SE2151356A1

SE2151356A1 - A method for long-term pool water disinfection

Info

Publication number: SE2151356A1
Application number: SE2151356A
Authority: SE
Inventors: Lisa Welander; Maria Forssell
Original assignee: Biocool Ab
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2023-05-06

Abstract

The invention relates to a method for long-term pool water disinfection comprising the steps of a first initial dosing of a composition consisting of 0.5 to 10 wt% didecyldimetylammonium chloride (DDAC), in at least one solvent, to pool water at an initial concentration of DDAC of 3 to 12 ppm, and subsequent dosing of said composition at a concentration of 0.25 to 0.5 times the initial concentration of DDAC for every subsequent bathing day.

Description

The invention relates to a long-term disinfection method for pool water comprising the steps of a first initial dosing of a composition consisting of 0.5 to 10wt% didecyldimetylammonium chloride (DDAC), in at least one solvent, to pool water at an initial concentration of DDAC of 3 to 12 ppm, and subsequent dosing of said composition at a concentration of 0.25 to 0.5 times the initial concentration of DDAC for every subsequent bathing day.

Background Sanitization of water in swimming pools and other recreational water systems, such asJacuzzis and bubble pools, is an important aspect of maintenance in order to keep the amount of microorganisms/microbes in the water below a threshold level, which is typically about 10000 - 100 000 CFU/100 ml (CFU = Colony Forming Units). The microbes that need to be managed are for instance bacteria, viruses, amoebae, algae, and fungi. A too high level of these microbes will render the water not fit to bathe in, or even hazardous to the health. The health hazard relates partly to the possible presence oforganisms that cause diseases but is not only related to the amount of microbes present, but also to toxins released by or from microbes.

Chlorine or hypochlorite salts have been used to disinfect water because it is effective against bacteria and viruses. However, due to health concerns regarding by-products (e.g. chloramines) of chlorinating pools, there have been efforts to find alternative methods of disinfection. Furthermore, chlorine solutions are corrosive to pools. Saltwater pools are an alternative that utilizes the formation of chlorine gas, which serves as the disinfectant via electrolysis. Unfortunately, the health issues associated with the chlorine by-products are not eliminated by the saltwater pools. Chloramines are still formed after chlorine gas is formed. Regardless of the means for providing chlorine, 1-5 ppm free chlorine is normally available in the pool water As an alternative to chlorine, various compositions of bromine, such as bronopol, and silver containing compositions have been used. When bromine containing compositions are used, such as bronopol, a concentration of 2-6 ppm is available in the pool water. However, these compositions also have environmental and health related concerns.

Besides, known disinfectant used today do not kill algae in the pool water. Therefore, didecyldimethylammonium chloride (DDAC) is commonly used in addition to known disinfectant, to manage algae in the pool water, when necessary. Alternatives to DDAC are for instance alkylbenzyldimethyl ammonium chloride, poly- quarternary ammonium compounds, a polymer of N-metyl-metanamin and chloromethyl(oxiran), and polyhexamethylene biguanide.

When DDAC is used as algaecide or fungicide, it is normally used at a concentration of 5-10 ppm. This is generally only done when signs of algae or fungi appear. At high concentrations, DDAC is corrosive, toxic, and environmentally hazardous and may inhibit reproduction. DDAC may be used in addition to the other disinfectants at a concentration of at least 1-3 ppm in the pool water. Thus, a total of 6-8 ppm active disinfectants may be present in the pool water when using standard methods for disinfection of pool water.

Even black algae, which are notoriously difficult to remove once such algae appear in the water of a swimming pool, will be removed by the use of DDAC.

DDAC is also used within the food production industry to disinfect equipment and tools. lt is for instance used to disinfect eggshells, milking equipment and udders, but then at much a lower concentration as compared to the algae treatment above. DDAC is classified as a food grade disinfectant. This means that it will not contaminate food with any harmful material on coming in direct contact or lying nearby. Thus, food grade DDAC, at low concentrations, is deemed harmless for human beings to even ingest. lt has an advantageous safety profile for use in swimming pools and other recreational water systems.

According to the harmonized classification and labelling CLP (EC) No 1272/2008, DDAC has the following classification: Acute Tox. 4, Skin Corr. 1B; H302, H314.

The concentration limit that triggers classification of mixtures containing DDAC as skin corrosion/skin irritation and serious eye damage/eye irritation is shown in table 1.

Table 1. Concentration limits that trigger the classification of DDAC according to CLP regulation No 1272/2008 Classification according Classification _ Converting according concentration in mg/kg classification accordingfor DDAC in Water to CLP regulation to CLP regulation 2 1% but<5% Skin irritation 2 10 000 but < 50 000 Skin corrosion 2 5% 2 50 000 Eye lrrltatlOn 2 1% but < 3% 2 10 000 but < 30 000 Serious eye damage 2 3% 2 30 000 Summary lt is an aim of the present invention to at least partly overcome the above problems, and to provide an improved method for disinfecting pool water, especially over a longer period of time. There is a need for a method for disinfecting water in swimming pools and other recreational water systems that is more environmentally safe, less health hazardous, and that minimizes chemicals used to manage the microbial quality of the water.

This aim is achieved by a method as defined in claim 1.

The invention relates to a method for long-term pool water disinfection comprising or consisting of the steps of - a first dosing of a composition consisting of 0.5 to 10wt% didecyldimetylammonium chloride (DDAC), in at least one solvent, to pool water at an initial concentration of DDAC of 3 to 12 ppm, or 4 to 10 ppm, or 4.5 to 9. 7 ppm, or 4.8 or 9.6 ppm, - a subsequent dosing of said composition at a concentration of DDAC of 0.25 to 0.5, or 0.3 to 0.4 or 0.30 to 0.35, or 0.33 times the initial concentration DDAC, for every subsequent bathing day. ln some aspects, DDAC is the sole disinfectant used in the method. ln some aspects, no further disinfectants are uses in the method. The method of the invention only uses DDAC as disinfectant, which is sufficient as the single anti-microbial agent for removal of all microbes, including bacteria e.g. legionella and pseudomonas aeruginosa), virus, amoebae, fungi and algae. There is therefore no need to add additional disinfectants.

By using the method of the invention for disinfecting and cleaning of pool water, an environmentally safe method is achieved, leaving out the environmentally hazardous chemicals usually used within the field. The chemical footprint is reduced by the method of the invention because less chemicals and a lower amount of chemicals are used, and health hazards and damage to materials are minimized.

The method of the invention can be used to reduce a variability in level of microbes present in pool water relative to the dosage amount of the disinfectant used. More specifically, the concentration of disinfectant in pool water kills microbes, or bacteria to an acceptable level according to microbiological parameters, thus providing an effective disinfection of pool water in EN 16713- 312016, Annex B. Annex B specifies that detection of pseudomonas aeruginosa in 100 ml should be 0.

The method provides for a safe use wherein the operating dosage in a pool water is within the limits, where DDAC is effective to kill microbes/bacteria and at the same time not causing skin or eye irritations or damages to equipment upon repeated usage. The concentration of the disinfectant in the initial dosage is optimized, such that the amount sufficiently kills microbes/bacteria and together with the subsequent dosages maintains an active level of disinfection of the pool water that is safe for the consumer of the pool water. ln some aspects, the composition consists of 1 to 10, or 2.5 to 7.5, or 2 to 6, or 3 to 6, or 4 to 6, or 4 to 5, or 4.9 wt% DDAC, in isopropanol at a weight ratio of isopropanol1DDAC of 0.3 to 0.611, or 0.4 to 0.511, and water up to 100 wt%. ln some aspects, the composition consists of 4,9 wt% didecyldimetylammonium chloride (DDAC), in isopropanol at a weight ratio of isopropanol1DDAC of 0.4 to 0.511, and water up to 100 wt%. ln some aspects, the subsequent dosing is added to the pool water every subsequent bathing day and the subsequent dosing is continued for 2 to 100, or 2 to 50, or 2 to 30, or 2 to 20, or 2 to 10 bathing days. ln some aspects, the pool water is heated to a temperature from 5 to 60°C, or 10 to 45°C, or 20 to 40°C. ln some aspects, the pool water has a chemical oxygen demand (COD) of 0.1 to 10, or 1 to 7, or 2.5 to 7.5, or 4 to 6, or about 5 mg/l. ln some aspects, the pool water comprises microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi. ln some aspects, the pool water comprises at least heterotrophic bacteria. ln some aspects, the pool water comprises pseudomonas aeruginosa. ln some aspects, the pool water comprises legionella. ln some aspects, the pool water comprises pseudomonas aeruginosa and legionella. ln some aspects, a material of a pool comprising the pool water is selected from the group comprising or consisting of steel, wood, plastic, ceramic, and any combination thereof. The use of DDAC in the method of the invention has no damaging effect on the materials of the pool. No corrosion of any material has been observed.

The invention also relates to a use of the method as defined anywhere above for removal of microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria or pseudomonas aeruginosa and/or legionella from pool water. The use may be for a period of 1 to 150 bathing days, or 2 to 100, or 2 to 50, or 2 to 30, or 2 to 20, or 2 to 10 bathing days. ln some aspects, the invention relates to a use of the method as defined anywhere above for prevention of skin burns, skin rashes, skin or eye infections and/or irritations and/or other injuries caused by use of disinfectants in pool water. ln some aspects, the invention relates to use of the method as defined anywhere above for prevention of pseudomonas folliculitis. ln some aspects, the disinfectant is DDAC. ln some aspects, the disinfectant is DDAC in the composition as defined above. ln some aspects, the invention relates to use of the method as defined anywhere above for prevention of damages to pool material or clothes caused by use of disinfectants in pool water. ln some aspects, the disinfectant is DDAC. ln some aspects, the disinfectant is DDAC in the composition as defined above. ln some aspects, the use(s) relate to prevention of skin burns, skin rashes, skin or eye irritations, infections and/or other injuries and damages to pool material or clothes causes by heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella. The method of the invention allows for effective disinfecting use of DDAC at an active DDAC level that is 250- 500 times lower than the EU defined threshold for a skin or eye irritant.

The invention further relates to a use of a composition consisting of 1 to 10, or 2.5 to 7.5, or 2 to 6, or 3 to 6, or 4 to 6, or 4 to 5, or 4.9 wt% didecyldimetylammonium chloride (DDAC), in isopropanol at a weight ratio of isopropanol:DDAC of 0.3 to 0.611, or 0.4 to 0.511, and water up to 100 wt%, for removal of microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water by applying the method as defined anywhere above.

The invention also relates to pool water treated with the method as defined anywhere above having a level of microbes, or bacteria or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from 10 000 to 100 000 CFU/100 ml pool water. The invention relates to pool water having a microbial level of 10 000 to 100 000 CFU/100 ml comprising or consisting of DDAC as the sole disinfectant. ln some aspects, the pool water has a microbial level of 10 000 to 100 000 CFU/100 ml over a period of 1 to 150 bathing days, or 1 to 60 bathing days or 1 to 30 bathing days, or 1 to 21 bathing days, or 1 to 9 bathing days. ln some aspects, the microbes are heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella.

The method ofthe invention provides for an effective concentration of DDAC as anti-microbial agent. The concentration in pool water is at least 3000 to 800, or 2400 times below the concentration of DDAC that might cause any burns, rashes, skin or eye irritations, infections and/or other injuries and damages to pool material or clothes, especially when caused by heterotrophic bacteria or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella.

Detailed description Aspects of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings. The method can, however, be realized in many different manners and should not be construed as being limited to the aspects set forth herein.

The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the invention. As used herein, the singular forms a , an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "pool water" as used herein means water in swimming pools, Jacuzzis, bubble pools, or any other recreational water systems in which humans bathe for recreational purposes. lt relates to any kind of pool, from large public swimming pools to smaller individual pools or hot tubs.

The term "microbe" as used herein means an organism that are too small to be seen without using a microscope, so they include things like bacteria, archaea, and single cell eukaryotes - cells that have a nucleus, like an amoeba or a paramecium.

The term "disinfectant" as used herein means a compound that has the ability to kill a microbe. The disinfectant may be in a liquid or solid form, pure or in a solvent.

The term "long term" as used herein means for a period of 1 to 150 bathing days.

The term "bathing days" as used herein means a day in which the pool water has been used by a customer. Pool water in e.g. jacuzzi is normally not used daily. After every use ofthe bath, the pool water must be disinfected, for example by adding a subsequent dosing of the composition using the method of the invention.

DDAC can be easily acquired by purchase from commercial providers, for instance from Nouyron under the name Arquad, or from I\/lerck Millipore under the product ID 814364.

The invention relates to a method for long-term pool water disinfection comprising or consisting of the steps of a first initial dosing of a composition consisting of 2 to 6 wt% didecyldimetylammonium chloride (DDAC), in at least one solvent, to pool water at an initial concentration of DDAC of 3 to 12 ppm, or 4 to 10 ppm, or 4.5 to 9.7 ppm, or 4.8 or 9.6 ppm and subsequent dosing of said composition of DDAC at a concentration of 0.25 to 0.5, or 0.3 to 0.4 or 0.30 to 0.35, or 0.33 times the initial concentration, for every subsequent bathing day.

The composition may consist of 0.1 to 10, or 2.5 to 7.5, or 2 to 6, or 3 to 6, or 4 to 6, or 4 to 5, or 4.9 wt% DDAC DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.3 to 0.611, or 0.4 to 0.5:1, and water up to 100 wt%. The composition may consist of 4,9 wt% DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.4 to 0.5:1, and water up to 100 wt%.

The subsequent dosing may be added to the pool water every subsequent bathing day, i.e. every day in which the pool water has been used by a customer. The subsequent dosing may continue for 2 to 100, or 2 to 50, or 2 to 28, or 2 to 21, or 2 to 10 or 2 to 9 bathing days or days. Normally, the pool water is refreshed every 2 to 5, or 3 months, or 60 to 150, or 90 days.

The invention may relate to a method for long-term pool water disinfection consisting of the steps of a first dosing of a composition consisting of 4,9 wt% DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.4 to 0.5:1, and water up to 100 wt%, to pool water at an initial concentration of DDAC of 4.5 to 9.7 ppm, or 4.8 or 9.6 ppm and subsequent dosing of said composition at a concentration of DDAC of 0.25 to 0.5, or 0.3 to 0.4 or 0.30 to 0.35, or 0.33 times the initial concentration, for every subsequent bathing day.

The subsequent dosing of said composition may be at a concentration of 0.1 to 5 ppm, or 0.5 to 4 ppm, or 1 to 3.5 ppm, or 1.6 to 3.2 ppm, or 1.6 or 3.2 ppm. lf the initial dosing is done at a concentration of 4.8 or 9.6 ppm, then the subsequent dosing may be done at a concentration of 1.6 and 3.2 ppm, respectively. Depending on the amount of microbes, the concentration ofthe subsequent dosing may need to be adjusted and may thus be slightly less or more than about one third of the concentration used for the first initial dosing.

The pool water may have a chemical oxygen demand (COD) of 0.1 to 10, or 1 to 7, or 2.5 to 7.5, or 4 to 6, or about 5 mg/l. The COD quantifies the amount of organic compounds or microbes in the water.

The pool water may comprise microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi. The microbe may be a heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella. The microbe may be black algae.

A material of a pool may be selected from the group comprising or consisting of steel, wood, plastic, ceramic, and any combination thereof.

The invention also relates to a use of the method as defined anywhere above for removal of microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water, especially heated pool water, at a temperature from 5 to 45°C, or 20 to 40°C, over a longer period of time, e.g. 1 to 150 bathing days, or 2 to 50, or 2 to 28, or 2 to 21, or 2 to 10 or 2 to 9 bathing days.

The invention relates to a use of the method as defined anywhere above for prevention of skin burns, skin rashes, skin or eye infections and/or irritations and/or other injuries caused by use of disinfectants or DDAC in pool water, especially heated pool water, at a temperature from 5 to 60°C, or 10 to 45°C, or 20 to 40°C, over a longer period of time, e.g. 1 to 150 bathing days, or 2 to 50, or 2 to 28, or 2 to 21, or 2 to 10 or 2 to 9 bathing days. Said use of the method as defined anywhere above may be for prevention of pseudomonas folliculitis caused by use of DDAC in pool water.

The invention relates to use of the method as defined anywhere above for prevention of damages to pool material or clothes caused by use of disinfectants or DDAC in pool water, especially heated pool water, at a temperature from 5 to 60°C, or 10 to 45°C, or 20 to 40°C, over a longer period of time, e.g. 1 to 150 bathing days, or 2 to 50, or 2 to 28, or 2 to 21, or 2 to 10 or 2 to 9 bathing days.

These use(s) may relate to prevention of skin burns, skin rashes, skin or eye irritations, infections and/or other injuries and damages to pool material or clothes causes by heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella .

The invention further relates to a use of a composition consisting of 1 to 10, or 2.5 to 7.5, or 2 to 6, or 3 to 6, or 4 to 6, or 4 to 5, or 4.9 wt% DDAC DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.3 to 0.6:1, or 0.4 to 0.5:1, and water up to 100 wt%, for removal of microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water by applying the method as defined anywhere above.

The invention further relates to a use of a composition consisting of 2 or 4.9 wt% DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.4 to 0.5:1, and water up to 100 wt%, for removal of microbes selected from the group comprising or consisting of bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water by applying a method for long-term pool water disinfection comprising or consisting of the steps of a first initial dosing of said composition to pool water at an initial concentration of 3 to 12 ppm, or4 to 10 ppm, or 4.5 to 9. 7 ppm, or 4.8 or 9.6 ppm and subsequent dosing of said composition at a concentration of 0.25 to 0.5, or 0.3 to 0.4 or 0.30 to 0.35, or 0.33 times the initial concentration, for every subsequent bathing day.

The invention also relates to pool water treated with the method as defined anywhere above having a level of microbes, or bacteria or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from 10 000 to 100 000 CFU/100 ml pool water, especially heated pool water, at a temperature from 5 to 60°C, or 10 to 45°C, or 20 to 40°C, over a longer period of time, e.g. 1 to 150 bathing days, or 2 to 50, or 2 to 28, or 2 to 21, or 2 to 10 or 2 to 9 bathing days.

Experiments Experiments were performed to understand the concentration needed and the effect of increasing the concentration on anti-bacterial activity as well as safety aspects. ln the first trials, a threshold concentration for an effective dosage was determined. ln the second trial, additional organic matter was added to replicate the effect of use of the pool water. Concentrations that demonstrated successful anti- microbial or anti- bacterial activity in the first trial were subjected to this additional organic matter followed by 9 subsequent supplemental additions of "disinfect my pool". This test was conducted over three weeks. Through these cycles, the anti-bacterial behaviour was quantified and the added DDAC concentration was calculated. The remaining unconsumed DDAC was analysed and quantified after the 9 supplemental addition ofdisinfectant. Both elevated concentrations tested in the series were demonstrated to be effective at killing bacteria. Finally, the unconsumed DDAC was at a safe concentration that was 2 orders of magnitude below any risk for DDAC be classified as an irritant.

The composition "Disinfect my Pool" (Art.nr : BDMP1000FS) manufactured by Biocool AB is a liquid composition for cleaning of spa baths and hot tubs. According to the EU Biocidal Products Regulation (BPR (EU) 528/2012) this is a product type 2 (PT2). The active substance is didecyldimetylammonium chloride (DDAC) with CAS 7173-51-5. The composition has a DDAC concentration of 20 g/kg solution (2wt%) and isopropanol:DDAC of 0.3 to 0.6:1, or 0.4 to 0.5:1, and water up to 100 wt%.

This study was also undertaken to reduce variability in composition's performance relative to the dosage amount. I\/|ore specifically, the concentration of DDAC in water should kill microbial/bacteria to an acceptable level according to microbiological parameters providing effective disinfection of the pool water in EN 16713- 312016, Annex B. Annex B specifies that detection of pseudomonas aeruginosa in 100 ml should be 0. One objective ofthese tests was to find the necessary dosage of DDAC needed to kill undesired bacteria commonly found in spa baths and hot tubs. The experimental trial was focused on the growth of pseudomonas aeruginosa since it can cause infections in humans, and in pool and spa environments it is often associated with infections in the ears and on the skin. Pseudomonas aeruginosa can cause a rash on the skin, especially on skin that has been covered with swimwear (called pseudomonas folliculitis). lt is commonly found in water and soil and the growth of pseudomonas aeruginosa increases in hot and ventilated pools.

Experimental Test Experimental baths, used to simulate the water quality in a hot tub as pool water, were created. Parameters such as disinfectant dosage, the presence of bovine serum albumin (BSA) and bacterial cultures (pseudomonas aeruginosa and other heterotrophic bacteria) were evaluated. The efficacy was analyzed by measuring the reduction of bacterial colonies present on a petri dish. Both short- and long-term studies were designed to understand the efficacy of DDAC and levels of unreacted DDAC from starting different concentrations of DDAC. Longer experiments where conducted over a three-week period to replicate a scenario where a hot tub is being used by consumers. During this period, the efficacy of killing bacteria and the accumulation of unreacted DDAC in the water was evaluated.

Optimizing the presence of organic compounds in the water A typical water quality from a consumer's hot tube has a chemical oxygen demand (COD) of 5 mg/l. COD quantifies the amount of organic compounds in the water. This can be obtained by adding BSA, which is often used as a protein concentration in lab experiments.

A calibration was evaluated using BSA at different concentrations to figure out at which concentration a COD of 5 mg/I would appear. The calibration showed a BSA concentration of 0.03 g/l to reach a COD of 5 mg/I in the bath water.

Dosage of disinfectant Table 2 shows the primary and maintenance dosages of disinfectant tested in 1 liter of water.

Table 2. Primary and maintenance dosages evaluated for efficacy and safety Product: Biocool Disinfect my pool Primary dosage (ml) Maintenance dosage (ml) 0.12 0.04 0.24 0.08 0.48 0.16 LC-MS analysis The amount of DDAC in the simulated repeated bath samples was determined by LC-|\/IS. The LC-|\/IS system consisted of a C18 column and pre column, the mobile was a gradient of a 95 % acetonitrile solution and a 0.2 % formic acid solution. The flow rate was 0.4 ml/min. Triplicate samples were analyzed and the results were averaged. The calibration was performed with a 4.5 mg/kg DDAC solution.

Tests without BSA A control series was performed to understand the effect of the composition in the absence of BSA. This series was conducted to understand the anti-bacterial efficacy of the normal dosage (0.12 ml to 1 litre of water). A test with the disinfectant and pseudomonas aeruginosa was performed and the CFU (colony- forming unit)/100 ml was analyzed after 1 hour and 24 hours. A similar test was conducted by doubling the normal dosage (0.24 ml to 1 litre of water). The results also shown in table 3. A dosage of 0.12 ml disinfectant to 1litre of water was not sufficient to kill normal levels of pseudomonas aeruginosa. However, doubling the concentration to 0.24 ml leads to a 5 log reduction of the pseudomonas aeruginosa, i.e. 99.999% of the bacteria were killed.

Table 3. Bath test results for reduction of pseudomonas aeruginosa (CFU/100 ml): normal dosage (0.12 ml to 1 litre of water) (left) and doubling (0.24 ml to 1 litre of water) of disinfectant (right).

Disinfectant dosage = 0.12 ml Added bacteria = 420 000 Disinfectant dosage = 0.24 ml Added bacteria = 420 000 CFU/100ml CFU/100ml Pseudomonas aeruginosa Pseudomonas aeruginosa CFU/100ml) CFU/100ml 0 hours 1 hour 24 hours 0 hours 1 hour 24 hours 420000 640000 420000 420000 1 0 Repeated bath tests After the initial tests indicated that the dosage of 0.12 ml was insufficient towards pseudomonas aeruginosa, doses of 0.24 ml and 0.48 ml were further evaluated. Experiments with the higher dosage showed promising anti-bacterial activity. A new experiment was undertaken over a three-week period. ln this time, a primary dosage was applied to the bath, followed by subsequent, smaller dosages to simulate proper bath maintenance. The goal was to keep the bacteria levels to 10 000 - 100 000 CFU/100 ml. The optimized concentration of BSA was also added to these tests to simulate organics residues that are emitted from human skin. The specifications for the pre-bath condition and every simulated bath are shown in table 4. The results of bacterial activity measured after 24 hours of adding the ingredients to the bath are also shown in table 4.

Table 4. Simulated bathing with a pre-bathing conditioning and 9 baths over a time period of 21 days Pre-bath BSA = 0.03 mg/L Added bacteria = 40 000 CFU/100ml Bacterial activity after 24 hours: Pseudomonas aeruginosa CFU/100ml = 0 BSA = 0.03 mg/L Added bacteria = 40 000 CFU/100ml Bacterial activity after 24 hours: Pseudomonas aeruginosa CFU/100ml = 0 conditioning Primary dosage (disinfectant) = 0.24 ml Primary dosage (disinfectant) = 0.48 ml 11 BSA = 0.03 mg/L ' Maintenance dosage (disinfectant)' 0.08 ml Bacterial activity after 24 hours: Pseudomonas aeruginosa _ CFU/100ml = 0 . Bath #1 . Added bacteria = 40 000 CFU/100ml. dded bacteria = 40 000 CFU/100ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =I\/|aintenance dosage (disinfectant) = 0.08 ml 0.16 ml Bacterial activity after 24 hour Bacterial activity after 24 hours: - Pseudomonas aeruginosa Pseudomonas aeruginosa CFU/100ml = 0 CFU/100ml = 0 Bath #2 Added bacteria = 38 700 CFU/100ml Added bacteria = 38 700 CFU/100ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 ' 0.08 ml ml ' Bacterial activity after 24 Bacterial activity after 24 hours: hours: Pseudomonas Pseudomonas aeruginosa aeruginosa CFU/100ml = 0 CFU/100ml = 0 _ Bath #3 _ Added bacteria = 40 200 CFU/100ml _ Added bacteria = 40 200 CFU/100ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 0.08 ml ml Bacterial activity after 24 Bacterial activity after 24 hours: hours: Pseudomonas Pseudomonas aeruginosa - aeruginosa - CFU/100ml = 0 CFU/100ml = 0 Bath #4 Added bacteria = 37 200 CFU/100ml Added bacteria = 37 200 CFU/100ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 0.08 ml ml ' Bacterial activity after 24 ' Bacterial activity after 24 hours: ' hours: Pseudomonas Pseudomonas aeruginosa aeruginosa CFU/100ml = 0 rirl |/1 nnml = n Bath #5 Added bacteria = 39 700 CFU/100ml Added bacteria = 39 700 CFU/100ml BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) = 0.16 ml Bacterial activity after 24 hours: Pseudomonas aeruginosa CFU/100ml = 0 12 Added bacteria = 30 000 CFU/100ml BSA = 0.03 mg/L Maintenance dosage (disinfectant) 0.08 ml Bacterial activity after 24 hours: Pseudomonas aeruginosa CFU/100m|=0 Bath #6 Added bacteria = 28 000 CFU/100ml Added bacteria = 28 000 CFU/l00ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 0.08 ml ml Bacterial activity after 24 Bacterial activity after 24 hours: hours: Pseudomonas Pseudomonas aeruginosa aeruginosa CFU/100ml = 0 CFU/100ml = 0 Bath #7 Added bacteria = 32 800 CFU/100ml Added bacteria = 32 800 CFU/l00ml BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 0.08 ml ml Bacterial activity after 24 Bacterial activity after 24 hours: hours: Pseudomonas Pseudomonas aeruginosa aeruginosa CFU/100ml = 0 CFU/100ml = 0 "at" "ö Added bacteria = 30 900 cFu/loomi Added bacteria = 30 900 cFu/loomi BSA = 0.03 mg/L BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) =Maintenance dosage (disinfectant) = 0.16 0.08 ml ml Bacterial activity after 24 Bacterial activity after 24 hours: hours: Pseudomonas Pseudomonas aeruginosa aeruginosa CFU/100ml = 0 CFU/100ml = 0 Batn #9 Added bacteria = 30 000 CFU/l00ml BSA = 0.03 mg/L I\/|aintenance dosage (disinfectant) = 0.16 ml Bacterial activity after 24 hours: Pseudomonas aeruginosa CFU/100ml = 0 The concentration of DDAC in the baths were calculated as below: Volume of bath = 1000 ml (1 litre) Concentration of DDAC in the disinfectant = 20 g/kg Volume of disinfectant = 0.24 ml (as an example) Conc. of DDAC in the bath = volume of disinfectant X conc. of DDAC in disinfectant Total Volum e (pool + disinfectant) Conc. of DDAC in the bath = 0.24 ml=l<20 g[kg = 0.0048 g/kg = 4.8 mg/kg = 4.8 ppm 0.24 ml + 1000 ml 13 The concentration of DDAC in the bath was calculated for each step and summarized in table Table 5. Concentrations for primary dosages and maintenance dosages calculated according to the equation above.

Primary dosage I\/|aintenance dosage Primary dosage I\/|aintenance dosage (disinfectant) = 0.24 (disinfectant) = 0.08 (disinfectant) = (disinfectant) = 0.16 ml ml 0.48 ml ml 4.8 mg/kg 1.6 mg/kg 9.6 mg/kg 3.2 mg/kg Sum of total added disinfectant in table 5: 4.8 Sum of total added disinfectant in table 5: mg/kg + 9*1-6 mg/kg = 19-2 mg/kg 9.6 mg/kg + 9*3.zmg/| The sum of total added disinfectant for 1 primary dosage and 9 maintenance dosages were also calculated.

The total concentration of DDAC in the baths would be 19.2 mg/kg and 38.4 mg/kg if all DDAC was unreacted. DDAC has likely reacted with the bacteria. An LC-MS analysis on the last bath sample will show if DDAC has been consumed and how much unreacted DDAC is left. Table 1 highlights that DDAC concentrations in the sample need to be 10 000 mg/kg to be skin or eye irritant. These concentrations are over 500 and 250 times below the threshold values for the concentrations used in the method of the invention (19.2 and 38.4 mg/kg, respectively). Furthermore, the values shown in table 5 were calculated to be a theoretical limit to the highest possible DDAC value. lt would not be possible to obtain DDAC levels higher than mentioned in table 5 (after 1 primary dosage and 9 maintenance dosages).

LC-MS analysis of bathing samples Samples from bath #9 were collected and evaluated for quantitative and qualitative LC-MS analysis of the active substance DDAC. The purpose was to see how much DDAC was consumed with the bacteria and the amount of DDAC remaining. The concentration of DDAC added was calculated for each bathing step. An LC-MS analysis was conducted after the final bath in the series to analyze remaining, unreacted DDAC. The amount of unreacted DDAC was expected to be somewhere between initial concentration and ending concentration (bath 9), see table 4. The measured DDAC concentration was within the expected values, and likely consumed due to the interactions with pseudomonas aeruginosa. Triplicate samples were analyzed and the average value reported. Results are shown in table 6.

Table 6. Expected/calculated concentration and LC-MS analysed concentration of DDAC in samples from bath #9 with the two different dosage scenarios.

Disinfecting dosage (ml): Calculated LC-MS Analysed concentration concentration (mg/kg) (mg/kg) 14 Primary dosage: 0.24 ml 4.8-19.2 12 Maintenance dosage: 0.08 ml Primary dosage: 0.48 ml 9.6-38.4 11 Maintenance dosage: 0.16 ml Conclusion lt was demonstrated that DDAC dosages of 0.24 and 0.48 ml were effective as a disinfectant. These two dosages were selected to replicate a more realistic environment for a consumer hot tube. This involved including BSA to the water, treating the bath with a primary dose of 0.24 or 0.48 ml of DDAC, followed by 9 maintenance doses. Both dosing levels demonstrated an ability to effectively kill bacteria and remain at an active DDAC level 250-500 times lower than the EU defined threshold for a skin or eye irritant. These active DDAC concentrations were also confirmed through LC-MS. The results demonstrate that it is possible to have a sufficient dosage of DDAC, neutralize bacteria and have many subsequent maintenance dosages while retaining safe usage.

The present invention is not limited to the embodiments disclosed but may be varied and modified within the scope ofthe following claims.

Claims

1. A method for long-term pool water disinfection comprising the steps of - a first dosing of a composition consisting of 0.5 to 10 wt% didecyldimetylammonium chloride (DDAC), in at least one solvent, to pool water at an initial concentration of DDAC of 3 to 12 ppm, or 4 to 10 ppm, or 4.5 to 9.7 ppm, or 4.8 or 9.6 ppm, - a subsequent dosing of said composition at a concentration of DDAC of 0.25 to 0.5, or 0.3 to 0.4 or 0.30 to 0.35, or 0.33 times the initial concentration DDAC, for every subsequent bathing day.

2. The method according to claim 1, wherein the composition consists of 2 to 6 wt% DDAC, in isopropanol at a weight ratio of isopropanol:DDAC of 0.3 to 0.611, or 0.4 to 0.511, and water up to 100 wt%.

3. The method according to claims 1 or 2, wherein the subsequent dosing is added to the pool water every subsequent bathing day and the subsequent dosing is continue for 2 to 100, or 2 to 50, or 2 to 30, or 2 to 20, or 2 to 10 bathing days.

4. The method according to any of the preceding claims, wherein the pool water has a chemical oxygen demand (COD) of 0.1 to 10, or 1 to 7, or 2.5 to 7.5, or 4 to 6, or about 5 mg/l.

5. The method according to any of the preceding claims, wherein the pool water comprises microbes selected from the group comprising bacteria, viruses, amoebae, algae and fungi.

6. The method according to any of the preceding claims, wherein the pool water comprises at least heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella.

7. The method according to any of the preceding claims, wherein a material of a pool comprising the pool water is selected from the group comprising steel, wood, plastic, ceramic, and any combination thereof.

8. Use of the method according to any of the preceding claims for removal of microbes selected from the group comprising bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water.

9. Use of a composition consisting of 1 to 3, or 2 wt% didecyldimetylammonium chloride (DDAC), in isopropanol at a weight ratio of isopropanol:DDAC of 0.3 to 0.611, or 0.4 to 0.511, and water up to 100 wt%, for removal of microbes selected from the group comprising bacteria, viruses, amoebae, algae and fungi, or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from pool water by applying the method according to any one of claims 1 to

10. Pool water treated with the method according to any of the claims 1 to 7 having a level of microbes, or bacteria or heterotrophic bacteria, such as pseudomonas aeruginosa and/or legionella from 10 000 to 100 000 CFU/100 ml pool water.