METHOD AND COMPOSITION FOR WATER TREATMENT

Abstract

The invention provides a method of microbial control in water comprising adding to the water one or more bromine-based biocide(s) and cis-2-decenoic acid or a salt thereof. Compositions in the form of liquid concentrates comprising bromine-based biocides and cis-2-decenoic acid or a salt thereof are also described.

Claims

1. A method of microbial control in water comprising adding to the water one or more bromine-based biocide (s) and cis-2-decenoic acid (CDA) or a salt thereof.

2. A method according to claim 1, wherein the microbial control comprises combatting planktonic bacteria and/or biofilm bacteria on a surface in contact with the water and/or inhibiting biofilm formation on a surface prone to biofilm growth.

3. A method according to claim 1, wherein the bromine-based biocide is a non-oxidizing biocide.

4. A method according to claim 3, wherein the non-oxidizing bromine-based biocide is selected from the group consisting of: 2-bromo-2-nitro-1,3-propanediol (Bronopol); and 2,2-dibromo-3-nitrilopropionamide (DBNPA).

5. A method according to claim 1, wherein the bromine-based biocide is an oxidizing biocide.

6. A method according to claim 5, wherein the oxidizing bromine-based biocide is 1,3-dihalo-5,5-dialkylhydantoin, wherein at least one of the halogen atoms is bromine, and the alkyl groups may be the same or different.

7. A method according to claim 6, wherein the 1,3-dihalo-5,5-dialkylhydantoin is selected from the group consisting of 1-bromo-3-chloro-5,5-dimethylhydantoin, 1-chloro-3-bromo-5,5-dimethylhydantoin, 1,3-dibromo-5,5-dimethylhydantoin and 1-bromo-3-chloro-methylethylhydantoin, or mixtures thereof.

8. A method according to claim 5, wherein the oxidizing bromine-based biocide is an on-site oxidized bromide source, which releases active bromine species in water.

9. A method according to claim 8, wherein the on-site oxidized bromide source is selected from the group consisting of: sodium bromide, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated; HBr, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated; ammonium bromide, which is oxidized on-site with hypochlorite, chlorine or electrochemically to produce its active form, to be added to the water system to be treated; and solution of HBr and urea, which reacts with hypochlorite, chlorine or electrochemically on-site to produce the bromourea active form, to be added to the water system to be treated.

10. A method according to claim 1, wherein the effective microbiocidal dosage of the bromine-based biocide(s) is from 1 to 100 ppm and the enhancement-inducing amount of cis-2-decenoic acid is from 0.005 to 5 ppm.

11. A method according to claim 1, wherein low purity CDA grade, of less than 95% purity as measured by gas chromatography, is added to the water.

12. A method according to claim 1, wherein the bromine-based biocide(s) and CDA are supplied to an industrial water stream in contact with an infested surface using multiple feed solutions, whereby the biocide and CDA are added sequentially or simultaneously to the water.

13. A method according to claim 1, wherein the bromine-based biocide(s) and CDA are supplied to an industrial water stream in contact with an infested surface using a single feed solution, whereby the biocide and CDA are added simultaneously to the water.

14. A method according to claim 13, wherein the bromine-based biocide is a non-oxidizing biocide, and wherein said biocide and CDA are formulated in a liquid concentrate supplied to the industrial water stream using a single feed solution.

15. A method according to claim 1, comprising combatting biofilm bacteria by adding to the water an effective microbiocidal dosage of the bromine-based biocide(s) and an enhancement-inducing amount of the cis-2-decenoic acid to achieve biofilm reduction which is at least 2 log units lower than the reduction achieved with the same dosage of the biocide acting alone.

16. A composition comprising one or more bromine-based biocides and cis-2-decenoic acid or a salt thereof.

17. A composition according to claim 16, which is a liquid concentrate comprising one or more bromine-based biocides and cis-2-decenoic acid or a salt thereof in a carrier, said carrier comprising water, water miscible solvent or a mixture thereof and optionally one or more of cosolvent(s), antifreeze(s) and stabilizer(s).

18. A liquid concentrate according to claim 17, comprising: from 10 to 50 wt. % of a non-oxidizing bromine-based biocide; from 0.05 to 1 wt. of cis-2-decenoic acid or a salt thereof; and a carrier comprising water, a glycol cosolvent and an antioxidant.

19. A liquid concentrate according to claim 18, comprising: from 10 to 35 wt % of 2-bromo-2-nitro-1,3-propanediol; from 0.05 to 0.5% of cis-2-decenoic acid; from 5.0 to 80.0% of water; from 10 to 70.0% of glycol selected from the group consisting of ethylene glycol, propylene glycol and dipropylene glycol monomethylether; and from 0.05 to 0.5% of an antioxidant.

20. A liquid concentrate according to claim 18, comprising: from 15 to 25 wt. % of 2,2-dibromo-3-nitrilopropionamide; from 0.05 to 0.5% of cis-2-decenoic acid; from 10 to 60.0% of water; from 40 to 60.0% of polyethylene glycol; from 0.05 to 0.5% of an antioxidant.

Description

EXAMPLES

Materials

[0051] Materials and reagents used in the experimental work are tabulated in Table 4.

TABLE-US-00004 TABLE 4 Typical Name of the dosage* Commercial name biocide (ppm) Source C-103 DBNPA 0.1-1000 Bromine compounds (ICL-IP) Bromotop0 Bronopol 0.1-1000 Bromine compounds (ICL-IP) Bactebrom0 Activated 0.1-100 Bromine compounds (ICL-IP) HBr:Urea active form prepared prior mixture to use, as described in (Bromourea) Preparation 1 Halogene® BCDMH 0.1-100 Bromine compounds (ICL-IP) Bromosol® Bromosulfamate 0.1-100 Bromine compounds (ICL-IP) Bromide plus Activated NaBr 0.1-100 Bromine compounds (ICL-IP); (Na0Br) Active form prepared prior to use Ammonium Bromide Activated AmBr 0.1-100 Bromine compounds (ICL-IP); active form prepared prior to use, as described in Preparation 2 HBr 48% Bromine compounds (ICL-IP) Sodium Sodium 0.1-100 F&C Israel hypochlorite 10- hypochlorite 12% Cis Decenoic CDA pure Carbosynth Ltd. (Compton - acid -97% pure Berkshire, United Kingdom) (GC) Chemodex (St. Gallen, Switzerland) Cis Decenoic CDA crude as described in preparation acid -90% pure 3 (GC) *As active biocide

Methods

[0052] GC measurement was done with AGILENT HP-5 19091J-413 30 m*0.32 mm*0.25 micron.

Method: 50° C.//2′//10° C./min//280° C.//5° C./min//300° C./2′.

Preparation 1

Biocide Preparation by Activation of HBr/Urea Solution

[0053] Stock solution 1-8.94 g of Bactebrom® from ICL-IP (HBr:urea solution) diluted with 241.06 g of distilled water.

[0054] Stock solution 2-NaOCl ˜1% prepared by 23.58 g of NaOCl 10.6% w/w diluted with 226.42 g of distilled-water.

[0055] Stock solution 2 (250.00 g of NaOCl 1.0%) was added gradually while stirring to the above diluted Bactebrom® solution (stock solution 1), to get the active biocide (orange solution)—total weight 500.00 g. Expected biocide concentration as determined by iodometric titration using Titroprocessor: Titrino 848 plus: ˜0.5% as Cl.sub.2 (˜5000 ppm as C12). The desired biocide concentration in each experiment was obtained by dilution with distilled water.

Preparation 2

Biocide Preparation by Activation of Ammonium Bromide

[0056] 975 μl 10.25 Wt % aq. NaOCl was diluted with distilled water to 100 ml in a volumetric flask. C12 concentration was ˜1000 ppm as C12 as determined by iodometric titration using Titroprocessor: Titrino 848 plus.

[0057] 213 mg NH.sub.4Br was diluted with distilled water to 100 ml in a volumetric flask.

[0058] Mix equal volumes of 5 ml as follows: add the NaOCl solution in one stroke to a mixed solution (using a magnetic stirrer) of the NH.sub.4Br solution at ambient temperature.

[0059] The concentration of the product (activated AmBr) was based on the concentration of the Na-Hypochlorite (˜1000 ppm as C12). Equal volumes of the reactants were mixed to obtain the concentration of the active chlorine in the mixture as 50% of the concentration of the reactant NaOCl, ˜500 ppm as C12. The desired biocide concentration in each experiment was obtained by dilution with distilled water.

Preparation 3

Synthesis and Purification of Cis-2-Decenoic Acid

[0060] Cis-2-decenoic acid was prepared according to the two-step synthetic pathway described in U.S. Pat. No. 8,748,486, replacing sodium hydroxide with lithium hydroxide in the second step (dehalogenation/rearrangement of 1,3-dibromo-2-decanone) as depicted below:

##STR00001##

[0061] The reaction mixture was worked-up using conventional techniques (such as silica gel and solvent extraction) to recover cis-2-decenoic acid with purity varying in the range from 60 to 97% (GC). In the studies reported below, 90% pure cis-2-decenoic acid was tested.

Example 1

[0062] Enhancing the Effect of Bromine-Based Biocide on 3-Day Biofilm with the Aid of CDA (Simultaneous Application)

[0063] The effect of bromine-containing biocide in combination with CDA on pre-grown biofilms was studied. The experiment was carried out utilizing (A) the P. aeruginosa strain PA14 and (B) mixed bacterial species derived from environmental and industrial water.

[0064] Three bromine-based biocides were tested in this study: bronopol, DBNPA and BCDMH.

Experimental Procedure:

[0065] Bacteria were cultured in EPRI medium supplemented with Hutners mineral solution and glucose (0.2%). Microorganisms were incubated at room temperature (22° C.), under aerobic conditions with shaking. The biofilm culture system used included polystyrene 24-well plates that were treated with protein to enhance attachment and growth of biofilm bacteria according to the method described in Davies D G, Marques C N, 2009, J Bacteriol 191:1393-1403.

[0066] Following inoculation with 1 mL bacterial culture, spent medium was removed and replaced with sterile medium every 24 hours for 3 days, and a final medium exchange was performed 3 hours prior to treatment. Additionally, the medium was exchanged prior to treatment in order to remove planktonic bacteria. Treatments consisted of 100 μL of 310 nM CDA and bromine-containing biocide, or bromine-containing biocide alone at concentrations used in commercial water treatment, water was used as a carrier, for a contact time determined by activity of each biocide and ranging from 1 hour to 24 hours, as detailed below for each of the tested biocides.

[0067] Following the treatment, the medium from each well was removed by pipet and 1 mL of DE neutralization broth was added in order to stop the treatment. Bacteria from each well were then removed by scraping the biofilm formed in the well with a sterile cell-scraper, and 1.0 mL of culture was transferred to chilled (4° C.) 9.0 mL DE neutralizing broth and homogenized for 15 seconds at 40,000 rpm on ice. Further dilutions were performed prior to enumeration (further neutralizing biocide activity). Recovery of bacteria were tested at different dilutions of biocide in water, in LB medium with thioglycolate and in DE neutralizing broth to ensure the active agent was properly neutralized. Viable bacteria were enumerated via the drop plate method. Each biocide was evaluated using 24-well plates with 8 wells each for the control cultures (Ctl) inoculated with P. aeruginosa but not treated, a CDA minus test (−CDA) treated with bromine-containing biocide only, and a CDA plus test treated with bromine-containing biocide and CDA (+CDA).

Results:

[0068] A) The results of the treatment of PA14 bacteria with 10 ppm of bronopol are presented in FIGS. 2A and 2B. FIG. 2A shows that the addition of CDA to the treatment with bronopol reduced the number of bacteria by more than an order of magnitude after only two hours of treatment. FIG. 2B demonstrates the effect achieved after 24 hours of treatment. It is seen that bronopol alone is very effective in reducing biofilm. Nevertheless, with the aid of added CDA, the treatment became much more effective and led to biofilm eradication.

[0069] B. The results of the treatment of PA14 bacteria with 15 ppm of DBNPA are presented in FIG. 3 in the form of a bar diagram. It is seen that a short treatment of two hours which included both DBNPA and CDA yielded an improved effect compared with a treatment which was carried under the same conditions with DBNPA alone.

[0070] C. The results of the treatment of PA14 bacteria with 10 ppm of BCDMH are presented in FIG. 4 in the form of a bar diagram. It is seen that a short treatment of just one hour, which included the application of both BCDMH and CDA, generated an improved result compared with treatment using BCDMH alone (log reduction=2).

[0071] D. The results of the treatment against undefined mixed microbial biofilm cultures derived from an environmental water source and against a biofilm culture derived from fresh-water mixed microbial community that were obtained from cooling tower water, with 15 ppm of DBNPA are presented in FIGS. 5A and 5B respectively. It is seen that a short treatment of two hours which included both DBNPA and CDA yielded an improved result, compared with a treatment which was carried out under the same conditions with DBNPA alone.

Example 2

Enhancing the Effect of Bromine-Based Biocide on 3-Day Biofilm with the Aid of CDA (Sequential Application)

[0072] The effect of the sequential addition of CDA and bromine-containing biocide was studied using P. aeruginosa biofilm grown in the CDC biofilm reactor on borosilicate glass coupons (test method E2562). The addition of the bromine-containing biocide took place 60 min after the addition of 310 nM CDA. See Sections A-F, corresponding to six bromine-based biocides that were tested. In section G herein below, two more bacteria strains (Staphylococcus aureus 6538, Bacillus mycoides 6462) were added to the reactor and contributed to the formation of a mixed biofilm. In section H herein below, two other CDA concentrations (31 nM and 3100 nM) were tested against P. aeruginosa biofilm.

[0073] Six bromine-based biocides were tested (DBNPA, sulfamate-stabilized bromine, BCDMH, bromourea, activated ammonium bromide and activated NaBr) in combination with two CDA products of different purity: commercial CDA (CV-CHEM, 97% by GC) and crude CDA (90% by GC) of Preparation 3, to evaluate the effect of the purity level of the CDA used.

Experimental Procedure:

[0074] The efficacy test on the coupons was performed according to the single tube method (E2871-13). This test method is used for growing a reproducible P. aeruginosa biofilm in a CDC Biofilm Reactor.

Biofilm Formation:

[0075] The biofilm was established by operating the reactor in batch mode (no flow of the nutrients) for 4 h. A steady state population was reached after the reactor operated for an additional 3 days with continuous flow of the nutrients. During the entire 3-day period, the biofilm was exposed to continuous fluid shear from the rotation of a baffled stir bar. At the end of the 3 days, the biofilm from the coupons was sampled as follows:

[0076] a. The coupons were rinsed to remove planktonic cells. The rods were oriented in a vertical position directly over a 50 mL conical centrifuge tube that contained 30 mL sterile buffered water. The rods were immersed with a continuous motion into the buffered water with minimal to no splashing, then immediately removed. A new 50 mL conical tube containing 30 mL sterile buffered water was used for each rod.

[0077] b. The rods were held with one of the randomly selected coupons centered over an empty, sterile 50 mL conical tube. The set screws were loosened, allowing the coupons to drop directly to the bottom of the tube.

[0078] Sequential addition of CDA and bromine-containing biocide:

[0079] a. Four mL of a solution containing either phosphate buffer (untreated control), a buffer with 310 nM CDA or a buffer with different concentrations of biocides were slowly pipetted into the tubes containing the coupons.

[0080] b. Each tube was tapped to release any air bubbles trapped below the coupon.

[0081] c. The tubes (containing the control, CDA or the biocide) were incubated at 20° C., under shaking at 200 rpm for one hour contact time.

[0082] d. After one hour contact time, 36 ml of a neutralizer was added to each tube.

[0083] e. The combined treatment was carried out as a sequential treatment in which CDA was introduced first, and after 1 hour contact time with CDA, the coupons were transferred to another tube containing the biocide and incubated at 20° C., under shaking of 200 rpm for another 1 hour contact time. After the one hour contact time, 36 ml of a neutralizer was added to each tube.

Removing and Disaggregating the Biofilm:

[0084] a. Each tube was vortexed using Vortex Genie-2 Model no. G560E on the highest setting for 30 s.

[0085] b. The tubes were sonicated at 45 kHz for 30 s.

[0086] c. The tubes were vortexed as described above, then sonicated and vortexed again.

[0087] d. The samples were serially diluted in buffered water.

[0088] e. Each dilution was cultured in duplicate (on R2A agar) for colony growth using the pour plating method.

[0089] f. The plates were incubated at 35° C. for 72 h.

[0090] g. The appropriate number of colonies were counted.

Results:

[0091] A. The effect of 10 ppm DBNPA (2,2-dibromo-3-nitrilopropionamide) on biofilms produced as described above, alone and in combination with 310 nM commercial CDA (CV-CHEM, 97% by GC) is illustrated in the bar diagram of FIG. 6. It is seen that CDA alone exhibits no useful effect compared to the control. The application of DPNPA alone reduces the biofilm. Notably, an enhancement of two log units was achieved by the combined treatment compared to the treatment with DBNPA alone (the combined treatment consists of addition of DBNPA to a sample that was in contact with CDA for one hour prior to the addition of the biocide).

[0092] B. The effect of 5 ppm sulfamate-stabilized bromine (Bromosol) on biofilms produced as described above, alone and in combination with 310 nM CDA (either of commercial source from CV-CHEM, 97% by GC or of Preparation 3, 90% by GC) is illustrated in the bar diagram of FIG. 7. It is seen that CDA alone exhibits no useful effect compared to the control, regardless of its source. The application of Bromosol alone reduces the biofilm (4 log units). The combined treatment (that is, CDA followed by the biocide) achieves a further biofilm reduction compared to the treatment with the biocide alone. It is worth noting that the use of commercial and crude CDA has led to 1 log unit and 2 log unit reduction, respectively, relative to the biocide alone.

[0093] C. The effect of 10 ppm BCDMH on biofilms produced as described above, alone and in combination with 310 nM CDA (either of commercial source from CV-CHEM, 97% by GC or of Preparation 3, 90% by GC) is illustrated in the bar diagram of FIG. 8. It is seen that CDA alone exhibits no useful effect compared to the control, regardless of its source. The application of BCDMH alone reduces the biofilm (˜3 log units). The combined treatment (that is, CDA followed by the biocide) achieves a further biofilm reduction compared to the treatment with the biocide alone; both commercial and crude CDA generated comparable enhancement, i.e., ˜2 log units reduction relative to BCDMH alone.

[0094] D. The effects of 5 ppm bromourea or 10 ppm bromourea on biofilms produced as described above, alone and in combination with 310 nM CDA (either of commercial source from CV-CHEM, 97% by GC, or of Preparation 3, 90% by GC) are illustrated in the bar diagrams of FIGS. 9A (5 ppm bromourea) and 9B (10 ppm bromourea). It is seen that CDA alone exhibits no useful effect compared to the control, regardless of its source. It is also worth mentioning that comparable biofilm reductions were measured for the solely applied 5 ppm bromourea and 10 ppm bromourea. That is, twofold increase of the concentration of the biocide (5 ppm.fwdarw.10 ppm) did not manifest itself in enhanced biofilm control. However, the combined treatment (application of CDA followed by application of 5 ppm biocide or 10 ppm biocide) achieves significant improvement compared to the treatment with the biocide alone; both commercial and crude CDA generated very good results i.e., ˜2-3 log units enhancement relative to 5 ppm bromourea alone, and 4-5 log units enhancement relative to 10 ppm bromourea alone.

[0095] E. The effect of 5 ppm activated ammonium bromide (AmBr) on biofilms produced as described above, alone and in combination with 310 nM commercial CDA (CV-CHEM, 97% by GC) is illustrated in the bar diagram of FIG. 10. It is seen that CDA alone exhibits no useful effect compared to the control. The application of the biocide alone reduces the biofilm. Notably, an enhancement of ˜4.5 log units was achieved by the combined treatment compared to the treatment with the biocide alone (the combined treatment consists of addition of the biocide to a sample that was in contact with CDA for one hour prior to the addition of the biocide).

[0096] F. The effect of 20 ppm activated NaBr on biofilms, alone and in combination with 310 nM commercial CDA (CV-CHEM, 97% by GC) is illustrated in the bar diagram of FIG. 11. It is seen that CDA alone exhibits no useful effect compared to the control. The application of activated NaBr alone reduces the biofilm. Notably, an enhancement of two log units was achieved by the combined treatment compared to the treatment with activated NaBr alone (the combined treatment consists of addition of activated NaBr to a sample that was in contact with CDA for one hour prior to the addition of the biocide).

[0097] G. The effects of either 5 ppm BCDMH or 10 ppm BCDMH on a biofilm formed by mixed bacteria (three different bacteria: S. aureus 6538, B. mycoides 6462 and P. aeruginosa 700888) produced as described above, alone and in combination with 310 nM commercial CDA (CV-CHEM, 97% by GC) are illustrated in the bar diagrams of FIGS. 12A and 12B (5 ppm BCDMH or 10 ppm BCDMH, respectively). It is seen that CDA alone exhibits no useful effect compared to the control. The application of the biocide alone reduces the biofilm, but the 10 ppm BCDMH treatment is not appreciably better than the 5 ppm BCDMH treatment. However, both the 5 ppm and 10 ppm BCDMH treatments benefit from the incorporation of CDA into the treatment. The combined treatment has been found to achieve enhancement of ˜2.5 and ˜5.5 log units compared to the treatment with the 5 ppm and 10 ppm biocide acting alone, respectively (the combined treatment consists of addition of the biocide to a sample that was in contact with CDA for one hour prior to the addition of the biocide).

[0098] H. The effect of 10 ppm BCDMH on biofilms produced as described above, alone and in combination with different concentrations (31 nM, 310 nM and 3100 nM) of commercial CDA (CV-CHEM, 97% by GC) is illustrated in the bar diagram of FIG. 13. It is seen that CDA alone at all tested concentrations exhibits no useful effect compared to the control. The application of BCDMH alone reduces the biofilm. Notably, an enhancement of 4.5 log units was achieved by the combined treatment of CDA (31 nM and 310 nM) and of 5.5 log units (3100 nM) compared to the treatment with BCDMH alone (the combined treatment consists of addition of BCDMH to a sample that was in contact with CDA for one hour prior to the addition of the biocide).

Example 3

Reducing Dosage Levels of Bromine-Based Biocides with the Aid of CDA

[0099] The goal of the study was to estimate to which extent the addition of CDA can offset a decrease of the dosage level of the bromine-based biocide. That is, to offer combined bromine/CDA treatment program that is equally effective as currently acceptable, high-dosage level, acting-alone bromine.

[0100] The biocide tested was BCDMH. CDA of commercial source, 97% purity grade, was used. The experimental protocol of Example 2 was repeated (i.e., sequential treatment, CDA followed by BCDMH, applied to biofilm grown for three days, 1 hour biocide contact time).

[0101] The results are presented in the form of a bar diagram in FIG. 14, showing that the effect of 5.0 ppm of BCDMH in the presence of 310 nM CDA is comparable to that of 10 ppm BCDMH in the absence of CDA. Hence, addition of very small amount of CDA to the water can offset a 50% decrease in the dosage level of the biocide.

Example 4

Reducing Dosage Levels of Bromine-Based Biocides with the Aid of CDA

[0102] CDA (commercial 97% purity grade) was applied in combination with varying quantities of bromine-based biocide (the dosage level of the biocide was varied in the range from 0 to 10 ppm) to investigate the ability of CDA to support the action of the biocide across a wide biocide concentration range. CDA was used at a constant concentration of 310 nM. The combined treatment bromine/CDA was compared to the solely applied bromine-based biocide.

[0103] The biocide tested was bromourea. The experimental protocol of Example 2 was repeated (i.e., sequential treatment, CDA followed by bromourea, applied to biofilm grown for three days, 1 hour biocide contact time).

[0104] The results are shown in FIGS. 15A and 15B, in the form of survival plots corresponding to the solely applied biocide (marked by rhombuses) and the combined biocide/CDA treatment. The concentrations of the biocide tested were 0.0, 0.625, 1.25, 2.5, 5 and 10 ppm. The results indicate the effectiveness of the combined treatment against 3-day biofilm over the entire biocide concentration range (i.e., >0.5 ppm), demonstrating LCso (biofilm concentration at which 50% of the culture is killed) biofilm control at treatment level of 2.5 ppm bromourea/310 nM CDA and biofilm eradication concentration as low as 5 ppm bromourea/310 nM CDA.

Example 5

Enhancing the Effect of Bromine-Based Biocide on Planktonic Bacteria with the Aid of CDA (Sequential Application)

[0105] 310 nM CDA and bromine-containing biocide were introduced into a mix of planktonic bacteria in a medium containing different levels of organic loading (TOC 10-3000 ppm). High organic loading tends to reduce the efficacy of antimicrobial agents in industrial applications and, therefore, the test was aimed to mimic these conditions. The test was performed in accordance with the modified European standard EN 1040: 2005: “Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics—Tests method and requirements (phase 1)”. 19 ml of phosphate buffer solution (pH=7) including tryptone in order to obtain a solution of TOC=30 ppm, and 1 ml of the tested bacteria suspension (consisted of E. Coli (ATCC 11229), S. aureus (ATCC 6538), Enterobacter aerogenes (ATCC 130489) and P. aeruginosa (ATCC 13388)), at a concentration of 1.5×10.sup.8-5×10.sup.8 CFU/ml, were placed into a container of suitable capacity. A stopwatch was started immediately and the container was placed in a water bath controlled at 30° C.

[0106] The activity was determined with 2.5 ppm BCDMH alone for a contact time of 3 hours. The combination of CDA (CV-CHEM) with 2.5 ppm BCDMH was tested when added in sequence (1 hour with CDA then additional 3 hours with BCDMH). At the desired contact time, 1 ml of the tested mixture was pipetted into a tube containing 9.0 ml neutralizer. Immediately after 5 sec of neutralization time, a sample of 1 ml was taken in duplicate and transferred to a Petri dish. TSA, cooled to 45±1° C., was added. The plates were incubated at 37±2° C. for 48 hours. Countable plates were counted and the number of colony-forming units was determined, for each plate.

[0107] The results are presented in FIG. 16. As can be seen, enhancement of 2 log units was obtained when CDA (CV-CHEM) and BCDMH (2.5 ppm) were introduced in sequence compared with the effect achieved by BCDMH in the absence of CDA.

Example 6

Room Temperature Storage-Stable Composition of DENPA and CDA

[0108] 22 mg of cis-2-decenoic acid (CiVentiChem, 96.72% purity) were charged into 50 ml flask, equipped with a stirrer 3.99 gr of C-103 (ICL, batch 640160147) were added, followed by 9.97 gr of PEG200 (Merck 8.07483.5000 Lot 56904983 451), 5.95 gr of DI water and 22 mg of BHT (Aldrich B1378-100G, BCBH9491V). The obtained solution was stirred till complete clarification; sonication at +30° C. is recommended if some particles are observed. Clear colorless solution of a total 30 gr and 20:50:30:0.1:0.1 weight ratio of C103-PEG.sub.200-H.sub.2O-CDA-BHT was obtained.

[0109] The solution was tested to determine the stability of the biocide and CDA and under storage at 25° C. over a two-month period. Neither DBNPA nor CDA underwent degradation in the 25° C./two-month test (analysis by HPLC).

Example 7

Room Temperature Storage-Stable Composition of Bronopol and CDA

[0110] 47.6 mg or cis-2-decenoic acid (CiVentiChem, 96.72% purity) were charged into 50 ml flask, equipped with a stirrer. 9.01 gr of Bronopol (ICL, batch 17101607) were added, followed by 18.03 gr of propylene glycol (Biolab 16200201 Lot 1007861), 3.0 gr of DI water and 31.2 mg of BHT (Aldrich B1378-100G, BCBH9491V). After stirring for several minutes, undissolved traces of BHT were filtered through filtering paper for BHT particle removal. Clear colorless solution of a total 30 gr and 30:60:10:0.158:0.1 weight ratio of Bronopol-PG-H.sub.2O-CDA-BHT was obtained.

[0111] The solution was tested to determine the stability of the biocide and CDA and under storage at 25° C. over a two-month period. Neither bronopol nor CDA underwent degradation in the 25° C./two-month test (analysis by HPLC).

Example 8

Bromine/CDA Versus Chlorine/CDA Effect on Biofilm

[0112] The purpose of the set of experiments reported in this Example was to check if the addition of CDA to bromine- and chlorine-based treatments generate comparable effects on the targeted biofilm. That is, whether CDA augments the action of bromine and chlorine on biofilm in equally effective manner. Sodium hypochlorite and BCDMH were chosen as illustrative chlorine and bromine biocides, respectively.

[0113] CDA of commercial source, 97% purity grade, was used. The experimental protocol of Example 2 was repeated (i.e., sequential treatment, CDA followed by the halogenated biocide, applied to biofilm grown for three days, 1 hour biocide contact time).

[0114] The bar diagram of FIG. 17 shows that sodium hypochlorite and BCDMH applied alone at dosage level of 5 ppm have comparable effect on biofilm. However, a surprisingly better effect was generated by the bromine/CDA combination of the invention on biofilm, compared with chlorine/CDA. The combination BCDMH/CDA applied at treatment level of 5 ppm BCDMH/310 nM CDA achieved 4.5 log units reduction versus the solely applied BCDMH treatment, whereas the corresponding hypochlorite/CDA treatment was able to improve to a lesser extent (2 log units reduction).

Example 9

Enhancing the Effect of Bromine-Based Biocide on Massive Biofilm with the Aid of CDA (Sequential Application)

[0115] The effect of the sequential addition of CDA and bromine-containing biocide was studied using P. aeruginosa biofilm grown in the drip flow tubes (according to a modification of standard ASTM 2647-13, standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow). This method produces massive biofilm resulting with harsh conditions that are very difficult to treat. The addition of the bromine-containing biocide took place 120 min after the addition of 310 nM CDA (the biocide that was tested in this study was DBNPA).

Experimental Procedure:

[0116] a) A continuous once-through tube system was configured by using silicone coated latex tubes, connected to a peristaltic pump via additional silicone tubing.

[0117] b) The silicone coated latex tubes were inoculated by syringe injection of overnight cultures of P. aeruginosa (ATCC 700888), 5*10.sup.7 CFU/ML in 10% TSB (Tryptic Soy Broth). The bacterial cells were allowed to attach (static incubation) to the tubing for 2 h (batch phase).

[0118] c) After 2 hours the system was moved into an incubator at 25° C. and a growth medium (10% TSB) was pumped at a flow rate of 10 mL/hr for ˜42 hours.

[0119] d) Sequential addition of CDA and bromine-containing biocide: The biofilm treatment was done by exchanging the medium with buffered water (control) or buffered CDA solution and then exchanging to a biocide solution.

[0120] The following treatments were conducted:

[0121] 1. Phosphate buffer for 5 hours (control)

[0122] 2. Phosphate buffer for 2 hours followed by biocide solution (at different concentrations) for another 3 hours.

[0123] 3. CDA buffer solution for 2 hours followed by biocide solution (at different concentrations) or buffer solution for another 3 hours (control).

[0124] e) After the end of the experiment, the silicone coated latex tubes were disconnected from the tubing and the biofilm was removed from within the tubes by inserting a specially made cleaning rod.

[0125] f) The biofilm was removed into a vial and disaggregated by vortex.

[0126] g) The sample was serially diluted. Each dilution was cultured in duplicate (on R2A agar) for colony growth using the pour plating method.

[0127] h) The plates were incubated at 36° C. for 17-20 h.

[0128] i) The appropriate number of colonies were counted.

Results

[0129] The results presented in FIG. 18 show that the effect of 30.0 ppm of DBNPA in the combined treatment of 310 nM CDA enhanced the biocidal effect of the biocide alone by ca 2 log orders.

Example 10

Enhancing the Effect of Bromine-Based Biocide on 3-Day Biofilm with the Aid of the Salt Form of CDA (Simultaneous Application)

[0130] Experimental stepwise acidification of CDA sodium/lithium salt indicates that cis-DA pKa value is in the range of 6.5-7.5. In several industrial applications the pH values of the treated water have higher alkalinity and therefore will promote the formation of the salt form of cis-DA. The effect of the simultaneous addition of CDA in its salt form and bromine-containing biocide was studied using P. aeruginosa biofilm grown in the CDC biofilm reactor on borosilicate glass coupons (test method E2562). The bromine-containing biocide tested was sulfamate-stabilized bromine (Bromosol), added simultaneously with 310 nM CDA for 60 min. The pH of the treated solution was in the range of pH 8-9—conditions which promote the formation of the salt of cis-DA.

Experimental Procedure:

[0131] The efficacy test on the coupons was performed according to the single tube method (E2871-13). This test method is used for growing a reproducible P. aeruginosa biofilm in a CDC Biofilm Reactor.

Biofilm Formation:

[0132] The biofilm was established by operating the reactor in batch mode (no flow of the nutrients) for 4 h. A steady state population was reached after the reactor operated for an additional 3 days with continuous flow of the nutrients. During the entire 3-day period, the biofilm was exposed to continuous fluid shear from the rotation of a baffled stir bar. At the end of the 3 days, the biofilm from the coupons was sampled as follows:

[0133] a. The coupons were rinsed to remove planktonic cells. The rods were oriented in a vertical position directly over a 50 mL conical centrifuge tube that contained 30 mL sterile buffered water. The rods were immersed with a continuous motion into the buffered water with minimal to no splashing, then immediately removed. A new 50 mL conical tube containing 30 mL sterile buffered water was used for each rod.

[0134] b. The rods were held with one of the randomly selected coupons centered over an empty, sterile 50 mL conical tube. The set screws were loosened, allowing the coupons to drop directly to the bottom of the tube.

[0135] Simultaneous addition of CDA and bromine-containing biocide:

[0136] a. Four mL of a solution containing either phosphate buffer (untreated control), a buffer with 310 nM CDA or a buffer with different concentrations of biocides were slowly pipetted into the tubes containing the coupons.

[0137] b. Each tube was tapped to release any air bubbles trapped below the coupon.

[0138] c. The tubes (containing the control, CDA or the biocide) were incubated at 20° C., under shaking at 200 rpm for one-hour contact time.

[0139] d. After one-hour contact time, 36 ml of a neutralizer was added to each tube.

[0140] e. The combined treatment was carried out as a simultaneous treatment in which CDA (310 nM CDA) and the biocide were added together to the tubes. The tubes were incubated at 20° C., under shaking at 200 rpm for one-hour contact time. The pH measured in the system was between 8 to 9.

[0141] After the one-hour contact time, 36 ml of a neutralizer was added to each tube.

Removing and Disaggregating the Biofilm:

[0142] a. Each tube was vortexed using Vortex Genie-2 Model no. G560E on the highest setting for 30 s.

[0143] b. The tubes were sonicated at 45 kHz for 30 s.

[0144] c. The tubes were vortexed as described above, then sonicated and vortexed again.

[0145] d. The samples were serially diluted in buffered water.

[0146] e. Each dilution was cultured in duplicate (on R2A agar) for colony growth using the pour plating method.

[0147] f. The plates were incubated at 35° C. for 72 h.

[0148] g. The appropriate number of colonies were counted.

Results:

[0149] The effect of 10 ppm sulfamate-stabilized bromine (Bromosol) on biofilms produced as described above, alone and in combination with 310 nM CDA is illustrated in the bar diagram of FIG. 19. It is seen that CDA alone exhibits no useful effect compared to the control. The application of Bromosol alone reduces the biofilm (2.5 log units). The combined treatment (that is, CDA with the biocide) achieves a further biofilm reduction compared to the treatment with the biocide alone, enhancement of 1.4 log units.