Microplastic ingestion in mussels from the East Mediterranean Sea: Exploring its impacts in nature and controlled conditions.

Microplastic ingestion poses a significant concern for a plethora of marine organisms due to its widespread presence in marine ecosystems. Despite growing scientific interest, the effects on marine biota are not yet well understood. This study investigates the ingestion of microplastics (MPs) by mussels from various marine environments and assesses the associated effects that can be induced by MPs and associated toxic chemicals. Biomarkers of oxidative stress (catalase, lipid peroxidation), biotransformation (glutathione S-transferase), genotoxicity (micronuclei frequency) and neurotoxicity (acetylcholinesterase) were employed. Mussels, considered reliable bioindicators of MPs pollution, were sampled by hand from diverse locations under varied anthropogenic pressures, including a highly touristic Marine Protected Area (MPA) in the Ionian Sea, a mussel farm and a fish farm in the Aegean Sea. The results revealed the highest MP ingestion in mussels from the fish farm [0.21 ± 0.04 (SE) MPs/g or 0.63 ± 0.12 (SE) MPs/Ind.], likely due to plastic aquaculture equipment use. Stereoscopic observation revealed fibers, as the predominant shape of ingested MPs across all sites, and µFTIR polymer identification revealed the presence of various types, with polyethylene (PE) and polyamide (PA) being the most abundant. Significant physiological alterations in mussels related to MP ingestion levels were observed through biomarkers indicative of oxidative stress and biotransformation, as well as the Integrated Biomarker Response (IBR index). However, laboratory experiments with mussels exposed to controlled increasing PE concentrations for four weeks, did not show significant effects triggered by the PE ingestion, possibly indicating other environmental factors, such as contaminants from aquaculture environments, may influence biomarker levels in the field. Despite the observed effects, MP ingestion rates in mussels from the field were relatively low compared to other studies. Future research should continue to investigate the interactions between MPs and marine organisms in diverse environments to better understand and mitigate their impacts.

Assessment of the impact of microplastic ingestion in striped red mullets from an Eastern Mediterranean coastal area (Zakynthos Island, Ionian Sea).

Monitoring microplastics (MPs) in the marine environment is an ongoing process, and our understanding of their impact on marine organisms is limited. The present study evaluates the effects of ingested MPs on the marine MP pollution bioindicator fish species Mullus surmuletus. The study follows a three-fold approach to assess the impact of MPs on marine organisms by investigating: 1) the ingestion of MPs, 2) the bioaccumulation of phthalate compounds as plastic additives, and 3) the evaluation of toxicological biochemical and cellular biomarkers. Striped red mullets were sampled in the marine protected area (MPA) of the National Marine Park of Zakynthos and coastal sites with high touristic pressure in Zakynthos Island in the Ionian Sea, Greece. Fewer ingested MPs and lower phthalate concentrations were found in fish inside the MPA compared to those sampled outside the marine park. However, no relationship was found between either phthalate concentrations or biomarker levels with the ingested MPs in the red striped mullets. Biomarker levels were influenced by season and site, but no effect could be attributed to the ingested MPs. The lack of association of biomarker responses and plasticizer bioaccumulation to MP ingestion can be explained by the low number of ingested MPs in the fish from Zakynthos coastal area as MP abundance ranged from 0.15 to 0.55 items per individual fish.

Microplastic ingestion and its effects οn sea urchin Paracentrotus lividus: A field study in a coastal East Mediterranean environment.

Microplastics (MPs) are recognized as an increasing threat to the marine environment, but little is known about their effects on benthic organisms, including sea urchins, when ingested. For this purpose, wild sea urchins (P. lividus) and seafloor sediment samples were investigated across three coastal areas of Zakynthos Island (Ionian Sea), each exposed to different anthropogenic pressures, revealing a consistent pattern in MP abundance, shape, and color. Biomarkers related to oxidative stress, neurotoxicity, and genotoxicity showed no significant effects of MP ingestion in the sea urchins, except for a positive correlation between GST activity and ingested MPs, suggesting a possible activation of their detoxification system in response to MP ingestion. While MP concentrations in sea urchins and sediments were within the low range reported in the global literature, it remains crucial to conduct further investigations in areas with MP pollution approaching predicted levels to fully comprehend the potential effects of MP pollution on marine organisms.

Monk seal faeces as a non-invasive technique to monitor the incidence of ingested microplastics and potential presence of plastic additives.

Anthropogenic debris, including plastics, has recently been identified as a major threat for marine mammals and the Marine Strategy Framework Directive aims to achieve the good environmental status of European waters by addressing among other criteria, the effects of marine litter on biota. This study implemented for the first time a non-invasive technique for collecting monk seal samples to assess microdebris ingestion in combination with identifying plastic additives and porphyrins biomarkers. A total of 12 samples of monk seal faeces were collected from marine caves in Zakynthos Island, Greece. A total of 166 microplastic particles were identified; 75 % of the particles were smaller than 3 mm. Nine phthalates and three porphyrins were detected. A strong correlation was found between the number of microplastics and the concentration of phthalates. The values of both phthalates and porphyrins were found lower than in other marine mammal tissues, suggesting that seals might not be impacted by them yet.

Exploring microplastic pollution in a Mediterranean river: The role of introduced species as bioindicators.

Studies of plastic contamination in freshwater ecosystems and their biota remain scarce, despite the fact that the vast majority of plastic waste initially passes through lotic ecosystems. Biomonitoring provides valuable information regarding plastic pollution and microplastic threats to biota and human health. The aim of this study was to explore the potential use of a non-indigenous fish species as a bioindicator of microplastic pollution in an Eastern Mediterranean River. Our study area is located in a heavily modified and vastly impacted urban river which flows through the largest part of the Metropolitan area of Athens, Greece. We used an introduced chub species (Squalius vardarensis) to assess microplastic ingestion in the river. The results indicated moderate occurrence and abundance of microplastics in the fish gastrointestinal tracts; one-third of specimens (35%) contained microplastics, although the average number of microplastics per specimen was relatively low (1.7 ± 0.2). Overall, the abundance of microplastics in the water confirmed the moderate level of microplastics contamination in our study area. The major polymer types of microplastics identified by FT-IR analysis were: polyethylene (PE), polyvinyl alcohol (PVA) and polypropylene (PP); reflecting the fragmentation of larger litter from industrial packaging and/or household goods. Surface runoff of the urban environment, via motorways and major road networks, could be the contributing factor to the reported microplastics. Our results suggest that generalist's non-indigenous species such as chubs could be used as bioindicators of microplastics in inland waters. Introduced fishes can be a feasible, nondestructive, and cost-effective option for the assessment of microplastics in freshwater ecosystems, while freshwater chubs' high abundance and omnipresence in European rivers further serve this scope. However, it is worth noting that the suitability of any particular species as a bioindicator of microplastics may depend on a variety of factors, including their feeding behavior, habitat, and exposure to microplastics in their environment.

Drivers of litter ingestion by sea turtles: Three decades of empirical data collected in Atlantic Europe and the Mediterranean.

Sea turtles are considered as bio-indicators for monitoring the efficiency of restoration measures to reduce marine litter impacts on health. However, the lack of extended and standardised empirical data has prevented the accurate analysis of the factors influencing litter ingestion and the relationships with individual health. Historic data collected from 1988 and standard data collected from 2016 were harmonised to enable such analyses on necropsied loggerhead turtles (Caretta caretta) in eight Mediterranean and North-East Atlantic countries. Litter was found in 69.24 % of the 1121 individuals, mostly single-use and fishing-related plastics. Spatial location, sex and life history stage explained a minor part of litter ingestion. While no relationships with health could be detected, indicating that all individuals can be integrated as bio-indicators, the mechanistic models published in literature suggest that the high proportion of plastics in the digestive contents (38.77 % per individual) could have long-term repercussions on population dynamics.

Interlaboratory comparison of microplastic extraction methods from marine biota tissues: A harmonization exercise of the Plastic Busters MPAs project.

In the framework of the Plastic Busters MPAs project, a harmonization exercise on two methods of microplastic extraction from biological samples i.e. 15% H2O2 digestion and 10% KOH digestion was carried out. The two methods were tested in four laboratories on fish gastrointestinal tracts and mussel tissues spiked with polyethylene, polypropylene and polyethylene terephthalate. The recovery percentage of microplastics for each method, species and polymer tested were overall similar among laboratories, and interlaboratory coefficient of variation was less than 11% for the majority of samples. Microplastic recovery rates for the two methods were similar for each sample tested, but overall mean interlaboratory recovery rate using KOH (96.67%) was higher than H2O2 (88.75%). Results validate the use of both methods for extracting microplastics from biota tissues. However, when comparing the two methods in terms of microplastic recovery rate, time consumed, technical difficulties and cost, digestion with 10% KOH is considered optimal.

Using Boops boops (osteichthyes) to assess microplastic ingestion in the Mediterranean Sea.

This study assesses microplastic ingestion in Boops boops at different geographical areas in the Mediterranean Sea. A total of 884 fish were caught at 20 coastal sites in Spain, France, Italy and Greece and analyzed using a common methodological protocol. Microplastics were found in 46.8% of the sampled fish, with an average number of items per individual of 1.17 ± 0.07. Filaments were the predominant shape type, while polyethylene and polypropylene were indicated by FTIR as the most common polymer types of ingested microplastics. The frequency of occurrence, as well as the abundance and proportion of types (size, shape, color and polymer) of ingested microplastics, varied among geographical areas. The spatial heterogeneity of the abundance of ingested microplastics was mainly related to the degree of coastal anthropogenic pressure at the sampling sites. Our findings further support the suitability of B. boops as bioindicator of microplastic pollution in the Mediterranean Sea.

Evidence of ingested plastics in stranded loggerhead sea turtles along the Greek coastline, East Mediterranean Sea.

Plastic debris has become a major threat to the marine environment and wildlife. Sea turtles are particularly vulnerable, and are known to ingest plastic debris globally; however, information from Greek waters is still absent. In this study, 36 stranded dead loggerhead turtles (Caretta caretta) were collected from the Greek coastline area, and their gastrointestinal content was analysed for ingested plastic debris. Twenty-six individuals (72%) were found to have ingested plastic, with an average of 7.94 ± 3.85 (SE) plastic items per turtle. In total, 286 plastic items were counted and categorised by size, shape, colour, and polymer type. Fourier Transform Infrared Spectrometry revealed that polypropylene and polyethylene were the dominant polymer plastic types found. Results indicated a variation in plastic ingestion amongst life stages of the loggerhead specimens. This study provides evidence of plastic ingestion by loggerhead turtles in Greek waters.

Assessment of organophosphate flame retardants in Mediterranean Boops boops and their relationship to anthropization levels and microplastic ingestion.

Plastic litter pollution is increasing in the seas and oceans worldwide, raising concern on the potential effects of plasticizer additives on marine fauna. In this study, muscle samples of 30 bogues (Boops boops; Linneaus, 1758) from the North Western Mediterranean Sea were analysed to assess the concentrations of 19 organophosphate flame retardant (OPFR) compounds and to inspect any relationship with microplastic ingestion and relative levels of anthropization. Out of the 19 OPFRs analysed, 6 compounds were detected, being tri-n-butyl phosphate (TNBP), 2-ethylhexyldiphenyl phosphate (EHDPP) and triphenylphosphine oxide (TPPO) the most abundant. As expected, OPFR concentrations were higher in samples collected off the most anthropized area of the city of Barcelona than in those from the Cap de Creus Marine Protected Area, while no significant correlation was detected between OPFR concentrations and microplastic ingestion. The results of this manuscript provide a first evidence of OPFR presence in the muscle of the bogue and identify the coastal area off Barcelona as a possible concentration area for contaminants, further supporting the use of the bogue as an indicator species of plastic pollution in the Mediterranean Sea.

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status.

The following protocol is intended to respond to the requirements set by the European Union's Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: "There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region", where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: "There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region".

Acute and sub-chronic toxicity bioassays of Olive Mill Wastewater on the Eastern mosquitofish Gambusia holbrooki.

Olive oil production generates large volumes of wastewaters mostly in peri-Mediterranean countries with adverse impacts on the biota of the receiving aquatic systems. Few studies have however documented its toxicity on aquatic species, with an almost total lack of relative studies on fish. We assessed the acute and sub-chronic OMW toxicity, as well as the acute and sub-chronic behavioural, morphological and biochemical effects of OMW exposure on the mosquitofish Gambusia holbrooki. LC50 values of the acute bioassays ranged from 7.31% (24 h) to 6.38% (96 h). Behavioural symptoms of toxicity included hypoactivity and a shift away from the water surface, coupled with a range of morphological alterations, such as skin damage, excessive mucus secretion, hemorrhages, fin rot and exophhalmia, with indications also of gill swelling and anemia. Biochemical assays showed that OMW toxicity resulted in induction of catalase (CAT) and inhibition of acetylcholinesterase (AChE) activities. The implications of our results at the level of environmental policy for the sustainable management of the olive mill industry, i.e. the effective restriction of untreated OMW disposal of in adjacent waterways, as well as the implementation of new technologies that reduce their impact (detoxification and/or revalorization of its residues) are discussed.

Determining suitable fish to monitor plastic ingestion trends in the Mediterranean Sea.

The presence of marine litter is a complex, yet persistent, threat to the health and biodiversity of the marine environment, and plastic is the most abundant, and ubiquitous type of marine litter. To monitor the level of plastic waste in an area, and the prospect of it entering the food chain, bioindicator species are used extensively throughout Northern European Seas, however due to their distribution ranges many are not applicable to the Mediterranean Sea. Guidance published for the Marine Strategy Framework Directive suggests that the contents of fish stomachs may be analyzed to determine trends of marine plastic ingestion. In order to equate transnational trends in marine plastic ingestion, the use of standardized fish species that widely occur throughout the basin is favoured, however for the Mediterranean Sea, specific species are not listed. Here we propose a methodology to assess how effective Mediterranean fish species, that are known to have ingested marine plastic, are as bioindicators. A new Bioindicator Index (BI) was established by incorporating several parameters considered important for bioindicators. These parameters included species distribution throughout the Mediterranean basin, several life history traits, the commercial value of each species, and the occurrence of marine litter in their gut contents. By collecting existing data for Mediterranean fish, ranked scores were assigned to each trait and an average value (BI value) was calculated for each species. Based on their habitat preferences, Engraulis encrasicolus (pelagic), Boops boops (benthopelagic), three species of Myctophidae (Hygophum benoiti, Myctophum punctatum and Electrona risso) (mesopelagic), Mullus barbatus barbartus (demersal) and Chelidonichthys lucerna (benthic), were identified as currently, the most suitable fish for monitoring the ingestion of marine plastics throughout the Mediterranean basin. The use of standardized indicator species will ensure coherence in the reporting of marine litter ingestion trends throughout the Mediterranean Sea.

Microplastics in mussels and fish from the Northern Ionian Sea.

Microplastic ingestion by marine organisms presents an emerging threat to marine ecosystems; microplastics in different marine species are currently reported worldwide. This study aims to assess microplastic ingestion in four, highly commercial, marine species from Greek waters in the Northern Ionian Sea (Mediterranean Sea). Microplastics were found in mussels (Mytilus galloprovincialis) and all three fish species (Sardina pilchardus, Pagellus erythrinus, Mullus barbatus) examined. The frequency of occurrence of ingested microplastics was 46.25% in mussels, while among fish species, S. pilchardus showed the highest frequency of microplastic ingestion (47.2%). Microplastic abundance ranged from 1.7-2 items/individual in mussels and from 1.5-1.9 items/individual in fish. The majority of ingested microplastics were fragments, while their color and size varied. Fourier Transform Infrared Spectroscopy (FT-IR) indicated polyethylene as the most common polymer type in mussels and fish. Results can be used to set baseline levels for the assessment of microplastic pollution in the Ionian Sea.

Assessment on marine litter ingested by fish in the Adriatic and NE Ionian Sea macro-region (Mediterranean).

This study presents data on the marine litter occurrence in the stomachs of fish species living in different marine habitats for the Adriatic and NE Ionian Sea macro-region. "Macro-litter" was examined in 614 specimens belonging to 11 species, while micro-litter in 230 specimens belonging to 7 species. The study highlights for the first time the presence of litter in the stomachs of the fish species Citharus linguatula. The occurrence of "macro-litter" in the guts of fish was <3% in both the NE Ionian and N Adriatic but reached 26% in the S Adriatic Sea. Micro-litter occurrence was 40 for the NE Ionian and increased to 87% in the N Adriatic (Slovenian Sea). The ingested "macro" and micro-litter differed among the areas. The marine habitat was found to affect the "macro"-litter ingestion but not the micro-litter.

Beach litter dynamics on Mediterranean coasts: Distinguishing sources and pathways.

We assessed amounts, composition and net accumulation rates every ~15days of beach macro litter (≥2.5cm) on 4 Mediterranean beaches, on Corfu island, N. Ionian Sea, taking into account natural and anthropogenic drivers. Average net accumulation rate on all beaches was found 142±115N/100m/15d. By applying a Generalized Linear Model (GzLM) it was shown that sea transport is the dominant pathway affecting the amount and variability in beach litter loadings. Principal Component Analysis (PCA) on compositional data and indicator items discerned two more pathways of beach litter, i.e. in situ litter from beach goers and wind and/or runoff transport of litter from land. By comparing the PCA results to those from a simple item to source attribution, it is shown that regardless their source litter items arrive at beaches from various pathways. Our data provide baseline knowledge for designing monitoring strategies and for setting management targets.

Bioindicators for monitoring marine litter ingestion and its impacts on Mediterranean biodiversity.

The Mediterranean Sea has been described as one of the most affected areas by marine litter in the world. Although effects on organisms from marine plastic litter ingestion have been investigated in several oceanic areas, there is still a lack of information from the Mediterranean Sea. The main objectives of this paper are to review current knowledge on the impact of marine litter on Mediterranean biodiversity, to define selection criteria for choosing marine organisms suitable for use as bioindicator species, and to propose a methodological approach to assessing the harm related to marine litter ingestion in several Mediterranean habitats and sub-regions. A new integrated monitoring tool that would provide the information necessary to design and implement future mitigation actions in the Mediterranean basin is proposed. According to bibliographic research and statistical analysis on current knowledge of marine litter ingestion, the area of the Mediterranean most studied, in terms of number of species and papers in the Mediterranean Sea is the western sub-area as well as demersal (32.9%) and pelagic (27.7%) amongst habitats. Applying ecological and biological criteria to the most threatened species obtained by statistical analysis, bioindicator species for different habitats and monitoring scale were selected. A threefold approach, simultaneously measuring the presence and effects of plastic, can provide the actual harm and sub-lethal effects to organisms caused by marine litter ingestion. The research revealed gaps in knowledge, and this paper suggests measures to close the gap. This and the selection of appropriate bioindicator species would represent a step forward for marine litter risk assessment, and the implementation of future actions and mitigation measures for specific Mediterranean areas, habitats and species affected by marine litter ingestion.

Anthropogenic microfibres pollution in marine biota. A new and simple methodology to minimize airborne contamination.

Research studies on the effects of microlitter on marine biota have become more and more frequent the last few years. However, there is strong evidence that scientific results based on microlitter analyses can be biased by contamination from air transported fibres. This study demonstrates a low cost and easy to apply methodology to minimize the background contamination and thus to increase results validity. The contamination during the gastrointestinal content analysis of 400 fishes was tested for several sample processing steps of high risk airborne contamination (e.g. dissection, stereomicroscopic analysis, and chemical digestion treatment for microlitter extraction). It was demonstrated that, using our methodology based on hermetic enclosure devices, isolating the working areas during the various processing steps, airborne contamination reduced by 95.3%. The simplicity and low cost of this methodology provide the benefit that it could be applied not only to laboratory but also to field or on board work.

Biochemical biomarker responses to pollution in selected sentinel organisms across the Eastern Mediterranean and the Black Sea.

Pollution effects were assessed by means of biochemical biomarkers (catalase, glutathione S-transferase and acetylcholinesterase activities, and metallothioneins content) in five species at selected coastal sites across the Eastern Mediterranean and the Black Sea. The mussel Mytilus galloprovincialis, a well-established sentinel species, was investigated in the Adriatic Sea, Aegean Sea, and Black Sea. The mussel Brachidontes pharaonis and the striped red mullet Mullus surmuletus were used in the Levantine Sea where M. galloprovincialis is not present. The white seabream Diplodus sargus sargus and the gastropod Rapana venosa were additionally sampled in the Adriatic and the Black Sea, respectively. Mussels showed catalase, glutathione S-transferase, and acetylcholinesterase responses to pollution in most geographical areas while the response of metallothioneins was restricted to a few sites. R. venosa showed marked responses of catalase and metallothioneins whereas both fish species did not generally exhibit variations in biomarker values among sites. The approach based on the reference deviation concept using the "Integrated Biological Responses version 2" index was useful for the interpretation of overall biomarker responses.