Karbonoan oinarritutako nanomaterialen eta mikro eta nanoplastikoen eragina kutsatzaile organiko iraunkorren garraiatzaile gisa organismo itsastarretan

The development of industry and technology has led to the synthesis of new materials with unique physico-chemical properties, among them, nanomaterials are becoming abundant. Nanomaterials are materials with at least one dimension at the nanoscale ('100 nm). At present, there is no specific regulation for nanomaterials in European law, although their physico-chemical properties are different in comparison to their bulk counterparts. Microplastics are plastic materials smaller than 5 mm while nanoplastics are smaller than 100 nm. Whilst the impacts of macroplastics are well studied, it was only in recent years that interest developed in understanding the effects of micro and nanoplastics. Nanomaterials, micro and nanoplastics originate from different sources and reach the sea. As a result, their effects on marine biota represent a major concern. Due to the high surface/volume ratio and hydrophobicity of nanomaterials, micro and nanoplastics, they pose an additional risk: they can adsorb persistent organic pollutants and act as their carriers, increasing bioavailability of these compounds to marine organisms. The purpose of this work is to review the potential effects caused by carbon based nanomaterials, micro and nanoplastics in different marine organisms at molecular, cellular and physiological levels and to compare these effects to those caused by nanomaterials, micro and nanoplastics with adsorbed persistent organic pollutants. For example, after feeding mussels with a diet containing microplastics, these have been found in gut lumen, digestive epithelium and connective tissue and in mussels fed with microplastics with adsorbed benzo(a)pyrene, benzo(a)pyrene, was accumulated in mussel tissues. Microplastics alone and with adsorbed persistent organic pollutants produce inmunotoxic, genotoxic and deleterious physiological effects. Thus, since pollutans do not appear isolated in the complex marine environment, this knowledge should be taken into account to ensure the health and safety of marine ecosystems.; Industriaren eta teknologiaren garapenak ezaugarri fisiko-kimiko bereziak dituzten material berriak sortzea eragin du, eta, horien artean, nanomaterialak gero eta ugariagoak dira. Nanomaterialak gutxienez nanoeskaladun ('100 nm) dimentsio bat duten materialak dira. Gaur egun, Europako legedian ez dago nanomaterialentzako araudi berezirik, nahiz eta, haien homologo makroskopikoekin alderatuta, ezaugarri fisiko-kimiko ezberdinak agertu. Mikroplastikoak 5 mm baino txikiagoak diren plastikozko materialak dira eta nanoplastikoak, 100 nm baino txikiagoak direnak. Plastiko makroskopikoen eraginak zabal ikertu diren arren, mikroplastiko eta nanoplastikoen inguruko hausnarketa duela gutxi hasi da egiten. Nanomaterialak, mikro eta nanoplastikoak, iturri desberdinetatik iristen dira itsasora eta, beraz, itsas organismoetan eragin ditzaketen kalteen inguruko kezka zabaldu da. Gainera, nanomaterialek, mikroplastikoek eta nanoplastikoek duten azalera/bolumen erlazio handia eta hidrofobizitatea dela eta, arrisku gehigarri bat dute: kutsatzaile organiko iraunkorren garraiatzaile gisa joka dezakete itsasoan. Hau da, nanomaterialek, mikroplastikoek eta nanoplastikoek, itsasoan dauden kutsatzaile organiko iraunkorrak adsorbatu ditzakete eta haien eskuragarritasuna emendatu organismo itsastarrentzat. Lan honen xedea da karbonoan oinarritutako nanomaterialek eta mikro eta nanoplastikoek hainbat espezie itsastarretan eragindako kalte fisiologikoak zein zelula eta molekula mailako kalteak berrikustea. Kalte horiek, kutsatzaile organiko iraunkordun karbonoan oinarritutako nanomaterialek, mikro eta nanoplastikoek, eragindako kalteekin alderatu ere egin nahi dira. Adibidez, mikroplastikodun mikroalgekin elikatutako muskuiluek mikroplastikoak agertu dituzte urdailaren argian, digestio epitelioan eta ehun konektiboan, eta bentzo(a)pirenoa moduko kutsatzaile organiko iraunkorra adsorbatuta duten mikroplastikodun mikroalgekin elikatutako muskuiluek bentzo(a)pirenoa metatzen dute ehunetan. Mikroplastikoek bakarrik zein kutsatzaile organikodun mikroplastikoek immunotoxizitatea, kalte fisiologikoak eta DNA mailako kalteak eragiten dizkiete muskuiluei. Hortaz, ingurune konplexu batean, hala nola itsasoan, kutsatzaileak ez direnez modu isolatuan agertzen, ezagutza hori kontuan hartu beharko litzateke, itsas ekosistemen osasuna eta segurtasuna bermatzeko

Polystyrene nanoplastics and microplastics can act as Trojan horse carriers of benzo(a)pyrene to mussel hemocytes in vitro

[EN]In this work we studied the ability of polystyrene (PS) nanoplastics (NPs) and microplastics (MPs) to transfer benzo(a)pyrene (BaP) to mussel hemocytes and to produce toxic effects in vitro. For this, intracellular fate and toxicity of PS NPs (0.05 μm) and MPs (0.5 and 4.5 μm) alone or with BaP and of BaP alone were assessed. Particles of 0.05 and 0.5 µm largely aggregated in the exposure medium whereas presence of BaP reduced particle aggregation. Cells internalized PS NPs and MPs alone or with BaP and these were found inside and outside lysosomes, depending on their size. PS particles alone or with BaP were cytotoxic to hemocytes only at the highest concentrations tested. The same was true for most sublethal endpoints except for increased phagocytic activity provoked by NPs and 0.5 μm MPs at lower concentrations. Plastic particles appeared to be the main drivers for reduced plasma membrane integrity and increased phagocytic and lysosomal activities whereas BaP appeared to contribute more to reduced cell viability and phagocytosis and increased ROS production and genotoxicity. Overall, PS NPs and MPs can act as carriers of BaP to mussel hemocytes, rising concerns about risks plastics associated to pollutants may pose to aquatic organisms.This work was carried out within the EU Project PLASTOX (JPI Oceans 005/2015) and was funded by the Spanish MINECO (NACE Project, CTM2016-81130-R) and Basque Government (Consolidated Research Groups Grant IT1302-19)

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

[ENG]Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programsWork granted by MIUR and Co.Ri.La. to PV. LV is currently supported by the FP7-KBBE-2010-4-266157 Bivalife project. Work in the laboratory of MPC is funded by grants to consolidated research groups (ref GIC07/26-IT-393-07, Basque Government), and to the unit of formation and research (UFI ref 11/37, University of the Basque Country) and projects Nanoretox (ref CP-FP 214478-2, UE 7th FP) and Nanocancer (ref CTM2009-13477, Spanish Ministry of Science and Innovation

Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50–60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol–gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230–260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.This research was funded by the Basque Government, grant numbers IT912-16 and IT-1302-19; Ministry of Economy and Competitiveness (MINECO), grant numbers MAT2017-83856-C3-3-P and CTM2016-81130-R; and the University of the Basque Country (UPV/EHU), grant number COLAB19/01

Effects of microplastics alone or with sorbed oil compounds from the water accommodated fraction of a North Sea crude oil on marine mussels (Mytilus galloprovincialis)

Microplastics (MPs) can adsorb persistent organic pollutants such as oil hydrocarbons and may facilitate their transfer to organisms (Trojan horse effect). The aim of this study was to examine the effects of a 21 day dietary exposure to polystyrene MPs of 4.5 mu m at 1000 particles/mL, alone and with sorbed oil compounds from the water accommodated fraction (WAF) of a naphthenic North Sea crude oil at two dilutions (25 % and 100 %), on marine mussels. An addi-tional group of mussels was exposed to 25 % WAF for comparison. PAHs were accumulated in mussels exposed to WAF but not in those exposed to MPs with sorbed oil compounds from WAF (MPs-WAF), partly due to the low concen-tration of PAHs in the studied crude oil. Exposure to MPs or to WAF alone altered the activity of enzymes involved in aerobic (isocitrate dehydrogenase) and biotransformation metabolism (glutathione S-transferase). Prevalence of oo-cyte atresia and volume density of basophilic cells were higher and absorption efficiency lower in mussels exposed to MPs and to WAF than in controls. After 21 days MPs caused DNA damage (Comet assay) in mussel hemocytes. In conclusion, a Trojan horse effect was not observed but both MPs and oil WAF caused an array of deleterious effects on marine mussels at different levels of biological organization.This work was funded by Spanish MINECO (NACE project CTM2016-81130-R), Basque Government (consolidated group IT1302-19 and IT1743-22 and predoctoral fellowship to NGS) and UPV/EHU (VRI grant PLASTOX). Work carried out within the EU project PLASTOX (JPI Oceans 005/2015) and the EU H2020-BG-2005-2 project GRACE (grant agreement number 679266)

Cell and tissue level responses in mussels Mytilus galloprovincialis dietarily exposed to PVP/PEI coated Ag nanoparticles at two seasons

Silver nanoparticles (Ag NPs) are present in numerous consumer products due to their antimicrobial and other unique properties, thus concerns about their potential input into aquatic ecosystems are increasing. Toxicity of Ag NPs in waterborne exposed aquatic organisms has been widely investigated, but studies assessing the potential toxic effects caused after ingestion through the food web, especially at low realistic concentrations, remain scarce. Moreover, it is not well known whether season may influence toxic effects of Ag NPs. The main objective of this study was to determine cell and tissue level responses in mussels Mytilus galloprovincialis dietarily exposed to poly-N-vinyl-2-pirrolidone/polyethyleneimine (PVP/PEI) coated 5 nm Ag NPs for 1, 7 and 21 days both in autumn and spring. Mussels were fed every day with microalgae Isochrysis galbana exposed for 24 h to a low dose (1 mu g Ag/L Ag NPs) in spring and to a higher dose (10 mu g Ag/L Ag NPs) in spring and autumn. Mussels fed with microalgae exposed to the high dose accumulated Ag significantly after 21 days in both seasons, higher levels being measured in autumn compared to spring. Intralysosomal metal accumulation measured in mussel digestive gland and time- and dose-dependent reduction of mussels health status was similar in both seasons. DNA strand breaks increased significantly in hemocytes at both exposure doses along the 21 days in spring and micronuclei frequency showed an increasing trend after 1 and 7 days of exposure to 1 mu g Ag/L Ag NPs in spring and to 10 mu g Ag/L in both seasons. Values decreased after 21 days of exposure in all the cases. In conclusion, PVP/PEI coated 5 nm Ag NPs ingested through the food web were significantly accumulated in mussel tissues and caused adverse cell and tissue level effects both in autumn and in spring.This work has been funded by the Spanish Ministry of Economy and Competitiveness (Nano Silver Omics project MAT2012-39372), Basque Government (SAIOTEK project S-PE13UN142 and Consolidated Research Group GIC IT810-13 and IT1302-19), the University of the Basque Country UPV/EHU (UFI 11/37 and PhD fellowship to N.D.) and French Ministry of Higher Education and Research (PhD fellowship to M.M.)

Fate and effects of graphene oxide alone and with sorbed benzo(a)pyrene in mussels Mytilus galloprovincialis

Graphene oxide (GO) has gained a great scientific and economic interest due to its unique properties. As incorporation of GO in consumer products is rising, it is expected that GO will end up in oceans. Due to its high surface to volume ratio, GO can adsorb persistent organic pollutants (POPs), such as benzo(a)pyrene (BaP), and act as carrier of POPs, increasing their bioavailability to marine organisms. Thus, uptake and effects of GO in marine biota represent a major concern. This work aimed to assess the potential hazards of GO, alone or with sorbed BaP (GO+BaP), and BaP alone in marine mussels after 7 days of exposure. GO was detected through Raman spectroscopy in the lumen of the digestive tract and in feces of mussels exposed to GO and GO+BaP while BaP was bioaccumulated in mussels exposed to GO+BaP, but especially in those exposed to BaP. Overall, GO acted as a carrier of BaP to mussels but GO appeared to protect mussels towards BaP accumulation. Some effects observed in mussels exposed to GO+BaP were due to BaP carried onto GO nanoplatelets. Enhanced toxicity of GO+BaP with respect to GO and/or BaP or to controls were identified for other biological responses, demonstrating the complexity of interactions between GO and BaP.This work was funded by the Spanish MINECO (NACE project CTM2016–81130-R) and the Basque Government (grants to consolidated research group IT1302–19 and IT1743–22, and predoctoral fellowship to NGS)

Screening of the Toxicity of Polystyrene Nano- and Microplastics Alone and in Combination with Benzo(a)pyrene in Brine Shrimp Larvae and Zebrafish Embryos

The occurrence of nanoplastics (NPs) and microplastics (MPs) in aquatic ecosystems and their capacity to sorb hydrophobic pollutants is nowadays an issue of great concern. This study aimed to assess the potential bioavailability and acute toxicity of polystyrene (PS) NPs (50 and 500 nm) and of MPs (4.5 µm), alone and with sorbed benzo(a)pyrene (B(a)P), in the embryo/larval stages of brine shrimps and zebrafish. Exposure to pristine plastics up to 50.1 mg PS/L did not cause significant impact on brine shrimp survival, while some treatments of plastics-B(a)P and all concentrations of B(a)P (0.1–10 mg/L) resulted acutely toxic. In zebrafish, only the highest concentrations of MPs-B(a)P and B(a)P caused a significant increase of malformation prevalence. Ingestion of NPs was observed by 24–48 h of exposure in the two organisms (from 0.069 to 6.87 mg PS/L). In brine shrimps, NPs were observed over the body surface and within the digestive tract, associated with feces. In zebrafish, NPs were localized in the eyes, yolk sac, and tail at 72 h, showing their capacity to translocate and spread into the embryo. MP ingestion was only demonstrated for brine shrimps. In zebrafish embryos exposed to plastics-B(a)P, B(a)P appeared in the yolk sac of the embryos. The presence of B(a)P was also noticeable in brine shrimps exposed to 500 nm NPs-B(a)P. In conclusion, NPs entered and spread into the zebrafish embryo and PS NPs, and MPs were successful vectors of B(a)P to brine shrimp and zebrafish embryos. Particle size played a significant role in explaining the toxicity of plastics–B(a)P. Our study provides support for the idea that plastics may pose a risk to aquatic organisms when combined with persistent organic pollutants such as B(a)P.This work was funded by UPV/EHU (predoctoral grant to IMA), Basque Government (consolidated research group IT810-13 and IT1302-19), Spanish MINECO project NACE (CTM2016-81130-R), French ANR (No.–10–IDEX-03-02 and Cluster of Excellence COTE (ANR-10-LABX 45). This work was performed within the framework of the Centre for Advanced Studies (CAS) project “H2020 CAS6 Nanoplastics” funded by the European Commission- Joint Research Centre (JRC/A/05)

Mugilid Fish Are Sentinels of Exposure to Endocrine Disrupting Compounds in Coastal and Estuarine Environments

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.This work has been funded through research projects of the Spanish MINECO (SEXOVUM AGL2012-33477), Basque Government (SAIOTEK OVUM-II S-PE12UN086 and Consolidated research groups IT-810-13) and UPV/EHU (UFI 11/37). IRB is recipient of a predoctoral grant of the Basque Government while CB is recipient of a grant of UPV/EHU

Uptake and Effects of Graphene Oxide Nanomaterials Alone and in Combination with Polycyclic Aromatic Hydrocarbons in Zebrafish

Because of its surface characteristics, once in the aquatic environment, graphene could act as a carrier of pollutants, such as polycyclic aromatic hydrocarbons (PAHs), to aquatic organisms. In this study we aimed to (1) assess the capacity of graphene oxide (GO) to sorb PAHs and (2) to evaluate the toxicity of GO alone and in combination with PAHs on zebrafish embryos and adults. GO showed a high sorption capacity for benzo(a)pyrene (B(a)P) (98% of B(a)P sorbed from a nominal concentration of 100 mu g/L) and for other PAHs of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil, depending on their log Kow (95.7% of phenanthrene, 84.4% of fluorene and 51.5% of acenaphthene). In embryos exposed to different GO nanomaterials alone and with PAHs, no significant mortality was recorded for any treatment. Nevertheless, malformation rate increased significantly in embryos exposed to the highest concentrations (5 or 10 mg/L) of GO and reduced GO (rGO) alone and with sorbed B(a)P (GO-B(a)P). On the other hand, adults were exposed for 21 days to 2 mg/L of GO, GO-B(a)P and GO co-exposed with WAF (GO + WAF) and to 100 mu g/L B(a)P. Fish exposed to GO presented GO in the intestine lumen and liver vacuolisation. Transcription level of genes related to cell cycle regulation and oxidative stress was not altered, but the slight up-regulation of cyp1a measured in fish exposed to B(a)P for 3 days resulted in a significantly increased EROD activity. Fish exposed to GO-B(a)P and to B(a)P for 3 days and to GO + WAF for 21 days showed significantly higher catalase activity in the gills than control fish. Significantly lower acetylcholinesterase activity, indicating neurotoxic effects, was also observed in all fish treated for 21 days. Results demonstrated the capacity of GO to carry PAHs and to exert sublethal effects in zebrafish.This work has been funded by University of the Basque Country (predoctoral grant to IMA PIFBUR15/15), Basque Government (consolidated research group IT810-13 and IT1302-19), Spanish Ministry of Economy and Competitiveness project NACE (CTM2016-81130-R), French National Research Agency (No.-10-IDEX-03-02) and Cluster of Excellence Continental To coastal Ecosystems-COTE (ANR-10-LABX 45). Thanks to staff at Driftslaboratoriet Mongstad, Equinor (former Statoil) for supplying the sample of crude oil used in the experiments. The authors thank for technical and human support provided by SGIker (UPV/EHU/ERDF, EU)