Risk of environmental genotoxicity in the Baltic Sea over the period of 2009e2011

assessed by micronuclei frequencies in blood erythrocytes of ounder

(Platichthys esus), herring (Clupea harengus) and eelpout (Zoarces viviparus)
Janina Bar

sien
_
e
a,
*
, Aleksandras Rybakovas
a
, Thomas Lang
b
, Wlodzimierz Grygiel
c
,
Laura Andreik
_
enait
_
e
a
, Aleksandras Michailovas
a
a
Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania
b
vTI Institute of Fisheries Ecology, Deichstrae 12, 27472 Cuxhaven, Germany
c
National Marine Fisheries Research Institute in Gdynia, 1 Kollataja Street, 81-332 Gdynia, Poland
a r t i c l e i n f o
Article history:
Received 25 November 2011
Received in revised form
20 January 2012
Accepted 24 January 2012
Keywords:
Biomarker
Micronuclei
MN background level
Genotoxicity
Fish
Flounder
Herring
Eelpout
Baltic Sea
a b s t r a c t
Environmental genotoxicity was investigated at 82 locations encompassing different regions of the Baltic
Sea. Micronuclei (MN) analysis was performed in erythrocytes of 1892 specimens of ounder Platichthys
esus, herring Clupea harengus and eelpout Zoarces viviparus, three of the most common native sh
species of the Baltic Sea collected in 2009e2011. MN background levels in sh were determined using
data obtained in 2001e2011 from 107 Baltic sites. Extremely high genotoxicity risk zones were found for
ounder at 11 stations out of 16 in 2009 and 33 stations of 41 in 2010e2011, for herring, at 5 of 18
stations in 2009 and 20 of 43 stations in 2010e2011, in eelpout only at one out of 29 stations. The
sampling stations were restricted mainly to the southern and eastern Baltic Sea offshore zones and in
most of them, MN frequencies in ounder and herring signicantly exceeded the reference and back-
ground levels of micronuclei. This is a rst attempt to evaluate the background MN responses, as well as
low, high and extremely high genotoxicity risk levels for native sh species.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
The Baltic Sea is one of the most contaminated marine ecosys-
tems. Summarizing results of eld studies carried out within the
framework of the EU funded pan-European BEEP project on bio-
logical effects of contaminants in organisms inhabiting the Baltic
Sea, Lehtonen et al., 2006 concluded that, although the loads of
some classical chemical toxic substances (e.g., PCBs, DDTs) have
been reduced over the last decades, chemical pollution by a wide
spectrum of hazardous substances may be assumed to be higher
nowadays than ever before. According to the results of the inte-
grated HELCOM CHASE assessment (HELCOM, 2010) based on data
from the period 1999e2007 for hazardous substances and selected
biological effects in the Baltic Sea, 137 out of the 144 areas assessed
were classied as being disturbed by hazardous substances,
including all open-sea areas of the Baltic Sea analyzed. In the
southern Baltic Sea, the Kiel and Mecklenburg Bights were classi-
ed as most polluted and ecologically worst areas (HELCOM, 2010).
In the HELCOM assessment, (HELCOM, 2010) it was pointed out
that a large number of different substances exceeded the threshold
levels in the different Baltic Sea sub-basins. In sh, mussels and bird
tissues, PCBs, dioxins, heavy metals, organometals, alkylphenols,
phthalates, brominated compounds, polycyclic aromatic hydrocar-
bons (PAHs), DDTs and chlorinated pesticides, and caesium-137
were found at the highest concentrations in relation to target
levels. The southern region of the Baltic Sea is polluted by all of the
above-mentioned substances (HELCOM, 2010). Many of these
substances inherently are genotoxic compounds and may exert
genotoxicity effects via direct action or the activation of toxic
metabolic mechanisms and are, thus, of concern regarding their
potential impact on aquatic organisms and human health. Chemical
substances with genotoxicity potential can be sub-divided into four
groups: (1) substances directly inducing DNA damage; (2)
substances the metabolites of which cause DNA damage; (3)
* Corresponding author. Nature Research Centre, Institute of Ecology, Akademijos
str. 2, 08412, Vilnius, Lithuania. Tel.: 370 6 8260979; fax: 370 5 2729257.
E-mail address: janbar@ekoi.lt (J. Barsien_ e).
Contents lists available at SciVerse ScienceDirect
Marine Environmental Research
j ournal homepage: www. el sevi er. com/ l ocat e/ marenvrev
0141-1136/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.marenvres.2012.01.004
Marine Environmental Research 77 (2012) 35e42
substances that increase the production of reactive oxygen species
(ROS) and free radicals, which can subsequently damage both DNA
bases and the deoxyribose backbone; (4) substances that inhibit
DNA synthesis and repair (Lee and Steinert, 2003). Genotoxic
compounds can bind to DNA causing the formation of DNA adducts,
single and double strand breakages, modications in DNA repair
and crosslink consistent pattern, as well as alterations of cell
functions, reproduction disturbances, growth inhibition, or even
carcinogenesis (Ohe et al., 2004). As a consequence, further
generations of the organisms can suffer from reduced tness,
fertility or embryonic viability (Russo et al., 2004). Non-repaired
genetic damage is considered important since it provides a funda-
mental early warning sign of adverse long-term effects of
contaminants at population and, furthermore, ecosystem levels
(Rybakovas et al., 2009). Furthermore, contaminants, usually dis-
charged in complex mixtures, can provoke interactions between
unknown substances and lead to the unpredictability in genotoxic
responses to pollution (Jha, 2008).
Environmental genotoxicity in the Baltic Sea was earlier
assessed in the Swedish part of the Gulf of Bothnia (Al-Sabti and
Hrdig, 1990) and in Danish waters in Kge Bay, Little Belt, Store
Belt and Kattegat (Wrisberg et al., 1992). Later studies, carried out
by the Institute of Ecology (Lithuania), covered the Lithuanian
economic zone (Barsien_ e and Barsyt _ e Lovejoy, 2000; Barsien_ e,
2002; Barsien_ e and Rybakovas, 2006; Barsien_ e et al., 2004,
2005a, 2006a, 2006b, 2008, in press), the Gulf of Gdansk (Barsien_ e
et al., 2006b; Kopecka et al., 2006; Napierska et al., 2009), Swedish
coastal sites Kvdfjrden and those in the Stockholm archipelago
(Barsien_ e et al., 2006b; unpublished data), the Wismar Bay
(Barsien_ e et al., 2006b; Schiedek et al., 2006) and 12 offshore areas
of the Baltic Sea (Rybakovas et al., 2009).
Based on these data, a large database on environmental geno-
toxicity in the Baltic Sea was established. In sh, the data collected
in the period of 2001e2011 were available for ounder (Platichthys
esus) from 75 stations, for herring (Clupea harengus) from 59
stations and for eelpout (Zoarces viviparus) from 35 stations. Data
for cod (Gadus morhua), plaice (Pleuronectes platessa) and turbot
(Psetta maxima) were collected from 25 stations. Environmental
genotoxicity was also evaluated in bivalve mollusks Mytilus edulis
(45 stations), Mytilus trossulus (6 stations) and Macoma balthica (28
stations). A validation of the micronucleus test was performed
repeatedly in a variety of laboratory exposure studies using
contaminants fromdifferent chemical groups (Barsien_ e et al., 2004,
2005b, 2006a, 2006b, 2010a, 2010b; Bagni et al., 2005; Barsien_ e
and Andreik_ enait _ e, 2007; unpublished data).
The existing large database allows dening the reference and
background levels of genotoxicity responses in different marine
sh and molluscs, i.e., the formation of micronuclei (MN), nuclear
buds (NB) and bi-nucleated cells with nucleoplasmic bridges (BNb).
These endpoints reect the action of aneugenic and clastogenic
substances in different species inhabiting various regions of the
Baltic Sea and other marine ecosystems.
The main goal of the present study was to assess environmental
genotoxicity levels in blood erythrocytes of three of the most
common native sh species of the Baltic Sea and to map genotox-
icity risk levels in different zones of the Baltic Sea in 2009e2011. As
indicator of genotoxicity, the formation of micronuclei in blood
erythrocytes, as a large lesion at a sub-cellular level, was evaluated.
The selection of sh blood erythrocytes as a target cell to investi-
gate genetic damage was based on the important role of blood in
the transfer of hazardous substances absorbed through skin, gill
and other tissues of the aquatic organisms.
2. Materials and methods
Material for the micronuclei (MN) analysis in ounder, herring
and eelpout was collected from June 2009 to March 2011 at a total
of 82 study stations located in different regions of the Baltic Sea.
The locations of the sh sampling stations are presented in Figs. 1
and 2. The list of the sh sampling stations and their
Fig. 1. Results of environmental genotoxicity risk assessment in ounder (Platichthys esus), herring (Clupea harengus) and eelpout (Zoarces viviparus) collected from different
regions of the Baltic Sea in 2009.
J. Barsien_ e et al. / Marine Environmental Research 77 (2012) 35e42 36
geographical coordinates is presented in Table 1. Samples were
obtained from the trawls research catches carried out by the RV
Walther Herwig III and the RV Baltica as well as from local
shermen. The analysis of MN was carried out following the
method described earlier by Barsien_ e et al. (2004). Only alive
specimens in good health condition of approximately the same size
were processed for further analysis. For age determination otoliths
were removed.
The analysis of MN was performed in ounder P. esus from 52
stations, in herring C. harengus from 59 stations, and in eelpout
Z. viviparus from 29 stations. Since in some study stations the
sampling of sh was done during two surveys in 2009, as well as in
2010, the MN analysis was carried out in a total of 168 sampling
groups (Table 2). The MN frequency was determined in 1892 sh
specimens: 714 ounder, 759 herring and 419 eelpout.
Assessment of the genotoxicity risk in each of 82 studied
stations was done on a basis of the established background
response (BR) of MN incidences in ounder (<0.23 MN/1000
erythrocytes in offshore, <0.29 MN/1000 erythrocytes in coastal
zones), in herring (<0.39 MN/1000 erythrocytes) and in eelpout
(<0.38 MN/1000 erythrocytes). The background level of MN
frequencies was calculated as the empirical 90% percentile (P90) in
different sh species collected in the 2001e2010 period from the
reference sites Kvdfjrden, Palanga, Leba, Prnu, 1a-1, 2a-1 and
2b-1, that are characterized by no known local sources of
contamination and no impact from human and industrial activity.
The P90 value separates the upper 10% of all MNvalues 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 belong to individuals that are unexposed, weakly-
exposed or non-responding or adapted to stressful conditions.
The establishment of BR was done following suggestions by Fenech
(1993), that the background frequency of micronuclei can be
dened as MN frequency observed in the reference sites with
absence of environmental risk or in experimental pre-exposure of
organisms to genotoxins.
In each of the study stations, the percentage of the sh speci-
mens with MN frequencies exceeding the background level of MN
was assessed and mapped in the GIS system. The study stations
were grouped into a 5-grade scale, i.e.,: 0.0e19% of specimens with
MN frequencies higher than the background level was indicated as
a low, 20e39% e as moderate, 40e59% eas increased, 60e79% e as
high and 80e100% e as exceptionally high genotoxicity risk level.
Since, at some study stations, two or three species were
collected, results from the ounder analysis are marked exactly
under the geographical coordinates of the sh sampling. The other
species are marked closely to the ounder. In the southern Baltic
Sea study stations B01, B12, B11, B10, SFI4, the sampling of sh was
done two to four times, therefore, on the left side, data of earlier
sampling (in June and September 2009) are presented and, on the
right side, of sampling in November and December of 2009. On the
map of 2009, also the results received in November and December
are marked, using black contour bookmarks. In the map of
2010e2011, such black contour bookmarks were used to express
the responses in sh collected in May and August 2010.
The statistical analysis was carried out using the GraphPad
PRISM 5.0 statistical package. Data normality and variance homo-
geneity was checked using the KolmogoroveSmirnov test. The non-
parametric ManneWhitney U-test was used to compare nuclear
alteration frequencies in the sh males and females. Pearsons
correlation was performed to illustrate possible relationships
between MN frequency and environmental variables or the sh
biometrical measurements. Genotoxicity risk zones were mapped
utilizing programme ArcGIS Desktop by using ArcMap application.
Fig. 2. Results of environmental genotoxicity risk assessment in ounder (Platichthys esus), herring (Clupea harengus) and eelpout (Zoarces viviparus) collected from different
regions of the Baltic Sea in 2010 and 2011.
J. Barsien_ e et al. / Marine Environmental Research 77 (2012) 35e42 37
3. Results
The assessment of environmental genotoxicity levels in 62
sampling groups of ounder, herring and eelpout collected in 2009
indicated 16 sampling groups attributable to a low, 9 to a moderate,
10 to an increased, 9 to a high and 18 to an extremely high geno-
toxicity risk levels. Study stations characterized being at extremely
high genotoxicity risk for ounder and herring were located
predominantly in the Gulf of Riga and the southern Baltic Sea.
Eelpout, in contrast, predominantly showed low genotoxicity risk
zones, such as the reference station Prnu, the Swedish and Danish
coastal waters and the Wismar Bay. Low and moderate levels of
environmental genotoxicity dominated in herring from the Gulf of
Finland. When comparing responses of the sh species from the
same study stations, interspecies differences were observed, and
the cytogenetic damage always was highest in ounder (Fig. 1).
In 2010e2011, the analysis and assessment of environmental
genotoxicity in 106 sampling groups of ounder, herring and
eelpout captured during various sampling campaigns indicated
only 7 sampling groups attributable to a low, 15 to a moderate, 17 to
an increased, 13 to a high and 54 to an extremely high genotoxicity
risk level (Fig. 2). It should be pointed out that in 2010e2011, 74% of
the ounder (34 of 46 sampling groups) and 42% (20 of 48 sampling
groups) of the herring sampling groups were classied as living in
areas with extremely high genotoxicity risk mainly located in the
southern and eastern Baltic Sea offshore zones. However, 42% (5 of
12 sampling groups) of eelpout groups were living in low geno-
toxicity risk zones located in the Gulf of Bothnia and the Roskilde
area in Denmark (Table 3) and (Fig. 2). The average means of MN
frequencies were also higher in ounder compared to herring or
eelpout.
Summarizing the results for all three sh species collected in
the period 2009e2011, it emerged that 72 of 168 analyzed
sampling groups (42.9%) were attributed to extremely high
genotoxicity risk zones and only 13.7% of them could be assigned
to low genotoxicity risk zones (Table 3). In 2009, 29.0% were
Table 1
The list of the sh sampling stations and their geographical coordinates (the list running fromthe Danish waters and nished at the Gulf of Bothnia in the northern Baltic Sea).
No Stations Latitude Longitude No Stations Latitude Longitude
1 2IV 55

43.08
0
N 11

47.05
0
E 35 3a 54

31.60
0
N 54

33.00
0
N 19

21.50
0
E 19

22.00
0
E
2 2FV 55

57.05
0
N 12

01.00
0
E
3 2RV 55

41.50
0
N 55

42.80
0
N 12

04.00
0
E 12

05.00
0
E 36 SFI4 54

52.53
0
N 55

01.59
0
N 17

21.97
0
E 17

29.62
0
E
4 2AV 55

12.00
0
N 11

12.50
0
E 37 SFI3 54

46.71
0
N 54

50.74
0
N 18

40.99
0
E 18

43.97
0
E
5 B01 54

32.10
0
N 54

40.90
0
N 10

25.80
0
E 10

47.40
0
E 38 SFI2 54

25.62
0
N 54

26.99
0
N 19

01.44
0
E 19

02.83
0
E
6 B12 54

13.92
0
N 54

27.00
0
N 11

23.22
0
E 11

46.91
0
E 39 SFI1 54

28.44
0
N 54

25.89
0
N 19

16.04
0
E 19

21.25
0
E
7 EW 53

56.67
0
N 11

22.35
0
E
8 WD 53

54.82
0
N 11

26.31
0
E 40 BP3 55

31.86
0
N 55

46.42
0
N 20

30.74
0
E 20

40.19
0
E
9 SH 54

02.48
0
N 11

32.40
0
E
10 J2 54

48.30
0
N 12

18.10
0
E 41 26 LT 55

48.50
0
N 20

04.00
0
E
11 B11 54

43.60
0
N 54

49.18
0
N 13

12.84
0
E 13

54.23
0
E 42 27 LT 55

47.60
0
N 20

11.50
0
E
43 28 LT 55

42.00
0
N 19

58.40
0
E
12 B10 54

37.95
0
N 54

52.25
0
N 14

01.63
0
E 14

02.47
0
E 44 30 LT 55

39.40
0
N 20

15.80
0
E
45 25 LV 56

28.00
0
N 20

12.40
0
E
13 B03 54

33.70
0
N 14

58.75
0
E 46 17 LV 56

38.30
0
N 20

42.80
0
E
14 B05 55

06.23
0
N 16

30.97
0
E 47 14 LV 57

13.00
0
N 20

42.60
0
E
15 19 54

26.20
0
N 54

26.40
0
N 15

09.50
0
E 15

12.00
0
E 48 15 LV 57

20.90
0
N 20

54.60
0
E
49 11 LV 57

28.00
0
N 21

01.60
0
E
16 21 54

27.50
0
N 54

27.20
0
N 15

37.40
0
E 15

40.00
0
E 50 6 LV 57

22.20
0
N 21

15.10
0
E
51 5 LV 57

30.10
0
N 21

25.20
0
E
17 22 54

23.00
0
N 54

22.90
0
N 15

46.30
0
E 15

48.80
0
E 52 GoR1 57

50.31
0
N 57

50.92
0
N 24

00.11
0
E 24

00.33
0
E
18 15a 54

39.40
0
N 54

39.50
0
N 15

09.80
0
E 15

08.90
0
E 53 GoR2 57

22.96
0
N 57

18.68
0
N 23

13.31
0
E 23

16.02
0
E
19 17a 54

34.00
0
N 54

34.80
0
N 15

38.50
0
E 15

36.80
0
E 54 GoR3 57

04.51
0
N 57

08.29
0
N 23

54.83
0
E 24

02.43
0
E
20 18a 54

33.30
0
N 54

33.90
0
N 15

24.60
0
E 15

22.30
0
E 55 SLK 56

52.00
0
N 16

25.00
0
E
56 Gaso 58

13.60
0
N 16

24.30
0
E
21 23 54

31.70
0
N 54

30.70
0
N 15

47.30
0
E 15

49.20
0
E 57 Marso 57

13.30
0
N 16

42.13
0
E
58 KVD 58

01.05
0
N 16

46.57
0
E
22 25 54

32.00
0
N 54

33.60
0
N 16

00.00
0
E 16

00.20
0
E 59 3 E 58

02.00
0
N 21

00.40
0
E
60 4 E 57

53.60
0
N 21

20.00
0
E
23 19a 54

39.20
0
N 54

38.90
0
N 15

34.10
0
E 15

31.70
0
E 61 SRV 58

02.00
0
N 22

16.00
0
E
62 TRM 57

56.00
0
N 24

17.00
0
E
24 21a 54

38.00
0
N 54

36.60
0
N 15

53.70
0
E 15

52.50
0
E 63 Parnu 58

16.00
0
N 24

20.00
0
E
64 KH 58

07.00
0
N 23

58.00
0
E
25 24 54

37.60
0
N 54

36.20
0
N 16

03.50
0
E 16

02.00
0
E 65 1b-1 59

15.00
0
N 59

15.26
0
N 23

07.04
0
E 23

04.30
0
E
26 22a 54

44.20
0
N 54

43.10
0
N 15

54.70
0
E 15

54.70
0
E 66 Nova 59

14.11
0
N 23

42.00
0
E
67 2b-1 59

36.70
0
N 24

12.68
0
E
27 28 54

52.60
0
N 54

52.80
0
N 16

39.50
0
E 16

41.90
0
E 68 2b-2 59

46.23
0
N 59

43.77
0
N 25

27.98
0
E 25

23.06
0
E
28 23a 54

48.70
0
N 54

47.50
0
N 16

01.00
0
E 15

59.30
0
E 69 3b-1 59

47.51
0
N 26

09.28
0
E
70 4b-1 59

36.34
0
N 27

02.76
0
E
29 28a 55

02.60
0
N 55

02.30
0
N 16

24.00
0
E 16

21.60
0
E 71 4a-4 60

12.15
0
N 27

12.73
0
E
72 3a-1 60

06.34
0
N 26

20.31
0
E
30 8a 55

23.10
0
N 55

23.00
0
N 17

19.60
0
E 17

17.00
0
E 73 2a-1 59

45.16
0
N 24

09.13
0
E
74 1a-1 59

37.64
0
N 23

13.67
0
E
31 7a 55

21.30
0
N 55

21.50
0
N 17

25.40
0
E 17

22.80
0
E 75 BS1/23 60

46.05
0
N 18

05.03
0
E
76 BS1/20 60

45.66
0
N 18

05.13
0
E
32 6a 55

15.80
0
N 55

15.90
0
N 17

23.30
0
E 17

20.90
0
E 77 BS1/24 60

47.11
0
N 18

05.17
0
E
78 BS1/22 60

49.70
0
N 18

05.41
0
E
33 3 55

06.40
0
N 55

06.30
0
N 18

42.90
0
E 18

45.70
0
E 79 BS1/21 60

49.70
0
N 18

04.43
0
E
80 BS2/27 61

35.10
0
N 17

48.36
0
E
34 4a 54

40.90
0
N 54

41.70
0
N 19

16.10
0
E 19

14.00
0
E 81 BS2/26 61

35.07
0
N 17

48.11
0
E
82 BS2/25 61

35.07
0
N 17

48.27
0
E
J. Barsien_ e et al. / Marine Environmental Research 77 (2012) 35e42 38
assigned to extremely high and 25.8% to low genotoxicity risk
zones, whilst in 2010e2011, the percentages were 50.9% and
6.6%, respectively. Comparing environmental genotoxicity levels
in sh from the same study stations between 2009 and 2010,
an increase of genotoxicity risk was found in sh collected in
2010.
Analysis of MN levels in ounder in 2009e2011 revealed that
90.9% of sampling groups could be attributed to high (21.2%) and to
extremely high (69.7%) genotoxicity risk levels. In herring, 39.7% of
the sampling groups showed high (9.0%) or extremely high (37.3%)
genotoxicity risk level. In eelpout, only 8.6% of the sampling groups
showed such characteristics (Table 3).
Assessing the numbers of ounder inhabiting sites character-
ized as extremely high genotoxicity risk zones in the Baltic Sea, it
emerged that 11 stations of 16 studied in 2009 and 33 stations of 41
studied in 2010e2011 were attributed to zones of extremely high
genotoxicity risk. For herring, there were 5 of 18 stations in 2009
and 20 of 43 stations in 2010e2011. A clearly different situation
appeared in eelpout because only one station located in the Gulf of
Riga, out of 20 stations studied, was characterized by an extremely
high genotoxicity risk in 2009, and none of those was found in
2010e2011 (Figs. 1 and 2).
Pearson correlation analysis of micronuclei frequency and
environmental variables (water temperature, salinity, oxygen
concentration, oxygen saturation and depth of sampling) showed
that the depth was the only inuencing factor for the formation of
MN in eelpout inhabiting the BS1 area in the Gulf of Bothnia.
Pearson correlation analysis of MN frequency and biometrical
variables (sh age, length, weight, liver weight) in sh from the 82
study stations showed strong relationships only in sh from
stations GoR3, GoR2 and SFI2 in 2009 and in sh collected from
stations 3EST, 4EST, 6LV, 7a, 15a and 17a in 2011. It should be
emphasized that station 7a is located closely to an oil platform, and
stations 15a and 17a close to known dumping site of chemical
munitions in the Polish Bornholm zone. Gender specic differences
were found only in sh from two stations. Signicantly higher MN
frequency was observed in females of herring collected only from
25LV (p 0.018; ManneWhitney U-test) and females of ounder
from 17a (p 0.017) station.
4. Discussion
The EU Baltic Sea Strategy Action document (Baltic Sea Strategy
Action, 2009) stresses that hazardous substances, including organic
Table 2
Materials for the analysis of micronuclei (MN) in peripheral blood erythrocytes of ounder, herring and eelpout collected from different zones of the Baltic Sea in 2009e2011
mainly during surveys of the RVs Walther Herwig III (Germany) and Baltica (Poland) (for location of sampling sites see Figs. 1 and 2).
Sampling date Flounder sampling stations
(number of specimens)
(66 sampling groups from 52 stations)
Herring sampling stations
(number of specimens)
(67 sampling groups from 59 stations)
Eelpout sampling stations
(number of specimens)
(35 sampling groups from 29 stations)
June 2009 e e SH (10), EW (10), WD (10), TRM (18), Prnu (9)
September 2009 B01 (10), B12 (10), B11 (10),
B10 (10), B05 (4), SFI4 (10),
BP3 (10), 1b-1 (10)
B11 (6), 4a-4 (11), 3a-1 (17), 2a-1 (10),
1a-1 (10), 4b-1 (10), 3b-1 (10), 2b-2 (10),
2b-1 (10), 1b-1 (10)
SFI4 (5), 3a-1 (10), 4b-1 (9), 1b-1 (5)
November 2009 e e 2R (20), 2F (16), 2A (15), 2I (10), SH (10),
EW (18), WD (10), SLK (10), Gaso (10),
Marso (10), KVD (10)
December 2009 2F (9), B01 (10), B12 (11), B11 (11),
B03 (10), SFI1 (10), SFI2 (10), SFI3 (10),
SFI4 (10), GoR1 (12), GoR2 (23), GoR3 (11)
B11 (11), B03 (14), SFI1 (10), SFI2 (10),
SFI3 (10), SFI4 (10), GoR1 (10),
GoR2 (10), GoR3 (10)
GoR1 (10), GoR2 (10), GoR3 (10)
May 2010 Kihnu (19), Nova (21) e 2I (12), 2F (10), 2R (10), Sorve (10), Nova (12)
August 2010 B01 (4), J2 (4), B12 (10), B11 (10), B10 (10) B01 (4), J2 (5), B12 (15), B11 (8), B10 (8) e
November 2010 3 (3), 19 (10), 21 (2), 22 (10), 23 (8),
24 (5), 25 (9), 28 (6)
3 (10), 19 (10), 21 (10), 22 (10), 23 (10),
24 (10), 25 (10), 28 (10)
e
December 2010 B01 (19), J2 (20), B12 (20), B11 (20),
B10 (20), SFI4 (20)
B01 (20), J2 (20), B12 (20), B11 (20), B10 (20),
SFI4 (20), BS1/20 (20), BS1/21 (10), BS2/25 (30)
BS1/20 (10), BS1/22 (7), BS1/23 (6), BS1/24 (6),
BS2/25 (18), BS2/26 (8), BS2/27 (4)
February 2011 3a (10), 4a (10), 6a (10), 7a (10), 8a (10),
15a (10), 17a (10), 18a (10), 19a (10),
21a (10), 22a (10), 23 (10)a, 28a (10)
3a (10), 4a (10), 6a (10), 7a (10), 8a (10),
15a (10), 17a (10), 18a (10), 19a (10),
21a (10), 22a (10), 23a (10), 28a (10)
e
March 2011 26LT (10), 27LT (10), 28LT (10), 30 LT(10),
25LV (10), 17LV (10), 15LV (10), 14LV (10),
6LV (10), 5LV (10), 4EST (10), 3EST (10)
26LT (10), 27LT (10), 28LT (10),
30LT(10), 25LV (10), 17LV (10), 15LV (10),
14LV (10), 11LV (10), 6LV (10), 5LV (10),
4EST (10), 3EST (10)
e
Table 3
Number of the sh sampling groups where the frequency of micronuclei (MN) was higher than the background level in the species.
Species Sampling year Number of the sh sampling groups and percentage of specimens (in brackets) Total number of the sh
sampling groups
0e19& 20e39& 40e59& 60e79& 80e100&
Flounder 2009 0 0 1 7 12 20
2010e2011 0 1 4 7 34 46
Herring 2009 5 4 5 0 5 19
2010e2011 2 11 9 6 20 48
Eelpout 2009 11 5 4 2 1 23
2010e2011 5 3 4 0 0 12
Total 2009 16 (25.8%) 9 (14.5%) 10 (16.2%) 9 (14.5%) 18 (29.0%) 62
2010e2011 7 (6.6%) 15 (14.2%) 17 (16.0%) 13 (12.3%) 54 (50.9%) 106
Total 2009e2011 23 (13.7%) 24 (14.2%) 27 (16.1%) 22 (13.1%) 72(42.9%) 168
Total Flounder 0 (0.0%) 1 (1.5%) 5 (7.6%) 14 (21.2%) 46 (69.7%) 66
Herring 7 (10.4%) 15 (22.4%) 14 (20.9%) 6 (9.0%) 25 (37.3%) 67
Eelpout 16 (45.6%) 8 (22.9%) 8 (22.9%) 2 (5.7%) 1 (2.9%) 35
J. Barsien_ e et al. / Marine Environmental Research 77 (2012) 35e42 39
contaminants and heavy metals, as well as chemical weapons
dumped in the Baltic Sea, may persist in the environment for very
long periods, may accumulate in marine organisms and, thus,
continue to be a risk for the ecosystem health. A large number of
pollution hotspots in the Baltic Sea have been dened, and, thus,
more attention has to be paid to reduce the use and the impact of
hazardous substances at an ecosystem level. The Baltic Sea area
could serve as a model area for the development of relevant novel
long-term management strategies and decision-making
approaches. In addition, it has been pointed out that adequate
research, applying indicators at the ecosystem level, should
endorse the progress towards a sustainable shery in the Baltic Sea
(Baltic Sea Strategy Action, 2009).
In the present study, a special focus was placed on the ability of
the micronucleus (MN) assay to identify and quantify environ-
mental genotoxicity in three common sh species, inhabiting 82
study stations located in different regions of the Baltic Sea
ecosystem. Data reported are based on a low cost, easy to perform
and non-destructive genotoxicity assay for in situ evaluation of
environmental risk to native Baltic Sea sh species. Frequencies of
MN were detected at differently polluted sites with the aim to
evaluate genotoxicity levels in different regions of the Baltic Sea.
This is also a rst attempt to evaluate the background MN
responses dened, as well as low, high and extremely high geno-
toxicity risk levels for native sh species.
The MN assay is a toxicogenetic technique considered to be
a sensitive and informative marker of environmental genotoxicity.
MN analysis widely has been used as marker of genotoxicity in
different organisms and the assay has extensively been applied to
identify adverse potential of various genotoxic agents. Formation of
MN reects chromosomal instability, disrupted cell cycle check-
point machinery, potential carcinogenesis, and a defective DNA
damage repair process in the cells. Recently it was pointed out that
micronuclear DNA content can be degraded and, consequently,
gene loss and chromosomal instability in general can be induced
(Terradas et al., 2010). Unlike DNA single strands breaks, MN
represents non-repaired genetic damage.
In the last decades, the use of sh as sentinel organisms in
monitoring programmes associated with the description of envi-
ronment contamination by heavy metals, PAHs and other
hazardous compounds has been shown to be an appropriate
methodology. The atsh ounder (P. esus) and the viviparous
eelpout (Z. viviparus) were used in the present study as sentinel
species distributed widely in the Baltic Sea and, due to their direct
contact to the sediment, are particularly exposed to multi-
contaminant mixtures. The utility of ounder and eelpout in
pollution monitoring within different zones of the Baltic Sea has
been conrmed during the pan-European project BEEP (Biological
Effects of Environmental Pollution on Marine Coastal Ecosystems,
2001e2004) and was continued within the BSRP, BONUS BEAST,
BONUS BALCOFISH and GENCITOX (Lithuanian Science Council)
projects in 2005e2011. The major advantages of these two species
are that they are locally abundant and comparatively stationary,
and, consequently, these species are representatives of regional
environmental conditions including contaminant exposure.
Herring (C. harengus) is one of the most abundant and main
economically important sh species, is a widely-distributed pelagic
species with migrating potential within a wide range of environ-
mental conditions, and, thus, reect cumulative effects fromlarger-
scale areas.
Our previous studies demonstrated elevated MN frequencies in
organisms collected from an oil terminal and marine port zones in
the Lithuanian waters of the Baltic Sea (Barsien_ e and Barsyt _ e
Lovejoy, 2000; Barsien_ e, 2002). Signicantly increased levels of
micronuclei, nuclear buds and fragmented-apoptotic cells were
found in sh and bivalves inhabiting the Baltic Sea after the oil spill
in the B uting_ e oil terminal in November 2001 and January 2008
(Barsien_ e et al., 2006c, 2006d, 2008, in press) and in the vicinity of
the Russian oil platform D-6 (Barsien_ e et al., unpublished data).
Increased environmental genotoxicity was detected in 2002e2004
in the Gulf of Gdansk (Barsien_ e et al., 2006b), in the Mecklenburg
and Kiel Bights, and in extensive ship trafc zones (Rybakovas et al.,
2009). In the present study, an increased numbers of ounder and
herring individuals with elevated levels of MN in blood erythro-
cytes were recorded at most of the study sites in the eastern and
southern parts of the Baltic Sea. At the stations characterized
having an increased (40e59% numbers of individuals exceeding
MN background level), high (60e79% individuals) and extremely
high genotoxicity risk (80e100% specimens possessing MN levels
higher than background response), there is evidence that sh
populations are signicantly exposed to genotoxins and their
habitats can, thus, be suspected to represent a poor or bad status of
ecosystem health. It is important to stress that in ounder from
most of stations attributed to extremely high genotoxicity risk
zones, the micronuclei incidences in 2010e2011 were at higher
levels than MN frequency in those with an impaired sh health.
In herring, MN frequencies higher than the background level
were found in 37.3% of studied samplings groups categorized as
having an extremely high genotoxicity risk level. Lowand moderate
levels of environmental genotoxicity predominated in herring
collected in September 2009 from nine stations of the Gulf of
Finland. However, at two stations (4a-1 and 3a-1) located in the
Gulf of Finland, 15.6% of herring specimens examined showed a MN
frequency up to 2500 times higher than the background level. This
indicates the existence of zones in the Gulf of Finland with an
extremely high genotoxicity risks, possibly associated with pollu-
tion by aneugenic and clastogenic compounds triggering an
extensive formation of micronuclei and the occurrence of irre-
versible genetic changes in the herring.
Summarizing the results of the present study, it should be
pointed out that in 2009e2011 ounder from 80.8% and herring
from 42.4% of the study stations in the Baltic Sea were living in
ecological conditions reecting extremely high level of environ-
mental genotoxicity. Since MN frequencies in sh were monitored
at a large number of study stations in 2001e2010, it was possible to
identify an increase in the level of environmental genotoxicity in
2009e2011. Long-term environmental genotoxicity studies
(2001e2011) in different zones of the Baltic Sea showed lower MN
levels in sh collected in 2001e2007. There were only some cases of
MN frequency elevation due to accidental spills of contaminants, as
well as in zones close to river estuaries or industrial activities
(Barsien_ e et al., 2004, 2008, in press; Kopecka et al., 2006; Schiedek
et al., 2006; Rybakovas et al., 2009; our data published in HELCOM,
2010). During earlier observations in the Baltic Sea (in 2001e2003),
MN frequencies exceeded the background level in 80e100% of
ounder examined only at seven coastal stations and none of the
offshore stations. These stations were located in the Wismar Bay (in
spring 2001), in the Gulf of Gdansk (autumn 2001 and spring 2003)
and off the Lithuanian coast (in September 2001 and June 2002)
(own unpublished data).
Stressful conditions in the Baltic Sea triggering signicant
increase of micronuclei levels in sh were evident in 2009e2011.
Pearson correlation analysis of micronuclei frequency and envi-
ronmental (temperature, salinity, bottom depth, oxygen saturation
and concentration) and sh biometrical (total length, weight, liver
weight and age) variables showed signicant relationships in sh
from 10 stations out of 82 stations studied. In general, the increase
of genotoxicity in 2009e2010 in sh fromthe most studied stations
was related strongly neither to hydrologic, nor to biological vari-
ables. The tendency was appeared especially in ounder from ten
J. Barsien_ e et al. / Marine Environmental Research 77 (2012) 35e42 40
study sites in the southern Baltic Sea (Barsien_ e et al., in prepara-
tion). Instead, chemical stress might be considered as one of
determinants provoking an environmental genotoxicity effects in
most of the locations studied. For instance in experimental expo-
sures, a strong and positive correlation (r 0.980) between total
metal concentrations and nuclear abnormality frequency has been
described in tilapias Oreochromis niloticus (Summak et al., 2010),
and a linear correlation (R
2
0.8444) between MN frequency and
PAH body burden in M. edulis (Sundt et al., 2011).
The application of cytogenetic methods can provide us with
information regarding organisms exposure to genotoxic agents in
situ and can be of great interest in the assessment of complex
mixtures effects. Analysis of MN in marine animals is one of the
methods recognized as particularly relevant for biological effects
monitoring purposes (Hylland et al., 2008; Brooks et al., 2011;
Sundt et al., 2011). Consequently, the application of the MNassay in
an integrated monitoring and assessment programme will help to
identify problems related to pollution and to dene future
management tasks, especially those identied in the HELCOM
Baltic Sea Action Plan, i.e. to achieve of good ecological status and
healthy wildlife in the Baltic Sea. The MN assay in sh would be
an informative indicator in the assessment of the Ecological Quality
Objectives Hazardous substances within the marine environment
shall not cause irreversible changes in the functioning of the
ecosystem and in humans and Toxic substances shall not cause
sub-lethal, intergenerational or transgenic effects to the health of
marine organisms (e.g., reproductive disturbances) as was
expressed in the Baltic Sea Action Plan.
5. Conclusions
This is a rst attempt to evaluate the background MN responses,
as well as low, high and extremely high genotoxicity risk levels for
native sh species inhabiting the Baltic Sea. Environmental geno-
toxicity risk was evaluated in three sh species collected from 82
study stations located in different regions of the Baltic Sea. The
frequencies of micronuclei (MN) were analyzed in blood erythro-
cytes of ounder P. esus (714 specimens), herring C. harengus (759
specimens) and eelpout Z. viviparus (419 specimens) collected in
2009, 2010 and 2011. Environmental genotoxicity risk was assessed
by using MN background levels in sh, developed using MN data
obtained in studies carried out in the period 2001e2011 from 107
study locations of the Baltic Sea. Extremely high genotoxicity risk
zones were found for ounder at 11 stations out of 16 in 2009 and
33 stations of 41 in 2010e2011, for herring, at 5 of 18 stations in
2009 and 20 of 43 stations in 2010e2011, in eelpout only at one out
of 29 stations. Study stations with an extremely high genotoxicity
risk were located mainly in the southern and eastern Baltic Sea
offshore zones. When comparing sh species collected at the same
station, it was found that there always was a higher percentage of
ounder with MN frequencies exceeding the background level
compared to herring and eelpout.
Conict of interests
None
Acknowledgements
We are thankful to Lars Frlin (Goteborg University, Sweden) for
providing material from four Swedish stations, Jens Gercken
(Institute for Applied Ecology, Germany) for providing material
fromthree stations in the Wismar Bay and Arvo Tuvikene (Estonian
University of Life Sciences, Estonia) for providing material fromve
stations at the Estonian coast. This study was funded mainly by
Lithuanian Science Council for the genotoxicity analysis in 55 study
stations (MIP-62/2010 GENCITOX project) and by BONUS BEAST
project (FP/2007e2013 under grant agreement no 217246) for the
analysis in 27 stations in the Little Belt, in the Gulfs of Gdansk, Riga,
Finland and Bothnia.
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