The document describes research being conducted at the Miller Laboratory located in Milwaukee, Wisconsin. It discusses three doctoral students and their projects: Lucas Beversdorf is studying regulation of hepatotoxins in cyanobacteria and factors leading to toxic blooms; Chelsea Weirich is investigating algal toxin production and occurrence in drinking water; and Sarah Bartlett is examining bioaccumulation of algal toxins in fish. It then provides details on methods used to study cylindrospermopsin and saxitoxin levels in water samples from Lagoa do Peri, Brazil.

1. Workshop
Laboratory for Aquatic
Microbiology and Chemistry
(The Miller Laboratory)
Milwaukee - Wisconsin

3. School of Public Health

4. Todd R. Miller

5. Lucas Beversdorf - Post Doctoral
Studying 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

6. Chelsea Weirich - Doctoral Student
Chelsea 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 cyanobacterial
toxin production

7. Sarah Bartlett - Doctoral Student
Sarah 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.

11. 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).
Cylindrospermopsis raciborskii
(Woloszynska) e Seenayya Subba Raju
Amostra da Lagoa do Peri – março/2014

12. 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)

13. Lagoa do Peri
Abastece 102 a 113 mil habitantes na Ilha de Santa Catarina (Pereira e Zanin, 2012)
Comunidade fitoplantônica apresenta dominância da cianobactéria
Cylindrospermopsis raciborskii (Woloszinska) Seenayya et Subba-Raju.
STX,
Neo-STX,
GTX-1,
GTX-2,
GTX-3,
GTX-4,
GTX-5,
dcGTX-2
dcGTX-3
Gonyautoxina
Decarbamoylsaxitoxina
(Grellmann, 2006; Melo Filho, 2006; Mondardo, 2009; Machado, 2011; Machado e Sens, 2012) .

14. Objetivo
Determinar a relação entre variáveis abióticas,
produção de cianotoxinas e densidade de
Cylindrospermopsis raciborskii “in locu”;
Sample STX_q Peak Area STX_q Peak Height
0 0,00E+00 0,00E+0,1 7,15E+02 5,49E+0,5 3,66E+03 3,07E+1 7,68E+03 6,15E+5 3,75E+04 3,40E+10 7,16E+04 6,00E+0 0,00E+00 0,00E+DB001FH 5,92E+03 6,18E+DB002FH 6,72E+03 7,17E+DB003FH 3,19E+03 2,75E+DB004FH 2,21E+03 2,48E+DB005FH 1,73E+03 2,32E+

15. Metodologia
Transporte
Identificação
Sample Name Date Received State Fraction Site Sample Date Note
DB001W 11/18/14Frozen Whole water Peri Lake 21/08/2013
DB002W 11/18/14Frozen Whole water Peri Lake 24/09/2013
DB003W 11/18/14Frozen Whole water Peri Lake 15/10/2013
DB004W 11/18/14Frozen Whole water Peri Lake 19/11/2013
DB005W 11/18/14Frozen Whole water Peri Lake 10/12/2013
DB006W 11/18/14Frozen Whole water Peri Lake 22/01/2014
…
DB001F 11/18/14Filter Filter Peri Lake 21/08/20132 X 300 ML
DB002F 11/18/14Filter Filter Peri Lake 24/09/20134 X 300 ML
DB003F 11/18/14Filter Filter Peri Lake 15/10/20134 X 300 ML
DB004F 11/18/14Filter Filter Peri Lake 19/11/20134 X 300 ML
DB005F 11/18/14Filter Filter Peri Lake 10/12/20134 X 300 ML
DB006F 11/18/14Filter Filter Peri Lake 22/01/20143 X 300 ML
DB007F 11/18/14Filter Filter Peri Lake 11/02/20141 X 300 ML
DB008F 11/18/14Filter Filter Peri Lake 18/02/20142 X 300 ML
…
DB002D 11/18/14Frozen Filtrate Peri Lake 24/09/2013
DB004D 11/18/14Frozen Filtrate Peri Lake 19/11/2013
DB006D 11/18/14Frozen Filtrate Peri Lake 22/01/2014
DB008D 11/18/14Frozen Filtrate Peri Lake 18/02/2014
DB012D 11/18/14Frozen Filtrate Peri Lake 25/03/2014

16. 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

17. Extraction of Microcystins & Cyanobacterial Peptides with 70% Methanol from
Whole Water Samples
Reagents:
Water with 0.1% formic acid
100% 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.

18. Cut Parafilm circles with razor blade so that they fall into the tubes; discard Parafilm
wrapped 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°C freezer and 50°C water bath;
turn on sonicating water bath during freeze-thaw cycles so it warms up.

19. 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 at
least 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 any
particulates as these could clog the LC lines.

20. Extraction of Neurotoxins & Cylindrospermopsin from Whole Water Samples
Reagents:
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 Parafilm
wrapped 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.

21. HILIC Extraction Method
1) Cut filters (300 mL per filter) in half with scissors – place each
half filter in each 15 mL Falcon tube to represent biomass from 150
mL water sample. Wash each filter with ethanol and Smartwater in
between each sample filter.
2) Add 4 mL HPLC water with 0.1% formic acid into the conical and
vortex to mix well.
3) Freeze (-80°C) the samples for 30 minutes and then thaw in a
50°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 for
15 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 5
μg/L) for 1) determination of difference between phenylalanine and
anatoxin-a retention time and 2) examining ion suppression among
LCMS runs.
C18 (Microcystin/Peptide) ExtractionMethod
1-5) Same steps that you did in HILIC
6) Add 8 mL 100% methanol to each tube so the total methanol
concentration is ~70%. Vortex and let this incubate on the benchtop
for one hour with intermittent vortexing (3-4 times or approx. every
15 minutes).
If having trouble resuspending with methanol addition, try
vortexing sample upside down and/or place samples in
sonicating water bath (45°C) for 5 minutes to try to release
pellet 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 for
15 minutes at maximum speed (5000 rpm).
9) Pour off supernatant into 20 mL scintillation vial and begin drying
down with heat and N2 gas.
10) Add 5 mL of 100% methanol to the 15 mL Falcon tube and let
incubate for one more hour on the benchtop with intermittent
vortexing.
11) Repeat steps 7-9.
12) Dry down entire extract to a residue in the bottom of the
scintillation 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

22. HighPerformance LiquidChromatography
CLAD = Cromatografia a Líquido de Alto Desempenho
CLAE = Cromatografia a Líquido de Alta Eficiência

23. 1
2
3
4
5

24. O objetivo da cromatografia é separar individualmente os diversos constituintes de uma
mistura 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 amostra
se distribuem entre as duas fases e viajam mais lentamente que a fase móvel devido ao
efeito retardante da fase estacionária.
O equilíbrio de distribuição determina a velocidade com a qual cada componente migra
através do sistema.

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

28. Cromatograma
Lagoa do Peri

29. Sample: DB019FD

30. Resultados
- Quantification Limit 0,005 μg.L-1; Detection Limit 0,0025μg.L-1
- STX
- Concentração intracelular
Sample
STX_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 NA
0,1 7,15E+02 5,49E+01< 0 NA NA NA NA
0,5 3,66E+03 3,07E+02 0,472NA NA NA NA
1 7,68E+03 6,15E+02 1,27NA NA NA NA
5 3,75E+04 3,40E+03 7,22NA NA NA NA
10 7,16E+04 6,00E+03 14NA NA NA NA
0 0,00E+00 0,00E+00N/A NA NA NA NA
DB001FH 5,92E+03 6,18E+02 0,922 150 4 0,026666667 0,024586667
DB002FH 6,72E+03 7,17E+02 1,08 150 4 0,026666667 0,0288
DB003FH 3,19E+03 2,75E+02 0,379 150 4 0,026666667 0,010106667
DB004FH 2,21E+03 2,48E+02 0,183 150 4 0,026666667 0,00488
DB005FH 1,73E+03 2,32E+02 0,0873 150 4 0,026666667 0,002328
DB006FH 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 NA
DB007FH 2,84E+03 3,19E+02 0,31 150 4 0,026666667 0,008266667
DB008FH 4,50E+03 4,53E+02 0,64 150 4 0,026666667 0,017066667
DB009FH 3,68E+03 3,73E+02 0,477 150 4 0,026666667 0,01272
DB012FH 2,65E+03 3,21E+02 0,272 150 4 0,026666667 0,007253333
DB013FH 3,40E+03 3,68E+02 0,421 150 4 0,026666667 0,011226667
DB014FH 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 NA
0 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

31. Thank you!