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Paine, Martin, Pianegonda, Nicole, Huynh, Tran, Manefield, Mike,MacLaughlin, Shane, Rice, Scott, Barker, Philip, & Blanksby, Stephen(2016)Evaluation of hindered amine light stabilisers and their N-chlorinatedderivatives as antibacterial and antifungal additives for thermoset surfacecoatings.Progress in Organic Coatings, 99, pp. 330-336.

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https://doi.org/10.1016/j.porgcoat.2016.06.009

Evaluation of Hindered Amine Light Stabilisers and their N-Chlorinated derivatives as 1

Antibacterial and Antifungal Additives for Thermoset Surface Coatings 2

3

Martin R. L. Paine1,8, Nicole A. Pianegonda1,5, Tran T. Huynh2, Mike Manefield2,3, Shane A. 4

MacLaughlin5, Scott A. Rice2,4,6, Philip J. Barker1,5 and Stephen J. Blanksby1,7 * 5

6

1ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, School of 7

Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia 8

2The Centre for Marine Bio-Innovation, University of New South Wales, Sydney, NSW 9

2052, Australia 10

3The School of Biotechnology and Biomolecular Sciences, The University of New South 11

Wales, Sydney, NSW 2052, Australia 12

4The School of Biological, Earth and Environmental Sciences, The University of New South 13

Wales, Sydney, NSW 2052, Australia 14

5BlueScope Research, The Innovation Labs, Old Port Road, Port Kembla, NSW 2502, 15

Australia 16

6The Singapore Centre on Environmental Life Sciences Engineering and the School of 17

Biological Sciences, Nanyang Technological University Singapore 18

7Central Analytical Research Facility, Queensland University of Technology, Brisbane QLD 19

Australia. 20

8School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 21

30332, USA. 22

23

*Corresponding author 24

Prof. Stephen J. Blanksby 25

Central Analytical Research Facility 26

Queensland University of Technology 27

Brisbane, QLD Australia 28

Email: stephen.blanksby@qut.edu.au 29

30

Abstract 31

32

N-Halogenated amines or ‘halamines’ have attracted recent attention as potential biocides for 33

materials and surface coatings application. Facile N-chlorination of the hindered amine light 34

stabiliser (HALS) Tinuvin®770, bis-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate, was 35

achieved by reaction with sodium dichloroisocyanurate. The chlorinated product was 36

incorporated into a polyester-based paint formulated for coil coating, applied to test panels 37

and subjected to high temperature curing conditions characteristic of the coil coating process 38

(55 seconds at 262°C). Rapid detection of N-chlorinated Tinuvin®770 in the cured coating 39

was confirmed, using liquid extraction surface analysis-mass spectrometry, by the 40

characteristic fragmentation patterns of the halamines observed upon collision-induced 41

dissociation. Antimicrobial activity of the coating was determined by testing against the 42

bacterium Pseudomonas aeruginosa and the fungus Cladosporium sp., two organisms that 43

are known to colonise both internal and external surfaces in building and cladding 44

applications. The activity of HALS and halamine containing coatings were compared against 45

a commercial product containing an antimicrobial additive as well as control surfaces without 46

additives. Significant activity against the bacterium, but not against the fungus was 47

demonstrated for the parent HALS and halamine containing coatings. The possibility of 48

regeneration of the halamines was also tested and confirmed by mass spectrometry, post-49

chlorination of samples showed no significant differences in activity between corresponding 50

pairs of samples. 51

52

Keywords: Biofilms, surface coatings, coil coatings, antimicrobial coatings, HALS, 53

halamines 54

1 Introduction 55

Coil coating is an industrial process whereby polymer-based surface coatings may be 56

applied, normally in liquid form, to rapidly moving steel or aluminium strip, then rapidly 57

cured and recoiled [1]. The painted product is uncoiled, then cut, slit and stamped or formed 58

into a variety of product types for wide-ranging final applications, e.g. exterior roofing and 59

walling, automotive body parts, whitegoods, insulated coolroom panels, fencing etc. Of this 60

broad spectrum of end uses, two in particular are subject to the build-up of microbial 61

infestation. These are exterior, non-vertical applications such as roofing and interior 62

applications, such as insulated coolroom panels for bulk food storage. 63

In non-vertical exterior applications such as roofing, there are two main mechanisms 64

for microbial build-up, where microbial growth (darkening of light coloured roofs, or patchy 65

discolouration across the roof body) may be observed [2]. Firstly, in the first five years of 66

service lifetime, failure to seal the building envelope, particularly the roof cavity, after 67

construction leads to leakage of moisture and other potential nutrients from the roof cavity. 68

This occurs particularly around the ridge-cap area, resulting in the build-up of visible fungal 69

darkening. Secondly, exposure to high temperatures in the presence of broad spectrum solar 70

radiation, humidity and oxygen for extended periods of time [3, 4] leads to thermo-photo-71

oxidative degradation of the polymer matrix which subsequently produces lower molecular 72

weight fragments with oxygenated structures (e.g. ketones, carboxylic acids and alcohols) 73

that increase the surface hydrophilicity of the surface coating [3]. This change in 74

hydrophilicity enhances the binding of moisture, thus increasing the roof surface 75

susceptibility to longer term (>10 years) microbial infestation [3], in extreme cases leading to 76

lichen growth. In a sector where long-term maintenance of aesthetic properties is paramount, 77

such infestations are a significant cause of concern throughout the supply chain from paint 78

companies, to coil-coaters, product manufacturers and end customers alike. 79

In interior applications such as bulk food cold-storage facilities, cool moist airflows 80

from climate control systems ensure a continuous circulation of microbes from the stored 81

products (particularly fruit and vegetables [5]). These can settle and proliferate on susceptible 82

surfaces such as coolroom walls and in air conditioning ducts, necessitating periodic 83

cleaning. 84

There are a number of existing applications of antimicrobial additives in the surface 85

coatings sector, for example, they are added to water-borne formulations to prevent microbial 86

paint spoilage in storage [6]; they are added to various paints and coatings to generate 87

antimicrobial surfaces in bathrooms and other wet areas [7]; and antimicrobial coatings have 88

been employed in the marine sector for decades [8]. However, there remains an urgent need 89

for effective additives relevant to coil coatings to combat the type of infestations mentioned 90

above. In coatings for roofing, for example, they must be non-toxic, as in many locations 91

around the world the roof is used for water collection for domestic or secondary applications. 92

Many commercial fungicides are, however, toxic organic (e.g., methylisothiazolinone) or 93

inorganic compounds (e.g., organozinc compounds) with environmental and health 94

consequences. In addition, functional additives (i.e., additives that function throughout the 95

service lifetime of a coating, rather than additives employed for storage or production) for 96

coil coatings must survive a vigorous rapid curing process with air temperatures over 300 °C: 97

conditions not always compatible with low molecular weight organic compounds. 98

One type of functional additive successfully formulated in coil coatings is the class of 99

chain breaking anti-oxidants known as hindered amine light stabilisers, HALS. These are 100

molecules which prolong the service life of surface coating by inhibiting free-radical 101

mediated degradation of the polymeric part of the surface coating [9]. Typically the active 102

moiety within a HALS additive is an N-substituted piperidine containing an >N-X bond, 103

where X may be a proton, alkyl, acyl or alkoxyl (H, R, (C=O)R, OR). Not only do these 104

molecules survive the curing process, but they can also be measured and studied, post curing, 105

by a range of experimental techniques [10]. 106

Facile chlorination of the simple unsubstituted secondary amine HALS molecules, 107

achieved using, for example, sodium dichloroisocyanurate (NaDCC), leads to the formation 108

of piperdinyl-N-chloroamines, or N-chloro-halamines, which themselves, over the past 109

decade, have begun to attract attention as potential antimicrobial compounds with varied 110

applicability [11] with the most recent applications including surface functionalised polymer 111

microspheres [12]. Hence, chlorination of specific HALS molecules has the potential to 112

produce antimicrobial additives which can not only survive the processing conditions of coil 113

coating, but also offer potential antimicrobial activity to coil-coated products. Moreover, in 114

coolroom applications, it may be possible to regenerate the chlorinated compounds, once 115

expired, simply using hypochlorite based cleaners, leading to longer activity period between 116

scheduled maintenance [13]. 117

In this study, the synthesis and incorporation of N-chloro-amines, based upon 118

commercial HALS structure (I), into coil coatings is described (Scheme 1). The quantitative 119

incorporation of unchlorinated parent HALS (I), mono- (II) and di-chlorinated (III) 120

halamines into the finished coating and, similarly, the generation/regeneration in situ, of N-121

chloro-halamine from the parent secondary amine (I) is demonstrated using mass 122

spectrometry. Finally, preliminary microbiological studies, describing the activity of the 123

surface coatings produced against two typical organisms of interest, the bacterium 124

Pseudomonas aeruginosa and the fungus, Cladosporium sp., are described. 125

126

Scheme 1 Compounds employed in this study 127

128

2 Experimental 129

2.1 Reagents 130

Methanol and formic acid were high performance liquid chromatography (HPLC) 131

grade (Crown Scientific, Minto NSW, Australia). Chloroform was analytical reagent (AR) 132

grade (Crown Scientific, Minto NSW, Australia). Sodium dichloroisocyanurate (96 %) and 133

anhydrous magnesium sulphate (97 %) were supplied by Sigma-Aldrich (Castle Hill, NSW, 134

Australia). Tinuvin®770, I, (bis-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate) was supplied by 135

BASF Gmbh, Ludwigshafen, Germany). 136

2.2 Preparation of N-chlorinated HALS 137

II and III were prepared by the chlorination of I with sodium dichloroisocyanurate 138

(NaDCC), similar to the method reported by Cao and Sun [14], but with lower chlorinating 139

reagent concentrations as to provide incomplete conversion to the chlorinated analogues. This 140

enabled the simultaneous qualitative investigation of all precursor and product HALS 141

compounds that would participate within the model coil coating systems while providing 142

enough chlorinated HALS material to observe any potential inhibition of biological growth 143

on the polymer surface. For the modified method, an aqueous solution of NaDCC (1 % w/w) 144

was added to a solution of I in chloroform (0.5 % w/w). The mixture was vigorously stirred 145

at room temperature for 1 h. After filtration, the chloroform layer was separated and dried 146

with magnesium sulphate. Magnesium sulfate was subsequently removed by filtration and the 147

chloroform evaporated to give the final crude product as a white powder at room temperature. 148

The crude product was added to the wet paint formulations (samples 7 and 8) to give a final 149

concentration of 2% w/w halamine based upon total resin solids. 150

‘Post-chlorination’ (samples 2, 4, 6 and 8 in Table 1) was achieved in situ by applying 151

an aqueous solution (10 % w/w) of NaDCC to test panels (described below). Panels were 152

then washed to remove excess NaDCC salt and dried with a clean cloth. 153

2.3 Preparation of Polyester-based coil coating 154

The topcoat paint employed in these studies was a white, solvent-borne, polyester-155

based system (colour: Surfmist®) incorporating a melamine-formaldehyde cross-linker and 156

formulated for typical coil paint-line application. As a wet paint, this sample was found to be 157

45% w/w resin solids by thermogravimetry (Perkin-Elmer TGA 7). Test samples 5 – 8 (Table 158

1) were laboratory prepared upon pre-primed (commercial chromated polyester primer) test 159

panels of a 0.6 mm thick GALVALUME®-type steel substrate. For each test panel the wet 160

paint was applied using a #28 wire-wound draw-down bar and the panel was transferred to a 161

fan-forced oven set at 262 °C for 55 s. Under these conditions, a 55 s bake cycle enabled the 162

substrate to attain a peak metal temperature of 232 °C, at which temperature the optimum 163

cure conditions for the coating had been attained (>100 double rubs with methylethylketone). 164

2.4 Sample Set 165

The complete sample set for analysis and microbial testing is shown in Table 1. 166

Sample 1 was from a coil paint-line production sample of Surfmist® and sample 2, was the 167

post-chlorinated version of it. Sample 3 was from coil paint-line production samples of 168

PERMAGARD® Steel, a product produced specifically for coolroom applications, with a 169

topcoat paint formulation largely similar to Surfmist® but also containing a commercial 170

antimicrobial additive. Sample 4 was the post-chlorinated version of sample 3. Sample 5 was 171

prepared from the Surfmist® paint, described above, with compound I added at 2% (w/w 172

based upon resin solids), while sample 6 was the post-chlorinated version of this sample. 173

Sample 7 was prepared from the Surfmist® paint with 2% of a mixture of II and III (w/w 174

based upon resin solids), while sample 8 was the post-chlorinated version of 7. 175

176

Sample Number Surface

1 Surfmist® 2 Surfmist® post-chlorinated

3 PERMAGARD® 4 PERMAGARD®post-chlorinated 5 Surfmist® 2% I 6 Surfmist® 2% I post-chlorinated 7 Surfmist® 2% III-Cl 8 Surfmist® 2% III-Cl post-chlorinated

Table 1 Sample set under study 177

2.5 Analysis by liquid extraction surface analysis-mass spectrometry (LESA-MS) 178

Samples with a polyester-based coating as described above, were cut into small 179

sections (25 x 75 mm) using hydraulic shears and placed on the sample stage. Positive ion 180

LESA-MS spectra were acquired using a TriVersa NanoMate® (Advion, New York, USA) 181

accessory coupled to a triple quadrupole QTRAP® 5500 mass spectrometer (AB Sciex, 182

California, USA) with Analyst® 1.5.1 software (AB Sciex, California, USA) used for spectral 183

acquisition. Typical LESA-MS experimental conditions for spray voltage (1.4 kV), delivery 184

gas (0.3 psi) and MeOH:CHCl3 (2:1) with 0.1 % formic acid (v/v) as the solvent system were 185

used throughout. A total volume of 2 µL of this solution was used for each experiment with 1 186

µL being dispensed 0.4 mm above the surface by the automated pipette tip, forming a liquid-187

surface junction and facilitating liquid extraction of analytes. The liquid junction was held in 188

place for 1 s before being aspirated back into the pipette tip. This process was repeated once 189

using the same volume of solvent to maximise analyte concentration in the aspirated solvent 190

prior to analysis. LESA-MS/MS spectra were acquired on the QTRAP 5500 by subjecting 191

mass-filtered ions to collision-induced dissociation (CID). Typical experimental parameters 192

include normalised CID energy (25-30 arbitrary units), declustering potential (100 V), 193

entrance potential (10 V), exit potential (2 V) and scan rate (200 Da/s). Baseline subtraction 194

was not used and mass spectra were averaged over a minimum of fifty scans. All mass 195

spectra were normalised to the most abundant ion in the spectrum. 196

2.6 Testing of coatings formulated with N-halamine under accelerated weathering 197

conditions 198

Artificial weathering was carried out in-house with a Q-SUN Xe-I xenon arc test 199

chamber (Q-Lab Corporation, Westlake, Ohio, USA) that utilises a xenon arc lamp to mimic 200

the full solar spectrum. The coatings were exposed for 1000 h on a continuous cycle of 8 h of 201

xenon arc light (750 mW/m2) at 70 °C; followed by 4 h of darkness at 50 °C. 202

2.7 Antimicrobial testing of coil coatings 203

Cultures of P. aeruginosa (PA01) and Cladosporium sp. were inoculated from 204

streaked plates and grown overnight at 37 °C in Luria (LB10) broth and at room temperature 205

for two days in peptone yeast glucose (PYG) media for PA01 and Cladosporium sp. 206

respectively. To simulate the growth and biofilm formation of microorganisms on pre-painted 207

steel surfaces exposed to external environmental conditions, 300 µL of the overnight culture 208

was inoculated directly onto discs (ca. 3 cm in diameter) punched from a test panel. Disks 209

were seated in a petri dish and incubated for 1 h at room temperature to allow for cell 210

attachment. Subsequently, 10 mL of M9 medium (47.6 mM Na2HPO4, 22 mM KH2PO4, 8.6 211

mM NaCl, 18.6 mM NH4Cl, 2 mM MgSO4, 0.1mM CaCl2, 0.03 mM FeCl2 supplemented 212

with 0.2% glucose) was added to the petri dish to provide a carbon source and nutrients and 213

was immediately tipped out to dry to simulate environmental conditions. At this stage, any 214

unattached cells were washed away. M9 medium was repeatedly added and immediately 215

tipped out every 2 d for 2 weeks. 216

Microbial colonization and biofilm formation was assessed using an Olympus 217

BX51W1 fluorescence microscope. Biofilms formed by both PA01 and Cladosporium sp. on 218

the discs were stained with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine). Five 219

areas were imaged on each disk and were processed using ImageJ software (ImageJ 1.46r, 220

USA) to calculate percentage surface coverage. For protein quantification, the Lowry protein 221

assay was used to determine the total protein amount. A pre-wetted PBS cotton bud was used 222

to swab the coupons to remove biomass, which was resuspended in 250 µL of 3M 223

trichloroacetic acid (TCA). The removal of biomass by swabbing was validated by image 224

analysis of coupons before and after swabbing. The biomass was collected following 225

centrifugation for 10 min at 16,000 x g. Biomass was then resuspended in 0.6 mL of 0.66 M 226

NaOH, and hydrolysed at 80 °C for 20 min. Bovine serum albumin (BSA) was used for 227

generation of the standard protein concentration curve. 228

Data analysis for three independent experiments was performed using single-factor 229

ANOVA followed by post-hoc testing and pairwise comparison using Tukey’s method. 230

231

3 Results and Discussion 232

3.1 Detection of N-chlorinated HALS in surface coatings 233

The direct ionisation technique, liquid extraction surface analysis-mass spectrometry 234

(LESA-MS), has recently been reported for the detection of polymer additives directly from 235

thermoset coil coatings [15]. LESA-MS conducts an automated solvent extraction of analytes 236

from the solid sample by forming a liquid microjunction between the investigated sample and 237

a pipette tip. The microjunction solvent performs the extraction and is aspirated back into the 238

pipette tip where it is subsequently infused into the mass spectrometer using a chip-based 239

nanospray ionisation source [16]. The in situ analyses of the test samples were conducted 240

using positive ion LESA-MS. The coating samples were formulated with the additive I, and 241

also with the crude reaction products following N-chlorination of I (a mixture of I, II, and 242

III). A representative spectrum, Figure 1 (a), shows all three chemical species are present 243

within the coating and are readily detected by this technique, with m/z 241.2 corresponding to 244

I [M+2H]2+, m/z 515.3 to II [M+H]+ and m/z 549.3 to III [M+H]+. Further supporting the 245

identification of II and III, peaks corresponding to the 37Cl isotopes of II and III were 246

observed at m/z 517.3 and 551.3, respectively (Figure 1 (a) inset). 247

To support the structural assignments inferred in Figure 1 (a) a rapid mass 248

spectrometric scanning protocol known as ‘precursor-ion scanning’ was employed. This 249

technique selectively detects all the precursor ions that produce a diagnostic fragment ion. 250

Previous work on the fragmentation of various HALS molecules shows that diagnostic 251

fragments, characteristic of the piperidinyl nitrogen substitution, can be readily detected [19]. 252

Fragmentation of I (>N-H), leads to the appearance of the propan-2-iminium ion at m/z 58. 253

Likewise, if N-methyl substitution (>N-CH3) were present, it would lead to the propan-2-254

methyliminium ion at m/z 72. Thus, in this instance, we can exploit precursor-ion scanning to 255

selectively detect all compounds that fragment to give either the 35Cl or 37Cl isotopologues of 256

the propan-2-chloroiminium product ions, at m/z 92 or 94, respectively. Figure 1 (b) shows a 257

positive-ion LESA precursor ion scan of the coating formulated with the crude reaction 258

products following N-chlorination of I targeting compounds that fragment to give the propan-259

2-chloroiminium product ions at m/z 92. The [M+H]+ ions corresponding to the 35Cl isotope 260

of protonated II (m/z 515.3) and III (m/z 549.3) dominate the spectrum. Concordantly, the 261

[M+H]+ ions corresponding to the 37Cl isotope of protonated II (m/z 517.3) and III (m/z 262

551.3) are readily detected in the precursor-ion scan for compounds that fragment to give 263

product ions at m/z 94 (data not shown). 264

The distribution of the three congeners based on relative ion abundances in Figure 1 265

(a) is similar for both the crude chlorination reaction product in solution (data not shown) and 266

the in situ analysis from the surface coating. The comparable ion distributions suggest that 267

the polymer curing process is not significantly reducing the degree of HALS chlorination 268

through chemical modifications and should therefore pose no problem to the formulation of 269

potentially biocidal coatings. The reaction conditions employed for chlorinated HALS 270

synthesis was tailored to provide incomplete conversion of the precursor to the chlorinated 271

analogues. The relative abundances of the three HALS compounds (I, II, and III) measured in 272

Figure 1(a) cannot be related directly to the quantitative ratio between these products due to 273

possible differences in ionization efficiencies, however their similarity in molecular structure 274

allows us to approximate the ratio between the three compounds I:II:II as being 1:3:1, 275

respectively. 276

277

Figure 1(a) Positive-ion LESA-MS spectrum from the surface of a test panel formulated with the crude reaction 278 products from N-chlorination of I showing, inset, an expansion of the m/z 510 – 560 region, demonstrating 279 isotopic ratios indicative of one and two chlorines (II and III, respectively). (b) The positive-ion LESA 280 precursor-ion scan targeting compounds that fragment to give the propan-2-chloroiminium product ions at m/z 281 92. 282

The use of N-halamines as biocidal additives is an attractive alternative to 283

conventional methods in part because of their ability to be “regenerated” in situ by treatment 284

with chlorinating agents. The application of dilute aqueous solutions of hypochlorite or 285

chlorinated isocyanurates to the surface of polymers containing N-halamines can replenish 286

the consumed chlorine content by re-establishing the >N-Cl functional group in the biocide 287

[17, 18]. To test the ability to generate and regenerate the >N-Cl group in situ for HALS 288

compounds already present in cured, thermoset coil coatings, 1 mL of an aqueous solution of 289

sodium dichloroisocyanurate (10 % w/w) was applied to the surface of a cured, thermoset 290

coil coating formulated with I (2 % w/w of resin solids). Due to the hydrophobic nature of the 291

coil coating surface, a 1 mL droplet of NaDCC solution forms a large contact angle with the 292

surface. Therefore 1 mL of solution was able to treat a localized spot on the surface 1 cm in 293

diameter and this area coverage was maintained until the solvent evaporated under ambient 294

conditions. Once the solution had completely evaporated the coating surface was thoroughly 295

rinsed with water to remove any residual salt. Positive ion LESA-MS analysis was performed 296

on the surface of the coating before and after chlorination. Figure 2(a) shows peaks 297

corresponding to fragmentation of I (m/z 241.2, [M+2H]2+), and chemical impurities 298

associated with I. The peak at m/z 248.2 represents the [M+2H]2+ ion following N-299

methylation of one piperidine group in I and the peak at m/z 342.2 is the [M+H]+ ion 300

following the loss of one piperidine group from I [19, 20]. These impurities are routinely 301

observed as by-products from the additive synthesis and the coil coating process. 302

Ions associated with II (m/z 515.3/517.3) and III (m/z 549.3/551.3) are not observed 303

in Figure 2 (a) but are clearly present in Figure 2 (b), after application of the 304

dichloroisocyanurate solution. This indicates successful in situ chlorination of I present 305

within the coating. Concordantly, the [M+2H]2+ ion associated with unchlorinated I (m/z 306

241.2) is the base peak in Figure 2 (a), while it is significantly reduced in relative abundance 307

post chlorination in Figure 2 (b). 308

309

Figure 2 Positive ion LESA-MS spectra acquired before (a) and after (b) the in situ chlorination of I present 310 within a cured, thermoset coil coating. 311

312

3.2 Accelerated weathering of coil coatings containing N-chlorinated HALS 313

We have shown that 2,2,6,6-tetramethylpiperidine-based HALS can be easily converted to its 314

N-chlorinated counterpart. However, the stability of the N-chlorinated additive when exposed 315

to weathering factors is also relevant to its longevity as an antimicrobial agent. To simulate 316

the typical conditions experienced by coatings in outdoor environments, coatings containing 317

either HALS I or N-chlorinated HALS II/III were subjected to artificial weathering, 318

exposing the coatings to elevated levels of heat and radiation in the UV-Visible spectrum. 319

Figure 3 shows the effect on the parent HALS I present within cured, thermoset coil coatings 320

as a result of accelerated weathering. Figures 3 (a) and (b) are positive ion LESA-MS spectra 321

acquired from a coating formulated with I (i.e., similar to Table 1 sample 5) before and after 322

exposure to 1000 h of artificial weathering, respectively. It is important to note that 323

commercial HALS products are often supplied un-purified (i.e., containing synthetic 324

precursors or by-products such as the compound corresponding to m/z 342.2) as these 325

compounds themselves also act as antioxidants. The difference between Figures 2(a) and 3(a) 326

is somewhat misleading as the absolute amount of compound corresponding to m/z 342.2 327

detected in both spectra is approximately the same. The difference arises from a change in the 328

relative intensities of the peaks corresponding to the singly and doubly charged species of the 329

precursor compound (I). In Figure 2(a) the doubly charged peak dominates, suggesting a 330

greater concentration of protons available for ionization. This may be due to a difference in 331

the coil coating formulation, for example, more acid catalyst being present. Regardless, the 332

comparison of molecular species before and after exposure to artificial weathering conditions 333

shown in Figures 3(a) and (b) occurs on the same sample thus allowing for a controlled, 334

qualitative comparison. The change in ion abundance at m/z 481.2 [M+H]+ and 241.2 335

[M+2H]2+ is indicative of the consumption of HALS as an antioxidant as a result of coating 336

degradation. Positive ion LESA-MS spectra acquired from a coating formulated with I and 337

post-chlorinated (evidently giving mainly II), are shown in Figures 3 (c), before and 3 (d), 338

after, exposure to artificial weathering. A comparison of these two spectra revealed the 339

[M+H]+ ion of II (m/z 515.3) decreased in relative abundance as the non-chlorinated I ions 340

(m/z 481.2 and 241.2) increased. This suggests that coatings formulated with N-chlorinated 341

HALS, if subjected to conditions promoting coating degradation, can revert back to the initial 342

non-chlorinated molecule, I. In this form, the molecule functions as an antioxidant but can, as 343

described above, be modified back to N-chlorinated HALS with the addition of a chlorinating 344

treatment. These results also indicate that II will be almost completely degraded by exposure 345

to typical outdoor conditions. Although it is possible to regenerate II/III via chlorination of I, 346

the rapid degradation experienced indicates that this additive is not suitable for outdoor 347

applications as a long-term low-maintenance antimicrobial solution. 348

349

Figure 3 Positive ion LESA-MS spectra acquired from a sample formulated with I (a) before and (b) after 350 exposure to 1000 h of Q-Sun artificial weathering. Positive ion LESA-MS spectra acquired from a cured, 351 thermoset coil coating formulated with I, then post-chlorinated in situ (c) before and (d) after exposure to 1000 h 352 of Q-Sun artificial weathering. 353

354

3.3 Antimicrobial activity of coatings containing N-chlorinated HALS 355

The antimicrobial activity of the eight samples described in Table 1 were assessed 356

against the simulated growth of a representative bacterial (P. aeruginosa, PA01) and fungal 357

(Cladosporium sp.) species. After two weeks of growth, surface coverage was quantified both 358

by visible surface coverage and a Lowry protein assay. Due to difficulties removing PA01 359

biomass, PA01 quantification was more suited to the visible surface coverage method (viz. 360

Figure 4). After two weeks, surface coverage of PA01 was assessed. Data were analysed 361

using ANOVA with post-hoc testing and pairwise comparison and are presented in Figure 4. 362

363

Figure 4 Level of infestation of PA01 by % visible surface area coverage. Data points shown as means ± SEM. 364 Tukey pairwise comparisons correspond to * p<0.05, ** p<0.005, *** p<0.0005. 365 366

Colony growth was significantly higher on the unmodified control sample, with 10 ± 367

2 % surface coverage. The difference between sample 1 and its corresponding post 368

chlorinated pair was not statistically significant. However, the differences between sample 1 369

and all the other samples (3-8) were statistically significant to varying degrees (*, ** and *** 370

corresponding to P <0.05, 0.005 and 0.0005 respectively). While the samples 3-8 show 371

significant activity compared with the unmodified sample, there was no significant statistical 372

difference between any of the possible pairs or between each parent sample and the 373

corresponding post-chlorinated twin (i.e., 3/4, 5/6, 7/8). 374

The apparent difference between 1 and 2 may indicate the coating surface was able to 375

bind chlorine during post-chlorination and that this process imparts some inhibitory activity. 376

The sites for in situ binder chlorination would most likely be associated with melamine cross-377

linker domains. While this particular formulation is proprietary, in-house experience with 378

these types of coating show that typically, melamine cross-linker levels are between 15% and 379

20% of total resin solids. These levels are able to provide the binder with optimum cross-link 380

density – in terms of yielding coatings with the necessary physical properties (hardness and 381

flexibility) for good post-production manufacturing and installation. 382

The results from pairs 5 and 6 are also worthy of further discussion. Not only did the 383

unmodified HALS appear to have some antibacterial activity (see discussion below), but also 384

post-chlorination of the parent secondary amine, I, shows a similar activity. This observation 385

confirms that when II and III are deactivated they can be regenerated to provide an active 386

chlorinated surface. While the authors are unaware of antimicrobial studies focussed upon 387

antimicrobial activity of this particular HALS molecule, structurally related piperidines are 388

historically important and potent biocides, for example, 2-propylpiperidine is the major toxin 389

in hemlock. Thus, there exist several classes of commercial bactericides, derived from both 390

synthetic [21] and natural [22] sources, where a piperidine or hindered piperidine motif is 391

part of the structure. Not all of these classes have piperidines as the active moiety, but several 392

certainly do. In these cases the piperidines may act either by protonation to quaternary 393

ammonium [23], or by oxidation to a nitroxyl free radical. Recent work suggests that both 394

mono- and di-nitroxyl radicals are not only mildly anti-bacterial [24], but also that simple 395

piperidinyl and indolinyl nitroxyls inhibit biofilm formation in flow systems by swarming 396

wild-type P. aeruginosa PA14 and also cause dispersal of established biofilms [25]. Similar 397

to the comparison between samples 1 and 2, sample 8 was included to investigate the effect 398

of potentially chlorinating components within the coil coating other than the HALS additives, 399

such as the melamine cross-linkers. The comparison between samples 7 and 8 indicates that 400

the majority of biocidal activity comes from the presence of pre-chlorinated HALS but there 401

is a small increase in biocidal activity due to the post-chlorination step. Chlorinated moieties 402

covalently bound to the polymer matrix are unfortunately not amenable to LESA-MS analysis 403

and will comprise a follow up study using alternative techniques. 404

Other bactericides that do not employ the piperidinyl group as an active part of the 405

structure utilise the motif as a docking group for attachment [26] or as an N-linked spacer 406

between active functional groups [21]. 407

Due to the propensity of Cladosporium sp. to grow vertically, image-based surface 408

coverage results were variable (data not shown) and protein quantification was deemed a 409

more suitable quantification method. The levels of Cladosporium sp. were found to be 410

similar, on all samples, at between 40 µg and 50 µg per test disk, and did not vary 411

significantly between the disks, indicating that neither the additives, nor their post-412

chlorinated counterparts yielded coatings with significant inhibitory effects on the level of 413

fungal growth. 414

415

4 Conclusions 416

Using the ambient ionisation technique LESA-MS, products following N-chlorination 417

of the polymer stabiliser I were detected directly from a thermoset polyester-based coil 418

coating substrate. This technique allowed for the rapid detection and characterisation of 419

additives formulated within the polymer without prior extraction, filtration, or sample 420

preparation. LESA-MS also revealed the ability to generate N-chlorinated HALS II/III in situ 421

by treating a coil-coated sample containing I with a dilute solution of sodium 422

dichloroisocyanurate, providing the opportunity to ‘recharge’ the coating. While additives II 423

and III appear to have significant antibacterial activity, the rapid loss of chlorine during the 424

accelerated weathering testing suggests that these compounds are not suitable for exterior 425

(roofing) applications. This work indicates that the halamines may be well-suited to coolroom 426

and other interior surface applications, or cases where bacterial biofilm formation is 427

confirmed as an early stage in the infestation. The ability to ‘recharge’ these coatings would 428

be important in these applications. 429

430

5 Acknowledgements 431

The authors acknowledge funding from the Australian Research Council (ARC) and 432

BlueScope Steel (LP 0775032 and LP110200322). S.J.B. has also been supported by the 433

ARC Centre of Excellence for Free Radical Chemistry and Biotechnology (CE0561607). 434

M.R.L.P. and N.A.P. were supported by Australian Postgraduate Awards. We thank A/Prof. 435

Todd Mitchell and Dr Simon Brown (UOW) for helpful discussions. 436

437

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