A study of the toxicity of Kuwait Crude Oil
to the copepod Tigriopus californicus (Baker, 1912).
Robert Dorit
Hopkins Marine Station
Biology 175 H
Spring, 197
ABSTRACT
The toxicity of several preparations obtained from Kuwait Crude oil
to Tigriopus californicus was investigated. Whole oil, sea water extracted
whole oil, the seawater extract, and similar materials obtained from oil
weathered for 40 days were compared. The LDg's may be ranked as follows;
The most toxic preparation was the seawater extract, followed by Whole Kuwait
oil, washed Kuwait oil, weathered oil and washed weathered oil, in order
of decreasing toxicity. Males were found to be twice as susceptible as females
to the whole oil preparation. Assays conducted in an attempted simulation of
the tidepool environment revealed higher toxicities than were cbtained
under more traditional methods of assay.
INTRODUCTION
A considerable body of information dealing with both field and laboratory
toxicity of oil and petroleum products to marine organisms has been generated
in the last decade (see Moore, 1973). Most of this work has tended to focus
on economically important species such as oysters or clams. Until recently,
little was known about the impact of oil pollution on representative copepods.
Mironov (1968, 1969) examined the response of Black Sea copepods to crude
cil, and reports impact of 1.0 ppm of crude oil on the survival of copepods.
More recent work by Kontgiannis and Barnett (1973,1975) studied the toxicity
of crude oil and crude oil fractions on Tigriopus californicus, concluding
that effects were due to both the blocking of oxygen transport across the
air-water interface and to toxic materials present in the oil.
The present study attempts to examine the toxic effect of Kuwait Crude
oil on a high tidepool organism, Tigriopus californicus. Whole fresh oil as well
as weathered oil were compared for toxicity. Sea water soluble and less
sea water soluble fractions of these materials were also studied. An attempt
at a closer simulation of tidepool conditions in toxicity tests is presented,
as well as an investigation of differential susceptibility of males and females
in the copepod population.
METHODS AND MATERIALS

T. californicus used in this study were collected from a high tidepool
located at Mussel Point in Pacific Grove, California. Animals were kept
in two large bowls of filtered seawater at room temperature. Water was
changed every two days. The diet supplied to the animals was Tetramin fish
food (Telia Werke, West Germany). Prior to any experiment, a rundown sample
of T. californicus was taken from the aquarium and placed under a dissecting
scope for removal of copulating pairs and larvae, along with dead and moribund
specimens. Ten animals were subjected to each dose of the test materials.
The fractions of oil used in this study are all derived from an original
one gallon sample of American Petroleum Institute Reference Kuwait Crude,
referred to in this paper as Whole Kuwait. This sample was generously pro¬
vided by Dr. John Martin of Moss Landing Marine Laboratory in Moss Landing,
California.
The Washed Kuwait fraction was prepared by mixing one part of Whole
Kuwait with three parts of fresh filtered seawater. The mixing was done
manually in a separating funnel by shaking vigorously for five minutes.
Separation was then allowed to proceed for approximately 90 minutes before
water was drained from the funnel. The process was repeated five times with
fresh filtered seawater. The water extracted after the fifth wash showed
no toxicity to Tigriopus c. over a 96 hour test period.
The soluble fraction of Kuwait Crude oil was obtained by extracting
one part of oil with two parts of seawater in a separating funnel, mixed
vigorously by hand for five minutes. The mixture was allowed to stand for
two hours and was then mixed again. This process was repeated a third time,
The mixture was then allowed to separate for approximately 12 hours before
the water was removed and filtered through Whatman l filter paper (Whatman
Manufacturers, England) to remove any microparticles of oil that might have
remained in suspension. This soluble fraction was tested immediately after
preparation.
The weathered oil was prepared by placing 100 ml. of Whole Kuwait cil
with 400 ml. of seawater in a large beaker that was then placed outdoors,
in full sunlight for 40 days. The residue was more viscous than the original
oil. Water was removed from the beaker as completely as was possible.
The washed weathered oil was obtained using the washing procedure
described above.
With the exception of the soluble fraction (administered by volume).
all other fractions were administered by weight and carefully placed on
the surface of the sea water containing the test animals. All tests were
carried out in 30 ml. glass stoppered bottles, with a final water volume
of 25 ml.
Animals were examined at regular intervals to record the number of deaths,
Any animal that did not respond to shaking was considered dead.
Oxygen determinations were carried out using the Winkler technique
(Strickland and Parsons, 1965).
The results of toxicity titrationswere analyzed using the approximate
probit method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
This method provides a factor f LD.. The product and quotient of this
factor with the LD yields 958 confidence limits for the LD5. Potency
ratios, P.R., may be obtained as a comparison between toxicants. The factor
Ipg must be less than P.R. for there to be a significant difference between
EDgo's.
Generally, log-dose effect plots were drawn using the results of two
independent toxicity titrations.The assays were terminated when no further
mortality was noted.
In all cases, control mortalities over the prescribed time period,
h dawe wene zero
RESULTS
Figures 1,2,3,4 and 5 show the lethal effect of various fractions
for Tigriopus californicus. Dose is shown in grams/liter of water vs. 9
mortality. Table 1 summarizes the information obtained from the previous
plots. The LDgo's, along with their 95 confidence limits show the varying
toxicities of the different fractions. Table 1 shows that the different
fraction used in this study exhibit different toxicities for T. californicus.
Apparent differences in the toxicity of the fractions point to the necessity
of a careful definition of the history of the oil when describing its
toxicity.
The soluble fraction, a seawater extract of the most water soluble components
of Kuwait Crude oil shows the highest toxicity to the copepod T. californicus.
It should be mentioned that the volume of soluble fraction that was administered
to the copepods was translated into a weight dose by using Anderson's
estimate of the solubility of Kuwait Crude oil in seawater (Anderson, et al,
1974). Although the technique used here differs substantially from the one
described by Anderson, the figure of 21.68 ppm of oil in solution was
considered the best available estimate of the solubility of this oil in sea¬
water.
Where the toxicant formed a surface film, oxygen determinations were
carried out at the end of the assays, showing no noticeable decrease in the
dissolved oxygen in experimental vessels; except a decrease to 40% of
saturation at the high doses of washed oil. Oxygen levels of as little as
203 saturation have been found to have little effect in survival of T.
californicus (Fahey, 1977).
Table 2 compares the toxicities of several different fractions using
the potency ratio as a quantitative measure. The factor fpp and the 95%
confidence limits for Potency Ratios are also presented.
The potency ratio of Whole Kuwait vs. Washed Kuwait is a measure of
the important loss of toxicity that occurs upon removal of the seawater
soluble fraction. The weathering of crude oil results in an almost seven
fold reduction in its toxicity to T. californicus. The potency ratio of
Washed Kuwait vs. weathered oil indicates that weathering is the more
effective mechanism for the reduction of toxic constituents. Subsequent
washing of the weathered oil does not yield a significantly less toxic
material.
A comparison of the susceptibility of adult male and female T. californicus
to Whole Kuwait Crude oil yielded the results presented in figure 6. Females
with external egg masses were excluded from the female test population. The
LDgo for females was .195 grams/liter, with 95% confidence limits of .123 and
.309. The LDg for males was .092 grams/liter with 95 confidence limits of
.Obl and .139. A comparison provided a potency ratio of 2.12 and an fpp
of 1.84, indicating a significant difference in the susceptibility of the
sexes. Females with internal eggs in darkened ovaries were observed to
survive 2-3 days longer than females with external egg masses or females
with empty ovaries.
A closer simulation of high tidepool conditions was attempted by con¬
ducting the toxicity titrations in the presence of sand and naturally
occuring drift algae. Approximately 7.5 mm. of washed sand were used to
cover approximately 2 grams of fresh Macrocystis pyrifera in the bottcm of
assay bottles. Washed oil was added and the toxicity compared to that obtained
in assays lacking sand and algae. Figure 7 presents the results. The
toxicity was greater under the simulating conditions. Without sand and algae,
the LD was .405 grams/liter with 958 confidence limits of .252 and .650.
In the simulations the LD was .080 grams/liter with 958 confidence limits
A comparison gave a potency ratio of 5.06 and an fy
of .051 and .12
of 1.89, indicating a significant potency difference in the two conditions.
Oxygen levels in both controls and experimental bottles were less than 10%
of saturation when the assay was terminated. There were no mortalities in
the controls as the animals managed to obtain oxygen by hovering near the
surface. Although animals behaved similarly in the bottles containing
oil, the oil film may have inhibited respiration at the surface. The
degree to which this enhancement of oil toxicity is due to interference
with oxygen diffusion is difficult to evaluate. The aqueous phase removed
from the assays was tested and proved not to be toxic to T. californicus
after a period of aeration.
DISCUSSION
It is known that the most soluble components of crude oil are the
short chain alkanes and light aromatics (Anderson, et al, 1974) Consequently
it appears that these seawater soluble compounds are highly toxic to T.
californicus. The steeper slope of the dose effect curve of the soluble
fraction may be an indication of the acute toxicity of the preparation.
Immediate sublethal effects include a marked decrease in the activity of
T. californicus. The narcotic effect of crude oil, in particular of the
soluble fraction has been mentioned before (Goldacre, 1968). Other workers
have identified soluble material as the acutely toxic fraction of oil, with
benzenes and napthalenes being singled out as particularly toxic to marine
invertebrates (Anderson, 1974; Blumer, 1970; Boylan, 1971; Ottaway, 1971;
Shelton, 1971).
It is probable that the large dilution volumes present in the open
ocean, combined with the relatively low solubility of these hydrocarbon
compounds may result in negligible concentrations of soluble aromatics
other organics in the ocean. The situation in the high tidepool, however,
may be considerably more critical, given that the smaller volumes of water
available for dilution may enable the levels of solubilized hydrocarbons in
the water column to reach lethal concentrations.
Whole Kuwait also exhibited considerable toxicity to T. californicus.
This whole oil is the source of the soluble hydrocarbons accounting for the
toxicity of the soluble fraction. However, the rate of diffusion of
aromatics and short chain saturates from a surface layer of oil is probably
relatively slow and results in low concentrations of solubilized hydrocarbons.
The uptake by T. californicus of any of the soluble components may result
in further release of substances from the oil film.
Washed Kuwait, although treated in a manner that removes all or most
of the readily soluble materials from crude oil, nevertheless exhibits toxicity
to T. californicus and probably indicates residual toxic materials not
removed by the wash procedure.
In attempting to ascertain the reasons for the toxicity of Whole
weathered oil, it should be kept in mind that the same short chain alkanes
and light aromatics that are seawater soluble are the most volatile
hydrocarbons present (Lockwood, 1976). The 40 day weathering period left
a residue that has been altered by volatilization, dissolution, microbial
action and autooxidation (Moore,1973 ; Butler, 1974). The first two processes,
volatilization and dissolution,remove the fraction of oil with boiling points
below. 250° C, probably within a matter of hours (Moore, 1973). Further loss
of toxicity may result from the shortening of certain alkane chains by auto¬
oxidation or microbial action, and consequently increase solubility and
volatility.
In addition to the differential susceptibility of males and females to
Kuwait Crude oil, it was noted that among the females, those with darkened
ovaries and oviducts,indicating the presence of ripe eggs but preceeding the
laying of the eggs, survived as long as 2 or 3 days more than females with
external egg masses or empty ovaries. Perhaps at least some of the lipid
soluble toxicants are deposited in the developing eggs, resulting iu a
decrease in the internal effective concentration of these toxicants in the
particular female. An investigation of the viability of larval forms born
to these females should prove interesting as a subject for further investigation.
Coroner noted a decrease in clutch size in the copepod Eurytemora affinis
following exposure to oil (Coroner, 1975). Therefore, there may be further
effects on fertility.
The attempts at simulation of some tidepool conditions in toxicity
titrations illustrate some of the deficiencies of the usual laboratory assays
as measures of toxicity in the natural environment. Other effects, such as
a decrease in photosynthetic efficiency in tidepools where surface is coated
with oil may result in further deleterious effects.
Although a fresh spill occuring near the shore seems to pose the most
immediate threat to the high tidepool environment and its inhabitants, the
effect of washed, weathered oil on Tigriopus californicus remains far from
negligible.
ACKNOVLEDGEMENT
I would like to extend my appreciation to the faculty and students of
the 175H class at Hopkins Marine station, and in particular to Dr. John
Phillips whose editing and suggestions were enormously useful. Thanks also
to Moss Landing Marine Labs for the sample of Kuwait Crude oil. Finally,
a special word of thanks to Gillian Kendall, without whom this quarter would
not have been quite the same.
BIBLIOGRAPHY

harateristics of dispersions and water
Anderson, J.W., et al. (1974).
soluble extracts of crude and refined oils and their toxicity to
estuarine crustaceans and fish. Marine Biology, 27, 75-88.
C.J., Kontogiannis, J.E. (1975). The effect of crude oil fractions
Barnett.
on the survival of a tidepool copepod, T. californicus. Environmental
Pollution, 8, 45-54
M., et al. (1970). Hydrocarbon pollution of edible shellfish by oil
Blumer,
spill. Marine Biology, 5, 195-202.
D.B., Tripp, B.W.(1971). Determination of hydrocarbons in seawater
Boylan
extracts of crude oil and crude oil fractions, Nature, 230, 44-47.
Butler, M.J.A., et al. (1974). Biological aspects of oil pollution in the
marine environment - a review. MSC Report, 22A.
E.D.S. (1975). Fate of fossil fuel hydrocarbons in marine animals.
Coroner,
Proc. R. Soc. Lond., B. 189, 391-413.
(1977). Some adaptations to anaerobiasis in T. californicus. Biology
Fahey, M.,
175 H, Hopkins Marine Station.
Goldacre, R.J. (1968). The effects of detergents and oils on the cell
membrane, in The biological effects of oil pollution on littoral
communities, Carthy, 0.D. and Arthur, D.R., eds., Field Studies
Council. 131-138.
Kiontogiannis, J.E., Barnett, C.J. (1973). The effect of oil pollution
on survival of the tidal pool copepod, T. californicus. Environmental
Pollution, 4, 69-79.
Litchfield, J.T., Wilcoxon, F. (1949). A simplified method of evaluating
dose-effect experiments. J. of Pharmacology, 96, 99-113.
Lockwood, A.P.M. (1976). Effects of pollutants on aquatic organisms,
Cambridge University Press, London.
O.G. (1968) Hydrocarbon pollution of the sea and its influence
Mironoy
on microorganisms. Helgolander Wiss. Meersunters, 17, 335-339.
O.G., (1969). Effect of oil pollution upon some representatives
Mironoy,
of Black Sea zooplanktcn. Zoologicheskii Zhurnal, 48, 980-984.
Moore, S.F., et al. (1973). A preliminary assessment of the envirormental
vulnerability of Machias Bay, Maine to oil supertankers. Report
No. MITSG 73-6, Sea Grant.
S. (1971), The comparative toxicities of crude oils in The ecological
Ottaway,
effects of oil pollution on littoral communiries, E.B. Cowell, ed.
Institute of Petroleum, 172-180.
R.G.J. (1971). Effects of oil and oil dispersants on the marine
Shelton,
environment, Proc. Roy. Soc. Lond. B., 177, 411-422.
Strickland,J.p., Parsons, T.R. (1965). Handbook of seawater analysis
Fisheries research board of Canada, Bulletin No. 125.
Figure 1. Log Dose vs. Percent Mortality of Whole Kuwait Crude Oil,
Each point represents the percent mortality observed using 10 T. californicus.
Two separate assays were used in obtaining the line and are distinguished by
open and closed circles. The LD is 130 grams/liter with 958 confidence
limits of .092 and .184.
1o MORTALITY
991
95
90
80
70
60
50
40
30
20
10
5
--
0
.03 .04
A
.20
—
.50
900
kk -
.10
—
Figure 2. Log Dose vs. Percent Mortality of Washed Kuwait Crude Oil.
Each point represents the percent mortality observed using 10 T. call
fornicus.
Two separate assays were used in obtaining the line and are distinguished by
opened and closed circles. The LDg is .405 grams/liter with 958 confidence
limits of .252 and .650.
2

k k-
staatatakakava-

L
Figure 3. Log Dose vs. Percent Mortality of The Soluble Fraction.
Each point rep
ents
percent mortality observed using 10 T. californicus,
Two separate assays were used in obtaining the line and are distinguished
by open and closed circles. The LDg is .008 grams/liter with 953
confidence limits of .007 and.009.
1o MORTALITY
99
95
90
80
70
60
50
40
30
20
101
o0

. . ..
0
.003
—
.005
—
.02
—
.05
Figure 4. Log Dose vs. Percent Mortality of Weathered Oil. Each
point represents the percent mortality observed using 10 T. californicus.
Two separate assays were used in obtaining the line and are distinguished
by open and closed circles. The LD is.900 grams/liter with 95%
confidence limits of .550 and 1.434.
2

—

aataaaaaata-

S
Figure 5. Log Dose vs. Percent Mortality of Washed Weathered Oil.
Each point represents the percent mortality observed using 10 T. californicus.
Two separate assays were used in obtaining
the line and are distinguished by
open and closed circles. The LDg is 3.001 grams/liter with 958 confidence
limits of .911 and 9.886.
O
2

3
kataatatatatavaa-
1

S
L
TABLE 1
COMPARISON OF EDgO'S FOR VARIOUS FRACTIONS OF KUWAIT CRUDE OIL
f EDg
958 LIMITS
E0
1.1234
SOLUBLE
.0086 g/1
.0076 g/1
FRACTION
.0068 g/1
1.4178
.184 g/1
WHOLE
.130 g/1
.092 g/1
KUWAIT
1.6506
.650 g/1
WASHED
.405 g/1
.252 g/1
KUWAIT
1.6374
1.474 g/1
.900 g/1
WHOLE
.550 g/1
WEATHERED OIL
9.886 g/1
3.2945
3.001 g/1
WASHED
.911 g/1
WEATHERED OIL
TABLE 2
POTENCY RATIOS OF VARIOUS FRACTIONS OF KUWAIT CRUDE OIL
958 LIMIT
P.R.
5.545
WHOLE KUWAIT/
1.78
3.115
WASHED KUWAIT
1.750
6.923
12.599
WHOLE KUWAIT/
1.82
3.804
WEATHERED OIL
WASHED KUWAIT/
4.377
1.97
2.222
WEATHERED OIL
1.128
WEATHERED OIL/
NOT SIGNIFICANT TO P=.05
WASHED WEATHERED OIL
—
Figure 6. Comparison of the susceptibilities of male and female
adults of the species Tigriopus californicus to whole Kuwait Crude Oil.
Open and closed squares represent two separate assays performed on male
T. californicus and yield an LD of .092 grams/liter with 95% confidence
limits of .139 and .061. Open and closed squares represent two separate
toxicity assays using female T. californicus. LD for females is .195
grams/liter, with 95% confidence limits of .309 and .123.
a
2
ktaaaaataata-
Figure 7. Comparison of the Log Dose vs. Percent Mortality plots
of washed Kuwait Crude Oil to T. californicus as assayed in a simulated
tidepool condition containing algae and sand,and containing only seawater.
Open and closed circles represent two separate assays in only seawater.
Squares represent toxicity titration performed in a simulated tidepool
situation. The LD for washed Kuwait Oil assayed in seawater is.405
grams/liter, with 95% confidence limits of .650 and .252. The LD., obtained
in the tidepool simulation is .080 grams/liter, with 958 confidence limits of
.125 and .051.
7o MORTALITY
991
95
90f
804
701
60
50
40
30
20
10


6
0
a o

.03 .04
kt-
10
20
50
—
0