*3.10. Extractive Procedure*

Each leaching water was divided in three portions and passed through an OASIS ® WAX 6cc cartridge according to the following procedure: the cartridge was conditioned with 6 mL of methanol, followed by 6 mL of water and equilibrated with 6 mL of 25 mM sodium acetate bu ffer acidified with acetic acid (pH 4); water samples were also acidified with sodium acetate bu ffer in order to obtain a pH of 4 and then passed through the cartridge; after drying, each cartridge was conditioned with 6 mL of a solution containing methanol and acetonitrile (20:80 *v*/*v*) with 2% of ammonium, followed by 2 mL of acetonitrile with 2% of ammonium, and finally 2 mL of acetonitrile; finally, the cartridge was washed with 8 mL of methanol and ammonium (90:10 *v*/*v*), followed by 2 mL of methanol basified with ammonium until a pH of 12, and the several solutions were collected in a glass vial. Solvents were reduced to dryness under nitrogen purge by a sample concentrator with a block heater at a temperature of 37 ◦C. After solvent evaporation, the dried compound was solubilized in 200 μL of UPW, passed through a nylon filter and injected into the IP-RP-HPLC system using a mobile phase containing an aqueous solution with 25 mM of TBA-Br and acetonitrile (50:50 *v*/*v*). Recoveries of 100% for this extractive procedure were previously obtained (Figures S8 and S9).

### *3.11. Anti-Settlement Activity Assessment of GAP Based Coatings*

For a preliminary assessment of the behavior/suitability of GAP as bioactive ingredient after its incorporation in coatings, a small-scale laboratory bioassay was developed, based on the bioassay previously used to assess GAP bioactivity against mussel (*Mytilus galloprovincialis*) plantigrades [24]. For this assay, the wells of 24-well microplates were coated with the di fferent GAP formulations to be tested (one formulation per column), and negative control columns were included for each formulation (AF agent-free coating system). The purpose of these bioassays was to determine the ability of the di fferent GAP-based coatings to prevent the fixation of mussel larvae, testing whether the GAP bioactivity towards this macrofouling species is maintained after DI and CI procedures in di fferent coating matrices.

Competent *Mytilus galloprovincialis* plantigrades were collected on Memory Beach (N 41◦1351.5", W 8◦4315.5") at low tide, and those showing exploratory behavior were selected in the laboratory and transferred to the coated 24-well microplates. All the coated wells were filled with 2.5 mL snSW (previously treated by UV light and carbon filters and mechanically filtered with 0.45 μM filter before use) to reduce any interferents in relation to mussel larvae fixation ability and health conditions. Each coating, including negative controls, was tested in four replicates (4 wells in a column) with five plantigrades per well in the darkness. After 15 and 40 h, the percentage of larval settlement was determined by the presence/absence of e fficiently attached byssal threads, produced by each individual in each condition. The reading times (15 h and 40 h) for larval attachment were selected based on previous trials for bioassay optimization, where 15 h is adequate for maximum thread production and efficient attachment in normal conditions, and 40 h is the maximum time to guarantee the good health status of larvae under bioassay conditions.
