Eman ta zabal zazu

Euskal HerrikoUnbertsitatea

Universidaddel País Vasco

PAHs, biomarker responses, PAHs, biomarker responses, histopathology, histopathology,

biochemical analysisbiochemical analysis of larvaeof larvae andand reproductive parameters in hake and reproductive parameters in hake and

anchovy anchovy after the Prestige oil spillafter the Prestige oil spill

Eman ta zabal zazu

Euskal HerrikoUnbertsitatea

Universidaddel País Vasco

G. Díez, E. Díaz, L. Arregi, P. Alvarez, U. Cotano, H. Murua, M. Santos, A. Alday, E. Saitua, A. Orbea,

R. Werding, U. Izagirre, I. Cancio, I. Marigómez, M. P. Cajaraville

G. Díez, E. Díaz, L. Arregi, P. Alvarez, U. Cotano, H. Murua, M. Santos, A. Alday, E. Saitua, A. Orbea,

R. Werding, U. Izagirre, I. Cancio, I. Marigómez, M. P. Cajaraville

OBJECTIVE:

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

 

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

Miño-Fisterra (MF)

Peñas-Ajo(PA)

Ajo-Bidasoa(AB)

North

South

2003-05

2003-05

 

Selected tools:

5.- analysis of historical series of fecundity parameters

4.- Biochemical and nucleic acid analysis, growth rates and K index

3.- Liver and gonad histopathology

1.- Chemical analysis of PAHs concentration in flesh

2.- Exposure and effect biomarkers

PPAHs average concentration (g/kg dry weight) in

flesh of anchovy (61 samples) and hake (107 samples)

1. Chemical analysis of PAH concentration in flesh

Gas Chromatography/Mass Spectrometry, analysis of 24 PAHs

anchovyaverage HAPs

0,1Chrysene0,5Fluorene0,5Anthracene1,41-methyl phenanthrene3,62-methyl naphthalene4,11-methyl naphthalene4,5Fluoranthene9,2Naphthalene11,0Phenanthrene16,0Pyrene

Average: 51.1 g/kg dry weight

hake

0,1Acenaphthylene0,8Anthracene0,82,6-dimethyl naphthalene1,7Fluoranthene2,1Phenanthrene2,2Pyrene3,0Biphenyl4,22-methyl naphthalene5,71-methyl naphthalene6,3Naphthalene

Average: 27.2 g/kg dry weight

average HAPs

2004

PCorrelation coefficient PAHs average concentration in

flesh hake and length (n:107)

1. Chemical analysis of PAH concentration in flesh

Samples of 2005 year in process

flesh hake

y = - 0,9188x + 65,762R2 = 0,0813

correlation coefficient : - 0,285135P= 0,0029

020406080

100120140160180

20 25 30 35 40 45 50 55 60length (cm)

PAH

s ug

/kg

dry

wei

ght

BIOMARKERS AS EARLY WARNING SIGNALS

2.- Exposure and effect biomarkers

*No previous data in selected especies

EXPOSURE BIOMARKERS

-EROD activity

-peroxisome proliferation: (Vvp and AOX activity)

EFFECT BIOMARKER

-Lysosomal membrane stability test

BEQUALM intercalibration programme- EROD activity in cod liver

- Protein concentration in cod liver and muscle

CYP1A1 induction: 7-ethoxyresorufin O-deethylase (EROD) activity

2.- Exposure and effect biomarkers

anchovyEROD activity in S9 fraction

0

1

2

3

Male North Male South Female North Female South

pmol

reso

rufin

/m

in/m

g pr

ot

2004

2005

Peroxisome proliferation

x100

2.- Exposure and effect biomarkers

Peroxisomes in anchovy

Male North

Male South

Female North

Female South

Palmitoyl CoA-oxidase Activity

0

0,5

1

1,5

2

AO

X (m

U/m

g pr

ot)

*Volume Density of Peroxisomes

0

0,001

0,002

0,003

0,004

VvP

(µm3 /

µm3 )

2004

2005

**

DAB

anchovy

anchovy

Lysosomal membrane stability2.- Exposure and effect biomarkers

Hake Anchovy

β-Gus (x 40)

0

2

4

6

8

10

Female South Male South Female North Male North

Lp (m

in)

2004

2005

acid phosphataseanchovy

Hake liver:Nematodes

Melanomacrophages

Inflammation

Nuclear polymorphism

3.- Liver and gonad histopathology

0%0%(n=23)(n=23)

0%0%(n=28)(n=28)

3%3%(n=68)(n=68)

Necrotic FociNecrotic Foci

30.4%30.4%

(n=23)(n=23)

50%50%(n=28)(n=28)

16.2%16.2%(n=68)(n=68)

HNPHNP

4.3%4.3%(n=23)(n=23)

17.9%17.9%(n=28)(n=28)

14.7%14.7%(n=68)(n=68)

IRLAIRLA

47.8%47.8%(n=23)(n=23)

25%25%(n=28)(n=28)

48.5%48.5%(n=68)(n=68)

MMCsMMCs

4.3%4.3%(n=23)(n=23)

7.1%7.1%(n=28)(n=28)

13.2%13.2%(n=68)(n=68)

Nematodes (microscopic)Nematodes (microscopic)

37.5%37.5%(n=24)(n=24)

10.7%10.7% (n=28)(n=28)

40.2% 40.2% (n=102)(n=102)

Nematodes (macroscopic)Nematodes (macroscopic)

ABABPA PA MFMF

MMCs: melanomacrophage centersIRLA: inflammatory response with lymphocyte accumulations HNP: hepatocellular nuclear polymorphism

3.- Liver and gonad histopathology

Hake liver: 20032003

Different colour indicates p<0.05 according to 2 test

Nematodes

MMCs and coccideans Inflammation

3.- Liver and gonad histopathology

Anchovy liver:Nuclear polymorphism

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

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

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

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

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

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

2004200420032003

MMCs: melanomacrophage centersIRLA: inflammatory response with lymphocyte accumulations HNP: hepatocellular nuclear polymorphism

3.- Liver and gonad histopathology

Anchovy liver:

p<0.05p<0.05

p<0.05p<0.05

--

p<0.05p<0.05

p<0.05p<0.05

p<0.05p<0.05

SOUTHSOUTH

3.- Liver and gonad histopathology

Degeneration of immature oocytes

Alveolo cortical atretic oocytes

hake gonad histopathology (prevalence of atretic oocytes)

vitellogenic atretic oocytes

3.- Liver and gonad histopathology

anchovy gonad histopathology (prevalence of atretic oocytes)

Atretic oocytes

3.- Liver and gonad histopathology

1,48Previtellogenic primary growth (n=68)2,25Cortical alveoli (n= 18)2,50Vitellogenic (n=10)

degenerated oocytes gonadal maturity (n=96)average of inmaturehake

stereological methods (Weibel lattice M168), 30 individuals, 20 fields, x 20

Checking of gonad histological

samples since 1996

Volume Density of atretic oocytes (2004)

0,27%4,8%

9,2%7,2%

0%

5%

10%

15%

20%

25%

30%

hake total hake Vitello. hake Corticalalv.

anchovy total

gonadal development

DV

(%)

- Somatic conditionK condition index <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 (average) R/D2000=5R/D2003=4,7

No significant differences (ANOVA p>0,05)

4.- Biochemical and nucleic acid analysis, growth rates and K index in anchovy larvae

- Growth rate

Age (days)

y = 0.5456x + 3.7933R2 = 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

% 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

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

10

2030

40

50

6070

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

4.- Biochemical and nucleic acid analysis, growth rates and K index in anchovy larvae

BEFORE THE SPILL (2000,2001)

n=118, p<.0001

% B

iom

olec

ule

AFTER THE SPILL (2003,2004)

n=47, p>.05Ln (legth mm+1)

Ln (legth mm+1)

Anchovy Batch Fecundity: (total number of eggs released in a batch)Spawning fraction (%): percentage of spawning mature females

-nothing extraordinary after the Prestige oil Spill

5.- Fecundity analysis

anchovy Batch fecundity & Spawning fraction

0

5.000

10.000

15.000

20.000

25.000

1987 1988 1989 1990 1991 1992 1994 1995 1996 1997 1998 2000 2001 2002 2003 2004

batc

h fe

cund

ity

0

5

10

15

20

25

30

35

Spaw

ning

frac

tion

Batch fecundity Spawning fraction (%)

Prestige spill

Hake Relative Batch Fecundity(total number of hydrated oocytes/female gutted weight)

5.- Fecundity analysis

-nothing extraordinary after the Prestige oil Spill

hake Relative Batch fecundity

139121123

165164

132

0255075

100125150175200225250

1996 1997 1998 1999 2000 2001 2002 2003 2004

Rela

tive

batc

h fe

cund

ity (e

ggs/

g)

Prestigespill

Summary-1-In 2004 the concentration of 24 PAHs in flesh was higher in anchovy than in hake. Negative correlation between PAHs concentration and length in hake.

-There were no differences in anchovy between years 2004 and 2005 in EROD activity and in LP. The volume density of Peroxisomas and AOX activity was decreased from 2004 to 2005. Could indicate recovery??

- Prevalence of histopathologies in anchovy decreased from 2003 to 2004, indicating a possible recovery of their health state. High prevalence (>40% in some cases) of parasitic infestations, melanomacrophages, inflammatory reactions, necrotic foci and hepatocellular nuclear polymorphism in hake.

-In anchovy larvae, no significant differences between 2000 and 2003 in growth rate and nucleic acid analysis have been found. The somatic index differed only in the younger larvae. Different pattern of biomolecular composition before and after spill

- There are no apparent differences in the annual fecundity for both species. Statistical comparative test among years and more biological parameters (size at maturity, duration of reproductive season…) are necessary to decipher whether there are differences in the reproductive capacity of populations.

Summary-2- The stereological preliminary results in 2004 show a 7.2% of atretic oocytes in hake and 0,26% in anchovy. It is necessary to check the historical series in order to test if there are significant differences between years.