Tracing the route: Using stable isotope analysis to understand microplastic pathways through the pelagic-neritic food web of the Tyrrhenian Sea (Western Mediterranean).

This study provides a pilot investigation of the relationship between microplastic ingestion and the trophic ecology of three pelagic fish species (Engraulis encrasicolus, Scomber scombrus, and Trachurus trachurus) from Anzio coast, Tyrrhenian Sea (Western Mediterranean). Stable isotope analysis has been performed to determine the trophic position and the isotopic niche of the three species. Then, data on the occurrence, abundance, and diversity of ingested microplastics have been analyzed considering the observed foraging patterns. The detected differences in the estimated trophic position (E. encrasicolus = 3.08 ± 0.18; S. scombrus = 3.57 ± 0.21; T. trachurus = 4.07 ± 0.21), together with the absence of overlap in the isotopic niches confirm that the three examined species cover different ecological roles within the coastal-pelagic food web. Results from the analysis of ingested microplastics show that the trophic position has no remarkable effects on the incidence of microplastic ingestion, with no significant differences detected in terms of both frequency of occurrence and number of ingested microplastics per individual. However, differences among species emerge when considering the diversity of ingested microplastic types in terms of shape, size, color, and polymer composition. Species at higher trophic levels have shown to ingest a greater diversity of microplastics, including a significant increase in the size of the ingested particles (median surface area: 0.011 mm2 in E. encrasicolus; 0.021 mm2 in S. scombrus; 0.036 mm2 in T. trachurus). The ingestion of larger microplastics might be due to the larger gape sizes but also to active selection mechanisms, likely stimulated by the similarity of these particles to natural or potential prey of both S. scombrus and T. trachurus. Overall, this study suggests that microplastic ingestion can be affected by the different trophic position of fish species, providing new insights about the impact of microplastic contamination on the pelagic community.

First record of microplastic in the environmental matrices of a Mediterranean marine cave (Bue Marino, Sardinia, Italy).

This study investigates for the first time the presence of microplastics in sediment, water, and benthic organisms (foraminifera) of a marine cave in the Gulf of Orosei (Sardinia, Italy). Microplastics were found in all water, and sediment samples with similar shapes, sizes, and compositions; identified items were mainly fragments and fibers constituted by PVC and polyethylene. Their provenance was supposed to be predominantly from the sea than from the seasonal freshwater supplies from the karst system. Foraminiferal assemblages were mainly constituted by calcareous hyaline taxa in the outer station, while in the inner ones, the agglutinated Eggerelloides advenus was dominant. FTIR analyses on agglutinated shells identified polyethylene. Microplastic items are collected by the foraminifers and sediment grains building the shell chambers. This is the first study providing evidence that marine caves may be collectors of microplastics and that, in these habitats, microplastics enter the biotic matrix at the protist's level.

New method for simultaneous determination of dissolved organic carbon and its stable carbon isotope ratio in liquid samples: environmental applications.

This study reports the development of an all-in-one elemental analyser isotope ratio mass spectrometry (EA-IRMS) system modified for simultaneous analysis of dissolved organic carbon (DOC) concentration and its stable carbon isotope footprint (δ13CDOC) in aqueous samples. The method involves a quantitative oxidation of DOC in a 200 µL liquid sample to CO2, after sample acidification and stripping by nitrogen. The detection limit of the method for DOC quantification was 0.2 mg C/L with an analytical precision of 12 %. Uncertainty of stable isotope determinations was 2 % at 0.2 mg DOC/L, while decreasing to 0.3 % at 20 mg DOC/L. Quantitative oxidation of DOC in aqueous samples was validated by using ring test water samples and Deep Sea reference seawater. The method performances of isotope analysis were evaluated by analysing different isotopic standard solutions. The applicability of the method was tested through the analysis of different environmental types of water, showing that δ13CDOC ranged from - 23.30 to -31.85 ‰, allowing to characterize samples of different environmental origin. The developed method offers several advantages including rapidity, use of small sample volumes and minimal sample pre-treatment, making it a valuable tool for routine DOC concentration measurements paired with isotopic characterization.

Snapshot of the Distribution and Biology of Alien Jellyfish Cassiopea andromeda (Forsskål, 1775) in a Mediterranean Touristic Harbour.

Harbors are hotspots for the introduction of alien species, and, usually, investigations on their host populations help fill the knowledge gap in their pathways of invasion and in their impacts on marine biodiversity and ecosystems. In 2014, the upside-down alien jellyfish Cassiopea andromeda invaded a Mediterranean touristic harbor ("Cala"), and its abundance has since increased over time. In the present study, the distribution and trophic behavior of C. andromeda in Cala were investigated for the years 2017-2018 through visual sampling, and GIS-based statistical and stable isotope analyses. Since Cala is a hard-to-reach area (with many anchor cables and boats), Megabenthos Underwater Video was used to count the number and estimate the size of jellyfishes. The variations in size throughout the study period suggest that the population of C. andromeda is quite established in Cala at depths lower than 7.5 m. The ranges of the environmental parameters recorded (temperature, salinity, and transparency) were consistent with the ideal conditions for maintaining a Cassiopea population, but they did not seem to influence aggregation. Additionally, the carbon and nitrogen isotopic signatures studied highlight the mixotrophic behavior of this species. These preliminary results confirm the capacity of C. andromeda to live and reproduce in heavily anthropized areas.

Environmental restoration by aquatic angiosperm transplants in transitional water systems: The Venice Lagoon as a case study.

The paper reports the results obtained after 4 years of aquatic angiosperm transplants in areas of the Venice Lagoon (North Adriatic Sea, Mediterranean) where meadows almost disappeared due to eutrophication, pollution and overexploitation of clam resources. The project LIFE12 NAT/IT/000331-SeResto, funded by the European Union, allowed to recolonize the Habitat 1150* (coastal lagoons) in the northernmost part of the lagoon, by extensive manual transplants of small sods or single rhizomes of Zostera marina, Zostera noltei, Ruppia cirrhosa and, in some stations also of Cymodocea nodosa. Over the 4 years of the project more than 75,000 rhizomes were transplanted in 35 stations with the support of local stakeholders (fishermen, hunters and sport clubs). Plants took root in 32 stations forming extensive meadows on a surface of approx. 10 km2 even if some failures were recorded in areas affected by outflows of freshwater rich in nutrients and suspended particulate matter. The rapid recovery of the ecological status of the involved areas was the result of this meadow restoration, which was in compliance with Water Framework Directive (WFD 2000/60/EC) objectives. Moreover, the monitoring of environmental parameters in the water column and in surface sediments allowed to identify the best conditions for successful transplants. Small, widespread interventions and the participation of local stakeholders in the environmental recovery, make this action economically cheap and easily transposable in other similar environments.

Preliminary multi analytical approach to address geographic traceability at the intraspecific level in Scombridae family.

Globalization of seafood product marketing caused the increase of request of an effective fish traceability that enhances the consumer confidence in food safety. In this study, an integrated multi analytical approach based on two different and independent analytical techniques (carbon and nitrogen stable isotopes and fatty acids analysis) was applied in order to identify different fish species and trace their geographical provenience. The investigation was focused on four species (Thunnus thynnus, Thunnus alalunga, Auxis rochei and Scomber scombrus) belonging to the Scombridae family. The DNA barcoding method confirmed genus and species for S. scombrus and A. rochei, but only genus for T. alalunga and T. thynnus. Carbon and nitrogen stable isotopes results evidenced different fish diets and trophic positions, whereas fatty acids analysis displayed that the unsaturated prevailed (∼60 %) over the saturated compounds with a variation among the species and the geographical area in particular for docosahexaenoic and eicosapentaenoic acids percentage. The principal component analysis applied to stable isotopes and fatty acids evidenced a good discrimination among species and their geographical catching area. This multi-disciplinary analytical approach could represent a promising tool to identify the commercial fish and trace their origin in order to guarantee the health of consumers.

Organotin compounds in touristic marinas of the northern Adriatic Sea: occurrence, speciation and potential recycling at the sediment-water interface.

Butyltin compound (BTC) contamination was evaluated in two north Adriatic marinas, San Rocco (Italy) and Lucija (Slovenia). BTC sedimentary concentrations (121 ± 46 and 352 ± 30 ng Sn g-1 in San Rocco and Lucija, respectively) evidenced the past use of antifouling paints, confirmed by the reduced tributyltin content (~ 46%) with respect to the sum of BTC. Elemental and organic carbon isotopic (δ13C) analyses of bulk sediments and its lipid and humic substances were performed in order to evaluate their role in BTC partitioning and preservation. The δ13C of sedimentary bulk and refractory organic matter suggested that diagenetic processes could play a role in the preservation or release of pollutants. No contamination was found in water collected from the benthic chamber and thus, fluxes at the sediment-water interface were not assessed, except for MBT efflux at Lucija (28.9 ng Sn m-2 day-1). Nevertheless, BTC concentrations in porewaters (up to 75 ng Sn l-1) and rather low sediment-porewater partitioning coefficients (Kd) with respect to the data reported in the literature would suggest a potential risk of the reintroduction of BTC into the water column at both sites: at Lucija, sedimentary contamination is high despite the greater Log Kd, whilst at San Rocco, the low BTC concentration is associated with a reduced sediment affinity.

Contribution of deltaic wetland food sources to coastal macrobenthic consumers (Po River Delta, north Adriatic Sea).

Estuaries are known to export huge quantities of terrigenous/riverine organic matter and nutrients to coastal areas, subsidizing food sources to their communities; however, this process is seldom investigated in complex multichannel delta-prodelta systems. Stable isotope analysis was used to investigate the contribution of organic sources originated from the Po River Delta, one of the widest deltaic wetlands in the northern Mediterranean Sea, to the diet of the nearby coastal macrobenthic consumers, and their influence on invertebrates' trophic structure and biomass. Following intense river flood events, macrofauna samples were collected in the prodelta area at increasing distance from the main river distributary mouth. Potential primary resources were collected within the delta and the prodelta areas. A terrestrial signal in the primary consumers' diet was evident as a shift in their δ13C, being this more 13C depleted near the main river distributary. The Bayesian mixing models indicated an important contribution of deltaic sources, in particular C4-grass detritus (25-57%), to primary consumers' diet, confirming a strong energetic link between delta and prodelta areas. Overall, most of the consumers' biomass were concentrated at sites close to the main distributary mouth, mainly as suspension and surface deposit feeders. A simplification of the macrobenthic community structure, accompanied by narrower transfer pathways was also evident at sites more distant from the main river distributary, in relation to changes in the quality of resources. Our data, although limited to winter season, suggest that during periods of low in situ productivity but high river flow energy, invertebrates are able to efficiently exploit terrigenous food sources, restraining the detrimental effect of increased turbidity and sedimentation. We conclude that riparian/wetland vegetation associated with river deltas can provide important food sources to marine primary consumers. These results furthermore highlight the need for integrated management and protection strategies of connected land-sea ecosystems.

Preliminary study to characterize plastic polymers using elemental analyser/isotope ratio mass spectrometry (EA/IRMS).

Plastic waste is a growing global environmental problem, particularly in the marine ecosystems, in consideration of its persistence. The monitoring of the plastic waste has become a global issue, as reported by several surveillance guidelines proposed by Regional Sea Conventions (OSPAR, UNEP) and appointed by the EU Marine Strategy Framework Directive. Policy responses to plastic waste vary at many levels, ranging from beach clean-up to bans on the commercialization of plastic bags and to Regional Plans for waste management and recycling. Moreover, in recent years, the production of plant-derived biodegradable plastic polymers has assumed increasing importance. This study reports the first preliminary characterization of carbon stable isotopes (δ13C) of different plastic polymers (petroleum- and plant-derived) in order to increase the dataset of isotopic values as a tool for further investigation in different fields of polymers research as well as in the marine environment surveillance. The δ13C values determined in different packaging for food uses reflect the plant origin of "BIO" materials, whereas the recycled plastic materials displayed a δ13C signatures between plant- and petroleum-derived polymers source. In a preliminary estimation, the different colours of plastic did not affect the variability of δ13C values, whereas the abiotic and biotic degradation processes that occurred in the plastic materials collected on beaches and in seawater, showed less negative δ13C values. A preliminary experimental field test confirmed these results. The advantages offered by isotope ratio mass spectrometry with respect to other analytical methods used to characterize the composition of plastic polymers are: high sensitivity, small amount of material required, rapidity of analysis, low cost and no limitation in black/dark samples compared with spectroscopic analysis.

DoE optimization of a mercury isotope ratio determination method for environmental studies.

By using the experimental design (DoE) technique, we optimized an analytical method for the determination of mercury isotope ratios by means of cold-vapor multicollector ICP-MS (CV-MC-ICP-MS) to provide absolute Hg isotopic ratio measurements with a suitable internal precision. By running 32 experiments, the influence of mercury and thallium internal standard concentrations, total measuring time and sample flow rate was evaluated. Method was optimized varying Hg concentration between 2 and 20 ng g(-1). The model finds out some correlations within the parameters affect the measurements precision and predicts suitable sample measurement precisions for Hg concentrations from 5 ng g(-1) Hg upwards. The method was successfully applied to samples of Manila clams (Ruditapes philippinarum) coming from the Marano and Grado lagoon (NE Italy), a coastal environment affected by long term mercury contamination mainly due to mining activity. Results show different extents of both mass dependent fractionation (MDF) and mass independent fractionation (MIF) phenomena in clams according to their size and sampling sites in the lagoon. The method is fit for determinations on real samples, allowing for the use of Hg isotopic ratios to study mercury biogeochemical cycles in complex ecosystems.

A fast and effective routine method based on HS-SPME-GC-MS/MS for the analysis of organotin compounds in biota samples.

This work validated an automated, fast, and low solvent- consuming methodology suited for routine analysis of tributyltin (TBT) and degradation products (dibutyltin, DBT; monobutyltin, MBT) in biota samples. The method was based on the headspace solid-phase microextraction methodology (HS-SPME), coupled with gas chromatographic separation and tandem mass-spectrometry (GC-MS/MS). The effectiveness of the matrix-matched signal ratio external calibration was tested for quantification purposes. The exclusion of matrix influences in the calibration curves proved the suitability of this versatile quantification method. The method detection limits obtained were of 3 ng Sn g(-1) dw for all the analytes. The analysis of references materials showed satisfying accuracy under optimum calibration conditions (% recovery between 87-111%; |Z-scores|<2). The repeatability RSD% and intra-laboratory reproducibility RSD% were lower than 9.6% and 12.6%, respectively. The work proved the remarkable analytical performances of the method and its high potential for routine application in monitoring organotin compounds (OTC).

Evidence of butyltin biomagnification along the Northern Adriatic food-web (Mediterranean Sea) elucidated by stable isotope ratios.

The biomagnification of tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), and total butyltins (ΣBT) was analyzed in the Northern Adriatic food-web (Mediterranean) considering trophodynamic interactions among species and carbon sources in the food-web. Although it is acknowledged that these contaminants bioaccumulate in marine organisms, it is still controversial whether they biomagnify along food-webs. A wide range of species was considered, from plankton feeders to top predators, whose trophic level (TL) was assessed measuring the biological enrichment of nitrogen stable isotopes (δ(15)N). Carbon isotopic signature (δ(13)C) was used to trace carbon sources in the food-web (terrestrial vs marine). At least one butyltin species was detected in the majority of samples, and TBT was the predominant contaminant. A significant positive relationship was found between TL and butyltin concentrations, implying food-web biomagnification. Coherently, the Trophic Magnification Factor resulted higher than 1, ranging between 3.88 for ΣBT and 4.62 for DBT. A negative but not significant correlation was instead found between δ(13)C and butyltin concentrations, indicating a slight decreasing gradient of contaminants concentrations in species according to the coastal influence as carbon source in their diet. However, trophodynamic mechanisms are likely more important factors in determining butyltin distribution in the Northern Adriatic food-web.