One-year variation in quantity and properties of microplastics in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from Aveiro lagoon.

As filter feeders, marine bivalves inhabiting estuarine and coastal areas are directly exposed to microplastics (MPs) in water. To assess whether MPs number, and their shape, size, colour, and polymer type present in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) varied over one year, bivalves were collected over the year of 2019 in the lower part of the coastal Aveiro lagoon, Portugal. After extraction from the bivalve's whole-body soft tissues, a subset of the visually inspected particles was randomly separated for identification using the Fourier-transform mid-infrared (FT-MIR) spectroscopy. A fraction of the inspected particles, 26-32% of particles >100 µm, and 59-100% of smaller ones were confirmed as MPs. Concentrations varied within the intervals of 0.77-4.3 items g-1 in mussels and 0.83-5.1 items g-1 in cockles, with the lowest values observed in January. In winter, the accumulation of large-sized fibers was composed of a mixture of plastic types, which contrasted against the most abundant MPs in summer consisting mainly of polyethylene of diverse size classes and shapes. Temperature decrease registered in winter might have triggered a lower filtration rate, resulting in lower MPs concentrations in the whole-soft body tissues of organisms. Different properties of MPs found in bivalves between January-February and August-September appear to reflect changes in the characteristics of MPs available in the Aveiro lagoon.

The influence of short-term experimental fasting on biomarker responsiveness in oil WAF exposed mussels.

Mussels are widely used in toxicological experimentation; however, experimental setups are not standardized yet. Although there is evidence of changes in biomarker values during food digestion and depending on the mussel nutritive status, the mode of feeding differs among toxicological experiments. Typically, mussels are fed with different diets in different long-term experiments, while fasting is the most common approach for short-term studies. Consequently, comparisons among experiments and reliable interpretations of biomarker results are often unfeasible. The present investigation aimed at determining the influence of fasting (against feeding with Isochrysis galbana) on biomarkers and their responsiveness in mussels exposed for 96 h to the water accommodated fraction (WAF) of a heavy fuel oil (0%, 6.25%, 12.5% and 25% WAF in sea water). PAH tissue levels in digestive gland and a battery of biomarkers were compared. WAF exposure led to decrease of cytochrome-C-oxidase activity, modulated glutathione-S-transferase activity, augmented lipid peroxidation, inhibited acetyl cholinesterase (AChE) activity, and led to lysosomal enlargement (VvLYS and S/VLYS) and membrane destabilisation, lipofuscin accumulation, and histopathological alterations (VvBAS, MLR/MET and CTD ratio) in the digestive gland epithelium; and were integrated as IBR/n (biological response index). Overall, no significant changes were recorded in AChE activity, S/VLYS and CTD ratio in any experimental treatment, while all the other biomarkers showed significant changes depending on the fasting/feeding condition, the exposure to WAF and/or their interaction. As a result, the integrated biomarker index IBR/n was higher at increasing WAF exposure levels both in fasted and fed mussels albeit the response was more marked in the latter. The response profiles were qualitatively similar between fasted and fed mussels but quantitatively more pronounced in fed mussels, especially upon exposure to the highest concentration (25% WAF). Therefore, it is highly recommended that mussels are also supplied with food during short-term, like during long-term toxicological experiments. This practice would avoid the interference of fasting with biological responses elicited by the tested chemicals and allow for reliable comparison with data obtained in long-term experiments and monitoring programmes.

Transcriptional and biochemical analysis of antioxidant enzymes in the mussel Mytilus galloprovincialis during experimental exposures to the toxic dinoflagellate Prorocentrum lima.

The genotoxic and cytotoxic effects of Diarrhetic Shellfish Poisoning (DSP) toxins have been widely investigated in bivalve molluscs, representing the main vectors of these compounds in the Atlantic coast of Europe. DSP toxins are produced by Harmful Algal Blooms (HABs) of Dinophysis and Prorocentrum dinoflagellates, being subsequently accumulated by marine organisms and biomagnified throughout trophic webs. Yet, bivalves display increased resistance to the harmful effects of these toxins during HAB episodes. While previous reports have suggested that such resilience might be the result of an increased activity in the bivalve antioxidant system, very little is still known about the specific mechanism underlying the protective effect observed in these organisms. The present work aims to fill this gap by studying transcriptional expression levels and biochemical activities of antioxidant enzymes in different tissues the mussel Mytilus galloprovincialis during experimental exposures to DSP toxins produced by the dinoflagellate Prorocentrum lima. Results are consistent with the presence of a compensatory mechanism involving a down-regulation in the expression of specific genes encoding antioxidant enzymes [i.e., SuperOxide Dismutase (SOD) and CATalase (CAT)] which is counterbalanced by the up-regulation of other antioxidant genes such as Glutathione S-Transferase pi-1 (GST-pi) and Selenium-dependent Glutathione PeroXidase (Se-GPx), respectively. Enzymatic activity analyses mirror gene expression results, revealing high antioxidant activity levels (consistent with a protective role for the antioxidant system) along with reduced lipid peroxidation (increasing the defense against oxidative stress).

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua).

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Exposure of Carcinus maenas to waterborne fluoranthene: accumulation and multibiomarker responses.

Fluoranthene (FLU) is a priority polycyclic aromatic hydrocarbon (PAH) commonly detected in estuarine sediments, water and biota. Despite this, information on FLU detection, accumulation and effects on marine crustaceans is scarce. This work investigated the accumulation of FLU in Carcinus maenas and the responses of several early-warning biomarkers after a 7-day laboratory exposure to five FLU concentrations (2.56 to 100 µg L(-1)). After exposure to FLU, sub-samples of the crabs' digestive gland and muscle were collected for biomarker determinations. The remaining digestive gland and muscle, together with the rest of the whole-body soft tissues, were analysed for FLU residues by gas chromatography-mass spectrometry (GC-MS). The biomarkers assessed were: i) the quantification of FLU-type compounds by fixed wavelength fluorescence (FF); ii) the activities of glutathione S-transferases (GST) and glutathione reductase (GR), and the levels of total glutathione (GT) and lipid peroxidation (LPO) for oxidative stress; iii) the activity of acetylcholinesterase (AChE) for neurotoxicity; iv) the activities of isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH) enzymes, and total protein, glycogen and lipids as indicators of changes in energy metabolism and storage; and v) the lysosomal membrane stability (LMS) as a measure of cell damage. The results showed strong (R(2)>0.95) concentration-dependent accumulation of FLU residues (as measured by GC-MS) in the remaining whole-body soft tissues and of FLU-type compounds (as measured by FF) in the digestive gland and muscle. A strong positive linear relationship (R(2)=0.91) between FLU residues and FLU-type compounds was also found. Comparing to controls, activities of GST and GR were significantly higher in crabs exposed to ≥16 and ≥40 µg L(-1) FLU, respectively. TG levels and IDH activity showed a significant trend to increase with FLU concentrations whereas AChE activity exhibited the opposite trend. FF measurements in the digestive gland and muscle proved to be an expeditious cost-effective method to assess the uptake and availability of FLU and its metabolites in C. maenas. The results suggest that under continuous environmental exposure, FLU may enhance detoxification and anti-oxidant defences, and cause alterations in the aerobic energy pathway, as well as neuromuscular toxic effects that may increase C. maenas risk of predation.

Two strategies to live in low chronic pollution estuaries: the potential role of lifestyle.

Although physiological and genetic adaptation to toxic stress has been the focus of recent research, the role of lifestyle in pollution tolerance has received less attention. In this study, copper tolerance of benthic and epi-benthic species of harpacticoid copepods was investigated. Concentration-response curves were generated for populations of both species but collected at contaminated and uncontaminated estuaries. The population of the benthic species from the contaminated site showed higher tolerance than its population from the uncontaminated site. The epi-benthic species showed no inter-specific differences in tolerance. The comparison of tolerance between field collected animals of the benthic species and animals from the same population but reared in the laboratory for two generations suggested the existence of a genetic inherited tolerance. Results revealed the importance that lifestyle may have on the generation of tolerance to toxic substances and highlight a potential unforeseen role of it in maintenance of biodiversity on contaminated habitats.

Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham.

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Biochemical effects and polycyclic aromatic hydrocarbons (PAHs) in senegal sole (Solea senegalensis) from a Huelva estuary (SW Spain).

Relations between several stress oxidative biomarkers and aromatic polycyclic hydrocarbon (PAH) concentrations have been studied in wild sole, Solea senegalensis collected in the vicinity of a petrochemical industry. Antioxidant enzyme activities in eco-toxicological studies constitute excellent markers for exposure to a large variety of pollutants. The 16 PAHs in sediment as well as oxidative damage (LPO), activity of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and PAHs type metabolites in sole liver were analysed. Significant correlations (p<0.05) were established between some biomarkers as GST, GPx and CAT and PAHs metabolites in liver (naphthalene, pyrene and phenanthrene) and PAHs concentrations in sediments (fluoranthene, acenaphthene, anthracene and chrysene). PAHs accumulated in the sediment and organisms are inducers of antioxidant defences. GST, GPx and CAT were robust biomarkers showing correlations with both PAHs in sediments and liver PAH metabolites showing different responses to low and high molecular weight PAHs.

Ecotoxicological tools for the tropics: Sublethal assays with fish to evaluate edge-of-field pesticide runoff toxicity.

This study proposes short-term sublethal assays for the tropics using the fish Poecilia reticulata. Assays were evaluated under realistic exposure scenarios by simulating a runoff over an agricultural area sprayed with deltamethrin (Decis). In situ assays were performed inside microcosms set up to simulate runoff water entrance in lentic systems. Laboratory assays were conducted with water samples from the microcosms. In both assays the biochemical parameters were similarly responsive to Decis, with a significant inhibition of acetylcholinesterase (30-46%) and induction of lactate dehydrogenase (33-67%) and glutathione S-transferases (48-176%) activities, observed as of the lowest Decis dose. Postexposure feeding was more sensitive in the laboratory assay than in situ. Among the additional potential stressors, only acetylcholinesterase was significantly influenced by the runoff per se. The proposed methodologies were well adapted for assays with P. reticulata, as organisms were easily deployed and retrieved and enzymatic activities and postexposure feeding were sensitive endpoints.

Blood delta-ALAD, lead and cadmium concentrations in spur-thighed tortoises (Testudo graeca) from Southeastern Spain and Northern Africa.

Mediterranean spur-thighed tortoise (Testudo graeca) is actually included in the IUCN as vulnerable species. Its main European population is located in southeastern Spain. Although a great deal of information has been acquired on the internal medicine and survey and even parasitological fauna on these animals, there are no references about contaminants levels in this species. The objectives of this study were to compare the levels of two metals (cadmium and lead) in the blood of spur-thighed tortoises from two different populations, one from Southeastern of Spain (n = 22) and the other from North of Africa (n = 39), kept in captivity at the Santa Faz Recuperation Centre (Alicante, Spain) and to investigate the relationship between their blood levels of lead and their blood delta-aminolevulinic acid dehydratase (delta-ALAD) activity. Blood lead and cadmium concentrations were higher in tortoises from African than in those from Spain. Moreover, a negative and significant correlation (P < 0.05) was found between delta-ALAD activity and blood lead levels, indicating the suitability of this enzyme as biomarker for lead in this species.

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: linking biomarkers to behaviour.

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC(50)s of copper and mercury (dissolved throughout metal concentrations) at 96h were 568 microg L(-1), and 62 microg L(-1), respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 microg L(-1) for copper and 3 microg L(-1) for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 microg L(-1), for copper and from 3 to 25 microg L(-1) for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.

Effects of natural and chemical stressors on Enchytraeus albidus: can oxidative stress parameters be used as fast screening tools for the assessment of different stress impacts in soils?

Enchytraeids are important organisms of the soil biocenosis. They improve the soil pore structure and the degradation of organic matter. These organisms are used in standardized testing, using survival and reproduction (6 weeks) as endpoints. The use of biomarkers, linked to ecologically relevant alterations at higher levels of biological organization, is a promising tool for Environmental Risk Assessment. Here, enchytraeids were exposed for different time periods (two days and three weeks) to different soils (OECD artificial soil, different compositions in its organic matter, clay or pH value, and LUFA 2.2 natural soil) and different chemicals (Phenmedipham and copper). The main question addressed in the present study was if the effects of chemicals and different soil properties are preceded by alterations at the sub-cellular level, and if these endpoints may be used reliantly as faster screening tools for the assessment of different stress conditions in soils. The parameters measured in E. albidus whole body were: lipid peroxidation (LPO), total glutathione (TG), as well as the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST). The results showed that biomarker responses in E. albidus were significantly affected by the soil type (GST, CAT, GPx, GR and LPO) and the duration of exposure in OECD artificial soil (GST, GPx, GR, CAT and LPO) but not in LUFA 2.2 natural soil. For the abiotic factors studied, after 2 days, low pH decreased significantly the TG levels and the activities of CAT and GR,and low OM also significantly decreased CAT and GR activities. After 3 weeks, differences in soil properties caused a decrease in GR and GPx activities, whereas increased GST activity was observed due to low organic matter and pH. Copper significantly increased the activities of CAT, GPx and GR, and decreased the activity of GST after 2 days as well as inscreasing LPO levels after 3 weeks. Phenmedipham increased LPO levels, associated with increased levels of TG as well as increased activities of CAT and GPx and decreased GST activity after 3 weeks exposure. This study shows that both abiotic and chemical stresses could be followed through biomarker analysis and that some of these determinations are potential endpoints in a quick soil contamination assessment procedure.

Transport and acclimation conditions for the use of an estuarine fish (Pomatoschistus microps) in ecotoxicity bioassays: effects on enzymatic biomarkers.

Acclimation of organisms for ecotoxicity testing is in general processed according to Organisation for Economic Co-operation and Development (OECD) and/or Environmental Protection Agency (EPA) guidelines, under controlled conditions. However, when organisms are collected in the field, their capture, transport and adaptation to laboratory conditions are factors of stress. In their natural habitat, estuarine fish are exposed to considerable fluctuations of environmental variables, while in laboratory they are acclimated to constant conditions and this can be per se a factor of stress that may influence biomarker responses. Therefore, it is important to investigate the effects of these procedures on estuarine fish performance before using them as test organisms in ecotoxicity bioassays. The goal of the present study was to investigate the effects of transporting the common goby, Pomatoschistus microps from the field (natural populations) to the laboratory and of its acclimation to laboratory conditions on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GSTs). Fish were collected in a reference site of the Minho River estuary (NW of Portugal) and the activities of the biomarkers were monitored before and after the transport of organisms to the laboratory and during the acclimation period (at 5, 10 and 15 days). The activities of all the enzymes indicated that capture and transport conditions had no effects on enzymatic activities. Furthermore, AChE, LDH and GST presented higher activities at the end of the acclimation period than at beginning, suggesting a physiological adaptation to laboratory conditions. This adaptation should be taken into consideration in the experimental design to avoid bias in the interpretation of effects of xenobiotics on biomarkers.

Ras gene in marine mussels: a molecular level response to petrochemical exposure.

Mussels are susceptible to numerous toxicants and are often employed as bioindicators. This study investigated the status of the ras proto-oncogene in Mytilus galloprovincialis following petrochemical exposure. A M. galloprovincialis homologue of the vertebrate ras gene was isolated, showing conserved sequence in regions of functional importance and a high incidence of polymorphic variation. Mutational damage was investigated in mussels chronically exposed to the water-accommodated fraction of #4 fuel-oil (WAF), and in mussels collected along the NW coast of Portugal in sites with different levels of petrochemical contamination. A ras gene point mutation was identified in the codon 35 of one individual exposed to 12.5% WAF. No mutations were detected in mussels from the WAF control or environmental samples. This represents the first report of a ras gene mutation, experimentally-induced by petrochemical exposure, in an invertebrate species.

Acute effects of Benzo[a]pyrene, anthracene and a fuel oil on biomarkers of the common goby Pomatoschistus microps (Teleostei, Gobiidae).

The objective of this study was to investigate the effects of two different PAHs and a complex petrochemical mixture on the common goby, Pomatoschistus microps, using selected biomarkers as effect criteria. Benzo[a]pyrene (BaP) and anthracene were used as reference substances, while the water accommodated fraction of #4 fuel-oil (#4 WAF) was used as an example of a petrochemical mixture. P. microps was used since it is both a suitable bioindicator and a good test organism. Groups of fish were exposed to different concentrations of each of the test substances for 96 h and the activities of several enzymes commonly used as biomarkers were determined at the end of the bioassays. All the substances inhibited P. microps acetylcholinesterase (AChE) indicating that they have at least one mechanism of neurotoxicity in common: the disruption of cholinergic transmission by inhibition of AChE. An induction of lactate dehydrogenase (LDH) activity was found in fish exposed to BaP or to anthracene, suggesting an increase of the anaerobic pathway of energy production. On the contrary, inhibition of LDH was found in fish exposed to #4 WAF, suggesting a distinct effect of the mixture. An induction of P. microps glutathione S-transferase (GST) activity was found in fish exposed to BaP or to #4 WAF, while an inhibition was observed after exposure to anthracene. These results suggest that GST is involved in the detoxification of BaP and #4 WAF, but not of anthracene. All the substances increased catalase activity and isolated PAHs also increased superoxide dismutase, glutathione reductase and glutathione peroxidase activities, while #4 WAF did not cause significant alterations on these enzymes. These results suggest that all the substances may induce oxidative stress on P. microps, with BaP and anthracene apparently having more oxidative stress potential than #4 WAF.

Behaviour and biomarkers of oxidative stress in Gambusia holbrooki after acute exposure to widely used pharmaceuticals and a detergent.

Pharmaceuticals are continuously dispersed into the environment, as a result of human and veterinary use, and have become a relevant environmental concern. In the present study, the acute toxicity of three therapeutic agents (diazepam, clofibrate, and clofibric acid) and a detergent, sodium dodecylsulphate (SDS), to the euryhaline fish Gambusia holbrooki was evaluated. Special attention was devoted to oxidative stress parameters. G. holbrooki males, captured in the estuary of the Minho River (NW Portugal), were exposed for 96 h to the selected compounds. The following oxidative stress biomarkers were evaluated in gills and liver tissues: reduced and oxidised glutathione, lipid peroxidation, and several antioxidant enzymes, namely (1) total and selenium-dependent glutathione peroxidase (GPx), (2) glutathione reductase (GRed), (3) copper-zinc superoxide dismutase (Cu-ZnSOD) and manganese superoxide dismutase (MnSOD), and (4) glutathione-S-transferases (GSTs). In the particular case of diazepam, swimming behaviour was also evaluated. The obtained results indicate an overall diminished oxidative stress response caused by SDS and diazepam. Oxidative-based alterations were observed after exposure to clofibrate and clofibric acid, with modifications of several enzymatic activities. Diazepam caused evident behavioural changes: animals showed dark pigmentation and also abnormal postures, namely lethargy and anomalous movement.

Fucus spp. as a mercury contamination bioindicator in costal areas (Northwestern Portugal).

Mercury has been considered as one of the most important pollutants in coastal and estuarine areas. Efforts have been made to detect, as early as possible, the effects of this and other metals in several species. Macroalgae, particularly Fucus spp., have been widely used as biomonitors of metal pollution. In this study, three Fucus species (F. spiralis, F. vesiculosus and F. ceranoides) were collected from several sampling sites in Portugal. The concentrations of mercury were determined in three structural parts (holdfast, stipe and receptacles). Two different techniques were used to determine mercury concentrations. Almost all mercury concentrations (in sediments and in water) were below national and international standards. Mercury concentration in the specimens (0.012-0.061 microg g(-1) for receptacles, 0.028-0.221 microg g(-1) for stipe and 0.029-0.287 microg g(-1) for holdfast) was always higher that those obtained for the sediment (0.001-0.112 microg g(-1)). With few exceptions the contrary was found for receptacles. In general, a good agreement between concentrations of mercury in sediment and Fucus was found. The results indicate that Fucus accumulate mercury and may be a suitable species for use in risk assessment for coast and estuarine areas, by providing valuable information regarding the levels of mercury that will be available for the consumers of Fucus spp.

Effects of copper and cadmium on cholinesterase and glutathione S-transferase activities of two marine gastropods (Monodonta lineata and Nucella lapillus).

With the view of using Nucella lapillus and Monodonta lineata as bioindicators in biomonitoring programs in the NW coast of Portugal, the sensitivity to copper and cadmium of these two common species in Atlantic coasts of Europe was investigated. Assays based on mortality and on the activity of the enzymes cholinesterases (ChE) and glutathione S-transferases (GST) were used, as these biomarkers have been used in biomonitoring studies in the area. ChEs present in foot muscle of both species were characterised and found to show properties of both typical acetylcholinesterase and pseudocholinesterase. Cadmium LC50s for N. lapillus and M. lineata were 2.64 and 2.44 mg/L, respectively, while the lowest observed effect concentration (LOEC) was 1.53 mg/L for both species. LOEC value for cooper was 0.075 mg/L for both. Cadmium in vivo exposure increased ChE activity of N. lapillus, but had no effects on M. lineata ChE. No in vitro effects of cadmium on ChE activity of any of the tested species was observed. Copper had no significant in vivo effects on ChE activity, although it inhibited the ChE of both species in vitro, the IC50s being 5.87 and 12.17 mg/L for N. lapillus and M. lineata, respectively. Cadmium had no significant effects on GST activity of either species, while copper caused a significant reduction of N. lapillus GST (LOEC=0.044 mg/L) but had no effect on M. lineata GST. Results indicate that (i) N. lapillus and M. lineata have a similar acute sensitivity to cadmium and copper; (ii) ChE and GST of these species are sensitive to cadmium (iii) ChEs of both species are inhibited by copper at concentrations in the mg/L range and therefore, its use is limited to heavily polluted sites; (iv) N. lapillus GST does not seem to be a suitable biomarker for copper, at least in the range of concentrations tested, since it was inhibited by copper, but no clear concentration-response relationship was observed.

Enzymatic biomarkers in the crab Carcinus maenas from the Minho River estuary (NW Portugal) exposed to zinc and mercury.

Zinc and mercury are common contaminants of estuaries and coastal ecosystems where they may induce adverse effects on the biota. Carcinus maenas is a key-species in several European estuaries, living in close association with the sediment where a considerable number of environmental contaminants, including zinc and mercury may accumulate. In the present study, the acute effects of zinc and mercury on C. maenas from the Minho River Estuary (NW Portugal) were investigated by using the activity of the enzymes cholinesterase (ChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GST) as effects' criteria. Crabs were exposed for 96h to several concentrations of Zn(2+) (0, 1.84, 3.71, 7.39 and 14.79mg/l) or Hg(2+) (0, 0.09, 0.19, 0.37 and 0.74mg/l). The choice of Zn(2+) test concentrations was based on the LC50 value (14.86mg/l) determined in a first part of the study, while the choice of Hg(2+) concentrations was based on values from the literature. At the end of the bioassays, eye, muscle and hepatopancreas tissues were isolated and used for ChE, LDH and GST determinations, respectively. Zinc significantly inhibited ChE activity (p<0.05, EC50=14.68mg/l), caused significant alterations of GST activity (p<0.05) and induced LDH activity (p<0.05) at the exposure of 14.79mg/l. Mercury significantly inhibited ChE activity (p<0.001, LOEC=0.09mg/l, EC50=0.235mg/l) and increased both GST (p<0.05, LOEC=0.774mg/l) and LDH activities (p<0.05, LOEC=0.119mg/l). These results suggest that both metals interfere with cholinergic neurotransmission in C. maenas by inhibiting ChE activity. In addition, they also interfere with GST activity and this may reduce the capacity of detoxification of some chemicals and/or to increase the probability of oxidative stress to occur. Furthermore, both metals increase LDH activity, suggesting an interference with energy production pathways. Therefore, the presence of zinc and mercury in estuaries at concentrations in the mg/l or mug/l range, respectively, may have a negative impact on C. maenas.

Impact of chemical exposure on the fish Pomatoschistus microps Krøyer (1838) in estuaries of the Portuguese Northwest coast.

Juveniles of the estuarine fish Pomatoschistus microps were collected from autumn 2001 to summer 2002 in five stations along the Portuguese Northwest coast with different types and/or levels of environmental contamination: two reference sites with low levels of contamination (R1 and R2) and three differently impacted areas with higher levels of contamination. UI is located in an estuary under the influence of urban and industrial effluents, AA in a channel that receives intensive agriculture run-off and IE in a highly impacted industrial area. The activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferases (GST) were used as environmental biomarkers on P. microps. A significant seasonality effect on all the enzymatic activities was found, lower levels being registered in winter and spring on AChE, in autumn on LDH, and in winter on GST and EROD. The battery of biomarkers used was capable of discriminating sites with different types and/or levels of contamination, R1 and UI being the highest discriminated (91.7% and 66.7%, respectively). At R1 significantly lower levels of AChE and LDH were found, and EROD was significantly inducted at UI. Furthermore, IE presented higher levels of GST, and R2 and AA an inhibition of AChE in winter and spring. The results indicated that the battery of biomarkers used in this study seems to be a useful tool to distinguish between different types of environmental contamination in estuarine systems, and that P. microps is a suitable species to be used as bioindicator.