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ECOTOXICITY OF THE WATER-SOLUBLE FRACTION OF THE PRESTIGE FUEL-OIL

ECOTOXICITY OF THE WATER-SOLUBLE FRACTION OF THE PRESTIGE FUEL-OIL. Laboratory for Ecotoxicology, Department of the Environment, INIA (Spanish National Institute for Agricultural and Food Research and Technology).

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ECOTOXICITY OF THE WATER-SOLUBLE FRACTION OF THE PRESTIGE FUEL-OIL

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  1. ECOTOXICITY OF THE WATER-SOLUBLE FRACTION OF THE PRESTIGE FUEL-OIL Laboratory for Ecotoxicology, Department of the Environment, INIA (Spanish National Institute for Agricultural and Food Research and Technology) José María Navas, Mar Babín, Pilar García, Paloma Heranz, Carlos Fernández, José Vicente Tarazona

  2. The Prestige oil spill, a laboratory study about the toxicity of the water soluble fraction: • Lack of General Toxicity • Possible Mechanisms causing Deleterious Effects

  3. OVERVIEW OF THE PRESENTATION • General Introduction • Mechanisms of Deleterious Effects of the spilled Fuel • Main Objective: toxicity of the water soluble fraction • Obtention of the water soluble fraction in the laboratory • General Toxicity Bioassays used and results obtained: • Cell Toxicity Tests • Alga Growth Inhibition Test • Daphnia Acute Toxicity and Reproductive Toxicity Tests • Induction of EROD activity

  4. Evident deleterious effects of the spilled fuel: Avoiding aeration of the water column: Short term organism dead, caused by asphyxia, starvation, inanition or poisoning By direct contact or ingestion:

  5. WORK CARRIED OUT IN INIA: Main Objective: To determine the toxicity of the water soluble fraction of the fuel oil, since it can affect organisms at long term and without direct contact with the spilled fuel

  6. Obtention of the water soluble fraction Spilled fuel taken directly from the beach Original fuel Always the same quantity: 5g 100 ml Of the corresponding culture medium (cells, algae, Daphnia…) 15 minutes sonication in order to disperse the material 48 hours stirring in darkness Filtration (for cells until sterilization) Exposure

  7. CYTOTOXICITY TESTS RTG-2 Dilutions of the water soluble fraction 48 h exposure ß-galactosidase Neutral red uptake Protein content

  8. RTG-2, cytotoxicity tests Results

  9. ALGA GROWTH INHIBITION TEST (OECD Test guideline 201) Protocol 72 h Measurements at 24, 48 and 72 h Chlorella vulgaris Increase in absorbance (450nm) Increase in fluorescence (Ex:430 nm Em:680 nm) Quantity of chlorophyll Culture medium Stirring 8000 lux Cellular counting (Neubauer)

  10. ALGA GROWTH INHIBITION TEST (following OCDE, Test guideline 201) • Several assays using: • Water soluble fraction of the spilled fuel • Water soluble fraction of the original fuel No significant effects were observed Example: results of extraction carried out on fuel using algae medium

  11. Daphnia, ACUTE IMMOBILISATION TEST (following OECD test guideline 202) Protocol 3 replicates per fuel dilution 10 individuals per replicate Daphnia magna 48 hours treatment Juveniles < 24h Counting Results: no toxicity was observed

  12. Daphnia, REPRODUCTION TEST (following OECD test guideline 202 ) Protocol 10 replicates per fuel dilution 1 individual per replicate Daphnia magna Exposure 21 days (medium change: m, w, f) Juveniles < 24h Surviving adults Living juveniles Dead juveniles Counting (m, w, f)

  13. Daphnia, reproduction test: Daphnia exposed to the water soluble fraction of the spilled fuel No effect (dil 1/1) Daphnia fed with algae exposed to the spilled fuel oil No effect (dil 1/1) Daphnia exposed to the water soluble fraction of the original fuel No effect (dil 1/1) Daphnia fed with algae exposed to the original fuel No effect (dil 1/1)

  14. Results about general toxicity: • No cytotoxicity observed on RTG 2 (protein content, neutral red uptake, ß-galactosidase induction) • No effect on alga growth (Chlorella vulgaris) was observed • No acute toxicity or reproductive toxicity was observed in Daphnia magna

  15. Mechanisms of specific toxicity: Strong induction of EROD activity (CYP1A) in RTG-2 cells. EROD induction, associated with an increase of transcription of CYP1A and an activation of AhR has been characterized in RTL-W1 Casado et al., in this symposium

  16. FUTURE WORK Antiestrogenic effect (reduction of VTG production in vitro) Navas et al., Aquatic Toxicology 2000 Anderson et al., Aquatic Toxicology 1996 Induction of EROD activity Alteration of hormonal control of reproduction in vivo Navas et al., Aquatic Toxicology 2004 To study the effects of the water soluble fraction of the fuel oil on reproductive performance and hormonal control of reproduction in Medaka

  17. Thank you very much

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