Quantifying the bioaccumulation of nanoplastics and PAHs in the clamworm Perinereis aibuhitensis

The impact of nanometer-scale plastics (<1000 nm nanoplastics, NPs) on the bioaccumulation of hydrophobic organic pollutants, and especially polycyclic aromatic hydrocarbons (PAHs), in marine organisms has become of urgent concern. However, simultaneous determinations of the bioaccumulation of NPs and PAHs have been hindered by the lack of an efficient digestion method that removes background interference from the tissue without altering the surface properties of the plastic and destroying the PAHs. To solve this problem, an enzymatic digestion-based protocol using proteinase K and subsequent quantification methods were developed on a typical marine benthic invertebrate - the clamworm Perinereis aibuhitensis. Enzymatic digestion removed 91% of the biological tissues, comparable to the amount removed using 65% HNO₃ (93% removed) and better than that removed using 30% H₂O₂ or 10% KOH digestion (76% and 66%, respectively). After enzymatic digestion, roughly 92% of the NPs and 88% of the amount of pyrene were recovered, without significant modification of the NPs or pyrene degradation. By contrast, the NP and pyrene recovery achieved with HNO₃ digestion was only 1.4% and 0.1%, respectively. The newly developed protocol was successfully applied to a 96-h bioaccumulation study. The use of radioactively labeled ¹⁴C-pyrene and fluorescently labeled NPs allowed the simultaneous quantification of NPs and PAHs in the clamworm and revealed a bioconcentration factor (BCF) of 1.96 ± 0.93 and 402.7 ± 47.0, respectively. The quantification of NPs and pyrene indicated that NP-adsorbed pyrene accounted for <1% of the total pyrene accumulation in the clamworm body when the concentration of NPs in seawater was as low as 0.4 mg/L. Our enzymatic digestion and dual-labeling technique thus provides the first reported BCF value of NPs in a marine benthic organism and new insights into the vector effects of these particles on the bioaccumulation of organic contaminants in a marine ecosystem.