The study investigated the inhibitory effects of the heavy metals Cd, Pb, Hg, Ag and Cr (as Cr3+ and Cr6+) on a nitrifying sludge. The aim was to assess the IC50 concentrations leading to 50% inhibition. The method is based on respiration of nitrifying sludge in the presence of these metals. Both O-2 consumption and CO2 production were taken into account. The order of the inhibitory effect was Ag > Hg > Cd > Cr3+ = Cr6+. Metal speciation was calculated in terms of free metal, inorganic metal complexes and bound metal. Pb largely precipitated and 50% inhibition was never reached. Ag was always in the form of the free ion or labile complexes. Hg had apparently a lower toxicity than Ag, since most of it was initially highly complexed with ammonia. Cd was present in the form of free ion and complexes which caused inhibition although a large part of them were precipitated. The inhibitory effects of trivalent chromium (Cr3+) and hexavalent chromium (Cr6+) were similar. The latter was present in the form of the anion CrO42- and was not taken up by biomass. The study highlighted that IC50 values alone do not have an explanatory power of inhibition unless speciation is also considered. (C) 2010 Elsevier B.V. All rights reserved.