Environmental Pollution 162 (2012) 406e412

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Environmental Pollution
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Tissue-specific assimilation, depuration and toxicity of nickel in Mytilus edulis

Geoffrey E. Millward a, *, Sandeep Kadam a, Awadhesh N. Jha b
a
Marine Institute, Portland Square, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom
b
School of Biomedical and Biological Sciences, Portland Square, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, United Kingdom

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

Article history: The tissue-specific accumulation and time-dependent depuration of radioactive 63Ni by the byssus, gut,
Received 14 July 2011 foot, gills, kidney, adductor muscle and faeces of Mytilus edulis has been investigated using a pulse-chase
Received in revised form technique. The rate and extent of depuration of 63Ni varied between tissues and, after 168 h, the
18 November 2011
concentration factors and assimilation efficiencies ranged from 1 to 35 L kg
1 and 5%e13%, respectively.
Accepted 24 November 2011
Mussels were also exposed to a range of environmentally-realistic concentrations of dissolved Ni, prior to
the analysis of biological endpoints. The clearance rate was concentration-dependent and at the highest
Keywords:
concentration decreased by 30%. Neutral red retention (NRR) assays indicated a cytotoxic response and
Marine mussels
Nickel
DNA strand breaks were observed in the haemocytes. The association of DNA damage with that of
Assimilation physiological and cytotoxic effects suggests that Ni exerts a significant impact on Mytilus edulis at cellular
Cytotoxicity and genetic levels.
Genotoxicity Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction 5  103 L kg
1 due to its tendency to complex with dissolved

Nickel (Ni) has increasing world-wide importance in applica-
tions of new technology, for example in Ni-metal hydride batteries
for electric and hybrid vehicles, in super-alloys for commercial and
military aircraft and in austenitic stainless steel for a new genera-
tion of nuclear power stations. Accordingly, there has been a surge
in the development of Ni mines and processing and refining plants
in the coastal regions of, for example, Brazil, Madagascar, New
Caledonia, Russia and western Australia (Salazar and McNutt, 2011).
Consequently, Ni enters coastal waters as mine tailings and during
ship-to-shore transfer operations of Ni ores, mainly laterite, leading
to elevated dissolved concentrations due to leaching by seawater
(Florence et al., 1994; Hédouin et al., 2007). High dissolved Ni
concentrations, in the range 0.6e30 mg L
1, have been detected in
industrialised estuaries (e.g. Martino et al., 2002; Prego et al., 2003;
Braungardt et al., 2007) and in sediment porewaters (e.g. Langston
et al., 1999; Prego and Cobelo-Garcia, 2003; Santos-Echeandia et al.,
2009). Accordingly Ni is included in the European Union (EU)
priority list of contaminants with an Environmental Quality Stan-
dard (EQS) for marine surface waters of 20 mg L
1 (Crane and Babut,
2007). Geochemically, Ni has a relatively low particle affinity and
distribution coefficients, KDs, in seawater are typically less than
* Corresponding author.
E-mail addresses: gmillward@plymouth.ac.uk (G.E. Millward), ajha@plymouth.
ac.uk (A.N. Jha).
organic matter (Martino et al., 2003).
Species of Mytilus have been used as biomonitors of industrial
metal contamination, including Ni, since the US Mussel Watch
Programme began in 1976 (Goldberg et al., 1983). The recom-
mended biological concentration factor for molluscs, CF [defined as
the ratio of the concentration of the metal in the tissue of the
organism in mg kg
1(dry weight) to that in seawater in mg L
1], for
Ni is 2  103 L kg
1 (IAEA, 2004). Although there has been extensive
research on the overall bioaccumulation of Ni in Mytilus spp., its
toxic impact on these organisms is not well understood. Laboratory
studies have elucidated, albeit to a limited extent, the impact of Ni
within Mytilus spp., using concentrations generally outside the
range found in estuaries and coastal waters mentioned above
(Table 1). Nickel compounds are classified as being carcinogenic to
humans (Beyersmann and Hartwig, 2008), although whether
aquatic organisms are also affected at the cellular and genetic level
is not clear. However, improved assessment of DNA damage can
now be achieved using highly sensitive single-cell gel electropho-
resis (i.e. the Comet assay) in both laboratory studies (Jha, 2008)
and as a biomarker in environmental monitoring for coastal
pollution (Rank et al., 2005; Klobu car et al., 2008).
Thus, there is scant evidence in the literature on the accumu-
lation and time-dependent depuration of Ni from specific tissues of
mussels. Furthermore, the impact of Ni on the genetic integrity of
mussels also lacks clear definition, requiring laboratory experi-
ments to be conducted at environmentally-realistic concentrations.
Here we adopt an integrated approach, in which the tissue-specific

0269-7491/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2011.11.034
 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412 407

Table 1
Biological or biomarker responses in Mytilus spp. exposed to dissolved and particulate Ni under laboratory and field conditions.

Organism Expoure Time, d [Ni]D, mg L
1 Biomarker Responses Reference

Laboratory Exposures
Mytilus edulis 28 13e102 N/A Friedrich and Filice, 1976
Mytilus edulis 84 5 & 10 N/A Zaroogian and Johnson, 1984
Mytilus edulis 2.3 0.4a N/A Punt et al., 1998
Mytilus galloprovincialis 28 20 & 200b SFG & GPX response related to Tsangaris et al., 2007
metal concentrations but not for AChE
Mytilus sp. 7 0.01e0.10c No MT induction at any concentration Amiard et al., 2008
Mytilus galloprovincialis 4 770 Changes in energy metabolism and Jones et al., 2008
amino acid profiles; no reduction of AChE
Mytilus galloprovincialis 28 3e3000 SFG & GPX response related to Ni Tsangaris et al., 2008
concentrations but not for AChE
Mytilus trossolus (larvae) 2 50e1000 Abnormalities occurred in embryo Nadella et al., 2009
larvae at EC50 ¼ 150 mg L-1
Mytilus galloprovincialis 8 135 & 770 MT; GST; MDA increased with time; Attig et al., 2010
AChE decreased

Field Exposuresd [Ni]P, mg g
1

Mytilus galloprovincialis 90 ND MT induction in whole soft tissues Mourgaud et al., 2002
containing Cd, Cu, Ni and Zn
Mytilus edulis N/A 0.2e2.1 Higher DNA damage (Comet assay) Rank et al., 2005
in gill vs haemolymph cells
Mytilus sp. 30 45 MT induction by Ni and V from Amiard et al., 2004
an oil spill
Mytilus galloprovincialis 30 48e240 Increase in MN frequency and %tail Klobu
car et al., 2008
DNA (Comet assay) at polluted sites

[Ni]D and [Ni]P are dissolved and particulate Ni, respectively. AChE ¼ acetylcholine esterase activity; EC50 ¼ half maximal effective concentration; GPX ¼ glutathione
peroxidase activity an indicator of ROS; GST ¼ glutathione S-transferase activity; MDA ¼ malondialdehyde accumulation; MN ¼ micronucleus; MT ¼ metallothionien;
SFG ¼ scope for growth; N/A ¼ not applicable.
a
Dissolved Ni added as 63Ni.
b
Experiments carried out with a mixture of Ni, Cr, Fe.
c
Dissolved Ni injected into the posterior adductor muscle.
d
Concentration of Ni in contaminated sediments which also contain other metals.

bioaccumulation and depuration of Ni is complemented with an
examination of its physiological, cytotoxic and genotoxic effects.
2. Materials and methods
2.1. Collection and acclimatisation of mussels
Adult mussels, in the size range 45e55 mm, were collected from a reference site
at Whitsand Bay, S.W. England, in early spring to avoid seasonal spawning effects.
Specimens were carefully detached from rocks to avoid damage to the byssus gland
(Rajagopal et al., 2005) and were cleaned of epibionts. In the laboratory, the mussels
were placed into filtered (<5 mm pore size) seawater (salinity ¼ 34) in a constant
temperature room (15  1  C). Mussels were fed daily with micro algae (Isochrysis
galbana, AlgaeÒ Reed Mariculture, Inc. USA) and the water was changed daily after
the feed. The mussels were acclimatised for 2 weeks prior to exposure.
63
2.2. Exposures to radio-labelled Ni
A pulse-chase experiment was designed to assess the tissue-specific
bioaccumulation and depuration of radio-labelled 63Ni by Mytilus edulis under
environmentally-realistic, controlled conditions of temperature, salinity and sus-
pended matter concentration used previously (Punt et al., 1998; Jaeschke et al., 2011).
The addition of a radiolabel has the advantage that, because liquid scintillation
counting is very sensitive, relatively low concentrations of Ni can be used. The pulse
of radioactive feed was for 2 h which approximates to the gut passage time allowing
better estimation of the assimilation efficiency, AE (Wang and Fisher, 1999).
2.2.1. Preparation of suspended solids and radio-labelling
Muddy, oxic surface sediment (0e1 cm) was obtained from the Tamar Estuary
(S.W. England). A three-stage separation procedure was used to isolate permanently
suspended particulate material (P-SPM): (1) the sediment was wet sieved to obtain
a sufficient quantity of the <63 mm grain size fraction, (2) the sieved sediment was
resuspended into 20 L tanks containing seawater and allowed to settle for 5 min
before the slow settling fraction was siphoned off and (3) the slow settling fraction
was then resuspended and allowed to stand for 2 h, prior to the P-SPM being siphoned
off. Similar aliquots of P-SPM were added to two plastic carboys, each containing 18 L
of seawater (salinity ¼ 34.5), to create suspensions of 5 mg L
1 which was determined
gravimetrically. The carboys were stirred and aerated in a constant temperature room
in the dark. One carboy was used as feed during 48 h of acclimatisation and the other
was spiked with 7.3 mL of 63NiCl2 solution (206 kBq mL
1) for use as feed during the
2 h pulse period. This gave an activity concentration of 8.35  104 Bq L
1 and a stable
Ni concentration of 36 ng L
1, which is significantly lower than concentrations in
coastal waters (Tappin et al., 1995). The carboy containing the 63Ni spike was allowed
to equilibrate with dissolved ligands and the P-SPM for 24 h by stirring in the dark in
the constant temperature room. After equilibration, analysis of the dissolved 63Ni
concentration had a KD ¼ 890  95 L kg
1 (n ¼ 3), indicating that 99% of the 63Ni was in
the dissolved phase.
2.2.2. Preparation and implementation of exposure
Fifty mussels were attached to the side of a 20 L feed tank using a fast setting
epoxy resin (Araldite Rapid). Mussels were mounted to expose the byssus threads and
with the posteriors angled downward, to allow faeces capture in a beaker. The tank
was filled with the unspiked P-SPM suspension and the mussels were acclimatised for
48 h under constant aeration. Subsequently, the unspiked suspension in the tank was
replaced with the 18 L of P-SPM spiked with 63Ni and the mussels fed for 2 h.
When the feeding period ended the spiked suspension was discarded and the
periostracums of each mussel carefully washed with seawater to remove residual
radioactivity. The mussels were then depurated for 168 h during which they were
fed clean, unspiked P-SPM suspension, refreshed from a reservoir using a continuous
flow system (Punt et al., 1998). At pre-determined time intervals at least 3 mussels
were removed from the tank and their associated faeces in the beakers were
removed with a Pasteur pipette. The mussels were stored at
20  C and the faeces
were filtered using pre-weighed cellulose nitrate filters of pore size 0.45 mm.
2.2.3. Post-exposure analyses
Mussels were dissected to obtain separate tissues of byssus, gut, foot, gills, kidney
and adductor muscle. Each tissue was washed with 5 mL of distilled water to remove
superficial 63Ni and the tissues, and the filter samples of faeces, were freeze-dried to
constant weight. Soluene-350 (PerkineElmer, Buckinghamshire, UK) was added
(1 mL) to each sample and placed in an oven held at 55  C for 48 h until the tissues
and filter membranes had solubilised. Glacial acetic acid (35 mL) was added to the
samples to neutralise any remaining Soluene-350. Scintillation cocktail (Ultima Gold,
PerkineElmer, Bucks, UK) was added (5 mL) to the solubilised tissues and the filters.
The samples were kept in the dark for 2 h to allow decay of chemiluminescence prior
to liquid scintillation counting (Beckman Coulter, UK) at a precision of 5%.
2.3. Exposure of mussels to stable Ni
Glass beakers (1 L) each containing a mussel and filtered (<5 mm pore size),
aerated seawater, were located in a constant temperature room (15  C). The mussels
were acclimatised for 48 h, after which the beakers were amended with Ni(II)
nitrate, made up in seawater at pH ¼ 8. There were five replicates for each of three
408 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412

Fig. 1. Assimilation of 63Ni (Bq g
1) after the 2 h pulse and its tissue-specific time-dependent depuration (a) byssus; (b) digestive gland. (c) gills: (d) foot; (e) kidney; (f) adductor
muscle. The filled circles positioned at
10 h indicate the activity concentrations of 63Ni in the controls (n ¼ 5) where the error bars are subsumed into the symbol.

dissolved Ni concentrations and five replicates acted as controls. Water samples
were taken from each beaker for analysis of temperature, salinity, pH and dissolved
oxygen, and dissolved Ni. The average nominal concentrations (n ¼ 5) of dissolved
Ni, as determined by inductively coupled plasma-mass spectrometry, over the
course of the experiment were 14  2, 31  4 and 120  11 mg L
1 and for the control
was 4.6  2.1 mg L
1. During the 120 h exposure the mussels were not fed.
2009). Approximately, 15  103 cells of algal suspension (Isochrysis galbana, AlgaeÒ
Reed Mariculture, Inc. USA) was added to the beakers. Immediately, 20 mL of
seawater was removed from each beaker and additional samples were taken at 15
and 30 min intervals. Samples were analysed on a Coulter Particle Counter (Beckman
Coulter, Z2) fitted with a 100 mm aperture tube and set to count particles between
4.0 and 10.0 mm in diameter.

2.4. Biomarker or biological responses 2.4.2. Haemolymph extraction

After exposure, the haemolymph was extracted from posterior adductor muscle
2.4.1. Clearance rate using a sterile 1 mL syringe fitted with a 21 gauge needle. Approximately 0.2 mL of
The clearance rate (CR, L h
1) was quantified after the exposure period by haemolymph was withdrawn and placed in an Eppendorf tube containing physio-
placing mussels in separate beakers, which contained filtered seawater (Canty et al., logical buffer saline (PBS; pH w 7.36) and stored at 5  C.
 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412 409

2.4.3. Neutral red retention (NRR) assay
The cytotoxicity in the haemolymph was determined by NRR, which is based on
the ability of cells to retain supravital neutral red dye. The method is adapted for
mussel haemocytes and is used routinely in our laboratory (Canty et al., 2009). A
stock solution of neutral red dye was made by dissolving the dye in dimethyl
sulphoxide (DMSO) from which a dilute working solution was prepared in PBS.
Samples of haemolymph (50 mL) were incubated, in flat bottomed microtitre plates,
to allow a monolayer of cells to adhere to the wells. Non-adhered cells were dis-
carded and the wells were washed with PBS. A solution of neutral red dye was added
to each well and incubated for 3 h at room temperature. The cells were washed with
a mixture of acetic acid and ethanol and the plate shaken for 10 min to solubilise the
dye. The solution was read at 560 nm, using a microplate reader (Optimax) and
protein concentrations were determined using a commercially-available bovine
serum albumin standard (Thermo Scientific, USA).
2.4.4. Comet assay
The genotoxicity in the haemolymph was determined, as the reliable parameter
tail %DNA, by Comet assays (Jha, 2008; Canty et al., 2009). Aliquots of the haemo-
lymphs were pelleted in a microfuge tube, the supernatant removed and resus-
pended in low melting-point agarose. This cell suspension was added to the frosted
ends of glass slides coated with dilute, normal melting-point agarose and placed in
refrigerator to allow the agarose to solidify. The coverslips were removed and placed
in a lysis solution to dissolve the cells. Alkaline unwinding by electrophoresis was
carried out at 4  C in a gel electrophoresis solution, after which the slides were
placed in a neutralising buffer. The slides were then stained with ethidium bromide
and viewed under an epifluorescence microscope (Leica, DMR). The slides were
analysed using the Komet 5.0 image analysis system (Kinetic Imaging, Liverpool,
UK).
3. Results and discussion
63
3.1. Tissue-specific accumulation and depuration of Ni
3.1.1. Accumulation
The tissue-specific concentrations of 63Ni, immediately after the
pulse, were significantly above the respective concentrations in the
controls (Fig. 1). The accumulation of 63Ni followed the sequence
byssus > gut > gills > foot > kidney > adductor muscle and activity
concentrations in the tissues varied over two orders of magnitude.
Mussels may accumulate metals in various tissues via three
processes: (a) absorption across the gastro-intestinal tract via the
intake of particulate matter comprising the diet, (b) absorption
across the surfaces of the body that may be exposed to the water, in
particular the gills, foot, byssus and adductor muscle and (c)
re-distribution of metals within the organism by metabolic
processes (Widdows and Donkin, 1992).
The tissue-specific accumulation of 63Ni within the Mytilus
edulis occurred, predominantly, in the byssus (69%), which is
significantly more than the w20% observed by Punt et al. (1998).
The threads, produced by the byssal gland within the foot, have
similar dimensions to human hair and are a sticky mixture of
keratin and polyphenolic proteins which enable mussels to adhere
to substrates. The attachment of the byssal threads to a substrate is
important because the sensitivity of Mytilus spp. to toxicants is
dependent on whether they are attached or not. If the byssus
is unattached the valve opens to allow extension of the foot to
re-attach the byssal threads, thereby exposing the internal soft
body parts to dissolved contaminants (Rajagopal et al., 2005).
Although attention was paid to maintaining the integrity of the
byssus while sampling, the mussels had to be carefully cut away
from their rocky holdfasts. During the 48 h acclimatisation, the
byssi were partially attached to the surface of the tank and thread
development may have taken place while the experiment was
underway. Therefore, we cannot rule out the possibility that
byssogenic activity was important in determining the byssus Ni
content during uptake and depuration. Increases in the 63Ni
content of the byssus could be due to complexation of dissolved
Ni with the organic molecules comprising existing, or nascent,
threads or to P-SPM, spiked with 63Ni, becoming trapped within
them. However, the byssus samples were carefully washed in a few
mL of Milli-Q water, prior to Soluene digestion, thereby reducing
the amount of particle-associated 63Ni. Mytilus spp. from the
southern Baltic Sea were analysed for their trace metal content and
were found to have 6- to 8-fold higher concentrations of Ni in the
byssus as compared to the body. It has been argued that elevated
concentrations of metals in the byssus arise because, rather than
the metals being adsorbed to the surface, the byssus is the main
conduit whereby metals are eliminated from the body of mussels
(Szefer et al., 2002).
The gut accounts for about 13% of the total 63Ni assimilated by
the mussels which confirms its importance as a pathway for Ni
uptake (Punt et al., 1998; Hédouin et al., 2007). The relative amount
of 63Ni assimilated from the food and/or the water cannot be easily
assessed from these experiments, except that the magnitude of the
KD indicated most of the radiolabel was in the dissolved phase. The
kidney only contained 4e7% of the total 63Ni taken up. However,
the kidneys are an important site for metal accumulation because
metals are immobilised by cellular compartmentalisation into
granules which, ultimately, may be excreted in the urine (George,
1983).
3.1.2. Depuration
The depuration of 63Ni from the tissues, with the exception of
the kidney, followed a general exponential decrease with time
(Fig. 1). After 168 h the order of the activity concentrations followed
the sequence byssus > gut > kidney > gill z foot > adductor
muscle, with the byssus and gut having retained about 68 and 13%,
respectively, of the total 63Ni. The net amount of 63Ni excreted by
the tissues after 168 h of depuration was relatively constant, in the
range, 64e81%, with the exception of the kidney (Table 2). This is in
broad agreement with Zaroogian and Johnson (1984) who found
that mussels depurated between 73 and 89% of the Ni assimilated.
The depuration of 63Ni from the byssus was irregular with an
initial increase followed by a decrease to 12 h, a sharp increase at
24 h and a subsequent decline (Fig. 1a). There was a delay of about
48 h before the start of depuration of 63Ni from the gut (Fig. 1b)
after which there was a slow loss of the radiolabel until 168 h, when
36% of the original 63Ni remained in the gut. The gills showed
a biphasic depuration profile with a rapid first stage (Fig. 1c), rep-
resenting the excretion of particles containing 63Ni that were
unavailable to the organism. This was followed by a slower second
stage represented by loss through phagocytosis of fine particles
channelled from the stomach, as proposed in a model for marine
bivalves (Wang and Fisher, 1999). Approximately 22% of the original
activity remained in the gut after 168 h. A general exponential
release was observed for the foot and adductor muscle (Fig. 1d and
e). The kidney had an initial uptake which, although less than the
other tissues, remained relatively constant over 168 h (Fig. 1f). The
faeces had an elevated activity concentration of 63Ni after 1 h,
Table 2
63
Biological constants for uptake and depuration of Ni by Mytilus edulis.
Tissue Extent of Depuration Concentration Assimilation
Depuration, % Half Life,a h Factor,b L kg
1 Efficiency,b %
Byssus 71 N/S 35 37
Gut 64 N/S 7 10
Gill 76 N/S 3 4.3
Foot 84 14 2 2.8
Kidney 27 N/S 4 6.3
Adductor 81 13 1 1.6
Muscle
Faeces 86 5 N/A N/A
a
N/S ¼ fit to the exponential depuration model, Eq. (1), not significant at p < 0.05.
b
From Eq. (2), after 168 h. N/A ¼ not applicable.
410 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412
Fig. 2. Time-dependent activity concentrations of 63Ni in the faeces. The filled circle
positioned at
10 h indicates the activity concentrations of 63Ni in the controls (n ¼ 5)
where the error bars are subsumed into the symbol.
followed by an exponential decrease (Fig. 2) and after 48 h they had
declined to relatively low values.
3.2. Biological constants
The time-dependent depuration trends in Figs. 1 and 2 were
fitted to a first-order exponential decay model:
½63 Nit ¼ ½63 Ni0 *e
kt (1)
where [63Ni]0 and [63Ni]t are the mean activity concentrations of 63Ni
in the organism at time t ¼ 0 and t ¼ t, respectively, and k is the decay
constant in h
1. The fit of the data to Eq. (1) was significant, at
p < 0.05, only for the foot, adductor muscle and faeces, where the
depuration half lives (t1/2 ¼ 0.69/k) were 14 h, 13 h and 5 h,
respectively (Table 2). The depuration from the foot and adductor
muscle fitted the single compartment model, in common with other
invertebrates (Kumblad et al., 2005; Hédouin et al., 2007). The foot,
and to a lesser extent the adductor muscle, are involved in the
manipulation of the newly forming byssus threads and may,
therefore, have proteinaceous coatings. These external surfaces,
exposed to dissolved 63Ni during the exposure phase, may complex
with the radiolabel. Since the t1/2 of faeces in the gut is 5 h, it is
anticipated that particles labelled with 63Ni would have been almost
completely evacuated by the mussels within about 30 h (i.e. 6  t1/2).
Metal retention in the gut is a common feature of bivalves, for
example retention of Co can last up to 72 h (Wang and Fisher, 1999)
and the relatively long dwell time for 63Ni in the gut epithelium,
observed in our experiments, may give rise to increased uptake and
dispersal to other tissues, resulting in enhanced toxic effects.
The CFs, on a dry weight basis, varied from 1 L kg
1 for the
adductor muscle to 35 L kg
1 for the byssus (Table 2). Values of CF
(dry weight) reported for Mytilus spp., and other molluscs, are
derived mainly from the laboratory experiments described in Table 1.
Thus, exposure of Mytilus edulis to various concentrations of dis-
solved Ni gave CFs for whole body tissue in the range 250e400 L kg
1
(Friedrich and Filice, 1976), whereas Zaroogian and Johnson (1984)
obtained values of 1640e2080 L kg
1. However, estimates of CF ob-
tained from experiments of 63Ni uptake by Mytilus edulis gave
significantly lower CFs of 100 L kg
1 for the byssus and 12 to 27 L kg-1
for the whole body tissue (Punt et al., 1998). For Mytilus
galloprovincialis the CFs in whole body tissue were dependent on the
dissolved Ni dose and were in the range 73e3170 L kg
1 (Tsangaris
et al., 2008), whereas studies of the digestive gland showed they
were from 11 to 43 L kg
1 (Attig et al., 2010). For clams and oysters
(Hédouin et al., 2007), estimated 63Ni CFs for various tissues in the
range 16e660 L kg
1, except for the combined visceral mass and
mantle of Mulleus regula where the CF was 1901 L kg
1. Thus, almost
all of the recent values of CF, with the exception of those resulting
from the study by Zaroogian and Johnson (1984), are significantly
lower than the recommended CF for Ni in molluscs of 2  103 L kg
1
(dry weight), which appears to be derived from relatively few envi-
ronmental measurements in Long Island Sound (IAEA, 2004). This
suggests that the recommended CF for Ni for a broad range of
molluscs needs to be re-examined and, possibly, revised downwards.
The assimilation efficiency, AE, is given by:
Atissue
AE ¼   (2)
Atissue þ Afaeces
where Atissue and Afaeces are the mean activity concentrations of 63Ni
in a tissue and the faeces at 168 h, respectively (Table 2). Assimilation
efficiencies for metals are reliant on several important factors related
to the biology of the organism, such as the rate of ingestion, gut
retention time and the relative importance of extra- and intra-
cellular digestion. Also, the bioavailability of the metals associated
with the food has a significant effect which is controlled, to a large
extent, by the strength of metal binding with the particle surface, i.e.
the KD. Metals that are strongly bound to food particles may not
desorb when attacked by the low pH environment, normally pH 5 to
6, in the gut of bivalves (Widdows and Donkin, 1992). Thus, for
Mytilus edulis AEs for Cr were persistently less than 10% because, in
seawater, the metal has a high KD, typically 104 to 105 L kg
1, and is
less bioavailable. Silver has a KD of a similar magnitude, and hence
a relatively low bioavailability, and typical AEs are of the order 5e20%
(Luoma and Rainbow, 2005). On the contrary, Cd has a KD of the order
103 L kg
1 and AEs in Mytilus edulis ranged from 10% to 30% indicating
that at ambient coastal SPM concentrations the organism may
acquire the metal from the dissolved and particulate phase. Our
tissue-specific AEs for Ni were in the range 5e35% (Table 2). Since Ni
also has a relatively low KD, it likely that a proportion of Atissue orig-
inated from the assimilation of Ni (as 63Ni) from the dissolved phase.
3.3. Physiological, cytotoxic and genotoxic effects
The values of CR decreased by about 30% from 2.2 L h
1 for the
controls to 1.54 L h
1 for a nominal dissolved Ni concentration of
150 mg L
1 (Fig. 3a). A regression analysis of CR against the dissolved
Ni concentrations showed a significant relationship (R ¼
0.998;
p ¼ 0.027). There was a major difference between the control and
the lowest concentration (p < 0.05, KruskaleWallis test), however
the differences between each of the concentrations were not
statistically significant.
Neutral red retention assays are designed to assess the viability of
the cells based on the penetration of a weakly cationic dye across
lysosomal membranes. These organelles accumulate a significant
fraction of environmental contaminants, including metals, which can
destabilise the lysosomal membrane. Since lysosomes play key role in
biological processes of the organism, any deleterious changes in their
function can affect cell growth, the maintenance of homeostasis and
the defence mechanism of the organism, which leads to an auto-
phagic loss of body tissue. The NRR values in this study ranged from
0.70 to 0.32 OD mg (protein)
1 for the control and the maximum
dissolved Ni concentration, respectively (Fig. 3b). The major decrease,
about 44% of NRR, occurred between the control and the lowest
 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412
Fig. 3. (a) Clearance rate of M. edulis following exposure to dissolved nickel; (b)
Cellular viability in M. edulis haemocytes after exposure to dissolved nickel; (c)
Induction of DNA strand breaks (represented as % tail DNA) in Mytilus edulis haemo-
cytes following exposure to dissolved nickel. Asterisks (*) denote a statistically
significant difference (p < 0.05).
dissolved Ni. Only minor decreases in NRR occurred as the doses
increased and there were no statistical differences between each
concentration. This could be due to the behaviour of Ni at a cellular
level indicating that at relatively low concentrations may be more
toxic than previously thought or the lysosomal compartments of the
cells had reached their saturation level. The loss in lysosomal stability
may also be due to an increase in calcium in the haemolymph since Ni
enters the cell via the calcium channel which causes the initial
decrease in Ca2þ, followed by its release from intra-cellular stores via
cell surface receptors (Denkhaus and Salnikow, 2002).
DNA damage in the haemolymph of mussels varied from 9.1% for
the controls to 55% for the maximum dissolved Ni concentration
(Fig. 3c). There was no statistically significant concentration
dependant increase for the induction of DNA damage (p < 0.05,
KruskaleWallis test). Multiple variance tests showed a significant
difference between the control and all three concentrations.
However, there were only minor increases in the DNA damage in
the mussels as the dissolved Ni concentration increased. The Comet
411
assays showed a 5-fold increase in percentage tail DNA between the
control and the lowest dissolved Ni concentration. When the dis-
solved Ni concentration was augmented by about an order of
magnitude only minor increases in percentage tail DNA, i.e.
approximately 22%, were observed. This indicates that accumula-
tion of Ni in Mytilus edulis has a significant genotoxic effect on the
integrity of the DNA in the cells. Dissolved Ni, particularly the free
ion Ni2þ, forms a variety of complexes with different ligands, such
as amino acids, peptides, proteins, and glutathione, some of which
are redox active and catalyse reactive oxygen species (ROS), such as
the hydroxyl radical. The ROS subsequently interact with DNA,
inducing damage to the bases of DNA leading to the generation of
strand breaks. Beyersmann and Hartwig (2008) report that Ni2þ
interacts with DNA repair systems, this mechanism may involve the
ability of Ni2þ to compete with magnesium and zinc ions in the
structure of DNA repair enzymes, in addition to causing
DNA-protein cross links. The relative contribution of these mech-
anisms for the generation of DNA strand breaks, under the exper-
imental conditions, is difficult to ascertain.
It is generally recognized that results obtained under laboratory
conditions cannot be compared directly with observations made in
the natural environment, where a wide range of contaminants are
present in a multiplicity of combinations and where biotic and
abiotic factors can modulate biological responses in natural biota
(Jha, 2008). Nevertheless, in laboratory experiments using Mytilus
galloprovincialis exposed a dissolved Ni concentration of 770 mg L
1
increases in glutathione-S-transferase activity and malondialdehyde
accumulation (Attig et al., 2010) and changes in energy metabolism
within the organism (Jones et al., 2008) were detected. Tsangaris
et al. (2008) showed that the scope for growth (SFG; an indicator
of the physiological health of the organism of which CR, as deter-
mined in this study, serves as a component) of Mytilus gallopro-
vincialis was seriously impaired as the dissolved Ni concentration
was raised from 300 to 3000 mg L
1 and that glutathione peroxidase
induction tended to occur predominantly at the highest dissolved Ni
concentration. Amiard et al. (2008) found little evidence for the
induction of metallothionein in Mytilus spp., although the dissolved
Ni was administered via the posterior adductor muscle, rather than
through feeding. For embryo larvae of Mytilus trossolus, the EC50 for
Ni was 150 mg L
1 in a 48 h exposure, although metal complexation
with dissolved organic carbon appeared to offer a “protective effect”
(Nadella et al., 2009). Important nerve transmission processes are
dependent on levels of acetylcholine (AChE) within the adductor
muscle and changes in AChE activity were generally not observed or
only at elevated dissolved Ni concentrations (Jones et al., 2008;
Tsangaris et al., 2007, 2008). The conclusion of these investigations is
that relatively high and environmentally-unrealistic concentrations
of dissolved Ni are required to induce response in the biomarkers
mentioned above and it is somewhat surprising that there is no
Comet assay or NRR data for any of the laboratory studies given in
Table 1. Thus, our results appear to be the first in respect of the
application of these sensitive techniques to laboratory investigations
of the effect of Ni on Mytilus edulis. Typical dissolved Ni concentra-
tions in estuaries and sediment porewaters cover the range
0.6e30 mg L
1 (Langston et al., 1999; Martino et al., 2002; Prego and
Cobelo-Garcia, 2003; Braungardt et al., 2007; Santos-Echeandia
et al., 2009) and <0.5 mg L
1 in coastal waters (Tappin et al., 1995).
Thus, more sensitive biomarkers such as DNA strand breaks and NRR
need to be employed to assess damage to marine organisms at
concentrations that are environmentally-realistic.
4. Conclusions
Mytilus edulis assimilated 63Ni preferentially in the byssus and it
was internalised especially in the digestive gland. The tissue-
412 G.E. Millward et al. / Environmental Pollution 162 (2012) 406e412
specific CFs for Ni were lower than the recommended value for
molluscs indicating the CF be revised downwards to aid improved
modelling of the bioaccumulation of stable and radioactive Ni.
Comparison of biological constants reported in the literature is
hampered by a lack of consistency in experimental conditions and
a more systematic approach to experimental design is required,
including animal husbandry particularly byssus attachment, accu-
mulation and depuration times and levels of dose. Cytotoxic and
genotoxic effects of dissolved Ni were detected at nominal
concentrations of 18 mg L
1 and above. More generally, under
chronic exposure from the dissolved Ni concentrations encoun-
tered in coastal waters it is likely there will be cytotoxic and gen-
otoxic impacts on marine mussels that may influence their short-
and long-term survival. Further study is required to correlate the
levels of elevated oxidative enzymes with that of DNA damage and
the mechanism of Ni-induced toxicity at different levels of orga-
nisation in marine biota is still not clear. Being regarded as
a priority hazardous substance by the international community,
and the growing importance of carcinogenic, mutagenic and
reproductive (CMR) impact assessments for contaminants, there is
a need for improved management of Ni discharges to estuaries and
coastal waters.
Acknowledgements
The work was funded, in part, by the European Regional
Development Fund, INTERREG IVA (Grant No. 4059) to ANJ. The
radiochemical experiments were carried out in the ISO9001
accredited Consolidated Radio-isotope Facility at Plymouth
University.
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