Mechanistic Insights from DGT and Soil Solution Measurements on the

May 22, 2014 - State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, People's R...
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Mechanistic Insights from DGT and Soil Solution Measurements on the Uptake of Ni and Cd by Radish Jun Luo,† Hao Cheng,‡ Jinghua Ren,† William Davison,‡ and Hao Zhang*,‡ †

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, People’s Republic of China ‡ Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, United Kingdom S Supporting Information *

ABSTRACT: This work tests the previously proposed hypothesis that plant uptake of metals is determined dominantly by diffusional controlled or plant limiting uptake mechanisms at, respectively, low and high metal concentrations. Radish (Raphanus sativus) was grown in 13 soils spiked with Ni (10 and 100 mg kg−1) and Cd (0.5 and 4 mg kg−1) for 4 weeks to investigate the mechanisms affecting plant uptake. Soil solution concentrations, Css, of Ni and Cd were measured, along with the DGT interfacial concentration, CDGT, and the derived effective concentration in soil solution, CE. Free ion activities, aNi2+ and aCd2+, were obtained using WHAM 6. Although there was a poor relationship between Ni in radish roots and either Css or aNi2+ in unamended soils, the distribution of data could be rationalized in terms of the extent of release of Ni from the soil solid phase, as identified by DGT and soil solution measurements. By contrast Ni in radish was linearly related to CE, demonstrating diffusion limited uptake. For soils amended with high concentrations of Ni, linear relationships were obtained for Ni in radish plotted against, Css, aNi2+, and CE, consistent with the plant controlling uptake. For Ni the hypothesis concerning dominant diffusional and plant limiting uptake mechanisms was demonstrated. Poor relationships between Cd in radish and Css, aCd2+, and CE, irrespective of amendment by Cd, showed the importance of factors other than diffusional supply, such as rhizosphere and inhibitory processes, and that fulfilment of this hypothesis is plant and metal specific.

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INTRODUCTION Metal uptake by plants can be governed by both soil and plant processes.1 If a plant removes metal from the soil more slowly than it can be supplied by diffusion, there is negligible depletion of metal at the root surface and the metal concentration in the plant can be expected to be governed by the free ion activity in solution.2 Uptake models based on free-ion activity (e.g., the biotic ligand model (BLM)) are then applicable. Sometimes there are good relationships between concentrations of metal in plants and in soil solutions. In these cases soil solution concentrations may be proportional to free ion activities. If uptake of metal by the plant is fast relative to diffusional supply, the concentration of the free ion is depleted at the interface of root and soil solution, which induces a resupply from the complexes in the soil solution and from the solid phase.3 Due to the metal depletion in the confined zone of the interface between root and soil solution, there are many processes that potentially affect the supply of metals to plants, including diffusional transport to the root, the available pool size of labile metal in the solid phase, and the kinetics of release from solid phase to solution. Under these conditions, free-ion based models and soil solution measurements generally fail to predict plant uptake of metals.4 The technique of DGT is proving to be a useful tool to study the dynamic availability of metals in waters, sediments and soils.5,6 Like a plant root, DGT removes metal from soil © 2014 American Chemical Society

solution and induces resupply from the solid phase. The magnitude of the flux measured by DGT is determined by the concentration in solution, the rate of diffusion and the rate of resupply from both the solid phase and complexes in solution. The resupply from solid phase is dependent on the amount of labile metal sorbed on the solid phase and the rate of its release. Therefore, the DGT measurement reflects the fundamental kinetic and capacity properties of the soil, as well as the concentration in soil solution.7 When uptake by the plant is rapid, such that processes in the soil limit uptake, DGT measurements of metal in soil generally correlate well with concentrations of the metal in plants.3,8 Degryse, et al.1 argued from a theoretical standpoint that at low concentrations of metals diffusional supply can be expected to be limiting, leading to good correlations between metals in plants and DGT measurements. However, at high metal concentrations, plant control is expected, leading to good correlations with free ion activities and in some cases soil solution concentrations. There are few measurements available that test this hypothesis directly, especially for plants grown in soils. A range of tests, including comparative measurements made using DGT, were Received: Revised: Accepted: Published: 7305

January 12, 2014 May 11, 2014 May 22, 2014 May 22, 2014 dx.doi.org/10.1021/es500173e | Environ. Sci. Technol. 2014, 48, 7305−7313

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Table 1. Properties of Soils Rdiff site

soil

pH

Shaftsbury Road Coventry Road Hawthorne House Boulevard Donegal Road Trinity Road Hall Hays Road Station Road Henlow Road Aldridge Road Olford Farm Chudleigh Road Win

SHR COV HH B DR TR HHR SR HR AR OF CR WIN

7.15 7.12 7.01 6.52 6.51 6.44 6.38 6.38 6.24 6.23 6.10 5.66 4.21

Ni (mg kg−1) 32.5 21.4 19.8 11.8 10.4 18.7 15.1 18.5 21.9 18.4 17.5 16.3 19.9

± ± ± ± ± ± ± ± ± ± ± ± ±

3.2 3.1 2.3 0.2 0.4 1.6 0.7 2.3 2.3 0.4 0.5 0.5 0.1

Cd (mg kg−1) 1.34 1.09 2.72 0.99 0.83 0.93 0.79 0.97 0.57 0.84 1.03 0.95 0.47

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.08 0.04 0.06 0.02 0.02 0.08 0.06 0.04 0.12 0.04 0.06 0.02

Zn (mg kg−1)

TOC (%)

DOC (mg L−1)

Ni

Cd

± ± ± ± ± ± ± ± ± ± ± ± ±

2.19 2.70 6.23 3.57 3.73 2.28 1.92 2.15 4.27 3.08 2.94 1.17 2.06

19.3 15.0 56.9 18.9 21.2 23.2 14.3 21.3 12.1 12.2 16.5 12.2 13.4

0.099 0.104 0.107 0.106 0.105 0.109 0.098 0.101 0.105 0.101 0.098 0.097 0.112

0.090 0.094 0.098 0.096 0.096 0.100 0.089 0.092 0.095 0.092 0.089 0.089 0.103

424 275 433 657 268 355 132 220 211 354 418 416 67

39 19 23 27 10 4 6 10 22 22 6 29 9

of each amended soil and then thinned to one seedling after germination. Each soil sample at a given metal concentration was in triplicate. All the plants were grown, without nutrient addition, in a glass house with the following conditions: 14 h/ 10 h day/night, 20 °C/16 °C day/night temperatures. Natural light was supplemented with several 1 kw SON-T lamps to maintain a minimum photon flux of 250 μmol m−2 s−1. Four weeks after germination, radish plants were harvested. After harvest, the plant samples were cleaned using MQ water (MilliQ, Millipore) and dried at 80 °C for 24 h.13 Samples of ground plant roots or shoots (∼0.20 g) were digested with 5 mL aqua regia (concentrated HNO3 and HCl (1:4 v/v)) taken to dryness.14 DGT Deployment and Soil Analysis. The cylindrical DGT piston devices had an exposure window of 2.54 cm2. Diffusion and resin gels were prepared using polyacrylamide solution with 0.3% by volume agarose-derived cross-linker and Chelex-100, 200−400 mesh (Bio-Rad), following the standard procedure for gel making.15 The thickness of the diffusive gel used in this study was 0.8 mm. The thickness of resin gel was 0.4 mm unless specified in the text. A 0.14 mm thick poly(ether sulfone) filter membrane (0.45 μm) (Pall) overlaid the diffusive gel. Eighty grams of each amended soil was wetted to 60% maximum water holding capacity (MWHC) and incubated for 2 days, then raised to 80% MWHC for 24 h prior to DGT deployment.16 DGT devices were carefully placed on the soil paste with gentle pressure to ensure complete contact between the filter membrane of the device and the soil and kept at 20 ± 1 °C for 24 h. Three replicates per soil were used. Upon retrieval, DGT devices were jet-washed with ultrapure water (Milli-Q, Millipore), to remove soil particles, and then disassembled.8 The resin gels were removed from the DGT devices, and immersed in 1 mL of 1 M HNO 3 in microcentrifuge PVC tubes for at least 24 h at 20 ± 1 °C before analysis. After DGT deployments, soil solution was extracted by centrifugation (5000g for 15 min at room temperature).12 The resulting soil solutions, after filtration through a 13 mm diameter, 0.45-μm, polysulfone filter, were divided into halves. One was used for DOC measurement (Dohrmann DC-190) and the other was acidified using 20 μL of 5 M HNO3 in 1 mL of soil solution. Soil pH was measured at a solid to water ratio of 1:5.17 Samples of ground dried soil (∼0.5 g) were digested

used for the uptake of Cd, Zn, and Ni by spinach, tomato, rapeseed, wheat, and the Cd hyperaccumulater Thlaspi caerulescens grown in solution with and without complexing ligands.9,10 They showed that generally Cd and Zn uptake was diffusion limited, whereas Ni uptake is limited by plant control. By comparing metal accumulated by DGT and plants as a function of added metal when three Thlaspi species were grown in two soils amended to a series of metal concentrations, Luo, et al.11 were able to conclude that diffusion limitation applied for Cd for both hyperaccumulator and nonhyperaccumulator plants, but that uptake of Ni was only diffusion limited for the Ni hyperaccumulator. Here we investigated uptake of Cd and Ni by radish (Raphanus sativus) grown in pots containing 13 different soils, with and without amendment of the metals at fairly low and moderately high levels. These metals were chosen because of their contrasting properties. Cd can be released rapidly from both solution complexes and the solid phase, while the release of Ni is kinetically limited.5,12 The relationship between metal concentrations measured by DGT and in soil solution was examined in detail to gain insight into soil processes. This knowledge was then used to aid understanding of how concentrations of metals in radish roots depend on DGTmeasured concentrations, concentrations in soil solution and calculated free ion activities. Mechanisms of uptake were deduced for each metal at background and elevated metal concentrations.

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MATERIALS AND METHODS Soil Preparation. Thirteen topsoils (0−20 cm) covering a range of soil types were sampled from allotments in the vicinity of Birmingham, UK (Table 1). They covered a wide range of pH (4.00−6.85) and organic carbon content (1.17−6.23%) (Table 1). All samples were air-dried and passed through a 2 mm sieve. The soils were amended with either Cd or Ni nitrate stock solutions at two concentration levels: 0.5 and 4 mg kg−1 for Cd and 10 and 100 mg kg−1 for Ni. The maximum amended concentrations were selected to be close to, but largely avoid, the threshold where toxicity effects are observed. These amended soils were left to equilibrate while maintaining moist conditions for 2 years before use in pot experiments. Amendments had little effect on soil pH (Supporting Information (SI) Table S1). Plant Growth and Plant Sample Analysis. Radish (Raphanus sativus) was directly sown in pots filled with 250 g 7306

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Figure 1. Dependency of effective concentrations (CE) of Ni and Cd measured by DGT on concentrations measured directly in extracted soil solution. Figures (a) to (c) represent Ni measurements for unamended soils and soils spiked with 10 and 100 mg kg−1 Ni, respectively. Figures (d) to (f) represent Cd measurements for unamended soils and soils spiked with 0.5 and 4 mg kg−1 Cd, respectively. In (a), the solid circles (●) represent soils B, TR, and SR (from left to right); the squares (□) represent soils DR, ShR and HH (from left to right). In (b), the solid circles (●) represent soils DR, B, TR, and SR (from left to right); the squares (□) represent soils ShR and HH (from left to right). In (c), the solid circle (●) represents soil HH. In (d) to (f), the solid circles (●) represent soil HH and the open circles (○) represent the remaining soils. All DGT data have error bars; where they do not show they are smaller than the symbols. Solution data do not have error bars because they were not replicated. Dotted lines show the 1:1 relationship.

with 10 mL aqua regia (concentrated HNO3 and HCl (1:4 v/ v)) for total metal extraction.18 Trace Metal Analysis. The total concentrations of Cd and Ni in the DGT eluates, acidified soil solutions and digests were determined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo X7) after appropriate dilution. Certified reference materials (SLRS-4, river water reference material for trace metals, National Research Council Canada) and blank samples were included in all analytical sets. Basis for Interpretation of DGT Data. As metal is taken up by DGT during deployment, its concentration at the surface of the device starts to decline. If there is a dynamic equilibrium between metal in the solid phase and metal in solution, metal will be resupplied from solid phase to solution. Depending on the rate and extent of this resupply, the concentration of metal

at the interface of the DGT device will be to a greater or lesser extent sustained. The time-averaged concentration in soil solution at the interface during the deployment time, usually referred to as DGT-measured concentration, CDGT, is calculated from the measured accumulated mass of metal using well established equations.12,15 There are two extreme cases. When supply is rapid and the solid phase has a large reservoir of labile metal, the concentration in solution at the interface will not decline appreciably. The ratio of CDGT to the bulk concentration of metals in soil solution, Css, known as R, (eq 1), is then close to 1. R = C DGT/Css , 0 < R < 1 7307

(1)

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organic complexes and the value for soil HH was substantially higher than for other soils (SI Table S3). The true extent of organic complexation is likely to be much higher than this predicted value of 25% because WHAM is known to substantially underestimate the complexation of Ni by organic matter.5,22 Although the concentrations of DOC and the percentages of Ni organic complexes in solutions of soils DR and SHR were among the highest, they were not exceptional. For the six soils with slopes ≤1 there is little if any supply from the solid phase, indicating that Ni is firmly bound. Apart from soil COV, these soils have the highest pH (≥6.38). It is well-known that at higher pH Ni is transferred to a slowly exchangeable Ni−Al layered double hydroxide and Ni−Al phyllosilicate.23,24 The total organic carbon of some of these soils is lower than the average, indicating that binding to solid phase organic groups is unlikely to account for the poor release of Ni to solution. When the soils were amended with 10 mg kg−1 of Ni, they could still be fitted to three different lines (Figure 1b) with slopes of 2.2, 1.0, and 0.6, similar to those found for the unamended soils. The main difference was that soil DR moved from having a very low slope to the group of soils B, TR, and SR, with a slope of 1. Amendment with Ni to a moderate concentration does not appear to change appreciably the extent of resupply from the solid phase. For soil DR a greater proportion of the added Ni appears to be available to the DGT sink, suggesting that the amount of added Ni exceeded the capacity of large heterogeneous ligands in solution or at least their strong binding sites. In the latter case more of the added Ni would occupy weaker binding sites, where the metal could be rapidly released and contribute to the DGT measurement. After amendment with 100 mg kg−1 of Ni, 12 soils could be represented by the same line with a slope of 2.3 (r2 = 0.97) (Figure 1c). Soil HH was the sole exception. For the five new soils on this line it appears that the strong, effectively irreversible, binding sites of the solid phase, that were available at lower Ni concentrations, were unavailable at these high concentrations, suggesting that these sites have a limited capacity. The slope for soil HH increased slightly to 0.92, close to the diffusion only case (Table 1). This may have been due to there being less complexation in solution, although the WHAM calculations do not suggest this (SI Table S3). More likely there was increased, albeit limited, supply from the solid phase. Except for soil HH, effective Cd concentrations measured by DGT were linearly related to those measured in extracted soil solution (Figure 1d−f). The regression equations (forced through zero) for unamended soil and soils amended with 0.5 mg kg−1 and 4 mg kg−1 of Cd were similar, with the same slopes of 3.0 and values of r2 of 0.84, 0.93 and 0.89, respectively. The virtually constant slope, irrespective of amendment by Cd salts, indicates that the proportional resupply from the soil solid phase was similar before and after amendment. This suggests that the distribution coefficients for labile exchange of Cd between solid phase and solution, Kdl, and the resupply rate constants, were unaffected by this range of Cd concentrations. As Cd release from the solid phase is known to be fast,7 there must be a small proportion of labile Cd in the solid phase to account for the slopes for most soils being much less than 10, unless Cd is predominantly bound to large solution ligands, which is unlikely. For the soil HH, slopes were just a little above 1 (1.2−1.3) for both unamended and amended soil (Figure 1d−f), suggesting that the Cd was mainly supplied by diffusion. The

If there is no resupply of labile metal species from soil to solution, the DGT device is supplied solely by diffusion of solutes from the soil solution, which become progressively depleted in the vicinity of the device. In this case, R is at its minimum possible value and is termed Rdiff (R = Rdiff). The Rdiff value is determined by the diffusion coefficient of labile metal species in soil solution, the geometry of the device and the deployment time. Clearly CDGT is generally less than Css. However, the flux of metal to DGT is generally greater than the flux if supply was only by diffusion of metal in soil solution. To accommodate this additional supply in terms of a concentration, Zhang et al. (2001) introduced the concept of effective concentration, CE. Conceptually CE is the concentration of metal that would have to be present in the soil solution to supply the same mass of metal accumulated by DGT. It is simply given by eq 2. C E = C DGT/R diff

(2)

Expressed more simply, it is the effective concentration of metal available to DGT from both soil solution and solid-phase labile pool. Rdiff was calculated using the 2D DIFS numerical model (2D DGT induced fluxes in soils).19 The input parameters used, included particle concentration (Pc, g/mL), soil porosity (φ) and diffusion coefficient in soil (Ds) are provided in the SI (Table S2).20 If supply is solely by diffusion, CE/Css has a value of 1. Values greater than 1 indicate some supply from the solid phase. A maximum possible value of 1/Rdiff, which in this study is ∼10, corresponds to the sustained case. Values less than 1 indicate that a fraction of the metal in soil solution is not measured by DGT due to the presence of species with (a) lower diffusion coefficients than the free metal (colloidal species and large labile complexes) and/or (b) slow rates of metal release (typically poorly labile complexes).

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RESULTS AND DISCUSSION DGT and Soil Solution Concentrations. When CE was plotted against the directly measured concentrations in soil solution, Css, the soils without Ni amendment and with the lower Ni amendment (10 mg kg−1) could be placed in three distinct groups characterized by different slopes (Figures 1a and 1b). For the soils amended with Ni to 100 mg kg−1 there were two distinct groups (Figure 1c). For each group with more than one soil, the data fitted the lines well, with good correlations (r2 > 0.9, forced through zero) (Figure 1a−c). In the soils without amendment of Ni, there are a group of seven soils with the largest slope of 2.1 (r2 = 0.95) (Figure 1a). This slope indicates that the metal concentrations in soil solution are partially buffered by resupply of metal from the solid phase. The other two slopes were only 1.0 (r2 = 0.92) and 0.5 (r2 = 0.93). The slope of 1 for the soils B, TR and SR showed that these three soils are close to the diffusion only case. The lower slope of 0.5 for soils DR, SHR and HH indicates some limitation of the supply from solution. Complexation in solution by organic matter or the presence of colloidal (nanoparticulate) species might contribute to a lower slope simply by increasing the effective diffusion coefficient of Ni species. The effects of organic complexation might especially apply to soil HH, which had a much higher DOC concentration (57 mg L−1) than the other soils (Table 1). It also had the highest concentration of Ni in soil solution, which, given its fairly high pH of 7.0, indicates that complexation in solution is important. WHAM21 was used to calculate the percentage of Ni species present as 7308

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Figure 2. Plots of Ni concentrations in roots of radish versus soil solution concentrations and effective concentrations (CE). Figures a and d show soils without amendment with Ni salts; Panels b and e show soils spiked with 10 mg kg−1 of Ni; Panels c and f show soils spiked with 100 mg kg−1 of Ni. The regression line in Panel c includes soil HH (●). All data of DGT measurement and root uptake have error bars; where they do not show they are smaller than the symbols. Solution data do not have error bars because they were not replicated.

Bioavailability of Ni and Cd to Radish. Metals measured in roots and shoots had similar concentrations and the graphs against soil measurements of concentration were similar. Data are only presented for roots as this is the edible part and less prone to plant effects. Ni concentrations in radish did not increase linearly with concentrations in solution for soils without Ni amendment (Figure 2a). For the three soils (HH, SHR, and DR), identified by DGT as having the lowest (diffusion only) supply, concentrations in roots were low compared to most other soils (Figure 2a). Concentrations measured in the soil solution included labile and nonlabile metal, so the data suggest a lower labile proportion in these three soils. Radish grown in soils HH, SHR, and SR after amendment with Ni at 10 mg kg−1, had lower than expected Ni concentrations compared to other soils

higher concentration of Cd in soil solution compared to other soils (except WIN and CR with pH < 6), the relatively low DGT measured concentration and the fact that soil HH had the highest DOC concentration of all 13 different soils, indicates that, for this soil, a large proportion of Cd may be bound as complexes with organic matter. WHAM calculations support this contention (SI Table S3). There is also the possibility that the high soil pH and organic matter content compared to other soils, strongly binds the cadmium in the solid phase, lowering both the proportion that is labile and the dissociation rate constant. We cannot exclude the pool size of readily exchangeable Cd being limited by other solid phases or the possibility that Cd exists as mineral colloids apparently in soil solution, but unavailable to DGT. 7309

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concentrations in both roots and shoots of radish. In the soils highly contaminated with Ni, the concentration in the plant correlated well with both soil solution (r2 = 0.80) and free ion activity (r2 = 0.87) (SI Figure S2). The correlation improved to (r2 = 0.96) in both cases when soil HH, the obvious outlier, was excluded. There was a good correlation (r2 = 0.93) for the comparable plot for CE including all data. In this case, where there is expected to be ample diffusional supply, the uptake of Ni by radish is probably controlled by the plant and therefore the free ion activity, consistent with the ideas of Degryse et al.1 However, it should be noted that, even if there was depletion of Ni at the root surface at high concentrations of Ni, limitation of supply by slow dissociation rates is unlikely to be important, as progressively weaker solid phase binding sites are occupied and most of the added Ni in solution will be present as the free ion or labile species. The good correlation between free ion and soil solution values has already been noted and the soil solution concentration is a major factor determining the values of CE, which accounts for the strong positive correlation between concentration in radish and CE. The marginally better correlation for CE rather than aNi2+ may be due to CE excluding colloids or inert complexes which would be measured in soil solution, producing an erroneously high value for aNi2+. Alternatively, as already mentioned, WHAM tends to underestimate complexation of aNi2+ by natural organic matter and therefore overestimate aNi2+. Previous mechanistic studies of Ni uptake involving DGT, performed hydroponically, have demonstrated that plant rather than diffusion control dominates for both tomato and spinach.9,10 Although no other work has considered Ni in radish along with Ni measured by DGT in soils, uptake of Ni by wheat has been studied. Strong correlations were observed between Ni in wheat shoots and Ni measured by DGT and in soil solution, in soils with Ni in the range 4.7−21 mg kg−1, suggesting that, here too, Ni uptake is under plant control.27 More generally the terrestrial biotic ligand model has been shown to be able to predict Ni toxicity, in terms of barley root elongation and tomato shoot yield, in contaminated soils.28,29To embrace all data, log−log plots were considered (SI Figure S3). While the regression equations logNiroot = 1.00logCE - 2.08 (r2 = 0.90) and logNiroot = 1.09logCss - 2.12 (r2 = 0.74) confirm the good relationships between Ni in radish and CE, they obscure the poor relationship between Ni in radish and Css at low concentrations of Ni. The main reason for the reasonable correlation coefficient of 0.74 for the plot against Css is that the concentration range extends over decades, due to the amendments. If only log−log plots of all data had been considered, as is customary, the mechanistic differences associated with concentration would have probably been overlooked. It is instructive to bring together the major findings that have emerged from consideration of the dependency of CE on Css, and of Ni in radish on Css and CE. The relationship between CE and Css enabled soils to be separated into two main groups, corresponding to appreciable and negligible resupply of Ni from the solid phase. In the latter case there was a subgroup where complexation in solution further limited supply. Amendment with a high concentration of Ni largely eliminated these distinctions between soils, possibly by reducing complexation in solution, but more probably by providing a labile solid phase pool. Although there was a poor relationship between Ni in radish roots and either Css or aNi2+ in unamended soils, the soils with a low uptake by radish relative to Css or aNi2+ were

(Figure 2b), indicating that a large proportion of added Ni in these soils was unavailable for plant uptake. It appears that the amount of added Ni did not exceed the capacity of the binding sites of the solid phase or the organic matter in solution, as also indicated by the DGT−soil solution plots (Figure 1). However, when the soils were spiked with Ni at 100 mg kg−1, the Ni concentration in the root correlated well (r2 = 0.80) with Ni in soil solution (Figure 2c), especially if soil HH was excluded from the regression (r2 = 0.96). In highly contaminated soils, it is unlikely that there is a depletion of metal in the zone around the plant roots, as transpirational fluxes of water are likely to bring more metal to the plant root surface than is required physiologically and taken up by the plant.25,26 When supply by diffusion is unimportant there can be no kinetic limitation from either soil or solution. This plentiful supply from soil solution does not apply to the exception of soil HH. Its deviation from the line is consistent with there being a higher proportion of nonlabile Ni (colloids and complexes) in soil solution compared to the other soils (Figure 2c), which fits with the DGT data (Figure 1) and this soil having the highest DOC and percentage of organic species of Ni in soil solution (Table 1 and SI Table S3). Apart from small deviations for soil HH, and to an even lesser extent soil SHR, the activities of Ni2+ and Cd2+, aNi2+, and aCd2+, were linearly related to Ni and Cd, respectively, in the soil solution of both unamended and amended soils (SI Figure S1). Consequently, plots of concentrations in roots versus aNi2+ in soil solution (SI Figure S2d, e) are similar to those versus Ni in soil solution, suggesting plant uptake cannot be explained simply by considering speciation of Ni in soil solution. The effective concentrations (CE) correlated well (r2 > 0.9) with concentrations of Ni in radish roots, irrespective of whether the soils were amended with Ni or not (Figure 2d−f). For unamended soils, soil HH was only a little away from the regression line. This difference from the other soils was less marked than for the plots versus soil solution (Figure 2a) or for plots of CE versus Css (Figure 1). However, the fact that the data point for soil HH does not lie on the Ni in plant versus CE line indicates that a proportion of Ni is available to DGT while not being taken up by the radish. The DGT measurement excludes inert Ni bound by both the solid phase and complexes in solution, but includes contributions from the free ion in soil solution and resupply of labile Ni from the solid phase and solution complexes. A dynamic competition for Ni2+ exists between the Chelex binding agent of DGT and ligands in solution.4 The high affinity of Ni2+ for Chelex, augmented by Chelex’s very high concentration in the binding layer, is likely to be greater than the affinity of Ni2+ for root surface sites. Consequently, some metal-complexes that are available to DGT might not be taken up by plant roots, as appears to be the case for soil HH. For the Ni-amended soils, the proportion of Ni which was available to both DGT and plant increased for soil HH, as shown by soil HH data points being close to the lines of Figures 2e-f. The concentration of Ni in radish root is linearly dependent on CE across a wide range of concentrations and soil types. For the lower range of Ni in the soils, the good correlation between plant uptake and CE is consistent with uptake being limited by diffusion and associated soil processes, including resupply from the solid phase, rather than plant processes. CE was linearly related to concentrations of Ni in both root and shoot (data not shown), implying that it can be used as a good predictor of Ni 7310

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transport at a common carrier in the plasma membrane of wheat root cells, consistent with previously observed inhibition of Cd accumulation in crop plants by Zn. However, multiple regressions including Zn did not provide appreciably improved relationships. Although the relationship between CE and Css was simpler for Cd than for Ni, there was a poor relationship between Cd in radish and soil measurements of Cd, irrespective of amended amounts. The hypothesis of Degryse, et al.1 of dominant diffusional and plant limiting uptake mechanisms at respectively low and high metal concentrations does not apply for uptake of Cd by radish. PlantYield. Amending soils with Cd did not significantly affect plant yields (dry weight), consistent with another study where radish yields were unaffected by Cd applications up to 20 mg kg−1.40 Except for soils OF, WIN, and CR amended at 100 mg Ni kg−1, the plant yields were unaffected by Ni amendments. The concentration of Ni in the soil solution of these three soils was greater than1.5 mg L−1. The yields (dry weight) of radish for soils WIN and CR at 100 mg Ni kg−1 were 1 /5 of those of unamended soils, whereas for soil OF they were about 1/2. Radish response in this study was comparable to that reported by Simon et al.,41 who observed toxicity of radish in soils containing 120 mg Ni kg−1. Although the Ni concentration in soil solution for soil HH was also about 1.5 mg L−1, the yield of radish did not changed significantly. This is consistent with the low concentration of Ni in radish for this soil, again indicating the probable effect of the high organic matter content in the soil and solution.

those identified by DGT as having a diffusion only supply and probably some complexation in solution. By contrast Ni in radish was linearly related to CE, demonstrating that, except for soil HH, DGT measurements mimic plant uptake and account for the differences between soils associated with resupply from the solid phase and complexation in solution. This demonstrates diffusion limited uptake. For soils amended with high concentrations of Ni, linear relationships were obtained for Ni in radish plotted against, Css, aNi2+, and CE, consistent with the plant controlling uptake. Therefore, these results for Ni provide strong support for the hypothesis of Degryse et al.1 Cadmium in roots generally increased with Cd free ion activity, its concentration in soil solution and CE, but the correlation coefficients were not high (r2 = 0.26 to 0.58) (SI Figure S4a−i). In contrast to the Ni case, there was no improvement in the relationships for the amended soils at either added concentration. However, the maximum amendment for Cd of 4 mg kg−1 was much less than the 100 mg kg−1 amendment for Ni where good correlations with soil solution concentrations were observed. Therefore, it is unlikely that the capacity of solution complexes or solid phase binding sites would have been exceeded. These data suggest that Cd uptake by radish is not simply related to either the free ion activity or the diffusional supply from soil solution augmented by resupply from the solid phase. Cadmium in roots increased systematically with the large increases in CE and Css caused by amendments, as shown by log−log plots (SI Figure S5): logCdroot = 0.84logCE − 1.28 (r2 = 0.74) and logCdroot = 0.78logCss − 0.81 (r2 = 0.73). Close inspection of these plots shows that the data for each amendment group would not be fitted by the regression line for all data. This shows that the relationship does not account satisfactorily for observed differences between soils, confirming the conclusion from the linear plots. Strong correlations between Cd in some plants and DGTmeasured Cd concentrations have been reported, including for wheat shoots,8 rice shoots and grain,16 and plantain shoots.30 However, poor relationships between Cd uptake by radish and either total Cd concentrations or Cd2+ in soil solution have been observed.31−33 For lettuce, neither Cd2+ (r2 = 0.44) nor CE (r2 = 0.50) could predict uptake of Cd well.34 There are several possible explanations for DGT and soil solution measurements failing to correlate well with plant uptake of Cd. (a) The plants might be affected by toxicity. However, there was no significant toxic response to Cd in this study (see next section “Plant yield”). (b) During the plant growth period soil microorganisms could facilitate oxidation of organic matter, causing release of some organically bound Cd to soil solution.35,36 A comparable process would not occur during the short DGT deployment time (about 24 h). Similarly, root exudates, which could complex Cd, would be absent from the DGT experiments. The special environment of the rhizosphere of radish was demonstrated by Lorenz, et al.,33 who reported that plant uptake of Cd correlated well with Cd in rhizosphere soil solution, but not with Cd in bulk soil solution. (c) Active physiological processes, such as changes in transporter affinity and density, and to the extent of root hairs, may be occurring to regulate Cd uptake and translocation.10,34 (d) Complexes of Cd may be taken up directly at high concentrations of ligands, especially if plants are stressed, but this is unlikely to occur for the soils used here.10,37 (e) There are many studies showing inhibitory effects of Zn on Cd uptake by higher plants or algae.38 Hart et al.39 demonstrated that Zn and Cd compete for

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ASSOCIATED CONTENT

S Supporting Information *

Tables of: pH of soils before and after amendment, soil parameters for Rdiff calculations and concentrations in soil solution with % present as organic complexes. Figures of: free ion activities versus soil solution concentrations, dependency of Ni concentrations in roots on aNi2+, log−log plots of concentrations in roots versus Css and CE for both Ni and Cd, linear plots of Cd in roots versus CE, Css and aCd2+. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: 0044-1524-593985; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Science Foundation of China (Grant No. 21207062), the Natural Science Foundation of Jiangsu Province (BK2012311), Specialized Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (20120091120016), an Engineering and Physical Sciences Research Council Dorothy Hodgkin Postgraduate Award (NE/C506999/1), and the Fundamental Research Funds for the Central Universities in China.

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