Luís Vieira

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.

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.

Zooplankton distribution in a temperate estuary (Mondego estuary southern arm: Western Portugal)

The composition and spatio-temporal distribution of zooplankton abundance and diversity were studied monthly from July 1999 to June 2000 in the southern arm of the Mondego estuarine system (Western Portugal). Two sampling stations with different salinity conditions were selected. Zooplankton samples were obtained using 63 and 125 μm mesh nets. In both taxocenosis densities were higher in autumn and late spring-summer with copepodits and adult copepods among the most abundant zooplankters, representing more than 90.0% of the total densities. Abundance increased with increasing temperature, salinity and chlorophyll a values. The most abundant species were Oithona nana, Acartia tonsa, Acartia clausi, Euterpina acutifrons, Oithona similis, Temora longicornis, Clausocalanus arcuicornis, Paracalanus parvus, and Acartia bifilosa var. inermis. In the 63 μm taxocenosis, diversity was higher closest to the mouth of the estuary, during late spring and summer, and in the inner estuary, during autumn and winter. Cluster analysis showed that spatial distribution dominated over seasonal patterns, i.e. the similarities between the clusters grouping the samples of different months is high, which was also confirmed by ANOVA analysis.

Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrarchus labrax (Linnaeus, 1758)

Microplastics pollution is a global paradigm that raises concern in relation to environmental and human health. This study investigated toxic effects of microplastics and mercury in the European seabass (Dicentrarchus labrax), a marine fish widely used as food for humans. A short-term (96 h) laboratory bioassay was done by exposing juvenile fish to microplastics (0.26 and 0.69 mg/L), mercury (0.010 and 0.016 mg/L) and binary mixtures of the two substances using the same concentrations, through test media. Microplastics alone and mercury alone caused neurotoxicity through acetylcholinesterase (AChE) inhibition, increased lipid oxidation (LPO) in brain and muscle, and changed the activities of the energy-related enzymes lactate dehydrogenase (LDH) and isocitrate dehydrogenase (IDH). All the mixtures caused significant inhibition of brain AChE activity (64-76%), and significant increase of LPO levels in brain (2.9-3.4 fold) and muscle (2.2-2.9 fold) but not in a concentration-dependent manner; mixtures containing low and high concentrations of microplastics caused different effects on IDH and LDH activity. Mercury was found to accumulate in the brain and muscle, with bioaccumulation factors of 4-7 and 25-40, respectively. Moreover, in the analysis of mercury concentrations in both tissues, a significant interaction between mercury and microplastics was found. The decay of mercury in the water increased with microplastics concentration, and was higher in the presence of fish than in their absence. Overall, these results indicate that: microplastics influence the bioaccumulation of mercury by D. labrax juveniles; microplastics, mercury and their mixtures (ppb range concentrations) cause neurotoxicity, oxidative stress and damage, and changes in the activities of energy-related enzymes in juveniles of this species; mixtures with the lowest and highest concentrations of their components induced different effects on some biomarkers. These findings and other published in the literature raise concern regarding high level predators and humans consuming fish being exposed to microplastics and heavy metals, and highlight the need of more research on the topic.

Uptake and effects of the antimicrobial florfenicol, microplastics and their mixtures on freshwater exotic invasive bivalve Corbicula fluminea

Microplastics and antimicrobials are widely spread environmental contaminants and more research on their toxicity is needed. The uptake and effects of the antimicrobial florfenicol, microplastics, and their mixtures on Corbicula fluminea were investigated. Bivalves were exposed for 96h to florfenicol (1.8 and 7.1mg/l), microplastics (0.2 and 0.7mg/l), or mixtures of the two substances. After 96h, all bivalves exposed to antimicrobial treatments had florfenicol in their body (e.g. 2±1μg/g). Microplastics were found in the gut, lumen of the digestive gland, connective tissue, hemolymphatic sinuses, and gills surface of animals. Florfenicol caused a significant inhibition of cholinesterase (ChE) activity (~32%). Animals exposed to 0.2mg/l of microplastics showed ChE activity inhibition (31%), and no other significant alterations. Mixtures caused feeding inhibition (57-83%), significant ChE inhibition (44-57%) and of isocitrate dehydrogenase activity, and increased anti-oxidant enzymes activity and lipid peroxidation levels. Overall, the results indicate that C. fluminea take up florfenicol and microplastics from the water and accumulated or at least retained it in their body for some time; both florfenicol (low ppm range) and microplastics (ppb range) were toxic to C. fluminea, with mixtures containing florfenicol and microplastics being more toxic. Thus, the risk of exposure and toxic effects of florfenicol to C. fluminea and other bivalves, and its predators increase in ecosystems contaminated with the antimicrobial and microplastics, as well as to humans consuming contaminated species from these ecosystems.

Population dynamics of Acartia clausi from a temperate estuary (Mondego Estuary, Western Portugal)

Summary The main purpose of this study was to estimate the distribution, production and production/biomass ratio values of Acartia clausi, one of the most representative taxa of the Copepoda community in the Mondego estuary. The following biomass/length relationship was estimated for specimens of Acartia clausi: AFDW = 2.27 BL 2.41. Length-weight relationships were used to estimate production taking into account cohort growth and mortality. The annual production was calculated at 63.44 mgC m−3y−1 and the production/biomass (P/Ƀ) ratio was estimated at 25.50. These results indicate that Acartia clausi may play a significant role in transferring energy to higher trophic levels in the Mondego estuarine system.

Single and combined effects of microplastics and mercury on juveniles of the European seabass ( Dicentrarchus labrax ): Changes in behavioural responses and reduction of swimming velocity and resistance time

Microplastics and mercury are environmental pollutants of great concern. The main goal of the present study was to investigate the effects of these pollutants, both individually and in binary mixtures, on the swimming performance of juvenile European seabass, Dicentrarchus labrax. Microplastics alone, mercury alone and all the mixtures caused significant reduction of the swimming velocity and resistance time of fish. Moreover, changes in behavioural responses including lethargic and erratic swimming behaviour were observed. These results highlight that fish behavioural responses can be used as sensitive endpoint to establish the effects of contamination by microplastics and also emphasizes the need to assess the combined effects of microplastics and other environmental contaminants, with special attention to the effects on behavioural responses in fish and other aquatic species.

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).

Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements.

Microplastics increase mercury bioconcentration in gills and bioaccumulation in the liver, and cause oxidative stress and damage in Dicentrarchus labrax juveniles

The presence of microplastics and several other pollutants in the marine environment is of growing concern. However, the knowledge on the toxicity of mixtures containing microplastics and other contaminants to marine species is still scarce. The main goals of this study were to investigate the oxidative stress and lipid oxidative damage potentially induced by 96 h of exposure to mercury (0.010 and 0.016 mg/L), microplastics (0.26 and 0.69 mg/L), and mixtures of the two substances (same concentrations, full factorial) in the gills and liver of D. labrax juveniles, and the possible influence of microplastics on mercury bioconcentration (gills) and bioaccumulation (liver). The results indicate that the presence of microplastics in the water increased the concentration of mercury in gills and liver of D. labrax juveniles. Microplastics and mercury, alone and in mixtures, caused oxidative stress in both organs. Based on the total induction of antioxidant enzymatic activity, the type of toxicological interaction in fish exposed to the mixture containing the lowest concentration of the two substances was addition in gills, and addition or synergism in the liver. These results stress the need to further address the role of microplastics in the bioconcentration, bioaccumulation, and toxicity of other environmental contaminants in different species.