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European Coatings Journal
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A challenge for biocides
Achieving long-term protection for colorants inpoint-of-sale dispensers.Point-of-sale tinting systems pose difficult challenges forbiocides, since they are open to air and very rarely cleanedout. Tests for biocide efficiency were carried out on asolvent-free tinter and it was confirmed that present additivepackages can control the growth of microorganisms intinting machines, but this environment is much moredemanding than in-can preservation.Elina Kähkönen*, Mervi Suominen, Katrina Nordström.The move from traditional propylene glycol-based colorantsto VOC-free technology, already used for more than adecade in Scandinavia, is now well under way in manyregions in Europe, with some areas in the Americas andAsia following. This change is driven partly by EU directive2004/42/EC, limiting the VOC emissions of decorativepaints, but mainly by the eco-labels used in paint marketing.The market for VOC-free colorants is growing in regions withwarmer climates, and higher temperatures both shorten theshelf-life of biocides and create a better growth environmentfor certain microbes that are known to contaminate paints.Universal colorants are used in many ways, in different painttechnologies and applications. The tightest criteria for thecomposition of VOC-free colorants in Europe are defined ineco-labels set by consumer organisations and by certainmarket requirements. Biocides are strictly regulated underall eco-labels. The criteria are updated frequently andmanufacturers must adjust colorant compositionaccordingly.To do this successfully, they need to know what is requiredto preserve the colorants during their shelf-life and use. Thisstudy examined different methods for evaluating andcomparing biocide efficacy in VOC-free colorants.
POS dispensers are vulnerable to microbial growthPoint-of-sale (POS) tinting is an application with specialrequirements for biocides in VOC-free colorants. Thesecolorants consist of water and polymers, combined withorganic and inorganic materials such as pigments and fillers,and their pH is neutral. The dispensers are 'open systems',where canisters are refilled without ever being emptied andcleaned.Spilt colorant forms semi-dry and dry films on the canisterwalls. The protection required in the canister itself and thedispensing circuit as a whole is different from in-canpreservation. Colorants are typically used at roomtemperature, but in hot climates the temperature can exceed30°C during the night when the air conditioning is off. Thestirring or circulation process supplies oxygen, whilehumidity causes water to condense on the lid and walls ofthe canister.The biocides used to preserve colorants are similar to thoseused to protect paints. From the hygiene point of view, themost critical limits set by the criteria concerning thecomposition of VOC-free colorants are those for VOCcontent, biocide chemistries and quantities:- VOC, < 700 ppm in Blue Angel (blauer Engel) eco label [1]- Isothiazolines (CIT, MIT, OIT and BIT)* < 500 ppm in EUEcolabel [2]- Specific isothiazolines (CIT and MIT) < 15 ppm in EUEcolabel [2]- Formaldehyde: not used in Scandinavia; classified as acarcinogen (particularly undesirable in interior paints)- Isothiazoline OIT, prohibition considered in interior paintsin Blue Angel during study
* CIT = 5-chloro-2-methyl-4-isothiazolin-3-oneMIT = 2-methyl-4-isothiazolin-3-oneOIT = 2-n-octyl-4-isothiazolin-3-oneBIT = 1,2-benzoisothazolin-3-oneRaw materials used in VOC-free colorants need to becarefully selected and controlled. They must not containresiduals classified as VOCs and each must meet hygienerequirements.The quality of the process and cleaning water must also bespecified and controlled. Every batch of VOC-free colorant issubjected to microbial quality control to ensure that it isclean. Once the colorant is poured into a dispenser canister,it comes into contact with the microbes in the environment.Biocide protection affects the shelf-life of colorants, so itsinfluence and the longevity of its preservative effect werestudied in the dispenser.The throughput of the colorant throughout the POS colorantdelivery and usage chain (Figure 1) influences the age ofthe colorant inside the canister. Colorant consumption issteadier when the dispensing system is carefully designedto meet the customer's colour area needs. Choosing adispenser size that is appropriate for usage requirementsalso maintains the freshness of the colorant in the canister.
Experimental
Microbial strains and test methodsCPS Color Oy produced the colorants used in the tests,based on an existing production formula for a VOC-freemagenta colorant containing pigment, filler, water,dispersing agents and rheology modifiers. The differentbiocidal compositions are shown in Table 1. The selection ofmicrobial strains used in the test took into account thecommonly identified causes of biodeterioration in paints andcoatings (Table 2). They also represent different types ofmicrobes.The Pseudomonas species are the most common cause ofpaint spoilage [3]. They are Gram-negative, strictly aerobicbacteria with an optimum growth temperature range of25-30°C [4]. Bacillus species are spore-forming,Gram-positive bacteria with optimum growth at 25-40°C [5].The Alcaligenes species are Gram-negative and areparticularly resistant to a number of chemicals, includingdisinfectants and biocides [6].The safety of the strains was a concern, due to the massiveinoculations used in the dispenser test. Geotrichum waschosen to represent fungi primarily for safety reasons. It is afilament-forming, yeast-like fungus, and is anotherwell-known contaminant in paints [7].The strains were stored on agar slopes at 4°C. Thebacterium inoculum was cultivated by suspending a culturetaken by a slope in 20 ml of nutrient medium ("Nutrient BrothNo.2" LAB MTM, U.K.). The suspension was incubated for 2days. In the case of the fungi, 3 ml of 0.9% NaCl solutionwas added on a G. candidum agar slant and the culture wassuspended in liquid. 10 ml of the suspension obtained wasmixed with 20 ml of each incubated bacteria suspension.This inoculation mix was used in challenge tests.
Dispenser tests simulate normal operationIn challenge tests, the biocide protection is repeatedlychallenged by inoculation. In these tests re-inoculation wascarried out six times, once a week. Samples were takenbefore each new inoculation. The 0 sample was also studiedtwo days after inoculation.The contamination was studied on malt extract and nutrient
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agar plate cultivations after 2 days' incubation. Tests werecontinued for 6 weeks with the exception of the 0-tests,which were ended after the first week due to heavycontamination. The dispenser circuit was disinfected withhot water and ethanol prior to testing. Adequate microbialcleanliness was verified by testing rinsing water dispensedon agar plates. Test colorants (1.7 l) were added to thecanister and the full dispensing circuit of the dispenser(Figure 2). Inoculation (3 ml of suspension) was carried outvia a hole in the lid (e in Figure 2) and the samples weredispensed from the nozzle (d in Figure 2).In each sampling, 20 ml of colorant was dispensed beforetaking the actual sample to ensure each cultivated samplewas representative. The colorant was pumped through thecircuit regularly (5 cycle/h). The ambient temperature wasmaintained at 26°-34°C and the humidity was increasedduring the daytime using moisturising equipment.
In-can and agar testsFor the can tests, 50g of the colorant was incubated in canswith their lids on at 30°C, while the stirring speed wasmaintained at 50 rpm. The frequency of inoculation andsampling was the same as in the dispenser.Two further analyses were also made. In one, quantities ofthe individual isothiazolines and the biocide2-bromo-2-nitropropane-1,3-diol ("Bronopol") in the sampleswere analysed using high-pressure liquid chromatography(HPLC). The CIT and "Bronopol" analyses are shown inFigure 4. Thor Specialities (Sweden) provided theseanalyses.The other involved an agar diffusion assay. 100 µl ofinoculums (containing 107-109 cfu (colony forming units)/ml)of each strain were cultivated separately on agar plates. Amaximum of 4 test discs ("Antibiotic Assay Disc", Whatman,)were placed on a plate and three drops of colorant (1.3-1.5g) were dropped by pipette onto the disks. The halo aroundeach disk was measured and the average size of the halosformed on different plates was calculated.
Can and dispenser results differ as expectedThe agar diffusion assay was tested to find an indicativemethod for a quick comparison of biocide efficacy incolorant. The efficacy results from the challenge test and theagar diffusion test were then compared to the quantitativeresults of the HPLC analysis.Before the dispenser was cleaned for the actual challengetest, the colorants inside the dispenser were examined. Thedispenser had been in normal retail use since 1998. Theage of the VOC-free colorants inside it was not known.Cultivations on malt and nutrient agar were made from boththe dispensed colorant and the semi-dry colorant film on thecanister walls. No growth was detected.The dispensing circuits, which contained heavilycontaminated 0-sample colorants, were cleaned after thechallenge test according to instructions given by CPS Color.The rinsing water samples, cultivated on malt and nutrientagar plates, were found to be clean.Table 3 sets out the contamination found during the testperiod. Apart from the 0-sample, the in-can test producedonly two random contaminations.In the dispenser, samples 2, 5 and 6, with reduced biocidelevels, became contaminated. The normal biocides survivedall 6 inoculations, even when 2 weeks' ageing at atemperature of 40°C was used. The expected logical trendwas for the contamination to continually increase, butvariations were found. Nevertheless, the frequentoccurrence of contamination indicated a decrease in biocideefficacy. The viable cells detected in the dispensed sampleshad been exposed to the biocides in the colorants as they
passed through the dispenser circuit.
Species differences observed in different locationsThe 0-samples became contaminated immediately at thefirst inoculation. Differences were observed between thespecies that had grown. P. fluorescens prevailed in thedispenser while A. faecalis was predominant in cans. Thisalso indicates that the conditions in the cans and in thedispenser were different. These differences could be relatedto the oxygen requirement of the species, as P. fluorescensis strictly aerobic, while A. faecalis is facultatively aerobic.This result then suggests that there is a greater supply ofoxygen in the dispenser.The results of the agar diffusion assay and HPLCmeasurements are presented in Figure 3 and 4. The knowndifferences between the protection levels at the beginning ofthe challenge test could be clearly seen in the halos. Therewas an evident difference between the full amount ofbiocides and 25% of the full amount.Due to the small number of species examined in this pilotstudy, it was not possible to directly compare the averagevalues of the measured halos between the different types ofprotection. The observable influence of the fungicide willchange according to the ratio of bacterial/fungal species inthe test set up.The decreasing quantity and efficacy of the biocide throughthe course of the inoculation cycles was examined usingagar diffusion assays and HPLC measurements of biocidelevels. The results obtained show a similar tendency. Thevalues are only indicative in all the results due to the smallnumber of parallel samples (two in agar diffusion and one inHPLC).
Main features of biocidal protectionThe biocide protection in the colorant remained activefollowing all 6 inoculations at raised temperature andhumidity. A typical ageing duration of two weeks at 40°Chad no effect.Steady colorant consumption under recommendedconditions is the basis for a good hygiene status, as shownby the fact that no contamination was found in the liquid andsemi-dry colorants in the dispenser, which had been innormal retail use for seven years.Dry film fungicides are an essential component of biocideprotection in VOC-free colorants. 95% of in-can preservationalone gave similar results to 25% of the normal biocides inboth the agar diffusion assay and in-dispenser challengetests. With both these types of protection, indications ofcontamination were found from the third week onwards.Though the in-can test showed contamination only in the0-samples, contamination was found in the dispensersamples with reduced quantities of biocide. The differencesin the species growing in the 0-sample could be due to adifference in oxygen supply or in moisture content in the testsystems. The cans contained only a small quantity ofcolorant, which appeared to dry during the test. It was alsomore difficult to mix the inoculum into the colorant.The agar diffusion test results were in line with both thein-dispenser challenge tests and with the HPLC analysis.The agar diffusion test could be further developed as anindicative method for a quick comparison of biocideefficacies in colorants.
ACKNOWLEDGEMENTSThe paper is based on Mervi Suominen's Master's Thesis[8]. The study was conducted at CPS Color Oy in closeco-operation with the Laboratory of Biochemistry andMicrobiology at Helsinki University of Technology, withstrong support from Thor Specialities (U.K. and Sweden),
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Rohm & Haas biocides (France), and Tikkurila Oy. Tekes(Finnish Funding Agency for Technology and Innovation)provided funding. Special thanks are due to colleagues inthe International Biodeterioration Research Group for theirinterest, comments and feedback.
REFERENCES[1] EU Eco label decision 2002/739/EC (to be updated 2007)[2] Blaue Engel (Germany) RAL-UZ 12a Varnishes (Feb2006), RAL-UZ 102 Wall Paints (Sep 2003)[3] P. S. Kappock, Biocides: Wet State and Dry Film, inHandbook of Coatings Additives, Eds. J. J. Florio, D. J.Miller, New York 2004, 261-295[4] M. T. Madigan, J. M. Martinko, J. Parker, Brock biologyof microorganisms, 10th edition, New Jersey 2003[5] Bergey's manual of systematic bacteriology 2, Baltimore1986, 1105-1139[6] Bergey's manual of systematic bacteriology 1, Baltimore1984, 361-367[7] J. M. Jay, M. J. Loessner, D. A. Golden, Modern FoodMicrobiology, 7th edition, New York 1992[8] M. Suominen, Development of biocide efficacydetermination methods for colorants - a Master's thesis (InFinnish), Espoo 2006
Results at a glance- Universal colorants are being increasingly formulated assolvent-free products, in response to eco-label requirementsand VOC restrictions.- However, point-of-sale (POS) tinting systems using thesecolorants pose challenges for biocides, as the systems areopen to the air and are topped up without being cleaned.- Microbiological challenge tests were carried out oncolorants having different biocide levels, both stored in cansand recirculated through a POS dispenser over a period ofsix weeks- Few examples of biological contamination were found inthe cans, even when the biocide level was greatly reduced,but only near-normal levels of biocide provided adequateprotection in the POS dispenser tests.- An agar diffusion test examined here appears to be apromising way to carry out simple and rapid indicative teststo compare biocide effectiveness.
The authors:-> Elina Kähkönen started her caree at CPS Color in 1997and has worked there in various positions. Presently she isstudying for a PhD at the Helsinki University of Technologyat the laboratory of Biochemistry and Microbiology.-> Katrina Nordström is Professor of Microbiology at theHelsinki University of Technology. She received her B.Sc.(Hons.) from the University of Surrey, UK, in 1979, and herPh.D. from the University of London, UK, in 1983.-> Mervi Suominen completed her Master's thesis for CPSColor Oy at Helsinki University of Technology in 2006.Currently, she is working as a research microbiologist atTikkurila Oy.* Corresponding Author. Contact: Elina Kähkönen, CPSColor Oy, Vernissakatu 1, 01301 Ventaa, Finland, Tel. +358207 188 075, Fax +358 207 188 021
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Figure 1: Colorant delivery and usage chain.
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Figure 2: Structure of the dispenser circuit in contact with the colorant: a) Canister(volume 4 l); b) Gear pump; c) Valve, which directs colorant to dispensing nozzle or to
recirculation; d) Dispensing nozzle; e) Lid with a hole for inoculation addition.
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Figure 3: Agar diffusion assay with G. candidum clearly shows the influence of dry filmfungicides. The average values of halos on different samples are shown in Figure 4..
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Figure 4: a) Size of agar diffusion inhibition halos (diameter of zone inhibition); b) CITlevels during challenge test; c) Bronopol levels during challenge test.
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