One Step Forward towards the Development of Eco-Friendly Antifouling Coatings: Immobilization of a Sulfated Marine-Inspired Compound

Marine biofouling represents a global economic and ecological challenge and few eco-friendly antifouling agents are available. The aim of this work was to establish the proof of concept that a recently synthesized nature-inspired compound (gallic acid persulfate, GAP) can act as an eco-friendly and effective antifoulant when immobilized in coatings through a non-release strategy, promoting a long-lasting antifouling effect. The synthesis of GAP was optimized to provide quantitative yields. GAP water solubility was assessed, showing values higher than 1000 mg/mL. GAP was found to be stable in sterilized natural seawater with a half-life (DT50) of 7 months. GAP was immobilized into several commercial coatings, exhibiting high compatibility with different polymeric matrices. Leaching assays of polydimethylsiloxane and polyurethane-based marine coatings containing GAP confirmed that the chemical immobilization of GAP was successful, since releases up to fivefold lower than the conventional releasing systems of polyurethane-based marine coatings were observed. Furthermore, coatings containing immobilized GAP exhibited the most auspicious anti-settlement effect against Mytilus galloprovincialis larvae for the maximum exposure period (40 h) in laboratory trials. Overall, GAP promises to be an agent capable of improving the antifouling activity of several commercial marine coatings with desirable environmental properties.


IP-RP-HPLC Method validation
The method developed to analyze a sulfated and polar compound in water was validated according to the International Council for Harmonisation (ICH) Guidance for Industry for parameters such as selectivity, linearity, range, accuracy, and precision [1].
To evaluate selectivity, several concentrations in the range of 5-300 % of the expected working range were prepared in both sample matrices (UPW and snSW), and their chromatographic signals analyzed under the same conditions (retention time and resolution), as well as spiked and non-spiked blank samples for comparative purposes. Standard snSW samples were pre-treated before injection, with a dilution of 60 mM of TBA-Br in a proportion of 1:3. Linearity was determined by calculation of a regression line from the peak area vs. concentration plot for five standard solutions (10, 25, 50, 100, 200, and 500 µM), analyzed in triplicate. This parameter has a coefficient of determination (R2) higher than 0.995. The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated based on the signal-to-noise approach by comparing measured signals from standard samples with known low concentrations of each analyte with those of blank samples and establishing the signal-to-noise ratio between 3:1 for LOD and 10:1 for LOQ (Table S1). Accuracy tests were performed in triplicate using three different known concentrations (30, 60, and 90 µM) and their percentage of recovery in both matrices were calculated. This parameter must be between 80-120 %. Relative standard deviation (RSD) values were calculated as a measure of precision. The intraday variability (repeatability) was determined by analysing each sample within 24 h, while the inter-day variability (reproducibility) was measured on three non-consecutive days. RSD values lower than 15 % are considered acceptable (Table S2).

Identification of gallic acid persulfate (GAP) obtained by an optimized synthesis
The chromatogram of the obtained product presented a retention time similar to the previous synthetised GAP, allowing the identification of the new obtained product as GAP ( Figure S1).

Density Functional Theory (DFT) calculations.
Density Functional Theory (DFT) calculations [1] were performed using the Amsterdam Density Functional (ADF) program [2][3][4]. Geometries were optimized without symmetry constraints, using the Local Density Approximation (LDA) of the correlation energy (Vosko-Wilk-Nusair) [5] and the Generalized Gradient Approximation (Becke's [6] exchange and Perdew's [7,8] correlation functionals) with gradient correction. Relativistic effects were treated with the ZORA approximation [9]. Triple-ζ Slater-type orbitals (STOs) were used to describe all the valence electrons of H, O, C, N, and S. A set of two polarization functions was added to H (single ζ 2s, 2p), O, C, N, and S (single ζ, 3d, 4f). Frequency calculations were performed to obtain the vibrational spectra and to check that intermediates were minima in the potential energy surface. Vibrational spectra were analyzed with Chemcraft [10].
The optimized structures of GAP and triaziridine crosslinker (TZA) are represented in Figure S2.

Nuclear Magnetic Resonance (NMR) Spectra
1 H and 13 C (APT) NMR spectra were recorded on Bruker Avance 400 spectrometer, operating at 293 K and a frequency of 400.13 MHz for 1 H NMR, 100.61 MHz for 13 C NMR. The 1 H NMR spectra of gallic acid persulfate (GAP), triaziridine propionate crosslinker TZA, and GAP-TZA derivative are shown in Figure  S2 (page 6). The 13 C (APT) NMR spectra of GAP and TZA are shown in Figure S3 (page 7) and Figure S4 (page 8), respectively, and the 13 C (APT) NMR spectrum of the GAP-TZA derivative is shown in Figure 5 (page 9).

Recovery of GAP
To analyze GAP recovered by the new extractive procedure using WAX cartridges, 300 µL of UPW containing 100 µM and 500 µM of GAP were dissolved in 10 mL of ASW and passed through the cartridges and subsequently extracted according the procedure described in section 2.10. The recovery rate of the extractive process was determined by comparing chromatographic signals of chromatograms with those resulting from the injection of the standard solutions before extraction. A recovery rate of 104 ± 2 % was obtained ( Figure S8 and Figure S9). Figure S8. Representative chromatograms of 500 μM of GAP dissolved in UPW before and after being passed through the WAX cartridge, with a chromatographic signal at 10 min at 236 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile (50:50 v/v) was used. 500 µM of GAP before extraction 500 µM of GAP after extraction Figure S9. Representative chromatograms of 100 μM of GAP dissolved in UPW before and after being passed through the WAX cartridge, with a chromatographic signal at 10 min at 236 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile (50:50 v/v) was used.