Cyanobacteria and their toxins: ecological and health risks Luděk Bláha, Blahoslav Maršálek and co. Masaryk University, Faculty of Science, RECETOX & Institute of Botany, Academy of Sciences Brno, Czech Republic www.recetox.muni.cz www.cyanobacteria.net Blue green algae (CYANOBACTERIA, CYANOPHYTA) • photosynthetic prokaryota • live at various biotops (water, soil, ice, rocks, lichens …) • cca 3 x 109 years old • formation of the oxygen atmosphere Cyanobacteria - current problem HUMAN ACTIVITIES (agriculture, waste waters…) EU/TROPHICATION (=increased concentration of nutrients) CYANOBACTERIAL MASS DEVELOPMENT Cyanobacterial water blooms – global problem Upper Saranac River, USA Bedetti Lake, Argentina Neuse River, USA Baltic sea, Europe Nové Mlýny, Czech Rep. Yellow sea, China Lake Mokoan, Australia South Africa Cyanos in space Space colonization, oxygen, fuel and biomass production, nutrient acquisition, and feedstock provisions. Astronauts retrieve cyanobacteria samples from the outside of the ISS Extremophiles Astrobiology Chamber used for simulating conditions on the martian surface. Cyanos from space Cyanobacterial bloom in Lake Erie (satellite image, Sept. 27 2011) Satellite sensing of harmful algal blooms in Lake Taihu, China. The Great Lakes over Europe Research on cyanotoxins Merel et.al (2013) Toxicon 76, 118-131 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 Open data for monitoring of lakes and drinking water supplies (US EPA) Google “Hot Searches”, August 2, 2014  Short – term measures: Continuous monitoring of water supplies for toxins  Long –term strategies: limit nutrient runoffs in lake Erie (mainly phosphorus) Toledo mayor, Dr. Michael Collins on August 4, when the water ban was lifted PAUL SANCYA / THE ASSOCIATED PRESS thestar.com Serbia: The Uzice case  In December 2013 there was a widespread bloom of Planktothrix rubescens in lake Vruci which 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. Water tank in Užice. Photo: Milos Cvetkovic Talking about „risks“ of cyanobacteria • RISK = probability of the occurrence of HAZARDOUS event • „Hazardous events“ resulting from eu/trophication of the environment • Primary damage to structure and functioning of ecosystems Secondary signs -> ecotoxicity and toxicity • Ecological „stability“ • Stable and functioning ecosystem • Complex and complicated structure (diversity) Many links (food networks) among organisms = ecosystem functioning • Including „ecosystem services“ to humans: • supplies, regulations, cultural / aesthetic, supporting Complex ecosystem Organisms exist together -> rich network of relationships CYANOBACTERIAL BLOOMS: RISKS BLOOM ECOLOGICAL RISKS TOXINS HUMAN health risks Ecological risk 1: Loss of phytoplankton biodiversity • Anthropogenic changes in the environment (more nutrients - P,N) -> advantage for „some“ phytoplankton organisms Complex communities replaced with „monoculture“ (often Microcystis aeruginosa, Planktothrix sp.) „Monocultures“ have secondary effects • • changes in hydrochemistry (higher pH, transparency) -> further indirect impacts on other organisms Ecological risk 1: Loss of phytoplankton biodiversity Ecological risk 1: Loss of phytoplankton biodiversity Ecological risk 2: Further ecosystem changes • Phytoplankton -> changes in the whole network • Reported examples … • Changes in the consumers communites zooplankton -> fish -> … • Makrophyte disappearance (reed) (shading -> no germination …) -> macrophytes = substrate for other organisms … • • New „expansive“ species • cyanobacterium Cylindrospermopsis raciborskii (?) Water blooms = substrate for „associated bacteria“ Ecological risk 3: Ecosystem catastrophes • Sudden disappearance of the producers „monoculture“ (rapid environmental changes, „infections“ by viruses/phages ) -> Ecosystem collapse • Seasonal changes • Cyanobacterial biomass lysis -> bacterial decay -> loss of O2 -> anaerobic conditions - collapse • Deaths of aquatic organisms (fish …) • Pathogens (anaerobic Clostridium botulinum) CYANOBACTERIAL BLOOMS: RISKS BLOOM ECOLOGICAL RISKS TOXINS HUMAN health risks Ecological risk 4: Cyanobacterial toxins • Cyanobacteria - evolutionary old and important organisms (atmospheric oxygen) G- bacteria (10 mil. Cells / mL) - G- : cell walls contain lipopolysaccharides (LPS, similar to E. coli, Salmonella sp…) Water blooms - several complex problems (see previous slides…) - just one of the problems = toxin production • • Cyanobacteria (Eu)trophication Water blooms Cyanotoxins COOH HO H3C H3 C O N NH O O NH OH OH O HO NH NH2 H N NH H NH + N H2 N H CH3 CH3 O N O H3 C CH3 O NH O NH NH NH O OH NH COOH NH NH NH NH2 C H3 C H3 O C H3 HN O OCH3 H3C O NH NH O NH NH2 CH3 O N NH H3C NH NH O C H3 CH3 O H2C CH3 COOH O OH HO H3 C OH O3SO H3C H H2N O O NH NH NH OH NH2+ OH PROBLEM Selected „known“ cyanotoxins H2N O H NH + N H2 N NH NH OH O NH NH2+ OH COOH NH OCH3 H3C O NH C H3 CH3 O NH O NH HN NH2 Saxitoxin O C H3 Anatoxin-a CH3 O N O H2C CH3 H3C NH NH O3SO H3C H H N H OH O NH O NH O C H3 CH3 NH NH NH Cylindrospermopsin Microcystins Nodularin … peptides COOH O Categorization of cyanotoxins 1. According to the chemical structure - cyclic and linear peptids - alkaloids - lipopolysaccharides 2. According to biological activity mechanisms of toxicity - hepatotoxicity, neurotoxicity, cytotoxicity, irritating, immunotoxicity, genotoxicity … Cyanobacteria Toxins produced Anatoxins, Microcystins, Saxitoxins, LPS's Microcystins, LPS's LPS's Saxitoxins, Cylindrospermopsins, LPS's Cylindrospermopsins, Saxitoxins, LPS's Microcystins, LPS's Aplysiatoxins, Lyngbiatoxin-a, LPS's Microcystins, LPS's Nodularin, LPS's Microcystins, LPS's Anabaena Anabaenopsis Anacystis Aphanizomenon Cylindrospermopsis Hapalosiphon Lyngbia Microcystis Nodularia Nostoc Phormidium (Oscillatoria) Anatoxin, LPS's Planktothrix (Oscillatoria) Anatoxins, Aplysiatoxins, Microcystins, Saxitoxins, LPS's Schizothrix Trichodesmium Umezakia Aplysiatoxins, LPS's yet to be identified Cylindrospermopsin, LPS's THE COMPARIOSON OF TOXICITY OF THE NATURAL TOXINS (i.p. injection, acute rat test, LD50 in µg/kg) Bacteria-cyanobacteria- animals- fungi- plants Amatoxin Amanita phalloides fungus 500 Muscarin Amanita muscaria fungus 1100 Aphanotoxin Aphanizomenon flos-aquae cyano 10 Anatoxin -A Anabaena flos-aquae cyano 20 microcystin LR Microcystis aeruginosa cyano 43 nodularin Nodularia spumigena cyano 50 botulin Clostridium botulinum bacteria 0,00003 tetan Clostridium tetani bacteria 0,0001 kobra Naja naja snake 20 kurare Chondrodendron tomentosum plant 500 strychnine Strychnos nux-vomica plant 2 000 Cyanobacterial EKOtoxicity ? • Isolated microcystins - many toxicological studies HOWEVER: Water blooms are more than microcystins - complex mixtures of many compounds (toxins, ipopolysaccharides, non-toxic components…) - ? accumulated toxicants (metals, POPs ???) • Many studies: tested complex water blooms BUT interpreted as „MCs“ Ecotoxicity of WATER BLOOMS to bacterioplankton - highly relevant question (MCs are evolutionary old … as well as bacteria) - only few studies - in general low toxicity observed Ecotoxicity of WATER BLOOMS to algae (phytoplankton) • Algae = competitors to cyanobacteria - limited data - weak direct toxicity only at high (nonrelevant) concentrations - some studies indicate allelopathy between cyanobacteria & algae (inhibition of growth, specific effects on dormant stages) Ecotoxicity of WATER BLOOMS to zooplankton invertebrates - lower sensitivity than vertebrates variable sensitivity of different (even closely related) invertebrate pecies one of the first hypotheses: „MCs are against predators“ not confirmed - several contras…) (? -> indirect BUT: zooplankton prefers nontoxic strains during feeding fects on development of toxic blooms ?) 20 20 Ecotoxicity of cyanobacteria 0 0 2 4 6 8 10 tim e c on c e ntration 405 m g /l 10 0 12 [day ] 14 16 18 20 22 Reproduction c ontro l BIOM ASS AQ.EXTRACT C18 PERM EATE C18 EL UATE SPINACH 0 0 2 4 Controls Nontoxic biomass (spinach) Complex water bloom c ontro l BIOM ASS AQ.EXTRACT C18 PERM EATE C18 EL UATE SPINACH 80 Fraction with MCs ! LOWER TOXICITY ! 40 natality (%) 20 60 Fraction w/o MCs HIGHER TOXICITY 0 0 Extract 2 4 6 8 10 tim e (d ay ) 12 14 16 18 20 22 Ecotoxicity of WATER BLOOMS to fish and amphibians Many studies … toxin accumulations + several effects observed (histhology, biochemistry…) Indirect effects (pH changes, oxygen content) more important in toxicology ! Ecotoxicity of WATER BLOOMS to birds deaths documented (with toxins in bird tissues) limited number of controlled experiments - low direct toxicity to model birds Water blooms stimulate effects of other agents (lead toxicity, immunosupressions) Summary Ecotoxicological risks • Only MCs studied (… results disputable …) • In general: Lower importance of „known“ isolated toxins (such as MCs) ! Complex bloom effects are more important ! ? ? ? ? ? CYANOBACTERIAL BLOOMS: RISKS BLOOM ECOLOGICAL RISKS TOXINS HUMAN health risks MICROCYSTINS • • The most studied and most important Produced and present inside cells: • Intracellular: • up to 10 mg/g d.w. of biomass 1% dw -> tons / reservoir • Extracellular (dissolved): up to 10 ug/L • Stable in water column, bioaccumulative (?) MICROCYSTINS Inhibit regulatory protein phosphatases -> tumor promoter -> hepatotoxic • • 70 variants: MC-LR only considered by WHO • chronic TDI: 0.04 ug/kg b.w./day • drinking water guidline recommendation: 1 ug/L Highly toxic to mammals and humans • Ecotoxicology ? Natural function ? • Microcystin synthesis  Non-ribozomal polyketide synthetases 7 genes: 7 enzymes Enzymatic complex catalyzing binding of aminoacids (!!! Very high ATP demand) Microcystin synthesis  Evolution of non-ribozomal polyketide synthetases Evolutionary old genes  Why remained?  Rantala et al. (2004) PNAS 101:568 Microcystins in the Czech Rep. M e d ia n ; B o x : 2 5 % - 7 5 % ; W h is k e r : N o n - O u tlie r R a n g e 8000 7000 4000 3000 2000 1000 0 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 (Water bloom biomass concentrations B P o t (S p re a s h e e t1 1 6 v 1 0 9 c ) … upo xto l several d mg/g dry* 1weight) MC-LR Total MCs ug/g d.w. 38 37 Seasonal variability 2004 dissolved MCs concentration dissolved MCs concentration (ug/L) (ug/L) • dissolved microcystins in the C.R. 19 (water concentrations) 18 5 19 4 36 20 2005 2004 18 3 15 7 2 1 4 0 3 -1 M e d ia n 2 5 % -7 5 % N o n - O u t lie r R a n g e O u t lie r s E x tr e m e s 2 1 6 7 8 m o n th s 9 10 11 2005 0 -1 6 7 8 m o n th s 9 10 11 M e d ia n 2 5 % -7 5 % N o n - O u t lie r R a n g e O u t lie r s E x tr e m e s Reservoir seasonal data In t e r s e a s o n a l v a r ia b ilit y b io m a s s M C ( u g / g d w ) 3500 3000 MC ug/g d.w. Nové Mlýny 2500 2000 Brno 1500 1000 500 0 1999 -5 0 0 Case 1 Case 5 2001 Case 9 2003 Case 13 2004 Case 17 2005 Case 21 medB medN Reservoir spatial variability Microcystins HUMAN HEALTH RISKS EXPOSURE ROUTES EXPOSURE ROUTES CEECHE – Bratislava, 2006 MICROCYSTINS • … brief reminder … 70 structural variants: MC-LR only (about 30-50% of MCs) considered by WHO • Human chronic TDI: 0.04 ug/kg b.w. daily • drinking water guideline recommendation: 1 ug/L (usually accepted in national laws worldwide, ncl. Czech Rep.) • High toxicity - safety risks: manipulation regulated nited Nations - Bacteriological and Toxin Weapons Convention zech Rep. - Law no. 281/2002 Sb. and 474/2002 Sb. MCs in drinking water reservoirs 37 MCs concentraion (µg/L) 36 4 3 2 1 0 ju l 2004: All reservoirs 2004: 27 DW reservoirs WHO recom. for tap waters 1 ug/L aug sept • Tap waters up to 8 ug/L (1999) Bláha & Maršálek (2003) Arch Hydrobiol MCs in drinking water reservoirs 4 .0 3 .5 3 .0 2 .5 2 .0 1 .5 1 .0 0 .5 0 .0 - 0 .5 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 M e d ia n 2 5 % -7 5 % N o n - O u t lie r R a n g e ) /L 4 .0 3 .5 3 .0 2 .5 2 .0 1 .5 1 .0 0 .5 0 .0 - 0 .5 4 5 6 R ok: 2004 R ok: 2005 MC (ug 7 8 9 10 11 4 5 6 7 8 9 10 11 R ok: 2006 R ok: 2007 “TOP” MCs in waters (Czech Rep. 2004-7) Lokalita Velké Žernoseky (pískovna) Nechranice Dubice, Česká Lípa Prostřední, Lednice Lučina České údolí VN Plumlov Dalešice Hracholusky Nechranice Skalka Novoveský Bláhová et al. (2007). CLEAN - Soil, Air, Water 35(4), 348-354. Datum odběru 1.8.2004 31.7.2004 8.9.2004 6.9.2005 19.7.2005 8.8.2005 15.8.2006 14.7.2006 21.8.2006 26.7.2007 22.8.2007 2.10.2007 MC [ug/L] 37.0 19.0 15.1 18.7 17.3 9.3 24.8 16.3 16.3 29.8 19.9 16.3 Risks of MCs in drinking water supplies SIGNIFICANT HEALTH RISKS EXIST ! • To minimize risk • Addopt appropriate technologies and treatments • Establish routine monitoring of MCs during the season • Accumulation of MCs in fish Silver carp Common carp Accumulation of MCs in fish muscle Silver carp liver Common carp Control 30 d 60 d Control 30 d 60 d Risk of MCs in edible fish Silver carp Max. conc. (dose) SC: liver muscle CC: liver 226 ng/g 68 ug 29 8.8 217 65 18.8 5.6 Max. HI 28 3.7 27 2.4 Average conc. (dose) 106 ng/g 32 ug 8.4 2.5 132 39 8.5 2.6 Average HI 13.2 1.1 16.5 1.1 Common carp muscle 100% of food from the contaminated source avg. person: 60kg, food - 300g TDI: 0.04 ug/kg/day MCs in fish [ng/g f.w.] (Czech Republic reservoirs, 2008) Pike perch Amur Carp Catfish Silver salmon • Liver Average Maximum 15.6 22.7 2.02 6.1 0.57 1.8 0 0 4.14 9.5 Muscle 0 0 0 0 0 Exposure to MCs from fish Less (if any) significant health risks RECREATIONAL EXPOSURE Contact dermatitis non-specific (!!!!) responsible agents (? MCs, LPS?) • • Toxins enter the body (MCs risk assessment possible) Risks of MCs: recreational exposure (US EPA R.A.methodology) • Recreation exposure -> significant risks of MCs Lipopolysaccharides ? • Pyrogenicity of LPS significant in water blooms (less in lab cultures) rnardová et al. 08 J Appl Toxicol Toxic cyanobacteria in recreational reservoirs (WHO approach - „preliminary caution“) Cells / mL b/ml 2500000 2000000 1000000 500000 High risks 100000 Swim. not recommended Risk for sensitive Warning HRAZ SOKOLAK 3.5. 15.5. 30.5. 12.6. 26.6. datum 10.7. 24.7. 7.8. 20.8. 3.9. 18.9. datum 100 20000 10000 1000 ROKLE Summary MCs and the health risks MCs present in 80-90% of reservoirs • High MCs concentrations • • All exposure routes pose significant health risks under certain scenarios Recreation, Drinking water Cs accumulated in fish - less important) How to „manage“ toxic blooms Successful solution: Always reservoir-specific Combinations of methods of PREVENTION + REMEDIATION How to manage toxic blooms? • Limit nutrient sources river basin (upstream) in the reservoir • Cyanocides • (chemical, natural - e.g. Humic acids) Flocculants Al(OH)3 … • Biological control (… planktophagous fish) • Others (mechanical removal, ultrasonic …) ample o reservoir rces of nobacteria onies ediment) urces nutrients n the ervoir diments to 3 m kness) Sediment thickness (cm) urces nutrients upstream veral small ns & villages WWTPs) • REMOVAL OF Phosphorus from river basin Lower contamination Bulding (P-free) + improvements of WWTPs Revitalizations (wetlands) • Phosphorus immobilization „within“ the lake Draining the lake: Surface chemistry at sediments Flocculation of P in the river or lake • Aeration towers Mechanical mixing, deep water oxidation • Biological control: manipulation of food chains • Cyanocide application POSSIBLE DRAWBACKS: accidental release of toxins from dead cells  drinking water! • Mechanical collection of water blooms CONCLUSIONS • Eutrophication causes complex risks with complicated management 1) Ecological risks Loss of diversity … followed by losses of functioning • Secondary changes in the environment - hydrochemistry (pH, O2) - loss on natural habitats (makrophytes…) - new conditions (associated bacteria - patogenic ?) • Susceptibility to catastrophes • Direct ecotoxicity of individual (known) cyanotoxins • seems to be less important CONCLUSIONS 2) HEALTH RISKS OF CYANOTOXINS • Lower importance - known toxins (MC) in food chains (fish) MC in drinking water - higher costs needed for management and control Important risk - recreation ! • • New and less explored risks - new toxins (and their mixtures) - LPS, CYN … - water blooms as „sorbents“ of other toxins (metals, POPs) •