Applicability of enzymatic and phenol–sulfuric acid methods for determination of cellulose nanofibers in ecotoxicity testing

In parallel with the development of novel materials, such as cellulose nanofibers (CNFs), their effects on aquatic organisms should be evaluated. In ecotoxicity testing, concentration measurements are important to obtain accurate exposure concentrations; however, few studies have measured the concentration and dispersion state of CNFs in test solutions. Therefore, we evaluated the applicability of enzymatic and phenol–sulfuric acid methods for CNF quantification in ecotoxicity testing. In the enzymatic method, CNF content was determined by quantifying the d-glucose produced from the CNFs using cellulase with a glucose assay kit. Three types of cellulases were purchased, and impurities such as d-glucose were identified and removed. The differences in CNF degradation efficiency among the cellulases were evaluated, and a cellulase suitable for CNF degradation was selected. In the phenol–sulfuric acid method, a microplate-based method was employed to handle a large number of samples. We demonstrated that both the enzymatic and phenol–sulfuric acid methods could quantify CNFs in media for ecotoxicity testing. The enzymatic method was able to measure lower CNF concentrations (~ 1 mg/L) than the phenol–sulfuric acid method (~ 10 mg/L) and had less interference with CNF quantification by algae and non-cellulosic polysaccharides.