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.

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.

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.

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.

Animal Personality Relates to Thermal Preference in Wild-Type Zebrafish, Danio rerio.

It has been widely supported that individual animals express different strategies to cope with environmental challenge. In ectothermic species such as fish, individuals must use behavioral thermoregulation mechanisms to optimize physiological performance. In the present study, thermal preference was tested in groups of wild-type zebrafish, Danio rerio, screened for proactive and reactive animal personalities. Three replicate groups of proactive, reactive, and naive randomly sampled non-screened controls were used for the experiments. The frequency distribution of the animals was recorded in a custom-built multichamber tank under both constant temperature (temperature restricted conditions: TR) and a continuous thermal gradient profile (temperature choice: TCh ranging from 21°C to 35°C). Proactive and reactive animal personalities expressed significantly different thermal preferences and general activity within the temperature gradient. Our results show that proactive fish, generally characterized as being more aggressive, bold risk takers, and prone to routine formation, have a preference for higher temperature environments. Reactive fish, which are shy, less risk-prone, and more flexible, favor medium colder temperatures. This is the first report of thermopreferendum in zebrafish where individual animal personality coupled to freedom of thermal choice has been applied to understand variation in individual preferences within a population.

Husbandry of zebrafish, Danio rerio, and the cortisol stress response.

The effect of common husbandry conditions (crowding, social environment, water quality, handling, and background color) on the cortisol stress response in adult zebrafish, Danio rerio, was investigated to check the usefulness of zebrafish as a model organism in aquaculture research. In addition, a noninvasive methodology for assessing stress was evaluated. Zebrafish showed a fast cortisol response with high values at 30 min that returned to basal levels within 2 h of poststress. There was a significant positive correlation between trunk cortisol concentrations and the free water cortisol rate (r(2)=0.829-0.850, p<0.001), indicating that measurement of the water-borne cortisol release rate may serve as a noninvasive and reliable stress indicator at the population level. Crowding resulted in 13- to 21-fold greater mean trunk cortisol concentrations compared with controls. However, even at low stocking density (2-5 fish/L), the maintenance cost was higher than the one at higher densities (10 fish/L) due to the formation of dominance hierarchies. The background color affected trunk cortisol concentrations, with fish exposed to brighter backgrounds (green and white) showing 3- to 8-fold greater mean trunk cortisol concentrations than fish exposed to a black background or transparent aquaria. Fish exposed to high stocking densities for 2 h or 5 days had similar high mean trunk cortisol levels, indicating that exposure of fish for the period of 2 h to a specific stressor may represent a chronic situation in zebrafish. It is concluded that adult laboratory zebrafish had a preference for a transparent or black background aquarium, at a number of 10 individuals per 2 L of available water volume, to express their normal behavior and avoid increased cortisol stress reaction.