Periostracum of bivalve mollusk shells for sampling engineered metal nanoparticles: A case study of silver-based nanoparticles in Canada's experimental lake

Given the ability of engineered metal nanoparticles to be transformed in natural waters in unpredictable manners, various sampling methods must be developed. Here, we took a novel approach to collection silver nanoparticles (AgNPs) that involved the use of the intact periostracum, the outer proteinaceous organic layer, of freshwater unionid mussels Pyganodon sp. Eight adult mussels were collected in August 2019 from a small boreal lake (L222) at the International Institute for Sustainable Development - Experimental Lakes Area (northwestern Ontario), which had been dosed with 15 kg of poly(vinylpyrrolidone)-coated silver nanoparticles (PVP-AgNPs) in 2014-2015. Additionally, three adult mussels were collected from a control lake (L375). Numerous silica (SiO₂) diatom frustules were adhered to periostracum of all mussels. Intact periostracum promotes the formation of layer composed of diatoms and sand grains. The Ag content in soft tissues and shells of the mussels from L375 was as low as ≤ 0.1 μg/g. In mussels from L222, Ag concentrations in the periostracum of five shells were in detectable amounts (1-4 μg/g); in three shells concentrations were as high as 86, 122, and 494 μg/g. The underlying mineral shell is depleted in Ag (<0.1 μg/g). The Ag content in soft tissue organs (whole body) ranged from 44 to 191 μg/g. AgNPs occur on the surface of both periostracum and diatoms. Single AgNPs (d = 20-60 nm) were partly sulfidized to Ag₂S. The observed AgNPs often form aggregates with an average and a maximal size of circa 100 nm and 1.5 μm, respectively. Scraping small fragments of intact periostracum of unionid shell is non-lethal to mussels, and is easy to do under field conditions. This simple sampling protocol could be used to detect metal-based nanoparticles (engineered or accidental) with the use of unionid and dreissenid bivalves.