Physicochemical characterization of synthetic amorphous silica (SAS) and the OECD representative nano-sized silica (NM-202). SEM micrographs of SAS in (A) water + 0.05% BSA and (B) feed, and of NM-202 in (C) water + 0.05% BSA and (D) feed. (E) SEM size distribution pattern of SAS and NM-202 in water + 0.05% BSA, showing larger sizes for NM-202. The size limit of detection lies at 25 nm. (F) EDX characterization of SAS and NM-202 agglomerates in water containing 0.05% BSA, demonstrating the presence of Si, O, and C on the surface of both materials. The peak representing nickel can be attributed to the nickel coated membrane that was used for sample preparation. (G) FTIR spectra of SAS and NM-202 showing mild C-H stretching vibrations for NM-202 (inset). (H) HDC-ICP-MS chromatogram showing the size distribution and concentration (area under the curve) of nano-sized silica in SAS, NM-202, and in control feed mixtures. (I) The fraction of silica in the nano-size range (i.e. with a size of 5–200 nm; measured by HDC-ICP-MS) given as a percentage of the total silica content (measured by ICP-MS) in the large intestinal (LI) contents, 24 hours after the last exposure (mean ± standard error of the mean; n = 5). * Significant difference versus the control (p < 0.05).