Transcriptional and cellular effects of paracetamol in the oyster Crassostrea gigas.

Acetaminophen (paracetamol) (PAR) is one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) with analgesic and antipyretic properties consumed worldwide and often detected in the aquatic environment. Due to the fact that PAR induces oxidative stress in mammals, the aim of this study was to evaluate if similar effects were observed in oysters Crassostrea gigas, given their economic and ecological importance and worldwide distribution. Oysters were exposed for 1, 4 and 7 days to two different sublethal PAR concentrations (0, 1 and 100µgL-1). Cell viability, DNA damage in hemocytes and enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferases (GST) were evaluated in oyster gills. In addition, changes at transcriptional level of Cu/Zn superoxide dismutase (SOD), catalase-like (CAT-like), cytochrome P450 genes (CYP30C1, CYP2AU2, CYP3071A1, CYP356A1), glutathione S-transferase isoforms (GST-ω and GST-π-like), cyclooxygenase (COX), fatty acid binding proteins-like (FABP-like), and caspase genes were evaluated in oyster gills and digestive gland. No changes in cell viability and DNA damage were observed in oysters exposed to both PAR concentrations. Similarly, no significant changes were detected in the major antioxidant enzymes (except for auxiliary enzyme GR) in oyster gills, suggesting that changes in GR activity are enough to counteract a potential oxidative stress in C. gigas gills under these experimental conditions. Furthermore, changes at transcriptional level are concentration and tissue dependent. PAR elicited an inhibition of CYP30C1, CYP3071A1 and FABP-like transcripts highlighting their role in drug metabolism, transport and detoxification of PAR in the gills. GST transcript levels were type, tissue and concentration-dependent. GST-π-like was down-regulated in oyster gills exposed to the lowest PAR concentration and up-regulated in the digestive gland of oysters exposed to the highest PAR concentration. However, GST-ω transcript levels were lower only in oysters digestive gland exposed to the lowest PAR concentration. Therefore, changes at transcriptional level were more sensitive to assess the exposure to PAR at environmental relevant concentrations.

Ecotoxicity of emerging contaminants in the reproductive organ of marine mussels Mytilus galloprovincialis.

Contaminants of emerging concern (CECs) present a new threat to the marine environment, and it is vital to understand the interactions and possible toxicity of CEC mixtures once they reach the ocean. CECs-such as metal nanoparticles, nanoplastics, and pharmaceuticals-are groups of contaminants some of which have been individually evaluated, though their interactions as mixtures are still not fully understood. To ensure a healthy and prosperous future generation, successful reproduction is key: however, if hindered, population dynamics may be at danger leading to a negative impact on biodiversity. This study aimed to understand the effects of silver (20 nm nAg, 10 µg/L), polystyrene nanoparticles (50 nm nPS, 10 µg/L), and 5-fluorouracil (5FU, 10 ng/L) individually and as a mixture (10 µg/L of nPS + 10 µg/L of nAg +10 ng/L of 5FU) in the gonads of Mytilus galloprovincialis. A multibiomarker approach, namely the antioxidant defence system (ADS; superoxide dismutase, catalase, glutathione peroxidases, glutathione - S - transferases activities), and oxidative damage (OD; lipid peroxidation) were analysed in the gonads of mussels. All exposure treatments after 3 days led to an increase of enzymatic activity, followed by an inhibition after 14 and 21 days. Thus, ADS was overwhelmed due to the generation of ROS, resulting in OD, except for nPS exposed mussels. The OD in Mix exposed mussels increased exponentially by 57-fold. When CEC mixtures interact, they are potentially more hazardous than their individual components, posing a major threat to marine species. To understand synergistic and antagonistic interactions, a model was applied, and antagonistic interactions were observed in evaluated biomarkers at all time-points, apart from a synergistic interaction at day 3 relative to LPO. Results indicate that the effects observed in Mix-exposed mussel gonads are mainly due to the interaction of nAg and 5FU but not nPS.

Polystyrene nanoplastics in the marine mussel Mytilus galloprovincialis.

Concerns about plastic pollution and its toxicity towards animals and people are growing. Polystyrene (PS) is a plastic polymer highly produced in Europe for packaging purposes and building insulation amongst others. Whatever their source-illegal dumping, improper waste management, or a lack of treatment for the removal of plastic debris from wastewater treatment plants-PS products ultimately end up in the marine environment. Nanoplastics (<1000 nm) are the new focus for plastic pollution, gaining broad interest. Whether primary or secondary, their small size permits nanoparticles to cross cellular boundaries, consequently leading to adverse toxic effects. An in vitro assay of Mytilus galloprovincialis haemocytes exposed to 10 µg/L of polystyrene nanoplastics (PS-NPs; 50 nm) for 24 h was used to test cellular viability along with the luminescence inhibition (LC50) of Aliivibrio fischeri bacteria to evaluate acute toxicity. Cellular viability of mussel haemocytes decreased significantly after a 24 h exposure and PS-NPs LC50 range from 180 to 217, µg/L. In addition, a 28-day exposure of the marine bivalve M. galloprovincialis to PS-NPs (10 µg/L; 50 nm) was performed to evaluate the neurotoxic effects and the uptake of these plastic particles in three bivalve tissues (gills, digestive gland, and gonads). The ingestion of PS-NPs was time- and tissue-specific, suggesting that PS-NPs are ingested through the gills and then translocated through the mussel bloodstream, to the digestive gland and gonads where the highest amount of ingested PS-NPs was reported. Ingested PS-NPs may compromise the digestive glands' key metabolic function and impair mussels' gametogenic and reproductive success. Data on acetylcholinesterase inhibition and those previously obtained on a wide range of cellular biomarkers were elaborated through weighted criteria providing a synthetic assessment of cellular hazard from PS-NPs.

Assessment of essential and nonessential metals and different metal exposure biomarkers in the human placenta in a population from the south of Portugal.

The general population is exposed to metals as trace amounts of metallic compounds are present in air, water, and food. Information on background exposures and biomarker concentrations of environmental chemicals in the general Portuguese population is limited. Therefore, the purpose of this study was to determine the levels of important nonessential metals with recognized toxicity cadmium (Cd) and lead (Pb) and essential metals copper (Cu), nickel (Ni), chromium (Cr), and zinc (Zn) in placentas of mothers living in south Portugal (Algarve). Due to the difficulty in establishing the effects of chemicals in a complex and variable environment, this study also aimed to examine the response of biomarkers, such as biochemical changes that occurs at subcellular levels in the presence of contaminants. The investigated biomarkers in placentas indicative of metal exposure or damage included the metallothioneins (MT), delta-aminolevulinic acid dehydratase (ALAD) (specific for Pb), and lipid peroxidation (LPO) as an index of oxidative stress damage. Moreover, HJ-BIPLOT was applied in order to identify and categorize mothers vulnerable to environmental contamination in this region. Metal concentrations in the placenta were not excessive but within the range found in most European studies. In general, the biomarkers MT and LPO were positively correlated with metal levels, while with ALAD the opposite occurred, indicating the selected battery of biomarkers were suitable to study the effects of metals on human placenta. Further, the application of multivariate analysis with HJ-BIPLOT showed that most significant factors contributing to maternal and fetal exposures via placenta were dietary and smoking habits.

Responses of CYP450 dependent system to aliphatic and aromatic hydrocarbons body burden in transplanted mussels from South coast of Portugal.

Mussels Mytilus galloprovincialis were cross-transplanted at South Portugal from a reference site (site 1) to a site more contaminated with hydrocarbon compounds (site 2), and vice versa, in an active biomonitoring (ABM) concept, to assess the biotransformation capacity catalyzed by the mixed function oxygenase (MFO) system. Total alkanes (TAlk), the unresolved complex mixture (UCM), and total polycyclic aromatic hydrocarbons (TPAHs) concentration increased respectively 6, 4.4 and 4.2 fold relatively to control, in mussels transplanted from site 1 to 2. In the cross-transplant, a 48, 57 and 62% depuration of TAlk, UCM and TPAHs concentrations occurred by the end of the 3-4th week. Petrogenic and biogenic (marine and terrigenous) sources of AHs, and petrogenic and pyrolitic (biomass and oil/fuel incomplete combustion) sources of PAHs were detected at both sites. CYP450, CYT b (5) and NADPH-RED in mussels transplanted from site 1 to 2 were induced from day 0 to 28, with a total increase of 35, 32 and 35%, respectively, while biochemical equilibrium to lesser environmental contamination occurs in mussels transplanted from site 2 to 1. A significant relationship between CYP450 and NADPH-RED was found with TPAH, with distinctive behavior at the two sites. MFO system components increase with exposure time at one site and decreases in the other, reflecting an adaptation to distinct environmental hydrocarbon loads. The ABM strategy proved to be useful to understand the environment real impact on the biochemical responses in mussels' local populations. In this study, CYP450 and NADPH-RED are a useful biomarker for hydrocarbon exposure.

Effects of microplastics alone and with adsorbed benzo(a)pyrene on the gills proteome of Scrobicularia plana.

Microplastics (MPs) are globally present in the marine environment, but the biological effects on marine organisms at the molecular and cellular levels remain scarce. Due to their lipophilic nature, MPs can adsorb other contaminants present in the marine environment, which may increase their detrimental effects once ingested by organisms. This study investigates the effects of low-density polyethylene (PE) MPs with and without adsorbed benzo[a]pyrene (BaP) in the gills proteome of the peppery furrow shell clam, Scrobicularia plana. Clams were exposed to PE MPs (11-13 µm; 1 mg L-1) for 14 days. BaP was analyzed in whole clams' soft tissues, and a proteomic approach was applied in the gills using SWATH/DIA analysis. Proteomic responses suggest that virgin MPs cause disturbance by altering cytoskeleton and cell structure, energy metabolism, conformational changes, oxidative stress, fatty acids, DNA binding and, neurotransmission highlighting the potential risk of this type of MPs for the clam health. Conversely, when clam gills were exposed to MPs adsorbed with BaP a higher differentiation of protein expression was observed that besides changes in cytoskeleton and cell structure, oxidative stress, energy metabolism and DNA binding also induce changes in glucose metabolism, RNA binding and apoptosis. These results indicate that the presence of both stressors (MPs and BaP) have a higher toxicological risk to the health of S. plana.

Do microplastic contaminated seafood consumption pose a potential risk to human health?

Microplastics are present in all parts of the ocean and can have deleterious effects on marine resources. The aim of this work was to map the presence of microplastics in commercial marine species such as bivalves (mussels Mytilus galloprovincialis and clams Scrobicularia plana), crabs (Carcinus maenas) as well as fish (Mullus surmuletus) to relate microplastics levels to pollution sources, assess possible impact on marine food chains and on human health. These species were collected from several sites of the Ria Formosa lagoon and along the south coast of Portugal. A quantitative assessment (number, size and color) and typology of microplastics were made in these species. Only one green fragment of polypropylene was detected in the gills of the crabs, while a blue polyethylene fragment was detected in the hepatopancreas of the mullets. Moreover, no microplastics were present in S. plana nor in the crabs whole soft tissues. Among mussels, 86% of microplastics were present from all sites and the number, size and color were site specific. Mussels from the west side of the coast (Sites 1-3) had the highest levels of MPs per mussel and per weight compared to the other sites, probably related to the impact of touristic activity, fishing gears, fresh water and sewage effluents along with the hydrodynamics of the area.

Perfluorooctane sulfonic acid (PFOS) adsorbed to polyethylene microplastics: Accumulation and ecotoxicological effects in the clam Scrobicularia plana.

Microplastics are widespread in the marine environment, whereby the uptake of these tiny particles by organisms, can cause adverse biological responses. Plastic debris also act as a vector of many contaminants, herein depending on type, size, shape and chemical properties, possibly intensifying their effects on marine organisms. This study aimed to assess the accumulation and potential toxicity of different sizes of microplastics with and without adsorbed perfluorooctane sulfonic acid (PFOS) in the clam Scrobicularia plana. Clams were exposed to low-density polyethylene microplastics (1 mg L-1) of two different sizes (4-6 and 20-25 µm) virgin and contaminated with PFOS (55.7 ± 5.3 and 46.1 ± 2.9 µg g-1 respectively) over 14 days. Microplastic ingestion, PFOS accumulation and filtration rate were determined along with a multi biomarker approach to assess the biological effects of microplastics ingestion. Biomarkers include oxidative stress (superoxide dismutase, catalase, glutathione peroxidases), biotransformation enzymes (glutathione-S-transferases activity), neurotoxicity (acetylcholinesterase activity), oxidative damage and apoptosis. Microplastics ingestion and PFOS accumulation was microplastic size dependent but not PFOS dependent and filtration rate was reduced at the end of the exposure. Reactive oxygen species in gills and digestive gland were generated as a result of exposure to both types of microplastics, confirming the disturbance of the antioxidant system. Larger virgin microparticles lead to stronger impacts, when compared to smaller ones which was also supported by the Integrated Biomarker Responses index calculated for both tissues. An anti-apoptotic response was detected in digestive glands under exposure to any of the MPs treatments.

Evaluation of oxidative DNA lesions in plasma and nuclear abnormalities in erythrocytes of wild fish (Liza aurata) as an integrated approach to genotoxicity assessment.

Genetic lesions (8-hydroxy-2'-deoxyguanosine (8-OHdG) and erythrocytic nuclear abnormalities (ENA) were seasonally quantified in the blood of Liza aurata caught at Ria de Aveiro (Portugal), a multi-contaminated aquatic system. Thus, five critical sites were assessed and compared with a reference site (Torreira). Oxidative DNA damage was found in Gafanha (harbour-water area), Laranjo (metal-contaminated) and Vagos (contaminated with PAHs) in the spring; Rio Novo do Principe (near a former paper-mill effluent) in the autumn; Rio Novo do Principe and Vagos in the winter. ENA were higher than Torreira at VAG (spring and winter). Torreira did not display seasonal variation neither in terms of 8-OHdG or total ENA. A positive correlation between 8-OHdG and ENA was found, suggesting oxidative stress as a mechanism involved in the formation of ENA. This study clearly demonstrates the presence of DNA-damaging substances in Ria de Aveiro and recommends the use of 8-OHdG and ENA as biomarkers of environmental contamination.

Golden grey mullet and sea bass oxidative DNA damage and clastogenic/aneugenic responses in a contaminated coastal lagoon.

Several xenobiotics or their metabolites have redox-cycling properties and potential to induce oxidative stress and DNA damage. The current work aimed to study, under environmental conditions, oxidative DNA damage (8-hydroxy-2'-deoxyguanosine (8-OHdG)) and its association with chromosomal damage measured as erythrocytic nuclear abnormalities (ENAs), in Liza aurata and Dicentrarchus labrax, caught at a costal lagoon (Ria de Aveiro, Portugal) having sites with different contamination profiles. The quantified parameters were also used to assess the lagoon's environmental status. Five critical sites were assessed comparing to a reference site. L. aurata displayed higher 8-OHdG levels and ENAs frequency, respectively, at Laranjo and Vagos. D. labrax 8-OHdG levels were higher at the sites where quantification was possible whereas no differences were found in terms of ENAs. No correlation was found in both species between 8-OHdG and ENAs. Despite no direct linkage between the two biomarkers was found, this study demonstrates species and site dependent genotoxic responses.

Assessing cadmium-based quantum dots effect on the gonads of the marine mussel Mytilus galloprovincialis.

This study assesses the sex-specific effects induced by CdTe QDs, on the marine mussel Mytilus galloprovincialis in comparison to its dissolved counterpart. A 14 days exposure to CdTe QDs and dissolved Cd was conducted (10 µg Cd L-1), analysing Cd accumulation, oxidative stress, biotransformation, metallothionein and oxidative damage in the gonads. Both Cd forms caused significant antioxidant alterations, whereby QDs were more pro-oxidant, leading to oxidative damage, being females more affected. Overall, biochemical impairments on gonads of M. galloprovincialis demonstrate that the reproductive toxicity induced by CdTe QDs in mussels are sex-dependent and mediated by oxidative stress and lipid peroxidation. It is crucial to acknowledge how gametes are affected by metal-based nanoparticles, such as Cd-based QDs. As well as understanding the potential changes they may undergo at the cellular level during gametogenesis, embryogenesis and larval development potentially leading to serious impacts on population sustainability and ecosystem health.

Metallothionein role in the kinetic model of copper accumulation and elimination in the clam Ruditapes decussatus.

In order to clarify the role of metallothioneins (MT) in copper (Cu) toxicity, this work aimed to assess the involvement of this protein in the accumulation and elimination strategies of Cu in the clam Ruditapes decussatus exposed to two sublethal concentrations (25 and 50 microgCul(-1)). The behaviour of MT in three different tissues of clams during the accumulation and depuration processes was also followed by gel-filtration chromatography to assess if Cu was bound to MT or to other cytosolic components. The 96 h LC50 for water-borne copper was 715 microgL(-1) in R. decussatus. The Cu accumulation pattern was dependent on Cu exposure concentrations. In clams exposed to 25 microgl(-1), total Cu accumulation in the three tissues increased linearly during the exposure period, while in those exposed to 50 microgl(-1) it followed the first order kinetic model. The greatest amount of Cu accumulated in all tissues is associated to the low molecular weight cytosolic fraction (>50%). The chromatographic assay indicated that Cu in the cytosolic fraction is bound to MT and MT levels increase with the increase of Cu exposure confirming the binding affinity of Cu to MT in all tissues. However, a smaller percentage of Cu seems to be bond to other ligands, such as GSH. Copper was exponentially eliminated (only studied in clams exposed to 25 microgl(-1)) and the estimated half-life was tissue dependent (9, 5 and 14 days for the gills, digestive gland and remaining tissues, respectively). Copper bound to the thermostable compounds was eliminated more quickly (t(1/2)=4-7 days) in all tissues than those bound to the thermolabile compounds (t(1/2)=7-18 days). Interestingly, MT is rapidly degraded (t(1/2)=7 and 18 days), suggesting that this protein is actively involved in the elimination of this metal, through the Cu-MT complex since MT and Cu are turning over simultaneously. Therefore, when Cu exposure is low, the clam can cope efficiently with the excess of Cu levels by increasing MT induction as well as rapidly eliminating this metal via the MT-Cu complex. Copper toxicity in the clam R. decussatus is associated to the limited capacity of MT induction at higher and environmental unrealistic Cu exposures especially in the gills and remaining tissues.

Wild juvenile Dicentrarchus labrax L. liver antioxidant and damage responses at Aveiro Lagoon, Portugal.

The Aveiro Lagoon, at the north-western coast of Portugal, has been under considerable anthropogenic pressure for the last 5 decades. In order to perform an adequate survey of the effects induced by the contaminants in presence, wild juveniles Dicentrarchus labrax (sea bass) were selected. Thus, sea bass was captured at five sites: Torreira (TOR, as reference site), Gafanha (GAF), Rio Novo Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) in autumn 2005. Liver defence responses such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), total non-protein thiols (NP-SH) and metallothioneins (MT) were measured. Liver damage was determined as lipid peroxidation (LPO) and DNA integrity loss. RIO, LAR and VAG presented lower CAT, GR, GST activities and NP-SH and GSHt depletion in comparison to TOR. VAG and LAR showed higher GPx activity when compared to TOR. The highest MT level was found at GAF and VAG. The NP-SH and DNA integrity decreased at GAF compared to TOR. This field study demonstrated that not only antioxidant induction but also inhibitory responses must be considered as a signal of contamination.

Incidence of intersex in male clams Scrobicularia plana in the Guadiana Estuary (Portugal).

Endocrine disrupting chemicals (EDCs) known to be present in the marine environment, have great potential to influence reproductive status in aquatic organisms. A widespread incidence of intersex linked with EDCs exposure has been reported in molluscs and fish species. The species Scrobicularia plana is inherently gonochoristic; nonetheless, the feminisation of male clams was described in estuaries from the United Kingdom. Thus, this study reports the presence of oocytes in the testis of male clams of S. plana collected in the Guadiana River (Portugal). Samples were collected between June and September (2007), and gonad tissue analysed by light microscopy. Intersex with several degrees of intensity was detected during the first three months of sampling, in which the percentage of male clams affected varied between 5.9, 58.3 and 71.4%. Little is known about the causes of intersex in S. plana, being the urban, industrial and agricultural discharges possible major sources of induction. In the Guadiana River, the presence of several EDCs may be directly linked to the incidence of the intersex condition detected in Scrobicularia plana.

Biomarkers of damage and protection in Mytilus galloprovincialis cross transplanted in Ria Formosa Lagoon (Portugal).

The study was carried out to check the water contamination in two sites of Ria Formosa Lagoon by assessing the damage responses versus protection in the gills and digestive gland (Dg) of mussels--Mytilus galloprovincialis Lmk. Damage was assessed as DNA integrity and lipid peroxidation (LPO) while protection was evaluated by measuring catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt) and metallothioneins (MT). Mussels were collected in March of 2007 at Ramalhete (R) as a reference site and Faro port (F) as harbour area. Cross transplantation was from R to F and vice versa performed during 0, 3 and 6 days. Gills DNA integrity decrease was observed in mussels transplanted to F (3 days) and R (6 days). Highest Dg LPO was found in mussels sampled at R. Elevated gills CAT, GPx, GR and GST was observed in mussels transplanted to F. Dg GSHt decreased in mussels transplanted to F. Gills and Dg mussels MT increased in mussels transplanted to R. M. galloprovincialis responses show their capability as early warning signals of the contaminants presence. The adopted approach, considering simultaneously protection responses and damaging effects, also revealed its usefulness on the pollution assessment.

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil.

Biomarkers as lipid peroxidation, metallothionein and delta-aminolevulinic acid dehydratase were determined in Cathorops spixii to compare the biological responses of this fish from estuaries with distinct anthropogenic influence. Three areas were selected in two estuaries in accordance with the levels of contamination for the polluted (Santos/São Vicente) and with the hydrodynamic characteristics for the non-polluted (Cananéia) estuary. Water characteristics and mercury levels in C. spixii confirmed a high human influence in the polluted system. In general, the biomarkers showed differences between the estuaries, suggesting disturbances in the specific cell mechanisms due to the presence of multiple xenobiotics in the contaminated system. Therefore, these biomarkers are recommended to promote more accurate information about the exposure to pollutants. Additionally, the study of the effect of the multiple xenobiotics on resident species such as the benthic fish C. spixii can favor a better assessment of the environmental quality of these systems.

Impacts of in vivo and in vitro exposures to tamoxifen: Comparative effects on human cells and marine organisms.

Tamoxifen (TAM) is a first generation-SERM administered for hormone receptor-positive (HER+) breast cancer in both pre- and post-menopausal patients and may undergo metabolic activation in organisms that share similar receptors and thus face comparable mechanisms of response. The present study aimed to assess whether environmental trace concentrations of TAM are bioavailable to the filter feeder M. galloprovincialis (100 ng L-1) and to the deposit feeder N. diversicolor (0.5, 10, 25 and 100 ng L-1) after 14 days of exposure. Behavioural impairment (burrowing kinetic), neurotoxicity (AChE activity), endocrine disruption by alkali-labile phosphate (ALP) content, oxidative stress (SOD, CAT, GPXs activities), biotransformation (GST activity), oxidative damage (LPO) and genotoxicity (DNA damage) were assessed. Moreover, this study also pertained to compare TAM cytotoxicity effects to mussels and targeted human (i.e. immortalized retinal pigment epithelium - RPE; and human transformed endothelial cells - HeLa) cell lines, in a range of concentrations from 0.5 ng L-1 to 50 µg L-1. In polychaetes N. diversicolor, TAM exerted remarkable oxidative stress and damage at the lowest concentration (0.5 ng L-1), whereas significant genotoxicity was reported at the highest exposure level (100 ng L-1). In mussels M. galloprovincialis, 100 ng L-1 TAM caused endocrine disruption in males, neurotoxicity, and an induction in GST activity and LPO byproducts in gills, corroborating in genotoxicity over the exposure days. Although cytotoxicity assays conducted with mussel haemocytes following in vivo exposure was not effective, in vitro exposure showed to be a feasible alternative, with comparable sensitivity to human cell line (HeLa).

Are shallow-water shrimps proxies for hydrothermal-vent shrimps to assess the impact of deep-sea mining?

Polymetallic seafloor massive sulphide deposits are potential targets for deep-sea mining, but high concentrations of metals (including copper - Cu) may be released during exploitation activities, potentially inducing harmful impact. To determine whether shallow-water shrimp are suitable ecotoxicological proxies for deep-sea hydrothermal vent shrimp the effects of waterborne Cu exposure (3 and 10 days at 0.4 and 4 µM concentrations) in Palaemon elegans, Palaemon serratus, and Palaemon varians were compared with Mirocaris fortunata. Accumulation of Cu and a set of biomarkers were analysed. Results show different responses among congeneric species indicating that it is not appropriate to use shallow-water shrimps as ecotoxicological proxies for deep-water shrimps. During the evolutionary history of these species they were likely subject to different chemical environments which may have induced different molecular/biochemical adaptations/tolerances. Results highlight the importance of analysing effects of deep-sea mining in situ and in local species to adequately assess ecotoxicological effects under natural environmental conditions.

Using biochemical and isotope geochemistry to understand the environmental and public health implications of lead pollution in the lower Guadiana River, Iberia: a freshwater bivalve study.

Lead is a natural component of aquatic ecosystems with no known biological role and is highly toxic. Its toxicity stems from its ability to mimic biologically important metals and to produce membrane damage through lipid peroxidation (LPO). Most lead poisoning symptoms are thought to occur by interfering with an essential enzyme, delta-aminolevulinic acid dehydratase (ALAD), the activity of which is markedly inhibited by lead. The purpose of this work was to study the levels and effects of lead pollution (responses of ALAD and oxidative stress biomarker LPO) in the freshwater bivalve Corbicula fluminea along the lower Guadiana River (Portugal and Spain); a major river system impacted by historic mining pollution and more recent anthropogenic inputs. The results show that the enzymatic activity of ALAD is negatively correlated with the total Pb concentration of the whole tissue suggesting that ALAD has considerable potential as a biomarker of lead exposure in C. fluminea. To identify the sources of lead to which bivalves have been exposed, high precision (206)Pb/(204)Pb, (207)Pb/(204)Pb, (208)Pb/(204)/Pb ratios for C. fluminea confirm that historical mining activities in the Iberian Pyrite Belt are the dominant source of lead pollution in the lower Guadiana River. The isotope patterns however exhibit marked seasonal and geographic variation in response to rainfall and river water management. Locally, other anthropogenic sources of lead have been detected in C. fluminea close to population centres, thus adding to its versatility as a freshwater bio-indicator. Overall, the study highlights the value of natural ecosystems as monitors of water quality and their importance for public health assessment and surveillance.

DNA damage and lipid peroxidation vs. protection responses in the gill of Dicentrarchus labrax L. from a contaminated coastal lagoon (Ria de Aveiro, Portugal).

The present research work aimed to investigate the damage vs. protection responses in gill of European sea bass (Dicentrarchus labrax) captured at a polluted coastal lagoon, Ria de Aveiro (Portugal), as a tool to evaluate the human impacts on environmental health. Damage was assessed as DNA strand breakage and lipid peroxidation (LPO) whereas protection was evaluated by measuring catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), thiols and metallothioneins (MT). Fish were caught at five locations: Gafanha (GAF), Rio Novo do Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) presenting each a different recognized source of contamination, and Torreira (TOR), assumed as reference site. Among the surveyed sites, gill damage was observed only at GAF, as measured by DNA integrity loss and LPO increase. An overall induction in enzymatic antioxidant protection was perceptible in fish from GAF and VAG, expressed as higher GPX, GR and GST activities. In addition, LAR fish showed elevated GST and CAT activities. Thiols content was higher in all study sites irrespective to the pollution spectrum, whereas GSHt increase was only observed at GAF and VAG. The highest MT level was detected in fish from VAG and the lowest level from RIO suggesting a low contamination degree on this particular site. Globally, the results expressed site-specific response patterns, signalling two critical areas - GAF and VAG. Additionally, a joint analysis (damage vs. protection) allowed the following ordering of surveyed sites according to the contamination degree and risk to fish health; GAF>VAG>LAR>RIO>TOR. D. labrax gill responses demonstrated their efficacy as early warning signals of the contaminants presence. Moreover, the adopted approach, considering simultaneously protection responses and damaging effects, also revealed its usefulness on the pollution extent assessment.