Send to: Clean Water Environmental, LLC 1939 Talamore Court Southeast, Grand Rapids, MI 49546 Dr. Dale Williams Result:COMPLETE Report Date: December 28 th, 2015 Customer Name: Clean Water Environmental, LLC Location of Testing: NSF Ann Arbor Description: Antimicrobial Efficacy Testing of SAFI in Drinking Water Test Type: Test Only Job Number: J-00183576 Project Number: 10006503 NSF Corporate: C0243887 Project Manager: J. Vantine Executive Summary: Clean Water Environmental s SAFI antimicrobial product was evaluated to determine its antimicrobial efficacy in respect to disinfecting drinking water contaminated with bacterial pathogens. Test water was prepared with a designated copper concentration of the SAFI product and then challenge populations were monitored for 24 hours. The efficacy studies demonstrated that the SAFI product, at an active copper concentration of 1ppm, achieved greater than a 4.28 log reduction of Shigella dysenteriae at 4 hr and 24 hr. The 10ppm concentration of the SAFI product also resulted in significant reduction in viability of Escherichia coli at 24 hour (>6.96 log). Thank you for having your product tested by NSF International. Please contact your Project Manager if you have any questions or concerns pertaining to this report. Report Authorization: Robert Donofrio Director, Applied Research Center Scope of Test Report
The antibacterial properties of the SAFI product were tested against two challenge organisms: Escherichia coli ATCC 8739 and Shigella dysenteriae ATCC 9361. E.coli is a representative gram negative bacterium and one of the most well-known fecal-coliforms. S. dysenteriae is also a gram negative rod and is the causative agent of bacillary dysentery. A suspension of 1 x 10 8 for each challenge organism was prepared and exposed to SAFI product with various Copper concentrations. The surviving populations of the microorganisms were then evaluated at specified time points. Methodology The following organisms have been included in the study. o E. coli ATCC 8739 o S. dysenteriae ATCC 9361 Organism Preparation Escherichia. coli ATCC 14033 o 4 days prior to the Time Kill Study- 0.1mL of the organism was transferred from freezer stock to 10mL Tryptic Soy Broth (TSB) and incubated at 35 C for approximately 24 hours. This is referred to as the Day 1 culture. o 3 days prior to the Time Kill Study- 1mL of the Day 1 culture was passed to two 250mL bottles of TSB and incubated at 35 C for approximately 24 hours. This is referred to as the Day 2 culture. o 2 days prior to the Time Kill Study- Both 250 ml cultures were submitted to centrifugation at 3500 rpm for 10 minutes. The pellet was then resuspended in 50 ml using sterile phosphate buffer (approximately 0.25M). Shigella dysenteriae ATCC 9361 o 2 days prior to the Time Kill Study- 0.1 ml the organism was passed to a bottle of 1L TSB and incubated at 35 C for approximately 24 hours. o 1 day prior to the Time Kill Study- The 1L culture was submitted to centrifugation at 3500 rpm for 10 minutes. The pellet was then resuspended in 50 ml sterile phosphate buffer (approximately 0.25M).
The bacterial challenge stocks were added to the test water so that a minimum concentration of 1 x 10 8 is achieved at the start of the study. Preparation of the Test Water NSF Chemistry lab prepared test water to match the following profile water characteristics: - Free of any chlorine or other disinfectant residual - ph 6.5 to 8.5 - Total Organic Carbon (TOC) 0.1 to 5.0 mg/l - Turbidity 0.1 to 5 NTU - Temperature - 20 C±5 C - Total Dissolved Solids (TDS) 50 to 500 mg/l NSF verified the aforementioned abiotic parameters of the test water using EPA and APHA standard methods. The reference methods used for the analytical testing are provided in the Reference section. Preparation of SAFI product NSF Chemistry lab prepared solutions of the SAFI product with the copper concentrations of 1 and 10 ppm. Concentrations of copper were determined with ICAP analysis based on EPA 200.7. Time Kill Study The Time Kill Study was based on the references USEPA Guide Standard for Testing Microbiological Water Purifiers, NSF Protocol P231 Microbiological Water Purifiers, and ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure. Modifications to these methods are contained in this report. NSF s Chemistry Laboratory delivered the prepared SAFI solutions (concentrations of 1ppm and 10ppm) to NSF s Microbiology Laboratory. A flask of test water was also prepared and delivered to NSF s Microbiology Laboratory to serve as a control water. Triplicate 100mL volumes of each type of test water were set up for both organisms and SAFI concentration combinations. The flasks were placed on a rotary shaker (200 rpm) and were held at 21.0 +/- 2 ºC. These flasks were inoculated with the appropriate amount of organism to achieve a target concentration of 1 x 10 8.
The sampling points for this study were as follows: 0 hour, 1 hour, 4 hours, and 24 hours. At each sampling point, a 1mL aliquot was removed from each flask for processing, immediately transferred to 9mL of Neutralization Broth (0.25M potassium phosphate, ph 7.2, 0.1% sodium thioglycollate, 0.6% sodium thiosulfate, 0.5% Tween 80, and 0.7% lecithin) and vortexed. Serial ten-fold dilutions were performed using Neutralization Broth and the samples were processed and plated onto the appropriate selective media, then incubated. Samples containing E. coli were processed and plated onto 3M Petrifilm E.coli/Coliform Count Plates in duplicate and incubated at 35 C ± 1 C for 48 hours ± 4 hours. Samples containing S. dysenteriae were processed using the spread plate method. These were plated onto Xylose Lysine Deoxychlorate (XLD Agar) in duplicate and incubated at 35 C ± 1 C for 24 ± 2 hours. Post incubation, plates were read and enumerated using a Quebec Colony Counter. Colony counts were converted to CFU per milliliter concentrations for each replicate plate. Statistical Analysis To determine the efficacy of the Client s product, log reduction in challenge organism viability was calculated at each exposure time point. The following formula was employed to calculate log reduction: Log Reduction = log10 (N / T) Where: N is the number of viable organism in the numbers control (untreated group) at time point x T is the number of viable organism in the treatment group (exposed to SAFI product) at time point x To determine the statistical significance of the log reduction data sets, a student s t-test using a 95% confidence interval (α=0.05) was performed, comparing each treatment group to the numbers control at each exposure time point.
Results and Discussion The results of the efficacy testing are presented in Tables 2 through 5. (Appendix A). For all experiments the media/reagent and organism growth controls indicated the tests were run under valid conditions. E. coli At copper concentration of 10ppm a significant log reduction of the E. coli challenge population was not observed until the 24 hour sampling time point. The challenge population was reduced by >6.96 log 10. At 1 and 4 hour time points 1ppm and 10ppm copper concentrations solutions showed limited challenge population reduction (less than a 2.34 log 10 ). S. dysenteriae A significant log reduction of the S. dysenteriae challenge population was not observed until the 24 hour sampling time point for both the 1ppm and 10ppm copper concentrations. At the 1ppm copper concentration there was a 4.28 log 10 reduction. At the 10ppm concentration there was a reduction of >5.9 log 10.
References USEPA Guide Standard for Testing Microbiological Water Purifiers NSF Protocol P231 Microbiological Water Purifiers ASTM E2315-03 will be used as the reference methods for testing. EPA Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma- Atomic Emission Spectrometry Method 200.7 EPA Determination of Turbidity by Nephelometry Method 180.1 SM 2540-C Solids Standard Methods for the Examination of Water and Wastewater 1997 EPA Approved Methodology SM 4500-Cl Chlorine (Residual) Standard Methods for the Examination of Water and Wastewater 2000 EPA Approved Methodology SM 4500-H+B ph Value in Water by Potentiometry Using a Standard Hydrogen Electrode Standard Methods for the Examination of Water and Wastewater 1997 EPA Approved Methodology SM 5310C Total Organic Carbon (TOC) Standard Methods for the Examination of Water and Wastewater 2000 EPA Approved Methodology
Scope of Work Revisions Scope of work authorized: August 8 th, 2015 NSF authorized / Quote received August 27 th, 2015 Revision o Client Authorized: September 2 nd, 2015 o ARC Program Authorized: September 2 nd, 2015 o Revisions made: Amended from Proposal Letter format to Attachment/Annex format to track changes. In Organisms Evaluations updated the bacteria to include ATCC. In Test Setup and Performance updated the concentrations to remove 0.5 ppm, per Client request. Updated Sample Requirements to include Certificate of Analysis. Updated Cost of Service chart details: Corrected Phase 1 to be Phase 2 (clerical oversight) Updated number of concentrations from 3 to 2 per Clients request. Updated (one control of 0ppm copper) to read (once control of tap water) this clarification was determined to better document what was being used and to acknowledge that the tap water may contain trace amounts of copper. Delete 1 test water assessed. Depending on the laboratory schedule for processing study it will be determined if 1 test or parent water will be assessed or 2. The laboratory will report test water characteristics. Updated footer to include page numbering new (project) version number 10014421 1
Appendix A- Tables and Figures
Table 1: Test water and SAFI product characteristics. Applicable water analysis methods are provided in the References section. E. coli ATCC 8739 Target Concentration Actual [Cu] Concentration Other Characteristics SAFI [Cu] of 1ppm 0.93 mg/l Solids, Total Dissolved 350 mg/l SAFI [Cu] of 10ppm 11 mg/l Turbidity 0.1 NTU Test water (Blank) 0.04 mg/l ph 6.9 S. dysenteriae ATCC9361 Temperature 21 deg C Total Organic Carbon 1.9 mg/l Total Residual Chlorine ND(0.05) mg/l SAFI [Cu] of 1ppm 0.93 mg/l Solids, Total Dissolved 400 mg/l SAFI [Cu] of 10ppm 9 mg/l Turbidity 0.1 NTU Test water (Blank) 0.02 mg/l ph 7.08 SAFI - [Cu] 67000 mg/l Temperature 19 deg C Total Organic Carbon 2 mg/l Total Residual Chlorine ND(0.05) mg/l
Table 2: Data for all replicate samples of E. coli in test water evaluated against 2 concentrations of SAFI product (1 ppm and 10 ppm). The experiment was conducted at 21.0 +/- 2 ºC and all flasks were placed on a rotary shaker set at 100 rpm for the entirety of the experiment. The limit of detection for this assay was 20. Hour 0 Numbers Control Replicate A Replicate B Replicate C Average Log 10 Average Log 10 Average Log 10 1.10E+08 1.10E+08 8.04 9.90E+07 1.00E+08 8.00 9.00E+07 1.01E+08 8.00 1.10E+08 1.01E+08 1.12E+08 1ppm 9.50E+07 9.75E+07 7.99 8.90E+07 9.80E+07 7.99 9.80E+07 1.01E+08 8.00 1.00E+08 1.07E+08 1.04E+08 10ppm 8.80E+07 8.80E+07 7.94 8.70E+07 8.95E+07 7.95 9.30E+07 9.45E+07 7.98 Hour 1 8.80E+07 9.20E+07 9.60E+07 Numbers Control 1.30E+08 1.21E+08 8.08 1.38E+08 1.30E+08 8.11 1.13E+08 1.24E+08 8.09 1.12E+08 1.22E+08 1.34E+08 1ppm 5.10E+07 5.05E+07 7.70 6.80E+07 6.60E+07 7.82 6.60E+07 6.55E+07 7.82 5.00E+07 6.40E+07 6.50E+07 10ppm 3.00E+06 4.15E+06 6.62 5.50E+06 6.00E+06 6.78 6.60E+06 6.20E+06 6.79 Hour 4 5.30E+06 6.50E+06 5.80E+06 Numbers Control 1.44E+08 1.47E+08 8.17 1.25E+08 1.09E+08 8.04 1.24E+08 1.24E+08 8.09 1.50E+08 9.20E+07 1.23E+08 1ppm 4.70E+07 4.10E+07 7.61 4.00E+07 3.50E+07 7.54 5.50E+06 5.45E+06 6.74 3.50E+07 3.00E+07 5.40E+06 10ppm 4.20E+05 4.40E+05 5.64 6.70E+05 6.75E+05 5.83 6.40E+05 6.20E+05 5.79 Hour 24 4.60E+05 6.80E+05 6.00E+05 Numbers Control 1.05E+08 1.03E+08 8.01 8.60E+07 8.60E+07 7.93 1.06E+08 8.90E+07 7.95 1.00E+08 8.60E+07 7.20E+07 1ppm 4.20E+04 5.90E+04 4.77 4.00E+05 4.55E+05 5.66 4.10E+05 4.55E+05 5.66 7.60E+04 5.10E+05 5.00E+05 10ppm <10 1.00E+01 1.00 <10 1.00E+01 1.00 <10 1.00E+01 1.00 <10 <10 <10
Table 3: Summary of log and percent reductions observed for E. coli in test water at 2 concentrations of SAFI product (1 ppm and 10 ppm). Hour 0 Log10 (Mean ± SD) Cellular Reduction (Log10 ) Statistical Significance (p Value, where α=0.05) Numbers Control 8.02 ± 0.02 NA NA 1ppm 7.99 ± 0.01 0.02 0.28 10ppm 7.96 ± 0.02 0.06 0.02 Hour 1 Numbers Control 8.10 ± 0.02 NA NA 1ppm 7.78 ± 0.07 0.32 <0.001 10ppm 6.73 ± 0.10 1.37 <0.001 Hour 4 Numbers Control 8.10 ± 0.07 NA NA 1ppm 7.30 ± 0.49 0.80 <0.01 10ppm 5.76 ± 0.10 2.34 <0.001 Hour 24 Numbers Control 7.96 ± 0.04 NA NA 1ppm 5.36 ± 0.51 2.60 <0.001 10ppm <1.00 ± 0.00 >6.96 <0.001 SD = Standard deviation
Table 4: Data for all replicate samples of S. dysenteriae in test water evaluated against 2 concentrations of SAFI product (1 ppm and 10 ppm). The experiment was conducted at 21.0 +/- 2 ºC and all flasks were placed on a rotary shaker set at 100 rpm for the entirety of the experiment. The limit of detection for this assay was 10. Hour 0 Replicate A Replicate B Replicate C Average Log 10 Average Log 10 Average Log 10 Numbers 1.00E+08 1.09E+08 8.04 1.01E+08 1.13E+08 8.05 1.15E+08 1.19E+08 8.07 Control 1.18E+08 1.25E+08 1.22E+08 1ppm 8.70E+07 9.95E+07 8.00 1.95E+08 1.61E+08 8.21 3.30E+07 5.10E+07 7.71 1.12E+08 1.27E+08 6.90E+07 10ppm 1.43E+08 1.10E+08 8.04 1.24E+08 1.14E+08 8.05 8.50E+07 1.15E+08 8.06 Hour 1 7.70E+07 1.03E+08 1.45E+08 Numbers 1.06E+08 1.21E+08 8.08 1.31E+08 1.27E+08 8.10 1.81E+08 1.82E+08 8.26 Control 1.35E+08 1.22E+08 1.82E+08 1ppm 8.10E+07 5.55E+07 7.74 6.90E+07 5.80E+07 7.76 3.70E+07 3.50E+07 7.54 3.00E+07 4.70E+07 3.30E+07 10ppm 4.10E+07 3.05E+07 7.48 2.29E+07 1.89E+07 7.28 1.67E+07 1.51E+07 7.18 Hour 4 2.00E+07 1.48E+07 1.35E+07 Numbers 2.58E+08 2.44E+08 8.39 1.38E+08 1.15E+08 8.06 1.11E+08 9.85E+07 7.99 Control 2.30E+08 9.10E+07 8.60E+07 1ppm 3.20E+06 5.35E+06 6.73 1.58E+07 1.42E+07 7.15 1.87E+07 1.70E+07 7.23 7.50E+06 1.26E+07 1.53E+07 10ppm 3.30E+06 3.90E+06 6.59 1.79E+06 2.34E+06 6.37 1.35E+06 8.80E+05 5.94 Hour 24 4.50E+06 2.89E+06 4.10E+05 Numbers 1.40E+06 2.40E+06 6.38 3.67E+07 2.85E+07 7.45 6.20E+06 8.55E+06 6.93 Control 3.39E+06 2.02E+07 1.09E+07 1ppm 4.00E+02 3.60E+02 2.56 1.61E+03 1.70E+03 3.23 1.40E+02 1.40E+02 2.15 3.20E+02 1.78E+03 1.40E+02 10ppm 1.00E+01 <1.00E+01 <1.00 <10 <1.00E+01 <1.00 2.00E+01 1.00E+01 1.00 <10 <10 <10
Table 5: Summary of log and percent reductions observed for S. dysenteriae in test water at 2 concentrations of SAFI product (1 ppm and 10 ppm). Hour 0 Log10 (Mean ± SD) Cellular Reduction (Log10 ) Statistical Significance (p Value, where α=0.05) Numbers Control 8.05 ± 0.02 NA NA 1ppm 7.97 ± 0.25 0.08 0.43 10ppm 8.05 ± 0.01 0.002 0.82 Hour 1 Numbers Control 8.15 ± 0.10 NA NA 1ppm 7.68 ± 0.12 0.46 <0.01 10ppm 7.31 ± 0.16 0.83 <0.001 Hour 4 Numbers Control 8.15 ± 0.21 NA NA 1ppm 7.04 ± 0.27 1.11 <0.01 10ppm 6.30 ± 0.33 1.85 <0.001 Hour 24 Numbers Control 6.92 ± 0.54 NA NA 1ppm 2.64 ± 0.55 4.28 <0.001 10ppm <1.00 ± 0.00 >5.92 <0.001 SD = Standard deviation