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Copper bioavailability in the rhizosphere of maize (Zea mays L.) grown in two Italian soils
Chemosphere 64 (2006) 1972–1979 www.elsevier.com/locate/chemosphere
Copper bioavailability in the rhizosphere of maize (Zea mays L.) grown in two Italian soils I. Cattani *, G. Fragoulis, R. Boccelli, E. Capri Istituto di Chimica Agraria ed Ambientale, sez. Vegetale, Universita` Cattolica del Sacro Cuore, Via Milano, 24, 26100 Cremona, Italy Received 7 March 2005; received in revised form 30 December 2005; accepted 3 January 2006 Available online 14 February 2006
Abstract In this study, potentially bioavailable copper was estimated in two soils (a fungicide polluted and a natural soil) using a passive sampling technique, DGT. As plants can alter copper mobility and bioavailability in the soil, the rhizosphere properties of Zea mays L. were investigated using rhizoboxes. Compared to the total concentration, the soluble and the potentially bioavailable copper concentration in the bulk soils were generally low (less than 0.20% and 0.06% respectively), with a sixfold increase in the rhizosphere of the polluted soil. Our results suggest that maize cultivation in a polluted vineyard soil could increase the potentially available fraction of copper. DGTs showed a good sensitivity to soil properties and to root-induced changes in the rhizosphere, but the potentially bioavailable copper could not be related to the copper concentration in the above ground parts of maize. The results suggest that DGT may be used to predict some effects of the cultivation of polluted soils, for example, metal mobility and increased availability, but they cannot mimic the uptake of a tolerant plant. For both soils, dissolved organic carbon (DOC) concentrations were threefold higher in the rhizosphere than in the bulk soil, whilst bioaccumulation in leaves and roots was not significant. DOC production, usually effective in ion mobilization and assimilation, may help also in the reduction of Cu uptake at toxic concentrations. The sequestration of available Cu in soil and soil solution by DOC seems to contribute to maize tolerance. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Copper; Bioavailability; DGT; Rhizon sampler; Rhizobox
1. Introduction The prevention of the accumulation of heavy metals, such as copper, in soil is one of the prerequisites of sustain-
Abbreviations: CuDGT, potentially bioavailable Cu measured by DGT; CuDTPA, extractable copper; CE, effective concentration; C0, initial dissolved concentration; Cusol, Cu concentration in soil solution; Cutot, total Cu; Dg, diffusion layer thickness; DGT, diffusive gradients in thin films; DIFS, DGT-induced fluxes in soils; D0, diffusion coefficient in water; DOC, dissolved organic carbon; Ds, diffusion coefficient in soil; /, soil porosity; Kd, distribution coefficient for labile metal; M, mass accumulated by DGT; Pc, soil particles concentration; R, ratio between CuDGT and Cusol; Tc, response time of the solid phase/solution equilibrium; WHC, water holding capacity. * Corresponding author. Tel./fax: +39 0372499122. E-mail address: [email protected] (I. Cattani). 0045-6535/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.01.007
able agricultural production (Witter, 1996). Risk assessment of copper contaminated soils needs to estimate its bioavailability to organisms and plants in order to understand the concentration to which they are exposed, and the potentially toxic effects (Ye et al., 2003). Copper is an essential element for every organism at low concentrations, however excess amounts may cause metabolic alterations, even in tolerant plants (Monni et al., 2000; Orsega et al., 2003). The ‘bioavailable’ copper can be best described as that portion of soil copper available for intake by a given organism. Consequently, the fraction bioavailable to plants is the amount that can be taken up by the roots of a given plant species. It is well known that copper bioavailability in soil depends on soil properties (pH, redox potential, CEC, amount and nature of organic matter, soil minerals). The Cu that crosses the plant membrane is considered as the part
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
that is ‘free’, i.e. not complexed with organic species in solution or absorbed to soil particles (Zhang et al., 2001) and intake occurs essentially via uptake by the roots from the soil solution (Marchner, 1995; Fitter, 1997; Hinsinger, 1998). The assessment of copper bioavailability is difficult, as the concentration of copper in the soil solution represents only a portion of exchangeable and slightly complexed metal and does not give a measure of the total labile pool (Qiao et al., 2003). Furthermore, plants can alter the mobility and bioavailability of copper in the rhizosphere (MacLaughlin et al., 1998; Chaignon et al., 2002; Chaignon and Hinsinger, 2003) by modifying pH (Hinsinger, 1998, 2001a,b; Chaignon et al., 2002) or by releasing soluble organic compounds to complex copper (Lombi et al., 2001; Chaignon and Hinsinger, 2002). These findings conflict with the existing Italian legislation (Ministerial Decree, 1999), that is based on a risk assessment procedure that takes into account only the total concentration of copper. The free ion concentration in soil solution and the labile amount in the solid phase represent the real potentially toxic amount (Sauve´ et al., 1996a,b), that is, the potentially bioavailable concentration of copper. Official analytical methods only provide a measurement of the DTPA/EDTA extractable copper concentration, which has been correlated with plant accumulation. In practice, however, this correlation is not always reliable (Marchner, 1995; MacLaughlin et al., 1998; Hinsinger, 2001a,b). In recent years it has been recognised that the measurement of free ion activity in soil solution may provide a better indication of metal availability and potential bioaccumulation (Sauve´ et al., 1997; Sauve´, 2002). Conversely, Ginocchio et al. (2002) and Song et al. (2004) reported a poor correlation between free Cu2+ activity and plant Cu concentration. They proposed rhizosphere-induced changes as a possible explanation of this discrepancy. Recently, Zhang et al. (2001) introduced the concept of effective concentration, CE, which represents both the soil solution concentration and the metal supplied from the solid phase. CE is measured using the technique of diffusive gradients in thin films (DGT) (Davison and Zhang, 1994; Zhang et al., 1998) and the authors showed that this value correlated very well with plant uptake. They considered that the DGT-soil system mimics diffusion and labile metal release and that CE represents bioavailable fraction and can be used to probe the dynamics of system. Song et al. (2004) also found a good correlation of both soluble Cu and CE with plant concentration. The aim of this work was to estimate the bioavailability of copper in the bulk soil and rhizosphere of two different soil types, in order to obtain a better understanding of copper bioavailability and subsequent crop uptake. 2. Materials and methods 2.1. Soil sampling and characterization Soil samples were collected from the top soil, to a depth of approximately 0.3 m, from sites in the locality of Cem-
1973
bra (46°10 0 4000 N, 11°13 0 5000 E), near Trento (Italy). The two soils were of the same geological origin but differ in terms of soil management (agricultural versus natural soil). Moist soil was passed through a 2 mm mesh sieve and stored at 4 °C prior to use in experiments. Soil properties, including pH, water holding capacity (WHC), texture, cation exchange capacity (CEC), organic matter (OM), total (Cutot) and DTPA–extractable copper (CuDTPA) content (Table 1), were measured using official analytical methods (SSIS, 2000), whereas dissolved organic carbon (DOC) was measured in soil solution using the UV absorbance method previously published by Brandstetter et al. (1996). 2.2. Soluble Cu (Cusol) measurement Rhizon soil moisture samplers (Rhizosphere Research Products, Wageningen, The Netherlands) were used to extract soil solution from 500 g of each soil at approximately weekly intervals. Measurements were made in triplicate. The samplers consist of 5 cm of inert porous plastic tubing (2.5 mm diameter), capped with nylon wire at one end. The capped end was inserted into each soil pot during filling. The other end was attached to a 30 cm long PVC/ PE tubing, joined to a female lock. A syringe needle was connected to the female lock and inserted into a 10 ml glass vacuum tube, in order to extract the soil pore water by vacuum. In this way, approximately 9 ml of soil solution was extracted overnight. Samplers were washed before use following the technique of Tiensing et al. (2001). The soil humidity was maintained at 80% of WHC during the experiment. The soluble Cu (Cusol), that includes the fraction of Cu potentially available to plants in soil solution, was measured using inductively coupled plasma atom emission spectrometry (ICP-OES, Spectro CIROSCCD) following nitric acid– hydrochloric acid microwave assisted digestion (ETHOS, Milestone, FKV). 2.3. Potentially available Cu (CuDGT) measurement The potentially available copper concentration (CuDGT) was determined using the DGT technique. DGT devices were deployed in duplicate and at 6 h intervals (from 24
Table 1 Experimental soils characteristics Soil
Forest
Vineyard
Texture pH WHC (%) Bulk density (g cm3) CEC (cmol kg1) OM (g kg1) DOC (mg l1 C) CaCO3 (g kg1) CuDTPA (mg kg1) Cutot (mg kg1)
Sand 7.89 35 1.3 16.8 36.6 27.4 1.2 4.3 18.4
Loamy sand 8.52 23 1.4 8.3 15.2 19.2 118.3 113 183
1974
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
to 310 h) in each soil, following the technique used by Hooda et al. (1999). The mass of soil used was 100 g. Soil moisture was kept at 80% of WHC and readjusted daily. All experiments were conducted at 30 °C. Copper bound by DGT resin was measured by ICP-OES, following acid microwave assisted digestion, and CuDGT values were calculated using the equations provided by DGT Research Ltd (Lancaster, UK, web: www.dgtresearch.com). DGT performance was evaluated according to the method used by Zhang and Davison (1995), in two replicates. After acid digestion of the DGT resin, the mean recovery of Cu in acid solution was 95%. The dynamic numerical model of DGT-induced fluxes in soils (DIFS) (Harper et al., 2000), was used to calculate the following variables, representative of the soil system dynamic: (1) the pool sizes of labile metal, (2) the distribution coefficient for labile metal (Kd) and (3) the response time (Tc) of the solid phase/solution equilibrium. The ratio R = CuDGT/Cusol represents the ability of the soil to resupply local pore water concentrations. In this work, R was calculated and Kd and Tc were derived from the best model fit of plots of R versus deployment time. The DIFS model assumes the first order exchange between solid phase and solution, and diffusional transport in both soil solution and the DGT device. After calibration for the Kd and Tc parameters, the model was used to quantify dynamic processes controlling metal uptake from soils. 2.4. Rhizosphere trials The experimental soils, previously sieved to 2 mm, homogenized and frozen at 18 °C, were equilibrated at room temperature for a week and placed into the rhizobox described by Fitz et al. (2003a). Three replicates were prepared. The bulk density of the vineyard soil was 1.4 g cm3, and the bulk density of the forest soil was 1.3 g cm3. The root compartment was separated from the rhizosphere–soil compartment by a 30 lm mesh size nylon, in order to allow elongation of the root hairs. We decided against the use of the slicing device to separate the rhizosphere into 0.5 mm root parallel sections because the soil texture made separation at this thickness very difficult and the amount of soil in each sample would be insufficient for analysis. The 2-mm section of soil close to the root compartment (rhizosphere) was separated from the remaining soil (bulk soil) using the previously cited slicing device (Fitz et al., 2003b). pH, DOC, CuDTPA, Cusol and CuDGT were measured in samples of both the rhizosphere and bulk soil. As the weight of the soil layers was only approximately 35 g, soil solution was extracted by centrifugation, and DGT experiment was performed for 24 h using an acrylic plate adaptor, described by Fitz et al. (2003b). Soil solution and DGT gel were digested and analyzed as previously described. Seeds of maize (Zea mays L.) were germinated in Petri dishes on wet cotton-wool in the dark for three days. Three plantlets were placed in the upper compartments of the rhizobox and, after about 10 days, the upper compartments
were placed and fixed on the lower compartment. The soil water content (determined immediately after harvest) was about 15% for the vineyard soil and 23% for the forest soil (approximately 65% of the maximum WHC of each soil). The experiment was performed in a greenhouse for a period of two months, at a temperature of approximately 20–22 °C during the day and 16–18 °C at night. Fertilization was avoided because it may affect copper mobility in soil. Harvested plants were divided into roots and aerian parts, weighed, dried for 24 h at 105 °C and analyzed following nitric acid digestion in the microwave. 3. Results and discussion 3.1. Kinetics of metal exchange between soil and soil solution without plants Table 2 shows the comparison of soluble with potentially bioavailable, total and extractable Cu, measured in each soil before the rhizobox experiment. Cusol and CuDGT are generally low in comparison with total and extractable Cu, but the vineyard soil exhibits higher concentrations of these parameters (365 and 85 lg l1 for the vineyard soil compared to 20 and 15 lg l1 for the forest soil). According to Zhang et al. (2001) and Song et al. (2004), these values would lead to the prediction of a higher Cu availability in the contaminated soil, even if free Cu activity was not estimated. Experimental data and DIFS simulation of R (CuDGT/ Cusol) for Cu in both soils are shown in Fig. 1. We used the model in both simulation and parameter estimation mode (Harper et al., 2000), to improve the fitting between the model simulation and experimental data. Tc and Kd were obtained as fitting parameters. Even though, theoretically, a large number of data points are needed to assess the goodness of fit, our experimental data show a good agreement with the simulated R for both soils (Fig. 1). R values for the vineyard soil are always lower than for the forest soil. For the vineyard soil, values of R are less than 0.25 (Table 2). In both soils, R decreases with time and, as Table 2 CuDGT concentrations (average of two values) at different sampling time (time measured at the DGT retrieval moment) CuDGT (lg l1)
Sampling time (h) 24 72 91 169 240 310
1
Cusol (lg l ) Cusol/Cutot (%) Cusol/CuDTPA (%)
Forest
R
Vineyard
R
14.5 11.5 10.1 8.1 6.9 6.1
0.72 0.57 0.50 0.40 0.34 0.30
85 63 54 49 52 43
0.23 0.17 0.15 0.13 0.14 0.12
Forest
Vineyard
20.2 0.11 0.47
365 0.20 0.32
R represents the ratio CuDGT/Cusol. Comparison of Cusol (mean values),
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
(a) forest soil 1.0 0.8
R-simulated DIFS R-measured DGT
R
0.6 0.4 0.2 0.0 0
50
100
150 200 Time (h)
250
300
350
0.8
R-simulated DIFS R-measured DGT
R
0.6 0.4 0.2 0.0 50
tially buffer its high copper content, probably by reducing the immediate bioavailability and so reducing the potentially acute toxicity. However, the R values obtained for the vineyard soil could be low simply because DGT does not measure Cu bound on colloids or trace metal complexes in soil solution, whereas Cusol includes these species. The fact that after 24 h of deployment the estimated R was more than 0.8 for the forest soil (Fig. 1), suggests that complexation was not important in this soil or in other soils with similar DOC content, including such vineyard soil. 3.2. Rhizosphere characteristics and copper uptake by maize
(b) vineyard soil 1.0
0
1975
100
150 200 Time (h)
250
300
350
Fig. 1. Experimental R time dependence on (a) forest soil and (b) vineyard soil. The line shows the optimal DIFS model fit. R represents the ratio CuDGT/Cusol (CuDGT, concentration accumulated by DGT; Cusol, soil solution concentration; DIFS, DGT-induced fluxes in soils).
expected, with a decrease in the amount of labile Cu bound to the solid phase. There is a peak in R (0.72 in the forest soil and 0.23 in the vineyard soil, corresponding to the highest values of CuDGT) during the first hours of deployment. This could be due to the initially higher flux to the diffusion layer from the soluble phase than from the solid phase. In the forest soil, the major depletion of soil solution concentration at the DGT interface (greater decrease in R) occurs within 92 h of deployment. In the vineyard soil, R decreases more slowly over time (50% after 310 h). Values of Kd and Tc and other input parameters needed for model simulation of R are presented in Table 3. As DGT takes Cu from soil solution, it appears that the forest soil has a short response time, with rapid resupply from the solid phase and a slow depletion over time of the available Cu pool. By contrast, the vineyard soil has a longer response time and so very little resupply from the solid phase, leading to a progressive depletion. It appears that the vineyard soil is able to par-
Fig. 2 shows the chemical characteristics (pH, DOC, CuDTPA, Cusol, CuDGT and ratio CuDGT/Cusol) measured in the rhizosphere and bulk soil of the forest and vineyard soils. It appears that in the rhizosphere of both soils, independent of their characteristics, the cation and anion uptake mechanisms of plants may decrease the soil pH, whereas rhizodeposition/exudation mechanisms may increase the DOC content. Comparing the rhizosphere data, Cusol is 200-fold and CuDGT is 50-fold greater (2250 and 452 lg l1 respectively) in the vineyard soil than in the forest soil. There is also a significant increase (600%) of both Cusol and CuDGT in the rhizosphere of the vineyard soil. As previous studies on copper toxicity (e.g. Ouzounidou et al., 1992; Giller et al., 1998) refer to free copper concentrations in soil solution, we are unable to establish if Cusol values in the rhizosphere of the vineyard soil could be potentially dangerous for soil fauna, microorganisms and plants. Nevertheless, the increase in potentially available Cu and the results of Song et al. (2004) suggest that intake and assimilation of copper could be increased with maize growing. If compared to the bulk soil, the R value for the vineyard soil rhizosphere is less than 25% (Fig. 2), as for the bulk soil. DGT has been described as a surrogate of the plant/soil system (Zhang et al., 2001). On this basis, our results suggest that mobilization from soil to soil solution is slower than root uptake, and it appears as though maize plants were able to partially reduce the increase in soluble Cu. By contrast, R increases in the rhizosphere of the forest soil, suggesting that Cusol is prevailingly free and not complexed. In our case, the depletion of CuDTPA in the rhizosphere (Fig. 2) is not very clear, and this parameter does not seem to be related to plant uptake and Cusol and CuDGT
Table 3 DIFS model input parameters of forest and vineyard soil for R simulation
Vineyard Forest
Tc (s)
Kd (cm3 g1)
Pc (g cm3)
D0 (cm2 s1)
Ds (cm2 s1)
C0 (nmol/ml)
/
Dg (cm)
1500 40
23 850
3.25 2.50
7.10E06 7.10E06
2.73E06 2.73E06
6.60 0.43
0.46 0.41
0.093 0.093
Tc = response time; Kd = distribution coefficient, equilibrium ratio of the sorbed to dissolved concentration; Pc = soil particle concentration; D0 = diffusion coefficient of Cu; Ds = diffusion coefficient in soil; C0 = initial dissolved concentration; / = porosity; Dg = diffusion layer thickness. Tc and Kd were previously obtained as fitting parameters.
1976
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
(a) pH
(b) CuDTPA BULK
BULK
RHIZOSPHERE 120
8.80
pH
8.00 7.60 7.20
5
80
4 3
60
2
40
1 0
20
6.80
0 FOREST
FOREST
VINEYARD
VINEYARD
(d) DOC
(c) Cusol BULK
BULK
RHIZOSPHERE
25
1600
20
DOC (mg l-1 C)
FOREST
2000 1200
15 10
800
5
400
0
150 120 90 60 30 0
0 FOREST
FOREST
VINEYARD
(e) CuDGT (24 h)
VINEYARD
(f) R (CuDGT /Cusol)
BULK
BULK
RHIZOSPHERE
RHIZOSPHERE
1.00
600 FOREST
500
25
400
20
0.80 0.60
10
200
5
R
15
300
100
RHIZOSPHERE
180
2400 Cusol (μg l-1)
FOREST
100
CuDTPA (mg kg-1)
8.40
CuDGT (μg l-1)
RHIZOSPHERE
0.40
0
0.20
0
0.00 FOREST
VINEYARD
FOREST
VINEYARD
Fig. 2. Maize rhizosphere (the first 2-mm soil section close to the root) and bulk soil characteristics of the experimental soils. Mean of three replicates. (a) pH, (b) CuDTPA, (c) Cusol, (d) DOC concentration in soil solution, (e) CuDGT (deployment 24 h) and (f) R, ratio of CuDGT/Cusol. Error bars represent standard deviation.
modifications. Our results are not in agreement with the experimental results of Chino et al. (1999), who found smaller Cu in soluble and assimilable forms in the presence of plants (Tamarix gallica and Cynodon dactylon). Comparing the copper content of maize leaves (Fig. 3), no significant difference between the two soils is evident (13 mg g1 in the vineyard soil compared to 9.9 mg g1 in the forest soil). The root concentration, however, is approximately fourfold greater in the vineyard soil compared to the forest soil (148 mg g1 compared to 39 mg g1). The roots contain more Cu than the shoots, particularly in the vineyard soil. This is a typical feature of a heavy metal excluder plant (Baker, 1981). In this work,
apoplastic Cu (Cu bound to the root cell walls) was not removed and root concentration is overestimated. Plant uptake data do not appear to be related to Cusol and CuDGT modifications in the rhizosphere. This is not in agreement with the good correlation between CuDGT and concentrations in aerial plant parts previously described by Zhang et al. (2001). Merrington et al. (2002) showed that Cu contaminated soils exhibit a significantly greater Cu available fraction and a significant reduction in biomass carbon compared to a reference site, although levels of total organic carbon were elevated and similar in both contaminated and reference soil. The results indicated that microbial populations
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
ROOT
LEAVES
160 140 Cu (mg kg-1)
120 100 80
1977
when it is excessive, like in the vineyard soil. According to Ouzounidou et al. (1992) the high Cusol concentration in this soil could be toxic for maize plants, unless it was partially complexed. DOC concentrations are comparable in the rhizosphere soil solutions of the two soils (Fig. 2), but it appears that DOC has opposing effects, depending on differences in the efficiency of uptake processes under deficient or excessive conditions.
60
4. Conclusions
40 20 0 FOREST
VINEYARD
Fig. 3. Maize Cu content (mean value) in root and leaves after harvest, at the end of rhizobox experiment.
in these soils were stressed. Ali et al. (2002) studied maize tolerance and proposed this plant as a possible solution for the stabilization and restoration of Cu-polluted soils. Zea mays is an example of a cultivated plant that can replace vines in vineyard soil, due to its agronomic interest and its tolerance. Additionally, it may create particularly good environmental conditions for soil microorganisms and microfauna (Lynch and Whipps, 1990; Schulin et al., 1995; Romkens et al., 1999). In general, the bioavailability of copper strongly depends on the soil, the plant, mineral deficiency and its chemical mobility, i.e. the ability of copper to leave the soil solid phase and enter the soil solution (Dı´az-Barrientos et al., 2003). In our case, CuDGT increases with decreasing soil pH but only in the rhizosphere of the vineyard soil and not in that of the forest soil. Although the forest soil contains twice as much organic matter and has a higher CEC than the vineyard soil (Table 1), it is maize plant growth that makes the key contribution to the observed effect. In the case of the forest soil, the CuDGT decrease close to the root may be explained by uptake, whereas the observed increase in the rhizosphere of the contaminated vineyard soil could be a secondary effect of H+ and DOC release. It is generally accepted that DOC facilitates metal transport through soils through the formation of organic complexes. Copper forms stronger complexes with DOC than other divalent cations (Pandley et al., 2000). Although Cu–DOC complexes are more labile in soils, copper has been shown to be less available for plant uptake (Nierop et al., 2002; Bolan et al., 2003; Cao et al., 2004). The copper tolerance of maize is probably due to three mechanisms: uptake and translocation reduction, particularly in the vineyard soil; segregation in the root cell wall (Ouzounidou et al., 1992) and, on the basis of our data, DOC production. DOC forms complexes with Cu. Complexed-Cu is more mobile and more rapidly available under deficient conditions, such as in the forest soil, where Cutot is low and CuDGT and Cusol values are decreased in the rhizosphere (Fig. 2). Conversely, complexed-Cu is hardly usable
Analysis of soils before the rhizobox experiment showed that the concentrations of soluble and potentially bioavailable copper were generally low compared to total copper concentration, even in the case of the vineyard soil, which presents the greatest Cusol and CuDGT values of the two soils considered. Copper sorption kinetics appear to be quite slow in the Cu-polluted soil, with low R values (CuDGT/Cusol) and slow as well as low resupply from the solid phase to the soil solution. As, in the presence of maize, the R value for the vineyard rhizosphere does not change, it seems that mobilization from soil to soil solution is slower than root uptake. It appears that the maize plant was able to partially reduce the increase in soluble Cu close to the root. Maize cultivation may also decrease soil pH and increase the DOC content in the rhizosphere of both soils, independent of their characteristics. The increase of Cusol and CuDGT in the rhizosphere of the vineyard soil and the drop in the rhizosphere of the forest soil may be caused indirectly by uptake mechanisms. Maize plants generate a similar increase in DOC in both soils, but Cutot and CuDGT are very different. DOC production, usually effective in ion mobilization and assimilation, may help also in the sequestration of Cu at toxic concentrations. The concentration of Cu in maize leaves is similar for both soils, whereas the concentration in the roots is higher in the vineyard soil. We think that the lower uptake, and, consequently, tolerance, is partially due to DOC. The results demonstrate that DGT-based experiments are very sensitive to soil properties and may allow the study of root-induced changes in rhizosphere. The devices may be very useful in the estimation of pollutant availability in contaminated soils and in investigating the environmental effects of cultivation of polluted soils, such as mobility and increasing availability with plant growth. In our case, plant uptake does not appear to be related to CuDGT. We therefore concluded that DGT cannot be successfully and easily used to forecast the uptake of copper into aerial parts of a tolerant plant. Acknowledgements George Fragoulis and Ilenia Cattani wish to thank the Marie Curie program (QLK5-CT-2002-51598) and the COST631 section programme respectively for the support given.
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Hinsinger, P., 2001b. Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237, 173–195. Hooda, P.S., Hang, H., Davison, W., Edwards, A.C., 1999. Measuring bioavailable trace metals by diffusive gradients in thin films (DGT): soil moisture effects on its performance in soils. Eur. J. Soil Sci. 50, 285–294. Lombi, E., Wenzel, W.W., Gobran, G.R., Adriano, D.C., 2001. Dependency of phytoavailability of metals on indigenous and induced rhizosphere processes: a review. In: Gobran, G.R., Wenzel, W.W., Lombi, E. (Eds.), Trace elements in the Rhizosphere. CRC Press, Boca Raton, London, New York, Washington, DC, pp. 4–23. Lynch, J.M., Whipps, J.M., 1990. Substrate flow in the rhizosphere. Plant Soil 129, 1–10. MacLaughlin, M.J., Smolders, E., Merckx, R., 1998. Soil root interface: physicochemical processes. In Chemistry and Ecosystem Health (Special Publication No. 52). Soil Science Society of America, Madison, USA, pp. 233–277. Marchner, H., 1995. In: Mineral Nutrition of Higher Plants, second ed. Academic Press, London, pp. 333–347. Merrington, G., Rogers, S.L., Van Zwieten, L., 2002. The potential impact of long-term copper fungicide usage on soil microbial biomass and microbial activity in an avocado orchard. Aust. J. Soil Res. 40, 749– 759. Monni, S., Salemaa, M., Millar, N., 2000. The tolerance of Empetrum nigrum to copper and nickel. Environ. Pollut. 109, 221–229. Ministerial Decree. 1999. Ministerial Decree (D.M. 471/99) 25 Oct. 1999, nr. 471. Ordinary supplement of Gazzetta Ufficiale, general series, nr. 293. p. 21. Nierop, K.G.J., Jansen, B., Vrugt, J.A., Verstraten, J.M., 2002. Copper complexation by dissolved organic matter and uncertainty assessment of their stability constants. Chemosphere 49, 1191–1200. Orsega, E.F., Agnoli, F., Cacco, G., Delaney, E., Argese, E., 2003. Effects of copper on Sambucus nigra L. seedlings studied by electron paramagnetic resonance and atomic absorption spectroscopies. Chemosphere 53, 263–268. Ouzounidou, G., Eleftheriou, E.P., Karataglis, S., 1992. Ecophysiological and ultrastructural effects of copper on Thlaspi ochroleucum (Cruciferae). Can. J. Botany 70, 947–957. Pandley, A.K., Pandley, S.D., Misral, V., 2000. Stability constants of metal humic acid complexes and its role in environmental detoxification. Ecotox. Environ. Safe. 47, 195–200. Qiao, X.L., Luo, Y.M., Christie, P., Wong, M.H., 2003. Chemical speciation and extractability of Zn, Cu and Cd in two contrasting biosolids-amended clay soils. Chemosphere 50, 823–829. Romkens, P.F.A.M., Bouwman, L.A., Boon, G.T., 1999. Effect of plant growth on copper solubility and speciation in soil solution samples. Environ. Pollut. 106, 315–321. Sauve´, S., 2002. Speciation of metals in soils. In: Allen, H.E. (Ed.), Bioavailability of Metals in Terrestrial Ecosystems: Importance of Partitioning for Bioavailability to Invertebrates, Microbes and Plants. Society for Environmental Toxicology and Chemistry, Pensacola, FL, pp. 7–39. Sauve´, S., McBride, M.B., Norvell, W.A., Hendershot, W.H., 1996a. Copper solubility and speciation of in situ contaminated soils: effects of copper level, pH and organic matter. Water Air Soil Pollut. 100, 133–149. Sauve´, S., Cook, N., Hendershot, W.H., Mc Bride, M.B., 1996b. Linking plant tissue concentration and soil copper pools in urban contaminated soils. Environ. Pollut. 94, 154–157. Sauve´, S., Mc Bride, M.B., Hendershot, W.H., 1997. Speciation of lead in contaminated soils. Environ. Pollut. 98, 149–155. Schulin, R., Geiger, G., Furrer, G., 1995. Heavy metal retention by soil organic matter under changing environmental conditions. In: Salomons, W., Stigliani, W.M. (Eds.), Biogeodynamics of Pollutants in Soils and Sediments: Risk Assessment of Delayed and Nonlinear Responses. Springer, Berlin, pp. 53–85.
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979 Song, J., Zhao, F., Luo, Y., McGrath, S., Zhang, H., 2004. Copper uptake by Elsholtzia splendens and Silene vulgaris and assessment of copper phytoavailability in contaminated soils. Environ. Pollut. 128, 307– 315. SSIS, Soil Science Italian Society, 2000. Metodi di analisi chimica del suolo, a cura di Pietro Violante. In: Sequi P. (Ed.), Collana di metodi analitici per l’agricoltura. Franco Angeli, Bologna, pp. 536. Tiensing, T., Preston, S., Strachan, N., Paton, G.I., 2001. Soil solution extraction techniques for microbial ecotoxicity testing: a comparative evaluation. J. Environ. Monitor. 3, 91–96. Witter, E., 1996. Towards zero accumulation of heavy metals in soils. An imperative or a fad? Fert. Res. 43, 225–233.
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Ye, Z.H., Baker, A.J.M., Wong, M.H., Willis, A.J., 2003. Copper tolerance, uptake and accumulation by Phragmites australis. Chemosphere 50, 795–800. Zhang, H., Davison, W., 1995. Performance characteristics of diffusion gradients in thin films for the in situ measurement of trace metals in aqueous solution. Anal. Chem. 67, 3391–3400. Zhang, H., Davison, W., Knight, B., McGrath, S., 1998. In situ measurement of solution concentrations and fluxes of trace metals in soils using DGT. Environ. Sci. Technol. 32, 704–710. Zhang, H., Zhao, F.J., Sun, B., Davison, W., McGrath, S.P., 2001. A new method to measure effective soil solution concentration predicts copper availability to plants. Environ. Sci. Technol. 35, 2602–2607.
Copper bioavailability in the rhizosphere of maize (Zea mays L.) grown in two Italian soils I. Cattani *, G. Fragoulis, R. Boccelli, E. Capri Istituto di Chimica Agraria ed Ambientale, sez. Vegetale, Universita` Cattolica del Sacro Cuore, Via Milano, 24, 26100 Cremona, Italy Received 7 March 2005; received in revised form 30 December 2005; accepted 3 January 2006 Available online 14 February 2006
Abstract In this study, potentially bioavailable copper was estimated in two soils (a fungicide polluted and a natural soil) using a passive sampling technique, DGT. As plants can alter copper mobility and bioavailability in the soil, the rhizosphere properties of Zea mays L. were investigated using rhizoboxes. Compared to the total concentration, the soluble and the potentially bioavailable copper concentration in the bulk soils were generally low (less than 0.20% and 0.06% respectively), with a sixfold increase in the rhizosphere of the polluted soil. Our results suggest that maize cultivation in a polluted vineyard soil could increase the potentially available fraction of copper. DGTs showed a good sensitivity to soil properties and to root-induced changes in the rhizosphere, but the potentially bioavailable copper could not be related to the copper concentration in the above ground parts of maize. The results suggest that DGT may be used to predict some effects of the cultivation of polluted soils, for example, metal mobility and increased availability, but they cannot mimic the uptake of a tolerant plant. For both soils, dissolved organic carbon (DOC) concentrations were threefold higher in the rhizosphere than in the bulk soil, whilst bioaccumulation in leaves and roots was not significant. DOC production, usually effective in ion mobilization and assimilation, may help also in the reduction of Cu uptake at toxic concentrations. The sequestration of available Cu in soil and soil solution by DOC seems to contribute to maize tolerance. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Copper; Bioavailability; DGT; Rhizon sampler; Rhizobox
1. Introduction The prevention of the accumulation of heavy metals, such as copper, in soil is one of the prerequisites of sustain-
Abbreviations: CuDGT, potentially bioavailable Cu measured by DGT; CuDTPA, extractable copper; CE, effective concentration; C0, initial dissolved concentration; Cusol, Cu concentration in soil solution; Cutot, total Cu; Dg, diffusion layer thickness; DGT, diffusive gradients in thin films; DIFS, DGT-induced fluxes in soils; D0, diffusion coefficient in water; DOC, dissolved organic carbon; Ds, diffusion coefficient in soil; /, soil porosity; Kd, distribution coefficient for labile metal; M, mass accumulated by DGT; Pc, soil particles concentration; R, ratio between CuDGT and Cusol; Tc, response time of the solid phase/solution equilibrium; WHC, water holding capacity. * Corresponding author. Tel./fax: +39 0372499122. E-mail address: [email protected] (I. Cattani). 0045-6535/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.01.007
able agricultural production (Witter, 1996). Risk assessment of copper contaminated soils needs to estimate its bioavailability to organisms and plants in order to understand the concentration to which they are exposed, and the potentially toxic effects (Ye et al., 2003). Copper is an essential element for every organism at low concentrations, however excess amounts may cause metabolic alterations, even in tolerant plants (Monni et al., 2000; Orsega et al., 2003). The ‘bioavailable’ copper can be best described as that portion of soil copper available for intake by a given organism. Consequently, the fraction bioavailable to plants is the amount that can be taken up by the roots of a given plant species. It is well known that copper bioavailability in soil depends on soil properties (pH, redox potential, CEC, amount and nature of organic matter, soil minerals). The Cu that crosses the plant membrane is considered as the part
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
that is ‘free’, i.e. not complexed with organic species in solution or absorbed to soil particles (Zhang et al., 2001) and intake occurs essentially via uptake by the roots from the soil solution (Marchner, 1995; Fitter, 1997; Hinsinger, 1998). The assessment of copper bioavailability is difficult, as the concentration of copper in the soil solution represents only a portion of exchangeable and slightly complexed metal and does not give a measure of the total labile pool (Qiao et al., 2003). Furthermore, plants can alter the mobility and bioavailability of copper in the rhizosphere (MacLaughlin et al., 1998; Chaignon et al., 2002; Chaignon and Hinsinger, 2003) by modifying pH (Hinsinger, 1998, 2001a,b; Chaignon et al., 2002) or by releasing soluble organic compounds to complex copper (Lombi et al., 2001; Chaignon and Hinsinger, 2002). These findings conflict with the existing Italian legislation (Ministerial Decree, 1999), that is based on a risk assessment procedure that takes into account only the total concentration of copper. The free ion concentration in soil solution and the labile amount in the solid phase represent the real potentially toxic amount (Sauve´ et al., 1996a,b), that is, the potentially bioavailable concentration of copper. Official analytical methods only provide a measurement of the DTPA/EDTA extractable copper concentration, which has been correlated with plant accumulation. In practice, however, this correlation is not always reliable (Marchner, 1995; MacLaughlin et al., 1998; Hinsinger, 2001a,b). In recent years it has been recognised that the measurement of free ion activity in soil solution may provide a better indication of metal availability and potential bioaccumulation (Sauve´ et al., 1997; Sauve´, 2002). Conversely, Ginocchio et al. (2002) and Song et al. (2004) reported a poor correlation between free Cu2+ activity and plant Cu concentration. They proposed rhizosphere-induced changes as a possible explanation of this discrepancy. Recently, Zhang et al. (2001) introduced the concept of effective concentration, CE, which represents both the soil solution concentration and the metal supplied from the solid phase. CE is measured using the technique of diffusive gradients in thin films (DGT) (Davison and Zhang, 1994; Zhang et al., 1998) and the authors showed that this value correlated very well with plant uptake. They considered that the DGT-soil system mimics diffusion and labile metal release and that CE represents bioavailable fraction and can be used to probe the dynamics of system. Song et al. (2004) also found a good correlation of both soluble Cu and CE with plant concentration. The aim of this work was to estimate the bioavailability of copper in the bulk soil and rhizosphere of two different soil types, in order to obtain a better understanding of copper bioavailability and subsequent crop uptake. 2. Materials and methods 2.1. Soil sampling and characterization Soil samples were collected from the top soil, to a depth of approximately 0.3 m, from sites in the locality of Cem-
1973
bra (46°10 0 4000 N, 11°13 0 5000 E), near Trento (Italy). The two soils were of the same geological origin but differ in terms of soil management (agricultural versus natural soil). Moist soil was passed through a 2 mm mesh sieve and stored at 4 °C prior to use in experiments. Soil properties, including pH, water holding capacity (WHC), texture, cation exchange capacity (CEC), organic matter (OM), total (Cutot) and DTPA–extractable copper (CuDTPA) content (Table 1), were measured using official analytical methods (SSIS, 2000), whereas dissolved organic carbon (DOC) was measured in soil solution using the UV absorbance method previously published by Brandstetter et al. (1996). 2.2. Soluble Cu (Cusol) measurement Rhizon soil moisture samplers (Rhizosphere Research Products, Wageningen, The Netherlands) were used to extract soil solution from 500 g of each soil at approximately weekly intervals. Measurements were made in triplicate. The samplers consist of 5 cm of inert porous plastic tubing (2.5 mm diameter), capped with nylon wire at one end. The capped end was inserted into each soil pot during filling. The other end was attached to a 30 cm long PVC/ PE tubing, joined to a female lock. A syringe needle was connected to the female lock and inserted into a 10 ml glass vacuum tube, in order to extract the soil pore water by vacuum. In this way, approximately 9 ml of soil solution was extracted overnight. Samplers were washed before use following the technique of Tiensing et al. (2001). The soil humidity was maintained at 80% of WHC during the experiment. The soluble Cu (Cusol), that includes the fraction of Cu potentially available to plants in soil solution, was measured using inductively coupled plasma atom emission spectrometry (ICP-OES, Spectro CIROSCCD) following nitric acid– hydrochloric acid microwave assisted digestion (ETHOS, Milestone, FKV). 2.3. Potentially available Cu (CuDGT) measurement The potentially available copper concentration (CuDGT) was determined using the DGT technique. DGT devices were deployed in duplicate and at 6 h intervals (from 24
Table 1 Experimental soils characteristics Soil
Forest
Vineyard
Texture pH WHC (%) Bulk density (g cm3) CEC (cmol kg1) OM (g kg1) DOC (mg l1 C) CaCO3 (g kg1) CuDTPA (mg kg1) Cutot (mg kg1)
Sand 7.89 35 1.3 16.8 36.6 27.4 1.2 4.3 18.4
Loamy sand 8.52 23 1.4 8.3 15.2 19.2 118.3 113 183
1974
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
to 310 h) in each soil, following the technique used by Hooda et al. (1999). The mass of soil used was 100 g. Soil moisture was kept at 80% of WHC and readjusted daily. All experiments were conducted at 30 °C. Copper bound by DGT resin was measured by ICP-OES, following acid microwave assisted digestion, and CuDGT values were calculated using the equations provided by DGT Research Ltd (Lancaster, UK, web: www.dgtresearch.com). DGT performance was evaluated according to the method used by Zhang and Davison (1995), in two replicates. After acid digestion of the DGT resin, the mean recovery of Cu in acid solution was 95%. The dynamic numerical model of DGT-induced fluxes in soils (DIFS) (Harper et al., 2000), was used to calculate the following variables, representative of the soil system dynamic: (1) the pool sizes of labile metal, (2) the distribution coefficient for labile metal (Kd) and (3) the response time (Tc) of the solid phase/solution equilibrium. The ratio R = CuDGT/Cusol represents the ability of the soil to resupply local pore water concentrations. In this work, R was calculated and Kd and Tc were derived from the best model fit of plots of R versus deployment time. The DIFS model assumes the first order exchange between solid phase and solution, and diffusional transport in both soil solution and the DGT device. After calibration for the Kd and Tc parameters, the model was used to quantify dynamic processes controlling metal uptake from soils. 2.4. Rhizosphere trials The experimental soils, previously sieved to 2 mm, homogenized and frozen at 18 °C, were equilibrated at room temperature for a week and placed into the rhizobox described by Fitz et al. (2003a). Three replicates were prepared. The bulk density of the vineyard soil was 1.4 g cm3, and the bulk density of the forest soil was 1.3 g cm3. The root compartment was separated from the rhizosphere–soil compartment by a 30 lm mesh size nylon, in order to allow elongation of the root hairs. We decided against the use of the slicing device to separate the rhizosphere into 0.5 mm root parallel sections because the soil texture made separation at this thickness very difficult and the amount of soil in each sample would be insufficient for analysis. The 2-mm section of soil close to the root compartment (rhizosphere) was separated from the remaining soil (bulk soil) using the previously cited slicing device (Fitz et al., 2003b). pH, DOC, CuDTPA, Cusol and CuDGT were measured in samples of both the rhizosphere and bulk soil. As the weight of the soil layers was only approximately 35 g, soil solution was extracted by centrifugation, and DGT experiment was performed for 24 h using an acrylic plate adaptor, described by Fitz et al. (2003b). Soil solution and DGT gel were digested and analyzed as previously described. Seeds of maize (Zea mays L.) were germinated in Petri dishes on wet cotton-wool in the dark for three days. Three plantlets were placed in the upper compartments of the rhizobox and, after about 10 days, the upper compartments
were placed and fixed on the lower compartment. The soil water content (determined immediately after harvest) was about 15% for the vineyard soil and 23% for the forest soil (approximately 65% of the maximum WHC of each soil). The experiment was performed in a greenhouse for a period of two months, at a temperature of approximately 20–22 °C during the day and 16–18 °C at night. Fertilization was avoided because it may affect copper mobility in soil. Harvested plants were divided into roots and aerian parts, weighed, dried for 24 h at 105 °C and analyzed following nitric acid digestion in the microwave. 3. Results and discussion 3.1. Kinetics of metal exchange between soil and soil solution without plants Table 2 shows the comparison of soluble with potentially bioavailable, total and extractable Cu, measured in each soil before the rhizobox experiment. Cusol and CuDGT are generally low in comparison with total and extractable Cu, but the vineyard soil exhibits higher concentrations of these parameters (365 and 85 lg l1 for the vineyard soil compared to 20 and 15 lg l1 for the forest soil). According to Zhang et al. (2001) and Song et al. (2004), these values would lead to the prediction of a higher Cu availability in the contaminated soil, even if free Cu activity was not estimated. Experimental data and DIFS simulation of R (CuDGT/ Cusol) for Cu in both soils are shown in Fig. 1. We used the model in both simulation and parameter estimation mode (Harper et al., 2000), to improve the fitting between the model simulation and experimental data. Tc and Kd were obtained as fitting parameters. Even though, theoretically, a large number of data points are needed to assess the goodness of fit, our experimental data show a good agreement with the simulated R for both soils (Fig. 1). R values for the vineyard soil are always lower than for the forest soil. For the vineyard soil, values of R are less than 0.25 (Table 2). In both soils, R decreases with time and, as Table 2 CuDGT concentrations (average of two values) at different sampling time (time measured at the DGT retrieval moment) CuDGT (lg l1)
Sampling time (h) 24 72 91 169 240 310
1
Cusol (lg l ) Cusol/Cutot (%) Cusol/CuDTPA (%)
Forest
R
Vineyard
R
14.5 11.5 10.1 8.1 6.9 6.1
0.72 0.57 0.50 0.40 0.34 0.30
85 63 54 49 52 43
0.23 0.17 0.15 0.13 0.14 0.12
Forest
Vineyard
20.2 0.11 0.47
365 0.20 0.32
R represents the ratio CuDGT/Cusol. Comparison of Cusol (mean values),
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
(a) forest soil 1.0 0.8
R-simulated DIFS R-measured DGT
R
0.6 0.4 0.2 0.0 0
50
100
150 200 Time (h)
250
300
350
0.8
R-simulated DIFS R-measured DGT
R
0.6 0.4 0.2 0.0 50
tially buffer its high copper content, probably by reducing the immediate bioavailability and so reducing the potentially acute toxicity. However, the R values obtained for the vineyard soil could be low simply because DGT does not measure Cu bound on colloids or trace metal complexes in soil solution, whereas Cusol includes these species. The fact that after 24 h of deployment the estimated R was more than 0.8 for the forest soil (Fig. 1), suggests that complexation was not important in this soil or in other soils with similar DOC content, including such vineyard soil. 3.2. Rhizosphere characteristics and copper uptake by maize
(b) vineyard soil 1.0
0
1975
100
150 200 Time (h)
250
300
350
Fig. 1. Experimental R time dependence on (a) forest soil and (b) vineyard soil. The line shows the optimal DIFS model fit. R represents the ratio CuDGT/Cusol (CuDGT, concentration accumulated by DGT; Cusol, soil solution concentration; DIFS, DGT-induced fluxes in soils).
expected, with a decrease in the amount of labile Cu bound to the solid phase. There is a peak in R (0.72 in the forest soil and 0.23 in the vineyard soil, corresponding to the highest values of CuDGT) during the first hours of deployment. This could be due to the initially higher flux to the diffusion layer from the soluble phase than from the solid phase. In the forest soil, the major depletion of soil solution concentration at the DGT interface (greater decrease in R) occurs within 92 h of deployment. In the vineyard soil, R decreases more slowly over time (50% after 310 h). Values of Kd and Tc and other input parameters needed for model simulation of R are presented in Table 3. As DGT takes Cu from soil solution, it appears that the forest soil has a short response time, with rapid resupply from the solid phase and a slow depletion over time of the available Cu pool. By contrast, the vineyard soil has a longer response time and so very little resupply from the solid phase, leading to a progressive depletion. It appears that the vineyard soil is able to par-
Fig. 2 shows the chemical characteristics (pH, DOC, CuDTPA, Cusol, CuDGT and ratio CuDGT/Cusol) measured in the rhizosphere and bulk soil of the forest and vineyard soils. It appears that in the rhizosphere of both soils, independent of their characteristics, the cation and anion uptake mechanisms of plants may decrease the soil pH, whereas rhizodeposition/exudation mechanisms may increase the DOC content. Comparing the rhizosphere data, Cusol is 200-fold and CuDGT is 50-fold greater (2250 and 452 lg l1 respectively) in the vineyard soil than in the forest soil. There is also a significant increase (600%) of both Cusol and CuDGT in the rhizosphere of the vineyard soil. As previous studies on copper toxicity (e.g. Ouzounidou et al., 1992; Giller et al., 1998) refer to free copper concentrations in soil solution, we are unable to establish if Cusol values in the rhizosphere of the vineyard soil could be potentially dangerous for soil fauna, microorganisms and plants. Nevertheless, the increase in potentially available Cu and the results of Song et al. (2004) suggest that intake and assimilation of copper could be increased with maize growing. If compared to the bulk soil, the R value for the vineyard soil rhizosphere is less than 25% (Fig. 2), as for the bulk soil. DGT has been described as a surrogate of the plant/soil system (Zhang et al., 2001). On this basis, our results suggest that mobilization from soil to soil solution is slower than root uptake, and it appears as though maize plants were able to partially reduce the increase in soluble Cu. By contrast, R increases in the rhizosphere of the forest soil, suggesting that Cusol is prevailingly free and not complexed. In our case, the depletion of CuDTPA in the rhizosphere (Fig. 2) is not very clear, and this parameter does not seem to be related to plant uptake and Cusol and CuDGT
Table 3 DIFS model input parameters of forest and vineyard soil for R simulation
Vineyard Forest
Tc (s)
Kd (cm3 g1)
Pc (g cm3)
D0 (cm2 s1)
Ds (cm2 s1)
C0 (nmol/ml)
/
Dg (cm)
1500 40
23 850
3.25 2.50
7.10E06 7.10E06
2.73E06 2.73E06
6.60 0.43
0.46 0.41
0.093 0.093
Tc = response time; Kd = distribution coefficient, equilibrium ratio of the sorbed to dissolved concentration; Pc = soil particle concentration; D0 = diffusion coefficient of Cu; Ds = diffusion coefficient in soil; C0 = initial dissolved concentration; / = porosity; Dg = diffusion layer thickness. Tc and Kd were previously obtained as fitting parameters.
1976
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
(a) pH
(b) CuDTPA BULK
BULK
RHIZOSPHERE 120
8.80
pH
8.00 7.60 7.20
5
80
4 3
60
2
40
1 0
20
6.80
0 FOREST
FOREST
VINEYARD
VINEYARD
(d) DOC
(c) Cusol BULK
BULK
RHIZOSPHERE
25
1600
20
DOC (mg l-1 C)
FOREST
2000 1200
15 10
800
5
400
0
150 120 90 60 30 0
0 FOREST
FOREST
VINEYARD
(e) CuDGT (24 h)
VINEYARD
(f) R (CuDGT /Cusol)
BULK
BULK
RHIZOSPHERE
RHIZOSPHERE
1.00
600 FOREST
500
25
400
20
0.80 0.60
10
200
5
R
15
300
100
RHIZOSPHERE
180
2400 Cusol (μg l-1)
FOREST
100
CuDTPA (mg kg-1)
8.40
CuDGT (μg l-1)
RHIZOSPHERE
0.40
0
0.20
0
0.00 FOREST
VINEYARD
FOREST
VINEYARD
Fig. 2. Maize rhizosphere (the first 2-mm soil section close to the root) and bulk soil characteristics of the experimental soils. Mean of three replicates. (a) pH, (b) CuDTPA, (c) Cusol, (d) DOC concentration in soil solution, (e) CuDGT (deployment 24 h) and (f) R, ratio of CuDGT/Cusol. Error bars represent standard deviation.
modifications. Our results are not in agreement with the experimental results of Chino et al. (1999), who found smaller Cu in soluble and assimilable forms in the presence of plants (Tamarix gallica and Cynodon dactylon). Comparing the copper content of maize leaves (Fig. 3), no significant difference between the two soils is evident (13 mg g1 in the vineyard soil compared to 9.9 mg g1 in the forest soil). The root concentration, however, is approximately fourfold greater in the vineyard soil compared to the forest soil (148 mg g1 compared to 39 mg g1). The roots contain more Cu than the shoots, particularly in the vineyard soil. This is a typical feature of a heavy metal excluder plant (Baker, 1981). In this work,
apoplastic Cu (Cu bound to the root cell walls) was not removed and root concentration is overestimated. Plant uptake data do not appear to be related to Cusol and CuDGT modifications in the rhizosphere. This is not in agreement with the good correlation between CuDGT and concentrations in aerial plant parts previously described by Zhang et al. (2001). Merrington et al. (2002) showed that Cu contaminated soils exhibit a significantly greater Cu available fraction and a significant reduction in biomass carbon compared to a reference site, although levels of total organic carbon were elevated and similar in both contaminated and reference soil. The results indicated that microbial populations
I. Cattani et al. / Chemosphere 64 (2006) 1972–1979
ROOT
LEAVES
160 140 Cu (mg kg-1)
120 100 80
1977
when it is excessive, like in the vineyard soil. According to Ouzounidou et al. (1992) the high Cusol concentration in this soil could be toxic for maize plants, unless it was partially complexed. DOC concentrations are comparable in the rhizosphere soil solutions of the two soils (Fig. 2), but it appears that DOC has opposing effects, depending on differences in the efficiency of uptake processes under deficient or excessive conditions.
60
4. Conclusions
40 20 0 FOREST
VINEYARD
Fig. 3. Maize Cu content (mean value) in root and leaves after harvest, at the end of rhizobox experiment.
in these soils were stressed. Ali et al. (2002) studied maize tolerance and proposed this plant as a possible solution for the stabilization and restoration of Cu-polluted soils. Zea mays is an example of a cultivated plant that can replace vines in vineyard soil, due to its agronomic interest and its tolerance. Additionally, it may create particularly good environmental conditions for soil microorganisms and microfauna (Lynch and Whipps, 1990; Schulin et al., 1995; Romkens et al., 1999). In general, the bioavailability of copper strongly depends on the soil, the plant, mineral deficiency and its chemical mobility, i.e. the ability of copper to leave the soil solid phase and enter the soil solution (Dı´az-Barrientos et al., 2003). In our case, CuDGT increases with decreasing soil pH but only in the rhizosphere of the vineyard soil and not in that of the forest soil. Although the forest soil contains twice as much organic matter and has a higher CEC than the vineyard soil (Table 1), it is maize plant growth that makes the key contribution to the observed effect. In the case of the forest soil, the CuDGT decrease close to the root may be explained by uptake, whereas the observed increase in the rhizosphere of the contaminated vineyard soil could be a secondary effect of H+ and DOC release. It is generally accepted that DOC facilitates metal transport through soils through the formation of organic complexes. Copper forms stronger complexes with DOC than other divalent cations (Pandley et al., 2000). Although Cu–DOC complexes are more labile in soils, copper has been shown to be less available for plant uptake (Nierop et al., 2002; Bolan et al., 2003; Cao et al., 2004). The copper tolerance of maize is probably due to three mechanisms: uptake and translocation reduction, particularly in the vineyard soil; segregation in the root cell wall (Ouzounidou et al., 1992) and, on the basis of our data, DOC production. DOC forms complexes with Cu. Complexed-Cu is more mobile and more rapidly available under deficient conditions, such as in the forest soil, where Cutot is low and CuDGT and Cusol values are decreased in the rhizosphere (Fig. 2). Conversely, complexed-Cu is hardly usable
Analysis of soils before the rhizobox experiment showed that the concentrations of soluble and potentially bioavailable copper were generally low compared to total copper concentration, even in the case of the vineyard soil, which presents the greatest Cusol and CuDGT values of the two soils considered. Copper sorption kinetics appear to be quite slow in the Cu-polluted soil, with low R values (CuDGT/Cusol) and slow as well as low resupply from the solid phase to the soil solution. As, in the presence of maize, the R value for the vineyard rhizosphere does not change, it seems that mobilization from soil to soil solution is slower than root uptake. It appears that the maize plant was able to partially reduce the increase in soluble Cu close to the root. Maize cultivation may also decrease soil pH and increase the DOC content in the rhizosphere of both soils, independent of their characteristics. The increase of Cusol and CuDGT in the rhizosphere of the vineyard soil and the drop in the rhizosphere of the forest soil may be caused indirectly by uptake mechanisms. Maize plants generate a similar increase in DOC in both soils, but Cutot and CuDGT are very different. DOC production, usually effective in ion mobilization and assimilation, may help also in the sequestration of Cu at toxic concentrations. The concentration of Cu in maize leaves is similar for both soils, whereas the concentration in the roots is higher in the vineyard soil. We think that the lower uptake, and, consequently, tolerance, is partially due to DOC. The results demonstrate that DGT-based experiments are very sensitive to soil properties and may allow the study of root-induced changes in rhizosphere. The devices may be very useful in the estimation of pollutant availability in contaminated soils and in investigating the environmental effects of cultivation of polluted soils, such as mobility and increasing availability with plant growth. In our case, plant uptake does not appear to be related to CuDGT. We therefore concluded that DGT cannot be successfully and easily used to forecast the uptake of copper into aerial parts of a tolerant plant. Acknowledgements George Fragoulis and Ilenia Cattani wish to thank the Marie Curie program (QLK5-CT-2002-51598) and the COST631 section programme respectively for the support given.
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