Environmental Arsenic Speciationdefinitely more than just arsenite and arsenate!

Grand Prismatic Hot Spring, Yellowstone NP

Arsenic the classical poison

Homemade elderberry wine

laced with Arsenic StrychnineArsenic, Strychnine and just a pinch of

CyanideCyanide

Joseph Kesselring, 1939

18401840

1836 M h t t1836 Marsh test

Principle of Marsh´s TestpDetermination of total As by hydride generation

As2O3 + 6Zn + 6H2SO4 → 2AsH3 + 6Zn(SO4)2 + 3H2O

+3 -30 +2+3 -30 +2

As

Arsenic speciesse c spec es

Arsenate As(V)

Arsenate As(V)

Arsenite As(III)

Arsenite As(III)

1970s Species-selective Hydride GenerationSeparating As(III) and As(V)

H3AsIIIO30 / H3AsVO4

0 + NaBH4 AsH3

8090

100H3AsO3H3AsO4As(III)“

0607080 H3AsO4

pK 2.2 pK 9.2

„As(III)

304050

p p

As(III)“

0102030 „As(III)

+ „As(V)“

00 1 2 3 4 5 6 7 8 9 10 11 12

Ion chromatography for speciation of arsenite and arsenate+ detection e g by atomic absorption spectroscopy

1.E+05

+ detection e.g. by atomic absorption spectroscopy

8.E+04

6.E+04

ity [c

ps]

HA O 2

4.E+04

al in

tens HAsO4

2-

arsenate

2.E+04

sign

a

arseniteH3AsO3

0

0.E+000 2 4 6 8 10 12 14 16 18 20 220 2 4 6 8 10 12 14 16 18 20 22

time [minutes]

Modeling arsenite and arsenate distribution

ArsenitepK1, pK2, pK3 (H3AsO3)

t t d+ protonated species H4AsO3

+

A tArsenatepK1, pK2, pK3 (H3AsO4)

link arsenite - arsenate

Traditional Arsenic Speciation

Arsenate07

06

Arsenate

05

04

03

01

03

Arsenite02

01

Gibbon Geyser Basin

Sulfidic environment affects arsenic speciation!

Arsenicup to 5 4 mg/L

Sulfide0 1 5 /L up to 5.4 mg/L0.1-5 mg/L

H2S

Grotto Geyser

Chromatographic separation revealed As species with retention times that did not match those of any commercial standards

1.E+05

times that did not match those of any commercial standards

?

8.E+04?

?

6.E+04

ity [c

ps]

4.E+04

al in

tens arsenate ?

2.E+04

sign

a

arsenite?

0.E+000 2 4 6 8 10 12 14 16 18 20 220 2 4 6 8 10 12 14 16 18 20 22

time [minutes]

Requires a more selective detector:I d ti l l d l t tInductively coupled plasma mass spectrometry

ELAN DRC II ICP-MS, Perkin Elmer

Samplepintroductionca.1 ml/min

aerosolaerosolH2AsO4

-

Plasma 6000-8000°C

atomAs, H, O

ionAs+, H+, O+

Detection of unknown As species

1.E+05sulfate S:As S:As

8.E+04

Arsenic AsO+Sulfur SO+

sulfate S:As0.95 ± 0.1n = 17

S:As2.99 ± 0.22

n = 26

6.E+04y [c

ps]

4 E+04

6.E 04

inte

nsity

arsenatesulfide

S:As1.92 ± 0.14

n = 21 S:As

2 E+04

4.E+04

sign

al

thiosulfateca.4

2.E+04 arsenite

0.E+000 2 4 6 8 10 12 14 16 18 20 22

time [minutes]

Thioarsenates or Thioarsenites?

Thioarsenites

OH SH SH SHOH OH SH SH

As AsAs As

OH OHOH SH

Mono~ Di~ Tri~ Tetra~

OH

OH – As = O

OH

OH – As = O

OH

SH – As = O

SH

SH – As = O

SH

SH – As = SOH As O

SH

OH As O

OH

SH As O

SH

SH As O

SH

SH As S

SH

Thioarsenates retention time match with synthesized standards

Electrospray Ionization MSElectrospray Ionization MSNo prior chromatographic separation!

No atomization of compoundp

• „Soft ionization“, rather transfer from solution to gasg• Solvent evaporation• Formation of desolvated aerosol

H A O ions by Coulombic fission (repellent forces by charged molecules)

H2AsO4-

molecules)

Thioarsenates or Thioarsenites? Identical molecular masses of thioarsenites and thioarsenates on low-resolution

Thioarsenites

mass spectrometry is a problem for electrospray ionization mass spectrometry

OH SH SH SHOH OH SH SH

As AsAs As

OH OHOH SH

Mono~ Di~ Tri~ Tetra~141 157 173

OH

OH – As = O

OH

OH – As = O

OH

SH – As = O

SH

SH – As = O

SH

SH – As = SOH As O

SH

OH As O

OH

SH As O

SH

SH As O

SH

SH As S

SH141 157 173

Thioarsenates retention time match with synthesized standardsm/z: 2 * O (2*16) S (32)

ESI-MS proofs for thioarsenates not thioarsenites

1. ES Q-TRAP MS molecular massesless deviations when assuming thioarsenate m/z ratios

Measured m/z

Thio-arsenate

expected m/z for thio-

rel. dev.

Thio-arsenite

expected m/z for thio-

rel. dev. [ppm]arsenate [ppm] arsenite [ppm]

140.914 H2AsO4- 140.9169 -20.6 H2AsO2S- 140.8991 +105.7

156 891 H AsO S- 156 8941 19 8 H AsOS - 156 8763 +93 7156.891 H2AsO3S 156.8941 -19.8 H2AsOS2 156.8763 +93.7

172.868 H2AsO2S2- 172.8712 -18.5 H2AsS3

- 172.8535 +83.9

188 848 H2AsOS3- 188 8484 -0 53188.848 H2AsOS3 188.8484 0.53

204.825 H2AsS4- 204.8255 -4.88

Wallschläger & Stadey Anal. Chem. 2007, 79, 3873-3880

ESI-MS proofs for thioarsenates not thioarsenites

1. ES Q-TRAP MS molecular masses

OH

less deviations when assuming thioarsenate m/z ratios

OH

SH – As = O2. ES-MS-MS fractionation patterns

(m/z 173 eliminated H2O SH SH

As

SHmust be H2AsO2S2-

H2AsS3- can not eliminate H2O!)

SH

trithio-ESI-MS proofs for thioarsenates not thioarsenites

arsenate

1. ES Q-TRAP MS molecular masses

dithio-arsenate

arsenate

less deviations when assuming thioarsenate m/z ratios

2. ES-MS-MS fractionation patterns(m/z 173 eliminated H2O must be H AsO S2-

monothio-arsenate

must be H2AsO2SH2AsS3

- can not eliminate H2O!)

3 Analysis of IC fractions by ES MStetratharsena

3. Analysis of IC fractions by ES-MS known As:S ratios from IC and molecular mass information from ES-MS

800 1000 1time [s]

S:As 1:1Monothioarsenite (m/z = 141)

Monothioarsenate (m/z = 157)

Thioarsenates predominate arsenic speciationat the geothermal sources

dot size = sum of thioarsenates [%]

Methylated (thio)arsenatesaccounted for all remaining missing species

CH3

CH3 – As = Oaccounted for all remaining missing species CH3 As O

SHSlightly different eluent:0- 3 min 2.5 mM3- 5 min 2.5 20 mM 1.0x104 dimethyl-

arsenitedimethyl-arsenate arsenate

ps]3 5 min 2.5 20 mM

5-10 min 20 mM10-20 min 20 100 mM20 24 i 2 5 M 3

monothio-arsenate

monomethyl-arsenate

monothio-arsenatey

AsO

+ [cp

20-24 min 2.5 mM

No suppressor

1.0x103

monomethyl- monothio- arsenate

arsenate

dithio-arsenatena

l int

ensi

ty

Additional As species: 13-20% DMA

1.0x102

arsenate

sign

13-20% DMA 1% MMA 5% DMMTA 200 400 600 800 1000 120

1.0x101

0.5% MMMTA time [s]

Why were thioarsenates notWhy were thioarsenates not detected in previous studies?p

Preservation of arsenic speciation by acidification90

10012.0

14.0

speciation by acidification

607080

cove

ry [%

]

8.0

10.0

H

304050

arse

nic

rec

4.0

6.0

pH

010

20

0.0

2.0

80

90

100

%]

12.0

14.0

90100

12.0

14.0

50

60

70

80

reco

very

[%

8.0

10.0

pH

607080

ecov

ery

[%]

8.0

10.0H

20

30

40

50ar

seni

c r

4.0

6.0

p

20304050

arse

nic

re

4.0

6.0

p

0

10

20

0.0

2.0

01020

0.0

2.0

Preservation of arsenic speciation by acidificationYellowstone: Trithioarsenate pH >6speciation by acidification

not suitable in sulfidic waters!

Monothioarsenate at all pH conditions

not suitable in sulfidic waters!

flash-freezing

1.2

1.4Monothioarsenate

Dithioarsenate

Trithioarsenate

0.8

1

[mg/

L]

Trithioarsenate

Tetrathioarsenate

0.4

0.6

arse

nic

0

0.2

02 3 4 5 6 7 8 9 10

pH

Analysis of arsenic by hydride generationnot suitable for arsenic speciation in sulfidic waters!

H3AsO30 / H3AsO4

0 + NaBH4 AsH3T t l d ith KI / bi

not suitable for arsenic speciation in sulfidic waters!

8090

100H3AsO3H3AsO4

„As(III)“

Totals: prered. with KI /ascorbicacid in 3.5 M HCl

40506070

pK 2.2 pK 9.2

Speciation: non-std. procedure

• different buffers

10203040 p p

„As(III)“

+ „As(V)“

• final pH 4 to 7

• batch vs. flow injection

Totals: thioarsenates arsenate arsenite

00 1 2 3 4 5 6 7 8 9 10 11 12

(diff. pH of sample / reductant prior to mixing)

Totals: thioarsenates arsenate, arsenite correct (if samples were not acidified for preservation and 1% KI is used as pre-reducing agent; not L-cysteine)

Species: „As(III)“: Arsenite, Di-, Tri-, and Tetrathioarsenate„As(V)“: Arsenate, Monothioarsenate

Traditional Arsenic Speciation

ArsenateImplications of existence

f hi

0706

Arsenate

of thioarsenates…05

04

03

01

03

Arsenite02

01

Gibbon Geyser Basin

Implications for speciation transformations and redox processesp

1.6 1.6

1 2

1.4

1 2

1.4measured byhydride generation

measured by AEC-ICP-MS

1.0

1.2

L]

1.0

1.2

L]

0 6

0.8

seni

c [m

g/L

0 6

0.8

seni

c [m

g/L

0.4

0.6ars

0.4

0.6ars

0 0

0.2

0 0

0.2

0.00 3 6 9 12 15 18 21 24 27 30 33 36

distance from source [m]

0.00 3 6 9 12 15 18 21 24 27 30 33 36

distance from source [m]

„Oxidation“ of thioarsenate to arsenite??1190

1070950

830

720

600

480

360

240

120

0

Thioarsenate / Arsenite ArsenateXX

0

-120

-240

-360

Arsenite + S0 donor Thioarsenate

X

XX

X-480

-600

-720

830

Arsenite + (Sulfide) / S0 donor ThioarsenateXX

-830

-950

intermediate S species determine As speciation

Influence of S-oxidizing microorganisms on arsenic redox transformations…?

• transformation rates are 40-500 times faster (k =

1/min, t1/2 = 0.6 min) than in abiotic control experiments

in the laboratory (k = 0 004/min t1/2 = 170 min)in the laboratory (k 0.004/min, t1/2 170 min)

• arsenate production coincides with the temperature-

dependent occurrence of organisms closely related to

Thermocrinis ruber a known sulfur-oxidizerThermocrinis ruber, a known sulfur oxidizer

Implications for toxicity

1.6 1.6

measured by measured by AEC-ICP-MSLab toxicity tests exposing Vibrio fischeri to thioarsenate standards:

1 2

1.4

1 2

1.4measured by

hydride generation

yto thioarsenate standards:

Exposure time 15 min:

1.0

1.2

L]

1.0

1.2

L]

p

trithioarsenate = arsenite > di >> mono

??

0 6

0.8

seni

c [m

g/L

0 6

0.8

seni

c [m

g/L

Exposure time 60 min:

trithioarsenate > arsenite

??

0.4

0.6ars

0.4

0.6arstrithioarsenate > arsenite

lack of adaptation for thioarsenates?

0 0

0.2

0 0

0.2in the presence of both arsenite and trithioarsenate sig lower toxicity0.0

0 3 6 9 12 15 18 21 24 27 30 33 36distance from source [m]

0.00 3 6 9 12 15 18 21 24 27 30 33 36

distance from source [m]

trithioarsenate sig. lower toxicity

antagonism trithioarsenate - arsenite?

Grenoble, FranceGrenoble, FranceESRF = European Synchrotron Radiation Facility

So what about the thioarsenites …?Synchrothron radiation main advantages: radiation with a large y g gspectrum (IR, VIS, UV, X-Ray), high intensity, sharp focus

844 m

X-ray absorption spectroscopy XAS

monochromator(monochromatic X-rays)

synchrotron radiation source

I0

(high energetic, precise radiation)

incoming beam

sample

absorption

fluorescenceI1

absorption

fluorescence

I2reference (e.g. Au0, As0)

Resulting data: XAS spectra2.5

XANES (E0 ± 50 eV) X ray Absorption Near Edge Spectroscopy

2.0

XANES (E0 ± 50 eV) X-ray Absorption Near-Edge Spectroscopy• oxidation state• local site symmetry and orbital occupancy

1.5

bsor

ptio

n µ absorption edge E0

As 11867 eV

1.0

mal

ized

ab

EXAFS

0.5

nor Extended X-ray Absorption Fine-Structure

50 – 1000 eV above E0, local coordination• bond distances R

0.011650 11850 12050 12250 12450 12650

• coordination number CN • type and number of neighbors

11650 11850 12050 12250 12450 12650

Energy [eV]

Interpretation of coordination number AsS from EXAFS vs. edge position from XANES

As(V)As(III) • First spectra of synthesized thioarsenate standards

upon acidification Thioarsenites?

4

Tetrathioarsenate thioarsenate standards

• First detection of thioarsenates and

2

3

elle

d C

N

Dithioarsenate

As2S3

A S

thioarsenites in the same solution

1

2

mod

e

Monothioarsenate

DithioarsenateAs4S4

011867 11868 11869 11870 11871 11872

Na-ArsenateNa-Arsenite

E0 [eV]

Can the occurrence of thioarsenates and thioarsenites be predicted by thermodynamic models?

Can the occurrence of thioarsenates and thioarsenites be predicted by thermodynamic models?

pH

be predicted by thermodynamic models?

TetrathioarsenateH 9 5 d 12 3

pH 6.3 TetrathioarsenateT ithi it

pH 5.8 Trithio-it

pH 2.3 Trithio-it + A S

1 00E-02

1.00E-012 3 4 5 6 7 8 9 10 11 12

pH pH 9.5 and 12.3Trithioarsenitearsenitearsenite + As2S3

1.00E-03

1.00E 02

H3AsS3As2S3 precipitation

1.00E-04

ion

[mol

/L] H2AsS3-

AsS4-3

AsO4-3

1.00E-06

1.00E-05

conc

entr

at

As tot

1.00E-07

1.00E-08

Projekte• Charakterisierung Thioarsenate/Thioarsenite mit IC-ICP-MS, XAS, RAMAN Elke

DFG Emmy Noether Förderung: Hydrogeochemische Speziierung von As, Au, Cu in Fe-S-Systemen unter Berücksichtigung abiotischer und mikrobiell katalysierter Wechselwirkungen

• Conny: Mikrobiell katalysierte Speziierung in As-Au-S Geothermalsystemen (Yellowstone, Neuseeland) Elke & April (DAAD RISE Praktikantin) As-S + Fe Ulli DA B d t i t diä S S i fü A R d dl tl th l Ulli: DA Bedeutung intermediärer S-Spezies für As-Redoxumwandlungen entlang geothermaler Drainagekanäle in Yellowstone NP (Photometrie vs. ionensensitive Elektrode)

• Abiotische Thioarsen-Speziierung im Fe(II)/Fe(III) System (Probenstabilisierung, Bildung von Fe-As-S Komplexen, Thioarsenat-Sorption/Remobilisierung auf Fe-Hydroxiden und Fe-Sulfiden)p p g y )

• Abiotische Reaktionen von Au- und Cu-Thioarsen-Spezies (Chile, etc.)

DFG Forschergruppe etrap• Identifikation von Polysulfiden und ihre Bedeutung als intermediäre Schwefel-Spezies für

Elektronentransferprozesse in anoxischen Aquiferen Sasan: Derivatisierung Polysulfide, Methoden-Setup RP-IC-ICP-MS Polysulfide als Elektronenshuttle bei indirekter FeOOH Reduktion durch Sulfurospirillum deleyianum Polysulfide als Elektronenshuttle bei indirekter FeOOH-Reduktion durch Sulfurospirillum deleyianum

Postdocs• Sasan: Organo-As-S-Verbindungen, As in Lebensmitteln / Toxikologie…?g g g• Nathaniel Wilson: Thioantimonate, Humboldt-Postdoc-Stipendium beantragt

MSc OS3 Toxizitätstests mit Vibrio fischeri für Thioantimonate