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Metabolic responses of Mytilus galloprovincialis to fullerenes in mesocosms exposure experiments Josep Sanchís, Marta Llorca, Mar Olmos, Gabriella Francesca Schirinzi, Cristina Bosch-Orea, Esteban Abad, Damia Barcelo, and Marinella Farre Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04089 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017
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Environmental Science & Technology
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Metabolic responses of Mytilus galloprovincialis to fullerenes in
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mesocosms exposure experiments
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Josep Sanchís1, Marta Llorca1, Mar Olmos1, Gabriella F. Schirinzi1, Cristina Bosch-Orea1,
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Esteban Abad1, Damià Barceló1,2, Marinella Farré1*
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Institute of Environmental Assessment and Water Research (IDAEA-CSIC), C/Jordi Girona, 18-26, 08034, Barcelona, Catalonia, Spain.
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Catalan Institute of Water Research (ICRA), C/ Emili Grahit, 101, 17003, Girona, Catalonia, Spain.
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In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed through the
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diet to fullerene soot, at three concentrations in parallel to a control group. Their
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metabolomics response was assessed by high-performance liquid chromatography coupled to
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high-resolution mass spectrometry (HPLC-HRMS). The experiments were conducted in
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marine mesocosms, during 35 days (7 days of acclimatization, 21 days of exposure and 7
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seven days of depuration). Real conditions were emulated in terms of physicochemical
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conditions of the habitat. Results confirmed the bioaccumulation of fullerenes, and the
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metabolome of the exposed organisms revealed significant differences in the concentrations
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of seven free amino acids when compared to the control group. An increase in small non-
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polar amino acids (e.g. alanine) and branched chain amino acids (leucine and isoleucine) were
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observed. Also, glutamine concentrations decreased significantly, suggesting the activation of
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facultative anaerobic energy metabolism. Branched chain amino acids, such as leucine and
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isoleucine, followed the opposite trend after the highest level of exposure, which can imply
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hormesis effects. Other significant differences were observed on lipids content, such as the
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general increase of free fatty acids, i.e. long chain fatty acids (lauric, myristic and palmitic
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acids) when increasing the concentration of exposure. These results were consistent with
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hypoxia and oxidative stress.
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Introduction
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Fullerenes are a class of carbon allotropes consisting of hollow polyhedron molecules
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composed of carbon atoms. Due to their unique chemical and physical properties, they have
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been the object of study since the discovery of C60 fullerene1.
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Energetic processes such as forest fires and volcanic eruptions can naturally emit fullerenes.
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However, currently, secondary emission from industrial combustion processes, diesel
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exhausts, and plane brakes seem to be primary environmental sources. Besides, during the last
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years, the residues of the nanotechnology industry should also be considered. The possible
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derivatization of the native structures has opened a new window to launching new materials.
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Pristine and functionalized fullerenes are currently used in solar cells2, 3, nanomedicine4,
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cosmetics5, 6 and microelectronics7. Therefore, an increase of the releases of fullerenes to the
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environment is expected, while their ecotoxicological potential remains unclear.
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Their small size and high specific surface areas enhance their potential to cross cell
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membranes. Moreover, aggregates may interact with proteins and nucleic acids, consequently
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disrupting vital processes such as enzyme functions and gene transcription/translation8, 9. On
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the other hand, due to their high chemical sorption potential, they could act as carriers for
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other contaminants, providing rapid and long-range transport10, 11, or just immobilizing them
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and enhancing their precipitation.
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The development of sensitive analytical methods has permitted to confirm the presence of
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fullerenes in different environmental compartments such as atmospheric aerosols12, 15, 16
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,
17-20
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soils
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presumed to be major sinks for nanomaterials (NMs) and several non-volatile anthropogenic
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contaminants. Therefore, their potential damages and transference to the food chain should be
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evaluated.
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The toxicological data and the measured environmental concentrations indicate that fullerenes
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are not acute toxicants at environmentally relevant levels 21, 22. However, the results of several
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studies call for a warning about their possible sub-lethal, long-term effects and the possible
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synergistic/antagonistic effects that they may establish with other contaminants in the same
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compartments
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nano-sized materials are aiming at elucidating the mechanisms of action at sub-lethal doses to
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aquatic organisms such as microcrustaceans (including bioaccumulation, alteration of their
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reproductive cycle and behavioral changes
, waters and sediments
. However, estuaries and coastal environments are
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. The recent trends for toxicological and ecotoxicological evaluations of
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) and to several species of fish (including the
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alteration of their fatty metabolism, oxidative stress and growth inhibition26-28. In this sense,
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metabolomics has great potential to increase our knowledge on ecotoxicology and to assess
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long-term effects at sub-lethal concentrations of exposure.
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Bivalve mollusks as mussels are tolerant to a certain degree of pollution and to environmental
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changes. They are also filter feeders, that can accumulate pollutants found in the water
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column, they are sedentary and they are easy to sample. For these reasons, mussels are widely
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used as sentinel organisms in bio-monitoring studies and, also, in metabolomics experiments
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performed under controlled conditions, being exposed to organic contaminants (as atrazine29,
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lindane29, polybromodiphenyl ethers30 and bisphenol A31), and often supported by analytical
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techniques such as 1H-NMR32-34. In addition, mussels have also been used in metabolomic
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studies performed in real polluted environments 35.
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The first aim of this study was to assess the potential uptake of fullerenes by filter-feeding
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organisms (mussel, Mytilus galloprovincialis) exposed through the diet. And second, we
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carried out high-resolution mass spectrometry (HRMS) based metabolomic studies to
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evaluate
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Chromatography coupled to HRMS instrumentation offers some advantages over other
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analytical methods (such as 1H-NMR) that makes it a powerful tool for metabolomics: for
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instance, isobaric isomers (diasteromers, enantiomers, etc.) can be potentially distinguished
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thanks to the liquid chromatography separation and the unsurpassed selectivity of HRMS.
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Besides, the formula of unknown metabolites can be identified via accurate mass
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measurements and their structure can be elucidated with MSn fragmentation. The high
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sensitivity of this technique allows also the identification of metabolites at ultra-trace levels
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and to determine their concentration with great accuracy via isotopic dilution.
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Exposure experiments of filter-feeding organisms were through algal feeding in marine
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mesocosms emulating real environmental conditions. The environmental metabolomics,
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based on HRMS, is a qualitative and quantitative approach that can be used to determine
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accurately metabolite concentration with high sensitivity.
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To our knowledge, this is the first study that has used HRMS-based metabolomics studies to
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assess sub-acute toxicological perturbations produced by carbon NMs on filter-feeding
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organisms in environmental conditions.
the
sub-lethal
toxicity
of fullerenes
to these
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filter-feeding
organisms.
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Materials and methods
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Chemicals Fullerene soot (76 % of C60 fullerene, 22 % of C70 fullerene and 2 % of higher-
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order fullerenes) produced by the Krätschmer-Huffman arc method was purchased from
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Sigma Aldrich (Steinheim, Germany). For the chemical analysis, to establish the
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accumulation of fullerenes, standards of the highest purity available were employed: C60
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(CAS: 99685-96-8, 99.9 % purity, reference 572500), C70 (CAS: 115383-22-7, 98 % purity,
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reference 482994) were purchased from Sigma Aldrich. Analytical standards of amino acids
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(ref. 09416-1EA), saturated fatty acids (ref. EC10A-1KT) and unsaturated fatty acids (ref.
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UN10-1KT) were obtained from Sigma Aldrich.
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13
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Corporation (Tucson, Arizona, USA).
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Toluene, ultrapure water and methanol Chromasolv® were supplied by Merck (Darmstadt,
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Germany). Nitrogen used as drying gas with 99.995% purity was acquired from Air Liquide
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(Barcelona, Spain).
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Dispersion of fullerene soot aggregates and characterization A mother suspension of
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fullerene soot was prepared by suspending fullerene soot in artificial estuary water at 10.0
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mg/l in an amber glass bottle. The dispersion was achieved by stirring with a magnetic
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nucleus during 40 days, without the use of ultrasounds or any organic solvent that may distort
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the properties of the aggregates. The resulting aggregates were characterized by Scan Electron
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Microscopy (SEM). A drop of suspension was dried onto a silica surface overnight, and SEM
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micrographs were acquired with a NOVA NanoSEM 230 (FEI), using an Everhart-Thornley
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and a back-scattered electron detector. The resultant micrographs showed highly polydisperse
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aggregates with diameters in the nanometers order (40-600 nm) that co-occurred with supra-
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aggregates, some of which visible at naked eye, as previously observed in Sanchís et al.
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(2015)36. All the aggregates were round-shaped. The smallest fraction of aggregates was
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characterized by Nanoparticle Tracking Analysis (NTA), Measurements were carried out by
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quintuplicate with an LM10-HS (Nanosight, UK), acquiring videos of 60-90 s and averaging
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the obtained size distributions. The final population presented a maximum at a hydrodynamic
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diameter of 246 nm and some relative maxima at 103 nm (65 % of absolute maximum), 126
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nm (~70 %), 292 nm (52 %) and 395 nm (38 %).
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Organisms collection and mesocosms set-up Adult Mytilus galloprovincialis were collected
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in January from a Mediterranean coastal mussel bed. Mussels were transported to the
C-labelled C60 fullerene (> 99% purity, reference MER613) was purchased from MER
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laboratory within 2 h. In this study, model communities were created in seawater aquaria.
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Fifty adult individuals of 5±0.1 cm were placed in four identical mesocosms in identical
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conditions. A control and three exposed groups were studied during five weeks (one week of
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acclimatization, 3 weeks of exposure and one week for detoxification) in a climate chamber
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laboratory following day/night cycles.
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The model communities in this experiment were formed to resemble a habitat of a coastal
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ecosystem, with natural abundances of the respective organisms at the sampling site at the
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north Catalan coast, close to the Aiguamolls de l’Empordà natural park. Each community was
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represented by: i) The studied organisms (M. galloprovincialis); ii) echinoderms
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(Paracentrotus lividus and Astropecten aranciacus,
