The EU, in October 2009, released the document “Nanosciences and Nanotechnologies: An action plan for Europe 2005-2009. First Implementation Report 2005-2007 COM(2009)607 final” [1] while in the US, the National Science and Technology Council released “Review of the Federal Strategy for Nanotechnology-Related Environmental, Health, and Safety Research” [2]. Both reports highlight some priorities for future research, at national, EU and international level, namely the need to: - Develop methods to detect nanomaterials in biological matrices - Develop methods for standardizing assessment of particle size, size distribution, shape, structure, and surface area - Develop methods to characterize a nanomaterial´s spatio-chemical composition, purity, and heterogeneity - Understand the effects of engineered nanomaterials (ENPs) in individuals of a species and the applicability of testing schemes to measure effects This project directly addresses these priorities and, like any robust approach to hazard identification for nanomaterials, considers three elements: physical-chemical characterization, in vitro assays and in vivo studies [3]. The physical-chemical characterization of the nanomaterials relies on the combination of FIFFF with inductively coupled plasma mass spectrometry (ICP-MS) . This is a well-established technique to study the composition of colloidal material [4] and is one of the most promising techniques to quantify and characterize metallic nanoparticles [5]. In parallel a battery of bioassays, both in vitro and in vivo, covering a wide range of endpoints, from citotoxicity and oxidative stress to growth and mortality. An experimental approach involving increased complexity of the systems being studied will be followed. Thus, chemistry studies start from simple systems to complex matrices (mixed systems) and while ecotoxicological studies move from tissues to organisms belonging to taxonomic groups with different levels of complexity and trophic functions. Two issues of importance are the low expected concentrations of ENPs and the large background of natural nanoparticles (NNPs) in environmental samples. In vitro assays allow the examination of specific biological responses and/or mechanisms of action under controlled conditions. Cytotoxic responses are usually evaluated in terms of morphological chances of cells and indicators of oxidative stress. This project enlarges this approach assessing cytotoxic and genotoxic effects of nanomaterials on human cells as well. The in vivo studies, on the other hand, will use aquatic organisms representing different trophic levels: decomposers, primary producers and primary and secondary consumers. Standard exposure protocols will be followed and adapted to the exposure to nanomaterials, as necessary, following recommendations of the OECD [6]. A line of human Epidermal cells (e.g. NHEK[CS1], NHEM) will be used as in vitro system to investigate the effects and the possible mechanisms of toxicity of ENPs on tissues from secondary consumers. The immobilization of Daphnia, the inhibition of the algae growth and the fish embryo test with zebrafish are incorporated in most environmental legislations, including the Guidelines for testing of chemicals of the OECD. However, the bioluminescence inhibition assay in Vibrio fischeri has only been adopted by some regulations for the characterization of effluents. Nonetheless, the four systems we propose to use for the ecotoxicological evaluation of nanomaterials in vivo, include bioluminescence inhibition of the bacterium V. fischeri (decomposer), the inhibition of the alga growth Chlorella vulgaris (first producer), the immobilization of the cladoceran Daphnia magna (primary consumer) and the fish embryo test with zebrafish (secondary consumer). The project aims to develop/validate and integrated methodological approach to characterize and assess the ecotoxicological effects and fate of silver nanoparticles (NP) and ionic silver. Standard endpoints like bioluminescence , growth inhibition, embryo development and mortality will be used together of indicators of citotoxicity, genotoxicity and oxidative stress. Our goal is not only to evaluate biological indicators of chemical stress but also characterize and quantify the silver ENPs in all bioassays to be able to generate functional dose-response curves. Effects from direct exposure to silver ENPs and to the ionic form they release will be assessed quantifying ionic silver. The project partners play a complementary role in the project. While the CSM partner has a strong chemical background and experience in the use of FIFFF combined with ICP-MS [4] the CESAM/UA partner have a strong background on the use of ecotoxicological bioassays (e.g. Barata et al. [7]), in vitro and in vivo bioassays [7-11] involving the assessement of citoxicity, genotoxicity and/or oxidative stress.

CESAM members on this project

António José Arsénia Nogueira
Coordinator (PI)

Helena Cristina Correia de Oliveira
Researcher

Researcher

Maria de Fátima T. Jesus
Research Fellow (BI)