Abstrac
etroleum products vary considerably in their
composition, and consequently, they differ in toxicity
to living systems. In this study, the effects of
various dilutions of different kinds of oils were
tested on the fertilization and early development of
the sea urchin, Strongylocentrotus purpuratus. Most
of the oils had little effect on the fertilization,
but many of them did inhibit early cleavage. Crude
oils and heavy bunker oils were the most toxic to
cleavage, even at concentrations of 6.25% saturation, while
the diesel and jet fuels were the least toxic.
Introduction
Pollution from crude oils and other oil products,
while not a recent phenomenon, has steeply increased
during the last decades. An estimated five to ten tons
of crude oil and its derivitives are released into
the ocean each year (Blumer et. al., 1971). Most of
the pollution occurs in shipping lanes and in biologically
productive coastal regions. World consumption of
petroleum derivitives represents 60% of the tonnage
of goods transported by sea (International Conference on
Oil Pollution of the Sea, 1968). This large scale
transport is being done with ever increasing bulk
carriers with an accompanying degree of unavoidable
spillage.
In the past decade, especially after the "Torrey
Canyon" spill in 1967, much research has been done on
the toxicities of the detergents used to clean up the
oil after the spill (Smith, 1968), but little has
been done on the actual toxicities of the oils themselves.
The aftermath of the "Tampico Maru" accident, in which
eight thousand tons of diesel fuel were spilled in a small
cove on the coast of Baja California in 1957, showed
that oil left without mechanical or chemical treatment
can still have serious toxic effects on the marine
Mitche
environment (North et. al., 1965; Anderson et. al., 1970).
Damage from oil pollution of the ocean has been
heaviest in the intertidal regions (Straughan, 1971).
Most of the species common to this region are sedentary
and for that reason cannot escape the noxious irritants
around them. Organisms that can migrate are indirectly
hurt when their food supplies in the intertidal communities
are depleted by excessive pollution.
Echinoderms are known to be extremely sensitive
to petroleum products. North et. al. (1965) pointed
out that after the "Tampico Maru" accident, the immediate
mortalities of echinoderms were considerable, and the
number of echinoderms was still noticeably reduced
seven years later. Sea urchins in particular were
effected, and North found that a 0.1% emulsion of the oil
inactivated the tube feet and was lethal in one hour.
Moreover, even weathered tanker oil reduced the success
of fertilization and caused abnormalities in the resulting
larvae (Elmhirst, 1922, as reported by Nelson-Smith, 1970).
Because of the urchin's sensitivity to water quality and
their common intertidal occurrence, I tested the sensitivity
of the development of this animal to the toxicities of
various petroleum products.
Methods and Materials
Oil samples were obtained from the Standard Oil
Company of California, the Union Oil Company of
California, and the Petrolite Corporation in Saint Louis.
Missouri. The kinds of petroleum products were:
Argentina Crude, Central Libyan Crude, Bunker C Fuel
Oil (Residual), Diesel Fuel 42, and Jet Fuel JP-8
from the Petrolite Corporation; Wilmington California
Crude, Cook Inlet Alaska Crude, Bunker Oil 46, Residual
Heating Oil (45 fuel oil), Distillate Heating Oil
(42 fuel oil), and Diesel Fuel 42 from the Union Oil
Company; and Waxy Crude, 4-Corner Crude, Bunker Fuel
Oil, Diesel Fuel Hi, and Jet Fuel A-50 from the Standard
Oil Company.
Twenty-five milliliters of each oil sample was
vigorously shaken with 500 ml. of sea water. After
shaking, most of the oil reformed as a film on top.
Nevertheless, fractions of the oils went into solution,
producing what was considered to be a saturated
solution, and these then were the fractions tested.
Samples of the saturated solution were drawn from
the bottom of separatory funnels.
Serial dilutions were set up in test tubes
using the saturated oil solution and sea water. The
concentrations of oil used were 100% (containing only the saturated
oil-in-sea water solution), 50%, 25%, 12.5%, and 6.25%.
These dilutions were then used to test the effects
of the petroleum fractions on the fertilization and
early development of the urchin.
Purple sea urchins, Strongylocentrotus purpuratus,
were obtained from Pigeon Point, California, and
maintained in running sea water at Hopkins Marine
Station. The urchins were spawned by injecting them
with 0.5 ml. of a 0.5 M solution of KCl. For the
fertilization tests, the sperm were added to the dilutions,
left five minutes, and then the eggs were added, so that,
in actuality, it was the ability of the sperm to survive
exposure to the test solutions and fertilize the eggs
that was being tested. Aliquots of the eggs were removed
after two minutes with a pipette, and the percentage of
fertilization versus non-fertilization out of one hundred
eggs was determined under a microscope. In testing for
effects on cleavage, the eggs were fertilized in less than
0.5 ml. of sea water, then added to 5 ml. of each of the
dilutions two minutes later. Cleavage versus non-cleavage
out of one hundred fertilized eggs was checked after
two hours, and the percentage of fertilized eggs at
the four cell stage or less was checked after four hours.
One hundred eggs were counted in each test. All experiments
were conducted at 15° C. Controls were run in sea water
with each series of dilutions.
Each experiment for each series of oil samples tested
was repeated three tines. The mean, variance, and
standard deviation was calculated for each oil sample
and the controls in each series. In all cases the standard
deviation was less than ten. Calculations were done on
each sample to determine what concentrations differed
from the control in their series at the 5% level of
uncertainty.
Results
Most of the oil samples did not seem to have much
of an effect on the ability of the sperm to fertilize
the eggs. Fertilization in the majority of the samples
was not significantly different from the controls even
at the highest oil concentrations. (Table I). The
one exception to this finding was the Residual Heating
Oil 45. Fourteen percent of the eggs in the 12.5%
dilution of this oil sample did not fertilize, and this
figure was significantly different from the percentages
of non-fertilization obtained with the controls. The
46 Bunker Oil also proved to be somewhat toxic to fertilization,
but only at 50% or greater oil saturation.
Cleavage was more sensitive to the toxic effects
of petroleum fractions. At the two hour stage, eleven
out of the sixteen different kinds of oil tested
showed effects significantly different from the controls
even in the most dilute concentration (Table II,
Figure I). In some instances, cleavage was totally
arrested. The more highly refined petroleum products
were less toxic than the heavier crudes and bunker oils.
Only the Jet Fuel JP-8 did not have an effect different
from the control.
Four hours after fertilization, fourteen out of the
sixteen oil samples showed effects different from the
controls at the 6.25% oil concentration. (Table III,
Figure II). In general, the samples that were highly
toxic to cleavage after two hours were still the most
highly toxic after four hours. Again, it was the crudes
and the bunker oils that had the most inhibiting effect
on cleavage, and the Jet Fuel JP-8 that had the least effect.
Discussion
Crude oils and petroleum products differ in
composition and consequently they differ in toxicity. Some
crude oils contain virtually only carbon and hydrogen
(Dean, 1968). The major non-hydrocarbons in other crude
oils are sulfur compounds, nitrogen compounds, and oxygen
compounds. Petroleum products contain four main classes
of hydrocarbons: alkanes, olephins (except in crudes).
naphthenes, and aromatics. The composition of oil also
effects the dispersal of oil at sea, and how readily it
forms emulsions. There are two basic types of petroleum
products, the persistent oils including crudes, residuals.
lubricating oils and sludges, and the light fuel oils
including kerosene and the various gasolines.
Alteration of these compounds occurs mainly through
dissolution and biological degradation. Many feel that
the effects of evaporation and biological degradation on
reducing the toxicities of oils have been over estimated.
Blumer (1970), even concluded that weathering increases
rather than decreases the toxicity of oils. His results
showed that degradation removes straight and branched chain
fractions, yet it is the remaining aromatics that are the
more highly toxic. Thus, as the straight and branched
fractions are removed by degradation, the toxicity
increases due to an increased aromaticity. However,
Shelton (1971) stated that the aromatics are rapidly lost
during weathering, and therefore, crude oils lose their
toxicity in time.
Of the two processes, fertilization and cleavage,
fertilization was the least sensitive to the toxic
effects of oils. In all my cleavage tests, I found
crude oils and heavy bunker oils to be more inhibiting
than the more highly refined petroleum products, possibly
because the process of refining itself removes many of
the more toxic fractions. This too, differs from the
report of Shelton (1971). He concluded that, although
the light refined products are the least persistent,
they are the most highly toxic. He also maintained that
apart from the light refined oils, petroleum products
are not highly toxic substances.
is interesting to note that many of the oil
samples that had the greatest toxic effect on fertilization
less
had the-least toxic effect on fertilization and vice
versa. For example, the 4-Corner Grude, which was the
most toxic sample at the two hour stage and still very
toxic at the four hour stage, was among the least toxi-
o fertilization. Bunker Fuel Oil and Waxy Crude Oil
were not very toxic as far as fertilization was concerned
but both were fairly toxic to cleavage. In contrast
the Bunker Oil K6 from the Union Oil Company seemed to
be highly toxic to both fertilization and to cleavage.
As was previously mentioned, the purple sea urchin
was chosen as the test organism because of its noted
sensitivity to pollutants in the marine environment.
This fact also explains why the urchin might not have
been the best animal to use. Due to the urchin's
sensitivity, it is difficult to determine gradations
of toxicity between samples, because most of the sample
appear to be highly toxic. The urchin, perhaps, would
e better used to detect subtle changes in toxicity
that occur in one particular sample over a period of
time due to weathering, or in very low dilutions.
In addition to investigating the initial toxicities
these petroleum products, it will be important to
letermine the toxic effects to urchins after exposure
to the oil over a considerable period of time. It may
well be that those fractions that do not appear to be
very toxic on an acute level are extremely toxic after
prolonged exposure. The chronic toxicities of residual
fractions are unknown. It would also be interesting
to test the oils on the larvae of these organisms
because they might be very sensitive to different adverse
environmental conditions than the gametes and embryos.
Summary
From these results the following conclusions
can be drawn:
1. Early cleavage in sea urchins is more sensitive to
the toxic effects of most petroleum products than
fertilization.
2. The crude oils and bunker oils have the most toxic
effects on cleavage, while the more refined diesel and
jet fuels have the least.
3. Many of the oils that were the most toxic to cleavage
Less
were the-least toxic to fertilization and vice versa.
4. Bunker Oil 46 from the Union Oil Company was very
toxic to both fertilization and cleavage.
Acknowledgements
I would like to thank the Standard Oil Company
of California, the Union Oil Company of California,
and the Petrolite Corporation for supplying me with
my oil samples, without which this project could not
have been undertaken.
I would also like to gratefully acknowledge the
advice and encouragement of my advisor, Dr. John S. Pearse,
who made himself available for consultation and
assistance at any time.
This work was done as an undergraduate research
project in Biology 175H at Hopkins Marine Station of
Stanford University.
Literature Citings
1. (Anderson, E., Mitchell, C., Jones, L., and North, W.
(1970) What Oil Does to Ecology. 'Journal of Water
Pollution Control Federation', 42:812.818.
2.
Blumer, M., Sass, J., Souza, G., Grassle, F., and
Hampson, G. (1970) The West Falmouth Oil Spill.
Woods Hole Oceanographic Institution. Unpublished
manuscript. Reerenc No. 10 -44
3. Blumer, M., Sanders, H., Grassle, J., and Hampson, G.
(1971) A Small Oil Spill. 'Environment', 13:2./2
Dean, R. (1968) The Chemistry of Crude Oils in
Relation to Their Spillage on the Sea. (In: The
Biological Effects of Oil Pollution on Littoral
communities, ed. by Carthy, J. and Arthur, D.)
pp. 1-0.
Nelson-Smith, A. (1970) The Problem of Oil Pollution
of the Sea. (In: Advances in Marine Biology, ed. by
Russell, F., and loung, M., 0:215-290.
6. North, W., Neushul, M., and Clendenning, K. (1965)
Successive Biological Changes in a Marine Cove
Exposed to a Large Spillage of Mineral Oil.do
Oo,'Symp. Poll..Mar. Micro-org. Prod. petrol,
pp. 355-354.
7. Shelton, R. (1971) Effects of Oil and Oil Dispersants.
on the Marine Environment. (In: Proceedings of the
Royal Society of London: A Discussion on Biological
Effects of Pollution in the Sea, ed. by Cole, H.) a.
177:7.
411-422
2
C
8. Smith, J. E., editor (1968) 'Torrey Canyon' Pollution and Marine
Life. Cambridge University Press. 196 pp.
9. Straughan, D. (1971) What has been the effect of the spill on the
ecology in the Santa Barbara Channel? Biological and Oceanographic
Survey of the Santa Barbara Channel Oil Spill 1969-1970. Vol. 1
Biology and Bacteriology. Alan Hancock Foundation, University of
Southern California. pp. 401-426
10. International Conference on Oil Pollution of the Sea. October 7-9
1968 at Rome. Report of Proceedings. 114 pp.
C
Table 1: % Non-fertilization
Ihe means and standard deviations of the sea urchin eggs
in different dilutions of various petroleum products. Differences
from the controls at the 5% level of uncertainty are to the
right of the dotted line.
Concentrations
Samples
100%
50%
25%
12.5%
6.25%
0%
Waxy Crude
315
32.5
.32.5
4-Corner Crude
1.32.9
.72.9
Bunker Fuel Oil
74.9
1.32.9
.71.9
7.32.9
Wilmington Calif
Crude
1.321.8
221.6
3.62.5
2.62.9
Bunker C Fuel Oil
(residual)
4.715.2
1.34.9
74.9
64.9
.61.9
Ook Inlet Alaska
Crude
4.642.4 4.621.8 4.621.8
2.621.4
341.8
211.6
Argentina Crude
32.9
521.8
5t2.4
211.6
321.8
Diesel Fuel +2
.74.9
644.3
Diesel Fuel +1
716.5
312.4
623.2
311.8
3.9
211.6
Jet Fuel JP-8
— — —
.7.9
8 2
Jet Fuel A-50
62.9
811.6
1.31.9
.71.9
Diesel Fuel +2
8.6.9
5.34.9
2.61.9
411.6
5.31.9
211.6
Central Libyan
Crude
1242.2 1223.7  8423.7
612.8
6.321.2 622.2
Distillate Heating
Oil +2
1012.4
3023.5 8424.3
6.3t1.2 821.6
1.6
Bunker Oil 16
—-
————
7614.5 9821.6
3412.8
1442.2
6.311.2 1222.9
Residual Heating
Oil 15
Table II: 2 Hr. % Non-cleavage
Ihe means and the standard deviations of the sea urchin embryos
in different dilutions of various petroleum products. Differences
from the control at the 5% level of uncertainty are to the
right of the dotted line.
Concentrations
Samples
25%
50%
12.5%
6.25%
0%
822
824
622
Jet Fuel JP-8
5.312.5
4925
73.329.2
Diesel Fuel 1
521
1226
6717
9511
2917
9.321.9
1223.3
Diesel Fuel +2
—
— ——
68.717.4
74.723.4
4215.9
Distillate Heating 8.321. 7
1527.3
Oil +2
8818.6
8327
2527
8317
5.312.5
Argentina Crude
6215.9
4614.3
51.323.4
3422
At Fuel A-50
91.32.9
87.326.2
5.312.5
6517
78.626.2
Bunker C Fuel Oil
(residual)
941.9
9214.8
2024
5412
5.322.5
Diesel Fuel +2
91.32.9
4812
7814
5.312.5
3315
Central Libyan
Crude
8113
6024
9624.3
4125
Waxy Crude
99.31.9
65.319.3 95.373.4
9821.6
8.311.7
Residual Heating
Oil 15
93.344.9
100
7612
7647.1
Bunker Fuel Oil
98.312.3
100
98
9.311.9
8625.6
Wilmongton Calif.
Crude
98
9.341.9 92.628.9 93.329.4 99.34.9
Cook Inlet Alaska
Crude
100
100
8.341.7 93.343.4 100
Bunker Oil 16
100
100
4-Corner Crude
99.3t.9 100
2
100%
1925
95.324.1
100
80.34.7
9021.6
8323
88.615.7
9711.8
9622.8
100
100
100
100
100
100
100
15
16
Table III: 4 Hr. 244 Cells
Ihe means and standard deviations of the sea urchin embryos
in different dilutions of various petroleum peoducts. Differences
from the control at the 5% level of uncertainty are to the
right of the dotted line.
for eac
licated
deviation
Concentrations
Samples

50%
100%
12.5%
25%
02
6.25%
9711
3222.5
3123
8747
2622.8
3222.8
JetFuel JP-8
—
—
9212.8
691
74.313.3
42.325.2
67.324.
45.726.3
Distillate Heating
Oil +2
954
8912.4
8626
8416
7824
Diesel Fuel +2
2612.8
95.312.5
92.71.9
93.314.1
83.612.6
6816.5
33.312.3
Diesel Fuel +2
8515.2
8813.3
912.9
8514
2612.8
8213.2
Argentina Crude
9211.6
93:2.5
88t2
8426
8612.8
25.313.4
Diesel Fuel 1
91.317.
89.345.2 9216.5
33.312.3 89.311.9 92.624.1
Wilmington Calif
Crude
9022
94:2
9212
8824
25.313.4 8814
Jet Fuel A-50
9025.9
9221.6
93.3t.8
33.312.3 93.324.1 89.321.9
Cook Inlet Alaska
Crude
98.6t1.
2642.8 81.325.2 8824.3 88.616.6 100
Central Libyan
Crude
91.345.2 90.744.9 98.721.9 100
25.343.4 8125
Waxy Crude

95.324.1 98.72.9 100
98.611.
42.345.2 8018
Residual Heating
Oil 15
26t2.8 91.342.5 9623.3 97.342.5 99.34.9 98
Bunker C Fuel Oil
(residual)
25.323.4 , 97.3t1.9 99.3t.9 99.32.9 99.34.9 100
4-Corner Crüde
99.32.9 100
25.343.4 97.31.9 99.32.9
100
Bunker Fuel Oil
100
100
100
42.325.2 9623.3 100
Bunker Oi1 46
Figure Captions
Figure I:
Ihis graph shows the mean percentages of non-cleavage
two hours after fertilization at the 6.25% concentration o
oil. The standard deviation is also indicated for each
sample. The samples are listed in order of increasing toxicity.
Figure II:
This graph shows the mean percentages of the cells at the four
cell stage or less four hours after fertilization. The
standard deviation is also indicated for each sample. The
samples are listed in order of increasing toxicity.
Control
Diesel Fuel /1
Fuel JP-8
Jet
Diesel Fuel 42
Distillate Heating
Oi1 42
Diesel
Fuel 42
Argentina Crude
Central Libyan
Crude
Jet Fuel A-50
Waxy Crude Oil
Bunker
C Fuel Oil
(residual)
Residual Heating
Oil 45
Bunker Fuel Oil
Wilmington Calif.
Crude
Cook Inlet
Alaska Crude
Bunker Oil 16
4-Corner Crude
Figure
.5


115
15
33
34
6.258 Concentration
Samples
u
65
65.3
P-Petrolite
U=Union Oil
76
86
92.6
1
050
) 9
LO
Control
Jet Fuel JP-8
Distillate Heating
Doil #2
Diesel Fuel +2
Diesel Fuel /2
Residual Heating
Oil 45
Waxy Crude Oil
Central Libyan
Crude
Argentina Crude
Diesel Fuel /1
Jet Fuel A-50
Wilmington Calif.
Crude
Bunker C Fuel Oil
(residual)
Cook Inlet Alaska
Crude
Bunker
Oil 46
4-Corner Crude
Bunker Fuel Oil
Figure II
6.25% Concentrations
Sample
4 hr. 844 cells
40
50.2

5.
P=Petrolite
U=Union Oil
Js
1e
80
l
181.3

ei

189.3
1o.
1.
6
1or3
y
20
80
100