Testing of SteriPEN™, a Portable Ultraviolet Light Water Purifier, On
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Testing of SteriPEN™, a Portable Ultraviolet Light Water Purifier, On
Environmental Consulting ~ Drinking Water Analysis ~ Radon Testing Testing of SteriPEN™, a Portable Ultraviolet Light Water Purifier, On 3 Liter Hydration Bladders to NSF International Protocol P248, Emergency Military Operations Microbiological Water Purifiers May 21st, 2008 Research Conducted For: Miles Maiden Hydro-Photon, Inc. 262 Ellsworth Road Blue Hill, Maine 04614 Jonathan T. Dyer Laboratory Director Rebecca L. Lebrun Quality Control Officer A & L Laboratory Inc. 3100 Hotel Road P.O. Box 1507 Auburn, Maine 04211-1507 Telephone: (207) 784-5354 Fax: (207) 782-5561 Email: allabs@adelphia.net NELAP CERT #250103 MAINE CERT #ME021 1 NH CERT #2501 Introduction SteriPEN™ is a portable, handheld device designed to disinfect water by using a short wave germicidal ultraviolet (UV) light. The device, unlike traditional flow through UV water purifiers, treats batches of water up to 1 liter. Though the method of treatment is slightly different the concept is the same. The SteriPEN™ produces ultraviolet energy that is used to destroy microorganisms, without the use of chemicals. The SteriPEN™ is submerged in the water, where microorganisms are exposed to a dose of ultraviolet light in the 254-nanometer range. Ultraviolet light in this wavelength inactivates a wide range of microorganisms including bacteria, viruses and protozoan cysts. This inactivation occurs as the ultraviolet light disrupts the organism's DNA structure, making reproduction impossible. The intensity of the ultraviolet light and the microorganism's exposure time to the ultraviolet light are factors that influence which microorganisms are inactivated [6]. This study will examine the effects of the SteriPEN™ on 3 Liter Hydration Bladders {Figure #1}. The bladders were tested as described in the NSF International Protocol P248. – Emergency Military Operations Microbiological Water Purifiers. The P248 Protocol calls for use of both General Test Water and a Challenge Test Water in the 3 Liter Hydration Bladders.. Test Organism MS2 Coliphage is the test organism designated by the P248 protocol. MS2 offers a high linear response over a wide range of UV dose levels, UV inactivation results are highly reproducible, it’s easily propagated to high titers, and it is non-pathogenic to humans [9]. The MS2 Coliphage was provided by Clancy Environmental Consultants, P.O. Box 314, St. Albans, VT, 05478. Thomas Hargy, Senior Scientist at Clancy Consultants ran a collimated beam study on samples of this MS2 to determine the virus’s UV dose response curve. Testing concluded that a 2.1-log reduction of MS2 correlated with a dose of 40mJ/sq.cm. (see attached collimated beam study). Test Procedure The testing procedure was done in accordance with the NSF International P248 Protocol – Emergency Military Operations Microbiological Water Purifiers. The complete protocol is found in the U.S. Army Center for Health Promotion and Preventive Medicine publication entitled “WATER SUPPLY MANAGEMENT PROGRAM NO. 31-EC-04TM, NSF PROTOCOL P248 PURIFIER SPECIFIC TEST PLAN, HYDRO-PHOTON STERIPEN”. Samples of both General Test Water (EPA Test Water # 1) and Challenge Test Water (EPA Test Water #4) were used to compare the effects of the SteriPEN™ on both visually clear water and water of known contaminant levels. The General Test Waters and the Challenge Test Waters were created from laboratory reagent water. The required physical and chemical characteristics of both waters are listed in Table #1. Neither water contained chlorine or any other disinfectant residuals. pH in both types of water was measured by a Denver Instruments pH-ISE Meter 2 model # 225. The pH was adjusted using a 1N solution of sodium hydroxide (NaOH) and/or hydrochloric acid (HCL). Total organic carbon (TOC) analyzed on a Shimadzu TOC-V Combustion Analyzer was adjusted in the challenge water using potassium hydrogen phthalate. The turbidity in the challenge water was achieved through the addition of A.C. Fine Test Dust Measurements of turbidity were taken on a Hach 2100A Turbidimeter. Total dissolved solids, measured by a YSI Conductivity Meter, were increased in both waters to the appropriate concentrations by the use of sea salts. The alkalinity value was obtained by following Standard Methods Number 4500-CO3. The UV absorption was measured with a Shimadzu UV-2501PC Spectrophotometer and then the percent transmittance was calculated. Proper water temperatures were monitored (Sper Scientific Infrared Thermometer 800048) and maintained throughout the entire experiment. Please refer to Table #2 for the actual readings of each parameter used in the test. Table #1. Required chemical and physical characteristics of test water per U.S.E.P.A . Guide Standard[7] Parameter General Test Water Challenge Test Water <0.1 mg/L <0.1 mg/L 6.5 - 8.5 6.5 - 8.5 <0.1 mg/L 10.0 – 15.0 mg/L 0.1 NTU - 5 NTU 30 NTU – 50 NTU Chlorine Residual pH Total Organic Carbon (TOC) Turbidity Temperature Total Dissolved Solids (TDS) 20ºC +/- 5ºC 4º +/- 1ºC 50 mg/L - 500 mg/L 1500 mg/L +/- 300 mg/L measure measure measure 80 mg/L – 120 mg/L measure 80 mg/L – 120 mg/L Color U.V. Absorption Color U.V. Transmittance Alkalinity Table #2. Actual chemical and physical characteristics of test water. Parameter General Test Water Challenge Test Water <0.10 mg/L <0.10 mg/L 7.3 6.9 Total Organic Carbon (TOC) <1 mg/L 11 mg/L Turbidity 1.3 NTU 42 NTU 20ºC 4ºC Chlorine Residual pH Temperature Total Dissolved Solids (TDS) Color U.V. Absorption Color U.V. Transmittance Alkalinity 92 mg/L 1670 mg/L 0.0125//cm 0.5400/cm 97.16% 98 mg/L 28.84% 106 mg/L 1. General Test Water - Bladder Test 1st Draw Sample (200ml to waste) and Running Sample (1,500mls) 3 Although the specified bladder (Source Vagabond MOLLE) is nominally 3.0L (3,000mls) it will only hold 2.85L (2,850mls) for this testing scenario with the pre-filter attachment on the bladder (Figure 1 and Figure 2). The SteriPENs™ and General Test Water were kept at 20°C. The General Test Water was spiked with test organism MS-2 coliphage (Escherichia coli bacteriophage ATCC® 15597-B1™). The hydration bladder was filled with 2.85L of the General Test Water. An air compressor with regulator and filter were attached to the bite valve on the end of the bite tube.. The water in the bite tube was blown back into the bladder. Once the tube was empty of water the bite valve was closed preventing water from re-entering the bite tube. No water re-entered the bite tube until sampling was initiated. A control sample was removed from each 3 liter bladder of water. Three UV doses for three liters of water (270 seconds total) were applied to each sample.. The UV dose was administered according to the manufacturers instructions for treating between 0.5 –1.0 liter of water [5]. The on/off button was pushed once to begin the treatment scenario.. The green LED flashed indicating the SteriPEN™ was ready for use. The UV lamp end was then inserted through the pre-filter adapter into the bladder sufficiently to submerge the water sensors.[Figure #3 and Figure #4] and creating a seal between the SteriPEN™ unit and the bladder. The bladder was then held in such a way as to rock it back and forth creating a wave motion for mixing [Figure #5 and Figure #6] for the 90 second cycle. At the end of the 90 second cycle the SteriPEN™ unit was removed, the on/off button was pressed again and the procedure was repeated again and then a third time for a total of three doses. Two samples were taken through the bite tube. The first sample is the “1st Draw” which extracts 200mls of water from the bladder to waste and prior to sampling. The cap was screwed tight and the bladder was inverted several times to mix before the second sample was taken. The second sample is the “Run” sample which extracts 1,500mls to waste and prior to sampling. These two aliquots were made into several dilutions and plated according to the agar layer method described by Adams using E. coli host (Escherichia coli ATCC® 15597™) [1]. The testing was run in triplicate (three bladders) with a total of three runs per bladder which equals nine (9) test sample points. 2. Challenge Test Water - Bladder Test 1st Draw Sample (200ml to waste) and Running Sample (1,500mls) Although the specified bladder (Source Vagabond MOLLE) is nominally 3.0L (3,000mls) it will only hold 2.85L (2,850mls) for this testing scenario with the pre-filter attachment on the bladder (Figure 1 and Figure 2). The SteriPENs™ and Challenge Test Water were kept at 4°C. The Challenge Test Water was spiked with test organism MS-2 coliphage (Escherichia coli bacteriophage ATCC® 15597-B1™). The hydration bladder was filled with 2.85L of the General Test Water. An air compressor with regulator and filter were attached to the bite valve on the end of the bite tube.. The water in the bite tube was blown back into the bladder. Once the tube was empty of water the bite valve was closed preventing water from re-entering the bite tube. No water re-entered the bite tube until sampling was initiated. 4 A control sample was removed from each 3 liter bladder of water. Three UV doses for three liters of water (270 seconds total) were applied to each sample.. The UV dose was administered according to the manufacturers instructions for treating between 0.5 –1.0 liter of water [5]. The on/off button was pushed once to begin the treatment scenario.. The green LED flashed indicating the SteriPEN™ was ready for use. The UV lamp end was then inserted through the pre-filter adapter into the bladder sufficiently to submerge the water sensors.[Figure #3 and Figure #4) and creating a seal between the SteriPEN™ unit and the bladder. The bladder was then held in such a way as to rock it back and forth creating a wave motion for mixing [Figure #5 and Figure #6) for the 90 second cycle. At the end of the 90 second cycle the SteriPEN™ unit was removed, the on/off button was pressed again and the procedure was repeated again and then a third time for a total of three doses. Two samples were taken through the bite tube. The first sample is the “1st Draw” which extracts 200mls of water from the bladder to waste and prior to sampling. The cap was screwed tight and the bladder was inverted several times to mix before the second sample was taken. The second sample is the “Run” sample which extracts 1,500mls to waste and prior to sampling. These two aliquots were made into several dilutions and plated according to the agar layer method described by Adams using E. coli host (Escherichia coli ATCC® 15597™) [1]. The testing was run in triplicate (three bladders) with a total of three runs per bladder which equals nine (9) test sample points. Results Table #3. Hydration Bladder with General Test Water (Three 90 Second doses) MS-2 coliphage Titer (PFU/ml)st logarithmic reductions and percent kill – 1 Draw Sample (200mls to waste) Trial #1 SteriPEN SteriPEN SteriPEN Trial #2 SteriPEN SteriPEN SteriPEN Trial #3 SteriPEN SteriPEN SteriPEN Mean Control Untreated (T=0) General Test Water Treated (T=270 Sec) #1 #2 #3 8.82E+05 8.51E+05 8.34E+05 6.46E+02 6.28E+02 6.39E+02 #1 #2 #3 8.88E+05 8.20E+05 9.33E+05 1.61E+03 1.71E+03 1.53E+03 #1 #2 #3 8.13E+05 8.46E+05 8.38E+05 2.21E+03 2.09E+03 2.03E+03 Log Reduction 3.1276 3.1352 3.1320 3.1157 2.7359 2.7416 2.6808 2.7852 2.5958 2.5649 2.6064 2.6160 2.8197 % Kill 99.9254% 99.9268% 99.9262% 99.9234% 99.8154% 99.8187% 99.7915% 99.8360% 99.7460% 99.7277% 99.7525% 99.7579% 99.8289% Table #4. Hydration Bladder with General Test Water (Three 90 Second doses) MS2 coliphage Titer (PFU/ml)logarithmic reductions and percent kill – Running Sample (1,500mls to waste) Control Untreated (T=0) Challenge Test Water Treated (T=270 Sec) 5 Log Reduction % Kill Trial #1 SteriPEN SteriPEN SteriPEN Trial #2 SteriPEN SteriPEN SteriPEN Trial #3 SteriPEN SteriPEN SteriPEN Mean #1 #2 #3 7.36E+05 7.61E+05 7.89E+05 2.11E+03 2.22E+03 2.31E+03 #1 #2 #3 8.38E+05 7.94E+05 7.83E+05 2.71E+03 2.56E+03 2.59E+03 #1 #2 #3 8.05E+05 8.38E+05 8.18E+05 1.93E+03 2.44E+03 2.23E+03 2.5370 2.5426 2.5350 2.5335 2.4874 2.4903 2.4916 2.4805 2.5735 2.6202 2.5359 2.5644 2.5329 99.7096% 99.7133% 99.7083% 99.7072% 99.6745% 99.6766% 99.6776% 99.6692% 99.7322% 99.7602% 99.7088% 99.7274% 99.7055% Table #5. Hydration Bladder with Challenge Test Water (Six 90 Second doses) MS2 coliphage Titer (PFU/ml)st logarithmic reductions and percent kill – 1 Draw Sample (200mls to waste) Trial #1 SteriPEN SteriPEN SteriPEN Trial #2 SteriPEN SteriPEN SteriPEN Trial #3 SteriPEN SteriPEN SteriPEN Control Untreated (T=0) General Test Water Treated (T=540 Sec) #1 #2 #3 1.36E+06 9.94E+05 1.27E+06 6.06E+03 6.33E+03 6.17E+03 #1 #2 #3 9.26E+05 9.14E+05 9.19E+05 6.23E+03 6.44E+03 6.36E+03 #1 #2 #3 9.61E+05 9.54E+05 9.35E+05 3.31E+03 3.05E+03 3.16E+03 Mean Log Reduction % Kill 2.2877 2.3523 2.1962 2.3145 2.1613 2.1721 2.1521 2.1599 2.4764 2.4629 2.4952 2.4711 99.4782% 99.5557% 99.3634% 99.5153% 99.3102% 99.3272% 99.2954% 99.3079% 99.6660% 99.6556% 99.6803% 99.6620% 2.3082 99.4845% Table #6. Hydration Bladder with Challenge Test Water (Six 90 Second Doses) MS2 coliphage Titer (PFU/ml)logarithmic reductions and percent kill – Running Sample (1,500mls to waste) Control Untreated (T=0) Challenge Test Water Treated (T=540 Sec) Trial #1 6 Log Reduction % Kill 2.1483 99.2867% SteriPEN SteriPEN SteriPEN Trial #2 SteriPEN SteriPEN SteriPEN Trial #3 SteriPEN SteriPEN SteriPEN Mean #1 #2 #3 1.52E+06 1.28E+06 1.41E+06 9.87E+03 1.02E+04 9.79E+03 #1 #2 #3 9.98E+05 9.92E+05 9.84E+05 6.14E+03 6.52E+03 6.43E+03 #1 #2 #3 9.52E+05 9.69E+05 9.97E+05 4.86E+03 5.75E+03 5.44E+03 2.1872 2.0991 2.1584 2.1925 2.2108 2.2820 2.1846 2.2606 2.2920 2.2267 2.2631 2.2004 99.3502% 99.2040% 99.3057% 99.3577% 99.3845% 99.3424% 99.3462% 99.4502% 99.4895% 99.4066% 99.4544% 99.3648% Conclusion The evaluation of SteriPEN™ on the General Test Water {Table #3} resulted in a 2.82-log reduction (99.829 %) of MS-2 coliphage after three doses (90 seconds each) and running 200mls of sample to waste. The use of SteriPEN™ on the Challenge Test Water {Table #5} resulted in a 2.31-log reduction (99.485%) of MS-2 coliphage after a six doses (90 seconds each) and running 200mls of sample to waste. The increased contaminants in the challenge test water slightly reduced the effectiveness of SteriPEN™ but not enough to decrease the log reduction to an inadequate level. The evaluation of SteriPEN™ on the General Test Water {Table #4} resulted in a 2.53-log reduction (99.706 %) of MS-2 coliphage after three doses (90 seconds each) and running 1,500mls of sample to waste. The use of SteriPEN™ on the Challenge Test Water {Table #6} resulted in a 2.20-log reduction (99.365%) of MS-2 coliphage after a six doses (90 seconds each) and running 1,500mls of sample to waste. The increased contaminants in the challenge test water slightly reduced the effectiveness of SteriPEN™ but not enough to decrease the log reduction to an inadequate level. The testing conducted on both General Test Waters (2.82- log reduction and 2.53-log reduction respectively) and Challenge Test Waters (2.31- log reduction and 2.20-log reduction respectively) indicates that SteriPEN™ delivered UV doses in excess of the required 40mJ/sq.cm. and therefore meets the requirements of the NSF International P248 Protocol – Emergency Military Operations Microbiological Water Purifiers. Jonathan T. Dyer/ Laboratory Director Rebecca Lebrun/ Quality Assurance Officer Figure #1 7 Hydration Bladder Unit Figure #2 Hydration Bladder Unit with SteriPEN™ Adapter Figure #3 8 Photograph of SteriPEN™ Inserted Into The Bladder Figure #4 Closer Photograph Of The UV light Inside The Bladder 9 Figure #5 Tipping the Hydration Bladder Down to Create “Wave” Motion Figure #6 Tipping the Hydration Bladder Up to Create “Wave” Motion 10 References 1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers, New York 2. Enriquez, C. and Gerba, c. 2001. Evaluation of the Steri-Pen® Water Treatment System According to the US Environmental Protection Agency Guide Standard And Protocol For Testing of Microbiological Water Purifiers. 3. Hanson, Anne 2000. Testing of Steri-Pen, a Hand-held Ultraviolet Water Treatment Device using MS2 Coliphage. 4. Hanson, Anne 2001. Testing of Steri-Pen, a Hand-held Ultraviolet Water Treatment Device using MS2 Coliphage on Visually Turbid Natural Water. 5. Hydro-Photon, Inc., 2005 SteriPEN™ Users Guide, Blue Hill, Maine http://www.hydro-photon.com/PDF/SteriPENEnglish.pdf 6. Ultraviolet Light Disinfection Technology In Drinking Water Application - An Overview. United States Environmental Protection Agency, Office of Water. EPA 811-R-96-002. September, 1996 7. U.S.E.P.A. - Task Force Report, 1987. Guide Standard and Protocol for Testing Microbiological Water Purifiers. United States Environmental Protection Agency, Registration Division, Office of Pesticide Programs and Criteria and Standards Division, Office of Drinking Water, Washington, DC 8. U.S. Army Center for Health Promotion and Preventive Medicine, 2005. Technical Information Paper; Ultraviolet Light Disinfection in the Use of Individual Water Purification Devices, Aberdeen Proving Ground, MD. 9. Wilson, B.R.P.F. Roessler, E. Van Dellen, M. Abbaszadegan and C.P. Gerba. Coliphage MS2 as a UV Water Disinfection Efficacy Test Surrogate for Bacterial and Viral Pathogens. University of Arizona, Tuczon, AZ 11 Appendix 12 CEC Clancy Environmental Consultants, Inc. Consulting and Microbiological Laboratory Services May 15, 2008 Mr. Miles Maiden Hydro-Photon P.O. Box 675 262 Ellsworth Rd. Blue Hill, ME 04614 Dear Miles, Please find enclosed the results of collimated beam testing performed on the coliphage MS2 lot that was shipped to A&L Laboratory for disinfection system testing. If you have any questions concerning this information, please contact me. Sincerely, CLANCY ENVIRONMENTAL CONSULTANTS, INC. Thomas M. Hargy Senior Scientist P.O. Box 314, St. Albans, VT 05478 Telephone (802) 527-2460 Fax (802) 524-3909 13 CEC Clancy Environmental Consultants, Inc. Consulting and Microbiological Laboratory Services MS2 UV Dose Response For Hydro-Photon Test date: May 1, 2008 Clancy Environmental Consultants, Inc. Purpose: This study was undertaken to determine the sensitivity of a specific lot of coliphage MS2 to UV irradiation. Methods: Challenge microorganism: MS-2 phage obtained from the American Type Culture Collection (ATCC #15597-B1) were propagated in the host bacteria Escherichia coli HS (pFamp)R (ATCC #700891) using a large volume liquid culture method.7 Bacteria were maintained at Clancy Environmental Consultants, Inc. (CEC) under liquid nitrogen for long-term storage. Working cultures were prepared regularly from archived sub-samples. All stocks were maintained on trypticase soy agar plates containing ampicillin and streptomycin. Stock MS2 phage was propagated in batches with titers of approximately 5 X 1011 plaque forming units (pfu) per mL. Collimated beam dose-response determination: Stock MS2 was diluted to a working volume of 100 mL containing approximately 1 x 106/mL, for dose response determination. The process, known as a collimated beam test (refer to Fig. 1) was carried out at CEC. The UV source used was a low-pressure mercury vapor lamp (Atlantic Ultraviolet G12T6L). This lamp was housed above a shutter. When the shutter was opened, light from the lamp passed through a 12 cm collimating tube to irradiate the test organisms suspended in a 6 cm diameter Petri dish. Prior to irradiations, the lamp was allowed to warm up for a minimum of 30 min. The UV incident to the surface of the Petri dish was then measured using a radiometer and detector (model X-911, Gigahertz Optik) calibrated at 254 nm. The incident irradiation across the surface of the Petri dish was measured at 5 mm intervals along an X-Y grid originating at the center of the dish. Overall irradiance distribution was then determined relative to the center reading, which was confirmed using a second radiometer (model 1400A, International Light). This value was then used in the calculation of average irradiation incident to the water surface. Factors influencing average irradiation to the entire volume include reflection from the water surface, divergence of the UV light, depth of the water, and UV absorption of the inoculated test water. The latter was measured at 254 nm by spectrophotometry (Spectronic Genesys 10uv™). UV dose was defined as the irradiation multiplied by the exposure time. P.O. Box 314, St. Albans, VT 05478 Telephone (802) 527-2460 Fax (802) 524-3909 14 CEC Clancy Environmental Consultants, Inc. Consulting and Microbiological Laboratory Services Figure 1 – Collimated Beam Apparatus Irradiations of sub-samples of the test water were made across a range of exposure times to provide UV doses of 0, 15 30, 45, 60, 75 and 90 mJ/cm2. Ten-milliliter subsamples were transferred to 6 cm diameter Petri dishes containing a 12 mm stir bar. After 1 min of stirring, the UV lamp shutter was opened and the suspension irradiated for a predetermined length of time. A 0 mJ/cm2 dose was run simultaneously with the irradiation test for the highest mJ/cm2 dose, in the absence of UV. This 0 dose control provides the base count for determination of log inactivation. Each dose was run in duplicate. Results: Concentrations of survivors and resulting log10 inactivations achieved at the above doses are given in Table 1 and Figure 2. The best fit equation for the average inactivation values is the polynomial equation: y = 2.831x2 + 13.19 +0.214 Thus if a UV disinfection test achieves 2.1 log inactivation of this MS2, a UV dose of 40.4 mJ/cm2 would have been delivered. P.O. Box 314, St. Albans, VT 05478 Telephone (802) 527-2460 Fax (802) 524-3909 15 CEC Clancy Environmental Consultants, Inc. Consulting and Microbiological Laboratory Services Table 1. MS2 surviving concentrations and log10 inactivations in collimated dose response test. Lot #: 120407F Replicate: A Dose PFU/mL CB date: log UV dose 01-May-08 Replicate: B Dose PFU/mL log UV dose UV dose A B average 0 1.2E+06 6.10 0 1.3E+06 6.12 0 0.00 0.00 0.00 15 1.5E+05 5.17 15 1.5E+05 5.19 15.0 0.93 0.93 0.93 30 2.9E+04 4.47 30 2.9E+04 4.47 30.0 1.63 1.65 1.64 45 6.6E+03 3.82 45 7.1E+03 3.85 45.0 2.28 2.27 2.27 60 1.7E+03 3.23 60 2.1E+03 3.32 60.0 2.87 2.80 2.83 75 5.3E+02 2.72 75 6.1E+02 2.78 75.0 3.37 3.34 3.36 90 1.8E+02 2.26 90 3.1E+02 2.49 90.0 3.84 3.63 3.73 Figure 2. UV Dose response of MS2 Lot 120407F UV dose response, Lot 120407F 90 80 70 60 UV Dose 50 2) 40 (mJ/cm 30 20 10 0 0.00 2 + 13.19x + 0.214 y = 2.831x 1.00 2.00 Average Log 3.00 4.00 Inactivation P.O. Box 314, St. Albans, VT 05478 Telephone (802) 527-2460 Fax (802) 524-3909 16