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www.eatonanalytical.com Algal Toxins – Analytical Issues and What’s on the Horizon? October 2015 Monrovia South Bend
www.eatonanalytical.com
Algal Toxins – Analytical Issues and What’s on the Horizon?
October 2015
Monrovia South Bend
Cyanotoxins
Produced by Cyanobacteria Surface issue - freshwater
and marine ecosystems Blue-green algae Must have high nutrient
concentrations and high light intensity to ‘bloom’
Water flow and temperature USEPA has issued Health
Advisories for Microcystin and Cylindrospermopsin
Microcystin LR Over 90 identified Microcystins Microcystin LR most toxic USEPA 10-Day Drinking Water Health
Advisory June 2015 (total microcystins) Based on body weight, consumption WHO Limit 1 ug/L (2003) Total Microcystins will
be in UCMR4
Microcystin LR
Cyanobacteria Sources Microcystis Anabaena Planktothrix Anabaenopsis Aphanizomenon
Cylindrospermopsin
Health Effects (same as Microcystin) Abdominal Pain Vomiting and diarrhea Liver problems Pneumonia Dermatitis Kidney Damage Potential Tumor Growth
Cylindrospermopsin
Cyanobacteria Sources Cylindrospermopsis Aphanizomenon Anabaena Lyngbya Rhaphidiopsis Umezakia
USEPA 10 day health advisoryWill be in UCMR4
Neurotoxins – Anatoxin ACauses tingling, burning, numbness, drowsiness, incoherent speech, salivation, respiratory paralysis leading to deathOn proposed CCL4 list for further evaluation (will be in UCMR4)Sources Anabaena Aphanizomenon Oscillatoria Phormidium Raphidiosis
Neurotoxins - Saxitoxins
Paralytic Shellfish poisonCaused by Shellfish eating dinoflagellates‘Red Tide’ eventsSources Aphanizomenon Lyngbya Cylindrospermopsis Anabaena
NodularinPeptide hepatotoxinSimilar effects as MicrocystinProduced by Nodularia spumigenaFound in brackish water
I have a bloom, but do I have a problem?
Algae Type Not all Algae produce Cyanotoxins
Taste and Odor May be MIB/Geosmin only
Be Proactive With proper treatment you can prevent
an event
Analytical Methods
Algae IdentificationAlgae Enumeration
Analytical Methods
1Enzyme-Linked ImmunoSorbent Assay 4Gas Chromatography/Mass Spectrometry2Protein-Phosphatase Inhibition Assay 5Liquid Chromatography/Mass Spectrometry3High Pressure Liquid Chromatography-Diode Array Detector 6Liquid Chromatography/Tandem Mass Spectrometry
Microcystins Nodularin Cylindrospermopsin Anatoxin-a Saxitoxin
ELISA1 X X X XELISA-ADDA XPPIA2 X
HPLC-DAD3
HPLC-FLUORESCENCE
X X X X
XGC/MS4 X X
LC/MS5 X X X X X
LC/MS/MS6 X X X X M
Sample Collection and Preparation
Collection, Preservation, and Storage Glass bottles preferred, but allow for expansion if freezing Quenching by adding Sodium Thiosulfate or Ascorbic Acid Other preservatives may be required – see EPA 544 and 545 Freeze or store at 4 degrees C Keep away from light
Toxins present in the water (free, dissolved, or extracellular) Without lysis you only determine extracellular toxins
Cell Lysis – Total Concentration Freeze-thaw cycles Sonication
Sample Collection and Preparation
Filtering Compounds may be absorbed
Matrix Interferences Organic matter
Solid Phase Extraction (SPE) Concentrates the samples for lower
Minimum Reporting Levels (MRLs)
Required by EPA 544
Analytical Methods - ELISA Cost – Relatively
inexpensive compared to LC/MS/MS
Specificity – May have false positives that need to be confirmed by LC/MS/MS
Interferences – suspended solids, other matrix effects
Samples serial diluted Sensitivity – 0.1-0.15 ug/L
ELISA Methods Have Different Cross Reactivity to Various MCs
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Assigned valuesTest n SW-LR DW-LR DW-LA SW-LA
LC-MS 1.99 1.22 0.61 0.6
ELISA-ADDA 1.77 1.25 1.1 1.24
ELISA-DM 1.94 1.24 0.58 0.59
ELISA-Envirologix 1.95 1.44 0.7 0.73
Ave % recoveryLC-MS 13 99% 102% 110% 100%
ELISA-ADDA 18 101% 100% 99% 101%
ELISA-DM 8 100% 102% 103% 100%
ELISA-Envirologix 8 99% 100% 103% 100%
Rel Std DeviatonLC-MS 25% 37% 49% 30%
ELISA-ADDA 19% 16% 27% 27%
ELISA-DM 17% 21% 24% 20%
ELISA-Envirologix 10% 20% 26% 16%
Analytical Methods - ELISA
Mean +/- 2 std deviations for 3 years worth of data
99 101111
98 96 95
139.2145.4 144.3
130.4123.0 124.9
59.7 55.8
77.064.7 68.0 65.9
0
20
40
60
80
100
120
140
160
LCS1 LCS2 LCS3 MRL_CHK MS MSD
Perc
ent R
ecov
ery
QC Type
Analytical Methods – LC/MS/MS
EPA Method 544 – Microcystins and Nodularin 6 Microcystins – LA, LF, LR, LY, RR, YR
Special preservatives• Buffering reagent – Trizma• Antimicrobial – 2-Chloroacetamide• Dechlorinating reagent – Ascorbic Acid• Metals inhibitor - EDTA
Surrogate added to 500 ml sample with cell lysing SPE used with concentration step Analysis by LC/MS/MS Sensitivity – 2.9-22 ng/L (0.0029-0.022 ug/L)
Analytical Methods – LC/MS/MS
EPA 544 Chromatogram
Analytical Methods – LC/MS/MS
EPA Method 545 – Anatoxin-a and Cylindrospermopsin Preservatives
• Acidic microbial inhibitor – Sodium Bisulfate• Dechlorinating agent – Ascorbic Acid
Internal Standards added prior to Direct Injection LC/MS/MS with Electrospray Ionization Sensitivity - 0.018-0.063 ug/L
Analytical Methods – LC/MS/MS
EPA 545 Chromatogram
Analytical Methods – LC/MS/MS
Combining SPE with on-line injectionHigher MDLs than SPE, but lower than ELISA
Initial Demonstration of Capability for Direct Injection
Analytical Methods – LC/MS/MS
Analytical Methods – LC/MS/MS
Anatoxin-a
Cylindrospermopsin
Microcystin-LR
Analyte Reporting Limit, ug/L
Anatoxin-a 0.02
Cylindrospermopsin 0.05
Microcystin-LA 0.1
Microcystin-LF 0.1
Microcystin-LR 0.1
Microcystin-LY 0.1
Microcystin-RR 0.1
Microcystin-YR 0.1
Nodularin 0.1
Direct injection of 3 cyanotoxins
Analytical Methods – LC/MS/MS
Calibration Curves for 3 CyanotoxinsCylindrospermopsin0.05 – 2 ug/L
Anatoxin-A0.02 – 1 ug/L
Microcystin-LR0.05 – 2 ug/L
Analytical Methods - Comparison
ELISALC/MS/MS
EPA 544 EPA 545
Characteristic Measure toxins in groups Measure individual variants
Quantification Semi-quantitative Quantitative
Sample volume <5 mL 500 mL <5 mL
MRLs 0.1-0.15 ug/L 0.003-0.02 ug/L 0.02-0.06 ug/L
Turnaround time Fast Slowest Slower
Initial investment Affordable Expensive
Personnel Training Less More
Other aspects Kits available New methods for UCMR4
What Is EPA Currently Thinking for UCMR4 and Algal Toxins?
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Source water MicrocystinsELISA by ADDA
Method
Positive
NDSTOP
LC-MS-MS Confirmation
Positive
NDSTOP
Finished waterLC-MS-MS (544)
How frequent?
What’s the QC?
Do you collect all samples and store them?
Any Questions?
Andy Eaton, PhD, [email protected]
Linda [email protected]
Eurofins Eaton Analytical, Inc.750 Royal Oaks Drive
Monrovia, CA 91016 USAwww.eatonanalytical.com
