Role of UV radiation and oxidation on polyethylene micro- and nanoplastics: impacts on cadmium sorption, bioaccumulation, and toxicity in fish intestinal cells.

This study investigated the role of ultraviolet (UV) radiation and oxidation in high-density polyethylene microplastics (2-15 µm) and nanoplastics (0.2-9.9 µm) (NMPs) on particle chemistry, morphology, and reactivity with cadmium (Cd). Additionally, toxicity of NMPs alone and with Cd was evaluated using RTgutGC cells, a model of the rainbow trout (Oncorhynchus mykiss) intestine. The role on NMPs on Cd bioaccumulation in RTgutGC cells was also evaluated. Dynamic light scattering indicated that after UV radiation NPs agglomerated size increased from 0.8 to 28 µm, and to 8 µm when Cd was added. Oxidized MPs agglomerated size increased from 11 and 7 to 46 and 27 µm in non-UV- and UV-aged oxidized MPs when adding Cd, respectively. Cd-coated particles exhibited generally significantly higher zeta potential than non-Cd-coated particles, while attenuated total reflectance-Fourier transform infrared spectroscopy showed that the functional chemistry of the particles was oxidized and modified after being exposed to UV radiation. Presence of NMPs resulted in a significant decrease in Cd bioaccumulation in RTgutGC cells (100.5-87.9 ng Cd/mg protein) compared to Cd alone (138.1 ng Cd/mg protein), although this was not quite significant for co-exposures with UV-aged NPs (105.7 ng Cd/mg protein). No toxicity was observed in RTgutGC cells exposed to NMPs alone for 24 h. Moreover, co-exposures with Cd indicated that NMPs reduce the toxicity of Cd. Altogether these results show that UV aging enhances NMP surface reactivity, increasing Cd absorption in solution, which resulted in a reduction in Cd bioavailability and toxicity.

Comparative assessment of microplastics and microalgae as vectors of mercury and chlorpyrifos in the copepod Acartia tonsa.

Microplastics (MPs) raise concerns not only as pollutants themselves, but also due to their ability to act as vectors of pollutants adsorbed from seawater, transferring them to marine organisms. However, the relevance of MPs as carriers of pollutants compared to microalgae needs further exploration. This study compared the role of MPs (2-10 µm non-oxidized and 10-15 µm oxidized high-density polyethylene) and natural organic particles (Rhodomonas lens microalgae, MA) as carriers of mercury (Hg, 2.3 µg Hg/L) and chlorpyrifos (CPF, 1.0 µg CPF/L) to adult Acartia tonsa copepods, after 24-48 h exposure. Dose-response experiments were first performed with adult female copepods exposed to oxidized MPs (0.25-4.0 mg/L), waterborne Hg (0.01-10.0 µg/L) and Ox MPs + Hg (0.25-4.0 mg oxidized MPs/L + 0.50-8.0 µg Hg/L) for 48 h, to complement previous studies that focused on the pesticide CPF. Effects were evaluated with four replicates for physiological and reproductive responses (6 females/replicate), biochemical techniques (40 individuals/replicate) and Hg/CPF bioaccumulation measurements (1000 individuals/replicate). Copepods accumulated Hg/CPF similarly from dissolved pollutants (6204 ± 2265 ng Hg/g and 1251 ± 646 ng CPF/g) and loaded MPs (3125 ± 1389 ng Hg/g and 1156 ± 266 ng CPF/g), but significantly less from loaded MA (21 ± 8 ng Hg/g and 173 ± 80 ng CPF/g). After 24-48 h, copepods exposed to MPs + Hg/CPF showed generally greater biological effects than those exposed to dissolved Hg/CPF or to MA + Hg/CPF, although differences were not statistically significant. MA + CPF had significantly lower AChE inhibition (1073.4 nmol min-1 mg-1) and MA + Hg lower GRx induction (48.8 nmol min-1 mg-1) compared to MPs + Hg/CPF and dissolved Hg/CPF (182.8-236.4 nmol min-1 mg-1 of AChE and 74.1-101.7 nmol min-1 mg-1 of GRx). Principal component analysis suggested different modes of action for Hg and CPF.

A recipe for plastic: Expert insights on plastic additives in the marine environment.

The production and consumption of plastic products had been steadily increasing over the years, leading to more plastic waste entering the environment. Plastic pollution is ubiquitous and comes in many types and forms. To enhance or modify their properties, chemical additives are added to plastic items during manufacturing. The presence and leakage of these additives, from managed and mismanaged plastic waste, into the environment are of growing concern. In this study, we gauged, via an online questionnaire, expert knowledge on the use, characteristics, monitoring and risks of plastic additives to the marine environment. We analysed the survey results against actual data to identify and prioritise risks and gaps. Participants also highlighted key factors for future consideration, including gaining a deeper understanding of the use and types of plastic additives, how they leach throughout the entire lifecycle, their toxicity, and the safety of alternative options. More extensive chemical regulation and an evaluation of the essentiality of their use should also be considered.

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.

Global climate change increases the impact of pollutant mixtures in the model species Paracentrotus lividus.

The goal of the present work is to study whether ocean- acidification (OA) and -warming (OW) could increase the toxicity of pollutants on P. lividus. We studied how model pollutants such as chlorpyrifos (CPF) and microplastics (MP), alone or in combination, impact the fertilisation process, and the development of larvae under conditions of OA (dissolved inorganic carbon increase of 126 × 10-6 mol per kg of sea water) and OW (temperature increase of 4 °C) predicted by FAO (Food and Agriculture Organization) for the next 50 years. Fertilisation was determined by microscopic examination after 1 h. Growth, morphology, and alteration level were measured after 48 h of incubation. Results showed that CPF has a marked effect on the growth of larvae, but less on the fertilisation rate. When larvae are exposed to both MP and CPF, the effect on fertilisation and growth is higher than when CPF is added alone. Larvae exposed to CPF tend to adopt a rounded shape which is detrimental to their buoyancy and the combination with other stressors aggravate this situation. The variables most influenced by CPF or its mixtures are those related to body length, body width, and higher levels of body abnormalities, which is consistent with the degenerative effects caused by CPF on sea urchin larvae. The PCA analysis showed that temperature has more influence when embryos or larvae are exposed to a combination of stressors, demonstrating that global climate change drastically increase the impact of CPF on aquatic ecosystems. Overall, in this work we demonstrated that global climate change conditions increase the sensitivity of embryos to MP and CPF. Our findings support the idea that global change conditions could have a severe impact on marine life, increasing the negative effect of toxic agents commonly present in the sea and their mixtures.

Influence of microplastics on the toxicity of chlorpyrifos and mercury on the marine microalgae Rhodomonas lens.

The growing use of plastics, including microplastics (MPs), has enhanced their potential release into aquatic environments, where microalgae represent the basis of food webs. Due to their physicochemical properties, MPs may act as carriers of organic and inorganic pollutants. The present study aimed to determine the toxicity of polyethylene MPs (plain and oxidized) and the model pollutants chlorpyrifos (CPF) and mercury (Hg) on the red microalgae Rhodomonas lens, to contribute to the understanding of the effects of MPs and associated pollutants on marine ecosystems, including the role of MPs as vectors of potentially harmful pollutants to marine food webs. R. lens cultures were exposed to MPs (1-1000 µg/L; 25-24,750 particles/mL), CPF (1-4900 µg/L), Hg (1-500 µg/L), and to CPF- and Hg-loaded MPs, for 96 h. Average specific growth rate (ASGR, day-1), cellular viability and pigment concentration (chlorophyll a, c2 and carotenoids) were measured at 48 and 96 h. No significant effects were observed on the growth pattern of the microalgae after 96-h exposure to plain and oxidized MPs. However, a significant increase in cell concentration was detected after 48-h exposure to plain MPs. A decrease of the ASGR was noticed after exposure to CPF, Hg and to CPF/Hg-loaded MPs, whereas viability was affected by exposure to MPs, CPF and Hg, alone and in combination. Chlorophyll a and c2 significantly decreased when microalgae were exposed to plain MPs and CPF, while both pigments significantly increased when exposed to CPF-loaded MPs. Similarly, chlorophyll and carotenoids content significantly decreased after exposure to Hg, whereas a significant increase in chlorophyll a was observed after 48-h exposure to Hg-loaded MPs, at the higher tested concentration. Overall, the presence of MPs modulates the toxicity of Hg and CPF to these microalgae, decreasing the toxic effects on R. lens, probably due to a lower bioavailability of the contaminants.

Microplastics and copper induce apoptosis, alter neurocircuits, and cause behavioral changes in zebrafish (Danio rerio) brain.

The knowledge regarding the neurological and behavioral toxic effects associated with microplastics (MPs) and heavy metals exposure is still scarce. The present study aimed to evaluate the potential chronic (30 days) toxic effects of MPs (2 mg/L) and copper (Cu, 25 µg/L), alone or combined, in the zebrafish (Danio rerio) brain antioxidant system, cell proliferation/death, cholinergic-, serotonergic- and dopaminergic pathways and, consequently, in locomotor, anxiety, and social behaviors. Our findings showed that MPs and Cu exposure modulated the antioxidant system of zebrafish brain, with superoxide dismutase (SOD) and glutathione reductase (GR) having higher activity in the Cu25 +MPs group, but glutathione peroxidase (GPx) being inhibited in MPs, Cu25 and Cu25 +MPs. Moreover, an increase in acetylcholinesterase (AChE) activity was observed in all exposed groups. When considering neurogenesis genes, a downregulation of proliferating cell nuclear antigen (pcna) was noticed in zebrafish exposed to the mixture treatment, while for dopaminergic system-related genes (th and slc6a3) an upregulation was observed in MPs, Cu25 and Cu25 +MPs groups. An increase in apoptosis-related genes expression (casp8, casp9 and casp3) was observed in the MPs exposed group. Changes in zebrafish behavior, particularly in mean speed, total distance moved, inactivity in the aquaria, and social/shoaling behavior was also observed in the MPs and Cu exposed groups. Overall, our results highlight the multiplicity of toxic effects of MPs, alone or combined with Cu, in zebrafish brain, namely apoptosis and alterations in adult neurogenesis, neurocircuits and, consequently, behavior.

Microplastics- and copper-induced changes in neurogenesis and DNA methyltransferases in the early life stages of zebrafish.

In this study, zebrafish embryos were exposed to microplastics (MPs, 2 mg/L) and copper (Cu, 60 and 125 µg/L), alone or combined, for 14 days, and the development of motor neurons was assessed through gene expression and immunohistochemistry. DNA methyltransferases (DNMTs) genes expression was also evaluated. The results showed a downregulation of neuronal proliferation (sox2, pcna), neurogenesis (neuroD, olig2), and motor neurons development (islet) related genes, implying potential deficits in the neurogenesis of the exposed zebrafish early life stages. Downregulation of the maintenance and de novo DNMTs expression was also found, indicating that the DNA methylation patterns could be modulated by MPs and Cu. A high relative volume of proliferating cell nuclear antigen (PCNA)-positive cells was found in the fish retina from the MPs exposed group, suggesting that MPs increased the rate of cellular division. In contrast, a significant decrease of PCNA-positive cells, and therefore a lower cell proliferation, was found in the retina and brain of zebrafish exposed to Cu and Cu + MPs, which could lead to cognitive and behavioral functions impairment. No alterations were found in the relative volume of ISL1&2-positive cells. This study contributes to the knowledge of the mechanisms by which MPs and Cu cause neurotoxicity, fundamental for a comprehensive and realistic ecological risk assessment in aquatic populations.

Oxidative stress, apoptosis and serotonergic system changes in zebrafish (Danio rerio) gills after long-term exposure to microplastics and copper.

Fish gills are in direct contact with the surrounding pollutants, and thus, potentially more vulnerable to microplastics (MPs) and heavy metals. The present study aimed to evaluate the long-term exposure effects of MPs and copper (Cu) in the gills of adult zebrafish (Danio rerio). To this end, zebrafish were exposed to MPs (2 mg/L), Cu (Cu25, 25 µg/L) and their mixture (Cu25 + MPs) for 30 days, and then oxidative stress, detoxification, antioxidant, metabolic and neurotoxicity enzymes/genes, as well serotonergic system and apoptosis genes, were evaluated in gills. In the mixture group, ROS levels were increased, while CAT and GPx activities were inhibited, indicating the induction of oxidative stress in zebrafish gills. This was followed by an increase of LPO levels and potential oxidative damage in zebrafish gills. The tryptophan hydroxylase 1a (tph1a) and caspase-3 (casp3) genes were significantly upregulated in Cu25 + MPs group, indicating a potential dysregulation of serotonin synthesis and apoptosis pathways, respectively. Overall, the present study contributes to improving the knowledge about the response of aquatic organisms to MPs and the potential ecological risk that these particles represent to the ecosystems.

Toxicity of microplastics and copper, alone or combined, in blackspot seabream (Pagellus bogaraveo) larvae.

Plastics pose serious risks for fish productivity and a potential constraint for food security. Newly hatched blackspot seabream larvae were exposed to microplastics (MPs), copper (Cu, 10-810 µg/L) and their mixtures (Cu+MPs), during 3 and 9 days. Biochemical biomarkers and the expression of antioxidant and neurotoxicity-related genes were evaluated. In the 3-day exposure, catalase and glutathione-S-transferase activities decreased in MPs, Cu and Cu+MPs groups, followed by an increase of lipid peroxidation in the Cu270 and Cu270 +MPs exposed larvae. In the 9-day exposure, ROS levels increased in MPs and Cu30 groups, but no significant oxidative damage was observed, suggesting that the antioxidant system overcome the induced oxidative stress. However, the acetylcholinesterase transcript was downregulated in MPs, Cu and Cu10+MPs groups, indicating that MPs effects in cholinergic neurotransmission may arise after longer exposures. Overall, MPs and Cu can reduce survival, induce oxidative stress, lipid peroxidation, neurotoxicity, and impact negatively fish larvae fitness.

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.

Comparative role of microplastics and microalgae as vectors for chlorpyrifos bioacumulation and related physiological and immune effects in mussels.

Microplastics (MP) are contaminants of concern per se, and also by their capacity to sorb dissolved chemicals from seawater, acting as vehicles for their transfer into marine organisms. Still, the role of MP as vehicles for contaminants and their associated toxicological effects have been poorly investigated. In this work we have compared the role of MP (high density polyethylene, HDPE, ≤22 µm) and of natural organic particles (microalgae, MA) as vehicle for chlorpyrifos (CPF), one of the most common pesticides found in river and coastal waters. We have compared the capacity of MP and MA to carry CPF. Then, the mussel Mytilus galloprovincialis has been exposed for 21 days to dissolved CPF, and to the same amount of CPF loaded onto MP and MA. The concentration of CPF in mussel' tissues and several physiological, energetics and immune parameters have been analyzed after 7 and 21 days of exposure. Results showed similar CPF accumulation in mussel exposed to MP and to MA spiked with CPF. This revealed that MP acted as vector for CPF in a similar way (or even to a lesser extent) than MA. After 21 days of exposure mussels exposed to MP spiked with CPF displayed similar or more pronounced biological effects than mussels exposed to dissolved CPF or to MA loaded with CPF. This suggested that the combined "particle" and "organic contaminant" effect produced an alteration on the biological responses greater than that produced by each stressor alone, although this was evident only after 3 weeks of exposure.

First evidence of ingestion and retention of microplastics in seahorses (Hippocampus reidi) using copepods (Acartia tonsa) as transfer vectors.

Microplastics (MPs) are a major concern for marine ecosystems since they can be ingested by a wide range of marine species and transmitted through the food web. However, the potential hazardous impact of MPs in fishes, especially in early developing stages, is relatively unknown. In the present study, we assessed for the first time the ingestion and retention of MPs in early developing seahorses Hippocampus reidi. Seahorses are vulnerable species that may also be affected by both the direct ingestion of MPs through their preys and the accidental ingestion of MPs particles present in the water (i.e., seahorses ingest the prey by suction). We used copepods as both preys for seahorse juveniles and transfer vectors of MPs. Fed or starved copepods previously exposed to polyethylene microspheres (1-5 µm in diameter; 10 and 100 µg L-1) for 60 min at 26 °C showed fast evacuation of microspheres. The presence of MPs in copepods was significantly higher in previously fasted copepods compared to fed copepods. Seahorse juveniles fed on copepods pre-exposed to MPs, accumulated MPs in the gut proportionally to the concentration of MPs in copepods. A lower concentration of MPs in seahorses was observed at the longer exposure time (60 min), especially in fish fed with fasted copepods. However, after longer exposure, MPs were mainly accumulated close to the anus both individually or forming aggregates. Further studies should be performed to assess secondary effects of MPs ingestion in seahorses since they are considered a flagship species for marine conservation.

Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure.

Microplastics (MPs, <5 mm) have been frequently detected in aquatic ecosystems, representing both health and ecological concerns. However data about the combined effects of MPs and other contaminants is still limited. This study aimed to evaluate the impact of MPs and the heavy metal copper (Cu) on zebrafish (Danio rerio) larvae development and behavior. Zebrafish embryos were subchronically exposed to MPs (2 mg/L), two sub-lethal concentrations of Cu (60 and 125 µg/L) and binary mixtures of MPs and Cu using the same concentrations, from 2-h post fertilization until 14 days post fertilization. Lethal and sub-lethal responses (mortality, hatching, body length) were evaluated during the embryogenesis period, and locomotor, avoidance, anxiety and shoaling behaviors, and acetylcholinesterase (AChE) activity were measured at 14 dpf. The results showed that survival of larvae was reduced in groups exposed to MPs, Cu and Cu+MPs. Regarding the behavioral patterns, the higher Cu concentration and mixtures decreased significantly the mean speed, the total distance traveled and the absolute turn angle, demonstrating an adverse effect on swimming competence of zebrafish larvae. Exposure to MPs and Cu, alone or combined, also affected avoidance behavior of zebrafish, with larvae not reacting to the aversive stimulus. There was a significant inhibition of AChE activity in larvae exposed to all experimental groups, compared to the control group. Moreover, a higher inhibition of AChE was noticed in larvae exposed to MPs and both Cu+MPs groups, comparatively to the Cu alone groups. Our findings demonstrate the adverse effects of MPs, alone or co-exposed with Cu, on fish early life stages behavior. This study highlights that MPs and heavy metals may have significant impacts on fish population fitness by disrupting locomotor and avoidance behaviors.

Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages.

The evaluation of the interaction between microplastics (MPs) and heavy metals is of special importance for risk assessment. In this study, zebrafish (Danio rerio) were exposed to MPs (2 mg/L), two sub-lethal concentrations of copper (Cu, 60 and 125 µg/L) and their mixtures (Cu60 + MPs, Cu125 + MPs), from 2-h post-fertilization (hpf) until 14-days post-fertilization (dpf). Lethal and sublethal endpoints were evaluated, along with a set of biochemical and genetic biomarkers between 2 and 14 dpf. Exposure to MPs and Cu, single or combined, induced high mortality and oxidative stress in zebrafish larvae, with data showing that the antioxidant enzymes were inhibited at 6 dpf, increasing thereafter until 14 dpf, due to the accumulation of reactive oxygen species. MPs and Cu, single or combined, caused neurotoxicity in larvae by inhibiting acetylcholinesterase activity. There was an increased and significant effect of Cu + MPs groups on the evaluated biomarkers, concerning the corresponding Cu groups, suggesting that MPs may have a synergistic effect in relation to Cu. The Integrated Biomarker Response (IBR) evidenced that a higher degree of stress occurred at the larval period. Our findings highlight that MPs can act as a vector for heavy metals, therefore, influencing their bioavailability and toxicity in the organisms.

Toxicological effects induced on early life stages of zebrafish (Danio rerio) after an acute exposure to microplastics alone or co-exposed with copper.

Data about the toxicological interactions of MPs and heavy metals in biota is limited, particularly in fish early life stages. This study aimed to evaluate the toxicological effects of MPs and copper (Cu), alone or combined, in zebrafish early life stages. Embryos were exposed from 2 until 96-h post-fertilization (hpf) to MPs (2 mg/L), three sub-lethal concentrations of Cu (15, 60 and 125 µg/L) and binary mixtures containing Cu and MPs (Cu15+MPs, Cu60+MPs, Cu125+MPs). Lethal and sub-lethal parameters, histopathological changes, biochemical biomarkers, gene expression and behavior were assessed. Our findings showed that Cu and Cu + MPs decreased embryos survival and hatching rate. Increased ROS levels were observed in larvae exposed to the two lowest Cu and Cu + MPs groups, suggesting an induction of oxidative stress. An increased CAT and GPx activities were observed in Cu and Cu + MPs, implying a response of the antioxidant defense system to overcome the metal and MPs stress. The sod1 expression was downregulated in all Cu groups and in the two highest Cu + MPs exposed groups. AChE was significantly inhibited in Cu and Cu + MPs groups, indicating neurotoxicity. A disruption of avoidance and social behaviors were also noticed in the Cu125 and Cu125+MPs exposed larvae. Evidences of Cu-toxicity modulation by MPs were observed in some endpoints. Overall, the findings of this study highlight that Cu alone or co-exposed with MPs lead to oxidative stress, neurotoxicity and ultimately behavioral alterations in early life stages of zebrafish, while MPs alone do not produce significant effects on zebrafish larvae.

Polyethylene microplastics increase the toxicity of chlorpyrifos to the marine copepod Acartia tonsa.

Ingestion of microplastics by marine organisms has been well documented, but their interaction with chemical pollutants has not been sufficiently addressed. The aim of this study was to determine the individual and combined effects of chlorpyrifos (CPF) and polyethylene microplastics (MP) on the survival, fecundity, feeding and egg viability of Acartia tonsa, a calanoid copepod widely distributed in planktonic communities. The median lethal concentration obtained for CPF was higher (LC50 = 1.34 µg/L) than for the combination with MP (LC50 = 0.37 µg/L), or CPF-loaded MP (LC50 = 0.26 µg/L). Significant effects were also observed for feeding and egg production (EC50 = 0.77 and 1.07 µg/L for CPF, 0.03 and 0.05 µg/L for CPF combined with MP, 0.18 and 0.20 µg/L for CPF-loaded MP). No significant effects were observed in the exposure to 'virgin' MP. This study confirms the role of MP as vectors of pollutants to marine organisms and supports the increased availability of certain toxicants carried out by MP. The effects observed in fitness-related responses suggest potential damage to A. tonsa populations. The comparison of the results obtained here with environmental concentrations indicates that the combined exposure to CPF and MP could constitute a risk to A. tonsa in the natural environment.

Metabolomic responses of mussel Mytilus galloprovincialis to fluoranthene exposure under different nutritive conditions.

Biomarkers are useful tools to assess biological effects of pollutants that are extensively used in monitoring programs to assess ecosystem health. However, they are strongly affected by mussel physiological state, especially nutritive status, which has led to the search of new biological indicators of chemical pollutants exposition. Environmental metabolomics is an approach for examining the metabolic responses (measurement of low molecular weight endogenous metabolites) of an organism to both natural and anthropogenic stressors that can occur in its environment. The aim of the present work was to assess the effect of the polycyclic aromatic hydrocarbon fluoranthene (FLU) exposure on the metabolomic profiles of mussel digestive glands under different nutritive conditions. To achieve this objective, mussels were reared, for a period of 56 days, under three different food rations in order to obtain a gradient of nutritive status (negative, zero and positive energy balance), and after that, they were exposed, during 3 weeks, to a nominal concentration of 3 µg FLU L-1. A total of 43 metabolites, including aminoacids (Ala, Val, Leu, Ile, etc.), energy metabolism related metabolites (ATP, AMP, etc.), organic osmolytes (taurine, etc.), redox metabolism (GSH, NADP+) and nucleotides, were identified and quantified in the digestive glands of the mussels. Principal Component Analysis (PCA) defined two principal components (PC1 and PC2) that explained 55.6% of the total variance, although the first component explains more than 80% of this variance, this being related to the mussel nutritive condition. The effect of the toxicant, explained by the PC2, is similar to that produced under conditions of food restriction, which masks the effect of the toxicant under these conditions. As the feeding conditions are more favorable, the toxic effect becomes more apparent. Therefore, the great influence of nutritive condition on mussel metabolome implies a handicap for the use of metabolomic biomarkers, as previously demonstrated for biochemical and other molecular biomarkers, in large-scale monitoring programs in which several food conditions coexist with pollution levels.

Extended imposex monitoring in N Atlantic Spain confirms punctual attainment of European environmental objectives for TBT.

Legislation in the European Union (EU) aimed at reaching by 2015 a Good Ecological Status in regard to tributyltin (TBT, the biocide used in traditional antifouling paints). With a view to check such an achievement in N Atlantic Spain, baseline monitoring of gastropod imposex (the recommended assessment tool) was extended up to that date. In Galicia (the Western part of the study area) the use of the rock snail Nucella lapillus since 1996 had shown this environmental objective to be met as soon as 2009, but new surveys reveal no further improvement thereafter. As for the Eastern Cantabrian coast, imposex levels in the mud snail Nassarius reticulatus progressively declined from 2006 to 2015, when records finally complied with expectations. Both data sets are confronted and discussed in relation to the diverse environmental factors that may be determining the distribution of gastropods in these regions.

Effect of mussel reproductive status on biomarker responses to PAHs: Implications for large-scale monitoring programs.

Biomarkers are useful tools to assess biological effects of pollutants and have been extensively used in monitoring programs to determine ecosystem health. In these programs, a wide range of environmental conditions are covered and sometimes, obtained data are difficult to interpret because of natural variables are affecting biomarker responses. Among these variables, mussels reproductive status has been considered one of the most changing variables between sites in a monitoring survey. Thus, the main aim of this work was to identify the effect that mussel reproductive status has on biomarker responses. For that purpose, mussels sampled at two periods in the reproductive cycle (reproductive and resting stages) were conditioned to the same laboratory conditions and exposed to fluoranthene (FLU) for three weeks. Studied biomarkers covering a wide range of organism responses were included: bioaccumulation, physiological rates (clearance rate -CR-, absorption efficiency -AE-, respiration rate -RR- and their integration in the scope for growth -SFG-), antioxidant enzyme activities (superoxide-dismutase -SOD-, catalase -CAT-, glutathione reductase -GR-, glutathione peroxidase -GPx-, glutathione-S-tranferase -GST-) and biochemical damage responses (lipid membrane peroxidation -LPO-). The results obtained evidenced that the levels of the biomarkers studied (RR, SOD, CAT and GPx) were higher at reproductive than at resting stage. On the other hand, the effect of toxicant was observed in SFG, CAT and GPx but this effect was only detected during the resting period. Moreover, there was a deterioration of mussel gonadal tissue with FLU exposure during reproductive stage. FLU accumulation in mussel tissues was also dependent of the reproductive status with higher internal concentrations during resting than reproductive period. In conclusion, there was a strong effect of reproductive status on studied biomarkers which seems to mask the effect of FLU at reproductive stage. The present study evidences the need to include the measurement of mussel biological status in marine pollution monitoring programs for a correct interpretation of biomarker data.