v> EPA United States Environmental Protection Agency Method Development for Unregulated Contaminants in Drinking Water: Public Meeting and Webinar Held June 6, 2018 USEPA, Office of Ground Water and Drinking Water Office of Water (MLK 140) EPA 815-A-18-001 June 2018 ------- Methods Development for Unregulated Contaminants in Drinking Water Methods Development for Unregulated Contaminants in Drinking Water ฆ Public Meeting and Webinar June 6, 2018 9:00 a.m. - 3:00 p.m. ET U.S. EPA Office of Water and Office of Research and Development Welcome & SDWA Regulatory Process Brenda Parris, U.S. EPA Office of Ground Water and Drinking Water Technical Support Center Page 1 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Participating by Webinar Listen-only mode Click on "+" next to "Questions" in the control panel (Figure 1) to submit questions/comments Type a question in the box; click send (Figure 2) Submit questions as soon as possible Questions will be answered at the end of the presentations !:igure 1 0 Polls (0/0) ft 0 Chat Figure 2 f1 Questions [Enter a question for staff] June 2018 U.S. Environmental Protection Agency USฎ (S) Slide 3 of 206 Agenda 8:30-9:00 Stakeholder Sign-In Welcome & SDWA Regulatory Process Overview of Method Development EPA Method 542 EPA Methods 524.2/524.3/524.4 and 525.3 EPA Method 556.1 ~10:15-10:30 Break EPA Method 540 & 543 EPA Methods 537 & 538 Method in Development: PFAS Method in Development 558: Ethyl carbamate (Urethane) and N-Methyl-2-pyrrolidone Method in Development: Nonylphenols ~11:45-12:45 Lunch Method in Development: Legionella Method in Development: Mycobacterium ~l:45-2:00 Break 2:00-3:00 Open Forum and Discussion Closing Remarks Page 2 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Overview Regulatory background for UCMR Safe Drinking Water Act (SDWA) authority Relationships to: Contaminant Candidate List (CCL) Unregulated Contaminant Monitoring Rule (UCMR) Regulatory Determination Six-Year Review June 2018 U.S. Environmental Protection Agency Slide 5 of 206 SDWA Enacted in 1974, SDWA authorized EPA to set enforceable health standards for contaminants in drinking water 8 National Primary Drinking Water Regulations (NPDWRs) 1986 SDWA amendments were the basis for the original UCMR State drinking water programs managed the original UCM program PWSs serving > 500 people were required to monitor 1996 SDWA amendments changed the process of developing and reviewing NPDWRs CCL UCMR Regulatory Determination Six-Year Review June 2018 U.S. Environmental Protection Agency Slide 6 of 206 Page 3 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water General Flow of SDWA Regulatory Processes * Public Review and Comment Regulatory Determination Rule Preliminary m Regulatory f Determinations 24 Final Regulatory ^^iionths^^ Determinations Research Needs Assessment Review UCMR Monitoring Results UCMR No further action if decision is to not regulate May develop health advisory Proposed Rule 1 (NPDWR) i l 1 18 1 n ^ months ! =>> Final Rule Six-Year Review of (NPDWR) Existing NPDWRs Increased specificity and confidence in the type of supporting data used (e.g., health, occurrence, treatment) is needed at each stage. June 2018 U.S. Environmental Protection Agency Slide 7 of 199 CCL SDWA 1412(b)(1)(B) established listing of contaminants for regulatory consideration: EPA shall publish a list of contaminants which are: not subject to any proposed or promulgated NPDWR, which are known or anticipated to occur in PWSs, and which may require regulation under SDWA The Final CCL 4 was published November 17, 2016 and included 97 chemicals or chemical groups and 12 microbes June 2018 U.S. Environmental Protection Agency Slide 8 of 199 Page 4 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water CCL4 1,1,1,2-Tetrachloroethane (502.2, 524.2, 524.3, 524.4) Erythromycin (542) Oxirane, methyl- 1,1-Dichloroethane (UCMR 3) Estradiol (17-beta estradiol) (UCMR 3) Oxydemeton-methyl (538) 1,2,3-Trichloropropane (UCMR 3) Estriol (UCMR 3) Oxyf 1 uo rfe n (UC M R 4) 1,3-Butadiene (UCMR 3) Estrone (UCMR 3) Perfluorooctane sulfonic acid (PFOS) (UCMR 3) 1,4-Dioxane (UCMR 3) Ethinyl Estradiol (17-alpha ethynyl estradiol) (UCMR 3) Perfluorooctanoic acid (PFOA) (UCMR 3) 17 alpha-Estradiol Ethoprop (UCMR 4) Permethrin (UCMR4) 1-Butanol (UCMR 4) Ethylene glycol P rofe nofos (UC M R 4) 2-Methoxyethanol (UCMR 4) Ethylene Oxide Quinoline (UCMR 4) 2-Propen-l-ol (UCMR 4) Ethylene thiourea RDX (UCMR 2) 3- Hyd roxycar bofu ra n (531.1, 531.2, 540, 543) Formaldehyde (556, 556.1) sec-Butyl benzene (502.2, 524.2, 524.3, 524.4) 4,4'-M et hy le ne d ia n i 1 i ne Germanium (UCMR 4) Tebuconazole (UCMR4) Ace p hate (538) Halon 1011 (bromochloromethane) (UCMR 3) Tebufenozide (540, 543) Acetaldehyde (556, 556.1) HCFC22 (UCMR 3) Tellurium Acetamide Hexane Thiodicarb Acetochlor (UCMR 1, UCMR 2) Hydrazine Thiophanate-methyl Acetochlor ethanesulfonic acid (ESA) (UCMR 2) Manganese (UCMR4) Toluene diisocyanate Acetochlor oxanilic acid (OA) (UCMR 2) Mestranol Tribufos (UCMR 4) Acrolein Methamidophos(538) Triethylamine Alachlor ethanesulfonic acid (ESA) (UCMR 2) Methanol Triphenyltin hydroxide (TPTH) Alachlor oxanilic acid (OA) (UCMR 2) Methyl bromide (Bro mo methane) (UCMR 3) Urethane (In Development) alpha-Hexachlorocyclohexane (UCMR 4) Methyl tert-butyl ether (UCMR 1) Vanadium (UCMR 3) Aniline Metolachlor(UCMR 2) Vinclozolin (525.3, 527) Bensulide (540, 543) Metolachlor ethanesulfonic acid (ESA) (UCMR 2) Ziram Benzyl chloride Metolachlor oxanilic acid (OA) (UCMR 2) Adenovirus Butylated hydroxyanisole (UCMR4) Molybdenum (UCMR 3) Caliciviruses(UCMR3) Captan Nitrobenzene (UCMR 1) Campylobacterjejuni Chlorate (UCMR 3) Nitroglycerin Enterovirus (UCMR 3) Chloromethane (Methyl chloride) (UCMR 3) N-Methyl-2-pyrrolidone (In Development) Escherichia coli (0157) Clethodim N-Nitrosodiethylamine (NDEA) (UCMR 2) Helicobacter pylori Cobalt (UCMR 3) N-Nitrosodimethylamine (NDMA) (UCMR 2) Hepatitis A virus Cumene hydroperoxide N-Nitroso-di-n-propylamine (NDPA) (UCMR 2) Legionella pneumophila (In Development) Cya not ox i ns (UC M R 4) N -N it rosod i p he ny la m ine Mycobacterium avium (In Development) Dicrotophos (538) N-Nitrosopyrrolidine (NPYR) (UCMR 2) Naegleria fowleri Dimethipin (UCMR 4) Nonylphenol (In Development) Salmonella enterica Diuron (UCMR 1) No ret hind rone (19-N o ret histe rone) Shigella sonnei Equilenin n-Propylbenzene (502.2, 524.2, 524.3, 524.4) Equilin (UCMR 3) o-Toluidine (UCMR4) ^ PRQ-^ General Flow of SDWA Regulatory Processes ^ Public Review and Comment CCL Regulatory UCMR Monitoring Results UCMR Determination Preliminary Regulatory Determinations Rule Final Regulatory Determinations No further action if decision is to not regulate May develop health advisory Proposed Rule (NPDWR) Review Six-Year Review of Existing NPDWRs ~ Increased specificity and confidence in the type of supporting data used (e.g., health, occurrence, treatment) is needed at each stage. June 2018 U.S. Environmental Protection Agency Slide 10 of 206 Page 5 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water UCMR SDWA section 1445(a)(2), established requirements for the UCMR Program: Issue list of no more than 30 unregulated contaminants, once every 5 years Require PWSs serving population >10,000 people as well as a nationally representative sample of PWSs serving <10,000 people to monitor Store analytical results in the National Contaminant Occurrence Database (NCOD) for Drinking Water EPA funds shipping/analytical costs for small PWSs EPA manages program in partnership with states June 2018 U.S. Environmental Protection Agency Slide 11 of 206 UCMR History UCMR 1 (2001-2005, 26 contaminants) UCMR 2 (2007-2011, 25 contaminants) UCMR 3 (2012-2016, 30 contaminants) UCMR 4 (2017-2021, 30 contaminants) Published in the FR on December 20, 2016 PWSs monitor 2018-2020 Each new UCMR cycle is established via a revision to the rule for the ongoing/preceding cycle. June 2018 U.S. Environmental Protection Agency Slide 12 of 206 Page 6 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Objective of UCMR Program Collect nationally representative occurrence data for unregulated contaminants that may require regulation under the SDWA Consider data collected as part of future EPA decisions on actions to protect public health Provide data to States, local governments and to the public for their use in decisions regarding public health protection June 2018 National occurrence data publically available: http://www.epa.gov/dwucmr/occurrence-data-unregulated- contaminarit-moriitoring-rule U.S. Environmental Protection Agency Slide 13 of 206 2* PRO^S General Flow of SDWA Regulatory Processes Public Review and Comment CCL 1*. Draft CCL t 1 f k ~j l Draft UCMR t Final UCMR 1 UCMR Monitoring Results UCMR Regulatory Determination Preliminary Regulatory Determinations Rule Review Final Regulatory Determinations No further action if decision is to not regulate May develop health advisory Proposed Rule (NPDWR) Six-Year Review of Existing NPDWRs Increased specificity and confidence in the type of supporting data used (e.g., health, occurrence, treatment) is needed at each stage. June 2018 U.S. Environmental Protection Agency Slide 14 of 206 Page 7 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water ,32, Regulatory Determinations Every five years, the Administrator shall, after notice of the preliminary determination and opportunity for public comment, for not fewer than five contaminants included on the CCL, make determinations on whether or not to regulate such contaminants. SDWA requires EPA to publish a maximum contaminant level goal (MCLG) and promulgate an NPDWR for a contaminant if the Administrator determines that: 2. 3. The contaminant may have an adverse effect on the health of persons; The contaminant is known to occur or there is substantial likelihood that the contaminant will occur in public water systems with a frequency and at levels of public health concern; and In the sole judgment of the Administrator, regulation of such contaminant presents a meaningful opportunity for health risk reduction for persons served by public water systems. June 2018 U.S. Environmental Protection Agency *SDWA Section 1412(b)(1) Slide 15 of 206 2* PRO^i General Flow of SDWA Regulatory Processes Public Review and Comment CCL . Regulatory Determination Ek Rule Preliminary Regulatory Determinations Review Final Regulatory Determinations Proposed Rule (NPDWR) Six-Year Review of Existing NPDWRs UCMR Monitoring Results UCMR No further action if decision is to not regulate May develop health advisory Increased specificity and confidence in the type of supporting data used (e.g., health, occurrence, treatment) is needed at each stage. June 2018 U.S. Environmental Protection Agency Slide 16 of 206 Page 8 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Six-Year Review Reviews existing NPDWRs, every six years, and determines if a revision is appropriate Includes the re-evaluation of new information on health effects, treatment technologies, analytical methods, occurrence and exposure, implementation and/or other factors that provide a health or technical basis to support a regulatory revision that will improve public health protection. Any revisions to existing NPDWRs must maintain protection or provide for greater health protection *SDWA Section 1412(b)(9) June 2018 U.S. Environmental Protection Agency Slide 17 of 199 CCL4 Contaminants Monitored in UCMRs 1,1-Dichloroethane (UCMR 3) Dimethipin (UCMR 4) Nitrobenzene (UCMR 1) 1,2,3-Trichloropropane (UCMR 3) Diuron (UCMR1) N-Nitrosodiethylamine (NDEA) (UCMR 2) 1,3-Butadiene (UCMR3) Equilin (UCMR3) N-Nitrosodimethylamine (NDMA) (UCMR 2) 1,4-Dioxane (UCMR 3) Estradiol (17-beta estradiol) (UCMR 3) N-Nitroso-di-n-propylamine (NDPA) (UCMR 2) 1-Butanol (UCMR 4) Estriol (UCMR 3) N-Nitrosopyrrolidine (NPYR) (UCMR 2) 2-Methoxyethanol (UCMR 4) Estrone (UCMR 3) o-Toluidine (UCMR 4) 2-Propen-l-ol (UCMR 4) Ethinyl Estradiol (17-alpha ethynyl estradiol) (UCMR 3) Oxyfluorfen (UCMR 4) Acetochlor (UCMR 1, UCMR 2) Ethoprop (UCMR 4) Perfluorooctane sulfonic acid (PFOS) (UCMR 3) Acetochlorethanesulfonicacid (ESA) (UCMR 2) Germanium (UCMR 4) Perf 1 uorooetanoic acid (PFOA) (UCMR 3) Acetochlor oxanilic acid (OA) (UCMR 2) Halon 1011 (bromochloromethane) (UCMR 3) Permethrin (UCMR 4) Alachlorethanesulfonic acid (ESA) (UCMR 2) HCFC-22 (UCMR 3) Profenofos (UCMR 4) Alachloroxanilic acid (OA) (UCMR 2) Manganese (UCMR 4) Quinoline (UCMR 4) alpha-Hexachlorocyclohexane (UCMR 4) Methyl bromide (Bromomethane) (UCMR 3) RDX (UCMR 2) Butylated hydroxyanisole (UCMR 4) Methyl tert-butyl ether (UCMR 1) Tebuconazole (UCMR 4) Chlorate (UCMR 3) Metolachlor (UCMR 2) Tribufos (UCMR 4) Chloromethane (Methyl chloride) (UCMR 3) Metolachlorethanesulfonicacid (ESA) (UCMR 2) Vanadium (UCMR 3) Cobalt (UCMR 3) Metolachlor oxanilic acid (OA) (UCMR 2) Caliciviruses (UCMR 3) Cyanotoxins (UCMR 4) Molybdenum (UCMR 3) Enterovirus (UCMR 3) June 2018 U.S. Environmental Protection Agency Slide 18 of 206 Page 9 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water CCL 4 Contaminants Not Yet Monitored in UCMR Method Available Method in Development No Method or Current Development Activity by EPA 1,1,1.2-Tetrachloroethane (502.2, 524.2, 524.3, 524.4) N-Methyl-2-pyrrolidone 17 alpha-Estradiol Adenovirus 3-Hydroxyca rbofuran (531.1, 531.2, 540, 543) Nonylphenol 4,4'-Methylenedianiline Campylobacter jejuni Acephate (538) Urethane Acetamide Escherichia coli (0157) Acetaldehyde (556, 556.1) Legionella pneumophila Acrolein Helicobacter pylori Bensulide (540, 543) Mycobacterium avium Aniline Hepatitis A virus Dicrotophos (538) Clethodim Naegleria fowleri Erythromycin (542) Equilenin Salmonella enterica Formaldehyde (556,556.1) Ethylene glycol Shigella sonnei Methamidophos (538) Ethylene thiourea n-Propylbenzene (502.2, 524.2, 524.3, 524.4) Hydrazine Oxydemeton-methyl (538) Nitroglycerin sec-Butylbenzene (502.2, 524.2, 524.3, 524.4) N-Nitrosodiphenylamine Tebufenozide (540, 543) Norethindrone (19-Norethisterone) Vinclozolin (525.3, 527) Oxirane, methyl- Tellurium Th iodicarb Thiophanate-methyl Triethylamine Triphenyltin hydroxide (TPTH) Ziram Benzyl chloride* Captan* Cumene hydroperoxide* Ethylene Oxide* Hexane* Mestranol* Methanol* Toluene diisocyanate* * Method Challenges Questions Page 10 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water General Guidelines Used in U.S. EPA Drinking Water Method Development and Application William A. Adams, Ph.D. U.S. EPA Office of Ground Water and Drinking Water Technical Support Center Overview General Method Development Process EPA Method 545 as example of approach June 2018 U.S. Environmental Protection Agency Slide 22 of 206 Andrew W. Bieidenbaeh Environmental Research Center Facility Page 11 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Drinking Water Method Attributes Preservation Dechlorination Storage Stability/Hold Time Studies Quality Control Quantitation Levels June 2018 U.S. Environmental Protection Agency Slide 23 of 206 S^tpRO^, 4/ Method Development Considerations Simplicity No overly complicated steps Relatively non-hazardous components Ease of sample collection Reasonable instrumentation Data Quality Focus on QC to ensure valid data especially for potentially regulated contaminants June 2018 U.S. Environmental Protection Agency Slide 24 of 199 Page 12 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Laboratory Quality Control Initial Demonstration of Capability (IDC) Demonstration of Low System Background Precision and Accuracy Minimum Reporting Level (MRL) Confirmation Quality Control Sample (QCS) from Second Source Ongoing QC Initial Calibration Continuing Calibration Check (CCC) Laboratory Reagent Blank (LRB) Laboratory Fortified Blank (LFB) Internal Standards (IS) Surrogates Standards (SUR) Laboratory Fortified Sample Matrix and Duplicates (LFSM, LFSMD) QCS at intervals June 2018 U.S. Environmental Protection Agency Slide 25 of 199 2* PRO"*5 Target analyte selection Storage Stability Study Tracks target analyte cc in preserved tap water for 5 weeks Multi-Laboratory Demonstration At least two outside laboratories June 2018 Instrument Optimization Based on scientific literature and preliminary experiments Instrument: Analytical column, eluent, temperature programs, flow, injection volume, assays Detectors: Target analyte MS tuning, detector settings, probes Precision and Accuracy Measurements Accuracy: Low: 50-150% Mid/High: 70-130% Precision: Low: <30% Mid/High: <20% Analyzed in three rr Submitted for EPA clearance U.S. Environmental Protection Agency System Background - Laboratory Reagent Blank (LRB) LCMRL Calculation - Lowest Concentration Minimum Reporting Level The lowest true concentration for which the future recovery is predicted to fall between 50% to 150% with 99% confidence General Method Development Slide 26 of 199 Page 13 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Method Performance Data Evaluation of Method Simplicity Data Quality Demonstration of Low System Background using a Laboratory Reagent Blank (LRB) LCMRL The lowest true concentration for which the future recovery is predicted to fall between 50% to 150% with 99% confidence Precision and Accuracy Study in Three Matrixes Meet %Rec and %RSD thresholds Storage Stability Study 35 Day study observing target analyte loss over time Second Laboratory Validation June 2018 U.S. Environmental Protection Agency Slide 27 of 199 Laboratory Method Flexibility Usually YES Usually NO Instrumental conditions Chromatography Detector Parameters Analytical Column Sample Collection and Preservation Sample Preparation (e.g. Extraction, Elution) QC Requirements Prescribed IS or SUR Different Instrumentation Additional IS or SUR Different Manufacturers Unless otherwise stated in the method Must verify method performance June 2018 U.S. Environmental Protection Agency Slide 28 of 199 Page 14 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Method 545 Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS) June 2018 U.S. Environmental Protection Agency Slide 29 of 199 2* PRO^i June 2018 M545 Target Analytes and IS o=s-o ,N_.NH HN NH h' * x y H 'NH O CylindrospermoDsin o D N Uracil-cf4 ~ H n w O Anatoxin-a L-phenylalanine-ds U.S. Environmental Protection Agency Slide 30 of 199 Page 15 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water M545 Preservation Store samples in refrigerator (<6 ฐC) 100 mg/L ascorbic acid Reduces residual chlorine present in tap water samples Easy to handle Solid can be added to bottles before sampling 1000 mg/L sodium bisulfate Acts as a microbial inhibitor pH less than 3 Solid can be added to bottles before sampling No observable interferences with direct injection June 2018 U.S. Environmental Protection Agency Slide 31 of 199 M545 Instrumental Method Waters Acquity Liquid Chromatography (LC) / Quattro Premier XE triple quadrupole MS (ESI) [equivalents acceptable] Waters XSeiectฎ HSS T3 2.1 x 150 mm, 3.5 p.m analytical column, 30 ฐC [equivalents acceptable] 100 mM acetic acid in reagent water (A) and 100% methanol (B) step gradient at a 0.2 mL/min flow (Mostly isocratic @ 90% aqueous) 50-jj.L injections Ionization for all analytes was achieved through protonation ([M+l]+) June 2018 U.S. Environmental Protection Agency Slide 32 of 199 Page 16 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water L-ph en yl al ani ne-c/5 M545 Method Performance Uracil-of4 Analyte Fortified Cone. (Mg/L) Day 0 Day 7 Day 14 Day 21 Dav 28 Avg. Meas. C'onc. (ua/L) %RSD %A %RSD %A %RSD %A %RSD %A %RSD cy lindr osp erinop sin 2.50 2.57 3.3 -5.4 2.4 -6.2 1.5 -5.1 1.2 -1.6 3.5 anatoxin-a 1.47 1.49 1.1 0.67 4.4 -7.4 2.4 -0.67 0.91 -1.3 2.1 ฆ cylindrospermopsin (0.100 pg/L) Banatoxin-a (0.059 pg/L) icylindrospermopsin (2.50 pg/L) Banatoxin-a (1.47 pg/L) 140.0 120.0 ฃ 100.0 | 80.0 S 60.0 S5 40.0 20.0 0.0 1400 T T cfa -I 120.0 ฆ S ซ i I ฆ i Reagent Water Ground Source Tap Surface Source Tap Water Water Reagent Water Ground Source Tap Surface Source Tap Water Water June 2018 U.S. Environmental Protection Agency Slide 33 of 199 Questions Page 17 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water EPA Method 542: Analysis Of Erythromycin and Other Pharmaceuticals by LC-MS/MS William A. Adams, Ph.D. U.S. EPA Office of Ground Water and Drinking Water, Technical Support Center U.S. EPA Method 542 Method 542: Determination of Pharmaceuticals and Persona! Care Products (PPCPj in Drinking Water by Solid Phase Extraction (SPE) and Liquid Chromatography Electrosprav Ionization Tandem Mass Spectrometry (LC/ESi-MS/MS) September 2016, EPA 815-R-15-012 June 2018 U.S. Environmental Protection Agency Slide 36 of 206 Page 18 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water EPA Method 542 (LC-MS/MS) Erythromycin Gemfibrozil Carbamazepine Naproxen Diazepam Phenytoin Diclofenac (sodium salt) Sulfamethoxazole Enalapril (maleate salt) Triclosan Fluoxetine (HCI) Trimethoprim Blue Fill: CCL4 with methods; Plain: Included in method, not on CCL4 13C-Naproxen-d3, Triclosan-d3, Carbamazepine-d10, chosen as internal standards; 13C-Trimethoprim-d3 and Diclofenac-^ chosen as surrogate standards June 2018 U.S. Environmental Protection Agency Slide 37 of 206 Technology Overview Variety of chemically unrelated arialytes Both ESI positive and ESI negative modes using separate injections and elution programs Analysis by LC-MS/MS using a 5 mM ammonium acetate and methanol gradient SPE (6 cc, 200 mg HLB cartridge) followed by concentration step (100:1) Preservation 8 Refrigeration, 100 mg/L ascorbic acid, 350 mg/L Ethylenediaminetetraacetic acid (EDTA), 9.4.g/L potassium citrate Solid preservatives can be added prior to sample collection June 2018 U.S. Environmental Protection Agency Slide 38 of 206 Page 19 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water June 2018 Erythromycin Antibiotic on CCL 4 At pH <7, water is removed and compound no longer exhibits antibiotic properties (Hirsch et al., 1999) For analysis, erythromycin is measured as erythromycin-H20 (717.0 > 158.3 m/z) HO-7-A-7 *0 HsC2"" CH3 Erythromycin U.S. Environmental Protection Agency Slide 39 of 206 Method 542 Performance Data Highlights No significant blank interferences Precision and Accuracy Study in three tap water matrixes Acceptable levels of matrix effects at low and middle concentrations Storage Stability Study Sample and extract hold times change less than 20% after 28 days Second Laboratory Validation External laboratories showed comparable results Sensitivity - LCMRL The lowest true concentration for which the future recovery is predicted to fall between 50% to 150% with 99% confidence S^JPRO^i 4/ June 2018 U.S. Environmental Protection Agency Slide 40 of 206 Page 20 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Analyte LCMRL (ng/L) Analyte LCMRL (ng/L) Carbamazepine Naproxen Diazepam Phenytoin Diclofenac Sulfamethoxazole Fluoxetine Trimethoprim Gemfibrozil Erythromycin Enalapril Triclosan EPA Method 542 LCMRL Slide 41 of 206 June 2018 U.S. Environmental Protection Agency EPA Method 542: Pharmaceuticals and Personal Care Products June 2018 U.S. Environmental Protection Agency Slide 42 of 206 Page 21 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water EPA Method 542: Pharmaceuticals and Personal Care Products June 2018 U.S. Environmental Protection Agency Slide 43 of 206 Questions ------- Methods Development for Unregulated Contaminants in Drinking Water Drinking Water Methods for Volatile and Semivolatile Compounds Paul E. Grimmett U.S. EPA Office of Ground Water and Drinking Water Technical Support Center U.S. EPA Method 524.4 EPA Method 524.4: Measurement of Puraeable Organic Compounds in Water by Gas Chromatography / Mass Spectrometry using Nitrogen Purge Gas May 2013, EPA 815-R-13-002 U.S. EPA Method 525.3 EPA Method 525.3: Determination of Semivolatile Organic Chemicals in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography /Mass Spectrometry (GC/MS) February 2012, EPA 600-R-12-010 June 2018 U.S. Environmental Protection Agency Slide 46 of 206 Page 23 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water EPA Methods 524.2/524.3/524.4 (GC/MS) 1,1,1,2-tetrachloroethane 1,4-dichloro benzene 1,1,2,2-tetrachloroethane Diisopropyl ether (DIPE) N-propyl benzene Benzene 1,1-dichloropropene Ethyl methacrylate Sec-butyl benzene Carbon tetrachloride 1,2,3-trichlorobenzene Hexach lorobutadiene 1,1-dichloroethane 1,2,4-trimethylbenzene Hexachloroethane 1,2,3-trichloropropane Bromoform 1,2-dibromoethane Isopropylbenzene 1,3-butadiene 1,3,5-trimethylbenzene Methyl acetate Bromochloromethane (Halon 1011) Chloroform 1,3-dichlorobenzene Methyl iodide Bromomethane (Methyl Bromide) Cis-l,2-dichloroethene 1,3-dichloropropane Naphthalene Chlorodifluoromethane (HCFC-22) Dibromochloromethane 1-chlorobutane N-butylbenzene Chloromethane (Methyl Chloride) Ethylbenzene 2-chlorotoluene Pentachloroethane Methyl-t-butyl ether (MtBE) 4-chlorotoluene T-amyl ethyl ether (TAEE) 1,1-dichioroethene M-xylene 4-isopropyltoluene T-amyl methyl ether (TAME) 1,1,1-trichloroethane cylene Allyl chloride T-butyl alcohol (TBA) 1,1,2-trichloroethane P-xylene Bromobenzene T-butyl ethyl ether (ETBE) 1,2,4-trichlorobenzene Tetrachloroethene Carbon disulfide T-butylbenzene Cis-l,3-dichloropropene Tetrahydrofuran 1,2-dichloro benzene Toluene Dibromomethane Trans-l,3-dichloropropene Dich lo rod ifluoromethane Trichlorofluoromethane Diethyl ether June 2018 Blue Fill: CCL 4; Green: Monitored under UCMR Orange: Regulated; Plain: Included in method, not on CCL 4 U.S. Environmental Protection Agency Slide 47 of 206 EPA Method 525.3 (GC/MS) PROl^X1 Vinclozolin Terbacil Chlorpyrifos Dimethipin Ethoprop Hexachlorocyclohexane, alpha (a-HCH) Oxyfluorfen Permethrin, cis- Permethrin, trans- Endrin Prof en of os Heptachlor Tebuconazole Heptachlor epoxide Tribufos Hexachlorobenzene (HCB) Acetochlor (UCMR 1) Hexachlorocyclohexane, gamma (y-HCH) (Lindane) Metolachlor Hexach lorocyclopentadiene (HCCPD) DDE, 4,4'- Methoxychlor Dinitrotoluene, 2,4- Pentachlorophenol Dinitrotoluene, 2,6- Simazine Disulfoton Toxaphene EPTC (S-Ethyl dipropylthiocarbamate) Polychlorinated Biphenyl (PCB) Congeners (IUPAC#) Molinate 74 Additional Contaminants Prometon Blue Fill: CCL 4; Green: Monitored under UCMR Orange: Regulated; Plain: Included in method, not on CCL 4 June 2018 U.S. Environmental Protection Agency Slide 48 of 206 Page 24 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water EPA Method 525.3 (GC/MS) Additional Contaminants (chlordane) cis-chlordane benzo (g,h,i) perylene dimethyl phthalate MGK264 (chlordane) trans-chlordane aenzo (k) fluoranthene di-n-butyl pthalate napropamide (chlordane) trans-nonachlor bromacil diphenamid nitrofen 2,2',3,4,4',5,5'-heptachlorobiphenyl butachlor disulfoton norflurazon 2,2',3,4,4',5'-hexachlorobiphenyl butyl benzyl phthalate endosufan I pebulate 2,2',3,4',5',6-hexachlorobiphenyl butylate endosufan II phenanthrene 2,2',3,5'-tetrachlorobiphenyl chlorfenvinphos endosufan sulfate phorate 2,2',5-trichlorobiphenyl chlorobenzilate ethion phosphamidon 2,3,3',4',6-pentachlorobiphenyl chloroneb ethyl pa rath ion prometryn 2,3',4,4',5-pentachlorobiphenyl ch lorotha Ion il etridiazole pronamide 2,3',4',5-tetrachlorobiphenyl chlorpropham fenarimol propachlor 2,4,4'-trichlorobiphenyl chrysene fluorene propazine 2,4'-dichlorobiphenyl cycloate fluridone pyrene 2-chlorobiphenyl dacthal (DCPA) hexachlorocyclohexane, beta simetryn 4-chlorobiphenyl DDD, 4,4' hexachlorocyclohexane, delta tebuthiuron acenaphthylene DDT, 4,4' hexachlorocyclohexane, gamma terbutryn aldrin DEET (N,N-diethyl-meta-toluamide) hexazinone tetrach lorvinphos ametryn di(2-ethyihexyl) phthalate indeno [l,2,3-c,d]pyrene triadimefon anthracene dibenz [a,h] anthracene isophorone trifluralin atraton dichlorvos methyl pa rath ion vernolate benzo (a) anthracene dieldrin metribuzin benzo (b) fluoranthene diethyl phthalate mevinphos June 2018 U.S. Environmental Protection Agency Slide 49 of 206 CCL 4 Compounds - VOCs (yet to be monitored) Volatile Organic Compounds ^ c|^ \ 1,1,1,2-tetrachloroethane CI CI - solvent used in wood stains and varnishes n-propylbenzene - solvent used in printing and dying sec-butylbenzene - solvent used in surface coating June 2018 U.S. Environmental Protection AgencySlide 50 of 206 Page 25 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water CCL 4 Compounds - Semivolatiles (yet to be monitored) CI Semivolatile Organic Compounds vinclozolin - fungicide used on various fruits/vegetables Ch3 -n r sฐh2 Yฐ CI o June 2018 U.S. Environmental Protection Agency Slide 51 of 199 Volatile Organic Compounds (VOCs) Volatile Organic Compounds Normally high vapor pressures and low boiling points Natural or synthetic Form a gas easily (volatilize) Analysis commonly done by Gas Chromatography- Purge and Trap June 2018 U.S. Environmental Protection Agency Slide 52 of 206 Page 26 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Sample inlet Helium in To trap Optional foam trap Purge & Trap Extraction M. Purge & Trap Extraction S^tPRO^, 4/ Trap: He From purge Desorb: From tank Trap 25ฐ C Trap 180ฐ C GC Heating drives the compound out of the trap and into the column VJ> MS Detector GC 0!) MS Detector Page 27 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Newer GC Methods for VOCs Method 524.3 and Method 524.4 GC/MS with purge and trap extraction Both approved through the expedited method approval process for monitoring regulated contaminants *Federal Register / Vol, 74, No. 147 / Monday, August 3, 2009, p. 38348 * Federal Register / Vol. 78, No. 105 / Friday, May 31, 2013, p. 32558 1,1,1,2-tetrachloroethane, n-propylbenzene, and sec-butylbenzene are included in Methods 524.3 and 524.4 June 2018 U.S. Environmental Protection Agency Slide 55 of 206 Updates to Method 524 * Method 524.3 - allows more flexibility in purge & trap parameters Parameter Rerom mended Allowable Minimum Maximum Minimum Maximum Sample temperature Ambient 10 ฐC Ambient 60 C Purse flow rate 40 mlJ\min 80 niL/min 20 ml ./mi n 200 mL/min Plir^e volume 360 ml. 520 nil. 240 mL 680 ml. Desorb time 1 min 2 min 0.5 min 4 min Purge volume + dry purge volume 360 ml. 720 mL 240 mL 880 mL Method 524.4 - allows for purging with nitrogen instead of helium Parameter Recommended Allowable Minimum Maximum Minimum Maximum Sample temperature Ambient 40 ฐC Ambient 60 ฐC Purge flow rate 40 mL/min 55 mL/min 20 mL/min 80 mL/min Purge volume 360 mL 520 mL 320 mL 520 mL Desorb time 1 min 1 min 0.5 min 2 min Purge volume + drv purge volume 360 mL 720 mL 320 mL 720 mL June 2018 U.S. Environmental Protection Agency Slide 56 of 206 Page 28 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Method 524 Comparison 524 2 I 524.3 524.4 0.32 to 0.75 mm internal diameter (i.d.) columns Cryogenic interface; no split; jet separator to MS 5 or 25-mL purge volume Any trap that meets method criteria Single concentrator 1 internal standard - fluorobenzene 0.18 to 0.25 mm i.d. columns * Split injection 5-mL purge volume Any trap that meets method criteria * Single or tandem concentrator * 3 internal standards - 1,4-difluorobenzene - chlorobenzene-ds - l,4-dichlorobenzene-d4 0.18 to 0.25 mm i.d. columns * Split injection 5-mL purge volume * Only traps containing synthetic carbon adsorbent media Single or tandem concentrator * 3 internal standards - 1,4-difluorobenzene - chlorobenzene-d5 - 1,4-dichlorobenzene-d, Semivolatile Organic Compounds (SVOCs) Semivolatile Organic Compounds Broad chemical properties and structural features - pesticides - flame retardants - PAHs, PCBs, etc. Natural or synthetic Higher boiling points and low vapor pressures Analysis commonly done by Gas Chromatography- Soiid Phase Extraction (SPE), among others June 2018 U.S. Environmental Protection Agency Slide 58 of 206 Page 29 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Solid-Phase Extraction CONDITIONING SAMPLE ADDITION WASHING ELUTION I i f I i r . ฆ i i 1 n r ฆ ป Analyte * Iriterferents ฆ June 2018 U.S. Environmental Protection Agency Slide 59 of 206 Solid-Phase Extraction Uses small volumes of solvent vs. traditional LLE / Sep Funnel methods Allows for large-scale concentration (better sensitivity) Less time consuming Minimal emulsion issues, i.e. no shaking June 2018 U.S. Environmental Protection Agency Slide 60 of 206 mm" Page 30 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Newer GC Method for SVOCs Method 525.3 SPE on polymeric sorberit (DVB), followed by GC/MS Approx. 130 analytes (pesticides, herbicides, PCBs, PAHs, etc) Published in February 2012., approved by expedited method approval process (Federal Register/Vol. 77, No. 125 / Thursday, June 28, 2012, p. 38523) Contains vinclozolin Currently using in UCMR 4 for monitoring 9 compounds June 2018 U.S. Environmental Protection Agency Slide 61 of 206 Method 525.3 Improvements Improved preservation scheme - citrate buffer vs. acid Updated surrogates and internal standards Addition of SIM option - better sensitivity New PCB screening procedure June 2018 U.S. Environmental Protection Agency Slide 62 of 206 Page 31 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Add internal standards Analyze extract by GC/MS Add surrogates to 1-L sample. Summary of Procedure (Method 525.3) DVB disk or cartridge Rinse, condition, wet: methylene chloride, ethyl acetate, methanol, and reagent water I Add 1-L water sample, extract at ~10 mL/min. Rinse with reagent water. Dry cartridge for 10 minutes. Elute with ethyl acetate & methylene chloride. Pass through Na2S04, and concentrate extract to 1 mL. Conclusion CCL 4 has a number of VOC and SVOC contaminants that have applicable EPA methods associated with them. 1) Method 524.3/524.4 Compound MRL (ng/L)* HRL (ng/L)** 1,1,1,2-tetrachloroethane 0.018 1 n-propylbenzene 0.030 5.83 sec-butylberizene 0.035 10.3 2) Method 525.3 Compound MRL (ng/L)* HRL (ng/L)** vinclozolin 0.028 0.549 * Multi-laboratory calculation, SIM ** Contaminant Information Sheets (CISs) for the Final Fourth Contaminant Candidate List - EPA 815-R-16-003 June 2018 U.S. Environmental Protection Agency Slide 64 of 206 Page 32 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Questions ~ PR0-^> EPA Method 556.1: Determination of Carbonyl Compounds in Drinking Water by Fast Gas Chromatography Steve Wendelken, Ph.D. U.S. EPA Office of Ground Water and Drinking Water Technical Support Center Page 33 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water U.S. EPA Method 556.1 EPA Method 556.1: Determination ofCarbonvl Compounds in Drinking Water by Fast Gas Chromatography September 1999 June 2018 U.S. Environmental Protection Agency Slide 67 of 206 Method 556.1 Target Analyte List Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal Octanal Nonanal Decanal Cyclohexanone Benzaldehyde Glyoxal (ethanedial) Methyl glyoxal (2-oxopropanal or pyruvic aldehyde) Blue Fill: CCL 4 with methods; Plain: Included in method, not on CCL 4 June 2018 U.S. Environmental Protection Agency Slide 68 of 206 Page 34 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water 556.1 Procedural Summary 20 mL sample adjusted to pH 4 Derivitization (2h) with (2,3,4,5,6-pentafluorobenzyl)- hydroxylamine (PFBHA) Derivatized analytes extracted with 4 mL hexane Acid wash with 3 mL 0.2 N sulfuric acid Extracts are analyzed by gas chromatography with electron capture detection Some targets form 2 or more isomers whose peaks must be summed June 2018 U.S. Environmental Protection Agency Slide 69 of 206 Precision & Accuracy Finished Drinking Water (n=7) Analyte Fortified Concentration (mg/L) Average Concentration (mg/L) Unfortified Sample (mg/L) Relative Standard Deviation Average Recovery Formaldehyde 5.0 8.45 3.40 3.3% 101% Acetaldehyde 5.0 6.53 1.76 2.7% 96% Propanal 5.0 5.68 0.620 2.2% 101% Butanal 5.0 5.73 0.390 2.4% 107% Pentanal 5.0 5.43 ND 2.6% 109% Hexanal 5.0 5.48 ND 2.8% 110% Cyclohexanone 5.0 6.02 0.650 4.2% 107% Heptanal 5.0 5.64 0.840 4.1% 96% Octanal 5.0 4.84 ND 6.4% 97% Benzaldehyde 5.0 4.92 ND 3.1% 98% Nonanal 5.0 5.25 0.250 8.5% 100% Decanal 5.0 5.78 ND 8.9% 116% Glyoxal 5.0 7.92 1.40 9.2% 130% Methyl Glyoxal 5.0 6.42 0.380 9.2% 121% June 2018 U.S. Environmental Protection Agency Slide 70 of 206 Page 35 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Minimum Reporting Levels (MRLs) 556.1 was developed prior to the LCMRL process being instituted The MRLs for formaldehyde and acetaldehyde will be dependent on the laboratories' ability to control background levels of these analytes The most successful techniques for generating aldehyde free water are exposure to UV light, or distillation from permanganate A Miilipore Elix 3 reverse osmosis system followed by a Milli-QTOC Pius polishing unit provided a background < 1 fig/L June 2018 U.S. Environmental Protection Agency Slide 71 of 206 Method Detection Limits Analyte Fortified Concentration (mg/L) Primary Column MDL (mg/L) Secondary Column MDL (mg/L) Formaldehyde 1.0 0.09 0.08 Acetaldehyde 1.0 0.18 0.12 Propanal 1.0 0.11 0.06 Butanal 1.0 0.09 0.06 Pentanal 1.0 0.09 0.06 Hexanal 1.0 0.10 0.04 Cyclohexanone 1.0 0.19 0.09 Heptanal 1.0 0.40 0.24 Octanal 1.0 0.22 0.84 Berizaldehyde 1.0 0.19 0.04 Nonanal 1.0 0.62 0.64 Decanal 1.0 0.46 0.35 Glyoxal 1.0 0.39 0.13 Methyl Glyoxal 1.0 0.26 0.12 June 2018 U.S. Environmental Protection Agency Slide 72 of 206 Page 36 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Questions Break ' - ซ 3:V (15 minutes) Page 37 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water United Stales Environmental Protection Agoncv Drinking Water Methods Meeting/Webinar - 2018 Development of Method 538, 540, 543 and 537 for the Analysis of Chemicals on U.S. EPA's Contaminant Candidate List Jody A. Shoemaker Disclaimer: Mention of trade names or commercial products does not constitute endorsement or recommendation for use. | Office of Research and Development National Exposure Research Laboratory SERA United Stales Environmental Protection Agency Method 538: DAI-LC/MS/MS (2009) Contains 11 Analytes acephate dicrotophos methamidophos oxyd e meto n-met h y I quinoline Method Analvtes aldicarb aldicarb sulfone diisopropyl methylphosphonate (DIMP) fenamiphos sulfone fenamiphos sulfoxide thiofanox Analytes in red are on CCL 4 ~ Most method analytes are pesticides (except for quinoline and DIMP) with the potential to contaminate drinking water sources ~ Quinoline is an industrial starting material, a pharmaceutical (anti- malarial) and a flavoring agent ~ DIMP is a chemical by-product in the production of sarin gas I Office of Research and Development I National Exposure Research Laboratory Page 38 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmontol Protection Agency M538 Direct Aqueous Injection Approach 40 mL sample^ 990 nL aliquot Pos ESI 50 |jl injection Source Detector 10 uL IS PDS acephate, d6 methamidophos, d6 oxydemeton-methyl, d( quinoline, d-f DIMP, d14 Antimicrobial: 64 mg/L sodium omadine Remove free chlorine: 20 mM ammonium acetate Selected Reaction Monitoring I Office of Research and Development I National Exposure Research Laboratory SERA United Stales Environmental Protection Agency 120 MOO 80 60 40 20 0 M538 Performance data (n=7) LFBs and LFSMs fortified at 0.99 - 43 |jg/L ฆ LFB bLFSM (surface water source) bLFSM (ground water source) 1*1 It I T 0) > o o 0) a: c TO 0) I I ฆ I I I ฆ I I ฆ sfP * / f /, & V jr /jr // I Office of Research and Development I National Exposure Research Laboratory QC criteria at mid-level cal: 70-130% Page 39 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental Protection Agency M538 Aqueous Holding Time Data (n=7) ฆ DayO ปDay7 ฆ Day 14 ~ sf ^ ^ ^ J* J" ///// /> * ** ฆ* yv r ^ <5S ^ o+A o<0 >0ฐ oP / / I Office of Research and Development I National Exposure Research Laboratory M538 LCMRLs and HRLs United Stales Environmental Protection Agency Analyte LCMRL (ng/L) HRL (ng/L) Methamidophos 32 2100 Acephate 44 4000 Aldicarb sulfoxide 88 Oxydemeton-methyl 19 910 Dicrotophos 39 490 Aldicarb 30 Quinoline 1500* 10 DIMP 22 Fenamiphos sulfoxide 42 Fenamiphos sulfone 11 Thiofanox 180 I Office of Research and Development I National Exposure Research Laboratory LCMRL range: 11-180 ng/L Page 40 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmontol Protection Agency Method 540: SPE-LC/MS/MS (2013) Contains 12 Analytes Method Analvtes 3-hyd roxycarbofu ran bensulide tebuconazole tebufenozide disulfoton sulfoxide fenamiphos fenamiphos sulfone fenamiphos sulfoxide methomyl chlorpryifos oxon phorate sulfone phorate sulfoxide Analytes in red are on CCL 4 all method analytes are pesticides or pesticide degradates with the potential to contaminate drinking water sources I Office of Research and Development I National Exposure Research Laboratory SERA United Stales Environmental Protection Agency Surrogates methomyl-13C2,15N tebuconazole-d6 Preservatives 2-chloracetamide ascorbic acid Trizma Internal Standards carbofuran-13C6 bensulide-d14 phorate-d10 inject Method 540 Analytical Procedure Surrogates & preservatives 250 mL sample Condition with 5 mL MeOI- & 10 mL reagent water Rinse cartridge with 5 mL reagent water after loading 150 mg Water Oasis HLB .T. Baker Speedisk H20-Philic DVB N2 blowdown + IS Elute with 5 mL MeOH Argon LC + ESI Source Detector LC/MS/MS - Selected Reaction Monitoring I Office of Research and Development I National Exposure Research Laboratory Page 41 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environment!!! Protection Agency I LFB M540 Performance Data LFBs & LFSMs fortified at 12.8-32 ng/L (n=4) ฆ LFSM (surface water source) ฆ LFSM (ground water source) 120 i > 100 r / ' * QC criteria at mid-level cal: 70-130% SERA United Stales Environmental Protect' Agency Aqueous Holding Time Study- Method 540 (n=4) DayO ฆ Day 7 ฆ Day 14 ฆ Day 23 ฆ Day 28 00 I Office of Research and Development I National Exposure Research Laboratory 7 day holding time for chlorpyrifos oxon Page 42 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental Protection Agency M540 LCMRLs & HRLs Analyte LCMRL ng/L URL ng/L methomyl 1.2 3-hyd roxyca rbofu ran 1.3 420 fenamiphos sulfone 1.0 fenamiphos sulfoxide 0.86 phorate sulfone 0.99 phorate sulfoxide 2.0 disulfoton sulfoxide 2.0 bensulide 1.2 35,000 tebufenozide 0.81 126,000 chlorpyrifos oxon 2.0 fenamiphos 0.64 tebuconazole 2.0 210,000 LCMRL range: 0.64-2.0 ng/L All analytes well below the HRLs. I Office of Research and Development I National Exposure Research Laboratory SEPA United Slales Environmfl ntol Protection Agency Method 543: On-line SPE-LC/MS/MS (2015) Contains 8 Analytes Method Analytes 3-hydroxycarbofuran fenamiphos bensulide fenamiphos sulfone tebuconazole fenamiphos sulfoxide tebufenozide I Office of Research and Development I National Exposure Research Laboratory Analytes in red are on CCL 4 Page 43 of 103 ali method analytes are pesticides or pesticide degradates (except quinoline) with the potential to contaminate drinking water sources concentration, elution, separation all done by automation 1-5 mL sample volume typical analysis time/sample is <20 min high throughput ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental P Agency Method 543 Procedure preservatives: 2-chloroacetamide ascorbic acid trizma 10 mL sample SPE cartridge 1 Oasis HLB ^3 SPE cartridge 2 Oasis HLB Detector I Office of Research and Development I National Exposure Research Laboratory LC/MS/MS - Selected Reaction Monitoring Method 543: On-line SPE Events 5 min gradient elution Re-equilibration Start 20 mM NH4OAc ACN Sample loading SPE wash 20 mM NH4OAc 20 mM NH4OAc Analytical Pump SPE Cartridge #1 Analytical Pump Loading Pump Loading Pump | I I I I | I I I I | I I I I | I I I I | I I I I | I M I | I I I I | I I I I | I I I I | I I I I | I I M | I M I | I I I I | I I I I | I I I I | I I I I |1 0 2 4 6 8 10 12 Start Re-condition > Loading Pump Re-equilibration 20 mM NH4OAc Loading Pump SPE Cartridge #2 1111111111111111111111111111111111111111111111111111 i i 1111 i i 11111111111111111111 (Time, min 0 2 4 6 8 10 12 I Office of Research and Development ACN acetotlitrile I National Exposure Research Laboratory Pos ESI UPLC- Source Argon -1r SPE 5-mL sample loop Internal Standards methomyl-13C2, 15N carbofuran-13C6 bensulide-d14 SPE injection port 2 rnL injections Page 44 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmontol Protection Agency M543 Performance data LFBs & LFSMs fortified at 20-50 ng/L (n=5) i LFB ฆ LFSM (surface water source) ฆ LFSM (ground water source) 120 > 100 8 80 0) * 60 Mi ^ jf & AO+ # / /" <ฆ? <$> & QC criteria: 70-130% I Office of Research and Development I National Exposure Research Laboratory SERA United Stales Environmental Protection Agency Aqueous Holding Time Study- Method 543 (n=5) ฆ Day 0 ฆ Day 7 ฆ Day 14 ฆ Day 22 ฆ Day 28 I Office of Research and Development I National Exposure Research Laboratory Page 45 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water *>EPA Pro M540 & 543 HRL and LCMRL Comparison United Stales Environmental Protection Analytes HRL, ng/L LCMRLs, ng/L Method 540 On-line 3-hyd roxycarbofu ran 420 1.3 1.7 bensulide 35,000 1.2 1.2 tebufenozide 126,000 0.81 0.47 tebuconazole 210,000 2.0 1.3 fenamiphos 0.64 0.27 fenamiphos sulfone 1.0 1.4 fenamiphos sulfoxide 0.86 1.2 LCMRLs obtained by on-line method are below the HRLs for all analytes and similar to Method 540. / \ 250 mL sample 2 mL sample 0.64-2.0 ng/L 0.27-1.7 ng/L I Office of Research and Development I National Exposure Research Laboratory SERA ssipmrt. Method 537: SPE-LC/MS/MS 14 Perfluorinated Alkyl Acids (PFAA) Perfluorocarboxylic acids (9) Perfluorosulfonates (3) Perfluorosulfonamidoacetic acids (2) Method Analvtes on CCL 4 PFOA- perfluorooctanoic acid PFOS - perfluorooctane sulfonic acid Method Analvtes in UCMR 3 PFOA - perfluorooctanoic acid PFHpA - perfluoroheptanoic acid PFNA - perfluorononanoic acid PFOS - perfluorooctane sulfonic acid PFHxS - perfluorohexanesulfonic acid PFBS - perfluorobutanesulfonic acid Challenges: wide range of water solubilities (C4-C14), laboratory and field blank contamination, LC contamination I Office of Research and Development I National Exposure Research Laboratory Page 46 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water United States *>EPA Pro Update to Method 537 (in progress) Potential PFAS additions Acronvm CAS# Perfluoro-2-propoxypropanoic acid GenX 13252-13-6 Nonafluoro-3,6-dioxaheptanoic acid NFDHA 151772-58-6 Perfluoro (2-ethoxyethane) sulfonic acid PFEESA 113507-82-7 Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 Potassium 11 -chloroeicosafluoro-3-oxaundecane-1 - sulfonate 11CI-PF30UdS 83329-89-9 Potassium 9-chlorohexadecafluoro-3-oxanone-1 -sulfonate 9CI-PF30NS 73606-19-6 Sodium dodecafluoro-3H-4,8-dioxanonate ADONA 958445-44-8 Challenge: Obtain performance data, write method, conduct multi-lab verification and peer review method ASAP I Office of Research and Development I National Exposure Research Laboratory SERA United Stales Environmental Protection Agency M537 Approach Preservative trizma Surrogates 13C2-PFHxA 13c2-pfda d5-N EtFOSAA 13C-GenX 10 Mi- injection Internal Standards 13C2-PFOA 13c4-pfos d3-NMeFOSAA Surrogates & trizma 250 mL Sample Rinse cartridge and bottle with styrene divinylbenzene cartridges reagent water Elution with Methanol: Bottle must be eluted Blowdown Neg ESI LC/MS/MS - Selected Reaction Monitoring I Office of Research and Development I National Exposure Research Laboratory Page 47 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA Unit&d Stales Environmental Protection Conclusions ~~~ Four published methods available/expected for usage in future monitoring for unregulated contaminants S Pesticides S Pesticide degradates ฆf Additional PFAS(s) ~~~ Methods contain preservation, holding times and QC ~~~ Performance data demonstrated ~~~ Update to Method 537 expected to contain GenX ฆ Office of Research and Development National Exposure Research Laboratory oEPA United States Environmentiปl Protection Agency Questions? I Office of Research and Development I National Exposure Research Laboratory Page 48 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Analysis of Select PFAS Compounds in Drinking Water Steve Wendelken, Ph.D. U.S. EPA Office of Ground Water and Drinking Water, Technical Support Center Initial Method Development Objectives Develop a SPE LC/MS/MS method for the analysis of "short chain" [perfluorinated acids, sulfonates and mono/poly perfluorinated ethers] in drinking water "short chain" representing no PFAS greater than C12 due to physicochemical disparities Initially targeting method reporting levels < 10 ng/L Extend the method to include surface water and non-potable groundwater as time and resources permit June 2018 U.S. Environmental Protection Agency Slide 98 of 206 Page 49 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Initial Target Analyte List Compound Abbreviation CAS Formula (anion) m/z (anion) 1 Perfluorobutanoic acid PFBA 375-22-4 fA 213 2 Perfluoro-3-methoxypropionic acid PFMPA 377-73-1 Wfii 228.974 3 Perfluoropentanoic acid PFPeA 2706-90-3 c5f9o2 263 4 Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 C5F903 278.97 5 Perfluoro (2-ethoxyethane) sulfonic acid PFEESA 113507-82-7 c4f9o4s 315 6 Nonafluoro-3,6-dioxaheptanoicacid NFDHA 151772-58-6 C5F904 295 7 Perfluoro-2-propoxypropanoic acid GenX 13252-13-6 C6Fii03 328.97 8 D od e caf 1 u o ro-3 H -4,8-d i oxa nonanoate ADONA 958445-44-8 C7HF12ฐ4 377 9 9-chlorohexadecafluoro-3-oxanonane-l-sulfonate 9CI 73606-19-6 c8f16so4ci 531/533 10 ll-chloroeicoafluoro-3-oxaundecane-l-sulfonate 11CI 83329-89-9 C10F 4^' 631/633 June 2018 U.S. Environmental Protection Agency Slide 99 of 206 Initial Weak Anion Exchange SPE Results with Blanks PFAS fortified "high TOC finished drinking water 50 ng/L PFAS fortification Sample preserved with 5 g/LTrizma 100 mL sample extraction on 200 mg UCT WAX SPE Eluted w/ 2X5 mL 2% NH40H in MeOH. Diluent 75% MeOH. All values in ng/L Rep A Rep B Rep C Matrix Blank LRB Average Recovery % %RSD PFBA 55 58 55 7 5 112 3.2 PFMPA 49 51 54 1 1 103 5.3 PFPeA 45 45 47 0 1 91 2.3 PFMBA 45 45 46 1 1 90 1.3 EESA 49 45 47 0 0 94 4.1 NFDHA 44 46 47 0 0 91 2.3 GenX 43 43 44 0 0 87 2.1 ADONA 45 47 46 0 0 92 1.6 9CI 50 50 51 0 0 101 1.6 11CI 49 50 51 0 0 100 1.7 June 2018 U.S. Environmental Protection Agency Slide 100 of 206 Page 50 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Expanding PFAS Target List Currently evaluating expanding the target list to create a more "universal" PFAS method that includes most Method 537 targets along with other analytically feasible PFAS, New method focused on highest analytical performance for priority short chain perfluorinated acids, sulfonates and mono/poly perfluorinated ethers. Any additional PFAS targets must have an available analytical standard, Final target list may include closer to two dozen or more PFAS, June 2018 U.S. Environmental Protection Agency Slide 101 of 206 General Method Procedure Samples collected in polypropylene bottles Samples preserved with Trizma SPE extraction with weak anion exchange media SPE eluted with basic Extracts analyzed by LC/MS/MS Target MRLs < 10 ng/L June 2018 U.S. Environmental Protection Agency Slide 102 of 206 Page 51 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Questions EPA Method in Development 558 Alan Zaffiro Ph.D. APTIM Contractor to U.S. EPA Page 52 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Method 558 (GC/MS) N-Methyl-2-pyrrolidone (in Development) Ethyl carbamate/Urethane (In Development) : CCL 4 June 2018 U.S. Environmental Protection Agency Slide 105 of 206 2* PRO^i Initial Contaminants Considered (Method 558) Water-soluble, low-molecular weight, and priority contaminants remaining on CCL 4 for analysis via GC/MS acetamide, aniline, ethyl carbamate (urethane), ethylene glycol, ethylene oxide, hydrazine, methanol, N-methyl-2-pyrrolidone, propylene oxide, triethylamine, N-nitrosodiphenylamine and 4,4'-methylenedianiline Investigated via literature search and laboratory experiments contaminant properties potential extraction techniques chromatographic efficiency programmed temperature and split/splitless injection June 2018 U.S. Environmental Protection Agency Slide 106 of 206 Page 53 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Selection of Target Analytes for Method 558 Proposed multi-analyte method for: 4,4'methylenedianiline, urethane, N-methyl-2-pyrrolidone, aniline, p-chloroaniline, 2,4-dichloroaniline, 2,4,6-trichloroaniline, and N- nitrosodiphenylamine Requires tandem Solid Phase Extraction (SPE) and results in 3 separate extracts for GC/MS Settled on two priority contaminants Ethyl carbamate and N-methyl-2-pyrrolidone Common SPE cartridge and GC column June 2018 U.S. Environmental Protection Agency Slide 107 of 206 Draft Method 558 Analytes ethyl carbamate (HRL = 0.035 ng/L) and N-methyl-2-pyrrolidone (HRL = 4,200 |ig/L) ABILITY TO MEET MONITORING GOALS: MRLs confirmed at 0.0175 (ig/Lin our laboratory for both contaminants without extract concentra tion Attempted 4:1 extract concentration as option to achieve lower MRL, but abandoned because of inconsistent performance Surrogate compounds Ethyl-d5 carbamate and N-methyl-2-pyrrolidone-d9 Internal standard l,4-dichlrobenzene-d4 June 2018 U.S. Environmental Protection Agency Slide 108 of 206 Page 54 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Method 558 Parameters Preservation: identical to EPA Methods 522 and 541 1 g/L NaHS04 and 50 mg/L Na2S03 SPE: 0.5 L Sx; neutralized with 25 mL x 0.8 M NaHC03; Waters (Miiford, MA) AC-2 (400 mg); 150 pi MeOH rinse followed by 5 min N2 dry @ 5 I /min; elution with 150 |i!_ MeOH followed by 2.3 mL ethyl acetate; 2-mL final extract volume Extract analysis: 30 m x 0.25 mm i.d. x 0.5 |im df WAX column; 1 |iL pulsed-pressure injection @ 200 ฐC inlet; temperature- programmed separation; MS detection in SIM mode June 2018 U.S. Environmental Protection Agency Slide 109 of 206 Measurement Ranges Analyte / QC Compound Range without extract concentration, |ig/L Ethyl carbamate 0.0175-2.0 N-methyl-2-pyrrolidone 0.0175-2.0 Ethyl-d5 carbamate, surrogate 0.25 N-methyl-2-pyrrolidone-dg, surrogate 0.25 June 2018 U.S. Environmental Protection Agency Slide 110 of 206 Page 55 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Future Work Method 558 Evaluate other activated carbon cartridges Must be reversible cartridge UCT (Bristol, PA) 500 mg cartridge in progress Collect method performance data for all extraction formats Waters AC-2 in progress Outside laboratory validation At least two laboratories, preferably three or more The following ion chromatograms illustrate the chromatographic peak profiles, the ions monitored, and demonstrate that at least one confirmation ion is detected at 0.035 |ag/L June 2018 U.S. Environmental Protection Agency Slide 111 of 206 ------- Methods Development for Unregulated Contaminants in Drinking Water Ion 82.00(61 70 lo 62.70* 01201005.D\datoms flton 45 00 (44 70 to 45 701 012010C5.D\daUms l*n 74 00 [73 70 to 74.70) 01201005 DVdaUrm "A. 83 00 (88 70 to 89 701 01201006D\dab*mt LFB fortified at the HRL (0.035 |ig/L) with ethyl carbamate. L=3jjl..l_jj_' Ion 99.00 (98.70 to 99.70) Ion 98.00 (97.70 to 98.70) Ion 42.00 (41.70 to 42.70) Ion 71 00 (70 70to 71 70) 00901013.D\data.m: 00901013. D \data.m: 00901013.D\data.rn: 00901013.DMata.rn N-methyl-2-pyrrolidone at 250 ng/mL in iCAL standard (1.0 ug/L aqueous equivalent) ; [1] Scan 2096 03311 min): 00901013.D\data.ms (-2065) (-) JpEgi' Page 57 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Ion 99-00(98.70 to 99.701 01201005.D\data.ms Ion 98 00 (97 70 to 98.70V 01201005D\eUu.im Ion 4200(41,70to42.70) 01201005.D\data.nปป Ion 71 00170.70 to 71 701 01201005DVd*aim N-methyl-2-pyrrolidone: LFB fortified at 0.035 ng/L (HRL is 4,200 ng/L) Window *6 Questions Page 58 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental Protection Afloncv DW Stakeholder June 6, 2018 Development of U. S. EPA Method 559 for the Analysis of Nonylphenol in Drinking Water by Solid Phase Extraction and LC/MS/MS Daniel R. Tettenhorst and Jody A. Shoemaker Disclaimers: Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. | Office of Research and Development ' National Exposure Research Laboratory &EPA Unitad Stales Environmental Protection Agency Method Development Goals ~70-130% recovery with <30% RSD ~Laboratory Reagent Blank (LRB) no more than 1/3 the Minimum Reporting Level (MRL) ~Preservation S Dechlorinating Agent S Antimicrobial ~Establish sample and extract holding times - ideally >14 days ~Lowest concentration minimum reporting limits (LCMRLs) goal - less than health reference level (HRL) ~HRL for Nonylphenol is 105 pg/L ฆ Office of Research and Development National Exposure Research Laboratory Page 59 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental Protection Agency What is Nonylphenol? 4 Nonylphenol (NP) is used to make plastics, detergents, paints, pesticides, personal care products ~ Many products have "down the drain" applications and are flushed into the water supply Technical, Branched Nonylphenol Linear Nonylphenol Mostly branched C9-alkyl phenols Linear C9 chain CAS# 84852-15-3 CAS# 104-40-5 Best represents commercially produced NP found in environment Laboratory generated chemical not found in environment Method will report technical, branched NP, CAS #84852-15-3 SERA United Stales Environmontol Protection Agency Drinking Water Procedure Amber Glass Bottle Preservatives Sodium bisulfate= antimicrobial < Ascorbic acid = dechlor Q Possible Surrogates 4-n-Nonylphenol-13C6(linear) 4-n-Nonylphenol-2,3,4,5-d4, OD (linear) 4-n-Octyl-d17-phenol (linear) 4-tert-Octylphenol-13C6(branched) 250 mL sample LC/MS/MS Neg ESI Rinse cartridge with reagent water and dry under vacuum Rinse and elute sample bottle with 2 X 2 mL acetone Add Adjust to 5 mL final volume with acetone Oasis HLB Bond Elut C18 Strata-X 100-200 mg Internal Standard 4-(1,3-Dimethyl-1 -ethylpentyl) phenol-13C6 Optimized Conditions Page 60 of 103 One example of a 4- branched-NP isomer ------- Methods Development for Unregulated Contaminants in Drinking Water oEPA Protection System Background Contribution NP is a common laboratory contaminant 'i \ Injected j 1 CAL1 NP peak Solvent Injection "Trapped" NP peak Trapping column used to separate LC system Trapping contamination away from injected NP peak Column SEPA Urritad Stoles Environmental Protection Agency Mobile Phase Conditions ~ Sensitivity increases as mobile phase pH approaches the pKa (10.28) ~ Ammonium hydroxide (0.01%) used to increase pH to 10 ~ Still 1-2 pH units below upper pH limit of C18 LC columns ~ Additional mobile phase modifiers resulted in loss of NP sensitivity Standard Concentration NP Area Summary of Conditions 150 M-g/L 2573 0.01% acetic acid in A/B 150 M-g/L 3621 5 mM ammonium acetate in A/B pH = estimate 6.5 150 M.g/L 18437 no modifiers/neutral pH 150 Mg/L 323825 0.01% ammonium hydroxide in A/B pH = 10.05 150 ug/L ??? 0.1-0.5 mM ammonium fluoride in A Mobile Phase A B Time (min) Dl water No modifiers Methanol No modifiers Initial 80 20 15 5 95 19 5 95 19.1 80 20 23 80 20 Thermo Hypersil Gold C18, 2.1 x 50 mm, 3 |jm, 0.3 mL/min flowrate, 10 mL injection Electrospray Conditions Polarity Negative Ion Capillary needle voltage -4.0 kV Sheath Gas 40 L/h Aux Gas 4 L/h Sweep Gas 2 L/h Ion Transfer Tube Temp 325 ฐC Vaporizer Temp 375 ฐC Page 61 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmental Protection Agency Degradation of C18 Analytical Column at pH 10 j 50 |jg/L f\ iNonylphenol / 1 4-n-NP-13C6 | ~ Narrower peaks represent injections on | new column \ ~ Wider peaks show damage caused in one month at pH 10 ~ Upper column pH limit M listed at pH 11 SERA United Stales Environmental Protection Agency Mean Recovery and Precision (n=4) of Laboratory Fortified Blanks with Various SPE Sorbents ฆ Oasis HLB, 150 mg ฆ Bond Elut, 200 mg ฆStrata-X, 100 mg 120 Samples fortified at 4 [jg/L 110 100 3> 90 80 70 60 50 Nonylphenol 4-n-Octyl-d17-phenol 4-n-NP-2,3,5,6-d4,OD 4-tert-Octylphenol-13C6 (SUR) (SUR) (SUR) Linear Linear Branched Page 62 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Stales Environmontol Protection Agency Mean Recovery and Precision (n=4) in Laboratory Fortified Drinking Water Samples ฆ Ground Water (4 pg/L) ฆ Ground Water (1 pg/L) ฆ Surface Water #1 (4 pg/L) ^Surface Water #1 (1 |jg/L) ฆ Surface Water #2 (4 pg/L) 120 110 100 I 90~| ฃ 80 1 70 60 50 Oasis HLB, 150 mg T T T T ฑTTiT TMT Nonylphenol 4-n-Octyl-d17-phenol (SUR) Linear 4-n-NP-2,3,5,6-d4,OD (SUR) Linear 4-tert-Octylphenol-13C6 (SUR) Branched Results meet DQOs of 70-130% Surface Water #1 TOC = 2.2 mg/L, Hardness = 103 mg/L Surface Water #2 TOC = 0.97 mg/L, Hardness = 120 mg/L Ground Water TOC = 0.55 mg/L, Hardness = 360 mg/L SERA United Stales Environmontol Protection Agency Preliminary Aqueous Holding Time Study Mean Recovery and Precision (n=4) Tap Water Fortified at 4 |Jg/L, Oasis HLB, 150 mg i Day 0 ฆ Day 8 ฆ Day 14 > 80 Nonylphenol Sodium bisulfate and ascorbic acid preservation Preliminary study to 14 days A longer study to 28 days will be performed Extract holding time will be studied as well Page 63 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water ~ Preliminary Detection Limit (DL) based on precision only ~ LCMRL to be developed based on precision and accuracy ฆ Office of Research and Development National Exposure Research Laboratory Guideline HRL Minnesota Department of Health Limit 105 Mg/i 20 [jg/L Analyte Nonylphenol SEPA United Slates Environmental Protection Detection Limit Reference Guidelines SEPA United Slalea Predicting Blank Contamination Problems ฆLaboratory Reagent Blanks ^^Calibration Blanks (Extracted Blank) (Not extracted, injected after highest CAL point) Low CAL standard = 0.40 |jg/L Predicted MRL range (2-5X DL) = 0.16-0.40 ng/L 53 0.2 1/3 of MRL 0.053 (jg/L i 9 i i ft i 9- LRBs (n=16) Calibration Blanks (n=16) Page 64 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water *>EPA Protection Summary United Stales Environmontol Protection Agency ~ Rugged, standardized, sensitive method developed for nonylphenol in drinking water ~Investigated best labeled SUR and IS standards for method, chose branched octylphenol for SUR and branched nonylphenol for IS ~Meets data quality objectives (DQOs) for several types of SPE cartridges, for a ground water source and surface water sources, and for blank recovery ~On target to easily meet HRL *>EPA Protection Future Work and Method Delivery ~Investigate ammonium fluoride as LC mobile phase modifier to increase sensitivity and evaluate its impact on ESI stability in different DW matrices ~Include octylphenol as an additional analyte provided DQOs can be met ~Final performance data including holding time study and LCMRL study to be performed spring-summer of 2018 ~Final peer reviewed, multi-laboratory validated method published by September, 2019 I Off ice of Research and Development National Exposure Research Laboratory Page 65 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SERA United Slaios Environmental Protection Agency Questions?? Office of Research and Development National Exposure Research Laboratory Lunch Page 66 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA United States Environmental Protection Agency Method in Development: Legionella Maura J. Donohue Ph.D. Disclaimer: The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Stakeholder Meeting, Cincinnati, Ohio: June 6,2018 Legionellaceae Legionella (Genus) Gram negative bacteria (Gammaproteobacteria) Flagelia rod (2 20 urn) Slow grower (3 to 10 days) Majority of species will grow in free-living amoebae Aerobic, L-cysteine and iron salts are required for in vitro growth, pH: 6.8 to 1, Temp: 25 to 43 ฐ C ~65 species Pathogenic or potentially pathogenic for human 134 Page 67 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water From Environmental Microorganism to Public Enemy 65 Species Legionella spp. Legionella pneumophila Brenner etal. 1979 2000-2014 All OUTBREAKS are associated with Legionella pneumophila Sgl (Garrison et al. 2016) 94% of all legionellosis cases are associated with Legionella pneumophila Sgl Legionellosis: Respiratory Disease Disease Legionellosis = pneumonia Legionaries' Disease (severe) Pontiac Fever (mild/lite) Signs/Symptoms Pneumonia (Signs/Symptoms) High Fever q q Headache q Muscle ฆฆ |l| Ache* 0*||*( National Notifiable Disease Surveillance System (NNDSS) 2012 National Reportable Disease List: contained the names of 110 Diseases/Microorganisms Number of Cases Reported in 2015: 5,100 cases legionellosis III! II IS 11111-1 swas ranked 23rd/H0 Annual Cost of Treatment in the U.S. Number of Hospitalization/year ฎ8,000-18,000 cases avg( 13,000) Marston, (1997) Total Hospitalization Cost: $433,758,000 Collier, SA. etal 2012: Direct healthcare costs of selected disease primarily or partially transmitted by water. Epidemiology Infection, 140, p2003-2013 Page 68 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water 1^=1 Exposure Routes: Environmental Sources Legionella pneumophila is a microorganism of the natural environment found in soil and water. Route Treated WATERRoi WATER Route fi.- "T"1 inside the Agency: Legionella Environmental microorganism Due to treatment the likelihood of Legionella presence in public supply water is low. Gram negative bacteria are inherently prone to chemical disinfection. Premise plumbing issue not a public supply issue. (cooling towers, decorate fountain, HVAC systems) Potential occurrence, maybe persistence, and never colonization. Distribution not premise plumbing (amount of water moved) IBS Page 69 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Inside the Agency: Legionella Surface Water Treatment Rule -1989 SWTRs is to reduce illnesses caused by pathogens in drinking water. The disease-causing pathogens include Legionella, Giardia lamblia, and Cryp tosp ori dium. ฆ Applies to all public water systems using surface water sources or ground water sources under the direct influence of surface water. ฆ Requires most water systems to filter and disinfect water Establishes maximum contaminant level goals (MCLGs) for viruses, bacteria and Giardia lamblia. ฆ Includes treatment technique (TT) requirements for filtered and unfiltered systems to protect against adverse health effects of exposure to pathogens. ฆ Legionella MCLG is 0 cfu ฆ Prescribes NO method nor monitoring requirements ฆ TT requirements (filtering and disinfection) ^tDSr> 3> iฎ, % ,c> 4* PR Legionella Methods Standard Methods BYCE CDC CVCC Colony Form Units Legionella spp. ISO GVCC Legionella pneumophila Legioleart Yes, ฆ - Most Probable Idexx Proprietary 1 = Number (MPN) Legionella DNA Legionella pneumophila Legionella pneumophila Sgl Target Number RNA Legionella spp. Sigma Aldrich: HybriScan Legionella pneumophila Antigen II - s P rese nce/Abse nee Legionella pneumophila Sgl Alere Page 70 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Legionella Methods- Time is of the Essence? Method Type Concentration Extractions Incubation Plus gionella identificatior Culture - CDC Standard Methods ISO Culture - Legioleart Urine Antigen Test DNA-qPCR RNA- Southern Blot Days Days 4 days + Days ^Davs Minutes Analysis 15 minutes ฆ Hฐur'- 4 hrs _ Hours _ Hours 3 hrs Parshionikar S, et a I. 2009 Method Validation of US EPA Microbiological Methods of Analysis Bustin SA, et al. 2009 The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry 55 611-622p. 142 iฃlateConti^[s Method Verification Process Page 71 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Jf The Molecular Magnifying Glass Quantitative Polymerase Chain Reaction (qPCR) L. pneumophila Cell *388%* Components in a qPCR Reaction DNA Extracted from Water (DNA Template) Free Base Pairs DNA Buffer dNTPs Polymerase w MgCI Primers & Probes -2 Fluorophore DNA Product The Molecular Detection of Legionella in Potable Water Method Membrane Filtration Polycarbonate 0.4 /jm DNA Extraction Bead Beating DNA precipitation qPCR 40 cycles Assays I. Genus: Gen-L (16S gene) 2. Species: Lp-16S (16S gene) 3. Species: Lp-Mip (MipAgene) Page 72 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water QC Samples for qPCR 1. Method Blank (negative control) Sterile molecular biology grade water filtered and processed at the same time in the same way as unknowns 2. Standards (positive control) Purified genomic DNA from target, serially diluted 3. No Template Control (negative control) Sterile molecular biology grade water added to qPCR reaction instead of DNA 4. Internal positive Control (IC) Commercially available kit (TaqMan Exogenous Internal Positive Control Kit, Life Technologies, Carlsbad, CA) Method in Development: Legionella (qPCR) Phase I: Standard Curve Generation Phase II: Extraction Proficiency Phase III: Sensitivity Study 146 Page 73 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Phase ^Standard Curve Generation Purpose: To establish assay performance measures and analyst competence Standard Curve (7 point of DNA dilution series). Each analyst will make four independent "Ass ay/Master mix mixes". Analyst will test each Assay/Master mix mix by analyzing each standard in triplicate. Three Internal Labs One External Lab Include a Non Template Control (NTC). Verification by: Lab 1 Lab 2 Lab 3 Lab 4 In total 96 Reactions were analyzed by each (Internal) (Internal) (Internal) (External) Assay analyst. Designation Gen L Lab 1 Lab 2 Lab 4 Lp Mip Lab 1 Lab 2 Lab 4 Lp 16S Lab 2 Lab 3 Lab 4 Phase I: Standard Curve Generation 100 Hi 100 Hi 100 hL 100 ^L 100 hL Measures Precision Accuracy Linearity Relative Standard Deviation Efficiency Limit of Detection Limit of Quantification Page 74 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Units of Measurement: qPCR Cq (quantitation cycle) = florescence light of the probe Cell Equivalence Cell (Log) Equivalence Amplification Plot "ป / // f / A mathematical formula generated from the standard curve (Regression Line). Cell Equivalence (log) transformed gji 8 i | ~ DNA extract quantified ~ Mass of the genome Higher Cq= Lower the Concentration Lower Cq= Higher the Concentration 149 Standard Curve: Genus (16S) Legionella Genus Assay t 1 9 012 3 45678 Standards 1 ID"! 10*2 10*3 10"4 10*5 10*6 mean r 37.55 34.35 30.98 2Z5i 23.96 20.54 16.86 STDev r 0.98 0.32 0.18 0.24 0.15 0.13 0.18 % Det 77% 100% 100% 100% 100% 100% 100% Cv 0.03 0.01 0.01 0.01 0.01 0.01 0.01 %RSD 2.62 0.93 0.60 0.87 0.65 0.64 104 Legionella spp. Legionella Genus Assay Number of Reactions 0 5 10 15 20 25 30 35 40 Std Std Std Std Std T t - std 150 Page 75 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Standard Curve: L. pneumophila (species) (16S) L. pneumophila (species) Standard Curve S B v = -3.3932x +42.735 Rz = n mean STDev % Det 01234567 Standards 1 10^1 10*2 10A3 10M 10*5 10A6 38.87 36.23 32.78 29.36 25.74 22.38 18.78 100% I 100% 100% 100% 100% 100% 1.84 1.94 L pneumophila 16S Assay Standard Curve Number of Reactions " ซ . ป- std 7 Standard Curve: L. pneumophila (species) (MIPA) L pneumophila MIP gene Standard Curve Standards LowestDNAConcentration HighestDNAConcentration L. pneumophila MIP gene Standard Curve Number of Reactions 1 10*1 10*2 10*3 iom"| 10*5 10*6 mean 38.75 37.62 34.13 31.00 27.39 24.05 20.46 STDev 1.48 0.73 0.28J 0.19 J o.28 n 0.43 0.53 %Det 36% 100% 100% 100% 100% 100% 100% Cv 0.04 0.02 0.01 0.01 0.01 0.02 0.03 %RSD 3.81 1.95J 0.82 J 0.63 1.04^ 1.81 2.60 I s std3 r~std4 * * ;ซ Std 6 Std 7 Page 76 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Phase I: qPCR Method Performance Criteria Leg-G Lp-16S Lp-MipA Precision (RSD) repeatability Analyst II 0.9-3.5 0.1-1.5 0.6-3.8 Analyst IV 0.3-2.1 0.4-2.1 0.6-4.8 Precision (RSD) reproducibility 0.6-2.6 1.0-2.9 Linearity (R2) 0.9997 0.9986 0.9897 Efficiency (E) 94.7 97.1 106 Limit of Detection (LOD) 10 ce/rx 10 ce/rx 100 ce/rx Limit of Quantification (LOQ) 100 ce/rx 100 ce/rx 100 ce/rx 153 Phase II: Extraction Proficiency Purpose: to define Extraction Proficiencv Spiked filters with known quantities are given to each Lab for extraction and analysis What: ~ 3 Concentrations + Method Blank ~ 3 spike filters/concentration (Total: 9 Extractions) ~ Each Extraction analyzed in triplicate. Who: ~ Three Internal Labs One External Lab Theoretical Amount Extracted Amount (N 0 M em brane a nd Ex traction) (MembraneandE xtracti on) Percent (%) Mean Recovery 154 Page 77 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Precipitation MPC Precipitation 500 nl_ RNAase A luL Isopropanol 850 |iL Ethanol 500 ML Add Lysis Buffer 500 piL Sample Extraction Extraction Efficiency: Legionella spp. Genus Extraction Efficiency: Legionella Genus - Cq Cq scatterplot at each cell spike -327477X + 35.977 10 *3 Dilution ฆ Analyst 1 ฆ Analyst 2 ฆ Analyst 4 156 Page 78 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Extraction Efficiency: Legionella spp. Genus-Cell Equivalence/200ml_ l.E+07 l.E+06 E ง l.E+05 8 l.E+04 l.E+03 ^ l.E+02 U l.E+01 1.E+00 ฆAnalyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 ฆTheoretical A6B Analyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 Extraction Efficiency: L. pneumophila- Cq/200 mL Recovery Efficiency: L. pneumophila 16S gene - Cq 100 E i I I I I D7 D4 D.S 10 1,110 io*2 Dilution Series 120 l| IIIIII D2 D4 D5 10 10 *3 io*2 "6 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 Scatterplot (Spread) by Cell Dilution L ""-ฆI l-.._ VI '*ฆฆฆ y =^7024x " 37.704 | 10 "2 10" 10 *6 Page 79 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Extraction Efficiency: L. pneumophila- Cell Equivalence/200mL I lit 11 10A6 10A3 10A2 ฆ Analyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 10 *6 10 *3 10 ฆ Theoretical ฆ Analyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 Extraction Efficiency: L. pneumophila MIP gene Cq/200mL Recovery Efficiency: L. pneumophila MIP gene - Cq III ill i.i Dilution Series Scatterplot (Spread) by Cell Dilution 1 = -3.487Sx + 39.778 t R2 = .9764 1 1 10 -2 10 "3 10 "6 ฆ Analyst 1 ฆ Analyst 2 ฆ Analyst 4 Page 80 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Extraction Efficiency: L. pneumophila MIP gene - Cell Equivalence/200ml_ ฆAnalyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 ฆ Theoretical ฆ Analyst 1 ฆ Analyst 2 ฆ Analyst 3 ฆ Analyst 4 Summary Phase II: Percent Recovery Cq Cell Equivalence GenL Analyst Analyst 2 Analyst Analyst 4 Gen L Analyst 1 Analyst 2 Analyst 3 Analyst 4 96 Recovery RSD 96 Recovery RSD Recovery RSD 96 Recovery RSD Theoretical Recovery 96RDS Recovery 96RDS Recovery 96RDS Recovery 96RDS D2 99.61 2.6 95.59 4.1 99.52 2.2 D2 952,004 931,099 35 572,635 54 862,986 30 D4 98.15 1.6 98.39 2.1 94.48 2.2 D4 8,833 6,471 27 6,975 38 3,303 48 D5 99.01 1.0 96.76 2.6 93.53 1.7 D5 846 700 20 474 41 216 39 Lpl6S Analyst Analyst 2 Analyst Analyst 4 Lpl6S Analyst 1 Analyst 2 Analyst 3 Analyst 4 96 Recovery RSD 96 Recovery RSD Recovery RSD 96 Recovery RSD Theoretical Recovery 96RDS Recovery 96RDS Recovery 96RDS Recovery 96RDS D2 92.92 1.44 94.82 3.47 98.51 2.5 D2 1,552,857 319,098 50 338,665 26 702,596 31 D4 96.09 0.64 95.14 1.74 92.57 1.9 D4 12,977 4,284 32 3,392 12 2,093 40 D5 95.73 0.82 96.07 2.31 95.84 1.5 D5 1,033 330 36 327 18 326 38 MIP A Analyst Analyst 2 Analyst Analyst 4 Mip A Analyst 1 Analyst 2 Analyst 3 Analyst 4 96 Recovery RSD 96 Recovery RSD Recovery RSD 96 Recovery RSD Theoretical Recovery 96RDS Recovery 96RDS Recovery 96RDS Recovery 96RDS D2 97.1 2.2 94.8 3.3 95.8 2.3 D2 1,138,959 706,657 38 501,326 55 557,594 44 D4 96.3 1.2 97.3 1.4 91.9 1.8 D4 6,852 2,951 23 3,833 29 1,039 47 D5 97.3 0.7 96.4 2.0 94.3 1.8 D5 591 300 18 256 41 148 57 162 Page 81 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Phase Ml: Compete Method- Sensitivity Study 105-felhw.tE 10" Cll.c-CE 10i3 calhorft 10 iปCE 10 atom Cell Spikes 200mL 200ml. 200mL 200ml 200mL cilia 163 Phase IN; Compete Method-Sensitivity Study What: 6 Concentrations + Method Blank 3 spike bottles/concentration (Total: 21 Extractions) Each Extraction analyzed in triplicate for each assy. Who: One Internal Lab One External Lab Theoretically Cell Spike: Quantified by spiking 50|iL directly into a bead tube. No Bottle No membrane filtration 164 Page 82 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Internal Lab ONLY Sensitivity Study: Legionella spp. (Genus) 10 Cells or CE 10 5 Cell 10 3 Cells Theoretical Spike 10 4 Cells or CE 10 z Cells or CE ZOOmL 200mL 200mL 200m L 200mL Recovery vlethod Blank 101% 93% No Detects Cell Equivalence 67% RSD 9 117% 10 3 Cells or CE 10 Cells or CE 200m L 10 Cells or CE 200mL 10 Cells or CE 200mL 10 Cells or CE 200m L Cell (Log) Equivalence 200m L Internal Lab ONLY Sensitivity Study: L. pneumophila species C16S) Theoretical 105ceiisorCE 10 4 Cells or CE 10 3 Cells or CE 10 2 Cells or CE 10 Cells or CE * 1) V, Spike 200mL 200mL ~ 200m L 200mL 200mL Recovery B r- ฆ 0 ฆMethod 1 Blank 1 9/9 rx 9/9rx 9/9rx 9/9rx 9/9 rx 9/9 rx Cq 97% 98% 1.0 100% 0.2 98 0.6 % 99% 2.4 No Detects Cell Equivalence 61% RSD 11 70% 17 103% 3.2 71% 100% 14 59 10 4 Cells or CE 10 3 Cells or CE 10 2 Cells or CE 10 Cells or CE 200m L 200mL 200mL 200m L 200mL 166 Page 83 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Internal Lab ONLY Sensitivity Study: L. pneumophila species MipA ll I m Theoretical 105 ceiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 2 CeiisorCE 10 CeiisorCE 1 Spike 200mL 200mL ~ 200m L >8- 200mL ~ 200mL Recovery jj L ฆ r ฆ ฆ g 9/9 rx 9/9rx 9/9rx 9/9rx 3/9 rx 9/9rx Cq 98% RSD 048 99% 1.04 100% 0.55 90% 93% B.14 0.87 No Detects Cell Equivalence 67% RSD 9 80% 21 111% 14 10% 19% 66 22 Cell (Log) Equivalence ceiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 CeiisorCE 10 CeiisorCE 200m L 200mL 200mL 200m L 200mL 167 Internal Lab Results ONLY Summary of Sensitivity Study Cell Spike per 200 mL 10 5 CeiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 2 CeiisorCE 10 CeiisorCE Cq 97% 98% 101% 98% 93% 97% 98% 100% 98% 99% 98% 99% 100% 90% 93% Cell Equivalence 67% 74% 117% 46% 17% 61% 70% 103% 71% 100% 67% 80% 111% 10% 19% Cell (Log) Equivalence io5 ceiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 CeiisorCE 10 CeiisorCE 200mL 200m L 200mL 200mL 200mL 10 5 CeiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 2 CeiisorCE 10 CeiisorCE 200mL 200m L 200mL 200mL 200mL 10 5 CeiisorCE 10 4 CeiisorCE 10 3 CeiisorCE 10 CeiisorCE 10 CeiisorCE 200mL 200mL 200mL 200mL 200mL Page 84 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Status All Three Phases have been completed by both internal and external labs, Find another external Lab for Second Lab Verification Data has been received from the External Lab for Phase III Sensitivity Study. Analyze data Holding time study Write Method 169 Water Type Source/Raw Water Potable Water Waste Water Rain Water Sediment Biofilm 170 Page 85 of 103 'sM ------- Methods Development for Unregulated Contaminants in Drinking Water Acknowledgements Dr. Stacy Pfaller Dawn King Dr. Jingrang Lu lanStruewing Dr. Myriam Medina-Vera Dr. Lindsey Stanek Dr. Eric Villegas Northeast Ohio Regional Sewer District Mr. Frank Greenland Rosemarie Read Dr. Maura Donohue ORD/NERIVEMMD/PHCB tfoa/ ' Q \ IWJ 4L PrO^ Questions Office of Research and Development National Exposure Research Laboratory, Exposure Methods and Measurement Division (EMMD) Page 86 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water oEPA Unitttd Stales Erjvironmontal Protnction Agency Detection Methods for Mycobacteria Stacy Pfaller Ohio River and Downtown Cincinnati, OH Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Background Environmental Protection Agoncy ~Waterborne illness caused by nontuberculous mycobacteria (NTM) cost nearly $1.8 B for in-patient and out-patient treatment in 2010 (aThomson et al, 2015). ~>Pulmonary NTM infections account for almost half of all NTM hospitalizations in the US, and are typically caused by Mycobacterium avium (MA) and M. intracellulars (Ml) ~Mn addition to pulmonary infections, can cause skin, soft tissue, lymph node, systemic infections, among others ~Primary source of human exposure: WATER :-CCL's 1 and 2: Mycobacterium avium Complex (MAC) ~CCL's 3 and 4: M, avium 4 subspecies: M. avium subsp. hominissuis M. avium subsp. avium M. avium subsp. silvaticum M. avium subsp. paratuberculosis aThomson et al (2015) Ann Am Thorac Soc, Vol 12:1425-1427 r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 174 Page 87 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA United Stales Environmental Protection Agency Culture and Molecular Methods ~Culture Method Standard Methods for the Examination of Water and Wastewater3 section 9260M with modifications described in Covert et al (1999) Appl Environ Microbiol 65:2492-2496 ~Quantitative PGR (qPCR) Beumer et al, (2010) Appl Environ Microbiol 76:7367-7370 and Figure S1, Supplemental Material http://aem.asm.org/content/76/21/7367/suppl/DC1 Chern et al, (2015) J Wat Health 13.1:131-139 aEaton, A. D., L. S. Clesceri, E. W. Rice, and A. E. Greenberg (ed.). 2005. Standard methods for the examination of water and wastewater, 21st ed. American Public Health Association, Washington, DC. - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division SERA United Slates Environmental Protection Agency Sample Collection ' Sample collection is identical for both culture and qPCR Bulk Water: collected in 1L sterile polypropylene bottles, NO preservative (Na2S203), according to sections 9060A and B of Standard Methods for the Examination of Water and Wastewater3 Samples transported back to lab on ice, stored at 4 ฐ C until processing, within 48 hours - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Page 88 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA United Slates Environmental Protection Agency Culture method ~1L water split into replicate aliquots (vol. depends on sample type) ~Samples filtered through 0.45 urn pore-size, 47 mm black-grid, cellulose ester filter by vacuum filtration, washing the filter with sterile deionized water, filter aseptically transferred to Middlebrook 7H10 agar containing 500 mg L1 cycloheximide ~ Plates are incubated a minimum 8 weeks at 37 ฐ C and inspected weekly for growth t Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division SERA United Slates Environmental Protection Agency QC samples for Culture method ~Sterile medium negative control - Performed when medium is made, in advance of samples arriving Incubation of un-inoculated medium to ensure sterility ~Method blank negative control Sterile deionized water filtered processed at the same time in the same way as unknowns t Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Page 89 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water United Slates Environmental Protection Agency SERA United Slates Environmental Protection Agency Benefits of Culture Method ~ Many species of NTM can grow on medium ~ Live only detection ~ Obtain a culture collection for future characterization Genotype Virulence genes Drawbacks of Culture Method ~ Method has not been characterized for specificity or sensitivity ~ medium is not selective for mycobacteria ~>Cetyl pyridinium chloride (CPC) disinfection may reduce recovery of target by 70% (Personal communication: Terry Covert) ~ Every colony is an unknown in need of identification ~ Only a subset of colonies can be chosen for identification ~ method is not quantitative ~ Months to years before results are obtained ~ Performs poorly on biofilm r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 180 Page 90 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water *>EPA Bulk water Biofilm United States Environmfflntol Protection Agency qPCR Method and/or * rv ~ 1 L water (per target assay) or biofilm slurry is vacuum filtered through 47.0 mm, 0.45 um polycarbonate membrane ~ Membrane rolled and placed in 2.0ml tube containing 0.3g glass beads and buffer ~ Microorganisms trapped on membrane lysed physically by bead beating ~ DNA from crude lysate extracted using WaterMaster kit reagents from (Epicenter Biotechnologies, Madison, Wl) MA/MI/MC-specific assay ~ DNA resuspended in sterile, molecular biology-grade water ~Three replicate qPCR reactions analyzed/ DNA extract Two replicates must be positive for a sample to be considered positive t Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Data Analysis Absolute Quantification SEPA United States Environmental Protection Agency Absolute Quantification from Master Standard Curves J'Generated from six independents series of 10-fold serial dilutions of purified genomic DNA from ATCC Type strains of MA, MI/MC J'Each dilution series contains eight standards, ranging in concentration from 10ฎ target copies to 1 copy, run in triplicate = 18 measurements/ standard >CT measurements plotted against log target number and analyzed by linear regression to generate line equation J'Target number in unknown sample estimated from line equation '. ฆ . 1 ป ป - . . f r-k * "V "IT** * A i t Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Page 91 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water v>EPA United Stales Environnrontol Protection Agency QC Samples for qPCR ~ Method Blank (negative control) Sterile molecular biology grade water filtered and processed at the same time in the same way as unknowns ~Extraction Control Sterile filter processed at the same time in the same way as unknowns ~Standards (positive control) Purified genomic DNA from target, serially diluted ~No Template Control (negative control) Sterile molecular biology grade water added to qPCR reaction instead of DNA extract ~Internal positive Control (IC) Commercially available kit (TaqMan Exogenous Internal Positive Control Kit, Life Technologies, Carlsbad, CA) r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 183 SEPA United Stales Environmentnl Protection Agency Benefits of qPCR Method ~Assays are specific for MA and MI/MC ~Quantitative ~Time to results = 3 days Drawbacks of qPCR Method ~Assays are specific for MA, MI/MC only ~Cannot distinguish between live and dead organisms though studies have demonstrated that DNA contained within chlorine disinfected cells does not typically persist in water with a chlorine residual Page et al, 2010, Appl Environ Microbiol, 29:2946-2954 Sen et al, 2010, Current Microbiol, 62:727-732 r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 184 Page 92 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water v>EPA United Stales Environnrontol Protection Agoncy Mycobacterium qPCR Method Verification ~Started December 2014 in coordination with Maura Donohue and the Legionella qPCR method verification ~Mwo Mycobacterium assays - M. avium - M. intracellularelchimaera ~Verification performed in three phases -Phase I: Characterizing LOD, LOQ from generation of DNA standard curves (First performed on LifeTech StepOne instrument, repeated on new LifeTech Quant 6 Studio) = complete - Phase II: Characterizing target extraction efficiency = needs repeating Options: Bioballs containing known DNA concentrations Known DNA concentrations spiked on filters - Phase III: Characterizing method sensitivity = not complete r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division SERA United Stales Environnrontol Protection Agency Characteristics of qPCR assays for drinking water and biofilm qPCR assay Target (copies/ genome) En Amplification Efficiency LODt Targets/qPCR reaction LOQt Targets/qPCR reaction Specificity ฃ Sensitivity6 Drinking Water M. avium 16S rDNA (1) 103% 10 10 100% Not determined M. intracellulare/ chimaera 16S rDNA (1) 92% 10 10 100% Not determined ''Amplification Efficiency = -1 + io<-1/slฐPe>. Acceptable range = 90 - 110%. *LOD = Limit of detection = lowest copy number/assay giving CT< 40 in 6/6 independent assays. +LOQ = Limit of quantification = lowest copy number/assay yielding a coefficient of variation < 25%. ฃSpecificity = Number of target testing positive/total number targets tested x 100. ง Sensitivity = lowest copy number detected when spiking serial dilutions of known cell quantities into actual tap water samples, processed as described. - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Page 93 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA Additional qPCR Methods United Slates Environmental Protection Agency ~The literature describes many qPCR assays for targeting various taxonomic levels within the genus Mycobacterium - Mycobacterium Genus-specific assays Bruijnesteijn van Coppenraet, E.S.. Lindeboom, J.A., Prins, J M Peeters, M.F., Claas, E.C.J, and Kuijper, E.J. (2004) J Clin Microbiol 42:2644-2660. Radomski, N., Lucas, F.8., Moilleron, R., Carnbau, E., Haenn, S., Moulin, L. (2010) Appl Environ Microbiol 76:7348-7351. - Mycobacterium species-specific assays ฆ M avium: Feazel, L.M., Baumgartner, L.K., Peterson, K. L., Frank, D.N., Kirk Harris, J., Pace, N.R. (2009) PNAS 106:16393-16399 M. tuberculosis. M. avium. M. intracellulars, M. kansasii, M. abscessus, M. massilense, and M. fortuitum'. Kim, K.I., Lee, H., Lee, M.-K, Lee, S.-A., Shim, T.-S., Lim, S.Y., Koh, W.-J., Yim, J.-J., Munkhtsetseg, B., Kim, W., Chung, S.-l., Kook, Y.-H., Kim, B.-J. (2010) J Clin Microbiol 48:3073-3080. Eighteen Mycobacterium species: Lim, S.Y., Kim, B.-J., Lee, M.-K., Kim, I.K., Lett Appl Microbiol (2008)46:101-106. r Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 187 SERA United Slates Environmental Protection Agency Identification of Isolates using Matrix Assisted Laser Desorption/ Ionization (MALDI) Protein Profiles ~ Performed on purified culture isolates ~ More rapid than DNA sequencing methods ~Two systems for bacterial identification: -Bruker MALDI Biotyper and Mycobacterium database: 164 species with unique profiles - Biomerieux Vitek MS and V3 database for molds, Nocardia, and Mycobacterium - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division n I It1 MU 1 ill J Lin ( tkrtwf Page 94 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA United States Environmental Protection Agoncy Best Method for Mycobacterium Detection for Finished Drinking Water? ~qPCR performs regardless of sample matrix (water or biofilm) and volumes up to 10 L are easily and rapidly analyzed ~Culture does not perform on microbiologically complex samples (water and biofilm) but does perform on samples where microbiological water quality is high (treated water before distribution) - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division SERA United Slates Environmental Protection Agency Acknowledgments Dawn King (NERL) Maura Donohue (NERL) Amy Beumer (NERL) Eunice Chern (NERL) Terry Covert (NERL) Jingrang Lu (NERL) Ian Struewing (NERL-Aptim) - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division 190 Page 95 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water SEPA United Stales Environmental Protection Agency Questions - Office of Research and Development National Exposure Research Laboratory, Systems Exposure Division Open Forum and Discussion Brenda Parris U.S. EPA Office of Ground Water and Drinking Water Technical Support Center Page 96 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Webinar Participant Questions Figure 1 Click on "+" next to "Questions" in the control panel (Figure 1) to submit questions/comments You may need to unhide the control panel to ask a question (Figure 2) Type a question in the box; click send (Figure 3) Figure 2 Figure 3 El Polls (0/0) @ ^estions 0 Chat [Enter a question for staff] fI Questions June 2018 U.S. Environmental Protection Agency Slide 193 of 206 2* PRO^i If You Have Questions Following This Meeting/Webinar Analytical Methods for Drinking Water Homepage: https://www.epa.gov/dwanalvticalmethods/analytical- methods-developed-epa-analysis-unregulated- contaminants Methods Presenters Webinar methodsdevelopment@cadmusgroup.com June 2018 U.S. Environmental Protection Agency Slide 194 of 206 Page 97 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Break -r --V " -i- X V (15 minutes) Open Forum and Discussion Page 98 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Closing Remarks Brerida Parris, U.S. EPA Office of Ground Water and Drinking Water Technical Support Center Abbreviations and Acronyms CCC - Continuing Calibration Check CCL-Contaminant Candidate List CDC - Centers for Disease Control and Prevention CIS - Contaminant Information Sheet Cq - Quantitation Cycle DAI - Direct Aqueous Injection DIMP - Diisopropyl methylphosphonate DL- Detection Limit DNA - Deoxyribonucleic Acid June 2018 U.S. Environmental Protection Agency Slide 198 of 206 Page 99 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Abbreviations and Acronyms dNTP - Deoxyribonucleotide Triphosphate DQO - Data Quality Objective DVB - Divinylberizene ESA - Ethane Sulfonic Acid ESI - Electrospray Ionization FR-Federal Register GC - Gas Chromatography GC - Gas Chromatography GenX- Perfluoro-2-propoxypropanoic acid June 2018 U.S. Environmental Protection Agency Slide 199 of 206 Abbreviations and Acronyms HLB - Hydrophilic Lipophilic Balanced HRL- Health Reference Level HVAC - Heating, Venting, and Air Conditioning i.d. - Internal Diameter IDC-Initial Demonstration of Capability IS - Internal Standard ISO -International Organization for Standardization IUPAC - International Union of Pure and Applied Chemistry LC-Liquid Chromatography June 2018 U.S. Environmental Protection Agency Slide 200 of 206 Page 100 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Abbreviations and Acronyms LCMRL - Lowest Concentration Minimum Reporting Level LFB - Laboratory Fortified Blank LFSM - Laboratory Fortified Sample Matrix LFSMD - Laboratory Fortified Sample Matrix Duplicate LLE- Liquid Liquid Extraction LOD - Limit of detection LOQ-Limit of quantification LRB - Laboratory Reagent Blank MCLG - Maximum Contaminant Level Goal June 2018 U.S. Environmental Protection Agency Slide 201 of 206 Abbreviations and Acronyms MDL- Method Detection Limit MRL- Minimum Reporting Level MRM - Multiple Reaction Monitoring MS - Mass Spectrometry MS/MS -Tandem Mass Spectrometry NCOD - National Contaminant Occurrence Database NDEA - N-Nitrosodiethylamine NOMA - N-Nitrosodimethylamine NDPA- N-Nitrosodipropylamine June 2018 U.S. Environmental Protection Agency Slide 202 of 206 Page 101 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Abbreviations and Acronyms NP - Nonylphenol NPDWRs - National Primary Drinking Water Regulations NPYR - N-Nitrosopyrrolidine NTM - Nontuberculous Mycobacteria OA-Oxanilic Acid PAH - Polycyclic Aromatic Hydrocarbons PCB- Polychlorinated Biphenyl PCR-Polymerase Chain Reaction PFAS - Per- and Polyfluoroalkyl Substances June 2018 U.S. Environmental Protection Agency Slide 203 of 206 Abbreviations and Acronyms PFOA - Perfluorooctanoic Acid PFOS - Perfluorooctane Sulfonic Acid PWS - Public Water System QC - Quality Control QCS - Quality Control Sample qPCR- Quantitative Polymerase Chain Reaction RNA- Ribonucleic Acid RSD - Relative Standard Deviation SDWA - Safe Drinking Water Act June 2018 U.S. Environmental Protection Agency Slide 204 of 206 Page 102 of 103 ------- Methods Development for Unregulated Contaminants in Drinking Water Abbreviations and Acronyms SIM - Select Ion Monitoring SPE-Solid Phase Extraction SUR - Surrogate Standard SVOC - Semivolatile Organic Compound TPTH -Triphenyltin hydroxide TOC-Total Organic Carbon TT- Treatment Technique UCMR- Unregulated Contaminant Monitoring Rule UPLC - Ultra Performance Liquid Chromatography June 2018 U.S. Environmental Protection Agency Slide 205 of 206 Abbreviations and Acronyms UV- Ultraviolet Light VOC - Volatile Organic Compound WAX SPE - Weak Anion Exchange Solid Phase Extraction June 2018 U.S. Environmental Protection Agency S^tPRO^, 4/ Slide 206 of 206 Page 103 of 103 ------- |