WaterMicro Workshop, 5-6 November 2014, Athens

Analysis of cyanotoxins and its importance in managing emergency events

Triantafyllos KaloudisChair, CYANOCOST (ES 1105 COST Action)

Chemical analysis of cyanotoxins

• What is the purpose ?

Presence of cyanotoxins or other characteristic (e.g toxicity)?

Which compounds? Targeted or non-targeted analysis? In what samples? Detection or quantitation or both? At what concentration levels? Identification? Confidence? Cost?

Chemical analysis: Incredible potential

Ability to detect, identify and quantify chemical compounds in water, at concentrations in the ng/L range.

Example:Lake Marathonas, Athens: 40 million m3.This is equal to about 30 day’s consumption of drinking water in Athens (4,3 million consumers).

Ability to detect 40 g of a compound dissolved and homogenized in the whole water volume of Lake Marathonas (1 ng/L).

Cyanotoxins

Methods of analysis

Localization of cyanotoxins

Cyanotoxins can be inside the cells (endo-toxins), dissolved in water, or adsorbed to suspended matter.

Different extraction/preconcentration/cleanup protocol may be needed for each case.

Analysis of cyclic peptide hepatotoxins:Microcystins (MCs) and Nodularins (NOD)

Microcystins

Nodularins

Analysis of cyclic heptide hepatotoxins(Microcystins and Nodularins)

Methods that can be used:

High Performance Liquid Chromatography with Ultra-Violet Detection (HPLC-UV, HPLC-PDA).

Liquid Chromatography coupled to Mass Spectrometry LC-MS, LC-MS/MS, LC-QTOF etc.

Enzyme Linked Immunosorbent Assays (ELISA)

Protein Phosphatase Inhibition Assay (PPIA).

With chromatographic methods (HPLC-UV, LC-MS) anextraction/preconcentration/cleanup step is needed fordeterminations at low concentrations (< 1 μg/L)

Microcystins-Nodularins in water (Chromatographic methods)

ISO 20179:2005. Water Quality –Determination of Microcystins –Method Using Solid Phase Extraction (SPE) and High Performance Liquid Chromatography (HPLC) with Ultraviolet (UV) Detection.

The sample preparation step is almost the same for LC-MS analysis.

Endotoxins are determined separately, after extraction of the filter with methanol/sonication and clean-up with SPE.

A 1000-fold pre-concentration can be achieved (500 ml to 0,5 ml).

Solid Phase Extraction

HPLC of Microcystins

Optimized fast HPLC chromatogram of a standard solution of MC-RR, MC-YR, MC-LR and MC-LA.Column: Zorbax XDB C18 50mm x 4.6mm, 1.8μm, flow rate=2ml/min, Gt = 4min, Rt (MC-LA) = 2,49 min, Resolution (YR-LR) = 1,57

Triantis et al. (2010), Toxicon 55, 979-989.

LC-MS/MS (triple quadrupole)

Picotti and Aebersold (2012), Nature Methods 9, 555–566

Selected Reaction Monitoring (SRM): Both Q1 and Q3 are set to a selected mass, allowing only a distinct fragment ion from a certain precursor ion to be detected. Multiple Reaction Monitoring (MRM): Q1 and/or Q3 is set to more than a single mass.Advantages: Maximum sensitivity (S/N), low detection limits, accuratequantification over wide concentration ranges, identification.

Microcystins and Nodularin by LC-MS/MS

(a) Standard mixture (b) Real sample (lake water).

LC-MS/MS method performance

Kaloudis et al. (2013), J. of Hazardous Materials, 263P, 105-115.

ELISA kits for MCs and NOD

• Intended for quantitative screening.

• Commercially available.

• Microplate or tube formats.

• No sample preparation.

• Limits of detection comparable to HPLC and LC-MS.

• Fast, multiple samples.

• Consumables is the main cost.

ELISA kits for MCs and NOD

Competitive ELISA assays.Anti-ADDA or anti-MC-LR antibodies.

ELISA kits for MCs and NODMatrix Effects

D= Ultra-pure water, T=Tap water, L= Surface Water

Kit A Kit B

ELISA kits for MCs and NODMatrix Effects

ELISA Methods for cyanotoxins (MCs, NOD, CYN, ANX).

Strong matrix effects (e.g. surface waters, methanolic extracts).

Cross-reactivity should be taken intoconsideration.

False positives / negatives can be >20%

In-house validation is needed.

Verification with LC-MS/MS is needed for “positive” samples especially where there is limited information about the sample (history or other analysis).

ELISA Methods for cyanotoxins: Caution !

Protein Phosphatase Inhibition Assay (PPIA) for MCs and NOD

PPIA is based on the inhibition of protein phosphatase (e.g. PP 1 or 2A) by MCs and NOD. Other PP inhibitors may be present (e.g. okadaic acid).

In the test, p-nitrophenyl-phosphate is converted to p-nitrophenol by PP2A. The product absorbs at 405 nm.

Available commercially as a kit, in microplate format.

No sample preparation for water samples.

LOQ = 0,25 μg/L.

Fast (less than 1 hour).

Intended for quantitative screening of samples.

Matrix effects, cross-reactivity should be considered.

Correlations between individual MCs,Total MCs by LC-MS/MS and PPIA resultsin Lake Marathonas samples.

Fitted line plot of PPIA vs Total MCs by LC-MS/MS (PPIA=0.04134+1.298 Total MCs,r2=0.899) for positive samples from LakeMarathonas.

PPIA vs LC-MS/MS in real samples:Correlation

Kaloudis et al. (2013), HAZMAT, 263P, 105-115.

Analytical protocols

Purpose: To maximize efficiency of monitoring.

Use of screening (ELISA and PPIA).

Limited number of samples to LC-MS/MS.

Protocol was validated.

Triantis et al. (2010), Toxicon 55, 979-989.

Determination of anatoxin-a in water by LC-MS/MS

Phenylalanine is isobaric to anatoxin-a, it has similar chromatographic retention characteristics and has a similar fragmentation pathway, producing isobaric secondary ions during MS/MS spectrometric detection.

Method summary:Extraction from water by SPE, using Porous Graphitic Carbon cartridges at pH=10,5.Isotopically labelled phenylalanine-d5 was used as internal standard.Separation of anatoxin-a and phenylalanine with C18 column.Limit of detection was 1 ng/L.Method meets validation criteria.Dimitrakopoulos et al. (2010), Anal Bioanal Chem 397:2245–2252

On-line systems for real-time monitoring

Shi et al. (2013), ES&T 47, 4434-4441.

Automated Online Optical Biosensing System for Continuous Real-Time Determination of Microcystin-LR with High Sensitivity and Specificity: Early Warning for Cyanotoxin Risk in Drinking Water Sources

What is measured? (Dissolved toxins, endo-toxins, adsorbed on suspended matter etc).

Traceability:

Purity of standards

Determination of concentration by absorbance rather than by weight.

Lack of reference materials.

No proficiency testing schemes.

Only one standardized method (ISO 20179).

Metrological issues in cyanotoxins analysis

Method Standardization - Laboratory Accreditation

Laboratory for Environmental Analysis - Services

The Laboratory of Environmental Analysis – Services of NCSR Demokritos achieved accreditation (ISO 17025) for the analysis of cyanotoxins in water.

The laboratory shares it’s expertise through the CYANOCOST network in order to contribute to harmonization of methods in Europe.

Brazil: The Itaparica dam case

Hydroelectric dam constructed in 1988. The town of Paulo Alfonso (ppl. 213000) used it as

drinking water supply (after filtration-chlorination). Epidemic of gastroenteritis and diarrhea started in

March 1988. 2000 gastroenteritis cases, 88 deaths reported

during a 42-day period.

Cyanobacteria (Anabaena, Microcystis) present at extremely high levels.

No other toxic agent was detected (pathogenic bacteria, viruses, pesticides, heavy metals etc).

No cyanotoxin analysis was carried out at the time.

Texeira et al., (1993), Bulletin of PAHO, 27(3), 244-253.

Brazil: The Caruaru tragedy In February 1996, in a dialysis clinic, 116 of 131 patients

dialyzed over 1 week had serious symptoms and 100 of them developed liver failure. 52 died from “Caruaru Syndrome”.

Microcystins (MCs) were detected in dialysis water and in the filters of the dialysis unit. MCs were detected in blood and liver samples postmortem. Later analysis showed also cylindrospermopsin (CYN) in the filters of the dialysis unit.

The water reservoir used for water supply had a long history of cyanobacterial blooms.

The clinic was not connected to the water supply network.

“Unfinished” water was trucked from the WTP to the clinic.

The Brazilian Health Ministry enforced the regulation of cyanotoxins in water quality control.

Jochimsen et al., (1998), NEJM, 338(13), 873-878.

Australia: The Palm Island “Mystery Disease”

In November 1979, Palm Island, Queensland, major human poisoning associated with drinking water.

148 people, mainly children, were hospitalized with serious symptoms of gastroenteritis.

The drinking water reservoir (Solomon Dam) had been previously treated with copper sulphate as a response to complaints about taste & odor due to a cyanobacterial bloom.

Presence of a new species Cylindrospermopsisraciborskii in the water reservoir.

Mouse bioassays showed that a potent hepatotoxic agent was present.

Cylindrospermopsin was identified and structurally characterized only in 1992.

Hawkins et al. (1985), Appl. Environ. Microbiol. 50, 1292-1295.Ohtani et al. (1992), J.Amer. Chem. Soc. 114, 7942-7944.De la Cruz et al. (2013), Environ. Science: Processes Impacts, 15, 1979-2003

Cylindrospermopsin

Water tank in Užice. Photo: Milos Cvetkovic

Serbia: The Uzice case

In December 2013 there was a widespread bloom of Planktothrix rubescens in lake Vruciwhich is an artificial water reservoir serving the city of Uzice (ppl. 70.000).

The use of water for drinking and preparation of food was forbidden.

The WTP switched to an alternative source of water (groundwater).

Data regarding the presence of cyanotoxins in water during the episode were not publicized.

MC-RR: Monoisotopic mass: 1037.565796

CH3

Desmethyl MC-RR: Monois. mass: 1023.550171

Analysis of Microcystins in P. rubescens blooms

More than 100 MC variants identified so far. Care should be taken not to miss any MC variants. Non-targeted MS strategies or complementary analysis (eg PPIA) can be valuable.

Planktothrix rubescens forms red colored blooms with possible release of Desmethyl-MC-RR

USA: The Toledo water crisis

On August 2, 2014, the City of Toledo, Ohio, issued a “Do Not Drink – Do Not Boil” water notice, due to the presence of Microcystins.

The notice affected more than 400.000 people. Toledo water utilities abstract water from lake Erie,

which suffers from cyanobacterial blooms.

Cyanobacterial bloom in Lake Erie (satellite image, Sept. 27 2011)

The water notice issued by the City of Toledo

USA: Response to Toledo water crisis

Toledo mayor, Dr. Michael Collins on August 4, when the water ban was lifted PAUL SANCYA / THE ASSOCIATED PRESS thestar.com

Google “Hot Searches”, August 2, 2014

Open data for monitoring of lakes and drinking water supplies (US EPA)

Short – term measures: Continuous monitoring of water supplies

Long –term strategies: limit nutrient runoffs in lake Erie (mainly phosphorus)

Occurrences of toxic cyanobacteria bloomsTo be continued….

Lake Kastoria, Sep. 2014. Photo: C. Avagianos

Climate change

Nutrient runoffs

Eutrophication

Agriculture

Invasive species

Emerging cyanotoxins

Our group:Dr. A. Hiskia, Dr. T. Triantis, Dr. T. Fotiou, Dr. C. Christophoridis, Sevasti Zervou, KorinaManolidi (NCSR Demokritos), Dr. T. Kaloudis (EYDAP SA)Collaborations – joint publications:Prof. D. Dionysiou (U. of Cincinnati), Dr. Armah De la Cruz (USEPA), Dr. M. Antoniou (CUT), Dr. Jussi Meriluoto (Abo Akademi), Prof. M. Moustaka (AUTH), Prof. K. Kormas (UTH), Prof. K. O’Shea (Florida IU), Prof. Virender Sharma (Florida IT), Prof. WeihuaSong (Fudan Univ. China), the CYANOCOST network.

AOP-NanoMat