cyanotoxin analysis - cyanocost · 2008 gc-ms 96 130 2008 lc-ms/ms 0 - 2009 lc-ms/ms 43 13 2010...
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Cyanotoxin analysis
Miquel Lürling and Els Faassen
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Why worry about cyanobacteria?
2
Human health risk
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Why worry about cyanobacteria?
3
Krienitz et al 2003
Cattle, pet and wildlife kills
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Cyanobacteria produce potent toxins
4
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Cyanobacterial toxins: Neurotoxins
5
(Homo)anatoxin-a
H2N
N
HN N
CH3
CH3
O
PO
OO
CH3
Anatoxin-a(s)
O
NR1
R4 H
NH
NH2+
+H2N N
R2R3
NH
OH
OH
Saxitoxins
OH
O
NH2HN
H3C
BMAA Involved in ALS,
Parkinsonism, dementia???
Acutely toxic
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Cyanobacterial toxins: Cytotoxins
6
Cylindrospermopsins
Irreversible inhibition protein synthesis, genotoxic, hepatotoxic
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Cyanobacterial toxins: Hepatotoxins
7
Microcystins
Nodularins
Cylindrospermopsins
OCH3
H3C CH3
H3C O
HN
NH
O
COOH
O
NR2
O
CH2
NH
H3C
O
NH
O
NH
COOH
R1
O
NH
NH
HN
NH2
CH3
CH3
(1)
(2)
(3)
(4)
(5)
(6)(7)
Affect liver, kidney, nervous system, some are tumour promotors
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Who is making what?
Toxin Variants
Anatoxins 2 Anabaena, Aphanizomenon, Cylindrospermum, Microcystis, Oscillatoria, Phormidium, Planktothrix, Raphidiopsis
Anatoxin-a(S) 1 Anabaena flos-aquae , An. lemmermannii, An. spirolides
BMAA 1 ??? (diatoms)
Cylindrospermopsins 3 Anabaena bergii, An. planctonica, An. lapponica, Aphanizomenon ovalisporum, Aph. flos-aquae , Aph. gracile, Aph. klebahnii , C. raciborskii, Lyngbya wollei, Oscillatoria sp., Raphidiopsis curvata , R. mediterranea, Umezakia natans
LPS All
Microcystins >80 Anabaena, Anabaenopsis, Aphanocapsa, Aphanizomenon, Cylindrospermopsis, Fischerella, Hapalosiphon, Lyngbya, Microcystis, Nostoc, Oscillatoria, Phormidium, Planktothrix, Rivularia, Snowella, Synechococcus, Woronichinia
Nodularins 7 Nodularia spumigena, N. sphaerocarpa, Nostoc sp. ‘Macrozamia riedlei 65.1’ , Nostoc sp. ‘Macrozamia serpentina 73.1’
Saxitoxins >30 Aphanizomenon, Cylindrospermopsis, Lyngbya, Planktothrix (Alexandrium, Gymnodinium catenatum, Pyrodinium bahamense)
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Toxic / non-toxic
strains
Biomass
Environment NHO
CH3
N NH
NH
-O3SO
H3CNHHN
O
OH
O
+
O
NR1
R4 H
NH
NH2+
+H2N N
R2R3
NH
OH
OH
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How to determine cyanobacterial toxicity
11
NOT
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Health risk CHL-a (µg l-1)
Cyanobacteria (cells ml-1)
Microcystin (µg l-1)
Low < 10 < 20.000 < 10
Moderate 10 -50 20.000 – 100.000 10 - 20
High 50 -5000 100.000 – 10.000.000 20 - 2000
Very high > 5000 > 10.000.000 > 2000
Dutch bathing water guidelines
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ATX < 0.1 µg/l STX < LOQ CYN < LOD NOD < LOD
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Cyanobacteria chlorophyll-a ( g l-1
)
0 50 100 150 200 4000 800012000
Mic
rocystin-c
once
ntr
atio
n (
g l-1
)
0
5
10
15
20
Cyanobacteria chlorophyll-a ( g l-1
)
0 50 100 150 200 4000 800012000
Mic
rocystin-c
once
ntr
atio
n (
g l
-1)
0
5
10
15
20
Col 1 vs Col 3
Cyanobacteria (cells ml-1
)
103 104 105 106 107 108 109
Mic
rocystin-c
once
ntr
atio
n (
g l-1
)
0
5
10
15
20
Low MC < 10
Moderate MC 10-20
<10
10-50
50-5000
>5000
CHLa
<2·104
2·104-105
105-107 >107
Cells ml-1
Microcystins vs cell counts/Chl-a
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Microcystins vs qPCR
mcyB copies g-1
sample dryweight
0 5.0x108 109 1.5x109 2.0x109 2.5x109 3.0x109 2.0x1011 4.0x1011
MIc
rocystin c
once
ntr
atio
n (
g g
-1)
0
20
40
60
80
100
120
Hautala et al., 2013
“However, the confirmation of the toxin presence and the determination of its concentration still relies on biochemical or chemical analytical methods, such as ELISA or LC-MS/MS.” (Pacheco et al 2016 Toxins)
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How to determine cyanobacterial toxicity
Don’t use estimates such as cell count, chlorophyll-a or qPCR:
● Often no relation with toxin concentration
● No discrimination between toxin variants
Immunoassays (ELISA) not always preferred, because:
● Possible cross reactivity with non-toxic compounds
● Too little cross reactivity with toxic compounds
● No information on toxin variants
16
NOT
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How to determine cyanobacterial toxicity
Use reliable analytical techniques
● Include different toxin variants
17
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Variants differ in their toxicity
18
41% 88% 87% 80% 93% 19% 17% 13%
Faassen and Lürling 2013
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Variants differ in their toxicity
19
Faassen and Lürling 2013
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How to determine cyanobacterial toxicity
Use reliable analytical techniques
● Include different toxin variants
● Consider possible bound forms of toxins
20
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Bound microcystins
21
Protein bound MC
Methanol soluble MC
Meissner et al 2013
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Bound BMAA
22
Faassen et al 2016
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How to determine cyanobacterial toxicity
Use reliable analytical techniques
● Include different toxin variants
● Consider possible bound forms of toxins
Perform proper sampling
23
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Inhomogeneous distribution of
cyanobacteria
24
Sampling point
Beach
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Inhomogeneous distribution of toxins
25 Wood et al 2010
(homo)anatoxin-a detected no (homo)anatoxin-a detected
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How to determine cyanobacterial toxicity
Use reliable analytical techniques
● Include different toxin variants
● Consider possible bound forms of toxins
Perform proper sampling
Use the analytical methods properly
26
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Publication year
Quantification method
% positive samples
[BMAA] in
positive samples µg/g DW
2005 LC-FLD 97 968
2008 LC-FLD 100 103
2008 LC-FLD 100 10
2008 GC-MS 96 130
2008 LC-MS/MS 0 -
2009 LC-MS/MS 43 13
2010 LC-MS/MS 0 -
2010 LC-MS/MS 100 0.01
2011 LC-MS 80 1.4
2011 CE-UV 100 402
2012 LC-FLD 100 14
2012 LC-FLD 100 0.29
2012 LC-MS/MS 0 -
2012 LC-MS/MS 0 -
2012 LC-FLD 38 28
2014 LC-MS/MS 100 4.4
The BMAA story in a nutshell
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Publication year
Quantification method
% positive samples
[BMAA] in
positive samples µg/g DW
2005 LC-FLD 97 968
2008 LC-FLD 100 103
2008 LC-FLD 100 10
2008 GC-MS 96 130
2008 LC-MS/MS 0 -
2009 LC-MS/MS 43 13
2010 LC-MS/MS 0 -
2010 LC-MS/MS 100 0.01
2011 LC-MS 80 1.4
2011 CE-UV 100 402
2012 LC-FLD 100 14
2012 LC-FLD 100 0.29
2012 LC-MS/MS 0 -
2012 LC-MS/MS 0 -
2012 LC-FLD 38 28
2014 LC-MS/MS 100 4.4
The BMAA story in a nutshell
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The BMAA story in a nutshell
n = 17
n = 10
n = 5
n = 4
n = 7
Faassen 2014
Appropriate techniques,
correctly used
Appropriate techniques,
not (shown to be) used
correctly
Inappropriate techniques used
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Cyanocost 2015 Workshop BMAA analysis
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Learn from each
other!
31