The prevalence of silver nanoparticles (AgNPs) requires a comprehensive understanding of their biological impacts especially in marine and estuarine environments. Nevertheless, the background Ag concentration in organisms may impede the accuracy of Ag detection if the net accumulated Ag is low over a short exposure period. Here, a radio-synthesizing method was employed to trace the behavior of AgNPs with two sizes (15 and 60 nm) and two coatings (humic acid and citrate) in an estuarine oyster Crassostrea hongkongensis. This method was sensitive to detect the bioaccumulation and depuration of AgNPs in the oysters over a short period of exposure, which was necessary given the significant changes of particle aggregation in saline water environments. Through radioactive AgNP tracing and biokinetic modeling, we for the first time demonstrated the differential uptake mechanisms of different-sized AgNPs in oysters. Specifically, the ingestion of particles dominated the uptake of 60 nm AgNPs, whereas dermal uptake and ingestion contributed equally to 15 nm AgNPs. Surface coating (humic acid vs citrate) did not significantly affect the uptake of AgNPs by the oysters. The depuration of AgNPs from the oysters was relatively faster than that for the Ag ion. The digestive gland was the key detoxification organ of AgNPs with the greatest loss of Ag by the end of depuration. The findings of this study provide fundamental knowledge for nano-specific risk assessment in marine and estuarine environments.

The extensive application of silver nanoparticles (AgNPs) requires a full examination of their biological impacts, especially in aquatic systems where AgNPs are likely to end up. Despite numerous toxicity studies from molecular to individual levels, it is still a daunting challenge to achieve in situ subcellular imaging of Ag and to determine the sites of AgNP interaction with organelles or macromolecules simultaneously. Here, by coupling high-resolution nanoscale secondary ion mass spectrometry elemental mapping with scanning electron microscopy ultrastructural characterization, we successfully visualized the subcellular localization and the potential toxicity effects of AgNPs in the oyster gill filaments. The stable isotope tracing method was also adopted to investigate the respective uptake and transport mechanisms of differently labeled 109 AgNPs and 107 Ag + ions. 109 Ag hotspots were colocalized with endosomes or lysosomes, proving an endocytosis-based entry of AgNPs which passed through the barrier of oyster gill epithelium. These 109 Ag hotspots showed a strong colocalization with 32 S − . For the first time, we provided visualized evidence of AgNP-induced autophagy in the oyster gill cells. We further identified two categories of hemocytes (blood cells) and illustrated their roles in AgNP transport and sequestration. The integration of morphological and functional aspects of Ag subcellular distribution in different target cells suggested that oysters were equipped with a specialized endolysosomal (epithelial cells) or phagolysosomal system (hemocytes) in regulating the cellular process of AgNPs, during which the lysosome was the most involved organelle and sulfur was the most relevant macronutrient element. This study highlighted not only the intracellular but also the intercellular AgNP translocation and transformation, providing important subcellular imaging of silver and reliable methodology regarding bio−nano interactions in natural environments.