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
 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 toxins Planktothrix 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
Anabaena Aphanizomenon

Microcystis Cylindrosperomopsis
Planktothrix
 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/
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 0.15-5 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 <0.15-5 ug/L
 Absorbance w photometer @ 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
 http://www.phytoxigene.com/products/
 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-a Cylindrospermopsin Microcystin Saxitoxin
Chlorine Not Effective Effective (at low pH) Effective* Somewhat Effective

Chloramine Not Effective Not Effective Not Effective at Inadequate

normal levels Information

Chlorine Not Effective at Not Effective Not Effective at Inadequate

normal levels normal levels Information
Dioxide

Potassium Effective Data ranges from Not Effective* Not Effective

Effective to Possibly Effective
Permanganate

Ozone Effective Effective Very Effective Not Effective

UV/advanced Effective Effective Effective at High UV Inadequate

Levels* Information
Oxidation

*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