Heavy metals in tissues of loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea

Heavy metals in tissues of loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea

Comparative Biochemistry and Physiology, Part C 138 (2004) 187 – 194 www.elsevier.com/locate/cbpc Heavy metals in tissues of loggerhead turtles (Care...

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Comparative Biochemistry and Physiology, Part C 138 (2004) 187 – 194 www.elsevier.com/locate/cbpc

Heavy metals in tissues of loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea Silvia Franzellittia, Clinio Locatellia, Guido Gerosab, Carola Vallinic, Elena Fabbria,* a

Interdepartment Centre for Research in Environmental Sciences, University of Bologna, via S. Alberto 163, Ravenna 48100, Italy b CHELON Marine Turtle Conservation and Research Program, Rome, Italy c A.R.C.H.E´. Research and Educational Activities for Chelonian Conservation, Ferrara, Italy Received 23 March 2004; received in revised form 11 July 2004; accepted 14 July 2004

Abstract Thirty-five specimens of Caretta caretta were collected dead along the Adriatic Sea coast from the Po Delta to the Reno mouth (Italy). Turtles were classified into four size categories ranging from 24.5 to 74 cm, by measuring the minimum straight-line carapace length (MSCL). Cd, Cu, Fe, Mn, Ni, and Zn levels were assessed in liver, lung, muscle and adipose tissue. Cd, Cu and Fe mainly accumulated in the liver (8.9, 23.7 and 1180 mg/kg dry mass [d.w.], respectively), and Mn in the lung (29.5 mg/kg d.w.). Levels of Ni were higher in adipose (22 mg/kg d.w.) than other tissues, while Zn concentrations were higher in muscle (about 140 mg/kg d.w.). Negative correlations with size were established for Zn in liver and Cu in adipose tissue, while positive correlations were observed for Mn and Ni in adipose tissue. Metal concentrations did not differ between males and females, nor between individuals found stranded and those victims of by-catch. On average, Cd, Cu, Mn and Ni concentrations in our specimens were higher than in loggerhead turtles and other species living in other areas. We hypothesize that trace metals could be used as bacquired markersQ to help investigate migration routes of C. caretta. D 2004 Elsevier Inc. All rights reserved. Keywords: Adriatic Sea; Atomic absorption spectrometry; Bioaccumulation; Caretta caretta; Environmental markers; Heavy metal; Loggerhead turtle; Strandings

1. Introduction Loggerhead turtles (Caretta caretta) are the most common turtle in the Mediterranean Sea. They nest on eastern basin shores, mainly in Greece, Turkey and Cyprus, although dispersed nestings are documented on north African and south Italian beaches. From nesting sites, hatchlings move to the open ocean foraging on the surface, then start a developmental migration towards near-shore and continental shelf waters, foraging at the bottom in the shallow fringes of the sea. Sexually mature turtles move to specific mating and nesting sites during the breeding season, and return thereafter to the foraging and wintering areas where they spend much of their life (Miller, 1997). * Corresponding author. Tel.: +39 0544600387; fax: +39 0544600411. E-mail address: [email protected] (E. Fabbri). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.07.008

The presence of loggerhead turtles has been documented in the northeastern (Lazar et al., 2000) and northwestern Adriatic Sea, in particular close to the Po Delta (Vallini et al., 2001). Due to the great availability of food and warm shallow waters, this area seems an ideal foraging and over-wintering environment. Nevertheless, turtles inhabiting the basin are severely threatened by human activities, and the impact of incidental captures during the fishing effort is high (Vallini et al., 2001). Pollution cannot be neglected as potential threat for marine turtles, and although evidence is not available, environmental contamination could be somehow related to the high number of strandings occurring along the northwestern Adriatic shores. Toxic effects of heavy metals have been reported for several marine vertebrates (Law, 1996; Franson, 1996). Heavy metal concentrations in turtle tissues have been

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quantified (Godley et al., 1998, 1999; Sakai et al., 2000a,b; Storelli et al., 1998), but no information on toxicological effects and detrimental threshold concentrations are available. Progress towards the understanding of the possible heavy metal impact on turtle health might be obtained with more data on accumulation and distribution of trace elements within their body. The present study aimed to quantify the concentrations of Cd, Cu, Fe, Mn, Ni, and Zn in liver, lung, muscle and adipose tissue of C. caretta collected dead along the northwestern Adriatic Sea coasts. The choice of these heavy metals has been made on the basis of previous studies on the Po Delta environment showing particularly high levels of Ni, Cu and Zn in sediments, Cd and Ni in the water, and Mn and Fe in bivalves known to be at the base of the C. caretta diet (Dinelli et al., 1996; Dinelli and Lucchini, 1999; Tankere et al., 2000; Fagioli et al., 1994). The possible relationship between the heavy metals accumulated by the animals and those measured in the environment is discussed.

2. Materials and methods 2.1. Sample collection Thirty-five loggerhead turtles were collected dead between May–November 2000 and May–September 2001 along northwestern Adriatic beaches (Fig. 1).

Sixteen of these were victims of by-catch, while 19 were found dead along the coast. Each turtle was measured (minimum straight-line carapace length, MSCL; Gerosa, 1996) and underwent a summary necroscopic examination. Sex was determined on the basis of morphological characters showing strong sexual dimorphism (Marquez, 1990). Liver was sampled from 30 individuals, lung from 13, pectoral muscle and abdominal adipose tissue from 17. Three 10-g aliquots of the various tissues were dissected out and stored in high-density polyethylene vials at 20 8C. 2.2. Chemical analysis Frozen samples were independently lyophilised, pulverised in liquid nitrogen, and 0.5 g aliquots were digested in 10% HCl, 13% HNO3 and 19% H2SO4 by volume, all reagents suprapure grade. The digested solutions were made up to a final volume of 100 ml with distilled water, and used for spectrometric determination with an atomic absorption spectrometer (AAnalyst 100, Perkin Elmer). Concentrations of Fe and Zn were determined by a flame atomization system (AS-90, Perkin Elmer), while for Cd, Cu, Mn, and Ni a graphite furnace (HGA-850, Perkin Elmer) was used. Values are expressed as mg/kg dry mass. Accuracy of measures was verified by inner standard method; percentage of recovery was between 95% and 106%, and precision was always lower than 5% (number of independent measures=5). The purity of the chemicals used in the analysis has been assayed by running a series of chemical blanks; there was no evidence of any contamination in these blanks. Quality Control (QC) of the analytical procedure was also checked by using a standard reference material, ISS-MURST-A2 (Istituto Superiore di Sanita`, Italy). The accuracy and precision of this method in triplicate analysis was more than 95% for all the elements. In the aqueous reference solution, the limits of detection (LOD) were obtained by the equation LOD=Ks y/x /b (Miller and Miller, 1984), where s y/x and b are the estimated standard deviation and the slope of the analytical calibration function of each element, respectively, with a 98% (K=3) confidence level (IUPAC, 1978). The limits of detection for each element, calculated as Ag/l and expressed as mg/kg d.w., were the following: 0.11 (Cd), 0.49 (Cu), 2.3 (Fe), 0.77 (Mn), 0.69 (Ni), 2.1 (Zn). 2.3. Statistical analysis

Fig. 1. Sampling area (from the Po Delta to the River Reno mouth, about 150 km) along the northwestern Adriatic Sea coast, Italy.

Data are presented as meanFstandard deviation (S.D.), and tested for significance by one-way ANOVA using the Program Systat (ver. 8.00, SPSS Science, Chicago, IL, USA); dependence of metal concentration with body size was examined using the Spearman rank correlation test with the SigmaStat software (SPSS). Differences were considered statistically significant at Pb0.05.

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3. Results and discussion 3.1. Age and sex composition Size distribution of the analysed individuals is shown in Table 1. MSCL of our sample ranged from 24.5 to 74 cm, and individuals were distributed into four size classes with benthonic sub-adults and adults representing the highestfrequency class, while juveniles and sexually mature adults accounted only for a smaller proportion of the total. No systematic difference between victims of by-catch and other stranded animals was observed. Females accounted for 44% of the total sample, and their mean MSCL was 55.4F14.5 cm; males accounted for 14% of the total, with a mean MSCL of 68.1F12.6 cm. The lack of sexually dimorphic features made sex determination impossible for about 36% of the turtles analysed. According to the adopted dimensional classification (Table 1), these individuals have been recognized as juveniles (mean MSCL=29.1F2.65 cm, n=5) and immature sub-adults (mean MSCL=38.4F6.4 cm, n=8). Taken together, these findings support the role of north Adriatic Sea as foraging and wintering area for adult loggerheads as previously reported (Margaritoulis, 1988; Argano et al., 1992), as well as a migration route for females. This would agree with previous observation on the migration of females from Zakynthos Island towards the north Adriatic Sea (Argano et al., 1992). However, the high proportion of sub-adults would also ascribe to the north Adriatic Sea an important role as developmental area for Mediterranean loggerhead turtles. Moreover, our data indicate that juveniles also undertake developmental migration towards the north Adriatic Sea. 3.2. Heavy metal concentrations Cd, Cu, Fe, Mn, Ni, and Zn concentrations in each tissue are reported in Fig. 2. Most analysed metals showed organotropism; in particular, a strong tendency towards a tissue-specific concentration has been detected for Cd and Cu in liver and for Fe in liver and lung, which were two to six times higher than those of remaining tissues. Lung was the main site of Mn accumulation, while the highest levels

Table 1 Size distribution of the north Adriatic loggerhead turtles (Caretta caretta) MSCL range (cm)

Life stage

% Individuals

13–32 32–51 51–70 z70

juvenile immature sub-adults benthonic sub-adults and adults sexually mature adults

11.1 25 55.5 8.3

Animals were grouped according to their minimum straight-line carapace length (MSCL) following a dimensional classification modified from Dodd (1988) based on the size distribution of Mediterranean loggerheads (Margaritoulis, 1988; Argano et al., 1992; Margaritoulis, personal communication).

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of Ni were measured in adipose tissue. As to Zn, the highest average value was found in muscle. Levels of heavy metals in tissues of C. caretta from the present and other studies are compared in Table 2. A mean water content of 68.3F5.5%, 82F10%, 77.3F6.2%, and 15.4F1.8% for liver, lung, muscle and adipose tissue, respectively, was calculated in our samples after lyophilisation, and used to convert data on a wet weight (w.w.) basis. Cadmium is a toxic transition metal that marine vertebrates seem to consume with the food (Nagle et al., 2001; Watanabe et al., 2002). As shown in Table 2, Cd accumulates mainly in the liver of turtles. Cd concentrations measured in liver and lung of C. caretta were in the range of those detected in the same tissues of loggerheads from other areas; its levels in muscle were instead lower than those reported for loggerheads from Cyprus but higher than those measured in other studies; differently, mean levels of Cd in adipose tissue were significantly higher than those reported in other individuals (Table 2). Copper is an essential element but toxic for mammals above threshold concentrations (Romero et al., 1996). On average, levels of Cu in liver of C. caretta from the northwestern Adriatic Sea were within the range reported for other turtle species and marine mammals (5–30 Ag w.w.; Sakai et al., 2000a, b), while Cu concentrations in lung, muscle and fat were significantly higher (Table 2). As a common feature shared by pulmonates with superior diving abilities, Fe was the metal present at the highest concentration in tissues of C. caretta (Watanabe et al., 2002). The measured concentrations were in the same range of those reported in other studies (Table 2). Manganese is an essential cofactor for some enzymes, but also toxic for mammals at high doses (Inoue and Makita, 1996; Romero et al., 1996); in tissues of C. caretta, its concentration was significantly higher than that reported in other studies (Table 2), but no information supporting detrimental effects of Mn on marine organisms is available. Rather homogeneous concentrations of nickel were found in liver, lung and muscle of C. caretta, while the highest accumulation was observed in fat. Few data are available as to the levels of this metal in turtles, and at present it is not possible to establish the reasons for such elevated concentrations of Ni in adipose tissue. In our samples, Ni concentrations were significantly higher than those reported for Japanese and British loggerhead turtles (Table 2), as well as for green turtle, Chelonia mydas (0.6 mg/kg w.w.; Sakai et al., 2000b). Toxic and carcinogenic effects of nickel compounds are associated with nickelmediated oxidative damage to DNA and proteins and to inhibition of cellular antioxidant defenses (Rodriguez et al., 1996); however, no evidence supports toxic effects of Ni in turtles. Nevertheless, the high levels of Ni found in the present study are of concern. Zn was rather homogeneously distributed in the tissues of C. caretta as reported also for turtles inhabiting other areas (Caurant et al., 1999; Aguirre et al., 1994; Sakai et al.,

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Fig. 2. Concentration of heavy metals in different tissues of C. caretta for the northwestern Adriatic Sea, 2000–2001. Data are expressed as mean valuesFS.D. Number of samples separately analysed are: 30 (liver), 13 (lung), 17 (muscle), and 17 (fat). Different letters (a, b, c, d) indicate statistically different data ( Pb0.05).

1995). The levels of Zn detected in this work were very similar to those measured in other studies (Table 2), suggesting that also in turtle, Zn body levels are under homeostatic regulation (Anan et al., 2002a). 3.3. Heavy metal accumulation with growth Scarce information is available on heavy metal accumulation in sea turtles of different sizes (Sakai et al., 2000a; Gordon et al., 1998; Anan et al., 2001). Although a validated method for age determination is not available, MSCL is currently considered a good indicator of turtle age (Bjorndarl et al., 2000), and it was used to investigate a potential relationship between heavy metal concentrations and growth. A negative correlation ( pb0.05) between MSCL and Zn was observed in liver, while Cd, Cu, Fe Mn and Ni showed widely variable values with no apparent correlation with MSCL (Fig. 3). Our data therefore are not in agreement with other reports which identified a negative correlation between Cd concentration and growth in several turtle species (Sakai et al., 2000b; Caurant et al., 1999; Gordon et al., 1998). However, Anan et al. (2001) did not find a significant decreasing tendency for Cd levels in tissues of C. mydas, and suggested it could be due to the variable rates of shift in the feeding habits among individuals.

No statistically significant correlation was noted in muscle between metal concentration and size of the individuals (Fig. 3). In adipose tissue, Cu levels decreased, and Mn and Ni levels increased with size ( pb0.05), while no significant correlation between the two parameters was observed for Cd, Fe and Zn (Fig. 3). To our knowledge, there are no reports dealing with growth-related changes of metal concentrations in adipose tissue besides the present study, and the pattern of metal accumulation observed cannot be readily explained. The lower metabolic rates reported for fat of green turtle with respect to the liver (Penick et al., 1996) could eventually support the Mn and Ni accumulation over time. In addition, decreasing trends observed for Zn in liver and Cu in adipose tissue are not easily understood; nevertheless, various hypotheses can be made on the basis of previous reports. Heavy metal variations with size are affected by various factors, among them feeding habits seem of primary importance (Sakai et al., 2000a), and the changes in habitat utilization and feeding behaviour between the pelagic-omnivorous juveniles and benthonic-carnivorous adults could modify the exposure to environmental contaminants. Godley et al. (1999) pointed out the function of egg deposition as an important route for metals excretion in females. Storelli et al. (1998) hypothesized that the lower

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Table 2 Heavy metal accumulation in tissues of Caretta caretta from different locations Tissue

Cd

Cu

Fe

Mn

Ni

Zn

Location

Reference

Liver n=30 n=12 n=7 n=4 n=6 n=7 n=8 n=1

2.84F0.72 2.24F1.78 2.58F4.12 8.64* 9.74F3.37 9.3F3.3 16.4* –

7.4F3.9 – 8.25F6.59 – 17.7F8.9 17.90F8.17 – –

377.4F211.2 – – – 604F401 649F385 – –

6.23F2.8 – – – 2.2F0.4 2.07F0.46 – –

4.38F1.43 – – – b0.03 – – b0.062

27.9F6.5 – 25.0F9.5 – 28.10F4.73 27.9F10.4 22.8* –

North Adriatic Sea South Adriatic Sea French Atlantic Coasts Cyprus Japan Japan Australia British Coasts

This study Storelli et al. (1998) Caurant et al. (1999) Godley et al. (1999) Sakai et al. (2000b) Sakai et al. (1995) Gordon et al. (1998) Godley et al. (1998)

Lung n=13 n=12 n=6

0.47F0.13 0.58F0.75 0.371F0.143

1.8F0.8 – 0.545F0.105

184.1F87.0 – 134F114

5.3F3.0 – 0.12F0.08

1.80F0.44 – b0.03

17.0F4.7 – 16.7F7.2

North Adriatic Sea South Adriatic Sea Japan

This study Storelli et al. (1998) Sakai et al. (2000b)

Muscle n=17 n=12 n=21 n=4 n=6 n=7 n=1

0.36F0.11 0.14F0.16 0.08F0.05 0.57* 0.06F0.03 0.06F0.03 –

1.5F0.4 – 0.73F0.45 – 0.81F0.28 0.83F0.26 –

60.9F38.3 – – – 19.80F8.7 20F8 –

2.7F1.2 – – – 0.28F0.11 0.3F0.1 –

2.76F0.60 – – – 0.083F0.026 0.58

30.9F8.0 – 19.6F5.7 – 25.0F3.5 24.2F3.8 –

North Adriatic Sea South Adriatic Sea French Atlantic Coasts Cyprus Japan Japan British Coasts

This study Storelli et al. (1998) Caurant et al. (1999) Godley et al. (1999) Sakai et al. (2000b) Sakai et al. (1995) Godley et al. (1998)

Fat n=7 n=6

2.33F0.52 0.07F0.04

3.4F1.7 0.11F0.03

132.3F121.0 9.92F5.61

8.4F2.5 0.12F0.09

18.77F4.40 b0.03

68.2F34.7 96.1F18.8

North Adriatic Sea Japan

This study Sakai et al. (2000b)

Values are reported in mg/kg of wet mass and expressed as meanFS.D. * S.D. was not reported by authors.

Cd levels detected in adults with respect to young individuals might be related to the onset of sexual maturity, when the increasing hormonal activity could affect the metabolic processes responsible for the uptake and organotropism of metals. Recent studies demonstrated the occurrence of metals bound to metallothioneins in the liver of turtles (Anan et al., 2002b; Rie et al., 2001), and since metallothionein levels have been reported to change with age (Law, 1996; Sakai et al., 2000a), this feature can be of relevance in determining trends of metal accumulation. Some investigations have also been carried out to test a possible relationship between content of heavy metal and sex, or apparent cause of death. According to our data, concentration did not differ between males and females, nor between individuals found stranded and those victims of bycatch (data not shown). 3.4. Relationship between heavy metal contents and environmental features We have observed that all tissues of C. caretta from the northwest Adriatic Sea have significantly higher Ni and Mn concentrations than those of turtles from other habitats; similarly, levels of Cd and Mn in lung, muscle and adipose tissue were significantly higher than those reported in other studies. We hypothesize that these findings are related to the coastal Adriatic Sea environmental features (i.e. water,

sediments, benthonic flora and fauna). A gradient of Cd concentration in water has been observed along the Adriatic Sea (Tankere and Statham, 1996), with minimum levels of 0.05 nM in the south, intermediate concentrations (0.06– 0.08 nM) in the central part of the basin, and about 0.15 nM Cd in the north. In particular, the marine environment close to the Po Delta and the Goro Bay is noted for its high content of heavy metals (Tankere et al., 2000), and concentrations as high as 3.3 Ag/l in water have been measured in this area (Fagioli et al., 1994). A progressive south–northward increase of Ni concentration has been found, with about 4 nM Ni in the southern Adriatic, 6 nM around the Yabuka Pit (Central Adriatic Sea), up to 30 nM in the northern part of the basin (Tankere and Statham, 1996). Ni levels as high as 170 nM have been measured in water from the Goro Bay (Fagioli et al., 1994). Similar trends have been recognized in surface sediments (Dinelli et al., 1996). Algae accumulate heavy metals readily from seawater, therefore reflecting the environmental features. Studies recently carried out in the Goro lagoon on Ulva laetevirens and Gracilaria verrucosa have shown high bioaccumulation of Cd Cu and Zn, in parallel with high levels of the same metals in the sediments (Roncarati, 2003). Several natural processes contribute to the high levels of heavy metals recognized in the north Adriatic Sea environment as well; among them, riverine inputs and remobilization from sediments play a pivotal role (Tankere

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Fig. 3. Relationship between heavy metal concentration in different tissues and the size classification of C. caretta, evaluated by minimum straight-line carapace length (MSCL). Each point corresponds to one individual analysed in triplicate. *Statistically significant correlation, Pb0.05.

et al., 2000). North Adriatic Sea is the final acceptor of a wide fluvial system composed of the Po River, its tributaries, and a series of secondary rivers flowing from the Italian border. Mass balance assessments demonstrated that metals supplied through north Italian rivers accounts for 3.2% (Mn), 2.8% (Fe), 9.8% (Zn), 12.6% (Cu) and 13.5%

(Ni) of the whole metal load of the north Adriatic Sea (Tankere et al., 2000). A geochemical characterization of the sedimentary yield for these rivers (Dinelli and Lucchini, 1999) has recently indicated high content of heavy metals in those rivers draining the western and the southern Alps, and the Ligurian–Emilian section of the northern Apennines.

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The presence of ultramafic lithological formations in these areas represents an important source of metals, such as Cr, Cu, Ni, released through weathering processes. Dinelli and Lucchini (1999) underlined that this ultramafic contribution to the sediment chemistry is particularly relevant for the Po River, with concentrations of Ni, Cu and Zn several times higher with respect to other rivers. The north Adriatic Sea also has a peculiar sedimentary environment. Sedimentary rates are very high compared with those of the middle Adriatic (5.3 vs. 1 kg m 2 year 1), and almost 97% Mn, 98% Fe, 93% Zn, 90% Cu and 88% Ni of the sedimentation flux is remobilized and diffused across the water column (Tankere et al., 2000). Direct measurements showed that such bbenthic fluxesQ are generated by the anoxic conditions commonly occurring at the water– sediment interface in late summer, when the surface circulation in this area is rather slow, and strong vertical temperature and salinity gradients produce a pronounced horizontal stratification of the water column (Zavatarelli et al., 1998). Several authors suggested that the main route of metal accumulation in sea turtles is the diet (Anan et al., 2001; Caurant et al., 1999), and, being at the top of its food web, C. caretta could be exposed to high levels of heavy metals resulting from bioamplification processes across the trophic chain. Bivalves could be a relevant source of metals, because they are at the base of the loggerhead diet (Bjorndarl, 1997), and they have the capacity to accumulate metals up to high body concentrations, particularly in contaminated environments. In fact, significant amounts of Ni, Cd and Zn have been found in bivalves collected in Goro Bay (Fagioli et al., 1994; Perdicaro et al., 1997), in agreement with the observed environmental background values.

4. Conclusions The present study supports a previous hypothesis that the Adriatic Sea is on the migratory routes of C. caretta. In particular, the rescue of both large and small size individuals along the north Adriatic coasts suggests that the basin provides suitable environments for both adult and sub-adult foraging, and also for development of younger individuals. Lung, muscle and fat of C. caretta inhabiting the northwestern Adriatic Sea showed significantly higher concentrations of Ni, Mn, Cd and Cu than turtles form other areas (Storelli et al., 1998; Caurant et al., 1999; Godley et al., 1998, 1999; Sakai et al., 1995, 2000b). The same is true for liver, although limited to Mn and Ni. At present, we cannot state whether such high values may have detrimental effects on loggerhead turtles health; nevertheless, we hypothesize that they are related to the peculiar environmental and ecological features of the northwestern Adriatic Sea, which is used by Mediterranean C. caretta populations as feeding grounds (Dodd, 1988; Argano et al.,

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1992). In agreement with previous reports (Stoneburner et al., 1980; Bowen et al., 1994, Nagle et al., 2001), we therefore suggest that specific heavy metals could be used as benvironmentally acquired markersQ to investigate the feeding area frequented by the sea turtles and also as indicators of the animal–environment interaction.

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