In the literature, sieving effects, charge interactions and adsorptive/hydrophobic interactions have been named as major mechanisms influencing the rejection of trace organic compounds (TOrCs). However, dominant mechanisms influencing TOrC rejection by fouled membranes are not yet fully understood. Most studies have attributed the decline in rejection to cake-enhanced concentration polarisation (CECP) effects and have overlooked the influence of flux decline and attractive membrane-TOrC interactions. Therefore, the aim of this study was to investigate the role of various factors in TOrC rejection for nanofiltration (NF) membranes fouled in cross-flow filtration. To further gain insight into the role of affinity interactions on TOrC rejection, membrane-solute interaction energies (Delta G(slm)) were computed from the contact angle measurements based on the Lifshitz-van der Waals/acid-base approach. A novel fouling approach (termed sequential fouling) is used to investigate CECP effects for different fouling layers. Sequential fouling results are validated using the CECP model. For a virgin membrane, TOrCs are rejected through sieving mechanisms and repulsive non-electrostatic membrane-TOrC interactions. Upon fouling (especially in the presence of calcium), there was an improvement in the rejection of some TOrCs (solute permeation decreased) due to an improvement in sieving effects. The rejection of some TOrCs decreased over time (solute permeation increased) but the rejection values were close to those of the virgin membrane at similar fluxes showing the influence of flux. When solute permeation increased as a result of fouling, TOrC transport was mainly influenced by non-electrostatic membrane-TOrC affinity interactions and CECP effects (as evidenced by novel sequential fouling experiments and the CECP model). (C) 2016 Elsevier B.V. All rights reserved.