the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Aug 2026
Optical and electrochemical measurements characterize different functional fractions of aquatic humic substances
Abstract. The extent to which the metal-binding functionality of dissolved organic matter (DOM) can be inferred from its conventional optical properties remains largely unknown. Optical measurements of dissolved organic matter (DOM), including chromophoric (CDOM) and fluorescent (FDOM) properties, are commonly used as proxies for humic substances, yet it remains unclear whether these approaches also reflect the same metal-binding fraction quantified by recently developed electrochemical techniques. In this study, electroactive humic substances (eHS), CDOM, FDOM, and dissolved trace metals were measured across a humic-rich tropical terrestrial-marine continuum, and the results were compared with a previous dataset from a less humified estuarine system. Principal component analysis revealed that eHS was decoupled from conventional optical indices, including the absorption coefficient at 254 nm (a254) and the humification index (HIX), but was closely associated with dissolved manganese and cobalt. Despite the contrasting environmental settings, a similar separation between eHS and optical DOM properties was observed, indicating this decoupling is a general feature rather than a site-specific phenomenon. These findings show that optical proxies alone do not adequately characterize the fraction of humic substances responsible for trace metal complexation and transport, whereas electrochemical measurements selectively quantify a chemically distinct, metal-reactive fraction of the DOM pool. Integrating optical and electrochemical approaches therefore provides complementary perspectives on DOM composition and functionality, offering a more complete framework for improving our understanding of trace metal-DOM interactions in aquatic environments.
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Status: open (until 27 Oct 2026)
- RC1: 'Comment on egusphere-2026-4066', Anonymous Referee #1, 02 Sep 2026 reply
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This manuscript presents a valuable investigation into the relationship between electroactive humic substances (eHS) and conventional optical proxies for dissolved organic matter (DOM) across a tropical peatland-to-marine continuum. The study addresses an important knowledge gap regarding whether optical measurements can effectively represent the metal-binding functionality of DOM. The experimental design, which includes a comparative analysis with a previous dataset from Otsuchi Bay, is robust and provides useful insights. The finding that eHS is decoupled from bulk optical properties like a254 and HIX is significant and supports the complementary use of electrochemical and optical methods.
However, before the manuscript can be accepted for publication, several critical issues, particularly concerning the data presented in Figure 3 and methodological choices, must be addressed by the authors. These issues, if left unresolved, could undermine the clarity and validity of the study's core conclusions.
Major Comments:
1. Inconsistency of Optical Data in Marine Waters (Figure 3): The data presented in Figure 3 reveal a substantial and concerning inconsistency with well-established biogeochemical paradigms and the authors' own classification scheme. According to the PCA classification, sites (S14, S15, S16, S18) represent "marine waters" with a strong marine influence. In such a setting, one would expect:
(1) Lowest DOM abundance: Due to dilution with low-DOM seawater, the absorption coefficient (a254) should be at its lowest in the marine endmember.
(2) Highest spectral slope (S275-295): This slope is inversely related to molecular weight and aromaticity. Marine DOM, which is more processed and photobleached, typically exhibits the highest S275-295 values.
(3) Lowest humification index (HIX): Marine DOM is generally less humified than its terrestrial counterparts.
Conversely, Figure 3 shows that the "marine waters" group has the highest a254, intermediate S275-295, and moderately elevated HIX. This pattern is not only contradictory to the authors' own classification but also to the classic behavior observed in other estuarine systems. This is a fundamental data contradiction that must be resolved. The authors need to:
(1) Re-examine the raw data for a254, S275-295, and HIX for the marine sites. Is it possible that there was a mislabeling of samples or an error in the calculations?
(2) Provide a clear and convincing explanation for why these samples, which are supposed to be dominated by marine water, exhibit such high terrestrial-like optical properties. If the data are correct, this finding in itself is a major discovery that warrants a detailed discussion, but the current interpretation of a "marine endmember" is invalid.
2. Choice of absorbance coefficient wavelength (a254 vs. a350/a355): In section 2.4, the authors report using the absorption coefficient at 254 nm (a254) as a proxy for CDOM abundance. However, in marine and estuarine waters, a254 is susceptible to interference from inorganic ions like nitrate and bromide. For studying DOM in saline environments, it is standard practice to use absorption coefficients at longer wavelengths (e.g., a350 or a355) to minimize these interferences and obtain a more reliable measure of CDOM. The authors should justify why a254 was selected over a350/a355, especially given that the study covers a wide salinity gradient. If a350/a355 data were collected, it would be highly beneficial to present them to support the conclusions drawn in this paper.
Minor Comments:
Line 87: The temperature for sample preservation is written as “-20°c”. This should be corrected to “-20 ℃” to use the correct degree symbol and a capital "C" for Celsius.