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Mitigation of Cyanotoxins

Mitigation of Cyanotoxins. Richard Lorenz City of Westerville & The Ohio State University, Stone Laboratory. IAFP, 2016. Grand Lake St. Marys . OH. The Great Lakes. Lake Erie Microcystis Bloom. Detroit. Stone lab. Toledo. Cleveland. Mitigation of Cyanotoxins Topics.

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Mitigation of Cyanotoxins

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  1. Mitigation of Cyanotoxins Richard Lorenz City of Westerville & The Ohio State University, Stone Laboratory IAFP, 2016 Grand Lake St. Marys. OH

  2. The Great Lakes

  3. Lake Erie Microcystis Bloom Detroit Stone lab Toledo Cleveland

  4. Mitigation of CyanotoxinsTopics • Source Water Detection • Indicators and Monitoring • Analytical Methods for Cyanotoxins • Prevention & Treatment Options • Risks to Food

  5. Source Water Detection • Visual Bloom • Not all blooms are Cyanobacteria • Not all Cyanobacteria blooms produce toxins • Not all blooms are visible or form scums BGA Bloom – No toxins Planktothrix Bloom - Toxins

  6. SourceWater Detection • Satellite • Visible light – true color Spectral Analysis -Phycocyanin

  7. Source Water Monitoring • Microscopic Examination • Grab or concentrated sample w plankton net • ID to Genus and enumeration • Automated FlowCam – particle imaging Anabaena Aphanizomenon Microcystis Cylindrosperomopsis Planktothrix

  8. Source Water Monitoring -Bloom Indicators • Changing Water Quality Parameters • Increased pH, chlorophyll, phycocyanin, turbidity, conductivity, dissolved oxygen, temperature • Grab samples • Continuous data - remotely with probes on Sondes • http://habs.glos.us/map/

  9. Source Water Monitoring • Relationship of Toxins to Taste and Odors • Toxins and T & O events can be related, but notlinked • Cyanobacteria can produce Geosmin and MIB • But so can other organisms - Actinomycetes • Not all Cyanobacteria blooms that produce T & O also produce toxins • Not all toxic blooms produce T & O compounds • T & O event warrants further investigation

  10. Toxin Analytical Methods • Various Options Based on Objective • Screening & field methods • Quantitative/Qualitative lab methods • Standard Methods Evolving • Lack of Standards • 6 of 100+ Mircocystin Variants • Reporting Levels Close to Health Advisories

  11. Sampling • Glass or PETG bottles • Sequestering agent for any oxidants • Refrigerate/Freeze • Preparation • Extracellular toxins • Filter • Intracellular toxins • Lyse: chemical, sonication, freeze thaw

  12. Common Analytical Methods • Enzyme-linked Immunosorbent Assay (ELISA) • Test Strips/tubes • Plate kits • Liquid Chromatography-Ultraviolet (HPLC-UV) • Liquid Chromatography with tandem Mass spectrometery (LC-MS/MS) • Quantitative Polymerase Chain Reaction (qPCR)

  13. Enzyme Linked Immunosorbent Assay (ELISA) Microcystin-ADDA Method • Measures Total Microcystins • Detects all variants based on ADDA group, highly selective • MC Variants not identified • Indirect measurement of antigen using an antibody

  14. ELISA, continued • Suitable for complex samples – Tap & natural waters • No concentration step • Sold in Kits, no high end equipment/expertise • Certified by USEPA, Ohio • Relatively Inexpensive • Quantification based on MC-LR, can over/under report other variants

  15. ELISA Microtiter Plate Kit • Generate Std. Curve • Plate reader, color is inversely proportional to MC conc. • Range 0.15-5 ppb, Reporting Level 0.3 ug/L • Quick ~4 hrs, operational needs • ~$500/kit (42 tests) • MC, Cylindrospermopsin, Anatoxin-a & Saxitoxin

  16. ELISA Based Field Test strips • Screening qualitative test • 30 minutes • Source water chemical lysing adds 20 minutes • 0-5 & 0-10 ug/L range • Strips for Anatoxin-a, MC

  17. ELISA Based Test Kit Tube • Screening Method, semi-quantitative • Lysing required for total MC • ~1 hour • $5-7/test (~$200/kit) • Generate std curve, Reporting Range<0.15-5 ug/L • Absorbance w photometer @ 450 nm • Results confirmed for regulatory use

  18. Liquid Chromatography-Ultraviolet (HPLC-UV) • LC separates components • MC UV absorption at 238nm • Non-selective detector, co-eluting interferences • Less expensive than MS • Less sensitive than MS ~0.3 ug/L

  19. Liquid Chromatography w Tandem Mass Spectrometry LC-MS/MS • Typically require solid phase extraction step • Only tap water • Sensitive to ~0.02 ppb • Variants can be ID, with standards • More expensive than ELISA, LC-UV • Highly skilled analysts • Standard Method USEPA 544 • Limited to 6 MC variants • Standard Method 545 • Anatoxin-a & Cylindrospermopsin

  20. LC-MS/MS MMPB Method • MMPB – 2-methyl-3(methoxy)-4-phenylbutyic acid • Chemically cleaves ADDA group • Total MC, all variants • Natural and tap waters • No freeze thaw or lysing • Quick ~2 hours • Sensitive 0.05 ppb • No standards needed • Confirms ELISA results

  21. Quantitative Polymerase Chain Reaction qPCR • Simultaneously quantifies Total Cyanobacteria along with Genes responsible for Toxin production • Total Cyanobacteria • Based on 16S rDNA gene - correlates with cell counts • Identifies Genes that produce • Microcystins/Nodularin • Cylindrospermopsin • Saxitotoxin • 2-3 hours limited availability • Specific - no gene = no toxin • Proven molecular diagnostic method, very sensitive, high sample throughput, costly equipment and reagents, can be inhibitors to PCR • http://www.phytoxigene.com/products/

  22. Mircocystins Method Study – Ohio EPA • 16 MC variants found • Most common variants: MC-YR, MC-LR, MC-RR • 91% samples had MC-variants not detected by USEPA Method 544 (LC-MS/MS) • LC-MS/MS under reported total MC • LC-MS/MS MMPB agreed with ELISA results - total MC

  23. Mitigation of Cyanotoxins in the Source Water Supply • Prevention • Nutrient control • external/internal loading • Water Column Mixing • Treatment of source water • Algaecide • Avoidance • Alternative source • Manipulating intake depth

  24. Mitigation of Cyanotoxins in the Water Supply • Treatment Options • Cell removal – keep cells intact • Coagulation, flocculation, clarification, filtration, micro or ultra membrane filtration • Toxin Removal/Destruction • Reverse Osmosis • Oxidation • Ozone,Free Chlorine, Permanganate, UV very high dose with hydrogen peroxide • Adsorption • Activated Carbon: PAC or GAC

  25. Effectiveness of Oxidants on Cyanotoxins *dependent on initial cyanotoxin concentration, pH, temperature, and presence of NOM Source: OAWWA/OEPA White Paper on Cyanotoxin Treatment

  26. Mitigation of Cyanotoxins in the Water Supply, continued • Optimize • Current Treatment Processes • Chemical Feed Capacity • Stop any Recycling • Multiple Barriers • Source Water • Cell Remove Intact • Adsorption • Increase Oxidant Dose and Contact Time

  27. Cyanotoxins Potential Impact On Foods • Fish and Shellfish • Organs & Mussel, exceed TDI 0.04 ug/kg/day • Not removed by cooking • Livestock Water, Forage and Feed • Cattle deaths • Crop Irrigation • Process Water • In Store Misting • Supplements

  28. Microcystins in Crops From Irrigation • Spray • Found both toxins and algal cells on leafy crops • Cells remained after 10 days & were not removed by washing • Ground • Root uptake with translocation to shoots in seedlings • MC found: • Lettuce, tomatoes, carrots, rice, rape seed • Forage crops, clover • Varies with crop

  29. Phytotoxic Effects of MircocystinsPhysiological & Morphological Impact • Reduced Seed Germination • Rice, rape seed, alfalfa, lentil, corn, wheat, pea • Reduced Seedling Growth • Potato, bean, cress, spinach, wheat, corn, rice, pea • Reduced Crop Quality and Yield • Inhibits regulatory enzymes (protein phosphatases) • Bioaccumlation

  30. Mitigation of Cyanotoxins Summary • Monitor - Source Water for blooms • Not all visible • Water Quality indicators • Toxin screening • Can occur year round • Test • Analytical methods evolving • Ohio – ELISA MC- ADDA – Total MC • LC-MS/MS – Saxitoxins, Cylindrospermopsin, Anatoxin-a and individual MC variants • may under report Total MC • MMPB capture Total MC • qPCR • Treat - reduce/eliminate Toxins • Remove cells intact • Oxidize - Free Chlorine • Absorb – Activated Carbon • Case by case basis

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