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.

Co-exposure to environmental carcinogens in vivo induces neoplasia-related hallmarks in low-genotoxicity events, even after removal of insult.

Addressing the risk of mixed carcinogens in vivo under environmentally-realistic scenarios is still a challenge. Searching for adequate biomarkers of exposure requires understanding molecular pathways and their connection with neoplasia-related benchmark pathologies. Subjecting the zebrafish model to realistic concentrations of two genotoxicants and carcinogens, cadmium and benzo[a]pyrene, isolated and combined, yielded low levels of DNA damage. Altogether, the organisms' mechanisms of DNA repair, oxidative stress and phases I and II were not overwhelmed after two weeks of treatment. Still, transcriptional responses related to detoxification (epoxide hydrolase and UDP-glucuronosyltransferase) were higher in animals subjected to the combination treatment, inclusively following depuration. Nonetheless, inflammation and formation of hyperplasic foci in fish epithelia were more severe in animals exposed to the combined substances, showing slower recovery during depuration. Additionally, the combination treatment yielded unexpected increased expression of a ras-family oncogene homologue after depuration, with evidence for increased tp53 counter-response in the same period. The findings indicate that oncogene expression, cell proliferation and inflammation, may not require noticeable DNA damage to occur. Furthermore, albeit absent proof for neoplasic growth, the removal of chemical insult may promote tissue recovery but does not entirely clear molecular and histopathological endpoints that are commonly associated to neoplasia.

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.

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast.

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies.

Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast.

Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts.

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.

Alterations in juvenile flatfish gill epithelia induced by sediment-bound toxicants: A comparative in situ and ex situ study.

Juvenile Solea senegalensis were exposed in the laboratory (ex situ) and field (in situ) to different sediments of a moderately impacted estuary (the Sado, Portugal) for 28 days. A qualitative histopathological screening yielded scant lesions to gills, albeit alterations such as epithelial hyperplasia being evident and more frequent in fish exposed ex situ. Fully quantitative traits, namely chloride and goblet cell count and size revealed differences between the two bioassay approaches, with ex situ experiments likely enhancing bioavailability of toxicants. Chloride cells endured autolytic processes that could, at least in part, relate to contamination by mixed metals and polycyclic aromatic hydrocarbons (PAHs). Goblet cells did not reveal changes in the chemistry of mucous. Still, their number and size was reduced in fish exposed ex situ to the sediments most contaminated by PAHs, with evidence for adaptation. Also, copper histochemistry revealed the potential role of mucocytes in the regulation of metals.

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.

An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responses.

The application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.

Hypocholesterolaemic pharmaceutical simvastatin disrupts reproduction and population growth of the amphipod Gammarus locusta at the ng/L range.

Simvastatin (SIM), a hypocholesterolaemic drug, is among the most widely used pharmaceuticals worldwide and is therefore of emerging environmental concern. Despite the ubiquitous nature of SIM in the aquatic ecosystems, significant uncertainties exist about sublethal effects of the drug in aquatic organisms. Therefore, here we aimed at investigating a multi-level biological response in the model amphipod Gammarus locusta, following chronic exposures to low levels of SIM (64 ng/L to 8 µg/L). The work integrated a battery of key endpoints at individual-level (survival, growth and reproduction) with histopathological biomarkers in hepatopancreas and gonads. Additionally, an individual-based population modelling was used to project the ecological costs associated with long-term exposure to SIM at the population level. SIM severely impacted growth, reproduction and gonad maturation of G. locusta, concomitantly to changes at the histological level. Among all analysed endpoints, reproduction was particularly sensitive to SIM with significant impact at 320 ng/L. These findings have important implications for environmental risk assessment and disclose new concerns about the effects of SIM in aquatic ecosystems.

May sediment contamination be xenoestrogenic to benthic fish? A case study with Solea senegalensis.

Within an environmental risk assessment framework of a moderately contaminated estuary (the Sado, SW Portugal), the present work intended to detect endocrine disruption in a flatfish, Solea senegalensis Kaup, 1858, and its potential relationship to organic toxicants. Animals were collected from two distinct areas in the estuary (industrial and rural) and from an external reference area. Hepatic vitellogenin (VTG) levels, cytochrome P450 (CYP1A) induction, ethoxyresorufin-O-deethylase (EROD) activity plus gonad histology were analysed. Males and females were sexually immature and showed no significant evidence of degenerative pathologies. However, hepatic VTG concentrations in males from the industrial area were higher than Reference, even reaching levels comparable to females, indicating low but measurable oestrogenic effects caused by the complex contaminant mixture in estuarine sediments. These individuals also presented elevated CYP1A induction and EROD activity, which is consistent with contamination by organic toxicants such as PAHs and other aryl hydrocarbon receptor (Ahr) -mediated toxicants.

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.

Comparative DNA damage and oxidative effects of carcinogenic and non-carcinogenic sediment-bound PAHs in the gills of a bivalve.

Polycyclic aromatic hydrocarbons (PAHs) regarded as carcinogenic and non-carcinogenic to humans are ubiquitous hydrophobic pollutants that tend to be trapped in aquatic sediments. As a consequence of their acknowledged toxicity and pro-mutagenic or even carcinogenic potential, PAHs are deemed prioritary in biomonitoring programmes. Still, the differences between the toxicity of carcinogenic and non-carcinogenic PAHs are poorly known especially, when aquatic organisms are exposed to ecologically-relevant concentrations of these compounds in sediments. Laboratory bioassays with sediments spiked with phenanthrene (Phe) and benzo[b]fluoranthene (B[b]F), non-carcinogenic and carcinogenic PAH, respectively, were conducted and the effects of exposure (related to DNA damage and oxidative stress) were analyzed in the gills of a burrowing clam, Ruditapes decussatus (Bivalvia, Veneridae). To ensure ecological relevance, two contaminant concentrations (termed "low" and "high") were selected in accordance with available PAH sediment quality guidelines. The results showed that, even in "low" concentrations, both compounds caused a likely genotoxic effect in the gills, which is in accordance with the link between PAHs in water. Glutathione S-transferase activity and glutathione biosynthesis appear to be associated with limited lipid peroxidation even though they were insufficient to prevent higher and faster genotoxicity induced by exposure to the carcinogenic B[b]F, comparative to Phe. Overall the findings indicate that low concentrations of sediment-bound PAHs, carcinogenic or not, may be rendered significantly bioavailable to benthic filter-feeders as to induce genotoxicity, revealing that even PAHs considered non-carcinogenic to humans detain a latent, albeit significant, pro-mutagenic hazard to bivalve molluscs.

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 of histopathological indices in a commercial marine bivalve (Ruditapes decussatus) to determine environmental quality.

Bivalve histopathology is an acknowledged tool in environmental toxicology studies, however geographically restricted, limited to a few species and still lacking the degree of detail needed to develop effective (semi)quantitative approaches. A first-time detailed histopathological screening was performed on grooved carpet shell clams collected from commercial shellfish beds in distinct coastal ecosystems of the Southern Portuguese coast: two parted sites within an impacted estuary (S(1) and S(2)), an inlet channel of a fish farm at a considered pristine estuary (site M) and a site allocated in a clean coastal lagoon (A). A total of thirty histopathological lesions and alterations were analysed in the gills and digestive glands following a weighted condition indices approach, including inflammation-related responses, necrosis, neoplastic diseases and parasites. Digestive glands were consistently more damaged than gills, except for animals collected from site M, where the most severe lesions were found in both organs, immediately followed by S(2). Clams from sites S(1) and A were overall the least damaged. Neoplastic diseases were infrequent in all cases. Inflammation-related traits were some of the most common alterations progressing in animals enduring severe lesions such as digestive tubule (diverticula) and intertubular tissue necrosis. Some alterations, such as lipofuscin aggregates within digestive tubule cells, did not relate to histological lesions. Granulocytomas only occurred in heavily infected tissues. Animals from M and A presented the highest infections in the digestive gland, especially by protozoa. Gill infections were more similar between sites. Still, the level of infection does not account for all histopathological lesions in either organ. Overall, the results are in accordance with environmental parameters, such as distance to pollution sources, sediment type and hydrodynamics, and show that the combination of multiple histopathological features in these clams provides good sensitivity for inter-site distinction even when low or moderate anthropogenic impacts are at stake.

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.

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.