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

Abstract

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.

Keywords:

• Standard tests
• adsorption
• carbon nanotubes
• graphene oxide

Acknowledgements

The authors acknowledge the essential technical assistance of Lisbet Støen and Inger Steinsvik. We thank Julian Tolchard for conducting the STEM analyses and Dag Altin for access to light microscopy equipment.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

Polish-Norwegian Research Programme operated by the National Centre for Research and Development under the Norwegian Financial Mechanism 2009?2014 in the frame of Project Contract No Pol-Nor/237761/98/2014.