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Microchemical Journal 130 (2017) 162167

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Experimental evaluation of sampling, storage and analytical protocols for

measuring arsenic speciation in sulphidic hot spring waters
Katrin Hug a,,1, William A. Maher b,, Simon Foster b, Frank Krikowa b, John W. Moreau a

Geomicrobiology Laboratory, School of Earth Sciences, University of Melbourne, Parkville, VIC 3010, Australia
Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, ACT 2601, Australia

a r t i c l e

i n f o

Article history:
Received 9 August 2016
Accepted 15 August 2016
Available online 28 August 2016
Arsenic speciation

a b s t r a c t
Recently developed analytical techniques allowed for the detection of a range of dissolved arsenic-sulphur species in sulphur-rich environments. These so called thioarsenates are unstable, however, and can degrade upon
handling and storage. An experiment evaluating the effect of exposure to air on arsenic and sulphur-enriched
geothermal waters demonstrated a near to complete loss of thioarsenate species to arsenite or arsenate during
short oxidation times. In contrast, thioarsenic standards were stable for the duration of analysis in spite of exposure to air. For samples containing thio-methylated arsenic species, the extent of oxidation varied for different
methylated arsenic species. This study recommends ash freezing of samples in liquid nitrogen immediately
after recovery and further storage under anaerobic conditions at 80 C. A second experiment to test the efciencies of different HPLC columns for separating arsenic species resulted in the preference for an IonPac column
with NaOH as the mobile phase when analysing arsenic thioanions, over the commonly used PEEK PRP-X100
anion exchange and Atlantis C18 reverse phase column with ammonium phosphate mobile phases. Distinct separation of thio-methylated arsenic species with the IonPac column, however, was not successful potentially due
to matrix components. Acceptable detection, separation and quantication of thio-methylated arsenic species
were only achieved with the Atlantis C18 column. This study shows that preservation and analysis of samples
is matrix dependent, which holds important implications for efforts to interpret arsenic speciation in geothermal
waters, especially those of low pH (23), low oxygen (49% saturation), low iron (5 mg L1) and high sulphur
concentrations (91 mg L1).
2016 Elsevier B.V. All rights reserved.

1. Introduction

The majority of geothermal waters, however, contain sulphur

(e.g., [6]), which has a higher afnity for arsenic than does oxygen
[18]. In the presence of sulphide, low pH and redox conditions, As-S species can be formed (Fig. 1).
Arsenite can be transformed by reaction with either sulphide or elemental sulphur into dissolved arsenic-sulphur species referred to as
thioarsenate species i.e. mono- di- and trithioarsenate, with sulphide
or elemental sulphur [14,21], e.g., as follows:

Accurate measurement of arsenic speciation is critical for understanding the arsenic biogeochemical cycle in hydrothermal systems.
Until recently the inorganic oxyanions arsenate and arsenite were believed to be the only dissolved arsenic species in hydrothermal waters
[1,8,22,25]. Arsenic bonds with free oxygen and forms arsenic
oxyanions in different oxidation states: arsenite (AsIIIO33 ) is stable
under reduced geochemical conditions and arsenate (AsVO3
4 ) under
more oxidising conditions [20]. An increase in redox potential promotes
the oxidation of arsenite to arsenate, while an increase in pH results in
deprotonation of protonated arsenic species.

H3 AsO3 HS H2 AsO3 S H2

H3 AsO3 S0 H2 AsO3 S H

Corresponding authors.
E-mail addresses: (K. Hug),
(W.A. Maher).
Present address: Institute of Groundwater Ecology, Helmholtz Center Munich,
German Research Center for Environmental Health, 85764 Neuherberg, Germany.

In addition to abiotic thioarsenate transformations, microorganisms

are also able to transform thioarsenate species directly via arsenic methylation, an arsenic resistance mechanism [7,10,11,16,19], or indirectly
via sulphur metabolism [6].
In order to identify and distinguish between abiotic and biotic arsenic transformation processes, it is essential to quantify all inorganic and
organic arsenic oxy- and thioanions present. In this respect, thioarsenic
species are more difcult to measure than arsenic oxyanions as a result
0026-265X/ 2016 Elsevier B.V. All rights reserved.

K. Hug et al. / Microchemical Journal 130 (2017) 162167


critical. Acidication and storage at 4 C is a commonly used preservation technique [25]. In sulphide- or sulphur-rich waters, however, acidication can potentially lead to total dissolved arsenic loss via induced
arsenic-sulphur precipitation under low pH [12] and storage of samples
at 4 C could lead to thioarsenate degradation, due to the rapid oxidation
of arsenic thioanions compared to oxyanions [12].
For analysis, high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLCICPMS) using an
IonPac column and a sodium hydroxide gradient is a well-established laboratory method for thioarsenate speciation [13,21,23], while an Atlantis
C18 column with phosphate buffer is commonly used to separate thiomethylated arsenic species [9]. These separation techniques, however, require an efcacy evaluation with different sample types. In this paper we
describe our experiences in preserving geothermal samples and the analysis of thioarsenates and methylated thioarsenic species in arsenicenriched hot spring waters by HPLCICPMS.
Fig. 1. EhpH diagrams for the AsOHS system (S = 1 104 M and As =
2 105 M) (modied from [2]). The diagram is based on the arsenic thioanions and
thermodynamic data is provided in [4].

of their lower stability with respect to potential oxidation. Previous

studies have described thioarsenic species' stability under a range of sulphide concentrations, pH values and oxygen saturations [2,4,14]. Helz
and Tossell [4] evaluated Gibbs free energy for the formation values of
thioarsenate formation reactions (1 atm, 298 K) and concluded that,
in the presence of sulphide, arsenate is the most stable arsenic species,
followed by arsenite, MTA, DTA, TriTA and TetraTA.

G0f H3 AsO3 640:03 kJ mol


G0f H3 AsO4 766:75 kJ mol

G0f H3 AsSO3 620:2 kJ mol

G0f H3 AsS2 O2 411:5 kJ mol

G0f H3 AsS3 O

222:2 kJ mol

G0f H3 AsS4 27:7 kJ mol

2. Experimental methods
2.1. Field site and water chemistry
The sampling sites are situated in the Taupo Volcanic Zone (TVZ) at
Waiotapu (Fig. 2) and were selected on the basis of distinctive physical
and chemical characteristics such as pH, temperature, redox potential
and sulphur concentration, all factors known to inuence arsenic speciation [12,14,21,26]. In close vicinity of Champagne Pool (CPr), the biggest and most prominent feature in the area, Alum Cliffs (AC), stream
to Frying Pan (FPs) and pool at Frying Pan (FPp) are characterised
by low pH and compared to Champagne Pool higher dissolved oxygen
saturation and redox potential, which indicates mixing of deep hydrothermal uids with oxygenated water close to the surface. Temperature,
pH, redox potential and dissolved oxygen saturation (DO%) were measured in situ using a Professional Plus multimeter (YSI, USA). The
pools in this area contain signicant sulphur but low iron concentrations, which were measured via inductively coupled plasma mass spectrometry (ICP-MS).
2.2. Sampling and storage
Water samples for arsenic speciation and quantication were collected in February 2012. Each suite of samples was stored in opaque
125 mL high-density polyethylene bottles (Nalgene, USA) washed

Oxic conditions deplete thioarsenate species very quickly via conversion to less thiolated arsenic species and eventually arsenite [12].
Planer-Friedrich et al. [14] showed that the [SH]:[OH] activity ratio
affects the stability of thioarsenic species. When [SH] N [OH] under
anaerobic conditions, the most stable arsenic species is thioarsenite,
whereas under aerobic conditions thioarsenate is more stable. When
[SH] b [OH] under either aerobic or anaerobic conditions, the most
stable arsenic species is arsenite, as thiolated arsenic species would be
unstable at excess OH. Arsenite oxidation, however, only starts to
occur in signicant levels when the thioarsenate species are almost
completely transformed to arsenite.
H2 AsOS3 2:5 O2 H2 AsO2 S
2 SO4

H2 AsO2 S
2 2:5 O2 H2 AsO3 S SO4


H2 AsO3 S H H3 AsO3 S0


H3 AsO3 O2 H2 AsO4


These artefacts produced by thioarsenate species oxidation, and to

some extent also arsenite oxidation, can occur during sampling and
storage [12]. Therefore, correct preservation of samples for analysis is

Fig. 2. Sampling sites at Waiotapu in the Taupo Volcanic Zone on the North Island of New
Zealand (photo credit: Google Earth). Alum Cliffs (AC), stream to Frying Pan (FPs), Frying
Pan pool (FPp).


K. Hug et al. / Microchemical Journal 130 (2017) 162167

with 1 M HCl and triple-rinsed with sterile water (Pall Corporation,

USA). The nal rinse immediately prior to sample collection was with
the respective hot spring water. The samples were ltered through sterile 0.22 m pore size Sterivex-GP polyethersulphone syringe lters
(Merck Millipore, Germany) into sample bottles, immediately ash frozen on liquid nitrogen, and stored in anoxic bags together with oxygenscrubbing GasPak EZ Anaerobe paper sachets (BD Biosciences, USA).
They were transported on dry ice to the laboratory, where they were
stored at 80 C until analysis.

2.3. Preparation of arsenic standards

Stock solutions of arsenite, arsenate, methylarsonic acid (MA) and
dimethylarsinic acid (DMA) were prepared as standards (1000 mg L1)
by dissolving sodium arsenite, sodium arsenate heptahydrate
(AJAX Laboratory Chemicals), disodium monomethylarsenic and sodium dimethylarsenic (Sigma-Aldrich, Australia), respectively, in
deionized water (Sartorius, Germany). Sodium monothioarsenate
(Na 3AsO3 S 7H2 O), sodium dithioarsenate (Na 3 AsO2 S 2 7H2 O),
sodium trithioarsenate (Na3AsOS3 10H2O) and sodium tetrathioarsenate
(Na3AsS4 8H2O) were synthesized as described by [17]. Na3AsO3S7H2O
was prepared by adding sulphur (0.045 mol S) to a mixture of As2O3
(0.05 mol As) and NaOH (0.15 mol Na) in 20 mL H2O and heating at
90 C for 2 h. The excess sulphur was ltered off and the solution cooled
down slowly to 4 C. The colourless crystals obtained were dried under
a vacuum for 1 h. Na3AsO2S2 7H2O was synthesized by adding sulphur
(0.18 mol S) to a mixture of As2O3 (0.05 mol As) and NaOH (0.15 mol
Na) in 20 mL H2O and heated up to 70 C for 2 days. The excess colloidal
sulphur was ltered off and the crude crystals were washed with ethanol
to remove all residual sulphur. The crude product was subsequently recrystallized from water and ne colourless needles were obtained at
4 C and dried under a vacuum for 1 h. All other thioarsenate compounds
were obtained using the general approach used by [26] by mixing an excess of sodium bisulphide (NaHS, formed by dissolving NaS2 in deionized
water) to sodium arsenite to obtain a mixed standard. A 1:1 ratio of As:S
yielded arsenite and monothioarsenate, whereas a 1:100 M ratio of As:S
yielded di-, tri- and tetrathioarsenate. All thioarsenate compounds were
stored under nitrogen at 4 C. Monomethylmonothioarsenate (MTMA)
was synthesised by adding a saturated sulphide solution (deionized
water purged with H2S for 1 h) to the monomethylarsenate (MA) standard and incubating for 30 min. Dimethylmonothioarsenate (MTDMA)
was synthesised using the protocol by Raml et al. [15]. The same procedure was applied to synthesise dimethyldithioarsenate (DTDMA), except
that the pH was adjusted to 6 and no ethylacetate extraction was performed. All thioarsenate standards were stored under nitrogen at 4 C.

2.4. Total arsenic and arsenic speciation analyses

Immediately prior to analysis, the ash-frozen samples were thawed
under nitrogen in an anaerobic chamber. In a nitrogen-purged glove
box a volume of 1 mL of each sample was transferred into Teon screw
cap glass vials for HPLCICPMS analysis. Dilution of samples was avoided
by use on precision injection aliquots down to 1 L. Total arsenic concentrations in water samples were measured in triplicate by electrothermal
atomic absorption spectroscopy with a Perkin Elmer AAnalyst 600 graphite furnace using a previously published protocol [3] with optimum concentrations of Pd/Mg (0.15 mol (Pd) + 0.4 mol (Mg)).
Arsenic species were measured via high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLCICPMS). Arsenic thio- and oxyanions were measured using
an IonPac AS16 Analytical Column (250 mm 4 mm) (Dionex, Sunnyvale, CA, USA) eluted with a NaOH gradient (1100 mM) at 25 C and
using a ow rate of 1 mL min 1 [13,21,23]. Mobile phase A was
deionised H2O and mobile phase B was 100 mM NaOH. The gradient
was 1% B to 10% B in 5 min, 10% B to 100% B in 15 min, hold 100% B
for 5 min, 1% B in 5 min, for a total analysis time of 30 min [9]. Thiomethylated arsenic species were measured using an Atlantis C18 column (150 mm 4.6 mm, 5 m). The mobile phase consisted of
20 mM ammonium phosphate buffer at pH 3, a ow rate of
1.0 mL min 1 and a column temperature of 25 C [9].
3. Results and discussion
3.1. Water chemistry
The inner rim of Champagne Pool (CPr) exhibited mildly acidic
(pH 5.5) as well as reducing conditions (75 mV) and a low DO saturation (20%). The highly acidic sites AC, FPs and FPp (pH 2.2 to 2.6) at
Waiotapu showed signs of oxidation with DO saturation of up to 49%
and redox values from +228 to +249 mV. The pools in this area contain signicant sulphur (N 91 mg L 1) but low iron concentrations
(Table 1).
3.2. Sampling and storage, arsenic speciation after exposure to oxygen
Thawed water samples from site AC contained 90% arsenic
oxyanions and 10% arsenic thioanions (Fig. 3). Arsenic oxyanions
consisted of 85% arsenite and 5% arsenate, and arsenic thioanions
consisted of 3% monothioarsenate (MTA) and 7% dithioarsenate (DTA)
as a proportion of the total arsenic concentration (Fig. 3). At site FPs,
water samples contained 70% arsenic oxyanions, 27% arsenic thioanions
and 3% methylated arsenic species (Fig. 3). Arsenic oxyanions consisted

Table 1
Temperature, pH, redox potential and dissolved oxygen saturation (DO%) at Waiotapu sites, New Zealand.
Temperature (C)
(0.2 C)

pH (0.2)

Redox potential (mV) (20 mV)

DO (%) (2%)

Fe concentration (mg L1)

Alum Cliffs





1.02 0.02


Stream to Frying Pan





1.97 0.02


Pool to Frying Pan





5 0.04


Rim Champagne Pool






Site ID

Site description



K. Hug et al. / Microchemical Journal 130 (2017) 162167


Table 2
MTA spiked environmental Champagne Pool sample CPr held at room temperature for the
duration of 4 days with measurements on the rst and fourth day over a period of 4 h.
MTA: monothioarsenate, DTA: dithioarsenate, TriTA: trithioarsenate, TetraTA:





% Total
peak area
Day 1
Day 4
Mean SD
% peak area (n = 6)
Retention time (n = 6)
Fig. 3. Arsenic speciation in water samples from Waiotapu, New Zealand before
and after oxygen exposure. MTA: monothioarsenate, DTA: dithioarsenate, MA:
monomethylarsenate, DMA: dimethylarsenate, MTMA: monomethylmonothioarsenate,
MTDMA: dimethylmonothioarsenate, DTDMA: dimethyldithioarsenate. AC: Alum Cliffs,
FPs: stream to Frying Pan.

of 46% arsenite and 24% arsenate, arsenic thioanions consisted of 6%

monothioarsenate (MTA) and 21% dithioarsenate (DTA), and methylated arsenic species consisted of 1% monomethylmonothioarsenate
(MTMA) and 2% dimethylmonothioarsenate (MTDMA) as a proportion
of the total arsenic concentration (Fig. 3).
After 3 h of oxygen exposure, the arsenic species in the water samples changed signicantly (Fig. 3). At AC, all thioarsenates disappeared
and 100% of the total arsenic concentration was represented by arsenic
oxyanions, with 69% arsenite and 31% arsenate. Thioarsenate concentrations that accounted for 10% of the total arsenic concentration at AC
oxidised to arsenite and eventually arsenate, which showed a six-fold
increase. At FPs, the thioarsenate species almost disappeared, with 2%
of MTA left in the sample. Arsenite remained nearly stable at 45% of
the total arsenic concentration, whereas arsenate increased to 43% of
the total arsenic concentration. The methylated arsenic species
MTDMA remained stable at 2% of the total arsenic concentration,
whereas MTMA disappeared, and 1% dimetyldithioarsenate (DTDMA),
5% monomethylarsenate (MA) and 3% dimethylarsenate (DMA) were
detected. Thioarsenate concentrations that accounted for 27% of the
total arsenic concentration oxidised to arsenite and eventually arsenate,
which showed a two-fold increase.
The experiment testing the stability of individual arsenic species
under oxygen exposure demonstrates the near to complete loss of
thioarsenates and their transformation into arsenite or arsenate in environmental samples. The remaining MTA after oxidation can be explained by the higher initial thioarsenate concentration at FPs
compared to AC. A possible explanation for the consistent arsenite concentration before and after oxidation at FPs is a faster transformation reaction of thioarsenate to arsenite compared to the subsequent slower
arsenite oxidation [12]. Sample oxidation also leads to methylated arsenic species transformation. MTMA transformed into MA, potentially via
sulphide oxidation, which breaks the bonds between sulphur and arsenic. MTDMA concentrations, however, were consistent throughout oxidation, which suggests that MTDMA is more stable than MTMA. The
appearance of DTDMA and DMA after oxidation, and a higher than expected MA concentration after MTMA oxidation might be an indication
for retention of methyltransferase activity derived from dead cells (i.e.
killed by ash freezing) during oxidation. An environmental sample
spiked with a MTA standard showed that if samples are thawed under
oxygen-free conditions no loss of thioarsenates occurs during chromatography (Table 2).











58 1
21 1
4.1 0.8
12.6 0.1 14.2 0.1 16 0.1

17 1
0.6 0.1
17.7 0.1 19.1 0.3

This experiment shows that oxidation has to be instantly and strictly

eliminated in arsenic and sulphur-rich water samples prior to arsenic
speciation analysis. General use of dry ice (78.5 C) for ash freezing
without the addition of ethanol or isopropanol to create an ultra-cold
slurry could still result in introduction of oxygen to the samples. In contrast, ash-freezing using liquid nitrogen ( 196 C) instantly freezes
the sample without crystallization and precludes artefacts caused by
the inux of oxygen. In general, liquid freezing agents are probably
more effective in excluding oxygen evenly across the sampling container. Problems with precipitation of iron in the sample during liquid freezing, illustrated by other authors, are unlikely to occur in this type of
samples, which are low in iron (b 5 mg L1). Additionally, geothermal
waters should be lled into sample containers with zero headspace
prior to ash freezing with liquid nitrogen, thereby minimizing oxidation potentials [5]. Optimized preservation can be accomplished when
using oxygen-scrubbing sachets, eliminating all oxygen within the

Fig. 4. HPLCICPMS chromatogram of arsenic species standards using the IonPac column.
Dimethylarsenate (DMA), dimethylmonothioarsenate (MTDMA), monomethylarsenate
(MA), monomethylmonothioarsenate (MTMA), monothioarsenate (MTA), dithioarseante
(DTA), and trithioarsenate (TriTA). cps: counts per second.


K. Hug et al. / Microchemical Journal 130 (2017) 162167

gas-tight sampling bags, in which sample containers were immediately

stored after ash freezing. The subsequent cryo-storage of samples even
at 20 C could lead to vitreous ice recrystallisation resulting in potential arsenic speciation transformations, which is expected to be negligible at 80 C. Slow oxidation reactions during storage in an 80 C
freezer, however, should be prevented by the oxygen-scrubbing sachets
that were placed into the gas-tight sampling bags containing the sample
containers. To prevent oxidation during sample preparation for HPLC
ICPMS analysis, the samples should be thawed under nitrogen.
3.3. Separation and detection of inorganic arsenic oxy- and thioanions, as
well as thio-methylated arsenic species via different HPLC-columns
3.3.1. IonPac
The chromatogram of arsenic standards using the IonPac column
showed good separation of arsenate, monomethylated arsenic and
thioarsenate species but no distinct separation of DMA, arsenite, and
MTDMA (Fig. 4). This issue is exacerbated in the environmental water
sample FPp (Fig. 5) due to the presence of major matrix components
such as sulphur species and co-eluting salts.
Baseline separation of thio-methylated arsenic species was, however, not achieved with all samples (Fig. 5), and represents a challenge for
arsenic species quantication. Optimisation of thio-methylated arsenic
species separation has been achieved by Wallschlger and London
[24], using an IonPac AG-16 column and NAOH gradient. However,
water samples, such as the ones used in this study, can contain appreciable amounts of cations and sulphur species that potentially co-elute
with thio-methylated arsenic peaks or cause them to split, making
quantication difcult.
Additionally, the common approach to dilute geochemical samples
containing high arsenic concentrations cannot be used, as reduction of
sulphide content in samples by dilution may cause the dissociation of
thioarsenate complexes. In this study, the approach taken was to inject
small sample amounts rather than dilution. The analysis of replicates for
an 500 g L1 As(III) and 1000 g L1 As(V) standards injection showed
an RSD of 2.94.9%, 2.34.2% and 1.62.7% for 1, 5 and 50 L injections
(n = 5) showing that good reproducibility was obtained (Fig. 6).
3.3.2. Atlantis C18 column
The use of the Atlantis column gave a reproducible baseline separation of the arsenic standards dimethylmonothioarsenate and

Fig. 6. Replicate injections (n = 5) of 500 g L1 As(III) and 1000 g L1 As(V) standard at

1 L volume. cps: counts per second.

Fig. 7. HPLCICPMS chromatogram of arsenic species standards using the Atlantis

C18 column. Dimethylarsenate (DMA), dimethylmonothioarsenate (MTDMA),
monomethylarsenate (MA), monomethylmonothioarsenate (MTMA). cps: counts
per second.

monomethylmonothioarsenate (Fig. 7), which was also visible in spiked

water samples (Fig. 8). Thio-methylated standards are stable for weeks
and no transformations occur during chromatographic runs [9]. The column appears not to be sensitive to the sample matrix (cations and sulphur species), as it does not appear to inuence separation efciencies.
4. Conclusions
Given the unstable character of arsenic with relatively low iron content in sulphidic geothermal waters, instantaneous ash freezing of
water samples in liquid nitrogen at 196 C in sample containers
with zero headspace with oxygen-scrubbing sachets in secondary gastight containment placed at 80 C, thawing of samples under nitrogen, and the renouncement of sample dilution is adequate in preventing
thioarsenate species conversion prior to analysis. As previously reported, separation and quantication of thioarsenates can be obtained on
an IonPac column, but separation and quantication of thiomethylated arsenic species requires the use of an Atlantis C18 column.

Fig. 5. HPLCICPMS chromatogram of arsenic species at Frying Pan pool (FPp) using the
IonPac column. Dimethylarsenate (DMA), dimethylmonothioarsenate (MTDMA),
monomethylmonothioarsenate (MTMA), monothioarsenate (MTA), dithioarsenate
(DTA), and trithioarsenate (TriTA). cps: counts per second.

We thank Waiotapu Wonderland and the Ngti Tahu Ngti Whaoa

Runanga for access to Champagne Pool, and ongoing logistical support
for this project. We also thank Matthew Stott and Karen Houghton at
GNS Science, New Zealand, for eld support during sampling. We acknowledge the funding provided for writing this publication by the

K. Hug et al. / Microchemical Journal 130 (2017) 162167


Fig. 8. HPLCICPMS chromatogram of a spiked water sample using the Atlantis C18 column. Dimethylarsenate (DMA), dimethylmonothioarsenate, monomethylarsenate (MA),
dimethylmonothioarsenate (MTDMA), monomethylmonothioarsenate (MTMA). cps: counts per second.

Albert Shimmins Memorial Fund (UTR7.220) to K.H. and Dyason Fellowship to J.W.M.
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