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
Persistent organic pollutants (POPs), metals, crude oil and marine
debris (e.g. marine litter or microplastics) represent the most common
marine pollutants globally (United Nations Environment Programme,
2021). The last decades there has been an increasing concern for the
effects of various pollutants on the marine environment, on marine or­
ganisms and subsequently on humans (e.g., Pourang, 1995; Kumar et al.,
2019; Salvaggio et al., 2019; Tiktak et al., 2020). As a result of various
anthropogenic activities such as mining, industry, agriculture etc.
(WHO, 2020), these pollutants enter and accumulate in the marine
environment, bio-accumulate in marine organisms and bio-magnifying
towards the upper levels of the marine trophic webs, including
humans. Among those, trace elements, have been identified as
significant stressors for the marine biota (Scheuhammer et al., 2015;
Ehnert-Russo & Gelsleichter, 2020), hindering as well potential risks for
humans (Barone et al., 2021), given that marine organisms are an
important source of protein for humanity (Dent and Clarke, 2015).
Compared to other marine vertebrates, elasmobranchs are consid­
ered very prone to biomagnification of trace elements (Mull et al., 2012;
Boldrocchi et al., 2021) due to their life characteristics. The majority of
the elasmobranchs are considered top to meso-predators (Heupel et al.,
2014) and are long-living species. This makes them particularly sus­
ceptible to contamination, especially of pollutants like trace elements.
Studies have shown that trace elements like Hg and Cd, can alter their
reproductive physiology, decrease, and alter the development of em­
bryos and pups and create alterations in their physiology (Tiktak et al.,
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: ioannis.giovos@isea.com.gr (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.
relation to their conservation status, is heavily understudied for elas­
mobranchs (Consales and Marsili, 2021), although they are among the
most threatened marine taxa globally with the most recent global IUCN
Red List of Threatened Species assessment estimated that 37% of the
species are threatened with extinction (Dulvy et al., 2021).
At the same time the demand for elasmobranch products is rising
(Dent and Clarke, 2015) as a result of (i) commercial fish stock over­
exploitation, (ii) the emerging demand for seafood and protein, and (iii)
the increasing finning regulations that are suspected to stimulate the
creation of new shark meat markets (Dent and Clarke, 2015). Only for
2011, global shark fisheries estimated around 1$ US billion with an
increasing trend, excluding the value of elasmobranch products
consumed domestically, which may comprise the majority of the catches
(Dulvy et al., 2017). In the Mediterranean, elasmobranch catches
represent only 1 % of the total landings (Statistic FAO 1980–2015), with
most of the species landed as valuable bycatch that are consequently
sold, primarily, domestically (Bradai et al., 2018; Touloupaki et al.,
2020). In Greece, the contribution of elasmobranchs to the total annual
Greek landings between 1990 and 2020 is <2 % (around 1000 tn
annually) (HELSTAT, 1990–2021). However, the country is among the
top elasmobranch markets of South Europe, with significant imports
from Italy and Turkey (Giovos et al., 2020), although the country’s
seafood per capita consumption remains still low (18.2 kg per capita/
year), significantly below the European average (EUMOFA, 2017). Thus,
elasmobranch products can be found in all fish markets, especially of
North Greece, with Greeks consuming a variety of elasmobranch species,
with Raja spp., Mustelus spp., and Dasyatis spp., being the most common
(Giovos et al., 2021). Like elsewhere (Bornatowski et al., 2013), mis­
labelling and illegal trade is common throughout the season (Giovos
et al., 2021) and thus consumers often buy shark and batoid products
without intention.
In this work, the levels of 12 trace elements in edible tissues of
elasmobranchs caught in the North Aegean Sea and sold in the markets
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.)
2
Marine Pollution Bulletin 184 (2022) 114129
of Northern Greece were evaluated, with the aim to understand the risk
for consumers in an area that traditionally consumes high amounts of
such products (Giovos et al., 2021), but also to provide a baseline in­
formation about the level of concentration in different elasmobranch
species for future studies. Several indicators were applied for measuring
the risk from the consumption of different species by adults and chil­
dren. The study has been based on: (i) genetic identification of the
samples and (ii) quantitative analysis of the trace elements’ levels in the
flesh.
2. Materials and methods
2.1. Sampling
Fish market surveys took place on a monthly basis in four selected
towns for two to five days each; Alexandroupoli, Kavala, Thessaloniki,
and Volos (Fig. 1) as part of the wider research project ByElasmoCatch
(Giovos et al., 2021). Through the project, the research team developed
relationships with fishmongers in these towns that facilitated trust full
answers and access to invoices for securing that all samples were
collected from specimens that caught in the North Aegean region that
was our target area. When the capture location was not certain and/or
could not validate no sample was collected. Tissue samples of products
sold as elasmobranchs (e.g. ray wings, shark fillets etc.; Fig. 2) were
taken for genetic and metal analysis along with pictures. There were no
specific target species for the sampling nor a specific age or sex when
choosing the specimen, rather the tissue samples were obtained through
a random selection process, and every sampling day different fish stores
were selected. The samples for the genetic analysis were obtained from
the product using a single-use surgical lancet to cut at least 3 g of tissue
that then deepened in an Eppendorf with 100 % alcohol. Using the same
tools two bigger samples that weighed at least 10 g each, were carefully
cut off the product to contain as much muscle tissue as possible and were
I. Giovos et al.
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
parafilm on top. All the samples from the same product were tied
together with a duct tape and a reference code indicating the town, the
date, the store and the sample, was added to each sample, along with the
code for the reference photos. The tweezers then were disinfected with
100 % alcohol for next use. During the sampling, the research team was
collecting all the required information from the fishmongers including
the capture location, the fishing gear and the depth when possible.
Additionally, the information about the town, the date, the number of
samples, were also recorded. The samples were then stored in a portable
refrigerator containing ice during the sampling, to be transferred and
stored in a freezer at − 18 ◦ C. When the sampling effort had finished the
samples were sent for analysis into dry ice.
2.2. DNA analysis
Genomic DNA was extracted from approximately 30 mg of fin or
wing clip tissue, preserved in 100 % ethanol, using the PureLink™
Genomic DNA Mini kit (Invitrogen, USA). The quality and quantity of
the isolated DNA were checked using a NanoDrop™ 1000 Spectropho­
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
mitochondrial (mtDNA) genes (16S rRNA and COI) were selected as
targets for the analysis, as both markers have been repeatedly used in
fish DNA barcoding studies, as they produce reliable amplification and
sequencing (Palumbi, 1996; Ivanova et al., 2007). A universal primer
pair (16SH: 5′ -CCGGTCTGAACTCAATCACG-3′ , 16SL: 5′ -
CGCCTGTTTAACAAAAACAT-3′ ) was used for the amplification of a
600 bp fragment from the mtDNA 16S rRNA gene (Palumbi, 1996).
Cycling conditions consisted of an initial denaturation step of 94 ◦ C for 3
min followed by 30 cycles of 94 ◦ C for 50 s, 50 ◦ C for 50 s and 72 ◦ C for
50 s, with a final extension at 72 ◦ C for 5 min. A universal primer pair
(FishF2: 5′ -TCGACTAATCATAAAGATATCGGCAC-3′ , FishR2: 5′ -ACTT­
CAGGGTGACCGAAGAATCAGAA-3′ ) was also used for the amplification
of a 670 bp segment of the mtDNA COI gene (Ward et al., 2005). The
PCR cycling conditions included an initial denaturation at 95 ◦ C for 2
min, followed by 35 cycles at 94 ◦ C for 30 s, 53 ◦ C for 30 s, 72 ◦ C for 1
min, and a final extension at 72 ◦ C for 10 min. For both genes, PCR was
performed in 10 μL volumes containing 8 μL (1×) of OneTaq® 2×
Master Mix (New England BioLabs, USA), 0.5 μL of each forward and
reverse primer (0.5 μM each) and 1 μL of template DNA (20 ng).
Negative controls were included in all PCR runs. A volume of 3 μL of
each PCR product was electrophoresed on a 1.5 % agarose gel stained
with Midori Green DNA stain (NIPPON Genetics Europe, Germany). The
quality and quantity of the fragments were checked against a FastGene
100 bp DNA Ladder (NIPPON Genetics Europe, Germany). When PCR
products showed a clear and single band of the correct expected length,
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Marine Pollution Bulletin 184 (2022) 114129
the whole PCR products were purified with the microCLEAN DNA
cleanup reagent (Gel Company, USA) according to the manufacturer’s
protocol. Finally, the purified PCR products were single-stranded
sequenced with BigDye Terminator v3.1 (Life Technologies, USA)
cycle sequencing methodology, on an ABI3500 Genetic Analyzer
(Applied Biosystems, USA), using the same primers as for PCRs (a for­
ward primer for each gene).
Sequences were manually checked and proofread in BioEdit v7.2.6
software (Hall, 1999). In addition, COI sequences were translated to
check for stop codons and detect possible mitochondrial pseudogene
presence (NUMTs). All sequences were compared with those available in
GenBank using the standard nucleotide BLAST (blastn) against the
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/),
through the Identification System (ID’s), for verification purposes. For
both databases, the identity threshold was set at 99 % and sequences
with high homology (≥99 %) were considered as acceptable and
consequently registered (see GenBank Accession Numbers in Appendix,
Table A1), whereas sequences below the threshold were disregarded
from further analysis.
2.3. Metal/metalloid extraction and analysis
Wet samples of muscle tissue were weighed (0.5 ± 0.14 g) using an
analytical balance (Mettler Toledo) and then mineralized in a micro­
wave oven (Ethos, TC, Milestone), equipped with Teflon vessels. We
performed an acid digestion with 8 mL of 67 % superpure nitric acid
(HNO3; Carlo Erba, Italy) for 30 min at 80 ◦ C. After acid digestion, the
contents of the vessels were decanted into Falcon tubes and double-
distilled water (Merck, USA) was added to the samples up to 50 mL.
10 mL from each digested sample was taken for analysis, and 50 μg/L of
internal standard (Yttrium, Y, and Rhenium, Re, 1000 mg/l Merck, USA)
was added as internal quality control. The quantification of Arsenic (As),
Cadmium (Cd), Cobalt (Co), Copper (Cu), Mercury (Hg), Manganese
(Mn), Nickel (Ni), Lead (Pb), Antimony (Sb), Selenium (Se), Vanadium
(V), Zinc (Zn) performed with an Inductively Coupled Plasma – Mass
Spectrometer (ICP-MS) Elan-DRC-e (Perkin Elmer, USA).
Concentrations were determined using standard solutions prepared
in the reagent matrix. Standards for the instrument calibration were
prepared based on mono element certified reference solution ICP Stan­
dard (Merck, USA). A seven-level calibration line was prepared from the
concentration of 0.5 μg/L to the concentration of 100 μg/L. The samples
were read as they were. If the determined concentrations exceeded the
last calibration point, they were diluted accordingly with double-
distilled water.
For the quality control, a sample for each batch of mineralization was
processed in duplicates; one was spiked with a multi-element solution of
I. Giovos et al.
25 mg/L with 50 μg/L (equivalent to 5 mg/kg in 0.5 g of sample) and we
obtained a mean recovery of 79.3–123 % for all the metals/metalloids.
The method detection limit (LOD) was calculated based on the 40
CFR 136, EPA procedure for digested analytical blanks using the
following equation:
LOD = t(df = n − 1, p = 0.99%) × SD (1)
where t is the one-tailed Student’s t distribution, df is the degrees of
freedom, n is the number of blank replicates, p is the probability and SD
is the standard deviation. Limit of detection (LOD) mg/Kg wet weight
(w.w.) calculated for the metals/metalloids analysed are the following:
As (0.077), Cd (0.006), Co (0.002), Cu (0.0111), Hg (0.013), Mn (0.027),
Ni (0.002), Pb (0.004), Sb (0.009), Se (0.088), V (0.006) and Zn (0.015).
For the statistical elaboration, LOD/2 was applied.
2.4. Risk from consumption
The daily intake of an element from food consumption is dependent
on the element concentration in food, the amount of food consumed and
the body weight. To understand if the food can pose a risk to human
health derived from consumption, the first step in risk analysis is to
compare the estimated daily intake (EDI) with the reference dose (RfDo)
set by US-EPA.
The Estimated Daily Intake (EDI) (μg/kg b.w.) is calculated based on
the following formula:
EDI = (C × IR)/BW (2)
where C is the concentration of trace elements in the edible fish tissue
(μg/Kg w.w.), IR the daily average consumption of the fish in the study
area 8.74 Kg/capita/day (FAO 2013) and BW is the body weight. For
this calculation we used the average weight of adult and child (70 Kg
and 16 Kg respectively).
The Target Hazard Quotient (THQ) is a complex parameter used for
the estimation of potential health risks associated with long term
exposure to chemical pollutants. In this work it was used to determine
health risks from the consumption of elasmobranch products. THQ value
below 1 means that the level of exposure is smaller than the reference
dose, which assumes that a daily exposure at this level is not likely to
cause any pernicious effects during lifetime for the human population. In
other words, a THQ below 1 means the adverse effects are negligible.
The THQ value was determined for both adults and children by the
following formula (US-EPA, 2000):
THQ = (C × IR × EF × ED)/(RfD × BW × AT) (3)
where C is the metal concentration (mg/kg), IR is the food ingestion rate
(0.00874 Kg/(capita * day) for Greek consumers, according to FAOSTAT
2013), EF is the exposure frequency (350 days/year), ED is the exposure
duration (26 years for adult and 6 years for child, according to Copat
et al., 2013), RfDo is the oral reference dose (mg/kg b.w. day), BW is the
average body weight (70 kg for adults and 16 Kg for child, according to
Copat et al., 2013), AT is the average exposure time for noncarcinogens
(350 days/year x ED). The following RfDo (mg/Kg/day) were used; As
= 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 =
0.0004, Se = 0.005, V = 0.0054, Zn = 0.3 (US-EPA, IRIS, 2017; FAO/
WHO 2004).
The EDI and THQ value for As was calculated assuming the inorganic
form as the 3 % of the total, being a pelagic fish (Ferrante et al., 2019).
3. Results
A total number of 53 specimens were collected, 27 from Alexan­
droupoli, 21 from Kavala, 4 from Thessaloniki and 1 from Volos. The low
sample number from Thessaloniki is related to the complexity of the fish
4
Marine Pollution Bulletin 184 (2022) 114129
market since Thessaloniki is the second largest town in Greece and re­
ceives fish products from all over the country and beyond. Thus, in
several cases, fishmongers were not certain about the capture location,
or in some cases even if the fish was imported. For Volos, we did not
manage to develop trustful relationships with fishmonger and although
they were willing to facilitate samples, they never showed us any invoice
that will allow us to see the fishing boat details. Therefore, the only
sample collected was a specimen that was sold from a local fisher to a
fishmonger when landing monitoring was performed by the research
team of the project. Amplification and sequencing were successful for all
DNA samples. In most samples (N = 46), amplification of the COI and
16S rRNA genes provided sufficient resolution for identifying the sam­
ples to the species level (Appendix, Table A1), while in three cases,
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
using the BOLD database. Eight different species were identified, with
Raja radula Delaroche, 1809 being the most reported (n = 17) followed
by Dasyatis pastinaca (Linnaeus, 1758), Myliobatis aquila (Linnaeus,
1758) (both n = 11), R. clavata Linnaeus, 1758 (n = 4), Dasyatis torto­
nesei Capapé, 1975 (n = 2) and Aetomylaeus bovinus (Geoffroy Saint-
Hilaire, 1817) (n = 1). While Hexanchus griseus (Bonnaterre, 1788) (n
= 3) and Centrophorus cf. uyato (n = 1) were identified visually because
the whole specimens were retrieved.
3.1. Risk from consumption
Concentrations of the selected metallic trace elements in the muscles
of the elasmobranchs are expressed in mg/kg (ww) and presented per
species in Table 1, and compared with (i) Commission Regulation (EC)
No 629/2008 and (ii) FAO (1983) limits. These numbers vary between
the samples of different species. Lead (Pb) was found to exceed the
maximum level (0.30 mg/kg) set by the Commission Regulation (EC) No
1881/2006 in 25 samples which refer to three species, namely
D. pastinaca, D. tortonesei and R. radula. Regarding Mercury (Hg), in 6
cases the samples exceeded the maximum level of 1 mg/kg set by
Commission Regulation (EC) No 629/2008 with 4 (out of the 6) cases
referring to 4 samples of shark species; H. griseus (n = 3) and C. uyato (n
= 1). The latter, bio-accumulated 11.1 mg/kg, far exceeding the
allowable levels. While, in none of the samples, Cd exceed the maximum
level (0,050 mg/kg) set by the Commission Regulation (EC) No 629/
2008.
The comparison between EDI and Reference Dose (RfDo) doesn’t
highlight any concern because all the value are below the maximum
acceptable oral daily dose (Table 2).
The lifetime risk evaluated with THQ values for investigated metals
and elasmobranchs are presented in Table 3. Individual THQ values
surpass 1 only for As and Hg. Arsenic is higher than 1 for A. bovinus (for
children) and R. clavata (for children) while Hg for R. clavata (for chil­
dren), H. griseus (for adults and children), C. uyato (for adults and chil­
dren) and A. bovinus (for adults and children) (Table 3).
Especially for A. bovinus, C. uyato and Dasyatis sp. the findings cannot
be considered conclusive given that one sample from only one specimen
was analysed.
4. Discussion
This is the first study that presents the levels of 12 trace elements in
the edible tissues of 10 different elasmobranch species caught in the
Greek waters. Up to date, only two studies exist regarding the concen­
tration of trace elements in the tissues of elasmobranchs in Greece. One
focused on the concentration of the mercury level in the tissues of the
spiny dogfish (Squalus acanthias) and the smooth-hound sharks (Mustelus
mustelus), with all specimens caught in Crete (South Greece) (Kousteni
et al., 2006) and the second on the concentration 27 metals and elements
in the tissues of 28 marine fish, which included one elasmobranch
 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
regarding the risk from the consumption of elasmobranchs for the Greek
consumers, is the work of Mol et al. (2018) that estimated the risk for the
Greek and the Turkish consumers based on specimens collected from the
Sea of Marmara. The increasing demand for shark meat globally (Nie­
dermüller et al., 2021), the high levels of mislabelling (Dulvy et al.,
2017) and fraudulent sales, stresses the need for more studies that focus
on the health risks from consumption of elasmobranchs. Based on the
latest global review on the topic (Tiktak et al., 2020), only a small
number of studies from the Mediterranean Sea exist, thus the basin has
been listed with “moderate scientific effort” on the topic, while studies
from the eastern Mediterranean are absent (Tiktak et al., 2020). Our
work aims to contribute towards this end, providing baseline informa­
tion about the levels of selected trace elements in different elasmo­
branch species and highlighting the risks from their consumption. Our
results are in accordance with the findings of Mol et al. (2018) for the
Greek consumers but also with other studies, reporting the risk from the
consumption of elasmobranchs (e.g. Storelli et al., 2011; Adel et al.,
2016; Adel et al., 2018; Kim et al., 2019; Boldrocchi et al., 2019, 2021;
Barcia et al., 2020) and in general of marine apex predators (e.g. Araújo

6
I. Giovos et al.
and Cedeño-Macias, 2016; Di Bella et al., 2020) and bony fish (Barone
et al., 2021; Vetsis et al., 2021; Storelli et al., 2022).
The biomagnification of trace elements can be affected by several
factors (Boldrocchi et al., 2021) like the species ecology (Shipley et al.,
2021), their diet and trophic level (Medina-Morales et al., 2020; Vetsis
et al., 2021), the trophic web complexity (Wang, 2002) and the human
activities in an area (Adel et al., 2018). In our work we detected similar
levels for most trace elements with the relevant global bibliography
(Tiktak et al., 2020) and the relevant Greek and Mediterranean studies
(Kousteni et al., 2006; Storelli et al., 2010; Mille et al., 2018; Vetsis et al.,
2021). In general, the concentrations of Cd found in all cases to be
within the limits set in the Commission Regulation (EC) No 1881/2006
and lower compared to the levels detected in other similar works (Tiktak
et al., 2020 and all references within). However, three species
(A. bovinus, C. uyato and H. griseus) were found to have high Hg levels
and three species (P. pastinaca, D. tortonesei and R. radula) were found to
have high Pb levels above the limits of Regulation (EC) No. 1881/2006.
Moreover, in the analyses performed to estimate the lifetime risk from
consumption, we found a high risk in highly commercialized species for
both adults and children with respect to Hg and for children only with
respect to As.
Mercury (Hg) occurs in deposits throughout the world and has been
widely used in a variety of devices (thermometers, barometers, ma­
nometers etc.) and processes. Through human activities, it can reach the
marine environment and once is methylated it bio-magnifies to the
upper trophic levels (Copat et al., 2012; Pappalardo et al., 2017; Storelli
et al., 2022) Mercury is listed as the third most toxic substance in the
Substance Priority List (Agency for Toxic Substances and Disease Reg­
istry (ATSDR), 2019), with symptoms from exposure varying, depending
on the type, dose, method, and duration of exposure, including muscle
weakness, poor coordination, numbness in the hands and feet, skin
rashes, anxiety, memory problems, trouble speaking, trouble hearing,
trouble seeing (Bernhoft, 2012) to even disruption of the fetus devel­
opment (Caserta et al., 2013). It is important to highlight that an
increased risk for both adults and children was found when consuming
some of the most commonly traded elasmobranch species in Greece,
such as R. clavata (Giovos et al., 2021), exceeding the mercury limit
before reaching the recommended amount of omega-3 fatty acids that
humans can ingest after consumption (Smith and Guentzel, 2010).
For arsenic (As) we estimated high lifetime risk from the consump­
tion of 2 species when children are exposed to long term consumption
(Table 3). The element is widespread in the aquatic ecosystems, occur­
ring naturally but also through human activities (Ferrante et al., 2019).
The element is listed as the most toxic substance in the Substance Pri­
ority List (Agency for Toxic Substances and Disease Registry (ATSDR),
2019) and increased level in the human organism can increase skin,
lung, liver and bladder cancer, as well as cause the occurrence of arterial
vascular disease (Sakuma et al., 2010 and references within). Again, it is
important to note that R. clavata is highly commercial in the country
(Giovos et al., 2021), however, we must highlight that the number of
samples for several species in our study is limited and further research is
required to investigate the bioaccumulated concentrations.
Lead (Pb), is a naturally occurring metal present in small amounts in
the earth’s crust. Anthropogenic activities such as fossil fuels burning,
mining, and manufacturing contribute to the release of high concen­
trations. In high levels, Pb is the most systemic toxicant that affects
several organs in the body including the kidneys, liver, central nervous
system, hematopoetic system, endocrine system, and reproductive sys­
tem of both adults and children (Wani et al., 2015) and for this reason is
listed as the second most toxic substance in the Substance Priority List
(Agency for Toxic Substances and Disease Registry (ATSDR), 2019). The
samples with high levels of Pb were detected in specimens caught in the
area of Kavala and close to Evros Delta (Fig. 1). Bio-accumulation of
trace elements is highly related to their existence and concentration in
the marine environment, which to a large extent is a result of human
activities in an area (Adel et al., 2018), while mining activities is well
7
Marine Pollution Bulletin 184 (2022) 114129
described that pose a serious threat to the marine environment (Kroll
et al., 2005), affecting even areas with low proximity to the facilities,
since pollutants can be transferred by the air and the waterways (Gusso-
Choueri et al., 2018 and references within). Across the coast of the
Thracean Sea (North Aegean), several mines exist, exploited for Au and
Pb–Ag both in ancient times and during the 70s (Vavelidis et al., 1997),
with the highest concentration near the towns of Kavala and Alexan­
droupoli. Most of those facilities are now abandoned, and no measures
have been taken for disposing their runoffs, potentially resulting to high
levels of the trace metal in the marine environment. Although in the
latest assessment for Water Framework Directive (Simboura et al., 2015)
in all sampling locations from the Thracean Sea, trace elements were
found within the limits set by Directive 2008/105/EC but this refers to
only three locations (Ierissos gulf, Kavala gulf, Thassos straights) and
none with proximity to the abandoned mines.
DNA analysis was deemed crucial for the study as identification is­
sues for some species exist (i.e., Dasyatis sp.), while for slices and ray
wings it would be impossible to identify otherwise, considering the
extend of mislabeling in Greek markets (Giovos et al., 2021). Finally,
considering the limited number of studies in the area, accurate trace
concentrations in a species level will help as reference for future studies.
We must highlight the risk from the consumption of H. griseus we
estimated for Hg both for adults and children. The species is protected in
Greece based on Presidential Decree no 67/1981 while the species is
commonly sold illegally in the fish markets of the country (Giovos et al.,
2020), mislabeled as “Galeos” that based on the Ministerial Decision No
1750/32219/2015 must refers to Mustelus spp. “Galeos” is generally
promoted in Greece as a cheap alternative to hake and a quality food
especially for children due to the lack of bones. Thus, consumers
remained unprotected from the lack of law enforcement and the erro­
neous mislabeling of a protected species (Giovos et al., 2020) with direct
implications to their health.
Nevertheless, the accumulation of trace elements has a negative ef­
fect on the health of elasmobranch species too (Depew et al., 2012;
Bezerra et al., 2019; Merly et al., 2019; Tiktak et al., 2020; Wosnick
et al., 2021). Few studies have evaluated the effects from the contami­
nation of specific trace elements on elasmobranch species health,
highlighting the provoked implications in osmoregulation (e.g. De
Boeck et al., 2010) or in cellular and fluid composition (e.g. Ballatori and
Boyer, 1996; De Boeck et al., 2010). In the case of Pb, its accumulation in
the gills, rectal gland, muscle and liver of elasmobranchs affect directly
the osmoregulation system of the species, their respiratory capacity and
metabolism (Eyckmans et al., 2013). While Hg has related to alternation
in the reproductive biology of the species (Bezerra et al., 2019). How­
ever, there are currently no toxic thresholds for tolerable concentrations
of metals and other elements in elasmobranchs (Merly et al., 2019),
while their impacts on the health and the well-being of elasmobranchs is
a poorly known and heavily understudied (Consales and Marsili, 2021).
5. Conclusions
Our work provides a preliminary information for the levels of
selected trace elements in the edible tissues of different elasmobranch
species caught in the North Aegean Sea. A national scale effort is
required to better understand the level of this phenomenon and the risks
that consumers might be facing from the consumption of elasmobranchs.
Relevant work must be conducted also for imported products given that
Greece is one of the main importers of elasmobranchs products in
Europe (Giovos et al., 2020), also from countries outside the European
Union that are not under the EU legislative framework. An important
step towards this direction could be done through the establishment of
an adequately staffed and equipped management authorities that would
ensure the proper implementation and enforcement of the EU
legislations.
Further research and a dedicated sampling design is required for
understanding better the potential influence of mines in the
I. Giovos et al. Marine Pollution Bulletin 184 (2022) 114129

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

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

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

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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