Effects of polymethylmethacrylate nanoplastics on the polychaete Hediste diversicolor: Behavioural, regenerative, and biochemical responses.

Plastics, particularly microplastics (MPs) and nanoplastics (NPs), have been regarded as pollutants of emerging concern due to their effects on organisms and ecosystems, especially considering marine environments. However, in terms of NPs, there is still a knowledge gap regarding the effects of size and polymer on marine invertebrates, such as benthic organisms. Therefore, this study aimed to understand, regarding behavioural, physiological, and biochemical endpoints (neurotransmission, energy metabolism, antioxidant status, and oxidative damage), the effects of 50 nm waterborne polymethylmethacrylate (PMMA) NPs (0.5 to 500 µg/L) on the marine benthic polychaete Hediste diversicolor, a key species in estuarine and coastal ecosystems. Results demonstrated that worms exposed to PMMA NPs had a shorter burrowing time than control organisms. Nevertheless, worms exposed to PMMA NPs (0.5 and 500 µg/L) decreased cholinesterase activity. Energy metabolism was decreased at 50 and 500 µg/L, and glycogen content decreased at all concentrations of PMMA NPs. Enzymes related to the antioxidant defence system (superoxide dismutase and glutathione peroxidase) displayed increased activities in H. diversicolor specimens exposed to concentrations between 0.5 and 500 µg/L, which led to no damage at the cell membrane and protein levels. In this study, polychaetes also displayed a lower regenerative capacity when exposed to PMMA NPs. Overall, the data obtained in this study emphasize the potential consequences of PMMA NPs to benthic worms, particularly between 0.5 and 50 µg/L, with polychaetes exposed to 50 µg/L being the most impacted by the analysed NPs. However, since sediments are considered to be sinks and sources of plastics, further studies are needed to better understand the impacts of different sizes and polymers on marine organisms, particularly benthic species.

The impact of chemical pollution on the European eel (Anguilla anguilla) from a Mediterranean hypersaline coastal lagoon.

The European eel (Anguilla anguilla) is a critically endangered species. The impact of environmental contamination on this species has been highlighted as contributing to the decline in recruitment. The Mar Menor hypersaline coastal lagoon (SE Spain) is one of the most productive fisheries of European eel in Europe, making it a critical habitat for species conservation. The present study aimed to provide an initial overview of the impact of organic chemical contaminants on the European eel and the potential sublethal effects of chemical pollution on pre-migrating eels in this hypersaline habitat. We investigated muscle bioaccumulation of main persistent and hazardous organic contaminants (including some current-use pesticides) and genotoxicity, neurotoxicity, and xenobiotic detoxification system responses. The findings show that lagoon eels were exposed to high levels of legacy organochlorine contaminants, recently banned pesticides (chlorpyrifos), and some emerging chemicals. Some individuals surpassed the maximum levels of CBs authorized by the European Commission for human consumption. In this species, residuals of chlorpyrifos, pendimethalin, and chlorthal dimethyl have been reported for the first time. This field study provides relevant data to stock management and human health consumption and provides the first biomarker responses in European eel under permanent hypersaline conditions. Furthermore, the high frequency of micronuclei in peripheral erythrocytes of lagoon eels indicates sublethal genotoxic effects on the organism. Overall, the European eels growing and maturing in the Mar Menor lagoon are exposed to toxic and carcinogenic chemicals. The lack of seafood safety regulations for human consumption for some legacy chemicals that were measured in high concentrations in our study requires special action. Further biomonitoring and research are recommended to protect the animal, public, and environmental health.

Does parental exposure to nanoplastics modulate the response of Hediste diversicolor to other contaminants: A case study with arsenic.

Plastic pollution is a serious problem in aquatic systems throughout the world. Despite the increasing number of studies addressing the impact of macro- and microplastics on biota, there is still a significant knowledge gap regarding the effects of nanoplastics alone and in combination with other contaminants. Among the aquatic contaminants that may interact with nanoplastics is arsenic (As), a metalloid found in estuarine and coastal ecosystems, pernicious to benthic organisms. This study aimed to understand how a parental pre-exposure to 100 nm polystyrene nanoplastics (PS NPs) would influence the response of Hediste diversicolor to exposure to arsenic in terms of behaviour, neurotransmission, antioxidant defences and oxidative damage, and energy metabolism. The obtained data revealed an increase in burrowing time and a significant inhibition in cholinesterase activity in all polychaetes exposed to As, regardless of the pre-exposure to PS NPs. Oxidative status was altered particularly in parentally exposed organisms, with damage detected in terms of lipid peroxidation at 50 µg/L and protein carbonylation at 50 and 250 µg As/L exposed organisms when compared to control. Overall, data shows that parental pre-exposure to plastics influences the response of aquatic organisms, increasing their susceptibility to other contaminants. Thus, more studies should be performed with other environmental contaminants, to better understand the potential increased risk associated with the presence of nanoplastics to aquatic ecosystems.

The influence of natural vs anthropogenic factors on trace metal(loid) levels in the Mussel Watch programme: Two decades of monitoring in the Spanish Mediterranean sea.

The spatial distribution and temporal trends of trace metals (i.e. Cd, Cu, Hg, Pb and Zn) and a metalloid (i.e. As) along the Spanish Mediterranean coast from 1993 to 2013 are presented with a new estimation of their background levels monitored using wild mussels. Over a 20 years period, yearly mussel monitoring was undertaken with a rigorous field sampling protocol using 3 pooled samples strategy (3 x n = 80, with 8 mussels in the 3.0 to 3.9 size categories at each site), obtained in the pre-spawning period (May-June) to minimize biological factors and seasonal variability, which is a fundamental element of the international programme. Spatial distribution was characterized every 5 years and temporal trends were determined in 11 locations. The main aims of the present long term study are to evaluate the environmental status of different coastal locations regarding trace metal levels and follow the evolution of these levels over time after the implementation of regulatory measures. Regarding spatial distribution, the highest values for Cd, Cu, Hg and Pb were found close to known highly anthopogenic cities or shipyard areas. However, As and Zn did not strictly follow this pattern, showing natural increased concentrations in the Levantine Balearic and Strait of Gibraltar-Alboran Sea demarcations respectively. These distributions are associated with the conjunction of two geological formation inputs (Massif Central in France and Iberian Pyritic Belt in Spain) and the oceanographic conditions in the adjacent coastal area. In the case of temporal trends, metal concentrations decreased significantly over time in most stations, confirming the effectiveness of the regulatory measures and prohibitions established under European legislation. Concentrations of Pb were above the established thresholds for human consumption in only 12-14% of the sampling areas. With the information obtained for this study, we estimate background concentrations and propose new Background Assesment Criteria (BAC) for the Spanish Mediterranean coast as a threshold criterion: 1.62 mg/kg d.w. for Cd, 8.75 mg/kg d.w. for Cu, 0.202 mg/kg d.w. for Hg and 2.83 mg/kg d.w. for Pb. Exceptions should exist for As and Zn, for which there should be different levels in each demarcation, due to the geological, hydrological and oceanographic peculiarities of the Spanish coast. For the Levantine-Balearic demarcation, the proposed background concentrations are 117 mg/kg d.w. for As and 200 mg/kg d.w. for Zn., whereas in the Strait of Gibraltar-Alboran Sea demarcation, they are 27.5 mg/kg d.w. for As, and 471 mg/kg d.w. for Zn. This work demonstrates the vital importance of defining the background levels of metal(loid)s at a regional or subregional level because, for areas not affected by anthropogenic causes which have high values as the result of natural processes, this would avoid the risk of constantly surpassing the levels proposed in directives.

Toxicity characterization of surface sediments from a Mediterranean coastal lagoon.

The occurrence of bioactive compounds and contaminant-associated effects was assessed by means of in vivo and in vitro assays using different extractable fractions of surface sediments from a contaminated coastal lagoon (Mar Menor, SE Spain). Sediment elutriates and clean seawater, previously exposed to whole sediment, were used for assessing the in vivo toxicity on embryo development of the sea urchin Paracentrotus lividus. Agonist and antagonist activities relating to estrogen and androgen receptors and agonist activities on aryl hydrocarbon receptor (expressed as ethoxyresorufin-O-deethylase (EROD) activities) were investigated in sediment extracts by using HER-Luc, AR-EcoScreenTM and fibroblast-like RTG-2 cell lines. Embryotoxicity effects were greater for sediment elutriates than those incubated in sediment-water interphase, implying that diffusion of bioactive chemicals can occur from sediments to sea water column, favoured by sediment disturbance events. In vitro results show the occurrence in extracts of compounds with estrogen antagonism, androgen antagonism and dioxin-like activities. Multidimensional scaling analysis classified the sampling sites into four sub-clusters according to their chemical-physical and biological similarities, relating in vitro bioactivity with the total organic carbon and known organic chemical load, with particular reference to total sum of PAHs, PCB 180, p,p-DDE and terbuthylazine. Overall, results pointed to the presence of unknown or unanalyzed biologically-active compounds in the sediments, mostly associated with the extracted polar fraction of the Mar Menor lagoon sediments. Our findings provide relevant information to be considered for the environmental management of contaminated coastal lagoons.

Biomarkers of general stress in mussels as common indicators for marine biomonitoring programmes in Europe: The ICON experience.

This study investigated whether general stress biomarkers in mussels can be applied as common first-tier biomarkers in regional biomonitoring programmes in the North Sea (including Iceland) and western Mediterranean Sea. Stress on Stress (SoS) and lysosomal membrane stability (LMS) biomarkers were analysed in resident mussels (Mytilus sp.) from 8 coastal sites and in transplanted mussels (Mytilus galloprovincialis) from two Spanish Mediterranean coastal sites. The assessment of results, as input to pollution monitoring strategies, was performed jointly for LMS and SoS data from the two regions. Contaminant body burden of the mussels was compared with biomarker results. The results demonstrated that these two general and non-expensive stress biomarkers in mussel can be applied throughout European waters, providing a cost-effective and harmonised approach to screen contaminant-related biological effects within the framework of wide-scale pollution biomonitoring programmes, such as that proposed by the European Union, i.e. the Marine Strategy Framework Directive.

Integrated indicator framework and methodology for monitoring and assessment of hazardous substances and their effects in the marine environment.

Many maritime countries in Europe have implemented marine environmental monitoring programmes which include the measurement of chemical contaminants and related biological effects. How best to integrate data obtained in these two types of monitoring into meaningful assessments has been the subject of recent efforts by the International Council for Exploration of the Sea (ICES) Expert Groups. Work within these groups has concentrated on defining a core set of chemical and biological endpoints that can be used across maritime areas, defining confounding factors, supporting parameters and protocols for measurement. The framework comprised markers for concentrations of, exposure to and effects from, contaminants. Most importantly, assessment criteria for biological effect measurements have been set and the framework suggests how these measurements can be used in an integrated manner alongside contaminant measurements in biota, sediments and potentially water. Output from this process resulted in OSPAR Commission (www.ospar.org) guidelines that were adopted in 2012 on a trial basis for a period of 3 years. The developed assessment framework can furthermore provide a suitable approach for the assessment of Good Environmental Status (GES) for Descriptor 8 of the European Union (EU) Marine Strategy Framework Directive (MSFD).

How can we quantify impacts of contaminants in marine ecosystems? The ICON project.

An international workshop on marine integrated contaminant monitoring (ICON) was organised to test a framework on integrated environmental assessment and simultaneously assess the status of selected European marine areas. Biota and sediment were sampled in selected estuarine, inshore and offshore locations encompassing marine habitats from Iceland to the Spanish Mediterranean. The outcome of the ICON project is reported in this special issue as method-oriented papers addressing chemical analyses, PAH metabolites, oxidative stress, biotransformation, lysosomal membrane stability, genotoxicity, disease in fish, and sediment assessment, as well as papers assessing specific areas. This paper provides a background and introduction to the ICON project, by reviewing how effects of contaminants on marine organisms can be monitored and by describing strategies that have been employed to monitor and assess such effects. Through the ICON project we have demonstrated the use of an integrating framework and gleaned more knowledge than ever before in any single field campaign about the impacts contaminants may have in European marine areas.

Integrated chemical and biological assessment of contaminant impacts in selected European coastal and offshore marine areas.

This paper reports a full assessment of results from ICON, an international workshop on marine integrated contaminant monitoring, encompassing different matrices (sediment, fish, mussels, gastropods), areas (Iceland, North Sea, Baltic, Wadden Sea, Seine estuary and the western Mediterranean) and endpoints (chemical analyses, biological effects). ICON has demonstrated the use of a framework for integrated contaminant assessment on European coastal and offshore areas. The assessment showed that chemical contamination did not always correspond with biological effects, indicating that both are required. The framework can be used to develop assessments for EU directives. If a 95% target were to be used as a regional indicator of MSFD GES, Iceland and offshore North Sea would achieve the target using the ICON dataset, but inshore North Sea, Baltic and Spanish Mediterranean regions would fail.

Assessing environmental quality status by integrating chemical and biological effect data: The Cartagena coastal zone as a case.

Cartagena coastal zone (W Mediterranean) was chosen for a practical case study to investigate the suitability of an integrated indicator framework for marine monitoring and assessment of chemicals and their effects, which was developed by ICES and OSPAR. Red mullet (Mullus barbatus) and the Mediterranean mussel (Mytilus galloprovincialis) were selected as target species. Concentrations of contaminants in sediment and biota, and contaminant-related biomarkers were analysed. To assess environmental quality in the Cartagena coastal zone with respect to chemical pollution, data were assessed using available assessment criteria, and then integrated for different environmental matrices. A qualitative scoring method was used to rank the overall assessments into selected categories and to evaluate the confidence level of the final integrated assessment. The ICES/OSPAR integrated assessment framework, originally designed for the North Atlantic, was found to be applicable for Mediterranean species and environmental matrices. Further development of assessment criteria of chemical and biological parameters in sediments and target species from the Mediterranean will, however, be required before this framework can be fully applied for determining Good Environmental Status (GES) of the Marine Strategy Framework Directive in these regions.

Assessment of contaminant concentrations in sediments, fish and mussels sampled from the North Atlantic and European regional seas within the ICON project.

Understanding the status of contaminants in the marine environment is a requirement of European Union Directives and the Regional Seas Conventions, so that measures to reduce pollution can be identified and their efficacy assessed. The international ICON workshop (Hylland et al., in this issue) was developed in order to test an integrated approach to assessing both contaminant concentrations and their effects. This paper describes and assesses the concentrations of trace metals, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls in sediments, mussels, and fish collected from estuarine, coastal and offshore waters from Iceland to the Mediterranean Sea. For organic contaminants, concentrations progressively increased from Iceland, to the offshore North Sea, to the coastal seas, and were highest in estuaries. Metals had a more complex distribution, reflecting local anthropogenic inputs, natural sources and hydrological conditions. Use of internationally recognised assessment criteria indicated that at no site were concentrations of all contaminants at background and that concentrations of some contaminants were of significant concern in all areas, except the central North Sea.

Toxicity profiling of marine surface sediments: A case study using rapid screening bioassays of exhaustive total extracts, elutriates and passive sampler extracts.

This study was carried out in the framework of the ICON project (Integrated Assessment of Contaminant Impacts on the North Sea) (Hylland et al., 2015) and aimed (1) to evaluate the toxicity of marine sediments using a battery of rapid toxicity bioassays, and; (2) to explore the applicability and data interpretation of in vitro toxicity profiling of sediment extracts obtained from ex situ passive sampling. Sediment samples were collected at 12 selected (estuarine, coastal, offshore) sites in the North Sea, Icelandic waters (as reference sites), south-western Baltic Sea and western Mediterranean during autumn 2008. Organic extracts using a mild non-destructive clean-up procedure were prepared from total sediment and silicone passive samplers and tested with five in vitro bioassays: DR-Luc bioassay, ER-Luc bioassay, AR-EcoScreen bioassay, transthyretin (TTR) binding assay, and Vibrio fischeri bioluminescence bioassay. In vitro toxicity profiling of total sediment and silicone passive sampler extracts showed the presence of multiple organic contaminations by arylhydrocarbon receptor agonists (e.g. polycyclic aromatic hydrocarbons) and endocrine-active compounds, as well as non-specific toxicity caused by organic contaminants, at virtually all sampling sites. In vitro responses to total sediment extracts from coastal/estuarine sites were significantly different from those in offshore sites (p < 0.05). Several bioassays of passive sampler extracts showed highest activity in some offshore sediment samples. Impact on embryogenesis success and larval growth in undiluted sediment elutriates was shown at some sites using the in vivo sea urchin embryo test. The observed toxicity profiles could only partially be explained by the chemical target analysis, indicating the presence of unknown or unanalysed biologically-active compounds in the sediments. In vitro bioassay testing with silicone passive sampler extracts of sediments is a promising tool to assess the toxic potency of the bioavailable fraction of hydrophobic sediment contaminants, but further work will be needed before it can be routinely applied for sediment quality assessment.

Integrated monitoring of chemicals and their effects on four sentinel species, Limanda limanda, Platichthys flesus, Nucella lapillus and Mytilus sp., in Seine Bay: A key step towards applying biological effects to monitoring.

The International workshop on Integrated Assessment of CONtaminants impacts on the North sea (ICON) provided a framework to validate the application of chemical and biological assessment thresholds (BACs and EACs) in the Seine Bay in France. Bioassays (oyster larval anomalies, Corophium arenarium toxicity assay and DR Calux) for sediment and biomarkers: ethoxyresorufin-O-deethylase (EROD) activity, acetylcholinesterase (AChE) activity, lysosomal membrane stability (LMS), DNA strand breaks using the Comet assay, DNA adducts, micronucleus (MN), PAH metabolites, imposex, intersex and fish external pathologies were analysed in four marine sentinel species (Platichthys flesus, Limanda limanda, Mytilus sp. and Nucella lapilus). Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals were analysed in biota and sediment. Results for sediment and four species in 2008-2009 made it possible to quantify the impact of contaminants using thresholds (Environmental Assessment Criteria/EAC2008: 70% and EAC2009: 60%) and effects (EAC2008: 50% and EAC2009: 40%) in the Seine estuary. The Seine estuary is ranked among Europe's most highly polluted sites.

Ingestion of microplastics by demersal fish from the Spanish Atlantic and Mediterranean coasts.

Microplastic pollution has received increased attention over the last few years. This study documents microplastic ingestion in three commercially relevant demersal fish species from the Spanish Atlantic and Mediterranean coasts, the lesser spotted dogfish Scyliorhinus canicula, the European hake Merluccius merluccius and the red mullet Mullus barbatus. Overall 212 fish were examined, 72 dogfish, 12 hakes and 128 red mullets. The percentage of fish with microplastics was 17.5% (15.3% dogfish, 18.8% red mullets and 16.7% hakes), averaging 1.56±0.5 items per fish, and the size of the microplastics ranged from 0.38 to 3.1mm. These fish species are used currently as biomonitors for marine pollution monitoring within the Spanish Marine Pollution Monitoring Programme (SMP), and may be as well suitable candidates for monitoring spatial and temporal trends of ingested litter. The data presented here represent a baseline for the implementation of the Marine Strategy Framework Directive descriptor 10 in Spain.

Integrated chemical and biological analysis to explain estrogenic potency in bile extracts of red mullet (Mullus barbatus).

A biological screening was performed to establish the total exposure to estrogenic compounds of red mullet (Mullus barbatus) collected at several sites along the Spanish Mediterranean coast by testing male fish bile extracts using the in vitro ER-LUC reporter gene assay. In addition, major metabolites were identified and measurements of OH-PAHs (1-naphthol, 9-phenantrol, 9-fluorenol, 1-pyrenol, 1OH-BaP and 3OH-BaP) and alkylphenols (4-n-nonylphenol (4-n-NP) and 4-tertoctylphenol (4-tert-OP)) in the same fish bile extracts were taken by gas chromatography-mass spectrometry in electron ionization mode (GC-EI-MS). Relative in vitro estrogenic potencies of the chemically quantified compounds were also tested. The highest biliary concentrations of 1-pyrenol, 9-fluorenol and 4-n-NP were found in fish from Barcelona and from the Mar Menor coastal lagoon. However, these concentrations can be considered relatively low compared to values reported in red mullet from other polluted waters in the Mediterranean Sea. The contribution of 1-pyrenol, 4-n-NP and 4-tert-OP to the total estrogenic potency measured in male fish bile was found to be negligible, indicating the presence of other estrogenic compounds in red mullet bile. Estrogenic potency in bile from male fish was markedly elevated in Mar Menor lagoon (234.8±5.7 pg E2EQ/µl), and further research will be necessary to explain whether the presence of natural and synthetic-hormones in the lagoon contributed to this finding. Values of approximately 15-16E2EQ pg/mg bile can be regarded as preliminary baseline levels of bile estrogenicity in male red mullet from the western Mediterranean Sea.

Health status of red mullets from polluted areas of the Spanish Mediterranean coast, with special reference to Portmán (SE Spain).

A suite of general physiological indicators and hepatic biomarker responses were determined in red mullet (Mullus barbatus) from priority pollution areas of W Mediterranean Sea, including the highly metal polluted area of Portmán (Cartagena, SE Spain). Concentrations of metals and persistent organic pollutants in fish muscle tissue and sediment samples were also analysed. Our results showed that fish from Portmán accumulated the highest concentrations of mercury, lead and arsenic and also of some polycyclic aromatic hydrocarbons and polychlorinated biphenyls congeners. In addition, they had significantly lower condition factor, muscle lipid content and gonadosomatic index, as well as the lowest levels of DNA integrity and the highest ethoxyresorufin-O-deethylase (EROD) activity in liver of the areas investigated. Contaminant body burden in fish only partly corresponded to chemical characteristics of the sediments in the areas in which they were collected. Our findings indicate that red mullets from Portmán had suboptimal health status that warrant further study.

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites.

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.