Mid-Atlantic elasmobranchs: Suitable metal scouts?

MPB-08358; No of Pages 12 Marine Pollution Bulletin xxx (2017) xxx–xxx

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Mid-Atlantic elasmobranchs: Suitable metal scouts? Paulo Torres a,c,⁎, Regina Tristão da Cunha a,c, Armindo dos Santos Rodrigues b,c a CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Pólo dos Açores - Departamento de Biologia, Universidade dos Açores. Rua Mãe de Deus, 58, 9500-801 Ponta Delgada, Azores, Portugal b IVAR, Instituto de Investigação em Vulcanologia e Avaliação de Riscos, University of the Azores, 9501-801 Ponta Delgada, Azores, Portugal c Faculty of Sciences and Technology, University of the Azores, 9501-801 Ponta Delgada, Azores, Portugal

a r t i c l e

i n f o

Article history: Received 25 November 2016 Received in revised form 20 January 2017 Accepted 25 January 2017 Available online xxxx Keywords: Heavy metals Bioindicator Raja clavata Galeorhinus galeus Prionace glauca Isurus oxyrinchus

a b s t r a c t Heavy metals are a hazard to marine fauna and human health. In this study we assess stable isotopes and metal content in Prionace glauca and Isurus oxyrinchus and analyse these results within and among other species and across regions and geographical areas. Also, we evaluate their suitability, together with Raja clavata and Galeorhinus galeus, as Mid-Atlantic bioindicators. Prionace glauca and I. oxyrinchus shared the same trophic level in a pelagic food web and did not present significant differences between genders or metals, except for As. Arsenic and Hg accumulated while Cd and Pb were not detected. One I. oxyrinchus presented Hg values above regulatory limits. A high Hg exposure was associated with I. oxyrinchus since its maximum weekly intake was exceeded. Elasmobranchs can be used as metal sentinels, each presenting different key features which defines a good marine bioindicator, allowing long-term monitoring at different temporal and spatial scales. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Marine ecosystems are seriously threatened by human activities which promote loads of a wide variety of toxic substances such as pesticides, hydrocarbons and heavy metals (Van der Oost et al., 2003). Metals, including heavy metals (high toxicity metals such as arsenic, cadmium, mercury and lead), are released into the environment through natural and/or anthropogenic processes and can negatively affect marine fauna and human health given their high toxicity and persistence in the environment (Storelli et al., 2003; Storelli and Marcotrigiano, 2004; Storelli, 2008; Barrera-García et al., 2013: Kibria and Haroon, 2015; Alves et al., 2016). While some of these elements are essential for physiological processes (e.g. selenium) in small quantities or up to certain concentrations, others can be harmful even at very low amounts without any known biological function (e.g. mercury). The degree of toxic effects depends on the duration of exposure, pathway of uptake and the magnitude of exposure (Ansari et al., 2004; Bosch et al., 2015). However, the concentrations and uptake of these metals by marine organisms also depend on environmental and species-specific biological factors as well as the chemical and physical form in which metals occur in the environment (Canli and Atli, 2003). As apex predators, elasmobranchs are exposed to environmental contamination through bioaccumulation and biomagnification processes, accumulating high levels of heavy metals in their tissues (Branco et ⁎ Corresponding author. E-mail address: [email protected] (P. Torres).

al., 2004; Domi et al., 2005; Hussey et al., 2012; Torres et al., 2014, 2016b; Alves et al., 2016). Given that these species have a wide distribution, assimilate the effects of pollutants within a complex food web, a long longevity, exhibit a slow growth and are capable of patrolling both small and larger areas, they can be considered as sentinel species of great potential for environmental pollution monitoring (Barrera-García et al., 2012, 2013; Alves et al., 2016; Torres et al., 2014, 2016b). Moreover, recent toxicological studies have pointed to their greater sensitivity to dissolved metal levels in seawater when compared to marine teleosts (De Boeck et al., 2001). Some species have a vast home range (migratory species) making it somewhat difficult to locate the source of contamination, while others have more restricted movements and can therefore provide more information on the contamination of a particular area (Alves et al., 2016; Torres et al., 2014, 2016b; Mckinney et al., 2016). As diet is the main source of these contaminants, it is important to study the feeding ecology of these top predators to identify potential pathways of metal transfer within marine food-webs (Taylor et al., 2014; Kiszka et al., 2015). The degree of piscivory (Adams, 2010), length prey size in regional diets (Suk et al., 2009) and geographic variability in bioavailability (Verdouw et al., 2011) influence accumulation rates and tissue concentration. Hence, information from stomach contents and stable isotope analyses, representing respectively short and long-term feeding habits, can be used to more comprehensively characterize these pathways, particularly in highly migratory predator fishes. The use of naturally occurring carbon and nitrogen stable isotopes provide chemical tracers to examine the ecology of organisms in a given

http://dx.doi.org/10.1016/j.marpolbul.2017.01.058 0025-326X/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

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P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

ecosystem. Over the last years this technique have been widely used in many elasmobranch studies to address and clarify several ecological issues, including trophic levels, diet and diet shifting and isotope turnover rates (Hussey et al., 2012). Consumption of elasmobranchs and related products (finsoup, filets, liver oil) represent a major dietary source of metals for humans (Pethybridge et al., 2010; Bosch et al., 2015). However, despite their historical and current commercial importance, information on metal content for many of these species is still scarce and/or incomplete since it is also essential to understand concentration variation within and among species and across regions (Branco et al., 2004, 2007; Pethybridge et al., 2010; Maz-Courrau et al., 2012; Mckinney et al., 2016). Additionally, the increased captures of elasmobranchs for human consumption emphasize the importance of their monitoring (Storelli et al., 2001). The Mid-Atlantic surrounds the relatively pristine Azores Archipelago covering the Mid-Atlantic Ridge (França et al., 2003), a volcanic and tectonically active seafloor reflected in active deep sea (Colaço et al., 2006) and shallow hydrothermal activities around the islands which can release metals into the water (Wallenstein et al., 2009; Dionísio et al., 2013). Few studies focused metal content in elasmobranchs in this area (Branco et al., 2004, 2007; Torres et al., 2014, 2016b). Hence, monitoring this area is vital considering its geological nature, the atmospheric and oceanic dispersal patterns of many contaminants, the future prospective mining activities in the region (according to local government) and the Transatlantic Trade and Investment Partnership (TTIP), a proposed economic trade agreement between the European Union and the United States, which is going to increase maritime traffic across the Atlantic. The objectives of this study were: 1) to access and analyse size-based and gender-specific variations combining previous stomach content information, stable isotope profiles (δ15N and δ13C) and metal content in muscle tissue of the blue shark Prionace glauca (Linnaeus, 1758) and the shortfin mako shark Isurus oxyrinchus Rafinesque, 1810; 2) to discuss implications of our results for both elasmobranch and human health; 3) to discuss their Mid-Atlantic environmental bioindicator potential together with the thornback ray Raja clavata Linnaeus, 1758 and the tope shark Galeorhinus galeus (Linnaeus, 1758), previously study by Torres et al. (2014, 2016b) from the same area, according to their different life-traits and some worldwide literature review; 4) provide a baseline for future monitoring in the Mid-Atlantic region. 2. Materials and methods

15 N/14N ratios in the samples were determined by continuous flow isotope mass spectrometry (CF-IRMS), on a Sercon Hydra 20–22 (Sercon, UK) stable isotope ratio mass spectrometer, coupled to a EuroEA (EuroVector, Italy) elemental analyser for online sample preparation by Dumas-combustion. Stable isotope ratios are expressed in δ notation as parts per thousand (‰) according to the following equation:

δX ¼ ½ðRsample=RstandardÞ−1  1000

ð1Þ

in which X is 13C or 15N, Rsample is the corresponding ratio 13C/12C or 15 N/14N and Rstandard represents the ratio for the respective standard. The standards used were IAEA-N1 and USGS-35 for nitrogen isotope ratio, and IAEA-CH6 and IAEA-CH7 for carbon isotope ratio; δ15N results referred to Air and δ13C to PeeDee Belemnite (PDB). Precision of the isotope ratio analysis, assessed through the standard deviation between 6 and 9 replicates of laboratory standard material interspersed among samples in every batch, was ≤0.2‰. Trophic position (TP) was calculated with a scaled Δ15N framework approach based on a dietary δ15N value-dependent Δ15N model (Hussey et al., 2014a, 2014b). Hence, knowing the δ15N value of a known baseline consumer (δ15N base), the δ15N value of the consumer of interest (δ15NTP), the dietary δ15N value at which 15N incorporation and 15N elimination are equal (δ15Nlim) and the rate at which the ratio between 15 N incorporation and 15N elimination changes relative to dietary δ15N averaged across the food-web (k), TP is calculated as follows:

TPSI ¼

Log ðδ15N lim−δ15NbaseÞ−Log ðδ15N lim−δ15NTPÞ þ TPbase k ð2Þ

The boarfish Capros aper (Linnaeus, 1758) was used as the δ15Nbase estimate (TP = 3) according to data obtained from Torres et al. (2016b) for this species in the Azores. Its abundance in the Mid-Atlantic, integrating the isotopic signature of the food web at a timescale large enough to minimise the effects of short-term variation justifies its selection. Furthermore, this species has been reported to be one of the main prey for other elasmobranchs studied in the region such as R. clavata, G. galeus (Morato et al., 2003) and even P. glauca (Clarke et al., 1996). A value of k = 0.14 and δ15Nlim = 21.9 were used according to experimental isotopic studies conducted by Hussey et al. (2014a, 2014b).

2.1. Sample collection and preparation 2.3. Metal analyses Thirty individuals (15 males and 15 females) of Prionace glauca and four individuals (3 males and 1 female) of Isurus oxyrinchus were caught between April and July 2013 as bycatch of the swordfish Xiphias gladius (Linnaeus, 1758) pelagic longline fishery in the Mid-Atlantic region located between 36 and 39°N 25–31°W, ICES division Xa2. At harbour, specimens were identified, counted, measured [total length (TL)] and sexed. Tissue muscle samples for stable isotope and trace metal analyses were excised from the block anterior to the first dorsal fin and stored frozen on arrival at the laboratory (−20 °C). 2.2. Stable isotope analyses and trophic position Lipids were extracted by soaking samples in triplicate 1:1 solutions of chloroform: methanol for 10 min (Beaudoin et al., 2001) and then rinsed with distilled water. The residue obtained was then stored at 50 °C for at least 48 h and reduced to powder. Approximately 1 to 2 mg of ground tissue was used to determine the 13C and 15N isotopic values. Stable isotope ratio analyses were performed at the Stable Isotopes and Instrumental Analysis Facility (SIIAF) of the Centre for Environmental Biology (CBA), University of Lisbon (Portugal). 13C/12C and

After being weighed and dried, muscle samples (approximately 2 g per sample) were digested at 95 °C for 2 h and then microwave digested (45 min + 1 h) inside closed vessels. Atomic absorption spectrophotometry (ARL 3510) was used to determine total arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), rubidium (Rb), selenium (Se), strontium (Sr) and zinc (Zn) concentrations. Total mercury (Hg) was also analysed by flameless atomic absorption spectrophotometry. Quality control was implemented and included reagent blanks, and reference materials were analysed for every batch which were only accepted if results were within certified range of values. The accuracy was N95% for the analysed elements, assessed with the analysis of eight standard reference materials: GXR-1, GXR-2, GXR-4, GXR-6, OREAS13P, SDC-1, SCO-1, NIST694 and DNC-1. Ultra-pure water was used to prepare the blanks and the calibration standards. The resulting solutions were diluted and analysed on a Finnegan Mat Element 2 High Resolution ICP/MS for quantification and all recoveries were within 10% of the certified values. All metal concentrations values were reported as mg·kg−1 of dry weight (dw) and converted to wet weight (ww) to allow for comparisons with legislated recommended maximum levels.

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

2.4. Data analyses A Student's t-test was used to compare values between males and females of P. glauca. Data on G. galeus and R. clavata was used from Torres et al. (2014, 2016b) to allow comparisons with other elasmobranch species from the same study area. A one-way ANOVA was used to compare isotopic profiles and heavy metal concentrations between the four species. When ANOVA assumptions, homogeneity of variances and normal distribution of residuals were not met, the data were transformed (log or square root). Accordingly, Tukey multi-comparisons tests were used if ANOVA showed significant treatment effect. All results were considered statistically significant at p b 0.05 level. Pearson correlation test (correlation factor rp) was used for correlation between metal concentration.

Table 2 δ15N and δ13C (‰) profiles, trophic position and metals concentration mean differences and p-values based on post hoc tests Tukey's tests for paired comparisons. Significant differences between species for each variable (p b 0.05) are in bold. Variables

Species

R. clavata

G. galeus

P. glauca

δ15N (‰)

G. galeus P. glauca I. oxyrinchus

−2.823, b0.01 −1.883, b0.01 −2.231, b0.01

0.940, b0.01 0.592, 0.310

−0.348, 0.712

δ13C (‰)

G. galeus P. glauca I. oxyrinchus

−0.052, 0.983 0.107, 0.830 −0.532, 0.103

Trophic position

G. galeus P. glauca I. oxyrinchus

−0.888, b0.01 −0.562, b0.01 −0.675, b0.01

0.327, b0.01 0.214, 0.283

−0.113, 0.720

As

G. galeus P. glauca I. oxyrinchus

0.209, b0.01 0.533, b0.01 1.311, b0.01

0.324, b0.01 1.101, b0.01

0.777, b0.01

Cr

G. galeus P. glauca I. oxyrinchus

−0.303, b0.01 0.477, b0.01 −0.086, 0.918

0.780, b0.01 0.218, 0.394

−0.562, b0.01

Cu

G. galeus P. glauca I. oxyrinchus

0.469, b0.01 0.294, 0.017 0.316, 0.309

−0.175, 0.277 −0.154, 0.796

0.022, 1.000

Hg

G. galeus P. glauca I. oxyrinchus

−0.601, b0.01 −0.747, b0.01 −1.125, b0.01

−0.146, 0.441 −0.524, 0.074

−0.378, 0.211

Rb

G. galeus P. glauca I. oxyrinchus

0.091, b0.01 0.295, b0.01 0.157, 0.569

0.204, b0.01 0.066, 0.966

−1.377, 0.659

Se

G. galeus −0.0696, b0.01 P. glauca −0.042, 0.186 I. oxyrinchus −0.197, 0.174

0.654, b0.01 0.499, 0.039

−0.155, 0.296

Sr

G. galeus P. glauca I. oxyrinchus

7.495, b0.01 7.519, b0.01 7.586, b0.01

0.024, 0.988 0.091, 0.520

0.067, 0.813

Zn

G. galeus P. glauca I. oxyrinchus

1.181, 0.131 3.463, b0.01 2.941, b0.01

2.282, b0.01 1.760, 0.073

−0.522, 0.810

3. Results Table 1 presents the mean (±SE) and range of δ15N, δ13C (‰), trophic position and metal (mg·kg−1·wet weight) profiles of the four elasmobranch species, including Prionace glauca and Isurus oxyrinchus individuals. Stable isotope and metal concentrations for P. glauca muscle tissue did not vary significantly between males and females (Student's t-test, p b 0.05) and, given the reduced number of I. oxyrinchus, sexes were analysed together for both species. δ15N did not vary significantly between these species, sharing the same trophic position, contrarily to δ13C (Tables 1 and 2). Cadmium and Pb were below detection levels and, except for As and Zn, while the other detected metals were present in small amounts and remained relatively stable with size (Fig. 1 and Table 1). Mercury and As showed significant increases with size for Prionace glauca (Fig. 1). Isurus oxyrinchus also seemed to show an increasing tendency of Hg with size, however there were few individuals. According to Table 2, only As and Cr had significant variations between both species. δ15N was not significant correlated with any metal and Hg was not correlated with Se. Concerning interspecific comparisons between the four elasmobranchs, Raja clavata δ15N values were significant lower compared to the other three species, contrarily to δ13C profiles which were similar

3

0.158, 0.575 −0.481, 0.160 −0.638, 0.025

Table 1 Average (±SE) and range of δ15N, δ13C (‰) profiles, trophic position according to Hussey et al. (2014a, 2014b) and metals concentration (mg·kg−1·wet weight) of elasmobranch species for muscle tissue. N is the number of individuals sampled. Species number Length (cm) Range δ15N (‰) Range δ13C (‰) Range TP Range As Range Cr Range Cu Range Hg Range Rb Range Se Range Sr Range Zn Range

Raja clavata N = 30 63.46 ± 1.73 48–79.5

Galeorhinus galeus N = 124 96.81 ± 2.28 61–154

Prionace glauca N = 30 145.32 ± 5.44 98–223

Isurus oxyrinchus N=4 178.75 ± 13.28 153–216

10.49 ± 0.14 9.85–11.47 −18.06 ± 0.08 −18.61/−17.57 3.51 ± 0.04 3.34–3.79 33.79 ± 2.39 19.85–59.30 0.98 ± 0.05 0.61–1.69 0.77 ± 0.09 0.36–2.94 0.07 ± 0.01 0.03–0.30 1.24 ± 0.06 0.92–2.22 0.41 ± 0.01 0.31–0.50 7.89 ± 1.79 0.29–27.32 7.42 ± 0.45 4.07–15.51

13.30 ± 0.17 12.35–14.67 −18.01 ± 0.09 −18.51/−17.24 4.40 ± 0.06 4.08–4.94 20.68 ± 1.52 11.99–30.24 1.28 ± 0.08 0.11–1.60 0.24 ± 0.01 0.13–0.40 0.29 ± 0.05 0.08–0.57 0.99 ± 0.03 0.59–1.26 1.10 ± 0.15 0.46–2.48 0.39 ± 0.02 0.23–0.55 6.24 ± 0.22 3.63–8.15

12.38 ± 0.11 11.2–13.9 −18.17 ± 0.07 −18.8/−16.9 4.07 ± 0.04 3.71–4.61 10.02 ± 0.69 3.65–17.91 0.50 ± 0.03 0.17–0.89 0.70 ± 0.21 0.13–4.99 0.33 ± 0.02 0.14–0.50 0.62 ± 0.02 0.43–0.84 0.45 ± 0.02 0.29–0.64 0.37 ± 0.03 0.07–0.82 3.95 ± 0.15 2.94–6.17

12.74 ± 0.22 12.2–13.3 −17.52 ± 0.29 −18.1/−16.7 4.19 ± 0.08 4.01–4.39 1.71 ± 0.43 0.87–2.90 1.06 ± 0.17 0.70–1.50 0.38 ± 011 0.17–0.69 0.84 ± 0.25 0.35–1.53 0.90 ± 0.16 0.45–1.21 0.60 ± 0.06 0.48–0.78 0.30 ± 0.05 0.15–0.35 4.48 ± 0.43 3.19–5.09

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

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P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Fig. 1. Metal concentrations in muscle tissue from Prionace glauca and Isurus oxyrinchus according to total length (cm). Results are expressed as mg·kg−1·wet weight. Black circles represent Prionace glauca and grey circles represent Isurus oxyrinchus. Only significant increases of metal concentrations with size are represented in the figure, with the respective regression equation and only for Prionace glauca.

with the exception of Isurus oxyrinchus (Tables 1 and 2). Arsenic, Hg, Sr, Se and Zn were the main metals that significantly varied among species, with As varying significantly among all species (Fig. 2 and Tables 1 and 2). Raja clavata presented a significant higher As, Sr and Zn and lower Hg content when compared to other species, with the exception of Zn concentration in Galeorhinus galeus (Fig. 2 and Table 2). Isurus oxyrinchus presented the higher Hg average value although not significant, while Galeorhinus galeus presented a significant higher value of Se (Fig. 2 and Table 2). Only one individual of Isurus oxyrinchus presented an Hg value that exceeded legal European Union legislated limit ML of 1 mg·kg−1·wet weight (European Commission 2008).

4. Discussion 4.1. Dietary tracers Stable isotope analysis suggested that P. glauca and I. oxyrinchus from the Mid-Atlantic occupy similar trophic positions in oceanicbased food webs, reflected by their depleted δ13C values which commonly decrease from productive continental shelf waters to pelagic systems (Hussey et al., 2011; Chouvelon et al., 2012). Surprisingly, G. galeus presented a significant higher δ15N value, although in the same order of magnitude (Torres et al., 2014), possibly attributed to individuals size/ age, given that only juveniles or subadults of P. glauca and I. oxyrinchus

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

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Fig. 2. Metal concentrations of Raja clavata, Galeorhinus galeus, Prionace glauca and Isurus oxyrinchus in muscle tissue (mg·kg−1·wet weight). Within the boxes, the line is the median, 1st and 3rd quartiles, maximum and minimum values. Different letters represent significant differences (p b 0.05).

were sampled. Most P. glauca males become sexually mature at about 220 cm and females between 200 and 220 cm (Henderson et al., 2001) and around 200 cm for I. oxyrinchus males and females (Maia et al., 2007), matching our size range for these species. Particularly, I. oxyrinchus is known to forage on large predatory fish, including small elasmobranchs and marine mammals (Cliff et al., 1990) which should reflect higher δ15N values (Estrada et al., 2003). However, Maia et al. (2006) reported a dominance of teleosts in the diet of individuals sampled off the southwest coast of Portugal, similar to G. galeus (Morato et al., 2003; Torres et al., 2014) and P. glauca (Markaida and SosaNishizaki, 2010) in the Mid-Atlantic region, reflecting a clearly more piscivorous diet. This degree of overlap among these three shark species is likely due to the homogeneity of carbon sources in oligotrophic openocean ecosystems, characterized by low productivity and little seasonality (Kiszka et al., 2015), such as the Azores region (Perez et al., 2006), reflecting depleted δ15N and δ13C prey sources. A similar pattern was previously reported for P. glauca and I. oxyrinchus in the Atlantic (Estrada et al., 2003) and in the Indian Ocean (Kiszka et al., 2015), contrarily to isotope profiles obtained by Meneses et al. (2016) in a study performed in the Pacific, more inshore, close to California.

Ontogenetic shifts in diet are known to occur in elasmobranchs (Lowe et al., 1996; Estrada et al., 2006; Borrell et al., 2011). However, although previous studies suggest a significant increase in δ15N and δ13C with size in P. glauca (Rabehagasoa et al., 2012), our data did not revealed that pattern, probably also due to the small length range of specimens sampled. Trophic position estimates provided slightly higher results than those based on stomach contents by Cortés (1999) and those based in other estimation models, mainly for G. galeus (Torres et al., 2014), probably due to the use of a more accurate scaled Δ15N framework (Hussey et al., 2014a, 2014b). As already reported by Torres et al. (2014, 2016b) TPSI can significantly vary depending on the selected baseline consumer species (δ15N base) and Δ15N. Hence, caution is advised when interpreting TP estimates, given that it is subjected to possible shifts in diet considering prey abundance or availability at a given time. 4.2. Biological and ecological factors influencing metal levels Metal content in elasmobranchs muscle tissue was most likely related to the volcanic nature of the area in the absence of a clear

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

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Species

Raja clavata

Galeorhinus galeus

N

Size (cm)

Location

As

30

Azores

33.79 ± 2.39

11

63.46 ± 1.73 73.5 ± 11.1

– 15

– 37.8 ± 1.31

NE Atlantic Turkey

0.05 0.07 ± 0.02

31 8 20

– 60.8 ± 2.8 46–71.3

Adriatic Adriatic North sea

0.02 ± 0.02 6.2–35.9

Azores

20.68 ± 1.52

124 96.81 ± 2.28 6 77.8 ± 18.3

Cd

Cr

Cu

0.98 ± 0.05

0.77 ± 0.09

Fe

2 –

108.5–111.0 Liverpool Bay – Argentina

30 20

145.32 ± 5.44 112–167

44

117–269

21 38

206.2 ± 52.8 113–287

37 27 23

84–239 172–265 80.5–212.0

5

203–219

Azores

0.97 ± 0.10

35.1 ± 1.08 1.10 ± 0.27 0.87 ± 0.52

1.28 ± 0.08 0.4 ± 0.2*

0.24 ± 0.01 1.1 ± 0.3* 0.44

Se

Sr

Zn

Reference

1.24 ± 0.06

0.41 ± 0.01

7.89 ± 1.79

7.42 ± 0.45

This study

9.31 ± 0.44

Chouvelon et al. (2012) Storelli et al. (2012) Türkmen et al. (2013)

0.17 0.58 ± 0.14

Storelli et al. (2003) Storelli (2008) Gieter et al. (2002)

0.03 ± 0.01

0.29 ± 0.05 14.5 ± 3.4* 3.14

Rb

0.99 ± 0.03

1.1 ± 0.6*

1.10 ± 0.15

0.39 ± 0.02

2.2 ± 0.5* 0.16

6.24 ± 0.22

This study

16.5 ± 2.4*

Domi et al. (2005)

2.12

Vas (1991) Pérez et al., (1986)

3.95 ± 0.15

This study

24.61 ± 15.51 6.10 ± 0.37

Alves et al. (2016)

0.52–1.13 10.02 ± 0.69

Southwest of 78.19 ± 21.98 0.01 ± 0.03 Portugal Pacific 6.66 ± 0.55 0.2 ± 0.12 (Mexico) Pacific (Mexico) Pacific (Mexico) Azores South Atlantic Mediterranean 7.20 ± 3.05 English Channel

1.02 ± 0.82* 0.52 0.36 ± 0.09

Pb

0.07 ± 0.01

Bay of Biscay

Celtic Sea

Hg

0.45

0.50 ± 0.03

0.70 ± 0.21

2.58 ± 3.27

1.15 ± 0.55 1.64 ± 0.13

0.24

0.33 ± 0.02 28.21 ± 26.17 27.39 ± 3.57

6.34

1.36 ± 0.83

0.62 ± 0.02 0.12 ± 0.11

0.45 ± 0.02 0.29 ± 0.93

1.03 ± 0.08

0.22 ± 0.02

1.96 ± 1.48* 1.39 ± 1.58

0.10 ± 0.05

0.22–1.3 0.68–2.5

0.084–0.30 0.23–0.46

0.37 ± 0.03

Barrera-García et al. (2012) Maz-Courrau et al. (2012) Escobar-Sanchez et al. (2011) Branco et al. (2007) Branco et al. (2007) Storelli and Marcotrigiano (2004) Vas (1991)

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

Table 3 Comparison of metal concentrations (mg·kg−1·wet weight) found in muscle tissues from different studies with these 4 elasmobranch species. Data is presented as mean, mean ± SD, mean ± SE or range, as found on the literature.* values in dry weight.

31

160–269

Madagascar

4

–

17 – 23 26 15 39

80.5–128 89–335 97–210 126–258 110 ± 20 100–400

Pacific (Mexico) Adriatic Tasmania Azores Canaries Pacific Chile

47 27

– 77–137

Brazil Brazil

0.20–0.89 0.27–1.20 0.16–1.20 0.16–1.84 0.55–7.0 0.014 ± 0.09 0.76 0.46–2.40

30

–

Brazil

0.012–1.15

4

Azores

4

178.75 ± 13.28 161–220

29

122–304

33 20

75–330 89–249

10 Isurus 19 oxyrinchus 7 32

85–251 90–215

69 24 4 20

5.30 ± 2.17* 0.82 ± 0.34

1.71 ± 0.43

1.06 ± 0.17

0.38 ± 0.11

13.43 ± 1.18* 5.96 ± 2.78* 0.15–2.90 0.4

0.06 4.43

0.35

12

Pacific New England

2.5–5.0 2.65 ± 1.16

Chile

127.1 ± 37.9 – 89–249

Pacific (Mexico) Brazil Isla Magdalena 0.1–0.16

0.006 ± 0.001 1.05 ± 0.82

Data from this study is in bold.

0.0001–0.55

Dias et al. (2008) De Carvalho et al. (2014) Mársico et al. (2007) 0.90 ± 0.16

0.60 ± 0.06

0.30 ± 0.05

4.48 ± 0.43

This study Mckinney et al. (2016) Kiszka et al. (2015) Suk et al. (2009) Velez (2009)

0.29

1.58 0.59–5.58

120 ± 10 199.5 ± 23.0 150–450

Maz-Courrau and López-Vera (2006) Storelli et al. (2001) Davenport (1995) Branco et al. (2004) Branco et al. (2004) Kim et al. (2016) Lopez et al. (2013)

2.244 ± 0.81

0.84 ± 0.25

South Africa (East) Madagascar California Pacific (Mexico) New Zealand South Atlantic

Kiszka et al. (2015)

0.38

4

Vlieg et al. (1993) Watling et al. (1981) Kim et al. (2016) Teffer et al. (2014)

0.848 ± 0.47

0.120–0.691 0.09–0.49 0.0001–1.11

Lopez et al. (2013) Maz-Courrau et al. (2012) Mársico et al. (2007) Veléz-Alavez et al. (2013)

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

Prionace glauca

7

8

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

anthropogenic source. As P. glauca and I. oxyrinchus are migratory and highly mobile species it could be difficult to clearly ascertain these metals origin. However, because these species present a low growth rate, the turnover of stable isotopes in muscle tissue is very slow (MacNeil et al., 2005, 2006; Hussey et al., 2012). Hence, the identification of similar isotopic profiles, specifically for δ13C (390–540 days in turnover rate for carbon isotope; MacNeil et al., 2005), seem to indicate that these oceanic species are in the area for a significant period, reflecting possible metal burden in the region. Furthermore, this region of the Mid-Atlantic appears to be an important breeding and nursery area, especially for P. glauca (Vandeperre et al., 2014) which also justifies the occurrence of juveniles in the area over their first years. Usually, studies on contaminants and pollutants in sharks and/or rays tissues compile a list of different species to compare and interpret results to draw out conclusions (Gieter et al., 2002; Barrera-García et al., 2012; Taylor et al., 2014; Gilbert et al., 2015). This type of approach should be performed with caution since pollutants, such as heavy metals, have an associated interspecific variability, reflecting differences in metabolic regulation and different physiological requirements including trophic level, and associated diet and ecology (McMeans et al., 2007; Pethybridge et al., 2010; Barrera-García et al., 2012; Taylor et al., 2014; Kiszka et al., 2015). In fact, looking at other available studies in the literature, in general, concentrations fluctuate not only among species, but also between individuals of the same species and captured in the same geographical area. Nevertheless, in general, muscular metal concentrations in this study are within the ranges previously described for these species worldwide (Table 3). Differences in metal levels between genders may be caused by factors such as energetic requirements, maturation condition and transference to eggs and fetuses (Walker, 1976), especially important in viviparous species such as Prionace glauca. Also, usually females have higher growth rates and are larger (sometimes up to 40%) than males (Daley et al., 2002), displaying sexual dimorphism, which can reflect a higher contaminant bioaccumulation rate. Nevertheless, in this study no significant differences were found between males and females of Prionace glauca consistent to many other works with other species (Penedo De Pinho et al., 2002; Endo et al., 2008; Suk et al., 2009; Pethybridge et al., 2010; Kiszka et al., 2015; Mckinney et al., 2016; Nicolaus et al., 2016), works performed for other elasmobranchs in the same area (Torres et al., 2014, 2016b), for the same species from different areas (Kiszka et al., 2015; Alves et al., 2016) and even from the same area (Branco et al., 2004, 2007). This suggests that maternal offloading of metals to offspring is not a strong driver (Le Bourg et al., 2014) and both genders studied do not have substantial differences in feeding habits or habitats. However, it is important to note that Torres et al. (2014) detected a significance difference for Se between larger males and females of G. galeus. The fact that only juveniles or subadults of Prionace glauca and Isurus oxyrinchus were analysed could reduce the likelihood of observing sex-based differences. Metal bioaccumulation with size/age and biomagnification are some of the main drivers studied in elasmobranchs, especially for the heavy metals As, Cd, Hg and Pb. Similarly to works performed by Torres et al. (2014, 2016b) with other elasmobranch species from the same area, Cd and Pb were not detected in muscle tissue of P. glauca and I. oxyrinchus. Arsenic and Hg content is usually related with diet and associated ecology, identified as one of the most important factors affecting differences between species (De Pinho et al., 2002; Pethybridge et al., 2010; Barrera-García et al., 2012; Torres et al., 2014, 2016b). However compared to R. clavata (Torres et al., 2016b) and G. galeus (Torres et al., 2014) from the same area, the two migratory shark species presented low concentrations of As, although Alves et al. (2016) reported a much higher value of As for P. glauca captured near mainland Portugal. Nonetheless, the majority of As found in fish is in the form of arsenobetaine, a quite stable and non-toxic compound (Gieter et al., 2002). Fish accumulate As predominantly via their diet, but in contrast to Hg, does not seem to biomagnified through the food web but instead

metabolized (Maeda et al., 1990). High concentrations of As are usually attributed to a crustacean based diet (Turoczy et al., 2000) which reflects the significant elevated content in R. clavata (Torres et al., 2016b). This heavy metal was the only one that presented significant differences between the four elasmobranchs analysed, reflecting their different feeding habits. In fact, considering their trophic level it is possible to notice that As does not magnify to higher trophic levels, contrarily to Hg which was higher in I. oxyrinchus. The few studies available on the influence of either age or body length on As accumulation in elasmobranchs did not report any correlation (Storelli and Marcotrigiano, 2004; Alves et al., 2016), contrarily to our study and the work performed on G. galeus (Torres et al., 2014) and R. clavata (Torres et al., 2016b). Mercury concentrations are typically correlated with size (weight, length) and age in fish, a pattern well documented in various studies (De Pinho et al., 2002; Branco et al., 2004, 2007; Barrera-García et al., 2012; Torres et al., 2014; Alves et al., 2016; Torres et al., 2016b). However, this relationship is influenced by other factors, as larger (older) sharks tend to feed on prey of a higher trophic position, have different habitats, habitat ranges or movements, relative to smaller (younger) sharks (Cortés, 1999; Speed et al., 2010), which is especially relevant for migratory species. Methylmercury (90% of total Hg found in fish muscles and the most toxic organomercury compound) binds to proteins whose content increases with fish age (Storelli et al., 2002). Average mercury levels of P. glauca and I. oxyrinchus were higher than those reported for G. galeus (Torres et al., 2014) and R. clavata (Torres et al., 2016b) from the same area, although not significant for G. galeus, reflecting their high trophic level. However, compared to other studies, these values were generally slightly lower (Table 3). Selenium has been reported in some studies as having a protective role against Hg toxicity (Storelli and Marcotrigiano, 2002; Endo et al., 2005; Branco et al., 2007) although the underlying mechanisms are still unclear. Although the results obtained in this study were insufficient to elucidate this issue, no correlation was found between both metals in muscle tissue, contrarily to G. galeus (Torres et al., 2014) and R. clavata from the same area (Torres et al., 2016b). Regarding Sr and Zn, although usually disregarded in elasmobranch studies, were significantly higher in R. clavata, except Zn in G. galeus, which is probably associated with the invertebrate component of their diet (Torres et al., 2014, 2016b). It is also worth mentioning that Zn, similarly to As, was detected in higher amounts by Alves et al. (2016) in P. glauca muscle tissue (Table 3).

4.3. Implications for elasmobranch and human health In recent years, a significant increase in consumer demand for seafood has occurred in many countries. In fact, Portugal is the largest fish consumer among all of the EU countries and one of the biggest in the world with an average consumption of 56.8 kg/year/inhabitant, while for EU it is estimated an average on 24.9 kg/year/inhabitant (European Commission, 2016). European Union countries play a major role in the international trade of sharks and shark meat, responsible for 56% of global shark meat imports and 32% of worldwide exports, justifying the need to evaluate the consumption safety of these products. Spain, Portugal, France and Germany, are the biggest European shark meat consumers (OCEANA, 2008). The four different species of this study are a valuable commercial resource and constitute an important component of Mid-Atlantic fisheries (Torres et al., 2016a). As elasmobranchs accumulate higher levels of pollutants it is of upmost importance to understand if and how these pollutants might affect the organisms, their physiology and natural behaviour, which can have repercussions in the entire ecosystem, including humans. Some studies have already been successful correlating metal concentrations with biochemical responses of oxidative stress in these species, resulting in DNA damage (Barrera-García et al., 2012, 2013; Alves et al., 2016).

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Arsenic legal limits vary between 0.1 mg kg− 1 ww (Venezuela) and 10 mg kg − 1 ww (Hong Kong). The Joint FAO/WHO Expert Committee (1983) has also set a limit of 0.1 mg kg− 1 ww. There is no legislation within the EU or EUA and each country has its own legislation and restrictions. Some countries legislate according to total As concentration, while others use the inorganic As fraction. Inorganic As is considered carcinogenic and is related mainly to lung, kidney, bladder, and skin disorders (ATSDR, 2003). This lack of formal As concentration norms make it difficult to evaluate the potential human risk related to its consumption. However, considering that its concentration can easily vary according to physiological and seasonal variations or changes in food habits, since it is metabolized, and its occurrence in non-toxic forms, As potential hazard for both elasmobranchs and humans is potentially low (Gieter et al., 2002). The European Commission has set maximum levels for Hg, Cd, and Pb in seafood for human consumption (European Commission, 2008). For Hg, the maximum limit in the edible parts of fish products is 0.5 mg kg−1 ww except for some fish species for which it is increased to 1 mg kg−1 ww (elasmobranchs belong to this group). For Cd, this regulation separates fishes from molluscs and crustaceans, fixing maximal permissible concentrations at 0.05 to 0.1 mg kg− 1 ww for most fish muscle and 0.3 mg kg−1 ww for swordfish muscle. Regarding Pb, the guideline values are 0.3 mg kg−1 ww for fishes. In the present study, Cd and Pb were below detection levels and regarding Hg almost all the samples evaluated were suitable for human consumption, except one I. oxyrinchus individual whose levels exceeded 1 mg kg−1 ww. However, as already stated, P. glauca and I. oxyrinchus specimens sampled were mainly juveniles and, given that Hg has a strong relation with size/age, this should raise some concern. Mercury has negative behavioural, neurochemical, hormonal and reproductive impacts on fish such as emaciation, cerebral lesions and impaired gonadal development (Silva et al., 1992; Scheuhammer et al., 2007). However, differences in Hg sensitivity should be expected among shark species, as has been observed for other vertebrates (Sandheinrich and Wiener, 2011). In humans, methylmercury penetrates cell membranes and attaches to nucleophilic groups in enzymes concerned with protein synthesis in the central nervous system resulting in serious brain damage, including psychological disturbance, impaired hearing, loss of sight, ataxia, loss of motor control and general debilitation (Storelli et al., 2003). Also, the absorption of methylmercury during the embryonic phase negatively affects children psychomotor development (Storelli et al., 2003). The Joint FAO/WHO Expert Committee on Food Additives established a PTWI (provisional tolerable weekly intake) of 300 μg of total mercury, equivalent to 5 μg of total mercury per kg of body weight (WHO, 2006; EFSA, 2009). Accordingly, considering an average body weight of 70 kg and the mean Hg concentration in muscle wet weight, the maximum allowable weekly intake would be 5, 1.21, 1.06 and 0.42 kg for R. clavata, G. galeus, P. glauca and I. oxyrinchus, respectively. Considering the 0.49 kg of fish consumed in average each week per capita in the EU, a high exposure is associated with the consumption of I. oxyrinchus since it already exceeds the maximum allowable weekly intake, regardless of legislated maximal permissible concentrations. If we only concentrate on Portugal, the average consumption of 56.8 kg/year/inhabitant implies that an average of 1.09 kg of fish is consumed weekly, which encloses an even greater risk. However, given that this weekly consumption estimate represents an average across the whole population and that it is unlikely that someone would consume the same species on a weekly basis, the estimated value might be overestimated. Nevertheless, the variability of Hg content in fish, including elasmobranchs in this study, suggests that the consumption of certain species can substantially affect the intake of these heavy metals, resulting in large differences in health risks.

9

4.4. Elasmobranchs: suitable metal bioindicators? Ideally, monitoring should provide early warning of any changes that could potentially affect individual species (including humans), populations, communities or ecosystems. Thus it is important to develop a set of key bioindicators, together with specific biomarkers, to easily assess status and trends within an ecosystem and, more specifically, the effects of anthropogenic activities in the environment (Burger, 2006). According to Burger (2006) indicators should be selected given their biological, methodological, and societal relevance. An indicator must reveal changes in response to a stressor, but not be so sensitive that changes occur when there is no cause for concern (no lasting reproductive, survival, or population effects). Also, it must be methodologically relevant; easy to measure, for managers to use, for conservationists to employ in species preservation, and for regulators to employ in compliance mandates. Finally, it needs societal relevance, because without public and governmental support, it will not be used over appropriate spatial and temporal scales that provide meaningful information. Hence, charismatic species are often used as indicators because there is sustained public and governmental interest. For marine pollution monitoring, bioindicator species used belong to numerous taxonomic groupings, each showing some special merits when compared to the others. Elasmobranchs, besides having specific life-traits that make them ideal bioindicator species for longterm monitoring, they have biological and methodological relevance, and a great advantage when compared to other species: societal relevance. However, to use elasmobranchs as bioindicators of marine pollution it is important to focus the same species and to outline specific areas to allow comparisons and even so caution is required since, as already stated, metal concentration also varies with size, gender, maturity stage, diet, season, health condition and tissue within species (Table 3). The four Mid-Atlantic elasmobranch species of this study have specific life traits, each one with different characteristics regarding the key features that defines a good marine bioindicator. Generally, their high trophic level associated with their wide distribution, low growth rates and longevity allows a reasonable assessment of ecosystem health and can easily be monitored over great periods of time. Accessibility and mobility are the main constrains on using these species. Regarding accessibility, unfortunately nowadays these species are easy to obtain as they are targeted by fisheries (Torres et al., 2016a). The increasing number of tagging studies, programmes and campaigns also enables accessing some species (e.g. P. glauca in the Mid-Atlantic, Vandeperre et al., 2014). Elasmobranchs mobility is perhaps the most relevant feature concerning monitoring of a given area or region. However, while some species are highly mobile as P. glauca and I. oxyrinchus (Vandeperre et al., 2014), others have much more restrained movements. Raja clavata has a much smaller home range (Walker et al., 1997) and recent studies revealed that even G. galeus have more restrained movements (Torres et al., 2014; Thorburn, 2015). Ironically, these two species have the disadvantage of being much less studied regarding metal levels than the migratory ones (Table 3). Of the four, G. galeus is perhaps the best bioindicator for the Mid-Atlantic region considering all key features; its low mobility, small home range, high trophic level and greater longevity and slower growth rate (Dureuil, 2013), although R. clavata would allow the monitoring of specific areas, of just a few square miles. Prionace glauca has the advantage of being everywhere, enabling comparisons between areas and regions and is by far the most studied species (Table 3). Recently Alves et al. (2016), working with P. glauca detected higher values of Mn and Cd in the liver and higher levels of Fe and Cu in liver and muscle tissue compared to a study by Vas (1991) performed with older individuals collected in the same ocean (Atlantic) and reached to the possibility of an increase in metal contamination in the Atlantic Ocean. However, both areas are separated by a couple of thousand

Please cite this article as: Torres, P., et al., Mid-Atlantic elasmobranchs: Suitable metal scouts?, Marine Pollution Bulletin (2017), http://dx.doi.org/ 10.1016/j.marpolbul.2017.01.058

10

P. Torres et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

miles, between southwest Portugal until the English Channel. In this study, the analysed sharks of P. glauca were captured in a specific area of the Mid-Atlantic similar to Branco et al. (2004, 2007), with approximately the same length, and it is possible to observe that the values remained relatively stable for mercury since 2004 (Table 3). Nevertheless, given its high mobility is almost impossible to associate metal levels to a specific limited area, unless the goal is to monitor a vast area as the North Atlantic. Another alternative is to use other techniques, such as stable isotopes or tagging studies, to allow a more reasonable temporal and spatial evaluation of a specific site. As top predators, another problem is that metal levels in these species may vary according to different diets. In our case, R. clavata reflected the existence of an important As source in the region (Torres et al.,2016b). Although the majority of studies still rely on one species to monitor ecosystem changes (Siddig et al., 2016), according to Lindenmayer and Likens (2011) no single species can reflect the complexity of the total environment. Hence, different elasmobranchs species with different life traits could be a solution. Also, as some metals as Cd or Zn preferentially accumulates in liver tissue (Barrera-García et al., 2013; Alves et al., 2016; Torres et al., 2016b) it would be advisable to use several tissues. 5. Final remarks Ecotoxicological baseline data on marine wildlife in the Mid-Atlantic region is particularly important due to its volcanic nature and potential increase in anthropogenic activity in the region. Heavy metal compounds found in elasmobranchs can severely damage the health of both marine organisms and humans. Thus it is crucial to develop biomarkers to monitor changes caused by the contaminants, before they cause an effect at higher complexity levels, communities or ecosystems. Generally, heavy metal content (As, Cd, Hg and Pb) was low compared with other worldwide studies and did not exceed EU legislated limits, except Hg content in one individual of I. oxyrinchus, which should raise some concern since the sampled migratory species were juveniles. As this area of the Atlantic is probably a breeding and nursery ground for some sharks, contamination in such an area could influence the health of entire Atlantic populations, emphasizing the need of its monitoring. Also, it is important to highlight that although heavy metals are legislated according to maximum accepted levels, it is undoubtedly more reliable to regulate the amount of seafood consumed on a regular basis. Elasmobranchs appear to be good long-term metal bioindicators for the Mid-Atlantic region given their overall biological traits and specific species features, allowing both broad and small temporal and spatial monitoring. Further studies are important to improve our knowledge of the bioaccumulation, organotropism, ontogenic and reproductive contaminant processes in these species. The analysis of multiple tissues, regarding both stable isotope and metal analysis, would provide a temporal perspective and help to determine the main accumulation and detoxification tissues/organs, clarifying main pathways for different metals. Acknowledgements The authors would like to thank Maria Ana Dionísio, Dr. Patrícia Garcia and Dr. Ricardo Camarinho for their logistical and technical assistance. António Mineiro & Andrade are thanked for providing a sampling facility and ray access. This research was funded by a FRC/M3.1.2/F/045/ 2011 grant and by Fundação Luso Americana para o Desenvolvimento (Project 275/2013). This work was also funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and by National Funds through FCT - Foundation for Science and Technology under the UID/BIA/50027/2013 and POCI-01-0145-FEDER006821.

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