Bioavailability Of Cu In Soil Research Articles

Exogenous iron alters uptake and translocation of CuO nanoparticles in soil-rice system: A life cycle study.

The abundant iron in farmland soil may affect the environmental fate of metal-based nanoparticles (MNPs). In this study, the effect of FeSO4 and nano-zero-valent iron (nZVI) as exogenous iron on the uptake and translocation of CuO nanoparticles (NPs) in soil-rice system was performed in a life cycle study. The results show that exogenous iron basically elevated the soil pH and electrical conductivity but lowered the redox potential. Moreover, the Cu bioavailability in soil was significantly increased by 86-269% with exogenous iron at the tillering stage, while was reduced by 15-45% with medium and high concentrations of Fe(II) at the maturation stage. Meanwhile, the addition of exogenous iron resolved the unfilling of grains caused by CuO NPs. Notably, except for highest Fe(II) treatment, both Fe(II) and nZVI reduced Cu accumulation from 31% to 84% in roots and leaves due to more iron plaque. Especially, medium Fe(II) level markedly decreased the Cu content in the brown rice. μ-XRF analysis suggests that high intensity of Cu was primarily located in the rice hull and embryo under Fe(II) treatment. The reduction of CuO NPs to Cu2O caused by Fe(II) can explain the positive effect of exogenous iron on controlling the environmental risk of MNPs.

Influence of selenium combined with rice straw on the inhibition of copper phytotoxicity to pak choi

ABSTRACT This study aimed to explore the effects of Se combined with rice straw (RS) on alleviating Cu toxicity through the change of Cu bioavailability in soil. The growth parameters of pak choi, the total Cu concentrations in pak choi, the changes of Cu fractions in soils before planting and after harvest, and the bioconcentration and the translocation factors were determined. Results showed that Se application may detoxify Cu at excessive concentrations (200 mg/kg) by transforming mobile Cu fractions into semi-mobile and non-mobile Cu fractions (FEM-Cu, OM-Cu, and RES-Cu). The Cu concentrations in pak choi decreased and pak choi growth increased. Se also increased the Cu concentration in pak choi and promoted pak choi growth at low Cu concentration (50 mg/kg). The RS supplementation in Cu–Se co-exposure treatments can limit the absorption Cu in pak choi and enhance pak choi growth than only Cu–Se co-exposure treatments. This combination only affected the uptake of Cu by pak choi tissue but did not change the translocation efficiency from root to shoot. This study suggested that Se combined with RS plays an important role in preventing Cu uptake by plant tissues at excessive concentrations through transformations into immobile Cu fractions.

Using vermiwash to enhance performance of small-scale vermifiltration for swine farm wastewater.

Pollution caused by swine wastewater is a growing concern in many countries. In the developing countries, swine wastewater is not properly collected and treated, the wastewater from swine farm pollutes the ecosystem. Especially for small swine farms, they could not afford to have wastewater treatment system. Therefore, farmers need cheap, sustainable technology for future mixed farming. Vermifiltration by earthworm has been introduced to be an answer for enhancing wastewater treatment. Vermiwash is the liquid gathered from vermicomposting that has high microbial activities and nutrients. This study was carried out on a small pilot scale to investigate swine wastewater treatment efficiency of vermifiltration system with and without vermiwash and compared with the geofiltration system. Vermiwash was incubated in vermifiltration and geofiltration systems for 1week before the treatment. The result showed improved efficiency of vermifiltration incubated with vermiwash in swine wastewater treatment for biological oxygen demand (BOD), chemical oxygen demand (COD) and total suspended solids (TSS) removal, which was highest followed by vermifiltration without incubated vermiwash, geofilter incubated with vermiwash and geofilter, respectively. Good performance of vermifiltration incubated with vermiwash compared with the geofilter treatment was demonstrated for removal of BOD (91.29 ± 9.89%, n = 10), COD (91.42 ± 6.34%, n = 10) and TSS (86.02 ± 10.45%, n = 10). Furthermore, the burrowing activity of the test earthworm (Eisenia fetida) promoted the aeration condition in vermifilter which led to more dissolved oxygen (DO) in effluent (61.28 ± 20.05%, n = 10). Moreover, the amount of copper (Cu) in effluent was decreased compared with influent by up to 88% in all treatment. After 10weeks of the experiment, the vermicompost that was incubated with vermiwash and produced from earthworm on the top layer was analyzed and showed that nutrients (nitrogen, phosphorus) and soil organic carbon were increased with vermifilter treatment (47.65, 81.61 and 31.79%, respectively) compared with geofilter treatment. In addition, bioavailability of Cu in soil in form of exchangeable Cu was decreased by increasing the bound to organic matter fraction. Transformation of Cu during vermifiltration happened and alleviated the mobility and availability of Cu. Copper in exchangeable form can change into non-toxic form. Therefore, vermifiltration process incubated with vermiwash could reduce the dispersion of copper in swine waste. In conclusion, vermiwash could enhance performance of vermifiltration for swine farm wastewater treatment. The available fraction of copper in vermicompost produced from vermifiltration decreased. Therefore, the farmer could produce vermicompost as the biofertilizer for agricultural production. Using vermifiltration for wastewater treatment in small swine farm could be the eco-solution for nutrient recovery, water resource recycles and minimize pollution.

Effects of sulfur on toxicity and bioavailability of Cu for castor (Ricinus communis L.) in Cu-contaminated soil.

The biogeochemical cycling of sulfur (S) in soil has an important impact on the bioavailability of heavy metals and affects the utilization of soil polluted by heavy metals. In addition, S-containing compounds are involved in heavy metal detoxification. This study investigated the effects of S on the toxicity and bioavailability of copper (Cu) in castor (Ricinus communis L.) grown in Cu-contaminated mine tailings. The results showed that the application of S reduced the accumulation of Cu in castor and promoted its growth. With the addition of S, the malondialdehyde (MDA) content of castor leaves decreased significantly compared with control plants, indicating the alleviation of oxidative stress. Superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) content decreased significantly with the alleviation of oxidative stress. The sequential extraction of Cu fractions showed that the application of S significantly reduced the reducible Cu fraction, and increased the oxidizable Cu fraction. It also increased the residual Cu fraction in the soil. The transformation of chemical speciation reduced the bioavailability of Cu in soil, which then reduced the accumulation of Cu in castor. Our results demonstrated that S application was effective at promoting castor growth by reducing the bioavailability and uptake of Cu in Cu-contaminated mine tailings.

Effects of single and combined exposures to copper and benzotriazole on Eisenia fetida

Benzotriazole (BTR), an emerging class of environmental pollutant, is widely used in industrial applications and household dishwashing agents. Despite the reported toxicity of BTR to aquatic organisms, little is known about its effects on terrestrial invertebrates. Copper (Cu) accumulates in agricultural soils receiving urban waste products, fertilizers, fungicides, and urban sewage. In this study, two different types of bioassays (acute toxicity test and behavioral toxicity test) were performed to evaluate the toxicity of Cu and BTR, both singly and together, on the earthworm (Eisenia fetida) in artificial soil. The results of avoidance behaviour tests showed that the EC50,48 h values for Cu and BTR were 1.47 and 0.46 mmol kg−1, respectively. The results of the acute toxicity tests showed that the LC50,7 d and LC50,14 d of Cu in earthworms were 9.19 and 5.28 mmol kg−1, respectively, and the LC50,7 d and LC50,14 d of BTR were 2.43 and 1.76 mmol kg−1, respectively. Toxicity analysis demonstrated that the binary BTR and Cu mixture had predominantly antagonistic effects on the avoidance behaviour and survival of earthworms. The Cu2+ activities and mortality of earthworms decreased significantly with increasing concentrations of BTR, while the solid-liquid distribution coefficient of Cu increased. These results indicated that the presence of BTR can reduce the toxicity as well as the bioavailability of Cu in soil with both BTR and Cu.

Cu Immobilization and Lolium perenne Development in an Acid Vineyard Soil Amended with Crushed Mussel Shell

AbstractCu pollution and erosion pose an important risk of degradation in vineyard devoted soils. Therefore, minimizing harmful effects of Cu in the soil and avoiding erosion are important challenges in vineyard soil management. The addition of low cost bio‐adsorbents to the soil which decreased Cu bioavailability in soil and/or increased cover crops development may be an important management practice for soil stabilization in vineyards. This study investigated the effect on Lolium perenne performance because of crushed mussel shell (CMS) amendment in an acid Cu‐polluted vineyard soil. Quadruplicate samples of a Cu polluted soil were amended with 4 doses (0, 6, 24 and 48 g kg−1) of CMS by in lab pots, where L. perenne was sowed and incubated for 51 days. The soil pH, as well as Cu bioavailability and exchangeable Cu, was analysed. The effect of CMS on L. perenne response was measured using plant germination, plant growth and Cu uptake. The plant growth was assessed in both roots and shoots by measuring length and biomass. Cu uptake by roots and shoots was also determined. The CMS amendment on the acid vineyard soil increased the pH and reduced the bioavailable Cu concentration. Moreover, shoot and root growth increased for low and intermediate CMS doses, and decreased for higher doses. The increase in plant growth corresponded with increases in soil pH up to 7 · 0, whereas the highest mussel shell dose caused a pH increase over the neutrality (7 · 8). The CMS amendment decreased Cu uptake by both, roots and shoots, and it was highly correlated with changes in Cu bioavailability in the soil. The CMS amendment on a Cu‐polluted acid soil presented positive effects on Cu immobilization and L. perenne growth. However, mussel shell application must be performed carefully, taking into account that high doses can lead to decreases on plant growth. Copyright © 2016 John Wiley & Sons, Ltd.

Response of Cu partitioning to flooding: A δ65Cu approach in a carbonatic alluvial soil

The mobility and bioavailability of Cu in soils depend on the chemical Cu forms and are strongly influenced by water-induced changes in soil redox conditions. We chose an experimental approach by flooding a carbonatic floodplain soil and incubating it in a glovebox for 35days. We used a five-step sequential chemical extraction to characterize the changes in chemical partitioning (F1–F5; NH4NO3-extractable, NaOAc-extractable, NH4Ox-extractable, hot H2O2/NH4OAc-extractable and residual fractions, respectively) of Cu and δ65Cu values in the soil during the 35days. After flooding, Cu concentrations decreased in F1–F3 and increased in F4–F5. Overall, 73% of the total Cu was redistributed among the five studied fractions. Before flooding (Day 0), δ65Cu values in F1–F4 followed the estimated bonding strengths of Cu in the respective fractions, indicating equilibrium distribution of Cu at the beginning of the experiment. The total variation in δ65Cu values among F1–F5 changed strongly from 0.83±0.18‰ on Day 0 to a maximum of 2.18±0.17‰ on Day 7. This change indicates the reduction of Cu2+ to Cu+ or Cu0 after flooding. The strongest variations in δ65Cu values occurred in F3 (0.09±0.07‰ to 1.43±0.13‰) and F4 (−0.24±0.07‰ to 0.55±0.07‰), while flooding had no or small effects on the δ65Cu values of F1, F2 and F5. Our results suggest a direct transfer of Cu from F3 to F4 because both concentration changes and changes in δ65Cu values were balanced between the two fractions. The responses of Cu partitioning and δ65Cu values to flooding are in line with the formation of CuxS or other reduced Cu species and reduction of Cu associated to Fe (oxyhydr)oxides. Our results emphasize the strong influence of redox conditions on Cu partitioning in soils and indicate a reduction in Cu availability under anoxic redox conditions.

Copper availability assessment of Cu-contaminated vineyard soils using black oat cultivation and chemical extractants.

Grapevines grown on acid soils with low fertility in southern Brazil are treated with intense foliar applications of copper (Cu) fungicides, resulting in an increased level of Cu in the soil and increased toxicity. The present study evaluated the accumulation and bioavailability of Cu, and soils with varying levels of Cu from the main producing regions of southern Brazil were collected. The forms of Cu present in the soil were assessed using chemical extractants; additionally, oat cultivation was performed, reflecting the use of the plant as an indicator of Cu bioavailability. Cu accumulated in the topsoil, mainly in bioavailable forms, and there was also an increase of Cu up to a depth of 0.4m. Cu was primarily found in the mineral fraction, with apparent saturation of the soil organic matter functional groups. Inceptisol and Alfisol soils with a long history of cupric fungicide application were found to have levels of Cu toxic to oat plants. Furthermore, accumulated copper in Alfisol soil from the Campanha Gaúcha region of the state of Rio Grande do Sul had higher bioavailability compared to Cu accumulated in Inceptisol soil from the Serra Gaúcha region. In addition, the copper concentration in roots was found to serve as an indicator of Cu bioavailability in soil, but not of copper phytotoxicity in plants.

Addressing Geographic Variability in the Comparative Toxicity Potential of Copper and Nickel in Soils

Comparative toxicity potentials (CTP), in life cycle impact assessment also known as characterization factors (CF), of copper (Cu) and nickel (Ni) were calculated for a global set of 760 soils. An accessibility factor (ACF) that takes into account the role of the reactive, solid-phase metal pool in the soil was introduced into the definition of CTP. Geographic differences in fate, accessibility, bioavailability, and terrestrial toxicity were assessed by combining the USEtox characterization model, empirical regression models, and terrestrial biotic ligand models. The median CTPs for Cu and Ni with 95% geographic variability intervals are 1.4 × 10(3) (1.7 × 10(2) to 2.0 × 10(4)) and 1.7 × 10(3) (2.1 × 10(2) to 1.1 × 10(4)) m(3)/kg · day, respectively. The geographic variability of 3.5 orders of magnitude in the CTP of Cu is mainly associated with the variability in soil organic carbon and pH. They largely influence the fate and bioavailability of Cu in soils. In contrast, the geographic variability of 3 orders of magnitude in the CTP of Ni can mainly be explained by differences in pore water concentration of magnesium (Mg(2+)). Mg(2+) competes with Ni(2+) for binding to biotic ligands, influencing the toxicity. Our findings stress the importance of dealing with geographic variability in the calculation of CTPs for terrestrial ecotoxicity of metals.

Remediation of copper polluted red soils with clay materials

Attapulgite and montmorillonite were utilized to remediate heavy metal polluted red soils in Guixi City, Jiangxi Province, China. The effects of clay minerals on availability, chemical distribution, and biotoxicity of Cu and Zn were evaluated. The results provided a reference for the rational application of clay materials to remediate heavy metal contaminated soils. From the sorption experiment, the maximum adsorbed Cu 2+ by attapulgite and montmorillonite was 1501 and 3741 mg/kg, respectively. After polluted red soil was amended with attapulgite or montmorillonite and cultured at 30 and 60 days, soil pH increased significantly compared to the control. An 8% increase in the amount of montmorillonite in soil and 30 days incubation decreased acid exchangeable Cu by 24.7% compared to the control red soil. Acid exchangeable Cu decreased with increasing amounts of attapulgite and montmorillonite, with best remediation effect reached at a dose of 8%. Results also showed that the Cu poisoning effect on earthworms was reduced with the addition of attapulgite and montmorillonite. Montmorillonite showed the best effect, with the addition of a 2% dose the mortality of earthworms decreased from 60% to zero compared to the control. Our results indicated that the bioavailability of Cu in soils was reduced more effectively with the application of montmorillonite than attapulgite.

Fractionation and bioavailability of Cu in soil remediated by EDTA leaching and processed by earthworms (Lumbricus terrestris L.)

Soil remediation with ethylenediamine tetraacetic acid (EDTA) leaching is capable of removing only part of the total metal concentration in the soil, mostly the labile, bioavailable metal species (metal bioavailability stripping). However, reintroduction of remediated soil in the environment exposes the soil to various environmental factors, which could potentially shift nonlabile residual metals back to labile bioavailable forms. We studied the effect of autochthonous earthworm species as model biotic environmental factor on the fractionation and bioavailability of Cu residual in soil after remediation. We used soil from a 50-year-old vineyard regularly managed and treated with CuSO(4)*5H(2)O (Bordeaux mixture) as fungicide. Soil containing 400 mg kg(-1) of Cu was leached with total 15 mmol kg(-1) EDTA. Remediated and nonremediated soil was processed by fully clitellated adult specimens of Lumbricus terrestris L., a prevailing autochthonous soil earthworm species. Cu fractionation, phytoavailability, and oral-bioavailability in processed and nonprocessed soil were determined using six-step sequential extraction, extraction with diethylenediamine pentaacetic acid, and in vitro physiologically based extraction test, respectively. EDTA leaching removed 41% of the pseudototal Cu, mostly from the soil Fe- and Mn-oxides, carbonates, and organic matter. A 2.7-fold decrease in Cu phytoavailability and a 4.4- and 2.8-fold decrease in Cu oral-bioavailability in the stomach and small intestine fractions, respectively, were achieved after remediation. In nonremediated soil, earthworms increased the share of nonlabile Cu in residual soil fraction, while in remediated soil they increased the share of Cu bound to carbonates. A statistically significant 1.1- and 1.7-fold increase in Cu phytoavailability and intestinal oral-bioavailability, respectively, was observed in earthworm processed remediated soil. Cu occurs in various soil "pools" of different solubilities with different chemical characteristics and consequently different functions. By removing the labile part of the metals from the soil during remediation, we disrupt the chemical equilibrium; the nonlabile residual metals left in soil after remediation might become more labile in time in tendency to re-establish that equilibrium. Earthworms alter the physical and chemical properties of soil affecting consequently the fractionation of metals. The increase in earthworm's gut pH due to the excretion of ammonia and/or calcium carbonate into the intestine could lead to the transbounding of metals into the carbonate fraction. However, their activity in remediated soil increased Cu phytoavailability and intestinal oral-bioavailability, and it would, therefore, be improper to generalize the influence of earthworms on metal availability in soil. The results presented here show that residual Cu in remediated soil is affected by environmental factors such as earthworms, which should be considered in evaluating the effect of Cu polluted soil remediation. Information on the behavior of residual metals in soil after its remediation is surprisingly scarce. The development of new effective remediation techniques should imply also the evaluation of postremediation effects on remediated soil. The results presented in this work indicate a possible tool for assessing the effect of biotic environmental factors on residual metals left in soil after its remediation.

The effect of earthworms on copper fractionation of freshly and long-term polluted soils

We investigated the effects of earthworm activity on the bioavailability of Cu in soil. The bioavailable fraction was estimated using sequential extraction, and the results of diethylenetriaminepentaacetic acid (DTPA) extraction were analyzed for comparison. Changes in the Cu fraction were compared in Cu-spiked soil (high bioavailability) and long-term polluted field soil (low bioavailability) with approximately equivalent total Cu concentrations. Earthworm activity decreased the bioavailable fraction in the Cu-spiked soil, where earthworm body Cu concentrations did not affect the bioavailable fraction. Soil pH was not a factor in the bioavailability differences between soils with and without earthworms in this study. The bioavailable fraction appears to be more heavily affected by biological and physical mechanisms than by soil pH. The two extraction methods showed different trends; the bioavailable fraction method was better than DTPA extraction, because the former gives clear insight into the aging process of Cu in soil.

Comparison of soil solution speciation and diffusive gradients in thin‐films measurement as an indicator of copper bioavailability to plants

The toxicity effect concentrations (10% effective concentration [EC10] and 50% effective concentration [EC50]) of total added Cu derived from barley root elongation and tomato growth assays varied widely among 18 European soils. We investigated whether this variation could be explained by the solubility or speciation of Cu in soil solutions or the diffusive gradients in thin-films (DGT) measurement. Solubility and Cu speciation varied greatly among the soils tested. However, the EC10 and EC50 of soil solution Cu or free Cu2+ activity varied even more widely than those based on the total added Cu, indicating that solubility or soil solution speciation alone could not explain intersoil variation in Cu toxicity. Estimated EC10 and EC50 of free Cu2+ activity correlated closely and negatively with soil pH, indicating a protective effect of H+, which is consistent with the biotic ligand model concept. The DGT measurement was found to narrow the intersoil variation in EC50 considerably and to be a better predictor of plant Cu concentrations than either soil solution Cu or free Cu2+ activity. We conclude that plant bioavailability of Cu in soil depends on Cu speciation, interactions with protective ions (particularly H+), and the resupply from the solid phase, and we conclude that the DGT measurement provides a useful indicator of Cu bioavailability in soil.

Chemical extractability of copper added to soils at two different concentrations in a batch experiment

The bioavailability of Cu in soil is dependent on the desorption into the soil solution of Cu from the surfaces of soil colloid materials (viz. organic matter, the clay fraction, Fe and Mn oxides). Six samples of natural soils of the Typic Eutrochrepts type were used in this work to determine Cu extraction by using 0.1M Na2EDTA and 1M NH4OAc solutions. The soil samples were spiked with 3 or 1.5 g Cu kg-1 soil and analysed in batch tests. Most of the Cu added was sorbed within 24 h. Both single-step extractions with 0.5M Na2EDTA, 0.5M Ca(NO3)2 and 1M NH4OAc solutions, and sequential extractions, were performed. The proportions of copper extracted by the NH4OAc solution were found to be higher than those extracted by the Na2EDTA and Ca(NO3)2 solutions. The amount of Cu EDTA formed exhibited a high, negative correlation with OC, Fed and Mnd at an added copper concentration of 3 g kg-1. There were no significant differences between the mean percentage of copper extracted by the Na2EDTA solution from spiked and natural soils. There were, however, significant differences between the amount of copper extracted by the NH4OAc solution at both added Cu concentrations and that extracted from natural soils. Sequential extraction was highly efficient at both Cu concentrations. Copper was found to be primarily associated to carbonates. The correlations between non-extractable Cu (residual fraction) and organic carbon, Fed and Mnd (crystalline oxides) were highly significant at the higher Cu added concentration (3 g kg-1). On the other hand, that between residual Cu (non-extractable Cu) and pH was negative and highly significant at the lower Cu added concentration (1.5 g kg-1). There were no significant differences between the mean percentages of copper sequentially extracted and copper extracted by the Na2EDTA, NH4OAc, Ca(NO3)2 solutions at the two added Cu concentrations (3 g kg-1 and 1.5 g kg-1).

Identification of copper-induced genes in Pseudomonas fluorescens and use of a reporter strain to monitor bioavailable copper in soil

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.

Identification of copper-induced genes in Pseudomonas fluorescens and use of a reporter strain to monitor bioavailable copper in soil

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 Tn 5:: 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.

Effect of manuring practices and increased copper concentrations on soil microbial populations

Addition of piggery manure to soils over 5 yr (manured soils) increased the amount of Cu extracted by EDTA and diethylenetriaminepentaacetic acid (DTPA) compared to selected reference soils. DTPA-extractable Cu ranged from about 3 to 10 μg Cug −1 soil in the manured soils and from about 1 to 2 μg Cu g −1 soil in the reference soils. Although the soil Cu concentrations in the manured soils were many times smaller than currently permitted, the increase in DTPA- or EDTA-extractable Cu was correlated with a 10- to a 1000-fold increase in the number of aerobic Cu-resistant bacteria. Although the Cu was mainly concentrated in the plough layer (15 cm) of the manured soils, Cu-resistant bacteria were also detected down to 110cm soil depth. In contrast, no such differences were found between the total number of colony forming units, total microbial biomass or the degree of Cu-resistance of fungi and anaerobic bacteria between manured and reference soils. It is suggested that the degree of Cu-resistance of the aerobic soil bacteria may provide a sensitive measure of Cu bioavailability in soil. In general, bacteria were more sensitive to Cu than fungi. All of the 42 Cu-resistant bacterial strains investigated were oxidase-positive and 50% of the strains were pigmented. In contrast, only 20% of the 37 Cu-sensitive bacterial strains investigated were oxidase-positive and none were pigmented. Cu-resistant bacteria exhibited more resistance to several antibiotics and heavy metals than Cu-sensitive bacteria.