Substrates of treatment wetlands, which were constructed at an operational lead-zinc mining facility (Tara Mines) in Ireland, were analysed over a 5-year period for metal content. Lead, zinc, iron and sulphate were removed from wastewater passing though the treatment systems and were significantly retained in the substrates. The degree to which these contaminants are bio-available depends on the biogeochemical conditions of the substrates, namely the pH, redox potential, amount of organic carbon present and the contaminant loading. Various sediment fractions (water-soluble, exchangeable, carbonate-bound, organic matter-bound and residual) were also analysed in these treatment wetlands, in order to estimate the fate of metals and sulphur over time. Results showed that the majority of metals and sulphur were retained in immobile residual forms, principally as metal-sulphides. Furthermore, the neutral-alkaline pH and reducing biogeochemistry of the treatment systems suggest that metals are not toxic under these conditions.