Bactericidal tests of Neutral Anolyte produced in Eurostel EE-90 units supplied by the Aquastel

Transcription

1 Gutachten Nr. G c.tmd vom Seite 1 Bactericidal tests of Neutral Anolyte produced in Eurostel EE-90 units supplied by the Aquastel Institut für Hygiene und Umwelt, Steinstraße 10, D Lollar, Germany May 2001 Dr. J. Prucha Client: Aquastel Group, Tallinn Approval No. G c.tmd Lollar,

2 Gutachten Nr. G c.tmd vom Seite 2 CLIENT & PATENTOWNER Aquastel Group, Müürivahe 27-1, 10140, Tallinn, Estonia Tel: Fax: aquastel@uninet.ee UNIT SPECIFICATION EUROSTEL EE-90 unit (serial no: 0003) and all other EE-90 or EE-160 Eurostel units designed to produce Neutral Anolyte supplied by the Aquastel Group incorporating the following patented diaphragmatic Electrolysers. DISINFECTANT TESTED Superoxidised water (Neutral Anolyte) produced in hereafter-mentioned diaphragmatic electrolysers accordingly the following methods Electrolysers: Electrochemical Plant (Patent Western Pacific Company Inc., Tallinn; No. 2285; RU ; WO98/23793; EP A1) Electrochemical Installation (Patent Harrisson Investments Ltd., Tallinn; No ; RU ; WO98/40536; EP A1) Methods: A Method for Sterilising water and device for realising the same (Patent Western Pacific Company Inc., Tallinn; No. 2286; RU ; WO98/25855, EP A1) A Method for electrochemical treatment of aqueous solutions and device for realising the same (Patent Western Pacific Company Inc., Tallinn; No. 2287; RU ; WO98/27012; EP A1)

3 Gutachten Nr. G c.tmd vom Seite 3 1. Introduction Eurostel EE-90 units produce Neutral Anolyte. The bactericidal properties of this Neutral Anolyte was tested accordingly the German Drinking water regulations (TVO) and the German Swimg pool regulations (DIN ). Drinking water The microbiological properties of drinking water in Germany are detered by the German Drinkingwater Standards (TVO: Trinkwasserverordnung, last edition: ). The Drinking Water Standard explains in chapter 1., 1. (properties of drinking water) the following: (1) Drinking water must be free of pathogens. This requirement is not fulfilled if in ml drinking water Escherichia coli is measured (Limit). Germs of E. coli are not allowed in ml (limit). Swimg pool water The microbiological properties of swimg pool water in Germany are detered by the German Industry Standard ( DIN: : Aufbereitung von Schwimm und Badebeckenwasser ). The DIN explains in Table 2 the following requirements: (1) Swimg pool and drinking water must be free of pathogens. This requirement is not fulfilled if in ml Swimg pool and Drinking water Escherichia coli and Pseudomonas aeruginosa is measured (Limit). Germs of E. Coli and Ps. Aerigunosa are not allowed in ml (limit). (2) Moreover, swimg pool and drinking water must be free of Legionella pneumophila. Germs of L. Pneumophila are not allowed in ml drinking water (limit). Germs of L. Pneumophila are not allowed in 1ml swimgpoolwater (limit).

4 Gutachten Nr. G c.tmd vom Seite 4 2. Execution of the tests and analyses Bacteria tested: To execute the tests Aquastel Europe BV supplied us with a Eurostel EE-90 unit. Several Neutral Anolyte solutions were produced by this Eurostel unit with an amount of free between 300 and 500 mg/l. With a view to simulate a practical application, the following pathogens - Escherichia coli und - Pseudomonas aeruginosa were used and isolated in water-samples and in caso-bouillon cultivated. By means of dilution of the pathogens with sterile physiological saline solutions were the needed concentrations produced. For all test samples tap water (drinking water) of the city of Lollar was used. Production of Neutral Anolyte A Eurostel EE-90 unit was used to produce Neutral Anolyte samples. During the production the current was set for ~30A. after one-hour production the current was still ~30A and Neutral Anolyte samples were taken for analyses. Free Chlorine: mg/l PH- Value: 8.2 Conductivity: µS/cm ORP: 790mV Analyses of bactericidal effect in laboratory During these tests samples of 2 litre of tap water of the city of Lollar contaated with bacteria was used. Freshly made Neutral Anolyte was added to these samples. The volume of dosing was dictated after several pre-tests. After adding Neutral Anolyte to the samples the -concentration was detered. Calculation of number of spores (bacteria) In order to count the exact number of spores in the test samples and at the same time prepare test samples with very high concentrations and very low concentration of spores, three different methods to count the spores were used. Count of very high number of spores Small volumes of undiluted test samples were injected on the surface of a nutrient agar plate and incubated at 36 o C, prior to counting and recalculated for ml. The used volumes varied between 0.05 and 0.25ml.

5 Gutachten Nr. G c.tmd vom Seite 5 Count of very low number of spores 10ml of the test samples were filtered through a sterile membrane filter, pore size 0.45µm. The filter was then placed (face up) on the surface of a nutrient agar plate and incubated at 36 o C, prior to counting and recalculated for ml. Count of spores in ml test-samples ml of the test samples were mixed with a double concentrated bouillon. Then this mixture was incubated at 36 o C, prior to counting. In case no spores were found in the test samples, these samples remained unchanged during the tests. In case at least one spore was found in the test samples, the spores will reproduce themselves. Something that can easily measured. This method ensures that all spores are inactivated and proves whether a disinfectant is effective. This method of analysing the test samples is accordingly the German Drinking Water Standards, in which is stated that ml of water must be free of Escheria coli. For swimg pools ml of water must also be free of Pseudomonas Aerigunosa.

6 Gutachten Nr. G c.tmd vom Seite 6 3. Analysing the degradability of free in Neutral Anolyte Neutral Anolyte produced by a Eurostel EE-90 unit was dosed into tap water of the city of Lollar at a dosing rate 1:0 and analysed during one hour. In the following table the degradation of free and redoxpotential is shown. Table 1: Degradation of free and Redoxpotential in Neutral Anolyte in relation to time. 1900ml tapwater +1.9ml Neutral Anolyte Contact time,. Redoxpotential, mv Free, mg/l Tapwater Results of chemical-physical and micro-biological (bactericidal) tests of Testsamples with a concentration of Escherichia coli During these tests two testsamples were used: A low concentration of spores: 500 CFU/ml A high concetration of spores: CFU/ml Neutral Anolyte was diluted with tap water of the city of Lollar to produce Neutral Anolyte samples with the following concentration of free. 0.1 mg/l 0.5 mg/l The following tables show the micro-biological criteria at different concentrations of

7 Gutachten Nr. G c.tmd vom Seite 7 Tab. 2: Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with Escherichia coli in relation of time Escherichia coli concentration: Chlorine concentration: 500 CFU/ ml 0,1 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l Bounded mg/l Total mg/l ml of testsample in Lactose- Bouillon pos pos pos pos pos pos Sample on Endo-Agar pos pos pos pos pos Pos Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml

8 Gutachten Nr. G c.tmd vom Seite 8 Tab. 3 Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with Escherichia coli in relation of time Escherichia coli concentration: Chlorine concentration: CFU/ ml 0,5 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l bounded mg/l total mg/l ml of testsample in Lactose- Bouillon pos neg neg neg neg neg Sample on Endo-Agar pos neg neg neg neg Neg Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml

9 Gutachten Nr. G c.tmd vom Seite 9 Tab. 4: Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with Escherichia coli in relation of time Escherichia coli concentration: Chlorine concentration: 500 CFU/ ml 0,5 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l bounded mg/l total mg/l ml of testsample in Lactose- Bouillon pos pos pos pos pos pos Sample on Endo-Agar pos pos pos pos pos pos Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml

10 Gutachten Nr. G c.tmd vom Seite Conclusion: testsamples with Escherichia coli Table 2 shows that the testsample with a low concentration of Escherichia coli and a very low concentration of 0,1mg/l was not free of spores. After one ute contact time no effect was observed and only after 5 utes a sporicidal effect was observed; number of spores was reduced to 40% of the initial concentration. After 10 ute the number of spores was reduced to 20% of the initial concetration. In the following 50Minutes no furter reduction in the number of spores was observed. Due to this result no further tests were executed with Neutral Anolyte samples with a free concentration of 0,1mg/l. Table 3 and 4 shows that the testsamples with a high and low concentration of Escherichia coli and a concentration of 0,5mg/l, no spores in ml could be measured after 1 Minute. Neutral Anolyte has proved to be an effective disinfectant. Table 4 shows that the testsamples with a free concentration of 0,1mg/l contains after one ute over 66% of bounded.

11 Gutachten Nr. G c.tmd vom Seite Results of chemical-physical and micro-biological (bactericidal) tests of testsamples with a concentration of Pseudomonas aerigunosa During these tests three testsamples were used: A low concentration of spores: 500 CFU/ml A medium concentration of spores: CFU/ml A high concetration of spores: CFU/ml Neutral Anolyte was diluted with tap water of the city of Lollar to produce Neutral Anolyte samples with the following concentration of free. 0.1 mg/l 0.5 mg/l The following tables show the micro-biological criteria at different concentrations of

12 Gutachten Nr. G c.tmd vom Seite 12 Tab. 6: Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with pseudomonas aerigunosai in relation of time Escherichia coli concentration: Chlorine concentration: 4000 CFU/ ml 0,1 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l bounded mg/l total mg/l ml of testsample in Lactose- Bouillon pos pos pos pos pos Pos Sample on Endo-Agar pos pos pos pos pos Pos Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml

13 Gutachten Nr. G c.tmd vom Seite 13 Tab. 7: Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with pseudomonas aerigunosa in relation of time Escherichia coli concentration: Chlorine concentration: 40 CFU/ ml 0,5 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l bounded mg/l total mg/l ml of testsample in Lactose- Bouillon pos neg neg neg neg neg Sample on Endo-Agar pos neg neg neg neg neg Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml

14 Gutachten Nr. G c.tmd vom Seite 14 Tab. 8: Chemical-physical and micro-biological (bactericidal) Parameter of a testsample with pseudomonas aerigunosa in relation of time Escherichia coli concentration: Chlorine concentration: 500 CFU/ ml 0,5 mg/l Parameter Unit Tap water after adding of bacteria Testsample of tap water after adding of bacteria and Anolyte After mixing After 1 After 5 After 10 After 30 after 60 free mg/l bounded mg/l total mg/l ml of testsample in Lactose- Bouillon pos neg neg neg neg Neg Sample on Endo-Agar pos neg neg neg neg neg Total bacteria CFU count in 1 ml at 36 C per 1 ml Escherichia coli in ml CFU Per ml Conclusion: testsamples with pseudomonas aerigunosa The testsample with a low concentration of Pseudomoans aerigunosa and a very low concentration of 0,1mg/l no bacteria in ml could be measured after 1 Minute. As in 1 ml of the testsample after 1 Minute no bacteria could be measured, it can be concluded that after 1 Minute in ml of the testsample the total bacteria count was less than CFU/ ml but imal 1 CFU/ ml is. In the testsamples with a medium and high concentration of Pseudomonas aeruginosa and initial concentrations of 0,5 mg/l, no bacteria in ml could be measured after 1 Minute. Neutral Anolyte has proved to be an effective disinfectant.

15 Gutachten Nr. G c.tmd vom Seite Conclusion and evaluation of the sporicidal effect of Neutral Anolyte The micro-biological tests with two spores Escheria coli and Pseudomonas aerigunosa showed that Neutral Anolyte dosed in to tap water up to a free concentration of 0,5mg/l a very safe and effective disinfectant is, even at concentrations of CFU/ml (4.1x10 2 ) Both spores Escheria coli and Pseudomonas aerigunosa, which are the main indicators whether water is suitable for drinking or swimg (accordingly the German Drinking water & Swimgpool Standards), were within a very short time completely inactivated. Neutral anolyte produced by Eurstel EE-90 units is an effective low-cost disinfectant that can be recommended to disinfect both drinking as Swimgpool water. Dr. J. Prucha Dr. W. Schulz