envi toxicity in baltic sea

5/20/2018 Envi Toxicity in Baltic Sea

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Risk of environmental genotoxicity in the Baltic Sea over the period of 2009e2011assessed by micronuclei frequencies in blood erythrocytes ofounder(Platichthys esus), herring (Clupea harengus) and eelpout (Zoarces viviparus)

Janina Barsien _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, Lithuaniab vTI Institute of Fisheries Ecology, Deichstrae 12, 27472 Cuxhaven, Germanyc 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

FlounderHerring

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 ofounderPlatichthys

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 2010 e2011, 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 theframework of the EU funded pan-European BEEP project on bio-logical effects of contaminants in organisms inhabiting the BalticSea, Lehtonen et al., 2006 concluded that, although the loads of

some classical chemical toxic substances (e.g., PCBs, DDTs) havebeen reduced over the last decades, chemical pollution by a widespectrum 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 datafrom the period 1999e2007 for hazardous substances and selectedbiological 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 thesouthern 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 outthat a large number of different substances exceeded the thresholdlevels in the different Baltic Seasub-basins.In sh,musselsand bird

tissues, PCBs, dioxins, heavy metals, organometals, alkylphenolsphthalates, brominated compounds, polycyclic aromatic hydrocar-bons (PAHs), DDTs and chlorinated pesticides, and caesium-137were found at the highest concentrations in relation to targe

levels. The southern region of the Baltic Sea is polluted by all of theabove-mentioned substances (HELCOM, 2010). Many of thesesubstances inherently are genotoxic compounds and may exergenotoxicity effects via direct action or the activation of toxic

metabolic mechanisms and are, thus, of concern regarding theirpotential impact on aquatic organisms and human health. Chemicasubstances with genotoxicity potential can be sub-divided into fourgroups: (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:[email protected](J. Barsien _e).

Contents lists available atSciVerse ScienceDirect

Marine Environmental Research

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c om / l o c a t e / m a r e n v r e v

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
mailto:[email protected]://www.sciencedirect.com/science/journal/01411136http://www.elsevier.com/locate/marenvrevhttp://dx.doi.org/10.1016/j.marenvres.2012.01.004http://dx.doi.org/10.1016/j.marenvres.2012.01.004http://dx.doi.org/10.1016/j.marenvres.2012.01.004http://dx.doi.org/10.1016/j.marenvres.2012.01.004http://dx.doi.org/10.1016/j.marenvres.2012.01.004http://dx.doi.org/10.1016/j.marenvres.2012.01.004http://www.elsevier.com/locate/marenvrevhttp://www.sciencedirect.com/science/journal/01411136mailto:[email protected]


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substances that increase the production of reactive oxygen species

(ROS) and free radicals, which can subsequently damage both DNAbases and the deoxyribose backbone; (4) substances that inhibitDNA synthesis and repair (Lee and Steinert, 2003). Genotoxic

compounds canbind to DNA causing the formation of DNA adducts,single and double strand breakages, modications in DNA repairand crosslink consistent pattern, as well as alterations of cellfunctions, reproduction disturbances, growth inhibition, or even

carcinogenesis (Ohe et al., 2004). As a consequence, furthergenerations of the organisms can suffer from reduced tness,fertility or embryonic viability (Russo et al., 2004). Non-repairedgenetic damage is considered important since it provides a funda-

mental early warning sign of adverse long-term effects ofcontaminants 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 genotoxicresponses to pollution (Jha, 2008).

Environmental genotoxicity in the Baltic Sea was earlierassessed in the Swedish part of the Gulf of Bothnia ( Al-Sabti and

Hrdig, 1990) and in Danish waters in Kge Bay, Little Belt, StoreBelt and Kattegat (Wrisberg et al., 1992). Later studies, carried out

by the Institute of Ecology (Lithuania), covered the Lithuanianeconomic 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 _eet 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 _eet al., 2006b;Schiedek et al., 2006) and 12 offshore areasof 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 (Platichthysesus) 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. Environmentalgenotoxicity was also evaluated in bivalve mollusks Mytilus edulis

(45 stations),Mytilus trossulus (6 stations) andMacoma balthica(28stations). A validation of the micronucleus test was performedrepeatedly in a variety of laboratory exposure studies usingcontaminants from different chemical groups (Barsien _eet al., 2004,

2005b, 2006a, 2006b,2010a, 2010b;Bagni et al., 2005; Barsien _eand Andreik _enait _e, 2007; unpublished data).

The existing large database allows dening the reference andbackground levels of genotoxicity responses in different marinesh and molluscs, i.e., the formation of micronuclei (MN), nuclearbuds (NB) and bi-nucleated cells with nucleoplasmic bridges (BNb).These endpoints reect the action of aneugenic and clastogenicsubstances 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 mostcommon native sh species of the Baltic Sea and to map genotox-

icity risk levels in different zones of the Baltic Sea in 2009e2011. Asindicator of genotoxicity, the formation of micronuclei in blood

erythrocytes, as a large lesion at a sub-cellular level, was evaluated.The selection ofsh blood erythrocytes as a target cell to investi-gate genetic damage was based on the important role of blood inthe transfer of hazardous substances absorbed through skin, gilland other tissues of the aquatic organisms.

2. Materials and methods

Material for the micronuclei (MN) analysis in ounder, herringand eelpout was collected from June 2009 to March 2011 at a totalof 82 study stations located in different regions of the Baltic Sea.The locations of the sh sampling stations are presented inFigs. 1

and 2. The list of the sh sampling stations and their

Fig. 1. Results of environmental genotoxicity risk assessment in ounder (Platichthysesus), 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) 35e4236


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geographical coordinates is presented in Table 1. Samples wereobtained 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 themethod described earlier by Barsien _e et al. (2004). Only alivespecimens in good health condition of approximately the same sizewere processed for further analysis. For age determination otolithswere removed.

The analysis of MN was performed in ounderP.esusfrom 52stations, in herring C. harengus from 59 stations, and in eelpoutZ. viviparus from 29 stations. Since in some study stations thesampling ofsh was done during two surveys in 2009, as well as in

2010, the MN analysis was carried out in a total of 168 samplinggroups (Table 2). The MN frequency was determined in 1892 shspecimens: 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 (


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3. Results

The assessment of environmental genotoxicity levels in 62

sampling groups ofounder, herring and eelpout collected in 2009indicated 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 extremelyhigh genotoxicity risk for ounder and herring were locatedpredominantly in the Gulf of Riga and the southern Baltic Sea.

Eelpout, in contrast, predominantly showed low genotoxicity riskzones, such as the reference station Prnu, the Swedish and Danishcoastal waters and the Wismar Bay. Low and moderate levels ofenvironmental genotoxicity dominated in herring from the Gulf of

Finland. When comparing responses of the sh species from thesame study stations, interspecies differences were observed, andthe 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 indicatedonly 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 2010e

2011, 74% ofthe ounder (34 of 46 sampling groups) and 42% (20 of 48 samplinggroups) of the herring sampling groups were classied as living in

areas with extremely high genotoxicity risk mainly located in thesouthern and eastern Baltic Sea offshore zones. However, 42% (5 of12 sampling groups) of eelpout groups were living in low geno-

toxicity risk zones located in the Gulf of Bothnia and the Roskildearea in Denmark (Table 3) and (Fig. 2). The average means of MNfrequencies were also higher in ounder compared to herring oreelpout.

Summarizing the results for all three sh species collected inthe period 2009e2011, it emerged that 72 of 168 analyzedsampling 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 thesh sampling stations and their geographical coordinates (the list running from the Danish waters and nished at the Gulf of Bothnia in the northern Baltic Sea).

No Stations Latitude Longitude No Stations Latitude Longitude

1 2IV 5543.080N 1147.050E 35 3a 5431.600N 5433.000N 1921.500E 1922.000E

2 2FV 5557.050N 1201.000E

3 2RV 5541.500N 5542.800N 1204.000E 1205.000E 36 SFI4 5452.530N 5501.590N 1721.970E 1729.620E

4 2AV 5512.000N 1112.500E 37 SFI3 5446.710N 5450.740N 1840.990E 1843.970E

5 B01 5432.100N 5440.900N 1025.800E 1047.400E 38 SFI2 5425.620N 5426.990N 1901.440E 1902.830E

6 B12 54

13.920

N 54

27.000

N 11

23.220

E 11

46.910

E 39 SFI1 54

28.440

N 54

25.890

N 19

16.040

E 19

21.250

E7 EW 5356.670N 1122.350E

8 WD 5354.820N 1126.310E 40 BP3 5531.860N 5546.420N 2030.740E 2040.190E

9 SH 5402.480N 1132.400E

10 J2 5448.300N 1218.100E 41 26 LT 5548.500N 2004.000E

11 B11 5443.600N 5449.180N 1312.840E 1354.230E 42 27 LT 5547.600N 2011.500E

43 28 LT 5542.000N 1958.400E

12 B10 5437.950N 5452.250N 1401.630E 1402.470E 44 30 LT 5539.400N 2015.800E

45 25 LV 5628.000N 2012.400E

13 B03 5433.700N 1458.750E 46 17 LV 5638.300N 2042.800E

14 B05 5506.230N 1630.970E 47 14 LV 5713.000N 2042.600E

15 19 5426.200N 5426.400N 1509.500E 1512.000E 48 15 LV 5720.900N 2054.600E

49 11 LV 5728.000N 2101.600E

16 21 5427.500N 5427.200N 1537.400E 1540.000E 50 6 LV 5722.200N 2115.100E

51 5 LV 5730.100N 2125.200E

17 22 5423.000N 5422.900N 1546.300E 1548.800E 52 GoR1 5750.310N 5750.920N 2400.110E 2400.330E

18 15a 5439.400N 5439.500N 1509.800E 1508.900E 53 GoR2 5722.960N 5718.680N 2313.310E 2316.020E

19 17a 5434.000N 5434.800N 1538.500E 1536.800E 54 GoR3 5704.510N 5708.290N 2354.830E 2402.430E

20 18a 5433.300N 5433.900N 1524.600E 1522.300E 55 SLK 5652.000N 1625.000E

56 Gaso 5813.600N 1624.300E

21 23 5431.700N 5430.700N 1547.300E 1549.200E 57 Marso 5713.300N 1642.130E

58 KVD 5801.050N 1646.570E

22 25 5432.000N 5433.600N 1600.000E 1600.200E 59 3 E 5802.000N 2100.400E

60 4 E 5753.600N 2120.000E

23 19a 5439.200N 5438.900N 1534.100E 1531.700E 61 SRV 5802.000N 2216.000E

62 TRM 5756.000N 2417.000E

24 21a 5438.000N 5436.600N 1553.700E 1552.500E 63 Parnu 5816.000N 2420.000E

64 KH 5807.000N 2358.000E

25 24 5437.600N 5436.200N 1603.500E 1602.000E 65 1b-1 5915.000N 5915.260N 2307.040E 2304.300E

26 22a 5444.200N 5443.100N 1554.700E 1554.700E 66 Nova 5914.110N 2342.000E

67 2b-1 5936.700N 2412.680E

27 28 5452.600N 5452.800N 1639.500E 1641.900E 68 2b-2 5946.230N 5943.770N 2527.980E 2523.060E

28 23a 5448.700N 5447.500N 1601.000E 1559.300E 69 3b-1 5947.510N 2609.280E

70 4b-1 5936.340N 2702.760E

29 28a 5502.600N 5502.300N 1624.000E 1621.600E 71 4a-4 6012.150N 2712.730E

72 3a-1 6006.340N 2620.310E30 8a 5523.100N 5523.000N 1719.600E 1717.000E 73 2a-1 5945.160N 2409.130E

74 1a-1 5937.640N 2313.670E

31 7a 5521.300N 5521.500N 1725.400E 1722.800E 75 BS1/23 6046.050N 1805.030E

76 BS1/20 6045.660N 1805.130E

32 6a 5515.800N 5515.900N 1723.300E 1720.900E 77 BS1/24 6047.110N 1805.170E

78 BS1/22 6049.700N 1805.410E

33 3 5506.400N 5506.300N 1842.900E 1845.700E 79 BS1/21 6049.700N 1804.430E

80 BS2/27 6135.100N 1748.360E

34 4a 5440.900N 5441.700N 1916.100E 1914.000E 81 BS2/26 6135.070N 1748.110E

82 BS2/25 6135.070N 1748.270E



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assigned to extremely high and 25.8% to low genotoxicity riskzones, whilst in 2010e2011, the percentages were 50.9% and6.6%, respectively. Comparing environmental genotoxicity levelsin sh from the same study stations between 2009 and 2010,

an increase of genotoxicity risk was found in sh collected in2010.

Analysis of MN levels in ounder in 2009e2011 revealed that90.9% 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 groupsshowed such characteristics (Table 3).

Assessing the numbers of ounder inhabiting sites character-ized as extremely high genotoxicity risk zones in the Baltic Sea, itemerged that 11 stations of 16 studied in 2009 and 33 stations of 41studied in 2010e2011 were attributed to zones of extremely high

genotoxicity risk. For herring, there were 5 of 18 stations in 2009and 20 of 43 stations in 2010e2011. A clearly different situationappeared in eelpout because only one station located in the Gulf of

Riga, out of 20 stations studied, was characterized by an extremelyhigh genotoxicity risk in 2009, and none of those was found in2010e2011 (Figs. 1 and 2).

Pearson correlation analysis of micronuclei frequency andenvironmental variables (water temperature, salinity, oxygenconcentration, oxygen saturation and depth of sampling) showedthat the depth was the only inuencing factor for the formation of

MN in eelpout inhabiting the BS1 area in the Gulf of BothniaPearson correlation analysis of MN frequency and biometricavariables (sh age, length, weight, liver weight) in sh from the 82study 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 beemphasized that station 7a is located closely to an oil platform, andstations 15a and 17a close to known dumping site of chemica

munitions in the Polish Bornholm zone. Gender specic differenceswere found only in sh from two stations. Signicantly higher MNfrequency was observed in females of herring collected only from25LV (p 0.018; ManneWhitney U-test) and females ofounder

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 ofounder, 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 seeFigs. 1 and 2).

Sampling date Flounder sampling stations

(number of specimens)

(66 sampling groups from 52 stations)

Herring sampling stations



Eelpout sampling 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)

Ma y 2010 K ih nu (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 3Number of the sh sampling groups where the frequency of micronuclei (MN) was higher than the background level in the species.

Speci es Sampli ng yea r Number of the sh sampli ng gro ups a nd perc entage of specimens (in bra ckets) To ta l n umber of the sh

sampling groups0e19& 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


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contaminants and heavy metals, as well as chemical weapons

dumped in the Baltic Sea, may persist in the environment for verylong 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 ofhazardous substances at an ecosystem level. The Baltic Sea areacould serve as a model area for the development of relevant novel

long-term management strategies and decision-makingapproaches. In addition, it has been pointed out that adequateresearch, applying indicators at the ecosystem level, shouldendorse 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 Seaecosystem. Data reported are based on a low cost, easy to performand non-destructive genotoxicity assay for in situ evaluation ofenvironmental risk to native Baltic Sea sh species. Frequencies of

MN were detected at differently polluted sites with the aim toevaluate genotoxicity levels in different regions of the Baltic Sea.

This is also a rst attempt to evaluate the background MNresponses 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 bea sensitive and informative marker of environmental genotoxicity.

MN analysis widely has been used as marker of genotoxicity indifferent organisms and the assay has extensively been applied toidentify adverse potential of various genotoxic agents. Formation ofMN reects chromosomal instability, disrupted cell cycle check-

point machinery, potential carcinogenesis, and a defective DNAdamage repair process in the cells. Recently it was pointed out thatmicronuclear 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 otherhazardous compounds has been shown to be an appropriatemethodology. The atsh ounder (P. esus) and the viviparouseelpout (Z. viviparus) were used in the present study as sentinel

species distributed widely in the Baltic Sea and, due to their directcontact to the sediment, are particularly exposed to multi-contaminant mixtures. The utility of ounder and eelpout inpollution monitoring within different zones of the Baltic Sea has

been conrmed during the pan-European project BEEP (BiologicalEffects 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 speciesare that they are locally abundant and comparatively stationary,and, consequently, these species are representatives of regionalenvironmental conditions including contaminant exposure.

Herring (C. harengus) is one of the most abundant and maineconomically important sh species, is a widely-distributed pelagicspecies with migrating potential within a wide range of environ-mental conditions, and, thus, reect cumulative effects from larger-scale areas.

Our previous studies demonstrated elevated MN frequencies inorganisms collected from an oil terminal and marine port zones inthe Lithuanian waters of the Baltic Sea (Barsien _e and Barsyt _eLovejoy, 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 spillin the Buting _e oil terminal in November 2001 and January 2008(Barsien _eet al., 2006c,2006d,2008,in press) and in the vicinity ofthe Russian oil platform D-6 (Barsien _e et al., unpublished data).

Increased environmental genotoxicity was detected in 2002e2004in the Gulf of Gdansk (Barsien _eet al., 2006b), in the Mecklenburgand Kiel Bights, and in extensive ship trafc zones (Rybakovas et al.,2009). In the present study, an increased numbers ofounder and

herring individuals with elevated levels of MN in blood erythro-cytes were recorded at most of the study sites in the eastern andsouthern parts of the Baltic Sea. At the stations characterizedhaving an increased (40e59% numbers of individuals exceeding

MN background level), high (60e79% individuals) and extremelyhigh genotoxicity risk (80e100% specimens possessing MN levelshigher than background response), there is evidence that shpopulations are signicantly exposed to genotoxins and their

habitats can, thus, be suspected to represent a poor or bad status ofecosystem health. It is important to stress that in ounder frommost of stations attributed to extremely high genotoxicity riskzones, the micronuclei incidences in 2010e2011 were at higher

levels than MN frequency in those with an impaired shhealth.

In herring, MN frequencies higher than the background level

were found in 37.3% of studied samplings groups categorized ashaving an extremely high genotoxicity risk level. Low and moderatelevels of environmental genotoxicity predominated in herring

collected in September 2009 from nine stations of the Gulf ofFinland. However, at two stations (4a-1 and 3a-1) located in theGulf of Finland, 15.6% of herring specimens examined showed a MNfrequency up to 2500 times higher than the background level. This

indicates the existence of zones in the Gulf of Finland with anextremely high genotoxicity risks, possibly associated with pollu-tion by aneugenic and clastogenic compounds triggering anextensive 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 inecological conditions reecting extremely high level of environ-mental genotoxicity. Since MN frequencies in sh were monitoredat a large number of study stations in 2001e2010, it was possible to

identify an increase in the level of environmental genotoxicity in2009e2011. 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 ofMN frequency elevation due to accidental spills of contaminants, aswell as in zones close to river estuaries or industrial activities(Barsien _eet al., 2004,2008,in press;Kopecka et al., 2006;Schiedek

et al., 2006;Rybakovas et al., 2009; our data published inHELCOM,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 theoffshore stations. These stations were located in the Wismar Bay (inspring 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 signicantincrease of micronuclei levels in sh were evident in 2009e2011.Pearson correlation analysis of micronuclei frequency and envi-

ronmental (temperature, salinity, bottom depth, oxygen saturationand concentration) and sh biometrical (total length, weight, liverweight and age) variables showed signicant relationships in shfrom 10 stations out of 82 stations studied. In general, the increase

of genotoxicity in 2009e2010 in sh from the most studied stationswas related strongly neither to hydrologic, nor to biological vari-

ables. The tendency was appeared especially in ounder from ten



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study sites in the southern Baltic Sea (Barsien _e et al., in prepara-

tion). Instead, chemical stress might be considered as one ofdeterminants provoking an environmental genotoxicity effects inmost of the locations studied. For instance in experimental expo-

sures, a strong and positive correlation (r0.980) between totalmetal concentrations and nuclear abnormality frequency has beendescribed in tilapias Oreochromis niloticus (Summak et al., 2010),and a linear correlation (R2 0.8444) between MN frequency andPAH body burden in M. edulis(Sundt et al., 2011).

The application of cytogenetic methods can provide us withinformation regarding organisms exposure to genotoxic agents insitu and can be of great interest in the assessment of complexmixtures effects. Analysis of MN in marine animals is one of the

methods recognized as particularly relevant for biological effectsmonitoring purposes (Hylland et al., 2008; Brooks et al., 2011;Sundt et al., 2011). Consequently, the application of the MN assay in

an integrated monitoring and assessment programme will help toidentify problems related to pollution and to dene futuremanagement tasks, especially those identied in the HELCOMBaltic Sea Action Plan, i.e. to achieve of good ecological statusand

healthy wildlifein the Baltic Sea. The MN assay in sh would bean informative indicator in the assessment of the Ecological Quality

Objectives Hazardous substances within the marine environmentshall not cause irreversible changes in the functioning of the

ecosystem and in humans and Toxic substances shall not causesub-lethal, intergenerational or transgenic effects to the health ofmarine 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 fornative 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 ofounderP.esus(714 specimens), herringC. harengus(759specimens) and eelpout Z. viviparus (419 specimens) collected in

2009, 2010 and 2011. Environmental genotoxicity risk was assessedby using MN background levels in sh, developed using MN dataobtained in studies carried out in the period 2001e2011 from 107study locations of the Baltic Sea. Extremely high genotoxicity risk

zones were found for ounder at 11 stations out of 16 in 2009 and33 stations of 41 in 2010e2011, for herring, at 5 of 18 stations in2009 and 20 of 43 stations in 2010e2011, in eelpout only at one outof 29 stations. Study stations with an extremely high genotoxicity

risk were located mainly in the southern and eastern Baltic Seaoffshore zones. When comparing shspecies collected at the same

station, it was found that there always was a higher percentage of

ounder with MN frequencies exceeding the background levelcompared 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 materialfrom three stations in the Wismar Bay and Arvo Tuvikene (EstonianUniversity of Life Sciences, Estonia) for providing material from ve

stations at the Estonian coast. This study was funded mainly by

Lithuanian Science Council for the genotoxicity analysis in 55 studystations (MIP-62/2010 GENCITOX project) and by BONUS BEASTproject (FP/2007e2013 under grant agreement no 217246) for theanalysis in 27 stations in the Little Belt, in the Gulfs of Gdansk, Riga

Finland and Bothnia.

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