Optimization of Fluorescence Humification Index Calculation for Low-Concentration Dissolved Organic Matter Solutions †

Abstract

The fluorescence normalized humification index (HIXn), defined as the ratio of the fluorescence intensity in the 435-480 nm region (I300-345) to the sum of the intensities in the 300-345 and 435-480 nm region (I435-480) under an excitation wavelength of 254 nm, has been widely adopted to quantify the degree of humification of dissolved organic matter (DOM). Nevertheless, in low-concentration DOM solutions (A254 < 0.3 cm-1), weak fluorescence signals at short excitation–emission wavelengths are susceptible to distortion by instrumental background noise, compromising the precision and accuracy of HIXn. This study systematically evaluates the effect of low-concentrations on the reliability of HIXn using natural solutions and dilution experiments analysed via three-dimensional fluorescence spectroscopy. Dilution experiments revealed that as dilution ratios increased, the precision of both I300-345 and I435-480 decreased, resulting in a corresponding reduction in the precision of HIXn (CV ~ 10.0%). In contrast, the fluorescence regional integration (FRI) parameters FRIⅣ and FRIⅤ exhibited significantly higher PREC (CV < 2.0%). Furthermore, strong correlations were observed between I300-345 (I435-480) and FRIⅣ (FRIⅤ) (R2 > 0.95). These findings indicate that the FRI method can effectively replace sectional integration analysis, leading to the development of an optimized index (HIXf). HIXf not only significantly improves the precision (CV < 1.6%) even at low-concentrations (0.3 cm-1>A₂₅₄ > 0.005 cm-1), but increase the accuracy (0.72±0.02, 2SE) by reducing uncertainty caused by the inner filter effect. We therefore propose HIXf as an alternative for assessing the DOM humification degree across broader aquatic ecosystems and wastewater treatment systems.