Influence of seasonal changes in organic matter pool on copper bioavailability in a stratified estuary

This work presents a detailed analysis of seasonal changes in copper (Cu) organic speciation and its bioavailability in the Krka River estuary, a highly stratified microtidal system characterized by low riverine input of dissolved organic matter (DOM) and trace metals but experiencing heightened anthropogenic Cu pressures during summer as a result of increased touristic activities. Total dissolved Cu concentrations ranged from 3.4 nmol L⁻¹ in winter up to 25.8 nmol L⁻¹ in summer, with substantial summer increase limited to water layer above the halocline. Two distinct ligand classes were identified in all samples, with conditional stability constants of logK’₁ = 13.2 ± 0.5 and logK’₂ = 11.2 ± 0.4. Dissolved organic carbon (DOC) and concentrations of both ligand classes were higher in summer than in winter, with strong indications of their in situ production driven by enhanced primary production and/or intensified microbial activity. In winter, despite the low DOC levels (avg 63.3 μmol L⁻¹), DOM above the halocline showed pronounced terrestrial signature with higher density of Cu-binding sites compared to DOM in summer with nearly twice as high DOC levels (avg 113.5 μmol L⁻¹). The results suggest that terrestrial humic substances (HS) dominated the ligand pool in these waters during winter (estimated 80 % of total ligand pool), possibly contributing to both ligand classes. While HS remained an important fraction of the ligand pool in the summer (estimated 30 % of total ligand pool), the terrestrial signature of DOM in this season was minimal, pointing to their production within the estuary. The summer increase in ligand concentrations above the halocline was accompanied by a reduction in their binding strengths (by approximately 0.5 log units) compared to winter, along with a decrease in DOM molecular weight, possibly influenced by solar irradiation. Seasonal anthropogenic Cu input was successfully buffered by the ambient ligand pool, keeping the concentration of free Cu-ions, its most bioavailable specie, well below documented toxicity levels for Cu (maximum of 2.54 pmol L⁻¹). The seasonal DOM variations resulted in a ∼ two-fold increase in the Cu-binding capacity in summer (33 nmol L⁻¹) compared to winter (17 nmol L⁻¹). Despite the increased buffering capacity in summer, Cu levels are approaching this safety limit, which may increase its bioavailability to a concerning level, a risk much lower in winter when Cu inputs are minimal. These results underscore the critical role of DOM in mitigating seasonal anthropogenic Cu inputs, while also highlighting the need for further investigation into the long-term resilience of estuarine systems under shifting environmental and anthropogenic pressures.