Advancing fish tissue reference material development for Ag nanoparticles through a perfusion approach

Abstract

There is a regulatory need for reliable methods to quantify nanoparticles in biological samples for the hazard assessment and bioaccumulation potential of engineered nanomaterials (ENMs). Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) is a promising technique, but its application is hindered by the lack of validated sample preparation procedures (e.g., solubilisation) and suitable biological reference materials. This study aimed to develop an in-house biological reference material for ENMs to support analytical method validation. Silver nanoparticles (Ag NPs) were introduced into rainbow trout via cardiac perfusion, with the gills and liver collected as target tissues. Tissues were homogenised using a blade grinder, followed by additional milling. Pre- and post-milling preparations were compared to evaluate the level of tissue processing required prior to SP-ICP-MS analysis. Tissue samples were solubilised using tetramethylammonium hydroxide before analysis. Background Ag NP concentrations in control tissues were low (0.2–0.7 × 10⁹ nanoparticles per g), consistent with previous studies. In contrast, fish exposed to Ag NPs showed a clear increase in particle concentration, reaching up to 33.8 ± 4.4 × 10⁹ nanoparticles per g, with median particle sizes of 74–81 nm. The additional milling significantly improved data quality. In the liver, nanoparticle concentrations increased from 10.7 ± 4.4 × 10⁹ nanoparticles per g before milling to 15.9 ± 1.4 × 10⁹ nanoparticles per g after milling. Precision also improved, with relative standard deviation (RSD%) reducing from 40.7% to 9.1%, and all nanoparticle metrics achieving RSD values below 15% after combined blade grinding and milling. These results demonstrate that perfusion, followed by blade grinding and milling, produces a homogenous in-house biological reference material containing ENMs. This approach is simple, cost effective, and ensures analytical robustness for SP-ICP-MS measurements. Future work should assess the material's shelf life and apply this approach in inter-laboratory testing to support broader method validation.