www.recetox.www.recetox.muni.czmuni.cz

www.cyanobacteria.netwww.cyanobacteria.net

CCyanobacteria and their toxins:yanobacteria and their toxins: ecological and health risksecological and health risks

Luděk Bláha, Blahoslav Maršálek and co.Luděk Bláha, Blahoslav Maršálek and co.

Masaryk University, Masaryk University, FacultFaculty of Science, RECETOXy of Science, RECETOX

& Institute of Botan& Institute of Botany, Academy of Sciencesy, Academy of Sciences Brno, Czech RepublicBrno, Czech Republic

Blue green algae Blue green algae (CYANOBACTERIA, CYANOPHYTA)(CYANOBACTERIA, CYANOPHYTA)

• photosynthetic prokaryota

• live at various biotops (water, soil, ice, rocks, lichens …)

• 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

Cyanobacteria Cyanobacteria -- current problemcurrent problem

Cyanobacterial water blooms Cyanobacterial water blooms –– global problemglobal problem

Upper Saranac River, USA

Lake Mokoan, Australia

Neuse River, USA

Baltic sea, Europe

South Africa Yellow sea, China

Nové Mlýny, Czech Rep.

Bedetti Lake, Argentina

Space colonization, oxygen, fuel and biomass production, nutrient

acquisition, and feedstock provisions.

Chamber used for simulating conditions on the martian surface.

Cyanos in space

Astronauts retrieve cyanobacteria samples from the outside of the ISS

Extremophiles

Astrobiology

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

RResearch on cyanotoxinsesearch on cyanotoxins Merel et.al (2013) Merel et.al (2013) ToxiconToxicon 76, 11876, 118--131131

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 for toxins

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

Water tank in Užice. Photo: Milos Cvetkovic

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.

Talking about „risks“ of Talking about „risks“ of cyanobacteria 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“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 eComplex ecocosystemsystem

Organisms exist together

-> rich network of

relationships

CYANOBACTERIAL BLOOMS: RISKSCYANOBACTERIAL BLOOMS: RISKS

BLOOM

HUMAN

health risks

TOXINS

ECOLOGICAL RISKS

Ecological risk Ecological risk 1: 1: Loss of Loss of phytoplankton phytoplankton biodiversitbiodiversityy

• 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 Ecological risk 1: 1: Loss of Loss of phytoplankton phytoplankton biodiversitbiodiversityy

Ecological risk Ecological risk 1: 1: Loss of Loss of phytoplankton phytoplankton biodiversitbiodiversityy

Ecological risk 2:Ecological risk 2: Further ecosystem changes 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:Ecological risk 3: Ecosystem catastrophesEcosystem catastrophes

CYANOBACTERIAL BLOOMS: RISKSCYANOBACTERIAL BLOOMS: RISKS

BLOOM

HUMAN

health risks

TOXINS

ECOLOGICAL RISKS

Ecological risk Ecological risk 4: 4: CCyanobacterial yanobacterial toxinstoxins

• 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

NH

NHO

O

CH3NH

NH

NHN

NH

O

O

OOCH3

CH3 CH3

H3C O

COOH CH3

COOH

H3C

O

H2C

CH3

CH3

NH

NH2HN

NH

NHO

O

CH3NH

NH

NHN

NH

O

O

OOCH3

CH3 CH3

H3C O

COOH CH3

COOH

H3C

O

H2C

CH3

CH3

NH

NH2HN

H2N

O

O

+H2N

H

OH

OH

NH2+

NH

NHN

NH

H2N

O

O

+H2N

H

OH

OH

NH2+

NH

NHN

NH

N NH NH NH

NH

O

O

OHHH

O3SO

H3C H

N NH NH NH

NH

O

O

OHHH

O3SO

H3C H

Microcystins Nodularin … peptides

Cylindrospermopsin

Anatoxin-a

Saxitoxin

Selected „known“ cyanotoxinsSelected „known“ cyanotoxins

Categorization of cyanotoxinsCategorization of cyanotoxins

11.. AccordingAccording toto thethe chemicalchemical structurestructure -- cycyccliclic aandnd linelinearar peptidpeptidss

-- alkaloidalkaloidss

-- lipopolysalipopolysacccharidcharideses

22.. AccordingAccording toto biologicalbiological activityactivity mechanismsmechanisms ofof toxicitytoxicity

-- hepatotoxicity, neurotoxicity, cytotoxicity,hepatotoxicity, neurotoxicity, cytotoxicity, irritating, immunotoxicity, genotoxicity …irritating, immunotoxicity, genotoxicity …

CCyanobacteriayanobacteria

Toxins producedToxins produced

AnabaenaAnabaena AnatoxinsAnatoxins, , MicrocystinsMicrocystins, , SaxitoxinsSaxitoxins, LPS's, LPS's

AnabaenopsisAnabaenopsis MicrocystinsMicrocystins, LPS's, LPS's

AnacystisAnacystis LPS'sLPS's

AphanizomenonAphanizomenon SaxitoxinsSaxitoxins, , CylindrospermopsinsCylindrospermopsins, LPS's, LPS's

CylindrospermopsisCylindrospermopsis CylindrospermopsinsCylindrospermopsins, , SaxitoxinsSaxitoxins, LPS's, LPS's

HapalosiphonHapalosiphon MicrocystinsMicrocystins, LPS's, LPS's

LyngbiaLyngbia AplysiatoxinsAplysiatoxins, Lyngbiatoxin, Lyngbiatoxin--a, LPS'sa, LPS's

MicrocystisMicrocystis MicrocystinsMicrocystins, LPS's, LPS's

NodulariaNodularia NodularinNodularin, LPS's, LPS's

NostocNostoc MicrocystinsMicrocystins, LPS's, LPS's

PhormidiumPhormidium ((OscillatoriaOscillatoria)) AnatoxinAnatoxin, LPS's, LPS's

PlanktothrixPlanktothrix ((OscillatoriaOscillatoria)) AnatoxinsAnatoxins, , AplysiatoxinsAplysiatoxins, , MicrocystinsMicrocystins, , SaxitoxinsSaxitoxins, LPS's, LPS's

SchizothrixSchizothrix AplysiatoxinsAplysiatoxins, LPS's, LPS's

TrichodesmiumTrichodesmium yet to be identifiedyet to be identified

UmezakiaUmezakia CylindrospermopsinCylindrospermopsin, LPS's, 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

CyanobacterialCyanobacterial EKOEKOtoxicity ?toxicity ?

• 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 Ecotoxicity of WATER BLOOMSWATER BLOOMS to to bacterioplanktonbacterioplankton

- 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 Ecotoxicity of WATER BLOOMSWATER BLOOMS to to algae (phytoplankton)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 Ecotoxicity of WATER BLOOMSWATER BLOOMS to to zooplanktonzooplankton

Ecotoxicity of cyanobacteria Ecotoxicity of cyanobacteria

Reproduction

Controls

Nontoxic biomass

(spinach)

Complex water bloom

Fraction with MCs

! LOWER TOXICITY !

Fraction w/o MCs

HIGHER TOXICITY

Extract

- Many studies … toxin accumulations

+ several effects observed (histhology, biochemistry…)

! Indirect effects (pH changes, oxygen content)

more important in toxicology !

Ecotoxicity of Ecotoxicity of WATER BLOOMSWATER BLOOMS to to fish and amphibiansfish 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)

Ecotoxicity of Ecotoxicity of WATER BLOOMSWATER BLOOMS to to birdsbirds

? ?

? ?

? ?

? ?

? ?

• Only MCs studied (… results disputable …)

• In general: Lower importance of „known“ isolated toxins (such as MCs)

! Complex bloom effects are more important !

Summary Summary EcotoxicologicalEcotoxicological risksrisks

CYANOBACTERIAL BLOOMS: RISKSCYANOBACTERIAL BLOOMS: RISKS

BLOOM

HUMAN

health risks

TOXINS

ECOLOGICAL RISKS

MICROCYSTINSMICROCYSTINS

• 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 (?)

MICROCYSTINSMICROCYSTINS

• 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 ?

MicrocystinMicrocystin synthesissynthesis

Non-ribozomal polyketide synthetases

7 genes: 7 enzymes

Enzymatic

complex catalyzing

binding of aminoacids

(!!! Very high ATP demand)

MicrocystinMicrocystin synthesissynthesis

Rantala et al. (2004) PNAS 101:568

Evolution of non-ribozomal polyketide synthetases

Evolutionary old genes

Why remained?

Box Plot (Spreadsheet1 16v*1109c)

Median; Box: 25%-75%; Whisker: Non-Outlier Range

konc. MC-LR (biomasa, µg/g DW) Outliers Extremes Suma ALL (biomasa, µg/g DW) Outliers Extremes

19931994

19951996

19971998

19992000

20012002

20032004

2005

rok odběru

0

1000

2000

3000

40007000

8000

MC-LR Total MCs

ug/g d.w.

Microcystins in the Czech Rep.Microcystins in the Czech Rep. (Water bloom biomass concentrations (Water bloom biomass concentrations

… up to several mg… up to several mg/g dr/g dry weight)y weight)

Seasonal variabilitySeasonal variability

• dissolved microcystins in the C.R. (water concentrations)

2005

Median

25%-75%

Non-Outlier Range

Outliers

Extremes6 7 8 9 10 11

months

-1

0

1

2

3

4

517

18

19dis

solv

ed M

Cs c

oncentr

atio

n (

ug/L

)

2004

Median

25%-75%

Non-Outlier Range

Outliers

Extremes6 7 8 9 10 11

months

-1

0

1

2

3

4

518

19

2036

37

38

dis

solv

ed M

Cs c

oncentr

atio

n (

ug/L

)

2004

2005

Interseasonal variability biomassMC (ug/g dw )

medBP

medNMCase 1

Case 3

Case 5

Case 7

Case 9

Case 11

Case 13

Case 15

Case 17

Case 19

Case 21

Case 23

-500

0

500

1000

1500

2000

2500

3000

3500

Reservoir Reservoir seasonal seasonal datadata MC

ug/g d.w.

Brno

Nové Mlýny

1999 2001 2003 2004 2005

Reservoir spatial variabilitReservoir spatial variabilityy

MicrocystinsMicrocystins

HUMAN HEALTH RISKSHUMAN HEALTH RISKS

EXPOSURE ROUTESEXPOSURE ROUTES

EXPOSURE ROUTESEXPOSURE ROUTES

CEECHE CEECHE –– Bratislava, 2006Bratislava, 2006

MICROCYSTINSMICROCYSTINS

… 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 reservoirsMCs in drinking water reservoirs

jul aug sept

0

1

2

3

4

36

37

MC

s c

oncentr

aio

n (m

g/L

)

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

MC

(ug

/L)

Rok: 2004

4 5 6 7 8 9 10 11-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Rok: 2005

4 5 6 7 8 9 10 11

Rok: 2006

4 5 6 7 8 9 10 11-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Rok: 2007

4 5 6 7 8 9 10 11

Median 25%-75% Non-Outlier Range

MCs in drinking water reservoirsMCs in drinking water reservoirs

“TOP” MC“TOP” MCss in waters in waters (Czech Rep. 2004(Czech Rep. 2004--7)7)

Lokalita Datum odběru MC [ug/L]

Velké Žernoseky (pískovna) 1.8.2004 37.0

Nechranice 31.7.2004 19.0

Dubice, Česká Lípa 8.9.2004 15.1

Prostřední, Lednice 6.9.2005 18.7

Lučina 19.7.2005 17.3

České údolí VN 8.8.2005 9.3

Plumlov 15.8.2006 24.8

Dalešice 14.7.2006 16.3

Hracholusky 21.8.2006 16.3

Nechranice 26.7.2007 29.8

Skalka 22.8.2007 19.9

Novoveský 2.10.2007 16.3

Bláhová et al. (2007). CLEAN - Soil, Air, Water 35(4), 348-354.

Risks of Risks of MCs in drinking water MCs in drinking water suppliessupplies

• SIGNIFICANT HEALTH RISKS EXIST !

• To minimize risk

• Addopt appropriate technologies and treatments

• Establish routine monitoring of MCs during the season

Accumulation of MCs in fishAccumulation of MCs in fish

Common carp

Silver carp

Accumulation of MCs in fishAccumulation of MCs in fish

Common carp

Silver carp

Control 30 d 60 d Control 30 d 60 d

muscle liver

Risk of Risk of MCs MCs in edible fishin edible fish

Common carp

Silver carp

Max. Max. conc. conc. (dose)(dose)

Max.Max. HIHI

Average Average conc. conc. (dose)(dose)

Average Average HIHI

SC: liverSC: liver 226226 ngng/g/g

68 ug68 ug

2828 106 ng/g106 ng/g

32 ug32 ug

13.213.2

musclemuscle 2929

8.88.8

3.73.7 8.48.4

2.52.5

1.11.1

CC: liverCC: liver 217217

6565

2727 132132

3939

16.516.5

musclemuscle 18.818.8

5.65.6

2.42.4 8.58.5

2.62.6

1.11.1

100% of food from the contaminated source avg. person: 60kg, food - 300g TDI: 0.04 ug/kg/day

MCs in fish MCs in fish [ng/g f.w.][ng/g f.w.] (Czech Republic reservoirs, 2008)(Czech Republic reservoirs, 2008)

Muscle

Average Maximum

Pike perch 15.6 22.7 0

Amur 2.02 6.1 0

Carp 0.57 1.8 0

Catfish 0 0 0

Silver salmon 4.14 9.5 0

Liver

• Exposure to MCs from fish

Less (if any) significant health risks

RECREATIONAL EXPOSURERECREATIONAL EXPOSURE

• Contact dermatitis

non-specific (!!!!) responsible agents (? MCs, LPS?)

• Toxins enter the body (MCs risk assessment possible)

Risks of Risks of MCsMCs:: recreational exposurerecreational exposure

(US EPA (US EPA R.A.R.A.methodology)methodology)

• Recreation exposure -> significant risks of MCs

Lipopolysaccharides ?Lipopolysaccharides ?

• Pyrogenicity of LPS significant in water blooms (less in lab cultures)

Bernardová et al. 2008 J Appl Toxicol

Toxic cyanobacteria Toxic cyanobacteria in recreational in recreational reservoirsreservoirs

(WHO approach (WHO approach -- „preliminary caution“)„preliminary caution“)

HRAZ

SOKOLAK

ROKLE

datum

100

1000

10000

20000

100000

500000

1000000

2000000

2500000

3.5. 15.5. 30.5. 12.6. 26.6. 10.7. 24.7. 7.8. 20.8. 3.9. 18.9.

b/ml

datum

Warning

Risk for sensitive

Swim. not recommended

High risks

Cells / mL

Summary Summary MCsMCs andand thethe healthhealth risksrisks

• 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)

HowHow to „to „managemanage“ “ toxictoxic bloomsblooms

Successful solution:

Always

reservoir-specific

Combinations

of methods

of PREVENTION

+ REMEDIATION

• 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?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)

• 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

• 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

CONCLUSIONSCONCLUSIONS

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)

CONCLUSIONSCONCLUSIONS