It is well established that anthropogenic contaminants occur in all probable combinations and therefore are not isolated in their threat to the aquatic environment. Recently there has been an increasing interest in measuring pollutants in environmentally realistic conditions to predict the potential detrimental outcomes on the ecosystem. This includes realistic levels of contamination concentrations, chronic exposures duration and mixtures of contaminants. With this perspective, in the present work, marine mussels, Mytilus galloprovincialis were exposed to concentrations of copper (5, 32 μg L-1), lead (5, 25 μg L-1) and tritiated water (HTO, 1.5, 5 MBq L-1) both individually and as a binary mixture. After a 14-days exposure period, ranges of endpoints at different levels of biological organisation were investigated, including an in-depth investigation into the mussel’s proteome. In addition, the results obtained for different endpoints (viz., DNA and chromosomal damage; acetylcholine and glutathione activities, protein carbonyl content, ‘clearance rate’) were analysed using network modelling to establish role of sub-lethal biological responses or biomarkers on overall health of the mussels following exposure to contaminants. Binary combinations of Cu, Pb and HTO demonstrated different impacts to genotoxic (i.e. DNA damage and chromosomal aberrations) enzymatic (acetylcholinesterase and glutathione activity, protein carbonyl content) and behavioural (clearance rate) compared to M. galloprovincialis exposed to the same contaminants singularly. In particular, the comet assay results, which suggests an antagonistic increase with the highest binary treatment in Cu-Pb, Cu-HTO and Pb-HTO (Cu-32 µg L-1, Pb- 25 µg L-1 and HTO- 5 MBq L-1). Whereas the induction of micronucleus were significantly lower in treatments combined with Pb-HTO compared to the single treatments. A number of influential biomarkers were found with the endpoints used in these studies, especially comet assay and glutathione activity when M. galloprovincialis are exposed to the combination of Cu and Pb. As determined in this work, environmental contaminants interact with each other, which affect an ecologically and economically important marine invertebrate differently compared to single exposures. This was investigated further by proteomic analyses, which revealed firstly, a number of proteins of interest that were altered when the mussels were exposed to binary combinations of Cu, Pb and HTO. These altered proteins also indicate that a number of biological processes, cellular components and molecular functions are potentially affected by these combinations of contaminants. The variation in biomarker responses found and alterations in the proteome of M. galloprovincialis exposed to mixtures of Cu, Pb and HTO demonstrate the biological complexity of these combination effects. This indicates the requirement for more environmentally realistic exposure conditions to facilitate the implementations of regulations for hazard and risk assessments for the protection of both environmental and human health.

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