Stigmastane and hopanes as conserved biomarkers for estimating oil biodegradation in a former refinery plant-contaminated soil

In the last decade, a refinery plant located in Lido Adriano, East Ravenna (Italy) has been subject to mineral oil contamination. The mineral crude oil, extracted from the offshore in Adriatic sea, consisted of 78% aliphatics, cyclic alkanes and saturated polycyclic hydrocarbons, 9% aromatics, polycyclic aromatic hydrocarbons (PAHs) and alkylated derivatives, and 13% of tars/asphaltenes. Analysis of soil after 10 years of natural attenuation revealed a complete depletion of linear (n-C(9)-C(24)), light aromatics (C1-C3/benzenes) and PAHs (C2/naphthalene, C1/phenanthrene); besides a substantial degradation of isoprenoids prystane and phytane, branched and cyclic alkanes. The remaining contaminants which withstood to natural degradation was saturated polycyclic hydrocarbons (perhydro-PAH derivatives), unsaturated polycyclic hydrocarbons (tetrahydro, dihydro-PAH derivatives), terpanes, steranes and unidentified compounds. Such residues resulted in 80% reduction of its concentration after two months of laboratory treatment. Samples were extracted by organic solvents, separated by silica/alumina gel column chromatography and analyzed by gas chromatography-mass selective detector (GC-MSD). Identification and quantification of aliphatic, cyclic alkanes, typical PAHs, terpanes and steranes were carried out to chromatograms of M/Z=85, 83, individual M/Zs, M/Z=191 and 217, respectively. The present work shows that, among numerous biomarkers present in the source oil, stigmastane and two isomers of hopane showed invariable concentrations after laboratory experiments that mimic natural biodegradation in the field, so they can be used as conserved internal biomarkers. These are very useful tools to assess alterations in less stable classes of saturated compounds contained in petroleum. Marked degradation of perhydro, tetrahydro, dihydro-PAH derivatives in the laboratory treatment has been evidenced.