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a Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
b Department of Microbiology, Danish Veterinary Institute, Bülowsvej 27, 1790 Copenhagen V, Denmark
* Corresponding author (oln{at}kvl.dk).
Received for publication December 20, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: [Cu]bio, bioavailable copper concentration • [Cu]tot, total copper concentration • DMM, Davis Minimal Medium • DOM, dissolved organic matter
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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When different soils are compared, the total Cu content may not correlate well with toxicity and bioavailability to microorganisms. A better correlation has been found between bioavailability and the activity of distinct Cu species, for example, Cu2+
determined by ion-specific electrodes or estimated by equilibrium computation (Sauvé et al., 1998; Vulkan et al., 2001). The results underline the fact that soil properties, such as the content of minerals, organic matter, and pH, exert a strong influence on Cu bonding and bioavailability (McLaren and Crawford, 1973; Sauvé et al., 1997; Römkens et al., 1999) pointing to the need for corresponding chemical and biological analysis.
Microbial toxicity tests provide a direct measure of Cu bioavailability, but the results can be influenced by the presence of other toxic metals, which often accompany Cu contamination in soil (McGrath et al., 1995; Preston et al., 2000). This problem can be circumvented by use of Cu-specific reporter organisms. The Pseudomonas fluorescens Cu-reporter strain DF57-Cu15 contains luxAB reporter genes controlled by a Cu-induced promoter and consequently emits bioluminescence in response to Cu that is available to this specific organism (Tom-Petersen et al., 2001). The strain has been tested on soil solutions from Cu-amended soils, where the bioluminescence response was highly correlated to the Cu concentrations used (Tom-Petersen et al., 2001).
Environmental risk assessment of heavy metals in soils is frequently based on toxicity tests performed on freshly spiked soil kept at a stable humidity. However it has been questioned whether these soil tests are representative of field soils where Cu has had a long time to distribute between different bonding forms and where diffusion of Cu into soil sorbents may restrict Cu bioavailability (van Gestel, 1997; McKenzie, 1980; Bruemmer et al., 1986; Swift and McLaren, 1991). Retention of Cu could potentially lead to a continuous decrease in bioavailability, which would be important to consider when predicting Cu toxicity in the long term.
The aim of the present work was to determine time- and moisture-dependent changes in total as well as in bioavailable Cu in water extracts from soil. Soil were amended with Cu and incubated under stable or variable moisture conditions for time periods ranging between 1 h and 220 d. Measurements of total Cu concentrations in water extracts were performed using furnace atomic absorption spectrometry. Correspondingly an in vitro assay employing the P. fluorescens DF57-Cu15 reporter strain was used to estimate Cu that was biologically available to the reporter strain under the defined assay conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Soil System Setup and Chemical Analysis
Each soil system consisted of 1 g of soil (dry weight) contained in 9-mL polyethylene centrifuge tubes (Ole Dich, Rødovre, Denmark). Reverse osmosis–purified water (Milli-Q; Millipore, Billerica, MA) containing different concentrations of Cu (as CuSO4; Merck, Darmstadt, Germany) was added on top of the soil with a pipette obtaining soil systems amended with 0, 100, 200, 400, and 800 µmol Cu kg–1 soil (6.4–51 mg Cu kg–1 soil). The soil was wetted with reverse osmosis–purified water to 80, 100, or 185% of the field capacity and incubated with caps to maintain a stable humidity throughout the experiment. For other soil systems humidity varied. Soil systems referred to as "dry" were initially wetted to 80% field capacity and incubated without caps allowing the soil to dry out. Soil systems denoted "variable" were wetted to 130% field capacity and incubated without caps. Every seventh day these soil systems were rewetted to 130% field capacity. Soil systems wetted to 100% field capacity were incubated in the dark at 20°C for time periods ranging between 1 h and 220 d, after which they were subjected to Cu extraction. Soil systems with other treatments were incubated under identical conditions and extracted after 4 wk.
Extraction of Cu from the soil was performed by adding up to 6 mL Milli-Q water to the soil, depending on the humidity of the soils at the time of extraction. Thereby a constant extraction volume of 6 mL and a constant solid to solution ratio of 1:6 was obtained for all systems. The capped soil system tubes were placed horizontally on a shaking-table operating at 150 rpm for 2 h at room temperature. After centrifugation at 10000 x g for 5 min at room temperature the supernatants were collected for further analysis.
Measurements of Cu in the supernatants involving the Cu-specific reporter strain were performed immediately after extraction while the supernatants used for analysis of total Cu concentration were conserved in 0.1 mol L–1 (final concentration) analytical-grade HNO3 (Merck) and stored at 5°C until analysis.
The pH values of soil systems were measured with a Model 91-02 electrode (Thermo Orion, Beverly, MA) using a Model PHM28 pH meter (Radiometer, Copenhagen, Denmark). Before measurements soil systems were suspended in 6 mL Milli-Q water, shaken for 2 h at 150 rpm and subsequently equilibrated for 1 d at room temperature.
Total Cu concentration in the water extracts, referred to as [Cu]tot, was determined by graphite furnace atomic absorption spectrometry (GFAAS) using a PerkinElmer (Wellesley, MA) 5100 with Zeeman correction. The detection limit for Cu was 2 nmol L–1.
The P. fluorescens DF57-Cu15 Reporter Assay
A reporter strain was used to estimate Cu fractions able to induce specific reporter gene expression. The strain, P. fluorescens DF57-Cu15, is a Tn5::luxAB mutant strain responding specifically to Cu as described in Tom-Petersen et al. (2001). Another P. fluorescens Tn5::luxAB mutant strain denoted DF57-40E7, which has a Cu tolerance comparable with DF57-Cu15 and expresses the luxAB genes constitutively (Tom-Petersen et al., 2001), was exposed to water extracts in parallel experiments. The bioluminescence signal from this strain was used as control for conditions inhibiting expression of bioluminescence. In brief, DF57-Cu15 and DF57-40E7 were grown at room temperature in Davis Minimal Medium (DMM; Difco, Detroit, MI) adjusted to pH 7.2 and supplemented with 0.4% glucose (Kragelund and Nybroe, 1994). Exponential phase-cells were harvested by centrifugation (5000 x g, 10 min, room temperature) and resuspended in fresh DMM containing 0.8% glucose to a final cell density of 2.5 x 108 cells mL–1. Cell suspensions (500 µL) were amended with soil supernatant (500 µL) from each system, and incubated for 1.5 h at room temperature before bioluminescence from the luxAB reporter construct was measured by luminometry (Model 1253; BioOrbit, Turku, Finland) essentially as described by Tom-Petersen et al. (2001). To normalize data from different experiments standard curves of bioluminescence obtained from DF57-Cu15 exposed to increasing concentrations of CuSO4 in Milli-Q water were established. These curves were used to convert bioluminescence values obtained from soil samples to estimates of concentration of Cu that is biologically available to the reporter strain under the specific assay conditions. We hereafter refer to this concentration as "bioavailable" copper ([Cu]bio) when making comparisons with the fraction of total Cu in water extracts ([Cu]tot).
Modeling and Statistics
All soil system experiments were made in duplicate, and all experiments were repeated independently two times. Significance of regression lines was tested using an f test. Significance of difference (P values) between values was tested using Student's t test (two-tailed) after an f test. Equilibrium speciation of test solutions was calculated using PHREEQC Version 2.6 and applying the MINTEQ database (Pankhurst and Appelo, 1999). Copper phosphate complexes not included in the database were included in the calculations; stability constants were taken from NIST (Martell et al., 1998).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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4–2 and the distribution of Cu among the different complexes remains constant with increasing [Cu]tot. Bioavailability of Cu to microorganisms depends on Cu speciation but is furthermore influenced by several other factors such as the concentration of other ions, and microbial assimilation of specific Cu–organo complexes (Campbell, 1995). At present there is no clear understanding of the bioavailability of different Cu species to Pseudomonas. However, it has been demonstrated that availability of some Cu–organo complexes to Pseudomonas spp. decreases with increasing stability of the Cu complex (Azenha et al., 1995; Teresa et al., 1997).
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The PHREEQC calculations were performed to test for the sensitivity of DMM toward changes in Cu speciation due to competitive ligands from a soil extract (L). Figure 2A shows the change in Cu speciation with an increasing concentration of L at constant stability constant of the Cu–L complex, while Fig. 2B shows the effect of increasing stability of the Cu–L complex at constant concentration of L. It appears that Cu speciation in DMM is not markedly affected at ligand concentrations below 10–4 mol L–1 and at log K values of less than 5. Low molecular weight carboxylic acids, phenols, and macromolecular humic substances (fulvic and humic acids) present in dissolved organic matter (DOM) represent important Cu ligands (Hodgson et al., 1966; McBride et al., 1997; Sauvé et al., 1997; Temminghoff et al., 1997). Ligands in the DOM fraction typically have log K values for Cu complexes in the range 2 to 7 (Strobel, 2001; Strobel et al., 2001). Soil solution concentrations of aliphatic carboxylic acids and humic substances above 1 mmol L–1 are quite common (Strobel et al., 2001) and therefore in general we may expect that ligands present in soil extracts may change Cu speciation in DMM and thus affect bioavailability to the DF57-Cu15 reporter strain. In concordance with this assumption, the current assay has been used to show that soil amendments with barley straw and pig manure decrease "bioavailable" Cu relative to the total Cu content in water extracts (Tom-Petersen et al., 2001).
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Time Effects on Copper Concentrations in Water Extracts
Soil systems amended with different levels of Cu and kept at a constant soil moisture of 100% field capacity were analyzed after incubations between 1 h and 220 d.
Chemical Analysis
At all incubation times [Cu]tot increased linearly with the amounts of Cu added to the soils producing regression lines with R2 > 0.99 (P << 0.01) (Fig. 3A)
. After 1 h of incubation only between 1.4 and 2.4% of the amended Cu was recovered in the water extracts indicating that the vast majority of Cu was rapidly removed from the soil solution (Fig. 3B). The decrease in [Cu]tot continued throughout the experimental period; the fraction of amended Cu recovered as water-extractable Cu showed a logarithmic decrease with time (R2 > 0.96; P < 0.01) (Fig. 3B). The [Cu]tot at the end of the 220-d period ranged between 25 and 29% of [Cu]tot obtained at the first sampling with larger proportional recoveries for the lower Cu amendments.
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The data in Fig. 3A are easily converted to sorption isotherms if [Cu]tot is used to represent "equilibrium" concentrations of Cu in solution. These isotherms are near linear, and show increasing slopes (approximately proportional to inverse slopes of lines in Fig. 3A) with increasing reaction time. As the slope of the sorption isotherms is a measure of the bonding strength or affinity it is inferred from Fig. 3A that the bonding strength of Cu increases by a factor of 3 when the reaction time increases from 1 h to 220 d. The linearity of the sorption isotherms also indicates that the sorption capacity for Cu is not reached even at the highest dose of Cu added. Linear isotherms have also been obtained by McLaren et al. (1981) and Gao et al. (1997).
The processes that lead to decrease in extractability of Cu and other heavy metals in soils are not fully understood. It has been proposed that decrease in solubility is mediated by diffusion of sorbed metal ions into micropores or internal sites in the structure of soil minerals (McKenzie, 1980; Bruemmer et al., 1986; Swift and McLaren, 1991). Diffusion-dependent sorption typically follows the parabolic law (Fick's second law), in which the sorbed amount is linearly correlated with the square root of time. Such a relationship has been observed for the retention of zinc in a calcareous soil (Ma and Uren, 1997). However, in our experiments plots of sorbed Cu versus square root of time gave poor linear relationships (R2 between 0.38 and 0.81, mean = 0.62), suggesting that other factors contribute considerably to the observed Cu retention. It is possible that diffusion into humus aggregates or microbial activity is of importance for the time-dependent Cu retention observed in this study.
The percentage of [Cu]tot relative to amended Cu is higher for the low Cu amendments than for the high Cu amendments (Fig. 3B). This feature is partly explained by the native Cu present in the soil. As can be observed in Fig. 3A the regression lines extrapolated to zero Cu amendment show positive intercepts with the y axis representing the water extractability of native Cu in the soil. The presence of ligands in soil solutions presumably is another reason for the higher proportion of Cu kept in solution at lower Cu amendments. As discussed above these ligands are mainly contained in the DOM fraction. Thus at low Cu amendments DOM possibly complexes a higher proportion of Cu than at higher amendments.
Corresponding Chemical and Biological Analysis
Figure 4A
shows the [Cu]bio in the water extracts measured with the DF57-Cu15 Cu reporter strain. As for [Cu]tot we found good linear correlations between added Cu and [Cu]bio (R2 > 0.99; P << 0.01) for each incubation time, while water extracts from control soil without added Cu did not induce the Cu reporter (data not shown). As for [Cu]tot, a time-dependent decrease in [Cu]bio could be seen (Fig. 4B). The decline could be described by logarithmic decreasing regression lines when [Cu]bio was plotted as function of time (R2 > 0.94; P < 0.01). The decline in [Cu]bio observed for each Cu amendment is probably primarily a consequence of the concomitant reduction in [Cu]tot. The [Cu]bio constituted between 0.94 and 1.06% of the added Cu at the first sampling after 1 h, and was reduced to between 0.33 and 0.38% after 220 d of incubation. In contrast to the higher proportional [Cu]tot recoveries from low Cu amendments compared with high Cu amendments (see Fig. 3B), proportional [Cu]bio recoveries showed a tendency to increase with increasing Cu amendments.
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The results indicate that soluble Cu is distributed among several chemical forms with varying degree of bioavailability to bacteria, and that the relative speciation of Cu in solution depends on the degree of Cu loading. According to the previous discussion a lower [Cu]bio to [Cu]tot ratio is explained as an effect of ligands from the soil extracts that form Cu complexes, which are less bioavailable than the Cu species in the DMM standards. The higher [Cu]bio to [Cu]tot ratio at higher Cu amendments is likely to be an effect of the limited complexation capacity of the soil-derived ligands.
It can be seen from Fig. 4C that the [Cu]bio to [Cu]tot ratio tends to increase with time, reflecting the fact that the concentration and/or bonding strength of ligands in the soil water extracts changes with time. In a comparable study of Cu-contaminated soil it was found that the proportion of Cu2+ ions increased relative to total soluble Cu with incubation time (Sanders, 1982). It is possible that the concentration of DOM decreases over time due to mineralization, and hence that a similar proportional increase in Cu2+ is responsible for the relative increase in [Cu]bio with time in our study.
In relation to ecotoxicity studies the time-dependent decrease in "bioavailable" Cu found in this study emphasizes the need to consider the time period from Cu amendment to exposure of added indicator microorganisms. Also the time-dependent decrease would be important to consider in risk assessments of short-term and long-term Cu effects on indigenous soil microorganisms living in the soil soluble phase.
Moisture Effects on Copper Concentrations in Water Extracts
To examine the effects of soil humidity, soil systems, which had been amended with increasing Cu concentrations, were sampled after incubation for 4 wk under different moisture conditions.
Chemical Analysis
Figure 5A
shows [Cu]tot versus the amount of added Cu for soil systems representing different moisture conditions. For all moisture conditions the curves were well fitted by linear regression (R2 > 0.93; P < 0.01), in agreement with previous measurements (Fig. 3A).
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Although humid conditions are needed for Cu to be immobilized, alternating drying and rewetting has been reported as a method to obtain rapid partitioning of heavy metals in soil (Bruemmer et al., 1986; Ma and Uren, 1997). However, our results do not indicate that shifts in humidity lead to a different distribution of Cu between soluble and solid phase when compared with incubations at stable humid conditions.
Corresponding Chemical and Biological Analysis
Figure 5B shows that the [Cu]bio to [Cu]tot ratio in the water extracts depended on the moisture conditions used. Soil systems with moisture contents of 80 or 100% field capacity had a higher [Cu]bio to [Cu]tot ratio than found for the remaining soil systems. Dry soil systems had a relatively low ratio of 0.4 to 0.6 and did not show a clear relationship (R2 = 0.36; P > 0.1) between [Cu]bio to [Cu]tot ratio and initial Cu amendments. For the remaining soil systems a general increase in the ratio with increasing amendments of Cu was observed and could be described by logarithmic regression lines (R2 > 0.76; P < 0.01). A ratio higher than 1.0 was observed at a single sampling reflecting that Cu bioavailability in the soil extracts was higher than in the DMM standard solution. This observation illustrates that [Cu]bio should not be interpreted as absolute units of bioavailability and implies that ligands from the soil solution may form complexes that are more bioavailable than the Cu species in the test medium.
"Bioavailable" Cu concentration was influenced by the humidity conditions under which the soils had been incubated. Generally, the [Cu]bio to [Cu]tot ratio decreased with decreasing humidity of the soil. Possibly a low DOM turnover in dry soil as a consequence of reduced microbial activity can partly explain a higher proportion of strongly complexed Cu in these soils. For the dry soils a lower [Cu]bio to [Cu]tot ratio would also be expected as consequence of a relative increase in organic matter solubility as discussed above. The water-saturated soil constituted a notable exception. Although [Cu]tot was approximately similar in extracts from 100% field capacity and water-saturated soils, a significantly (P < 0.05) lower [Cu]bio to [Cu]tot ratio was observed in the water-saturated soil solutions for each of the different Cu loads added. It is possible that anaerobic conditions occurred in the water-saturated soils, which facilitated the formation of less bioavailable Cu species, for instance complexes with organic acids formed by fermentative processes or with carbonate as consequence of increased CO2 levels (Dassonville and Renault, 2002).
The impact of soil moisture on Cu "bioavailability" indicates that incubation conditions for Cu-spiked soil samples can be important for the outcome of ecotoxicity tests performed in soil extracts.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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used for production of bioluminescence standard curves (1 Davis Minimal Medium [DMM
] to 1 CuSO4 aqueous solution).
