Optimization of fluorescence humification index calculation for low-concentration dissolved organic matter solutions

The fluorescence normalized humification index (HIX_n), defined as the ratio of the fluorescence intensity in the 435-480 nm region (I_300-345) to the sum of the intensities in the 300-345 and 435-480 nm regions (I_435-480) at 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 HIX_n. This study systematically evaluates the effect of low concentrations on the reliability of HIX_n using natural solutions and dilution experiments analysed via three-dimensional fluorescence spectroscopy. Dilution experiments revealed that as the dilution ratios increased, the precisions of both I_300-345 and I_435-480 decreased, resulting in a corresponding reduction in the precision of HIX_n (CV ∼10.0%). In contrast, the fluorescence regional integration (FRI) parameters FRI_IV and FRI_V exhibited significantly higher precision (CV < 2.0%). Furthermore, strong correlations were observed between I_300-345 (I_435-480) and FRI_IV (FRI_V) (R² > 0.95). These findings indicate that the FRI method can effectively replace sectional integration analysis, leading to the development of an optimized index (HIX_f). HIX_f not only significantly improves the precision (CV < 1.6%) even at low concentrations (0.3 cm^-1 > A₂₅₄ > 0.005 cm^-1) but also increases the accuracy (0.72 ± 0.02, 2SE) by reducing the uncertainty caused by the inner filter effect. We therefore propose HIX_f as an alternative for assessing the DOM humification degree across broader aquatic ecosystems and wastewater treatment systems.