United States

Environmental Protection

AgencV

Environmental Research

Laboratory

Narragansett RI 02882

EPA600/3-80-058a

July 1980

&EPA

Research and Development

Fate and Effect of

Oil in the Aquatic

Environment

Gulf Coast Region
RESEARCH REPORTING SERIES

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aquatic, terrpstrial, iJnd atmospheric environments

This document is available to the fJublic through the National Technical Information Service, Springfield, Virginia 22161
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
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
FOREWORD

The Environmental Research Laboratory of the U.S. Environmental Protection 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
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 wi~l reduce

the rate of conversion of the grass into utilizable detritus by'up ;:0 50%. ';0\- ~{~:'l:~'." ,1-

Oil in the marsh grass systems serves as a mechanism for reentry o!f".:q,U

into the estuarine food web.

Oil, even at levels which would result in a slick only 0.01 rom 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

enzymo10gica1 and fatty acid analyses) in these organisms are evident and

appear to be a stress response.

iv
Long-t~~~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 St,te 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

TABLE OF CONTENTS

ABSTRACT.

FIGURES

ACKNOWLEDGMENTS

INTRODUCTION •

CONCLUSIONS

RECOMMENDATIONS

MATERIALS AND METHODS

LABORATORY RESULTS

A. Fate Studies

1. Volatility.

2. Fate in Water and Sediments

3. Fate of Empire Mix Crude Oil With or Without

4. Microbial Degradation of Oil

B. Phytoplankton

1. Toxicity Tests

a. 96-Hour Toxicity Testsb. 12-Day Toxicity Tests- •

c. Chronic Toxicity Test.s2. Bioaccumu1ation

C. Zooplankton

1. Toxicity Tests

D. Marsh Grass

1. Greenhouse Studies

2. Field Studies

vii

Page

iv

x

xi

1

4

6

8

21

21

21

21

Aeration 23

23

24

24

24

25

25

25

26

26

27

27

28
E.

F.

G.

Mullet

1. Acute Toxicity

2. Behavior

3. Enzymology

4. Fatty Acids

5. Oil Uptake

6. Histology

7. Laboratory Studies on Disease

Shrimp

1. Acute Toxicity

2. Behavior

3. Enzymology

4. Fatty Acids

5. Oil Uptake

6. Histology and Disease

a. Acute Studiesb. Laboratory Feeding ExperimentsOysters

1. Acute Toxicity

2. Behavior

3. Depuration

4. Enzymology

5. Fatty Acids

6. Oil Uptake

7. Histology and Disease

29

29

29

30

31

32

32

33

34

34

35

35

37

38

38

38

39

39

39

40

40

40

42

42

43

FIELD RESULTS

A. Tidal-Pond Study

1. Description of the System

2. Fate of Oil in Sediments

3. Phytoplankton/Zooplankton

4. Marsh Grass

5. Animals

B. First Pilot-Plant Ecosystem Study

1. Description of the System

2. Conduct of the Oil Spill

3. Key Events

4. Environmental Data

5. Fate Studies

6. Microorganisms

7. Phytoplankton/Zooplankton

a. Core Sampling8. Marsh Grass

viii

44

44

44

44

46

47

47

47

47

53

55

56

56

59

59

60

60
61

61

61

62

62

63

66

66

67

67

67

68

68

69

69

69

69

70

70

70

72

72

73

73

73

73

74

75

75

76

76

77

78

78

(Pond 2)-

with Saudi Arabian Crude Oil (Pond 3)-

with Nigerian Crude Oil (Pond 4)-

Crude Oil (Pond 5)- .

,.

b.

c.

a.

a.

Description of the System

Environmental Data

Fate Studies

Control Pond

Pond Treated

Pond Treated

d. Pond Treated with Empire Mix

Phytoplankton/Zooplankton

Core Samp1ingMicroorganisms

Primary Productivity of Phytoplankton Community

Marsh Grass

Mullet Pathology.

Shrimp Pathology.

Oysters - Pathology

Second Pilot-Plant Ecosystem Study •

1-

2.

3.

4.

9. Oil Uptake •

a. Mu11etb. Shrimpc. Oysters-.

10. Enzymes

a. Mu11etb. Shrimpc. Oysters11. Fatty Acids

a. Mu11etb. Shrimpc. Oystersd. Conc1usions12. Pathology

a. Mu11etb. Shrimpc. Oysters-.

13. Disease in Mullet

5.

6.

7.

8.

9.

10.

C.

SUMMARY 82

DISCUSSION 92

REFERENCES 99

APPENDIX Tabular and graphic data available separate1y--see

INTRODUCTION.

ix
FIGURES

FIGURE 1. Aerial photograph of the pilot-plant ecosystem located

at the National Science Technology Laboratory in Hancock County, Mississippi • • • • • • • • • • • • • • • 48

FIGURE 2. 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 • • • • • • • • 49

FIGURE 3. Photograph showing clumps of Juncus growing along the

edge of one experimental pond in the pilot-plant

ecosystem • • • • • • • • • • • • • • • • • • • 51

FIGURE 4. Tidal-Simulation System (TSS) installed in Pump House 52

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

FIGURE 6. Photographs of mullet with various degrees of fin erosion. A and B--normal, C and D--very heavily eroded

fins . . . . . . . . . . . . . . . . . 71

FIGURE 7. Photographs of whole and sectioned shrimp eyes.

A--normal eye; B--eye with white spot; C--photomicrograph of a normal section of eye; D--photomicrograph

with necrotic area visible just under corneal layer 79

x
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 interpretation of the results. All of these individuals worked on the project for

the entire period of the contract and their contribution is hereby acknowledged with gratitude. These scientists and their affiliation are Armando

A. de 1a 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. McIlwain 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. L~dner, Lance10t 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

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 representative samples, etc., seriously limit the value of data regarding identification 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 complexities 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,

1
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

2
Empire Mix crude oil in a naturally~occuring estuarine tidal pond, (2) monitoring 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) fo1iowing 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)

microfiche from NTIS only, National Technical

Royal Road, S~ringfield, Virginia 22161.

3

are available in hard copy or

Information Service, 5285 Port
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 enzymo10gica1 and fatty acid

analyses) in these organisms are evident and appear to be a stress response.

4
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.

5
SECTION III

RECOMMENDATIONS

The results obtained in this investigation clearly indicate the desirability 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) determining 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 (~.~. speckled trout), one or

more scavengers (~.~., crabs) and an increased quantity of marsh grasses.

These studies should extend over at least two years and should include a

6
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.~., 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.

7
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. [1]*

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 Specimen!

Mullet, Shrimp and OystersAll 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

8
Coast area. For laboratory studies, the organisms were held for at least 7

cays before being employed in bioassays.

PhytoplanktonPhytoplankton were collected from the Mississippi Sound in a 200 mesh

plankton net (iJ25 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 samp1eswererefrige~ated,returned to the laboratory 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 0/00.

ZooplanktonZooplankton 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 transported 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 rom I.D.) and subjected to a variety of conditions

for growth and maintenance.

9
Marsh PlantsAll 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 CC14 were

added to 1 liter of water in a separatory funnel. The sample was shaken vigorously for 1 min, the phases allowed to separate and the CC14 extract passed

through a column containing a 2.54 cm layer of anhydrous Na2S04 (CC14 washed).

This procedure was repeated four times and the extracts combined. Oil was

-1 measured at an absorption band of 2930 cm using a 10 mm path length cell and

compared to a standard CC14 solution containing a known amount of oil.

.pil 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 ala (1975). Analyses were conducted using a Beckman GC-45 gas chromatograph with a flame ionization detector with a 183 cm x 0.32 cm aD 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 ~-C16 peak.

10
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:lO rnl) (for samples composed 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 mI. This concentrated extract was transferred to a small vial,

evaporated under nitrogen, dissolved in 10 ~l of hexane and analyzed

using a Waters Associates Model 202/401 liquid chromatograph with a UV

detector (wavelength, 277 nrn). A 2.54 x 0.63 em OD micro Bondapak

c

18/corasil column was employed with a MeOH:H2

0 (70 ml:30 ml) solvent

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 HewlettPackard automatic integrator. Corrections for control absorbance were

made. Results were calculated as ~g oil by comparison to standards.

11
Oil in Mullet, Shrimp and Oysters

The samples were homogenized in TenBroeck Tissue grinders 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-l00-~-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 SO 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
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 HCl to about pH 3. The water layer was extracted

three. times with 30 ml CHC13 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 mls of water were added to the flask, the

contents transferred to a separatory funnel and the water layer extracted 4

times with ZO-30 mls of hexane each time. Each extraction was drained

through NaZS04 into another roundbottom flask and rotovaped to 1-2 rols

volume. The extracted fatty acid methyl esters (FAME) were run through 8-

18 cm of silica gel column with 45-85 rols of benzene and collected in a

flask. The benzene was rotovaped down to 1 m1 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 m1 solvent. Approximately 1 ~l 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 (DEGS)

supported on 80/100 mesh chromasorb WAW. Operating conditions were as

follows: Carrier flow (NZ)' 22 cc/min; Column temperature, 190 C; Detector

temperature, ZZ5 C; Inlet temperature, ZOO C; Hydrogen flow rate, 50 cc/min;

and Air flow rate, 34 liters/min.

13
Qualitative Identification of PeaksPurified 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 positively 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 multiplication 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
Microbiological Methods

Routine Counting ProceduresMedia 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 gIl 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 OilThese tests were conducted in 14-liter fermentor jars containing 10

liters of the following medium: 40.00 g of (NH4)2S04' 5.06 g of KH2P04 ,

14.94 g of K2HP04 , 171.30 g of Ri1a 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 Laboratory Fermentor (Model No. MF-114) or a Model No. FS-6l4 (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
Bioassay Procedures

Preparation of OilThe 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 AquariaAll glass aquaria (114 liters) were filled with artificial sea water

(RUa Mix), salinity of 15 0/00. 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 TanksMany of the laboratory tests concerned with the effects of low levels

of oil over a prolonged period of time were conducted in 1,895-lite~ tanks.

A 1,895-liter tank was filled with artificial sea water (15 0/00) 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.

PhytoplanktonBoth 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 0/00 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 0/00. Culture

vessels were 16 by 150 mm test tubes or 125 m1 Erlenmeyer flasks for tests

involving emulsified oil and 125 m1 Erlenmeyer flasks and aquaria for tests

employing oily sediments. Appropriate dilutions of emulsified oil in the

medium were dispensed in 10 m1 amounts in the test tube and 25 ml amounts

16
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 (stripcount method).

ZooplanktonFive 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 consisted 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 0/00. 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 PhytoplanktonHeinle 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 0/00 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 TechniquesCores 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 BBM 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 0/00 and 20 0/00 were employed. All plating was

done in duplicate or triplicate. Two m1 of the mixture was added to 75 m1

BBM liquid culture and 75 ml Heinle's media @2 0/00 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 SamplesBrain, 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 KCl 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 Disruptor, Model W185, micro-tip (Heat

18
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 superna,tant 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 KCl.

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 KCl and

again centrifu~ed at 110,000 x g for '60 min. The pellet was resuspended at

500 mg equivalents/ml 0.15 M KCl. This microsomal suspension was used in

all subsequent microsomal oxidase assays.

Enzyme AnalysesAll 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, et al., 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 £: 02 oxidoreductase; 1.9.3.1). The

procedure followed was described by Whorton and Tzagoloff (1967).

Glutamic Oxalacetic Transaminase--(L-aspartate: 2-oxoglutarate aminotransferase; 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 al. (1961).

Alkaline Phosphatase--(orthophosphoric monoester phosphohydrolase;

3.1.3.1). The procedure followed was described by Bessey, et ale (1946).

19
Acid Phosphatase--(orthophosphoric monoester phosphohydrolase; 3.1.3.2).

The procedure followed was described by Andersch and Szczypinski (1947).

a-Glucuronidase--(a-D-glucuronide glucuronohydrolase; 3.2.1.31). The

procedure followed Sigma kit 325-A and Fishman (1965).

Leucine aminopeptidase--(3.4.l.l). 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 4 Reductase--(NADH2:cytochrome £ oxidoreductase;

1.6.2.1). The procedure followed was described by Mackler (1967).

NADH-Cytochrome ~5 Reductase--(NADH2:cytochrome bS 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, et a1. (1962),

and Masters, et a1. (1967).

NADPH-Dich1oropheno1indopheno1 Reductase--The procedure followed was

described by Ernster, et a1. (1962) and Masters, et a1. (1967).

Cytochrome bS--The procedure was essentially the method described by

Ernster, et a1. (1962).

Cytochrome P-4S0--The procedure followed was described by Omura and

Sato (1964).

20
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. [2J

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). [2J

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
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. [3-7J

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 (~-C2l - ~-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 ~-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. [8-11]

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, ~lcohol 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
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 inpristane/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 experimental 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%. [12J

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

8 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
phytane were relatively resistant to attack. The more polar aromatic

constituents were metabolized before the less polar compounds. No accumulation of aliphatics occurred in the bacterial cells. [13-21]

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 TestsEighty six 96 ± 3 hr algal toxicity tests consisting of 272 replicates

were performed using 5 crude oils on unialgal ~solates 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 (EC50). Cell growth was measured in terms of cell numbers.

Experimental oil exposure levels ranged from 0 to 100 mg/l assuming 100%

emulsification. Actual oil levels present in the growth media 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. 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. L. galbana was the most sensitive followed by ~. costatum,

L. undulatum, f. curvisetus, Rhizosolenia calcar, Asterionella japonica and

T. decipiens. Carteria chuii was the least sensitive of the 8 algal species

tested with Empire Mix. [25-42]

24
12-Day Toxicity TestsExperiments 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 ECSO values by more than 5 mgll in 5 of 7 of the

species tested. For~. galbana, extended exposure had little effect on the

ECSO ' but T. decipiens became more tolerant with extended exposure, and the

ECSO value increased by 5 mg/l. 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 TestsThirty-two 96-hr experiments were performed in which f. curvisetus, I.

galbana, L. undulatum and T. decipiens were tested against Empire Mix and

Saudi Arabian. S. costatum was tested against all 5 oils. In these studies,

phytoplankton exposed to crude oil at concentrations between 0 and 100 mgll

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. L. undulatum consistently

failed to recover or recovered at a greatly decreased rate in control and

treated cultures. [51]

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 mgll Empire Mix

(approximately 1110th 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 oiltreated samples showing oil levels which could account for 40 to 70% of the

25
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 m

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 0/00. 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. [54-59J

When wild A. tonsa (collected from the pilot-plant ecosystem) were

exposed to Empire Mix in the laboratory, the resultant TL value was the

m

same as that for cultured organisms, indicating that laboratory data on

cultured Acartia can be extrapolated to Acartia in the pilot-plant

ecosystem. [60J

26
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. [61] m

Euterpina acutifrons is the most prominent pelagic harpactacoid copepod

found in the Mississippi Sound. As ~th A. tonsa, 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 cyc10poid 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
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). [73-74J

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 mortality as well as reduced growth the following year ranging from 40%

(250 ml/m2) to 90% (1500 ml/m2). [75-78J

(2) Multiple spills of doses of 250 ml or greater, 12 mo apart, significantly 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
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/l) and Saudi Arabian (100% mortality at 350'mg/l) were the most toxic,

Iranian and Venezuelan were intermediate (TL 's between 400-800 mg/l) and

m

Empire Mix (TL > 800 mg/l) was the least toxic. [90] m

Based on these results, laboratory treatment levels of 75 mg/l 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/l) 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/l to the less

toxic oils (!...~., 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 l14-liter aquaria at concentrations ranging from 200

to 400 mg/l, 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
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/l 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) , S-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 intraperitoneal injection with 100 ~l 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/! 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
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/l of Empire Mix or Saudi Arabian for 96 hra.

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, particularly by Saudi Arabian; this apparent decline represents the failure of

protein to increase at the same rate as liver size. Both cytochromes bS

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 £ reductase following exposure to Empire

Mix; these increases .may have been activations rather than inductions.

NADH-cytochrome £ and NADH-cytochrome bS 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.

8-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/l 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/l oil

31
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 predominance 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 partitioning 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 negative 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
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 I

due to the short (96 hr) duration of the majority of these tests. Consequently, 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/l to 75 mg/l 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 microf10ra 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
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 hemolytic 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 connection 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 tail 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
the most toxic as 10 mg/l resulted in 100% mortality of the test shrimp.

The TL values were estimated to be between 15 and 25 mg/l for Empire Mix, m

Saudi Arabian and Iranian, and between 35 and 45 mg/l 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/l 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/l 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 swinunerettes which sometimes resulted in "scooting" movements

but usually caused no forward motion. The swinuning 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/l emulsified Empire Mix for 12 hrs:

acid phosphatase (ACP), malic dehydrogenase (MDH), glutamic oxaloacetic

transaminase (GOT), glutamic pyruvic transaminase (GPT), S-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
Shrimp were exposed to 4 mg/l 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 supernatant CYT OX were decreased after oil exposure; mitochondrial and supernatant GOT and GPT, supernatant CYT RD and mitochondrial CYT OX were elevated

after oil exposure; ACP, ALP, and GLU, were not consistently affected. [110]

Shrimp were exposed to 10 mg/l emulsified Empire Mix or Saudi Arabian

for 24 hrs. The activities of the following enzymes were determined: ACP,

ALP, CYT OX, CYT RO, GLU, GOT, GPT, GGTP, LAP, MDH, a-hydroxybutyric dehydrogenase (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 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 ~.)

exposed to 50 mg/l emulsified Empire Mix for up to 12 days: MDH, LDH,

36
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/l 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/l 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/l 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/l 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 S-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 percentages of short-chain acids and less long-chain fatty acids. Differences

37
in the fatty acid pattern decreased with increased exposure time, indicating

some form of compensation or acclimation to the stress. This decrease I

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/l) 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 demonstrate the presence of oil in the hepatopancreas. [123-124]

Histology and Disease

Acute StudiesHistological 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/l Empire Mix in l14-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. Histologically, 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
prior to testing; in this instance it appeared that a bacterium (tentatively

identified as Vibrio ~.) appeared to be responsible. In conclusiont 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 ExperimentsA series of experiments were conducted using aerated t l14-liter aquaria

with sand covered bottoms in which test shrimp were fed 5 g of oil-treated

feed (250 mg/l) per day while the control shrimp were fed 5 g of feed with

no oil added. At the termination of the experiment t some of the shrimp

were sacrificed and examined histologically. A number of abnormal areas

were observed in the area of the mandiblest 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 t 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
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 solub1es

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/l 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

(eHE) , acid phosphatase (ACP) , alkaline phosphatase (ALP), cytochrome

oxidase (CYT OX), glutamic oxa10acetic transaminase (GOT), mitochondrial

leucine aminopeptidase (LAP) and supernatant malic dehydrogenase (MDH)

A number of enzyme activities from oysters exposed to Empire Mix were

40
statistically different from those of controls. However, no clear trend as

to stimulation or inhibition was observed. At 7 days, the t3-g1ucuronidase

(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/l 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 significant 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
NADPH-cytochrome reductase was studied in fractions of whole body

homogenates of oysters exposed to 75 mg/l emulsified Empire Mix, Saudi

Arabian, Venezuelan, Nigerian and Iranian. There were statistically significant increases in NADPH-cytochrome £ reductase activity after exposures to

Empire Mix and Nigerian. [133J

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 hypothesized 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/l 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-l35J

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 hra. Data on

oil uptake in oysters were extremely erratic apparently due to variations

42
in pumping habits. Neverthe1esst the trend was toward a constant increase

in oil concentration with increased length of exposure to the oil. In a

second systemt 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 ~pire 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 cob1d be directly associated with their treatment with Empire

Mix.

43
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/l oil

at low tide. A similar adjoining pond served as control. The pre-spill

conditions of the ponds were: salinity 10-12 0/00, temperature 30-37 C,

and dissolved oxygen 3.7 mg/l at low tide and 12.2 mg/l at high tide. The

dikes were vegetated by Juncus roemerianus, Spartina cynosuroides, Spartina

alterniflora and Distichlis 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/l.

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 ~-C10 - ~-C20 hydrocarbons as compared to

the n-C2l - n-C33 hydrocarbons had dropped approximately 8% in the top 2 cm

44
of sediments. The ratios of n-C17/pristane and ~-C18/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 ~-C18/phytane ratios in the deeper core sections

had increased as compared to the top sections. [139-l40J

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 ~-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-C17/pristane, nC18/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 ~-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. [14lJ

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. [142J

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/l 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
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 in 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-l44J

46
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 populations 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

~.) and grass shrimp (Pa1aemonetes ~.). Present in relatively low

numbers were the bay anchovy (Anchoa mitchilli), the si1verside (Menidia

bery11ina), the striped mullet (Mugil cephalus), the pinfish (Lagodon

rhomboides) and the ladyfish (E1ops 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 1adyfish. [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 responsible 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
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 (approximately salinity 28 0/00) from the Gulf of Mexico mixed with fresh well

water to adjust the final salinity to about 14 0/00.

Clumps of Juncus mixed occasionally with other species (~~, 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 rna 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 approximately 1.2-1.5 m tall and had completely covered the pond banks. Additionally, 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 0/00 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 available; consequently, it was decided that the salinity would be increased by

the addition of commercial salts. Calculations were made to determine the

50
amounts of Na, K, Ca, Mg, Cl and S04 required to increase the salinity to

12 Q/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, CaC12, MgS04,

MgC12 and KC1.

In November 1973, approximately 360 mullet, 750 brown shrimp and 360

oyste~s 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
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
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 rom 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/l. 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/l.

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 ~he control ponds. It was possible to see for considerable 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 0/00).

55
11 June 1974

24 June 1974

17 July 1974

19 July 1974

28 July 1974

19 August 1974

24 September 1974

6 November 1974

9 January 1975

11 March 1975

6 May 1975

16 May - 2 June 1975

Environmental Data

Third Pre-spill sampling

Salts added to ponds

11.3 1 of Empire Mix added

to each test pond (3 & 4)

another 11.3 1 of Empire Mix

added to each test pond (3 & 4)

First Post-spill sampling

Second Post-spill sampling

Third Post-spill sampling

Fourth Post-spill sampling

Fifth Post-spill sampling

Sixth Post-spill sampling

Seventh Post-spill sampling

Experiment terminated

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.

~he salinity was maintained between 6 and 10 0/00 for the duration of

the study by the additions of seawater and/or commercial salts. The' dissolved

oxygen ranged between 6 and 10 mg/l.

Fate Studies

The first pre-spill core samples of the sediment showed a fairly large

concentration of straight-chain hydrocarbons below ~-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
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, C3l). These observations were probably 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 hydrocarbons remained the same; however, there was an increase in the concentration 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
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 apparent 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-l64J

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/l, whereas the total hydrocarbons in

sediments opposite the pump yielded 1080 mg/l. 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, ~-C23,

~-C25, ~-C27, ~-C29, ~-C31) predominated. In the sample opposite the pump,

however, the envelope contained only a few resolved peaks with no oddcarbon dominance. In Pond 4 (oil), the sediments nearest the pump contained

654 mg/l total hydrocarbons, whereas the sediments opposite the pump contained 100 mg/l. 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.~. strong

58
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 - ~-C25 molecular weight

range. The unresolved envelope was not in the same molecular weight range

as that of Empire Mix, and there was little ~vidence 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/sulfatereducing 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.

Phytoplankton/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 attributed 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. Immediately 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
Acartia populations with a subsequent shift to a Brachionus dominated

population must be attributed to the presence of the Empire Mix crude

oil. [168-l72J

Core SamplingCore samples were taken from the ponds on a regular basis beginning in

September of 1974. Prior to that timet 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 t and Heinle's Media at a salinity of

20 0/00 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/m~.

Agar plates were counted as to number of colonies on the plate.

PresumablYt 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 t 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 pondst and that a decline in population in one area

represented a decline in the entire benthic population. This was disproved

by taking 2 cores I m apart on several sampling dates and comparing

the results. No relationship was observed between the numbers of algae

grown from duplicate cores.

In conclusiont the areas of concentration and paucity of algal populations 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 bankst no meaningful conclusions could be

drawn from this phase of the study. There was very little plant mortality

60
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 postspill 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 om. While the number of samples in most

cases was small, certain tentative conclusions can be drawn.

MulletBrain samples collected throughout the 9 l/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.

ShrimpThe data on the oil content of shrimp hepatopancreas and muscle were

inconsistent, due in part to the small number of samples available for

61
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]

OystersThe 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 rna only 25-33%

of the oysters from the test ponds had any traces of oil in them, thus confirming 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),

S-glucuronidase (GLU) , glutamic pyruvic transaminase (GPT) , malic dehydrogenase (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 indications 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
MulletIn 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,l:.iver 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 immediately 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
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

64
test ponds were caused by an oil-related phenomenon and not general nutritional state. However, total length to body weight ratios were not consistent 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 tha~ 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]

eHE 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
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 proportional to the degree of stress the organisms were experiencing. [198J

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. Als~ 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.

ShrimpIn 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 agreement 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. [199-203J

OystersGenerally, the oysters from the oil ponds did not have significantly different 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 eHE, 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. [204-208J

fi6
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).

MulletOf 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 ~hrimp 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

l6C fatty acid to the l8C 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]

ShrimpOf 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 temperature 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 conditions. 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 l8-carbon fatty acid. When

the sum of the 18:2 and 18:3 acids was plotted, there seemed to be a direct

67
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. [2ll-2l3J

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 hepatopancreas tissue of shrimp, no statistical differences of biological variation

could be found between control and oil-exposed tissue.

OystersIn 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 inconsistent 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. [2l4-2l6J

ConclusionsThere 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, et 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 statistically (P<05) greater extent than control tissue.

68
Pathology

MulletDuring the early part of the first field study, no histological chang~s

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 addition, nearly all of the livers from treated mullet had enl&rged 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. [217J

ShrimpDuring 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.

OystersNo 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 (prespill) 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
into the treatment period it was noted that the general health of the

oysters in all ponds was declining. Their tissues were clear and w~tery,

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 (lOO-lOOO/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 observation by all personnel involved that the oil-treated fish (even late in the

study when they outwardly appeared to be recovering) were much more susceptible 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 ob~ained 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
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 initially to approximately 9.5 ppt at the conclusion of the study. The dissolved

oxygen ranged between 6 and 9 mg/l .

. 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.

72
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. [221]

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 recognizable 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
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 oiltreated 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. [225-228J

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/I). However, by June of

1975 Brachionus had greatly increased in numbers (>100 organisms/l - control;

50 organisms/l - oil-treated) while the Acartia populations remained at

about the same level as the summer of 1974 «10 organism/l - all

ponds). [225-228J

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 Mixtreated pond immediately following the spill and then recovered by late

August. [225-228J

74
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 SamplingCulture 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 0/00, and Heinle's Media at 3 0/00.

Liquid media were not used for counting.

Two days after the oil spill, a decline was noted in the algal populations 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 simultaneous 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 individual 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 environment which is not generally as rich in organic matter as the oil-treated

ponds were during the study period. [233-236]

75
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/l) reduced phytoplankton growth under laboratory

conditions. [237-238]

These observations are reflected further in the total pigment concentrations 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 postspill, and persisted to be lower than Ponds 2 and 5 during subsequent postspill samples. It appeared that oil stimulated the growth of phytoplankton

when present at low levels. [239]

Marsh Grass r

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]

76
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 th~

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 rom in length and averaged 15 g in weight. When harvested 9

1/2 mo later, the average lengths and weights were: (1) control mullet-lSI

mm; 65 g (2) Nigerian-treated mullet-129 rom; 35 g (3) Saudi Arabiantreated mullet-124 rom;. 42 g (4) Empire Mix-treated mullet-ISO rom; 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-treaned 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 t~ but less severe tha~ 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 Nigeriantreated 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.

77
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 nonfunctional. 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 syndrome 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 constructed primarily to determine if there existed a relationship between the

78
Figure 7. Photographs of whole and sectioned shrimp eyes.

A--normal eye; B--eye with white spot; C--photomicrograph of a normal section of eye; D--photomicrograph

with necrotic area visible just under corneal layer.

79
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 Arabiantreated, 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

80
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.

81
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 i~ or disappearance fro~ 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 EC50 values ranging from 5.6 mg/1

for Nigerian crude oil (tested against Ske1etonema costatum) to greater

than 58.0 mg/1 for Empire Mix crude oil (tested against Carteria chiuu).

82
3. In general the zooplankton species found in the Gulf Coast environment are more susceptible to crude oil than are the phytoplankton with TL

m

values for Acartia tonsa ranging from 0.55 mg/l for Nigerian to 1.62 mg/l

for Venezuelan.

4. From the marsh grass studies it is estimated that (A) a single oil

spill with Empire Mix at a dosage of 250-1500 m1 of oil/m2 of marsh will

reduce marsh plant productivity by 400-900 g/m2• (B) Repeated spills with

Saudi Arabian oil at 600-6000 ml of oil/m2 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 de60mposed 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 marshestuarine 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/l; 100% mortality at 200 mg/l) , Saudi

m

Arabian the next most toxic (TL less than 350 mg/l; 100% mortality at 350

m

mg/l) , Iranian and Venezuelan intermediate (TL between 400-800 mg/l) and

m

Empire Mix the least toxic (TL 800 mg/l). At laboratory treatment levels 'm

of 25 and 75 mg/l 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 oc~urring 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

83
histological 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/l) , Empire Mix, Saudi Arabian, and

m

Iranian intermediate (TL 15-25 mg/l) and Venezuelan the least toxic (TL m m

35-45 mg/l). 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 hepatopancreas 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/l Empire Mix crude) for a period of 18 mo.

84
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/l) 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 contribution of biogenic a1iphatics 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 pi1otplant 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 em every 12 hrs then reversed.

Salinity was established and maintained between 6-12 ppt through the addition

85
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

86
than 10 mg/l 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/l) 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 checkerboard 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 depur?tion. 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 S-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.

87
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 oilexposed 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

88
were restocked by

For this study

ponds.

removed from the ponds after the first test and the ponds

the addition of mullet, shrimp and oysters to each pond.

the oysters were placed in wire baskets suspended in the

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 essentially the same as in the previous laboratory and pilot-plant tests indicating

a high tolerance to crude oil.

3. The response of zoop1ankters 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

89
mullet averaged 82 rom 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 rom; 64 g, (3) Saudi Arabiantreated mullet: 124 mm; 42 g , and (4) Nigerian-treated mullet: 129 rom;

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

90
cones and the structures associated with the ommatidia were completely

destroyed. No other damage to the eye was observed.

91
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 (Ga1tstaff, 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 recolonization. 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, et 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

92
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 seminatura1 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 0/00 salinity) diluted

with freshwater to a salinity of 14 0/00. Decreases in salinity occurred

as a result of dilution with rainwater but was maintained at 6.0 - 14.0

0/00 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 0/00,

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.

93
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 me 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 reintroduced 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 functioned 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

94
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 interpreting 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 concentration 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 unreported 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

95
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 S 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 oilexposed 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 LDSO determinations. For example,

the high sulfur crudes like Saudi Arabian are usually thought to be more

96
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 different 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.

97
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 pilotplant ecosystem studies produced results which clearly indicated the debilitating 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.

98
SECTION VIII

REFERENCES

Anderson, M. A. and A. J. Szczypinski. 1947. Use of £-nitrophenylphosphate

as the subs~rate in determination of serum acid phosphatase. Amer.

J. Clin. Path. 17:571-574.

Bergmeyer, N. V. and E. Bernt. 1965. Malic dehydrogenase, In: Methods of

Enzymatic Analysis, Ed. by H. V. Bergmeyer, Academic Press, New York,

pp. 757-760.

Bergmeyer, H. V. and E. Bernt. 1965. Glutamate-pyruvate transaminase. In:

Methods of Enzymatic Analysis, Ed. by H. V. Bergmeyer, Academic Pres~

New York, pp. 846-853.

Bernt, E. and H. V. Bergmeyer. 1965. Colorimetric determination of leucine

aminopeptidase in serum. In: Methods of Enzymatic Analysis, Ed. by

H. V. Bergmeyer, Academic Press, New York, pp. 833-836.

Bessey, O. A., O. H. Lowry, and M. J. Brock. 1946. A method for the rapid

determination of alkaline phosphate with five cubic millimeters of

serum. J. BioI. Chern. 164:321-329.

Blumer, M. and J. Sass. 1972. Oil pollution: persistence and degradation

of spilled fuel oil. Science, 1976:1120-1122.

Blumer, M., J. Sass, G. Souza, H. Sanders, F. Grassle, and G. Hampson. 1970.

The West Falmouth oil spill. Woods Hold Oceanographic Institution.

Reference 70-44, pp. 1-32.

Blumer, M., L. L. Sanders, J. F. Grassle, and G. R. Hampson. 1971. A small

oil spill. Environment. 13:2-12.

Caroll, K. K. 1961.

chromatography.

Quantitative estimation of peak areas in gas-liquid

Nature. 191:377-378.

Ellman, G. L., K. D. Courtney, V. Andes, Jr., and R. M. Featherstone. 1961.

A new and rapid colorimetric determination of acetylcholinesterase

activity. Biochem. Pharmacol. 7:88-95.

99
1935. EfBull. U.

Ernster, L., P. Siekevitz, and G. E. Palade. 1962. Enzyme-structure relationships in the endoplasmic reticulum of rat liver. J. Cell. BioI.

15:541.

Fishman, W. H. 1965. S-Glucuronidase. In: Methods of Enzyme Analysis,

Ed. by H. V. Bergrneyer, Academic Press, New York, pp. 869-874.

Galtsoff, P. S., H. F. Prythersh, R. O. Smith, and V. Koehring.

fects of crude oil pollution on oysters in Louisiana waters.

S. Bur. Fish., 48:142-210.

Klenk, E. and G. Keemer. 1960. Biogenesis of C20 and C22 polyenoic acids

in vertebrates. Z. Physiol. Chern. 320:111-125.

Knipprath, W. A. and J. F. Mead. 1960. Influence of temperature on the

fatty acid pattern of mosquitofish (Gambusia affinis) and guppies

(Lebistes reticulatus). Lipids. 1:113-117.

Kruppa, R. B. and R. S. Henly. 1971. Gas chromatography injection technique.

Amer. Lab., (May issue).

Lind, O. T. and R. S. Campbell. 1969. Comments on the use of liquid scintillation for routine determination for l4C activity in producation

studies. Limnol. Oceanogr. 14:787-789.

Lytle, J. S. 1975. Fate and effect of crude oil on an estuarine pond.

In: Proceedings of Joint Conference on Prevention and Control of Oil

Spills, American Petroleum Institute, Washington, D. C., pp. 595-600.

Mackin, J. and S. Hopkins. 1961. Studies on oyster morality in relation to

natural environments and to oil fields in Louisiana. In: Studies on

oysters in Relationship to the Oil Industry. Publ. Inst. Mar. Sci.,

University of Texas, 7:1-131.

Mackin, J. and A. Sparks. 1961.

oils loss from a wild well.

A study of the effects on oysters of crude

Publ. Inst. Mar. Sci., 7:230.

Mackler, B. 1967. Microsomal DPNH-cytochrome reductase. In: Methods

in Enzymology. Ed. by R. W. Estabrook and M. E. Pullman. Academic

Press, New York, Vol. X:55l-553.

Masters, B. S. S., C. H. Williams, Jr., and H. Kamin. 1967. The preparation

and properties of microsomal TPNH-cytochrome reductase from pig liver.

In: Methods in Enzymology. Ed. by R. W. estabrook and M. E. Pullman,

Academic Press, New York, Vol. X:565-573.

Miles, H. D., M. J. Coign, and L. R. Brown. 1975. The estimation of the

amount of Empire Mix crude oil in mullet, shrimp and oysters by liquid

chromatography. In: Proceedings of Joint Conference on Prevention and

Control of Oil Spills, American Petroleum Institute, Washington, D. C.,

pp. 149-154.

100
Nelson-Smith, A. 1973. Oil pollution and marine ecology. Plenum Press, New

York, pp. 23-41.

Rosenfeld, W. D. 1960. Petroleum prospecting method. U. S. Patent No.

2,921,003.

Strittmatter, P. 1967. NADH-cytochrome Es reductase. In: Methods in

Enzymology, Ed. by R. W. Estabrook and M. E. Pullman, Academic Press,

New York, pp. 565-573.

Szasz, G. 1969.

peptidase.

A kinetic photometric method for serum y-g1utamy1 transC1in. Chem. 15:124-136.

Whorton, D. C. and A. Tzago1off. 1967. Cytochrome oxidase from beef heart

metochondria. In: Methods in Enzymology, Ed. by R. W. Estabrook and

M. E. Pullman, Academic Press, New York, Vol X:846-853.

Wroblewski, F. and J. S. LaDue. 1965.

blood. Proc. Soc. Exp. Bio1. Med.

Lactic dehydrogenase.

90:210-213.

Activity in

Vale, G. L., G. S. Sidhy and W. A. Montgomery. 1970. Studies on a kerosenelike taint in mullet (Mugi1 cepha1us). J. Sci. Fd. Agric. 21:429.

Vollenweider, R. A. 1974. Primary production in aquatic environments.

Burgeso and Son (Abingdon) Limited, pp. 225.

101
TECHNICAL REPORT DATA

(Please read Ins./rUctions on the reverse before completing)

1. REPORT NO.

/2.

3. RECIPIENT'S ACCESSION-NO.

EPA-600/3-80-058a.

4. TITLE AND SUBTITLE 6. REPORT DATE

Fate and Effect of Oil in the Aquatic Environment-- JULY 1980 ISSUING DATE

Gulf Coast Region 6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.

Lewis R. Brown

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.

Mississippi State University lBA608

Mississippi State, Mississippi 39762 11. CONTRACT/GRANT NO.

68-01-0745

12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED

Environmental Research Laboratory -- Narr. , RI 'lfi n." 1

Office of Research and Development 14. SPONSORING AGENCY CODE

U. S. Environmental Protection Agency EPA/600/05

Narragansett. Rhode Island 02882

16. 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

a. DESCRIPTORS b.IOENTIFIERS/OPEN ENDED TERMS c. COSATI Field/Group

Toxicology 0620 Fate

Pollution 1302 Degradation 06, F

Crude oil 1108/0807

Oyster 0603

Shrimp 0603

Plankton 0603

Marine fishes 0603

18. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES

Release Unlimited

Iunc1assified 114

20. SECURITY CLASS (This page) 22. PRICE

tunc1assified

EPA Form 2220-1 (9-73) 102

* u.s. GOVERNMENT PRINTING OFFICE: 1980--657-165/0099