Fullerene (C-60) aggregation mechanisms in aqueous media require considerable attention in the near future due to the heavy use and application of fullerene-based products within the context of nanotechnology. Such intensive development will result in the release of massive amounts of C-60 in aqueous environmental systems in the aggregate form (nC(60)). In that sense, the aggregation mechanisms need to be fully determined to better evaluate the environmental fate and behavior of C-60. To fulfil these needs, the aim of this work was to extensively characterize the aggregation mechanisms of fullerene aggregates in aqueous media by asymmetrical flow field fractionation (AF4) coupled to static light scattering (SLS). We developed a sequential ultrafiltration method that allows the fractionation of the whole nC(60) size distribution into different size classes (1-100-200-450-800 nm). Following a preliminary analysis by dynamic light scattering (DLS), we optimized several AF4 separation methods to allow screening of these colloidal size classes of nC(60) with high efficiency and resolution. The fractal dimension (D-f) of this entire size class was characterized directly on-line according to the radius of gyration through a combination of angle-dependent light scattering and fractal dimension analysis. We demonstrate the possible formation and persistence of colloidal populations of nC(60) in aqueous media from a few nanometers up to 800 nm. Df values ranging from 1.2 to 2.8, based on the nC(60) colloidal size range, strongly depend on the method of the sample filtration. (C) 2017 Elsevier Inc. All rights reserved.