Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508

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Comparative Biochemistry and Physiology, Part A

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Metal accumulation and metallothionein induction in the spotted dogfish

Scyliorhinus canicula☆
G. De Boeck ⁎, M. Eyckmans, I. Lardon, R. Bobbaers, A.K. Sinha, R. Blust
University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry, and Toxicology, Groenenborgerlaan 171, 2020 Antwerp, Belgium

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

Article history: Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts.
Received 21 November 2009 Accumulation rates can be high, which despite the fact that normal background levels for metals in the
Received in revised form 16 December 2009 marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals
Accepted 17 December 2009
of the present study were to examine differences in accumulation rates and toxicity of a range of metals at
Available online 4 January 2010
equimolar concentrations (10 µM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this
Keywords:
purpose, we exposed the dogfish to Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L), and Ag
Elasmobranch (1079 µg/L and two additional exposures at 10 µg/L and 1 µg/L) for one week and measured total metal
Chondrichthyes accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the
Shark high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than
Nickel exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any
Cadmium osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the
Lead kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb
Silver
accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal
Copper
toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.
Urea
© 2010 Elsevier Inc. All rights reserved.

1. Introduction exposures (Jeffree et al., 2008). In polar elasmobranches, both Hg and

Metal toxicity has been studied extensively in freshwater fish, but
much less is known about the effect of metals on marine fish and more
in particular elasmobranches. Data on baseline metal concentrations
in elasmobranch tissues, especially muscle, exist since several species
are consumed by man (data for As, Cd, Cu, Fe, Hg, Mn, Ni, Pb and Zn in
shark reviewed by Turoczy et al., 2000). Particularly Hg seems to be of
concern, especially in large adult shark, and high concentrations in
muscle tissues have recently lead to recalls of batches of shark from
the market (Marcovecchio et al., 1986; Blanco et al., 2008; Endo et al.,
2008; Petroczi and Naughton, 2009). In a recent study comparing the
bioaccumulation of Am, Cd, Co, Cr, Cs, Mn and Zn from seawater in
spotted dogfish Scyliorhinus canicula and turbot Psetta maxima all
radiotracers accumulated at a faster rate in S. canicula than in
P. maxima with the exception of Cs (Jeffree et al., 2006). Concentration
factors for Am and Zn were two and one order of magnitude greater in
S. canicula, respectively. Also egg cases of spotted dogfish accumulated
Hg and Pb rapidly, which led to a subsequent chronic exposure of the
embryos within the cases, causing for concern even after short
☆ This paper derives from a presentation at the session entitled ‘Biology of
Elasmobranchs: from Genes to Ecophysiology and Behaviour’ at the Society for
Experimental Biology's Annual Main Meeting, Glasgow, 28 June–1 July, 2009.
⁎ Corresponding author. Tel.: +32 3 2653 478; fax: +32 3 2653 497.
E-mail address: gudrun.deboeck@ua.ac.be (G. De Boeck).
Cd accumulations seem to be an issue (Zauke et al., 1999; McMeans
et al., 2007). High baseline levels of Ag have been noted in tropical,
temperate and arctic waters (Cornisa et al., 2007; Webb and Wood,
2000; De Boeck et al., 2001; McMeans et al., 2007).
Dietary intake of metals seems to be a more important route of
uptake than aqueous uptake for both marine teleosts and elasmo-
branchs (Mathews and Fisher, 2009) although it is no clear whether
biomagnification through the food chain is really important (Mathews
et al., 2008). However, these authors noted that when comparing an
elasmobranch (S. canicula) to a marine teleost (P. maxima), aqueous
uptake for Co, Am, Zn and Cd was proportionally more important than
dietary intake to the elasmobranch (Mathews and Fisher, 2009),
partly due to the high affinity of elasmobranch skin for metals (Jeffree
et al., 2006), but also due to its unique physiology which leads to
lower absorption rates from food, likely due to the secretion of urea
into the intestinal chime (Wood et al., 2007).
Elasmobranches have an alternative osmoregulatory strategy, and
are slightly hyperosmotic to the environment because they use high
levels of urea and trimethylamine oxide (TMAO) as osmolytes (Hazon
et al., 2003 for review). Therefore, and in contrast to marine teleosts,
these fish do not have to drink, which also reduces the role of the
intestinal tract as a target organ for metal uptake. Gills are, again
compared to marine teleosts, less important in ionoregulation since
excess NaCl can be excreted by the rectal gland. As a result gills are
perhaps less a target for metal toxicity, but rectal gland might be an

1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2009.12.014
504 G. De Boeck et al. / Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508
additional target organ. Gill membranes have proven to be very tight
and impermeable, but a continuous loss of urea cannot be prevented
due to the huge urea gradient that exists between the internal and
external environment of an elasmobranch fish. (Part et al., 1998, Fines
et al., 2001; Hill et al., 2004; reviewed by Walsh and Smith, 2001).
Moreover, since water diffuses into the fish at a steady rate, kidneys
are continuously producing urine which increases the risk of urea loss.
Therefore, specific urea back-transporters (UT) are crucial both in the
kidney (Hyodo et al., 2004) and the gill. In the kidney, these
transporters show high amino acid identity with mammalian UT-A2
(N60%), while in gill homologues could be related to mammalian UT-
B2, both belonging to the SLC14 gene family (Smith and Wright, 1999;
Janech et al., 2006). At the gill, these UT are located in the basolateral
membrane and are sodium-coupled (Part et al., 1998; Fines et al.,
2001; Hill et al., 2004). Some metals, such as Cu and Ag can act as
sodium analogue and/or inhibit Na transporting processes (Wood,
2001; Grosell et al., 2002). It is not known whether they also affect
urea transporters, but disturbance of urea retention potentially could
be an additional effect of metal toxicity in elasmobranches.
These differences between marine teleosts and elasmobranches
warrant further investigation of metal toxicity in marine elasmo-
branches. The few available earlier toxicological studies indicate that
elasmobranches can tolerate relatively high levels of some metals but
are surprisingly sensitive to some others. Lethal concentrations for the
Mediterranean or spotted dogfish S. canicula for Cu (24 h LC50:
16 mg/L, 48 h LC50: 4 mg/L, Torres et al., 1987), Zn (24 h LC50:
180 mg/L, 48 h LC50: 80 mg/L, Crespo and Balasch, 1980) and Cd (24 h
LC50: 200–250 mg/L, Tort and Torres, 1988) are in the milligram per
liter range. However, in a study examining the effects of metals on
Squalus acanthias, silver appeared to be 10 times more toxic to
spiny dogfish than to similarly sized marine teleosts. 96-h LC50 values
for silver for marine teleosts vary between 183 to 1200 µg/L,
compared to freshwater 96-h LC50 values of 5 to 70 µg/L (Hogstrand
and Wood, 1998; Wood et al., 1998). In fact, sensitivity approached
that of freshwater teleosts and the 96 h LC50 was estimated to be
close to 100 µg/L (De Boeck et al., 2001). This sensitivity coincided
with extremely high Ag accumulation rates in gill and other tissues
(Webb and Wood, 2000, De Boeck et al., 2001). As in teleosts, toxicity
appeared to be related to osmoregulatory disturbance. However this
disequilibrium was not so much caused by disturbance of NaCl
transport, but by the loss of urea. Cu, which usually exerts similar
effects as Ag, did not induce this high toxicity or the extremely high
accumulation rates in spiny dogfish, with an estimated 96 h LC50 just
below 1 mg/L (De Boeck et al., 2007), much more comparable with the
data obtained in Mediterranean dogfish (Torres et al., 1987).
Nevertheless, when comparing Cu uptake directly between long
nosed skate (Raja erinacea) and the sculpin (Myoxocephalus octode-
cemspinosus), branchial accumulation rates of the elasmobranch were
10 to 15 times higher (Grosell et al., 2003).
Sublethal effects of Ag included mainly osmoregulatory failure with
influx of salts and loss of urea. Na+/K+ ATPase activities in gill and rectal
gland were only reduced at acutely toxic Ag levels. Respiratory
disturbance was observed as well, with hyperventilation and blood
alkalosis at low Ag concentrations and anaerobic metabolism and
acidosis at high Ag levels (De Boeck et al., 2001). For Cu, increases in
plasma ammonia were observed at sublethal exposure levels (Grosell
et al., 2003), as well as blood acidosis and lactate accumulation (De
Boeck et al., 2007). Again, reductions in gill, but not rectal gland Na+/K+
ATPase activities were only observed at acutely toxic exposure levels.
Both plasma urea and TMAO dropped proportionally, indicating that the
general impermeability of the gills was compromised at high exposure
levels (De Boeck et al., 2007). Erythrocyte swelling and haemolysis can
occur at high Cu levels, followed by haemodilution, again suggesting
osmoregulatory disturbance at acutely lethal doses (Tort et al., 1987).
Subacute and acute Zn treatment did not seem to cause an effect on
plasma Na or Cl (Crespo et al., 1982) but did affect haematological
parameters (Torres et al., 1986). Zn exposure also increased the number
of chloride cells on the gills (Crespo et al., 1981). Also Cd exposure
induced changes in haematological parameters (Tort and Torres, 1988),
and it is able to disrupt chloride transport in the rectal gland (Forrest
et al., 1997). Hg exposure disrupts cell volume regulation by inhibiting
apical chloride channels (Ratner et al., 2006), Na–K–2Cl co-transporters
(Kinne-Saffran and Kine, 2001), and by increasing sodium permeability
(Ballatori and Boyer, 1996).
Despite the fact that normal background levels for metals in the
marine environment are low, the differences in response between
marine teleosts and elasmobranches are intriguing, and some metals
seem to accumulate considerably despite the low background levels.
Therefore, the goals of the present study were to examine differences in
accumulation rates and toxicity of a range of metals at equimolar
concentrations in the Mediterranean or spotted dogfish, S. canicula. For
this purpose, we exposed the dogfish to equimolar concentrations
(10 µM) of Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L),
and Ag (1079 µg/L) for one week and measured metal accumulation,
metallothionein induction, and parameters related to osmoregulation.
Since spotted dogfish responded with a similar sensitivity to Ag
exposure as spiny dogfish, two additional groups of fish were exposed
to Ag levels that were 100 and 1000 times lower (10 and 1 µg/L). At all
sublethal levels, metal accumulation as well as physiological indices of
metal toxicity related to osmoregulatory disturbances were measured.
Elasmobranches also posses metal binding proteins such as metal-
lothionein (MT) (Bonwick et al., 1990; Cho et al., 2005), which besides
playing a role in trace element homeostasis can also bind non-essential
toxic metals such as Cd (Hidalgo et al., 1985; Hidalgoa and Flosa, 1986a,
b; Planas et al., 1991). Therefore, a final goal of this study was to assess
whether these MT could be induced by metal exposure at sublethal
levels.
2. Material and methods
European dogfish (S. canicula) were caught by trawling in the North
Sea at the east coast of England in August 2008. Dogfish weighing 75.25±
18.83 g were acclimatized per group of 6 in 8 well aerated 200 L tanks in
artificial seawater (HW Marinemix Professional, HW Aquaristic, Krefeld,
Germany) at a salinity of 34‰ and a temperature of 16.0 ±0.1 °C for
1 month before experiments started. Each tank was provided with a
biological filter, and oxygen saturation levels remained above 90%, and
ammonia levels below 0.1 mg/L at all times. Dogfish were fed 3 times a
week at 1% body weight with defrosted pollack (Pollachius pollachius).
2.1. Metal exposure
Fish were exposed to a variety of metals (Ni: 587 µg/L, Cd: 1124 µg/L,
Pb: 2072 µg/L, Cu: 635 µg/L, and Ag: 1079 µg/L) for 1 week in the tanks
described above. Since all dogfish in the Ag exposure died within 24 h,
two extra tanks of dogfish were exposed to 10 µg/L and 1 µg/L of Ag
respectively. Filters were shut off during the static exposure, and 50% of
the water was renewed on a daily basis. Metals were added as chloride
or nitrate salts of analytical grade (NiCl2, Cd(NO3)2, Pb(NO3)2, AgNO3, Cu
(NO3)2) dissolved at a concentration of 1 mM in 1% HNO3 (Merck,
Darmstad, Germany). Addition of 1 ml stock solution per liter seawater
did not affect water pH (8.0 ± 0.1) significantly. Water samples were
taken before and after each water renewal, acidified to 1% using
analytical grade 69% HNO3 (Merck, Darmstad, Germany) and frozen for
later metal analysis. Water quality was checked daily and oxygen
saturation levels remained above 90%, and ammonia levels below
0.1 mg/L at all times. Fish were not fed during the exposure period. Two
groups of control fish was treated in the same way.
After 7 days of exposure, fish were killed in an overdose of anesthetic
(MS-222, 2 g/L) and immediately sampled for blood, gill, liver, kidney,
rectal gland, intestine, muscle and skin tissue. Tissues were divided in 2
subsamples, instantly frozen in liquid nitrogen and later stored at
 G. De Boeck et al. / Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508
−80 °C for later analysis. Blood samples were centrifuged for 3 min
(11.000 rpm at 4 °C) and plasma was frozen in liquid nitrogen and
stored at −80 °C as well.
2.2. Sample analysis
Plasma samples were analysed for electrolytes (Na, K, Ca, and Cl)
using an AVL 9180 Electrolyte Analyser (AVL, Roche Diagnostic,
Belgium) and for urea using the photometric diacetyl monoxime
method (Price and Harrison, 1987). A subsample of the plasma samples
was diluted with 1% HNO3 made with analytical grade 69% HNO3
(Merck, Darmstad, Germany) and Milli-Q grade water (Millipore,
Bedford, MA, USA) for metal analysis.
One of each of the tissue subsamples was dried in a 60 °C drying
oven for a minimum of 1 week, cooled in a desiccator, weighed,
dissolved with 69% HNO3 (Merck, Darmstad, Germany) and 27% H2O2
(Normapur, VWR International, Belgium) and placed in a microwave
oven until total digestion had occurred. They were then diluted with
Milli-Q grade water (Millipore, Bedford, MA, USA). Standard curves
were made by standard addition. Total metal concentrations of water,
plasma and tissues were determined using ICP-MS (Varian, Mulgrave-
Victoria, Australia).
The other tissue subsamples were used for the determination of
metallothionein. They were homogenized on ice with 4 volume of
buffer A (10 mM Tris–HCl, 86 mM NaCl, pH 7.4) at 4 °C with a Qiagen
TissueRuptor (Qiagen, Hilden, Germany). Tissue homogenates were
centrifuged at 16,000 × g at 4 °C for 20 min and supernatant fractions
were taken and kept at −80 °C for further processing. They were later
analysed following the procedure described by Klein et al. (1994). This
method allows the quantification of total MT in biological materials,
including the oxidized and aggregated MT and Cu binding MT via
109
Cd saturation. It is a modification of an earlier described procedure
specially developed to quantify Cu-containing metallothionein (Klein
et al., 1990). The main features of the procedure are that oxidized MT
is converted into native MT with 2-mercaptoethanol as a reducing
agent and Zn2+ as a metal donor, and MT is subsequently quantified
via Cd saturation. High molecular weight Cd-binding compounds are
denatured with acetonitrile, Cu bound to MT is removed with
ammonium tetrathiomolybdate and excessive tetrathiomolybdate
and its Cu complexes are removed with DEAE-Sephacel (Sigma, St.
Louis, MO). Subsequently apothionein is saturated with 109Cd-labeled
CdCl2-solution (Amersham Pharmacia Biotech, Buckinghamshire,
England: 50 ppm of 37 MBq/mg Cd in 0.1 M HCl), and excessive Cd
is bound to Chelex 100 (Bio-Rad, Munich, Germany). The precipitate
was removed by centrifugation and the supernatant counted for 1 min
in a gamma counter (Minaxi γ , Canberra Packard, Frankfurt,
Germany). The MT concentration was calculated assuming a molar
ratio of Cd/MT of 7.
2.3. Statistical analysis
Data are presented as mean± S.D. Differences in metal and MT
concentrations were determined by one-way ANOVA and followed by
Tukey–Kramer multiple comparisons test if significant differences were
found, using Graph-Pad InStat (GraphPad Software, San Diego, CA).
Statistical significance was assigned at P b 0.05.
3. Results
3.1. Exposure conditions
All exposures were sublethal except for the 1079 µg/L Ag exposure
where all dogfish died within the first day. Therefore two new exposures
were performed at 10 and 1 µg/L of Ag. Measured total exposure
concentrations were slightly lower than nominal concentrations with
1034 ± 315 µg/L for Cd, 578 ± 89 µg/L for Cu, 505 ± 117 µg/L for Ni,
505
1533 ± 995 µg/L for Pb, and 9.7 ± 6.5 µg/L and 0.97 ± 43 µg/L for Ag
respectively. Pb levels were most variable, and fish seemed more
distressed and lethargic just after each water renewal when new metal
stock solution was added.
3.2. Metal accumulation
Overall, all control metal levels of the non-essential metals (Ni, Cd,
Pb, and Ag) in the different tissues were below 1.2 µg/g dry weight
except for Ni which had accumulated in skin and rectal gland to
control values of 3.47 ± 0.08 µg/g and 3.99 ± 3.28 µg/g respectively
(Fig. 1A–H). The highest metal value in plasma was also recorded for
Ni with levels of 0.02 ± 0.05 µg/ml. Cu control levels were in general 1
to 2 orders of a magnitude higher than control levels of any other
metal with 1.63 ± 0.19 µg/ml in plasma and between 5 and 18 µg/g in
the different tissues.
Of all metals, only Ag in the 10 µg/L exposure accumulated
significantly in all tissues and plasma despite the fact that exposure
levels were 100 times lower compared to the other metals. At the
1 µg/L exposure level, Ag still accumulated significantly in plasma and
all tissues except rectal gland. Also Pb accumulated significantly in
plasma and all tissues. Other metals accumulated more selectively,
with Ni and Cd accumulating both in gill and kidney, but not liver
tissue, and Cu in gill, liver and rectal gland, but not kidney. Cu and Ni
also accumulated in plasma and Cd in muscle and skin.
When comparing at the tissue levels, all metals accumulated in the
gills (Fig. 1A). Except for Cu (26.70 ±6.64 µg/g), Ag accumulated to the
highest extend (7.46± 9.83 and 9.51± 3.73 µg/g) despite the low
exposure levels. In plasma (Fig. 1B), Ni did not only have the highest
control levels of the non-essential metals, but it also increased most
significantly during exposure, from 0.02± 0.05 µg/ml to 0.16±0.03 µg/
ml. Cd was the only metal that did not show an increase in plasma. Liver
(Fig. 1C) clearly accumulated Cu which increased 13-fold to 66.1 ±
39.1 µg/g. Also Ag levels clearly increased in liver. Both these metals also
showed relatively high control levels, which can be expected for Cu since
it is an essential metal, but which is more surprising for Ag. Pb levels more
than doubled, but remained really low. Cd did not accumulate at all in
liver during this one week exposure. Cu did not accumulate in kidney
(Fig. 1D), but levels of all other metals roughly doubled (Ni, Cd) or tripled
(Pb, Ag). The same metals that accumulated in the liver, also accumulated
in the rectal gland (Fig. 1E), but this time with huge increases in Cu
(6-fold increase to 84.70±65.13 µg/g) and Pb (20-fold to 15.97 ±
9.74 µg/g) and although Ag also increased 4 and 12-fold, actual
concentrations remained relatively low with 0.53 ±0.44 µg/g. Ni showed
relatively high control values (3.99±3.28 µg/g), but no increase was
noted what so ever during exposure. Intestinal tissue (Fig. 1F) showed
only a moderate increase in Pb and again a dose dependent increase in
Ag. In muscle tissue (Fig. 1G), Ag levels increased more than 20-fold both
in the 1 µg/L and 10 µg/L exposure group. Cd and Pb showed modest
increases, and Ni and Cu did not increase at all. Skin primarily
accumulated Pb, with a 15-fold increase (Fig. 1H). Also Cd and Ag levels
were elevated in the skin, although for Ag this did not happen in a dose
dependent way.
3.3. Physiological effects
Metallothionein induction only occurred in Cu exposed fishes,
both in gill and liver tissue (Fig. 2). None of the other metal exposures
could significantly induce MT. Also osmoregulatory disturbances were
limited (Table 1). Plasma Na and Cl were not affected by any of the
exposure scenarios. Plasma K levels increased in Ni and Cd exposed
fish and were decreased in Cu exposed shark. Ca levels were increased
in Cd exposed dogfish. Urea, an important osmolyte in elasmobranchs,
was increased in Ni and Ag exposed fish.
506 G. De Boeck et al. / Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508

Fig. 1. Metal accumulation in (A) gill, (B) plasma, (C) liver, (D) kidney, (E) rectal gland, (F) intestine, (G) muscle and (H) skin of spotted dogfish Scyliorhinus canicula after one week
under control conditions (white bars) or after one week exposure to sublethal levels of Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L) (black bars) and Ag at 1 µg/L
(light grey bar) and 10 µg/L (dark grey bar) (N = 6, Mean ± S.D., *: P b 0.05, **: P b 0.01, ***: P b 0.001).
 G. De Boeck et al. / Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508
4. Discussion
Our study confirms the high toxicity for Ag in elasmobranch fish as
was previously seen in the spiny dogfish, S. acanthias (De Boeck et al.,
2001), as well as the high Ag accumulation rates even at very low
exposure concentrations compared to marine teleosts (Pentreath, 1977;
Webb and Wood, 2000; De Boeck et al., 2001). Not only is Ag taken up
easily, it also accumulated quickly in every single organ measured,
mostly in a dose dependent way (except for skin and muscle). Even in
control fish or in the wild, baseline Ag concentrations can be detected
(Webb and Wood, 2000; De Boeck et al., 2001; McMeans et al., 2007)
despite the low environmental levels present. This seems to be a
consequence of the rapid uptake combined with a long biological half
time in sharks (Pentreath, 1977), rather than biomagnification through
the foodweb. High Ag levels have been found in other large marine
predators such as dolphins and especially Beluga whales (Mackey et al.,
1996; Woshner et al., 2001; Dehn et al., 2006a; Seixas et al., 2009) but
there seems to be no clear correlation between Ag accumulation and
size or age for either shark or marine mammals (Woshner et al., 2001;
Dehn et al., 2006a; Cornisa et al., 2007; Seixas et al., 2009), and studies
focussing on the biomagnification of trace elements in marine foodwebs
did not find an indication for Ag biomagnification either (Campbell et al.,
2005; Dehn et al., 2006b).
At the Ag lower exposure concentrations in this study, we did not
see any indication of an increased permeability of the membranes.
Plasma electrolyte levels were stable, and in contrast with earlier
findings in spiny dogfish (De Boeck et al., 2001) urea levels increased,
possibly due to a stress induced breakdown of protein. Despite the
high accumulation rates in gill and liver, there was no induction of MT
synthesis in these organs.
Fig. 2. Metallothionein levels in (A) gill and (B) liver tissue of spotted dogfish Scylior-
hinus canicula after one week under control conditions or after one week exposure to
sublethal levels of Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Ag (10 µg/L) and Cu
(635 µg/L) (N = 6, Mean ± S.D., **: P b 0.01).
507
Table 1
Plasma electrolyte levels (Na, Cl, K, and Ca) and plasma urea levels of spotted dogfish
Scyliorhinus canicula after one week under control conditions or after one week
exposure to sublethal levels of Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Ag
(10 µg/L and 1 µg/L) and Cu (635 µg/L) (N = 6, Mean ± S.D., *: P b 0.05, **: P b 0.01,
***: P b 0.001).
mM
Na Cl K Ca Urea
Control 244.4 ± 14.5 238.2 ± 13.8 3.4 ± 0.8 2.7 ± 0.2 370.4 ± 33.4
Ni 234.4 ± 4.3 229.7 ± 8.5 4.3 ± 0.3* 2.8 ± 0.1 459.5 ± 10.7***
Cd 258.0 ± 9.2 251.7 ± 5.6 4.4 ± 0.2** 3.0 ± 0.0** 424.2 ± 51.0
Pb 239.0 ± 10.3 232.0 ± 9.8 3.8 ± 0.3 2.6 ± 0.1 410.0 ± 18.5
Ag (1 µg/L) 236.0 ± 6.2 230.4 ± 6.2 3.1 ± 0.5 2.8 ± 0.1 446.2 ± 25.5**
Ag (10 µg/L) 245.2 ± 11.2 238.0 ± 9.4 4.1 ± 0.2 2.8 ± 0.1 459.0 ± 46.4***
Cu 233.2 ± 11.2 220.8 ± 11.1 2.2 ± 0.4** 2.4 ± 0.3 387.2 ± 24.3
The only other metal that accumulated significantly in all tissues,
albeit at exposure levels that were 100 to 1000 times higher than for Ag,
was Pb. Not much is known about Pb and elasmobranch fishes, although
a study did indicate that it is absorbed by eggs of spotted dogfish and
that it accumulated in the embryo's thereafter (Jeffree et al., 2008). In
the wild, Pb levels often remain under the detection limit (Zauke et al.,
1999; Turoczy et al., 2000; Blanco et al., 2008), except in some shark
caught near polluted areas in the U.K. (Vas, 1987, 1991). This confirms
that Pb only accumulates to significant levels at relatively high exposure
levels. Although we did note behavioural changes with less activity after
each water renewal, physiological disturbances were not seen.
Apparently the high accumulation rates in the ionoregulatory organs
such as gill and rectal gland did not compromise their function. Pb
seemed to absorb really well to the skin and so did, to a lesser extent, Cd
and Ag. This high affinity of dogfish skin was also observed before in
S. canicula and is contributed to its high collagen content and placoid
scales (Jeffree et al., 2006, Mathews and Fisher, 2009). The two metals
that did not accumulate significantly in the skin in our study were Ni and
Cu, but both of these metals were already present in relatively high
concentrations in the skin of control fish (3.5 and 8.8 µg/g respectively)
indicating that they do accumulate in the skin easily.
Although Ni accumulated in gill and plasma demonstrating that it
was taken up by the dogfish, kidney was the only other organ where it
appeared, suggesting that excretion through the kidney might be
important and sufficient to prevent accumulation in any other organ. Ni
also caused osmoregulatory disturbance, with increases in plasma K and
urea, but not Na or Cl. Cd also appeared in the kidney, and not liver, but
excretion rates did not seem to be sufficient for this metal, since it also
accumulated in muscle and skin. This corresponds with the radiotracer
study Jeffree et al., 2006, where Cd accumulated primarily in kidney as
well, and not so much in liver. In contrast, Cu and Ag primarily
accumulated in the liver, but only Cu seemed able to induce an increase
in the synthesis of detoxifying protein such as MT both in liver and gill.
From our data we cannot conclude whether dogfish MT only respond to
changes in essential metals like Cu and thus only play a role in the
homeostasis of these essential trace elements, or whether the toxicity of
the exposure levels used for the non-essential metals were too low to
induce a protective response. After all, Cu was the only metal that
accumulated in liver to levels that were at least 20 times higher than any
of the other metal concentrations. Previous studies on elasmobranch
MT's did confirm that Cd can bind to shark MT, but these studies did not
quantify total MT induction (Hidalgo et al., 1985; Hidalgoa and Flosa,
1986a,b; Planas et al, 1991).
In conclusion, our study confirms the high toxicity of Ag for
elasmobranch fish, even at levels 100 to 1000 times lower than
exposure levels of other metals. Ag accumulation occurs fast, and
throughout all organs examined in this study, a trait shared with Pb
albeit at higher exposure concentrations. In contrast, Ni and Cd seem to
accumulate primarily in the kidney while Cu mainly accumulates in
508 G. De Boeck et al. / Comparative Biochemistry and Physiology, Part A 155 (2010) 503–508
liver. Only Cu succeeded in initiating a protective response by inducing
MT synthesis in liver and gills.
Acknowledgements
This research was financially supported by projects from The
University of Antwerp Research Council (UA-BOF-NOI) and the
Science Foundation-Flanders (FWO- 1.5.199.09N). M. Eyckmans, I.
Lardon and A.K. Sinha are research fellows supported by IWT-
Flanders, by UA-BOF-ID, and by FWO-Flanders respectively.
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