Chemosphere 189 (2017) 171e179

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Chemosphere
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Characterisation of artisanal mine waste on Buru Island, Indonesia and

toxicity to the brittle star Amphipholis squamata
Amanda J. Reichelt-Brushett a, b, *, Bernard Thomas b, Pelli L. Howe a, b, Yusthinus Male c,
Malcolm W. Clark a, b
a
Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW, Australia
b
School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW, Australia
c
Department of Chemistry, Faculty of Mathematics and Natural Science, Pattimura University, Ambon, Indonesia

h i g h l i g h t s

Mercury contaminated artisanal mine tailings from Buru Island, Indonesia were investigated.

Novel toxicity tests were completed using the marine brittle star Aphipholis squamata.

Toxicity test species potentially useful for deep sea risk assessment.

The 96 h LC50 was estimated at 6.7% v/v tailings or 4.0 mg/kg total mercury.

An estimated 48 h EC50 was 7.3% v/v tailings or 14.4 mg/kg total mercury.

a r t i c l e i n f o a b s t r a c t

Article history: Artisanal small-scale gold mining (ASGM) using mercury (Hg) amalgamation commenced on Buru Island,
Received 13 January 2017 Indonesia, in 2012, but was halted in 2015 due to concerns of widespread Hg contamination. Much of the
Received in revised form Hg used in the mining process is lost to trommel waste which is disposed of in settlement ponds that
17 August 2017
drain into adjacent waterways and into Kayeli Bay. Several thousand unmanaged trommel sites and
Accepted 12 September 2017
Available online 12 September 2017
associated tailing ponds exist on Buru Island. This study shows that waste from the Marloso trommel at
the Gogrea site contained 203 mg/kg total Hg (THg), with a negligible proportion present as bioavailable
Handling Editor: Jian-Ying Hu methyl Hg (MeHg) and a low total organic carbon content. There are currently very few tools available for
ecotoxicological risk assessment of mine tailings for tropical marine ecosystems, and we support the
Keywords: development of Tailings Toxicity Tests (TTTs) and describe laboratory toxicity test methods using the
Mercury cosmopolitan benthic echinoderm Amphipholis squamata. Undiluted trommel waste caused 100% mor-
Sediment toxicity test tality of A. squamata within 48 h, and a 96-h LC50 of 6.7% w/w trommel waste (4 mg/kg THg) was
Tropical estimated. Sub-lethal effects on the water vascular system of the brittle star were assessed by quanti-
Marine
cation of the Ability to Right Itself (ARI), and a 48-h EC50 of 7.3% w/w trommel waste (14.4 mg/kg THg)
Gold mining
was estimated. The results show that trommel waste produced on Buru Island is highly contaminated
with THg and is acutely toxic, raising serious concern for receiving ecosystems where Hg methylation to
more toxic and bioavailable forms is likely.
2017 Elsevier Ltd. All rights reserved.

1. Introduction

1.1. Artisanal small-scale gold mining (ASGM) on Buru Island

In December 2011, gold (Au) was found on Mount Botak, Buru

* Corresponding author. Marine Ecology Research Centre, School of Environment,
Science and Engineering, Southern Cross University, Lismore, NSW, Australia.
E-mail address: amanda.reichelt-brushett@scu.edu.au (A.J. Reichelt-Brushett).
Island, Indonesia (Fig. 1) (Male et al., 2013). This prompted the
illegal erection of several thousand ad-hoc mines in the region, and
at a second site to the north at Gogrea in late 2012 (Fig. 1). In
artisanal small-scale gold mining (ASGM) operations such as these,

http://dx.doi.org/10.1016/j.chemosphere.2017.09.053
0045-6535/ 2017 Elsevier Ltd. All rights reserved.
172 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179
Marloso trommel
Fig. 1. Location of the Marloso trommel sample site, Buru Island, Indonesia (3 390 S, 126 780 E). Adapted from Male et al. (2013).
ore is processed in simple processing plants, locally referred to as
trommels, using whole ore mercury (Hg) amalgamation (Male et al.,
2013). This process involves bringing all the mined ore material into
contact with Hg, either by adding Hg to ball or rod mill grinders, or
passing a the nely ground rock slurry over Hg-coated copper
plates (Male et al., 2013). The ore may be processed several times,
extracting up to 30% of Au (Schmidt, 2012), however an estimated
20 g of Hg are consumed (i.e. not recovered) for each troy ounce of
Au extracted in Indonesian ASGM operations (Telmer and Stapper,
2007; Telmer and Veiga, 2009). On Buru Island, trommel waste is
washed into settlement ponds which typically drain into local
waterways and Kayeli Bay (Fig. 1).
Mercury is a contaminant of global concern (WHO, 2007), and in
July 2012 total Hg (THg) concentrations up to 838 mg/kg were
measured in waste settlement ponds on Buru Island, and concen-
trations in river and marine sediments in the catchment far
exceeded the Indonesian Standard (SNI) and the Australian and
New Zealand (ANZECC and ARMCANZ, 2000) high trigger value of
1 mg/kg THg (Male et al., 2013). Although THg concentrations are of
concern the greater risk to organisms are the organometallic
complex methyl Hg (MeHg), and particularly monomethyl Hg
(MMeHg) (Aggarwal et al., 2014; Broussard et al., 2002). Both MeHg
and MMeHg have a high water solubility, are lipophilic, bio-
concentrates in organisms, and biomagnies in food chains (Atwell
et al., 1998; Bowles et al., 2001; Kehrig et al., 2010; Lavoie et al.,
2013; Morel et al., 1998; Watras et al., 1998). Male et al. (2013)
showed that < 5% of THg present in trommel waste were bioavail-
able, organo-chelated Hg species, but that this availability
increased up to 50% in estuarine and marine sediments down-
stream of the Mount Botak site on Buru Island, showing trans-
formation of inorganic Hg in receiving environments. More recent
studies showed that in trommel waste and nearby estuarine and bay
sediment contained no greater than 0.12% MeHg yet with the high
Total Organic Carbon measured at some sites transformation of
inorganic mercury to methyl mercury was likely (Reichelt-Brushett
et al., 2017).
Mercury is persistent in the environment and Hg-contaminated
sediments may remain highly toxic for long periods after initial
contamination (Weiner and Suchanek, 2008) and be dispersed over
great distances through many abiotic and biotic means (Bank (Ed.),
2012; Brosset, 1981). Considering the magnitude and intensity of
the ASGM operations on Buru Island, and evidence of extremely
high THg levels in surrounding aquatic sediments (Male et al.,
2013), and in locally-caught seafood (Reichelt-Brushett et al.,
2017), the potential short- and long-term risks to ecosystems, as
well as humans, warrant urgent investigation. Moreover, increases
in Hg bioavailability raised serious concern regarding the long-term
potential for Hg distribution, changes in speciation from inorganic
to more bioavailable forms, and bioconcentration and bio-
magnication through food chains (Male et al., 2013).
Methylation of inorganic Hg occurs primarily in sediments
which directly exposes benthic organisms, particularly those that
burrow and/or ingest sediment, and inhabit the sediment water
interface (Merritt and Amirbahman, 2009), to higher MeHg con-
centrations than pelagic or surface-dwelling organisms (Coelho
et al., 2008). However, contaminant effects on benthic tropical
marine organisms are not well understood (Adams and Stauber,
2008), and effective tools for ecotoxicological risk assessment
relevant to tropical marine environments are generally lacking (van
Dam et al., 2008). This lack is despite the rapid increase in
contamination threats to marine ecosystems throughout the tro-
pics (Burke et al., 2011; Reichelt-Brushett, 2012), and considerable
differences in contaminant behaviour and toxicity in tropical
compared to temperate environments (Cairns et al., 1975). For
example, higher temperatures increase MeHg accumulation in
killsh (Fundulus heteroclitus; Dijkstra et al., 2013), bacterial Hg
methylation rates, and Hg consumption by plankton (Costa et al.,
2012).
1.2. Mine tailings toxicity tests
Threats to ecosystems from intentional (usually chronic) and
accidental (usually acute) release of mine tailings into freshwater,
estuarine, coastal and deep-sea environments are increasing along
with the volumes of tailings being produced (Reichelt-Brushett,
2012). As available land for tailings disposal diminishes in many
regions, direct deposition into surface and sub-surface aquatic en-
vironments is considered the most viable options for disposal of
 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179
mine waste, particularly in low or middle income countries where
legislative frameworks are often weak, or poorly enforced (e.g.
Brewer et al., 2012; Hughes et al., 2015; Vogt, 2013). Ecological
effects of tailings disposal vary depending on the composition of
the material, treatment and disposal methods, and biotic and
abiotic characteristics of the deposition environment and will
change as the material ages and disperses (Holloway et al., 2009;
Sunderland et al., 2006; Vogt, 2013). Furthermore, with
increasing interest in seabed mining a new and unique set of risk
assessment challenges exist (Reichelt-Brushett, 2012; Wedding
et al., 2015).
Laboratory toxicity tests have several important limitations in
their ability to assess ecological risk (Chapman, 1983), however
they have many benets and remain an important line of evidence
in ecotoxicological risk assessments (ANZECC and ARMCANZ,
2000). Whole sediment toxicity test methods have previously
been applied using mine tailings (e.g. Bori et al., 2016; Farkas et al.,
2017), and a method has been developed assessing tailings water
extracts toxicity on the bacterium E. coli (MetPAD; Boularbah
et al., 2006). For marine ecosystems, numerous standardised
aquatic sediment toxicity tests have been developed using
temperate species (e.g. Adams and Stauber, 2008), while fewer exist
for tropical marine species, including 10-d survival of the crab
Diogenes sp., the bivalve Donax cuneata and the polycheate Scolo-
plos sp. (Black, 2003; cited in Adams and Stauber, 2008). There is
value in developing a suite of standardised tests designed for mine
tailings, i.e. Tailings Toxicity Tests (TTTs), using organisms (such as
the brittle star Amphipholis squamata) that are relevant to potential
tailings disposal areas and interact with sediment, thus providing
tools for ecotoxicological risk assessments for mining operations
and tailings disposal.
1.3. Whole sediment toxicity tests using Amphipholis squamata
The brittle star A. squamata is a benthic ophiuroid that has been
identied as a useful toxicity test species for coastal and deep-sea
ecotoxicology (Black et al., 2015), however to our knowledge has
not been used in toxicity tests on sediment or solid waste material
prior to this study. A. squamata is a small (<5 mm disc), her-
maphroditic, brooding ophiuroid echinoderm with a cosmopolitan
distribution throughout benthic habitats in all non-polar oceans to
depths to 1300 m (Le Gac et al., 2004). A. squamata is tolerant of
laboratory culturing and testing conditions, and is relatively sen-
sitive to waterborne copper exposures compared to standard ma-
rine toxicity test species (Black et al., 2015).
Brittle stars use a water vascular system for movement, and a
promising sub-lethal endpoint has been developed for A. squamata
in this species based on the organism's Ability to Right Itself (ARI)
following manipulation to an inverted position (Black et al., 2015).
This ARI endpoint was reasonably sensitive to waterborne Cu
exposure, is easily quantied, and is ecologically relevant due to the
sensitivity to deteriorations in the functioning of the water vascular
system, which is necessary for motility, feeding, burrowing, and
predator avoidance among other activities.
This study investigates the geochemical characteristics of
trommel waste after ore processing using mercury amalgamation
Au extraction from the Gogrea ASGM site on Buru Island, and as-
sesses the acute lethal and sub-lethal toxicity of the material to
A. squamata in tropical conditions.
2. Methods
2.1. Test organism culturing
A. squamata cultures were maintained in aquaria at the Marine
173
Ecology Research Centre, Southern Cross University, for several
years prior to this study. Culture tanks were tted with live rock,
ne sediments, sand, small gravel and stones and provided with
continual aeration and a 12: 12-h photoperiod using 30-W uo-
rescent aquarium lighting. Dissolved Oxygen (DO), pH, EC and
temperature were recorded weekly prior to water exchanges
(10e50%), and ranged between 6.94 and 8.34 mg/L, 7.92e8.38,
50.1e57.9 mS/cm, and 20.3e26.3  C, respectively. Seawater used to
maintain culturing tanks was collected from Broken Head, New
South Wales (28 42016.0300 S, 153 360 55.9100 E) and was aerated until
use within 48-h of collection. Culturing tanks were provided with
Artemia sp. (Crustacea:Branchiopoda) twice weekly as a food
source.
2.2. Collection of trommel waste material
In May 2013, representative ASGM tailings were collected from
the surface of a tailings settlement pond adjacent to a trommel
processing plant in the Marloso area at the Gogrea mine site, Buru
Island, Indonesia (3170 06.500 S, 1271028.600 E), transported on ice to
the University of Pattimura, Ambon, and immediately frozen. In July
2013, tailings were dried at 35e40  C for 48 h prior to trans-
portation to Southern Cross University, Lismore, NSW, Australia. A
low drying temperature is preferred in order to minimise any
volatilisation of Hg and other potential chemical changes (e.g.
Hojdova et al., 2015). For quarantine purposes samples were
gamma irradiated at 50 kGy (60Co, 1.32 MeV); gamma irradiation is
preferred to other treatments as there is little physical or chemical
disturbance of the sample including minimal temperature change,
minimising Hg speciation changes and volatilisation compared to
other sterilisation processes such as chemical treatment or auto-
claving (Yu and Yan, 2003) although biological activity would be
halted. Following sterilisation, samples were dried a further 48 h at
40  C and stored at 24  C in individual polyethylene zip lock bags in
the dark.
2.3. Trommel waste analyses
Sub-samples (<15 g) of trommel waste were homogenised using
a Labtechnics LM 1 laboratory mill. Duplicate 0.20 0.01 g sub-
samples of the homogenate and 5 mL of 69% Aristar HNO3 were
placed in Teon tubes and digested for 20 min at 300  C using a
MARSXpress microwave system. Prepared samples, duplicate
blanks and certied reference materials (Montana 2711a, and the
Australian National Measurement Institute reference sediment
AGAL-12) were analysed for As, Au, Hg, Se and Ag concentrations
using a Perkin Elmer NexION 300D dynamic reaction cell induc-
tively coupled plasma-mass spectrometer. Recovery was within
10% of the certied values for all metals tested except Se (77e87%).
Sub-samples, blanks and reference materials were analysed for
total organic carbon (TOC) using a LECO CNS at the NATA registered
Environmental Analysis Laboratory, Southern Cross University.
Methylmercury speciation of sub-samples and CRM (DORM-1) was
conducted at the CSIRO Centre for Environmental Contaminants
(Land and Water), Lucas Heights, Australia, following the method C-
221 Methylmercury by GC-AFS. Mineralogy was determined on 15 g
samples by x-ray diffraction (XRD) using a Bruker D4 Endeavor XRD
operating at 40 kV and 40 mA with a Co lamp (Ka1 1.78897 )
with Lynx Eye position sensitive detector (PSD). A PSD divergence
slit of 0.300 mm was used with scans generated from 5 to 80 2q at
a step size of 0.0357 2q/s. International Centre for Diffraction Data
(ICDD) and EVA 3.1 software were used for phase identication
and semi-quantitative estimations. Bulk elemental analyses were
generated by X-ray uorescence (XRF) using a PANalytical Epsilon 3
XRF analyser; elemental concentrations were calculated utilising
174 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179

the Omnian standards in Panalytical Epsilon 3 Software and matrix
corrections. Grain size analyses were conducted using a Mastersizer
2000 (Malvern) with calgon and organics pre-treatment (Vaasma,
2008).
2.4. Preparation of test sediments
Clean sand was collected from the upper dunes of Broken Head
beach (28 42016.0300 S, 153 360 55.9100 E) and dried for 48 h at 40  C
prior to use as a dilutent material for the trommel waste, providing
treatment sediments with concentration ratios by weight. Grain
size analyses were carried out using the Hydrometer Analysis
method with modication (Klute, 1986). Treatment sediment
mixtures were placed into 50 mL polycarbonate containers and
shaken for at least 2 min, and 3 g samples of each treatment
mixture were retained for Hg analyses. Following the pilot study
and the denitive test, water overlaying treatment sediments, and
source seawater were analysed by ICP-MS for THg concentration.
All glassware was soaked in a 1 M HNO3 acid bath for 3 d then
triple-rinsed with Milli-Q water, and soaked in fresh seawater for 7-
d prior to use in tests. A test temperature between 22 and 25  C was
maintained by water baths tted with 50-W heaters and a 1000 L/h
pump. Treatments were prepared 24-h prior to test commence-
ment to allow sediments to settle.
2.5. Toxicity test procedure
evaporation while allowing air ow. Treatment sediments were not
renewed. Dissolved oxygen, conductivity, pH and temperature of
test solutions were measured at 0, 48 and 96 h, and observations of
mortality and sub-lethal effects were recorded every 24 h. Mor-
tality was recorded if the organism failed to respond to gentle
stimulation. A. squamata were not fed during tests, and has previ-
ously been shown to tolerate extended periods without food (Black
et al., 2015).
2.5.1. Sub-lethal effects
2.5.1.1. ARI. The ARI determined was for live organisms measured
at 0, 48 and 96 h in the denitive test and only these organisms
were included in the analysis. This sub-lethal endpoint involved
gently inverting the organism and recording the time taken to re-
turn to an oral disc down position. Previous studies have shown
that healthy A. squamata return to a normal position within 10 s
(Black et al., 2015), and hence a time of >10 s was recorded as a
binary sub-lethal response.
2.6. Statistical analyses
Mortality and sub-lethal data were analysed using Probit Anal-
ysis (ToxCalc ver 5.0). Statistically signicant differences from
control mortality were determined using the Shapiro-Wilks
method (p < 0.05). Bivariate correlation of treatment sediment
and treatment water Hg content was performed using SPSS Ver.
22.

Three static acute toxicity tests were conducted sequentially, 3. Results

using trommel waste concentrations based on results of the previ-
ous test. In the initial pilot test, A. squamata were exposed to
treatment sediments with trommel waste concentrations between
0 and 100% by weight (0e203 mg/kg THg); this test was terminated
at 58 h due to 100% mortality in all non-control treatments. Sub-
sequently a range-nding test was conducted using treatment
sediments with trommel waste concentrations between 0 and 15.0%
by weight (5.2e31.1 mg/kg THg); the range nding test was
terminated at 48 h due to 100% mortality in all non-control treat-
ments. The nal 96-h denitive test used a similar concentration
range as the range-nding test, but with increased replication and
measured THg concentrations (Table 1).
Juvenile (1e2 mm disc) A. squamata were placed in 50 mL
beakers containing 40 mL of freshly-collected seawater and
allowed to acclimate to physical test conditions for 48 h prior to
tests. Treatments were prepared by adding 3 g of treatment sedi-
ment and 40 mL of seawater to 50 mL replicate test beakers, and
allowing sediment to settle for several hours. At test initiation, a
single brittle star was placed in each pre-prepared treatment
sediment, and a watch glass was placed on each beaker to minimise
Table 1
Measured percentage by weight of trommel waste and measured THg concentrations
(mg/kg) in each treatment sediment (Control, T1e T4) used in the acute pilot, range-
nder and denitive toxicity test.
Control T1 T2 T3
3.1. Trommel waste analyses
In addition to expected elevation of Hg (203 mg/kg) and Au
(0.32 mg/kg) concentrations in trommel waste compared to the
Average Crust Content (ACC), concentrations of both Ag (0.98 mg/
kg) and As (92.14 mg/kg) were greater than 1300% of the ACC and Se
(0.27 mg/kg) was somewhat enriched at 540% of the ACC (Fig. 2).
3.1.1. Total organic carbon and mercury speciation
A TOC concentration of 500 mg/kg was measured in trommel
waste. The methylmercury (MeHg) concentration of 0.0094 mg/kg
was negligible as a proportion of THg (i.e. 0.00004%), which is ex-
pected in processed ore material (Table 2).
3.1.2. XRD, XRF and grain size analyses
XRD analyses identied SiO2 as the primary constituent of
trommel waste, with 5e30% albite and clay NaeK aluminosilicates,
and trace amounts of Ferrihydrite (FeO(OH)3) and Goethite
(FeOOH). These results were supported by XRF analyses, measuring
concentrations of 82.15% SiO2, 9.21% Al2O3 and 4.29% Fe2O3. Grain
size analyses showed that the trommel sediments were ner than
the dilution sand, although with a lower clay content (Table 2).
3.2. Toxicity tests
T4

Pilot (5 reps.)
There were no mortalities or obvious loss of condition in con-
% trommel waste 0 17.9 45.0 78.4 100.0 trols in any test, and controls were maintained after the denitive
THg concentration (mg/kg) 0 36.5 91.6 159.5 203.5 test. Test temperatures of 22e25  C were maintained.
Range-Finder (3 reps.)
% trommel waste 0 2.5 5.0 10.0 15.0
3.2.1. Treatment sediment and treatment water Hg concentration
THg concentration (mg/kg) 0a 5.2a 10.4a 20.8a 31.1a
Denitive (10 reps.) Relationships between treatment sediment THg content and
% trommel waste 0 3.1 5.4 10.3 14.0 overlying water THg content were assessed using the Pearson
THg concentration (mg/kg) 0 6.4 11.3 21.5 29.2 product-moment correlation co-efcient. In both the pilot study
a
Indicates unmeasured THg concentrations which are estimated based on the and the denitive test there were positive correlation between THg
THg content in 100% trommel waste. concentrations in treatment sediment, and concentrations in
 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179 175

1000000

100000

% of ACC 10000

1000

100

10

1
Hg Au As Ag Se
Metal
Fig. 2. Metal concentrations in trommel waste as a % of the average crust content (ACC). Note: log scale on y-axis.

Table 2
Trommel waste and dilution sand characteristics. nm - not measured.

Material TOC (mg/kg) Hg concentration (mg/ Grain Size (%)

kg)

THg MeHg Clay <2 mm Silt 2e20 mm Sand 20 mm-2mm Gravel>2 mm

Trommel waste 500 203.5 0.0094 2.3 22.9 74.8 0.0

Dilution sand 300 nm nm 4.0 1.1 94.9 0.0

overlying water after up to 96 h in static conditions (R2 0.978 and
0.968 and p 0.004 and 0.002, respectively). This shows some Hg
loss from the sediment and indicates a potential additional expo-
sure pathway, however this loss was minimal and consistently
<0.03% of sediment THg concentration.
3.3. Mortality
In the pilot test 100% mortality occurred in all non-control
treatments within 72 h (Fig. 3). The range-nder test was termi-
nated after 48 h due to high mortality in all treatment sediments
within 48 h, with 100% mortality in all treatments with trommel
waste concentrations >7.5%. In the denitive test 100% mortality
occurred in the 14.0% w/w trommel waste treatment sediment
(29.2 mg/kg THg), 90% in the 10.3% w/w treatment (21.5 mg/kg
THg) and 10% in the lower treatments (3.1 and 5.4% w/w trommel
waste; 6.4 and 11.3 mg/kg THg; Fig. 4) within 96 h.
100% mortality occurred in all trommel waste treatments within

100 Trommel waste

concentra ons (% v/v)
in treatment sediments
80
0
% mortality

17.9
60 45.0
78.4
100.0
40

20

0
0 10 20 30 40 50 60
Exposure me (h)
Fig. 3. Amphipholis squamata mortality during 58-h exposure to Hg-contaminated trommel waste in the pilot test. Trommel waste concentrations in treatment sediments are
equivalent to THg concentrations of 36.5, 91.6, 159.5 and 203.5 mg/kg.
176 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179

100
Trommel waste
concentra on (% w/w) in
treatment sediments
80
0
3.1
% mortality

5.4
60 10.3
14.0

40

20

0
0 24 48 72 96
Exposure me (h)
Fig. 4. Amphipholis squamata mortality during 96-h exposures to Hg-contaminated trommel waste in the denitive test. Trommel waste concentrations are equivalent to THg
concentrations of 6.4, 11.3, 21.5 and 29.2 mg/kg.

Table 3
Toxicity estimates for trommel waste and total Hg (THg) for the 96-h denitive test. EC50 values are based on the ability to right itself (ARI). Lower and upper CL values are 95%
condence limits.

Toxicity estimates THg (mg/kg) % w/w trommel

LC/EC50 Lower CL Upper CL LC/EC50 Lower CL Upper CL

LC50
72 h 14.8 11.3 18.8 7.1 5.4 9.0
96 h 14.0 10.8 17.6 6.7 4.5 7.5
EC50
48 h 14.4 10.6 19.5 7.3 5.1 10.4

48 h in the pilot test. Analysis of mortality data from the range-
nder test (based on nominal concentrations) resulted in an esti-
mated 48-h LC50 value of 7.6% w/w trommel waste (15.4 mg/kg
THg) with unreliable condence limits due to low replication. The
results of the denitive test were similar to 96-h LC50 values of 14.0
(10.8e17.6; 95% condence limits) mg/kg THg, or 6.7 (3.5e7.5)% w/
w trommel waste, were estimated using measured concentrations
(Table 3).
3.4. Sub-lethal responses
3.4.1. EC50 values based on ability to right itself
The number of organisms that exhibited the ability to right itself
in 10 s was <100% in all treatments except controls within 48 h in
the denitive test; 10, 11, 20, 78 and 100% of surviving A. squamata
were unable to return to the oral disc down position within the
predetermined time of 10 s (Fig. 5). A. squamata took considerably
longer to return to oral disc up position in the 10.3% trommel waste
treatment and high mortality occurred in the 14% trommel waste
treatment (Fig. 6). Analyses of ARI data resulted in a 48-h EC50 of
7.3 (5.1e10.3)% w/w trommel waste, equivalent to 14.4 (10.6e19.4)
mg/kg. Effects on the ARI were greater after 96-h exposure (Fig. 5),
however a reliable 96-h EC50 was not calculable due to high
mortality; a 96-h Lowest Observed Effect Concentration (LOEC) of
3.2% w/w trommel waste (6.44 mg/kg THg) was estimated.
3.4.2. Other potential sub-lethal endpoints
Other common sub-lethal responses were observed in this study
and have potential for development as sub-lethal endpoints,
including; oral surface up orientation (Fig. 7b), autotomy (Fig. 7c),
arm curling (Fig. 7d), burial and climbing. Arm curling was
observed in all non-control treatments, and usually in several ap-
pendages simultaneously, and is likely linked to effects on the
water vascular system. The observation of exposed A. squamata
maintaining an oral disc up position (without manipulation) during
this study was not seen in controls, has not been observed in
healthy organisms during long-term culturing, and has previously
been reported as a toxicological effect and a potential sub-lethal
endpoint in this species (Black et al., 2015).
4. Discussion
This study shows that trommel waste associated with ASGM
operations on Buru Island was highly contaminated with THg, and
was acutely toxic to A. squamata. The material was highly enriched
in Hg at a concentration of 203.5 mg/kg THg (Fig. 2). This is more
than 200 times the ANZECC and ARMCANZ (2000) high-effect
interim sediment quality guideline (ISQG) trigger value of 1 mg/
 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179 177

100

80 48 h
96 h
60

40

20

0
0 2 4 6 8 10 12 14
Trommel

Fig. 5. The percentage of surviving Amphipholis squamata that exhibited an Ability to Right Itself (ARI) in 10 s after 48- and 96-h exposures to Hg-contaminated treatment
sediments containing trommel waste.

18 raises serious concern for long term ecosystem contamination from

the deposition of trommel waste into aquatic environments on Buru
15
Mean ARI (seconds)

Island.
12 Trommel waste was also enriched in As, with the concentration
of 92 mg/kg exceeding the high-effect ISQG trigger value of 70 mg/
9 kg. However total As concentrations in uncontaminated marine
sediments range from 5 to 40 mg/kg (Cited in Neff, 1997), and no
6
observed effect concentrations of 25e29 mg/kg As in sediment
3 have been reported for some marine organisms (Mamindy-Pajany
et al., 2013). As cannot be discounted as contributing to some
0
* toxicity, however these previous studies support the hypothesis
0.0 3.1 5.4 10.3 14.0 that Hg is the major toxicant of concern in trommel waste in this
study. The Ag concentration was below ANZECC and ARMCANZ
Trommel waste concentra on (% w/w) in treatment
(2000) trigger values. Little is known about the toxicity of Se to
sediments marine invertebrates (e.g. Xie et al., 2016) and there is no ANZECC
Fig. 6. Average time taken for Amphipholis squamata to right itself (ARI) after 48-h and ARMCANZ (2000) interim trigger value for sediments, how-
exposure to Hg-contaminated treatment sediments containing trommel waste. Error ever, it is known to interact antagonistically with Hg (Pelletier,
bars show the standard error. * 100% mortality in highest treatment. 1986). Trommel waste was lower in TOC (500 mg/kg; Table 2)
than is typical of marine sediments (e.g. Hyland et al., 2005) and
lower than has been measured in Cu mine tailings (1000e2000 mg/
A kg; Boularbah et al., 2006).
B C

4.1. Trommel waste toxicity to Amphipholis squamata

D Undiluted trommel waste caused 100% mortality of A. squamata

Fig. 7. Image of A) healthy Amphipholis squamata, and examples of common sub-lethal
responses observed during 96-h exposures to Hg-contaminated Trommel waste; B)
inversion (oral disc up position), C) autotomy, and D) leg curling.
kg; inorganic Hg is speculated to be the major toxicant in this study.
Trommel waste had lower THg concentrations than have been
measured at abandoned gold mining sites in California, where THg
concentrations between 400 and 1200 mg/kg have been reported
(Slowey et al., 2007). Such evidence of the potential for Hg to persist
in sediments for many decades or even centuries after a source is
removed (Wong et al., 1999; review Weiner and Suchanek, 2008),
within 24 h (Fig. 3), and the 96-h LC50 of 6.7% w/w trommel waste
(14.0 mg/kg THg) shows that the diluted material remained highly
toxic. Compared to similar studies, toxicity tests on three temperate
marine species commissioned by Nussir ASA (Arctic Minerals
Norway) showed much lower toxicity of Cu mine tailings; no sig-
nicant effects occurred in tailings treatments up to 32% w/w
(Lillicrap et al., 2011). A terrestrial study on the earthworms Pher-
etime sp. and Pheretime guillelmi was similar and reported no effects
at Pb/Zn mine tailing concentrations up to 20% w/w, and 14-d LC50
values between 81 and 100% w/w (Ma et al., 2002). Toxicity tests on
tailings intended for ocean disposal, commissioned by the Batu
Hajiu Cu and Au mine in Indonesia, reported no acute or chronic
toxicity to marine sh and diatoms (cited in Vogt, 2013). More
similarly to the current study, 10-d exposures to contaminated
marine sediment containing 11.55 mg/kg THg caused between 35
178 A.J. Reichelt-Brushett et al. / Chemosphere 189 (2017) 171e179
and 68%.mortality of marine amphipods and isopods
(Annicchiarico et al., 2007).
Toxicity estimates based on THg concentrations in this study
(Table 3) are much less sensitive than are generally reported for
marine organisms exposed to Hg species such as aqueous mercuric
chloride (HgCl2) and MeHg (see review Boening, 2000). The lower
toxicity of THg in this study was likely due to the presence of
negligible MeHg concentrations in trommel waste (Table 2), which
was expected given the geochemical characteristics of processed
ore materials which do not facilitate Hg methylation (e.g. a lack of
organic matter and absence of biological activity; Lambertsson and
Nilsson, 2006; Merritt and Amirbahman, 2009).
Sediments with high TOC loads and biological activity, however,
such as depositional wetland, river, estuarine and coastal envi-
ronments downstream of ASGM operations on Buru Island, may
readily facilitate Hg methylation (e.g. Holloway et al., 2009; Merritt
and Amirbahman, 2009; Sunderland et al., 2006). Male et al. (2013),
as well as a more recent study (Reichelt-Brushett et al., 2017)
showed that freshwater, estuarine and marine environments
downstream of ASGM operations on Buru Island are facilitating Hg
methylation, resulting in much higher proportions of bioavailable
Hg in aquatic sediments compared to trommel waste. This study
showed that unaged trommel waste was toxic, however we
recommend that further development of TTTs should incorporate
methods of articially aging tailings (e.g. Meunier et al., 2011; Tang
et al., 2007) to reect the potential changes in toxicity of the ma-
terial following deposition and aging in the environment (e.g.
Jubileus et al., 2013).
4.1.1. Test endpoint ability to right itself
Effects of trommel waste exposures on the ARI shows that this
endpoint effectively captured sub-lethal toxicological effects on
A. squamata prior to mortality; the 48-h EC50 of 7.3% w/w trommel
waste is comparable to the 96-h LC50 of 6.7% w/w (Table 3). This
result is similar to aqueous Cu toxicity tests using A. squamata,
where the 96-h EC50 value based on the ARI was approximately
half that of the 96-h LC50 (Black et al., 2015). These results
demonstrate that effects on the water vascular system causing an
inability to right itself are evident only when A. squamata are
severely affected and may not be the most sensitive quantiable
sub-lethal endpoint in this species. Observations of leg curling,
inversion and autonomy made during this study may be useful in
the further development of A. squamata as a routine toxicity test
species and may also be potentially useful in eld monitoring
studies (Fig. 7). Other reported sub-lethal effects in brittle stars
including tube foot retraction and movement, normal curling
behaviour (Black et al., 2015), burrow emergence (Bowmer et al.,
1986) and decreased bioluminescence (Deheyn and Jangoux.,
1999) are also worthy of further investigation as endpoints in this
species.
4.2. Tailings toxicity tests
In addition to the unregulated release of tailings from ASGM
operations such as those on Buru Island, deep sea tailings place-
ment (DSTP) is increasingly being used as a means of tailings
disposal in many regions (Ramirez-Llodra et al., 2015), including
throughout the Indo-Pacic (Angel et al., 2013; Reichelt-Brushett,
2012). This process directly exposes benthic organisms to volumi-
nous loads of processed ore material, often over many decades of
operation. Despite considerable resources being directed towards
establishing new DSTP operations, and requirements for mining
operations to conduct meaningful ecological risk assessments,
there are currently very few tools available to assess mine tailings
toxicity and no routine tests relevant to deep sea environments.
This study shows that A. squamata is a valuable test species for
ecotoxicological risk assessments relevant to mine tailings disposal
in coastal environments, and because it is found to a depth of
1300 m, may also be useful for DSTP and deep sea mining toxicity
assessments.
5. Conclusions and recommendations
ASGM operations on Buru Island have generated large volumes
of highly contaminated and toxic trommel waste. Samples collected
from settlement ponds at the Gogrea site contained 203 mg/kg THg
and low TOC content, and inorganic Hg is implicated as the primary
source of acute toxicity of the waste material to the brittle star
A. squamata. Exposure of A. squamata to trommel waste caused
rapid mortality and sub-lethal effects on the water vascular sys-
tems, resulting in a 96-h LC50 of 6.7% w/w trommel waste and a 48-
h EC50 of 7.3% w/w trommel waste (14.0 and 14.4 mg/kg THg,
respectively). The high toxicity of the unaged processed ore mate-
rial, containing a low proportion of bioavailable MeHg, raises
concern regarding the potential for long term ecosystem contam-
ination and toxicity to aquatic organisms, as well as humans, and
warrants urgent further investigation.
The newly-developed test method using A. squamata has po-
tential for inclusion in TTT suites for ecotoxicological risk assess-
ments relevant to benthic coastal habitats, and has applications for
deep-sea ecotoxicology relevant to DSTP and seaoor mining.
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