Marine Environmental Research 171 (2021) 105471

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Marine Environmental Research

journal homepage: www.elsevier.com/locate/marenvrev

Elevated toxicity of resuspended mine tailings over time

Keshtav S. Ramdial *, 1, Richard Abell , Kim S. Last
The Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Resuspension of disposed mine tailings is an important secondary source of heavy metal pollution in affected
Mine tailings regions. UK copper mine tailings were continuously resuspended in seawater over 14 days to understand po­
Resuspension tential metal dissolution and associated ecotoxicological impacts. Aqueous concentrations of arsenic increased
Heavy metals
859%, nickel 85%, manganese and cadmium over 40%, and vanadium and zinc over 20%. Green sea urchin
Dissolution
Ecotoxicology
(Psammechinus miliaris) gametes were exposed to 18-h (18hr) and 14-day (14d) resuspension elutriates. Fertil­
Sea urchin isation success dropped from 72% (18hr) to 21.06% (14d). Toxicity Index (TI) values (reduced fertilisation
membrane quality and fertilisation success) increased from 1.6 (18hr) to 2.8 (14d). Embryo polyspermy fre­
quencies increased from 17.58% (18hr) to 21.13% (14d). Higher TI values and polyspermy rates indicate impacts
may go beyond standard bioassay endpoints. Current predictions of mine tailing impacts may be underestimated.
This is important for ecosystem resilience when considering other anthropogenic stressors such as climate
change.
Capsule: Disturbance of metal contaminated sediments over prolonged periods results in ever increasing disso­
lution concentrations, and elevated toxicity of the water medium to biological receptors.

1. Introduction
This study aimed to investigate the dissolution of metals from mine
tailings when continuously agitated for a prolonged event. Tailings from
a copper mine in the UK were used to represent mine waste that can be
transported to or disposed into coastal and/or marine locations, natu­
rally via rivers, accidently from mining waste spills, or intentionally
from mining operations. Mining wastes are recognised as a major con­
stituent of contaminated sediment in many estuarine and coastal envi­
ronments worldwide as summarised by Koski (2012) and in detail for
various locations – northern Chile (Correa et al., 1999; Ramirez et al.,
2005; Andrade et al. 2006), Alaska, USA (Kline and Stekoll 2001),
Greenland (Josefson et al., 2008), Cornwall, UK (Pirrie et al. 2002,
2003), Papua New Guinea (Hughes et al., 2015) and Spain (Mestre et al.,
2017; Bourrin et al., 2021). Mine tailings in the receiving aquatic en­
vironments create allochthonous sediments which act as the primary
sink and source for metals (Miller 1997; Leblanc et al. 2000). For stark
examples of mine tailings forming contaminated sediment in coastal and
marine locations, Koski (2012) highlights the case of: 1) Marinduque
Island, Phillipines where 200 million tonnes were discharged over 16
years and cover 60 km2 of seafloor; 2) Chañaral Bay and Caleta Palito,
Northern Chile where 150 million tonnes were released over 50 years
leading to deposition down to 15 m in depth and; 3) Portman Bay, Spain
where 60 million tonnes were discharged into the Bay over roughly 34
years and constitutes 10% of the Bay’s sediment content. The metal
enrichment in this Portman Bay is stated as 10–60 times higher than
other coastal sediments in the Mediterranean Sea (Mestre et al., 2017).
In environments subjected to contaminated sediments from mine tail­
ings the biodiversity of various trophic groups is often markedly lower
than unpolluted sites (Correa et al., 1999; Ramirez et al., 2005; Josefson
et al., 2008; Hughes et al., 2015).
Bourrin et al., (2021) and Mestre et al., (2017) described the his­
torical impacts of mine tailings deposition and disposal in Portman Bay
and investigated the results of a disturbance event (trawling) on the
sediment. Both studies highlighted how these activities can result in the
resuspension of contaminated sediments leading to increased aqueous
and bioavailable metal concentrations over prolonged periods of time
(hours to days). Resuspended contaminated sediments (RCS) may be
caused by natural processes (waves, tidal currents and storms) or
anthropogenic perturbations (dredging, trawling, ship traffic and
anchoring). These activities become important secondary sources of
contamination (de Carvalho et al., 1998; Latimer et al. 1999; Roberts

* Corresponding author.
E-mail addresses: keshtavr@coastaldynamics.com (K.S. Ramdial), kim.last@sams.ac.uk (K.S. Last).
1
Present affiliation: Coastal Dynamics Limited, Unit 5, Lot 2C, Chootoo Trace, El Socorro, San Juan, Trinidad and Tobago.

https://doi.org/10.1016/j.marenvres.2021.105471
Received 29 March 2021; Received in revised form 29 July 2021; Accepted 27 August 2021
Available online 30 August 2021
0141-1136/© 2021 Elsevier Ltd. All rights reserved.
K.S. Ramdial et al.
2012) that liberate metals, via desorption/dissolution from particulate
matter, and/or by mixing of released interstitial water, into the over­
lying water column (Kalnejais et al., 2007; van den Berg et al., 2001).
This potentially releases contaminants in states which are once again
bioavailable to organisms in aquatic ecosystems (Weyhenmeyer 1998).
It also represents a pathway by which different compartments of an
ecosystem, besides benthic habitats, can be exposed to previously
sediment-bound contaminants.
Resuspension mechanisms vary greatly in temporal extent and fre­
quency making RCS a complex form of pulsed pollution (Roberts 2012).
For example, dredging is an infrequent event where sediment plumes
usually dissipate shortly after cessation of disposal (John et al., 2000;
Peddicord 1987). However, natural processes such as wave action and
tides may continuously resuspend sediments, or even enhance previ­
ously initiated sediment resuspension, propagating effects across spatial
and temporal scales (Vale et al. 1998; Roberts 2012). The persistence of
RCS could facilitate dissolution of metals from mine tailing sediments
beyond time scales investigated by standard methods. As such, there is a
need to understand the effects of RCS for such sediments across pro­
longed time frames as previously explored by Saulnier and Mucci
(2000), van den Berg et al. (2001) and Mestre et al. (2017).
The dissolution of metals by resuspension may not only be influenced
by the type and duration of the perturbation event. Factors controlling
the quantities, fate and speciation of metals after resuspension are
extensively described by Eggleton and Thomas (2004), Atkinson et al.
(2007) and Roberts (2012). These factors include changes in sediment
redox, lowering of pH and dissolved oxygen concentrations, and the
scavenging ability of metal oxides/hydroxides. Furthermore, partition­
ing effects of metals in mineral structures (Torres and Auleda 2013) and
insoluble sulphide fractions (Mcgrath et al. 2002) play key roles in
regulating the availability of sediment-bound metals.
While the effects of abiotic factors on metal remobilisation are
relatively well understood, delineating potential impacts and setting
environmental standards require a biological context, and the biological
effects of RCS, especially in environments impacted by mine tailings,
have been less researched (Hughes et al., 2015). Yet research is vital for
quantifying potential impacts and establishing monitoring guidelines for
activities such as coastal reclamation, construction and dredging, the
management of historically metal contaminated nearshore habitats, or
the exploitation of exotic and vulnerable seabed habitats for deep-sea
mining (K. A. Miller et al., 2018).
Sea urchins are commonly used as a test species to investigate the
effects of contaminants with well-developed toxicity assays (Dinnel et al.
(1987), and have been incorporated into various environmental pro­
tocols (USEPA 2002; 2012; Environment Canada 2011). The sea urchin
was chosen as the specimen for investigating the impacts of metal
remobilisation from copper mine tailings since it is abundant in coastal
ecosystems, not only around the UK, but globally. It can therefore be
considered a likely receptor of contaminants from tailings deposited in
coastal waters. Echinoderms such as sea urchins are favoured as bio­
indicators of pollution due to the sensitivity of their early life stages to
contaminants (G. Pagano et al., 1986; N. Kobayashi 1995) and their
relative ecosystem performance in modulating ecosystems as keystone
species (Brusca and Brusca 1990). The toxicity of whole sediments
and/or their associated pore water (Geffard et al., 2001; Volpi Ghir­
ardini, Arizzi Novelli, and Tagliapietra 2005; Fabbrocini, di Stasio, and
D’Adamo 2010) or that of specific metals such as cadmium (Vaschenko
et al., 1999) and copper (Schäfer et al. 2009) have been previously
investigated by assessing the fertilisation success rates of sea urchin
gametes.
This study aims to determine the dissolution of metals into the
aqueous phase from the resuspension of mine tailings under general
conditions, and whether remobilised metals inhibit sea urchin fertil­
ization success in the green sea urchin Psammechinus miliaris. The pro­
tocol and general conditions for the dissolution of metals were followed
as outlined in Organisation for Economic Cooperation and Development
2
Marine Environmental Research 171 (2021) 105471
(OECD 2002). The USEPA (2002) Sea Urchin, Arbacia Punctulata,
Fertilization Test Method 1008.0. EPA-821-R-02-014 was used to test
the impact of remobilised metals on green sea urchin fertilization. This
framework aims to provide a broad understanding of metal dissolution
from resuspended mine tailings and the potential ecotoxicological im­
pacts over time.
2. Material and methods
2.1. Mine tailing samples and bulk trace metal concentrations
Copper mine tailings samples were obtained on April 16th, 2013
from the Wheal Maid Mine, Cornwall, UK by members of the Camborne
School of Mines. Samples were transported on ice by courier to the
Scottish Association for Marine Science (SAMS) where they were stored
at 4 ◦ C until use. Ground tailings, 0.1000 ± 0.001 g, were transferred
into Teflon tubes CEM Omni XP 1500 vessels. Contents were acidified
with 6 ml nitric acid (15.8 M HNO3), 3 ml hydrochloric acid (11.8 M
HCl) and 2 ml hydrofluoric acid (28.4 M HF). All acids used in this study
were of high purity trace metal analysis grade (VWR ARISTAR). All
samples were digested in a CEM Mars microwave digester under pres­
sure for a total digestion time of 1.5 h in three stages as follows: 20 min
at 100 ◦ C, 30 min at 150 ◦ C and 40 min at 190 ◦ C until tailings were
dissolved (Hughes et al., 2015). Post digestion, samples were evaporated
and diluted to 5% v/v HNO3 for trace metal concentration analysis via
Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Complete
digestion was assured by monitoring concentrations of the target metals
in a certified reference marine sediment material GBW07315 (China
National Analysis Center for Iron and Steel).
In order to understand the inherent environmental risk potentially
posed by the mine tailings, the bulk metals concentrations were
compared against appropriate criteria. Long et al. (1995) provides effect
range-low (ERL) and effect range-median (EMR) values for specific
metals of interest in marine and estuarine sediments, as selected from an
extensive analysis of predicting toxicity impacts on benthic organisms
based on sediment chemistry data (Long et al. 1998). At concentrations
below the ERL adverse biological effects are rarely expected to occur;
between the ERL and ERM adverse effect may occasionally occur; and
above the ERM adverse effects may frequently occur. Action Level
criteria devised by the Center for Environment, Fisheries and Aquacul­
ture (CEFAS) is used as guidance to assess the suitability of dredged
material for disposal to sea. Two Action Levels (1 and 2) are applied
whereby contaminant levels lower than Chemical Action Level 1 (cAL1)
are of no concern, but those greater than Action Chemical Level 2 (cAL2)
are deemed unsuitable for disposal at sea (MMO 2015). A risk charac­
terisation ratio (RCR) (Manuel Nicolaus et al., 2015) was used here to
evaluate the risk for bulk tailings metal concentrations based on
respective ERL, ERM or cAL2 criteria value (Eqn. (1)). RCR values > 1
indicate adverse impacts may be likely when compared to ERL and ERM
values, or the tailings would be deemed unsuitable for sea disposal when
compared to the cAL2 criteria (assuming a scenario where the tailings
are proposed for dredging or extraction).
Bulk tailings concentration
RCR = (1)
ERL or ERM or cAL2
2.1.1. Laser particle size analysis (LPSA)
Tailings were homogenised using a large plastic stirrer and added to
six 50 ml centrifuge tubes up to the 5 ml mark. Each tube was made up to
25 ml with 5 ml of calgon solution (sodium hexametaphosphate) and 20
ml deionised (DI) water (18.2 MΩ). Clumps were broken up by vigor­
ously mixing on a Stuart Vortex mixer for 1 min and then in a Clifton
ultrasonic water bath for 15 min. Small portions of the tailings suspen­
sion were incrementally pipetted into a Beckman Coulter LS230 Laser
Diffractometer until the Polarization Intensity Differential Scattering
(PIDS) read between 40% and 45%. Tailing samples were analysed
K.S. Ramdial et al.
alongside Coulter LS Control BG500 nominal 500 μm glass beads. LPSA
outputs from the diffractometer were exported to GRADISTAT (Blott and
Pye 2001) which returned the grain size distribution.
2.2. Total organic content (TOC)
Approximately 100 g wet weight of homogenised tailings were
frozen and dried using an Edward Modulyo vacuum pump for 48 h.
Dried samples were re-weighed and the % water content determined. A
Retsch Planetary Ball Mill PM 400 ground the dried tailings into a fine
texture. Six glass ampoules were filled with ~0.02 ± 0.002 g of the
ground tailings along with 0.8 ml sulphurous acid (1.6 M H2SO3) to
dissolve calcium carbonates (CaCO3). All ampules were frozen and
vacuum dried to eliminate excess H2SO3. Dried contents were trans­
ferred and folded into Sercon Ltd. pressed tin capsules. TOC analysis was
performed using a Costech International Elemental Combustion System
(ECS) 4010 operating at 1000 ◦ C in the left combustion furnace, 650 ◦ C
in the right reaction furnace and 50 ◦ C in the gas chromatographic
separation oven. Six standards of 0.1 mg–3.0 mg acetanilide (BDH
Chemicals – 10.36% Nitrogen, 71.09% Carbon) were analysed alongside
the tailings samples.
2.3. ‘Available’ metal fractions
Available metal fractions were targeted for extraction from the
tailings samples. These fractions can be considered those metals which
are loosely bound to soil/sediment particles and are readily remobilised
via dissolution or chemical exchange. Available fractions include those
which are soluble metal complexes, adsorbed onto organic matter, or are
in the form of Fe-Mn oxides or carbonates. Tailings were subjected to
Sequential Extraction Procedures (SEP) targeting water soluble and
exchangeable fractions of the tailings by adapting methods from Dold
(2003), and Torres and Auleda (2013). For extraction of the
water-soluble fraction (SEP1), secondary sulphates were primarily dis­
solved by adding 0.5 ± 0.05 g of freeze-dried tailings to acid cleaned
centrifuge tubes and made up to 50 ml with oxygen (O2) free Filtered
Seawater (FSW). A tube with only O2 free FSW acted as a control.
All centrifuge tubes were agitated on a VWR ADV3500 shaker table
for 1 h at 100 revolutions per minute (rpm) and 15 ± 1 ◦ C in a controlled
temperature room. They were subsequently centrifuged at 3000 rpm for
15 min also at 15 ± 1 ◦ C. Filtered elutriate was collected by first using
acid cleaned 15 ml plastic syringes to extract 9 ml of supernatant from
each tube. Each syringe was then fitted with an acid rinsed 0.2 μm nylon
filter before dispensing the supernatant into an acid cleaned 15 ml
plastic vial. Triplicate 2.85 ml samples were pipetted, placed in acid
cleaned narrow-mouthed 4 ml LDPE bottles, acidified to 5% v/v HNO3,
and stored in a fridge at 4 ◦ C until ICP-MS analysis.
The remaining supernatant from each tube was decanted and the
solid residue was treated with 20 ml of 1 M ammonium acetate (acidified
to pH 4.5 using 100% glacial acetic acid) to extract the second
exchangeable monosulphide and carbonate targeted fraction (SEP2). In
SEP2, metals associated with monosulphide and carbonate minerals
were targeted. Tubes were agitated for 2 h on the shaker table, centri­
fuged, sampled, filtered, acidified and stored as previously described.
2.4. Resuspension of tailings and dissolution of metals
Resuspension of the tailings was conducted over 14 days (14d)
following OECD (2002). Tailings were homogenised with 20 ± 0.1 g wet
weight subsamples each placed in four acid cleaned 2 L wide mouthed
LDPE bottles. Each bottle was topped off with 2 L of FSW. One additional
bottle was filled with 2 L of only FSW as a negative control. Resus­
pension of the tailings was achieved by agitation on an orbital VWR
ADV3500 shaker table at 100 rpm and 15 ± 1 ◦ C in a controlled tem­
perature room. The resuspension conditions outlined in OECD (2002)
are stated as representative of those generally occurring in the
3
Marine Environmental Research 171 (2021) 105471
environment with a standardized agitation. A total of 4 bottles were
placed on the shaker - 3 bottles with Continuously-agitated Tailings (CT)
and 1 bottle with FSW only as the negative Continuously-agitated
Control (CC). A bottle with tailings and FSW, which was not shaken,
represented the positive control i.e., Un-agitated Tailings (UT).
2.5. Leachate sampling
The supernatant seawater in each bottle was sampled at four time
points: start time zero (0hr); 18 h (18hr), 7 days (7d) and 14 days (14d).
At each time point (excluding 0hr), agitation was stopped at least 1hr
prior to sampling to allow any particulate matter to settle out. Elutriate
of the supernatant was collected, acidified and stored as previously
described. At 18hr, one of the 3 bottles containing tailings on the shaker
was randomly selected. For the sea urchin bioassay an elutriate sample
of 60 ml was drawn from the random bottle into acid cleaned 30 ml
narrow-mouthed LDPE bottles, stored at 4 ◦ C, and used within 48 h of
collection. After the required elutriate was collected, each bottle was
replenished with a volume of FSW equal to that drawn. The bioassay
elutriate sampling was also repeated after 14d from the same bottle
sampled at 18hr. This allowed a comparison of elutriate toxicities for
18hr and 14d. The USEPA (2008) Toxicity Characteristic Leachate
Procedure (TCLP), which is regularly employed in commercial labora­
tory bioassays to obtain test solutions for use in fertilisation tests, ex­
tracts leachates from sediment after 18hr agitations. During each
sampling event, water temperature, pH, salinity and dissolved oxygen
levels in each bottle were recorded using a WTW Multi 340i Universal
Pocket meter.
2.6. ICP-MS analysis
ICP-MS was used for all trace metal analyses. The acid digested bulk
sediment samples were analysed alongside the digested reagent blank
and marine sediment CRM GBW07315. Long term data using this
digestion and analytical technique (n = 58) gives a recovery of metals in
this study between 92 and 103% with the exception of As, which has a
recovery of 119%.
For seawater samples, the instrument was preconditioned in a tuning
solution prepared in a seawater matrix. This allowed for the ICP-MS
analysis to be calibrated and the seawater samples to be quantified,
using a standard addition technique similar to those described by
Engstron (1988). The aim of the standard addition technique was to
reduce measurement interference in the ICP-MS analysis caused by
matrix effects due to the seawater medium. Seawater samples were
analysed along reagent blanks and nearshore seawater CRM (CASS-4
National Research Council Canada). All standards, blanks and sediment
sample solutions were prepared using high purity water (18.2 MΩ-cm
resistivity), obtained from an Elga LabWater purification system. The
accuracy for sediment and seawater CRMs runs were generally <6%
RSD.
2.7. Preparation of equipment and seawater
All glass and plastic equipment were acid cleaned in a 10% HNO3
bath for 48 h and rinsed in deionised (DI) water prior to use. The 0.2 μm
filters however were only rinsed in 10% HNO3 and DI water immedi­
ately before use. FSW was obtained by passing stored seawater through a
Perspex gravity pump fitted with 150 mm diameter Grade 1:11 μm
medium flow filter paper. O2 free water was necessary to prevent
oxidation of reduced species and was achieved by bubbling nitrogen gas
(N2) through FSW overnight.
2.8. Sea urchin bioassay
2.8.1. Gamete preparation
Aquaria raised adult green sea urchins, Psammechinus miliaris, from
K.S. Ramdial et al. Marine Environmental Research 171 (2021) 105471

SAMS, were kept in aerated water baths (15 ± 1 ◦ C) and fed kelp at least
48 h before spawning was induced. Spawning, gamete collection and a
gamete quality pre-test were carried out as outlined by Environment
Canada (2011). If pre-test fertilization success rates >90% were ob­
tained, then use of gametes for the definitive bioassays were confirmed.
Fertilisation success as identified by elevation of the fertilisation mem­
brane around an egg is shown in Fig. 1 by images (a) – (g). To ensure
consistency of sea urchin bioassay tests, a sperm-egg ratio of 2000:1 was
prepared as outlined by Environment Canada (2011). The egg stock was
prepared as outlined in USEPA (2002) with sperm added to stock just
before the inoculation of test vials.
2.8.2. Fertilisation success test
Elutriate from the randomly selected shaker bottle after 18hr and
14d was progressively diluted by a factor of 0.5 with FSW to create 5 ml
test solutions of 5 different concentrations following USEPA 2002 pro­
tocols: 100% (whole elutriate); 50%, 25%, 12.5%, 6.25%. A negative
control of only FSW (0% elutriate) was also prepared. Each concentra­
tion had 5 replicates and was prepared in 15 ml plastic vials. Four of the
5 test vials for each dilution were inoculated with 0.1 ml of the sperm
suspension. Sperm were exposed for 60 min after which 1 ml of the
adjusted egg stock was added and incubated a further 20 min (USEPA
2002; 2012). Eggs only were added to the 5th vial of each concentration
and represented a positive control to check for sperm contamination in
the egg stock.
After the 20-min incubation period, the test was terminated by
adding 250 μl of 10% buffered formalin in FSW to each vial. The per­
centage fertilisation success was calculated from the observation of 100
eggs at random. Samples from each vial were viewed on a Sedgewick-
Rafter chamber under a Wild Heerbrugg Ltd. M20 microscope with a
x20 phase objective lens in a dimmed bright field. Since unfertilised eggs
may adhere to the base of the vials biasing observations (USEPA 2012)
eggs in each vial were gently re-suspended prior to sampling and
counting. The USEPA (2012) recommends that if fertilisation mem­
branes described by images (e), (f) and (g) are copious in samples, then
all samples should be examined for any dose-related impact. The USEPA
method recommends against counting distorted eggs to prevent a
skewed positive bias of unfertilised egg counts in case the egg was
already compromised.
Based on the proportions of normal or abnormal development of
fertilisation membranes observed within a sample, an additional method
of quantifying elutriate toxicity was applied. Eqn (2). defines a Toxicity
Index (TI) score from Carballeira et al. (2012) for the larval development
of the urchin, Paracentrotus lividus. The relative frequencies of each
fertilisation membrane type for each elutriate concentration contributes
to the TI score which ranges between 0 (no toxicity – normal fertilisation
membranes dominant) and 3 (high toxicity – unfertilised eggs
dominant).
TI = [(0 x %Type 0) + (1 x %Type 1) + (2 x %Type 2) + (3 x %Type 3)]/100
(2)
The TI score was adapted to rank fertilisation membranes for
P. miliaris as described in Fig. 1. The occurrence of polyspermic embryos
(i, j), indicated by unusual division or rupturing of egg surface, was also
recorded but not applied to Eqn. (2). Immature or misshapen eggs (k, l)
were not counted even if they showed a fertilisation membrane. This
was done to prevent a positive bias of unfertilised egg counts if the egg
was already compromised or incapable of being fertilised.
2.9. Statistical analysis
The relationships between the water quality in the treatment bottles,

Fig. 1. Fertilisation Membrane Types based on USEPA (2012). Normal fertilisation with a well elevated membrane and an inner hyaline membrane (a); displaced
zygote touching the outer membrane after preservation (b); full but slender elevation of either membrane (c); folded or collapsed elevation (d); hemispheric elevation
(e); sliver (f); sporadic or blistered elevation (g); unfertilised egg (h); polyspermy as indicated by abnormal development of zygote (i) or bubbling/rupturing of
surface (j); immature/unviable eggs: clearly smaller or oversized, translucent or heavily darkened bodies, vacuolated bodies (k) and distorted shapes (l) were not
counted to prevent skewed results if eggs were not capable of fertilisation or producing a viable embryo. Toxicity rankings adapted from Carballeira et al. (2012).
Images captured using a Minolta DiMAGE 7 digital camera at x2 zoom and ISO 800 viewed on a Sedgewick-Rafter chamber under a Wild Heerbrugg Ltd. M20
microscope with a x20 phase objective lens in a dimmed bright field. Image source: Keshtav Ramdial.

4
K.S. Ramdial et al. Marine Environmental Research 171 (2021) 105471

the aqueous metal concentrations and resuspension times were tested

Metal concentrations in bulk tailings, SEP1 and SEP2 fractions and 14d dissolution elutriate. Highest 14d concentrations are presented for metals which underwent dissolution irrespective of bottle treatments. Un-agitated

adjustment for seawater density). <LOD – below Limit of Detection where LOD values (ppm) are as follows: Fe (0.00031), As (0.0001), Zn (0.00015), Cu (0.00107), Pb (0.00005), Mn (0.00007), Ni (0.0001), V (0.00012),
Tailings (UT), Continuously-agitated FSW Control (CC) or Continuously-agitated Tailings (CT). n = number of replicates. *Metal concentrations from the SEP aqueous fractions were converted to mg/kg (with an

±1 standard deviation (μg/L)

using a Principal Component Analysis (PCA). PCA was done on XLSTAT

concentrations and bottle

(Addinsoft 2015) to determine which abiotic variables best explained
metals concentrations in the treatment bottles following resuspension.

20.396 ± 0.150 (UT)

88.121 ± 14.85 (CT)
0.689 ± 0.002 (UT)

0.202 ± 0.004 (UT)

1.353 ± 0.152 (CC)
0.588 ± 0.025 (CT)
The Effective Concentration (EC) of elutriate that can be attributed to ≤

14d dissolution
50% fertilisation success, or EC50, was derived from dose response
curves using the drc add-on package (Ritz and Streibig 2005), for the R

Cr (0.00014) and Cd (0.00002). N/A (Not applicable – could not be calculated due to < LODs in the SEP fractions). (-) No dissolution after 14 days i.e., net negative concentration after 14d vs 0hr.

treatment

(n = 9)
statistical package version 3.0.1 (R Core Team 2012). The TI score and %
polyspermy results were modelled to best fit curves in order to deter­

–
–

–
mine response type of the observations after 18hr and 14d. Curve fitting
was also done in XLSTAT and the best models were chosen based on the

concentration (%)
lowest Akaike information criterion (AIC) scores.

SEP2 fraction of

bulk tailings

8.9842E-07

6.5714E-04
6.2481E-07
8.0671E-06
2.8419E-06
1.1741E-05

4.3127E-05
2.9376E-05
3. Results

total

N/A

N/A
3.1. Water content, TOC and grain size distribution

Gravimetric water content of the bulk tailings was 18.9% after an

SEP1 fraction of total bulk

tailings concentration (%)
initial wet weight of 108.91 g and post dried weight of 88.3 g. TOC was
calculated as the % carbon weight of the total ampoule sample weight
and averaged 0.07%. Grain size distribution by LPSA classed the tailings
as ‘very fine sand’ with a mean particle size of 103.83 ± 12.6 μm. The

5.3387E-08

4.5752E-06
2.5411E-07

9.8796E-05
standard deviation was small across all subsamples indicating that the
tailings can be described as homogenous with respect to particle size.

N/A
N/A

N/A
N/A
N/A

N/A
3.2. Bulk and SEP metal concentrations

bulk tailings* (mg/kg)

SEP2 concentration in
Only Mn was higher in the SEP1 (water soluble fraction) than in the
SEP2 (monosulphide and carbonate targeted fraction). V, Cr (III), Ni, Cu,
Cd and Pb were not detected in the SEP1 (water soluble) fraction. Fe, Zn,
As, V, Cr (III), Cu and Pb were all higher in SEP2. Metal concentrations in

0.2747

1.6073
0.0008
0.0103
0.0017
0.0020

0.0012
0.0006
the SEP aqueous fractions were converted to mg/kg (with an adjustment

N/A

N/A
for seawater density) and compared to the respective levels found in the
bulk tailings (Table 1). The SEP1 and SEP2 concentrations for all metals
were <1% of their respective bulk tailings concentration. For all metals,
bulk tailings* (mg/kg)
SEP1 concentration in

the majority of their bulk concentrations remained trapped within the

mineral lattice framework of the tailings. RCR values for the bulk con­
centrations against respective ERL/ERM and cAL2 values are presented
in Table 2. The RCRERM values were >1 for As, Cu, Zn, Pb and Ni (in
0.0163

0.0112
0.0003

0.0170
order of high to low) and these metals could elicit adverse effects, while N/A
N/A

N/A
N/A
N/A

N/A
Cd and Cr (III) pose low to no risk respectively. Arsenic would be of
particular concern with a RCRERM value of 38.1. RCRcAL2 results were
±1 standard deviation

similar with As, Cu, Zn and Pb (in order of high to low) having ratios >1,
1647.436 ± 32.575

with As at 26.67.
281.614 ± 6.211

10.519 ± 0.334
0.847 ± 0.001

1.775 ± 0.322
2.071 ± 0.062

1.261 ± 0.030
0.624 ± 0.085
(μg/L) (n = 9)
SEP2 aqueous
concentration

3.3. Long term tailings resuspension

<LOD

<LOD

3.3.1. Water quality parameters in bottle treatments

Dissolved oxygen and pH levels in each bottle declined on average
±1 standard deviation

over time by 1.92 mg/L and 0.26 units respectively. The decline in pH is
within acceptable ranges (6.0–8.5) defined by OECD (2002). Tempera­
16.734 ± 3.357

11.470 ± 0.630

17.426 ± 0.808

ture fluctuated within 1 ◦ C over the course of the experiment. Salinity

0.344 ± 0.032
(μg/L) (n = 9)
SEP1 aqueous
concentration

dropped negligibly from 33.2 psμ at 0hr to 33.1 psμ from 18hr onwards.
<LOD
<LOD

<LOD
<LOD
<LOD

<LOD

3.3.2. Change in aqueous metal concentrations after 14d resuspension

Arsenic recorded the greatest dissolution (net positive % change)
after 14d, with a concentration increase of 859.2% in the Continuously-
±1 standard deviation

2445.823 ± 159.214

1272.080 ± 141.890

agitated Tailings (CT), followed by Ni at 86.0%. Aqueous concentrations

1322.462 ± 89.617
Mean bulk tailings

609.377 ± 66.112
(mg/kg) (n = 10)

172.080 ± 1.102
100.449 ± 4.955

of Mn and Cd increased over 40.0% while V and Zn increased over

28.535 ± 0.970
20.736 ± 1.102

3.391 ± 0.285
305810.000 ±
concentration

20.0%. Cr (III), Fe, Cu and Pb however, were removed from the aqueous
phase during the resuspension period resulting in a net negative con­
5518.052

centration after 14d.

It was further found after 14d Mn, Zn and Cd concentrations were
highest in Un-agitated Tailings (UT). The 14d Ni concentrations were
Table 1

(III)
Metal

highest in the CT bottle but similar concentrations were recorded in the

Mn
Cu

Cd
Zn

Pb
As
Fe

Ni

Cr
V

UT bottle. V concentrations after 14d were highest in the Continuously-

5
K.S. Ramdial et al. Marine Environmental Research 171 (2021) 105471

Table 2
RCR values for maximum bulk tailings concentrations based on ERL and ERM values from Long et al., (1995), and CEFAS Action Level 2 (cAL2) criteria (MMO, 2020).
RCRERL/ERM values > 1 indicate potential for metal to cause adverse effects. RCRcAL2 values > 1 indicate material (dredged) is generally unsuitable for sea disposal.
Metal Maximum bulk tailings concentration (mg/kg) ERL mg/kg ERM (mg/kg) CEFAS cAL2 (mg/kg) RCRERL RCRERM RCRcAL2

As 2666.69 8.2 70 100 325.21 38.10 26.67

Cu 1434.65 34 270 400 42.20 5.31 3.59
Zn 1452.31 150 410 800 9.68 3.54 1.82
Pb 718.32 47 218 500 15.38 3.30 1.44
Ni 108.35 20.9 51.6 200 5.18 2.10 0.54
Cd 3.96 1.2 9.6 5 3.30 0.41 0.79
Cr (III) 22.19 81 370 400 0.27 0.06 0.06

agitated FSW Control (CC). Dissolution curves for select metals are
illustrated in Fig. 2. The error bars (±1 standard deviation) shown in
Fig. 2 for the average of the triplicate concentrations measured for the
UT and CT treatments generally did not overlap with the error bars from
the previous sampling points. This indicates the change in concentra­
tions between sampling timepoints are greater than the uncertainty of
the measurements/analysis and the trends are meaningful. However, the
exception is the 14d Zn concentrations in the CT treatment where the
standard deviation overlapped with those from the 18hr and 7d
concentrations.
3.3.3. 14d dissolution vs. SEP concentrations
The 14d metal concentrations which experienced dissolution were
compared to respective concentrations extracted from each SEP fraction
(Table 1). SEP1 represented those metals highly labile in the aqueous
FSW medium (the same medium used in all resuspension treatments).
All 14d metal concentrations were found to be greater than their
respective SEP1 levels. It is likely that portions of these metals linked to
the SEP2 fraction also underwent dissolution during resuspension. This
is shown where 14d concentrations of V, Mn, Ni and Cd were greater
than their respective SEP1 and SEP2 concentrations combined. Zn and
As dissolutions after 14d did not attain concentrations predicted by SEP1
and SEP2 combined, or by SEP2 only.

Fig. 2. Dissolution curves for select metals from time zero hours (0hr) to 14 days (14d) with error bars of ±1 standard deviation. Treatments: Continuously-agitated
FSW Control (CC); Un-agitated Tailings (UT); Continuously-agitated Tailings (CT).

6
K.S. Ramdial et al.
3.3.4. PCA of variables affecting dissolution
PCA yielded 3 Principal Components (PCs) with eigenvalues >1,
with a cumulative variance = 93.58% (Fig. 3). PC1 explained 68.24% of
the total variance where V had a weak loading, Cd, pH and salinity had
moderate loadings and all other abiotic variables had strong loadings.
PC2 explained 17.53% of the total variance where V had a strong
loading and salinity had a moderate loading. PC3 explained 7.81% of the
total variance where all variables had weak loadings except pH which
had a moderate loading. For PC1 the metals Mn, Zn, As and Cd displayed
statistically significant positive correlations with time (Pearson n =
0.898, 0.932, 0.774, 0.981 and 0.583 respectively; p = <0.05).
Conversely, As and Cd had statistically significant negative correlations
with pH (Pearson n = − 0.580 and − 0.628 respectively; p = <0.05). All
metals had statistically significant negative correlations with dissolved
oxygen (Pearson n ranged between − 0.769 and − 0.923; p = <0.05).
Note that dissolved oxygen and pH values decreased over time during
tailings resuspension.
3.4. Sea urchin bioassay – fertilisation success, toxicity index and
polyspermy
Bioassay results for P. miliaris gametes revealed that the 14d elutriate
markedly reduced fertilization success compared to the 18hr elutriate. A
fertilisation success rate of 72.0% was observed for the pure (100%)
18hr elutriate, but drastically drops to 21.1% for the pure 14d elutriate.
The decrease in fertilisation success was mirrored by the lowering of the
estimated EC50 concentration from >100% (18hr) to 51.53% (14d).
Fig. 4 illustrates the lower sea urchin fertilisation success rate (height­
ened toxic response) when P. miliaris gametes were exposed to the 14d
elutriate. The inflection points for the 14d elutriate dose response curve
occur at lower concentrations and are more pronounced compared to
the 18hr dose response curve. From the EC50 value derived for the 14d
elutriate sample respective EC50 concentrations were estimated for
metals that underwent dissolution. These concentrations represent the
levels at which they may have contributed to 50% inhibition of fertil­
isation success in P. miliaris. The estimated EC50 concentration for the
metals investigated ranged between 0.002 μg/L (Pb) and 45.409 μg/L
(As). Refer to Table 3.
The USEPA Aquatic Life Criteria Tables (USEPA 2017) provides a
Fig. 3. Principal Component Analysis (PCA) correlation circle illustrating
relationship between metals concentrations following resuspension (dissolu­
tion) vs. abiotic variables (time, temperature, dissolved oxygen, pH and
salinity). PCA performed using XLSTAT (Addinsoft 2015).
7
Marine Environmental Research 171 (2021) 105471
Criteria Maximum Concentration (CMC), concentration above which
toxicological effects are likely to occur, for various contaminants of in­
terest. The CMCs are the highest concentrations recommended without
causing unacceptable impacts to aquatic life (USEPA, 2017). Table 3
provides a comparison of EC50 concentrations of the 14d tailings
elutriate with existing study and literature review EC50 concentrations
listed by Novelli et al. (2003) and against CMCs for salt-water envi­
ronments. The tailings EC50 concentrations for metals investigated in
this study were below those previously noted by Novelli et al. (2003)
and outlined by the USEPA CMC (where available). The calculated EC50
value for As was the highest of the metals investigated suggesting it had
the lowest effective dose. However, this EC50 value was closest to the
respective CMC value outlined by the USEPA and it may be reasonable to
suggest As exerted the greatest influence on the acute toxic response
observed for fertilisation success rates based on the sheer dissolution
rate observed after 14d (859.2%).
TI scores were calculated for the 18hr and 14d elutriates based on the
frequency of normal and abnormal fertilisation membranes. Trends
between the TI scores and concentrations of 18hr and 14d elutriate test
solutions are shown in (Fig. 5). For the 100% elutriate samples the TI
scores increased from 1.6 (18hr) to 2.8 (14d). The increase in TI scores
followed a typical sigmoidal (S-curve) dose-response for the 14d
elutriate but not for the 18hr elutriate. In the 14d elutriate solutions type
1 and type 2 fertilisation membranes were more frequent at concen­
trations ≥25% with an absence of type 0 (normally developed) fertil­
isation membranes recorded in 3 of the 4 replicates for the 50% and
100% solutions.
Higher embryo polyspermy percentage observations were recorded
after 14d (21.1%) vs after 18hr (17.58%) for the 100% elutriate solu­
tions. The proportion of fertilised eggs exhibiting polyspermy increased
with higher concentrations of the 18hr and 14d elutriate test solutions
with a spike in the 50% solution for both the 18hr and 14d elutriates.
This resulted in both responses fitting best to Gaussian curves with a
predicted peak in percentage polyspermy between 60% and 70%
elutriate concentrations (Fig. 5). At higher elutriate concentrations there
is a drop in polyspermy occurrence and expectedly mirrors decreased
fertilisation success rates at these concentrations (refer to Fig. 4).
4. Discussion
Copper mine tailings were subjected to continuous resuspension in
seawater over 14 days (14d). Aqueous concentrations of As increased
859%, Ni increased over 85%, Mn and Cd increased over 40% while V
and Zn increased over 20%. These increases after 14d were found to be
substantial compared to concentrations measured after 18 h (18hr). V
concentrations were highest in the Continuously-agitated FSW Control
(CC) bottle. It was noted that at 14d Mn, Zn and Cd concentrations were
highest in the Un-agitated Tailings (UT). This shows the dissolution
potential once the soluble phase concentrations are greater than that of
the receiving medium, even in the absence of a significant disturbance
event. Thus, remobilisation of metals from contaminated sediments into
coastal or marine waters can potentially occur for a very long time, even
in calm conditions and undisturbed seabeds, until an equilibrium is met.
The ecological implications of contaminated sediments even in low
energy unagitated waters, may therefore be severe.
To determine levels of toxicity due to mobilisation of the mine tail­
ings green sea urchin (Psammechinus miliaris) gametes were exposed to
18hr and 14d sample elutriates with fertilisation success dropping from
72% to 21% respectively as illustrated shown in Fig. 4. Toxicity Index
values for fertilisation membrane and embryo quality increased for the
14d elutriate. This meant reduced fertilisation membrane quality and
greater occurrence of embryo polyspermy were recorded over time
leading to reduced fecundity. These findings reveal that the biological
impacts due to remobilised metals can go beyond standard fertilisation
bioassay endpoints. Consequently, current predictions of the impacts of
RCS such as from mining wastes or dredge spoils may be
K.S. Ramdial et al. Marine Environmental Research 171 (2021) 105471

Fig. 4. Dose response curves for the inhibition of P. miliaris fertilisation success by elutriate of tailings resuspended for 18hr and 14d.

Table 3
Comparison of EC50 concentrations (μg/L) of the mine tailings vs. those from
Novelli et al. (2003) and the associated literature review, and USEPA (2017)
CMCs. Tailings EC50 was calculated as 51.53% of metal concentration in bottle
CT3 after 14d. N/A (Not Available).
Metals Calculated Sperm cell Lit review Saltwater CMC
tailings EC50 toxicity test EC50 (μg/L) (acute) (μg/L) (
(μg/L) EC50 (μg/L) ( Novelli et al. USEPA, 2017)
Novelli et al., (2003)
2003)

As 45.409 3940 N/A 69

Mn 10.402 N/A N/A N/A
Cu 0.457 57 10–60 4.8
V 0.441 N/A N/A N/A
Zn 0.309 210 280–380 90
Ni 0.303 5130 N/A 74
Fe 0.183 N/A N/A N/A
Cd 0.076 8400 8000–38000 40
Cr 0.040 3220 2070 and N/A
(III) 341800
Pb 0.002 16210 1300–19000 210

underestimated. This is important when considering community/eco­

systems resilience especially for habitats already compromised, or are
likely to be compromised, by other anthropogenic stressors such as
ocean acidification (Atkinson 2007; Roberts 2013).
The 14d resuspension experiment determined that the dissolution of
various metals from the mine tailings into the overlying seawater me­
dium, generally increased with the length of time resuspended. The
dissolution of various metals can be traced back to their composition and
associations with mineral complexes in the bulk tailings. The composi­
tion and levels of metals within the tailings were characteristic of Cor­
nish mine wastes. Pirrie et al. (2002) lists the maximum levels of As, Cu,
Zn and Pb ranging between 144 ppm–6100 ppm, 241 ppm–4300 ppm,
207 ppm–21200 ppm and 53 ppm–16000 ppm for each metal respec­
tively in various Cornish estuaries and where As contamination is
characteristic of the region (Beeston et al. (2008). RCRERM values indi­
cate metal fractions for As, CU, Zn, Pb and Ni within the bulk mine
tailings can cause adverse effects in marine environments. Additionally, Fig. 5. Response curve fitting for percentage elutriate concentration vs toxicity
RCRcAL2 for As, Cu, Zn and Pb also indicate the mine tailings would be index (upper) and percentage elutriate vs polyspermy concentration (lower) for
18hr (white circles/dotted line) and 14d (asterisks/solid line) resuspended
unsuitable for disposal at sea. A brief comparison of metal levels
tailings. The 14d toxicity index values were best fitted to a typical sigmoidal
detected in the copper mine tailings used in this study against impacted
dose response curve while the 18hr toxicity index values did not suggest a dose
coastal/marine locations found concentrations can vary depending on response. The percentage polyspermy responses were best fitted to gaussian
the source of the mine tailings (Cu vs Pb-Zn mines for example), see bell-shaped curves for both 18hr and 14d treatments.
Table 4. However, the general trend observed shows contamination in
mine tailings for enriched metals are in the ‘000 s mg/kg compared to in
the ‘00 s mg/kg for a bay historically contaminated by industrial wastes

8
K.S. Ramdial et al. Marine Environmental Research 171 (2021) 105471

(Bagnoli-Coroglio, Naples, Italy). Therefore, an investigation into the

Comparison of maximum (unless otherwise stated) metal concentrations (mg/kg) for mine tailings used in this study vs. mine tailings found in estuarine/coastal contaminated regions and an example industrially

Armiento et al. (2020)

mobilisation potential and ecotoxicological impact of metals from mine

(Bagnoli-Coroglio,
tailings into the water column due to resuspension is merited as detailed
in this study.
The comparison of metal concentrations derived from acid digestion

Industrial
of the bulk tailings and SEP (available) fractions indicated that the

Italy)

0.96
87.1
713
285
majority of metals present in the tailings were locked within less reac­

84
60

28
tionary fractions usually associated with crystalline oxyhydroxide and

Pb-Zn Mine tailings (average

oxide structures, organic matter, pyrite compounds, and silicate re­
siduals (Dold 2003; Torres and Auleda 2013). The association of As
(Portman Bay, Spain)

concentrations with the SEP2 fraction suggested that their dissolution

Mestre et al., (2017)

would be promoted by acidic conditions. Tessier, Campbell and Bisson

(1979) and Inmaculada et al. (2004) have both previously discussed
how the lowering of pH in solution media can promote the dissolution of
values)

trace metals adsorbed onto sediments. The concentrations of As in the

3772
1259
321

5.6
11

32

48
bottle resuspension tests increased substantially with agitation after
14d. However, the largest drop in pH in any of the resuspended tailings
bottle treatments was 0.26 units. This drop is small compared to what
(Dold 2006)(Chanaral

was required in the SEP2 test to lower the pH of the ammonium acetate
Cu mine tailings

leaching solution from pH7 to pH4.5 (drop of 2.5 units). Hence, disso­
lution concentration of As did not attain levels predicted by SEP2 and
Bay, Chile)

much more of their ‘available’ fraction in the tailings may have been
24100

N/A
N/A
N/A

locked within the Acid-Volatile Sulphide (AVS) fraction. Metals such as

281

223
26

Cd, Ni, Cu, Pb and also Zn are also bound onto insoluble sulphide forms
such as mono- and secondary sulphides and pyrites which constitute
Hudson-Edwards et al., 1999 in Dold

AVS fractions (Mcgrath et al. 2002).

AVS fractions have been recognised as a limiting factor in the
availability and potential toxicity of soils and sediments contaminated
(2006) (Rio Tinto, Spain)

with metals (Morse 1994). If the AVS fraction is greater than the
Cu/pyrite Mine tailings

opposing labile states the toxicity of the source sediment is low (Mcgrath
et al. 2002). Changes in the redox states of sediments can release metals
from their AVS sites. Resuspension events can oxidise sulphides into
sulphur species which readily desorb their previously bound metals.
This can explain the partial release of As and Zn from their assumed AVS
5500
4300
1300
2500
N/A
N/A
N/A

fraction state, since tailings were added to oxygenated FSW in agitated

treatments. Decreasing dissolved oxygen concentrations over the course
of resuspension may have moderated total release of As and Zn but the
Ramirez et al., (2005) (Caleta

14d concentrations of As and Zn were still far greater than at earlier

sampling points.
Pb-Zn Mine tailings

The effects of pH and changing redox potentials may also apply to

Palito Bay, Chile)

Mn, Ni and Cd which showed appreciable dissolution after 14d in our

study. This relationship was suggested by the PCA results (Fig. 3).
Atkinson, Jolley, and Simpson (2007) described Mn dissolution from
1.085
1985

21.2
7.57

RCS being promoted by lower pH levels, with dissolution of other metals

N/A

N/A
236

being inversely related to declines in dissolved oxygen concentrations.

Our study found dissolved oxygen levels dropped by 1.92 mg/L and pH
levels dropped by 0.26 units after 14d respectively. It is reasonable to
assume continued resuspension beyond 14d could have led to lower
Pirrie et al., (2003) (Fal
estuary, Cornwall, UK)

Mine tailings (various)

dissolved oxygen and pH levels, and further dissolution of metals. A

recent study (Ruocco et al., 2020) found O2 enriched mesocosms (which
contained RCS) resulted in a lower occurrence of malformations in the
purple sea urchin (Paracentrotus lividus) embryos and plutei compared to
mesocosms with no O2 enrichment (natural gas exchange), and where
2803
5073
6600

N/A
N/A
N/A
902

dissolution of metals was the main driver of embryo malformations as

opposed to PAHs. This inferred active oxygenation of contaminated
Wheal Maid Cu mine tailings,

sediments and the overlying water column could be an effective reme­

diation tool for inhibiting redox processes that promote dissolution, and
thereby mitigating adverse ecological impacts.
Liberated Fe oxides are strong scavenging agents of other released
Cornwall, UK

metals. This occurs via flocculation onto Fe oxides/hydroxide pre­

contaminated region.

cipitates which draws metals out of solution within short time periods
2666.69
1434.65
1452.31
718.32
108.35

(Jones-Lee and Lee 2005). Our study found Cu was rapidly removed
22.19
3.96

from dissolution between 0hr and 18hr and maintained low levels
thereafter; possibly due to the scavenging effect of Fe oxide/hydroxide
Table 4

(III)
Metal

precipitates. This relationship was observed where Fe was also rapidly

Cu

Cd
Zn
Pb
As

Ni

Cr

removed from dissolution between 0hr and 18hr in our study.

9
K.S. Ramdial et al.
Scavenging may also occur with hydroxides of Mn (Saulnier and Mucci
2000) but may have played a much smaller role compared to Fe scav­
enging. The results of our study noted Mn underwent significant disso­
lution during the resuspension study. Mn was found the have the second
highest estimated EC50 concentration, and likely played a role in the
decreased fertilisation success response in the 14d elutriate.
Resuspended metals may become complexed by organic matter
(OM). The negligible proportions of TOC in the tailings indicate OM
complexes may not have been crucial in metal dissolution or re-
adsorption. Interestingly, although fine grained particles usually bind
metals strongly to the sediment phase and restrict their availability even
in conditions of lower pH levels (van den Berg et al., 2001), Hansen et al.
(2005) found that Cu was more easily leached from the smallest particle
sizes (<38 μm) of tailings from a Chilean copper mine. Larger sized
particles had higher relative fractions of insoluble copper sulphides. If
this holds true for tailings from copper mines in general, then it would
explain the poor dissolution data observed for Cu.
Given the notable rise in metals dissolution after resuspension in our
study, a greater toxicological effect on sea urchin fertilisation success
was unsurprisingly induced by the 14d elutriate vs. 18hr elutriate. The
elutriate contains metals that are simultaneously interacting with the
sea urchin gametes with presumably synergistic, additive and even
antagonistic reactions. For a mixture of contaminants, in this case trace
metals, the overall assumption for interactions is the receptor will
respond firstly and greatest to the most toxic contaminant. We can
compartmentalise the tailings elutriate into toxic units for each metal
investigated where the toxic unit is equated to the EC50 estimated from
the dose response curves. Arsenic (As) had the markedly highest esti­
mated EC50, hence highest toxic unit and greatest toxic potential, sug­
gesting this metal alone could explain the majority of the fertilisation
success inhibition. Mn was found to have the second highest EC50 and
may have played a role in the toxic response with As or with other
metals. For example, Kobayashi and Okamura (2005) noted additive
interactions between Mn, Zn, and Ni created higher rates of anomalous
development in sea urchin embryos. This was concluded when ZnCl2 test
solutions diluted up to 50-fold with sample seawater near an abandoned
mine resulted in poorly developed or no fertilisation membranes as
observed for the undiluted solution, and confirmed the influence of
other metals in the ecotoxicological response.
Lowered pH levels have previously been identified as a potential
constraint on fertilisation success (Pagano et al., 1986). The decline in
pH levels observed during the resuspension experiment may have also
had a role in the ecotoxicological response. Given the vulnerability of
certain groups of marine organisms to elevated atmospheric CO2 driven
ocean acidification, any further reductions to pH levels will also serve to
diminish these organisms’ resilience to pollution (Roberts et al., 2013).
Additionally, the availability and toxicity of contaminants may be
increased by ocean acidification (Atkinson et al. 2007; Roberts et al.,
2013) which can compound the impacts of RCS under predicted climate
change scenarios.
The observed degradation of fertilisation membranes and higher
polyspermy with increasing metal concentrations after the 14d resus­
pension raises the issue of quantifying physiological and sub-cellular
burdens on sea urchin eggs, and those of broadcast spawners in gen­
eral. It is expected that within unimpacted natural habitats, the vast
proportion of fertilised embryos would fall under the TI rank 0 with
well-developed fertilisation membranes, as pictured in Fig. 1 (images a
and b). This would facilitate the development of viable embryos in
sufficient numbers to enable the recruitment rates needed to support
resilient and genetically diverse populations. A decrease in the quality of
the fertilisation membranes due to pollution, as illustrated for TI rank 1
(images c to g), could result in cascade effects as the embryo develops.
Severe pollution events, whether episodic or continuous, are likely to
result in the dominance of eggs representative of TI rank 2 with very
poor membranes and TI rank 3 (image h) where no membrane has
formed due to failure of fertilisation. A proportion of highly impacted
10
Marine Environmental Research 171 (2021) 105471
eggs may also express polyspermy upon fertilisation (images i and j)
resulting in unviable eggs. Therefore, Fig. 5 highlights how the TI rank
index can be used as a response curve tool for understanding the rela­
tionship between a toxicant, membrane quality and fertilisation success.
Fig. 5 also highlights how polyspermy occurrence can be present as a
confounding factor to embryo viability when the gametes of a broadcast
spawner are released into contaminated environments. Polyspermy
occurrence could be a result of heightened sperm activity/encounters
combined with the inhibition of fast blocks at the egg wall with
increasing metal concentrations. The percentage polyspermy occurrence
expectedly drops off beyond a threshold elutriate concentration where
the solution has become saturated with dissolved metals which either
inhibit sperm activity and/or fertilisation success at egg sites.
Loss of viable embryos and degraded embryo quality arising out of
compromised fertilisation may amplify the likelihood for abnormal
development beyond what is investigated in current standard bioassays.
While the inhibition of normal larval development in sea urchins has
been widely used in a range of ecotoxicological contexts (Beiras et al.,
2003; Carballeira et al., 2012), there are limited studies that link
membrane quality after fertilisation success assays with subsequent
larval development. The transmissible effects of exposed sperm on em­
bryo development have been previously investigated (Warnau et al.,
1996; Pagano et al., 2002; Manzo et al. 2008). However, exposure to a
toxicant is not restricted to any one gamete. This is particularly impor­
tant given that the ‘fast’ and ‘slow’ blocks to polyspermy in sea urchins
are activated by influxes of sodium (Na+) and calcium (Ca2+) ions
respectively. Ionic imbalances created outside and within eggs by
intense dissolution of metal ions (and possibly compounded by lowered
pH) could play a pivotal role in fertilisation membranes development,
polyspermy incidence and development of larvae into adults. Manzo,
Buono, and Cremisini (2010) investigated the impacts of Cu, Cd and Pb
and its mixtures on P. lividus offspring quality. Skeletal malformations
were observed even at low concentrations but the toxicants represented
a small subset of contaminants that can be found in polluted sediments.
A mechanism that can also be explored is the impact on sperm exposure
to elutriates of resuspended sediments on curvilinear velocity and swim
path trajectories and frequency of non-motile sperm. Fabbrocini, di
Stasio, and D’Adamo (2010) describe methods for a sperm motility
bioassay. Very recent work by Ruocco et al., (2019) found sediments
contaminated from historical industrial inputs (Bagnoli-Coroglio, Gulf
of Naples, Italy) resulted in higher rates of embryo and plutei skeletal
malformations for P. lividus. This was after adults and fertilised embryos
were exposed to the resuspended sediment in mesocosms. Toxicity
analysis of the whole life-cycle would provide a better understanding of
potential population bottlenecks, causes and impacts on recruitment in
already polluted environments, or habitats likely to be impacted by
resuspension of disposed mine tailings, or mining activities in deep-sea
area targeted for their enriched mineral content (Mestre et al., 2017).
Notable limitations were encountered during this study. Firstly,
resuspension did not arrive at a dissolution endpoint after 14d for most
of the metals encountered and where concentrations become saturated
within the seawater media (typically signalled by plateaued S-curves).
Further studies should aim to extend the resuspension timeframes until
such endpoints are reached. This would provide a better understanding
of the total contaminant concentrations that may be expected from mine
tailing sediments after a disturbance event. Secondly, variations in
salinity, water temperature, water depth (nearshore vs. offshore e.g.
deep-sea mining regions), and reduction in seawater pH due to ocean
acidification could influence dissolution behaviour and cumulative im­
pacts experienced by organisms, and should be explored in similar
studies.
Lastly, the continuous nature of the experimental resuspension may
also have influenced the dissolution curves observed. The remobilisation
of contaminants associated with resuspension events may significantly
vary depending on the properties of the resuspension event (weak/
strong, periodic/continuous, brief/prolonged). Therefore, the type of
K.S. Ramdial et al.
resuspension event should also be heavily considered when deriving
meaningful sediment guidelines and methods (Roberts 2012) especially
for managing sediment historically contaminated by industrial waste
from mining activities. Natural tidal environments would be more
complex than the resuspension created by a shake table on a single
continuous rpm setting, and are likely to be punctuated by events of
severe sediment disturbance such as the tidal spring/neap tidal cycles
and storm seasons. For examples of other studies replicating disturbance
events, Ruocco et al. (2019) configured resuspension mesocosms to
mimic two different turbulence scenarios generated by the passage of
storms over 30 days where resuspension was initiated by a centrifugal
pump (capacity 4500L/h). Pelusi et al. (2020) derived sediment elutri­
ates by stirring on an orbital shaker at 300 rpm for 1 h based on (USEPA
1991) for evaluation of dredge material for disposal. Rial and Beiras
(2012) obtained elutriate for 60 rpm for 30 min for an Ecological Risk
Assessment of resuspended estuarine sediment for viaduct construction.
Note that our study carried out resuspension on a shaker table at 100
rpm over 14 days as a representation of general agitation conditions
(OECD, 2020). A dedicated study towards understanding different
resuspension events and applying appropriate mechanical forcing (rpm)
and disturbance frequencies to reflect temporal variabilities would help
inform which criteria should be applied to better represent site specific
conditions.
This study provides an insight into the possible contaminant release
pathways and ecological consequences of RCS from disposed mine
tailings, with many experimental design permutations that make this
approach applicable for estimating such effects in various receiving
environments. While several Lines of Evidence (LOE) were explored
(chemical levels in the tailings and elutriate; fertilisation bioassay; TI
index and polyspermy), additional methods to assess risks posed by
resuspended mine tailings should also be considered. Such methods
include bioavailability of metals to test organism and/or gametes/em­
bryos and sub-cellular biomarker testing (Morroni et al., 2020).
Sequential Extraction Procedures (SEPs) can also be modified to better
replicate realistic pH and redox conditions in the sediment under
investigation, and derive bioavailability potentials for metals undergo­
ing dissolution as additional LOE for assessing contaminated sediments.
SEPs can also be tailored to investigate the availability of Rare Earth
Elements (REE) where these elements maybe of concern in site specific
scenarios due to their increasing rates of extraction for use in modern
technology (Mittermüller et al. 2016). Such methods will improve the
Weight of Evidence (WOE) generated and facilitate a more holistic
approach to understanding impacts, mechanisms and effective man­
agement tools for sediments affected by industrial wastes. As a case
study, Ruocco et al., 2019, can be used to further investigate active
oxygenation of contaminated sediments and the overlying water column
as an effective remediation tool for inhibiting redox processes that
promote dissolution, thereby mitigating adverse ecological impacts.
5. Conclusion
The dissolution of several metals from copper mine tailings into
seawater markedly increased over time with no definitive equilibrium
endpoints. The increase in concentrations was reflected in the decline in
fertilisation success in sea urchin gametes, and the increase in degraded
fertilisation membranes and frequencies of polyspermy. This high­
lighted the possible underestimation of impacts inferred from existing
sediment toxicity bioassays where exposure is typically terminated after
short time-periods. These findings underline the potential impacts of
RCS in coastal or marine sediment historically impacted by large scale
disposal of tailings. Not only are the total concentrations of each metal
after a given sampling point important, but also their ratios to (and in­
teractions with) other metals in dissolution over time. This underscores
why the application of extraction techniques and sediment guideline
criteria (ERMs and cALs) should be coupled with bioassays to make
sound management decisions on 1) the contaminant pathways from
11
Marine Environmental Research 171 (2021) 105471
resuspended material and 2) the potential ecological risks posed by
contaminated (or naturally enriched) sediments at risk for disturbance,
or currently experiencing natural or anthropogenic resuspension events.
Funding
This work was supported by the Scottish Association for Marine
Science (SAMS) through an internal small grant for the ICP-MS analysis.
CRediT authorship contribution statement
Keshtav S. Ramdial: Conceptualization, Methodology, Investiga­
tion, Writing – original draft, Writing – review & editing. Richard Abell:
Methodology, Formal analysis, Validation, Resources, Writing – review
& editing, Supervision. Kim S. Last: Conceptualization, Methodology,
Writing – review & editing, Supervision, Project administration, Fund­
ing acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was completed as part of an MRes in Ecosystem-based
Management of Marine Systems run jointly between the Scottish Asso­
ciation for Marine Science (SAMS) and the University of St Andrews, UK.
Dr. David Roberts (James Cook University, QLD, Australia), David
Hughes and Dr. Helena Reinardy (SAMS), and anonymous reviewers
contributed to improved manuscripts of this research. Gratitude is
extended to SAMS staff in particular - Tim Brand, Leah Morrison and
Gangi Ubbara who greatly assisted with the ICP-MS analysis; Lars
Brunner provided sea urchins and guidance in spawning methods; Peter
Lamont, Dr. Phil Kerrison and Chris Beveridge granted access to mi­
croscopy and camera equipment; and sadly and posthumously, to John
Kershaw for use and troubleshooting of the aquarium and controlled
temperature facilities. Terrie Sawyer and Kim Moreton from the Cam­
borne School of Mines kindly provided the mine tailing samples. Special
thanks to Shazara Ali, Richard Cottrell and Tara Beardmore.
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