Plant induced changes to rhizosphere characteristics affecting supply

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Characterization of Natural and Affected Environments

Plant induced changes to rhizosphere characteristics affecting supply of Cd to Noccaea caerulescens and Ni to Thlaspi goesingense Jun Luo, Daixia Yin, Hao Cheng, William Davison, and Hao Zhang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Environmental Science & Technology

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Plant induced changes to rhizosphere characteristics affecting supply of Cd to Noccaea caerulescens and Ni to Thlaspi goesingense

4 5

Jun Luo1*, Daixia Yin1, Hao Cheng2, William Davison2, Hao Zhang2*

6 7

1

8

Environment, Nanjing University, Nanjing, Jiangsu 210023, China

9

2

10

State Key Laboratory of Pollution Control and Resource Reuse, School of the

Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, United

Kingdom

11 12 13

*Corresponding

authors,

J.

Luo,

H.

Zhang,

14

[email protected];

15

[email protected]

Fax: Tel:

0086-25-89680632,

e-mail:

0044-1524-593899;

e-mail:

16 17 18

ACS Paragon Plus Environment


19

ABSTRACT

20

Changes in soil rhizosphere properties after growing the Cd hyperaccumulator

21

Noccaea caerulescens and the Ni hyperaccumulator Thlaspi goesingense were

22

investigated. Dissolved organic carbon content increased in the rhizosphere, but there

23

were no significant changes in the solution concentrations of Cd and Ni.

24

Concentrations of these metals extracted by NH4Cl and EDTA decreased in the

25

rhizosphere, as did DGT-measured concentrations, indicating a depletion of labile

26

metal in the solid phase. The results could be explained by the increased DOC in the

27

rhizosphere maintaining a higher proportion of the labile metal in solution through

28

complexation, with the overall depletion of metals only manifest in the solid phase.

29

The DGT induced fluxes in soils and sediments (DIFS) model was used to provide

30

key kinetic information on soil processes and labile pool size. These data showed that

31

the more limited metal supply in the rhizosphere after the growth of

32

hyperaccumulators was due to both depletion of the solid phase pool and a lower rate

33

constant of supply from solid phase to solution. The effect on the rate constant, which

34

could be rationalized by the plant sequentially accessing and consuming the more

35

labile pools of metal, was most marked for Cd, which had the highest accumulation

36

factors.


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INTRODUCTION

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Contamination of metals in soils is a serious environmental issue, but remediation

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is difficult.1 Phytoremediation is the name given to the technology that uses green

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plants, especially hyperaccumulator plants, to clean up contaminated soils.2 Metals

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are taken up from soil into the above-ground, harvestable tissues of plants that are

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grown in the soil. It is considered to be an environment-friendly, low-cost, and

43

sustainable remediation technology,3 compared to physical and chemical approaches,

44

which are expensive and often severely impact the structure and fertility of soils.4

45

Although hyperaccumulator plants are capable of accumulating more than 100 times

46

higher concentrations of metals in plant tissues than normal plants, their relatively

47

small biomass has hindered their wider applications in soil remediation. One

48

exception is the arsenic hyperaccumulator Pteris vittata, which can grow to 1-2

49

meters high and has been proved to be effective at removing As from contaminated

50

soils.5

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Although phytoremediation has advantages, its efficiency decreases with increasing

52

plant growth time. Accumulated Cd in the shoots of Cd hyperaccumulator (Sedum

53

plumbizincicola) decreased significantly with successive harvests over a two-year

54

period.6 Similarly, a field study showed that7 the As hyperaccumulator (Pteris vittata)

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removed greater amount of arsenic in the first year than in the second year (15.7 g vs.

56

10.7 g). This reduction in uptake with time may be due to reduction of the labile pool

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size of targeted metals on the solid phase in the rhizosphere as phytoremediation

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proceeds.8 Root-induced chemical changes in the rhizosphere, which is spatially

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defined as a small volume of soil surrounding the living roots that is influenced by

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their activity,9 may be particularly pronounced for hyperaccumulator plants.10,

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Reactions occurring in the rhizosphere, such as increased solubility of the soil mineral


11


62

components and changes in pH, can affect the concentrations of the macro- and

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micro-nutrients in soil solution and hence their uptake by the plant.12 The main

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constituents of root exudates are considered to be low molecular weight organic acids,

65

which can affect metal speciation and availability, but some plant species release

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specific metal-chelating or reducing compounds into the rhizosphere in their strategy

67

to mobilize metals.13 Other physical, chemical, and biological properties of the

68

rhizosphere can also be affected by plants.14

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Given its importance, there is a need to assess how the rhizosphere affects

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phytoremediation efficiency. There are many processes potentially affecting metal

71

supply to plants, including diffusional transport to the root, the available pool size of

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labile metal from the solid phase, and the kinetics of release from solid phase to

73

solution.15 Traditional chemical extraction procedures, including isolation of soil

74

solution have been used to investigate the chemical changes in the rhizosphere of

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hyperaccumulator plants.16, 17 However, it has been recognized that metals released by

76

these extraction procedures rarely correlate well with plant metal concentrations,18

77

and that such approaches do not consider possible changes in the dynamics of metal

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supply within the soil.3 If there is a fast uptake by the plant (i.e. hyperaccumulator

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plants), the concentration of the free metal is depleted at the interface of plant soil

80

solution, which induces a resupply from the complexes in the soil solution and from

81

the labile component of the solid phase. Therefore, both the available pool size of

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labile metal and the kinetics of supply of free metal are potentially important factors

83

influencing uptake.

84

In recent years the DGT (diffusive gradients in thin-films) technique has been used

85

to obtain information on bioavailability and the resupply dynamics of metals from

86

solid phase to solution.15, 19 Significant correlations between metals in plants and


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those measured using DGT have been reported for a range of metals and soil types20-22

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and the mechanisms of DGT and plant uptake have been systematically compared23.

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DGT has also been used to determine root-induced changes in labile pool fractions in

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soils, as shown for As.24

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The mass of metals accumulated by DGT depends collectively on the metal

92

concentration in the soil solution, the rate of diffusional supply and the extent and rate

93

of release of metal from complexes in solution and from the solid phase. DGT is best

94

regarded as a tool for conducting in situ perturbation experiments by introducing a

95

localized metal sink, rather than being a device for measuring metal concentrations in

96

soil solution. The DGT induced fluxes in soils and sediments (DIFS) model

97

developed by Harper et al.25 and upgraded as 2D DIFS by Sochaczewski et al.26

98

provides a numerical simulation of the interaction between the DGT device and soils.

99

Kinetic information on soil processes and the labile pool size can be obtained from

100

DGT measurement by using the 2D DIFS model. The DIFS kinetic models that have

101

been used to interpret DGT measurements to obtain soil parameters, such as the

102

distribution coefficients for labile Cd, Ni, Zn (Kdl) and exchange kinetics between

103

solid phase and solution.27 Bravin et al.28 used DIFS to study the effect of wheat

104

growth on the rhizosphere changes of Cu. They found plant uptake had little impact

105

on Cu in the rhizosphere.

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Previously,29 systematic comparison of DGT and soil solution measurements with

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plant uptake showed that Cd uptake by the hyperaccumulator (Noccaea caerulescens)

108

and Ni uptake by the hyperaccumulator (Thlaspi goesingense) is not predicted by the

109

biotic ligand model, but is controlled by the rate of supply from the soil. These two

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hyperaccumulator species were grown in pots containing five different soils with

111

different contents of Cd and Ni for this study. The objectives were: i) to assess



112

rhizosphere characteristics of these hyperaccumulators using DGT and other chemical

113

extraction procedures; ii) to investigate the effect of chemical changes in the

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rhizosphere on the resupply dynamics of metals; iii) to highlight the importance of

115

pool size and resupply kinetics in evaluating phytoremediation efficiency. This is the

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first study to investigating the plant-induced changes in the rhizosphere of Cd

117

hyperaccumulator (Noccaea caerulescens) and Ni hyperaccumulator (Thlaspi

118

goesingense) using the well-established in situ dynamic technique of DGT.

119

EXPERIMENTAL SECTION

120

Soils sampling. Five different soils were collected in the delta region of the

121

Yangtze River at Nanjing (soils MX and BX), Wuxi (soils WX and WG), and

122

Zhenjiang (soil ZY) in Jiangsu Province, China. This area is highly industrialized with

123

some severe metal pollution of agricultural soils. They were chosen to cover a

124

reasonable range of soil texture, pH, TOC and Cd and Ni concentrations. All the soil

125

samples were air-dried and passed through a 2-mm sieve. The physico-chemical

126

characteristics of these five bulk soils are shown in Table1.

127

Pot experiment. Noccaea caerulescens and Thlaspi goesingense were grown in

128

pots that were filled with 200 g air-dried soil. Six germinated plant seedlings (about

129

2-3 cm high) were transplanted into each pot. All the plants were grown in a glass

130

house with the following conditions: 14 h/10 h day/night, 20 ºC /16 ºC day/night

131

temperatures, and natural light supplemented with several 1 kw SON-T lamps to

132

maintain a minimum photon flux of 250 µmol m-2 s-1. The plants were watered using

133

MQ water (Milli-Q, Millipore) every day. All the plants were harvested 9 weeks after

134

transplant when they had achieved sufficient biomass. After collection of rhizosphere

135

soil samples (see below), the plant samples were cleaned using MQ water and dried in

136

the oven at 80 ºC for 24 hours. About 0.20 g of ground plant shoot sample were


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transferred to an acid-cleaned Pyrex tube. After adding 5 ml aqua regia (concentrated

138

HNO3 and concentrated HCl mixture (1:4 v/v)) to each tube the samples were

139

digested using a heating block in a fume cupboard.15

140

Rhizosphere soil sampling and soil analysis. The rhizosphere soils exist in the

141

root mesh (pore size < 2 mm) of the plant roots. After removing the bulk soil, the

142

rhizosphere soil was collected by first carefully shaking by hand to harvest the loose

143

soil. Soil that adhered to the roots was collected using a clean plastic spatula. After

144

plant harvest, bulk soil was collected and mixed well prior to sample analysis. Soil

145

samples (both rhizosphere and bulk soil) were air-dried and sieved through a 2 mm

146

nylon mesh. The available metals were extracted by both 1 M NH4Cl (soil: solution =

147

1: 6, 16 hours, end-over-end shaker) and 0.05 M EDTA (soil: solution = 1: 5, 1 hours,

148

end-over-end shaker) to study the effect of plant growth on the pool size of labile

149

metal. Dissolved organic carbon (DOC) in soils was analyzed with a TOC analyzer

150

after water extraction (soil: water = 1: 10, 16 hours, end-over-end shaker). Soil pH

151

was measured in 0.01 M CaCl2 solution (soil: water = 1: 2.5) after shaking for 30

152

min.30 The total concentrations of metals were determined with ICP-MS after

153

HNO3-HClO4-HF (1:1:1) digestion.31 All samples and measurements were in

154

triplicate.

155

DGT measurements. The plastic mouldings of the DGT devices for soils

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comprised a backing base and a front cap with a round window of 2.54 cm2 area. A

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resin gel layer was placed on the base with the side containing the resin (Chelex-100,

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200-400 mesh, Bio-Rad, USA) facing upward. A 0.8 mm thick diffusive gel layer was

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placed directly on the resin gel layer. On the top of the diffusive gel there was a

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0.14-mm-thick, 0.45-µm hydrophilic polyethersulfone membrane, which prevents

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adherence of the soil particles and has been shown to have similar diffusion



162

characteristics to the diffusive gel layer.32 Detailed information on the standard

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procedure for gel making has been published.30

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Before DGT deployment on soils, air-dried soil samples (rhizosphere and bulk soils)

165

were brought to 60% MWHC (maximum water holding capacity) and incubated for 2

166

days, then raised to 80% MWHC for 24 hours. Soil and water were mixed thoroughly

167

using a plastic spatula when adjusting the moisture content. Thereafter, two DGT

168

devices were exposed to the incubated soils (dry weight 6 g) for 24 hours, ensuring

169

good contact between the soil and the device. After DGT retrieval, soil solution was

170

collected from soil samples by centrifuging at 4600 rpm for 15 min. The DGT devices

171

were jet-washed with MQ water to remove soil particles and then disassembled. The

172

resin gels were removed from the DGT device and immersed in 1 M HNO3 in

173

micro-centrifuge PVC tubes for at least 24 hours at 20±1 oC. The eluate and soil

174

solution were stored at 4 oC prior to analysis by ICP-MS.

175 176

Interpretation of DGT measurements. The Supporting Information outlines the already published approach to interpreting DGT measurements.

177

Heavy metal analysis. The concentrations of Cd and Ni in the eluate, soil solution,

178

extraction solution and plant digests were determined by inductively coupled plasma

179

mass spectrometry (ICP-MS, Thermo X-Series X7, USA). Certified reference

180

materials, SLRS-4 (River water reference material for trace metals, National Research

181

Council Canada) were used. The measured values (0.013±0.001 and 0.687±0.047 µg

182

L-1 for Cd and Ni) were within the range of the certified values (0.012±0.002 and

183

0.67±0.08 µg L-1). To ensure analytical quality control they were included with blank

184

samples in all analytical sets, including those from DGT deployments, digestions, and

185

extraction experiments.

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RESULTS AND DISCUSSION ACS Paragon Plus Environment

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After 9 weeks growth in five different soils, Noccaea caerulescens and Thlaspi

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goesingense were close to maturity, but not yet flowering. The physico-chemical

189

characteristics of the five soils (bulk soils) and accumulation factors (AF,

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concentration ratio of metal in shoots to metal in soil) for the two hyperaccumulators

191

are presented in Table 1. The AF of Noccaea caerulescens for Cd ranged from 70 to

192

237, similar to previously reported values.33 The accumulation factor of Thlaspi

193

goesingense for Ni was about 3.3 for these five soils, comparable to the ratio (AF =

194

5.4) reported by Puschenreiter et al.34

195 196

pH changes in the rhizosphere. There were small, but significant (p < 0.05)

197

increases in the pH of the rhizosphere compared to bulk soils after growth of both

198

Noccaea caerulescens and Thlaspi goesingense (Fig. 1). The maximum increase in

199

pH of 0.33 was found for the Ni hyperaccumulator in soil WX. The observed modest

200

increases in pH in rhizosphere soils agrees with several previous studies,17,

201

involving growth of Noccaea caerulescens and Thlaspi goesingense. Increases in pH

202

have been attributed to the release of hydroxyl ions during mineral dissolution35,

203

especially when the soil pH was lower than 7.3.

204

pH was ascribed to the plants taking up N primarily in the NO3-N form, with

205

concurrent excretion of OH- ion.37 Rhizosphere alkalization has also been found in

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subterranean clover,

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al.40 found that there was a small decrease of soil pH after growth of Noccaea

208

caerulescens and Thlaspi ochroleucum, which is a non-hyperaccumulator. Although

209

there were pH changes in rhizospheres after growth of these Cd (N. caerulescens) and

210

Ni

211

hyperaccumulation by these two species did not involve rhizosphere acidification.

(T.

38

28, 36

33, 34

The increase in the rhizosphere

and in some crops (millet, cowpea).39 In contrast, McGrath et

goesingense)

hyperaccumulators,

the

authors


suggested

that

the


212

Generally, rhizosphere pH changes have been found to be related to the ion charge

213

balance of cation and anion uptake, and not to the specific metal being

214

hyperaccumulated.41 Some plants acidify the soils around their roots,12 inducing

215

increased uptake of metals as they are liberated by the protons. The acidification is

216

considered to be mainly caused by a release of H+ by roots in response to an ion

217

charge imbalance caused by the uptake of metal ions.42 Given the overall small

218

increase in pH, acidification is an unlikely mechanism for the hyperaccumulation of

219

metals for the plants considered here.

220

DOC in the rhizosphere. Significant changes in DOC between rhizosphere and

221

bulk soils following the growth of the two hyperaccumulator plants are shown in Fig.

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2. The increases in DOC in the rhizosphere, which can promote complex formation

223

and increase the migration velocity of metals,43 ranged from 15% to 41% for Noccaea

224

caerulescens and 14% to 86% for Thlaspi goesingense. We recognize that a

225

substantial part of the measured DOC in the rhizosphere soil may be of microbial

226

origin and so without direct evidence the observed increases cannot be solely

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attributed to exudates. However, these changes in DOC are consistent with the

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Thlaspi family generally producing substantial exudates during plant growth.

229

Puschenreiter et al.34 reported increases of 27.1% in DOC in the rhizosphere

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compared to bulk soils following growth of Noccaea caerulescens, and a 61%

231

increase following growth of Thlaspi arvense (non-hyperaccumulator). The authors

232

concluded that there was no evidence for DOC being responsible for the

233

hyperaccumulation of metal by Noccaea caerulescens. The increased DOC in the

234

rhizosphere of Thlaspi arvense might suggest a protective mechanism to decrease

235

phytotoxicity by reducing uptake of metals as the free ion concentration will decrease.

236

However, an effect of increased DOC concentration is to shift equilibrium towards


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more dissolved metals as well as promoting DOM adsorption on the surface of

238

metals-bearing minerals.35 Although specific mechanisms involving organic ligands

239

that might affect metal uptake have been considered,17,36,41,44 there is a lack of data

240

that directly assesses how the rate of supply of labile metals might influence

241

phytoremediation.

242

Soil extraction measurements. After growth of Noccaea caerulescens, the

243

difference in soil solution concentrations between rhizosphere and bulk soil was not

244

significant as it was below 10% for Cd (Figure 3). The difference in soil solution Ni

245

between rhizosphere and bulk soils after growth of Thlaspi goesingense was also

246

below 10% (Figure 4). The generally sustained concentrations of metals in soil

247

solution in the rhizosphere is in keeping with previous findings. Puschenreiter et al.17

248

found that Ni extracted by water increased in the rhizosphere after the growth of

249

Thlaspi goesingense. An arsenic hyperaccumulator (Pteris vittata) did not decrease

250

the As concentration in the soil solution in the rhizosphere.3 Given the substantial

251

uptake by hyperaccumulators, the sustained concentration in soil solution suggests

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there is substantial resupply of metal from the solid phase pool during plant growth,

253

bringing about an effective re-equilibration.

254

Cd extracted by NH4Cl and EDTA was 15 to 33% less in the rhizosphere than in

255

bulk soils after growth of Noccaea caerulescens (Table S1 and Figures 3). There were

256

similar significant changes (from 14 to 50%) in NH4Cl and EDTA extractions of Ni

257

from the rhizosphere following growth of Thlaspi goesingense. NH4Cl extraction

258

normally targets the easily desorbed metals bound to the solid phase while EDTA

259

extraction may additionally include some strongly bound fractions. The effect of plant

260

growth on the pool size of labile metals can be investigated using two different

261

extractions by reagents with different binding strengths. Similar reductions in Cd and



262

Ni extractable by NH4Cl and EDTA have been observed previously after growth of

263

the appropriate Thlaspi species.17, 35

264

If simple equilibrium between metal in the labile solid phase pool and in soil

265

solution of both Noccaea caerulescens and Thlaspi goesingense had been maintained,

266

this depletion of the labile pool of metal would be expected to be reflected in a similar

267

depletion in the concentrations of metal in soil solution. However, as shown above,

268

concentrations of metals in soil solution were not significantly depleted. Therefore,

269

either part of the metals extracted from the solid phase pool is not in equilibrium with

270

metal in solution or there must be a mechanism within the rhizosphere that brings

271

about some mobilization of metal to soil solution. Although some studies have

272

suggested that root exudates are not involved in the hyperaccumulation of metals in

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soils, the possible role of enhanced DOC in the rhizosphere on plant uptake by

274

hyperaccumulators has been previously considered.3, 35 It is known that organic acids

275

added to soils are likely to be rapidly adsorbed onto mineral surfaces.44 Consequently,

276

large quantities of organic compounds may need to be exuded by roots for there to be

277

a substantial increase in the solution DOC concentration within the rhizosphere. These

278

organic compounds may then be involved in the dissolution of metals from the solid

279

phase.45, 46 One possible mechanism is that some of the components of the exudates

280

are relatively low molecular weight chelates that favor the solution phase and so can

281

increase the proportion of metal in solution. Then, the concentration of metal in

282

solution could be maintained while the total concentration of the labile metal pool is

283

lowered through plant uptake.

284

Such a mechanism would be less likely to be effective if the concentration of metal

285

in solution was well buffered by humic and fulvic acids in solution. The Windermere

286

humic aqueous model (WHAM 6) was used to calculate the speciation of metals in


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soil solution making the standard assumption for the application of WHAM where

288

only DOC information is available that 50% of the DOC was fulvic acid and that

289

fulvic acid contained 50% carbon.47 According to these calculations (Table S2), the

290

percentage of metals present as organic species was very low, which allows scope for

291

organic exudates to have a significant effect on metal concentrations. Calculations for

292

different assumed percentages of DOC as fulvic acid (Table S2) using WHAM model

293

showed that proportions of complexed Ni were relatively unaffected, but if >100% of

294

the DOC was fulvic acid, complexation of Cd by humic material was sufficiently

295

strong that the exudated organics were less likely to affect Cd concentrations in soil

296

solution.

297

DGT-interpreted properties of the rhizosphere. DGT-measured concentrations

298

of Cd and Ni in the rhizosphere were lower than those in bulk soils where Noccaea

299

caerulescens (Cd) and Thlaspi goesingense (Ni) had been grown (Figures. 3 and 4).

300

Fitz et al.3 reported a reduction in the DGT concentration of the metalloid As in the

301

rhizosphere of an As hyperaccumulator (Pteris vittata), indicating depletion of the

302

labile As pool. The ratio R can be used as an index of the soil’s ability to resupply a

303

metal from solid phase to solution provided there are negligible proportions in

304

solution of metal organic complexes with substantially lower diffusion coefficients

305

than those of inorganic species.32 The most likely type of high molecular weight

306

complexes present is humic substances. According to calculations using WHAM 6

307

(Table S2), the percentage of metals present as fulvic acid complexes was very low,

308

allowing calculation of R directly from the DGT and soil solution measurements. The

309

first derivation of kinetic data from DGT data using the dynamic model DIFS

310

measured R as a function of time and obtained estimates of both Kdl and the kinetic

311

parameter, TC, from curve fitting19. TC is related directly to the rate of metal supply



312

from solid phase to solution. However, if the distribution coefficient for labile metal

313

(Kdl) is known, DIFS can be used directly to obtain a value of TC from the DGT

314

measured value of R.26 The EDTA-extractable metal and the concentration in soil

315

solution was used to estimate Kdl. Use of this Kdl value in the DIFS model represents

316

the largest uncertainty in this work, as there is a presumption that the metal extracted

317

by EDTA accurately represents the metal that is able to interact with the metal in the

318

solution phase. Justification for this approach comes from previous studies which

319

have successfully used it 48 and the good agreement obtained when Kdl values deduced

320

from DGT measurements using the DIFS model have agreed with values obtained by

321

extraction.27 When the response time (TC) is smaller than a threshold value, it is

322

difficult to estimate its value precisely, as changing the value of TC has little effect on

323

the R value.30, 49 Therefore, a threshold value was calculated using the DIFS model,

324

based on the minimum value of TC required to change the value of R by 10%. The

325

threshold value varied with different soils and metal combinations. As shown in Table

326

S3, apart from Cd in soil WG, all the directly estimated TC values from measured Kdl

327

were larger than the corresponding threshold values. We recognize that there will be

328

errors in TC and derived rate constants associated with the fitting procedures, the

329

assumptions of the model and most importantly the use of a value of Kdl obtained

330

using another technique. However, in the paper the kinetic parameters are being used

331

relatively rather than in an absolute sense. There will also be some chemical changes

332

associated with air drying and subsequent rehydration. Although air-drying affects

333

microbes, it has less effect on metals mobilization. For example, Kjærgaard et al.50

334

observed that drying-induced mobilization of colloids is limited to the initial phase of

335

re-wetting (about 2-3 h). Knowing that our major aim was to compare chemical and

336

particularly kinetic parameters measured on rhizosphere soils to those measured on


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bulk soils we were concerned that our measurements on these two soil types were

338

comparable. The approach we took ensured that the size range of material, the water

339

content and the contact between soil and DGT were controlled. Drying and

340

rehydration most probably did affect the absolute values of our derived parameters,

341

but it should have affected both soil pools similarly so our comparative use of the data

342

is still valid.

343

Values for TC are presented in Table 2, which also includes the derived values of

344

the rate constant for release, k-1, along with Kdl and R values. The slightly lower R

345

values for the rhizosphere of hyperaccumulators compared with bulk soils imply a

346

poorer metal supply from the solid phase to the soil solution in the rhizosphere. This

347

reduction in the rate of supply can be brought about by the concentration of labile

348

metal in the solid phase being lowered and/or a change in the rate constant for release.

349

In all cases, values of Kdl in the rhizosphere are lower than the comparative values for

350

bulk soils, consistent with the solid phase pool being smaller. However, the increased

351

response time (Tc) for the rhizosphere indicates that the intrinsic rate of release of

352

metals from the solid phase is lowered after the growth of hyperaccumulators. This is

353

easier to appreciate in the consistently lower values for the rate constant for metal

354

release, k-1, which can be derived from Tc and Kdl (eq S5). The proportional decline in

355

Kdl was generally lower than the proportional decline in k-1 for Cd (13±3% vs.

356

56±20%), but similar for Ni (26±5% vs. 24±10%). This observation is consistent with

357

the rate constant for Ni release from bulk soil being lower than for Cd release, as

358

observed previously.15, 27 The higher rate constant for release of Cd appears to be

359

more prone to modification by the processes operating in the rhizosphere than the

360

lower rate constant of Ni.



Page 16 of 29

361

We recognize a potential problem with the comparison of rhizosphere and bulk soil

362

chemistry reported above: that the drying and rewetting approach is likely to modify

363

the soil chemistry from that pertaining in the soils prior to air-drying. As our primary

364

concern was examining differences between chemical measurement on rhizosphere

365

and bulk soils we took steps to ensure comparability by controlling the size range of

366

material, the water content and the contact between soil and DGT. However, we

367

appreciate that these precautions would not compensate for differences in carbon

368

mineralization between rhizosphere and bulk soils. Fortunately we performed a

369

similar

370

none-hyperaccumulator plant. None of the measurements of metals we made showed

371

any

372

none-hyperaccumulator cases, that is Ni and N. caerulescens and Cd and T.

373

goesingense (Table S4). Therefore, we could conclude there was no decrease in Kdl

374

and Tc between bulk-soil and rhizosphere. This strengthens the claim that the

375

observed changes in Kdl and Tc were due to hyperaccumulation.

set

of

significant

measurements

differences

where

between

each

metal

rhizosphere

was

paired

and bulk

soils

with

for

the

the

376

In considering possible processes that might bring about a change in the rate

377

constant, it is important to appreciate the meaning of k-1. It has been obtained by

378

applying the DIFS model which assumes that the rate of release from the solid phase

379

to solution is a first order process and therefore equal to the product of the

380

concentration of labile metal associated with the solid phase and the release rate

381

constant. This simplified representation of the ongoing processes was used to allow

382

the application of a minimally parametrized model that is appropriate for the limited

383

kinetic information manifested in the measured R term. In reality there is likely to be a

384

distribution of solid phase pools of metal, each with their own release rate constants,

385

and this release may not necessarily be first order. Models along these lines have been


Page 17 of 29


386

proposed,49, 51 but the additional parameterization cannot be justified by the limited

387

data available here. However, it is reasonable to presume that as plants uptake metal

388

from the rhizosphere the resupply from the solid phase will initially occur from the

389

most labile pools (with the highest values of k-1). Therefore, after plant growth, these

390

most labile pools will be depleted. It means that after early stage uptake of metals by

391

hyperaccumulators the residual metals would become more kinetically limited for

392

phytoextraction and therefore potentially reduce the phytoremediation efficiency,

393

indicating the importance of these two factors controlling plant uptake.

394

This analysis indicates that both labile pool size and the rate of metal release are

395

important in the study of phytoremediation and the assessment of its efficiency. When

396

DGT is deployed in a soil where plants are already established (in our case the

397

rhizosphere soil), the reservoir of labile metal is lower than that in the original or bulk

398

soil and the effective rate constant of release is lower. These two factors both lower

399

the value of R, which is the resupply term obtained directly from the DGT

400

measurement. DGT measurement, allied to application of the DIFS model, can

401

provide insights into changes between rhizosphere and bulk soils after plant growth in

402

the lability of solid phase metals and may be more helpful to assess the

403

phytoremediation efficiency than other chemical methods used in this study.

404 405

Environmental

Implications.

Due

to

high

uptake

of

metals

by

406

hyperaccumulators, the metals extracted by NH4Cl and EDTA were significantly

407

depleted in the rhizosphere, illustrating the decreasing pool size of labile metals which

408

are available to phytoextraction. Concentrations of metals in soil solution were

409

affected less markedly, possibly due to their association with root exudates, which

410

enhanced the DOC content in the rhizosphere, increasing the proportion of readily



411

exchangeable metals in the soil solution of the rhizosphere. In two different soils,

412

uptake of Cd by Noccaea caerulescens and of Ni by Thlaspi goesingense were shown

413

to be limited by the rate of supply from the soil rather than regulated according to the

414

biotic ligand model29. We assume this is also the case for the soils of this study. One

415

way of assessing the efficiency of remediation is to consider how the chemical

416

availability of the metals changes after plant growth. With this definition, in our case

417

of uptake limited by supply, measurement of soil solution may not efficiently reflect

418

the changes in availability of metals.

419

The combination of DGT and DIFS model has provided for the first time the key

420

information on the kinetics of exchange between solid phase and soil solution in the

421

rhizosphere. The rate of supply of both Cd and Ni from solid phase to solution was

422

diminished in rhizosphere soils. For Cd, the major factor limiting metal supply after

423

the growth of hyperaccumulators was a lower rate constant of supply from solid phase

424

to solution, but for Ni the reduction in the concentration of labile metal associated

425

with the solid phase was equally important in affecting the rate. Differences in Kdl and

426

k-1 between rhizosphere and bulk soils is consistent with the plants taking up the

427

kinetically most available metals in the early stages of growth with the continuing

428

supply of residual metal being more kinetically limited with time. This study

429

indicated the importance of both pool size of labile metals in the solid phase and

430

supply kinetics of metals from solid phase to solution in the assessment of availability

431

of metals and therefore remediation efficiency. These findings provide new

432

mechanistic insight into the process occurring during phytoremediation. The holistic

433

approach of DGT technique could have a significant impact in the field of soil

434

remediation by phytoextraction as DGT gives more informed assessment of

435

availability of metals after phytoremediation and the efficiency of the remediation.


Page 18 of 29

Page 19 of 29


436

ASSOCIATED CONTENT

437

Information on the probability of significant differences in chemical parameters

438

between rhizosphere and bulk soil, percentage of metals in soil solution in each soil

439

present as organic species and comparison of threshold and estimated values of TC in

440

rhizosphere and bulk soils after growth of hyperaccumulators. This information is

441

available from the website of ACS Publications.

442

ACKNOWLEDGMENTS

443 444 445 446 447

This work was funded by the National Natural Science Foundation of China (No. 21477053) and the Engineering and Physical Sciences Research Council (EPSRC) Dorothy Hodgkin Postgraduate Award (NE/C506999/1).

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

REFERENCES

.

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41. McGrath, S.; Zhao, F.; Lombi, E., Plant and rhizosphere processes involved in phytoremediation of metal-contaminated soils. In Interactions in the Root Environment: An Integrated Approach, Springer: 2002; pp 207-214. 42. Huang, Y.; Tao, S.; Chen, Y. J., The role of arbuscular mycorrhiza on change of heavy metal speciation in rhizosphere of maize in wastewater irrigated agriculture soil. J. Enviro. Sci. 2005, 17, (2), 276-280. 43. Christensen, J. B.; Jensen, D. L.; Christensen, T. H., Effect of dissolved organic carbon on the mobility of cadmium, nickel and zinc in leachate polluted groundwater. Water Res. 1996, 30, (12), 3037-3049. 44. Delle Site, A., Factors affecting sorption of organic compounds in natural sorbent/water systems and sorption coefficients for selected pollutants. A review. J. Phys. Chem. Ref. Data 2001, 30, (1), 187-439. 45. Pérez-Esteban, J.; Escolástico, C.; Masaguer, A.; Vargas, C.; Moliner, A., Soluble organic carbon and pH of organic amendments affect metal mobility and chemical speciation in mine soils. Chemosphere 2014, 103, 164-171. 46. Tapia, Y.; Cala, V.; Eymar, E.; Frutos, I.; Gárate, A.; Masaguer, A., Chemical characterization and evaluation of composts as organic amendments for immobilizing cadmium. Bioresource technology 2010, 101, (14), 5437-5443. 47. Tipping, E., Cation binding by humic substances. Cambridge University Press: Cambridge, UK, 2002. 48. Zhao, F. J.; Rooney, C. P.; Zhang, H.; McGrath, S. P., Comparison of soil solution speciation and diffusive gradients in thin‐films measurement as an indicator of copper bioavailability to plants. Environ. Toxicol. Chem. 2006, 25, (3), 733-742. 49. Lehto, N.; Davison, W.; Tych, W.; Zhang, H., Quantitative assessment of soil parameter (K D and T C) estimation using DGT measurements and the 2D DIFS model. Chemosphere 2008, 71, (4), 795-801. 50. Kjaergaard, C.; Moldrup, P.; De Jonge, L. W.; Jacobsen, O. H., Colloid mobilization and transport in undisturbed soil columns. II. The role of colloid dispersibility and preferential flow. Vadose Zone Journal 2004, 3, (2). 51. Lehto, N. J.; Davison, W.; Zhang, H.; Tych, W., An evaluation of DGT performance using a dynamic numerical model. Environ. Sci. Technol. 2006, 40, (20), 6368-6376.


Page 22 of 29

Page 23 of 29


607 608 609 610

Table 1. The physico-chemical characteristics of bulk soils and accumulation factors (AF) for the two hyperaccumulators

TOC Soil

Texture

pH

Total Cd

Total Ni

MWHC

Noccaea -1

%

AF for

-1

(mg kg )

(mg kg )

caerulescens

AF for Thlaspi goesingense

MX

Sandy loam

7.75

0.70

39.4%

4.79

17.5

108

3.0

BX

Silty loam

7.06

1.33

40.5%

0.22

32.5

237

3.1

WX

Silty clay

6.39

2.00

57.2%

5.88

180

70

3.8

WG ZY

611 612 613

Silty clay 5.83 1.32 57.6% 3.23 29.6 184 3.3 loam Silty clay 7.14 2.33 60.5% 0.38 16.3 96 3.5 loam pH values are the average of pH in bulk soil for Cd hyperaccumulator and in bulk soil for Ni hyperaccumulator. MWHC refers to the maximum water holding capacity; AF is the concentration ratio of metal in shoots to metal in soil.



614

Table 2. DGT-interpreted properties of the rhizosphere and bulk soil CDGT (µg L-1) Plant

N. caerulescens

T. goesingense

615 616 617 618

Page 24 of 29

Metal

Cd

Ni

Kdl (L kg-1)

R

k-1 (s-1) (x 10-8)

Tc (s)

Site rhizosphere

bulk

rhizosphere

Bulk

rhizosphere

bulk

rhizosphere

bulk

rhizosphere

bulk

MX

2.24

2.63

0.64

0.76

812

956

567

116

68

280

BX

0.025

0.032

0.55

0.63

4009

4682

1070

597

7.5

12

WX

2.00

2.48

0.67

0.79

377

442

449

75

270

1400

WG

3.21

3.76

0.78

0.88

187

210

50*

24*

4900*

9000*

ZY

0.018

0.028

0.46

0.56

1832

1987

2112

1145

12

21

MX

0.70

0.87

0.20

0.24

58

87

20000

12000

27

30

BX

0.20

0.33

0.14

0.20

899

1157

45000

22707

0.8

1.2

WX

115.9

137.2

0.55

0.60

121

153

1250

727

300

410

WG

4.21

5.30

0.25

0.31

59

80

13889

8497

56

67

ZY

0.51

0.63

0.18

0.23

218

307

34963

18342

6.0

9.0

Note: CDGT is the concentrations measured by DGT at the interface of the DGT device and the soil. R is the ratio of CDGT to concentration of soil solution. Kdl represents the distribution coefficient of the labile solid phase pool defined by EDTA to soil solution concentration. Tc refers to the response time of the soil to metal depletion. k-1 is the first-order rate constant of metal supply from solid phase to solution. Asterisk means that estimated value were below the threshold value where estimates of kinetic parameters are less reliable.


Page 25 of 29


619

Fig. 1

620 8.0

8.0

*

7.5

bulk

rhizosphere

7.5

*

* 7.0

**

pH

7.0

bulk

*

**

pH

* 6.5

6.5

**

*

6.0

6.0

5.5

621 622 623

*

rhizosphere

5.5

MX

BX

WX

WG

Cd hyperaccumulator

ZY

MX

BX

WX

WG

ZY

Ni hyperaccumulator

624

Figure 1. pH in rhizosphere and bulk soil after growth of hyperaccumulator plants in

625

different soils. * and ** represent significance levels p < 0.05 and p < 0.01,

626

respectively.

627 628



629

Page 26 of 29

Fig. 2

630 631 632 90

90 rhizosphere

rhizosphere

*

bulk

DOC (mg/kg)

DOC (mg/kg)

bulk

*

60

30

* 0

633 634 635

* *

60

* 30

*

0

MX

BX

WX

WG

Cd hyperaccumulator

ZY

MX

BX

WX

WG

ZY

Ni hyperaccumulator

636

Figure 2. DOC in rhizosphere and bulk soil after growth of hyperaccumulator plants

637

in different soils. * and ** represent significance levels p < 0.05 and p < 0.01,

638

respectively.

639 640


Page 27 of 29


641

Fig. 3

642 643 4.00 Cd - NH4Cl Extraction (mg/kg)

Cd - Soil solution (µ µ g/L)

5.00 Rhizosphere Bulk

4.00 3.00 0.10 0.08 0.06 0.04 0.02 0.00

BX

WX

WG

0.04 0.02 MX

BX

WX

WG

ZY

4.00

4.0

Rhizosphere Bulk

3.00

*

2.0

**

1.0 0.3

Cd - DGT (µ g/L)

Cd - EDTA Extraction (mg/kg)

*

0.06

Rhizosphere Bulk

*

*

0.2

*

0.1 0.0

645 646

1.00

ZY

5.0

3.0

* 2.00

0.00

MX

644

Rhizosphere Bulk

3.00

*

* 2.00

0.04

* 0.02 0.00

MX

BX

WX

WG

ZY

MX

BX

WX

WG

ZY

647

Figure 3. Rhizosphere and bulk soil characteristics for Cd after growth of Cd

648

hyperaccumulator plants (N. caerulescens) in different soils. * and ** represent

649

significance levels p < 0.05 and p < 0.01, respectively.

650



651

Page 28 of 29

Fig. 4

652 653 8.00 Rhizosphere Bulk

Ni - NH4Cl Extraction (mg/kg)

Ni - Soil solution (µ g/L)

250

200

150 20 15 10 5 0 BX

Rhizosphere Bulk

20 2

WX

WG

2.00 0.02

*

*

0.01

*

* MX

BX

140

*

120

* **

1

**

Rhizosphere Bulk

WX

WG

ZY

*

100 6

*

4

2

*

* 0

655 656

4.00

ZY

Ni - DGT (µ g/L)

Ni - EDTA Extraction (mg/kg)

30

*

Rhizosphere Bulk

0.00 MX

654

6.00

0 MX

BX

WX

WG

ZY

MX

BX

WX

WG

ZY

657

Figure 4. Rhizosphere and bulk soil characteristics for Ni after growth of Ni

658

hyperaccumulator plants (T. goesingense) in different soils. * and ** represent

659

significance levels p < 0.05 and p < 0.01, respectively.

660 661


Page 29 of 29


662 663 664

TOC Art only

665


Plant induced changes to rhizosphere characteristics affecting supply

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