Cyanotoxin analysis

Miquel Lürling and Els Faassen

Why worry about cyanobacteria?

2

Human health risk

Why worry about cyanobacteria?

3

Krienitz et al 2003

Cattle, pet and wildlife kills

Cyanobacteria produce potent toxins

4

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

Cyanobacterial toxins: Cytotoxins

6

Cylindrospermopsins

Irreversible inhibition protein synthesis, genotoxic, hepatotoxic

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

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)

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

How to determine cyanobacterial toxicity

11

NOT

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

ATX < 0.1 µg/l STX < LOQ CYN < LOD NOD < LOD

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

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)

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

How to determine cyanobacterial toxicity

Use reliable analytical techniques

● Include different toxin variants

17

Variants differ in their toxicity

18

41% 88% 87% 80% 93% 19% 17% 13%

Faassen and Lürling 2013

Variants differ in their toxicity

19

Faassen and Lürling 2013

How to determine cyanobacterial toxicity

Use reliable analytical techniques

● Include different toxin variants

● Consider possible bound forms of toxins

20

Bound microcystins

21

Protein bound MC

Methanol soluble MC

Meissner et al 2013

Bound BMAA

22

Faassen et al 2016

How to determine cyanobacterial toxicity

Use reliable analytical techniques

● Include different toxin variants

● Consider possible bound forms of toxins

Perform proper sampling

23

Inhomogeneous distribution of

cyanobacteria

24

Sampling point

Beach

Inhomogeneous distribution of toxins

25 Wood et al 2010

(homo)anatoxin-a detected no (homo)anatoxin-a detected

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

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

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

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

Cyanocost 2015 Workshop BMAA analysis

Learn from each

other!

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