New solutions for in-can preservation in the making for Europe

Transcription

1 New solutions for in-can preservation in the making for Europe ABSTRACT Anders Carlsen* and Gerhard Tiedtke*, *Dow Microbial Control, Buchs SG, Switzerland Due to increasing restrictions on many traditional active ingredients used as in-can preservatives (BPD, ecolabels, exposure limitations, PR pressure) the remaining choices decrease. At the same time, the global development towards environmentally more acceptable products in general (VOC regulations, residual monomer restrictions, natural ingredients) results in a higher susceptibility towards microbial spoilage. The few most frequently used active ingredients, however, are limited in efficacy spectrum, speed-of-kill and/or chemical stability. Therefore, it gets increasingly difficult to achieve robust preservation. Dow Microbial Control developed and notified Methyl-BIT, a new active substance, which is an additional building block for powerful in-can preservation. INTRODUCTION Dithio-2,2 -bis-benzmethylamide (DTBMA) is a well known biocide which has been on the market for over 30 years. It was recommended for both wet state preservation applications (in-can) and dry-film preservation alike due to its broad spectrum of effectiveness versus moulds, yeasts and bacteria. It has long been known that DTBMA hydrolyses in alkaline media to N-methylbenzisothiazolinone (see Fig 1). More recently 2-Methyl-1, 2-Benzisothiazol-3(2H)-one (Methyl-BIT or simply MBIT, CAS N ) has come to be recognised as the biocidal active component in DTBMA and a mircobicide in its own right. Many waterborne systems including paints are susceptible to microbiological contamination and spoilage and require the inclusion of an in-can preservative to provide protection during manufacture and an appropriate shelf life [1]. Biocidal active ingredients (ai s) possess a range of both physicochemical and biological properties which make them more or less suitable for certain formulation types and manufacturing processes [2]. The selection of an appropriate in-can preservative is an important factor in the development of a paint formulation and failure to prevent spoilage due to microbiological growth can result in the development of foul odours, discolouration, loss of structure and the generation of gasses that might distort / damage the final packaging. The protection provided includes the interval during manufacture as well as storage both within the plant and prior to sale. The protection should be sufficient to provide a shelf life suitable for the product and may be extended to allow storage of partused containers by the end user.

2 Fig 1. MBIT is formed by hydrolysis of 2,2'-dithiobis-N-methylbenzamide (DTBMA). MBIT has much higher water solubility than DTBMA. This is not a trivial thing, since MBIT does not form salts. When DTBMA is formulated in a solvent together with surfactants, such formulations are generally in the form of emulsive concentrates, emulsions, microemulsive concentrates, or microemulsions. Emulsive concentrates form emulsions upon the addition of a sufficient amount of water. Microemulsive concentrates form microemulsions upon the addition of a sufficient amount of water. The DTBMA component having low water solubility also can be formulated in the form of a dispersion. The solvent component of the dispersion can be an organic solvent but is preferably water. For formulations with DTBMA to be stable they have to be either emulsions or dispersions, whereas MBIT can be formulated as clear liquid. Liquid formulations of MBIT have lower viscosity than emulsions/dispersions allowing dosing through pipes without the need to add solvents; making liquid formulations with MBIT ideal for use in low VOC paint / latex etc. In Table 1 are shown conversion rates for DTBMA to MBIT in different systems. In all systems where water is present and available the conversion begins very fast. In this experiment only DTBMA (~200ppm) was added and within few hours (0 days) about 10-15% of DTBMA is converted to MBIT. Slowest is the conversion in 100% DMSO, exemplifying the need for presence of water for the conversion to take place. The rapid conversion of DTMBA to MBIT together with the much higher water solubility of MBIT than DTBMA demonstrates that MBIT is the actual biocidal active component in DTBMA.

3 0 days 1 day 3/4 days 14 days DTBMA MBIT DTBMA MBIT DTBMA MBIT DTBMA MBIT Masonry Paint based on acrylic binder 166 ph styrene-acrylic emulsion ph Styrene-acrylic paint ph Binder (Vinyl-acetate/ester emulsion) ph Pure water % water, 10% Tetrahydrofuran % DMSO NM NM Table 1. Conversion rates for DTBMA to MBIT in different systems. Concentrations are in ppm. NM = not measured. The isothiazolones are structurally quite similar, the tendency being that the more traditional bactericides have shorter chains and the fungicides have longer chains (see Fig 2). So based alone on the structure one might guess that MBIT (Methyl-BIT) be a better fungicide than BIT and a better bactericide than BBIT (Bu-BIT). Indeed several studies have been carried out which show that MBIT is both an effective bactericide and fungicide. Fig 2. Structural similarities between isothiazolone based bactericides and fungicides. On the right are the longer chained: DCOIT, OIT, and BBIT (Bu-BIT) which are known as fungicides with a modest efficacy against bacteria. On the left side, the typical short/no chained isothiazolone bactericides: MIT, BIT, and the broadband CMIT (effective against bacteria and fungi alike). MBIT (Methyl-BIT) is structurally very similar to both the bactericide BIT and the fungicide BBIT, which could explain why it works better versus fungi than BIT and better versus bacteria than BBIT. BIT can be de-protonized (pk b ~8.5) and the de-protonized form of the molecule has much lower efficacy as a bactericide. MBIT does not de-protonize and so does not form salts. The consequence of this is that MBIT as a molecule can be considered a more potent biocide than BIT at alkaline ph.

4 BIT is lipophilic with a low solubility in water (< 0.1%) whereas MBIT is more hydrophilic with a higher solubility in water (~2%). This makes MBIT rather more available in the aqueous phase than BIT which is essential for contact with microorganisms. EXPERIMENTAL Minimum inhibitory concentration (MIC) is the most common method to determine the intrinsic antimicrobial of a biocide. The MIC test is used to determine the lowest level of biocide that inhibits or prevents the growth of microorganisms under controlled laboratory conditions. It is applicable to bacteria, algae, mould and yeasts, and other organisms and may be adapted to various sample sizes, matrices or test containers. MIC tests against representative bacteria and fungi were conducted using a high resolution MIC method in 96-well microtiter plates (405 μl final volume). Varying amounts of stock solutions of MBIT in DMSO solvent were added automatically to the first to the twelfth column of wells in amounts typically 5-20% of the total volume. The biocide was then serially diluted ten-fold in the growth media from column to column to get a series of closely spaced endpoints. The amount of biocide in the plate can be altered by changing the concentration of the biocide stock solution Cultures of microorganisms were added automatically to all wells in amounts typically 9 % of the final volume. All media and transfers used sterile supplies and aseptic techniques. Controls included samples with no biocide added to growth media (columns 5 and 12). The injection of the various reagents and subsequent inoculation with the strain studied onto the microtiter plate was performed by the Biomek 1000 automated workstation according to an appropriate program. All media and transfers used sterile supplies and aseptic techniques. Controls included samples with no biocide added to growth media. MIC values were determined after 24 hours, 3, and/or 5 days incubation period at 30 C ± 2 C for bacteria and 2, 3, and/or 7 days at 25 C± 2 C for fungi. The MIC value was visually determined as the lowest concentration where no visible growth (turbidity, or mycelia development on the surface of the broth) was observed. Values are reported as the mean of replicate samples. Summary analyses of MIC values and minimum lethal concentrations (MLC) results for MBIT alone are reported in Table 2. It can be observed that the MLC and the MIC values are quite similar and in some cases the reported MLC is slightly higher than the MIC. This is believed to be due to experimental differences like using different growth medium, since the two values were determined by different experiments. Bacteria strain used T+ 24 hours T+ 120 hours (5 days) MIC (MBIT) MLC (MBIT) - Escherichia coli ATCC # ± ± Klebsiella pneumoniae ATCC # ± ± Enterobacter gergoviae ATCC # ± ± Burkholderia cepacia ATCC # ± ± Fungal strain used T+ 168 hours (7 days) MIC (MBIT) MLC (MBIT) - Penicillium ochrochloron ATCC # ± Geotrichum candidum ATCC # ± Rhodotorula rubra ATCC # ± Table 2. Mould/Yeast results. Minimal inhibitory concentration (MIC), Minimal Lethal Concentration (MLC). Concentrations in ppm. Medium used for MIC: M9G + 0.1% Yeast extract. ph 5.0; Medium used for MLC: Sabouraud Agar media. In some cases, commercial biocides cannot provide effective control of microorganisms, even at high use concentrations, due to weak activity against certain types of microorganisms, e.g., those tolerant or resistant to some biocides. Combinations of different biocides are sometimes used to provide overall control of microorganisms in a particular end-use environment. For example, a combination of 2- methylisothiazolin-3-one and 1,2-benzisothiazolin-3-one is disclosed in U.S. Pat. No. 6,361,788. However, there is a need for additional combinations of biocides having enhanced activity against various strains of microorganisms to provide effective control of the microorganisms that is both quick and long lasting.

5 The problem is here addressed to provide such additional combinations of biocides, looking at synergistic effect of MBIT in combination with MIT. The synergism of the combinations was demonstrated by testing a wide range of concentrations and ratios of the compounds. Synergism was determined by an industrially accepted method [3] using the ratio determined by the formula: Qa/QA + Qb/QB = Synergy Index ("SI") Wherein: QA = concentration of compound A (first component) in ppm, acting alone, which produced an end point (MIC of Compound A). Qa = concentration of compound A in ppm, in the mixture, which produced an end point. QB = concentration of compound B (second component) in ppm, acting alone, which produced an end point (MIC of Compound B). Qb = concentration of compound B in ppm, in the mixture, which produced an end point. When the sum of Qa/QA and Qb/QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated, and when less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. The minimum inhibitory concentration (MIC) of a biocide is the lowest concentration tested under a specific set of conditions that prevents the growth of added microorganisms. Synergy tests were conducted using standard microtiter plate assays with media designed for optimal growth of the test microorganism. Minimal salt medium supplemented with 0.2% glucose and 0.1% yeast extract (M9GY medium) was used for testing bacteria; Potato Dextrose Broth (PDB medium) was used for testing yeast and mold. In this method, a wide range of combinations of biocides was tested by conducting high resolution MIC assays in the presence of various concentrations of MIT. High resolution MICs were determined by adding varying amounts of biocide to one column of a microtitre plate and doing subsequent ten-fold dilutions using an automated liquid handling system to obtain a series of endpoints ranging from 2 ppm to ppm active ingredient. P. aeruginosa (ATCC #15442) in M9GY MIC at 72 hours S. aureus (ATCC #6538) in M9GY MIC at 24 hours MIC (MIT) MIC (MBIT) SI MIC (MIT) MIC (MBIT) SI C. albicans (ATCC #10231) in PDB MIC at 48 hours A. niger (ATCC #16404) in PDB MIC at 72 hours MIC (MIT) MIC (MBIT) SI MIC (MIT) MIC (MBIT) SI Table 3. MIC values for MIT and MBIT versus bacteria and fungi individually. The synergy of combinations of MIT and MBIT was determined against two bacteria, Staphylococcus aureus (S. aureus ATCC #6538) or Pseudomonas aeruginosa (P. aeruginosa ATCC #15442), a yeast, Candida albicans (C. albicans ATCC 10231), and a mold, Aspergillus niger (A. niger ATCC 16404).

6 The bacteria were used at a concentration of about bacteria per ml and the yeast and mold at fungal spores per ml. These microorganisms are representative of natural contaminants in many consumer and industrial applications. The plates were visually evaluated for microbial growth (turbidity) to determine the MIC after various incubation times at 25 C (yeast and mould) or 30 C (bacteria). The results are shown in Table 3 as MIC for MIT alone, MBIT alone and MIC for different concentrations of MIT and MBIT in combination. These results demonstrate synergy at least for ratios of MIT:MBIT ranging from 1:6 (10 ppm MIT:60 ppm MBIT vs. P. aeruginosa) to 750:1 (150 ppm MIT:0.2 ppm MBIT vs. C. albicans). The susceptibility of a coating formulation to microbiological spoilage and the potential efficacy of an in-can preservation system are usually determined using a microbiological challenge test [4]. A relatively limited number of standard test protocols have been developed over the last few decades and some operators have tried to employ methods based on those described in the various pharmacopoeia for cosmetics and pharmaceutical products although these have been found to be far from satisfactory. The International Biodeterioration Research Group (IBRG) has been developing a test protocol for testing the in-can preservation of paints and varnishes [5]. Although still under development, it is the most common method employed by workers in the field although in many cases some modification is made to the method described. The method uses a consortium of microorganisms, which have been demonstrated to grow in water-based paints, to challenge a paint formulation on a number of occasions. This study reports the results of several studies on the efficacy of MBIT either alone or in combination with one other mircobicide as an in-can preservative for water based paints using a challenge test based on the current version of the IBRG in-can test method. Five repeat challenges were employed in the study to simulate the introduction of a bioburden at different stages of a products life such as to provide a balance between the removal of populations introduced during manufacture and use and preventing subsequent growth in the formulation; the intention was to simulate preservation rather than disinfection of a spoiled product. 50 g aliquots of the test systems were treated with the biocidal active substances and incubated at room temperature (approximately 22 C). These test samples were inoculated on the following day and in weekly intervals with one ml of the mixed inoculum suspension (see below). Seven days after each inoculation, the surviving microorganisms were evaluated by the number of colony forming units (CFU) present in each sample by streaking 100 µl aliquots onto Trypticase Soy Agar and Malt Extract Agar and the plates were incubated for 3 to 7 days (at 30 C ±2 C and 25 C ±2 C respectively) prior to counting. Table 4 shows the scale used for rating microbial growth. Rating 0 = 0 cfu / plate no detectable growth 1 = cfu /plate slight growth 2 = cfu / plate moderate growth 3 = > 1000 cfu / plate heavy growth Table 4. Growth rating scale, used for assessing microbial growth for in-can challenge tests; cfu = colony forming units. The bacterial inoculum was prepared from 4 day old cultures in Trypticase Soy Broth; the fungal inoculum was prepared by washing 7 days old cultures on Malt Extract Agar with sterile saline solution. All microorganisms were finally mixed to a single inoculum suspension containing about 10 9 cfu/ml of bacteria and 10 7 cfu/ml of fungal spores. The inoculum was added into the test systems within 2 hours after preparation. The aerobic bacterial pool consisted of: Burkholderia cepacia ATCC 25416, Escherichia coli ATCC #8739, Enterobacter gergoviae ATCC #33028, Klebsiella pneumoniae ATCC #10031, Proteus vulgaris ATCC #13315, Pseudomonas aerugionsa ATCC #15442, Staphylococcus aureus ATCC #6538. The fungal pool consisted of: Aspergillus niger ATCC #16404, Candida albicans ATCC #10231, Geotrichum candidum ATCC #12784, Penicillium ochrochloron ATCC #9112, Rhodotorula rubra ATCC #9449. Results are reported in Table 5 for efficacy of MBIT alone in acrylic paint. Unpreserved paint proved susceptible to bacterial and fungal attack. In acrylic paint samples (ph 8.6), ppm active ingre-

7 dient of MBIT protects against microbial attack during 2 weekly inoculations. Samples preserved with 150 to 200 ppm a.i MBIT resists against 4 successive inoculations. MBIT (ppm) 1 th. inoc. 2 nd. inoc. 3 rd. inoc. 4 th. inoc. 5 th. inoc. B F B F B F B F B F Unpreserved Table 5. Challenge test of MBIT in an acrylic paint formulation (ph 8.6). B = Bacteriocidal activity; F = Fungicidal activity. Fifty grams of a styrene acrylate binder (ph 8-8.5) were inoculated repeatedly following the described method but with rising concentrations of inoculum. This way the 4 th challenge uses 3-fold concentration inoculum and the 5 th uses 10-fold concentration. This treatment is carried out once a week and a total of five inoculation cycles are carried out. The results are presented in Table 6 and the rating scale for evaluation of microbial growth is shown in Table 4. ppm AI MIT BIT BNPD OIT DTBMA Inoculations / ratings 1 inoc. 2 inoc. 3 inoc. 4 inoc. 5 inoc. * * ** # Table 6. Challenge test of a styrene acrylate binder, ph using a mixed inucolum of bacteria, moulds, and yeast. The ratings given are totals for the combined inoculum. *) inoculated with 3-fold concentration; **) inoculated with 10-fold concentration; #) number of successive challenges to pass. MIT (145 ppm) withstands the 4 th inoculation whereas 97 ppm MIT does not; same does 97 ppm MIT ppm bronopol (BNPD), 97 ppm MIT + 20 ppm OIT, 100 ppm MIT ppm BIT, and 25 ppm MIT 18.8 ppm DTBMA.

8 Only three of the presented combinations to withstand all 5 challenges including the 5 th super challenge are: MIT (145 ppm) + OIT (75 ppm), MIT (50 ppm) + DTBMA (37.5 ppm), and MIT (100 ppm) + DTBMA (75 ppm) the later being of no surprise since half the concentration also passes all 5 challenges. The results in Table 6 show how well the combination of 50 ppm MIT and 37.5 ppm MBIT is preserving a material in wet state in this case a styrene acrylate binder; ph By comparison 150 to 200 ppm MBIT alone withstood 4 successive inoculations in an acrylic paint formulation; ph 8.6 (Table 5). So MBIT is a effective biocide in its own right, and furthermore works very well in a synergistic combination with other actives in this case MIT. SUMMARY MBIT is the active component in DTBMA, and being able to formulate using MBIT rather than DTMBA holds a number of advantages. Being soluble in water MBIT can be formulated as stable, flow able clear liquid rather than a more viscous dispersion. MBIT is an effective bactericide and fungicide for wet state preservation applications as the structural similarities between isothiazolone based bactericides and fungicides certainly hint at. Due to differences in water solubility MBIT is more bio-available in the aqueous phase than BIT which is critical for contact with microorganisms. At alkaline ph MBIT can be considered a more potent biocide than BIT for a different reason; since the presence of the methyl group effectively prevents the salt formation of BIT. We have carried out many experiments demonstrating that MBIT is indeed effective both as a bactericide and a fungicide against several common spoilage organisms as a single active. And moreover demonstrated true synergism between MBIT and other actives, for which data have here been presented for MBIT/DTBMA and MIT. REFERENCES 1. Downey, A (1995), The Use of Biocides in Paint Preservation, Handbook of Biocide and Preservative Use, Ed. Rossmore H W, Blackie Academic and Professional, London. 2. Paulus, W (2005) Directory of Microbicides for the Protection of Materials: A Handbook, Kluwer Academic Publishers 3. Kull, F C, Eisman, P C, Sylwestrowicz, H D and Mayer, R L, in Applied Microbiology 9: (1961) 4. Askew P D (2006) Coatings and Coatings Materials in Springer Handbook of Materials Measurement Methods Eds Czichos H, Saito T, Smith L, Springer Science and Business Media 5. Lunenberg-Duindam J, Lindner W (2000). In-Can Preservation. European Coating Journal. 03,