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ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL SPILL

Report of the NOAA-CNEXO Joint Scientific Commission

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ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL SPILL

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Report of the NOAA-CNEXO Joint Scientific Commission

October 1982

4Wt, U. S. DEPARTMENT OF COMMERCE

1%^KA- Malcolm Baldnge, Secretary ^B^ National Oceanic and Atmospheric Administration John V. Byrne, Administrator

CENTRE NATIONAL POUR I'EXPLOITATION DES OCEANS

DISCLAIMER

Mention of a commercial company or product does not constitute an endorsement by NOAA Environmental Research Laboratories. Use for publicity or advertising purposes of information from this publi- cation concerning proprietary products or the tests of- such products is not authorized.

ii

TABLE OF CONTENTS

Page Preface v

I. Physical, Chemical, and Microbiological Studies After the AMOCO CADIZ Oil Spill

ATLAS, R.M.

Microbial Degradation within Sediment Impacted by

the AMOCO CADIZ Oil Spill 1

BALLERINI, D. , DUCREUX, J., and RIVIERE, J.

Laboratory Simulation of the Microbiological

Degradation of Crude Oil in a Marine Environment . . 27

BOEHM, P.D.

The AMOCO CADIZ Analytical Chemistry Program .... 35

DOU, H., GIUST, G., and MILLE, G.

Studies of Hydrocarbon Concentrations at the

He Grande and Baie de Lannion Stations Polluted

by the Wreck of the AMOCO CADIZ 101

DUCREUX, J.

Evolution of the Hydrocarbons Present in the

Sediments in the Aber Wrac'h Estuary Ill

MARCHAND, M., BODENNEC, G. , CAPRAIS, J.-C, and PIGNET, P.

The AMOCO CADIZ Oil Spill, Distribution and

Evolution of Oil Pollution in Marine Sediments . . . 143

WARD, D.M., WINFREY, M.R., BECK, E., and BOEHM, P. AMOCO CADIZ Pollutants in Anaerobic Sediments: Fate and Effects on Anaerobic Processes 159

II. Biological Studies After the AMOCO CADIZ SPILL

GLEMAREC, M. and HUSSENOT, E.

Reponses des Peuplements Subtidaux a la Perturbation

Creee par 1 'AMOCO CADIZ dans les Abers Benoit et

Wrac'h 191

CABIOCH, L., DAUVIN, J.-C, RETIERE, C, RIVAIN, V. and

ARCHAMBAULT, D.

Les Effets des Hydrocarbures de 1 'AMOCO-CADIZ sur

les Peuplements Benthiques des Baies de Morlaix et

de Lannion d'Avril 1978 a Mars 1981 205

in

Page

BOUCHER, G., CHAMROUX, S., LE BORGME, L. , and MEVEL, G.

Etude Experimental e d'une Pollution par Hydrocarbures

dans un Microecosysteme Sedimentaire. I: Effet de

i Contamination du Sediment sur la Meiofaunr- 22 "

BODIN, P. and BOUCHER, D.

Evolution a floyen-Terme du Meiobenthos et du

Microphytobenthos sur Quelques Plages Touchees par

la Maree Noire de 1 'AMOCO -CADIZ 245

NEF and HAENSLY, W.E.

Long-Term Impact of the AMOCO CADIZ Crude Oil

Spill on Oysters Crassostrea gigas and Plaice

: ieuronectes platessa From Aber Benoit and Aber

Wrac'h, Brittany, France. I. Oyster Histopathology.

II Hetrcleum Contamination and Biochemical

Indices of Stress in Oysters and Plaice . . . 269

LEVASSEUR, J.E. and JORY, M.-L.

Retablissement Naturel d'une Vegetation de Marais

Mari times Alteree par les Hydrocarbures de 1 'AMOCO

CADIZ: Modalites et Tendances 329

SENECA, E.D. and BROOME, S.W.

Restoration of Marsh Vegetation Impacted by the

AMOCO CADIZ Oil Spill and Subsequent Cleanup

Operations at He Grande, France 363

LE CAMPION-ALSUMARD, T. , PLANTE-CUNY, M.-R., and

VACELET, E.

Etudes Microbiologiques et Microphytiques dans

les Sediments des Marais Maritimes de l'lle Grande

a la Suite de la Pollution par 1 'AMOCO CADIZ 421

CHASSE, C. and GUENOLE-BOUDER, A.

1964-1982, Comparaison Quantitative des Populations

Benthiques des Plages de St Efflam, St Michel-en-

Greve Avant, Pendant et Depuis le Naufrage de

1 'AMOCO-CADIZ 451

iv

PREFACE

At approximately 11:30 p.m. on Thursday, March 16, 1978, the super- tanker Amoco Cadiz went aground on a rock outcropping 1.5 km offshore of Portsall on the northwest coast of France. The vessel contained a cargo of 216,000 tens of crude oil and 4,000 tons of bunker fuel. At 6:00 a.m. on Friday, March 17, the vessel broke just forward of the wheelhouse and thus started the largest oil spill in maritime history. During the course of the next 15 days, the bunker fuel and contents of all 13 loaded cargo tanks, which contained two varieties of light mideastern crude oil, were released into the ocean. The oil quickly became a water-in-oil emulsion (mousse) of at least 50% water, and heavily impacted nearly 140km of the Brittany coast from Portsall to He de Brehat. At one time or another, oil contamination was observed along 393 km of coastline and at least 60 km offshore. Impacted areas included recreational beaches, mariculture impoundments, and a substantial marine fishery industry.

h arcr, .3, r. »,ilmot N. Hess, Director of the . v.v , - /omental Research Laboratories (ERL) of the National Oceanic and Atmospheric Administration (N0AA), contacted Dr. Lucien Laubier, Director of the Centre Oceanologique de Bretagne (COB) of the Centre National pour 1 'Exploitation des Oceans (CNEX0), the French national oceanographic organization. Dr. Hess and Dr. Laubier arranged for participation by United States scientists in a joint Franco-American investigation of physical and chemical manifestations of the spill. On March 24, the agreement was expanded to include cooperative biological investigations through contacts initiated by Dr. Eric Schneider, Director of the Environmental Protection Agency's Environmental Research Laboratory in Narragansett, Rhode Island.

N0AA personnel arrived on March 19 to join the investigation initiated on March 17 by several French scientific teams. Initial photographic over-flights and active beach sampling began on Tuesday, March 21, followed by initial chemical sampling by vessel on Friday, March 24. The team was supplemented with EPA biological observers on Sunday, March 26. Sampling has continued by some segments of this original team until the present time.

Throughout the period of investigation, active interaction and coordination with the French scientific community have taken place under the auspices of C0B/CNEX0. All sampling has been coordinated with the general ecological impact study designed by the French Ministry of Environment, organized, ,by CNEX0, and operated by several scientific institutions in France^ , making possible a more thorough evaluation of the effects of the incident than would otherwise have been possible.

- National Museum of Natural History, National Geographic Institute, French Institute of Petroleum, Scientific and Technical Institute of Marine Fisheries, University of Western Brittany, University P. and M. Curie, Paris VI, and the National Center for the Exploitation of the Oceans.

About three months after the oil spill the U.S. team prepared a "Preliminary Scientific Report on the Amoco Cadiz Oil Spill" covering data up to May 15, 1978. This document covered only the period of acute effects. A one-day symposium on the Amoco Cadiz spill was held in Brest on June 7, 1978, and published soon after. It was obvious from these initial observations that a period of years would be required to under- stand what had happened to these portions of the coast where the oil had settled in and not been cleansed promptly.

During this early period of study of the spill Mr. Russ Mallatt of the Amoco Trading Company had several discussions with Drs. Hess, Laubier and Schneider. Mr. Mallatt was the General Manager for Environmental Conservation and Toxicology of Amoco. Discussion with Mr. Mallatt during the first two months after the spill identified Amoco's interests in carrying out long-term studies of the effects of the oil spill. These early contacts were followed up by substantial discussions between Mr. John Linsner of Amoco and Mr. Eldon Greenberg, General Counsel of NOAA. These discussions culminated with an agreement being signed by Amoco and NOAA to carry out long-term studies of the effects of the spill. The study would cover three years and would be a joint French-U.S. activity. A Joint NOAA/CNEXO Scientific Commission was established through another agreement between the two agencies signed June 2, 1978. Amoco would transfer money to NOAA and the Joint Commission, chaired by Drs. Hess and Laubier, would determine the research program to be carried out, the investigators to do the research, and the funding levels. The Joint Commission would also monitor the progress of the studies and be responsible for making the final report. One of its major goals was to make U.S. and French scientific teams work together in a common effort to better understand the consequences of the wreckage.

The Joint Commission first met in Brest at the CNEXO Laboratory on July 18, 1978. Taking into account the French program to assess the long-term ecological impact of the oil spill funded by the Ministry of Environment, it determined that the most important areas for research were:

1. Heavily impacted subtidal areas like the Abers and the Bays of Morlaix and Lannion.

2. Heavily impacted intertidal areas such as St. Efflam and the salt marsh at He Grande.

3. The detailed chemical evolution of the petroleum hydrocarbons.

4. Biodegradation of petroleum.

The second meeting of the Joint Commission, held in Washington, D.C., on October 12, 1978, reviewed the work carried out during the first months of the first year and planned the research program for the second year's study.

VI

In November 1979, an international conference was held in Brest sponsored by CNEXO. Investigators sponsored by the Joint NOAA/CNEXO Scientific Commission, as well as a number of other scientists, gave papers at this conference. The proceedings of this conference entitled "Amoco Cadiz: Fates and Effects of the Oil Spill" make a very good summary of the first one and one-half year study after the spill.

Following the second meeting of the Joint Commission, Dr. Hess left NOAA and was replaced as co-chairman by Dr. Joseph W. Angelovic from the Office of Ocean Programs in NOAA.

The third meeting of the Joint Commission was held in Paris, France, October 28, 1980, in conjunction with the meeting of the U.S. -French Cooperative Program in Oceanography. The previous work was reviewed and the final year of the research program was planned.

Now the three-year study is over and attempts are being made to bring together the findings of the investigators. A workshop was held in Charleston, South Carolina, on September 17-18, 1981, to report on the physical and chemical studies. A second workshop was held in Brest, France, on October 28-30, 1981, to report on the biological effects studies. This document is the report of those workshops and forms the body of the final report to Amoco from the Joint NOAA/CNEXO Scientific Commission.

Speaking for all who worked on the spill, we would like to thank the Amoco Transport Company for sponsoring this three-year study of the effects of the spill. Without Amoco 's help, we would be nowhere near our present state of knowledge of what the effects of the spill were or how the recovery back to normal conditions has proceeded. Other studies have been carried out, sponsored by the French Government and other sources, but an important part of the work has been sponsored by Amoco.

Mr. Russ Mallatt, Dr. James Marum, Mr. John Lamping, Ms. Carol Cummings and others from Amoco attended meetings of the Joint Commission and the scientific sessions. They were always helpful and supportive of the Commission's work and never intruded on the design or conduct of the program.

We have, through this cooperative effort, obtained more detailed and more useful knowledge of the effects of this oil spill than of any other large oil spill in history. A major reason for this is that the biological communities present before the spill had been studied in great detail by French scientists.

Today many of the areas impacted by the spill appear to the casual observer to be recovered from the effects of the oil. However, investi- gations have shown that differences still exist between some of the current ecosystems and those present prior to the spill. Hopefully other studies will continue to watch and document the recovery processes.

Vll

These studies have added substantially to man's knowledge about oil spills. We can only hope that others will follow and build on the understanding of oil spill effects accumulated through these studies.

Lucien Laubier Wilmot Hess Joseph Angelovic

viii

CNEXO-NOAA Joint Scientific Commission

MEMBERSHIP

L. Laubier, Cochairman

Centre National pour l1 Exploitation des Oceans

Paris, FRANCE

Wilmot N. Hess, Cochairman

National Oceanic & Atmospheric Administration

Boulder, Colorado

Joseph W. Angelovic, Cochairman NOAA Office of Ocean Programs Rockville, Maryland

Jack Anderson

Battel le Pacific Northwest Laboratory

Sequim, Washington

J. Bergerard Station Biologique Roscoff, FRANCE

Edward S. Gilfillan Bowdoin College Bowdoin, Maine

I. R. Kaplan

University of California, Los Angeles

Los Angeles, California

R. l.etaconnoux

Institut des Peches Maritimes

Nantes, FRANCE

J. M. Peres

Station Marine and Endoume

Marseille, FRANCE

Philippe Renault

Institut Francais du Petrole

Rueil Malmaison, FRANCE

Douglas A. Wolfe

NOAA Office of Marine Pollution Assessment

Boulder, Colorado

IX

PART I

Physical, Chemical, and Microbiological Studies After the AMOCO CADIZ Oil Spill

Edited by E. R. Gundlach

Research Planning Institute, Inc.

Columbia, South Carolina, U.S.A. 29201

MICROBIAL HYDROCARBON DEGRADATION WITHIN SEDIMENT IMPACTED BY THE AMOCO CADIZ OIL SPILL

by

Ronald M. Atlas Department of Biology University of Louisville Louisville, Kentucky 40292

INTRODUCTION

The wreck of the AMOCO CADIZ in March 1978 released over 210,000 tons of oil into the marine environment. As much as one third of the spilt oil may have been washed into the intertidal zone. The spill occurred during storm surges, thereby spreading the oil throughout the intertidal zone. Two years after the AMOCO spill, the wreck of the tanker TANIO resulted in another oil spill that contaminated much of the same Brittany shoreline impacted by the AMOCO CADIZ. This study was undertaken to determine the fate of petroleum hydrocarbons within surface sediments along the Brittany coast with reference to the role of microorganisms in the oil weathering process.

METHODS

Sampling Regime

Duplicate samples were collected at intertidal sites along the Brittany coast which had received varying degrees of oiling from the AMOCO CADIZ spillage (Fig. 1). The sampling sites included the salt marsh at lie Grande, a beach near Portsall in the vicinity of the wreck site, a mudflat in Aber Wrac'h, a beach at St-Michel-en-Greve near where a large bivalve kill had been reported, a relatively lightly oiled reference site at Trez Hir and a site at Tregastel which was not oiled by the AMOCO CADIZ spill, but was later oiled by the spill from the tanker TANIO (Table 1). Surface sediment samples (upper 5 cm) were collected with a 3 cm diameter soil corer.

Samples were placed in metal cans for hydrocarbon analyses and in Whirlpak bags for microbial analyses. Samples were collected during December, 1978; March, 1979; August, 1979, November, 1979, March 1980, July, 1980 and June, 1981; 9, 12, 17, 20, 24, 28 and 39 months after the spillage, respectively. During November, 1979 sediment samples were also collected at four offshore sites in the Bay of Morlaix.

Samples for microbiological analyses were processed within four hours of collection. For hydrocarbon analyses, samples were frozen and shipped to Energy Resources Company (ERCO) for extraction and analysis

13

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FIGURE 1. Location of intertidal and subtidal sampling sites.

TABLE 1 - Description of sampling sites,

Site

Description

1 lie Grande - sandy - low energy - NE of bridge - relatively

unoiled.

2 lie Grande - sandy - low energy - SW of bridge - near end of

excavation area.

3 lie Grande - soil - heavily oiled - amid Juncus - above

excavation area.

4 St-Michel-en-Greve - sandy - high energy - near low tide mark.

5 Aber Wrac'h - mud - 100m offshore at Pcrros.

6 Aber Wrac'h - mud - 200m offshore at Perros.

7 Portsall - sandy - high energy - near wreck site - below high

tide line.

8 Portsall - sandy - high energy - near wreck site - rear rocks

- 100m below high tide line.

9 Trez Hir - sandy - moderate energy - reference site - below

high tide.

10 Trez Hir - sandy - moderate energy - reference site - 20m

below high tide line.

11 Tregastel - sandy - low energy - Tanio spill site - 20m below

high tide line.

12 Tregastel - sandy - low energy - Tanio spill site - 50m below

high tide line.

by silica gel column chromatography, weight determination, glass capillary gas chromatography and mass spectrometry.

Enumeration of Microbial Populations

Total numbers of microorganisms per gram dry weight of sediment were determined by direct count procedures. Portions of collected sediment samples were preserved with formalin. Microorgansims in the preserved samples were collected on a 0.2 mm pore size Nuclepore filter which had been stained with irgalan black. The microorganisms were stained with acridine orange and viewed using an Olympus epif luorescence microscope. Cells staining orange or green were counted in 20 randomly selected fields and the mean concentration determined.

Hydrocarbon utilizing microorganisms were enumerated using a three tube Most Probable Number (MPN) procedure. Serial dilutions of sediment samples, prepared using Rila marine salts solutions, were inoculated into sealed serum vials containing 10 ml BushnjeAl Haas broth (Difco) and 50 ml of Arabian crude oil spiked with C hexadecane (sp. act. 1 mCi/ml) . After 14 days incubation at 15°C, the C02 (if any) in the head space was collected by flushing and trapping in oxifluor CO .and quantitated by liquid scintillation counting. Vials showing CO production (counts significantly above background) were scored as positive and the Most Probable Number of hydrocarbon utilizers per gram dry weight calculated from standard MPN tables.

Biodegradation Potentials

Portions of sediment samples were placed into serum vials containing 10 ml Bushnell Haas broth. and 50 ml light Arabian crude oil spiked with either., C hexadecane, C pristane, C naphthalene, C benzanthracene or C 9-methylanthracene. After 14 days incubation, microbial hydrocarbon degrading activities were stopped by addition of KOH. The " C0„ produced from mineralization of the radiolabelled hydrocarbon was determined by acidifying the solution, flushing the headspace, trapping the CO in oxifluor C0„ and quantitating the C by liquid scintillation counting. The residual undegraded hydrocarbons and biodegradation products were recovered by extraction with hexane. The L C in each solvent extract was determined and fractionated, using silica gel column chromatography, into undegraded hydrocarbon fractions (hexane + benzene eluates) and degradation product fractions (methanol eluate + residual non-eluted counts). A 0.75 cm diameter X 10 cm column packed with 70-230 mesh silca gel 60 was used. Radiolabelled material in each fraction was quantitated by liquid scintillation counting. Sterile controls were used to correct for efficiency of recovery and fractionation. Triplicate determinations were made for each sample and radiolabelled hydrocarbon substrate combination. The percent hydrocarbon mineralization was calculated as C02 produced (above sterile control)/ C hydrocarbon, added. The .percent hydrocarbon biodegradation was calculated as ' C0„ produced +, C methanol fraction + C residual (all above sterile control)/ C hydrocarbon added. Carbon balances generally accounted for approximately 90% of the radiolabelled carbon added to the sediment (except for naphthalene where volatility losses prevented efficient recovery).

Chemical Hydrocarbon Analyses Performed at ERCO

For hydrocarbon analyses the samples were thawed, dried with methanol and extracted by high energy shaking with a mixture of methylene chloride-methanol (9:1). The extract was fractionated into an aliphatic (f ) fraction and an aromatic (f ) fraction using silica gel/alumina column chromatography. A 1 cm diameter X 25 cm column (1 cm alumina on top of 15 cm silica gel) was used. The f. fraction was eluted with 18 ml hexane; the f fraction subsequently was eluted with 21 ml of a 1:1 mixture of hexane-methylene chloride. After reducing the volume of solvent by evaporation, the gross amount (weight) of hydrocarbon in each fraction was determined gravimetrically from an evaporated and dried aliquot of the extract. The extracts were subjected to quantitative glass capillary-gas chromatographic (GC) analysis. Selected aromatic fractions also were analysed by combined glass capillary gas chromatographic/mass spectrometric (GC/MS) analysis for qualitative identification of individual compounds and quantification of minor components. Participation in an intercalibration exercise under the direction of the National Analytical Laboratory indicated that these analyses were at the state^ of the art with repeatable ± 20% detection of hydrocarbons in the ng g dry weight sediment range. The details of GC and GC/MS analysis employed are as follows:

CC: Hewlett Packard 5840A reporting GC with glass; splitless injection inlet system; 30 m glass capillary column coated with SE-30 (s 100,000 theoretical plates); FID detector; temperature programmed at 60-275°C min ; helium carrier gas 1 ml min ; transmission of integrated peak areas and retention time through HP 18846A digital communications interface to a PDP-10 computer for storage, retention index and concentration calculations. Deuterated anthracene (f.) and androstane (f ) were used as internal standards and response factors were determined with known concentrations of the reported compounds. GC analysis was used to quantitate components of the f. fraction.

GC/MS: Hewlett Packard 5985 quadrapole system (GC/MS Computer); mass spectrometer conditions: ionization voltage=70 eV, electron multiplier voltage=2200 V, scan conditions 40 amu to 500 amu at 225 amu s~ . Quantification of components of the f fraction was accomplished by mass f ragraentography wherein the stored GC/MS data is scanned for parent ions (m ) . The tabulated total ion currents for each parent ion is compared with deuterated anthracene (internal standard) and an instrumental response factor applied. Authentic polynuclear aromatic hydrocarbon standards were used to determine relative response factors (when no standard was available a response factor was assigned by extrapolation) .

In vitro Biodegradation

Sediment was collected at sites 6 and 7 in November, 1979 for in vitro biodegradation experiments. Replicate one hundred gram portions of sediment were placed into 250 ml flasks to which 50 ml of a sterile solution containing 0.5% KNO + 0.5% KH2P04 and 0.5 ml of light Arabian crude oil were added. The flasks were agitated on a rotary shaker at

100 RPM. After two, four, and six weeks of incubation at 15°C, the oil remaining in replicate flasks (two at each sampling time) was extracted and analysed as described below.

Additionally, replicate 100 g portions of sediment were placed into 1 liter stainless steel buckets. The containers were continuously flushed with a solution of Rila marine salts supplemented with 10 ppm KNO + 10 ppm KH PO . The height of the water level was adjusted to be 3 cm above the surface of the sediment layer. The flow rate was adjusted to 10 ml/h. After two, four, and six weeks of incubation at 15°C the oil remaining in replicate sediment portions was extracted and analysed as described below.

Analyses of ^in vitro Experiments

Residual oil was recovered from samples by extraction with sequential portions of diethyl ether and methylene chloride. The sediment was shaken at 200 RPM with repetitive portions of solvent. The extracts were subjected to column chromatography to split the extracts into aliphatic (f,) and aromatic (f~) fractions. Columns were prepared by suspending silica gel 100 (E. M. Reagents, Darmstadt, W. Germ.) in CH„C1„ and transferring the suspension into 25 ml burets with teflon stopcocks to attain a 15 ml silica gel bed. The CH„C1? was washed from the columns with three volumes of pentane. Portions of the extracts in pentane were applied to the columns, drained into the column bed, and allowed to stand for three to five minutes. The aliphatic fraction (f ) was eluted from the column with 25 ml pentane. After 25 ml pentane had been added to the column, 5 ml of 20% (v/v) CH„C1„ in pentane was added and allowed to drain into the column bed. Fraction f was 30 ml. The aromatic fraction (f ) was eluted from the column with 45 ml of 40% (v/v) CH2C1„ in pentane.

The fractions of each extract were then concentrated to about 5 ml at 35°C and transferred quantitatively to clean glass vials. Fractions f. and f were prepared for analysis by gas chromatography or gas chromatography mass spectrometry. An internal standard, hexamethyl benzene (Aldrich Chem. Co., Milwaukee, WI.), was added to each sample. In fraction f . , hexamethyl benzene (HMB) was present at 12.6 ng/ml; in fraction f„, HMB was present at 25.2 ng/ml.

Fraction f. was analyzed by GC on a Hewlett-Packard 5840 reporting GC with FID detector. The column was a 30 m, SE54 grade AA glass capillary (Supelco, Bellefonte, PA.). Conditions for chromatography were injector, 240°C; oven 70°C for 2 min. to 270°C at 4°C/min. and hold for 28 min.; FID, 300°C; and carrier, He at 25 cm/sec. A valley-valley intergration function was used for quantitative data acquisition. Response factors were calculated using n-alkanes, (C -C ) , pristane and phytane standards.

Fraction f„ was analyzed with a Hewlett-Packard 5992A GC-MS. Conditions for chromatography were injector, 240°C; oven 70°C for 2 min. to 270°C at 4°C/min. and hold for 18 min. Data was acquired using a selected ion monitor program. Thirteen ions were selected for representative aromatic compounds. The ions monitored were 128, 142,

147, 156, 170, 178, 184, 192, 198, 206, 212, 220, and 226. The representative compounds were naphthalene, methyl naphthalene, HMB as an interanal standard, dimethyl naphthalene, trimethyl naphthalene, phenanthrene, dibenzothiophene, methyl phenanthrene, methyl dibenzothiophene, dimethyl phenanthrene, dimethyl dibenzothiophene, trimethyl phenanthrene, and trimethyl dibenzothiophene, respectively. The dwell time per ion was 10 msec. Instrument response factors were calculated by injecting known quantities of unsubstituted and C and C„ substituted authentic aromatic hydrocarbons and determining the integrated response for each compound. These values were used to extrapolate for quantitation of isomers and C_ substituted compounds.

For analysis of the polar fraction including microbial degradation products, three samples were selected for analysis by the University of New Orleans Center for Bio-organic Studies. The samples were: 1) flow through, 6 week incubation from site 6; 2) flow through, 6 week incubation from site 7; 3) agitated flask, 6 week incubation from site 7. Frozen samples were sent for analysis. At the Center for Bio-organic Studies the samples were extracted with successive portions of CH OH, CH OH/CH CI and CH CI . The extracts were fractionated using silica gel and the f_ fraction was collected, methylated and analysed by high resolution GC-MS .

RESULTS AND DISCUSSION

The enumeration of hydrocarbon utilizing microorganisms indicated that numbers of hydrocarbon utilizers in the intertidal sediments increased significantly in response to hydrocarbon inputs (Table 2). Site 3, which is covered with seawater only at times of extreme high tide, showed very high populations of hydrocarbon utilizing microorganisms even three years after the AMOCO CADIZ spillage. Sites 5 and 6 (located within Aber Wrac'h) and Sites 7 and 8 (located near Portsall) showed variable, but apparently elevated, numbers of hydrocarbon utilizers for up to two years following the spill. It appears that hydrocarbons contained within the mud sediments of Aber Wrac'h continued to exert a selective pressure on the microbial community that favored elevated populations of hydrocarbon utilizers for a longer period of time than sites on high-energy sand beaches. Site 2 showed evidence that the TANIO spill impacted the lie Crande region. This site did not show elevated numbers of hydrocarbon utilizers in December 1978 or at later sampling times as a result of the AMOCO CADIZ spill, but in July of 1980, several months after the wreck of the TANIO, numbers of hydrocarbon utilizers were greatly elevated. A year later, however, the numbers of hydrocarbon utilizers had returned to background levels at this site. The unoiled control sites 9 and 10 and sites 1 and 4, which were impacted by the AMOCO CADIZ spill, did not show any evidence of elevated hydrocarbon-utilizing populations during the sampling period. Similarly, the offshore sites A-D in the Bay of Morlaix did not appear to be elevated at the time of sampling in November 1979. Sites 11 and 12 were added following the wreck of the TANIO and showed obviously elevated populations of hydrocarbon utilizers that persisted for over a year.

TABLE 2. MPN-Hydrocarbon Utilizers. ,3

Site 2-78 3-79

(// X 10 /g dry wt.) 8-79 11-79 3-80

7-80

6-81

1

0.

2

0.5

1

0.7

5

16

1

2

5

7

1

14

30

45000

1

3

2200

14000

41000

13000

160000

48000

24000

4

2

0.4

2

7

1

4

5

5

8

18

8

450

19

10

15

6

9

390

20

27

190

11

17

7

40

1900

1

2

12

2

2

8

57

350

150

8

3

1

10

9

0.

7

0.4

3

1

4

1

4

10

0.

1

0.2

4

1

2

2

3

11

-

-

-

-

19000

140000

24000

12

-

-

-

-

920000

140000

24000

A

-

-

-

66

-

-

-

B

-

-

-

32

-

-

-

C

-

-

-

13

-

-

-

D

-

-

-

13

-

-

-

The elevation in hydrocarbon utilizing populations, when detected, represented a shift within the microbial community. There generally was no evidence that total microbial biomass increased as a result of oiling although there generally was a tenfold variation in the microbial biomass between different sampling times (Table 3).

Site

12-78

TABLE 3. Direct Count. (// X !08/g dry wt.) 3-79 8-79 11-79 3-80

7-80

6-81

1

3

1

4

3

1

1

3

2

4

2

16

7

3

40

2

3

10

6

220

18

24

40

38

4

2

1

3

0.4

0.5

0.6

2

5

3

2

19

12

17

2

15

6

6

7

150

20

27

24

26

7

3

1

8

1

1

1

2

8

1

4

1

1

2

2

4

9

1

0.

5

2

1

0.3

1

4

10

0.5

0.

4

13

1

0.4

2

7

11

-

-

-

-

5

1

4

12

-

-

-

-

39

36

40

A

-

-

-

15

-

-

-

B

-

-

-

16

-

_

C

-

-

-

3

_

_

D

-

-

-

10

_

_

The microbial hydrocarbon biodegradation potential measurements showed that following the AMOCO CADIZ oil spillage, indigenous microbial populations in the sediment at all sampling sites were capable of degrading both aliphatic and aromatic components of crude oil (Tables 4-8). The variability in the results is not indicated in these tables, but the standard error was less than 4% for the percentage degraded and less than 10% for the percentage mineralized in all cases. The biodegradation potentials indicated that n-alkanes were preferentially degraded and that pristane was degraded at approximately half the rate of n-hexadecane. For aliphatic hydrocarbons approximately 30% of the amount of hydrocarbon biodegraded was converted to C0„ (mineralized) . Methodological difficulties in handling naphthalene made it difficult to assess the true extent of biodegradation for this compound. It is apparent, though, that the indigenous microbial populations were capable of degrading light aromatic hydrocarbons. The rates of degradation of the 3- and 4-ringed polynuclear aromatic hydrocarbons were lower than for branched and straight chained aliphatic hydrocarbons. In the case of the polynuclear aromatic hydrocarbons, a very low proportion of the amount of hydrocarbon degraded was converted to CO .

TABLE 4. Hexadecane biodegradation showing % degraded and (% mineralized).

Site 12-78 3-79 8-79 11-79 3-80 7-80 6-81

1 40 41 21 17 10 25 17

10

(8)

(11)

(1)

(15)

(2)

(12)

(10)

43

38

26

22

25

19

6

(ID

(13)

(8)

(13)

(17)

(12)

(7)

45

46

29

23

51

19

33

(15)

(15)

(8)

(18)

(39)

(14)

(26)

36

48

21

25

8

26

17

(14)

(13)

(7)

(14)

(6)

(19)

(10)

42

46

25

35

32

24

23

(14)

(14)

(13)

(14)

(20)

(18)

(15)

34

47

29

26

36

18

20

(11)

(12)

(11)

(20)

(18)

(11)

(13)

31

45

13

31

2

20

28

(10)

(13)

(3)

(21)

(1)

(14)

(19)

40

43

21

35

3

17

34

(15)

(11)

(5)

(19)

(2)

(12)

(20)

28

32

22

35

7

27

34

(12)

(3)

(3)

(14)

(3)

(15)

(24)

37

30

21

45

8

22

25

(10)

(3)

(10)

(32)

(3)

(14)

(17)

TABLE 5. Pristane biodegradation showing % degraded and

(% mineralized).

Site 12-78 3-79

8-79 11-79

3-80

7-80

6-81

1

18

22

12

17

18

17

27

(3)

(3)

(1)

(3)

(2)

(3)

(3)

2

23

22

16

15

17

24

24

(3)

(4)

(3)

(5)

(3)

(3)

(1)

3

19

21

16

14

18

20

24

(2)

(4)

(3)

(5)

(5)

(3)

(6)

4

26

23

16

18

17

19

20

(3)

(4)

(2)

(4)

(1)

(3)

(2)

5

21

28

21

17

16

22

25

(3)

(6)

(4)

(5)

(4)

(3)

(6)

6

21

30

19

19

17

20

21

(3)

(6)

(3)

(5)

(4)

(4)

(3)

7

25

24

16

25

12

23

23

(3)

(4)

(1)

(4)

(1)

(2)

(4)

8

31

23

21

18

20

21

23

(3)

(4)

(1)

(5)

(1)

(2)

(4)

9

27

20

22

19

17

22

24

(3)

(2)

(1)

(5)

(2)

(2)

(7)

0

29

-

21

20

18

21

20

(2)

(-)

(3)

(5)

(2)

(2)

(8)

TABLE 6. Biodegradation of naphthalene showing % degraded and (% mineralized).

Site

3-79

8-79

11-79 3-80

1

3(2)

2(1

I 3(31)

KD

2

9(7)

5(3

) 2(2)

2(2)

3

12(10)

5(3

> 2(2)

6(6)

4

7(6)

Kl

) 5(5)

KD

5

8(6)

Id

) 7(7)

7(7)

6

11(10)

Kl

1 KD

2(2)

7

10(9)

1(1

> 6(6)

KD

8

9(7)

1(1

> 7(7)

KD

9

HI)

2(1]

1 KD

KD

0

-

2(1

1 KD

KD

TABLE 7. Biodegradation of 9-methylanthracene showing % degradation and (% mineralization).

Site 3-79 8-79 11-79 3-80 7-80 6-81

1

10

-

1

5

6

9

(0)

(0)

(0)

(0)

(0)

(0)

2

19

8

6

8

10

9

(0)

(0)

(0)

(0)

(0)

(0)

3

18

18

6

7

7

10

(0)

(0)

(0)

(0)

(0)