Cyanotoxin Analysis Methods Oregon Cyanotoxin Rule Considerations - - PowerPoint PPT Presentation

Cyanotoxin Analysis Methods

Oregon Cyanotoxin Rule Considerations Webinar August 22, 2018 Heather Raymond Ohio EPA HAB Coordinator

Analytical Method Comparison & Microcystin Variant Evaluation

• 11 Sites: 4 up‐ground reservoirs, 2 in‐stream

reservoirs, 2 Lake Erie locations, 2 canal‐feeder lakes, and 1 river source

• 22 samples from 2014 selected to help evaluate

spatial and temporal variability within source waters

• Variety of cyanobacteria genera represented
• Each sample analyzed using 5 separate analytical

methods

Methods Evaluated

• Enzyme‐Linked ImmunoSorbent Assay (ELISA)

Microcystin‐ADDA Method

• Liquid Chromatography (LC) – Ultraviolet (UV)
• Liquid Chromatography(LC) –Tandem Mass

Spectrometry (MS/MS) (individual variants)

• LC‐MS/MS (MMPB)
• LC‐UV and LC‐MS (scan for variants without

standards)

Microcystins Testing

• Over 300 microcystin variants
• Standards not available for majority of variants

No “Perfect” Analytical Method for Detecting TOTAL Microcystins

ELISA Microcystins‐ADDA

Enzyme‐Linked ImmunoSorbent Assay (ELISA) Microcystin‐ADDA Method

(detection of antigen using an antibody) – Measures total microcystins (all variants/congeners, based on ADDA) – Highly selective/specific (for ADDA) – Certified by ETV Program – Moderately sensitive (RL: 0.30ug/L) – Suitable for raw & finished water – Quick (~four hours), useful for operational adjustments – Relatively inexpensive – Does not require high end equipment or expertise to run (can (can be used in water system lab) – Does not require pre‐concentration solid phase extraction (SPE) step – Does not provide concentrations of specific microcystin variants – Is an indirect measure of the toxin – Non‐linear curve: may require sample dilution and reanalysis if results out of range – Ohio EPA Standard Method 701.0 & Lab Certification – U.S. EPA Method 546

Liquid Chromatography (LC) – Ultraviolet (UV)

LC‐UV

‐ Liquid Chromatography separates components ‐ Microcystins have UV absorption maxima at 238 nm ‐ Non‐selective detector; co‐eluting interferents prevent accurate identification of components and quantitation ‐ Less expensive than mass spectrometry ‐ Less sensitive than mass spectrometry (average LOQ ~ 0.3 µg/L) ‐ ISO 20179 Standard Method

Liquid Chromatography(LC) –Tandem Mass Spectrometry (MS/MS)

• LC/MS/MS

– Highly specific identification of components (based on standards) – MS can identify a component in the presence of co‐eluting interferents but quantitation may be compromised

• Presence of co‐eluting interferents can act to suppress or enhance response

resulting in analytical bias

• Sensitive (LOQ ~ 0.02 µg/L)

– “Weak” product ion abundance limits sensitivity. Requires pre‐ concentration with SPE to augment sensitivity (LOQs < 0.02 µg/L)

• Preconcentrates NOM too

– U.S. EPA Method 544

• Standard Method‐ includes QA/QC protocols and reduces variability in results

between labs

• Limited to 6 microcystin variants and finished water only

– Expensive and requires highly skilled analysts – Issues with standard availability, purity, and variability

Use of Standard Addition to Account for Matrix Effects in LC‐MS/MS Analysis

LC‐MS/MS MMPB Method

– MMPB (2‐methyl‐3(methoxy)‐4‐phenylbutyic acid) method analyzes the chemically cleaved Adda group common to all microcystin variants – Measures total microcystins (all variants, based on ADDA) – Quick (~2 hours, does not require freeze/thaw or sonication) – Sensitive (0.05 ug/L) – Does not require standards for individual variants – Utilizes 4 PB internal standard – Suitable for raw water, some limitations with finished water – Does not provide data on individual variants – Requires oxidation step – Potential for detection of microcystins disinfection byproducts

Toxicon 104 (2015) 91-101 (Foss & Aubel): Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS)

LC‐UV/PDA & LC‐MS Scan

Uses two LC‐based methods in tandem to independently confirm presence

• f microcystins

– Can detect microcystin variants without standards – No standard methods, expensive, requires complex data‐interpretation, time‐consuming Source: Greenwater Labs

* LC-UV data presented does not include false-positives that were eliminated from total (Based on lack of confirmation with LC-MS methods). Sample # 14 was non-detect using LC-UV.

Results of Method Comparison

Ohio-EPA-10x-166165-E-Fork-Camp-Beach... 3/16/2015 4:38:28 PM Kinetex C18

RT: 3.98 - 20.00 SM: 15G 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 7.27 4.84 4.76 7.53 7.09 4.40 8.51 14.28 14.59 13.29 12.87 12.41 16.69 11.86 16.11 8.36 17.45 17.74 19.28 6.19 15.38 18.95 10.23 9.80 10.96 5.15 5.71 6.71 9.41 NL: 5.49E2 TIC MS Ohio-EPA-10x- 166165-E-Fork- Camp-Beach- MC-MSMS- 031615-2 RT: 4.23 - 19.56 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) 2000 4000 6000 8000 10000 12000 14000 16000 uAU 16.65 17.13 16.14 17.81 15.47 18.27 15.04 8.14 4.40 7.27 5.06 8.79 7.51 7.14 4.78 9.70 9.27 6.56 14.33 6.08 14.03 5.76 10.06 10.74 11.17 13.61 11.70 12.11 NL: 1.73E4 Channel A UV Ohio-EPA-10x- 166165-E-Fork- Camp-Beach-MC- MSMS-031615-2

UV chromatogram with multiple peaks, most not corresponding to MCs (SPE was used) MS/MS TIC & individual variant chromatograms Analysis: Amanda Foss, Greenwater Labs

Results of LC‐MS/MS MMPB and Individual Variant Analysis Compared to ELISA

Inland Lake Microcystin Variants (Planktothrix) MC‐Variant Site 1 6/16/14 Site 2 6/16/14 Site 2 9/2/14 [DAsp3] MC‐RR 5.3 6.1 17.5 [Dha7] MC‐LR 1.1 1.4 1.5 MC‐YR 0.2‐0.6 0.2‐0.6 1.2 MC‐RR 0.1‐0.3 Inland Lake Microcystin Variants (Mixed Bloom) MC‐Variant Site 1 6/18/14 Site 2 6/18/14 Site 2 7/9/14 Site 3 6/30/14 [Dha7] MC‐RR 2.9 3‐9 1.0 0.08 MC‐RR 1.4 39 1.0 0.01‐0.03 MC‐YR 1.1 15 1.0 MC‐LR 4.0 67 2.4 0.55 [DAsp3] MC‐LR 0.6 18 0.4 0.03 [Dha7] MC‐LR 3.6 1.0 0.05 MC‐WR 0.2‐0.6 0.2‐0.6 MC‐LA 0.2‐0.6 MC‐LY 0.2‐0.6 6 0.2‐0.6 0.10

MC‐Variant Lake Erie Upground Reservoir In‐stream Reservoir Canal Lake Stream MC‐YR

Y Y Y Y

[Dha7] MC‐LR

Y Y Y Y

[DAsp3] MC‐RR

Y Y Y Y

MC‐LR

Y Y Y

MC‐RR

Y Y Y Y

MC‐LY

Y Y Y Y

MC‐WR

Y Y Y

[DAsp3] MC‐LR

Y Y

MC‐HilR

Y

MC‐LA

Y Y

[Dha7] MC‐RR

Y Y

MC‐FR

Y

[DAsp3] MC‐FR

Y

6.9 min 1049.5 m/z

Y

7.5 min 1029.5 m/z

Y

8.7 min 1043.5 m/z

Y

Microcystin (MC) Variant Distribution by Source Type

MC‐LF and Nodularin, which are included in USEPA Method 544, were not detected (MC‐LF and additional MC variants have been detected in follow‐up studies).

Key Findings

• 16 different MC‐variants were detected
• MC‐LR was only detected at 5 of 11 sites (45%)
• Most common variants were: MC‐YR, [Dha7] MC‐LR and

[DAsp3] MC‐RR

• LC‐PDA methods prone to interference, potential for false

positives and false negatives

• LC‐MS/MS MMPB method helped confirm ELISA results
• 91% of samples had MC‐variants not detectable by U.S. EPA

Method 544 (including dominant MC‐variant in some samples)

• LC‐MS/MS individual variant analysis under‐reported total

microcystins, based on MMPB and LC‐UV/MS scan data

ELISA MC‐ADDA Matrix Interference Studies

Treatment Chemical Microcystins – ADDA ELISA Assay Tolerance (< / = ) Sodium Carbonate (Soda Ash) ≤25 gpg Sodium Hexametaphosphate ≤250 ppm Sodium Silicofluoride ≤10 ppm Aluminum Sulfate1 ≤100 gpg (with pH adjustment within assay tolerance) Calcium Oxide (Lime)1 ≤2000 gpg (with pH adjustment to within assay tolerance) Potassium Permanganate2 ≤10 ppm (with quenching using 1 mg sodium thiosulfate per 1 ml sample) Sodium Chlorite2 ≤10 ppm (with quenching using 1 mg sodium thiosulfate per 1 ml sample) Carbon3 ≤2 ppm with filtering at time of sampling

1Natural pH of solution outside assay tolerance, tolerance levels determined after pH adjustment 2 Oxidizers degrade microcystins, tolerance determined after quenching 3 Tolerance level due to effect of carbon on toxin, not assay performance

Lisa Kamp, et. at, 2016. The effects of water sample treatment, preparation, and storage prior to cyanotoxin analysis for cylindrospermopsin, microcystin and

• Saxitoxin. Chemico‐Biological Interactions.

ELISA MC‐ADDA Matrix Interference Studies

Studies by U.S. EPA as part of ELISA MC‐ADDA Method Development for UCMR 4:

• Storage Stability – Holding Times
• Sample Preservation and Container Studies
• Matrix Interference Studies

‐Microcystins Variant Fortified Sample Studies (finished water, raw water, reagent water with chemical addition, etc.) ‐Dilution Experiments (real world raw/finished water samples)

• U.S. EPA Method Validation & Interlab Validation

LC‐MS/MS MMPB Method Finished Water Matrix Evaluation:

• Concern regarding detection of microcystins disinfection byproducts

Analytical Methods Utilized by Ohio EPA

Microcystins (μg/L) Cylindro‐ spermopsin (μg/L) Saxitoxins (μg/L) Anatoxin‐a (μg/L)

Surveillance sampling

ELISA (MC‐ADDA) ELISA ELISA LC‐MS/MS

Repeat sampling in response to a finished water detection

ELISA (MC‐ADDA) LC‐MS/MS LC‐MS/MS LC‐MS/MS ELISA: Enzyme‐Linked Immunosorbent Assay LC‐MS/MS: Liquid Chromatography followed by tandem Mass Spectrometry

Cyanobacteria Screening: Molecular Methods (Multiplex qPCR)

• Multiplex quantitative polymerase chain reaction (qPCR) assay –

identifies and quantifies the presence of genes unique to:

• Cyanobacteria: 16S rDNA
• Microcystins and Nodularin production: mcyE gene
• Cylindrospermopsin production: cyrA gene
• Saxitoxins production: sxtA gene

– Fast: 2‐3 hours – Scalable – Cost‐effective – Utilizes certified reference material – Specific

• Ohio EPA DES 705.0 Method and lab certification
• www.phytoxigene.com (current assay in use)
• Ohio EPA method evaluation study

• Microcystins detected in raw water at 45 PWSs (38%) and mcyE

detected at 57 PWSs (48%)

• Out of 1850 paired PWS samples:
• 100% of microcystins >1.6 µg/L had paired mcyE gene detections.
• 100% of microcystins >5 µg/L had mcyE detections > 5gc/µL
• 90% of microcystins detections >1.6 ug/L had mcyE detections

>5 gc/µL

• Less than 2% of samples (22 sites, 32 samples) had microcystins

detections without mcyE detections:

• 19 of the 22 sites had gene detections in either prior
• r post sampling events
• The remaining three PWSs had only one low level

(0.35 – 0.44 µg/L) microcystins detection in 2016; all had trace mcyE gene copies

qPCR as Screening Tool for Microcystins

Example: Source Water With Consistently High Microcystins & mcyE Concentrations

Microcystins, mcyE

1 10 100 1000 10000 6/1/16 7/11/16 8/20/16 9/29/16 11/8/16 12/18/16

mcyE gene (gc/µL) Microcystins (µg/L)

Example: Phytoplankton Enumeration versus qPCR

500 1000 1500 2000 2500 0.5 1 1.5 2 2.5 3

5/30/16 7/19/16 9/7/16 10/27/16 12/16/16 mcyE (GC/µL) Microcystins (µg/L) Total Cyanobacteria qPCR (GC/µL) Total Cyanobacteria count (cells/µL) ND ND

Microcystins, mcyE Total Cyanobacteria, 16S

5000 10000 15000 20000 25000

1 2 3 4 5 6 7 8 9 10

Jun‐16 Jul‐16 Jul‐16 Aug‐16 Sep‐16 Oct‐16 Nov‐16 Dec‐16

Microcystins, mcyE Total Cyanobacteria, 16S

Microcystins (µg/L) mcyE (GC/µL) 16S (GC/µL)

Example: 16s and mcyE trends

• Microcystins trend with mcyE genes
• Non‐toxic cyanobacteria bloom (16s) in August

ND ND

• sxtA detections triggered response sampling at 33 PWSs

(22%), including many with no historic saxitoxins

• ccurrence and three Lake Erie intakes.
• 15 of those PWSs detected saxitoxins in raw water (12%)
• 6 PWSs had finished water detections (none above thresholds)
• Less than 1% percent of samples had saxitoxins detections

without corresponding gene detections (includes 168 paired inland lakes samples)

• Only one cyrA detection, no cylindrospermopsin

detections in 2016

• cyrA detections at 2 PWSs and cylindrospermopsin

detections at one of those PWSs in 2017

qPCR as Screening Tool for Saxitoxins and Cylindrospermopsin

0.01 0.1 1 10 100 1000 10000 100000 5 10 15 20 25 30 5/30/16 7/19/16 9/7/16 10/27/16 12/16/16

Microcystins (µg/L) mcyE (GC/µL) 16S (GC/µL) Saxitoxins (µg/L) sxtA (GC/µL)

Microcystins, mcyE Saxitoxins, sxtA, 16S

ND

Example: Simultaneous Saxitoxins and Microcystins

0.01 0.1 1 10 100 50 100 150 200 250 300 350 Cylindrospermopsis Planktothrix Aphanizomenon Dolichospermum (Anabaena) Saxitoxins sxtA

Site A B C D1 D2 D3 D4 E1 E2 F

Cyanobacteria (cells/µL) Saxitoxins (µg/L) & sxtA (GC/µL)

Inland Lake Examples: Saxitoxin‐producing Cyanobacteria, sxtA, and Saxitoxins

• Low concentrations of saxitoxins detected in drinking water

(below Ohio EPA threshold) from late July – September, 2015.

• Extracellular saxitoxins predominated all samples.
• 10 different potential saxitoxin‐producing genera found in multiple

habitat zones (pelagic, benthic, periphyton, etc.) in multiple (all sampling) locations.

• Follow‐up qPCR analysis revealed highest sxtA gene copies in

benthic samples.

• qPCR data used to target algaecide application.

Example: Use of qPCR to Help Identify Source

• f Saxitoxins and Target Reservoir Management

qPCR Summary

• mcyE is an effective, specific, screen for microcystins

and may be useful as an early warning tool.

• sxtA is an effective, specific, screen for saxitoxins.
• Multi‐plex functionality of assay works.
• 16S is not an effective screen for cyanotoxins, but

potentially useful for assessing susceptibility

• qPCR is superior to phytoplankton enumeration as a

screen for saxitoxins and provides a more targeted screen for microcystins.

• qPCR data can help inform reservoir management

strategies.

Questions?

Heather.Raymond@epa.ohio.gov (614) 644‐2752 epa.ohio.gov/ddagw/HAB.aspx

Adapted from: Detection and Monitoring of Microcystins in Theory & Practice

Sheela Agrawal, Ph.D., NEORSD Judy Westrick, Ph.D., Wayne State University

NEORSD vs WSU

y = 1.1038x + 1.0934 R² = 0.831

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18

Total MC µg/L by WSU Total MC µg/L NEORSD

Total MC Data WSU vs NEORSD by LC/MS/MS

• WSU > NEORSD
• Could be a result of

method flexibility modifications, standard accuracy, purity, etc

NEORSD

Method 701.0/546 vs. 544

2 4 6 8 10 12 14 16 18 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Method 701.0/546 vs. NEORSD and WSU Method 544

Microcystin 701.0/546 NEORSD 544 WSU 544

MC concentration (ppb) Sample ID

Standard Accuracy

Company Name MC Standard APExBIO MC-LR Beagle BioProducts MC-LR, RR, YR, LA Cayman Chemicals MC-LR, LA, RR Cyano BioTech GmbH MC-LR, RR, YR, LW, LF, [D-Asp3, E-Dhb7]MC-RR National Resource Council Canada MC-LR, RR Sigma-Aldrich MC-YR, RR, LA, LW [Dha7]MC-LR, [D-Asp3, E- Dhb7]MC-RR

• Known issues:
• evaporation
• degradation
• methylation
• Non‐uniform dissolution
• Standardize MC concentrations using

UV238

• Found discrepancies between labelled

and measured concentrations, between lots

• Could result in over/underestimation
• f congener concentrations

20% 35% 50% 65% 80% 95% 110% 125% 140% 155% 170% 2211406 2211406 2211406 2211406 2211406 2211406 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 2211407 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251411 7251412 7251412 7251412 7251412 7251412 7251412 L221407 L221407 L29994 L29994 L29994 L29994 L30354

Standard Analysis MC‐RR

% Recovery of MC Standard

Lot number

Standard Purity

Enzo Standard Lot Number Observed Impurities % Impurities MC-LR 02211428 DAsp3MC--RR 1% MC-LW L30332 MC-YR L30158 MC-HtyR 2% DAsp3MC-RR L30311 MC-HtyR L30287 MC-YR, MC-LR, MC-D- Asp3MC-LR 14% Nodularin 0614420 MC-LY L30309 DAsp3MC-LR L30336 MC-LA L30307 MC-RR 0221407 MC-D-Asp3-RR 2% MC-LF L30373 MC-LW 2% MC-HilR L30372 MC-WR L30310

Enzo Life Science standard purity summary

ELISA Cross‐Reactivity

• Individual congeners run via

ELISA; measured in MC‐LR equivalents

• Congeners potentially
• verestimated/

underestimated relative to MC‐ LR; differed from kit correction factors (Issue with cross‐ reactivity? Standard accuracy?)

• 0.6 ppb MC‐LA, 1.5 ppb MC‐RR

reported as 1 ppb MC‐LR