Are fluorescence-based chlorophyll quantification methods suitable for algae toxicity assessment of carbon nanomaterials?

Using a multiwalled carbon nanotube (MWCNT) and graphene oxide (GO) as representative test materials, we evaluated the applicability of in vivo and in vitro chlorophyll-a (Chl-a) fluorescence quantification methods, which are used in standard algae ecotoxicity tests such as OECD 201 and ISO 8692. In vivo quantification of Chl-a from Raphidocelis subcapitata indicated a significant reduction in Chl-a fluorescence in the presence of MWCNTs due to shading, but a significant autofluorescence from GO caused an overestimation of Chl-a concentration. In vitro Chl-a quantification methods employing a modified acetone and an ethanol extraction protocol reduced the influence of shading and autofluorescence, but both resulted in a significant loss of fluorescence signal in the presence of 100 mgL-1 MWCNTs (99-100%) and GO (21-52%). Chl-a reduction was dose dependent for both tested carbon-based MNMs (CNMs), but effects were more pronounced for MWCNT, which caused a significant fluorescence reduction (16 ± 0.3%) already at 1 mgL-1. Further study of the CNM-algae-Chl-a interaction processes revealed that CNM can not only interact with live algae, but also efficiently adsorb extracted Chl-a. Our results showed that within 10 min, 95-100% of Chl-a extracted from two algae concentrations were adsorbed to MWCNT, while 35-60% of Chl-a was adsorbed to the GO. This study shows that Chl-a quantification by fluorescence determination is not a suitable method for ecotoxicity testing of CNM. However, a quick screening test for individual MNMs is recommended to determine whether Chl-a adsorption is a significant process prior to selection of a quantification method.