Differential bioavailability of copper complexes to bioluminescent Pseudomonas fluorescens reporter strains.

Increased levels of Cu in agricultural soils are of concern, because Cu toxicity may adversely affect important soil microorganisms, including pseudomonads. Because total metal concentrations correlate poorly with bioavailability and toxicity, a need exists for more information linking Cu speciation, bioavailability, and toxicity. The objective of the present study was to determine the bioavailability of different Cu complexes to Pseudomonas spp. A Cu-specific bioluminescent Pseudomonas fluorescens reporter strain was used to determine bioavailable Cu, which was operationally defined as those Cu species that induced expression of bioluminescence. Another strain of P. fluorescens, which continuously expressed bioluminescence, was used as a toxicity reporter. Experiments were performed in a defined aqueous medium containing 0.04 microM Cu, which was amended with ethylenediaminetetraacetic acid (EDTA), citrate, or a well-characterized pool of dissolved organic matter (DOM). Bioluminescence emitted by the biosensors was related to data for Cu speciation obtained by geochemical modeling. Changes in Cu bioavailability in the presence of EDTA coincided with modeled changes in Cu2+ activity, indicating that Cu-EDTA complexes were not bioavailable to the Cu-specific reporter. In contrast, changes of Cu bioavailability in the presence of citrate did not correspond to changes in Cu2+, indicating that Cu-citrate complexes were fully bioavailable to the reporter strain. Finally, the response of the Cu-reporter strain to Cu in the presence of DOM indicated that Cu formed bioavailable as well as unavailable complexes with DOM. We conclude that free Cu2+ activity is a poor predictor of Cu bioavailability to Pseudomonas spp. in samples containing organic ligands.

Time and moisture effects on total and bioavailable copper in soil water extracts.

Environmental risk assessment of heavy metals in soil frequently involves testing of freshly spiked soils kept under stable humidity conditions, but it has been questioned whether these assessments are representative of the field situation. Furthermore, the poor correspondence that is often found between total metal content and metal toxicity calls for integrated chemical and biological analysis. The aim of this work was to determine time- and moisture-dependent changes in total water-extractable Cu as well as bioavailable Cu in soil water extracts. Measurements of total water-extractable copper ([Cu]tot) were performed using furnace atomic absorption spectrometry. An in vitro assay employing a Cu-specific Pseudomonas fluorescens reporter strain was used to estimate Cu that was biologically available to the reporter strain. We refer to this copper fraction as "bioavailable," [Cu]bio. We found a time-dependent decrease in [Cu]tot and [Cu]bio during incubation for up to 220 d at field capacity. Hence the [Cu]bio was reduced to between 32 and 40% of the initial values. Furthermore, the [Cu]bio to [Cu]tot ratio correlated positively with the amount of added Cu and tended to increase with time. The moisture content of the soil was important for Cu retention. Dry soil had higher [Cu]tot concentrations than humid soil, but the [Cu]bio to [Cu]tot, ratio was lower in the dry soil. Alternating drying and wetting did not lead to a more rapid Cu retention than observed under constant humid conditions. Our observations underline the need for considering both time and moisture effects when interpreting short-term toxicity studies and when making predictions concerning possible long-term effects of Cu in the soil environment.

Effects of copper amendment on the bacterial community in agricultural soil analyzed by the T-RFLP technique.

Abstract The impact of copper amendment on the bacterial community in agricultural soil was investigated by a 2-year field experiment complemented by short-term microcosm studies. In the field, the amendments led to total copper contents that were close to the safety limits laid down by European authorities. In parallel, bioavailable copper was determined with a copper-specific bioluminescent Pseudomonas reporter strain. The amounts of total Cu as well as of bioavailable Cu in the field declined throughout the experiment. Bacterial community structure was examined by terminal restriction fragment length polymorphism (T-RFLP) analysis of community DNA amplified with primers specific for 16S rDNA from the Bacteria domain, the Rhizobium-Agrobacterium group and the Cytophaga group. Similarity analysis of T-RFLP profiles from field samples demonstrated an impact of copper at the domain level and within the Rhizobium-Agrobacterium group. Comparable Cu effects were observed for microcosms, but in addition an impact on community structure within the Cytophaga group was observed.