55, L fvnlp

ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL

*^rt s '

U. S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

CENTRE NATIONAL POUR l'EXPLOITATION DES OCEANS

Digitized by the Internet Archive

in 2012 with funding from

LYRASIS Members and Sloan Foundation

http://www.archive.org/details/ecologicalstudyOOnoaa

■* ..* : . .-

ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL SPILL

,-sSSy^

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Report of the NOAA-CNEXO Joint Scientific Commission

October 1982

,^gï&, U. S. DEPARTMENT OF COMMERCE

| Malcolm Baldrige, Secretary W&' National Oceanic and Atmospheric Administration

John V. Byrne, Administrator

CENTRE NATIONAL POUR l'EXPLOITATION DES OCEANS

U. S. Depository Copy

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

Preface

Page 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.

Réponses 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

m

Page

BOUCHER, G., CHAMROUX, S., LE BORGNE, L. , and MEVEL, G. Etude Expérimentale d'une Pollution par Hydrocarbures dans un Mi croecosys terne Sedimentaire. I: Effet de la Contamination du Sediment sur la Meiofaune 229

BODIN, P. and BOUCHER, D.

Évolution a Moyen -Terme du Meiobenthos et du

Microphytobenthos sur Quelques Plages Touchées par

la Marée Noire de 1 'AMOCO -CADIZ 245

NEFF, J. M. and HAENSLY, W.E.

Long-Term Impact of the AMOCO CADIZ Crude Oil

Spill on Oysters Crassostrea gigas and Plaice

Pleuronectes platessa From Aber Benoit and Aber

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

II. Petroleum Contamination and Biochemical

Indices of Stress in Oysters and Plaice ........ 269

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

Rétablissement Naturel d'une Vegetation de Marais

Maritimes Altérée par les Hydrocarbures de 1 'AM0C0-

CADIZ: Modalités 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 Mi crophy tiques dans

les Sediments des Marais Maritimes de l'Ile Grande

a la Suite de la Pollution par T 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

TAMOCO-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 tons 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 wheel house 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 Ile 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.

On March 18, Dr. Wilmot N. Hess, Director of the Environmental Research Laboratories (ERL) of the National Oceanic and Atmospheric Administration (N0AA), contacted Dr. Lucien Laubier, Director of the Centre Océanologique de Bretagne (COB) of the Centre National pour l'Exploitation des Oceans (CNEX0), the French national océanographie 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. activityc 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. Biodégradation 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. Angel ovic 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 i.s 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 Angel ovic

viii

CNEXO-NOAA Joint Scientific Commission

MEMBERSHIP

L. Laubier, Cochai rman

Centre National pour l'Exploitation des Oceans

Paris, FRANCE

Wilmot N. Hess, Cochai rman

National Oceanic & Atmospheric Administration

Boulder, Colorado

Joseph W. Angel ovic, Cochai rman NOAA Office of Ocean Programs Rockville, Maryland

Jack Anderson

Battel le Pacific Northwest Laboratory

Seçiuim, Washington

J. Bergerard Station Biologique Roscoff, FRANCE

Edward S. Gil fil Ian Bowdoin College Bowdoin, Maine

I. R. Kaplan

University of California, Los Angeles

Los Angeles, California

R. Letaconnoux

Institut des Pèches Maritimes

Nantes, FRANCE

J. M. Peres

Station Marine and Endoume

Marseille, FRANCE

Philippe Renault

Institut Français du Pétrole

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-Crève 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 iron the tanker TANIO (Table 1). Surface sediment samples (upper 5 en) were collected with a 3 cm diameter soil corer.

Samples were placed in metal cans for hydrocarbon analyses and in Whirlpak bags for nicrobial analyses. Samples were collected during December, 1978; March, 1979; August, 1979, November, 1979, March L980, 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

ST MICHÊU- EN -GREVE (4»

FIGURE 1. Location of intertidal and subtidal sampling sites.

Sit<

1

2

3

4 5 6 7

9

10 11 12

TABLE 1 - Description of sampling sites.

Description

Ile Grande - sandy - low energy - NE of bridge - relatively

unoiled. Ile Grande - sandy - low energy - SW of bridge - near end of

excavation area, lie Grande - soil - heavily oiled - amid Juncus - above

excavation area. St-Michel-en-Grève - sandy - high energy - near low cide mark. Aber Wrac'h - mud - 100m offshore at Perros. Aber Wrac'h - mud - 200m offshore at Perros. Portsall - sandy - high energy - near wreck, site - below high

tide line. Portsall - sandy - high energy - near wreck site - near rocks

- 100m below high tide line. Trez Hir - sandy - moderate energy - reference site - below

high tide. Trez Hir - sandy - moderate energy - reference site - 20m

below high tide line. Tregastel - sandy - low energy - Tanio spill site - 20m below

high tide line. 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 Bushnell 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 C0? 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.

Biodégradation 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, l 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 radiolabeled hydrocarbon was determined by acidifying the solution, flushing the headspace, trapping the L CO^ in oxifluor CO» and quantitating the C by liquid scintillation counting. The residual undegraded hydrocarbons and biodégradation products were recovered by extraction with hexane. The 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 biodégradation 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).

3

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-raethanol (9:.l). 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 A dry weight sediment range. The details of GC and GC/MS analysis employed are as follows:

GC: Hewlett Packard 58A0A 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 Che 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 fragmentography 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 Biodégradation

Sediment was collected at sites 6 and 7 in November, 1979 for in vitro biodégradation 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% KH PO and 0.5 ml of light Arabian crude oil were added. The rlasks 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 KH2P0,. 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. \\. 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^Cl^ 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) CH2C12 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, Belief onte, 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 ri-alkanes, (C n-C?R) , pristane and phytane standards.

Fraction f2 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 CL 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-0H/CH2C12 and CH2C12. 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 Grande 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. (# X 103/g dry wt.)

Site 2-78 3-79

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

À

- -

-

-

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).

TABLE 3. Direct Count. (// X 108/g dry wt.)

Site

12-78 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 biodégradation 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 Che percentage mineralized in all cases. The biodégradation potentials indicated that ti-alkanes were preferentially degraded and that pristane was degraded at approximately half the rate of ri-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 biodégradation 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 C0?.

TABLE 4. Hexadecane biodégradation 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

8

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)

8

TABLE 5. Pristane biodégradation 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)

10

29

-

21

20

18

21

20

(2)

(-)

(3)

(5)

(2)

(2)

(8)

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

Site

3-79

8-79 11-79 3-80

I

3(2)

2(1)

3(31)

KD

2

9(7)

5(3)

2(2)

2(2)

3

12(10)

5(3)

2(2)

6(6)

4

7(6)

KD

5(5)

KD

5

8(6)

KD

7(7)

7(7)

6

11(10)

KD

KD

2(2)

7

10(9)

KD

6(6)

KD

8

9(7)

KD

7(7)

KD

9

HO

2(1)

KD

KD

0

2(1)

KD

KD

TABLE 7. Biodégradation 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)

(0)

4

23

2

2

1

10

6

(0)

(0)

(0)

(0)

(0)

(0)

5

17

4

2

7

21

6

(0)

(0)

(0)

(0)

(0)

(0)

6

19

1

3

4

11

5

(0)

(0)

(0)

(0) .

(0)

(0)

7

15

7

3

2

9

5

m +

(0)

(0)

(0)

(0)

(0)

(0)

8

21

2

4

1

6

5

(0)

(0)

(0)

(0)

(0)

(0)

9

15

11

1

4

11

4

(0)

(0)

(0)

(0)

(0)

(0)

0

6

3

13

5

4

<-)

(0)

(0)

(0)

(0)

(0)

TABLE 8.. Biodégradation of benzanthracene showing

% degradation and (% mineralization).

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

8

10

5

21

18

2

5

10

13

(0)

(0)

(0)

(0)

(0)

(0)

(0)

8

17

10

-

4

2

6

(0)

(0)

(0)

(0)

(0)

(0)

(0)

11

15

7

7

8

fc

12

(0)

(0)

(0)

(-)

(0)

(0)

(0)

4

5

6

2

6

3

5

(0)

(0)

(0)

(0)

(0)

(0)

(0)

8

13

14

2

10

11

8

(0)

(0)

(0)

(0)

(0)

(0)

(0)

4

8

11

2

4

1

6

(0)

(0)

(0)

(0)

(0)

(0)

(0)

11

3

6

7

4

3

6

(0)

(0)

(0)

(0)

(0)

(0)

" (0)

6

5

5

4

1

7

4

(0)

(0)

(0)

(0)

(0)

(0)

(0)

2

8

5

7

2

13

5

(0)

(0)

(0)

(0)

(0)

(0)

(0)

1

-

14

8

11

12

4

(0)

(-)

(0)

(0)

(0)

(0)

(0)

10

Based on the changes in the composition of the microbial community, as evidenced by elevations in numbers of hydrocarbon utilizing microorganisms and based on the microbial biodégradation potentials, it can be stated that biodégradation appears to have been a very important process that had the potential' for significantly altering the composition of the hydrocarbon mixture that impacted the sediments of the Brittany Coast following the AMOCO CADIZ spill. With time the residual hydrocarbon mixture should contain increasingly high proportions of complexed branched and condensed-ring hydrocarbon compounds that are degraded relatively slowly by the indigenous microorganisms .

The weight of the extractable hydrocarbons confirmed the occurrence of contaminating hydrocarbons at site 2 in July L980, presumably as a result of the TANIO spillage (Table 9). Similarly, high concentrations of hydrocarbons were found in at Sites 11 and 12 which were closer to the TANIO wreck. The levels of hydrocarbons at Site 3 remained high throughout the sampling program. Sites 1, 4, 9, and 10 showed a general lack of significant hydrocarbon concentration that would be indicative of petroleum pollution. Sites 5, 6, 7, and 8, in contrast, showed somewhat elevated hydrocarbon concentrations.

TABLE 9. Weights of extractable hydrocarbons (ug/g) .

1 λ

2

2 '

2

3 .'

2

4 Ï

2 l2

2

7 '

2

8 '

2

2 10 f

"|i

2

12 t{

t2

* i

2 2

2-78

3-79

8-79

11-79

3-80

7-80

6-81

27

9

5

17

8

20

4

57

5

16

58

37

25

22

52

21

42

147

50

2370

9

53

11

47

136

50

2160

29

272

2232

121092

108000

33000

16600

3680

338

2537

70329

95833

22500

32400

8080

21

22

9

20

35

12

8

57

17

11

19

29

17

49

122

140

56

213

65

68

21

103

82

99

233

73

156

42

178

458

177

536

874

109

56

226

416

209

556

830

281

75

91

72

152

80

58

23

15

75

59

123

63

39

26

29

179

382

164

243

98

34

31

148

298

135

194

83

24

59

7

32

77

20

21

43

13

3

34

78

32

21

36

25

8

29

13

36

23

23

21

11

20

' 29

96

31

48

74

-

-

515000

60800

320

-

-

-

512000

36300

440

-

-

-

-

73200

67

-

-

-

-

14300

109

-

-

-

102

-

-

-

-

-

-

88

-

-

-

-

-

-

210

-

-

'-

-

-

-

210

-

-

-

-

-

13

-

-

-

-

-

-

21

-

-

-

-

-

-

21

-

-

-

10

-

-

11

The detailed gas-chromatographic and mass-spectral analyses of the samples collected at each site indicated a lack, of significant petroleum hydrocarbons throughout the study at Sites 1, 4, 9, and 10 (Tables 10, 13, 18, 19). Site 2 showed some evidence of weathered hydrocarbons in

1978 and- a significant input of fresh petroleum hydrocarbons in July 1980 (Table 11). Site 3 had significant concentrations of weathered petroleum origin throughout the study (Table 12). Sites 5 and 6 showed an alteration of the hydrocarbon mixture with time that indicated the occurrence of biodégradation (Tables 14, 15). Samples at Sites 7 and 8 continued to show the presence of a relatively unweathered hydrocarbon mixture up to two years following the AMOCO CADIZ spill (Tables 16, 17). It appears that undegraded hydrocarbons were seeping into the surface sediments at Site 8 and it is postulated that either shifts in the sediment were repeatedly exposing hydrocarbons that had been protected from microbial degradation and/or that some oil continued to be washed ashore from the sunken AMOCO CADIZ vessel. Site 11 showed clear evidence of heavy oiling from the TANIO spill which persisted for a year following the spill (Table 20). The offshore sites sampled in November

1979 in thé' Bay of Morlaix failed- to show the presence of AMOCO CADIZ oil.

TABLE 10. Hydrocarbon concentration ng/g.

SITE 1 C-# 12-78 3-79 8-79 11-79 3-80 7-80 6-81

14

0

0

1

2

0

-

2

15

20

0

125

250

23

1

123

16

2

0

14

12

3

3

9

17

65

15

438

86

156

5

91

pristane

62

22

27

76

43

30

102

18

3

2

3

5

8

4

10

phytane

11

2

2

5

6

3

2

19

0

2

2

3

7

3

5

20

5

2

2

4

7

3

4

21

6

1

3

4

8

2

7

22

6

2

3

4

8

3

9

23

9

2

4

. 6

9

5

18

24

9

2

3

5

8

2

24

25

17

2

5

14

9

13

34

26

8

2

2

2

6

3

28

27

10

1

4

10

7

6

35

28

7

1

1

2

5

2

21

29

18

5

6

10

10

18

38

30

8

1

7

6

3

4

35

alkanes:

1.2

0.9

20.0

2.3

5.6

6.0

2.3

isoprenoids

pristane:

5.6

7.8

13.5

-

7.3

1.6

50.0

phytane

"

12

TABLE 11. Hydrocarbon concentration ng/g.

SITE 2

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

0

3

6

0

0

11700

3

15

44

115

J31

154

39

18200

111

16

0

11

18

0

4

19800

11

17

65

40

169

- 68

18

22000

93

priscane

27

38

160

1100

6

19800

46

18

8

9

8

181

5

22600

10

phycane

17

126

32

151

15

25900

15

19'

30

26

5

35

2

23400

6

20

18

16

9

9

8

18600

9

21

14

40

18

15

5

16700

13

22

16

29

7

10

9

12800

14

23

14

11

8

38

8

10900

23

24

12

11

7

6

13

10000

22

25

45

70

30

64

146

7120

35

26

22

12

6

10

16

6450

30

27

34

15

23

36

24

6320

33

28

53

40

6

29

10

4780

15

29

51

47

9

39

53

6040

36

30 --

31

95

71

116

55

4380

20

alkanes:

m

1.5

2.5

0.3

3.2

1.3

3.5

isoprenoids

priscane:

0.4

0.8

5.1

7.3

0.4

0.8

3.0

phytane

TABLE 12. Hydrocarbon concentration ng/g,

SITE 3

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

19

_

3525

1040

1390

_

15

-•

-

11025

0

633

-

-

16

17

121

9350

0

400

-

-

17

32

12

9550

3440

200

-

-

priscane

213

809

145150

47900

104

-

-

18

48

86

20250

3600

106

-

-

phycane

985

3088

275900

130000

673

-

-

19

255

948

63075

29200

283

825

-

20

149

247

36000

L1900

0

-

1270

21

31

241

17975

6210

508

2103

2380

22

25

12

8875

1440

130

-

3050

23

38

56

Il 100

0

0

-

3280

24

54

48

7925

3510

0

-

4640

25

-

-

12600

21300

2340

-

5 700

26

-

160

14900

2540

329

-

5190

27

172

898

45250

0

1160

516

10800

28

81

934

18600

0

109

-

2460

29

41

2025

31250

2090

3330

1692

12120

30

-

400

75775

0

118

13900

3120

alkanes:

0.1

..

0.1

0.1

0.8

_

_

isoprenoids

priscane:

0.5

0.3

0.5

0.4

0.2

-

1.9

phycane

13

TABLE 13. Hydrocarbon concentration ng/g.

SITE 4

c-//

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

0

0

0

0

i

T

15

0

-%

0

0

3

13

8

16

0

2

0

0

5

8

7

17

18

4

4

4

12

15

8

priscane

60

6

0

2

3

4

3

18

37

3

C

2

10

6

8

phvcane

153

18

0

8

8

6

11

19'

68

9

2

5

9

7

6

20

37

10

2

3

8

7

6

21

30

8

4

4

12

5

9

22

21

5

1

3

7

1

8

23

27

6

2

6

8

7

17

24

20

4

0

3

5

3

17

25

23

6

l

3

14

3

27

26

42

7

0

3

4

6

16

27

37

7

10

7

17

11

48

28

60

9

3

3

3

3

7

29

47

19

9

18

28

22

61

lu-

43

1

30

1

50

2

12

--

alkanes:

0.2

0.7

^

0.6

2.8

4.1

2.3

isoprenoids

priscane:

0.4

0.4

•c

0.4

0.6

0.3

phytane

TABLE

14.

Hydre

Dcarboi

a conce

intrati

,on nq/q

SITE 5

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

19

37

2

16

«B

5

3

15

20

65

167

59

11

35

29

16

-

-

18

0

-

12

8

17

25

112

122

0

95

18

585

priscane

24

150

II

885

8

175

108

18

16

50

17

0

4

2

4

phytane.

158

293

43

L58

12

36

20

19

64

140

12

16

4

3

8

20

43

5

17

19

16

7

9

21

57

50

23

208

8

13

17

22

39

18

19

14

6

18

11

23

25

36

23

30

20

18

20

24

4

30

15

18

9

13

20

25

22

5

39

115

99

111

100

26

46

5

11

24

21

11

21

27

51

93

30

87

48

29

62

28

70

64

6

13

7

13

10

29

96

'.36

17

113

13û

86

122

30

17

57

80

10

20

0

23

alkanes:

0.3

0.5

5.1

0.3

5.5

2.7

4.4

isoprenoids

priscane:

0.2

0.5

0.3

12.0

-

0.7

5.4

phytane

14

TABLE 15. Hydrocarbon concentration ng/g.

SITE 6

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

82

0

10

_

4

i

15

8

61

28

111

68

28

30

16

-

-

0

0

-

9

9

17

23

117

14

164

200

6

101

priscane

102

125

15

71

70

0

12

18

-

8

0

28

-

6

7

phytane

360

489

135

289

225

23

">

19

101

121

20

20

-

0

15

20

35

12

0

42

-

2

10

?l

86

180

59

223

35

31

30

22

-

149

4

48

38

9

6

23

39

58

16

81

80

28

61

24

5

-

5

37

27

5

10

25

109

159

80

128

484

47

85

26

128

151

10

50

53

6

30

27

68

126

88

135

255

48

124

28

97

56

13

105

28

12

21

29

L59

319

55

200

590

122

245

30 " '

36

140

254

342

30

18

55

alkanes:

0.1

0.3

0.2

0.8

0.9

1.5

2.7

isoprenoids

pristane:

0.3

0.3

0.1

0.3

-

0.0

0.5

phytane

TABLE 16.

Hyd:

rocarb<

on conc

entrât

ion na/

a .

STTE 7

c-#

12-78

3-79

8-79

U-79

3-80

7-80

6-81

14

9

4

9

0

2

14

43

15

7

7

43

11

11

29

77

16

51

10

58

18

32

29

64

17

109

20

96

27

63

38

85

pristnne

154

24

102

25

47

35

7

18

121

33

113

36

39

41

64

phytnne

256

65

159

43

86

43

39

19"

54

46

139

55

44

47

65

20

122

36

124

23

56

35

67

21

83

25

106

30

49

29

42

22

108

24

94

30

58

0

45

23

84

23

86

28

59

24

43

24

6)

18

80

46

47

24

54

25

111

27

89

42

62

25

32

26

105

27

62

25

42

22

35

27

54

20

110

48

30

17

35

28

126

42

52

15

28

14

17

29

92

28

62

28

49

26

44

30

112

36

356

152

29

37

29

nlkanes :

0.6

0.7

1.0

1.4

1 .1

1.6

3.3

isoprenoids

priscane:

0.6

0.4

0.6

0.6

0.6

0.8

0.2

phycane

15

TABLE 17.

Hydrocarbon concentration ng/g.

SITE 8

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

14

160

34

16

24

«

8

12

15

368

77

66

73

7

21

80

16

518

87

47

73

22

23

29

17

640

113

80

104

44

31

64

priscane

941

427

98

135

41

38

108

18

818

219

70

108

50

34

40

phytane

1839

955

116

171

74

54

46

19

1122

352

85

158

57

54

42

20

849

203

81

97

51

35

29

21

530

121

62

66

44

28

27

22

430

135

50

64

38

24

30

23

314

85

47

59

34

32

36

24

272

85

42

68

30

43

21

25

54

190

52

42

56

18

25

26

489

234

33

46

28

19

28

27

328

197

71

31

26

41

22

28

431

292

24

19

11

12

11

29

362

326

15

30

63

27

33

30"

315

411

170

10

218

14

29

alkaness

0.7

0.3

l.l

L.l

1.0

l.l

1.2

ispprenaids

priscane:

0.5

0.4

0.8

0.8

0.6

0.7

2.3

phycane

TABLE 18.

Hvdi

rocarfcx

Dn conc

entrât

ion nq/<

3*

SITE 9

c-#

12-78

3-79

8-79

11-79

3-80

7-80

6-81

u

0

0

0

0

-

28

-

15

0

0

3

8

-

42

-

16

0

0

3

3

-

21

-

17

3

II

5

22

8

36

19

priscane

1

4

6

2

-

0

1

18

3

6

3

5

6

9

9

phycane

2

4

2

3

-

0

5

19

4

3

4

4

8

6

12

20

4

3

0

6

11

6

12

21

4

6

4

6

II

4

15

22

4

7

3

5

9

6

12

23

4

9

3

10

8

7

19

24

4

8

1

£

5

4

4

15

25

7

13

8

15

7

62

29

26

8

23

l

3

1

2

15

27

8

12

3

11

3

14

44

28

11

34

0

3

1

0

12

29

11

22

0

12

15

24

65

30

6

8

13

1

1

3

25

alkanes:

1.8

2.0

1.6

7.1

5.7

isoprenoids

priscane:

0.6

0.2

2.6

0.9

-

mm

phycane

16

TABLE 19. Hydrocarbon concentration ng/g

SITE 10

c-#

12-78

3-79 8-79

11-79

3-80

7-80

6-8!

14

0

1 2

_

25

7

L5

0

5 33

3

-

42

74 7'

16

l

5 29

3

-

19

17

17

3

Il 53

14

8

41

228

pristane

l

3 7

3

-

7

4

,18

3

7 9

10

4

8

10

phytane

2

0 4

L.

-

2

5

19

3

11 5

6

1

7

il

20

4

8 4

6

3

6

8

21

3

4 5

6

4

246

14

22

2

10 5

3

6

15

23

2

8 7

4

10

31

24

1

4 5

2

1

21

25

3

226 13

24

7

55

76

26

2

13 6

2

2

33

27

1

11 22

21

5

31

137

28

1

11 8

5

7

23

29

0

5 38

33

14

53

194

30.

0

3 23

4

34

alkanes:

1.9

9.0

4.0

63.6

isoprenoids

pristar.e:

0.5

1.9

-

-

3.5

0.8

phytane

TABLE 20.

Hydrocarbon concentration

ng/g.

SITE II

C-ff

3-80

7-80

6r8l

14

605000

73200

69

15

661000

88200

285

16

625000

89100

83

17

636000

93600

110

pristane

342000

66900

418

18

643000

110700

76

phytane

231000

53400

.

372

19

629000

129000

80

20

680000

142000

128

21

724C00

132000

122

22

719000

141000

141

23

719000

142000

196

24

724000

140000

209

25

685000

133000

202

26

718000

155000

224

27

669000

167000

207

28

627000

192000

108

29

620000

168000

152

?0

479000

190000

318

Alkanes: Isop

renoids

0.2

2.4

0.3

Pristane:Phv

tane

0.7

1.3

1.1

17

The significant features of the chemical changes that were observed included a marked decrease in the proportion of n_-alkanes relative to isoprenoid hydrocarbons, the transient occurrence of an increase in unresolved hydrocarbons within the first year following the AMOCO CADIZ spillage, and the decreased importance of unsubstituted polynuclear aromatic hydrocarbons relative to dibenzothiophenes and the comparable or substituted forms of the polynuclear aromatic hydrocarbons (Figs. 2 and 3) >

The _in vitro hydrocarbon biodégradation experiments confirmed the fact that the indigenous microbial populations were capable of rapid and extensive degradation of Arabian crude oil. Much greater rates of biodégradation were observed in agitated compared to flow through experiments (Figs. 4-9). Both the in vitro experiments and the analysis of field experiments support the hypothesis that mixing energy has a very significant effect on the rates of hydrocarbon biodégradation. Rates of biodégradation appear to be environmentally influenced by the turbulence of mixing which can ensure a continued supply of nutrients and oxygen as well as dispersing the oil so as to establish a favor- able surface area to volume ratio for rapid microbial hydrocarbon biodégradation. The similarity of changes, observed in the composition of the hydrocarbon mixture in_ vitro compared to the analysis of field samples also suggests that nutrients were not a limiting factor that determined the rates of hydrocarbon biodégradation.

The analysis of the polar fractions from the in vitro experiments showed some surprising results (Table 21). There was a lack of oxygenated aromatic compounds. It had been predicted that there would be a greater accumulation of polar products from aromatic biodégradation since less C0? was being produced than from aliphatic biodégradation where a significant proportion of the hydrocarbon that was biodegraded was released as CO,. . There were significant accumulations of polar compounds that appear to be biodégradation products of aliphatic hydrocarbons, especially as C.^-C.q acids. Interestingly, the major polar products included unsaturated acids. As a rule, the predominant biochemical pathway for the biodégradation of straight chained hydrocarbons does not involve the formation of unsaturated compounds, although a> biochemical pathway has recently been elucidated for some bacteria that does introduce a double bond into the hydrocarbon. It appears that the microbial populations indigenous to the sediment of the Brittany Coast possess such a biochemical capability.

18

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FIGURE 2. Changes in the relative concentrations of aliphatic hydrocarbons at sites 3, 5, and 7.

19

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Nl'HlHl PHENAN OISENfO NAPHTHA PHENAN- DIBEKZO'

I.ENE THBENE THlOPHENE LENE ThRENE rHMPHENE

FIGURE 3. Changes in the relative concentrations of aromatic hydrocarbons at. sites 3, 5, and 7.

20

5-

4-

3-

Z2.

<

1 -

Z UJ

u

z

o

UJ

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FLOW THROUGH 2 WEEKS SITE 7

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FLOW THROUGH 6 WEEKS SITE 7

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FLOW THROUGH 2 WEEKS SITE 6

ll

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4 WEEKS SITE 6

u

11 12 13 14 IS 16 1 7 PR 1 8 PH 19 20 2 1 22 23242$ 262/28

H 17 Ll 14 l> io 1' PH 18 PH 19 20 21 22 2] 24 25 26 27 28

FIGURE 4. (Left column) Changes in the relative concentrations of- aliphatic hydrocarbons in flow-through experiment with sediment from site 7

FIGURE 5. (Right column) Changes in the relative concentrations of aliphatic hydrocarbons in f Low-through experiment with sediment from site 6.

21

3-

Z

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r

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N C, C2C3P C, C2C30 C, CjC3

n n' h p p p b d o o

T B S 8

T T T

4

■1

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U

tmlil

2 WEEKS

rh-TTh-TL I

J

4 WEEKS

6 WEEKS

11 12 13 14 is is t7 PR is PH 19 20 21 2 2 23 24 2S 28 27 28

FIGURE 6. (Left column) Changes in Che relative concentrations of aromatic hydrocarbons in flow-through experiment with sediment from site 6

FIGURE 7. (Right column) Changes in the relative concentrations of aliphatic hydrocarbons in flask experiment with sediment from site 7.

22

< s

' FLASK

^ __ 0 TIME

' ' ' SITE 6

4- '"",

r n

3. 1

2-

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11 12 13 14 IS 16 17 PR 18 PH 19 20 21 22 23 24 25 26 27 28

N C,C, C,P C,C, C,0 C, C,C, F c, NNN PPP80D0 F

T 8 8 S T T T

FIGURE 8. (Left column) Changes in Che relative concentrations of aliphatic hydrocarbons in flask experiment with sediment from site 6.

FIGURE 9. (Right column) Changes in the relative concentrations of aromatic hydrocarbons in flask experiment with sediment from site 6.

23

TABLE 21. Concentrations and Identities of Polar Compounds ng/g

SITE SITE SITE

7 6 7 FLASK FLOW FLOW THROUGH THROUGH

dodecanoic acid tetradecanoic acid methyltetradecanoic acid pentadecanoic acid hadecenoic acid hexadecanoic acid isoheptadecanoic acid heptadecanoic acid octadecenoic acid octadecanoic acid nonadecanoic acid eicosanoic acid tetracosanic acid hexacosanic acid octacosanic acid

16.5

2.3

4.7

22.2

13.7

24.5

15.0

19.7

43.3

10.5

13.5

24.0

47.0

129.0

217.0

73.9

109.0

157,0

8.3

7.8

11.9

3.2

8.6

4.3

59.3

76.4

99.2

44.4

34.2

44.6

10.1

25.4

_

9.8

25.4

55.8

14.5

3.0

13.8

7.7

1.4

10.8

15.8

13.3

15.6

isocyclopropaneoctanoic acid

15.5

25.5

42.2

methyloctahydrophenanthrene- carboxylic acid (tent.)

21.3

2.0

24

CONCLUSIONS

Microbial degradation appears to have played a very important role in the weathering of oil spilled from the AMOCO CADIZ. Microbial hydrocarbon degradation potentials are in general agreement with the observed changes in the composition of oil stranded within the littoral zone. The chemical evolution of the hydrocarbon mixture within intertidal sediments led to a relative enrichment in isoprenoid alkanes, a transient complex unresolved mixture, and a relative enrichment of dibenzothiophenes and alkylated phenanthrenes.

There was a general, but variable decline in concentrations of hydrocarbons over the three year period following the AMOCO CADIZ spill within Aber Wrac'h. The concentrations of hydrocarbons also declined at sites that were regularly covered by tides. At the one site in lie Grande, which is not subject to daily tidal washing, the concentrations of hydrocarbons remained high even three years following the spill. At nearby sites within the He Grande salt marsh, which were physically cleansed of , AMOCO CADIZ oil, there was little chemical or microbial evidence of any impact from the AMOCO CADIZ spill at any of the sampling times. The incurrence of oil from the TANIO wreck was apparent even at sites that had been oiled as a result of the AMOCO CADIZ spill.

The microbial population levels generally reflected the relative degrees of persistence of petroleum hydrocarbons. The microbial community at all of the sites studied had essentially the same potential capability for degrading hydrocarbons and as such the differences in the hydrocarbon concentrations and composition recovered from the field samples probably reflect the initial rates of oiling and environmental influences. The indigenous microbial community retained the capability of responding to a second incursion of oil resulting from the TANIO spill.

Both the field experiments and the Jji vitro studies suggest that mixing energy, related to nutrient and oxygen availability, was extremely important in permitting the high rates of observed oil weathering. The occurrence of both saturated and unsaturated acids in the sediments studied _in vitro suggest that several biochemical pathways were active in the biodégradation of the aliphatic hydrocarbon fraction. The hydrocarbon biodégradation potential suggested that relatively high concentrations of oxygenated aromatic hydrocarbons should accumulate, but for unexplained reasons the analyses of the polar fraction generally failed to show such accumulations.

25

LABORATORY SIMULATION OF THE

MICROBIOLOGICAL DEGRADATION OF CRUDE OIL

IN A MARINE ENVIRONMENT

by

D. Ballerini(l) , J. Ducreux(l) and J. Rivière(2)

(1) Institut Français du Pétrole - Direction de Recherche "Environnement et Biologie Pétrolière"

1 et 4 avenue de Bois-Préau - 92506 RUEIL-MALMAISON - FRANCE

(2) Institut National Agronomique, Paris-Grignon

16, rue Claude Bernard - 75231 PARIS 05 - FRANCE

This study essentially intends to quantify the biodégradation process of a crude oil in optimum conditions compatible with the marine environment.

Experiments were conducted in the Laboratory reactors (batch and continuous cultures), with perfect monitoring, of all physicochemical parameters such as pH (pH 8.1), temperature at 20°C, mixing rate 600 rpm, and aeration velocity (1 liter of air/liter of medium per hour) .

The composition of the mineral medium was defined by taking the

mean composition of salts in the Atlantic Ocean as a basis, and

enriching it with nitrogen (235 mg.1-1), phosphorus (26.7 mg. 1-1) and iron (0.4 mg.1-1).

In order to reproduce conditions prevailing at sea as closely as possible, in which the evaporation of light products is not negligible Arabian Light Crude was employed (ALC 240+) from which all fractions distilling below 240°C were removed by low pressure distillation.

The analytical methodology employed to observe the crude oil biodé- gradation process is shown schematically in the following- figure.

The gas flow was passed through a trap containing CC14, which retained the evaporated hydrocarbons, and then through a second trap containing a known quantity of 1 N KOH, which retained the carbon dioxide. The hydrocarbons were then determined by infrared spectrometry. The C02 produced was determined by titrimetry.

Liquid samples were taken during fermentation.

The first sample was centrifuged to separate the hydrocarbon phase -from the aqueous phase, which was then filtered (filter pore diameter 0.22 u) to eliminate fine particles in suspension. The following were analyzed in this perfectly clarified aqueous phase:

total organic carbon (Dohrman DC. 50 instrument),

dissolved C02 using the Warburg equipment,

27

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Figure 1

28

residual phosphorus,

residual ammoniacal nitrogen and the intracellular nitrogen concen- tration (Kjeldahl's method); these two analyses served to determine the quantity of biomass formed.

The residual hydrocarbons were extracted from a second liquid sample. Asphaltenes were precipitated from the hydrocarbon residue using hot heptane for one hour, dried and weighed.

The residue obtained after evaporation of the heptane was processed to separate the three main families of hydrocarbons in crude oil: saturates, aromatics and resins, by thin layer chromatography (50 mg samples) or liquid chromatography (samples weighing about 1 g) .

The sum of the weights of the three fractions thus recovered, using liquid chromatography, compared with the initial rate of the hydrocar- bons deposited on the column, always accounted for a proportion between 90 and 100 %.

The loss percentage increased when the test samples' were taken at increasingly long culture times, hence with samples that underwent the longest biodégradation times. These losses are iikely to be due largely to the retention of polar compounds of the resins on the column, compounds that are formed during oxydation reactions, or pos- sibly by biochemical co-oxydation, and whose concentration increases with biodégradation time.

Using the different fractions obtained (saturates, aromatics, resins), we performed more detailed analyses by gas phase chromatography (Varian 3700 chromatograph) equipped with "Splitless" injection and flamme ionization detector) , a combination of gas phase chromatography and mass spectrometry (Varian CH5DF spectrometer), proton NMR that yielded the fraction of hydrogen- belonging to methyl groups in the sa- turates family, ^C NMR, which yields the percentage of aromatic carbon in comparison with total carbon in the aromatic fraction, and by infra- red spectrometry on the resins.

1. BATCH CULTURES

1.1. Biodégradation of hydrocarbon families and sub-families in ALC 240+.

We selected a mixed culture of bacteria from samples of muds and slud- ges collected on places hit by crude oil spills.

The experiment was conducted with ALC 240 in an initial concentration of 2.65 g.l""l, over a period of 48 hours.

Of the 2.65 g.l-1 of initial hydrocarbons, 1.08 g.l"1 were consumed, representing 41 % degradation. It appears clearly that the saturates fraction is most sensitive to biodégradation, because 67 % of this fraction were consumed, whereas only 27 % of the aromatics fraction were degraded. The quantity of hydrocarbons evaporated was negligible.

From the standpoint of reproducibility of results, a previous experi- ment yielded the following results: hydrocarbons consumed 44 %, satu- rates degraded 63.1 %, aromatics disappeared 48.6 %.

29

The saturated hydrocarbons were most rapidly biodegraded At the end of the culture, the disappearance o/ aromatic compounds is

lltTrZ , lan QTlrichment of the aqueous phase in organic carbon, the concentration of which may reach 250 mg.1-1. This observation

di^H h% that a lar§e Part °f the aromatics a™ only partly oxi- dized before passing into the aqueous phase.

The resins were only slightly attacked if at all, and the asphaltene concentrations at the start and end of the batch culture were absolu»

1'7arabe' demonstrating total insensitivity of these substances to Diochemical processes.

The determination of n-alkanes <C14-C35) and detectable isoprenoids ^C16-C23) by gas phase chromatography showed that these compounds disappeared almost totally by the end of the culture.

The mass spectrometry analysis of the "saturates" fraction showed that the alkanes were mainly biodegraded, as 88.9 % disappeared at the end of the culture. This enables us to postulate that, in addition to the n-alkanes and isoprenoids, which only account for 14.8 % of the

saturates" fraction, the bulk of the iso-alkanes present in the crude oil was consumed by microorganisms.

Among the naphtenic compounds, the 1- and 2-cycle naphtenes were mainly consumed, with respective biodégradation rates of 44 and 47 %.

Proton NMR analyses giving the CH3/CH2 ratio, conducted on the satu-

Inrt^Hr It k in?icate any significant difference between the start and end of the batch culture.

mainlvea?f^ T "ar0matics" faction, the action of microorganisms mainly affected the mono- and di-aromatic compounds. At the end of the

ïhin^'/iV m0n°" and di-aromatics «ith a number of carbons less than 16 had disappeared.

Among the mono-aromatics, the substances most sensitive to microbial action were the alkylbenzenes, of which 67.7 % disappeared at the end "of 46%Cy Th6,Han4 ^ benzo^cl°Paraffins, with a consumption rate lurr7, ;, -^ erences measured for benzodicycloparaff ins were not sufficiently wide to be meaningful..

As for di-aromatic compounds, the microorganisms displayed a very clear et feet on the residual concentration of naphtalenes, of which 50 % disappeared after 48 hours of culture.

Through a second experiment, we investigated the changes in composition of the aromatics fraction, by drawing a distinction between sulfu- compounds and other aromatics.

tlZlZT th°Se fth a r°Ugh f°rmula CnH2n-l°S. the sulfur-containing

* o? 0 orV? atKa°!:ed by bacteria" Th* aromatics/sulfur-compounds ratio of 0.98 before biodégradation decreased to 0.82 after biodegra-

tnnT" SH0Hin? *!!at " WaS mainly the "°"-sulfur-oontaining aromatics sulf,^ ?n \t * disaPPeared- I" addition, the weight percentage of

sulfur in the aromatics fraction increased with time from 4.05 to 4.15 confirming the enrichment of this fraction in sulfur^containing sub- '

30

13 The " C NMR analyses used to quantify the aromatics C/total C ratio

failed to reveal any significant difference before (43.4 %) an after

(43.7 %) biodégradation.

With respect to the resins, part of the polar compounds of this frac- tion formed during biodégradation remained absorbed on the liquid chro- matography column, and consequently the analyses performed on the eluted resin fraction were not truly representative. This retention of polar compounds of the liquid chromatography column was confirmed by elemental analysis, showing oxygen to drop from 2.75 % (by weight) at the start of the batch culture to 2.35 % at the end of the culture.

The determination of molecular weights of the resins yielded the following results: 690 at the start of the batch culture, 740 at the end of the batch, namely very slightly differing molecular weights.

1.2. Examination of oxidation products.

Analyzing the aqueous phase sampled at the end of the batch culture (volume sampled = 1 liter), centrifuged and filtered, we found a total organic carbon concentration of 260 mg.1-1.

We carried out an esterification (BF3-CH3OH) of the compounds of this aqueous phase. The organic extract was evaporated and weighed. The weight of the extracted compounds, related to one liter of culture, was 120.2 mg. In the acidified residual aqueous phase, initial extrac- tion with CHpClpi followed by a second extraction with benzene, yielded a new organic phase that contained polar compounds such as alcohols, ketones and phenols, which represented 7.58 mg/1 of aqueous phase after evaporation.

Identification by GC/MS coupling of compounds separated by gas phase chromatography was difficult because of the presence of a strong background of poorly resolved constituents, which could be hydrocarbons Despite these problems, we succeeded in identifying normal and iso acidic compounds in the aqueous phase, in the form of their correspon- ding esters, obtained after esterification of the aqueous phase.

The GC/MS coupling enabled us to observe the masses m/e = 74 characte- ristic of n-esters, and m/e = 88 characteristics of iso-esters.

1.3. Changes in microbial flora with time.

Three samples were taken during the batch culture, the first at the start of growth, the second during the active biodégradation phase (after 15 hours of culture), corresponding to consumption of the satu- rated hydrocarbons, and the third after 25 hours, in the slowdown period of the biodégradation process, corresponding to microbial attack of the aromatics.

In the three different stages investigated, different dominant strains were found, belonging to two genera only, Pseudomonas and Moraxella, confirming that changes in the crude oil during a biodégradation pro- cess are accompanied automatically by changes in the microbial flora. At the start and middle of the batch culture, we chiefly identified

31

bacteria of the genus Moraxella, indicating t-h=t- ♦* ,. .

fectly adapted to the hydrocarbons Present "a TîLl \ ?"* "* Per"

the culture and, beine dominant h! ? * Partlcular time in

tially consumed ^he^dro^roons ^vF^g^J* "«'j"—

. types of constituents were bioripar^^ ! saturates fraction, as these

the culture, h^Z K2,M2 ^^ At *' ^ °f

microorganisms, only the Pseudomo^^ins^L^nant?30^ * *"

This investigation again confirms fhaf u

degradation of hyc*ocarbons in r=rude\n contalnin^^1""*

of compounds, a mixed culture of bacteria it? I ? "lde varie'y

than a pure bacteria of .hi 1 L ! , S certainly ">ore effective

given type of constituent -tabolism is only adapted to a

1.4. Toxicity analysis of oxidation products.

During the different ALC 240* crude „u n^. j ...

always observed a substantial rise in L£? ^ * ^ exPariraen^ , we concentration in the aqueous phase with th~ n^"1' T b°" (T°C) regular final concentration around 200 mga"? &* °f ''"' W"h a

^-^^

dation products of certain hydrocarbons present L'he crude oil?""

^::rs sss^Sori's samph^ was determi-d * «-

Research i975, ^ p 4MM 1 "f T 'V" dStaU in Mutation

the fiv.S^PleS W6re tSSted in a ran*e fr°m 0-1 to 500 Ml on three of

order " ££™t£ ™î" *"? ^ teSt (TAa538' TA'98 and "A^°> ^ detect the mutagenicity of products such as HAP, for example.

No mutagenic activity was detected in these .two samples.

effect on0;heftn«eystr:inrith:rH°fTtheSe tWO Sampl" had ^ *o*c on cne three strains tested (TA. 1538, TA. 98 and TA. 100).

1.5. Study of the biodégradation of a mixture of pure hydrocarbons.

Ind o^cos'anl "one T^^T ""t^ °f *"° "-*■»"• he*adaCa"a two mono-aromatics n I ' /?? ""' * tv°~^S naphtene, decaline. dimethyl-naohtal^: P_Cym!n and dodecylbenzene, one di-aromatic, containing = ! ' '«-aromatic, phenanthrene , and two sulfur- containing aromatic, benzothiophene and dibenzothiophene

32

Experiments were conducted in batch culture in the same conditions as those described for Arabian Light Crude. The mixed culture of bacteria used was the mixture of the strains Pseudomonas and Moraxella isolated and purified, described in Section 1.3. By sucessive cultures in flasks with the pure hydrocarbon mixture as the only carbon substrate, the mixed culture was progressively adapted to grow on these ten hydrocar- bon compounds .

The culture was carried out in batch for 61 Vz hours, and we observed the changes in the biomass, total hydrocarbons, and each compound, and also the organic substances that passed into the aqueous phase.

Following a lag phase of about 10 hours, a growth acceleration phase was observed up to the 25th hour, then a linear phase from the 25th to the 35th hour, and finally the slowdown of bacterial growth. At the end of the batch, the dry cell weight was 0.5 g.l--1-. The biodégradation process of total hydrocarbons perfectly matched the microorganism growth pattern. After 61 Vz hours, 82.8 % of the hydrocarbons were degraded.

It appears that the three most volatile compounds, paracymene, decaline and benzothiophene, could not be found after extraction, from the very outset of the experiment. These three products must therefore disappear chiefly during extract evaporation operations. However, a small propor- tion passes very rapidly into the aqueous phase in the marine environ- ment, because the latter contained oxidation products of p-cymene among others, as well as benzothiophene.

The two n-alkanes , n-hexadecane and octâcosane, and the dodecylbenzene were consumed first. For these three products, which practically disappeared by the end of the batch, their respective biodégradation rates after 37 Vz hours only of culture were 93 %, 87.5 % and 80 %. Pristane only started being attacked after 24 Vz hours, and was 69.2 % consumed at the end of the culture. During the last 20 hours, while practically no alkanes or alkylbenzene remained in the reactor, dimethyl- naphtalene was biodegraded (disappearance rate 67 %) . Phenanthrene and dibenzothiophene were consumed very little if at all.

These results perfectly confirm those found with Arabian Light Crude, which showed that alkanes and isoprenoids were attacked first, follo- ■wed by mono- and di-aromatics. Similarly, it was observed that tri- aromatics and sulfur-containing aromatics were only slightly sensitive or insensitive to the action of microorganisms. The fact that dodecyl- benzene disappeared fairly rapidly is explained by the presence of the linear chain which, like the n-alkanes, is readily accessible to bacteria.

The total organic carbon concentration (TOC) measured in the medium was. 385 mg.l"1. After esterif ication and evaporation of the organic extract, esters and some other polar compounds were found in a concentration of 221.5 mg.l"1.

We carried out analyses by gas phase chromatography and GC/MS coupling" in an attempt to identify these products.

Since many products were present in trace amounts, and several of them were eluted simultaneously and combined in a single peak, we encountered considerable difficulty in identifying them on the mass spectrometer.

33

2. CONTINUOUS CULTURES

In continuous culture, since this technique serves to check the concen- trations of all the nutritive elements at all times, and to adjust these concentrations to limit thresholds, thus closely approaching conditions encountered at sea, we attempted to quantify the nitrogen, phosphorus and oxygen requirements for the biodégradation of given quantities of hydrocarbons present in ALC 240+ .

The following operating conditions were used:

-1

Dilution rate

D = 0.04 h

Temperature

20°C

Reactor volume

2 liters

Agitation

520 rpm

Ph of culture

8.1

GHSV

1

(except for quantification of the oxygen requirements, where the GHSV was varied' from 1 to 0.25).

ALC 240 concentration entering reactor always about 2.5 g.l

For a given concentration of an element (nitrogen, phosphorus or oxy- gen) entering the reactor, the experimental time was about one week. Upon each alteration in operating conditions, it was necessary to wait another week for equilibrium to be re-established.

When the residual concentration of nitrogen was in excess, the bacterial consumption of this element per mg of hydrocarbons degraded ranged from 0.1 to 0.11 mg. However, when the nitrogen reached a limit with residual contents around 1 mg/liter, the nitrogen requirements dropped to 0.07 mg.

The same occurence was observed with phosphorus. In conditions of non- limitation, the biochemical consumption of phosphorus, around 0.012 to 0.013 mg/mg of hydrocarbons consumed, declined to only 0.005 mg/mg of hydrocarbons consumed when the residual concentration of elemental P reached a limit ( < 1 mg.l""^).

With respect to oxygen, microorganism requirements fluctuated between 1.4 and 1.9 mg oxygen per mg of biodegraded hydrocarbons, for residual dissolved oxygen concentrations between 50 and 7 % of the saturation value.

34

THE AMOCO CADIZ ANALYTICAL CHEMISTRY

PROGRAM

by

Paul D. Boehm, Ph.D.

Environmental Sciences Divisions, ERCO (Energy Resources Company, Inc.), 185 Alewife Brook Parkway, Cambridge, Massachusetts 02138

TABLE OF CONTENTS

1 . INTRODUCTION

2. METHODS AND MATERIALS

2.1 Sediments and Sediment Cores (Extraction and Processing)

2.2 Plant and Animal Tissue

3.' RESULTS AND DISCUSSION

3.1 Overall Findings

3.1.1 Weathering of AMOCO CADIZ Oil

3.1.2 Persistence of Marker Compounds

3.1.3 Residues in Tissues

3.1.4 Environmental Var iab il ity

3.2 Surface Sediments (Atlas, University of Louisville)

3.3 Offshore Sediments (Marchand, CNEXO)

L'Aber Benoit Sediments (Courtot, U. West Brittany)

3.4 Sediment Cores (Ward, Montana State University)

3.5 Oysters and Plaice (Neff, Battelle)

3.6 Oysters and Fish (Michel, ISTPM)

3.7 Seaweed and Sediments (Topinka, Bigelow Laboratory for Ocean Sciences)

4. Conclusions

5. REFERENCES

35

INTRODUCTION

All fate and effects studies of oil spills in the marine environ- ment depend on analytical chemical information concerning the distribu- tion and composition of the spilled oil. This includes petroleum hydrocarbon concentrations and compositions in water, sediment, and tissue samples. In turn, this information can be used to deduce the nature of the weathering process (including evaporation, dissolution, and biodégradation) , biological assimilation and depuration, and the mass budget of the oil. Thus the analytical chemistry component of the AMOCO CADIZ research program provides crucial information to many other components of the program in the investigation of the time- dependent fate and effects of this spill.

During the six weeks following the grounding of the supertanker AMOCO CADIZ on March 16, 1978, oil came ashore along 320 kilometers of the Brittany coastline (Gundlach and Hayes, 1978) . Various shoreline types were impacted (e.g., rocky shores, sand flats, coastal embay- ments, tidal mud flats and salt marshes) . During the early stages of the spill, oil was transported offshore and deposited in the benthic environment. The fate of petroleum residues deposited in these impact- ed areas was and continues to be affected by coastal processes which dictate such factors as wave energy and sediment transport, and create environments of differing substrate character (e.g., grain size), chemical status (oxidizing versus reducing) , and biological activity (e.g., microbiological biomass) . All of these factors and others (e.g., light intensity) combine to determine the weathering character- istics of the residual petroleum assemblage.

Biological populations initially impacted by the spilled oil may be subject to chronic exposure to petroleum hydrocarbons associated with, (and released from) the substrate to which they are closely linked, or they may undergo rapid or slow depuration of initial resi- dues if no longer exposed to oil, via transplantation or due to flush- ing by "clean" seawater. Such differential exposure histories have been previously observed to profoundly affect the spilled oil residual body burdens in marine organisms (Boehm et al., 1982).

Although oil spills have received increasing attention from the scientific community during the past decade, there have been few opportunities to examine the chemical compositional changes in beached or sedimented oil in a variety of coastal environments, over a signifi- cant period of time and to examine uptake (impact) and depuration (recovery) of petroleum by marine organisms. A detailed examination of the chemical changes in oiled substrate suggests both the anticipated residence time of deposited oil, and the potential for biological damage of the petroleum residues. Rashid (1974) examined compositional changes of Bunker C oil from the ARROW spill in Nova Scotia at dif- ferent coastal locations. Other than this study only site-specific studies of the geochemistry of petroleum weathering (e.g., Mayo et al., 1978; Blumer et al., 1973; Teal et al., 1978) have been undertaken.

36

Uptake and depuration by organisms have been the subjects of many laboratory experiments (e.g. Neff et al., 1976; Roesijadi et al., 1978) but relatively few real spill scenarios (e.g. Boehm et al., 1982; Grahl-Nielsen et al., 1978).

This report is intended to present an overview of the chemistry program along with enough supporting data and interpretations for each program element to make this a self contained document. After a methods section, a summary of the general findings is presented. Discussions of the analytical chemical and biogeochemical findings of each of the six specific investigations follow; the last section draws conclusions from the study as a whole. Much of the raw analytical data has been omitted here for brevity. Tabulations of analytical data are available either from the individual, principal investigators or from the chemistry group. This data has formed the basis of several publications to date (Calder and Boehm, 1981, Boehm et al., 1981, Atlas et al., 1981, Winfrey et al. 1981) as well as several manuscripts in preparation. Additional interpretative details are found in these manuscripts. *-

METHODS AND MATERIALS

As part of the NOAA/CNEXO research program to examine the long- term fates and effects of the spill, we obtained samples of frozen intertidal surface sediment, benthic sediment, sediment cores, oysters, flatfish and macroalgae from a number of U.S. and French investigators (Table 1).

TABLE 1. Summary of AMOCO CADIZ chemistry program.

1 - Chemical Composition, Weathering, and Concentrations in

Surface Intertidal Sediments (Atlas; Calder): 1978-1981

2 - Chemical Composition, Weathering, and Concentrations in

Subtidal Sediment (Marchand, Cour tot ) : 1978-1979

3 - Chemical Composition, Weathering, and Concentrations in * Intertidal Cores (Ward) : 1978-1980

4 - Chemical Concentrations and Composition of Oil in Oysters

and Flatfish from Abers (Neff) : 1978-1980

5 - Chemical Concentrations and Composition of Oil in Variety of

Fish and Oyster Tissues (Michel): 1978-1979

6 - Chemical Concentrations and Composition of Oil Associated

with Seaweeds (Topinka) : 1978-1980 /

37

2.1 Sediments and Sediment Cores (Extraction and Processing)

Samples of surface sediment or specific depth interval sections of sediment cores were solvent-extracted and fractionated according to an ambient temperature solvent drying and solvent extraction procedure based on that of Brown et al. (1980) as revised by Atlas et al. (1981) and Boehm et al. (1981). The procedure, involving methanol drying and ambient temperature extraction with a methylene chloride/raethanol azeotrope, is illustrated in Figure 2.1. The concentrated extract is displaced with hexane and charged to a glass absorption chromatography column (1 cm i.d.) containing 10 g fully activated (150 °C) 80-100 mesh silica gel topped with 1 g 5% deactivated alumina and 1 g activated (i.e. acid washed) copper powder. The column, which is wet packed in methylene chloride, is rinsed with this solvent followed by hexane. A 0.5 ml volume of extract is charged to the column and eluted with hexane (17 ml, f|) , hexane: methylene chloride (21 ml, f 2) » and methanol (20 ml, f 3) . The fractions are collected separately, reduced in volume, desulfurized using an activated (1 N HC1) copper powder slurry, and an aliquot weighed on a Cahn electrobalance. The f]_ and f2 fractions are then analyzed by fused silica capillary gas chroma- tography (FSCGC flame ionization detector) and a selected set further scrutinized by gas chromatographic mass spectrometry. FSCGC analysis determined the overall composition of the sample by appraisal of the distribution of resolved (peaks) and unresolved (hump) features, as well as the specific quantities of individual n-alkane (C^q to Ç32) and isoprenoid (C^5 to C20) compounds. GC/MS/computer analyses focused on the list of saturated and aromatic compounds presented in Table 2 to confirm the identities of compounds or to quantify minor, but important "marker" compounds.

Details of the GC and. GC/MS analytical procedures are presented in Table 3 .

Quantification of GC traces was according to the internal standard method wherein quantities of individual hydrocarbons are computed. Several other GC-derived parameters were routinely calculated on sample data. One of these was the n-alkane to isoprenoid ratio (ALK/ISO) in the C]^3 - C^9 range:

ALK/ISO = n - d A ■*■ n-d g + n-Ci ^nC17> n-d 8 ' 1380 + 1450 + 1650 + 1710 + 1812a

aGC retention indices of isoprenoids: 1450 = farnesane, 1710 - pristane, 1812 = phytane.

This ratio, beginning at -v7 in the reference oil is quickly decreased due to preferential bacterial degradation of n-alkanes versus the branched isoprenoids.

The carbon preference index (CPI) , the ratio of odd chain alkanes to even chain alkanes in the n-C26 to n-C3i range, is defined as follows :

„„„ 2(n-C?7 ± n-C?Q)

CPI = n-C26 4- 2n-C28 + ""^30

38

Sedimant

3ucard

Sadiment Samoa

Vlatnanci <Vasn

Cnad Sediment

! Tl SOq n Teflon jar or centrifuge :uoe t2) interna» standards •;31 CH^OH^CH^Cj 11:9» i4> rtuan v»/N^

(S) Shane at imoient :emoerature -0 ^our» witn solvent «nance irtar 1 S ind 24 .tour*

CH2C2 *nd

CH3OH/CH7C2

Extract

(1) NaCJ saturated)

(2) Acioiry *<Hçi

3) Extract 3x v/CH^Cj

Metnviene

CMonoe CH2C2'

Aqueous Mernanai

111 Camome

:2) Orv over NasSO* 3) Concentrate ro 1G0

rteign

Giscard

Cancan tratad extract

Hexane (M

1 Weigh Aliouat)

! 1 ) Oismace *itn Hexana

Alumina/ Silica Ga< Cdiumn Ciromatogrscnv

Hexane/ Metnviene Chtortaa 'fji

Wetgn -Aliouot)

Matnanoi ^3) Wetgn Aliquot)

Saturated

Hvcrocarocns

Aromarc Hycrocarooni

?oiar NSC Comooundj

1

GC2 GC2 MS

GC2 GC2 MS

1

Store

FIGURE 2.1. Analytical scheme for sediment samples.

39

TABLE 2. Focus of GC/MS analyses

Saturated hydrocarbons

Pentacyclic triterpanes (hopanes)

Aromatic hydrocarbons

Alkylated benzenes (C4, C5, Cg)

Naphthalene and alkylated naphthalenes (C^, C2, C3, C4)

Fluorene and alkylated fluoeenes (Cj_, C2, C3)

Phenanthrenes and alkylated phenanthrenes (C^, C2, C3, C4)

Fluoranthene

Pyre ne

Benzanthracene

Chrysene

Benzo fluor anthenes

Benzo(a)pyrene

Benzo (e)pyrene

Perylene

Arpmatic heterocyclics

Dibenzothiophene and alkyl dibenzothiophenes ("C^, C2, C3)

TABLE 3. GC and GC/MS conditions.

GC

GC/MS/COMPUTER

A.

Instrument

HP 5840A

HP 5985

B.

Column

lo Liquid phase

SE-30 (saturates) SE-52 (aroraatics)

SE-52 "

2. Type

Fused silica

(J&tf Scientific)

Fused silica

(J&W Scientific)

3. Diameter

0.25 id

0.25 id

4. Length

30 m

30 m

5. Carrier

Helium 9 1 ml/mi n

Helium @ 1 ml/rain

C.

Temperatures

1. Oven

40-290 § 3*/min

40-290 9 3Vmin

2. Injector

250* C

250* C

3. Detector

300* C (FID)

300 (ion source)

0.

Ionization voltage

-

70 ev

E.

Electron multiplier voltage

2200 volts

Scan conditions

40-500 amu § 225 amu/sec (1 scan/ 2 seconds minimum of 5 spectra per peak)

40

The CPI ranges from values of 1, where oil is present, to values greater than 1 if odd chain biogenic terrigenous n-alkanes dominate the higher boiling n-alkanes.

Quantification of aromatic hydrocarbons was accomplished using the technique of mass fragmentography wherein the computer stored raw GC/MS data is searched for parent ions (ra+) and the total ion currents for these ions is integrated and tabulated. Retention times of the parent ion mass fragmen tog rams obtained were compared with authentic standards. The total ion current for each parent ion is compared with that for the internal standard (deuterated anthracene) and instrumental response factors applied. Where authentic polynuclear aromatic hydrocarbon (PAH) standards were not available for relative response factor determination, a response factor was assigned by extrapolation.

All of the above techniques were applied successfully to the analyses of replicates of a NOAA intercalibration sediment sample, Duwaraish I,, prior to commencement of the program and to Duwamish II during the program. Additionally, the EPA "megamussel" intercalibra- tion sample was successfully analyzed for PAH levels.

2.2 Plant and Animal Tissues

All specimens of wet tissue, freeze dried tissue, and plant material were thawed and homogenized, or in the case of the seaweed tissue were cut into small pieces, prior to placement in a digestion flask. The samples were added to 250 ml Teflon screw top jars. The digestion, extraction, and fractionation schemes were similar to those developed by Warner (.1976) except that the digestion was performed using a 0.5 N KOH/distilled water/distilled methanol system heated in a boiling water bath for 4 hours to achieve complete digestion and hence release of hydrocarbons from the cellular matrix (Boehm et al., 1982). Internal standards were added prior to digestion and carried through the entire procedure (Fig. 2.2) (f^ androstane; f2 deuterated anthracene or phenanthrene) .

« *

The digestate was extracted three times with distilled hexane in the jar, the mixture being centrifuged between extractions. The extracts were combined, concentrated to 0.5 ml, weighed on a Cahn electrobalance, and fractionated on an alumina over silica gel column (see previous section) . Two fractions corresponding to the saturated or f]_ (hexane eluate) and the aromatic/olef inic or f2 (hexane :methyl- ene chloride eluate) hydrocarbons were obtained for gas chromatographic and combined gas chromatographic/mass spec trorae trie analyses (GC/MS) .

41

Hssue Samoie â 50 grams Wat)

(1) Dissect slesn

(2) Homogamza

(3) Aod to Teflon Jar

(4 1 Add Internal Hydrocarbon Stanaaras

(5) 4M kQh (aa)

(6) Flush Jar witn N-? v7) Saai

(8) Qigast (Saoonify» Overmgnt at Room

i emoerature

Digestate

1 1 ) Transfer to Seoaratorv Punne? !2> Add Saturated NaC

!3) Extract 3 Times with Hexane

Combined Haxana extracts

(15 Concentrate

<2) Dry Ova* Na2S04

yw

Concentrated Extract

(1) Alumina C

(2) Metnviene (3! Qisotace w

Alumina/ Sliea G«i Column Ch rematograc

leanuo

Chiortde slution itn Hexane

ny

Haxana (f i ) (Weigh)

*

Hexana/MeC>2(f) (Weigh)

(1) Resapenify or

(2) Gat Permeation HPLC Geanuo

Metnanoi Ifg) (Wetgn)

Saturated

Hydrocartaon»

(1) Aromatic Mydrocaroons

(2) PC3

Poiar NSO Comoounas

i

GC2

I

GC2 GC2/MS

I

Store

Fiaure 2.2. Analytical scheme for tissue samples (after Warner, 1976; Boehm et al., 1982).

42

3. RESULTS AND DISCUSSION

3.1 Overall Findings

Several general trends in the data presented in the following sections should be noted here along with several considerations of the use of marker compounds as "fingerprints" to trace aged AMOCO CADIZ oil in environmental samples.

3.1.1 Weathering of AMOCO CADIZ Oil

The chemical composition of spilled oil from the tanker changed markedly over the first days to weeks, both at sea and once associated with sediment (Atlas et al., 1981; Calder and Boehm, 1981; Boehm et al., 1981). The changes are well documented in Figures 3.1 and 3.2 and are summarized in Table 3A. For comparison, the background saturated and aromatic hydrocarbon composition of sediment samples is illustrated. The non-impacted sediments contain: 1) an unresolved complex mixture (UCM) of hydrocarbon material in both fractions, 2) terrigenous n- alkanes (odd chain) in the saturated fraction, and 3) pyrogenic PAH compounds in the aromatic fraction. Weathered AMOCO CADIZ oil is identified as such in the sections that follow based on the following:

1) The presence of large UCM in f^ and fractions with residual tri terpenoid peaks.

2) The presence of isolated isoprenoid hydrocarbon compounds in the resolved (peak) part of the GC trace (in samples during the first year post-spill only) .

3) The dominance of alkylated phenanthrene (C2* C3, C4) , di- benzothiophene, naphthalene, and fluorene (in earlier samples) compounds in the aromatic fraction and a dominance of these aromatics versus pyrogenic PAH (i.e. fluoranthene, pyrene, benzanthracene, chrysene, benzopyrenes, etc.).

3.1.2 Persistence of Marker Compounds

The most persistent compounds in the saturated (£±) fraction are the pentacyclic triterpanes (PCT) ; in the aromatic (f2) fraction the alkylated phenanthrenes (P) and dibenzothiophenes (DBT) are most persistent. To examine the PCT compound distribution, GC/MS analysis of the f^ fraction was necessary (e.g. Figs. 3.3 and 3.4). This results in a "terpanogram,, yielding information on the relative concen- tration of eight PCT compounds used by several investigators as indica- tors of presence and origin of petroleum (e.g. Dastillung and Albrecht 1976; Pym et al., 1975) .

Two PCT time series (Fig. 3.5) reveal that the PCT fingerprint is rather constant throughout the December 1978 to March 1980 time period

43

a *6s£a*NCE MC.S3S S«:ufiiM «■..:: oc*oo**i

l 3

z i ! ! :

■iZ

PW

Jl

III : *

3 STACC 1 .lC*r-*€WiNG S*turat« «■•«roejrooMi

7**s"««*:

wui^

C STACI. 2 «(ATMCAlMi iSimim »»«œjrwi<

« *<

J*1

J

e*

.V

.*■

uCJ

Hjii

9 STAGi .1 *e*n«e»iN<j iSwvkh mvgurioni

»0 ■*».«»«-» <M«»CI

I STACK «*A1>e»lN« tevuMHiinonani

^Uw>1^

UCM

* SACXG'QCNO iStcu/»(M H*ofac4roami

.-*.! •^-L-

L3;

ill

lit

^J

FIGURE 3.1.

Weathering patterns of saturated hydrocarbons in AMOCO CADIZ oil.

44

A REFERENCE MOUSSE lAromjtieil

z z

'JIM*.*'!***'*'*""' ,

S STAGE I WEATHERING lAromwiei» *„■

» OiT

IK

t .^

C STAGE 2 WEATHERING ,Afom«iejl

>

y

iV

UCM *

0 STAGE 2 WEATHERING lAramaticst

y

flfbw***,

UCM

»

J*'

$

S PVRQLYTIC PAH SOURCE lAramirci

_) . iL)**

A>

0

RMJL

Wi

3A*atnxantnrar*nt

CHV-Chryttn»

3F •9t«ito'iuo'»nt'"Mi« 9EP 3AP"atn:oov'f«i 38»"'a»"ioo«rvi«n» 3«3«njo«uOf»^«

FIGURE 3.2. Weathering patterns of aromatic hydrocarbons in AMOCO CADIZ oil.

45

TABLE 3A. Weathering of AMOCO CADIZ oil.

RAPID

1. Loss of volatile (<n-C^5) hydrocarbons due to evapora- tion of:

a. alkanes

b. aroraatics - benzenes, naphthalenes, biphenyl (one- to two-ring aroraatics)

2. Relative and/or absolute increase in unresolved complex mixture.

MODERATE

1. Microbial degradation of n-alkanes; preferential attack of n-alkanes versus branched alkanes (i.e., decrease in ratio of alkanes to isoprenoids) .

2. Loss (to solution or other processes) of most resolved saturated hydrocarbon GC traces.

3. Emergence of triterpanes as major molecular markers in saturated fraction.

4. Increase in UCM, with formation of secondary (bimodal) UCM distribution.

5. Loss of fluorenes (two aromatic rings, one saturated ring) and alkyl naphthalenes.

6. Increase in abundance of polar fraction. LONG

1. Persistence of alkylated phenanthrenes and alkylated dibenzothiophenes.

2. Increase in polar fraction.

3. Loss of long chain n-alkanes and isoprenoids.

46

FIGURE 3.3.

•t'IMNCS V.OLSSZ

t»l.rf_JJ

•A*

,,*.. ( OpU^^U^

U

WJCJ

VM;\i

JJ

iTAj

^Ll^Ll 1 1 I i i i ^

X ■•« 1< « 4j «« jj •! -^ «, «J

un

.V— .L.

JlJnf

Uu

U/*

i^UJ~l

n

i m 1 1 1 1 1 1 1 , 1 1 1 , i .

GC/MS selected ion searches for pentacyclic triterpanes (ho- panes) in AMOCO CADIZ reference and November 1978 weathered oil in sediments.

191 .0

Tl

UtAl ION

"~ T 1 1 1 1 I 1 1 1 1 1 1 1 I 1 I 1 1

fi4 fig fifi fi7 SO fiq 70 71 73 71 74 75 TR 7-7_lJ5 -_ZS _Bki_ .OU

FIGURE 3.4

Triterpane (m/e 191) mass chromatogram of weathered oil. 1,2 = Trisnorhopane (C2 7Hi»6), 3 = Norhopane (C29H50), 4 = Hopane (C30H52K 5,6 = Homohopanes (C31H51+), 7,8 = Bishomo- hopanes (C2 3HS6 ) .

47

n*»l»ii»iicr Moim*

IJccrnil«*i 19 7R

.ii<iv 19/9

M.uili I ••ill)

1 ? 3 1 5 6 7 8

1 ? 3 4 5 G 7 8 I ? 3 4 S G / (1

STATION 3- ILE GRAND MAnSH

i > i 4 5 r. 7 n

n*feiMv» Momse

Drcnnlwr 1978

July 1979

M.i.ch 1900

17 3 4 5 6 7 8 17345078 17 3 40678 I ? 3 4 '5 fi 7 8

STATION G- AHCn WOACH

FIGURE 3.5. 191 terpanograms . (1,2 = Trisnorhopanes, 3 = Norhopane, 4 Hopane, 5,6 = Homohopanes, 7,8 = B ishorno hopane s )

at the two stations. Note that the PCT fingerprints of AMOCO CADIZ and TANIO oils are quite distinct (Fig. 3.6), notably in the ratios of compounds 1, 2 and 5, 6. Thus it appears that PCT fingerprints offer a good means to trace AMOCO oil in highly weathered samples when most other identifiable molecular characteristics have been lost.

I 2 3 4 5 6 ! 8

AMOCO CAOI*

I 2 3 < 5 fi ; n

TANK) ST A I ION II

FIGURE 3.6. 191 terpanograms of crude oils. 48

As the most persistent aromatic compounds, the P and DBT compounds (mainly C2r C3, and C4) mark AMOCO CADIZ oil in tissue (oysters) and sediments through mid-1980. The final (June 1981) sediment sampling failed to reveal significant P and DBT levels in any of the stations. The latest (1981) status of the oyster P and DBT levels is unknown. However, through most of the data to compounds dominate the distribution. C3P/C3DBT, used by Overton et al. (1981) spill remain in the 0.3-0.6 range. The in the text.

be discussed, the P and DBT

The ratios of C2P/C2DBT and

to differentiate oils, in this

use of this ratio is discussed

3.1.3 Residues in Tissues

As stated, the P and DBT compounds are most readily associated with oyster tissue samples in the two-year period following the spil- lage. The branched alkanes (isoprenoids) also persist throughout this period.

3.1.4 Environmental Variability

A major question in oil spill studies and for that matter environ- mental studies in general is the question of patchiness of pollutant distributions and the variability due to patchiness in chemical meas- urements. To shed some light on this subject two sets of measurements are available. Two principal investigators (Atlas and Ward) obtained samples at the same time and location in several instances, Atlas sampling the top 3-5 cm, Ward sampling an entire sediment core but subdividing the top 0-5 cm section. The total hydrocarbon values (Table 4) reveal wide disparities where contamination is very heavy (pooling of oil in the lie Grande) but reasonable to excellent agree- ment in most cases. (Note also that additional replicate analyses are available for sediment samples in Section 3.2 as well).

TABLE 4. Analysis of sampling variability.

DATE

TOTAL HYDROCARBONS

(fi + f2) (pg/g)

STATION

ATLAS (SURFACE)

WARD (0-5 cm)

lie Grande

12-78

650/1300a

1,100

3-79

4,700

700

L'Aber Wrac'h

12-78

400/400a

770

3-79

870

1,100

7/8-79

390

290

11-79

1,100

1,100

aReplicate samples.

49

3.2 Surface Sediments (Atlas, University of Louisville)

The frequency of sampling for surface sediment (0-3 cm) is shown in Table 5. Ten primary locations were sampled repeatedly (Fig. 3.7). Results of total hydrocarbon determinations for the ten stations, as these concentrations varied with time, are presented in Figures 3.8 through 3.12. Also included in these figures are source evaluations for each sample hydrocarbon assemblage, based on GC information. The biogenic (B) category indicates that terrigenous odd chain n-alkanes dominate the f^ GC trace. The pyrogenic (P) category signifies an important abundance of combustion-related polynuclear aromatic hydro- carbons (PAH) in the fraction as well as the presence of some unresolved material (UCM) in both the f^ and f2« In those samples labeled B or B/P the primary sources of hydrocarbons are as indicated although a small fraction of the hydrocarbons may consist of petroleum. Figure 3.13 summarizes these source criteria. Only GC/MS analysis of each sample would definitely eliminate the small chance of a false negative (i.e. not finding AMOCO oil where there were traces) .

The error bars in the figures indicate that two determinations were made for the December 1978 samples (Table 6) . All other determin- ations were based on one replicate. Note that the coefficient of variation ranges from .01 (1%) to .94 (94%). The higher variability is observed in samples with the lowest and highest (^1000 ppm) absolute concentration levels, the former due to natural patchiness, the latter owing to "pooling" of oil in heavily impacted stations.

GC/MS results are available for stations 3, 5 and 7 throughout the study period and are presented graphically in Figures 3.14 through 3.33. These semi-log plots illustrate quantitatively the aromatic composition of all samples normalized to C3 dibenzothiophene or where C3DBT is absent to pyrene. C3DBT was used to normalize the data as it is assumed that these compounds are the slowest to weather of all of the aromatic hydrocarbons.

All AMOCO CADIZ -impacted stations illustrate a normal weathering sequence (i.e. see Fig. 3.1). However, fresh inputs of petroleum were observed to impact the region of stations 7 and 8 in the form of tar chips during November 1979 and stations 2, 11 and 12 in the form of oil from the TANIO spill in August of 1980 (Fig. 3.34).

Although a wide range of residual oil concentrations appear in the various samples, several trends in the data seem apparent. Stations 1, 9, and 10 remain unimpacted by the spill throughout the study. Station 2 remains unimpacted until a secondary petroleum input influences its hydrocarbon chemistry in November of 1979 (the timing of the secondary tar impact at stations 7 and 8 also is probably related to leakage from the sunken tanker) and again in August of 1980, the latter relating to the TANIO spill, also readily detected at Stations 11 and 12 at this time. Through March of 1980 weathered AMOCO CADIZ oil is readily detected at Stations 3, 4, 5, 6, 7 and 8. However, the results of the August 1980 samplings indicate that inputs of non-AMOCO CADIZ hydro- carbons (i.e. background) at Stations 6 and 8 become dominant. At stations 3 and 7 where GC/MS data exists, the main AMOCO CADIZ aromatic

50

IE UIIANIIl ew\

Ht Me. Ml I fNMII-Vt 141

FIGURE 3.7. Surface sediment "sampling locations (Atlas).

TABLE 5. AMOCO CADIZ chemistry program; surface sediments (Atlas)

Frequency

April 1978-October 1978 (Calder)

December 19 78

March 1979

July 1979

November 1979

March 1980

May 1981

Total

20 10 12 15 11 12

80

Locations

Ten Primary Stations

1,2,3

lie Grande

4

St. Michel -en-Gr eve

5,6

L'Aber Wrac'h

7,8

Portsall

9,10

Trez-Hir

11-14 Other Impact Stations

GC/MS

Stations 3 (lie Grande), 5 (L'Aber Wrac'h), 7 (Portsall)

51

100 -

ao - _

I

%

A

CorWOll

9 BIOGENIC

«Y010GÏNIC .CM«0NIC1

AC AMOCO OIL

1 I L_L

iLt GHANOI 'Control!

■«•Oil "TANlO""

J L

I I I I I I I I I I I I I

I 000 -

200 -

STATION 3

"91K *M3K

STATION 4

•I" «J3K

IK

ilESKANO Cik.E0>

a biogenic

» >v*0CINIC iCMNONIC) AC AMOCO OIL

I I I I I I I I I I

ST «ICMiLENGKCVt

'2 78 3.7» 7 117* ISO Si» «.il

I J. 17»

h •» xao sao 4 si

FIGURE 3.8. (Left) lie Grande' (control) sediment time series. FIGURE 3.9. (Right) lie Grande (oiled) and St. Michel- en- Grève time series (note scale difference between the two plots) .

<

5

| l 300

3

- *3E» vJ->C M ^ALi6»-A AOkcS-'00»ACtS ATLAS-9

a siogcnic

•-««OGENIC C»«ONiCi AC AMOCO 311

STATION

?

_L

L AMW AWAÇH

c*tJE«-a

*ot«c-;oo'*cïs

aTwAS-a

«OS- «Jt» ,V»AÇ>

i 7i : -a : -9 -i '-9 j ao i jc * s:

STATION 7

AC

■QftTSftU.

700

s aiocf Nic

'M

- AC >,

Vac

» = ""«OGINIC OH0NIC1 AC AMOCO OIL

AC

V AC

«

\*c

<0

1 1 1

1 1

1 1

1

*■ - ac a

i ' i i i i

STATION S

•0«TSALL

500

wo

'AC

100

AC

AC

:oo

~

V AC

■00

! 1 1

1

1 1

i i

-J L

3 »

1 1 i l 1 1

11/71 3.-7» 171 11.71 .1 ao oil

FIGURE 3.10. Aber Wrac'h sediment time series (left). FIGURE 3.11. Portsall sediment time series (right).

52

FIGURE 3.12.

3 ï

station»

1 9I0CINIC

» »v»OG£„.C ICHKONlCl

*c> amocooii.

J I I L_i

STATION 10

_L

J I I L

I ' '

'2/7* J/7» i.7» UT* ISO 1*0 «.Il

Trez Hir sediment time series.

AC

J L

a

•**, W**iO<«<l ttl— ■«— ■•» !'««««•

1

J «

1

i

1

1

1

. 1 '

Jill

ill

L

J!

J,

.'.•i.

' ' - - - .

-•

FIGURE 3.13. Hydrocarbon compositions forming the basis of source

classification categories.

53

TABLE 6. Replication of hydrocarbon concentration data (based on December 1978 analyses of two replicates) .

STATION

1 2 3 4

5 6 7 8 9 10

56

113

1,000

135

401

217

159

358

18

11

cr/x

0.57 0.08 0.52 0.60 0.01 0.06 0.10 0.21 0.71 0.94

i.o -

2.130 nq g

tTit>MtfM»Tt

IJKUMMOPQBST

II II IL

s c

i i i e f g m

_l I

z **

FIGURE 3.14.

Aromatic hydrocarbons, station 3, December 1978; normalized to C3DBT.

(A = napthelenes, B = CiN, C = C;N, D = C3N, E = C£N, F = biphenyl, G = fluorenes, H = CiF, I = C2F, J = C3F, K = phenanthrenes, L = CiPh, M = C?Ph, N = C3Ph, O = Ci+Ph, P = dibenzothiophenes, Q = ClDBT, R = C2DBT, S = C3DBT, T = fluorene, U = pyrene, V = benzo (a) anthracene , W = chrysene , X = benzof luoranthene , Y = benzo (a)pyrene, Z = benzo (e)- pyrene , AA = perylene)

54

10-

11.00Ong.-g

tiiititTiiiifff»tf!tff ,---,-

ABCOEFGHIJKLMNOPORSTUVWXTZM

J L

JL

JL

1.9-

410 ng. g

I I I I I f

^ i 1 1 1 1 1 1 1 i i 1 i i 1 i i 1 i i 1 r ABCDEFGH I J K LMNOPORSTUVWX T Z M

J L

JL

JL

JL

t.o-

iiiiTiiiypii»r?if

ABCOeFaHIJKUMNOPQRSrUVWXYZA*

I I I II II II I

FIGURE 3.15. (Top) Aromatic hydrocarbons, station 3, March 1979; normal- ized to C3DBT.

FIGURE 3.16. (Middle) Aromatic hydrocarbons, station 3, July 1979; nor- malized to C3DBT.

FIGURE 3.17. (Bottom) Aromatic hydrocarbons, station 3, November 1979;

normalized to C3DBT.

(See Figure 3.14 for key.)

55

1.0-

S.100nç,e

V.0-

A8C0EFGH I J K UMNO PQBSTUVWï f ZM

77ng^

J L

JL

1.0-

5346.4 nfl/9

»»i»tiiiflf»>ffitrîiiii r— t i—r

AaCOEFGHIJKLMNOPORSTUVWXVZAA I 1 I Il Il Il |

FIGURE 3.18. (Top) Aromatic hydrocarbons, station 3, March 1980; normal- ized to C3DBT.

FIGURE 3.19. (Middle) Aromatic hydrocarbons, station 3, June 1981; nor- malized to C3DBT.

FIGURE 3.20. (Bottom) Aromatic hydrocarbons, station 5, April 1978; nor- malized to C3DBT.

(See Figure 3.14 for key.)

56

1.0-

4.160 ng/g

ffff\\t\fffff

I— f"

l I r— i I i I r

ABCOEFGHIJKLMNOPORSTUVWXYZAA I I I II II II I

1.0-

440 ng/g

-I IfTTitttfttfllTTfTl 1— i i i 1 i— r

ABCOEFGHIJKLMNOPQRSTUVWXYZM I I I II II II I

1.0-

880ng/g

fftffi f y r M t

i f r r i i i i i i

ABCDèFàHijKLMNOPQRSTUVWXrZ I I I II II II 1

FIGURE 3.21. (Top) Aromatic hydrocarbons, station 5, October 1978; nor- malized to C3DBT.

FIGURE 3.22. (Middle) Aromatic hydrocarbons, station 5, December 1978;

normalized to C3DBT.

FIGURE 3.23. (Bottom) Aromatic hydrocarbons, station 5, March 1979; nor- malized to C3DBT.

(See Figure 3.14 for key.)

57

1.0-

f f I It?

t t I I I

94n«/g

"+■

i— r

A S C 0 E f G H I J K UMMOPQHSTUvWiyjA* 1 I II II || I

1.0-

460 fig.g

i ' i 1 I I I 1 I I T f f t t I f i r~ -

*BC0EFGMIJKLMM0PQ«STUVWXV2AA

J L

JL

JL

JL

1.0-

i i i i i i i i i i l f y y i i i i i < I J i t f i \

ABCOEFGHIJKLMNOPORSTUVWXTZAA

J L

JL

JL

FIGURE 3.24. (Top) Aromatic hydrocarbons, station 5, July 1979; normal- ized to C3DBT.

FIGURE 3.25. (Middle) Aromatic hydrocarbons, station 5, November 1979;

normalized to C3DBT.

FIGURE 3.26. (Bottom) Aromatic hydrocarbons, station 5, March 1980; nor- malized to C3DBT.

(See Figure 3.14 for key.)

58

1.0-

140r

A B C 0 E F G M I J K LMNOPQRSTUVWX Y Z AA

J L

JL

JL

JL

1.0-

* H ' i i * *

C3O8T' 280na/9

» ! 'I I » \ 1 1 1 1 1

À B C 0 È F G M ij K LMNOPOP.STUV*XYZ I I I II H II 1

10-

C3OBT < 130 119/g

f 1 \ tt

« I *

» T t 1 r— t

iiifiTiitTir

ABCDEFGH IJ K LMNOPOP.STUVWXYZ

L

J L

JL

JL

JL

FIGURE 3.27. (Top) Aromatic hydrocarbons, station 5, June 1981; normal- ized to pyrene .

FIGURE 3.28. (Middle) Aromatic hydrocarbons, station 7 , December 1978;

normalized to C3DBT.

FIGURE 3.29. (Bottom) Aromatic hydrocarbons, station 7, March 1979; nor- malized to C3DBT.

(See Figure 3.14 for key.)

59

1.0-

C308T=177n9,g

ABCOEFGM IJKLMNOPQRSTUVWXTZM

JL

JL

JL

.vo-

C308T>75ng/g

I I I I I I I I I I I > f t I I y ABCOEFGH IJKLMMOPORSTUVWXYZM

I S L II II II 1

C3O8T! 11ng g

, t > i i i i i i i r r i f 1 1 1 r r r . f f f 1

ABCOEFGH IJKUMNOPOnSTUVWXYZM I I I II II II 1

FIGURE 3c 30. (Top) Aromatic hydrocarbons, station 7, July 1979; normal- ized to C3DBT.

FIGURE 3.31. (Middle) Aromatic hydrocarbons/ station 7, November 1979;

normalized to C3DBT.

FIGURE 3.32. (Bottom) Aromatic hydrocarbons, station 7, March 1980; nor- malized to C3DBT.

(See Figure 3.14 .for key.)

60

220n«,9

1.0-

i i i i i i i i i i i i i i i i i l i r f f i l i I i

ABCOEFGHIJKUMNOPQRSTUVWXVZM

L

J L

JL

JL

JL

FIGURE 3.33. Aromatic hydrocarbons, station 7, June 1981; normalized to

pyrene. (See Figure 3.14 for key.)

Jîi'îUw

A\

%

\

p

•.v.

A- SATURATES

•ti;

.i i.

....J-.aIIjw"*-"1-'"'

JiUi

IP*

JiLJ^"

■".■JD*

/M4Ï-.':

;i»«w

S:'

*\

V,

a AHOMATICS

FIGURE 3.34. TANIO oil.

61

marker compounds, the C3 dibenzothiophenes, and C3 and C4 phenanthrenes, persist but the pyrogenic PAH compounds have replaced any AMOCO CADIZ oil traces at Station 5 in L'Aber Wrac'h.

The last sampling, June 1981, reveals total disappearance of traces of AMOCO CADIZ aromatic marker compounds at stations 3, 5, and

7. By June 1981 the only unequivocal presence of AMOCO oil is seen at station 3 in He Grande, although it has been extremely weathered. Only pentacyclic triterpanes can be linked to the residual AMOCO oil. GC patterns suggest that petroleum still affects stations 4, 6, 7, and

8, but in only minor quantities relative to other inputs.

Thus, for the most part, less than three and one half years has been required to allow normal background inputs to resume their sedi- mentary dominance at all but the most heavily impacted (in terms of post cleanup oil concentrations) and lowest energy (i.e. most protected from waves) environments (i.e.. station 3 in the He Grande).

Further interpretive details are presented in Atlas et al. (1981).

3.3 Offshore Sediments (Marchand, CNEXO)

L'Aber Benoit Sediments (Courtot, U. West Brittany)

In this phase of the analytical chemical program the levels, the persistence, and the precise chemical nature of petroleum hydrocarbons in the offshore sediments of the Bays of Morlaix and Lannion were examined as well as those of L'Aber Benoit sediments (November 1978 only) . A summary of the samples analyzed appears in Table 7 and in Figure 3.35.

TABLE 7. AMOCO CADIZ chemistry program; 2. Offshore surface sediments (Marchand) and Aber Benoit sediments (Courtot) .

Frequency:

April 1978 6

July 1978 14

November 1978 13+7

February 1979 13

TOTAL 53

Locations :

Aber Benoit (November 1978) Baie de Morlaix Baie de Lannion

GC/MS:

Four Time Series (18 Samples)

62

FIGURE 3.35

Offshore surface sediment and l'Aber Benoit sampling locations (Marchand, Courtot) .

Hydrocarbon concentrations and source classifications for the entire data set are shown in Table 8. Individual aromatic hydrocarbon determinations by GC/MS appearv for several time series in Tables 9 through 13 and for two of the L'Aber Benoit samples in Table 14.

An instructive way of viewing the time series information is presented in Figures 3.36 and 3.37. At both the Terenez/ Morlaix and lie Grande time series, concentrations increased between April and July 1978. In the case of the Terenez samples, the increase is due to offshore transport of weathered oil as evidenced by 1) an increase in absolute concentrations, 2) a decrease in the ALK/ISO ratio, and 3) an increase in phenanthrenes (total P, C^, C^* C3, C4P) and dibenzothio- phenes, without an accompanying increase in the pyrogenic PAH compounds (m/e 202). However, the lie Grande benthic samples show an increase in total hydrocarbons along with increases in the aromatics including the pyrogenic PAH. This latter finding indicates that both petroleum hydrocarbons and combustion-related PAH material are being transported to and deposited in the offshore sediments near lie Grande by a similar mechanism, most likely in association with suspended matter from riverine plumes. Figure 3.38, a plot of phenanthrene and its alkyl homologues at the Morlaix Station (Station B) , reveals that while the source of the phenanthrenes is petroleum in July 1978, as evidenced by the greater abundance of alkylated compounds versus the parent (unsub- stituted) compounds, the input in February of 1979 is largely pyrogenic (i.e. greater amounts of parent phenanthrene). This illustrates both the usefulness of detailed GC/MS-derived data and their subsequent presentation in alkyl homologue distribution plots.

63

TABLE 8. AMOCO CADIZ sediment sample, source

classification (GC) (offshore sediments)

SAMPLE NO.

( mj 4 )

(U'j.'-J)

AC100 AC369 AC426 AC10 3 AC 365 AC429 AC42

Acna

AC371

AC453

AC 56

AC139

AC381

AC4S8A

AC458B

AC127

AC370

AC452

AC132

AC362

AC141

AC196

AC432

AC134

AC4S1

AC44

ACUl

AC384

ACU2

AC389

AC445 AC107

AC376

AC436

AC53

ACU8

AC377 '

AC438

AC51

ACIL4

AC379

AC440

AC48

AC125

AC378

AC479

4/58

34. L

1/4

90.4

3/33

252.9

3

3.172.5

4/5B

35.5

3

133.6

4/2/SB

200.6

4/2

397.0

4/ SB

107.9

4/2

503.4

SB/ 4

37.3

5/3

31.6

I

109.0

2/1

123.7

2/4

408.8

2/4

502.8

4/a

19.4

2/4

97.7

2/4

142.5

2/4

36.0

2

47.2

. 2

86.0

4

5.9

4/2

17.2

2

54.4

2

119.3

4

5.0

5

8.1

4

4.4

5

7.9

2

36.9

2/4

58.5

4/2

13. 6

3/4/2

48.8

2/4

13.8

4

41.5

2/4

16S.8

4/2

211.7

2/4

24.5

4/2

44.7

2

15.6

2

39.2

4/ SB

6.0

3/4

13.6

4

35.4

4/2

26.9

5B/4

78.3

2/4

171.9

4/2

42.2

4/2

50.7

1

38. S

2

69.1

2

17.5

2

44.8

2

32.1

2/1

173.6

2/4

122. S

2

239.3

2/4

56.4

2/4

151.1

2/4

29.0

2/4

64.8

2

53.2

2/4

176.7

2/4

5.9

2/4

8.9

2

27.7

2

60.6

1

28.9

2

31.5

2/4

82.2

2/4

97.2

4

31.9

4

132.1

2/4

IS. 8

4

32.1

1/2

96.4

1/2

102.4

2/4

56.4

2/4

249.6

2/4

27.2

2/4

80.7

2

103.5

2/4

104.5

2/1

21.0

4

14.3

2

59.1

2

27.8

2

63.3

2

181.0

2

36.5

2

37.9

AC40 and 50 series sampled 4/78 AC100 secies sampled 7/78 AC300 series sampled 11/78 AC400 secies sampled 2/79

L'Aber Benoit

Sediments:

ABT

2

158.2 2

180.8

ACC

4

25.1 4

29.3

AB25

2

29.2 2

29.7

AS 29

4

13.6 4

L7.5

AB16

2

22.2 2

28.6

AB21

2

455.1 2

440.7

AB4

2

70.6 2

76.9

64

TABLE 9. Terenez/Morlaix time series (station A)

SAMPLE

DATE

ALIPHATICS (uq/q)

MJt/ISO

ARGMATICS

<w/q>

TOTAL

RESOLVED

TOTAL

RESOLVED

AC 42

4/78

109.0

6.1

0.79

123.7

7.4

AC 138

7/78

408.8

11.7

0.10

502.8

17.2

AC 371

11/78

19.4

1.3

0.10

97.7

2.4

AC 453

2/7»

142.5

2.8

0.51

36.0

1.6

H

Cl"

CjM

CjM

C4N

T

«V

c2'

<Ô'

P

V

Ci'

<Ô'

c«'

AC

42

nd

10.3

40.9

LIS. S

161.0

16.8

20.1

54.3

198.9

125.

7

95.4

153.3

274.8

136.3

AC

138

2.9

6.3

62. S

423.2

730.7

nd

37.4

230.2

905.8

20.

4

151.4

593.5

1678.9

892.9

AC

171

nd

nd

nd

nd

nd

nd

nd

4.4

38.0

7.

4

9.0

12.4

41.9

27.6

AC

«S3

2.1

4.C

9.7

10.6

18.8

nd

1.0

7.8

14.9

5.

2

5.9

15.0

22.0

21.9

OBT

CtOBT C2OBT C3D8T

PL

PYR CHRY

BP 9<«>P 3(i(P

PERL

AC 42 AC 138 AC 371 AC 453

19.2 113.6

12.4 383.9

.ni 4.0

2.3 6.7

714.4

3363.0

54.9

63.8

1088 6388 179.0 93.5

195.0 3.0 9.1 4.8

171.4

47.1

7.6

4.2

265.7

77.4

16.4

9.6

298.9 83.5

120.8 87.2

20.9 9.9

9.1 6.0

64.8

45.4

3.7

3.4

24.5

20.3

1.8

1.5

KEY 1 nd - non* detected .

N napthalene. C^-C^N alkylated naphthalene*, P fluorene, C^P-CjP alkylated fluorene», P pnenanthren*. C1-C4P alkylated phenanthrenee. DBT Otbenzoth tophene, C^reT-CjOBT alkylated dlbenzothLophenes, PI fluoranthene, PYR pyrene, CHRY Ccyaene, BP - benzof luocanthene, 8(e)P Benzo(e) pyrene, B(a)P Bento<a)pyren«, PERL » perylene.

TABLE 10. Morlaix time series (station B) .

ALIPHATICS (pq/q)

AROMATICS (pq/q>

SAMPLE

DATE

TOTAL

RESOLVED

ALK/ISO

TOTAL

RESOLVED

AC 103

7/78

200.6

:

12.6

2.5

397.0

7.4

AC 365

11/78

107.9

5.3

5.0

503.0

7.5

AC 429

2/79

37.3

2.0

0.02

31.6

11.9

N

Cl"

C2"

C,N

V

P

ctP c2p

V

P

C1P

V

V

V

AC 103

17

18.

9

49.6

135.9

220.7

16.3

23.8 72,

,3

301.0

U7.8

95.4

160.1

312.5

273.9

AC 365

9

14.

4

33.9

55.0

111.2

9.9

14.9 36.

9

81.3

114.2

65.3

58.9

143.3

103.6

AC 429

6

10.

0

19.7

28.0

50.2

16.8

11.6 23.

3

7.4

195.6

109.6

52.6

26.0

34.9

AC 103 AC 365 AC 429

OBT

C.DBT

15.2 100.8

13.2 32.7

11.3 7.7

CjDBT

772.3

235.2

10.3

C DBT

1348 440.3 5.0

PL

PYR

240.2 202.2 325.9

203.8

175.6 302.9

CHRY

345.2 254.0 207.4

BP

B(e)P 8<a)P

350.7 324.6

202.5

146.4 166.3 115.0 130.9

112.5 115.0

PERL

87.0 65.4 37.7

KEY: nd » none detected.

H - napthalene, C1-C4N - alkylated naphthalenes.

F fluorene, C1F-C3F alkylated fluorenes, P » ph*nanthrene.

cl"c4p " alkylated phenanthrenes, DBT Dlbenzothlophene. ClDBT-C208T alkylated dibenzoch tophenea, Fl fluoranthene, PYR pyrene, CHRY Ccysene, BF » benzof luoranthene, 8(e)P » Oenzo(e) pycene, B(a)P Benzol a) pyrene. PERL perylene.

65

TABLE 11. St. Michel en Greve/Lannion time series (station C) .

UATB

ALIPHATICS (W/g)

ALK/ISO

AROMATICS

<w/g)

SAMPLE

TOTAL

RESOLVED

TOTAL RESOLVED

AC 44

4/78

38. S

1,

9

0.38

69,

.1

8.6

AC 121

7/78

17.5

0.

2

0.09

44

.3

0.6

AC 184

11/78

32.1

0,

5

0.14

:

173

.6

1.6

H

V

C2H

C,N

c4«

F

cir

V

C3F

P

CiP

C2P

CjP

V

AC 44

8.6

6.3

12.6

25.0

51.7

nd

3.3

12.4

89.5

10.9

29.8

48.3

115.0

79.4

AC 121

6.2

4.8

10.2

14.0

12.4

1.6

2.2

13.7

57.5

19.8

17.7

55.6

75.4

33.9

AC 384

2.4

2.1

6.3

59.6

121.1

nd

4.2

35.8

73.1

2.8

28.7

30.6

76.6

36.4

DBT

C DBT

C DBT

C DBT

n.

PYR

CURY

BF

B(«)P

B(a)P

PERL

AC 44

8.7

26.6

253.5

378.»

17.2

10.2

22.7

31.4

21.5

11.1

6.9

AC 121

3.0

21.2

211.1

348.7

22.1

17.1

34.3

35.1

IS. 3

10.8

3.2

AC 184

3.3--

86.2

337.8

322.5

3.0

3.5

7.5

6.7

3.8

1.5

0.5

KEY: nd non* detected.

H napthalene, C^C^N alkylated naphthalenes, P tluocene, C^P-CjP alkylated Cluorenes, P ° phenanthrene, C^-C4P alkylated phenanthcenea, DBT Dlbenzothiophene, C]_DBT-C2DBT - alkylated dtbenzothlophcnea, PI » fluoranthen* , PYR pycene, CIIRY Ccyaene, BP « benzoCluoranthene, B(e)P Benzole) pytene, B(a)P Benzol a) pycene, PERL pecylene.

TABLE 12. lie Grande time series (station D) .

SAMPLE

DATE

ALIPHATICS (l«?/g)

TOTAL

RESOLVED

ALK/ISO

APOMATICS (yg/g)

TOTAL RESOLVED

AC 48 AC 125 AC 378 AC 4J9

4/7S

7/78

11/78

2/79

21.0 59.1 63.3 36.5

0.3 0.9 1.9 0.8

3.4 0.6 0.4 5.4

14.3

27.8

181.0

37.0

0.6

0.3

12.9

0.1

Cl"

C2H

C3M

<V

V

V

V

V

V

V

V

AC 48 nd nd nd nd ml nd

AC 125 11. 8 5.1 143.7 686.2 825.1 12.8

AC 578 4.2 3.1 112.2 472.5 569.0 nd

AC 439 0.8 <1.0 <1.0 1.0 nd nd

nd 9.9 25.1 14.9

68.0 286.0 674.) * 63.3

20.2 152.4 386.7 1J.9

nd ml nd 3.7

16.5 20.2 35.2 21.3

309.9 539.7 729.0 229.0

127.8 251.7 473.0 227.9

5.9 5.1 4.5 2.4

DBT

C DBT

C DBT

C DBT

FL

PYR

CHRY

BP

B(e)P B(a)P

PERL

AC 48 nd 13.2 80.7

AC 125 1033 785.4 2420

AC J78 27.3 274.0 1481

AC 439 nd 2.5 12.4

145.6 19.8

2917 55.2

1945 3.8

15.5 <l.O

18.3 34.1

39.4 107.7 9. 4 na

<1.0 na

17.2

99.0

15.0

1.0

16.7 6.1

52.7 16.0

19.3 2.2

1.0 1.0

44.7

6.1

2.0

nd

KEY: nd none detected.

na « not analyzed. M napthalene, C^-C^M alkylated naphthalenes, F ' fluocene. O^F-CjF » alkylated Cluocenes, P phenanthcen», Cj-C^P « alkylated phenanthcenea, DBT Dlbenzothiophene, CiOBT-C^OBT " alkylated dihenzothiophenes, Fl » riuoranthene, PYR « pycene, CHRY - Ccysene, Br - benzoduocanthene, B(e)P » Benzole) pycene, BlalP - Benzo I a) pycene, PERL petylene.

66

TABLE 13. Lannion I and II time series.

LAHNIOM I TIME SERIES (STATION E)

SAMPLE DATE

N

CtH

C2H

CjM

C4N

r

V

V

C3r

P

ClP

V

V

°4P

AC 107 7/78 AC 43S 2/79

nd 1.6

8.7 1.9

32.8 2.9

87.4 1.9

167.8 nd

4. S

nd

10.1

nd

34.1

nd

95.3 nd

22.8 3.7

41.3 5.9

60.9 5.1

117.7 2.3

71.1 nd

DBT

C DBT

C DBT

C DBT

rt

PYR

CHRY

BP

B(e)P

8(a>P

PERI,

AC 107 AC 436

9.9

nd

75.4 l.S

295.6 22.4

416.4 40.2

23

1.6

19.6 1.9

na

na

42.6

12.3

20.6 3.7

19.4 2.5

9.5 2.0

LAHHIOM II TIME SERIE3 (STATION P)

SAMPLE

DATE

N

Cl"

C2H

C,N

C4N

P

v

V

V

P

V

V

V

V

AC 118 AC 377

7/78 11/78

5.8 '

nd 4.1

9.5

7.3

16.3 6.1

26.3 nd

1.0 1.5

2.2 1.3

$'.0 2.3

18.6 9.0

7.9 12.5

9.3 7.3

19.8 7.8

37.1 17.9

21.0 7.8

DBT

C.DBT

C DBT

C.DBT

PL

PYR

CHRY

BP

B(a|P

B(a)P

PERL

AC AC

118 377

nd

nd

10.2

nd

90.1

nd

181.2

nd

10.6

nd

8.5

nd

na

na

22.6 nd

12.8 nd

6.7 nd

2.2

nd

RETt nd non* detected.

na not analyze*). N napthalena, Cj-C4N alkylated napnthalanea, P fluorene, C^P-CjP - alkylated fluotenea, P phenanthcen» , C1-C4P alkylated phananthrerves, DBT Olbanrothlophene, C^DBT-CjOBT * alkylated dtbenzotnlophenes, PI fluoranthene, PYR pyrene, CHRY Cryaene, BP banni luocanthene, B(elP Benzo (e) pyrene, B(a)P Benzo (a) pyrene, PERL parylana.

67

TABLE 14. L'Aber Benoit GC/MS results.

AB 25

AB 21

N

nd

3

C]N

nd

11

C2N

4

104

C3N

30

220

C4N

55

307

F

-

4

C1F .

4

25

C2P

11

113

C3P

59

311

P

3

14

Cl?

30

50 '

C2P

54

440

C3P

84

800

C4P

54

450

DBT

5

29

C^BT

34

200

C2DBT

166

1350

C3DBT

195

2000

Fluor an thene

6

19

Pyrene

4

23

Benzanthracene

nd

69

Chrysene

15

53

Benzo fluor an thene

11

43

Benzo(e) pyrene

7

24

Benzo (a) pyrene

4

12

Perylene

2

10

F1 (Total)

29

460

F2 (Total)

30

440

KEY: nd none detected.

68

I.OOO

ÎOO-

;w-t

i i

FIGURE 3.36. FIGURE 3.37. FIGURE 3.38.

(Upper left) Terenez/Morlaix sediment time series. (Upper right) Offshore lie Grande sediment time series (Bottom) Alkyl homologue distributions of phenanthrene series .

69

3.4 Sediment Cores (Ward, Montana State University)

An extensive series of sediment cores was obtained and analyzed by GC and selected samples analyzed by GC/MS for detailed aromatic hydro- carbon profiles. Samples were analyzed in support of anaerobic petro- leum biodégradation experiments (e.g. Winfrey et al., 1981). Three impacted sites and three control sites representative of beach, aber (estuarine) and marsh environments were selected (Table 15, Pig. 3.39).

TABLE 15.

AMOCO CADIZ chemistr tidal cores (Ward) .

y program, in te

Frequency:

December 1978

16

■-

March 1979

28

August 1979

16

November 1979

10

May 1980

12

Total

82

Locations:

Oiled :

AMC-4 (Portsall) - beach L'Aber Wrac'h - estuary lie Grande (South-Oiled) - marsh

Unoiled:

lie Grande (North-Control) marsh

Trez Hir - beach

Aber Ildut - estuary

Other Stations: Station 11 Station 12 Baie de Morlaix Port de Concarneau

GC/MS:

Several selected cores

70

MUM*

"A* V

'y

1

i M 4 NI k i .

\ >

/

itir.HANne A*f\ f\A I

*>y z^r^^ /

0 fs- «Alt Ot MUMIAIX

f*"^ / >»J^

^T*^ j?

t -^ V

's>*'7_ K.

V-*v vv'

■^i

"ft" \ f'

/X-c-

5c L AUtM WHACII

7K \t

-

irOHISAl 11

amhiluui

III».- IIIH'

-^JL

ALSO

ronr ue cuMCAHNtAu

FIGURE 3.39. Sediment core sampling locations (Ward).

The basic set of data, where GC and GC/MS data exist, is illustra- ted by the data in Table 16. However, secondary data products are presented here to illustrate the basic findings of this program segment,

Illustrative GC traces from March of 1979 are shown in Figures 3.40 and 3.41. While the hydrocarbon composition of the control estuary (L'Aber Ildut) is comprised mainly of biogenic compounds the marsh mudflat and beach both contain anthropogenic inputs. The lie Grande "control" has been impacted by the AMOCO oil as its GC profiles closely resemble those for weathered oil. However, the Trez Hir (beach) site consists mainly of compounds of a pyrogenic origin. The impacted sites all illustrate AMOCO oil in various states of weather- ing, the lie Grande (marsh) site containing the best "preserved" oil. This is also indicated by secondary treatment of some of the core aromatic data (Fig. 3.42) where oil in both L'Aber Wrac'h and lie Grande appears to be less weathered at depth.

Data from the three impacted cores and the control core are shown in Figures 3.43. 3.52, 3.58, and 3.63. Each figure depicts the depth of penetration of AMOCO CADIZ oil throughout the December 1978 to May 1980 time period. The C3P/C3DBT ratio is presented as is the level of the non-petrogenic fluoranthene + pyrene (m/e = 202) total.

Accompanying these figures are graphs of the down-core variations in gross hydrocarbon parameters (i.e. f]_, f2r ALK/ ISO ratio) and detailed aromatic compound families. Figures 3.44 to 3.51 depict details of the L'Aber Wrac'h cores, Figures 3.53 to 3.57 the lie Grande

71

TABLE 16. L'Aber Wrac ' h sediment core (March 1979).

ALIPHATICS (uq/q)

AROMATICS (uq/q)

SAMPLE

DATE

TOTAL

RESOLVED

0-S cm

3/79

530.0

27.8

5-10

3/79

113.7

8.4

10-15

3/79

95. «

2.8

15-20

3/79

4.4

1.1

20-25

3/79

10.1

1.6

ALK/ISO

TOTAL

RESOLVED

565.0

16. 4

318.4

19.4

118.1

4.0

46.5

4.4

C,M

C-.N

C,N

CaH

ClF

c,f

c,f

c,p

<V

CiP

C*P

0-5

20.4

23.0

42.5

292.2

288.9

18.9

53.7

137.2

434.4

100.6

200.5

294.0

S16.6

120

5-10

2.6

11.9

50.3

181.6

426.7

13.4

40.8

226.3

440.2

156.5

185.0

278.4

348.7

102

10-15

4.9

10.7

21.8

87.4

159.1

9.3

24.5

91.2

176.1

134.1

101.3

165.6

153.6

117

15-20

5.8

11.5

20.5

36.7

78.0

10.6

13.0

14.3

86.0

146.1

80.7

50.5

21.3

35

20-25

nd

2.6

14.3

17.9

nd

6.9

7.0

25.0

25.5

100.0

67.4

29.3

10.1

26

DBT

CjOBT

CiDBT

C3D8T

FL

PYR

CHRY

BF

B(e)P B<a)P

PERL

0-5

40.0

87.5

1466

1598

116.6

157.7

170.4

136.3

89.0

64.8

24.5

5-10

24.3

135.6

1360

830.7

132.8

172.2

185.2

219.7

124.2

128.2

33.7

10-15

17.4

93.7

498.4

491.6

1S6.4

178.6

174.4

230.9

118.6

137.0

36.2

15-20

. 10.7

16.7

32.3

65.2

164.3

155.7

169.6

215.3

100.2

122.3

29.5

20-25

s.o

8.0

10.1

5.0

98.9

74.9

93.2

76.7

94.3

105.6

24.4

KEY: nd » none detected.

N napthalene, C^-C^N « alkylated naphthalenes, F fluorene, C^r-CjP alkylated fluorenes, P <- phenanthrene ,

C1-C4P alkylated phenanthrenes, DBT Dibenzothlophene, C^DBT-C2DBT * alkylated dibenzothiophenes, FI «

f îuoranthene, PYR pyrene, CHRY » Crysene, BF » benzot Iuoranthene, B(e)P » Benzo(e)pycenef B(a)P » Benzo (a) pyrene,

PERL » pecylene.

IAI llil.il I Mu. 11 v M111lll.il

'Ml'. >'.■%• KM III* III, I*.*»

I '. I..I.....I "il M.ltail

«j^^UA^

(CI Oilnl '-ill M.vsli Miiilll.il

J||%^l,,

iH Oil.il BB,.|,

> I

P

llll I iiiilml I iln.iiy Miiilll.il

ll I

L.IU"

Ml

1 h

1

(01 Ciiiiliul S.1I1 M.n Ji M111lll.il

)i I .. ,. IJ

ID (..1111111I Spacli

...J

|lM^ |N P.I

FIGURE 3.40. Saturated hydrocarbons in sediments from oiled and control

sites.

72

(A| OiImI tsiuarv Mu.llUi

AllftlMI

ll>nui«lltH*l A

* S >«4«trw« SIJ..L-.I

!..

IC) Oilwl &■!( Mwili MwllUl

Wfc>lMlll

Itl Oiliii 8eji.li

181 COMIHll ElllUlV Minllln

JiU

l*U...t

101 Cntiiroi SjM MjmjIi Mo.lfl.il

yjjil.^4.,.

'UtiWi*!

FIGURE 3.41. FIGURE 3.42.

|F1 CuuiKil Hi-jcIi

:sa

AMOCO CA0I2 3IL _ *«*tft«e» Meuiu _ I 30-'«>

ï \

1 I

X t

.5 I I.

id

i

Aon! 27 1979

•» CiCjCjCt » C.CjCjC 0 C.C;Cj I I I I

j£y

m

,sol n.saaANoeisouTMi

100

s 1

S u

3 50-

Mwoi 1979

3-5 em

4

2^00 M

I i

1111

mm 1 1 m WmWIH»W SiMuta-

A86» '.VB AO-

Mwei '979

0— Set"

'JOOo

-ad

Ri

:$0"»

: too

% «■

ILsG»AN0E ISOUT»! Uaren 1979 t5-J0em

n=s

I ' '

ABEA WRACM

U»en 1979

10-ISem |

I

Pi

jjHlk

N N

« CiCzCjC* » CiCjCjCi 0 CiCjCj

I I I I

n CiCiC]Ca » CiC;CjC« 0 C. :;Cj

^.w— n»***»— 3.0«f<0-

(Top) Aromatic hydrocarbons in sediments from oiled and con- trol sites.

(Bottom) Comparative aromatic compound concentration pro- files derived from GC/MS-histogram presentation.

73

OÏCEMM* 197)

VHRCH '579

0-5

»

S-iO tO-lS

!«-2I 11-2» 29-31 )t-M

' ' ' i, i F

,V . S, . ,

kV',lvs''j

17

a

17

M

39 39

900

290

'90 -300 -MO

44 4) tjoar

0-5 S- 10 10-19 19-20 20-29

:

-

^J

a

ggg

a.m».^.»

v'v'O'-' -"

! 'X UO 200 50 30

23 -12 11

JJ 20Î

392, .-* 4,91

-274

-105 -3JS

220

•74

AUGUST 1979

N0V«M9j€S 1979

»K Cl» 202Kt4>4>

w»«i Cjoar

If** Cl» 202l<-»«

u4.1l C)097

fiS3gS3 270 0 79 1 100

t**4i Cj08T

0-1

3-4

«-5

5-3

I! 300

920

■P

-300 300 340 390

-300 "0

4MOC0CAOIZ0IL,

iiL

■^;\^ W,"0MM,C SIOGENlC

FIGURE 3.43.

Aber Wrac'h sediment cores

200

400

UB/B

600

800

lOr I

220

. Saturât»*! j-

Aromatlci ------- l

AU/ISO : ,0.

\

\

20

\

0.50

ALK ISO

4.75

1000

Bfl/a

2000

3000, 8000

D8T

202 2?8"2S2

FIGURE 3.44. (Left) Aber Wrac'h core, December 1978. FIGURE 3.45. (Right) Aber Wrac'h core, December 1978.

200

400 I|/| 600

8QQ

1000

fifl/B

2000

3000

Aromalics

10

220

Saturatis : ALK/ISO : iq

N OBT

J 25J-

202 228 252

0.50

1.0

1.50

2.0

5.0

ALK/ISO

FIGURE 3.46. (Left) Aber Wrac'h core, March 1979. FIGURE 3.47. (Right) Aber Wrac'h core, March 1979.

74

200

jg/g 400 600

200

400 MQ'9 600

800

10 I

\

1

10

20

Aronutics

Sit ont is -1 20

■ALK/ISO

Aromtlct

•Sitantit -

0.50

1.0

l.SO

ALK/ISO

0.50

1.0

1.50

2.0

2.50

ALK/ISO

ALK/ISO

FIGURE 3.48. (Left) Aber Wrac'h core, August 1979.

FIGURE 3.49. (Right) Aber Wrac'h core, November 1979.

200

400 a8/« 600

800

ALK/ISO

-10

20

0.50

1.0

Arsmitics

1.5

FIGURE 3.50 FIGURE 3.51

ALK/ISO

(Left) Aber Wrac'h core, November 1979. (Right) Aber Wrac'h core, May 1980.

OECEMSER 197S

VtASCH 1979

IPHC C3P 20209.91

<W9y9> C3O8T

28

2S

r

a. C

20-25 :

;phc Cjf

■«19:91 CjOBT

22 45 so 37 30

203O9.9'

J5

2*0

3SO

310 230

Sitintu ALK ISO

2.0

AUGUST 1979

;phc Cjf 20209,91

iu«9l C3O8T

o-s

S&>§8

1.100

550

S— 10

>>.-w/i' e

iO— IS

440

1 1

15-20

too

22 «2

130 170

AMOCO CAO II OIL

PYflOGENIC BIOGENIC

MAY 1980

;?HC C3P

U.9/9) C3O8T

0—2 mm 2—10 mm 1-2 em 2-3 cm 3—1 cm 4-5 cm 5-l0cm

,!! ,11.

! Il-i

a.

: i ; 1

j-1

e

w

1 t 1

iil'i, '

5.900 48

3 400 42

3.300

3.700

22.000 52

3300

3.310

75

FIGURE 3.52

20209.91

330 I 100

1 300

lie Grande (oiled) sedi- ment cores.

5 20

m

400 "/8 600

0.50

1.0

t. 50

ALK/iSO

800

2.0

Afiaiticj

Salanlis ALK/ISO -

200

O.SO

jjQ/g 400 600

Aromatics

Saturates

AU ISO

1.0

l.SO

AU ISO

1000 "/«2000

3000.8000

OIT——

zoTzTiisz

■r

10

S 20

/

2000

ji/i

0.50

1.0

Aromatic*

1.50

2.0

ALK/ISO

200 400 J|/,60fl

m » > " F

800

4000 «"■ 8000 8000 J5000

Sitiratit

ALK/ISO

2.50

0.50

Aromatic*

SKantis

ALK/ISO

t.O

1.5

ALK/ISO

2.0

83.28

FIGURE 3.53. FIGURE 3.54. FIGURE 3.55. FIGURE 3.56. FIGURE 3.57.

(Upper left) Ile Grande south core, December 1978. (Upper right) Ile Grande south core, March 1979. (Middle left) lie Grande south core, March 1979. (Middle right) lie Grande south core, August 1979. (Bottom) lie Grande south core, May 1980.

76

r

FIGURE 3.58. AMC-4 sediment cores.

0ECEM8EP 1978

\»A«CH :979

IPMC C]P 2021*9 9'

W9 9' CjOBT

0-5

. ! ,

5-10

10-IS

•': ;

15-20

;. ' ,;

20-2S

^^

:30

140

200 15 «S

37

;?HC C3P 202.", gi

uj9 91 CjOBT

0-5 5-10 10-15 15-17

"1

3

;

a. ■w 2

. i; i

&

«S

200 150 130 80

30

38

AUGUST 1979 INEW .NPUT1

MOVEM8EH 1979

0-5

5-10 10-IS

15-19

;phc C]> 202109/») i#a<gi C3OBT

w»'»i C3O8T

33 35

«8.000

8800

880

1.100

AMOCO CADIZ Oit

PYftOCENIC

BIOGENIC

25 25

0-5

II' I

28

32

MAY 1980

0-5

•-13

nil

1

11

i

IPHC C3> 2021-9 9' <UVV C3OBT

55

24

cores, Figures 3.59 to 3.62 the AMC-4 cores and Figure 3.64 a L'Aber Ildut core. Most of the cores were subdivided into sections of 3-5 cm in depth. However, two finer subdivisions from L'Aber Wrac'h - Novem- ber 1979 (1 cm segments down to 5 cm) , lie Grande - May 1980 (top 10 mm subdivided plus 1 cm sections down to 5 cm) were made.

Penetration of oil was observed down to 10-15 cm in L'Aber Wrac'h sediments with concentrations decreasing with depth when viewed in 5 cm sections. Note however, that while petroleum aromatics were decreasing in concentration with depth, the pyrogenic PAH compounds increased with depth. Finer subdivisions of the core indicate greater variation within the core than the 5 cm sections would indicate (Fig. 3.52).

An increase in vertical penetration of oil was observed for the lie Grande site between December 1978 and March 1979. A fresher layer of oil is found at the 15-20 cm depth (see Figs. 3.53 and 3.54) where naphthalenes, dibenzothiophenes, and to a lesser extent phenanthrenes, are more abundant than in surrounding layers. The gross hydrocarbon concentration changes at this level are not nearly as dramatic as are the petroleum aromatics, thus confirming that the "bulge" in Figure 3.42 is due to the less weathered nature of the buried oil. The finely divided May 1980 core (Fig. 3.52) indicates a higher petroleum content probably owing to a secondary input or to sampling variability which resulted in much higher levels (5-10. parts per thousand) during May 1980. The down core distribution of hydrocarbons is quite non-uniform a3 well with a preserved layer of fresher oil at 3-4 cm.

77

The AMC-4 cores appear dominated by well mixed AMOCO oil through- out the 0-20 cm depths. Lesser amounts of pyrogenic PAH vis-a-vis the Aber and marsh sediments are due to the sandy nature of the AMC-4 samples. A new large input of oil is seen in August 1979 resulting in some down-core concentration variation.

Chemical descriptions of the "control" site cores are shown in Figure 3.63. Note that non-petroleum PAH are widely observed in these sediments and that non-AMOCO CADIZ-impacted sediments contain 50-300 ppm of chronic hydrocarbon pollutants.

200

400

ug/g

600

800

~z?-

- ioj- '!

1.0

Aronitlcs

1.5

Salantes - 20 ALU/ISO ■! \

2.0

UK/ISO

jio/g

200 400 600

0.50

\

\

1.0

Aromatics

Saturates

ALKIS0

1.50

AIXIS0

_10^ '/

20L

1000

ng/g

2000

P --

0BT

202 IFa 252

20

200

400

iig/g

3000

-N-

5000 40000

Aromatics

Saturates -

0.50

1.0

.50

ALK ISO

2.0

ALK/ ISO

FIGURE 3.59.

FIGURE 3.60.

FIGURE 3.61.

FIGURE 3.62.

(Upper "left) AMC-4 core, December 1978, (Upper right) AMC-4 core, March 1979. (Lower left) AMC-4 core, March 1979. (Lower right) AMC-4 core, August 1979.

78

A3E» 'LOuT

ICE GHANOE ;NORTu.

VIA3CH 1979

MASCH '979

0-5 S-tO 10-15

■s-:o

20-25 F 25-29

If»t«C C3P M2(i9-ji

91 C3O8T

MO " â M

:oo

TAO

HO

:5o

230 970

n

, ; i

L ■'■ J

'0-

IS f ■)

15-

.J: | 1

1

'D-

* 1

:*HC C3" 202.*» «1

iwjgl C3O8T

250

200

100 - -

100 10

-961 Ml»

MARCH 1979

IS»MC C3» 202HW -1 V C3O8T

-s 0-5

£>N>^

= 5-10

-»■

a.

"" 10-15

59

30

91

2S SAO

K^

AMOCO CA0I2 OIL PYflOGENiC

BIOGENIC

FIGURE 3.63. Miscellaneous sediment cores

1000 "fl/9 2000

10

20

l\

\

OBT

202 223 252

FIGURE 3.64. Aber Ildut, March 1979.

79

3.5 Oysters and Plaice (Neff, Battelle)

Samples of oysters and plaice from several impacted regions (L'Aber Wrac'h, L'Aber Benoit and Baie de Morlaix) and two supposedly unimpacted locations (Brest and Loctudy) were analyzed (Table 17, Figure 3.65) .

The results of the oyster time series analyses are summarized in Table 18. Both the "gross" hydrocarbon parameters as well as the petroleum-associated aromatic hydrocarbons are presented. Though not "clean", the control (Brest) oysters are several times lower in gross concentration throughout the time period and an order of magnitude lower in aromatic hydrocarbon content than either of the impacted sites. It is not apparent if the levels have decreased substantially in either of the Abers, though aromatic levels are 3 to 4 times lower a year and a half after the spill. For comparison, levels of several of the non-petrogenic PAH components (i.e. m/e 252) are presented.

TABLE 17. AMOCO CADIZ chemistry program; oysters and plaice (Neff).

Frequency:

December 1978

4

April 1979

6

July 1979

7

February 1980

9

June 1980

11

Total

37

Location:

L'Aber Benoit - Oysters; Plaice Muscle/Liver

L'Aber Wrac'h - Oysters; Plaice Muscle/Liver

Loctudy - Plaice Muscle/Liver; Oysters (7/79 only)

Brest - Oysters

Baie de Morlaix - Oysters (7/79 only)

GC/MS

L'Aber Benoit Oysters L'Aber Wrac'h Oysters Control Oysters

80

ALSO LOCIUOV

FIGURE 3.65. Oysters and Plaice sampling locations.

TABLE 18. *- Petroleum hydrocarbons in oysters (Crassostrea gigas).

PETROLEUM

a

DBT

f^

HYDROCARBONS

Pa

252

LOCATION

DATE

(ug/g)

(ug/g)

(ug/g)

(ug/g)

L'Aber Wrac'h

12/78

660

12

22

0.04

4/79

1,200

15

12

0.02

7/79

590

5

10

0.03

2/80

820

10

16

0.60

6/8.0 (#1)

440

4

6

0.40

>

6/80 (#2)

560/570d

-

-

-

Brest

12/78

260

4

4

0.07

(control)

4/79e

1,100

11

10

0.01

7/79

91

0.3

0.3

-

2/80

150

0.4

1.1

0.6

6/80

93

0.6

0.7

0.2

L'Aber Benoit

12/78

690

-

-

-

4/79

800

15

15

1.0

7/79

-

-

-

-

2/80

430

14

9

1.1

6/80

520

3

5

0.2

aSum of phenanthrene and alkyl phenanthrenes.

^Sum of dibenzothiophenes and alkyl dibenzothiophenes

cSura of m/e = 252.

^Replicate analyses.

eOrigin of sample unclear.

81

The GC traces for the impacted oysters are consistent throughout the study. The aromatic hydrocarbons (Figs. 3.66, 3.67) are dominated by the alkylated dibenzothiophenes and alkylated phenanthrenes through- out. The alkyl naphthalenes and fluorenes, significant in December of 1978, are removed from the tissues by June 1980. Aromatic hydrocarbons in the control oysters (Fig. 3.67), while less concentrated, are dominated by the same compound series, though the compositions in the controls remain consistent with time (i.e. no loss of fluorenes or naphthalenes) . GC/MS traces of the oysters confirm the importance of the dibenzothiophene series (Fig. 3.68).

Saturated hydrocarbon GC traces are illustrated in Figures 3.69 and 3.70 for impacted and control oysters respectively. The saturates of the L'Aber Wrac'h samples are dominated by branched alkanes (e.g. isoprenoids) and a large low boiling UCM (C]^ - C20) The UCM in the controls is less pronounced yet significant, and while the isoprenoids are abundant indicating some weathered petroleum, a higher boiling smooth n-alkane distribution (i.e. paraffins, ÏI-C20 ~ n~C3Q) is of equal importance. Figures 3.71 to 3.76 show some representative aroma- tic and saturated fraction data from oyster samples taken from L'Aber Wrac'h and the control station.

The results of the plaice analyses are summarized in Table 19. The absolute concentration data does not address the source of the observed levels which for the most part are not linked to AMOCO CADIZ oil. The muscle tissues exhibit some petroleum-like GC traces includ- ing some UCM material and smooth n-alkane distributions with the presence of UCM material primarily responsible for the higher levels shown in Table 19. Liver tissue in all samples is much higher in absolute hydrocarbon content (Figs. 3.77 and 3.78). The f^ (saturated) traces are characterized by a high molecular weight UCM (cycloalkanes) , and an n-alkane distribution in the C22 to ^23 region, while the f2 traces are characterized by polyolefinic material, including the biosynthesized compound squalene. These f]_ and f2 distributions are characteristic of fish livers from many geographic regions (Boehm, 1980; Boehm and Hirtzer, 1981) and are probably not related to any particular spill event.

82

1

I?.: Vli..

r"'i

*>%

■■&<■

"

m

fit.

HM.

FIGURE 3.66.

Aber Wrac'h impacted oysters - aromatic hydrocarbons; A December 1978; B - June 1980.

f

t

Alkvl P

»«l C1HI