45
:30
:20
:25
:47
:06
:17
:13
:07
:09
:35
:27
:23
:05
:40
:20
:35
:20
:20
:17
:11
:30
:30
:38
:15
:05 :05
:25 :22
:04 :07
TO 1 C
« X O * JL O
:1S :10
:12 :20
:17 :28
:07
:26
:10
:08
:31
:15
:30
-92-
-------
APPENDIX TABLE III cont,
CRUDE, WAITING PHASE
Animal Number Days pre-oil Days post-oil
1 2345 6789 10
1
2
3
4
5
6
7
:12
:21
:07
:04
:20
:05
:07
:10
:17
:04
:08
:53
:05
:40
:12
:00
:00
:07
:00
:08
:20
:00
:65
:00
:25
:00
:18
:15
:00
:72!V
:02
:54
:11 .
:14
:65
:78
:45
:03
:35
:03
:27
:98
:55 :10
:46 :40
:22 :11
:25 :44
:22 :13
:17 :10
: 130: 30
:12
:58
:10
:45
:15
:92
:47
:10
:60
:15
:70
:08
:13
:47
-93-
-------
APPENDIX TABLE III cont.
CRUDE, SEARCHING PHASE
Animal Number
1
2
3
4
5
6
7
Days pre-611
12 3 45
Days post-oil
7 8 9 10
:06
:38
:15
:4S
:07
:07
:09
:04 :12
:10 :12
:10 :25
:61 :38
:13 :12
:10 :04
:08 :05
:04
:12
:07
:08
:33
:08
:22
:09
:24
:22
:37
:08
:05
:16
:09
:07
:29
:13
:12
:07
:34
:07
:18
:12
:35
:09
:06
:06
:15 :03
:12 :07
:10 :03
:11 :26
:06 :08
:04 ;08
:11 :05
:03
:06
:30
:57
:04
:07
:05
-94-
-------
APPENDIX TABLE III cont.
SOLUBLE, ALERTING PHASE
Animal Number Days pre-oil Days post-oil
2345 6789 10
:25 :08
:28 :40 :15
:27 :14 :07
:105
:65
:24
:25
:20
:35
:22
:26
:22
:20
2
3 :24 :30 :50 :10 :35 :25 :12 :13 :22 :21
4 :28 :14 :15 :08 :15 :15 :12 :20 :07 :07
5 :18 :27 :29 :07 :14 :12 :32 :14 :15 :08
6 :23 :13 :15 :15 :12 :15 :10 :20 :13 :07
7 :28 :26 :26 :60 :36 :09 :06 :13 :12 :13
-------
APPENDIX TABLE III cont.
SOLUBLE, WAITING PHASE
Animal Number Days pre-oil Days post-oil
12345 6789 10
2
3
4
5
6
7
:42
:25
:08
:12
:17
:17
:16
:58
:10
:26
:20
:21
:04
:09
:32
:43
:26
:10
:09
:21
:33
:45 :15
:80 :13
:23 :58
:06 :04
:12 :02
:07 :11
:06 :15
:100
:84
:18
:40
:20
:14
:31
:08
:68
:13
:21
:19
:07
:46
:26
:101
:17
:46
:16
:06
:37
:38
:39
:03
:34
:14
:19
:40
:14
:30
:05
:48
:14
:20
:30
-96-
-------
APPENDIX TABLE III cont.
SOLUBLE, SEARCHING PHASE
Animal Number Days pre-oil Days post-oil
1 2 .34 5 6789 10
2
3
4
5
6
7
:05
:04
:08
:06
:15
:18
:08
:04
:09
:13
:05
:31
:13
:09
:22
:1S
:22
:06
:32
:07
:18
:04
:17
:07
:06
:05
:15
:15
:12
:05
:12
:06
:06
:06
:18
:87
:1S
:07
:11
:06
:13
:07
:U
:04
:08
:07
:09
:26
:10
:20
:07
:08
:08
:04
:08
:07
:06
:02
:05
:07
:06
:04
:26
:09
:08
:07
:08
:08
:14
:28
97~ »U.S. GOVERNMENT PRINTING OFFICE:1973 M4-156/356
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
W
INTERACTION BETWEEN MARINE ORGANISMS AND OIL POLLUTION
5, K -artDs.f
6.
H. Pt.' formic r Oigasi ztion.
Blumer, Max; Hunt, John M.; Atema, Jelle, and Stein, Lauren
Woods Hole Oceanographic Institution
Woods Hole, Massachusetts 02543
18080 EBN
13.
Period Coveted
12. Sponsoring Or
Environmental Protection Agency report
number EPA-R3-73-042, May 1973
Part I of this project has established that fossil hydrocarbons can be distinguished
from biogenic hydrocarbons in living organisms. Hydrocarbons are stable in marine
organisms and sediments and can move unaltered through several trophic levals. Only
very low levels of organic stimuli are necessary for chemical communicationa
mechanism especially prone to interference by pollutants.
Part II has established that a low level of crude oil (0.9 milliliters/liter)
interferes with the timing of feeding behavior in the lobster (Homarus americanus).
Water soluble fractions (in the 50 ppb range) did not affect feeding behavior.
Added oil reduced the lipids as well as alkane and alkene-aromatic content of
aquaria. Degradation of added oil followed the usual pathways of evaporation,
dissolution, oxidation, polymerization, and metabolism.
-*Behavior,*Oil, Analytical Techniques, Gas Chromatography, Degradation (Decomposition)
*Path of Pollutants, Hydrocarbon, Feeding
t"b. Identifiers
>7<: f*-WKP 'field A- Group 0SC
19. S'~>-at!tyC;3.s$,
(Report)
20 Security Class.
21. If a, of
Pages
22 P:ice
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 2O24O
C. S. Hegre
National Marine Water Quality Lab.
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