Office of Research and DevelopmentNational Exposure Research Laboratory

Jody A. Shoemaker, Daniel R. Tettenhorst, Armah de la Cruz

EPA Drinking Water Method 544

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

The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.

OAWWA-2016

Office of Research and DevelopmentNational Exposure Research Laboratory

Outline

• Method Development Background–Compliance methods–Methods for Unregulated Contaminant Monitoring Regulations

(UCMR)

• General Method Development Process

• EPA Method 544–SPE-LC/MS/MS method for 6 MCs and nodularin in drinking water

Office of Research and DevelopmentNational Exposure Research Laboratory

CCL & UCMR Background The 1996 amendments to the SDWA required EPA to establish a

Drinking Water Contaminant Candidate List (CCL)

The first CCL was published in 1998 - updated every 5 years

CCL 3 was published in October 2009 and contains 104 chemicals or chemical groups; Draft CCL 4 proposed in January 2015

Nationwide occurrence data needed on CCL chemicals and/or contaminants of emerging concern

EPA’s OGWDW collects occurrence data under the Unregulated Contaminant Monitoring Regulation (UCMR)

Rugged, standardized EPA methods for drinking water contaminants needed for UCMR

If contaminants are regulated, methods usually suitable for compliance monitoring

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Toxin Release (extract intracellular toxin from bacterial cells)

Optimize Instrumentation (chromatography, mass calibrate, tune, evaluate instrument stability, calibration including IS and SUR)

Method Development Process

YESWrite Method

Potential Use inFuture UCMR

Multi-Lab Verification

Determine Aqueous & Extract Holding Times

(ideally ≥14 days)

Revise Technical Approach (re-evaluate sensitivity,

precision, accuracy, interferences and challenging

matrices)

Does Method Meet DQOs? (70-130% REC <30%

RSD)

NO

Consider Analyte Properties and Prior Work(chemical: volatile/nonvolatile, polar/nonpolar, etc.physical: solubility, boiling pointliterature?)

Determine Preservatives(select antimicrobial, dechlor, buffer)

Determine Interferences (determine tolerance limits of method against predetermined QC criteria in challenging matrices)

Internal and External Peer Review

Develop SPE(optimize extraction, accuracy and precision in reagent water)

Determine LCMRL (ideally less than HRL)

HRL = health reference level

LCMRL = Lowest concentration minimum reporting level

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Development ofCyanotoxin LC/MS/MS Methods

Cyanobacteria & cyanotoxins have become a worldwide concern

Blooms are ecologically disruptive, have economic impacts and produce toxins at relevant health levels

Lack of rugged, multi-lab verified, standardized methods for cyanotoxins

MC-LR, cylindrospermopsin and anatoxin-a on CCL 3

What’s the problem?

Solution Method 544 – SPE-LC/MS/MS method for 6 MCs and nodularin

Method 545 – direct aqueous injection LC/MS/MS method for cylindrospermopsin and anatoxin-a

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Method 544 Challenges Lack of certified standards – quality control standard (QCS)

difficulties

Release of intracellular toxins with a 500-mL sample MCs are largely intracellular - need to extract toxins from cyanobacteria Conventional cell lysis - 3 freeze/thaw cycles Cannot perform freeze/thaw cycles efficiently on large volume samples

Extract composition – extract composition affects some recoveries

Lack of sufficient isotopically-labeled MCs to use as internal standards and surrogates

Effect of mobile phase on MC recoveries in tap water (matrix effects)

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Method 544 Toxin Release Procedure

Filtrate collected

Filter collected

SPE after toxin release

Freeze 1-16 h at -20 ºC

2 mL 80:20 MeOH:DI

500-mL water sample filtered using a 0.4

micron polycarbonate filter

Filter

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0

20

40

60

80

100

120M

ean

Rec

over

y, %

Glass Fiber (n=4) Polyethersulfone (n=2) Polycarbonate (n=4)

Evaluation of Filter Types

Results: Glass fiber filters disintegrated during F/T cycles Polycarbonate overall highest recoveries

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Effectiveness of Toxin Release ProcedureCells + H2O

3 F/T in H2O

MeOH:H2O

Freeze 1 h

ELISA (n=3) 0.206

MeOH:H2O

Freeze 1 h

0.258

Cells on FilterCells + H2O

MeOH:H2O

Freeze 1 h

0.276

Demonstrates: A single freeze of the

filter in MeOH:H2O is equivalent to 3 freeze/thaw (F/T) cycles of a water sample

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Method 544 SPE Procedure

10 µL injection

Elution with 9:1 MeOH:water

Oasis HLBcartridge

N2 Evaporate to dryness Add 1 mL

9:1 MeOH:water

Surrogate & preservatives

Preservatives2-chloroacetamide

ascorbic acidEDTAtrizma

SurrogateC2D5-MC-LR

Rinse cartridge and bottle withreagent water

LC/MS/MSExternal Calibration

500 mLSample

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Recovery & Precision Acid & Neutral pH Mobile Phases (MP)

preserved and fortified at 0.2-1 µg/L (n=4)

Demonstrates: Importance of evaluating challenging matrices

Neutral MP/LFSMNeutral MP/LFBAcidic MP/LFSMAcidic MP/LFB

0

20

40

60

80

100

120

% R

ecov

ery

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MC-YR

MC-YR

m/z200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050

%

0

100

Acidic vs Neutral pH LC Mobile PhaseTap water extract - Full Scan MS

Normalized to max intensity of 7.7x108

mass spectrumm/z 200-1050

pH ~3

pH ~6

Demonstrates: LC parameters can

affect recoveries in matrices

Modifications, without evaluating challenging matrices, may affect ruggedness

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40

50

60

70

80

90

100

110

8/29

/13

9/3/

13

9/17

/13

9/18

/13

10/2

2/13

10/2

3/13

11/2

/13

11/1

3/13

11/2

6/13

1/7/

14

1/16

/14

2/18

/14

3/6/

14

3/17

/14

% R

ecov

ery

Fortified DI water extract CCC

Demonstrates: Ruggedness is

not determined in a day or a week!

MC-LW Ruggedness Issues

CCC= continuing calibration check standard

MC-LW not included in Method 544

Office of Research and DevelopmentNational Exposure Research Laboratory

0

20

40

60

80

100

120

140

MC

-YR

Nod

ular

in-R

MC

-RR

MC

-LR

MC

-LA

MC

-LY

MC

-LF

C2D

5-M

C-L

R (S

UR

)

% R

ecov

ery

LFB LFSM-GW source LFSM-SW source

Method 544 Performance Datafortified at 18.8-100 ng/L

LFB QC CriteriaLFSM QC Criteria

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Aqueous & Extract Holding Time Study (n=4)Chlorinated tap water, fortified at 188-1000 ng/L

0

20

40

60

80

100

120

MC-YR Nodularin MC-RR MC-LR MC-LA MC-LY MC-LF

Mea

n R

ecov

ery,

%

Day Zero Aqueous Day 14 Aqueous Day 28 AqueousDay Zero Extract Day 14 Extract Day 28 Extract

Demonstrates: MCs stable with preservatives for 28 days in aqueous samples MCs stable in extracts for 28 days after extraction

Office of Research and DevelopmentNational Exposure Research Laboratory

Method 544 DLs and LCMRLs (ng/L)Analyte

ng/LDL LCMRL HRL

MC-LR 4.3 6.6 21MC-RR 1.2 5.6MC-LA 4.0 2.9MC-YR 4.6 22 MC-LY 2.2 4.6MC-LF 3.4 3.5NOD 1.8 7.3

DLs based on precision only 7 Laboratory fortified blanks (LFBs) Calculated using student t-test For compliance methods, labs have to

demonstrate detection limits (DLs)

LCMRLs based on precision and accuracy 4 LFBs at 7 concentrations Lowest true concentration for which the future recovery is predicted

to fall, with high confidence (99%), between 50 and 150% recovery Multi-lab LCMRLs used to determine minimum reporting level (MRL) For UCMR, labs do not demonstrate LCMRL - only confirm accuracy

and precision at MRL

LCMRL = Lowest concentration minimum reporting level

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Multi-Laboratory Study

Typically 4-6 labs participate Voluntary participation

Labs provide performance data using draft method LRBs LFBs LFSMs LCMRLs

Evaluate multi-lab data Analytes may be dropped or QC criteria adjusted if

necessary May illuminate where method clarifications needed

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Conclusions

Many parameters evaluated to produce rugged standardized methods that are accurate and precise for use in the UCMR or compliance purposes

Drinking water methods take 2-3 years to develop

Method published on-line at https://www.epa.gov/water-research/epa-drinking-water-research-methods

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EPA TOUR SNEAK PEAK:Cyanotoxin Ambient Water Methods

EPA Method 544 Modifications for Ambient Water6 Microcystin congeners and Nodularin in DW 13 Microcystin congeners and NodularinSPE-LC/MS/MS Filter pore size increased

Sample volume reduced by 5xInternal Standard added

EPA Method 545 Modifications for Ambient WaterCylindrospermopsin and Anatoxin-a in DW No major modificationsDirect aqueous injection LC/MS/MS (no extraction)

Effects of Copper Sulfate and Oxidant Exposure on Cyanobacterial Cells; Methods Employed Include Toxin Analyses by ELISA, Chlorophyll-a Analyses by Fluorometry, and Cell Analyses by Flow Cytometry

Nick Dugan

ADDA ELISA, Cylindrospermopsin ELISA and MMPB Drinking Water Method Development Steve Wendelken

Cultivation of Cyanobacteria & in vitro Toxicity Assay of Cyanotoxins Armah de la Cruz

Application of the MMPB Method for Measurement of ‘Total’ Microcystin Congeners Toby Sanan

LC/MS/MS Methods for Cyanotoxins in Ambient Water Jody Shoemaker