Contaminants from dredged sediments alter the transcriptome of Manila clam and induce shifts in microbiota composition.

BACKGROUND: The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam's microbial communities. RESULTS: Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments' chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities. CONCLUSIONS: Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon.

Different ecological histories of sea urchins acclimated to reduced pH influence offspring response to multiple stressors.

End-of-the-century predictions on carbon dioxide (CO2) driven ocean acidification and the continuous leakage of pesticides from inland to coastal areas are of concern for potential negative effects on marine species' early life stages which are the most vulnerable to environmental changes. Variations in seawater chemistry related to human activities may interfere with the normal development from embryo to juvenile/adult stage. However, transgenerational studies suggest that the parental generation can influence the offspring phenotype, and thus their performances, based on the environment experienced. Here we compared the transgenerational responses to a multiple stressor scenario in sea urchins (Paracentrotus lividus) that experienced different environments since their settlement: i.e., animals from a highly variable environment, such as the Venice lagoon, versus animals from a coastal area with prevailing oligotrophic conditions in the Northern Adriatic Sea. After long-term maintenance (2 and 6 months) of adult sea urchins at natural and -0.4 units reduced pH, the F1 generations were obtained. Embryos were reared under four experimental conditions: natural and -0.4 pH both in the absence and in the presence of an emerging contaminants' mixture (glyphosate and aminomethylphosphonic acid at environmentally relevant concentrations, 100 µg/L). A significant detrimental effect of both the parental and the filial pH was highlighted, affecting embryo development and growth. Nonetheless, sea urchins from both sites were able to cope with ocean acidification. The 6-months F1 response was better than that of the 2-months F1. Conversely, the F1 response of the sea urchins maintained at natural conditions did not change sensibly after more prolonged parental exposure. An additive but mild negative effect of the mixture was observed, mostly in lagoon offspring. Results suggest that long-term exposure to reduced pH leads to transgenerational acclimation but does not affect susceptibility to the tested pollutants.

Toxicological effects and bioaccumulation of fullerene C60 (FC60) in the marine bivalve Ruditapes philippinarum.

Fullerene C60 (FC60), with its unique physical properties, has been used in many applications in recent decades. The increased likelihood of direct release into the environment has raised interest in understanding the biological effects of FC60 to aquatic organisms. Nowadays, only few studies have analysed FC60 effects and bioaccumulation in marine organisms following in vivo exposure. To provide new data about FC60 toxicity, Ruditapes philippinarum was selected as target species to assess potential adverse effects of the contaminant. Clams were exposed for 1, 3 and 7 days to predicted environmental concentrations of FC60 (1 and 10 µg/L) and cellular and biochemical responses were evaluated in clams' gills, digestive gland and haemolymph. The FC60 content in gills and digestive gland was determined in all experimental conditions after 7 days of exposure. Results showed an increase in oxidative stress. In particular, a significant modulation in antioxidant enzyme activities, and changes in glutathione S-transferase activity were observed in gills. Moreover, damage to lipids and proteins was detected in FC60-treated (10 µg/L) clams. In digestive gland, slighter variations in antioxidant enzyme activities and damage to molecules were detected. CAT activity was significantly affected throughout the exposure, whereas damage to lipids was evident only at the end of exposure. FC60 accumulation was revealed in both gills and digestive gland, with values up to twelve-fold higher in the latter. Interestingly, haemolymph parameters were slightly affected by FC60 compared to the other tissues investigated. Indeed, only Single Cell Gel Electrophoresis and Neutral Red uptake assays showed increased values in FC60-exposed clams. Moreover, volume and diameter of haemocytes, haemocyte proliferation, and micronucleus assay highlighted significant variations in treated clams, but only in the first phases of exposure, and no changes were detected after 7 days. Our results suggested clam gills as the target tissue for FC60 toxicity under the exposure conditions tested: the high damage detected to lipids and proteins could contribute to long-term problems for the organism.

A multibiomarker approach in clams (Ruditapes philippinarum) for a toxicological evaluation of dredged sediments.

The Lagoon of Venice is often dredged for channel maintenance. To avoid harmful consequences to the ecosystem, a proper disposal of bottom sediments requires a preliminary evaluation of its potential toxicity before excavation. Here we evaluated the effects of polluted sediments on clams (Ruditapes philippinarum) using a multibiomarker approach. Bivalves were exposed for 3 and 14 days to five sediment samples collected along a navigation canal between Venice historical centre and the industrial area of Porto Marghera. Immunological, antioxidant, detoxification, and neurotoxicity biomarkers were analysed in haemolymph, gill, and digestive gland. As a control, sediment collected far from pollution sources was used. Two experiments were performed to assess potential seasonal/gametogenic influence in clam sensitivity. A different response of clam biomarkers was observed during the two experiments and among sampling sites. Clams' digestive gland resulted to be the most sensitive tissue analysed showing significant differences among sites in all biomarkers analysed. Greater differences were present due to seasonality rather than exposure. The concentrations of metals and organic pollutants increased from the city centre to the industrial area, highlighting the influence that industrial activities had on the lagoon ecosystem. However, bioaccumulation in clams did not follow the same clear pattern, suggesting low bioavailability of compounds due to relatively high organic matter content. Biomarkers modulation was mainly driven by metals, both present in sediments and bioaccumulated. In comparison, effects of organic pollutants on the biomarkers tested were negligible. Other sources of contamination not investigated (e.g. pesticides) were suggested by neurotoxicity biomarkers alteration.

Echinoid skeleton: an insight on the species-specific pattern of the Paracentrotus lividus plate and its microstructural variability.

The skeletal plates of echinoids consist of a peculiar lightweight structure, called stereom, which is organized in a porous three-dimensional lattice-like meshwork. The stereom is characterized by an extremely complex and diverse microarchitecture, largely varying not only from species to species but also among different test plates. It consists of different basic types combined in extremely different ways according to specific functional needs, creating species-specific structural patterns. These patterns can lead to specific mechanical behaviours, which can inspire biomimetic technology and design development. In this framework, the present study aimed to characterize the species-specific pattern of the Paracentrotus lividus interambulacral plate and the main microstructural features regarding its geometrical variability and mechanical responses. The results achieved quantitatively highlighted the differences between the analysed stereom types providing new insights regarding their topological configuration and isotropic and anisotropic behaviour. Interestingly, data also revealed that the galleried stereom present at the tubercle is significantly different from the one located at the suture. These analyses and findings are encouraging and provide a starting point for future research to unravel the wide range of mechanical strategies evolved in the echinoid skeletal structure.

Development, growth and metabolic effects in stage IV lobster (Homarus americanus) following chronic exposure to sediments spiked with commercial formulations of deltamethrin and permethrin.

Coastal and estuarine ecosystems are environments heavily influenced by natural and anthropogenic activities. Chemicals used for pest control in agriculture and aquaculture may accumulate in natural coastal environments. Pyrethroids are common pesticides that are used on crops as well as applied to aquaculture pens and then may disperse in the surrounding ocean once treatment is complete. This study observed the sublethal effects of two pyrethroids, permethrin and deltamethrin (within commercially available formulations), on post-larval stage IV American lobster (Homarus americanus) using growth parameters and metabolic rate as indicators. Observed effects on growth parameters were a decrease in size increment and specific growth rate as well as an increase in intermolt period in stage IV lobsters exposed to 100 µg/kg permethrin. No significant differences were found for intermolt period, size increment, or specific growth rate in deltamethrin-exposed stage IV lobsters. Metabolic rates were not significantly different between deltamethrin-exposed and control lobsters, however, this sublethal effect warrants further investigation. Collectively, these results represent the first examination of the sublethal effects of exposure to pyrethroids formulations in post-larval lobsters, highlighting the potential for effects on non-target marine organisms.

Zinc oxide, titanium dioxide and C60 fullerene nanoparticles, alone and in mixture, differently affect biomarker responses and proteome in the clam Ruditapes philippinarum.

Continuous release of nanoparticles (NPs) into marine coastal environments results in an increased risk of exposure to complex NP mixtures for marine organisms. However, to date, the information on the effects at molecular and biochemical levels induced by the exposure to NPs, singly and as a mixture, is still scant. The present work aimed at exploring the independent and combined effects and the mechanism(s) of action induced by 7-days exposure to 1 µg/L nZnO, 1 µg/L nTiO2 and 1 µg/L FC60 fullerene in the Manila clam Ruditapes philippinarum, using a battery of immunological and oxidative stress biomarkers in haemolymph, gills and digestive gland. In addition, proteomics analyses were performed in gills and the digestive gland, where NP bioaccumulation was also assessed. Increased bioaccumulation of single NPs and the mixture was linked with increased oxidative stress and higher damage to proteins, lipids and DNA in all tissues analysed. The proteomics approach highlighted protein modulation in terms of abundance and damage (higher redox-thiol and carbonylated groups content). In particular, the modulated proteins (16 in gills and 18 in digestive gland) were mostly related to cytoskeleton and energetic metabolism. The digestive gland was the tissue more affected. For all biomarkers measured, increased detrimental effects were observed in the mixture compared to single NP exposures.

When site matters: Metabolic and behavioural responses of adult sea urchins from different environments during long-term exposure to seawater acidification.

CO2-driven ocean acidification (OA) affects many aspects of sea urchin biology. However, even in the same species, OA effects are often not univocal due to non-uniform exposure setups or different ecological history of the experimental specimens. In the present work, two groups of adult sea urchins Paracentrotus lividus from different environments (the Lagoon of Venice and a coastal area in the Northern Adriatic Sea) were exposed to OA in a long-term exposure. Animals were maintained for six months in both natural seawater (pHT 8.04) and end-of-the-century predicted condition (-0.4 units pH). Monthly, physiological (respiration rate, ammonia excretion, O:N ratio) and behavioural (righting, sheltering) endpoints were investigated. Both pH and time of exposure significantly influenced sea urchin responses, but differences between sites were highlighted, particularly in the first months. Under reduced pH, ammonia excretion increased and O:N decreased in coastal specimens. Righting and sheltering were impaired in coastal animals, whereas only righting decreased in lagoon ones. These findings suggested a higher adaptation ability in sea urchins from a more variable environment. Interestingly, as the exposure continued, animals from both sites were able to acclimate. Results revealed plasticity in the physiological and behavioural responses of sea urchins under future predicted OA conditions.

First Evidence of In Vitro Effects of C6O4-A Substitute of PFOA-On Haemocytes of the Clam Ruditapes philippinarum.

Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated-for the first time-on haemocytes of the clam Ruditapes philippinarum. Cells were exposed to three concentrations of C6O4 (0.05, 0.5, 5 µg/mL) and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and ß-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.