Cyanobacterial toxins profiling in the Subalpine lakes

Leonardo Cerasino, PhD

Centro Ricerca ed Innovazione – Fondazione Edmund Mach

Istituto Agrario di San Michele all’Adige (TN)

Mail: [email protected]

Cyanobacterial toxins profiling in

the Subalpine lakes

Harmful Algal Blooms (HABs)

• An algal bloom is a rapid increase in the population of algae in an aquatic system.

Typically only one or a few phytoplankton species are involved and some blooms may

be recognized by discoloration of the water.

• Algal blooms may also be of concern as some species of algae produce toxins.

• Causes: denaturalization, eutrophication, global changes.

2/12

Anabaena lemmermannii

Planktothrix rubescens

Toxic Cyanobacteria in the southern subalpine lakes

First records of Harmful Algal Blooms in the deep southern subalpine lakes.

2005 2005 Maggiore Maggiore 1999 1999 2006 2006 1980 1980--8585 Lugano Lugano 1980s and 2000 1980s and 2000--20032003 2006 2006 Como Como 2006 2006 1998 1998 1997 1997 Iseo Iseo 1990 1990--19921992 1990 1990 Garda Garda Aphanizomenon Aphanizomenon flos

flos--aquaeaquae

Microcystis

Microcystis

aeruginosa/

aeruginosa/flosflos--aquaeaquae

Planktothrix Planktothrix rubescens rubescens Anabaena Anabaena lemmermannii lemmermannii 3/12

Garda

Levico

Ledro

Idra

Iseo

Como

Pusiano

Lugano

Maggiore

Cyanotoxins

4/12

Alkaloids

Saxitoxins

>20

Anatoxins

3

Cylindrospermopsins

2

Cyclic peptides

Microcystins (heptapeptides)

>70

Nodularin (pentapeptides)

1

Lipopolysaccharides

(undefined)

Secondary metabolites

Neurotoxins

Hepatotoxins

(tumor promotors)

Dermatotoxins

Anatoxin-a

_Saxitoxin

Anatoxin-a(S)

Cylindrospermopsin

Microcystin-LR

Cyanotoxins extraction and analysis

5/12

Water Sample

Sequential extraction: 1) Methanol 2) water

filtration

water

particulate

LC-MS

analysis

extract

Sample treatment

Sample

Alkaloids

Cyclic

peptides

LC-MS Analysis

Microcystins Analysis

6/12 CH₃ Microcystin-

LR

1

2

3

4

5 6 7

Commercial

standards:

LR [D-Asp3_]LR RR [D-Asp3_]RR YR LA LF LW LY WR NOD-R

LC separation – Reverse Phase

Inj. Volume: 8 µµµµl

Eluents: A= Water, B= ACN (both containing 0.1% FA)

Flow rate: 0.25 ml/min

Gradient: B from 30% (0 min) to 80% (5 min), to 30 % (6 min)

Column: Acquity BEH C18 (1.7 µµµµm) 2.1x50mm, 40°C

Product ions 135.1 and 213.1 can be conveniently used for

identification (and possibly quantitation) of MC with different AA 2, 3, 4. List of MC screened: Molecular ion DP (V) CE (V) LR / [D-Asp3_{]LR [M+2]}2+ _{50 20} RR / [D-Asp3_{]RR [M+2]}2+ _{100 43} YR / [D-Asp3_{]YR [M+2]}2+ _{50 20} LA / [D-Asp3_{]LA [M+1]}+ _{70 80} LY / [D-Asp3_{]LY [M+1]}+ _{90 100} LW / [D-Asp3_{]LW [M+1]}+ _{90 90} LF / [D-Asp3_{]LF [M+1]}+ _{90 100} WR / [D-Asp3_{]WR [M+1]}+ _{50 20}

MS detection: ESI+, MRM scanning mode

Transition 1 [M+1]+_{/135.1 (or [M+2]}2+_/135.1)

Transition 2 [M+1]+_{/213.1 (or [M+2]}2+_/213.1)

General settings: Ion Spray 5000V

Entrance Pot. 10V

Cell Exit Pot 10V

Interface Heater T 300°C

7/12

Microcystins Analysis

XIC of +MRM (25 pairs): 513.0/135.0 amu Expected RT: 1.4 ID: RRdm 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), S... Max. 1.4e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 In te n s it y , c p s 1.36 MC-RRdm

XIC of +MRM (25 pairs): 520.1/135.0 amu Expected RT: 1.4 ID: RR 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), Smo... Max. 3.6e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 In te n s it y , c p s 1.45 MC-RR

XIC of +MRM (25 pairs): 826.1/70.0 amu Expected RT: 1.7 ID: NODR 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), S... Max. 4.6e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 In te n sit y , c p s 1.74 NOD-R

XIC of +MRM (25 pairs): 523.6/135.0 amu Expected RT: 1.9 ID: YR 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), Smo... Max. 9.7e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 1.0e4 2.0e4 3.0e4 4.0e4 5.0e4 6.0e4 7.0e4 8.0e4 9.0e4 9.7e4 In te n s it y , c p s 1.92 MC-YR

XIC of +MRM (25 pairs): 498.6/135.0 amu Expected RT: 2.0 ID: LR 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), Smo... Max. 2.8e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 2.8e5 In te n sit y , c p s 2.00 MC-LR

XIC of +MRM (25 pairs): 911.1/135.0 amu Expected RT: 3.0 ID: LA 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), Smo... Max. 2.7e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 2.7e4 In te n s it y , c p s 2.91 MC-LA

XIC of +MRM (25 pairs): 1026.1/135.0 amu Expected RT: 3.6 ID: LW 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), Sm... Max. 1.5e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.5e4 In te n s it y , c p s 3.54 MC-LW

XIC of +MRM (25 pairs): 491.6/135.0 amu Expected RT: 2.0 ID: LRdm 1 from Sample 5 (STANDARD IRSA005) of DataSET1.wiff (Turbo Spray), S... Max. 1.9e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 1.6e5 1.8e5 1.9e5 In te n s it y , c p s 2.03 MC-LRdm

XWC of DAD Spectral Data: 238.0 nm from Sample 5 (STANDARD IRSA005) of DataSET1.wiff Max. 0.1 mAU.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 0.107 A b s o rb a n c e , m A U 6.90 6.19 3.46 1.79 4.83 3.33 2.70 1.51 4.68 4.08 1.23 2.61 0.57 0.39 2.28 1.14 1.01 MC-RRdm MC-RR NOD-R MC-YR MC-LR MC-LRdm MC-LA MC-LW MC-LF

UV, 238 nm

Conc. = 0.5 µg/L

Multistandard solution 0.5

µ

g/l

Linearity range:

0.1-100 µµµµg/l

LOQ (

µ

g/l):

LR / [D-Asp3]LR 0.1 RR / [D-Asp3]RR 0.1 YR / [D-Asp3]YR 0.5 LA / [D-Asp3]LA 1.0 LY / [D-Asp3]LY 1.0 LW / [D-Asp3]LW 2.0 LF / [D-Asp3]LF 2.0 WR / [D-Asp3_{]WR 2.0} NOD-R 1.0

Alkaloids Analysis

8/12

LC separation - HILIC

Inj. Volume: 8 µµµµl

Eluents: A= 0.05M NH₄F, B= MeOH

Flow rate: 0.25 ml/min

Gradient: B from 20% (0 min) to 80% (5 min), to 20 % (6 min)

Column: Acquity HILIC (1.7 µµµµm) 2.1x50mm, 30°C

MS detection: ESI+, MRM scanning mode

General settings: Ion Spray 5000V

Entrance Pot. 10V

Cell Exit Pot 10V

Interface Heater T 300°C Commercial standards: Anatoxin-a Cylindrospermopsin

Anatoxin-a

Anatoxin-a(S)

Cylindrospermopsin

List of alkaloids screened:

Transition 1 Transition 2 DP(V) CE(V)

Ana-a 166/149 166/91 70 30 Dihydro-Ana-a 168/151 70 30 Dihydroxymetoxy-Ana-a 198/181 70 30 Epoxy-Ana-a 182/165 70 30 Hydroxyl-Ana-a 182/165 70 30 HomoAna-a 180/163 70 30 Dihydro-homoAna-a 182/165 70 30 Dihydroxymetoxy-homoAna-a 212/195 70 30 Epoxy-homoAna-a 196/179 70 30 Hydroxy-homoAna-a 196/179 Cylindrospermopsin 416/194 416/176 90 53 Desoxy-cylindrospermopsin 400/194 90 53 Phenylalanine 166/120 70 30

9/12

Alkaloids Analysis

2 standards solution, 10

µ

g/l

Linearity range:

2 - 100 µg/l

LOQ (

µ

g/l):

Anatoxin-a

2.0

cylindrospermopsin

4.0

Anatoxin-a

_{Cylindrospermopsin}

Garda

Levico

Ledro

Idra

Iseo

Como

Pusiano

Lugano

Maggiore

10/12

n.d.

n.d.

70

70

-

-

460

460

530

530

-

-

3493

3493

Pusiano

Pusiano

n.d.

n.d.

1.4

1.4

15

15

Idro

Idro

n.d.

n.d.

375

375

4600

4600

Ledro

Ledro

n.d.

n.d.

4.5

4.5

-

-

78

78

16

16

-

-

278

278

Levico

Levico

120

120

12

12

-

-

34

34

26

26

-

-

76

76

Maggiore

Maggiore

n.d.

n.d.

15

15

-

-

17

17

84

84

-

-

98

98

Lugano

Lugano

400

400

-

-

1700

1700

15

15

-

-

38

38

80

80

-

-

200

200

Como

Como

60

60

-

-

400

400

14

14

-

-

17

17

90

90

-

-

112

112

Iseo

Iseo

45

45

-

-

2000

2000

0.03

0.03

-

-

10

10

0.3

0.3

-

-

96

96

Garda

Garda

ANA

ANA

-

-

a

a

MC tot (

MC tot (

LReq

LReq

)

)

MC tot

MC tot

Min-max cyanotoxins concentrations (

ng/l

) in the trophogenic layer

(0-20m). Data refer to samplings conducted in June - October 2009.

Results

• MC are widely distributed in the lakes.

• Highest MC concentrations in the most

eutrophic lakes.

• Values are always below the WHO limit

of 1

µµµµ

g/l

MC-LR for drinking waters.

• ANA-a is present only in few lakes and

is related to the presence of A.

Garda

Levico

Ledro

Idra

Iseo

Como

Pusiano

Lugano

Maggiore

11/12

50

50

150

150

100

100

200

200

LD

50

in mice

98%

98%

1

1

5

5

16

16

77

77

Pusiano

100%

100%

-5

5

-95

95

Idro

99%

99%

-1

1

1

1

97

97

Ledro

100%

100%

1

1

27

27

60

60

13

13

Levico

99%

99%

37

37

6

6

8

8

47

47

Maggiore

94%

94%

8

8

10

10

1

1

75

75

Lugano

98%

98%

9

9

4

4

8

8

77

77

Como

98%

98%

1

1

8

8

25

25

64

64

Iseo

99%

99%

2

2

3

3

3

3

92

92

Garda

Sum

a

LR

[D-Asp

3

_]LR

YR

[D-Asp

3

_]RR

%

Results

Abundances of individual MC.

Minor variants are LF, LW, [D-Asp

3

_]YR.

• There is a wide MC diversity among

lakes.

• The most toxic variant LR

represents a small percentage (with

one exception).

• Different lakes have different

“toxic potentials”.

12/12

Thank you for your kind attention

[email protected]