Bioaccumulation of BDE-47 and effects on molecular biomarkers acetylcholinesterase, glutathione-S-transferase and glutathione peroxidase in Mytilus galloprovincialis mussels.

Mussels, Mytilus galloprovincialis, showed a high bioaccumulation ability when exposed to waterborne tetrabromodiphenyl ether (BDE-47), with a bioconcentration factor of 10,900 L Kg(-1) wet weight, and slow depuration rates in clean seawater. Kinetic and concentration-response experiments were performed measuring in the exposed mussel the activities of three molecular biomarkers: glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE). The long term (30 days) exposure of mussels to all concentrations (2-15 µg L(-1)) of BDE-47 significantly inhibited the AChE and GST activities, a result that supports the suitability of these biomarkers in marine pollution monitoring programs. However, GPx activity showed a less consistent pattern of response depending on the concentration and the duration of exposure.

The particle effect: comparative toxicity of chlorpyrifos in combination with microplastics and phytoplankton particles in mussel.

Lately, the role of microplastics (MP) as vectors for dissolved contaminants and as vehicle for their transfer to aquatic organisms has received attention. Similarly to MP, other inorganic and organic particles may act as passive samplers. However, limited comparative knowledge exists at this respect. In the present study we have comparatively investigated the risk for mussel of MP and the pesticide chlorpyrifos (CPF) alone and in combination with MP and phytoplankton particles of microalgae (MP-CPF and MA-CPF, respectively). We selected MP and microalgae of similar size to expose mussel to the same volume of particles (≈1.5 mm3L-1 ≈ equivalent to 1.5 mg MP L-1) and the same concentration of contaminant (CPF, 7.6 µg L-1). MP were virgin HDPE microparticles (≤10 µm) while the microalgae species was Isochrisis galbana (4-8 µm). Mussels were exposed for 21 days to MP, CPF, MP-CPF and MA-CPF. Then, a suite of neurotoxicity, oxidative stress and oxidative damage biomarkers were measured in samples collected at day 7 and 21. Additionally, these biochemical markers were assessed in an integrated manner with others measured at physiological, immune and cell component level in the same organisms, previously published. Overall, MP did not elicit significant alterations on the majority of parameters measured. In contrast, mussels exposed to CPF, MA-CPF and MP-CPF showed evidence of neurotoxicity and oxidant imbalance at day 7, added to a detrimental physiological condition and immune imbalance at day 21. At the latter time MP-CPF mussels showed greater alterations than CPF or MA-CPF mussels. This suggested a synergistic toxicity of MP combined with CPF greater than that produced by the contaminants alone (MP or CPF) or by MA combined with CPF.

Filtration of biopolymer PHB particles loaded with synthetic musks does not cause significant bioaccumulation in marine mussels.

The role of the biopolymer polyhydroxybutyrate (PHB, <250 µm) as a vehicle of a synthetic musks mixture (celestolide, galaxolide, tonalide, musk xylene, musk moskene and musk ketone) to Mytilus galloprovincialis was investigated. For 30 days, virgin PHB, virgin PHB+musks (6.82 µg g-1) and weathered PHB+musks, were daily spiked into tanks containing mussels, followed by a 10-day depuration period. Water and tissues samples were collected to measure exposure concentrations and accumulation in tissues. Mussels were able to actively filter microplastics in suspension but the concentration of the musks found in tissues (celestolide, galaxolide, tonalide) were markedly lower than the spiked concentration. Estimated Trophic Transfer Factors suggest that PHB will only play a minor role on musks accumulation in marine mussels, even if our results suggest a slightly extended persistence in tissues of musks loaded to weathered PHB.

Microplastics increase the toxicity of mercury, chlorpyrifos and fluoranthene to mussel and sea urchin embryos.

The objective of this study was to determine whether and to what extent microplastics (MPs) enhance the toxicity of pollutants as well as whether pollutant-loaded MPs act as relevant vectors of chemical pollutants. With this aim, the toxicity for mussel and sea urchin embryos of: 1) three dissolved pollutants (Pol): chlorpyrifos (CPF), fluoranthene (FLT) and mercury (Hg); 2) their mixture with Microplastics (MP + Pol); and 3) pollutant-loaded MPs (MPPol), was assessed. Analyses of CPF, FLT and Hg were also performed to evaluate the transfer among dissolved and particulate phases. In general, the 'MP + Pol' treatments were more toxic as 48-h EC50 (µg/L) than the 'Pol' treatments for sea urchin or mussel. The 48-h and 120-h EC50s (µg/L) for sea urchin showed little variation for CPF and MP + CPF, and no clear pattern was found for FLT and MP + FLT. The performed chemical analysis in the MPPol tests indicated that desorption was the main route to explain the observed toxicity of Hg and a relevant route for CPF and FLT. This study contributes to improve the knowledge about the interactions between MPs and chemical pollutants, which is fundamental for a more realistic ecological risk assessment in aquatic ecosystems.

Microplastic occurrence in deep-sea fish species Alepocephalus bairdii and Coryphaenoides rupestris from the Porcupine Bank (North Atlantic).

Microplastic occurrence in marine biota has been reported in a wide range of animals, from marine mammals and seabirds to invertebrates. Commercial and shallow-water fish have been the subject of numerous works on microplastic ingestion, given their importance in human diet and accessibility. However, little is known about microlitter occurrence in fish species inhabiting the dark ocean, in the bathyal zone and there is a high degree of uncertainty about microplastic distribution in offshore areas and the deep sea. In this study, bathydemersal species Alepocephalus bairdii and Coryphaenoides rupestris from the Porcupine Bank caught between 985 and 1037 m depth were inspected for microdebris. The stomach contents were digested by the alkaline method plus ethanol addition to avoid clogging. A filament of Polyethylene Terephthalate (PET) was found in the stomach of a specimen of A. bairdii, representing 4% of the total sampled specimens of this species (i.e. prevalence in n = 25). However, when considering potential microplastics, the prevalence increased to 28% in both, A. bairdii and C. rupestris. This work provides the first baseline study of microplastic items in fish from such depths in the Atlantic and suggests these species might be used as biomonitors in future research.

Linking biochemical and individual-level effects of chlorpyrifos, triphenyl phosphate, and bisphenol A on sea urchin (Paracentrotus lividus) larvae.

The effects of three relevant organic pollutants: chlorpyrifos (CPF), a widely used insecticide, triphenyl phosphate (TPHP), employed as flame retardant and as plastic additive, and bisphenol A (BPA), used primarily as plastic additive, on sea urchin (Paracentrotus lividus) larvae, were investigated. Experiments consisted of exposing sea urchin fertilized eggs throughout their development to the 4-arm pluteus larval stage. The antioxidant enzymes glutathione reductase (GR) and catalase (CAT), the phase II detoxification enzyme glutathione S-transferase (GST), and the neurotransmitter catabolism enzyme acetylcholinesterase (AChE) were assessed in combination with responses at the individual level (larval growth). CPF was the most toxic compound with 10 and 50% effective concentrations (EC10 and EC50) values of 60 and 279 µg/l (0.17 and 0.80 µM), followed by TPHP with EC10 and EC50 values of 224 and 1213 µg/l (0.68 and 3.7 µM), and by BPA with EC10 and EC50 values of 885 and 1549 µg/l (3.9 and 6.8 µM). The toxicity of the three compounds was attributed to oxidative stress, to the modulation of the AChE response, and/or to the reduction of the detoxification efficacy. Increasing trends in CAT activity were observed for BPA and, to a lower extent, for CPF. GR activity showed a bell-shaped response in larvae exposed to CPF, whereas BPA caused an increasing trend in GR. GST also displayed a bell-shaped response to CPF exposure and a decreasing trend was observed for TPHP. An inhibition pattern in AChE activity was observed at increasing BPA concentrations. A potential role of the GST in the metabolism of CPF was proposed, but not for TPHP or BPA, and a significant increase of AChE activity associated with oxidative stress was observed in TPHP-exposed larvae. Among the biochemical responses, the GR activity was found to be a reliable biomarker of exposure for sea urchin early-life stages, providing a first sign of damage. These results show that the integration of responses at the biochemical level with fitness-related responses (e.g., growth) may help to improve knowledge about the impact of toxic substances on marine ecosystems.

Aquatic toxicity of chemically defined microplastics can be explained by functional additives.

A novel, systematic approach to relate plastic toxicity with chemical composition is undertaken. Using industrial methods, three petroleum-based polymers, low-density polyethylene (PE), polyvinyl chloride (PVC), and polyamide (PA), and the biopolymer polyhydroxybutyrate (PHB) were manufactured in different formularies including conventional and alternative additives, and microplastics of two sizes (<250 and <20 µm) were obtained with the aim to relate their composition with environmental impact in aquatic environments. Internationally accepted standard tests of regulatory use with marine organisms representative of microalgae (Tisochrysis lutea population growth), crustaceans (Acartia clausi larval survival), and echinoderms (Paracentrotus lividus sea-urchin embryo test) support the following conclusions. Aquatic toxicity of microplastics made from conventional oil-based polymers is due to leaching of chemical additives, and not to ingestion of microplastics. Use of alternative formulations based on natural rather than synthetic chemical additives did not consistently reduce aquatic toxicity except for the replacement of triclosan by the alternative biocide lawsone. In contrast, the biopolymer tested, PHB, seemed to impact marine plankton through different mechanisms associated to the higher abundance of plastic particles within the nanometric range found in this resin and absent in other materials.

Proteomic analysis and biochemical alterations in marine mussel gills after exposure to the organophosphate flame retardant TDCPP.

Organophosphate flame retardants (OPFRs) are (re-)emergent environmental pollutants increasingly being used because of the restriction of other flame retardants. The chlorinated OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is among those of highest environmental concern, but its potential effects in the marine environment have rarely been investigated. We exposed a widely used sentinel marine mussel species, Mytilus galloprovincialis, to 10 µg L-1 of TDCPP during 28 days and studied: (i) the kinetics of bioaccumulation and elimination of the compound, (ii) the effect on two molecular biomarkers, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities, and (iii) proteomic alterations in the gills, following an isobaric labeling quantitative shotgun proteomic approach, at two exposure times (7 and 28 days). Uptake and elimination of TDCPP by mussels were very fast, and the bioconcentration factor of this compound in mussels was 147 L kgww-1, confirming that this compound is not very bioaccumulative, as predicted by its chemical properties. GST activity was not affected by TDCPP exposure, but AChE activity was inhibited by TDCPP at both 7 and 28 days of exposure. Proteomic analysis revealed subtle effects of TDCPP in mussel gills, since few proteins (less than 2 % of the analysed proteome) were significantly affected by TDCPP, and effect sizes were low. The most relevant effects detected were the up-regulation of epimerase family protein SDR39U1, an enzyme that could be involved in detoxification processes, at both exposure times, and the down-regulation of receptor-type tyrosine-protein phosphatase N2-like (PTPRN2) after 7 days of exposure, which is involved in neurotransmitter secretion and might be related to the neurotoxicity described for this compound. Exposure time rather than TDCPP exposure was the most important driver of protein abundance changes, with 33 % of the proteome being affected by this factor, suggesting that stress caused by laboratory conditions could be an important confounding factor that needs to be controlled in similar ecotoxicology studies. Proteomic data are available via ProteomeXchange with identifier PXD019720.

Bioaccumulation of UV filters in Mytilus galloprovincialis mussel.

In this study the bioaccumulation kinetics of organic UV filters, such as 4-MBC, BP-3, BP-4, OC and OD-PABA in wild Mytilus galloprovincialis mussels was investigated. The uptake and accumulation of waterborne 4-MBC, BP-4 and OC was very rapid, and after only 24 h of exposure to 1 µg L-1, the tissular concentrations were 418, 263 and 327 µg kg-1d.w., respectively. The kinetics of bioaccumulation of BP-4 and OC significantly fitted to an asymptotic model with BCF values of 905 L kg-1 and 2210 L kg-1, respectively. Measured bioaccumulation of the hydrophilic chemical BP-4 was much higher than predicted by Kow-based bioconcentration models, which would lead to a marked underestimation of actual risk. On the other hand, the patterns of uptake found for BP-3 and OD-PABA suggest biotransformation ability of mussels for these two chemicals.

Bioaccumulation of PCB-153 and effects on molecular biomarkers acetylcholinesterase, glutathione-S-transferase and glutathione peroxidase in Mytilus galloprovincialis mussels.

In this study, PCB-153 bioaccumulation kinetics and concentration-response experiments were performed employing wild Mytilus galloprovincialis mussels. In addition, the activity of three enzymatic biomarkers: glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE), were measured in the mussel gills. The experimental data fitted well to an asymptotic accumulation model with a high bioconcentration factor (BCF) of 9324 L kg(-1) and a very limited depuration capacity, described by a low excretion rate coefficient (Kd = 0.083 d(-1)). This study reports by first time in mussels significant inhibition of GST activity and significant induction of GPx activity as a result of exposure to dissolved PCB-153. In contrast, AChE activity was unaffected at all concentrations and exposure times tested. The effects on both enzymes are time-dependent, which stresses the difficulties inherent to the use of these biomarkers in chemical pollution monitoring programs.

Bioaccumulation of 4-nonylphenol and effects on biomarkers, acetylcholinesterase, glutathione-S-transferase and glutathione peroxidase, in Mytilus galloprovincialis mussel gilla.

Wild marine mussels, Mytilus galloprovincialis showed a moderate bioaccumulation ability when exposed to waterborne 4-nonylphenol (4-NP), with a bioconcentration factor (BCF) of 6850 L Kg(-1) (dry weight). Kinetic and concentration-response experiments were performed and three enzymatic biomarkers in mussel gills were measured: Glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE). Exposure of mussels to environmentally relevant concentrations (25-100 µg L(-1)) of 4-nonylphenol significantly inhibited the AChE activity and induced the GST and GPx activities. GST induction was dose dependent whilst GPx activity showed a less consistent pattern, but in both cases the induction remained after a 10 d depuration period. Mussels seem capable of eliminating 4-NP from their tissues through a mechanism involving GST induction.

Combined use of chemical, biochemical and physiological variables in mussels for the assessment of marine pollution along the N-NW Spanish coast.

This study undertakes an overall assessment of pollution in a large region (over 2500 km of coastline) of the N-NW Spanish coast, by combining the use of biochemical (AChE, GST, GPx) and physiological (SFG) responses to pollution, with chemical analyses in wild mussel populations (Mytilus galloprovincialis). The application of chemical analysis and biological techniques identified polluted sites and quantified the level of toxicity. High levels of pollutants were found in mussel populations located close to major cities and industrialized areas and, in general, average concentrations were higher in the Cantabrian than in the Iberian Atlantic coast. AChE activities ranged between 5.8 and 27.1 nmol/min/mg prot, showing inhibition in 12 sampling sites, according to available ecotoxicological criteria. GST activities ranged between 29.5 and 112.7 nmol/min/mg prot, and extreme variability was observed in GPx, showing activities between 2.6 and 64.5 nmol/min/mg prot. Regarding SFG, only 5 sites showed 'moderate stress' (SFG value below 20 J/g/h), and most sites presented a 'high potential growth' (>35 J/g/h) corresponding to a 'healthy state'. Multivariate statistical techniques applied to the chemical and biological data identified PCBs, organochlorine pesticides and BDEs as the main responsible of the observed toxicity. However, the alteration of biological responses caused by pollutants seems to be, in general, masked by biological variables, namely age and mussel condition, which have an effect on the mussels' response to pollutant exposure.

Practical procedures for selected biomarkers in mussels, Mytilus galloprovincialis--implications for marine pollution monitoring.

Biomarkers are required to assess the biological effects of pollutants on marine organisms in order to monitor ecosystem status, but their use is often limited by their strong variability due to environmental and/or intrinsic biological factors. Accordingly, the main aim of this work was to set up practical procedures for a battery of widely used biomarkers in mussels (Mytilus galloprovincialis). Antioxidant enzymes (catalase [CAT] and glutathione peroxidase [GPx]), a phase II detoxification enzyme (glutathione S-transferase [GST]) and a neurotransmitter catabolism enzyme (acetylcholinesterase [AChE]), were considered. Several relevant aspects were studied in order to obtain a more realistic interpretation of biomarker responses, including the calculation of the minimum sample size required to estimate the population mean with a fixed error margin, the selection of the specific organ or tissue where the enzymatic activity is higher for each biomarker, and the influence of tidal height and temperature on the basal enzymatic activity. GST and CAT activities needed a minimum sample size of 12, whereas for GPx and AChE activities a minimum sample size of 14 was required. The gills were the organ with higher GST, GPx and AChE enzymatic activities, whereas the digestive gland showed the highest CAT activity. Also, the low inter-tidal was the recommended tide level whilst no significant effect of temperature was observed on GST, GPx and CAT, and no clear pattern could be identified for AChE. The implications for environmental monitoring are discussed.

Integrated use of antioxidant enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain).

In the present work, we investigated the potential use of several antioxidant enzymes in wild mussels (Mytilus galloprovincialis) as biomarkers of marine pollution. The enzymatic activity levels of glutathione S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) were measured in gills and digestive gland. Those enzymes participate in the cellular defense system that is involved in the adaptive response of organisms to chemical pollution. Adult mussels were collected at five sampling sites located at the Ría de Pontevedra and Ría de Vigo. Seasonal variations in the control site were observed for the CAT activity, but no significant variability was found for GST and GPx. Mussels from the most polluted sites exhibited a significantly greater GST activity compared to the control site (p<0.05) during the sampling period, whereas GPx and CAT activities have not such a marked pattern. Trace metals, PAHs, PCBs and DDT contents in mussels at sampling sites showed highly significant positive correlations with the GST activity. This study supports the use of GST as a useful biomarker for long-term pollution monitoring in marine coastal ecosystems, whilst more research is needed for GPx and CAT.