1. INTRODUCTIONDetection and monitoring of environmental contamination in the Mediterranean, an almost closed sea, are matters of concern, particularly in heavily urbanized and industrialized areas [1]. The major sources of pollution of surface waters include effluent discharges by industries, urban discharge, atmospheric depositions of pollutants and occasional accidental spills of toxic chemicals. Trace metals are regarded as serious pollutants of the aquatic environment because they are persistent, potentially toxic and naturally bioaccumulated [2,3]. There is currently a great interest in the use of living organisms as pollution biomonitors in aquatic ecosystems [4,5]. Among these, the seagrasses Zostera marina L. and Posidonia oceanica L. (Delile), key species of the coastal ecosystems, have been used as a metal bioindicator for several decades [6-16]. Arsenic can be taken up, bioaccumulated and biotransformed. Its toxic effects depend on the chemical form of its compounds and there is a growing interest in the quantification and identification of the arsenic in matrices related to human and animal food (e.g. sediments, algae, seagrasses, shellfish). Also it has been observed that P. oceanica could “record” past metal contamination [17-23]. Annual heavy metal accumulation can be analyzed by dating fragments of rhizomes and scales (dead sheaths of the leaves) according to plant annual life cycle [17]. Therefore, the time course of such a contamination can be reconstructed over a period ranging between few to many years. Since the early 1950’s, the area of Crotone (South-Eastern Mediterranean-Ionian Sea-Calabria, Italy), has become one of the most important petrochemical sites in the South of Italy, with several petrochemical plants and many other industrial installations. About the metal contamination of this zone, there are very few published data [24]; in accordance with Italian law 208 of 30/12/2008, the same industrial area was declared an ‘‘area at high risk of environment crisis” and must be subjected to environmental restoration for serious contamination by heavy metals (overall As, Zn, Cd, Cr). In this context, we performed a lepidochronological analyses by sampling P. oceanica from meadows growing in two different sites (referred as Disturbed and Control, see section below) in order to dected, for the first time at a local scale, the the level of the trace metals As, Cd, Cu, Pb. The distribution of these metals was also verified with respect to the leaf axis (base vs apical portion) as well as the stage of the leaf development (juvenile vs adult leaf), in order to suggest some differences along a gradient of leaf age. The total arsenic occurrence was furthermore analysed in P. oceanica during a relatively long period (nine years) demonstrating the capability of the scales as a preferential organs to biotransfer this toxic contaminant.
2. MATERIAL AND METHODS2.1. Sampling and Tissues PreparationPlants of P. oceanica were collected during the summer of 2004 by scuba diving at 10 ± 1 m depth from meadows growing in two sites of the East coast of Calabria (Ionian Sea, Italy) (Figure 1). Probably, the two sampling sites were differently impacted: the first meadow was at Località Tonnara (38˚54.881'N 16˚59.845'E), a site very close to the industrial harbour of Crotone, characterized by petrochemical traffic and a metallurgical plant, therefore this site was considered as disturbed (D); the second meadow was located at Capo Bianco (38˚55.014'N 17˚07.857'E), away from the bay of Crotone, in the Marine Protected Area (MPA) of Capo Rizzuto (Figure 1); this area is usually considered a pristine region with widespread P. oceanica meadows [25] and was considered as a reference site (control: C). Plants of P. oceanica were collected in each meadow along five different transects that were 50 meters separate; twenty plants were collected in each area with orthotropic rhizomes (vertical growth) at a distance of at least 50 - 100 cm apart in order to collect plants belonging to different individuals. The associated superficial sediments were also collected to a depth of about 5 cm.
The macroscopic epiphytes were then carefully removed by gently scraping and washing and the leaves were separated into adult, intermediate and juvenile leaves according to Giraud (1979) [26]. Each leaf was fractionated in three different portions along the main leaf axis: the tip (last 3 cm), the basal portion (the growing one, including part of the blade and the sheath) and the intermediate one. For the lepidochronological analyses, the dead sheaths (scales) and rhizomes were fractioned according to Pergent (1990) [27]. The collected tissues were dried at 105˚C for 48 h, milled to obtain particles of approximately 100 µm in size and then processed for metal analyses. Also the sediment samples, after sieving on 2-mm meshes, were dried at 105˚C for 48 h.
2.2. Trace Metal AnalysesThree subsamples of the ground material (500 mg∙dw) were hot digested in a microwave oven with a mixture of 5 ml of HNO3 65% and 2 ml of H2O2 30%. As, Cd, Cu and Pb concentrations were determined in all samples by an Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Agilent Technologies, 7500 CE Octopole Reaction System). The accuracy of the method was evaluated by its calibration versus an international standard (Stock Standard, Environmental Calibration Standard Agilent; Internal Standard Mix Agilent). A parallel analysis of blanks was also carried out to quantify possible contamination. The obtained data were analysed using the Student’s t-test for the paired sample and the results expressed as mean ± S.E. of three independent replicates for every metal analysed. The trace metals content in different tissue, sites and years were compared by 2-way ANOVA using the software package “R” (The R Foundation for Statistical Computing ISBN 3-900051- 07-0, version 2.14.1).
3. RESULTS3.1. Lepidochronological AnalysesThe arsenic content detected in the scales was tenfold higher than in the rhizomes, over all sampled years and in both sites (Figures 2(A) and (B); Tables 1, 2). In particular, the scales sampled at the D site showed a significant higher level of As compared with the C one in the period 1995-2004, with a peak of 22.3 µg∙g∙dw−1 at the D site after which a significantly decreasing trend was detected (Figure 2(A), Table 1). In the same lepidochronological year (1997) a peak of As (2.26 µg∙g∙dw−1) was also detected in the rhizome, after which a significantly downward trend was observed (Figure 2(B), Table 2). For Cd, comparable levels between scales and rhizomes were measured (Figures 2(C) and (D)) with the highest values (1.89 µg∙g∙dw−1) in the rhizome in 1995 at the D
site (Figure 2(D)); both in the scales as in the rhizome a significantly temporal fluctuation was observed (Figures 2(C) and (D); Tables 1 and 2). The content of Cu in the rhizomes was higher than in the scales with a clear trend to increase during the whole lepidochronological period reaching the maximum values (29.71 µg∙g∙dw−1) in 2004 in the rhizome of the both sites (Figures 2(E) and (F); Table 2). The concentration of Pb was significantly higher in the scales than the rhizomes (Figures 2(G) and (H); Tables 1 and 2). The highest Pb concentration (6.2 µg∙g∙dw−1) was recorded in the scales in the lepidochronological year 1996 at the D site; after which a constant significantly decline was detected (Figure 2(G); Table 1); otherwise, the Pb level measured in the rhizomes was approximately constant along the whole lepidochronological period, except a peak in the D site in the year 1998 (Figure 2(H); Table 2).
3.2. Trace Metals in Different Compartments of P. oceanica PlantsTable 3 shows the distribution of As, Cd, Cu, Pb in P. oceanica specimens and in the upper 5 cm of sediments sampled at the Disturbed and Control site respectively. The distribution of metals in the plant varied considera-