AbstractThe presence of chloride ions in the Microtox® test can cause problems when testing metal toxicity, both due to extraction of metals from solid samples and formation of chloro complexes of metals in the liquid phase. To investigate alternatives to NaCl in the Microtox test, the toxicity of Cu, Cd, Pb, and Zn toPhotobacterium phosphoreumwas tested in 28 osmotic surrogates for NaCl. It was found that Na+must be present to keep the blank luminescence stable for 30 min. The results point to NaClO4as the most satisfactory surrogate solution as it has an inert behaviour and does not form complexes with any metal of environmental interest. Raw, digested, and reference sewage sludges were tested in the osmotic surrogates. The EC50 values for sludges were lower in solutions of NaNO3, Na2SO4, and NaClO4, and higher in sucrose, mannitol, and KCl, compared to NaCl. NaClO4can be recommended as an osmotic surrogate for sewage sludge testing. Another problem with the Microtox assay is the lack of pH control in the cuvette. Copper toxicity tests were carried out in Tris buffer and KH2PO4at two different concentrations and at pH 7 and 8. The results show that 1 mM phosphate buffer, pH 7.0, can be recommended for solid samples. However, owing to formation of KClO4a buffer containing potassium is not recommended in combination with NaClO4