1. Introduction
Biofouling can be defined as the accumulation of micro- and macro-organisms on artificial surfaces immersed in seawater. To control biofouling, antifouling (AF) paints have been developed and commonly used for several decades. They contain polymers as binders, toxic compounds, called biocides, which are leached from the paint matrix, and additives (thixotropic agents, pigments, viscosity modifiers). Biocides are based on copper compounds (copper oxide or copper thiocyanate) associated with booster biocides. These organic biocides are intended to be environmentally less harmful than the organotin biocides used in the 1970s. However, alternative strategies are researched because problems of toxicity for marine species and an accumulation of substances in seawater persist [
1,
2]. Among them, the use of natural antifouling compounds has received a lot of attention. For example, papain, butenolide or cardenolides recently showed an interesting efficiency against biofouling [
3,
4,
5]. However, several restrictions limit the use of these products: large scale production, degradation, compatibility with paint matrix, release characteristics and costs are the main difficulties impeding their development [
6]. Moreover, recent regulation concerning the use of biocides (EU Regulation No 528/2012) known as Biocide Product Regulation (BPR) also restricts their industrial development for reasons concerning costs. Actually, only a few biocides are authorized by European Union and commercialized: 3 copper derivatives (copper, copper thiocyanate and dicopper oxide), 5 booster biocides (DCOIT, Zineb, copper pyrithione, zinc pyrithione and a substituent of copper called Tralopyril). Currently, antifouling paint researchers have to look for a compromise between efficiency of coatings and their impact on the environment.
The aim of this work is to study paints based on a copper derivative such as copper thiocyanate, but devoid of booster biocides. Copper is an effective biocide against algae and mollusks. Moreover, a lower amount of copper thiocyanate is needed than copper oxide for the same level of efficiency [
7]. However, to improve its efficiency and to enlarge the activity spectrum of paints, an additive acting as surface modifier could be added. Its role is not to produce a biocidal effect but instead the promotion of an anti-adhesive effect by modifying wettability of surface and paint surface/organisms interactions. Fouling release (FR) coatings based on poly(dimethylsiloxane) (PDMS) rely on this principle by decreasing surface energy [
8]. However, several publications have mentioned the combination of antifouling and fouling release concepts to develop new hybrid materials effective in marine antifouling protection. Azemar and al. have proposed a hybrid system based on triblock copolymer poly(ε-caprolactone)-block-poly(dimethylsiloxane)-block-poly(ε-caprolactone) to mix the properties of erosion/biocide release used in antifouling systems and hydrophobicity properties through the use of PDMS [
9]. Afterwards, Yang et al. have confirmed the need to combine the concepts of “antifouling” and “fouling release” [
10].
The surface wettability plays a major role in antifouling performance [
10]. It can be modulated by the use of surfactants. For example, Tween 20 has improved the antifouling characteristics of membranes by adsorption at interfaces [
11,
12]. In antifouling applications, we have previously shown that Tween 85 disturbed interactions between colonizing organisms and surfaces by decreasing their hydrophobicity and thus a physical repelling of Tween 85 has been hypothesized [
13]. Another strategy concerns the use of grafted surfaces. Surface-grafted poly(ethylene glycol) (PEG) molecules are known to prevent protein adsorption and coatings based on PDMS-g-PEG have been studied in seawater [
8,
14,
15]. In this study, bacteria and diatoms adhesion were inhibited. Recently, Jimenez-Pardo et al. have proposed hydrophilic self-replenishing coatings based on polycarbonate-poly(ethylene glycol) methyl ether polyurethane exhibiting low proteins adhesion values [
16].
Hence, this work has studied the effect of three additives incorporated in a copper paint: Tween 80 and Span 85, two no ionic, hydrophilic and hydrophobic surfactant respectively and a PEG-silane. The last one is considered as a surface modifier because of its properties such as hydrophilicity, flexibility, high exclusion volume in water and non toxicity [
17]. The paint had an additive free formulation. Parameters as surface hydrophobicity, paint hydration and copper release were evaluated. The anti-microfouling and anti-macrofouling efficiencies of paints were studied as well as their toxicity.
3. Materials and Methods
3.1. Chemical Products
Tween 80 (Polyethylene glycol sorbitan monoleate) and Span 85 (Sorbitan trioleate) were purchased from Sigma-Aldrich (Saint Louis, MI, USA). PEG-silane (Silquest A1230, molecular weight = 546 g·mol
−1) and ingredients of formulation (Solvents, plastifiant, calcium carbonate and fillers) were supplied from Momentive and Nautix Company respectively. The binder was a biodegradable polymer called poly(ε-caprolactone-co-δ-valerolactone) (P(CL-VL) 80-20) synthetized by Mäder (Lille, France) following the protocol described in Loriot et al. [
25]. The molecular weight (Mn) of the polymer was 19,000 g·mol
−1 with a polydispersity of 1.5.
3.2. Paints Formulation and Coupons Preparation
Binder was solubilized in solvent during 24 h (25 °C, 70 rpm) then paints were formulated by dispersing all ingredients (
Table 3) under mechanical vigorous agitation (PBD40, Bosch) at 600 rpm. Once all ingredients added, the agitation was maintained for 15 min at 1000 rpm. Then the paints were filtered through a sifter (100 µm).
A layer of wet film (200 µm thick) was deposited with an automatic film applicator (ASTM D823 Sheen instrument) on a polycarbonate support. Then, the specimens were dried at 20 °C for one week.
A commercial paint called A4T was furnished by Nautix Compagny (Guidel, France).
3.3. Karl Fisher Titration
Paints plates were immersed in Artificial SeaWater (ASW, 33 g·L−1, Sigma Aldrich). Pieces of films (2 cm in diameter) were cut off in order to quantify the water amount present in films. The Karl-Fisher titration was performed with a Coulometer Methrom KF831 equipped with an Oven Methrom 860KF Thermoprep (150 °C) under an air flow of 60 mL·min−1. The reactant used was Hydranal-coulomat AG. The experiment was conducted in three triplicates for each sample.
3.4. Contact Angles Measurement
Measurements were obtained at room temperature with a contact angle Digidrop GBX (Dublin, Irland) equipped with a syringe, a video camera, and an acquisition of angle measurement. Five water droplets of 2 µL were measured at 0, 15 and 30 s after contact between the drop and the paint surface. The indicated values are an average of 5 measurements obtained on different areas of films.
3.5. Copper Thiocyanate Release
The cyclic voltammetric stripping (CVS) studies were carried out in determination mode on a software (Viva 2.0) connected to Metrohm 884 Professional VA. The voltammetry cell consists of a three electrodes assembly and a stirrer with hanging mercury drop electrode as a working electrode (Multi Mode Electrode pro, Metrohm; 6.0728.120 and 6.1246.1) a platinum wire (Metrohm; 6.0343.100) as auxiliary electrode leading the electric current to the working electrode and Ag/AgCl (satured KCl 3.0 M) electrode (Metrohm; 6.1204.50) as a reference electrode with a constant potential.
Analysis were carried out using the standard addition method. Thus 4 mL of sample solution were transferred into the electrolysis cell, containing 10 mL water HPLC grade (VWR) and 1 mL of electrolyte solution (21.6 g KCl, 50 mL NaOH at 30%, 28.4 mL of acetic acid and water up to 1 L with a pH = 4.6 ± 0.2). The solution was purged with pure nitrogen during 5 min. The accumulation potential was applied to a new mercury drop (5 mm) while the solution was stirred at 2000 rpm for 60 s. At the end of the accumulation period, the stirring was stopped and a 60 seconds rest period was allowed for the solution to become quiescent. Then the voltammogram was recorded by scanning the potential toward the positive direction over the range –0.9 to +0.2 V. Copper was detected around −0.1 V. The standard solution of Cu (VWR) at 2 mg·L−1 was prepared from standard solution at 1 g·L−1. The volume of the standard solution was 100 µL. All measurements were made at room temperature.
3.6. Anti-Bacterial Activity
The marine bacteria used (
Bacillus sp. (4J6),
Pseudoalteromonas sp. (5M6) and
Paraccous sp. (4M6)) were grown on a Zobell medium: Artificial Seawater 30 g/L, Tryptone 4 g/L, Yeast Extract 1 g/L. Bacterial cultures were incubated at 10
6 cfu/mL during 48 h under agitation. Planktonic cultures were maintained at 20 °C whilst shaking. These bacteria were used because they are pioneer adherents. Strains were isolated from the surface of a glass cover immersed in natural seawater (Morbihan gulf, France) for 6 h [
26]. 5M6, a Gram negative bacteria, was affiliated to the
Pseudoalteromonas genus. The Gram positive bacteria 4J6 clustered with the genus
Bacillus (100% similarity) and 4M6 was affiliated to
Paracoccus sp.
The zone of inhibition assay on solid media was used for determination of the antimicrobial effects of paints against Bacillus sp. (4J6), Pseudoalteromonas sp. (5M6) and Paraccous sp. (4M6). 10 mL of molten Zobell agar was inoculated by 1 mL of bacterial cultures (colony count of 1 × 107 UFC/mL). Coupons of paints (2 cm diameter) were placed on the bacterial carpets and incubated at 20 °C for 48 h in an appropriate incubation chamber. The plates were examined, and the diameter of the inhibition zone was measured (in centimeters). These experiments were repeated three times for each sample.
3.7. Anti-Microalgal Activity
Three marine strains Cylindrotheca closterium (Diatomophyceae, AC515), Porphyridium purpureum (AC122) and Exanthemacrysis gayraliae (AC15) were used. Microalgae were obtained from the Culture Collection of Algae of the University of Caen (France). Microalgae were grown in an ASW-based culture medium with Guillard’s F/2 Marine Enrichment Basal Salt Mixture (Sigma Aldrich, Saint Louis, MO, USA), in sterile conditions at 20 °C. Guillard’s F/2 was added after sterilization and the culture medium was stored at 4 °C before use.
The zone of inhibition assay on solid media was used. 10 mL of molten medium agar was inoculated by 1 mL of microalgal cultures (1 × 105 cells/mL). Coupons of paints (1.5 cm diameter) were placed on the microalgal carpets and incubated at 20 °C for five days in phytotronic chambers (controlled illumination of 150 µmol. photons.m−2·s−1 white fluorescent lamps with a 16h:8h light:dark cycle). The plates were examined, and the diameter of the inhibition zone was measured (in centimeters). These experiments were repeated three times for each sample.
3.8. Anti-Microfouling Properties
Paints (2 × 5 cm) were exposed in natural seawater, at a depth of 50 cm (Atlantic Ocean, W 47°43’8.39” N 3°22’7.38”, Larmor Plage, France). The study began in April 2017. The seawater characteristics were in
Table 4. Coupons were sampled over 9 weeks and observed by CLSM microscopy, as described above [
27]. For each sample time, five observations were realized. Biovolumes and average thicknesses values were determined with COMSTAT program to compare paints between them [
28]. The significance test was conducted using one-way analysis of variance (ANOVA).
3.9. Anti-Macrofouling Properties
Paints were applied onto panels (8 × 12 cm). Paints were observed monthly during immersion in natural seawater. The Antifouling performance was assessed according to a modified protocol of the French Standard (NFT34-552 September 1996). Paints were classified using an efficiency parameter N which is expressed as
N = Σ
I.G where I stand for the intensity of fouling and G the severity of fouling as shown in
Table 5.
N was determined at each observation time by visual inspection (determination of the surface coverage by each type of fouling) following by a determination of efficiency parameter N referring to
Table 5. The lower the
N value was, the more efficient was the paint.