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). ICES CM 2007/I:05 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