This article was downloaded by: [Swedish Institute for Food and biotechno] On: 30 September 2011, At: 01:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Biofouling Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gbif20 Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings Emiliano Pinori a b , Mattias Berglin a , Lena M. Brive b , Mats Hulander a , Mia Dahlström a & Hans Elwing a a Department of Cell and Molecular Biology, University of Gothenburg, Gothenburg, Sweden b SP, Technical Research Institute of Sweden, Chemistry and Materials Technology, Borås, Sweden Available online: 20 Sep 2011 To cite this article: Emiliano Pinori, Mattias Berglin, Lena M. Brive, Mats Hulander, Mia Dahlström & Hans Elwing (2011): Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings, Biofouling, 27:9, 941-953 To link to this article: http://dx.doi.org/10.1080/08927014.2011.616636 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [Swedish Institute for Food and biotechno]On: 30 September 2011, At: 01:17Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
BiofoulingPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/gbif20
Multi-seasonal barnacle (Balanus improvisus)protection achieved by trace amounts of a macrocycliclactone (ivermectin) included in rosin-based coatingsEmiliano Pinori a b , Mattias Berglin a , Lena M. Brive b , Mats Hulander a , Mia Dahlström a &Hans Elwing aa Department of Cell and Molecular Biology, University of Gothenburg, Gothenburg, Swedenb SP, Technical Research Institute of Sweden, Chemistry and Materials Technology, Borås,Sweden
Available online: 20 Sep 2011
To cite this article: Emiliano Pinori, Mattias Berglin, Lena M. Brive, Mats Hulander, Mia Dahlström & Hans Elwing (2011):Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin)included in rosin-based coatings, Biofouling, 27:9, 941-953
To link to this article: http://dx.doi.org/10.1080/08927014.2011.616636
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a
macrocyclic lactone (ivermectin) included in rosin-based coatings
Emiliano Pinoria,b, Mattias Berglina, Lena M. Briveb, Mats Hulandera, Mia Dahlstroma and Hans Elwinga*
aDepartment of Cell and Molecular Biology, University of Gothenburg, Gothenburg, Sweden; bSP, Technical Research Institute ofSweden, Chemistry and Materials Technology, Boras, Sweden
(Received 12 March 2011; final version received 17 August 2011)
Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization bybarnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leachingrate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7 ng cm72 day71. This low leachingrate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to thematrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersedin the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the meanbarnacle base plate area on the controls was 13 mm2, while on the ivermectin coating it was 3 mm2. After 388 days,no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a meanof 60 mm2. In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycoland glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacleperformance of the coatings. An increased release rate (3 ng cm72 day71) and dispersion of ivermectin, determinedby fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixturein the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in furtherstudies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxicationoccurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for themode of action and this is under investigation.
Marine biofouling can be defined as the colonization ofman-made surfaces immersed in seawater by micro-scopic and macroscopic organisms. This phenomenoncan result in loss of function and effectiveness both forcruising ships as well as for static constructions.Reduced hydrodynamic properties around the shiphull result in increased drag (Schultz 2007) and thusincreased fuel consumption (Schultz et al. 2011).Marine platforms, offshore rigs, underwater pipelinesand constructions can be damaged. Of special concernis the disruption of the corrosion protective layer bythe action of hard foulers such as barnacles (Eashwaret al. 1992; Sangeetha et al. 2010). To protect surfaces,biocides that act against marine biofouling organismshave been used for centuries. With recent environ-mental concerns and after the ban on tributyltin (TBT)coatings, new ways of preventing the accumulation ofbiofouling are sought (Yebra et al. 2004).
Antifouling (AF) coatings, based on the release ofbiocides such as copper oxide are still the market
leaders. Environmental concerns about copper, to-gether with its increasing market price, have promotednew ideas and the development of novel compoundsincluding those with a biocidal mode of action. Amongthese are (a) new environmentally benign candidatebiocides with novel modes of action such as medeto-midine and Econea1 (eg Dahlstrom et al. 2000; Bressyet al. 2010; Fay et al. 2010; Thomas and Brooks 2010),(b) numerous biomimetic or natural substances listedin comprehensive reviews (Qian et al. 2010; Scardinoand deNys 2011), enzyme antifoulings (Pettitt et al.2004; Kristensen et al. 2008, 2010), (c) optimization ofbiocide release profiles by improved formulations (Kiilet al. 2001; Fay et al. 2006, 2007; Bressy andMargaillan 2009; Larsson et al. 2010) or by biocideencapsulation in nano- or meso-sized carriers to avoidthe unwanted initial boost in release of the biocidefrom newly immersed coating (Fay et al. 2008;Nordstierna et al. 2010), (d) and the use of mixturesof biocides, whereby substances with different modesof action are combined, thus a lower concentration of
each substance is used to produce good efficacy whilereducing the risk of the development of tolerance bythe target organisms (Arrhenius et al. 2006).
All these AF strategies based on the release ofchemical biocides show similarities with pharmaceu-tical drug delivery systems. The goal of paints thatcontain these types of compounds, as for the drugdelivery systems, is to reach and maintain a concentra-tion of an active ingredient that is sufficient to give thedesired effect. This is referred to as the minimuminhibition concentration (MIC) in the AF. The releaserate needed to maintain the MIC at the surface isrelated to and influenced by many factors, includingthe affinity of the biocide for the surface. If the biocideis easily dissolved and dispersed by water flowing alongthe hull surface, a higher release rate is needed than ifthe biocide has a strong affinity to the solid surface ofthe matrix. A typical example of a biocide with highaffinity for the surface is medetomidine (Dahlstromet al. 2004) where 0.1% loading provides full protec-tion against barnacles despite a low release rate.
An interesting new possibility in AF technology,inspired by observations in the field and literature, wasthe starting point of this study. The ability of barnaclesto penetrate some coating films, observed in the fieldand already described by Barenfanger (1939), togetherwith enhanced post-settlement mortality of barnaclesobserved on Fucus evanescens described by Wikstromand Pavia (2004), provided inspiration for a possiblenew AF perspective, which was not based on release ofthe biocide. The aim of this study was thus toinvestigate if a biocide with high affinity to the matrixand loaded at a low concentration, could show anti-barnacle efficacy by acting from inside the film.
Taking advantage of pharmaceutical science, it waspossible to use the Higuchi equation to select a modelbiocide with a low predicted release rate. Higuchi(1961, 1963) described the release rate of a drug froman insoluble matrix as:
where, RR is the amount of drug released in time t, Dis the diffusion coefficient, S is the solubility of drugin the dissolution medium, e is the porosity of thematrix, A is the drug content per cubic centimetre ofmatrix, and Kh is the release rate constant of theHiguchi model. The release rate of a biocide or activeingredient (AI) from a paint film to the water can begoverned by the Higuchi model and/or as generallyachieved in a marine paint system, by the erosion ofthe matrix itself. The first situation will further bediscussed.
The release rate is dependent on the physico-chemical properties of both the biocide and the matrix.Biocide concentration (A) and water solubility (S) areobviously important factors when controlling therelease rate. Affinity between the biocide and thematrix (or some other components of the matrix)(Gerstl et al. 1998) influences the diffusion coefficient(D). Another important property is the molecularvolume of the biocide, affecting the diffusion of themolecule trough the matrix porosity (e) (Kristl et al.1991; Wesselingh 1993). Matrix porosity can have alarge effect on diffusion, especially in crystalline ordense matrix. It can be concluded that a biocide at lowconcentration, with low water solubility, high affinityto the matrix and high molecular volume will have avery low release rate.
In the present study, the model biocide was selectedfrom the family of avermectins, a series of macrocycliclactones produced by Streptomyces avermitilis, anactinomycete. In contrast to the macrolide or polyeneantibiotics, avermectins lack antibacterial or antifun-gal activity, but exhibit activity against a broad rangeof nematodes and arthropod parasites (Hotson 1982).Among the different forms of avermectins, ivermectin(22,23-dihydroavermectin) (Figure 1) was selected asthe model biocide for this investigation. Ivermectinpresents low water solubility (4 mg l71), high Koc
(12660–15700) (Halley et al. 1989), indicating a highaffinity to organic molecules such as rosin and high
Kow (1651) (Bloom and Matheson 1993). All theseproperties suggest a low rate of diffusion from thepaint film when the Higuchi model is considered.
Ivermectin has been used for several decades inmany different fields; as an antiparasitic agent inveterinary medicine and fish farming, as a pesticideagent in agriculture and even in human healthapplications (Fox 2006; Omura 2008). In the literature,there are reports of high toxicity of ivermectin againstcrustaceans (Burridge and Haya 1993). Preliminaryresults from a surface plasmon resonance (SPR)surface affinity study (unpublished data), confirmed ahigh affinity to the solid surface of a rosin matrix.Laboratory tests confirmed ivermectin toxicity atnanomolar levels against all life stages of Balanusimprovisus (unpublished data). Other macrocycliclactones, such as Spinosad, have shown AF activityagainst barnacles and have been tested and evaluatedin static field tests (Kritikou 2010).
In the initial field experiment, Ivomec1 1% wasmixed with a rosin-based, copper-free paint formula-tion and applied to a boat’s hull and panels. Ivomec1
is a veterinary injection preparation used for antipar-asitic treatment of cattle and household animals.It contains 1% (w/v) ie 10 mg ml71 ivermectin,dissolved in a cosolvent mix of propylene glycol andglycerol formal (60:40). Initial colonisation and meta-morphosis of small barnacles were not affected bythe incorporation of ivermectin in the coating.However, no adult barnacles were observed on thetreated sides of the panels, compared with control sideswhich were covered by barnacles by the end of thesummer season.
Encouraged by these promising results, an ex-tended field experiment to study the colonisationpattern of barnacles on static panels was designed. Inorder to study and discriminate the influence on thebarnacle settlement process, each of the differentcomponents present in the Ivomec1 1% solution wasevaluated. Ivermectin powder was added, dissolved inxylene and, in a separate experiment, dissolved in thecosolvent mix present in the Ivomec1 1% solution.Finally the cosolvent mix itself, ie without ivermectin,was added to the rosin-based paint and tested. In thisarticle, the fouling efficacy against barnacles (denotedanti-barnacle efficacy), the release rate of ivermectindetermined by LC/MS-MS, together with variousmechanical and structural studies on the experimentalcoatings are presented.
Material and methods
Avermectins and cosolvent
A commercially available injection solution for cattleand swine called Ivomec1 1% was purchased from
(Merial Norden A/S, Denmark) and used in the boatexperiments and in the preliminary static field test.Pure ivermectin (CAS no 70288-86-7) and abamectin(CAS no 71751-41-2) were purchased from WuhanYuancheng Technology Development Co., China.
To make the stock solutions, ivermectin wasdissolved at a concentration of 10 mg ml71 in bothxylene (Sigma Aldrich AB, Sweden) and in a solventmixture of propylene glycol (Fischer scientific AB,Sweden) and glycerol formal (Lambiotte & Cie,Belgium) in a 60:40 ratio, denoted as cosolvent mix.The cosolvent mix ratio was the same as for theIvomec1 1%. The stock solutions were used whenpreparing formulations for the field tests.
Rosin-based model paints
Several copper-free, rosin-based AF paints were usedin this study. TF1 and Mark51 and a rosintransparent lacquer were purchased from Lotrec AB,Lidingo, Sweden. The composition of CruiserEco1
(International Paint AB, Sweden) is presented inTable 1. This formulation was used in the extendedstatic field experiments.
The choice of the model paint formulation is animportant issue in AF field experiments. A laboratorymodel formulation gives the advantage of control onall the components that could influence the results. Onthe other hand, the probability of loss of data is high ifthe model binder fails the field test conditions duringimmersion. Using a commercial paint as a model for afield test can be regarded as more reliable, although insome cases this will make the evaluation process morecomplicated. A commercial paint usually containsmany different components, and many of those couldinteract and alter the final results. A compromisebetween these two alternatives could be the employ-ment of a commercial paint, resistant to field condi-tions for the duration of the experiment, but with asfew components as possible. Copper-free, rosin-based
Table 1. Ingredients present in CruiserEco1 according toSafety Data Sheet (YMA269) Version No. 4. Revision date:05/07/03.
paint that is resistant to most fouling except barnaclesis advantageous when testing a new biocide directlytargeting barnacles. Moreover, applying the base paint(control) and treated paint beside each other on eachpanel greatly simplifies the evaluation process. It givesthe opportunity of normalizing results and eliminatesthe effects of other components in the base paint. Atthe same time, this experimental set-up compensatesfor local variation in fouling pressure arising fromfluctuation of natural factors as immersion depth, lightpenetration and food availability.
For the extended field test study, the commerciallyavailable CruiserEco1 was chosen. CruiserEco1 is acopper-free rosin-based AF paint that is allowed forprotection against fouling in the Baltic Sea, wherespecial regulations are in force. When CruiserEco1 isused in water with higher fouling pressure than theBaltic Sea, such as the West coast of Sweden, itbecomes easily fouled, especially by barnacles. Crui-serEco1 is the copper-free version of the morecommonly used copper containing AF paint calledCruiser1. Thus, this study also evaluated whethercopper containing paints could be replaced by applyingivermectin at low loading. Currently *140 tons ofcopper are used every year in Sweden for AF purposes(KEMI 1998–2007).
Boat experiments
Ivomec1 1% was added to TF1, Mark51 orCruiserEco1 to a 10% (v/v) concentration, giving a0.1% final concentration of ivermectin in the paint.The resulting paint preparations were then applied onthe hull according to instructions given by themanufacturer. Control surfaces were also included inthe experiments. The control coating was the samepaint used for the experimental area, but without theaddition of 1% Ivomec1. The experiments werecarried out throughout the years 2006–2009.
Static field tests
Early field test
Plexiglas1 panels with dimension of 110 mm 6 110 mmwere used as the substratum. One half of each panelsurface was painted with TF1 containing 10% (v/v)Ivomec1 and the other half was painted with TF1
without the addition of Ivomec1.
Extended field test
The two stock solutions containing ivermectin at1% were added at 10% (v/v) to 50 ml of CruiserEco1,and mixed using a vortex at 2500 rpm for 2 min.The resulting formulations, containing a final
concentration of 0.1% (w/v) ivermectin were appliedto the test panels. Finally, the cosolvent mix was alsoadded in the same way to CruiserEco1. The experi-mental series of boats and panels was made inaccordance with exemptions issued by the SwedishChemicals Agency (Diary number 750-482-09). Theresulting paint preparations listed in Table 2 wereapplied with brushes resulting in a wet film thickness ofca 200 mm and a dry film thickness of ca 100 mm onPlexiglas1 panels (200 mm 6 150 mm obtained fromDAF screen AS, Denmark). Each panel (triplicates,n ¼ 3) was coated both with treated and controlformulation as described above. Panels were deployedby hanging them with a tie strap from a rack, usingpre-drilled holes on the corners of the panel. Thepanels of both the early and the expanded field testwere attached to aluminium frames (180 cm 6 200cm). Each frame consisted of six rows of panels with adistance of ca 4 cm from each other. The frames wereimmersed at the Sven Loven Centre for MarineBiology situated at þ 588 150 0.0600 N; þ 118 260
48.8400 E. The panels were randomly distributed inwater depth ranging from 25 to 180 cm. The field studystarted (T0) on 6 June 2009. The panels were inspectedvisually and photographed at three different dates: T1(10 July 2009) after immersion for 35 days; T2 (18August 2009) after immersion for 73 days and T3 (29June 2010) after immersion for 388 days.
Fouling evaluation
At each time point the panels were removed from thewater, observed and photographed. B. improvisus wasthe only macrofouler present that was visible to thenaked eye. The images were analysed with the help ofimage editor software ‘ImageJ’ (Rasband 1997–2009).Using the contrast between the B. improvisus brightcalcareous shells and the dark paint, the software coulddistinguish all the barnacles as confirmed by directobservation. The software reported the number ofbarnacles present on the surface and the area ofindividual barnacles.
The coverage of barnacles was calculated as thepercentage of the area occupied by barnacles related to
Table 2. Description of the four paint formulations used inthe static field test.
the total area examined (20 cm 6 7.5 cm). This valuewas also normalized with respect to the coveragereported for the control area of the same panel.
Determination of leached ivermectin
A 77 cm2 area was painted with Ive, IveCos and Cosformulations (Table 2), on Plexiglas1 panels using a120 mm thick wet film applicator. The panels were leftto dry at room temperature for one week. Each of thepanels was immersed for two days in 2 l of Milli-Qwater in order to equilibrate the release rate, and thensoaked for 10 days in separate Petri dishes with 100 mlof ‘substitute ocean water’ (SOW) prepared accordingto ASTM D1114-98 (2008). The soaking was per-formed under constant rocking (1 revolution s71), indarkness at room temperature. After 10 days, 10 mlaliquots were collected and frozen for future analysisof the total accumulated ivermectin using LC/MS–MS.
The frozen samples were thawed and concentratedby solid phase extraction (SPE) using an Isolute1
200 mg C18 column (Biotage, Sweden). The columnwas activated with 1 ml þ 1 ml methanol followed by1 ml þ 1 ml Milli-Q water. A 10 ml thawed samplewas applied to the column, which was then washedwith 1 ml þ 1 ml Milli-Q water, and finally ivermectinwas eluted with 0.5 ml þ 0.5 ml 2 mM abamectin(Sigma Aldrich AB, Sweden) in acetonitrile. Abamec-tin was used as an internal standard.
Recovery of ivermectin from the leaching experimentset-up
A volume of 99.5 ml SOW was spiked with 500 mlivermectin solution in acetonitrile to obtain a finalconcentration of 5 mM Ivermectin in SOW. At T0 a10 ml sample was collected and frozen for futureanalysis. The remaining 90 ml were added to a Petridish containing a PMMA panel painted on a 10 cm2
area with negative control paint. After soaking for 10days (T10) 10 ml were collected and frozen for futureanalysis. This experiment was performed in triplicate.The recovery rate was calculated as the ratio betweenthe ivermectin concentration at T10 and at T0, theanalysis was performed by LC/MS-MS with the sameextraction and concentration procedure as for the totalleached ivermectin determination described below.
Quantification of ivermectin by LC/MS–MS
Ivermectin in SOW was quantified by an externalstandard curve ranging from 0.0012–1.12 pmol ml71
ivermectin and using abamectin (5 pmol ml71) asinternal standard. The limit of quantification by LC/MS–MS was 0.002 pmol ml71 and the limit of
detection was not determined. The samples andstandards (20 ml) were separated on an XBridge C-182.5 mm (30 6 2.1 mm, Waters Milford, Massachu-setts) column. The linear gradient was from 50%water þ 0.2% formic acid and 50% acetonitrile 0.2%formic acid to 4% water þ 0.2% formic acid and96% acetonitrile þ 0.2% formic acid at a flow rate of0.5 ml min71 with an Alliance 2695 HPLC pump(Waters, Milford, Massachusetts).
The eluate was analysed on-line by electro-spray/MS on a QuattroUltima (Waters Milford, Massachu-setts) using the MRM-mode. Ivermectin was detectedby the mother ion [MþNa]þ at m/z ¼ 897.36 amu anddaughter ion at m/z ¼ 753.24 amu ([M-MeO-pentoseþNa]þ) and abamectin using the mother ion[MþNa]þ at m/z ¼ 895.28 amu and daughter ion atm/z ¼ 751.51 amu ([M-MeO-pentoseþNa]þ) usingcapillary voltage ¼ 2 kV, cone voltage ¼ 50 mV andcollision energy ¼ 45. The data were collected by thesoftware MassLynx 4.1 (Waters, Milford, Massachu-setts) and integration and quantification was sup-ported by the QuanLynx software (Waters, Milford,Massachusetts).
The average release rates expressed as mass cm72
day71 during the 10 days of the experiment have beencalculated from the ivermectin measured at T10 by LC/MS-MS. This value has been corrected by theconcentration factor arising from the purification onSPE column and for the loss of ivermectin occurringduring the leaching experiment.
Fluorescence microscopy
Fluorescent ivermectin was obtained by reactingivermectin with 1-methylimidazole (MI), trifluoroace-tic anhydride (TFAA), triethylamine (TEA) andtrifluoroacetic acid (TFA) as described by Berendsenet al. (2007). All the reagents were purchased fromSigma-Aldrich (Steinheim, Germany). The fluorescentproduct hereby denoted as ‘ive-flu’ was stored in thedark at 7188C prior to use.
Transparent lacquer obtained from Lotrec AB,Lidingo, Sweden was used to study the difference indispersion of the ‘ive-flu’ in the dry rosin film; ‘ive-flu’was added and dissolved in xylene or cosolvent mix.The coloured pigments are not wanted in thisparticular case because of the interference withfluorescence microscopy. The experimental conditionsare not fully representative of the real dispersionpattern of ivermectin inside CruiserEco1, but give anindication of the mechanism. The final concentrationin the transparent lacquer in both cases was 0.1% (w/v). A volume of 100 ml of each two resulting lacquersformulations was allowed to dry on microscope glassslides. When dry they were studied by fluorescence
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microscopy using a Leica DM RXA microscopecoupled to a Hamamatsu C–474295 camera connectedto a computer running ‘QFluoro1’ software versionV1.2.0. The DAPI fluorescence filter was used, as it fitthe expected excitation and emission wavelengths ofthe ‘ive-flu’ reported in Berendsen et al. (2007)(excitation 365 nm–emission 470 nm).
Hardness and erosion test
In order to test the influence of the cosolvent mix onthe physical properties of the coating, an indentationtest and an erosion test were made. To test the degreeof hardness of the paint, the Buchholz indentation test(DIN EN ISO 2815) was used. Plastic panels werepainted with Ive and IveCos using a film applicatorwith a 120 mm film thickness. The panels were left todry for 10 days prior to analysis. After analysis thecoatings were immersed in 2 l of Milli-Q water forthree days and hardness was re-measured. The test wasperformed in a constant climate chamber where thetemperature and humidity were 23 + 28C and50 + 5%, respectively.
The erosion test was made on panels similar to theones utilised in the extended static field test. The panelswere painted with Ive and IveCos and placed verticallyin a plastic beaker exposed to running seawater at theSven Loven centre for two months. Erosion wasdetermined by measuring the thickness of paint layerwith a micrometre. Although this method is not fineenough to detect small and precise differences in theerosion rate as demonstrated previously using QCM-D(Berglin and Elwing 2008), it was considered a morerealistic method to demonstrate the feasibility ofadding the cosolvent mix into a typical commercialrosin-based paint without compromising the resistanceof the film over a long period (60 days).
Results
Boat experiments
According to local boat owners, the barnacle, B.improvisus, is the major problem in terms of marinefouling on the Swedish west coast. This opinion wasthe main reason for choosing avermectin, which isknown to be toxic to crustaceans, has low toxicity tomammals and is cheap enough to make experimentalcoatings for full size boat trials. A general observationwas that the addition of 10% (v/v) Ivomec1 1% didnot affect the wetting and adhesion properties of thepaint. After the summer season, barnacles andsecondary attached blue mussels were the onlydominating biofouling organisms observed on thecontrol surface, in addition to some algae growing atthe water line of the boat hull. Addition of 10% (v/v)
Ivomec1 1% in the paint formulations resulted in asurface free from adult barnacles. A representativeexample of a full size boat experiment after one fullfouling season is shown in Figure 2.
Early field test
After immersion of static panels for three summermonths, the surfaces treated with 10% (v/v) Ivomec1
1%, were free from adult barnacles compared with the100% fouled control coatings (Figure 3a). At highermagnification (Figure 3b) the presence of smallbarnacles with calcareous shells can be seen even onthe Ivomec1 1% surfaces. This result indicated that amore systematic field test was needed with severalvisual inspections at different time points in order tofollow the pattern of colonisation.
Extended field test
Representative panels showing the accumulation ofbarnacles at three inspections (T1 ¼ 35 days; T2 ¼ 73days and T3 ¼ 388 days) on the three differentformulations are shown in Figures 4–6.
The percentage of the area covered by barnacles isshown in Figures 7–9 for the treated and control sidesof the panels studied. The ratio between the areacovered by barnacles for the treated and control areasfor each formulation is reported as the normalizedanti-barnacle efficacy in Figure 10. As the efficacy ofivermectin against barnacles was the purpose of thisstudy, further evaluations were focused on thisorganism. The efficacy of the formulation will refer
Figure 2. The yacht shown had been sailing for foursummer months along the Swedish west coast. The majorpart of the hull was painted with CruiserEco1 with theaddition of 0.1% (w/v) ivermectin solubilised in a cosolvent-mix (see Materials and methods). The control areas are thetwo areas painted with only CruiserEco1 as indicated by thewhite arrows.
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to anti-barnacle performance from here on because noother macroorganisms were observed on the panels, aswas expected for CruiserEco1.
The efficacy of the Ive-formulation compared to thecontrol was 13% (+29%) after immersion for 35 days(Figure 10). The average size of barnacles was ca1 mm2 on both the sides with ivermectin and thecontrol coatings.
After immersion for 73 days, the efficacy of the Ivetreatment was 81% (+10%). Towards the periphery ofthe control panels (Figure 4b) barnacles reached ca13 mm2. This edge effect on the growth rate was notpresent in Ive painted areas. On the Ive treated panels,
barnacles reached a size limit of about 3 mm2
regardless of position with respect to the edge of thepanels. Water flow at the edge of the panels affectscyprid settlement (Rittschof et al. 1984; Mullineauxand Butman 1991) and is also related to the postsettlement growth and survival as described in Larssonand Jonsson (2006). Thus, the edge effect on thecontrol panels is attributed to hydrodynamics.
After immersion for 388 days, no barnacles wereobserved on the Ive coating (Figure 4c,f), thusindicating 100% efficacy. Since the rate of erosion ofthe Ive and control coatings did not vary, the loss ofsmall barnacles from Ive is attributed to a biocidalinfluence on the adhesion strength of the barnacles,which may also be connected with the absence ofgrowth.
The other ivermectin containing formulation,IveCos, showed an efficacy of 60% (+10%) at thefirst inspection, ie after 35 days (Figure 10). After 73days, efficacy reached 77% (+9%). No increase inbase plate size could be observed between T1 and T2on the side painted with IveCos. After 388 days, nobarnacles were present on the treated side, thusresulting in 100% efficacy (Figure 5c,f). As for theIve paint, barnacles that were present at T1, wereabsent from the treated panels at T2. This result ismost likely due to a biocidal influence on adhesionstrength, causing fouling-release, probably correlatedwith the absence of growth of the small barnacles.Healthy barnacles on the control sides were stillattached and had reached the stage of adults.
Finally, the formulation without ivermectin, Cos-formulation, was tested to check for possible effects ofthe cosolvent mix on the settlement and growth ofbarnacles (Figure 6). After immersion for 35 days, theefficacy was 79% (+32%), meaning that on somepanels more barnacles were found on the Cos treatedside compared to the control side. However, thisdifference was not statistically significant. Again, nosubstantial difference in efficacy (19% + 13%) wasfound after immersion for 73 days. After 388 days, thereduction in barnacles reached 35% (+19%). How-ever, when images were studied at higher magnification(Figure 6c,f), ‘footprints’ of large barnacles dislodgedfrom the coatings could be seen. These ‘footprints’were of the size of the largest barnacles present on thecontrol paint. Thus, no influence in the ability to growcould be attributed to the Cos formulation. Rather, thephenomenon seems to be related to a partial fouling-release effect due to a change in the mechanicalproperties of the Cos coating compared with thecontrol paint CruiserEco1. The effect of the cosolventmix 10% (v/v) on the mechanical properties ofCruiserEco1 will be discussed in more detail in thehardness and erosion test results.
Figure 3. (a). Effect of ivermectin on the settlement andgrowth of barnacles. The –A side of the panel was painted awith rosin-based paint (Mark51). The þA side was paintedwith the same paint containing 0.1% (w/v) ivermectin. Thepanel had been in water for three summer months. (b).Magnified area of the panel shown in (a). On the –A side, theblue colour on the barnacle shells is visible due to penetrationof the paint. On the þA side, barnacles that have not grownwere still present after three summer months (labelled smallbarnacle).
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Release of ivermectin
The total amount of ivermectin released from panelsafter soaking for 10 days in 100 ml of SOW in Petridishes was determined by LC/MS-MS analysis. Theamount released was found to be 26 ng + 28% for Iveand 114 ng + 13% for IveCos. No ivermectin wasdetected for the Cos formulation (Table 3). Therecovery of ivermectin from the soaking and leachingexperiment was found to be 5%. Thus the totalamount of released ivermectin corrected for recoverywas found to be 0.52 mg + 28% for the Ive-formula-tion and 2.27 mg + 13% for the IveCos-formulation.Using these values average release rates of0.68 + 0.2 ng cm72 day71 for Ive and2.95 + 0.38 ng cm72 day71 for IveCos were obtained.Whether the release of ivermectin is a consequence ofboth diffusion and erosion or mainly through erosionis still under investigation. One way to investigate thiswould have been to use a non-erodible model paint,but for the reasons explained in the Materials andmethods section, it was preferred not to use alaboratory-produced model-coating.
Ivermectin distribution in the film as determined byfluorescence microscopy
To study the distribution of ivermectin in thepaint film, the ivermectin molecule was transformedby an intramolecular reaction in a fluorescentderivative (Berendsen et al. 2007) and dissolved intwo stock solutions, one in xylene and one in thecosolvent-mix. These solutions were then added tothe Mark51 transparent lacquer until the finalconcentration of 0.1 mg ml71 was reached. Figure 11shows the distribution of fluorescent ivermectin inthe dried lacquer film. When the fluorescent iver-mectin was added to the Mark51 lacquerdissolved in xylene, it formed aggregates anddid not form a homogeneous pattern in the drylacquer film (Figure 11a). When fluorescent ivermec-tin was added into propylene glycol/glycerol for-mal 60:40, an emulsion-like pattern was observed inthe dried lacquer film, and the fluorescence wasstronger and more homogeneously distributedaround small ‘bubbles’ of *50 mm diameter(Figure 11b).
Figure 4. A representative panel from the field test photographed after different immersion times. The left side of the panel waspainted with the Ive formulation. The right half was painted with the control formulation. (a) After immersion for 35 days; (b)after immersion for 73 days; (c) after immersion for 388 days. Areas labelled (d), (e) and (f) are magnifications of (a), (b) and (c),respectively.
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Hardness and erosion tests
To study influence of the cosolvent mix (propyleneglycol:glycerol formal 60:40) on the mechanical prop-erties of the coatings, an indentation resistance test, theISO 2815 standard Buchholz test, was performed onIve and IveCos coated panels. The mechanical proper-ties of the Ive formulation for CruiserEco1 with 0.1%(w/v) ivermectin were assumed to be the same as forthe CruiserEco1 paint. By contrast, IveCos containedthe same concentration of ivermectin but 10% (v/v) ofthe cosolvent-mix. The resistance to indentation aB wascalculated as described in the Materials and methodssection. For the IveCos aB ¼ 60.1 + 1.69 compared toIve aB ¼ 71.2 + 3.6. Thus, the addition of the cosol-vents made the rosin-based coatings softer. Thischange of softness could explain the release of theadult barnacles from the edge of the cos-formulationpainted panels after immersion for 388 days. Largerbarnacles are exposed to higher shear stresses and withslightly softer coatings they might release more easily,ie a fouling-release effect induced by the cosolvent-mix.An accurate study of the lateral force involved inremoving barnacles from the coatings needs to bedone.
A preliminary erosion test on the Ive and IveCosformulations showed that the thicknesses of the twoformulations were both reduced by about 2 6 1075
cm day71. Even though these values are preliminary, itis possible to conclude that the cosolvent mix presentin IveCos did not influence the erosion rate to any greatextent.
Discussion
A major finding in this study was that the presence ofivermectin in the coatings had no effect on thesettlement and metamorphosis of barnacles. After afew weeks, the presence of ivermectin resulted in asignificant enhancement of post-settlement mortalityand/or release of the newly metamorphosed barnacles.This delayed AF effect could be explained by a chronictoxicity effect caused by ivermectin slowly leachingfrom the paint. This aspect is currently underinvestigation. Nevertheless, contact intoxication ofthe barnacles occurring in the inner layer of the filmappears as to be a possible alternative explanation.
The phenomenon of enhanced post-settlementmortality, rather than settlement inhibition, occurringafter the metamorphosis, opens up the possibility of
Figure 5. A representative panel from the field test photographed after different immersion times. The left side of the panel waspainted with the IveCos formulation. The right half was painted with the control formulation. (a) After immersion for 35 days;(b) after immersion for 73 days; (c) after immersion for 388 days. Areas labelled (d), (e) and (f) are magnifications of (a), (b) and(c), respectively.
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Figure 6. A representative panel from the field test photographed after different immersion times. The left side of the panel waspainted with the Cos formulation. The right half was painted with the control formulation. (a) After immersion for 35 days; (b)after immersion for 73 days; (c) after immersion for 388 days. Areas labelled (d), (e) and (f) are magnifications of (a), (b) and (c),respectively.
Figure 7. Percentage area of the test panels covered bybarnacles at three different inspections. Results are theaverage values for triplicate panels (n ¼ 3). Barnaclecoverage of the Ive treated side (dark yellow) and thecontrol side (light yellow) are shown after immersion for 35days (T1); 73 days (T2); and 388 days (T3).
Figure 8. Percentage area of the test panels covered bybarnacles at three different inspections. Results are theaverage values of triplicates panels (n ¼ 3). Barnaclecoverage for the IveCos treated side (dark red) and thecontrol side (light red) are shown after immersion for 35 days(T1); 73 days (T2); and 388 days (T3).
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new perspectives in AF research. That barnacles aftermetamorphosis can penetrate the surfaces of certaincoatings is known from the literature (Barenfanger1939) and has been observed here (Figure 3a,b).Penetration into the inner part of the coatings anddirect contact with a biocide with high affinity to thematrix could result in the possibility of developing AFformulations not based on the release of the biocide.This would have a great impact on the environmentaland economic aspects of AF paints.
A similar phenomenon of enhanced post settle-ment mortality has been seen on fronds of the brownalga Fucus evanescens. This alga allows normalcolonisation by cyprid larvae, but inhibits the growthto full adults (Wikstrom and Pavia 2004). Themechanism behind this phenomenon is presentlyunknown. The similarity in anti-barnacle strategiesbetween the low loaded ivermectin and F. evanescensis striking.
What is surprising is the long lasting efficacy ofthe formulations studied, even considering the lowamount of biocide measured in leachates over thefirst 10 days. It is important to stress that acommonly used biocide such as cuprous oxide(ACE 2002) is released at an order of 30,000 timeshigher than in the case studied here. This ishighlighted in the fact that the rate of release ofAF biocides is expressed in mg cm72 day 71 and notin ng cm72 day71 as in this study.
Table 3. Ivermectin leached as studied by LC/MS-MS.
Note: The concentration measured by LC/MS-MS expressed as pmolml71 was used to calculate the total amount of ivermectin released bythe coatings tested in the 10 day soaking study in the laboratory.
Figure 11. Fluorescence of a rosin based film of a driedMark51 transparent lacquer film with 0.1% (w/v) offluorescent ivermectin ‘flu-ive’. In (a) the flu-ive wasdissolved in xylene and added to 0.1% (w/v); in (b) the flu-ive was dissolved in cosolvent-mix and added to 0.1% (w/v)(propylene glycol–glycerol formal 60:40).
Figure 9. Percentage area of the test panels covered bybarnacles at three different inspections. Results are theaverage values of triplicates panels (n ¼ 3). Barnaclecoverage for the Cos treated side (dark blue) and thecontrol side (light blue) is shown after immersion for 35days (T1); 73 days (T2); and 388 days (T3).
Figure 10. Anti-barnacle efficacy as a % normalized againsteach respective control of the test panels at three differentinspections. Result are the average of triplicate panels (n ¼ 3)with the SD as error bars. Anti-barnacle efficacy is reportedfor the Cos treated sides (dark blue), the Ive treated sides(dark yellow) and the IveCos treated sides (dark red) afterimmersion for 35 days (T1); 73 days (T2); and 388 days (T3).
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The difference in the rate of release is not soinformative if no data on comparative toxicity are alsopresented. Another member of the avermectin family isEmamectin Benzoate (EB), which is approved for usein salmon farming to treat sea lice under thecommercial name of Slice1. A comparative study ofthe toxicity of copper oxide and EB to sedimentdwelling organisms (Mayor et al. 2008) reported aLC50 of EB between 300 and 700 times lowerthan copper oxide. The 0.1% (ca 6300 lower)loading and 630,000 slower release for EB coatingscompensates for even the worst case scenario of hightoxicity to non-target organism. Furthermore, becauseof the low loading, the economic aspect is alsofavourable for EB compared to both Cu-oxide andother approved biocides. Preliminary field tests haveshown similar efficacy and mechanism of action for EBin CruiserEco1 (unpublished) as for ivermectin.Furthermore, there may be molecules similar toavermectin that are better suited to AF applicationsfrom an ecotoxicological point of view since othermacrocyclic lactones have been shown to have anti-barnacle activity (Kritikou 2010).
This study has focused on protecting surfaces frombarnacles and the results are positive. However, theexperimental set up cannot predict if ivermectin wouldhave efficacy against other fouling organisms. Never-theless, the interest in controlling barnacles is highsince the detrimental effects of fouling of ships’ hulls bybarnacles are undisputed.
Conclusions
The addition of 0.1% (w/v) ivermectin in a commer-cial, copper-free, rosin-based AF paint provided 100%protection against barnacles for several fouling sea-sons. The release rate measured was in the range of 0.7to 3 ng cm72 day71. Unfortunately, due to difficultiesin controlling all the variables in a field test, theauthors cannot rule out a possible effect of chronicintoxication caused by the biocide. Nevertheless, theresults suggest a new mechanism of inhibition invol-ving contact between barnacles and biocide inside thepaint film. This fascinating hypothesis could havemany positive consequences in terms of new AFstrategies and will be studied further.
Acknowledgments
The authors gratefully acknowledge financial support for thisresearch from the Nordic Innovation Center NICe in theframe of the MARINORD Project.They also thank all thevolunteer boat-owners who participated in the field test;the enthusiasm received from their feedback was the best fuelfor this study.
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