Marine Pollution Bulletin 68 (2013) 106–116

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Marine Pollution Bulletin

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Environmental genotoxicity and cytotoxicity levels in fish from the North Sea
offshore region and Atlantic coastal waters
Janina Baršienė a, Aleksandras Rybakovas a, Thomas Lang b, Laura Andreikėnaitė a,⇑,
Aleksandras Michailovas a
a
Nature Research Centre, Akademijos Str. 2, 08412 Vilnius, Lithuania
b
vTI Institute of Fisheries Ecology, Deichstraße 12, 27472 Cuxhaven, Germany

a r t i c l e i n f o a b s t r a c t

Keywords: In the framework of the ICON project, environmental genotoxicity and cytotoxicity levels were assessed
Genotoxicity in blood erythrocytes of dab (Limanda limanda) and haddock (Melanogrammus aeglefinus) collected at 25
Cytotoxicity stations in the North Sea and near the coast of Iceland in August–October 2008. Micronuclei, nuclear buds
Dab and bi-nucleated cells with nucleoplasmic bridges were assessed as environmental genotoxicity bio-
Haddock
markers, and the frequency of fragmented-apoptotic and bi-nucleated erythrocytes were assessed as
North Sea
Atlantic
environmental cytotoxicity biomarkers. The lowest frequencies of genotoxic and cytotoxic abnormalities
were detected in fish from the Icelandic study stations. The highest frequencies of abnormalities were
recorded in dab from the Dogger Bank and the German Bight, in haddock from the Egersund Bank and
from an area off the Firth of Forth (North Sea). In fish from the Icelandic reference area, frequencies of
genotoxicity and cytotoxicity responses were significantly lower than in fish from most areas of the North
Sea.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Thousands of new chemicals annually enter the European mar-
ket, which in total constitutes approximately 100,000 substances
(OSPAR, 2010). A large proportion of anthropogenic chemicals are
hazardous to aquatic organisms. They are persistent, able to accu-
mulate in living organisms, toxic, able to mimic hormones or inter-
fere with enzyme-controlled biological processes and are capable
of modifying genetic material. In total, approximately 300 sub-
stances are considered to be of possible concern for the marine
environment, of which 40 substances and groups of substances
were identified as chemicals requiring priority action (OSPAR,
2010) aimed at preventing and reducing pollution. However, these
substances often occur below the detection limit but may still act
as genotoxins or carcinogens, even at very low concentrations. Fur-
thermore, contaminants are usually discharged in complex mix-
tures, and interactions between unknown compounds can lead to
unpredictable genotoxicity responses to pollution (Jha, 2008).
Genotoxic compounds or their metabolites can bind to DNA mole-
cules and trigger a damaging chain of biological changes, such as
impaired enzyme function or general metabolism, cytotoxicity,
immunotoxicity, reproduction disturbances, growth inhibition or
carcinogenesis (Ohe et al., 2004). Such changes can be passed on
⇑ Corresponding author. Tel.: +370 6 8716939; fax: +370 5 2729257.
E-mail address: laura@ekoi.lt (L. Andreikėnaitė).
to ensuing generations and, in some cases, may lead to a loss of ge-
netic diversity (Dixon et al., 1999; Jha, 2004).
Environmental genotoxicity studies enable the description of
the particular impacts of pollution in areas of concern, and geno-
toxicity biomarkers can serve as early warning signals of environ-
mental pollution by chemicals able to modify the DNA of
organisms. The implementation of the EU Marine Strategy Frame-
work Directive (EC, 2008/56/EC) delineates the development of
common criteria for the assessment of Good Environmental Status
(GES) and environmental monitoring across Europe. The joint ICES/
OSPAR Study Group on Integrated Monitoring of Contaminants and
Biological Effects (SGIMC) developed an integrated approach to the
monitoring of chemical contaminants and biological effects and
assessment criteria for Descriptor 8 of Good Environmental Status
under the Marine Strategy Framework Directive. The micronucleus
(MN) assay was included in a core set of measurements of GES and
the background document on the MN assay as a tool for assessing
cytogenetic/DNA damage in marine organisms is presented in the
ICES SGIMC report (ICES, 2011).
The micronucleus (MN) test is one of the most frequently used
assays and has served as an indicator of cytogenetic damage for
over 30 years (Fenech et al., 2003). It has proven to be a sensitive
and rapid technique to detect structural and numerical chromo-
somal alterations induced by clastogenic and aneugenic agents
(Heddle et al., 1991). The formation of nuclear buds (NB) reflects
the unequal capacity of organisms to expel from the nucleus

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 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
damaged, amplified, replication-failed or improperly condensed
DNA and chromosome fragments without telomeres or centro-
meres (Lindberg et al., 2007). Bi-nucleated cells with nucleoplas-
mic bridges (BNb) are formed from dicentric chromosomes in
which centromeres have been dragged to the opposite poles of
the dividing cell at the anaphase, followed by formation of a nucle-
ar membrane around the chromosomes. BNb formation is a marker
of DNA damage (Fenech and Crott, 2002) that has been applied to
genotoxicity studies in fish (Summak et al., 2010). In addition,
environmental cytotoxicity endpoints reflecting nuclear abnormal-
ities related to cell death (fragmented-apoptotic cells, FA) and de-
fects in cytokinesis (bi-nucleated cells, BN) were analyzed in the
present study. Integrated analysis of micronuclei and other nuclear
abnormalities is a widely applied method for the in situ assessment
of marine environmental genotoxicity (Carrasco et al., 1990; Dolc-
etti and Venier, 2002; Pacheco and Santos, 2002; Çavasß and Er-
gene-Gozukara, 2005; Frenzilli et al., 2004; Napierska et al.,
2009; Baršienė and Andreikėnaitė, 2007; Rybakovas et al., 2009).
Information on environmental genotoxicity and cytotoxicity in
coastal and offshore areas of the North Sea is limited. In a study
by Bresler et al. (1999), frequencies of MN as well as alkaline and
acidic DNA unwinding were evaluated in blue mussels (Mytilus
edulis) from the German Bight. The highest genotoxicity levels
were found in mussels collected in areas impacted by the waters
of the Elbe River, and the lowest were found in mussels collected
near the shore of the island of Helgoland. In order to assess envi-
ronmental genotoxicity levels along a pollution gradient in the
Firth of Forth region of the North Sea, the genotoxicity effects
MN, other nuclear abnormalities and DNA strand breaks (Comet
assay) were evaluated in blood erythrocytes of butterfish (Pholis
gunnellus). Elevated frequencies of MN as well as lobed and bleb-
bed erythrocytes were detected in fish from industrially contami-
nated areas (Bombail et al., 2001). Levels of environmental
contamination with PCBs, PAHs and metals correlated with forma-
tion of DNA strand breaks in erythrocytes and liver cells of dab
(Limanda limanda) from the eastern English Channel, in the areas
of the Seine and Somme Bays. Higher levels of genotoxicity bio-
markers were observed in adult dab compared to juvenile fishes,
with females being more sensitive than males (Akcha et al., 2003).
Increased levels of DNA strand breaks in blood erythrocytes and
high concentrations of bile PAH metabolites were detected in eel-
pout (Zoarces viviparus) collected in Göteborg harbor 3 weeks after
a bunker oil spill (10–100 tons). Significant recovery was observed
in fish from the area 5 months after the accidental oil spill (Frenzilli
et al., 2004). Levels of DNA adducts, up to 27 nmol/mol and up to
239 nmol/mol nucleotides, respectively, were observed in Atlantic
cod (Gadus morhua) and corkwing wrasse (Symphodus melops)
caught in the Karmsund area of the North Sea affected by pollution
with combustion-related PAH compounds produced in connection
with aluminum production (Aas et al., 2001). In blue mussel gill
cells sampled in the harbor areas of Fiskaatangen (Norway) and
Reykjavik (Iceland), levels of DNA adducts were up to 10 nmol/
mol nucleotides. Levels of the DNA adducts correlated with PAH
levels in the tissues and were higher in the gills than in digestive
glands of the mussels (Skarphéðinsdóttir et al., 2007). A signifi-
cantly elevated level of DNA damage (Comet assay) was observed
in blue mussels collected in coastal areas of western Denmark
potentially affected by anthropogenic pollution originating from
chemical dumping sites (Rank et al., 2007). In the same study,
the observed levels of genotoxicity effects were in accord with
the results of heavy metal analysis, and caging of mussels in the
contaminated area induced formation of DNA strand breaks after
64 days of exposure.
In comparison to a reference station, increased frequencies of
MN in peripheral blood erythrocytes as well as higher levels of
macroscopically visible neoplastic liver changes were observed in
107
North Sea flounder (Platichthys flesus) from the German Bight influ-
enced by contamination of the river Elbe. Increased MN formation
was recorded in flounder with macroscopic liver neoplasms as well
as in older individuals (Köhler and Ellesat, 2008). Frequencies of
MN observed in liver erythrocytes of Atlantic cod and hematocytes
of blue mussels caged in the vicinity of Norwegian oil platforms
(Statfjord B/C and Troll B) correlated with distance to the platforms
(Hylland et al., 2008). Produced water from the North Sea Ekofisk
oil field induced genotoxic effects in blue mussels during labora-
tory exposure and caging in situ (Sundt et al., 2011). Increased lev-
els of micronuclei, nuclear buds and fragmented apoptotic
erythrocytes were observed in dab collected in North Sea areas
close to oil and gas platforms, in zones with extensive shipping
and in German Bight areas impacted by contaminants transported
with waters of the rivers Elbe, Weser and Ems (Rybakovas et al.,
2009).
A number of studies have stated that, because of the long-range
transport of anthropogenic contaminants in marine areas around
Europe, it is difficult to select a ‘true’ reference location with a pris-
tine, uncontaminated environment (Baršienė et al., 2006a; Broeg
and Lehtonen, 2006; Gercken et al., 2006; Kopecka et al., 2006;
Lang et al., 2006; Schiedek et al., 2006). However, for the assess-
ment of results from studies on biological effects of contaminants,
it is very important to determine reference levels for the parame-
ters studied. The main objective of the present study was, there-
fore, to evaluate the level of environmental genotoxicity and
cytotoxicity in areas which are away from possible sources of pol-
lution (Icelandic coastal sites) and to compare it with the levels
determined in different areas of the North Sea considered to be im-
pacted by contaminants. Additional tasks were to evaluate envi-
ronmental geno-cytotoxicity differences observed in fish
collected at different stations of the same study area and to com-
pare the results of the present study with results of previous stud-
ies performed in 2004 (Rybakovas et al., 2009). During the
implementation of the ICON project, a wide range of biomarkers,
including liver histopathology, lysosomal membrane stability,
DNA adducts, Comet assay, analysis of external fish diseases, PAH
metabolites, EROD and acetylcholinesterase activity, were ana-
lyzed in the same specimens. In addition, chemical analysis of or-
ganic compounds, metals and methyl mercury was performed.
Thus, the results of the present study will supplement the out-
comes of the integrated assessment of these parameters. Analysis
of biomarker responses will provide a link between environmental
contamination and impact on aquatic organisms. The responses of
five biomarkers were used for the assessment of geno-cytotoxicity
at 25 sampling stations located in 11 areas: the frequency of micro-
nuclei (MN), nuclear buds (NB) and bi-nucleated erythrocytes with
nucleoplasmic bridges (BNb) in blood erythrocytes of two fish spe-
cies (dab and haddock) were assessed as the endpoints of environ-
mental genotoxicity, and the frequency of fragmented-apoptotic
(FA) and bi-nucleated erythrocytes (BN) were measured as markers
of environmental cytotoxicity. For environmental geno-cytotoxic-
ity assessment in the North Sea and coastal areas of Iceland, had-
dock was used as a bioindicator species for the first time.
2. Materials and methods
2.1. Sampling and sample preparation
Samples were collected in August–September 2008 during
cruise 315 of the RV Walther Herwig III, organized by the vTI Insti-
tute of Fisheries Ecology. Additional sampling in the Egersund Bank
area was performed in October 2008 by the RV Scotia (organized
by the Marine Laboratory, Marine Scotland). Dab (L. limanda) and
haddock (Melanogrammus aeglefinus) were sampled in nine
offshore areas (17 stations) of the North Sea and in two coastal
108 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
areas (eight stations) near Iceland (Fig. 1). The Iceland 2 area (sta-
tions Iceland 2/18 and Iceland 2/20) was chosen as a reference be-
cause it is located comparatively far from intense industrial and
municipal pollution zones, and there is no site-specific contamina-
tion observed in the area.
Information about the sampling areas, geographical coordinates
of each station, as well as data about the numbers of specimens
studied are presented in Table 1. In total, 197 dab and 68 haddock
were used for the analysis. Fish from two sampling stations within
each of the areas Iceland 1, Iceland 2, P02, P01 and N06 were ana-
lyzed separately, whereas in areas N01 and N04, a combined anal-
ysis of genotoxicity and cytotoxicity was carried out in fish from
three sampling stations. Dab and haddock were caught by bottom
trawling using a 140-foot trawl with a towing time of 30–60 min.
Sample preparation was performed either immediately after
catching or after keeping fish in tanks with running seawater at
ambient water temperature. Only living specimens in good condi-
tion of approximately the same sizes (dab: 20–24 cm; haddock:
28–32 cm) were processed for further analysis. After blood sam-
pling from the caudal vessel using a heparinized syringe, each fish
was sacrificed by a blow to the head, followed by decapitation. To-
tal weight, total length and weights of livers and gonads were mea-
sured immediately. For age determination, otoliths were removed.
For each fish, a drop of blood was directly smeared on a glass
slide and air-dried. Smears were fixed in methanol for 10 min
Fig. 1. Locations of sampling stations in the North Sea offshore zones and near the coast of Iceland.
and stained with a 5% Giemsa solution in phosphate buffer, pH
6.8, for 40 min. The stained slides were analyzed using either an
Olympus BX51 or a Nikon eclipse 50i light microscope at a final
magnification of 1000.
As markers of environmental geno-cytotoxicity, the frequencies
of micronuclei (MN), nuclear buds (NB), fragmented-apoptotic (FA)
and bi-nucleated (BN) cells were assessed in peripheral erythro-
cytes of fish from all of the study stations. The frequencies of bi-
nucleated erythrocytes with nucleoplasmic bridges (BNb) were
analyzed in dab from areas Iceland 1, Iceland 2, N01, N04, GB1
and GB3.
2.2. Identification criteria
Micronuclei (MN) were identified according to the following
criteria: round and ovoid-shaped non-refractory particles in the
cytoplasm, color and structure similar to chromatin, diameter of
1/3–1/20 of the main nucleus and completely separated from the
main nucleus. Nuclear buds (NB) were identified as extruded nu-
clear material that appeared similar to micronuclei with narrow
or obvious nucleoplasmic connections to the main nucleus. Nucle-
oplasmic bridges (BNb) were identified as uninterrupted nucleo-
plasmic links between the nuclei in bi-nucleated cells with the
same staining characteristics as either of the main nuclei; the
width of a nucleoplasmic bridge usually does not exceed 1/4 of
 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116 109

Table 1
Number of fish collected during cruise 315 of RV Walther Herwig III (29 August–19 September 2008) from different study sites of the North Sea and Icelandic coast.

Sampling areas (short description) Sampling Dab Limanda limanda Haddock Melanogrammus aeglefinus
stations
Number of Latitude Longitude Number of Latitude Longitude
specimens specimens
analyzed analyzed
Iceland 1 – reference area (distant from potential sources IS1/14 10 63°46.400 N 16°30.540 W
of pollution) IS1/15 10 63°46.710 N 16°30.430 W
IS1/16 10 63°46.710 N 16°30.430 W
IS1/17 12 63°45.730 N 16°30.530 W
Iceland 2 – reference area (distant from potential sources IS2/18 10 64°10.000 N 22°24.880 W
of pollution) IS2/19 7 64°10.390 N 22°17.250 W
IS2/20 10 64°06.480 N 22°22.280 W
IS2/21 11 64°07.210 N 22°18.700 W
GB1 area (extensive ship traffic, influence of the Elbe GB1 12 54°04.690 N 07°45.870 E
river)
GB3 area (extensive ship traffic, influence of the Elbe GB3 14 54°55.640 N 06°16.180 E
river)
N01 German Bight (extensive ship traffic, influence of N01/35 10 54°23.310 N 07°35.680 E
the Elbe river) N01/36 7 54°18.770 N 07°30.410 E
N04 Dogger Bank (gas extraction area) N04/09 8 54°49.210 N 02°19.930 E
N04/10 7 54°47.630 N 02°16.050 E
P02 Ekofisk area (offshore oil drilling zone) N04/11 13 54°38.240 N 02°16.810 E
P02/28 15 56°21.160 N 03°02.030 E
P02/29 10 56°25.360 N 03°08.530 E
P01 Dan Field area (offshore oil/gas drilling zones) P01/05 14 55°41.830 N 05°03.360 E
P01/06 14 55°30.070 N 04°58.320 E
Egersund banka (comparatively distant from potential Egersund 10 57°30.450 N 04°42.020 E 10 57°30.450 N 04°42.020 E
sources of pollution)
N06 Firth of forth (extensive ship traffic, influence of N06/22 8 56°17.240 N 02°03.290 W
pollution from the Edinburgh region) N06/23 9 56°17.190 N 02°04.620 W
N06/24 8 56°18.540 N 02°04.910 W
N06/25 6 56°17.560 N 01°57.540 W
N11 Danish offshore area (potentially influenced by the N11 10 55°30.910 N 07°01.130 E
plume of the river Elbe and pollution from Denmark)
a
sampling was performed in October 2008 by RV Scotia.

the diameter of the nuclei. Fragmented-apoptotic cells (FA) in early
stages were identified by the presence of chromatin condensation
within the nucleus and intact cytoplasmic and nuclear boundaries.
Late apoptotic cells were identified as those that exhibited nuclear
fragmentation into smaller nuclear bodies within an intact cyto-
plasm. To be identified as BN, the two nuclei of a bi-nucleated cell
(BN) should not overlap; they should be approximately equal in
size, staining pattern and staining intensity, have intact nuclear
membranes and be situated within the same cytoplasmic mem-
brane. The morphological features of the nuclear abnormalities
studied were identified using criteria described by Fenech et al.
(2003) and are shown in Fig. 2. In total, 4000 peripheral blood
erythrocytes were examined for each fish specimen. The final re-
sults were expressed as the mean frequency (‰) of the analyzed
individual lesions assessed in 1000 erythrocytes per fish collected
from every study station (Baršienė et al., 2004).
Assessment of the genotoxicity risk at the stations studied was
assessed on the basis of the established background response of
MN incidence in dab (<0.37 MN/1000 erythrocytes) and haddock
(<0.30 MN/1000 erythrocytes). The background level of MN fre-
quencies was calculated as the empirical 90th percentile (P90) in
the two fish species collected during the 2004–2008 period from
the reference sites at the Icelandic coast and N11, which are char-
acterized by having no known local sources of contamination and
no impact from human and industrial activity. The P90 value sep-
arates the upper 10% of all MN values in the group of data from the
lower 90%. In general, an elevated MN frequency lies above the P90
percentile, whereas the majority of values below the P90 value be-
long to individuals that are unexposed, weakly exposed or non-
responding or adapted to stressful conditions. Additionally, an in-
creased level of the MN values was calculated as the empirical
99th percentile and demarcated an extremely high level of MN re-
sponses exceeding the P99 percentile values of the MN frequencies.
The study stations were grouped according to a 4-grade scale,
i.e., the reference level of MN indicating no risk, the background
P90 percentile value of MN reflecting low risk, the P99 percentile
value of MN reflecting increased risk and the MN values higher
than the P99 percentile indicating exceptionally high genotoxicity
risk for the studied fish species. Genotoxicity risk zones were
mapped to the GIS system utilizing the program ArcGIS Desktop
and the ArcMap application.
2.3. Statistical analysis
The GraphPad PRISM 5.0 statistical package was used for statis-
tical analysis. Means and standard errors were calculated for each
station studied. The non-parametric Mann–Whitney U-test was
used to compare the frequencies of nuclear abnormalities observed
in dab at the reference station Island 2/18 and the other stations.
One-way analysis of variance (ANOVA) was applied to analyze
the level of variance in the genotoxicity and cytotoxicity responses
in fish from the different sites. Pearson’s correlation analysis was
performed to identify possible relationships between nuclear
abnormalities and biometrical measurements of the fish or be-
tween biomarker responses and environmental variables.
3. Results
3.1. Nuclear abnormalities in dab
In the blood erythrocytes of the dab, the frequencies of micro-
nuclei (MN/1000 cells) varied from 0.09‰ to 1.37‰ and of nuclear
buds (NB/1000 cells) from 0.09‰ to 3.13‰ (Fig. 3). The frequencies
of fragmented-apoptotic cells (FA/1000 cells) varied from 0.03‰ to
0.74‰ and that of bi-nucleated erythrocytes (BN/1000 cells) from
110 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
0.0‰ to 0.53‰ (Fig. 4). The lowest MN, NB, BN and FA values were
recorded in dab from the stations in the Iceland 2 reference area. In
fish collected from sampling stations Iceland 2/19, N01/35 and
N01/39, no BN cells were detected. The highest levels of MN and
BN were found in dab collected from station N04/11, and the high-
est levels of NB and FA were in fish from station N01/36.
Comparing levels of parameters studied in dab from the refer-
ence station Iceland 2/18 with those from the other stations, an
8-fold elevation of MN was recorded at station N04/11 and a 7-fold
elevation at the Egersund station as well as at stations N04/09 and
N04/10. Comparing frequencies of NB, high levels were found in
fish collected at stations N01/36 and N01/39 (35-fold increase),
GB1 (17-fold increase) and N04/10 (14-fold increase).
Statistically significant differences were detected between
genotoxicity and cytotoxicity responses in dab from the reference
station Iceland 2/18 and those from most other stations (Figs. 3
and 4). One-way ANOVA revealed significant differences in MN
(p < 0.0001, F = 4.909), NB (p < 0.0001, F = 4.842), FA (p < 0.0001,
F = 3.801) and BN (p < 0.001, F = 2.564) frequencies. There were dif-
ferences between the induction of NB in fish from the reference
station Iceland 2/18 and from stations Iceland 1/16 (p = 0.0129)
and Iceland 1/17 (p = 0.0036). Similarly, significant differences in
NB frequencies in dab were recorded between the study stations
of areas N01 and N06 (p = 0.0409 between N01/35 and N01/39
and p = 0.0050 between N06/22 and N06/25).
The analysis of bi-nucleated erythrocytes with nucleoplasmic
bridges (BNb) revealed an increased induction in dab from the
Fig. 2. Nuclear abnormalities in fish peripheral blood erythrocytes: (a) micronu-
cleus in dab erythrocyte, (b) micronucleus in haddock erythrocyte, (c) nuclear bud,
(d) nucleoplasmic bridge, (e) fragmented-apoptotic and (f) bi-nucleated dab
erythrocytes.
N01 area, with the highest expression and individual variation at
station N01/39 (Fig. 3). In dab from four stations at the Iceland
coast, BNb were observed only at station Iceland 1/17. One-way
ANOVA showed differences between the stations (F = 2.688,
R2 = 0.2161, p = 0.0115).
A comparison of frequencies of fragmented-apoptotic erythro-
cytes in dab from the reference station with those at the other sta-
tions revealed the highest induction in fish from the N01 and N04
areas: 25-fold higher levels at the N01/36, 24-fold at the N04/09
and 22-fold at the N04/11 stations. Furthermore, significant differ-
ences were found between dab collected at the reference station
Iceland 2/18 and at most of the other study stations (Fig. 4). The
FA levels differed significantly in dab collected at the reference sta-
tion Iceland 2/18 and at Iceland 1/17 (p = 0.048).
In dab from three stations (Iceland 2/19, Iceland 1/17 and N01/
35), the frequency of bi-nucleated erythrocytes (BN) was equal to
zero. The highest levels of BN were observed in fish from stations
N04/11, GB1, N04/09 and P01/05. However, statistically significant
differences were recorded only between dab from the reference
station and N04/11 (p = 0.0067). One-way ANOVA showed signifi-
cant differences in BN among dab from the stations studied
(p = 0.0002, F = 2.564).
Pearson’s correlation was applied to assess relationships be-
tween environmental geno-cytotoxicity and dab age, length, total
weight, liver and gonad weight, condition factor (CF), liver somatic
index (LSI) and gonadosomatic index (GSI). Pearson’s correlation
analysis was performed using dab from each study station. Positive
or negative correlations were found only in dab from the uncon-
taminated Icelandic coast and areas GB3 and N06, i.e., in six of
the 19 stations studied (Table 2). In fish collected from these sta-
tions, comparatively low levels of biomarker responses were ob-
served. No relationships between geno-cytotoxicity levels and
fish condition factor were recorded.
3.2. Nuclear abnormalities in haddock
In blood erythrocytes of haddock from the reference station Ice-
land 2/20, a very low frequency (0.03‰) of MN was assessed. In
haddock from the Egersund area in the North Sea, the MN level
was 25 times higher than at the reference station. The frequency
of NB at the reference station Iceland 2/20 was 5-fold lower than
in fish from area N06. The lowest levels of environmental cytotox-
icity were also recorded in haddock from the Iceland study stations
(Fig. 5).
Applying Pearson’s correlation test, relationships between envi-
ronmental geno-cytotoxicity responses and haddock weight, liver
weight, condition factor (CF), liver somatic index (LSI) and gonado-
somatic index (GSI) were identified. Positive or negative correla-
tion was found only in fish from stations at the uncontaminated
Icelandic coast, as well as strong correlations between MN or NB
induction and biometrical variables in haddock collected from
the Egersund area (Table 3).
The assessment of environmental genotoxicity risk levels in dab
and haddock indicated no risk for both species inhabiting the Ice-
landic coastal stations 2/19 and 2/20. At all of the other Icelandic
study stations, there was a background level of MN frequencies
reflecting a low level of genotoxicity risk. The background level
of MN frequency was also found in both species collected from
the N06 area (excepting flounder from station N06/25) and in
flounder from stations N11, GB1, GB3 and P02/28. Moderate risk
was determined for flounder collected from stations N01/35,
N01/36, P01/05 and N06/26. The assessment of the numbers of sta-
tions characterized as extremely high genotoxicity risk zones in
the North Sea indicated that the N04 (Dogger Bank) and the Egers-
und Bank areas were zones of extremely high genotoxicity risk
(Fig. 6).
 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116 111

Fig. 3. Mean frequencies (and SE) of micronuclei (MN) and nuclear buds (NB) and erythrocytes with nucleoplasmic bridge (BNb) in peripheral blood of dab (L. limanda)
collected at different North Sea and Icelandic study stations. Asterisks indicate statistically significant differences between stations and the reference station Iceland 2/18
(Mann–Whitney U-test); p < 0.01, p < 0.001, p < 0.0001.

Fig. 4. Mean frequencies (and SEs) of fragmented-apoptotic and bi-nucleated erythrocytes in dab (L. limanda) collected at North Sea and Icelandic study stations. Asterisks
indicate statistically significant differences between stations and the reference station Iceland 2/18 (Mann–Whitney U-test); p < 0.01, p < 0.001, p < 0.0001.

4. Discussion
The results of the present study reveal marked regional differ-
ences in the frequencies of the genotoxicity and cytotoxicity bio-
markers studied. At the North Sea study stations, frequencies of
MN in dab were up to 8 times higher than in fish from the refer-
ence station, frequencies of NB in dab differed up to 35 times
and frequencies of FA cells up to 25 times. In the haddock inhabit-
ing the Egersund area of the North Sea, the level of MN was 25
times higher than in those at the reference stations. These findings
highlight a huge genotoxicity and cytotoxicity impact to the North
Sea that is possibly caused by anthropogenic contamination.
Near the coast of Iceland, genotoxicity and cytotoxicity effects
in dab and haddock were assessed for the first time. The lowest
levels of the studied biomarkers were recorded in the dab from
the Iceland 2/19 station (MN – 0.09‰, NB – 0.09‰, BNb – 0.0‰,
FA – 0.03‰ and BN – 0.0‰) and in the haddock from the Iceland
2/20 station (MN 0.03‰, NB 0.18‰, FA 0.12‰ and BN 0.06‰).
Although levels of the studied parameters were comparatively
low in fish from all the Icelandic stations, the frequencies of NB
and FA differed significantly among the stations. Bi-nucleated
erythrocytes with nucleoplasmic bridges were also observed only
in erythrocytes of the dab collected from the Iceland 1/17 station,
even though the Iceland 2 area is located closer to ship traffic roads
and Reykjavík harbor than is the Iceland 1 area.
The highest frequencies of MN (1.37‰) and BN (0.53‰) eryth-
rocytes were found in the dab from the N04/11 station, which is lo-
cated in the area of the Dogger Bank close to the gas extraction
platforms. This MN frequency is the absolute highest value ob-
served in the dab from the 30 study stations (including monitoring
at some stations – 43 analyses) in the North Sea and Atlantic
Ocean. Increased environmental genotoxicity and cytotoxicity
112 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
levels were assessed in the dabs from the Ekofisk P02 and Dan Field
P01 stations, which are located close to the oil/gas drilling plat-
forms. These results are in accord with those found at the Dogger
Bank and Ekofisk areas in 2003 and 2004 (Rybakovas et al.,
2009). Furthermore, the assessment of environmental genotoxicity
risk using the MN background levels in two fish species indicated
an extremely high risk for fish inhabiting the Dogger Bank and
Egersund Bank areas of the North Sea in 2008. It is interesting to
note that, in particular, the Dogger Bank has been a hotspot area
for the occurrence of neoplastic and pre-neoplastic liver lesions
in dab since the late 1980s (Lang, unpublished data, Stentiford
et al., 2009); thus, the role of chronic genotoxicity effects cannot
be excluded.
The Ekofisk oil field has been exploited for a long time (since
1971). The discharges of produced water in the area have been
comparatively high and reached approximately 8  106 L/day in
2006 (Sundt et al., 2011). Caging of blue mussels (M. edulis) for
48 days near produced water outfall installations of the Ekofisk
oil platform and laboratory exposure to produced water caused
petrogenic PAH tissue residues in the mussels. Analysis of MN in
the mussel hemocytes showed a gradient related to the distance
from the discharge, a dose response under laboratory treatment
and a linear correlation between MN levels and PAH metabolites
measured in mussel soft tissues (Sundt et al., 2011).
According to an OSPAR (2010) report, concentrations of PAHs,
one of the most widespread organic pollutant classes in the marine
environment, in the North Sea region are still widely above back-
ground values. The PAHs are entering the North Sea from offshore
oil and gas installations, accidental and operational spills and ship-
ping accidents, in river effluents and through atmospheric input.
Approximately 8000 tons of oil is released annually with produced
water into the North Sea (Sundt et al., 2009). Some of the hazard-
ous substances, such as heavy metals, aromatic hydrocarbons,
alkylphenols and radionuclides in the produced water are originat-
ing from the reservoirs, while other components, such as corrosion
inhibitors and demulsifiers, are residues of chemicals used in
extraction processes (OSPAR, 2010). Analysis of the chemical com-
position of produced water from the Norwegian sector of the North
Sea oil platform showed that its dominant organic components are
phenanthrenes, dibenzothiophenes, naphthalenes, benzene, tolu-
ene, ethylbenzene, xylene and PAHs (such as fluorine, pyrene, flu-
oranthene, chrysene, benz(a)anthracene, benzo(a)pyrene), organic
Table 2
Results of Pearson’s correlation analysis performed between biomarker responses and biometrical measurements in dab (L. limanda) from different North Sea and Icelandic study
stations.
Station Length Age Weight
Iceland 2/19 NB FA NB
r = 0.892 r = 0.847 r = 0.836
p = 0.007 p = 0.016 p = 0.019
Iceland 1/16
Iceland 1/17 FA FA FA
r = 0.690 r = 0.766 r = 0.696
p = 0.001 p = 0.013 p = 0.004
GB3 BN
r = 0.544
p = 0.044
N06/22
N06/25
acids, alkylphenols and phenols (Utvik, 1999). During implementa-
tion of a water column monitoring program in 2001, 2003 and
2004, a number of chemical and biological parameters were ana-
lyzed in Atlantic cod (G. morhua) and blue mussels (M. edulis) caged
at different distances from produced water discharges of oil plat-
forms located in the Norwegian sector of the North Sea (Hylland
et al., 2008). Analysis of the ensuing biological effects reflected gra-
dients of exposure. Higher frequencies of MN were detected in
immature liver erythrocytes of the cod and hematocytes of the
blue mussels caged close to the platforms. Even at comparatively
low exposure levels (campaign of 2004), a gradient-related in-
crease in the frequencies of MN in the mussel hemocytes was ob-
served (Hylland et al., 2008). Dispersed crude oil from the North
Sea Statfjord B platform and a model concentration of oil spiked
with an additional mixture of PAHs and alkylphenols induced
genotoxicity and cytotoxicity effects in turbot (Scophthalmus max-
imus) and Atlantic cod. After a 3-week exposure, species-specific
and tissue-specific biomarker responses were observed (Baršienė
et al., 2006b).
A study performed in 2003 and 2004 revealed that, in fish col-
lected at different stations of the same study area (P01 and P02), lev-
els of genotoxic and cytotoxic effects could be statistically
significant. Higher levels of the observed effects were found in fish
collected closer to the oil platforms (Rybakovas et al., 2009). There-
fore, one of the objectives of the present study was to evaluate the
geno-cytotoxicity differences between closely located stations of
the same study area. Such differences were observed only in the
frequencies of NB in erythrocytes of the dab from stations N06/22
and N06/25 (Mann–Whitney U-test, p < 0.01). Such differences
may have been caused by a heterogeneous distribution of the pollu-
tants in area N06, which can be potentially affected by discharges
from the Edinburgh region. Similarly marked environmental geno-
toxicity and cytotoxicity differences were observed in coastal
stations of the Baltic Sea, where significant differences of MN fre-
quencies were observed in gill cells of mussels (Mytilus trossulus)
and blood erythrocytes of flounder (P. flesus) collected at closely
located stations in the Gulf of Gdansk during a sampling campaign
in 2001–2003 (Kopecka et al., 2006). Significant differences in MN,
NB and FA were also observed in flounder erythrocytes from the
same study stations in September 2005 (Napierska et al., 2009).
In the present study, the highest frequencies of NB, BNb and FA
were found in peripheral blood erythrocytes of the dab collected
Liver wt Gonad wt LSI GSI
MN
r = 0.778
p = 0.039
MN
R = 0.734
p = 0.016
FA NB MN
r = 0.576 r = 0.585 r = 0.616
p = 0.050 p = 0.046 p = 0.033
NB
r = 0.608
p = 0.036
BN MN BN MN
r = 0.792 r = 0.926 r = 0.850 r = -0.807
p = 0.019 p = 0.001 p = 0.008 p = 0.015
MN
r = 0.857
p = 0.029
 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116 113

Fig. 5. Mean frequencies (and SE) of micronuclei (MN), nuclear buds (NB) and fragmented-apoptotic (FA) and bi-nucleated (BN) cells in blood erythrocytes of haddock (M.
aeglefinus) collected at North Sea and Icelandic study stations. Asterisks indicate statistically significant differences between studied locations compared to the reference
station Iceland 2/20 (Mann–Whitney U-test); p < 0.01, p < 0.001.

Table 3
Results of Pearson’s correlation analysis performed between studied biomarker treatment with nonylphenol also induced formation of BN and
responses and biometrical measurements in haddock from different North Sea and FA in turbot kidney erythrocyte cells (Baršienė et al., 2006b).
Icelandic study stations. Distribution of polyfluoroalkyl compounds (PFCs) was analyzed
Station Weight Liver wt CF LSI GSI in bile fluids of dab collected during the same sampling campaign
Iceland 2/ MN as the one presented in this study (Ahrens and Ebinghaus, 2010).
20 r = 0.831 Among the PFC compounds, perfluorooctane sulfonate (PFOS)
p = 0.003 was dominant, with the highest concentrations observed in the
Iceland 2/ MN German Bight (N01, GB1) and along the Danish coast. Concentra-
21 r = 0.666
tions of PFOS in the other areas of the North Sea and near Iceland
p = 0.025
BN were significantly lower. Studies performed earlier by Ahrens et al.
r = 0.746 (2009) in the German Bight area indicated the highest RPFC con-
p = 0.008 centrations were in water samples collected from the estuaries of
Iceland 1/ MN
the rivers Elbe, Weser and Ems (up to 31.2 ng L1). In the offshore
15 r = 0.692
p = 0.027
areas, the concentrations of RPFC decreased 2–3-fold. It should be
Egersund MN MN MN MN noted that perfluorinated carboxylic acids were distributed almost
r = 0.834 r = 0.974 r = 0.989 r = 0.986 homogeneously in the North Sea areas studied, and this distribu-
p = 0.003 p = 0.0001 p = 0.0001 p = 0.0001 tion can be explained by the long-range transportation of these
NB NB NB NB
compounds (Ahrens and Ebinghaus, 2010). Fish exposure to per-
r = 0.725 r = 0.703 r = 0.703 r = 0.695
p = 0.018 p = 0.023 p = 0.023 p = 0.026 fluorooctane sulfonate causes multiple changes in gene expression,
including genes related to oxidative stress, heat shock protein re-
sponses, steroid homeostasis and apoptosis (Liu et al., 2007; Krovel
from the German Bight station N01/36. In dab from site GB1, an- et al., 2008). Exposures to PFOS disrupted normal homeostasis in
other area of the German Bight, the frequencies of NB and FA cells bottlenose dolphin skin cells, promoting expression of protective
were also significantly higher than those registered at the refer- stress response genes and altering expression of genes responsible
ence station. Sites N01 and GB1 are characterized by very extensive for translation, transcription and cellular proliferation (Mollenhau-
ship traffic. A number of studies also demonstrated a considerable er et al., 2009).
river influence on the spatial distribution of contaminants in the According to an OSPAR (2010) report, accumulated concentra-
German Bight (Bester et al., 2001; Xie et al., 2006; Ahrens et al., tions of cadmium, mercury and lead in fish, shellfish tissues and
2009). Studies performed by Bester et al. (2001) indicated that con- sediments in most areas of the North Sea are still above back-
centrations of nonylphenols in water extracts ranged from ground levels. In addition, it has been determined that concentra-
33 ng L1 in the estuary of the Elbe River to less than 1 ng L1 in tions of cadmium and mercury in fish and shellfish collected from
the central part of the North Sea. The highest total concentrations the N04 area in the Dogger Bank are increasing. Concentrations of
of nonylphenols (up to 1400 pg L1) were observed in water sam- lead, cadmium and mercury in the sediments of the Dogger Bank
ples collected near the plume of the Elbe, and a decreasing concen- and the German Bight are at levels that can pose a risk of pollution
tration profile was found with increasing distance from the coast to effects for marine organisms. It is important to stress that high
the central part of the North Sea. The concentrations of nonylphe- concentrations of cadmium bioaccumulated in tissues of fish
nols in the open sea areas were comparatively low and often lower caught near Iceland may have originated from natural factors, such
than the detection limit of 10 pg L1 (Xie et al., 2006). In the open as volcanic activity (OSPAR, 2010). The genotoxicity and cytotoxic-
sea, an increased concentration (55 ng g1) of nonylphenols was ity potential of cadmium to fish have been demonstrated in a num-
detected only in sediments collected 30 km from Helgoland Island ber of studies (Metcalfe, 1989; Risso-de Faverney et al., 2001;
(Bester et al., 2001). Experimental treatment of turbot (S. maximus) Chandra and Khuda-Bukhsh, 2001, 2004; Cambier et al., 2010).
with 30 ppb of nonylphenol significantly induced formation of MN Cd induces DNA strand breaks and apoptosis in rainbow trout
in peripheral blood and cephalic kidney erythrocytes. The hepatocytes (Risso-de Faverney et al., 2001), and cadmium
114 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
Fig. 6. Results of environmental genotoxicity risk assessment in dab (L. limanda) and haddock (M. aeglefinus) collected from different zones of the North Sea and Icelandic
coast.
chloride causes formation of chromosome aberrations, abnormal
red blood cell nuclei and alterations in sperm morphology of the
fish Oreochromis mossambicus (Chandra and Khuda-Bukhsh,
2004). CdCl2 also triggers formation of DNA mutations (point
mutations and large rearrangements) and other structural effects
in the zebrafish Danio rerio (Cambier et al., 2010). A study by San-
chez-Galan et al. (1999) demonstrated that injections of CdCl2
(1.7 mg kg1 body weight) induced MN formation in the European
minnow Phoxinus phoxinus and in the brown trout Salmo trutta.
Exposure to Cd may inhibit the DNA repair system. Proteins partic-
ipating in DNA excision and mismatch repair are sensitive to the
toxic action of cadmium. This metal is also able to inhibit enzymes
involved in oxidative stress responses and thereby increase forma-
tion of reactive oxygen species and free radicals (Risso-de Faverney
et al., 2001; Waisberg et al., 2003; Giaginis et al., 2006). Cd induces
some proto-oncogenes (Giaginis et al., 2006) and affects apoptosis
(Risso-de Faverney et al., 2001).
Different concentrations (1 lg L1, 5 lg L1 and 10 lg L1) of
mercury chloride and lead acetate induce formation of micronuclei
and nuclear buds, as well as changes in the ratio of polychromatic
and normochromatic erythrocytes in the fish Carassius auratus. MN
frequencies in gill cells of both control and treated goldfish were
higher than in peripheral blood erythrocytes and epithelial fin cells
(Çavasß, 2008). Mercury and methylmercury induce dose-depen-
dent formation of MN in binucleated blocked hepatic cells of trout
after one cycle of cell division (Al-Sabti, 1995). Short-term (up to
96 h) exposure of the fish Prochilodus lineatus to lead nitrate
induced time-dependent formation of DNA strand breaks in blood,
liver and gill cells as well as nuclear abnormalities, such as kidney-
shaped nuclei, segmented nuclei and lobed nuclei, in erythrocytes,
but did not increase MN frequencies (Monteiro et al., 2011). Inor-
ganic lead and mercury salts act as indirect genotoxins, provoking
formation of aneugenic MN after interactions with proteins of the
cytoskeleton such as tubulin and/or kinesin (Their et al., 2003).
Treatment with lead is related to the production of reactive oxygen
species, thus damaging DNA and depleting the antioxidant defense
system of the cell (Farmand et al., 2005).
According to ICES (2010), in biota and sediments of the Firth of
Forth region (comparatively close to the N06 area) and areas close
to the island of Heligoland in the German Bight (N01, GB1), PCBs
are at levels considered to pose the risk of chronic effects in marine
organisms. PCBs are very persistent, able to concentrate in fatty tis-
sues and are toxic, genotoxic and carcinogenic substances (Silber-
horn et al., 1990; Knerr and Schrenk, 2006; Marabini et al., 2011).
The genotoxicity of PCBs in fish cells may appear via oxidative
stress and the production of ROS, lipid peroxidation, loss of gluta-
thione content and reduced activity of antioxidant enzymes
(Strathmann et al., 2006; Marabini et al., 2011).
In the present study, correlation between environmental geno-
cytotoxicity responses and host-specific factors (age, length, total
weight, liver and gonad weight, CF, LSI and GSI) were assessed.
Pearson’s correlation analysis revealed that the frequencies of FA
and NB correlated with age and biometrical measurements of bot-
tom-dwelling dab mainly in samples from the reference areas near
Iceland, while in dabs from the Firth of Forth region of the North
Sea (station N06/22), the frequency of bi-nucleated erythrocytes
 J. Baršienė et al. / Marine Pollution Bulletin 68 (2013) 106–116
was positively correlated with liver weight and LSI, and the fre-
quency of MN was negatively correlated with gonad weight and
GSI. In pelagic haddock from the Egersund study area, the frequen-
cies of the genotoxicity biomarkers (MN and NB) were negatively
correlated with fish weight and CF and positively correlated with
LSI and GSI. The influence of host-specific factors was described
in a study performed by Talapatra et al. (2010); after treatment
of Channa punctatus with metronidazole, frequencies of MN and
BN erythrocytes showed strong negative correlations (at p < 0.05)
with fish body weight. In sticklebacks (Gasterosteus aculeatus L.)
from three water bodies that differed in the amount of sewage-
treatment effluent received, frequencies of MN in blood erythro-
cytes were negatively correlated with the lengths of the fish,
whereas the correlation of DNA strand breakage with this factor
was positive (Wirzinger et al., 2007). Studies of dab (L. limanda)
collected in situ in the Seine Bay and the Somme Bay (Eastern Eng-
lish Channel) revealed significant age and sex effects on the forma-
tion of DNA strand breaks in blood erythrocytes (Akcha et al.,
2004). These results show the importance of studying the effects
of host-specific factors on the genotoxic and cytotoxic endpoints
for correct assessment of anthropogenic impact.
From the results of the present study, it can be concluded that
the Icelandic sampling sites can be used as reference sites for com-
parison to other North Atlantic and North Sea areas. Levels of geno-
toxicity and cytotoxicity found in the Icelandic dab and haddock
could potentially be employed as baselines for environmental
genotoxicity and cytotoxicity impact and risk assessment. In the
present study, frequencies of genotoxic and cytotoxic abnormali-
ties in haddock from the North Sea and near the coast of Iceland
were assessed for the first time. For future marine monitoring pro-
grams, sizeable efforts should be made to identify baseline levels of
biomarker responses in other bioindicator species. Comparison of
the results of the present study with those obtained in September
2004 (Rybakovas et al., 2009) revealed consistent patterns of envi-
ronmental geno-cytotoxicity in oil/gas extraction areas P01 (Dan
Field) and P02 (Ekofisk) and increased biomarker responses in
areas N06 (off the Firth of Forth) and N04 (Dogger Bank). In dab
from areas N06 and N04, MN frequencies determined in 2008 were
more than 3 times higher than those detected in 2004, and NB and
FA frequencies were more than 2 times higher. Therefore, there is
an evident need to perform long-term monitoring in areas of in-
tense anthropogenic impact.
Acknowledgement
This study was funded by the Research Council of Lithuania
through the GENCITOX project (MIP-10123).
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