www.recetox.muni.cz www.cyanobacteria.net 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 nadpis MU in Brno Flos Aquae Foundation cyanophyta_anabaena_upr BRNO10_99 Blue green algae (CYANOBACTERIA, CYANOPHYTA) • photosynthetic prokaryota ANABFA cyanophyta_cylindrospermum_upr cyanophyta_anabaena_upr cyanophyta_oscillatoria_upr • live at various biotops (water, soil, ice, rocks, lichens …) flmcy5b cyanophyta_microcystis_upr • cca 3 x 109 years old • formation of the oxygen atmosphere HUMAN ACTIVITIES (agriculture, waste waters…) EU/TROPHICATION (=increased concentration of nutrients) CYANOBACTERIAL MASS DEVELOPMENT VRANOV99 bloomUpperSaranac MUSOV_NMB sin_nove-mlyny-hraz3 Cyanobacteria - current problem Cyanobacterial water blooms – global problem Upper Saranac River, USA Microcystis aeruginosa bloom, Lake Mokoan, Victoria, Austral Lake Mokoan, Australia neuse_cyano_bloom Neuse River, USA Baltic sea, Europe pic1lrg South Africa bloomUpperSaranac2 algpit13 yellow sea Yellow sea, China Nové Mlýny, Czech Rep. earth3 bedetti_lake_argentina Bedetti Lake, Argentina sin_nove-mlyny-hraz 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 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“ • •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) Ecological risk 3: Ecosystem catastrophes 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 Cyanotoxins PROBLEM (Eu)trophication Water blooms N H N H O O C H 3 N H N H N H N N H O O O O C H 3 C H 3 C H 3 H 3 C O C O O H C H 3 C O O H H 3 C O H 2 C C H 3 C H 3 N H N H 2 H N N N H N H N H N H 2 H O O O H N H H O O H O H 3 C H 3 C O H 3 C N H 2 H O N H N O O C H 3 C H 3 N H O O H N H C O O H O O H H 3 C C H 3 H 2 N O O + H 2 N H O H O H N H 2 + N H N H N N H N N H N H N H N H O O O H H H O 3 S O H 3 C H N H O C H 3 Cyanobacteria Microcystins Nodularin … peptides Cylindrospermopsin Anatoxin-a Saxitoxin Selected „known“ cyanotoxins Categorization of cyanotoxins 1. According to the chemical structure - cyclic and linear peptids - alkaloids - lipopolysaccharides n 2. According to biological activity mechanisms of toxicity - hepatotoxicity, neurotoxicity, cytotoxicity, irritating, immunotoxicity, genotoxicity … n n Cyanobacteria Toxins produced Anabaena Anatoxins, Microcystins, Saxitoxins, LPS's Anabaenopsis Microcystins, LPS's Anacystis LPS's Aphanizomenon Saxitoxins, Cylindrospermopsins, LPS's Cylindrospermopsis Cylindrospermopsins, Saxitoxins, LPS's Hapalosiphon Microcystins, LPS's Lyngbia Aplysiatoxins, Lyngbiatoxin-a, LPS's Microcystis Microcystins, LPS's Nodularia Nodularin, LPS's Nostoc Microcystins, LPS's Phormidium (Oscillatoria) Anatoxin, LPS's Planktothrix (Oscillatoria) Anatoxins, Aplysiatoxins, Microcystins, Saxitoxins, LPS's Schizothrix Aplysiatoxins, LPS's Trichodesmium yet to be identified Umezakia Cylindrospermopsin, LPS's THE COMPARIOSON OF TOXICITY OF THE NATURAL TOXINS (i.p. injection, acute rat test, LD50 in mg/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 gallerycobra Strychnos_nux-vomica-4 Clostridium%2520botulinum%2520spores amanita Anatoxin-A, Anatoxin-A(S) nneurotoxic alkaloids n nproduced by a number of cyanobacterial genera including Anabaena, Oscillatoria and Aphanizomenon. n nLD50s from 20 µg kg-1 (by weight, I.P. mouse) making them more toxic than microcystins. A diagram of anatoxin-a homoanatoxin A diagram of anatoxin-a(s) SAXITOXINS nneurotoxic alkaloids nalso known as PSP's - paralytic shelfish poisons - due to their accumulation in seafood n nProduced by marine dinoflagellates and cyanobacteria (but also in others such as Aphanizomenon sp.) n nNumber of STX variants exist saxitoxin 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 Rantala et al. (2004) PNAS 101:568 nNon-ribozomal polyketide synthetases n nEvolutionary old genes nWhy remained? n nHorizontal gene transfer • MC-LR Total MCs ug/g d.w. Microcystins in the Czech Rep. (Water bloom biomass concentrations … up to several mg/g dry weight) Seasonal variability •dissolved microcystins in the C.R. (water concentrations) 2004 2005 Reservoir seasonal data MC ug/g d.w. Brno Nové Mlýny 1999 2001 2003 2004 2005 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, incl. Czech Rep.) •High toxicity - safety risks: manipulation regulated United Nations - Bacteriological and Toxin Weapons Convention Czech Rep. - Law no. 281/2002 Sb. and 474/2002 Sb. • MCs in drinking water reservoirs WHO recom. for tap waters 1 ug/L 2004: 27 DW reservoirs 2004: All reservoirs •Tap waters up to 8 ug/L (1999) Bláha & Maršálek (2003) Arch Hydrobiol MCs in drinking water reservoirs “TOP” MCs in waters (Czech Rep. 2004-7) Bláhová et al. (2007). CLEAN - Soil, Air, Water 35(4), 348-354. 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 Cyprinus%20Carpio4 Common carp Hypophthalmichthys%20(tolstolobik) Silver carp P1010467 P1010521 Accumulation of MCs in fish Cyprinus%20Carpio4 Common carp Hypophthalmichthys%20(tolstolobik) Silver carp Control 30 d 60 d Control 30 d 60 d muscle liver Risk of MCs in edible fish Cyprinus%20Carpio4 Common carp Hypophthalmichthys%20(tolstolobik) Silver carp Max. conc. (dose) Max. HI Average conc. (dose) Average HI SC: liver 226 ng/g 68 ug 28 106 ng/g 32 ug 13.2 muscle 29 8.8 3.7 8.4 2.5 1.1 CC: liver 217 65 27 132 39 16.5 muscle 18.8 5.6 2.4 8.5 2.6 1.1 100% 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) •Exposure to MCs from fish Less (if any) significant health risks RECREATIONAL EXPOSURE 0F1gac009 •Contact dermatitis non-specific (!!!!) responsible agents (? MCs, LPS?) Lipopolysaccharides ? •Pyrogenicity of LPS significant in water blooms (less in lab cultures) • • • • • • • Bernardová et al. 2008 J Appl Toxicol Toxic cyanobacteria in recreational reservoirs (WHO approach - „preliminary caution“) Warning Risk for sensitive Swim. not recommended High risks Cells / mL RECREATIONAL EXPOSURE 0F1gac009 •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 Summary I - 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 (MCs accumulated in fish - less important) Cyanobacterial EKOtoxicity ? •Isolated microcystins - many toxicological studies • •HOWEVER: Water blooms are more than microcystins - complex mixtures of many compounds (toxins, lipopolysaccharides, 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 •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 algae (phytoplankton) - invertebrates - lower sensitivity than vertebrates - variable sensitivity of different (even closely related) invertebrate species - one of the first hypotheses: „MCs are against predators“ (not confirmed - several contras…) BUT: zooplankton prefers nontoxic strains during feeding (? -> indirect effects on development of toxic blooms ?) Ecotoxicity of WATER BLOOMS to zooplankton Ecotoxicity of cyanobacteria Reproduction Controls Nontoxic biomass (spinach) Complex water bloom Fraction with MCs ! LOWER TOXICITY ! Fraction w/o MCs HIGHER TOXICITY JAREK-predni-strana Extract - Many studies … toxin accumulations + several effects observed (histhology, biochemistry…) ! Indirect effects (pH changes, oxygen content) more important in toxicology ! Ecotoxicity of WATER BLOOMS to fish and amphibians - 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) flam opisthotonos Ecotoxicity of WATER BLOOMS to birds ? ? ? ? ? •Only MCs studied (… results disputable …) • •In general: Lower importance of „known“ isolated toxins (such as MCs) ! Complex bloom effects are more important ! Summary II - Ecotoxicological risks Cylindrospermopsin (CYN) Risks of both MCs and CYN are comparable (CYN not regulated, concentrations unknown…) MC CYN LD50 (acute oral toxicity) 6000 mg/kg 5000mg/kg NOAEL 40 mg/kg/den 30 mg/kg/den TDI 0.04 mg/kg 0.03 mg/kg Limit pro pitnou vodu 1 mg/L* 1 mg/L * * 15 mg/L * * * ? … emerging toxins - discovered in tropics (Australia, Florida, New Zealand …) - now reported from Europe … including C.R. Cylindrospermopsin in the C.R. Bláhová et al. 2008 Toxicon • Limit nutrient sources • Cyanocides (chemical, natural - e.g. Humic acids) • Flocculants Al(OH)3 … • Biological control (… planktophagous fish) • Others (mechanical removal, ultrasonic …) river basin (upstream) in the reservoir How to manage toxic blooms? No ideal and universal approach exists - combinations of methos - locality-specific approach How to manage toxic blooms? Example Brno reservoir sources of cyanobacteria (colonies in sediment) Sediment thickness (cm) Sources of nutrients … in the reservoir (sediments up to 3 m thickness) Sources of nutrients … upstream - several small towns & villages (no WWTPs) cov •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) CONCLUSIONS