Interim report of the HELCOM CORESET project

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main nuclei were not counted as MN. The area to be scored should fi rst be examined under low magnifi cation
to select the part of the slide showing the highest quality (good staining, non overlapping cells). Scoring
of MN should then be undertaken at 1000x magnifi cation.
Confounding factors
Earlier studies on MN formation in mussels have disclosed a signifi cant infl uence of environmental and
physiological factors (Dixon et al. 2002). Therefore, the role of the confounding factors should be considered
prior to the application of MN assay in biomonitoring programs, as well as in description of genetic
risk zones, or ecosystem health assessments.
Water temperature. MN induction is a cell cycle-related process and depends on water temperature, which
is a confounding factor for the mitotic activity in poikilotherm animals. Several studies have demonstrated
that baseline frequencies of MN in mussels are related to water temperature (Brunetti et al. 1988, 1992;
Kopecka et al. 2006). Baseline frequencies of MN are regarded as the incidence of MN observed in the
absence of environmental risk or before exposure to genotoxins (Fenech 1993). In fi sh MN frequencies
showed also seasonal differences in relation to water temperature with lower MN levels in winter than in
autumn (Rybakovas et al. 2009). This was assumed to be an effect of higher mitotic activity and MN formation
due to high water temperatures in the autumn (Brunetti et al. 1988). Additionally, it has been reported
that increases in water temperature (4 - 37ºC) can increase the ability of genotoxic compounds to damage
DNA (Buschini et al. 2003).
Cell type. MN may be seen in any type of cell, both somatic and germinal and thus the micronucleus test
can be carried out in any active tissue. Nevertheless there are some limitations using different types of cells,
for example, agranular and granular haemocytes in mussels. There are also differences between MN induction
level in mussel haemolymph and gill cells, mainly because gills are primary targets for the action of
contaminants. The anatomical architecture of the spleen in fi sh does not allow erythrocytes removal in the
spleen (Udroiu et al. 2006), though, in mammals this process go.
Salinity. The infl uence of salinity on the formation of MN was observed in mussels from the Danish coast
located in the transitional zone between the Baltic and North Sea. No relationship between salinity and
MN frequencies in mussels could be found for mussels from the Wismar Bay and Lithuanian coast. Similar
results were found for Macoma balthica from the Baltic Sea in the Gulfs of Bothnia, Finland, Riga and in
the Lithuanian EEZ (Baršienė et al., unpublished data).
Individual size. Since the linear regression analysis of animal’s length and induction of MN shows that the
size could be a confounding factor, sampling of organisms with similar sizes should take place (Baršienė et
al., unpublished data). It should also be noted that size is not always indicative of age and therefore age
could also potentially affect the response of genotoxicity in the fi sh.
Diet. Results have shown that MN formation was not infl uenced in mussels who were maintained under
simple laboratory conditions without feeding (Baršienė et al. 2006d).
Ecological relevance
Markers of genotoxic effects refl ect damage to genetic material of organisms and thus get a lot of attention
(Moore et al. 2004). Different methods have been developed for the detection of both double- and
single-strand breaks of DNA, DNA-adducts, MN formation and chromosome aberrations. The assessment
of chemical induced genetic damage has been widely utilized to predict the genotoxic, mutagenic and
carcinogenic potency of a range of substances, however these investigations have mainly been restricted
to humans or mammals (Siu et al. 2004). MN formation indicates chromosomal breaks, known to result
in teratogenesis (effects on offspring) in mammals. There is however limited knowledge of relationships