1. Biodegradation of the Prestige fuel oil under simulated conditions S. DÍEZ a, J. SABATÉ b, M. VIÑAS b, J. M. BAYONA a, A. M. SOLANAS b and J. ALBAIGÉS a a Instituto Investigaciones Químicas y Ambientales de Barcelona-CSIC. B Universidad de Barcelona. Departamento de Microbiología

2. The fate of spilled oil in the marine environment Fuente: Albaigés 1989

3. OBJECTIVES To evaluate the biodegradation extent of Prestige fuel oil in vitro experiments times by two consortia. –Aliphatics, aromatics and molecular markers. Effect of biodegradation on: –source and weathering indices To assess weathering effects on biodegradation. To evaluate the effect of oleophilic nutrients.

4. METHODOLOGY

5. Microbial Consortia Enrichment AMTD Inocula: crude oil contaminated sand + 5-PAHs ( 3-4 aromatic ring) Inocula: diesel contaminated Soil + diesel oil Mineral medium 5 years

6. Biodegradation Experiments S200 : microemulsion urea + oleic acid + phosphate ester +2-butoxy-1-ethanol AM 50 mg AM 50 mg TD 50 mg original fuel weathered fuelfuel + S200 C h e m i c a l a n a l y s i s 20 days 40 days AM 50 mg TD 50 mg TD 50 mg original fuel 50 mg C h e m i c a l a n a l y s i s

7. Chemical Analysis Incubated fuel pH < 2.5 LLE (CH 2 Cl 2 ) organic extract Fractionation SPE (SiO 2 -C 3 CN) Aliphatics GC-FID GC-MS Aromatics GC-MS surrogates

8. HYDROCARBON BIODEGRADATION

9. Biodegradation Aliphatics

10. Time (min) 0 5 10 15 20 25 30 35 40 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 0 5 10 15 20 25 30 35 40 N N1 N2 N3 P P1 P2 P3 Biodegradation Aromatics original fuel biodegraded fuel 20 days

11. Weathering Effect

12. Strain Efficiency

13. Oleophilic Nutrient Effect

14. (II) (I) m/z 106 m/z 92 (I) (II) m/z 106 alkyltoluenes m/z 92 alkylbenzenes Alkylbenzenes ORIGINAL FUEL BIODEGRADED (TD) 1-methyl-3-phytanylbenzene phytanylbenzene

15. C 2 & C 3 -Phenanthrenes ORIGINAL BIODEGRADED ORIGINAL BIODEGRADED m/z 206 m/z 220 C 2 -Phenanthrenes (P2) C 3 -Phenanthrenes (P3)

16. C 2 & C 3 - Dibenzothiophenes ORIGINAL BIODEGRADED ORIGINAL BIODEGRADED m/z 212 m/z 226 C 2 - Dibenzothiophenes (D2) C 3 - Dibenzothiophenes (D3)

17. Source Indices Hopanes Steranes

18. BIODEGRADATION MOLECULAR MARKERS Acyclic isoprenoids Diasteranes C 27 -steranes  -Steranes Homohopanes Monoaroamtic steranes Triaromatic steranes biodegradation

19. Effect of Biodegradation on Source Diagnostic Ratios IndexOriginal oil Biodegraded oil AM 20 days AM+S200 40 days Pr/Ph0.980.97--- %23  636270 %24TT11 7 %27Ts222122 %29  43 42 %27d363929 %29  S 4549 %29  474957 %27  37 7 hopanes steranes isoprenoides

20. Source Indices 206+220 D2/P2 D3/P3

21. Effect of Biodegradation on Source Diagnostic Ratios IndexOriginal oil Biodegraded oil AM 20 days AM+S200 40 days D2/P234 25 D3/P3403733

22. Weathering Indices 212+226 D2 D3 D2/C2 D3/C3 256+270 C2 C3

23. Effect of Biodegradation on Weathering Diagnostic Ratios IndexOriginal oil Biodegraded oil AM 20 days AM+S200 40 days D2/C2675912 D3/C370 51

24. N-alkanes, alkylbenzenes low MW parent PAHs and mono and di-alkylated derivatives are degraded by both consortia in 20-40 days incubation. Degradation rates of PAHs decrease according with the alkyl substitution and number aromatic rings. Weathering slow down biodegradation. Oleophilic nutrient enhance the biodegradation of the more refractory PAHs and some molecular markers. Source indicators can be modified when oleophilic nutrient are added to the incubation medium. CONCLUSIONS

25. ACKNOWLEDGEMENTS Financial support was obtained from the MCYT (VEM2003-20068-CO5-01). Dr. S. Diez acknowledges a Ramon & Cajal contract.