Evidence of internalized microplastics in mussel tissues detected by volumetric Raman imaging.

Microplastics are a global ecological concern due to their potential risk to wildlife and human health. Animals ingest microplastics, which can enter the trophic chain and ultimately impact human well-being. The ingestion of microplastics can cause physical and chemical damage to the animals' digestive systems, affecting their health. To estimate the risk to ecosystems and human health, it is crucial to understand the accumulation and localization of ingested microplastics within the cells and tissues of living organisms. However, analyzing this issue is challenging due to the risk of sample contamination, given the ubiquity of microplastics. Here, an analytical approach is employed to confirm the internalization of microplastics in cryogenic cross-sections of mussel tissue. Using 3D Raman confocal microscopy in combination with chemometrics, microplastics measuring 1 µm in size were detected. The results were further validated using optical and fluorescence microscopy. The findings revealed evidence of microplastics being internalized in the digestive epithelial tissues of exposed mussels (Mytilus galloprovincialis), specifically within the digestive cells forming digestive alveoli. This study highlights the need to investigate the internalization of microplastics in organisms like mussels, as it helps us understand the potential risks they pose to aquatic biota and ultimately to human health. By employing advanced imaging techniques, challenges associated with sample contamination can be overcome and valuable insights into the impact of microplastics on marine ecosystems and human consumers are provided.

Environmental risk assessment of PPP application in European soils and potential ecosystem service losses considering impacts on non-target organisms.

The use of Plant Protection Products (PPPs) is leading to high exposure scenarios with potential risk to soil organisms, including non-target species. Assessment of the effects of PPPs on non-target organisms is one of the most important components of environmental risk assessment (ERA) since they play crucial functions in ecosystems, being main driving forces in different soil processes. As part of the framework, EFSA is proposing the use of the ecosystem services approach for setting specific protection goals. In fact, the services provided by soil organisms can be impacted by the misuse of PPPs in agroecosystems. The aim of this work was to assess PPPs potential risk upon ecosystem services along European soils, considering impacts on earthworms and collembola. Four well-known (2 insecticides-esfenvalerate and cyclaniliprole- and 2 fungicides - picoxystrobin and fenamidone-) worst case application (highest recommended application) were studied; exploring approaches for linked observed effects with impacts on ecosystem services, accounting for their mode of action (MoA), predicted exposure, time-course effects in Eisenia fetida and Folsomia sp. and landscape variability. The selected fungicides exerted more effects than insecticides on E. fetida, whereas few effects were reported for both pesticides regarding Folsomia sp. The most impacted ecosystem services after PPP application to crops appeared to be habitat provision, soil formation and retention, nutrient cycling, biodiversity, erosion regulation, soil remediation/waste treatment and pest and disease regulation. The main factors to be taken into account for a correct PPP use management in crops are discussed.

Molecular confirmation of pearl formation in arctic mussels (Mytilus edulis) caused by Gymnophallus bursicola (Odhner 1900) metacercariae.

In recent field studies, suspected gymnophallid metacercariae were histologically located in the mantle of mussels from the Norwegian Sea. Mussels from the sites in which that infection was detected also presented abnormally high pearl numbers. It has been previously described that gymnophallid metacercariae could cause pearl formation processes in mussels, as a host reaction to encapsulate these metacercariae. Given the pathological host reaction these parasites elicit, a study was performed to identify gymnophallid metacercariae found in mussels collected from Tromsø at morphological and molecular level and to assess, by the use of molecular tools, the relationship between the parasite and the biological material inside the pearls. As a result, Gymnophallus bursicola metacercariae infecting Norwegian Mytilus edulis were identified according to morphological characters, along with the first 18S rDNA and COI sequences for this trematode species. In addition, parasite DNA from the core of the pearls was extracted and amplified for the first time, confirming the parasitological origin of these pearls. This procedure could allow identifying different parasitic organisms responsible for the generation of pearls in bivalves.

Integrated assessment of biological responses to pollution in wild mussels (Mytilus edulis) from subarctic and arctic areas in the Norwegian sea.

North Atlantic and Arctic Oceans contain large amount of undiscovered oil and gas reserves. Therefore threat of oil spills and its hazardous ecological consequences are of great importance to the marine environment. Although mussels (Mytilus sp.) respond clearly to contaminants, biomarkers have shown variability linked to biological and environmental changes. In order to help avoiding misinterpretation of biological responses the aim of this study was to reveal the effect of natural variability in the responsiveness to pollution of a battery of cell and tissue-level biomarkers in mussels. Mussels were collected in relatively non-impacted and potentially impacted sites at ports and the vicinity of a waste water treatment plant in Trondheim and Tromsø in autumn of 2016. Although the battery of biomarkers used herein proved to be useful to discriminate impacted and non-impacted mussel populations, some confounding factors altering the biological responses were identified. Geographical/latitudinal factors seemed to be critical regarding the reproductive cycle, reserve material storage and the prevalence of parasites such as Gymnophallus cf. Bursicola trematodes. Mussels from the reference site in Tromsø displayed general stress responses at different levels, which could be influenced by the pathogenic effect of the Gymnophallus cf. Bursicola trematode and by a more advanced gametogenic developmental stage compared to the mussels from Trondheim, which could lead to misinterpretation of the reasons behind the measured stress levels in those mussels. Despite these confounding effects, the use of integrative tools such as IBR index helped to discriminate mussel populations from chemically impacted and non-impacted sites. Overall, this work serves as an anchor point both as a reference of the baseline level values of the analyzed endpoints in the studied geographical area and time of the year, and as an indication of the potential extent of the environmental confounding factors in monitoring programs causing stress on the analyzed mussel populations.

Biomarker responses in mussels (Mytilus trossulus) from the Baltic Sea exposed to water-accommodated fraction of crude oil and a dispersant at different salinities.

Oil spills pose significant environmental risks, particularly in cold seas. In the Baltic Sea, the low salinity (from 0 to 2 up to 18) affects the behaviour of the spilled oil as well as the efficiency and ecological impacts of oil spill response methods such as mechanical collection and the use of dispersants. In the present study, mussels (Mytilus trossulus) were exposed under winter conditions (5 °C) to the water-accommodated fraction (WAF) of Naphthenic North Atlantic crude oil prepared by mechanical dispersion or to the chemically enhanced fraction (CEWAF) obtained using the dispersant Finasol OSR 51 at salinities of 5.6 and 15.0. Especially at the lower salinity, high bioaccumulation of polycyclic aromatic hydrocarbons was recorded in mussels in the CEWAF treatments, accompanied by increased biomarker responses. In the WAF treatments these impacts were less evident. Thus, the use of dispersants in the Baltic Sea still needs to be carefully considered.

Time-course distribution of fluorescent microplastics in target tissues of mussels and polychaetes.

The majority of the plastic produced in the last century is accumulated in the environment, leading to an exacerbated contamination of marine environments due to transport from land to the ocean. In the ocean, mechanical abrasion, oxidation, and photodegradation degrade large plastics into microplastics (MPs) - 0.1 µm to 5 mm (EFSA, 2016) which are transported through water currents reaching the water surface, water column, and sediments. Further, they can be accumulated by aquatic and benthic species, entering the trophic chain and becoming a potential threat to humans. In the present research, we aimed to decipher the accumulation and distribution time-courses between different organs or target tissues of organisms inhabiting coastal areas such as mussels Mytilus galloprovincialis and polychaetes Hediste diversicolor. Both were exposed in microcosm experiments to fluorescent polystyrene MPs (1 µm) which were spiked at two doses (103 and 105 particles/mL) for 1, 4, 24, and 72 h. Mussels and polychaetes were digested with 10% KOH and filtered to quantify the number of MPs incorporated. Different anatomical parts of the body were selected and processed for cryosectioning and posterior microscopic localisation of MPs. Both species accumulate MPs spiked in water column, mainly after exposure to the highest dose. In mussels, particles were found in distinct parts of the digestive tract (stomach, digestive diverticula, ducts) and gills. Even if the majority of MPs were localised in the lumen of the digestive tract, in some cases, were inside the digestive epithelium. The identification of MPs and their internalization in the digestive system was studied using Raman spectroscopy. A decreasing trend with time regarding MPs number in the digestive tract (stomach) of mussels was observed while the opposite was recorded for polychaetes and sediments. The combination of microscopical observations of frozen sections and Raman, appeared to be accurate methodologies to address MPs abundances and to reveal their localisation in different organs. This work has enabled to understand the distribution and fate of MPs in different environmental compartments and it could contribute to gain knowledge about their impact after ingestion by coastal organisms.

Predicting environmental concentrations and the potential risk of Plant Protection Products (PPP) on non-target soil organisms accounting for regional and landscape ecological variability in european soils.

Plant Protection Products (PPP) raise concerns as their application may cause effects on some soil organisms considered non-target species which could be highly sensitive to some pesticides. The European Food and Safety Authority (EFSA), in collaboration with the Joint Research Centre (JRC) of the European Commission, has developed guidance and a software tool, Persistence in Soil Analytical Model (PERSAM), for conducting soil exposure assessments. EFSA PPR Panel has published recommendations for the risk assessment of non-target soil organisms. We have used PERSAM for calculating PPPs predicted environmental concentrations (PECs); and used the estimated PEC for assessing potential risks using Toxicity Exposure Ratios (TER) for selected soil organisms and good agricultural practices. Soil characteristics and environmental variables change along a latitudinal axis through the European continent, influencing the availability of PPP, their toxicity upon soil biota, and hence, impacting on the risk characterization. Although PERSAM includes as input geographical information, the information is aggregated and not further detailed in the model outputs. Therefore, there is a need to develop landscape based environmental risk assessment methods addressing regional variability. The objective was to integrate spatially explicit exposure (PECs) and effect data (biological endpoints i.e. LC50, NOEC, etc.) to estimate the risk quotient (TER) of four PPP active substances (esfenvalerate, cyclaniliprole, picoxystrobin, fenamidone) on non-target species accounting European landscape and agricultural variability. The study was focused on the effects produced by the above-mentioned pesticides on two soil organisms: E. fetida earthworms and Folsomia sp. collembolans. After running PERSAM assuming a worst case application of PPPs, PECs in total soil and pore water were obtained for different depths in northern, central and southern European soils. With this data, soil variability and climatic differences among soils divided in three large Euroregions along a latitudinal transect (Northern, Central, Southern Europe) were analysed. Summarising, a trend to accumulate higher PECs and TERs in total soil was observed in the north decreasing towards the south. Higher PECs and TERs could be expected in pore water in southern soils, decreasing towards the north. The risk disparity between pollutant concentrations at different soils compartments should be taken into account for regulatory purposes, as well as the potential landscape variabilities among different Euroregions.

Variability and distribution of parasites, pathologies and their effect on wild mussels (Mytilus sp) in different environments along a wide latitudinal span in the Northern Atlantic and Arctic Oceans.

Histopathological examination in mussels can provide useful information for the diagnosis of ecosystem health status. The distribution of parasites in mussels can be conditioned by several environmental factors, including mussels collecting sites or the presence/absence of other species necessary to complete the complex life cycle of certain parasites. Thus, these variables could not only govern the parasitic burden of mussels but also the presence of pathologies associated to parasitism. The aim of this study was to identify the histopathological alterations which could be indicative of a health status distress along a wide latitudinal span in the Northern Atlantic and Arctic Oceans in mussels of two size-classes sampled in clean and impacted sites. A latitudinal gradient is clearly observed in gamete developmental stages as northern and southern mussels presented different conditions at the same period. Furthermore, mussels of the same size in different latitudes presented differences in the reproductive cycle and the appearance of related pathologies, which probably meant the age of individuals was different. In addition, specific parasitic profiles ruled by latitudinal conditions and the settlement of mussels in the shore (horizontal/vertical) have been demonstrated to be significantly influential in the health condition of mussels. Furthermore, the present work provides the first histological description of Gymnophallus cf. bursicola parasite causing a considerable host response in Tromsø and Iceland plus the report of grave histopathological status that included high prevalence of granulocytomas in Scotland and Germany.

Infection Rate in Seabasses Fed with Viscera Parasitised by Anisakid Larvae.

PURPOSE: It has been suggested that the removal of infected viscera on board is responsible for the high prevalence of anisakid larvae present in wild fish species. The aim of this work is to assess the re-infection capacity of anisakid larvae in European seabasses, a natural host species for the parasite by feeding with pieces of parasitised hake liver under controlled experimental conditions. METHODS: To prove this potential link between manipulation and re-infestation, 50 farmed seabasses free of anisakid nematodes were fed with fresh hake liver pieces naturally infested with anisakid larvae. RESULTS: After digestion periods from 4 to 21 days, the seabasses showed a prevalence of Anisakis of 6%, and a low retention rate of 0.11 larvae/seabass after four days' digestion, and 0.0021 after 21 day digestion. Two nematodes were found in the intestine and in the visceral cavity, and 13 Anisakis were found partially digested in the stomach of one same individual after 4 day digestion. Results showed that only a small number of Anisakis ingested with the viscera were able to reinfect the seabasses, as most of the larvae seemed to be quickly digested or defecated. CONCLUSION: it seems that the availability of larvae that could re-enter the life cycle and re-infect a fish after the removal and discarding the infected viscera on board could be much less important than commonly believed.

Impacts of sewage sludges deposition on agricultural soils: Effects upon model soil organisms.

During years sewage sludges have been worldwide poured in agricultural soils to enhance vegetal production. The "Landfill 17" located in Gernika-Lumo town (43°19'28.9"N 2°40'30.9"W) received for decades sewage sludges from the local Waste Water Treatment Plant (WWTP) with agricultural purposes. To this WWTP, several pollutants as heavy metals (Cd, Cr, Ni, Pb), PAHs (benzo(a)pyrene among many others) and pesticides (i.e. dieldrin) could have arrived from local industry and be widespread all over the landfill. Soil invertebrates like earthworms and plants are of special interest due to their close contact with the polluted matrix and their potential effects by the presence of pollutants. In this context, the aim of the present work was to determine the health status of landfill soils by evaluating the effects on model soil organisms exerted by long-lasted pollutants after on site deposition of WWTP active sludges. With such a purpose, different standard toxicity tests and cellular level endpoints were performed on lettuce and earthworms. Indeed, germination (EPA 850.4100) and root elongation (EPA 850.4230) tests were carried out in Lactuca sativa, while OECD acute toxicity test (OECD-204), reproduction test (OECD-222) and Calcein-AM viability test with coelomocytes were applied in Eisenia fetida worms. For the exposure, soils collected in the landfield containing low, medium and high concentrations of pollutants were selected, and as reference LUFA 2.3 natural standard soil was chosen. While no differences were shown in the assays with L. sativa, significant differences between sludge exposed groups and control group were recorded with E. fetida, with lower coelomocyte number and viability and higher tissue metal accumulation after 28 days of exposure to polluted soils. These results confirmed the impact of contaminants to soil biota even after long periods of time.

Sea Bass Primary Cultures versus RTgill-W1 Cell Line: Influence of Cell Model on the Sensitivity to Nanoparticles.

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz's L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures.

Application of in situ bioremediation strategies in soils amended with sewage sludges.

Increasing soil loss and the scarcity of useful land requires new reusing strategies. Thus, recovery of polluted soils recovery offers a chance for economic and social regeneration. With this objective, different soil cleaning technologies have been developed during the last few decades. On one hand, classical physical and/or chemical technologies can be found which are efficient, but have high costs and impacts upon ecosystems. On the other hand, biological methods (such as phytoremediation, bioremediation and vermiremediation) are relatively cost effective and eco-friendly, but also more time-consuming. These biological methods and their yields have been widely studied but little is known about the interaction between different soil cleaning methods. The combination of different biological strategies could lead to an improvement in remediation performance. Hence, in the present work, different micro-, vermi- and phyto-remediation combinations are applied in a sewage sludge polluted landfill in Gernika-Lumo (Basque Country) which was used as a disposal point for decades, in search of the treatment (single) or combination (dual or triple) of treatments with best remediation yields. Eight experimental groups were applied (n=3) placing earthworms (E), bacteria (B), plants (P), bacteria+earthworms (B+E), bacteria+plants (B+P), plants+earthworms (P+E) plants+bacteria+earthworms (P+B+E) and a non-treated (N.T.) group in the experimental plot (Landfill 17), for 12 months. In order to assess the efficiency of each treatment, a complete characterization (chemical and ecotoxicological) was carried out before and after remediation. Results showed high removal rates for dieldrin (between 50% and 78%) in all the experimental groups. In contrast, removal rates around 20-25% were achieved for heavy metals (Cd 15%-35%; Ni 24%-37%; Pb 15%-33%; Cr 7%-39%) and benzo(a)pyrene (19.5%-28%). The highest reductions were observed in dual (P+E, B+E) and triple (P+B+E) treatments. The best elimination yields were obtained after P+B+E treatment, as highlighted by the battery of ecotoxicological tests and bioassays performed with earthworms, plants and bacteria.

Effects of elevated temperatures and cadmium exposure on stress biomarkers at different biological complexity levels in Eisenia fetida earthworms.

Several ecotoxicological studies assessed metal toxicity upon soil biota and other communities but were mainly focused on the study of a single chemical and usually under optimal conditions of temperature. Meanwhile an increasing global warming is leading to new scenarios by combining different stress factors; chemical stress and thermal stress. Presently, this study aims to assess the joint effects produced by cadmium and elevated temperature on earthworms different levels of biological complexity. Eisenia fetida earthworms were maintained at 19 °C and 26 °C and simultaneously exposed to four Cd concentrations (1.25, 2.5, 25 and 125 mg Cd/Kg soil) for 14 (Short term exposure) and 56 days (reproduction test). Endpoints were addressed at different levels of biological complexity: reproductive impairment (cocoons and juvenile productions), Cd tissue accumulation, mortality of adults, weight loss and cytotoxic effects (coelomocyte viability). In the Short term exposure, increase in temperature produced a larger accumulation of Cd. Hence, earthworms exposed to 125 mg Cd/kg soil under heat stress (26 °C) showed a two fold higher Cd accumulation comparing to those at 19 °C. Earthworms exposed to moderate-high concentrations of Cd (2.5-125 mg Cd/kg) and maintained at 26 °C showed severe weight loss and high mortality rates. The neutral red uptake capacity of coelomocytes extruded from earthworms exposed to the highest Cd concentration decreased after 14 d at 19 °C, and more markedly at 26 °C. The reproduction impairment (decreased number of cocoons) was enhanced after exposure to concentrations higher than 2.5 mg Cd/kg at 26 °C, and after exposure to 125 mg Cd/kg at 19 °C. Earthworm reproduction capability is highly vulnerable to the effect of toxicants at elevated temperatures and sublethal concentrations.

Organotropism and biomarker response in oyster Crassostrea gigas exposed to platinum in seawater.

Platinum (Pt) is a technology critical element (TCE) for which biogeochemical cycles are still poorly understood. This lack of knowledge includes Pt effects on marine organisms, which proved to be able to bioconcentrate this trace element. Oysters Crassostrea gigas were exposed to stable Pt isotope spiked daily in seawater for 35 days. Seawater was renewed daily and spiked (with Pt(IV)) to three nominal Pt concentrations (50, 100, and 10,000 ng L-1) for two replicate series. Organotropism study revealed that gills, and to a lesser extent mantle, are the key organs regarding Pt accumulation, although a time- and concentration-dependent linear increase in Pt levels occurred in all the organs investigated (i.e., digestive gland, gonads, gills, mantle, and muscle). In oysters exposed to Pt concentrations of 10,000 ng L-1, significant biomarker impairments occurred, especially at cellular levels. They reflect altered lipofuscin and neutral lipid contents, as well as intralysosomal metal accumulation. These observations were attributed to activation of excretion/detoxification mechanisms, including Pt elimination through feces and clearly support the importance of the digestive gland in the response to direct Pt exposure. Despite relatively constant condition index, the integrative biological response (IBR) index suggests a generally decreasing health status of oysters.

Influence of season-depending ecological variables on biomarker baseline levels in mussels (Mytilus trossulus) from two Baltic Sea subregions.

For reliable mussel monitoring programmes based on biomarkers, regionally relevant reference values and their natural variability need to be known. The Baltic Sea exhibits high inter-regional and seasonal variability in physical factors such as salinity, temperature and primary production. The aim of this pilot study is to depict the effects of season-related environmental factors in a selected battery of biomarkers in two environmentally different subregions of the Baltic Sea to help establishing reference data for biochemical, cellular and tissue-level biomarkers. In order to achieve that, mussels were collected from reference sites in Kiel (Germany) and Tvärminne (Finland) during three seasons: summer and autumn 2016, and spring 2017. Finally, in order to characterize the ecological situation, analysis of the chemical tissue burden was performed and chlorophyll­a and particulate organic carbon concentration and temperature changes were analyzed at each sampling locality using satellite remote sensing images. An integrated biomarker response index was performed to summarize the biomarker responses of each locality and season. The biochemical endpoints showed seasonal variability regulated by temperature, food supply and reproductive cycle, while among the cellular endpoints only lipofuscin accumulation and lysosomal structural changes showed slight seasonal variation. Seasonal changes in tissue level biomarkers were observed only at the northern Baltic Sea site Tvärminne, dictated by the demanding energetic trade-off caused by reproduction. In conclusion, the characterization of the ecological variables and physico-chemical conditions at each site, is crucial to perform a reliable assessment of the effects of a hypothetical pollution scenario in the Baltic Sea. Moreover, reference levels of biomarkers and their responses to natural environmental conditions must be established.

Evaluation of the effectiveness of a bioremediation process in experimental soils polluted with chromium and lindane.

Bioremediation using actinobacterium consortia proved to be a promising alternative for the purification of co-contaminated environments. In this sense, the quadruple consortium composed of Streptomyces sp. M7, MC1, A5, and Amycolatopsis tucumanensis AB0 has been able to remove significant levels of Cr(VI) and lindane from anthropogenically contaminated soils. However, the effectiveness of the bioremediation process could not be evaluated only by analytical monitoring, which is complex mainly due to the characteristics of the matrix, producing non-quantitative analyte recoveries, or interferences in the detection stage and quantification. However, the effectiveness of the bioremediation process cannot be evaluated only through analytical monitoring, which is complex due mainly to the characteristics of the matrix, to the recoveries of non-quantitative analytes or to interferences in the detection and quantification stage. For this reason, it is essential to have tools of ecological relevance to assess the biological impact of pollutants on the environment. In this context, the objective of this work was to establish the appropriate bioassays to evaluate the effectiveness of a bioremediation process of co-contaminated soils. For this, five model species were studied: four plant species (Lactuca sativa, Raphanus sativus, Lycopersicon esculentum, and Zea mays) and one animal species (Eisenia fetida). On plant species, the biomarkers evaluated were inhibition of germination (IG) and the length of hypocotyls/steam and radicles/roots of the seedling. While on E. fetida, mortality (M), weight lost, coelomocyte concentration and cell viability were tested. These bioindicators and the battery of biomarkers quantified in them showed a different level of sensitivity, from maximum to minimum: E. fetida > L. esculentum > L. sativa > R. sativus ≫>Z. mays. Therefore, E. fetida and L. esculentum and their respective biomarkers were selected to evaluate the effectiveness of the bioremediation process due to the capability of assessing the effect on the flora and the fauna of the soil, respectively. The joint application of these bioindicators in a field scale bioremediation process is a feasible tool to demonstrate the recovery of the quality and health of the soil.

Bioaccumulation, tissue and cell distribution, biomarkers and toxicopathic effects of CdS quantum dots in mussels, Mytilus galloprovincialis.

The bioaccumulation, cell, tissue distribution, and biological effects of 5 nm glutathione-capped CdS quantum dots (CdS QDs) in mussels was compared to bulk and aqueous Cd forms through a two-tier experimental approach. In the 1st tier, mussels were exposed for 3 d to 0.05, 0.5 and 5 mg Cd/l (QDs, bulk, aqueous), bioaccumulation, distribution and lysosomal responses were investigated. In the 2nd tier, mussels were exposed for 21 d to the same forms at the lowest effective concentration selected after Tier 1 (0.05 mg Cd/l), biomarkers and toxicopathic effects were investigated. Accumulation was comparable in QDs and aqueous Cd exposed mussels after 3 d. After 21 d, QDs exposed mussels accumulated less than mussels exposed to aqueous Cd and localised in the endo-lysosomal system and released to the alveoli lumen (21 d) after exposure to QDs and aqueous Cd. Intracellular levels of Cd increased on exposure to QDs and aqueous Cd, and to a lesser extent to bulk, and accompanied by the up-regulation of metallothionein 10 (1 d) and 20 (1, 21 d). Lysosomal membrane destabilisation depended on Cd2+ released by all forms but was marked after exposure to aqueous Cd (1 d). Toxicopathic effects (vacuolisation, loss of digestive cells and haemocytic infiltration) were evident after exposure to QDs (1 d) and aqueous Cd (21 d). Toxicity most likely depended on the ionic load resulting from Cd2+ release from the different forms of Cd; yet nanoparticle-specific effects of QDs cannot be disregarded.

Changes in metallothionein transcription levels in the mussel Mytilus galloprovincialis exposed to CdTe quantum dots.

Quantum dots (QDs) are a class of engineered nanoparticles (ENPs) with several biomedical, industrial and commercial applications. However, their metabolism and detoxification process in aquatic invertebrates and environmental health hazards remain unclear. This study investigate the transcriptional changes of metallothioneins (MTs) isoforms (mt10IIIa and mt20IV) induced by CdTe QDs, in comparison with its dissolved counterpart, in the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and to the same Cd concentration (10 µg Cd L-1) of dissolved Cd for 14 days and mt transcription levels were measured by real time quantitative PCR (qPCR). Tissue specific mt transcription patterns were observed in mussels exposed to both Cd forms, wherein the gills were a more sensitive organ compared to the digestive gland. No significant changes were observed in mt10IIIa transcription levels in mussels exposed to both Cd forms. In contrast, transcription of mt20IV was tissue and exposure time dependent, with higher mt20IV mRNA levels in mussels exposed to QDs and dissolved Cd when compared to unexposed mussels. Multivariate analysis indicates particle-specific effects after 14 days of exposure and a dual role of MTs in the QD metabolism and in the protection against oxidative stress in mussels exposed to Cd-based ENPs.

Chemical contamination assessment in mangrove-lined Caribbean coastal systems using the oyster Crassostrea rhizophorae as biomonitor species.

This paper aims to contribute to the use of mangrove cupped oyster, Crassostrea rhizophorae, as a biomonitor species for chemical contamination assessment in mangrove-lined Caribbean coastal systems. Sampling was carried out in eight localities (three in Nicaragua and five in Colombia) with different types and levels of contamination. Oysters were collected during the rainy and dry seasons of 2012-2013 and the tissue concentrations of metals, polycyclic aromatic hydrocarbons (PAHs), and persistent organic pollutants (POPs) were determined. Low tissue concentrations of metals (except Hg) and PAHs; moderate-to-high tissue concentrations of Hg, hexachlorocyclohexanes (HCHs), and dichlorodiphenyl-trichloroethanes (DDTs); detectable levels of chlorpyrifos, polychlorinated biphenyls (PCBs) (mainly CB28, CB118, CB138 and CB 153) and brominated diphenyl ethers 85 (BDE85); and negligible levels of musks were recorded in Nicaraguan oysters. A distinct profile of POPs was identified in Colombia, where the tissue concentrations of PCBs and synthetic musk fragrances were low to moderate, and Ag, As, Cd, Pb, and PAHs ranged from moderate to extremely high. Overall, the values recorded for HCHs, DDTs and PCBs in Nicaraguan mangrove cupped oysters greatly exceeded the reference values in tissues of C. rhizophorae from the Wider Caribbean Region, whereas only the levels of PCBs were occasionally surpassed in Colombia. Different contaminant profiles were distinguished between oysters from Nicaragua and Colombia in radar plots constructed using the main groups of contaminants (metals, PAHs, musks, PCBs, and organochlorine pesticides (OCPs)). Likewise, integrated pollution indices revealed differences in the levels of contaminants. Moreover, the profiles and levels in oyster tissues also varied with season. Thus, principal component analysis clearly discriminated Nicaraguan and Colombian localities and, especially in Colombia, seasonal trends in chemical contamination and differences amongst localities were evidenced. The geographical and environmental disparity of the studied scenarios may represent to a large extent the diversity of mangrove-lined Caribbean coastal systems and therefore the present results support the use of C. rhizophorae as suitable biomonitor species at Caribbean regional scale, where seasonal variability is a major factor controlling pollutant mobility and bioavailability.

Tracing platinum accumulation kinetics in oyster Crassostrea gigas, a sentinel species in coastal marine environments.

Platinum Group Elements (PGEs) are extremely scarce in the Earth's Crust and of strong interest for high-end technologies due to their specific properties. They belong to the Technology Critical Elements (TCEs) for which use is forecast to increase, implying growing emissions into the environment in the following years. In particular, with the intensive use of platinum (Pt) in car catalytic converters, the anthropogenic geochemical cycle of this element has surpassed the natural cycle. Yet, environmental Pt levels are still in the sub picomolar range, making its analytical detection a challenge. Few studies cover the behavior of Pt in marine waters in terms of speciation, reactivity and possible transfer to the biota. In this study, oysters (Crassostrea gigas) from an unpolluted estuary were exposed to the stable isotope 194Pt in seawater at a range of concentrations during 35days. Seawater was renewed daily and spiked to three nominal Pt concentrations (50, 100, and 10,000ng·L-1) for two replicate series. In addition, control conditions were monitored. Five oysters from each tank were dissected after 3, 7, 14, 21, 28, 35days of Pt exposure, and analyzed by ICP-MS. Accuracy of this analytical method applied to biological matrix was checked by an inter-method comparison with a voltammetrical technique. A concentration-dependent accumulation of Pt in oysters increasing with exposure time occurred. After 28days, oyster Pt accumulation from low and intermediate exposure conditions reached a plateau. This was not the case of the highest exposure condition for which oyster tissues showed increasing concentrations until the last day of the experiment. A linear correlation exists between seawater concentrations and Pt content in oysters for low and intermediate exposure concentrations i.e. closer to environmental concentrations. By showing high Pt accumulation potential, oysters may serve as sentinels, ensuring biomonitoring of Pt concentrations in marine coastal waters.