Metal and metalloid concentrations in the tissues of dusky Carcharhinus obscurus, sandbar C. plumbeus and white Carcharodon carcharias sharks from south-eastern Australian waters,

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Highlights

�Metals were analysed in 12 sandbar, 12 dusky and 6 great white sharks. �Most samples have high concentrations of Hg and As, some higherthan reported elsewhere (e.g. >80 mg kg�1 ww). � Two 120-gram serves per week of either commercial species exceeds the FSANZ PTWI forHg and As. � Hg concentrations are significantly positively correlated with length in all species. � A 1.5 m size limit is recommended to ensurea product safe for human consumption.

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3 Metal and metalloid concentrations in the tissues of dusky Carcharhinus4 obscurus, sandbar C. plumbeus and white Carcharodon carcharias sharks5 from south-eastern Australian waters, and the implications for human6 consumption

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9 Jann M. Gilbert a,b, Amanda J. Reichelt-Brushett b,⇑, Paul A. Butcher a,c, Shane P. McGrath c,10 Victor M. Peddemors d, Alison C. Bowling e, Les Christidis a

11 a National Marine Science Centre, Southern Cross University, Coffs Harbour, New South Wales 2450, Australia12 b Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, Lismore, New South Wales 2480, Australia13 c Fisheries NSW, NSW Department of Primary Industries, National Marine Science Centre, Coffs Harbour, New South Wales 2450, Australia14 d Fisheries NSW, NSW Department of Primary Industries, Sydney Institute of Marine Science, Mosman, New South Wales 2088, Australia15 e School of Health and Human Sciences, Southern Cross University, Coffs Harbour 2450, Australia

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20 Article history:21 Available online xxxx

22 Keywords:23 Carcharhinus obscurus24 Carcharhinus plumbeus25 Carcharodon carcharias26 Metals contamination27 Shark fisheries28

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30Shark fisheries have expanded due to increased demand for shark products. As long-lived apex predators,31sharks are susceptible to bioaccumulation of metals and metalloids and biomagnification of some such as32Hg, primarily through diet. This may have negative health implications for human consumers. Concentra-33tions of Hg, As, Cd, Cu, Fe, Se and Zn were analysed in muscle, liver and fin fibres (ceratotrichia) from34dusky Carcharhinus obscurus, sandbar Carcharhinus plumbeus, and white Carcharodon carcharias sharks35from south-eastern Australian waters. Concentrations of analytes were generally higher in liver than in36muscle and lowest in fin fibres. Muscle tissue concentrations of Hg were significantly correlated with37total length, and >50% of sampled individuals had concentrations above Food Standards of Australia38and New Zealand’s maximum limit (1 mg kg�1 ww). Arsenic concentrations were also of concern, partic-39ularly in fins. Results warrant further investigation to accurately assess health risks for regular consump-40tion of shark products.41� 2015 Published by Elsevier Ltd.42

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45 Over recent decades global shark fisheries have expanded46 (Myers et al., 2007). The increase is primarily due to demand for47 fins in Asia (Walker, 1998; Clarke et al., 2007; Worm et al.,48 2013), although shark flesh (muscle tissue) also provides an impor-49 tant source of protein in many parts of the world (Simpfendorfer50 et al., 2011). Demand for other shark products such as squalene51 and cartilage, for use in a variety of applications including cosmet-52 ics and pharmaceuticals, also continues to increase (Walker, 1998;53 Lack, 2014; Momigliano and Harcourt, 2014).54 Australian shark fisheries have expanded in response to the55 increased demand. This is demonstrated in the �200% increase in56 New South Wales (NSW) shark catches in the Ocean Trap and Line57 Fishery (OTLF), operating off the east coast of Australia. Reported58 landings between 1997/8 and 2005/06 leapt from an annual

59average of 165–440 t in 2006/07 (Macbeth et al., 2009). The rise60was directly attributed to an increase in targeted fishing effort61for large ‘whaler’ sharks (Carcharhinidae) including dusky Carcha-62rhinus obscurus and sandbar Carcharhinus plumbeus, which together63accounted for almost 60% of observed catches (Macbeth et al.,642009). These two species have been primarily targeted for their fins65(Macbeth et al., 2009; Geraghty et al., 2013). In 2009, to prevent66overfishing of undefined and potentially vulnerable stocks, a67160-t total allowable catch (TAC) (landed weight) for pelagic68sharks was implemented across the OTLF. Subsequently, this has69reduced the catch of both species in the NSW sector of the OTLF,70with 2.4 t of dusky shark (McAuley et al., 2014a) and 5.6 t of sand-71bar shark (McAuley et al., 2014b) harvested in calendar year 2013.72The east coast stock of both dusky and sandbar sharks remain73undefined (McAuley et al., 2014a,b) and sustainability of the shark74fishery remains unknown (Bruce, 2010; Rowling et al., 2010).75The overall fisheries-induced negative impact on shark stocks76is, ironically, also of concern for human health, primarily because

http://dx.doi.org/10.1016/j.marpolbul.2014.12.0370025-326X/� 2015 Published by Elsevier Ltd.

⇑ Corresponding author at: Marine Ecology Research Centre, School ofEnvironment, Science and Engineering, Southern Cross University, PO Box 157,Lismore, NSW 2480, Australia. Tel.: +61 2 6620 3250.

E-mail address: [email protected] (A.J. Reichelt-Brushett).

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Marine Pollution Bulletin xxx (2015) xxx–xxx

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Marine Pollution Bulletin

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Please cite this article in press as: Gilbert, J.M., et al. Metal and metalloid concentrations in the tissues of dusky Carcharhinus obscurus, sandbar C. plumbeusand white Carcharodon carcharias sharks from south-eastern Australian waters, and the implications for human consumption. Mar. Pollut. Bull. (2015),http://dx.doi.org/10.1016/j.marpolbul.2014.12.037

77 it is driven by demand for shark products that are destined for78 direct or indirect human consumption. This is despite evidence79 that, as apex predators, sharks bioaccumulate and biomagnify80 certain metals and metalloids in their tissues (Ratkowsky et al.,81 1975; Gray, 2002; Endo et al., 2008), which may be harmful to82 human health (FSANZ, 2011; Mamtani et al., 2011). Susceptibility83 to bioaccumulation and biomagnification of some metals and met-84 alloids is believed to be a consequence of the longevity, size, slow85 growth and low fecundity of many shark species (Walker, 1988;86 Branco et al., 2007; Pethybridge et al., 2010). This is coupled with87 the uptake of metals and metalloids through a diet that consists88 predominantly of large predatory fish and/or marine mammals in89 larger sharks (Cortes, 1999; Endo et al., 2008; Hussey et al.,90 2012). A lipid-rich liver, designed for buoyancy (Last and Stevens,91 2009), also makes sharks particularly susceptible to the uptake92 and accumulation of lipophilic metal species such as methylmer-93 cury (MeHg) (Domi et al., 2005; Mull et al., 2012).94 Mercury (Hg) and other metals such as cadmium (Cd), copper95 (Cu), iron (Fe) and zinc (Zn), and metalloids including arsenic96 (As) and selenium (Se), occur naturally in the environment in var-97 ious forms and some are considered essential to many biological98 processes (Islam and Tanaka, 2004; Mamtani et al., 2011; Brewer99 et al., 2012). While many metals and metalloids are required in

100 trace concentrations, potentially harmful effects may occur if101 biological requirements are exceeded or they are present in certain102 forms and quantities (Haynes and Johnson, 2000; Mamtani et al.,103 2011; van Dam et al., 2011). The severity of toxic effects depends104 on the duration of exposure, pathway of uptake and the magnitude105 of exposure (Mamtani et al., 2011).106 Consumption of shark muscle tissue and shark products is107 believed to represent a major dietary source of metals and metal-108 loids for human consumers (Adams and McMichael, 1999;109 Pethybridge et al., 2010; FSANZ, 2011). For this reason, metal and110 metalloid concentrations in shark species of commercial value111 (e.g. blue shark Prionace glauca, and school shark Galeorhinus112 galeus) have been investigated, particularly for Hg and MeHg con-113 centrations (e.g. Walker, 1988; Branco et al., 2004; Maz-Courrau114 et al., 2012). Subsequently, a maximum limit of 0.5–1.0 mg Hg kg�1

115 wet weight (ww) in fish tissue has been set for human consump-116 tion in most countries including Australia, and maximum limits117 exist for some of the other metals and metalloids analysed in this118 study (FSANZ, 2011; US EPA, 2014).119 Despite the historical and current commercial importance of120 sharks there is still relatively little information on metals and met-121 alloids in many species that are harvested globally including com-122 mercial, artisanal and recreational catches (Stevens and Brown,123 1974; Marcovecchio et al., 1991; Mull et al., 2013). Windom124 et al. (1973) highlighted a need to identify Hg and other element125 concentrations in non-commercial species of finfish in order to126 understand if high levels in predatory commercial species were127 environmentally and/or physiologically mediated. Four decades128 later, the question remains largely unanswered. Indeed, regardless129 of the recognised high consumption of shark muscle tissue in130 Australia, few studies have investigated metals or metalloids in131 sharks, and the studies that do exist are generally dated (e.g.132 Walker, 1976; Lyle, 1984; Turoczy et al., 2000).133 The principal aim of the present study was to quantify accumu-134 lated metals and metalloids in the muscle, liver tissue and fin fibres135 of two important commercial shark species, the dusky and sandbar136 shark, and one protected (EA, 2002) apex predator, the white shark137 Carcharodon carcharias. The main objectives were to: (i) enable138 comparisons of metal and metalloid concentrations with previous139 shark studies; (ii) compare the concentrations found in two140 commercially important shark species from Australian waters with141 Food Standards Australia New Zealand (FSANZ) guidelines; and (iii)142 provide a baseline for future monitoring.

143Shark tissue samples were provided by Fisheries NSW (NSW144Department of Primary Industries) (DPI) from the NSW OTLF145(dusky and sandbar sharks), and from the NSW Shark Meshing146Program (SMP) (white sharks) (Fig. 1). The OTLF is a multi-species147commercial fishery that deploys a number of different types of gear148(mainly setlines and trotlines) to target large sharks (Macbeth149et al., 2009). The NSW SMP was introduced in 1937 and now150includes 51 popular swimming beaches from Wollongong to151Newcastle, NSW. The nets are set demersally from September to152April, inclusive, at a distance of approximately 400–500 m from153the shore in 10–12 m of water (Reid et al., 2011).154Muscle, liver and fin samples from 12 dusky and 12 sandbar155sharks were collected from demersal long-lines set in the Pacific156Ocean between Nambucca Heads (30� 340S, 153� 130E) and Wooli157(29� 560S, 153� 260E), off northern NSW, between January and April1582013. Tissue samples from six white sharks were collected from159animals caught in SMP nets set in the Pacific Ocean between Cole-160dale (34� 170S, 150� 570E) and Warriewood (33� 420S, 151� 180E), off161southern NSW, between March 2011 and January 2013 (Fig. 1). Fin162samples from four white sharks were not collected.163Following capture of each shark, tissues were collected by164scientific personnel (from Fisheries NSW) using standard scientific165collection methods (OSPAR, 2012). Specifically, a sample (approxi-166mately 10 � 5 � 1 cm) of muscle (from in front of the dorsal fin),167liver (posterior left lobe) and the whole lower caudal fin was168collected with a clean ceramic knife. Each sample was then placed169into a clean plastic ziplock bag, sealed, and put on ice for up to 48 h170before being frozen (�17 �C) in the laboratory. For each shark, the171location, date and depth (m) of capture as well as the total length172(TL cm), total weight (kg) and gender were recorded.173Duplicate sub-samples of individual shark tissues were174collected from stored samples using a ceramic knife. These were175prepared following OSPAR, 2012 guidelines. Between processing176of each sample all equipment was cleaned with 70% ethanol fol-177lowed by 10% HNO3, and then rinsed with Milli-Q ultra-pure water.178Wet samples of liver and muscle were weighed to 5.0 g (±0.1 g) and179freeze-dried in a Labconco freeze dryer at �80 �C, 0.045 mBar until180no moisture remained. Muscle samples were then ground with a181mortar and pestle, and liver samples were homogenised in the182vials using an Omni International 240-watt Tissue Master 125183laboratory homogeniser. Fins were also freeze-dried and approxi-184mately 2 g of fin fibres were removed for use in analyses.185From each dried, homogenised sample, 0.2 g (±0.003 g) was186weighed into an acid-cleaned Teflon digestion tube. In each tube,1875 mL of 70% analytical-grade HNO3 was added and the tubes were188sealed. After pre-digestion for 30 min the tubes were sealed and189placed in a closed, high-pressure microwave system (MARS5,190CEM Corporation, Matthews, NC). Following digestion, tubes were191left to cool in a fume hood for approximately 30 min. Material from192each digestion tube was made up to 25 mL with Milli-Q ultra-pure193water. Diluted material was stored in sealed and labelled polypro-194pylene sample vials at 4 �C until analyses.195Analyses of metals were completed at the Environmental Anal-196ysis Laboratory (EAL) at Southern Cross University (SCU). Analytes197measured in the sample digests included Hg, As, Cd, Cu, Fe, Se and198Zn. Concentrations in the sample digests were measured using an199inductively coupled plasma-mass spectrometer (ICP-MS; Perkin200Elmer NexION 300D). The instrument was calibrated for each ele-201ment using a three-point calibration curve, prepared from certified202stock solutions, to provide an R2 coefficient of 0.9999 or greater.203Calibration standards were analysed at regular intervals to ensure204the instrument maintained acceptable linearity and sensitivity205criteria (EAL, 2013; Kalantzi et al., 2013). ICP-MS detection limits206(limits of reporting) for the seven metals analysed were207Hg < 0.0002, As < 0.0007, Cd < 0.00004, Cu < 0.0002, Fe < 0.005,208Se < 0.00025 and Zn < 0.003 mg L�1 (EAL, 2013). Further quality

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209 control measures included the use of duplicate blanks and interna-210 tionally certified reference material (CRM) (DORM-4 fish protein,211 National Research Council of Canada) in each analytical run of 20212 samples. Recoveries of analytes from DORM-4 fish protein are213 given in Table S1.214 To enable comparison of data with other studies (e.g. Walker,215 1976; Storelli et al., 2003; Endo et al., 2008), wet weight to dry216 weight ratios were calculated for each tissue type. This was done217 by measuring the moisture loss from wet weight in four different218 samples of each of the tissue types, from each species. Wet weight219 to dry weight ratios were comparable in each tissue type across220 species so a mean ratio was calculated for each tissue type. Dry221 weight to wet weight ratios were 6.67:1 g for muscle tissue,222 1.91:1 g for liver tissue and 2.27:1 g for fin fibres.223 Dry weight is believed to provide the best basis for comparison224 of substance concentrations in different tissues (Clark, 2001),225 therefore, dry weight concentrations (mg kg�1) of a suite of seven226 metals and metalloids (Hg, As, Cd, Cu, Fe, Se and Zn) were statisti-227 cally analysed for each tissue. Given the small sample size and the228 variance in the data, non-parametric statistical analyses were per-229 formed with PERMANOVA+ (Anderson, 2005; PRIMER 6, PRIMER-E230 Ltd, Plymouth, UK). Three types of analyses were conducted:

231 (1) A 3 � 3 mixed between-within subject factorial design with232 species and tissue type as factors was used to test for differ-233 ences between metal and metalloid concentrations, and for

234interactions between these factors. To follow-up significant235main effects and identify the source of variation, post hoc236pairwise comparisons were also conducted.237(2) Differences between genders were investigated using a238one-way PERMANOVA design with the data for dusky and239sandbar shark species pooled, and data for white sharks240omitted due to the smaller sample size and gender informa-241tion on only five individuals of this species.242(3) Relationships between TL and metal and metalloid concen-243trations were investigated using correlations. Given the use244of non-parametric tests medians are reported, with error245bars representing the 25th and 75th percentiles.246

247Significance was at the <0.05 level unless otherwise stated.248A significant positive correlation between Hg concentrations in249muscle tissue and TL was found, with TL explaining 74% of the var-250iation in Hg concentrations for all species combined (r = 0.86,251n = 30, p < 0.001). For individual species, this relationship was most252significant in white (r = 0.99, n = 6, p < 0.001) and dusky sharks253(r = 0.96, n = 12, p < 0.001), where 98% and 93% of the variation,254respectively, was explained via TL (Fig. 2). The relationship was255less strong in sandbar sharks, although still significant (r = 0.85,256n = 12, p = 0.001, 72%; Fig. 2). Further species-specific, significant257correlations between TL and other analytes in muscle and liver tis-258sue were also found. Results for these are presented in Table 1.259Notably, muscle and liver tissue concentrations of As were

Fig. 1. Map of the study area where sample collection occurred. Area A represents the area of capture in the NSW Ocean Trap and Line Fishery (OTLF) for dusky Carcharhinusobscurus, and sandbar Carcharhinus plumbeus sharks in the present study. Area B represents the area of capture in the NSW Shark Meshing Program (SMP) for whiteCarcharodon carcharias sharks in the present study. Source: NSW Department of Primary Industries (DPI).

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260 negatively correlated with TL for dusky and sandbar sharks, and261 Cd, Se and Zn concentrations were positively correlated. There262 was also a stronger correlation between analyte concentrations263 and TL in females (r = 0.95, n = 12, p < 0.001, 90%) than males264 (r = 0.89, n = 12, p < 0.001, 79%) although the difference was not265 significant.266 There was no significant main effect of species for Hg concen-267 trations, although the two larger species of shark, dusky and white,268 exhibited higher median concentrations in muscle tissue (Fig. 3A).269 There was a significant main effect of species for As concentrations,270 Pseudo-F (2,54) = 6.98, p = 0.009, with sandbar sharks having271 higher overall As concentrations than the other two species272 (Fig. 3B). Likewise, for Cd concentrations the main effect of species273 was significant, Pseudo-F (2,54) = 5.68, p = 0.007, and post hoc274 comparisons showed sandbars also had higher overall Cd concen-275 trations (Fig. 3C). Differences between males and females in metal276 and metalloid concentrations were only significant for Fe concen-277 trations, which were higher in males (median = 344.57) than278 females (median = 123.04).279 There was a significant main effect of tissue type on concentra-280 tions of Hg, Pseudo-F (2,81) = 3.26, p = 0.03. Post hoc comparisons281 showed muscle and liver tissue had significantly higher Hg concen-282 trations than fin fibres, but Hg concentrations in the muscle and283 liver did not differ substantially (Fig. 3A). Tissue type was also sig-284 nificant in explaining As concentrations, Pseudo-F (2,54) = 34.28,285 p = 0.001. Specifically, muscle and liver tissue had significantly286 higher concentrations than fin fibres (Fig. 3B). A significant main287 effect of tissue type was also evident for concentrations of Cd,288 Pseudo-F (2,54) = 7.72, p = 0.003 (Fig. 3C); Cu, Pseudo-F289 (2,54) = 4.99, p = 0.009 (Fig. 3D); Fe, Pseudo-F (2,54) = 30.54,290 p < 0.001 (Fig. 3E); and Se, Pseudo-F (2,54) = 5.73, p = 0.005

291(Fig. 3F). Post hoc comparisons for these analytes showed that liver292tissue concentrations were higher than muscle tissue and fin fibres,293which also differed from one another. There was no significant294effect of tissue for Zn concentrations, however, unlike other ana-295lytes, median Zn concentrations in dusky and white sharks were296higher in fin fibres than liver or muscle tissue (Fig. 3G).297The species x tissue interaction was significant for Hg concentra-298tions, Pseudo-F (4,81) = 2.14, p = 0.05, with higher concentrations in299the liver tissue of sandbar sharks than the other two species300(Fig. 3A). Likewise, a significant species x tissue interaction for Cd301concentrations, Pseudo-F (4,54) = 5.60, p = 0.001, also showed302higher concentrations in liver tissue of sandbar sharks (Fig. 3C).303No species x tissue interaction was found for other analytes.304In summary, differences in most analyte concentrations were305greater between the three types of tissue than between species306or genders, and the significant species x tissue interaction for Hg307and Cd concentrations indicates that these analytes varied differ-308entially in the three species across the three tissue types.309Results from the present study are generally comparable to310those found in sharks from other oceanic waters (Fig. 4), and also311highlight the wide variation in metal concentrations between312species, individuals and tissues (de Pinho et al., 2002; Endo et al.,3132008; Escobar-Sanchez et al., 2010; Bendall et al., 2014). Several314cases of extremely high concentrations of metals and metalloids315were found in the present study (Fig. 2A–G), and were considerably316higher than those reported in the literature (Vas, 1991; Storelli317et al., 2003; Endo et al., 2008; Pethybridge et al., 2010; Mull318et al., 2012).319Trophic level, and associated diet and ecology, has been identi-320fied as one of the most important factors affecting differences in321metal and metalloid concentrations between species (Vas and322Gordon, 1993; Turoczy et al., 2000; Pethybridge et al., 2010).323Carcharhinid and Lamnid sharks are ranked with high mean tro-324phic levels as a result of specific prey preferences (Cortes, 1999).325Elevated concentrations of Hg and other metal and metalloids have326been associated with a diet consisting of large carnivorous teleost327fish, other elasmobranchs and marine mammals (Walker, 1976; de328Pinho et al., 2002; Barrera-Garcia et al., 2012). The greater percent-329age of these prey items in the diet of dusky and, more so, white330sharks (Cortes, 1999; Hussey et al., 2012) may explain the higher331Hg muscle tissue concentrations found in these two species when332compared with sandbar sharks. Moreover, the variation in Hg con-333centrations between species serves to illustrate the elevated levels334found in higher trophic-level predators (Vas, 1991; Storelli et al.,3352003; Branco et al., 2007) with sandbar, dusky and white sharks336each ranked at successively higher trophic levels (Cortes, 1999).337The higher Hg concentrations in sandbar sharks overall, how-338ever, does not fit this trend and may be influenced by this species’339close association with sediments (Last and Stevens, 2009), where340many contaminants are sequestered (Clark, 2001) and subse-341quently transferred to the food chain through both biotic and abi-342otic processes (Domi et al., 2005; Brewer et al., 2012). Bendall et al.,

Fig. 2. Relationships between median mercury (Hg) concentrations in muscletissue and total length for dusky Carcharhinus obscurus (n = 12), sandbar Carcharhi-nus plumbeus (n = 12) and great white Carcharodon carcharias sharks (n = 6). Thedotted line indicates the Food Standards Australia New Zealand (FSANZ) maximumlimit of 1.0 mg Hg kg�1 ww in fish tissues.

Table 1Pearson product-moment correlations between total length and analyte concentrations in muscle and liver tissue of dusky Carcharhinus obscurus and sandbar Carcharhinusplumbeus sharks.

Species n Analyte Tissue Pearson correlation Significance (2-tailed) % of var. shared

Dusky 12 Arsenic Muscle –0.59 0.04 35Carcharhinus obscurus Cadmium Muscle 0.75 0.005 55

Liver 0.57 0.05 33Zinc Muscle 0.72 0.008 52

Sandbar 12 Arsenic Muscle –0.62 0.03 39Carcharhinus plumbeus 10 Cadmium Muscle 0.79 0.002 62

Liver 0.76 0.01 57Selenium Liver 0.74 0.01 55

Q8Q9

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Fig. 3. Median analyte concentrations in muscle and liver tissue, and fin fibres (ceratotrichia) of dusky Carcharhinus obscurus (n = 12), sandbar Carcharhinus plumbeus (n = 12)and white Carcharodon carcharias sharks (n = 6 for muscle and liver tissue, n = 2 for fin fibres). (A) Mercury (Hg). (B) Arsenic (As). (C) Cadmium (Cd). (D) Copper (Cu). (E) Iron(Fe). (F) Selenium (Se). (G) Zinc (Zn). A dotted line indicates the Food Standards Australia New Zealand (FSANZ) maximum limit in fish tissues and a dashed line indicates theupper level of intake (UL) per day (mg/kg body weight) for adults. Where the UL was above the sample values, no line is shown. NB: dotted and dashed lines indicate the dryweight value equivalent to the FSANZ wet weight maximum limit or UL.

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Please cite this article in press as: Gilbert, J.M., et al. Metal and metalloid concentrations in the tissues of dusky Carcharhinus obscurus, sandbar C. plumbeusand white Carcharodon carcharias sharks from south-eastern Australian waters, and the implications for human consumption. Mar. Pollut. Bull. (2015),http://dx.doi.org/10.1016/j.marpolbul.2014.12.037

343 2014 found a similar trend in porbeagle sharks Lamna nasus, which344 occupy the same trophic level as sandbar sharks (Cortes, 1999).345 The porbeagles in their study from north-east Atlantic waters346 had substantially lower mean concentrations of all metals and347 metalloids in comparison to the present study, with the exception348 of Cd, Cu and Fe (Bendall et al., 2014). This may be due to a differ-349 entiation in diet between sandbar and porbeagle sharks, with the350 latter species’ diet incorporating a higher percentage of fish and351 cephalopods but no marine mammals and few other chondrichth-352 yans (Cortes, 1999; Joyce et al., 2002). Moreover, the majority of353 teleosts consumed consist of groundfish (Joyce et al., 2002), which354 would likely reduce the effect of biomagnification in porbeagle355 sharks.356 High concentrations of As and Cd have also been attributed to357 diet; the former to one rich in crustaceans rather than fish358 (Turoczy et al., 2000), and the latter to one rich in cephalopods359 (Bustamante et al., 1998). This could contribute to the significantly360 higher As and Cd concentrations found in the sandbar shark, which361 is reported to have a relatively large proportion of crustaceans362 (25.3%; Cortes, 1999) and cephalopods (13–21%; Cortes, 1999;363 McAuley et al., 2006) in its diet. However, neither Cliff et al.364 (1988) nor McAuley et al. (2006) found this proportion of crusta-365 ceans in the diet of sandbar sharks from South African or West366 Australian waters, respectively. Cortes (1999) also reported the367 dusky shark to have a higher percentage of cephalopods in its diet368 than those sampled from South African waters (Dudley et al., 2005)369 (22.8% and 15.3%, respectively) but this species had lower median370 concentrations of Cd than the sandbar. These variations may, how-371 ever, be the result of regional or species-specific diet preferences,372 prey availability, and the age/size of sharks in each study373 (Walker, 1988; Pethybridge et al., 2010).374 Anthropogenic pollution from urbanised coastlines has been375 associated with elevated concentrations of metals and metalloids376 in shark tissues (Walker, 1988; Branco et al., 2004; Mull et al.,377 2012) and this may partly explain the high concentrations of Hg378 and As, and relatively high concentrations of Cu in the three shark379 species examined here. All three sharks occur from coastal waters380 to insular and continental shelf waters (Last and Stevens, 2009).381 While sharks are known to be wide-ranging foragers, it is likely382 that these animals accumulated most of their contaminant loads383 in Australian waters (or from prey in Australian waters). The avail-384 able data for metal and metalloid concentrations in coastal waters385 of northern NSW and south-eastern Australia ranks concentrations386 as some of the lowest in the Southern Hemisphere (DEST, 1995;387 Apte et al., 1998) so high natural concentrations does not seem a388 likely explanation for the elevated levels found in these sharks.

389Moreover, Turoczy et al. (2000) found high muscle tissue concen-390trations of several metals (including Hg and As), relative to their391average concentration in seawater, in their study of deep-water392sharks (the golden velvet dogfish Centroscymnus crepidater,393Owston’s dogfish Centroscymnus owstonii, and the brier shark394Deania calcea) from south-eastern Australian waters.395Significantly higher Fe concentrations in males could be the396result of gender-specific diet preferences or foraging locations397(i.e. coastal or pelagic) (Walker, 1976; Portnoy et al., 2010;398Maz-Courrau et al., 2012) or different nutritional needs399(Pethybridge et al., 2010; Mull et al., 2012). Lower concentrations400in females may also indicate depuration of Fe for reproductive pur-401poses. Viviparous reproduction in dusky and sandbar sharks402enables the transfer of nutrients and contaminants with maternal403blood supply (de Pinho et al., 2002; Lowe et al., 2012; Olin et al.,4042014), which may explain the tendency for lower concentrations405of most analytes found in sexually mature females in the present406study. Maternal transfer has been documented previously in407Carcharhinus species (Lyle, 1984; Adams and McMichael, 1999;408de Pinho et al., 2002) and in white sharks (Lowe et al., 2012;409Mull et al., 2012), although frequency of reproduction and age at410onset of sexual maturity would also influence the rate of maternal411transfer (Adams and McMichael, 1999).412A trend for higher concentrations of metals and metalloids in413males has generally been attributed to slower and faster growth414rates in males and females, respectively (e.g. Walker, 1976;415Marcovecchio et al., 1986; de Pinho et al., 2002). Geraghty et al.416(2013), however, reported that male dusky and sandbar sharks in417south-eastern Australian waters grew more rapidly than females418in the juvenile phase, and slower growth in males was only419observed after this phase. This growth pattern for males and420females is evident in the stronger correlation between Hg concen-421trations and TL in females in the present study. Geraghty et al.422(2013) also found that females were generally larger at any given423age, meaning that males of the same size as females would be older424and would therefore have had longer to accumulate contaminants425(Lyle, 1984; de Pinho et al., 2002). The lack of a significant effect of426gender for other analytes has been reported in other studies427(Hueter et al., 1995; Branco et al., 2004; Maz-Courrau et al.,4282012), and may be the result of differences in habitat, diet, age429and maximum size (Turoczy et al., 2000; Maz-Courrau et al., 2012).430The high Hg concentrations in muscle tissue of larger sharks in431the present study have been found in other commercial and non-432commercial species (Walker, 1988; Storelli et al., 2003; Mull433et al., 2012), as has the positive correlation between Hg muscle tis-434sue concentrations and TL (Walker, 1976; Adams and McMichael,

Fig. 4. A comparison of mean Hg muscle tissue concentrations in commercial shark species from previous studies and the present study. 1. Walker (1976). 2. Walker (1988).3. Marcovecchio et al. (1991). 4. Adams and McMichael (1999). 5. Turoczy et al. (2000). 6. de Pinho et al. (2002). 7. Storelli et al. (2003). 8. Endo et al. (2008). 9. Pethybridgeet al. (2010). 10. Barrera-Garcia et al. (2012). 11. Present study. The dotted line indicates the Food Standards Australia New Zealand (FSANZ) maximum limit of1.0 mg kg�1 ww in fish tissues.

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435 1999; Endo et al., 2008). This suggests an age-related accumulation436 (Walker, 1976; Marcovecchio et al., 1991; Pethybridge et al., 2010).437 Endo et al. (2008) reported an age- or growth-related pattern of438 metal and metalloid tissue distribution with higher concentrations439 in the muscle and liver tissue of juvenile and adult tiger sharks440 Galeocerdo cuvier, respectively. Concentrations generally increased441 in muscle tissue with increasing TL but showed a rapid increase in442 liver tissue approaching or following maturity (Endo et al., 2008).443 This is consistent with the findings of the present study for Hg,444 Cd and Se. In general, concentrations of these analytes in dusky445 and sandbar sharks were higher in muscle than liver tissue until446 maximum length for each species was approached (365 and447 240 cm, respectively), after which, liver concentrations were448 approximately three- to four-times higher. This trend was not449 evident in white sharks, all of which were juveniles, nor in concen-450 trations of Zn, which continued to increase in dusky and sandbar451 shark muscle tissue only. The reason for this is unclear but may452 be related to the different energy or nutritional requirements of453 larger sharks (Endo et al., 2008; Mull et al., 2012), ontogenetic454 changes in diet (Stevens and Brown, 1974) or the high turnover455 and metabolic activity of liver tissue (McMeans et al., 2007).456 An age- or growth-related trend and elevated concentrations in457 the liver of older animals indicates that accumulation rates of some458 metals such as Hg and Cd exceed metabolism and excretion rates459 (Turoczy et al., 2000), particularly following the juvenile phase.460 This accumulation is likely to reflect ontogenetic changes in diet461 without the dilution effect of more intense growth (Endo et al.,462 2008). Moreover, the slow growth rates and long lifespan of the463 dusky, sandbar and white shark (Last and Stevens, 2009;464 Geraghty et al., 2013; Hamady et al., 2014) mean that these metals465 can be accumulated over a considerable period of time (Walker,466 1976; de Pinho et al., 2002; Mull et al., 2012). The largest dusky467 and sandbar sharks used in the present study were up to 34 and468 28 years, respectively, (approximated from Geraghty et al., 2013)469 highlighting the substantial time available for accumulation of470 Hg and Cd. The significant negative correlation between As and471 TL in dusky and sandbar sharks, despite being a reversal of the gen-472 eral trend, is further evidence for ontogenetic diet changes in these473 species (McElroy et al., 2006; Bornatowski et al., 2014). Neonates474 and juveniles have been reported with a greater proportion of475 crustaceans in their diet (Medved et al., 1985; Bornatowski et al.,476 2014), which may explain the higher As muscle tissue concentra-477 tions in juveniles (compared with adults) recorded in the present478 study.479 High liver tissue concentrations of Hg and Cd in dusky and480 sandbar sharks approaching maximum length (and a positive rela-481 tionship with TL) could indicate the role of the liver as the end-482 point of tissue distribution (Endo et al., 2008) for non-essential ele-483 ments. Liver tissue concentrations have not previously been of484 interest in terms of human consumption but they have been used485 to describe the accumulation of metals and metalloids in older,486 high trophic level predators e.g. the smooth hammerhead Sphyrna487 zygaena (Storelli et al., 2003) and tiger shark (Endo et al., 2008).488 Concentrations of metals and metalloids in fin fibres in the489 present study were, in most cases, significantly lower than other490 tissues. Zn was the exception with similar concentrations in all491 tissues, and higher concentrations (but not significantly) in fin492 fibres than other tissues in dusky and white sharks. The reasons493 for this are unclear, although Vas (1991) found the highest Zn con-494 centrations in his study on skin, which is similarly collagenous in495 nature (Kemp, 1977). Only one previous study has investigated496 metal and metalloid concentrations in fin fibres (Escobar-Sanchez497 et al., 2010), and these authors reported on Hg and Se only. Their498 results for concentrations of these elements in sharks off the499 Mexican Pacific Ocean were generally comparable with those500 found in the present study.

501FSANZ has developed guidelines for the consumption of shark502muscle tissue based on what is considered a provisional tolerable503weekly or monthly dietary intake (PTWI and PTMI, respectively)504of individual metals and metalloids (FSANZ, 2011; NSWFA, 2013).505Mean Hg muscle tissue concentrations in the two commercial spe-506cies in the present study exceeded the FSANZ regulatory standard,507with 75% of dusky and 58% of sandbar shark samples above the508maximum limit. Given that Hg concentrations in both commercial509species approached the FSANZ maximum limit at �150 cm, target-510ing larger sharks in the OTLF means that muscle tissue sold511for human consumption could potentially contain high Hg concen-512trations. These concentrations are high enough to cause concern513(Storelli et al., 2003; Kojadinovic et al., 2006; Dorea, 2008), partic-514ularly if consumed regularly by children or pregnant women or for515regular adult consumers of shark muscle tissue (Turoczy et al.,5162000; FSANZ, 2011). FSANZ’s current reference health standard517for the PTWI of Hg and MeHg combined is 5.6 ug kg�1 body weight518(bw) (Table 2). Using the highest concentration found in muscle519tissue in the two commercial species (because this is what con-520sumers may ingest), based on 70 kg bw, two 120 g serves per week521of either dusky or sandbar shark muscle tissue (containing 7.71522and 6.62 ug Hg kg�1 bw, respectively) would be enough to exceed523the FSANZ PTWI.524Excluding Cu, Se and Zn, other analyte concentrations in liver525tissue in the present study were above the FSANZ maximum limit526or upper level intake (mg kg�1 bw) for adults. This could be of527concern given the increasing human consumption of ‘fish oil’ sup-528plements (PCRM, 2008), which are often made from squalene529(shark liver oil; Foran et al., 2003). There is relatively little research530on metal and metalloid concentrations in these supplements and531the implications for human health remain undefined (Foran532et al., 2003; PCRM, 2008).533All muscle and liver tissue, and fin fibre samples in the three534species studied had As concentrations above the recently-with-535drawn FSANZ maximum level of 2.0 mg kg�1 ww. Despite with-536drawing the maximum limit for As because it was not possible to537establish a safe level of exposure, FSANZ continues to issue a health538warning regarding As exposure to people who consume large539amounts of seafood (FSANZ, 2011). One 120-gram serve of muscle540tissue per week from any species in the present study would con-541stitute between �21–65 ug As kg�1 bw for a person weighing54270 kg. The previous PTWI for As was 21 ug kg�1 bw (FSANZ,5432002). Storelli et al. (2003) considered As concentrations in the544muscle tissue of the smooth hammerheads in their study to be545‘notable’, given that concentrations above 10 mg kg�1 ww were546rarely reported in the muscle tissue of sharks. This makes some547of the extremely high concentrations of As found in sharks in the548present study quite remarkable, and identification of the ratio of549organic:inorganic As and its potential toxicity will be important550in further investigations (Glover, 1979).551The high concentrations of As in most samples of fin fibres in552the present study are also of concern particularly since implica-553tions for regular consumers of these products (i.e. shark fin soup)554are unknown. Considering the value of large Carcharhinid fins to555the shark fin trade, the results of the present study imply that con-556sumers may be exposed to high concentrations of As. These, and557other recent findings of a highly potent neurotoxin (BMAA) in558shark fins (Mondo et al., 2012), highlight the requirement for fur-559ther investigation into the effects of consumption of these products560and the potential negative human health implications they pose.561Regular monitoring of metal and metalloid concentrations in562sharks would be beneficial in developing an accurate assessment563of the potential impacts of shark consumption on human health.564Responsible management of shark fisheries has generally focused565on sustainability and prevention of overfishing, however, it also566assumes some guarantee of quality, and a consistent and safe

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567 product for human consumption. The variation in results between568 individual sharks in the present study makes it clear that health569 advisories and regulations on consumption of shark products need570 to be conservative to account for this variation. In the absence of571 regular monitoring of shark tissues for metal and metalloid con-572 centrations, the 1.5 m size limit applied in Queensland and Victoria573 appears to be a logical form of regulation since concentrations for574 Hg in the present study and others from Australian waters575 approach or reach the FSANZ maximum limit at that size.

576 Acknowledgements

577 Thanks are due to NSW Department of Primary Industries (DPI)578 for provision of samples from the OTLF through research con-579 ducted via support from the Fisheries Research and Development580 Corporation on behalf of the Australian Government, and the Shark581 Meshing Program. Sample collection was approved by NSW DPI582 Animal Care and Ethics Committee (Refs. 12/19 and 10/06), and583 approval for this study was granted by Southern Cross University584 Animal Care and Ethics Committee (Ref. 13/12). This study was585 also generously supported by grants from the Norman Wettenhall586 Foundation and the George Lewin Foundation, and in-kind support587 from EAL. Additional funding was provided by the School of Envi-588 ronment, Science and Engineering, and the Marine Ecology589 Research Centre, Southern Cross University.

590 Appendix A. Supplementary material

591 Supplementary data associated with this article can be found, in592 the online version, at http://dx.doi.org/10.1016/j.marpolbul.2014.593 12.037.

594 References

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Table 2Food Standards Australia & New Zealand (FSANZ) reference health standards formetals analysed in the present study. Source: FSANZ.

Metal/metalloid

Reference health standard

Arsenic None (previously 3 ug kg�1 bw day�1, PTWIa 21 ug kg�1 bwfor inorganic As)

Cadmium 25 ug kg�1 bw PTMIb

Copper AIc 0.2–1.7 mg day�1; ULd 1–10 mg day�1

Iron UL 20–45 mg day�1

Mercury Mean level of 1.0 mg kg�1 for shark flesh; 5.6 ug kg�1 bwPTWI

Selenium UL 60–400 mg day�1

Zinc UL 5–40 mg day�1

bw: body weight.a PTWI: provisional tolerable weekly intake.b PTMI: provisional tolerable monthly intake.c AI: adequate intake.d UL: upper level of intake.

Q3

Q4

Q5

Q6

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Please cite this article in press as: Gilbert, J.M., et al. Metal and metalloid concentrations in the tissues of dusky Carcharhinus obscurus, sandbar C. plumbeusand white Carcharodon carcharias sharks from south-eastern Australian waters, and the implications for human consumption. Mar. Pollut. Bull. (2015),http://dx.doi.org/10.1016/j.marpolbul.2014.12.037