Part-financed by the European Union (European Regional Development Fund)
Health Status of Cod (Gadus morhua) at Dumpsites for Chemical Warfare Agents
in the Baltic Sea T. Lang1, N. Fricke1, M. Faber1, M. Brenner2, U. Bickmeyer2, K. Broeg2, K. Lehtonen3, R. Turja3,
J. Baršiene4
1Thünen Institute of Fisheries Ecology (DE) 2Alfred-Wegener-Institute of Polar and Marine Research (DE) 3Finnish Environment Institute, Marine Research Centre (FI)
4Nature Research Centre, Institute of Ecology (LT)
Part-financed by the European Union (European Regional Development Fund)
Aim of the study:
to assess the health status of cod at known and suspected chemical warfare agents (CWA) dumpsites and in reference areas of the Baltic Sea
by applying a batterie of physiological, pathological, cellular and subcellular health indicators
Cod health status at Baltic Sea CWA dumpsites
Part-financed by the European Union (European Regional Development Fund)
Some words about cod (G. morhua) in the Baltic Sea: Two cod stocks in the Baltic Sea:
– western stock – eastern stock
Borderline between stocks: ~ Bornholm
Eastern stock spawn in summer in the Bornholm Basin and in the Gotland basin (mainly because of favourable salinity)
Western stock spawns in early spring in the Arkona Sea and further west
Fishery: approx 70,000 t/year
Cod in the Baltic Sea
Part-financed by the European Union (European Regional Development Fund)
Cod sampling areas
Bornholm Basin CWA
Gdansk Deep CWA
Gotland Basin CWA
Little Belt CWA
western stock eastern stock
Main dumping site
Reference site
Part-financed by the European Union (European Regional Development Fund)
Sampling: Dec. 2011: Bornholm Basin/Gdansk Deep and reference areas
May 2012: Gotland Deep and reference areas
Dec. 2012: Gotland Deep, Bornholm Basin and reference areas
Sept. 2013: Bornholm Basin and reference areas
Fishing: pelagic and bottom trawling
Platform: FRV Walther Herwig III (DE)
Sampling of cod
Part-financed by the European Union (European Regional Development Fund)
Vertical distribution of cod: impact of hydrography (T, S, 02)
Fishing depth Dec. 2011
Water depth: 95 m Fishing: 40-60 m
Water depth: 105 m Fishing: 60-80 m Water depth: 60 m
Fishing: bottom
Bornholm Basin (B13) Gdansk Deep (B15) Gdansk (B09, ref.)
Part-financed by the European Union (European Regional Development Fund)
Health indicators:
Cod health indicators measured
Fitness • Condition factor (CF) • Liver somatic index (LSI) • Spleen somatic index (SSI) • Gonadosomatic index (GSI)
Disease & pathology • Gross diseases and parasites • Liver histopathology • Head kidney pathology • Lysosomal membrane
stability • Differential blood cell
counts
Cyto-/genotoxicity • Morphological alterations in
red blood cells
Oxidative stress • Antioxidant defence
enzymes (e.g., catalase, glutathione reductase)
Neurotoxicity • Acetylcholinesterase (AChE)
inhibition
Part-financed by the European Union (European Regional Development Fund)
Cod fitness indices
0.100.120.140.160.180.20
B01 B11 B10 B13 B09 B15 B14
mea
n SS
I +/-
95
% C
.I.
Sampling area
Spleen Somatic Index gutted weight
Dec. 2011
Dec. 2012
* *
3.00
5.00
7.00
9.00
B01 B11 B10 B13 B09 B15 B14mea
n LS
I +/-
95
% C
.I.
Sampling area
Liver Somatic Index gutted weight
Dec. 2011
Dec. 2012
* * * * *
0.002.004.006.008.00
10.00
B01 B11 B10 B13 B09 B15 B14
mea
n G
SI +
/- 9
5 %
C.I.
Sampling area
Gonadosomatic Index gutted weight
Dec. 2011
Dec. 2012
* * *
0.60
0.80
1.00
1.20
B01 B11 B10 B13 B09 B15 B14mea
n CF
+/-
95
% C
.I.
Sampling area
Condition Factor gutted weight
Dec. 2011
Dec. 2012*
* * *
Results: some significant differences between sampling years; CF higher in western cod (B01, B11, B10), GSI higher in eastern cod (B13, B09); no significant differences between CWA areas (B13, B14, B15) and reference area (B09)
Part-financed by the European Union (European Regional Development Fund)
Externally visible diseases of cod
Disease/parasite Aetiology
Skin ulcerations Bacterial
Skeletal deformities Multifactorial
Pseudobranchial swelling
Parasitic (Amoeba-like)
Fin rot/erosion Bacterial
Epidermal hyperplasia/ papilloma
Viral, skin
Cryptocotyle lingua Digenean trematode, skin
Loma branchialis (= morhua)
Microspora, gills
Lernaeocera branchialis Copepode, gill chamber
Examinations based on standardised methodologies for fish disease monitoring (ICES, BEAST project)
Part-financed by the European Union (European Regional Development Fund)
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
B01 B11 B10 B13 B09 B15 B14
mFD
I +/-
95%
C.I.
Sampling area
Fish Disease Index (FDI)
Dec. 2011
Dec. 2012
*
Externally visible diseases of cod: Fish Disease Index
Results: some significant differences in the FDI between sampling years and between areas (only 2011); highest value in fish from the Bornholm CWA dumpsite (B13) in 2011 but not in 2012; overall, no clear indication of effects of CWA on the status of external diseases
Fish Disease Index (FDI) combines disease data into one numeric figure per fish and considers: • Presence/absence of all diseases
• Severity grades (1-3)
• Disease weights (based on known or suspected effects on affected hosts)
• Adjustment factors for effects of size, sex and season (derived from empirical long-term data)
FDI is an established ICES/OSPAR analysis and assessment tool and was adapted to Baltic cod
The maximum FDI value is 100; high FDI values indicate poor health
Part-financed by the European Union (European Regional Development Fund)
Liver diseases/histopathology in cod
Myxozoa Nematoda
Infiltration/ Regeneration
Macrovesicular Steatosis
Results: Non-specific liver lesions with no clear link to pollution are dominating, neoplastic lesions (tumours and pre-stages) are very rare
Examples of non-specific liver lesions
Part-financed by the European Union (European Regional Development Fund)
Liver diseases/histopathology in cod: Liver Disease Index
Results: some differences in LDI between areas and sampling years; no significant differences between CWA areas (B13, B14, B15) and reference area (B09)
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
B01 B11 B10 B13 B09 B15 B14
LDI +
/- 9
5 %
C.I.
Sampling area
Liver Disease Index
Dec. 2011
Dec. 2012
*
LDI combines data on the presence and severity of all liver lesion types into one numeric figure per fish
Part-financed by the European Union (European Regional Development Fund)
Lysosomal stability in head kidney of cod
Results LMS-Test: Differences of the reference site (B09) are significant for both peak 1 and 2 (Kruskal-Wallis-Test, p<0.05, n=60)
Tim
e of
des
tabi
lisat
ion
[min
]
peak 1 peak 2
Lysosomal Membrane Stability (LMS) – Head Kidney Dec. 2011
Part-financed by the European Union (European Regional Development Fund)
Head kidney pathology in cod
Results Dumping sites: Tissue less dense, haematopoietic cell (HC) cluster small with less number of cells and higher number of histopathological alterations Results reference area: Dense tissue, cluster of HC surrounding blood vessels, less alterations
Histopathological Alterations
1 Vacuolisation
Reference site
1
2
3
4
3
4 1
Exposed sites
2 Karyomegaly 3 Hypertrophy 4 Apoptotic bodies
Part-financed by the European Union (European Regional Development Fund)
Head kidney pathology of cod
Results: Significantly more alterations at dumping site B13 compared to reference site B09 (Kruskal-Wallis-ANOVA-Test, p<0,05, n=30).
*
Hist
opat
holo
gica
l alte
ratio
ns [n
]
Histopathological Alterations
Dec. 2011
Part-financed by the European Union (European Regional Development Fund)
Geno- and cytotoxicity in cod red blood cells
Genotoxicity
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
B01 B11 B10 B13 B09 B15 B14
Sampling area
MN
+NB
+BN
b/10
00 e
ryth
rocy
tes Dec. 2011
Dec. 2012
Results: significant differences in geno-/cytotoxicity levels between areas and sampling years; indication of elevated levels in CWA areas (B13, B14, B15) compared to the reference area (B09)
Cytotoxicity
***
00.2
0.40.60.8
11.21.4
1.61.8
B01 B11 B10 B13 B09 B15 B14
Sampling area
FA+B
N+8
/100
0 er
ythr
ocyt
es Dec. 2011Dec. 2012
Fragmented apoptosis, bi-nucleated cells, 8-shaped nuclei Micronuclei, nuclear buds, nucleoplasmic bridges
Micronucleus Apoptosis
Part-financed by the European Union (European Regional Development Fund)
Enzymatic biomarkers in cod: oxidative stress, neurotoxicity
Results: Some significant differences in GR activity between dumpsite (B13, B13 dump) and reference area (B09); no differences in GST, CAT and AChE (data from Dec. 2011)
0.00100.00200.00300.00400.00500.00
B13 B13 dump B09 B14
GST
(nm
ol m
in−
1 mg
prot
− 1 )
+
/- 9
5% C
.I.
Sampling area
Glutathione S-transferase
0.00
50.00
100.00
150.00
200.00
B13 B13 dump B09 B14CAT
(µm
ol m
in−
1 mg
prot
− 1 )
+/
- 95%
C.I.
Sampling area
Catalase
0.005.00
10.0015.0020.0025.0030.00
B13 B13 dump B09 B14GR
(µm
ol m
in−
1 mg
prot
− 1 )
+/
- 95%
C.I.
Sampling area
Glutathione reductase
* *
0.0020.0040.0060.0080.00
100.00
B13 B13 dump B09 B14AChE
(µm
ol m
in−
1 mg
prot
− 1 )
+/
- 95%
C.I.
Sampling area
Acetylcholinesterase
Part-financed by the European Union (European Regional Development Fund)
Fish Health Index A integrates 5 health parameters with 3 grades each:
a) Condition factor b) Liver somatic index c) External diseases d) Liver histopathology e) Lymphocyte ratio
Cod health: Fish Health Index
1.50
2.00
2.50
3.00
B01 B11 B10 B13 B09 B15 B14
mea
n FH
I +/-
95
% C
.I.
Sampling area
Fish Health Index A (5 parameters)
Dec. 2011
Dec. 2012
FHI = (a(1,2,3) +b(1,2,3) +c(1,2,3) +d(1,2,3) +e(1,2,3))*5-1
FHI range: 1 (good) – 3 (bad)
* * * *
Results: some significant differences in FHI-A values between cod from the reference area (B09) and other areas. However, no indication that health of cod from CWA areas (B13, B14, B15) is significantly affected.
Part-financed by the European Union (European Regional Development Fund)
Health status of cod in CWA areas: Conclusions (1)
Single health parameters measured in cod reveal differences between CWA dumpsites and reference areas.
Responses were stronger on tissue and cell level and less pronounced on individual or population level (early warning?)
However, when combining all health parameters into a simple Fish Health Index (FHI-A/FHI-B), the results do not indicate major differences in health status of cod from CWA dumpsites compared to cod from reference areas.
A possible reason for the inter-annual variability could be changes in hydrographic conditions between 2011 and 2012 (influx) that may have affected the general health status or the vertical distribution of cod.
Part-financed by the European Union (European Regional Development Fund)
Hydrography: 02 concentrations in the Bornholm Basin
Oxygen deficiency in Oct./Nov. 2011 But: Oxygen increase in Feb. 2012
(http://www.io-warnemuende.de/suboxische-und-anoxische-bereiche-im-tiefenwasser-der-ostsee.html)
(Cruise Report R/V "ALKOR" Cruise- No. AL-385B ( 06AK1202 ) 02.02 - 14.02 2012)
Fehmarn (>7 ml/L)
Bornholm Basin (>2 ml/L) Gotland Basin
(<1 ml/L)
Results: strong variations (seasonal/annual) in hydrographic conditions (e.g. O2 concentrations in deep water layers), largely depending on water influx from the west
Part-financed by the European Union (European Regional Development Fund)
Vertical distribution of cod: impact of T, S and 02
Horizontal and vertical distribution patterns of adult cod (Gadus morhua) along a hydroacoustic transect (15°45’00’’E) in the central Bornholm Basin in April (top panel), June (middle panel) and August (lower panel) 2007 and 2008. Black dots represent indi-vidual cod identified by hydroacoustic single-fish tracking. Dotted line, salinity 11 halo-cline; solid line, 1 mL L-1 oxycline (Schaber et al. 2012, Fish. Oceanogr. 21:1: 33–43.)
Results: Cod in the Bornholm Basin prefer salinities of >11 PSU and oxygen concentrations of > 1 ml/L. However, they can be found even in deep water layers < 1 ml/L O2 (e.g. June 2007 and 2008); if O2 concentrations are higher, fish aggregate at the bottom (e.g. April 2008).
Part-financed by the European Union (European Regional Development Fund)
Health status of cod in CWA areas: Conclusions (2)
There is evidence that cod prefers deep water layers even at low O2 concentrations and may, thus, get in direct contact with dumped munitions and CWA, increasing the risk of adverse health effects.
However, due to the inconsistent pattern and the marked inter-annual variability of responses measured so far, there is no clear picture yet regarding the extent of such effect.
Data on more parameters (biological and chemical) and over a longer period of time are required for a more conclusive assessment of acute and chronic CWA effects.
In a variable environment like the Baltic Sea, it cannot be excluded that health risks posed by CWA vary on the short and the long term and, thus, regular monitoring and assessment of CWA risks should be considered.
Part-financed by the European Union (European Regional Development Fund)
... the end
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