Zn Speciation and Stable Isotope Fractionation in a Contaminated

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Zn speciation and stable isotope fractionation in a contaminated urban wetland soil- Typha latifolia system Anne-Marie Aucour, Jean-Philippe Bedell, Marine Queyron, Romain Tholé, Aline Lamboux, and Géraldine Sarret Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02734 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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Environmental Science & Technology

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Zn speciation and stable isotope fractionation in a contaminated urban wetland soil- Typha latifolia system Anne-Marie Aucour1*, Jean-Philippe Bedell2, Marine Queyron2, Romain Tholé2, Aline Lamboux3, Géraldine Sarret4 1

Université de Lyon, Université Lyon 1, ENS de Lyon, CNRS, UMR 5276 LGL-TPE, F-69622 Villeurbanne, France 2 Université de Lyon, ENTPE, CNRS, UMR 5023 LEHNA, 2 Rue Maurice Audin F69518 Vaulx-en-Velin, France 3 Université de Lyon, ENS de Lyon, Université Lyon 1, CNRS, UMR 5276 LGL-TPE, F-69364 Lyon Cedex 7, France 4 ISTerre, Université Grenoble Alpes, CNRS, F-38058 Grenoble, France

* Corresponding author, [email protected]

tel.:

(33)679116770,

fax:

ACS Paragon Plus Environment

(33)472448593,

e-mail:


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2 19

Abstract

20

Wetlands play a key role in the immobilization of metallic contaminants. In this context

21

the mechanisms of Zn sequestration and Zn transfer and storage in Typha latifolia L. colo-

22

nizing a frequently flooded contaminated soil were studied. A combination of EXAFS

23

spectroscopy, micro X-ray fluorescence (µXRF) and Zn isotope measurements was applied

24

to soil, plant organs and decaying biomass. Zn was present in the soil as Zn-layered double

25

hydroxide, as tetrahedral and octahedral sorbed Zn species, and as ZnS. Octahedral and

26

tetrahedral Zn (attributed to symplastic Zn-organic acid and apoplasmic Zn-cell wall com-

27

plexes, respectively) and Zn-thiol species were observed in the roots, rhizomes and stems.

28

Iron plaque was present on the rhizomes and roots. Enrichment in light isotopes for Zn

29

sorbed on the plaque relative to the soil (∆66Znplaque-soil = -0.3 to -0.1‰) suggested the dis-

30

solution of ZnS (enriched in light isotopes) in the rhizosphere with subsequent Zn2+ sorp-

31

tion on the root plaque. Furthermore, enrichment in light isotopes of stems relative to

32

leaves (∆66Znstem-leaves = -0.2 ‰) suggested the remobilization of Zn via the phloem, from

33

leaves back to the stems. Overall these data highlight the role of thiols in controlling Zn

34

speciation during its transfer and storage in T. latifolia.

35 36 37


Page 3 of 37


3 38

Introduction

39

Plants play a key role in the immobilization of metals in contaminated environments.

40

Wetland plants are of particular interest because they are essential components of con-

41

structed wetlands, increasingly used to manage urban waste- and storm-waters1. Many wet-

42

land plants are metal tolerant species2 and are thus particularly suitable for metal stabiliza-

43

tion. The dynamics of metals in the soil-to-plant continuum in contaminated wetlands are

44

still poorly understood. Several studies have been conducted on the speciation and seques-

45

tration of metals in contaminated sediments and flooded soils, e.g.3-6. However, there are

46

few data available on metal speciation and dynamics in the soil-root-shoot continuum. The

47

pattern of low metal concentrations being maintained in leaves, whereas their concentra-

48

tions in sediments and associated roots are high, has been described as contributing to met-

49

al tolerance7. Certain previous studies focused on the role of root iron plaque7-11, a coating

50

of Fe oxyhydroxides, which is due to the oxidation of Fe2+ in anoxic, waterlogged condi-

51

tions at the root surface by O2 diffusing through the aerenchyma. The presence of a root

52

plaque was shown to influence metal accumulation in the roots of wetland plants11. It has

53

been suggested that the root plaque increases metal tolerance by forming a barrier against

54

these elements4, although, it has been reported instead to increase metal availability under

55

flooded, alkaline conditions12.

56

Combining metal isotope measurement with the study of metal speciation by extended

57

X-ray absorption fine structure (EXAFS) spectroscopy can provide new information on

58

metal sequestration and translocation mechanisms in soil-plant systems in contaminated

59

wetlands. The present study focuses on Zn, a metal present at high to toxic concentrations

60

in mining, urban and industrial environments. Previous studies have shown the occurrence



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4 61

of large Zn stable isotope fractionations from the substrate (soil or synthetic nutrient me-

62

dium) to the plant organs13-19. These fractionations help in identifying plant uptake and

63

translocation mechanisms while speciation by EXAFS provides a snapshot of the major

64

forms of Zn in the soil20-21 and in plant organs22.

65

Constructed urban wetlands generally present steep hydric gradients towards a main in-

66

flowing water source. Waterlogging and redox conditions are expected to be prime factors

67

controlling Zn speciation, sequestration and availability in soils and at the soil-root inter-

68

face1,3,23. Our objective in the present study was to investigate the mechanisms of metal

69

sequestration in the soil and of metal transfer and storage in Typha latifolia L. growing in a

70

frequently flooded soil from an urban wetland. Specific attention was given to the soil-root

71

interface by combining micro X-ray fluorescence, selective chemical extraction, EXAFS

72

spectroscopy and Zn isotope measurements. The results obtained were then compared to

73

results obtained in a previous study on Phalaris arundinacea L. growing in a drier zone of

74

the same basin16.

75 76

Material and methods

77

Description of the site

78

The infiltration basin studied is located at Chassieu (45°N 44.163’; 4°E 57.478’), in the

79

eastern suburbs of Lyon (France). It is situated at the outlet of an urban industrialized wa-

80

tershed covering 185 ha drained by a separate stormwater system. Before entering the infil-

81

tration basin, stormwater flows through a detention/retention basin covering a surface area

82

of about 1 ha. The surface area of the infiltration basin is about 7 ha.


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5 83

The infiltration basin had been spontaneously colonized by plants24. Two zones can be

84

distinguished: a meadow and a wetland. The wetland covers 53.4 % of the surface area of

85

the infiltration basin. The two most abundant species of the wetland are Typha latifolia and

86

Phalaris arundinacea25. The T. latifolia plants selected for this study grow in the zone di-

87

rectly fed by the water inflow, where the soil is frequently flooded and is usually water

88

saturated (Fig. S1, Supporting Information). The pH of the soil was near-neutral, 7.3 to 7.6.

89

Other physico-chemical properties are given in Supporting information. Based on pH and

90

presence of usual metallic contaminants, we believe that the site studied is representative

91

of urban wetlands in temperate conditions.

92 93

Sampling and conditioning, chemical extractions

94

For each compartment studied (stems, leaves, rhizomes, adventive roots, dead leaves,

95

litter (here fallen litter covering the soil), soil), we made composite samples, which were

96

subsampled for both EXAFS and isotope analysis. The procedure used to obtain the com-

97

posite samples is detailed in Supporting information. Briefly, the aerial parts were collect-

98

ed on June 5th 2012, the dead leaves (collected on standing shoots) in February 2012 and

99

the litter in March and June 2012, from a surface area of one square meter. The upper layer

100

of the substrate, between 0 and 20 cm in depth, which will be referred to as “soil”, was

101

sampled for a surface area of 400 cm2 in duplicate on June 5th 2012. The fresh soil was

102

separated from the rhizomes and roots, then sieved at 8 mm. One aliquot was used for

103

EXAFS and elemental/isotope analysis whereas other aliquots were subjected to triplicate

104

CaCl2 and DTPA extractions in accordance with previously reported protocols16,25 given in



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6 105

Supporting information. All the soil samples were then freeze-dried. Note that the contact

106

of the fresh soil with air was minimized during handling (see Supporting information).

107

The organs of living plants (adventive roots, rhizomes, stems, leaves), dead leaves, de-

108

caying stems and leaves, and litter were rinsed several times with deionized water. For

109

EXAFS analysis, plant organs were frozen in liquid nitrogen and kept at -80°C while they

110

were air-dried for elemental and isotopic analysis. Dead leaves, decaying leaves, and litter

111

were freeze-dried.

112

In order to extract Zn adsorbed by roots, fresh adventive roots (2g) were soaked either

113

(1) twice in 10 mM CaCl2 solution (50 ml) for 15 min26 or (2) in 1M HCl solution for three

114

min, then in 10mM HCl for 5 min27. All these extraction steps were performed using

115

cooled reagents (5°C).

116 117

Zn elemental and stable isotope analysis

118

The crushed plant material and soil and the soluble extracts were digested and the sam-

119

ple digests were extracted by anion exchange chromatography in AG-1X8 chromatograph-

120

ic columns following previously reported protocols15,16 given in Supporting information.

121

Elemental analysis was performed using quadrupole inductively coupled plasma mass

122

spectrometry (ICP-MS). Zn and Fe were measured in the soluble extracts, plant and soil

123

digests. The precision for Zn and Fe analysis was ± 5%. Accuracy and precision of the Zn

124

concentration measurement was checked through replicate analysis of lichen certified ref-

125

erence material BCR-CRM 482.

126

Zn isotope ratios of the Zn obtained after column purification was measured at least in

127

duplicate over different mass-spectrometry analytical sessions by multicollector inductive-


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7 128

ly coupled plasma mass spectrometry (MC-ICP-MS) using a Nu plasma 500 HR (Nu in-

129

strument) at the ENS de Lyon. Mass spectrometry and data processing procedures were

130

performed as per previously described methods28. The overall analytical reproducibility of

131

± 0.05 ‰ (2σ) was obtained from six full replicate analyses, including digestion, chroma-

132

tographic separation and mass spectrometry, of the lichen standard BCR-CRM 482. Full

133

replicates (including subsampling, digestion, chromatographic separation, mass spectrome-

134

try) for selected samples generally fell within the overall analytical reproducibility of

135

0.05‰ obtained on BCR-CRM 482 (Table S1).

136 137 138

Zn isotopic compositions were expressed as the relative deviation from the “Lyon” Zn standard JMC 3-0749 L in ‰: δxZnsample=[(xZn/64Zn)sample/(xZn/64Zn)standard – 1)103

(1)

139

where x = 66, 67 or 68. To express the isotope fractionation between two components, A

140

and B, we used ∆66ZnA-B equal to the difference between δ66ZnA and δ66ZnB.

141 142

Bulk EXAFS

143

EXAFS measurements were performed on the FAME beamline at the ESRF (European

144

Synchrotron Radiation Facility, Grenoble, France), equipped with a Si(220) double crystal

145

monochromator and a 30-element solid-state Ge fluorescence detector (Canberra, France).

146

Frozen plant samples were ground in liquid N2, pressed as frozen hydrated pellets and kept

147

frozen until analysis. Freeze-dried soil, dead leaves and litter samples were ground in an

148

agate mechanical grinder and pressed as 5 mm diameter pellets. Zn K-edge EXAFS spectra

149

for these samples were recorded at room temperature, and dead leaves and litter samples

150

were recorded both at room temperature and at 10 K. Zn K-edge EXAFS spectra were rec-



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8 151

orded at 10 K using a He cryostat. Depending on the Zn signal, four to eight spectra of 40

152

min each were averaged. EXAFS data were treated using principal component analysis

153

(PCA) and linear combination fits (LCFs) as described previously16. LCFs were performed

154

over a k range of 2.3–11.2 Å-1 using a database of Zn reference spectra, as described previ-

155

ously 22,29,30.

156 157

Micro X-ray fluorescence (µ µXRF)

158

Root and rhizome cross-sections were studied using lab-based µXRF. Fresh roots were

159

embedded in optimal cutting temperature resin, frozen in liquid nitrogen and cut into 30

160

µm thick sections using a cryo-microtome. Thin sections were then freeze-dried before

161

µXRF analysis. Rhizomes were freeze-dried and cut into 2-mm thick sections using a razor

162

blade. Root and rhizome sections were analyzed under vacuum using an EDAX Eagle III

163

XRF spectrometer, equipped with a Rh anode and a poly-capillary that focuses the X-ray

164

beam down to 30 or 100 µm full width at half maximum (FWHM). An EDX detector with

165

a resolution of 140 eV was used to measure the X-ray fluorescence. The spectrometer was

166

operated at 20 kV, and 300 to 400 µA. The µXRF spectra of 250 s were recorded at differ-

167

ent positions along the root and rhizome radius. Quantification was performed using the

168

ZAF method after background subtraction, using Vision32 ©software.

169

170

Results

171

Zn concentration, isotope composition and speciation in the soil

172

The soil had a high Zn concentration (ca. 2100 mg kg-1, Table S2 in Supporting infor-

173

mation, Fig.1), in agreement with a previous study reporting Zn particulate/dissolved ratios


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9 174

and sources in inlet waters16. CaCl2 (0.1 M) extraction was used to evaluate the pool of Zn

175

exchangeable via cation exchange reactions, and DTPA extraction to mimic the effect of

176

strong organic ligands25. The Zn extracted by CaCl2 represented a small fraction of total

177

soil Zn (1.6‰). Its δ66Zn value (0.17‰) was close to that of the soil (0.12‰). The Zn ex-

178

tractable by DTPA accounted for a much larger fraction of soil Zn (ca. 19%). It was rela-

179

tively enriched in heavy isotopes (with δ66Zn of 0.28‰).

180

The chemical form of Zn in the various compartments of the soil-plant system was stud-

181

ied by Zn K-edge EXAFS spectroscopy. Figure 2 shows a selection of standard spectra and

182

the whole set of sample spectra. Sample spectra were treated by PCA and LCFs. Four

183

components were necessary to reconstruct the spectra (Table S3, Supporting information).

184

Target transformation allowed the identification of four groups of Zn species: (1) Zn pre-

185

sent in layered minerals (Zn layered double hydroxide, Zn-LDH23 and Zn included in the

186

octahedral sheet of phyllosilicate, Zn-phyllosilicate), (2) sorbed or complexed tetrahedral

187

Zn including Zn sorbed on ferrihydrite (Zn-ferrihydrite), Zn phosphate and Zn complexed

188

to plant cell wall (Zn-cell wall), (3) sorbed or complexed octahedral Zn including Zn

189

sorbed on goethite (Zn-goethite) and Zn complexed to COOH-OH groups of organic acids

190

in solution (Zn-OAs), and (4) ZnS and/or Zn-thiol complexes (Fig. S2, Supporting infor-

191

mation). As explained previously16, considering the relationship between Zn speciation and

192

soil properties21 and the pH and Zn concentration ranges, the presence of Zn-LDH is more

193

likely than that of Zn phyllosilicate. The fact that the site studied is most often waterlogged

194

should also favor the presence of Zn-LDH. Indeed, waterlogging combined with soil respi-

195

ration may result in partial pressures of CO2 100 to 1000 times higher than the atmospheric

196

CO2. This increase in CO2 should favor the formation of Zn-LDH, which contains bicar-



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10 197

bonate in the interlayer at neutral pH and a chloride concentration typical of soil solu-

198

tions (1 mM)23. Nonetheless, although Zn-LDH is a more likely species compared to Zn

199

phyllosilicate, its proportion should be considered as an upper limit and may include a cer-

200

tain fraction of Zn phyllosilicate.

201

The LCFs results are presented in Figure 3 and Table S4 (Supporting information). The

202

results for suspended particulate matter (SPM) present in the water inlet and for an alter-

203

nating wet-dry soil under P. arundinacea from the same wetland, already published in a

204

previous paper16, were considered for comparison with the soil under T. latifolia. The SPM

205

contained sorbed/complexed tetrahedral Zn as a major species, and Zn-LDH and ZnS. In

206

contrast to the soil under P. arundinacea, the soil under T. latifolia contained a proportion

207

of ZnS and/or Zn thiol (9%). Due to the low proportion of this species, it is not possible to

208

distinguish ZnS from Zn-thiol, but ZnS, identified in the SPM, is more likely.

209

The presence of ZnS in the T. latifolia soil was also attested by the comparison of the

210

XANES spectra, which showed an increase in the amplitude and a shift to higher energy of

211

the peak maximum in the order ZnS < SPM-baseflow < soil under T. latifolia < soil under

212

P. arundinacea < Zn-LDH (Fig. S3, Supporting information). Besides 9% ZnS, the soil

213

under T. latifolia contained Zn-LDH (48%) and sorbed/complexed tetrahedral Zn (45%).

214

The concentrations in Zn species were obtained by multiplying the LCFs percentages

215

with total Zn concentration (Table S5, Supporting information). The DTPA treatment de-

216

creased the sorbed/complexed tetrahedral Zn pool (from 922 to 170 mg kg-1), produced

217

408 mg kg-1 sorbed/complexed octahedral Zn and did not significantly affect the Zn-LDH

218

and ZnS pools.

219


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Zn and Fe concentration in plant organs and root extracts

221

Zn was more concentrated in the underground parts (350-550 mg kg-1) than in the aeri-

222

al parts (50-150 mg kg-1) (Fig. 1a). The leaves presented the lowest concentrations. The

223

stock of Zn stored in the plant organs (and in soil and soil chemical extracts) was estimated

224

by using the Zn concentration and the biomass (or mass) per unit area (Fig. 1c). The

225

aboveground parts represented the largest fraction of the biomass but the largest Zn reser-

226

voir in the plant was the rhizome. Compared to the soil, the plant was a minor reservoir of

227

Zn that accounted for only 0.2% of total Zn in the soil-plant system. The litter was also a

228

minor reservoir (0.2% of total Zn) as was the exchangeable CaCl2 fraction of the soil (ca. 1

229

to 2 %), whilst the soil Zn extractable by DTPA accounted for a much larger fraction of

230

total Zn in the soil-plant system (ca. 16%).

231

In the adventive roots, two types of extraction were used to extract Zn sorbed or precipi-

232

tated on the outer surfaces and cell walls. CaCl2 extraction is assumed to extract cation

233

exchangeable metal sorbed on the outer surfaces and cell walls, whereas the HCl treatment

234

is assumed, in addition, to extract metal more strongly bound to or precipitated on root

235

surfaces and cortical cell walls. The potassium content in the extracts was used as an indi-

236

cator of the possible damage to cell membranes during HCl extraction, since cell breakage

237

releases intracellular K27. K concentrations were low for both the CaCl2 and HCl extracts

238

(Fig. S4, Supporting information), but this approach may be not sensitive to cell breakage

239

restricted to external root layer. As expected, a lower fraction of Zn was removed by the

240

CaCl2 treatment (7%) than by the HCl extraction (16%). Fe measurements showed higher

241

Fe release for HCl treatment and a high total Fe concentration in the underground parts

242

(3360 mg kg-1 in roots, 1697 mg kg-1 in the rhizome) (Table S2, Supporting information).



Page 12 of 37

12 243

It is noteworthy that the Fe extracted with HCl represented only a small percentage of

244

total Fe in roots (ca. 2%). The latter also presented a high Fe/Zn ratio (9.0), similar to that

245

of the soil. These findings suggest the presence of iron plaque for T. latifolia roots and rhi-

246

zome.

247 248

Localization of Fe, Zn and S in rhizomes and roots

249

µXRF analyses of rhizome and root sections confirmed the presence of an iron plaque

250

(Fig. 4). In the rhizome, Fe was mostly detected in the epidermis, whereas in roots it was

251

detected in the epidermis and in the most external part of the cortex. Zn was detected in the

252

iron plaque of the rhizome and roots. It should be noted however that laboratory-based

253

µXRF is sensitive to elevated concentrations only (detection limit of about 1000 mg kg-1

254

local concentrations). The cortex and central cylinder, although containing Zn in lower

255

concentration, represented a large fraction of the total Zn in the roots, as shown by the

256

CaCl2 and HCl extractions. S was detected in all tissues of the rhizome. Data on S in roots

257

could not be interpreted because the sample holder used for the root sections contained

258

some S.

259 260

Zn isotope composition of plant organs

261

Compared to the soil, the plant organs were generally enriched in light isotopes (Fig.

262

1b). The isotope composition of the whole T. latifolia plant (δ66Znplant) calculated from the

263

proportion of Zn and isotope composition of each plant organ was -0.36‰. The Zn isotope

264

composition presented a wide variation within the plant. The adventive roots were the

265

heaviest compartment while the stem was the lightest. Regarding the shoot, the leaves were


Page 13 of 37


13 266

slightly enriched in heavy isotopes compared to the stem (by 0.16‰). The δ66Zn values

267

in dead biomass increased in parallel with the Zn concentration from dead leaves to decay-

268

ing shoot and finally to litter. The litter δ66Zn values (0.24‰) were close to that of the soil

269

DTPA exchangeable Zn (0.28‰).

270

The root CaCl2-exchangeable Zn (with δ66Zn value of -0.20%) was depleted in heavy

271

isotopes compared to the soil CaCl2-exchangeable Zn and to the bulk soil (0.17 and

272

0.12‰, respectively). The Zn extracted from the root by HCl, and which is likely more

273

tightly bound on cell walls, was heavier than the root CaCl2-exchangeable Zn (by ca. 0.2

274

‰) and comparable to the Zn found in the untreated root. It was slightly depleted in heavy

275

isotopes compared to the bulk Zn in the soil (by ca. -0.1‰) and even more depleted com-

276

pared to the DTPA-extractable Zn in the soil (by -0.3 ‰).

277 278

Zn speciation in plant organs

279

LCFs of EXAFS spectra indicated the presence of tetrahedral and octahedral Zn com-

280

plexes and of ZnS/Zn-thiols. In the rhizome and stem, the LCFs with Zn-thiols was better

281

(residue (NSS) decreased by 25%) than with ZnS, and Zn-thiols accounted for 32 to 33% of

282

Zn in roots, rhizome and stem (Fig. 3, Table S4). In the roots, equivalent fits were obtained

283

with ZnS or Zn-thiols, and they represented 20% of Zn (Fig. 3). The HCl extraction per-

284

formed on the roots removed roughly half of the sorbed/complexed tetrahedral and of oc-

285

tahedral Zn and doubled the amounts of ZnS/Zn-thiols (Table S5, Supporting information),

286

consistently with previous findings24. In this case, the LCF was clearly better with ZnS

287

(NSS decreased by 50%), so the HCl treatment led to the formation of ZnS. The origin of

288

sulfide, necessary for the formation of ZnS, remains unclear. It is unlikely that sulfide is



Page 14 of 37

14 289

present in millimolar concentration inside the root or root plaque, so this species may

290

have been formed during the extraction.

291

No good fit was obtained for T. latifolia leaves, possibly because of the lack of specific

292

Zn species in our database, so Zn speciation in this organ remained undetermined. Tetra-

293

hedral Zn complexes attributed to Zn bound to polymeric cell-wall like compounds repre-

294

sented ca. 60% of the total Zn in the dead T. latifolia leaves, 100 % in the litter. The dead

295

leaves also contained octahedral Zn complexes attributed to hydrated Zn-organic acid

296

complexes (ca. 40%).

297 298

Discussion

299

Soil Zn speciation and extraction

300

The

major

soil

constituent

was

Zn-LDH.

The

rest

included

tetrahedral

301

sorbed/complexed forms and a small proportion of ZnS. The soil samples were in contact

302

with air during handling and grinding. Contact with air was minimized (see above) and we

303

did not observe any oxidation of ZnS reference material, although the mineral was finely

304

ground under atmospheric conditions. However, the possibility of oxidation of ZnS phases

305

in the whole procedure cannot be discarded and the abundance of ZnS detected represented

306

a minimum value. Note also that in-situ conditions were already partly oxic since the soil

307

was densely colonized by rhizomes and roots (which bring oxygen to the soil) and no re-

308

duced sulphur smell was detected.

309

A previous study3 showed that under reducing, low sulfate conditions, sulfide phases

310

preferentially incorporated Cu, whereas Zn and Fe were hardly sequestered in sulfide pre-

311

cipitates. The concentration in exchangeable sulfate in the latter study (2.3 mmol kg-1) was


Page 15 of 37


15 312

similar to that in water-exchangeable sulfate for the soils studied here (0.6 to 3.6 mmol

313

kg-1

314

study might be related to a higher Zn concentration (2000-2500 mg kg-1 versus 1361 mg

315

kg-1 in3).

33

), as well as total Cu concentration (ca. 300 mg kg-1). The presence of ZnS in our

316

The results for a drier zone of the same basin colonized by P. arundinacea16 were com-

317

pared here with the flooded T. latifolia zone (Fig. 5) and are provided in the Supporting

318

information (Tables S2, S3). The P. arundinacea zone is not directly submitted to the wa-

319

ter inflow, and the soil undergoes wet-dry cycles. Zn-LDH was the major constituent and

320

presented similar abundances in the two soils. In the soil under P. arundinacea, the re-

321

maining Zn was a mixture of sorbed/complexed tetrahedral and octahedral Zn. The ab-

322

sence of ZnS in the soil under P. arundinacea is likely due to more oxic conditions prevail-

323

ing in that zone.

324

In the soil under T. latifolia, EXAFS analysis showed that tetrahedral complexes were

325

preferentially extracted during the DTPA extraction whilst Zn-LDH and ZnS were essen-

326

tially preserved and octahedral sorbed/complexed species seemed to be newly formed. In

327

addition, the heavy isotopes were preferentially removed from the soil under T. latifolia by

328

the DTPA treatment. A similar result was observed in the soil under P. arundinaceae16

329

where DTPA extraction preferentially removed heavy Zn isotopes and tetrahedral com-

330

plexes and did not significantly affect the LDH pool. The enrichment in heavy isotopes of

331

the DTPA extract is consistent with previous studies reporting that tetrahedral Zn com-

332

plexed with carboxyl/hydroxyl groups are enriched in heavy isotopes versus free Zn2+ or

333

octahedral sorbed species34,35 and that Zn-LDH36 and ZnS37 should preferentially sequester

334

light isotopes. Theoretical calculations of isotope fractionations between hydrated Zn ion



Page 16 of 37

16 335

and hydrated sulfide species predicted depletion in heavy isotopes of the sulfide species

336

(by -0.2 to -1.1‰ in ∆66Zn at 298 K)37. The reason for the formation of octahedral

337

sorbed/complexed Zn species during DTPA extraction of the soil under T. latifolia remains

338

unclear. It could be due to some changes in the mineralogy of Fe-bearing phases induced

339

by the modifications in the redox conditions. Further investigations into Fe speciation are

340

required to clarify this point. Overall, the above results confirmed that DTPA extracts pref-

341

erentially tetrahedral Zn complexes, which are enriched in heavy isotopes in comparison to

342

octahedral O-coordinated Zn (LDH, sorbed/complexed forms) and ZnS forms.

343 344

Zn at the soil-root and soil-rhizome interface

345

The presence of iron plaque for T. latifolia rhizome and roots is consistent with the fact

346

that the substrate is frequently flooded. Various assemblages of Fe minerals including fer-

347

rihydrite, goethite, siderite, lepidocrocite and amorphous hydroxides were reported in iron

348

plaques10. The nature of these assemblages seems to depend on the plant species and phys-

349

ico-chemical conditions of the medium. In the present study, the mineralogy of T. latifolia

350

iron plaque was not determined.

351

µXRF and HCl extraction showed that a significant fraction of Zn in the roots and rhi-

352

zome of T. latifolia was present in the iron plaque. Furthermore, both tetrahedral and octa-

353

hedral species were partly removed from the roots by HCl treatment. These findings sug-

354

gest that the T. latifolia root plaque contained various Fe minerals, which sorbed Zn. The

355

light isotope enrichment of the root CaCl2 extract in comparison to the HCl extract is con-

356

sistent with previous reports indicating that aqueous Zn2+ was lighter than tetrahedral com-


Page 17 of 37


17 357

plexes with ferrihydrite (by -0.5 to -0.3‰34,35,38) and octahedral complexes on goethite

358

(by -0.3‰33), although it was also reported as being heavier in the latter case39.

359

The presence of iron plaque has been reported to influence metal accumulation in the

360

roots of wetland plants1,2. It is clear here that the isotope fractionation between the Zn

361

sorbed on root and soil differs from that previously observed for P. arundinacea roots in a

362

drier soil and without iron plaque16. P. arundinacea plants had similar isotopic composi-

363

tions of CaCl2 exchangeable Zn in roots and soil, which suggested exchange and equilibri-

364

um between Zn weakly sorbed on soil and roots. A similar pattern was observed for Zn

365

more strongly sorbed on soil and roots, as shown by the δ66Zn values of root HCl and soil

366

DTPA extracts16. In contrast, T. latifolia plants had lighter isotopic compositions of Zn

367

sorbed on roots, either weakly or strongly bound. The distinct Zn isotope behavior on the

368

root surfaces of the two plants is also expressed by the ∆ value between the HCl root ex-

369

tract and bulk soil (Fig. 5). Zn sulfides are expected to present rather large enrichment in

370

light isotopes in comparison to O-coordinated octahedral and tetrahedral Zn species37,40.

371

Thus oxidation/solubilization of ZnS present in the soil, due to O2 release by roots, would

372

be a source of light Zn2+ at the soil-root interface. This hypothesis is supported by a study

373

on sulfur isotopes indicating that under wetland conditions sulphide is a likely source of

374

sulfur for T. latifolia41.

375 376

Zn in the plant

377

The enrichment in light isotopes from soil to plant (with ∆66Znplant-soil of ca. -0.5 ‰) can

378

be attributable to the light δ66Zn value of the phyto-available pool and/or to the preferential

379

uptake of light isotopes from this pool. Soil DTPA extracts were enriched in heavy iso-



Page 18 of 37

18 380

topes compared to the bulk soil. DTPA, a synthetic hexadentate ligand containing three

381

carboxylates and three amines, is structurally close to siderophores and nicotianamine.

382

Thus, the light isotope enrichment of the plant confirms that Zn uptake is not mediated by

383

phytosiderophores and indicates that the pool extracted by DTPA does not actually corre-

384

spond to the pool that is taken up by the plant. The soil CaCl2 extract is a fraction that is

385

very easily mobilised by plants. Its isotopic composition was close to that of the bulk soil

386

and cannot account for the large isotope effect observed from the soil to the plant. Howev-

387

er, the root CaCl2 extract, with a ∆66Zn of ca. -0.30‰ (relative to the soil), points towards

388

the occurrence of light Zn2+ in the vicinity of roots possibly contributing to the light iso-

389

tope composition in the plant. P. arundinacea plants growing in the same basin were also

390

Zn-isotopically lighter than the soil, with ∆66Znplant-soil value of ca. -0.6 ‰ (Fig. 5) but in

391

this case the isotopic composition of the root and soil CaCl2 extract fell close to each other

392

and were only slightly depleted relative to bulk soil (by ca. 0.05‰). The light isotope

393

composition of both plants suggests a role of low-affinity uptake transport systems (such as

394

ion channels) that should preferentially take up light Zn isotopes18,42.

395

Enrichment of the heavy isotopes in roots in comparison to shoots has already been re-

396

ported in previous studies11,15-19. Coupled stable isotope/speciation investigation showed

397

large heavy isotope enrichment in adventive roots of P. arundinacea, with ∆66Znroot-shoot of

398

ca. 0.8‰, associated with Zn binding to cell-wall tetrahedral sites and as Zn-OAs in the

399

vacuole16. The lesser heavy isotope enrichment in the roots of T. latifolia in comparison to

400

the shoots, with ∆66Znroot-shoot of ca. 0.4‰, is consistent with cellular storage as Zn-thiol, in

401

addition to vacuolar Zn-OAs and Zn-cell wall complexes. Theoretical calculations of iso-

402

tope fractionations between Zn hydrated cation and amino-acid complexes indeed indicat-


Page 19 of 37


19 403

ed that light isotopes tend to bind preferentially to S-donor ligands, heavy isotopes to O-

404

donor and N-donor ligands. They led to the depletion of heavy isotopes in Zn-cysteine and

405

-methionine relative to complexes with O- and N- donor ligands of up to -0.6‰40 which

406

corroborates the fact that root Zn is less enriched in heavy isotopes (relative to shoot) when

407

root storage includes Zn-thiol.

408

Concerning the aerial parts, the same trends in Zn concentration and δ66Zn values were

409

observed for T. latifolia and P. arundinacea, i.e., a two-fold lower Zn concentration and a

410

higher δ66Zn value in leaves compared to the stem; however the drop in Zn from under-

411

ground parts to leaves was more drastic in T. latifolia. The enrichment in light isotopes of

412

Zn in the stem (compared to leaves) indicates that Zn in the stem follows a more complex

413

pathway than unloading through yellow stripe-like (YSL) proteins 43,44 or adsorption onto

414

cell-wall from the ascending transpiration stream45. The chelation of free Zn2+ in xylem sap

415

by nicotianamine prior to YSL transfer at the symplasm membrane as well as Zn2+ adsorp-

416

tion onto the cell wall are indeed expected to favor heavier isotopes18. Furthermore, sym-

417

plastic storage predominated over cell wall storage in T. latifolia (with symplastic Zn-thiol

418

and -OAs) and P. arundinacea (with symplastic OAs16). We thus propose that a phloem

419

reallocation of light Zn occurs from leaf to stem in both species.

420

The evolution of Zn content, isotope composition and speciation from fresh to dead

421

leaves/shoot and to litter was similar in T. latifolia and P. arundinacea, with a progressive

422

enrichment in total Zn and in heavy isotopes, and the formation of tetrahedral Zn. These

423

findings indicate the fixation of Zn essentially by cell wall-like components implying a

424

preferential sorption of heavier isotopes in the dead leaves and a transfer of Zn from the

425

underlying horizon to litter.



Page 20 of 37

20 426 427

Role of thiols in Zn tolerance in T. latifolia

428

This study showed the presence of Zn-thiol complexes in the roots, rhizome and stem of

429

T. latifolia, accounting for 20, 32 and 33% respectively, of total Zn in the organ. Many

430

studies have shown a link between cysteine-rich peptides (glutathione and phytochelatins)

431

in plants and the tolerance, accumulation or detoxification of metals46-47. Although these S-

432

rich peptides act as ligands for Cd, Hg, Cu, they are not the most common ligands for Zn48.

433

However, several recent studies have identified Zn-thiol complexes in plants49-51. Moreo-

434

ver, Typha sp. seems to be particular in terms of S metabolism. Typha domigensis Pers.

435

showed tolerance to relatively high sulfide concentrations52. Sulfide is highly toxic to plant

436

tissue and tolerance to sulfide should depend on the species’ ability to rapidly metabolize

437

sulfide to thiol and cysteine53. Cysteine synthesis was reported to be much higher in T.

438

latifolia roots and shoots than in Phragmites communis Trin.. The activity of O-

439

acetylserine (thiol) lyase, which catalyzes cysteine synthesis, increased in response to Cd

440

treatment in both species but was much higher in T. latifolia54. Based on elemental map-

441

ping and quantification in cross-sections, S-containing compounds were proposed as bind-

442

ing sites for Pb and Zn in T. latifolia roots and rhizomes55. These various studies are con-

443

sistent with the presence of Zn-thiol complexes in T. latifolia observed here.

444 445

Environmental implications

446

Constructed wetlands are used for a variety of effluents, waste- or storm-waters, and

447

have diverse sizes and designs. Alternating wet and dry conditions and zoning with drier

448

and wetter areas are often observed. The results of this study and of a previous one14 show


Page 21 of 37


21 449

common trends in Zn isotopic fractionation and speciation in two soil-plant systems

450

from the same infiltration basin, but marked differences due to waterlogging and also to

451

plant species, with a prominent role of sulfur in the waterlogged system. The ZnS present

452

in the frequently flooded soil under T. latifolia seems to be partly dissolved in the rhizo-

453

sphere but T. latifolia accumulates lesser Zn in leaves than P. arundinacea, which thrives

454

under drier conditions in the same basin. Thus, the formation of sulfides does not appear to

455

be an efficient stabilization mechanism. Instead, the plant species that have colonized the

456

site by natural selection are able to cope with Zn toxicity. The specificity of T. latifolia in

457

this respect is the use of reduced sulfur to bind part of the Zn in the plant. In case of wet-

458

lands with planted macrophytes (as opposed to naturally colonized ones), the selection of

459

species adapted to the local hydric and redox conditions is of crucial importance.

460 461

Acknowledgments

462

We are grateful to ESRF for providing beam time, to Denis Testemale, Valérie Magnin and

463

the FAME beamline staff for their help in collecting data, to Philippe Telouk for his assis-

464

tance in isotope analysis and to Andreas Scheinost and Michel Schlegel for providing Zn

465

reference spectra. This work was funded by the CNRS/INSU/EC2CO program. The au-

466

thors are also grateful to the OTHU, Greater Lyon for their logistic and data support.

467

ISTerre is part of Labex OSUG@2020 (ANR10 LABX56).

468 469

Supporting Information



Page 22 of 37

22 470

Additional information on the sampling and soil extraction protocols, Zn purification

471

and isotope measurements, a map of the filtration basin with the T. latifolia and P. arundi-

472

nacea zones, the elemental (Zn, Fe), isotopic (δ66Zn) and speciation results for the T. lati-

473

folia and P. arundinacea soil-plant systems, and the chemical composition (K, Fe, Zn) of

474

T. latifolia root extracts are presented in a supplementary document.

475 476

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the expansion of Typha domigensis (cattail) in the Florida everglades? Freshwater Biol.

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2009. 9, 1909-1923.

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(53) Lamers, L.P.M.; Govers, L.L.; Janssen, I.C.J.M.; Geurts, J.J.M.; Van der Welle,

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M.E.W.; Van Katwijk, M.M.; Van der Heide, T.; Roelofs, J.G.M.; Smolders, A.J.P. Sul-

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fide as a soil phytotoxin, a review. Front. Plant Sci. 2013, 4, 268.

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(54) Fediuc, E.; Lips, S.H.; Erdei, L. O-acetylserine (thiol) lyase activity in Phragmites and

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Typha plants under cadmium and NaCl stress conditions and the involvement of ABA in

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the stress response. J. Plant Physiol. 2005, 162, 865-872.

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(55) Lyubenova, L.; Pongrac, P.; Vogel-Mikus, K.; Mezek, G.K.; Vavpetic, P.; Grlj, N.;

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Kump, P.; Necemer, M.; Regvar, M.; Pelicon, P.; Schröder, P. Localization and quatifica-

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tion of Pb and nutrient in Typha latifolia by micro-PIXE. Metallomics 2012, 4: 333-341.

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Figure captions

642

Figure 1: Zn concentration (a), Zn isotope composition (b) and Zn partitioning (c) in the

643

various compartments of the Typha latifolia L. soil-plant system. Error bars for δ66Zn val-

644

ues represent the overall analytical uncertainty (2 σ = 0.05‰). For concentration data, they

645

fall within the symbols for most samples.

646

Figure 2: Zn K-edge EXAFS spectra for selected Zn reference spectra and for the suspend-

647

ed particulate matter, plant and soil samples. Reference spectra include ZnS (sphalerite),

648

Zn cysteine31, Zn-layered double hydroxide (Zn-LDH)30, Zn-reacted phosphate30, Zn-

649

sorbed goethite, Zn-plant cell wall complex (Zn-cell wall) and Zn plus three organic acids

650

in solution (Zn-OAs)22. Dashed lines represent linear combination fits.

651

Figure 3: Proportion of Zn species (in % of total Zn) in the various samples of the Typha

652

latifolia L. soil-plant system. The uncertainty on the percentage is estimated at 10% as pre-

653

viously described32. For some of the samples, it was possible to distinguish ZnS from Zn-

654

thiols as indicated on the yellow bar. For the abbreviations of compounds, see caption of

655

Fig. 2.

656

Figure 4: Elemental profiles obtained by µXRF on rhizome (a) and root (b) sections of

657

Typha latifolia L. µXRF spectra were recorded along a transect from the epidermis to the

658

center of the rhizome or root. Representative µXRF spectra recorded along the root section

659

are shown in (c). The optical image of the rhizome section is shown in the background.

660

Atomic percentages were calculated over the sum of the elements detected, including Si, P,

661

S, Cl, K, Ca, Ti, Fe and Zn. ep.: epidermis, end.: endodermis.


Page 31 of 37


31 662

Figure 5: Synthesis of results obtained for Typha latifolia L. (this work) and Phalaris

663

arundinacea L.16 soil-plant systems from the same infiltration basin. Common features

664

between the two systems are in black, those specific to T. latifolia in blue, and those spe-

665

cific to P. arundinacea in brown.

666

layered double hydroxide.

IV

Zn: tetrahedral Zn,

VI

Zn: octahedral Zn, Zn-LDH: Zn-



Figure 1 144x161mm (300 x 300 DPI)


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Figure 2 135x235mm (300 x 300 DPI)



Figure 3 149x168mm (300 x 300 DPI)


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Figure 4 149x198mm (300 x 300 DPI)



Figure 5 205x218mm (300 x 300 DPI)


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TOC 75x46mm (300 x 300 DPI)


Zn Speciation and Stable Isotope Fractionation in a Contaminated

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