Forensic Toxicol (2007) 25:92–95
DOI 10.1007/s11419-007-0033-7
SHORT COMMUNICATION
Thiosulfate in human urine following minor exposure to hydrogen sulfide:
implications for forensic analysis of poisoning
Michael Durand · Philip Weinstein
Received: 3 May 2007 / Accepted: 4 July 2007 / Published online: 15 September 2007
© Japanese Association of Forensic Toxicology and Springer 2007
Abstract Thiosulfate is a sulfide metabolite and a bio-
logical marker, especially in urine, of exposure to hydro-
gen sulfide gas (H2S). In many suspected poisoning cases,
victims are known to be exposed to low concentrations
of H2S, but it is difficult to establish the degree of expo-
sure to H2S responsible for the poisoning. In such cases
it is necessary to account for a possible chronic exposure
signal by subtracting any background thiosulfate from
the measured total. However, no data exist on the back-
ground levels of thiosulfate in individuals exposed to
relatively low levels of H2S. We obtained preexposure
and postexposure urine thiosulfate data from eight indi-
viduals exposed to H2S in the ppb to low ppm range.
Mean thiosulfate concentrations in urine increased from
4.6 to 11.5 µmol/l following exposure (P ≤ 0.05). When
normalized against creatinine, mean thiosulfate increased
from 7.2 to 9.8 µmol/mol (P ≤ 0.05). However, positive
changes were not consistent between individuals. Whilst
these findings support the idea that relatively minor
exposures to H2S can be confirmed by thiosulfate in
urine, it also suggests that the response of this biomarker
to minor H2S exposure may vary significantly between
individuals.
Keywords Hydrogen sulfide poisoning · Thiosulfate ·
Geothermal gas · Minor exposure to H2S · Biomarker ·
Rotorua
M. Durand (*)
Natural Hazards Research Centre, University of Canterbury,
Private Bag 4800, Christchurch 8140, New Zealand
e-mail: michaeldurand2004@yahoo.co.uk
P. Weinstein
School of Population Health, University of Western Australia,
Crawley, Western Australia, Australia
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Introduction
The occupational and community hazard posed by
exposure to hydrogen sulfide (H2S) gas exists in the fol-
lowing three ways. First, very rapid loss of consciousness
after exposure to H2S at concentrations over 1000 ppm,
often called the “knockdown” or the “slaughterhouse
sledgehammer” effect. Knockdown is the result of H2S
binding to mitochondrial c cytochrome oxidase in cellu-
lar metabolism, which then inhibits oxidative phosphor-
ylation and causes cellular hypoxia [1,2]. Second, loss of
mobility and/or consciousness when victims are exposed
to ∼200–500 ppm H2S or more for several minutes. These
cases are often fatal because secondary effects can be
respiratory arrest, hypoxia, pulmonary edema, and coma
[3]. Most of what is known about H2S exposure–response
relationships in humans has been learned from the
many fatal or near-fatal accidental poisonings that have
occurred in industrial settings [3–8]. The third hazard
type is chronic and low-level exposures that are typical
of “acceptable” occupational exposures and community
exposure situations near factories or geothermal features
emitting the gas [9–11]. In community and occupational
exposure settings, health ailments have been observed
including neurological and respiratory problems such as
fatigue, poor memory, dizziness, and elevated rates of
asthma [12–18]. Epidemiological studies have stressed
the potentially dangerous effects of low-level exposures,
despite the fact that clear exposure–response relation-
ships for these exposures are yet to be established in
toxicological studies.
In cases of suspected poisoning by H2S, biological
markers can be useful tools to resolve exposure to H2S
as a contributory factor. Sulfide in blood is a reliable
indicator of poisoning in fatal cases [5,19–21]. In one
Forensic Toxicol (2007) 25:92–95
case, postmortem sulfide levels measured in the blood of
four victims who were exposed to H2S levels of more
than 4000 ppm were elevated by up to 190 times above
the normal levels of unexposed individuals [6]. In non-
fatal poisonings, sulfide is rapidly metabolized and
thiosulfate in urine is a better indicator of exposure to
H2S. In laboratory experiments, a volunteer exposed to
18 ppm H2S for 30 min excreted thiosulfate in urine at a
rate that increased linearly to a peak of 30 µmol/mol
creatinine 15 h following the exposure; this level is 18 to
75 times the normal level [22]. Through animal experi-
ments and analyses of fatal and nonfatal human poison-
ings, Kage et al. [5,23,24] stressed that thiosulfate in
urine “is the only indicator to prove hydrogen sulfide
poisoning in nonfatal cases” [5].
However, a diagnostic problem with thiosulfate is
that its presence in urine can be a signal of either acute
poisoning or chronic exposure, or both. In many occu-
pational exposure situations, poisoning victims may
have been exposed to low levels of H2S in the short or
long term prior to exposure to high and potentially poi-
soning concentrations in a short term. Therefore, in
investigating a suspected acute exposure case it is neces-
sary to subtract any possible background thiosulfate
level from the measured urine thiosulfate level. However,
no data exist on the response of this biomarker to low-
level H2S exposure. To obtain such baseline data, we
exposed eight volunteers to H2S in the high ppb to low
ppm range for 2 days in an urbanized geothermal area.
In this short report, we describe thiosulfate levels in
human urine obtained from individuals before and after
exposure to H2S.
Materials and methods
Exposure of humans to H2S
In this experiment, eight volunteers were exposed to H2S
in Rotorua (New Zealand), a city built upon an actively
degassing geothermal field. H2S emissions from the field
are known to elevate H2S levels in ambient air and
indoors [11,18,25]. The volunteers, who lived elsewhere
in New Zealand, visited the city for 2 days and were
exposed to H2S in two ways: (1) indoors and outdoors
through normal activities at their hotel accommodation
and in its surroundings (located within the main geother-
mal area), and (2) by two periods of exposure to elevated
gas concentrations: a visit to a nearby commercial spa
pool complex (<1 h) and a short walk (<25 min) in an
area of sulfur flats where thermal features emitting H2S
are concentrated. Subjects 5, 6, and 8 did not attend the
spa pool. All subjects refrained from vigorous exercise
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during the experiment. None were habitual smokers or
suffered from any chest conditions. Their exposure was
monitored through the experiment with passive H2S
detector tubes (Dositubes No. 4D; Gastec, Kanagawa,
Japan) worn on the lapel of each participant (except for
during the spa pool visit, when tubes were not worn) and
two real-time gas sensors (ToxiRAE II and QRAE Plus;
RAE Systems, San Jose, CA, USA). Further details of
the field site, subjects, exposure and exposure monitor-
ing will be published in another report (Durand et al., in
preparation).
Ethical consent for the human experiments was given
by the New Zealand Ministry of Health Upper South A
Ethics Committee. The volunteers gave informed consent
and agreed to take part after its implications had been
explained.
Urine collection and analysis
Urine samples were taken within 30 min of arrival in
Rotorua; following the main exposure periods they were
also taken 1–2 h before departure. Samples were frozen
immediately after collection. Urine collection was timed
to coincide with our hypothesized “peak flow” of thio-
sulfate after H2S exposure at the spa pool, which was
suggested by Kangas and Savolainan [22] to occur not
later than 15 h following the exposure. Thiosulfate
was later measured using a modified method based on
that of Kage et al. [26]. Thiosulfate was converted to
tetrathionate by reaction with iodine. The tetrathionate
was then further converted to bis(pentafluorobenzyl)di
sulfide by reaction with pentafluorobenzyl bromide in
acetone/ethyl acetate. 1,3,5-Tribromobenzene was used
as an internal standard (IS). The resulting bis(pentaflu
orobenzyl)disulfide and the IS were analyzed by gas
chromatography-mass spectrometry with selected ion
monitoring using ions at m/z 181 and 426 for bis(pent
afluorobenzyl)disulfide and m/z 235 and 314 for the
IS.
A series of standards for calibration were prepared by
spiking water with sodium thiosulfate to give a range of
thiosulfate concentrations from 4.2 to 114 µmol/l. These
standards were put through the same method along with
the urine samples being analyzed. From each of the urine
samples, 0.2 ml was taken and analyzed in duplicate. The
urine samples were also analyzed for creatinine levels by
an enzyme multiplied immunoassay (EMIT) method.
The Wilcoxon signed-rank test for matched pairs [27], a
nonparametric test for dependant variables, was used to
determine the statistical significance of the postexposure
changes in thiosulfate.
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