A reagent-free fully automated flow injection analysis (FIA) system coupled to atomic fluorescence spectrometry (AFS) for mercury (Hg) quantification is reported, using active nano-structured gold collectors for direct preconcentration of dissolved mercury species from natural waters. Recently we had shown the potential of such an approach for Hg analysis in seawater. This paper now describes the optimisation and validation of the proposed method including the investigation of possible limitations arising with matrix constituents, such as dissolved organic carbon (DOC). A broad variety of water matrices (seawater, river water, moorland water, effluent from wastewater treatment plant) were investigated in order to check the feasibility of the proposed method for total dissolved Hg determination in natural waters. All FIA parameters were optimised by checking Hg recovery in real water samples. Figures of merit of the proposed method - working range, carry over effects, detection limit, reproducibility, etc. - were determined. The method provides a high sensitivity (detection limit: 0.2pgHg) and very good reproducibility (RSD 1.1%, [Hg]=5ngL(-1), n=10). It offers several advantages because no reagents are needed for species conversion, preconcentration, or desorption and therefore the risk of contamination and blank values are lowered, reagent and time consumption are minimized. The system was successfully validated by measurement of a series of recoveries in real waters (all >96%) and in the certified standard reference material BCR 579 (mercury in coastal sea water, recovery 100.5%). Furthermore, the proposed method was applied to 15 real water samples for Hg ultra trace analysis.