# Overview of the Contaminant Candidate List and the Unregulated Contaminant Monitoring Rule

1 

# Steven C. Wendelken, Ph.D.

Office of Groundwater and Drinking Water

Standards and Risk Management Division

Technical Support Center

# How Are Drinking Water Standards Developed?

• 1996 SDWA amendments changed the process of dev eloping and reviewing NPDWS

– Contaminant Candidate List (CCL)

– Unregulated Contaminant Monitoring Rule (UCMR)

– Regulatory Determination

– Six

- Year Review

2

# Contaminant Candidate List (CCL)

• SDWA requires EPA to list unregulated

con taminants that may require a national drinking water regulation in the future

• Every five years CCL defines unregulated

co n taminants for which EPA needs

– Occurrence data

– Analytical methods

– Potential health effects

– Evaluation of treatment techniques

3

# UCMR Objective

• Collect occurrence data for suspected drinking
water contaminants that do not have health

based standards set under SDWA

• Occurrence information is used to support future

re g ulatory decision

- making

– Supports the Administrator’s determination of whether (or not) to regulate a contaminant under the drinking water program

4

# Three Regulatory Determination Criteria

SDWA requires EPA to consider the following criteria in evaluating whether to regulate a contaminant:
1) The contaminant may have an adverse effect on the health of persons; 2) 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 3) 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

*SDWA Section 1412(b)(1)

5

# Status and Next Steps for Regulatory Determinations 3 (RD 3)

• Published Third Contaminant Candidate List (CCL 3) in Oct 2009, which lis ted 116 contaminants:

– 12 microbes (e.g., viruses, bacteria)

– 104 chemicals (pesticides, industrial chemicals, pharmaceuticals, inorganics)



## • Evaluating the health and occurrence information to identify which CCL 3

con tam inants have sufficient information to make to the preliminary regulatory determinations

• Expect to publish preliminary RD 3 for public comment in 2013

• After considering public comments, expect to publish final RD3 in 2014

6

# Six Year Review 3

• 1996 SDWA Amendments require EPA to review and, if
app ropriate, revise existing National Primary Drinking Water Regulations (NPDWRs) every six years

– In 2003, EPA completed the 1st Six Year Review of 69 NPDWRs; made dec ision to revise TCR

– In 2010, EPA completed the 2nd Six Year Review of 71 NPDWRs and made deci s ions to revise tetrachloroethylene (PCE), trichloroethylene (TCE), acrylamide and epichlorohydrin



# • Occurrence analysis is a key component in the Six Year Revi ew process

• Expect to complete Six Year Review 3 by 2016

7

# General Flow of SDWA Regulatory Processes

Draft CCL

Final CCL

Final Rule (NPDWR) Six Year Review of Existing NPDWRs No further action if make decision to not to regulate (may develop health advisory). 

### Preliminary Regulatory Determinations

Final Regulatory

Determinations Proposed Rule (NPDWR) Public review and comment



### Draft UCMR

Final UCMR

UCMR Monitoring Results



## At each stage, need increased specificity and confidence in the type of supporting data used (e.g. health, occurrence, treatment).

8

# Generation of CCL 3

• EPA considered approximately 7,500 potential chemical and microb ial contaminants

• Screening process based on a contaminant’s potential to occur in public water systems (PWSs) and the potential for public health concerns

• Further detailed evaluations, public input, and expert judgment a nd review are the final contaminant selection tools

• Final CCL 3 published September 2009

– 104 chemicals or chemical groups and 12 microbiological contaminants 9

# Unregulated CCL 3 Contaminants 104 Chemicals and 12 Microbes

1,1,1,2

- Tetrachloroethane 1,1

- Dichloroethane 1,2,3

- Trichloropropane 1,3

- Butadiene 1,3

- Dinitrobenzene 1,4

- Dioxane 17 alpha

- Estradiol 1- Butanol 2- Methoxyethanol 2- Propen -1- ol 3- Hydroxycarbofuran (degradate) 4,4'

- Methylenedianiline Acephate Acetaldehyde Acetamide Acetochlor Acetochlor ethanesulfonic acid (ESA) Acetochlor oxanilic acid (OA) Acrolein Alachlor ethanesulfonic acid (ESA) Alachlor oxanilic acid (OA) alpha

- Hexachlorocyclohexane (former) Aniline Bensulide Benzyl chloride Butylated hydroxyanisole Captan Chlorate (also D

- DBP) Chloromethane (Methyl chloride) Clethodim Cobalt Cumene hydroperoxide C yanotoxins (3) Dicrotophos Dimethipin Dimethoate Disulfoton Diuron Equilenin Equilin Erythromycin Estradiol (17

- beta) Estriol Estrone Ethinyl Estradiol (17

- alpha) Ethoprop Ethylene glycol Ethylene oxide Ethylene thiourea Fenamiphos Formaldehyde (formerly) Germanium Halon 1011 (Bromochloromethane) HCFC

- 22 Hexane Hydrazine Mestranol Methamidophos Methyl bromide (Bromomethane) Methyl tert

- butyl ether Metolachlor Metolachlor ethanesulfonic acid (ESA) Metolachlor oxanilic acid (OA) Molinate Mol ybdenum Nitrobenzene Nitroglycerin N- Methyl -2- pyrrolidone N- Nitrosodiethylamine (NDEA) N- nitrosodimethylamine (NDMA) N- Nitroso

- di -n- propylamine (NDPA) N- Nitrosodiphenylamine N- nitrosopyrrolidine (NPYR) Norethindrone (19

- Norethisterone)

n- Propylbenzene

o- Toluidine Oxirane, methyl Oxydemeton

- methyl Oxyfluorfen Perchlorate Perfluorooctane sulfonic acid (PFOS) Perfluorooctanoic acid (PFOA) Permethrin Profenofos Quinoline RDX

sec

- Butylbenzene Strontium Tebuconazole Tebufenozide Tellurium Terbufos Terbufos sulfone Thiodicarb Thiophanate

- methyl Toluene diisocyanate Tribufos T riethylamine Triphenyltin hydroxide (TPTH) Urethane Vanadium Vinclozolin Ziram

Adenovirus Caliciviruses Campylobacter jejuni Enterovirus Escherichia coli (0157) Helicobacter pylori Hepatitis A virus Legionella pneumophila Mycobacterium avium Naegleria fowleri Salmonella enterica Shigella sonnei

10

# UCMR 3

• Final rule published May 2, 2012

• http://water.epa.gov/lawsregs/rulesregs/sdwa/ucmr/u cmr3/index.cfm

• Monitoring will occur from 2013 -15

• 28 chemi cals and 2 viruses

• Contaminants include hormones, perfluorinated
comp o unds (e.g., PFOS/PFOA), VOCs, metals (including Cr -6 and total Cr), 1,4- dioxane, chlorate and pathogens

11

# UCMR 3 – Monitoring Required

• Assessment monitoring (List 1)

– All systems serving >10,000 people

– 800 representative systems serving < 10,000 people



# • Screening Survey (List 2)

– All systems serving >100,000 people

– 320 representative systems serving 10,001 to 100,000 people

– 480 representative systems serving < 10,000 people

12

# UCMR 3 – Monitoring Required (cont.)

• Pre

- Screen Testing (List 3)

– Selected 800 systems serving < 1000 people that do not disinfect. Systems with wells that are located in areas of karst or fractured bedrock



# • EPA pays for analysis of all samples from systems serving < 10,000 and arranges for collection of List 3 samples

13

# UCMR 3 – Contaminants: Assessment Monitoring (“List 1”)

• Perfluorinated Chemicals (EPA Method 537)

– perfluorooctanonic acid (PFOA)

– perfluorooctane sulfonic acid (PFOS)

– perfluoroheptanoic acid (PFHpA)

– perfluorononanoic acid (PFNA)

– perfluorobutane sulfonic acid (PFBS)

– perfluorohexane sulfonic acid (PFHxS)

• Metals ( EPA Method 200.8)

– cobalt

– molybdenum

– strontium

– vanadium

– (total) chromium • EPA Method 218.7

- hexavalent chromium

• Volatile Organic Compounds (EPA

Method 524. 3)

- 1,1

- dichloroethane

- 1,2,3

- trichloropropane

- 1,3

- butadiene

- bromochloromethane

- chlorodifluoromethane

- chloromethane

- methyl bromide

• EPA Method 522

- 1,4

- diox ane

• EPA Method 300.1

- chlorat e

14

# UCMR 3 – Contaminants: Screening Survey (“List 2”) and Pre- Screen Testing (“List 3”)

Hormones (EPA Method 539) – List 2

- 17

- α

- ethynylestradiol

-

-

-

-

-

- 17

- β

- estradiol equi lin estriol estrone testosterone 4- androstene

- 3,17

- dion

15 

### Viruses – List 3

– enterovirus (qPCR & cell culture)

– n orovirus (qPCR)

– “Indicator organisms”

• Total coliform

• E. coli

• enterococci

• coliphage

• aerobic spores

# Unregulated CCL 3 Contaminants 104 Chemicals and 12 Microbes

1,1,1,2

- Tetrachloroethane 1,1

- Dichloroethane 1,2,3

- Trichloropropane 1,3

- Butadiene 1,3

- Dinitrobenzene 1,4

- Dioxane 17 alpha

- Estradiol 1- Butanol 2- Methoxyethanol 2- Propen -1- ol 3- Hydroxycarbofuran (degradate) 4,4'

- Methylenedianiline Acephate Acetaldehyde Acetamide Acetochlor Acetochlor ethanesulfonic acid (ESA) Acetochlor oxanilic acid (OA) Acrolein Alachlor ethanesulfonic acid (ESA) Alachlor oxanilic acid (OA) alpha

- Hexachlorocyclohexane (former) Aniline Bensulide Benzyl chloride Butylated hydroxyanisole Captan Chlorate (also D

- DBP) Chloromethane (Methyl chloride) Clethodim Cobalt Cumene hydroperoxide Cyanotoxins (3) Dicrotophos Dimethipin Dimethoate Disulfoton Diuron Equilenin Equilin Erythromycin Estradiol (17

- beta) Estriol Estrone Ethinyl Estradiol (17

- alpha) Ethoprop Ethylene glycol Ethylene oxide Ethylene thiourea Fenamiphos Formaldehyde (formerly) Germanium Halon 1011 (Bromochloromethane) HCFC

- 22 Hexane Hydrazine Mestranol Methamidophos Methyl bromide (Bromomethane) Methyl tert

- butyl ether Metolachlor Metolachlor ethanesulfonic acid (ESA) Metolachlor oxanilic acid (OA) Molinate Molybdenum Nitrobenzene Nitroglycerin N- Methyl -2- pyrrolidone N- Nitrosodiethylamine (NDEA) N- nitrosodimethylamine (NDMA) N- Nitroso

- di -n- propylamine (NDPA) N- Nitrosodiphenylamine N- nitrosopyrrolidine (NPYR) Norethindrone (19

- Norethisterone)

n- Propylbenzene

o- Toluidine Oxirane, methyl Oxydemeton

- methyl Oxyfluorfen Perchlorate Perfluorooctane sulfonic acid (PFOS) Perfluorooctanoic acid (PFOA) Permethrin Profenofos Quinoline RDX

sec

- Butylbenzene Strontium Tebuconazole Tebufenozide Tellurium Terbufos Terbufos sulfone Thiodicarb Thiophanate

- methyl Toluene diisocyanate Tribufos Triethylamine Triphenyltin hydroxide (TPTH) Urethane Vanadium Vinclozolin Ziram

Adenovirus Caliciviruses **Campylobacter jejuni** Enterovirus **Escherichia coli (0157) Helicobacter pylori**  Hepatitis A virus **Legionella pneumophila Mycobacterium avium**  **Naegleria fowleri Salmonella enterica**  **Shigella sonnei**

Red = No EPA Method

Blue = Current Method Development

16

# Contaminant Candidate List 4 (CCL 4)

• Spring 2012 - Publishe d FR notice requesting nominations of contaminants to be considered for inclusion on CCL 4

• Summary of Nominations:

• 59 unique contaminants were nominated by 10 organizations and individ uals

• 5 microbes and 54 chemicals

• 8 contaminants were nominated more than once



## • EPA is currently evaluating the new data provided for nominated chemical s and microbes, to determine the appropriateness of inclusion on the CCL 4

• The nomination letters and web site submittals can be found in the CCL 4 docket (EPA

- HQ

- OW -2012- 0217) at www.regulations.gov

17

# Current Status

18 

# • 2013 - Expect to publish Draft CCL 4 for publ ic review and comment

• 2014 - Expect to publish Final CCL 4

# Method Development Challenges

• Lack of suitable standards, internal standards and

surr ogate compounds

• Compounds with extremely high water solubility

• Low detection limits are often required

• Timeframe imposed by continual CCL/UCMR cycle

• Lack of previous method development work

19

## Office of Research and Development National Exposure Research Laboratory 

# Development of LC/MS/MS Methods for the Analysis of Chemicals on U.S. EPA’s Contaminant Candidate List

Jody A. Shoemaker and Daniel R. Tettenhorst

Disclaimer: Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 

### CCL/UCMR Methods Webinar -2013Office of Research and Development National Exposure Research Laboratory 

### NO YES Does Method Meet DQOs ? 70

- 130% recovery

with <30% RSD



### Revise Technical Approach Write Method

Use in Future UCMR Optimize Instrumentation (chromatography, mass calibrate, tune, evaluate instrument stability)

Determine Preservatives (select antimicrobial & dechlor) Determine Best Calibration (linear/quadratic, internal standards)

Determine Interferences (Are DQOs met in various difficult matrices?)

Determine Aqueous & Extract Holding Times Optimize SPE (select sorbent, surrogates, solvents , solvent volumes, sample volume, evaporation parameters)

Multi

- Lab Verification 

# Method Development ProcessOffice of Research and Development National Exposure Research Laboratory 

# ORD/NERL LC/MS/MS Methods

 LC/MS/MS Drinking Water Methods
  Method 535 – 12 acetanilide degradates (6 CCL)

- UCMR 2 

# Method 537 – 14 perfluorinated alkyl acids (2 CCL)

- UCMR 3 

#  Method 538 – 11 chemicals (4 CCL)



#  LC/MS/MS Methods Under Development

  Method 540 – 12 pesticides (5 CCL) 

# Method 543

– 8 pesticides (6 CCL ) 

# Method 544 – 8 cyanotoxins (1 CCL) http://www.epa.gov/nerlcwww/ordmeth.htmOffice of Research and Development National Exposure Research Laboratory 

# Method 538: DAI

- LC/MS/MS Contains 11 Analytes

Method Analytes acephate aldicarb dicrotophos aldicarb sulfone methamidophos diisopropyl methylphosphonate (DIMP) oxydemeton

- methyl fenamiphos sulfone quinoline fenamiphos sulfoxide thiofanox
Analytes in red are on CCL 3



###  Most method analytes are pesticides (except for quinoline and DIMP) with the potential to contaminate drinking water sources

 Q uinoline is an industrial starting material, a pharmaceutical (anti

malar ial) and a flavoring agent

 DIMP is a chemical by

- pro duct in the production of sarin gasOffice of Research and Development National Exposure Research Laboratory 

# M538 Direct Aqueous Injection Approach

Selected Reaction Monitoring Detector Source LC Pos ESI 50 μ L injection 

### 40 mL sample

10 μL IS PDS acephate, d 6 quinoline, d 7 methamidophos, d 6 DIMP, d 14 oxydemeton

- methyl, d 6 

### Antimicrobial:

64 mg/L sodium omadine Remove free chlorine: 20 mM ammonium acetate

990 μL aliquotOffice of Research and Development National Exposure Research Laboratory 

# M538 Performance Data
Fortified at 0.99 – 43 μg/L (n=7)

0 20 40 60 80 100 120mean % recovery 

### Groundwater source Surface water source



### LCMRL range: 11

- 180 ng/L (quinoline = 1.5 μg/L) HRLs: 250 ng/L – 175 μg/L (quinoline 10 ng/L)Office of Research and Development National Exposure Research Laboratory 

# Method 540: SPE

- LC/MS/MS Contains 12 Analytes

Method Analytes on CCL 3 3- hydroxycarbofuran fenamiphos sulfone f enamiphos fenamiphos sulfoxide b ensulide methomyl t ebuconazole chlorpryifos oxon t ebufenozide phorate sulfone disulfoton sulfoxide phorate sulfoxide

Analytes in red are on CCL 3



###  all method analytes are pesticides or pesticide degradates with the potential to contaminate drinking water sourcesOffice of Research and Development National Exposure Research Laboratory 

# Method 540 Analytical Procedure

inject Elute with 5 mL MeOH 150 mg Water Oasis HLB J.T. Baker Speedisk H2O

- Philic DVB

N 2 blowdown

+ IS



### Det Source C



## LC/MS/MS - Selected Reaction Monitoring Argon



### ector L + ESI Rinse cartridge with 5 mL reagent

w ater after loading Surrogates & preservatives Surrogates methomyl

- 13 C 2 , 15 N tebuconazole

- d 6

Preservatives 2- chloracetamide ascorbic acid Trizma 250 mL

s ample

Condition with 5 mL MeOH & 10 mL reagent water

Internal Standards carbofuran

- 13 C 6 bensulide

- d 14 p horate

- d 10Office of Research and Development National Exposure Research Laboratory 

### 0 20 40 60 80 100 120mean % recovery 

### Groundwater source 

# M540 Performance Data
Fortified at 12.8 – 32 n g/L (n=4)

Surface water source



## LCMRL range: 0.64

– 2.0 ng/L HRLs: 420 ng/L – 210 μg/LOffice of Research and Development National Exposure Research Laboratory 

# Method 543: On

- line SPE

- LC/MS/MS Contains 8 Analytes
Method Analytes on CCL 3 3- hydroxycarbofuran fenamiphos sulfone f enamiphos fenamiphos sulfoxide bensulide quinoline tebuconazole tebufenozide Analytes in red are on CCL 3



###  all method analytes are pesticides or pesticide degradates (except quinoline ) with the potential to contaminate drinking water sources

 concentration, elution, separation all done by

automa tion
 1- 5 mL sample volume typical
 anal y sis time/sample is <20 min
 high t hr oughputOffice of Research and Development National Exposure Research Laboratory SPE injection port 2.5 mL injections UPLC injection port 1- 10 μL range

SPE 5

- mL sample loop Analytical sample loop 10 μL

SPE cartridge 1 Oasis HLB
SPE cartridge 2 Oasis HLB



## LC/MS/MS - Selected Reaction Monitoring Argon
Detector Source UPLC Pos ESI 20 mL

s ample vial



### Internal Standards

quinoline- d 7 

### carbofuran- 13 

### C 6 

### tebuconazole- d 6 

### bensulide- d 14 IS(s) & preservatives: p otassium citrate ascorbic acid 

# Method 543 Preliminary ProcedureOffice of Research and Development National Exposure Research Laboratory 

# Method 543: On

- line SPE Events

0 Time
2 4 6 8 10 12 Start

Loading

3 min/5mL SPE wash 5 min gradient elution Re

- equilibration



### SPE Cartridge #1

0 Time
2 4 6 8 10 12 Start Re

- equilibration



### SPE Cartridge #2 Re

- condition 2% NH 4 OH 2% FA 0.5% FA ACN + 0.5% FA

ACN +

0.5% FA 2 % FA Quat Pump Quat Pump

Quat Pump Quat Pump Binary Pump Binary PumpOffice of Research and Development National Exposure Research Laboratory 

## On

- line SPE

- LC/MS/MS Preliminary Performance Data Fortified at 4 – 10 n g/L (n=3)

0 20 40 60 80 100 120mean % recovery 

### Surface water source 3 mg/L Ohio River NOM



## LCMRL range: TBD HRLs: 10 ng/L – 210 μg/LOffice of Research and Development National Exposure Research Laboratory 

# Method 544: SPE

- LC/MS/MS Potentially 8 Cyanotoxins

Method Analytes MC- LR MC

- RR MC-YR MC- LA MC- LW MC

- LF MC- LY Nodularin

 widespread occurrence of blooms pot ential for occurrence in finished drinking water toxic – HRL is 21 ng /L for MC

- LR unique problems in development of robust analytical methods – availability of multiple sources of high purity standards, lack of isotopically labeled IS and SUR, matrix interferences   

Analytes in red are on CCL 3Office of Research and Development National Exposure Research Laboratory 

# Preliminary Method 544 Analytical Procedure

inject Elute with 10 mL MeOH Water Oasis HLB others?

N 2 blowdown

+ IS



## LC/MS/MS - Selected Reaction Monitoring Argon



### Detector Source LC + ESI Rinse cartridge with 15 mL reagent

w ater after loading Surrogates & preservatives
Surrogates TBD e.g., leucine

- enkephalin cyclo

- RADFV Preservatives 2- chloracetamide ascorbic acid Trizma 250

- 500 mL

s ample

Condition with MeOH & reagent water

Internal Standards TBD Current : Me

- MC

- LR, CD 3 Lysing of the cyanobacterial cells in the sample will be investigatedOffice of Research and Development National Exposure Research Laboratory 

# Method 544 Preliminary Performance Data
Fortified at 200 – 400 ng /L except MC

- LA at 1000 ng/L (n=3)

0 0 0 0

0 20 40 6 8 10 12mean % recovery 

### Groundwater source Surface water source (External Cal) (Internal Cal)



## LCMRL range: TBD HRL MC

- LR: 21 ng/LOffice of Research and Development National Exposure Research Laboratory 

# Summary of ORD/NERL LC/MS/MS Methods

 2 LC/MS/MS methods completed and ready for potential use in UCMR 4 – total of 9 CCL 3 chemicals

• Method 538

• Method 540



#  2 methods under development for potential use in UC MR 4 – one additional CCL 3 analyte

• Method 543 (optional method for Method 540 analytes)

• Met hod 544 (M C

- LR)Office of Research and Development National Exposure Research Laboratory High water solubility Aniline
N- Methyl -2- pyrrolidone Triethylamine

Attempted but failed DQOs 4,4’

- methylenedianiline Thiodicarb Nitroglycerin

Clethodim Not suitable for MS analysis Triphenyltin hydroxide Ziram

ETU

ETU=ethylenethiourea DQOs=data quality objectives Aqueous instability Captan Hexane Benzyl chloride N- Nitrosodiphenylamine Thiophanate

- methyl Toluene diisocyanateOxirane, methylEthylene oxide 

# Remaining CCL 3 Chemicals – Challenges (as of May 2013)Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 Photo image area measures 2” H x 6.93” W and can be masked by a collage strip of one, two or three images. The photo image area is located 3.19” from left and 3.81” from top of page. Each image used in collage should be reduced or cropped to a maximum of 2” high, stroked with a 1.5 pt white frame and positioned edge

- to

- edge with accompanying images.

Disclaimer: Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official agen cy policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 

# Drinking Water Methods Development by Gas Chromatography

- Mass Spectrometry (GC

- MS)

Paul E. GrimmettOffice of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 1 

## Background

• 

### The Safe Drinking Water Act, as amended in 1996, requires EPA to execute a program that identifies, monitors and considers for regulation new and emerging chemicals that may pose a risk to drinking water consumers.

• 

### EPA notifies the public of chemicals it is investigating by publishing the drinking water Contaminant Candidate List (CCL).

• 

### In order for EPA’s Office of Water to evaluate CCL chemicals for regulation, it is vital that nationwide occurrence data for these chemicals in drinking water are obtained.

• 

### Nationwide occurrence data are obtained through Unregulated Contaminant Monitoring (UCM).

• 

### Rugged, accurate, and sensitive methods are needed for UCM.Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268



## Method Development Goals

•

•

•

•

• Determine appropriate surrogates and internal standards

Establish Data Quality Objectives (DQOs): normally 70

- 130% recovery with < 30% RSD

Preservation scheme to allow for sample holding: antimicrobial, dechlorinat ing agent, and pH buffer

Establish sample and extract holding times – ideally ≥14 days

Establish detection limits (DLs) and lowest concentration minimum reporting l evels (LCMRLs) below health reference levels (HRLs)

2Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 3 

## Recently Published GC Methods by ORD

- NERL

1. 

### Method 522: 1,4

- dioxane

– SPE on coconut carbon sorbent, followed by GC/MS

– allows for selected ion monitoring (SIM)

– two sampling/extraction options (100 mL and 500 mL samples)

– method currently used for occurrence data in UCMR 3 (2013

- 2015)Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Recently Published GC Methods by ORD

- NERL (cont.)

2. 

### Method 525.3: Semi

- volatiles

– published in February 2012

– approx. 130 analytes (pesticides, herbicides, PCBs, PAHs, etc)

– includes 16 CCL 3 compounds and 17 regulated contaminants

– SPE on polymeric sorbent (DVB), followed by GC/MS

– improved preservation scheme, updated surrogates and internal standards, addition of SIM option, and new PCB screening procedure

4Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 5 

## Current Work
Development of drinking water method for o

- toluidine, quinoline, dimethipin, captan, and butylated hydroxyanisole (BHA)

• 

### o

- Toluidine and quinoline (both industrial precursors) have basic structures, which led to poor sorbent recovery using Method 525.3 preservatives (acidic scheme)

• 

### BHA (a food and packaging preservative) suffered from randomly occurring extraction inefficiency and was removed from Method 525.3

• 

### Dimethipin was included in EPA Method 525.3, but with limited sorbent options

• 

### Captan was removed from Method 525.3 in the evaluation phase, due to hydrolysis (half life in water is less than 1 day)

• 

### All 5 compounds are being evaluated for an EPA method using a neutral pH preservation schemeOffice of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

# Current SPE Extraction Technique - Cartridges

Add surrogates:

o

- toluidine

- d 9 quinoline

- d 7 Add preservatives : ascorbic acid Trizma buffer (pH 7.0) EDTA DZU 

### Rinse & condition: methylene chloride methanol water

Add 1 L water sample

ex tr ac t at ~10 mL/min Rinse with RW Dry cartridge for 10 min

Elute with methylene chloride

Add IS(s): acenaphthene- d 10 

### phenanthrene- d 10 

### chrysene- d 12 

### Dry and concentrate extract to 1 mL

GC/MS: Full scan option or SIM option Multiplehydrophilicmodified sorbent optionsOffice of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 7 

# GC/MS Full Scan Analysis Specs

Column : Restek RXI-5sil- MS and J&W DB -1701 30 m x 0.25 mm x 0.25 μm column Injector : 275 ° C (splitless mode), 20 psi pulse Inj. vol : 1 μL

Flow : 1 mL/min, helium carrier gas

Oven : 60 ° C for 1 min, to 300 ° C at 10

° C/min, hold 2 min. Total time = 27 min.

MS Transfer Line: 275 ° C

MS : scan 50-350 amu , full scanOffice of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 8 

# GC/MS SIM Analysis Specs (Preliminary)

Column : Restek RXI-5sil- MS Injector : 275 ° C (splitless mode), 20 psi pulse Inj. vol : 1 μL

Flow : 1 mL/min, helium carrier gas

Oven : 60 ° C for 1 min, to 300 ° C at 10 ° C/min, hold 2 min. Total time = 27 min.

MS Transfer Line: 275 ° C

MS SIM : 6 windows ( amu , dwell time) WIN1, 5.00 min: (106.10, 25) (107.10, 25) (112.10, 25)(114.10, 25) WIN2, 7.64 min: (102.00, 25) (108.10, 25) (129.10, 25)(136.10, 25) WIN3, 10.62 min: (137.10, 25) (162.10, 25) (164.10, 25)(180.10, 25) WIN4, 13.76 min: ( 54.10, 75) (118.00, 75) (188.10, 25) WIN5, 17.16 min: ( 79.00, 75) (149.00, 75) WIN6, 20.37 min: (236.20, 25) (240.20, 25)Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Sample Total Ion Chromatogram (TIC) with Mass Spectra, 5 μg/mL standard

9 Internal Standards acenaphthene

- d 10 , phenanthrene

- d 10 , and chrysene

- d 12 labeled as ISTD 1

, ISTD 2 , and ISTD 3

, respectively.Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Quality Control Data at Various Concentrations, Drinking Water Matrices

• 

### Three different water matrices were fortified with CCL 3 compounds of interest, then extracted and analyzed.

1. Drinking water – surface water source

2. Drinking water – groundwater source (hardness ~ 350 mg/L)

3. Natural Organic Material NOM water – lab water spiked with concentrated organic matter from the Ohio River (TOC @ 1.8

- 2.2 mg/L)

• 

### Fortification performed at 5 μg/L, 1 μg/L, and 0.1 μg/L.

• 

### Each matrix was extracted in 4x replicates

10Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Quality Control Data from Matrix Extracts at High Level Fortification (Full Scan GC/MS)

11 Lower and upper limit bars are set at 70% and 130%, respectively.Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Quality Control Data from Matrix Extracts at Mid Level Fortification (Full Scan GC/MS)

12 Lower and upper limit bars are set at 70% and 130%, respectively



### .Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 13 

## Quality Control Data from Matrix Extracts at Low Level Fortification (Full Scan GC/MS)

Lower and upper limit bars are set at 50% and 150%, respectively



### .Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 

## Remaining Laboratory Work with Current Method

• 

### Perform sample and extract holding time study

- GOALS :14-28 day holding time for samples; 14- 28 days for extracts

- Special attention to captan (hydrolysis issues) and o -toluidine (solvent stability issues)

• 

### LCMRL and MDL analysis and calculations

• 

### Repeat sample matrix challenges, holding time studies, and LCMRL/MDL calculations in SIM ( work has been initiated )

- quinoline: HRL of 0.010 μg/L

• 

### Multi- laboratory verification

14Office of Research and Development National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 15 

## Future Research and Potential Challenges
Review of Select Remaining CCL 3 Compounds:

CCL3 Compound Comments

Acetamide Not suitable for generic SPE, highly water soluble (2000g/L); Too small molecular weight (59.1) for LC/MS or EI

- GC/MS

Acrolein Volatile, water solubility > 10%. Stability issues in water.

Cumene hydroperoxide Safety concerns due to high reactivity , low water solubility

Ethylene glycol Too small molecular weight (62.1) for EI

- GC/MS or LC/MS; miscible with water, not suitable for generic SPE

Ethylene oxide Too small molecular weight (44.0) for EI

- GC/MS or LC/MS/MS; gas at room temperature
Hydrazine Too small molecular weight (32.0 ) for EI

- GC/MS or LC/MS; volatile

Methanol Too small molecular weight (32.0) for EI

- GC/MS or LC/MS; volatile

Nitroglycerin Not amenable to GC due to thermal instability ; cannot ionize by positive ion ESI

- LC/MS/MS

N

- Methyl -2- pyrrolidone Miscible with water , not suitable for generic SPE

Oxirane, methyl

- (propylene oxide) Too small molecular weight (58.1) for EI

- GC/MS or LC/MS; volatile

Toluene diisocyanate Reacts immediately with water, stability issues

Urethane Not suitable for GC ; Too small molecular weight (89.1) for LC/MSA World of Solutions TM 

# METHOD DEVELOPMENT PROCESS FOR DRINKING WATER ANALYSIS

CB&I
Alan ZaffiroA World of Solutions TM 1 

# W HA T WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATIONA World of Solutions TM 2 

# WHAT WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANAL YTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATIONA World of Solutions TM 3 

# 











 INITIAL ANALYTE GROUPING

CONSIDERATIONS

M AXIMIZE # OF COMPOUNDS ANALYZED BY A SINGLE TECHNIQUE
PHYSICAL

- CHEMICAL PROPERTIES REACTIVITY / FORM

MONITORING LIMITS
USE OR ENVIRONMENTAL IMPACT
RELEVANCE
EXTEND EXISTING DRINKING WATER METHOD

# WHAT WE DO TO CREATE A

PLANNING 

# M ETHOD

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

A World of Solutions TM 4A World of Solutions TM 5 

## 

















 

# L IT ERATURE SEARCH AND PARTNERS

SOURCES

E PA METHODS PUBLISHED BY OTHER DIVISIONS
JOURNALS METHODS PUBLISHED BY OTHER GOVERNMENT AGENCIES, e.g., USGS
APPLICATION NOTES
VENDORS
UNIVERSITIES
PERSONAL COMMUNICATION
PAST TSC ATTEMPTS
PRELIMINARY LAB WORK VOLUNTARY CONSENSUS STANDARD BODIES (e.g. ASTM, STANDARD METHODS)

# WHAT WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

A World of Solutions TM 6A World of Solutions TM 7 

# 







 RESEARCH PLAN

ELEMENTS

L ITERATURE SEARCH RESULTS
PROPOSED TARG ET LIST PROPOSED QC COMPOUNDS

– INTERNAL AND SURROGATE STANDARDS 

# PROPOSED TECHNIQUE

– PRESERVATION

– DIRECT INSTRUMENTAL ANALYSIS?

– SAMPLE PREPARATION (EXTRACTION, DERIVATIZATION, BUFFER) 

# PROPOSED CONDITIONS

# WHAT WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

A World of Solutions TM 8A World of Solutions TM 9 

## 





 

# METHOD DEVELOPMENT

BENCH WORK TO OPTIMIZE CONDITIONS

W ORK BACKWARDS ITERATIVELY

– INSTRUMENTAL DETERMINATION>Sx PPEP>PRESERVATION

– REAGENT WATER>FIELD SAMPLES


## GOALS

– ROBUST, REPEATABLE, TRANSLATABLE METHOD

– 70 TO 130% RECOVERY; + 30% PRECISION

– AT LEAST 14 DAYS STORAGE STABILITY

– DETECTION LIMITS MEET MONITORING / REGULATORY OBJECTIVES


## MINIMIZE COST AND HAZARDOUS MATERIALS
UTIL IZE GENERIC LABORATORY MATERIALS

# WHAT WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

A World of Solutions TM 10A World of Solutions TM 11 

# 

 DEMONSTRATION

VERIFY PERFORMANCE AT EPA AND MULTIPLE OUTSIDE LABORATORIES

S INGLE LABORATORY

-- MOST EXTENSIVE SCOPE

– LCMRL , P&A (CHALLENGING MATRIXES), MRL CONFIRMATION

– STORAGE STABILITY WITH MICROBIAL INOCULANT PRESENT

– DATA PUBLISHED IN SECTION 17 DRINKING WATER METHODS


# MULTI

- LABORATORY VALIDATION

– LCMRL , P&A (ONE MATRIX), MRL CONFIRMATION

– DATA PUBLISHED IN RESEARCH SUMMARY

– LABORATORIES RECOGNIZED IN METHOD

# WHAT WE DO TO CREATE A METHOD

PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

A World of Solutions TM 12A World of Solutions TM 13 

# 

 REVIEW

METHOD DOCUMENT AND PERFORMANCE DATA

P EER REVIEW OF METHOD

– CONCURRENT WITH MULTI

- LABORATORY VALIDATION


# AGENCY

– INTERNAL ( CB&I )

– EPA PO

– EPA MANAGEMENT

– EPA HEADQUARTERS

– VERIFY PDF CONVERSION (SECTION 508 COMPLIANT)

# WHAT WE DO TO CREATE A METHOD

World of Solutions TM 14 

## PLANNING

AGENCY DIRECTION ( e.g.,CCL3 )

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN (NEW OR UPDATE EXISTING METHOD) 

## EXECUTION

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION

AA World of Solutions TM 15 

## 



 

# CCL3 LO W MW COMPOUNDS REMAINING

12 CANDIDATES



# INITIAL ANALYTE GROUPING

Ethylene oxide, propylene oxide, methanol, 1

- butanol, allyl alcohol, 2- methoxy ethanol, ethylene glycol, acrolein, hexane, o

- toluidine, triethylamine, benzyl chloride SMALL, POLAR MOLECULES POSSIBLY AMENABLE TO GC
MOST POSSIBLY ISOLATED FROM H 2 

## O USING CARBON - OR CMS- BASED EXTRACTION MEDIAA World of Solutions TM 16 

## 





 

# RESEARCH PLAN

12 CANDIDATES



# LITERATURE SEARCH

PH YSICAL PROPERTIES

– HENRY’S LAW CONSTANT

– MW

– BOILING POINT


## HRL
METHOD S PUBLISHED BY OTHER GOVERNMENT AGENCIES TECHNIQUES REPORTED IN:

– APPLICATION NOTES

– JOURNALS

– PROCEEDINGS OF CONFERENCES

– PERSONAL COMMUNICATIONA World of Solutions TM 17 

# RESEARCH PLAN

Table 1. Physical properties, published methods, and literature summary.

a. Reference: ethanol (1.9 x 10 2 ), benzene (1.8 x 10 - 1 ), 1

- propanol (1.4 x 10 2 ), MtBE (1.7), 1,4

- dioxane (2.1 X 10 2 ) , TBA (8.4). b. Azeotropic distillation

c. Purge and trap technique Analyte(s) Henry’s Law Constant, M/atm a MW bp, °C HRL, μg/L SW846 Literature techniques (author reference in parentheses) Ethylene oxide Propylene oxide 7.1 5.2 44 58 10.7 34 0.1 0.2 5031 b , 8015D (direct aqueous injection

— DAI) Air and pharmaceutical residual methods

Methanol 2.3 x 10 2 32 64.7 3,500 8015D DAI P&T w/FID Ether extraction (Woo) Headspace in blood (numerous citations) Derivatization to alkyl nitrite/extraction/GC/ECD (Nguyen) SPME (Supelco application notes) 1

- Butanol 1.2 x 10 2 74 118 700 5031, 5030C c w/heat, Ether extraction (Woo) Allyl alcohol 2 x 10 2 58 97 35 5031, 5030C w/heat, 8015D DAI 2

- Methoxyethanol 12.1 76 125 21 Not found SPE

- carbon molecular sieve ( Supelco application notes)

Ethylene glycol 1.7 x 10 4 62 197 14,000 8015B DAI SPE

- carbon molecular sieve (Supelco), DAI

- GC

- FID (Turner, Restek, Teske)

Acrolein 7

– 8 56 53 3.5 5030C, 8015D DAI, 8261A vacuum distillation EPA Method 603 (heated purge and trap)

Hexane 5.5 x 10 - 4 86 69 420 Not found P&T (524.3 Research Summary) Ortho

- toluidine Not available 107 200 0.19 8015D DAI SPME (Supelco), IC (Zhu) Triethylamine Not available 101 90 2.3 8015D DAI SPME (Alltech application note) Benzyl chloride 1.6 127 179 0.2 5030C P&T (524.2) 

### LITERATURE SEARCH SUMMARY TABLEA World of Solutions TM 18 

## 





 

# RESEARCH PLAN

12 CANDIDATES



## INITIAL LAB WORK AND PROPOSED METHOD

PRIORITY C ONTAMINANT: 2-methoxyethanol
LIMITED INFORMATION OR EXPENSIVE INSTRUMENTS NEEDED

– methanol, hexane, acrolein, triethyl amine, o

- toluidine (not practical) 

## INITIAL LAB INVESTIGATION REQUIRED

– EtO AND ptO : HEADSPACE, EXTRACTION, AND P&T FAILED

– THESE TWO ELIMINATED


## REMAINING 5 COMPOUNDS AMENABLE TO EXTRACTION

– SIMILAR TECHNIQUE TO EPA METHOD 522

– ORD

- NERL ORIGINALLY PROPOSED MULTIPLE TARGETS FOR THIS METHODA World of Solutions TM 19 

# 





 RESEARCH PLAN

6 CANDIDATES



# PROPOSED METHOD (CONT.)

PROPOSE EXTENDING M

- 522 bey ond 1,4 -dioxane
NEW TARGETS: n

- butanol, allyl alcohol, 2- methoxyeth anol , ethylene glycol, benzyl chloride

– CONTAINS PRIORITY CONTAMINANT 

# QC COMPOUNDS

– LABELED ISOTOPES OF 1,4

- dioxane, n

- butanol, allyl alcohol 

# PROPOSED CONDITIONS

– CARBON

- EXTRACTION, GC

- MS DETERMINATIONA World of Solutions TM 20 

# 







 METHOD DEVELOPMENT

6 CANDIDATES



# BENCH WORK TO OPTIMIZE CONDITIONS

ETHYLENE GLYCOL NOT RETAINED BY ANY EXTRACTION MEDIA EV ALUATED
BENZYL CHLORIDE FAILED PRELIMINARY STORAGE STABILITY
ONLY ONE OF THE EXTRACTION MEDIA IN M

- 522 SUITABLE FOR REMAINING COMPOUNDS
MODIFICATION OF M

- 522 EXTRACTION SOLVENT REQUIRED TO RECOVER ADDITIONAL TARGETS 1,4

- dioxane, n

- butanol, allyl alcohol , 2

- methoxyethanol

# DEMONSTRATION/VALIDATION

A World of Solutions TM 21 

# 



 4 ANALYTES 

# VERIFY PERFORMANCE AT EPA AND MULTIPLE OUTSIDE LABORATORIES

SUCCESSFULLY DEMONSTRATED AT EPA
METHOD CURRENTLY BEING WRITTEN
CURRENTLY RECRUITING OUTSIDE LABORATORIESA World of Solutions TM 22 

# CONCLUSION SLIDE 1

ABILITY TO MEET MONITORING GOALS

MRL’s and HRLs

Analytes MRL * CCL3 HRL 1,4

- dioxane 0.2 μg/L 3.0 μg/L (cancer) allyl alcohol 1.0 μg/L 35 μg/L

n

- butanol 1.0 μg/L 700 μg/L
2

- methoxyethanol 1.0 μg/L 21 μg/L
* This is the aqueous concentration equivalent to the low calibration standard.A World of Solutions TM 23 

# 



 CONCLUSION SLIDE 2

METHOD PARAMETERS



# Preservation: ident ical to EPA method 522

SPE: 0.1 L Sx; neutralized with 5 mL x 0.8 M NaHCO 3 

# ; Supelco Envi

- Carb Plus (400 mg); 60

- min air dry in reverse direction ; elution 20% MeOH:DCM ; 2

- mL extract volume

Extract analysis : 30 m x 0.25 mm i.d . x 0.5 μm d f 

# column (Agilent P/N CP9222 VF

- WAXms ); 1 μL injection @ 200 ° C inlet; temperature - programmed separation; MS detection in SIM modeA World of Solutions TM 24 

# CONCLUSION SLIDE 3

ANALYTE LIST AND CALIBRATION RANGES

Analytes Range, μg/L Range, ng/mL (equivalent extract concentration)
I.S.: 1,4

- dioxane

- d 8 

### a dded to extra ct 250
I.S.: chlorobenzene

- d 5 

### added to extra ct 250
Surr: allyl alcohol

- d 6 

### 5.0 250 (also serves as isotope dilution internal standard)
Surr: n

- butanol

- d 10 

### 5.0 250 (also serves as isotope dilution internal standard) 1,4

- dioxane 0.10

— 8.0 5.0

— 400 allyl alcohol 0.50

— 20 25

— 1000 allyl alcohol* 0.50

— 20 25

— 1000

n

- butanol 0.50

— 20 25

— 1000

n

- butanol* 0.50

— 20 25

— 1000
2

- methoxyethanol 0.50

— 20 25

— 1000 * Reported with isotope dilution quantitationA World of Solutions TM 25 

# 



 CONCLUSION SLIDE 4

CHALLENGES



# W ATER MANAGEMENT

– EXTRACTION MEDIA IS DIFFICULT TO DRY

– WET EXTRACTS CAUSE RETENTION TIME SHIFTS (COMPRO M ISING QUALITATIVE ID) AND CAN AFFECT ANALYTE RESPONSE (QUANTITATIVE AND QC)

– EXCESSIVE DRYING CAUSES LOW RECOVERY


# ONLY 1 EXTRACTION MEDIUM AVAILABLE FOR THE ANALYS IS OF THESE ANALYTES
LOT TO LOT VARIATION

– LOADING TIME

– DRYING TIMEA World of Solutions TM 

# ANALYSIS OF ERYTHROMYCIN AND OTHER PHARMACEUTICALS BY LC

MS/MS

CB&I William A. Adams, Ph.D.A World of Solutions TM 1 

## 







 

# PHARMACEUTICAL TARGET ANALYTES

Carbamazepine, Diazepam, Diclofenac (sodium salt),
E nalapril (maleate salt), Fluoxetine (HCl), Gemfibrozil, Naproxen, Phenytoin, Sulfamethoxazole, Triclosan, Trimethoprim, and Erythromycin ( measured as Erythromycin –H 2 

## O )

Variety of chemically unrelated analytes

– Both ESI positive and ESI negative modes



## Analysis by LC

- MS/MS using an 5 mM ammonium acetate and methanol gradient

SPE (6 mL, 200 mg HLB cartridge)

13 

## C- Naproxen

- d 3 

## , Triclosan

- d 3 

## , Carbamazepine

- d 10 

## , chosen as internal standards; 13 

## C- Trimethoprim

- d 3 

## and Diclofenac

- d 4 

## chosen as surrogate standardsA World of Solutions TM 2 

## 

 

# CCL3 AND PRIORITY TARGET ANALYTES

Erythromycin and triclosan
Onl y erythromycin listed on CCL3

Erythromycin Tricl osanA World of Solutions TM 3 

##  Erythromycin

– At pH <7, water is

remove d and compound no longer exhibits antibiotic properties (Hirsch et al., 1999)

– For analysis, erythromycin is measured as erythromycin –H 2 

### O (717.0 > 158.3 m/z ) 

# Erythromycin

ErythromycinA World of Solutions TM 4 

## 





 

# Preservation

Store samples in refrigerator (6 °C)

100 mg/L ascorbic acid

– Reduces free chlorine present in tap water samples

– Easy to handle

– Solid can be added to bottles before sampling



## 350 mg/L EDTA

– Chelates metals in tap water samples

– Prevents metal hydrolysis

– Solid can be added to bottles before sampling



## 9.4 g/L potassium citrate

– Acts as a microbial inhibitor

– pH ~3.8

– Solid can be added to bottles before samplingA World of Solutions TM 5 

## 

 

# SAMPLE PREPARATION

Solid phase extraction (SPE)

– 6 cc, 200 mg HLB cartridges

– Vacuum manifold

– 1 liter samples extracted and eluted with 5 mL 1:1 methanol/acetone



## Dilution

– 5 mL reagent water added to extract

– 100:1 sample to extract concentration factorA World of Solutions TM 6 

# INSTRUMENTAL METHOD

CHROMATOGRAPHIC CONDITIONS

ESI positive

HPLC Column : Waters Xterra® MS C18, 2.1 x 150 mm, 3.5 μm Column temperature: 30 o C Column flow rate: 0.200 mL/min Autosampler temperature: 10 O C Injection volume: 10 μL Gradient :

Time (min) %5 mM ammonium acetate in 10%

MeOH/90% reagent water %MeOH

0.00 90 10 0.50 90 10 0.51 50 50 8.00 25 75 8.01 0 100 10.00 0 100 14.00 90 10 24.00 End Run ESI negative

HPLC Column : Waters Xterra® MS C18, 2.1 x 150 mm, 3.5 μm Column temperature: 30 o C Column flow rate: 0.200 mL/min Autosampler temperature: 10 O C Injection volume: 50 μL Gradient :

Time (min) %5 mM ammonium acetate in 10%

MeOH/90% reagent water %MeOH

0.00 90 10 0.50 90 10 0.51 40 60 8.00 0 100 11.00 0 100 15.00 90 10 25.00 End RunA World of Solutions TM 7 

# INSTRUMENTAL METHOD

MS CONDITIONS

MS Parameter HPLC

- MS/MS Polarity Positive ion electrospray Capillary Voltage, kV 2.50 Source Temperature, o C 120 N 2 Desolvation Temperature, o C 400 N 2 Desolvation Gas Flow, L/hr 900 Cone Gas Flow, L/hr 50 Extractor Lens, V 2.00 RF Lens, V 0.2 MS Parameter HPLC

- MS /MS Polarity Negative ion electrospray Capillary Voltage, kV 2.50 Source Temperature, o C 120 N 2 Desolvation Temperature, o C 400 N 2 Desolvation Gas Flow, L/hr 900 Cone Gas Flow, L/hr 50 Extractor Lens, V 1.00 RF Lens, V 0.1A World of Solutions TM 8 

# ESI POSTIVE CHROMATOGRAM

# ESI NEGATIVE CHROMATOGRAMS

A World of Solutions TM 9A World of Solutions TM 10 

# LCMRL

Analyte LCMRL (ng/L) a Carbamazepine 2.4 Diazepam 0.27 Diclofenac 1.1 Enalapril 0.60 Fluoxetine 0.98 Gemfibrozil 1.4 Naproxen 4.5 Phenytoin 1.4 Sulfamethoxazole 0.28 Triclosan 3.4 Trimethoprim 4.1 Erythromycin 5.0

# PRECISION AND ACCURACY

A World of Solutions TM 11 

## 





 Precision and accuracy measured in different matrixes (e.g.

re a gent water, high TOC tap water, hard tap water)

Measured at two concentrations

– Low and Mid/High of calibration range



## Accuracy

– Low: 50

– 150% recovery

– Mid/High: 70

– 130% recovery



## Precision

– Low: ≤30% RSD

– Mid/High: ≤20% RSDA World of Solutions TM 12 

##  

# PRECISION AND ACCURACY

Reagent water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

Analyte Low Concentration High Concentration
Fortified Concentration (ng/L) Avg. %Recovery %RSD Fortified Concentration (ng/L) Avg. %Recovery %RSD

Carbamazepine 4.94 87.9 4.0 31.3 91.5 0.88 Diazepam 0.340 72.9 7.8 20.0 92.1 3.4 Diclofenac 2.04 97.2 4.2 102 95.1 1.2 Enalapril 1.22 105 6.0 40.4 89.7 1.6 Fluoxetine 1.24 70.3 8.2 104 84.6 2.3 Gemfibrozil 3.00 92.7 1.4 100 95.6 2.1 Naproxen 9.20 101 4.0 100 98.5 1.2 Phenytoin 2.42 76.0 4.5 30.7 90.2 4.1 Sulfamethoxazole 0.410 92.2 8.0 29.4 90.9 0.51 Triclosan 7.00 91.5 5.0 311 99.7 1.5 Trimethoprim 8.80 90.5 4.2 30.4 91.1 2.4 Erythromycin 5.60 80.4 6.2 40.0 83.9 1.7 Diclofenac

- d 4 100 94.0 2.4 100 93.9 1.8

13 C 3 - Trimethoprim 20.0 86.6 2.3 20.0 87.3 3.3A World of Solutions TM 13 

# PRECISION AND ACCURACY

 Ground source tap water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

– Free chlorine: 0.76 mg/L

– To tal chlorine: 0.97 mg/L

– Har dness: 331 mg/L

– TOC: 0.75 ppm

Analyte Low Concentration High Concentration
Fortified Concentration (ng/L) Avg. %Recovery %RSD Fortified Concentration (ng/L) Avg. %Recovery %RSD

Carbamazepine 4.94 93.0 2.5 31.3 95.3 1.2 Diazepam 0.340 73.5 13 20.0 97.7 1.8 Diclofenac 2.04 104 7.6 102 97.4 1.4 Enalapril 1.22 94.3 2.9 40.4 94.4 1.1 Fluoxetine 1.24 86.1 6.4 104 94.9 2.4 Gemfibrozil 3.00 92.7 4.1 100 96.4 2.0 Naproxen 9.20 89.0 3.8 100 104 2.1 Phenytoin 2.42 83.0 5.9 30.7 80.2 2.1 Sulfamethoxazole 0.410 87.8 12 29.4 93.8 1.3 Triclosan 7.00 88.5 10 311 96.7 1.0 Trimethoprim 8.80 92.4 5.2 30.4 94.3 1.2 Erythromycin 5.60 69.8 7.3 40.0 78.7 5.5 Diclofenac

- d 4 100 98.9 1.5 100 88.9 2.2

13 C 3 - Trimethoprim 20.0 98.4 1.8 20.0 97.7 1.8A World of Solutions TM 14 

# PRECISION AND ACCURACY

 Surface source tap water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

– Free chlorine: 1.16 mg/L

– Total chlorine: 1.65 mg/L

– Hardness: 124 mg/L

– TOC: 4.23 ppm

Analyte Low Concentration High Concentration
Fortified Concentration (ng/L) Avg. %Recovery %RSD Fortified Concentration (ng/L) Avg. %Recovery %RSD

Carbamazepine 4.94 83.7 2.7 31.3 91.1 2.8 Diazepam 0.340 54.7 27 20.0 95.7 0.92 Diclofenac 2.04 71.8 5.6 102 73.6 1.4 Enalapril 1.22 99.5 8.8 40.4 94.1 0.42 Fluoxetine 1.24 96.6 4.3 104 105 1.8 Gemfibrozil 3.00 104 3.3 100 111 0.91 Naproxen 9.20 92.7 4.8 100 98.6 1.9 Phenytoin 2.42 95.4 6.6 30.7 95.4 1.6 Sulfamethoxazole 0.410 92.2 28 29.4 93.3 0.96 Triclosan 7.00 93.5 12 311 99.2 0.94 Trimethoprim 8.80 69.6 5.2 30.4 72.5 2.1 Erythromycin 5.60 85.0 6.3 40.0 87.2 2.0 Diclofenac

- d 4 100 72.3 0.92 100 75.3 1.7

13 C 3 - Trimethoprim 20.0 80.2 1.6 20.0 81.4 1.3A World of Solutions TM 15 

# HOLDING TIME - SAMPLE

 Surface source tap water

Analyte Fortified Conc. (ng/L) Day 0 Day 7 Day 14 Day 21 Day 28

Avg. %Rec %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD

Carbamazepine 7.82 106 6.1 -1.3 6.6 -2.2 3.0 -6.7 3.4 -8.4 0.46 Diazepam 5.00 114 1.1 -8.9 1.5 -6.7 1.8 -6.7 1.8

- 11 1.7 Diclofenac 25.6 105 0.84

- c

- c 4.3 1.8 3.3 0.92 -13 1.2 Enalapril 10.1 90.8 2.3 0.29 2.9 -2.6 2.8 -1.3 0.78 1.1 2.2 Fluoxetine 26.0 113 2.4 0.56 7.8 15 41 -6.6 3.6 -4.1 1.9 Gemfibrozil 25.0 104 1.2 7.3 2.3 3.3 2.1 4.1 1.1 -5.3 2.5 Naproxen 25.0 106 1.2 3.4 2.0 2.5 2.7 0.39 2.7 0.50 2.2 Phenytoi n 7.68 94.8 11

- c

- c -30 3.4 -15 9.2 -18 2.8 Sulfamethoxazole 7.34 93.0 3.3 -1.2 5.5 -2.7 0.53 -7.8 0.56 -6.1 2.6 Triclosan 77.8 103 2.8 3.1 3.5 5.1 2.2 1.5 1.3 -1.1 0.76 Trimeth oprim 7.60 74.8 2.0 1.7 4.1 -10 3.0 -10 4.5 -16 3.4 Erythromycin 10.0 102 1.9 -1.3 22 -6.2 4.5 -0.62 1.5 -3.7 2.8

Surrogate Fortified Conc. (ng/L) Avg. %Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD

Diclofenac

- d 4 100 85.5 1.2 103 5.1 97.8 1.7 99.7 0.55 82.2 0.68

13 C 3 - Trimethoprim 20.0 79.4 0.84 78.9 0.84 74.7 0.43 76.7 2.4 79.1 1.9

# HOLDING TIME - EXTRACT

 Surface source tap water

A World of Solutions TM 16 Analyte Fortified Conc. (ng/L) Day 0 Day 7 Day 14 Day 21 Day 28

Avg. %Rec %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD % Change

from Day 0 %RSD

Carbamazepine 7.82 106 6.1 -10 3.8 -9.4 3.5 -12 2.3 -14 3.5 Diazepam 5.00 114 1.1 -14 2.5 -12 4.1

- 11 2.4 -13 3.0 Diclofenac 25.6 105 0.84 -6.4 1.1 -6.4 3.2 -0.74 1.0 -17 1.1 Enalapril 10.1 90.8 2.3 4.4 2.2 0.69 0.50 2.4 0.79 4.3 1.5 Fluoxetine 26.0 113 2.4 13 2.2 2.6 4.6 8.8 4.9 18 6.0 Gemfibrozil 25.0 104 1.2 -2.5 1.8 -3.3 1.7 3.0 0.41

- 11 0.63 Naproxen 25.0 106 1.2 -0.48 1.3 3.6 3.1 4.0 1.8 3.2 2.8 Phenytoin 7.68 94.8 11

- - -35 7.4 -20 4.3 -16 13 Sulfamethoxazole 7.34 93.0 3.3 -12 4.0 -9.6 1.2 -7.6 2.2 -8.2 1.6 Triclosan 77.8 103 2.8 -1.0 1.8 -0.10 1.1 1.5 1.6 -4.1 2.7 T

rimethop rim 7.60 74.8 2.0 -3.1 3.0 -12 3.1 -9.7 0.41

- 11 3.4 Erythromycin 10.0 102 1.9 -24 6.6 -14 4.5 -12 1.8 -16 4.8 Diclofenac

- d 4 100 85.5 1.2 -0.94 2.4 -1.8 5.2 10 5.1

- 11 4.2

13 C 3 - Trimethoprim 20.0 79.4 0.84 11 3.7 -1.9 4.5 1.6 2.8 1.2 2.0A World of Solutions TM 17 

## 







 

# CONCLUSIONS

Various pharmaceuticals/personal care products (PPCPs) analyzed in both ESI positive and ESI negative modes

Erythromycin measured as erythromycin –H 2 

## O

– Likely transforms prior to analysis



## Extraction necessary to reach desired concentration levels

– HLB cartridges found to provide best overall extraction recovery for both positive and negative analytes



## All analytes meet precision and accuracy QC requirements in three differ ent matrixes

Sample and extract hold times change less than 20% after 28 daysA World of Solutions TM 

# ANALYSIS OF CYLINDROSPERMOPSIN AND ANATOXIN

- A BY LC

- MS/MS

CB&I William A. Adams, Ph.D.

# CC L 3 ALGAL TOXIN TARGET ANALYTES

A World of Solutions TM 1 

### Anatoxin -a

Cylindrospermopsin 

## 





 Cylindrospermopsin and anatoxin

- a

– Toxins produced by blue

- green algae



## Water soluble, polar molecules

Analysis by LC

- MS/MS using a high aqueous composition eluent

– Cylindrospermopsin: 416.2 > 194.0 m/z

– Anatoxin

- a: 165.8 > 148.8 m/z



## Direct injection

# SE LE CTION OF INTERNAL STANDARDS

A World of Solutions TM 2 

## 





Uracil L- phenylalanine 

## Isotopically labeled standards not available for cylindrospermopsin or anatoxin -a

Cylindrospermopsin is a uracil derivative

– Deuterated uracil readily available and elutes near retention time of cylindrospermopsin (Uracil

- d 5 

## )

Anatoxin is isobaric and structurally similar to L

- phenylalanine

– Deuterated phenylalanine is readily available and appears to track well with anatoxin

- a (L

- phenylalanine

- d 4 

## )A World of Solutions TM 3 

# CHROMATOGRAPHIC CONDITIONS INSTRUMENTAL METHOD

HPLC Column : Waters XSelect® HSS T3, 2.1 x 150 mm, 3.5 μm Column temperature: 30 o C Column flow rate: 0.200 mL/min Autosampler temperature: 10 o C Injection volume: 50 μL Gradient :
Time (min) %100 mM acetic acid in reagent water %MeOH

0.00 100 0 0.50 90 10 8.50 100 0 13.50 End Run



# MS CONDITIONS

MS Parameter HPLC

- MS/MS Polarity Positive ion electrospray Capillary Voltage, kV 2.50 Source Temperature, o C 120 N 2 Desolvation Temperature, o C 400 N 2 Desolvation Gas Flow, L/hr 900 Cone Gas Flow, L/hr 50 Extractor Lens, V 2.00 RF Lens, V 0.2A World of Solutions TM 4 

## 



 

# Preservation

Store samples in refrigerator

100 m g/L asc orbic acid

– Reduces free 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

# CHR O MATOGRAM – 5 ng/mL ANALYTES

A World of Solutions TM 5 Uracil

- d 4 L- phenylalanine

- d 5

CYN ANAA World of Solutions TM 6 

## 



 

# MRL TARGETS

HRL values FROM CCL3

– Cylindrospermopsin – 0.21 μg/L

– Anatoxin -a – 3.5 μg/L

Calibration range

– Cylindrospermopsin – 0.050 to 10.0 μg/L

– Anatoxin -a – 0.050 to 10.0 μg/L

MRL confirmed at 0.100 μg/L for both analytesA World of Solutions TM 7 

# LCMRLA World of Solutions TM 8 

## 





 

# PRECISION AND ACCURACY

Precision and accuracy measured in different matrixes (e.g.

re a gen t water, high TOC tap water, hard tap water)

Measured at two concentrations

– Low and Mid/High of calibration range



## Accuracy

– Low: 50

– 150% recovery

– Mid/High: 70

– 130% recovery



## Precision

– Low: ≤30% RSD

– Mid/High: ≤20% RSDA World of Solutions TM 9 

##  

# PRECISION AND ACCURACY

Reagent water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

Reagent Water Low Compound Samples (ug/L ) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 0.117 0.128 0.117 0.103 0.100 0.094 0.102 0.109 0.012 0.000 0.100 11.136 108.7 anatoxin -a 0.108 0.116 0.108 0.106 0.102 0.127 0.111 0.111 0.008 0.000 0.100 7.391 111.1

Reagent Water High Compound Samples (ug/L ) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 2.837 2.609 2.834 2.767 2.614 2.681 2.664 2.715 0.097 0.000 2.500 3.588 108.6 anatoxin -a 2.757 2.689 2.810 2.580 2.688 2.798 2.709 2.719 0.079 0.000 2.500 2.898 108.7A World of Solutions TM 10 

##  

# PRECISION AND ACCURACY

Ground source tap water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

– Free chlorine: 0.69 mg/L

– Total chlorine: 0.97 mg/L

– Hardness: 325 mg/L

– Conductivity: 798 μS

– pH: 7.76

Ground Water Low Compound Samples (ug/L) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 0.137 0.132 0.120 0.128 0.119 0.122 0.125 0.126 0.007 0.000 0.100 5.247 126.1 anatoxin -a 0.102 0.080 0.087 0.099 0.092 0.096 0.093 0.093 0.007 0.000 0.100 8.016 92.7

Ground Water High Compound Samples (ug/L) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 2.406 2.516 2.413 2.435 2.380 2.487 2.508 2.449 0.054 0.000 2.500 2.206 98.0 anatoxin -a 2.316 2.394 2.370 2.324 2.382 2.419 2.424 2.376 0.043 0.000 2.500 1.790 95.0A World of Solutions TM 11 

##  

# PRECISION AND ACCURACY

Surface source tap water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

– Free chlorine: 0.64 mg/L

– Total chlorine: 1.14 mg/L

– Hardness: 142 mg/L

– Conductivity: 344 μS

– pH: 7.20

– TOC 3.04 mg/L

Surface Water Low Compound Samples (ug/L) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 0.139 0.124 0.111 0.112 0.119 0.109 0.104 0.117 0.012 0.000 0.100 10.070 116.9 anatoxin -a 0.106 0.091 0.096 0.101 0.101 0.093 0.090 0.097 0.006 0.000 0.100 6.178 96.9

Surface Water High Compound Samples (ug/L) Mean (ug/L) SD Unfortified blank (ug/L) Theoretical spike (ug/L) %RSD %Rec ESI (+) 1 2 3 4 5 6 7
cylindrospermopsin 2.762 2.768 2.699 2.714 2.715 2.649 2.679 2.712 0.043 0.000 2.500 1.570 108.5 anatoxin -a 2.450 2.506 2.383 2.585 2.484 2.565 2.548 2.503 0.071 0.000 2.500 2.833 100.1A World of Solutions TM 12 

## 







 

# CONCLUSIONS

C ylindrospermopsin and anatoxin

- a show goo d response with method

Labeled analogs of target analytes not available

– Internal standards chosen based on tracking ability and response

– Uracil

- d 4 

### has shown some inconsistency in dirtier matrixes and may be removed



## Precision and accuracy are within the acceptable range in three different matrixes

M RL is confirmed below HRL of both analytes and LCMRL gives

reasona b le results

Method development will continue with storage/stability study and second l ab oratory demonstrationA World of Solutions TM 

# Update to Method 539

CB&I
Steven D. WinslowA World of Solutions TM 1 

# 

 Update to Method 539

E PA Method 539: LC

- MSM S analy sis , multiple reaction monitoring (MRM) , SPE extraction Original Target Analyte List

Method 539 CASRN

4- Androstene

- 3,17

- dione 63

- 05 -8 Equilin 474

- 86 -2 17β

- Estradiol 50

- 28 -2
Estriol 50

- 27 -1
Estrone 53

- 16 -7
17α

- Ethynylestradiol 57

- 63 -6
Testosterone 58

- 22 -0A World of Solutions TM 

### β -estradiol

2 

### Estriol Estrone

Ethynylestradiol Androstenedione

Equilin Testosterone 

# Update to Method 539

 E PA METHOD 539 TARGET ANALYTESA World of Solutions TM 3 

#  Update to Method 539

 Proposed additional hormones to Method 539


# Five compounds, introduced late in Method 539 development , wer e initially rejected from method

COMPOUNDS CAS# ISSUES HRL (ug/L) alpha- Estradiol 57-91-0 M539: enhancement issue 0.35 Equilenin 517-09-9 M539: recovery in finished ground water failed QC 0.35

Mestranol 72-33-3 M539: did not work with ESI 0.28

Norethindrone 68-22-4 M539: ESI+ internal standard did not track 0.04
Progesterone 57-83-0 M539: ESI+ internal standard did not track Nominated for CCL4 ---



# Internal standards now available

– Norethindrone

- 2,2,4,6 ,6,10

- d 6



## – Progesterone

- 2,3,4

- 13 

## C 3A World of Solutions TM 4 

### Equilenin

α -Estradiol Norethindrone Mestranol 

# Update to Method 539

 Structures of new hormones

ProgesteroneA World of Solutions TM 5 

#  Update to Method 539

Bisphenol A 

# Also add bisphenol A to method

– Nominated for CCL4

– Deuterated bisphenol A used as surrogate M539

• Extraction not an issue



# – But, bisphenol A contamination from instrument and solvent at least 8 ppb

– Quantitation level can be no lower than 3 times LRB level

• MRL would be above 24 ppbA World of Solutions TM 6 

# Update to Method 539



# 

 Bisphenol A 

# Instrument manufacturer suggests use of an “isolator” column for bi sphenol A

– Contamination temporarily retained in short column placed between solvent mixer & pumps

– Contamination separated on chromatogram by later elution 

# Would bisphenol A require too many precautions for a “hormone” m ethod? S eparate method?A World of Solutions TM 7 

#  Update to Method 539
Proposed target analyte list for Method 539 revision with additional five hormones & bisphenol A (in bold)
CAS#
Androstened ione 63

- 05 -8 Bisphenol A 80

- 05 -7 Equilenin 517

- 09 -9 Equilin 474

- 86 -2

α - Estradiol 57

- 91 -0 β -Estradiol 50

- 28 -2
Estriol 50

- 27 -1
Estrone 53

- 16 -7

α

- Ethynylestradiol 57

- 63 -6
Mestranol 72

- 33 -3 Norethindrone 68

- 22 -4 Progesterone 57

- 83 -0
Testosterone 58

- 22 -0A World of Solutions TM 8 

# 





 Update to Method 539

Initial chromatographic challenges

– alpha - & bet a

- estradiol m ust be well separated, because precursor and product ions are same

– Must use high water content (90%) in initial segment of gradient mobil e phase program to focus bisphenol A contamination Develop calibration range

– Test for reproducibility

– Estimate lowest calibration level 

# Extract spiked samples with C18 disks

– If needed, use Oasis HLB disks 

# Check for contamination or interference in Laboratory Reagent Blank s ( LRBs) that contain preservation reagents, sodium omadine & sodium thiosulfateA World of Solutions TM 9



#  First 6 Norethindrone 299.52 > 108.88



### Androstenedione 287.53 > 96.92



### Equilin 267.46 > 143.08



### Equilinen 265.44 > 221.32



### Bisphenol A 227.45 >212.28



### Estriol 287.48 > 171.18 

# Update to Method 539A World of Solutions TM



#  Last 7 Mestranol 311.53 > 121.10



### Progesterone 315.55 > 96.85



### Testosterone 289.58 > 96.89



### alpha

- ethynylestradiol 295.48 > 145.09



### beta

- estradiol alpha

- estradiol 271.48 > 145.08



### Estrone 269.48> 145.11

10 

# Update to Method 539A World of Solutions TM 11 

#  Update to Method 539

Collect data for demonstration of capability

– Determine Lowest Concentration Minimum Reporting Level (LCMRL)

– Run Precision & Accuracy studies at a low and a mid level in reagent water, surface water and ground water

– Conduct holding time study for samples with preservatives

• Holding time study for extracts



## – Conduct Multi

- Laboratory Validation

• At least two other labs

• Use data to calculate MRL for method

# Microbiology Methods Overview

Sandhya Parshionikar, Ph.D.

Office of Ground Water and Drinking Water

Standards and Risk Management Division Technical Support Center

May 15, 2013

5/10/2013 1 U.S. Environmental Protection Agency

# Candidate Contaminant List 3

Microbial Contaminant Name Information

Adenovirus Virus

- respiratory and gastrointestinal illness Caliciviruses Virus (includes Norovirus) Virus - gastrointestinal illness
Campylobacter jejuni Bacterium - gastrointestinal illness

Enterovirus Group of viruses - mild respiratory illness

Escherichia coli (0157) Toxin

- producing bacterium - gastrointestinal illness and kidney failure
Helicobacter pylori Bacterium - ulcers and cancer
Hepatitis A virus Virus - liver disease and jaundice
Legionella pneumophila Bacterium

- lung diseases when inhaled
Mycobacterium avium Bacterium - lung infection in those with underlying lung disease, and disseminated infection in the severely immuno compromised Naegleria fowleri Protozoan parasite

- warm surface and ground water causing primary amebic meningoencephalitis

Salmonella enterica Bacterium

- gastrointestinal illness
Shigella sonnei Bacterium

- gastrointestinal illness and bloody diarrhea
5/10/2013 U.S. Environmental Protection Agency 2

# Challenges to Microbial monitoring under UCMR

• Pathogen presence in biofilms

– Biofilm sampling presents a challenge for UCMR



# • Could be present in low concentrations

– Need to sample large volumes of water

• Presents logistical challenges for UCMR



# • Pathogen presence in unconventional locations

– Hospital and nursing home hot water tanks and storage tanks

• Public health concern but challenging to monitor



# • Some pathogens hard to grow in culture

• Risk assessment with PCR data is a challenge

5/10/2013 U.S. Environmental Protection Agency 3

# Method Development Rationale

• Depends on which pathogen to target for UCMR monito ring

– What is the likelihood of a pathogen being found in finished drinking water?

– Which pathogen has a validated method ready?

– Which public health issue to address during UCMR monitoring?

• Outbreaks

• Other public health concerns

– Under what conditions do we monitor?

• Frequency

• Volume of water

• Seasonality

• Type of public water system
5/10/2013 U.S. Environmental Protection Agency 4

# Current Method Development Efforts

• H.pylori

• E.coli O157:H7

• Ade n ovirus

• Mycobacterium

5/10/2013 U.S. Environmental Protection Agency 5

# Development of Methods to detect H. pylori and E. coli O157:H7 in Water

CCL Methods Meeting/Webinar
Keya Sen, Technical Support Center, SRMD, OGWDW, OW US EPA May 15, 2013

5/10/2013 1 U.S. Environmental Protection Agency

# Facts about H. pylori

• H. pylori a gram negative bacteria

– Causes chronic active and persistent gastritis

– Stomach and duodenal ulcers and in few of these cases, gastric can cer

• Approximately 25 Million Americans suffer from the disease

– Each year 500,000 to 850,000 new cases

• Culturable from stomach but unculturable from water

– ne cessitates the use of PCR based methods

5/10/2013 U.S. Environmental Protection Agency 2

# H. Pylori Method Development

• Analyzes 1 liter of sample collected

• Detects a segment of the gene ure A,

cons e rved in all H. pylori , by qPCR

• Uses an internal control which is a process
cont r ol and is added to every sample - a Bioball

– Confirms whether a sample is a true negative

5/10/2013 3 U.S. Environmental Protection Agency

# Detection of H. pylori from Water

Collect 1 liter of drinking water. Spike with 10

- 1000 cells of H. pylori . Add 1 Bioball

Filter through 47mM, 0.4 micron

5/10/2013 4 U.S. Environmental Protection Agency 

## Extract filter with lysis solution

Remove solution to centrifuge tube.

Extract DNA into 33 μ l w ater. Use 11 μ l (x3) for qPCR

# Inter

- laboratory validation of qPCR method for H. pylori detection

• Five volunteer laboratories selected

• Three drinking water samples (1L) tested

• One sterile reagent grade water (1L)

• 2 levels of spike used

– 15 or 100 cells of H. pylori

• BioBall

- KS 10 containing 30 cells of E. c o li used as matrix spike (process control)

5/10/2013 U.S. Environmental Protection Agency 5

# Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria 1



### Performance test* **E. coli** KS10 BioBall TM acceptance criteria
Initial precision and recovery (IPR)

• 

### Mean percent recovery 25% - 379 % (8 -114 gene copies) 2

• 

### Precision (as maximum relative standard deviation) 107% Ongoing precision and recovery (OPR) as percent recovery Detect - 482% (145 gene copies)

1 IPR requires the analysis of 4 reagent

- grade water samples; OPR requires the analysis of a single reagent water sample

2 One gene copy can be considered to be equivalent to one cell since each E. coli KS10 cell has the target cloned into a single copy plasmid.

5/10/2013 6 U.S. Environmental Protection Agency

# E. Coli O157:H7 Method Development

5/10/2013 7 U.S. Environmental Protection Agency

# Facts about E. coli O157:H7

• Gram negative bacteria that lives in the guts of ruminants

• Causes severe illness

– bloody diarrhea and vomiting

– About 10 % develop hemolytic uremic syndrome (HUS) that can

resu lt in non

- functioning kidneys

• About 95,400 cases each year from E. c o li O157:H7 in the US

• Bacteria can be cultured from water but can be difficult to identify

– Needs specialized approach

5/10/2013 U.S. Environmental Protection Agency 8

# Traditional Methods for Detection of E. coli O157:H7

5/10/2013 9 U.S. Environmental Protection Agency Filter Water 

### • 0.1L

- 1L

Enrich filter in broth 

### • Tryptic Soy or Buffered Peptone Water

Immunomagnetic separation 

### • Specific Antibodies

Grow in CT

- SMAC or Rainbow Agar with antibiotics 

### • select colonies with characteristic morphology

Verify by PCR/qPCR • Pr imers and Probes to virulence factors Primers and Probes to Structural genes •

# Issues With Detection of E. coli O157:H7 by Traditional Methods

5/10/2013 10 U.S. Environmental Protection Agency 

# False Positives obtained • Closely related pathogenic E. coli serotypes having similar phenotypic and genotypic properties Growth of O157:H7 suppressed •



# Takes 3

- 5 days

Labor intensive

# •

•

•

•

• 

# Rapid method 

# Goals for EPA Method for E. coli O157:H7
Detect in source and drinking water within a day

5/10/2013 11 U.S. Environmental Protection Agency 

# Highly Sensitive Able to detect 1

- 5 cells



# Highly specific

Able to detect viable cells No false positives Appropriate QC reagents
Allow better risk assessment

# Strategy

• Add an enrichment medium directly to water sample

• Provide conditions for stressed cells (starvation, chlorinat ion induced), if present , to recover and grow

• Perform qPCR before and after enrichment without isolation of E. co li cells

– Change in Ct value from pre to post enrichment will indicate viable cells

– A decrease of 3.3 Ct value corresponds to 1 log increase in gene co pies, if the amplification efficiency is 100%

5/10/2013 12 U.S. Environmental Protection Agency

# Development of E. coli O157: H7 qPCR assays

• Searched literature for primers and probes

• Tested primers and probes with an ATCC strain

• Tested 4 commercially available kits with an ATCC strain

• Obtained 61 strains from USDA, FDA and in -ho use

co lle ction

– Tested kits and probes

5/10/2013 U.S. Environmental Protection Agency 13

# Selection of Targets/kits

• ABI MicroSeq kit p roduced no false positives

– Targets two non

- coding r egions on the E. coli genome

– Has an internal control for qPCR



## • Additional triplex qPCR reaction optimized

– Targets genes stx1, stx2, eae

– Developed MGB probes



## • Additional monopl ex assay that targets rfb E gene optimized for confirmation

– rfb E specific to O157 serotype

– Devel oped MGB probes

( Environmental Science and Technology (2011): 45, 2250

- 2256 Sen, K 1 

### . Sinclair 1 

### , J, Boczek , L 2 

### , Rice, E.G 3 

### .)

5/10/2013 U.S. Environmental Protection Agency 14

# Demonstration of Viability

• Bacterial Cells starved for 12 - 14 day s

• Dilutions made, 4

- 400 cel l s

• Spiked water samples with different dilutions

• Added 6X Presence

- Abs en ce broth to sample

- 1 mL aliquot removed and stored at – 20 0 

## C (pre

- enrichment).



# • A llowed to grow:35 0 

# C for 18

- 23 hrs

– 1 mL aliquot removed



# • DNA extracted from both aliquots

– Centrifuge to pellet cells

– Add 100 μ L of Pre p Man buffer; Heat at 99 0 

## C



# • Perform 3 qPCR assays with 3 μ L extract.

• Examine whether there is a shift in Ct values from pre to post enrich m ent samples

5/10/2013 U.S. Environmental Protection Agency 155/10/2013 U.S. Environmental Protection Agency 16 

### Stx1 Stx2 eae 18 hr

0 hr 

# Demonstration of Viability

 Detects less than 10

c ells

 Examines only 30 μ L of sample

# Addition of Process Control

• Bioball

- KS10 used for H. pylori considered good for the purpo se since they are E. coli cells but not expected to occur naturally

• Optimized the number of cells to add

• BTF

- Biomerieux packaged 5000 cells into Bioball.

• Bio ball added at the time of extraction

• Detected in a separate Monoplex assay,

– Used same primers and probe used in H. py lori assay

5/10/2013 U.S. Environmental Protection Agency 175/10/2013 U.S. Environmental Protection Agency 18 

### S S ID 

# Collection to Detection

Collect 1L water

Add 200 mLPresence /Absence broth

Remove 1 mL. Store at -20 0 

### C Grow 1199 mL x 18- 23 h r s at 35 0 

### C till yellow



# . Remov e 2 x 1 mL

Add 1 Bioball- 5000 to all three tubes Centrifuge all tubes Remove supernatant Suspend Pellet in PrepMan Buffer

Heat to 95 0 

### C x 10 mins Centrifuge tubes x 2 mins Use 3 μ L of supernatant from each tube in 3 qPCR assays

rfbE ABI MicroSeq ( Triplex 1) tx 1, Stx2, eae (Triplex 2) PC (Monoplex)

# Testing Method With Field Samples

• Six Surface water tested

– Four had pathogenic E. coli

– Only one sample had E. coli O157:H7

19 5/10/2013 U.S. Environmental Protection Agency

# Inter

- laboratory validation of qPCR method for detection

• Five volunteer laboratories selected

• Three drinking (ground) water samples (1L) tested by all 5 la b s

• One sterile reagent grade water (1L) by all 5 labs

• One spike level used <10 cells

5/10/2013 U.S. Environmental Protection Agency 20

# Initial and Ongoing Demonstration of Capability (IDC and ODC) Acceptance Criteria

5/10/2013 U.S. Environmental Protection Agency 21 

### Performance Test Acceptance Criteria Initial Demonstration of Capability ( IDC) (as Ct difference between pre and post enrichment samples). IPR requires the analysis of 4 reagent

- grade water samples Target Ct difference Precision (as standard deviation of Ct difference )

MicroSEQ FAM 15.86 - 25.21 1.79 MicroSEQ VIC 17.51

- 23.90 1.49

eae 15.11 - 22.85 2.46
stx1 16.42

- 25.02 2.38
stx2 10.40

- 29.01 3.85 Ongoing Demonstration of Capability ( IDC) (as Ct difference between pre and post enrichment samples). OPR requires the analysis of a single reagent water sample Target Ct difference

MicroSEQ FAM 15.74

- 25.33

MicroSEQ VIC 17.44

- 23.97

eae 13.40

- 24.56

stx1 14.95

- 26.49

stx2 10.55

- 28.86

# Conclusions and Next Steps

• Both methods have been developed and validat ed

• Currently being processed for EPA publication

5/10/2013 U.S. Environmental Protection Agency 22Office of Research and Development National Exposure Research Laboratory, Cincinnati, Ohio May 2013 

## G. Shay Fout, Jennifer L. Cashdollar, and Nichole E. Brinkman U.S. Environmental Protection Agency, Cincinnati, OH 

# A denovirus Methods: Method 1615 Modification and Small Volume Approach1 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Presentation Outline

•

•

• Method 1615 

  Rationale for the development and enhancement of Method 1615 Method overview Water Cluster modifications  Method performance Small Volume Approaches

 Method overview and performance Future development2 

## •

•

•

•



 

# EPA Method 1615 Rationale

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Groundwater

- Borne Disease Outbreaks in the USA

From 1971 to 2008 t here w ere nearly 846 outbreaks associated with an infectious agent in drinking water; about 50 % of the outbreaks were attributable to groundwater
During the same period, 8% of the outbreaks were caused by viruses and anothe r 43% had a likely viral cause

Numerous studies have found human enteric viruses in about 25% of wells stud ied Two studies focused on small community systems that use untreated groundw a ter A study of communities in Southeast Michigan found virus presence in 24% of the wells (Francy et al., 2004) A study of communities in Wisconsin found virus in 95% of the wells (Bor c hardt et al. 2012)3 

# Virus Types, Frequencies, and Concentrations in Tap Water

Virus Type Number qPCR Positive Samples Virus Concentration Genomic copies/L Number Culture Positive Samples Mean Maximum
Adenovirus 157 (13%) 0.07 9.5 40/157 (25%)

Enterovirus 109 (9%) 0.8 851.1 31/109 (28%)

GI Norovirus 51 (4%) 0.6 115.7

GII Norovirus 0 (0%) 0 0 Hepatitis A 10 (1%) 0.006 4.1

Rotavirus 1 (0.1%) 2 x 10 -5 

### 0.03

All Viruses 287 (24%) 1.5 853.6

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

### N = 1,204 samples from 14 Communities; Infectivity was measured on qPCR positive samples only; Reference: Borchardt et al . 2012

.4 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# EPA Method 1615 Rationale

• The Wisconsin study demonstrated that 22% of the AGI in the study communities was from virus

- contaminated tap water (Borchardt et al. 2012. Environmental Health Perspectives 120:1272

- 1279)

• For children < 5 yrs in the spring of 2006, the fraction of AGI from norovir u s in drinking water was 63% !5 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# EPA Method 1615 Rationale

Virus in U.S. Source Waters Percent Cell Culture Positive Samples Plant Intakes Region East 28 85 South 22 86 Midwest 21 90 West 20 83

Totals 24 87

Average titer all samples: 1.5 MPN/100 L (range: 0 -1974)6 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# EPA Method 1615 Enhancement

Adenovirus in Wastewater

Adenovirus Genomic Copies/Liter Raw/Primary Effluent 2,000 – 800,000,000 Tertiary Effluent 930 – 8,900,000

References:
Bofill

- Mas et al., 2006 Katayama et al., 2008 Dong et al., 2009 Kuo et al., 2010 Hewitt et al., 2011 Sidhu et al., 2013 Brinkman et al., submitted7 

# EPA Method 1615 Overview
Filtration/Sample Concentration
Samples: Controls : NanoCeram filters (1800 L of groundwater) PT and PE: Filters containing unknown amounts of virus (10 L spiked) QC: Filters containing 0 or 500 MPN of virus (10 L spiked) Matrix: Filters containing 1,000 MPN of virus (1790 + 10 L spiked)



## Elution

2 × 500 mL 1.5% beef extract, 0.05 M glycine, pH 9.0


## Organic flocculation pH 3.5 for 30 min Resuspend floc in 30 mL 0.15 M sodium phosphate, pH 9.0 Subsample 1: approx. 9 mL 

## Cell Culture

Quantal assay MPN/L 

### Sub sample 3 Approx. 12 mL

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Archive Subs ample 2: approx. 9 mL 

## Tertiary Concentration

Vivaspin 20, 30 kDa MWCO



## RNA Extraction QIAamp DNA Blood Mini Kit



# RT

- qPCR
Genomic copies/L References: Fout et al . 2012. EPA/600/R

- 10/181, January 2012 revision Cashdollar et al . 2012. Applied Environmental Microbiology 79:215

- 2238 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

## Total Culturable Virus Assay

BGM cells showing early cytopathic effect from poliovirus

QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET Sample Number: Sample 1

Sample Number Replicates
Inoculated Number with CPE (1) MPN/mL (2) 95% Confidence Limits/mL
Lower Upper Undiluted 10 6

1.1 0.4 2.2 1:5 Dilution 0
1:25 Dilution 0
1:125 Dilution 0



## D S M = M L m L 

# = (1.1 MPN/mL * 8.33 mL)/1800 L = 0.02 MPN/L9 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Additional Concentration for the Molecular Assay

Tertiary Concentration with Vivaspin Concentrators

• Fill Vivaspin 20 unit with PBS, 0.2% BSA

• Soak overnight at 4 ° C. Dis car d PBS

• Rinse with sterile water. Discard water

• Add an amount of Subsample 2 = S

• Centrifuge at 3,000 – 6, 000 x g t o a volume less than 0.4 mL

• Add 1 mL of 0.15 M sodium phosphate, pH 7- 7.5

• Centr ifuge at 3,000 – 6,000 x g to a volume less than 0.4 mL

• Add 1 mL of 0.15 M sodium phosphate, pH 7- 7.5

• Centr ifuge at 3,000 – 6,000 x g to a volume less than 0.4 mL

• Transfer sample to a 1.5 mL microcentrifuge tube

• Bring t o a volume of 400 ± 2 μ L10 

# Tertiary Concentration

Recovery of Poliovirus and Murine Norovirus Using Vivaspin 20

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

### Poliovirus Murine Norovirus 30,000 MWCO 80.9% 45.6%

100,000 MWCO 55.1% 26.1%11 

### Subsample 2 

# Reverse Transcription – Quantitative PCR

Vo lume = S Tertiary Concentration

Nucleic Acid Extraction 0 .2 mL Volume = 0.4 mL

Volume = 100 μ L
RNA 6. 7 μ L per replicate 16.5 μ L RT Master Mix 1

RT 1 RT2 RT 3

Step 10.4.9 99 °C 99 °C 99 °C

16.8 μ L RT Master Mix 2

Step 10.4.12 42 °C 42 °C 42 °C

qPCR qPCR qPCR

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH12 

# Reverse Transcription

Reverse Transcription

• Prepare master mixes

• Add 16.5 μ L RT Mix 1

• Add 6.7 μ L of sample

• Add 6. 7 μ L PCR

- g rade water as no template controls

• Heat at 99 ° C fo r 4 min

• Quench on ice

• Add 16.8 μ L R T

Mix 2

• Run at 25 ° C for 15 min, 42 ° C for 60 min, 99 ° C for 5 min, hold of 4 ° C

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH Ingredient Volume per reaction (1) Final concentration Volume per Master Mix (2)

RT Master Mix 1 Random primer (Item 7.5.11) 0.8 10 ng/μL 84.0 Hepatitis G Armored RNA (3) (Item 7.5.12) 1.0 105.0
PCR grade water (Item 7.5.2) 14.7 1543.5 Total 16.5 1732.5 RT Master Mix 2

10X PCR Buffer II (Item 7.5.13) 4.0 10 mM tris, pH 8.3, 50 mM KCL 420.0

25

- mM MgCl 2 (Item 7.5.13) 4.8 3 mM 504.0

10

- mM dNTPs (Item 7.5.14) 3.2 0.8 mM 336.0

100

- mM DTT (Item 7.5.15) 4.0 10 mM 420.0

RNase Inhibitor (Item 7.5.10) 0.5 0.5 units/μL 52.5

SuperScript II RT (Item 7.5.16) 0.3 1.6 units/μL 31.5

Total 16.8 1764.0

(1) The volumes given are for 40

- μL RT assays. (2) Reagent amounts sufficient for a 96

- well PCR plate are given. The volumes shown were calculated by multiplying the volume p er reaction amount by the number of assays to be performed, plus an additional 9 assays to account for losses during transfer of th e master mix to plates (Item 6.6.15) using items 6.6.9 and 6.6.12. The amount of additional assays to add can be reduced if experienc e s hows that lower amounts are adequate. (3) Hepatitis G Armored RNA is supplied as an untitered stock. The amount to use must be determined for each lot, as described in Step 13.6.1.13 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Quantitative PCR

Ingredient Volume per reaction (μL) (1) Final concentration Volume per Master Mix (μL) (2)

2X LightCycler 480 Probes Master Mix (Item 7.4.17) (3) 10.0 Proprietary 1050.0

ROX reference dye (Item 7.4.18) (4) 0.4 0.5 mM 42.0

PCR grade water (Item 7.4.2) 1.0 105.0

10 μM EntF 0.6 300 nM 63.0

10 μM EntR 1.8 900 nM 189.0

10 μM EntP 0.2 100 nM 21.0

Total 14.0 1470.0

(1) The volumes given are for using 6 μL of cDNA from Step 12.5.3 in a qPCR assay using a total qPCR volume of 20 μL. (2) Reagent amounts sufficient for a 96

- well PCR plate are given. The volumes shown were calculated by multiplying the volume per reaction amount by the number of assays to be performed, plus an additional 9 assays to account for losses during transfer of the master mix to tubes or plates. The amount of additional assays to add can be reduced if experience shows that lower amounts are adequate. (3) 10X PCR Buffer II , 25

- mM MgCl 2 , and AmpliTaq Gold can be substituted for the LightCycler 480 Probe Master Mix. (4) This reagent is necessary for use with Applied Biosystems and similar instruments. It should be substituted with PCR grade water for use with the LightCycler and similar instruments.14 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Quantitative PCR
Standard Curves Using Armored RNA® EPA

- 1615

• All results must be based upon standard curves run in duplicate on
every RT

- qPCR plate

• Standard curves are prepared at concentrations of 2.5 x 10 8 

## to 2.5 x 10 3 

## , giving 502,500 to 5 genomic copies per standard

• Acceptable standard curves must have a R 2 

## value of > 0.97 and an efficiency of 80

- 110%

• R 2 

## and slope are calculated by the thermocycler

• Efficiency is calculated by the thermocycler or as shown in the
examp l e below

• For example, a curve having a slope of

- 3.2 9 has a % efficiency of 100 x (10

- 1/

- 3.29 

## - 1) = 101.3%15 NorGIA: y =
- 3.2032x + 41.422 R2 = 0.9931

Nor GIB: y =
- 3. 3328x + 41.144 R2 = 0.9969 20.00 25.00 30.00 35.00 40.00 45.00

0 2 4 6Ct Value

Log Copies/Reaction NorGIA

NorGIB 

# Armored RNA® EPA

- 1615 Norovirus GI Standard Curve

Target

NorGIA NorGIB E fficiency

105.4%

99.7% Theoretical Detection Limit (# Particles) 3 2

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH16 

# Mean Recovery (%) of Spiked Poliovirus and Murine Norovirus from Ground, Surface, and Reagent

- Grade Water Samples

Assay Groundwater
n=7 Reagent-Grade Water (High Titer b 

### )
n=6 Reagent- Grade Water (Low Titer c 

### )
n=6

Recovery CV a 

### Recovery CV a 

### Recovery CV a



### Total Culturable Virus Assay 58 79 42 34 122 96

Poliovirus RT

qPCR 20 64 48 36 39 29

Murine
Norovirus RT

qPCR 30 75 0.6 100 8 83

a CV is % coefficient of variation

b High titer spike at 1000 MPN of poliovirus and 1000 PFU of murine norovirus per 10 L sample

c Low titer spike at 300 MPN of poliovirus and 300 PFU of murine norovirus per 10 L sample



### Reference: Cashdollar et al., 2012

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 1617 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Issues with EPA 1615

• Filter clogging in turbid waters

• An assay for adenoviruses was not included in the method

• Poor virus recovery from turbid waters

• Turbid waters have RT

- q PCR i n hibitors18 

## Subobjective 1.A.i . Prefilter and electropositive filter options: Enhance ability to collect required sample volume when testing turbid waters
Subobjective 1.A.ii. Ultrafiltration versus NanoCeram – Determine the relative effectiveness of using ultrafiltration versus the NanoCeram electropositive filter for concentration of secondary and tertiary effluent
Subobjective 1.A.iii. Celite versus NanoCeram – Determine the relative effectiveness of the NanoCeram versus the small volume method
Subobjective 1.A.iv. Addition of Sea Salts – Determine whether the addition of sea salts can enhance recovery of norovirus recovery
Subobjective 1.A.v. Sonification, Dispersants, and Surfactants – Determine if sonication or treatment with dispersants and surfactants will improve recovery as measured by plaque assay and RT

- qPCR Project P1.5.3A.D.1: Improvements, Field Evaluation, and Commercialization of EPA Method 1615 

# Water Cluster Modifications

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH

## Subobjective 1.B. Dilutions for Samples with High Virus Levels – quantify the loss of precision resulting from the use of 10

- fold rather than 5

- fold dilutions when assaying samples with high virus titer
Subobjective 1.C. Tertiary Concentration - Large Volume Extraction Option

– Examine if virus recoveries can be improved by replacing the tertiary concentration followed by a small volume RNA extraction approach with a large volume RNA extraction
Subobjective 1.D. Inhibitor Removal - Proprietary versus EPA Options: improve ability to remove RT

- qPCR inhibitors from turbid waters
Subobjective 2.A. and 2.B. Add an adenovirus culture and molecular assay to 1615

Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Water Cluster Modifications20 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Water Cluster Modifications

Objective 3 . Develop a RT

- qPCR Commercial Kit and compare it to our current approach
Objective 4. Develop an Infectious Virus RT

- qPCR assay using PMA
Objective 5. Measurement of Virus Occurrence

• Measure virus in primary, secondary, and tertiary effluents at AAA Wastewat er Services, Hamilton Water Reclamation Facility (biological with chlorine treatment), Sycamore Wastewater Treatment Plant (nutrient removal plant), Twin Creek Preserve, and Upper Mill Creek Regional Water Reclamation Facility (biological with UV)

• Compare virus occurrence with indicator occurrence21 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Addition of Adenovirus to Method 1615

Adenovirus Culture Assay using A549 cells

Process Control % Recovery Negative - Positive 1 18 Positive 2 32 Positive 3 28 Positive 4 12 Positive 5 7 Positive 6 3 Positive 7 12 Positive 8 96 Positive 9 29

Mean Recovery 26 CV 10822 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Addition of Adenovirus to Method 1615

AdV PCR

AdV PCR AdV PCR23 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Adenovirus PCR

Prepare Standard Curves for Adenovirus using quantified Adenovirus 5 from OD260, Inc.

y = - 3.363x + 36.003 R2 = 0.9951 Efficiency = 98.3%

0 5 10 15 20 25 30 35 40

0 1 2 3 4 

### Standard Curve for Adenovirus24 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Small Volume Method

Celite Method

1. Add 1.5g of celite to the eluent

2. Drop the pH to 4 and mix for 10 -30 minutes

3. Col lect celite on a sterile pre

- fi lter

4. Elute virus from celi te with 40- 80 ml of 0.15M sodium phosphate

5. Adjust pH to 7 -7.5. Fi l ter sterilize. Freeze for analysis
Recoveries range from 60

- 90% for enteroviruses and 65- 69% for adenovirus25 Office of Research and Development National Exposure Research Laboratory, Cincinnati, OH 

# Future Development

• As part of the Water Cluster Project

 Improve virus detection for turbid waters

 Test robustness of EPA Method 1615 for measuring virus

oc cur r ence at wastewater “End

- of

- Pipe” settings and in recreational settings

• Test whether the small volume method is scalable to 10 L of secondar y or tertiary effluent

# Questions?

Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division Photo image area measures 2” H x 6.93” W and can be masked by a collage strip of one, two or three images. The photo image area is located 3.19” from left and 3.81” from top of page. Each image used in collage should be reduced or cropped to a maximum of 2” high, stroked with a 1.5 pt white frame and positioned edge

- to

- edge with accompanying images.

May 15, 2013 

# Detection Methods for Mycobacteria Stacy Pfaller

Ohio River and Downtown Cincinnati, OH

Disclaimer: This presentation does not necessarily reflect official U.S. EPA policyOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 1 

## Background









 

### Waterborne illness caused by nontuberculous mycobacteria (NTM) costs the US nearly $500 M in hospitalizations per year ( a 

### Collier et al, 2012). Pulmonary NTM infections account for almost half of all NTM hospit alizations in the US, and are typically caused by Mycobacterium avium (MA) and M. intracellulare (MI) In addition to pulmonary infections, cause skin, soft tissue, lymph node, sys temic infections, among others Primary source of human exposure: WATE R a 

### CCL’s 1 and 2: Mycobacterium avium Complex (MAC) CCL 3: M. avium 4 subs pecies: M. avium subsp. hominissuis M. avium subsp . avium M. avium subsp . silvaticum M. avium subsp . paratuberculosis

a Collier et al (2012) Epidemiology and Infection, 140: 2003

- 2013Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 2 

## Culture and Molecular Methods

 

### Culture Method Standard Methods for the Examination of Water and Wastewater a 

### section 9260M Modifications described in Covert et al (1999) Appl Environ Microbiol 65:2492

- 2496

 

### Quantitative PCR (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 

### Primers and probe for M. avium and M. intracellulare have been submitted for publication

a Eaton, 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, Microbial and Chemical Exposure and Assessment Research Division 3 

# Sample Collection






 

## Sample collection is identical for both culture and qPCR Bulk Water: collected in 1L sterile polypropylene bottles

acc or ding to sections 9060A and B of Standard Methods for the Examination of Water and Wastewater a 

## , except NO preservative (Na 2 

## S 2 

## O 3 

## ).

Biofilm: collected by swabbing surface of tap, pipe, tubing, etc.

Samples transported back to lab on ice, stored at 4 ° C unti l processing, within 48 hours.

a 

### Eaton, 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, Microbial and Chemical Exposure and Assessment Research Division 4 

# Culture method











 

### 1L water split into 2 x 500 ml aliquots or some volume of biofilm slurry (1 – 2 ml typically) cetyl pyridinium chloride (CPC) is added to a final

conc entration of 0.04% for reduction of background organisms Samples shaken and incubated at room tem perature for 30 min Samples filtered through 0.45 um pore

- size, 47 mm bl ack

- grid, cellulose ester filter by vacuum filtration, washing the filter with sterile deionized water.

filter aseptically transferred to Middlebrook 7H10 agar containing 500 mg L -1 

### cycloheximide Plates are incubated a mi nimum 8 weeks at 37

° C and inspected weekly for growthOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## 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

 

## No CPC control , if possible

5Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 6Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 7



# Benefits of Culture Method
   Many 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** CPC disinfection may reduce recovery of target by 70%

(P ersonal 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 biofilmOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

### 











8 

### I/MAP- specific

ta Analysis te Quantification 

# qPCR Method and/or 

### r Biofilm

MA/M assay

Da Absolu 

### Bulk wate

Water/biofilm slurry is vacuum filtered through 47.0 mm, 0. 45 um poly carbonate membrane (no CPC treatment necessary) Membrane rolled and placed in 2.0ml tube containing 0.3g glas s beads and buffer Microorganisms trapped on membrane lysed physically by bead beati ng DNA from crude lysate extracted using WaterMaster kit reagents from ( EpiCenter Biotechnologies, Madison, WI) DNA resuspended in sterile, molecular biology

- grade wat er Three replicate qPCR reactions analyzed/ DNA extract

• Two replicates must be positive for a sample to be consider ed positiveOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## Absolute Quantification from Master Standard Curves







 

### Generated from six independents series of 10

- fold serial dilutions of purified genomic DNA from ATCC Type strains of MA, MI, and MAP Each dilution series contains eight standards,

r anging in concentration from 10 6 

### target copies to 1 copy, run in triplicate = 18 measurements/ standard C T 

### measurements plotted against log target number and analyzed by linear regression to generate line equation Target number in unknown sample estimated from l ine equation

9Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division



# QC Samples for qPCR



•



• 

•



• 

### Method Blank (negative control) Sterile molecular biology grade water or sterile swab filtered and pr ocessed 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 D NA extract Internal positive Control (I C) Commercially available kit (TaqMan Exogenous Internal Positive Control K it, Life Technologies, Carlsbad, CA)

10Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 11



## Characteristics of qPCR assays for drinking water and biofilm qPCR assay Target (copies/ genome) 

### E ¶ Amplification Efficiency 

### LOD ‡
Targets/qPCR reaction 

### LOQ †
Targets/qPCR reaction Specificity £ Sensitivity § 

### Drinking

Water Sensitivity § 

### Biofilm

M. avium 16S rDNA (1) 1.92 10 10 100% 100 cells/L 1000 cells/swab

M. avium subsp. paratuberculosis IS 900 (14

- 20) 2.01 1.8 1.8 100% 100 cells/L Not determined

Beumer et al

, 2010, Appl Environ Microbiol Target 251 (1) Not determined Not determined Not determined 95% Not determined Not determined

M. intracellulare 16S rDNA (1 ) 1.91 10 10 100% 10 cells/L 1000 cells/swab

¶ Amplification Efficiency = 10(1/

- slope). If E = 2, the reaction is 100% efficient. ‡ 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%.

£ Specificity = Number of target testing positive/total number targets tested x 100.
§ Sensitivity = lowest copy number detected when spiking serial dilutions of known cell quantities into actual tap water samples, processed as described in Beumer et al, 2010, Appl Environ Microbiol, 76:7367

- 70Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 12 

# Benefits of qPCR Method

   A ssays are specific for MA and MI (CCL) No CPC treatment Time to results = 3 days



# D rawbacks of qPCR Method

  A ssays are specific for MA , MI, and MAP only C annot 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

- 732Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## National Occurrence Study using Culture




 

### Covert et al (1999) Appl Environ Microbiol, 65:2492

- 2496 139 samples

– D rinking water (ground and surface water sources)

– Bottled water

– Cistern

– Ice

– Reservoir Results

– 1/ 139 samples positive for MA (drinking water from surface water

sourc e)

– 5/139 samples positive for MI (drinking water from surface water

sourc e)

13Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## Occurrence Revisited MJ Study: National Survey of MA and MI Occurrence using Culture and qPCR, 2009

- 2010


 

### 40 sites across the US 68 taps (1 to 2 taps/site) ground + no treatment = 2 ground + chlorine = 6 ground + chloramine = 1 surface + no treatment = 0 surface + chlorine = 15 surface + chloramine = 12 mixed + chlorine = 2 mixed + chloramine = 2
 

### Samples collected 3 times in 2009, once in 2010

14 PROffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## qPCR and Culture Results by Site

15 Number of Sites N = 40 

# qPCR Culture

Number of Sites N = 40 

# 9 5 19

7 + MI Only + MA Only

+ MA & MI

- MA & MI 

# 6 8 6

19 + MI Only + MA Only

+ MA & MI

- MA & MIOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## qPCR and Culture Results by Tap

16 Number of Taps N = 68 

# qPCR Culture

Number of Taps N = 68 

# 15 9 23

21 + MI Only + MA Only

+ MA & MI

- MA & MI 

# 7 11 5

45 + MI Only + MA Only

+ MA & MI

- MA & MIOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## Persistence of MA and MI at taps

17 Number Times Positive Number Times PositiveNumber Taps Positive

0 10 20 30 40 50 60

MA MI

Culture

0 Times 1 Time 2 Times 3 Times 4 Times 0 Times 1 Time 2 Times 3 Times 4 Times qPCRNumber Taps Positive

0 10 20 30 40

MA MIOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

# Agreement Between Culture and qPCR

Target No. Samples P ositive Both Methods Culture Positive O nly qPCR Positive Only
M. avium 12 16 37
M. intracellulare 11 4 67

18Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## PHASE II Study Leads Susan Glassmeyer(USEPA), Ed Furlong (USGS), and Dana Kolpin (USGS): Occurrence of Contaminants of Emerging Concern at Drinking Water Treatment Plants (DWTPs)

19 EPA Region Number of DWTPs

1 1

2 1

3 4

4 5

5 3

6 5

7 1

8 2

9 2

10 1Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

### 



20 

## DWTPs positive by qPCR

Twelve DWTPs were positive for MA, MI, and/or MAP by qPCR

Estimated concentrations of MA, MI, and MAP are higher in untreated source water than finished treated water 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Estimated Targets L-1

1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6

MA MI MAP UNTREATED

Drinking Water Treatment Plant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Estimated Targets L-1

1e+0 1e+1 1e+2 1e+3 1e+4 1e+5

MA MI MAP TREATEDOffice of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## Description of qPCR Positive Samples

target N untreated untreated N treated treated

geometric mean (target copies/L) maximum ( target copies/L) geometric mean (target copies/L) maximum ( target copies/L)

M. avium 4 900 5200 2 240 300

M. intracellulare 3 1600 5300 3 40 50 M. avium subsp. paratuberculosis 0 3 20 300

21Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

# Culture: NTM isolates from treated water at DWTPs

 

### No NTM isolated from untreated source water samples.

• 

### All samples lost prior to 8 week minimum incubation period for MAC

 

### Thirty one isolates obtained from treated finished water samples from 6 DWTPs.

• 

### None are MAC though 71% (22/31) have been putatively identified as other clinically relevant NTM species by 16S sequencing

22 6 (27%)

8 (36%) 5 (23%) M. septicum M. mucogenicum M. phocaicum M. triplex

M. fortuitum

M. lentiflavum

1 ( 5%)

1 (5%) 1 (5%)Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division



# Method for **Mycobacterium** Detection for UCMR?




 

# Both, if possible, as both provide important information qPCR performs well regardless of sample matrix (water

o r biofilm)

Culture does not perform well on microbiologically

c omplex samples (contaminated water and biofilm) but does perform well on samples where microbiological water quality is good ( i.g . treated water before distribution)

23Office of Research and Development National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 

## Acknowledgments

•

•

•

• 

### Dawn King (NERL)

•

•

•

•

• 

### Amy Beumer (NERL) Eunice Chern (NERL) Terry Covert (NERL)

Maura Donohue (NERL) Jatin Mistry (EPA Region 6)

Susan Glassmeyer (NERL) Ed Furlong (USGS) Dana Kolpin (USGS)

24