the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 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.
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Status: open (until 24 Apr 2026)
- CC1: 'Comment on egusphere-2026-216', Xuegang Li, 12 Feb 2026 reply
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RC1: 'Comment on egusphere-2026-216', Xiaosong Zhong, 10 Apr 2026
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This manuscript addresses an important and timely topic: how estuarine mixing reshapes the partitioning, composition, and potential bioavailability of organic nitrogen. The distinction among PON, CON, and tDON is meaningful, and the comparison between biologically active mixing (BAM) and biologically inhibited mixing (BIM) is a potentially strong feature of the study. The analytical framework is also relatively comprehensive, combining TFF-based fractionation, fluorescence signatures, FTIR, stable isotopes, and ancillary particle and enzyme measurements. Overall, the study has clear potential and is built on a potentially publishable dataset.
Specific Comments
- The central message should be sharpened.At present, the manuscript tries to emphasize too many things at once: ON partitioning, DON bioavailability, PON refractoriness, nitrogen sequestration, eutrophication risk, and a broadly generalized estuarine framework. This weakens the paper’s main contribution. In my view, the strongest and most defensible message is that estuarine mixing generates a dual response: humic-like ON is preferentially transferred into particulate/refractory pools through adsorption and flocculation, while biological processing simultaneously alters the residual dissolved pool toward a more potentially labile composition. I encourage the authors to reorganize the abstract, discussion, and conclusions around this narrower and clearer core advance.
- The manuscript interprets increasing protein-like fluorescence, higher FI and BIX, lower SUVA254, and NH4+ accumulation as evidence for enhanced DON bioavailability. These are useful and suggestive indicators, but they remain indirect proxies. The study does not include direct DON bioassays, uptake experiments, or response experiments demonstrating that the transformed dissolved nitrogen pool is more readily used by microbes or phytoplankton. Therefore, I suggest that the manuscript consistently distinguish between demonstrated transformation and inferred increases in potential bioavailability.
- L84 provides coordinates for the riverine and marine endmembers and notes that the seawater site was located approximately 40 km downstream of the river mouth, but there is no figure showing the sampling region, the river mouth, and the relative positions of the two endmembers. A map is a basic but important element for a study of this type. It would help readers assess the geographic setting, the representativeness of the chosen endmembers, and the realism of the experimental river–sea mixing framework. I strongly recommend adding a location figure.
- L92-93 states that river water and seawater were mixed at 10:0, 8:2, 6:4, 5:5, 4:6, 2:8, and 0:10, but the manuscript does not explain why these particular ratios were selected. Were they chosen to represent specific salinity zones in the estuary, to match field observations, or simply to generate a broad mixing gradient? This matters because the interpretation of non-conservative behavior may depend on how well the critical salinity range is resolved. The authors should provide a clear rationale for this design choice.
- Figure 4 presents direct experimental observations, including FI, SUVA254, BIX, FTIR, POC, and C/N changes along the salinity gradient. They are primary results. Moving Figure 4 to the Results would also improve the structure of the paper.
- The manuscript ends by linking its findings to coastal eutrophication risk and bloom development. This is a reasonable broader implication, but it extends beyond what is directly shown in a short-term controlled mixing experiment based on one summer sampling event. The transformed ON pool may indeed have ecological implications, but the paper does not directly demonstrate downstream biological responses in the receiving coastal system. I recommend framing this as a likely implication or testable hypothesis, not as an outcome already established by the present study.
Technical Corrections
- L87 “dissolved oxygen (DO) were measured in Table S1”
- L171-172 “While in the BAM treatments” it should almost certainly refer to BIM？
- There is an apparent inconsistency regarding Figure 4d: the figure caption says FTIR is shown for BAM only, whereas the Discussion interprets the FTIR trends as occurring in the BIM.
- Supplement
(1) Text S1the TFF cleaning protocol appears to omit the concentrations for NaOH and HCl (“mol L-1 NaOH” and “mol L-1 HCl”)
(2) “Pdl” should presumably be “PdI,” and “Phl” also appears to be a typo or mislabeled parameter. Please check both the text and the figure labeling.
(3) The supplement still contains typographical errors such as “doenotes” in the Figure S8 caption.
(4) Table S1 “Sampling data” should be “Sampling date.”
(5) Table S2 in the supplement incorrectly refers to “truly dissolved organic carbon (tDON)” and should be corrected to nitrogen.
Citation: https://doi.org/10.5194/egusphere-2026-216-RC1 -
RC2: 'Comment on egusphere-2026-216', Shengwei Cao, 14 Apr 2026
reply
General comments
This manuscript investigates organic nitrogen transformation and bioavailability during estuarine mixing using a dual-treatment design (BAM vs. BIM) to separate biological from physicochemical processes. The topic is relevant to BG's scope, and the experimental approach — combining tangential flow filtration, EEM-PARAFAC, stable isotopes, FTIR, and enzyme assays — is comprehensive. The three-pathway conceptual model and the "enhanced nitrogen pump" framework are valuable contributions.
Scientific significance: The BAM/BIM design effectively isolates biological and physicochemical contributions to ON fate, which is difficult to achieve in field studies. The global estuarine ON synthesis (Fig. 5, Table S4) adds breadth.
Scientific quality: The analytical methods are sound, but several issues in data interpretation weaken key arguments — notably an apparent inconsistency between the FTIR description and figure caption (Line 257–258), a questionable zeta potential argument (Line 189–192), and insufficient support for the claimed humic-to-protein transformation pathway (Line 260–275).
Presentation quality: Generally well-organized, but inconsistent abbreviations, ambiguous method descriptions, and a disproportionately long limitations section reduce clarity. The Conclusions section lacks focus.
Specific Comments
Lines 119–122: The description of Zetasizer measurements immediately follows FTIR analysis of freeze-dried DON, making it unclear what matrix was actually measured for particle size and zeta potential. The measurement target should be specified.
Lines 142–143: The statement that declining CON and tDON concentrations cause the DON/ON ratio to decrease is logically incomplete — the ratio also depends on concurrent PON changes.
Lines 189–192: The claim that negative zeta potentials (ζ < 0) promote DON adsorption onto SPM is inconsistent with colloidal chemistry, where strongly negative ζ indicates greater electrostatic repulsion and colloidal stability. The roles of surface adsorption and colloidal destabilization/flocculation need to be distinguished.
Lines 257–258: FTIR evidence is attributed to the BIM treatments, but the Fig. 4d caption states these spectra are from BAM only. This contradiction undermines a key argument in Section 4.2.
Lines 260–275: The strong negative correlation between C2 and (C1+C3) does not establish that humic-like components are directly transformed into protein-like components. Alternative explanations (e.g., de novo microbial production of C2) could be considered.
Text S1 describes a two-step TFF (10 kDa + 1 kDa) yielding three size fractions, but only two are reported. The rationale for merging the >10 kDa and 1–10 kDa fractions is not explained.
Section 4.5 (Limitations) is disproportionately long and risks overshadowing the study's strengths. The Conclusions section partly repeats the Discussion and could be more concise and quantitative.
Technical Corrections
Line 30: "where critically mediate" — missing subject.
Line 95–96: "to inhibited" — should be "to inhibit."
Line 127 and Fig. S2 caption: "Phl" — should be "PdI."
Line 188: "are govern by" — should be "are governed by."
Line 309: "This study highlight" — should be "highlights."
Line 351: "component effects" — unclear; perhaps "compound effects"?
Fig. 2 legend: "phasa" — should be "phase."
Fig. S2a: Treatment labels "BEM"/"APM" are inconsistent with the defined abbreviations "BIM"/"BAM."
Citation: https://doi.org/10.5194/egusphere-2026-216-RC2 -
RC3: 'Comment on egusphere-2026-216', Yixi Qiu, 15 Apr 2026
reply
Manuscript egusphere‑2026‑216 employs laboratory incubation experiments to investigate the transformation of dissolved organic nitrogen (DON) and particulate organic nitrogen (PON), as well as nitrogen bioavailability. The study offers valuable insights with clear relevance to future research on nitrogen biogeochemical cycling in coastal waters. Overall, the experimental design is robust and the results are interesting and potentially impactful. However, several aspects require further clarification:
- Between line 50 and 55: is there a citation for “Humic-like (terrestrial and microbial sources) and protein-like components are considered as the dominant terrestrial DON components entering the sea”?
- Between line 110 and 115: authors mentioned the PN was considered equivalent to PON because PIN is less than 5%. Wonder how much would it affect the isotopic ratios? Especially if the nitrogen isotopic values of PIN vary along the salinity gradient, would it affect the observed trend?
- Between line 115 and 120: does the S in “Additional water quality parameters including S, pH, SPM, and chlorophyll-a (Chl-a) were measured” stand for sulfur? Why is sulfur collected and how would affect nitrogen? I don’t think I have seen the relevant discussion in the manuscript.
- In results, the manuscript mentioned the DON indicated a non-conservative mixing and PON indicated a conservative mixing. Do you have data to support it? All I can see from the paragraph and figures is that DON decreases with increasing salinity, which looks like conservative for DON.
- Between line 155 and 160: what are the theoretical fluorescence values?
- Between line 170 and 175: numbers from −28.3‰ to −26.5‰, 1.1‰ to 8.8‰, −26.1‰ to −25.9‰, and 6.2‰ to 7.2‰, are them statistically different? If not, it would be a stretch to say they decline along the salinity gradient, especially Figure 3 didn't look like these numbers decreased when the salinity increased.
- Between line 225 and 230: How does the Fig S5a indicate the decrease in the intensity of C1? I didn't found a decrease trend in the figure and also decrease with what? With time, treatment numbers, or salinity?
- Between line 230 and 235: what are the fluorescence index and biological index and how are them measured? You would want to address them in the methods section first.
Citation: https://doi.org/10.5194/egusphere-2026-216-RC3
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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).