Development of a method for the detection of polystyrene microplastics in paraffin-embedded histological sections.

The concerns about the presence of microplastics (MPs) in marine ecosystems have widely increased in the past years. This is reflected in a growing number of studies addressing the effects of exposure to these materials in indigenous, farmed and even laboratory marine animals subjected to toxicity-oriented bioassays. There have been, however, many constraints in the detection of MPs in biological tissues, as routine histological techniques tend to degrade these materials, which are especially sensitive to organic solvents. This issue hinders the application of standard histopathological procedures based on convenient paraffin wax-embedding protocols, with consequences for biomonitoring and bioassay procedures. The method described here was developed and validated for the detection of polystyrene microplastics in biological tissue processed for paraffin-based histology. The strategy was developed and tested from whole-soft body sections of marine mussels that internalised the MPs following dedicated bioassays. The protocol is based on the replacement of xylenes with isopropanol for the purpose of intermediate infiltration and deparaffinization. Special modifications for staining, mounting and archiving are needed and are detailed as well. The protocol is shown to be a highly cost- and time-effective procedure compatible with formalin-based fixatives plus standard sectioning and staining, yielding complete preservation of MPs and optimal tissue conditioning. The method also produced excellent results with pre-stained MPs, with fluorochromes included, altogether providing excellent localisation of polystyrene MPs in paraffin-processed biological tissue.

Applying quantitative and semi-quantitative histopathology to address the interaction between sediment-bound polycyclic aromatic hydrocarbons in fish gills.

Even though PAHs are considered priority marine pollutants, information on the interaction effects between these compounds is scarce, furthermore under ecologically-relevant circumstances. Semi-quantitative and quantitative histological analyses were enforced on the gills of the seabass (Dicentrarchus labrax), exposed to two model PAHs, single or combined, through a series of 28-day laboratory bioassays. Fish exposed to sediments contaminated with either PAH (250-800ngg(-1)), isolated or combined, exhibited most significant gill histopathological alterations after 28 days of exposure, as determined through weighted condition indices, especially in animals exposed to the potential carcinogen benzo[b]fluoranthene (B[b]F) and to mixtures of this compound with its lower, non-carcinogenic counterpart Phenanthrene (Phe). Negative correlations between interlamellar hyperplasia (the most remarkable alteration) and goblet cell counts suggest that fish exposed to sediments contaminated with B[b]F or mixed PAHs increased the thickness of epithelial cells as a response to insult, albeit compromising cell differentiation, to which is likely added impaired gas exchange and osmotic balance. In contrast, animals exposed to Phe increased the number of chloride and goblet cells relatively to control fish at early stages of exposure, suggesting then a more efficient protective mechanism. The results also showed that histopathological alterations in mixture-exposed animals do not match the expected additive effects. Overall, the findings indicate that chronic exposures to sediment-bound PAHs, under realistic scenarios, may induce lesions in gills that may imply significant hindering of basal metabolic/homeostatic pathways in marine fish whose interpretation may be hindered by complicated interaction effects and unknown factor involving, more that dose-response, time-dependent effects.

Comparing the genotoxicity of a potentially carcinogenic and a noncarcinogenic PAH, singly, and in binary combination, on peripheral blood cells of the European sea bass.

Research on the toxicological mechanisms of polycyclic aromatic hydrocarbons (PAHs) deemed carcinogenic and noncarcinogenic has mostly been developed for individual compounds even though, in the environment, PAHs invariably occur in mixtures. The present work aimed at understanding the interaction effects of two model PAHs, the potentially carcinogenic benzo[b]fluoranthene (B[b]F) and the noncarcinogenic phenanthrene (Phe) to a marine fish (the sea bass Dicentrarchus labrax). The study endeavoured an ecologically-relevant scenario with respect to concentrations and contaminant matrix, sediments, which are the main reservoirs of these substances in the environment, due to their hydrophobic nature. For the purpose, 28-day laboratorial bioassays with spiked sediments (with individual and combined PAHs at equitoxic concentrations) were conducted. Genotoxicity was determined in peripheral blood through the "Comet" assay and by scoring erythrocytic nuclear abnormalities (ENA). The results showed that exposure to either PAHs induced similar levels of DNA strand breaks, although without a clear dose- or time-response, likely due to the low concentrations of exposure and potential shits in PAH bioavailability during the assays. However, clastogenic/aneugenic lesions were only observed in fish exposed to B[b]F-spiked sediments. Conversely, the combination assays revealed a supra-additive effect especially at chromosome level, linked to concentrations of PAHs in water. A decrease in DNA-strand breakage was observed over time during all assays, revealing some ability of fish to cope with this DNA lesion. Overall, the findings show that low-moderate concentrations of sediment-bound mixed PAHs may significantly increase the hazard of mutagenesis even when the individual concentrations indicate low risk, especially considering that chromosome-level damage is unlikely to be repaired, leading to the fixation of DNA lesions upon prolonged exposures. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1307-1318, 2016.

Effects of carcinogenic versus non-carcinogenic AHR-active PAHs and their mixtures: lessons from ecological relevance.

Polycyclic aromatic hydrocarbons (PAHs) are priority environmental mutagens and carcinogens that occur in the aquatic environment as mixtures rather than the individual compounds for which guidelines are issued. The present work aimed at understanding the interaction effects between carcinogenic and non-carcinogenic PAHs in a model marine fish (Dicentrarchus labrax) in realistic scenarios. Laboratory assays under ecologically-relevant parameters were conducted for 28 days with sediments spiked with low-moderate concentrations (250-800ngg(-1)) of two model PAHs, phenanthrene (non-carcinogenic) and benzo[b]fluoranthene (carcinogenic to experimental animals). Both PAHs induced hepatic histopathological changes that indicate metabolic failure and inflammation, especially in animals exposed to mixtures. Phenanthrene elicited biochemical changes better related to oxidative stress (lipid peroxidation, glutathione and glutathione S-transferase activity) and CYP function, whereas B[b]F disrupted metabolic responses and defences to toxicological challenge. Conversely, mixed PAHs yielded lesions and responses that, altogether, are compatible with the AHR dependent pathway (the basis of PAH mutagenicity), potentially generating supra-additive effects. Nonetheless, the low, ecologically-relevant, concentrations of PAHs diluted dose and time-response relations. Overall, although seemingly predicting the risk of individual PAHs, environmental guidelines may not apply to mixtures by underestimating adverse effects, which calls for a redefinition of standards when determining the true risk of toxicants under realistic circumstances.

A study on the digestive physiology of a marine polychaete (Eulalia viridis) through microanatomical changes of epithelia during the digestive cycle.

As for many invertebrates, the gut of marine polychaete species has key physiological functions. However, studies integrating microanatomical descriptions with physiological processes are scarce. The present investigates histological, histochemical and cytological changes in the alimentary canal during the digestive cycle of the marine annelid Eulalia viridis, a species that combines opportunist scavenging, predation and cannibalistic behavior. The gut is comprised of an eversible pharynx, esophagus, intestine and rectum. Three main phases of digestion were identified, namely, resting/secretory, absorptive and excretory. The intestinal epithelium is complex and exhibited the most significant changes regarding intracellular digestion, excretion and storage. Conversely, the pharynx and esophagus were chiefly important for enzyme secretion. The results also indicate the existence of two distinct types of secretory cells in the intestine, with likely distinct physiological roles. Some similarities have been found between the intestinal epithelia and the molluscan (especially cephalopod) digestive gland, as, for instance, the shedding of apical corpuscles by digestive cells at posterior stages of digestion. The findings indicate that the digestive process in this worm is complex and related to the many physiological roles that cells need to play in the presence of reduced organ differentiation.

Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations.

Acrylamide is an amide used in several industrial applications making it easily discharged to aquatic ecosystems. The toxicity of acrylamide to aquatic organisms is scarcely known, although previous studies with murine models provided evidence for deleterious effects. To assess the effects of acrylamide to freshwater fish, goldfish (Carassius auratus L.) were exposed to several concentrations of waterborne acrylamide and analysed for genotoxic damage, alterations to detoxifying enzymes and histopathology. Results revealed a dose-dependent increase in total DNA strand breakage, the formation of erythrocytic nuclear abnormalities and in the levels of hepatic cytochrome P4501A (CYP1A) and glutathione S-transferase (GST) activity. In addition, acrylamide induced more histopathological changes to pancreatic acini than to the hepatic parenchyma, regardless of exposure concentration, whereas hepatic tissue only endured significant alterations at higher concentrations of exposure. Thus, results confirm the genotoxic potential of acrylamide to fish and its ability to induce CYP1A, probably as a direct primary defence mechanism. This strongly suggests the substance's pro-mutagenic potential in fish, similarly to what is known for rodents. However, the deleterious effects observed in the pancreatic acini, more severe than in the liver, could indicate a specific, albeit unknown toxic mechanism of acrylamide to fish that overran the organism's metabolic defences against a chemical agent rather than causing a general systemic failure.

Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans.

BACKGROUND: Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called "universal" primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. RESULTS: Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first-time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. CONCLUSIONS: These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory.

Ecological risk assessment of impacted estuarine areas: integrating histological and biochemical endpoints in wild Senegalese sole.

The analysis of multiple biomarker responses is nowadays recognized as a valuable tool to circumvent potential confounding factors affecting biomonitoring studies and allows a better understanding of the mechanisms underlying exposure to natural mixtures of toxicants. For the purpose of an environmental risk assessment (ERA) in an impacted estuary in SW Europe (the Sado, Portugal), juvenile Solea senegalensis from commercial fishing areas were surveyed for histopathological liver alterations and biochemical biomarkers. Although the findings revealed moderate differences in the patterns of histopathological traits between urban/industrial- and agricultural-influenced areas within the same estuary, no significant distinction was found between the cumulative alterations in animals from the two sites. The overall level of histopathological injury was low and severe traits like neoplasms or pre-neoplastic foci were absent. While metallothionein induction and lipid peroxidation could relate to histopathological condition indices, the activity of anti-oxidant enzymes appeared to be impaired in animals collected off the estuary's heavy-industry belt (the most contaminated site), which may partially explain some degree of hepatic integrity loss. Overall, the results are consistent with low-moderate contamination of the estuary and indicate that oxidative stress is the most important factor accounting for differences between sites. The study highlights the need of integrating multiple biomarkers when multiple environmental stressors are involved and the advantages of surveying toxicity effects in field-collected, foraging, organisms.

Development and application of a novel histological multichrome technique for clam histopathology.

A novel and expedient histological tetrachrome technique was developed and applied to whole-body sections of the clam Ruditapes decussatus (L. 1758). The technique involves fixation in Carnoy's fluid followed by immediate embedding in paraffin with staining with a combination of Alcian Blue, Periodic Acid-Schiff's, Haematoxylin and Picric Acid. Fixation and staining was perfect for all tissues and resolved good identification of Perkinsus sp. infection and high structural detail. Among the surveyed fixatives, Bouin-Hollande's fluid also provided good results, however, fixation is potentially longer, polysaccharide staining was less intense and fibres appeared to be better preserved by Carnoy's.

Impact of remobilized contaminants in Mytilus edulis during dredging operations in a harbour area: bioaccumulation and biomarker responses.

Dredging operations in harbours are recurrent to maintain accessibility and navigational depths. One of the main environmental risks of these operations is the remobilization of contaminants trapped in the sediments, rendering them more bioavailable to the biota. However, regulatory policies regarding the contamination risk of dredging chiefly apply to the disposal of dredged materials rather than the direct impact of the procedure itself. In order to assess the ecotoxicological risk of harbour dredging operations in a polluted estuary (the Tagus, W Portugal), the present study compared bioaccumulation and biomarker responses in field-deployed mussels before and after the beginning of operations, complemented by sediment characterization and risk analysis based on standardized sediment quality guidelines. The results revealed a very significant increase in genotoxicity and oxidative stress from the beginning of dredging onwards, which was accompanied by increased bioaccumulation of toxicants, especially polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Overall, the results indicate the importance of surveying the direct impacts of these procedures on local contamination, especially considering these sediments had been previously classified as "trace contaminated", according to normative guidelines, and therefore safe for disposal. This study shows the importance of obtaining both chemical and biological data in standard monitoring procedures and that the remobilization of contaminants by dredging operations may be grossly underestimated, which calls for caution when assessing the impact of these activities even in low to moderately polluted areas.

Hepatic proteome changes in Solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey.

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.

Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral blood: laboratory versus in situ studies.

Juvenile Senegalese soles (Solea senegalensis) were exposed to estuarine sediments through 28-day laboratory and in situ (field) bioassays. The sediments, collected from three distinct sites (a reference plus two contaminated) of the Sado Estuary (W Portugal) were characterized for total organic matter, redox potential, fine fraction and for the levels of metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorines, namely polychlorinated biphenyls (PCBs) and dichloro diphenyl tricholoethane plus its main metabolites (DDTs). Genotoxicity was determined in whole peripheral blood by the single-cell gel electrophoresis (SCGE or "comet") assay and by scoring erythrocytic nuclear abnormalities (ENA). Analysis was complemented with the determination of lipid peroxidation in blood plasma by the thiobarbituric acid reactive substances (TBARS) protocol and cell type sorting. The results showed that exposure to contaminated sediments induced DNA fragmentation and clastogenesis. Still, laboratory exposure to the most contaminated sediment revealed a possible antagonistic effect between metallic and organic contaminants that might have been enhanced by increased bioavailability. The laboratory assay caused a more pronounced increase in ENA whereas a very significant increase in DNA fragmentation was observed in field-tested fish exposed to the reference sediment, which is likely linked to increased lipid peroxidation that probably occurred due to impaired access to food. Influence of natural pathogens was ruled out by unaltered leukocyte counts. The statistical integration of data correlated lipid peroxidation with biological variables such as fish length and weight, whereas the genotoxicity biomarkers were more correlated to sediment contamination. It was demonstrated that laboratory and field bioassays for the risk assessment of sediment contamination may yield different genotoxicity profiles although both provided results that are in overall accordance with sediment contamination levels. While field assays may provide more ecologically relevant data, the multiple environmental variables may produce sufficient background noise to mask the true effects of contamination.

Transcriptomic analyses in a benthic fish exposed to contaminated estuarine sediments through laboratory and in situ bioassays.

The transcription of contaminant response-related genes was investigated in juvenile Senegalese soles exposed to sediments from three distinct sites (a reference plus two contaminated) of a Portuguese estuary (the Sado, W Portugal) through simultaneous 28-day laboratory and in situ bioassays. Transcription of cytochrome P450 1A (CYP1A), metallothionein 1 (MT1), glutathione peroxidase (GPx), catalase (CAT), caspase 3 (CASP3) and 90 kDa heat-shock protein alpha (HSP90AA) was surveyed in the liver by real-time PCR. CASP3 transcription analysis was complemented by surveying apoptosis through the TUNEL reaction. After 14 days of exposure, relative transcription was either reduced or decreased in fish exposed to the contaminated sediments, revealing a disturbance stress phase during which animals failed to respond to insult. After 28 days of exposure all genes' transcription responded to contamination but laboratory and in situ assays depicted distinct patterns of regulation. Although sediments revealed a combination of organic and inorganic toxicants, transcription of the CYP1A gene was consistently correlated to organic contaminants. Metallothionein regulation was found correlated to metallic and organic xenobiotic contamination in the laboratory and in situ, respectively. The transcription of oxidative stress-related genes can be a good indicator of general stress but caution is mandatory when interpreting the results since regulation may be influenced by multiple factors. As for MT1, HSP90 up-regulation has potential to be a good indicator for total contamination, as well as the CASP3 gene, even though hepatocyte apoptosis depicted values inconsistent with sediment contamination, showing that programmed cell death did not directly depend on caspase transcription alone.

Dressing liposomal particles with chitosan and poly(vinylic alcohol) for oral vaccine delivery.

Liposomes have been used as adjuvants since 1974. One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our aim was to combine liposomes that could encapsulate antigens (i.e., Dtxd, diphtheria toxoid) with chitosan, which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3:1) can be sandwiched with chitosan (Chi) and stabilized by PVA (poly-vinylic alcohol), which are biodegradable, biocompatible polymers. Round, smooth-surfaced particles of REVs-Chi (reversed-phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on the REVs surface was accompanied by an increase of ζ-potential. In contrast, PVA adsorption on the REVs-Chi surface was accompanied by a decrease of ζ-potential. The presence of Dtxd increased the Chi surface-adsorption efficiency. The PVA affinity by mucine was 2,000 times higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new, stabilized particles were able to be adsorbed by intestinal surfaces, resisted degradation, and controlled antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant.

Diphtheria toxoid conformation in the context of its nanoencapsulation within liposomal particles sandwiched by chitosan.

Chitosan (α-(1-4)-amino-2-deoxy-ß-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, PO(2-)(4). This ion was used to prepare a stable Dtxd and immunologically recognized REV-Chi-PVA formulation in the presence of 50 mM of PO(2-)(4). Under these conditions, the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH(3)CO(2)C(2)H(5) to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough, because a simple solution, such as salt, addition avoided heterologous protein use.

Evaluation of the potential of the common cockle (Cerastoderma edule L.) for the ecological risk assessment of estuarine sediments: bioaccumulation and biomarkers.

Common cockles (Cerastoderma edule, L. 1758, Bivalvia: Cardiidae) were subjected to a laboratory assay with sediments collected from distinct sites of the Sado Estuary (Portugal). Cockles were obtained from a mariculture site of the Sado Estuary and exposed through 28-day, semi-static, assays to sediments collected from three sites of the estuary. Sediments from these sites revealed different physico-chemical properties and levels of metals and organic contaminants, ranging from unimpacted (the reference site) to moderately impacted, when compared to available sediment quality guidelines. Cockles were surveyed for bioaccumulation of trace elements (Ni, Cu, Zn, As, Cd and Pb) and organic contaminants (PAHs, PCBs and DDTs). Two sets of potential biomarkers were employed to assess toxicity: whole-body metallothionein (MT) induction and digestive gland histopathology. The bioaccumulation factor and the biota-to-soil accumulation factor were estimated as ecological indices of exposure to metals and organic compounds. From the results it is inferred that C. edule responds to sediment-bound contamination and might, therefore, be suitable for biomonitoring. The species was found capable to regulate and eliminate both types of contaminants. Still, the sediment contamination levels do not account for all the variation in bioaccumulation and MT levels, which may result from the moderate metal concentrations found in sediments, the species' intrinsic resistance to pollution and from yet unexplained xenobiotic interaction effects.

Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction.

Juvenile Solea senegalensis were exposed to fresh sediments from three stations of the Sado estuary (Portugal) in 28-day laboratory assays. Sediments revealed distinct levels of total organic matter, fine fraction, redox potential, trace elements (arsenic, cadmium, chromium, copper, nickel, lead and zinc) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide: dichloro diphenyl trichloroethane). Organisms were surveyed for contaminant bioaccumulation and induction of two hepatic biochemical biomarkers: metallothionein (MT) and cytochrome P450 (CYP1A), as potential indicators of exposure to metallic and organic contaminants, respectively. Using an integrative approach it was established that, although bioaccumulation is in general accordance with sediment contamination, lethality and biomarker responses are not linearly dependent of the cumulative concentrations of sediment contaminants but rather of their bioavailability and synergistic effects in organisms. It is concluded that metals and organic contaminants modulate both MT and CYP1A induction and it is suggested that reactive oxygen species may be the link between responses and effects of toxicity.

Toxicokinetics of waterborne trivalent arsenic in the freshwater bivalve Corbicula fluminea.

Arsenite (As(III)) uptake and elimination kinetics were studied in a freshwater bivalve, Corbicula fluminea, exposed to several nominal concentrations of As(III) (0, 100, 300, 500, and 1000 microg L(-1)) in a static 28-day assay, followed by a depuration stage of 14 days. At the end of each sampling time (days 0, 7, 28, and 42) whole-body portions were surveyed for total As concentrations and, complimentarily, surveyed for whole-body metallothionein (MT) induction to assess its role as a defense mechanism against exposure to As(III). Histochemical evaluation of the digestive gland was performed to verify As deposition and elimination in the tissue. Results show a significant increase in whole-body total As after 28 days of exposure for all treatments, followed by a decrease at the end of the depuration phase. Biodynamic kinetic models for As uptake and elimination were obtained from bioaccumulation data during the exposure phase, for all As treatments, by estimating uptake and elimination rate constants. Bioconcentration factors (BCFs) were estimated by the ratio of these constants. Results revealed that exposure to higher concentrations of As(III) causes a decrease in BCFs, suggesting that C. fluminea triggers effective regulatory mechanisms when exposed to higher concentrations of the metalloid. Significant induction of MT was detected during the exposure phase, followed by a decrease in MT concentration to control levels after depuration for all treatments. No significant differences in MT concentrations were observed between treatments. This finding may confirm the role of MT as part of the As regulation process, but its independence relative to concentrations of As(III) in water suggests that MT induction is not dose dependent. The histochemical evaluation provided clear evidence that As was effectively accumulated in the digestive gland during exposure and eliminated during depuration. The present work demonstrated that C. fluminea is capable of regulating As, even at exposures as high as 1000 microg L(-1) of waterborne As(III).

An assessment of the ability to ingest and excrete microplastics by filter-feeders: A case study with the Mediterranean mussel.

Plastic debris has been recognized as a growing threat to marine biota due to its widespread distribution and possible interactions with marine species. Concerns over the effects of plastic polymers in marine ecosystems is reflected in the high number of toxicological studies, regarding microplastics (<5 mm) and marine fauna. Although several studies reported that organisms ingest and subsequently eliminate microplastics (MP), the potential effects at organ and tissue level remain unclear, especially considering exposure to different microplastic sizes and concentrations. The present study aimed at investigating potential pathophysiological effects of the ingestion of MP by marine filter-feeders. For the purpose, Mediterranean mussel (Mytilus galloprovincialis) was exposed to spherical polystyrene MP (2 and 10 µm Ø) over short- and medium-term exposure periods, under single and combined concentrations that represent high, yet realistic doses (10 and 1000 MP mL-1). Overall, results suggest rapid MP' clearance from water column by filtering, regardless of MP size. Ingestion occurred, identified by MP in the lumen of the gut (mostly in midgut region), followed by excretion through faeces. However, no MP were found in gills or digestive gland diverticula. Biochemical indicators for oxidative stress were generally irresponsive regardless of organ and time of exposure. Small foci of haemocytic infiltration in gastric epithelia were found, albeit not clearly related to MP ingestion. Globally, no evident histopathological damage was recorded in whole-body sections of exposed animals. The present findings highlight the adaptative ability of filter-feeding bivalves to cope with filtration of suspended MP, resulting in rapid elimination and reduced internal damage following ingestion of spherical MP. Nevertheless, the fact that the animals are able to translocate MP to the gut reveals that filter feeding organisms may indeed became a target of concern for fragmented materials with smaller, mixed sizes and sharper edges.

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): effects of metallic and organic contaminants.

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T(14) and T(28), while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T(28) only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.