Aims: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. Methods and Results: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gramreaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple ( ‡3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple ( ‡2) antibiotics than corresponding isolates from control plots. Significance and Impact of the Study: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.

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.

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.

Kunito et al., 1999). Biological assays determining bioavailability and toxicity of Cu to soil micro-Environmental risk assessment of heavy metals in soil frequently organisms typically measure the impact that the metal involves testing of freshly spiked soils kept under stable humidity conditions, but it has been questioned whether these assessments are

Copper (Cu) is an essential trace metal required by living organisms but it is potentially toxic at higher concentrations. Cu salts are used as bactericides in agriculture and as growth promoters in pig production. They are consequently introduced to the environment, e.g. through manure, and might negatively affect the bacterial soil community. In this study, our goal was to develop a specific and stable Cu reporter construction harboured by an indigenous soil bacterium to measure bioavailability of Cu in soil. Following mutagenesis of Pseudomonas fluorescens strain DF57 with a Tn5::luxAB promoter probe transposon, we identified four mutants with elevated luxAB expression in response to Cu. Two of the mutated loci encoded proteins homologous to Cop proteins conferring Cu resistance to Pseudomonas syringae, while a third displayed homology to metal‐transporting ATPases. In the fourth mutant, DF57‐Cu15, the gene interrupted by the transposon encoded a protein carrying a Cu‐binding domain but with low homology to known proteins. DF57‐Cu15 was the most suitable Cu reporter due to its high specific response and tolerance to Cu in pure culture. DF57‐Cu15 responded to soil solutions from Cu‐amended soil microcosms in a concentration‐dependent manner. The chelator EDTA reduced the availability of Cu to P. fluorescens in soil. This showed that complex‐bound Cu is not necessarily available to bacteria. We compared chemical analysis of soluble Cu and the reporter assay on soil solutions from Cu‐containing soil microcosms supplemented with either manure or straw. Organic matter increased the amount of soluble Cu but not the amount of bioavailable Cu. Probably, Cu binds with high affinity to organic constituents in pig manure or barley straw. Hence, determination of soluble Cu by chemical analysis cannot always predict the bioavailability of Cu to a specific bacterium.

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.

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