The samples investigated were taken during the expedition on board the JOIDES Resolution and kept frozen until analysis. Because the sampling was done on board, no detailed sampling strategy was applied. Sample information is given in Table T1.
Sediment samples were lyophilized and finely ground in an agate mortar and subsequently serially extracted ultrasonically using three 40-mL aliquots of methanol (MeOH), three 40-mL aliquots of dichloromethane (DCM):MeOH (1:1, v/v), and three 40-mL aliquots of DCM, each for 5 min. For each sample, the extracts were combined and concentrated with a rotary evaporator at 35°C. Salts were removed by washing with double-distilled H2O in a separatory funnel and extraction of the lipids with three aliquots of DCM. For total lipid analysis, known aliquots of the extracts with an added amount of standard (2,3-dimethyl-5-1´,1´-d2-hexadecyl-thiophene) were methylated with diazomethane after drying with anhydrous Na2SO4. Before silylation with bis(trimethyl-silyl)trifluoro-acetamide in pyridine (1 hr at 60°C), very polar compounds were removed on a silica gel column eluted with ethyl acetate. Before fractionation, extracts were mixed with known amounts of thiophene and chroman standards and separated on a 4-cm column packed with activated Al2O3. Apolar fractions were collected by elution with four column volumes of hexane:DCM (9:1, v/v) and polar fractions by stripping the column with MeOH:DCM (1:1, v/v).
One sample, 175-1084A-12H-6, 140-143 cm, was further separated by thin-layer chromatography (TLC). The plate was developed with diisopropylether:acetic acid (96:4, v/v) to 75% of the height of the plate and redeveloped with petroleum ether 40-60:ether:acetic acid (89:10:1, v/v) (Skipski et al., 1965). Eight fractions were scraped off the TLC plate and ultrasonically extracted using three aliquots of hexane for TLC fractions 1 and 2 and three aliquots of ethyl acetate for other fractions. Fractions were cleaned on a small column filled with nonactivated alumina by elution with hexane:DCM (9:1, v/v) and subsequently methylated and silylated before analyses. The polar fraction of Sample 175-1084A-12H-6, 140-143 cm, was desulfurized with Raney nickel (Sinninghe Damsté et al., 1988) followed by hydrogenation.
Gas chromatography (GC) was performed on a Hewlett Packard 5890 series II chromatograph equipped with an on-column injector and fitted with a fused silica capillary column (25 m × 0.32 mm) coated with CP Sil 5 (film thickness = 0.12 µm). Helium was used as the carrier gas, and the oven was programmed from 70°C to 130°C at 20°C/min, followed by 4°C/min to 320°C (10 min hold time). Effluents were detected using a flame ionization detector (FID). GC-mass spectrometry (GC-MS) was performed using the same type of gas chromatograph with the same conditions described above. The chromatographic column was directly inserted into the electron impact ion source of a VG Autospec Ultima mass spectrometer operated with an ionization energy of 70 eV and scanned over a mass range of m/z 50-800 with a cycle time of 1.8 s. Compound identifications are based on comparison of relative GC retention times and mass spectra with those in the literature. Quantification of long-chain alkenones, alkyldiols, and biphytanediols was performed by integration of their peak areas and those of internal standards in FID chromatograms. Data were acquired and integrated using Atlas analytical software. Other biomarkers were quantified using characteristic fragment ion abundances in mass chromatograms. Resulting abundance values were converted to concentrations by compound-specific correction factors determined in samples in which these compounds could be identified in FID traces. The relative precision of the entire analytical procedure, based on duplicate sediment extractions, was between 10% and 15%. Concentrations are calculated in micrograms per gram of total organic carbon (TOC). TOC contents were measured after decalcification of samples on a Carlo Erba NA-1500 elemental analyzer using flash combustion at 1050°C. Standard deviations of duplicate measurements were better than 0.3%.