Diapositiva 1

1
PAHs, biomarker responses, histopathology,
biochemical analysis of larvae and reproductive
parameters in hake and anchovy after the
Prestige oil spill
2
OBJECTIVE

-Selected species hake (M. merluccius) and
anchovy (E. encrasicolus)
- commercial importance in local fisheries -
representative of demersal and pelagic habitats
North
Peñas-Ajo (PA)
Ajo-Bidasoa (AB)
South
Miño-Fisterra (MF)
3
Selected tools

1.- Chemical analysis of PAHs concentration in
flesh
2.- Exposure and effect biomarkers
3.- Liver and gonad histopathology
4.- Biochemical and nucleic acid analysis,
growth rates and K index
5.- analysis of historical series of fecundity
parameters
4
Gas Chromatography/Mass Spectrometry, analysis of
24 PAHs
2004
anchovy
hake

Average 51.1 ?g/kg dry weight
Average 27.2 ?g/kg dry weight
5

Samples of 2005 year in process
6
BIOMARKERS AS EARLY WARNING SIGNALS
EXPOSURE BIOMARKERS -EROD activity -peroxisome
proliferation (Vvp and AOX activity)
EFFECT BIOMARKER -Lysosomal membrane stability
test
No previous data in selected especies
7
CYP1A1 induction 7-ethoxyresorufin O-deethylase
(EROD) activity
anchovy EROD activity in S9 fraction
3
2
pmol resorufin/ min/mg prot
1
0
Male North
Male South
Female North
Female South
BEQUALM intercalibration programme - EROD
activity in cod liver - Protein concentration in
cod liver and muscle
8
Palmitoyl CoA-oxidase Activity
2

Peroxisome proliferation
anchovy
1,5

AOX (mU/mg prot)
1
0,5
DAB
0
Volume Density of Peroxisomes
0,004
anchovy

0,003
x100
0,002
Peroxisomes in anchovy
0,001
0
Male North
Male South
Female North
Female South
9
Lysosomal membrane stability
ß-Gus (x 40)
Hake
Anchovy
acid phosphatase
10
anchovy
8
6
2004
Lp (min)
2005
4
2
0
Female South
Male South
Female North
Male North
10
Hake liver
Nematodes
Inflammation
Melanomacrophages
Nuclear polymorphism
11
2003
Hake liver
AB
PA
MF
Nematodes (macroscopic)
37.5 (n24)
10.7 (n28)
40.2 (n102)
Nematodes (microscopic)
4.3 (n23)
7.1 (n28)
13.2 (n68)
MMCs
47.8 (n23)
25 (n28)
48.5 (n68)
IRLA
4.3 (n23)
17.9 (n28)
14.7 (n68)
HNP
30.4 (n23)
50 (n28)
16.2 (n68)
Necrotic Foci
0 (n23)
0 (n28)
3 (n68)
MMCs melanomacrophage centers IRLA inflammatory
response with lymphocyte accumulations HNP
hepatocellular nuclear polymorphism
Different colour indicates plt0.05 according to
?2 test
12
Anchovy liver
Nematodes
Nuclear polymorphism
MMCs and coccideans
Inflammation
13
Anchovy liver
SOUTH
2004
2003
0 (n59)
46 (n50)
Nematodes (microscopic)
plt0.05
10.2 (n59)
22 (n50)
Coccideans
plt0.05
11.9 (n59)
52 (n50)
MMCs
plt0.05
1.7 (n59)
10 (n50)
IRLA
plt0.05
16 (n50)
HNP
13.5 (n59)
-
0 (n59)
8 (n50)
Necrotic Foci
plt0.05
MMCs melanomacrophage centers IRLA inflammatory
response with lymphocyte accumulations HNP
hepatocellular nuclear polymorphism
14
hake gonad histopathology (prevalence of atretic
oocytes)
Degeneration of immature oocytes
Alveolo cortical atretic oocytes
vitellogenic atretic oocytes
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anchovy gonad histopathology (prevalence of
atretic oocytes)
Atretic oocytes
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stereological methods (Weibel lattice M168), 30
individuals, 20 fields, x 20

Checking of gonad histological samples since
1996
17
- Somatic condition
- RNA/DNA (average)
R/D20005
No significant differences (ANOVA pgt0,05)
R/D20034,7
- Growth rate
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BEFORE THE SPILL (2000,2001)
n118, plt.0001
Proteins -46,628 47,024 ln(length1) R2
,326
Carbohydrates 54,574 - 19,323 ln(length1)
R2 ,17
Lipids 92,054 - 27,701 ln(length1) R2
,164
Biomolecule
AFTER THE SPILL (2003,2004)
1,6
1,8
2
2,2
2,4
2,6
2,8
Ln (legth mm1)
n47, pgt.05
Biomolecule
Proteins 23,44 12,754 ln(length1) R2
,056
Carbohydrates 16,715 - 3,598 ln(length1)
R2 ,04
Lipids 59,845 - 9,156 ln(length1) R2
,025
Ln (legth mm1)
19
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
20
Hake Relative Batch Fecundity (total number of
hydrated oocytes/female gutted weight)
-nothing extraordinary after the Prestige oil
Spill
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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 (gt40 in some cases) of parasitic
  infestations, melanomacrophages, inflammatory
  reactions, necrotic foci and hepatocellular
  nuclear polymorphism in hake.

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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.

• 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.

23
Thanks