1. TRIALS OF IN SITU BIOREMEDIATION ON THE BEACH OF SORRIZO (SPAIN) P. Fernández-Álvarez, J.M. Garrido and J.M. Lema Department of Chemical Engineering Universidade de Santiago de Compostela (Spain) VERTIMAR 2005 Vigo, July 14 th 2005 J. Vila and M. Grifoll Department of Microbiology Universitat de Barcelona (Spain)

2. 1. OBJECTIVE 2. INTRODUCTION 3. MATERIALS AND METHODS 4. RESULTS AND DISCUSSION 5. CONCLUSIONS Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain)

3. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 1. OBJECTIVE  Who: Department of Chemical Engineering of the University of Santiago de Compostela (Spain). Department of Microbiology of the University of Barcelona (Spain).  When: From April 2003 on.  Where: Beach of Sorrizo (A Coruña, NW Spain). The assessment of the efficiency of several bioremediation strategies on a beach seriously affected by the oil spill of the Prestige.

4. Field experiments with several bioremediation agents following the Prestige accident 1. INTRODUCTION Shoreline clean-up Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 2. INTRODUCTION SANDY BEACHES Manual recovery. Specialist beach cleaning machinery ROCKY SHORES Manual recovery. Low/high pressure flushing. These techniques are frequently expensive and aggresive to the fauna and flora.

5. Field experiments with several bioremediation agents following the Prestige accident 1. INTRODUCTION Definition: Act of adding materials to contaminated environments to cause an acceleration of the natural biodegradation processes. Approaches: Bioaugmentation: oil-degrading bacteria are added to supplement the existing microbial population. Biostimulation: the growth of indigenous oil degraders is stimulated by the addition of nutrients. Requirements: Establish and maintain conditions that favour enhanced oil biodegradation rates. A large percentage of oil components are readily biodegradable. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 2. INTRODUCTION Bioremediation

6. Composition of the fuel of the Prestige Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 2. INTRODUCTION

7. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS The experimental site: the beach of Sorrizo SPAIN GALICIA Arteixo Galicia Beach of Sorrizo Beach of Sorrizo (Arteixo, A Coruña, NW Spain) 43º18’N, 8º34’ W Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

8. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS The experimental site: the beach of Sorrizo Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

9. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Nitrophoska Supreme (NPK): commercial nutrients formulation (Compo, Germany; 20% N, 5% P 2 0 5 ). B350: commercial bioaugmentation product (Bio-systems, USA). L1800: commercial bioaugmentation product with biodegradable surfactants (Bio-systems Co, USA). Autochthonous culture (UB): obtained by enrichment of fuel- degrading populations from a beach affected by the oil spill of the Prestige. Developed by the Department of Microbiology of the University of Barcelona (Spain). Assessed products The applications took place between April and October 2003, after a preliminary clean-up of the beach. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

10. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Assessed products ZoneTreatment 1Nitrophoska 2Blank 3B350 4B350 + L1800 5Autochtonous culture 6Blank The beach was divided into six sections that were treated with the different products. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

11. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Experiments with granite tiles Experiments with granite tiles were performed to minimize the heterogeneity of the environmental samples. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

12. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Experiments with granite tiles The bioremediation agents were applied with the same frequency and doses used on the beach Supra-littoral tiles NPK Blank B350 L1800 UB Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS

13. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Experiments with granite tiles Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS B350 UB B350 UB Intertidal tiles

14. Field experiments with several bioremediation agents following the Prestige accident 3. MATERIALS AND METHODS Monitoring of the beach Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 3. MATERIALS AND METHODS Seawater and interstitial water: Nutrients Hydrocarbon-degrading populations PAH Sand: Residual oil PAH Percentages of degradation Rocks: Percentages of degradation Granite tiles: Percentages of degradation Oxygen monitoring

15. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Level of nutrients Applications Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

16. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Level of nutrients Applications Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

17. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Hydrocarbon-degrading populations Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION 10-40%

18. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Hydrocarbon-degrading populations Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION 5-20%

19. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Polycyclic aromatic hydrocarbons Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION Date Concentration of PAH (  g/L) SeawaterZone 1Zone 2Zone 3Zone 4Zone 5 14/04/030.107-0.0410.0557.149- 30/04/030.1610.0490.1272.0543.4260.15 28/05/030.0480.2870.040.1050.7370.33 18/06/030.1620.2140.3280.2621.350.323 21/07/030.0850.0750.1030.2020.450.072 25/08/030000.040.0150 24/09/030000.070.0150 24/10/030000.0310.0550 10/12/03000000 12/02/04000000

20. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Concentration of residual oil in sand Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

21. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Concentration of PAH in sand Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

22. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Degradation in sand Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

23. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Degradation in rocks Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

24. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION Degradation of aromatics in tiles

25. Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION Oxygen monitoring in tiles

26. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Persistent pollution in rocks 18 months after the oil spill the rocks were still coated with fuel oil Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

27. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Biodiesel Sunflower oil methyl esters produced through the transesterification of sunflower oil. It is easily incorporated into oil and reduces its apparent viscosity, increasing the surface area of the oil and accelerating microbial degradation. It may also induce increased microbial activity as a source of bioavailable carbon. How it acts It is readily biodegraded in the environment. No long-term toxicity or damage has been documented. It is cheap. Its spraying is a low-cost operation. Advantages Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

28. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Biodiesel Biodiesel-treated face The experiments with biodiesel started in March 2004 on a polluted rock from the beach Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION

29. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Biodiesel 0 days 150 days 80 days Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION 470 days

30. Field experiments with several bioremediation agents following the Prestige accident 4. RESULTS AND DISCUSSION Biodiesel Date Saturates (%) Aromatics (%)Polars (%) Before the applicationMar-049.734.356.0 Non-treated faceSep-0411.132.656.3 Biodiesel-treated faceSep-049.324.166.6 It did not only acted as a cleaning agent but also as a BIOREMEDIATION agent Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 4. RESULTS AND DISCUSSION Percentages of the fractions of the oil from the rock

31. Field experiments with several bioremediation agents following the Prestige accident 5. CONCLUSIONS Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) 5. CONCLUSIONS  The environmental parameters were appropriate for biodegradation processes (nutrients, microorganisms).  None of the products enhanced the biodegradation rates in sand and rocks with regard to natural attenuation.  It seemed that photooxidation significantly influenced the degradation processes in the rocky substrate.  The application of the oleophilic agent biodiesel improved the appearance of polluted rocks and seemed to accelerate the biodegradation processes.

32. Field experiments with several bioremediation agents following the Prestige accident 5. CONCLUSIONS Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) Trials of in situ bioremediation on the beach of Sorrizo (Spain) ACKNOWLEGMENTS “FUNDACIÓN ARAO” (ARAO FOUNDATION) FOR THE FUNDS PROVIDED TO DO THIS RESEARCH AUTHORS WISH TO THANK:

33. Field experiments with several bioremediation agents following the Prestige accident 5. CONCLUSIONS Vigo, July 14th 2005 VERTIMAR 2005 Paula Fernández Álvarez Universidade de Santiago de Compostela (Spain) TRIALS OF IN SITU BIOREMEDIATION ON THE BEACH OF SORRIZO (SPAIN) THANKS FOR YOUR ATTENTION AND… ANY QUESTIONS?