Declining sea ice cover and political and economical interests in the vast potential of oil and hard mineral reserves in the Greenlandic underground are all drivers for Greenland’s aspirations concerning the future exploitation of mineral recourses, including iron (Fe), gold (Au), zinc (Zn), lead (Pb), gas and oil (Nuttall 2014, Rosing et al. 2014).
In Greenland, mining activities have been conducted for over a century with well-documented long-term environmental impacts, including heavy metal contamination of fjords and organisms (Søndergaard et al. 2011, Søndergaard et al. 2014). For example, at an abandoned mining site in South-Greenland, Johansen et al. (2008) found Pb concentrations in blue mussels (Mytilus edulis) elevated to 200-500 times above background concentration, and Loring & Asmund (1989) reported Pb tissue concentrations exceeding 800 µg/g (background 3 µg/g) in mussels collected close to the Maarmorilik mine in West Greenland. Because of previous environmental contamination, recent oil and mineral exploration in the Arctic region have lead to growing concerns about uncontainable pollution of the Arctic marine ecosystems (AMAP 2010). Heavy metal waste from increasing mining activities is an environmental threat due to potential pollution and contamination of the sensitive marine flora and fauna (Reichelt-Brushett 2012, Søndergaard 2013).
In the Arctic, contaminated organisms simultaneously get exposed to multiple stressors, both chemical and physical. Chemical contamination may under some conditions reduce the cold tolerance of invertebrates, leading to increased mortality when low temperature is combined with contamination (Holmstrup et al. 2008, Bindesbøl et al. 2009a, b). Still, in traditional ecotoxicological laboratory studies organisms are usually exposed to a single chemical, while held at otherwise optimal conditions. Risk-assessment and environmental monitoring methods often consist of point sampling and transplant studies with the focus of estimating uptake/release rates of contaminants, mortality and metal tissue content (Søndergaard et al. 2011, Zimmer et al. 2011).
However, such studies have limitations, as they do not consider synergetic stress interactions of multiple stressors acting simultaneously on organisms in natural environments. Both physical and chemical stressors can interact synergetically and in the Arctic, extreme temperatures, increasingly acidic oceans (AMAP 2013), additive effects of multiple contaminants, food scarcity during winter and other relevant stressors are factors worth considering when conducting risk assessment in this region. Also, such traditional studies often do not include sub-lethal effects. Sub-lethal concentrations of metals may affect growth, and enzyme activities of plants and animals (Naimo 1995, Ouyang et al. 2012). Consequently, as both chemical and physical induced stress is known to
have chronic and (sub)-lethal effects, multi-factorial studies investigating synergetic interactions between multiple stressors are crucial to incorporate in future risk-assessments of chemicals in the Arctic (Heugens et al. 2001).
In marine environments a number of species are frequently included when conducting risk-assessments (Søndergaard et al. 2014). One key indicator organism is the blue mussel (M. edulis) because it is relatively long-lived, sessile and abundant with potential for concentrating and assimilating metals directly from seawater and suspended particles (Zimmer et al. 2011, Søndergaard et al. 2014). In subarctic Greenland, intertidal blue mussels are widely distributed (Blicher et al. 2013). Here they create unique habitats with a rich associated fauna and they are an important food source for higher trophic levels such as fish, sea birds and mammals (Gosling 2003).
In the intertidal zone, sub-zero air temperatures are a primary factor influencing blue mussels distributions-patterns (Blicher et al. 2013). With minimum winter temperatures down to -25ºC (Blicher et al. 2013), cold stress and freezing mortality is common among intertidal blue mussels in the Arctic region (Bourget 1983). When acclimatizing to sub-zero temperatures, ectotherms modify the lipid composition of their cell membrane. The phase behaviour and physical properties of membrane phospholipid fatty acids (PLFAs) are extremely sensitive to temperature changes (Hazel
& Williams 1990). Fully functional membranes are in a liquid-crystalline phase, but if membranes are cooled to a threshold temperature [the phase-transition-temperature (Tm)], the membrane enters a dysfunctional rigid gel phase (Hazel 1995). To lower Tm and thereby ensure the proper fluidity at low temperatures, ectotherms regulate cholesterol content and the proportion of unsaturated fatty acids, mainly they up-regulate the content of polyunsaturated fatty acids (PUFAs) (Cossins &
Raynard 1987, Hazel 1995). Such changes in cell membrane PLFA composition are probably important for freeze tolerance in cold adapted ectothermic animals (Hazel 1995, Kostal et al. 2003).
When blue mussels are exposed to sub-zero temperatures (approximately -7°C depending on body size (Sejr, unpublished data)), ice forms in extracellular fluids. Since only water molecules form the ice, the concentration of extracellular solutes increases. This causes cells to dehydrate as intracellular water moves down the osmotic gradient into the extracellular space. Excessive cell dehydration is lethal as it disrupts membrane function and protein structures (Meryman 1971). To increase freezing tolerance, blue mussels change the intracellular free amino acid concentration, altering the freezing point of intracellular fluids and preventing an intolerable degree of dehydration (Williams 1970, Aarset 1982).
When the hazardous heavy metal lead (Pb) is released into marine environments, it may be assimilated into marine organisms. Filter-feeding bivalves are exposed to large quantities of water during feeding and respiration, and in this process they accumulate Pb in both soft tissues and the shell (Sericano 2000). On a cellular level, Pb contamination has been shown to cause mitochondrial swelling, deformation and decreased membrane permeability (Goyer & Krall 1969, Raghavan et al.
1981). Heavy metal contamination with various metals has been demonstrated to increase reactive oxygen species (ROS) production, which accelerates lipid peroxidation, destabilises cell membranes and alternate PLFA composition (Christie & Costa 1984, Gallego et al. 1996, Valko et al. 2005). Increased ROS production has also been demonstrated with Pb contamination (Lawton &
Donaldson 1991, Verma & Dubey 2003). ROS affects both monounsaturated (MUFAs) and polyunsaturated fatty acids, but PUFAs are particularly susceptible to oxidation by ROS (Nyska &
Kohen 2002). PUFAs are vulnerable to peroxidation since double bonds weaken the adjacent monoallylic C-H bonds and facilitate the reaction of –H with an oxidizing agent, resulting in lipid radicals and eventually lipid chain termination (Marcelo 2004). Lipid peroxidation will hereby alter the overall degree of unsaturation from a highly polyunsaturated cell membrane to a more ordered and rigid membrane structure. This would in theory increase the phase-transition-temperature (Tm) of the membrane, which leads to a local reduction in membrane fluidity (Hazel & Williams 1990).
If ambient temperature decreases to Tm it can lead to a loss of vital functions and selective properties (Hazel & Williams 1990). Since both sub-zero temperatures and Pb possibly stress cell membranes by making it inadequately fluid for full functioning, a synergetic effect of these stressors can possibly exist.
In the present study we test the hypothesis that synergetic interactions between Pb at ecological relevant tissue concentrations and realistic sub-zero air temperatures will increase mortality due to freezing in the arctic blue mussel. Further, to understand the importance of altering cell membrane fluidity in the freezing tolerance strategy of blue mussels, we test the possibility that Pb contamination destabilizes the cell membrane by modifying the PLFA composition, and hereby cause blue mussels to become more vulnerable to the sub-zero temperatures of the arctic intertidal zone.