2.4.3. Marine Coating Formulations

The main goal was to achieve compatible behavior between the GAP compound and the selected coating systems (DI and CI), to determine the limiting GAP content supported by each system. Several formulations of the coatings were optimized in an iterative process considering different GAP contents, solvent compatibilities, and incorporation of additional paint additives. Finally, the optimum GAP concentration was selected as the one that did not damage the main paint properties, i.e., the formulation looked the same as the control (coating system without the immobilization of GAP), Figure S7. The most promising optimized formulations are presented in Table 2 and were selected to pursue further evaluations.

**Table 2.** Marine coating formulations with gallic acid persulfate (GAP).


CI: chemically immobilized; DI: directly incorporated; PDMS: polydimethylsiloxane; PU: Polyurethane; TZA: triaziridine propionate crosslinker.

### 2.4.4. GAP Quantification in Leaching Water Samples

To evaluate the e ffectiveness of the CI strategy in minimizing the release of GAP from the coatings into the aquatic environment, leaching tests were performed using polyvinylchloride (PVC) plates coated with the selected marine coatings formulations. After a period of 45 days, the released GAP was extracted from leaching water samples through an optimized weak anionic exchange (WAX) methodology and then dissolved in UPW. The WAX stationary phase possesses weak groups (secondary amines), which are activated at a reduced working pH, creating a reversible interaction with strongly acidic, acidic and neutral compounds, allowing their concentration and extraction. Therefore, a low pH loading step (pH < 5) will ionize the stationary phase to facilitate the retention of GAP in the leaching water, while the remaining impurities are discarded. The elution of GAP is achieved by increasing the pH to >11, neutralizing the stationary phase. Table 3 shows the amount of detected GAP after quantification by the previously established IP-RP-HPLC method.

**Table 3.** Gallic acid persulfate (GAP) amount released from marine coatings prepared by direct incorporation (DI) and chemical immobilization (CI) strategies into artificial seawater (ASW), after an immersion period of 45 days.


(n): number of replicates; PDMS: polydimethylsiloxane; PU: polyurethane; PVC: polyvinyl chloride. \* mean values ± standard deviation of two independent experiences.

After a period of 45 days, the content of GAP present in the several leaching water samples, after DI in PU and PDMS coatings, was approximately 20% (Table 3). However, after CI, the percentage of GAP released from PDMS coating was only 11% (two times lower than DI in PDMS coatings), and from PU coatings 4% (almost five times lower than DI in PU coatings). These results demonstrate the high e ffectiveness of the chemical immobilization to retain this water-soluble compound in marine coatings, compared to the DI strategy. This phenomenon is also clearly more pronounced for a polyurethane-based matrix, which may be explained by the higher chemical compatibility of the GAP–TZA derivative functionality with a polyurethane system, associated with the intrinsic reactivity of aziridine and its possible cross-link promotion with the highly alkoxy-reactive polyurethane system, thus allowing us to reach higher agen<sup>t</sup> content in the formulations (Table 3) [17].

### 2.4.5. Anti-Settlement Behavior of GAP after Incorporation in Coatings

In order to evaluate the ability of the di fferent GAP-containing marine coatings to prevent the attachment of mussel larvae, the anti-settlement response was evaluated at lab scale in a bioassay using *Mytilus galloprovincialis* larvae (Figure 5).

PU marine coating containing CI GAP seems to be e ffective against the settlement of mussel larvae, inhibiting larval settlement after both 15 h (−50%) and 40 h (−80%), in wells with this coating, compared to the control (PU). Although the individual variability of the larvae responses does not allow significantly di fferent results against the control, the obtained settlement inhibition rates still represent a good indicator of the GAP AF potential in marine coatings. In contrast, PU marine coating containing DI GAP did not behave di fferently from the control. Since this conventional insoluble PU-based coating matrix acts mainly through a leaching e ffect [38], this result may indicate that the leaching rate and/or available GAP concentration in test media and on the outer surface of the coating is not enough to provide an e ffective AF action. The antifouling mechanisms involved, for instance, following leaching and/or contact strategies, would require a deep reformulation of coatings and further characterization, which go beyond the scope of this work.
