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

The Great Lakes

Lake Erie Microcystis Bloom Cleveland Detroit Toledo Stone lab

Mitigation of Cyanotoxins Topics Source Water Detection Indicators and Monitoring Analytical Methods for Cyanotoxins Prevention & Treatment Options Risks to Food

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 toxinsPlanktothrix Bloom - Toxins

Source Water Detection Satellite Visible light – true color Spectral Analysis -Phycocyanin

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

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

Source Water Monitoring Relationship of Toxins to Taste and Odors Toxins and T & O events can be related, but not linked 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

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

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

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)

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

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

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

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

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 < ug/L Absorbance w 450 nm Results confirmed for regulatory use

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

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

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

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

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

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

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

Effectiveness of Oxidants on Cyanotoxins Anatoxin-aCylindrospermopsinMicrocystinSaxitoxin Chlorine Not EffectiveEffective (at low pH)Effective*Somewhat Effective Chloramine Not Effective Not Effective at normal levels Inadequate Information Chlorine Dioxide Not Effective at normal levels Not EffectiveNot Effective at normal levels Inadequate Information Potassium Permanganate EffectiveData ranges from Not Effective to Possibly Effective Effective*Not Effective Ozone Effective Very EffectiveNot Effective UV/advanced Oxidation Effective Effective at High UV Levels* Inadequate Information * dependent on initial cyanotoxin concentration, pH, temperature, and presence of NOM Source: OAWWA/OEPA White Paper on Cyanotoxin Treatment

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

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

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

Phytotoxic Effects of Mircocystins Physiological & 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

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