Pollutants bioavailability and toxicological risk from microplastics to marine mussels

Microplastics represent a growing environmental concern for the oceans due to their potential capability to adsorb different classes of pollutants, thus representing a still unexplored source of exposure for aquatic organisms. In this study polystyrene (PS) microplastics were characterized for their capability to adsorb pyrene (PYR) as model compound for polycyclic aromatic hydrocarbons, and transfer this chemical to filter feeding mussels Mytilus galloprovincialis. Gene expression analyses of Mytilus galloprovincialis exposed to polystyrene (PS) microplastics and to polystyrene contaminated with pyrene (PS-PYR) have been performed trough a DNA microarray platform. For the exposure of mussels to microplastics, specimens of Mytilus galloprovincialis (5 ± 1 cm shell length) were obtained from a local farm (Numana, Ancona) and acclimatized for 10 days to laboratory conditions with aerated seawater and 18± 1 °C, salinity at 28–30 ‰. Contaminated plastics were prepared by maintaining a solution of <100µm microplastics (20g/L) with pyrene (50µg/L) in rotating conditions for 6 days. A total of 150 organisms were distributed into fifteen 6L- glass-beakers and exposed to virgin or contaminated plastics for 7 days: three replicates were performed for each of 3 treatments; Control (CNTR), Polystyrene (PS), Pyrene-treated Polystyrene (PS-PYR).Water was changed daily and both virgin and pyrene-treated particles re-dosed at a nominal concentration of 1.5 g/L. Four independent digestive glands pools for CNTR, PS and PS-PYR groups, each consisting of three individuals, were constituted for each treatment to perform DNA microarray analysis. Due to technical problems during the hybridization step, one pool belonging to PS group has been excluded by the dataset used for gene expression analyses. Gene transcription analyses of 12 digestive glands pools (four pools for each treatment composed by 3 digestive glands; CNTR, PS and PS-PYR) were performed using an 8X60K Agilent oligo-DNA microarray platform. Microarrays were synthesized in situ using the Agilent non-contact ink-jet technology including default positive and negative controls. Total RNA was isolated using Extract-all (Eurobio) procedure. RNA quality and integrity was controlled on the Agilent bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80°C until further use. Gene expression profiling was performed using an Mytilus galloprovincialis oligo-DNA microarray of 59,971 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.