Paraffin waxes in the North-Western Mediterranean Sea: A comprehensive assessment in the Pelagos Sanctuary, a Specially Protected Area of Mediterranean Importance.

Paraffin waxes are widely recognized as emerging marine pollutants, even their classification by the recent monitoring programs and the knowledge of their occurrence, and sources of contamination in marine ecosystems are poorly defined and reported. Wax presence and distribution have been evaluated in different environmental compartments in the Pelagos Sanctuary (Mediterranean Sea) floating on the sea surface and stranded on beaches, focussing on their characterization, accumulation areas and pollution inputs. More than 2500 yellow paraffin residues were detected and analysed in the study area showing a prevailing dimension smaller than 5 mm. The Genoa Canyon and the waters facing Gorgona Island resulted in the more polluted areas representing two distinct hotspots of wax accumulation potentially related to the high density of tanker vessels sailing to and from the harbour of Genova and Livorno. Higher concentrations of beached particles were found along the Tuscan coast (11 items/100 m) and on Pianosa Island (110 items/m2). This study gives valuable insights into paraffin wax pollution in the Pelagos Sanctuary, emphasizing the need for harmonized monitoring and detection methods to elucidate the potential impacts on marine organisms. Moreover, mitigating actions are crucial to prevent and curb the waxes pollution of marine ecosystems.

Assessing marine litter in a highly polluted area in the Mediterranean: A multi-perspective approach in the Saronikos Gulf, Greece.

Marine litter poses a significant environmental challenge in the Saronikos Gulf, Greece. Employing an integrated approach, data from both beach and underwater sites were analyzed. The average litter density on beaches was 2.61 items m-2, with plastic being the most common material, notably small polystyrene fragments and cigarette butts. The western part of the Gulf exhibited higher litter density, mainly due to surface circulation patterns. Most beaches fell short of meeting Good Environmental Status criteria for marine litter. Higher litter densities were observed in autumn. Benthic litter density decreased with depth, being 23 times higher in shallower waters, with plastic being the predominant type. This extensive study offers crucial insights into the pollution status and litter distribution in the Saronikos Gulf, contributing significantly to the global understanding of marine litter distribution on coastal ecosystems. Such information is crucial for raising awareness, informing policy decisions, and driving environmental actions.

Characterization of floating microplastic contamination in the bay of Marseille (French Mediterranean Sea) and its impact on zooplankton and mussels.

Microplastics (MPs) were sampled in three seasons from 2016 to 2018 in the Bay of Marseille, northwestern Mediterranean Sea, adjacent to a highly urbanized area. Six sites were selected according to their different characteristics (river mouth, treatment plants, protected marine area). Surface floating MPs were characterized (number, weight, typology and polymer) as was zooplankton. In addition, mussels were submerged and used to investigate ingestion. Finally, a hydrodynamic model was used to improve understanding of dispersion mechanisms. The annual averages of floating MPs values ranged from 39,217 to 514,817 items/km2. The MPs collected were mainly fragments principally composed of polyethylene and polypropylene. The mean abundance ratio (MPs/zooplankton) was 0.09. On average 87% of mussel pools were contaminated and ingested 18.73 items/100 g of flesh. Two hydrodynamic patterns were identified: the first retaining the MPs in the harbor, and the second dispersing them outside.

Minerals as additives for decreasing the toxicity of Mediterranean contaminated dredged sediments.

The management of dredged sediments is a priority issue in the Mediterranean sea where sediments are historically polluted. The aims of this study were to evaluate the toxicity of port sediment samples and the effect of three mineral additives (hematite, zerovalent iron (ZVI) and natural zeolite (NZ)) on sediment elutriate toxicity. Four sediments (A, B, C and D) were provided by port authorities after composting procedure; particle size, particulate organic carbon, metals and organic pollutants (TBT, PAHs, PCBs) were determined in whole sediments. Elutriates from these composted sediments were analyzed by determining toxicity level using oyster (Crassostrea gigas) larvae bioassay, metal and dissolved organic carbon concentrations. Toxicity, measured on undiluted elutriates (250 g/L), decreased as follows: A≥B>C∼D. The treatment of sediments with mineral additives (5%) revealed that hematite tends to decrease the elutriate toxicity in all samples, particularly in samples B and C. This effect may be related to metal concentration decrease in elutriates, in particular Cu and Zn, that have a significant toxic effect on oyster larvae. ZVI and NZ have a variable influence on elutriate toxicity. Results suggest that hematite may be a possible candidate for decreasing chemical concentration and improving the quality of elutriates. Hematite could be used for sediment stabilization prior to the deposit in a specific site or landfill.

Lessons learned from an intercalibration exercise on the quantification and characterisation of microplastic particles in sediment and water samples.

An intercalibration exercise on the characterisation of microplastics in marine sediment and water samples was carried out among five laboratories involved in the implementation of the Marine Strategy Framework Directive (MSFD) in their country. The samples were prepared by mixing cleaned natural sediment and sea water with microplastics sets made of particles of various polymers, shapes and colours. Overall, the errors on total counts were under 25% in absolute value. The risk of non-detection and loss of particles is greater than the risk of contamination during sample analysis. Significant differences are observed among particle types. It appears difficult to obtain reliable and comparable data on the colour of microplastics. A comparison of the errors with regards to the protocols used led to recommend NaCl [1.2 g/cm3] density separation for sediment and one filtering step (200 µm). The operators' experience appears as a key factor for the quality of the results.

Assessment of the environmental quality of French continental Mediterranean lagoons with oyster embryo bioassay.

In order to better understand environmental disturbances in the French coastal Mediterranean lagoons, we used an ecotoxicological approach based on the measurement of the toxicity of the sediments using oyster embryo bioassay that provides a basis for assessing the effects on the fauna of contaminants adsorbed on the sedimentary particles. The study covers all of the main lagoons of the French Mediterranean coasts of Languedoc Roussillon, Camargue, and Provence (Berre and Bolmon lagoons), where 188 stations were sampled. The toxicity tests provide evidence of variable levels of toxicity in sediments. Contaminated lagoons such as La peyrade, Le canet, and Ingrill and locally affected lagoons such as Bages-Sigean, Vaccares, Bolmon, and Berre have sampling stations with 100% of larval abnormalities during 24-h development. In all of the lagoons, the toxicity was mainly located close to local harbors and rivers. Salses Leucate (Languedoc roussillon) lagoon was found very clean, with no important toxicity. The results are discussed in terms of environmental disturbances of the coastal lagoons and with regard to the long-term monitoring of the impact of contaminants on the coastal environment.

Seafloor litter from the continental shelf and canyons in French Mediterranean Water: Distribution, typologies and trends.

Seafloor litter has been studied both on the continental shelves (by trawling during 24 years) and in canyons (by ROV) of the French Mediterranean sea Water (FMW). On the continental shelf, mean densities range from 49.63 to 289.01 items/km2. The most abundant categories were plastic, glass/ceramics, metals and textiles. Trend analysis shows a significant increase in plastic quantities during the study period. Plastics accumulate at all depths, with heavier items being found in deeper areas, while the continental slope-break appears as a clean area. The spatial distribution of litter revealed the influence of geomorphologic factors, anthropic activities, shipping route, river inputs. All the canyons are affected by debris but coastal canyons (Ligurian Sea and Corsica) were more impacted than offshore canyons in the Gulf of Lion. The FMW appears to be highly polluted with regard to values found in other areas, but lower than those observed in the Eastern Mediterranean.

Tools and constraints in monitoring interactions between marine litter and megafauna: Insights from case studies around the world.

Adverse impacts of marine litter is documented on >1400 species, including marine megafauna (fish, birds, sea turtles and mammals). The primary impacts include ingestion and entanglement, and there is increasing concern about chemical contamination via ingestion. Numerous survey approaches and monitoring programs have been developed and implemented around the world. They may aim to provide data about parameters such as species distribution and interactions with anthropogenic activities. During the Sixth International Marine Debris Conference, a session was dedicated to the tools and constraints in monitoring interactions between litter and megafauna. In the present paper, we summarize 7 case studies which discuss entanglement and ingestion including macro- and micro-debris in several taxa and across multiple geographic regions. We then discusses the importance of tools and standardizing methods for assessment and management purposes, in the context of international environmental policies and marine litter strategies.

Optimising beached litter monitoring protocols through aerial imagery.

The monitoring of beached litter along the coast is an onerous obligation enshrined within a number of legislative frameworks (e.g. the MSFD) and which requires substantial human resources in the field. Through this study, we have optimised the protocol for the monitoring of the same litter along coastal stretches within an MPA in the Maltese Islands through aerial drones, with the aim of generating density maps for the beached litter, of assisting in the identification of the same litter and of mainstreaming this type of methodology within national and regional monitoring programmes for marine litter. Concurrent and concomitant in situ monitoring of beached litter enabled us to ground truth the aerial imagery results. Results were finally discussed within the context of current and future MSFD monitoring obligations, with considerations made on possible future policy implications.

The degradation potential of PET bottles in the marine environment: An ATR-FTIR based approach.

The dominance and persistence of plastic debris in the marine environment are well documented. No information exists in respect to their lifespan in the marine environment. Nevertheless, the degradation potential of plastic litter items remains a critical issue for marine litter research. In the present study, polyethylene terephthalate bottles (PETs) collected from the submarine environment were characterized using ATR-FTIR in respect to their degradation potential attributed to environmental conditions. A temporal indication was used as indicative to the years of presence of the PETs in the environment as debris. PETs seem to remain robust for approximately fifteen years. Afterwards, a significant decrease of the native functional groups was recorded; some even disappear; or new-not typical for PETs-are created. At a later stage, using the PET time series collected from the Saronikos Gulf (Aegean Sea-E. Mediterranean), it was possible to date bottles that were collected from the bottom of the Ionian Sea (W. Greece). It is the first time that such a study has been conducted with samples that were actually degraded in the marine environment.

A MSFD complementary approach for the assessment of pressures, knowledge and data gaps in Southern European Seas: The PERSEUS experience.

PERSEUS project aims to identify the most relevant pressures exerted on the ecosystems of the Southern European Seas (SES), highlighting knowledge and data gaps that endanger the achievement of SES Good Environmental Status (GES) as mandated by the Marine Strategy Framework Directive (MSFD). A complementary approach has been adopted, by a meta-analysis of existing literature on pressure/impact/knowledge gaps summarized in tables related to the MSFD descriptors, discriminating open waters from coastal areas. A comparative assessment of the Initial Assessments (IAs) for five SES countries has been also independently performed. The comparison between meta-analysis results and IAs shows similarities for coastal areas only. Major knowledge gaps have been detected for the biodiversity, marine food web, marine litter and underwater noise descriptors. The meta-analysis also allowed the identification of additional research themes targeting research topics that are requested to the achievement of GES.

The micronucleus assay in Crassostrea gigas for the detection of seawater genotoxicity.

The micronucleus (MN) test was performed in vivo and in vitro on the oyster Crassostrea gigas to evaluate the genotoxic effect of the marine environment. In vitro tests were carried out on adult and young (spat) specimens exposed to benzo[a]pyrene (BaP: 0.5, 5, 500 and 1000 micrograms.l-1) and an effluent (5, 50, 75 and 100%) of Seine Bay, one of the most highly contaminated sites in France. MN frequency observed after 48 h exposure to the two pollutants was much greater in adults than spats. A preliminary test of the genotoxic effect of BaP (0.05, 0.5, 1 and 500 micrograms.l-1), cupric sulfate (10, 25, 50 and 100 micrograms.l-1) and a paper mill effluent (1, 3, 10 and 30 mg.l-1) was performed in C. gigas heart cells cultured for 6 days. Comparison of the MN assay with the C. gigas larva test showed the clastogenic action of BaP and the toxic effect of cupric sulfate on culture cells as well as the slighter toxic effect of paper mill effluent on spats. An in vivo study was conducted in an oyster-farming area contaminated by cadmium and copper. MN frequency was not very sensitive to a pollution gradient but showed high interindividual variability. The absence of precise criteria for MN identification in mollusks and the identification of highly basophilic spherical inclusions in the cytoplasm of gill tissue hemocytes in oysters during viral infection are handicap for application of the micronuclei assay in the marine environment. Another limitation of the assay is the particularly onerous requirement for manual observation. Optimization of the assay by automated analysis is necessary but can only be achieved if cytologic preparations are of good quality.

Evidence of p53 mutation in an early stage of liver cancer in European flounder, Platichthys flesus (L.).

A number of flounders dwelling in highly contaminated coastal areas of Northern Europe develop liver tumours. In order to increase our understanding of the molecular pathogenesis of these sporadic tumours, we examined p53 mutations in eleven hyperplasia and six adenoma. p53 introns 4 to 8 were first sequenced to allow individual amplification of exons 5 to 8. DNA extracted from formalin-fixed livers was amplified and PCR products were directly sequenced. Two major results were obtained. (i) Flounders from different geographical areas displayed a high rate of sequence variation. Base substitutions were identified in both tumour and normal tissues and thus may be considered as polymorphic variations in individuals. (ii) One mutation was detected in two hyperplastic foci from the same flounder. This mutation was a T:A to A:T transversion at codon 147, resulting in the replacement of valine for glutamic acid. This residue took place in the L2 loop of the DNA binding surface. Its substitution by an hydrophilic and charged residue could thus impair p53 (protein) biological activity.

cDNA cloning and expression analysis of flounder p53 tumour suppressor gene.

The screening of a flounder cDNA library with a rainbow trout p53 probe allowed the isolation of a 2.8-kb fragment homologous to human (50%) and rainbow trout (57%) p53 coding sequences. The fragment contains a single open reading frame coding for a 366-amino acid protein. The predicted amino acid sequence is relatively divergent from other p53 proteins but it displays the main p53 features: five highly conserved domains, an acidic N-terminus, a hydrophilic and charged C-terminus, a penultimate serine residue and a putative nuclear localization signal. Furthermore, conservation of critical amino acids and comparable distribution of charge and hydrophobicity suggest that flounder p53 properties could be similar to those in mammals. Northern blot analysis revealed a single transcript of about 3 kb in the flounder ovary tissues. In fact, RT-PCR showed an ubiquitous but very low expression of p53 gene in all flounder tissues.

A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis.

A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601-893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum. This procedure involves the use of all tissues of P. linearis and live cell suspensions of H. ostreria or S. costatum, as DNA templates, without any further purification of DNA. As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species. Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene. The method was then used to screen mutations in partial psbA genes of 23 samples of P. linearis collected at four different stations along the mid-Atlantic coast of France. An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance. All mutations found were silent. However, genetic polymorphism discriminated between samples collected from two stations. The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae. Its application to other genes and species is considered.

Production of a polyclonal antibody raised against recombinant flounder p53 protein.

The cDNA encoding the wild type p53 protein from flounder, Platichthys flesus, was expressed in Escherichia coli using the GST fusion protein system. Several milligrams of recombinant p53 protein were purified. This protein displayed an apparent molecular weight of 45,000 Da, a value which is very similar to Xenopus p53, but significantly greater than was expected based on the length of the open reading frame. Immunization of rabbits against purified p53 protein allowed the production of high titre polyclonal antiserum. This new polyclonal antibody recognized recombinant flounder p53 protein in Western blot. Cross reaction was also observed with recombinant Xenopus p53 protein but not with human p53 protein. Immunoblotting of the total protein extract from normal flounder ovaries did not reveal any p53 expression.

Tributyltin along the coasts of Corsica (Western Mediterranean): a persistent problem.

Despite optimistic forecasts by various scientists after regulatory measures were taken in the 1980s, coastal tributyltin (TBT) contamination is still a major problem. The present study concerning Corsica (Western Mediterranean) shows that contamination is not limited to harbour areas, but extends along the coast, involving protected nature reserves. The concentrations measured in harbours, which can reach 200 ng TBT l(-1), tend to incriminate both pleasure craft and ferries providing regular service between the island and the continent. Contamination as high as 7 ng TBT l(-1) has been measured in waters of the Scandola nature reserve, which is quite excessive given the no effect concentrations (NOEC) for marine fauna are around 1-2 ng TBT l(-1). The inadequacy of current regulations and their application are a major factor in this situation.

Monitoring the impact of litter in large vertebrates in the Mediterranean Sea within the European Marine Strategy Framework Directive (MSFD): constraints, specificities and recommendations.

In its decision (2010/477/EU) relating to the European Marine Strategy Framework Directive (MSFD, 2008/56/EC), the European Commission identified the following points as focuses for monitoring: (i) 10.1.1: Trends in the amount, source and composition of litter washed ashore and/or deposited on coastlines, (ii) 10.1.2: Trends in the amount and composition of litter in the water column and accumulation on the sea floor, (iii) 10.1.3: Trends in the amount, distribution and composition of micro-particles (mainly microplastics), and (iv) 10.2.1: Trends in the amount and composition of litter ingested by marine animals. Monitoring the impacts of litter will be considered further in 2014. At that time, the strategy will be discussed in the context of the Mediterranean Sea, providing information on constraints, protocols, existing harm and research needed to support monitoring efforts. The definition of targets and acceptable levels of harm must take all factors into account, whether entanglement, ingestion, the transport and release of pollutants, the transport of alien species and socio-economic impacts. It must also reflect on the practical deployment of "ingestion" measures (10.2.1). The analysis of existing data will reveal the potential and suitability of some higher trophic level organisms (fish, turtles, birds and mammals) for monitoring the adverse effects of litter. Sea turtles appear to be useful indicator species, but the definition of an ecological quality objective is still needed, as well as research on alternative potential indicator species.

A comparative study of marine litter on the seafloor of coastal areas in the Eastern Mediterranean and Black Seas.

In the present work, abundance, spatial distribution and qualitative composition, of benthic marine litter, were investigated in five study areas from the Eastern Mediterranean and Black Seas (Saronikos, Patras and Echinades Gulfs; Limassol Gulf; Constanta Bay). Surveys were performed using the monitoring protocol proposed by the Technical Group for Marine Litter. Densities ranged from 24items/km(2) to 1211items/km(2), with the Saronikos Gulf being the most affected area. Plastics were predominant in all study areas ranging from 45.2% to 95%. Metals and Glass/Ceramics reached maximum values of 21.9% and of 22.4%. The size distribution of litter items showed that ⩾50% fall into medium size categories (10×10cm, 20×20cm) along with an elevated percentage of small-sized (<5×5cm) plastic litter items. The comparative analysis of the data highlighted the dependence of the marine litter problem on many local factors (human sources and oceanographic conditions) and the urgent need for specific actions.

Plastic litter in the sea.

On June 2013 a workshop at the University of Siena (Italy) was organized to review current knowledge and to clarify what is known, and what remains to be investigated, concerning plastic litter in the sea. The content of the workshop was designed to contribute further to the European Marine Strategy Framework Directive (MSFD) following an inaugural workshop in 2012. Here we report a number of statements relevant to policymakers and scientists that was overwhelming agreement from the participants. Many might view this as already providing sufficient grounds for policy action. At the very least, this early warning of the problems that lie ahead should be taken seriously, and serve as a stimulus for further research.