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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