Toxic mineral elements in Mytilus galloprovincialis from Sicilian coasts (Southern Italy)

Abstract We assessed the relationship between V, Cr, Mn, Hg, As, Cd, Sn, Sb and Pb concentrations in Mytilus galloprovincialis samples from the coasts of Sicily and the expression of metallothioneins. Toxic mineral elements assessment was carried out by A.A. Spectrometry and ICP-MS. The metallothioneins expression was performed by q-PCR method. Low metals’ levels were found in the mussel samples examined, in comparison with what was reported in literature. The highest mean values of toxic mineral elements were found in Gela (Cr 0.178 ± 0.03 mg/Kg, Mn 4.325 ± 0.012 mg/Kg, As 3.706 ± 0.009 mg/Kg, Sn 0.148 ± 0.014 mg/Kg, Sb 0.009 ± 0.004 mg/Kg e Pb 0.364 ± 0.01 mg/Kg). Significant levels of Hg were found in samples from Catania (0.014 ± 0.005 mg/Kg). Only vanadium and lead concentrations showed significant differences between sampling areas (p < 0.05). Molecular analysis verified a basal expression of Mt1 and the absence of over-expression of Mt2, confirming the low mineral’s concentrations found in the samples examined. Graphical Abstract


Introduction
Pollution by toxic metals in marine environment increased considerably because of anthropogenic activities, leading to an increase of the number of studies on the presence of these elements in different species of the marine ecosystem Di Bella et al. 2015;Naccari et al. 2015;Giangrosso et al. 2016;Cammilleri et al. 2018). Toxic metals bioaccumulates in the aquatic food chain and reach the highest concentration in the upper trophic levels (Di Bella et al. 2010Bella et al. , 2017. At present, many studies have focused on the application of innovative techniques for the detection of organic and inorganic contaminants in the environment for biomonitoring purpose (Campone et al. 2016(Campone et al. , 2018a(Campone et al. ,b, 2019. Biomonitoring, as proved strategy to detect inorganic contaminants in Mediterranean Sea, has widely employed some species representative of different trophic levels . Mytilus galloprovincialis is a bivalve mollusc belonging to the Mytilidae family, often used as a sentinel organism to monitor the biological effects associated with the presence of toxic metals (Catsiki and Florou 2006;Fasulo et al. 2008). These contaminants can produce alterations of biochemical and physiological processes that can be quantified by estimating biological parameters whose variations may be related to the physiological state of animals (Bolognesi et al. 1996;Fasulo et al. 2008). Among the detoxification mechanisms, metallothioneins (Mt) induction in response to toxic metals exposure is certainly the most well documented (Nordberg 1998;Domouhtsidou et al. 2004). The biological response may also depend from seasonal changes, which in turn can influence the sensitivity to pollutants (Ivankovi c et al. 2005).
Considering that the Mt production is induced by exposure to toxic metals, the purposes of this work are to estimate a correlation between V, Cr, Mn, As, Cd, Sn, Sb and Pb levels in M. galloprovincialis samples from Sicilian coasts and the metallothioneins expression by molecular methods.

Results and discussion
The linearity test of the ICP-MS method for V, Cr, Mn, Cu, As, Cd, Sn, Sb and Pb analysis gave satisfactory results (r 2 > 0.999). The LOD and LOQ values obtained (Table S3) were in accordance with the regulation (CE) n. 333/2007. Recoveries studies showed values between 96% and 105%. All the results were satisfactory for limit of repeatability (metrological approach). All concentration levels showed Horrat values of repeatability and reproducibility lower than 2 as specified by the Commision Regulation n 836/2011. The DMA-80 has been used according to the US EPA method 7473 (Di  and the validation results of the Hg determination method are shown in Table S4. The method developed was able to determine the Hg concentration of the samples in a wide range of concentrations (0.050-2.00 mg/kg) with a mean recovery of 103%.
The average toxic mineral elements concentrations obtained from M. galloprovincialis samples sorted by sampling area are shown in Figure S1. All the samples analysed showed V, Cr, Mn, Cu, As, Cd, Sn, Sb and Pb values above the limits of the quantification of the method. Whereas, only 29% of the samples examined showed Hg values below the LOQ.
The highest mean values were reached in samples from Trappeto for V (0.635 ± 0.057 mg/Kg), Gela for Cr (0.178 ± 0.03 mg/Kg), Mn (4.325 ± 0.012 mg/Kg), As (3.706 ± 0.009 mg/Kg), Sn (0.148 ± 0.014 mg/Kg), Sb (0.009 ± 0.004 mg/Kg) and Pb (0.364 ± 0, 01 mg/Kg), Barcellona PG for Cd (0.201 ± 0.025 mg/Kg) and Catania for Hg (0.014 ± 0.005 mg/Kg). The highest values were found in Barcelona P.G. for V (0.701 mg/ Kg), Catania for Cr (0.178 mg/Kg), Messina for Mn and Pb (13.644 mg/Kg and 0.402 mg/ Kg), Catania for As, Cd and Sb (3.706 mg/Kg, 0.05 mg/Kg and 0.03 mg/Kg), Milazzo for Sn (0.222 mg/Kg) and Syracuse for Hg (0.027 mg/Kg). All the M. galloprovincialis samples from Gela, Licata, Palermo and Trappeto showed Hg levels below the quantification limit of the method. The Kruskal-Wallis test revealed statistically significant differences between sampling areas (p < 0.05) for V and Pb levels, whereas no differences were found between sampling areas (p > 0.05) for Mn, Cr, As, Cd, Sn, Sb and Hg levels. Dunn's post-hoc test showed that samples from Palermo and Trappeto contribute predominantly to the differences between data groups for V, whereas Gela and Catania for Cr and Pb. The average values of toxic mineral elements obtained in this study are lower than the limits imposed by the EC Reg. 1881/2006, suggesting the absence of important risks in the sampling areas studied. The values obtained in this study are also significantly lower than those found by Maanan (2007) in mussel samples from Morocco, with average concentrations of Cd, Pb, Hg, Cr and Mn from 7 to 10 times higher than those of our study. Furthermore, our Cd, Pb, Hg, Cr and Mn values are 3 to 10 times lower than what showed by Orescanin et al. (2006) in the Adriatic coasts of Croatia. The samples from Gela revealed the highest levels of chromium, manganese, arsenic, tin and antimony, probably due to the intense industrial activity registered in this area (Ministero dell'Ambiente e della Tutela del Territorio e del Mare et al. 2008).
The highest mercury concentrations were found in mussels samples from Catania coast; these values could be related to the Etna volcanic activity, considered one of the major natural sources of mercury release into the environment (Martin et al. 2012). These considerations are further confirmed by the highest concentrations of mercury in samples of mussels taken along the eastern coast of Sicily (Messina, Milazzo and Syracuse). The mRNA expression study in mussels examined showed no significant changes in the pollution response genes. Our findings are in contrast to what was found by Fasulo et al. (2008) on the expression of methallothioneins in M. galloprovincialis samples from Faro lake (Messina, Southern Italy) probably due to the low concentrations of toxic mineral elements found in our work and the absence of significant differences between sampling areas of the major inductors of metallothioneins expression (Cr, Cd, Pb and Sn). Mussels and other marine invertebrates tend to accumulate pollutants in their tissues (Odẑak et al. 1994;Salvo et al. 2016), without showing any apparent harmful effect, probably because they react to the presence of high concentrations of xenobiotics. The results of this study showed an overall absence of risks in the sampling areas examined. As far we know, this is the first report on the presence of V, Cr, Mn, Sn and Sb in M. galloprovincialis samples from central Mediterranean sea, suggesting that further studies on the use of stress indicator markers are needed to have a comprehensive evaluation of inorganic pollutants presence in Mediterranean Sea.

Experimental
See the supplementary material for the experimental section.