Table 5. Advantages and limitations of the selected test assays based on the experience gained by the partners of the NANOVALID project.
| Test, organism | Model for | Advantages | Limitations |
|---|---|---|---|
| Bacterial growth inhibition assay (Escherichia coli, Staphylococcus aureus, Pseudomonas sp. Bacillus subtilis) | Medically important pathogenic bacteria;Environmentally relevant bacteria | Simple and rapid tests showing whether the tested NMs exhibit general toxicity to the given bacterial strains. | Growth inhibition assays are conducted in organics-rich media that may sorb or interfere with NMs (agglomeration/dissolution etc). |
| Vibrio fischeri bioluminescence inhibition assay | Marine bacteria | The assay with V. fischeri is rapid and reliable standardized assay (ISO, 2010). | Test is sensitive to pH (the test medium is not buffered). High salinity (2% NaCl). |
| Yeast (Saccharomyces cerevisiae) viability assay | The simpliest model for eukaryotic cell | The use of DI water as the test environemnt eliminates the speciation-related effects of metallic NMs. | DI water is not a natural environment and may trigger osmotic stress in cells. This may change the susceptibility of cells to NMs. |
| Algal (Raphidocelis subcapitata) growth inhibition assay | Environmentally relevant freshwater primary producer | Algal growth inhibition assay (OECD, 2011) is among the most sensitive ecotoxicological tests. | NMs may interfere with optical density measurements and shade the light necessary for the growth of algae. |
| Protozoan (Tetrahymena thermophila) viability assay | Environmentally relevant eukaryotic organism | T. thermophila can internalize NMs by phagocytosis. | DI water is not a natural environment and may trigger osmotic stress in cells. This may change the susceptibility of cells to NMs. |
| Crustacean (Daphnia magna) acute immobilization assay | Environmentally relevant freshwater organism | One of the most sensitive organisms used in ecotoxicity assessment of chemicals and D. magna test (OECD, 2004) is among the assays that are required by the REACH guidelines for the ecotoxicity assessment.Particle-feeding organism and thus, very relevant model for NMs. | Test medium may induce agglomeration and precipitation of NMs.High sample volume (50 ml per concentration in one experiment) is required. |
| Isopod (Porcellio scaber) cell membrane integrity assay (AO/EB) | Environmentally relevant terrestrial organism | Isolated digestive gland is the model for tissue cell membrane permeability. The test provides very specific data on the cell membrane stability. | Accurate training of staff is required. The method is not robust. The physiological state of the gland need to be precisely described. |
| Zebrafish (Danio rerio) embryo toxicity assay | Environmentally relevant freshwater organism | The test is robust and highly valuable for NM testing in aquatic environment. Fish embryos are transparent allowing direct observation of malformations. | The set-up of rearing the zebrafish embryos is technically challenging. |
| Human mesenchymal stem cell membrane integrity (PI) and mitochondrial activity (MTT) assays | Non-cancerous primary derived human cells | Test endpoints are based on fundamental cellular processes, i.e., mitochondrial activity (MTT, WST-1) and membrane integrity (NRU and PI).NRU assay with murine fibroblasts is standardized toxicological assay for chemical testing (OECD, 2010). | Some NMs may interfere with the fluorescence and absorbance-based viability assays. |
| Murine fibroblast BALB/c 3T3 membrane integrity (NRU) and mitochondrial activity (WST-1) assays | Non-cancerous mammalian cell line | ||
| Rat alveolar macrophage NR8383 mitochondrial activity assay (WST-1) | Non-cancerous mammalian macrophages | Does not require washing after exposure prior to the assay, and is therefore suitable for suspension cells. Very fast and easy to perform. | Some NMs may interfere with the assays. |
AO/EB: acridine orange/ethidium bromide; NRU: neutral red uptake; PI: propidium iodide; WST-1: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium.