Environ Monit Assess (2015) 187:360 DOI 10.1007/s10661-015-4434-5
Metals content in otoliths of Dicentrarchus labrax from two fish farms of Sicily A. Traina & E. Oliveri & D. Salvagio Manta & M. Barra & S. Mazzola & A. Cuttitta
Received: 11 September 2014 / Accepted: 12 March 2015 # Springer International Publishing Switzerland 2015
Abstract Otoliths of cultured sea bass (Dicentrarchus labrax) from two different fish farms of Sicily were collected and analyzed by using inductively coupled plasma optical emission spectroscopy. Metal content (Ba, Cd, Fe, Mg, Mn, Sr, and Zn) was measured in order to test the potential use of biogenic carbonates as proxies of dissimilar environmental conditions since the fish farms are implanted in opposite coastal marine areas (Gulf of Castellammare and Gulf of Gela) characterized by different oceanographic features and human activities. Cluster analysis discriminates samples as different groups on the basis of metal content. Results show that concentrations of Sr in the otoliths have a similar range of distribution and not significantly different between the two farms. Otherwise, Fe, Mg, Mn, and Cd show higher concentrations in otoliths collected from fish reared in the farm in the southern coast (Gulf of Gela), an area subject to a great anthropogenic pressure. Zn is the only element with higher values in the otoliths from the farm in the northern coast (Gulf of Trappeto) probably due to industrial effluent. In this work, obtained data confirm the high potential of trace elements
A. Traina : E. Oliveri : D. Salvagio Manta : S. Mazzola : A. Cuttitta (*) Institute for Coastal and Marine Environment (IAMC)-CNR, Via del Mare, 3, Torretta Granitola, 91021 Campobello di Mazara, TP, Italy e-mail: [email protected] M. Barra Institute for Coastal and Marine Environment (IAMC)-CNR, Calata Porta di Massa, Naples 80133, Italy
measurements in these biogenic carbonates as proxies of different environmental conditions. Keywords Dicentrarchus labrax . Otoliths . Trace elements . Fish farm . Metal exposure
Introduction Otoliths are calcified structures located inside the ear inner cavity of teleost fish and form part of the organs that provide hearing and sense of balance (Campana 1999; Campana and Thorrold 2001). They are composed mainly by aragonite and a small fraction of organic molecules (Borelli et al. 2001; Campana 1999) and are formed through a specific biomineralization process that produce a particular structure, characterized by an alternation of concentric layers of aragonite crystals within a protein matrix (Campana 1999; Elsdon et al. 2008). Trace elements present in seawater can be incorporated as vicariant to Ca2+ in the crystal lattice of aragonite or be complexed by organic-ligand in the protein, in direct proportion to the availability of these elements in the surrounding environment (Campana 1999). The route of the ions from environment to otolith involves different barriers and compartments: for example, an efficient exchange of elements and contaminants occurs through the gills, which allow breathing through the extraction of oxygen dissolved in water. As the dissolved elements are absorbed and transported from the bloodstream and deposited in the organs (e.g., heart, liver, and gonads), it is likely that the same elements are
360
Page 2 of 11
transported to the plasma through a process of osmoregulation and thus to the otic structure, then from endolymph to the otolith. Otoliths grow daily throughout the life of a fish and they are metabolically inert (Campana and Jones 1998; Campana and Thorrold 2001), hence they chronologically sequence and maintain chemical signals in the growth layers (Panfili et al. 2002; Secor et al. 1998; Radtke 1989; Hoff and Fuiman 1995; Limburg 1995; Ranaldi and Gagnon 2008a). Given this capacity, the otolith chemical composition may represent a reliable natural tag of the environment in which the organism lives. The uptake mechanism of trace elements in otolith is not completely understood as the behavior of different metals is complex and the incorporation differs substantially between metals (Ranaldi and Gagnon 2010). Furthermore, the biomineralization process can be a function of different biological and environmental factors (Secor et al. 1998; Limburg 1995; Sadovy and Severin 1992; Gillanders and Kingsford 2003; Kalish 1989; De Pontual et al. 2003) as well as physiologic differences or speciesspecific ontogenies (Kalish 1989; Elsdon and Gillanders 2002; Gillanders and Kingsford 2003; Swearer et al. 2003; Martin and Thorrold 2004; Vasconcelos et al. 2007; Reis Santos et al. 2008; Ranaldi and Gagnon 2010). Although the study of elements incorporation from water to carbonate structure may be complicated, it is undeniable that chemical analysis of otoliths is a valuable tool used to identify individual from different sites and thus get information about their habitat use (Thorrold et al. 1997; De Pontual et al. 2003; Rooker et al. 2008). Otolith chemical composition has been actually widely used to discriminate different stocks and connectivity of populations (Campana and Thorrold 2001; Campana et al. 2000; Gillanders 2001; Gillanders and Kingsford 2000; Rooker et al. 2003), to reconstruct environmental histories and migratory patterns of fish (Kafemann et al. 2000; Secor and Rooker 2000; Tsukamoto and Arai 2001; Thorrold et al. 1997; Patterson 1999; Gillanders and Kingsford 2000; Campana et al. 2000), to certificate natal nursery habitats of the adults (Rooker and Secor 2004; Thorrold et al. 1997; Patterson 1999; Campana et al. 2000; Gillanders and Kingsford 2000; Zimmerman 2005; Yang et al. 2006; Brown and Severin 2008), and recently as proxy of marine pollution (Sarimin and Mohamed 2012; Ranaldi and Gagnon 2008b, 2010; Saquet et al. 2002; Yamashita et al. 2000; Bath et al. 2000; Elsdon and Gillanders 2003).
Environ Monit Assess (2015) 187:360
Trace elements (Ba, Cd, Fe, Mg, Mn, Sr, and Zn) were analyzed in otoliths of cultured Dicentrarchus labrax from two fish farms in Sicily widely different for rearing and ambient conditions. The European sea bass (D. labrax) is a demersal specie with a high commercial value, and due to some of its ecological features (e.g., large distribution, eurythermal and euryahaline specie), it is considered a good model organism for environmental monitoring study (Kerambrun et al. 2012). Actually, many researchers have performed on D. labrax for ecotoxicological investigations by monitoring its physiological status under different rearing systems (Maricchiolo et al. 2011; Kalantzi et al. 2013), exposure to heavy metals (Vazzana et al. 2009, 2014), or stress conditions (Celi et al. 2012). The aim of this study was to evaluate the impact of different environmental systems on otoliths elemental signature of D. Labrax and thus investigate the use of this biogenic carbonate as a valuable indicator of environmental condition.
Materials and methods Study areas and sampling collection Specimens of D. labrax were sampled from two farms of Sicily (Italy) located on southern and northern coast, respectively. Because of their geographical position, they are exposed to different hydrochemical and hydrophysical characteristics hence influencing their farming conditions. The farm of Trappeto is an off shore plant located in the Gulf of Castellammare (Tp) in the northwest coast of Sicily, in front of the city of Trappeto (Fig. 1). Cages are placed from 1,400 to 2,000 m far from the coast, with a seabed of about 30 m and therefore exposed to the northern winds and to high hydrodynamics. In particular, the hydrodynamic regime of the cage area is characterized by a dominant current, with an average speed of 12±7.5 cm s−1, coming from the third and fourth quadrants (along a west–east axis) for most of the year (CEOM 2002). Different kinds of industrial and agriculture activities affect the Gulf and a high density of population concentrated in the surrounding area contribute to the eutrophication of this seawater environment. Otherwise, the fish farm of Licata is an in shore plant inside the Gulf of Gela (southern Sicily), an area that covers about 21,000 m2. The cages are located in the confined area of the outer harbor of Licata, characterized
Environ Monit Assess (2015) 187:360
Page 3 of 11 360
Fig. 1 Location of sampling sites for D. labrax. The two fish farms are located in the northern and southern coast of Sicily
by 14 m depth and limited water circulation. Breeding structures are made up of 24 floating cages 7×14 m in which nylon grids 7 m deep are fixed. Licata is one of the most important commercial centers of Sicily, and the coastal marine area is affected by different human activities and potential pollutant sources: industrial, agricultural, zootechnical activities, intensive maritime commercial traffic, and improperly treated urban sewages. Furthermore, Licata is located on the right bank of the outfall of Salso River, which outfall has often been monitored for its trophic status compromised by the contribution of municipal wastewater. A total of 70 specimens of D. labrax (12–14 months old) were sampled from the cages of the farms. After collection, fish were killed in ice and transferred to the laboratory. Morphometric measures for each specimen were calculated. Particularly, total length (TL), standard length (SL), and total weight (TW) were measured by using a balance and an ichtiometer, respectively (Table 1). Fish head were cut horizontally parallel to the preopercle in order to expose the sagittal otolith (Sarimin and Mohamed 2012). The right sagitta was extracted from sea bass specimens using plastic forceps and cleaned with deionized water. After that, otoliths were dried under hood flow and finally placed in precleaned plastic tubes. Table 1 Morphometric data of D. labrax collected from the different fish farms (mean±SD) No. of specimens
Total length (mm)
Standard length (mm)
Weight (g)
Trappeto
35
30.38±2.05
26.15±1.82
0.34±0.07
Licata
35
27.95±0.92
24.11±0.83
0.25±0.02
Chemical and statistical analyses Each otolith was carefully decontaminated, soaked in Milli-Q water with 3 % hydrogen peroxide for 5 min to dissolve any remaining biological residue, and successively rinsed with Milli-Q water, according to the protocol of Rooker et al. (2001). The entire otoliths were dissolved with concentrated nitric acid and then diluted with Milli-Q water to a final acid concentration of 1 % HNO3. The solutions were analyzed by ICP-AES (Varian Vista MPX) to determine Ba, Ca, Fe, Mg, Mn, Sr, and Zn concentrations, and by ICP-MS (Varian) for Cd measurements. Quantitative analysis was performed using matrix-matched external calibration standards method. Procedural blanks (about 20 % of total number of samples) were concurrently prepared and analyzed to estimate detection limit (dl) and verify any contamination during preparation procedure. Analytical precision was routinely better than 6 % (RSD%, n=3) for all the analyzed elements. The results were standardized to calcium and expressed as concentration ratios (element/Ca: μmol/mol) to allow comparisons with the majority of other studies on otoliths chemistry. A non-parametric test for pairwise comparisons was preferred as assumptions required to run classical t test (normality and homoscedasticity) were violated. Specifically Mann–Whitney U test (Mann and Whitney 1947) was used to assess if otoliths collected in two different areas were significantly different in terms of trace metal concentrations. Also cluster analysis (k-means method) was used to evaluate the group classification based on elements content. As k-means method requires as input parameter the number of cluster to identify, it was objectively calculated by means of v-fold cross-validation method.
360
Page 4 of 11
Environ Monit Assess (2015) 187:360
Results and discussion
of samples to group in different clusters, exactly corresponding to the specimens from Trappeto and Licata farms respectively (Fig. 2), points out the extraordinary skill of otolith chemistry to discriminate different environmental conditions in which fish live. The accumulation of elements in fish otoliths depends on different factors, including the trace element concentration in water environment, bioavailability, the affinity of the calcium carbonate for different metals, and also the physiological state of individual (Hoff and Fuiman 1995; Campana 1999; Ranaldi and Gagnon 2010). Since factors influencing metal accumulation in otoliths are several and different, it is necessary to consider the potential impacts that fish farm can have on the surrounding environment as a further factor. Many studies have shown that aquaculture activities can affect the physicochemical conditions of the water column, the bottom sediment, and natural marine communities dynamics in the farm and in the surrounding zones (Troell and Norberg 1998; Naylor et al. 2000; Holmer 1991; Findlay and Watling 1997; Hargrave et al. 1997; Pergent et al. 1999; Pearson and Black 2001; La Rosa et al. 2001; Mirto et al. 2002; Alongi et al. 2003). Farm activities indeed release organic matter in the marine system (e.g., uneaten feed, fecal material, and other products of excretion) influencing natural environmental conditions (Ackefors and Enell 1990; Holmer 1991; Iwama 1991; Troell and Norberg 1998; Naylor et al. 2000; Sarà et al. 2004; Schendel et al. 2004; Salazar and Saldana 2007). Furthermore, other chemical substances such as antibiotics used as feed supplement are released
A cluster analysis (k-means method) was performed on the entire dataset in order to verify natural trend of grouping on the basis of the otolith microchemistry. Interestingly, two principal clusters that actually coincide with the two groups studied were identified. Such result highlighted that individuals belonging to different areas showed quite different values for considered trace metals and that in each area trace metals concentrations were homogeneous making impossible to identify subgroups. A line plot of clusters means (Fig. 2) showed the presence of strong differences between the two groups for all considered variables except Sr/Ca. In order to establish which element/Ca ratios determine the difference between the otoliths from the two farms, a Mann–Whitney U test was performed on the dataset. Basic statistics of element/Ca ratios distribution patterns and significant differences are shown in the box–whisker plots in Fig. 3. Except for the Sr/Ca ratio, the two group of otoliths (Trappeto and Licata) significantly differ for all element/Ca ratios (p level
Metals content in otoliths of Dicentrarchus labrax from two fish farms of Sicily A. Traina & E. Oliveri & D. Salvagio Manta & M. Barra & S. Mazzola & A. Cuttitta
Received: 11 September 2014 / Accepted: 12 March 2015 # Springer International Publishing Switzerland 2015
Abstract Otoliths of cultured sea bass (Dicentrarchus labrax) from two different fish farms of Sicily were collected and analyzed by using inductively coupled plasma optical emission spectroscopy. Metal content (Ba, Cd, Fe, Mg, Mn, Sr, and Zn) was measured in order to test the potential use of biogenic carbonates as proxies of dissimilar environmental conditions since the fish farms are implanted in opposite coastal marine areas (Gulf of Castellammare and Gulf of Gela) characterized by different oceanographic features and human activities. Cluster analysis discriminates samples as different groups on the basis of metal content. Results show that concentrations of Sr in the otoliths have a similar range of distribution and not significantly different between the two farms. Otherwise, Fe, Mg, Mn, and Cd show higher concentrations in otoliths collected from fish reared in the farm in the southern coast (Gulf of Gela), an area subject to a great anthropogenic pressure. Zn is the only element with higher values in the otoliths from the farm in the northern coast (Gulf of Trappeto) probably due to industrial effluent. In this work, obtained data confirm the high potential of trace elements
A. Traina : E. Oliveri : D. Salvagio Manta : S. Mazzola : A. Cuttitta (*) Institute for Coastal and Marine Environment (IAMC)-CNR, Via del Mare, 3, Torretta Granitola, 91021 Campobello di Mazara, TP, Italy e-mail: [email protected] M. Barra Institute for Coastal and Marine Environment (IAMC)-CNR, Calata Porta di Massa, Naples 80133, Italy
measurements in these biogenic carbonates as proxies of different environmental conditions. Keywords Dicentrarchus labrax . Otoliths . Trace elements . Fish farm . Metal exposure
Introduction Otoliths are calcified structures located inside the ear inner cavity of teleost fish and form part of the organs that provide hearing and sense of balance (Campana 1999; Campana and Thorrold 2001). They are composed mainly by aragonite and a small fraction of organic molecules (Borelli et al. 2001; Campana 1999) and are formed through a specific biomineralization process that produce a particular structure, characterized by an alternation of concentric layers of aragonite crystals within a protein matrix (Campana 1999; Elsdon et al. 2008). Trace elements present in seawater can be incorporated as vicariant to Ca2+ in the crystal lattice of aragonite or be complexed by organic-ligand in the protein, in direct proportion to the availability of these elements in the surrounding environment (Campana 1999). The route of the ions from environment to otolith involves different barriers and compartments: for example, an efficient exchange of elements and contaminants occurs through the gills, which allow breathing through the extraction of oxygen dissolved in water. As the dissolved elements are absorbed and transported from the bloodstream and deposited in the organs (e.g., heart, liver, and gonads), it is likely that the same elements are
360
Page 2 of 11
transported to the plasma through a process of osmoregulation and thus to the otic structure, then from endolymph to the otolith. Otoliths grow daily throughout the life of a fish and they are metabolically inert (Campana and Jones 1998; Campana and Thorrold 2001), hence they chronologically sequence and maintain chemical signals in the growth layers (Panfili et al. 2002; Secor et al. 1998; Radtke 1989; Hoff and Fuiman 1995; Limburg 1995; Ranaldi and Gagnon 2008a). Given this capacity, the otolith chemical composition may represent a reliable natural tag of the environment in which the organism lives. The uptake mechanism of trace elements in otolith is not completely understood as the behavior of different metals is complex and the incorporation differs substantially between metals (Ranaldi and Gagnon 2010). Furthermore, the biomineralization process can be a function of different biological and environmental factors (Secor et al. 1998; Limburg 1995; Sadovy and Severin 1992; Gillanders and Kingsford 2003; Kalish 1989; De Pontual et al. 2003) as well as physiologic differences or speciesspecific ontogenies (Kalish 1989; Elsdon and Gillanders 2002; Gillanders and Kingsford 2003; Swearer et al. 2003; Martin and Thorrold 2004; Vasconcelos et al. 2007; Reis Santos et al. 2008; Ranaldi and Gagnon 2010). Although the study of elements incorporation from water to carbonate structure may be complicated, it is undeniable that chemical analysis of otoliths is a valuable tool used to identify individual from different sites and thus get information about their habitat use (Thorrold et al. 1997; De Pontual et al. 2003; Rooker et al. 2008). Otolith chemical composition has been actually widely used to discriminate different stocks and connectivity of populations (Campana and Thorrold 2001; Campana et al. 2000; Gillanders 2001; Gillanders and Kingsford 2000; Rooker et al. 2003), to reconstruct environmental histories and migratory patterns of fish (Kafemann et al. 2000; Secor and Rooker 2000; Tsukamoto and Arai 2001; Thorrold et al. 1997; Patterson 1999; Gillanders and Kingsford 2000; Campana et al. 2000), to certificate natal nursery habitats of the adults (Rooker and Secor 2004; Thorrold et al. 1997; Patterson 1999; Campana et al. 2000; Gillanders and Kingsford 2000; Zimmerman 2005; Yang et al. 2006; Brown and Severin 2008), and recently as proxy of marine pollution (Sarimin and Mohamed 2012; Ranaldi and Gagnon 2008b, 2010; Saquet et al. 2002; Yamashita et al. 2000; Bath et al. 2000; Elsdon and Gillanders 2003).
Environ Monit Assess (2015) 187:360
Trace elements (Ba, Cd, Fe, Mg, Mn, Sr, and Zn) were analyzed in otoliths of cultured Dicentrarchus labrax from two fish farms in Sicily widely different for rearing and ambient conditions. The European sea bass (D. labrax) is a demersal specie with a high commercial value, and due to some of its ecological features (e.g., large distribution, eurythermal and euryahaline specie), it is considered a good model organism for environmental monitoring study (Kerambrun et al. 2012). Actually, many researchers have performed on D. labrax for ecotoxicological investigations by monitoring its physiological status under different rearing systems (Maricchiolo et al. 2011; Kalantzi et al. 2013), exposure to heavy metals (Vazzana et al. 2009, 2014), or stress conditions (Celi et al. 2012). The aim of this study was to evaluate the impact of different environmental systems on otoliths elemental signature of D. Labrax and thus investigate the use of this biogenic carbonate as a valuable indicator of environmental condition.
Materials and methods Study areas and sampling collection Specimens of D. labrax were sampled from two farms of Sicily (Italy) located on southern and northern coast, respectively. Because of their geographical position, they are exposed to different hydrochemical and hydrophysical characteristics hence influencing their farming conditions. The farm of Trappeto is an off shore plant located in the Gulf of Castellammare (Tp) in the northwest coast of Sicily, in front of the city of Trappeto (Fig. 1). Cages are placed from 1,400 to 2,000 m far from the coast, with a seabed of about 30 m and therefore exposed to the northern winds and to high hydrodynamics. In particular, the hydrodynamic regime of the cage area is characterized by a dominant current, with an average speed of 12±7.5 cm s−1, coming from the third and fourth quadrants (along a west–east axis) for most of the year (CEOM 2002). Different kinds of industrial and agriculture activities affect the Gulf and a high density of population concentrated in the surrounding area contribute to the eutrophication of this seawater environment. Otherwise, the fish farm of Licata is an in shore plant inside the Gulf of Gela (southern Sicily), an area that covers about 21,000 m2. The cages are located in the confined area of the outer harbor of Licata, characterized
Environ Monit Assess (2015) 187:360
Page 3 of 11 360
Fig. 1 Location of sampling sites for D. labrax. The two fish farms are located in the northern and southern coast of Sicily
by 14 m depth and limited water circulation. Breeding structures are made up of 24 floating cages 7×14 m in which nylon grids 7 m deep are fixed. Licata is one of the most important commercial centers of Sicily, and the coastal marine area is affected by different human activities and potential pollutant sources: industrial, agricultural, zootechnical activities, intensive maritime commercial traffic, and improperly treated urban sewages. Furthermore, Licata is located on the right bank of the outfall of Salso River, which outfall has often been monitored for its trophic status compromised by the contribution of municipal wastewater. A total of 70 specimens of D. labrax (12–14 months old) were sampled from the cages of the farms. After collection, fish were killed in ice and transferred to the laboratory. Morphometric measures for each specimen were calculated. Particularly, total length (TL), standard length (SL), and total weight (TW) were measured by using a balance and an ichtiometer, respectively (Table 1). Fish head were cut horizontally parallel to the preopercle in order to expose the sagittal otolith (Sarimin and Mohamed 2012). The right sagitta was extracted from sea bass specimens using plastic forceps and cleaned with deionized water. After that, otoliths were dried under hood flow and finally placed in precleaned plastic tubes. Table 1 Morphometric data of D. labrax collected from the different fish farms (mean±SD) No. of specimens
Total length (mm)
Standard length (mm)
Weight (g)
Trappeto
35
30.38±2.05
26.15±1.82
0.34±0.07
Licata
35
27.95±0.92
24.11±0.83
0.25±0.02
Chemical and statistical analyses Each otolith was carefully decontaminated, soaked in Milli-Q water with 3 % hydrogen peroxide for 5 min to dissolve any remaining biological residue, and successively rinsed with Milli-Q water, according to the protocol of Rooker et al. (2001). The entire otoliths were dissolved with concentrated nitric acid and then diluted with Milli-Q water to a final acid concentration of 1 % HNO3. The solutions were analyzed by ICP-AES (Varian Vista MPX) to determine Ba, Ca, Fe, Mg, Mn, Sr, and Zn concentrations, and by ICP-MS (Varian) for Cd measurements. Quantitative analysis was performed using matrix-matched external calibration standards method. Procedural blanks (about 20 % of total number of samples) were concurrently prepared and analyzed to estimate detection limit (dl) and verify any contamination during preparation procedure. Analytical precision was routinely better than 6 % (RSD%, n=3) for all the analyzed elements. The results were standardized to calcium and expressed as concentration ratios (element/Ca: μmol/mol) to allow comparisons with the majority of other studies on otoliths chemistry. A non-parametric test for pairwise comparisons was preferred as assumptions required to run classical t test (normality and homoscedasticity) were violated. Specifically Mann–Whitney U test (Mann and Whitney 1947) was used to assess if otoliths collected in two different areas were significantly different in terms of trace metal concentrations. Also cluster analysis (k-means method) was used to evaluate the group classification based on elements content. As k-means method requires as input parameter the number of cluster to identify, it was objectively calculated by means of v-fold cross-validation method.
360
Page 4 of 11
Environ Monit Assess (2015) 187:360
Results and discussion
of samples to group in different clusters, exactly corresponding to the specimens from Trappeto and Licata farms respectively (Fig. 2), points out the extraordinary skill of otolith chemistry to discriminate different environmental conditions in which fish live. The accumulation of elements in fish otoliths depends on different factors, including the trace element concentration in water environment, bioavailability, the affinity of the calcium carbonate for different metals, and also the physiological state of individual (Hoff and Fuiman 1995; Campana 1999; Ranaldi and Gagnon 2010). Since factors influencing metal accumulation in otoliths are several and different, it is necessary to consider the potential impacts that fish farm can have on the surrounding environment as a further factor. Many studies have shown that aquaculture activities can affect the physicochemical conditions of the water column, the bottom sediment, and natural marine communities dynamics in the farm and in the surrounding zones (Troell and Norberg 1998; Naylor et al. 2000; Holmer 1991; Findlay and Watling 1997; Hargrave et al. 1997; Pergent et al. 1999; Pearson and Black 2001; La Rosa et al. 2001; Mirto et al. 2002; Alongi et al. 2003). Farm activities indeed release organic matter in the marine system (e.g., uneaten feed, fecal material, and other products of excretion) influencing natural environmental conditions (Ackefors and Enell 1990; Holmer 1991; Iwama 1991; Troell and Norberg 1998; Naylor et al. 2000; Sarà et al. 2004; Schendel et al. 2004; Salazar and Saldana 2007). Furthermore, other chemical substances such as antibiotics used as feed supplement are released
A cluster analysis (k-means method) was performed on the entire dataset in order to verify natural trend of grouping on the basis of the otolith microchemistry. Interestingly, two principal clusters that actually coincide with the two groups studied were identified. Such result highlighted that individuals belonging to different areas showed quite different values for considered trace metals and that in each area trace metals concentrations were homogeneous making impossible to identify subgroups. A line plot of clusters means (Fig. 2) showed the presence of strong differences between the two groups for all considered variables except Sr/Ca. In order to establish which element/Ca ratios determine the difference between the otoliths from the two farms, a Mann–Whitney U test was performed on the dataset. Basic statistics of element/Ca ratios distribution patterns and significant differences are shown in the box–whisker plots in Fig. 3. Except for the Sr/Ca ratio, the two group of otoliths (Trappeto and Licata) significantly differ for all element/Ca ratios (p level
