Abstract
Existing standard (eco)toxicity test guidelines (TGs) have been found to exhibit significant limitations in the reliable assessment of manufactured nanomaterials (MNMs). These include the preparation of MNM dispersions, dispersion stability during exposure and interference with measurement techniques e.g. through the presence of MNMs and their aggregates. This has resulted in significant challenges in establishing a functional risk assessment framework for MNMs. Recently, international efforts have been undertaken to address these MNM-related challenges and propose modified TGs. In this study, we evaluated existing standard TGs and some proposed modified TGs for freshwater algae ecotoxicity assessment using two carbon-based nanomaterials (CNMs); multi-walled carbon nanotubes (MWCNT) and graphene oxide (GO). Interactions of MWCNT and GO with chlorophyll-a (Chl-a) quantification were studied using (i) OECD 201 TG with in vivo Chl-a determination, (ii) ISO 10260 TG with in vitro Chl-a determination, and (iii) OECD 201 TG with a modified in vitro Chl-a determination.
The tested CNMs caused a high level of interference in in vivo Chl-a determination, resulting from shading of Chl-a fluorescence by CNMs and their aggregates (MWCNTs). Furthermore, GO caused a high background noise through autofluorescence and MWCNTs exhibited algal adsorption. Direct signal interference and autofluorescence were found to be sufficiently reduced using in vitro Chl-a determination approaches. However, both CNMs caused a concentration dependent loss of fluorescence signal, where algae were extracted directly after CNM exposure. Furthermore, we also found a significant reduction of Chl-a fluorescence when MNMs were added after the extraction process, or shortly before fluorescence measurements. This indicates that the CNMs used in this study directly reduce the amount of free Chl-a. This likely due to adsorption processes as CNM feature high surface areas and strong adsorptive properties. While both materials significantly reduced the amount of measured Chl-a, the more hydrophobic MWCNTs exhibited stronger adsorption of Chl-a compared to GO. Owing to their specific physicochemical properties, toxicity assessment of CNM with algae remains challenging and this study emphasizes the need for further research towards the development and establishment of standard TG which are suitable for all MNM types.
The tested CNMs caused a high level of interference in in vivo Chl-a determination, resulting from shading of Chl-a fluorescence by CNMs and their aggregates (MWCNTs). Furthermore, GO caused a high background noise through autofluorescence and MWCNTs exhibited algal adsorption. Direct signal interference and autofluorescence were found to be sufficiently reduced using in vitro Chl-a determination approaches. However, both CNMs caused a concentration dependent loss of fluorescence signal, where algae were extracted directly after CNM exposure. Furthermore, we also found a significant reduction of Chl-a fluorescence when MNMs were added after the extraction process, or shortly before fluorescence measurements. This indicates that the CNMs used in this study directly reduce the amount of free Chl-a. This likely due to adsorption processes as CNM feature high surface areas and strong adsorptive properties. While both materials significantly reduced the amount of measured Chl-a, the more hydrophobic MWCNTs exhibited stronger adsorption of Chl-a compared to GO. Owing to their specific physicochemical properties, toxicity assessment of CNM with algae remains challenging and this study emphasizes the need for further research towards the development and establishment of standard TG which are suitable for all MNM types.