Marine Pollution Bulletin 184 (2022) 114129

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Trace elements in edible tissues of elasmobranchs from the North Aegean

Sea (Eastern Mediterranean) and potential risks from consumption
Ioannis Giovos a, b, c, *, 1, Maria Violetta Brundo d, Nikolaos Doumpas a, Zoi Kazlari e,
Dimitrios Loukovitis e, f, Dimitrios K. Moutopoulos b, Roxani Naasan Aga Spyridopoulou a,
Athina Papadopoulou a, Maria Papapetrou e, Francesco Tiralongo d, g, Margherita Ferrante h,
Chiara Copat h, 1
a
iSea, Environmental Organisation for the Preservation of the Aquatic Ecosystems, Thessaloníki, Greece
b
University of Patras, Department of Animal Production, Fisheries & Aquaculture, Mesolongi, Greece
c
Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy
d
Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy
e
Lab of Agrobiotechnology and Inspection of Agricultural Products, School of Agriculture, International Hellenic University, Sindos, Thessaloniki, Greece
f
Research Institute of Animal Science, ELGO Demeter, 58100 Paralimni, Giannitsa, Greece
g
Ente Fauna Marina Mediterranea, Avola, Italy
h
Department of Medical, Surgical and Advanced Technologies “G.F. Ingrassia”, University of Catania, Via Santa Sofia 87, Catania 95123, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Trace elements have the potential to bioaccumulate in marine organisms and to biomagnify towards the upper
Chondrichthyans levels of marine trophic webs, resulting in a range of negative effects on organisms. Elasmobranchs are highly
Trace metals susceptible to bioaccumulation of trace metals, while their consumption by humans is increasing worldwide.
Marine pollution
Therefore, it is important to monitor the trace metal content in the edible tissues of elasmobranchs. This work
Mediterranean Sea
reveals the content of 12 trace metals in the edible tissues of 10 elasmobranch species caught in Greek waters.
Levels above the permissible limits for Hg and Pb were found in some species, while analysis of the lifetime
consumption risk for adults and children using the Target Hazard Quotient (THQ), revealed a high risk for two of
the most toxic substances on the priority list for substances, namely As and Hg. These are preliminary results, and
further research is required to understand better the issue.

1. Introduction significant stressors for the marine biota (Scheuhammer et al., 2015;
Ehnert-Russo & Gelsleichter, 2020), hindering as well potential risks for
Persistent organic pollutants (POPs), metals, crude oil and marine humans (Barone et al., 2021), given that marine organisms are an
debris (e.g. marine litter or microplastics) represent the most common important source of protein for humanity (Dent and Clarke, 2015).
marine pollutants globally (United Nations Environment Programme, Compared to other marine vertebrates, elasmobranchs are consid­
2021). The last decades there has been an increasing concern for the ered very prone to biomagnification of trace elements (Mull et al., 2012;
effects of various pollutants on the marine environment, on marine or­ Boldrocchi et al., 2021) due to their life characteristics. The majority of
ganisms and subsequently on humans (e.g., Pourang, 1995; Kumar et al., the elasmobranchs are considered top to meso-predators (Heupel et al.,
2019; Salvaggio et al., 2019; Tiktak et al., 2020). As a result of various 2014) and are long-living species. This makes them particularly sus­
anthropogenic activities such as mining, industry, agriculture etc. ceptible to contamination, especially of pollutants like trace elements.
(WHO, 2020), these pollutants enter and accumulate in the marine Studies have shown that trace elements like Hg and Cd, can alter their
environment, bio-accumulate in marine organisms and bio-magnifying reproductive physiology, decrease, and alter the development of em­
towards the upper levels of the marine trophic webs, including bryos and pups and create alterations in their physiology (Tiktak et al.,
humans. Among those, trace elements, have been identified as 2020). However, compared to other fish species, this topic, especially in

* Corresponding author at: iSea, Environmental Organisation for the Preservation of the Aquatic Ecosystems, Thessaloníki, Greece.
E-mail address: [email protected] (I. Giovos).
1
These authors contributed equally to this work

https://doi.org/10.1016/j.marpolbul.2022.114129
Received 29 June 2022; Received in revised form 8 September 2022; Accepted 9 September 2022
Available online 13 October 2022
0025-326X/© 2022 Elsevier Ltd. All rights reserved.
I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

relation to their conservation status, is heavily understudied for elas­ of Northern Greece were evaluated, with the aim to understand the risk
mobranchs (Consales and Marsili, 2021), although they are among the for consumers in an area that traditionally consumes high amounts of
most threatened marine taxa globally with the most recent global IUCN such products (Giovos et al., 2021), but also to provide a baseline in­
Red List of Threatened Species assessment estimated that 37% of the formation about the level of concentration in different elasmobranch
species are threatened with extinction (Dulvy et al., 2021). species for future studies. Several indicators were applied for measuring
At the same time the demand for elasmobranch products is rising the risk from the consumption of different species by adults and chil­
(Dent and Clarke, 2015) as a result of (i) commercial fish stock over­ dren. The study has been based on: (i) genetic identification of the
exploitation, (ii) the emerging demand for seafood and protein, and (iii) samples and (ii) quantitative analysis of the trace elements’ levels in the
the increasing finning regulations that are suspected to stimulate the flesh.
creation of new shark meat markets (Dent and Clarke, 2015). Only for
2011, global shark fisheries estimated around 1$ US billion with an 2. Materials and methods
increasing trend, excluding the value of elasmobranch products
consumed domestically, which may comprise the majority of the catches 2.1. Sampling
(Dulvy et al., 2017). In the Mediterranean, elasmobranch catches
represent only 1 % of the total landings (Statistic FAO 1980–2015), with Fish market surveys took place on a monthly basis in four selected
most of the species landed as valuable bycatch that are consequently towns for two to five days each; Alexandroupoli, Kavala, Thessaloniki,
sold, primarily, domestically (Bradai et al., 2018; Touloupaki et al., and Volos (Fig. 1) as part of the wider research project ByElasmoCatch
2020). In Greece, the contribution of elasmobranchs to the total annual (Giovos et al., 2021). Through the project, the research team developed
Greek landings between 1990 and 2020 is <2 % (around 1000 tn relationships with fishmongers in these towns that facilitated trust full
annually) (HELSTAT, 1990–2021). However, the country is among the answers and access to invoices for securing that all samples were
top elasmobranch markets of South Europe, with significant imports collected from specimens that caught in the North Aegean region that
from Italy and Turkey (Giovos et al., 2020), although the country’s was our target area. When the capture location was not certain and/or
seafood per capita consumption remains still low (18.2 kg per capita/ could not validate no sample was collected. Tissue samples of products
year), significantly below the European average (EUMOFA, 2017). Thus, sold as elasmobranchs (e.g. ray wings, shark fillets etc.; Fig. 2) were
elasmobranch products can be found in all fish markets, especially of taken for genetic and metal analysis along with pictures. There were no
North Greece, with Greeks consuming a variety of elasmobranch species, specific target species for the sampling nor a specific age or sex when
with Raja spp., Mustelus spp., and Dasyatis spp., being the most common choosing the specimen, rather the tissue samples were obtained through
(Giovos et al., 2021). Like elsewhere (Bornatowski et al., 2013), mis­ a random selection process, and every sampling day different fish stores
labelling and illegal trade is common throughout the season (Giovos were selected. The samples for the genetic analysis were obtained from
et al., 2021) and thus consumers often buy shark and batoid products the product using a single-use surgical lancet to cut at least 3 g of tissue
without intention. that then deepened in an Eppendorf with 100 % alcohol. Using the same
In this work, the levels of 12 trace elements in edible tissues of tools two bigger samples that weighed at least 10 g each, were carefully
elasmobranchs caught in the North Aegean Sea and sold in the markets cut off the product to contain as much muscle tissue as possible and were

Fig. 1. With the white pins the sampling locations in Northern Greece are presented. The red cycle represents the capture location near Kavala and the green cycle
the capture location near Evros Delta, where all the majority of the analysed specimens from the fish markets were captured. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

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I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

Fig. 2. A. Slices of Mustelus mustelus offered in Kavala street market. B. Wings of Raja clavata in a fish market in Alexandroupoli.

carefully placed in test glass tubes. The tubes then were sealed by adding the whole PCR products were purified with the microCLEAN DNA
parafilm on top. All the samples from the same product were tied cleanup reagent (Gel Company, USA) according to the manufacturer’s
together with a duct tape and a reference code indicating the town, the protocol. Finally, the purified PCR products were single-stranded
date, the store and the sample, was added to each sample, along with the sequenced with BigDye Terminator v3.1 (Life Technologies, USA)
code for the reference photos. The tweezers then were disinfected with cycle sequencing methodology, on an ABI3500 Genetic Analyzer
100 % alcohol for next use. During the sampling, the research team was (Applied Biosystems, USA), using the same primers as for PCRs (a for­
collecting all the required information from the fishmongers including ward primer for each gene).
the capture location, the fishing gear and the depth when possible. Sequences were manually checked and proofread in BioEdit v7.2.6
Additionally, the information about the town, the date, the number of software (Hall, 1999). In addition, COI sequences were translated to
samples, were also recorded. The samples were then stored in a portable check for stop codons and detect possible mitochondrial pseudogene
refrigerator containing ice during the sampling, to be transferred and presence (NUMTs). All sequences were compared with those available in
stored in a freezer at − 18 ◦ C. When the sampling effort had finished the GenBank using the standard nucleotide BLAST (blastn) against the
samples were sent for analysis into dry ice. nucleotide collection (nr/nt) database (http://blast.ncbi.nlm.nih.gov/
Blast.cgi). COI sequences were also compared to the BOLD database
(Species Level Barcode Records, http://www.boldsystems.org/),
2.2. DNA analysis through the Identification System (ID’s), for verification purposes. For
both databases, the identity threshold was set at 99 % and sequences
Genomic DNA was extracted from approximately 30 mg of fin or with high homology (≥99 %) were considered as acceptable and
wing clip tissue, preserved in 100 % ethanol, using the PureLink™ consequently registered (see GenBank Accession Numbers in Appendix,
Genomic DNA Mini kit (Invitrogen, USA). The quality and quantity of Table A1), whereas sequences below the threshold were disregarded
the isolated DNA were checked using a NanoDrop™ 1000 Spectropho­ from further analysis.
tometer (Thermo Fisher Scientific, USA). Subsequently, the DNA con­
centration of each sample was adjusted to 20 ng/μl through dilution
with ultrapure water and arrayed into 96-well PCR plates. Two different 2.3. Metal/metalloid extraction and analysis
mitochondrial (mtDNA) genes (16S rRNA and COI) were selected as
targets for the analysis, as both markers have been repeatedly used in Wet samples of muscle tissue were weighed (0.5 ± 0.14 g) using an
fish DNA barcoding studies, as they produce reliable amplification and analytical balance (Mettler Toledo) and then mineralized in a micro­
sequencing (Palumbi, 1996; Ivanova et al., 2007). A universal primer wave oven (Ethos, TC, Milestone), equipped with Teflon vessels. We
pair (16SH: 5′ -CCGGTCTGAACTCAATCACG-3′ , 16SL: 5′ - performed an acid digestion with 8 mL of 67 % superpure nitric acid
CGCCTGTTTAACAAAAACAT-3′ ) was used for the amplification of a (HNO3; Carlo Erba, Italy) for 30 min at 80 ◦ C. After acid digestion, the
600 bp fragment from the mtDNA 16S rRNA gene (Palumbi, 1996). contents of the vessels were decanted into Falcon tubes and double-
Cycling conditions consisted of an initial denaturation step of 94 ◦ C for 3 distilled water (Merck, USA) was added to the samples up to 50 mL.
min followed by 30 cycles of 94 ◦ C for 50 s, 50 ◦ C for 50 s and 72 ◦ C for 10 mL from each digested sample was taken for analysis, and 50 μg/L of
50 s, with a final extension at 72 ◦ C for 5 min. A universal primer pair internal standard (Yttrium, Y, and Rhenium, Re, 1000 mg/l Merck, USA)
(FishF2: 5′ -TCGACTAATCATAAAGATATCGGCAC-3′ , FishR2: 5′ -ACTT­ was added as internal quality control. The quantification of Arsenic (As),
CAGGGTGACCGAAGAATCAGAA-3′ ) was also used for the amplification Cadmium (Cd), Cobalt (Co), Copper (Cu), Mercury (Hg), Manganese
of a 670 bp segment of the mtDNA COI gene (Ward et al., 2005). The (Mn), Nickel (Ni), Lead (Pb), Antimony (Sb), Selenium (Se), Vanadium
PCR cycling conditions included an initial denaturation at 95 ◦ C for 2 (V), Zinc (Zn) performed with an Inductively Coupled Plasma – Mass
min, followed by 35 cycles at 94 ◦ C for 30 s, 53 ◦ C for 30 s, 72 ◦ C for 1 Spectrometer (ICP-MS) Elan-DRC-e (Perkin Elmer, USA).
min, and a final extension at 72 ◦ C for 10 min. For both genes, PCR was Concentrations were determined using standard solutions prepared
performed in 10 μL volumes containing 8 μL (1×) of OneTaq® 2× in the reagent matrix. Standards for the instrument calibration were
Master Mix (New England BioLabs, USA), 0.5 μL of each forward and prepared based on mono element certified reference solution ICP Stan­
reverse primer (0.5 μM each) and 1 μL of template DNA (20 ng). dard (Merck, USA). A seven-level calibration line was prepared from the
Negative controls were included in all PCR runs. A volume of 3 μL of concentration of 0.5 μg/L to the concentration of 100 μg/L. The samples
each PCR product was electrophoresed on a 1.5 % agarose gel stained were read as they were. If the determined concentrations exceeded the
with Midori Green DNA stain (NIPPON Genetics Europe, Germany). The last calibration point, they were diluted accordingly with double-
quality and quantity of the fragments were checked against a FastGene distilled water.
100 bp DNA Ladder (NIPPON Genetics Europe, Germany). When PCR For the quality control, a sample for each batch of mineralization was
products showed a clear and single band of the correct expected length, processed in duplicates; one was spiked with a multi-element solution of

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I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

25 mg/L with 50 μg/L (equivalent to 5 mg/kg in 0.5 g of sample) and we market since Thessaloniki is the second largest town in Greece and re­
obtained a mean recovery of 79.3–123 % for all the metals/metalloids. ceives fish products from all over the country and beyond. Thus, in
The method detection limit (LOD) was calculated based on the 40 several cases, fishmongers were not certain about the capture location,
CFR 136, EPA procedure for digested analytical blanks using the or in some cases even if the fish was imported. For Volos, we did not
following equation: manage to develop trustful relationships with fishmonger and although
they were willing to facilitate samples, they never showed us any invoice
LOD = t(df = n − 1, p = 0.99%) × SD (1)
that will allow us to see the fishing boat details. Therefore, the only
where t is the one-tailed Student’s t distribution, df is the degrees of sample collected was a specimen that was sold from a local fisher to a
freedom, n is the number of blank replicates, p is the probability and SD fishmonger when landing monitoring was performed by the research
is the standard deviation. Limit of detection (LOD) mg/Kg wet weight team of the project. Amplification and sequencing were successful for all
(w.w.) calculated for the metals/metalloids analysed are the following: DNA samples. In most samples (N = 46), amplification of the COI and
As (0.077), Cd (0.006), Co (0.002), Cu (0.0111), Hg (0.013), Mn (0.027), 16S rRNA genes provided sufficient resolution for identifying the sam­
Ni (0.002), Pb (0.004), Sb (0.009), Se (0.088), V (0.006) and Zn (0.015). ples to the species level (Appendix, Table A1), while in three cases,
For the statistical elaboration, LOD/2 was applied. species were identified as Dasyatis sp. (one case) and as Squalus blain­
ville/Squalus acanthias/Squalus megalops (two cases). All identification
results for COI marker were verified through sequence comparisons
2.4. Risk from consumption using the BOLD database. Eight different species were identified, with
Raja radula Delaroche, 1809 being the most reported (n = 17) followed
The daily intake of an element from food consumption is dependent by Dasyatis pastinaca (Linnaeus, 1758), Myliobatis aquila (Linnaeus,
on the element concentration in food, the amount of food consumed and 1758) (both n = 11), R. clavata Linnaeus, 1758 (n = 4), Dasyatis torto­
the body weight. To understand if the food can pose a risk to human nesei Capapé, 1975 (n = 2) and Aetomylaeus bovinus (Geoffroy Saint-
health derived from consumption, the first step in risk analysis is to Hilaire, 1817) (n = 1). While Hexanchus griseus (Bonnaterre, 1788) (n
compare the estimated daily intake (EDI) with the reference dose (RfDo) = 3) and Centrophorus cf. uyato (n = 1) were identified visually because
set by US-EPA. the whole specimens were retrieved.
The Estimated Daily Intake (EDI) (μg/kg b.w.) is calculated based on
the following formula: 3.1. Risk from consumption

EDI = (C × IR)/BW (2)

Concentrations of the selected metallic trace elements in the muscles
of the elasmobranchs are expressed in mg/kg (ww) and presented per
where C is the concentration of trace elements in the edible fish tissue
species in Table 1, and compared with (i) Commission Regulation (EC)
(μg/Kg w.w.), IR the daily average consumption of the fish in the study
No 629/2008 and (ii) FAO (1983) limits. These numbers vary between
area 8.74 Kg/capita/day (FAO 2013) and BW is the body weight. For
the samples of different species. Lead (Pb) was found to exceed the
this calculation we used the average weight of adult and child (70 Kg
maximum level (0.30 mg/kg) set by the Commission Regulation (EC) No
and 16 Kg respectively).
1881/2006 in 25 samples which refer to three species, namely
The Target Hazard Quotient (THQ) is a complex parameter used for
D. pastinaca, D. tortonesei and R. radula. Regarding Mercury (Hg), in 6
the estimation of potential health risks associated with long term
cases the samples exceeded the maximum level of 1 mg/kg set by
exposure to chemical pollutants. In this work it was used to determine
Commission Regulation (EC) No 629/2008 with 4 (out of the 6) cases
health risks from the consumption of elasmobranch products. THQ value
referring to 4 samples of shark species; H. griseus (n = 3) and C. uyato (n
below 1 means that the level of exposure is smaller than the reference
= 1). The latter, bio-accumulated 11.1 mg/kg, far exceeding the
dose, which assumes that a daily exposure at this level is not likely to
allowable levels. While, in none of the samples, Cd exceed the maximum
cause any pernicious effects during lifetime for the human population. In
level (0,050 mg/kg) set by the Commission Regulation (EC) No 629/
other words, a THQ below 1 means the adverse effects are negligible.
2008.
The THQ value was determined for both adults and children by the
The comparison between EDI and Reference Dose (RfDo) doesn’t
following formula (US-EPA, 2000):
highlight any concern because all the value are below the maximum
THQ = (C × IR × EF × ED)/(RfD × BW × AT) (3) acceptable oral daily dose (Table 2).
The lifetime risk evaluated with THQ values for investigated metals
where C is the metal concentration (mg/kg), IR is the food ingestion rate and elasmobranchs are presented in Table 3. Individual THQ values
(0.00874 Kg/(capita * day) for Greek consumers, according to FAOSTAT surpass 1 only for As and Hg. Arsenic is higher than 1 for A. bovinus (for
2013), EF is the exposure frequency (350 days/year), ED is the exposure children) and R. clavata (for children) while Hg for R. clavata (for chil­
duration (26 years for adult and 6 years for child, according to Copat dren), H. griseus (for adults and children), C. uyato (for adults and chil­
et al., 2013), RfDo is the oral reference dose (mg/kg b.w. day), BW is the dren) and A. bovinus (for adults and children) (Table 3).
average body weight (70 kg for adults and 16 Kg for child, according to Especially for A. bovinus, C. uyato and Dasyatis sp. the findings cannot
Copat et al., 2013), AT is the average exposure time for noncarcinogens be considered conclusive given that one sample from only one specimen
(350 days/year x ED). The following RfDo (mg/Kg/day) were used; As was analysed.
= 0.0003 (set for inorganic As), Cd = 0.001, Co = 0.0003, Cu = 0.04, Hg
= 0.0001 (set for MeHg), Mn = 0.14, Ni = 0.011, Pb = 0.0035, Sb = 4. Discussion
0.0004, Se = 0.005, V = 0.0054, Zn = 0.3 (US-EPA, IRIS, 2017; FAO/
WHO 2004). This is the first study that presents the levels of 12 trace elements in
The EDI and THQ value for As was calculated assuming the inorganic the edible tissues of 10 different elasmobranch species caught in the
form as the 3 % of the total, being a pelagic fish (Ferrante et al., 2019). Greek waters. Up to date, only two studies exist regarding the concen­
tration of trace elements in the tissues of elasmobranchs in Greece. One
3. Results focused on the concentration of the mercury level in the tissues of the
spiny dogfish (Squalus acanthias) and the smooth-hound sharks (Mustelus
A total number of 53 specimens were collected, 27 from Alexan­ mustelus), with all specimens caught in Crete (South Greece) (Kousteni
droupoli, 21 from Kavala, 4 from Thessaloniki and 1 from Volos. The low et al., 2006) and the second on the concentration 27 metals and elements
sample number from Thessaloniki is related to the complexity of the fish in the tissues of 28 marine fish, which included one elasmobranch

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 I. Giovos et al.
Table 1
Average concentration (and standard deviation) of selected trace elements concentration (expressed in μg/g w.w.) in the muscle tissue of different elasmobranch species. In bold italics are given all values surpassing the
total allowable limits based on (i) Commission Regulation (EC) No 629/2008 and (ii) FAO. (1983).
Trace elements A. bovinus (N.1) C. cf. uyato (N. 1) D. pastinaca (N.11) Dasyatis sp. (N.1) D. tortonesei (N.2) H. griseus (N.3) M. aquila (N.11) R. clavata (N.4) R. radula (N.17) Squalus spp. (N.2)

As Average ± S. 32.546 25.989 5.629 ± 5.397 0.749 14.301 ± 7.583 10.845 ± 3.194 10.300 ± 6.299 38.158 ± 41.180 5.874 ± 1.986 1.879 ± 0.675
D.
Range 18.162–1.331 8.939–19.662 13.502–7.302 22.963–4.213 90.352–2.483 9.551–2.744 2.356–1.402
Cd Average ± S. 0.022 0.019 0.003 ± 0.000 0.008 0.003 ± 0.000 0.007 ± 0.004 0.009 ± 0.012 0.004 ± 0.002 0.005 ± 0.005 0.003 ± 0.000
D.
Range 0.003 0.003 0.010–0.003 0.043–0.003 0.007–0.003 0.021–0.003 0.003
Co Average ± S. 0.004 0.003 0.014 ± 0.006 0.007 0.011 ± 0.006 0.006 ± 0.003 0.020 ± 0.013 0.008 ± 0.005 0.009 ± 0.003 0.011 ± 0.006
D.
Range 0.025–0.003 0.016–0.007 0.008–0.002 0.042–0.006 0.015–0.004 0.014–0.010 0.015–0.006
Cu Average ± S. 0.638 0.323 0.463 ± 0.101 0.491 0.414 ± 0.041 0.357 ± 0.077 0.488 ± 0.104 0.460 ± 0.239 0.302 ± 0.073 0.436 ± 0.058
D.
Range 0.616–0.265 0.384–0.443 0.434–0.281 0.663–0.335 0.809–0.266 0.452–0.213 0.477–0.395
Hg Average ± S. 2.737 11.094 0.081 ± 0.082 0.023 0.151 ± 0.135 1.983 ± 0.588 0.116 ± 0.129 0.600 ± 0.704 0.023 ± 0.007 0.032 ± 0.005
D.
Range 0.303–0.016 0.246–0.056 2.579–1.403 0.473–0.036 1.501–0.027 0.044–0.017 0.036–0.028
Mn Average ± S. 0.174 0.154 1.184 ± 0.645 1.333 0.424 ± 0.148 0.231 ± 0.052 1.482 ± 1.510 1.071 ± 0.608 1.111 ± 0.384 1.235 ± 0.895
5

D.
Range 2.373–0.161 0.528–0.319 0.263–0.172 4.244–0.166 1.854–0.381 1.806–0.418 1.868–0.602
Ni Average ± S. 0.190 0.072 0.166 ± 0.055 0.138 0.074 ± 0.048 0.040 ± 0.017 0.100 ± 0.057 0.153 ± 0.114 0.160 ± 0.044 0.131 ± 0.095
D.
Range 0.247–0.080 0.108–0.040 0.059–0.029 0.246–0.017 0.320–0.069 0.243–0.070 0.199–0.064
Pb Average ± S. 0.035 0.088 0.392 ± 0.179 0.170 1.008 ± 1.082 0.192 ± 0.082 0.211 ± 0.238 0.109 ± 0.100 0.644 ± 0.518 0.297 ± 0.290
D.
Range 0.703–0.176 1.774–0.243 0.102–0.263 0.823–0.022 0.226–0.024 1.742–0.158 0.503–0.092
Sb Average ± S. 0.016 0.005 0.016 ± 0.033 0.010 0.0180 ± 0.011 0.005 ± 0.000 0.026 ± 0.040 0.032 ± 0.051 0.015 ± 0.024 0.005 ± 0.000
D.
Range 0.115–0.005 0.026–0.010 0.005 0.112–0.005 0107–0.005 0.107–0.005 0.005
Se Average ± S. 0.799 3.297 0.792 ± 0.160 0.650 0.689 ± 0.088 0.382 ± 0.101 0.648 ± 0.249 0.554 ± 0.132 0.301 ± 0.042 0.726 ± 0.131
D.
Range 1.109–0.547 0.751–0.626 0.499–0.319 1.067–0.362 0.708–0.393 0.411–0.247 0.818–0.634
V Average ± S. 0.038 0.027 0.031 ± 0.010 0.020 0.041 ± 0.026 0.029 ± 0.010 0.044 ± 0.021 0.019 ± 0.006 0.046 ± 0.019 0.030 ± 0.002
D.

Marine Pollution Bulletin 184 (2022) 114129

Range 0.053–0.020 0.060–0.023 0.035–0.018 0.093–0.013 0.028–0.015 0.098–0.020 0.031–0.634
Zn Average ± S. 8.685 5.509 8.046 ± 1.546 9.037 5.213 ± 0.310 5.923 ± 0.979 8.386 ± 1.439 6.628 ± 1.531 7.019 ± 1.179 10.026 ± 0.680
D.
Range 10.441–4.812 5.432–4.994 7.046–5.250 10.060–6.359 8.622–4.936 9.808–5.393 10.507–9.546
I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

Table 2
Estimated daily intake (EDI μg/kg b.w.) based on the average daily pelagic fish intake for individual metals/metalloids calculated for adults and children; with bold
italics EDI values surpassing (RfDo μg/kg b.w).
Trace EDI (μg Squallus R. radula R. clavata M. aquila H. griseus D. tortonesei Dasyatis D. pastinaca C. uyato A. bovinus RfDo
elements /Kg b. spp. spp. (μg/kg
w.) b.w.)

Asx Adult 7.04E− 03 2.20E− 02 1.43E− 01 3.86E− 02 4.06E− 02 5.36E− 02 3.67E− 02 2.11E− 02 9.74E− 02 1.22E− 01 0.3
Child 3.08E− 02 9.62E− 02 6.25E− 01 1.69E− 01 1.78E− 01 2.34E− 01 1.60E− 01 9.22E− 02 4.26E− 01 5.33E− 01 0.3
Cd Adult 3.75E− 04 6.54E− 04 5.05E− 04 1.08E− 03 8.65E− 04 3.75E− 04 5.88E− 04 3.75E− 04 2.39E-03 2.77E− 03 1
Child 1.64E− 03 2.86E− 03 2.20E− 03 4.74E− 03 3.78E− 03 1.64E− 03 2.57E− 03 1.64E− 03 1.04E− 02 1.21E− 02 1
Co Adult 1.36E− 03 1.15E− 03 9.73E− 04 2.45E− 03 7.17E− 04 1.37E− 03 1.23E− 03 1.70E− 03 3.65E− 04 4.74E− 04 0.3
Child 5.96E− 03 5.04E− 03 4.20E− 02 1.07E− 02 3.10E− 03 6.01E− 03 5.39E− 03 7.40E− 03 1.60E− 03 2.07E− 03 0.3
Cu Adult 5.40E− 02 3.80E− 02 5.70E− 02 6.10E− 02 4.50E− 02 5.17E− 02 5.50E− 02 5.80E− 02 4.00E− 02 8.00E− 02 40
Child 2.38E− 01 1.65E− 01 2.51E− 01 2.66E− 01 1.95E− 01 2.26E− 01 2.40E− 01 2.53E− 01 1.77E− 01 3.48E− 01 40
Hg Adult 4.00E− 03 3.00E− 03 7.50E− 02 1.50E− 02 2.48E− 01 1.89E− 02 1.40E− 02 1.00E− 02 1.39E+00 3.42E− 01 0.1
Child 1.70E− 02 1.26E− 02 3.28E− 01 6.36E− 02 1.08E+00 8.25E− 02 5.90E− 02 4.45E− 02 6.06E+00 1.50E+00 0.1
Mn Adult 1.54E− 01 1.39E− 01 1.34E− 01 1.85E− 01 2.90E− 02 5.29E− 02 9.10E− 02 1.48E− 01 1.90E− 02 2.20E− 02 140
Child 6.75E− 01 6.07E− 01 5.85E− 01 8.10E− 01 1.26E− 01 2.32E− 01 3.97E− 01 6.47E− 01 8.41E− 02 9.49E− 02 140
Ni Adult 1.60E− 02 2.00E− 02 1.90E− 02 1.20E− 02 5.00E− 03 9.24E− 03 1.20E− 02 2.10E− 02 9.00E− 03 2.40E− 02 11
Child 7.18E− 02 8.73E− 02 8.33E− 02 5.45E− 02 2.18E− 02 4.04E− 02 5.22E− 02 9.05E− 02 3.90E− 02 1.04E− 01 11
Pb Adult 3.70E− 02 8.00E− 02 1.40E− 02 2.60E− 02 2.40E− 02 1.26E− 01 9.10E− 02 4.90E− 02 1.10E− 02 4.00E− 03 3.5
Child 1.62E− 01 3.52E− 01 5.93E− 02 1.15E− 01 1.05E− 01 5.51E− 01 3.98E− 01 2.14E− 01 4.82E− 02 1.91E− 02 3.5
Sb Adult 5.62E− 04 1.92E− 03 3.93E− 03 3.27E− 03 5.62E− 04 2.25E− 03 1.91E− 03 2.05E− 03 5.62E− 04 1.94E− 03 0.4
Child 2.40E− 03 8.30E− 03 1.72E− 02 1.43E− 02 2.46E− 03 9.83E− 03 8.37E− 03 8.90E− 03 2.46E− 03 8.48E− 03 0.4
Se Adult 9.10E− 02 3.80E− 02 6.90E− 02 8.10E− 02 4.80E− 02 8.60E− 02 8.40E− 02 9.90E− 02 4.12E− 01 1.00E− 01 5
Child 3.96E− 01 1.65E− 01 3.03E− 01 3.54E− 01 2.09E− 01 3.76E− 01 3.69E− 01 4.32E− 01 1.80E+00 4.36E− 01 5
V Adult 4.00E− 03 6.00E− 03 2.00E− 03 6.20E− 02 4.00E− 03 5.12E− 03 4.00E− 03 4.00E− 03 3.00E− 03 5.00E− 03 5.4
Child 1.64E− 02 2.52E− 02 1.04E− 02 2.45E− 02 1.59E− 02 2.24E− 02 1.86E− 02 1.68E− 02 1.50E− 02 2.09E− 02 5.4
Zn Adult 1.25E+00 8.76E− 01 8.27E− 01 1.05E+00 7.40E− 01 6.51E− 01 8.10E− 01 1.01E+00 6.88E− 01 1.08E+00 300
Child 5.48E+00 3.83E+00 3.62E+00 4.58E+00 3.24E+00 2.85E+00 3.54E+00 4.40E+00 3.01E+00 4.74E+00 300
x
The value of EDI was calculated for Inorganic Arsenic by assuming 3 % of the total.

Table 3
Target Hazard Quotients (THQs) for individual metals/metalloids caused by the consumption of different elasmobranch species calculated for adults and children; in
bold italics THQ values surpassing 1.
Trace elements THQ Squalus spp. R. radula R. clavata M. aquila H. griseus D. tortonesei Dasyatis spp. D. pastinaca C. cf. uyato A. bovinus

Asx Adult 2.35E− 02 7.33E− 02 4.76E− 01 1.29E− 01 1.35E− 01 1.79E− 01 1.22E− 01 7.03E− 02 3.25E− 02 4.04E− 01
Child 1.03E− 01 3.21E− 01 2.08E+00 5.63E− 01 5.92E− 01 7.81E− 01 5.34E− 01 3.08E− 01 1.42E− 01 1.77E+00
Cd Adult 3.75E− 04 6.54E− 04 4.99E− 04 1.09E− 03 8.74E− 04 3.75E− 04 5.83E− 04 3.75E− 04 2.37E− 03 2.75E− 03
Child 1.64E− 03 2.86E− 03 2.19E− 03 4.77E− 03 3.82E− 03 1.64E− 03 2.55E− 03 1.64E− 03 1.04E− 02 1.20E− 02
Co Adult 4.37E− 03 3.79E− 03 3.12E− 03 8.21E− 03 2.36E− 03 4.58E− 03 4.16E− 03 5.71E− 03 1.25E− 03 1.66E− 03
Child 1.91E− 02 1.66E− 02 1.37E− 02 3.59E− 02 1.03E− 02 2.00E− 02 1.82E− 02 2.50E− 02 5.46E− 03 7.28E− 03
Cu Adult 1.36E− 03 9.42E− 04 1.44E− 03 1.52E− 03 1.11E− 03 1.29E− 03 1.37E− 03 1.45E− 03 1.01E− 03 1.99E− 03
Child 5.95E− 03 4.12E− 03 6.28E− 03 6.67E− 03 4.87E− 03 5.65E− 03 6.00E− 03 6.33E− 03 4.41E− 03 8.71E− 03
Hg Adult 4.00E− 02 2.91E− 02 7.49E− 01 1.45E− 01 2.48E+00 1.89E− 01 1.36E− 01 1.02E− 01 1.39E+01 3.42E+00
Child 1.75E− 01 1.27E− 01 3.28E+00 6.36E− 01 1.08E+01 8.25E− 01 5.94E− 01 4.46E− 01 6.06E+01 1.50E+01
Mn Adult 1.10E− 03 9.91E− 04 9.55E− 04 1.32E− 03 2.06E− 04 3.78E− 04 6.48E− 04 1.06E− 03 1.37E− 04 1.55E− 04
Child 4.82E− 03 4.34E− 03 4.18E− 03 5.78E− 03 9.03E− 04 1.65E− 03 2.84E− 03 4.62E− 03 6.01E− 04 6.79E− 04
Ni Adult 1.49E− 03 1.82E− 03 1.73E− 03 1.13E− 03 4.54E− 04 8.40E− 04 1.08E− 03 1.88E− 03 8.17E− 04 2.16E− 03
Child 6.53E− 03 7.95E− 03 7.57E− 03 4.96E− 03 1.99E− 03 3.67E− 03 4.73E− 03 8.23E− 03 3.58E− 03 9.44E− 03
Pb Adult 1.06E− 02 2.30E− 02 3.86E− 03 7.54E− 03 6.85E− 03 3.60E− 02 2.60E− 02 1.40E− 02 3.14E− 03 1.25E− 03
Child 4.64E− 02 1.00E− 01 1.69E− 02 3.30E− 02 3.00E− 02 1.57E− 01 1.14E− 01 6.12E− 02 1.37E− 02 5.46E− 03
Sb Adult 1.56E− 03 4.83E− 03 9.91E− 03 8.26E− 03 1.56E− 03 5.62E− 03 4.79E− 03 5.22E− 03 1.56E− 03 4.99E− 03
Child 6.83E− 03 2.11E− 02 4.34E− 02 3.61E− 02 6.83E− 03 2.46E− 02 2.09E− 02 2.28E− 02 6.83E− 03 2.19E− 02
Se Adult 1.81E− 02 7.52E− 03 1.38E− 02 1.62E− 02 9.55E− 03 1.72E− 02 1.69E− 02 1.98E− 02 8.23E− 02 2.00E− 02
Child 7.93E− 02 3.29E− 02 6.06E− 02 7.09E− 02 4.18E− 02 7.53E− 02 7.38E− 02 8.65E− 02 3.60E− 01 8.73E− 02
V Adult 7.43E− 04 1.14E− 03 4.77E− 04 1.11E− 03 7.18E− 04 1.02E− 03 8.51E− 04 7.61E− 04 6.69E− 04 9.41E− 04
Child 3.25E− 03 5.00E− 03 2.09E− 03 4.87E− 03 3.14E− 03 4.44E− 03 3.72E− 03 3.33E− 03 2.93E− 03 4.12E− 03
Zn Adult 4.17E− 03 2.92E− 03 2.76E− 03 3.49E− 03 2.47E− 03 2.17E− 03 2.70E− 03 3.35E− 03 2.29E− 03 3.61E− 03
Child 1.83E− 02 1.28E− 02 1.21E− 02 1.53E− 02 1.08E− 02 9.49E− 03 1.18E− 02 1.47E− 02 1.00E− 02 1.58E− 02
x
The value of THQ was calculated for Inorganic Arsenic by assuming 3 % of the total.

(Torpedo torpedo) (Vetsis et al., 2021). The only relevant bibliography been listed with “moderate scientific effort” on the topic, while studies
regarding the risk from the consumption of elasmobranchs for the Greek from the eastern Mediterranean are absent (Tiktak et al., 2020). Our
consumers, is the work of Mol et al. (2018) that estimated the risk for the work aims to contribute towards this end, providing baseline informa­
Greek and the Turkish consumers based on specimens collected from the tion about the levels of selected trace elements in different elasmo­
Sea of Marmara. The increasing demand for shark meat globally (Nie­ branch species and highlighting the risks from their consumption. Our
dermüller et al., 2021), the high levels of mislabelling (Dulvy et al., results are in accordance with the findings of Mol et al. (2018) for the
2017) and fraudulent sales, stresses the need for more studies that focus Greek consumers but also with other studies, reporting the risk from the
on the health risks from consumption of elasmobranchs. Based on the consumption of elasmobranchs (e.g. Storelli et al., 2011; Adel et al.,
latest global review on the topic (Tiktak et al., 2020), only a small 2016; Adel et al., 2018; Kim et al., 2019; Boldrocchi et al., 2019, 2021;
number of studies from the Mediterranean Sea exist, thus the basin has Barcia et al., 2020) and in general of marine apex predators (e.g. Araújo

6
I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

and Cedeño-Macias, 2016; Di Bella et al., 2020) and bony fish (Barone described that pose a serious threat to the marine environment (Kroll
et al., 2021; Vetsis et al., 2021; Storelli et al., 2022). et al., 2005), affecting even areas with low proximity to the facilities,
The biomagnification of trace elements can be affected by several since pollutants can be transferred by the air and the waterways (Gusso-
factors (Boldrocchi et al., 2021) like the species ecology (Shipley et al., Choueri et al., 2018 and references within). Across the coast of the
2021), their diet and trophic level (Medina-Morales et al., 2020; Vetsis Thracean Sea (North Aegean), several mines exist, exploited for Au and
et al., 2021), the trophic web complexity (Wang, 2002) and the human Pb–Ag both in ancient times and during the 70s (Vavelidis et al., 1997),
activities in an area (Adel et al., 2018). In our work we detected similar with the highest concentration near the towns of Kavala and Alexan­
levels for most trace elements with the relevant global bibliography droupoli. Most of those facilities are now abandoned, and no measures
(Tiktak et al., 2020) and the relevant Greek and Mediterranean studies have been taken for disposing their runoffs, potentially resulting to high
(Kousteni et al., 2006; Storelli et al., 2010; Mille et al., 2018; Vetsis et al., levels of the trace metal in the marine environment. Although in the
2021). In general, the concentrations of Cd found in all cases to be latest assessment for Water Framework Directive (Simboura et al., 2015)
within the limits set in the Commission Regulation (EC) No 1881/2006 in all sampling locations from the Thracean Sea, trace elements were
and lower compared to the levels detected in other similar works (Tiktak found within the limits set by Directive 2008/105/EC but this refers to
et al., 2020 and all references within). However, three species only three locations (Ierissos gulf, Kavala gulf, Thassos straights) and
(A. bovinus, C. uyato and H. griseus) were found to have high Hg levels none with proximity to the abandoned mines.
and three species (P. pastinaca, D. tortonesei and R. radula) were found to DNA analysis was deemed crucial for the study as identification is­
have high Pb levels above the limits of Regulation (EC) No. 1881/2006. sues for some species exist (i.e., Dasyatis sp.), while for slices and ray
Moreover, in the analyses performed to estimate the lifetime risk from wings it would be impossible to identify otherwise, considering the
consumption, we found a high risk in highly commercialized species for extend of mislabeling in Greek markets (Giovos et al., 2021). Finally,
both adults and children with respect to Hg and for children only with considering the limited number of studies in the area, accurate trace
respect to As. concentrations in a species level will help as reference for future studies.
Mercury (Hg) occurs in deposits throughout the world and has been We must highlight the risk from the consumption of H. griseus we
widely used in a variety of devices (thermometers, barometers, ma­ estimated for Hg both for adults and children. The species is protected in
nometers etc.) and processes. Through human activities, it can reach the Greece based on Presidential Decree no 67/1981 while the species is
marine environment and once is methylated it bio-magnifies to the commonly sold illegally in the fish markets of the country (Giovos et al.,
upper trophic levels (Copat et al., 2012; Pappalardo et al., 2017; Storelli 2020), mislabeled as “Galeos” that based on the Ministerial Decision No
et al., 2022) Mercury is listed as the third most toxic substance in the 1750/32219/2015 must refers to Mustelus spp. “Galeos” is generally
Substance Priority List (Agency for Toxic Substances and Disease Reg­ promoted in Greece as a cheap alternative to hake and a quality food
istry (ATSDR), 2019), with symptoms from exposure varying, depending especially for children due to the lack of bones. Thus, consumers
on the type, dose, method, and duration of exposure, including muscle remained unprotected from the lack of law enforcement and the erro­
weakness, poor coordination, numbness in the hands and feet, skin neous mislabeling of a protected species (Giovos et al., 2020) with direct
rashes, anxiety, memory problems, trouble speaking, trouble hearing, implications to their health.
trouble seeing (Bernhoft, 2012) to even disruption of the fetus devel­ Nevertheless, the accumulation of trace elements has a negative ef­
opment (Caserta et al., 2013). It is important to highlight that an fect on the health of elasmobranch species too (Depew et al., 2012;
increased risk for both adults and children was found when consuming Bezerra et al., 2019; Merly et al., 2019; Tiktak et al., 2020; Wosnick
some of the most commonly traded elasmobranch species in Greece, et al., 2021). Few studies have evaluated the effects from the contami­
such as R. clavata (Giovos et al., 2021), exceeding the mercury limit nation of specific trace elements on elasmobranch species health,
before reaching the recommended amount of omega-3 fatty acids that highlighting the provoked implications in osmoregulation (e.g. De
humans can ingest after consumption (Smith and Guentzel, 2010). Boeck et al., 2010) or in cellular and fluid composition (e.g. Ballatori and
For arsenic (As) we estimated high lifetime risk from the consump­ Boyer, 1996; De Boeck et al., 2010). In the case of Pb, its accumulation in
tion of 2 species when children are exposed to long term consumption the gills, rectal gland, muscle and liver of elasmobranchs affect directly
(Table 3). The element is widespread in the aquatic ecosystems, occur­ the osmoregulation system of the species, their respiratory capacity and
ring naturally but also through human activities (Ferrante et al., 2019). metabolism (Eyckmans et al., 2013). While Hg has related to alternation
The element is listed as the most toxic substance in the Substance Pri­ in the reproductive biology of the species (Bezerra et al., 2019). How­
ority List (Agency for Toxic Substances and Disease Registry (ATSDR), ever, there are currently no toxic thresholds for tolerable concentrations
2019) and increased level in the human organism can increase skin, of metals and other elements in elasmobranchs (Merly et al., 2019),
lung, liver and bladder cancer, as well as cause the occurrence of arterial while their impacts on the health and the well-being of elasmobranchs is
vascular disease (Sakuma et al., 2010 and references within). Again, it is a poorly known and heavily understudied (Consales and Marsili, 2021).
important to note that R. clavata is highly commercial in the country
(Giovos et al., 2021), however, we must highlight that the number of 5. Conclusions
samples for several species in our study is limited and further research is
required to investigate the bioaccumulated concentrations. Our work provides a preliminary information for the levels of
Lead (Pb), is a naturally occurring metal present in small amounts in selected trace elements in the edible tissues of different elasmobranch
the earth’s crust. Anthropogenic activities such as fossil fuels burning, species caught in the North Aegean Sea. A national scale effort is
mining, and manufacturing contribute to the release of high concen­ required to better understand the level of this phenomenon and the risks
trations. In high levels, Pb is the most systemic toxicant that affects that consumers might be facing from the consumption of elasmobranchs.
several organs in the body including the kidneys, liver, central nervous Relevant work must be conducted also for imported products given that
system, hematopoetic system, endocrine system, and reproductive sys­ Greece is one of the main importers of elasmobranchs products in
tem of both adults and children (Wani et al., 2015) and for this reason is Europe (Giovos et al., 2020), also from countries outside the European
listed as the second most toxic substance in the Substance Priority List Union that are not under the EU legislative framework. An important
(Agency for Toxic Substances and Disease Registry (ATSDR), 2019). The step towards this direction could be done through the establishment of
samples with high levels of Pb were detected in specimens caught in the an adequately staffed and equipped management authorities that would
area of Kavala and close to Evros Delta (Fig. 1). Bio-accumulation of ensure the proper implementation and enforcement of the EU
trace elements is highly related to their existence and concentration in legislations.
the marine environment, which to a large extent is a result of human Further research and a dedicated sampling design is required for
activities in an area (Adel et al., 2018), while mining activities is well understanding better the potential influence of mines in the

7
I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

concentration of trace elements in the North Aegean and in the tissues of Declaration of competing interest
different marine species. This will help to alert the local and national
authorities in an effort to take appropriate measures. Ioannis Giovos reports financial support was provided by OceanCare.
Last, no conclusion can be derived about the health implications that Ioannis Giovos reports financial support was provided by Shark Foun­
elasmobranch may face in the studied area from the bioaccumulation of dation/Hai Stiftung. The authors have no conflict of interest to declare.
trace elements, but we suggest that this threat should be considered and
explored more in the near future for these species. Data availability

CRediT authorship contribution statement Data will be made available on request.

Each author has participated sufficiently in the work to take public Acknowledgements
responsibility for the content of the paper and approved the final version
of the manuscript. We would like to warmly thank all fishers, traders, and fishmongers
1. Conception and design of the study: Ioannis Giovos, Chiara for the great collaboration and the Central Markets and Fishery Orga­
Copat, Maria Violetta Brundo, Francesco Tiralongo nizations (CMFO SA) for granting us access to all facilities in the North
2. Generation, collection, assembly, analysis and/or interpretation Aegean. In addition, we would like to thank OceanCare and Shark
of data: Ioannis Giovos, Nikolaos Doumpas, Roxani Naasan Aga Spyr­ Foundation/Hai Stiftung (Grant number: 2019-01-04/1) for funding this
idopoulou, Dimitrios Loukovitis, Zoi Kazlari, Maria Papapetrou, Athina work, which was conducted in the context of the PhD thesis of IG in the
Papadopoulou, Francesco Tiralongo, Chiara Copat, Maria Violetta University of Patras in collaboration with the University of Padova
Brundo, Dimitrios K. Moutopoulos under the research permit 158976/1756 from the Hellenic Ministry of
3. Drafting or revision of the manuscript: Ioannis Giovos, Chiara Environment and Energy.
Copat, Dimitrios K. Moutopoulos
4. Approval of the final version of the manuscript: all co authors

Appendix A
Table A1
Identified species using COI and 16S rRNA genes with GenBank Accession Numbers.

Species COI gene 16S gene

1 Dasyatis tortonesei MW535732

2 Myliobatis aquila MW535741 MW543943
3 Dasyatis pastinaca MW549014-MW549015 MW543449
4 Aetomylaeus bovinus MW549016-MW549017
5 Raja clavata MW549018-MW549019
6 Raja radula MW549004-MW549007

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