integrated assessment of the effects of the prestige oil spill in galicia and the biscay gulf:...

Integrated assessment of the effects of the Prestige oil spill in

Galicia and the Biscay Gulf: toxicological, ecological, productive

and socioeconomical aspects (PRESTEPSE)

*Funded by the Spanish Ministry of Science and Technology, Basque Government and UPV/EHU

M.P. Cajaraville, I Díez, JM Gorostiaga, N

Etxebarria, I Astorkiza, JM Navas, JV Tarazona &

G Díez

Eman ta zabal zazu

Euskal HerrikoUnbertsitatea

Universidaddel País Vasco

PRESTEPSE

Integrated assessment of the impact of the “Prestige” oil spill in Galicia and in the Gulf of

Biscay: toxicological, ecological, productive and socio-economical aspects

PHYSICAL IMPACT(short-term)

ECOLOGICALIMPACT

(intermediate/long-term)

PRODUCTIONIMPACT


SOCIO-ECONOMICIMPACT


TOXICOLOGICAL IMPACT

(short/intermediate-term)

Conceptual approachBased on the combination of three parallel

approaches:

1. STANDARD APPROACH• Assessing the toxicity of the WAF of aged and

non-aged fuel using algae (Chlorella) and invertebrate (Daphnia) standard tests

2. THE MECHANISTIC APPROACH• Effects associated to non-polar narcosis• Effects associated to other mechanisms,

focusing on endocrine disruption

3. THE HIGHER TIER APPROACH• Direct exposure to the fuel mixed with sediment

in aquatic microcosms resembling realistic exposure conditions. Population dynamics, reproductive potential

INIA Ecotoxicological assessment

1. STANDARD APPROACH• Very low toxicity for the WAF of aged and non-

aged fuel on algae and invertebrates, including food chain exposure to invertebrates

2. THE MECHANISTIC APPROACH• No cytotoxic effects on RTG-2 cells• Specific induction of CYP1A1 expression and

EROD activity in RTL-W1 cells through activation of AhR

• The WAF did not interact with ER in a yeast gene reporter assay

3. THE HIGHER TIER APPROACH• No clear dose/responses observed from direct

exposure to the fuel in microcosms

Preliminary results


Preliminary results


Control

1/5121/256

1/1281/64

1/321/16 1/8 1/4 1/2 1

0

5

10

15

20

25

* ** * *

Dilución de la fracción soluble

ER

OD

(p

Mo

l/mg

pro

t/m

in)

Fracción soluble del chapapote en medio de cultivo

ß-actin

CYP1A

½ 1/8 1/32 1/128 1/512 Control

Metanol

EROD activity

Dilution of the soluble fraction

EROD activity induction, higher at

lower dilution except at lowest dilution

CYP1A1 expression induced even at lowest

dilution

Results are compatible with two main hypothesis:

• The aged fuel (“chapapote”) toxicity must be described by a complex toxicodynamics where non-polar narcosis does not play the main role

• The environmental effects are related to the variability of individual exposures which is observed even in controlled experimental conditions, suggesting direct contact with the fuel droplets as the only relevant exposure route

Additional experimental studies (fish, microcosm) are programmed

A new conceptual probabilistic risk assessment model is being developed in parallel

Explanations and future work


Aim of the study: To assess the impact of the Prestige oil spill on coastal and platform ecosystems of Galicia and the Bay of Biscay using sentinel molluscs (mussels) and commercially important fish species (hake, anchovy).

Approaches:

Molecular processes

Cellular processes

Tissues

Organism

Population

Community

Ecosystem

early-warning signalsExposu

re

•Biomarkers: exposure and effect biomarkers, genotoxicity, endocrine disruption

•Histopathology

•Proteomics and genomics

UPV-Cell Biol Impact assessment

Mussels 2003

30 Histopathology/Biometry/ Gonad development/Planimetry/ Vvbas

20 AOX

10 LP, B-GUS, ORO

FEBRUARY 04

APRIL 03/04/05

JULY 03/04/05

SEPTEMBER O3

OCTOBER 04/05

1 ESPOSENDE2 ONS3 CIES4 OIA5 AGUIÑO6 CALDEBARCOS7 CAMELLE8 SEGAÑO9 ESTACA10 LLANES11 SAN VICENTE12 SUANCES13 PEDREÑA14 LAREDO15 MUSKIZ16 ARRIGUNAGA17 GORLIZ18 BAKIO19 MUNDAKA20 MUTRIKU21 ORIO22 HONDARRIBIA

Mussels 2004

10 Histopathology/Biometry/ Gonad development/ Planimetry/ Vvbas

20 AOX/ALP

10 LP, β-GUS, ORO

50 Proteomics

7 Genomics

6 Genotoxicity

July

Mussel sampling

UPV-Cell Biol + Anal Chem Impact assessment-MUSSELS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 22

0

5

10

15

20

25

30

LP

(m

in)

JULY 04


0

5

10

15

20

25

30

LP

(m

in)

JULY 03

Izagirre, 2002

(Plentzia)

Lysosomes (LMS)

UPV-Cell Biol Impact assessment-MUSSELS

VvB

AS

(µ

m3/µ

m3)

APRIL 030.5

0.4

0.3

0.2

0.1

00.5

0.4

0.3

0.2

0.1

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 22

APRIL 04

VvB

AS

(µ

m3/µ

m3)


Méndez, 1993

(Abra)

UPV-Cell Biol Impact assessment-MUSSELS

Histopathology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

25000

20000

15000

10000

500

0

tPA

Hs

(ng

/g)

July 04

February 04

Abril 04

October 04

1 ESPOSENDE2 ONS3 CIES4 OIA5 AGUIÑO6 CALDEBARCOS7 CAMELLE8 SEGAÑO9 ESTACA10 LLANES11 SAN VICENTE

12 SUANCES13 PEDREÑA14 LAREDO15 MUSKIZ16 ARRIGUNAGA17 GORLIZ18 BAKIO19 MUNDAKA20 MUTRIKU21 ORIO22 HONDARRIBIA

Total PAH concentrations

in mussel tissues

UPV-Anal Chem

SVM02

ARM02

HOM02

LAM02

MUM02

DEM02

PEM02BAM02PLM02SUM02

MZM02ORM02

LLM02

GA102GA202GA302GA402

GA502GA602

SVM04

ARM04

HOM04

LAM04

MUM04

DEM04

PEM04

BAM04

PLM04

SUM04

MZM04ORM04

LLM04

GA104

GA204

GA304

GA404GA504GA604GA704

GA804GA904

SVM08

ARM08

HOM08LAM08

MUM08DEM08 PEM08

BAM08

PLM08

SUM08MZM08

ORM08

LLM08

GA108GA208GA308GA408GA508GA608GA708GA808GA908

SVM10

ARM10

HOM10

LAM10

MUM10

DEM10

PEM10

BAM10

PLM10

SUM10

MZM10

ORM10

LLM10

GA110GA210GA310GA410GA510

GA810

GA910JAI1LP

SJU1LPPN1LPKO1LP IGLP

ORLPLELP

ENLP

SJLP

KOLP

ITLP

LELP

MOLP

PNLP

ELLP

GELP

LAS02><PES02>

PES02<

PES04>

PES04<

R 2 = 0.7472

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

L-PAH

H-P

AH

Petrogénico

Pirolítico

Most of the samples show a petrogenic origin

UPV-Anal Chem PAH concentrations-MUSSELS

Spearman tPAHs (1) tPAHs (2)

LP 0,140 -0,257

P=0,897 P=0,042

VvL 0,480 -0,208

P=0,735 P=0,138

S/VL -0,194 0,327

P=0,167 P= 0,018

ORO 0,240 -0,240

P=0,029 P= 0,059

BAS 0,095 0,549

P=0,391 P<0,001

MET -0,142 -0,334

P= 0,199 P= 0,007

MLR/MET 0,330 0,433

P= 0,002 P<0,001

GI -0,188 0,160

P=0,087 P=0,211

AOX -0,336 -0,396

P= 0,001 P= 0,001

MN 0,188 -0,442

P= 0,603 P= 0,200

Correlation betweenbiomarkers and

PAH levelsin mussel tissues

(2004)(1) PAH tissue levels

versus “same-sampling” biomarkers

(2) PAH tissue levels versus “next-sampling” biomarkers

Significant Spearman´s correlation coefficient

UPV-Cell Biol + Anal Chem

-5

0

5

10

-5 0 5 RESULT1, X-expl: 12%,9%

4.000 4.750 5.500 6.250 7.000

OiaAguiño

CaldebarcosCamelleSegaño

Estaca

Llanes

San vicenteSuancesPedreña

Laredo

Muskiz

Arrigunaga

GorlizBakio

MundakaMutriku

Orio

Hondarribia

EsposendeOns

Cies

Oia

AguiñoCaldebarcos

Camelle

Segaño

EstacaLlanes

San vicenteSuances

Pedreña

LaredoMuskizArrigunaga

Gorliz

BakioMundaka

Mutriku

OrioHondarribia

EsposendeOns

Cies

OiaAguiño

Caldebarcos

Camelle

Segaño

Estaca

Llanes

San vicenteSuances

Pedreña

Laredo

Muskiz

Arrigunaga

Gorliz

Bakio

Mundaka

MutrikuOrioHondarribia

Esposende

OnsCies

Oia

AguiñoCaldebarcos CamelleSegaño

Estaca

Llanes

San vicente

Suances

Pedreña

Laredo

Muskiz

Arrigunaga

Gorliz

Bakio

Mundaka Mutriku

Orio

Hondarribia

PC2

PC3 Scores

PCA analysis: stations can be clustered according to the sampling campaing

UPV-Cell Biol + Anal Chem PCA analysis-MUSSELS

Selected species : hake (M. merluccius) and anchovy (E. encrasicolus)

Aim of the study: to assess the effects of the Prestige oil spill in commercial fish

species in the Bay of Biscay

- commercial importance in local fisheries- representative of demersal and pelagic habitats

2003

20032003

2003-04

2003-04

2003-04

Miño-Fisterra

Peñas-Ajo Ajo-Bidasoa

Adour-Garonne

UPV-Cell Biol + AZTI Impact assessment-FISH

Biomarkers:

Peroxisome proliferation

x100

Peroxisomes in anchovy

Male North

Male South

Female North

Female South

Palmitoyl CoA-oxidase Activity

0

0,5

1

1,5

2

AO

X (

mU

/mg

pro

t)*

Volume Density of Peroxisomes

0

0,001

0,002

0,003

0,004

VvP

(µ

m3 / µ

m3 )

2004

2005

**

DAB

UPV-Cell Biol Impact assessment-FISH

10.2% (n=59)22% (n=50)Coccideans

0% (n=59)8% (n=50)Necrotic Foci

13.5% (n=59)16% (n=50)HNP

1.7% (n=59)10% (n=50)IRLA

11.9% (n=59)52% (n=50)MMCs

0% (n=59)46% (n=50)Nematodes (microscopic)

20042003

MMCs: melanomacrophage centers

IRLA: inflammatory response with lymphocyte accumulations

HNP: hepatocellular nuclear polymorphism

Liver histopathology, anchovy:

p<0.05

p<0.05

-

p<0.05

p<0.05

p<0.05

SOUTH

UPV-Cell Biol Impact assessment-FISH

1,48Previtellogenic primary growth (n=68)

2,25Cortical alveoli (n= 18)

2,50Vitellogenic (n=10)

degenerated oocytes gonadal maturity (n=96)

average of inmaturehakeChecking of gonad

histological samples since 1996

In progress

Volume Density of atretic oocytes

0%

5%

10%

15%

20%

25%

30%

35%

hake total hake Vitello. hake Corticalalv.

anchovy total

gonadal development

DV

(%

)AZTI Impact assessment-FISH

Gonad histopathology, anchovy:

Atresia in 2004 samples:

Hake 7.2%

Anchovy 0.26%

- Somatic conditionK condition index 0-5 6-10 10-15 15-20 > 202000 0.012 0.010 0.015 0.019 0.0202003 0.016 0.014 0.018 0.020 0.023t student (p) 0.043 0.003 0.021 0.547 0.127

days

- RNA/DNA

R/D2000=5 R/D2003=4,7 No significant differences (ANOVA p>0,05)

- Growth rate

Age (days)

y = 0.5456x + 3.7933

R2 = 0.8653

y = 0.6517x + 2.8199R2 = 0.8731

0.0

5.0

10.0

15.0

20.0

25.0

0 5 10 15 20 25 30

SL

(m

m)

2000 2003ANCOVA, p> 0,05

AZTI Impact assessment-FISH

Biochemistry and condition of anchovy larvae

1,6 1,8 2 2,2 2,4 2,6 2,80

10

20

30

40

50

60

70

80

90

100

Proteins % = 23,44 + 12,754 * ln(length+1); R^2 = ,056

Carbohydrates % = 16,715 - 3,598 * ln(length+1); R^2 = ,04

Lipids % = 59,845 - 9,156 * ln(length+1); R^2 = ,025

Proteins % = -46,628 + 47,024 * ln(length+1); R^2 = ,326

Carbohydrates % = 54,574 - 19,323 * ln(length+1); R^2 = ,17

Lipids % = 92,054 - 27,701 * ln(length+1); R^2 = ,164

% B

iom

olec

ule

AFTER THE SPILL (2003,2004)

n=47, p>.05

% B

iom

olec

ule

1,6 1,8 2 2,2 2,4 2,6 2,80

10

20

30

40

50

60

70

80

90

100

BEFORE THE SPILL (2000,2001)

n=118, p<.0001

Ln (legth mm+1)

Ln (legth mm+1)


Biochemistry and condition of anchovy larvae

Batch Fecundity: (total number of eggs released in a batch)

Spawning fraction (%): percentage of spawning mature females

anchovy Batch fecundity & Spawning fraction

0

5.000

10.000

15.000

20.000

25.000

1987

1988

1989

1989

1990

1990

1991

1992

1994

1995

1996

1997

1998

2000

2001

2002

2003

2004

bat

ch f

ecu

nd

ity

0

5

10

15

20

25

30

35

Sp

awn

ing

fra

ctio

n

Batch fecundity Spawning fraction (%)

Prestige spill


Fecundity of anchovy

Primary objective

UPV-Economics Economic impact on fisheries

Evaluate the potential existence of a ““Prestige Prestige EffectEffect”” over the activity and the economic results of the Basque coastal (bajura) fleet.

•Evaluate the pertinence of structuring the fleet at different, but more homogeneous segments/strata.

•Evaluate how the oil spill affected at each segment/stratum of this fleet.

Secondary objectives

Reference information


A database, containing microeconomic information, was created from an inquiry of a panel of skippers, representing all the strata of the coastal fleet from two time periods: “a control year” 1999 (“average year” previous to the oil spill) and the year 2003

(after the spill).Exploratory Analysis

Allows to search for regularities within the sample data and identifies possible errors, extreme values, weird behavior in the data,

unexpected variability, etc.

Inference Statistics

Allows to choose between parametric and non-parametric procedures to compare averages

(medians) among the different clusters.

Methodology

Conclusions


•The main economic indicators used in this analysis support the splitting of the coastal fleet into three typologies/clusters. As the typologies are found relevant, their incorporation to subsequent analisis provides a higher precision to the results.

•When we take the whole coastal fleet as the object for analysis, we observe that during 2003, the vessels have worked less days, earned less, spent less, embarked less crew and their main activity (fishing) proportioned them less profits. This lead us to assume the existence of a “Negative Prestige EffectPrestige Effect”, upon the coastal fleet.

Conclusions


•Nevertheless this negative impact has been (temporarily) counteracted with the incomes perceived for the cleaning of the “galipot”, which allowed the Basque fishermen to attain similar income and profitability levels than 3 years before (1999).

•The analytical results allowed for the confirmation that the negative effects of the blackened/polluted sea were not supported by any fleet stratum/typology in special, but, rather, have been spread in the entire Basque coastal fleet.

2004 Work in progress

PHYTOBENTHOS• Functional groups • Life-forms: Perennial/Annual

Floristic and Faunistic changes?• Communities composition (species level)

• Species abundance

Physiognomic or structural changes?

• Species Diversity•Algal Cover

• Faunal Cover

ZOOBENTHOS• Taxonomical groups • Trophic strategies

Corallina elongata community

Chthamalus spp.community

Target communities as damage indicators

Mid-intertidalzone

Low-intertidalzone

Aim: assessment of the effects of the Prestige oil spill in the hard bottom intertidal communities of the Basque coast

UPV-Benthos Ecologic impact

Objective and approach

0

5

10

15

0

5

10

15

20

25

30

0

10

20

30

40

50

60

0

2

4

6

Life Forms

Diversity

AUTUMN ‘04

AUTUMN ‘03

SPRING ‘04

Nº

spp/

sam

ple

Fis

her’s

Perennial

Annual

50

60

70

80

90

Samplings0

10

20

30

40

Samplings

Cov

er (

%)

Cov

er (

%)

Algal Cover (%)

Samplings Samplings

Cov

er (

%)

Cov

er (

%)

0

20

40

60

80

100

120

Functional groups

ArticulatedcalcareousPolysiphonous


Phytobenthos: low intertidal

0

10

20

30

40

50

60

Nº

of s

pp.

0

1

2

3

4

5

6

7

8

9 Cover(%)

0

2

4

6

8

10

12

14

16

Cover(%)

0

1

2Cover (%)

Samplings0

2

4

6Cover (%)

Samplings

Cover (%)

0

2

4

6

8

10

12

Samplings

Trophic structure

DiversityFauna

Herbivores

Suspension-feeders

Detritivores

AUTUMN ‘03

SPRING ‘04

AUTUMN ‘04

Limpets

3

4

5

Nº

spp.

/ sa

mpl

e


Zoobenthos: low intertidal

50

52

54

56

58

60

10

12

14

16

18

20

11

12

13

14

Nº

of s

pp.

4,0

4,5

5,0

Nº

spp.

/ sa

mpl

e

Diversity

AUTUMN ‘03

SPRING ‘04

AUTUMN ‘0460

62

64

66

68

70

72

74

cover(%)

0

2

4

6

8

10

12

14cover(%)

Fauna

Cover (%) Cover (%)

Trophic structure

50

51

52

53

54

55

56

57

58

cover(%)

SamplingsSamplings

Herbivores

Suspension-feeders

Barnacles

Limpets


Zoobenthos: mid intertidal

PRESTEPSE

Integrated assessment of the impact of the “Prestige” oil spill in Galicia and in the Gulf of

Biscay: toxicological, ecological, productive and socio-economical aspects

PHYSICAL IMPACT(short-term)

ECOLOGICALIMPACT


PRODUCTIONIMPACT


SOCIO-ECONOMICIMPACT


TOXICOLOGICAL IMPACT

(short/intermediate-term)

Final ConclusionsFinal ConclusionsCAUSALITY? How to relate observed changes to

the Prestige oil??

1.- Comparison with data before the oil spill

2.- Comparison of historical series

3.- Prestige´s chemical fingerprint and correlations between biological effects and PAH

levels in biota

4.- Controlled laboratory experiments

Eskerrik asko!!!Eskerrik asko!!!

Thank you!!!Thank you!!!

Torrey Canyon

1967

Urquiola 1976

Amoco Cádiz 1978

Exxon Valdez 1989

Aegean Sea 1992

Erika 1999

Prestige 2002

NEXT???

What did we learn from the Prestige?

Need for a regular, integrated biomonitoring programme

Conclusions Biol CellConclusions Biol Cell• Mussels health was significantly deteriorated in 2003

after the Prestige oil spill, especially in Galicia

• No evidence of genotoxic effects of the oil spill in mussels

• Some biomarkers (LP, Vvbas) gave signs of recovery of mussels health starting in mid 2004

• Biomarker responses were significantly correlated with total PAH concentrations in mussels

• Decreasing prevalences of parasitosis and histopathological alterations (HNP) in fish from 2003 to

2004 also point to a possible recovery

• This possible recovery trend must be confirmed in future samplings

• Proteomics and genomics can be applied to perform alternative measurements of the same biomarkers and

also to develop new ones

integrated assessment of the effects of the prestige oil spill in galicia and the biscay gulf:...

Documents

standard approach

nonaged fuel

mechanistic approach

conceptual approach

lowest dilution slide

waf of aged

aged fuel chapapote

fuel droplets