Inferring Class of organisms in the Central-East Atlantic from eco-toxicological characterization

Regional Studies in Marine Science 35 (2020) 101190

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Regional Studies in Marine Science journal homepage: www.elsevier.com/locate/rsma

Inferring Class of organisms in the Central-East Atlantic from eco-toxicological characterization ∗

Enrique Lozano-Bilbao a , , José María Espinosa b , Alba Jurado-Ruzafa c , Gonzalo Lozano a , Arturo Hardisson d , Carmen Rubio d , Dailos González Weller e , Ángel J. Gutiérrez d a

Departamento de Biología Animal y Edafología y Geología, Unidad Departamental de Ciencias Marinas, Universidad de La Laguna, 38206 La Laguna, Santa Cruz de Tenerife, Spain b Fundación del Sector Público Estatal Observatorio Ambiental Granadilla (Unidad Técnica), 38001 Santa Cruz de Tenerife, Spain c Instituto Español de Oceanografía. Centro Oceanográfico de Canarias, Dársena Pesquera s/n, 38180, Santa Cruz de Tenerife, Spain d Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Universidad de La Laguna, 38200 La Laguna, Santa Cruz de Tenerife, Spain e Servicio Público Canario de Salud, Laboratorio Central, Santa Cruz de Tenerife, Spain

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Article history: Received 2 October 2019 Received in revised form 8 January 2020 Accepted 18 February 2020 Available online 24 February 2020 Keywords: Class Heavy metal Trace element Ecological Marine ecosystem

a b s t r a c t All marine organisms bio-accumulate metals and trace elements in a different way, and there are great differences depending on the level of taxonomic classes. A total of 660 samples of 22 species of different taxonomic class types have been used for this study, all samples were measured with atomic emission spectrometry with inductively coupled plasma (ICP-OES), the mammalian class has the highest concentration of Zn (17.215 ± 13.750 mg/kg w.w) and of Fe (86.215 ± 49.495 mg/kg w.w), these values being much higher than the other classes, and the cephalopods have the highest concentration in heavy metals such as Cd (0.963 ± 1.056 mg/kg w.w) and Pb (0.466 ± 0.909 mg/kg w.w). All the classes presented significant differences among each other, each class presents some ranges of each metal different to the other classes. Based on the present study, the ecological preferences and the taxonomical classification give differential metal content patterns for marine organisms, thus showing the importance of this study for the preparation of environmental plans to check the state of the marine ecosystem. © 2020 Elsevier B.V. All rights reserved.

1. Introduction The oceans have been contaminated by an active anthropic action for many decades, in such a way that chemical compounds discharged into the ocean enter the food chains where they are assimilated and accumulated by marine organisms, from primary producers such as phytoplankton to predators (Raimundo et al., 2005; Storelli and Marcotrigiano, 2005; Bustamante et al., 2008; Lozano et al., 2010; Kucuksezgin et al., 2011; Wu and Wang, 2011; Rosa et al., 2015; Fort et al., 2016; Lozano-Bilbao et al., 2018a). Many of these compounds are discharged through submarine outlets and coastal runoff from agricultural fields and the coastal areas with the largest populations which have the highest concentration of metals and trace elements. These elements, in small concentrations, can cause a fertilization effect in the environment, but when the concentrations increase above a certain threshold they cause harmful effects in the organisms and the ecosystem (DeForest et al., 2007; Dolenec et al., 2007; ∗ Corresponding author. E-mail address: [email protected] (E. Lozano-Bilbao). https://doi.org/10.1016/j.rsma.2020.101190 2352-4855/© 2020 Elsevier B.V. All rights reserved.

Ramírez-Alvarez et al., 2007; Ruilian et al., 2008; Morar et al., 2011; Santana-Casiano et al., 2013; Tornero et al., 2014; Žvab Rožič et al., 2015; Riba et al., 2016; Rouane-Hacene et al., 2017; Lozano-Bilbao et al., 2017a, 2018b). All marine organisms bio-accumulate metals and trace elements in a different way, and there are great differences depending on the level of taxonomic classes. Gastropods found on the coasts are used as bio-indicators of pollution, but they should not be used as sole indicators since they contain hemocyanin they have high levels of copper which would affect the monitoring of metals (Zhong et al., 2016; Aydin-Önen and Öztürk, 2017; Reguera et al., 2018). Cephalopods are carnivorous organisms in which the accumulation of heavy metals has a great impact, since they are active predators and have high concentrations of metals such as cadmium, lead and mercury (Raimundo et al., 2005; Storelli and Marcotrigiano, 2007; Bustamante et al., 2008; Kojadinovic et al., 2011; Lacoue-Labarthe et al., 2011; LozanoBilbao et al., 2017b). Actinopterygian fish have variable life forms ranging from pelagic to demersal and their feeding habits are carnivorous, herbivorous and omnivorous. For this reason, each species has different bio-accumulation patterns (Hornung et al.,

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E. Lozano-Bilbao, J.M. Espinosa, A. Jurado-Ruzafa et al. / Regional Studies in Marine Science 35 (2020) 101190

1994; Cornish et al., 2007; Tzikas et al., 2007; Company et al., 2010; Vieira et al., 2011; Lozano et al., 2016; Afonso et al., 2017, 2018). Anthozoans are considered good bio-indicators of contamination (for trace elements, heavy metals or N15 ), and this is due to the fact that they are found on the coast, they contain zooxanthellae and incorporate all these compounds from the environment (Main et al., 2010; Horwitz et al., 2014a; Lozano et al., 2016; González-Delgado et al., 2018; Lozano-Bilbao et al., 2018a). Malacostraca are benthic organisms, except the planktonic species of malacostraca or when some species have a planktonic stage before becoming benthic when are adults, and that is why they will be affected by anthropic contamination if they inhabit areas close to the coasts (Devineau and Amiard Triquet, 1985; Lorenzon et al., 2000; Lozano et al., 2010, 2016). Mammals accumulate many pollutants because their diet is carnivorous and their trophic position is high. For example, a large part of the odontocetes feed on large cephalopods or tunas, and the bioaccumulation of many heavy metals in their fat is a normal and proven fact (Liu et al., 2015; Bilandžić et al., 2016; Peltier et al., 2016; Ball et al., 2017). Based on the different bioaccumulation patterns, the aim of the present study is to test the starting hypothesis that it is possible to differentiate groups of marine organisms by their heavy metal and trace metal contents from taxonomical and ecological perspectives. 2. Material and methods In order to conduct this study, 660 samples of 22 different species were collected around the Canary Islands (Spain), (Fig. 1), taking 30 samples for each species: Scomber colias Gmelin, 1789, Trachurus picturatus (Bowdich, 1825), Sardina pilchardus (Walbaum, 1792), Serranus cabrilla (Linnaeus, 1758), Mullus surmuletus (Linnaeus, 1758), Diplodus sargus (Linnaeus, 1758), Sarpa salpa (Linnaeus, 1758), Chelon labrosus (Risso, 1827), Sparisoma cretense (Linnaeus, 1758), Anemonia sulcata (Pennant, 1777), Sepia officinalis Linnaeus, 1758, Octopus vulgaris Cuvier, 1797, Loligo vulgaris Lamarck, 1798, Abraliopsis morisii (Verany, 1839), Pyroteuthis margaritifera (Rüppell, 1844), Patella aspera Röding, 1798, Patella candei crenata D’Orbigny, 1840, Palaemon elegans Rathke, 1837, Plesionika narval (Fabricius, 1787), Physeter macrocephalus Linnaeus, 1758, Stenella frontallis (Cuvier, 1829) and Tursiops truncatus (Montagu, 1821). The fish samples were taken with artisanal fishing techniques, as well as the crustaceans, gastropods and cephalopods, the cetacean samples were taken from the biopsies of beached specimens in the archipelago, finally the Antozoan samples were taken in the intertidal areas of the islands 2.1. Sample preparation The analytical sample consisted of a muscle portion of around 5–10 g. The samples were dried in an oven where they remained at a temperature of 70 ◦ C for 24 h. They were then placed in a muffle furnace (FP | 1000 o C Witeg) for 48 h at 450 o C ± 25 o C, until white ashes were obtained. If after this time the total mineralization of the samples had not been achieved, 65% HNO3 R (Suprapur⃝ MERCK)was added to them in the fume hood, and then evaporated on a heating plate at 70–90 o C. Once treated, they were re-incinerated in a muffle furnace at 450 ± 25 o C until white ashes were obtained. After white ashes were obtained, they were filtered with a 1.5% HNO3 solution, made up to 25 ml for further reading by atomic emission spectrometry with inductively coupled plasma (ICP-OES; Belonging to the Canary Public Health Service, Central Laboratory, Santa Cruz de Tenerife, Spain), in such a way that the concentration of the metals Al, Cd and Pb and the trace elements: B, Cr, Cu, Fe, Li, Ni, V and Zn in the analyzed tissues was obtained (Afonso et al., 2018).

Table 1 Mean value (±standard deviation) of the metal contents (mg/kg) in wet weight for all the organisms by ‘‘Habitat’’. Habitat

Al B Cd Cr Cu Fe Li Ni Pb V Zn

Coastal

Pelagic

4.797 ± 5.615 0.586 ± 0.893 0.080 ± 0.160 0.142 ± 0.179 0.786 ± 0.498 19.121 ± 32.544 0.449 ± 0.401 0.181 ± 0.585 0.127 ± 0.532 0.105 ± 0.173 4.237 ± 2.958

4.728 ± 3.242 0.297 ± 0.414 0.501 ± 0864 0.235 ± 0.356 2.088 ± 2.097 18.408 ± 28.526 0.648 ± 0.708 0.343 ± 0.579 0.266 ± 0.660 0.175 ± 0.642 9.096 ± 6.348

2.2. Statistical analysis In order to investigate possible differences in the content and relative composition of heavy metals and trace metals among the analyzed samples, a statistical analysis was performed, using a distance-based permutational multivariate analysis of variance (PERMANOVA) with Euclidean distances (Anderson and Braak, 2003). A one-way design was used with the fixed factor of ‘‘Habitat’’ with two levels of variation: Pelagic = S. colias, T. picturatus, S. pilchardus, L. vulgaris, A. morisii, P. margaritifera, P. macrocephalus, S. frontallis, Tursius truncatus; Coastal = S. cabrilla, M. surmuletus, D. sargus, S. salpa, C. labrosus, S. cretense, A. sulcata, S. officinalis, O. vulgaris, P. aspera, P. candei crenata, P. elegans, P. narval. A one-way design was used with the fixed factor ‘‘Class’’ with 6 levels of variation, according to the taxonomic class of the analyzed species: Actinopterygii = S. colias, T. picturatus, S. pilchardus, S. cabrilla, M. surmuletus, D. sargus, S. salpa, C. labrosus, S. cretense; Anthozoa = A. sulcata; Cephalopod = S. officinalis, O. vulgaris, L. vulgaris, A. morisii, P. margaritifera; Gastropod = P. aspera, P. candei crenata; Malacostraca = P. elegans, P. narval; Mammal = P. macrocephalus, S. frontallis, T. truncatus. The following metal contents were included in the analysis: Al, B, Cd, Cr, Cu, Fe, Li, Ni, Pb, V and Zn. Relative dissimilarities among the fish feeding habits groups were studied using a principal coordinate analysis (PCoA) where metals that best explained data variability were represented as vectors. Finally, metals that best explained the variability of the data found in samples were further investigated by means of univariate assessments. One-way permutational analyses of variance with Euclidean distances of raw data were performed using the same design with the factors ‘‘Habitat and Class’’ as previously described. In all analyzes, 4999 permutations of exchangeable units and a posteriori pairwise comparisons were used to verify the differences between the levels of the significant factors (p-value < 0.01) (Anderson, 2004). The statistical packages PRIMER 7 & PERMANOVA + v.1.0.1 were used for the statistical analyses. 3. Results The values obtained for the analyzed metals by habitat and taxonomic class are shown in Tables 1 and 2, respectively. Table 3 shows the limits of instrumental detection and quantification, which were estimated based on the instrument’s instrumental response via the analysis of 15 targets under conditions of reproducibility (see Table 3). The PERMANOVA showed significant differences in the content and relative composition of heavy metals and trace metals among the samples of the different groups of organisms analyzed according to their habitat (Table 4). These results can be clearly

E. Lozano-Bilbao, J.M. Espinosa, A. Jurado-Ruzafa et al. / Regional Studies in Marine Science 35 (2020) 101190

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Fig. 1. Map of the Canary Archipelago.

Fig. 2. Principal coordinate analysis (PCoA) showing the first two axes (87.4% of variability), based on Euclidean distances of square-root-transformed data of heavy metal and trace element content in the groups of class with contrasting habitats. Table 2 Mean value (±standard deviation) of the metal contents (mg/kg) wet weight for all the organisms by ‘‘Class’’. Class

Al B Cd Cr Cu Fe Li Ni Pb V Zn

Actinopterygii

Anthozoa

Cephalopod

Gastropod

Malacostraca

Mammalia

3.931 0.172 0.032 0.163 0.823 7.660 0.476 0.144 0.087 0.105 5.804

9.774 ± 9.797 2.001 ± 1.976 0.007 ± 0.009 0.109 ± 0.141 0.328 ± 0.343 10.775 ± 19.079 0.236 ± 0.365 0.977 ± 1.842 0.115 ± 0.132 0.052 ± 0.073 1.824 ± 1.910

2.939 0.408 0.963 0.216 3.023 6.853 0.810 0.174 0.466 0.140 8.692

9.955 ± 5.924 1.425 ± 0.526 0.328 ± 0.225 0.273 ± 0.263 1.197 ± 0.510 74.339 ± 34.408 0.535 ± 0.314 0.336 ± 0.184 0.213 ± 0.179 0.356 ± 0.242 3.780 ± 1.046

2.710 0.161 0.009 0.109 0.940 2.824 0.281 0.545 0.232 0.018 1.502

5.314 ± 4.386 0.532 ± 0.479 0.099 ± 0.146 0.072 ± 0.063 1.446 ± 1.062 86.215 ± 49.495 0.560 ± 0.447 0.325 ± 0.524 0.042 ± 0.068 0.008 ± 0.021 17.215 ± 13.750

± ± ± ± ± ± ± ± ± ± ±

3.654 0.106 0.102 0.276 0.522 5.695 0.44 0.4 0.549 0.534 3.649

± ± ± ± ± ± ± ± ± ± ±

1.713 0.571 1.056 0.289 2.623 9.131 0.877 0.196 0.909 0.173 3.993

seen in the PCoA analysis, which accounted for 87.4% of the total variability of the data recorded (Fig. 2). The ordination of samples in the graph showed a clear difference in the content of heavy metals between the coastal and the pelagic group, with a certain degree of overlap. Metals that best explained the variability found in the data are represented as vectors in the PCoA, which shows

± ± ± ± ± ± ± ± ± ± ±

2.115 0.061 0.009 0.147 0.571 3.335 0.259 0.840 0.294 0.019 0.931

a clear pattern of increase of Al, Fe, Ni and Cd (Fig. 3), Cu, Zn, B and V in the coastal group, despite the great variability among samples in the pelagic group (Fig. 2). Regarding the taxonomic classes, the PERMANOVA also showed significant differences in the content and relative composition of heavy metals and trace metals among the samples

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E. Lozano-Bilbao, J.M. Espinosa, A. Jurado-Ruzafa et al. / Regional Studies in Marine Science 35 (2020) 101190 Table 3 Detection limits and quantification. Metal and wavelength

Detection limit (DL) (mg/L)

Quantification limit (QL) (mg/L)

Al (167.0 nm) B (249.7 nm) Cd (226.5 nm) Cr (267.7 nm) Cu (327.3 nm) Fe (259.9 nm) Li (670.8 nm) Ni (231.6 nm) Pb (220.3 nm) V (310. 2 nm) Zn (206.2 nm)

0.004 0.003 0.0003 0.003 0.004 0.003 0.005 0.0007 0.0003 0.001 0.002

0.012 0.012 0.001 0.008 0.012 0.009 0.013 0.003 0.001 0.005 0.007

Fig. 3. Box plot for Cd (mg/kg). Table 4 Results of the PERMANOVA, main test. Results of the one-way PERMANOVA analyzing the variation in the content of heavy metals and trace elements in the ‘‘Class’’ groups with contrasting habitats. Source df Fe Res Total

SS

MS

Pseudo-F P(perm) Unique perms P(MC)

1 22862 22862 46.832 592 2.89E+05 488.18 593 3.1186E+05

0.0002* 4984

0.0002

*p-value <0.01 Table 5 Results of the PERMANOVA, main test. Results of the one-way PERMANOVA analyzing the variation in the content of heavy metals and trace elements in the groups of class. Source df Fe Res Total

SS

MS

Pseudo-F P(perm) Unique perms P(MC)

5 1.3474E+05 26949 89.464 588 1.7712E+05 301.23 593 3.1186E+05

0.0002* 4975

0.0002

Table 6 Results of the pairwise analysis. Results of pairwise tests examining the significant factor of ‘Class’ obtained in the one-way PERMANOVA analyzing the variation in the content of heavy metals and trace elements in the class. Groups

t

P(perm)

Unique perms

P(MC)

Actinopterygii, Anthozoa Actinopterygii, Cephalopod Actinopterygii, Gastropod Actinopterygii, Malacostraca Actinopterygii, Mammal Anthozoa, Cephalopod Anthozoa, Gastropod Anthozoa, Malacostraca Anthozoa, Mammal Cephalopod, Gastropod Cephalopod, Malacostraca Cephalopod, Mammal Gastropod, Malacostraca Gastropod, Mammal Malacostraca, Mammal

6.6842 7.3571 16.413 5.8355 8.788 7.3977 10.237 4.8053 6.9067 14.057 6.1751 7.3801 15.092 5.2262 8.6245

0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002*

4980 4995 4988 4985 4985 4986 4983 4982 4990 4988 4984 4987 4986 4989 4988

0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002

*p-value <0.01

*p-value <0.01

of the different groups of organisms analyzed (Table 5). Twoto-two post-hoc analyses showed significant differences at the level of all the fish groups (Table 6). These results are supported by the PCoA analysis, which accounted for 87.4% of the total variability of the data recorded (Fig. 4). The ordination of samples in the graph showed clear differences in the content of heavy metals between the gastropods and mammals. Malacostraca,

actinopterigii and cephalopods appeared to have more similar heavy metal contents, as shown by a smaller segregation between points belonging to each group (Fig. 4). Metals that best explained the variability in the data are represented as vectors in the PCoA, which shows a clear pattern of increase of Al, Fe, Ni, Cd, Cu, Zn, B and V (Figs. 5, 6, 7), (Table 7).

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Fig. 4. Principal coordinate analysis (PCoA) showing the first two axes (87.4% of variability), based on Euclidean distances of square-root-transformed data of heavy metal and trace element content in the groups of class.

Fig. 5. Box plot for Al (mg/kg).

4. Discussion Although the contents and the relative concentrations of the analyzed metals did not follow clearly differentiable patterns between the groups considered, it was possible to identify significant differences between the pelagic and coastal species (Table 4), since the mean concentrations of the metals in the coastal organisms were clearly higher for Al, B and Fe and, in pelagic organisms, for Cd, Cr, Cu. Li, Ni, Pb, V and Zn. It should be mentioned that, in general, the pelagic species included in the study are larger in size than the coastal species chosen (with the exception of A. morisii and P. margaritifera, which are small squids), thus as pelagic species are in a high trophic position this could explain the higher concentration in most metals (with the exception of Al, B and Fe, which are metals used in factories and agriculture). Therefore, they can be found in greater concentrations near the coast (Hydes and Liss, 1977; Shahid, 2011; Bundy et al., 2015; Zhang et al., 2015; Mamun et al., 2018). A great variability in the concentrations of heavy metals and trace metals was also observed by taxonomic class. Cephalopods

had the highest concentration of toxic the heavy metals Cd (0.963 ± 1.056 mg/kg) and Pb (0.466 ± 0.909 mg/kg); they also had a higher concentration of Cu (3.023 ± 2.623 mg/kg), and Li (0.810 ± 0.877 mg/kg) (Raimundo et al., 2005; Storelli and Marcotrigiano, 2007; Bustamante et al., 2008; Kojadinovic et al., 2011; Lacoue-Labarthe et al., 2011; Lozano-Bilbao et al., 2017a). Fig. 4 shows these differences, observing that all classes are clearly separated. The highest concentrations of Fe (86.215 ± 49.495 mg/kg) and Zn (17.215 ± 13.750 mg/kg) were observed in the mammal class. Although it could be expected that mammals would have the highest concentration in all metals, the samples included in the present study were taken from muscle tissue and not from fat. The cetaceans have sophisticated mechanisms of bioaccumulation of heavy metals, trace elements and toxins that are stored in the fat so that it is not toxic for them, and the high concentration of Fe is due to the fact that they have a higher amount of hemoglobin in blood and myoglobin (Noren and Williams, 2000; Ball et al., 2017; Barone et al., 2013; Ai et al.,

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E. Lozano-Bilbao, J.M. Espinosa, A. Jurado-Ruzafa et al. / Regional Studies in Marine Science 35 (2020) 101190

Fig. 6. Box plot for Cd (mg/kg).

Fig. 7. Box plot for Cu (mg/kg).

2014; Barón et al., 2015; Liu et al., 2015; Bilandžić et al., 2016; Hansen et al., 2016; Peltier et al., 2016; Fago et al., 2017). Based on the predatory behavior of the cetaceans T. truncatus and S. coeruleoalba on pelagic and demersal fish described by Queiros et al. (2018), it is plausible that they have higher concentrations than the actinopterygians, except for Cr and Pb in this case. The high concentration in Pb could be due to the fact that it is a toxic heavy metal with greater presence on the coasts (due to anthropic factors) and that this metal accumulates in the fat and not in the muscle (Hansen et al., 2016). The actinopterygians did not have the highest concentration for any metal, and this may be due to the wide diversity of habitats they live in and the different feeding habits of the species in the study. In this regard, species of different trophic levels (from herbivore species to predators) were included, which bioaccumulate and require trace elements in different ways (LozanoBilbao et al., 2019a). The actinopterygies are vitally important

specimens in the marine ecosystem, since without them, many organisms would be left without their basic nutritional material (Pauly et al., 1998; Begg et al., 1999; Walters et al., 1999; Fujiwara, 2012; Raimundo et al., 2013; Watson et al., 2013; Mariani et al., 2017; Lozano-Bilbao et al., 2019b). Anthozoa were only represented by a species of anemone A. sulcata. This species lives in the intertidal pools and are more abundant when they are close to anthropogenic zones (LozanoBilbao et al., 2018b). Their largest energy contribution comes from zooxanthellae, which provides compounds containing N, with greater concentrations, in this study, of the metals B (2.001 ±1.976 mg/kg) and Ni (0.977 ± 1.842 mg/kg) (Roberts et al., 1999; Dolenec et al., 2007; Morar et al., 2011; Arántzazu and Marrero, 2012; Gumara, 2014; Horwitz et al., 2014b; Lozano et al., 2016; González-Delgado et al., 2018; Lozano-Bilbao et al., 2018a).

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Table 7 Results of pairwise tests examining the significant factor ‘Class’ obtained in one-way ANOVAs analyzing the variation in the content of the main heavy metals and trace elements by class groups. Actinopterygii, Anthozoa Actinopterygii, Cephalopod Actinopterygii, Gastropod Actinopterygii, Malacostraca Actinopterygii, Mammal Anthozoa, Cephalopod Anthozoa, Gastropod Anthozoa, Malacostraca Anthozoa, Mammal Cephalopod, Gastropod Cephalopod, Malacostraca Cephalopod, Mammal Gasteropod, Malacostraca Gastropod, Mammal Malacostraca, Mammal

Al

B

Cd

Cr

Cu

Fe

Li

Ni

Pb

V

Zn

0.0002* 0.0478 0.0002* 0.091 0.1196 0.0002* 0.6594 0.0002* 0.003* 0.0002* 0.1812 0.003* 0.0002* 0.0002* 0.0088

0.0002* 0.0002* 0.0002* 0.8272 0.0002* 0.0002* 0.31 0.0002* 0.0002* 0.0002* 0.0814 0.0214 0.0002* 0.0002* 0.0002*

0.1322 0.0002* 0.0002* 0.4466 0.0002* 0.0002* 0.0002* 0.0844 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002*

0.1158 0.0562 0.0002* 0.0338 0.0164 0.0312 0.0002* 0.6882 0.2584 0.0144* 0.0116* 0.0036* 0.0002* 0.0002* 0.5418

0.0002* 0.0002* 0.0002* 0.311 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.004* 0.0038* 0.239 0.016*

0.4848 0.0014* 0.0002* 0.0002* 0.0002* 0.0906 0.0002* 0.0004* 0.0002* 0.0002* 0.0024* 0.0002* 0.0002* 0.3506 0.0002*

0.0002* 0.0002* 0.025 0.0104 0.4872 0.0002* 0.0002* 0.105 0.003* 0.0386 0.0002* 0.119 0.0002* 0.5392 0.0196*

0.0002* 0.008* 0.0002* 0.0002* 0.0082* 0.0002* 0.0044* 0.0802* 0.018* 0.0002* 0.0064* 0.1232 0.5454 0.02 0.5164

0.0534 0.0002* 0.0002* 0.0006* 0.1904 0.019 0.001* 0.206 0.0002* 0.1454 0.141 0.0012* 0.3288 0.0002* 0.0012*

0.2434 0.002* 0.0002* 0.015 0.0034* 0.004* 0.0002* 0.0086* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.002*

0.0002* 0.0002* 0.0004* 0.0002* 0.0002* 0.0002* 0.0002* 0.9652 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002*

*p-value <0.01

The gastropods studied here belonged to the genus Patella, which are benthic and herbivore organisms and they are frequently the first to provide proof of alterations in the environment. They live in the intertidal and the supratidal zones and are more affected by anthropogenic pollution, which would explain the higher concentrations of Al (9.955 ± 5.924 mg/kg) and V (0.356 ± 0.242 mg/kg), compared to the other classes (Aydin-Önen and Öztürk, 2017; Reguera et al., 2018). The malacostraca class was represented by two species of benthic shrimp, one deep water species P. narval and another, P. elegans, found in intertidal and supratidal waters and did not have greater metal concentrations than the other taxonomical classes. Lozano et al. (2010) studied the metal content in species of the genus Palaemon and obtained values of Fe much higher than those in the present study. Palaemon species are found in coastal waters and can be quickly affected by anthropic and natural phenomena as they inhabit intertidal pools (Kurun et al., 2007; Lozano et al., 2016). Based on the present study, give differential metal content patterns for marine organisms. The inclusion of metals and trace elements of anthropic character gives more strength to the study, because thanks to these you can perform studies in different areas and know if they have been anthropically altered. Therefore, when selecting bio-indicators to monitor marine pollution, not only should feeding habits (Lozano et al., 2016) be taken into account, but ecological preferences and taxonomical classification and these issues should also be carefully considered, along with other factors. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Afonso, A., Gutiérrez, Á.J., Lozano, G., González-Weller, D., Lozano-Bilbao, E., Rubio, C., Caballero, J.M., Revert, C., Hardisson, A., 2018. Metals in Diplodus sargus cadenati and Sparisoma cretense—a risk assessment for consumers. Environ. Sci. Pollut. Res. 25, http://dx.doi.org/10.1007/s11356-017-0697-4. Afonso, A., Gutiérrez, A.J., Lozano, G., González-Weller, D., Rubio, C., Caballero, J.M., Hardisson, A., Revert, C., 2017. Determination of toxic metals, trace and essentials, and macronutrients in Sarpa salpa and Chelon labrosus : risk assessment for the consumers. http://dx.doi.org/10.1007/s11356-0178741-y. Ai, W.-M., Chen, S.-B., Chen, X., Shen, X.-J., Shen, Y.-Y., 2014. Parallel evolution of IDH2 gene in cetaceans, primates and bats. FEBS Lett. 588, 450–454. http://dx.doi.org/10.1016/j.febslet.2013.12.005.

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