Algal growth inhibition test with TEMPO-oxidized cellulose nanofibers

Ecotoxicity data on cellulose nanofibers (CNFs) are limited despite their wide potential applications prospects, such as structural and packaging materials, filters, coatings, foods, and cosmetics. In this study, toxicity tests of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (TEMPO-CNFs), which are one of the major CNF products commercially available in Japan, on the green alga Raphidocelis subcapitata were conducted. As nanomaterials are considered difficult-to-test substances, the Organisation for Economic Co-operation and Development has released a guidance document that provides considerations regarding ecotoxicity tests of nanomaterials. In the algal growth inhibition tests of TEMPO-CNFs, there were specific issues to be examined, including the effects of medium components on the characteristics of TEMPO-CNFs, CNF interference with algal density measurements, algal interference with CNF measurements, and the effects of ion concentration changes in the test medium by the addition of CNFs on algal growth. To examine these issues, we conducted preliminary studies and established a suitable test method for algal growth inhibition tests of TEMPO-CNFs. We confirmed that the components in the medium for algal growth inhibition tests had negligible effects on the characteristics (zeta-potential, viscosity, and morphology) and concentration stability of TEMPO-CNFs and that in vitro and in vivo fluorescence measurements were applicable for estimating the algal densities, without interference by TEMPO-CNFs. In contrast, we observed that the grown algae interfered with the CNF concentration measurements. Therefore, we established a method to correct the measured CNF concentrations by estimating the algal contribution. Furthermore, we found that the nutrient salt concentrations in the medium changed due to interactions with CNFs; however, this change did not affect algal growth. Based on the results of the preliminary studies, algal growth inhibition tests of TEMPO-CNFs were conducted using in vitro and in vivo fluorescence measurements, along with measurements of CNFs and ion concentrations in the test dispersions. The test results showed that no growth inhibition was observed on growth rate or yield even at the maximum CNF concentration of 100 mg/L, suggesting that the ecological effect of TEMPO-CNFs on algae was relatively low. The results of this study will be valuable for conducting ecotoxicity assessments on additional CNFs and comparable nanomaterials in future studies.