Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula☆
Introduction
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 exposures (Jeffree et al., 2008). In polar elasmobranches, both Hg and 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 elasmobranchs (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 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 (> 60%), 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 elasmobranches. 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 octodecemspinosus), 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 metallothionein (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, Hidalgoa and Flosa, 1986b, 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.
Section snippets
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
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, 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
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
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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2021, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :Sharks play important roles in marine ecosystems as key predators in the structuring of marine food webs. Recent studies, however, have demonstrated that elasmobranchs show increased susceptibility to metal uptake and biomagnification, as they incorporate these compounds very efficiently and eliminate them slowly [1–3]. Because of this and known species-specific life history parameters, these animals are often considered sentinel species regarding environmental contamination [4–6].
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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.