Workshop

Laboratory for Aquatic Microbiology and Chemistry

(The Miller Laboratory)

Milwaukee - Wisconsin

School of Public Health

Todd R. Miller

Lucas Beversdorf - Post DoctoralStudying the regulation of hepatotoxin synthesis in cyanobacteria and factors

leading to the formation of toxic cyanobacterial blooms in lakes using automated samplers and mass spectrometry

Chelsea Weirich - Doctoral StudentChelsea is investigating factors leading to algal toxin production in lakes and their subsequent occurrence in drinking water. She is leading a project with the Global

Lakes Ecological Observatory Network to examine global patterns in cyanobacterialtoxin production

Sarah Bartlett - Doctoral StudentSarah is investigating bioaccumulation and occurrence of algal toxins, primarily anatoxin-a in fish at all trophic levels. She is also investigating the relationship

between gross primary productivity and algal toxin production.

Cylindrospermopsis raciborskii(Woloszynska) e Seenayya Subba Raju

Amostra da Lagoa do Peri – março/2014

Mutagenicidade in vitro e carcinogenicidade in vivo (Humpage et al, 2000; Falconer e Humpage, 2001).

Bioacumulação em peixe (Saker e Eaglesham, 1999), em moluscos (Saker et al., 2004) e em Cladóceros(Nogueira et al., 2004). Em mamíferos provoca lesão necrótica generalizada, afetando fígado, rins,pulmões, baço, intestino, timo e coração (Chorus e Bartmann, 1999).

Bloqueia os canais de sódio na membrana do axônio e induzir disfunção do nervo,causando paralisia e então a morte por insuficiência respiratória (van Apeldoorn et al., 2007).Altamente tóxicas para os animais e, em doses elevadas o suficiente, letais para humanos(Landsberg, 2002).

Fatores abióticos e produção de toxinas por C. raciborskii

Fator Abiótico Observação Referências Bibliográficas

CYL

Temperatura Forte correlação negativa Saker e Griffiths (2000)

STX

Temperatura Não há variação entre 19 e 25oC

Em 25oC produz dcSTX

Castro et al. (2004)

N e P Correlação positiva com altas relações N:P Chislock et al. (2014)

Dureza Correlação positiva Carneiro et al. (2013)

Lagoa do Peri

Abastece 102 a 113 mil habitantes na Ilha de Santa Catarina (Pereira e Zanin, 2012)

Comunidade fitoplantônica apresenta dominância da cianobactériaCylindrospermopsis raciborskii (Woloszinska) Seenayya et Subba-Raju.

STX, Neo-STX,

GTX-1, GTX-2, GTX-3, GTX-4, GTX-5,

dcGTX-2 dcGTX-3

(Grellmann, 2006; Melo Filho, 2006; Mondardo, 2009; Machado, 2011; Machado e Sens, 2012) .

Gonyautoxina

Decarbamoylsaxitoxina

Objetivo

Determinar a relação entre variáveis abióticas,produção de cianotoxinas e densidade deCylindrospermopsis raciborskii “in locu”;

Sample STX_q Peak Area STX_q Peak Height

0 0,00E+00 0,00E+00

0,1 7,15E+02 5,49E+01

0,5 3,66E+03 3,07E+02

1 7,68E+03 6,15E+02

5 3,75E+04 3,40E+03

10 7,16E+04 6,00E+03

0 0,00E+00 0,00E+00

DB001FH 5,92E+03 6,18E+02

DB002FH 6,72E+03 7,17E+02

DB003FH 3,19E+03 2,75E+02

DB004FH 2,21E+03 2,48E+02

DB005FH 1,73E+03 2,32E+02

Metodologia

Transporte

Identificação

Sample Name Date Received State Fraction Site Sample Date NoteDB001W 11/18/14Frozen Whole water Peri Lake 21/08/2013DB002W 11/18/14Frozen Whole water Peri Lake 24/09/2013DB003W 11/18/14Frozen Whole water Peri Lake 15/10/2013DB004W 11/18/14Frozen Whole water Peri Lake 19/11/2013DB005W 11/18/14Frozen Whole water Peri Lake 10/12/2013DB006W 11/18/14Frozen Whole water Peri Lake 22/01/2014…DB001F 11/18/14Filter Filter Peri Lake 21/08/20132 X 300 MLDB002F 11/18/14Filter Filter Peri Lake 24/09/20134 X 300 MLDB003F 11/18/14Filter Filter Peri Lake 15/10/20134 X 300 MLDB004F 11/18/14Filter Filter Peri Lake 19/11/20134 X 300 MLDB005F 11/18/14Filter Filter Peri Lake 10/12/20134 X 300 MLDB006F 11/18/14Filter Filter Peri Lake 22/01/20143 X 300 MLDB007F 11/18/14Filter Filter Peri Lake 11/02/20141 X 300 MLDB008F 11/18/14Filter Filter Peri Lake 18/02/20142 X 300 ML…DB002D 11/18/14Frozen Filtrate Peri Lake 24/09/2013DB004D 11/18/14Frozen Filtrate Peri Lake 19/11/2013DB006D 11/18/14Frozen Filtrate Peri Lake 22/01/2014DB008D 11/18/14Frozen Filtrate Peri Lake 18/02/2014DB012D 11/18/14Frozen Filtrate Peri Lake 25/03/2014

Extração

Amostra líquida(whole and fitrated)

H=Hydrophilic Interaction Liquid Chromatography

(HILIC) column/extraction

for anatoxin-a, homoanatoxin-a,

saxitoxin, cylindrospermopsin

C=C18 (18-Carbon chain)

column / extraction for microcystins

and peptides

Filtro(filter)

HILIC C18

Extraction of Microcystins & Cyanobacterial Peptides with 70% Methanol from Whole Water SamplesReagents:Water with 0.1% formic acid100% Methanol

Lyophilize 20 mL of each water sample for 48 hours. Pipet 20 mL sample into a 50 mL Falcon tube.Wrap one-and-a-half squares of Parafilm around the top of each tube.Use a small pipet tip to poke holes in the tops of the Parafilm.Cap and freeze on an angle in -80°C freezer for >1 hour.Samples are lyophilized for 24-48 hours at -50°C until complete dryness. This is to concentrate and preserve cells and dissolved toxins.

Cut Parafilm circles with razor blade so that they fall into the tubes; discard Parafilmwrapped around tubes. Letting Parafilm that covers the top of the tube duringlyophilization fall into the tube allows it to be “washed” for maximum extractionrecovery of toxins.

Add 1 ml of water/0.1% formic acid (vortex)

Three 30-minute freeze-thaw cycles between the -80°C freezer and 50°C water bath;turn on sonicating water bath during freeze-thaw cycles so it warms up.

Add 2 mL 100% methanol (vortex; final concentration methanol ~70%).

Sonicate in the 45°C sonicating water bath for 10 minutes – make sure the bath is atleast 2/3 of the way full with distilled water (vortex).

Centrifuge at maximum speed (11,000 rpm) for 15 minutes.

Transfer 1000 μL of the supernatant to a labeled LC vial; make sure not to suck up anyparticulates as these could clog the LC lines.

Extraction of Neurotoxins & Cylindrospermopsin from Whole Water SamplesReagents:Water with 0.1% formic acid

Lyophilize 20 mL of each water sample for 48 hours. Pipet 20 mL sample into a 50 mL Falcon tube.Wrap one-and-a-half squares of Parafilm around the top of each tube.Use a small pipet tip to poke holes in the tops of the Parafilm.Cap and freeze on an angle in -80°C freezer for >1 hour.Samples are lyophilized for 24-48 hours at -50°C until complete dryness. This is to concentrate and preserve cells and dissolved toxins.

Cut Parafilm circles with razor blade so that they fall into the tubes; discard Parafilmwrapped around tubes. Letting Parafilm that covers the top of the tube during lyophilization fall into the tube allows it to be “washed” for maximum extraction recovery of toxins.Add 1 mL of water/0.1% formic acid (vortex)Three 30-minute freeze-thaw cycles between the -80°freezer and 50°C water bath; turn on sonicating water bath during freeze-thaw cycles so it warms up.Sonicate in the 45°C sonicating water bath for 10 minutes – make sure the bath is at least 2/3 of the way full with distilled water (vortex).Centrifuge at maximum speed (11,000 rpm) for 15 minutes.Transfer 750 μL of the supernatant to a labeled LC vial; make sure not to suck up any particulates as these could clog the LC lines.

HILIC Extraction Method

1) Cut filters (300 mL per filter) in half with scissors – place eachhalf filter in each 15 mL Falcon tube to represent biomass from 150mL water sample. Wash each filter with ethanol and Smartwater inbetween each sample filter.

2) Add 4 mL HPLC water with 0.1% formic acid into the conical andvortex to mix well.

3) Freeze (-80°C) the samples for 30 minutes and then thaw in a50°C water bath.

4) Vortex and place in a sonicating water bath at 45°C for 5 minutes,then vortex again.

5) Repeat steps 3 and 4 two more times.

6) Place 15 mL tubes in a swinging bucket rotor and centrifuge for15 minutes at maximum speed.

7) Transfer 1 mL of supernatant to LC vial for LC-MS/MS analysis.

8) Add 5 μL of 1000 μg/L 13C6-phenylalanine (extracts spiked at 5μg/L) for 1) determination of difference between phenylalanine andanatoxin-a retention time and 2) examining ion suppression amongLCMS runs.

C18 (Microcystin/Peptide) Extraction Method

1-5) Same steps that you did in HILIC

6) Add 8 mL 100% methanol to each tube so the total methanolconcentration is ~70%. Vortex and let this incubate on the benchtopfor one hour with intermittent vortexing (3-4 times or approx. every15 minutes).

If having trouble resuspending with methanol addition, tryvortexing sample upside down and/or place samples insonicating water bath (45°C) for 5 minutes to try to releasepellet from sides of tube.

7) Place in sonicating water bath for 5 minutes.

8) Place 15 mL tubes in a swinging bucket rotor and centrifuge for15 minutes at maximum speed (5000 rpm).

9) Pour off supernatant into 20 mL scintillation vial and begin dryingdown with heat and N2 gas.

10) Add 5 mL of 100% methanol to the 15 mL Falcon tube and letincubate for one more hour on the benchtop with intermittentvortexing.

11) Repeat steps 7-9.

12) Dry down entire extract to a residue in the bottom of thescintillation vial. Resuspend each residue in 1 mL 70% methanol.

13) Sonicate the scintillation vials in the water bath for 5 minutes(tightly capped) to help remove the residue stuck to the glass.

14) Transfer 1 mL of suspended toxin extract into 1.5 mL mini-centrifuge tube, and centrifuge at max speed for 12 minutes.

15) Transfer 750 μL of supernatant to LC vial for LC-MS/MS analysis.

Filters

High Performance Liquid Chromatography

CLAD = Cromatografia a Líquido de Alto DesempenhoCLAE = Cromatografia a Líquido de Alta Eficiência

1

23

4

5

O objetivo da cromatografia é separar individualmente os diversos constituintes de umamistura de substâncias.

Tal separação dá-se através da migração da amostra através de uma fase estacionária(FE) por intermédio de um fluido (fase móvel - FM).

Após a introdução da amostra no sistema cromatográfico, os componentes da amostrase distribuem entre as duas fases e viajam mais lentamente que a fase móvel devido aoefeito retardante da fase estacionária.

O equilíbrio de distribuição determina a velocidade com a qual cada componente migraatravés do sistema.

DESCRIÇÃO GERAL DE HPLC•A amostra é dissolvida em um solvente e introduzida na coluna cromatográficapreenchida com a fase estacionária (FE)•Um solvente (FM) é bombeado com vazão constante e desloca os componentesda mistura através da coluna. Esses se distribuem entre as duas fases de acordocom suas afinidades•As substâncias com maior afinidade com a FE movem-se mais lentamente. Já assubstâncias com pouca afinidade com a FE movem-se mais rapidamente.•Ao sair da coluna, os componentes passam por um detector que emite um sinalelétrico o qual é registrado, constituindo um cromatograma.

CromatogramaLagoa do Peri

Sample: DB019FD

Resultados

- Quantification Limit 0,005 μg.L-1; Detection Limit 0,0025μg.L-1

- STX- Concentração intracelular

SampleSTX_q Peak Area

STX_q Peak Height

Calculated Concentration_q

Volume (Filtered or Lyophilized)

Final Volume

Dilution factor

Lake Concentration (ug/L) STX_q

0 0,00E+00 0,00E+00N/A NA NA NA NA0,1 7,15E+02 5,49E+01< 0 NA NA NA NA0,5 3,66E+03 3,07E+02 0,472NA NA NA NA

1 7,68E+03 6,15E+02 1,27NA NA NA NA5 3,75E+04 3,40E+03 7,22NA NA NA NA

10 7,16E+04 6,00E+03 14NA NA NA NA0 0,00E+00 0,00E+00N/A NA NA NA NA

DB001FH 5,92E+03 6,18E+02 0,922 150 4 0,026666667 0,024586667DB002FH 6,72E+03 7,17E+02 1,08 150 4 0,026666667 0,0288DB003FH 3,19E+03 2,75E+02 0,379 150 4 0,026666667 0,010106667DB004FH 2,21E+03 2,48E+02 0,183 150 4 0,026666667 0,00488DB005FH 1,73E+03 2,32E+02 0,0873 150 4 0,026666667 0,002328DB006FH 4,08E+03 4,61E+02 0,557 150 4 0,026666667 0,014853333

0 0,00E+00 0,00E+00N/A NA NA NA NADB007FH 2,84E+03 3,19E+02 0,31 150 4 0,026666667 0,008266667DB008FH 4,50E+03 4,53E+02 0,64 150 4 0,026666667 0,017066667DB009FH 3,68E+03 3,73E+02 0,477 150 4 0,026666667 0,01272DB012FH 2,65E+03 3,21E+02 0,272 150 4 0,026666667 0,007253333DB013FH 3,40E+03 3,68E+02 0,421 150 4 0,026666667 0,011226667DB014FH 3,42E+03 3,63E+02 0,425 150 4 0,026666667 0,011333333

10 4,67E+04 4,05E+03 9,04NA NA NA NA0 0,00E+00 0,00E+00N/A NA NA NA NA

DB015FH 4,03E+03 4,33E+02 0,546 150 4 0,026666667 0,01456

Thank you!