ECOTOXICITY OF THE WATER-SOLUBLE FRACTION OF THE PRESTIGE FUEL-OIL Laboratory for Ecotoxicology, Department of the Environment, INIA (Spanish National.

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Presentation transcript:

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

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

1.General Introduction Mechanisms of Deleterious Effects of the spilled Fuel Main Objective: toxicity of the water soluble fraction 2.Obtention of the water soluble fraction in the laboratory 3.General Toxicity Bioassays used and results obtained: Cell Toxicity Tests Alga Growth Inhibition Test Daphnia Acute Toxicity and Reproductive Toxicity Tests 4. Induction of EROD activity OVERVIEW OF THE PRESENTATION

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

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

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

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

RTG-2, cytotoxicity tests Results

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

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

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

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

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

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

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

FUTURE WORK Induction of EROD activity Antiestrogenic effect (reduction of VTG production in vitro) Navas et al., Aquatic Toxicology 2000 Anderson et al., Aquatic Toxicology 1996 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

Thank you very much