Main cyanobacterial genera that produce cyanotoxins: Dolichospermum sp. Source: Public Health Authority of the Slovak Republic

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1 The Application of the Chromatographic Methods for the Cyanotoxins Analysis Kurejová E., Nagyová V., Drastichová I., Chomová L., Perczelová E.

2 CYANOBACTERIA Known as blue-green algae, are widely distributed, especially in surface waters Some species produce toxins as secondary metabolites These cyanobacterial toxins are called cyanotoxins Main cyanobacterial genera that produce cyanotoxins: 1. Microcystis sp. 2. Aphanizomenon sp. 3. Anabaena/ 4.Cylindrospermopsis sp. Dolichospermum sp. Source: Public Health Authority of the Slovak Republic

3 CYANOTOXINS According to Chemical Structure cyclic peptids microcystins (MCST), nodularins alkaloids anatoxin, saxitoxin lipopolysaccharides According to Influence of the Organism neurotoxins - anatoxin, saxitoxin hepatotoxins - MCST, nodularins dermatotoxins lipopolysaccharides cytotoxins cylindrospermopsin (CYN)

4 Public Health Authirity of the Slovak Republic participate in the COST Action ES1105: Cyanobacteria booms and toxins in water resources: Occurrence, impacts and mangement (CYANOCOST) Cyanocost Website:

5 Working Groups WG1: Occurrence of cyanobacteria and cyanotoxins including methods for monitoring and analysis WG2: Fate, impact and health effects WG3: Prevention and control measures WG4: End-user and outreach tools, materials and products PREPARING : Handbook 1 Cyanobacterial Monitoring and Cyanotoxins Analysis Handbook 2 Molecular methods Handbook 3 Drinking water treatment and cyanotoxins Handbook 4 Prevention, control and mitigation of cyanobacteria in freshwaters

6 Main Task of UVZ SR: Cyanobacteria Quality of water blooms (Species ofcyanobacteria) Chlorophyll-a Toxicity of blooms and water ph Total nitrogen Total phosphorus Cyanotoxins Microbiological water quality Bloom sample of cyanobacteria, Reservoir Hriňová,

7 Cyanotoxin analysis Cyanobacteria: Diversity of genera-species that are possible cyanotoxin producers. Not all genera species and strains are toxic. There is a shift to molecular biology methods for identification. LC-MS techniques more suitable for analysis.

8 Sample Preparation Water Samples Extracellular toxins Solid phase extraction MCST: SPE columns Supelclean LC-18 CYN: SPE columns ENVI- Carb and Supelclean LC-18

9 Sample Preparation Water Bloom Samples Intracellular toxins Freeze-drying - cell wall disruption Toxin extraction - with an aqueous solution of methanol (90 %) in the ultrasonic water bath

10 HPLC analysis Sample Concentration Filtration of Samples HPLC Analysis

11 HPLC analysis HPLC coupled with diode-array detection 239 nm - microcystins 262 nm - cylindrospermopsin Analytical column: reversed phase C 18 VARIAN ChromSep 250 x 4,60 mm, Particle Size 5 µm Mobile phase: A- acetonitrile+0,05 % (v/v) TFA B - water+0,05 % (v/v) TFA Flow: 1,0 ml/min Column temperature: 20 C Injection volume: 100 µl Run time: 40 min

12 59,2 40,0 Chromatograms 09_29_2009 #3 [m odified by PC1] st.2 UV_VIS_1 m AU WVL:239 nm Inhbit = Off Inhbit = On 3 - M CYST LR - 12, M CYST YR - 11,817 Inhbit = Off Inhbit = On Inhbit = Off 30,0 20,0 1 - M CYST RR - 10, M CYST LA - 18, M CYST LW - 26, M CYST LF - 27,767 10,0 0,0-17,0 m in 7,2 8,8 10,0 11,3 12,5 13,8 15,0 16,3 17,5 18,8 20,0 21,3 22,5 23,8 25,0 26,3 27,5 28,8 30,0 31,3 32,3 FJ #25 [modified by PC1] 2909/2008a UV_VIS_1 180 mau WVL:239 nm : MCYST RR : MCYST YR 3: MCYST LR min -20 0,1 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 Figure 1: Separation of MCST 1,0 µg/l standard Figure 2: Trace of Bloom sample from lake Počúvadlo

13 ND = non-detected

14 Main objective of Cyanocost project is to: a) Manage risk of cyanobacteria and cyanotoxins b) Establish a strong collaboration c) Develop of European Database about this field Microcystins content in treated water in Slovak reservoirs were not detected Highest content of microcystins in raw water and water bloom was detected in reservoir Hriňová In reservoirs Nová Bystrica, Bukovec no water bloom observed

15 1. Jussi Meriluoto and Geoffrey A. Codd: TOXIC Cyanobacterial Monitoring and Cyanotoxin Analysis, Åbo Akademis förlag Åbo Akademi University Press, Tavastgatan 30 C, FIN ÅBO 2005, Finland 2. Dr Triantafyllos Kaloudis: Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management. CYANOCOST - COST Action ES1105, Joint COST WssTP Strategic Conference, April 2012, Brussels