ICES CM 2007/I:05
Not to be cited without prior reference to the authors
ICES CM 2007/I:05 Theme Session on Effects of hazardous substances on ecosystem health in coastal and brackish-water ecosystems: present research, monitoring strategies, and future requirements (I)
Environmental genotoxicity studies in marine fish and mussels
Janina Baršienė, Aleksandras Rybakovas, Laura Andreikėnaitė
Institute of Ecology of Vilnius University, Akademijos 2, 08412 Vilnius, Lithuania, [tel: +370 68260979, fax: +370 52729257, e-mail: [email protected]]
Abstract: A growing concern over the presence of genotoxins in marine media, there is a rising need to elaborate sensitive methods for the assessment genetic damage in indigenous organisms. It has been developed different methods for the detection of both double- and single-strand breaks of DNA, DNA-adducts, micronuclei formation, chromosome aberrations. The micronucleus (MN) test has been widely used in vivo assay and was proved as simple to perform, sensitive enough and fast test to detect genomic alterations due to clastogenic effects and impairments of mitotic spindle caused by aneuploidogenic poisons. Main objective of the present study is to identify regularities of genotoxicity in marine indigenous organisms in situ, under experimental caging, deployment and laboratory conditions. Peculiarities of MN formation were investigated in various cells of fish and mussel species inhabiting geographically and ecologically different zones of the Baltic and North Seas. Active monitoring approach (fish and mussel caging) applied to assess MN induction in certain polluted areas of the North Sea. MN test validation was performed in multiple controlled exposures at IRIS (Norway) marine experimental center. The wide-range MN investigations indicated specific responses in relation to species, tissue, environmental temperature, contaminant type and concentration, duration of exposure, distance from contamination source. Furthermore micronuclei formation in the blue mussels was approximately 10-fold higher than in studied fish species (cod, flounder, turbot, perch, eelpout and wrasse). This variation in the levels of MN can be explained by the differences in invertebrate and vertebrate metabolism, DNA repair and in the rate of damaged cell recruitment.
Keywords: genotoxicity, micronuclei, Baltic Sea, North Sea, mussels, fish,
Introduction Many environmental contaminants exert their effects via genotoxic and metabolically toxic mechanisms simultaneously causing carcinogenesis, embryotoxicity and implicit a long term alterations in organisms by being active through several generations (Jha et al., 2000). It has been developed different methods for the detection of both double- and single-strand breaks of DNA, DNA-adducts, micronuclei formation, and chromosome aberrations. One of the most popular and promising is the micronucleus (MN) test. It is a marker of cytogenetic damage usually caused by clastogenic or aneugenic compounds. The assessment of cytogenetic damage has been presented as ICES CM 2007/I:05 very important assay in identification of pollution hazards in marine environment (Dixon et al., 2002). Micronuclei are produced from chromosomal fragments or whole chromosomes that lag at the cell division due to the lacking or damage of the centromere or a defect in cytokinesis. These small secondary structures of chromatin are surrounded by membranes located in the cytoplasm and have no detectable link to the cell nucleus. Although originally the micronucleus test was developed for the application in mammals (Boller, Schmid, 1970; Heddle, 1973), it was subsequently modified and used in fish (Hooftman, de Raat, 1982). Afterwards, the analysis of MN was increasingly used for assessing environmental genotoxicity in fish (Al-Sabti, Härdig, 1990; Al-Shabti, Metcalfe, 1995; Hayashi et al., 1998; Ayllon et al., 2000; Ayllon, Garcia-Vazquez, 2000; Bombail et al., 2001; Pietrapiana et al., 2002; Rodriguez-Cea et al., 2003; Baršienė et al., 2004, 2005, 2006a, 2006b, 2006c; Venier, Zampieron, 2005; Pacheco et al., 2005; Bolognesi et al., 2006a, 2006b). The micronuclei assay is one of the best biomarkers that clearly correlate with pollution load, as it has been shown in a number of studies (Al-Sabti, Hardig, 1990; Bolognesi et al., 1996, 2004, 2006; Pietrapiana et al., 2002; Baršienė et al., 2002, 2004, 2005, 2006a, 2006b, 2006c, 2006d, 2006e; Cavas, Ergene-Gozukara, 2005). Contaminants in marine environment can seriously impact DNA of filter-feeding bivalve populations (Hamoutene et al., 2002). Significant elevation of micronuclei level in mussels 30 days post-oil spill and persistence of the cytogenetic damage up to 100 days (Parry et al., 1997) and 8 months later (Baršienė et al., 2004, 2006a) has been described. Interestingly to stress, that statistically significant increase of micronuclei levels has been found in oysters and fish caged in Haven oil spill zones 10 years after the oil spill (Bolognesi et al., 2006a). Higher frequency of MN has been detected in mussels from oil terminal and marine port zones in the Baltic Sea (Baršienė, Baršytė Lovejoy, 2000; Baršienė, 2002), in Mediterranean commercial port zone (Magni et al., 2006), in polluted by aromatic hydrocarbons zones of the Venice lagoon (Venier, Zampieron, 2005). Cells with micronuclei were found to increase in the gills or hemolymph of marine molluscs treated with benzo(a)pyrene (Burgeot et al., 1995; Venier et al., 1997; Siu et al., 2004), dimethylbenz(a)antracene (Bolognesi et al., 1996), with crude oil from the North Sea (Baršienė et al., 2007). The results of the Comet and MN assays have been presented evidences on clear dose- and time-dependent responses to benzo(a)pyrene exposure in mytiliid bivalve Perna viridis (Siu et al., 2004). The main objective of the present study was to describe the peculiarities of environmental genotoxicity in fish and mussels from different sites of the Baltic Sea and the North Sea, as well as to show the pattern of micronuclei induction after caging of the organisms in polluted zones and after treatment with various genotoxic compounds. At present, only limited information is available on environmental genotoxicity in the Baltic Sea and in the North Sea.
Materials and methods Sampling in the Baltic Sea The peculiarities of environmental genotoxicity in the Baltic Sea were described in gill cells of the blue mussels (Mytilus spp.), in erythrocytes of flounder (Platichthys flesus), perch (Perca fluviatilis) and eelpout (Zoarces viviparous). The target species inhabited 5 study sites – the reference Kvadofjarden (Sweden), Stockholm archipelago, Lithuanian coast, Gulf of Gdansk (Poland) and Wismar Bay (Germany) (Fig. 1). The blue mussels were sampled in spring and autumn 2001 and 2002 from 12 stations and fish – from 21 stations located in the coastal zone of the Baltic Sea. In spring 2003-2006, mussels and flounder were sampled from 3 locations in the Lithuanian coast (Table 1).
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Sampling in the North Sea Material for the genotoxicity studies in the North Sea was collected from mussels (Mytilus edulis), cod (Gadus morhua), wrasse (Symphodus melops), eelpout (Zoarces viviparous), flounder (Platichthys flesus) and turbot (Scophthalmus maximus). Gills in mussels, erythrocytes in fish were used for the micronuclei analysis. In experimental treatments, as target cells were explored additionally immature erythrocytes from cephalic kidney and liver. Mussels and fish were collected from 12 locations in south-western part of the North Sea - Karmsund (Norway) and Gothenburg (Sweden) areas. Experimental treatments with crude oil and organic contaminants were performed using mussels, cod and turbot (Table 2). In 2002-2006, big amount of samples was collected from mussels and cod in oil industrial zones, like, Statfjord, Troll, Ekofisk, in gas fields in northern Norway. To study genotoxicity of produced water and crude oil there were performed experimental treatments of cod and mussels. In total, genotoxicity in oil industrial zones was analyzed in 1207 samples of cod and in 408 haemolymph samples of mussels.
Micronuclei analysis In fish for the analysis of MN in mature erythrocytes, a drop of blood from caudal vessels was directly smeared on slides. Smears from cephalic kidney or liver tissue (for the analysis of MN in immature erythrocytes) were prepared directly on the slides. The slides were air-dried, fixed in methanol for 10 min and stained with 10% Giemsa (Sigma) solution for 8-15 min. The frequency of MN was evaluated by scoring at a 1000 × magnification of 5000 intact mature or immature erythrocytes in each fish specimen. In mussels, the gill cell suspension was prepared in a drop of 3:1 ethanol acetic acid and smeared on clean microscopic slides. After that the slides were air-dried, fixed in methanol for 10 min and stained with 5% Giemsa solution in a phosphate buffer (pH = 6.8). 2000 cells with intact cytoplasm were scored in each studied specimen of mussels. The stained slides were analysed under a light microscopes (Olympus BX51, Olympus CX31, and Nikon Eclipse 50i) at a final magnification of 1000×. MN were identified according to the following criteria: (1) spherical or ovoid-shaped extra nuclear bodies in the cytoplasm, (2) a diameter of 1/3 - 1/20 of the main nucleus, (3) non-refractory bodies, (4) colour, texture and optical features resembling those of the nucleus, and (5) the bodies are completely separated from the main nucleus (Fig. 2). The final results were expressed as mean value (‰) of sums for the analyzed individual lesions scored in 1000 cells per mussel or fish collected from every study location. The statistical analysis (ANOVA and Mann-Whitney U test) was performed using PRISM statistical package.
Results Genotoxicity in the Baltic Sea In flounder inhabiting coastal areas, the frequency of micronuclei varied from 0.12 MN/1000 erythrocytes to 1.45 MN/1000 erythrocytes. The study results showed the highest environmental genotoxicity in the Lithuanian coast, the lowest – in reference Kvadofjarden site (in autumn 2001). Comparatively high frequency of micronuclei (1.01 MN/1000 erythrocytes) was found in flounder from Wismar Bay (spring 2001). 5-10-fold increase of micronuclei frequency was marked in Klaipėda-Būtingė locations in September 2001 compare to the values, measured in June 2001. High level of responses were observed 2002 after the oil spill in Būtingė oil terminal (November 2001) in locations which were affected by oil pollution – in Palanga and Būtingė zones (Fig. 3). Further reduction of genotoxicity levels was appeared in flounder from the Lithuanian coast in 2003-2006 (Fig. 4). ANOVA analysis revealed significant differences in genotoxicity levels between all studied sites and also between reference (Kvadofjarden) site and Wismar Bay. Flounders from Kvadofjarden, Lithuanian and Polish coast did not show significant differences. ICES CM 2007/I:05
Frequency of MN in flounder from Wismar Bay differs from the fish species collected in Lithuanian coast and in Gulf of Gdansk. The frequencies of micronuclei were studied in perch inhabiting 15 coastal locations in Sweden, Lithuania and Poland. The highest incidences of MN (1.15 MN/1000 erythrocytes) was counted in perch from Lithuanian coast by the Nemirseta (autumn 2001). Two-fold less micronuclei incidence (0.60 MN/1000 erythrocytes) was in those collected from Mechelinki station located in the Gulf of Gdansk (Fig. 5). In Stockholm archipelago the elevated level of micronuclei (0.45 MN/1000 erythrocytes) was observed only in perch from the closest zone to the city center. Statistically significant differences (P<0.0001) were expressed between genotoxicity in perch collected from reference (Kvadofjarden) site and Lithuanian coast as well as from Gulf of Gdansk. Data on the micronuclei frequencies in perch from sites in Sweden (Kvadofjarden and 9 stations in Stockholm archipelago) did not showed significant differences. The range of MN variation was from 0.02 to 0.66 MN/1000 erythrocytes in eelpout collected in 2001 and 2002 from Kvadofjarden (Sweden) and from 3 locations in Wismar Bay (Germany). The highest and lowest mean values were observed in eelpout from German coast. The highest value was 5-fold higher that a baseline level of micronuclei incidence in the Baltic fish. In the reference site Kvadofjarden there was low genotoxicity level (Fig. 6). Statistically significant differences (P<0.0001) were detected compare genotoxicity level in fish from the reference and other studied sites. From the same locations in the Baltic Sea were studied MN frequencies in gill cells of mussels. MN incidences varied in a range from 0.37 MN/1000 cells (Kvadofjarden) to 6.7 MN/1000 cells (Sopot) (Fig. 7). The levels of MN in mussels from the reference Kvadofjarden site were significantly lower (P<0.05) than in individuals from Lithuanian coast, from Gulf of Gdansk and Wismar Bay. MN frequency in mussels from the Lithuanian coast ranged from 1.2 (Palanga) to 3.85 MN/1000 cells (Būtingė), with a gradient Palanga < Nemirseta < Būtingė in June 2001. However after the oil spill (23 November 2001), the local reference location by the Palanga was covered with oil and in June 2002 and 2003, MN incidences were significantly increased (P < 0.0001) in resident mussels. Very high frequencies of MN were observed mussels inhabiting the Gulf of Gdansk. Exception was only samples collected in early spring, 21-24 March 2002 (Fig. 7). Genotoxicity level in Wismar Bay was higher in mussel samples collected in 2001 compared to those of 2002. MN frequency varied from 1.14 (Salzhaff, autumn 2002) to 5.06 MN/1000 cells (Wendorf, spring 2001). A clear gradient regarding the MN incidences was observed with significantly higher MN frequencies in mussels from Eggers Wiek (P < 0.05 to 0.001) and Wendorf (P < 0.001 to 0.0001), the location closest to Wismar harbour (Fig. 7).
Genotoxicity in the North Sea Analysis of environmental genotoxicity in some south-western Norwegian fjords and coastal zones showed relationships between contamination level and micronuclei incidences in mature erythrocytes of wrasse (Fig. 8) and in gill cells of mussels. The highest frequencies of the micronuclei were found in mussels and wrasse inhabiting the most polluted Hogevarde and Bukkoy localities. The average frequency in mussels from Hogevarde reached 6.9 MN/1000 cells, from Bukkoy – 7.2 MN/1000 cells (Fig. 8). In mussels from other 5 stations the values of MN varied from 1.1 to 3.4 MN/1000 cells. In Atlantic cod blood, there were no clear relationships. Studies of MN in flounder from the Gothenburg harbor indicated that the highest frequency of MN was in fish from Jordhammarwik location (0.45 MN/1000 erythrocytes) and in fish from Nya Alvsborg (0.33 MN/1000 erythrocytes). Very low responses were observed in flounder inhabiting Fjallbacka and Nordre Alvsborg locations (Fig. 9). The highest level of MN incidences was found in mussels from Ringhals and Hjuvik locations (Fig. 10). Consider a current concern of contamination from oil industry, there were performed a number of studies on genotoxic influence of certain compounds as well as caging experiments in different oil ICES CM 2007/I:05 fields in the North Sea. In mussels and Atlantic cod, a gradient towards high genotoxicity in organisms caged closer to platform in the Statfjord oil field was detected. Statistically significant differences were observed between mussels caged in reference and polluted locations (Fig. 11). Similar decrease of genotoxic responses with a distance from oil platform was marked in cod (Fig. 12). Eight experimental groups of Atlantic cod were explored to analyze effects of alkylphenols, mixture of PAHs and alkylphenols, crude oil and produced water. Cod was exposed to three different concentrations (2000, 10 and 2 ng/l) of four alkylated phenols (C4-C7: 4-tert-butylphenol, 4-n- pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol), to nine alkylated phenols mixture (AP9 mixture: 2-methylphenol, 4- methylphenol, 3,5-dimethylphenol, 2,4,6-trimethylphenol, 4-tert- butylphenol, 4-tert-butyl-2-methylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n- heptylphenol), to produced water (PW) diluted 1:1000 and 1:200, to complex mixture of oil, alkylphenols and PAHs and to 0.2 ppm of crude oil from the North Sea Oseberg C platform. Control group of cod was maintained in clean marine water. Induction of micronuclei was detected in immature erythrocytes from cod liver and cephalic kidney. The genotoxicity of studied compounds was higher in cod cephalic kidney (Fig. 13). MN induction in cod control groups and in treated with 0.06 ppm, 0.25 ppm, 1 ppm of oil and 1 ppm of spiked by alkylphenols and PAHs crude oil was analyzed three times in time scale from 3 to 27 days and after 3- and 14-days of cod recovery in clean filtrated seawater. The study results revealed time- and spike-dependent formation of MN in cod liver immature erythrocytes. Highest level of genotoxicity was marked after 14-day exposure and slight decrease of MN incidences – in 24-day exposure group. Extensive 14-day recovery process up to control level of MN was observed in 38-day exposure groups after treatment with 0.06ppm, 0.25ppm and spiked 1ppm of crude oil from the Stafjord B platform (Fig. 14). Significant increase of fragmented-apoptotic cells was determined in 24-day exposure groups (Fig. 15).
Discussion The results of the wide-range study showed that lowest level of genotoxicity exists in organisms sampled from in uncontaminated marine environment sites. In the Baltic Sea, the spontaneous level of genotoxicity was observed in fish and mussels from the reference Kvadofjarden site, Salzhaff location in the Wismar Bay and from Palanga location before the oil spill in the Lithuanian coast, as well as in organisms inhabiting Forlandsfjorden, Bleivik, Alvestad and Visnes locations in the North Sea. The highest micronuclei incidences were found in most contaminated zones, which are affected by the discharge from industry, marine traffic, heavy metals, PAHs and other organic pollutants. Only one exception was appeared in the North Sea, in a former copper-mining site Visnes. Although a low response in indigenous organisms, experimental caging of mussels in Visnes location indicated exceptionally high level of genotoxicity and pointed on adaptation process in chronically polluted areas. Genotoxic properties of heavy metals are related mainly to accumulation of DNA damaging free radicals, clastogenic process or simultaneously to clastogenic and aneugenic action in aquatic organisms (Nepomuceno et al. 1997). Many studies describing the biological effects of heavy metals under laboratory and field conditions have a point that at the certain concentrations they are genotoxic in fish (de Lemos et al. 2001; Sanchez-Galan et al. 2001; Ferraro et al. 2004; Arkhipchuk & Garanko, 2005; Porto et al. 2005; Ergene-Gozukara et al. 2007). The induction of micronuclei has been shown in common carp (Cyprinus carpio), Prussian carp (Carassius gibelio) and Peppered cory (Corydoras paleatus) after 3-week treatment with 0.01–0.25 mg/L copper (Cavas et al. 2005). The study results indicated significant increase of genotoxicity after accidental oil spill in Lithuanian coast of the Baltic Sea and in organisms inhabiting or caged in zones of oil and gas industry in the North Sea. Genotoxicity of produced water components was also shown. The extensive development of oil and gas industry in the North Sea has resulted in a large release of ICES CM 2007/I:05 produced water. The produced water contains minor amounts of dispersed oil droplets (typically 10- 20 ppm) but also a complex aqueous mixture of PAHs, alkylphenols and other organic and inorganic compounds. From an environmental perspective, the PAHs and the alkylphenols (APs) have particular attention because of their potential mutagenic and carcinogenic properties of some PAHs and the possible endocrine disrupting potencies of some APs. It is known that hazardous effects of different PAHs arise typically as a result of oxidative biotransformation producing highly DNA-reactive metabolites or associated with appearance of reactive oxygen species which can injure fish DNA, proteins and lipids (Lemaire et al., 1994). Mechanisms of PAH metabolic transformation have been widely studied and genotoxic potency of metabolites was confirmed in various fish species (Pacheco, Santos, 1997, 2001; Harvey et al., 1999; White, 2002; Maria et al., 2002a, 2002b; Gravato, Santos, 2002, 2003; Brown, Steinert, 2003; Teles et al., 2003). Increase level of environmental genotoxicity was determined in zones affected by different oil spills (Parry et al., 1997; Harvey et al., 1999; Pietrapiana et al., 2002; Perez-Cadahia et al., 2004; Baršienė et al., 2004; 2006a, 2006b; Frenzilli et al., 2004; Laffon et al., 2005; Bolognesi et al., 2006a; Martinez-Gomez et al., 2006). Significant induction of micronuclei was in Atlantic cod and turbot after treatment with crude oil from the North Sea Statfjord B platform (Baršienė et al., 2006c; Bolognesi et al., 2006b). In erythrocytes of eelpout sampled from the Göteborg harbor after a bunker oil spill, levels of DNA integrity (Comet assay) was at the similar level to European eel exposed to 1mg/kg benzo(a)pyrene (Frenzilli et al., 2004). Short-term exposure in situ of eel (Anguilla anguilla) to harbors water contaminated by PAHs increased induction DNA strand breaks and nuclear abnormalities in blood, kidney and liver erythrocytes (Maria et al., 2003). A time- related potency of naphtalenes to induce micronuclei in liver has been observed in juvenile Dicentrarchus labrax (Gravato, Santos, 2002). In eel Anguilla anguilla, treatment with 0.3, 0.9 and 2.7 μM of naphtalene resulted in the induction of micronuclei and other nuclear abnormalities (Teles et al., 2003). Genotoxicity of 10 polycyclic aromatic hydrocarbons (anthracene, benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, dibenz[a,h]anthracene, dibenz[a,c]anthracene, 3-methylcholanthrene, benzo[a]pyrene, benzo[e]pyrene, chrysene and pyrene) in mice skin cells and was pointed that genotoxicity of these compounds in general correlated with their reported carcinogenicity (Nishikawa et al., 2005). About 400 million tones of produced water effluents including approximately 8,000 tones of dispersed oil annually are released from Norwegian and UK oil platforms in the North Sea (Utvik, 1999). Therefore assessment of genotoxic effects caused by complex mixture of crude oil and produced water effluents should provide new information towards the validation of environmental risk estimates the discharges from offshore oil industry. The study demonstrates that micronuclei analysis is a cost-effective and rapid approach to detect harmful effects of environmental pollution in situ. The endpoint is well characterized, can easily be recognized and used cytogenetic technique is convenient to apply in field samplings by following standard procedures and protocols.
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Fig. 1 Sampling sites in the Baltic Sea: 1 – Kvadofjarden, 2 – Stockholm archipelago, 3 – Lithuanian coast, 4 – Gulf of Gdansk, 5 – Wismar Bay ICES CM 2007/I:05
A B
C D
Fig. 2. Micronucleated erythrocytes in Atlantic cod blood (A), cephalic kidney (B) and liver.
E – micronuclei in mussel haemocyte. ICES CM 2007/I:05
1,8 1,6 1,4
1,2 2001 spring 1 2001 autumn 0,8 2002 spring 0,6 2002 autumn 0,4 MN/1000 erythrocytes MN/1000 0,2 0 a t o et w s e isch ir langa z f fjarden Sopo al o Butinge chelinki es em Pa e bi Offentief W vad N M o K S
Fig. 3 Frequency of micronuclei in flounder from different coastal sites of the Baltic Sea: Kvadofjarden (Sweden); Nemirseta, Palanga, Butinge (Lithuanian coast); Mechelinki, Sopot, Sobieszewo (Gulf of Gdansk, Poland); Offenntief, Walfisch (Wismar Bay, Germany).
0,8 *** ** 0,7
0,6 ** 2001 0,5 2002 0,4 2003 2004 0,3 2005 2006 MN/1000 erythrocytes MN/1000 0,2
0,1
0 Nemirseta Palanga Būtingė
Fig.4 Frequency of micronuclei in flounder collected from the Lithuanian coast in June 2001-2006 ICES CM 2007/I:05
1,4
1,2
1
0,8
0,6
0,4 MN/1000 erythrocytes MN/1000
0,2
0
A A A A A A 1 1 3S 02S 02S 03S n 0 n 02A 01 01 0 n 0 e e n 01 o ta 01 ki e a d d e lm n r r nd e ewo ng a a ss i eli inge fj fj u V kho irs fjard sz ut o o l ch d d S Lo Pala B a va Nem Me vado Kv K K Sobie
Fig. 5 Frequency of micronuclei in perch collected in 2001-2002 from different locations in the Baltic Sea – Kvadofjarden, Slussen, Vindo, Lokholmen (Sweden); Nemirseta, Palanga Butinge (Lithuanian coast), Mechelinki (Gulf of Gdansk, Poland). 01A – autumn 2001, 02A – autumn 2002, 02S – spring 2002,03S – spring 2003.
1 0,9 0,8 0,7 Kvadofjarden 0,6 Wismar 0,5 Salzhaff 0,4 Eggers Wiek 0,3
MN/1000erythrocytes 0,2 0,1 0 2001 2001 2002 2002 spring autumn spring autumn
Fig. 6. Frequency of MN in erythrocytes of eelpout from coastal zones in Sweden and Germany ICES CM 2007/I:05
7
6
5
4 2001 S 3 2001 A
MN/1000 cellsMN/1000 2002 S 2 2002 A
1
0
n a i f e ta ot k rs or wo lin e ard lang rse ze Sop fj he Egg end o Pa Butinge ies c Salzhaff W vad Nemi ob Me K S
Fig. 7 Frequency of MN in gill cells of mussels from different locations in the Baltic Sea ICES CM 2007/I:05
0,9
0,8
0,7
0,6
0,5
0,4
0,3 MN/1000erythrocytes 0,2
0,1
0
y ik nes koy landsf r uk Bleiv Salvo Vis B Alvestad Fo Hogevarde
Fig. 8 MN frequency in wrasse from Karmsund zone of the North Sea ICES CM 2007/I:05
0,6
0,5
0,4
0,3
0,2 MN/1000erythrocytes
0,1
0
nd rg ka c arwik vsborg sbo m v inghals Al R nungsu e Al e a Fjallba St Ny Jordham Nordr
Fig. 9 Micronuclei frequency in flounder from Gothenburg harbor of the North Sea
3
2,5
2
1,5
1 MN/1000 cells
0,5
0
ik ls w vik a ju h acka H g in R Fjallb Stenungsund Jordhammar
Fig. 10 Frequency of MN in gill cells of mussels from Gothenburg harbor ICES CM 2007/I:05
10
9 ***
8
7
6 *
5
4
3 MN/1000 haemocytes MN/1000
2
1
0 Before Reference 500 m 1000 m 10000 m caging
Fig. 11 Induction of MN in mussel haemocytes after 5-week caging in Statffjord B oil platform zone (North Sea)
0,8
0,7
0,6
0,5
0,4
MN/1000 cells MN/1000 0,3
0,2
0,1
0 Before Reference 500 m 1000 m 10000 m caging
Fig. 12 Induction of MN in Atlantic cod liver erythrocytes after 5-week caging in Statffjord B oil platform zone (North Sea) ICES CM 2007/I:05
0,8 ** 0,7 *** ** ** * 0,6 ** *** *** *** 0,5 Kidney 0,4 Liver 0,3 MN/1000 cells MN/1000 0,2
0,1
0
w m gh ix. lo u i m 7 :1000 Control ,2ppm AP9 0 4-C 4-C7 h PW 1:200 il C C PW 1 O+PAH+APO C4-C7 medi
Fig. 13 Induction of MN in Atlantic cod immature erythrocytes from liver and cephalic kidney after exposure to crude oil (Oil 0.2 ppm), produced water (PW), mixture of different alkylphenols (C4- C7; AP9 mix.) and to mixture of crude oil, PAHs and alkylphenols (O+PAH+AP). Asterisks show statistically significant differences compared to control group of cod: *P<0.01, ** P<0.001, *** P<0.0001. ICES CM 2007/I:05
8 7 6 1 ppm+spike 5 1 ppm 4 0,25 ppm 3 0,06 ppm 2 Control
MN/1000 erythrocytes MN/1000 1 0 3days 14days 24days 27days 38days
Fig. 14 Induction of MN in cod liver after treatment with crude oil (3-, 14-, 24-days) and in recovery (27-, 38-days) process.
6 5 1 ppm +spike 4 1 ppm 3 0,25 ppm 2 0,06 ppm erythrocytes
Apoptotic/1000 Control 1 0 3 days 14 days 24 days 27 days 38 days
Fig. 15 Frequency of fragmented-apoptotic cells in cod liver after treatment with crude oil (3-, 14-, 24-days) and after recovery (27-, 38-days)
ICES CM 2007/I:05
Table 1 Samples for the micronuclei analysis in mussels and fish from the Baltic Sea
Species Kvadofjar Lithuanian Gulf of Wismar Stockholm Offshore Total den coast (3) Gdansk Bay (3) archipelago (5) (9) Mussels 30 300 259 120 709 Flounder 80 252 253 147 455 1187 Perch 82 38 24 188 332 Eelpout 109 42 307 458 Total 301 590 578 574 188 455 2686
Table 2 Samples for the micronuclei analysis in mussels and fish from the North Sea
Localities Mussels Wrasse Cod Flounder Eelpout Turbot Total Karmsund area Salvoy 20 19 39 Visnes 20 20 40 Bonk 20 19 13 52 (Alvestad) Frolandsfjorden 20 20 11 51 Hogevarde 20 16 19 55 Hogevarde B 20 20 Bukkoy 20 16 20 56 Bleivik 8 10 18 Goteborg area Jordhammarvik 20 20 13 30* 103 Stenungsund 20 4 9 18* 51 Norde alvsborg 9 19* 28 Nya alvsborg 9 5 18* 32 Hjuvik 20 18 1 26* 65 Skalkorgarna 12* 12 Ringhals 20 20 20 14 20* 94 Fjallbacka 18 11 30* 59 ICES CM 2007/I:05
Localities Mussels Wrasse Cod Flounder Eelpout Turbot Total Experiments 2002 Control 21 16** 12** 49 0.5ppm oil 20 14** 12** 46 0.5ppm oil+AP 23 12** 17** 52 Nonylphenol 8 15** 10** 33 Experiments 2003 Control 15 12** 10** 37 Bisphenol A 15 14** 11** 40 50ppb Diallyl 15 15** 11** 41 phthalate 50 ppb Tetrabromodiph 15 16** 13** 44 enyl ether 5ppb 0.5ppm oil 50 50 (1,2,4,8-day exposure Caging in 40 40 Visnes Cu gradient Total 460 158 258 62 173 96 1207 +114kd +173kd +96kd +383kd +114lv +96kd +210lv
Without asterisk – samples from fish blood and from mussel gills; * - samples from fish blood and cephalic kidney (kd); ** - samples from fish blood, cephalic kidney (kd) and liver (lv)