Na/JA/CNfiXO 0<+ 82- ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL SPILL Report of the NOAA-CNEXO Joint Scientific Commission WHOI DOCUMEN" COLLECTION a — "™ — i" *'^rts o. r U. S. DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration CENTRE NATIONAL POUR I'EXPLOITATION DES OCEANS WHO! tpCUMEHT Co^cr,ON Oi PI -IS 00- Sf : o ; CD ; a- : <=> ! ° D ECOLOGICAL STUDY OF THE AMOCO CADIZ OIL SPILL *^iE Report of the NOAA-CNEXO Joint Scientific Commission October 1982 4Wt, U. S. DEPARTMENT OF COMMERCE 1%^KA- Malcolm Baldnge, Secretary ^B^ National Oceanic and Atmospheric Administration John V. Byrne, Administrator CENTRE NATIONAL POUR I'EXPLOITATION DES OCEANS DISCLAIMER Mention of a commercial company or product does not constitute an endorsement by NOAA Environmental Research Laboratories. Use for publicity or advertising purposes of information from this publi- cation concerning proprietary products or the tests of- such products is not authorized. ii TABLE OF CONTENTS Page Preface v I. Physical, Chemical, and Microbiological Studies After the AMOCO CADIZ Oil Spill ATLAS, R.M. Microbial Degradation within Sediment Impacted by the AMOCO CADIZ Oil Spill 1 BALLERINI, D. , DUCREUX, J., and RIVIERE, J. Laboratory Simulation of the Microbiological Degradation of Crude Oil in a Marine Environment . . 27 BOEHM, P.D. The AMOCO CADIZ Analytical Chemistry Program .... 35 DOU, H., GIUST, G., and MILLE, G. Studies of Hydrocarbon Concentrations at the He Grande and Baie de Lannion Stations Polluted by the Wreck of the AMOCO CADIZ 101 DUCREUX, J. Evolution of the Hydrocarbons Present in the Sediments in the Aber Wrac'h Estuary Ill MARCHAND, M., BODENNEC, G. , CAPRAIS, J.-C, and PIGNET, P. The AMOCO CADIZ Oil Spill, Distribution and Evolution of Oil Pollution in Marine Sediments . . . 143 WARD, D.M., WINFREY, M.R., BECK, E., and BOEHM, P. AMOCO CADIZ Pollutants in Anaerobic Sediments: Fate and Effects on Anaerobic Processes 159 II. Biological Studies After the AMOCO CADIZ SPILL GLEMAREC, M. and HUSSENOT, E. Reponses des Peuplements Subtidaux a la Perturbation Creee par 1 'AMOCO CADIZ dans les Abers Benoit et Wrac'h 191 CABIOCH, L., DAUVIN, J.-C, RETIERE, C, RIVAIN, V. and ARCHAMBAULT, D. Les Effets des Hydrocarbures de 1 'AMOCO-CADIZ sur les Peuplements Benthiques des Baies de Morlaix et de Lannion d'Avril 1978 a Mars 1981 205 in Page BOUCHER, G., CHAMROUX, S., LE BORGME, L. , and MEVEL, G. Etude Experimental e d'une Pollution par Hydrocarbures dans un Microecosysteme Sedimentaire. I: Effet de i Contamination du Sediment sur la Meiofaunr- 22 " BODIN, P. and BOUCHER, D. Evolution a floyen-Terme du Meiobenthos et du Microphytobenthos sur Quelques Plages Touchees par la Maree Noire de 1 'AMOCO -CADIZ 245 NEF and HAENSLY, W.E. Long-Term Impact of the AMOCO CADIZ Crude Oil Spill on Oysters Crassostrea gigas and Plaice : ieuronectes platessa From Aber Benoit and Aber Wrac'h, Brittany, France. I. Oyster Histopathology. II Hetrcleum Contamination and Biochemical Indices of Stress in Oysters and Plaice . . . 269 LEVASSEUR, J.E. and JORY, M.-L. Retablissement Naturel d'une Vegetation de Marais Mari times Alteree par les Hydrocarbures de 1 'AMOCO CADIZ: Modalites et Tendances 329 SENECA, E.D. and BROOME, S.W. Restoration of Marsh Vegetation Impacted by the AMOCO CADIZ Oil Spill and Subsequent Cleanup Operations at He Grande, France 363 LE CAMPION-ALSUMARD, T. , PLANTE-CUNY, M.-R., and VACELET, E. Etudes Microbiologiques et Microphytiques dans les Sediments des Marais Maritimes de l'lle Grande a la Suite de la Pollution par 1 'AMOCO CADIZ 421 CHASSE, C. and GUENOLE-BOUDER, A. 1964-1982, Comparaison Quantitative des Populations Benthiques des Plages de St Efflam, St Michel-en- Greve Avant, Pendant et Depuis le Naufrage de 1 'AMOCO-CADIZ 451 iv PREFACE At approximately 11:30 p.m. on Thursday, March 16, 1978, the super- tanker Amoco Cadiz went aground on a rock outcropping 1.5 km offshore of Portsall on the northwest coast of France. The vessel contained a cargo of 216,000 tens of crude oil and 4,000 tons of bunker fuel. At 6:00 a.m. on Friday, March 17, the vessel broke just forward of the wheelhouse and thus started the largest oil spill in maritime history. During the course of the next 15 days, the bunker fuel and contents of all 13 loaded cargo tanks, which contained two varieties of light mideastern crude oil, were released into the ocean. The oil quickly became a water-in-oil emulsion (mousse) of at least 50% water, and heavily impacted nearly 140km of the Brittany coast from Portsall to He de Brehat. At one time or another, oil contamination was observed along 393 km of coastline and at least 60 km offshore. Impacted areas included recreational beaches, mariculture impoundments, and a substantial marine fishery industry. h arcr, .3, r. »,ilmot N. Hess, Director of the . v.v , - /omental Research Laboratories (ERL) of the National Oceanic and Atmospheric Administration (N0AA), contacted Dr. Lucien Laubier, Director of the Centre Oceanologique de Bretagne (COB) of the Centre National pour 1 'Exploitation des Oceans (CNEX0), the French national oceanographic organization. Dr. Hess and Dr. Laubier arranged for participation by United States scientists in a joint Franco-American investigation of physical and chemical manifestations of the spill. On March 24, the agreement was expanded to include cooperative biological investigations through contacts initiated by Dr. Eric Schneider, Director of the Environmental Protection Agency's Environmental Research Laboratory in Narragansett, Rhode Island. N0AA personnel arrived on March 19 to join the investigation initiated on March 17 by several French scientific teams. Initial photographic over-flights and active beach sampling began on Tuesday, March 21, followed by initial chemical sampling by vessel on Friday, March 24. The team was supplemented with EPA biological observers on Sunday, March 26. Sampling has continued by some segments of this original team until the present time. Throughout the period of investigation, active interaction and coordination with the French scientific community have taken place under the auspices of C0B/CNEX0. All sampling has been coordinated with the general ecological impact study designed by the French Ministry of Environment, organized, ,by CNEX0, and operated by several scientific institutions in France^ , making possible a more thorough evaluation of the effects of the incident than would otherwise have been possible. - National Museum of Natural History, National Geographic Institute, French Institute of Petroleum, Scientific and Technical Institute of Marine Fisheries, University of Western Brittany, University P. and M. Curie, Paris VI, and the National Center for the Exploitation of the Oceans. About three months after the oil spill the U.S. team prepared a "Preliminary Scientific Report on the Amoco Cadiz Oil Spill" covering data up to May 15, 1978. This document covered only the period of acute effects. A one-day symposium on the Amoco Cadiz spill was held in Brest on June 7, 1978, and published soon after. It was obvious from these initial observations that a period of years would be required to under- stand what had happened to these portions of the coast where the oil had settled in and not been cleansed promptly. During this early period of study of the spill Mr. Russ Mallatt of the Amoco Trading Company had several discussions with Drs. Hess, Laubier and Schneider. Mr. Mallatt was the General Manager for Environmental Conservation and Toxicology of Amoco. Discussion with Mr. Mallatt during the first two months after the spill identified Amoco's interests in carrying out long-term studies of the effects of the oil spill. These early contacts were followed up by substantial discussions between Mr. John Linsner of Amoco and Mr. Eldon Greenberg, General Counsel of NOAA. These discussions culminated with an agreement being signed by Amoco and NOAA to carry out long-term studies of the effects of the spill. The study would cover three years and would be a joint French-U.S. activity. A Joint NOAA/CNEXO Scientific Commission was established through another agreement between the two agencies signed June 2, 1978. Amoco would transfer money to NOAA and the Joint Commission, chaired by Drs. Hess and Laubier, would determine the research program to be carried out, the investigators to do the research, and the funding levels. The Joint Commission would also monitor the progress of the studies and be responsible for making the final report. One of its major goals was to make U.S. and French scientific teams work together in a common effort to better understand the consequences of the wreckage. The Joint Commission first met in Brest at the CNEXO Laboratory on July 18, 1978. Taking into account the French program to assess the long-term ecological impact of the oil spill funded by the Ministry of Environment, it determined that the most important areas for research were: 1. Heavily impacted subtidal areas like the Abers and the Bays of Morlaix and Lannion. 2. Heavily impacted intertidal areas such as St. Efflam and the salt marsh at He Grande. 3. The detailed chemical evolution of the petroleum hydrocarbons. 4. Biodegradation of petroleum. The second meeting of the Joint Commission, held in Washington, D.C., on October 12, 1978, reviewed the work carried out during the first months of the first year and planned the research program for the second year's study. VI In November 1979, an international conference was held in Brest sponsored by CNEXO. Investigators sponsored by the Joint NOAA/CNEXO Scientific Commission, as well as a number of other scientists, gave papers at this conference. The proceedings of this conference entitled "Amoco Cadiz: Fates and Effects of the Oil Spill" make a very good summary of the first one and one-half year study after the spill. Following the second meeting of the Joint Commission, Dr. Hess left NOAA and was replaced as co-chairman by Dr. Joseph W. Angelovic from the Office of Ocean Programs in NOAA. The third meeting of the Joint Commission was held in Paris, France, October 28, 1980, in conjunction with the meeting of the U.S. -French Cooperative Program in Oceanography. The previous work was reviewed and the final year of the research program was planned. Now the three-year study is over and attempts are being made to bring together the findings of the investigators. A workshop was held in Charleston, South Carolina, on September 17-18, 1981, to report on the physical and chemical studies. A second workshop was held in Brest, France, on October 28-30, 1981, to report on the biological effects studies. This document is the report of those workshops and forms the body of the final report to Amoco from the Joint NOAA/CNEXO Scientific Commission. Speaking for all who worked on the spill, we would like to thank the Amoco Transport Company for sponsoring this three-year study of the effects of the spill. Without Amoco 's help, we would be nowhere near our present state of knowledge of what the effects of the spill were or how the recovery back to normal conditions has proceeded. Other studies have been carried out, sponsored by the French Government and other sources, but an important part of the work has been sponsored by Amoco. Mr. Russ Mallatt, Dr. James Marum, Mr. John Lamping, Ms. Carol Cummings and others from Amoco attended meetings of the Joint Commission and the scientific sessions. They were always helpful and supportive of the Commission's work and never intruded on the design or conduct of the program. We have, through this cooperative effort, obtained more detailed and more useful knowledge of the effects of this oil spill than of any other large oil spill in history. A major reason for this is that the biological communities present before the spill had been studied in great detail by French scientists. Today many of the areas impacted by the spill appear to the casual observer to be recovered from the effects of the oil. However, investi- gations have shown that differences still exist between some of the current ecosystems and those present prior to the spill. Hopefully other studies will continue to watch and document the recovery processes. Vll These studies have added substantially to man's knowledge about oil spills. We can only hope that others will follow and build on the understanding of oil spill effects accumulated through these studies. Lucien Laubier Wilmot Hess Joseph Angelovic viii CNEXO-NOAA Joint Scientific Commission MEMBERSHIP L. Laubier, Cochairman Centre National pour l1 Exploitation des Oceans Paris, FRANCE Wilmot N. Hess, Cochairman National Oceanic & Atmospheric Administration Boulder, Colorado Joseph W. Angelovic, Cochairman NOAA Office of Ocean Programs Rockville, Maryland Jack Anderson Battel le Pacific Northwest Laboratory Sequim, Washington J. Bergerard Station Biologique Roscoff, FRANCE Edward S. Gilfillan Bowdoin College Bowdoin, Maine I. R. Kaplan University of California, Los Angeles Los Angeles, California R. l.etaconnoux Institut des Peches Maritimes Nantes, FRANCE J. M. Peres Station Marine and Endoume Marseille, FRANCE Philippe Renault Institut Francais du Petrole Rueil Malmaison, FRANCE Douglas A. Wolfe NOAA Office of Marine Pollution Assessment Boulder, Colorado IX PART I Physical, Chemical, and Microbiological Studies After the AMOCO CADIZ Oil Spill Edited by E. R. Gundlach Research Planning Institute, Inc. Columbia, South Carolina, U.S.A. 29201 MICROBIAL HYDROCARBON DEGRADATION WITHIN SEDIMENT IMPACTED BY THE AMOCO CADIZ OIL SPILL by Ronald M. Atlas Department of Biology University of Louisville Louisville, Kentucky 40292 INTRODUCTION The wreck of the AMOCO CADIZ in March 1978 released over 210,000 tons of oil into the marine environment. As much as one third of the spilt oil may have been washed into the intertidal zone. The spill occurred during storm surges, thereby spreading the oil throughout the intertidal zone. Two years after the AMOCO spill, the wreck of the tanker TANIO resulted in another oil spill that contaminated much of the same Brittany shoreline impacted by the AMOCO CADIZ. This study was undertaken to determine the fate of petroleum hydrocarbons within surface sediments along the Brittany coast with reference to the role of microorganisms in the oil weathering process. METHODS Sampling Regime Duplicate samples were collected at intertidal sites along the Brittany coast which had received varying degrees of oiling from the AMOCO CADIZ spillage (Fig. 1). The sampling sites included the salt marsh at lie Grande, a beach near Portsall in the vicinity of the wreck site, a mudflat in Aber Wrac'h, a beach at St-Michel-en-Greve near where a large bivalve kill had been reported, a relatively lightly oiled reference site at Trez Hir and a site at Tregastel which was not oiled by the AMOCO CADIZ spill, but was later oiled by the spill from the tanker TANIO (Table 1). Surface sediment samples (upper 5 cm) were collected with a 3 cm diameter soil corer. Samples were placed in metal cans for hydrocarbon analyses and in Whirlpak bags for microbial analyses. Samples were collected during December, 1978; March, 1979; August, 1979, November, 1979, March 1980, July, 1980 and June, 1981; 9, 12, 17, 20, 24, 28 and 39 months after the spillage, respectively. During November, 1979 sediment samples were also collected at four offshore sites in the Bay of Morlaix. Samples for microbiological analyses were processed within four hours of collection. For hydrocarbon analyses, samples were frozen and shipped to Energy Resources Company (ERCO) for extraction and analysis 13 1 LE GRANOE^Vn. X-^' S d.2,3] «> z_r^^ / BAY of /[_£>!* MORLAIX 9~\ /*& £^r-£ 9~vJ* ST MICHEL-EN-GREVE 9~^t^ %■ l'aber wrach ^ 15.6) (41 / PORTSALL / 1^.8] V TREZ-HIR V *J9,10' w- > /*tr^ FIGURE 1. Location of intertidal and subtidal sampling sites. TABLE 1 - Description of sampling sites, Site Description 1 lie Grande - sandy - low energy - NE of bridge - relatively unoiled. 2 lie Grande - sandy - low energy - SW of bridge - near end of excavation area. 3 lie Grande - soil - heavily oiled - amid Juncus - above excavation area. 4 St-Michel-en-Greve - sandy - high energy - near low tide mark. 5 Aber Wrac'h - mud - 100m offshore at Pcrros. 6 Aber Wrac'h - mud - 200m offshore at Perros. 7 Portsall - sandy - high energy - near wreck site - below high tide line. 8 Portsall - sandy - high energy - near wreck site - rear rocks - 100m below high tide line. 9 Trez Hir - sandy - moderate energy - reference site - below high tide. 10 Trez Hir - sandy - moderate energy - reference site - 20m below high tide line. 11 Tregastel - sandy - low energy - Tanio spill site - 20m below high tide line. 12 Tregastel - sandy - low energy - Tanio spill site - 50m below high tide line. by silica gel column chromatography, weight determination, glass capillary gas chromatography and mass spectrometry. Enumeration of Microbial Populations Total numbers of microorganisms per gram dry weight of sediment were determined by direct count procedures. Portions of collected sediment samples were preserved with formalin. Microorgansims in the preserved samples were collected on a 0.2 mm pore size Nuclepore filter which had been stained with irgalan black. The microorganisms were stained with acridine orange and viewed using an Olympus epif luorescence microscope. Cells staining orange or green were counted in 20 randomly selected fields and the mean concentration determined. Hydrocarbon utilizing microorganisms were enumerated using a three tube Most Probable Number (MPN) procedure. Serial dilutions of sediment samples, prepared using Rila marine salts solutions, were inoculated into sealed serum vials containing 10 ml BushnjeAl Haas broth (Difco) and 50 ml of Arabian crude oil spiked with C hexadecane (sp. act. 1 mCi/ml) . After 14 days incubation at 15°C, the C02 (if any) in the head space was collected by flushing and trapping in oxifluor CO .and quantitated by liquid scintillation counting. Vials showing CO production (counts significantly above background) were scored as positive and the Most Probable Number of hydrocarbon utilizers per gram dry weight calculated from standard MPN tables. Biodegradation Potentials Portions of sediment samples were placed into serum vials containing 10 ml Bushnell Haas broth. and 50 ml light Arabian crude oil spiked with either., C hexadecane, C pristane, C naphthalene, C benzanthracene or C 9-methylanthracene. After 14 days incubation, microbial hydrocarbon degrading activities were stopped by addition of KOH. The " C0„ produced from mineralization of the radiolabelled hydrocarbon was determined by acidifying the solution, flushing the headspace, trapping the CO in oxifluor C0„ and quantitating the C by liquid scintillation counting. The residual undegraded hydrocarbons and biodegradation products were recovered by extraction with hexane. The L C in each solvent extract was determined and fractionated, using silica gel column chromatography, into undegraded hydrocarbon fractions (hexane + benzene eluates) and degradation product fractions (methanol eluate + residual non-eluted counts). A 0.75 cm diameter X 10 cm column packed with 70-230 mesh silca gel 60 was used. Radiolabelled material in each fraction was quantitated by liquid scintillation counting. Sterile controls were used to correct for efficiency of recovery and fractionation. Triplicate determinations were made for each sample and radiolabelled hydrocarbon substrate combination. The percent hydrocarbon mineralization was calculated as C02 produced (above sterile control)/ C hydrocarbon, added. The .percent hydrocarbon biodegradation was calculated as ' C0„ produced +, C methanol fraction + C residual (all above sterile control)/ C hydrocarbon added. Carbon balances generally accounted for approximately 90% of the radiolabelled carbon added to the sediment (except for naphthalene where volatility losses prevented efficient recovery). Chemical Hydrocarbon Analyses Performed at ERCO For hydrocarbon analyses the samples were thawed, dried with methanol and extracted by high energy shaking with a mixture of methylene chloride-methanol (9:1). The extract was fractionated into an aliphatic (f ) fraction and an aromatic (f ) fraction using silica gel/alumina column chromatography. A 1 cm diameter X 25 cm column (1 cm alumina on top of 15 cm silica gel) was used. The f. fraction was eluted with 18 ml hexane; the f fraction subsequently was eluted with 21 ml of a 1:1 mixture of hexane-methylene chloride. After reducing the volume of solvent by evaporation, the gross amount (weight) of hydrocarbon in each fraction was determined gravimetrically from an evaporated and dried aliquot of the extract. The extracts were subjected to quantitative glass capillary-gas chromatographic (GC) analysis. Selected aromatic fractions also were analysed by combined glass capillary gas chromatographic/mass spectrometric (GC/MS) analysis for qualitative identification of individual compounds and quantification of minor components. Participation in an intercalibration exercise under the direction of the National Analytical Laboratory indicated that these analyses were at the state^ of the art with repeatable ± 20% detection of hydrocarbons in the ng g dry weight sediment range. The details of GC and GC/MS analysis employed are as follows: CC: Hewlett Packard 5840A reporting GC with glass; splitless injection inlet system; 30 m glass capillary column coated with SE-30 (s 100,000 theoretical plates); FID detector; temperature programmed at 60-275°C min ; helium carrier gas 1 ml min ; transmission of integrated peak areas and retention time through HP 18846A digital communications interface to a PDP-10 computer for storage, retention index and concentration calculations. Deuterated anthracene (f.) and androstane (f ) were used as internal standards and response factors were determined with known concentrations of the reported compounds. GC analysis was used to quantitate components of the f. fraction. GC/MS: Hewlett Packard 5985 quadrapole system (GC/MS Computer); mass spectrometer conditions: ionization voltage=70 eV, electron multiplier voltage=2200 V, scan conditions 40 amu to 500 amu at 225 amu s~ . Quantification of components of the f fraction was accomplished by mass f ragraentography wherein the stored GC/MS data is scanned for parent ions (m ) . The tabulated total ion currents for each parent ion is compared with deuterated anthracene (internal standard) and an instrumental response factor applied. Authentic polynuclear aromatic hydrocarbon standards were used to determine relative response factors (when no standard was available a response factor was assigned by extrapolation) . In vitro Biodegradation Sediment was collected at sites 6 and 7 in November, 1979 for in vitro biodegradation experiments. Replicate one hundred gram portions of sediment were placed into 250 ml flasks to which 50 ml of a sterile solution containing 0.5% KNO + 0.5% KH2P04 and 0.5 ml of light Arabian crude oil were added. The flasks were agitated on a rotary shaker at 100 RPM. After two, four, and six weeks of incubation at 15°C, the oil remaining in replicate flasks (two at each sampling time) was extracted and analysed as described below. Additionally, replicate 100 g portions of sediment were placed into 1 liter stainless steel buckets. The containers were continuously flushed with a solution of Rila marine salts supplemented with 10 ppm KNO + 10 ppm KH PO . The height of the water level was adjusted to be 3 cm above the surface of the sediment layer. The flow rate was adjusted to 10 ml/h. After two, four, and six weeks of incubation at 15°C the oil remaining in replicate sediment portions was extracted and analysed as described below. Analyses of ^in vitro Experiments Residual oil was recovered from samples by extraction with sequential portions of diethyl ether and methylene chloride. The sediment was shaken at 200 RPM with repetitive portions of solvent. The extracts were subjected to column chromatography to split the extracts into aliphatic (f,) and aromatic (f~) fractions. Columns were prepared by suspending silica gel 100 (E. M. Reagents, Darmstadt, W. Germ.) in CH„C1„ and transferring the suspension into 25 ml burets with teflon stopcocks to attain a 15 ml silica gel bed. The CH„C1? was washed from the columns with three volumes of pentane. Portions of the extracts in pentane were applied to the columns, drained into the column bed, and allowed to stand for three to five minutes. The aliphatic fraction (f ) was eluted from the column with 25 ml pentane. After 25 ml pentane had been added to the column, 5 ml of 20% (v/v) CH„C1„ in pentane was added and allowed to drain into the column bed. Fraction f was 30 ml. The aromatic fraction (f ) was eluted from the column with 45 ml of 40% (v/v) CH2C1„ in pentane. The fractions of each extract were then concentrated to about 5 ml at 35°C and transferred quantitatively to clean glass vials. Fractions f. and f were prepared for analysis by gas chromatography or gas chromatography mass spectrometry. An internal standard, hexamethyl benzene (Aldrich Chem. Co., Milwaukee, WI.), was added to each sample. In fraction f . , hexamethyl benzene (HMB) was present at 12.6 ng/ml; in fraction f„, HMB was present at 25.2 ng/ml. Fraction f. was analyzed by GC on a Hewlett-Packard 5840 reporting GC with FID detector. The column was a 30 m, SE54 grade AA glass capillary (Supelco, Bellefonte, PA.). Conditions for chromatography were injector, 240°C; oven 70°C for 2 min. to 270°C at 4°C/min. and hold for 28 min.; FID, 300°C; and carrier, He at 25 cm/sec. A valley-valley intergration function was used for quantitative data acquisition. Response factors were calculated using n-alkanes, (C -C ) , pristane and phytane standards. Fraction f„ was analyzed with a Hewlett-Packard 5992A GC-MS. Conditions for chromatography were injector, 240°C; oven 70°C for 2 min. to 270°C at 4°C/min. and hold for 18 min. Data was acquired using a selected ion monitor program. Thirteen ions were selected for representative aromatic compounds. The ions monitored were 128, 142, 147, 156, 170, 178, 184, 192, 198, 206, 212, 220, and 226. The representative compounds were naphthalene, methyl naphthalene, HMB as an interanal standard, dimethyl naphthalene, trimethyl naphthalene, phenanthrene, dibenzothiophene, methyl phenanthrene, methyl dibenzothiophene, dimethyl phenanthrene, dimethyl dibenzothiophene, trimethyl phenanthrene, and trimethyl dibenzothiophene, respectively. The dwell time per ion was 10 msec. Instrument response factors were calculated by injecting known quantities of unsubstituted and C and C„ substituted authentic aromatic hydrocarbons and determining the integrated response for each compound. These values were used to extrapolate for quantitation of isomers and C_ substituted compounds. For analysis of the polar fraction including microbial degradation products, three samples were selected for analysis by the University of New Orleans Center for Bio-organic Studies. The samples were: 1) flow through, 6 week incubation from site 6; 2) flow through, 6 week incubation from site 7; 3) agitated flask, 6 week incubation from site 7. Frozen samples were sent for analysis. At the Center for Bio-organic Studies the samples were extracted with successive portions of CH OH, CH OH/CH CI and CH CI . The extracts were fractionated using silica gel and the f_ fraction was collected, methylated and analysed by high resolution GC-MS . RESULTS AND DISCUSSION The enumeration of hydrocarbon utilizing microorganisms indicated that numbers of hydrocarbon utilizers in the intertidal sediments increased significantly in response to hydrocarbon inputs (Table 2). Site 3, which is covered with seawater only at times of extreme high tide, showed very high populations of hydrocarbon utilizing microorganisms even three years after the AMOCO CADIZ spillage. Sites 5 and 6 (located within Aber Wrac'h) and Sites 7 and 8 (located near Portsall) showed variable, but apparently elevated, numbers of hydrocarbon utilizers for up to two years following the spill. It appears that hydrocarbons contained within the mud sediments of Aber Wrac'h continued to exert a selective pressure on the microbial community that favored elevated populations of hydrocarbon utilizers for a longer period of time than sites on high-energy sand beaches. Site 2 showed evidence that the TANIO spill impacted the lie Crande region. This site did not show elevated numbers of hydrocarbon utilizers in December 1978 or at later sampling times as a result of the AMOCO CADIZ spill, but in July of 1980, several months after the wreck of the TANIO, numbers of hydrocarbon utilizers were greatly elevated. A year later, however, the numbers of hydrocarbon utilizers had returned to background levels at this site. The unoiled control sites 9 and 10 and sites 1 and 4, which were impacted by the AMOCO CADIZ spill, did not show any evidence of elevated hydrocarbon-utilizing populations during the sampling period. Similarly, the offshore sites A-D in the Bay of Morlaix did not appear to be elevated at the time of sampling in November 1979. Sites 11 and 12 were added following the wreck of the TANIO and showed obviously elevated populations of hydrocarbon utilizers that persisted for over a year. TABLE 2. MPN-Hydrocarbon Utilizers. ,3 Site 2-78 3-79 (// X 10 /g dry wt.) 8-79 11-79 3-80 7-80 6-81 1 0. 2 0.5 1 0.7 5 16 1 2 5 7 1 14 30 45000 1 3 2200 14000 41000 13000 160000 48000 24000 4 2 0.4 2 7 1 4 5 5 8 18 8 450 19 10 15 6 9 390 20 27 190 11 17 7 40 1900 1 2 12 2 2 8 57 350 150 8 3 1 10 9 0. 7 0.4 3 1 4 1 4 10 0. 1 0.2 4 1 2 2 3 11 - - - - 19000 140000 24000 12 - - - - 920000 140000 24000 A - - - 66 - - - B - - - 32 - - - C - - - 13 - - - D - - - 13 - - - The elevation in hydrocarbon utilizing populations, when detected, represented a shift within the microbial community. There generally was no evidence that total microbial biomass increased as a result of oiling although there generally was a tenfold variation in the microbial biomass between different sampling times (Table 3). Site 12-78 TABLE 3. Direct Count. (// X !08/g dry wt.) 3-79 8-79 11-79 3-80 7-80 6-81 1 3 1 4 3 1 1 3 2 4 2 16 7 3 40 2 3 10 6 220 18 24 40 38 4 2 1 3 0.4 0.5 0.6 2 5 3 2 19 12 17 2 15 6 6 7 150 20 27 24 26 7 3 1 8 1 1 1 2 8 1 4 1 1 2 2 4 9 1 0. 5 2 1 0.3 1 4 10 0.5 0. 4 13 1 0.4 2 7 11 - - - - 5 1 4 12 - - - - 39 36 40 A - - - 15 - - - B - - - 16 - — _ C - - - 3 — _ _ D - - - 10 — _ _ The microbial hydrocarbon biodegradation potential measurements showed that following the AMOCO CADIZ oil spillage, indigenous microbial populations in the sediment at all sampling sites were capable of degrading both aliphatic and aromatic components of crude oil (Tables 4-8). The variability in the results is not indicated in these tables, but the standard error was less than 4% for the percentage degraded and less than 10% for the percentage mineralized in all cases. The biodegradation potentials indicated that n-alkanes were preferentially degraded and that pristane was degraded at approximately half the rate of n-hexadecane. For aliphatic hydrocarbons approximately 30% of the amount of hydrocarbon biodegraded was converted to C0„ (mineralized) . Methodological difficulties in handling naphthalene made it difficult to assess the true extent of biodegradation for this compound. It is apparent, though, that the indigenous microbial populations were capable of degrading light aromatic hydrocarbons. The rates of degradation of the 3- and 4-ringed polynuclear aromatic hydrocarbons were lower than for branched and straight chained aliphatic hydrocarbons. In the case of the polynuclear aromatic hydrocarbons, a very low proportion of the amount of hydrocarbon degraded was converted to CO . TABLE 4. Hexadecane biodegradation showing % degraded and (% mineralized). Site 12-78 3-79 8-79 11-79 3-80 7-80 6-81 1 40 41 21 17 10 25 17 10 (8) (11) (1) (15) (2) (12) (10) 43 38 26 22 25 19 6 (ID (13) (8) (13) (17) (12) (7) 45 46 29 23 51 19 33 (15) (15) (8) (18) (39) (14) (26) 36 48 21 25 8 26 17 (14) (13) (7) (14) (6) (19) (10) 42 46 25 35 32 24 23 (14) (14) (13) (14) (20) (18) (15) 34 47 29 26 36 18 20 (11) (12) (11) (20) (18) (11) (13) 31 45 13 31 2 20 28 (10) (13) (3) (21) (1) (14) (19) 40 43 21 35 3 17 34 (15) (11) (5) (19) (2) (12) (20) 28 32 22 35 7 27 34 (12) (3) (3) (14) (3) (15) (24) 37 30 21 45 8 22 25 (10) (3) (10) (32) (3) (14) (17) TABLE 5. Pristane biodegradation showing % degraded and (% mineralized). Site 12-78 3-79 8-79 11-79 3-80 7-80 6-81 1 18 22 12 17 18 17 27 (3) (3) (1) (3) (2) (3) (3) 2 23 22 16 15 17 24 24 (3) (4) (3) (5) (3) (3) (1) 3 19 21 16 14 18 20 24 (2) (4) (3) (5) (5) (3) (6) 4 26 23 16 18 17 19 20 (3) (4) (2) (4) (1) (3) (2) 5 21 28 21 17 16 22 25 (3) (6) (4) (5) (4) (3) (6) 6 21 30 19 19 17 20 21 (3) (6) (3) (5) (4) (4) (3) 7 25 24 16 25 12 23 23 (3) (4) (1) (4) (1) (2) (4) 8 31 23 21 18 20 21 23 (3) (4) (1) (5) (1) (2) (4) 9 27 20 22 19 17 22 24 (3) (2) (1) (5) (2) (2) (7) 0 29 - 21 20 18 21 20 (2) (-) (3) (5) (2) (2) (8) TABLE 6. Biodegradation of naphthalene showing % degraded and (% mineralized). Site 3-79 8-79 11-79 3-80 1 3(2) 2(1 I 3(31) KD 2 9(7) 5(3 ) 2(2) 2(2) 3 12(10) 5(3 > 2(2) 6(6) 4 7(6) Kl ) 5(5) KD 5 8(6) Id ) 7(7) 7(7) 6 11(10) Kl 1 KD 2(2) 7 10(9) 1(1 > 6(6) KD 8 9(7) 1(1 > 7(7) KD 9 HI) 2(1] 1 KD KD 0 - 2(1 1 KD KD TABLE 7. Biodegradation of 9-methylanthracene showing % degradation and (% mineralization). Site 3-79 8-79 11-79 3-80 7-80 6-81 1 10 - 1 5 6 9 (0) (0) (0) (0) (0) (0) 2 19 8 6 8 10 9 (0) (0) (0) (0) (0) (0) 3 18 18 6 7 7 10 (0) (0) (0) (0) (0) (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 (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. Biodegradation of benzanthracene showing % degradation and (% mineralization). Site 12-78 3-79 8-79 11-79 3-80 7-80 6-81 1 5 21 18 2 5 10 13 10 (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 2 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 biodegradation potentials, it can be stated that biodegradation 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 1980, 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) . Site 12-78 3-79 8-79 11-79 3-80 7-80 6-81 3 h 4 E' 2 5 ' 2 6 f . 1 i\ 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 2 10 f 2 12 fl r2 A f , - - 102 t - - - 88 B f , - - - 210 f* - - - 210 C f - 13 f - - - 21 D f - 21 t\ - - 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 biodegradation (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 the 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 phy tane 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 I 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 44 115 331 154 39 18200 111 16 0 11 18 0 4 19800 11 17 65 40 169 68 18 22000 93 pristane 27 38 160 1100 6 19800 46 18 8 9 8 181 5 22600 10 phvcane 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: _ 1.5 2.5 0.3 3.2 1.3 3.5 isoprenoids pristane: 0.4 0.8 5.1 7.3 0.4 0.8 3.0 phytane TABLE 12. Hydrocarbon conce intration ng/g. SITE 3 C-tf 12-78 3-79 8-79 11-79 3-80 7-80 6-81 14 19 _ 3525 1040 1390 - - 15 - - 1 1025 0 633 - - 16 17 121 9350 0 400 - - 17 32 12 9550 3440 200 - - pristane 213 809 145150 47900 104 - - 18 48 86 20250 3600 106 - - phytane 985 3088 275900 130000 673 - - 19 255 948 63075 29200 283 825 - 20 149 247 36000 11900 0 - 1270 21 31 24 1 17975 6210 508 2103 2380 22 25 12 8875 1440 130 - 3050 23 38 56 11100 0 0 - 3280 24 54 48 7925 3510 0 - 4640 25 _ - 12600 21300 2340 - 5700 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 pristane: 0.5 0.3 0.5 0.4 0.2 - 1.9 phytane 13 TABLE 13. Hydrocarbon concentration ng/g . SITE 4 C-D 12-78 3-79 8-79 1 1-79 3-80 7-80 6-81 14 0 0 0 0 - 1 i 15 0 -> 0 0 3 13 8 16 0 2 0 0 5 8 7 17 18 4 4 4 12 15 8 pristane 60 6 0 2 3 4 3 18 37 3 C 2 10 6 8 phvtane 153 18 0 8 8 6 11 19" b8 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 1 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 30 43 1 30 1 50 2 12 alkanes : 0.2 0.7 _ 0.6 2.8 4.1 2.3 isoprenoids pristane: 0.4 0.4 _ _ 0.4 0.6 0.3 phytane TABLE 14. Hydrocarbon concentration ng/g. SITE 5 C-lt 12-78 3-79 8-79 11-79 3-80 7-80 6-81 14 19 37 2 16 - 5 3 15 20 65 167 59 1 1 35 29 16 - - 18 0 - 12 8 17 25 1 12 122 0 95 18 585 pristane 24 150 11 885 8 175 108 18 16 50 17 0 4 2 4 phvtane 158 293 43 158 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 2 2 39 18 19 14 6 18 1 1 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 17 113 130 86 122 30 17 57 80 10 20 0 23 alkanes: 0. 3 isoprenoids pristane: phy tane 0.2 0.5 0.5 5. 1 0.3 0.3 12.0 5.5 2.7 0.7 4.4 5.4 14 TABLE 15. Hydrocarbon concentration ng/g. SITE 6 C-ll 12-78 3-79 8-79 11-79 3-80 7-80 6-81 14 _ 82 0 10 _ 4 T 15 8 61 28 111 68 28 30 16 - - 0 0 - 9 9 17 23 1 17 14 164 200 6 101 pristane 102 125 15 71 70 0 12 18 - 8 0 28 - 6 7 phy tane 360 489 135 289 225 23 i n 19 101 121 20 20 - 0 15 20 35 12 0 42 - 2 10 ?1 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 29 159 319 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. Hydrocarbon concentration ng/g. site 7 C-lt 12-78 3-79 8-79 11-79 3-80 7-80 6-81 14 9 4 9 0 1 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 p r i s t .1 n e 154 24 102 25 47 35 7 18 121 33 113 36 39 41 64 phvtnne 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 61 18 80 46 47 24 54 25 1 1 1 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 1 12 36 356 152 29 37 29 nlkanes: 0.6 0.7 1.0 1.4 1.1 1.6 3.3 isoprenoids pristane: 0.6 0.4 0.6 0.6 0.6 0.8 0.2 phytane 15 TABLE 17. Hydrocarbon concentration ng/g. SITE 8 C-« 12-7S 3-79 8-79 11-79 3-80 7-80 6-81 24-8 12 73 7 21 80 29 64 108 40 46 42 29 27 30 36 21 25 28 22 11 33 10 218 14 29 alkanes: 0.7 0.3 1.1 1.1 1.0 1.1 1.2 isoprenoids pristane: 0.5 0.4 0.8 0.8 0.6 0.7 2.3 phytane TABLE 18. Hydrocarbon concentration ng/g. SITE 9 C-ll 12-78 3-79 8-79 11-79 3-80 7-80 6-81 14 160 34 16 15 368 77 66 16 518 87 47 17 640 113 80 pristane 941 427 98 18 818 219 70 phytane 1839 955 116 19 112? 352 85 20 849 203 81 21 530 121 62 22 430 135 50 23 314 85 47 24 272 85 42 25 54 190 52 26 489 234 33 27 328 197 71 28 431 292 24 29 362 326 15 30 315 411 170 73 22 23 104 44 31 135 41 38 108 50 34 171 74 54 158 57 54 97 51 35 66 44 28 64 38 24 59 34 32 68 30 43 42 56 18 46 28 19 31 26 41 19 11 12 30 63 27 14 0 0 0 0 - 28 - 15 0 0 3 8 - 42 - 16 0 0 3 3 - 21 - 17 3 11 5 22 8 36 19 pristane 1 4 6 2 - 0 1 18 3 6 3 5 6 9 9 phytane 2 4 L 3 - 0 5 19 4 3 4 4 8 6 12 20 4 3 0 6 11 6 12 21 4 6 4 6 11 4 15 22 4 7 3 5 9 6 12 23 4 9 3 10 8 7 19 24 4 e 2 5 4 4 15 25 "7 13 8 15 7 62 29 26 8 23 1 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 pristane: 0.6 0.2 2.6 0.9 - phytane 16 TABLE 19 Hydrocarb on concentration ng/q. SITE 10 c-# 12-78 3-79 8-79 11-79 3-80 7-80 6-81 1A 0 1 2 _ _ 25 7 15 0 5 33 3 - A2 7A7 16 1 5 29 3 - 19 17 17 3 11 53 1A 8 Al 228 pristane 1 3 7 3 - 7 A 18 3 7 9 10 A 8 10 pliytane 2 0 A 2 - 2 5 19 3 11 5 6 1 7 11 20 A 8 A 6 3 6 8 21 3 A 5 6 A 2A6 1A 22 2 10 5 5 3 6 15 23 2 8 7 7 A 10 31 2A 1 A 5 5 9 1 21 25 3 lib 13 2A 7 55 76 26 2 13 6 7 2 2 33 27 1 11 1 1 21 5 31 137 28 1 11 8 7 5 7 23 29 0 5 38 33 1A 53 19A 30 0 3 23 3 - A 3A alkanes: 1.9 - 9.0 - - A.O 63.6 isoprenoids pristar.e: 0.5 - 1.9 - - 3.5 0.8 phytane TABLE 20. Hydrocarbon concentration ng/g. C-» 3-80 7-80 6-81 IA 605000 73200 69 15 661000 88200 285 16 625000 89100 83 17 636000 93600 110 pristane 3A2000 66900 A18 18 6A3000 110700 76 phytane 231000 53A00 372 19 629000 129000 80 20 680000 142000 128 21 72AC00 132000 122 22 719000 1A1000 IA1 23 719000 1A2000 196 2A 72A000 1A0000 209 25 685000 133000 202 26 718000 155000 22A 27 669000 167000 207 28 62/000 192000 108 29 620000 168000 152 ?0 A79000 190000 318 Alkanes: Isoprenoids 0.2 2. A 0.3 Pristane:Phytane 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 biodegradation experiments confirmed the fact that the indigenous microbial populations were capable of rapid and extensive degradation of Arabian crude oil. Much greater rates of biodegradation 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 biodegradation. Rates of biodegradation 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 biodegradation. The similarity of changes, observed in the composition of the hydrocarbon mixture i_n vitro compared to the analysis of field samples also suggests that nutrients were not a limiting factor that determined the rates of hydrocarbon biodegradation. The analysis of the polar fractions from the iri 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 biodegradation since less CO was being produced than from aliphatic biodegradation 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 biodegradation products of aliphatic hydrocarbons, especially as C.,-C „ acids. Interestingly, the major polar products included unsaturated acids. As a rule, the predominant biochemical pathway for the biodegradation 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 TTTl n rrffl KMIIMCi WOU^I ! I .Dm. FIGURE 2. Changes in the relative concentrations of aliphatic hydrocarbons at sites 3, 5, and 7. 19 rrtnrert yrenranrrnrrr -t ■ "* Q. «rt(" W«*CH NOvCWBER >'9 JL HE '.H»NCJf NOVEMBER 19'9 nnJ NCCCCPCCCCDCCC NCCCCpCCCCOCCC nccccpccccdccc 1234 1234 1J3 1JJ* 1 J 3 4 1 I J l J 3 « i 2 3 4 « 2 3 N4PHTHA PHENAN DIBENiO NAPNtHA PhENAN OlBEN/O NAPHTHA PhENAN 0«BENJO 1 t NE fMHENE fMiQPMENE iCHt TmRENE ImiOPmENC LENC MRENE THlOPHENE FIGURE 3. Changes in the relative concentrations of aromatic hydrocarbons at sites 3, 5, and 7. 20 5- 3- o z o UJ >4 UJ - cc FLOW THROUGH 2 WEEKS SITE 7 ■ I 2- FLOW THROUGH 6 WEEKS SITE 7 ll - 3 FLOW THROUGH 2 WEEKS SITE 6 ll 4 WEEKS SITE 6 ll II 1213 14 15I6WPRI8PHI9 20 21 22232425262/28 M 1? 1.1 14 IS lb i; PR 18 PH 19 20 21 22 23 24 25 26 2/ 28 FIGURE 4. (Lett column) Changes in the reLative concent rations of aliphatic hydrocarbons in f low-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 < cr O FLOW THROUGH 3 - SITE 6 2 WEEKS 2- 1 o o i-| N C,C, CjP C,C2C3D C,C?C3 NNN PPPBDDD T B B B T T T 1 r 4 WEEKS 1 n r- 6 WEEKS < 3- _l 111 K 2" 1 - r f -r-Th FLASK 0 TIME SITE 7 a. rmd] rT"rfTTtf^ J II 12 13 14 IS 16 w PR 18 PM 19 20 21 7 2 23 24 25 26 2 7 28 FIGURE 6. (Left column) Changes in the 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 4. 3_ 2- z o t- 1 _ < K FLASK O TIME SITE 6 Ul o z o 1_ o 2 WEEKS £1 on 12 1 □ FLASK SITE 6 JJ Mi-m-L 1_ 4 WEEKS i I I n-i-n-r-i idl jD Ha 5.1 n 4ppml _d i. j 6 WEEKS x£H 11 12 13 14 15 16 17 PR 18 Ph 19 20 21 22 23 24 25 26 27 28 N C,C, CjP C,C, C,D C, C,C, F C, NNN PPPBDDD F T B B B TIT FIGURE 8. (Left column) Changes in the 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 methyloctahydrophenanthrene- carboxylic acid (tent.) 15.5 25.5 42.2 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 lie 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 i_n 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 biodegradation of the aliphatic hydrocarbon fraction. The hydrocarbon biodegradation 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. Riviere(2) (1) Institut Francais du Petrole - Direction de Recherche "Environnement et Biologie Petroliere" 1 et 4 avenue de Bois-Preau - 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 biodegradation 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 biode- 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 K0H, 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 jj) 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 C-AS sr\ RgSVOVALQl '"•[KrX>RCCA^^;0"<; LIQUID 5AK?lE CCNTftit-'JGAriOH + FILTKATIOM fC.12f^ J phase" LIQUID ^MPL£ £>:tkact(onCCmc'j^ r- / H'/D*OCflR30N PHASE V EVAT^oRATioH organic CAP. 15 ON KL'GICOAL E KcSlDU/.L H;T>KO-.,^.7..0fi.- ?.ICMASS v/A£,t:iri& I .-J prr 4/ WfiwHTj eXTRACTION AS PH ALT ENS S ^.rASFHALTeHES I I ' THIN LAVCR CHRGMftTOGP-ATrfY OR LIQUID 0,R<"*v\A*-OG^AVHY r.Ar'j?.AVE75 HY-D&/ CAP.2>r>N> MASS SPeCTROMf T<7 J'--^. CMSO-.AroSM'V IS, 1 o«CL .wr.cLMs KtcrrJiMjrfcy p ft.';MYi"S ANAl/CiO i 1 I C .< N o I Fl SURE 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 biodegradation times. These losses are likely 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 biodegradation 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, 13C 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. Biodegradation 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-1, 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 biodegradation, 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 of aromatic compounds is accompanied by an enrichment of the aqueous phase in organic carbon, the concentration of which may reach 250 mg.l--'-. This observation tends to show that a large part of the aromatics are 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- tely comparable, demonstrating total insensitivity of these substances to biochemical processes. The determination of n-alkanes (C14-C35) and detectable isoprenoids (C15-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 biodegradation rates of 44 and 47 %. Proton NMR analyses giving the CH3/CH2 ratio, conducted on the satu- rates, failed to indicate any significant difference between the start and end of the batch culture. With respect to the "aromatics" fraction, the action of microorganisms mainly affected the mono- and di-aromatic compounds. At the end of the culture, all the mono- and di-aromatics with 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 the culture, and the benzocycloparaf f ins , with a consumption rate of 46.2 %. The differences measured for benzodicycloparaf f ins were not sufficiently wide to be meaningful. As for di-aromatic compounds, the microorganisms displayed a very clear effect 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. Apart from those with a rough formula CnH2n-10S, the sulfur-containing compounds were not attacked by bacteria. The aromatics/sulfur-compounds ratio of 0.98 before biodegradation decreased to 0.82 after biodegra- dation, showing that it was mainly the non-sulfur-containing aromatics (mono- and di-) that disappeared. In 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- tances. 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 %) biodegradation. With respect to the resins, part of the polar compounds of this frac- tion formed during biodegradation 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 esterif ication (BF3-CHgOH) 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 CHpClp, 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 esterif ication 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 biodegradation 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 biodegradation 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 biodegradation 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 that these strains are per- fectly adapted to the hydrocarbons present at that particular time in the culture and, being dominant, they therefore naturally and preferen- tially consumed the hydrocarbons of the "saturates" fraction, as these types of constituents were biodegraded during this period. At the end of the culture, however, when the "aromatics" fraction was attacked by the microorganisms, only the Pseudomonas strains were dominant. This investigation again confirms that, to observe a significant degradation of hydrocarbons in a crude oil containing a wide variety of compounds, a mixed culture of bacteria is certainly more effective than a pure bacteria, of which the metabolism is only adapted to a given type of constituent. 1.4. Toxicity analysis of oxidation products. During the different ALC 240+ crude oil biodegradation experiments, we always observed a substantial rise in total organic carbon (TOC) concentration in the aqueous phase with the passage of time, with a regular final concentration around 200 mg.l . We decided to evaluate the potential toxicity of these solubilized products in the aqueous phase, enriched mainly in aromatics and oxi- dation products of certain hydrocarbons present in the crude oil. In particular, the mutagenicity of two samples was determined by the Ames test, the procedure of which is described in detail in Mutation Research 1975, 31, pp. 347-364. The first sample was taken at the start of the culture (with a TOC of 30 mg.l--1-), and the second at the end of the batch culture (sample with a TOC of 210 mg.l-1). The correlation between carcinogenic properties and mutagenic properties of 300 compounds was pointed out in Proc . Natl. Acad. Sci., (USA), 1975, 72, pp. 5135-5139. The principle of the Ames test is to measure the mutagenic properties of compounds that may be carcinogenic in Salmonella bacteria. The two samples were tested in a range from 0.1 to 500 jul on three of the five strains used in the Ames test (TA.1538, TA.98 and TA.100) in order to detect the mutagenicity of products such as HAP, for example. No mutagenic activity was detected in these two samples. It was shown finally that neither of these two samples had any toxic effect on the three strains tested (TA.1538, TA.98 and TA.100). 1.5. Study of the biodegradation of a mixture of pure hydrocarbons. The mixture of pure hydrocarbons consisted of two n-alkanes, hexadecane and octacosane, one isoprenoid, pristane, a two-ring naphtene, decaline, two mono-aromatics , p-cymene and dodecylbenzene, one di-aromatic, dimethyl-naphtalene , one tri-aromatic , phenanthrene, and two sulfur- containing aromatics, 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 biodegradation process of total hydrocarbons perfectly matched the microorganism growth pattern. After 61 Y2 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 octacosane, and the dodecylbenzene were consumed first. For these three products, which practically disappeared by the end of the batch, their respective biodegradation rates after 37 Y2 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 biodegradation of given quantities of hydrocarbons present in ALC 240+ . The following operating conditions were used: • 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 ( C 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 Variability 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 biodegradation) , 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, Courtot) : 1978-1979 3 - Chemical Composition, Weathering, and Concentrations in v 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/methanol 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^g to C32) and isoprenoid (C15 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 C13 - C19 range: ArK/Tsn = n ~ C14 + "-Cis + n-Ci e. + n C, 7 + n-Ci a ' 1380 + 1450 + 1650 + 1710 + 1812a aGC retention indices of isoprenoids: 1450 = farnesane, 1710 = pristane, 1812 = phytane. This ratio, beginning at ^7 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-c31 range, is defined as follows: 2(n-C?7 + n-Coq) CPI = n-C26 '+ 2n-C2Q + n-C30 38 3«dim«nt Ducard Stdimtnt 5*fci« Vlatnanoi Wijn Cn«d Sadiment Methanol 1 1 1 SOq in Teflon ;ar or ctntrifuge :uoa 2) Internal stjnaaraj 3)CH30H,CH2C2 11:9) '41 rlutfl WfN^ «SI Shake it amoient -.emotrature 40 hours .vitri aoivent cnanoa after 16 ana 24 iourj CH3OH/CH2C2 ixtractl (1) NaC! saturated) (2) Aodifv v»jXU>*'^> Ai t 9*.C*G»*Gi.NC 'S*t^'»<« H»0"*«*'W«P 1 llllfc Wj FIGURE 3.1. Weathering patterns of saturated hydrocarbons in AMOCO CADIZ oil. 44 A REFERENCE MOUSSE lAromji.cii , * * W«*Jw»','-tW''*"* STAGE 1 WEATHERING iAfomanci) Alkti : DBT J" • ^Ji'VWWvv H, C STAGE 2 FEATHERING .Aromit.cn ^ >**** V y 0 STAGE 3 FEATHERING lAiomarcil ..>' ^ WP / jijilW/rtw'W / C PYPOLYTlC PAH SOURCE lAfOPULCi 1 lilt piW,,i; PV-PY'«n« 3A«8tnz»ntn'in«na CHV-Chry«ft aF-Benio'iucant^'ef1* SEP 9AP"8enioovef,«i 3Ber'"6enioo«'v>en« 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 ('^04M -* rtrfr^^-rrVrV^^1^ 1 h WJCJ ,^^VVV( MM III I l ri mil Vt FIGURE 3.7. Surface sediment ' sampling locations (Atlas) TABLE 5. AMOCO CADIZ chemistry program; surface sediments (Atlas). Frequency April 1978-October 1978 (Caldec) December 1978 March 1979 July 1979 November 1979 March 1980 May 1981 Total 20 10 12 15 11 12 80 cations Ten Primary Stations 1,2,3 He Grande 4 St. Michel-en-Greve 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 a •BIOGENIC P • PYROGENlC iCHRONlCl AC - AMOCO OIL J I I I L_ wOh TANlO' _l I I I I I L_ STATION J •lllX •:oj* • 17X ILE GRAND C'cEO' J9« 4C 0900 a •BIOGENIC 8 000 P • PvboGENiC iCHftONiCi AC • AMOCO O'L 9 000 AC 4000 2000 1 1 1 1 I I 1 1 I 1 1 I 1 1 i STATION t ST M.CMEL £S GREvE ~ :oo Il »60 _ .AC '» : 30 - j AC \ ac a p aC 40 i i I P^^ 1 ' i 1 1 AC 1 , . Ill • PVROGENIC iCHBONiCi ac ■ AMOCO OIL J I I I L 12 TS J,T9 8.79 1 1 79 3 80 980 6.8) Trez Hir sediment time series. AC ,F /' Mm ! j , , i.i * < ill J l« 1 J! J. ■ •■i. I. k 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 5? cr/x 1 56 0.57 2 113 0.08 3 1,000 0.52 4 135 0.60 5 401 0.01 6 217 0.06 7 159 0.10 8 358 0. 21 9 18 0.71 10 11 0.94 2 130 nq g -t-t- -t-T- t f » J L FIGURE 3.14. Aromatic hydrocarbons, station 3, December 1978; normalized to C3DBT. (A = napthelenes, B = CiN, C = C:N, D = C3N, E = C4N, 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 = CiDBT, 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 11 OOOng g t— i — i — i t I i — I » I I i t , i t i — i i t r i i » r t i t f ABCDEFQHIJKLMNOPORSTUVWXrZM 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 -I — i i i i i i i i — i — i — I t ! t — r—r 1 i i i i i ABCDEFGHIJKIMNOPQRSTUVWXYZ J L 77ng ^ i i i i — i — i— i — i — i — i — i — i i i — r— i — i — I — r ABCOEFGH I J K L M N 0 P 0 R S T Li V w « I Z t t I I I J L 5346 4 ng g * t i * » i — i — i t t t t I f I — i t r I — i — i — i — i — i — r . • BCOEFGH IJKLMNOPQRSTUVWXTZM J L 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 4.160 r>g g r t t f f i t f f t t i i i i ■ i i CDEFGH1JKLMNOPORSTUVWKYZAA I f M J L 480 ng g t 1 t i t t t I t t i — I I I l — l — l — r ABCDEFGHIJKLMNOPOflSTUVWXrZM J L ffftfi tff -M- T— f" — I 1 I I I 1 P ABCDEFGMijKl-MNOPQflSTUVWXYZA* 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 ti I hn I ii I 94 n,, , ABCOEFGHIJKLMNOPQBSTUV* I 1 I II II IL | 460 fig g i i i i i — i — i — i — i — i f 1 t f f i t | | — f— M — i T 1 — I— f ABCOEFGHIJKLMNOPORSTUVWXrZU I 1 I II II II | t t T T-+ BCOEFGHIJKLMNOPORSTUVWXTZ J L 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.) 58 140 ng* -I— I 1 I I I I 1 I I I I I I I I I I I ' ABC0EFGMIJKLMNOPQRSTUVWXYZAA C30BT.280n9g W- +-r Ibcoefghijklmnoporstuvwxtz I I I II II II 1 C3OBT 1 130 rig g 1 t \ t t 1 I 1 t \ t t I t t t t 1 I ABCDEFGHIJKLMNOPQRSTUWWXYZAA I I I II II II 1 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 C30BT:177ng g 1 * ? » I — I — I I I I 1 I I 1 I — I » 1 I 1 T I I I I I I ABCDEFGHIJKLMNOPQRSTUVWirZAA J I IL C3DBT:75ng 8 I I I i I — r— i — i — i i I f f I I I f ABCDEFGHIJKIMNOPORSTUVWKYZ 4— r-f J L C30BT: ling g i i i — i — i— i — ii i i r t t r i — i i i r r f , f f i i — r ABCOEFGH IJ K LMNOPQRSTUVWXT 2W I I I II II II 1 FIGURE 3.30. (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 . i i i i i i i i — i i i i i — i i I — r t r T i T — i — i — p ABCOEFGM ijk LMNOPOBSTUVWXY zm I I I II II II 1 FIGURE 3.33. Aromatic hydrocarbons, station 7, June 1981; normalized to pyrene. (See Figure 3.14 for key.) JjJHA A A SATURATES ... n|L.. *{!*<•> ,illL jp i?.!ii^ H *\ £** B ABOMATICS 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 lie 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 lie 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 Fiqure 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 appear 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, CL, C2, 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. (J.J $1 (J.) ■}) AC100 AC 36 9 AC426 AC10 3 AC 365 AC4 29 AC42 AC138 AC371 AC4 53 AC56 AC139 AC 381 AC458A AC458B AC127 AC 370 AC4 52 ACL 3 2 AC362 AC141 AC 396 AC4 3 2 AC13 4 AC451 AC44 ACL21 AC 384 AC112 AC389 AC445 AC10 7 AC376 AC436 AC53 ACU8 AC377 AC4 38 AC51 ACU4 AC379 AC440 AC48 AC125 AC 378 AC479 4/5B 84.1 ] 4 90.4 3. 5a 252 .9 3 3,172.5 4/5B 35.5 3 133.6 4,'2/SB 200.6 4'2 39 7.0 4/5B 107.9 4/2 50 3.4 5B'4 37.3 5/3 31.6 1 109.0 2'1 121.7 2/4 408.8 2/4 502.8 4 '2 19.4 2/4 97. 7 2/4 142.5 2/4 16.0 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 18.6 3/4/2 48.8 2/4 13.8 4 41.5 2/4 165.8 4/2 211.7 2/4 24.5 4/2 44. 7 2 15.6 2 39.2 4/5B 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 I 38.5 2 69.1 2 17.5 2 44.8 2 32.1 2/1 173.6 2/4 122.5 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 15.8 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 17.9 AC40 and 50 series sampled 4/ AC100 series sampled 7/78 AC300 series sampled 11/78 AC400 series sampled 2/79 L'Aher Benoit Sediments: ABT ACC AB25 AB29 AB16 AB21 AB4 2 158.2 180.8 4 25.1 1 29. 3 2 29.2 29.7 4 11.6 < 17.5 2 22.2 28.6 2 455.1 440. 7 2 7J.6 76.9 64 TABLE 9. Terenez/Morlaix time series (s'tation A) ALIPHATICS Oiq/1) AROMATICS (p9/g) AC 42 AC 138 AC 371 AC 453 4/78 7/78 11/78 2/79 109.0 408.8 19.4 142. 5 6.1 11.7 1.3 2.8 0.79 0.10 0.10 0.51 123.7 502.8 97.7 36.0 7.4 17.2 2.4 1.6 ac 42 AC 138 AC 371 AC 453 nd 2.9 Cl« 10.3 6.3 C2H ClP 40.9 62.5 115.5 423.2 161.0 730.7 16.8 nd nd nd 20.1 37.4 nd 1.0 54.3 230.2 4.4 7.8 198.9 905.8 88.0 14.8 125.7 20.4 7.4 5.2 95.4 151.4 9.0 5.9 C3p C4P 15 3. 3 593.5 12.4 15.0 274.8 138.3 1678.9 882.9 41.9 27.6 22.0 21.9 DBT CiDBT C2DBT CjDBT B(e)P B(j)P AC 42 AC 138 AC 371 AC 453 19.2 12.4 nd 2.3 113.6 383.9 4.0 6.7 714.4 3363.0 54.8 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 9.6 298.9 120.8 20.9 9.1 83.5 87.2 9.8 6.0 3.7 3.4 24.5 20.3 1.8 1.5 FEYi nd - none detected. N - napthalene, C^-C^N - alkylated naphthalenes, P • fluorene, C^P-CjP • alkylated Fluorenes, P • phenanthrene. C1-C4P • alkylated phenanthrenea, DBT - Dlbenzoth lophene, CjDBT-C2DBT • alkylated dlbenzoth lophenes , Fl ■ Eluoranthene , PYB ■ pytene, CHRY - Cryaene, BP - benzof luotanthene, B(e)P ■ Benzo (e> pyrene, Bta)P • Benzot a) pyrene, PERL - perylene. TABLE 10. Morlaix time series (station B) DATE ALIPHATICS (U9/g] ALK/ISO AROMATICS ((»j/9> SAMPLE TOTAL RESOLVED TOTAL RESOLVED AC 103 7/78 200.6 12 6 2.5 397.0 7.4 AC 165 11/78 107.9 5 . 3 5.0 50 3.0 7.5 AC 429 2/79 37.3 2 0 0.02 31.6 11.9 N V C2N C]N C4N F Cl" C2F C3F 301.0 P V V C1P V AC 103 17 18.9 48.6 135.9 220.7 16. 3 23.8 72.8 117.8 95.4 160.1 312.5 273.9 AC 365 9 14.4 33.8 55.0 111.2 9.9 14.9 36.9 81.1 114.2 65.3 58.9 14 3.3 101.6 AC 429 6 10.0 19.7 28.0 50. 2 16.8 11.6 23. 3 7.4 185.6 108.6 PERL 52.6 26.0 34.9 DBT C DBT C2DBT C DBT FL PYR CHRY BF B(e)P BlalP AC 103 15.2 100.8 772. 1 1 348 240.2 201.8 345.2 350.7 146.4 166. 1 87.0 AC 365 11.2 32.7 215.2 440. 3 202.2 175.6 254.0 324.6 115. 0 110.9 65.4 AC 429 11.3 7.7 10.3 5.0 125.9 302.9 207.4 202.5 112.5 115.0 17.7 KEY: nd • none detected. N - napthalene, Cl-C4H = alkylated naphthalenes, F - fluorene, C1F-C3F - alkylated fluorenes, P <-l~L41' " alkylated ph^nanthrenes , f luoranthene, PYR * pyrene, CHRY PERL - perylene. phonanthrene , DBT - Dihenzothlophene, CjDBT-C^D alkylated d ib«*nzoth lophenes , Fl - Crysene, BF = benzof luoranthene , B(e)P ■ Denzo ( e) pyren*> , BlalP " Benzo) a) pyrene , 65 TABLE 11. St. Michel en Greve/Lannion time series (station C) ALIPHATICS 1)19/9) TOTAL RESOLVE! AROMATICS (iaj/91 AC 14 4/18 38.5 1. 9 0.38 61 1 8.6 AC 121 7/78 17.5 0 2 0.09 44 3 0.6 AC 384 11/78 32.1 0. 5 0.14 173 .6 1.6 44 N Cl" C2N C3H c4» F V V v P cxp V C3P V AC 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 80.6 76.6 36.4 EST C DBT C DBT C DBT PL PYR CURY BP B y p..^. u 11 1 1 Hi.. , Ail* V K.\M.\ FIGURE 3.40. Saturated hydrocarbons in sediments from oiled and control sites. 72 (A| <>ii«i Estuary Mu.ilidt I S Inlmvl ^l.i-la.l J IC) Oilwl '-..ill Maul. M1..111..1 IUhU 1J-" tEl Oilwl Bej.li IBI Control Esiujty MihIIIaI 1 jJppJUilJUUL. , (Dl Cnnlrol Sjll Ma.sJi Mu.lll.il L...jjwI t F J Ciit.litil Uracil m*&*\ iHUm M, : -00-, id AMOCO CA0I2 O'L „, ^•••'•«ct Mount t 30-161 n T AflE3 .V9ACM Aor-i 27 1978 !0 Ik N CiCjC3C4 » C,CjC]Ci 0 C,C;Cj -1 I I I P>i*n*nfftr«n*t O'0»«*»- i-m-Hiiun IL6 GSANDE 'SOUTH Mire* 1979 0-Scm n M » CCjCjC, • C,C;CiCi r> C,C,Cj ' ' ' I ABEfl '.VBACM M«rcri '979 0-5 em \ N CiCjCjC* » C|C;C]Ci 0 CiCjCj I I 1 1 ^TMlil 'BOO 000 \ s s \ ' C,CjCjC« * CCJCJC4 O C,CjCj ■'Mtf'ntt^n »*.«»Aff>r.* ILE GRANDE (SOUTH Mircn 1979 15-ZOcm ns n 1 S r1 ^ Mfc 11 ABES WRaCh Mire* 19:9 10-15 cm LU : N C1C3C3C4 * CiCjCjCj O C.CiCj * C,CiC]C« » CiCjCaCi O CiCrCj FIGURE 3.41. FIGURE 3.42. (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 occEMeea 1979 WAflCM '979 -300 -300 -300 C)" ; . . AUGUST -979 NCvEWBE" '9'9 CjC »Jl«ft)i CjDBT r^ 4M0C0 CADIZ OIL PYRQGENiC BIOGENIC FIGURE 3.43. Aber Wrac ' h sediment cores. 200 400 ug g 600 800 !•[ Aromallci Salurale< f — ALK ISO |0 220 \ 20 \ \ 0 50 1.0 \ ALK ISO 4.75 1000 no g 2000 3000, 8000 DBT — 202 228 TS2 FIGURE 3.44. (Left) Aber Wrac'h core, December 1978. FIGURE 3.45. (Right) Aber Wrac'h core, December 1978. 200 400 "S ° 600 800 1000 ng g 2000 3000 Aroma lie s 10r Salurilis ALK ISO : io = S20 N- DBT- 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 10 20 r \ 10- . Saturates 0.50 Aromitics 1.0 1.50 ALK/ISO 20 400 "a " 600 800 Aromitics Sitariln- ALK ISO FIGURE 3.48. (Left) Aber Wrac'h core, August 1979. FIGURE 3.49. (Right) Aber Wrac'h core, November 1979 ALK/ISO ALK/ISO 0.50 1.0 1-50 2.0 2.60 10 = 20 1000 '"'MOO 3000 P DBT 0.50 1.0 1.50 202 228 252 2.0 200 400 "' ■ 600 800 1 \ ' -10 ^ ^ 1 ! 20 Aromitics - " ~ ALK ISO i 0.50 1.0 1.5 2.0 "■ FIGURE 3.50. FIGURE 3.51. ALK/ISO ALK/ISO (Left) Aber Wrac'h core, November 1979. (Right) Aber Wrac'h core, May 1980. DECEMBER 1978 MARCH 1979 IPHC CjP 2021^9 V .>.g. 91 C3O8T 1PHC C3P 202119 9 vq 9) C3O8T 350 310 230 FIGURE 3.52. AUGUST 1979 IPHC C3P 202lng,g> iiig/91 C3OBT 0-5 ^M 5-10 10-15 15-20 Mv1' I 100 550 100 AMOCO CADIZ OIL PYROGENIC BIOGENIC 130 170 0-2 mm ,1 -10 mm 1-2 cm 2-3 cm ^ ^ 3—1 cm 1-5 cm 5-lOcm I?MC tpg.gl 5900 3100 3 300 3 700 22000 3300 8810 C3P 202(^99) C3O8T 330 1 100 lie Grande (oiled) sedi- ment cores. 75 no g 400 600 Silaralis ALX ISO ALU ISO ALK ISO 1000 "B/» 2000 OBT ' I 202 228 2?2 200 400 *' B 600 800 Aramllci Silinlii ' ALK/ISO • 0.50 1.0 1.5 2.0 63.25 1 1— »M ' ALK/ISO 10 120 2000 4000 J|/| 8000 6000 ,15000 0.50 Annuities Silinlii ALK/ISO . 1.0 1.50 2.0 2.50 ALKISO FIGURE 3.53. FIGURE 3.54. FIGURE 3.55. FIGURE 3.56. FIGURE 3.57. (Upper left) lie Grande south core, December 1978. (Upper right) lie Grande south core, March 1979. (Middle left) lie Grande south core, March 1979. (Middle right) He Grande south core, August 1979. (Bottom) He Grande south core, May 1980. 76 FIGURE 3.58. AMC-4 sediment cores. DECEMBER 1979 IPHC 0-5 5-10 10-15 15-20 20-25 « C30BT MARCH ■■" IPMC Uig 9) 200 150 130 SO CjP C3O8T 30 38 202. ng g, 0-5 5-10 10-15 15-17 : 1 - a. 3 - v i\ 12 AUGUST 1979 |NEW .NPUTl NOVEMBER '979 0-5 III ■ ,i:;li:l 5-10 10-15 15-19 ii III: SM AMOCO CADIZ OIL PYflOGENIC BIOGENIC IPHC C3P 2021 lfl'*l IPHC C3P 202iig Sl wg g) C3OBT 33 25 a. 0-5 Wg'9» CjOBT 28 46 000 ■ ||i: . 1 1 120 32 8 800 35 2S 680 - - 1,100 - - MAY 1980 o-s 8-13 R » \> IPMC C3P 202i^g 31 lpg'91 C3O6T 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 as 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 "8/| 600 800 Arositle* Saluratas - 20 UK ISO - 1.0 1.5 2.0 ALK ISO 200 jo g 400 600 \ Aromalics - Saturates ALK ISO 0 50 1.0 150 ALK ISO ng g 1000 2000 200 400 ut ' 3000 5000. 40000 10 1 . 20- P -0BT 202 228 252 10 20 Aromalics Salurilis ALK ISO 0.50 1.0 1.50 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 A3£B 'LOUT iLE GAANDE ,NOPTh MAflC" '979 wascm 1979 IPHC C3P 202f»9-9l iu9 9) C3DBT IPHC C3P 202,^gi \uq gl C3OBT MARCH 1979 -PHC C3P 202lnq,g iitq 9) C3D8T 0-5 . 5-10 10-15 ^ 2 6 640 AMOCO CADIZ OIL PYHOGENIC BIOGENIC FIGURE 3.63. Miscellaneous sediment cores. .000 ng/0 2000 \ 10 l\ \ V - lilt \ \ *\ P- N DBT . 202 228 252 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 LUCIUtl. FIGURE 3.65. Oysters and Plaice sampling locations. TABLE 18. Petroleum hydrocarbons in oysters (Crassostrea gigas) PETROLEUM DBT 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 6/80 (#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. bSum of dibenzothiophenes and alkyl dibenzothiophenes. cSum 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 (Cn - C20) • Tne U^M 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, n-C2o ~ n~C3o) 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 C23 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 FIGURE 3.66. Aber Wrac'h impacted oysters - aromatic hydrocarbons; A December 1978; B - June 1980. t ,, |... . J» if \,k^ P JpftJ **d ■ ' i- ill 1 1 ill I FIGURE 3.67. Brest control oyster - aromatic hydrocarbons; A - December 1978; B - June 1980. 83 b c J l| I "II mi ill j lit- 1 |!''*Ui v«vn f Wj CvWluia . i i i i i i i i i i i i i i i i ; i i i i i i i i i i , i i i i i i i S id IS Si_ 52 £3 £5 2a m/e m/8 m/e Lja.i t: E f aujIl La U U 1 \K Y_^rar>* N<"\AaWV/u_ A\- 43 -4 9 ga Si S? FIGURE 3.68. Section of GC/MS total ion chromatogram of aromatic fraction of oyster sample illustrating major alkyl dibenzothiophene (DBT) components (a = CiDBT: b - C2DBT; C = C3DBT). 84 *"" -' . 'IGURE 3.69. Aber Wrac'h impacted oysters December 1978; B - June 1980. saturated hydrocarbons; A •.w» ■if, 3» lit, Km >d 5f M~.., jM JiJL^ JMI U iiiii FIGURE 3.70. Brest control oysters - saturated hydrocarbons; A ber 1978; B - June 1980. Decem- 85 -*■ I I I I ' I I I I I I I I I I I r I f I I t f f I f f f ABCDEFGH IJ K LMNOPQRSTUVWXYZU ABCDEFGH IJ K LMNOPQRSTJVWXYZU FIGURE 3.71. (Left) Aber Wrac'h, Crassostrea gigas, aromatic hydrocarbons, December 1978. (See Figure 3.14 for key.) FIGURE 3.72. (Right) Aber Wrac'h, Crassostrea gigas, aromatic hydrocar- bons, June 1980. (See Figure 3.14 for key.) TABLE 19. Summary of Plaice hydrocarbon data. HYDROCARBONS STATION DATE TISSUE (pg/3 dry wt) L'Aber Wrac'h 5/79 Muscle 90 7/79 Muscle 33 2/80 Muscle 77 6/80 Muscle 186 7/79 Liver 1,350 2/80 Liver 1,200 6/80 Liver 1,640 L'Aber Bene- it 5/7 9 Muscle 147 7/79 Muscle 17 2/80 Muscle 104 6/80 Muscle 48 7/79 Liver 1,030 2/80 Liver 1,860 6/80 Liver 2,500 Loctudy 4/79 Muscle 2/80 Muscle 41 6/80 Muscle 38 2/80 Liver 1, 300 6/80 Liver 1,900 86 I I I I I I I I A8C0EFGH IJ K LMNOPQBSTJVWXYZi Cio ■ No'""» A.jn* C -10 C|i ■ Nof-n* Xtktnt C— 1 1 etc FAR :j -titrt PBlS • ?• nana •MY asvunc 1330 '550 • 'ioO'fnO'Oi r r r i f r r i i i p p f > i i i i i ABCDEFGHIJ KLMNOPQRSTUVWXrZ AA 68 I , I I I I M I I ABCDEFGHIJ KLMNOPQRSTUVWIVZAABB 1 M > P t P M f P f I f I I f P I P f P P T P I I i I ABCDEFGHIJ KLMNOPORSTUVWXYZAABB FIGURE 3.73. (Upper left) Brest, Crassostrea gigas, aromatic hydrocarbons, December 1978. (See Figure 3.14 for key.) FIGURE 3.74. (Upper right) Aber Wrac'h, Crassostrea gigas, saturated hydro- carbons, December 1978. FIGURE 3.75. (Lower left) Aber Wrac'h, Crassostrea gigas, saturated hydro- carbons, June 1980. (See Figure 3.74 for key.) FIGURE 3.76. (Lower right) Brest, Crassostrea gigas, saturated hydrocar- bons, December 1978. (See Figure 3.74 for key.) 87 AROMA IK S & ¥^L i SATURATES 4-1 j! M FIGURE 3.77. Plaice Liver, control. AHUMAl ICS I , j. J III., j IuIm»»-» '*' •i'.1 ■»^ '.AlunAi is J; m i a M)„ , FIGURE 3.78. Plaice Liver, oil-impacted. 88 3.6 Oysters and Fish (Michel, ISTPM) An analytical chemical program in support of the early post-spill (March 1978 - March 1979) programs of the Institute Scientifique et Technique des Peches Maritimes (ISTPM) was undertaken (Tables 20 to 22 and Fig. 3.79). Samples of freeze-dried oysters and fish (various species) were analyzed by GC and several samples by GC/MS. The results of the analyses are tabulated in Tables 23 to 25. Based on the nature of the GC traces, sources of observed hydrocarbon distributions are derived: fresh AMOCO CADIZ oil, weathered oil, and biogenic hydrocar- bons. Often combined sources are apparent (e.g. weathered oil/biogenic hydrocarbons) . Two oyster time series, summarized in Table 26, indicate that initial heavy oil impacts on the tissues are reduced over time but certainly not eliminated. GC traces illustrating the change in aroma- tic hydrocarbon composition with time (Fig. 3.80) show that again the alkylated phenanthrene (P) and dibenzothiophenes (DBT) dominate the assemblage through February of 1979. Fish tissues do not reveal significant oil impacts. For the most part the hydrocarbons consist mainly of biogenic compounds (e.g. olefins) with an occasional UCM and again the presence of DBT and P compounds probably, though not definitely, related to AMOCO CADIZ oil (see Table 24) . An attempt at decontamination via oyster transplantation yielded lower levels of hydrocarbons (Table 23; sample 143) indicating that once removed from a polluted substrate the oysters can depurate their oil burden significantly. Thus the oysters exhibit similar area-wide uptake of AMOCO oil, initially at the 3000 ppm level, rapidly reduced to the 300-700 ppm level and to the 50-200 ppm level a year after the spill. However, identifiable oil residues remain. Fish samples show only sporadic uptake of any oil indicating that the oil has not significantly impac- ted coastal fish, or that once impacted the fish rapidly depurate and/or metabolize petroleum FIGURE 3.79. Oysters and fish sampling locactions. 89 TABLE 20. AMOCO CADIZ chemistry program, f reeze-dried fish and oysters (Michel, ISTPM) . Frequency March 1978 1 Oyster April 1978 1 Oyster + 1 1 Fish May 1978 1 Oyster + 6 i Fish June 1978 3 Oysters + 1 Fish July 1978 4 Oysters September 1978 4 Oysters October 1978 3 Oysters + 4 Fish November 1978 3 Oysters December 1978 4 Oysters + 5 Fish January 1979 1 Oyster February 1979 2 Ovsters March 1979 3 Oysters TOTAL 30 + 23 = 53 Locations Various TABLE 21. ISTPM oyster sample summary. No. Date Sampling Location 5 5.4. 1978 71 23.3. 1978 143 24.5. 1978 176 22.6. 1978 178 20.6. 1978 184 22.6. 1978 212 20.7. 1978 223 21.7. 1978 234 18.7. 1978 242 18.7. 1978 295 20.9. 1978 297 20.9. ,1978 311 20.9. ,1978 327 18.9, ,1978 349 19.10. .1978 357 19.10, .1978 359 19.10, ,1978 399 16.11. ,1978 400 16.11, ,1978 406 16.11, .1978 420 15.12, .1978 436 15.12, .1978 440 15.12, .1978 442 15.12. .1978 446 31.1, .1979 471 27.2, .1979 473 27.2. .1979 514 30.3, .1979 517 30.3, ,1979 518 30.3, ,1979 Aber Benoit - Prat ar Coum Baie de L' Argue non Essai de decontamination Baie de Morlaix - Penze R.G. (Le Ven) Aber Benoit - Prat ar Coum Baie de Morlaix - Calot (transfert) Aber Benoit - Prat ar Coum Baie de Morlaix - Le Frout (Le Ven) Baie de Morlaix - Penze R G (Le Ven) Baie de Morlaix - Penze (B.I. Brannelec) Baie de Morlaix - Penze R D (Cablet) Baie de Morlaix - Penze R G (Le Ven) Baie de Morlaix - Penze R D (Gallion) Aber Benoit (Garo - Hanssen) Aber Benoit (Garo - Hanssen) Baie de Morlaix - Penze R D (Kerarmel) Baie de Morlaix - (B I Brannelec) Baie de Morlaix - Penze R D (V. Bernard) Baie de Morlaix - (B I Brannelec) Baie de Morlaix - Penze (Cadoret) Baie de Morlaix - Penze R G (Le Ven) Baie de Morlaix - Penze R D (Cadoret) Baie de Morlaix - Penze R G (Vallegant) Baie de Morlaix - R D lie Noire (Kerarmel) Baie de Morlaix - Penze R D (V. Bernard) Baie de Morlaix - Penze R D (Gallion) Baie de Morlaix - Penze R D (Vallegant) Baie de Morlaix - Penze R D (Cadoret) Baie de Morlaix - Penze R D (Ker Armel) Baie de Morlaix - Penze R D (Cadoret) 90 TABLE 22. ISTPM fish sample summary, No. Nature 36-3 lieu jaune 40-7 roussette 41-3 lieu noir 58-1 lieu jaune 93-2 mulet 100 flet 118 lieu jaune 151 maquereau 170-1 lieu jaune 170-2 lieu jaune 198 tacaud 200 lieu jaune 203 maquereau 209 mulet 377-2 plie 379-3 mulet 415-2 sole 419-2 grond in 446-4 plie 449-2 sole 454-1 plie 454-6 sole 455-4 plie Date Sampling Location 13.04.1978 Portsall (3' H Amoco) 13.04.1978 Roscoff (Bank ac Forest) 13.04.1978 Portsall (8' N Amoco) 13.04.1978 Portsall (2' E Amoco) 27.04.1978 Portsall 24.04.1978 Baie de Lannion 29.04.1978 Portsall (3' £ Amoco) 23.05.1978 Baie de Douarnenez 11.05.1978 Riviere de Trequier 11.05.1978 Riviere de Trequier 16.05.1978 Baie de Lannion 16.05.1978 Baie de Lannion 3.05.1978 Baie de Lannion 30.06.1978 Aber Wrac'h 26.10.1978 lie de Batz 24.10.1978 Baie de Morlaix 6.12.1978 Baie de Lannion 6.12.1978 Baie de Lannion 15.10.1978 Baie de Morlaix 15.10.1978 Baie de Morlaix 5.12.1978 Aber Benoit 5.12.1978 Aber Benoit 20.12.1978 Aber Wrac'h TABLE 23. Results of ISTPM oyster analyses. Total Hydrocarbons Sample No.a (f, + £?; ug/g dry wt) 5 2700 71 610 143 180 176 530 178* 1600 184 380 212 270 223 400 234 640 24 2 80 295 340 297 270 311 290 327 630 349 420 357 320 359 50 399 100 400 95 406 70 420 150 436* 1000 440 90 442 150 446* 105 471 50 473 140 514 140 517 170 518 140 Source (from GC)b 1 2/1 2/3 2 2 2 2 2/3 2 2/3 2 2 2/3 2 2 2 2 2 2 2 2 2 3/2 2/3 3/2 3/2 2 2 2 2 GC/MS results available (Table 25). aSee Table 21 for location and data of each sample number. bl = fresh AMOCO CADIZ oil 2 = weathered oil 3 = biogenic hydrocarbons 91 TABLE 24. Results of ISTPM fish analyses. Sample No. 36-3 40-7 41-3 58-1 93-2 LOO 118 151* 170-1 170-2* 198 200 203 209 377-2 379-3 415-2 419-2 449-2 454-1 454-6 455-4 Tot.)L Hydrocarbons 1 r_L * (2: u'' 9 ,!.rX wt-' 15 39 29 18 45 70 45 170 18 31 31 69 37 30 8 25 154 15 13 21 20 6 r Je (_f r_om GCJ ' 3 3 3 3 2/3 3/2 3/2 3/2 3 2 3 3/2 2/3 3 3 3 3 3/2 3 3 3 * GC/MS results available (Table 25) . aSee Table 22 for location and data of each sample number. bl • fresh AMOCO CADIZ oil 2 ■ weathered oil 3 » biogenic hydrocarbons TABLE 25. GC/MS results of selected analyses of oyster and fish tissues (ng/g) . Oysters 1178 1436 1446 1151 1170-2 N nd nd nd nd 2 C,N nd nd nd nd nd C?N nd nd nd nd nd C,N 290 nd 52 nd nd C4N 1400 nd 150 nd nd N 2190 nd 202 nd 2 P 220 30 85 17 40 ClP 1400 nd 220 30 70 C?P 4600 130 350 30 60 C,P 9500 200 1300 20 50 C4P 10000 100 1300 10 40 p 25720 460 3170 107 260 DBT 180 nd 16 nd 2 C1DBT 1400 nd 100 nd 24 C2DBT 6400 320 480 nd 120 C3DBT 9600 580 1410 nd 100 DBT 17580 900 2006 nd 246 m/e 202 700 100 320 30 41 m/e 228 900 30 330 nd 20 m/e 252 500 nd 400 nd nd N ■ naphthalenes P - phenanthrenes DBT - dibenzothiophenes 202 ■ fluocanthene + pyrene 228 =• benzanthracene ♦ chrysene 25 2 - benzof luoranthenes + benzopyrenes 92 TABLE 26. Petroleum hydrocarbons in oysters. LOCATION DATE Z PETROLEUM (ppm) Aber Benoit Baie de Morlaix April 5, 1978 2,700 June 20, 1978 1,600 July 20, 1978 270 September 18, 1978 620 September 19, 1978 410 June 2, 1978 530 July 1978 70-600 October 19, 1978 60-230 February 27, 1979 240 March 30, 1979 150 FIGURE 3.80. Baie de Morlaix impacted oysters - aromatic hydrocarbons; A - 5 April 1978; B - 27 February 1979. 93 3.7 Seaweed and Sediments (Topinka, Bigelow Laboratory for Ocean Sciences) In support of an investigation on the impact of the spill on macroalgal population recovery and growth, a series of plant and adjacent sediment samples was analyzed by GC to determine if and to what extent AMOCO oil was associated with the plants (Table 27). The data presented in Table 28 in conjunction with a consideration of Figures 3.81 and 3.82 illustrate that while several of the plant samples do contain weathered oil (see Fig. 3.81) the n-alkane, penta- decane (n-C^) , is the most abundant biogenic component in all sam- ples. The distribution of biogenic components in general (Fig. 3.82) can be seen as contributing markedly to the total hydrocarbon levels even in the "oil-impacted" tissues. GC/MS results of an "oil impacted" plant's aromatic hydrocarbon fraction (Table 29) indicate that again the P and DBT family series are the most abundant aromatic compounds present. In this sample the P compounds are, in total, more abundant than the DBT series, but the C3DBT are still the most abundant group (8400 ppb) . TABLE 27. AMOCO CADIZ chemistry program; seaweed samples (Topinka). Frequency June 1979 15 Plant + 7 Sediment August 1979 2 Plant May 1980 3 Plant Summer 1980 12 Plant TOTAL 32 Locations Var ious GC/MS One Seaweed Sample 94 TABLE 28. Summary of analytical results; seaweeds, summer 1980. Gravimetr ic Sample tx (pg/g) f2 (ug/g) HC-4-1 41 HC-4-2 10 HC-4-3 11 HC-5-1 31 HC-5-2 61 HC-5-3 61 HC-5-4 11 HC-5-5 39 HC-5-6 17 HC-5-7 10 HC-5-8 12 HC-5-8 8 (Repeat) HC-5-9 74 16 14 29 72 52 12 23 16 7 9 12 15 GC n_c15 (P9/9) Status 38.5 10.7 4.8 5.0 35.0 25.0 21.3 58.0 25.3 7.2 34.0 9.0 3.7 1/2 2 2 1/2 1/2 1/2 2 2 2 2 2 2 Status codes: 1 = weathered petroleum 2 = biogenic TABLE 29. GC/MS results of seaweed aromatic fraction analysis (sample HC-5; Tregolonou, Fucus vesiculosis ; 4 June 1979) . Compound Cj-f luorene Phenanthrene (P) CjP c2p C3P C4P ip Dibenzothiophene (DBT) C^BT C2DBT C3DBT I DBT m/e 202 ( f luoranthene + pyrene) m/e 252 (benzof luoranthenes + benzopyrenes) Concentration (ng/dry weight plant) 610 420 . 920 2400 6400 5300 15,440 40 300 4000 8400 12,740 930 1800 95 p w ma. \i SATlJHA t tS FIGURE 3.81. HC-4-1 seaweed hydrocarbons - oil-impacted. IWU.i 1 1 III i 1 1 1 \ i .III w 1 , \ i i. j; l *lU I SAf UHArtb 'I l! Jjiiil'Ailni LJi ill Ml J J «ttl' A.U.I. , Ij (WjJ, .1^ -I I U AHIjMA I l( ^ FIGURE 3.82. Seaweed hydrocarbons control. 96 CONCLUSIONS A number of specific conclusions concerning the levels of AMOCO CADIZ petroleum hydrocarbons in various environmental compartments, the changing chemistry of the hydrocarbon assemblages, and the persistence of petroleum in these compartments are presented here. 1) Upon introduction into the environment, the oil weathered rapidly with evaporation and biodegradation changing the oil's chemistry markedly even prior to landfall. 2) Oil impacted a variety of intertidal sedimentary types and a number of secondary impacts were noted at many stations. 3) Oil was buried in most sedimentary environments with burial and/or penetration down to 15 cm in fine-grained sediments and deeper (^20-30 cm) in sandy sediment. 4) Oil remained less biodegraded in sandy beach environments than in fine-grained sediments in which heavily biodegraded oil was characteristic. 5) The presence of UCM material, pentacyclic triterpanes, and alkylated phenanthrene and dibenzothiophene compounds remain as characteristic chemical features of AMOCO CADIZ oil in sediments. 6) Less weathered oil appeared to be buried (10-20 cm) in fine- grained sediments as evidenced in samples taken one year after the spill. 7) Offshore sediments were impacted after the shoreline impact, probably through processes involving beaching, sorption on intertidal sediments, and offshore transport of these sedi- ments. Samples taken after the spill in April 1978 do not reveal AMOCO CADIZ oil, thus indicating a lag (weeks to months) in offshore deposition. 8) Surface intertidal sediments taken in June 1981 show that "normal" background inputs, both of biogenic and chronic pollutant origins, have replaced AMOCO CADIZ oil as major components of the hydrocarbon geochemistry. Only at the most impacted stations at lie Grande marsh and within the sandy beach sediment at AMC-4 (Portsall) do identifiable AMOCO residues persist. At lie Grande the aromatic marker compounds are absent, but hopanoid compounds (triterpanes) and a large UCM persist. 97 9) Oysters were initially heavily impacted by oil (several thousand ppm) and after two years (June 1980) traceable AMOCO CADIZ residues are still evidenced by homologous series of isoprenoid alkanes, phenanthrenes and dibenzothiophenes. Petroleum residues persist approximately at the 100 ppm level . 10) Fish do not appear to have been directly impacted (chemical- ly) by the spillage to any significant extent. 11) Compositional profiles traceable to AMOCO CADIZ oil are likely to "disappear" from all sediments within another year (i.e. 1982; four years after the spill) although this should be confirmed by direct measurements and attention to molecular marker compound distributions. REFERENCES Atlas, R. M. , P. D. Boehm, and J. A. Calder, 1981, Chemical and biolog- ical weathering of oil from the AMOCO CADIZ oil spillage, within the littoral zone: Estuarine Coastal Mar. Sci., Vol. 12, pp. 589- 608. Blumer M., M. Ehrhardt, and J. H. Jones, 1973, The environmental fate of stranded crude oil: Deep Sea Res., Vol. 20, pp. 239-259. Boehm, P. D., 1980, Gulf and Atlantic Survey (Gas I): Atlantic survey for selected pollutants: Final Report, NOAA/NMFS Contract NA-90- FA-C-00046, National Marine Fisheries Service, Sandy Hook, New Jersey. Boehm, P. D. and P. Hirtzer, 1981, Gulf and Atlantic survey (GASI) Ches- apeake Bay to Port Isabel, Texas: survey for selected organic pollutants in finfish: Draft Final Report, NOAA/NMFS, Sandy Hook, New Jersey. Boehm, P. D. , D. L. Fiest, and A. A. Elskus, 1981, Comparative weather- ing patterns of hydrocarbons from the AMOCO CADIZ oil spill observed at a variety of coastal environments: Jji AMOCO CADIZ - Fates and Effects of the Oil Spill, Proceedings of the Interna- tional Symposium, 19-22 November 1979, pp. 159-173. Boehm, P. D. , J. E. Barak, D. L. Fiest, and A. A. Elskus, 1982, A chem- ical investigation of the transport and fate of petroleum hydrocar- bons in littoral and benthic environments: the TSESIS oil spill: Marine Environ. Res., (in press). Brown, D. W. , L. S. Ramos, M. Y. Uyeda, A. J. Friedman, and W. D. Mac- Leod, Jr., 1980, Ambient temperature contamination of hydrocarbons from marine sediment - comparison with boiling solvent extractions: in L. Petrakis and F. T. Weiss (Eds.), Petroleum in the Marine Environment, Advances in Chemistry Series No. 185, American Chem- ical Society, Washington, D.C., pp. 313-326. 98 Calder, J. A. and P. D. Boehm, 1981, The chemistry of AMOCO CADIZ oil in the L'Aber Wrac'h: jji AMOCO CADIZ: Fates and Effects of the Oil Spill, Proceedings of the International Symposium, 19-22 November 1979, Brest, France, pp. 149-178. Dastillung, M. and P. Albrecht, 1976, Molecular test for oil pollution in surface sediments: Mar. Poll. Bull., Vol. 7, pp. 13-15. Grahl-Nielsen, O. , J. T. Staveland, S. Wilhelmsen, 1978, Aromatic hydro- carbons in benthic organisms from coastal areas polluted by Iranian crude oil: J. Fish. Res. Bd. Canada, Vol. 35, pp. 615-623. Gundlach, E. R. and M. 0. Hayes, 1978, Investigations of beach pro- cesses: _in The AMOCO CADIZ Oil Spill, A Preliminary Scientific Report, NOAA/EPA Special Report, Washington, D.C., pp. 85-197. Mayo, D. W. , D. S. Page, J. Cooley, E. Sorenson, F. Bradlev, E. S. Gil- fillan, and S. A. Hanson, 1978, Weathering characteristics of petroleum hydrocarbons deposited in fine clay marine sediments, Searsport, Maine: J. Fish. Res. Bd. Canada, Vol. 35, pp. 552-562. Neff, J. M. , B. A. Cox, D. Dixit, and J. W. Anderson, 1976, Accumulation and release of petroleum derived aromatic hydrocarbons by four spe- cies of marine animals: Mar. Biol., Vol. 38, pp. 279-289. Overton, E. B. , J. McFall, S. W. Mascarella, C. F. Steele, S. A. An- toine, I. R. Politzer, and J. L. Laseter, 1981, Petroleum residue source identification after a fire and oil spill: jji Proceedings 1981 Oil Spill Conference, American Petroleum Institute, Washing- ton, D.C., pp. 541-546. Pym, J. G., J. E. Ray, G. W. Smith, and E. V. Whitehead, 1975, Petroleum triterpane fingerprinting of crude oils: Anal. Chem., Vol. 47, pp. 1617-1622. Rashid, M. A., 1974, Degradation of bunker C oil under different coastal environments of Chedabucto Bay, Nova Scotia: Estuarine Coastal Mar. Sci., Vol. 2, pp. 137-144. Roesijadi, G. , D. L. Woodruff, and J. W. Anderson, 1978, Bioavailability of naphthalenes from marine sediments artificially contaminated with Prudhoe Bay crude oil: Environ. Pollut., Vol. 15, pp. 223-229. Teal, J. M., K. Burns, and J. Farrington, 1978, Analyses of aromatic hydrocarbons in intertidal sediment resulting from two spills of No. 2 fuel oil in Buzzards Bay, MA: J. Fish. Res. Canada, Vol. 35, pp. 510-520. Warner, J. S., 1976, Determination of aliphatic and aromatic hydrocar- bons in marine organisms: Anal. Chem., Vol. 48, pp. 578-583. Winfrey, M. R. , E. Beck, P. Boehm, D. Ward, 1981, Impact of the AMOCO CADIZ oil spill on sulfate reduction and methane production in French intertidal sediments: Mar. Environ. Res., (submitted). 99 STUDIES OF HYDROCARBON CONCENTRATIONS AT THE ILE GRANDE AND BAIE DE LANNION STATIONS POLLUTED BY THE WRECK OF THE AMOCO CADIZ Henri Dou, Gerard Giusti, and Gilbert Mille Laboratoire de Chimie Organique A, Associe au CNRS n°126, Centre de St. Jerome 13397 Marseilles Cedex 13, France INTRODUCTION A study of the hydrocarbon concentrations in district no. 7 has been made since December 1978 in collaboration with the Marine Station of Endoume (Mes- dames Vacelet, Plante, and Lecampion) . The first series of analyses was made outside of the CNEXO-NOAA framework, while our second study was supported by them. Results of the two studies are herein combined. METHODS Nature of the Samples Samples were collected at sites indicated in Figure 1. Sample sites A, D, and F are located in a very polluted zone; B, C, and E are located in a zone where the pollution level is lower since a dam was erected under the bridge to prevent the spreading of oil. Subscripts indicate specific areas samples: 1 - marsh, 1 = tidal creek, and 3 = upper mud flat (see Figs. 1 and 2). Samples were collected in December 1978, March 1979, November 1979, and May 1980, using a plexiglass corer (ID = 26 mm). The 5 mm superficial layer was subsampled with a steel spatula. Sediments were immediately frozen, flown to Marseilles, and kept at -30 C. Analytical Techniques To yield the maximum amount of information, we have chosen the systematic soxhlet extraction following Farrington's method. This method is expensive and time-consuming but, for the biologists, it is the only one which gives satisfactory results. Moreover, reproducibility was tested several times and was found to be satisfactory. To avoid the very long separation of alumina- or silica-packed columns, we developed a micromethod of separation using Sep-Pak of Waters. This rapid technique is described in Analytica Chemica Acta. The general analytical scheme is as follows: 101 pn SOIL Od 1 "*l scMOHne: AI,DI,CI,BI □ MUD ON SANDY MUD AI£2,A3,B3,E3 SAMPLING SITES REFERENCE STATIONS -CI, Bl SCHORRE(SALT MARSH) C2 TIDAL CREEK POLLUTED STATIONS « AI.DI SCHORRE(SALT MARSH ) A2 TIDAL CREEK A3 SLIKKE(MUD SLOPE) FIGURE 1. lie Grande marsh sampling sites. M W- CHENAL tidal creek UHAUTE-SLIKKE — higher mud slope SCHORRE - solt meadow FIGURE 2. Detailed map of the sampling area. 102 sample 1 - extraction (toluene methanol-soxhlet) 2 - pentane, water + sodium chlorida weight before saponification AVSP saponification with KOH weight after saponification APSP separation with SEP PAK hexane elution chloroform elution fraction hydrocarbons. FA polar fraction FB Each fraction was weighted by gravimetry (Balance: Perkin Elmer AD2Z, 1/10 of ug) . In each case, the fraction FA was chroma tog raphed on a capillary column (OV1 or SE52) with a Girdel or a Carlo Erba Fractovap 4160. The fractions FA and FB were also analyzed by high-pressure liquid chromatograph (column RP 18, radial pressure, Waters-type detector). Since the FA fraction may contain saturated, unsaturated, or aromatic hydrocarbons, fluorescence spectrometry (Perkin Elmer 3000) was used especially during the 1980 survey when biodegradation rates were higher. Infrared spectroscopy (Perkin Elmer 125 and 225) was also used systematically to show the absence of carbonyl functions on the FA fractions. .During the 1980 campaign, it appeared that some of the chromatograms of the FA fractions were not sufficiently resolved due to the absence of saturated hydrocarbons. However, as they represented a substantial weight (A. * 257 g; A, ■ 14 g) , NMR spectroscopy (C 13 and proton 250 Mz Cameca) was used to denote the presence of heavy-weight compounds with fused 103 rings (aromatic and nonaromatic) . The structure of these complex mixtures was not elucidated at this time, but could be studied in the future. RESULTS Results are presented in Tables 1, 2, and 3. The weight of the compounds AVSP (before saponification) and APSP (after saponification) are indicated. The value of the ratio AVSP/APSP is characteristic of the capacity of the medium to be biodegraded. A value close to 1 shows poor bacterial activity. As the value increases, better biodegradation is indicated. This fact is due to the functionalized intermediate compounds made by the bacteria and extract in basic medium. The fraction FB is constituted by unsaponif iable compounds. Comparison of FA and FB is not interesting. Only the comparison of FB values for different sampling times is relevant. When biodegradation increases (less linear hydrocarbons) , the FB fraction decreases. At Al and A2 station (in very polluted zones) , the concentrations of hydrocarbons (linear or substituted) were about null in 1980, but complex organic compounds of heavy molecular weight, which are extractable with hexane (since present in the FA fraction) , remain in the sediment. The molecular structure of these compounds has not been established (the pet role urn- type asphaltenes are not extracted at the very beginning by pentane) . CONCLUSIONS Station C. In 1980, in spite of the presence of 0.54 g in the FA fraction, its chromatogram was no longer characteristic of a petroleum-polluted zone (C17, C18, C19 predominant). However, light petroleum pollution is indicated at 20 cm below the surface. Station B_ In 1980, various deposits did not allow correct sampling; therefore, only 1978 and 1979 values were determined. In 1979, there seemed to an indication of return to normal state, especially at the rhizome level (bottom) . Station A. In 1980, half of the hydrocarbons of 1979 remained. This fraction did not show the presence (by chromatogram) of linear or substituted hydrocarbons; however, its weight cannot be explained only by the unbiodegradable cyclanes or aromatics (33 g) . 104 TABLE 1. Hydrocarbon content of lie Grande marsh sediments. SAMPLE WET AV-SP WEIGHT (q) C22 + 2C24 +2C26 + C28 aliphatic hydrocarhons xx = Nonextractable su = Superficial part; some centimeters rh = Rhizosphere cs = Sandy layer Pr = Pristane Ph = Phytane zr = Reduced zone ca = Clay layer 107 Station D, This site is subjected to a flowing stream which must have facilitated the deposit of hydrocarbons in large quantity, since between 1978 and 1980, an important increase was measured (95 g in 1978 to 230 g in 1980) . Station C« This site indicates that substantial biodegradation is in progress. Station A, In the FA fraction, we again notice the presence of organic compounds which are neither linear nor substituted. The ratio FA 1978/FA 1980 is very close to that of station Al. Stations B and E These are reference stations in the less polluted zone. Some remaining pollution is visible, indicating that the dam was not able to prevent oil from spreading to this side of the bridge. In 1980 at B_, the chromatogram of the FA fraction did not indicate petroleum type, and there was a return to normal levels. At E. in 1980, some minor petroleum pollution remained. Station A3 There are large variations in the hydrocarbon concentrations, primarily at the sediment surface. Two separate samplings in 1980 (5 mm depth) yielded 0.27 g and 15 g of oil. However, at -10 cm, the values became equal to 0.50 g. The analysis of the 15-g fraction shows that linear and substituted hydrocarbons have disappeared. Our results are in good agreement with those noted by biologists. The less polluted zones are returning to a normal state, but a seemingly important residue remains in very polluted sites after linear, substituted, and light aromatic hydrocarbons have disappeared. 108 1A _jiU' Pr i IB •JlIw 2A 2B P* 1r- y ' izJj^^'- '3 L L _> U4JjJUuUJ^^~^_ Chromatogram 1) Station A3, March 1979. (A) surface, (B) bottom. 2) Station A2 , March 1979. (A) surface, (B) bottom. 3) Station A3, 1980, surface skim. 4) Station D, 1980, 0-3 cm surface. 109 EVOLUTION OF THE HYDROCARBONS PRESENT IN THE SEDIMENTS OF THE ABER WRAC ' H ESTUARY by Jean DUCREUX Institut Francais du Petrole 92506 Rueil-Malmaison - FRANCE Following the Amoco Cadiz accident, the Institut Francais du Petrole analyzed various samples in an effort to learn the physico- chemical characteristics of the oil pollutant released, and to observe its evolution over time. These studies dealt principally with samples of "chocolate mousse" and of beach-sand type surface sediments taken at various depths. In March 1978 a study of the physico-chemical evolution of the hydrocarbons trapped in the subtidal sediments of the Aber Wrac'h estuary was undertaken with the collaboration of the Centre Oceanolo- gique de Bretagne. INITIAL CHARACTERIZATION OF THE POLLUTANT The description of the Amoco Cadiz's cargo was undertaken on the basis of samples of foam ("chocolate mousse") taken at Portsall on March 22 and 23, 1981. This method of identification is based on the work of Pelet and Castex, and operates according to a breakdown by chemical family, which has already been used by J. Roucache to make a geochemical study of the organic matter extracted from sediments. The pollutant was identified as a mixture of two crude oils — Iranian light and Arabian light — physico-chemical characteristics of which are fairly similar ; they contain 45 to 47 % saturated hydro- carbons, 31 to 34 % aromatic hydrocarbons, 16 to 17 % polar compounds, and 4 to 5 % asphalt compounds (Fig. 1). The ratio of saturated hydro- carbons to aromatic hydrocarbons is on the order of 1.3. The saturated fraction is constituted of normal paraffins, iso- paraffins of isoprenoid compounds (pristane, phytane, etc..) and cyclic and polycyclic alkanes or cycloparaf fins . With gas-phase chromatography, n-alkanes in the sample taken at Portsall 23 March follow a regular distribution curve ; her top corresponds to the n-Cl5 and n-Cl6, after which it tapers off regularly to the n-C35 (Fig. 1). It is likely, incidentally, that compounds over n-C35 exist, but they have not yet been detected. Ratios of pristanes/n-C17 and phytanes/n-C18 in crude oil are, respectively, 0.37 and 0.51. To take into account evaporation pheno- mena and compare with evolved samples, this oil sample was topped at 340°C. An unresolved complex mixture (UCM) appears under the n-alkanes, constituted of isoparaffins and cycloparaf fins. Alkane (n+iso) 111 - f 2104 "*. vli . ! i » ■ -JOJ34 *'. W«Iilf •— -Iai3i *ilnmt i- c 10 •H a < c (0 I CD o 0. _^ -^. < % < r" =r ^ 5 N i 1 ? c IT) •rH C (0 00 O U) •U Vl 0 0< (/) c o u U o n >, r. r 112 contents determined by mass spectrometry are on the order of 5 3 %. Cyclane contents decrease gradually from 14.2 % for the single- membered cycloparaf fin rings, to 2.3 % for six-membered rings. The combination of gas-phase chromatography with mass spectro- metry (GPC/MS) made it possible to distinguish the components of the aromatic fraction : monoaromatic, diaromatic, and triaromatic hydro- carbons . Aromatic sulfur compounds of the thiophenic type are the benzo- thiophenes, dibenzothiophenes, and naphthobenzothiophenes. Polar compounds (resins) contain oxygenated functions (princi- pally hydroxyls and carboxyls) shown by infra-red spectrometer ana- lysis. Elemental analysis of this resin fraction made it possible to assess contents of the following elements : C = 78 % H = 8.9 % N = 1.5 % 0 = 4.5 % In this case, the oxygen content is not calculated by subtracting the total of the other contents from 100 %, but is titrated by the Unterzaucher method. Asphaltene compounds, separated from the oil by cold-hexane precipitation, are known to have very complex laminated structures. Metal (such as nickel and vanadium) and sulfur contents were determined on a dry extract before deasphalting (Fig. 2). Metals are essentially present in heavy fractions of crude (resins and asphal- tenes) as chelate compounds. Their contents range from 14 to 16.5 ppm for nickel and from 45 to 60 ppm for vanadium, with a ratio Ni/V of 0.27 to 0.31. Sulfur contents are on the order of 2.35 % by weight. These determinations will eventually serve as a point of depar- ture in following the pathways of the pollutant. It is to be noted that this study is not intended to cover the light evaporated and/or dissolved third of the cargo, of which about 25,000 tons is light aromatic hydrocarbons. Benzene, toluene, and xylene are estimated at respectively 3,300, 4,600, and 3,000 tons. EVOLUTION OF THE PHYSICO-CHEMICAL PARAMETERS OF THE HYDROCARBONS We followed the chemical evolution of the hydrocarbons in three different types of samples taken from three areas of pollution : - subtidal sediments (Aber Wrac'h) ; - water surface-stable emulsions (hydrocarbons with water, or "choco- late mousse") ; - intertidal sediments (beaches) . The Subtidal Sediments of the Aber Wrac'h A detailed study of these sediments was made by DUCREUX and MARCHAND (1979) (collaboration IFP/COB) . The evolution of the 113 [HiLJJLr..\l] L V 0 r ii ! L 1 S A T I 0 EXTRACTION /CI! CI , U-pMi ENLEVEMENT D U S LIBRE I 1 R ITf.cur c. KZ ict-uz/ A IB A extrait se: (Teneur cr. .V" trzcuz/ D E S A S P H V L T A C EXTRAIT DES'SRH^LT: C C "I HC. SATURES S M C C HC. AROMATIQl'ES C G RE SINES I R FIGURE 2. The program of analysis. hydrocarbons trapped in the subtidal sediments of the Aber Wrac'h was followed from March 31, 1978 to October 22, 1979 at nine stations (Fig. 3) in the estuary, taking into account the fairly pronounced marine character of the environment and the sandy, muddy, or sandy/ muddy nature of the sediments. Over a second period, from January 17, 1980 to June 24, 1980, three study sites, Stations 5, 6 and 8, which were deemed representa- tive of the different evolutionary patterns of the hydrocarbons, were again put into operation. The studies undertaken at these three stations only, are included in this report. To simplify the evolutions observed in the Aber Wrac'h, the figures included here are limited to samplings taken on March 31, 1978, November 22, 1978, June 20, 1979, January 17, 1980, and June 24, 1980. Station 5 A reduction was noted in the pollution of this station, situated in the outer area of the Aber where the marine environment is parti- cularly pronounced (Fig. 4). It was very slight, however, since the concentrations observed in June 1980 were over 670 mg of hydrocarbons per kilogram of sediment. This may be explained by the slightly muddy character of the sediment. 114 Aaen wrac'h • Plougu£rnaao #LAnnili« 1690 0 — mg.HC/kgSEO 1000_ 100. 3t 03 7B FIGURE 3. The Aber Wrac'h. 7T 20 06-79 17-oS-BO JToV ■SO FIGURE 4. Evolution of hydrocarbon contents in the sediments of the Aber Wrac'h. 115 The relative contents of saturated and aromatic hydrocarbons decreased perceptibly (Fig. 5) in nearly constant proportions, the ratio SAT/ARO decreased slightly probably because of a significant increase in resins -- from 17 to 40 % by weight. After January 1980, however, contents of all these substance remained about the same. 4 40. y * ' = 7'Sl! 71 3 30. Jk STS 20. — • SIS "~* SI8 in i i i 1 AW 31-3-71 %AR0 AW 22 II 78 AW ?C E 7S AW 17 1-80 AW 24-6-80 FIGURE 5. Evolution of the various chemical families (saturated hydrocarbons, aromatic hydrocarbons, polar compounds). The increase in resin contents results from an oxidation degra- dation of the crude in the medium, reflected in a perceptible upward curve of the absorption bands under infra-red at the level of the hydroxyls (3,600 to 2,900 cm"1) and the carbonyls — including esters, and a qreat predominance of Carboxylic acids (1,740 -> 116 1,700 cm-1; (Figure 6). The elemental analyses of the resins confirm this tendency toward oxidation over time (oxygen levels in March 1978 were 5.4 % ; in June 1980, 7 %). 31 03-78 22-11-78 20 06-79 17-01-80 24-06-80 3400 3000 1700 1100 700 cm 1600 Station 5 - FIGURE 6. Infra-red spectrometry of the resins. The distribution of saturated hydrocarbons determined by gas phase chromatography (Fig. 7) demonstrates the evolution which led to the degradation of the n-alkanes (5.99 % to 3.33 %) to n-C30 (Table 1). It should be noted that by March 31, 1978, this degradation appears to have begun. Thus there was an increase in the ratios of pristane/n-Cl7 and phytane/n-Cl8 (Table 1). The isoprenoids degraded less easily than the n-alkanes (TISSOT and al . ) , but by June 1980 these compounds could no longer be detected. As degradation advanced, the contribution of the biogenic hydrocarbons increased, and n-alkanes of odd carbon numbers between n-C25 and n-C35 -- characteristic of the cuticle waxes of higher plants (Eglington and Hamilton - 1963) — appeared, confirming the ter- restrial contribution to the contents of organic matter in the sediments. Moreover, the disappearance of the n-paraffins points up a relative enrichment in the unresolved complex mixture of the isoalkane and cyclo- alkane compounds, and generally in the compounds around n-C30 . It was possible to demonstrate these last compounds by GPC/MS by measuring the masses : m/e 217 being characteristic of the tetracyclic 117 v> _ p 0 3 r- cn 3 3 3 — T> 3 3 .... 3 — CO -^ 3 X o CM o o ^n 3 cn -r o p- o a o o ' in O ' ry.Mfi.-i 1 O n f* in in C O O O O o o o o :■ V -■v in u"! a O O en in o O ^T Lf! P* CO -:r r- ^n l.1 in jt > c I T ^ >n T *r *r ** in O ^T -n — ^ 1 1 l lD O CTt ^T f CO cr \ T ft in a an — . p^. r-i o CO O vO T Q O *T lO T pm -4 0" i m pm fi pm cn pm o 1 -^ -^ PM 1 t 1 *T — • f! PM PM Z CI T n CD o r- O in m cn cm 3 O CN PM CD 71 O O O — O pm TJ m f*1 in r- iTl kfl y) rj CO lO f» v^ .N T co cn x pm o f cn a i PM CM ft CJ fl CM PM ,-m T PM PM PM PM -j n -i ,M i .-j r .** • Ul ^ in u -a O —i cn ^r in -• a. (T* m 3 CJ> r- cn a "M cn 1 1 1 1 - i i i i i i i * pm CN in n ^ rn 0> c V x) in ^r vO PM 10 r» Au lTI u-i Ul — — a 0> lO E-\ i i till 1 1 1 1 - 1 1 i i i i >• V: o o H PM PM CM o PM ^ \ a, \ \ ^ p- r- o o fN \Q o o t-\o m r*i PM o \D T wi — 01 VL i 1 1 1 1 1 till * i i i i i i a: \ O o « •"■ ■• ~ o PM V £ „ *T O r- . J in PM CN t\ > co *T 21 O Lf ^0 "J •^T 1 1 1 1 i 1 1 1 1 1 1 » | 1 1 1 1 1 o O O o , o ' O a V \ o o CT* CO _ r* X CO ■- O in v0 n PM O O PM CO i pm cn r- -*i in 3 — \* O m X cn cn co r- j> cr> CT« o CO p^ r* r- .3 O o co o x x x r* •\< ^\ o 3 O O O 3 o o O O O O 3 a — O O 3 3 o o < \ •/} \ J to o PM o o 3 O O O O _ .. a o 3 3 3 O a . O O O O 3 3 3 < TJ 0 o n cn in t in ^T T v0 o sO ^-t — :n CD T t in co cn pm >n t r a. a. T T T CO T j~> .n r- t r> s o r> •^ ^T -" T 0* Ti tr - t? t r- i/1 iP Ul * < in JJ Jl 3 o o 3 3 3 3 3 3 o o o a O 3 3 3 3 .. o o o o o o o Z TJ .N a 3 3 X i 3 _ ul CO — PM 3 „-i -j pm ~> cn cn i£ W r- 3 3 P- T1 O ■ r 3 cn r* r* r- r*- 3 in o pm r- o "T • 3 3 ' ™ ^ ~ ~~ .N r^ »->* n ^ -. PM ~* r^ n * *> T f *N fl f f> T '*"1 f o « n o 3 o O O 3 O O o ■ 3 3 O 3 O O „ .'' O O O 3 O O O m tj l/1 ~ CO r* N 3 r^ >n p* r- -i rg a — 3 r-» *T 5 — pm r* o r- r- \o -^ -T *T in r* 3 "J "i T* J = ■T u-> 3 r- CT* J* CD *T 3 cn r- — —• pm p- p^ y - -n r*"l r*i ^ T ■^ -i c-j r^ ' —i ft 1 "! M -J ■ . ' PI PM fl ft PM CN N t- ,1 .. o a 3 3 ^333 3 O O 3 3 O 3 3 D O 3 O 3 O O 3 < "3 T ■n --. 3 3 CO r^ o k/» -r t T a cn o o n in 3 co r, c JT — i-^ m *33 c T O p- 3 3 PM :' cn*TP^ ocn no y - T f T n -1 n 'N - J i m f! PM PM P-l '"' '" pm cn cn rj — i r*i 4J CO u ra lj r* t r* J f> — —i - « > O O T N T j"> -a r*- CO PM ■fl- wi o r- CO -N " T -^ 3 r- X -01 ■ i> . — •01 PM -« CM u 3 a* — lo J -3 z o S* i V) to H C-, in -J X* 4 X X H o O 01 i 0. I/) M 0) 1 x: ft 0) o c o o > w CQ < E-« 118 31-03-78 jimuiuiiiJi 1«7 ■kj 22-11-78 20-06-79 17-1-80 JS J'l 2S *s J! 24-6-80 _*>- ^ J3 27 25 '■N CG STATION 5 FIGURE 7. Evolution of the saturated hydrocarbons. sterane compounds (CnH2n-G) eluted between n-C26 and n-C31, and m/e 191 characteristic of pentacyclic triterpane compounds (CnH2n-8) eluted over n-C30 (Fig. 8 and 9) . This relative enrichment in cyclo- paraffins is confirmed by the mass spectrometry analysis of the fraction (Hood and O'Neal - 1958). Among the saturated hydrocarbons, (n+iso) -alkanes show a rapid diminution after March 31, 1978, and a stabilization thereafter ; cycloparaf f ins , principally 2-, 3- and 4- membered rings show an enrichment, and 5- and 6-membered rings are fairly stabilized (Fig. 10) . 119 TJ 0 C £1 (0 U HJ >i U J= 0 O, U H3 T3 U >, Cf £ 0 ■P tJ i C M •U <4-t 0) 0 s 0 c u o -u •H U • 4J -( (0 >i U A 0 D4 U i CT> £ O •P Tl m , o> u 4-> 14-1 0) O £ O c u O 4J ■M u • 4J OJ c 13 a 0 c w ■A ^ U •i s O STATION 5 FIGURE 11. Evolution of aromatic hydrocarbons. 122 and phytane/n-Cl8 were among the highest and that this degradation was among the most advanced (Table I). In figure 5, a continual decrease in saturated hydrocarbons is noted over time (33 % on March 31, 1978 — 22 % on June 24, 1980). In spite of a maximum aromatic hudrocarbons content observed in June 1979, they underwent the same type of evolu- tion (34.3 % on March 31, 1978 -> 24.8 % on June 24, 1980). The result is an increase in the relative contents of resins ; but it should be noted that in March 1978, polar compound contents (24.6 %) were higher at this station, which is located in the outer part of the Aber, than at Station 5 (17.2 %) where the marine character is more pronounced. This level held true until June 1979, when the increase became the same as at Station 5 . This phenomenon can be explained by the presence of polar com- pounds of terrestrial origin deposited by the river which flows into the Aber. This contribution of autochthonous organic matter is seen in the presence of n-alkanes of odd carbon numbers (n-C25, 27, 29, 31, 33) in the aliphatic fraction, all the more accentuated by the signi- ficant acceleration in the degradation of the normal fossil paraffins (Fig. 12) The result is a relative enrichment in cyclic satured hydro- carbons (cycloparaf fins) — particularly of 3-, 4-, and 5-membered rings around n-C30 — which are less easily degradable . Chromatogram profiles of the aromatic hydrocarbons indicate how polluted the station is, principally in the FDP response (Fig. 13), where the persistence of dibenzothiophene and its alkylate derivatives (and also of naphthobenzothiophenes) may be noted. The stability of these compounds made it possible for us to use these chromatograms as the "fingerprints" of the pollution. As a matter of fact, according to the DGMK. report, biogenic hydrocarbons of terrestrial origin (recent sediments) are poor in aromatic compounds (< 5 %) , and particularly low in sulfurous com- pounds of the thiophenic type. The evolutions observed in the resins are similar to those at Station 5 ; an oxidizing degradation increases the oxygen contents (8.5 % in June 1980), and an upward curve is noted in the infra-red absorption bands of the hydroxyls and carbonyls, the latter of which are predominantly carboxylic acid compounds (Fig. 14). Metal and sul- fur contents remain fairly constant over time. Station 8 This station was distinguished in being the farthest from the sea, in an area of sandy mud. Hydrocarbon contents show that the decontamination process at this station was virtually nil (Fig. 4) . An anomaly is noted in January 1980, when the hydrocarbon con- tents went over 5,000 mg-/kg of sediment. As we shall see below, this is due to pollution from petroleum cuts or fuel oil which was later deposited on top of the pollution under study (Fig. 15 and 16). Al- through information is lacking on some samples, which were too small, it can be seen that metal and sulfur contents remained very stable over time. 123 31-03-78 L 22-11-78 20 - 06 - 79 17- 1-80 3331 Ll%, M_J^O, % 24 6 80 '-jU^i "X CG STATION G FIGURE 12. Evolution of the saturated hydrocarbons. Examination of the chromatograms of the "saturated" fractions shows a quantity of biogenic hydrocarbons of terrestrial origin which is not negligible, and which is characteristic of the upstream sta- tions on the Aber which receive large quantities of deposits from the soils. The distribution of n-alkanes is in fact typical of that ob- served in the extracts from recent sediments in the predominance of odd-carbon-numbered n-alkanes from n-C25 through n-C35 (Fig. 16). These quantities of biogenic hydrocarbons present in the ali- phatic hydrocarbons are demonstrated in the ratio R29-31 developed 124 ^^^^ 4 » I t " FID ~^ \ *\ \ ) 1 i 1 1 ! j \i'i'V/., ! i 1 1 i ] i > /Jiw*J X s /*^| j i • 1 FPD ^^ 1 • * l . i < ^ PORTSALL 23-3-78 AW 31-3-78 FID ^\ ,1 ""V )', \ *V li 1 \ \V ' ;| i \w 11 S2 i i! 1 I 1 ;l / FPD ^/ > ■ Vi J y ^ i i i • 1- AW 20 6 -79 A AW 22-11-78 \ A — i v' ; ■ FID ^N, "\aW ' ) \ W(: i ! i 1 Li li' 1 f> i 1 i FPD _^/ i j i ~"T i i ^^r '1 » ? i AW 17-1-80 AW 24-6-80 STATION 6 FIGURE 13. Evolution of aromatic hydrocarbons. 125 i — r 3400 3000 1600 STATION 6 31-03-78 FIGURE 14. Infra-red spectrometry of the resins. by Tissot et al ; they were calculated for the earliest samples only 2(C29 + C31) R 29-31 = C28 + 2C30 + C32 The relationship shows that in this case, the n-C29 and n-C31 predominate. Where the value is over 1, the odd-numbered carbons pre- dominate. In Table 2, a compilation of these values for all the stations, it is seen that R is much higher than 1 at Stations 6 and 8. Even in March 1978, this presence of natural compounds modified the distribution of hydrocarbons by family : - saturated hydrocarbon contents were low ,- - polar-compound contents (resins and asphaltenes) high. While the ratio of SAT/ARO decreased slightly, as at Stations 5 and 6 (Fig. 17), degradation of n-alkanes and isoprenoids (pristane and phytane) was observed throughout the period, showing clearly which polycyclic alkanes are most resistant to degradation. The chroma tograms of the aromatic hydrocarbons have very marked profiles under photometric detection (FPD) , and a persistence in the unresolved complex mixture (FID) (Fig. 16). Comparison of the results of the GPC with those of the high resolution MS undertaken on the re- ference samples of March 23, 1981 at Portsall, and the sampling taken 126 31-03-78 22-11-78 -^ 20-06-79 17-1-80 29 35. « 1 . 1 27 «,a»% V 24-S-IO ,# IS % CG STATION 8 FIGURE 15. Evolution of the saturated hydrocarbons. on June 24, 1980 at this station, shows a degradation (or dissolution) of the alkylated cycloparaf fins to C3 and C4 . Concerning the thio- phenic derivatives, some benzothiophenic alkyls disappear to become C5, while the initial alkylate derivatives of dibenzothiophene become C3 ; the naphthobenzothiophenes persisted. In the sampling taken in January 1980, an abnormally high res- ponse is noted — by Flame Ionization Detection (FID) and by Flame 127 PORTSALL 23-3-78 FPD 1/ --sj % AW 22-11-78 irf**4 n~r AW 20 6 -79 '■si FPD "vm FID AW 17-1-80 SK J -». ~t\ AW 24-6-80 STATION 8 FIGURE 16. Evolution of aromatic hydrocarbons. 128 AW 31-3-71 FIGURE 17. AVV 20 6 79 AW 22-11-78 „„ „ ..„ AW ,,.,.,„ 4W n t |D Evolution of the ratio of saturated hydrocarbons to aromatics. TABLE 2. Values of the R29-31 ratio. 2 (C29 + C3i) Valeurs de R29-31 = C28 + 2C3o + C 12 Station 31.03.78 2.05.78 1 1,02 1,23 2 1,13 - 3 1,10 1,05 4 1,36 1,07 5 1 ,23 1,22 6 2,34 1,35 7 1,26 1,59 8 4,00 2,78 9 1,06 1,12 129 Photometry Detection (FPD) as well. This is caused by the presence of a light cut (gasoline, fuel oil, etc..) deposited on top of the Amoco Cadiz pollution (Fig. 16) . Here again the infra-red resin spectrum shows an increase in absorption of hydroxyls and the transformation of esters to acids (Fig. 18). The oxygen content of these polar compounds (7.4-7.5 %) confirms the degradation by oxidation. 31 03 78 3400 3000 1700 1100 ;oo cm 1600 Station 8 FIGURE 18. Infra-red spectrometry of the resins. Stable Hydrocarbon/Water Emulsions or "Chocolate Mousse" When petroleum spreads over a marine environment, the movement of the waves, the wind, and the currents causes a very rapid emulsion with the sea water which is called "chocolate mousse". These stable emulsions are constituted by dispersing sea water in the hydrocarbons (inverse emulsions) . This type of emulsion was sampled during the first month after the accident (Fig. 19, Table 3). A program of analysis which is different from that used for the sediments and sands was employed (Fig. 20) . After purifying the emul- sions (of sand, algae, etc...), they were distilled to measure the water contents of those emulsions with boiling points below and those above 340°C. This method is described by Pelet and Castex. The results are assembled in Table 4. 130 BRIGNOGAN d ROSCOFF C^ • LANNION £v TREOnrAN ^_j(Z^S~^ -**—>>» GUISSENY HEROENIEL J"**" ( PORTSALL * HoRUAIX ( BBESIW^ 22, 23 mars 78 3 avrll 7B A avrll 78 18 octobre 78 31 Janvier 79 28 mars 79 PORTSALL GUISSENY ROSCOFF PORTSALL GUISSENY BRIGIIOGAN KERDENIEL TREOMPAN KEROENIEL TREOMPAN FIGURE 19. Sampling stations. TABLE 3. Characteristics of the samples. Lie-jx Dates Ces r'"1--- ■ -'"cr - - P0=TSALL v,= :s=ll Z-.lll'i'- 3~'rTrrr :2.::.7e Z3.:3.7- ■T -t ?a 3 . 1~ . ?z tt rcc--:rs. 1 ; - : *. . _ <= c f "• - * SRISIIXW \i'o:._\-\;\r: .:,. [f-rof. - 35 z-) ,,„,„_... ►.E^3£-.:c.L K;=2EMEL (Prof. ■ 2E cm) re.::. 79 26.G3.79 1 Moreover, on these fractions a simulated distillation curve was plotted (TBP or True Boiling Point) by gas phase chromatography accor- ding to the method of Petroff et al . (1981). Results of the distillation (Table 4) and the TBP on the PI-340°C (Table 5) indicate a loss by evaporation and dissolution of about 7- 8 % of the light- hydrocarbons between March 22, 1978 and April 4, 1978. 131 r PIODUI7S F.ECUPEF.ES BRUT FArLE PTi.LUE ! I n DECANTATION FILTRATION CE'.'TPIFUGATION ZXTR-CT]rt\ "50SHLET CITJ , KSKYDRATATIOK Na:S3. PRODUIT EPURE P3DDUIT EPURE ETETACE 340°C '. POIDS S, Ni, V C — ) CUD (EE5> — © ASPHALTENES I POIDS PA8 CKRO'LATOGRAPHIE C3UCHE MINCE 1 1' HC.SATURES H: . ARO'IATIPJES RESIDES C G IS V IR FIGURE 20. The program of analysis TABLE 4. Results of distillations of samples of emulsified crude, 1 ^- — L'r *; Icvumcnt s PORTSALL PORTSALL GUISSENY ROSCOFF PORTSALL GUISSENY BRIGNOGAN | r< 22 mars 78 23 mnrs 1978 3 jvril 78 4 •vril 1978 * |>olilft e.iu Jans ■ ttiu Lsitiit 1 71 ,7 68,7 67,7 51 60 57 57 n < lio'c 24,3 20,0 8.9 13,15 15,95 13,05 8,7 : |iuid< lie IK - J40*C 75,7 80,0 91,1 R4.05 86.95 91 ,3 86, 10 Two samples (Guisseny, March 23, 78, and Brignoqan, April 4, 1978), lost considerably more. The latter was initially the point of highest contents. Its chromatographic profile (TBP) is also very different from that of the samples taken at Portsall on March 22 and 23 (Fig. 132 TABLE 5. Simulated distillations of PI-340°C. Z poids Temperatures 'C cunules PORTSALL PORTSALL GUISSENY 1 PORTSALL CUISSENY R0SC0FT BRICNOGA.N 22.3. 23.3 23.3 4.4 4.4 3.4 4.4 1 162.2 199,1 223.6 216,3 210.2 227.3 239,2 I 5 193,4 218,6 248,2 233,4 229,7 245.5 172,9 10 207,8 228.7 260,6 246,7 245,9 257,8 285,9 20 224,6 245.0 273,8 264,2 266,2 271,4 300,5 j 30 237,0 256,1 285,9 275,8 279,9 284,8 310,5 40 251,4 271,7 292,4 286,3 290,7 292,9 f 319,6 50 267,9 281 ,7 300,6 294,7 300,9 301,6 326,5 60 282,7 290,6 306,5 303,5 308,9 308,9 J35.0 i 70 295,2 301,3 317,0 313,2 319,2 318,8 343,6 80 310,6 313,2 326,0 322,9 329,9 328,1 353.9 90 330,6 331,3 343,0 340,7 346,8 343.6 371,0 100 392,1 390,3 400,9 459,8 439,3 422,3 460,0 ei- ! :. ^:;:--y— -j- f-"|^-----i--= ■=£•■-( •": ' i i-'i-': : r.lr?~S=~~j"".=S ^1 1 la- -!-.V .-- f- -. - :-=i- r-'-K'-T- j- -■' \ i- ■---^r. 1 \i,\ ' t ■ ^iM . fiT .-;-; r-f \\\k 33-^- :-:.!•;: i-'i •: 1 ! '" :. ::•..! II Mil ; ■ ; . ji ■■, ■ ■ 1 i ! r r r- I I I I a I - J-i-i- ! 'j f. -m_ii4- _.__!. l- .1 i ' ;V FIGURE 21. "TBP" PI-340°C. 133 The chromatogram of the saturated hydrocarbons shows that the n-paraffins had disappeared (Fig. 22) . Although there are divergences FIGURE 22. Chromatogram of the saturated hydrocarbons taken at the Brignogan station April 4, 1978. in such parameters as metal and sulfur contents, the infra-red spec- trum definitely confirms that it is crude from the Amoco Cadiz. We believe that these differences may be due to the fact that we were dealing with oil sheets that had been treated to a greater or lesser degree. These observations should be compared with those made by Aminot et al. at the same time at a station just offshore from this one, which showed an abnormal loss of dissolved oxygen. He explained it as in-situ biodegradation of the hydrocarbons, which appears to be the only logical explanation. Distillation of the 340+ fractions shows little in the way of interpretable differences (Table 6) . The sulfur and metal (nickel and vanadium) contents show, by their stability, how important these com- pounds are as pollution markers (Table 7) . TABLE 6. Simulated distillations of 340+ residues. 1 Z poids discilUi P0RTSALL 22.3 P0RTSM.L 22.3 CUI'SEW 22.3 nperatures PORTSALl 4.4 C cuissncY 4.4 ROSCOFF 3.4 BRICNOGAN 4.4 1 291,4 283,8 281,8 305,1 303,2 295,8 266,6 5 346,0 325.2 327.1 338,9 342,9 336,1 318,1 10 370,2 346.9 349.6 359.4 367,9 360,8 342,2 20 408.8 383,3 386.2 394,2 405.3 399.5 386,3 30 446,0 420,3 422,6 431,3 443.8 439,5 421,7 40 484,1 457,5 458.5 463,7 476.3 472,7 458.8 50 526,9 497,8 497.2 499,3 5,4.6 515,7 503,6 60 543,4 540,2 539.3 558.6 560,1 1 54,35 Z i 547,3 61 Z 1 547,5 62,1 Z 1 548,6 62,3 Z i 549,6 61.8 Z a 568,6 56.8 Z 1 548 61.7 Z 1 569,4 The breakdown by chemical family is shown in Table 8. It appears that the evolution of the crude in all of the emulsions is a sLow process. A slight decrease in the ratio of saturated to aromatic 134 TABLE 7. Sulfur, nickel and vanadium contents. S Ni V Ni/V Z pds Ug/g ug/g PORTSALL 22.03.78 2,33 18,5 62 0,27 PORTSALL 23.03.78 2,38 14,0 45 0,31 *» «•"! CUISSENT 23.03.78 2,38 16 50 0,32 ■ 3 ROSCOJT 3.04.78 2,22 14 48 0,29 ■ PORTSALL 4.04.78 2,30 16 58 0,28 B£ CUISSENT 4.04.78 2,18 20 65 0,31 BRICNOGAN 4.04.78 2,30 22 68 0.32 TABLE 8. Evolution by chemical family. PrSlevesents lies J pds H ,C . satures Z pds nC aronat. ' pds Resines I pds Asphaltene: SAT/AROS. ■ PORTSALL PORTSALL 22.03.78 23.03.78 38,06 37,28 35.66 34,12 21,71 24,28 4,57 4,32 1,06 1,09 CUISSENT 23.03.78 41,69 34,11 16,40 7,80 1 .22 PORTSALL 22.03.78 47,65 31,29 16,77 4,28 (21,05) 1,52 1 1 PORTSALL 23.03.78 45,12 34,55 15,70 4,60 (20,30) 1,30 i CUISSENT 23.03.78 39.45 31 24,90 4,60 (29,50) 1.27 I u 0 PORTSALL 4.04.78 46,75 30,12 19,18 3,95 C3. 13) 1,55 j id S3 CUISSENT 4.04.78 46,38 31,76 17,65 i 4.21 (21.86) 1,46 BRIGNOCAX 4.04.78 34,10 31,50 25,60 1 8,80 (34,40) 1,08 RO SCOFF 3.04.78 43,69 34,01 16,96 5,34 (22,30) 1,28 KERDENIEL (35 cm) 18.10.78 40,00 33,50 19.50 7,00 (26.50) 1,19 ■ II a ■ TREOMPAN 31.01.79 33.90 39 19.20 | ' 7,90 (27,1) 0,87 ■ TREOVTAN 28.03.79 31,10 36,90 22,60 7,40 '30.0) 0,80 i ffl U KERDENIEL 28.03.79 38.40 36.90 18,10 6,60 (24,70) 1 .04 KERDENIEL (25 ca) 28.03.79 27,60 26,30 35,90 1 10,20 (^6.10) 1,05 hydrocarbons is seen, however, as well as a slight relative increase in the polar compounds. The infra-red spectra of these compounds show, in fact, a slight upward curve in the absorption band of the carbonyls 135 between the end of March and early April 1978 (Fig. 23) . These results corroborate those of Roussel and Gautier at Antifer . ( 1979) . PORTSUl 22.3.78 P0RT55LL 23.3.73 GUISSEHY 23.3.. 78 ROSCOFF 3.4.73 PORTSALL 4.4.78 / G'JISSENY 4.4.78 BRIG'iOGV* 4.4.7c 1 1 1 3600 3200 2600 n 1700 1600 — I — 1100 750 cm FIGURE 23. Infra-red spectrometry of samples of crude (R 340+) and extracts. 136 The pathways followed by the crude are shown in the triangular diagram of the saturated and aromatic hydrocarbons, and the polar compounds (resins + asphaltenes) (Fig. 24). P, PortsMl 22.?3.7!< P, Portsoll 23.C3.7P t} Portiall 4. CM. 79 G^ Gui99nny 23.C?.?" Cj Gulssany 4.G1.7*! 5 Drlgnopan i."i.7° rc noicirr 3.r3.7i in / ••• /■■■■"> SM. 00&mm§ RES.+ ASPH.) FIGURE 24. Triangular diagram of the distribution by chemical family. For the sake of comparison, we have added the corresponding values in samples of polluted sands and a subtidal sediment taken in the Aber Wrac'h (AW 9) . Intertidal Sediments - Polluted Beach Sands The program of analysis employed for the study of these sands is identical to that used for the subtidal sediments of the Aber Wrac'h (Fig. 2). The characteristics of these samples are seen in Table 3. The sulfur contents of these polluted sands (2.5-2.6 %) are all slightly higher than those of the emulsions — even the most advanced (2.3-2.4 %) , but are about the same as those in samples taken from the Aber Wrac'h. This evolution is explained by the disappearance of 137 certain chemical species which are easily degradable and/or soluble, such as n-alkanes, light aromatics, etc... leaving a higher relative concentration of sulfurous species, which are more resistant to degra- dation. Metal concentrations (nickel and vanadium) , and their ratio, did not vary significantly through the one-year pariod of the study (Table 9) . This behavior was noted above in the discussion of the Aber Wrac'h samples. TABLE 9. Sulfur, nickel and vanadium contents. S Ni V Ni/V 5 pds Ug/g Ug/g KERDENIEL 16. IP. 78 2,55 14 45 0,31 (35 cm) TREO".FAN 31 .01 .79 2.65 17,5 83 0,21 u TREO^AN 26.03.79 2,66 19 65 0,29 s KTRDr.NIEL 28.03.79 2,69 20 85 0,23 u KERDENIEL (25 en) 28.03.79 18 65 0,28 As we did with the samples of emulsified mousse, as described above (Fig. 24), we recorded the saturated and aromatic hydrocarbons, and the polar compounds (resins and asphaltenes) on the triangular diagram (Table 8) . The most notable evolution took place in the latest samplings of polluted sand. But it is difficult to isolate the factors contri- buting to evolution in the beach sands : time, extent of dispersion of' the crude, how long the oil was on the sea, the support material (sand, mud, rocks) . This is all the more true of a sampling taken a year after the catastrophe, which may have undergone a very complex history of burial before being picked up again by the water during a storm or a spring tide. Even with these reservations, however, it seems that the triangular diagram shows that the crude follows several pathways in its evolution : - a very short and stable pathway, as we saw above in emulsions on free water ; - a pathway in which a relatively slow disappearance of saturated hydrocarbons (n-alkanes) and aromatics (Mono- and diaromatics) results in a moderate increase in polar products, when the crude is trapped in sand ,- - an evolving pathway followed by crude which is trapped in more or less muddy subtital sediments of the Aber Wrac'h, as demonstrated above . The chromatograms of the saturated hydrocarbons in the polluted samples taken in 1979 all show a general degradation of n-paraffins to n-C30, confirmed by an increase in the ratios of isoprenoids to n-alkanes (n-C17 and n-C18). This degradation seems slower in polluted beach sands than in the sediments of the Aber Wrac'h. The mass spectrometry study of the (n+iso) distribution, and that of the 1- to 6-membered rings of cycloparaf f ins confirms this 138 evolution (Table 10, Fig. 25). But a slight alteration is seen in sur- face samples (Kerdeniel, March 28, 1979), which may be explained by the reemergence of masses of only slightly degraded crude, which have been trapped in sand, during a storm. TABLE 10. Distribution of the cycloparaf f ins by the mass spectrometry. I vol. Z vol de cyclanes a Prelevements paraf fines (n + iso) 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles P0RTSA1L 52.92 14.20 13.07 8,20 5.87 2,81 2.33 23.03.78 Gl'ISSEKY 55.66 14,29 13,04 8.05 5.30 2,07 1.59 C3 .03. 78 5JSC0FF 53,13 14,09 13.50 9, I1 6,22 2.69 1.19 3.0- .76 ?0RTSALL 47,49 14,44 14,80 10, 16 7,46 3,31 2,34 4.04.78 GCISSINY 46,33 16,88 15,78 10,43 7,73 2,29 0,55 4. 04. 76 BRIC7N0GAN 31,90 22,15 19,72 12,24 8,52 3,64 1,83 4.04.78 TRIOS AN 35 17,12 18,13 13,20 9,74 4,26 2.54 31 .01.7? TRIOSAN 30,93 17,75 19,42 14,24 10,21 4.59 2,86 28.03.79 KERTEXIEL 44,52 15,58 16,17 10,92 7,52 3,1 1 2,18 28.03.79 KER3ENIEL 31,88 11,23 15.97 16,07 13,99 6,80 4,06 28.03.79 (25 ex prof ) "T» MCI" rTr,iirL .'f ,i" . '-■ '.r"DrNIFL ?n.'n.?'< T'lFO^'A'l 31.01. '9 PTin\"ir.n\ i.n.;n GIJISSFNY 1.04,/n I"31TSA.IL 1."'1.'1 P'-ttt J. (14. 711 CUI5PFMV ?l.'n.71 -*■ Nomhro dn nnynu« (n«pht>lnml FIGURE 25. Distribution of the cycloparaf fins by the mass spectrometry. 139 It should be noted that a sample taken at the same time and same place, but at a depth of 25 cm, shows an advanced stage of degradation, comparable to that observed in samples from the Aber Wrac'h at the same time. Compared with these subtidal sediments, oxidation degradation of the trapped crude in beach sands is slight and slow. This is seen in the infra-red spectra of the resins, where the absorption bands of the carbonyls are less marked (Fig. 26) . KERDENIEL 18.10.78 treo:ip*:i 31.1.79 TREO'IPAN 28.3.79 KERDENIEL 28.3.79 t r 3600 3200 FIGURE 26. Infra-red spectrometry of the resins, CONCLUSION The samples studied fall into three categories : - subtidal sediments (Aber Wrac'h) ; - oil/water emulsions or "chocolate mousse" ; - intertidal sediments (beach sands) . In the slightly muddy sediments of fine sand in the stations lo- cated in the outer part of the Aber Wrac'h, where the marine character is pronounced, a decrease in global contents of extractable compounds is observed, whereas in those located in the upper part of the Aber, the decontamination process is slow, probably inhibited by the muddy nature of the sediments. 140 The degradations observed in these sediments results in : - the progressive disappearance of saturated hydrocarbons, principally the normal paraffins ; - the disappearance of the light aromatic hydrocarbons ; - the oxidation of the polar compounds (esters, acids, etc...). The compounds which persist are : - the saturated polycyclic hydrocarbons and the heavy aromatics ; - sulfurous aromatic hydrocarbons of the thiophenic type ; - resins and asphaltenes, resulting in stable metal (nickel and vana- dium) and sulfur contents. In the sediments samples in the area of the Aber, terrigenic de- posits are superposed on the Amoco Cadiz crude, resulting in : - an increase in polar compounds — resins and asphaltenes. The in- crease in asphaltene contents is due to the presence of pigment (green) of chlorophyllaceous origin ; - the appearance of n-alkanes of odd carbon numbers (n-C25 through n-C33) . The most striking evolution in the "chocolate mousse" samples is the loss of light hydrocarbons due to evaporation and dissolution. Volatile compounds under C14 were not considered in this report. The samples of polluted sand taken from the beaches a year after the accident show a degradation phenomenon principally affecting the saturated hydrocarbons, and among these, principally the n-paraffins. There is an increase in the contents of polar compounds. But our in- formation is not adequate to state at what point in the history of these samples, the degradation was most intense. 141 REFERENCES CITED Aminot, A., 1981, Actes du colloque, Brest, Nov. 1979 : Amoco Cadiz , Consequences d'une pollution accidentelle par les hydrocarbures , CNEXO, Paris, pp. 223-242. Rapport DGMK. Rapport de recherche 150. Methode de dif ferenciation des hydrocarbures biogenes et des hydrocarbures d'origines petrolieres. Ducreux, J., Marchand, M., 1981, Actes du colloque, Brest, Nov. 1979 : Amoco Cadiz , Consequences d'une pollution accidentelle par les hydrocarbures, CNEXO, Paris, pp. 175-216. Eglington, G., Hamilton, R. J., 1963, The distribution of alkanes. Chemical Plant Toxonomy. T. Swain Ed., Acad. Press, pp. 187-217. Hood, A., O'Neal, M. J., 1958, Preprint of I. P., Hydrocarbon Research Group and ASTM. Committee E14 Joint Conference on Mass Spectro- metry, University of London Senate House, Pergamon Press. Pelet, R., Castex, H., Juillet 1972, Atlas de references de pollutions petrolieres. Rapport IFP n° 22 422. Petroff, N., Colin, J. M., Feillens, N., Follain, G., Juillet-Aout 1981, Revue de l'Institut Francais du Petrole, Ed. Technip, Vol. 36, n° 4, pp. 467-484. Roucache, J., Hue, A. Y., Bernon, M., Caillet, G., Da Silva, M., 1976, Application de la chroma tog raphie couche mince a 1' etude quanti- tative et qualitative des extraits de roches et des huiles. Revue IFP, Vol. XXXI, pp. 67-98. Roussel, J. C, Gautier, R., 1981, Actes du colloque, Brest, Nov. 1979 : Amoco Cadi z , Consequences d'une pollution accidentelle par les hydrocarbures, CNEXO, Paris, pp. 135-147. Tissot, B., Pelet, R., Roucache, J., Combaz, A., Utilisation des al- canes comme fossiles geochimiques indicateurs des environnements geolog iques . Rapport IFP, ref. 2 3 440. Unterzaucher, 1940, Ber. Deut. Chem. Ges., 73 B, pp. 391. 142 THE AMOCO CADIZ OIL SPILL DISTRIBUTION AND EVOLUTION OF OIL POLLUTION IN MARINE SEDIMENTS by Michel Marchand, Guy Bodennec, Jean-Claude Caprais, and Patricia Pignet Centre Oceanologique de Bretagne - CNEXO BP 337, 29273 BREST CEDEX, France INTRODUCTION In March 1978, the supertanker AMOCO CADIZ was stranded on shallow rocks off Portsall (north Brittany), 2.5 km from the coast. Two hundred twenty-three thousand tons of a mixture of Arabian light crude oil (100,000 t) and Iranian light crude oil (123,000 t) flowed into the sea without interruption from 17 March to 30 March. The maximum extent of the oil slicks is presented in Figure 1. At this point, about 360 km of coastline were polluted by the oil. The analyses of oil in seawater, measured by UV fluorescence spec- troscopy (Marchand and Caprais, 1981), revealed that the oil spill «°W »° *° 30 jo a Ou«'ne 1 ^ ^JT^^OfiQURE i g* 5o t> v> ■> JO . ?» FIGURE 1. Maximum extent of oil slicks on the sea surface, 17 March to 26 April 1978. 143 affected a very large section of the western English Channel. One month after the AMOCO CADIZ wreck, the most polluted areas were located in the coastal zones, such as the Aber zone (38.9 + 6.7 ug/l) , the Bay of Morlaix (11.5 + 5.1 ug/1), and the Bay of Lannion (10.7 + 3.0 ug/1) . Analysis of samples from various depths revealed that the contamination extended throughout the water column. The 49°N parallel roughly con- stituted the northern limit of pollution. Beyond this limit, oil in surface seawater was not observed (1.6 + 0.5 jug/1) . In March 1979, one year after the AMOCO CADIZ stranding, hydrocarbon concentrations returned to a normal level (below 2.0 ug/1) ; however, some residual traces of pollution were still observed near the Abers and at the bottom of the Bay of Lannion (about 2.0 ug/1). We also began a chemical follow-up study of oil pollution in marine sediments. Some data have already been presented during the interna- tional symposium held in Brest (France) in November 1979 (CNEXO, 1981; Ducreux and Marchand, 1981; Marchand, 1981; Marchand and Caprais, 1981) . In this document, results of our study are presented in three parts: (1) oil pollution in sediments collected from the western English Channel one month after the wreck, (2) specific study in the Bays of Morlaix and Lannion to determine the distribution of oil pollution in surface sediments and at various depths, and the evolution of oil contamination over one year, and (3) specific study of the Aber Wrac'h to determine oil evolution from 1978 to 1981. MATERIAL AND METHODS Surface marine sediments were collected in the western English Channel with a Shipek grab. In coastal areas, small Ekman and Hamon grabs were used. The samples, after freezer storage, were dried by using a Soxhlet apparatus or by stirring with chloroform. The organic extract was concentrated to dryness, then dissolved with 10 ml of carbon tetrachloride. A first indication of petroleum pollution in sediment was obtained through a direct analysis of nonpurified extracts by IR spectrophotometry (Perkin Elmer 397). Quantitative measurements were carried out at 2920 cm corresponding to the presence of hydrocarbons and polar compounds. The data also reflect coextracted natural substances (fats, fatty acids, etc.) from sediments. The IR spectrophotometer was calibrated with a mixture of Arabian and Iranian light crude oils. Hydrocarbons were analyzed after cleanup of organic extracts on activated Florisil (200°C) in glass columns (15 cm x 0.6 cm i.d.). Hydrocarbons were eluted with 15 ml of carbon tetrachloride and measured by IR spectrophotometry. For some samples, organic carbon was determined with an auto-analyzer LECO WR-12. In a joint study with the French Petroleum Institute concerning the Aber Wrac'h sediments (Ducreux and Marchand, 1981), we compared the gravimetric determinations and the IR spectrophotometr ic analysis of nonpurified organic extracts. Results of the two methods are similar (Fig. 2). We also compared the IR spectrophotometric results obtained on nonpurified and purified organic extracts from some Aber Wrac'h sediments. In this case, correlation was significant (Fig. 3) . 144 EXT tPPMJ 15000.00 I0B2H 0$ 5B0B.0E. RMDCD CRDIZ SEDIMENTS DE L HBER HRHCH ME5URE DE5 EXTRA ITS DRSHNIOJES SRRVIHETRIE CEXT: i SPECTROPHOTOMETR I E IR CHCJ a.ra 0.0a FIGURE 2. IB0B0.B0 hc cppm: 15000.00 gflflfl .00 Correlation between gravimetric determinations and IR spec- trophotometry measurements for organic extracts. CHC1 PURIFIES CPPM3 I SBB . 03 . iB3a.ua. 500.08 .. RMOCD CHDIZ SEDIMENTS DE L FIBER WRROH MESURE DE5 EXTRP, I TS DRGRN I QUES PRR 5PECTRDPHTDMETR I E I . R EXTRHIT5 NDN PURIFIES ET PURIFIES 5UR FLDRISIL CHC3 NDN PURIFIfS Ci:r.M] 3 FIGURE 3. Correlation between IR spectrophotometr ic measurements for nonpurified and purified organic extracts. 145 OIL POLLUTION IN THE WESTERN ENGLISH CHANNEL (APRIL 1978) One month after the wreck, sediments were collected during an oceanographic cruise (R/V SUROIT) to assess sea bottom contamination of the western English Channel. The sampled sediments were coarse- to medium-grained calcareous sands (more than 70% CaCo,) . In the Bays of Morlaix and Lannion and near the Aber zone, the content of calcium carbonate in the sands was somewhat lower (50-70% CaCO-) . Organic carbon content was generally low, from 0.02 to 0.6 percent (m = 0.18% + 0.13%). The oil concentrations in the sediment ranged from 10 to 1,100 ppm (nonpurified organic extracts) (Marchand and Caprais, 1981). Generally, the zone of contaminated sediments reflected offshore and coastal areas impacted by the drifting slicks (Fig. 4). The pollution of the sea bottom was a result of the diffusion of oil into the water column. Off Sept-Iles, a gradient was observed from the coast to the open sea (210, 52, 42, 34 ppm). At the 49°N parallel, from west to east, one could observe an increasing and decreasing gradient (21, 19, 48, 102, 54, 52, 24 ppm). The highest petroleum accumulation in marine sediments were located in the coastal and sheltered zone of the Abers (100 to more than 10,000 ppm) and in the Bays of Morlaix and Lannion (10 to more than 1,500 ppm) (Fig. 5). FIGURE 4. Oil pollution in marine sediments (April 1978) Concentrations expressed in ppm. 146 115 { • 116 » V^. nOSCO*' n A>i..5. •'M'u'i ■ 107 ■ 117 ^—— R.MEL • 106 'l3C > ) "'45 , •'» r*U V • 112 \ J BAiE PE W»l»ll ■'J" I ^-/ .HI .124 J V /? "'2' r* \. -lOi^wj .110 ">23 / ? (? ",32«, / >-* — v f*>* "Aocouir 7 / .UlNVlf C»tLO' / j**** "'21 J. Si aaiCmEl EN GHEvES • ( v s ■^ I "120 / Si fOl Of LEON/ ** J 6*N^V " S^— -vS ).£/(^ "All t-^"^ \ !03» \ \ '02. \ V^ 101"«\ ^%iOO \ ' /^ • MOALA1I FIGURE 6. Sampling stations in the Bays of Morlaix and Lannion. TABLE 1. Average hydrocarbon concentrations in surface sediments in the Bays of Morlaix and Lannion (July 1978) . AREA SEDIMENT Number of observations Hydrocarbon concentrations (.ppinj BAY OF vnRL < 2 - 4 Al 1 14 1 .3 medium to coarse sand and maerl - (5) < 2 - 4 Al 1 JJ 6.8 fine sand - 8 <*> < 2 A I 1 :4 5.5 fine to coarse sand - 8 (*> < 2 - 6 Al i :" 2.1 medium to fine sand (8) 14 t 9 Al i ?: 1 .5 maerl and silt (S)<« 26 i 4 3 (*) < 2 Al 1 3S 2. 1 fine sand 7 (« 14 t 7 - 1 v, 1 vj 2.7 fine to coarse sand - 6 (*» < 2 ■ Al 1 i - 2.0 medium to fine sand (9) 10 * 10 i M 1 ". 1 2.0 fine to coarse sand - (8) < 2 - 5 1 A' Id 3 2. 1 medium to fine sand (7) 13 t 3 - ! Al ISU 2.3 coarse to fine sand (7) 26 • 6 1 '"'' P.. 1 2.5 medium to fine sand (7) 10 1 6 1 A' J 3 7 ,., fine sand to sandy mud - (6) < 5 (*) : excepted surface layer, (n) : number of observations, (EXT) : IR spectrophotometry determinations of non-purified organic extracts, (HC) : IR spectrophotometry determinations of purified organic extracts on florisil. ABER WRAC'H (1978 TO 1981) The two Abers (Benoit and Wrac'h), located 8 km east of Portsall, were heavily impacted during the spill, estuaries, 10-15 km long and about 1 km wide to March 1979 (Marchand and Caprais, 1981) sediments throughout the Abers were heavily were more than 100 ppm and, as in a muddy sometimes reached higher than 10,000 ppm. Caprais (1981) showed that the natural These Abers are small A study from March 1978 revealed that the bottom polluted. Concentrations area in the Aber Benoit, After one year, Marchand and decontamination process was related to the nature of the sediment and the energy level of the geographic zone. The fine- and medium-grained sands located in the exposed, downstream part of the Aber Benoit were well decontaminated (average hydrocarbon content reduced from 700 to 27 ppm) . On the other hand, in mud-dominated areas, the sediment acted as an oil trap (oil content above 10,000 ppm) and decontamination was not observed. For the Aber Benoit, oil pollution of mud-dominated zones such as Loc Majan will be long term. In the Aber Wrac'h, the evolution of oil pollution in the bottom sediments has been followed since March 1978. The location of sampling stations is presented in Figure 7; analyses for hydrocarbon and organic carbon concentrations are presented in Tables 7 and 8, respectively. Organic carbon concentrations ranged from 0.08 to 3.32 percent. Sediments are much more homogeneous than those of the Aber Benoit. Composition, with the exception of station 3 located at the mouth of the Aber, varied from slightly muddy to muddy sands. 152 FIGURE 7. Sampling stations in the Aber Wrac'h. ABER WRAC'H TABLE 7. AMOCO CADIZ oil pollution in the sediments of the Aber Wrac'h. Data collected from 1978 to 1981. OC = organic carbon; CaCO- = carbonate calcium; EXT = gravimetric determination of organic extract; OIL = IR spectrophotometric determination of nonpurified organic extract; HC = hydrocarbons, IR spectrophotometric determination of purified organic extract; (S) = surface; (P) = 10-15 cm depth. DATE T (months) SAMPLING STATION 1 2 3 4 5 6 7 8 9 Hatch 31, 1978 0,5 0C (Z) EXT (ppm) OIL (ppm) 0.53 2130 2051 2.42 11220 12000 0.2J 71 1 773 0.96 2185 24 50 1 .06 3160 3706 1.03 765 839 0.66 2259 1.94 397 953 0.78 1951 206.1 j Kay 5, 1978 1.5 CaCO Z 0C F.XT OIL 0.66 2400 307 3 - 0.34 800 1020 1.59 1 1490 1 1750 18.8 0.71 2000 2970 20.8 0.78 1660 1380 7.0 0.61 1 190 1236 8.3 0.55 460 503 is. j ; o.ao i 2I6U i 22 16 November 22, 1978 8.25 EXT OIL 990 1'.39 4030 4144 130 148 3740 3598 2600 2481 710 781 1600 1 105 680 67 1 856 j 872 February 22, 1979 1 1.25 OC f.XT OIL 0.56 1 1 '.0 1589 1.53 2660 2679 80 1 1) 0.75 1 140 1301 0.85 1360 I26B 1.12 3020 25 56 1 .00 1050 14 10 0.60 870 I2h6 1 I78H . 1677 ', June 20, 1979 15.25 FXT OIL 1550 14 58 1200 1 124 80 74 480 4 12 1450 1 178 6070 4900 1260 1325 1450 1445 8 30 ; ■128 i October 22, 1979 19.25 OIL 715 1374 80 1237 1047 2712 2496 485 5„9 | 1 January 20, 1980 22.25 OIL 1408 2567 - 1796 1473 1861 1399 5694 iiw; ' June 1980 27 OC OIL HC (ppm) 0.44 442 175 1.74 1892 995 0.08 42 1. 15 1225 724 0.81 1075 348 2.40 I7BH _ 1 12 2 2.70(S ?.nl(l' 2 320(S 1 7HM(P 83KS i iur 1 .90 1280 602 1. 12(S p. I<)(P (3167(S p 369(1' ,I644(S jMHlir 1 .67 1628 .1.00 j 81.6 1 . .0 i January 1981 14 OC OIL HC 11.83 321 62 0.84 562 255 0.90 34 7 1 105 0. 711 4 56 205 0. litS 2. M(P 1 17b(S 112 1(1' 6 JO ( S 4 20(1' 1 i.m :! ,,: >i ; 1 214 1 ! 0|9(S1 i I437U') ) 3I2(S> ; j 707U'l ' March 16, 1981 36 OIL HC 432 229 I07H(S 30J(P 43KS 90(1' ) 25 ) ) - ) !970(S) 651(S> 627(1') |l*7l(l' 55KS) |28I(S) 403(P) |787(1') I5I7(S 20201 P 674(S 722(P 180 40 9iO(S 1807(1' 4I5(S 642(P June 23, 1581 39,2 5 Oil. i 308 HC 50 B5KS 404 (P 362(S 71 (P ) - ) ) - ) 411(S) |864(S) 195(P) 1541(P 10B(S) kl'6(S) 42(P) Il486(r I623(S )I320(P 740 (S ) 660(P 540(S 961(P 200 (S ) 4I7(P H7(S)j 411 ! 33I(P)| , 35(S) 326 I3I(P)I 153 TABLE 8. Organic carbon content of the Aber Wrac'h sediments, number of observations. (n) = SAMPLING STATION. n 0C(l) m t s 1 5 0.60 ± 0.15 (25 %) 2 4 1.63 ± 0.65 (40 1) 3 3 0.22 ± 0. 13 (59 1) 4 5 1 .07 ± 0.32 (30 \) 5 5 0.83 ± 0.15 (17 % ) 6 6 1.16 i 0.88 (54 '.) 7 6 1 .50 ± 1.14 (76 %) 8 6 1.22 + 0.87 (71 %) 9 4 0.90 ± 0.12 (14 %) In this discussion, three areas of Aber Wrac'h are described in terms of oil degradation:: (1) the mouth of the estuary (fine-grained sands, station 3), (2) the downstream part (stations 1, 2, 4, and 5), and (3) the upstream part (stations 6, 7, 8, and 9). The evolution of oil pollution in sediments from 1978 to 1981 for each station is given in Figure 8; the change in average oil concentrations for each of the three defined areas is given in the Table 9 and Figure 9. In March 1978, 15 days after the AMOCO CADIZ wreck, concentrations ranged from 773 ppm to 12,000 ppm. At the mouth of the Aber which is well exposed to high marine energy, hydrocarbon content dropped from 773 ppm in March 1978 to 25 ppm in March 1981, illustrating a fairly rapid decontamination of these fine-grained sands. Since the sediments of the Aber Wrac'h are relatively homogeneous, the decontamination process is mainly related to the energy level of the zone. In the downstream part of the Aber, the sediments were more polluted in March 1978 (average about 5,000 ppm) than the sediments collected in the upstream part (average about 1,500 ppm). For the first 39 months after the spill, a natural but slow decontamination was observed with some temporary increases in January 1980 (about 1,800 ppm) and March 1981 (about 780 ppm). In June 1981, the average residual oil content was about 600 ppm. In the upper part of the Aber, the sediments were initially less polluted, but since this is a low-energy area, a decrease in hydrocarbon content was not observed until January 1981. As had been observed in the downstream portions of the Aber, significant increases in hydrocarbon levels were observed during January 1980 in upstream areas. Since March 1981, oil levels have decreased; average residual oil concentrations were about 670 ppm. Three years after the wreck, the petroleum pollution of the Aber Wrac'h sediments seems to be relatively uniform. Figure 10 gives the observed decontamination rate of the sediments. At first approximation, station 3 (located at the mouth of the Aber) is well decontaminated (3% of the initial residual oil level observed in March 1978). However, sediments within Aber Wrac'h remain quite polluted with about 20 percent of the residual oil content still remaining in March 1978. 154 IBB 000,- HC CPPM] ib me. lass. 100.. IB EvaurnoN des teneurs residueu.ee d hydrdcrrbures DHNS LE5 SEDIMENTS DE L RBER HRHOH 12 IB 21 2H 27 30 33 36 T CMDIS] t > 42 HS FIGURE 8. Evolution of residual oil pollution in Aber Wrac'h sediments, TABLE 9. Evolution of oil pollution (ppm) in the Aber Wrac'h sediments from 1978 to 1981. ~~~^ (month: March 51 ) 1 'J 78 May 5. 1978 Nov. 22, 1978 l-'chr .22, 1979 June 20, 1979 Oct. 22, 1979 Jan. 20 1980 June 1980 January 1981 March 16 1981 June 23 1981 LOCATIOV 0.5 1 .5 8.25 11.25 IS. 25 19.25 19. 2S 22.25 34 36 39.25 Month (st. 31 T73 1020 148 1 1 J 74 80 - 42 - 25 - Downs t 1 l it'i ;i i t t "il)5l S914 29 15 1709 1043 109 3 18 11 1 158 421 783 609 1st. I , : , i . :. i ■ loss 15053 1 1203 16U2 = 445 1285 = 531 1595 H 10 !29S !290 Upst r c.nn |i.. i i is:b 1340 857 I721 2149 1SDS 2512 1390 1 747 901 672 (st. 0. ;,i,"i ! 7 30 •708 •184 = 578 • 184b ! 1 202 !2I43 1409 H2S0 !5S0 !6S7 All stations ol the M.el ki.ii Mi 3: 'jo 3 300 1886 1718 1590 1329 2161 1274 1084 842 640 ( st . 1 , J, 1. ,". • !99%), and [1- C]- heptadecene, 18.5 mCi/mmole (97%) from ICN. AMOCO CADIZ mousse was obtained from the NOAA National Analytical Facility and was collected at Ploumanach on April 30, 1978 (44 days after the spill). Light Arabian crude oil (SX//0308) was obtained from Exxon Corp., Baytown, Texas. The crude oil was weathered by evapora- tion at 25 C for 8 and 48 h. All oil samples were stored at 4 C until used. RESULTS Physical-Chemical Comparison of Sites Beach cores consisted of medium grained sand, while estuary and marsh cores consisted of fine grained silt and clay. It was possible to determine Eh profile for muddy sediments (Fig. 3). In all sediments Eh (mv) + IOO + 200 +300 -100 SALT MARSH MUDFLATS + 100 +200 +300 -1- -£r-' ' / \ « VAW A AI / / \ ABER MUDFLATS Figure 3. Eh profiles in muddy sediments. Bars indicate the range of measurements on duplicate cores. pH increased with depth from 7.5 to 8.2 in He Grande, from 6.7 to 7.2 in Aber lldut, from 5.9 to 7.5 in Aber Wrac'h and from 7.0 to 8.1 in the lie Grande oiled site. conditions became more reducing with depth. The steepest Eh profile was observed in the He Grande oil site where a brown layer approxi- mately 2 mm thick covered black sediment. Marsh mudflat sediments 166 showed steeper Eh profiles than estuarine sediments and oiled sediments were more reducing and showed steeper Eh profiles than unoiled sedi- ments of the same type. The large Eh change with depth (300mv over 1 cm (lie Grande oiled site) or 2 cm (Aber Wrac'h) suggested that sedi- ments below these depths are likely anoxic. Chloride (data not presented) was relatively constant at all depths in all sites and was near seawater chloride levels (20,000 mg/liter). The concentrations of dissolved sulfate and methane with depth in each site are reported in Table 1. No major differences in sulfate concen- tration with depth or between cores were observed, _and levels were similar to seawater values (approximately 800 mgSO, -S/l ). Methane TABLE 1. Sediment Chemistry3 SULFATE CHEMISTRY** DEPTH AMC4 TREZ-HIR ABER WRAC'H ABER ILDUT ILE GRANDE ILE GRANDE (cm) (oiled) (control) 0-5 720 720 860 840 840 760 5-10 850 770 900 830 790 800 10-15 820 820 870 970 750 760 15-20 1070 1190 820 810 740 790 20-25 -- -- 860 920 800 710 METHANE CHEMISTRY 0-5 0.17 0.16 0.74 1.57 3.36 0.24 5-10 0.52 0.84 0.80 0.89 2.89 1.09 10-15 0.64 0.67 0.79 0.66 3.34 0.26 15-20 — — 0.99 0.93 4.26 0.35 20-25 -- -- 0.50 0.81 4.36 0.36 Sediments collected in March 1979 Results expressed in pg SO, -S/ml porewater p Results expressed in (jmoles CH./l sediment 167 concentrations were extremely low (less than 5 (Jmoles/1) and relatively constant with depth. Methane concentrations were significantly higher at the lie Grande oiled site (p < .001). Sediment Hydrocarbons Sediments collected during December 1978 and March 1979 were ana- lyzed for hydrocarbon content (Table 2) and type (Figs. 4 and 5) . Sur- face sediments (0-5 cm) at all sites oiled with AMOCO CADIZ oil exhi- bited a composition indicative of highly weathered oil residues. The saturate fractions were comprised of a degraded hydrocarbon assemblage with greater degradation in estuary and marsh mudflat samples than in the beach sample as evidenced by the relative dominance of the branched isoprenoid hydrocarbons (Fig. 4). Residual alkylated phenanthrenes , and dibenzothiophenes in the aromatic/unsaturate fractions (Fig. 5) also indicated the presence of weathered petroleum. All samples known to be impacted by AMOCO CADIZ oil exhibited a characteristic unresolved complex mixture (UCM) in both saturate and aromatic/unsaturate frac- tions indicative of weathered petroleum (Farrington and Meyers, 1975). Qualitative and quantitative differences existed between oiled and unoiled control sediments in the 0-5 cm depth interval. Hydrocarbon content was always higher in oiled sediments (Table 2) . The control estuary sediment exhibited a small UCM and hydrocarbons indicative of biogenic origin in the saturate (odd chain alkanes n-C to n-C_ ) and aromatic/saturate (polyolef inic material) fractions. Tne control marsh sediment exhibited a mixture of hydrocarbons of biogenic (odd-chain al- kanes n-C.,. to n-C„ ) and petroleum (UCM) origin, with low concentra- tions of residual aromatic/unsaturate hydrocarbons. The control beach sediment exhibited a n-alkane series (n-C ,. to n-C„~) and UCM in the saturate fraction, and polynuclear aromatic components originating from combustion of fossil fuel (eg. , nonalkylated 3-5 ringed polynuclear aromatics) (Youngblood and Blumer, 1975). The types and amounts of hydrocarbons were consistent with the known degree of impact from the AMOCO CADIZ oil spill. It is clear that in control beach and marsh sediments impact by hydrocarbons of petroleum or other anthropogenic sources had occurred. Evidence for degraded Amoco Cadiz oil at various sediment depths is summarized in Table 2. At the beach station AMC-4 , AMOCO CADIZ oil was evident in the hydrocarbon assemblage down to the 10-15 cm interval in a sample collected in December 1978, and to the 15-20 cm interval in a sample collected in March 1979. At Aber Wrac'h there was evidence of AMOCO CADIZ oil to the 10-15 cm interval at both collection dates. The amount of oil decreased with depth as evidenced by the total hydrocar- bon concentration and the increasing contribution of native sediment hydrocarbons (e.g., plant derived saturate and aromatic/saturate com- pounds) which dominated in the deepest layers as in the entire Aber Ildut core (see Figs. 4, 5). Similar results were found at the lie Grande oiled site where AMOCO CADIZ oil was detected in the 5-10 cm layer on both sampling dates and biogenic compounds dominated deeper layers. 168 TABLE 2. Preliminary Results of Total Hydrocarbon Levels in Brittany Sediments (AC=AMOCO CADIZ Oil Indicated by GC-MS Data) Sediment Type Depth Interval Tot; »1 Hydrocarbons (mr/r) 0: Lied Control (cm) 12/78 3/79 3/79 Beaches AMC- -4 Trez-Hir 0-5 295 AC 217 AC 110 5-10 158 AC 181 AC 46 10-15 244 AC 162 AC 130 15-20 72 128 AC 20-22 123 Abers Aber W rac'h Aber Ildut 0-5 977 AC 1095 AC 690 5-10 590 AC 630 AC 530 10-15 47 AC 307 AC 305 15-20 80 118 204 20-25 33 103 115 25-30 25 346 30-35 45 Salt Marshes He Grande He Grande 0-5 1137 AC 863 AC 465 5-10 144 AC 439 AC 365 10-15 28 134 217 15-20 32 220 154 20-25 74 54 Evidence for Weathering of Sediment Hydrocarbons It was evident that oil was present at depths where extremely re- ducing conditions indicated the lack of oxygen (Aber Wrac'h and the He Grande oiled site), as well as in surface sediments which were more likely exposed to oxygen. This provided an opportunity to compare weathering patterns in sediments with markedly different exposure to oxygen. Since the actual amount of oil could vary between sites, evi- dence of weathering was sought by comparing the relative amounts of hydrocarbons extracted from single samples known to be polluted with AMOCO CADIZ oil. Because of the rapid and extensive biodegradation which apparently followed the AMOCO CADIZ spill (Atlas, et al, 1981; Ward et al, 1980) the comparison of n-alkanes to the more recalcitrant isoprenoid alkanes of similar volatility was not possible as an index of biodegradation. By the first sampling date (December 1978), n-C17/ 169 (A) Oiled Estuary Mudtiat cms "■ ■ i ■ l'M> Ph.linr I i lnli«r\JI 5tjr«lJ>(l >>iolv«d . . " .■ (B) Control Estuary Mudtiat ,ilUi. jw li i i iCI O.led Sail Marsh Muddat ID) Control Salt Marsh Mutlflat I. I .. > A IE) O'led Beach (F/ Control Beach XU>\ i 3 r^iMh , FIGURE 4. Gas chromatograms of saturate fraction of hydrocarbons ex- tracted from Brittany sediments collected in March 1979 (0-5 cm depth interval) . (A) Oiled Estuary Mudtlat r«h«nan|H.en when added either at the time of anaerobic tubing or 38 hours after dark anaerobic incuba- tion began. Similar results were found for [methyl- C]-toluene. It was conceivable that the radiolabelled gases might have been produced from contaminants rather than from the hydrocarbons them- selves. When an attempt was made tcL recover the added C in, long-term radiolabelling experiments with [1- C] -heptadecane and Jl- C] -hepta- decene, it was noted that the total amount of CO + CH, produced during anaerobic incubations was similar to the level of impurities measured in C-labelled hydrocarbons recovered from formalin controls or from unpurified stocks of added radioisotopes (Table 5). Stock solutions were chromatographically separated into f , f„ ^nd f„ com- ponents which were then tested separately as sources of C-gases in dark anaerobic incubations with anaerobic sediments. The results of such experiments are presented in Eigure 7. The repurified f frac- tions of [1- C] hexadecane and [ 1- , C] -heptadecane were clearly sig- nificant sources for production of CO during dark anaerobic incuba- tions with a slurry of lie Grande oiled 3-6 cm sediment. Increases in CO with time following a lag of 5-15 days also suggested that oxi- dation did not result from any oxygen which might have been introduced accidentally during tubing. Similar results were observed with repuri- fied f of [1- C] -heptadecene. A final control was run to test the possibility that slow diffu- sion of oxygen through the vessels containing incubating samples could account for the obsexved metabolism. Darle .anaerobic incubations of repurified f of [1- C] -hexadecane and [1- C] -heptadecane were car- ried out with a slurry of mud from the 3-6 cm interval of Aber Wrac'h sediment. The individual vials were incubated inside an anaerobic 173 TABLE 4. 11*C02 + 1rensen et al, 1981; Banat and Nedwell, per- sonal communication). The chemistry of hydrocarbons present in the various sediments one year after the spill indicated the presence of oil highly altered by evaporation and biodegradation. The levels observed in the environment were also lower (0.1-1 mg/g) than the levels added in our experiments to simulate heavy oiling (50-250 mg/g). It is possible that a tempor- ary inhibition of acetate oxidation could have resulted from very heavy oiling of relatively fresh oil. Such conditions could have existed at all polluted sites immediately following the AMOCO CADIZ spill, al- though rapid loss of volatile compounds probably occurred between spillage and beaching of oil (Dowty, et al, 1981; Ward, et al, 1980). Any inhibitory effect would then have been reduced as cleanup or trans- port of hydrocarbons out of the sediments decreased hydrocarbon amount, and as evaporation, dissolution and biodegradation altered the remain- ing sediment hydrocarbons. By the time site comparison experiments could be performed, recovery from any negative effects which might have occurred had apparently taken place. The inhibitory effects on acetate oxidation we observed may be significant in extremely cold regions where slow rates of evaporation would occur. ACKNOWLEDGEMENTS We are indebted to the Centre Oceanologique de Bretagne at Brest and the Station Biologique at Roscoff for providing laboratory space and assistance. We also thank Bob Clark of the NOAA National Analyti- cal Facility for supplying AMOCO CADIZ mousse, George Ward of Exxon Corp. for supplying the light Arabian crude oil, and Dale Meland and Melinda Tussler for technical assistance. This study was part of a joint effort undertaken by the Centre Na- tional pour 1' Exploitation des Oceans (CNEXO) of the French Ministry of Industry and the NOAA of the U.S. Department of Commerce to study the ecological consequences of the AMOCO CADIZ oil spill. It was financed by funds given to NOAA (contract NA 79RAC00013) by the Amoco Transport Company and by the NOAA Outer Continental Shelf Environmental Assess- ment Program, through an interagency agreement with the Bureau of Land Management. 186 REFERENCES American Public Health Association. 1976. Standard methods for the examination of water and wastewater, 14th ed. American Public Health Association, Inc. New York. Atlas, R. M. 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiological Rev. 45: 180-209 . Atlas, R.M. and A. Bronner. 1981. Microbial hydrocarbon degradation within intertidal zones impacted by the AMOCO CADIZ oil spillage, pp. 251-256 in AMOCO CADIZ: Fates and Effects of the Oil Spill. Proc. of the Int. Symp . Centre Nationale pour L'Exploitation des Oceans, Paris. Atlas, R. M., P. D. Boehm and J. A. Calder. 1981. Chemical and biolo- gical weathering of oil, from the AMOCO CADIZ spillage, within the littoral zone. Est. Coastal Mar. Sci. 12:589-608. Bailey, N. J. L., H. H. Krouse, C. R. Evans, and M. A. Rodgers. 1973. Alteration of crude oil by waters and bacteria - evidence from geochemical and isotope studies. Am. Assoc. Pet. Geol. Bull., 57:1276-1290. Boehm, P. D. , D. L. Fiest, and A. Elskus. 1981. Comparative weather- ing patterns of hydrocarbons from the AMOCO CADIZ oil spill ob- served at a variety of coastal environments. pp. 159-173 In AMOCO CADIZ: Fates and Effects of the Oil Spill, Proc. of the Int. Symp. Centre National pour L'Exploitation des Oceans, Paris. Brown, D. W. , L. S. Ramos, M. Y. Vyeda , A. J. Friedman and W. D. Macloed. 1980. Ambient temperature extraction of hydrocarbons from marine sediments: comparison with boiling-solvent extrac- tions. In L. Petrakis and F. Weiss (eds), Petroleum in the Marine Environment, Advances in Chemistry series no. 185, American Chemi- cal Society, Washington, D.C. Bryant, M. P. 1976. The microbiology of anaerobic degradation and methanogenesis with special reference to sewage, pp. 107-118 In Microbial energy conversion, H. G. Schlegel, G. Gottschalk, and N. Pfennig (eds.) E. Goltze, K. G. Gottingen. Burkholder, D. 1963. Some nutritional relationships among microbes of the sea sediments and waters, pp. 1133-1150 in Symposium on ma- rine microbiology, C. H. Oppenheimer (ed.). Thomas Springfield, 111. Calder, J. A., J. Lake, and J. Laseter. 1978. Chemical composition of selected environmental and petroleum samples from the AMOCO CADIZ oil spill, pp. 21-84 in The AMOCO CADIZ Oil Spill, a preliminary scientific report. W. N. Hess (ed.). NOAA/EPA Special Report, U.S. Govt. Printing Office. Washington, D.C. 187 Centre Nationale pour 1 ' Exploitation des Oceans, Institut Francais du Petrole, and Institut Geographique National. 1979. Remote sens- ing of hydrocarbon pollution, preliminary report. Centre Nation- ale pour l'Exploitation des Oceans, 55 Avenue d'iena, 75016, Paris. DeLaune, R. D. , W. H. Patrick, Jr., and M. E. Casselman. 1981. Effect of sediment pH and redox conditions on degradation of benzo(a)- pyrene. Mar. Pollut. Bull. 12:251-253. Dowty, B. J., J. W. Brown, F. N. Stone, J. Lake and J. L. Laseter. 1981. GC-MS analysis of volatile organics from atmospheres im- pacted by the AMOCO CADIZ oil spill. pp. 13-22 in AMOCO CADIZ: Fate and Effects of the Oil Spill. Proc. Int. Symp . on the AMOCO CADIZ Centre Nationale pour L'Exploitation des Oceans, Paris. Farrington, J. W. and P. A. Meyers. 1975. Hydrocarbons in the marine environment. In Environmental chemistry, Vol. 1, G. Eglinton (ed.) The Chemical Society Special Report No. 35. London. Fenchel, T. M. and B. B. J, les sables dunaires (DUi) presentent un peuplement qui semble normal (etape 0), tous les autres peuplements sont en desequilibre (etapes 1, 2 et 2-4) et , dans les aires envasees , la decontamination commence a peine (etape 6-2). Ensuite les processus dynamiques sont plus lents et , au cours du troisieme hiver (t33), les sables dunaires ( DU2 et DU3 ) atteignent 1' etape 2, les sables fins et les sables heterogenes envases l'etape 2-4. Les dragages mecaniques qui ont ete preconises pour resorber les poches de vase et d'hydrocarbures ont eu lieu en avril 1980, a proximite des sables heterogenes envases. Leur peuplement temoigne d'un accroissement passager de la perturbation, etape 6 a t29- H faut moins de deux mois en effet pour que prolifere une nouvelle generation de Capitella capitata. Cette etape 6 est apparue comme une elevation dans 1 'evolution logique de l'ecosysteme ; elle est provoquee par une intervention anthropique . II est important de noter que les conditions hydrodynamiques ne sont pas aussi efficaces dans l'Aber Wrac'h que dans l'Aber Benoit, ce qui se traduit par une decontamination qui n'est pas si- multanee dans les abers . L' evolution regressive de la faune oppor- tuniste n'est pas aussi rapide dans l'Aber Wrac'h et nous pouvons observer qu'au meme moment (t25 par exemple ) , les memes peuplements restent plus perturbes que dans l'Aber Benoit : DU2 SHV SF VS_ Abers Benoit 1 2 2-4 6-2 Wrac'h 4-2 4 4-2 4 A t33 cette difference disparait : DU2 SHV SF VS Abers Benoit 2 2-4 2-4 6-2 Wrac'h 2 2-4 2-4 2-6 DISCUSSION Independemment des fluctuations cycliques annuelles, Involu- tion temporelle des differents groupes le long des gradients spa- tiaux que constituent les abers, montre une evolution acyclique et des processus chronologiques tout a fait similaires de ceux decrits par Le Moal (1981) dans la zone intertidale de ces abers. lis sont resumes sur la Figure 5a. II y a d'abord regression quasi-totale des 198 populations initiales durant la premiere periode to~t8- Le groupe II ne fluctuant pas significativement ou etant seulement dominant lors- que les autres groupes disparaissent (etapes 3 et 5), n'est pas repre- sent^ sur les figures . Entre tg et t^3, c'est le commencement de la seconde phase et la faune opportuniste s'installe. Le groupe IV est abondant partout a partir de t8 dans l'Aber Wrac'h, ses densites sont maximales au printemps , c'est-a-dire a ti3 et t25- Son importance est decroissante dans les peuplements de l'Aber Benoit apres t8, ti3 ou t]_7 selon 1 ' hydrodynamisme . Le groupe V apparait de facon significative seulement dans les vases sableuses de l'Aber Benoit, mais , sur un plan general, le de- veloppement de la faune opportuniste IV et V est maximal entre to et t20- Le groupe III reapparait a ti3 et son developpement est tres important partout durant le deuxieme hiver. De facon simultanee, le groupe I reapparait , un an apres la maree noire , mais apres deux annees son importance est encore limitee par le developpement anormal du groupe III qui semble entrer en competition. Avec le developpement des groupes I ou III, essentiellement apres t203 commence done la phase de reconstitution . A cote de ce schema general d' evolution, nous avons regroupe les differents sce- narios d'evolution temporelle : celui des vases sableuses de l'Aber Benoit est evoque plus haut (Fig. 5b), celui des vases sableuses de l'Aber Wrac'h montre d'abord la predominance du groupe V, remplace ensuite par celui du groupe IV (Fig. 5c). Pour les autres sediments de l'Aber Wrac'h, il y a deux pics successifs du groupe IV separes par un maximum du groupe III a t21- Le cas aberrant des sables hete- rogenes envases de l'Aber Benoit est illustre par la recrudescence du groupe V, apres celui du groupe IV. Pour 1' ensemble des sediments dunaires des deux abers , les Fi- gures 5d, e et f, illustrent les differentes possibilites ou la com- petition entre les groupes I et III apparait de facon tout a fait evidente . Ces scenarios sont etablis sur les densites relatives des diffe- rents groupes, mais la communaute sera jugee en etat d'equilibre lorsque les caracteristiques essentielles (A = abondance relative des especes ; S = nombre d'especes ; B = biomasse) restent relati- vement inchangees, hormis les fluctuations saisonnieres . Qualitati- vement , 1' ensemble de ces peuplements est encore en desequilibre et 1' analyse simultanee des trois parametres S, A, B, suggere des faits complementaires qui meritent d'etre suivis dans le temps. On notera que c'est dans le cas des sediments d'aval de l'Aber Benoit que l'on est encore le plus eloigne d'une certaine stabilisation de ces trois facteurs . Au contraire , c'est dans le cas des sediments envases que l'equilibre semble atteint le plus vite . Nous avons deja expose (Glemarec et al. , 1981) comment le modele methodologique , mis au point dans le cas des effluents urbains arri- vant en mer, pouvait etre utilise dans le cas de catastrophes petro- lieres, et l'echelle de temps des phenomenes observes semble avoir ete la meme dans le cas du Tanio (Aelion et Le Moal, 1981). 199 Modele general ( intertidal par ex.) — i-* 12 VS vs w SHV^ SF w FVW SHV8 < m Er— »-iz: nr- sf B DU w DU,B du3s DU,8 12 FIGURE 5. Principaux modeles de succession temporelle des differents groupes I a V au sein des peuplements des Abers Wrac'h (W) et Benoit (B). Le modele general (Fig. 5a) est synthetique ; il illustre les trois phases apres une telle perturbation : mortalite, substitution, reconstitution. 200 vs w DU w VS/ ' \ / \ / \ SHVW FVW DU,B FIGURE 6. Evolution des parametres synthetiques , nombre d especes S en trait plein, A abondance des individus en poxntille, B biomasse en tirete. La stabilisation simultanee dans le temps de ces trois parametres n'est pas acquise dans le cas des peuplements d'aval de l'Aber Benoit . 201 L' accident de l1 Amoco-Cadiz s'est revele pour nous une expe- rience d'ecologie experimentale tout a fait exceptionnelle . Elle permet d'apporter des elements de reflexion quant aux mecanismes qui mettent en place de telles sequences, probleme pose recemment par Connell et Slatyer (1977). Dans la succession decrite , les premiers stades correspondent a des especes a vie courte , de type opportuniste , capables de sup- porter une proportion de matiere organique encore import ante dans les sediments . Cette premiere phase de recolonisation par substitu- tion est mieux expliquee par les caracteristiques biologiques des especes en cause que par quelque propriete emergente de la commu- naute toute entiere (Sousa, 1980). Si ces premieres especes ne faci- litent pas le retour des especes caracteristiques des stocks ulte- rieurs (modele de facilitation), il leur est difficile d'inhiber la reapparition des especes a strategie differente qui s'installent plus lentement , mais de facon plus durable. Les phenomenes de competition existant , peu a peu les premieres especes disparaissent . Ce type de succession secondaire peut correspondre au modele de tolerance de Connell et Slatyer, dont il n'existe jusqu'ici que peu d'exemples connus . Les premieres etapes ont done ete relativement rapides, pour les suivantes e'est plus long. La reconstitution, si elle est quali- tative, doit aussi etre quantitative, energetique. L'approche que nous avons developpee semble plus efficace que lf etude dynamique de certaines populations. Elle s'est inspiree de la recherche des indicateurs biologiques bien connus en milieu ter- restre et d'eau douce. Merne si les parametres ecologiques abiotiques semblent normaux, ces bioindicateurs peuvent reveler des perturba- tions dans les ecosystemes , qu'il est impossible de detecter par une analyse des parametres physiques. REFERENCES CITEES Aelion, M. et Y. Le Moal , 1981, Impact ecologique de la maree noire du "Tanio" sur les plages de Tregastel (Bretagne nord-occiden- tale) : Rapport Contrat CNEXO , n° 80/6295 Bellan, G., Bellan-Santini D. et J. Picard, 1980, Mise en evidence des modeles ecobiologiques dans des zones soumises a perturba- tions par matieres organiques : Acta Oecologica , Oecol. Applic , vol. 3 (3), pp. 383-390 Cabioch, L., Dauvin J.C., Mora-Bermudez J. et C. Rodriguez-Babio , 1980, Effets de la maree noire de 1' "Amoco-Cadiz" sur le ben- thos sublittoral du nord de la Bretagne : Helgolander wiss . Meeresunters. , vol. 33 (1-4), pp. 192-208 Chasse, C, 1978, Impact ecologique dans la zone cotiere concernee par la maree noire de 1 '"Amoco-Cadiz" : Mar. Poll. Bull., vol. 11, pp. 298-301 Chasse, C, L'Hardy-Halos M.T. et Y. Perrot , 1967, Esquisse d'un bi- lan des pertes biologiques provoquees par le mazout du "Torrey- Canyon" : Penn ar Bed, vol. 6 , pp . 107-112 202 Connell, J.H. et R.O. Slatyer, 1977, Mechanisms of succession in na- tural communities and their role in community stability and or- ganisation : The Amer. Naturalist., vol. 3 (982), pp. 1119-1144 Gentil, F. et L. Cabioch, 1979, Premieres donnees sur le benthos de l'Aber Wrac'h (Nord-Bretagne ) et sur 1' impact des hydrocarbures de 1 ' "Amoco-Cadiz" : J. Rech. Oceanogr., vol. IV (1), pp. 35 Glemarec, M. et C. Hily, 1981, Perturbations apportees a la macro- faune benthique de la baie de Concarneau par les effluents ur- bains et portuaires : Acta Oecologica, Oecol . Applic . , vol. 3, pp. 139-150 Glemarec, M., C. Hily, E. Hussenot , C. Le Gall et Y. Le Moal , 1981, Recherches sur les indicateurs biologiques en milieu sedimen- taire marin : Colloque "Recherches sur les indicateurs biolo- giques", A.F.I.E., Grenoble Glemarec, M. et E. Hussenot, 1981, Definition d'une succession eco- logique en milieu meuble anormalement enrichi en matiere orga- nique a la suite de la catastrophe de 1' "Amoco-Cadiz" : In "Amoco-Cadiz, Consequences d'une pollution accidentelle par les hydrocarbures", CNEXO Ed., pp. 499-512 Le Moal, Y. , 1981, Ecologie dynamique des plages touchees par la maree noire de 1' "Amoco-Cadiz" : These 3e cycle, Universite de Bretagne Occidentale , 131 pp. Le Moal, Y. et M. Quillien-Monot , 1981, Etude des populations de la macrofaune et de leurs juveniles sur les plages des Abers Benoit et Wrac'h : In "Amoco-Cadiz, Consequences d'une pollution acci- dentelle par les hydrocarbures", CNEXO Ed., pp. 311-326 Marchand, M., 1981, Bilan du Colloque sur les consequences d'une pol- lution accidentelle par les hydrocarbures : In Rapport Scient . et Techn., CNEXO, 44, pp. 1-86 Marchand, M. et M.R. Caprais, 1981, Suivi de la pollution de 1' "Amoco- Cadiz" dans l'eau de mer et les sediments marins : In "Amoco- Cadiz, Consequences d'une pollution accidentelle par les hydro- carbures", CNEXO Ed., pp. 23-54 Pearson, T.H. et R. Rosenberg, 1978, Macrobenthic succession in rela- tion to organic enrichment and pollution of the marine environ- ment : Oceanogr. Mar. Biol. Ann. Rev., vol. 16, pp. 229-311 Sousa, W.P., 1980, The responses of a community to disturbance : the importance of successional age and species' life histories : Oecologia (Berl.), vol. 45, pp. 72-81 Ce travail a ete realise avec l'aide financiere de la N.O.A.A. (Contrat C.N.E.X.O. 79/6180). II a fait partiellement l'objet d'une communication presentee au 16eme European Marine Biology Symposium de Texel, Septembre 1981. 203 LES EFFETS DES HYDROCARBURES DE L1 AMOCO-CADIZ SUR LES PEUPLEMENTS BENTHIQUES DES BAIES DE MORLAIX ET DE LANNION D'AVRIL 1978 A MARS 1981 par Louis CABIOCH1, Jean-Claude DAUVIN1, Christian RETIERE2, Vincent RIVAIN2, et Diane ARCHAMBAULT 3, 1) Station Biologique de Roscoff, 29211, Roscoff, France 2) Laboratoire Maritime de Dinard, 35801, Dinard, France 3) Universite de Laval, Quebec. 1) INTRODUCTION Les premieres nappes d'hydrocarbures de 1 'Amoco-Cadiz atteignent le littoral de la region de Roscoff et les cotes orientales de la baie de Morlaix le 21 mars, quatre jours apres l'echouage du petrolier sur les roches de Portsall. Alors que les masses d'eau chargees en hydro- carbures plus toxiques transitent rapidement sur 1 'ensemble de la re- gion, des particules oleosedimentaires contaminent les fonds sublitto- raux. Elles se deposent preferentiellement dans les zones calmes et forment localement des poches de mazout residuel; en baie de Morlaix, on note leur presence le 3 avril dans les sables fins peu envases de la Pierre Noire par 20 metres de profondeur et le 13 avril dans les chenaux des rivieres de Morlaix et de Penze (CABIOCH et al., 1978). Durant la meme periode ce phenomene est observe par MARCHAND etCAPRAIS (1981) en baie de Lannion. La connaissance de la composition et de la distribution des com- munautes benthiques de cette region depuis 1968 (CABIOCH) et celle de la dynamique du peuplement des sables fins de la Pierre Noire engagee depuis 1977 (DAUVIN, 1979) permettent une meilleure evaluation des consequences de la pollution sur le macrobenthos subtidal. Ces etudes, financees par la NOAA (contrat 78/5830, 80/6145), entreprises immediatement apres la catastrophe, ont deja fait l'objet de publications : DAUVIN (1979 a, b et sous presse) , CABIOCH et al. (1980, 1981 et sous presse) . 2) LES PEUPLEMENTS ETUDIES En Manche les sequences bio-sedimentaires sont principalement sous le controle de l'intensite des courants de maree; en consequence del 'en- tree vers le fond des baies, les peuplements des sediments grossiers sont progressivement relayes par des peuplements de sediments fins plus ou moins envases. Certains termes de cette sequence ont une distribution spatiale discontinue; tel est le cas des peuplements des sables tins separes par de vastes etendues de nature differente. 205 Les unites cenotigues correspondant aux principaux maillons de cette succession bio-sediraentaire ont ete regulierement echantillon- nees : - les sables gross iers a Venus fasciata - Amphioxus lance- olatus de la baie de Morlaix (au large de la Pointe de Primel; carte 1, P.P.), peu classes avec pour mode la classe 1000 a 5000 microns (55 a 70% du sediment total) : releves trimestriels d'aout 1977 a aout 1980; - le maerl envase de Trebeurden en baie de Lannion (carte 1, L6), tres peu classe, avec pour mode la classe 250 a 500 microns (7 a 58% du sediment total) : releves trimestriels d'avril 1978 a mai 1979; - les sables fins faiblement envases a Abra alba - Hyalino- eaia bilineata des baies de Morlaix et de Lannion (carte 1, P.N., L7, L8) , bien classes avec pour mode la classe 125 a 250 microns (42 a 62% du sediment total) : releves mensuels d'avril 1977 a mars 1981 (P.N.) et trimestriels d'avril 1978 a fevrier 1981 (L7 et L8) ; - les sables tres fins peu vaseux a Tellina fabula - Abra alba en baie de Lannion (sous Saint-Ef f lam; carte 1 : Ll, L2, L3, L4 et L5) , tres bien classes avec pour mode la classe 63 a 125 microns (70 a 78% du sediment total) : releves trimestriels d'avril 1978 a fevrier 1981 sauf L4 d'avril 1978 a mai 1979; - les vases sableuses a Abra alba - Melinna palmata de la riviere de Morlaix (carte 1 : R.M.) ou domine la classe des parti- cules inferieures a 63 microns (47 a 74% du sediment total) accompa- gnee d'une importante fraction de sables tres fins et fins : releves trimestriels d'aout 1977 a fevrier 1981. Les stations de la baie de Morlaix ont ete etudiees par J.C. DAUVIN depuis 1977; Ch. RETIERE et V. RIVAIN ont observe les stations de la baie de Lannion avec la contribution de D. ARCHAMBAULT pour 1' etude de la fin du 3ieme cycle annuel. L" echantillonnage a ete effec- tue parallelement au moyen d'une benne Smith Mc Intyre et d'une benne Hamon (releves : 10 prelevements a la benne Smith Mc Intyre et 4 ou 5 a la benne Hamon; surface echantillonnee lm2 ou plus ) ;le tamisage a ete realise sur une maille circulaire de 1mm. 3) RESULTATS Nous avons suivi 1' evolution des parametres ecologiques richesse specifique, densite et biomasse. Les richesse specifique et densite sont actuellement connues pour la totalite des sites et jusqu'au prin- temps de 1981. II en est de meme pour les biomasses relatives aux stations de Primel et de la riviere de Morlaix; par contre a la Pierre Noire elles ont ete mesurees entre avril 1977 et novembre 1978, puis calculees pour les autres releves ; elles n'ont pas encore ete quanti- fiees pour les stations de la baie de Lannion. 3.1) Caractere du stress Les effets du stress n'ont pu etre evalues ayec precision que sur 206 Ha» Carte 1 - Repartition des stations d'echantillonnages en baie de Morlaix et en baie de Lannion. A B - C D - E Gal fonds rocheux communaute des sediments grossiers a Amphioxus - Venus fasciata, relativement independante de l'etagement (C : facies d'epifaune a Sabellaria spinulosa) . communaute du maerl (D : facies a Lithothamnium corallioides var. corallioides; E : facies a L. covaltioxd.es var. minima] . communaute des sables dunaires fins a Abra prismatica- Glycymeris glycymeris. communaute des sediments fins a Abra alba (G : facies sableux a Hyalinoecia bilineata; H : facies envase a Melinna palmata; I : facies heterogene envase a Vista cvistata ) . communaute des cailloutis et graviers prelittoraux cotiers : facies a Ophiothrix fragilis et a Bryozoaires dominants dans 1 ' encroutement . communaute des cailloutis et graviers prelittoraux du large : facies d ' ensablement a Porella conoinna. 207 les peuplements de la baie de Morlaix pour lesquels nous disposions d' observations juste avant la pollution. Richesse specif ique. (fig. 1) -<- Cycle normal— ►-« 1°Cycle ►-« 2°Cycle — N.especes -yd 100- 50- A.C J A i 1 977 y V "A 'J' A" 'O' 'Dl 1978 ' A' 'O' 'Dl 1979 TTT ^rv 1980 Figure 1 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata : evolution de la richesse specifique des releves (4 prelevements a la benne Hamon) d'avril 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydro- carbures de 1 '"Amoco Cadiz"). A la Pierre Noire la richesse specifique totale passe de 8<> es- peces le ler mars a E ;j cu R. a £- a *■» ^ Co O r-i o -CD "-> tc cy 3.-RT Cj ^ -W ,£: -W 03 •t-. CO *t-i to S. ^_, :; 0 P a O CO O..C O « c a ■*-. U !j tl V O ?. •^ *,J *r4 t3 Ct O R. R. R. ij *> S . <3: ^ ^ <: ^ ^ I I I I I t 1 » V H 3> a rtl CQ E O o ■» ■O Ci) CO CO c cr a O O +> •^ cu •s: a a^ co Co _ 3! C O: ,Q l-l *fJ R. Q l~ CD ^ '<-> rC] ,CA E O O v 1 'Pi <3- Q co co » e <» ^ „ cm J3 TS Cu C3 cj co e f^ O r, r, cd s,a a ^ CJ « 0«^3 ? { (1 c< o o o D. 3)-^ Q, R. R. O fcr i--i i-~> CM a-QM . i K (X to 3 R. R. si R, +i •^ CO / I I I I I I I 1 1 \ I i3 a cu *-> +i 5, CO "" — _ co T-J -ri CD '^ CO CD C3. t^ -Cj 3* !. '»-.'-> K s a r, a +» a 'r-i CO R\ O (1 A tl V c- -— n i i I i v i i n, R7 « a>^ O^S: X u 3 3 CO co ^^ 6-8 OG O O) C^ *- /« u a) 01 •H I— < u CO > M y a CO 3 » • co XI c 4-1 co CO a 4J 01 0 cfl u * 4J cu cu ca CO u c J2 •H o o 0 o CO z, CO 01 01 i — i "0 u u u 01 0) C ^ •H o CO Hi TD U o CO co u c 0) 0 u 01 ■rl »ai tl U) • [X S, J ^ CI R* r-^ ■r- CI -*J ■ ■rl p CI t -•: • - .-: U 3 CO a) i—i Xi CO H 210 -Cycle normal>"< 1 Cycle -►-< 2°Cycle »~< 3uCycle ► 10 000 ■ W 'J' "s' W |j' M W 'J"sf V IJ1 M M 'j' 'sf V |j' W W 'J' 's' 'n' Ij' 'M 1977 1978 1979 1980 'igure 2 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata de la Pierre Noire : evolution de la densite totale d'avril 1977 a fevrier 1981 avec mise en evidence de la part des trois especes d' Ampelisaa tres dominantes (A. a. : Ampelisca avmovioana ; A.s. : Ampelisaa sarsi; A.t. : Ampelisaa tenuiaornis) . (A.C. : debut de la pollution par les hydrocarbures de l'"Amoco Cadiz"). 3.2.1) Peuplement des sediments grossiers a Venus fasaiata - Amphioxus lanceolatus . Pollution (tabl. 2 ) Des le 27 avril les sediments sont contamines par les hydrocarbu- res; aux fortes teneurs enregistrees jusqu'en novembre (231 ppm) suc- cede, en fin d'hiver, une phase de depollution rapide. Richesse specifique (fig. 3) Le flechissement des valeurs de la richesse specifique au cours du premier cycle annuel suivant la pollution semble surtout lie a la disparition de crustaces et de polychetes; cependant, de 1978 a 1980, on note globalement, abstraction faite des fluctuations saisonnieres, un accroissement graduel auquel contribuent largement les amphipodes (48 taxons en aout 1978 contre 80 en aout 1980; 4 especes d' amphipodes en aout 1978 contre 19 en aout 1980) . Densites et biomasses Les densites tres faibles varient de 100' a 290 individus par m2; maximales en aout, minimales en fevrier, les valeurs sont du meme or- dre de grandeur d'une annee a 1' autre. 211 o o> a. • H 0> 1 u i — C 4-1 >w^ o »0> — 1 e • > — o t-i 4-1 Ci B o '_ ~ -K> a c. to •H 01 01 3 -n 0 en 01 to 01 p 01 •H t- >-l M ctj - 0 >-i X! ^H 60 M o o cfl -n 1 — 1 u 0 •H o X ^ )-i 4J -71 •-J jj >, h cfl fi CO D. C - — i 01 01 c | CO 0 Cfl t-l 1— 1 J-l • 3 01 M •~t 01 H9 • i-( i — c i — I 00 01 U) 1—4 0> 4-1 CU • H ■ — t)D e Cfl 3 n 01 0 c Ptj -a u 1 0) i— i c CO CO ►J o U 01 to •H IH » 3 X >ai W cfl 0) c 3 cfl 01 T— I cfl H 212 -<— avant N.especes 100 1°Cycle- ►-«-20Cycle 50 -3uCycle- n. individus • . N T— i — I — r— A O Dl "T 1977 'j' 'a' 'o' 'dI V 'A' 'J' 'a"o' 'Ol V 'A' 'J' 1978 1979 1980 1000 500 Figure 3 - Peuplement des sables grossiers a Venus fasciata - Amphioxus lanceotatus : evolution de la richesse specif ique des releves et du nombre d' individus (10 prelevements a la benne Hamon) d'aout 1977 a aout 1980 (A.C. : debut de la pollution par les hydrocarbures de l'"Amoco Cadiz"). Les biomasses des differentes composantes faunistiques sont tres inegales; par exemple, le lamellibranche Glyoymeris glyoymeris repre- sente 75 % de la biomasse totale du peuplement. Apres avoir depasse 25 g. par m2 en aout et novembre 1977, les valeurs n'oscillent ensuite qu'entre 8 et 12 grammes. Toutefois en novembre 1979, a la suite d'une recolte plus importante de Glyoymeris glyoymeris, elle culmine a pres de 24 g. par m2 rejoignant les valeurs donnees par HOLME (1953) et RETIERE (1979) pour des peuplements analogues de la Manche occidentale. 3.2.2) Peuplement de maerl envase Pollution La texture heterogene des sediments a favorise le piegeage et la retention des particules oleosedimentaires pendant -une longue periode. Richesse specifique Cette biocenose definie par CABIOCH (1968) comme un maerl a Lithothamnium corallioides a evolue vers un nouveau facies, vraisembla- blement sous l'effet d'un ensablement lie a des extractions industriel- les; elle est actuellement tres comparable a celle du maerl de Ricard, en baie de Morlaix : maerl a Lithothamnium oorallioides var. minima. Le caractere cenotique dominant est son extreme appauvrissement; aucune espece d'epifaune sessile n'est en effet presente dans nos echantillons preleves apres la pollution, ce qui atteste sans doute une profonde perturbation. 213 Nous n'avons recolte dans ces fonds qu'un petit nombre d' especes de l'endofaune. On passe toutefois de 23 especes en avril 1978 a 34 en fevrier 1979 et parmi les plus abondantes il convient de citer trois annelides polychetes : Goniada maculata, Staurooepnalus kefersteini, Heteromastus filiformis et deux peracarides : Nototropis swammerdani et Periooulodes longimanus. De plus il faut noter que les deux especes electives du facies heterogene envase, Sthenelais boa et Vista cristata, presentes dans les echantillons d'avril 1978, ont ensuite disparu. Densites L'evolution de la densite globale du peuplement reflete principa- lement celle de quelques populations d' annelides polychetes, en par- ticulier Goniada emerita et Staurooephalus k efersteini dont les re- crutements surmaille de 1 mm s'observent de facon synchrone en au- tomne . La premiere espece benef icierait de la proximite de biotopes servant de "reservoirs" a partir desquels se realiserait la disper- sion des larves pelagiques. En conclusion il est important de rappeler que depuis 1968, le peuplement macrobenthique de ces fonds a evolue, vraisemblablement sous l'effet d'un ensablement lie a 1' extraction du maerl. Ne dis- posant d'aucun etat de reference juste avant la catastrophe il est extremement difficile de connaitre la part qui revienta la pollution par les hydrocarbures, dans la modification de cette communaute. Pour cette raison son suivi ecologique a ete rapidement abandonne. 3.2.3) Peuplements des sediments fins a Abra alba 3.2.3.1) Peuplement des sables fins a Abra alba - Hyalinoecia bilineata Pollution Apres une phase initiale de forte pollution, que nous n'avons pu mesurer, les teneurs en hydrocarbures (mesurees en spectrophotometrie aux infra-rouges, exprimees en mg par kilogramme de sediment sec ou ppm) atteignent 1000 ppm a la station L8 au cours de l'ete 1978 alors qu'elles ne depassent pas 50 ppm aux deux autres stations (L7 et P.N.), puis elles diminuent fortement au cours de l'automne dans 1' ensemble des stations. Ensuite une remobilisation, lors des tempetes automnales et hivernales, des stocks d'hydrocarbures pieges en zone intertidale entraine une augmentation des teneurs en hydrocarbures au printemps 1979 sur 1' ensemble des stations, (ces teneurs depassent 200 ppm a la station de la Pierre Noire) ; elles decroissent rapidement ensuite, sauf en L8 ou elles se maintiennent a des valeurs voisines de 100 ppm jus- qu'a l'automne 1979. Enfin, dans l'ensemble des stations, apres une phase de recontamination au cours de l'hiver 1979-1980, les teneurs de- viennent inferieures a 50 ppm a partir de mai 1980. Richesse specifique Apres le stress on note dans les trois stations un enrichissement progressif en especes qui aboutit en 1980, a la Pierre Noire, a des va- 214 leurs du meme ordre qu'avant la pollution. Cette remontee provient d'une part de la reapparition d'especes eliminees lors du stress, d'au- tre part de 1* augmentation de la frequence de quelques especes de poly- chetes et de raollusques. Les memes phenomenes s 'observent en baie de Lannion (L7 et L8) , mais avec des valeurs inferieures. La comparaison des presences d'especes dans les trois stations apporte a cet egard des informations signif icatives dans le cas des amphipodes, groupe le plus affecte par le stress initial. Certaines especes, qui ont survecu en baie de Morlaix, sont absentes en baie de Lannion immediatement apres la maree noire, mais elles y apparaissent au cours du premier cycle annuel suivant le stress (Leuaothoe incisa, Urothoe grimaldii, Tryphosites lon- gipes, Bathyporeia tenuipes) , du deuxieme cycle (Ampelisca sarsi) ou du troisieme (Bathyporeia elegans, Synohelidium maculatum) .Cet- te constatation temoigne de leur presence probable en baie de Lan- nion avant la pollution et renforce l'hypothese d'une plus grande intensite de 1' impact initial dans cette baie par rapport a la baie de Morlaix (CABIOCH et at., 1981) . Des especes eliminees temporairement par le stress en baie de Morlaix et qui s'y reinstallent au cours du premier cycle pertur- be (Megaluropus agilis, Ampelisca spinipes) ou de deuxieme (Chei- rocratus intermedins) (DAUVIN, 1981) ne sont rencontrees dans les sables fins plus lonquement pollues de la baie de Lannion qu'au cours du troisieme cycle. Ces introductions comme les precedentes, inter- viennent generalement plus tot en L7 qu'en L8, station la plus pol- lute des deux. Les especes dont on est certain de 1 ' "insularite" figurent parmi celles qui se reinstallent plus tardivement dans l'une ou 1' autre des baies (Ampelisca tenuicornis 3 A. brevicornis, A. ar- moricana) ou qui n'ont pas encore reapparu (Photis longicaudata) . A 1' oppose, Ampelisca spinipes, espece non insulaire, commune dans les sables grossiers avoisinant la zone polluee, repeuple les sables fins de la Pierre Noire des le premier cycle. En conclusion, les effets eliminateurs du stress, plus ou moins accuses selon les localites d'un meme type de peuplement sem- blent accompagnes de reimplantations d'autant plus tardives que la pollution residuelle du sediment est durable et intense. La con- jugaison de 1' insularite geographique des populations de certaines especes et de leur mode direct de reproduction a aussi un effet re- tardateur . Densites et biomasses En baie de Morlaix, a la station de la Pierre Noire la densi- te moyenne du peuplement passe de 19450 individus par m2 lors du cycle normal a 2135 au cours du premier cycle apres la pollution. Cette tres grande difference est le fait de la disparition et de la forte reduction d'effectifs de trois especes d' Ampelisca largement dominantes avant la pollution. Durant les deuxieme et troisieme cy- cles annuels les densites moyennes atteignent les valeurs respectives de 3650 et 4110 individus par m2. 215 Parallelement a la forte decroissance des densites,la biomasse subit une reduction de pres de 50 % pendant le premier cycle annuel apres la pollution (4.4 g. par m2 contre 8.1). Des le second cycle elle retrouve des valeurs comparables a celles du cycle normal (8.0 g. par m') et les depasse meme au cours du troisieme cycle an- nuel (8.7 g. par m2) . Ainsi, trois ans apres le stress, bien que les densites soient encore inferieures, d'environ 80 %, a celles observees avant la pollution, les valeurs de biomasse sont du meme ordre de grandeur que celles du cycle normal. En effet les especes subsistant apres le stress, dont les densites sont passees de 2100 a 3860 individus par m2 du premier au troisieme cycle annuel apres la pollution, ont des poids individuels moyens tres super ieurs a celui des ampeliscides . En baie de Lannion 1' evolution de la densite globale du peuple- ment est fortement marquee par les variations d'abondance, d'ailleurs difficiles a interpreter, d'une seule espece, Paradoneis armata. La densite de celle-ci diminue progressivement a la station L8 pour at- teindre 500 individus par m2 en Janvier 1981 et se maintient a un ni- veau compris entre 500 et 600 individus par m2 a la station L7 pendant la periode d'observation. II faut toutefois noter qu'au cours des trois cycles annuels la densite correspondant a 1 'ensemble des autres especes tend a s'accroitre. Les reintroductions d'especes temporairement eliminees appor- tent tres peu a cette croissance de la densite a moyen terme apres le le stress. Elle resulte principalement de quelques cas de £f52lo.ni§5~ tion par des especes reduites en effectifs pendant le premier cycle perturbe et de P£2life£gtion d'especes non affectees par la pollution. Les autres especes poursuivent des cycles annuels peu differents du cycle normal. Les recolonisations signif icatives sont le fait de trois espe- ces : Ampelisca sarsi, Ampharete aoutifrons, Nephtys hombergtt. Alors que la communaute des sables fins de la Pierre Noire a pu heberger jusqu'a 40.000 Ampelisaa par m2, l'espece subsistante, A. sarsi ne re- colonise cette vaste niche ecologique vacante qu'a une cadence res- treinte (fig. 4) de par la conjonction de sa distribution "insulaire" et de ses caracteres biologiques (reproduction directe printaniere et estivale, femelles porteuses de 8 a 20 embryons seulement et ne se reproduisant qu'une fois, vie breve ne depassant guere un an (DAUVIN, 1979)). Limitee par 1' insular ite au seul potentiel reproducteur de sa population residuelle, l'espece multiplie cependant son effectif ma- ximum annuel par un facteur de 5 a 9 d'une annee sur 1' autre ce qui temoigne du succes de la reproduction directe. Ampharete aautifrons, (fig. 5) insulaire, de duree de vie inferieure a deux ans, a larve presque immediatement benthique, suit un schema de recolonisation len- te du meme type. Par contre, le repeuplement de Nephtys hombergii (vie longue, non insularite, larves pelagiques pendant plus d'un mois) s'effectue rapidement (fig. 6); des 1980, les effectifs estivaux, qui ne depassaient pas 30 individus par m2 en 1978, atteignent 170 par in2, valeur superieure a celle observee avant pollution (90 par m2) . En ce qui concerne les proliferations, la breve poussee d' Hete- rocirrus alatus a l'automne de 1978 (fig. 7) est suivie, au cours du deuxieme et du troisieme cycle par des accroissements importants de Chaetozone setosa, (Fig. 7), Spio filioornis, Saoloplos avmiger, Thyasira flexuosa, Abra alba. Ainsi se dessine probablement un pre- mier element d'une serie de "successions" (PEARSON & ROSENBERG, 1978) , phenomene moins immediat et moins accuse ici que sur les fonds sublittoraux bien plus pollues des Abers (GLEMAREC & HUSSENOT, 1981; GLEMAREC & HUSSENOT, sous presse) . 216 N/m 10 10 10 10 • -* Cycle normals — >~-< TCycle Figure 4 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata de la Pierre Noire : evolution de la densite d1 Ampelisca sarsi d'avril 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de 1 '"Amoco Cadiz"). -*- — CYCLE NORMAL - *••• N.m-2 1'CYCLE 2'CYCLE »-• 3'CYCIE - •- 200 . 100 - AC- \ \ \ I \ / \ ' / \ ' \ I a l\ I \ '\-l I \ /M\ i i i i i i i i i i i < M J S M I J 1977 ■ i rnVVi rr '."Fo-r. *• I-*' 7 AAkZ/ v~.\Ll J s'- • " v.PN I I I I I I I IT I I I I I I I I I I I I I I I I I I I I I I I J snIjmvj inIj MMJ $ N Ij 1978 1979 1980 Figure 5 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata : evo- lution de la densite d1 Ampharete grubei d'avril 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de l"'Amoco Cadiz") 217 -Cycle normal-*-- <- 1°Cycle >— «— 2° Cycle >~< 3°Cycle >- N .m 150- 100- 50 - rvr1 W 'J' 's' V ij' M M 'j' 's' 'n' Ij" W M 'j' 's' W Ij' 'm' W 'j' 's' 'n' 'j' 'iW 1977 1978 1979 1980 Figure 6 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata : evolu- tion de la densite de Nephtys hombergii d'avril 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de 1 '"Amoco Cadiz") ■"< 1°Cycle »— < 2°Cycle >— * 3°Cycle 1000 - 100 - m' tv? 'j' y W Ij' M W '/ 's' ' '*' |j' W W 'j' 's' W |j' 'm' W 'j' 's' W I 'f' 1977 1978 1979 1980 Figure 7 - Peuplement des sables fins a Abra alba - Hyalinoecia bilineata : evolu- tion schematique de la densite d' Abra alba (A. a.), de Chaetozone setosa (C.s.), d' ' Heterocirrus alatus (H.a.), de Scoloplos armiger (S.a.), de Spio filicornis (S. f.) et de Thyasira flexuosa (T.f.). (A.C. : debut de la pollution par les hydrocarbures de 1 ' "Amoco Cadiz"). 218 3.2.3.2) Peuplement des sables tres fins a Tellina fabula - Abra alba Pollution Les teneurs en hydrocarbures apres un pic passager en aout 1978 (premieres mesures) redeviennent fortes de fevrier a mars 1979 (valeurs comprises entre 80 et 300 ppm) . A la faveur d'une recontamination du sediment au cours de l'automne 1979, les teneurs depassent de nouveau 50 ppm en novembre (70 a 16 5 ppm) elles se maintiennent a ce niveau en L2 et L3 au cours de l'hiver 1980, puis redeviennent inferieures a par- tir de mars 1980 dans 1' ensemble des trois stations. Richesse specifique et densites4 On sait indirectement que ce peuplement a subi une perturbation considerable lors du stress; les immenses quantites d' Eehinocardium oordatum et de mollusques de diverses especes, rejetes rnorts sur la greve de St-Efflam en mars-avril 1978 en temoignent (CHASSE & GUENO- LE-BOUDER, 1981) . Les phenomenes observes a la suite du stress pre- sentent de grandes analogies avec ceux que nous venons de decrire : croissance a moyen terme de la richesse specifique (fig. 8) et de la densite totale, phenomenes de recolonisation. ~* CYCLE NORMAL N especes I'CYCLE 2'CYCLE h 3'CYCLE - »• SO. 25 AC I I I I I r r < J I N ~n i ■ i i i i ~i r i i r T7 • r i i j • Figure 8 - 1977 1978 I9>« 1980 Peuplement des sables tres fins a Tellina fabula - Abra alba : evolution de la richesse specifique des releves (4 prelevements a la benne Hamon) d'avril 1978 a fevrier 1981 (A.C. : debut de la pollution par les hydro- carbures de 1 '"Amoco Cadiz"). *Les resultats du suivi de la station Ll dont le peuplement presente un caractere nettement intertidal ne sont pas integres dans ce travail qui a pour objet 1' etude des communautes subtidales. De meme le suivi de la station L4 a ete abandonne en mai 1979, le depouillement des donnees n'apportait pas d' informations complementaires de celles recueillies aux stations L3 et L5. 2iq La croissance de la densite est principalement liee a la proli- feration, depuis la perturbation, du Capitellidae Mediomastus fragilis (fig. 9) dont les effectifs a la station L3 passent de 100 individus par m2 en avril 1978 a plus de 7000 par m2 en mai 1980. N.m-2 104-, •m 1'CTCLE J'cyci r 3*CYCLE „ 10J- 10 . AC I ' i ' 'l 1 I I I I I 1978 i i I i — r i i i i — r j s /L 1979 I I I I 1 I 1980 Figure 9 - Peuplement des sables tres fins a Tellina fabula - Abra alba : evolution de la densite de Mediomastus fragilis d'avril 1978 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de l"'Amoco Cadiz"). Les recolonisations signif icatives sont ici le fait de trois es- peces : Nephtys hombergii, Glycera convoluta et Tellina fabula; mais alors que les deux premieres voient leurs effectifs augmenter progres- sivement d'annee en annee (respectivement de 63 et 80 individus par m2 en 1978 a 162 et 360 en 1981) 1 ' abondance de Tellina fabula qui n'a pas depasse 250 individus par m2 en 1978 et 1979, s'eleve brusquement a plus de 1000 individus par m2 pendant le troisieme cycle. 3.2.3.3) Peuplement des vases sableuses a Abra alba - Melinna palmata Pollution Les teneurs en hydrocarbures sont tres elevees jusqu'en juillet 1979 (elles depassent toujours 100 ppm sauf en fevrier 1979 et elles atteignent meme 3000 ppm en mars 1979) ; ensuite on observe une depol- lution graduelle. 220 Richesse specifique (fig. 10) D'avril 1978 a avril 1980 la richesse specifique est stable; elle s'accroit considerablement au cours de l'ete 1980, diminue ensuite et se maintient durant l'hiver suivant a un niveau plus eleve que celui des hivers precedents. L1 augmentation de la richesse specifique provient a la fois de la reintroduction d'especes d'amphipodes eliminees lors du stress et de 1' accroissement du nombre d'especes de polychetes . •m CTCLI nohmai - «»4— t'C»CLI - • 2'CYCll «-• ■ - 3'ctcif - •• N especes 1 00 50. AC • ■ _. i i i i i i i w W J s 1977 I J M I I I I I J 1978 I J k I I I I I I 1 H J S I 1979 T-r i i i i i i i i 1980 -m Figure 10 - Peuplement des vases sableuses a Abra alba - Melinna palmata : evolution de la richesse specifique des releves (10 prelevements a la benne Smith Mc Intyre) d'aout 1977 a mars 1981. Les reapparitions d'amphipodes se realisent de maniere graduelle : les premiers exemplaires de Cheirocratus intermedins et Ampelisca breviaornis sont recoltes plus d'un an apres leur disparition, ceux d' Ampelisca tenuicornis seulement au cours du second cycle; les especes Ampelisca armoricana et Ampelisca spinimana n'ont pas encore ete retrou- vees. En ce qui concerne les polychetes on observe conjointement la cap- ture plus frequente d'especes sporadiques avant la pollution et 1' intru- sion d'especes constantes, pour la plupart, dans le peuplement des sa- bles fins a Abra alba - Hyalinoecia bilineata de la Pierre Noire. Densites et biomasses La densite qui n'est pas modifiee lors du stress croit au cours du premier cycle annuel perturbe : 4467 individus par m.2 en moyenne contre 2855 durant le cycle normal avant la pollution. Cette difference est due essentiellement aux fluctuations d'effectifs d'especes presentes, avant la pollution, en densites faibles (Mediomastus fragilis, fig. 12 et Tharyx marioniy fig. 13) ou fortes (Chaetozone setosa, fig.il). 221 N .m Cycle normal 2 -1° Cycle ->—< 2°Cycle ->~<- 3° Cycle- 6000 5 000 4 000 3 000 2 000- 1000 1979 1980 Figure 11 - Peuplement des vases sableuses a Abra alba - Melinna palmata : evolution de la densite totale d'aout 1977 a mars 1981 avec mise en evidence de la part de Chaetozone setosa (C.s.) (A.C. : debut de la pollution par les hydrocarbures de 1 '"Amoco Cadiz"). Au cours du second cycle, la densite redevient voisine de celle du cycle normal, puis augmente a nouveau durant le troisieme cycle : 3724 individus par m2; cette derniere valeur s'explique par le haut niveau d'abondance de Mediomastus fragilis et Tharyx marioni, par une elevation de la densite d'autres especes de polychetes notamment Lanice conohilega et Melinna palmata et enfin par la recolonisation de Nephtys hombergii et Ampelisaa tenuicovnis . — - CYCLE NORMAL- «--• I* CYCLE »--. 2'CYCLE ,-2 3'CYCLE - •- io a 250 . A-C- i i i i i * J s 1977 I I I I 1 I I I I I I I I I I Mj snIjmuj 1978 1979 I I I TT I I I I 1 I I I | S N I J M U.J S 1980 Figure 12 - Peuplement des vases sableuses a Abra alba - Melinna palmata : evolution de la densite de Mediomastus fragilis d'aout 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de 1 '"Amoco Cadiz"). 222 -• CYCLE NORMAL N.m-2 300^ 250 I'CYCLE 2*CYCLE TCYCLE «- 200- 150 100 50 / A-C. ' ' i i i ' ' ■ I n i r i i i i i i i [ t i i i i i i i i i i r i i i i i i i i i i i i i i ■' * 1 S H I J M M J InIjUUJ t m I j UMJ ( m I j 1177 1978 ' 3 .'9 {'ISO Figure 13 - Peuplement des vases sableuses a Abra alba - Melinna palmata .- evolution de la densite de Tharyx mavioni d'aout 1977 a fevrier 1981 (A.C. : debut de la pollution par les hydrocarbures de 1' "Amoco Cadiz"). L1 accroissement des biomasses moyennes correspondant aux quatre cycles annuels d 'observations est lie surtout a 1' installation progres- sive de Lanice conchilega dans le peuplement. De 1977 a 1980 les valeurs relatives aux observations effectuees entre les mois d'aout et avril sont respectivement 9.0, 13.0 et 12.4 g. par mz; pour la periode comprise entre aout 1980 et mars 1981 la biomasse moyenne atteint 16.8 g. par m . Les valeurs moyennes de densite (3425 individus par m2) et de bio- masse (12.9 g. par m2) s'accordent avec celles donnees par les auteurs travaillant sur des peuplements analogues (RETIERE, 1979) . Au terme de 1' etude des peuplements de sables fins vaseux il im- porte de souligner que les modalites quantitatives des divers pheno- 223 menes qui se succedent dans le peuplement a Abra alba - Melinna pal- mata de la rade de Morlaix different profondement de celles observees dans le peuplement a Hyalinoecia bilineata de la Pierre Noire. Dans le oremier, ies especes sensibles aux hydrocarbures sont en effet peu representees avant la pollution, si bien que les mortalites initiales sont faibles et n'alterent que legerement la structure du peuplement, contrairement a la modification structurale considerable intervenue a la Pierre Noire. L'evolution ulterieure des deux peuplements a moyen et long terme off re un contraste d'une autre nature. La communaute subsistante de la Pierre Noire poursuit une dynamique de reconstitu- ting dans un milieu benthique rapidement decontamine, et naturelle- ment oligotrophe. Au contraire, le peuplement de la rade de Morlaix vit dans un milieu benthique naturellement eutrophe, plus durablement charge d'hydrocarbures et de matieres organiques. On assiste alors a un developpement plus important des populations de detritivores , de- ja abondants en conditions naturelles (Chaetozone setosa) et aussi a de veritables proliferations d'especes reellement opportunistes (Medio- mastus filiformis et Tharyx marioni sp.), presentes seulement a l'etat latent au cours du cycle normal precedant la pollution. 4) CONCLUSIONS GENERALES Le recul qu'apportent trois annees d' observations permet de distin- guer parmi les phenomenes qui ont affecte les peuplements subtidaux de la region de Roscoff ceux lies au stress proprement dit de ceux qui se sont succedes au cours des cycles annuels suivants et d'en interpreter les differentes modalites : - 1' elimination des especes lors du stress est selective; elle est fonction a la fois de 1 ' eco-ethologie des especes et des con- centrations du milieu en hydrocarbures toxiques dissouts. Cette phase de mortalite est relativement limitee dans le temps (quelques semaines); - l'intensite des perturbations dues au stress varie d'une communaute a 1' autre le long du gradient edaphique et a 1' inter ieur de la mime unite de peuplement. Alors que les peuplements des sedi- ments grossiers et des sables vaseux ont ete peu modifies qualitative- ment et quantitativement par le stress, ceux des sables fins et tres fins ont ete intensement perturbes. Ces divers degres d' alteration sont fonction du nombre et de l'abondance des especes sensibles aux hydrocarbures; dans le cas extreme du peuplement des sables fins a Abra alba - Hyalinoea-ia bilineata on assiste a une reduction respecti- ve de 80 et 50 % des valeurs initiales de densite et de biomasse. Tou- tefois il convient de noter que ce n'est pas necessairement sur le peu- plement ou le stress a ete le plus devastateur que les effets secondai- res sont les plus marques; - dans les biotopes ou les effets du stress se sont faits sentir severement les valeurs de la richesse specifique, de la densite et de la biomasse restent faibles pendant le premier cycle annuel apres la pollution; on n'enregistre pas, au cours de cette periode, de morta- lites massives d'adultes mais 1' absence de recrutement chez un certain nombre d'especes freine considerablement la recolonisation du milieu. Dans la plupart des cas, durant cette premiere phase, les effets sont 224 propor tionnels aux quantities d'hydrocarbures residuels; - le deuxieme cycle annuel marque globalement le debut de la reintroduction des especes eliminees et de la recolonisation des fonds par celles dont les populations ont ete affectees par le stress. Ces phenomenes s'accelerent et s'accentuent au cours du troisieme cy- cle. La vitesse de reintroduction et le taux de recolonisation de certaines d'entre elles sont d'ailleurs limites par le caractere insu- laire de leur distribution et l'absence de phase pelagique; - les cycles de densite de la plupart des especes qui n'ont pas ete affectees par le stress se deroulent a peu pres normalement; par contre un petit nombre d' especes regroupant surtout des Cap-itelli- dae et Cirratulidae proliferent soit au cours du premier cycle, soit plus tardivement, constituant probablement les amorces d'une succes- sion ecologique; - trois ans apres la pollution par les hydrocarbures la ri- chesse specifique du peuplement le plus perturbe , c'est-a-dire celui des sables fins peu envases, a retrouve son niveau initial et bien que sa densite soit toujours beaucoup plus faible qu'elle ne l'etait aupa- vant les valeurs de biomasse sont a nouveau tout a fait comparables a celles de 1977. II semble done que ce peuplement evolue vers un "nou- vel equilibre"; En outre, de cette etude se degagent un certain nombre d'enseigne- ments : - les cartes bio-sedimentaires, support des etudes dynami- ques, constituent un etat de reference dont l'interet est evident dans le cas de pollutions accidentelles du type "Amoco-Cadiz" ; - sur 1' ensemble du secteur touche par la pollution les premieres investigations doivent etre engagees tres rapidement; au sein des communautes presumees les plus sensibles il est indispensable de selectionner plusieurs stations, le suivi de certaines d'entre elles pouvant etre abandonne a la lumiere des premiers resultats; - le suivi ecologique doit s'etaler sur une periode suffi- samment longue pour qu'au dela du bruit de fond des fluctuations natu- relles a plus ou moins long terme on puisse percevoir les phenomenes reellement dependants de la perturbation. 225 BIBLIOGRAPHIE BESLIER, A., J.L. BIRRIEN, L. CABIOCH, C. LARSONNEUR S L. IE BORGNE, 19B0. 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GENTIL, C. RETIERE & V. RIVAIN, 1981. Perturbations induites dans la composition et le f onctionnement des peuplements ben- thiques sublittoraux sous l'effet des hydrocarbures de 1' Amoco Cadiz. In : Consequences d'une pollution accidentelle par les hydrocarbures. Centre National pour 1 ' Exploitation des Oceans, Paris, pp. 513-525. CABIOCH, L., J.C. DAUVIN, C. RETIERE, V. RIVAIN & D. ARCHAMBAULT, 1982. Evolution a long terme (1978-1981) de peuplements benthiques des fonds sedimentai- res de la region de Roscoff, perturbes par les hydrocarbures de 1' Amoco Cadiz. Neth. J. Sea Research (sous presse) . CHASSE, C. & A. GUENOLE-BOUDER, 1981. Comparaison quantitative des populations benthiques des plages de St Eff lam et St Michel-en-Greve avant et depuis le naufrage de l'Amoco Cadiz. In : Consequences d'une pollution acciden- telle par les hydrocarbures. Centre National pour 1 ' Exploitation des 0- ceans, Paris, pp. 513-524. DAUVIN, J.C, 1979a. Impact des hydrocarbures de l'Amoco Cadiz sur le peuplement inf ralittoral des sables fins de la Pierre Noire (Baie de Morlaix). J. Rech. oceanogr.. Vol. 4 (1), pp. 28-29. DAUVIN, J.C, 1979b. Recherches quantitatives sur le peuplement des sables fins de la Pierre Noire, Baie de Morlaix, et sur sa perturbation par les hy- drocarbures de l"Amoco Cadiz". These 3eme cycle Oceanographie Biologique, Universite P. & M. Curie, 251 pp. DAUVIN, J.C, 1981. Evolution a long terme des populations d'Amphipodes des sa- bles fins de la Pierre Noire (Baie de Morlaix) apres 1' impact des hydro- carbures de l'Amoco Cadiz. J. Rech. oceanog., Vol. 6 (1), pp. 12-13. DAUVIN, J.C, 1982. Impact of Amoco Cadiz oil spill on the muddy fine sand Abra alba and Melinna palmata community from the Bay of Morlaix. Estua. coast, Shelf Science (in press). 226 Den HARTOG, C. 8 R.E.W.H. JACOBS, 1980. Effects of the Amoco Cadiz oil spill on an eelgrass community at Roscoff (France) with special reference to the mobile benthic fauna. - Helgoland, wiss. Meeresunters, Vol .33 , pp. 182- 191. ELMGREN, R., S. HANSSON, U. CARSSON & B. SUNDELIN, 1980. Impact of oil on deep soft bottoms. In : J.J. KINEMAN, R. ELMGREN & S. HANSSON. The Tsesis oil spill U.S. Department of Commerce, NOAA, pp. 97-126. ELK.AIP1, B., 1981. Effets de la maree noire de 1' Amoco Cadiz sur le peuplement sublittoral de l'estuaire de la Penze. In : Consequences d'une pollution accidentelle par les hydrocarbures. Centre National pour 1 'Exploitation des Oceans, Paris, pp. 527-537. GENTIL, F. S L. CABIOCH, 1979. Premieres donnees sur le benthos de l'Aber Wrach (Nord Bretagne) et sur 1 ' impact des hydrocarbures de 1' Amoco Cadiz. J. Rech. oceanogr.. Vol. 4 (1), pp. 33-34. GLEMAREC, M. S E. HUSSENOT, 1980. Definition d'une succession ecologique en mi- lieu anormalement enrichi en matieres organiques a la suite de la ca- tastrophe de l'Amoco Cadiz. In : Consequences d'une pollution acciden- telle par les hydrocarbures. Centre National pour 1 ' Exploitation des Oceans, Paris, pp. 499-512. GLF-VlAREC, H. & E. HUSSENOT, 1982. Ecological survey for the three years after Amoco Cadiz oil spill in Benoit and Wrac'h Abers. Neth. J. Sea Research ( in press] . HOLME, N.A., 1953. The biomass of the bottom fauna in the English Channel off Plymouth. J. mar. biol. Ass. U.K., Vol. 32, pp. 1-49. LEE, W.J. & J.A.C. NICOL, 1978. Individual and combined toxicity of some petro- leum aromatics to the marine Amphipod Elasmopus pectenicrus. Mar. Biol.. Vol. 48, pp. 215-222. LEE, W.H., M.F. WELCH & J.A.C. NICOL, 1977. Survival of two species of amphipods in aqueous extracts of petroleum oils. Mar. Pollut. Bull ..vol .8, pp. 92-94. LINDEN, 0., 1976. Effects of oil on the amphipod Gammavus oceanious. Environ. Pollut., vol. 10, pp. 239-250. MARCHAND, Y\. & V\.P. CAPRAIS, 1981. Suivi de la pollution de l'Amoco Cadiz dans 1 ' eau de mer et les sediments marins. In : Consequences d'une pollution accidentelle par les hydrocarbures. Centre National pour 1' Exploitation des Oceans, Paris, pp. 23-54. PEARSON, T.H. 8 R. ROSENBERG, 1978. Macrobenthos succession in relation to orga- nic enrichment and pollution of the marine environment. Oceanogr. mar. Biol. Ann. Rev., Vol. 16, pp. 229-311. RETIERE, C, 1979. Contribution a 1' etude des peuplements benthiques du golfe normanno-bretcn. These doctorat d'Etat, Sci. Nat., Univ. Rennes,370 pp. 227 SANDERS, H.L.. J.F. GRASSLE & G.R. HAMPSON, 1972. The West Falmouth oil spill. Woods Hole oceanogr. Instn. Tech. Rep. 1-72-20, 48 pp. SANDERS, H.L., J.F. GRASSLE, G.R. HAMPSON, L.S. MORSE, S. GARNER-PRICE S C. JONES, 1980. Anatomy of an oil spill : long-term effects from the grounding of the barge Florida off West Falmouth, Massachusetts. J. mar. Res. , Vol. 38, 265-380. 228 ETUDE EXPERIMENTALE D'UNE POLLUTION PAR HYDROCARBURES DANS UN MICROECOSYSTEME SEDIMENTAIRE. I : EPPET DE LA CONTAMINATION DU SEDIMENT SUR LA MEIOFAUNE par Boucher G., Chamroux S., Le Borgne L. et Mevel G. Station Biologique de Roscoff 29211 (FRANCE) RESUME Les consequences de deux niveaux de contamination par hydrocarbu- res ont ete analysees, par rapport a un temoin, dans des microecosyste- mes experimentaux en circuit clos contenant 100 litres de sable fin sublittoral. L'evolution des caracteristiques du peuplement de meio- faune (Nematodes et Copepodes) a ete choisie pour caracteriser 1 ' im- pact des hydrocarbures. Les densites des Nematodes augmentent sensi- blement par rapport au temoin pendant les deux premiers mois de la pollution puis regressent lentement sans qu'il soit possible de dis- tinguer les effets d'une forte pollution de ceux d'une faible pollu- tion. Les densites des Copepodes harpacticoides sont d'autant plus faibles que le sediment est plus contamine. Le rapport Nematodes/Co- pepodes parait etre un indice significatif du degre de pollution. La composition faunistique des Nematodes est profondement roodi- fiee dans le module le plus pollue apres 3 mois d' experience. Cette degradation se manifeste par une chute brutale de la biomasse et de la diversite specifique. Des petites especes opportunistes connues pour leur association avec 1 ' enrichissement en matiere organique, de- viennent dominantes. Le module faiblement pollue ne presente aucune degradation interpretable, par rapport au temoin, des parametres du peuplement. * Ce travail a ete realise avec l'aide d'un contrat n° 80/6189 passe entre : le Centre National de la Recherche Scientif ique, le Centre National pour 1 'Exploitation des Oceans et la National Oceanographic and Atmospheric Agency (USA). II a fait l'objet d'une presentation au Seminaire AMOCO CADIZ CNEX0-N0AA : "Bilan des etudes biologiques de ta pollution de I' Amoco Cadiz" organise au Centre Oceanologique de Bretagne Brest (France) les 28 et 29 octobre 1981. 229 INTRODUCTION Les consequences des pollutions sur 1 'environnement sont souvent diff icilement interpretables car leur dilution dans un milieu complexe peut provoquer des alterations .plus ou moins discernables des fluctua- tions naturelles, apparaissant immediatement apres contamination ou differees dans le temps. L' experimentation en laboratoire de la toxicite des polluants sur des organismes isoles de leur environnement naturel a montre sou- vent ses limites car elle ne prend pas en compte les effets cumulatifs. Par contre, les simulations sur des microecosystemes complexes appeles microcosmes ou mesocosmes selon leur taille, permettent de mieux cer- ner les consequences des perturbations des ecosystemes et souvent de completer les observations realisees dans le milieu naturel. L'utili- sation des microcosmes permet en outre d'integrer les interactions entre les niveaux trophiques d' organisation des ecosystemes par exem- ple et de realiser des manipulations et des replications. Quelques rares simulations au laboratoire de contaminations par hydrocarbures ont jusqu'ici ete realisees soit pour envisager les vi- tesses de degradation des hydrocarbures en milieu sedimentaire com- plexe (Johnston, 1970; Delaune et coll. 1980; Wade & Quinn 1980) soit pour comprendre les effets sur les organismes dans les differents ni- veaux d' organisation de l'ecosysteme (Lacaze 1979; Elmgren et coll. 1980; Grassle et coll. 1981; Elmgren & Frithsen, sous presse) . A la suite de la pollution petroliere de 1' "Amoco Cadiz" sur les cotes de Bretagne Nord (Manche occidentale) , nous nous sommes attaches parallelement a 1 'etude in situ des consequences de la contamination des sables fins sublittoraux (Boucher 1980 et 1981; Boucher, Chamroux et Riaux 1981), a realiser une simulation du phenomene dans des micro- ecosystemes en circuit clos. MATERIEL ET METHODES Trois modules experimentaux en circuit clos dont le principe a ete fourni dans Boucher et Chamroux (1976) ou Mevel (1979) sont uti- lises (Figure n° 1). Chaque bac comporte trois compartiments dont les niveaux sont regules par contacteur electrique (500 litres d'eau de mer du large). Le compartiment principal comporte 100 litres de sable reparti sur un double fond sur une surface de 0,41 m2 et une hauteur de 25 cm environ,, et percole par difference de niveau entre les compar- timents a une vitesse de filtration de l'ordre de 15 l/m2/heure. Le sediment est preleve a la benne Smith-Mclntyre en milieu sublittoral par - 19 metres de profondeur (Station de la Pierre Noire). Sa media- ne est de 136 ± 5 p. La temperature pendant le duree de 1' experience a varie entre 10 et 15°C. Les trois mesocosmes ont ete nourris tous les deux jours par des casaminoacides de DIFCO a des doses correspon- dant a 50 g d'Azote/an/m2 . L'un des problemes importants a resoudre etait le mode d' intro- duction des hydrocarbures dans les modules experimentaux. Respective- ment 100, 10 et 0 g. d 'hydrocarbures Arabian light»etetes a 240°C > 230 I. Pump2 Pumpl HL_Q I § ,1 i I 1 ■ * Water 500 1. Lsulslqasulqasul/ rid iqo I. I cooler amplifier pH_rH recorder pH_rH 0,66x0, 98m = 0, 41 m 2 FIGURE 1. Schema d'un bac experimental ou mesocosme utilise pour les simulations de pollution par hydrocarbures (Volume de sable 100 litres, Volume d'eau : 500 litres). (fournis par l'IFP) ont ete melanges a 1 kg de sable sec et homogenei- ses avec 100 ml de tetrachlorure de Carbone. Apres evaporation totale, le sediment ainsi traite a ete ajoute respectivement dans chacun des trois modules appeles : Bac fortement pollue, Bac faiblement pollue et Temoin. Le coulage des hydrocarbures a ete ainsi quasi immediat et l'essentiel des particules contaminees s'est reparti a la surface du substrat. Une faible fraction est restee a la surface de l'eau conte- nue dans le compartiment a sable du bac le plus pollue pendant quel- ques jours. Les prelevements dans chacun des mesocosmes ont ete realises a l'aide de tubes de carottages en plexiglass de 5,72 cm* Trois prises simultanees ont ete effectuees pour les hydrocarbures et les compta- ges de meiofaune avec une frequence hebdomadaire puis mensuelle, pen- dant plus de six mois du 17 mars 1981 au 29 septembre 1981. Chaque carottage a ete fractionne en trois niveaux : 0-4; 4-8; 8-12 centime- tres pour analyse de la repartition verticale des parametres. Les hydrocarbures ont ete extraits au tetrachlorure de Carbone a partir de 10 grammes de sediment seche a 60°C a l'etuve. Apres pas- sage sur Fluorisil, destine a eliminer les fractions oxydees et les hydrocarbures endogenes, l'extrait a ete lu au spectrophotometre in- frarouge UNICAM SP 1100, a une longueur d'onde comprise entre 2500 et 3000 cm" , avec calage du pic caracteristique a 2925 cm-1. Les resul- tats ont ete exprimes en ppm (mg/kg sable PS) d' apres l'abacle reali- see sur l'Arabian light IFP. La filtration sur Fluorisil provoque une retention sur le fil- tre de l'ordre de 60.4% du poids du produit d'origine. 231 Les organismes de la meiofaune ont ete fixes au formol 4%, colo- res au rose bengal et tries apres passage sur tamis de 40 u et elutria- tion. Des lots de 100 Nematodes ont ete mesures et identifies pour la determination de la biomasse et de la composition specifique. RESULTATS Evolution des hydrocarbures Les quantites d 'hydrocarbures introduites dans les modules fai- blement et fortement pollues correspondent a des teneurs initiales theoriques de 223 et 2236 ppm puisque seuls les quatre premiers centi- metres du sediment (soit 17.7 kg) sont contamines et que le passage de l'extrait tetrachlorure sur Fluorisil entraine une perte de 60.4% au dosage. En effet, 1 'analyse de la repartition verticale des hydrocarbu- res dans la colonne sedimentaire en utilisant ce type de dispositif experimental revele une penetration quasiment nulle du polluant sous la surface. Seule la tranche 0-4 centimetres contient des hydrocarbu- res en quantite notable et une penetration limitee dans la tranche 4-8 centimetres n'apparalt episodiquement qu'a partir du 106eme jour. Le temoin n'a jamais montre la moindre trace d' hydrocarbures. La figure n° 2 fournit 1' evolution des teneurs au cours du temps dans les bacs faiblement et moyennement pollues. L' evolution des teneurs dans le module faiblement pollue indique une degradation extremement faible au cours des six mois d'experience avec une heterogeneite des teneurs mesurees tres legerement plus accen- tuee en debut d'experience. Par contre, 1' evolution des teneurs dans le module fortement pollue met en evidence une tres forte heterogeneite des concentrations jusqu'au 23eme jour de prelevement qui reflete la repartition en aggre- gats des hydrocarbures a la surface du substrat ainsi qu'il a ete pos- sible de l'observer en plongee in situ sur les sables d'origine de la Pierre Noire. Cette heterogeneite tend a se reduire ensuite considera- blement du fait de la bioturbation. Le pic d'abondance des hydrocarbu- res observe au 23eme jour reste compatible avec les incertitudes de l'intervalle de confiance a la moyenne. II est lie* probablement aussi au delai necessaire au coulage de toutes les particules mazoutees ayant partiellement tendance a flotter a la surface de l'eau du compartiment principal en debut d'experience. La disparition des hydrocarbures entre 23 et 93 jours semble sa- tisfaisante puisqu'elle indique une evolution de 2979 ± 1672 ppm a 248 ± 52 ppm soit 38.5 mg HC/kg sable/jour (24 mg HC/kg sable/jour si l'on tient compte d'une valeur initiale theorique de 2236 ppm. II est cepen- dant impossible de considerer cette valeur comme un taux de degrada- tion realiste du fait de 1 'heterogeneite des teneurs initiales mesurees mais aussi d'une remontee incomprehensible des teneurs en hydrocarbu- res au 106eme et 124eme jour. 232 3000 1500 1000 - ISO Day* FIGURE 2. Evolution des teneurs en hydrocarbures et de leur ecart a la moyenne, dosees par la methode infra-rouge apres passage sur Fluorisil, dans le sable des modules fortement pollues (100 g HC : • •) et faiblement pollues (10 g HC : o -"-'o). Les hydrocarbures sont presque toujours concentres dans les quatre premiers centimetres du sediment. La difficulte d' interpretation des resultats des dosages effec- tues par infrarouge apres passage de l'extrait au CCli, sur Fluorisil montre done 1' extreme heterogeneite de la repartition des hydrocarbu- res dans le sediment, meme a l'echelle de quelques dizaines de centi- metres. II apparait difficile de realiser des calculs de biodegrada- tion dans des microcosmes ou l'on ne preleve qu'un faible aliquot du volume de sable contamine. Evolution des densites de la Meiofaune Les densites initiales du Meiobenthos dans le mesocosme temoin et dans ceux contamines par les hydrocarbures etaient comparables (non signif icativement differentes au niveau 5% par le test de Kruskall Wal- lis) . Les valeurs initiales trouvees de 1218 ± 13 Nematodes/10 cm2 et de 198 ± 26 Copepodes harpacticoides/10 cm2 , deux groupes qui consti- tuent la quasi totalite des organismes meiobenthiques recenses, peuvent etre favorablement comparees avec la densite relevee dans le milieu na- turel a la meme date (1357 Nematodes et 105 Copepodes/10 cm2) dans les douze premiers centimetres du sediment en mars 1981. Bien que 1' analyse de la repartition verticale montre qu1 environ 70% des nematodes et 44% des copepodes sont concentres dans les quatre premiers centimetres du sediment, les imperatifs de temps de tri ont conduit a estimer les densites seulement dans les quatre premiers cen- timetres. _.. 180 Days FIGURE 3. Evolution des densites (et de leur ecart a la moyenne) des Nematodes dans les quatre premiers centimetres du sediment pendant une periode de six mois. Temoin : 0 -- 0; Module faiblement pollue : o -.- o; Module fortement pollue : • • • Quel que soit le module considere, les densites de nematodes ont tendance a decroitre en circuit clos (Fig. 3). Cependant, il est pos- sible de distinguer : - une periode initiale de deux mois environ ou les valeurs trouvees dans les bacs pollues sont generalement plus for- tes que dans le temoin; - une periode ulterieure ou les densites dans ces modules contamines ont tendance a etre legerement plus faibles par rapport au temoin. II apparait done que la phase de pollution primaire serait ca- racterisee par une proliferation des nematodes (1,5 a 2 fois le niveau du temoin) mais que rapidement apparaitrait un declin lent (0,5 a 0,8 fois la valeur du temoin dans le bac le plus pollue, 0,7 fois a une valeur comparable au temoin dans le bac faiblement pollue). Ces obser- vations sont compatibles avec celles relevees dans le milieu naturel (Elmgren 1980 a; Boucher et al. 1981). 234 L'evolution des Copepodes harpacticoides (Fig. 4) montre au contraire un effet depressif des hydrocarbures sur le niveau de den- site du peuplement. Les abondances observees dans le temoin restent toujours superieures a celles des bacs polities malgre des fluctua- tions importantes des densites au cours de 1 'experience. Dans le bac le plus pollue, les densites de Copepodes deviennent faibles apres le 21eme jour. 20 40 00 00 100 120 140 100 IIODayi FIGURE 4. Evolution des densites (et de leur ecart a la moyenne) des Copepodes harpacticoides dans les quatre premiers centime- tres du sediment pendant une periode de six mois. Temoin : 0 0; Module faiblement pollue : o -.- o; Module forte- ment pollue : • •• Evolution du rapport Nematodes/Copepodes L'utilisation de ce rapport dans les etudes de pollution vient recemment d'etre proposee par Raffaelli et al (1981). Cette proposi- tion seduisante derive de deux considerations : - d'une part les Copepodes sont apparemment plus sensibles que les Nematodes au stress des pollutions; - d'autre part l'utilisation de la meiofaune dans les etudes d1 impact ne se justifie que si celle-ci repond avant que les effets ne deviennent visibles sur la macrofaune. L'un des obstacles majeurs a 1' interpretation de cet indice reside dans le fait que celui-ci est correle negativement avec la mediane granulometrique du sediment. L' ex- perimentation permet d'eliminer ce facteur qui obscurcit 1' interpreta- tion des resultats. 235 N/C 20 40 60 80 100 120 140 160 180 Days FIGURE 5. Evolution du rapport Nematodes /Copepodes dans les quatre premiers centimetres du sediment pendant les six mois d' ex- perience. Temoin : 0 0; Module faiblement pollue : o o; Module fortement pollue : • •. La figure 5 montre l'evolution de ce rapport dans les trois mo- dules experimentaux. Les valeurs sont d'autant plus fortes que le bac est plus pollue par les hydrocarbures . Ainsi le temoin presente tou- jours des valeurs faibles comprises entre 1.07 et 12.40 (moyenne : 5.04 ±0.50 par 33 mesures en six mois). Le bac faiblement pollue presente des valeurs legerement plus fortes et plus variables comprises entre 1.64 et 20.07 (moyenne : 7.81 ± 0.78 par 32 mesures). Enfin le module fortement pollue presente des valeurs nettement plus elevees mais fluctuantes. Deux periodes corres- pondant a des fortes valeurs peuvent etre distinguees entre 22 et 50 jours (N/C = 37 a 51) et entre 124 et 188 jours (N/C = 16 a 71) sepa- rees par une periode de faible valeur a 71 jours (N/C = 5 a 14). 236 La sensibilite de cet indice semble done se confirmer puisqu'un accroissement sensible est discernable apres 25 jours d'experience dans le bac fortement pollue du fait de la quasi disparition des Cope- podes, alliee a une proliferation des Nematodes. La generalisation de son utilisation demande cependant de preciser les modalites de ces fluctuations dans differentes conditions experimentales en tenant compte des changements de la composition specifique aussi bien des Nematodes que des Copepodes et de leur niveau de competition pour la nourriture par exemple (Warwick 1981). II est probable que le retour de ce rapport a des valeurs faibles dans le bac le plus pollue corres- pond a un changement de la composition faunistique des Copepodes. Evolution des parametres Abondance, Biomasse et Diversite. La plupart des etudes utilisant les modifications de la macro- faune pour mettre en evidence 1' impact des pollutions preconisent l'emploi de parametres simples tels le nombre d'especes, 1' abondance et la biomasse (Pearson et al. 1978; Glemarec et al.1981 entre autres) Cette approche pose pour 1' instant de serieux problemes methodologi- ques en ce qui concerne la Meiofaune. Les progres realises dans la systematique des groupes dominants des Nematodes et des Copepodes permettent d'envisager raisonnablement la determination de routine du nombre d'especes dans un echantillon representatif de la popula- tion (100 a 300 individus) malgre la grande diversite de ces groupes. II n'en est pas de meme en ce qui concerne 1' evolution de la biomasse du meiobenthos dans un echantillon donne. En effet, les methodes ac- tuellement utilisees posent generalement l'hypothese que la biomasse moyenne ne varie pas au cours du temps, ce qui n'est bien sur pas le cas. Elles consistent par consequent soit a evaluer les biovolumes a la chambre claire d'un microscope en utilisant des formules d'equiva- lence (Andrassy 1956; Wieser 1960; Juario 1975), soit a peser a la microbalance de precision un unique lot de quelques centaines a quel- ques milliers d' individus (De Bovee 1981; Guille et al. 1968). Les resultats presentes dans cet article doivent etre conside- red comme la mise au point d'une methode d' evaluation des indices vo- lumiques sur la meiofaune qui permet d'analyser rapidement chaque pre- levement et evite les incertitudes des methodes de pesee. Chacun des lots de 100 specimens montes entre lame et lamelle pour la determina- tion a ete grossi cent fois grace a un projecteur de profil. Les con- tours de chaque individu identifie ont ete traces puis analyses a la table digitalisante d'un analyseur d' images. Le volume a ete calcule et exprime en poids sec en utilisant une valeur de densite de 1,13 (Wieser 1960) et un rapport Poids sec/Poids frais = 0,25. Les autres parametres classiquement utilises tels que nombre d'especes (S) , Indice de diversite de Fisher et al. (a), Indice de diversite de Shannon (H) et Equitabilite de Pielou (J) ont ete aussi calcules au temps zero, 36 jours (avant la chute de densite) et 93 jours (apres la chute de densite) de l'experience dans chacun des trois modules sur des echantillons de 100 individus (Tableau I). Dans le bac temoin, 1 'evolution du poids sec individuel n'in- dique pas de fluctuations signif icatives puisque les limites des in- tervalles de confiance de la moyenne se recoupent (0.088 a 0.207 ug PS/individu) . 237 36 93 653 485 0.150 ± 0.057 (97) 0.101 ± 0.012 (101) 98.1 49.1 33 33 17.19 17.19 4.62 4.46 0.92 0.88 831 365 0.071 ± 0.0009 (90) 0.103 ± 0.035 (101) 59.1 37.7 34 34 18.15 18.15 4.35 4.15 0.86 0.82 955 418 0.125 ± 0.029 (95) 0.019 ± 0.003 (90) 119.6 7.8 28 17 12.91 5.88 4.05 2.19 0.84 0.54 TO 633 0.125 ± 0.015 (97) 79.3 29 13.71 3.97 0.82 1007 0.130 ± 0.028 (100) 131.2 29 13.71 4.46 0.92 1016 0.107 ± 0.015 (101) 108.9 23 9.35 3.71 0.82 en | 1 1 0) 1 1 CJ 1 CO 1 CO 1 CO •hi z di pq w a s h i zMam as'n i zfxmcoarE1-} XI 1 1 I CI 1 M 1 1 Modules Temoin Pollution faible Pollution forte 01 1 1 cO C s- 6 M X> 0) a Z II o c 0) EC XJ 3 • * •3 4-1 C r-l ^_^ 0) « B oa OI 4-1 '—' 1-4 0) OI 3 M en 0) 0) 05 ax CO U) B 3 "rl o X) fc •l-l -O <0) O) 4.) XI co • i-l i— i CO OI >-l O O) O) -i-l X) > X3 ■H C ■1 XI l-l •~ S CO 0) II Cu X) s-' to C 0) • 01 a /-s >> •H 0) . o XI >-l 3 6 C 3 O M O i-l o 'i— ) 0> 01 en m •I-l W OI CT. X) P-4 en 4-1 OI •3 4-1 OI XJ • f-i OI O vO >0J o. y-v ro u CO •H 3 s_^ #. i—l XI o • i-l tn (-4 XI #» 3 «o> CO •—s XJ N 4-1 2 • l-l ^— ' "I-l s—^ > 3 •i-i tn CT CO •a au »0> C B ■u •iH 01 II •r-( 4J U] o •"} C O 3 01 <- XI • #\ X) G Vj tn () 0) 3 >-i C O) O 3 C X3 CU O cd U x c tn CO o OI 3 • l-l CJ cfl II 4-1 /O) -a 3 CX tn i—l tn oi 01 o OI X) u > - o • l-l w X) 4-1 XI 3 < W 53 238 La biomasse est sensiblement plus forte au temps zero et sur- tout a 36 jours qu'a 93 jours essentiellement du fait de densites plus fortes. Le nombre d'especes identifiers est assez constant (29 a 33) ainsi que les divers indices de diversite. Dans le bac faiblement pollue, le poids sec individuel apres 93 jours n'est pas signif icativement different de celui de l'etat initial. La decroissance de la biomasse 131,2 a 37,7 ug PS) est surtout liee a la chute des densites (1007 a 365). Le nombre d'especes recense a ten- dance a legerement augmenter (29 a 34) ce qui provoque une augmenta- tion parallele de l'indice de Fisher et al. (13,71 a 18,15). La dimi- nution lente de l'indice de Shannon et de 1 'equitabilite indique l'ap- parition d'une hierarchisation plus marquee au cours du temps. Dans le bac fortement pollue, 1 'evolution des densites est si- milaire a celle du module faiblement pollue. Apres 36 jours, les va- leurs des parametres demeurent comparables a celles de l'etat initial. Apres 93 jours, par contre, le poids sec moyen chute fortement (0.019 ± 0.003 ug PS) d'ou une reduction brutale de la biomasse (7.8 ug/10 cm2) Celle-ci est liee au remplacement du peuplement d'origine par quel- ques especes de petite taille caracteristiques des milieux riches en matiere organique (Leptclaimus tripavillatus Boucher 1977, Monkystera aff. disjuncta Bastian i 865 ; Monhystera pusilla Boucher 1977) et mises en evidence dans des experiences prealables d'eutrophisation (Boucher 1979). DISCUSSION Ces simulations de pollutions en microecosysten-es sediment ai- res soulignent la difficulte d' interpreter les phenomenes de degrada- tion des hydrocarbures dans les sediments. La disparition de ceux-ci n'est pas detectable pendant la duree de l'experience dans le module faiblement pollue; elle est anarchique dans le bac fortement containi- ng. II ne semble done pas possible de caracteriser aisement une pol- lution par le niveau du polluant dans le milieu avec la methode em- ployee. L'utilisation d'organismes sensibles au polluant (indicateurs biologiques) integrant 1' ensemble des consequences du stress parait plus fiable pour caracteriser un impact. Elle suppose, pour etre ef- ficace, que ceux-ci repondent avant que la perturbation devienne evi- dente. Si certains organismes de la macrofaune benthique repondent de facon nette au stress primaire de la pollution par hydrocarbures (Dau- vin 1979 a et b et 1981) la duree des cycles (1 a 10 ans) rend proble- matique l'analyse des effets differes (Chasse et al. 1981). Du fait de la rapidite de reproduction, la meiofaune, dont les Nematodes et les Copepodes constituent l'essentiel des organismes, est un materiel prometteur pour comprendre les mecanismes regissant la destructuration et la restructuration d'un ecosysteme. Ces experiences de contamination brutale par hydrocarbures ne suggerent pas un effet tres important sur le niveau des densites des Nematodes. Leur augmentation entre 21 et 50 jours ne semble pas liee 239 a un developpement d'opportunistes necrophages comme le suggere Chasse (1978) puisque la composition faunistique reste tres comparable a celle du temoin. La chute des densites observee ulterieurement dans le bac le plus pollue est conforme aux resultats obtenus experimentalement in situ par Bakke et al. (1980) ou en mesocosmes par Elmgren et al. (1980 b). Elle s'accompagne d'un changement tres perceptible de la com- position faunistique avec reduction du nombre d'especes et diminution de la biomasse. Contrairement a 1 ' idee generalement admise, le groupe des Nema- todes peut done constituer un indicateur biologique fiable des modifi- cations de l'ecosysteme (Piatt & Warwick, 1980) puisque leurs possibili- tes adaptatives permettent a certaines especes de se maintenir quelles que soient les conditions de milieu, a d'autres de proliferer en quel- ques semaines pour occuper la niche laissee vide. La determination ex- perimental de groupes de Nematodes a comportement similaire vis-a-vis de 1 'eutrophisation ou des pollutions, apparait done comme une voie de recherche prometteuse pour caracteriser 1'etat ou la dynamique des eco- systemes perturbes. SUMMARY Experimental study of hydrocarbon pollution in a sand microecosystem I. Effect of the sediment contamination on meiofauna. The effects of hydrocarbon pollution, at two different intensi- ties with respect to a control, on the microecosystems were studied using recirculating experimental tanks containing 100 liters of subti- dal fine sand. Changes in the population characteristics of meiofauna (nematodes and copepods) were chosen to follow the effects of oil pol- lution. Irrespective of the intensity of pollution, the density of ne- matodes in the experimental tanks increased at a significantly higher rate than in the control tank during the first two months after pollu- tion and then decreased slowly. The density of harpacticoid copepods was negatively related to the intensity of oil pollution. It appears that the nematodes /copepods ratio would be an useful indicator of the degree of oil pollution. After 3 months of experimental duration, the faunal composition of the nematodes in the highly polluted tank was drastically modified. This change is evident from a sharp fall in biomass and species diver- sity; small opportunistic nematode species, known for their associa- tion with eutrophicated environment, became dominant. Changes in the meiofauna population parameters in the slightly polluted experimental tank did not show any significant variation from those in the control tank. REMERCIEMENTS L'ensemble des tris de la meiofaune a ete realise par Melle L. Cras, technicienne CNRS que nous tenons plus particulierement a remer- cier avec toutes les personnes ayant collabore a ce travail. 240 BIBLIOGRAPHIE Andrassy, I., 1956, Die Rauminhalts-und gewichtstimmung der Fadenwiir- mer (Nematoden) . Acta zool. Acad. Sci. hung. Vol. 2, pp. 1-15. Bakke, T. 1 T.M. Johnsen, 1979, Response of a subtidal sediment commu- nity to low levels of oil hydrocarbons in a Norvegian fjord. In Proc. 1979 oil spill Conf., Los Angeles, CA (USA). Publ. AmerT" Petrol. Inst. Washington, D.C., pp. 633-639. Boucher, G. , 1979, Evolution des caracteristiques chimiques et biologi- ques des sediments en circuit clos. II. Effet de la matiere orga- nique circulante sur la meiofaune de systemes polytrophes. Collo- que national Ecotron. Publ. Sc. Tech. CNEXO, Actes Colloq, pp. 31- 47. Boucher, G. 1980, Impact of Amoco Cadiz oil spill on intertidal and sub- littoral meiofauna. Mar. Pollut. Bull., Vol. 11 (4), pp. 95-100. Boucher, G. , 1981, Effets a long terme des hydrocarbures de 1 'Amoco Cadiz sur la structure des communautes de Nematodes libres des sables fins sublittoraux. I_n "Amoco Cadiz. Consequences d'une pollution accidentelle par les hydrocarbures". Actes Colloq. CNEXO pp. 539-549. Boucher G. & S. Chamroux, 1976, Bacteria and meiofauna in an experimen- tal sand ecosystem. I. Material and preliminary results. J. exp. mar. Biol. Ecol., Vol. 24, pp. 237-249. Boucher, G. S. Chamroux & C. Riaux, 1981, Etude d'impact ecologique de la pollution petroliere de 1 'Amoco Cadiz dans la region de Ros- coff et de la Baie de Morlaix. Effet a long terme sur la structu- re des ecosystemes sedimentaires. Rapport d' execution de contrat d'etudes environnement CNEXO/Universite Paris 6 n° 79/5973, 51 pp. Chasse, C, 1978, The ecological impact on an near shores by the Amoco- Cadiz oil spill. Mar. Pollut. Bull., Vol. 9 (11), 298-301. Chasse, C. & A. Guenole-Bouder, 1981, Comparaison quantitative des popu- lations benthiques des plages de St Efflam et St Michel en Greve avant et depuis le naufrage de 1 'Amoco Cadiz. In "Amoco Cadiz. Consequences d'une pollution accidentelle par les hydrocarbures". Actes Colloq. CNEXO, pp. 513-525. Dauvin, J.C., 1979 a, Impact des hydrocarbures de l'"Amoco Cadiz" sur le peuplement inf ralittoral des sables fins de la Pierre Noire (Baie de Morlaix). J. Rech. Oceanogr. , Vol. 4 (1), pp. 28-29. Dauvin, J.C., 1979 b, Recherches quantitatives sur le peuplement des sables fins de la Pierre Noire, Baie de Morlaix, et sur sa per- turbation par les hydrocarbures de 1 'Amoco Cadiz. These Doctorat 3e cycle Univ. Pierre et Marie Curie Paris 6, 251 pp. Dauvin, J.C., 1981, Evolution a long terme des populations d'Amphipodes des sables fins de la Pierre Noire (Baie de Morlaix) apres 1' im- pact des hydrocarbures de 1 'Amoco Cadiz. J. Rech. oceanogr. Vol. 6 (1), pp. 12-13. 241 Delaune, R.D., G.A. Hambrick & W.H. Patrick, 1980, Degradation of hy- drocarbons in oxidized and reduced sediments. Mar. Pollut. Bull. Vol. 11, pp. 103-106. De Bovee, F. , 1981, Ecologie et dynamique des Nematodes d'une vase sublittorale (Banyls-sur-Mer) . These Doc. Etat Sci. Nat. Univ. Pierre et Marie Curie Paris 6, 194 pp. Elmgren, R. , S. Hansson, U. Larsson & B. Sundelin, 1980 a, Impact of oil on deep soft bottoms. Tn "The Tsesis oil spill, ed. by J. Kineman, R. Elmgren & S. Hansson, U.S. Dept. of Commerce NOAA outer Continental Shelf Environmental Assessment Program, pp. 97-126. Elmgren, R. , G.A. Vargo, J.F. Grassle, J. P. Grassle, D.R. Heinle, G. Langlois & S.L. Vargo, 1980 b, Trophic Interactions in experi- mental marine ecosystems perturbed by oil. In Microcosms in e- cological Research ed. by J.O. Giesy Jr. Publ. by U.S. Techni- cal Information Center, U.S., Dept Energy, Symposium Series 52 (Conf. 781101) pp. 779-800. Elmgren, R. & J.B. Frithsen (in press), The use of experimental eco- systems for evaluating the environmental impact of polluants : a comparison of an oil spill in the Baltic sea and two long term low level oil addition experiments in microcosms. I_n Reeve, M. & G. Grice (eds) , Proc. Symp. Enclosed Experimental Ecosystems, Sidney, B.C. 12-16 Aug. 1980. Glemarec M. & C. Hily, 1981, Perturbations apportees a la macrofaune benthique de la Baie de Concarneau par les effluents urbains et portuaires. Acta Oecologia/Oecologia Applicata, Vol. 2 (2), pp. 139-150. Grassle, J.F., R. Elmgren & J. P. Grassle, 1981, Response of benthic communities in MERL experimental ecosystems to low level, chro- nic additions of n° 2 fuel oil. Mar. Environ. Res., Vol. 4, pp. 279-297. Guille, A. & J. Soyer, 1968, La faune benthique des substrats meubles de Banyuls-sur-mer. Premieres donnees qualitatives et quantitati- ves. Vie Milieu, vol. 19 (2-13), pp. 323-359. Johnston, R. , 1970, The decomposition of crude oil residues in sand co- lumns. J. Mar. Ass. U.K., Vol. 50, pp. 925-937. Juario, J., 1975, Nematode species composition and seasonal fluctuation of a sublittoral meiofauna community in the german Bight. Veroff Inst. Meeresforsch. Bremerh., Vol. 15, pp. 283-337. Lacaze, J.C., 1979, Experimentations en ecosystemes marins controles. Application a la pollution par ] es produits petroliers. Oceanis, Vol. 4, pp. 423-611. Mevel, G., 1979, Conditions de nitrification de la matiere organique dans les sediments marins en systeme clos. Applications aux ele- vages de Peneides. These Doc. 3eme cycle Oceanogr. Biol., Univ. Bretagne Occidentale, 130 pp. 242 Pearson & Rosenberg, 1978, Macrobenthic succession in relation to or- ganic enrichment and pollution of the marine environment. Ocea- nogr. Mar. Biol. Ann. Rev., Vol. 16, pp. 229-311. Piatt, H.M. & R.M. Warwick, 1980, The significance of free-living ne- matodes to the littoral ecosystem in the shore environment, vol. 2 : Ecosystems. ed. by J.H. Price, D.E.G. Irvine and W.F. Farnham, Acad. Press London, pp. 729-759 Raffaelli, D.G. & Mason, C.F., 1981, Pollution monitoring with meio- fauna, using the ratio of nematodes to copepods. Mar. Pollut. Bull., vol. 12, pp. 158-163. Wade, T.L. & Quinn, J.G., 1980, Incorporation, distribution and fate of satured petroleum hydrocarbons in sediments from a controlled marine ecosystem. Marine Environ. Res., Vol. 3 (1), pp. 15-33. Warwick, R.M. , 1981, The Nematode /Copepod ratio and its use in pollu- tion ecology. Mar. Pollut. Bull., Vol. 12 (10), pp. 329-333. Wieser, W. , 1960, Benthic studies in buzzards bay. II. The meiofauna. Limn. Oceanogr. Vol. 5 '2), pp. 121-137. 243 EVOLUTION A MOYEN-TERME DU MEIOBENTHOS ET DU MICROPHYTOBENTHOS SUR QUELQUES PLAGES TOUCHEES PAR LA MAREE NOIRE DE L 'AMOCO- CADIZ par Philippe BODIN et Denise BOUCHER Universite de Bretagne Occidentale , Laboratoire d'Oceanographie biologique, 6 avenue Le Gorgeu, 29283 Brest Cedex, France. ABSTRACT The ecological follow-up undertaken after the Amoco-Cadiz oil spill, on the beaches Brouennou and Corn ar Gazel (mouth of Aber Benoit) and Kersaint (near Portsall), was continued untill november 1980. Chlorophyll pigments have suffered little quantitatively from the direct effect of pollution, but the study of temporal variations in the meiofaunal densities revealed disturbances in seasonal cycles. Other factors, e.g. hydrodynamic fluctuations and macrofaunal preda- tors, could act as regulating mechanisms on the evolution of the po- pulations . The effects of pollution are particularly obvious in some fau- nistic imbalances, as the study of harpacticoid copepods showed. However, particular evolutionary trends between and within ecologi- cal groups of species implied that recovery was nearly complete, at least on exposed beaches . The conclusions drawn to date are tentative because of the lack of reference data, and it is intended to continue the survey annual- ly in spring. Key words : Pollution , Amoco-Cadiz, Chlorophyll pigments, Meiofauna, harpacticoids , Beaches. RESUME Le suivi ecologique mensuel entrepris , a la suite de la catas- trophe de 1 'Amoco-Cadiz. sur les plages de Brouennou et Corn ar Gazel, a 1' entree de l'Aber Benoit, et de Kersaint pres de Portsall, a ete maintenu jusqu'en novembre 1980. Alors que les pigments chlorophyl- liens ne semblent pas avoir souffert de l'action directe de la pol- lution, 1' etude des variations temporelles de la densite de la meio- faune revele une perturbation des cycles saisonniers . D'autres fac- teurs , tels que l'hydrodynamisme et les predateurs de la macrofaune , peuvent intervenir en tant que mecanismes regulateurs . Les effets de la pollution sont surtout sensibles au niveau de certains desequilibres faunistiques , comrae le montre 1 'etude des Co- pepodes Harpacticoides . Cependant , une certaine evolution des groupes ecologiques permet de penser qu'un processus de retour a l'etat ini- tial est en cours d 'achievement , du moins sur les plages de mode battu. En fait, 1' absence d'etats de references nous oblige encore a la prudence dans 1 'interpretation des resultats , et il est envisage une poursuite des recherches sous forme de "veille" ecologiques. Mots-cles : Pollution, Amoco-Cadiz, Pigments chlorophylliens, Meio- f aune , Harpacticoides, Plages. 245 INTRODUCTION Dans le cadre du suivi ecologique entrepris , a la suite du nau- frage de 1"'AM0C0-CADIZ" , par les Laboratoires de l'Institut d'Etudes Marines de l'Universite de Bretagne Occidentale , la meiofaune sensu lato et le microphytobenthos de la zone intertidale ont ete l'objet d'une etude realisee par le Laboratoire d'Oceanographie biologique . Apres une recherche de site effectuee sur la cote nord-Finistere au cours des mois de septembre et octobre 1978, deux plages a la sor- tie de l'Aber Benoit (Fig. 1), Corn ar Gazel au SW et Brouennou au NE, ont ete retenues pour cette etude. Ces deux plages, situees dans une zone particulierement eprouvee par la pollution due aux hydrocar- bures de 1' "AMOCO-CADIZ" , sont egalement etudiees au point de vue physico-chimique et au point de vue de la macrofaune (Le Moal , 1981) dans le cadre de ce suivi. II a malheureusement ete impossible de trouver dans cette region une plage ecologiquement homologue mais non polluee af in de servir de temoin . Une etude parallele , mais por- tant uniquement sur la meiofaune sensu stricto , a ete realisee sur la plage de Kersaint (pres de Portsall). 4*40- 4*M- FIGURE 1. Localisation des stations. Sur chacune des plages, une station (Quadrat) situee en dessous de la mi-maree, dans l'etage mediolittoral , est l'objet de preleve- ments mensuels depuis mars 1978 pour la plage de Kersaint, novembre 1978 pour les plages de Brouennou et Corn ar Gazel. Sur cette der- niere , deux prelevements "de reference" ont pu etre realises le 17 mars 1978, avant l'arrivee de la nappe d'hydrocarbures . Une premiere publication (Bodin et Boucher, 1981) faisait etat des resultats acquis en juillet 1979. La presente note les complete par les donnees obtenues jusqu'en novembre 1980 et tente une re- flexion sur l'ensemble de ce suivi ecologique. 246 Les techniques de prelevement et de traitement des echantillons , ainsi que les principaux parametres edaphiques , ont deja ete exposes dans la premiere publication, nous ne les reprenons done pas ici . Nous rappelons simplement quelques caracteristiques granulometriques essentielles sous forme de courbes ponderales cumulatives (Fig. 2). De plus , nous presentons le prof il topographique de deux des plages prospectees (Fig. 3) et les variations temporelles de la temperature et de la vitesse du vent (Fig. 4). i/ 75- 1 1 if i j KERSAINT 50- / 3/80 — t p 0 I 1 Md 190 pm ' So 1,15 25- i ji / li // 6/80 p 0 % Md 190 pm So 1,09 ij it ij It ,'/ CORN AR GA2EL ,/ 3/80 - i P 0 l i/ Md 130 '/ So 1,2 pn rf 6/80 - ' p 0 1 7 Md 130 ,7 So 1,1£ fjm 6 3 80 100 125 ISO 200 63 SO 100 12S 160 200 63 BO 100 12S 160 ?00 FIGURE 2. Granulometrie : courbes ponderales cumulatives. Teneur en pelites (p %), Mediane (Md), Indice de triage (So). BROUENNOU supralittoral Imediolittoral PMMEC40) TORAL supralittoral Imediolittoral PMME(40) CORN AR GAZEL ~-» Galets INFRALITTORAL BMME (40) FIGURE 3. Profil topographique des plages. Limites des etages bathy- metriques. Emplacement des quadrats (Q). \ \/ / \ n'd| j'f'm' »'m' j' j' a' s O n O I J f 1979 5 O N Dp /v r~' \ I / 1980 ndTj fmamj j a so no 1979 SON Ow 1980 FIGURE 4. Temperature de l'air (a), vitesse du vent (b) : variations des moyennes mensuelles . Une correction doit cependant etre apportee (Bodin et Boucher, 1981, p. 328) : a la place de P.M.M.E. il faut lire B.M.M.E., et a la place de B.M.M.E. il faut lire B.M.V.E. 247 RESULTATS Pigments chlorophylliens Un depouillement additionnel de carottes pour les prelevements anterieurs a septembre 1979 modifie legerement les chiffres prece- demment obtenus et publies (Bodin et Boucher, 1981). Le nombre de carottes utilise a permis l'utilisation de tests statistiques : test U de Mann-Whitney et test de Kruskall-Wallis ; hypothese nulle rejetee au niveau 5 %. Brouennou Dans les premiers centimetres d'epaisseur du sediment on observe une decroissance tres rapide des teneurs en pigments chlorophylliens, ce qui nous a permis de limiter 1' etude aux quatre premiers centi- metres . Pour la chlorophylle a, ce gradient est tres marque (on retrouve en moyenne 12 % de la teneur superficielle sous 4 cm d'epaisseur) et regulierement observe dans les prelevements (a 1' exception des mois de decembre 1978 et 1979). L'heterogeneite spatiale est importante et , de ce fait, les te- neurs moyennes calculees pour les deux premieres couches (0-0,2 cm et 0,2-1 cm) ne sont pas significativement differentes pour la majo- rite des prelevements mensuels , alors que la presence du film super- ficiel, plus riche en chlorophylle a, est constatee dans 85 % des carottes . Pour les pheopigments , le gradient est moins accentue (26 % de la teneur superficielle sont presents en moyenne sous 4 cm d'epais- seur) et moins frequemment observe (absent en novembre et decembre 1978, de decembre 1979 a fevrier 1980 et de juillet a septembre 1980) que dans le cas de la chlorophylle a, ce qui peut etre du en partie a ses plus faibles teneurs et done a la moindre precision du dosage. L 'enrichissement superficiel en pheophytine n'est rencontre que dans 63 % des carottes . La chlorophylle a est le pigment largement dominant surtout au sein des deux premiers centimetres d'epaisseur, la ou se limitent les variations temporelles. Sa teneur relative elevee (71 % de la somme Ca + Pheo) est un indice de la presence d'une active popula- tion de microphytes dans cette zone correspondant a l'epaisseur maximale de la couche oxygenee . L ' amplitude des variations temporelles est maximale au niveau de la couche superficielle et s'attenue rapidement dans l'epaisseur du sediment . La chlorophylle a presente, les deux annees, un cycle annuel de type saisonnier (Fig. 5a). Dans la couche superficielle, le minimum de decembre est suivi par un fort accroissement pendant les mois d'hiver ; il aboutit a un "plateau printanier" entre mars et juillet 1979 (22,1 Mg/g) et entre fevrier et juillet 1980 (15,6 yg/g, en excluant le mois de juin). Entre juillet et aout , une nette decroissance est observee ; elle est suivie par un "plateau automnal" entre aout et novembre 1979 (14 ug/g). Au cours de ces deux cycles il faut noter le minimum esti- va 1 esquisse en juin 1979, tres prononce en juin 1980, phenomene deja observe en zone intertidale (Colijn et Dijkema, 1981) mais non expli- cite . 248 20 10 _ P9'9 a 0.0 _ 0.2 cm a 0.2 _ 10 cm • 1.0 _ 1.8 cm 1 8_26 cm ■ 2.6 _ 3.4 c m o 3.4_4 2 cm ¥ "CN- V ■ — " ■-■ i"^ ^•D~" N> /■ I NDIJ FMAMJ J ASON Dlj F MAMJ J A S *+ 1979 ^ 1980 10 . wg/g Ull A ■ A ' A ■ V A ■ A ' A ' A ' n — nr~ ' A| A ' A ' A ■ * A ' A ' A ' A ' A ND. JFMAMJJASONDJFMAMJ JAS 1979 1980 FIGURE 5. Variation des moyennes mensuelles de la chlorophylle a (a) et de la pheophytine (b) a Brouennou. Dans la couche sous-jacente les fluctuations sont similaires . Le plateau printanier, situe entre fevrier et aout . correspond a une teneur moyenne de 15,8 yg/g en 1979 et de 11 yg/g en 1980 et le pla- teau automnal, entre septembre et novembre 1979, a une teneur moyenne de 9,4 yg/g. Les deux cycles annuels de la pheophytine (Fig. 5b) different essentiellement par le minimum estival tres accuse en 1980, les te- neurs moyennes des plateaux. atteints de mars a novembre 1979 (8,8 yg/g) et de fevrier a mai 1980 (8,5 yg/g), etant semblables . Les variations temporelles de ces deux pigments sont paral- leles , sauf au cours de l'automne ou elles tendent a s'inverser, faisant diminuer la teneur relative en chlorophylle a. Les fortes diminutions de concentration sont accompagnees d'une disparition ou d'une attenuation du gradient dans l'epaisseur du sediment. Cette homogeneisation des couches superficielles , tres 249 apparente en decembre 1978 et aout 1979, moins prononcee en decembre 1979 et juin 1980, peut etre attribute a une erosion de la pellicule superficielle et a un brassage du sediment sous 1' action des forces hydrodynamiques . C'est a la suite de ces decroissances que se situent les plus forts accroissements relatifs (100 % entre decembre 1978 et Janvier 1979, 7*4 % entre decembre 1979 et Janvier 1980, 77 % entre juin et juillet 1980). Ces accroissements sont du meme ordre de grandeur en hiver et en ete, et il semble done que les facteurs climatiques (temperature, eclairement) ne soient pas limitant . La comparaison des resultats obtenus en 1979 et en 1980 montre pour la chlorophylle a, qu'il n'y a pas de differences significatives entre les moyennes mensuelles de ces deux annees des mois de Janvier a mars, tandis que celles-ci sont toujours plus elevees en 1979 du mois d'avril au mois de juillet. Corn ar Gazel La distribution des pigments au sein des 12 premiers centimetres de sediment s'etant revelee tres homogene , nous avons etudie trois couches successives de 4 cm d'epaisseur. Les teneurs moyennes en chlorophylle a et en pheophytine varient peu entre les trois couches (variation environ de 10 %). On reconnait cependant , surtout pour la chlorophylle a , deux types de distribu- tion : dans le premier type , la teneur est maximale dans la couche superficielle puis decroit regulierement , dans le deuxieme type la teneur est maximale dans la couche intermediaire (4-8 cm). Sur 1' ensemble des prelevements , la teneur moyenne mensuelle en chlorophylle a du sediment est la plus faible dans la couche la plus profonde . Entre les deux premieres couches, comme a Brouennou, la difference observee n'est pas, le plus souvent , significative du fait de l'heterogeneite spatiale ; elle est cependant corroboree par la frequence, dans le prelevement mensuel, de chacun des deux types de distribution verticale . La teneur relative en chlorophylle a du sediment ne varie pas entre les trois couches . Les teneurs moyennes mensuelles en chlorophylle a (Fig. 6a) de la couche superficielle (0-4 cm) s 'accroissent de Janvier a novembre 1979. Apres la brutale diminution de decembre 1979, les moyennes mensuelles mesurees en 1980 sont, a 1 'exception du mois d'avril, toujours inferieures a celles de l'annee precedente. Dans la couche sous-jacente la variation des teneurs moyennes mensuelles presente la meme tendance, mais son amplitude est plus faible. Pour la pheophytine on remarque une elevation en automne (oc- tobre 1979 - septembre 1980) (Fig. 6b) des teneurs moyennes des deux premieres couches et , pour 1 'ensemble des deux annees, une augmenta- tion des moyennes mensuelles au cours de l'annee 1980. S'il n'y a pas de cycle de type saisonnier apparent au niveau des variations des teneurs moyennes mensuelles au sein de chacune des couches sedimentaires etudiees, un tel cycle se presente (plus nettement pour la chlorophylle a) sous la forme d'une succession reguliere des deux types de distribution verticale. Un enrichissement subsuperficiel est constate pendant 1' hiver (de novembre a avril) alors qu'en ete c'est la couche superficielle qui est la plus riche en pigments, ce qui peut etre du a la difference saisonniere de sta- bility sedimentaire . 2=;n 10 _ \ hP\ A** \ V -TTI 1 — I

< 5- pg/g i a 0_4 cm » • 4_8 cm I ■ 8_12cm A s&§ a a ' ' i-1 '&4A | x i i— i z i m — i— i — rj — tx 1 — n-x — r* ' — a~] — x— i — i— i — j—) xxix — rx — n — r— i — » M A ii S ii NdIj FMAMJJ ASONdIj FMAMJJ AS 1978 m 1979 M 1980 FIGURE 6. Variation des moyennes mensuelles de la chlorophylle a (a) et de la pheophytine (b) a Corn ar Gazel. Discussion La comparaison des deux plages montre que les teneurs pigmen- taires dans les couches superficielles , au moment du minimum hiver- nal, sont tres peu differentes (10 pg/g environ). Elles peuvent etre assimilees a la valeur intrinseque de Hartwig (1978) et resultent ici de l'identite des medianes granulometriques . De la meme f agon , la faible difference existant au niveau des valeurs moyennes du taux de chlorophylle a (81 % et 71 %) et de l'indice de diversite pigmen- taire (voisin de 2) dans la couche superf icielle , peut etre reliee a l'identite du taux de pelites . La difference de nature et d' action des forces hydrodynamiques agissant sur la stabilite et 1 'oxygenation du sediment se reflete dans la difference observee pour la distribution des pigments dans l'epaisseur du sediment (Fig. 7a, b, c), ainsi que l'ont constate de nombreux auteurs depuis Steele et Baird (1968). 251 Pheo (vg/q ) Ca (pg/g) 5 0 0 5 to 15 \ o PheO {uQ,tq) Pneo j U Ca lug/g / FIGURE 7. Repartition de la chlorophylle a et de la pheophytine a Brouennou (a) et a Corn ar Gazel (b, c) dans l'epaisseur du sediment . Sur la plage de Corn ar Gazel, la distribution homogene des differentes caracteristiques pigmentaires , mise en place par un brassage sous 1' action des vagues, peut se maintenir dans un milieu interstitiel presentant de bonnes conditions d' oxygenation (Gargas, 1970 ; Mclntyre et at. , 1970 ; Hunding, 1971) assurees par l'exis- tence de houle et de courants de maree. Dans ces milieux instables ou les microphytes sont passivement distribues, l'essentiel de la flore est generalement constitue par de petites Diatomees liees aux grains (Amspoker, 1977). Sur la plage de Brouennou la stabilite de la surface sedimen- taire permet le developpement dans la zone photique d'un film super- ficiel, tandis que, sous les deux premiers centimetres, en milieu non oxygene , on observe un enrichissement relatif en pigments de degradation, ceci pouvant etre du a la combinaison entre la migra- tion des formes mobiles vers la couche superficielle et la mort des cellules en milieu fortement reduit (Gargas, 1970). La teneur en pigments chlorophylliens du sediment est en moyenne deux fois plus elevee a Brouennou qu'a Corn ar Gazel lorsque sont considerees les 'pellicules superf icielles ; elle est au contraire deux fois plus faible lorsque les dix premiers centimetres sont pris en compte. II est done important de preciser l'epaisseur utilisee pour 1' evaluation de la biomasse. Pour une comparaison avec la meio- f aune , a Brouennou, ce sont les valeurs mesurees dans le premier cen- timetre, zone ou se concentrent les meiobenthontes , qui representent la quantite de nourriture disponible et traduisent la stabilite de cette zone. L' etude des variations saisonnieres , dans le cas de sediments instables comme a Corn ar Gazel, montre qu'un cycle quantitatif n'est pas apparent sur une annee et e'est essentiellement l'insta- bilite sedimentaire qui limite la biomasse des microphytes , la pro- duction dans la zone photique devant etre essentiellement exportee apres erosion et remise en suspension. Lorsque la surface sedimentaire est stable, comme cela est le cas a Brouennou, il apparait au contraire un cycle quantitatif reproductible. Toutes les etudes menees en zone intertidale montrent ce type de cycle annuel des que le sediment presente une fraction 252 fine importante (signe de sediment stable) (Cadee et Hegeman , 1977 ; Admiraal et Peletier, 1980 ; Colijn et Dijkema, 1981). Ce type de cycle se rencontre egalement en milieu infralittoral (Boucher, 1975). La biomasse pigmentaire est constante au cours des plateaux de prin- temps et d'automne, ce qui laisse presumer soit d'une productivity plus faible qu'en hiver, soit de l'etablissement d'un seuil regit par les conditions moyennes de stabilite sedimentaire d'une part, et d'activite de nutrition du maillon secondaire d' autre part. La pre- miere hypothese n'est pas confirmee par les differentes etudes menees en milieu intertidal. On ne peut l'etayer, en effet , ni par une photo- inhibition (Cadee et Hegeman, 1974 ; Colijn et Van Buurt , 1975), ni par un effet limitant des concentrations en sels nutritifs (Admiraal, 1977), ni par une saturation de la zone photique (Admiraal et Peletier, 1980), car la constitution de denses colonies de micro- phytes n'est pas suggeree par les teneurs observees (lors de la for- mation de croutes de microphytes, des teneurs superieures a 100 yg sont mesurees ; Plante-Cuny et at., 1981). La deuxieme hypothese est en accord avec 1 'observation de la tendance a un accroissement en pheophytine au cours de cette periode . La comparaison des resultats obtenus au cours de ces deux annees successives (Tableau 1) montre , a Brouennou comme a Corn ar Gazel , une diminution globale au cours de la deuxieme annee des teneurs en chlorophylle a, alors que la teneur en pheopigments reste inchangee ou est en augmentation. Cette variation resulte soit d'un effet secondaire de la pollution due aux hydrocarbures de 1' "AMOCO-CADIZ" , soit de la variation naturelle pluriannuelle (Cadee et Hegeman, 1974). TABLEAU 1. Valeurs moyennes au sein de chaque tranche de sediment calculees pour les periodes de Janvier a septembre 1979, 1980, et pour la duree totale de 1' etude. Ca : chlorophylle a (yg/g) - Pheo : pheophytine (yg/g) Ca % : Ca x 100 / (Ca + pheo) - DI : indice de diversite pigmentaire Epaisseur (an) BROUENNOU JAW. 1979 - SEPT. 1979 JAW. 1980 - SEPT. 1980 NOV. 1978 - SEPT. 1980 Ca Pheo Ca '. DI Ca Pheo Ca % DI Ca Pheo Ca % DI 0 -0,2 0,2-1 ,0 1 -1,8 1,8-2,6 2,6-3,4 3,4-4,2 18,6 14,1 8,1 4,5 2,6 1,5 6,7 4,9 2,9 2,0 1 ,3 1 73 74 74 69 67 60 1,99 2,26 2,76 3,20 3,62 3,97 13,2 10,1 7,1 3,6 2,6 2,0 6 3,6 2,8 3 2 1,7 69 74 72 54,5 56,5 54 2,3 2,49 2,72 3,19 3,67 3,97 14,9 .11,1 7,2 4,1 2,7 1 ,9 6,1 4,2 2,8 2,7 I ,8 1 ,6 71 72,5 72 61 60 54 2,14 2,44 2,81 3,19 3,64 3,89 Epaisseur (cm) CORN AR GAZEL JAW. 1979 - SEPT. 1979 JAW. 1980 - SEPT. 1980 NOV. 1978 - SEPT. 1980 Ca Pheo Ca % DI Ca Pheo Ca % DI Ca Pheo Ca % DI 0- 4 4- 8 8-12 12,9 12,2 10 2 1,5 1,5 87 87 87 2,24 2,30 2,42 8,4 8,2 7,55 2,75 3,0 2,1 76 74 78 2,40 2,51 2,64 1 1 10,7 9,1 2,05 1,9 1,8 82 78 82 2,29 2,41 2,54 253 Les conditions meteorologiques (facteurs climatiques et hydro- dynamiques ) ne peuvent etre retenues comme facteurs determinants de cette variation, n'ayant pas ete plus particulierement defavorables au cours de la deuxieme annee , si ce n'est en automne , alors que les deux plages, pourtant d' exposition differente, ont reagi simi- lairement et ceci des le mois d'avril. La reprise des activites de grazing est un des facteurs biolo- giques qui intervient au niveau de 1' evolution saisonniere et qui peut expliquer la difference observee entre les deux annee s . II est possible en effet de rapporter ces variations de la biomasse pig- mentaire a 1' evolution de la macrofaune au sein du processus de decontamination (Le Moal , 1981). Ainsi, la reapparition de l'Amphi- pode Bathyporeia sur la plage de Corn ar Gazel peut etre pour partie responsable de la diminution de la teneur en chlorophylle a, son activite de "brouteur" etant reconnue ( Sundback et Persson, 1981). Meiofaune : resultats quantitatifs La Figure 8 et le Tableau 2 montrent 1' evolution temporelle de la densite (nombre d'individus/10 era de surface) des Copepodes Har- pacticoides (echelle * 100), des Nematodes et de la meiofaune totale (sensu striato) dans les differents prelevements recueillis aux trois stations prospectees . Les Tableaux 3 et 4 indiquent 1' evolution tem- porelle (en pourcentage de la meiofaune totale) des autres groupes du meiobenthos vrai et de la meiofaune temporaire . II est evident que cette evolution est differente d'une station a 1' autre. Brouennou La densite moyenne (8 033 ind./lO cm2) y est extremement elevee en comparaison des donnees de la litterature pour la zone interti- dale (Hicks, 1977), et la meiofaune est composee essentiellement de Nematodes. Les variations saisonnieres sont assez nettes et a peu pres conservees d'une annee a 1' autre, avec des minima en fevrier- mars et en septembre (1979) ou juillet (1980), et des maxima en avril-Tnai et en decembre -Janvier ou octobre (1980). Cependant , la densite moyenne des Harpacticoides est nettement plus faible durant la seconde periode (1979-80) : 282 ind./lO cm2 (contre 593 precedem- ment ) . Durant cette seconde periode , le rapport Nematodes/Copepodes oscille entre 11 (juin 1980) et 172 (Janvier 1980). Les autres groupes du meiobenthos vrai representent un pourcen- tage relativement modeste de la meiofaune (maximum : 26,7 % en oc- tobre 1979). De plus, ce pourcentage est en regression : il ne de- passe pas 3,5 % depuis juillet 1980. Les Annelides constituent l'essentiel du meiobenthos temporaire, ce qui correspond aux donnees de la macrofaune (Le Moal, 1981). Corn ar Gazel La densite moyenne de la meiofaune (3 013 ind./lO cm2) y est beaucoup plus faible qu'a Brouennou. De plus, e'est a cette station que la difference avec la periode etudiee precedemment est la plus nette du point de vue quantitatif : la densite moyenne passe de 4 697 a 1 666 ind./lO cm2. Cette regression n'est pas le fait des 254 Meiofaune totnli Nema; A. 13000- 10000- N 7000- 5000- 3000- BROUENNOU Q i \ i \ i \ \a i—\\ 1 1 li a a A i i Harpacdcoides N/10cm' 1200 li \\ p. I \ • * 8 . m a a & // / *•/"» / / '"». U. ! \\ ! ..-Wtf TdI -i r— ~r--i*- FMAMI JASON D[tFMAMI JASON A / i COftN • AR - GAZEL a ' •--•-"•1^ S 0 N D| I F M A M I )ASOND[IFMAMI I A S 0 N KERSAINT "I' • fV-. i -1 1 1" MAMI I A S 0 N D I FMAMI.lASONDM F M A M I , I A S 0 N '1978 I 1979 I 1980 ii 'ii ' i i PRINTEMPS ETE AUTOMNE HIVER PRINTEMPS ETE AUTOMNE HIVER PRINTEMPS ETE -20 500 200 h BOO 500 200 0 FIGURE 8. Evolution temporelle des densites de la meiofaune aux trois stations. Copepodes Harpacticoides , mais celui des Nematodes et des autres groupes : de 53,8 % en aout 1979, ces derniers ne constituent plus que 11,5 % du meiobenthos vrai en aout 1980. Les variations saisonnieres sont encore plus ou moins marquees durant la seconde periode , avec un minimum classique en fevrier mais aussi un maximum en septembre (1979) et deux legers pics en avril et aout 1980). Dans ce biotope mieux oxygene , le rapport Nematodes/ Copepodes varie dans des limites plus etroites : 2,3 (septembre 1980) a 15 (octobre 1980) . Parmi la meiofaune temporaire , les Amphipodes constituent un groupe particulierement interessant a cette station ou ils avaient subi de lourdes pertes des le debut de la marie noire : ils ont ete absents durant tout l'hiver 1979-80, mais ont ete regulierement 255 TABLEAU 2. Evolution temporelle des densites de la meiofaune aux trois stations (N/10 cm?). 1 9 7 9 1 9 8 0 BROUENNOU Nematodes Harpacticoides Meiofaune totale CORN AR GAZEL Nematodes Harpacticoides Meiofaune totale KERSAINT Nematodes Harpacticoides Meiofaune totale 6/8 21/9 8/10 5/11 20/12 17/1 19/2 18/3 30/4 14/5 12/6 10/7 11/8 11/9 23/10 20/11 3928 200 4257 7/8 1312 33 1574 20/9 6163 224 9038 9/10 5840 325 7654 6/11 561 1 149 6781 21/12 9960 58 11022 18/1 5179 63 7102 18/2 3444 68 4858 19/3 9155 336 13721 29/4 8624 611 12104 13/5 6995 630 10667 13/6 5293 440 6140 11/7 6368 432 7141 12/8 8261 160 8968 12/9 7531 512 9499 22/10 7904 400 8859 11/7 585 189 1870 7/8 1596 351 4166 20/9 1184 272 2283 8/10 845 209 1264 6/11 917 200 1444 21/12 776 83 1073 18/1 213 32 305 18/2 796 123 1 181 19/3 1480 180 1818 29/4 1 124 260 1726 14/5 867 332 1387 13/6 1021 283 1434 11/7 1579 409 2313 12/8 924 407 1527 12/9 968 65 1198 22/10 20/11 603 208 2954 727 297 2309 427 181 1252 719 82 3068 472 425 3101 348 112 2352 832 123 1859 104 31 704 83 27 759 200 56 1259 336 39 3623 341 160 3168 376 232 1903 599 48 1295 783 77 1529 660 39 865 433 289 1256 TABLEAU 3. Evolution temporelle des autres groupes de la meiofaune et des nauplii (%). BROUENNOU ROTIFERES TARDIGRADES CASTROTRICHES OSTRACODES TURBELLARIES DIVERS TOTAL NAUPLII 19 7 9 19 8 0 6/8 21/9 8/10 5/11 20; i: 17/1 19/2 18/3 30/4 14/5 12/6 10/7 11/8 11/9 23/10 20/11 > + 0,8 ♦ 5,9 + 10,4 12,1 + + 14,6 2,9 + 9,2 7,2 + + + 5,4 + 0,6 1,5 5,3 0.5 0,7 + 1.5 20,0 + 1, 3 + 1,8 15,1 0,9 3,5 * + 2,9 16,2 3,4 3, 1 ♦ 1,8 7,9 0.5 0,5 + 1.6 20,5 + + + 1,0 0,7 + 0,5 + 0,8 0,7 ♦ + 0,6 2,9 + + * ♦ 1,2 0,6 ♦ ♦ 0,5 6,7 0,8 10,4 0,7 26,7 0,9 12,1 4,0 12,6 7,4 0.7 22,7 2,5 18,2 8,5 23,5 6,4 16,2 5,4 23,1 3,3 1,7 1.6 2,0 0,7 3,5 0,8 1.2 12,6 1 ,1 CORN AR GAZEL ROTIFERES TARDIGRADES CASTROTRICHES OSTRACODES TURBELLARIES DIVERS TOTAL NAUPLII 7/8 20/9 9/10 6/11 21/12 18/1 18/2 19/3 29/4 13/5 13/6 11/7 12/8 12/9 22/10 ) 2.3 1,2 2,4 47,9 0,9 1,0 6,9 2,7 39,0 + 10,9 1,2 21,9 0,7 4,8 0,7 4." 2,0 14,1 + 3,1 + 1 .5 12,3 + 1,4 3,3 6,6 4,3 4,3 3,0 2,3 3,1 12,5 2.0 1.3 2,0 + + 4,1 1,1 1,0 0,7 1,3 ♦ 4,9 1.7 1.7 1.0 + 4,5 0.6 1.8 0.6 3,6 + 1.9 0.6 0,8 9.3 + 1.4 + 0,8 4,7 + 1.4 ♦ + 2,6 0.8 7,4 + 53,8 2,9 50,5 2,1 34,0 0,7 10,6 0,6 19,2 18,5 20,5 20,9 0,6 6,9 1,5 10,6 3.2 7,5 6.1 1 1 ,5 6,9 10,8 KERSAINT ROTIFERES TARDIGRADES CASTROTRICHES OSTRACODES TURBELLARIES DIVERS TOTAL NAUPLII 11/7 7/8 20/9 8/10 6/11 21/12 18/1 18/2 19/3 29/4 14/5 13/6 11/7 12/8 12/9 22/10 20/11 0,9 14.3 13.8 *34,8 1,1 5.7 1.6 13,8 31,9 1,6 8,1 ♦ 8,0 30,3 1 ,0 5,6 7,2 2,4 56,0 2,0 2.6 + 0,9 64,5 4,5 1.3 1,0 1.4 71,0 1,3 7,6 11,9 2,6 20,4 3,5 + + 2,3 23.4 46,2 5,8 11,9 5,4 2,7 13,9 43,7 3,4 + 2,9 3,1 18,7 18,8 25,5 + 1,3 5,9 4.2 30,9 44,6 2.7 2,0 3.2 18,7 18,1 27,7 + 5,1 3,1 39,3 5,4 2,8 1.0 2,3 16.4 20,3 0,9 3,5 1,4 8,9 4.2 3.0 11,5 8,5 1.4 1.8 6,1 2,0 1.4 2,4 4,6 7,4 5,4 3,9 1 .1 3,2 71,2 0,8 54,1 48,0 1,6 72,2 1.1 70.0 79,2 0,5 47,3 77,7 81,0 4,2 69,0 9,6 86.9 2,1 72.4 11.2 55,7 10,2 44,4 3,8 37,5 3.2 15.1 25,6 13,9 TABLEAU 4. Evolution temporelle de certains groupes du meiobenthos temporaire (%). BROUENNOU Anne I ides Gasteropodes 1 9 1 9 19 8 0 6/8 21/9 8/10 5/11 20/12 17/1 19/2 18/3 30/4 14/5 12/6 10/7 11/8 11/9 23/10 0,8 2,8 1,5 3,3 1,8 0.8 + 0,7 0,7 0,7 2.2 2,6 3,1 1.4 0,6 ♦ + 00RN AR GAZEL 7/8 20/9 9/10 6/11 2 1 / 1 2 18/1 18/2 19/3 '•> 1 13/5 13/6 11/7 12/8 12/9 22/10 Anne 1 ides Tanaldaces Cumaces 3,8 + + + 1.3 3,3 1,3 + * ♦ 3.8 2,3 + 3,5 ♦ 1.4 ♦ 0,8 0.5 + ♦ 3.9 + KERSAINT Anni-lides Tanaldaces Gai t ,■ t opodei Cumaces 11/7 r/8 20/9 8/10 6/11 21/12 18/1 18/2 19/3 29/4 14/5 13/6 11/7 12/8 12/9 22/10 20/11 0,5 0,9 1,3 ♦ ♦ 2,2 0,8 0,9 0,6 1.7 0.8 1.7 0,6 + 2,7 2,0 + 1.0 : 1.4 + 1 ,5 1,8 1.3 1,0 2,9 3,8 1 .7 256 presents de mai a octobre 1980, confirmant la reinstallation de ce groupe tres important au niveau de la macrofaune de Corn ar Gazel (Le Moal , 1981). Les Cumaces ont a peu pres le meme comportement que les Amphipodes : presque toujours presents entre mai et octobre 1980, ils atteignent 3,9 % de la population totale en septembre . Kersaint Suivie mensuellement depuis le 17 mars 1978 , la meiofaune de cette station presentait une veritable explosion demographique en juin, juillet et aout 1978. Ce phenomene ne s'est pas reproduit par la suite, et l'on est revenu a des variations saisonnieres faible- ment accentuees, avec un minimum en fevrier-mars (comme aux deux autres stations) et un maximum en octobre-novembre 197 9 (comme a Brouennou) et en mai-juin 1980. La densite moyenne (2 063 ind./10cm2) est la plus faible des trois , ce que laissaient prevoir les carac- teristiques du sediment. L'evolution temporelle de la meiofaune de cette station a ete marquee par une inversion du rapport Nematodes/ Copepodes a partir de mai 1978, date depuis laquelle les Nematodes sont devenus preponderants et le sont restes : depuis le mois de juillet 1979, ce rapport oscille entre 1,1 (decembre 1979) et 16,9 (novembre 1980). La densite moyenne des Harpacticoides est d'ailleurs passee de 366 ind./lO cm2, entre mars 1978 et juin 1979, a 157 entre juillet 1979 et novembre 1980, en raison principalement du pic "anormal" de juin 1978. C'est a Kersaint que les autres groupes du meiobenthos vrai sont proportionnellement les plus importants : ils constituent pres de 87 % de la population en mai 1980, et les valeurs depassant 70 % ne sont pas rares . Les Ostracodes (tous a des stades tres jeunes) constituent 39,3 % de la population en juillet 1980, et la propor- tion, des Gastrotriches s'eleve a 16,4 % en aout de la meme annee ; mais le groupe le plus important et le plus regulierement present est celui des Turbellaries . Parmi le meiobenthos temporaire , les Tanaidaces sont toujours presents (2,7 % au maximum en fevrier 1980), les Annelides de- viennent de plus en plus rares a partir de fevrier 1980, alors qu'au contraire les Gasteropodes reapparaissent depuis juillet 1980 (3,8 % de la meiofaune totale en octobre). Discussion En 1' absence de donnees anterieures au 17 mars 1978, il est bien difficile de dire qu'elle est la periode la plus proche de la "normale" du point de vue quantitatif . Les fortes densites observees durant la premiere periode a Corn ar Gazel et Kersaint pourraient correspondre a une phase d'eutrophisation "anormale" consecutive a 1' accumulation de matiere organique dans le sediment, accumulation resultant elle-meme de la pollution par les hydrocarbures . Dans ces biotopes a "haute energie", 1 'hydrodynamisme intense a pu provoquer un retour a 1 'oligotrophie durant la seconde periode, alors qu'a Brouennou la stabilite du milieu maintenait une certaine eutrophi- sation. Malheureusement , nous ne disposons pas de donnees sur la teneur en matiere organique des sediments pour etayer cette hypo- these . 257 On peut aussi expliquer , du moins en partie, les chutes de densites de la seconde periode par la reinstallation dans le bio- tope de predateurs provisoirement elimines par l'arrivee des hydro- carbures ; en tout cas , cette reinstallation est evidente au niveau des Amphipodes . Evolution comparee de la meiofaune et du microphytobenthos La comparaison des resultats obtenus, aux deux stations de Brouennou et Corn ar Gazel, pour les pigments chlorophylliens de la couche superficielle (0-1 cm) et la meiofaune, montre que 1 'ampli- tude des variations et les valeurs maximales de la densite et de la teneur pigmentaire sont plus elevees a Brouennou, biotope le plus stable. A cette station, des relations de type trophique entre mi- crophytes et meiofaune sont fortement suggerees . On observe en effet un relais entre la phase d'accroissement des pigments chlorophyl- liens (decembre a mars) et celle de la meiofaune (avril-mai), relais suivi d'une phase d'equilibre relatif. L'accroissement des pigments chlorophylliens apparait done en hiver, alors que l'activite des meiobenthontes est ralentie et leur densite en diminution. Les fac- teurs climatiques n'etant pas ici limitants pour les microphytes, on est en droit de penser que e'est une diminution du "grazing" qui favorise leur accroissement . A Corn ar Gazel, 1' amplitude des variations saisonnieres des pigments chlorophylliens et de la meiofaune, surtout depuis juin 1979, est fortement limitee par 1' action de l'hydrodynamisme . II est possible d'etablir une coincidence entre la distribution verticale des pigments et la valeur du rapport Nematodes/Copepodes : ce rap- port presente ses plus fortes valeurs en hiver, periode pendant la- quelle les Copepodes, assez infeodes ici a la surface (au contraire des Nematodes), sont moins nombreux et ou il y a aussi moins de pig- ments. L' instabilite de la couche superficielle semble done etre le facteur limitant de la biomasse primaire et secondaire a cette sta- tion et , de ce fait, une possible relation trophique est masquee . Les Copepodes Harpacticoides : etude qualitative Comme nous avons deja eu 1' occasion de le montrer (Bodin et Boucher, 1981), une etude qualitative est souvent plus revelatrice des perturbations d'un peuplement qu'une simple etude quantitative. Variations temporelles des differents groupes ecologiques Apres determination, les especes d'Harpacticoides ont ete regroupees par affinites ecologiques (Tableau 5) d' apres nos ob- servations personnelles et les donnees de la litterature , operation toujours delicate en raison des incertitudes qui pesent sur l'eco- logie de certaines especes. La comparaison de deux annees consecu- tives : novembre 1978 a octobre 1979 et novembre 1979 a octobre 1980, met en evidence une certaine evolution des groupes ecologiques au niveau de chaque station. Pour chacune des deux annees et pour chaque groupe , deux variables ont ete calculees : la somme des den- sites des especes concernees (E densites) et la dominance generale moyenne (D.g.m.) (Bodin, 1977). 258 TABLEAU 5. Liste des especes recoltees aux trois stations entre aout 1979 et novembre 1980 (s = sabulicole , v = vasicole , p = phytophile, e = eurytope , m = mesopsammique) . BROUENNOU CORN AR GAZEL KERSAINT (6/8/79 au 20/11/80) (7/8/79 au 22/10/80) (11/7/79 au 20/11/80) Canuelia &uicigeAa Jroupe ecol . D.g.m. Frequence % V.g.m. Frequence D.g.m. Frequence % V + 6 R CanuelZa peAptexa s 12,0 100 C SO, 3 1 00 c 0,4 35 F Hatecti.no ioma heAdman*. s + 12 R + 7 R + 12 R Pieudobiadya bedcu.ua s + 7 R Kie.no ietetta. sp. m 0,9 18 R TackidiuA di&cipeA e 0,6 19 R + 7 R 0,8 29 F HictoaAthAidion ie.du.ctum V + 12 R Thompionula kyaenae s 1,1 20 R HaApacticuA falexuA s 13,7 100 C 0,2 7 R 6,7 18 R Ti&be sp. p + 13 R PoAathateAtAii dovi p + 6 R + 7 R Vactylopodia sp. p + 6 R PaAoi tenheJiia tpinoia butboia p 0,1 6 R + 6 R Stenhetia [del. ) patxi&tAXA b-c4p v 0,5 25 F PobeAXAonia cettica p 24,9 100 C + 6 R Sutbamplujaicui urui e 0,1 37 F AmpliiaA cui valiani P + 7 R Amphiaicui longaAticulatixi s + 7 R AmplrUa&coZdeA iubdebitii P + 6 R Amphiai colder debitib s. str. e 39,9 100 C Amptu.ai colder deb-cU* tunccotui V 0,6 69 FF SckizopeAa sp. P + 6 R Apodopiyttui aienicobit, m + 6 R 10,9 100 C KtLopiytluA conitAsLCtub s. str. m 5,5 47 F Inteimedopiyttui intelmedua m + 12 R PaAateptaataaxi t>pinicau.da m 0,2 12 R M 20 R 55,4 100 C MeiocliAa pygmaea e + 7 R Enkydloioma piopinquum V + 19 R RktzothAix. minata s + 6 R 2,2 80 C 1,9 71 FF Huntemannia jadeniii V 0,3 37 F Hetelotaophonte itAorru. s. str. p 5,9 81 C + 7 R HeXeAotaophonte tUXoiatii p + 6 R Panataophonte bieviA06tAU> s.str. p + 6 R + 7 R PoAonychocamptuA cuAt-tcaudatui s + 6 R AiettopiiA kis.pi.da s + 6 R AieZiopi-U, intermedia s 0,4 44 F 15, i 100 C 16,9 88 C 259 A Brouennou, quatre groupes ecologiques peuvent etre distin- gues : les sabulicoles, les vasicoles, les phytophiles et les eury- topes . D'apres les D.g.m., ces groupes se repartissent de la fagon suivante : Periode du 3/11/78 Periode du 5/11/79 au 8/10/79 au 23/10/80 E densites D.g.m. E densites D.g.m. Sabulicoles 942 18,4 917 25,1 Vasicoles 477 9,2 62 1,6 Phytophiles 1 628 31,8 1 043 28,5 Eurytopes 1 525 3 153 29,9 61,7 1 620 2 663 44,4 Phytophiles + Eurytopes 72,9 La premiere annee , les phytophiles dominent , avec pres de 32 % des Harpacticoides ; viennent ensuite les eurytopes, puis les sabu- licoles et, enfin, les vasicoles. La seconde annee, les eurytopes deviennent largement preponderants , avec plus de 44 %, et les phyto- philes passent en seconde position. L'ensemble phytophiles + eury- topes progresse de plus de 11 % . Les sabulicoles et les vasicoles evoluent en sens inverse, c'est-a-dire que les sabulicoles pro- gressent de pres de 7 %, alors que les vasicoles sont reduits d'au- tant (Fig. 9). A Corn ar Gazel, ces quatre memes groupes ecologiques sont representes la premiere annee , alors que les vasicoles et les eury- topes disparaissent la seconde annee. Mais, a cette station, les sabulicoles rassemblent toujours environ 99 % de la population : Periode du 15/11/78 Periode du 6/11/79 au 9/10/79 au 22/10/80 X densites D.g.m. E densites D.g.m. Sabulicoles 3 247 98,6 2 305 99,8 Vasicoles 4 0,1 - - Phytophiles 2 0,1 4 0,1 Eurytopes - Eurytopes 27 29 0,8 0,9 - - Phytophiles h 4 0,1 II n'est done plus question de variations entre les groupes, mais il est interessant de noter ici une variation a l'interieur du groupe des sabulicoles. Celui-ci est compose essentiellement de deux especes : Asellopsis intermedia et Canuella perplexa. La premiere annee, A. intermedia est preponderante , avec une D.g.m. de 71,5 % contre 18,2 % a C. perplexa. L' annee suivante, e'est C. perplexa qui redevient largement dominante ( comme e'etait le cas en mars 1978) avec 88,7 % de la population, contre seulement 9 % a A. intermedia (Fig. 10) A Kersaint , station de sable pratiquement pur, les especes vasi- coles sont evidemment absentes . Avec une mediane de pres de 200 ym, ce sable est propice a 1 ' installation des formes typiquement inters- titielles ; il devient alors necessaire de distinguer, parmi les Harpacticoides, un groupe d'especes sabulicoles mesopsammiques et 260 N Ul o n t ■ ■ • ' ♦— — ♦ Sabulicotes • • Phyloph le I \ [I => rv •^ i/ji \p—\ T->*»~«8 .-7-fc^^-^.-.<-,-. N D J 1978 FIGURE A 500 FMAMJJASOND 1979 J FMAMJ J ASON 1980 9. Brouennou : evolution temporelle de la densite des Harpac- ticoides regroupes par affinites ecologiques. ■ — ■ ■ — ' > -' M::S0 N » J F M A M J J A S 0 N OJ F M A MJJA S 0 1978 ' 1979 I 1980 FIGURE 10. Corn ar Gazel : evolution temporelle de la densite des principales especes d'Harpacticoides . 950 900 800 700 , 600 500 400 300 200 10O 50 M I \ i I I i ;' i ; i / i * / D 0 0 MfSMpsarnmlquf! D D e„. . tnoop..n A A !»»»>"■ • " '*■ .'< ■K » J J A S 0 » 1978 ^T ■■• :■■*-,•»■:? -,-^5|"B |" , g. jr f-,-rw-flT. ■ -rj-t-.,-fcv,;a- ,-g-,-n-,^, n,— o,». J F M A M J J A S 0 N D I J F M A H J J A S 0 » 1979 1980 FIGURE 11. Kersaint : evolution temporelle de la densite des Har- pacticoides regroupes par affinites ecologiques. 261 un groupe de sabulicoles epi- et endopsammiques . Par ailleurs, comme elles sont peu nombreuses et peu abondantes , les especes phytophiles et les especes eurytopes sont regroupees dans un seul et meme groupe Sabulicoles mesopsammiques Sabulicoles epi-endopsammiques Phytophiles + Eurytopes Periode du 21/11/78 au 8/10/79 £ densites 1 120 1 296 20 D.g.m. 45,9 53,3 0,8 Periode du 6/11/79 au 22/10/80 Z densites 1 181 260 15 D.g.m. 81,2 17,8 1,0 L'evolution de ces groupes est assez significative : durant la premiere annee, les formes epi- et endopsammiques dominent avec plus de 53 % de la population, alors que, 1 'annee suivante , les formes mesopsammiques reprennent largement la predominance avec plus de 81 % (Fig. 11). Diversite D'une periode a 1' autre, on observe une chute importante de la richesse specifique : 51 especes avaient ete recensees dans les trois stations jusqu'en juillet 1979, on n'en compte plus que 36 entre aout 1979 et novembre 1980 (Tableau 5). De plus, le nombre d'especes dominantes (D.g.m. > 1 %) diminue aux trois stations : au total, on passe de 28 especes dominantes du- rant la premiere periode a 15 durant la seconde. Parallelement , les especes principales voient leur dominance generale moyenne augmen- ter. A Brouennou, la D.g.m. de Amphi as coi.de s debilis s. str. passe de 23 a 10 i A Corn ar Gazel, la D.g.m. de A. intermedia etait de 63 % durant la premiere periode etudiee , celle de C. perplexa est de 80 % durant la seconde periode. A Kersaint , durant la premiere periode, la D.g.m. de A. intermedia etait de 26 %, celle de Kliop- syllus aonstriatus s. str. de 25 %, celle de Paraleptastaaus spini- oauda de 22 % ; durant la seconde periode, la D.g.m. de P. spiniaau- da passe a plus de 55 %. Enfin, le cas de K. aonstriatus est interessant a considerer : cette espece avait une position tout a fait preponderante jusqu'en juin 1978 ; elle est restee frequente par la suite, mais sa D.g.m. est tombee de 24,7 a 5,5 %. Cependant , on observe une recrudescence de cette forme mesopsammique en novembre 1980, ou sa dominance par- tielle est de 48,2 %, ce qui nous rapproche de la situation initiale de mars 1978. Discussion Dans 1' ensemble, on assiste done a une progression des especes sabulicoles et a une regression des vasicoles. A Corn ar Gazel et a Kersaint, l'evolution aboutit meme a une situation proche de celle qui prevalait en mars 1978 ; la diminution de A. intermedia et 1' augmentation du stock des mesopsammiques laissent supposer une depollution du milieu, depollution facilitee par un hydrodynamisme plus intense a ces stations. Mais, a Brouennou, la progression des eurytopes est encore plus nette que celle des sabulicoles, grace a certaines especes telles que A. debilis s. str. qui occupent encore largement le biotope. 262 Doit-on considerer ces especes (A. intermedia et A. debilis comme des "opportunistes" au sens ou l'entendent Bellan (1967) et Glemarec et Hily (1981) pour la macrofaune ? II est sans doute en- core trop tot pour l'af firmer, car nous manquons d'etats de refe- rences de ce type en meiofaune. Du point de vue de la richesse specif ique , c'est la station de Kersaint qui a perdu le plus d' especes (7) par rapport a la premiere periode etudiee (en juin 1978, 18 especes etaient presentes a Ker- saint ; en juin 1980, il n'y en avait plus que 6) ; Brouennou en a perdu 5 et Corn ar Gazel en a gagne 2. Mais le phenomene le plus significatif , a notre avis, est la reduction du nombre des especes dominantes de chaque station et la tendance a la concentration de la faune harpacticoidienne sur quelques especes particulierement bien adaptees au biotope. A Corn ar Gazel, cette tendance est poussee a l'extreme, c'est-a-dire qu'on a un peuplement presque monospeci- f ique , correspondant a un biotope tres selectif d'ou les especes qui avaient envahi le milieu a la suite de la pollution disparaissent peu a peu. CONCLUSION Le microphytobenthos est tres vite apparu, sur ces deux plages, peu sensible a 1' action directe de la pollution (dosages de pigments et observations microscopiques in vivo realises en avril 1978) mais , partie integrante de l'ecosysteme , il reagit au desequilibre provo- que dans celui-ci. II est un revelateur des caracteres edaphiques du biotope et represente un maillon du reseau trophique benthique sous sa forme active (chlorophylle a) ou detritique (pheophytine ) . Son etude apporte des elements dans la distinction entre des fluctua- tions naturelles provoquees par 1 'hydrodynamisme et une reaction a la pollution des peuplements animaux interstitiels , ce qui explique- rait la difference constatee entre les deux annees . Le meiobenthos, en tant que niveau trophique essentiellement lie au substrat, est particulierement sensible aux fluctuations des parametres ecologiques, comme l'ont montre de nombreux auteurs (Gray, 1971 ; Arlt , 1975 ; Giere , 1979, Frithsen et Elmgren , 1979 ; Coull et Bell, 1979 ; Renaud-Mornant et Gourbault , 1980 ; Boucher et ail . , 1981). Le meiobenthos est particulierement precieux dans le cas des biotopes pauvres en macrofaune (Kersaint). Du point de vue quantitatif, on peut constater qu'il n'y a pas eu d' "hecatombe" dans la meiofaune, comme ce fut le cas en d'autres circonstances (Wormald, 1976). Mais on observe des perturbations au niveau des cycles saisonniers. A Kersaint, par exemple , il semble que la presence d'hydrocarbures ait provoque une regression des peu- plements (en particulier des Copepodes Harpacticoides ) jusqu'en mai 1978, ce qui a eu pour effet de retarder de deux mois le pic de printemps . Ce decalage n'est plus que de un mois l'annee suivante et il est completement resorbe en 1980. L'elevation temporaire des densites observee au bout de quelques mois peut etre une autre con- sequence de la pollution liee a une eutrophisation inhabituelle du milieu provoqueepar un eventuel apport de matieres organiques . Dans les milieux de mode battu, 1 'hydrodynamisme a agit rapidement pour, au bout de 12 a 16 mois, operer un retour a l'oligotrophie habi- tuelle de ces milieux. D'une certaine maniere , on retrouve ici le 263 schema des mecanismes regulateurs des ecosystemes etudies en baie de Morlaix par Boucher et at. (1981). Mais, en l'absence d'etats de re- ferences anterieurs a la maree noire pour ces stations, nous reste- rons prudents dans 1 'interpretation des chutes de densites observees a Corn ar Gazel et Kersaint depuis juillet 1979, ou intervient pro- bablement aussi la reapparition des predateurs de la macrof aune . Beaucoup plus revelatrices sont les perturbations au niveau qua- litatif observees chez les Copepodes Harpacticoides . Apres 1' afflux d'especes qui a fait suite a la maree noire (juin 1978 a Kersaint), une diminution de la richesse specifique jointe a une certaine evo- lution des groupes ecologiques montrent qu'un processus de retour a l'etat initial est sur le point d'aboutir, en novembre 1980, sur les plages de mode battu. Par contre , une plage de mode abrite telle que Brouennou semble a un stade de depollution moins avance , a moins que ce ne soit la son etat normal... Encore une fois , l'absence de refe- rences ne nous permet pas de nous prononcer avec certitude. En tout etat de cause, 1' evolution de la meiofaune en milieu pollue par les hydrocarbures est done liee essentiellement a 1' oxy- genation du sediment et , par consequent, a l'intensite de l'hydrody- namisme . 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Pollut., vol. 11, pp. 117-130 Ce travail a ete en partie rialisi grace au Contrat NOAA/CNEXO n° 79/6184. 267 LONG-TERM IMPACT OF THE AMOCO CADIZ CRUDE OIL SPILL ON OYSTERS Cvassostvea gigas AND PLAICE Pleuroneotes 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 by 1 2 Jerry M. Neff and William E. Haensly 1) Battelle New England Marine Research Laboratory, Washington Street, Duxbury, MA 02332, USA 2) Texas A&M University, Department of Vetinary Anatomy, College Station, TX 77843, USA INTRODUCTION On the evening of 16 March 1978, the. Liberian-registered super- tanker Amoco Cadiz (233,680 tons deadweight) ran aground and subse- quently broke up on Men Goulven rock, Roches de Portsall, approximately 2 km off Portsall on the Breton coast of France. Over a period of several days the complete cargo of the supertanker, which consisted of 120,000 metric tons of light Iranian crude oil, 100,000 tons of light Arabian crude oil and 4,000 tons of bunker fuel was spilled into the coastal waters. By mid April the oil had spread to and contaminated in varying degrees 375 km of the north and west coasts of Brittany (Hess, 1978; Spooner, 1978; Southward, 1978). At the time, it was the largest oil spill in maritime history. There have been two larger spills since then. Two estuaries in the heavily impacted area, l'Aber Benoit 6 km east of the spill and l'Aber Wrac'h 9 km east of the spill, face west and became heavily contaminated with spilled oil. Aber Benoit and Aber Wrac'h are biologically rich and before the spill supported large oyster mariculture operations and other commercial fisheries. It was therefore of considerable economic and hygenic impor- tance to accurately assess the progress of the long-term recovery of the estuarine biota from the impact of the oil spill. Several factors relating to this spill, including the large volume of oil spilled, the prevailing winds and currents which drove much of the oil ashore, adverse weather conditions and large tidal prisms which resulted in the incorporation of large amounts of oil into bottom sedi- ments, and the extreme biological richness of the impacted area, all 269 conspired to create a "worst case" scenario for marine oil pollution. Therefore, the Amoco Cadiz spill offered a unique opportunity to study in detail the long-term impact and timecourse of biological recovery from a catastrophic pollution incident. While we already know that the immediate biological effects of the spill were very serious in some areas (Cross et al. , 1978; Chasse, 1978; Chasse and Morvan, 1978), there was very little information upon which to base estimates of the rate at which the impacted area would be returned to pre-spill biological productivity. We have used several biochemical parameters and histopathological examination in an ongoing biological survey to assess the health and rate of recovery of marine animals from the two heavily polluted estuaries. The primary objective of this research program was to assess the degree of chronic sublethal pollutant stress experienced by representa- tive species of benthic fauna from Aber Benoit and Aber Wrac'h. Two indices of stress were used. These are histopathology and biochemical composition. We expected the fauna of these severely impacted estuaries to exhibit an elevated incidence of various histopathological lesions directly or indirectly related to oil pollution stress. As the estuaries recovered from the spill the incidence of these lesions was expected to diminish. Similarly, the concentrations of certain diagnostic biochemical components of the severely stressed fauna were expected to deviate sig- nificantly from normal. These diagnostic biochemical indices were expected to return to normal as the estuaries recovered and the resident fauna became less severely stressed. The results of this investigation provide valuable information for assessing the biological recovery of these severely polluted estuaries. They also provide a means of diagnos- ing, pollutant stress in other polluted environments. I. Histopathology of Oysters Crassostrea gigas Marine animals readily accumulate petroleum hydrocarbons in their tissues from dispersion or solution in sea water and to a lesser extent from petroleum-contaminated sediments and food (see recent reviews by Neff et al., 1976 a,b; Lee, 1977; Varanasi and Malins, 1977; Neff, 1979; Neff and Anderson, 1981). The accumulated hydrocarbons and in particular the more toxic aromatic hydrocarbons interact with cellular membranes and interfere with membrane-mediated biological processes (Roubal and Collier, 1975). Two types of histopathological lesions may result from chronic contamination of marine animals with oil. 270 The first type is due to the direct toxic effects of petroleum hydrocarbons and associated heavy metals on cells. These compounds may produce a variety of histopathological lesions in the affected organ systems. There are several reports that exposure to sublethal concentrations of oil in laboratory or field studies resulted in epi- thelial sloughing and discharge of mucus glands in the gills of teleost fish (Blanton and Robinson, 1973; Gardner, 1975; Hawkes, 1977; McKeown and March, 1978). McCain et al. (1978) reported severe hepatocellular lipid vacuolization in English sole Parophrys ventulus following exposure for four months to experimentally oiled (Alaskan North Slope crude oil) sediments. Rainbow trout fed Prudhoe Bay crude oil-contaminated food showed several histopathological changes in the liver (Hawkes, 1977). These included glycogen depletion, proliferation of the endoplasmic reticulum and focal necrosis with connective tissue infiltration in necrotic regions. We have described a wide variety of histopathological lesions to embryos and fry of the killifish Fundulus heteroclitus exposed chronically during embryonic development to the water-soluble fraction of No. 2 fuel oil (Ernst et al. , 1977). In a recent laboratory study of the effects of water soluble fractions of crude oil on marine fish, one of us (Eurell and Haensly, 1981) observed a variety of histopatho- logic changes in liver and gill tissues. Little research has been published on the histopathological effects of petroleum in benthic marine invertebrates. However lesions similar to those described in fish can be expected in the analogous organs of marine invertebrates. Although crude oil contains known carcinogens such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, petroleum- induced cancer has not been unequivocally demonstrated in any marine species (Neff, 1979). However, there are several reports of increased incidence of cancer-like lesions in natural populations of marine invertebrates and fish from hydrocarbon polluted sites (See recent symposium volumes edited by Dawe et al. , 1976 and Kraybill et al. , 1977). The second type of histopathological lesion resulting from chronic exposure to sublethal concentrations of oil is caused by elevated suscep- tibility of contaminated animals to bacterial, virus or parasite infection. This increased susceptibility may result from damage to protective epi- thelia in the affected animals or to deleterious effects of the pollutant hydrocarbons on the immune system of the animal (Hodgins et al. , 1977; Sinderman, 1979). Marine animals which have been subjected to chronic sublethal oil pollution stress can be expected to exhibit an elevated incidence of disease in comparison to non-contaminated animals. 271 MATERIALS AND METHODS Oysters Crassostrea gigas were collected during five sampling trips to France. Dates of these trips were December 1978, April 1979, July- August 1979, February 1980, and June-July 1980. In Aber Benoit, oysters were obtained from commercial oyster pare owners in St. Pabu and Prat Ar Coum. Oysters from Aber Wrac'h were obtained from a commercial opera- tion near Paluden. Aber Benoit oysters were not available in August 1979. Reference oysters were obtained from several places. None were completely uncontaminated with oil. On the first two trips, December 1978 and April 1979, the oyster pare operator at St. Pabu had oysters from the Rade de Brest (supposedly uncontaminated) which he was holding for later sale. We used these as reference oysters. Subsequent hydro- carbon analysis revealed that these oysters were as heavily contaminated with petroleum as Aber Benoit oysters. They had probably become contam- inated during brief holding in the contaminated water of the Aber, as Michel and Grizel (1979) subsequently showed in transplant experiments. On the third trip, August 1979, reference oysters were obtained from the CNEXO mariculture field station at lie Tudy. On the fourth and fifth trips, February 1980 and June 1980, reference oysters were obtained from a commercial oyster pare owner on the Rade de Brest at Plougastel. As soon as possible after collection, the oysters were shucked and the soft tissues fixed whole in freshly prepared Helly's fixative. The visceral mass was incised to insure rapid penetration of the fixative. After fixation the oysters were washed, dissected into several organs or body regions, dehydrated in ethyl alcohol and embedded in paraffin embedding medium. Organ systems processed for histopathological examination included: visceral mass (includes digestive tract, digestive gland, kidney and gonad), gill, and mantle. Sections were cut a 6 ym with a rotary microtome and stained with hematoxylin-eosin. All tissue blocks and prepared microslides of oyster tissues were labeled, inventoried and archived. Tissue sections were evaluated qualitatively. The qualitative pro- cedures included a description of the average and limits of normal for the histological status of each tissue. All histopathological lesions were described in full. The incidence of different types of lesions in each tissue was recorded. The incidence of different types of lesions in each tissue was recorded. These data for the three populations (2 oil-contaminated stations and one control station) were compared. Seasonal and temporal differences in the incidence of pathological lesions were also recorded. A photographic record of normal tissue histology and of all types of histopathological lesions was made and archived. 272 RESULTS Tissues from 134 specimens of Cvassostvea gigas from four sites were examined for histopathologies over five sampling trips. From the speci- mens collected, tissue samples of 131 adductor muscles, 127 stomach/ intestines, 129 digestive glands, 130 gonads, 134 gills, and 130 mantles were examined for a total of 781 tissues out of a possible 804. A total of nine types of pathologies were found with an incidence of 241 occurrences (Table 1). Five-hundred and ninety of the 781 (75.6%) tissues examined were free of pathologies; or, 191 of the 781 (24.3%) tissues examined bore one or more pathologies. Of the 241 pathologies found, 77 (32.0%) were various types of symbioses, while 164 (68.0%) cases apparently were not correlated with symbioses. Table 2 summarizes the distribution of pathologies among the tissues examined. Adductor muscle had the lowest incidence (3.8%). Digestive gland tissue had the highest incidence (23.9%) followed by gill (22.0%), mantle (21.4%), gut (17%), and gonad (11.9%). The number of tissues with pathologies was nearly evenly distributed among the collecting sites. Oysters from reference stations had a higher incidence of lesions, particularly in gonad and gill, than oysters from oil-polluted sites. Thirty percent of the oyster tissues from both Aber Wrac'h and Aber Benoit bore one or more pathologies. Forty percent of the tissues from Rade de Brest and lie Tudy combined contained one or more pathologies. Overall, mantle bore the lowest number of pathology types (3) while digestive gland contained the most types of pathologies (9) followed by gut (7), gill (6), gonad (5), and muscle (4). Pathologies and their distributions among organs and sites are described below. 1. Muscle. - Muscle tissues were examined from 131 C. gigas. Samples for microscopic examination were dissected from the adductor muscle and both fast and catch muscles were examined when possible. Generally, two tissue samples were taken from each muscle and oriented to give both longitudinal and cross sections. Histopathologies occurred in 4.6% (6 of 131) of the muscle samples examined. There were a total of 9 incidences of. the three pathologies described below. Muscle from reference stations contained the widest variety of pathologies. No pathologies were found in muscles from Aber Benoit. 273 Table 1. Types of pathologies, total incidence of each, affected organ and collecting site of occurrence Pathology Incidence Organ* Site+ Amoebae Ciliates Sporozoans Copepods Nematodes Degeneration Necrosis General leucocytosis Focal leucocytosis Total 241 * Mil - Muscle GU - Gut DG - Digestive gland GO - Gonad GI - Gill MA - Mantle + C - Control (Rade de Brest and He Tudy) W - Aber Wrac' h B - Aber Benoit 3 DG.MA C 21 GU.DG.GI W,B,C 29 MU,GU,DG,G0,GI,MA W.B.C 23 GU.DG.GI B.C 1 DG W 10 MU,GU,DG,G0 W,C 9 GU.DG.GO W.B.C 93 MU,GU,DG,G0,GI,MA W,B,C 52 MU,GU,DG,G0,GI,MA W,B,C 274 Table I. Distribution of patholocies in tissues of oysters Crassostrea gigas from two oil contaminated estuaries and from reference stations, with sampling times combined. Organ Digestive Station Muscle Gut Gland Gonad Gill Mantle Total Reference 5 17 20 15 21 18 96 Aber Benoit 0 18 19 6 14 17 74 Aber Wrac'h _§._£]£ 7 18 17 71 Total 10 41 57 28 53 52 241 275 Abnormally high numbers of eosinophilic leucocytes (general leucocytosis) were apparent in 1.5% (2 of 131) of the adductor muscle samples exmained. Leucocytes were generally spread throughout the muscle rather than being in focal aggregations. Aggregated eosinophilic leucocytes were present in 1 of 131 adductor muscles examined. For the purposes of this report, this aggregation was classified as a focal leucocytosis although there was no central core or tight concentric arrangement of leucocytes as reported from Crassostrea vivg-iniaa (Armstrong et al., 1980). This may be an inflammatory response to what appears to be a foreign body, possibly a nematode, at the edge of the aggregation. Five (3.8%) of the muscles examined contained areas of degenerated muscle bundles. This condition was characterized by a breakdown or liquefaction of the cellular integrity. Degenerated areas contained amorphous, light staining debris and fibers. No pyknotic nuclei were present in surrounding whole muscle fibers and no inflammation (increased number of leucocytes) was apparent. Unidentified sporozoans in the plasmodial stage were found in 1 of the 131 muscles examined. 2. Digestive Gland. - The digestive glands of 129 C. gigas were examined. Generally, two samples were taken from each specimen at differ- ent levels (anterior and posterior) of the digestive gland. Histopathologies were noted in 44.2% (57 of 129) of the digestive gland samples examined. There were a total of 64 incidences of the 9 types of pathologies described below. Twenty-seven of these or 42.2% apparently were not attributable to symbioses, while 37 (57.8%) were a type of symbiont or were clearly attributable to symbioses (i.e. inflam- mation). The distribution of these histopathologies among sampling sites is summarized in Table 3. Digestive gland samples from Aber Benoit con- tained more pathologies than samples from the other two sites. All digestive gland samples from the December, 1978 collection at Aber Benoit bore one or more pathologies. Samples from other sites over the five collections had no more than 62% incidence of pathologies. Abnormally high numbers of eosinophilic leucocytes were dispersed throughout the leydig tissue between diverticula in 5 (3.7%) of the 129 samples examined. In some, leucocytes were also invading the diverti- cular epithelium. These cases could have been inflammatory responses to parasites such as copepods which were not included in the sectioned material. That is, the sections could be at the edge of an inflammatory response as described below. 276 r— IT) ■— a. B o i- d *ri -U CnE 3 C U QJ 4- C O +J l/l 4-> OJ T ■1— ai rr t/) O a; t- o CI c 01 4J u rtl Q tn o OJ cu ■<- O i— E — 277 Aggregates of eosinophilic leucocytes, were present in 12.4% (16 of 129) of the digestive glands examined. Almost all cases were in specimens from Aber Wrac'h or Aber Benoit (Table 3). For the purpose of this study, these were termed focal leucocytoses. They differed from a general leuco- cytosis in that the leucocytes were in a dense clump, sometimes focal, rather than being dispersed throughout the tissues. General leucocytosis may possibly, in some cases, be a part of a focal inflammation viewed some distance from the focal foreign body or parasite. Some cases of focal leucocytosis appeared to be confined to the leydig tissue surround- ing the diverticulae and were not totally "focal". In most cases, however, the condition involved mass invasion of the lumina by leucocytes and/or phagocytes with large numbers of leucocytes and/or phagocytes massed in the surrounding leydig tissue. Decomposed portions of copepods were present in the lumina of two specimens and no doubt were responsible for the mass inflammation. Copepods were not apparent in the leucocytic inflammations in the digestive glands of the other specimens. These inflammations, or focal leucocytoses, may also have been responses to copepods as they were identical in all aspects except for the observed presence of copepods in the section. In one case, well-formed focal aggregates were present in the leydig tissue adjacent to the digestive gland. In one, the leucocytes were con- fined to a well-formed "pocket", while in another the leucocytes were also spread from the "pocket" to adjacent leydig tissue. A massive pocket of leucocytes was present in one of the digestive glands examined. Leucocytes were confined to the large "pocket". Adjacent leydig cells were compressed. A degeneration of two or three diverticula was observed in one digestive gland and was associated with a copepod parasite. This involved a breakdown of the diverticular epithelia and basal membranes with leucocytic inflammation. Five (3.9%) of the samples examined bore small necrotic areas on one to four diverticula. These areas were characterized by a breakdown of cellular integrity accompanied by light staining cellular debris and a limited number of leucocytes. Necrosis appeared to be minor. Amoebae were present in the digestive gland of one C. gigas. Digestive glands of 16 (12.4%) of the C. gigas examined contained ciliates. Ciliates were evenly distributed among Aber Wrac'h, Aber Benoit and Rade de Brest oysters and were sometimes quite numerous in the diverticular lumina. Ciliates were oblong, with a somewhat pointed 278 antenai, and longitudinal spiral rows of short, stout cilia. They did not appear to damage the diverticula. Sporozoa were present in the diverticular epithelium of 16 (12.4%) of the specimens examined. All but two of the cases were from Aber Benoit or Rade de Brest and lie Tudy (Table 3). Sporozoans were spher- ical and stained very intensely. They were often surrounded by a clear (lysed ?) zone. The digestive gland of one specimen bore a nematode which elicited an inflammatory response, an aggregation of eosinophilic leucocytes. Remains of copepods were noted in the diverticula of 3 C. gigas from Aber Benoit. They were accompanied by heavy leucocytic inflammation and were being phagocytized as evidenced by the presence of leucocytes in the copepods. 3. Gut. - Samples consisting of stomach, intestine and often esoph- ageal and rectal tissues were examined from 127 C. gigas. Generally, two tissue samples were taken from each specimen (anterior and posterior portions of the visceral mass). Histopathologies were noted in 32.2% (41 of 127) of the gut samples examined. There were 62 cases of the 6 pathology types discussed below. Thirty-five percent (22) involved a symbiont while 40 cases (65%) apparently were not symbiotic in nature, although there may be some question about this. Specimens from the combined reference stations bore more than two and a half times the pathologies as Aber Wrac'h specimens. Oysters from Aber Benoit contained slightly fewer pathologies than those from the reference stations and twice the number of pathologies as speci- mens from Aber Wrac'h. Abnormally high numbers of eosinophilic leucocytes (general leuco- cytosis) were noted in the intestinal epithelium, and sometimes surround- ing leydig tissue, of 15% (19 of 127) of the C. gigas examined. Almost all cases were in oysters from Rade de Brest. This condition was diffi- cult to judge. Oyster intestinal epithelium normally has some leucocytes between columnar cells. However, the large number of leucocytes in the intestinal epithelium of these 14 specimens appeared abnormally high, the number was considered abnormally high if the basal portion of columnar cells was completely, or almost completely, obscured by leucocytes. However, there still is some doubt about whether- this is a "pathology" or a normal condition. Except for the large number of leucocytes, the intestinal tissues appeared very healthy. 279 Focal aggregates of leucocytes were present in the intestinal epi- thelium, or adjacent to it, in 14.2% (8 of 127) of the gut tissues examined. One case involved a large, loose aggregation of leucocytes in the leydig tissue beneath the basement membrane. Another involved small clumps of leucocytes between the columnar epithelial cells. Like the general leucocytosis , this condition was difficult to judge. Leuco- cytes are normally present in the epithelium, but more or less scattered about. These clumps could be normal phagocytosis, although no foreign matter was ever observed in such clumps. The intestinal epithelium containing the above clumps appeared otherwise very healthy. Focal necrotic areas were present in the gut epithelium of 3 (2.3%) oysters examined. In two incidences, the gastric shield was involved. This condition was characterized by a breakdown of the structure of the gastric shield and/or epithelium, a concentration of debris at the affected area, and leucocytic inflammation of the gastric shield and/or epithelium. Ciliates were present in the gut lumen of one C. gigas. These ciliates were the same type as described above in the digestive gland. The plasmodial stage of an unidentified sporozoan was noted in the epithelium of a single C. gigas. The Plasmodium was amoeboid in appear- ance with several nuclei. The gut otherwise appeared in very good condition. Copepods were present in the stomach of 15.7% (20 of 127) of the specimens examined. No oysters from Aber Wrac'h bore copepods. None of the copepods observed were being phagocytized as was the case in the digestive gland. Up to three copepods were observed in some sections. 4. Gonad. - Gonadal tissues of 130 C. gigas were examined. Gener- ally, two tissue samples were taken from each specimen (anterior and psoterior visceral mass). Gonadal tissues from C. gigas were a very difficult tissue type to assess for non-symbiotic pathologies. Possible histopathologies were noted in 38% (9 of 130) of the gonadal tissues examined. There were 50 incidences of the three non-symbiotic pathology (?) types and one symbi- otic pathology discussed below. Only six of the 50 conditions were of an apparent symbiotic nature. Half (36 of 71) of the female gonadal tissues examined exhibited moderate to heavy aggregations, both focal and general, of eosinophilic leucocytes. This presented a perplexing problem in determining if this 280 represented an inflammatory response to a stressful condition and therefore a pathology due to such stress, or if it was a normal condi- tion in the reproductive cycle of C. gigas. This condition was present in only one Cvassostvea virginica from South Louisiana oil platforms (Armstrong et al., 1980) but was observed in other bivalve species (10% of specimens examined in association with degeneration or necrosis of the gonad). Eight female C. gigas from the Pacific Northwest (Sequim, Washington) were examined for comparison. All eight appeared to be in a post-spawn condition and all had heavy aggregation of leucocytes in the gonadal tissues. The spawning cycle of the C. gigas from France could not be defin- itely determined. Undifferentiated (could not determine if it was male or female), undeveloped, developing (immature), ripe (mature) and spawned stages were present in samples from all five of the collecting periods (December 1978, April 1979, August 1979, February 1980, and June 1980). The majority of the specimens from December 1978, however, appeared to be of the spawned stage at Aber Wrac'h, ripe at Aber Benoit, and undiffer- entiated at Rade de Brest. In April 1979, the majority of the specimens appeared to be in the developing stage at all three sites. The majority of the specimens taken during August 1979 and June 1980 appeared to be of the ripe stage at all three sites, although there were some spawned- appearing specimens from Aber Wrac'h in August 1979. The February, 1980 collection yielded more undifferentiated and developing specimens. This does somewhat indicate an early winter spawn, but as already stated, all reproductive stages were present in samples from all five collection periods. In the C. gigas from France, 13.8% (18 of 130) of the gonads examined contained large numbers of leucocytes dispersed throughout the tissues. All 18 incidences were in female gonads (18 of 76 or 23.8%). This condition was present in undeveloped, developing (immature), ripe and post spawn ovaries. In some cases it could not be determined if the ovary was in a developing stage or a post-spawn stage because of the large numbers of leucocytes present. In some, the gonad appeared fully spawned (entire gonad examined contained only a few ova and ovacytes, follicles largely empty), while in others part of the ovary was packed with ova (ripe) and the other part contained few ova and ovacytes (spawned) and many leucocytes. In gonads with large numbers of leuco- cytes, all or almost all ova appeared normal (not degenerating or lysing) , The 29 normal ovaries (no aggregations of leucocytes present) included the undeveloped, developing (immature), ripe and spawned stages. Twenty-two of the 130 (16.6%) gonads examined contained compact clumps of leucocytes ranging from foci in the follicular wall to large 281 clumps in the ovary to foci in the leydig tissue of the testes. Nine- teen (86.4%) of the cases were in female gonads. Reproductive stages varied from undeveloped to spawned. Three of the cases were found in testes. In the general and focal leucocytoses discussed above, the differ- ence between the two was in the extent (small area, tight clump vs. general dispersion over several follicles) of inflammation, but this was sometimes difficult to ascertain and the two may blend together. Necrotic appearing areas were noted in 3.1% (4 of 130) of the gonads examined. These areas were characterized by cellular debris, degenerating ova, and leucocytosis. Sporozoa were present in 4.6% (6 of 130) of the specimens examined. Sporozoans were spherical, densely staining, and were embedded in the gonadal tissue. The specimens appeared to be surrounded by a small lysed "halo" area. 5. Gill. - Gills from 134 C. gigas were examined for pathologies. Generally, three pieces of gill (consisting of both lamellae) were dissected from one side and oriented (when possible) to give both longi- tudinal and transverse sections. Histopathologies were noted in 31.3% (42 of 134) of the gills examined. There were a total of 49 cases of the six pathology types described below. Forty-three (87.8%) were apparently not symbiotic or related to a symbiotic condition. . Abnormally high numbers of eosinophilic leucocytes were present in 31.3% (42 of 134) of the gills examined. In most incidences, the leuco- cytes were dispersed throughout several plica, but four cases appeared to be more focally organized in one or two plica. Amoebae were noted in the gills of one C. gigas. The infection appeared to be light as only two amoebae were found. The amoebae were circular in outline with a hyaline cytoplasm. The nucleus occupied approximately one-third of the cell. A small, spherical inclusion body was adjacent to the nucleus. The gills of four (3%) specimens examined harbored ciliates in their water tubules. Ciliates were somewhat crescent-shaped with tufts of stout cilia extending downward from the two tips. The arms of the crescent were sometimes turned inward so that the tips of the cilia were touching, giving a partially hollow, circular shape to the ciliate. Two 282 lateral nuclei were present. The ciliates apparently provoked an inflammatory response as most were surrounded by eosinophilic leuco- cytes in the water tubules, or the surrounding tissues contained abnormally high numbers of lucocytes. One specimen contained the plasmodial stage of a sporozoan. Multi- nucleate Plasmodia were subspherical to ovate. Numerous plasmodia were dispersed throughout the gill, but most heavily in the leydig tissue of the interlamellar area. A single copepod was found on a gill filament of one C. gigas. No necrotic areas were observed on the gills examined and the outer columnar epithelium of the specimens examined appeared healthy. The number of mucous glands in sections of randomly selected plica and term- inal grooves were counted in an effort to determine if specimens from Aber Wrac'h and Aber Benoit contained more active glands than those from Rade de Brest and lie Tudy. The results were inconclusive. The number of mucus cells per unit area of gill was not statistically significantly different among the three populations. 6. Mantle. - Sections of mantle from 130 specimens were examined. Two or three pieces of mantle were dissected from specimens and oriented to give a transverse section across the tri-lobed edge. Histopathologies were noted in the mantle of 31.5% (41 of 130) of the specimens examined. There were a total of 47 of the three pathology types described below. The distribution of the pathologies among sampling sites was nearly equal. Abnormally high numbers of eosinophilic leucocytes were noted in 35.4% (46 of 130) of the specimens examined. Thirty-eight of the inci- dences involved large numbers of leucocytes dispersed beneath the epi- thelium or in the leydig tissue. Eight cases, however, involved leuco- cytes which were more aggregated in clusters. Sporozoans were found in the mantle of a single specimen. No necrotic areas were found on any of the mantles examined. All epithelial cells appeared healthy. In an effort to determine if the mantle epithelium of oysters from Aber Wrac'h and Aber Benoit contained significantly more mucous cells than specimens from Rade de Brest and lie Tudy, the number of mucous cells in a high power field were counted at a level even with the circumpallial nerve and an area three fields higher. Specimens from Aber Wrac'h contained slightly more (average of 27.5 to 34 for the five collections) mucous cells than those from Aber 283 Benoit (average of 20 to 32) and Rade de Brest and lie Tudy (average of 22 to 31). The differences were not statistically significant. CONCLUSIONS In general, oysters Cvassostvea gigas from all five collections and all four sampling stations appeared to be extremely healthy as determined by histopathological examination. Incidence of parasitic infestation was very low, especially when compared to incidence of para- sitism in C. vivginioa from the northwest Gulf of Mexico. The low incidence of parasitism in C. gigas from Brittany may be due to the fact that they are a recently-introduced mariculture species in the area. There probably has not been enough time for their parasites to catch up with them. According to Henri Grizell (personal communication), parasitism and disease are increasing in these oysters. The most prevalent pathologic lesion in C. gigas from Brittany was leucocytosis. In mollsucs, this condition is usually a response to chemical or physical irritation. It is an inflammatory defensive response. However, size and distribution of leucocyte populations varies greatly in different mollusc species under different environmental conditions. C. gigas generally seems to have more leucocytes than the closely-related C. vivginioa. Thus, the extent to which observed leucocytoses in C. gigas were normal or pathologic is uncertain. In any event, incidence of leucocytosis was similar in oysters from oil-contaminated Aber Benoit and Aber Wrac'h and from reference stations in the Rade de Brest and at lie Tudy. Necrosis was observed several times but no definitive cases of hyperplasia, neoplasia or other precancerous conditions was noted in any of the four oyster populations. There were no consistent temporal trends in incidence of pathology in the oysters from oiled and reference stations. Oysters collected in December 1978, nine months after the spill, had an incidence of patho- logical conditions similar to that in oysters collected in June 1980, twenty-seven months after the spill. One difference that may have obscured other effects was size. By June 1980, oysters which had been in the Abers at the time of the spill had grown to very large size. During the first year after the spill, there was little evidence of growth in oysters from the two Abers. During the second year, growth appeared normal or even accelerated. 284 There was also some indication, based on observations of gonadal condition, that oysters from the Abers had an altered reproductive cycle compared to reference oysters, possibly including near complete reproduc- tive suppression for one year after the spill. Sample sizes and frequen- cies were not great enough to demonstrate this convincingly. II. Petroleum Contamination and Biochemical Indices of Stress in Oysters and Plaice The most obvious immediate biological effect of the Amoco Cadiz spill was a very large kill of benthic estuarine and coastal marine organisms (Cross et al. , 1978). The rate of recovery of these benthic communities would depend on the rate and success of reproduction by the surviving animals in the affected area and on the success of recruitment from adjacent unpolluted areas. The resident benthic fauna in the oil- impacted area which survived the spill were undoubtedly severely stressed. Because of the heavy contamination of the estuarine sediments with oil it is highly probable that the surviving resident benthic fauna would continue for some time to be stressed and potential immigrants to the estuaries would be subjected to stress as they settled there. Considerable research has been conducted in recent years on sub- lethal physiological stress responses of marine animals to oil and other types of pollution (Neff et al. , 1976a; Anderson, 1977; Johnson, 1977; Patten, 1977; Neff, 1979; Thomas et al., 1980; Neff and Anderson, 1981). A variety of sublethal physiological and biochemical responses to pollutant stress have been described. In an ecological perspective, the net effect of chronic pollutant stress on marine organisms is to shunt limited energy resources away from growth and reproductive processes to maintenance and homeostatic functions. The result is decreased growth, fecundity and reproductive success in the stressed population. A variety of biochemical parameters are altered in stressed animals and reflect the stress-induced changes in energy balance and partitioning. These biochemical parameters can be used as an index of pollutant stress in marine animals. Biochemical indices of pollutant stress chosen for use in this investigation include hemolymph glucose concentration and adductor muscle-free amino acids in oysters; and blood glucose and cholesterol, liver glycogen and ascorbic acid, and muscle-free amino acids in plaice. We have discussed elsewhere the rationale for using these parameters as indices of pollutant stress (Thomas et al. , 1980, 1981 a,b). 285 When exposed to petroleum, marine molluscs and teleost fish readily accumulate hydrocarbons in their tissues (Neff et al. , 1976b; Varanasi and Malins, 1977; Neff and Anderson, 1981). Molluscs tend to release accumulated hydrocarbons relatively slowly when concentrations in the ambient medium are reduced. However, under similar conditions, teleost fish release hydrocarbons very rapidly. Differences in hydrocarbon release rate by molluscs and fish can be attributed to differences in ability to convert hydrocarbons to polar more readily excreted metabo- lites by the cytochrome P-450 mixed function oxygenase system and related pollutant-metabolizing enzyme systems (Varanasi and Malins, 1977; Neff, 1979). In the present investigation, aliphatic and aromatic hydrocarbons were analyzed in oysters and plaice from oiled and reference stations to assess patterns of hydrocarbon accumulation and release and to allow for correlations between levels of hydrocarbon contamination of animals and histopathological/biochemical responses. MATERIALS AND METHODS Oysters Crassostrea gigas were collected for biochemical analysis on the first three sampling trips. Sampling sites were as described earlier in the section on oyster histopathology. Oysters were shucked and a sample of hemolymph was collected immediately from the heart or the adductor muscle and stored frozen until analyzed. Adductor muscle was also sampled and stored at -60°C until analyzed. Plaice Pleuroneotes platessa were collected by otter trawl from oil-contaminated Aber Benoit and Aber Wrac'h. Reference stations for plaice samples were as follows: December 1978, Baie de Douarnenez; April 1979, Loc Tudy; August 1979, February 1980, June 1980, He Tudy. Fish from the Baie de Douarnenez and Loc Tudy were collected by otter or beam trawl. Fish from He Tudy were captured by net at the sluice gate of the CNEXO mariculture pond and held in large circular holding tanks with flowing seawater until sampled. Samples were taken as soon as possible after capture and while the fish were still alive. Tissue samples included blood, muscle and liver. Blood samples were centrifuged to remove red blood cells. Serum, muscle, and liver were frozen immediately in liquid nitrogen and kept frozen at -60° until analyzed. Samples from 5-10 animals from each station and each trip were analyzed biochemically. Blood glucose and liver glycogen were measured with a Yellow Springs Instruments automatic glucose analyzer, Model 23A. 286 This method, based on the glucose oxidase enzymatic reaction, is highly specific for glucose and required only 25 ul of serum. Replicate deter- minations of each serum sample were performed. Total and esterified cholesterol in serum was determined by a cholesterol oxidase assay system which is both highly snesitive and specific. For tissue-free amino acid analysis, muscle tissue was thawed, weighed and homogenized in distilled water using a 2/1 ratio of distilled water/wet weight. Homogenates were deproteinized with 12.5% trichloro- acetic acid and then centrifuged. The supernates were frozen, thawed, and centrifuged again to remove additional TCA precipitates. The super- nates were then evaporated to dryness on a rotary evaporator and the residue dissolved in 0.2 M Citrate buffer adjusted to pH 2.2. The extracts were analyzed with a Beckman automatic amino acid analyzer. The amino acid composition of the extract and the concentration of individual amino acids in it were determined. Taurine/glycine molar ratios were computed. Variations in amino acid compositions and concen- trations among fish and oysters from different sampling stations were analyzed statistically. Plaice liver was analyzed for ascorbic acid. Tissue samples were thawed, weighed and homogenized in 3% metaphosphoric acid-8% acetic acid solution. After centrifugation, the supernates were analyzed immediately by the a,a-diperidyl technique of Zannoni et al. (1974). Oysters and plaice samples for hydrocarbon analysis were taken at the same times and places as samples for biochemical/histopathological analysis. Ten to twelve whole oysters were pooled for each sample. They were shucked and tissues were rinsed in distilled water, blotted dry, wrapped in hexane-cleaned aluminum foil and frozen at -60°C until analysis. For the April 1979 sample, whole fish were used. For sub- sequent samples, pooled samples of liver and muscle from 5-10 fish were used. Fish tissue samples were handled like oyster samples. Hydrocarbon analyses were performed by Dr. Paul Boehm, ERCO, Cambridge, Massachusetts using capillary gas chromatography /mass spectrometry. 287 RESULTS AND DISCUSSION Petroleum Hydrocarbons Concentrations of total aliphatic and aromatic hydrocarbons in tissues of oysters Crassostvea gigas from Aber Benoit and Aber Wrac'h, heavily contaminated with Amoco Cadiz oil, and from supposedly clean reference stations are summarized in Table 4. Reference oys-ters for the first two collections were Rade de Brest oysters which had been held for a -short period of time in concrete holding tanks on the shore of Aber Benoit at St. Pabu. These reference oysters were heavily contaminated with Amoco Cadiz oil as were the authentic Aber Benoit and Aber Wrac'h oysters. "Hydrocarbon status" of samples was determined by comparing GC peak profiles of f\ and t^ hydrocarbon fractions of tissue extracts to GC profiles of authentic weathered Amoco Cadiz oil. Apparently, sufficient oil was still leaching from the sediments of the Aber 13 months after the spill to allow rapid and heavy contamination of oysters exposed to waters of the bay. Michel and Grizel (1979) reported similar rapid hydrocarbon contamination of oysters transplanted to stations in Aber Benoit and Aber Wrac'h. Subsequent reference oyster samples were obtained from sites which had not received Amoco Cadiz oil. They con- tained low levels of petroleum hydrocarbons not of Amoco Cadiz origin. Concentrations of total aliphatic and aromatic hydrocarbons in oysters from Aber Benoit and Aber Wrac'h did not vary substantially over the time-course of this investigation (up to 27 months after the spill) . The persistence of petroleum hydrocarbons in tissues of oysters probably represents, in part, a continuous recontamination with hydrocarbons leaching gradually into the water from the heavily contaminated sediments of the Abers. Oysters from the Baie of Morlaix, east of Aber Wrac'h and less heavily contaminated with Amoco Cadiz oil than the Abers, collected 17 months after the spill, contained about half the aromatic hydrocarbons of Aber Wrac'h oysters. It is interesting to note that Aber Benoit oysters collected in December 1978 and April 1979 had a distinctly oily taste. Oysters sampled in August 1979 and later did not taste oily. Apparently, 200 ppm aromatics is not readily detected by taste, whereas 500 ppm is. More detailed analysis of the aliphatic fraction of the oyster samples revealed some interesting trends (Tables 5-7). In all but one case (Aber Wrac'h, April 1979), the aliphatic fraction of Aber Benoit and Aber Wrac'h oysters was dominated by the low boiling aliphatics, C10 ~ C20> inciting n-alkanes, branched and isoprenoid compounds. This is quite unlike weathered Amoco Cadiz oil or oil in the Aber 288 Table 4 . Concentrations of total aliphatic and aromatic hydrocarbons (measured gravimetrically) in oysters Crascoctrca gigas from reference stations and from two estuaries contaminated with Amoco Cadiz oil. Status determined according to pattern and identity of GC peaks. Date/Sample December 1978 (9) Rade de Brest (reference) Aber Benoit Aber Wrac'h Hydrocarbon Fraction (pg/g dry tissue) Aliphatics Aromatics 47.8 136.7 115.4 208.0 552.2 540.0 Status AC oil AC oil AC oil April 1979 (13) Rade de Brest (reference) Aber Benoit Aber Wrac'h 153.9 1001.0 114.9 690.0 225.8 986.1 AC oil AC oil AC oil August 1979 (17) He Tudy (reference) Baie de Morlaix Aber Wrac'h 39.3 51.9 134.6 206.4 101.0 485.4 Other oil Other oil AC oil February 1980 (23) Rade de Brest (reference) Aber Benoit Aber Wrac'h 62 154 217 87 275 599 Other oil AC oil AC oil June 1980 (27) Rade de Brest (reference) Aber Benoit Aber Wrac' h 33 238 132 60 283 430 Other oil AC oil/Other oil AC oil a' AC oil - Amoco Cadiz oil; other oil - definitely petroleum, but cannot be identified as Amoco Cadiz oil. ' month after the Amoco Cadiz oil spill, 16 March 1978. 289 Concentration of aliphatic hydrocarbons in tissues of oysters Craccoetrra tjifjar. from Aber fcenoit, Brittany Trance collected at different times after the Amoco Cadis oil spill. Values are in n<]/g dry weight (parts per billion). Samp le Date Compound Dec 197B (9)a Apr 1979 (13) Aug 1979 (17) Feb 1980 (23) Jun 1920 (27) n"cio NO 32 NS ND NO n-C„ 316 328 NS 162 ND «-e„ 43 47 ND 44 NO n-Cu 99 31 NS 7 ND nC14 394 18 NS 16 20 Farnesane 1,343 776 NS 66 92 n"C15 22 84 NS 68 10 n"C16 44 46 NS 62 62 "C17 184 61 NS 144 22 Pristane 376 232 NS 40 22 n"C18 ND ND NS 51 ND Phytane 613 375 NS 39 122 n'_C,9 47 68 NS 17 42 n C20 77 57 ND 7 iTC,, ND ND NS 18 49 nC22 ND ND NS 10 53 nC23 24 NO NS 20 57 nC24 43 ND NS 21 53 nC25 55 ND NS 21 17 nC26 51 ND NS 9 24 n"C27 53 NO ns 37 52 n C28 37 NO NS 12 11 n"C29 72 ND NS 18 66 nC30 ND ND NS 74 343 n-C„ ND ND NS 13 27 nC32 ND ND NS 12 134 nC33 ND ND NS ND 49 nC34 ND ND NS ND 12 Total Resolved Ali- phatics 3,893 2,155 NS 981 1,346 16 March 1978 ND, not detected NS, no sample available. 290 Table 6 . Concentration of aliphatic hydrocarbons in tissues of oysters O\ifwoatfca (jiyac from Aber Wrac'h, Brittany Franc- collected at different tunes after the fltnoco Cadi* oil spill. Values are in ng/g dry weight (ports per billion). Sampl ing Date Dec 1978 Apr 1975 Aug 1979 Feb 1980 Jun 1980 Compound (9)a (13) (17) (23) (27) n"cio 46 ND 110 47 84 n"Cn 383 55 759 170 255 n-C12 86 365 ND 126 63 n"C13 39 70 32 NO 15 n~C14 36 650 12 519 35 Farnesane 222 617 953 370 148 n'C15 13 NO 172 56 13 n"C16 53 ND 098 198 31 n"C,7 37 177 577 218 NO Pristane 319 52 39 44 78 n"C18 NO NO ND . 2' ND Phytane 571 242 141 194 64 ""C,, 187 NO ND 12 ND "~C20 74 NO ND 12 ND n'C2, ND NO NO ND ND n C22 NO NO ND ND NO n"C23 ND 30 NO 141 ND n"C24 15 135 ND NO ND n"C25 15 222 NO 19 12 n"C26 14 289 ND 27 ND n"C27 11 230 ND 19 ND n C28 NO 241 ND 23 ND n"C29 ND 219 ND 18 ND nC30 NO 132 ND 16 ND n"C31 ND NO ND NO ND n"C32 ND ND ND (ID ND Total Resolved Al i- 2,121 3,776 2,893 2,256 798 phatics ' months after the Amoco Cadiz oil spill, 16 March 1978 ND, not detected. 291 Table 7 . Concentration of aliphatic hydrocarbons in tissues of oysters rtvjnjocerv.: ™£.-;a." fron reference stations on the Brittany coast of France collected at different tin^s after the AnofO C: ;";':: oil spill. Values are in nj/n. dry weight (parts per Mllion). Sampl ing Date Corpound Dec 1978a Apr 1979a Aug 1979b Feb 1920c Jun 1930c (<.')J (13) (17) (23) (27) n~C10 53 ND ND e6 226 n"Cn 360 441 ND 339 4C6 n"C12 15 49 II D 86 112 iTC,, 17 111 ND 18 9 n"C,4 52 29 ND 33 18 Farnesane 11 1,346 4 45 35 n"C15 172 123 29 122 66 n"C16 139 55 17 69 41 n"C]7 252 228 61 81 46 Pristane 18 331 ND ND ND n"C18 39 HO NO 58 45 Phytane 117 529 17 NO 13 n"C,9 40 35 ND 22 28 n"C20 43 73 ND 20 24 n"C2, 42 20 ND 16 22 n"C22 40 47 NO 15 23 n"C23 42 85 21 16 36 n"C24 49 135 29 15 45 n"C25 50 173 33 16 47 n"C26 56 204 44 15 55 n-C2? 65 207 54 20 60 n"C28 64 167 36 16 48 n"C2, 66 165 64 29 47 n"C30 22 100 54 50 42 n"C31 NO 71 59 13 22 n'C32 ND ND ND ND 8 Total Resolved Ali- phatics 1.824 4.724 522 1,200 1,604 ' from Rade de Brest, but maintained in Aber Benoit before sampling • from oyster rjriculture ponds of CNEX0 at lie Tudy ' from a commercial oyster pare in the Rade de Brest ' months after the fmoeo Cadi: oil spill, 16 March 1978 ND, not dectected. 292 sediments which is dominated by higher boiling saturated hydrocarbons. This phenomenon is unexplained and could represent selective accumula- tion and/or retention of lighter aliphatics or more rapid metabolism and excretion of, heavier aliphatics. The most likely explanation is that oysters were being contaminated with hydrocarbons leaching from bottom sediments into the water column. Lighter aliphatics, because of their slightly higher aqueous solubility than heavy aliphatics, are desorbed more readily from sediments and therefore are more available for uptake by the oysters. Aliphatic hydrocarbon fractions of reference oysters were more uniform (Table 7). Relative abundances of C^q to C32 aliphatics were similar. There were no consistent differences in characteristics cf the ali- phatic hydrocarbon fraction between reference oysters and oysters from oil-polluted Aber Benoit and Aber Wrac'h (Table 8). With one exception (April 1979), alkane/isoprenoid ratios were higher in oysters from reference stations than in those from oil-polluted stations. Pristane/ phytane ratios were quite variable and without pattern. All but two carbon preference indices were near one indicating a petroleum origin for the high molecular weight aliphatic fraction. Composition of the aromatic fraction of oysters, as determined by gas chromatography /mass spectrometry, revealed a great deal about the origin of the hydrocarbon contamination of the oysters (Tables 9-11) . High concentrations of alkyl naphthalenes through alkyl dibenzothiophenes are characteristic of samples contaminated with crude oil. Amoco Cadiz oil was particularly rich in alkyl phenanthrenes and alkyl dibenzothio- phenes. These were the most abundant aromatics/heterocyclics in oyster samples from oil-contaminated Aber Benoit and Aber Wrac'h. Aromatic hydrocarbon assemblages of crude oil origin are dominated by alkylated species, whereas aromatic assemblages of pyrogenic origin are dominated by the unalkylated parent compound (Neff, 1979). Thus we can conclude that oysters from Aber Benoit and Aber Wrac'h at all five sampling times, and reference oysters from the December 1978 and April 1979 collections were heavily contaminated with crude oil, resembling the Amoco Cadiz oil. The other three reference samples contained some oil, but it did not resemble Amoco Cadiz oil. In oysters from the two Abers, there was a general trend for the concentration of aromatics/heterocyclics in the alkyl naphthalenes to alkyl dibenzothiophenes series to decrease slowly with time. The February 1980 samples contained higher concentra- tions of alkyl phenanthrenes and alkyl dibenzothiophenes than expected. It is possible that winter storms in December and January resuspended oil-contaminated sediments causing recontamination of resident oysters. 293 Table 8 . Characteristics of the aliphatic hydrocarbon fraction of oysters Cracsostrea gigas from reference stations and from two estuaries contaminated with Amoco Cadiz oil Date/Sample December 1980(9) Pristane/Phytane Carbon Prefer- Alkanes/Isoprinoids ence Index (C26"C30) Rade de Brest (reference) Aber Benoit Aber Wrac'h April 1979(13) Rade de Brest (reference) Aber Benoit Aber Wrac'h August 1979(17) He Tudy (reference) Baie de Morlaix (reference) Aber Benoit Aber Wrac'h February 1980(23) Rade de Brest (reference) Aber Benoit Aber Wrac'h June 1980(27) Rade de Brest (reference) Aber Benoit Aber Wrac'h 0.15 0.61 0.56 0.63 0.62 0.21 ND ND NS 0.28 ND ND 0.23 ND 0.18 1.68 2.34 0.18 0.07 0.12 0.13 1.00 5.15 ND NS 0.'48 4.37 1.30 0.81 2.64 0.28 0.19 1.27 2.0 1.57 1.16 ND 1.10 1.39 0.93 NS ND 1.00 1.02 ND 1.10 0.61 0.93 294 Table 9 . Concentration of aromatic hydrocarbons in tissues of oysters Ci'assostrca gigas from Aber Benoit, Brittany, France at differ- ent times after the Amoco Cadiz oil spill. Values are in ng/g tissue (parts per billion). Dec 1978 Sampl ing Date Apr 1979 Aug 1979 Feb 1980 Jun 1980 Compound (9)a (13) (17) (23) (27) Alkyl naphthalenes NA 1,243 NS ND 300 Alkyl fluorenes NA 2,230 NS 891 850 Phenanthrene NA 590 NS 43 64 Alkyl phenanthrenes NA 17,345 NS 6 ,088 3 ,014 Dibenzothiophene NA 123 NS ND ND Alkyl dibenzothiophenes NA 15,380 NS 8 ,860 5 ,420 Fluoranthene NA 665 NS 150 84 Pyrene NA 600 NS 150 87 Benz[a]anthracene NA 263 NS 200 ND Chrysene NA 490 NS 600 180 Benzofluoranthenes NA 570 NS 670 100 Benzopyrenes NA 339 NS 413 80 Perylene NA 80 NS ND ND Total Resolved Aromatics NA 39,918 NS 18 ,065 10 ,179 a' months after the Amoco Cadiz oil spill, 16 March 1978. NA, sample not analyzed by GC/MS NS, no sample available ND, not detected 295 Table 10. Concentration of aromatic hydrocarbons in tissues of oysters Crassoctrea gigar from Aber Wrac'h, Brittany, France at differ- ent times after the Amoco Cadiz oil spill. Values are in ng/g tissue (parts per billion). Compound Dec 1978 (9)a Apr 1979 (13) Sampling Date Aug 1979 Feb 1980 (17) (23) Jun 1980 (27) Alkyl naphthalenes 781 594 150 ND 10 Alkyl fluorenes 2,203 1,453 980 560 ND Phenanthrene 89 69 ND ND 170 Alkyl phenanthrenes 12,114 14,989 5,030 10,089 4,550 Dibenzothiophene 24 ND ND ND ND Alkyl dibenzothiophenes 21,748 11,521 9,900 15,820 5,530 Fluoranthene 258 58 50 150 70 Pyrene 291 105 65 190 90 Benz[a]anthracene Chrysene ] 557 1.330 ND 300 ) 1 , 1 00 63 230 Benzof 1 uoranthenes ND 237 260 410 190 Benzopyrenes ND 161 50 170 140 Perylene ND ND ND ND 50 Total Resolved Aromatics 38,065 29,517 16,785 28,489 11,093 a' months after the Amoco Cadiz oil spill, 16 March 1978; ND, Not detected. 296 Table 11 . Concentration of aromatic hydrocarbons in tissues of oysters Cransoatrea qigaa from "reference" stations on the Brittany coast of France at different sampling times after the Amoco Cadiz oil spill. Values are in ng/g tissue (parts per billion). Sampling Date Dec 1978a Apr 1979a Aug 1979° Feb 1980c Jun 1980c COn,pOUnd (9)d (13) (17) (23) (27) Alkyl naphthalenes 327 467 ND ND 180 Alkyl fluorenes 689 1,562 ND ND 180 Phenanthrene 129 85 5 180 350 Alkyl phenanthrenes 4,375 10,679 285 527 630 Dibenzothiophene 30 56 ND ND 20 Alkyl dibenzothiophenes 3,668 10,590 283 975 675 Fluoranthene 220 171 130 180 200 Pyrene 170 98 130 180 90 Benz[a]anthracene Chrysene \ 252 V290 ND 410 140 350 58 170 Benzofluoranthenes 88 48 350 410 160 Benzopyrenes 64 65 140 182 83 Perylenes ND ND ND ND ND Total Resolved Aromatics 10,012 24,111 1,733 3,124 2,796 ' from Rade de Brest, but maintained in Aber Benoit before sampling * from oyster mariculture ponds of CNEX0 at He Tudy c, d, ' from commercial oyster pare in the Rade de Brest months after the Amoco Cadiz oil spill, 16 March 1978 ND, not detected. 297 Higher molecular weight aromatics, flupranthene through perylene, although present in small amounts in crude oil, are more characteristic of pyrogenic hydrocarbon assemblages (Neff, 1979). Concentrations of these aromatics were similar in reference and Aber oysters and there was no consistent pattern of temporal change. These hydrocarbons probably have a similar origin in all three populations, namely from particulate organic matter derived from smoke of wood and fossil fuel combustion. Several of these aromatics, including benz[a]anthracene, benzofluoranthenes, and benzopyrenes, are known carcinogens. Their presence in tissues of oysters at relatively high concentration could be cause for concern. Whole fish and muscle samples of plaice Pleuvoneates platessa contained low concentrations of aliphatic and aromatic hydrocarbons (Table 12). Most of the muscle samples contained aliphatic hydrocarbon distributions characteristic of oil (Tables 13-15). Nearly tenfold higher concentrations of aliphatics were found in liver samples than in muscle samples of reference plaice and plaice from the oil-polluted Abers. In the August 1979 samples, some of this was identified as petroleum. In later samples, no petroleum-derived hydrocarbons were detected in liver samples. The aromatic fraction showed a distribution pattern similar to that of the aliphatic fraction. Liver aromatic fractions were dominated by biogenic squalene. Liver samples also contained high concentrations of what appeared to be naphthenic (cyclic alkanes) hydrocarbons. Aliphatic fractions from all liver samples were dominated by hydro- carbons in the C21 - C30 molecular weight range. In the three liver samples from Aber Benoit, two of the three samples from Aber Wrac'h, and one reference sample, dominant aliphatics were C27 and C2g. In the remaining two samples, dominant aliphatics were C^r and C2g. With few exceptions light aliphatics, C^q - C20» were present at low or non- detectable concentrations in the plaice livers. Plaice muscle contained 1-10% of the concentration of aliphatics that liver did. Alkane distribution patterns in muscles varied consid- erably. In most cases alkanes above C24 were dominant. Concentrations of aliphatic hydrocarbons in muscle and liver were higher in summer (August 1979 and June 1980) than in winter (February 1980) , suggesting a seaonal cycle of tissue hydrocarbon concentration. This seasonal pattern was not correlated with seasonal changes in total lipid content of plaice tissues (Table 18). As in the oysters, there was no consistent difference between reference plaice and plaice from oil-contaminated Aber Benoit and Aber Wrac'h with respect to pristane/phytane ratio, alkane/ isoprenoid ratio, or carbon preference index (Table 16). 298 Table 12. Concentrations of total aliphatic and aromatic hydrocarbons (measured gravimetrically) in tissues of plaice ricuroneates platessa from reference stations and from two estuaries con- taminated with Amoco Cadiz oil. Status determined according to pattern and identify of GC peaks. Date/Sample April 1979 (13)D Whole Fish Loc Tudy (reference) Aber Benoit Aber Wrac'h Hydrocarbon Fraction (ug/g dry tissue) Al iphatics Aroma tics 2.9 38.0 7.1 91 24 83 Status Biogenic Biogenic Small U.C.M. August 1979 (17) Muscle Aber Benoit Aber Wrac'h Liver Aber Benoit Aber Wrac' h 7.7 19.9 801.9 1034.0 9.0 12.7 235.6 317.9 Other oil/biogenic Other oil/biogenic Other oil/biogenic Other oil/biogenic February 1980 (23) Muscle lie Tudy (reference) Aber Benoit Aber Wrac'h Liver lie Tudy (reference) Aber Benoit Aber Wrac'h 23 83 66 736 1510 831 19 22 12 548 352 355 Other oil/biogenic Other oil/biogenic Other oil/biogenic Biogenic Biogenic Biogenic June 1980 (27) Muscle lie Tudy (reference) Aber Benoit Aber Wrac'h 16 38 146 23 17 41 Biogenic Other oil/biogenic Other oil Liver He Tudy (reference) Aber Benoit Aber Wrac'h 1210 1810 1130 723 682 511 Biogenic Biogenic Biogenic a" biogenic - probably of biological origin; small U.C.M. - small unresolved complex mixture, typical of weathered oil; other oil - definitely petroleum but cannot be identified as Amoco Cadiz oil. b' months after the Amoco Cadiz oil spill, 16 March 1978. 299 Table 13. Concentration of aliphatic hydrocarbons in tissues of plaice I'leitroncotea ' . Iroin Aber Ocnoit, Erittariy Irance, collected at different times after tne i\noeo CsJir. oil spill. Values are in ng/g dry weight (parts per billion). Date/Sample Compound Apr 1979(13) Whole Fish Aug 1979(17) Muscle Liver Feb 1980(23) Muscle Liver Jun 1930(27) Muscle Liver n"C10 MO ND ND ND ND ND MD nC,, 3 15 NO 6 531 NO 208 nC12 ND ND ND ND 324 MO NO nC,3 ND ND ND NO ND 4 MD nC,4 NO ND ND ND ND 8 MO Farnesane ND ND ND ND ND NO MD n"C15 3 6 ND 13 NO 8 MD nC16 3 12 ND 14 132 8 MD n"C„ 9 44 ND 31 336 13 ND Pristine 18 12 ND 16 NO 5 2.530 n"C18 4 47 ND 21 299 13 MO Phytane 31 20 ND 2? NO 5 MD n"C,g 3 24 ND 15 331 11 .'.0 n"C20 6 13 ND 29 459 22 MD nC21 4 9 555 39 425 50 1,130 n"C22 3 10 1.927 44 321 120 3,470 "C23 3 13 3,695 51 206 221 6.460 nC24 2 15 5,109 61 152 324 9,030 n"C25 4 21 6,476 73 452 436 10.500 nC26 7 23 8.315 92 2 ,530 486 13.700 nC27 15 31 14,363 95 6 ,660 503 19,600 nC28 8 28 10.552 84 4 .100 421 15,200 nC29 22 42 14.282 87 6 .770 359 23,500 nC30 ND 43 4,573 57 750 299 9.040 nC31 10 43 4,425 99 1 .430 197 9,150 nC32 ND 36 1 ,414 20 NO 117 3.280 nC33 ND NO NO ND NO 86 MD "CM NO ND ND ND NO 51 MD Total Resolved A1 i - phatics 158 507 76,586 991 :•: .208 3,767 126.798 months after the Amoco Cadiz oil spill, 16 March 1978. 300 Table 14. Concentration of aliphatic hydrocarbons in tissue of plaice . tvemnatrx nlntcar-a from Aber Wrae'h, Urittany France collected at different times after the Aioco Cadiz, oil spill. Values arc in ng/g dry weight (parts per billion). Date/Sample Apr 1979(1 3)a Aug 1979(17) Feb 1980(23) Jun 1980(27) Compound U'hole Fish Muscle Liver Muscle Liver Muscle Liver nC10 IID nC„ ND nC,2 ND n C13 NO " C,4 ND Farnesane ND n"C15 ND nC16 ND n C„ 8 Pristane 10 n~C18 3 Phytane 15 n"C19 ND nC20 ND nC21 3 nC2? 3 nC23 2 "C„ 2 nC25 3 nC26 4 nC27 7 n C28 4 n C2g 6 "C30 ND nC3, n-C32 ND ND nC33 ND nC34 ND Total Resolved Ali- 70 phatics 16 ND 105 NO ND NO ND ND NO HD NO ND 6 ND 8 ND 16 ND ND NO 7 ND ND ND ND ND ND NO ND ND ND ND 9 ND 14 207 23 4,722 34 1 ,467 64 3,781 76 3,883 147 6,625 18S 3,032 237 1,747 241 ND 237 ND 176 NO 1,605 25,464 157 3,914 16 152 46 ND 16 NO ND ND ND HD ND ND ND NO 9 NO 28 151 12 ND 22 ND 17 NO 6 ND 23 NO 45 1, 040 91 3, ,410 162 6, ,460 223 9, ,150 335 10,600 328 16 .300 280 20 ,100 255 16 .600 266 21 .700 254 10 ,800 80 10 ,900 100 3 ,130 18 2 .050 17 5S6 2,649 133 ,129 months after the Ar.oco Cadiz oil spill, 16 March 1978. 301 Table 15 . Concentration of aliphatic hydrocarbons in tissues of plaice Plvuivmcetee platcvua from reference stations on the Brittany coast of France at different times after the A"-ovo Cadiz oil spill. Values are In ng/g dry weight (parts per billion). Date/Sample Compound Apr 1979(13)a Whole Fish Feb 1930(23) fluscle Liver Jun 1980 (27) Kuscle Liver "~C10 NO 17 99 37 231 n cn ND 44 275 75 618 nC)2 ND 11 53 17 130 n C,3 ND 1 ND ND ND " c,4 ND 3 ND 1 ND Farnesane ND 4 ND ND ND n"C15 ND 11 NO 7 ND n_t16 2 16 ND 8 ND n C1? 5 32 ND 16 ND Pristane 3 10 110 3 ND n"C18 4 21 ND 18 ND Phytane 2 18 ND 6 ND n'C,, 3 8 ND 7 ND n"C20 3 7 80 9 187 nC2, 2 8 302 17 1.740 nC22 2 7 283 41 5.510 n"C23 2 9 412 76 1,060 nC24 1 10 571 no 14,500 n C26 2 7 771 141 23,600 n~C26 2 12 1,150 158 20,000 nc2? 3 12 2,380 158 21.400 nC28 2 10 2.060 134 16,200 n C„ 6 9 5,270 112 24,200 n"C30 NO 5 1,320 82 10,500 n"C3, 1 5 3,730 58 12,500 n C32 ND ND 749 34 482 nC33 ND ND ND ND 402 "C34 ND ND ND ND 1.160 Total Resolved Al i- phatics 45 297 19,505 1,325 154,420 months after the Amoco Cadiz oil spill, 16 March 1978. 302 Table 16. Characteristics of the aliphatic hydrocarbon fraction of plaice Plcuronectcs platessa from reference stations and from two estuaries contaminated with Amoco Cadiz oil. Date/Sample April 1979(13) Whole Fish Loc Tudy (reference Aber Benoit Aber Wrac'h Prtstane/Phytane Al kanes/lsoprenoids 1.40 0.59 0.66 2.34 0.39 0.45 Carbon Preference Index {C„ '26 C30> 3.72 3.11 2.41 August 1979(17] Muscle Aber Benoit Aber Wrac'h Liver Aber Benoit Aber Wrac'h 0.61 ND ND ND 3.38 6.14 ND ND 1.19 1.12 1.64 1.70 February 1980(23) Muscle lie Tudy (reference) Aber Benoit Aber Wrac ' h Liver He Tudy (reference) Aber Benoit Aber Wrac'h 0.58 0.55 0.64 ND ND ND 2.37 1.77 4.54 ND ND ND 1.13 1.11 1.27 2.32 2.34 2.65 June 1980(27) Muscle lie Tudy (reference) Aber Benoit Aber Wrac'h Liver lie Tudy (reference) Aber Benoit Aber Wrac'h 0.56 1.00 0.68 ND ND ND 5.22 5.00 2.07 ND ND ND 1.07 1.06 1.00 1.45 1.62 1.38 a' months after the Amoco Cadiz oil spill, 16 March 1978. 303 The hydrocarbon data demonstrate convincingly the dramatic differences in patterns of petroleum hydrocarbon contamination of oysters and plaice from the same oil-contaminated Abers. Oysters contained high concentrations of alkanes, dominated by low molecular weight compounds, while in plaice, the dominant alkanes in liver samples were the higher molecular weight compounds. Oysters contained abundant petrogenic and pyrogenic aromatic hydrocarbons spanning a wide molecular weight range. Plaice on the other hand contained little true aromatic hydrocarbon. These differences undoubtedly reflect the markedly different capabilities of bivalve molluscs and teleost fish to metabolize and actively excrete petroleum hydrocarbons. Most teleosts studied to date have a highly active and inducible cytochrome P-450 mixed function oxygenase system capable of converting aromatics and some aliphatics to polar and more easily excreted matabolites (Neff, 1979). This enzyme system is absent altogether or present at very low activity in bivalve mollusc tissues. Biochemical Indices of Stress Total lipid concentration in tissues of oysters and plaice, deter- mined in connection with hydrocarbon analyses, showed no consistent patterns in relation to station or season (Tables 17-18). In June 1980, but not at other sampling times, oysters from the two oil-contaminated Abers contained 2-3 times as much lipid as oysters from the reference station. It is quite possible that this is related to differences between reference and Aber oysters in state of reproductive ripeness, and not directly to oil- induced effects. Heniolymph glucose concentrations in oysters were low, highly variable, and showed no relationship to station (Table 19). No statis- tically significant differences were noted in values for reference and Aber oysters. There was a trend at all stations toward increasing hemo- lymph glucose concentration between December 1978 and August 1979. Some patterns did emerge in serum glucose concentrations of plaice (Table 20). In December 1978, April 1979 and August 1979, with one exception, serum glucose concentrations of plaice from oil-contaminated Aber Benoit and Aber Wrac'h were lower than values for reference plaice. Two of these differences were statistically significant. The collecting technique (otter trawl) is highly stressful, and maximal hyperglycemic stress response occurs rapidly in fish (Thomas et al., 1980). The data suggest, not that Aber plaice were less stressed than reference plaice, but that they had become refractory — perhaps due to chronic stress — to capture-induced hyperglycemia. Inability to respond bio- chemically to stress has been demonstrated in plaice held in the 304 Table 17 . Concentration of total lipids (determined gravimetrical ly) in whole oysters Cracsostrea gigas from reference stations and from estuaries contaminated by Amoco Cadiv, oil. Values are in yg/g dry tissue. Station August 1979 Feb ruary 1980 June 1980 9,775 6,650 5,580 NS 9,150 15,900 6,151 4,860 11,200 6,188 NS NS Reference Aber Benoit Aber Wrac'h Baie de Morlaix NS, no sample available. Table 18. Concentration of total lipids (determined gravimetrically) in tissues of plaice {Pleuronectes platessa) from reference stations and from two estuaries contaminated by Amoco Cadiz oil. Values are in yig/g dry tissue. Station Tissue August 1979 Feb ruary 1980 June 1980 Reference Muscle NS 2,310 1,870 Liver NS 11,300 8,770 Aber Benoit Muscle 1,921 2,170 1,740 Liver 16,729 12,700 11,200 Aber Wrac' h Muscle 3,278 1,750 1,810 Liver 14,725 20,300 4,380 NS, no sample available. 305 Table 19. Hemolymph glucose concentration in oysters Craoaostrea gigas from reference stations and from oil-polluted Aber Benoit and Aber Wrac'h. Values and standard deviations are in mg glucose/ 100 ml hemolymph. n = 8 replicates. Sampl ing Date Station Dec 1978(9)a April 1979(13) Aug 1979(17) Reference 5.12+3.1 13.05+2.9 23.53+4.0 Aber Benoit 3.00+1.8 12.57+2.4 NS Aber Wrac'h 4.80+2.6 11.25+3.8 23.87+2.8 NS, no sample analyzed a' months after the Amoco Cadiz oil spill, 16 March 1978. Table 20. Serum glucose concentration in plaice Pleuronectec platesaa from reference stations and from oil-polluted Aber Benoit and Aber Wrac'h. n = 10 replicates. Values and standard deviations are in mg glucose/100 ml serum. Sampling Date Station Dec 1978(9)a Apr 1979(13) Aug 1979(17) Feb 1930(23) Jun 1980(27) Reference 158.1+11.6 149.6+23.5 160.4+36.9 27.1+19.2 37.2+12.8 Aber Benoit 118.3+32.9 57.0+32.9* 93.7+27.3* 85.9+33.2*147.3+46.0* Aber Wrac'h NS 125.6+19.5 168.4+31.3 135.0+28.7*135.0+55.4* significantly different from reference at a = 0.05 NS, no sample analyzed a' months after the Amoco Cadiz oil spill, 16 March 1978. 306 laboratory (Wardle, 1972). In the last two samples, February 1980 and June 1980, reference plaice were sampled very rapidly a^ter capture and their blood glucose values represent the normal unstressed values. Plaice from the Abers were stressed by capture and showed a hyper- glycemic response, suggesting some recovery of physiological function with time. These data show some of the difficulties in using blood glucose concentration as an index of stress in fish. If blood samples cannot be taken immediately after the fish are captured, capture- induced responses may obscure any due to pollution. Liver glycogen concentrations in fish from the last two collections were highly variable (Table 21). Because of extremely large standard deviations, no patterns could be discerned. Total cholesterol and high density lipoprotein (HDL) cholesterol concentrations in the blood of plaice were measured in sampels from the last two collecting trips (Table 22). The general trend was for total cholesterol to be elevated and HDL cholesterol concentration to be depressed in fish from the two oil-contaminated Abers. Several of these differences were statistically significant. As a result, HDL cholesterol as percent of total cholesterol was lower in plaice from Aber Benoit and Aber Wrac'h than in plaice from the reference station at lie Tudy. Concentration of liver-free ascorbic acid was measured in plaice from all five sampling trips (Table 23). In all but the February 1980 sample, liver ascorbate concentrations in plaice from oil-contaminated Aber Benoit and Aber Wrac'h were substantially lower than concentrations in livers of plaice from reference stations. In four cases, the differ- ence was statistically significant. In the February collection, the pattern was reversed. Reference fish contained hepatic ascorbate concen- trations significantly lower than concentrations in livers of fish from the two Abers. At this time, all the reference fish were gravid females ready to spawn. Only a few of the fish from the Abers were in this condi- tion. It is highly likely that the extreme depletion of liver ascorbate reserves in the reference fish is the result of ascorbate mobilization for gonadal maturation and ovogenesis. These gravid reference fish also had relatively low hepatic glycogen reserves (Table 21) . Adductor muscle-free amino acid profiles and concentrations were measured in oysters from the first three collecting trips (Tables 24-26). Total free amino acid concentrations were always lower in adductor muscles of oysters from oil-contaminated Aber Benoit and Aber Wrac'h than in adductors of oysters from reference stations. This difference cannot be attributed to differences in seawater salinity between Aber and reference stations, since all stations had salinities in the 30-34 o/oo 307 Table 21. Concentrations of glycogen in the liver of plaice Pleuronectes platessa from a reference station at lie Tudy and from oil- polluted Aber Benoit and Aber Wrac'h. Values are in mg glycogen/ g wet weight. Station February 1930(23)a June 1980(27) Reference 4.81 + 9.05 8.45 + 7.03 Aber Benoit 0.48 + 0.39 6.68 + 7.56 Aber Wrac'h 14.37 + 17.42 12.35 + 12.0 a' months after the Amoao Cadiz oil spill, 16 March 1978. 308 >> c -o -.- c 3 *— O) k. r~ ai id o r— i- •— i l/l oj at •*•> ■»-> Z> %A A3 r— ■ + 1 o + 1 r— 0> -O o ■*- S- -*-> ■U -O o> <3 CO 3 O +i r— O 4- fc ■— , — O Q) o +-J fD l/i 1- n. E 01 -*-> o C iu i- o a> u ■»- -cz u u c r Wrac'h 0. 68 + 0. 11 0. 25 + 0. 03 5. 67 + 1. 05 63. 91 + 3. 41 0, 41 + 0. 12* 2, ,83++ 3, ,05 + 0. .16 5 .14 + 0. .09* 24 .01 + 3, ,01 26 .54 + 2. .88* 10 .15 + 0 .49 0 .19 + 0 ,06+ 0 .18 + 0 .09+ 0 .10 + 0 .03+ 0 .19 + 0 .01 + 1.02 + 0.16* 0.53 + 0.09* 6.68 + 0.29 68.70 + 7.99 0.78 + 0.06* 3.93 + 0.52 7.59 + 1.09 40.82 + 9.97* 30.56 + 7.82* 13.50 + 2.90 0. 31 + 0. 21 0. 12 + 0. 06+ 0. 41 + 0. 21 + 0, .33 + 0. 18+ 0 ,22++ 2, .74 + 0 ,33 2.33 + 0.19 175.50 + 31.52 143.30 + 11.54 -- , not detected ' detected in two samples ' detected in one sample significantly different from reference at a = 0.05 311 Table 25 • Concentration of free amino acids in the adductor muscle of oysters Crassor.trca yitiac from a reference station in the Rade de Brest and from oil-polluted stations in Aber Benoit and Aber Wrac'h. n = 5 unless otherwise stated. April 1979 (thirteen months after spill) FAA Concentration (uM/g wet weight and standard deviation) Amino Acid Rade de Brest Aber Benoit Aber Wrac'h 0.79 + 0.15 1.04 + 0.46 0.24 + 0.07 0.33 + 0.10 4.31 + 0.65 4.64 + 0.72 64.39 + 6.19 65.58 + 3.87 1.31 + 0.58 0.92 + 0.39 3.59 + 0.92 2.76 + 0.46 10.27 + 1.21 10.37 + 1.11 20.76 + 9.29 6.76 + 5.28* 25.62 + 20.46 26.09 + 4.38 10.33 + 2.87 • 10.94 + 0.96 0.170++ 0.26 + 0.03+ 0.21 + 0.05+ 0.11 + 0.06+ 0.13 + 0.01+ 0.11 + 0.03+ 0.26 + 0.02+ 0.21 + 0.06+ 0.134++ 1.82 + 0.62 1.87 + 0.44 LYS 0.91 + 0.15 HIS 0.29 + 0.14 ARG 5.06 + 0.84 TAU 57.41 + 9.77 ASP 1.46 + 0.58 THR — SER 2.40 + 0.82 GLU 9.89 + 2.08 PRO 26.15 + 9.53 GLY 28.02 + 4.38 ALA 11.59 + 3.74 CYS -- VAL — MET 0.10 + 0.02+ ILE 0.15 + 0.06+ LEU 0.27 + 0.11+ TYR 0.163++ PHE -- NH3 2.44 + 0.79 Total FAA 146.94 --, not detected detected in two samples ++i . 143.60 129.86 detected in one sample significantly different from reference at a = 0.05. 312 Table 26. Concentration of free amino acids in the adductor muscle of oysters Crausoc.trca girtas from a reference station at He Tudy and from an oil-polluted station in Aber Wrac'h. n = 5 unless otherwise stated. LYS HIS ARG TAU ASP THR SER GLU PRO GLY ALA CYS VAL MET ILE LEU TYR PHE NH3 Auqust 1979 (sixteen months after spill) FAA Concentration (u!Vq wet weiqht and standard deviation! Amino Acid lie Tudy Aber Wrac'h 0.43 + 0.20 0.35 + 0.17 3.82 + 0.27 62.09 + 6.54 2.83 + 0.92 1.59 + 0.71 8.07 + 4.59 33.46 + 25.72 41.11 + 16.13 11.92 + 5.31 0.29 + 0.02 0.03 + 0.034 0.11 + 0.074 2.44 + 1.13 0.21 + 0.27 0.28 + 0.13 6.67 + 1.83 63.32 + 5.58 0.97 + 0.49* 2.73 + 1.23 7.66 + 2.13 28.57 + 9.74 26.11 + 11.52 14.09 + 3.72 0.23 + 0.13 0.23 + 0.30+ 0.19 + 0.003+ 2.43 + 0.56 Total FAA 166.12 151.77 --, not detected detected in two samples *. significantly different from reference at a = 0.05. 313 range. Dominant tissue-free amino acids in all samples were taurine (TAU) , glycine (GLY) , proline (PRO) and alanine (ALA) . In all samples from all collections and stations, taurine concentration was maintained nearly constant (range of means, 57.4 - 68.7 uM/g wet weight). There was a trend for glycine a.nd asp ar tic acid concentrations to be lower in adductors of oysters from the two oil-contaminated Abers than in adductors of reference oysters. The result was that free taurine :- glycine molar ratios (a recommended index of pollutant stress) were significantly higher in adductor muscles of oysters from Aber Benoit and Aber Wrac'h than in adductors of reference oysters in all but one instance (Table 27). Jefferies (1972) has suggested that taurine:- glycine ratios higher than about 2.0 in mollusc tissues may be a good index of stress. As indicated above, the high taurine: glycine ratios are attributed almost exclusively to a decrease in free glycine concen- tration. This, in turn, may be attributed to poorer nutritional status or altered patterns of amino acid metabolism in oil-stressed oysters. Similar patterns were observed in free amino acid profiles and concentrations in skeletal muscle of plaice (Table 28-32). Total free amino acid concentrations were much lower in plaice muscle than in oyster muscle, reflecting the well-developed capability of plaice to regulate body fluid concentration hypoosmotic to the ambient seawater medium. As in oyster muscle, taurine, glycine and alanine were the dominant free amino acids in plaice muscle. Concentrations of several free amino acids were statistically significantly different in muscle of plaice from Aber Benoit and/or Aber Wrac'h than in muscle of refer- ence plaice. However, there was no consistent pattern of change. Free glycine concentration was lower in muscle of plaice from the Abers than in muscle of plaice from reference stations in December 1978 and August 1979. In February and June 1980, free taurine concentration in muscle of Aber Wrac'h plaice was lower than in muscle of reference fish. In February 1980, it was higher. Despite these as yet unexplained varia- tions, in seven out of nine cases where comparative data were available, mean free taurine: glycine molar ratios in muscle of plaice from Aber Benoit and Aber Wrac'h were statistically significantly different from ratios in muscle of reference fish (Table 33) . Because of seasonal variations in free taurine: glycine ratios in muscle tissue of oysters and plaice, it is important when using this parameter as an index of stress to compare values for pollutant- impacted and reference animals collected at the same time from nearby locations. Several biochemical parameters were evaluated as potential indices of pollutant stress in oysters and plaice from oil-contaminated Aber Benoit and Aber Wrac'h. Values of some of these parameters were statistically significantly different in populations from the 314 Table 27 ■ Mean free taurine:glycine molar ratios in adductor muscle of oysters Crassostrea gigas from reference stations (Rade de Brest or lie Tudy) and from oil-contaminated estuaries (Aber Benoit and Aber Wrac'h). Seven replicate samples from each station were analyzed. Sampling Date Station Dec 1978(9)a April 1979(13) July 1979(16) Reference 0.93 2.05 1.51 Aber Benoit 2.25* 2.51 NS Aber Wrac'h 2.41* 2.51* 2.42* *. significantly different from reference sample at a = 0.05. NS, no sample analyzed. a' months after the Amoao Cadiz oil spill, 16 March 1978. 315 Table 28 Concentration of free amino acids in skeletal muscle plaice Plcuronectes platecaa from a reference station in Baie de Douarnenez and from oil-polluted Aber Benoit. n = 5 unless stated otherwise. December 1973 (nine months after spill) FAA Concentration (pM/q wet weight and standard deviation) Amino Acid Baie de Douarnenez Aber Benoit LYS 0.28 + 0.10 1.47 + 0.76* HIS 0.40 + 0.04 0.53 + 0.14 ARG 0.37 + 0.09 0.26 + 0.08 TAU 11.33+2.68 11.23+3.06 ASP THR 0.84 + 0.19 0.66 + 3.06 SER 0.92 + 0.68 0.71 + 0.18 GLU 0.35 + 0.13 0.15 + 0.06+ PRO 0.27 + 0.11 0.48 + 0.18 GLY 11.86 + 4.81 5.58 + 1.48* ALA 2.91 + 0.99 1.34 + 0.09* CYS VAL 0.12 + 0.01+ 0.23 + 0.17+ MET 0.07+0.01+ 0.07+0.02 ILE 0.06 + 0.01+ 0.11 + 0.07 LEU 0.11 + 0.01+ 0.13 + 0.08 TYR PHE NH, 6.36 + 0.39 6.29 + 0.34 Total FAA 29.89+9.86 22.95+6.58 --, Not detected * two samples ' significantly different from reference at a = 0.05. 316 Table 29 • Concentration of free amino acids in skeletal muscle of plaice Pleuroncatec platessa from a reference station at Loc Tudy and from oil -polluted Aber Benoit and Aber Wrac'h. n = 5 unless otherwise stated. April 1979 (thirteen months after spill) FAA Concentration (^M/g wet weight and standard deviation) Amino Acid Loc Tudy Aber Benoit Aber Wrac'h 0.18 + 0.11 0.56 + 0.35 8.94 + 2.43* 0.06 + 0.02 0.59 + 0.27 0.66 + 0.16 0.30 + 0.05 0.56 + 0.31 8.86 + 3.01 1.15 + 0.24* 0.12 + 0.01+ 0.06 + 0.02+ 0.07 + 0.01+ 0.06 + 0.05+ 5.30 + 1.24 LYS 0.47 + 0.23 0.88 + 0.21* HIS 0.92 + 0.31 0.99 + 0.21 ARG -- 0.21 + 0.12+ TAU 14.67 + 3.19 11.41 + 1.38 ASP 0.13 + 0.06 0.05 + 0.03 THR 0.89 + 0.31 1.21 + 0.58 SER 0.77 + 0.19 0.97 + 0.09 GLU 0.29 + 0.14 0.29 + 0.07 PRO 0.25 + 0.03 0.87 + 0.24* GLY 7.38 + 0.47 9.81 + 1.50 ALA 1.77 + 0.28 1.28 + 0.18 CYS -- 0.12++ VAL — 0.11 + 0.01+ MET 0.49 + 0.01+ 0.06 + 0.02+ ILE 0.09 + 0.03+ 0.04 + 0.03+ LEU 0.08 + 0.03+ 0.11 + 0.02+ TYR -- -- PHE -- -- NH3 4.69 + 0.74 5.07 + 0.61 Total FAA 28.21 28.41 --, not detected detected in two samples 22.18 detected in one sample significantly different from reference at o = 0.05 317 Table 30. Concentration of free amino acids in skeletal muscle of plaice Pleuroncctes platessa from a reference station at lie Tudy and from oil-polluted Aber Benoit and Aber Wrac'h. n = 5 unless otherwise stated. August 1979 (seventeen months after spill) FAA Concentration (tiM/g wet weight and standard deviation) Amino Acid He Tudj, Aber Benoit LYS 0.96 + 0.65 0.64 + 0.33 HIS 0.97 + 0.66 0.66 + 0.12 ARG 0.17 + 0.02+ TAU 9.28 + 1.22 10.08 + 1.32 ASP 0.03 + 0.01+ 0.05 + 0.03" THR 0.36 + 0.15 0.48 + 0.07 SER 0.27 + 0.22 0.41 + 0.22 GLU 0.10 + 0.01 0.18 _ 0.05+ PRO 0.50+0.03+ 0.74+0.20+ GLY 9.96 _ 3.40 6.57 + 3.14 ALA 1.67 + 0.81 0.86 + 0.26 CYS -- 0.18+ VAL MET 0.03 + 0.04+ 0.39 + 0.53+ ILE 0.41 + 0.03+ 0.07 + 0.03+ LEU 0.04 + 0.001+ 0.10 + 0.08+ TYR PHE NH, 5.65 + 0.58 5.38 + 2.19 6.34 + 1.27 Aber Wrac ' h 1. 40 + 0. 70 1. 50 + 0. 21 0. 21 + 0. 01 + 8. 01 + 1. 16 0. 06 + 0, .05 0, .65 + 0. ,24 0. .47 + 0, .25 0 .17 + 0 ,11 1, .78 + 1 .41 3 .70 + 1 .56* 1 .27 + 0 .29 0 .16 + 0 .15+ 0 .17 + 0 .09+ 0 .11 + 0 .09+ 0 .16 + 0 .n + Total FAA 24.40 21.39 20.29 --, Not detected ' detected in two samples detected in one sample *» significantly different from reference at a = 0.05. 318 Table 31. Concentration of free amino acids in skeletal muscle of plaice Plcuroneateo platcr.sa from a reference station at He Tudy and from oil-polluted Aber Benoit and Aber Wrac'h. n = 8 to 10 unless otherwise stated. February 1930 (23 months after spill) FAA Concentration (pM/g wet weight and standard deviation) Amino Acid lie Tudy Aber Benoit Aber Wrac' h LYS 0.57+0.40 0.95+0.58 0.55+0.42 HIS -- 0.50 + 0.21{1)^ 0.70 + 0.39 ARG TAU 7.55+2.90 12.57+3.38* 12.36+2.33* ASP 0.20 + 0.15(8) THR 0.33+0.11 0.63+0.20* 0.78+0.42* SER 0.70+0.52 0.93+0.50 0.79+0.36 GLU 0.57+0.20 0.24+0.13* 0.27+0.10* PRO — — 1.51 + 0.72(3) GLY 7.42+2.64 15.49+7.96* 18.61+5.32* ALA 3.70+0.74 1.63+0.59* 1.53+0.40* CYS VAL MET 0.32 + 0.08(5) — 0.03(1) ILE 0.21 + 0.14(7) LEU 0.29 + 0.17(7) — 0.16(1) TYR PHE NH3 NA NA NA Total FAA 20.86 32.94 38.80 --, not detected NA, not analyzed * ' significantly different from reference at a = 0.05 ' number of samples in which amino acid was detected. 319 Table 32 . Concentration of free amino acids in skeletal muscle of plaice Plcuronectes platcvsa from a reference station at lie Tudy and from oil-polluted Aber Benoit and Aber Wrac'h. n = 8 to 10 unless otherwise stated. June 1980 (twenty-seven months after spill) FAA Concentration (uM/g wet weight and standard deviation) Amino Acid He Tudy Aber Benoit Aber Wrac'h --, not detected NA, not analyzed * ' significantly different from reference at a = 0.05 ' number of samples in which amino acid was detected. LYS 0.34+0.30 0.63+0.52 1.05+0.32* HIS 0.29 + 0.13 1.39 + 0.63* 1 .87 jf 0.46* ARG TAU 16.92+3.96 12.37+3.63 11.81+2.30* ASP 0.13 + Q.}A{7)} 0.08+0.03 0.06+0.02 THR 0.33+0.17 0.87+0.37 0.77+0.35 SER 0.80+0.32 0.59+0.38 0.63+0.37 GLU 0.26+0.19 0.25+0.08 0.19+0.06 PRO 0.24+0.28 1.73+2.34(9) 2.30+2.33(9) GLY 2.50+2.08 14.65+6.95* 8.77+4.52* ALA 2.26+0.60 1.28+0.43* 1.07+0.32* CYS VAL 0.34+0.37(3) 0.15+0.02(6) 0.16+0.02(6) MET 0.15+0.08 0.12+0.02(9) 0.14+0.06(9) ILE 0.12+0.18 0.08+0.04 0.09+0.02(8) LEU 0.16+0.25 0.16+0.05 0.16+0.03(7) TYR PHE NH3 NA NA MA Total FAA 24.84 35.37 29.07 320 Table 33. Mean free taurine: glycine molar ratios in skeletal muscle of plaice Pleuroncates platessa from reference stations (Baie de Douarnenez , Loc Tudy, or He Tudy) and from oil-contaminated estuaries (Aber Benoit and Aber Wrac'h). Seven or ten replicate samples from each station were analyzed. Sampling Date Station Dec 1978(9)a Apr 1979(13) Aug 1979(17) Feb 1980(23) Jun 1980(27) Reference 0.96 1.99 0.93 0.66 1.35 Aber Benoit 2.10* 1.16* 1.53* 0.81 0.84 Aber Wrac'h NS 1.01* 2.16*. 1.02* 6.77* significantly different from reference sample at a - 0.05. NS, no sample analyzed. a' months after the Amoco Cadiz oil spill, 16 Mc.rch 1978. 321 oil-polluted Abers and from nearby reference stations. These may be useful indices of pollutant stress. They include blood cholesterol/ HDL cholesterol, liver ascorbic acid, and skeletal muscle-free amino acid ratios in fish; and adductor muscle-free amino acid ratios in oysters. Blood glucose also has potential as an index of stress in fish, if the fish can be captured and blood samples taken very quickly. Alternatively, useful information can be obtained if degree and duration of capture- induced stress can be standardized for reference and experi- mental fish. In such a case, the index of chronic pollutant stress is hypoglycemia, reflecting a loss or diminuation of the capacity of the hypophyseal-interrenal system to respond to stress. Several of the alterations in biochemical parameters in oil-polluted fish and oysters are indicative or symptomatic of poor nutritional status (e.g., depressed muscle glycine, depletion of liver glycogen and ascor- bate, etc.). This may be related to histopathological lesions, reported by Haensly and Neff in this publication in the gut and liver of plaice from the oil-contamianted Abers. One difficulty in using biochemical and histopathological parameters as indices of pollutant stress is that one is not always certain that animals from the impacted and reference sites are from the same popula- tion and therefore can be compared biochemically and histopathologically. The only way to establish convincingly that differences observed in indi- cator parameters in reference and impacted populations are due solely or primarily to the pollution incident under investigation, is to have comparative data collected before the pollution incident. This usually is not available. The oysters used in this investigation are a recently introduced species Crassostrea gigas and are from a common breeding stock throughout Brittany. Genetic differences between reference oysters and oysters from the Abers are therefore extremely unlikely. However, the extent to which plaice from the west and south coast of Brittany (reference sites) mix and interbreed with plaice from the northwest coast (site of the Abers) is not known. Some intermixing undoubtedly occurs. It seems likely, therefore, that many of the differences we have reported between oysters and plaice from the Abers and those from reference stations are attributable directly or indirectly to impacts of the Amooo Cadiz oil spill. There has been substantial improvement in condition of oysters and plaice in the Abers during the timecourse of this investigation (up to 27 months after the spill). Recovery is still not complete, however . 322 A most interesting observation from our investigations is that oysters, which were heavily contamianted by the oil spill and remained so for the duration of the investigation, showed little evidence of histopathological or biochemical damage, whereas plaice from the Abers, although not heavily contaminated with oil, showed evidence of serious and progressive histopathological and biochemical damage. This may be due to differences in the sensitivity of molluscs and fish to petrol- urn. However, an alternative hypothesis is that the metabolites of petroleum hydrocarbons, particularly of the polycyclic aromatic hydro- carbons, are much more toxic than the unmetabolized parent compounds and cause much of the damage in a chronic pollution situation. It is well-established that the phenolic, epoxide and diol metabolites of polycyclic aromatic hydrocarbons are much more elctrophilic and bio- logically reactive than the unoxygenated parent compounds (Neff, 1979). Since oysters have little or no capability to oxygenate polycyclic aro- matic hydrocarbons to reactive metabolites, they are quite tolerant to oil. Fish on the other hand have a highly active mixed-function oxygen- ase system and so rapidly convert polycyclic aromatics to reactive metabolites which cause tissue damage. 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Fate and Effects of Petroleum Hydrocarbons in Marine Organisms and Eco- systems. New York: Pergamon Press. 325 McCain, B.B., H.O. Hodgins, W.D. Gronlund, J.W. Hawkes, D.W. Brown, M.S. Myers, and J.H. Vandermeulen , 1978, Bioavailability of crude oil from experimentally oiled sediments to English sole (Paro- pkrys vetulus) , and pathological consequences. J. Fish. Res. Bd. Can. 35:657-667. McKeown, B.A. and G.L. March, 1978, The acute effect of bunker C oil and an oil dispersant on: 1 serum glucose, serum sodium and gill morphology in both freshwater and seawater acclimated rainbow trout (Salmo gairdneri) . Water Res. 12:157-163. Michel, P. and H. Grizel, 1979, Etude de-impact de la pollution petroliere de 1-Amooo Cadiz sur les huitres-Mars 1978/Avril 1979. CNEXO con- tract #78/5719. Centre Oceanologique de Bretagne. Brest, France. 32 pp. Neff, J.M., 1979, Polycyclic aromatic hydrocarbons in the aquatic environment: sources, fates and biological effects. Applied Science Publishers. 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Collier, 1975, Spin-labeling techniques for studying mode of action of petroleum hydrocarbons on marine organisms. Fish. Bull. 73:299-305. 326 Sinderman, C. J. , 1979, Pollution-associated diseases and abnormalities of fish and shellfish: a review. Fish. Bull. 76:717-749. Southward, A., 1978, Marine life and the Amoco Cadiz. New Scientist, 20 July 1978, 174-176. Spooner, M.F., 1978, Editorial introduction. Amoco Cadiz oil spill. Mar. Pollut. Bull., £:281-284. Thomas, P., M. Bally, and J.M. Neff, 1982, Ascorbic acid status of mullet, Mugil cephatus Linn, exposed to cadmium. J. Fish Biol. 20 (in press) . Thomas, P., R.S. Carr, and J.M. Neff, 1981, Biochemical stress responses of mullet Mugil cephatus and polychaete worms Neanthes vivens to pentachlorophenol. pp. 73-103. In: J. Vernberg, A. Calabrese, F.P. Thurberg, and W.B. Vernberg (eds.) Biological Monitoring of Marine Pollutants. New York: Academic Press. Thomas, P., B.R. Woodin, and J.M. Neff, 1980, Biochemical responses of striped mullet Mugil cephatus to oil exposure. I. Acute responses- interrenal activation and secondary stress responses. Mar. Biol. 59:141-149. Varanasi, U. and D.C. Malins, 1977, Metabolism of petroleum hydrocarbons: accumulation and biotransformation in marine organisms, pp. 175- 270 In: D.C. Malins (ed.) Effects of Petroleum on Arctic and Subarctic Marine Environments and Organisms. Vol. II. Biological Effects. Academic Press, New York. Wardle, C.S., 1972, The changes in blood glucose in Pleuronectes platessa following capture from the wild: a stress reaction. J. Mar. Biol. Ass. U.K. 52:635-651. Zannoni, V.C., M. Lynch, S. Goldstein, and P. Sato, 1974, A rapid micro- method for the determination of ascorbic acid in plasma and tissues. Biochem. Med. 11:41-48. 327 RETABLISSEMENT NATUREL D'UNE VEGETATION DE MARAIS MARITIMES ALTEREE PAR LES HYDROCARBURES DE L' AMOCO-CADIZ :MODALITES ET TENDANCES par Jacques E. LEVASSEUR et Marie-L. JORY Laboratoire de Botanique Generale Campus Scientifique de Beaulieu 35042 -RENNES Cedex - France RESUME Le retabLissement de La vegetation des marais de I'lle Grande partieL- Lement detruite par Les hydrocarbures est signi f i cativement engage et ce depuis 1980. Les modaLites et La chronoLogie du retabLissement sont fonction de La domi- nance reLative, en chaque point, de deux processus : regeneration in situ d'indi- vidus perennes, germination de graines et semences produites sur pLace ou dans Le voisinage. La coLonisation est surtout Le fait d'especes annueLLes, aLors que La germination des especes perennes est tres peu frequente, sauf dans Les zones abri- tees a substrat meubLe et propre. ELLe est toutefois raLentie ou nuLLe dans Les secteurs exposes aux effets directs de La maree et/ou pietines intensement Lors du nettoiement de 1978. CeLa justifie Les efforts de restauration voLontaire, au moyen de pLantation^ tentes dans de teLs sites et dont un des interets est d'ac- ceLerer Les phenomenes de depot des sediments et des semences. D'autre part, des especes initiaLement "resistantes" presentent actueL- Lement une sensibiLite marquee a La poLLution endogee toujours activecequi se tra- duit par Le decLin et, a terme, par La disparition sur de Larges espaces de popu- Lations entieres (cf. Juncus mari timus Lam.), Ainsi, ces processus, agissant simuLtanement ou successivement condui- sent-iLs a des sequences de retabLissement variees, a des stades transitoires (?) marques par une redistribution spatiaLe des especes qui s'ecarte notabLement de La distribution anterieure. ABSTRACT Recovery of iLe Grande saLt-marsh vegetation, partiaLLy destroyed by hydrocarbons has been signi f i cant Ly started up since 1980. Ways and timing of re- covery are due to the reLative dominance, in each point, of two processes viz. in situ regeneration of perenniaL individuaLs, germination of seeds producted near or on the site. CoLonisation is mainLy due to annuaL species whi Le germination of perenniaLs is a rare event, except in shades pLaces with Loose and cLean substrate. However, it is impeded either in tide exposed points or in formerLy heavi Ly tram- pLed pLaces. So, efforts of voLontary restoration are justified in such Locations ; pLanting acts besides by the speeding up of the aggregation of sediments and seeds cLose to the transpLants. 329 In an other hand, some species, initially "resistant" show a marked sensibility to underground actual pollution and consequent ly, large populations may decline or even die (cf. Juncus maritimus Lam.). Finally, these processes, acting in simultaneous or successive manners, will lead to varied recovery sequences, to transitory (?) stages characterized by a spatial species redistribution which may be quite different from the origi- nal pattern. MOTS-CLES: Retabli ssement, regeneration, restauration, successions secon- daire et primaire, pollution par les hydrocarbures, nettoiement, vegetation de marais maritimes. KEY WORDS: Recovery, regeneration, restoration, secondary and primary succession^ hydrocarbons pollution, cleaning up, salt marsh vegetation. 330 INTRODUCTION Le retablissement d'un couvert vegetal perturbe est un processus complexe qui recouvre des realites et presente des modalites tres diver- ses, d'autant que les causes perturbantes n'ont pas eu le meme impact suivant les lieux et suivant les especes composant le tapis vegetal (Baker, 1979 ; Levasseur et al , , 1981). Les marais maritimes constituent un ensemble heterogene, qui quoi- que fondamentalement organise en habitats Stages, aux conditions meso- logiques varices, supporte une vegetation qualitativement ou dans les espaces intrazonaux, quantitativement variee. Cependant, etant donne qu'il s'agit d'environnements physiquement determines, surtout dans les par- ties moyennes et basses des marais, la diversite specif ique est f aible, ce qui signifie qu'une perturbation peut avoir un effet drastique sur des communautes vegetales et ceci d'autant plus qu'elles seront pauci- specifiques et/ou particulierement sensibles, de par leurs composantes, a une cause perturbatrice particuliere, Ayant dans un travail anterieur (Levasseur et al., 1. c . ) detaille cet aspect des choses, nous ne presenterons, dans cette communication, que quelques donnees relatives au retablissement de la vegetation au cours des trois annees ecouleesjet ceci aussi bien dans les espaces non modifies par l'homme que dans ceux qui ont ete transformes du fait des operations de nettoiement. Pour ce faire, nous utiliserons les documents suivants, quoique non exhaustifs des differents cas de figures rencontres : - cartographie chronologique d'un marais choisi pour sa diversite intrinseque initiale, mais aussi pour la diversite des perburba- tions 1 'ayant affecte depuis mars 1978 ; - transects permanents, regulierement releves depuis 1971? et desti- nes, au plan*populations vegetales, a illustrer a la fois la chro- nologie des reprises, le developpement vegetatif ulterieur, les reorganisations spatiales interclones etla colonisation directe par les individus nouveaux. LE RETABLISSEMENT : DEFINITION ET PROCESSUS GENERAUX Definition II y a lieu de distinguer entre : 1 - le retablissement dans un marais donne du couvert vegetal, qui se traduit par une cicatrisation se deroulant non necessairement lineai- rement dans le temps et non synchroniquement dans l'espace et dont la duree probable, pour etre menee a son terme,est fonction de nombreux pa- rametresyessentiellement : 331 - le degre de destructuration et/ou de destruction initiales ; - les nouvelles conditions ecologiques (p. ex. la permanence, dans et sur le sol, et sous differentes formes, de quantites importantes de petrole est un nouveau facteur de l'environne- ment). Cette cicatrisation (i. e. gains en recouvrement) est independan- te des voies su ivies et des moyens mis en oeuvre. Elle peut quelquefois avoir pour consequence la constitution d'un peuplement vegetal qualitativement et/ou structuralement different du peuplement d'origine-mai's la dimension temps manque pour evaluer le de- gre de permanence de 1'etat atteint au moment du constat car, en cette matiere, tous les etats sont conditionnels et contractuels ! 2 - le retablissement d'une communaute vegetale particuliere, en qualite et en structure, dans un lieu donne, Cet etat, necessitant des references anterieures precises est beaucoup plus difficile a. evaluer que le premier cite, immediatement apprehendable car il s'agit du degre de recouvrement par la vegetation, au temps t, d'un espace donne. Cependant, le retablissement apres perturbation d'une vegetation peut etre estime, lorsqu'il est compris comme etant la reparation natu- relle des dommages subis, au moyen de constats etablis a intervalles reguliers. Processus generaux. Le retablissement est a la fois un processus et un resultat, lors- que l'on consider e qu'il est mene a terme. De ce point de vue il est largement engage en de nombreux sites et meme localement acheve. Mais sous ce phenomene,en depit des expressions spatiales et des chronolo- gies si diverses actuellement, se retrouvent les memes mecanismes. Ceux— ci sont fondamentalement au nombre de deux auxquels il faudrait ajouter les actions volontaires de restauration par plantations : Successions secondaire d'une part, primaire d' autre part. En fait, la distinction dans un lieu donne de ces deux processus est loin d'etre nette car frequemment ils agissent synchroniquement et non sequentielle- ment et de plus ils inter-et retroagissent quelquefois continuement . La reparation naturelle des destructions peut se faire soit dans un premier temps a partir de la regeneration in situ d' elements vivaces ayant survecu, que ceux-ci soient situes a l'interieur de la zone attein- te ou peripheriquement , soit, dans un second temps, par des implantations nouvelles a partir de migrules provenant d'individus ou de clones situes en dehors de la zone interessee^ou d'individus ou de portions de clones autochtones ayant pu poursuivre ou retrouver un cycle phenologique nor- mal ou ayant retrouve cette capacite apres un delai plus ou moins long de survivance en vie ralentie. Dans ce cas, il s'agit alors d'une colo- nisation interstitielle et/ou sequentielle puisque commandee spatiale- ment par l'ordre de reapparition, la localisation, le nombre et la na- ture des individus vivaces ayant survecu, raais aussi par les conditions ecologiques regnant dans le lieu. Ainsi, un processus de regeneration qui a notre sens se rapporte d'abord aux especes perennes implique la poursuite normale du cycle vege- tatif d'individus epargnes et la reprise de developpement epige d'indi- vidus survivants du fait de dispositions morphologiques particulieres ou de conditions d'habitats plus favorables. 332 Nous distinguerons alors les phases suivantes 1 - Apres une periode plus ou mo ins longue de vie ralentie, repri- se du cycle phenologique normal 2 - Extension vegetative eventuellement centrifuge consecutive ou concomittante de la premiere phase et/ou formation de graines et senten- ces viables 3 - Poursuite du processus si les conditions mesologiques restent adequates et si les conditions d' interactions coenotiques (concurrence) le permettent. Le second processus de colonisation (1, e^ succession primaire s_^ 1.) implique : 1 - La formation de graines et sentences dans, en peripheric ou a. l'exterieur de *zone concernee 2 - La non-exportation pour celles produites sur place ou inverse- ment l'accessibilite <*es liaux pour les autres 3 - Des conditions de germination et de developpement lavorables 4 - Le maintien de ces conditions auxquelles vont s'aj outer les con- ditions coenotiques, les unes et les autres variant avec le temps, dans l'espacejdu fait du processus fondamental de retroaction. La colonisation peut aussi etre le fait de fragments vegetatifs de- taches de pieds-meres, dans le lieu ou y ayant acces par le jeu des cou- rants. (cf. dispersion actuelle,qui utilise ces deux modalitesjde Sparti- na cf. anglica, dans le marais 2, mais aussi, a. l'Ouest du pont, dans le marais 4) . • e mblai s localisation des transects Figure 1. Carte de localisation des marais de l'lle Grande. 333 INVENTAIRE SOMMAIRE DES SITUATIONS HERITEES A la fin de 1978, les cinq situations suivantes ont ete distinguees, sur la base du recouvrement ou non par les hydrocarbures, de l'intensite du pietinement ou du passage repete d'engins lors du nettoiement interve- nu en 1978, des operations connexes de ce nettoiement telles le decapage au bulldozer de la couche superf icielle du sol ou l'etablissement de remblais a 1' emplacement des fosses de stockage du petrole, Groupe A : 1) zones non touchees ou touchees seulement marginalement par l'epandage d' hydrocarbures, mais qui ont pu etre secondairement pie- tinees. Groupe B : zones- ayant ete soumises a 1' impact direct du petrole. 2) zone petrolee mais non nettoyee Intensivement ( i . e . sans pietinement important ayant entraine une compaction durable des couches superieures du sol) . 3) zone petrolee et nettoyee intensivement. Groupe C : zones profondement modifiees par rapport a leur statut anterieur : 4) zones remblayees (mort-terrains, sediments meubles) 5) zones etrepees au bulldozer. Caracterlstiques generales de ces zones. Situation 1) Secteurs non atteints ou peu atteints par les hydrocarbures du fait de leur situation topographique ou des dispositions prises, immediatement apres la catastrophe (cf . marais 1 et 2, a l'Est du pont) , Localisation : Parties internes des marais ou dunes hordieres Processus en cours : Succession secondaire de cicatrisation dans les zones pietinees. Situation 2) Territoires pollues mais non ou peu pietines. Depot initial de petrole sur les parties aeriennes des plantes et sur le sol formant ensuite sur celui-ci un revetement coherent qui se desquame localement avec le temps, dans les sites exposes (modalite 1). Depot intrasedimentaire de petrole ; celui-ci encore actuellement sous forme semi-liquide dans les chenaux, dans la partie haute de la slikke, mais aussi en haut-schorre, dans les zones saturees en eaux douces par les sources venant du domaine terrestre et qui constituent des marecages supra- littoraux saumatres comme dans le marais 6 (modalite 2) . Processus en cours : Tres variables. Successions secondaires ayant debute des l'autom— ne 1978 et qui sont caracterisees essentiellement par une regeneration in situ d'especes perennes epargnees et survi- vantes. Lorsque la destruction du tapis vegetal a ete plus complete, il y a possibilite de colonisation directe par des elements allochtones si les conditions s'y pretent. II y a ainsi possibilite d'une succession primaire inter- stitielle. 334 Situation 3) Territoires fortement pietines et/ou sourais a. des passages d'engins, notamment d'engins chenilles qui detruisent les organes endoges de perennance. Destruction quasi-totale de la vegetation ; survivance d'un tres faible pourcentage(quelquefois inferieur a 5 %) d'itidivi'dus de type geophyte a rhizome et hemicryptophyte a souche. Compaction secondaire forte avec pour consequences le tassement du sol et la penetration forcee du petrole dans ses premiers centimetres. Peu de changements en trois ans de cet etat, sauf en mode expose ou des delitations et desquamations se produisent,sauf encore dans les zones pro— ximales soumises a sedimentation. Localisation : Cette situation se rencontre surtout dans les parties des marais les plus proches des chenaux et criques, la ou etait effec- tue le pompage du petrole. Les communautes les plus frequem- ment destructurees ou detruites sont les suivantes : peuplement a Spartina maritima, " a dalicornia perennis, " a Halimione portulacoides, a. Puccinellia maritima et Trlglochin maritima, p.p. " a Juncus maritimus du schorre moyen (vegetation a Limonium vulgare et Plantago mari'tima.f*) Processus en cours : Le retablissement de la vegetation, par des voies naturelles y est tres lent sinon nul, actuellement encore ; succession secondaire possible a partir des elements epargnes, mais ceux-ci sont en quantite insu.f f isante pour permettre une cicatrisation rapide de ces lieux. En fait la reprise de la vegetation y est quasi-nulle, Le retablissement d'un cou- vert vegetal ne peut etre que la. consequence d'une succes- sion primaiTe en quelque sorte obligatoire dans le lieu, mais qui pour de nombreux sites n'est encore que potentielle, ou bien encore d'operations de restauration par plantations ad hoc d'especes vivaces. Situation 4) Remblais Les parties de marais remblayees sont localisees a. l'empla- cement de fosses, ce qui explique les tassements ulterieurs observes depuis 1980, et la reintegration de certains de ces espaces dans le domaine maritime s^. JL. (marais 2 p. ex.) Processus en cours : Succession primaire obligatoire. D'ailleurs tres rapidement ini- tiee et qui, en 1981, en est deja au stade developpement ve— getatif horizontal d'especes vivaces- surtout Puccinellia maritima. II faut noter que dans ces lieux la germination d'especes vivaces a ete observee et que la colonisation s'est faite a partir de graines et semences ayant eu acces au site et ayant pu germer sur place, alors que ces germi- nations n'ont pas ete observees dans les autres sites des marais a sediments non meubles. (*)Nomenclature d'apres Abbayes H.des et al.(1970) 335 Situation 5) Territoires etrepes. Solution la plus extreme de nettoiement puisque la roche- mere, ici limons quaternaires decalcifies, est mise a nu. A part quelques exceptions tres locales, a la fois le con- tingent de graines produites avant 1978, et 1' ensemble de la masse vegetale epi - et endogee a ete detruite sur de vastes espaces comme par exemple dans l'estuaire de Ker- lavos, situe a quelques kilometres a l'Est de l'lle Grande. Ailleurs (marais i, 3, 4, 5, 6, 7) c'est essentiellement la partie proxiaale des marais qui a ete amputee de la sorte. Processus en cours : Initiation d'une nouvelle pedogenese. Le retablissement d'un couvert vegetal ne peut provenir que d'une succession primaire obligatoire ef f ectivement enga- gee apres une periode de latence de 1 an via 1' installa- tion de populations therophytiques essentiellement cons- titutes de differentes especes annuelles du genre Salicor- nia. CARTOGRAPHY CHRONOLQGIQUE D'UN MAPvAIS PRIS COMME EXEMPLE : le MARAIS NORD D'AN INIZIGO, Les caracteres particuliers de ce marais dont la localisation est precisee sur la figure 1, sous le numero 6, et qui nous l'ont fait choi- sir comme exemple reside dans la variete des habitats contigiis, parti- culierement 1' existence de prairies saumatres supra-littorales dans sa partie N, dominees soit par des roselieres a Scirpus tabernaemontani et Scirpus maritimus soit par des prairies a Juncus maritimus, espece quasi- exclusive sur de grands espaces. Morphologiquement, ce marais se releve peripheriquement , le reseau hydrographique convergeant en son centre se resoud en deux chenaux ma- jeurs orientes Est-Ouest. Sa partie Ouest a ete partiellement etrepee tandis que sa partie Sud a fortement ete pietinee, le sous-ensemble Nord etant de ce point de vue, peu touche. La figure 2 renseigne schematiquement sur la distribution spatiale des agressions qu'il a subi en 1978 : absence de pollution, pollution sans nettoyage, pollution puis nettoyage, etrepage proximal a l'Ouest. La partie Nord-Est a ete remblayee a differents moments depuis 1978. Aitisi, les cinq situations: precitees se retrouvent ici, mais les trois premieres dominent. Les figures 3, 4, 5 se rapportent a. des constats etablis en Aout 1979, 1980 et 1981. II ne s'agit pas a. proprement parler de cartes de vegetation, puisque 1' aspect qualitatif n'est pas le but de ces repre- sentations. Celui-ci en effet, est de fixer,, a iin moment donne, l'etat de la vegetation d'un double point de vue : - progres de la cicatrisation, - niveau de reconstitution des communautes vegetales. 336 Plus precisiment le codage correspond : - Pour le premier groupe (A) a. des peuplements recouvrant la quasi- totalite du sol, au moment de la cartographie ; le caractere differentiel intergroupe etant celui de la diversite specif ique a ce moment la, - Pour le second groupe (B) , il s'agit de peuplements en cours de regeneration ou soumis a. succession primaire. La destructuration, la de- nudation a pu y avoir ete tres forte et le recouvrement (hormis en niveau 6 pour les especes annuelles) est toujours inferieur a 50 % de la surface. Code des figures 3, 4 et 5, Groupe A : recouvrement des especes perennes pouvant atteindre 100 % Niveau 1 : Vegetation plurispecif ique, non touchee par les hydrocarbures ou vegetation ayant recouvre, a la fois sa diversite originelle, mais aussi sa structure anterieure. Dans ce cas on parlera de retablissement acheve. Niveau 2 : Vegetation paucispecif ique (n inf. a 3 especes). Niveau 3 : Vegetation monospecif ique. Ce resultat peut etre atteint de deux fa^ons ; la regeneration est le fait d'une seule espece ou le fait du retablisse- ment complet d'un clone (cf . roselieres) . Groupe B : recouvrement des especes perennes inferieur a 50 % te specifique indifferente : diversi- '/Z$yP%tti Niveau 4 : Recouvrement compris entre 50 et 25 % [J] Niveau 5 : Recouvrement compris- entre 25 et 5 % Niveau 6 : Recouvrement inferieur a. 5 % pour les especes perennes, mais pouvant depasser 80 % en ce qui concerne les therophytes. Dans ce dernier cas, il s'agit d'un recouvrement saisonnier . ] Niveau 7 : Recouvrement nul aussi bien pour les especes pe- rennes que pour les especes annuelles. II est ainsi possible, en un lieu donne, de suivre |es cbangements de statuts, et de comparer d'un site a 1' autre, 1 'amplitude de ces chan- gements et leur vitesse relative, et ceci pour une mime gamme d'habitats (cf, ci-apres). La figure 6, quant a elle, est. plus syntbetique puisqu'elle indique les ecarts des etats constates en Aout 1981 par rapport a. ceux d'Aout 1979. 337 Code de la figure 6 Pas de changement [.-.•. •. -, -~| Passage du niveau 7 au niveau 6 (i. e. acquisition d'un cou- vert phanerogamique saisonnier a. therophytes) . Changement d'etat correspondant a 1 niveau [] Changement d'etat correspondant a 2 niveaux Changement d'etat correspondant a 3 niveaux et ce, jusqu'a la categorie 4 incluse. Passage d'un niveau inferieur a 3 a. un niveau 3, 2 ou 1 ] Passage du niveau 3 au niveau 2, de celui-ci au niveau 1 1 1| | j | |p Passage du niveau 3 au niveau 1 cas particuliers : BjBi|||||| Evolution regressive, mais qui peut correspondre a des gains spatiaux d'une espece sociale, au detriment d'un une vegetation non concurrentielle. /££'' ] Secteurs plantes (zone de restauration de l'equipe "ameri- caine" Pr. Seneca-Raleigh). 338 \ y I •• < 7 1 Figure 2. Marais 6 -Extension de la pollution et modal it es 'de nettoiement • 339 Figure 3. Marais 6-Etat en 1979 (legende dans le texte) 340 Figure 4. Marais 6-Etat en 1930 (legende dans le texte) 341 Figure 5. Marais 6-Etat en 1981 (legende dans le texte) 342 :§§§' Figure 6. Marais 6- Distribution spatiale des ecarts de niveaux observes entre 1979 et 19*1 .dsgende dans le texte) . 343 Commentaires Mis a. part un petit nombre de points depourvus de toute vegetation, y compris therophytique, tous les autres ont vu leur couvert vegetal, meme fragmentaire, evoluer avec le temps. Ces transformations sont, dans la plupart des cas et selon nos conventions, progressives - i.. e.. tendent vers une cicatrisation des espaces denudes. Celle-ci peut etre accompa- gnee par une augmentation de la diversite specif ique, lorsqu'il y a eu destruction selective d'une partie du contingent specifique de depart, mais non denudation extensive. Cependant des tendances regressives s'ob- servent plus localement. II existe une certaine opposition entre les parties Nord et Sud du marais (gauche et droite sur les figures 2 a 6) , de part et d'autre des deux chenaux majeurs medians. L'etat de la vegetation, en secteur Sud, ne depasse pas le stade peuplement therophytique, ce qui correspond, 43 mois apres le depot de petrole, a. la destruction effective presque totale des especes perennes dans- ces zones bordieres. On constate neanmoins (cf .figure 6) que la cicatrisation a partir de regenerations autochtones est comparativement assez rapide le long de la route bordant An Inizigo au Nord. En trois ans, l'amplitude du changement est de l'ordre de deux ou trois niveaux, Une cause possible de cette regeneration plus rapide, qui interesse surtout les hemicryp- tophytes a souche et les chamaephytes ligneuses mais aussi les hemicryp- tophytes stolonif eres telles Puccinellia maritima, est a. rechercher dans le ruissellement permanent ou la percolation laterale de sources provenant de l'ile. C'est ef f ectivement en tete des chenaux et sur les marges que ce processus est le plus rapide. Lorsque l'on compare les figures 2 et 6, on remarque que ce sont les zones pietinees qui presentent le plus faible taux de reprises, exprime par le progres, en recouvrement , des especes vivaces. II faut noter toutefois que la vegetation initiale, dans la haute slikke, le bas-schorre et le schorre moyen etait pauci - ou monospecif ique. Le pietinement, ajoute a l'effet immediat du petrole sur des plantes a appareil vegetatif essentiellement epige a des effets drastiques et surtout durables. Ainsi ces deux facteurs, l'un initial, 1 'autre conse- cutif, mais encore operant actuellement , constituent-ils, en premiere hypothese, la raison majeure du retard dans le retablissement d'une vegetation, du fait de leurs effets multiplies, directs ou indirects. L' exposition aux effets dynamiques de la maree joue egalement, aussi bien sur le plan de la reprise que celui de 1' installation d'in- dividus nouveaux dans les espaces tres denudes. L'exposition favorise, etant donne l'absence de couverture vegetale, l'erosion des berges, ce qui conduit a des ef fondrements locaux qui, a terrae, peuvent entrainer des modifications dans les drainages, par occultation des chenaux les plus etroits ou les moins. profonds. Mais cette exposition, comme nous 1 avons dit plus haut, a pour consequence une destruction acceleree de la couche coherente de petrole deposee sur les sediments, meme lorsque celle-ci a ete tassee. Des souches survivantes ainsi liberties ont pu alors reprendre leur developpement , par formation de nouvelles pousses aeriennes. a partir de bourgeons adventifs. Par contre, en ce qui concerne la colonisation de ces espaces, l'exposition aux effets directs de la mer joue comme un facteur concrai- 344 re. II n'en est pour preuve que 1' installation reussie, dans des situa- tions mesologiques homologues, des especes annuelles, en l'absence de ce facteur (i. e. dans les zones abritees, en position plus interne). sons La partie Nord du marais se differencie des autres pour deux rai- - sa physiographie, - la nature de sa couverture vegetale. La topographie et 1' existence de nombreuses sources qui ont assure un auto-nettoyage precoce du petrole depose dans ces secteurs distaux font que le degre de destruction et de destructuration du couvert vege- tal y a ete comparativement plus faible. Structuralement en effet, les geophytes a rbizomes dominent et les parties aeriennes, qui de toutes fagons, ont une duree de vie limitee, ont joue le role de piege a. petro- le, sans que les organes endoges de perennance n'aient ete immediatement atteints. Seule, la sous-strate des peuplements, composee d'heraicrypto- phytes stolonif eres, a ete endommagee, mais la reconstitution de ce ta- pis inter stitiel est en cours et meme localement, dans les zones de stagnation des eaux continentales, presque achevee, ri ressort de ces observations que dans un meme marais les condi- tions et les modalites de retablissement de la vegetation sont tres va- rices. Si la regeneration, a proprement parler, est engagee, a des de- gres divers et pour des raisons et avec des moyens divers, 1' installa- tion de nouveaux individus, par colonisation directe ne s'observe pas, hormis le cas des especes annuelles dans les lieux proteges. Aussi peut- on dire que le retablissement est assure par le developpement vegetatif des seuls individus ou clones survivants, sans qu'il y ait ajout quan- titatif ulterieur signif icatif . Les evolutions ulterieures sont cependant dans la majorite des cas conditionnelles et dependantes de la resistance des populations regene- rees a la pollution remanente diffuse ou directe, de la. les delais va- riables observes dans 1' initiation du processus ! (cf . figure 6). ANALYSE DIACHRONIQUE DE QUELQUES TRANSECTS PERMANENTS. Parmi les transects etablis en 1979 et regulierement suivis depuis, nous en avons selectionne trois, deux dans les marais de l'lle Grande, le dernier dans l'estuaire de Kerlavos, La localisation des deux premiers est indiquee sur la figure 1. - A Kerlavos, il s'agit d'un schorre moyen a. Armeria maritima et Plan— tago maritlma, etrepe par decapage des dix premiers centimetres du sol. Le substrat plan est const itue de limon. Le mode d' exposition est abri- te. En utilisant le code des figures 3 a 5, le niveau de depart, en 1979 est 7. - Marais 5-Est d'An Inizigo. Transect d'orientation ENE-WSW, etabli dans une zone exposee en partie aux actions dynamiques de la maree et qui fut a la fois tres polluee, tres pietinee et soumise egalement au passage d'engins. Deux petits chenaux recoupent ce transect qui se rele— ve legerement vers 1'Est. Selon les sections de celuivci, les niveaux de depart sont de 4 ou 5. _,,. - Marais 3. Ce transect, situe en mode tres abrite, est oriente NW- SE. Son point de depart haut est situe sur une levee artificielle non touchee par le petrole, mais tres pietinee. II se tennine dans une zone marecageuse occupee par une prairie a. Juncus maritimus, encore actuelle- ment tres polluee, mais non pietinee. Les niveaux de depart vont de 1 a 4. Ces transects illustrent ainsi les differents cas de figures ren- contres, que ce soit sur le plan de la vegetation, des habitats, des destructions. Legendes des figures 7A - 7B, 8A-8B , 8C, 9 A - 9B. Figures 7A, 8A, 9A. Les transects sont constitues de carres contigils de 0,50 x 0,50 m subdivises chacun en 25 cases de 0,10 x 0,10 m, Les presences specif iques sont relevees dans chacune des cases. L'e— chelle des hauteurs, dans le diagramme est etahlie comme ci-apres : ,0 ,^15-2(^^5 1< l-sS^ioJg^i ^ Presence de l'espece dans n cases Afin de faciliter la comparaison des distributions lineaires et des recouvrements sur la ligne, les donnees relatives a chaque annee, pour une espece donnee, sont rapprochees. L'ordre de presentation des especes, dans les diagrammes est con- ventionnel. II s'appuie sur les types suivants : - plante a. organe de resistance endoge. rgeophyte a. rhizome *- hemicryptophyte a souche - plante sans organe de resistance en- p chamaephyte ligneuse doge L hemicryptophyte stolonifere - therophytes (y compris especes biannuelles) Figures 7B, 8B, 9B : sont indiques: - le nombre de cases par carre occupees par la vegetation, toutes especes confondues. - le nombre d' especes presentes dans chaque carre. Figure 8C : Pour ce qui est du transect 5, nous avons presente differem- ment les donnees, par la distinction des especes annuelles et pe- rennes et leur importance, exprimee comme la somme des cases occu- pees par chacune des especes. Ces sommes sont ensuite cumulees, de la les depassements possibles dans le cas de cooccurence ou meme de debut de stratification. La somme des recouvrements individuels peut ainsi etre superieure aux 100 % d'un carre elementaire. 346 1 I i 1 V a. Q- > e lO V ■p a u w — b E l_. I i CD I o 00 CO CO en i .-' ..J L 'f. I, [ L ,--- i i a 6 a CO m (M CO o CM - T" o t/1 CO OJ i-l en CO !j o co u CO P. 0) o f- r*. co CO 05 01 0) i i i D 0 d a, c CD o a w -h OJ 4-1 6 o CO « e co o CO — h o « CJ X Figure 7. Transect permanent -Estuaire de T'erlavos. A-Distribution spatiale des especes P-T'ariation longitudinale du recouvrenent et de la diversite. 347 TRANSECT 'VVATS 5 Figure 8. Transect permanent -Marais 5 (cf .figure 1) A-Distribution spatiale des espaces. 348 r i — i i LJTL&J r— — — i r- i I _, i 1 L_. L_ "J I I in 2 H U U C/l z as l__j g o 2 I I -.J r ( r . — J 1 J P" L. . ' I L"] r i — i , I i i i i n r ' i i _l r- ~i j — — ;*■*•-*« «■ L. 00 C5 e . , , J I b L L 4 ,. I f~ i i rw L, H ,-J L_. I r-i CO co CO Figure 8. Transect permanent-Marais 5 -"-Variation longitudinale du recouvrement et de la diversite 349 m M 1 H U Ed C/3 05 (11 O ; c > cd ^i 1 I o CM 00 £ lCM CM o 'CM i I i I i I I I I I ■ ■ i ■ i 1111 n in co O co in »M Transect permanent-f'arais S-C-xrariation longitudinale du recouvrement des plantes annuelles et vivaces (cf . texte) . 350 LTl to 00 E eg 'cm H U W to .o CM f • • • • i i if fc*«^« »***««* «»*»**»»*«« FTFt IV III I I I I I I I I *i'i*i'.*iSii*i*j*f*i*t i'i^'I'I'I -'■*-*-'-"-'•" ■'-'•'^' ■'■*■*•*■'•*■'• <»»»»»»» ++ »»»»%**■.» itt'i-n i i i i r i i 1 * * .■ y .- - • • i'.'i' i' .' i"ri'i I "T CO CD w r-H C 1-1 C > c3 ■• •• n vi i . i . i . rr- 1 1 1 1 1 1 1 1 1 , . . , t . 1 1 . i .' '■'*.''.''.' '■''.' .''.'.''■| ;'/'X^''/.^i/if'.''j' ' ' i'i'i'i'i'i'i'i t '•'•'iVVV'i'.i'ii' i'.i' •|" ■ ' ' H o CO o to o — T" o o CO o CM Figure 8. Transect permanent-Marais 5- C -Variation longitudinale du recouvrement des plantes annuelles et vivaces (cf .texte) 351 'igure 9 Transect permanent-Marais 3 (cf .figure 1) A-nistribution spatiale des especes 352 CD en o 00 oo U W CT 2 1 I 1 L. r I •8 \j >-i CO o >-i CO o. en at o / L L I I \ r" i_ c E r-J J L i i I r~ L_ i I r- i_ *-» L CO o » ^ ^— — — ^li iw. »« i^? _rJL Figure 9. Transect permanent-Marais 3 B-Variation longitudinale du recouvrement et de la diversite 353 Commentaires - Estuaire de Kerlavos (figures 7A, 7B) Les especes vivaces ont un recouvrement presque nul en 1979, tandis que s'installent quelques pieds de Salicornia ramosiss^a et Salicornia gr. herbacea (non distinguees sur les transects !). Le fait remarquable est la rapidite avec laquelle, en moins de trois ans, les therophytes ont sature lespace disponible, ce peuplement saison- nier ayant un recouvrement qui peut atteindre 100 %. II s'agit d'une co- lonisation primaire active (stade I - especes "opportunistes" dites a. strategie "r"). II faut noter 1' Importance des centres de dispersion ini- tiaux (cf .Brereton, 1971). Les pieds-meres etablis en 1979 ont produit des graines qui n'ont pas ete exportees etant donne le niveau topogra- phique des lieux et leurs positions tres internes dans 1' estuaire. II y a ainsi capitalisation locale des graines qui germent pratiquement sur place. La presence saisonniere d'un revetement algal micropnytique exer- ce une influence aussi Men dans le piegeage des graines deposees sur le sol que sur leur germination, au printemps suivant (protection ther- mique, hydrlque, photique, mais aussi protection contre les agents dyna- miques) . - Marais 5 (E-An Inizigo) (figures 8A, 8B et 8C) Augmentation, entre 1979 et 1981 de la richesse specif ique dans certaines sections du transect, notamment celles correspondant au schor- re moyen. Cette augmentation vient de reprises relativement tardives d'hemicryptophytes a souche et rosette telles Plantago maritima, Limo- nium vulgare et Armeria maritima dont 1' importance numerique reste nean- moins tres faible. De nouvelles therophytes apparaissent en 1981 : Coch- lear ia anglica et Parapholis strigosa. En ce qui concerne les plantes perennes, et si l'on met a part Juncus maritimus, il y a gain dans le recouvrement de chaque population. Le phenomene interessant est celui du decalage progressif des optimums, d'une annee sur l'autre, ce qui peut traduixe deux faits : . 1' importance de la competition interspecif ique, du fait de la mise en contiguite de clones regeneres, et qui se sont ensuite develop- pes lateralement d'une fagon centrifuge. . la biologie de la regeneration qui favorise un developpement plus actif des parties du clone les moins endommagees. Si cette partie est en situation marginale, par rapport au clone ancien, il peut y avoir double mouvement, normalement vers l'exterieur, mats aussi vers l'inte- rieur (developpement centripete) assurant ainsi une reconquete des posi- tions anterieures. Un clone epargne, a. developpement plagiotropique dominant, se cica- trise lui-meme tout en gagnant des espaces, a sa peripherie, qu'il n'oc- cupait pas precedemment du fait de l'occupation des lieux par d'autres especes ou par des clones de la meme espece (on peut rattacher ce pheno- mene a celui du "die-back" observe chez les especes a extension vegetati- ve). C'est ainsi que ce processus peut conduire a des redistributions spatiales de dominance, apres perturbation, telles celles signalees par Baier (1973). 354 De ce point de vue, des especes stolonif eres ou radicantes comme Puccinellia maritima et Halimione portulacoides ont un developpement spatial qui s'accelere avec le temps et qui est rapide compte-tenu du nombre de pieds survivants au depart. Comme dans la section du transect ou ces deux especes se developpent, le terrain est plan, done la pente, via les durees et frequences de submersion n'est pas un facteur limitant, l'aire actuelle de ces plantes tend a rejoindre leur aire potentielle , en 1' absence de concurrence. L' occupation de l'espace par les annuelles montre la meme tendance qu'a Kerlavos. Ilya saturation des espaces interstitiels . Le meme pro- cessus de capitalisation a partir des pieds-meres s'observe encore ici. De plus, apparalt un phenomene de nucleation (Yarranton et Morrisson, 1974). En effet, toute vegetation dans un espace intertidal soumis a submersion est un obstacle pour les particules sedimentaires-et les se- mences-vehiculees par l'eau. Dans un second temps, ces semences qui peu- vent provenir de plantes perennes egalement, germent dans le lieu ou elles se sont deposees a la f aveurdetet dans les sediments meubles depo- ses a la base de l'obstacle. On observe de la sorte,dans les secteurs ou ce phenomene se produit, a la fois une acceleration autoentretenue de la sedimentationjet, d'une fagon concomittante, une acceleration de la colo- nisation accompagnee d'une augmentation de la diversite specifique loca- le. Cette phase d' augmentation transitoire de la diversite est bien con- nue : transitoire car elle est suivie (cf. marais 2, remblai artificiel, non etudie dans cette communication) par une legere decroissance de la richesse specifique, consequence du comportement "imperialiste" de cer- taines especes "couvre-sol" . L'observation de la figure 8B montre, a un autre niveau le phenome- ne deja decrit a Kerlavos de saturation du plan -toutes especes confon- dues - egalement 1' augmentation de la diversite dans certaines sections, en meme temps que la variation horizontale de celle-ci, entre 1979 et 1981, traduisant l'heterogeneite, a grande echelle d'un tel espace. L'explication est plus a rechercher du cote de la competition interspeci- fique que de celui de contraintes mesologiques (types biomorphologiques compatibles ou incompatibles, cf . discussion in Levasseur et al. , I.e. ) . La figure 8C montre la part prise, des 1980, par les therophytes, part croissant tres brutalement en 1981. Mais 1 'acceleration la plus forte se tient precisement la ou une vegetation vivace deja installee fait protection alors que dans la partie gauche du transect, plus expo- see, la regeneration d' especes vivaces est moins active ou la des- truction initiale de celles-ci plus complete 'les annuelles sont nume- riquement moins abondantes. - Marais 3 dit de Notenno (figures 9A, et 9B) Ce transect est d'une interpretation plus delicate que les precedents etant donne l'heterogeneite des situations presentes et les tendan- ces en quelque sorte inverses qui s'y developpent depuis 1980. Ce qui frappe a premiere vue dans la figure 9A est le desequilibre entre les parties gauche et droite du diagramme pour ce qui est de la diversite specifique en chaque point. Deux raisons peuvent etre evoquees : 355 1 - appel a la notion de composition floristique initiale (cf . Egler, 1954), fonction, dans ce cas particulier, de gradients mesologi- ques le long de cette toposequence induisant en particulier une riches- se specif ique maximale dans la partie moyenne du gradient topographi- que, mais avec cooccurence des limites basses. 2 - 1' opposition peut aussi etre interpretee comme le resultat des perturbations dif f erenciees qui se sont exercees et qui continuent de s'exercer dans ces lieux, notamment dans les parties deprimees du tran- sect et qui marquentl' influence effective de l'epandage du petrole, et ce jusqu'a la limite superieure de depot. On notera a ce propos que la cicatrisation a ete rapidement effective au dessus de cette limite et ceci des 1980, alors que le recouvrement vegetal reste discontinu en dessous (cf . figure 9B). Le fait a retenir reste cependant le remplacement de la population primitive a Juncus maritimus, seule espece rescapee en 1978 par une es- pece de type biomorphologique different, Halimione portulacoides -cha- maephyte ligneuse a tiges radicantes- qui etend son aire, dans les par- ties basses, depuis 1980. Dans l'espace disponible libere, au moins au niveau et au dessus du sol, le meme phenomene de colonisation en nap- pe par les annuelles s' observe, de la part de Salicornia du groupe her- bacea, tandis que les autres therophytes -y compris des formes annuelles d' Aster tripolium- s' installent , a leur niveau bionomique habituel sur les parties moyennes et hautes du transect. L' evolution des performances de Juncus maritimus, geophyte a rhi- zome est interessante car tres representative de la tendance generale au declin qui affecte, en divers lieux, et ce depuis 1980, des populations entieres de cette espece. Celle-ci, rappelons-le, est une des plantes qui occupait et qui occupe encore la plus grande partie des marais 3, 4, 5 et 6 et qui a ete consideree par nous en 1979 comme une plante re- sistante. Comme son role physionomique, structural et coenotique est con- siderable, toutes modifications dans sa distribution et son abondance spatiale pourront avoir, a plus long terme, des incidences certaines. Les donnees quantitatives suivantes (figures 10 et 11) illustrent de telles tendances regressives. La figure 10 presente 1' evolution sai- sonniere du rapport biomasse epigee sur necromasse aerienne exprimees en poids sec. Les donnees de base sont le resultat de fauches effectuees au ras du sol, dans trois quadrats de 0,50 x 0,50 m, pris au hasard a trois niveaux topographiques, les recoltes etant ensuite sechees 48 heures a 65°C On notera 1' inversion du rapport a partir de la fin de l'annee 1980 et a terme, ceci conduira a la disparition de l'espece dans ce lieu. D'une fa^on concomittante, les capacites de reproduction sont alte- rees et se traduisent, selon les cas, par une reduction du nombre de ti- ges fertiles, ^par des malformations de 1? inflorescence et, pour ces der- nieres $ par une diminution du nombre de rameaux floriferes, par 1' absence d'etamines ou la non-formation de capsules et de graines ou seulement par la production d'un petit nombre de graines avortees. Dans le meme temps, les pieces du perianthe peuvent revetir un aspect brac- tiforme (cf. figure 12). De plus, 1' observation de coupes transversales de rhizomes montre une necrose des parenchymes; ceci, ajoute a un dessechement des apex vegetatifs, pose le probleme de la nutrition hydrique et minerale de (*) cjf.figure 11. 356 CO OJ \ •H 0] w CU a) r-H e H CO to 3 a. U c 4-1 3 c '-> i-i cu CD 4J T3 4-1 to CO c •H o 3 ■H (H •U OJ 01 y rH 3 4-1 a. CO E o CU E o a. o CU E o CO u CJ o o cu c V40 r* 13 cu o o 1 1 cu •• u ro II > CM •H CO M3J o M h-l M 4-1 •H l-l o 1— 1 M 01 03 t— 1 H rH 1-1 CU OJ 03 T3 4-1 M 6 c OJ CU 3 CU •H • < TD .H CU o '. 3 ■> •H :■ 1 4J CO •H cm t •H C c CM j 1 4-1 03 CU ■-{ T3 CU o ! i < ►J o • 4-1 M o ex a. CO fXi V -Z5 i /I f -co A / ; l\ \ 1 I I I '1 T 1 / ■*i y -5 * < / '■■■ \ ' \ \ m ■5 ■i > ■"-5 .q8 / V v. < v. •z \y S ,A j «r \ M ■C/l \ \ \ \ / \ s ' 1 T ■*5 Is ..-•■ i i i 4 ■5 _____\k_J ! -<8 fr\ 4 zff '•■ i / ■o . y -co ■ 1 ! 1 CU •M C CO C CO cu •H C M O (CU ■H cfl o > cu CO CO 03 e CU M 3 60 •H (Zm c/j Id Z z H w < CO K U w Q < H O H W (A e O z < g O Pm p- <: co K ►J M H a w CO w w Q Z o 01 3 1 4J •H en h 0) to ^H £ M tfl CO 3 Q, CJ C 4-1 3 C >-) •H OJ U-l •H t4 CO (OJ C4H 4-1 OJ M '■M H M 1— 1 CO *CD OJ i-i M i-l C OJ o M 0) c >-i o T3 o t 4 ■ ■H 3 0) 1 4) t-H ro •a | T3 -H CJ 3 4J > en 1 4-1 c •H OJ 1 •H CO e VOJ ] j 4-1 JS iH O OJ CO • •< ■ «! 'OJ rH e I / / . / / n, ' i \ \ \ / ■ K t ! \ \ •Qi 2 -H H o P- o PC PU ;• < \ ■zR ■o f" -'"' "*f \ 7 ■ co \ \ \ • i i * 4 I i 4 \ '< L \ ► < = 2 03 C 01 LT .H c ■H ■ 4-1 O k4 II CJ en V4H N— ' Dl If, CJ OJ M c •H e 4-1 CD •H 0) M T5 \0J CO 0) M to .fi 01 E CO 0 •H C 4-1 3 OJ XI Xi CJ OJ U IM /OJ X ■H F C 0 c c o in 3 ■H nj aj en 4-1 u C o o a •H a. 4-1 m 3 U O u > CO W c. —1 OJ M 3 XI 8 2 » 358 Figure 12. T'orphologie comparee d' inflorescences de Juncus maritimus Lam, provenant d'un meme clone situe au S du marais A (cf. figu- re 1) prelevees en Aout 1079 et Aout 1981. 359 ces organes, done des effets a long terme du petrole, toujours present, sur la physiologie et sur certains metabolismes de la plante. Apparem- ment, il n'y a pas renouvellement des reserves dans le rhizome : celles subsistant apres 1978 ont maintenant ete consommees. II faut rappeler a ce propos les conclusions de Baker (l.c . ) relatives a la sensibilite de cette espece a des pollutions chroniques par les hydrocarbures. L'observation simultanee des deux courbes montre la realite d'une tendance exprimee de deux facons differentes et qui ne touche pas seu- lement l'appareil reproducteur ! Rappelons cependant qu'une "allocation d'energie" plus forte en faveur de l'appareil vegetatif est consideree comme classique par Ranwell (1972) chez les plantes des marais mari- times. La veritable question relative a la nature et aux delais de reta- blissement de la vegetation, dans les marais de l'lle Grande, est la. II est vraisemblable que ce retablissement aura lieu, naturellement mais aussi avec l'aide des plantations volontaires; mais, sur de grands espa- ces encore occupes par le Jonc, il aura lieu sans lui, ce qui pourra mo- difier passablement le paysage vegetal, mais aussi les strategies de res- tauration, celle-ci ne pouvant alors etre uniquement focalisee sur les seuls secteurs actuellement denudes. CONCLUSIONS Les quelques exemples presentes montrent qu'au dela de la variete constatee, un processus de retablissement du couvert vegetal est effec- tif en de nombreux lieuxj les gains en recouvrement, par rapport a la situation de 1978, representent environ 35 %. II est vraisemblable que localement on assistera a une acceleration du phenomene puisque par nu- cleations en chaine, le nombre de points d'agglutination des sediments et des sentences va croltre d'une fagon non lineaire. II reste encore des sites ou la destruction de la vegetation a ete totale. Pour differentes raisons ils demeurent steriles, en ce sens que des germinations ne peuvent s'y effectuer. Aussi un retablissement natu- rel y est-il peu probable au mo ins dans un avenir proche. II semble alors que des restaurations au moyen de plantations soient la seule voie realiste possible, comme en temoignent les succes enregistres, a la sui- te des deux annees d' experimentations menees dans ces secteurs par le Dr. Seneca et son equipe. En effet, 1' introduction de boutures, selon les cas, avec ou sans sol, leur reprise et leur developpement ulterieur, montre a contrario que e'est la phase "germination" qui est inhibee et done qu'en la court-circuitant, on accelere la cicatrisation. De la meme maniere, comme ces boutures font obstacle, d'autres especes peuvent alors s'implanter naturellement, mais dans un second temps, dans ces lieux, retablissant une diversite specif ique qui n'existait pas au depart lors de 1' experience. Notons qu'un phenomene similaire peut etre initie par les nappes de therophytes qui marquent frequemment la premiere phase de la recolonisation. Si l'on compare l'etat actuel de la vegetation avec l'etat primitif, on constate que le retablissement de celle-ci passe en de nombreux lieux par une redistribution spatiale des especes, au profit d'un petit nombre d'entre elles. Cette redistribution, qui peut quelquefois aller jusqu'a la monopolisation (transitoire ?) d'un espace peut avoir deux causes : 360 - les plantes survivantes -initialement "resistantes" ne possedent pas des capacites d' extension vegetative suffisantes pour cicatriser les espaces interstitiels denudes, alors que d'autres especes, "sensi- bles" celles-la aux premieres perturbations presentent ces qualites,de part leur organisation et leur ethologie; de la l'ecart actuellement observe entre la composition floristique initiale du site et la compo- sition du moment. - des especes tout-a-fait resistantes, telles Juncus maritimus et dans une moindre mesure, Tri^lochin maritima, deviennent sensi- bles a la pollution chronique qui affecte maintenant ces marais. LeurS populations, en declin, sont envahies peripheriquement par des especes autrefois cantonnees, du fait de la saturation de l'espace par les pre- mieres , en dehors des clones les plus denses. Ce sont d'ailleurs les mimes especes qui jouent ce role dans les deux cas, a savoir Puccinellia maritima et Halimione portulacoides, toutes deux capables, par stolons ou tiges radicantesj de couvrir le sol, meme lorsque celui-ci est encom- bre, a un niveau endoge,par des souches ou des rhizomes qui se maintien- nent longtemps apres la mort de la plante. La resistance d'une plante est done une notion tres relative, elle est en quelque sorte individuelle et thematique mais 1' organisation fu- ture d'un couvert vegetal apres perturbation doit autant aux plantes dites resilientes qu'a des especes "resistantes" en nombre insuffi- sant ou devenant sensiblesa d'autres causes que celles qui avaient au- torise la resistance de depart. La soi-disant robustesse d'un tel ecosysteme tient plus a ses ca- pacites de cicatrisation via des colonisations peripheriques ou implan- tations directes, lorsqu'elles sont possibles, qu'a la resistance alea- toire, a plus long terme, d'autres especes. Mais encore y a-t-il une nuance fondamentale entre la reaction vis-a-vis d'une perturbation except ionnelle, mais finie dans le temps et une perturbation qui devient chronique et qui n'a pas ete integree dans le passe par exemple au moyen d'une selection particuliere d'especes. C'est peut-etre ce qui est en train de se dessiner actuellement. Encore faudrait-il a^iner le concept d' ecosysteme littoral. II ae p-Tusijeurs r i , . , vaut mieux en effet parler ecosystemes superposes ou inclus dont les caracteres qualitatifs, structuraux et dynamiques sont differents au travers des types biomorphologiques representes, de leur abondance rela- tive,de leur distribution spatiale. Une meme perturbation s'exercera alors d'une fagon selective et differenciee sur les elements composant une vegetation locale, dela les delais et les modalites differentes du retablissement consecutif. Celui-ci pourra meme ne pas etre possible : une Spartinaie alteree ne pourra etre reconstitute que par la Spartine elle-meme. Plus fondamentalement , la nature, 1' abondance relative, la dis- tribution spatiale des especes presentes ou apparaissant pendant la succession pourront etre soumis a variation, changeant dans un premier temps la composition mais aussi la structure des peuplements en cours de retablissement, ceci a l'interieur de certaines limites imposees par l'environnement mesologique. Ces ecarts et ces divergences, par rap- port a l'etat ancien, ne constituent pas des phenomenes "anormaux" et/ ou eventuellement inquietants. lis representent seulement la materiali- sation instantanee du processus fondamental qui conduit a une saturation par la vegetation de l'espace disponible, lorsque l'opportunite s'y pre- te, comme c'est le cas en ce moment. 361 REFERENCES Abbayes, H. des, G. Claustres, R. Corillion et P. Dupont, 1971, Flore et vegetation du Massif armoricain. I. Flore vasculaire, P.U.B. St-Brieuc, 1226 pp. Baker, J.M. , 1973, Reco very of salt marsh vegetation from successive oil spillages : Environ. Pollut., vol. 4, pp. 223-230. Baker, J.M., 1979, Responses of salt marsh vegetation to oil spills and refinery eff luents:_in Jefferies R.L. and A.J. Davy (eds.), Ecological processes in coastal environments, Blackwell Scien- tific Publ., Oxford, 684 pp. Brereton, A. J., 1971, The structure of the species populations in the initial stages of salt-marsh succession : J. Ecol., Vol. 59, pp. 321-338. Egler, F.E., 1954, Vegetation science concepts : I. Initial floristic composition, a factor in old field vegetation development : Ve- getatio ., vol. 4, pp. 412-417. Levasseur, J., M.-A. Durand et M.-L. Jory, 1981, Aspects biomorphologi- ques et floristiques de la reconstitution d'un couvert vegetal phanerogamique doublement altere par les hydrocarbures et les operations subsequentes de nettoiement (cas particulier des ma- rais maritimes de l'lle Grande, Cotes du Nord) : in AMOCO-CADIZ, Consequences d'une pollution accidentelle par les hydrocarbures, C.N.E.X.O., Paris, 881 pp. Ranwell, D.S., 1972, Ecology of salt marshes and sand dunes, Chapman and Hall, London, 258 pp. Yarranton, G.A. and R.G. Morrisson, 1974, Spatial dynamics of a prima- ry succession : Nucleation : J. Ecol., Vol. 62, pp. 417-428. 362 RESTORATION OF MARSH VEGETATION IMPACTED BY THE AMOCO CADIZ OIL SPILL AND SUBSEQUENT CLEANUP OPERATIONS AT ILE GRANDE, FRANCE Ernest D. Seneca and Stephen W. Broome' INTRODUCTION General Tidal salt marshes functon to stabilize estuarine shorelines, to exchange nutrients with sediments and the surrounding waters, to provide energy as detrital material to the estuarine food web, and to serve as nursery grounds for many commercially important fish and shellfish. Because competing land uses have resulted in a decrease in areal extent of these valuable resources in the past, there have been concerted efforts recently to preserve the remaining marshlands and to reestablish marshes at selected sites. Techniques and procedures have been developed to: (1) reestablish marsh in areas where Man has destroyed natural marsh, (2) reestablish marsh along shorelines where storms have damaged or destroyed natural marsh, (3) establish marsh along canal banks and shorelines to stabilize the substrate and retard erosion, and (4) establish marsh on dredged material (Woodhouse et al., 1974; Garbisch et al., 1975; Seneca et al., 1976). Our research efforts in marsh establishment along the southeastern coast of the United States led us to respond to an invitation from the joint scientific commission of the National Oceanic and Atmospheric Administration (NOAA)/Centre National pour l'Exploration des Oceans to study the effects of the Amoco Cadiz oil spill. We developed a proposal for restoring marsh at the lie Grande site adapting techniques and procedures developed for Spartina alternif lora Loisel. in North Carolina (Woodhouse et al . , 1974; Seneca et al., 1976) to restoration of a part of the lie Grande marsh using vegetation indigenous to that region. This interim report contains results from 2 years' marsh rehabilitation research at lie Grande and a nearby estuary at Kerlavos. Literature Review The effects of oil pollution on salt marsh vegetation have been studied and reported by European researchers. Based on observations of Welsh salt marshes affected by oil spills from the Chryssi P. Goulandris in January 1967 and the Torrey Canyon in March 1967, Cowell (1969) rated susceptibility of marsh plants to crude oil and concluded that salt marshes dominated by Spartina townsendii H. and J. Groves and Puccinellia maritima (Huds.) Pari, were most subject to damage. Stebbings (1970) studied the effects of oil from the Torrey Canyon spill on salt marshes in Brittany and found that these marshes were 1 ) Department of Botany and Department of Soil Science, respectively North Carolina State University, Raleigh, North Carolina U.S.A. 27650 363 able to withstand 2 to 10 cm of oil with only slight, short-term, floral composition changes. Apparently, most of the toxic fractions had been lost from the Torrey Canyon oil, since it had been weathered at sea for 2 to 18 days. Stebbings noted that oil appeared to form an impervious layer on the substrate preventing gaseous interchange between soil and air, causing reducing conditions in the mud, and ultimately chlorotic symptoms in plants. Stands of Agropyron pungens (Pers. ) R. and S. , Festuca rubra L. , Juncus maritimus Lam., and Scirpus maritimus L. were extremely vigorous and seemed to derive some nutritional benefit from the breakdown products of this Torrey Canyon oil. Cowell and Baker (1969) noted that populations of annuals such as Suaeda maritima (L. ) Dum. and Salicornia spp. near Pembroke, Southwest Wales, were reduced initially but were recovering a year after oiling from the Chryssi P. Goulandris. Halimione portulacoides (L.) Aell. was the plant most badly damaged. In June 1968 the plant species with the greatest coverage in the upper, middle, and lower marsh (Festuca rubra , Puccinellia maritima, and Spartina townsendii, respectively) had recovered completely (Cowell and Baker, 1969). Baker (1971a-i) reported on several aspects of the effects of oil pollution on salt marsh and concluded that single oil spillages do not cause long-term damage to marsh vegetation (Baker, 1971a). These studies indicate that marsh vegetation is resilient and often can recover from single oil spills. Baker (1971e) suggests that it is best to let an oiled marsh recover naturally. However, persistent oil pollution has killed Spartina marsh at Southampton Water (Ranwell, 1968). Such sites may develop extremely anaerobic conditions in the mud so that higher plants can no longer grow on them. Cowell (1969) states that repeated contamination is likely to have increasingly serious effects if anaerobic conditions are created due to bacterial use of oxygen in the biological oxidation of the oil. We found no account of marsh recovery after removal of the upper layer of marsh substrate and vegetation. Study Sites The lie Grande site is a relatively protected estuary with a mean tide range of ca . 6 m, a spring tide range of ca. 8 m, and a mean tide level of ca. 5 m. Our first visit to lie Grande was in December 1978. Our NOAA liason representative, Douglas Wolfe, indicated that the marsh west of the bridge at lie Grande was to be our primary study site (Fig. 1 ) . There were extensive stands of Juncus maritimus on both sides of the estuary with lesser stands composed of a mixture of species including Puccinellia maritima, Triglochin maritima L., Limonium vulgare Mill., Spartina maritima (Curtis) Fern., and Halimione portulacoides . There were vast areas with no vegetation cover, the result of cleanup operations by the French military to rid the marsh of Amoco Cadiz oil. In many areas only the aboveground marsh vegetation and associated oil had been removed and in other areas the entire marsh surface including the root mat had been removed to a depth of over 30 cm. The intertidal creek banks were almost completely lacking in vegetation cover. A limited number of substrate samples from the disturbed sites were taken which subsequently indicated a 364 FIGURE 1. Marsh west of the bridge at lie Grande. Area without vegetation is due to removal of oil and vegetation during Amoco Cadiz cleanup operations. material which was sandy loam in texture and low in nitrogen and phosphorus. Marsh vegetation adjacent to the disturbed sites indicated that prior to the oil spill the natural marsh was composed primarily of Juncus maritimus, Puccinellia maritima, Triglochin maritima, Limonium yulgare, with lesser amounts of Spartina maritima. Halimione portulacoides was dominant along the creek banks. We noted considerable variation in the relative dominance of these species and others within marshes in the vicinity. Spartina anglica C E. Hubbard was present only at a single site at lie Grande as a small clump less than 3 m in diameter. This species is abundant in the Bay of Mt. St. Michel some 125 km to the east of lie Grande. Juncus stands generally occupied the highest elevations of the marsh relative to the other species mentioned. Subsequent observations indicated that the Juncus marsh is flooded for about 3 days each spring tide cycle. Above the level of Juncus there was in some areas a narrow fringe of Festuca rubra and Agropyron pungens with associated species. Many salt marsh ecologists consider this vegetation to be a part of the marsh. This higher zone of vegetation which extends up to ca. 1m above the Juncus marsh is flooded relatively infrequently on extremely high storm tides and spring tides. It lies above the marsh impacted by Amoco Cadiz oil and cleanup operations. Our marsh rehabilitation efforts were confined to elevations from 0.8 m below to 0.3 m above that of the Juncus marsh. 365 Because we wanted to compare the marsh at lie Grande with other marshes in the vicinity, we also visited the marsh in the estuary at Kerlavos ca . 5 km from lie Grande (Fig. 2). This marsh contained less Juncus, no Spartina, and was dominated by Puccinellia maritima, Armeria maritima (Mill.) Willd. , and Triglochin maritima along with Plantago maritima L. , Cochleria officinalis L. , Halimione portulacoides and Aster tripolium L. There was evidence of marsh removal by cleanup operations in the Kerlavos marsh also, but it appeared that the marsh was much less heavily impacted than that at lie Grande. We chose to use this marsh area as a supplemental study site. FIGURE 2. Marsh in estuary at Kerlavos. Areas without vegetation represent sites of marsh removal during Amoco Cadiz oil cleanup operations. PROCEDURE Substrate Substrate samples were taken at the transplant source sites for each species and also at the experimental planting sites each year. These samples were analyzed for elemental concentrations, pH, organic matter, and volume weight by the North Carolina Department of Agriculture Soil Testing Division using their routine methods. 366 1979 Plantings Based on our preliminary plantings made in December 1978 and the nutrient analysis of initial substrate samples, we established 9 experimental plantings in May 1979, using primarily Puccinellia maritima (Fig. 3), to a lesser extent Juncus maritimus (Fig. 4), and to a lesser extent still because transplants were not locally abundant, Spartina maritima (Fig. 5). These experimental plantings were designed to determine transplant response to conventional ammonium sulfate + concentrated superphosphate and slow release (Mag Amp and Osmocote) fertilizer materials at different rates over a wide range of tidal elevations. All transplants were taken from the natural marshes at lie Grande and Kerlavos. Digging of transplants was confined to small areas along narrow drainageways (Fig. 6) and protected sites so as to impact the marsh as little as possible. Half of the 2900 May transplants were plugs (10 to 15 cm deep cores from 5 to 7 cm in diameter composed of root material with attached substrate) and half were sprigs (root material only) (Figs. 7, 8, 9). Holes for the transplants were made with a 6.5-cm diameter soil auger (Fig. 10). Transplants were spaced 0.5 m apart and the appropriate amount of fertilizer material was placed into the transplant hole prior to insertion of the transplant (Fig. 11). Planting was conducted just prior to the spring tide cycle so that transplants would be flooded shortly after planting. FIGURE 3. Puccinellia maritima. 367 FIGURE 4. Juncus maritimus. FIGURE 5. Spartina maritima. 368 FIGURE 6. Digging Puccinellia along a narrow drainageway. M f J 1 i M I FIGURE 7. Transplants of Puccinellia: sprig on left, plug on right. 369 J ■ ■ wk FIGURE 8. Plug type transplants of Juncus . j|i I FIGURE 9. Pluy type transplants of Spartina. 370 FIGURE 10. A 6.5-cm diameter soil auger used to make holes for transplants in experimental plantings. FIGURE 11. Osmocote (a slow release fertilizer material) + concentrated superphosphate. Black cup measures a dose of fertilizer (2.8 g N + 1.2 g P) per transplant. Holes for transplants are spaced 0.5 m apart. 371 FIGURE 12. Triglochin maritima. FIGURE 13. Plug type transplants of Triglochin. 372 In September, we made 9 additional plantings of Juncus maritimus, Puccinellia maritima, and Spartina maritima and established initial plantings of another species, Triglochin maritima (Figs. 12, 13). Although not recognized as such on our initial visits to the site, the latter species appears to be a common pioneer species on disturbed sites alone or with Puccinellia maritima. Both Puccinellia and Triglochin appear to invade by seed. Height, number of stems, cover (a measure of spread) and aboveground dry weight per transplant were determined in September 1979, 4 months after planting. Cover determinations were made by measuring the average maximal diameter and the average minimal diameter of the transplant and using these dimensions in the formula for the area of an ellipse. Percent survival by transplant type and species was also assessed at this time and at each subsequent visit. Because our major objective was to establish vegetation, destructive sampling for biomass determinations was held to a minimum of three samples per treatment per location. A photographic record of all plantings was initiated. 1980 Plantings Based on results of our 1979 plantings, we established 14 additional plantings at higher elevations in May 1980 utilizing plugs of Puccinellia, Juncus, Spartina, and Triglochin and sprigs of Halimione (Figs. 14, 15). Remains of stems and intact root systems of Halimione indicated that this species was the dominant along the creek banks prior to the Amoco Cadiz oil (Fig. 16). Consequently, we began preliminary tests of reestablishing this species along the creek banks (Fig. 17) and included it in an experiment to determine the feasibility of nursery production for transplants. Like the earlier plantings, these 1980 plantings were designed to determine transplant response to fertilizer materials at different rates over a range of substrate and exposure conditions. Cover was determined for selected plantings. All experimental plantings were surveyed to determine relative elevations, i.e. relative to the natural marshes (Fig. 18). In September we made 8 additional plantings using primarily Puccinellia and Halimione with some Spartina. Based on results from our earlier plantings, further planting of Triglochin seemed impractical. As in September 1979, all earlier plantings were assessed for survival, height and cover with sampling for dry weight determinations limited to only two plantings. Photographic surveillance was considered even more important because we did not conduct intensive destructive sampling. 373 FIGURE 14. Halimione portulacoides . FIGURE 15. Sprig type transplants of Halimione. 374 FIGURE 16. Creek bank without vegetation as a result of removal of oil and vegetation in cleanup operations. Old root systems of Halimione are visible on lower portion of banks. Note lack of natural marsh plant invasion of these sites at time of photo, May 1980. FIGURE 17. Making transplant holes along creek banks, May 1981. Experimental plantings made in May 1980 are visible in background on creek bank. 375 FIGURE 18. Surveying to determine the elevation of our plantings in relation to that of the natural marsh at lie Grande, May 1981. Note transplants on creek banks between surveyors and on right creek bank toward the village from the white stake . 1981 Plantings Based on results of all earlier plantings, we established 2 1 additional experimental plantings in May 1981 utilizing about 4900 transplants at lie Grande. Most of the planting effort was concentrated on establishing cover on the bare creek banks which were at this time beginning to erode due to decay of the binding root mat and undercutting by tidal waters (Fig. 19). Two rows of Halimione transplants (sprigs) were planted on the edge of the creek banks with two rows of Puccinellia transplants (plugs) adjacent to and toward the marsh along several intertidal creeks (Fig. 20). Many other areas, still bare of vegetation 2 years after the catastrophe, were planted to increase the probability of revegetation (Figs. 21, 22, 23). All transplants were spaced 0.5m apart. Cover was determined for selected plantings. All experimental plantings were surveyed to determine relative elevations, and the photographic record was continued. 376 FIGURE 19. Eroding creek bank at He Grande, May 1981 i**:-. 'f/V^-w • /! if^V FIGURE 44. Preliminary planting of Halimione sprigs along a creek bank at lie Grande in May 1980. ■ ,. Sffl FIGURE 45. Same site as shown in Figure 44 in May 1981, 1 year after planting. 406 Jfitjl i m-Jfc FIGURE 46. Experimental planting of Puccinellia along a creek bank at lie Grande in May 1980 just after planting. FIGURE 47. Same site as shown in Figure 46 in May 1981, 1 year after planting. 407 Transplant Time Requirement It is difficult to determine the time involved in the transplanting operations when experimental plantings are being established. In May 1981 we kept records of the time required for four persons to dig and transplant sprigs of Halimione and plugs of Puccinellia. Halimione plants were dug, separated into sprigs and put into plastic bags for transport to the planting site at the rate of about 180 per person hour. Puccinellia plants were dug, cut into plugs and put into a container for transport to the planting site at the rate of about 75 per person hour. These rates indicate that Halimione sprigs can be obtained about 2.4 times faster than Puccinellia plugs. The planting operation includes opening the transplant hole with a soil auger, inserting the appropriate amount of fertilizer, and inserting the transplant and firming the substrate around it. Both types of transplants can be planted at the rate of about 40 per person hour. These time requirements for digging and planting make no allowance for travel, supplies, and equipment, which must also be considered in the total cost of a planting operation. Based on our digging and planting time requirements only, the time required to plant 1 ha of Halimione on a 0.5 m spacing (40,000 transplants) would be about 1,220 person hours (220 person hours to dig sprigs + 1,000 person hours to plant). The time required to plant 1 ha of Puccinellia on a 0.5 m spacing would be about 1,530 person hours (530 person hours to dig + 1,000 person hours to plant). These cost estimates indicate that it would take four persons working 8 hour days about 38 days to plant 1 ha of Halimione on a 0.5 m spacing and about 48 days to do the same using Puccinellia. Recovery of Transplant Source Sites From the beginning of our restoration efforts we were aware of the potential for impact to the natural marsh in digging transplants for the plantings. Consequently, we confined our digging of plants in the natural marsh to areas adjacent to narrow drainageways (Fig. 6) or to small areas (0.25 m2) in the marsh. All Puccinellia transplant source sites were replanted and those areas that were dug in 1979 and 1980 were almost completely revegetated by May 1981' (Figs. 48, 49). In a further attempt to lessen the pressure for obtaining transplants from the natural marsh, we have initiated nursery areas for Halimione and Puccinellia. These combined actions will help keep impact to the natural marsh to a minimum and serve as a model for others who may engage in similar activities in the future. Nursery Plantings The Puccinellia nursery area at Kerlavos was established in May 1979 and now contains about 300 plants that can be dug and separated into transplants. Although the plants vary in size, the average cover 408 FIGURE 48. Site where Puccinellia transplants were dug in May 1980. The area was replanted and was becoming rapidly revegetated in September 1980, 4 months after digging. FIGURE 49. Same site as shown in Figure 48 in May 1981, 1 year after digging. Vegetation cover is almost complete. 409 is about 540 cm^ or over 20 times that of a plug type transplant. To determine the actual number of plugs that could be obtained from a sample of plants, we dug 11 plants from the row nearest the estuary in May 1981 (Fig. 50). One of the largest plants yielded 50 plugs (Figs. 51, 52, 53), but the average number of plugs per plant dug was 20, which agreed well with what we predicted based on the average cover. Since the nursery area contains about 300 plants, we can predict that it could have yielded a minimum of 6,00 0 plug type transplants in May 1981 . It seems reasonable to assume that cover will increase from 50 to 100% by our next major planting effort in May 1982. This assumption translates into a conservative estimate of about 10,000 plug type transplants or enough to plant 0.25 ha on a 0.5 m spacing. The Halimione nursery area at lie Grande was established in May 1980 and added to in May 1981. It contains about 200 plants that can be dug and separated into transplants (Fig. 54). In May 1981 we dug a sample of seven plants to determine the average number of sprig type Halimione transplants that could be obtained per plant dug. We obtained an average of five sprigs from each plant dug (Fig. 55). Based on 20 0 plants in the nursery area, we estimate that there were about 1,000 Halimione transplants available in May 1981. We estimate that the increase in cover by May 1982 will result in about 1,500 to 2,000 Halimione sprigs available for digging at that time which would plant about 0.05 ha on a 0.5 m spacing. FIGURE 50. Row of 2-year old Puccinellia transplants nearest estuary in the nursery area at Kerlavos, May 1981. the 410 *■& /* <* < ■ FIGURE 51 Sample Puccinellia plant that was dug for transplants from the same row of plants shown in Figure 50, May 1981 . FIGURE 52. Cutting the same plant shown in Figure 51 into plug type transplants. 411 FIGURE 53. Plant shown in Figure 51 yielded 50 plug type transplants. ■ '"' 'J$mM FIGURE 54. A 1-year old Halimione transplant in the nursery area at lie Grande. 412 FIGURE 55. Halimione sprigs being dug from the nursery area at lie Grande. Invasion of Plantings by Other Plants Observations at lie Grande and Kerlavos indicate that other marsh plants invade our experimental plantings more rapidly than they colonize areas that still lack vegetation cover as a result of cleanup operations. In one of our May 1979 experimental plantings of Puccinellia at Kerlavos (Figs. 56, 57, 58), 97% of the transplants in the 60 m^ area had been invaded by at least one other species by May 1981 (Fig. 59). Of these transplants which had been invaded, 66% were invaded by two or more other species. The most abundant invader was an annual species of Salicornia which was present in 94% of the transplants sampled. Other invading genera in the order of their percentage of presence per transplant sampled were Cochleria (49%), Halimione (24%), Spergularia (10%), and Armeria (1%). 413 FIGURE 56. Experimental planting of Puccinellia at Kerlavos in May 1979 just after planting. t- £*~'^r FIGURE 57. Same experimental planting as shown in Figure 56 in May 1980, 1 year after planting. 414 FIGURE 58. Same experimental planting as shown in Figure 56 in May 1981, 2 years after planting. FIGURE 59. A 2-year old Puccinellia transplant from the experimental planting shown in Figure 56 with two invading marsh plants: Cochle.ia (white flowers) and Salicornia (left center near cluster of white flowers). 415 ACKNOWLEDGEMENTS Cooperation with our French colleagues (Madames Le Campion-Alsumard, Plante-Cuny, and Vacelet from Marseille and Monsieur Levasseur and Mademoiselle Jory from Rennes) has been invaluable. In fact, our work would have just about been impossible without their help- They have gone out of their way to help us while we were in France, such as arranging a meeting with the Mayor of Pleumeur-Bodou, making observations on our experimental plots during the interim of our visits, and providing laboratory facilities for processing samples. Presently we are cooperating with Monsieur Levasseur on nursery production of transplants and monitoring of our plantings until November 1982. He has also agreed to serve as the major professor for a graduate student from the United States who is a candidate for a Fulbright Scholarship. Among his tasks, this student would follow our plantings and document the invasion of these plantings by other marsh plants subsequent to our active involvement in the project. In summary, the association with our French colleagues has been very beneficial from our standpoint and we cannot overemphasize the vital part they have played in making our research proceed smoothly. We thank Amoco Oil for providing the funds for our research through NOAA Contract No. NA79RAC00018. We thank NOAA personnel especially Drs . W.N. Hess and D.A. Wolfe for providing us the opportunity to conduct this very timely and environmentally beneficial research and for their cooperation throughout the period of the project. Last, special thanks for technical assistance in field work and in data analysis go to Messrs. C.L. Campbell and L.L. Hobbs who made the overall research effort a success. SUMMARY Experimental plantings of Halimione portulacoides , Juncus maritimus, Puccinellia maritima, Spartina maritima, and Triglochin maritima have been made at lie Grande and Kerlavos, France in an attempt to rehabilitate salt marsh that was impacted by the Amoco Cadiz oil spill and subsequent cleanup operations. Over 61 experimental plantings including over 11,000 transplants have been established to test two types of transplants, conventional and slow release fertilizer materials over a wide range of substrate and elevation conditions and to develop nursery areas. Spartina transplants survived at lower elevations than those of any other species tested, but the best growth of transplants of all species tested occurred within + 0.3 m of the elevation of the natural marsh in the vicinity. Survival and growth data indicate that transplants of Puccinellia with a core of root and substrate material intact (plugs) were superior to those transplants with roots only (sprigs). 416 Although there was considerable variation in response to fertilizer materials and rates, both nitrogen and phosphorus were required for good transplant growth on the disturbed sites tested. Slow release fertilizer materials produced better growth over a wide range of substrate types than did the conventional, more soluble fertilizer materials. Higher survival and better growth were obtained with Halimione and Puccinellia transplants than with those of the other three species tested. Aboveground growth of the best experimental plantings of Puccinellia spread radially at the rate of about 10 cm annually. At this rate of spread, these experimental plantings would achieve complete substrate cover in about 3 years after planting. Refertilization at various periods after planting produced a significant increase in cover. Halimione sprigs were dug at the rate of about 180 per person hour and plugs of Puccinellia at the rate of about 7 5 per person hour. Transplants of both species were planted and fertilized at the rate of about 40 per person hour. Sites in the natural marsh from which Puccinellia transplants were dug, were replanted and became almost completely revegetated within 1 year. Nursery areas were established for both Halimione and Puccinellia and estimates indicated that in May 1981 they contained about 6,000 transplants of Puccinellia and 1,000 of Halimione. Preliminary data indicate that other marsh plants invade our plantings more rapidly than they invade unplanted disturbed sites. 417 LITERATURE CITED Baker, J. M. , 1971a, The effects of a single oil spillage: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 16-20, Applied Science Publ . , Bristol, England, 250 pp. Baker, J. M. , 1971b, Successive spillages: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 21-32, Applied Science Publ., Bristol, England, 250 pp. Baker, J. M. , 1971c, Refinery effluent: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 33-43, Applied Science Publ., Bristol, England, 250 pp. Baker, J. M. , 1971d, Seasonal effects: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 44-51, Applied Science Publ., Bristol, England, 250 pp. Baker, J. M. , 1971e, Effects of cleaning: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 52-57, Applied Science Publ., Bristol, England, 250 pp. Baker, J. M. , 1971f, Oil and salt marsh soil: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 62-71, Applied Science Publ., Bristol, England, 2 50 pp. Baker, J. M. , 1971g, Growth stimulation following oil pollution: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 72-77, Applied Science Publ., Bristol, England, 250 pp. Baker, J. M. , 1971h, Comparative toxicity of oils, oil fractions and emulsifiers: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 78-87, Applied Science Publ. , Bristol, England, 250 pp. Baker, J. M. , 1971i, The effects of oils on plant physiology: in E.B. Cowell (ed.), The Ecological Effects of Oil Pollution on Littoral Communities, pp. 88-98, Applied Science Publ., Bristol, England, 250 pp. Cowell, E. B., 1969, The effects of oil pollution on salt-marsh communities in Pembrokeshire and Cornwall: J. Appl. Ecol . , Vol. 6, pp. 133-142. Cowell, E. B. and J. M. Baker, 1969, Recovery of a salt marsh in Pembrokeshire, Southwest Wales, from pollution by crude oil: Biol. Conserv. , Vol. 1, pp. 291-295. Garbisch, E. W. , P. B. Woller, and R. J. McCallum, 1975, Salt marsh establishment and development: U.S. Army, Coastal Engineering Research Center, Fort Belvoir, Virginia, Tech. Memo. 52, 110 pp. 418 Ranwell, D. S. , 1968, Extent of damage to coastal habitats due to the Torrey Canyon incident: in J.D. Carth and D.R. Arthue (eds.)/ The Biological Effects of Oil Pollution on Littoral Communities, pp. 39-47, Field Studies Council, London, England, 198 pp. Seneca, E. D., S. W. Broome, W. W. Woodhouse, Jr., L. M. Cammen, and J. T. Lyon, III, 1976, Establishing Spartina alternif lora marsh in North Carolina: Environ. Conserv. , Fol. 2, pp. 185-189. Stebbings, R. E. , 1970, Recovery of salt marsh in Brittany sixteen months after heavy pollution by oil: Environ. Poll., Vol. 1, pp. 163-167. Woodhouse, W. W. , Jr., E. D. Seneca, and S. W. Broome, 1974, Propagation of Spartina alternif lora for substrate stabilization and salt marsh development: U.S. Army, Coastal Engineering Research Center, Fort Belvoir, Virginia, Tech. Memo. 46, 155 pp. 419 ETUDES MICROBIOLOGIQUES ET MICROPHYTIQUES DANS LES SEDIMENTS DES MARAIS MARITIMES DE L'lLE GRANDE A LA SUITE DE LA POLLUTION PAR L* AMOCO CADIZ par Therese Le Carapion-Alsumard, Marie-Reine Plante-Cuny et Eveline Vacelet Station Marine d'Endoume et Centre d'Oceanographie 13007 - Marseille, France. PRESENTATION SOMMAIRE DES BIOTOPES ET STATIONS ETUDIES Trois des biotopes caracteris tiques des raarais maritimes - schorres, chenaux de schorre, et haute-slikke - ont ete retenus pour cette etude dans deux sites, differant tres nettement quant a 1' importance de la pol- lution par le petrole de l'Amoco Cadiz (Fig. 1A - site Sud, Sud-Ouest tres pollue ; site Est, Nord-Est peu pollue). Le bloc diagramme (Fig. IB) roontre la difference de niveau altitu- dinal entre les 3 biotopes, entrainant evidemraent des differences de dur6e d' immersion. Les Schorres Les schorres de l'lle Grande sont des pres-sales a Juncus maritimus et Halimionc portulacoi.de s qui presentent une surface plus ou moins tabu- laire et un reseau de drainage variable (Fig. 1A, schorre Dl mieux drain^ que le schorre Al). Les deux stations de reference pour le "biotope schorre" sont Bl et CI (site Est, Nord-Est, Fig. 1A) faiblement atteintes par la pollution du fait de la mise en place d'un barrage de protection sous le pont reliant l'lle Grande a la terre. Les schorres tres pollues, Al et Dl , sont done situes dans la par- tie Sud, Sud-Ouest. Cette partie du marais servit pendant un certain temps de zone de stockage d'hydrocarbures issus du nettoyage des plages et ro- chers. 421 Les Chcn.iux Ccs scliorres sont draines par des chenaux presque constamment immer- gcs, dont le sediment est une vase fluide (station de reference C2 ; sta- tion tres polluee A2 - Fig. 1A et B) . Le Biotope Haute-Slikke Le biotope de vase sableuse intertidale ou haute-slikke, de part et d'autre du chenal central, a ete etudie en A3 pour le site pollue et on B3 pour le site peu pollue. Cette derniere station fut remplacee a par- tir de mars 1980 par E3 (meme type de biotope) lorsque les travaux de sur- cri'iiscmcnt du cbenal central eliminerent la station B. FIGURE 1 (OPPOSITE) A - Schema de localisation des stations Stations de reference = C , B]f schorres (Est, Nord-Est du pont) C2 , chenal B_, E , haute-slikke Stations tres polluees = A , D , schorres (Sud, Sud-Ouest du pont) A , chenal A-, haute-slikke B - Bloc diagramme representant les 3 types de biotopes et le niveau des hautes-mers. 422 A SWIRLING SITES Reference stations = CI. Bl schorres (salt meadows) C2 tidal creek B3, E3 5LIKKE (mud slope) Polluted stations = Al. Dl schorres (salt meadows) A2 tidal creek A3 slikke (hud slope) B HMMME NTHWL Lhaute-slikke — higher mud slope schorre - salt meadow 423 ETUDES REALISEES DANS CES BIOTOPES ET METHODES UTILISEES Neuf missions d'echantillonnage pour 1' etude des hydrocarbures et des peuplements bacteriens et microphytiques ont ete realisees entre de- cembre 1978 et novembre 1980 (une derniere mission est programmee en no- vembre 1981, ce qui donnera un "suivi" de 4 ans) . Divers parametres physiques et chimiques (temperatures, salinites, pH, Eh) ont ete mesures dans l'eau et les sediments. Les Hydrocarbures Les hydrocarbures (HC) ont ete doses et leur composition analysee dans des fractions de carottes a differents niveaux (Tab. 1, 2, 3). Les concentrations en hydrocarbures totaux (HCT) exprimees en g. kg~l de sediment sec sont definies comme etant la fraction FA apres les traitements suivants appliques aux echantillons de sediments : - extraction de la matiere organique au toluene-methanol sur echan- tillon humide (Farrington et Tripp, 1975). - elimination du soufre (pesee avant saponification — > poids AV- SP). - saponification a la potasse (pesee apres saponification >• poids AP-SP). - fractionnement du produit de la saponification par la methode dite au "Sep-pak"(micro-colonne de silice a compression radiale) (Giusti et al., 1979) . - elution par trois solvants successifs : 1° hexane fraction FA = HCT 2° chloroforme fraction FB = fraction polaire 3° methanol fraction FC L'extrait a 1 'hexane (FA = HCT) est ensuite analyse en chromatogra- phic liquide haute pression (HPLC reverse-phase) puis en chromatographie gazeuse capillaire (CPG) et infra-rouge lorsque la concentration est suf- fisante. Une analyse plus fine par spectrometrie de masse couplee avec la CPG est effectuee si necessaire. Etude Bacterienne Pour l'etude bacterienne, 1 'echantillonnage des sediments etait effectue par carottages. Les fractions etudiees etaient : dans les schorres (carotte de 60 cm environ) - couche de surface (su) - rhizosphere (rh) - couches plus profondes (couche argileuse : ca ; couche sableuse : cs) ; 424 dans les chenaux (carottes de 30 a 40 cm) - couche de surface (su) - zone reduite (zr) - couches plus profondes (ca ou cs) ; dans les slikkes (carottes de 10 a 40 cm) - memes couches que les chenaux. L' etude bacterienne de sous-echantillons comprenait : - denombrement de la microflore heterotrophe par MPN sur eau de mer pep- tonee a 5 g.l-' ; - estimation de l'activite bacterienne sur les memes ensemencements ; etablissement d'une courbe d'activite qui depend de la presence de sou- ches a croissance rapide ; - denombrement des germes capables de degrader les hydrocarbures par MPN sur milieu mineral contenant du petrole "Arabian light" comme source de carbone ; - enzymologie : mise en evidence des differentes hydrolases presentes et estimation comparative de leur activite par la methode APIZYM. A la surface des carottes, l'activite enzymatique est due a l'ensem- ble du peuplement "bacteries + microphytes". Dans les couches plus profon- des, seule intervient l'activite bacterienne. Les peuplements microphytiques Les peuplements microphytiques etaient Studies sur des carottes plus courtes (3 premiers centimetres d'epaisseur) . Aspect Quantitatif L' aspect quantitatif du peuplement est essentiellement apprehende par 1' estimation d'un indice chlorophyllien de biomasse que nous abrege- rons en ICB : extraction a 1' acetone et mesures (avant et apres acidifi- cation des extraits) des concentrations en chlorophylle a (Chla ou ICB) et en produits de degradation de la chlorophylle (pheopigments = Pheo.), methode de Lorenzen (1967) modifiee par Plante-Cuny (1974) pour les sediments Resultats exprimes eniug.g-! de sediment sec. Rapport Chla/Chla + Pheo : indice de vitalite des peuplements s'il y a preponderance de la chlorophylle (rapport ^ 0,5). i Aspect Qualitatif L' aspect qualitatif du peuplement consiste en une etude ecotaxino- mique des principales especes de diatomees et cyanophycees presentes. Note II est evident que dans la presente synthese tous les resultats obtenus dans les etudes microbiologiques et microphytiques n'ont pu etre pris en compte. lis feront l'objet de rapports separes. (Voir aussi Vacelet et al . , et Plante-Cuny et al . , 1981). 425 SYNTHESE DES PRINCIPALS RESULTATS Les resultats concernant 1' analyse fine des hydrocarbures et de leur eventuelle degradation font l'objet d'un rapport separe x. Des resultats succints seront donnes ici seulement pour servir a 1' interpretation des phenomenes biologiques. Differents Degres de Pollution au Debut des Observations II faut noter ici 1' absence de "point zero" concernant l'etat des marais de l'lle Grande avant l'echouage de l'Amoco Cadiz en mars 1978. Aucune etude prealable n'existait sur ce site et il nous a ete impossible de trouver dans une region avoisinante un marais maritime de merae type, indemne, pouvant servir de reference. De sorte que, les stations choisies presentent en fait differents degres de pollution en decembre 1978, au debut de nos observations (HCT en surface, exprimes en g. kg-' de sediment sec). Les valeurs ci-dessous, et notamment celles de Al et Dl, sont tres elevees en comparaison des valeurs donnees par Marchand (1981) pour des sediments profonds (15 a 100 metres) a la suite du naufrage du "Bohlen". Rappelons que le site de l'lle Grande etait un lieu de stockage des hy- drocarbures apres nettoyage d'autres sites. - schorres encore couverts de mazout en decembre 1978 : Al : 32,97 ; Dl : 94,68 - schorre avec traces de pollution : CI : 4,17 - schorre apparemment indemne : Bl : 1,9 - chenaux drainant les schorres : tres pollue, A2 : 7,69 * apparemment indemne, C2 : 3,26 - haute-slikke bordant le chenal central : visiblement tres polluee, A3 : 5,56 * apparemment indemne, B3 : 0,50 Les concentrations evaluees en A2 et A3 (*) ont ete mesurees sur des echantillons provenant de 1' extreme pellicule superf icielle du sedi- ment (moins de 1 cm d'epaisseur) deja colonisee par des microphyte's. Evolution des Concentrations en Hydrocarbures et des Peuplements Bacteriens et Microphytiques a Partir de Decembre 1978 Dans chacun des 3 biotopes - schorres, chenaux et slikkes - seront faites des comparaisons entre les stations tres polluees a differents x Etudes realisees par Henri Dou, Gerard Giusti et Gilbert Mille. Laboratoire de Chimie Organique A et LA 126 CNRS - Faculte des Sciences de Saint Jerome - 13397 Marseille cedex 13. 426 degres d'une part, et entre les stations tres polluees et les stations peu polluees d' autre part. On expose dans chaque cas : - 1' evolution des concentrations en HCT (g.kg~l), et de leur eventuelle degradation ; - l'evolution de l'activite bacterienne, du nombre de germes degradant les HC, de l'activite enzyma- tique ; - l'evolution quantitative et qualitative des peuplements micro- phytiques . Evolution dans les schorres Schorres tres pollues, Dl et Al . Schqrre_ Dl . En surface, la station Dl (Tab. 1) presente, presque deux ans apres l'echouage, la meme concentration en HCT (94,51) qu'en decembre 1978. II existe dans ce site des zones encore plus polluees (prelevement intention- nel dans une tache d'hydrocarbures en mai 1980 : 230,60). II semble y avoir, en ces points, une accumulation en surface par drainage du schorre environnant, lui-meme encore visiblement tres pollue en 1980. Le rapport AV/AP (Tab. 2), indicateur presume d'une biodegradation, augmente legerement en 1979 ainsi que l'activite bacterienne : un debut de degradation aurait eu lieu entre 1978 et 1979. On note parallelement une augmentation du nombre de germes degradant les HC jusqu'en novembre 1979 (10^ a 10? germes. ml~l de sediment) suivi d'une regression en 1980 (Fig. 2). L'activite enzymatique de 1' ensemble de la microflore a ete impor- tante en decembre 1978, s'est prolongee jusqu'en 1979 ce qui suggere la possibility d'un effet favorisant des HC. Cette activite regresse en 1980 indiquant peut-etre un appauvrissement de la microflore bacterienne (re- gression de chymotrypsine, trypsine et hydrolases des glucides) (Fig. 5). Les microphytes (Fig. 8A,1>1 )totalement elimines et encore absents en novembre 1978, ont recolonise peu a peu la surface du sol et ont pre- sente un maximum non negligeable en juillet 1979 (50yug Chla.g-'). Ensui- te, la preponderance au printemps 80 des pheopigments indique un etat peu florissant de la population. Une reprise est amorcee en novembre 1980 (40yUg Chla.g-'). II est possible que cette population vegetale comportant un fort pourcentage de cyanophycees reputees riches en hydrocarbures natu- rels en C'7 (Han et Calvin, 1969 ; Saliot, 1981) contribue a l'augmenta- tion observee du rapport cl7/Pr (Tab. 3) ce qui rend difficiles les inter- pretations quant a l'etat de degradation des HC. Dl : Dans la rhizosphere (cf. schemas des carottes, Tab. 1), on note dans le temps une diminution (de 8,13 a 0,23) de la quantite d'HCT et une augmentation du rapport AV/AP (1,20 a 2,06) pouvant indiquer une forte biodegradation. En effet, la concentration en germes degradant les HC croit de 102 a 106 germes. ml-1 jusqu'en 1980 (Fig. 2). Quant a l'acti- vite enzymatique, elle est fluctuante et plus faible en general en 1980 (Fig. 5). 427 Dl : Dans les couches plus profondes (30 cm), les concentrations en HCT, faibles au depart (0,48) diminuent en 1979 (0,10). Le nombre des germes degradant les HC a cru jusqu'en avril 1980 puis a diminue (Fig. 2). L'activite enzymatique regresse depuis decembre 1978. Tous les groupes d'hydrolases sont concernes (Fig. 5). Sc_horre_ _A1 . En surface, contrairement au schorre precedent, ou persistent de fortes concentrations en HCT, les valeurs passent de 32,97 a 18,84 de 1978 a 1979, raais la biodegradation parait peu importante (AV/AP C^c 1 ) . En 1980, la concentration tombe a 14,98. Les resultats concernant la rhi- zosphere en 1979 tendraient a prouver qu'il y a eu percolation. L'analyse montre en 1980, 1' absence d'alcanes lineaires et la persistance des HC satures ramifies et aromatiques. On observe egalement que la densite des germes degradants les HC augmente jusqu'en novembre 1979 et regresse en 1980 (Fig. 2), soit faute de substrat (alcanes lineaires), soit par sui- te d'un phenomene climatique general (voir Bl et CI). L'activite enzymatique en Al en surface, est comparable a celle de Dl (accroissement en 1979, regression en 1980) (Fig. 5). Les microphytes ont ete elimines sur le sol Al par l'arrivee du mazout et etaient encore absents en decembre 1978, comme en Dl (Fig. 8A) . Par contre, une tres legere colonisation seulement etait amorcee en 1979, suivie d'une diminution en 1980 (quelques ug Chla.g ' seulement). Les pigments degrades sont dominants en toutes saisons, traduisant le peu de vitalite de la population (Chla/Chla + Pheo. : 0,2 en moyenne) . Al : Dans la rhizosphere du schorre Al, il y a augmentation de 0,47 a 3,68 des concentrations en HCT indiquant probablement une perco- lation en 1979, accompagnee d'une degradation importante (AV/AP : 1,47 et 1,26, Tab. 2). Ensuite, la pollution diminue en 1980. La microflore degradant les HC etait en densite maximale en 1979, puis a regresse for- tement en 1980, davantage qu'en surface (Fig. 2). L'activite enzymatique est en augmentation depuis 1978, la pollu- tion etant proportionnellement moins forte que dans le schorre Dl (Fig. 5). Conclusion sur les schorres tres pollues. Ces deux biotopes, tres pollues au depart, ont reagi differemment puisque le plus pollue (Dl) n'est pas le moins recolonise par les micro- phytes. II faut sans doute y voir 1' influence benefique d'une plus forte humectation : Dl, mieux draine done plus souvent inonde, est plus rapi- dement recolonise du fait de l'apport de particules argileuses favorisant la fixation des microphytes. Dans les rhizospheres, la degradation parait se faire convenable- ment, meme en cas de percolation. C'est done a la surface des schorres les plus eleves (ou les plus souvent exondes) que se restaure le plus lentement le peuplement micro- biologique. 428 Schorres peu polities, CI et Bl ■ Scho_rre_ CI . En surface, sur le schorre CI, raoins pollue (4,17 en 1978) les con- centrations en HCT ont rapidement diminue (0,54 en 1980). Les germes de- gradant les HC sont restes en nombre stable (10^ a 10^ germes. ml~l de sediment (Fig. 2)). L'activite enzymatique a ete constamment elevee. La degradation semble done s'effectuer normalement (Fig. 5). Dans la rhizosphere, il y a forte diminution des HCT avec le temps et forte degradation (AV/AP = 1,25). Les germes degradant les HC se sont maintenus en nombre stable (10 a 10-^ germes .ml-1 ) . L'activite enzymatique a decru en 1980. Dans les couches les plus profondes, les concentrations en HCT sont tres faibles en 1980 (0,05) et l'activite enzymatique constante. Schorre _B] . En surface, le deuxieme schorre de reference, peu pollue (1,90 en decembre 1978) n'a pratiquement pas raontre de variations jusqu'en 1979 (1,75). Le nombre de germes degradant les HC- et les indices de biodegra- dation (1,34) tendent a prouver qu'il y aurait eu forte degradation (Fig. 2). Les HCT doses en 1979 n'ayant pas les caracteres d'HC degrades (C'7/pr = 4,43), nous emettons les hypotheses que de faibles apports chro- niques d'HC se produisent en cette station, ou plutot que les microphytes, tres abondants sur ce site, seraient responsables d'apports d'HC biogenes (Predominance d'imparite ^ 1). Contrairement aux faits observes sur les schorres tres pollues, les microphytes n'ont pas ete ici elimines au depart et ne paraissent pas af- fectes par une faible pollution, tout au moins en 1978 et 1979. Une densi- te maximale du peuplement est observee en juillet 1979 avec 130 jag Chla. g~l (Fig. 8A) et un rapport Chla/Chla + Pheo. de 0,75 indice de bon fonc- tionnement de la population. Tout au long de l'annee, et contrairement aux schorres pollues, cet indice est toujours superieur a 0,5. Le peuplement de cyanophycees et diatomees est toute l'annee bien diversifie et les especes caracteristi- ques des marais maritimes y sont presentes . En 1980, cependant, un ICB moyen de 50 ug Chla.g-^ est plus faible que celui des annees precedentes, mais comparable a celui du schorre CI. On ne peut, dans ces deux stations, exclure 1' influence nefaste eventuelle d'un facteur climatique en 1980. Dans la rhizosphere Bl, l'activite bacterienne et l'activite enzy- matique ont ete importantes et les concentrations en HCT, faibles au de- part, ont diminue encore. Conclusions sur le biotope "schorre". Dans les stations tres polluees au depart : 429 1° Une depollution se produit peu a peu, sauf si de nouveaux apports par drainage (?) maintiennent des taux de concentrations eleves. 2° La degradation des HC est toujours plus faible, en surface et dans la rhizosphere, que dans les schorres peu pollues. Cette degradation est, dans les sites peu humectes, pratiquement stoppee. 3° Les concentrations en germes degradant les HC sont toujours supe- rieures de 1 a 2 ordres de grandeur a celles des schorres peu pollues. 4° Par contre, l'activite enzymatique est 2 a 3 fois plus faible que dans les schorres peu pollues. 5° Apres avoir totalement disparu, les microphytes recolonisent tres lentement les schorres "asphaltes", un peu plus rapidement les schor- res pollues mais plus humectes. Par contraste, les schorres peu pollues sont tres florissants (ICB 10 a 20 fois superieur) . Evolution dans les Chenaux Chenal tres pollue, A2 . En surface, les concentrations en HCT ont diminue de 10,78 a 2,57. Les indices adequats prouvent qu'une forte degradation a eu lieu. Les chromatogrammes des HC restant en 1980 ne presentent plus les pics des alcanes lineaires. La degradation rapide parait terminee et les autres fractions resteront probablement en l'etat. Les germes degradant les HC ont augmente en nombre jusqu'en 1979 (10^) puis ont decru en 1980 (10^ germes. ml-', Fig. 3), ce qui corrobore l'hypothese precedente de la non poursuite de la degradation mais n'exclut pas l'hypothese du facteur cli- matique. L'activite enzymatique est plus faible que dans la station non polluee (Fig. 6). Les microphytes ont recolonise tres rapidement la surface de la vase et ont atteint en novembre 1980, un maximum de 588 /ig Chla.g-1, maximum absolu dans toutes les stations etudiees (Fig. 8B) . En 1978, cette microflore etait paucispecif ique (Phormidium et Amphiprora alata) . En 1980, la diversite specif ique d'une vase normale equivalente (Carter, 1933) est retrouvee. Pourtant un raclage effectue en mai 1980 montre encore une concentration d'HC de 14,20 g.kg~l. Les chromatogrammes montrent l'absence totale d' alcanes lineaires (Tab. 3). Dans la zone reduite (Tab. 1 : zr) , les concentrations en HCT ayant augmente jusqu'en 1980, on peut evoquer la possibility d'une perco- lation. A ce meme niveau, en 1980, tous les alcanes lineaires sont degra- des. La concentration mesuree concerne done des HC plus resistants. On observe en outre que le nombre de germes degradant les HC decroit forte- ment (10 germes .ml-' ) . L'activite enzymatique decroit egalement. Chenal peu pollue, C2. En surface, les concentrations en HCT sont passees de 3,26 a 0,70. La biodegradation est importante (AV/AP eleve) . Le nombre de germes degra- dant les HC est stable (103 a 10^ germes .ml"1) . 430 L'activite enzymatique dans cette station "propre" est aussi impor- tante que dans les schorres peu polities. Elle est en nette augmentation en 1980, ce qui est peut-etre a relier aux travaux d'amenagement effectues a cet endroit (voir ci-dessous) . Les microphytes ont manifeste un maximum de developpement en ete 1979 suivi d'une decroissance. Les variations saisonnieres paraissent done tres differentes dans les deux stations de type "chenaux", mais le chenal ou se trouve la station C2 a ete obture momentanement, en mai 80, par des travaux de surcreusement du chenal central. La composition des peuplements de microphytes a ete nettement perturbee et s'est appauvrie en especes et en individus. La zone reduite et la couche sableuse sous-jacente sont fortement depolluees (0,09 et 0,05). Le norabre de germes degradant les HC est sta- ble. Dans la zone reduite, une diminution de l'activite enzymatique se poursuit depuis decembre 1978, indice possible de la restauration d'un etat d'origine (disparition des lipases, augmentation des activites este- rases, aminopeptidases, chymotripsines et glucidases). Conclusions sur le biotope "chenal de schorre". 1° Dans le cas de pollution forte, la biodegradation, apres avoir ete active, s'est ralentie ou arretee. Les HC encore presents ont peu de chances d'etre degrades rapidement. Dans le cas de pollution faible, la degradation a ete presque complete. 2° Parallelement, le nombre de germes degradant les HC a augmente puis diminue dans le cas de forte pollution. II est stable ailleurs. 3° L'activite enzymatique est moins importante, en surface, en cas de pollution. Elle est toujours plus faible en zone reduite. 4° Les microphytes se developpent en surface de facon luxuriante dans les chenaux pollues. Les concentrations en Chla sont en 1980 nette- ment superieures a celles du chenal non pollue. Evolution dans les slikkes Slikke tres polluee, A3. En surface, a l'endroit precis ou nous avons situe la station A3, on peut dire que les concentrations en HCT sont passees dans la pelli- cule superf icielle (ps = quelques millimetres d'epaisseur) de 5,56 a 0,27 g.kg-1 entre decembre 1978 et mai 1980 (Tab. 1). Dans la couche sous-jacente (su : 2 a 3 cm d'epaisseur) les concen- trations sont passees de 24,95 a 0,60 entre 1979 et 1980. II y a done eu depollution en surface. Dans le raeme temps, les indices de biodegradation ont augmente jusqu'en 1979 (Tab. 2) et le nombre de germes degradant les HC egalement (Fig. 4) . Dans la couche sableuse, par contre, les concentrations en HC ont augmente de 0,65 a 2,40 en 1979, puis diminue en 1980 (0,50). Les indi- ces de biodegradation sont faibles. L'activite enzymatique est reduite (Fig. 7). II semble y avoir eu percolation, surtout en novembre 1979. 431 Devant les difficulties d' interpretation des resultats dans un tel biotope (problemes d' echantillonnages) , des prelevements par raclages ont ete faits en mai 1980. lis ont permis d'effectuer des dosages a partir d' un materiel abondant et de dif ferencier, d'une part la pellicule superfi- cielle constitute essentiellement de particules argileuses compactees par les microphytes, et, d'autre part, la couche sableuse visiblement encore tres polluee. Les resultats sont eloquents : 0,27 dans le premier cas, 15 g.kg~l dans le second. Ce sont des hydrocarbures d'origine "Arabian light" dont les alcanes lineaires certes sont degrades, mais dont les autres consti- tuents sont toujours presents /Tab ■4,'b)- Notre premiere hypothese du "piegeage" du petrole sous la matte vegetale se trouve confirmee. En effet, les filaments de cyanophycees et les diatomees compactant les particules argileuses avaient tres rapidement colonise ces vases im- pregnees d'HC puisqu'en decembre 1978, on observait un ICB de 140 ^ig Chla. g~l (Fig. 8C) , et tres peu de pheopigments (rapport Chla/Chla + Pheo. de 0,96 , le plus eleve de cette etude) indiquant une population jeune. Ce peuplement se revelait paucispecif ique (Phormidiwn, deux especes de Nitz- sohia3 Amphipleura, Rhopalodia) mais se diversifiait tres rapidement. Les deux cycles annuels de 1979 et 1980 presentent tous deux un maximum en automne ou en hiver, tout comme la vase du chenal pollue, avec des ICB presque aussi eleves et toujours tres peu de pheopigments. En 1980, le peuplement est tres riche et tres diversifie (presence de nombreuses especes caracteristiques de ces milieux) . La matte algale recouvre done un sediment dans lequel une depollu- tion a eu lieu, tout au moins dans la partie superf icielle, mais dont la couche sableuse est toujours irapregnee d'HC dont la degradation parait tres ralentie. Cette matte semble toujours jouer un role de frein a une depollution mecanique par le jeu des marees . Slikkes peu polluees, B3 et E3. La station B3 nous a paru devoir etre remplacee, en mars 1980, par une nouvelle station de reference (E3) , la slikke centrale ayant ete per- turbee par 1' obturation momentanee du pont, apres l'echouage du Tanio (mars 1980) puis par des travaux d'amenagement . Les concentrations en HCT dans ces stations sont faibles. La bio- degradation a ete importante. Le nombre de germes degradant les HC est stable (Fig. 4) . L'activite enzymatique de surface est en augmentation en 1980 (Fig. 7). Le peuplement microphytique est florissant, particulierement en E3, ou un indice C17/Pr de 4,47 en mai 1980 pourrait traduire, comme dans les schorres non pollues Bl et CI, la presence d'un hydrocarbure biogene par- ticulierement abondant chez certaines cyanophycees (Fig. 8C) . Contrairement a la slikke polluee, les couches sous-jacentes ici ne renferment pas d' hydrocarbures . 432 Conclusions sur le biotope "slikke". Ces conclusions sont tres voisines de celles qui concernent les chenaux : 1° En cas de pollution grave, la biodegradation, active d'abord, s'est ralentie ou meme arretee. 2° Le nombre de germes degradant les HC a diminue depuis 1978. Dans les stations peu polluees il est stable. 3° L'activite enzymatique parait toujours freinee en surface en cas de pollution forte. 4° Les microphytes se sont developpes rapidement sur les sediments pollues, piegeant des particules argileuses et constituant une "matte al- gale" plus compacte que dans les chenaux, pellicule qui freine la depollu- tion de ces sediments. 433 CONCLUSIONS Nous avons essaye au cours de cette etude de nous attacher a compren- dre les interrelations qui pouvaient exister entre l'etat de degradation des hydrocarbures et 1' evolution des peuplements bacteriens et microphy- tiques des marais maritimes. La complexite de ces milieux et les problemes d'echantillonnage qui en decoulent rendent parfois difficile la comprehen- sion du f onctionnement d'un tel ecosysteme perturbe. En ce qui concerne l'ensemble des biotopes. 1° 11 apparait tout d'abord que les marais maritimes de l'lle Grande restent tres pollues malgre une biodegradation importante. 2° Les hydrocarbures presents actuellement a la surface des sediments ou dans des couches plus profondes (percolation ou "piegeage") sont a un stade tel (disparition totale des alcanes lineaires) que la degradation ne parait pas se poursuivre actuellement. Ce ralentissement peut avoir plusieurs causes : persistance des seules fractions les plus resistantes des HC, toxicite, pour le peuplement bacterien, de certains produits de de- gradation, facteur climatique defavorable a l'activite bacterienne. 3° L'evolution de ces milieux s'est averee differente suivant le degre initial de pollution : - dans les stations tres polluees, les concentrations en germes de- gradant les hydrocarbures ont ete tres elevees. Puis leur nombre a decru en 1980. L'activite enzymatique a ete moins elevee en surface et dans les rhizospheres . - dans les stations peu polluees, 1' impact sur les peuplements micro- phytiques et bacteriens a ete peu perceptible, et la degradation a ete plus poussee, mettant en evidence l'existence probable d'un seuil de con- centration en HC en-dessous duquel la "restauration" est possible. En ce qui concerne chaque biotope en particulier, le deversement massif du petrole "Arabian light" n'a pas eu les memes consequences dans les sols du pre-sale, le plus souvent exonde (schorres) que dans les se- diments fins, le plus souvent immerges (chenaux et slikk.es). L' aspect mi- crobiologique et 1' aspect mecanique sont a prendre en compte dans les differences de depollution. Les vases intertidales Elles ont ete probablement plus vite nettoyees par le jeu des marees que la surface des schorres. Mais, par ailleurs, elles ont ete tres rapi- dement recolonisees du fait de l'apport de particules argileuses favori- sant 1' installation d'un peuplement paucispecif ique de cyanophycees et de diatomees qui, par la suite, s'est diversifie. Le mazout restant s'est trouve ainsi plus ou moins piege sous cette "croute" algale et pourrait s'y maintenir longtemps . 434 Les schorres Les schorres, par contre, ont ete moins rapidement depollues par les marees, certains presentant meme des zones d1 accumulation. Apres avoir totalement disparu, les microphytes recolonisent tres lentement les sols, d'autant plus lentement qu'ils sont moins souvent immerges . La colo- nisation est actuellement environ 10 fois inferieure a la normale sur le sol des schorres a Juncus, observation qui concorde avec les resultats obtenus par Levasseur et Seneca sur la flore macrophytique (voir contri- butions de ces auteurs) . 435 TAULKAU 4 EV0UT10N DtS CONCENTRATIONS FN IIVCROCARJtUKES TOTAUX I)AKS LES SEDIMENTS DES HAKAIS MAk HIKES DE L'lI.E CRA1JDE . Eiotcpcs D f { orcnlj HC totaux B-ke"1 dc ccdirocnt tec (t u i vt aux Stations dui.s la IC1 IC? ,C6 5/80 carotte 12/78 3/79 11/79 Schorrc tj 32,97 18,84 14,98 A. th ca 0.4? 3,68 0,03 0.04 iU 94,68 94.51 230.60* ", rh 8.13 0.73 17.78* ca 0,48 0,10 O.I6» B. th 1.90 0.17 1.75 0,10 (U «.U 0,43 0,54 c. rh e«(1t-32 cd) 0,26 0.03 0.15 0,05 CHcnfttjx ft 7,89 14,20 •u 10 78 6,59 7.57 A? ir 0,48 0 22 0,29 1.14 ca 0, 10 0,03 0,08 - 28-36 (a 0,08 [u 3.76 1.41 0,70 C2 c t 0,77 0,23 0.09 0,05 llaau Sljfckc P* 5.56 0,27-15** A3 IU 24 95 3,45 0,60 cs 0,65 2,40 0,50 cu 0,89 S ir ca su 0,52 0,08 0,03 0,56 B3 /r 0.19 0.72 i ca 0,16 ill t i rh i, 1 '»! if : ti i rh tu l i rh (I I ts J i ?r I 1 I is K \ n d ' en i/l uiii» r 1 \;ui •.!. ** Rem i .'■.,.. • oni «' I <: i*f fc*i luo* dan* 1c cln * Cellr v- ■• d'hydrc.. i *1 Ir i," j ;■: uLurcw .ii ii ..I i.-.i i *» dan* b»p CAclte i;*.'[>,r..d.u i»>n. nj] central . i i D j r m T ps : pclliculc suporf icicllc, prtMcvcrccnC par i .■». 1 .(•> iu : pjit ic uupci'f iciollr, quvlqucs cent irti'i re t» rli : rlti^OKpn^ro es : roue he s.iM eusc icr : r.owc reJu i t v ca : t puclw ar*.' i 1 eusc 436 TABLEAU 2 IIYDROfARUUKES DANS l.LS SEDIMENTS UES MAKAIS MAR1TIMES DE L'lLE CRANDE (POLLUTION ]'AR L' AMOCO CADIZ) ANALYSE CII1MIQUE stations Poids r del fchant i t ucoide 1 Ions AV-SP E*E~ AT fi- -SP AV/AP FB . -1 g. kg Hydioca totaux B- kg'1 rbures (FA) IC, IC6 1C, IC6 le. 1C6 IG, ,C6 IC, 1C6 IC, ,G6 12/73 11/79 12/78 11/79 12/78 11/79 12/78 11/79 12/78 11/79 12/78 11/79 Schorre Al 6U 13.40 25,10 73,60 43,48 67,60 39,58 1.09 1,10 35,70 16,93 32,97 18,84 rli 66,70 99,50 2,00 8.92 1,37 7,10 1,47 1,26 0,90 2,99 0,47 3,68 6U 12,55 14,60 173,90 243,45 162,50 210,50 1,07 1,15 67,90 102,12 94,68 94,51 Dl rh 59,15 90,10 19,78 1,98 16,50 0,96 1,20 2,06 8,40 0,46 8,13 0,23 CJ 24,25 74,80 1,89 0,24 1,48 0,19 1.26 1.26 1,00 0,06 0,48 0, 10 Bl B" 13,30 10,85 8,00 10,29 5.15 8,98 1,34 1,14 4,00 5,56 1,90 1,75 rh 53.50 97,80 2,12 1,99 1,30 0,89 1.63 2,24 1.06 0,58 0,17 0, 10 CI su 15,35 19,85 13.74 2,43 12,27 1,93 1.12 1,26 8,10 1 , 19 4,17 0,43 rh 58,80 132,50 2,41 0,15 1,59 0,12 1,52 1,25 1,33 0,05 0,26 0,03 Oienaux de schorre PS 51,20 6,10 18,52 21,49 16,70 12,41 1,11 1,73 9,00 4,25 7,69 « 6,59 A? zr 83.50 120,60 1,78 0.95 1,42 0,60 1,25 1,58 0,91 0.23 0,48 0,29 ca 23,10 139,20 0,63 0,17 0,31 0,07 2,03 2,43 0,21 0,02 0, 10 0,03 EU 10.50 15,90 14,07 6,32 8,27 4,28 1,70 1 ,48 5,00 2,17 3.26 1,41 Cz zr 39,80 41,20 3,07 1,89 2,26 0,94 1,36 2,01 1,50 0,66 0.77 0,09 cs 18,65 76,90 0,60 0,92 0,45 0,40 1,33 2,3 0,22 0,25 0,23 0,05 Haul c- si ikkc A PS A3 cs 37, eo 1,05 11,48 16,91 1 1,44 12,91 1,00 1,31 5,88 8 5,56« 3,45 106.50 7,43 6,66 1,12 3,54 2,40 n P3 B3 cs 7,50 6,18 2,66 2.32 1,34 0,52 112, 30 3,43 1,76 1,95 1,14 0, 19 Arabian light 100 c 9', f. 1,07 32X 68% AV-SP : avJint Riiponi fi ration AP-5)' : ajirrii hflpnnif . rat jnn jmj3~VaV , fOutlor. HC CI 3 \h : fr.nii'.n A. M ut 3 m, \wr.n\\r : liy.lf it- ;n t.ni ■ i> \t*\u yn : |.r-M ImjIi- » i»|..tI h i-llc ( t n ( mi. i ni r«i i • -t.K «.iit .'t<' r-v/ilu'cM nur ifrn ■VUnl illrvtm XX Irn rlir m«m oj;rnwnti*n lie nioiitrmt p)u<. )n pii'i.n.r d'/ilt I i nffll ri • . hu - |, i r i i . r ,i,.. i 1 i . i . 1 1 , : ijih-lt|ii>H i t-iti inn* trf'ii ill - rlii 7 ■ »;p!i« I i ( r. - . rut- )..- b .1.) use VI * / ■ .;.. ■ I t'/' u j t t IT - plJMIIM 'rt m ' '■'>' I"- i'i ,- 1 I « m'io pi. - |.h> !/.•„. 438 12-14 )uiH 79 2-7 oct 79 23-25 nov 79 I 1 • T 1-2 avrll 80 19-20 mal 80 nov 80 Ci 1 FIGURE 2. Norabre de germes (log) degradant les hydrocarbures a differentes profondeurs dans les schorres tres pollues (Al et Dl) et moins pollues (Bl et CI) des marais niaritimes de l'lle Grande. i - . . coucbe d'hydrocarbure epuche a microphytes rhizosphere 4% argile couche reduite sable 439 12-14 juia 79 1-2 avril 60 19-20 mai EO 24-25 nov 00 B2 a o ° illllll 1 1 FIGURE 3. Nombre de germes (log) degradant les hydrocarbures a differentes profondeurs dans les chenaux tres pollues (A2) et moins pollues (B2) - meme legende que figure 2 -. 12-14 Juill 79 2-7 ocl 79 1-2 avril 80 19-20 mal 80 24-25 nov 80 B3 FIGURE 4. Nombre de germes (log) degradant les hydrocarbures a differentes profondeurs dans les slikkes tres polluees (A3) et moins pollu- tes (B3) - meme legende que figure 2) -. 440 .-■*. 3 o4 ,3 ex o u u a E •►" V- v.. * — K lissMs mmmm. m < $0 twj x <■ s gggggm o en C CO a 0) 1-1 CO ex \o> 3 3 0) r^ CX r-l o CO Gl- 0) Tl en a a) • H i-i 0 J3 B a 4) 4-1 en 01 fl cu *~N m- ■ 1 1-1 a 0) 4-1 X) 01 ^N _ CU < T3 •^ o x: en 4J \> o N .3 3 o • r4 en 4-1 C O a < 13 441 . - K a - u «« ? • s • » n a a - 1 t • ►.. o n " ■ **». o I c v.. ■•*.. u 4J 33 en VI) 3 O P. to 3 • H i a) CM < 3 O tfl /0) M 4-1 3 0) x; o (0 01 CO a •3 P. N 3 0) VU 4-1 • i-l > • r-l 4J o dm X> 3 O 442 "►, CI Ml) 3 o ex CO C I 4-1 0) J % ,1 V r» ^ c< > V..„v. < (A 3 O P. to u CO % c • 3 . ■ • • Ci *" o . o „ ** 1- * IS h >-- o *^ * ■ ** s ■ CO CU CO a s a '•CD 4J > ■ H 4-1 a m 3 M 443 FIGURE 8 Evolution temporelle des concentrations en chlorophylle a (trait plein) et en pheopigments (pointilles) a la surface des sediments (en ug-g de sediment sec) . A - Stations de schorres B , C peu pollues D , A tres pollues B - Stations de chenaux C peu pollue A tres pollue C - Stations de slikkes B , E , peu polluees A tres polluee 444 A 150 - Chi. a Pheo. 100 - 50- -i — i— I — r~ i — i — T — [ — i — i — [ — i — i — i — i — i — i — i — i 1979 1980 t— t — i — r 50- 50- s£ A1 t — r— i — i — i — i — t — i — r — i — i — I — i — r 1979 445 i — r— i — i — i — i 1981 150- 100 50- ■*o 150- 100- 50 446 c Chi. i Pheo. 150 100 - 50- t"°-j — i — i — i — r~~i — i — r — i — r~i — i — i — r 1979 t — r — r 1980 1 — i — i — i 150 - 100 — 50 1 1 1 1 i~ T 1 447 REFERENCES BIBLIOGRAPHIQUES Atlas, R.M. et A. Bronner, 1981, Microbial hydrocarbon degradation within the intertidal zones impacted by the Amoco Cadiz oil spillage : in Amoco Cadiz. Fates and effects of the oil spill. Proc. Internat. Symposium. C.O.B. Brest (France) novembre 19-22, 1979, pp. 251-256. Butler, J.N. and E.M. Levy, 1978, Long term fate of petroleum hydrocar- bons after spills. Compositional changes and microbial degradation : J. Fish. Res. Board Can., Vol. 35(5), pp. 604-605. Carter, N., 1933, A comparative study of the alga flora of two salt mar- shes : J. Ecol., Vol. 21, pp. 128-208, 385-403. Colwell, R.R., A.L. Mills, J.D. Walker, P. Garcia- Tello, et P.V. Campos, 1978, Microbial ecology studies of the Metula spill in the straits of Magellan : J. Fish. Res. Board Can., Vol. 35(5), pp. 573-580. Ducreux, J., et M. Marchand, 1981, Evolution des hydrocarbures presents dans les sediments de l'Aber Wrac'h, d'avril 1978 a juin 1979 : in Amoco Cadiz. Fates and effects of the oil spill. Proc. Internat. Symposium. C.O.B. Brest (France) novembre 19-22, 1979, pp. 175-216. Farrington, J.W., and B.W. Tripp, 1975, A comparison of analysis methods for hydrocarbons in surface sediments : in T.M. Church (ed.), Marine Chemistry in the Coastal Environments, Amer. Chem. Soc. Symp. Series n° 18, Washington, D.C., pp. 267-284. Fujisawa, H., M. Murakami, et T. Manabe, 1977, Ecological studies on hy- drocarbons oxidizing bacteria in Japanese coastal waters. I. Some methods of enumeration of hydrocarbon oxidizing bacteria : Bull. Jap. Soc. Sc. Fish., Vol. 43(6), pp. 659-668. Fujisawa, H., M. Masatada et M. Takehiko, 1978, Ecological studies on hy- drocarbons oxidizing bacteria in the oil polluted areas caused by the Mizushima oil refinery accident (Seta Inland Sea) : Bull. Jap. Soc. Sc. Fish., Vol. 44(2), pp. 91-104. Giusti, G., E.J. Vincent, H.J.M. Dou, et R. Faure, 1979, Etude par RMN de la concentration et de la nature des hydrocarbures presents dans les sediments cotiers superficiels de l'lle des Embiez : La Vie Marine, 1, pp. 24-29. Han, J. and M. Calvin, 1969, Hydrocarbon distribution of algae and bacte- ria and microbiological activity in sediments : Proc. nat. Acad. Sci. U.S.A., Vol. 64(2), pp. 436-443. Lorenzen, C.J., 1967, Determination of chlorophyll and Pheo-pigments : Spectrophotometric equations : Limnol. Oceanogr., Vol. 12(2), pp. 343-346. 448 Marchand, M. et J. Roucache, 1981, Criteres de pollution par hydrocarbu- res dans les sediments mar ins . Etude appliquee a la pollution du "Bohlen" : Oceanol. Acta, Vol. 4(2), pp. 171-183. Plante-Cuny, M.R., 1974, Evaluation par spectrophotometrie des teneurs en chlorophylle a fonctionnelle et en pheopigments des substrats meubles marins : Doc.~Sci. Mission ORSTOM, Nosy-Be, Vol. 45, pp. 1-76. Plante-Cuny, M.R., T. Le Campion-Alsumard, et E. Vacelet, 1980, Influence de la pollution due a 1' Amoco Cadiz sur les peuplements bacteriens et microphytiques des marais maritimes de l'lle Grande, 2, Peuple- ments microphytiques : i_n Amoco Cadiz. Fates and effects of the oil spill, Proc. Internat. Symposium, C.O.B., Brest (France) novembre 19-22, 1979, pp. 429-442. Saliot, A., 1981, Natural hydrocarbons in sea water : in E.K. Duursma and R. Dawson (ed.), Marine Organic Chemistry, Amsterdam, pp. 327-374. Thompson, S. and G. Eglinton, 1979, The presence of pollutant hydrocarbons in estuarine epipelic diatom populations, II, Diatom slimes : Estua- rine and Coastal Marine Science, Vol. 8, pp. 75-86. Traxler, R.W., et J.H. Vandermeulen, 1981, Hydrocarbon -utilizing micro- bial potential in marsh, mudflat and sandy sediments from North Brittany : in Amoco Cadiz. Fates and effects of the oil spill. Proc. Internat. Symposium. C.O.B., Brest (France) november 19-22, 1979, pp. 243-249. Vacelet, E., T. Le Campion-Alsumard, et M.R. Plante-Cuny, 1981, Influence de la pollution due a 1' Amoco Cadiz sur les peuplements bacteriens et microphytiques des marais maritimes de l'lle Grande, 1, Peuple- ments bacteriens : _in Amoco Cadiz. Fates and effects of the oil spill. Proc. Internat. Symposium. C.O.B., Brest (France) november 19-22, 1979, pp. 415-428. Ward, D.M., 1981, Note de synthese. Microbial responses to Amoco Cadiz oil pollutants : in Amoco Cadiz. Fates and effects of the oil spill. Proc. Internat. Symposium. C.O.B., Brest (France) november 19-22, 1979, pp. 217-222. 449 1964-1982, COMPARAISON QUANTITATIVE DES POPULATIONS BENTHIQUES DE ST-EFFLAM ET DE ST-MICHEL-EN GREVE AVANT, PENDANT ET DEPUIS LE NAUFRAGE DE L' AMOCO CAPIZ par C. CHASSE et A. GUENOLE-BOUDER Laboratoire d' Oceanographie Biologique, Institut d' Etudes Marines, Universite de Bretagne Occidentale, 6, avenue Le-Gorgeu, 29283 Brest cedex, France Resume La baie de Lannion, largement ouverte face a la progression des nappes de petrole de 1 'AMOCO CAPIZ, fut parti cul i erement souillee : 60 millions de cadavres echoues furent denombres sur les deux plages du fond de la baie de St-Efflam et Locquemeau. Des etudes anterieures sur l'estran de St-Efflam, representati f des nombreuses plages de sable fin de Bretagne, ont servi de reference a ce tra vai 1 . L'impact du petrole est tres variable sur les diverses especes d'une meme station. La partie Est de la plage, plus contaminee, montre une plus forte mortalite. Le haut et le bas de l'estran sont plus affectes que la partie intermediate. Deux processus semblent intervenir : - les nappes d'echouages en haut, - le petrole dissout ou en emulsion dans la masse d'eau en bas et dans tout 1 ' i nf rati dal . A la mortalite immediate s'ajoutent des mortalites et des effets patho 1 ogi ques a long terme. Certaines especes continuent a regresser meme en 1981 et les recrutements souvent inexistant en 1978 s'amorcent en 1980, pour certains timidement encore en 1981. L'Est de la plage reste fortement touche bien que des signes de recouvrance certains apparaissert sur le reste de la plage mais les gros peuplements du bas de la plage a Solcn, Eniii, lchinocan.dlu.rn, iu.tn.ania, h\actxa coniallina ne sont pas reapparus. 451 INTRODUCTION Les nappes d'emulsion petroliere de 1' AMOCO CADIZ poussees a la surface de la Manche, le long de la cote, par les vents d'Ouest ont ete freinees en se heurtant sur les saillants successifs du rivage. Quatre zones principales d'obstacles se sont dressees face a leur progression vers l'Est. Ce sont les roches et les ilots des abers et de la presqulle Ste-Marguente, d'abord ; le champ de roches de Hie de Batz, de Santec, de RoscolL ensuite ; puis les roches de Pnmel et du Guersit ; enfin, celles des rebords Est de la baie de Lannion avec Tile Grande. En chacun de ces lieux, et surtout dans les criques, les estuaires et les baies de sable fin les plus proches qui les precedent le petrole s'est abon- damment accumule en provoquant de lourdes mortalites donnant lieu a d'importants echouages de cadavres de poissons, d'oiseaux et de coquillages. La baie de Lannion, deja fortement souillee, en 1967, par le petrole du Torrey Canyon, a ete atteinte par les nappes de V AMOCO CADIZ des le 5ejour apres le naufrage. Sur les deux grandes plages de sable fin du fond de la baie nous avons recense 60 millions d'indi- vidus morts. dont la moitie sur la Grande Greve (St-Efflam). Le cinquieme seulement des cadavres etait accumule dans le spectaculaire et nauseabond cordon d'echouage du niveau des hautes mers ; la fraction la plus importante, bien que plus discrete, etait eparpillee en nuages a la surface de 1'ensemble des plages. Par des transects de plages, realises avec des gabarits metalliques d'1/4 de m2, par 5 equipes d'etudiants operant durant 3 jours, nous avons obtenu le decompte suivant pour les principales especes : ESPECES Mb re d'individus (cadavres) Poids en matiere organique seche(C) Poids brut ( t ) Eehinoaardiurr aordatum 20. 106 4 260 Cardium edule 16. I06 5 70 Mactra corcllina 14. I06 4 50 Pharus lejumen 5.106 10 100 Ensis siliqua 1 . 106 2,5 25 Lutraria lutraria 0, 1 .106 0,8 10 Donax vittatus 1 . !06 0,04 0,6 Tellina fabula 0,03. 106 0,001 0,01 Tellina tenuis 0,02. 106 0,001 0,01 SOMME 57.15.I06 25,366 515,62 A cette mortalite initiale. brutale, s'est ajoutee une importante mortalite ulteneure plus discrete. L'observation des phenomenes dans leur ensemble geographique ne nous a pas. permis de cerner cette mortalite durant les premiers mois. Deux campagnes trimestrielles. avec seulement 10 stations regulierement suivies a St-Efflam. ont pu etre effectuees. Ce n'est qu'en Janvier 1979. dans le cadre d'un contrat avec la NOOA. que nous avons pu mettre en place un reseau d'observations mensuelles mais qui ne couvrait 1'ensemble de la plage qu'en 6 mois. Les peuplements des plages de la Grande Greve (St-Michel et St-Efflam) et de Locque- meau ont ete etudies qualitativement depuis un siecle par les chercheurs et les etudiants de la station biolog'.que de RoscolT. Des etats de references quantitatifs. etablis sous forme de cartes d'isobiomasses, de 1965 a 1968 (C. Chasse, 1972), donnaient, pour la plage de St-Efflam. des points de comparaison utiles a la fois pour le suivi des principales especes et pour l'impact des hydrocarbures deja evalue lors du naufraae du Torrey Canyon. en 1967. 452 453 L'ensemble de la nouvelle canographie des peuplements de la plage de St-Efflam a ete realise avec une centaine de stations quantitatives dont 65 ont ete etudiees d'une maniere plus approfondie. Chaque station a fait l'objet de 3 prelevements de sediment effectues a la benne a mam de 1 16 de m2 sur 20 cm de profondeur. La faune est recueillie sur place par tamisage sous l'eau sur maille de 1 mm, elle est determinee. comptee et pesee. Des cartes d'isobiomasses des principals especes ont ete dressees, comparables aux cartes anteneures realisees avant et apres le naufrage du Torrey Canyon. Dix stations caracteristiques des pnncipaux peuplements. situees au bas et au centre de la plage, et suivies durant toutes les operations, ont permis d'etablir revolution des especes dans le temps. LE MILIEU Sur la cote Nord de Bretagne, s'ouvrant sur la baie de Lannion. face aux vents dominants de secteur Nord-Ouest, a quelque 20 km a l'Est de la baie de Morlaix, la « Lieue de Greve » est une vaste plage de 5 km2 tapissee de sable fin de 100 a 130 u, emergeant presque entie- rement aux grandes basses mers. Elle est profondement encaissee entre des collines elevees. large de 2 km au niveau des basses mers, elle atteint 4 km au niveau des hautes mers, d'oii son nom de « Lieue de Greve ». II y a 1,6 km en moyenne entre ces deux niveaux. Une butte enorme. Roc'al haz, haute de 99 m. s'avance legerement en compartimentant faiblement le fond de la baie, separant les localites de St-Efflam, a l'Ouest, de St-Michel-en- Greve. a l'Est. Six ruisseaux issus des coteaux eleves qui bordent le fond de la baie coulent en convergeant, a basse mer, vers l'entree de la baie entre les pointes de Plestin. a l'Ouest et de Beg-an-Fourn, a l'Est. Les trois ruissellements de l'anse onentale sont les plus importants. Par Ieur action de dessalure ils sont responsables de l'appauvnssement considerable des peuplements des niveaux moyens de cette anse, liee. par ailleurs. a l'expo- sition maximale aux houles des vents dominants du secteur Nord-Ouest. L'anse occi- dental ne recoit que des apports d'eau douce tres limites (une legere salure estivale apparait). Elle est relativement abntee des vents de Nord-Ouest par la pointe de Plestin. et partiellement protegee des vents importants de Nord-Est par les pointes de Beg-an- Fourn. Locquemeau, et la cote Est de la baie de Lannion. Aussi, le sediment y est-il legerement plus fin, moins permeable et moins oxygene ; c'est la zone la plus ricliement peuplee. Elle presente un petit massif de roches metamorphiques noires, dures et tour- mentees : le « Rocher Rouge ». qui, a 1 km du fond de l'anse. couvre 1 ha et s'eleve depuis les basses mers moyennes jusqu'au niveau des pleines mers de vives-eaux. II offre un abn permettant le developpement de sediments legerement envases en arriere. Un maigre herbier de Zostera nana s'etend au niveau des basses mers de mortes-eaux ; il ne modifie que tres peu le sediment, la biomasse des Zostera y etant faibie (250 a 600 g frais au m2, moyenne 350). Notons son leger deplacement vers l'Ouest, depuis 1968. Le phenomene de sursalure estivale qui s'y produit, du a l'evaporation du film d'eau qui n'a pas le temps de s'ecouler durant la basse mer, est insuffisant pour modifier les peuplements. a moins que Ton puisse lui attribuer la presence tres clairsemee de quelques .Wereis dicersicolor. Un aspect important particulier a cette greve est 1'accumulation croissante, d'annee en annee, d'algues non fixees, d'ectocarpales brunes libres au niveau des basses mers, mais surtout d'L ka laauca verte au debut du printemps et a 1'automne. au-dessus du niveau de la mi-maree. Ces algues couvrent a basse mer. d'un revetement parfois continu. de tres larges etendues de sable ; elles sont animees d'un balancement pendulaire au gre des marees et des vents. Ces « marees vertes » proviennent de l'eutropmsation croissante de l'impluvium des bassins de drainage des ruisseaux par les nitrates, les phosphates, la potasse. les pesticides et les lisiers d'ongine agncole. Elles jouent un role trophique impor- tant notamment par la liberation des spores et gametes et par le support qu'elles consti- tuent pour une riche faune d'Harpacticoides, de Foraminiferes, de Cilies et d'Amphipodes [Dexarmne spinosa). Elles sont partiellement consommees par les Talitres des hauts niveaux. La destruction des Amphipodes par le petrole explique peut-etre partiellement la particuliere abondance des « marees vertes » de 1978 et 1979. 454 EFFETS DE LA MAREE NOIRE SUR LES PEUPLEMENTS SED I MENTAI RES Sur les cartes suivantes sont reportes 4 etats successifs cie distribution pour chaque espece : 1964-1968, 1979, 1980 puis 1981. Sur les cartes A, on lit les zones de forte densite des principales especes. Nous comparons 4 periodes, les especes sont representees par leurs initiales : Ba. : BatkypoA2A.a pilot* a, i>aA6i it guMULamAonniayia. {notiej> AeApzctivrnznt V, S) We : NeAinn cJJVuxXalxx^ Un. : LiAothoz bnzviconniA Aa. : AAznicola maAina Ow : OwaYiia. ftuAifco Amii> It : Tzttina tznaiA T£ : Ttltina. fabula. Vo : Vonax vittat.uA Opk •. AcAocnida bAackiata En&iA e.yu>ii> oX Evu>iA iiliqua. MactAa. coAattina PhcuiuA ligamzn lutACL'UjX lutAOAJjX Les especes etudiees reagissent de maniere differente comme en temoigne la courbe de 1 'evolution des biomasses et le tableau Notons qu'il s'agit la de l'evolution moyenne de 10 stations. Des depl acements des zones de peuplements visibles sur les cartes n'ont pas pu etre pris en compte dans le tableau. Les cartes B a 0 presentent les courbes d'isovaleurs en cal/m des biomasses des principales especes de la macrofaune endogee. Les facteurs de conversion sont : 1 cal = 1 g de matiere organique fraiche = 0,2 g de matiere organi que seche . Les populations de quelques especes n'ont apparemment pas ete touchees , elles paraissent meme s'etendre : par exemple, les deux polychetes errantes Nzphty* hombzAgii (Cartes B) et SigaZion mathildaz (Carte C) ce dernier ayant tendance a coloniser maintenant 1 e haut de la pi are . 455 Le crabe Platyoni.cka6 latipei, (cartes D) dont les noyaux se decalent vers l'Ouest de la greve ou ils s'etendent en densite, et le bivalve Te.lli.na tenui.6 (carte E), qui apres une tres forte progression jusqu'en 1980, voit ses effectifs amorcer une diminution sur les noyaux Est et Ouest mais avec le maintien du noyau central f i xe . D'autres especes par contre ont beaucoup regresse apres la catastrophe . En opposition a T ell-ina tenui.6 , Telltna faabula (cartes F) qui occupait en 1968 tout le niveau de basse mer, continue a regresser dans les tres bas niveaux ou elle disparait actuel 1 ement a l'est. L'ophiure kcn.o cni.da bn.achi.ata (cartes G) est de moins en moins presente. On assiste a une diminution du nombre des noyaux plus etal es vers 1 ' Ouest . Les polychetes sedentaires An.zni.cola man.ina (cartes H) et Owenla ^ubl^ofimii, (carte I) ont regresse apres la maree noire, mais a partir de 1980 ; les deux especes se developpent a nouveau sur le cote Est de 1 ' Estran, bi en que pour Owen-la on constate une legere diminution en 1981. Le bivalve Vonax. vi.ttatu.6 preponderant en 1968, a probablement bien diminue avant 1978 puis a completement disparu apres 1' "AMOCOCADIZ" . Apres une timide reapparition en 1980, le noyau central prend de 1 'importance , s'etend vers le bas de la plage et le noyau de l'Est se consolide (carte J). En 1979 on trouve les Amphipodes Ba£hypoie.tado ne-c-6 afimata, Etzonz falava zt Capita men, tu.6 (cartes 0) ont beaucoup diminue en 1981. Notons que Capi.toma.6tu6 minima* est significatif d'un milieu pollue (LE M0AL Y.et QUI LLIEN-M0N0T , 1979 ) CONCLUSIONS 1- PERTES VE BJ0MASSE VIFFEREES La contamination des organismes n'a pas toujours ete immedi atement mortelle. Dans les sediments restes longtemps contamines, certaines especes qui avaient bien survecues au printemps de la maree noire ont vu leurs populations s'effondrer en 6 mois, voire un an plus tard. Le tableau suivant, portant sur 10 stations de sable fin du bas de la plage de St Efflam en baie de Lannion, milieu bien representati f par sa nature et par son degre de contamination, montre que le taux de survie ultime pour les especes qui avaient bien survecues est nettement plus faible que celui enregistre a la fin du printemps 1978 pris comme reference, soit apres la maree noire. ESPECES PRINTEMPS 1978 ETE 1978 1ER SEMESTRE 1979 2eme SEMESTRE 1980 1ER SEMESTRE 1981 TELLINA FABULA TELLINA TENUIS OWENIA FUSIFORMIS ARENICOLA MARINA NEPHTYS HOMBERGII 0,20 0,65 0,75 0,72 2 0,20 1,39 0,32 1,67 0,33 0,035 1 0,34 3,30 0,42 0,09 0,88 0,36 3,11 0,68 BI0MASSE T0TALE 1 0,77 1,18 1,02 0,95 Le facteur mul ti pi i cati f est proche des valeurs ulterieures les plus faibles des dates variees rencontrees dans ce tableau soit 0,09 ; 0,6 5 ; 0,3 2 ; 0,7 2 ; 0,33. 457 On peut estimer que les especes qui avaient bien survecues initialement accusent une mortalite addi ti onnel 1 e raisonnable proche de : (0,o9 + 0,65 + 0,32 + 0,72 + 0,33) /5 soit 0,4 La mortalite totale ultime etant 1,4 fois plus elevee que celle calculee pour la fin du printemps 1978. 2- PlfERSITE VES C0MP0RTEMENTS SPECIFIQUES Le comportement relatif des diverses especes est tres variable et assez imprevisi ble . En ce qui concerne les effets immediats pour une meme station certaines especes resistent parfai tement (T zllina te.nu.iA , Ouiznia fau.Aifaon.miA) d'autres sont presque i ntegral ement detruites {Vonax vittatuA , Can.dium zdulz, Ba.tkypon.zia, Eckinocan.diu.rn co fidatam , ?ha.nu.& Izgum&n, Eni>li> znAi.6, Eni>ii> Alliqua, Mact>ia coAall-ina, Lu.tia.tA.CL lu.tna.tiia.. Seules les trois premieres especes de cette liste sont timidement reparues aujourd'hui. A plus long terme, les comportemen ts sont aussi disparates : Te.llA.na te.nu.i6 non affecte a prospere jusqu'en 1980 et amorce une diminution de meme pour Ue.phtyi> homben.Qll. Tzllina &abula, 0we.nla fau.Aihon.mi6, kn.znic.ola. mafiina peu affectes initialement ont consi derabl ement regresses en 1979, quelquefois tres tardivement bien que certains signes d'un retabl i ssement certain apparaissent {An.znic.ola maxina) en 1981. Un.oth.oz zt Bath.ypon.zia qui avaient initialement disparus sont reapparus mais seulement dans la partie la plus occidentale et sans encore atteindre les densites initiales. Depuis 1980 on assiste a la reapparition de certaines especes qui avaient completement disparu apres la maree noire, Vonax vittatui se consolide alors que Nzninz cin.n.atu.lu.6 , EnAiA znbiA zt Can.dium zdalz ont du mal a se reimplanter. Des especes nouvelles pour la localite apparues en 1980 dont certaines seraient si gni f i cati ves d'une pollution residuelle, commencent a diminuer en 1981. On doit done considerer que les peuplements presentent encore en fin 1981 surtout dans la partie Est de la plage un desequilibre ecologique profond alors qu'a l'Ouest des signes d'une recouvrance avancee apparaissent. . co H JO 3- EVOLUTION GLOBALE II semble s'amorcer une derive qualitative generale des peuplements de sables fins bien calibres tres typiques a Vonax v-ittatu-i, , Ttlllna fcabula, Ec.klyioca.fidlu.rn co nda.tu.m et grands Solznldaz vers des peuplements plus banalises de sables fins plus eutrophises qu'envases a Kfie.yilc.ola matiina. La maree noire n'est sans doute pas seule en cause ("irarees vertes") mais elle a accelere cette evolution regressive des peuplements originels. Les parties hautes et surtout basses de 1 ' estran, pi us que les niveaux medians, sont les plus touchees. Ceci coTncide avec une plus forte accumulation des echouages des nappes dans le haut de la plage et dans la moitie Est, suivie d'une persistance des hydrocarbures dans l'epaisseur du sediment. Ceci est conforme a ce qui a ete observe sur tout le littoral en matiere d ' accumul ation d ' hydrocarbures sous l'influence des vents d'Ouest dominants. La forte regression constatee dans le basde l&pl age , confirmee par la nature essentiel 1 ement infratidale de la grande masse des cadavres retrouves dans les echouages, souligne un autre fait majeur en dehors des hauts de plage directement atteints par les nappes, 1'essentiel de la mortalite est a imputer au petrole disperse ou dissout au sein de la masse d'eau. Au niveau biomasse totale, on constate une chute importante en 1978 et 1979 par rapport a 1964-1968. Des af f ai bl i ssements des noyaux de peuplements de la partie Est par rapport a ceux de la partie Ouest, et du bas par rapport au haut, sont tres notables. Cette distribution se maintient en 1980 puis 1981 en s ' appauvri ssant encore. Notons que la progression des Atiznico la, se fait au depend d'especes de petite tail 1 e plus productives. II en resulte done une baisse generale de la fertilite de l'ensemble de la baie par rapport a 1968 de 1'ordre de pres des deux tiers. Le retabl i ssement encore tres incomplet des peuplements demanderaib des etudes ulterieures. 459 BIBLIOGRAPHIE Beauchamp P, 1914. — Les greves de Roscoff. Le Chevalier ed. Paris. Chasse CL L'Hardy-Halos M. T, Perrot Y, 1967. — Esquisse d'un bilan des pertes biologiques provoquees par le mazout du Torrey-Canyon sur le littoral du Tregor. Perm ar Bed, 6, 50, pp. 107-112. Chasse O. et colL 1967. — La maree noire sur la cote Nord du Finistere. Perm ar Bed, 6, 50, pp. 99- 106. Chasse CL, 1972. — Economie sedimentaire et biologiquc (production des estrans meubles des cotes de la Bretagne). These d'etat. Paris VL 1-293. Chasse CL 1978. — Bilan ecologique provisoire de rimpact de 1'echouage de V AMOCO CADIZ. Inventaire et evaluation de la mortalite des especes touchees. Public. CSEXO. Chasse CL 1978. — Un indice malacologique pour mesurer rimpact ecologique de la maree noire de VAMOCO CADIZ sur le littoral Haliothis PV : 9. n° 2, pp. 127-135. Chasse CI, 1978. — Esquisse d'un bilan ecologique provisoire de rimpact de la maree noire de VAMOCO CADIZ sur le littoral. Public. CNEXO serie actes de colloques. P, n° 6, pp. 1 15-135 Congres AMOCO CADIZ Brest 7 juin 1978. Chasse CL Morvan D, 1978. — Six mois apres la maree noire de I' AMOCO CADIZ, Bilan pro- visoire de I'impact ecologique. Pern ar'Bed. PV, n° 93, pp. 31 1-338. Chasse CL 1979. — Bilan ecologique de I" AMOCO CADIZ. Evaluer pour dissuader. J. rech. Oceano. n° 1, pp. 25-26. Toulmond A, 1964. — Les Amphipodes des facies sableux intertidaux de RoscofT. Apercus faunis- tiques et ecologiques. 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T) 1 — 1 -a in C CO in (1) th- e c l-H - o m 01 •o --< •n c. 4J •1^ 01 CO e u u o e JO 00 •-H •TH E 4- PH o o C3 a c — •-H l- in oi a ro V > pH -— ' 461 COUREE DE L EVOLUTION DES BIOMASSES Moyenne de 10 stations de bas de plage. Les nombres d' individus au m* et de la biomasse en cal/n pour tous les especes examines (44 especes differentes) sont disponibles sur demande de l'auteur. |000_ BIOMASSE (col/rn ) x +1 500- 100- Tt Tellina tenuis Tf Dv Donax vittatus Nh Am : Arenicola marina Ow Bath: Bathyporeia Uro Tellina fabula Nephtys hombergil Owenia fusiformis U rot hoe 50 -U •^/ 1963 JANV JANV. 80 JANV. 81 AMOCO (mart) CADIZ TEMPS (mois) A62 Morphologie di 1 'escran Penneab Lite A Kt& Ist Et,L'™ 1 en dare j&E& Aj Af^^' ** • \ {'r""l-m/ ^7T^7f\\\ TZ^?\_ *^H '\\ A**^ \\*K L / ' \J\ / \\ • "T "H — =i- —1—1/ V IBM ' — . •* ^ — — — '/^V (J 1 / ' \ \ 'a 10 b- //^j*~-l A B\\ ■- S \ \ -W- ^^--yLTy^W SI jS \\ if (V^f // rJ \ v=- — C y^J/ ifl "ar^^" f 1 KM B vr~~ —^i. — — w~ *" -~ EMPLACEMENT DES STATIONS DE PRELEVEHENTS . 1 RUISSEAtfX. 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