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Applied Surface Science 341 (2015) 75–85

Contents lists available at ScienceDirect

Applied Surface Science

jou rn al h om ep age: www.elsev ier .com/ locate /apsusc

ransport and antifouling properties of papain-based antifoulingoatings

afael S. Peresa,b,∗, Elaine Armelinb,c, Juan A. Moreno-Martínezd, Carlos Alemánb,c,arlos A. Ferreiraa

LAPOL/PPGE3M, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc alves 9500, 91501-970 Porto Alegre, BrazilDepartament d’Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Av. Diagonal 647, 08028 Barcelona, SpainCentre for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C’, C/Pasqual i Vila s/n, Barcelona E-08028, SpainLaboratori d’assaigs no destructius, Departament de Ciència i Enginyeria Nàutiques, FNB, Universitat Politècnica de Catalunya, Plac a Palau 18,arcelona 08003, Spain

r t i c l e i n f o

rticle history:eceived 26 January 2015eceived in revised form 3 March 2015ccepted 3 March 2015vailable online 7 March 2015

eywords:ntifoulingoatings

a b s t r a c t

The aim of this work is to study the antifouling performance and water uptake behaviour of coatings for-mulated with papain (an environmentally friendly pigment). Antifouling coatings have been formulatedusing rosin (natural resin) as matrix and papain adsorbed in activated carbon as pigment. Electrochemi-cal impedance spectroscopy (EIS) measurements were used to evaluate the behaviour of the formulatedcoatings in the marine environment and to calculate the apparent water coefficient of diffusion (D). FTIRand XPS analyses confirm the presence of papain adsorbed inside the activated carbon pores and therelease of papain in water. Immersion tests in the Mediterranean Sea were carried out for 7 monthsto verify the degree of biofouling of the tested coatings. These field assays clearly indicate the excel-

apainroteasenvironmentally friendly

lent behaviour of papain-based antifouling coatings; the results being similar to those achieved usinga commercial coating. Additionally, the EIS technique is shown to be a great tool to predict the coatingdiffusivity of antifouling coatings before immersion tests. Furthermore, the use of biodegradable papainas a nature-friendly antifouling agent can eliminate the negative environmental impact caused by metalsand chemical biocides typically used in current commercial formulations.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Biofouling has a significant impact on the naval industry dueo biofilms attached to immersed structures [1,2]. These films areormed by micro- and macro-organisms, the dominant speciesepending on the geographical localization [1,3,4]. The presence ofouling increases the roughness and weight of the hull, contributingo raising the costs of maintenance and fuel consumption of ships5]. Stationary structures and hydroelectric plants are also nega-ively impacted by biofouling due to corrosion and obstruction ofropeller-type turbines and pipes [6].

Although many solutions have been proposed to avoid foul-ng attachment, the application of antifouling paints is the

ost used method due to both economic aspects and its high

∗ Corresponding author. Tel.: +55 51 3308 9412.E-mail addresses: rafael.s.peres@gmail.com, peres.rafael@outlook.com

R.S. Peres).

ttp://dx.doi.org/10.1016/j.apsusc.2015.03.004169-4332/© 2015 Elsevier B.V. All rights reserved.

efficiency [2]. In the past, self-polishing paints with tributyltin (TBT)biocide were used as efficient antifouling paints [2]. However, seri-ous environmental problems were associated with the TBT, leadingthe International Marine Organization to ban such biocides [7–9].Currently, the majority of antifouling paints have cuprous oxideas an antifouling agent combined with a soluble or self-polishingmatrix [4]. Several other biocides such as zinc pyrithione, Diuron®and Irgarol® 1051 are also used together with copper pigment,even though its behaviour can be harmful to the marine environ-ment [10]. Diuron® and Irgarol® 1051 are toxic to phytoplanktonorganisms, impacting the food chain [11]. The use of Diuron® wasforbidden in the United Kingdom and Irgarol® 1051 was limitedto watercrafts of length less than 25 m [12]. Currently, less toxicantifouling agents are being used and studied by several investiga-tors [13–16].

The study of water uptake in antifouling paints is of funda-mental importance to predicting the behaviour of these paintsin the marine environment [17]. The presence of water insidethe polymeric film can release the active antifouling agent

7 rface

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6 R.S. Peres et al. / Applied Su

ppropriately, improving the coating efficiency [17]. Some studiesave reported the use of electrochemical methods to determineater uptake in polymeric films [17–19]. Within this scenario,

lectrochemical impedance spectroscopy (EIS) has become annteresting technique due to the possibility of estimating waterptake in polymeric films by the Brasher–Kingsbury method19,20].

The mechanisms of mussel and barnacle adhesion are veryell described in the [21] review. Proteins are the compounds

esponsible for the adhesion of mussels and barnacles to the sub-trate surface [21]. In the blue mussel (Mytilus edulis) adhesive,ve types of proteins, abbreviated as Mefps, contain differentmount of 3,4-dihydroxyphenylalanine (DOPA) in their primaryequence [21–24]. Mefp-3 and Mefp-5 are responsible for the con-act between the mussel adhesive and substrate, showing largemounts of DOPA [21,23–25]. Thus, the concentration of DOPAs crucial to the quality of the mussel adhesion [25]. Greaterhan 90% of the adhesive of barnacles is composed of pro-eins, lipids, ash and carbohydrate being the minor constituents21,26]. According to Cheung et al. [27] the adhesive of bar-acles has a fast curing time, becoming a rigid and extremelyesistant material called cement [21]. Some algal spores, diatomsnd bacteria also used proteins to attach to many substrates28–31].

The use of enzymes can cleave proteins, thus an enzymes antifouling agent can cleavage the proteins of fouling adhe-ives, avoiding attachments by marine organisms on substrates28,32]. According to Dobretsov et al. [28], fouling settlement byugula neritina can be inhibited by using the right concentra-ions of certain enzymes, such as amylase, galactosidase, papainnd trypsin. An interesting feature about enzymes is their readyecomposition in the marine environment to CO2 and H2O byhe action of microorganisms [28]. Some studies have reportedhe use of enzymes as antifouling agent in coatings [33,34], evenhough the great variability in matrix paints, water conditionsnd enzyme stability make the practical application of these for-ulations to real situations difficult [28]. Kristensen et al. [33]

sed glucoamylase and hexose to produce an antifouling coatingith good efficiency for up 97 days of immersion in the North

ea. Kim et al. used pronase and �-chymotrypsin together witholy(dimethylsiloxane), reporting a reduction in protein adsorp-ion [34].

Papain is a proteolytic enzyme extracted from the latex ofhe papaya fruit (Carica papaya L.) [35]. Brazil is among theargest producers of papaya in the world, with a production of,650,000 ton/year [36]. The papain is used for different applica-ions including cosmetics [37], meat tenderization [38] and woundare [39]. On the other hand, different investigators have pro-osed immobilization processes to improve the stability and usef enzymes in different media [40–43]. The immobilization pro-ess ensures the reusability of the enzymes, which is important forndustrial applications [43]. Silva et al. [40] immobilized papainnd pancreatin in activated carbon and alumina to prepare lowost dietary supplements. Afaq and Iqbal [41] immobilized papainn a chelating sepharose, obtaining good regeneration of thisnzyme.

The aim of this work is to prepare an antifouling coating usingapain adsorbed in activated carbon as an antifouling pigmentnd evaluated the EIS technique to predict the water diffusiv-ty in the antifouling coating. It should be noted that the stepf adsorption has been proposed due to the high solubility ofapain in water. Furthermore, adsorption is also expected to

mprove the papain’s stability in different environments. Theesults indicate that the coating fabricated using papain adsorbed inctivated carbon allied to a rosin matrix shows excellent antifoulingroperties.

Science 341 (2015) 75–85

2. Experimental procedure

2.1. Materials

Activated carbon powder (Delaware, Brazil), papain enzymepowder 6000 USP (Delaware, Brazil), Na2HPO4 (Synth, Brazil),and NaH2PO4 (Synth, Brazil) were used in the preparation ofantifouling pigment, and NaCl (Synth, Brazil) and NaOH (Synth,Brazil) in electrolyte preparation. Coatings were prepared usingmethyl ethyl ketone (MBN chemicals, Brazil) as the solvent, oleicacid (Sigma–Aldrich, USA) as plasticizer and WW rosin (RB Sul,Brazil) and commercial acrylic resin (Águia Química, Brazil) asthe matrix. The commercial antifouling coating Micron® Premium(Akzo Nobel, USA) was used as a reference to compare the antifoul-ing activity and the two-component epoxy primer Intergard 269(Akzo Nobel, USA) was used as an anticorrosive primer and blank.

2.2. Antifouling pigment preparation

The papain antifouling pigment was prepared as described Silvaet al. [40,44] even though the procedure was modified accord-ing to our conditions. First, 1000 mL of a phosphate buffer wasprepared with 11.525 g of Na2HPO4 and 2.257 g of NaH2PO4 dis-solved in deionized water. Then, 8 g of papain was dissolved in thebuffer solution prepared previously. After complete dissolution ofthe papain, 40 g of activated carbon was added to the buffer solu-tion and the mixture was stirred for 1 h. Afterward, the activatedcarbon was filtered in a Büchner funnel and dried for 48 h at roomtemperature.

In order to characterize the pigment, Fourier transform infraredspectra (FTIR) were recorded using a FTIR 4100 Jasco spectropho-tometer coupled with an attenuated total reflection accessory(Specac model MKII Golden Gate Heated Single Reflection DiamondATR). The spectra were obtained after 32 scans at a resolution of4 cm−1, in a spectral range of 600–4000 cm−1, in transmittancemode.

2.3. Coating preparation

The dispersion of all coating components was carried out bya Dispermat N1 (VMA-Getzmann GMBH of Reichshof, Germany)disperser coupled with a Cowles disk and jacketed reactor.

PAP1: Prior to the dispersion process, 48 g of rosin had previouslybeen dissolved in 50 mL of methyl ethyl ketone (MEK). As a firststep, the dissolved rosin was added to the jacketed reactor. In orderto achieve homogenization, the rosin was dispersed for 20 minbefore adding papain/activated carbon. Then, 24 g of antifoulingpigment was added together with 50 mL of MEK and the coatingwas dispersed at 4000 rpm for 50 min. The entire dispersed prod-uct was then put on a Dispermat SL-12 ball mill (VMA-GetzmannGMBH of Reichshof, Germany) to mill the pigments. More MEK wasalso added to adjust the coating’s viscosity according to the sys-tem’s demand. The milling process was halted when the pigmentsreached a size between 15 and 25 �m (6 and 7 Hegman). The pig-ment volume concentration (PVC) of the formulated antifoulingcoating was 45 vol.%. According to Kill et al. [45] and Gitlitz andLeiner [46], typical PVC values in coatings formulated with solublematrices should be between 30% and 45% to improve the antifoulingefficiency.

PAP2: The procedure and materials were the same as describedfor PAP1, except that the binder used was a mixture of 36 g of rosin,

12 g of commercial acrylic resin (co-binder) and 3 g of oleic acidas plasticizer. This procedure was carried out to verify the influ-ence of the co-binder in the outcome and to improve the aspectand mechanical properties of the formulated coating. For the sake

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R.S. Peres et al. / Applied Su

f simplicity, FTIR and XPS analysis were carried out only on theoating without co-binder and plasticizer (PAP1).

.4. Panel and sample preparation

Before application of antifouling coatings, the surfaces of steelanels (AISI 1010) with sizes of 25 cm × 20 cm × 1 mm were pre-ared using the following four-step procedure: (i) degreasing withcetone and methyl ethyl ketone; (ii) polishing with abrasive sand-aper (grain size #150); (iii) rinsing with deionized water; andiv) degreasing again. After these procedures, the two sides of eachample (panel) were painted with anticorrosive primer. The com-ercial (COM) and papain-based antifouling coating (PAP) were

pplied only on one side of the sample (panel). All samples wereainted with a brush and dried at room temperature for 48 h before

mmersion. The thicknesses of all coating films were measured with Byko-7500 test unit (BYK Gardner, Germany). The thickness waseasured in different areas of each sample (12 different places)

nd the mean ± standard deviation was calculated. The averagehickness obtained for blank, COM, PAP1 and PAP2 coatings was8 ± 4 �m, 220 ± 23 �m, 265 ± 27 and 278 ± 26, respectively.

For electrochemical tests, the primer layer was not used, theamples being painted only with the antifouling coatings. Theurface preparation of these samples was the same describedbove, and the average thickness used in the EIS experiments was07 ± 3 �m (COM), 104 ± 4 �m (PAP1) and 101 ± 6 �m (PAP2).

.5. Assays in the natural marine environment

All samples were immersed in the Mediterranean Sea atadalona Nautic Port (41◦26′08.4′′N, 2◦14’33.0′′E) in Spain betweenpril and November of 2013. The immersion tests were carried outuring the warm months in Spain, which coincides with high activ-

ties of Balanus amphitrite, bacterial and diatom biofilms [47,48].The tested samples were drilled and fixed with nylon straps on

support built with poly(vinyl chloride) tubes. Before fixation, theubes were covered with polyurethane foam to protect the sam-les. The support was tied with a nautical rope in distinct pointsnd immersed approximately 50 cm deep, according to previousnstructions [49]. Samples were inspected every month to verifyhe degree of fouling attachment. Distances less than 1.3 cm fromhe edges were not considered [49].

.6. Measurements

Electrochemical impedance spectroscopy (EIS) was carried outsing an AUTOLAB PGSTAT 302N potentiostat coupled to a fre-uency response analyser. All measurements were performed inotentiostatic mode at the open circuit potential with an ampli-ude signal of 10 mV, with frequencies ranging from 100 kHz to0 mHz. The electrolyte used in the EIS measurements was a solu-ion of 3.5% NaCl (w/v) with the pH adjusted to 8.2 with a solutionf 0.1 M NaOH. The water uptake, coating behaviour and appar-nt water coefficient of diffusion (D) were analysed by EIS plotsnd parameters. The area of the working electrodes (samples) waselimited by an electrochemical cell (4 cm2). A saturated calomellectrode (SCE) was used as reference and a platinum wire (spiralormat) used as an auxiliary electrode.

To characterize the coatings before and after the immersionests, scanning electron microscopy (SEM) analyses were carriedut using a focused ion beam Zeiss Neon 40 microscope operating

t 5 kV. Coatings were fixed on a sample holder with a double-sideddhesive carbon disk and sputter-coated with a thin layer of carbonraphite. Optical microscopy images were obtained by a Dino-liteodel AD7013MT USB digital microscope.

Science 341 (2015) 75–85 77

The coatings before and after immersion tests were also char-acterized by X-ray photoelectron spectroscopy (XPS) performed ina SPECS system equipped with a high-intensity twin-anode X-raysource XR50 of Mg/Al (1253 and 1487 eV, respectively) operatingat 150 W, placed perpendicular to the analyser axis, and using aPhoibos 150 MCD-9 XP detector. The X-ray spot size was 650 �m.The pass energy was set to 25 and 0.1 eV for the survey and thenarrow scans, respectively. For the flood gun, the energy and theemission current were 0 eV and 0.1 mA, respectively. Spectra wererecorded with a pass energy of 25 eV in 0.1 eV steps at a pressurebelow 6 × 10−9 mbar. The C1s peak was used as an internal refer-ence with a binding energy of 284.8 eV. High-resolution XPS spectrawere acquired by Gaussian/Lorentzian curve fitting after S-shapebackground subtraction.

FTIR analyses were also carried out in PAP coating before andafter immersion tests with an FTIR 4100 Jasco spectrophotome-ter coupled with an attenuated total reflection accessory (Specacmodel MKII Golden Gate Heated Single Reflection Diamond ATR).

3. Results and discussion

3.1. Mechanism of action of the antifouling coating

The mechanism of action proposed for the PAP antifouling coat-ing is described in Fig. 1. In this work, we assumed that papaincan react with the proteins in fouling adhesives and thus prevent,its attachment to substrates [28,32]. Fig. 1 shows a hypotheticalscheme of the temporal evolution of PAP in two stages. The firststage (Fig. 1a) represents the initial immersion time in the marineenvironment when the fouling organisms approach the coating,encountering a surface rich in papain. The second stage, which isrepresented in Fig. 1b, corresponds to the launching of the first PAPlayer into the marine environment and the appearance of a new PAPlayer. The use of rosin as matrix in the formulation of PAP makesthe coating soluble in the marine environment [50] due to the for-mation of soluble resinates with certain metallic ions in water (Kand Na) [51].

Fig. 1c represents the activated carbon with papain adsorbedinside its pores while Fig. 1d illustrates the mechanism of foulingdetachment through the reaction of papain and fouling adhesive.Thus, Fig. 1a and d indicate that water penetrates inside the acti-vated carbon pores, releasing papain protease that reacts with thefouling proteins (adhesive).

3.2. Transport properties

3.2.1. Behaviour of antifouling coatingsNyquist and bode plots for the PAP1, PAP2 and COM coatings

after 1 h and 1 day of immersion are given in Fig. 2. The inset figuresrepresent the equivalent circuit used to simulate the data. Accord-ing to the Nyquist plots and Table 1, the overall resistance (Zoverall),given by the sum of R1 and R2, decreased after 1 day of immer-sion in all samples, which means penetration of water inside thecoating films that is necessary to release the papain. If the Nyquistdiagram remains stable after some hours or days of immersion, thismeans that coating is resistant to water penetration (not suitablefor a water-soluble formulation).

Fig. 2a (Nyquist plots) shows the presence of a Warburg (W)impedance for the PAP1 coating, which is associated with masstransfer phenomena [52] (water penetration into the polymericfilm). Other parameters are: (Rs) the ohmic resistance between

the sample (working electrode) and SCE (reference electrode); (R)the charge transfer resistance; (Z′′) is the imaginary part of theimpedance, (Z′) is the real part of the impedance; and (Q) the con-stant phase element [52]. The Zoverall for the PAP1 coating decreased

78 R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85

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3c

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ig. 1. Hypothetical schematic mechanism of the action of papain-based antifoulioluble coating action; (c) activated carbon with papain adsorbed inside of its pores;

pproximately 4 fold and the maximum phase angle decreasedrom around −35◦ to −10◦ (Fig. 2b) after one day of immersion in.5% NaCl solution that means water penetration into the coatingdiffusional process).

Fig. 2c shows Nyquist plots for the PAP2 coating after oneour and one day of immersion. The Zoverall for the PAP2 coatingecreased approximately 2.6 fold after one day of immersion andhe maximum phase angle decreased from −54◦ to −52◦ (Fig. 2d). Asxpected, the decreases in these electrochemical parameters wereess pronounced in PAP2 than PAP1 due to the presence of an insolu-le co-binder (acrylic resin), which means a decrease in the coatingolubility.

Fig. 2e shows Nyquist plots for the COM coating after 1 h andne day of immersion. The Zoverall for the COM coating decreasedpproximately 2 fold after one day of immersion and the maximumhase angle shifted from 1 Hz to 150 mHz (Fig. 2f).

.2.2. Apparent water coefficient of diffusion in the antifoulingoatings

The calculus of the apparent water coefficient of diffusion is nec-ssary to verify if the values are consistent with the literature for

able 1lectrochemical and transport parameters obtained by EIS technique.

Samples EIS Фc vs. time

Immersion time (h) Zoverall (k� cm2) Fitting

PAP1 1 86.06 Фc = −0.45exp(−t/42.93) +24 22.97

PAP2 1 64.85 Фc = −0.45exp(−t/48.12) +24 24.56

COM 1 127.10 Фc = −0.17exp(−t/28.73) +24 63.02

ting in the marine environment. (a) Initial immersion time; (b) representation ofechanism of fouling detaching through the reaction of papain and fouling adhesive.

antifouling coatings. Values higher than those reported may indi-cate the formulation of a low durability coating (high solubility). Onother hand, values lower than those reported may indicate the for-mulation of a low solubility coating, which may impair the releaseof antifouling agent.

The EIS technique was used to calculate the apparent water coef-ficient of diffusion (D) in all antifouling coatings. First of all, thevolume fraction of water uptake in the antifouling coating (Фc) wascalculated according to Eq. (1) [20,53].

˚c =log

(−Z ′′

2�f ((Z ′−Rs)2+)Z ′′2 x 1Co

)

log(80)(1)

where Z′′ is the imaginary part of the impedance at 19.5 kHz, Z′

is the real part of the impedance at the same frequency, Rs is theresistance related to the electrolyte (3.5% NaCl (w/v)), and Co is thecapacitance at the initial time of immersion [17]. The frequency at19.5 kHz was used because the water uptake is detected at high

frequencies [19].

The Фc values were obtained for several times of immersionand a curve of Фc versus time was plotted (Fig. 3a). As observed byBressey et al. [17], in the initial stage of immersion the curves follow

Фc vs.√

t × l−1 Transport parameter

R2 R2 D (m2 s−1)

0.440.97 0.96 0.87 × 10−13

0.450.99 0.97 0.70 × 10−13

0.160.99 0.97 1.35 × 10−13

R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85 79

Fig. 2. EIS spectra after different times of immersion in 3.5% NaCl (w/v) solution: (a) Nyquist and (b) Bode plots of PAP1 after 1 h (black circles) and after 1 day (grey circles);( ircles)c lectric

Fats

c) Nyquist and (d) Bode plots of PAP2 after 1 h (black circles) and after 1 day (grey circles). Lines are the fitting data and the inset of Nyquist plots are the equivalent e

ick’s law due to the linear relation between Фc and t0.5l−1 (Fig. 3bnd Table 1 where the R2 are between 0.96 and 0.97) that allowshe determination of water sorption in the coating. After reachingaturation, Фc becomes constant (Fig. 3a) due to the antifouling

; (e) Nyquist and (f) Bode plots of COM after 1 h (black circles) and after 1 day (greyal circuit.

coatings being saturated with water. The fitting data for the curvesshown in Fig. 3a and b is given in Table 1 and shows an excellentcorrelation between experimental and fitting data (R2 between 0.97and 0.99).

80 R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85

Fig. 3. (a) Evolution with time of water uptake in the PAP1 (grey circle), PAP2 (black star) and COM (black circle) coatings. (b) Water uptake versus t0.5 l−1 in the PAP1 (greycircle), PAP2 (black star) and COM (black circle) coatings.

F on process of papain. (b) FTIR spectra of (3) rosin, (4) PAP1 coating before immersion and(

(

D

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piafis

ig. 4. (a) FTIR spectra of (1) papain powder, (2) activated carbon after the adsorpti5) PAP1 coating after 7 months of immersion in the Mediterranean Sea.

The apparent water coefficient of diffusion (D) is given in m2 s−1

Table 1) by Eq. (2) [17,54]:

= 0.196 · L2

thalf(2)

here L is the antifouling coating thickness (in meters) and thalf ishe half time (in seconds) corresponding to water saturation (Фsat)f the antifouling coatings (Fig. 3a) [17].

As can be seen in Table 1, D values were within the range (10−13)eported in the literature for antifouling coatings [17]. As expected,he D value decreased (reduction in water uptake) with the pres-nce of an insoluble co-binder (acrylic resin) in the PAP2 coating,ut was still within the range found in related studies [17]. Accord-

ng to the EIS study, the use of rosin and a blend of rosin and acrylicesins provided good results in the formulation of new antifoulingoatings.

As mentioned before, the EIS can be a powerful technique toredict antifouling coating behaviour before in situ immersion test-

ng, which requires time-consuming experiments (several weeks)nd continuous control of the samples. Thus, adjustments in thenal coating formulation can first be carried out in laboratory scale,aving both time and expense.

Fig. 5. Comparison of the XPS survey spectra for the PAP1 antifouling coating beforeand after 7 months of immersion in Mediterranean Sea.

R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85 81

of im

3

3

icsmb

Fig. 6. High resolution XPS spectra of PAP1 coating before and after 7 months

.3. Pigment and surface characterization

.3.1. Fourier transform infrared spectroscopy (FTIR)The adsorption of papain inside the activated carbon was ver-

fied by FTIR. Fig. 4a shows the spectra of papain and activated

arbon after the adsorption process. The papain spectrum (Fig. 4a1)hows a broad absorption band centred at 3300 cm−1, whichatches the N–H stretching of the secondary amide bond [55]. The

ands at 1637 and 1551 cm−1 are associated with –CONH amides

mersion in Mediterranean Sea: (a, b) C1s; (c, d) O1s; (e, f) N1s and (g, h) S2p.

I [56] and II [57], respectively. The band at 2924 cm−1 is typicallyassociated with –CH2– asymmetric stretching [58], while peaks at1150, 1076 and 852 cm−1 can be attributed to sulphide and disul-phide –CS stretching [55].

The spectrum of activated carbon after adsorbing papain

(Fig. 4a2) is very similar to that of papain (Fig. 4a1), as evidencedby the presence of the amide I, amide II, sulphide and disulphideabsorption bands. This similarity shows the presence of papain intothe activated carbon. Together with the results of Silva et al. [40,44],

82 R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85

F f imma k (a, b2

wb

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ig. 7. Photographs of samples before immersion (left column), after two months ond after seven months of inmersion (right column) in Mediterranean Sea for: blan5 cm × 20 cm × 1 mm.

hich describes the adsorption of papain into the activated carbony similar method, the adsorption of papain was inferred.

The spectra of the rosin and PAP1 before and after 7 months ofmmersion testing in the Mediterranean Sea are shown in Fig. 4b.he rosin spectrum (Fig. 4b3) displays the characteristic peak at400 cm−1 associated with the hydroxyl absorption band [59]. Theand at 2927 cm−1 corresponds to asymmetric –CH2– stretchinghile the shoulder at 2865 cm−1 can be attributed to symmet-

ic –CH3 stretching of the methyl group [59]. The sharp peak at692 cm−1 corresponds to the carbonyl group of diterpernic resins60]. The absorptions at 1384 and 1447 cm−1 are associated withCH3 bending vibrations [60]. The peak at 1180 cm−1 correspondso saturated C–C or –CH in aromatic rings, whereas those at 1240nd 965 cm−1 are associated with carboxylic groups. Finally, thebsorptions at 890, 826 and 700 cm−1 are related to the vibrations

f aromatic groups [61].

The spectrum of PAP1 before immersion testing (Fig. 4b4) showsot only the rosin peaks but also very weak absorption bands asso-iated with amide II (1552 cm−1) and –CS stretching from sulphides

ersion (middle-left column), after four months of inmersion (middle-right column), c, d), PAP1 (e, f, g, h) and PAP2 (i, j, k, l) samples. The dimension of each sample is

and disulphides (1150 cm−1) [55]. These bands are almost imper-ceptible due to the entrapment of pigment (i.e. papain adsorbedin activated carbon) inside the rosin matrix. When the immersiontime was increased to 7 months (Fig. 4b5), part of the rosin wassolubilized in water and the bands related to the amide and sul-phur groups became more defined. According to El-Sayed et al.[62] the peak at ∼1023 cm−1 (Fig. 4b4–5) is related to the oxidationof disulphide bonds, which gives rise to the formation of –SSO3

−

groups (Buntë salt). This oxidation process could be originate inthe sample preparation process because of the prolonged exposureof non-adsorbed papain to the air (i.e. papain is unstable at hightemperatures).

3.3.2. X-ray photoelectron spectroscopy (XPS)XPS analyses were carried out to monitor chemical changes on

the surfaces of the antifouling coating layers after contact with sea-water and to corroborate the release of papain. As can be seen inFig. 5, the rosin-based antifouling film did not present a signifi-cant contribution by nitrogen atoms and zero release of sulphur

R.S. Peres et al. / Applied Surface Science 341 (2015) 75–85 83

F 7 mo7

attwattlswo

aNna[oTawot

ig. 8. Optical Micrographs of blank (a, c and e), COM (b) and PAP1 (d) samples after months of immersion in the Mediterranean Sea is given in figure (f).

t the initial time (before immersion), which is consistent withhe assumption that the active soluble pigment was located insidehe coating. This result was also supported by FTIR spectroscopy,hich enabled the detection of very weak signals for sulphur-

nd nitrogen-containing groups (Fig. 4b4). After 7 months of field-esting, both nitrogen and sulphur could be clearly detected inhe leached layer. These results indicate that papain enzyme waseaching gradually with time. The atomic percentages found foramples before immersion were C (80.2%), O (19.3%) and N (0.45%);hereas samples extracted after 7 months showed a composition

f C (75.5%), O (21.7%), N (2.4%) and S (0.4%).To investigate the chemical bonds present on the surface of the

ntifouling coatings in detail, high-resolution spectra of C1s, O1s,1s and S2p were registered (Fig. 6). In both cases, the C1s sig-al was deconvoluted in three peaks corresponding to C–C/C–Ht 284.6 eV, C O at 286.3 eV and C–N at 288.3 eV (Fig. 6a and b)63,64]. The C–N bond was attributed to backbone peptide bondsf the papain, which also appeared in the FTIR spectra (Fig. 4b4–5).he detection of C–N before immersion suggests the presence of

very small amount of papain at the surface of the PAP1 coating,hich could originate either from exposed activated carbon pores

r solubilized papain in the rosin matrix. It should be remarked thathis small content of papain at the surface is not enough to allow the

nths of immersion in the Mediterranean Sea. SEM micrograph of PAP1 sample after

detection of disulphide bonds by XPS since, although these proteinsare relatively large with 212 amino acid residues [65], they containthree disulphide bonds.

Before immersion, O1s showed one peak at 532.5 eV, whichwas attributed to the C–O bond from the rosin resin (Fig. 6c).After 7 months of immersion, an increase in the amount of oxy-gen adsorbed could be observed and thus other bond contributionscould be deconvoluted (Fig. 6d). The three obtained peaks were at528.6 eV (S–SO3

–), 532.2 eV (N–C–O) and 533.4 eV (C O) [66,67].Therefore, changes in the spectra are evidenced by the oxidationof disulphide bonds between cysteine residues during the courseof the leaching reaction or sample preparation process. These XPSresults are in excellent agreement with the FTIR observations, con-firm the formation of –S–SO3

– on the surface of the PAP2 coating.High-resolution spectra of S2p (Fig. 6g and h) corroborate

the observations obtained for O1s. Solubility and leaching ofpapain molecules from the paint cause opening of the disul-phide bonds [62]. Accordingly, oxidation of disulphide bonds isreflected by the high contribution by S–O linkages at 169.5 and

170.7 eV from sulphonated groups [62]. Moreover, no contributionfrom the S2p signal at ca. 164 eV, corresponding to the nor-mal disulphide bond in cysteine [62], was observed in the XPSspectra.

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Nitrogen was essentially attributed to secondary aminesC–N–H linkages at ca. 400 eV) in the paint films after dissolutionf the papain (Fig. 6f). However, N1s was also detected at a lowoncentration in the control sample (before immersion) becausef the possible presence of a very small concentration of papainolubilized in the rosin matrix (Fig. 6e). The XPS results supporthat the enzyme is still present at the surface of the PAP1 coatingven after 7 months of immersion. The presence of papain afterong exposure times suggests that this enzyme is a potential envi-onmentally friendly antifouling agent when used in rosin-basedoatings.

.4. Immersion tests

The place where the samples were immersed in the Mediter-anean Sea has high salinity (between 37.5 and 39.5 psu) andemperatures in the west basin between 12.8 and 13.5 ◦C [68]. Thegures on the COM sample are supplied in our previous study (bothtudies were carried out at same time) [69].

Fig. 7 shows photographs of the samples before and after immer-ion testing. According to Fig. 7b, the blank panel shows severalhite points (fouling) over the entire surface after two months

f immersion in the sea. In contrast, no sign of heavy fouling isetected in panels coated with PAP1 (Fig. 7f), PAP2 (Fig. 7j) or COM69] after the same time of immersion. After 4 months of immer-ion, the fouling over the blank sample (Fig. 7c) is more pronouncedhile the PAP1 (Fig. 7g), PAP2 (Fig. 7k) and COM [69] samples

emain without any sign of heavy fouling. The surface of the blankample (Fig. 7d) is severely degraded after 7 months of immersiont Mediterranean Sea. This fact reflects the intense fouling activityt Badalona port (Mediterranean Sea).

The COM [69] still did not present any heavy fouling after 7onths of immersion. Indeed, only coating failures were found.

imilarly, the samples coated with PAP1 (Fig. 7h) and PAP2 (Fig. 7l)id not exhibit heavy fouling over the surface after 7 months of

mmersion, even though light fouling (small limbs or branchesttributed to certain algae species) was detected in some points onhe surface. These observations corroborate the antifouling prop-rty of the PAP1 and PAP2 coatings. In all immersed samples, theouling on the back of each panel (the part that was not paintedith an antifouling coating) was very high.

A comparison between PAP1 and PAP2 antifouling efficiencieshows that both coatings have similar behaviour. In terms of aspectfilm integrity), PAP2 was less deteriorated after 7 months, probablyue to the presence of a co-binder (acrylic resin) and plasticizer. Asentioned in our previous study [50], the rosin has a dissolution

ate of 50 �g cm−2 day−1, while that of the mixture of rosin/acrylics ∼44 �g cm−2 day−1.

.4.1. SEM and optical imagesThe surfaces of the COM, PAP1 and blank samples after 7 months

f immersion in the Mediterranean Sea were analysed by SEM andptical microscopy. Fig. 8a and c, which show the surface of thelank sample after immersion, clearly reflect the presence of heavyouling on the surface of the sample. The presence of polychaeteubeworms was detectable as a major type of fouling (Fig. 8a and). Barnacles and mussels were also found in some places on theanel but in small amounts. The thickness of the fouling layer wasery irregular, reaching values close to 4 mm on the blank panel, asan be seen in Fig. 8e.

Fig. 8b shows the surface of the COM2 coating without foul-ng, while Fig. 8d shows an area of the PAP1 coating with an algae

pecies present. Fig. 8f shows a SEM micrograph of the PAP1 coatingfter 7 months of immersion in the Mediterranean Sea. The expo-ure of activated carbon on the surface of the PAP1 coating suggestshat the rosin matrix was solubilized in aqueous environment. This

Science 341 (2015) 75–85

mechanism facilitated the release of papain into the water enablingits antifouling activity.

4. Conclusion

The EIS technique is shown to be a great tool for predicting thebehaviour and water diffusivity in the antifouling coatings evenbefore immersion testing, saving time and reducing costs in pre-liminary tests. The PAP coatings had apparent water coefficientsof diffusion in the same range as those reported in Ref. [17]. Theelectrochemical behaviour of the PAP coatings was also consistentwith that of porous films (water migrating inside the coating films),especially after long times of exposure in a marine environment.

The performance of the papain-based antifouling coatings wastested in a natural marine environment. The pigment used as anantifouling agent was papain adsorbed inside activated carbonpores. XPS analyses evidenced the release of papain in the marineenvironment and confirmed the presence of papain inside the acti-vated carbon pores, the latter also being corroborated by FTIRspectroscopy. Immersion testing in the Mediterranean Sea during7 months showed the excellent antifouling action of the papain-based antifouling coatings in this environment. Optical microscopyimages reflected the presence of polychaete tubeworms as a majorfouling organism in the Mediterranean Sea, even though barnaclesand mussels were also identified.

In summary, immersion tests and microscopy images indicatethat the antifouling efficiencies of the papain-based coatings weresimilar to that of a commercial one. However, the great novelty ofPAP is the replacement in the coating formulation of metals andother toxic biocides, which are present in COM, by a natural andbiodegradable (papain) antifouling agent [28]. Therefore, the neg-ative environmental impact caused by metals and many chemicalbiocides can be eliminated by using papain as a nature-friendlyantifouling agent that is easily biodegraded in the marine environ-ment.

Acknowledgments

The authors thank the Brazilian government agencies CNPq andCAPES (process BEX 13736124) which provided the financial sup-port for this study and the scholarship for R.S Peres. Financialsupport for E.A. and C.A. comes from MICINN and FEDER (MAT2012-34498), and the Generalitat de Catalunya (research group 2009 SGR925) is gratefully acknowledged. Support for the research of C.A.was received through the “ICREA Academia” prize for excellence inresearch funded by the Generalitat de Catalunya.

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