Natural and anthropogenic processes that introduce chemical substances above background ecosystem levels and that may cause ecosystem deterioration are considered as sources of pollution in the marine environment (Clark 2001). It is well known that coastal areas receive large amounts of trace metal inputs from various sources, and some metals are regarded as serious pollutants due to their anthropogenic origin, bioaccumulation and toxicity to marine organisms (Lafabrie et al. 2008). Metal contaminants can be directly or indirectly accumulated in different marine species and cause several biological effects, ranging from molecular dysfunctions to ecological changes depending on the exposure and tolerance levels (Van der Oost et al. 2003). Particularly, filter-feeder marine bivalve molluscs (e.g. mussels, oysters, etc.) accumulate metals either directly from dissolved concentrations in the water column or indirectly via ingestion of metal-bounded suspended particles from the surrounding environment. Bivalves have been shown to be valuable ‘sentinel organisms’ because of their limited metabolic capacity of metal excretion and their worldwide distribution (Goldberg 1975, Goldberg et al. 2000), and they have therefore been used to monitor and assess coastal pollution for decades (O’Connor and Beliaeff 1995, O’Connor and Launstein 2006, Benedicto et al 2011, Besada et al. 2014, Neira et al. 2015).

Many studies on the occurrence of metals in different species and habitats of the Mediterranean Sea have been investigated primarily using the bivalve mollusc Mytilus galloprovincialis (Andral et al. 2004, Deudero et al. 2009, Benedicto et al. 2011) and benthic invertebrate species such as Venus verrucosa, Litophaga litophaga, Ostrea edulis and Chamelea gallina (Deudero et al. 2007). Similarly, following the transport, distribution and fate of metal species in the marine environment, many benthic organisms could accumulate them and also be targeted as sentinels of metal contamination. Thus, benthic animals and plants are clearly under the influence of anthropogenic pressures in the marine environment (Solis-Weiss et al. 2004, Deudero et al. 2007). In this context, the species Pinna nobilis Linnaeus 1758 is a benthic species endemic to the Mediterranean Sea that inhabits seagrass meadows, especially those of P. oceanica, from shallow areas down to 60 m bottom depth (García-March 2003). In the Balearic Archipelago, a high bioaccumulation of polyaromatic hydrocarbons in P. nobilis has been confirmed in the framework of a post–oil spill environmental assessment evaluation (Sureda et al 2013).

The family Pinnidae Leach 1819 are filter-feeder marine bivalve molluscs with a long triangular shape and fragile shells with the pointed end anchored in the substrate using the byssus threads. P. nobilis is the largest Mediterranean bivalve and one of the biggest worldwide (Zavodnik et al. 1991), with shell lengths usually of 15-35 cm and exceptionally up to 120 cm (Schultz et al. 2013). This family includes two genera (Pinna and Atrina, Lemer et al. 2014) with 61 species described worldwide (WoRMS 2014). With regard to substrate types and habitats, Pinnidae species have been found in mud, silt, sand, coral, rocks, seagrass meadows and mangrove swamps (Schultz et al. 2013). Pinnids are distributed from the shoreline, at very shallow depths of a few decimetres, to the bathyal zone down to 600 metres. In the Mediterranean Sea, P. nobilis is a protected species under both Annex II of the Barcelona Convention (SPA/BD Protocol 1995) and Annex IV of the EU Habitats Directive (EU Directive 2007), so scientific studies are required to understand and assess the current status of P. nobilis populations for future management and conservation strategies.

The datasets of metal concentrations are scarce and spread over a few geographical locations in the Mediterranean basin. So far, five studies have reported the accumulation of metals in P. nobilis in the Mediterranean: two were carried out in Greece (Catsiki and Katsilieri 1992, Catsiki et al. 1994), one in France (Mathew et al. 1996), and two recently in Tunis (Rabaoui et al. 2014, Jebali et al. 2014). These studies determined various metals in different parts of the specimens within a variety of field experiment approaches. The present study aims to contribute to the existing knowledge by (i) determining the concentration of metals in the soft tissues of P. nobilis in the Balearic Archipelago; and (ii) providing the first bibliographic review on levels of metals for Pinnidae species by reviewing worldwide datasets from the literature.

The present study provides the first dataset on metal levels in P. nobilis in the Balearic Archipelago (western Mediterranean Sea). The overall results evidence high metal concentrations between sampling sites with significant spatial differences. However, it is noteworthy that metal concentrations reviewed in the scientific literature also varied up to three orders of magnitude for some metals between specimens and locations, so our comparisons need to be taken with caution. Unfortunately, in most of the studies nothing is stated, for example, about the number of replicates, shell length (allometric parameters), collection depth or habitat types, or about the sample treatments, making evaluations even more difficult. Therefore, it should be stressed here, that sample collection, transport and laboratory methodologies for performing chemical contaminant analysis of large marine biota specimens such as P. nobilis require future agreed scientific protocols, especially for endangered species in the Mediterranean Sea.

Regarding metal concentrations, Cd, Hg and Pb levels were higher in S. Maria Bay, the MPA sampling site (Cabrera Archipelago), than in Majorca Island. However, the mean Cd levels at this site (26.4±6.9 mg kg^–1 dry weight (DW), Table 2) were much lower (1/5) than those found in Vietnam for P. bicolour (Khristoforova et al. 2007), but almost three times higher than P. muricata collected in the United Arab Emirates (de Mora et al. 2004). For Hg and Pb, S. Maria Bay shows the same pattern as Magaluf, though slightly higher Hg levels were found in S. Maria without significant differences (0.96±0.13 and 0.80±0.22 mg kg^–1 DW, respectively). Rabaoui et al. (2014) studied different tissues of P. nobilis and found the highest Hg values in gills (up to 0.169 mg kg^–1 DW). Correspondingly, similar mean Pb values determined in S. Maria Bay and Magaluf (23±5 and 20±6 mg kg^–1 DW, respectively) were about three times lower than the highest levels found in Vietnam for P. bicolour by (Khristoforova et al. 2007). These Hg and Pb levels are the highest in the literature (Table 2). In contrast, the P. nobilis specimens collected in the Port of Andratx (Majorca Island) showed much lower values for Hg and Pb than the other two sampled sites (0.30±0.04 and 0.9±0.2 mg kg–1 DW, respectively).

The highest mean Zn level (4034.3±358.7 mg kg^–1 DW) was found in Andratx and is one of the highest reported in the literature for Pinnidae species, with the exception of the Zn concentrations of up to 5080 mg kg^–1 DW observed by Reid and Brand (1989) in the cytosol fractions in gills of P. bicolour. The study, performed in pristine, human-impacted and harbour areas in Western Australia, reports the highest Cu concentrations in the literature (up to 235 mg kg^–1 DW), which are close to the mean Cu level (197.85±88.08 mg kg^–1 DW) found at the Magaluf sampling site (Table 2). However, these concentrations in P. bicolor are not given for the whole soft-tissue but for gills, so the specific metal content tissue variability should be considered. Nevertheless, the levels found in P. nobilis at these two sites (Andratx and Magaluf) of Majorca Island point to a high occurrence of Zn and Cu in the surrounding environment available for benthic filter-feeders.

In our study areas, the sampling sites were selected taking into account potential differences regarding metal inputs and levels. Both Andratx and Magaluf might be influenced by land, industrial (to some extent), and coastal seasonal tourism uses. Andratx has a recreational port (the Port of Andratx) and Magaluf town discharges treated sewage, mostly from domestic origin. Domestic sewage has been reported to be a source of metal contamination (Wei and Yang 2010), including copper (Santos-Echeandia 2008), and perhaps is the cause of the high Cu values at the Magaluf site. The high levels of Zn might be related to the influence of the Port of Andratx, as in the study by Jebali et al. (2014), who exposed P. nobilis in a harbour, resulting in increased Zn concentrations. At the S. Maria bay site, located in the MPA of Cabrera Marine-Terrestrial National Park, there are no evident metal inputs at present, but there was a Spanish military zone used as weapons shooting range until 1991. Jung et al. (2010) studied the contamination and dispersion of trace metals in intertidal sediments around a military weapon shooting range, and found substantial contamination of sediments by Pb, Cd, Cu and Zn, suggesting that those metals were from anthropogenic origin. These high levels of Cd, Hg and Pb might be due to legacy contamination distributed in the benthic habitats of the Cabrera Archipelago.

On the other hand, the levels of metals (expressed as wet weight concentration, ww) found in P. nobilis in the present study for the S. Maria Bay dataset (mean concentration: 5.02 mg Cd kg^–1 wet weight (WW); 0.18 mg Hg kg^–1 WW; 4.37 mg Pb kg^–1 WW; 19% dry/wet ratio applied) exceed the recommendations of the World Health Organization (WHO 1989) for bivalve molluscs (except for Hg) but are below the Food and Agriculture Organization (FAO 1992) standards, despite the large reference range for the latter (1.0/0.005-5.5 mg kg^–1 WW [WHO 1989/FAO 1992, respectively] for Cd; 0.5 mg kg^–1 WW [WHO 1989 and FAO 1992] for Hg; and 2.0 / 0.5-6.0 mg kg^–1 WW [WHO 1989/FAO 1992]). It should be pointed out here that international standards are set in the context of human health protection for shellfish consumption of commercial species, and this is not the case for P. nobilis in the Mediterranean Sea. The high metal levels bioaccumulated in P. nobilis in the Balearic Archipelago indicate different sources of metal contamination, but the potential biological effects in the P. nobilis populations and in the benthic ecosystem are still unknown. Elevated metal concentrations could cause a severe reduction or elimination of intolerant species, thereby having a significant effect on the diversity and trophic structure of the biological community (Boening 1999). Metals such as Cu and Zn have biological functions and are essential elements for many organisms, although they could be toxic above background levels. The excess of Zn accumulation in living organisms gives rise to severe metabolic disorders (Du et al. 2011), whilst non-bioactive heavy metals with no known biological functions such as Cd, Pb and Hg can be toxic even at low concentrations (Corbin and Wade 2004). In the case of P. nobilis, high population densities had been found in S. Maria Bay (Vázquez-Luis et al. 2014), where the highest heavy metal levels were found in this study, so the medium- and long-term consequences are uncertain. Surely, some biochemical responses and toxicological effects on the P. nobilis could be expected due to the high values of essential and non-essential elements, which should be investigated through a combined chemical and toxicological approach for this species. Metallothionein proteins (MTs) do appear to play roles in the routine metabolic handling of essential Cu and Zn and also in the detoxification of intracellular excess amounts of these metals and of non-essential Cd, Ag and Hg. Some authors have noted that in a single species the populations that live in a medium polluted by metals have higher concentrations of MT (e.g. Stuhlbacher et al. 1992, Ross et al. 2002); therefore, less sensitive stages corresponded to those with the highest MT concentrations (Pavicic et al. 1994). Different isoforms of MT probably play different physiological roles, and the dependence on MT in detoxification processes varies environmentally and between zoological groups (Amiard et al. 2006). In many species (annelids, molluscs, crustaceans, fish, etc.) induction of metallothionein synthesis by metal contaminants (Ag, Cd, Cu, Hg, etc.) has been demonstrated, with the exception of Pb. In fact, in the case of the bivalve Mytilus edulis, Cd and Zn induce MT, and after 12 h of exposure to Cd and Zn the MT-10 mRNA levels increased by 3.5 and 4.65, respectively (Soazig and Marc 2003). For Ruditapes decussatus the high capacity of metal bioaccumulation in the digestive gland of caged mussels may be related to the high metallothionein synthesis and the rapid sequestration of metals into lysosomes (Serafim and Bebianno 2009). In the case of Pinna nobilis nothing is known about MT, but it could be expected that the high bioaccumulation of metals found in this species may be related to high levels of MT that could be providing protection against toxicity, since a experimental study by Jebali et al. (2014) concluded that P. nobilis seems to be a tolerant bivalve species, showing the ability of metal elimination through depuration.

Regarding possible ways of metal uptake in Pinna nobilis, this bivalve is a filter-feeder organism which assimilates nutrients in both dissolved and particulate matter from the surrounding environment. Many studies have demonstrated that it feeds from different food sources, such as the detritus from the water column or from benthic habitats (epiphytes of P. oceanica leaves, Lophocladia lallemandii, suspended particulated matter, sedimentary organic matter [SOM] and seagrass) (Kennedy et al. 2001, Cabanellas-Reboredo et al. 2010, Davenport et al. 2011). Recently, Najdek et al. (2013) showed an increasing contribution of phytoplankton and zooplankton food components in the water column detritus with increasing shell size, and concluded that smaller individuals are less food-selective. Further, Trigos et al. (2014) stated that muddy detritus is easily ingested by P. nobilis, whilst nearly half of the organic matter contained in the detritus is retained or metabolized, so it is a major source of SOM, whereas phyto- and zooplankton would provide the necessary complements for a healthy diet. Therefore, P. nobilis could alternate feeding strategies during seasons typically poor in plankton biomass in the Mediterranean Sea (e.g. winter). However, Alomar et al. (2015) point to a strict pelagic rather than benthic origin of the diet. From these recent studies, it is difficult to figure out the exact routes and sources of metals in P. nobilis, although in sites with legacy pollution (such as S. Maria Bay in this study) the benthic routes should also be considered and further investigated. Hence, resuspension processes influencing the higher organic matter ingestion by P. nobilis (Cabanellas-Reboredo et al. 2010) may also intensify the uptake of sediment-associated contaminants, so the differences in water column depth and benthic substrate may influence the bioaccumulation processes. The filtration rate in P. nobilis is unknown, but it has been estimated that oysters are able to reach rates of filtration of 22×10⁵ L h^–1 in populations with a density of 157.6 individuals m^–2 (zu Ermgassen et al. 2013). Given the size and characteristics of P. nobilis, the filtration rates are expected to be high.

When the trace metal levels found in P. nobilis (Pinnidae) in this study are compared with those found in other bivalve mollusc species, such as Mytilus galloprovincialis (Mytilidae), a standard bioindicator of the coastal trace metal pollution, benthic P. nobilis shows concentrations 30 to 40 times greater for Cd, Cu and Zn, up to 4 times greater for Hg and 7 times greater for Pb in the Balearic Archipelago (Deudero et al. 2007, 2009). It was also determined in a previous study (Sureda et al. 2011, 2013) that polycyclic aromatic hydrocarbon levels in the soft tissues of P. nobilis were more than 20 times higher than those in the common M. galloprovincialis. In conclusion, the huge differences in metal bioaccumulation between these two bivalve species inhabiting the same coastal environment should be explained mostly in terms of two factors, the specific-species bioaccumulation factors and their feeding behaviours, though metal concentrations and bioavailability are expected to be high in the benthic environment. Further, detritivorous and scavenger gastropods could be accumulating even greater concentrations of metals due to these high concentrations. At present, there are insufficient chemical and toxicological datasets (including distributions of metals in marine sediments) to reach better conclusions. However, future investigations of P. nobilis populations in the Mediterranean Sea should agree on methodological scientific protocols to avoid “destruction sampling” of endangered and protected species and allow comparisons at regional and global scales.

Our results highlight the importance of improved monitoring strategies to assess the current status of the marine environments. In Europe, under the Marine Strategy Framework Directive (EU MSFD 2008), the good environmental status of the marine environment needs to be defined. Among the EU MSFD 11 ecosystem descriptors, Descriptor 8 refers to contaminants, including concentrations and biological effects, and it is therefore necessary to evaluate the concentrations of contaminants through an ecosystem-based approach. Until now, we considered ‘surrogate species’ in the marine ecosystem assessments (e.g. M. galloprovincialis), and therefore underestimated the anthropogenic impact in the ecosystems as a whole, as shown in this study. However, because the entire changing environment cannot be monitored, newer and refined indicators and ‘status ecological indices’ should be investigated and proposed, in order to help protect and predict human-induced marine ecosystem changes.