## 3 Results and Discussion
### Significance of Colloidal Metal Fraction in the Dissolved Phase-Comparison with Other Coastal Areas
#### 3.4.2 Metals Associated with Dissolved Organic Matter (Cu, Co)  
When considering the total metal fraction, Cu showed the highest affinity to colloids among studied metals. In the total fraction, on average, 29% of Cu was present as colloidal compounds, which is considerably more than for other studied metals (Table 1). Unlike Pb, Fe and Al, which relied on both the colloidal and particulate fraction, a small amount of Cu was bound to >0.1 um particles (<11%), highlighting the importance of the colloidal fraction to Cu distribution in the estuary. In the dissolved fraction, on average, 32% of Cu was present as colloidal compounds, similar to the results obtained in the Gulf of Trieste [37] and Loire estuary [51]. When comparing the importance of colloidal Cu in various estuarine systems, disparate results are obtained (Table S1). In the Xin'an River, Guandang River estuaries [24] and San Francisco Bay [5], 86-92% of dissolved Cu was found in the truly dissolved phase, whereas in the Penze estuary [6], Ob estuary [52] and Fal estuary [11], the colloidal fraction dominated the Cu size speciation. Cu complexes with dissolved organic ligands have been widely reported to dominate the Cu speciation in the dissolved fraction, so the differences in its size distribution between different studies could be related to differences in the organic matter composition within these systems [51; 53; 54; 55; 56]. A competition between high and low molecular weight organic ligands could drive the Cu size distribution in estuarine systems [10], such as the increase of colloidal Cu in the FSI observed in our study (Figure 2). [PERSON] et al., 2001 [57] showed that, in Galveston Bay, colloidal Cu-binding ligands have, on average, 60 times higher conditional stability constants than truly dissolved ligands. Similarly, in the Loire estuary, the colloidal Cu-binding ligands were estimated to be 4 to 260 times stronger than those found in the truly dissolved phase [51]. In coastal waters, macromolecular Cu-binding ligands in the colloidal size range could be allochthonous humic aggregates or freshly produced high molecular weight biomolecules, such as proteins or polysaccharides [6]. In the Krka estuary, the dissolved Cu concentration showed the highest correlation with protein-like (tryptophan-like) fluorescent substances (Figure 3A) (PARAFAC component C3: \(\lambda_{\text{ex}}/\lambda_{\text{em}}=275/344\); detailed in [PERSON] et al., 2020 [30]). Protein-like fluorophores are considered to be derived from freshly produced autochthonous organic matter and showed an evident relationship with elevated biological activity [58; 59]. Similarly to the Krka estuary, in the Gulf of Trieste, among 13 analyzed metals (including Mn, Fe, Co, Ni, Zn, Cd and Pb), Cu (along with Hg) showed the highest affinity to colloidal organic matter that mostly originated from phytoplankton activity [37]. Proteinaceous substances bear sulfur functional groups that strongly bind Cu; thus, they can play a major role in the estuarine Cu biogeochemistry [56; 60]. Biogenic reduced sulfur species can also enhance the formation of polysaccharide aggregates and Cu uptake into the colloidal fraction [10; 61]. In the Galveston Bay, colloidal Cu-organic complexes mediate the partitioning between particles and solution [57]. In this system, reduced sulfur species from biological sources accounted for most of the Cu-complexing ligands [57]. In addition, in the Loire estuary, proteinaceous colloidal material was most likely the cause of the change of Cu speciation in the middle of the estuary [51]. On average, 18% of Co in the dissolved fraction was associated with colloids (Table 1), similar to Galveston Bay where the colloidal fraction (1 kDa-0.45 um) averaged 19% of the filter-passing pool [62] (Table S1). In the Amazon and Para River estuaries, on the other hand, up to 86% of colloidal Co (0.015-0.2 um) was found in the dissolved fraction in the low-salinity range and, in contrast to the Krka River estuary, showed a general decrease, with salinity down to 16% [63].