Distribution, speciation and composition of humic substances in a macro-tidal temperate estuary

Highlights

• A simple method for the determination of humic substances in suspended particulate matter is developed and validated.

• The method allows the quantification and the characterization of particulate humic substances along the entire land-sea continuum of a temperate estuarine system.

• Internal abiotic and biotic estuarine processes alter the chemical composition of humic substances during estuarine mixing.

Abstract

In aquatic environments, the term humic substances (HS) encompasses terrestrial and autochthonous refractory organic matter. HS are one of the main fractions of natural organic matter and are important chelators of trace elements. In environmental studies, the determination of HS is often restricted to the dissolved fraction, and the content of HS in the suspended particles remains unknown. In this work, we present the composition and dynamics of HS along the mixing gradient of a macrotidal estuary in both the dissolved and particulate fraction. After the isolation of particulate HS using a solid-liquid alkali extraction method, HS were characterized by size exclusion chromatography (SEC) and electrochemical methods. The method, validated using a certified reference material, demonstrated a low detection limit (μg-C L⁻¹), a good repeatability (7.7%) an excellent reproducibility (1.3%) and was poorly prone to contamination (filter blank < 1 μg-C). Analyses of environmental samples showed a particulate fraction contributing significantly to the total humic pool in the estuary (3–20%) and representing up to 35% of electroactive HS. Phase transfers from the dissolved to the particulate fraction were observed and the electroactive HS were strongly affected in the maximum turbidity zone. Multi-detection SEC analyses showed differences in the molecular composition between the dissolved and particulate fractions. Particulate HS were more nitrogen-rich and electroactive than dissolved HS. The non-conservative changes in the composition of HS along the land-sea continuum evidence the existence of abiotic and biotic processes that altered HS during their transit from river to marine waters.

Introduction

Humification is the process that transforms detritic organic matter originating from plant and animal decomposition into “humus”. It is a complex process not well understood. The most commonly accepted hypothesis is that it would be controlled by physicochemical, biochemical and microbiological reactions that lead to the formation of aromatic and aliphatic compounds: humic substances (HS) (Stevenson, 1994). HS are traditionally recovered from soils via successive chemical processing and extractions. After extraction, they can be operationally defined in three groups of compounds: humins are insoluble at any pH, humic acids (HA) are insoluble at pH < 1 and fulvic acids (FA) soluble at any pH (Thurman and Malcolm, 1981; Moreda-Piñeiro et al., 2006). In aquatic ecosystems, HS accounts for up to 20% of the dissolved organic matter in ocean waters (Hessen and Tranvik, 1998; Dulaquais et al., 2018a) and between 30 and 80% in estuarine waters (Dittmar and Kattner, 2003; Waeles et al., 2013; Dulaquais et al., 2018b). HS are therefore one of the main fractions composing dissolved organic matter playing a key role in aquatic biogeochemistry. Among others, HS increase the solubility of organic contaminants (Tanaka et al., 1997; Blasioli et al., 2008) and stimulate the growth of some dinoflagellates (Gagnon et al., 2005). Moreover there is a growing interest in their important role as complexing ligands of essential trace metals such as iron (Laglera et al., 2011; Bundy et al., 2015; Dulaquais et al., 2018b; Whitby et al., 2020) and copper (Voelker and Kogut, 2001; Shank et al., 2004; Waeles et al., 2015, Dulaquais et al., 2020). Quantitative analysis of HS is not easy. It involves the determination of a class of operationally defined compounds. Moreover the definition of HS is also method dependent (e.g. fluorescent vs chromatographic isolation) making it difficult to compare results between different studies using different analytical methods. The study of HS can also be confusing to follow due to a complex terminology thereby the terminology used in this study is synthetized in Table 1. In this study we In order to overcome this issue, the scientific community has implemented the use of common standards for calibration (Fillela, 2014). Those standards are provided by the International Humic Substance Society (IHSS) with known elemental composition and chemical properties.

Different analytical techniques allow the quantification of HS in natural waters. The most commonly used are size exclusion chromatography (SEC) (Hubert et al., 2011; Abbt-Braun et al., 2004; Dulaquais et al., 2018b), UV–visible and fluorescence spectroscopy (Senesi, 1990; Miano, 1992; Coble, 1996; Parlanti et al., 2000; Leenheer and Croué, 2003) as well as electrochemical methods (Quentel et al., 1986, Chanudet et al., 2006, Laglera et al., 2007; Whitby and van den Berg, 2015, Sukekava et al., 2018). While SEC methods allow the determination of total HS carbon concentrations, fluorescence spectroscopy monitors the transformation of HS, and electrochemical methods provide information on the capacity of HS to complex trace elements. The development of these methodologies led to the publication of a large number of studies on the behaviour of dissolved HS in riverine, estuarine and coastal waters. In the case of macrotidal temperate estuaries, Waeles et al. (2013) studied the seasonal variations of dissolved electroactive HS (deHS) in an estuary prone to a high anthropogenic pressure from agricultural activities. Their results demonstrated that high levels of deHS (>3 mg-C L⁻¹) were associated with periods of the first seasonal high floods confirming the terrestrial origin of estuarine HS and the significant mobilization of eHS from soil by drainage in early fall. Waeles et al. (2013) also showed a quasi-conservative behaviour of deHS along the estuary most of the year but proposed that flocculation processes could induce non-conservative distributions. Similar results were reported in several estuarine systems by analysing either deHS or total dissolved HS (dHS) (Sholkovitz et al., 1978; Ertel et al., 1986; Huguet et al., 2009; Asmala et al., 2014; Marie et al., 2017). To date, the analysis of HS in natural waters has been carried out mainly in the dissolved phase and only a restricted number of studies have focused on the particulate phase. Calace et al. (2006) investigated particulate HS (pHS) in the Venice canals and in the Ross Sea (2010). These authors determined pHS concentrations as the difference between unfiltered and filtered samples and the extraction used to recover pHS was long, arduous and may not be the most appropriate technique for large-scale studies.

Quantification of pHS in estuarine and marine waters is of particular interest for several reasons. First, to our knowledge, there are only few published data on the levels and distribution of pHS along the entire land-sea continuum (Hair and Bassett, 1973; Harvey and Mannino, 2001) and there are so far no published data on peHS in estuaries. Secondly, studies conducted in estuarine ecosystems highlighted the extended role of deHS on the complexation and thus on the speciation of trace metal elements (Laglera et al., 2011; Bundy et al., 2015; Abualhaija et al., 2015; Dulaquais et al., 2019). Hence, understanding phase transfers of HS and eHS along the salinity gradient will help to better constrain the estuarine biogeochemistry of trace metals. Recent works in the oceanic environment have also shown that deHS account for between 4 and 15% of dHS and play a key role in the biogeochemical cycle of trace metals such as iron (Dulaquais et al., 2018a; Sukekava et al., 2018; Whitby et al., 2020). Unfortunately, without any data the role of peHS on trace element biogeochemistry in the marine environment cannot be established.

In this context, this work presents a simple method combining solid-liquid alkali extraction followed by electrochemical and multi-detection SEC analyses for the quantification of humic matter in suspended particulate matter. Our results provide the first speciation study of HS within (electroactive versus total) and between (dissolved versus particulate) fractions along the land-sea continuum of a macro-tidal estuary.