International Journal of Marine Biology and Research

Cadmium, mercury, Lead, Copper and Polycyclic Aromatic Hydrocarbons, in liver, kidney, muscle and heart samples of 5 loggerhead turtles Caretta caretta (Linneaus, 1758) stranded along the north Tunisian coast were measured. Analyses were performed by using the Atomic Absorption Spectrometry (AAS, VARIAN 220Z) for trace metals and the mass chromatography for PAH. The results demonstrated that the kidney and the liver are the main concentrated organs and muscles generally display the lowest trace metals and PAH concentration. By comparing with some bibliographic data, the mean trace metals concentrations are about identical to loggerhead turtles’ tissues of others localities in Mediterranean Sea. In addition, obtained results show that the kidney is relatively the most concentrated organ in cadmium, lead and PAH, the liver and the heart are relatively the two most organs concentrated in copper and the highest concentrations of mercury were found in the liver. This is the first assessment into metal and polycyclic aromatic hydrocarbons distribution in tissues of loggerhead turtles from Tunisian Mediterranean Coastline.

Key words: Trace metals; polycyclic aromatic hydrocarbons; loggerhead turtles; stranding; Tunisia;

Three species of marine turtles: the leatherback turtle Dermochelys coriacea (Vandelli, 1761), the loggerhead turtle Caretta caretta (Linnaeus, 1758) and the green turtle Chelonia mydas (Linnaeus, 1758) are encountered in the Mediterranean. The leatherback turtle is a visitor from the Atlantic and can be found all over the basin, although it does not breed in the Mediterranean [9,24]. The other two species reproduce in the Mediterranean and have evolved local populations with a genetic divergence from the Atlantic populations (Casale & Margaritoulis 2010, and references therein). The main identified threats at sea to these two Mediterranean populations are incidental catch, collision and intentional killing while the impact of other potential threats like chemical contaminants and debris is not clear yet [28].

All three marine turtle species mentioned were reported in Tunisian waters and were protected by several international conventions [25]. Loggerhead turtles, the most common sea turtles found on the marine habitats off the Tunisian coasts [22], are the only species that nest mainly in the Kuriat Islands [10,22,23].

Since 1988, the data of first record of the loggerhead sea turtles nesting in Tunisia, a Tunisian Sea Turtle Program (TunSTP) included in the activities of the National Institute of Sea Sciences and Technology (INSTM) was launched in order to identify appropriate conservation measures for these species, which are listed in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [15]; this program include (i) the monitoring of the loggerhead turtles nesting sites, (ii) the by-catch surveys and (iii) the national stranding network [10]. Moreover, a specialized sea turtle rescue centre has been established in the National Institute of Marine Sciences and Technologies at Monastir in 2004.

Following industrialization, high quantities of chemical pollutants have been released and continue to be released into the sea altering the natural biological equilibrium [20] and the sanitary of aquatic animals, particularly those long living species such as marine mammals, sea birds and sea turtles which have the potential to accumulate organic and inorganic contaminants from food, sediment and water in their tissues [4,18]. Therefore, the concentrations of these contaminants may vary depending on biological factors such us age and migration habits.

Trace elements are natural components of rocks and soil. The erosions favour their release in aquatic ecosystem. These elements occur naturally in very low concentrations in the environment. They are not necessarily toxic but many anthropogenic activities increase their natural concentrations causing pollution [37]. Some of them persist in the tissues of animals and can biaccumulate over time. Trace elements can be either non essential (e.g. Cd, Pb, Hg and Cs) or essential (e.g. Co, Cu, Fe, Mn) to living organisms [3] and above a threshold concentration, they are considered toxic.

Polycyclic Aromatic Hydrocarbons (PAH) are compounds containing two or more fused aromatic rings in linear, angular or clustered arrangements. PAH have high molecular weight and low volatility and are known to be highly persistent in the environment [35]. Hydrocarbons may arise from petroleum hydrocarbons pollution and incomplete combustion of organic materials such as wood, coal and oils. The health hazard posed by these compounds has been extensively studied by several authors [6,42]. Several polycyclic aromatic hydrocarbons were studied, including sixteen that are reported in the priority list of pollutants of the US Environmental Protection Agency (EPA). PAHs are bio available to fish and other marine organisms through the food chain, as waterborne compounds and from contaminated sediments. PAHs uptake always depends on their bioavailability as well as the physiology of the organisms [31]. Such compounds have adverse effects on health (carcinogenic and/or mutagenic activity) and ecosystem [27]. Numerous studies indicate that one-, two- and three-ring compounds are acutely toxic, while higher molecular weight are considered to be genotoxic [32,33].

Despite the extensive literature regarding the concentrations of contaminants in loggerhead turtles in the Mediterranean Sea [1,17,21,30,37], there is currently no available information on chemical elements in sea turtles stranded along Tunisian coast.

Considering the importance of pollution as a threat to sea turtles [1,11,14,29,38], the present study aims to assess levels of four trace metals Cadmium (Cd), Mercury (Hg), Lead (Pb), Copper (Cu) and Polycyclic Aromatic Hydrocarbons (PAH), in the tissues of the loggerhead turtles from the coast of Tunisia and to compare these data with those reported from other locations in Mediterranean Sea.

Tissues samples were taken during necropsies of five loggerhead turtles stranded along the north east Tunisian coasts (Figure 1) between July 2006 and November 2008. The Curved Carapace Length (CCL), measured to the nearest 1 cm using a flexible meter tape. Necropsies were performed and samples were taken only from fresh dead or slightly decomposed individuals. Sex was determined via visual examination of the gonads and in case of doubt confirmed by histology. Liver, kidney, muscle and heart were sampled and stored in bottles to avoid any contamination. All organs of all individuals were frozen at -20 °C until the chemical analysis was carried out.

Trace elements: Once in the laboratory, samples were freeze-dried and then grounded on an agate mortar to obtain a fine homogeneous powder. Separate sub-samples were analysis in three replicates. All element concentrations (μg/g) were expressed on a dry weight basis.

Digestion: To analyse Cd, Pb and Cu, the digestion of 200 mg of each organ was carried out in a Teflon bombs with 5 ml HNO3 using a microwave (Millestone type Ethos) digestion at 100% power with pressure set at 120 psi for 20 min, the overall digestion time for the one cycle was 40 min. Blank acid mixtures were digested in the same way [39]. Mercury was treated separately using 300 mg biota samples in 5 ml HNO3 using a water bath at a temperature of 90°C to dryness. The samples were then diluted to 50 ml with milli Q water [40].

Atomic Absorption Spectrometry (AAS): The dilutions of the acid extracts from both procedures were analysed by Atomic Absorption Spectrometry (AAS, VARIAN 220Z) using the graphite furnace technique and back ground corrections for cadmium and lead. The mercury concentration was determined by cold vapor technique using stannous chloride as a reducing agent and VGAAAS (Varian AA10) system.

The instrument was calibrated with diluted solution prepared from known stock solutions of each element. Accuracy of the determinations was checked by the analysis of the international standard reference material Nist 2976. The analytical precision was generally better than 5%.

Polycyclic Aromatic Hydrocarbons (PAH): Analysis of tissue samples was performed according to the technique proposed by Villeneuve, 1995. Appropriate blanks were analysed with each set of analyses. Sample comparisons were made with reference material (IAEA 406) for quality control purposes. Recoveries are ranged from 71% to 89% and the method detection limits are ranged from 0.05 to 0.25 ng g-1. Extraction of tissue was realised by a Soxhlet extractor during 8 h with Methanol. Internal standards 9, 10-dihydroanthracene, n-octadecene (C18:1) and n-dotriacontane (n-C32) were added to tissue samples before Soxhlet extraction to determine the recoveries during the analytical procedure. A saponification step was realised with addition of potassium hydroxide in a Soxhlet extractor during two hours to eliminate fatty acids. The mixture of KOH/MeOH was extracted twice with hexane into a glass separating funnel with a Teflon stopcock and then concentrated. Separation of PAH was performed with silica gel and alumina column. A mixture of hexane and dichloromethane (90:10 v/v) was used to obtain the PAH fraction. Analysis of PAH was performed by gas chromatography coupled with high resolution mass spectrometry (GC-HRMS) Varian 4000. All trace elements and PAH concentrations were expressed as (μg/g) dry weight (dw).

Mean trace elements and PAH concentrations were expressed with coefficients of variation. The comparison of the concentrations between organs for each metal was made with comparison t-test. Significant differences were considered at p < 0, 05.