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 beachOriginal 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 reduptake

Protein content

48 h exposure

RTG-2, cytotoxicity tests

Results

ß-galactosidase

Dilution

Flu

ore

scen

ce (

ex 3

60 n

m;

em

465

nm

)

0

2000

4000

6000

8000

10000

12000

Neutral Red Uptake

Dilution

Ab

so

rba

nc

e (

55

0 n

m)

0.0

0.1

0.2

0.3

0.4

0.5

Protein

Dilution

Ab

so

rba

nc

e (

62

0 n

m)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Protocol

Culture mediumStirring

8000 lux

72 hMeasurements 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

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

t=0h t=24h t=48h t=72h

Flu

ore

sce

nc

e

Control

1/1

1/4

1/16

1/64

1/256

Example: results of extraction carried out on fuel using algae medium

0

250

500

750

1000

t=24 t=48 t=72

inc

rem

en

to n

r d

e c

élu

las

Control

1/1

1/4

1/16

1/64

1/256

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 dilution1 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)

No effect (dil 1/1)

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

Dilution of the water soluble fraction

Ctrl

1/10241/512

1/2561/128

1/641/32

1/16

ER

OD

act

ivit

y (p

Mo

l/m

g/m

in)

0

5

10

15

20

25 ED50 = 1/ 227NOED = 1/ 1024

FUTURE WORK

Induction of EROD activity

Antiestrogenic effect (reduction of VTG production in vitro)

Navas et al., Aquatic Toxicology 2000Anderson 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