Priming Nanoplastics with Phenanthrene Compounds

The custom-synthesized nanoplastic particles were primed with either unlabelled phenanthrene or uniformly ¹³C-labelled phenanthrene ([U-¹³C]phenanthrene), following previous protocols^{72 (link),73 (link)}. Unlabelled phenanthrene (99.5% purity) and [U-¹³C]phenanthrene were from Sigma-Aldrich (Sigma, Dorset, UK). Briefly, a known mass of the compound (unlabelled or [U-¹³C]-labelled was dissolved in acetonitrile in a vented glass container and left for 48 h to allow the solvent to evaporate. To the residual crystallised phenanthrene, sdH₂0 was added to produce a final aqueous phenanthrene solution of 1.1 mg L⁻¹, which was then used to prime the nanoplastic particles. For this, 1 mg each of the nanoplastic sizes (500 nm or 1000 nm) was added to 10 mL of the prepared unlabelled or [U-¹³C]-labelled phenanthrene solution and allowed to incubate for 24 h with gentle rotatory shaking (~ 80 rpm) at 21 °C in the dark. The plastic particles were recovered by centrifugation (10,000×g for 4 h at 20 °C) and resuspended in sdH₂O. The nanoplastic suspensions with the unlabelled compounds were used for the agglomerate experiments, whereas the nanoplastic suspensions with the [U-¹³C]-labelled compound were used for the SIP experiment (described below). Before using these nanoplastic suspensions in their respective experiments, sub-samples were taken to re-measure and confirm the size and ζ-potential of the particles. For the 500 nm plastic particles, adsorption of the [U-¹³C]phenanthrene was examined by visual inspection using a Zeiss (Axio Scope.A1) epifluorescence microscope fitted with a Zeiss digital fluorescence imaging camera (AxioCam MRm) with excitation and emission wavelengths at 250 nm and 366 nm, respectively^{74 (link)}. In addition, sub-samples of the [U-¹³C]phenanthrene solution were taken prior to the addition of the plastic particles, and also after their removal, in order to determine phenanthrene concentrations (see below) as further confirmation that phenanthrene had become adsorbed onto the surface of the 500 nm nanoplastic particles.