Experiments were carried out at 5 m depth in the Carteau Cove (Gulf of Fos, Mediterranean Sea). The experimental site was divided into two similar fields, as described elsewhere [6]. At this site, the temperature varies along the year from 5 °C in winter to 25 °C in summer.

Surface sediments (top 20 cm) were collected with an Orange Peel Grab at the experimental site. After sieving (1 mm), the sediment was mixed with Arabian Light Crude Oil (A.L. Brut) to reach a concentration of 10 g kg⁻¹ wet sediment. This increased the hydrocarbon content of the initial sediment by more than 75 times which represented a high level of petroleum contamination. The sediment-oil mixture was then poured into PVC cores (25 cm length, 11 cm diameter). Analysis of the bacterial finger-print in the sediment-oil mixture by molecular techniques (RISA) did not show any change of the major bacterial communities compared with the original sediment (P. Cuny, pers. comm.). Following the addition of luminophores (6 g) at the surface, the cores were kept frozen until their implantation into the experimental site.

Four frozen cores were inserted into each field. After 6, 12, 18 and 24 months of in situ incubation, two cores (one per field) were collected by divers. Sediment was extruded from each collected core and sliced as follows: 0–3 cm in 0.5 cm intervals, 3–10 cm in 1 cm intervals and 10 to bottom in 2 cm intervals. Samples were immediately frozen, freeze-dried and carefully homogenised.

The presence of luminophores was determined under UV light in five replicates of each sediment section. Aliphatic hydrocarbons were extracted from a known amount of lyophilised sediment using a Soxhlet apparatus and isolated by column chromatography. Individual alkanes were identified by GC/MS and quantified by GC using two internal standards: hexamethylbenzene (HMB) for C₁₄₋₂₃ alkanes and squalane (Sq) for C₂₄₋₃₄ alkanes [6].

Fig. 1 shows a depth profile of luminophores in a core incubated for six months. Since these inert particles were initially deposited on top of the cores, their detection into deeper sediment layers demonstrates the reworking of the sediment. This suggests that the cores were recolonised by benthic macro- and meiofauna during the first six months of the experiment. Indeed, it was previously shown that, in the absence of macrofauna, the luminophores are hardly buried into the sediment [5]. The thickness of the reworked layer varied between the four analysed cores certainly due to differences in macrofaunal composition and/or activity: the luminophores were detected down to 160- and 100-mm depth respectively in the two cores after six months, and down to 100- and 120-mm depth respectively after 24 months. The absence of luminophores below these reworked layers indicated that the deeper sediments were not reworked and thus were never in contact with oxygen throughout the 24-month experiment. These were accordingly considered as anoxic zones.

Fig. 2 shows examples of chromatograms of the saturated hydrocarbon fraction (F1) of the oil-sediment mixture at the beginning of the experiment and below the reworked layer in a core incubated in situ for 24 months. The hydrocarbons distribution showed a marked decrease in ‘short-chain’ alkanes (<n-C₂₅) after 24 months. The depletion of individual hydrocarbons was followed through the study of C₁₇, C₁₈ and C₃₀ n-alkanes and of isoprenoid hydrocarbons (pristane and phytane). In order to demonstrate the degradation of these hydrocarbons, the 17α,21βC₃₀ hopane (C₃₀H) was used as an inert internal reference ([8]; Table 1). The degradation of the individual alkanes seemed to depend on their chain length and on the presence of branching. A statistical comparison (Student's t-Test, n=5 or 6, α=0.05) of the averages of degradation observed for different anoxic layers showed that n-C₁₇, n-C₁₈ and Pr were significantly degraded after six months of incubation (0<p<0.016). Their extents of degradation by the end of the experiment (24 months incubation) were 57, 56.5 and 50%, respectively. n-C₃₀ appeared to be significantly degraded (51%) only after 24 months of incubation (p=0.0001), whereas no significant degradation of phytane could be established throughout the experiment (Table 1). Phytane degradation could be observed (up to 38%), however, in some individual anoxic sedimentary layers (results not shown).

This structure-dependent reactivity of alkanes under anaerobic conditions is in good agreement with the results of Giger et al. [4], obtained under laboratory conditions. In addition, a similar scale of reactivity was observed when kinetics of hydrocarbon degradation was studied in the whole reworked layer [6]. Although the pathways for the anaerobic biodegradation of isoprenoid hydrocarbons remain unknown, the lower reactivity of phytane compared to pristane may be due to the ante-iso structure of one end of the carbon skeleton of the phytane molecule. Alternatively, a production of phytane from the phytyl side-chain of chlorophyll-a present in the original sediment cannot be ruled out and might have compensated the degradation of this compound in some anoxic sediment layers [2].

The preliminary results of this in situ experiment show that below the bioturbated zone, linear and isoprenoid acyclic petroleum hydrocarbons can be biodegraded under anoxic conditions, after 6 or 24 months of incubation, depending on the compound. Although the individual alkanes showed varying reactivity, it is clear that such compounds cannot be considered as recalcitrant biomarkers in recent marine anoxic sediments.