| 09 Feb 2026
Estuarine Mixing Drives Organic Nitrogen Transformation and Bioavailability Dynamics
Abstract. Estuaries act as critical transition zones for nitrogen transport, where the dynamics of inorganic nitrogen has been extensively studied. In contrast, organic nitrogen (ON), encompassing particulate organic nitrogen (PON) and dissolved organic nitrogen (DON), is strongly influenced by estuarine mixing of freshwater and seawater. However, the mechanisms driving ON transformation and their implications for bioavailability remain poorly understood. Here, estuarine mixing experiments are conducted across salinity gradients to explore ON transformation and changes in nitrogen bioavailability driven by physicochemical and biological processes. Using tangential flow filtration, optical signatures, and stable isotopes (δ¹³C, δ¹⁵N), we quantified ON composition and molecular characteristics. DON dominated the ON pool (> 71 %) throughout the mixing process, with the low-molecular-weight (LMW) fraction accounting for 49 ± 7.8 %. Salt-induced flocculation and adsorption (i.e. physicochemical processes) preferentially transferred a large fraction (63 ± 11 %) of humic-like components, mainly terrestrial refractory compounds, into the particulate phase, thereby increasing PON. Meanwhile, biological activity promoted the degradation of residual humic-like components (especially microbial C3), producing labile LMW-DON and ammonium. This conversion was evidenced by a strong negative correlation between humic-like and protein-like components in control treatments. The isotopic enrichment (δ¹³C, δ¹⁵N) and elevated C/N ratios in PON further suggested the re-adsorption of biologically-modified and δ¹⁵N-enriched DON onto particles, enhancing PON refractoriness. Overall, estuarine mixing drives conversion of humic-like ON into refractory particulate forms, while simultaneously enhancing the bioavailability of the residual dissolved nitrogen pool, thereby influencing nitrogen cycling and eutrophication risks at the river-sea interface.
General Comments:
Estuaries act as critical filters for terrestrial nutrients transporting to the ocean. With the increasing anthropogenic nitrogen loading worldwide, understanding the transformation and bioavailability of organic nitrogen (ON) in estuarine mixing zones is becoming crucial for predicting coastal eutrophication. However, most previous studies focused on inorganic nitrogen, while the dynamics of the organic fraction remains less understood. This manuscript investigates the ON transformation and bioavailability dynamics during estuarine mixing, which addresses an important gap. The experimental design is logical, and the data is solid. It is a meaningful paper.
Specific Comments:
1. In Introduction, add quantitative data or recent estimates regarding the global flux of estuarine ON to highlight the significance of estuarine organic nitrogen.
2. The incubation experiments were conducted for 3 days. While this effectively captures rapid mixing processes, the manuscript discusses "long-term" nitrogen sequestration. It would be better to briefly mention the limitations of this short-term incubation in the Discussion section to ensure the extrapolation to long-term sinks is robust.
3. Since phytoplankton uptake of DIN involves isotopic fractionation, could this process also contribute to the elevated δ15N values in PON?
4. Figure 7 presents an "Enhanced nitrogen pump model." What exactly is "enhanced"?
5. The authors mention using parametric tests (e.g., t-tests or ANOVA) to compare treatments. It would be rigorous to briefly clarify whether the normality of the data distribution was checked.
6. I noticed several grammatical and formatting errors that should be corrected:
Line 127: “p” is not italic. Line 188: "The non-conservative changes... are govern by..." should be "are governed by".
Line 223: "...particle characteristics , typically..." There is an extra space before the comma.
Line 309: "This study highlight that..." should be "highlights".
Line 424: "Crucially, our isotopic evidence that biologically-modified DON re-adsorbs onto particles..." This sentence appears to be a fragment or grammatically incomplete (missing a main verb for "evidence" or "that" should be removed).