Abstract:
The present invention relates to an antimicrobial composition comprising an isothiazolinone, such as 1,2-benzisothiazolin-3-one, and a zinc compound selected from zinc salts, zinc oxides, zinc hydroxides or combinations thereof. Useful zinc salts include for example, oxides, sulfates, chlorides, and combinations thereof. In use, the zinc from the zinc compound enhances the antimicrobial activity to the isothiazolin-containing composition. This enhancement permits achieving the desired antimicrobial activity at a lower usage rate than is achieved using the isothiazolinone in the absence of the zinc compound. The antimicrobial composition can also contain co-biocides, such as pyrithiones, including zinc pyrithione or copper pyrithione.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a division of U.S. application Ser. No. 11/805,779, filed on May 24, 2007; PCT application PCT/US07/12518 filed on May 25, 2007; and provisional application 60/808,697 filed on May 26, 2006 the entire contents of each of which are hereby incorporated in total by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Isothiazolinones, such as 1,2-benzisothiazolin-3-one (also referred to as “BIT”), are know to be effective antimicrobials. BIT, as disclosed for example in formulations described in European patent EP 0 703 726, is widely used as a bactericide in a variety of applications. Illustratively, British patent publication GB2,230,190A discloses a preservative composition containing BIT and an adduct of zinc chloride (“ZC”), together with 2,2′-dithiopyridine-1,1′-dioxide (“DTP”). Example 3 of &#39;190A publication compares compositions containing BIT plus ZC plus DTP against a control example containing only BIT plus ZC. The results given in Example 3 and Table 3 of the publication indicate that inclusion of DTP allows decreased usage of BIT in the composition. Nonetheless, the amount of BIT (2.5 ppm) employed in the control example has been found by the present inventors to be insufficient for antimicrobial efficacy in real-world applications. 
     While BIT has proven useful in a wide range of applications, the useful amount of BIT that can be added to a commercial product is limited by efficacy and economic considerations and to a lesser extent by environmental and toxicological concerns. Accordingly, alternative antimicrobial compositions are needed for wet state applications that are cost-effective and minimize the likelihood of adverse environmental and toxicological effects. The present invention provides one such alternative. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present invention relates to an antimicrobial composition comprising (a) at least one isothiazolin-3-one and (b) at least one zinc compound selected from the group consisting of zinc salts, zinc oxides, zinc hydroxides, and combinations thereof. Illustrative zinc salts are zinc chloride, zinc sulfide, zinc carbonate, basic zinc carbonate, and combinations thereof. The zinc compound provides a source of metal ion in the antimicrobial composition. In the composition, the isothiazolin-3-one is present in an amount of from 1 to 500 ppm (preferably from 5 to 500 ppm), the zinc compound is present in an amount from 5 to 200,000 ppm (preferably from 5 to 500 ppm), and the weight ratio of the isothiazolin-3-one to the zinc compound is from 1:2000 to 100:1 (preferably from 1:100 to 100:1). Optionally, the antimicrobial composition additionally comprises a pyrithione salt or pyrithione acid. Useful pyrithione salts include copper pyrithione, zinc pyrithione, sodium pyrithione, pyrithione disulfide, and combinations thereof. 
     In another aspect, the present invention relates to an antimicrobial composition concentrate that, upon dilution with water, provides antimicrobial efficacy in a functional fluid. The concentrate comprises (a) at least one isothiazolin-3-one and (b) at least one zinc compound selected from the group consisting of zinc salts, zinc oxides, zinc hydroxides, and combinations thereof. In the concentrate, component (a) is present in an amount of from 1 to 95% w/w, and component (b) is present in an amount of from 1 to 50% w/w based on the total weight of the concentrate, with the proviso that the weight percent of (a) plus (b) does not exceed 100% by weight. 
     In yet another aspect, the present invention relates to functional fluid that comprises a base medium plus (a) at least one isothiazolin-3-one and (b) at least one zinc compound selected from the group consisting of zinc salts, zinc oxides, zinc hydroxides, and combinations thereof. Illustrative zinc salts are zinc chloride, zinc sulfide, zinc carbonate, basic zinc carbonate, and combinations thereof. In the composition, the isothiazolin-3-one is present in an amount of from 1 to 500 ppm (preferably from 5 to 500 ppm), the metal compound is present in an amount of from 5 to 20,000 ppm (preferably from 5 to 500 ppm), and the weight ratio of the isothiazolin-3-one to zinc ion is from 1:2000 to 100:1 (preferably from 100:1 to 1:100). Optionally, the antimicrobial composition additionally comprises a pyrithione salt or pyrithione acid. Useful pyrithione salts include copper pyrithione, zinc pyrithione, sodium pyrithione, and pyrithione disulfide. The base medium for the functional fluid can be, for example, a polymer useful in polymer emulsions. Examples of polymer systems are lattices, such as acrylic and substituted (meth)acrylates, styrene/butadiene, ethylene vinyl acetate, polyvinyl acetate, styrene/butadiene/N-methylol acrylamide, nitrile and copolymers of the aforementioned. Typical functional fluids include coating compositions, such as paints, adhesives, sealants, caulks, mineral and pigment slurries, printing inks, agricultural pesticide formulations, household products, personal care, metal working fluids and other aqueous based systems. 
     These and other aspects will become apparent upon reading the following detailed description of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  provides a graphical depiction of an Isobologram showing Antimicrobial Activity of Mixtures of 1,2-Benzisothiazolin-3-one (“BIT”) and Zinc Chloride against  Ps. aeruginosa  based on Fractional Inhibitory Concentration values. 
         FIG. 2  provides a graphical depiction of an Isobologram showing Antimicrobial Activity of Mixtures of Kathon® (a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (“CIMIT”) and 2-methyl-4-isothiazolin-3-one (“MIT”)) and Zinc Chloride against  Ps. aeruginosa  based on Fractional Inhibitory Concentration values. 
         FIG. 3  provides a graphical depiction of an Isobologram showing Antimicrobial Activity of Mixtures of MIT and Zinc Chloride against  Ps. aeruginosa  based on Fractional Inhibitory Concentration values. 
         FIG. 4  provides a graphical depiction of an Isobologram showing Antimicrobial Activity of Mixtures of N-(n-butyl)-1,2-benzisothiazolin-3-one (“BBIT”) and Zinc Chloride against  Ps. aeruginosa  based on Fractional Inhibitory Concentration values. 
         FIG. 5  provides a graphical depiction of an Isobologram showing Antimicrobial Activity of Mixtures of BIT and Sodium Chloride against  Ps. aeruginosa  based on Fractional Inhibitory Concentration values. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It has now been surprisingly found in accordance with the present invention that an antimicrobial composition containing an isothiazolin-3-one, plus a zinc compound selected from the group consisting of zinc salts, zinc oxides, zinc hydroxides, and combinations thereof, exhibits enhanced antimicrobial efficacy, as compared to isothiazolin-containing compositions that do not contain the metal compound. Enhanced efficacy are suitably provided in antimicrobial compositions over a wide range of pHs of from 3 to 12. 
     The antimicrobial composition of the present invention is suitably incorporated into a functional fluid. The functional fluid suitably comprises a base medium plus (a) at least one isothiazolin-3-one and (b) at least one zinc compound selected from the group consisting of zinc salts of organic acids, zinc salts of inorganic acids, zinc oxides, zinc hydroxides, and combinations thereof. Illustrative zinc salts include zinc chloride, zinc sulfide, zinc carbonate, basic zinc carbonate (also known as “hydroxy-containing zinc carbonate”, also know as “zinc hydroxy carbonate” which is further identified by the empirical formula Zn 5 (OH) 6 (CO 3 ) 2 ), and combinations thereof. 
     The isothiazolinone useful in the present invention is preferably an isothiazolin-3-one that is selected from: 1,2-benzisothiazolin-3-one (“BIT”), N-(n-butyl)-1,2-benzisothiazolin-3-one (“BBIT”), 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (“DCOIT”), 2-methyl-4-isothiazolin-3-one (“MIT”), mixtures of 5-chloro-2-methyl-4-isothiazolin-3-one (“CIMIT”) plus MIT (available from Rohm and Haas Company, Philadelphia, Pa. under the tradename Kathon®) dithio-2,2′-bis(benzmethylamide), and combinations thereof. Particularly preferred isothiazolins are BIT, MIT and BBIT, and combinations thereof. 
     In the composition, the isothiazolin-3-one is present in an amount of from 1 to 500 ppm (preferably from 5 to 500 ppm), the zinc compound is present in an amount of from 5 to 200,000 ppm (preferably from 5 to 500 ppm), and the weight ratio of the isothiazolin-3-one to the zinc compound is from 1:2000 to 100:1 (preferably from 100:1 to 1:100). 
     Optionally, the antimicrobial composition additionally comprises a pyrithione salt or pyrithione acid. Useful pyrithione salts include copper pyrithione, zinc pyrithione, sodium pyrithione. If used, the pyrithione salt or pyrithione acid is suitably present in the composition in an amount of from about 0.001% w/w to about 1.0% w/w. 
     In one embodiment, the combination of antimicrobial components for the antimicrobial composition can be provided in the form of an antimicrobial composition concentrate that, upon dilution with water, provides antimicrobial efficacy in a functional fluid. The concentrate comprises (a) at least one isothiazolin-3-one and (b) at least one zinc compound selected from the group consisting of zinc salts, zinc oxides, zinc hydroxides, and combinations thereof. In the concentrate, component (a) is present in an amount of from 1 to 95% w/w, and component (b) is present in an amount of from 1 to 50% w/w. 
     The antimicrobial compositions of the present invention are suitably used in functional fluids such as polymer emulsions, or other coating compositions, to impart both wet state and dry film preservation. The base medium can be, for example, a polymer useful in polymer emulsions wherein the polymer is selected from the group consisting of acrylic and substituted (meth)acrylates, styrene/butadiene, ethylene vinyl acetate, polyvinyl acetate, styrene/butadiene/N-methylol acrylamide, nitrile and copolymers of the aforementioned. Typical functional fluids include coating compositions, such as paints, adhesives, sealants, caulks, mineral and pigment slurries, printing inks, agricultural pesticide formulations, household products, personal care, metal working fluids and other aqueous based systems. 
     The invention is further described in the Examples given below. All percentages given herein are weight percents based on the total weight of the composition, unless otherwise stated. All patents referred to in this application are incorporated herein by reference in their entirety. 
     Example 1 
     In this example, samples of a commercial water based acrylic latex, trade name REVACRYL 1A, supplied by the Harlow Chemical Company, pH 8.1, containing antimicrobial additives in amounts as identified in Table I below, were challenged with a bacterial suspension consisting of 
       Pseudomonas aeruginosa , NCIMB 8295 
       Providencia rettgeri , NCIMB 10842 
       Serratia marcescens , NCIMB 9523 
       Aeromonas hydrophila , NCIMB 9233 
       Alcaligenes  spp. (Wet State Preservation isolate) 
       Burkholderia cepacia , ATCC 25416 
       Pseudomonas putida , NCIMB9494 
     All test species were cultured onto nutrient agar and incubated for 24 hours at 30° C. After this period individual suspensions of each test organism were prepared at a concentration of 10 6  colony forming units per ml in saline solution by means of a Thoma counting chamber, achieved by mixing equal volumes of the individual suspensions. The concentration of bacteria in each aliquot after each challenge was 2×10 6  per ml. 
     The antimicrobial additive-containing paint samples were stored at 40° C. for 7 days prior to commencing the test. The paint samples were challenged with 1% v/v of bacterial suspension as described above. All test samples were incubated at 30° C. for the duration of the test and examined for viable bacteria after 1, 3 and 7 days after each challenge. Viable bacteria were detected by streaking aliquots onto nutrient agar followed by incubation at 30° C. for 2 days. 
     Table I presents the results obtained after the third inoculation days 1, 3 and 7. 
     
       
         
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE I 
               
             
             
               
                   
                   
               
               
                   
                 3 rd  inoculation 
               
             
          
           
               
                   
                 Sample 
                 BIT/ppm 
                 Day 1 
                 Day 3 
                 Day 7 
               
               
                   
                   
               
             
          
           
               
                   
                 BIT/ZnO 
                 50 
                 C 
                 C 
                 C 
               
               
                   
                   
                 100 
                 1+ (92) 
                 1+ (27) 
                 − 
               
               
                   
                   
                 150 
                 1+ (95) 
                 S (7) 
                 − 
               
               
                   
                   
                 200 
                 S (10) 
                 T (4) 
                 − 
               
               
                   
                   
                 250 
                 1+ (89) 
                 1+ (22) 
                 − 
               
               
                   
                   
                 300 
                 1+ (77) 
                 1+ (12) 
                 − 
               
               
                   
                 BIT/ZnCl2 
                 50 
                 C 
                 C 
                 C 
               
               
                   
                   
                 100 
                 ++ 
                 1+ (30) 
                 − 
               
               
                   
                   
                 150 
                 1+ (34) 
                 1+ (21) 
                 − 
               
               
                   
                   
                 200 
                 1+ (51) 
                 1+ (14) 
                 − 
               
               
                   
                   
                 250 
                 ++ (185) 
                 1+ (25) 
                 − 
               
               
                   
                   
                 300 
                 1+ (58) 
                 1+ (17) 
                 − 
               
               
                   
                 PROXEL 
                 50 
                 C 
                 C 
                 C 
               
               
                   
                 BD20 
                 100 
                 C 
                 C 
                 C 
               
               
                   
                   
                 150 
                 C 
                 C 
                 C 
               
               
                   
                   
                 200 
                 C 
                 C 
                 C 
               
               
                   
                   
                 250 
                 1+ (75) 
                 − 
                 − 
               
               
                   
                   
                 300 
                 1+ (54) 
                 − 
                 − 
               
               
                   
                   
                 400 
                 1+ (47) 
                 − 
                 − 
               
               
                   
                   
               
               
                   
                 Assessments are rated as: 
               
               
                   
                 − no growth detected 
               
               
                   
                 T 0-5 colonies present 
               
               
                   
                 S 5-10 colonies 
               
               
                   
                 1+ 20-100 colonies 
               
               
                   
                 + light growth - no specified number of colonies 
               
               
                   
                 ++ moderate growth - colonies visible, some coalescence 
               
               
                   
                 +++ heavy growth - coalescing colonies visible throughout 
               
               
                   
                 C confluent heavy growth 
               
             
          
         
       
     
     Results demonstrate a potentiating effect of BIT in combination with a zinc containing salt when compared against a commercial product, namely PROXEL BD20 a product of Arch Chemicals, Inc. containing only BIT for the preservation of a commercial latex in that only 100 ppm of BIT is required in the presence of a zinc containing salt compared to 250 ppm of BIT for sample containing only BIT. 
     Example 2 
     Investigation of Potentiation Between 1,2-Benzisothiazolin-3-one (BIT) and Zinc Chloride (ZnCl 2 ) Against  Pseudomonas aeruginosa    
     Bacteria 
       Pseudomonas aeruginosa  (NCIB 10421) maintained on nutrient agar, were used to study the synergy between BIT and ZnCl 2 . 
     Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures 
     Bacteria were grown to stationary phase (approximately 24 hours) in nutrient broth (approximately 10 9  organisms per ml). A 0.1% (v/v) inoculum was used to seed fresh medium and 100 μl of the inoculum was then added to each well of a microtitre plate, except for the first well which contained 200 μl. 
     Using doubling dilutions, the concentration of the compounds under investigation were varied in each well along the ordinate axis. The presence or absence of growth was recorded by visual inspection after 24 hours incubation at 37° C. 
     Calculation of Antimicrobial Activity Against Mono-Cultures 
     Microtitre plates were used for this assay. A simple matrix was constructed with varied concentrations of the two compounds from 2×MIC (minimum inhibitory concentration) down to zero concentration in a 10×10 array. As the microtitre plate has only 96 wells, the combinations of the two compounds that made up the extreme concentrations (highest and lowest) were omitted. Solutions were made up in broth at two times the final concentrations after pre-dissolving the compounds in distilled water. 
     The mixture (100 μl) was added to the plate so that the total volume in each well was 200 μl. Nutrient broth was used for  Ps. aeruginosa . Plates were incubated for 16-24 hours at 37°. The presence or absence of growth was determined by visual inspection. 
     Results for Example 2 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Table 2: Antimicrobial Activity of Compounds Under Investigation 
               
             
          
           
               
                   
                 MIC (ppm) against  Ps. aeruginosa   
                   
               
             
          
           
               
                   
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
                   
               
             
          
           
               
                   
                 BIT 
                 30 
                 42 
                 42 
                 30 
               
               
                   
                 ZnCl 2   
                 300 
                 450 
                 375 
                 450 
               
               
                   
                   
               
               
                   
                 BIT is 1,2-benzisothiazolin-3-one available from Arch Limited PROXEL ® GXL. 
               
             
          
         
       
     
     Calculation of Activity Against Mono-Cultures 
     The Minimum Inhibitory Concentration (MIC) is the lowest concentration of biocide which showed growth inhibition when used alone. For the purpose of Fractional Inhibitory Concentration (FIC) calculations, if a single biocide did not control growth, the MIC was taken as the highest concentration used. A Fractional Inhibitory Concentration is the concentration of biocide which controlled growth in the mixture divided by the MIC of that biocide. FIC values for both compounds in the mixture were calculated and the results are shown in Table 3. The sum of these two figures gives an indication of the action of the two biocides. A value less than one indicates an enhanced effect, if the total is unity or greater the action is additive and if the value is greater than two the biocides are antagonistic. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Fractional Inhibitory Concentrations for 1,2-Benzisothtazolin-3-one 
               
               
                 (BIT) and Zinc Chloride (ZnCl 2 ) against  Ps. aeruginosa   
               
               
                 Table 3 
               
             
          
           
               
                   
                 FIC VALUES 
               
             
          
           
               
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 BIT 
                 1 
                 0.20 
                 0.20 
                 0.40 
                 0.14 
                 0.14 
                 0.29 
                 0.29 
                 0.43 
                 0.14 
                 0.20 
                 0.40 
                 0 
               
               
                 ZnCl 2   
                 0 
                 0.25 
                 0.50 
                 0.25 
                 0.33 
                 0.50 
                 0.17 
                 0.33 
                 0.17 
                 0.60 
                 0.33 
                 0.17 
                 1 
               
               
                 Total 
                 1 
                 0.45 
                 0.70 
                 0.65 
                 0.47 
                 0.64 
                 0.46 
                 0.62 
                 0.60 
                 0.74 
                 0.53 
                 0.57 
                 1 
               
               
                   
               
             
          
         
       
     
     If a graph with the axes representing the biocide Fractional Inhibitory Concentrations for the two biocides on linear scales is constructed, when the combination is additive the isobole (i.e. the line joining the points that represent all combinations with the same effect including the equally effective concentrations of the biocides used alone) is straight, enhanced combinations give concave isoboles and antagonistic combinations give convex isoboles. As shown in  FIG. 1 , the combination of BIT and ZnCl 2  has an enhanced effect against  Ps. aeruginosa  as confirmed by the concave isobole on the graph of  FIG. 1 . 
     Example 3 
     Investigation of Potentiation Between Kathon® (CIMIT/MIT) and Zinc Chloride (ZnCl 2 ) Against  Pseudomonas aeruginosa    
     Bacteria 
       Pseudomonas aeruginosa  (NCIB 10421) maintained on nutrient agar, were used to study the synergy of Kathon® and ZnCl 2 . 
     Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures 
     Bacteria were grown to stationary phase (approximately 24 hours) in nutrient broth (approximately 10 9  organisms per ml). A 0.1% (v/v) inoculum was used to seed fresh medium and 100 μl of the inoculum was then added to each well of a microtitre plate, except for the first well which contained 200 μl. 
     Using doubling dilutions, the concentration of the compounds under investigation were varied in each well along the ordinate axis. The presence or absence of growth was recorded by visual inspection after 24 hours incubation at 37° C. 
     Calculation of Antimicrobial Activity Against Mono-Cultures 
     Microtitre plates were used for this assay. A simple matrix was constructed with varied concentrations of the two compounds from 2×MIC (minimum inhibitory concentration) down to zero concentration in a 10×10 array. As the microtitre plate has only 96 wells, the combinations of the two compounds that made up the extreme concentrations (highest and lowest) were omitted. Solutions were made up in broth at two times the final concentrations after pre-dissolving the compounds in distilled water. 
     The mixture (100 μl) was added to the plate so that the total volume in each well was 200 μl Nutrient broth was used for  Ps. aeruginosa . Plates were incubated for 16-24 hours at 37°. The presence or absence of growth was determined by visual inspection. 
     Results of Example 3 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Table 4: Antimicrobial Activity of Compounds Under Investigation 
               
             
          
           
               
                   
                 MIC (ppm) against  Ps. aeruginosa   
                   
               
             
          
           
               
                   
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
                   
               
             
          
           
               
                   
                 Kathon 
                 0.9 
                 0.9 
                 0.9 
                 0.53 
               
               
                   
                 ZnCl 2   
                 294 
                 294 
                 210 
                 600 
               
               
                   
                   
               
             
          
         
       
     
     The mixture of CIMIT and MIT is available from Rohm and Haas Company, Philadelphia, Pa. under the tradename Kathon®. 
     Calculation of Synergy Against Mono-Cultures 
     The Minimum Inhibitory Concentration (MIC) is the lowest concentration of biocide which showed growth inhibition when used alone. For the purpose of Fractional Inhibitory Concentration (FIC) calculations, if a single biocide did not control growth, the MIC was taken as the highest concentration used. A Fractional Inhibitory Concentration is the concentration of biocide which controlled growth in the mixture divided by the MIC of that biocide. FIC values for both compounds in the mixture were calculated and the results are shown in Table 5. The sum of these two figures gives an indication of the action of the two biocides. A value less than one indicates an enhanced effect, if the total is unity or greater the action is additive and if the value is greater than two the biocides are antagonistic. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Fractional Inhibitory Concentrations for Kathon 
               
               
                 (CIMIT/MIT) and Zinc Chloride (ZnCl 2 ) against  Ps. aeruginosa   
               
               
                 Table 5 
               
             
          
           
               
                   
                 FIC VALUES 
               
             
          
           
               
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 Kathon 
                 1 
                 0.17 
                 0.17 
                 0.33 
                 0.33 
                 0.17 
                 0.33 
                 0.33 
                 0.14 
                 0.28 
                 0.28 
                 0 
               
               
                 ZnCl 2   
                 0 
                 0.14 
                 0.29 
                 0.14 
                 0.29 
                 0.29 
                 0.14 
                 0.29 
                 0.50 
                 0.38 
                 0.50 
                 1 
               
               
                 Total 
                 1 
                 0.31 
                 0.46 
                 0.47 
                 0.62 
                 0.46 
                 0.47 
                 0.62 
                 0.64 
                 0.66 
                 0.78 
                 1 
               
               
                   
               
             
          
         
       
     
     If a graph with the axes representing the biocide Fractional Inhibitory Concentrations for the two biocides on linear scales is constructed, when the combination is additive the isobole (i.e. the line joining the points that represent all combinations with the same effect including the equally effective concentrations of the biocides used alone) is straight, enhanced combinations give concave isoboles and antagonistic combinations give convex isoboles. As shown in  FIG. 2 , the combination of Kathon® and ZnCl 2  has an enhanced effect against  Ps. aeruginosa  as confirmed by the concave isobole in  FIG. 2 . 
     Example 4 
     Investigation of Potentiation Between MIT and Zinc Chloride (ZnCl 2 ) Against  Pseudomonas aeruginosa    
     Bacteria 
       Pseudomonas aeruginosa  (NCIB 10421) maintained on nutrient agar, were used to study the synergy between MIT and ZnCl 2 . 
     Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures 
     Bacteria were grown to stationary phase (approximately 24 hours) in nutrient broth (approximately 10 9  organisms per ml). A 0.1% (v/v) inoculum was used to seed fresh medium and 100 μl of the inoculum was then added to each well of a microtitre plate, except for the first well which contained 200 μl. 
     Using doubling dilutions, the concentration of the compounds under investigation were varied in each well along the ordinate axis. The presence or absence of growth was recorded by visual inspection after 24 hours incubation at 37° C. 
     Calculation of Antimicrobial Activity Against Mono-Cultures 
     Microtitre plates were used for this assay. A simple matrix was constructed with varied concentrations of the two compounds from 2×MIC (minimum inhibitory concentration) down to zero concentration in a 10×10 array. As the microtitre plate has only 96 wells, the combinations of the two compounds that made up the extreme concentrations (highest and lowest) were omitted. Solutions were made up in broth at two times the final concentrations after pre-dissolving the compounds in distilled water. 
     The mixture (100 μl) was added to the plate so that the total volume in each well was 200 μl. Nutrient broth was used for  Ps. aeruginosa . Plates were incubated for 16-24 hours at 37°. The presence or absence of growth was determined by visual inspection. 
     Results of Example 4 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Table 6: Antimicrobial Activity of Compounds Under investigation 
               
             
          
           
               
                   
                 MIC (ppm) against 
                   
               
               
                   
                 
                   Ps. aeruginosa 
                 
               
             
          
           
               
                   
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
                   
               
             
          
           
               
                   
                 MIT 
                 9 
                 6 
                 10.5 
                 8 
               
               
                   
                 ZnCl 2   
                 210 
                 210 
                 450 
                 450 
               
               
                   
                   
               
             
          
         
       
     
     Calculation of Synergy Against Mono-Cultures 
     The Minimum Inhibitory Concentration (MIC) is the lowest concentration of biocide which showed growth inhibition when used alone. For the purpose of Fractional Inhibitory Concentration (FIC) calculations, if a single biocide did not control growth, the MIC was taken as the highest concentration used. A Fractional Inhibitory Concentration is the concentration of biocide which controlled growth in the mixture divided by the MIC of that biocide. FIC values for both compounds in the mixture were calculated and the results are shown in Table 7. The sum of these two figures gives an indication of the action of the two biocides. A value less than one indicates an enhanced effect, if the total is unity or greater the action is additive and if the value is greater than two the biocides are antagonistic. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Fractional Inhibitory Concentrations for (MIT) and Zinc Chloride (ZnCl 2 ) against  Ps. aeruginosa   
               
               
                 Table 7 
               
             
          
           
               
                   
                 FIC Values 
               
             
          
           
               
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 MIT 
                 1 
                 0.17 
                 0.33 
                 0.33 
                 0.50 
                 0.25 
                 0.50 
                 0.75 
                 0.14 
                 0.29 
                 0.17 
                 0 
               
               
                 ZnCl 2   
                 0 
                 0.57 
                 0.29 
                 0.43 
                 0.14 
                 0.43 
                 0.29 
                 0.14 
                 0.33 
                 0.17 
                 0.50 
                 1 
               
               
                 Total 
                 1 
                 0.74 
                 0.62 
                 0.76 
                 0.64 
                 0.68 
                 0.79 
                 0.89 
                 0.48 
                 0.46 
                 0.67 
                 1 
               
               
                   
               
             
          
         
       
     
     If a graph with the axes representing the biocide Fractional Inhibitory Concentrations for the two biocides on linear scales is constructed, when the combination is additive the isobole (i.e. the line joining the points that represent all combinations with the same effect including the equally effective concentrations of the biocides used alone) is straight, enhanced combinations give concave isoboles and antagonistic combinations give convex isoboles. As shown in  FIG. 3 , the combination of MIT and ZnCl 2  has an enhanced effect against  Ps. aeruginosa  as confirmed by the concave isobole in  FIG. 3 . 
     Example 5 
     Investigation of Potentiation Between BBIT and Zinc Chloride (ZnCl 2 ) Against  Pseudomonas  aeruginosa 
     Bacteria 
       Pseudomonas aeruginosa  (NCIB 10421) maintained on nutrient agar, were used to study the synergy between BBIT and ZnCl 2 . 
     Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures 
     Bacteria were grown to stationary phase (approximately 24 hours) in nutrient broth (approximately 10 9  organisms per ml). A 0.1% (v/v) inoculum was used to seed fresh medium and 100 μl of the inoculum was then added to each well of a microtitre plate, except for the first well which contained 200 μl. 
     Using doubling dilutions, the concentration of the compounds under investigation were varied in each well along the ordinate axis. The presence or absence of growth was recorded by visual inspection after 24 hours incubation at 37° C. 
     Calculation of Antimicrobial Activity Against Mono-Cultures 
     Microtitre plates were used for this assay. A simple matrix was constructed with varied concentrations of the two compounds from 2×MIC (minimum inhibitory concentration) down to zero concentration in a 10×10 array. As the microtitre plate has only 96 wells, the combinations of the two compounds that made up the extreme concentrations (highest and lowest) were omitted. Solutions were made up in broth at two times the final concentrations after pre-dissolving the compounds in distilled water. 
     The mixture (100 μl) was added to the plate so that the total volume in each well was 200 μl. Nutrient broth was used for  Ps. aeruginosa . Plates were incubated for 16-24 hours at 37°. The presence or absence of growth was determined by visual inspection. 
     Results of Example 5 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Table 8: Antimicrobial Activity of Compounds Under Investigation 
               
             
          
           
               
                   
                 MIC (ppm) against 
                   
               
               
                   
                 
                   Ps. aeruginosa 
                 
               
             
          
           
               
                   
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
                   
               
             
          
           
               
                   
                 BBIT 
                 315 
                 360 
                 42 
                 180 
               
               
                   
                 ZnCl 2   
                 525 
                 525 
                 600 
                 600 
               
               
                   
                   
               
               
                   
                 BBIT is N-(n-butyl)-1,2-benzisothiazolin-3-one. 
               
             
          
         
       
     
     Calculation of Synergy Against Mono-Cultures 
     The Minimum Inhibitory Concentration (MIC) is the lowest concentration of biocide which showed growth inhibition when used alone. For the purpose of Fractional Inhibitory Concentration (FIC) calculations, if a single biocide did not control growth, the MIC was taken as the highest concentration used. A Fractional Inhibitory Concentration is the concentration of biocide which controlled growth in the mixture divided by the MIC of that biocide. FIC values for both compounds in the mixture were calculated and the results are shown in Table 9. The sum of these two figures gives an indication of the action of the two biocides. A value less than one indicates an enhanced effect, if the total is unity or greater the action is additive and if the value is greater than two the biocides are antagonistic. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Fractional Inhibitory Concentrations for BBIT 
               
               
                 and Zinc Chloride (ZnCl 2 ) against  Ps. aeruginosa   
               
               
                 Table 9 
               
             
          
           
               
                   
                 FIC values 
               
             
          
           
               
                 COMPOUND 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 BBIT 
                 1 
                 0.29 
                 0.25 
                 0.14 
                 0.71 
                 0.25 
                 0 
               
               
                 ZnCl 2   
                 0 
                 0.43 
                 0.43 
                 0.50 
                 0.38 
                 0.38 
                 1 
               
               
                 Total 
                 1 
                 0.71 
                 0.68 
                 0.64 
                 1.09 
                 0.63 
                 1 
               
               
                   
               
             
          
         
       
     
     If a graph with the axes representing the biocide Fractional Inhibitory Concentrations for the two biocides on linear scales is constructed, when the combination is additive the isobole (i.e. the line joining the points that represent all combinations with the same effect including the equally effective concentrations of the biocides used alone) is straight, enhanced combinations give concave isoboles and antagonistic combinations give convex isoboles. As shown in  FIG. 4 , the combination of BBIT and ZnCl 2  has an enhanced effect against  Ps. aeruginosa  as confirmed by the concave isobole shown in  FIG. 4 . 
     Comparative Example A 
     Investigation of Potentiation Between 1,2-Benzisothiazolin-3-one (BIT) and Sodium Chloride (NaCl 2 ) Against  Pseudomonas aeruginosa  as Compared to the Synergy Between 1,2-Benzisothiazolin-3-one (BIT) and Zinc Chloride (ZnCl 2 ) Against  Pseudomonas aeruginosa  (Example 2) 
     Bacteria 
       Pseudomonas aeruginosa  (NCIB 10421) maintained on nutrient agar, were used to study the efficacy of the combination of BIT with sodium chloride. 
     Calculation of Minimum Inhibitory Concentrations Against Mono-Cultures 
     Bacteria were grown to stationary phase (approximately 24 hours) in nutrient broth (approximately 10 9  organisms per ml). A 0.1% (v/v) inoculum was used to seed fresh medium and 100 μl of the inoculum was then added to each well of a microtitre plate, except for the first well which contained 200 μl. 
     Using doubling dilutions, the concentration of the compounds under investigation were varied in each well along the ordinate axis. The presence or absence of growth was recorded by visual inspection after 24 hours incubation at 37° C. 
     Calculation of Antimicrobial Activity Against Mono-Cultures 
     Microtitre plates were used for this assay. A simple matrix was constructed with varied concentrations of the two compounds from 2×MIC (minimum inhibitory concentration) down to zero concentration in a 10×10 array. As the microtitre plate has only 96 wells, the combinations of the two compounds that made up the extreme concentrations (highest and lowest) were omitted. Solutions were made up in broth at two times the final concentrations after pre-dissolving the compounds in distilled water. 
     The mixture (100 μl) was added to the plate so that the total volume in each well was 200 μl. Nutrient broth was used for  Ps. aeruginosa . Plates were incubated for 16-24 hours at 37°. The presence or absence of growth was determined by visual inspection. 
     Results of Comparative Example A 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Table 10: Antimicrobial Activity of Compounds Under Investigation 
               
             
          
           
               
                   
                 COMPOUND 
                 MIC (ppm) against Ps. aeruginosa 
               
               
                   
                   
               
               
                   
                 BIT 
                 30 
               
               
                   
                 NaCl 2   
                 None detected (greater than 675) 
               
               
                   
                   
               
               
                   
                 BIT is 1,2-benzisothiazolin-3-one available from Arch Limited as PROXEL RTM  GXL. 
               
             
          
         
       
     
     Calculation of Potentiation Against Mono-Cultures 
     The Minimum Inhibitory Concentration (MIC) is the lowest concentration of biocide which showed growth inhibition when used alone. For the purpose of Fractional Inhibitory Concentration (FIC) calculations, if a single biocide did not control growth, the MIC was taken as the highest concentration used. A Fractional Inhibitory Concentration is the concentration of biocide which controlled growth in the mixture divided by the MIC of that biocide. FIC values for both compounds in the mixture were calculated and the results are shown in Table 11. The sum of these two figures gives an indication of the action of the two biocides. A value less than one indicates an enhanced effect, if the total is unity or greater the action is additive and if the value is greater than two the biocides are antagonistic. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Table 11: Fractional Inhibitory Concentrations for 1,2-Benzisothiazolin-3- 
               
               
                 one (BIT) and Sodium Chloride (NaCl 2 ) against  Ps. aeruginosa   
               
             
          
           
               
                   
                 COMPOUND 
                 FIC VALUES 
               
               
                   
                   
               
             
          
           
               
                   
                 BIT 
                 1 
                 1.00 
                 0 
               
               
                   
                 NaCl 2   
                 0 
                 0.33 
                 1 
               
               
                   
                 Total 
                 1 
                 1.33 
                 1 
               
               
                   
                   
               
             
          
         
       
     
     If a graph with the axes representing the biocide Fractional Inhibitory Concentrations for the two biocides on linear scales is constructed, when the combination is additive the isobole (i.e. the line joining the points that represent all combinations with the same effect including the equally effective concentrations of the biocides used alone) is straight, enhanced combinations give concave isoboles and antagonistic combinations give convex isoboles. As shown in  FIG. 5 , the combination of BIT and NaCl 2  is additive against  Ps. aeruginosa  as confirmed by the straight isobole in  FIG. 5 . Additionally, the results of this Example further illustrate that the combination of an isothiazolin-3-one such as BIT and a zinc compound such as ZnCl 2 , as shown in Example 2, show unexpected potentiation results against  Ps. aeruginosa.