Authors R. E. Danielson 1, J.R. Truscott BioVir Laboratories, Inc.; Benicia CA 2. Wastewater Management Div., City of Fresno, CA

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1 Validation of large volume MPN techniques using a modification of US EPA method 1601: detecting low concentrations of coliphage from secondary effluent substrata infiltration systems Authors R. E. Danielson 1, J.R. Truscott 1 S. Hogg 2, R. Staggs 2, and T. Adams 2 1. BioVir Laboratories, Inc.; Benicia CA 2. Wastewater Management Div., City of Fresno, CA

2 Fresno-Clovis Regional Wastewater Reclamation Facility (RWRF) 70 million gallons domestic sewage/dy 70% domestic, 30% commercial & industrial Activated Sludge Secondary Treatment Facility Majority of Effluent discharged into 1300 acres of infiltration ponds

3 Fresno-Clovis Regional Wastewater Reclamation Facility Network of 21 reclamation wells established since 1975 From February November the extracted water sent to Fresno Irrigation District to mix with surface water for agricultural purposes 25,000 acre-ft/yr from the network

4 HOW FRESNO S WASTEWATER MANAGEMENT SYSTEM WORKS

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8 Fresno-Clovis Regional Wastewater Reclamation Facility CA DPH required RWRF to evaluate the soil treatment as part of meeting CA Title 22 Disinfected tertiary recycled water RWRF met this criteria for total coliforms and turbidity

9 Fresno-Clovis Regional Wastewater Reclamation Facility However, RWRF not able to demonstrate 5-log reduction of viruses Bacteriophage used as measure for viruses Typical phage concentration from RWFW effluent < 5-log 5 reduction at the wells using standard methods

10 Viruses Viruses are obligate intracellular parasites Bacteriophage are viruses that infect bacteria AKA Phage CA DPH requested testing for the MS-2 bacteriophage

11 Viruses (con t) MS-2 2 phage require a specific bacterial host for multiplication Bacteria (e.g., E. coli) with a specialized appendage (pilus)) are the natural host MS-2 2 phage 0.03 microns dia.

12 US EPA Method 1601 US EPA developed Method 1601 to detect low levels of phage in groundwater It was designed for 1 L sample volumes RWFW phage concentration too low to demonstrate 5-log 5 reduction with 1-L 1 volumes in an MPN format

13 Most-Probable-Number (MPN) MPN commonly applied to bacterial test for coliforms Standard Methods 9221 Concentration can be estimated from the number of tubes with a positive response within a dilution series Refer to a Table with MPN outcomes

14 MPN MPN can be calculated based on the formula of Thomas (SM online) MPN/ ml = 1 x P/ (A x B)½ P = number of positive results A = volume of negative responding vessels B = volume of total sample

15 MPN For coliforms, typical dilution series: 5-tube, 3-dilutions 3 (10 ml, 1 ml, 0.1 ml) Results reported as MPN/100 ml For the sensitivity needed for phage by RWFW 100 fold more 3-tube, 3-dilution 3 (10 L, 1L, 0.1 L)

16 Increase Method Sensitivity Look at greater volumes 33.3 L samples MPN/ ml = 1xP/ (A x B)½ If 1 10 L portion is positive 1/(23,000 ml x 33,000) 1/ MPN/mL 1/2 = Equivalent to (-)4.44( log phage

17 Increase Method Sensitivity If secondary effluent has 10 pfu/ml, then method will demonstrate 5.44 log reduction 1 log phage effluent (-)) 4.44 = 5.44 log rduct If secondary effluent has 100 pfu/ml, then the method will demonstrate 6.44 log reduction. 2 log phage effluent (-)) 4.44 = 6.44 log rduct

18 That s the theory What s the reality? CA DPH requested that BioVir Laboratories demonstrate that the modification to 1601 (larger volume) can detect phage at this low concentration If one 10 L portion is positive, that would be equivalent to MPN/mL or 1.2 MPN / 33.3L

19 Validation of EPA 1601 for Larger Volumes Method 1601 is performance-based method Validation procedures for single-lab lab modifications (Tier-1 1 PBMS) Initial Demonstration of Capability 10 Reagent Grade Water Spikes 10 Matrix Spikes Associated Positive & Negative Controls

20 Method 1601 Water sample distributed to 9 vessels 3 10 L 3 1 L L Add MgCl,, Antibiotics, Host Bacteria, and Growth media

21 Method 1601 Incubate o/n at 35 o C to amplify phage in bacterial host

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23 Method 1601 Amplified phage detected by transferring to agar plate with host bacteria Those vessels that have phage will interact with the host in the agar and lyse them, creating a clear zone

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25 Phase 1 Spike Estimate If about 1 phage / 33.3 L required, need to demonstrate variability in delivering this low concentration Beginning with known concentration, made dilution series, enumerated by plate count Plated out 10 replicates near extinction point.

26 Phage Spike Variability pfu/0.1 ml Trial Phage

27 Phase 1 Spike Estimate About 30% variance in spikes at low concentration (< 10 pfu) This will make hitting the 1 pfu/33.3 L difficult

28 Phase 2 Reagent Grade Water 10 Samples needed that can demonstrate desired response with low concentration of phage 9 challenge runs (2-3 3 samples per run) Spiking with pfu/100 L 38 total samples processed Including +/- controls

29 Phase 2 Reagent Grade Water Sample Spike pfu/33.3l MPN Result ( ) 11, 12, , 17, , , pfu avg MPN (phage/ml) < < < < < <

30 Phase 2 - RGW Avg of 1.3 pfu/33.3 L 4 out of 10 samples (+) EPA 1601 Validation criteria At least 2 out of 10 samples (+)

31 Phase 3 Matrix Spikes 200 gal tank employed to transport necessary volume back to lab

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33 Phase 3 Matrix Spikes 10 Samples needed that can demonstrate desired response with low concentration of phage 8 challenge runs (2-3 3 samples per run) Spiking with pfu/100 L 38 total samples processed Including +/- controls

34 Phase 3 Matrix Spikes Sample Spike pfu/33.3l MPN Result ( ) 41, 42, , 57, MPN (phage/ml) < < < < < , 75, pfu avg <

35 Phase 3 Matrix Spike Avg of 1.5 pfu/33.3 L 4 out of 10 samples (+) EPA 1601 Validation criteria At least 2 out of 10 samples (+)

36 Validation of 1601 Tier 1 Single Laboratory Validation for larger volumes (33.3 L) demonstrated CA DPH: each site will need to re- validate matrix spike to determine detection limits in that specific matrix

37 Phase 4 Larger Volumes Large bulk volumes cumbersome and expensive to ship Large volumes limit sample # assay Not enough incubator space Sometimes 33.3 L not sensitive enough Increase in sensitivity must be 10-fold to be significant

38 Phase 4 Filter Concentration Virus filter concentration? Current virus filter very good at binding phage Not good at releasing phage Argonide Nano-Ceram filter Net (+) charge

39 Phase 4 - Filter Concentration Is it necessary to release the virus for a presence/absence assay? Filter unit relatively easy to work and transport Of course, another validation study is necessary

40 Phase 4 Lab Validation 3- Tube MPN with filters

41 Process Filter Remove Filter from Housing, transfer to Zip-Lock Bags, collect residual water Add MgCl,, Antibiotics, Host Bacteria, and Growth media 24 hr incubation Confirm on Plates

42 Results 4/10 (+) Sample # Spike (pfu/300l) Result (+) Control (-) Control / /3 3 0/3 4 0/3 5 1/3 6 1/3 7 0/3 8 0/3 9 1/3 10 0/3

43 Assay Sensitivity MPN/mL = 1/(200,000 x 300,000)½ = 4.0 x MPN/mL or 4.0 x MPN/100mL Single positive (1/3) = (-)( ) 3.4 Log In order to demonstrate 5-log 5 reduction there needs to be a minimum of 40 pfu/100ml Log 40 (-)3.4 Log = 5.0 Log

44 Laboratory Validation Large Volume Modification with 3-filters 3 has meet Single Laboratory Validation with reagent water (Tier 1 - PBMS) Summer 2010 Matrix Spikes Two Central Valley Locations

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