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

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

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

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
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 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
•
http://i.huffpost.com/gen/1950953/original.jpg
BLOOM
HUMAN
health risks
TOXINS
ECOLOGICAL 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“

•
•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

CYANOBACTERIAL BLOOMS: RISKS
•
http://i.huffpost.com/gen/1950953/original.jpg
BLOOM
HUMAN
health risks
TOXINS
ECOLOGICAL 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

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

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
Ecotoxicological risks

CYANOBACTERIAL BLOOMS: RISKS
•
http://i.huffpost.com/gen/1950953/original.jpg
BLOOM
HUMAN
health risks
TOXINS
ECOLOGICAL 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
nNon-ribozomal
polyketide
synthetases
Výsledek obrázku pro ribosome translation
7 genes: 7 enzymes
Enzymatic
complex catalyzing
binding of aminoacids
(!!! Very high ATP demand)

Microcystin
synthesis
Rantala et al. (2004) PNAS 101:568
nEvolution
of non-ribozomal
polyketide
synthetases
n
nEvolutionary
old genes
nWhy remained?
n

•
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?)
•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)
•
•
•
•
•
•
•
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

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
(MCs accumulated in fish - less important)

How to „manage“ toxic blooms
https://classconnection.s3.amazonaws.com/812/flashcards/710812/jpg/cfans_asset_2491631319402378972.
jpg
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?

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

•REMOVAL OF Phosphorus from river basin
•
Lower
contamination Bulding
(P-free) + improvements
of WWTPs Revitalizations
(wetlands)
Výsledek obrázku pro bezfosfátové prací prášky Výsledek obrázku pro dishwasher cartoon Výsledek
obrázku pro rekonstrukce ČOV Výsledek obrázku pro mokřad

•Phosphorus immobilization „within“ the lake
•
http://hgf10.vsb.cz/546/Ekologicke%20aspekty/cviceni/cviceni_lenticky/apli_hydroxi_fe.jpg
http://i.lidovky.cz/09/072/lnorg/PKS2c76c6_Vypustena_Brnenska_prehrada..jpg
Draining the lake:
Surface chemistry at sediments
Flocculation of P
in the river or lake
Výsledek obrázku pro sinice aerační věž

•Aeration towers
Výsledek obrázku pro sinice aerační věž
http://www.asio.cz/img/_/aeracni-veze.foto/grafika1_v8.jpg
http://img.ct24.cz/cache/616x411/article/17/1680/167970.jpg?1274883141
Mechanical mixing,
deep water oxidation

•Biological control: manipulation of food chains
http://lh5.ggpht.com/-3zZvZTCRJdo/U_S8QblX4eI/AAAAAAAAZXY/9rtDJwukmpc/image%25255B6%25255D.png?imgm
ax=800

•Cyanocide application
http://www.uva.nl/binaries/content/documents/personalpages/v/i/p.m.visser/en/tab-one/tab-one/cpitem
%5B7%5D/uva%3Apersonalimage?1355372834724
POSSIBLE DRAWBACKS:
accidental release of toxins from dead cells à drinking water!

•Mechanical collection of water blooms
http://www.envimarket.cz/picture.php?id=1226&w=80&h=80 Výsledek obrázku pro cyanobacterial bloom
surface removal

•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