Microplastics (≤ 10 µm) bioaccumulation in marine sponges along the Moroccan Mediterranean coast: Insights into species-specific distribution and potential bioindication.

Microplastics (MPs) are pervasive in marine environments and widely recognized as emerging environmental pollutants due to the multifaceted risks they exert on living organisms and ecosystems. Sponges (Phylum Porifera) are essential suspension-feeding organisms that may be highly susceptible to MPs uptake due to their global distribution, unique feeding behavior, and sedentary lifestyle. However, the role of sponges in MP research remains largely underexplored. In the present study, we investigate the presence and abundance of MPs (≤10 µm size) in four sponge species, namely Chondrosia reniformis, Ircinia variabilis, Petrosia ficiformis, and Sarcotragus spinosulus collected from four sites along the Mediterranean coast of Morocco, as well as their spatial distribution. MPs analysis was conducted using an innovative Italian patented extraction methodology coupled with SEM-EDX detection. Our findings reveal the presence of MPs in all collected sponge specimens, indicating a pollution rate of 100%. The abundance of MPs in the four sponge species ranged from 3.95×105 to 1.05×106 particles per gram dry weight of sponge tissue, with significant differences observed among sampling sites but no species-specific differences. These results imply that the uptake of MPs by sponges is likely influenced by aquatic environmental pollution rather than the sponge species themselves. The smallest and largest MPs were identified in C. reniformis and P. ficiformis, with median diameters of 1.84 µm and 2.57 µm, respectively. Overall, this study provides the first evidence and an important baseline for the ingestion of small MP particles in Mediterranean sponges, introducing the hypothesis that they may serve as valuable bioindicators of MP pollution in the near future.

Embryotoxicity of polystyrene microplastics in zebrafish Daniorerio.

In the recent years, increasing scientific and societal concern has been raised over the presence and accumulation of plastic debris in the environment and the effects of microplastics (MPs) that can easily interact with biota. In order to elucidate the impact of MPs at the critical development stages of freshwater fish species, a fish embryo toxicity test was herein performed on the zebrafish Danio rerio, exposed to 10 µm polystyrene MPs at 200 particles/mL for 120 hpf. After exposure, accumulation of MPs in larvae was measured, survival, hatching and larvae development were monitored and the oxidant/anti-oxidant responses and cellular detoxification evaluated. No impact on survival of developing zebrafish was revealed, but a moderate delay in hatching was observed. Alterations in larvae development were recorded with zebrafish exhibiting serious deformities, mainly at the level of column and tail, as well as a compromised integrity of the visual structure of the eyes. Moreover, increased levels of gene transcription involved in the oxidative stress (sod1, sod2 and cat) and in cellular detoxification (gst and cyp) were also detected in MPs-exposed zebrafish larvae. Overall, this research work provides new insights on the ecotoxicological impact of polystyrene MPs on the critical developmental stages of a freshwater fish species, therefore enhancing the current knowledge of the environmental risk posed by MPs to the aquatic ecosystem.

Time-dependent metabolic disorders induced by short-term exposure to polystyrene microplastics in the Mediterranean mussel Mytilus galloprovincialis.

In the modern society, plastic has achieved a crucial status in a myriad of applications because of its favourable properties. Despite the societal benefits, plastic has become a growing global concern due to it is persistence and bioavailability as microplastics (MPs) to aquatic biota. In order to provide mechanistic insights into the early toxicity effects of MPs on aquatic invertebrates, a short-term (up to 72 h) exposure to 3 µm red polystyrene MPs (50 particles/mL) was conducted on marine mussels Mytilus galloprovincialis, selected as model organism for their ability to ingest MPs and their commercial relevance. The use of protonic Nuclear Magnetic Resonance (1H NMR)-based metabolomics, combined with chemometrics, enabled a comprehensive exploration at fixed exposure time-points (T24, T48, T72) of the impact of MPs accumulated in mussel digestive glands, chosen as the major site for pollutants storage and detoxification processes. In detail, 1H NMR metabolic fingerprints of MP-treated mussels were clearly separated from control and grouped for experimental time-points by a Principal Component Analysis (PCA). Numerous metabolites, including amino acids, osmolytes, metabolites involved in energy metabolism, and antioxidants, participating in various metabolic pathways significantly changed over time in MP-exposed mussel digestive glands related to control, reflecting also the fluctuations in MPs accumulation and pointing out the occurrence of disorders in amino acid metabolism, osmotic equilibrium, antioxidant defense system and energy metabolism. Overall, the present work provides the first insights into the early mechanisms of toxicity of polystyrene MPs in marine invertebrates.

1H NMR-based metabolomics investigation on the effects of petrochemical contamination in posterior adductor muscles of caged mussel Mytilus galloprovincialis.

Environmental metabolomics is a high-throughout approach that provides a snapshot of the metabolic status of an organism. In order to elucidate the biological effects of petrochemical contamination on aquatic invertebrates, mussels Mytilus galloprovincialis were caged at the "Augusta-Melilli-Priolo" petrochemical area and Brucoli (Sicily, south Italy), chosen as the reference site. After confirming the elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and mercury (Hg) in Augusta sediments in our previous work (Maisano et al., 2016a), herein an environmental metabolomics approach based on protonic nuclear magnetic resonance (1H NMR), coupled with chemometrics, was applied on the mussel posterior adductor muscle (PAM), the main muscular system in bivalve molluscs. Amino acids, osmolytes, energy storage compounds, tricarboxylic acid cycle intermediates, and nucleotides, were found in PAM NMR spectra. Principal Component Analysis (PCA) indicated that mussels caged at the polluted site clustered separately from mussels from the control area, suggesting a clear differentiation between their metabolic profiles. Specifically, disorders in energy metabolism, alterations in amino acids metabolism, and disturbance in the osmoregulatory processes were observed in mussel PAM. Overall, findings from this work demonstrated the usefulness of applying an active biomonitoring strategy for environmental risk assessment, and the effectiveness of metabolomics in elucidating changes in metabolic pathways of aquatic organisms caged at sites differentially contaminated, and thus its suitability to be applied in ecotoxicological studies.

Sex steroids and metabolic responses in mussels Mytilus galloprovincialis exposed to drospirenone.

Drospirenone (DRO) is a synthetic progestin derived from 17α-spironolactone with a pharmacological mechanism of action similar to progesterone. Despite its wide use as pharmaceutical and consequent continuous release into the aquatic environment, DRO effects have been poorly investigated on aquatic biota. In order to unravel the toxicity mechanisms of DRO, mussels Mytilus galloprovincialis were exposed for 7 days to different concentrations of DRO, namely 20ng/L (Low; L), 200ng/L (Medium; M), 2000ng/L (High; H) and 10µg/L (Super High; SH) nominal doses. Following exposure, no significant effect was observed on gonad maturation of treated and untreated mussels. The levels of progesterone (P4) and testosterone (T) were measured in mantle/gonad tissues and no significant alteration detected after exposure. However, the application of a protonic nuclear magnetic resonance (1H NMR)-based metabolomics approach enabled a comprehensive assessment of DRO effects in mussels. Specifically, 1H NMR metabolic fingerprints of digestive glands of DRO treated mussel groups were clearly separated from each other and from controls through a principal component analysis (PCA). Moreover, a number of metabolites involved in different metabolic pathways were found to significantly change in DRO-exposed mussels compared to control, suggesting the occurrence of alterations in energy metabolism, amino acids metabolism, and glycerophospholipid metabolism. Overall, despite no changes in gonad maturation and steroids levels were recorded in mussels after DRO exposure, the metabolomics approach demonstrated its effectiveness and high sensitivity in elucidating DRO-induced metabolic disturbances in marine mussels, and thus its usefulness in the environmental risk assessment of pharmaceuticals.

Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island.

In aquatic environments, bivalve molluscs are used as sentinel species for environmental biomonitoring. In this study Pinna nobilis specimens, the biggest Mediterranean bivalve, were collected in the Magaluf bay (Mallorca), a touristic location and in a pristine area of the Cabrera National Park as the control location. Histological and histochemical analysis in gills of specimens sampled from Magaluf exhibited evident tissue alterations with high presence of haemocytes. Lower acetylcholinesterase (AChE) activity and protein expression were also found in the gills of specimens collected from Magaluf compared with the control area. The determination of antioxidant enzyme activities, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, showed a higher activities of these antioxidant enzymes and total glutathione content in samples from Magaluf bay than in Cabrera. In conclusion, the present study demonstrated that human activities result in morphological tissue alterations and a reduced AChE activity in gills of P. nobilis. Moreover, these stressful environmental conditions induced an adaptive response in P. nobilis as evidenced by increased antioxidant defences and a decreased AChE activity. CAPSULE: The human activities induce oxidative stress in P. nobilis as evidenced by increased antioxidant defences and a decreased acetylcholinesterase activity.

Metabolomic and biochemical disorders reveal the toxicity of environmental microplastics and benzo[a]pyrene in the marine polychaete Hediste diversicolor.

Recently, the abundance of environmental microplastics (MPs) has become a global paramount concern. Besides the danger of MPs for biota due to their tiny size, these minute particles may act as vectors of other pollutants. This study focused on evaluating the toxicity of environmentally relevant concentrations of MPs (10 and 50 mg/kg sediment) and benzo[a]pyrene (B[a]P, 1 µg/kg sediment), alone and in mixture, for 3 and 7 days in marine polychaete Hediste diversicolor, selected as a benthic bioindicator model. The exposure period was sufficient to confirm the bioaccumulation of both contaminants in seaworms, as well as the potential capacity of plastic particles to adsorb and vehiculate the B[a]P. Interestingly, increase of acidic mucus production was observed in seaworm tissues, indicative of a defense response. The activation of oxidative system pathways was demonstrated as a strategy to prevent lipid peroxidation. Furthermore, the comprehensive Nuclear Magnetic Resonance (NMR)-based metabolomics revealed significant disorders in amino acids metabolism, osmoregulatory process, energetic components, and oxidative stress related elements. Overall, these findings proved the possible synergic harmful effect of MPs and B[a]P even in small concentrations, which increases the concern about their long-term presence in marine ecosystems, and consequently their transfer and repercussions on marine fauna.

Internalization of nano- and micro-plastics in human erythrocytes leads to oxidative stress and estrogen receptor-mediated cellular responses.

Plastic material versatility has resulted in a substantial increase in its use in several sectors of our everyday lives. Consequently, concern regarding human exposure to nano-plastics (NPs) and micro-plastics (MPs) has recently increased. It has been shown that plastic particles entering the bloodstream may adhere to the erythrocyte surface and exert adverse effects following erythrocyte aggregation and adhesion to blood vessels. Here, we explored the effects of polystyrene nano-plastics (PS-NPs) and micro-plastics (PS-MPs) on human erythrocytes. Cellular morphology, binding/internalization of PS-NPs and PS-MPs, oxidative stress parameters, as well as the distribution and anion exchange capability of band 3 (anion exchanger 1; SLC4A1) have been analyzed in human erythrocytes exposed to 1 µg/mL PS-NPs or PS-MPs for 3 and 24 h, respectively. The data obtained showed significant modifications of the cellular shape after exposure to PS-NPs or PS-MPs. In particular, a significantly increased number of acanthocytes, echinocytes and leptocytes were detected. However, the percentage of eryptotic cells (<1 %) was comparable to physiological conditions. Analytical cytology and confocal microscopy showed that PS-NPs and PS-MPs bound to the erythrocyte plasma membrane, co-localized with estrogen receptors (Erα/ERß), and were internalized. An increased trafficking from the cytosol to the erythrocyte plasma membrane and abnormal distribution of ERs were also observed, consistent with ERα-mediated binding and internalization of PS-NPs. An increased phosphorylation of ERK1/2 and AKT kinases indicated that an activation of the ER-modulated non-genomic pathway occurred following exposure to PS-NPs and PS-MPs. Interestingly, PS-NPs or PS-MPs caused a significant production of reactive oxygen species, resulting in an increased lipid peroxidation and protein sulfhydryl group oxidation. Oxidative stress was also associated with an altered band 3 ion transport activity and increased oxidized haemoglobin, which led to abnormal clustering of band 3 on the plasma membrane. Taken together, these findings identify cellular events following the internalization of PS-NPs or PS-MPs in human erythrocytes and contribute to elucidating potential oxidative stress-related harmful effects, which may affect erythrocyte and systemic homeostasis.

Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments.

Multiple anthropogenic activities present along coastal environments may affect the health status of aquatic ecosystems. In this study, specimens of European sea bass (Dicentrarchus labrax) were exposed for 30 days to highly contaminated sediment collected from the industrial area between Augusta and Priolo (Syracuse, Italy), defined as the most mercury polluted site in the Mediterranean. The aim was to evaluate the responses of juvenile D. labrax to highly contaminated sediments, particularly enriched in Hg, in order to enhance the scarce knowledge on the potential compensatory mechanisms developed by organisms under severe stress conditions. Apoptotic and proliferative activities [cell turnover: Proliferating Cell Nuclear Antigen (PCNA) and FAS Ligand (FasL)], onset of hypoxic condition [hypoxia: Hypoxia Inducibile Factor-1α (HIF-1α)], and changes in the neuroendocrine control mechanisms [neurotransmission: Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT), Acetylcholinesterase (AChE), 5-Hydroxytryptamine (5-HT) and 5-Hydroxytryptamine receptor 3 (5-HT3)] were investigated in sea bass gill tissues. In the specimens exposed to the polluted sediment, the occurrence of altered cell turnover may result in impaired gas exchange that leads to a condition of "functional hypoxia". Changes in neurotransmission pathways were also observed, suggesting a remodeling process as an adaptive response to increase the O2-carrying capacity and restore the normal physiological conditions of the gills. Overall, these findings demonstrated that although chronic exposure to heavy metal polluted sediments alters the functioning of both the nervous and endocrine systems, as well as plasticity of the gill epithelium, fish are able to trigger a series of physiological adjustments or adaptations interfering with specific neuroendocrine control mechanisms that enable their long-term survival.

Histomorphological Changes in Fish Gut in Response to Prebiotics and Probiotics Treatment to Improve Their Health Status: A Review.

The gastrointestinal tract (GIT) promotes the digestion and absorption of feeds, in addition to the excretion of waste products of digestion. In fish, the GIT is divided into four regions, the headgut, foregut, midgut, and hindgut, to which glands and lymphoid tissues are associated to release digestive enzymes and molecules involved in the immune response and control of host-pathogens. The GIT is inhabited by different species of resident microorganisms, the microbiota, which have co-evolved with the host in a symbiotic relationship and are responsible for metabolic benefits and counteracting pathogen infection. There is a strict connection between a fish's gut microbiota and its health status. This review focuses on the modulation of fish microbiota by feed additives based on prebiotics and probiotics as a feasible strategy to improve fish health status and gut efficiency, mitigate emerging diseases, and maximize rearing and growth performance. Furthermore, the use of histological assays as a valid tool for fish welfare assessment is also discussed, and insights on nutrient absorptive capacity and responsiveness to pathogens in fish by gut morphological endpoints are provided. Overall, the literature reviewed emphasizes the complex interactions between microorganisms and host fish, shedding light on the beneficial use of prebiotics and probiotics in the aquaculture sector, with the potential to provide directions for future research.

Morphological and functional alterations in hemocytes of Mytilus galloprovincialis exposed in high-impact anthropogenic sites.

The work aimed to study the induction of morphological alterations in M. galloprovincialis in the field and its suitability to be integrated into a sensitive, simple, and cost-effective cell-based multimarker approach for the detection of the stress status induced by pollution in coastal marine environments in view of ecotoxicological biomonitoring and assessment application. Cellular morphometric alterations was paralleled by the analysis of standardized biomarkers such as lysosomal membrane destabilization, and genotoxocity biomarkers such as micronuclei and binuclated cells frequencies were investigated. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two multi-impacted coastal sites of the central Mediterranean area, Bagnoli in the eastern Tyrrhenian Sea and Augusta-Melilli-Priolo in the western Ionian Sea. Capo Miseno (NA) for the Tyrrhenian area and Brucoli (ME) for the Ionian area were chosen as control sites. Hemocyte enlargement and filopodial elongation increased frequencies were observed in organisms exposed to the impacted sites. These morphometric alterations showed strong agreement with the lysosomal membrane destabilization and biomarkers of genotoxicity, suggesting their usefulness in detecting the pollutant-induced stress syndrome related to genotoxic damage.

Short-term exposure to polystyrene microplastics hampers the cellular function of gills in the Mediterranean mussel Mytilus galloprovincialis.

Plastic is undoubtedly the most useful and versatile polymeric material that man has developed in the last two centuries Despite the societal benefits, plastic is now a serious global issue because it is persistent and may bioaccumulate into aquatic biota as microplastics (MPs). This study was designed to evaluate the daily uptake and cellular effects due to a short-term (up to 72 h) exposure to 3 µm red polystyrene MPs (50 beads/mL) in the gills of the Mediterranean mussel Mytilus galloprovincialis, chosen as model species for its ecological and commercial relevance. After measuring the daily uptake of MPs and detecting their presence within the branchial epithelium at all the exposure time-points (T24, T48, T72), some cleaning mechanisms were observed by neutral and acid mucous secretions at mussel gills. The protonic Nuclear Magnetic Resonance (1H NMR)-based metabolomics, combined with chemometrics, allowed to comprehensively explore the time-dependent metabolic disorders triggered by MPs in mussel gills over the short-term trial. Specifically, the clear clustering between MP-treated mussel gills and those from control, together with the grouping for experimental time-points as depicted by the Principal Component Analysis (PCA), were due to changes in the amino acids and energy metabolism, disturbances in the osmoregulatory processes, as well as in the cholinergic neurotransmission. Moreover, as evidenced by enzymatic assays, even the oxidative defense systems and lipid metabolism were hampered by MP exposure. Overall, these findings provides the first insights into the early time-dependent mechanisms of toxicity of polystyrene MPs in marine mussels, and underline the potential environment and human health risk posed by MPs contamination.

Metabolomic investigation of Mytilus galloprovincialis (Lamarck 1819) caged in aquatic environments.

Environmental metabolomics was applied to assess the metabolic responses in transplanted mussels to environmental pollution. Specimens of Mytilus galloprovincialis, sedentary filter-feeders, were caged in anthropogenic-impacted and reference sites along the Augusta coastline (Sicily, Italy). Chemical analysis revealed increased levels of PAHs in the digestive gland of mussels from the industrial area compared with control, and marked morphological changes were also observed. Digestive gland metabolic profiles, obtained by 1H NMR spectroscopy and analyzed by multivariate statistics, showed changes in metabolites involved in energy metabolism. Specifically, changes in lactate and acetoacetate could indicate increased anaerobic fermentation and alteration in lipid metabolism, respectively, suggesting that the mussels transplanted to the contaminated field site were suffering from adverse environmental condition. The NMR-based environmental metabolomics applied in this study results thus in it being a useful and effective tool for assessing environmental influences on the health status of aquatic organisms.

Toxicological impact of environmental microplastics and benzo[a]pyrene in the seaworm Hediste diversicolor under environmentally relevant exposure conditions.

Nowadays, marine ecosystems are under severe threat from the simultaneous presence of multiple stressors, including microplastics (MPs) and polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P). In addition to their presence in various marine compartments, there are increasing concerns on the potential capacity of MPs to sorb, concentrate and transfer these pollutants in the environment. Although their ecotoxicological impacts are currently evident, few works have studied the combined effects of these contaminants. Therefore, the major purpose of this work was to assess the toxicity of environmental relevant concentrations of MPs (<30 µm) and B[a]P, alone and in mixture, in the seaworm Hediste diversicolor by exploring their accumulation and hazardous biological effects for 3 and 7 days. Environmental MPs were able to increase B[a]P in a time-dependent manner. The obtained results showed that individual treatments, as well as co-exposure to contaminants, caused cytotoxicity and genotoxicity in the cœlomic fluid cells, while oxidative stress effects were observed at tissue and gene levels associated with alteration in neurotransmission. Overall, our findings provide additional clues about MPs as organic pollutant vectors in the marine environment, and contribute to a clearer understanding of their toxicological risk to aquatic invertebrates.

Histological endpoints and oxidative stress transcriptional responses in the Mediterranean mussel Mytilus galloprovincialis exposed to realistic doses of salicylic acid.

Despite the availability of analytic data, little is known about the toxicity of salicylic acid (SA) on aquatic non-target organisms. The present study aimed at evaluating the impact of SA through a short-term exposure of the Mediterranean mussel Mytilus galloprovincialis to five environmentally relevant concentrations of SA. A set of suitable biomarkers was applied at selected time-points on mussel digestive glands, including histological observations and expression of oxidative stress related genes. The obtained results showed a conspicuous hemocytic infiltration among mussel digestive tubules, as confirmed also by a flow cytometric approach that revealed an increase of halinocytes and granulocytes. Interestingly, a significant dose and time dependent decrease in the expression levels of oxidative stress related genes was found in mussels exposed to SA except for the glutathione S-transferase gene that was significantly up-regulated in a time-dependent manner confirming its important role against oxidant species and in the metabolism of pharmaceuticals.

Autophagic event and metabolomic disorders unveil cellular toxicity of environmental microplastics on marine polychaete Hediste diversicolor.

Although the hazards of microplastics (MPs) have been quite well explored, the aberrant metabolism and the involvement of the autophagy pathway as an adverse response to environmental MPs in benthic organisms are still unclear. The present work aims to assess the impact of different environmental MPs collected from the south coast of the Mediterranean Sea, composed by polyethylene (PE), polyethylene vinyl acetate (PEVA), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP) and polyamide (PA) on the metabolome and proteome of the marine polychaete Hediste diversicolor. As a result, all the microplastic types were detected with Raman microspectroscopy in polychaetes tissues, causing cytoskeleton damage and induced autophagy pathway manifested by immunohistochemical labeling of specific targeted proteins, through Tubulin (Tub), Microtubule-associated protein light chain 3 (LC3), and p62 (also named Sequestosome 1). Metabolomics was conducted to further investigate the metabolic alterations induced by the environmental MPs-mixture in polychaetes. A total of 28 metabolites were differentially expressed between control and MPs-treated polychaetes, which showed elevated levels of amino acids, glucose, ATP/ADP, osmolytes, glutathione, choline and phosphocholine, and reduced concentration of aspartate. These novel findings extend our understanding given the toxicity of environmental microplastics and unravel their underlying mechanisms.

Assessment of the effectiveness of a novel BioFilm-Membrane BioReactor oil-polluted wastewater treatment technology by applying biomarkers in the mussel Mytilus galloprovincialis.

Petrochemical industries and oil refineries are sources of hazardous chemicals into the aquatic environments, and often a leading cause of reduced oxygen availability, thus resulting in adverse effects in biota. This study is an expansion of our previous work on the assessment of the BioFilm-Membrane Bioreactor (BF-MBR) to mitigate the impact of oil-polluted wastewater on marine environments. Specifically, this study evaluated the reduction of selected chemical constituents (hydrocarbons and trace metals) and toxicity related to hypoxia and DNA damage to mussels Mytilus galloprovincialis, before and after treatment of oil-polluted wastewater with the BF-MBR. The application of a multidisciplinary approach provided evidence of the efficiency of BF-MBR to significantly reducing the pollutants load from oily contaminated seawaters. As result, the health status of mussels was preserved by a hypoxic condition due to oily pollutants, as evidenced by the modulation in the gene expression of HIF-1α and PHD and changes in the level of hypotaurine and taurine. Moreover, ameliorative effects in the energy metabolism were also found in mussel gills showing increased levels of glycogen, glucose and ATP, as well as a mitigated genotoxicity was revealed by the Micronucleus and Comet assays. Overall, findings from this study support the use of the BF-MBR as a promising treatment biotechnology to avoid or limiting the compromise of marine environments from oil pollution.

Acute and Sub-Chronic Effects of Microplastics (3 and 10 µm) on the Human Intestinal Cells HT-29.

Due to ingestion of contaminated foods, the human gastrointestinal tract is the most likely site of exposure to microplastics (MPs) with gut barrier dysfunction and intestinal inflammation. Aimed to assess the effects induced by MPs with different granulometry (polystyrene (PS) 3 and 10 µm), we performed an in vitro study by using the human intestinal cell line HT29. As a novelty, we assessed the sub-chronic exposure extending the treatment up to 48 days simulating the in vivo situation. In the range of 100-1600 particles mL-1, both the PS suspensions had moderate cytotoxicity after 24 h with percentages of mortality between 6.7 and 21.6 for the 10 µm and 6.1 and 29.6 for the 3 µm PS. Microscopic observation highlighted a more pronounced lysosomal membrane permeabilization in HT29 exposed to PS 3µm. Reactive oxygen species production was higher in cells exposed to PS 10 µm, but sub-chronic exposure highlighted the ability of the cells to partially neutralize this effect. Comet-assay confirmed the temporary oxidative damage that was PS-induced. Overall, considering the very fast turnover of intestinal cells, the increase in cell mortality, equal to 25% and 11% for 3 and 10 µm PS-MPs for each time point, could trigger intestinal disorders due to prolonged exposure.

Uptake, accumulation and associated cellular alterations of environmental samples of microplastics in the seaworm Hediste diversicolor.

The ubiquitous distribution of microplastics (MPs) in the marine environment raises global concern to understand their impact. Environmental MPs have been shown to exhibit different physicochemical properties during their life cycles. However, the body of knowledge regarding their accumulation and biological effects is still significantly limited compared to manufactured MPs. To evaluate the hazardous effects of a mixture of environmental MPs collected along the Tunisian beaches, their accumulation and cellular effects were investigated in Hediste diversicolor. MP sample was composed of polyethylene (PE), polyethylene vinyl acetate (PEVA), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP) and polyamide (PA) analyzed using Raman microspectroscopy (RM). The concentrations of MPs in seaworm tissues increased over time, following the order 1.2-0.45 µm > 3-1.2 µm > 100-3 µm. The ingestion of MPs by H. diversicolor reduced their survival and growth, affected the neuro-transmission and antioxidant pathways. Our data emphasised that the toxic effects of environmental MPs were closely related to the exposure dose and period. The results also demonstrated that the size distribution of MPs in seaworms was mainly correlated with biochemical markers. This study highlights the ecological risk in the ingestion and accumulation of environmental MPs by biota that threatens their functional parameters.

Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants.

When a contaminant interacts with biotic components of a marine ecosystem, it causes a series of changes that can compromise an entire community (Stebbing, 1985). This present study wants to focus on changes in the gills of a bioindicator benthic organism, Coris julis, collected in Milazzo (Messina, Italy), characterized by a strong anthropical impact), compared with individuals from the control site (Marinello, Messina). RT-PCR has been used for both MT and HSP70, and the respective mRNAs have been visualized by FISH. MT and HSP70 expression levels increased in individuals collected in Milazzo. The presence of numerous apoptotic and proliferating cells and the analysis of several neuronal markers by immunohistochemical method give information about the adaptation to a heavy metal mixture. The obtained results show that, in stressed fishes, defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.