Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes

Abstract

Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 μg Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO₃), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after Tier 1 (0.75 μg Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 μg Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 μg Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (<10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag⁺ions released from NPs, although the metal was not substantially accumulated.

Keywords:

• Ag NPs
• nanosize
• toxicity
• metal form
• biomarkers
• histopathology
• digestive gland
• gills

Acknowledgements

This work was funded by EU 7th FP (NanoReTox project, CP-FP 214478-2), Spanish Ministry (NanoCancer project CTM2009-13477), Basque Government (consolidated research group CIG12/IT810-13) and University of the Basque Country (UFI 11/37 and PhD fellowship to AJR). SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This work was funded by EU 7th FP (NanoReTox project, CP-FP 214478-2), Spanish Ministry (NanoCancer project CTM2009-13477), Basque Government (consolidated research group CIG12/IT810-13) and University of the Basque Country (UFI 11/37 and PhD fellowship to AJR). SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged.