## 1 Introduction  
Sediment dissolved organic matter (DOM) plays an essential role in the global biogeochemical cycling of carbon and nutrients [1, 2]. The chemical properties of the sediment humic substance (SHS) are dynamic and are affected by environmental conditions and the source of organic matter [3, 4, 5]. Moreover, SHS is a highly heterogeneous and complex organic mixture with a broad molecular weight range [5]. The chemical structure and composition of SHS and the molecular weight are significant properties that associate pollutants distribution, transport, and biotoxicity in an aquatic environment [6, 7, 8, 9, 10]. High molecular weight DOM and SHS have high specific surface areas and form aggregated structures by inter- or intra-molecular interactions within the DOM and SHS. The properties provide the particular sorption sites for hydrophobic organic compounds and heavy metals [6, 8, 9, 11, 12, 13, 14]. However, other studies also reported low molecular weight DOM having significant sorption capacity for organic pollutants and heavy metals [15, 16, 17, 18]. The chemical structure and composition of SHS and DOM might be the significant factor that influences the pollutants distribution, transport, and binding capacity [3, 6, 18, 19, 20, 21].  
Fluorescence and ultraviolet spectroscopy are sensitive and fast techniques that are widely used to investigate the composition and structure of SHS [11, 22, 23, 24, 25]. The aromaticity index (SUVA\({}_{254}\)) and the humification index (HIX) have been widely employed to investigate the chemical properties of DOM and SHS [2; 5; 9; 10; 18; 19]. Moreover, the fluorescence regional integration (FRI) method divides the excitation and emission matrix (EEM) into five regions and utilizes the fluorescence intensity at all EEM wavelength pairs for sample characterization [22; 26; 27; 28]. The FRI has been successfully and extensively utilized to characterize DOM in water and SHS in sediment and soil samples [27; 28; 29; 30]. The optical indicators and FRI-coupled EEM technology are time-saving and require minimal sample pre-treatment compared with typical physicochemical methods [22; 27; 31].  
Additionally, collected sediment generally has been pre-treated with air-drying (AD) or freeze-drying (FD). These two pre-treatment methods have resulted in different SHS quantities and qualities [10; 32; 33; 34], due to biological activity. [PERSON] et al. [10] reported that AD pre-treated sediment had slightly lower pH, organic matter, and total organic carbon compared to the FD pre-treated sediment. Since the SHS is a complex organic mixture with a wide-range molecular weight, size-fractionated SHS can be used as an effective method to investigate the differences between the chemical properties of SHS pre-treated by AD and FD [10]. This study investigated the SHS extracted from river sediment; it was pre-treated with both AD and FD methods. The extracted bulk SHS solution was separated into three size fractions. The bulk and size-fractionated SHS quantities were measured with dissolved organic carbon (DOC) concentrations, and the chemical characteristics were analyzed with UV-Vis and fluorescence spectroscopy. The indicators SUVA\({}_{254}\) and HIX were used to distinguish aromaticity and the extent of humification of the bulk and size-fractionated SHS. The chemical composition of the size-fractionated SHS was investigated with the FRI method.