One of the challenges in using in vitro data to understand the potential risks of engineered nanomaterials 22 (ENMs) is that results often differ or are even contradictory among studies. While it is recognized that 23 numerous factors can influence results produced by nanobioassays, there has not yet been a consistently used conceptual framework to identify key sources of variability in these assays. In this paper, we use 25 cause-and-effect analysis to systematically describe sources of variability in four key in vitro 26 nanobioassays: the DCF (2',7'-dichlorofluorescein) assay, an enzyme-linked immunosorbent assay (ELISA) 27 for measuring interleukin-8, a flow cytometry assay (Annexin V/Propidium Iodide), and the Comet assay. 28 These assays measure endpoints that can occur in cells impacted by ENMs through oxidative stress, a 29 principle mechanism for ENM toxicity. The results from this analysis identify control measurements to test 30 for potential artifacts or biases that could occur during conduct of these assays with ENMs. Cause-and-31 effect analysis also reveal additional measurements that could be performed either in preliminary 32 experiments or each time the assay is run to increase confidence in the assay results and their 33 reproducibility within and among laboratories. The approach applied here with these four assays can be 34 used the support the development of a broad range of nanobioassays. 35

Inhalation of particulate matter (PM) from residential biomass combustion is epidemiologically associated with cardiovascular and pulmonary diseases. This study investigates PM0.4–1 emissions from combustion of commercial Miscanthus straw (MS), softwood chips (SWC) and beech wood chips (BWC) in a domestic-scale boiler (40 kW). The PM0.4–1 emitted during combustion of the MS, SWC and BWC were characterized by ICP-MS/OES, XRD, SEM, TEM, and DLS. Cytotoxicity and genotoxicity in human alveolar epithelial A549 and human bronchial epithelial BEAS-2B cells were assessed by the WST-1 assay and the DNA-Alkaline Unwinding Assay (DAUA). PM0.4–1 uptake/translocation in cells was investigated with a new method developed using a confocal reflection microscope. SWC and BWC had a inherently higher residual water content than MS. The PM0.4–1 emitted during combustion of SWC and BWC exhibited higher levels of Polycyclic Aromatic Hydrocarbons (PAHs), a greater variety of mineral species and a higher heavy metal content than PM0.4–1 from MS combustion. Exposure to PM0.4–1 from combustion of SWC and BWC induced cytotoxic and genotoxic effects in human alveolar and bronchial cells, whereby the strongest effect was observed for BWC and was comparable to that caused by diesel PM (SRM 2 975), In contrast, PM0.4–1 from MS combustion did not induce cellular responses in the studied lung cells. A high PAH content in PM emissions seems to be a reliable chemical marker of both combustion efficiency and particle toxicity. Residual biomass water content strongly affects particulate emissions and their toxic potential. Therefore, to minimize the harmful effects of fine PM on health, improvement of combustion efficiency (aiming to reduce the presence of incomplete combustion products bound to PM) and application of fly ash capture technology, as well as use of novel biomass fuels like Miscanthus straw is recommended.