Leonardo Cerasino, PhD
Centro Ricerca ed Innovazione – Fondazione Edmund Mach
Istituto Agrario di San Michele all’Adige (TN)
Mail: leonardo.cerasino@iasma.it
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
4/12Alkaloids
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
5/12Water Sample
Sequential extraction: 1) Methanol 2) waterfiltration
water
particulate
LC-MS
analysis
extract
Sample treatment
Sample
Alkaloids
Cyclic
peptides
LC-MS Analysis
Microcystins Analysis
6/12 CH3 Microcystin-LR
12
34
5 6 7Commercial
standards:
LR [D-Asp3]LR RR [D-Asp3]RR YR LA LF LW LY WR NOD-RLC 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/lLOQ (
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 1.0Alkaloids 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
10/12n.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
11/1250
50
150
150
100
100
200
200
LD
50in 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
aLR
[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