Knowledge of heavy metal speciation and its relationship with biological responses is important for the derivation of effects-based soil quality criteria. We determined soluble Cu concentrations and free Cu 2+ activities in the pore waters from 22 soils with total Cu varying from 19 to 8645 mg kg -1 . Pore water pCu 2+ () -log (Cu 2+ activity)) varied from 3.9 to 10.5 and was controlled by soil pH and total Cu concentration. The percentage of free Cu 2+ in total soluble Cu varied from 0.02 to 96% and was influenced strongly by pore water pH and, to a lesser extent, by dissolved organic C. In the pore waters with pH >6, the percentage of free Cu 2+ in total soluble Cu was lower than 1%. Using the default data base and with the fulvic acid content of DOC optimized at 69%, the equilibrium speciation program WHAM/Model VI gave estimates of pCu 2+ that agreed closely with measured values. Pore water samples were analyzed by two bioluminescence-based bacterial biosensors: Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586r pUCD607. The response of P. fluorescens correlated more closely with soil pore water pCu 2+ than with soluble Cu concentration, whereas pCu 2+ and soluble Cu fitted the response of E. coli equally well. The effect concentrations (EC 25 and EC 50 values) of pCu 2+ for the two biosensors were about 5.8 and 5.0, respectively. This is the first time that threshold values for Cu have been obtained for bacterial biosensors exposed to soil pore water from well-equilibrated contaminated soils.

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A long-term incubation experiment was established to assess the solid Ð solution equilibria of (Cu 2þ ), (Pb 2þ ) and (Ni 2þ ) in soil pore water. The experiment comprised 23 soils spiked with 135, 300 and 75 mg kg À1 of Cu, Pb and Ni, respectively, added as nitrate salts in solution. Samples of soil pore water were extracted several times during the incubation period of 818 days and concentrations of Cu, Pb, Ni, dissolved organic carbon and major cations and anions were measured. Similar measurements were carried out on a smaller selection of historically contaminated soils to extend the range of data and assess compatibility of the measurements with the incubated soils. The chemical speciation program 'WHAM VI' was used to speciate metal ions in solution. A pH-dependent Freundlich equation was used to describe free ion activities, p(M 2þ ), for Cu, Pb and Ni using total adsorbed metal, soil pH, soil organic carbon content and ionic strength as determining variables. For all three metals the greatest improvements in model fit were found when metal ions were assumed to be adsorbed exclusively on soil organic carbon rather than on the 'whole soil'. Further improvements in the description of p(Cu 2þ ) and p(Ni 2þ ) were found when the ionic strength of the soil pore water was included within the model formulation. Residual standard deviations (-log 10 (M 2þ ) units) for the best-fit models were 0.36, 0.53 and 0.29 for p(Cu 2þ ), p(Pb 2þ ) and p(Ni 2þ ), respectively. The effects of progressive fixation of Pb, Cu and Ni on model parameterization, during the course of the experiment, were found to be small. Independent datasets from both published and unpublished sources were used to compare experimental protocols and validate the model for the determination of (M 2þ ) in soil pore water.

The toxicity of Zn, Cu, and Cd, alone or in combination, was assessed using two luminescence‐based microbialbiosensors from different ecological niches: Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586 pUCD607. Significant synergistic interactions occurred between the toxic effects of the Zn and Cu and Zn and Cd combinations and the response of E. coli (i.e., toxicities of combinations of pollutants were greater than predicted from addition of individual toxicities). Significant synergistic interactions were also observed between the toxicities of Cd and Cu combinations and the response of P. fluorescens. A longer exposure time was shown to significantly increase the sensitivity of E. coli to the metal pollutants. P. fluorescens was observed to significantly decrease in its sensitivity toward Zn and Cd with a longer exposure time. It was shown that the toxicity of combinations of metals could not be modeled on the basis that their toxic action was independent of each other. The application of different models to describe interactions between combinations of metals is discussed. The importance of considering the test species and the exposure period selected for toxicity assessment was highlighted, as was the need to further investigate the toxicity of combinations of pollutants.