United States
Environmental Protection
Agency
Environmental Research
Laboratory
Narragansett Rl 02882
EPA-600/3-80-058a
July 1980
Research and Development
Fate and Effect of
Oil in the Aquatic
Environment
Gulf Coast Region
, EPA/600/3-80/058a
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and ()evelopmenl, U.S. t nvironrnental
Protection Agency have been grouped into nine1 S(;nc's. These nine broad cate-
gories were established to facilitate further development and application ol en
vironrnental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
I Environmental Health Llfeets Research
? Environmental Protection Technology
3 E.cological Research
A Environmental Monitoring
h. Socioeoonomio Environmental Studios
(i Scientific and technical Assessment Reports (STAR)
/. Intoragency Energy Environment Research and Development
H. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESFARCEI series. This series
describes research on the effects of pollution on humans, plant and animal spe
cies, and materials Problems are assessed for their long and short term influ-
ences. Investigations include formation, transport, and pathway studies to deter
mine the fate of pollutants and their effects. Phis work provides the technical basis
for setting standards to mmimi/e undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-80-058a
July 1980
FATE AND EFFECT OF OIL IN THE
AQUATIC ENVIRONMENT - GULF COAST REGION
by
Lewis R. Brown
Mississippi State University
Mississippi State, Mississippi 39762
Contract No. 68-01-0745
Project Officer
C. Stanford Hegre
Environmental Research Laboratory
Narragansett, Rhode Island 02882
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND 02882
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Narragansett, Rhode Island, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection Agency,
nor does mention of trade names or commercial products constitute endorsement
or recommendation for use.
ii
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FOREWORD
The Environmental Research Laboratory of the U.S. Environmental Pro-
tection Agency is located on the shore of Narragansett Bay, Rhode Island.
In order to assure the protection of marine resources, the laboratory is
charged with providing a scientifically sound basis for Agency decisions
on the environmental safety of various uses of marine systems. To a great
extent, this requires research on the tolerance of marine organisms and their
life stages as well as of ecosystems to many forms of pollution stress.
In addition, a knowledge of pollutant transport and fate is needed.
This report describes a multidiciplinary study of the distribution
and alterations of crude oil in marine systems of the coastal Gulf of
Mexico. Biological uptake of and response to spilled, mechanically dispersed,
and absorbed oil was followed. Studies include both laboratory and field
systems used to assess chronic effects and ecosystem recovery.
Tudor T. Davies
Director
iii
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ABSTRACT
The purpose of this research investigation was to determine the fate
and effect of crude oil in the aquatic environment of the coastal Gulf of
Mexico. The project was multi-disciplinary and multi-institutional in
scope and involved both laboratory and field sized pilot-plant ecosystem
studies. Emphasis was placed on the long-term, low-level chronic effects
of oil pollution on the ecosystem. Of the five crudes employed in the
investigation, Empire Mix crude was studied most intensively.
The following conclusions were drawn from the investigation.
After an oil spill, the oil only remains in the water column for a
short period of time (days) and migrates into the sediments where it' persists
for long periods of time (years). Surface oil which migrates into the
marshes will reduce marsh plant productivity by up to 88% and will reduce
the rate of conversion of the grass into utilizable detritus by up to 50%.
.vv*$r •.''•»;'/,
Oil in the marsh grass systems serves as a mechanism for reentry of .oll
into the estuarine food web.
Oil, even at levels which would result in a slick only 0.01 mm in
thickness, drastically reduces the zooplankton population (particularly
Acartia) but recovery of the population to pre-spill levels occurs within
10-15 days. The major initial impact of a spill of this magnitude on
higher members of the food chain (shrimp and mullet) is an alteration in
behavior which will make them susceptible to predation for the period of
time they are exposed to the oil. Physiological changes (as evidenced by
enzymological and fatty acid analyses) in these organisms are evident and
appear to be a stress response.
iv
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Long-term chronic effects of oil on the ecosystem probably will occur
but the severity of the impact will vary from oil to oil and the environmental
conditions. Uptake of oil immediately after a spill or constant exposure
to low levels of oil in the environment (sediments and detritus) has a
debilitating effect on the higher elements of the ecosystem which may
manifest itself in one or more of the following ways: increased disease
(fin rot), histological changes and decreased growth in fish; increased
disease in shrimp; and decreased growth and increased mortality in oysters.
This report was submitted in fulfillment of Contract No. 68-01-0745 by
Mississippi State University under the partial sponsorship of the U.S.
Environmental Protection Agency. This report covers a period from June 28,
1972, to February 29, 1976, and the work was completed as of April 15, 1976.
The University of Southern Mississippi and the Gulf Coast Research Laboratory
served as subcontractors to Mississippi State University on this Contract.
v
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TABLE OF CONTENTS
ABSTRACT iv
FIGURES x
ACKNOWLEDGMENTS xi
INTRODUCTION 1
CONCLUSIONS . . 4
RECOMMENDATIONS 6
MATERIALS AND METHODS 8
LABORATORY RESULTS 21
A. Fate Studies 21
1. Volatility 21
2. Fate in Water and Sediments 21
3. Fate of Empire Mix Crude Oil With or Without Aeration ... 23
4. Microbial Degradation of Oil 23
B. Phytoplankton 24
1. Toxicity Tests 24
a. 96-Hour Toxicity Tests- 24
b. 12-Day Toxicity Tests- 25
c. Chronic Toxicity Tests- 25
2. Bioaccumulation 25
C. Zooplankton 26
1. Toxicity Tests 26
D. Marsh Grass 27
1. Greenhouse Studies 27
2. Field Studies 28
vii
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E. Mullet 29
1. Acute Toxicity 29
2. Behavior 29
3. Enzymology 30
4. Fatty Acids 31
5. Oil Uptake 32
6. Histology 32
7. Laboratory Studies on Disease 33
F. Shrimp 34
1. Acute Toxicity 34
2. Behavior 35
3. Enzymology 35
4. Fatty Acids 37
5. Oil Uptake 38
6. Histology and Disease 38
a. Acute Studies- 38
b. Laboratory Feeding Experiments- 39
G. Oysters 39
1. Acute Toxicity 39
2. Behavior 40
3. Depuration 40
4. Enzymology 40
5. Fatty Acids 42
6. Oil Uptake 42
7. Histology and Disease 43
FIELD RESULTS 44
A. Tidal-Pond Study 44
1. Description of the System 44
2. Fate of Oil in Sediments 44
3. Phytoplankton/Zooplankton 46
4. Marsh Grass 47
5. Animals 47
B. First Pilot-Plant Ecosystem Study 47
1. Description of the System 47
2. Conduct of the Oil Spill 53
3. Key Events 55
4. Environmental Data 56
5. Fate Studies 56
6. Microorganisms 59
7. Phytoplankton/Zooplankton 59
a. Core Sampling- 60
8. Marsh Grass 60
viii
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9. Oil Uptake 61
a. Mullet- 61
b. Shrimp- 61
c. Oysters- 62
10. Enzymes 62
a. Mullet- 63
b. Shrimp- 66
c. Oysters- 66
11. Fatty Acids 67
a. Mullet- 67
b. Shrimp- 67
c. Oysters- 68
d. Conclusions- 68
12. Pathology 69
a. Mullet- 69
b. Shrimp- 69
c. Oysters- 69
13. Disease in Mullet 70
C. Second Pilot-Plant Ecosystem Study 70
1. Description of the System 70
2. Environmental Data 72
3. Fate Studies 72
a. Control Pond (Pond 2)- 73
b. Pond Treated with Saudi Arabian Crude Oil (Pond 3)- . . 73
c. Pond Treated with Nigerian Crude Oil (Pond 4)- 73
d. Pond Treated with Empire Mix Crude Oil (Pond 5)- .... 73
4. Phytoplankton/Zooplankton 74
a. Core Sampling- 75
5. Microorganisms 75
6. Primary Productivity of Phytoplankton Community 76
7. Marsh Grass . , 76
8. Mullet - Pathology 77
9. Shrimp - Pathology 78
10. Oysters - Pathology 78
SUMMARY 82
DISCUSSION 92
REFERENCES 99
APPENDIX Tabular and graphic data available separately—see
INTRODUCTION.
ix
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FIGURES
FIGURE 1.
FIGURE 2.
FIGURE 3.
FIGURE 4.
FIGURE 5.
FIGURE 6.
FIGURE 7.
Aerial photograph of the pilot-plant ecosystem located
at the National Science Technology Laboratory in Han-
cock County, Mississippi .
48
Ground level photograph taken midway in the development
of the pilot-plant ecosystem ponds used in this study.
At the time this picture was taken, only one pumping
system was functional and the marsh grass had not been
planted around the edges of the ponds
Photograph showing clumps of Juncus growing along the
edge of one experimental pond in the pilot-plant
ecosystem
Tidal-Simulation System (TSS) installed in Pump House
Photograph taken shortly after oil spillage in one of
the pilot-plant ecosystem ponds. Note ring of oil
near the three floats in the center of the pond . . .
Photographs of mullet with various degrees of fin ero-
sion. A and B—normal, C and D—very heavily eroded
fins
Photographs of whole and sectioned shrimp eyes.
A—normal eye; B—eye with white spot; C—photomicro-
graph of a normal section of eye; D—photomicrograph
with necrotic area visible just under corneal layer
49
51
52
54
71
79
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ACKNOWLEDGMENTS
This contract involved a multi-disciplinary, multi-institutional team
of scientists working in a coordinated program. The senior scientific
staff was responsible for the conduct of all experiments and the interpreta-
tion of the results. All of these individuals worked on the project for
the entire period of the contract and their contribution is hereby acknowl-
edged with gratitude. These scientists and their affiliation are Armando
A. de la Cruz, James R. Heitz, D. Howard Miles, David Wesley and James D.
Yarbrough from Mississippi State University; Billy J. Grantham and George F.
Pessoney, III, from the University of Southern Mississippi; and William J.
Demoran, Julia S. Lytle and Thomas D. Mcllwain from the Gulf Coast Research
Laboratory. The work of other professionals is gratefully acknowledged,
particularly the following individuals: Joe R. Broome, Beth Cade, Michael F.
Callahan, Jack Carrol, Janice E. Chambers, Mary J. Coign, Robert C. Giles,
Ann Heitz, Richard A. Johnson, Cornell M. Ladner, Lancelot A. Lewis, Wendell
Lorio, Fred McCorkle, C. Douglas Minchew, Georgia Monnerat, Syed M. Nagvi,
Shirley Randle, Bettaiya Rajana, Steve Reed and Brenda Schumpert from
Mississippi State University; Ben T. Barr, George D. Fain, Kennard A. Grimes,
John J. Johnson and Eugene P. Leftwick from the University of Southern
Mississippi; and Jenny Christmas, Kay McGraw, Larry Nicholson and Sharon
Walker from the Gulf Coast Research Laboratory.
The work of Graham Wells of the Mechanical Engineering Department at
Mississippi State University and Richard A. Johnson of Johnson and Associates,
Gulfport, Mississippi, in designing and constructing the tidal simulation
system is gratefully acknowledged. Sincere appreciation is given to
Dr. C. D. Minchew and Mrs. Carol Sprayberry for their assistance in the
preparation of this document.
XI
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SECTION I
INTRODUCTION
The cause and effect relationship between oil pollution and its acute
effects on the aquatic ecosystem is easily established because of the brief
time span between the impact of the oil and the observed effect. To the
contrary, the chronic effects of oil on the ecosystem are more difficult to
establish, because the onset of observable effects may require months or
years.
Most reports on the effects of oil pollution involve either studies
conducted in the environment after major oil spills or laboratory studies
on individual members of the ecosystem. Both of these types of studies
have shortcomings in terms of estimating the true impact of oil on the
aquatic environment. In the first case, lack of adequate pre-spill data,
use of other chemicals in clean-up procedures, inability to obtain representa-
tive samples, etc., seriously limit the value of data regarding identifica-
tion of chronic effects or establishing the effects of low-level oil pollution.
In laboratory studies there is always the question of the validity of
extrapolating the results to the natural environment because of the complexi-
ties of the system.
The use of a pilot-plant ecosystem seems to be one of the ways of
obtaining meaningful data on the chronic effects of low-level oil pollution.
It affords an opportunity to verify laboratory data on a manageable field
scale, and the results should be more applicable to the natural environment.
It is obvious that a study involving the total biota of the ecosystem
would be economically impossible and scientifically unmanageable. Similarly,
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since crude oils vary widely in composition, extensive testing with all
types of oil would be economically impractical. Therefore, certain decisions
had to be made at the outset in regard to which crude oil should be employed
and which members of the ecosystem would be investigated. As a result of
these considerations, the following decisions regarding the proposed study
were made at the outset. A majority of this investigation was performed
with Empire Mix crude oil, since it typifies the oil produced in the Gulf
Coastal area and thus would be typical of the oil spilled during a drilling
accident (the most prevalent type of oil spill).
The salt marsh estuarine ecosystem is essentially a detritus-based
system. Of the three groups of primary producers in the system, the bulk
of the organic matter biomass is contributed by marsh plants. Phytoplankton
and diatoms also are important primary producers and thus were included in
this investigation. Since zooplankters represent the first link in the
food chain above the primary producers, they likewise were investigated in
the study.
Mullet are considered to be omnivorous filter feeders but, to a large
extent, are herbivorous and were included since they might readily reflect
changes in the lower members of the food chain.
The economically important oyster was selected as a representative of
sessile, inshore benthic animals.
Shrimp are one of the most valuable fisheries resource in the Gulf
Coast area, and their food habits and dependence upon the estuaries make
them an excellent choice for detecting subtle changes in the estuarine
environment.
This report is a summary of the results of a three and one-half year
study of the fate and effect of oil on the aquatic environment of the Gulf
Coast Region. Phase I involved laboratory studies on selected members of
the ecosystem and the establishment of an estuarine pilot-plant ecosystem.
Phase II consisted of (1) following the results of a heavy spill using
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Empire Mix crude oil in a naturally-occuring estuarine tidal pond, (2) moni-
toring the results of a low-level spill using Empire Mix crude oil in the
estuarine pilot-plant ecosystem for a period of eleven months under conditions
simulating tidal action and (3) following the effects of three different
crude oils (Empire Mix, Saudi Arabian, and Nigerian) on separate estuarine
pilot-plant ecosystems over a period of nine months in the absence of tidal
action.
All graphic and tabular data (Appendix ) are available in hard copy or
microfiche from NTIS only, National Technical Information Service, 5285 Port
Royal Road, Springfield, Virginia 22161.
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SECTION II
CONCLUSIONS
The use of a tidal-simulation pond system as a mini-ecosystem seems
eminently sound as a complement to laboratory studies. The system afforded
an opportunity to study a partially reconstructed ecosystem under conditions
more closely resembling the natural environment than those achieved in the
laboratory and yet the system is manageable while the natural environment is
not.
Based upon two pilot-plant ecosystem studies, the following conclusions
were drawn in regard to the fate and effect of oil in the aquatic environment
of the Gulf Coast region.
After an oil spill, the oil remains in the water column for a short
period of time (days) and then migrates into the sediments where it persists
for long periods of time (years). Surface oil which migrates into the
marshes will reduce marsh plant productivity by up to 88% and will reduce
the rate of conversion of the grass into utilizable detritus by up to 50%.
Oil in the marsh grass systems serves as a mechanism for reentry of oil
into the estuarine food web.
Oil, even at levels which would result in a film only 0.01 mm in
thickness, drastically reduces the zooplankton population (particularly
Acartia) but recovery of the population to pre-spill levels occurs within
10-15 days. The major initial impact of a spill of this magnitude on
finfish and Crustacea is an alteration in behavior which will make them
susceptible to predation for the period of time they are exposed to the
oil. Physiological changes (as evidenced by enzymological and fatty acid
analyses) in these organisms are evident and appear to be a stress response.
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Long-term chronic effects of crude oil on the estuarine ecosystem
would be expected to occur on the basis of our investigation. The severity
of the impact would vary with the specific oil employed and the environmental
conditions existing at the time. Uptake of oil immediately after a spill
or constant exposure to low levels of oil in the environment (sediments and
detritus) was found to have a debilitating effect on the fish, shellfish
and Crustacea which manifested itself in one or more of the following ways:
increased disease (fin rot), histological changes and decreased growth in
fish; increased disease in shrimp, and decreased growth and increased
mortality in oysters.
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SECTION III
RECOMMENDATIONS
The results obtained in this investigation clearly indicate the desira-
bility of conducting some additional pilot-plant ecosystem studies directed
toward answering certain key questions regarding the effect of oil pollution
on the estuarine ecosystem. The studies should be mission-oriented in that
the information obtained would be utilizeable by management in making
decisions on such matters as (1) selecting the clean-up method for oil
spills which would have the least damaging effect on the ecosystem, (2) deter-
mining if and what environmental reclammation procedures could be employed
in polluted areas and (3) more accurately assessing damages in oil spill
cases.
One study should be directed toward determining whether the chronic
effects of oil on shrimp, mullet and oysters were the result of the initial
exposure to the oil or the constant exposure to oil and oil degradation
products in the environment (sediment and detritus). One experiment would
be designed to subject specimens to low levels (as employed in the present
investigation) of oil for two to four days and periodically monitoring them
in a clean environment for a period of one to two years. In a companion
experiment, the system should be subjected to the same level of oil for
seven to fourteen days prior to the introduction of the test shrimp, mullet
and oysters.
A second pilot-plant ecosystem study should be conducted in a system
expanded to include one or more predator fish (e»g_« speckled trout), one or
more scavengers (e.g., crabs) and an increased quantity of marsh grasses.
These studies should extend over at least two years and should include a
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comparison between (1) oil allowed to remain on the surface as long as
possible and (2) oil entrained into the water column through mechanical
means (e_. g_., increased turbulence) .
Some additional laboratory studies would also seem to be justified.
Specifically, the following areas should be addressed: (1) the mixed
function oxidases in mullet liver as they relate to oil metabolism, (2) the
role of oil in fin rot disease in fish and (3) white eye syndrome in shrimp.
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SECTION IV
MATERIALS AND METHODS
MATERIALS
Crude Oils
The Empire Mix crude oil employed for a majority of the work performed
under this contract was kindly supplied by the Standard Oil Co., Pascagoula
Refinery, Pascagoula, Mississippi. Representative analyses of the oil also
were supplied by the Refinery. [l]*
The other crude oils employed in this investigation were kindly supplied
by Exxon Co. from the following sources: Saudi Arabian crude oil from the
Baytown, Texas, Refinery; Venezuelan (La Rosa) crude oil and Nigerian (LT)
crude oil from the Exxon Refinery at Bayonne, New Jersey; and the Iranian
(heavy crude) crude oil from the Exxon Refinery at Benicia, California.
Collection and Treatment of Biological Specimens
Mullet, Shrimp and Oysters-
All specimens of mullet (Mugil cephalus), shrimp (Penaeus aztecus) and
oysters (Crassostrea. virginica) were collected from the Mississippi Gulf
*Numbers in brackets at the end of a paragraph refer to items in Appendix
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Coast area. For laboratory studies, the organisms were held for at least 7
cays before being employed in bioassays.
Phytoplankton-
Phytoplankton were collected from the Mississippi Sound in a 200 mesh
plankton net (#25 Turtox), using a boat speed of 5 knots or less and a
towing time of 3 to 5 min. An aliquot, preserved in 10% formalin, was
counted under standard procedures (strip-count method) for both numbers and
species diversity.
Living phytoplankton samples were refrigerated, returned to the labora-
tory and placed in enrichment media (Heinle or N.H.). Single cells of
representative organisms were obtained from the enrichment cultures, washed
in sterile sea water and placed in test tubes of Heinle or the N.H. medium.
These cultures were grown and maintained as stock cultures and transferred
every 12 days.
As a rule the salinity in the collection areas range from 20-28 o/oo.
Zooplankton-
Zooplankton samples were collected in a 125 mesh net (#20 Turtox),
using a boat speed of 5 knots or less and a tow time of 2 min or less.
Samples were preserved in 10% formalin and counted by placing concentrated
samples in a petri dish and observing them with a stereo microscope.
Living samples were maintained at the collection temperature in 19-
liter oxygenated plastic sacks, placed in a styrofoam ice chest and trans-
ported to the laboratory. Cultures were maintained in 20-40 liter aquaria
at ambient temperature. Individuals were withdrawn from the aquaria with
glass tubes (at least 6 mm I.D.) and subjected to a variety of conditions
for growth and maintenance.
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Marsh Plants-
All marsh plants were collected from the Mississippi Coastal areas
(particularly the Bay St. Louis area and the Gulf Coast Research Laboratory
area). Plants for laboratory analyses were clipped at the base about 5 cm
from the surface of the mud, thoroughly cleaned of debris and silt and
air dried. Plants for transplanting were dug in clumps of about 60 cm
diameter and 60-90 cm deep to collect the rhizomes and roots.
ANALYTICAL METHODS
Oil in Water by Infrared Analysis
Five g of NaCl, 2.5 ml of 1:1 (v/v) sulfuric acid, and 25 ml CC1, were
added to 1 liter of water in a separatory funnel. The sample was shaken vigor-
ously for 1 tnin, the phases allowed to separate and the CC1, extract passed
through a column containing a 2.54 cm layer of anhydrous Na-SO, (CC1. washed).
This procedure was repeated four times and the extracts combined. Oil was
measured at an absorption band of 2930 cm using a 10 mm path length cell and
compared to a standard CCl, solution containing a known amount of oil.
.Oil in Water by Gas Chromatography
An 800 ml sample of water was placed in a 1 liter separatory funnel and
extracted 3 times with 100 ml of hexane and treated as described by
Miles et al. (1975). Analyses were conducted using a Beckman GC-45 gas chroma-
tograph with a flame ionization detector with a 183 cm x 0.32 cm OD stainless
steel column packed with 3% SE-30 on 80-100 mesh Chromasorb W. The injector
and detector temperatures were 300 C and the column temperature was programmed
from 100 to 300 C at a rate of 3 C per min. Qualitative identification
of the components was achieved by comparison to retention times of known
standards. The quantitative measurement of the total oil in the sample
was calculated from the area of the _n-C1 , peak.
10
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Oil in Sediments by Gas Chromatography
The gas chromatographic procedure for determining oil in sediments was
carried out in accord with the method described by Lytle (1975).
Oil in Sediments by Liquid Chromatography
The sediment sample (100 g wet wt) was soaked in 100 ml of MeOH (for heavy
mud samples) or in 100 ml of hexane and MeOH (90 ml:10 ml) (for samples com-
posed largely of shells or rocks). The sample was filtered through a
Buchner funnel containing Whatman #43 filter paper, washed with 100 ml
t
of hexane and both extracts combined and evaporated in vacuo to approximately
1 ml. This concentrated extract was transferred to a small vial,
evaporated under nitrogen, dissolved in 10 pi of hexane and analyzed
using a Waters Associates Model 202/401 liquid chromatograph with a UV
detector (wavelength, 277 nm). A 2.54 x 0.63 cm OD micro Bondapak
Cno/corasil column was employed with a MeOH:H00 (70 ml:30 ml) solvent
J.O <£
system at a flow rate of 2.0 ml/min.
Qualitative identification of the components was achieved by comparing
the retention times of oil components with known standards.
Estimation of total oil was made by integrating the total area for all
peaks obtained by analysis of the aromatic hydrocarbon fraction from the
control and test organisms. Integration was performed with a 3388 Hewlett-
Packard automatic integrator. Corrections for control absorbance were
made. Results were calculated as yg oil by comparison to standards.
11
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Oil in Mullet. Shrimp and Oysters
The samples were homogenized in TenBroeck Tissue grinder s at 10% (w/v)
in 0.2 M sodium phosphate and 0.25 M sucrose. For every gram of tissue, 10
ml of 0.75 M KOH in 2% aqueous methanol solution was added, and the resulting
mixture was refluxed for 15 hrs. The saponified samples were extracted
4 times with hexane (the amount of hexane used for each extraction was
1/4 to 1/3 the amount of the volume of total saponified solution). The
hexane extract was washed with water, dried with sodium sulfate and evaporated
in vacuo. The material was washed onto a chromatographic column (with a
70-100-vi-fritted disc packed with 4.5 g 60-200 mesh Activity I silica gel and
topped with 2.5 g 80-200 mesh Activity I neutral alumina) with 5 ml of
hexane and eluted with 75 ml of hexane followed by 85 ml of benzene. Each
fraction was evaporated in vacuo to 1 ml and transferred to a small vial,
where the remaining solvent was removed under nitrogen. The hexane eluate
was employed for analyses of aliphatic compounds as described in the section
on Oil in Water by Gas Chromatography. The benzene fraction was employed
for analyses of aromatic compounds as described by Miles et al., (1975) or
as described in the section on Oil in Sediments by Liquid Chromatography.
Oil in Marsh Plants
The marsh grass samples were pre-washed with soap and water and wiped
with a hexane-soaked cloth. Five grams of the finely ground material were
soaked over night in 50 ml of hexane, filtered and evaporated in vacuo.
The samples were then treated in the same manner as the animal samples
(beginning with the addition of KOH).
Determination of Dissolved Oxygen
Both the Azide Modification of the Winkler Method ("Standard Methods
for the Examination of Water and Wastewater", 13th Ed.) and a YSI Model 54
Oxygen Meter were employed for determining dissolved oxygen.
12
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Fatty Acids in Mullet, Shrimp and Oysters
After saponification of the tissue as described in the section on
Oil in Mullet, Shrimp and Oysters, the water layer containing the fatty
acids was acidified with 6 N HC1 to about pH 3. The water layer was extracted
three times with 30 ml CHC1» and the extract rotovaped to dryness. The
sides of the flask were rinsed with a small amount of MeOH and diazomethane
added until the reaction was complete, then rotovaped to dryness in a
roundbottom flask. Thirty mis of water were added to the flask, the
contents transferred to a separatory funnel and the water layer extracted 4
times with 20-30 mis of hexane each time. Each extraction was drained
through Na-SQ, into another roundbottom flask and rotovaped to 1-2 mis
volume. The extracted fatty acid methyl esters (FAME) were run through 8-
18 cm of silica gel column with 45-85 mis of benzene and collected in a
flask. The benzene was rotovaped down to 1 ml and transferred to small
.weighed vials. The sides of the flask were washed down with ether and
added to the sample in the vials. The solvent was evaporated under nitrogen
leaving the purified FAME. The sample was placed in a desiccator for at
least 1 hr and reweighed. The purified FAME were stored under nitrogen in
the refrigerator until analyzed by gas chromatography.
The purified samples were diluted with pesticide grade hexane, the
ratio being 1 mg sample to 10 ml solvent. Approximately 1 yl was injected
through the rubber septum into the vaporization chamber of the inlet with a
Hamilton 701 RN syringe. The solvent flush technique of Kruppa (1971)
proved to be the most successful method of sample introduction.
All FAME were analyzed with a MT-220 GLC equipped with a hydrogen
flame ionization detector. The columns used were 3.17 cm by 1.83 m aluminum
or stainless steel packed with 15-20% diethylene glycol succinate (DECS)
supported on 80/100 mesh chromasorb WAW. Operating conditions were as
follows: Carrier flow (N2), 22 cc/min; Column temperature, 190 C; Detector
temperature, 225 C; Inlet temperature, 200 C; Hydrogen flow rate, 50 cc/min;
and Air flow rate, 34 liters/min.
13
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Qualitative Identification of Peaks-
Purified FAME standards from the Hormel Institute were compared with
the retention times of the FAME for identification of individual peaks on
the chromatogram. The fatty acid methyl ester standards were: 14:0, 15:0,
16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:3, 19:0, 20:0, 20:4, 20:5 and 22:6.
Identification of some unsaturated and saturated fatty acids was
accomplished by plotting the log of the retention times versus the number
of carbons in the chain. This procedure allowed the tentative establishment
of the number of carbons and the number of double bonds for the fatty acids
contributing peaks to the chromatographic record.
Further identification of the unsaturated fatty acids was obtained by
hydrogenation. Hydrogenation converted the unsaturated acids to saturated
acids, and thus the chain length of the various acids was confirmed.
Hydrogenation of the unsaturated fatty acids was accomplished using Adam's
platinum oxide catalyst. "Spiking" of the sample served to identify posi-
tively such fatty acids as 14:0 and 18:3.
The method of Carroll (1961) was used for the quantitative estimation
of peak areas in the fatty acid spectrum. This method involves the multipli-
cation of the peak height by the retention time of each fatty acid. The
area under the peak represents the relative amount of that component in the
mixture.
Histological Examination
Immediately upon collection, specimens were placed in 10% formalin
solution for preservation.
Shrimp, mullet and oyster tissues were fixed in either buffered neutral
formalin or Bouin's or Davidson's fixatives. After a minimum of 24 hrs in
fixative, the tissues were processed in an Autotechnicon Mono tissue
processor and embedded in paraffin. Four to six micron thick sections were
then stained with hematoxylin eosin for routine histological study.
14
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Microbiological Methods
Routine Counting Procedures-
Media and methods employed in the microbiological monitoring programs
were as follows. All plate counts were conducted using the conventional
spread plate technique and media containing 17.13 g/1 Rila Marine Mix.
Total bacterial counts were made using nutrient agar (2% agar, w/v) with
incubation for 2 and 7 days. Total yeast counts were made using potato
dextrose agar (2%, pH = 3.5) with incubation for 5 days. Total fungal
counts were made using Cooke's Rose Bengal Agar (pH 7.0) with incubation
for 5 days (observations for actinomycetes were made after: 10 days).
Estimates of hydrocarbon-utilizing, nitrate-reducing microorganisms
and hydrocarbon-utilizing, sulfate-reducing microorganisms were conducted
using a modification of the method described by Rosenfeld (1960) wherein
Empire Mix crude oil was employed as the carbon source and the 3-tube MPN
technique employed. Incubation was for 10 and 21 days.
Microbiological Degradation of Oil-
These tests were conducted in 14-liter fermentor jars containing 10
liters of the following medium: 40.00 g of (NH,)2SO,, 5.06 g of KH2PO,,
14.94 g of K2HP04, 171.30 g of Rila Salt mix and 10 liters of distilled
water.
After solubilization of the salts, the medium was filtered to remove
insoluble precipitates. Fifty grams of Empire Mix crude oil was then added
and incubation carried out at room temperature on either a Microferm Labora-
tory Fermentor (Model No. MF-114) or a Model No. FS-614 (both from New
Brunswick Scientific Co). Agitation was set at 200 rpm's and aeration at
10 psi of air.
Ten grams of mud (from the Bay St. Louis area of Mississippi) and 300
ml physiological saline were blended at low speed for three minutes, allowed
to settle and 100 ml of supernatant withdrawn for use as an inoculum.
Samples were withdrawn as desired for analyses.
15
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Bioassay Procedures
Preparation of Oil-
The crude oil was emulsified using an ultrasonic dismembrator at 70
watts for 3 min. The oil-water emulsion was then introduced into the test
aquaria.
Mullet, Shrimp and Oysters in 114-liter Aquaria-
All glass aquaria (114 liters) were filled with artificial sea water
(Rila Mix), salinity of 15 o/oo. Aeration was accomplished using a subsurface
Dynaflow® underwater filtering system in which there was continuing aeration
through the Dynaflow® filter. The temperature was held constant at 20 C.
Water samples were taken routinely for dissolved oxygen and oil determination.
In general 6-8 animals were placed in each aquarium and the emulsified
oil preparation mixed into the test aquarium. Animals were placed in the
aquaria 24 hr before testing.
Mullet, Shrimp and Oysters in 1,895-liter Tanks-
Many of the laboratory tests concerned with the effects of low levels
of oil over a prolonged period of time were conducted in 1,895-liter tanks.
A 1,895-liter tank was filled with artificial sea water (15 o/oo) and two
pumps used for circulation and filtration of the water. The pump circulated
the water at 0.7 revolution/min or 947.5 liters/hr. Water samples were
taken routinely for dissolved oxygen and oil determinations.
Phytoplankton-
Both Heinle medium and N.H. medium were employed routinely. Heinle
medium has been found to be superior for those organisms requiring salinities
of less than 20 o/oo while N.H. medium was employed for the higher salinities.
Standard growth conditions for toxicity tests consisted of a 12-12 hr
light-dark cycle, 20 C, pH 8.0-8.4 and salinities of 20-28 o/oo. Culture
vessels were 16 by 150 mm test tubes or 125 ml Erlenmeyer flasks for tests
involving emulsified oil and 125 ml Erlenmeyer flasks and aquaria for tests
employing oily sediments. Appropriate dilutions of emulsified oil in the
medium were dispensed in 10 ml amounts in the test tube and 25 ml amounts
16
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in the flasks, inoculated and incubated. Sterility, if desired, was effected
by autoclaving. For tests with oily sediments, enough of the oily sediments
were added to the system to saturate the aqueous phase. Cell counts were
made employing 1 ml aliquots and a Sedgewich-Rafter counting cell (strip-
count method).
Zooplankton-
Five copepods were obtained from an aquarium (after acclimation) and
placed in 50 ml of filtered natural aerated sea water containing various
dilutions of the oil. Standard growth conditions for toxicity tests con-
sisted of a 12-12 hour light-dark cycle and a temperature of 20 C. Usually
the pH of the medium was in the range of 8.0-8.4 and the salinity in the
range of 15-28 o/oo. The culture vessels employed were 150 ml beakers.
Observations for mortality and natality were made using either a hand lens
or a stereo microscope.
Other Methods
Mass Culture Techniques for Phytoplankton-
Heinle medium and/or N.H. medium were employed routinely. Standard
growth conditions consisted of a 12-12 hour light-dark cycle, 20 C, pH 8.0-
8.4, 20-28 o/oo salinity and a light intensity of approximately 2152 meter
candles. The culture vessels were 4 or 9 liter inverted serum bottles aerated
with filtered air. Length of incubation varied with the culture and ranged
from approximately 4 days to 12 days. After incubation, 3.25 liters of the
culture were withdrawn for use as a copepod food or for chemical analyses.
Three liters of sterile medium were added to the culture and incubation
repeated. If contamination were found, the culture was discarded.
Core Sample Culturing Techniques-
Cores were taken from all four ponds from the same area of each pond
at two week intervals. Because the depth of each sample was difficult to
regulate, 17 g of material were removed 8 cm from the top (soil-water
interface) rather than from the "top" and "bottom" of the sample. Soil (17
g) was mixed with 20 ml BUM and agitated. Two ml of the mixture was placed
on an agar plate and spread with a sterile glass rod. BBM agar and Heinle's
17
-------
agar at salinities of 3 o/oo and 20 o/oo were employed. All plating was
done in duplicate or triplicate. Two ml of the mixture was added to 75 ml
BBM liquid culture and 75 ml Heinle's media @ 2 o/oo and cultured for two
wks. The colonies developing on the plates were counted and identified.
The algae in the liquid culture were identified.
Enzymological Analyses
Preparation of Samples-
Brain, gill, liver and muscle of mullet and hepatopancreas and muscle of
shrimp were homogenized in TenBroeck tissue grinders at 10% (w/v) in 0.1 M
sodium phosphate + 0.32 M sucrose buffer (pH 7.6). Homogenate was centrifuged
at 800 x g for 10 min at 0 C. The pellet was discarded. The supernatant
of this centrifugation was designated as the "homogenate fraction". The
supernatant of this centrifugation was spun at 8,000 x g for 15 min at 0 C.
The pellet was the "mitochondrial fraction" and was resuspended to make a
10% equivalent. The supernatant was saved and was subsequently referred to
as "supernatant fraction". The above centrifugations were performed on an
International Refrigerated Centrifuge, Model B20A with a type 873 rotor.
For preparation of sample for NADPH-cytochrome £ reductase assay, the
above supernatant fraction was centrifuged at 110,000 x g for 60 min at 0
C. The pellet was suspended in 5 ml of 0.15 M KC1 and centrifuged at
110,000 x g for 30 min at 0 C. The pellet was resuspended in 0.05 M potassium
-4
phosphate buffer in 10 M EDTA (pH 7.7) at a 40% equivalent. This was the
"microsomal fraction". These centrifugations were performed in a Beckman
Ultracentrifuge, Model L2-50, Type 50 rotor.
Oysters were removed from the shells, blotted dry and the whole organism
was used. The oyster was ground initially in a Sorvall Omni-Mixer at 50%
(w/v) in 0.1 M sodium phosphate + 0.32 M sucrose buffer (pH 7.6). This
mixture was then homogenized in a TenBroeck tissue grinder. A portion of
this homogenate was diluted to 10% with the same buffer and subjected to
the centrifugation procedures described above. Another portion of the
homogenate was diluted to 25% with the same buffer and sonified at 35 watts
for 0.5 min with a Sonifier® Cell Disrupter, Model W 185, micro-tip (Heat
18
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Systems - Ultrasonics, Inc.). This homogenate was then centrifuged at
110,000 x g for 30 min in a Beckman Ultracentrifuge, Model L2-50, Type 50
rotor, at 0 C. The supernatant was used and was referred to as the "sonified
soluble fraction."
For microsomal mixed function oxidases in mullet liver, the liver was
excised and homogenized in a TenBroeck tissue grinder in cold 0.15 M KC1.
Homogenates were centrifuged at 9000 x g for 15 min in an International
Refrigerated Centrifuge, Model B20A with a type 873 rotor. The supernatant
was then centrifuged at 110,000 x g for 60 min in a Beckman Ultracentrifuge,
Model L2-50, type 50 rotor. The pellet was resuspended in 0.15 M KC1 and
again centrifuged at 110,000 x g for '60 min. The pellet was resuspended at
500 mg equivalents/ml 0.15 M KC1. This microsomal suspension was used in
all subsequent microsomal oxidase assays.
Enzyme Analyses-
All enzyme specific activities were expressed as mU/mg protein, where
1 mU is defined as 1 millimicromole of product formed/min. All cytochrome
levels were calculated as nmoles/g. Protein concentrations were determined
using the Folin-phenol reagent (Lowry, jit_ ad., 1951).
Lactate Dehydrogenase—(L-lactate: NAD oxidoreductase; 1.1.1.27).
The procedure followed Boehringer Mannheim kit 15948 and Wroblewski and
LaDue (1965).
Malate Dehydrogenase—(L-malate: NAD oxidoreductase; 1.1.1.37). The
procedure followed Boehringer Mannheim kit 15981 and Bergmeyer and Bernt
(1965).
Cytochrome Oxidase—(cytochrome ^: 0., oxidoreductase; 1.9.3.1). The
procedure followed was described by Whorton and Tzagoloff (1967).
Glutamic Oxalacetic Transaminase—(L-aspartate: 2-oxoglutarate amino-
transferase; 2.6.1.1). The procedure followed Boehringer Mannheim kit
15923 and Bergmeyer and Bernt (1965).
Acetylcholinesterase—(acetylcholine acetylhydrolase; 3.1.1.7). The
procedure followed was described by Ellman, _et^ ad. (1961).
Alkaline Phosphatase—(orthophosphoric monoester phosphohydrolase;
3.1.3.1). The procedure followed was described by Bessey, et _al. (1946).
19
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Acid Phosphatase—(orthophosphoric monoester phosphohydrolase; 3.1.3.2),
The procedure followed was described by Andersch and Szczypinski (1947).
g-Glucuronidase—(3-D-glucuronide glucuronohydrolase; 3.2.1.31). The
procedure followed Sigma kit 325-A and Fishman (1965).
Leucine aminopeptidase—(3.4.1.1). The procedure followed Boehringer
Mannheim kit 15793 and Bernt and Bergmeyer (1965).
y-Glutamyl Transpeptidase—The procedure followed Boehringer Mannheim
kit 15794 and Szasz (1969).
NADH-Cytochrome .g. Reductase—(NADH2:cytochrome £ oxidoreductase;
1.6.2.1). The procedure followed was described by Mackler (1967).
NADH-Cytochrome h? Reductase—(NADH2:cytochrome b_,- oxidoreductase;
1.6.2.2). The procedure followed was described by Strittmatter (1967).
NADPH-Cytochrome £. Reductase—(NADPH2:cytochrome £ oxidoreductase;
1.6.2.3). The procedure followed was described by Ernster, £t aL. (1962),
and Masters, j* al. (1967).
NADPH-Dichlorophenolindophenol Reductase—The procedure followed was
described by Ernster, e* al. (1962) and Masters, et^ a!L. (1967).
Cytoehrome b^—The procedure was essentially the method described by
Ernster, jt al. (1962).
Cytoehrome P-450—The procedure followed was described by Omura and
Sato (1964).
20
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SECTION V
LABORATORY RESULTS
FATE STUDIES
Volatility
The 5 crude oils in decreasing order of volatility were: Nigerian,
Saudi Arabian, Iranian, Empire Mix and Venezuelan. [2]
Saudi Arabian, Nigerian and Iranian crude oils have similar pristane/
phytane ratios (0.65 - 0.74) as do Empire Mix and Venezuelan crude oils
(1.77 - 1.80). [2]
Fate in Water and Sediments
Saudi Arabian crude oil precipitated or was absorbed out of the water
column at a slightly faster rate than was Empire Mix crude oil. Using
emulsified Empire Mix crude oil in the presence of sediments, a drastic
change in total hydrocarbon distribution in the water column occurred
within 96 hours. After 3 hrs exposure, the total aliphatics appeared to
have the same hydrocarbon distribution as immediately after emulsion.
However, with time, the lower molecular weight straight-chain hydrocarbons
were drastically reduced relative to the isoprenoids, pristane and phytane.
After 96 hrs, there were a great number of unidentified chromatographic
peaks that were not present in the original emulsified oil. In sediments
exposed to oil in the water column, alkanes of low molecular weight (n-Cll -
n-C20) were present in very low concentrations. The alkanes above n-C20
showed an odd/even dominance that is a typical hydrocarbon distribution for
Holocene sediments. The amount of crude oil adsorbed into the sediment
from the water column was below the concentration needed to be detected at
the natural concentration level of the sediment hydrocarbons (0.0024%).
21
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Both pristane and phytane were present in the same ratios found in Empire
Mix crude oil. Transfer of emulsified oil from water column to sediments
took place within 96 hrs, but at very low levels. [3-7]
In studies on the transport of oil into the water column from sediments
mixed with oil, the oil concentrations in the water remained fairly constant
after 3 hrs although the distribution of the hydrocarbons in the water
column did change. The lower molecular weight straight-chain hydrocarbons
were substantially reduced, relative to pristane and phytane concentrations,
and the higher molecular weight hydrocarbons (n-C21 - n-C36) were increased.
There was no odd/even hydrocarbon preference which indicates that the
distribution of hydrocarbons was not influenced by sediment hydrocarbons.
There was an inexplicable 50-fold increase of n-C25 alkane. After 96 hrs,
the oil concentration remaining in the sediments completely masked any
natural sediment hydrocarbons. Further, there was little change in the
distribution and composition of the oil that had been slurried and left in
the sediments for 96 hrs. [8-ll]
Three classes of compounds, fatty acids, fatty alcohols and hydrocarbons,
were chosen to assess the role of sedimentary processes and to investigate
their interconversion. Since saturated C15 - C20 isoprenoid hydrocarbons
are found in ancient sediments and in petroleums but not in recent sediments,
the presence of pristane and phytane, isoprenoids C19 and C20, respectively,
were used as indicators of oil pollution in these studies. Sediments from
"clean" coastal bays were treated with simulated petroleum pollutants
14
spiked with a C labeled C18 acid, alcohol and hydrocarbon. Degradation
14
of the C labeled compounds was significant after 60 days. Of the original
labeled fatty acids, 50% of the activity was found in the alcohol fraction,
30% in the alkane fraction, and 10% in the fatty acid fraction. Of the
original labeled alcohols, 50% of the label was found in the alkane fraction
and the remainder in the alcohol fraction. Extracts from the simulated
oxygenated sediments showed decreased hydrocarbon/total lipid weight ratio
and changed hydrocarbon distribution when compared to the reduced sediments
22
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Fate of Empire Mix Crude Oil With or Without Aeration
The influence of marsh grass (Spartina) on the fate of Empire Mix
crude oil incubated with or without aeration was studied for 9 mo. The
degradation of straight-chain hydrocarbons was much greater than that of
the isoprenoids, and no changes in pristane/phytane ratios were observed
under either set of conditions during the first 7 months. After 9 mo,
however, the pristane/phytane ratio dropped from an average of 1.80 to 1.57
under aerobic conditions. The n-C17/pristane ratio remained constant in
both control and experimental containers without aeration at approximately
1.16. With aeration this ratio dropped drastically with time in the experi-
mental container containing Spartina (0.487 after 9 mo) suggesting increased
aerobic microbial activity. Since the ratio with aeration but without
Spartina did not change appreciably, it appears that the degradation of n-C17
in relation to pristane by microorganisms was altered by the Spartina. The
n-C18/phytane ratio showed similar trends to those described above with
the ratio without aeration remaining fairly constant at 1.60, while the
ratio with aeration dropped 65%. [12]
Microbial Degradation of Oil
In laboratory studies on the microbial degradation of Empire Mix crude
oil, degradation was minimal after 120 hrs of incubation in the absence of
added nitrogen and phosphorus. In the presence of added nitrogen and
phosphorus, maximum growth, respiration and aliphatic utilization occurred
during the first 24 hrs. Visible turbidity and obvious changes in the oil
were seen within 48 hrs. The total microbial population experienced an
Q
initial decrease during the first 8 hrs, then reached a count of 4 x 10
at 24 hrs and remained essentially constant for the rest of the 120 hr
period. Although the stationary phase of growth was reached in 24 hrs, the
visual degradation of the oil was not obvious until after 48 hrs. There
was a continual decrease in the number and quantity of straight chain
aliphatic hydrocarbons with time, and after 66 hrs only the CIS, C17, C18,
C19 and C20 remained. Only C17, C18 and C19 were present after 120 and 336
hrs and no even or odd chain preference was observed. The branched-chain
hydrocarbons decreased but were still present after 120 hrs and pristane and
23
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phytane were relatively resistant to attack. The more polar aromatic
constituents were metabolized before the less polar compounds. No accumula-
tion of aliphatics occurred in the bacterial cells. [l3-2l]
Three bacterial cultures were selected for further study on the basis
of numerical predominance relative to the total microbial population.
Decomposition of filter-sterilized oil by the isolates was studied by
analyzing oil after 108 hrs of incubation. Oxygen consumption during the
degradation of crude oil was essentially the same for all three isolates,
and no alkane specificity was observed. One isolate synthesized pristane
and phytane. [22-24]
PHYTOPLANKTON
Toxicity Tests
96-hr Toxicity Tests-
Eighty six 96 + 3 hr algal toxicity tests consisting of 272 replicates
were performed using 5 crude oils on unialgal Isolates of 8 representative
species of marine phytoplankton. Toxicity was measured in terms of the
concentration of crude oil required to reduce cell growth 50% with respect
to the controls (EC,.,.). Cell growth was measured in terms of cell numbers.
Experimental oil exposure levels ranged from 0 to 100 mg/1 assuming 100%
emulsification. Actual oil levels present in the growth media were quanti-
tated by IR spectrophotometric analyses of aliquots taken from each bioassay.
These values were used to calculate the percentage of emulsion that actually
occurred. Nigerian crude oil was the most toxic. Iranian and Empire Mix
were intermediate and Saudi Arabian and Venezuelan were the least toxic.
In comparing the oils, Empire Mix was approximately twice as toxic as Saudi
Arabian to the 5 algae tested with both oils. All 8 species were tested
using Empire. _!. galbana was the most sensitive followed by j>. costatum,
_L. undulatum, C^. curvisetus, Rhizosolenia calcar, Asterionella japonica and
Tl_. decipiens. Carteria chuii was the least sensitive of the 8 algal species
tested with Empire Mix. [25-42]
24
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12-Day Toxicity Tests-
Experiments were conducted to determine if extending the exposure time
to 12 days would increase the toxic effects of Empire Mix. By extending
the exposure time of the organisms to 12 days the Empire Mix increased in
toxicity thus lowering the EC5Q values by more than 5 mg/1 in 5 of 7 of the
species tested. For _!. galbana, extended exposure had little effect on the
ECcn, but T. decipiens became more tolerant with extended exposure, and the
DU —
EC,-0 value increased by 5 mg/1. The greatest effect of extended exposure
time was seen at the upper oil levels where cell mortality reached 100% in
many cases. [43-50]
Chronic Toxicity Tests-
Thirty-two 96-hr experiments were performed in which C_. curvisetus, Jt.
galbana, L. undulatum and T\ decipiens were tested against Empire Mix and
Saudi Arabian. j>. costatum was tested against all 5 oils. In these studies,
phytoplankton exposed to crude oil at concentrations between 0 and 100 mg/1
for 96 hrs were subcultured into an oil-free medium. Four of the five
species recovered after exposure both to Empire Mix and Saudi Arabian at
all concentrations tested. Signs of recovery usually were noted within 14
days with complete recovery within 28 days. Ij. undulatum consistently
failed to recover or recovered at a greatly decreased rate in control and
treated cultures. [5l]
Bioaccumulation
Sixteen experiments were performed using the 7 representative species
of marine phytoplankton. Unialgal cultures were grown in inverted, aerated
4-liter serum bottles containing 3 liters of Heinle medium. In each
experiment the algae in one bottle were exposed to 4 mg/1 Empire Mix
(approximately l/10th of the average amount of oil required to produce 50%
reduction in growth in the acute toxicity tests), while the algae in the
other bottle were left untreated. Concentrated samples from each bottle
were analyzed by LC and GC. Oil-treated algae samples showed consistently
higher levels of oil than did the control samples, with some of the oil-
treated samples showing oil levels which could account for 40 to 70% of the
25
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total amount of oil introduced into the bottles. It is not possible at
this time to state whether the oil was being bioaccumulated, adsorbed or
absorbed on the algal cells, or whether the algal cells were biologically
synthesizing natural oils in response to exposure to Empire Mix. Synthesis
appears less probable than either adsorption or absorption. Data suggest
that the ability to bioaccumulate, adsorb or absorb oil may be species
dependent with _!. galbana and T_. decipiens being the most efficient. [52]
ZOOPLANKTON
Toxicity Tests
Data for these tests were based on 98 valid bioassays carried out
under static conditions using emulsified oil. Criteria for bioassay validity
were: 70% or greater control survival, a 50% Tolerance Limit (TL ) included
m
within the range of concentrations in the test, and the use of the same
dilution scheme as used in all experiments with that test organism. Actual
oil levels present in the water were quantitated by IR spectrophotometric
analyses of aliquots taken from each bioassay. These values were used to
calculate the percentage of emulsion that actually occurred. Toxicity was
expressed as TL , the level of oil causing 50% mortality of the population
in 96 hrs. [53]
_A. tonsa, the major primary consumer among the zooplankters, is by far
the most abundant copepod in the Mississippi Sound and surrounding Gulf
waters, thriving in salinities of 1.1-36.5 o/oo. Of the copepods tested,
A^ tonsa was the most susceptible to crude oil. Of the 5 oils used on
cultured A. tonsa, Nigerian was the most toxic, Empire Mix, Saudi Arabian
and Iranian were intermediate, and Venezuelan was the least toxic. All TL
m
values obtained in these tests were low enough that these concentrations of
oil can realistically be expected in the water column following a major oil
spill. [54-59]
When wild A. tonsa (collected from the pilot-plant ecosystem) were
exposed to Empire Mix in the laboratory, the resultant TL value was the
same as that for cultured organisms, indicating that laboratory data on
cultured Acartia. can be extrapolated to Acartia in the pilot-plant
ecosystem. [60]
26
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Eurytemora affinis is another pelagic copepod found in the estuary of
the Mississippi Sound. On the basis of 4 bioassays, this species had an
average TL of 7.8 mg/1 for Empire Mix. [6l]
Euterpina acutifrons is the most prominent pelagic harpactacoid copepod
found in the Mississippi Sound. As with A., tonga, Nigerian was the most
toxic and Venezuelan the least toxic to cultured Euterpina acutifrons, with
Empire Mix, Saudi Arabian and Iranian intermediate. [62-67]
Cyclops viridis is the predominant species of cyclopoid copepod,
usually inhabiting low to moderately saline waters in the estuary. Nigerian
t
was the most toxic of the oils to C_. viridis. However, the toxicity of
the other 4 oils could not be determined reliably, since the concentration
of oil had to be so high that the emulsions could not be maintained long
enough for testing. [68]
MARSH GRASS
Greenhouse Studies
The majority of the studies on the uptake of oil by marsh vegetations
was conducted using potted plants in the greenhouse. When clumps of Juncus
roemerianus and Spartina cynosuroides (the dominant species in the Mississippi
marshes) were exposed to Empire Mix, they showed an increase in hydrocarbon
content, both aliphatic and aromatic. The small amount in the control
plants presumably was due either to the natural waxy substances synthesized
by plants or to prior exposure to oil. There was a greater concentration
of aromatic compounds than there was aliphatic compounds in control plants.
On the basis of aliphatic hydrocarbons, it appeared that Spartina did not
readily take up oil. These and other later data from similar experiments
using Saudi Arabian also indicated that, in general, Juncus took up oil
more readily than Spartina. The higher level of residues detected in dead
plants and in roots of live plants suggested that oil was being absorbed
rather than bioaccumulated. [69-72]
27
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Field Studies
A series of plot experiments were conducted on a natural marsh located
on a marsh island west of St. Louis Bay, Mississippi. The effects of
single and multiple exposures of marsh plants to doses of 250, 600, 750 and
1500 ml of Empire Mix for various periods, ranging from 4 mo to 2 yrs, was
2 2
studied using 1 m plots subdivided into 4 subplots (0.25 m ). [73-74]
From the results of these experiments, a number of conclusions were
drawn concerning the effects of the exposure of marsh plants to Empire
Mix:
(1) Single exposures to doses of 250 ml or above caused some plant mor-
tality as well as reduced growth the following year ranging from 40%
(250 ml/m2) to 90% (1500 ml/m2). [75-78]
(2) Multiple spills of doses of 250 ml or greater, 12 mo apart, signi-
ficantly reduced plant growth the following growing season from
23% (250 ml/m2) to 94% (1500 ml/m2). The degree of reduction
increased as the dosage increased. [79-83]
(3) Following single exposures to doses of 250 ml and greater, marsh
plants contained detectable oil residues up to 9 mo after exposure.
Following oil exposure, the roots of live marsh plants contained
more oil residues than the shoots; the bottom of the shoots
contained more oil residues than the tops of the shoots; and
dead marsh plants contained more oil residues than live
plants. [84-88]
(5) The decomposition of dead marsh plants subjected to the
ebb and flow of natural tides was reduced as much as 40 to 50%
after 1 yr of exposure. The oil-contaminated plant material will
remain in the environment longer, thus increasing the opportunities
for oil to enter the estuarine food chain via the detritus
pathway. [89]
28
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MULLET
Acute Toxicity
A number of toxicity tests using 130-160 mm mullet were conducted to:
(1) compare the relative toxicities of Empire Mix, Saudi Arabian, Venezuelan,
Nigerian and Iranian crude oils to mullet; (2) determine the levels of
Empire Mix which would be used for short-term laboratory tests to study the
effects of oil on the enzyme system of mullet; and (3) determine the dosage
of Empire Mix to be spilled on the pilot-plant ecosystem during the chronic
field studies.
Of the oils tested against mullet, Nigerian (100% mortality at 200
mg/1) and Saudi Arabian (100% mortality at 350 mg/1) were the most toxic,
Iranian and Venezuelan were intermediate (TL 's between 400-800 mg/1) and
m
Empire Mix (TL > 800 mg/1) was the least toxic. [90]
Based on these results, laboratory treatment levels of 75 mg/1 or less
were chosen for short-term enzymological studies. In addition, it was
estimated that the levels being considered for use in the field studies (5-
20 mg/1) would allow long-term survival of the mullet.
Behavior
There was a distinct difference in the behavior of mullet when exposed
to different crude oils. During exposures of up to 800 mg/1 to the less
toxic oils (i.e.., Empire Mix, Venezuelan and Iranian), the movements of the
mullet appeared generally normal to somewhat suppressed (they were often in
a stationary position near the top of the tank). When exposed to Nigerian
and Saudi Arabian in 114-liter aquaria at concentrations ranging from 200
to 400 mg/1, the mullet almost immediately became hyperactive. During
early stages of exposure they swam rapidly in a straight line, often hitting
the sides of the test tanks. Rapid swimming movements often were followed
by a pause during which the mullet rapidly flexed their bodies laterally in
a shaking motion. Near death, there was a loss of equilibrium during which
mullet sank to the bottom of the test tank.
29
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Enzymology
Mullet enzymes typically were assayed in mitochondrial or supernatant
fractions, or whole homogenates of brain, gill, liver and muscle tissues.
More than 1700 individual enzyme assays were conducted, using over 1000
mullet.
The following enzymes were studied in juvenile mullet exposed to
75 mg/1 emulsified Empire Mix for 96 hrs: lactic dehydrogenase (LDH),
malic dehydrogenase (MDH), NADPH-cytochrome c_ reductase (CYT RD), cytochrome
oxidase (CYT OX), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic
transaminase (GPT), acetylcholinesterase (CHE), alkaline phosphatase (ALP),
acid phosphatase (ACP), B-glucuronidase (GLU) and leucine aminopeptidase
(LAP). With the exception of GLU, the enzymes did not appear to be affected
by this oil exposure. Although muscle and brain were the tissues expected
to be least affected by oil exposure, they were the 2 tissues in which
there was a change in enzyme level, with a statistically significant decrease
in mitochondrial GLU activity from muscle and brain. There were no differences
observed following oil exposure for any enzymes studied in either liver or
gill tissue. It had been expected that gill and liver would be the tissues
most likely to be affected since these tissues are involved with initial
contact (gill) and detoxification (liver). [91-94]
Several enzymes were studied in juvenile mullet 96 hrs after intra-
peritoneal injection with 100 yl of Empire Mix. There were indications of
changes in the activities of CYT OX, CYT RD, GOT, GPT and LAP in some
tissues. No changes were noted in GLU, LDH, MDH or a-hydroxybutyric acid
dehydrogenase. [95]
To determine if any direct interaction between molecular components of
oil and the enzyme molecule existed, which could lead to activation or
inhibition, studies of in vitro time-dependent and time-independent effects
were conducted. Empire Mix in concentrations up to 80 mg/1 was incubated
with homogenates of mullet liver, and the enzyme GOT was monitored. This
enzyme was selected since it was part of the assay system for MDH, which
30
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had shown positive responses to oil in other studies. No in vitro inhibition
or activation of GOT in the presence of oil was observed. [96-97]
During the wide spectrum enzyme studies discussed above, one microsomal
oxidase, NADPH-cytochrome £ reductase, gave indications of induction in
livers of mullet following a one-week exposure to Empire Mix. To test the
inductive potential of crude oil on microsomal hydroxylation enzymes,
mullet were exposed to 75 mg/1 of Empire Mix or Saudi Arabian for 96 hrs.
Liver weight to body weight ratios and hepatic protein were also measured.
Liver weight increased by about 50% in response to both oils. Concentrations
of both total hepatic protein and microsomal protein were depressed, parti-
cularly by Saudi Arabian; this apparent decline represents the failure of
protein to increase at the same rate as liver size. Both cytochromes bc
and P-450 were induced above control levels, particularly cytochrome P-450.
NADPH-dichlorophenolindophenol reductase was increased following exposure
to both oils and NADPH-cytochrome c^ reductase following exposure to Empire
Mix; these increases .may have been activations rather than inductions.
NADH-cytochrome c_ and NADH-cytochrome ID,, reductase activities were not
affected, but remained proportional to the protein level. Although Saudi
Arabian had a greater effect on liver size and hepatic protein content,
Empire Mix had a greater effect on enzyme levels. Therefore, both crude
oils exert physiological effects with potential pharmacological significance
in mullet. [98]
In conclusion, the most consistent effects following oil exposure in
mullet were in those enzymes associated with stress and/or detoxication.
$-Glucuronidase was depressed and some microsomal oxidases were activated
or induced following exposure to crude oil.
Fatty Acids
Mullet were exposed to 10, 20, 30 and 75 mg/1 emulsified Empire Mix
for 96 hrs. Fatty acids 14:0, 16:0, 16:1 and 22:6 showed some changes in
the oil-treated mullet, but no consistent dosage-response pattern of either
increases or decreases was evident. The mean percent fatty acid composition
of the shorter-chain fatty acids up to 18:0 in fish exposed to 75 mg/1 oil
31
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were higher than the control fish. However, the longer-chain fatty acids
were lower in the treated fish than in the control fish. In addition,
there were differences between laboratory controls and wild mullet for 11
of the 25 fatty acids reported. [99-101]
Seasonal variation was observed with higher percentages of long-chain
polyunsaturated fatty acids present during the colder months, and predomi-
nance of short-chain fatty acids present during the warmer months. [102-103]
Oil Uptake
The LC and GC results of numerous oil uptake tests exposing mullet to
various concentrations of emulsified Empire Mix crude oil for varying periods
of time were extremely erratic. Even so, certain trends were evident. Fish
which were dead when removed from the test tanks usually were higher in oil
content than live fish exposed under similar conditions. Insofar as parti-
tioning of the oil into various tissues is concerned, gill and gut tissues
almost always contained oil. Approximately 60% of the liver tissues examined
contained measurable amounts of oil. It should be noted that a number of
the brain samples contained oil. Muscle tissues were generally low or nega-
tive in oil content. As would be expected the gill tissues taken early in
the experiments were higher in oil content than those taken later. [104]
For all of the above tests, oil in the tissues was calculated using
both the LC and GC analyses. The amount of oil found in each sample by the
two methods differed since the calculation of oil was based on the aromatic
hydrocarbons for the LC analyses and on the aliphatic hydrocarbon fraction
for the GC analyses. However, the trends of oil uptake and depuration for
each tissue were similar using either method, with the exception that for a
given sample low levels of oil could often be detected using the LC, while
none could be detected from a portion of the same sample using the GC.
Histology
No histological changes could be attributed directly to the effect of
either acute or short-term chronic exposures of mullet to Empire Mix and
Saudi Arabian. However, bacterial infections (accompanied by the pathological
32
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conditions associated with them) were observed in the majority of tests
that lasted more than two wks.
Laboratory Studies on Disease
One unexpected result observed when oil was spilled on the pilot-plant
ecosystem was the sudden and dramatic outbreak of fin rot which occurred in
the oil-treated ponds. This was especially surprising since fin rot had
not been observed during numerous acute laboratory tests nor had reference
to such an occurrence in response to oil been encountered in the literature.
The lack of disease in the laboratory experiments was thought to be
due to the short (96 hr) duration of the majority of these tests. Conse-
quently, chronic laboratory experiments were initiated in an attempt to
determine if the occurrence of fin rot in the first pilot-plant study was
indeed related to the exposure of mullet to oils or simply a unique happening
due to some peculiar order of events or circumstances. In these control
laboratory studies both Empire Mix crude oil and Saudi Arabian crude oil in
concentrations ranging from 14 mg/1 to 75 mg/1 were shown to cause the
disease. Overall, the incidence of infection was 7.1%, 97.1% and 100% for
control, Empire Mix-exposed and Saudi Arabian-exposed mullet,
respectively. [105]
Bacteriological examinations from both field and laboratory mullet
(approximately 425 fish sampled) indicate that primarily 5-6 different
colonial types of bacteria constitute the normal microflora on the exterior
of the mullet in the control situations while only 1-2 colonial types are
predominant in the microflora on the exterior of the mullet exposed to oil.
It is important to note that the suspected pathogen identified as Vibrio is
also found on the exterior of some control fish, as well as the oil-tested
fish but in much fewer numbers. [106]
Bacteriological examination of the kidneys of the mullet indicated that
under normal conditions the kidney was sterile. Only from kidneys of fish
which were heavily infected with fin rot were any bacteria isolated. The
33
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kidney isolate was always in pure culture and had identical characteristics
of the predominant isolate (Vibrio) from the exterior of the infected mullet.
Results from the microbial examination of the intestines of the mullet
were less clear cut. There appeared to be no normal bacterial flora in the
intestines and the number of organisms present seemed to vary from fish to
fish.
Pure culture isolates from both healthy and diseased mullet were examined
for their ability to utilize crude and degraded oil and for their hemolytic
ability. Interestingly, the organism identified as Vibrio was the only hemoly-
tic organism isolated which was capable of utilizing either crude or degraded
oil. Although this did not fully establish the relationship between microbial
utilization of oil and the ability to cause disease, it did suggest a connec-
tion between the two observations. [107]
The data seem to indicate that the potentially pathogenic bacteria are a
normal part of the mullets' microflora and only cause a problem when one or
more of the mullets' defense mechanisms are rendered inoperative. To further
test this theory, the mucus was removed by mechanical means from the tails of
a number of mullet. These test organisms contracted fin rot in 6-10 days.
In addition, the tall of a number of mullet were scraped near their base
causing superficial lesions. Again these mullet contracted fin rot.
In summary, these limited laboratory tests clearly establish the
relationship between oil exposure and the occurrence of fin rot in mullet.
However, the mechanism has not as yet been elucidated.
SHRIMP
Acute Toxicity
The purposes for running acute toxicity tests with shrimp were the
same as those stated for the acute toxicity tests using mullet.
The ranking of the 5 oils in decreasing toxicity to shrimp was:
Nigerian, Empire Mix, Saudi Arabian, Iranian and Venezuelan. Nigerian was
34
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the most toxic as 10 mg/1 resulted in 100% mortality of the test shrimp.
The XL values were estimated to be between 15 and 25 mg/1 for Empire Mix,
Saudi Arabian and Iranian, and between 35 and 45 mg/1 for Venezuelan. [108]
When the toxicity tests with mullet, -oysters and shrimp were evaluated,
it was obvious that shrimp were the most sensitive to all 5 oils. Therefore,
a calculated concentration of about 4 mg/1 was chosen as the concentration
of Empire Mix to be spilled in the pilot-plant ecosystem, since the majority
of the shrimp population should survive this dosage. In addition, 2, 4 and
8 mg/1 were chosen as the dosages of choice for laboratory studies using
shrimp.
Behavior
Generally, the first and the most characteristic behavioral response
of shrimp exposed to crude oils was a spiraling motion from the bottom to
the top of the tank followed by a loss of equilibrium. After falling back
to the bottom of the tank they often remained on their sides, rapidly
moving their swimmerettes which sometimes resulted in "scooting" movements
but usually caused no forward motion. The swimming motions of the treated
shrimp often were rapid and erratic. These reactions were essentially the
same for all oils studied; however, their onset came early and were more
pronounced with Nigerian than with the other crude oils when tested at the
same dosage. This was to be expected since, to shrimp, Nigerian was the
most toxic of the 5 oils.
Enzymology
Enzymes were assayed in mitochondrial and supernatant fractions of
hepatopancreas homogenates. In excess of 1100 Individual enzyme assays
using more than 750 shrimp were conducted. The following enzymes were
assayed from shrimp exposed to 8 mg/1 emulsified Empire Mix for 12 hrs:
acid phosphatase (AGP), malic dehydrogenase (MDH), glutamic oxaloacetic
transaminase (GOT), glutamic pyruvic transaminase (GPT), 6-glucuronidase
(GLU), cytochrome oxidase (CYT OX) and NADPH-cytochrome £ reductase (CYT
RD). There were no detectable effects from oil exposure on the enzymes
assayed. [109]
35
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Shrimp were exposed to 4 mg/1 emulsified Empire Mix for 24 and 48 hrs.
The following results were indicated: mitochondrial and supernatant
y-glutamyl transpeptidase (GGTP) and leucine aminopeptidase (LAP), and super-
natant CYT OX were decreased after oil exposure; mitochondrial and super-
natant GOT and GPT, supernatant CYT RD and mitochondrial CYT OX were elevated
after oil exposure; ACP, ALP, and GLU, were not consistently affected. [llO]
Shrimp were exposed to 10 mg/1 emulsified Empire Mix or Saudi Arabian
for 24 hrs. The activities of the following enzymes were determined: ACP,
ALP, CYT OX, CYT KD, GLU, GOT, GPT, GGTP, LAP, MDH, a-hydroxybutyric dehydro-
genase (HBDH) and lactic dehydrogenase (LDH). Enzyme activity responses
were different following the in vivo exposure to the two crude oils.
Exposure to Empire Mix resulted in no significant differences in the enzyme
activities between oil-treated and control groups. However, some dramatic
results were observed with Saudi Arabian. In the mitochondrial fraction,
ALP, GGTP and CYT OX activities were decreased. In the supernatant fraction,
ALP, GGTP and LAP activities were decreased. The following activities were
increased in the supernatant fraction: HBDH, GOT, GPT, LDH, GLU, and MDH.
Mitochondrial HBDH activity was increased. The other enzymes were unaffected
by oil exposure. Although supernatant increases in activity were observed
for several enzymes, in most cases concomitant decreases in mitochondrial
specific activities were not observed. [111-112]
No statistically significant induction of the microsomal oxidase,
NADPH-cytochrome c^ reductase, was observed in shrimp hepatopancreas following
up to 24 hrs of exposure to either Empire Mix or Saudi Arabian. [113]
In conclusion, the enzyme activities of the hepatopancreas do not seem
to be greatly affected following in vivo exposure of brown shrimp to Empire
Mix, whereas many of the same enzyme activities are greatly altered following
exposure of the shrimp to Saudi Arabian.
The following enzymes were assayed in mitochondrial and supernatant
fractions of whole body homogenates of grass shrimp (Palaemonetes spp.)
exposed to 50 mg/1 emulsified Empire Mix for up to 12 days: MDH, LDH,
36
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HBDH, GPT, GGTP, LAP and creatine phosphokinase (CPK). There were no
statistical differences observed with the 7 enzymes tested. When ratios of
supernatant/mitochondrial specific activities for each sample were compared
statistically, only the creatine phosphokinase ratios showed a statistical
difference. [114-115]
Fatty Acids
Fatty acid concentrations were determined in muscle tissue of shrimp
exposed to 15 mg/1 emulsified Empire Mix for 96 hrs. Fatty acids 16:1 and
18:3 were higher and 20:5 was lower in oil-exposed shrimp muscle than in
control shrimp muscle. [116-118]
Shrimp were exposed to 2, 4 or 8 mg/1 Empire Mix for 12-24 and 48-96
hrs, and fatty acids of muscle and hepatopancreas were determined. In muscle
tissue of shrimp exposed to 8 mg/1 Empire Mix for 12-24 hrs, the fatty
acids 14:1, 15:1 and 17:1 were higher than in control shrimp muscle. Fatty
acids 14:0, 14:1, 15:0, 16:0, 17:0 and 18:1 showed a progressive increase
in mean percentage with increased oil dosage. Fatty acid 17:1 was higher
in muscle of shrimp from all three oil doses than in control tissue. There
were fewer differences in fatty acids between control and oil-exposed
shrimp at 48-96 hrs than at 12-24 hrs. At 48-96 hrs of exposure, fatty
acid 15:1 of muscle from shrimp exposed to 2 mg/1 was higher than the
control. In hepatopancreas samples, fatty acids 18:1, 18:2 and 20:0 were
significantly higher and 20:1 was significantly lower in shrimp exposed to
these doses of oil for 12-24 hrs. No significant differences were observed
after 48-96 hrs of exposure. [119-122]
In shrimp exposed to oil, there was a pattern of higher mean percentages
for short-chain fatty acids and lower mean percentages for long-chain
unsaturated fatty acids following exposure. Possible explanations are:
(1) oil disrupted the normal chain elongation saturation-desaturation
process; (2) organisms were stressed, and selectively 3-oxidized the longer
chain fats for energy; and (3) oil could cause an enzymatic catabolism of
the bonds in the longer-chain carbon acids, resulting in higher mean per-
centages of short-chain acids and less long-chain fatty acids. Differences
37
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In the fatty acid pattern decreased with increased exposure time, indicating
some form of compensation or acclimation to the stress. This decrease
correlated positively with the decline in hydrocarbon content of the tissues
with time.
Oil Uptake
Shrimp were exposed to different levels of emulsified Empire Mix for
96 hrs with samples taken every 12 hrs for the first 24 hrs and at 24 hr
intervals thereafter. By LC, shrimp muscle contained higher levels of oil
at higher dosage levels (2-8 mg/1) during the first 24 hrs, followed by
lower tissue levels of oil uptake for the next 72 hrs at all dosage levels.
*
The level of oil as determined by LC in the shrimp hepatopancreas increased
for the first 24-48 hrs, after which time a decrease in oil content was
observed. Using GC analyses the data were very erratic and did not demon-
strate the presence of oil in the hepatopancreas. [123-124]
Histology and Disease
Acute Studies-
Histological findings as a result of short-term laboratory exposure of
shrimp to Empire Mix were inconclusive. However, a number of interesting
observations were made.
The exposure of shrimp to 2 mg/1 Empire Mix in 114-liter test aquaria
for 72-96 hrs resulted in the development of black spot gill syndrome.
Grossly, the gills of the treated shrimp were dark brown to black in color.
The gills of moderately involved specimens had a mottled appearance; while
in more severely affected shrimp, the entire gill appeared dark. Histologi-
cally, there were numerous branchiae which appeared to be swollen and/or
fused. In these areas the epithelial lining of the lamellae were thickened,
and the cytoplasm appeared to be swollen with a brownish infiltrate. Some
of the dark areas, especially near the base of the branchiae, were very
dense with little structural detail. It was thought that this syndrome was
caused directly by the oil treatment. However, additional tests at the
same treatment levels failed to consistently cause the syndrome. In addition,
the black gill syndrome also developed in shrimp which were being held
38
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prior to testing; in this instance it appeared that a bacterium (tentatively
identified as Vibrio spp.) appeared to be responsible. In conclusion, it
is not possible at this time to state whether the development of the black
gill syndrome in the treated shrimp was a direct response to oil or the
indirect effect of the oil acting as a stressor stimulating a bacterial
infection which caused the syndrome. However, it seems probable that the
syndrome is a general response of the gill tissue which can be triggered by
a number of factors. [125]
Laboratory Feeding Experiments-
A series of experiments were conducted using aerated, 114-liter aquaria
with sand covered bottoms in which test shrimp were fed 5 g of oil-treated
feed (250 mg/1) per day while the control shrimp were fed 5 g of feed with
no oil added. At the termination of the experiment, some of the shrimp
were sacrificed and examined histologically. A number of abnormal areas
were observed in the area of the mandibles, gastric mill and along the
inner edge of the carapace, especially ventrally near the muscles supporting
the digestive track attached to the body wall. The lesions typically
involved the keratinized layers and were characterized by a break or erosion
of the carapace sometimes accompanied by a poliferation of cells from
beneath the carapace.
In all cases, the necrotic areas were stained brownish and very closely
resembled the necrotic areas observed in the black gill syndrome. The
necrotic areas were often located either between the two layers of the
lining of the digestive tract or just beneath it. It is not known by what
means these lesions arise. Since only one lesion was observed in the
controls, it would seem likely that they were caused, at least indirectly,
by oil. Since the food was granular it could have caused mechanical abrasion
thus allowing oil to get into sensitive areas. [126]
OYSTERS
Acute Toxicity
Acute 96-hr toxicity tests indicated that oysters were very tolerant
to crude oil. There was no significant mortality caused by Empire Mix at
39
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exposure levels of up to 800 mg/1, nor was there significant mortality
caused by Saudi Arabian, Nigerian, Iranian or Venezuelan at exposure levels
of up to 400 mg/1. There was some random low-level mortality of controls
and test organisms not associated with oil exposure. [127]
Behavior
The only noticeable behavior observed in oysters when tested with
crude oil was their tendency to close when initially exposed. Later, as
the oil began to degrade, and after the concentration of water solubles
decreased, some oysters would open and begin to pump. According to the
results obtained in oil uptake studies, some oysters remained closed or did
not pump significantly during the duration of the test. This lack of
consistency complicated the interpretation of the data obtained in uptake
and depuration studies. It was thought that this behavior could be countered
by pegging the oysters open. However, the uptake data were still erratic,
indicating that some did not pump even when forced to remain open.
Depuration
Although Empire Mix was not acutely toxic to oysters in concentrations
which could be expected in the environment; the oil, if taken in, could be
expected to affect the taste of the oyster and thus potentially to impact
the economically important commercial oyster industry. Oysters were exposed
to 300 mg/1 Empire Mix for 96 hr and then placed in baskets in a clean
estuary. Routine taste tests of these oysters indicated that they retained
an oily taste for at least 9 wks.
Enzymology
More than 2400 individual enzyme assays were conducted on more than
300 oysters. Enzymes were assayed in fractions of whole body homogenates
of oysters exposed to 75 mg/1 emulsified Empire Mix for up to 7 days. No
statistically significant differences were observed between control and
oil-treated oysters in the specific activities of acetylcholinesterase
(CHE), acid phosphatase (AGP), alkaline phosphatase (ALP), cytochrome
oxidase (CYT OX), glutamic oxaloacetic transaminase (GOT), mitochondrial
leucine aminopeptidase (LAP) and supernatant malic dehydrogenase (MDH)
A number of enzyme activities from oysters exposed to Empire Mix were
40
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statistically different from those of controls. However, no clear trend as
to stimulation or inhibition was observed. At 7 days, the g-glucuronidase
(GLU) activity was significantly lower than the activity at 4 days. With
glutamic pyruvic transaminase (GPT), the supernatant activity peaked at day
2, and the mitochondrial enzyme activity was significantly higher at day 7.
The soluble LAP activity was lowest on day 2 of treatment. Mitochondrial
MDH activities dropped on day 2, but by day 7 were in the range of the
control activity. This pattern was the reverse of what was observed with
the other enzymes. The enzyme patterns observed may be the result of the
normal response of an organism to an environmental challenge. Another
possible explanation may be related to the very rapid decrease in the
available concentration of oil in the water column. [128-129]
Oysters were exposed to a surface film equivalent to 100 mg/1 of
Empire Mix daily for 7 days and were then sampled at 2, 3, 4, 5, 7 and 8
days after cessation of oil additions. There appeared to be an elevation
of both mitochondrial and supernatant GOT and GPT at 2 days after cessation
of oil addition. Mitochondrial LAP appeared elevated at 2, 5, 7 and 8
days. Supernatant LAP and creatine phosphokinase (CPK) appear unaffected
by oil exposure. Although certain elevations and depressions in enzyme
activity were observed, the only clear pattern of response was the general
increase noted at 2 days post-treatment for all enzyme activities except
supernatant LAP. [130]
Enzyme activities were measured in oysters exposed to 75 mg/1 Saudi
Arabian, Nigerian, Iranian and Venezuelan for 4 days. The following enzymes
were studied: ACP, ALP, CHE, CYT OX, GLU, GPT, GOT, LAP and MDH. Overall,
there were minimal effects on the enzymes tested. No statistically signifi-
cant differences were observed between the controls and organisms exposed
to Saudi Arabian. Nigerian caused decreases in supernatant activities of
GOT, GPT and LAP. Iranian caused an increase in mitochondrial GPT and
supernatant LAP. Venezuelan caused no statistically significant
changes. [131-132]
41
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NADPH-cytochrome c^ reductase was studied In fractions of whole body
homogenates of oysters exposed to 75 mg/1 emulsified Empire Mix, Saudi
Arabian, Venezuelan, Nigerian and Iranian. There were statistically signifi-
cant increases in NADPH-cytochrome c^ reductase activity after exposures to
Empire Mix and Nigerian. [133]
In conclusion, oysters that had been treated with Empire Mix, either
emulsified or a surface film, showed few significant effects on the activities
of the enzymes tested. Of the enzymes that were affected by acute oil
exposure, most are related to carbohydrate metabolism. MDH is involved in
both aerobic and anaerobic metabolism in the oyster, and it may be hypo-
thesized that under conditions of acute oil exposure the oyster closes and
functions anaerobically. The trend of decreased activity at 2 and 4 days
of exposure with the return to normal at 7 days probably represents a
normal compensation by the oyster to oil stress. Carbohydrate (tricarboxylic
acid cycle) and amino acid metabolism are enzymatically connected by GOT
and GPT. GLU is involved also in carbohydrate metabolism. Increased
levels of LAP in the oyster could indicate that under anaerobic conditions
there is mobilization of tissue proteins for energy.
Enzyme activities of oysters exposed to Nigerian and Iranian demonstrated
some changes, whereas those from oysters exposed to Saudi Arabian and
Venezuelan were unaffected. Induction of a microsomal oxidase, of potential
significance in detoxication, was caused by Empire Mix and Nigerian.
Fatty Acids
The fatty acid composition of oysters exposed to 75 mg/1 emulsified
Empire Mix for 24 and 72 hrs were determined. There were statistical
differences between groups for acids 17:1 and 22:1, but no outstanding
trends between control and oil-exposed oysters were apparent. [134-135]
Oil Uptake
The oysters were exposed to 75 ppm Empire Mix oil for 72 hrs, then
transferred to a clean tank and allowed to depurate for 96 hrs. Data on
oil uptake in oysters were extremely erratic apparently due to variations
42
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in pumping habits. Nevertheless, the trend was toward a constant increase
in oil concentration with increased length of exposure to the oil. In a
second system, the oysters were exposed to different amounts of both Empire
Mix and Saudi Arabian oils. The concentration of oil increased with an
increase in concentration with the uptake of Empire Mix being higher than
Saudi Arabian. The concentration of oil in the oysters was calculated
almost totally by LC since the GC was unable to detect oil in such low
amounts. [136-137]
Histology and Disease
Laboratory exposure of oysters to Empire Mix produced no histological
changes that cotild be directly associated with their treatment with Empire
Mix.
43
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SECTION V(a)
FIELD RESULTS
TIDAL-POND STUDY
Description of the System
On July 25, 1973, 57 liters of Empire Mix crude oil were spilled on a
9 x 9 m shallow tidal pond situated at the Gulf Coast Research Laboratory,
Ocean Springs, MS. The spill dosage was calculated to yield 250 mg/1 oil
at low tide. A similar adjoining pond served as control. The pre-spill
conditions of the ponds were: salinity 10-12 o/oo, temperature 30-37 C,
and dissolved oxygen 3.7 mg/1 at low tide and 12.2 mg/1 at high tide. The
dikes were vegetated by Juncus roemerianus, Spartina cynosuroides, Spartina
alterniflora and Distichlijs spicata.
Within 2 hrs after the spill, the pond was completely covered by oil.
After 5 days, the oil had dissipated and was concentrated along the bank,
on the mud and on the marsh plants. Infrared spectrophotometry of the
water 1 day after the spill indicated an oil concentration of 32.5 mg/1.
Observations on the fate of oil in the sediments and effects on floral
and faunal populations were made for 18 mo.
Fate of Oil in Sediments
During the first 18 days following the oil spill, the oil migrated
downward from the sediment surface to a depth of 42 cm. No changes occurred
in the less volatile, higher molecular weight aliphatic hydrocarbons which
were detectable by the technique employed. [138]
After 4 mo the percentage of ti-CIO - n-C20 hydrocarbons as compared to
the n.-C21 - n-C33 hydrocarbons had dropped approximately 8% in the top 2 cm
44
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of sediments. The ratios of n-C!7/pristane and n.-Cl8/phytane had decreased
by 12%. In the 25-34 cm core section, the ratio of percentage of low
molecular weight to high molecular weight hydrocarbons was reduced about
35%. The n-C17/pristane and ri-C18/phytane ratios in the deeper core sections
had increased as compared to the top sections. [139-140]
Only small changes occurred in the composition of the crude oil between
4 mo and 7 mo. Using the known ratios of n.-C17/pristane and n.-C18/phytane
in the crude oil and the hydrocarbon distribution between n-Cll and n-C23
as indicators of the presence of oil, the top section of the core (1-13 cm)
still demonstrated considerable evidence of oil. The n-Cl7/pristane, n-
C18/phytane, and pristane/phytane ratios were close to those in the Empire
Mix crude oil itself. The sediments contained slightly less oil at 7 mo
than at 4 mo. The n-C17/pristane and n,-C18/phytane ratios increased in
these core sections, indicating that the natural level of n-C17 and n-C18
hydrocarbons present were more dominant than those in oil. The deepest
core section analyzed (30-42 cm) contained only trace amounts of oil. [l4l]
Samples collected 12 mo after the oil spill indicated that the percent
hydrocarbon/lipid weight decreased with depth in both ponds as expected in
recent sediments. The oil-spill pond sediments showed a slightly higher
concentration of hydrocarbons than the control pond sediments, although the
level of crude-oil hydrocarbons in the oil-spill pond sediments after 1 yr
was extremely low. The relative percent of hydrocarbons below n-C22 in
both control and oil-spill pond was less than 10 percent. The oil-spill
pond hydrocarbons had a significantly higher pristane/phytane ratio than
the control pond hydrocarbons. The pristane/phytane ratio of the oil-spill
pond was similar to that in Empire Mix crude oil. This ratio, though
different in each pond, remained fairly constant to a depth of 42 cm. [142]
The final core samples, taken from the experimental pond 18 mo after
the oil spill, showed little evidence of crude oil in either the top 13 cm
or the bottom 13 cm. Both sections contained 19 mg/1 aliphatic hydrocarbons
(based on dry sediment weight). The top section of sediment contained a
higher percentage of total aromatic hydrocarbons than the bottom section.
45
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Phytoplankton/Zooplankton
Observations 30 days after the spill indicated a lower phytoplankton
population and a higher zooplankton population in the oil ponds than in the
control ponds. The immediate killing of the oil-sensitive zooplankton by
the initial oil spill allowed phytoplankton to replicate without predation.
As the oil dissipated from the ponds, the zooplankton recovered in the
presence of an abundant standing crop of phytoplankton. The zooplankton
population then rapidly increased, reducing the phytoplankton population
appropriately. This left an abundant zooplankton population composed of
animals in their developing stages. These observations were supported in
part by the large numbers of copepod naupliars found in the samples and
were consistent with the laboratory data. Six and seven mo after the oil
spill, the zooplankton populations in both ponds were similar in respect to
numbers and diversity of species. Phytoplankton, however, were more abundant
and slightly more diverse in the control pond. By 9-10 mo after the oil
spill, zooplankton and phytoplankton were in greater abundance and diversity
in the oil pond than in the control pond. By 12 mo after the oil spill,
phytoplankton were more abundant in the oil pond (15 species) than in the
control pond (11 species). An opposite trend was seen with zooplankton,
with greater abundance found in the control pond. No difference in diversity
was seen.
Primary productivity measured by jLn situ light and dark bottle technique
revealed a 43-65% reduction in the oil planktonic community 16 days after
the spill; respiration values were also reduced by 16-18%. The decrease in
primary productivity may be due to a decrease in phytoplankton biomass up
to 30 days after the spill rather than to any physiological inhibition of
photosynthesis. About 2 mo after the oil spill, the difference in primary
productivity of the plankton community between oil and control ponds was
less than 20%, which was about the condition of the two ponds prior to the
oil spill. [143-144]
46
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Marsh Grass
The results obtained during this tidal pond study paralleled the
results reported for the field studies discussed earlier (SECTION V).
Plants from both the control pond and the treated pond contained aliphatic
and aromatic hydrocarbons. The oil caused plant mortality and reduced the
plant growth in subsequent mo. The hydrocarbon content of the dead plant
material was greater than that in the live plants, and the roots of live
plants contained more hydrocarbon than did the shoots. [145-146]
Animals
The amount of meaningful data obtainable on the changes in the popula-
tions of organisms present in the tidal ponds at the time of the oil spill
was extremely limited because the ponds were stocked by natural recruitment
prior to their enclosure and because they were subject to flooding during
periods of high water. Seine samples prior to the oil spill indicated that
the most predominant species present in the ponds were menhaden (Brevoortia
sjjp.) and grass shrimp (Palaemonetes spp.). Present in relatively low
numbers were the bay anchovy (Anchoa mitchilli), the silverside (Menidia
beryllina), the striped mullet (Mugil cephalus), the pinfish (Lagodon
rhomboides) and the ladyfish (Elops saurus). Seine samples following the
spill indicated an initial drop in the menhaden and grass shrimp populations
followed by a repopulation after about 2 mo. The most dramatic effect on
the fish population appeared to be the predation of menhaden (which were
swimming erratically in response to the oil) by the ladyfish. [147]
Significantly higher concentrations of oil were found in oil-treated
organisms than in control organisms. The molluscs showed the highest
uptake (mussels and snails), crabs the next highest, and fish and shrimp
the least. The difference in feeding habits of these animals may be respon-
sible for the variation in oil uptake. [148]
FIRST PILOT-PLANT ECOSYSTEM STUDY
Description of the System
The pilot-plant ecosystem (Figures 1 and 2) was built at the NASA
National Space Technology Laboratories (NSTL) in Hancock County, Mississippi.
47
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It consisted of 4 ponds measuring 46 x 46 m at the top and narrowing to
30 x 30 m at the bottom. An overflow pipe was located approximately 2.5 m
from the bottom. In June 1972, the ponds were filled using seawater (approxi-
mately salinity 28 o/oo) from the Gulf of Mexico mixed with fresh well
water to adjust the final salinity to about 14 o/oo.
Clumps of Juncus mixed occasionally with other species (e.gj> Spartina,
Scirpus and Distichlis) were dug from preselected areas in St. Louis Bay
marsh, transported to NSTL and immediately transplanted along one side of
each pond. The clumps included the natural muddy substrate and all the
benthic fauna and microflora associated with it. Figure 3 is a photograph
of the marsh grass 6 mo after transplantation. Two additional sides of
each pond were planted with marsh plants in November 1974. The marsh grass
grew extremely well, and at the termination of the project it was approxi-
mately 1.2-1.5 m tall and had completely covered the pond banks. Addi-
tionally, a rich organic sediment had developed in the ponds prior to their
use.
A tidal simulation system (TSS) was designed and installed between
ponds 2 and 5 which were to be used as control ponds and 3 and 4 which were
to be used as treatment ponds (Figure 4). The TSS was operated in such a
fashion that 46 cm of water was pumped from one pond to the other every 12
hrs and then the flow was reversed.
The overflow pipes in the two ponds to which oil was to be added were
fitted with a wooden box containing polyurethane to absorb any oil contained
in run-off waters.
The salinity in all ponds eventually dropped to about 6 o/oo as the
result of rainfall and the continual mixing caused by the operation of the
TSS.
Barges originally planned for obtaining seawater were no longer avail-
able; consequently, it was decided that the salinity would be increased by
the addition of commercial salts. Calculations were made to determine the
50
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amounts of Na, K, Ca, Mg, Cl and SO, required to increase the salinity to
12 o/oo based on the ratios of these ions in Rila Sea Salts Mix which was
used routinely in the laboratory. The salts added were NaCl, CaCl~, MgSO,,
MgCl2 and KCl.
In November 1973, approximately 360 mullet, 750 brown shrimp and 360
oysters were added to each pond. The oysters were placed on the bottom
along one side of each pond.
Conduct of the Oil Spill
On 17 July 1974, 11.3 liters of Empire Mix were added to each test pond
(ponds 3 and 4). The additions of oil were made in the following manner.
Three 3.8 liter plastic jugs without tops and tied to a large brick were filled
with oil, perforated in the bottom and sunk in the middle of pond 3 at
10:45 a.m. A small amount of the oil was spilled onto the surface during
the additions. For a period of hrs following the spill, oil seeped up to
the surface from the sunken jugs and was distributed around the pond by
wind action. At the outset all of the oil was blown almost directly to the
northwest corner of the pond. Within 3 hrs, the wind had shifted to a
southeasterly direction and the oil slick moved across the pond. By late
afternoon approximately 3/4 of the pond surface was covered with a thin
layer of oil, and oil was visible around edges of the pond. Similarly, oil
was added to pond 4 at 3:00 p.m. on the same day. At that time, and for
several hrs thereafter, no wind was evident; and the oil spread rather
evenly in all directions from the center of the pond (Figure 5).
On 19 July, oil was visible around the edges of the pond but very
little remained on the surface; the small quantities of oil visible on the
surface were located predominantly on the south side of the pond. Only a
small amount of residual oil was left in the jugs when they were removed
from the ponds. Using the procedure described above, an additional 11.3 liters
of oil was added to pond 3 at 10:00 a.m. and 11.3 liters of oil was added to
pond 4 at 10:30 a.m. The ponds were continually monitored for dissolved
oxygen content and no observable differences were noted between the treated
and control ponds.
53
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Figure 5. Photograph taken shortly after oil spillage in one of the
pilot-plant ecosystem ponds. Note ring of oil near the
three floats in the center of the pond.
54
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Infrared analysis of water samples taken immediately prior to the
second oil spill indicated a concentration of between 0.16 and 0.32 mg/1
total oil in the water column. In terms of the amount of oil added to the
ponds, the following calculations should be of interest. If the total
amount of oil added to the ponds had been evenly distributed onto the
surface, an oil slick of 0.01 - 0.02 mm in thickness would have resulted.
On the other hand, if all of the oil added to the ponds had been equally
distributed throughout the water column, the concentration of oil in the
water column would have been approximately 4.0 mg/1. Based upon the infrared
analyses, if a uniform concentration of oil throughout the ponds could be
assumed, the concentration of oil in the ponds would have been 0.2 mg/1.
Small patches of oil were visible on the surfaces of the ponds for
7 days after the final oil additions were made. Furthermore, oil was
noticeable on the edges of the ponds and on the marsh grass. It is highly
significant to note that the waters in the two oil ponds became extremely
clear as compared to the control ponds. It was possible to see for consider-
able distances under water in these ponds while visibility was limited to
0.3-0.6 m in the control ponds. With time, very little evidence of the oil
spill was observable although small oil-stained areas remained on the banks
for several weeks after the oil spill. The presence of oil in the sediments
of the ponds was confirmed when the cast net was dragged on the bottom of
the ponds; small bubbles of oil rose to the surface and then quickly
dissipated.
Overall, it was concluded that the oil spill operation, as conducted,
was highly successful in terms of getting reasonably uniform distribution
of oil in the ponds.
Key Events
The chronological order of key events during this study were:
12 December 1973 First Pre-spill sampling
7 February 1974 Second Pre-spill sampling
1 June 1974 Approximately 475,000 1 of water was
removed from each of the four ponds
and replaced with seawater (salinity,
14 o/oo).
55
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11 June 1974 Third Pre-spill sampling
24 June 1974 Salts added to ponds
17 July 1974 11.3 1 of Empire Mix added
to each test pond (3 & 4)
19 July 1974 another 11.3 1 of Empire Mix
added to each test pond (3 & 4)
28 July 1974 First Post-spill sampling
19 August 1974 Second Post-spill sampling
24 September 1974 Third Post-spill sampling
6 November 1974 Fourth Post-spill sampling
9 January 1975 Fifth Post-spill sampling
11 March 1975 Sixth Post-spill sampling
6 May 1975 Seventh Post-spill sampling
16 May - 2 June 1975 Experiment terminated
Environmental Data
Daily recordings of barometric pressure, temperature, relative humidity
and rainfall were obtained from NASA for the immediate area. No unusual
events occurred during the test period.
The salinity was maintained between 6 and 10 o/oo for the duration of
the study by the additions of seawater and/or commercial salts. The dissolved
oxygen ranged between 6 and 10 mg/1.
Fate Studies
The first pre-spill core samples of the sediment showed a fairly large
concentration of straight-chain hydrocarbons below n-C23 in all four ponds,
indicating a contribution from source material other than terrestrial
plants. One likely source would be algae, which contribute large amounts
of n-C17 to the organic matter in the sediments. Pristane and phytane were
present in all sediments, but the pristane/phytane ratios were not typical
of most crude oils. Pond 2 (control) exhibited a completely different
profile from the other three ponds. All ponds contained trace quantities
of hydrocarbons as shown by infrared spectrophotometry; Ponds 2 (control), 4
(oil) and 5 (control) had 0.025 mg/1 hydrocarbons while Pond 3 (oil) had
0.038 mg/1 hydrocarbons. [149-153]
56
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Six mo later (just prior to the oil spill) surface grab samples of
sediment taken from the ponds in the vicinities of the first core samples
were black and contained hydrogen sulfide indicating that they were in
extremely reducing environments.
Hydrocarbon analyses of sediments from Pond 2 (control) showed large
concentrations of n-C15 and _n-C17 and a greater concentration of higher
molecular weight alkanes (C25, C27, C29, C31). These observations were pro-
bably a reflection of the occurrence of an algal bloom and the presence of
considerable marsh plant debris.
In Pond 5 (control) the distribution of higher molecular weight hydro-
carbons remained the same; however, there was an increase in the concentra-
tion of lower molecular weight hydrocarbons. This pond (5) contained the
highest total hydrocarbon concentrations of any of the 4 ponds.
The vast quantities of grasses, observed growing in Pond 3 (oil) were
reflected in the hydrocarbon analyses. Also, the low molecular weight
hydrocarbons usually contributed by the blue-green algae had decreased.
The samples from Pond 4 (oil) had a rather thick algal mat (3-4 cm)
and n-C17 was the dominant hydrocarbon. There appeared to be a loss of
higher plant contribution to this pond.
Even though some changes in total hydrocarbon concentrations and
distributions occurred in the ponds, it is interesting to note that the
percent lipid/dry weight of sediment was about the same in all four
ponds. [154-158]
Eleven days after the initial oil spill there was a slight oil sheen
at the edge of both treated ponds. Core samples oozed oil. The mud samples
taken from the east bank of the ponds were black and contained hydrogen
sulfide. Hydrocarbon content was exceedingly high in the sediments from
Pond 4 (oil). Gas chromatographic analysis indicated petroleum hydrocarbons
57
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in Pond 4 (oil) whereas the chromatograms for sediments in Pond 3 (oil)
indicated the presence of high concentrations of algal mat material.
Sediment samples taken from the middle of the ponds one mo after the
oil spill indicated that a small percentage of the crude oil had reached
these sediments since the percent hydrocarbon/lipid weight was slightly
higher in the test ponds than it was in the control ponds. It became appa-
rent that the oil was not evenly distributed throughout the sediments and
consequently it was impossible to accurately follow the fate of the oil in
the sediments. Apparently, the actions of the pumps greatly influenced the
results. [159-164]
Consequently, the final sediment samples were composites of 4
separate samples taken 9 mo after the oil spill. Sediments from the
half of the pond nearest the pump were combined, and those opposite the
pump were combined. In Pond 3 (oil), the total hydrocarbons in the sediments
nearest the pump represented 42 mg/1, whereas the total hydrocarbons in
sediments opposite the pump yielded 1080 mg/1. The organic matter near the
pump was sparse and was in an oyster shell hash, while samples from the
opposite side were a dark clay mud. Neither the mud nor the shell hash had
a high amount of extractable lipid material (both less than 1%), although
the sediments opposite the pump contained 5 times as much as the sediments
near the pump. The chromatograms contained a very large envelope with only
a small percentage of normal hydrocarbons resolved. The sample nearest the
pump showed a distribution of aliphatic hydrocarbons more characteristic of
young sediments. In this sample the odd-numbered carbons _(n-C21, n-C23,
_n-C25, n-C27, n-C29, n-C31) predominated. In the sample opposite the pump,
however, the envelope contained only a few resolved peaks with no odd-
carbon dominance. In Pond 4 (oil), the sediments nearest the pump contained
654 mg/1 total hydrocarbons, whereas the sediments opposite the pump con-
tained 100 mg/1. Again, the percent of extractable lipid material represented
less than one percent in these samples. GC results indicated that the
sediments opposite the pump were the least changed from the last sampling
period. These sediments yielded the most typical odd/even-carbon number
ratio of hydrocarbons in the higher molecular weight range, i. e_. strong
58
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odd-carbon number preference. These sediments also appeared to contain a
significant amount of algae (n-C17 present in high concentration). A large
unresolved envelope was present in the n-C17 - n-C25 molecular weight
range. The unresolved envelope was not in the same molecular weight range
as that of Empire Mix, and there was little evidence that Empire Mix was
present in these sediments. GC results indicated large amounts of unresolved
hydrocarbon material. [165]
Microorganisms
The total bacterial population in the water column of the ponds and
the sediments fluctuated from sample to sample, but there was no discernable
difference due' to the oil spill. Furthermore, no significant shifts in the
composition of the microbial population were observed as determined by tests
conducted for yeasts, fungi, actinomycetes, hydrocarbon-utilizing/sulfate-
reducing bacteria and hydrocarbon-utilizing/nitrate-reducing bacteria. The
ratio of hydrocarbon-utilizers to total microbial population was essentially
the same for all 4 ponds and was not affected by the oil spill. [166-167]
Microbiological observations made in association with disease in
mullet are reported in a later section on Mullet Disease.
Phy toplankton /Zooplankton
No quantitative differences were observed in the phytoplankton populations
existing in the control and oil-treated ponds as a result of the spilling
of Empire Mix on 17-19 of July 1974. Increases in numbers of phytoplankton
were observed in all 4 ponds after the oil spills. Since these increases
occurred in the control ponds as well as the oil ponds, they must be attri-
buted to some environmental factor other than the oil. [168-172]
Prior to the oil spill there was a mixed zooplankton population in all
ponds with Acartia (copepod) and Brachionus (rotifer) predominating. Imme-
diately following the oil spill there was a sharp decline in the total
zooplankton population in the oil-treated ponds followed by a gradual
return to pre-spill levels during the next 20 days. Significantly, after
this time Brachionus was clearly the dominant species. Since these changes
did not occur in the control ponds, the decline in the Brachionus and
59
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Acartla populations with a subsequent shift to a Brachionus dominated
population must be attributed to the presence of the Empire Mix crude
oil. [168-172]
Core Sampling-
Core samples were taken from the ponds on a regular basis beginning in
September of 1974. Prior to that time, a few core samples had been taken
at irregular intervals. Over 115 total core samples were taken.
The samples taken from Ponds 2 (control) and 4 (oil) after the first
oil spill were grown in liquid and agar media. Bold's Basal Media (BBM)
was used to support freshwater algae, and Heinle's Media at a salinity of
20 o/oo was used for culture of saltwater algae. Mud was taken from the
top and bottom of each core. Liquid cultures were counted as to cells/ml.
Agar plates were counted as to number of colonies on the plate.
Presumably, each colony represented a single cell falling on the agar when
mud was sprayed on the plate.
The analysis of the core sample data indicated little or no pattern of
algal population growth. Though cores were always taken in the same corner
of each pond, no relationship existed between the numbers of algae in cores
taken two weeks apart. It had been assumed that algae were spread evenly
in the mud of the ponds, and that a decline in population in one area
represented a decline in the entire benthic population. This was disproved
by taking 2 cores 1 m apart on several sampling dates and comparing
the results. No relationship was observed between the numbers of algae
grown from duplicate cores.
In conclusion, the areas of concentration and paucity of algal popula-
tions present in the mud are rather like a checkerboard in their distribution.
It is not known if this situation is unique to the experimental ponds.
Marsh Grass
Due to the small quantities of Empire Mix spilled and due to its
uneven distribution along the pond banks, no meaningful conclusions could be
drawn from this phase of the study. There was very little plant mortality
60
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associated with the spill; and, while the plants in the oil-treated ponds
contained more oil than the plants from the control pond, there was a
considerable difference in the amounts depending upon the location in the
treated ponds from which the plants were taken. By 125 days there was no
significant difference in the oil residue levels contained in the plants
taken from the treated or control ponds.
Oil Uptake
The analyses of several shrimp and mullet samples collected just prior
to the addition of the second 11.3 liters of oil to the test ponds indicated oil
uptake. All samples collected after that time, including the first post-
spill sampling period, failed to indicate oil in any of the samples of
shrimp, mullet or oysters by the analytical techniques employed (GC analyses
and LC analyses using a fixed wavelength UV detector at 254 nm). Subsequent
samples were archived and analyzed later using a Schoeffel GM-770 variable
wavelength UV detector at 277 nm. While the number of samples in most
cases was small, certain tentative conclusions can be drawn.
Mullet-
Brain samples collected throughout the 9 1/2-mo study all contained
increased concentrations of aromatic compounds while very few gill samples
had measurable increases in aromatics. Increased concentrations of aromatics
were evident in liver samples collected 11 days after the spill but were
not noticeable in the liver samples collected for the following 4 mo probably
due to the small number of samples analyzed. Liver samples collected for
the remainder of the study showed considerable increases in aromatics. [173]
It should be pointed out that the aromatic hydrocarbon profile of the
mullet tissue samples changed during the course of the study. There was a
general shift to shorter retention times for the predominant peaks in the
samples from the oil-treated ponds thus indicating an increase in polarity
of those compounds. This could be a reflection of the degradation of the
aromatics into phenolic, diol, or epoxy-like compounds.
Shrimp-
The data on the oil content of shrimp hepatopancreas and muscle were
inconsistent, due in part to the small number of samples available for
61
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analysis. Of 21 post-spill muscle samples (pooled samples, 5 organisms per
sample) oil was indicated in 4 samples. Of 22 post-spill hepatopancreas
samples (pooled samples, 5 per sample) 8 samples showed an increased
amount of aromatic compounds. The significance of these data is the fact
that oil was found in the hepatopancreas of shrimp, 5 1/2 mo after the oil
spill. This would indicate that the shrimp are obtaining the oil from the
environment (sediments). [174]
Oysters-
The concentration of oil in the oysters, even at the first sampling
period was low. This was not surprising since the concentration of oil in
the water column was extremely low. However, after 3 1/2 mo only 25-33%
of the oysters from the test ponds had any traces of oil in them, thus con-
firming the generally accepted fact that the oysters purge themselves of oil
with time. [175]
Enzymes
For the majority of the 10 sampling periods, the following enzymes
were assayed: acetylcholinesterase (CHE), alkaline phosphatase (ALP),
g-glucuronidase (GLU), glutamic pyruvic transaminase (GPT), malic dehydro-
genase (MDH) and lactic dehydrogenase (LDH). Enzymes were assayed in
mitochondrial and post-mitochondrial supernatant fractions of whole oysters,
shrimp hepatopancreas, and mullet brain, gill, liver and muscle homogenates.
These enzymes were selected on the basis of laboratory data indicating
effects related to oil treatment. It was hoped that CHE might give indica-
tions of nervous and muscular function, ALP changes in membrane transport,
GLU conjugate breakdown and hormone usage, GPT energy source shifts and
protein metabolism, MDH aerobic metabolism and LDH anaerobic metabolism.
Much variability was apparent with many of the samples. Also some
seasonal variation in specific activities occurred with most of the enzymes
studied.
62
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Mullet-
In mullet, elevations of muscle CHE immediately after the oil spill
could indicate increased nervous and muscular activity in response to the
presence of the noxious chemicals. Occasional decreases in brain CHE
activity may be a reflection of the oil-treated animals' poor health. [176-177]
There were generally no effects of oil exposure on supernatant ALP
activities from any of the organisms studied. Mitochondrial activity in
brain, liver and gill appeared depressed on several occasions, while muscle
mitochondrial activity showed both elevations and depressions during the
course of the experiment. The results do not correlate with the changes
seen in the individual mullet experiment, which will be discussed below.
The significance of these changes in activity is not clear. Similarly, the
laboratory experiments did not result in changes in ALP activities in
mullet following oil exposure. [178-181]
There was no effect on GLU by oil exposure in brain or the supernatant
fraction of gill. Gill mitochondrial activity was depressed very late in
the experiment. Liver activity in both fractions appeared depressed imme-
diately after the oil spill and may have been elevated very late in the
experiment. Muscle supernatant GLU may have been elevated on several
occasions following the oil spill. Elevations in activity could indicate
stress in that the enzyme could activate circulating hormone conjugates at
the tissue level. GLU is the only enzyme in mullet shown to be affected by
acute oil exposure in the laboratory in which muscle mitochondrial levels
were decreased and brain mitochondrial levels increased. Differences in
laboratory and field results might very well reflect the difference between
acute and chronic effects and the time required for hormonally-mediated
reactions to stress to become manifest. [182-185]
There were no oil-mediated effects on MDH in gill mitochondria and
brain, gill and liver supernatant. On a few occasions, mitochondrial MDH
from brain and muscle appeared elevated, supernatant MDH from muscle appeared
depressed and mitochondrial MDH from liver showed both an increase and
63
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decrease. The changes observed may indicate some effect upon the animals'
rate of aerobic metabolism. [186-189]
LDH was not affected by oil in any of the tissues observed. This
correlates with the laboratory results. [190-193]
GPT levels were not affected in brain or liver mitochondria or gill or
muscle supernatant. GPT levels in brain supernatant and gill and muscle
mitochondria were elevated during the early samplings after the oil spill.
Liver supernatant GPT appeared rather consistently depressed following the
oil spill. Those latter results could indicate alterations in nitrogen
metabolism in the oil-treated organisms. [194-197]
Typically, the results of the pond oil exposures regarding changes in
enzyme activities in mullet were not dramatic. This correlated with the
results of laboratory studies. The results here on pooled tissue samples
are not as clear-cut as the results obtained with individual mullet, where
the individual health and condition could be better correlated with enzyme
activities. Some of the particularly aberrant data points can be correlated
with mullet samples that show very pronounced disease symptoms. In general,
the results indicate that some of the enzyme systems of the mullet are
responding to their stressful situation.
Twenty days after the first oil spill, enzymes were assayed in tissues
of individual mullet in an attempt to provide closer correlations among
biochemical, physiological and pathological parameters than was possible
with the usual pooled samples.
Liver weight to body weight ratios were increased in the organisms
from the oil-treated ponds over those from the control ponds. Variability
was low between the two ponds within each group. Similar increases were
observed in laboratory studies and are consistent with observations of
liver hypertrophy induced by certain xenobiotics in other organisms. The
lack of differences between the two ponds in each group with regard to liver
weight to body weight ratios indicates that the increases observed in the
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test ponds were caused by an oil-related phenomenon and not general nutri-
tional state. However, total length to body weight ratios were not consis-
tent between the two groups. The ratios from the oil-treated ponds were
higher than the ratio from Pond 2 (control) but lower.than that from Pond 5
(control). The ratios, which should give an insight into the nutritional
condition and/or general health of the fish, indicate that the fish from
Pond 5 may have been suffering from a nutritional deficiency, and that the
fish from Pond 2 were the healthiest. [198]
*
ALP was increased over control values in gill and muscle. The enzyme
appeared decreased in liver mitochondria and unaltered in liver supernatant.
The fish from one of the control ponds (5), which may have been under a
nutritional stress, had higher ALP in the gill and muscle than the fish
from the other control pond (2). Likewise, the fish from Pond 4 (oil),
which had a more severe disease condition, had higher ALP in the gill and
muscle than the fish from the other oil pond (3). These elevated ALP
levels in the oil-treated fish could indicate an effect of oil on membrane
permeability. In addition the enzyme could be an index of the degree of
stress the organism is experiencing, since changes in this enzyme have been
noted in stress-related work in mammalian systems. [198]
GLU was not affected by oil in either the gill or the liver. In both
fractions of muscle the activities of the organisms from the oil-treated
ponds were higher than those from the control ponds. Muscle mitochondrial
GLU from one control pond (5) fish was higher than that taken from the
other control pond (2). GLU, a hormonally-controlled enzyme, could be an
indication of stress in muscle tissue. GLU hydrolyzes glucuronic acid
conjugates, which may allow the circulating forms of some hormones to enter
the effector tissues. Thus, elevated GLU activities could indicate a
greater utilization of stress-mediated hormones in peripheral tissues. [198]
CHE in the muscle was not affected by oil exposure. [198]
MDH was not affected in the gills or in the liver mitochondria. MDH
was depressed in the liver supernatant and elevated in both fractions of
65
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muscle in the oil-treated fish. These results indicate changes in the
metabolic activities of tissue and their level of aerobic metabolism in
response to stress, although the changes in MDH do not seem directly propor-
tional to the degree of stress the organisms were experiencing. [198]
Generally, the results of the individual mullet tissue enzyme assays
showed some differences not apparent in the pooled mullet tissue enzyme
assays. There may have been a masking effect by pooling tissues. Although
some trends were indicated, no definite correlations between biochemical
and pathological parameters could be made. Also, differences in mullet
samples from the pond were more pronounced than were observed with mullet
in laboratory studies. It is possible that two of the enzymes studied
here, ALP and GLU, are potential indices of stress phenomena.
Shrimp-
In shrimp hepatopancreas, there were no oil effects on ALP, MDH, or GPT
activities. GLU and LDH were depressed on some sampling occasions in the
organisms exposed to oil. No significant effects of treatment with Empire
Mix on enzymes in shrimp were observed in the laboratory which is in agree-
ment with the few enzyme effects observed in these field tests. However,
these few changes observed may be an indication of a long-term effect on some
physiological process in shrimp by exposure to Empire Mix. [199-203]
Oysters-
Generally, the oysters from the oil ponds did not have significantly dif-
ferent enzyme activities than those from the control ponds. Differences
between control and oil-treated oysters at a few of the sampling periods were
observed with CHE, ALP, MDH and GPT. There was no effect on GLU. This is
unlike the laboratory studies in which the majority of crude oil-mediated
effects on enzyme activities were demonstrable in oysters. However, the
changes observed indicate that certain biochemical and physiological processes
within oysters may be affected for several mo after exposure to Empire
Mix crude oil. [204-208]
-------
Fatty Acids
Fatty acid methyl ester weight data from all tissue samples were
highly variable and precluded their use for analysis. For this reason, all
analyses are based on fatty acid percent composition of samples using a
Duncan's New Multiple Range Test (DNMRT).
Mullet-
Of the mullet tissues examined, only liver showed any appreciable
change in fatty acid profile. Fatty acid 16:0 decreased as in shrimp
muscle tissue but with no difference found between control and oil-exposed
fish. Unlike shrimp muscle tissue, there was no increase in the percent
concentration of 16:1 fatty acid. The mullet muscle also differed from the
shrimp muscle tissue in that there was an increase in 18:0 with no real
difference in control and treatment tissues. These reactions of mullet
tissue are unexplainable, unless the trend was chain elongation from the
16C fatty acid to the 18C fraction. When the sum of all saturated fatty
acids was examined and compared to control liver tissue, there was a variable
but general decrease for both control and oil-treatment tissue. [209-210]
Shrimp-
Of the shrimp tissue examined in the pond studies, only shrimp muscle
revealed any real differences between control- and oil-exposed organisms. In
chain length there was a trend toward chain elongation upon oil exposure.
Klenk and Kremer (1960) saw a chain elongation procedure in fish in certain
conditions, and Knipprath and Mean (1966) showed elongation under tempera-
ture stress. The true significance of this finding is not clear but could
be a stress response.
Perhaps more pronounced than the above phenomenon was the trend toward
unsaturation of fatty acids upon chronic exposure of the shrimp to oil condi-
tions. The tissue from oil-exposed organisms showed a more rapid decline in
the concentration of the 16:0 fatty acid than tissue from control organisms.
There was a similar reduction in the saturated 18-carbon fatty acid. When
the sum of the 18:2 and 18:3 acids was plotted, there seemed to be a direct
67
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relationship with the reduction in 18:0. This observation suggested an
increased conversion of 18:0 to 18:2 and 18:3 upon chronic exposure to oil.
The pattern toward greater unsaturation may be a stress response. [211-213]
There was also evidence of an increase in 20:2, 20:4 and 20:5 fatty acids
with increased time after exposure, but the increase did not appear to be
related to oil exposure or with any decrease in 20:0 or 20:1 acids.
When chain length and degree of saturation were examined in the hepato-
pancreas tissue of shrimp, no statistical differences of biological variation
could be found between control and oil-exposed tissue.
Oysters-
In the examination of fatty acids from oysters, the same trend toward
a decrease in 16:0 and 18:0 acids was found. One interesting but inconsis-
tent phenomenon, when all organisms were compared, was the initial increase
in these acids after oil was spilled. After the third post-spill sampling,
the percent concentration of these two FA's was on a straight-line decrease.
No single unsaturated fatty acid could be singled out that could account
for the decrease in percent content of the saturated acids, but when total
saturation was tabulated, the decrease was highly significant. [214-216]
Conclusions-
There was a definite statistical decrease in 16:0 acid in oysters,
shrimp muscle and mullet liver tissue. The importance of this phenomenon
is not known, but others (Klenk and Kremer, 1960; Knipprath and Mead, 1966;
Vale, &t^ al_., 1970) have seen a similar pattern in response to stress. The
oyster and shrimp muscle tissue also showed a decrease in 18:0 FA. Oddly,
the mullet liver showed an increase in the 18:0 fatty acid. Vale, et al.
(1970) saw a similar response in tainted fish fillets. In general, there
was a movement toward unsaturation of fatty acids during the period of the
pond studies. This trend was seen in both the control- and oil-treated
organisms. The oil-exposed shrimp muscle showed this tendency to a statisti-
cally (P<05) greater extent than control tissue.
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Pathology
Mullet-
During the early part of the first field study, no histological changes
were observed which could be associated with the exposure of the mullet to
oil except the normal conditions associated with a bacterial infection
(described below). Subtle changes may have appeared in the gill and liver
tissues about 4-6 mo post-spill. However, when the ponds were harvested,
after 11 mo, 20 mullet from each pond were preserved for histological
study. Two significant conditions were observed. There was a high incidence
of enlarged, swollen, clubbed and fused gills in the treated fish. In addi-
tion, nearly all of the livers from treated mullet had enlarged hepatocytes
accompanied by a reduction in sinusoidal spaces. There also appeared to be
a loss of granulation with increased vacuolation as compared to the control
mullet. It seems significant that these two conditions were not observed
until late in the study, especially since the uptake studies indicated that
"oil" began to appear in the mullet liver samples late into the study
period. [217]
Shrimp-
During this estuarine-pond study in which Empire Mix was spilled, no
disease conditions directly attributable to oil were found in the shrimp.
The shrimp were in the ponds for 2 winters. During the early part of the
second winter, numerous shrimp with black spots on their bodies were observed
in all ponds. No shrimp were caught during the early spring sampling or
when the ponds were harvested in May.
Oysters-
No pathological conditions associated with exposure to Empire Mix were
observed in oysters during these field studies. The lack of response
during this study cannot be considered conclusive because of complications
with the experimental design. The oysters had been placed on the bottom on
the sloping sides of the ponds. They did extremely well for 6-8 mo (pre-
spill) as they were fat and had new shell growth. However, with time they
settled into the mud which apparently interfered with their feeding. Mid-way
69
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into the treatment period it was noted that the general health of the
oysters in all ponds was declining. Their tissues were clear and watery,
and mortality was high. All pond studies on oysters were terminated in
January (7 mo post-spill) due to the above conditions.
Disease in Mullet
An unexpected occurrence following the oil spill was the outbreak of a
severe case of fin rot in the population of mullet in the oil-treated
ponds (Figure 6). By the first sampling (13 days post-spill), all fish
from the oil-treated ponds had moderate to severely eroded fins, loose
scales, numerous petechiae and small lesions as well as active sites of
infection; most were very emaciated. The microbial counts in the water of
all four ponds were low (100-1000/ml), but 40-55% of the organisms
obtained from the oil ponds (3 and 4) were hemolytic while only 23-24% of
the organisms obtained from the control ponds were hemolytic. By the 28th
day post-spill and during subsequent samplings the fish from the treated
ponds no longer had active sites of infection, and most had fins in various
stages of regeneration. Hemorrhaging was less evident; however, their
scales dislodged much easier when handled than did the controls. When the
experiment was terminated (11 mo), the majority of the fish harvested were
found to have regeneration marks on their fins indicating that the original
infection included most of the oil-exposed mullet. It was a general observa-
tion by all personnel involved that the oil-treated fish (even late in the
study when they outwardly appeared to be recovering) were much more suscep-
tible to handling stresses. Fish taken from all ponds at this latter stage
of the study would initially appear normal. However, after an hr or so in
aerated holding vats the oil-exposed fish would develop numerous hemorrhages
and would often die while the fish from the control pond remained normal in
appearance. [218-220]
SECOND PILOT-PLANT ECOSYSTEM STUDY
Description of the System
It was decided that more information might be obtained in a second
pilot-plant ecosystem study by testing 3 different crude oils rather
than repeating the original study using only Empire Mix crude oil.
70
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Figure 6. Photographs of mullet with various degrees of fin erosion. A and B—normal,
C and D—very heavily eroded fins.
-------
Accordingly, all remaining mullet were removed from the ponds, the salinity
of the ponds increased to 13-14 ppt by the addition of salts, and the tidal
simulation system shut down. Each pond was stocked with 350 mullet, 1400
shrimp and 400 oysters in June 1975. In an effort to obviate the problems
encountered during the first field study, oysters were placed in galvanized
baskets suspended on stilts approximately 60 cm beneath the pond surface.
One hundred oysters were placed in each corner of each of the 4 ponds.
On 25 July 1975, 11.3 liters of oil was spilled in each of ponds 3, 4, and 5;
Saudi Arabian crude was spilled in pond 3, Nigerian crude in pond 4, and
Empire Mix crude in pond 5. The procedure was repeated on 27 July 1975, so
that each test pond received a total of 22.6 liters of oil. Pond 2 was left
untreated to serve as control.
During this second field study the following samples were taken on a
routine basis: (1) water and sediments for oil analysis, (2) phytoplankton
and zooplankton for species diversity counts, (3) oysters for oil analysis
and bacterial counts, and (4) mullet and shrimp for gross pathological
examinations.
Environmental Data
Daily recordings of barometric pressure, temperature, relative humidity
and rainfall were obtained from NASA for the immediate area. No unusual
events occurred during the test period.
The salinity showed a steady decline in all ponds from 13-14 ppt ini-
tially to approximately 9.5 ppt at the conclusion of the study. The dissolved
oxygen ranged between 6 and 9 mg/1.
Fate Studies
The ponds were sampled on June 24, July 29 and October 6, 1975.
The last 2 samples were taken after the addition of a small amount of
various crude oils were spilled on Ponds 3, 4, and 5. Samples were taken
from the middle of each pond, and only Pond 5 (Empire Mix) contained oil in
the samples.
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Control Pond (Pond 2)-
This was originally a control pond and was retained as a control
pond for the second study. The aliphatic hydrocarbon distribution was
unchanged throughout this period, but it was apparent that large amounts
of the planted marsh grasses around the pond's edge had increased the
natural biogenic organic carbon load in this pond over the two-year
period. [22l]
Pond Treated with Saudi Arabian Crude Oil (Pond 3)-
Pond 3 was an Empire Mix treated pond in the first field study. In
July of 1975, Saudi Arabian crude oil was spilled in this pond. Chemical
analysis one year after the first spill indicated that the oil had degraded
to the degree that oil hydrocarbons (as determined by GC) were not recog-
nizable in sediment samples with the exception of very small areas of
sediment along one edge of the pond which apparently accumulated a heavier
load of oil. Pond 3 has had a much greater input of organic matter into
the sediments from marsh plants and grasses growing in the pond than the
other three ponds. [222]
Pond Treated with Nigerian Crude Oil (Pond 4)-
Pond 4 was also an Empire Mix treated pond in the first field study.
In July of 1975, Nigerian crude oil was spilled in this pond. During the
year after the initial spill, several of the hydrocarbon analyses indicated
fairly high concentrations of crude oil hydrocarbons. However, the oil was
not evenly dispersed over the sediments; and, therefore, some core analyses
did not indicate oil. Thus, assessment of oil in the sediments was dependent
on sampling area. [223]
Pond Treated with Empire Mix Crude Oil (Pond 5)-
Pond 5 was a control pond in the original pond study. Empire Mix was
spilled in this pond in July of 1975. Prior to the spill the hydrocarbon
analyses of the pond sediment were very similar and showed little change
either in distribution or concentration of hydrocarbons. After the spill
the hydrocarbon analysis of the sediment changed drastically and indicated
oil pollution even in the sample taken from the middle of the pond. Possibly
73
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more mixing occurred in this pond, causing some of the oil to reach the
bottom sediments in the middle of the pond. On the other hand, there was
more grass in the middle of the other ponds which might have adsorbed any
oil coming in contact with it, reducing the amount of oil eventually reaching
the sediments. [224]
Phytoplankton/Zooplankton
Again, as in the first chronic study, no quantitative differences were
seen between the phytoplankton populations in the control pond and the oil-
treated ponds. The apparent lack of effect of Empire Mix, Saudi Arabian
and Nigerian on the phytoplankton populations in the respective ponds is in
agreement with laboratory studies which predict little or no reduction in
cell growth at the concentrations of oil found in the experimental ponds.
The changes seen in the phytoplankton of the ponds during the study reflect
seasonal and environmental changes rather than changes due to the acute or
chronic effects of the crude oils. [225-228]
Prior to the second pilot-plant study (July 1975) zooplankton samples
indicated a shift to a Brachionus (rotifer) population in all ponds. In
June of 1974 (pre-first pilot-plant study) the population consisted of a
near equal mixture of Brachionus and Acartia tonsa (copepod), both of which
were present in relative low levels (<10 organism/1). However, by June of
1975 Brachionus had greatly increased in numbers (>100 organisms/1 - control;
50 organisms/1 - oil-treated) while the Acartia populations remained at
about the same level as the summer of 1974 (<10 organism/1 - all
ponds). [225-228]
Immediately after the July 1975 oil spill, the Acartia populations
declined sharply in the Saudi Arabian, Nigerian and Empire Mix treated
ponds while remaining stable in the control pond. By late August (4-5
wks), the copepods in the oil-treated ponds had recovered to pre-spill
levels. The Brachionus population remained stable in the control, Saudi
Arabian and Nigerian-treated ponds while it declined in the Empire Mix-
treated pond immediately following the spill and then recovered by late
August. [225-228]
74
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The zooplankton populations (Acartia and Brachionus) in all 4 ponds
began a gradual decline about the first of September. This decline coincided
with a heavy Anacystis (blue-green) bloom which occurred at that time.
This factor prevented a further assessment of the impact of the test oils
on the zooplankton populations. [225-228]
Core Sampling-
Culture procedures were modified for the second oil spill. One core
sample was taken from each of the 4 ponds and mud taken 8 cm from the
top of the core. This mud was used to inoculate agar media. Three kinds
of media were used: BBM, Heinle 20 o/oo, and Heinle's Media at 3 o/oo.
Liquid media were not used for counting.
Two days after the oil spill, a decline was noted in the algal popula-
tions in all the oiled ponds. Four days after the spill, all 3 oiled
ponds showed an increase in algae. This was the only instance of a simulta-
neous change recorded in different ponds. Six days after the spill, the
random patterns of growth and decline again appeared on the graphs, indicating
the benthic population had returned to normal. [229-232]
In all, 224 liquid cultures and 1028 agar cultures were counted. Most
of the algae seen in Heinle's Media were diatoms. BBM showed a wide range
of genera throughout the experiment.
Microorganisms
An attempt was made during the second field study to relate changes in
the microbial population in the oysters at various post-spill times to oil
uptake. During the study period a total of 188 oysters were sampled from
each pond. Plate counts and oil analyses were carried out for each indivi-
dual oyster. No relationship was found between microbial plate counts and
oil uptake by the oysters. The results may have been complicated by the
high organic content in the ponds which apparently had a greater influence
on the microflora than did the oil. These findings do not negate the
possibility of a relationship between the two factors in the natural environ-
ment which is not generally as rich in organic matter as the oil-treated
ponds were during the study period. [233-236]
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Primary Productivity of Phytoplankton Community
The primary productivity of the phytoplankton community in the 4 ponds
was determined by the carbon-14 technique described by Lind and Campbell
(1969) and Vollenweider (1974). A total of 2 pre-spill and 6 post-spill
samples were taken and measured for phytoplankton primary productivity.
Pre-spill measurements showed that Ponds 2 and 5 (control ponds in
first study) had slightly lower phytoplankton production than Ponds 3 and 4
(ponds treated with Empire Mix crude oil in first study). Five days after
the first respill, primary production in all 4 ponds was comparable.
During subsequent post-spill measurements, primary production persisted to
be generally higher in Ponds 3 (Nigerian) and 4 (Saudi Arabian) than in
Ponds 2 (control) and 5 (Empire Mix). In addition, emulsified Empire Mix
crude oil (10 and 100 mg/1) reduced phytoplankton growth under laboratory
conditions. [237-238]
These observations are reflected further in the total pigment concentra-
tions which also were found to be lower in Ponds 2 and 5 during pre-spill
measurements, comparable among the four ponds 5 days after the first post-
spill, and persisted to be lower than Ponds 2 and 5 during subsequent post-
spill samples. It appeared that oil stimulated the growth of phytoplankton
when present at low levels. [239]
Marsh Grass
On the fifth day following the initiation of the second field study,
Juncus and Scirpus robustus were harvested from the control, Empire Mix,
Saudi Arabian and Nigerian ponds. Following routine processing, these
plants were analyzed for their oil content. In general, oil uptake in
Juncus was higher than in Scirpus, higher in dead tissues than in live
tissues and higher in the bottom parts than in the top parts of the plants.
Oil was detected in plants from all the oil ponds while none was detected
in plants from the control pond. It appears that Juncus has a greater
affinity for Empire Mix than for Saudi Arabian or Nigerian. [240]
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Mullet - Pathology
The results of the second pond study were different in many ways from
those in the first study. With mullet the most striking difference was the
lack of visible disease in the second study. As in the first study, the
oil-exposed mullet were observed to be much more sensitive to handling and
holding procedures than the controls; however, they did not develop fin
rot. In the first study there was no impact on the growth of mullet.
However, at the end of the second study there was a striking difference in
the size of the mullet from the different ponds. When stocked, the mullet
averaged 82 mm in length and averaged 15 g in weight. When harvested 9
1/2 mo later, the average lengths and weights were: (1) control mullet-151
mm; 65 g (2) Nigerian-treated mullet-129 mm; 35 g (3) Saudi Arabian-
treated mullet-124 mm; 42 g (4) Empire Mix-treated mullet-150 mm; 64 g.
From the data, it is obvious that the growth of the Nigerian- and Saudi
Arabian-treated fish was adversely affected, while the growth of the Empire
Mix-treated mullet was almost identical to the growth of the control mullet.
This reduction in growth of the mullet and a similar reduction in the growth
of oysters (see below), seems significant in the light of the uniformity of
food supplies in all ponds.
The histological examination of the mullet samples taken at the end of
the second study produced the following observations: (1) Mullet livers
from all 3 oil-treated ponds had enlarged hepatocytes and indistinct cord
structures. These conditions were similar to, but less severe than, those
reported in the first study and were more localized. (2) The incidence of
severely clubbed and fused gill filaments observed in the first study was
not observed in samples from the Empire Mix- nor Saudi Arabian-treated
ponds but was observed to a much lesser extent in samples from the Nigerian-
treated pond.
When comparing the chronic effects observed in the first and second
pilot-plant studies, it should be noted that the first study was terminated
after 11 mo while the second lasted for nearly 9 1/2 mo.
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Shrimp - Pathology
During the second field study in which 3 oils were spilled in separate
ponds, an unusual condition was observed which was not previously encountered
during this study nor cited in the literature. Oil was spilled in mid-July
1975. During the October sampling period, shrimp were obtained from the
Empire Mix pond which had whitish spots at various places on their eyes.
This condition has been termed the "white eye syndrome". Some had very small
spots on the ventral side towards the posterior edge of the eye which were
hardly visible, while others had large spots covering nearly half of their
eyes (Figure 7). In sectioned material, the crystalline cones and the
structures associated with the ommatidia (pigment cells, retinula cells,
rhabdomes, etc.) were eroded away. Some brownish material, probably the
remains of the pigment material which normally surrounds the ommatidia, could
be seen in the lesions (Figure 7). These diseased areas were clearly non-
functional. Usually both eyes were affected but not always to the same
degree. Another sample was taken the first of November to make certain this
was a commonly occurring problem. Significantly, nearly all of the shrimp
from the Empire Mix pond had the "white eye syndrome" (18 out of 19) while
only 6 out of 13 in the Saudi Arabian pond, and 7 out of 15 in the Nigerian
pond had the "white eye syndrome". The pathology associated with the syn-
drome was much more severe in shrimp from the Empire Mix-treated ponds than
from the Nigerian and Saudi Arabian ponds. Shrimp from the Nigerian and
Saudi Arabian ponds often had only one or two very small spots on one eye
while those from the Empire Mix pond almost always had large and small spots
on both eyes. A total of 24 shrimp were obtained from the control ponds and
all were normal. Additional samples could not be obtained because the
weather turned cold and the shrimp buried in the mud. No additional samples
were obtained from any of the ponds due to cold weather. Apparently the
shrimp were unable to survive the cold winter, as none were obtained in the
early spring nor during the final harvest.
Oysters - Pathology
While the experimental design of this portion of the study was con-
structed primarily to determine if there existed a relationship between the
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Figure 7. Photographs of whole and sectioned shrimp eyes.
A—normal eye; B—eye with white spot; C—photomicro-
graph of a normal section of eye; D—photomicrograph
with necrotic area visible just under corneal layer.
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resident microflora and oil uptake by the oysters, the observation was made
during the third and fourth sampling periods that the oysters from the
Saudi Arabian- and Nigerian-treated ponds were in poorer health than those
from the Control and Empire Mix-treated ponds. Based on this observation
and on the lack of positive data from the microbial assays, routine sampling
was suspended and the remaining oysters left undisturbed for the duration
of the study. Unfortunately, by the time this decision was made a large
number of the oysters in each pond (180 out of 400/pond) had been removed.
Nevertheless, there was a distinct difference in the overall state of health
and in the percent mortality of the surviving oysters in the various ponds
at the time of harvest (9 1/2 mo exposure).
The oysters from the control pond were in very good condition. They
were generally fat, had good color and 2 to 5 mm of new shell growth. The
oysters in the Empire Mix pond did not appear as healthy as those in the
control pond but were still in good condition; they had less color and were
slightly watery. There had been some new shell growth, but in all cases it
was less than 2 mm. The surviving oysters in the Nigerian and Saudi Arabian
ponds were in very poor condition. They were clear, watery and appeared
emaciated. Histologically, they resembled recently spawned oysters. One
unusual histological alteration was the occurrence of fibrinoid degeneration
of the connective tissue of the mouth and food groove of most surviving
Nigerian-exposed oysters and a few Empire Mix-exposed oysters. [241]
If all oysters stocked (400/pond) are considered, the percent mortalities
for the oysters stocked in each pond were: control, 23%; Saudi Arabian-
treated, 54%; Nigerian-treated (50%); Empire Mix-treated, 31%. If those
oysters removed for microbial analysis are not considered (180/pond), the
percent mortalities would be: control, 42%; Saudi Arabian-treated, 85%;
Nigerian-treated, 91%; Empire Mix-treated, 58%. In either case the percent
mortalities for oysters in the Saudi Arabian- and Nigerian-treated ponds
were much higher than those for the control or Empire Mix-treated ponds.
If the mortality data is considered in conjunction with the observations
made on the state of health of the surviving oysters, it must be concluded
that the chronic exposure to low levels of Saudi Arabian and Nigerian
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crudes had an adverse effect on the state of health of the oysters. These
observations seem even more significant when it is considered that the
oysters were suspended in cages and consequently were only subject to
impact by substances obtained from the water column directly or via the
food chain.
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SECTION VI
SUMMARY
INTRODUCTION
The format to be followed in the SUMMARY will parallel the RESULTS
SECTION in that the Laboratory, Tidal-Pond and Pilot-plant studies will be
summarized separately. A discussion of the merits of the pilot-plant
system and an interpretation of the results will be presented in the
DISCUSSION SECTION.
LABORATORY RESULTS
All laboratory experiments were carried out under controlled conditions
with the exception of some of the marsh grass tests which were conducted in
a natural estuarine environment. The following results in regard to the
fate and effect of crude oil in the aquatic environment of the Gulf Coast
Region have been obtained.
1. Empire Mix crude oil remains in the water column for only a short
period of time during which some of it migrates into the sediments.
Further, the degradation in, or disappearance from, the sediments is a slow
process which is influenced by the oxygen content, by the presence of other
sources of organic matter and by the availability of essential nutrients
(phosphorus and nitrogen sources). Aromatic compounds are considerably
more persistent than aliphatic compounds.
2. Phytoplankton species found in the estuarine environment vary in
their susceptibility to crude oils with EC^Q values ranging from 5.6 mg/1
for Nigerian crude oil (tested against Skeletonema costatum) to greater
than 58.0 mg/1 for Empire Mix crude oil (tested against Carteria chiuu).
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3. In general the zooplankton species found in the Gulf Coast environ-
ment are more susceptible to crude oil than are the phytoplankton with TL
values for Acartia tonsa ranging from 0.55 mg/1 for Nigerian to 1.62 mg/1
for Venezuelan.
4. From the marsh grass studies it is estimated that (A) a single oil
2
spill with Empire Mix at a dosage of 250-1500 ml of oil/m of marsh will
2
reduce marsh plant productivity by 400-900 g/m . (B) Repeated spills with
2
Saudi Arabian oil at 600-6000 ml of oil/m of marsh at the rate of 600
2
ml/mo will reduce marsh plant productivity by 500-900 g/m . (C) Dead marsh
plants (Juncus) contaminated (by soaking) with Empire Mix and Saudi Arabian
crude oils decomposed 40 to 50% slower than the control (unoiled) after 323
days in the field; therefore, natural degradation of marsh plants to detritus
is hindered by oil contamination. (D) Oiled dead plants and the detrital
material which is eventually formed from them serves as a mechanism for the
long term entry of oil and/or oil degradation products into the marsh-
estuarine food chain.
5. Accumulation of crude oil by marsh grass and phytoplankton was
shown to occur, but the data suggest that this is more a physical than a
biological phenomenon.
6. Crude oil was not highly toxic to mullet in 96-hr bioassays.
However, the 5 crude oils varied in their toxicity to mullet with Nigerian
the most toxic (TL less than 200 mg/1; 100% mortality at 200 mg/1), Saudi
Arabian the next most toxic (TL less than 350 mg/1; 100% mortality at 350
mg/1), Iranian and Venezuelan intermediate (TL between 400-800 mg/1) and
Empire Mix the least toxic (TL 800 mg/1). At laboratory treatment levels
of 25 and 75 mg/1 both Empire Mix and Saudi Arabian caused severe fin rot
outbreaks. Fish with some fin erosion were generally observable beginning
at about 8-10 days after treatment, with 100% mortality occurring after 18-
25 days. A Vibrio was tentatively identified as the causative agent.
Behaviorally, changes were restricted to hyperactivity which was more
pronounced following exposure to the more toxic oils. Some oil uptake by
the tissues was observed, and changes in fatty acids were noted although no
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histologlcal changes were evident. Changes In enzymes associated with
stress and/or detoxication were observed following exposure to crude oil.
In addition, certain microsomal oxidases were induced in livers following
short-term exposures to Empire Mix and Saudi Arabian.
7. Bioassay tests with shrimp using different crude oils showed that
Nigerian was the most toxic (TL 10 mg/1), Empire Mix, Saudi Arabian, and
Iranian intermediate (TL 15-25 mg/1) and Venezuelan the least toxic (TL
m m
35-45 mg/1). Behaviorally, the most significant response of shrimp to
crude oil was their tendency to "spiral". In nature, this "spiralling"
would most likely result in predation thus eliminating the possibility of
recovery. Enzymatically, there were no significant changes in the hepato-
pancreas following exposure of shrimp to Empire Mix. However, treatment
with Saudi Arabian resulted in great alterations in the activities of
certain enzymes of the hepatopancreas. Uptake of oil by the shrimp tissue
was highest during the first 24 hrs of exposure. The fatty acid profiles
changed after exposure of the shrimp to oil but tended to normalize with
time. Histologically, abnormalities were noted in the gills, mandibles,
gastric mill and inner lining of the carapace of shrimp exposed to Empire
Mix.
8. The general response of oysters to the addition of crude oil at
the levels tested was to close and cease pumping. This response precluded
the acquisition of data on lethality and complicated the interpretation of
other data. No histological changes or changes in fatty acid composition
could be attributed to exposure to oil. Some alterations of enzyme activity
were noted, which could be a reflection of the long periods of closing.
TIDAL POND STUDY
Two naturally stocked ponds (one control and one treated) adjacent to
Davis Bayou, which adjoins Gulf Coast Research Laboratory in Ocean Springs,
Mississippi, were chosen as sites for the tidal-pond study. A levee was
built up with sandbags to a height above the high tide level thus entrapping
a natural population of organisms. The ponds were monitored prior to and
following the oil spill (250 mg/1 Empire Mix crude) for a period of 18 mo.
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This tidal-pond study produced the following results concerning the fate
and effect of crude oil in the aquatic environment of the Gulf Coast Region.
1. High concentrations (32.5 mg/1) of oil in the water column were
found 1 day after the spill followed by entrainment of the hydrocarbons
into the sediment. On the basis of aliphatic components, the crude oil was
still evident in the sediments 12 mo later. After this time, the contribu-
tion of biogenic aliphatics appeared to mask the contribution of crude oil
aliphatics, but the top portion of the sediments still retained an increased
aromatic content.
2. Oil-sensitive phytoplankton were killed, as evidence by a 43-65%
reduction in primary productivity within 16 days. The primary productivity
returned to normal in 2 mo and there was an increased abundance and
diversity of phytoplankton in the oil pond (as compared to the control
pond) 1 yr later.
3. Oil-sensitive zooplankton were killed initially. As the oil
dissipated, there was a rapid increase in zooplankters, and after 6 mo
the population was similar to that in the control pond in both abundance
and diversity and exceeded those in the control pond in abundance after 12 mo.
4. Oil uptake by and decreased productivity of marsh grass was observed.
5. Under conditions of the test, data on mortalities of the macrofaunal
population were impossible, but predation of erratically swimming menhaden
by ladyfish was observed.
FIRST PILOT-PLANT ECOSYSTEM STUDY
The major thrust of this contract was to construct and employ a pilot-
plant ecosystem to study the long-term effects of a small quantity of crude
oil on the ecosystem. Physically the system consisted of two sets of ponds
(46 x 46 x 2.5 m) connected by means of a tidal simulation system which
lowered the water level in one pond by 46 cm every 12 hrs then reversed.
Salinity was established and maintained between 6-12 ppt through the addition
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of seawater and salts. Marsh grass planted along three sides of each pond
was intended to contribute nutrients for the shrimp, mullet and oysters in
the pond.
Overall performance of the system was excellent. The marsh grass
flourished, and the phytoplankton and zooplankton populations that developed
were representative of the estuarine areas of the Gulf Coast. The mullet
(in the control ponds) appeared healthy, and normal growth was obtained.
The shrimp exhibited excellent growth, survived the first winter which was
mild but failed to survive the second winter. Oysters did extremely well
for the first yr and displayed growth rates in excess of those normally
found in nature. The gradual accumulation of silt resulted in the loss of
all oysters during the experiment.
The emphasis in this first study was directed toward (1) assessing the
value of laboratory data when tested On a pilot-plant basis under conditions
resembling the environment and (2) ascertaining if studies on a pilot-plant
basis would yield results not observed in the laboratory.
The first test using the system involved two additions of 11.3 liters
of Empire Mix crude oil to each test pond, 48 hrs apart. The following
information was obtained from this 11 mo study.
1. The results confirmed the laboratory findings in respect to the
long residence time of the Empire Mix crude oil in the sediments and the
persistence of the aromatic compounds in the system.
2. The level of oil employed in this study did not have a measurable
effect on the number of bacteria, yeasts, fungi or actinomycetes. The
only observed impact of oil on the population was the increase in hemolytic
bacteria in the oil-treated ponds during the first 13 days following the
spill.
3. The response of the phytoplankton and zooplankton to Empire Mix
crude oil was in agreement with the laboratory data, namely that (1) less
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than 10 mg/1 stimulated the phytoplankton, (2) its effects varied depending
on the phytoplankters involved; for example Oscillatoria, a cyanophycophytan,
accounted for the majority of the observed phytoplankton increase while the
chlorophycophytan and chrysophycophytan species present increased very
little following the oil spill, (3) low levels (less than 10 mg/1) caused a
drastic reduction in specific zooplankters (Acartia; Brachionus), and (4)
the zooplankters recovered to pre-spill levels within a short period of
time (10-15 days). There was no measurable change in sedimentary algal
populations as a result of exposure to Empire Mix crude oil. It should be
noted, however, that these algal populations were distributed in a checker-
board fashion rather than distributed evenly which made the interpretation
of data difficult.
4. The oysters grew exceptionally well during the early stages of the
study but were subsequently lost because of silting; thus, no specimens were
available after 4 mo. The oysters demonstrated an uptake of low levels of
oil followed by depuration. Although no statistically significant differences
in the level of enzymes were found, some of the changes suggested that
certain biochemical and physiological processes may be affected for several
mo after the oil spill. Data on fatty acid content was in agreement with a
stress response. No abnormal pathological conditions were noted but general
poor health of the oysters was observed.
5. Since very few of the shrimp which had been placed in the ponds 8
mo prior to the spill survived the winter, the availability of specimens
was decreased. Thirty-six per cent of the pooled hepatopancreas samples
after the oil spill had an elevated aromatic content; this suggested that
the shrimp obtained oil from the sediments or their food rather than from
the water column. The fatty acids in oil-treated shrimp showed a trend
toward chain elongation and unsaturation. This trend could be attributed
to a stress response. Changes in 3-glucuronidase may be an indication of a
long-term effect on some physiological process in the shrimp. No pathological
abnormalities attributable to oil were observed.
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6. Oil uptake in mullet varied according to the tissue sampled. No
significant amounts of oil were found in gill samples at any time during
the study. All brain samples contained low levels of oil. Liver samples
taken 11 days after the spill had detectable but low levels of oil while
those samples taken shortly thereafter had no detectable oil. However,
samples taken about 5 mo after the spill as well as all samples taken
thereafter contained "oil." It should be noted that the chromatographic
characteristics of those aromatic hydrocarbons peaks in these liver tissue
samples differed from those found in the Empire Mix crude oil itself.
There was a general shift to shorter retention times for the
predominant peaks in the samples from the oil-treated ponds thus indicating
an increase in polarity of those compounds present. In general the enzyme
data reflected a stress response or the general poor health of the oil-
exposed fish. Fatty acid analyses reflected a pattern characteristic of a
stress response. Histologically, chronic exposure (9-10 mo) resulted in
severe hyperplasia, clubbing and fusion of the gill filament accompanied by
a large increase in the number of mucoidal cells. The hepatocytes of the
oil-exposed fish were enlarged causing a decrease in the sinusoidal spaces
and a distortion of the normal appearance of the hepatic cords. In addition,
there appeared to be a general loss of glycogen from the cells with an
increase in fat vacuoles. These changes were subtle in the earlier stages
of the study (4-6 mo) but were very pronounced and were present in a majority
of the samples (20/pond) obtained from the treated ponds when the test was
terminated (11 mo). These conditions were rarely observed in control fish
and when they were, they were localized as opposed to very general in the
liver tissues of treated fish.
7. By far the most dramatic finding during the first pilot-plant
study was the development of fin rot in essentially 100% of the fish in the
oil-treated ponds.
SECOND PILOT-PLANT ECOSYSTEM STUDY
The second test using the pilot-plant ecosystem involved monitoring
the effect(s) of low levels of Saudi Arabian, Nigerian and Empire Mix
without the influence of tidal simulation. A majority of the mullet were
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removed from the ponds after the first test and the ponds were restocked by
the addition of mullet, shrimp and oysters to each pond. For this study
the oysters were placed in wire baskets suspended in the ponds.
Since the emphasis in this test was to determine if the different
crudes elicited different responses, 22.6 liters of Empire Mix was added to one
pond, 22.6 liters of Nigerian added to a second pond, and 22.6 liters of Saudi
Arabian added to a third pond. The fourth pond served as a control.
This second static pilot-plant ecosystem study yielded the following
significant results.
1. The persistence of oil in the sediments was confirmed. There was
a more even distribution of petroleum hydrocarbons in the sediments probably
reflecting the discontinuation of the use of the tidal simulation system.
2. The response of phytoplankters to the 3 oils tested was essen-
tially the same as in the previous laboratory and pilot-plant tests indicating
a high tolerance to crude oil.
3. The response of zooplankters varied depending upon which oil was
employed: Empire Mix caused a significant reduction in the Brachionus
(rotifer) and Acartia (copepod) populations. This is in agreement with the
first pilot-plant study. However, the Brachionus population was unaffected
by Nigerian and Saudi Arabian while the Acartia was drastically reduced by
both oils. All changes in the zooplankton populations were short-term.
4. Marsh plant data generally agreed with the data from previous
studies in that uptake was higher in roots than in shoots and higher in
dead tissue than in live tissue. New observations made during this study
were (1) oil uptake by Juncus was higher than by Scirpus and (2) Juncus had
a greater affinity for Empire Mix than for Nigerian or Saudi Arabian.
5. Data from the second field study indicated a difference in the
impact of the 3 test oils on the growth of mullet. When stocked the
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mullet averaged 82 mm in length and 15 g in weight. When harvested 9 1/2
mo later the average lengths and weights were: (1) control mullet: 151;
65 g, , (2) Empire Mix-treated mullet: 150 mm; 64 g ., (3) Saudi Arabian-
treated mullet: 124 mm; 42 g , and (4) Nigerian-treated mullet: 129 mm;
35 g. Histologically, the changes observed in the hepatocyte cells of the
mullet from all 3 oil-treated ponds were similar by less severe than
those observed during the first pilot-plant study. However, of the 3
crudes tested Nigerian was the only one which caused the gill pathology
observed in the first study. Here too, the pathology was less severe. The
outbreak of fin rot observed in the first pilot-plant study did not occur
during this second study. However, there was an impact on growth as
indicated by the significant reduction in the lengths and weights of the
mullet in the Nigerian- and Saudi Arabian-treated ponds as compared to the
Empire Mix-treated and control ponds.
6. The suspension of the oysters in wire baskets eliminated the
problem of high mortalities associated with silting which was encountered
in the first pilot-plant study. The original intent of this phase of the
study was to determine if there was a correlation between microbial counts
and oil uptake in the oysters. While no correlation was found, the following
observations were made on those oysters remaining in the ponds at the
termination of the study: (1) The control oysters were fat, had good color
and 2.5 mm of new shell growth. There was a total mortality of 97 out of
400; (2) The oysters in the Empire Mix-treated pond were not as fat, had
less color and were slightly watery. There was a total mortality of 127
out of 400; and (3) The oysters in the Nigerian and Saudi Arabian ponds
were emaciated, lacked any color and were very watery. The total mortalities
were 201 and 186, respectively.
7. A heretofore unreported disease, designated as the "white eye
syndrome" was observed in the shrimp exposed to crude oil. The prevalence
of the syndrome varied with the oil employed and ranged from 95% for Empire
Mix, to 46% for the Saudi Arabian and Nigerian. Grossly, the diseased
areas appeared as white opaque spots varying from very small to very large
(covering up to 1/2 of the involved eye). Histologically, the crystalline
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cones and the structures associated with the ommatidia were completely
destroyed. No other damage to the eye was observed.
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SECTION VII
DISCUSSION
The pollution of the marine environment by petroleum products has been
documented since 1754 (Nelson-Smith, 1973) but has not been of major concern
because of man's belief that the vastness of the oceans and seas is such
that they have an unlimited ability to absorb pollutants. Early studies
revolved around the impact of oils on specific economically important
species such as the oyster (Galtstaff, et^ al., 1973; Mackin and Sparks,
1961; Mackin and Hopkins, 1961) and often were initiated in response to
charges that crude oils were adversely affecting the industries related to
these organisms. While some laboratory data indicated possible deleterious
effects, the observations could not be confirmed in the field and were
generally considered questionable. Then in 1967 the Torrey Canyon incident
incited public outrage due to its direct impact on man's activities at a
time when the general public was sensitized to environmental considerations.
This outrage resulted in voluminous survey type studies on the effects of
this and other major oil spills which followed. Most of these studies of
necessity involved surveys of the living and dead organisms following the
spills with follow-up surveys to determine the time required for recoloniza-
tion. They were complicated by the use of emulsifiers which were sometimes
more deleterious than the oils themselves. In general, these studies
concluded that there was a potential for severe short-term impact of oil
especially on intertidal organisms and aquatic birds. However, the marine
ecosystems were believed to recover in a short time with no long-term
impact. One exception to this general trend was the studies surrounding
the West Falmouth spill (Blumer, ^t al., 1970; Blumer, et, al., 1971; Blumer
and Sass, 1972) which indicated that No. 2 fuel oil not only was acutely
toxic to a wide range of organisms but remained in the system essentially
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unaltered for a considerable length of time. Data obtained in these and
other studies surrounding this spill indicated that the environment was
altered by the presence of the fuel oil for a number of years.
These seemingly opposing views about the long-term effects of oil
precipitated much needed scientific and governmental interest in clarifying
the long-term effects of oil under natural or seminatural conditions. The
tremendous variability in the marine environment and the complex nature of
crude oils and refined petroleum products resulted in many arguments in the
scientific community as to the best approach to utilize in attacking the
problem.
The need for complementing laboratory studies and field investigations
with pilot-plant ecosystem studies has become evident. Basically, the
system should include a complete but limited food chain which would operate
without additional manipulation. The system would be employed to (1)
verify laboratory findings on a more realistic scale, (2) establish effects
not observed in laboratory studies and (3) study long-term chronic effects
in a system simulating the natural environment. One of the major questions
regarding the establishment of a large scale pilot-plant ecosystem is how
well it simulates the natural environment.
In the present investigation, pre-spill data on the pilot-plant ecosystem
and the performance of the control ecosystem indicate that not only did the
system perform adequately, but it also simulated the ecosystem of the
estuarine area of the Gulf Coast. The ecosystem ponds employed in this
investigation were filled with natural seawater (28 o/oo salinity) diluted
with freshwater to a salinity of 14 o/oo. Decreases in salinity occurred
as a result of dilution with rainwater but was maintained at 6.0 - 14.0
o/oo through the addition of salts or natural seawater. While the salinity
in the estuaries of the Gulf Coast generally range from 0.0 to 24.0 o/oo,
the narrower range employed in the ponds was felt to be advantageous to the
present investigation. The planktonic community was typical of the estuaries
and exhibited natural fluctuations in response to changes in salinity and
season.
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Although no sediments were added to the ponds, a rich organic sediment
developed within the first 15-18 mo. The contribution of organic
detritus from the marsh grass, phytoplankton and other sources to the
sediment was clearly evident from the chemical analyses. The bacterial
flora of the water column and sediments was typical of the Gulf Coast
estuaries in numbers and diversity. The newly developed Tidal Simulation
System functioned extremely well and was probably responsible for transporting
the marsh grass detritus into the system.
The mullet, shrimp and oysters which were introduced into the system
thrived and appeared to be in excellent health. In fact, during the first
several mo in the system, the growth of the oysters exceeded that
normally occurring in the estuaries. These findings are especially important
in view of the fact that artificial feeding was limited to a small amount
of feed added for "training" the fish and shrimp to congregate to facilitate
sampling. Two major problems were encountered. First, the shrimp survived
the first winter (unusually mild) but failed to survive the second winter.
In studies spanning a year or more, shrimp would probably have to be reintro-
duced each spring. The second problem was concerned with the death of the
oysters after they became buried in the sediments. This problem was overcome
in the second study by placing the oysters in wire baskets suspended beneath
the surface of the ponds. Not only did the oysters do well under these
conditions, but histological examinations indicated that they underwent
normal sexual development.
In essence, the artificially constructed pilot-plant ecosystems func-
tioned extremely well throughout the course of the investigation and proved
to be a valuable tool in assessing the effect of long-term, low-level oil
exposure on an ecosystem basis. For example, the pilot-plant ecosystem
studies confirmed the laboratory findings in regard to the effect of oil on
the planktonic community, thus increasing confidence in extrapolating these
results to the natural environment. These studies also verified the fact
that zooplankters were the most oil-sensitive members of the ecosystem and
further demonstrated that their recovery was rapid even in the absence of
an exogenous source of new organisms. This recovery indicated not only
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that a sufficient number of organisms survived the spill, but also that the
system itself had recovered to the point that it was no longer inhibitory
or toxic to these forms of life. There was, however, a shift in population
from the copepod Acartia to the rotifer Brachionus. Further, the studies
confirmed the long residence time of oil in the sediments and clearly
demonstrated the problems of acquiring representative samples and inter-
preting results of hydrocarbon analyses in the presence of other sources of
hydrocarbons. The failure of the crude oil to cause shifts in the microbial
population was probably a reflection of the small amount of oil employed
and the presence of a large quantity of other organic compounds. These
results indicate that the fate of oil in the environment will be influenced
by the quantity of biologically available organics as well as the concentra-
tion of the oil.
While histological changes were observed in oil-exposed shrimp in the
laboratory studies, none were observed in the specimens in the pilot-plant
ecosystem. This was probably a reflection of the problem of obtaining
representative samples of the shrimp from the ponds.
The pilot-plant ecosystem did not contain predators for the mullet,
shrimp or oysters; therefore, the predation which was observed in the tidal
pond studies or which may well be observed in any field study, did not
occur in the pilot-plant system. The inclusion of predators, such as lady
fish, speckled trout, etc., would have afforded the opportunity to make
these observations in the pilot-plant system. However, the exclusion of
predators from the system resulted in the finding of two heretofore unre-
ported effects of oil pollution: namely, fin rot in fish and "white eye
syndrome" in shrimp. Both of these effects would not be observed in field
investigations, since the obvious impairments of the affected organisms
would probably have resulted in their demise through predation. The exact
cause of these two conditions has not been established but the results
clearly indicate that they are either directly or indirectly caused by oil.
Use of the pilot-plant ecosystem also led to several significant
findings relative to the long-term effect of oil pollution. Histological
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changes in the liver of mullet began to appear 4-6 mo after the oil spills.
Since these changes were not observed in the livers of control fish it was
concluded that they were caused by oil. The question as to whether these
changes were a result of uptake of oil immediately after the spill or
whether they were brought about by the continual exposure to oil or oil
degradation products is not known. Aromatic hydrocarbon profiles from the
livers of mullet 5 or more mo after the oil spill were suggestive of polar
(oxidized) metabolites of oil components. These polar materials could be
the result of either uptake and storage of microbial degradation products
or oxidation of oil components by the fish themselves. The latter option
is a distinct, although unconfirmed, possibility based on laboratory data
indicating the presence of certain microsomal hydroxylation enzymes in
mullet livers, which are inducible by crude oil.
The exact mechanism responsible for the decreased growth of oil-
exposed mullet during the second pilot-plant ecosystem study is not known.
The increased mortality of the oysters during the second pilot-plant study
did not occur until several mo after the oil spill. Since these organisms
are sessile and were suspended in the water column only materials suspended
and/or dissolved in the water column could have been responsible. It is
possible that any or all of these effects could have been a result of a
dietary deficiency brought about by the impact of the oil on lower members
of the food chain or by interference with the oysters' ability to utilize
their food, although no evidence to this effect was obtained during the
study. A clearer understanding of the cause and effect relationship of oil
to these observations is urgently needed. For example, if these findings
are the result of the uptake of oil immediately after the spill, the damage
to these members of the ecosystem would be limited to those organisms in
the immediate area of the spill. On the other hand, if these problems are
a result of oil in the sediments or in the detritus, the magnitude of
damage to the ecosystem could be many times greater.
There has been a tendency to rank oils according to their toxicity on
the basis of acute toxicity tests such as LDc0 determinations. For example,
the high sulfur crudes like Saudi Arabian are usually thought to be more
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toxic than the low sulfur crudes like Empire Mix. The present investigation
tends to substantiate this opinion in most cases but was reversed in a few
cases. The more volatile components of the crudes are considered to be
more toxic than the less volatile constituents and the results in the
present investigation tends to support this view. In summary, crude oils
do vary in toxicity, and further organisms vary in their response to differ-
ent crudes. It should also be emphasized that while Empire Mix crude oil
would have to be considered the least toxic of the 5 crudes tested in
this investigation from the standpoint of the short-term experiments, it
did cause both short-term and chronic effects even when employed in low
concentrations.
Not only does the specific type of crude oil make a difference in the
results but also the environmental conditions under which the test is
conducted influences the findings. For example, an outbreak of fin rot
occurred in the first test but not the second test. At first this seems
surprising since the same amount of oil was spilled in both studies.
However, the fact that the second test was in a static, not a flowing test
system, could well account for the difference. The static system by reducing
the mixing allowed the mullet the opportunity to avoid the oil. In addition,
the lack of mixing in the second study allowed the oil to remain on the
surface longer, facilitating the volatilization of more of the lighter
fractions of the oil. This would reduce the likelihood of physical or
chemical actions of these components on the mullet. Also, the mullet in
the second study were smaller (45-80 mm) than those used in the first study
(130-170 mm). This size difference may account for behavioral differences
observed in the mullet population in the ponds during the two study periods.
During the first spill the larger mullet were constantly observed hanging
at the surface (piping), generally swimming in and around the oil slick.
Contrary to this, however, the mullet in the second study were rarely
observed at the surface prior to or during the spill. This behavioral
difference could have limited the contact of the smaller mullet with the
oil slick. This contention was supported by the observation that exposure
to Empire and Saudi Arabian crudes in aerated aquaria and vats (thus mixed)
consistently caused fin rot in mullet in 8-10 days.
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Another significant difference between the two chronic studies was the
occurrence of the unusual "white eye syndrome" in shrimp only during the
second study. No reason for this difference can be given. The static
conditions seemed to have resulted in a more uniform distribution of oil in
the sediments which increased the opportunity of the shrimp to contact the
oil.
Finally it should be emphasized that overall the results of the pilot-
plant ecosystem studies produced results which clearly indicated the debili-
tating effect of even a low level of oil on higher members of the estuarine
food chain.
In fairness, it must be pointed out that these observations were made
in a closed system and, whether or not the same kinds and magnitudes of
effects would occur in nature, is obviously unknown. It would be fair,
however, to predict that if a natural system is subjected to levels of oil,
as employed in these experiments, whether or not as a single dose or a
multi-dose situation, that similar effects could be anticipated.
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SECTION VIII
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-80-058a
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Fate and Effect of Oil in the Aquatic Environment—
Gulf Coast Region
5. REPORT DATE
JULY 1980 ISSUING DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Lewis R. Brown
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Mississippi State University
Mississippi State, Mississippi 39762
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
68-01-0745
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory — Narr., RI
Office of Research and Development
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/05
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The purpose of this research investigation was to determine the fate and effect
of crude oil in the aquatic environment of the coastal Gulf of Mexico. The
project was multi-disciplinary and multi-institutional in scope and involved both
laboratory and field sized pilot-plant ecosystem studies. Emphasis was placed on
the long-term, low-level chronic effects of oil pollution on the ecosystem. Of
the five crudes employed in the investigation, Empire Mix crude was studied most
intensively.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Toxicology 0620
Pollution 1302
Crude oil 1108/0807
Oyster 0603
Shrimp 0603
Plankton 0603
Marine fishes 0603
Fate
Degradation
06, F
18. DISTRIBUTION STATEMENT
Release Unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
114
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
102
U.S. GOVERNMENT PRINTING OFFICE: 1980--657-165/0099
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