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.

Impact of the marine litter pollution on the Mediterranean biodiversity: A risk assessment study with focus on the marine protected areas.

In this paper a novel methodology to assess the risk of marine litter (ML) pollution in the Mediterranean Sea is implemented. In this approach, the hazard component is estimated using a state-of-the-art 3D modeling system, which allows the simulation of floating and sinking ML particles; the exposure component is defined from biodiversity estimates; and the vulnerability is related to ML ingestion rates of each species. The results show that the hot-spots for the ML risk concentrate in the coastal regions, and are mainly conditioned by the biodiversity in the region. A dedicated analysis on the marine protected areas shows that the risk therein is controlled by the proximity to ML sources and that their present-day protection levels are not effective in the case of ML pollution. Only a reduction of ML at the sources could reduce the impact of ML pollution in protected areas.

Relevance of current PCB concentrations in edible fish species from the Mediterranean Sea.

Legal restrictions and bans have led to a steady decrease in PCB environmental concentrations. Yet, in recent years PCBs have been found at very high levels in the Mediterranean Sea, for instance, in some apex predators. This work aimed to investigate current PCB (eighteen congeners: #28,52,77,81,101,105,114,118,123,126,138,153,156,157,167,169,180,189) concentrations in the Mediterranean Sea and their relevance today, focusing on their occurrence in edible fish species typically consumed in the Mediterranean diet. In spring 2017, a total of 48 fish samples from the Northern Thyrrenian Sea were collected: 16 specimens of sardine (Sardina pilchardus), 16 of anchovy (Engraulis encrasicolus) and 16 of bogue (Boops boops). PCBs were quantified in the muscle of the animals by means of GC-QqQ-MS. They were found in all samples at the greatest concentrations (ng/g w.w.) in sardine (4.15-17.9, range), and very similar values between anchovy (1.01-7.08) and bogue (1.46-7.22). WHO-TEQ PCB values followed the same order, i.e. sardine (0.410-1.24, range in pg/g w.w.) > anchovy (0.0778-0.396) ~ bogue (0.0726-0.268). These concentrations lied below the European limits of 75 ng/g (w.w.) for the six indicator PCBs and 6.5 pg/g WHO-TEQ for dioxins and dioxin-like PCBs in muscle meat of fish. Additionally, estimated weekly intakes (EWI, in pg WHO-TEQ/Kg/week) for sardine (1.2), anchovy (0.29) and bogue (0.35) scored below the safe value proposed by EFSA of 2 pg WHO-TEQ/Kg/week. When comparing with data reported for the same species in previous Mediterranean studies, values found here were lower than those surveyed in the late 90s and early 2000s; however, they were often not notably different from concentrations reported in last years. This builds up on the concept of a current slow decrease of PCBs in the Mediterranean Sea, likely linked to new inputs and/or remobilization of burdens, and reinforces the need of continous monitoring of these legacy contaminants still ubiquitous today.

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.

Effects of copper on larvae of the marbled crab Pachygrapsus marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses.

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbors. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC50 value of 0.5 mg/L of Cu2+. The LC50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells.

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.

Evaluation of PCDD/Fs, dioxin-like PCBs and PBDEs in sperm whales from the Mediterranean Sea.

Numerous studies to date have reported concentrations of Persistent Organic Pollutants (POPs) in different marine mammal species worldwide. Yet data on sperm whales are scarce from rich and unique biodiverse areas such as the Mediterranean Sea. This work aimed to assess levels of dioxin-like polychlorinated biphenyls (dl-PCBs), polybrominated diphenyl ethers (PBDEs), and polychlorodibenzo-p-dioxins and furans (PCDD/Fs) in blubber of sperm whales stranded along the Italian coast between 2008 and 2016. POP mean concentrations (dl-PCBs: 6410 ng/g l.w.; PBDEs: 612 ng/g l.w.; PCDD/Fs: 57.8 pg/g l.w.) were mostly in line with what has been previously reported on the same species in the Mediterranean environment and tended to be higher than those reported from other geographical regions. The relative abundance followed the order dl-PCBs > PBDEs ≫ PCDD/Fs. Interestingly, the non-ortho dl-PCB pattern (126 > 169 > 77) was similar to that described in other studies worldwide and different from what is described in its main prey. This could be linked to particular metabolic activities in sperm whales against these highly toxic contaminants. Total TEQs ranged from 275 to 987 pg/g l.w. and showed the pattern Σnon-ortho-dl-PCBs > Σortho-dl-PCBs > PCDDs > PCDFs, with PCBs' contribution about 96%. These findings highlight the high abundance of PCBs still found in the Mediterranean environment despite having been banned for decades. All sperm whales analyzed in this study surpassed the threshold of 210 pg WHO-TEQ/g l.w. proposed as starting point of immunosuppression in harbour seals; a level of contamination that may have contributed to an impairment of their immune system.

The Gulf of Ambracia's Common Bottlenose Dolphins, Tursiops truncatus: A Highly Dense and yet Threatened Population.

The common bottlenose dolphin (Tursiops truncatus) is the only cetacean present in the semiclosed waters of the Gulf of Ambracia, Western Greece. This increasingly degraded coastal ecosystem hosts one of the highest observed densities in the Mediterranean Sea for this species. Photo-identification data and tissue samples collected through skin-swabbing and remote biopsy sampling techniques during boat-based surveys conducted between 2006 and 2015 in the Gulf, were used to examine bottlenose dolphin abundance, population trends, site fidelity, genetic differentiation and toxicological status. Bottlenose dolphins showed high levels of year-round site fidelity throughout the 10-year study period. Dolphin population estimates mostly fell between 130 and 170 with CVs averaging about 10%; a trend in population size over the 10 years was a decline of 1.6% per year (but this was not significant). Genetic differentiation between the bottlenose dolphins of the Gulf and their conspecifics from neighbouring populations was detected, and low genetic diversity was found among individuals sampled. In addition, pesticides where identified as factors posing a real toxicological problem for local bottlenose dolphins. Therefore, in the Gulf of Ambracia, high dolphin density does not seem to be indicative of favourable conservation status or pristine habitat.

Could feeding habit and migratory behaviour be the causes of different toxicological hazard to cetaceans of Gulf of California (Mexico)?

In this work, a suite of diagnostic biomarkers was applied to seven cetacean species to evaluate the role of the feeding habits and migratory behavior in the toxicological status of these species from the Gulf of California, Mexico. We investigate the interspecific differences in cytochrome P450 1A1 and 2B (CYP1A1 and CYP2B, respectively), aryl hydrocarbon receptor and E2F transcription factor 1 and the contaminants levels [organochlorine compounds, polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs)] in four odontocete species (common bottlenose dolphin, long-beaked common dolphin, sperm whale and killer whale) and three mysticete species (blue whale, fin whale, and Bryde's whale) using skin biopsy. Differences in contaminant levels and molecular biomarker responses between the odontocete and mysticete species have been pointed out. The canonical discriminant analysis on principal component analysis factors, performed to reveal clustering variables, shows that odontocete are characterised by the highest levels of lipophilic contaminants compared to the mysticete, with the highest levels of polychlorinated biphenyls, dichlorodiphenyltrichloroethanes and PBDEs detected in killer whale and the lowest levels in Bryde's whale. The biomarker data show interspecific differences amongst the seven species, revealing highest CYP1A and CYP2B protein levels in the mysticete fish-eating species (Bryde's whale). In conclusion, three main factors seem to regulate the biomarker responses in these species: (a) the inductive ability of persistent organic pollutants and PAHs; (b) the different evolutionary process of the two CYPs related to the different feeding habits of the species; (c) the migratory/resident behaviour of the mysticete species in this area.

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.

The Italian wall lizard (Podarcis sicula) as a bioindicator of oil field activity.

The aim of this study was to develop and to validate a methodology based on biomarker responses and residue analysis on the terrestrial lizard Podarcis sicula to assess the ecotoxicological effects associated with on-shore oil extraction. The oil treatment plant investigated is located in Val d'Agri (southern Italy). Italian wall lizards were sampled on four stations along a transect determined on the basis of prevailing winds downwind of the oil plant. Cytochrome P450 1A1 activities (EROD and BPMO), AChE activity, PAH bile metabolites and contaminant levels (PAHs and trace elements) were measured. Major results in the evaluation of toxicological impact of oil field activity in the Italian wall lizards were obtained for Cd, Hg, total and carcinogenic PAH levels, and PAH metabolites in bile. Results obtained validate, for the first time, P. sicula as a terrestrial bioindicator for the assessment of the toxicological impact of on-shore extraction activity.

Potential toxicological hazard due to endocrine-disrupting chemicals on Mediterranean top predators: state of art, gender differences and methodological tools.

Man-made endocrine-disrupting chemicals (EDCs) range across all continents and oceans. Some geographic areas are potentially more threatened than others: one of these is the Mediterranean Sea. Levels of some xenobiotics are much higher here than in other seas and oceans. In this paper we review the final results of a project supported by the Italian Ministry of the Environment, in which the hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs was investigated. We illustrate the need to develop and apply sensitive methodological tools, such as biomarkers (Vitellogenin, Zona Radiata proteins and CYP1A activities) for evaluation of toxicological risk in large pelagic fish top predators (Swordfish, (Xiphias gladius), Bluefin Tuna (Thunnus thynnus thynnus)) and nondestructive biomarkers (CYP1A activities and fibroblast cell culture in skin biopsy), for the hazard assessment of threatened marine mammals species (Striped Dolphin, (Stenella coeruleoalba), Bottlenose Dolphin (Tursiops truncatus), Common Dolphin (Delphinus delphis) and Fin Whale (Balaenoptera physalus))exposed to EDCs. Differential gender susceptibility to EDCs is also explored both in large pelagic fish and in cetaceans. In cetaceans, male specimens showed higher cytochrome P450 induction (BPMO in skyn biopsies, CYP2B in fibroblasts cell cultures) by xenobiotics with respect to females.

Use of biomarkers to investigate toxicological effects of produced water treated with conventional and innovative methods.

The aim of this study was to develop and apply a multi-biomarker system to assess the toxicological effects of produced water (PW) from a Mediterranean off-shore oil platform. The selected bioindicator organism, mosquitofish (Gambusia affinis), was exposed in the laboratory to high concentrations of different PW: PW before treatment (BT), after conventional treatment (ACT) and after innovative treatment with zeolites in a prototype system (AIT). A set of biomarkers (benzo(alpha)pyrene monooxygenase, ethoxyresorufin-o-deethylase, vitellogenin, porphyrins, PAH bile metabolites, esterases, catalase, micronuclei) and PAH concentrations were measured in the experimental organism. The methodology proved to be appropriate and biomarker responses (CYP 1A1, PAH bile metabolites, micronuclei, esterases, porphyrins) affected by BT were less affected by ACT. PW treated with zeolites (AIT) had the lowest toxicological impact. The results obtained applying this multi-biomarker approach suggest that the system using zeolites is effective for treating produced water.

Endocrine disruptors in marine organisms: approaches and perspectives.

Organic pollutants exhibiting endocrine disrupting activity (Endocrine Disruptors--EDs) are prevalent over a wide range in the aquatic ecosystems; most EDs are resistant to environmental degradation and are considered ubiquitous contaminants. The actual potency of EDs is low compared to that of natural hormones, but environmental concentrations may still be sufficiently high to produce detrimental biological effects. Most information on the biological effects and mechanisms of action of EDs has been focused on vertebrates. Here we summarize recent progress in studies on selected aspects of endocrine disruption in marine organisms that are still poorly understood and that certainly deserve further research in the near future. This review, divided in four sections, focuses mainly on invertebrates (effects of EDs and mechanisms of action) and presents data on top predators (large pelagic fish and cetaceans), a group of vertebrates that are particularly at risk due to their position in the food chain. The first section deals with basic pathways of steroid biosynthesis and metabolism as a target for endocrine disruption in invertebrates. In the second section, data on the effects and alternative mechanisms of action of estrogenic compounds in mussel immunocytes are presented, addressing to the importance of investigating full range responses to estrogenic chemicals in ecologically relevant invertebrate species. In the third section we review the potential use of vitellogenin (Vtg)-like proteins as a biomarker of endocrine disruption in marine bivalve molluscs, used worldwide as sentinels in marine biomonitoring programmes. Finally, we summarize the results of a recent survey on ED accumulation and effects on marine fish and mammals, utilizing both classical biomarkers of endocrine disruption in vertebrates and non-lethal techniques, such as non-destructive biomarkers, indicating the toxicological risk for top predator species in the Mediterranean. Overall, the reviewed data underline the potential to identify specific types of responses to specific groups of chemicals such as EDs in order to develop suitable biomarkers that could be useful as diagnostic tools for endocrine disruption in marine invertebrates and vertebrates.

Theoretical models to evaluate hazard due to organochlorine compounds (OCs) in Mediterranean striped dolphin (Stenella coeruleoalba).

Many studies document the chemical stress related to organochlorine (OC) xenobiotics in Mediterranean cetaceans. The aim of this study was to establish a theoretical model to evaluate the hazard to Mediterranean striped dolphins (Stenella coeruleoalba) due to HCB, DDTs and PCB congeners. Differences in OC levels in blubber of stranded and free-ranging specimens enabled us to evaluate the hazard associated with different chlorinated xenobiotics, taking the live population as control sample, assumed to be in good health. For the most toxic compounds, with teratogenic, mutagenic, carcinogenic and endocrine disrupting capacity, we indicate levels beyond which there can be toxicological hazard for the striped dolphin. Using a mathematical formula derived from knowledge of the length and age of 62 stranded specimens, the age of dolphins was estimated and sexual maturity was identified at nine years. This evaluation was important for understanding differences in contaminant burden between males and females.

Biomarkers for endocrine disruptors in three species of Mediterranean large pelagic fish.

The hypothesis that Mediterranean top predator species, such as large pelagic fish, are potentially at risk due to endocrine disrupting chemicals (EDCs), is investigated. The potential estrogenic effects of PHAHs in three fish species of commercial interest, the top predators bluefin tuna (Thunnus thynnus thynnus), swordfish (Xiphias gladius), and Mediterranean spearfish (Tetrapturus belone), were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and mixed function oxidases (EROD, BPMO) as diagnostic tools. High induction of Vtg and Zrp was detected by western blot and ELISA techniques in adult males of X. gladius and T. thynnus thynnus, suggesting that these species are at high toxicological risk in the Mediterranean sea. Comparison of BPMO and EROD activities in the three species indicated, both in male and female, much higher MFO activity in bluefin tuna. This data suggests high exposure of this species to lipophilic xenobiotic contaminants in the Mediterranean environment.

Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean "whale sanctuary.".

The aim of this study was to propose a suite of biomarkers (BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase. esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multi-disciplinary diagnostic tool for assessment of the health status of the Mediterranean "whale sanctuary". Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h. On the other hand larger differences between sites were found for reductase activities. Esterases (AChE), porphyrins (Copro-, Uro-, Proto-porphyrins) vitellogenin and zona radiata proteins were also detectable in this zooplanctonic species. Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w. Total PAHs ranged from 860.7 to 5,037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w., HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w.

The use of porphyrins as a non-destructive biomarker of exposure to contaminants in two sea lion populations.

This study was carried out in two populations of Otariaflavescens, living in a polluted and in a reference site, in order to validate the use of porphyrins as a non-destructive biomarker of exposure to environmental contaminants. Analysis of porphyrins was carried out in the feces, blood and fur of free ranging sea lions and in the liver and kidney of stranded specimens. The results show that: (a) all biological materials used were useful for porphyrin determinations; (b) no clear seasonal variations in porphyrin accumulation and excretion were found; (c) differences in fecal porphyrin levels existed between the two colonies.

Evaluation of acetylcholinesterase activity in several zooplanktonic crustaceans.

Zooplankton are an essential component of the marine and estuarine food chains. The ecotoxicological risk to zooplanktonic communities, estimated through the use of the biomarkers, can be used as an early warning signal of a potential alteration of the ecosystem health. The aim of this project was to evaluate the potential use of several biomarkers (esterases, mixed function oxidases, porphyrins) in zooplanktonic organisms. The acetylcholinesterase activity (AChE) was determined in homogenates of whole organisms of 10 different zooplanktonic crustaceans. Mean activity of AChE was 10.05 micromol/min/g for Acartia margalefi; 3.30 for Acartia latisetosa; 79.70 for Siriella clausi; 49.97 for Diamysis bahirensis; 7.48 for Siriella armata; 14.20 for Mysidopsis gibbosa; 4.49 for Euphausia crystallorophias; 1.66 for Euphausia superba; 2.74 for Streetsia challengeri; 13.26 for Meganycthiphanes norvegica. The species moreover showed a linear increase in enzyme activity in relation to the increase in sample concentration. The key result of this study concerns the different AChE activity basal values of the crustaceans' different species.

Preliminary results of biomarker responses in zooplankton of brackish environments.

Zooplankton is an essential component of the food chains of marine and brackish environments. Ecotoxicological risk to zooplankton, evaluated by the biomarker approach, can be used as an early warning signal of risk to the health of marine ecosystems. The aim of this project is to evaluate the application of certain biomarkers in zooplankton. In the present paper, laboratory and field studies were performed with the copepods Acartia margalefi, Acartia latisetosa and the mysid Siriella clausi. Acetylcholinesterase activity (AChE) was determined in homogenates of whole organisms. Mean AChE activity was 10.05 micromol min g(-1) for A. margalefi, 3.30 micromol min g(-1) for A. latisetosa and 79.70 micromol min g(-1) for S. clausi. A. margalefi and S. clausi also showed a linear increase in enzyme activity with increasing concentrations of samples. Preliminary laboratory work was also carried out with specimens of A. latisetosa, which were exposed to an organophosphorus insecticide (parathion). Moderate inhibition of AChE (19%) with respect to controls was observed. AChE activities were two orders of magnitude higher in the zooplankton than in hemolymph samples of the decapod Carcinus aestuarii (Fossi et al., 1996) indicating that these species have a high metabolic rate, which makes them suitable for biomarker studies.