Вы находитесь на странице: 1из 45

Accepted Manuscript

Uptake, accumulation and metabolization of 1-butyl-3-methylimidazolium bromide by ryegrass from water: Prospects for phytoremediation

Nuzahat Habibul, Jie-Jie Chen, Yan-Yun Hu, Yi Hu, Hao Yin, Guo-Ping Sheng, Han- Qing Yu

PII:

S0043-1354(19)30229-5

Reference:

WR 14502

To appear in:

Water Research

Received Date: 2 December 2018

Revised Date:

Accepted Date: 13 March 2019

12 March 2019

Revised Date: Accepted Date: 13 March 2019 12 March 2019 Please cite this article as: Habibul,

Please cite this article as: Habibul, N., Chen, J.-J., Hu, Y.-Y., Hu, Y., Yin, H., Sheng, G.-P., Yu, H.- Q., Uptake, accumulation and metabolization of 1-butyl-3-methylimidazolium bromide by ryegrass from water: Prospects for phytoremediation, Water Research (2019), doi: https://doi.org/10.1016/

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphic Abstract

A CCEPTED MANUSCRIPT Graphic Abstract

ACCEPTED MANUSCRIPT

1 Uptake, accumulation and metabolization of 1-butyl-3-methylimidazolium

2 bromide by ryegrass from water: Prospects for phytoremediation

3

4

5

6

7

Nuzahat Habibul

a,bǂ

, Jie-Jie Chen

aǂ

, Yan-Yun Hu , Yi Hu , Hao Yin , Guo-Ping Sheng

c

a

c

Han-Qing Yu a

a*

,

a CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied

8 Chemistry, University of Science and Technology of China, Hefei, 230026, China

9

10

11

12

13

b College of Chemistry and Chemical Engineering, Xinjiang Normal University,

Urumqi, 830054, China

c Hefei National Laboratory for Physical Sciences at Microscale, University of Science

and Technology of China, Hefei, 230026, China

14 ǂ These authors contributed equally to this work

15

16 * Corresponding author:

17 Prof. Guo-Ping Sheng

18 Fax: +86-551-63601592

19 E-mail: gpsheng@ustc.edu.cn

20

1

ACCEPTED MANUSCRIPT

21 Abstract

22 The unique properties of ionic liquids make them attractive for a wide range of

23 industrial applications, which makes it easy to be released into the environment and

24 cause water or soil pollution. Phytoremediation of organic contaminants is a safe and

25 important process for removing persistent pollutants from the environment. However,

26 due to they are very chemically stable and potentially toxic to plants, whether they

27 can be removed, assimilated and metabolized by plants remains unknown during

28 phytoremediation process. In this study, ryegrass, Lolium perenne L., was used for

29 imidazolium ionic liquid (1-butyl-3-methylimidazolium bromide, [C mim]

4

+ ) removal

30 from water. The results show that [C mim]

4

+ could be taken up, accumulated and

31 metabolized by plants in vivo with a high removal efficiency. Most of the [C mim] +

4

32 was accumulated in the root tissue, with the root concentration fraction factors

33 ranging

from

4.9

to

51.5.

Two

hydroxylated

metabolites

34 1-(4-hydroxybutyl)-3-methylimidazolium,

 

and

35 1-(n-butyl)-3-(hydroxymethyl)-imidazolium, and two secondary metabolites were

36 detected in the ryegrass after [C mim]

4

+ uptake. The metabolic mechanism was

37 clarified using density functional theory calculations. Furthermore, [C mim]

4

+ at a high

38 concentration was found to be high toxic to inhibit the growth of ryegrass markedly.

39 In response, some oxidative stress was observed in the metabolic process, as indicated

40 by increasing of catalase, super dismutase and peroxidase activities. Our results

41 suggested that phytoremediation was an efficient technique for ionic liquids treatment

42 from water.

2

ACCEPTED MANUSCRIPT

43

44

Keywords:

density

functional

theory;

imidazolium;

ionic

liquids;

metabolic

45

mechanism; phytoremediation

 

3

ACCEPTED MANUSCRIPT

46 1. Introduction

47 Ionic liquids are molten salts that have a negligible vapor pressure and

48 relatively low melting point (<100°C) and have been classified as a “green solvent”

49 to replace the traditional solvents that are used in many important chemical

50 processes (Amde et al., 2015). Due to their numerous advantages, such as chemical

51 and thermal stability, outstanding solvation potential, high electric conductivity and

52 high water solubility, ionic liquids have been widely used for a broad range of

53 applications, including organic synthesis, extraction, catalysis, electrochemistry and

54 biotechnology (Petkovic et al. 2011). However, such a wide use of ionic liquids has a

55 risk of leaking into the environment, which may result in water or soil pollution. The

56 toxic effects of ionic liquids on plants and organisms in terrestrial and aquatic

57 environments have been recognized (Matzke et al. 20 08; Yu et al. 2009; Markiewicz

58 et al., 2013; Thamke et al., 2017). From the currently available data on the toxicity

59 of ionic liquids to different organisms, ionic liquids are obviously not very “green”

60 or “environmentally friendly” chemicals (Pham et al. 2010; Thamke et al., 2017).

61 Owing to the high stability of ionic liquids, the available treatment for

62 removing ionic liquids from water are based on chemical processes such as advanced

63 oxidation (Czerwicka et al. 2009; Spasiano et al., 2016), electrochemical degradation

64 (Stolte et al. 2008) and photodegradation (Zhou et al. 2013; Calza et al., 2017). The

65 chemical treatment process can effectively convert non-biodegradable and persistent

66 ionic liquids into biodegradable organic or/and inorganic compounds to eliminate

4

ACCEPTED MANUSCRIPT

67 them (Pan et al., 2014; Spasiano et al., 2016). However, once the ionic liquids enter

68 the ecosystem, it is difficult to remove ionic liquids from aquatic or terrestrial

69 environments. Therefore, it is important to develop an environmental nondestructive

70 bioremediation

technology

to

removal

them

from

aquatic

conditions.

71 Phytoremediation of organic contaminants is a safe and important process for

72 removing persistent pollutants from the environment (van Aken et al., 2004;

73 Prakashmaran et al., 2018). However, due to the specific properties of ionic liquids,

74 the removal aspects of ionic liquids by plants have rarely been studied to date.

75 Previous studies show that ionic liquids have phytotoxic effects on plants

76 (Matzke et al., 2008). Plants can assimilate and interact with organic compounds,

77 and thereby affect the fate of many organic pollutants. The phytoremediation of

78 organic contaminants is a safe and important process for removing persistent

79 pollutants from the environment (van Aken et al. 2004). However, due to the specific

80 properties of ionic liquids, the removal aspects of ionic liquids by vegetative plants

81 have rarely been studied to date. Thus, it is necessary to investigate the possibility of

82 the transport and metabolization of ionic liquids in plant tissues as well as their

83 stability and susceptibility to the accumulation or degradation processes in plants for

84

the

phytoremediation

of

ionic

liquids.

In

fact,

in

a

theoretical

study

some

85 imidazolium ionic liquids are predicted to have a tendency to be incorporated into

86 the membrane and bio-accumulated in cells and were subsequently broken down

87 metabolically by cytochrome P450 (CYP450) (Jastorff et al. 2003). However, the

88 potential accumulation and metabolization of ionic liquids such as imidazolium in

5

ACCEPTED MANUSCRIPT

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

vegetative plants remain unrevealed. Lolium perenne L. (ryegrass), a species of

economical feed, possesses a fibrous root system, with thick main roots and thinner

lateral branches. This dense root system making it easily interwoven floating straw

mats over surface of water. Due to the high biomass production rate, relatively high

tolerance to the adverse environmental stresses, and most importantly, its great

potential for contaminant remediation (Ruan et al. 2016; Duan et al. 2017), ryegrass

has been chosen as a candidate plant for wastewater treatment.

Therefore, the aim of this study was to investigate the phytoremediation of

imidazolium

ionic

liquid

1-butyl-3-methylimidazolium

([C mim]

4

+

)

by

ryegrass

(Lolium perenne L.). The uptake of [C mim]

4

+ into ryegrass roots and its subsequent

translocation and transformation in ryegrass were explored. Also, the toxic effects of

[C

4 mim]

+ on the physiological characters of ryegrass were investigated. In addition,

density functional theory (DFT) calculations were performed to elucidate the

possible transformation mechanism of [C mim]

4

ryegrass.

2. Materials and methods

+ by the active site of CYP450 in

2.1 IL and ryegrass cultivation

An imidazolium, [C

4 mim]Br (99%), was purchased from the Chengjie Chem.

Co. Ltd. (Shanghai, China) and used as the model ionic liquid in this study. Ryegrass

(Lolium perenne L.) was used in this study for [C mim]

4

+ uptake. The experiments

were conducted in a greenhouse at 26±1°C. The ryegrass seeds were germinated on a

6

ACCEPTED MANUSCRIPT

110 bed of sterilized cotton gauze with deionized water. The photoperiod was 16 h per

111 day, and the light intensity was 2100-2500 Lux. The grasses were transferred into

112 150 mL of glass conical flasks with half-strength Hoagland nutrient solution when

113 they grew to 5 cm high. After that, when the seedlings grew to 8-10 cm high

114 (excluding the roots), the healthy and actively growing ryegrass plants with white

115 color vigorous roots, strait stem and dark green leaf shapes were utilized for further

116 experiments.

117 2.2 Hydroponic exposure experiments

118 To investigate the removal efficiency of [C mim]

4

+ by plants, a final exposure

119 concentration of [C mim]

4

+ of 5 mg/L was dosed into each conical flask, and each

120 conical flask was planted with 15 ryegrass plants. The deionized water, the Hoagland

121 solution and the conical flasks were autoclaved to prevent any possible influence of

122 microorganisms on the [C mim]

4

+ removal. Deionized water was added to the conical

123 flasks every day to compensate for the transpiration losses according to the water

124 level. Three replicated conical flasks planted with ryegrass grown in Hoagland

125 solution without [C mim]

4

+ were used as the untreated plant control. Three replicated

126 conical flasks planted with only fresh roots (without shoots) acted as the excised

127 ryegrass control, and an additional three plants were air dried to act as the dead

128 control.

ryegrass

Two

replicated

blank

controls

without

plants

were

used

to

129 the

determine

contribution

of

biodegradation

possibly

occurring

inside

the

130 hydroponic solution on [C mim]

4

+ removal. All conical flasks were wrapped with

7

ACCEPTED MANUSCRIPT

131 aluminum foil to keep the root zone dark and to eliminate the photodegradation of

132

133

134

[C

4 mim]

+ . Four conical flasks were taken at 1, 2, 4, 8, 12, 24, 48 and 72 hours after

the dose of [C mim]

4

+ for plant exposure, and the water samples were collected to

analyze the residual [C mim]

4

+

concentrations.

135 The growth inhibition test experiments were performed in glass flasks with

136

dosages of various concentrations of [C mim]

4

+ (0, 1, 2, 5, 10 and 15 mg/L). Three

137 replicates at each concentration were conducted. After the ryegrass was planted, the

138 weights of the root, leaf elongation and fresh ryegrass were recorded every 3 days.

139

140

At the end of the experiment, all solutions were collected, and the [C mim]

4

+

concentrations were analyzed to investigate the influence of [C mim]

4

+

concentration

141 on its removal efficiency.

142

The

[C mim]

4

+

concentrations

in

water

samples

from

all

treatments

were

143 quantified by HPLC (Agilent 1260) with a C

144 shown in the Supporting Information).

18 (150×4.6 mm, 5 µm) column (details

145 2.3 [C 4 mim]

+ uptake kinetics by ryegrass

146

147

148

The

effects

of

ionic

liquid

initial

concentrations

on

plant

uptake

were

investigated. The uptake kinetics were estimated by measuring [C mim]

4

+

depletion

at various [C mim]

4

+ initial concentrations (0.5, 1, 2, 5, 10, 15 and 20 mg/L). After

149 four hours of treatment, the water samples were collected and analyzed, and the plant

150 root samples were weighed. The experiments were replicated three times under each

151 condition.

8

ACCEPTED MANUSCRIPT

152 The uptake kinetic parameters

V max and K m of [C 4 mim]

+ of ryegrass could be

153

estimated

by

fitting

the

experimental

data

during

154 Michaelis-Menten model equation (Brix et al. 2002):

155

V =

(C

V

max

K

+

C

C

min

C

min

)

depletion

to

the

modified

 

(1)

156

where C is the [C 4 mim] + concentration in the medium, V and V max

are, respectively,

157

the uptake rate and the maximum uptake rate at [C mim]

4

+ initial concentration C;

158

C

min

is [C mim]

4

+

concentration at which there is no net uptake, and a low

C

min

value

159

implies a good uptake ability at low [C mim]

4

+

initial concentrations; K and

K

m

is the

160 respective saturation and the half-saturation constant; and

K m is the [C 4 mim]

+

161

162

concentration where the uptake rate is the half of the maximum rate and

K

m

=K+C min

.

The phytoextraction ability of ryegrass for [C mim]

4

+ was assessed by the root

163 concentration factor (RCF) and the translocation factor (TF), which could be

164 calculated by Eq. (2) and Eq. (3), respectively (Felizeter et al. 2012):

165

166

RCF

=

TF =

[

C mim

4

]

+ concentration in roots

(

165 166 RCF = TF = [ C mim 4 ] + concentration in roots (

mg g

)

[

C mim

4

]

+ concentration in solution

[ ] ( / )

(

4 ] + concentration in solution [ ] ( / ) ( mg mL ) [

mg mL

)

[ ] ( / )

167 2.3 Growth inhibition test

(3)

(2)

168

The toxicity of [C mim]

4

+ towards ryegrass was determined using the modified

169 version of the Organization for Economic Co-operation and Development (OECD)

170 guidelines 201 (OECD, 1984).

171

The

average

specific

growth

rates

9

µ

of

ryegrass

at

various

[C 4 mim] +

ACCEPTED MANUSCRIPT

172 concentrations were calculated as the logarithmic increase in biomass using Eq. (4):

173

174

where µ

T

T

=

ln

B

t

j

j

i

B

i

ln

t

is the average specific growth rate from time t

i

to

t

j

, and B

i

and

B

j

(4)

are the

175 weights of the biomass at time t

i to t j , respectively.

176

The percent inhibition ratio (I) of the ryegrass growth rate by [C mim]

4

+

was

177 calculated using Eq. (5):

178

179 where µ

I %

0 T = 0
0
T
=
0

100

0 is the mean value of µ for the untreated plant control.

(5)

180 2.4 Analysis of chlorophyll content and antioxidant enzyme activities

181 Biochemical tests were performed to evaluate the destruction by ionic liquids of

182 photosynthetic activity and changes in antioxidative enzyme activities of ryegrass.

183 The chlorophyll in plants was extracted using 80% acetone and determined using a

184 spectrophotometric method at 646 nm and 663 nm according to a previous study

185 (Aibibu et al. 2010).

186 Before antioxidative enzyme activity measurements, the plant samples (root or

187

leaf) were homogenized in 0.1 M KH PO

2

4 buffer (pH 7.0) under liquid nitrogen

188 protection and then centrifuged at 15000 rpm for 10 min at 4°C. The supernatant was

189 collected as the crude extract for the enzyme assay. The catalase enzyme activity

190

(CAT) was determined by monitoring the H O

2

2 degradation at 240 nm (Gallego et al.

191 1996). A nitro blue tetrazorium (NBT) method was used to measure the super

10

ACCEPTED MANUSCRIPT

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

dismutase enzyme activity (SOD) (Giannopolitis and Ries 1977). The peroxidase

enzyme activity (POD) was determined on the basis of guaiacol oxidation by

hydrogen peroxide (Yin et al. 2008). Reactive oxygen species (ROS) levels of the

ryegrass tissues were detected by the 2,7-dichlorofluorescin diacetate (DCFH-DA)

method (Domey et al. 2015).

2.5

[C 4 mim]

+ and its metabolite extraction from ryegrass

Prior to analysis, the plants after [C mim]

4

+ uptake were separated into roots and

leaves, and then the samples were ground in liquid nitrogen with a ceramic mortar

and pestle. The plant samples were mixed and homogenized with 10 mL of

phosphate buffer with 0.5% triethylamine and methanol (7:3, v/v) and were then

shaken vigorously overnight to extract [C mim]

4

+ from plant samples. The extracts

containing [C mim]

4

+ were harvested after centrifugation at 10000 rpm for 20 min,

evaporated to dryness, and then re-dissolved in 1 mL of methanol. Subsequently, the

solution was filtered through a 0.45 µm membrane for subsequent HPLC- mass

spectrometer (MS) analysis (details shown in the Supporting Information).

2.6 Molecular models and computational methods

Because the crystal structure of CYP450 of Lolium perenne L. has not been

experimentally

solved,

homology

modeling,

currently

the

most

accurate

computational method, was used to build this three-dimensional model from its

amino acid sequence on the basis of alignment with a similar protein with known

11

ACCEPTED MANUSCRIPT

212 structure (Bordoli et al. 2009). The CYP450 of Bacillus megaterium (Girvan et al.

213 2009) with a sequence identity of 40.78% was selected as the template. On the basis

214 of the target-template alignment, the CYP450 model of Lolium perenne L. was

215 generated. All the above steps were completed in SWISS-MODEL workspace

216 (Arnold et al. 2006) and the molecular graphics program PyMOL. The catalytically

217 active heme group with a central iron atom is linked to the peptide chain by the

218 thiolate sulphur atom of a cysteine residue. The iron (IV)-oxo porphyrin radical

219 cation in CYP450 termed Compound I (Cpd I) is thought to be highly reactive. A

220 cluster model of Cpd I is used to mimic the active center of CYP450 (Rittle and

221 Green 2010) for probing the mechanism of CYP-dependent transformation of the

222 cation of ionic liquids.

223

The energy changes in the metabolic pathways for [C mim]

4

+ biotransformation

224 on the Cpd I cluster in aqueous solution were studied using DFT computations. An

225 all-electron method within the Perdew, Burke, and Ernzerhof (PBE) (Perdew et al.

226 1996) functional form of generalized gradient approximation (GGA) (Perdew et al.

227 1992) for the exchange-correlation term was employed in combination with the

228 double

precision

numerical

basis

sets

including

p

polarization

(DNP),

as

229 implemented in the DMol

3 code (Delley 2000). The Grimme method (Grimme 2006)

230

is

adopted

for

dispersion

corrections

with

the

PBE

functional

methods.

The

231 conductor-like screening model (COSMO) was used to describe the solvent effect

232 (Klamt and Schuurmann 1993), where the solute molecule forms a cavity within the

233 dielectric continuum of permittivity that represents the solvent. The energy in each

12

ACCEPTED MANUSCRIPT

234 geometry optimization cycle, the maximum displacement, and the maximum force

235

was

converged

to

within

1×10

-5

Hartree,

5×10

-3

Å,

and

2×10

-3

Hartree/Å,

236 respectively. The transition state (TS) structure and the activation energy (E

a ) were

237

acquired for the metabolic pathways of [C mim]

4

+ on Cpd I by the linear synchronous

238 transit/quadratic synchronous transit methods (Halgren and Lipscomb 1977).

239 3. Results and discussion

240 3.1 [C 4 mim]

+ Removal by Ryegrass

241

242

Figure 1 shows the profiles of [C mim]

4

+ removal from water at various

cultivation times. No significant change in peak area corresponding to [C mim]

4

+

243 concentration

was

observed

in

the

blank

control,

indicating

that

the

abiotic

244

245

degradation of [C mim]

4

+ did not occur under our test conditions (Table 1). The

adsorption of [C mim]

4

+ to the plant root surface was small because the dead root

246

247

248

control showed a negligible removal of [C mim]

4

+ (<5%) from the solution (Table 1).

Only 7.6% of [C mim]

4

+ was removed from the excised ryegrass control reactor,

which indicated that only a small part of [C mim]

4

+ could be adsorbed or assimilated

249 by the roots during the treatment. However, in the experiments with planted ryegrass,

250

[C

4 mim]

+ assimilation occurred. The concentration dropped from an initial 5 mg/L to

251 0.18 mg/L after 72 hours uptake, suggesting that approximately 96.5% of [C 4 mim] +

252

253

was removed by ryegrass (Figure 1a). The [C mim]

4

+ removal by ryegrass was found

to

follow

pseudo-first-order kinetics

with

a

high

coefficient

of determination

254 (R 2 =0.996) (Figure 1). The kinetic constant was 0.037/h, and the average [C 4 mim] +

13

ACCEPTED MANUSCRIPT

255 removal rate by ryegrass was 0.209 mg/d during the experiment with 5 mg/L of

256

257

258

[C

4 mim] +

treatment. The effect of [C mim]

4

+ initial concentration on its removal

efficiency by ryegrass was also investigated. At the [C mim]

4

+ initial concentration <

2 mg/L, most of the [C mim]

4

+ could be assimilated by ryegrass, and its removal

259 efficiency was approximately 100% (Figure 1b). However, the removal efficiency

260 decreased

with increasing

[C 4 mim] +

concentration,

and

it

decreased

to

261

approximately 66% from an initial [C mim]

4

+ concentration of 15 mg/L (Figure 1b).

262 Plant uptake kinetic parameters were estimated by a non-linear regression parameter

263

using the Michaelis-Menten model (Eq. 1) to estimate the values of

V

max

and

C

min

.

264 These constants can be considered important criteria for selecting plant species for

265

266

pollutant removal from contaminated water. The values of

V

max

and

C

min

for

[C

4 mim]

+ uptake by ryegrass were estimated to be 3.316 mg/L/h/g-fresh weight (FW)

267 and 0.0001 mg/L, respectively (Figure 1c). A good fit of the experimental data to Eq.

268

2

(1) (R =0.99) suggested that the uptake of [C mim]

4

+ and transport into ryegrass was

269 an active process. Such a low

C mim indicates that the uptake of [C 4 mim] + by ryegrass

270

was more efficient, even at a very low initial [C mim]

4

+ concentration. Therefore, the

271 uptake kinetic characteristics would be useful for selecting plant species for pollutant

272 removal from contaminated water.

273 3.2 [C 4 mim]

+ uptake and translocation in ryegrass tissues

274

275

plant

After 15-day exposures of ryegrass to various concentrations of [C mim]

4

+

, the

samples

were

collected

for

[C 4 mim] + analysis

to

investigate

[C 4 mim] +

14

ACCEPTED MANUSCRIPT

276 phytoaccumulation

in

different

ryegrass

tissues.

The

results

demonstrate

that

277

278

[C

4 mim]

+ could be assimilated by ryegrass roots and transported to aboveground

parts of the plants (Figure S1; Table 2). [C mim]

4

+ accumulation in the root tissue

279 was much higher than that in the leaf tissue. For example, it was found that

280

281

282

283

approximately 85.5% of assimilated [C mim]

4

+ was accumulated in the roots of the

plants exposed to 15 mg/L [C mim]

4

+ , but the translocation to the leaves was only

14.5% (Table 2). The [C mim]

4

+ contents in the roots and leaves of the plants were

concentration-dependent and increased with an increase in the dosage of [C mim]

4

+

.

284 RCF and TF can be utilized in phytoremediation studies to describe how many

285 contaminants could be taken up by plants and translocated to foliage. Ryegrass

286

exhibited a very high effectiveness to phyto-extract the [C mim]

4

+ from water with a

287 high root concentration factor (Table 2). However, the average translocation factors

288

289

at different initial [C mim]

4

+ concentrations were much less than 1, suggesting that

the transfer of [C mim]

4

+ from root to leaves was not favored.

290 Our results demonstrated that from a phytoremediation point of view, the highest

291

fraction

of

[C mim]

4

+

could

be

assimilated

by ryegrass,

some

of

which

was

292 translocated to the leaves of the plants. This resulted in a high removal efficiency of

293

[C mim]

4

+

from

contaminated

water

by

ryegrass.

Most

of

the

[C mim]

4

+

was

294 accumulated in the root tissue, whose content in the roots was approximately 4.9

295 times higher than that in the leaves after a 15-day exposure to 15 mg/L [C 4 mim] +

296 (Figure 1, Table 2). These results suggested that the main process of [C 4 mim] +

297

removal by plants was root uptake and that the translocation of [C mim]

4

15

+

from roots

ACCEPTED MANUSCRIPT

298 to leaves was considerably less efficient.

299 Based on the calculated root concentration factor (RCF) and the translocation

300 factor (TF) data, ryegrass had a high potential for successful phytoaccumulation to

301

remove ionic liquids (e.g., [C mim]

4

+ ) from contaminated water, but their transfer

302 from root to leaves was not favored. In contrast to other neutral organics, the

303 ionizable characteristics of ionic liquids may affect their partitioning behaviors in

304 terms of plant uptake, accumulation and translocation (Carter et al. 2014). After the

305 ionic liquids are taken up into the plant roots, their translocation from the roots to

306 other parts of the plant may depend on two factors: partitioning into xylem sap and

307 cell membrane permeability (Hsu et al. 1990). The negative electrical potential of the

308

309

plasmalemma membrane could favor cation accumulation in the cell (e.g., [C mim]

4

+

)

(Eggen et al. 2011), resulting in a relatively high uptake rate by plants. [C mim]

4

+

310 uptake was mainly driven by the water flow through the root apoplast to the

311 Casparian strip and subsequent transportation into xylem sap. However, because of

312

its high hydrophilicity, [C mim]

4

+ crosses the lipophilic cell membrane of the plant

313 endodermis more slowly, and its efficiency of translocation is consequently curtailed

314

(Sicbaldi et al. 1997). As consequence, [C mim]

4

315 tissues was decreased.

316 3.3 Metabolic pathway of [C 4 mim]

+ in ryegrass

+ translocation to the aboveground

317 Once the contaminants are taken up, the plant tissue will accumulate the

318 chemicals and transform them to other new chemicals by lignification (Schnoor et al.

16

ACCEPTED MANUSCRIPT

319

320

1995). Notably, in this study, the mass balance calculation of [C mim]

4

+

indicated that

approximately 11-15% of assimilated [C mim]

4

+ might be metabolized in ryegrass

321 tissues (Table 2). Further analysis of metabolization intermediates of [C 4 mim] +

322 would

be

useful

not

only

to

characterize

the

mechanisms

of

uptake

and

323 metabolization but also to evaluate the secondary pollution and risk associated with

324

325

[C

[C

4

4

mim]

mim]

+ uptake by plants. Identification of the metabolization intermediates of

+ in ryegrass tissues was confirmed by the highly sensitive mass spectrum

326 value (m/z). Four metabolites were clearly identified in plant tissues (Table 3). The

327

328

hydroxylated derivative (M1 and/or M2) ([C 8 H 15 N 2 O]

+ ) was observed in the root and

leaf samples. The exact mass spectrum was consistent with [C H

8

12

N O ]

2

2

+

fragment

329 (M3) as a secondary metabolite of 1-(4-hydroxybutyl)-3-methylimidazolium (M1).

330 The other two metabolic products, m/z 125.1073 (M4) and m/z 83.0599 (M5), were,

331 respectively, identified as butyl-imidazole and methylimidazole.

332

333

Based on the identified metabolization intermediates of [C mim]

4

+

, the possible

metabolic pathways of [C mim]

4

+ in ryegrass are proposed (Figure 5). Hydroxylation

334 products (M1 and M2) preferentially occurred in ryegrass as a direct oxidative

335

336

metabolism of [C mim]

4

+ . A previous study also suggested that imidazolium ionic

liquids

could

be

oxidized

by

CYP450

in

microorganisms

to

hydroxylation

337 metabolites in different positions of the alkyl side chains and could be metabolically

338 broken down to imidazole and biodegradable fatty acids (Neumann et al. 2010). The

339 observation of M3 might be due to the subsequent oxidation of the hydroxyl group at

340 C4 of the butyl chain into a carbonyl group (M1). Methylimidazolium (M5) was

17

ACCEPTED MANUSCRIPT

341 another oxidation product from 1-(4-Hydroxybutyl)-3-methylimidazolium (M1). The

342 intermediate M4 would be generated by a secondary oxidation process from another

343 hydroxylation product, 1-(n-butyl)-3-(hydroxymethyl)-imidazolium (M2). As no

344 standard for the metabolization intermediates were available, the quantification of

345 these metabolites in ryegrass tissue extracts was not conducted. Although the

346 obtained metabolic information for [C mim]

4

+ in ryegrass in this study is preliminary,

347 the identification of the intermediate supports that the ionic liquids could potentially

348 be metabolized and transformed by plants during phytodegradation process.

349 To gain further insights into the possible transformation mechanism of [C mim]

4

+

350 by CYP450 in ryegrass, the thermodynamic and kinetic properties of two metabolic

351 scenarios

(Figure

5)

were

calculated.

CYP450

belongs

to

the

family

of

352 monooxygenase enzymes, which catalyze hydrocarbon hydroxylation by inserting

353 one oxygen atom into the carbon-hydrogen bond of their substrates (Ortiz de

354 Montellano 2010). The intermediate products (M1 and M2) were generated from

355 hydroxylation of the butyl and methyl side chains of [C mim]

4

+ , respectively. The

356 geometrical structures of the optimized initial reactants, transition states, and key

357 intermediate products are illustrated in Figure 6 (c-h) for two proposed metabolic

358 pathways. Because the active center is exposed to the solvent (Figure 6a), the

359 calculations are performed under aqueous conditions (water, ε = 78.39). The cysteine

360 ligand (Cys77) coordinated on the iron atom of the porphyrin ring determines the

361

electron

reduction

potential

of

the

active

center

(Fu

et

al.

2016).

In

this

362 CYP-dependent transformation of the cation of ionic liquids, the negative Gibbs free

18

ACCEPTED MANUSCRIPT

363 energy (G

Ө ) values (Table S1) indicate that the formation of both M1 and M2 is

364 thermodynamically favorable, which promotes the following transformation. In

365 addition, the hydroxylation of the methyl side chain to form M2 has a 4 times higher

366 energy barrier (E

a ) than does the generation of M1; thus, the M1 formation process

367 in Pathway 1 would have a higher reaction rate. Therefore, the metabolic process of

368 imidazolium cation with ryegrass CYP450 might tend to occur on the tail of the long

369 saturated alkyl chain. This theoretical study provided molecular-level insights into

370 the metabolic mechanisms of ionic liquids.

371 3.4 Inhibition of [C 4 mim]

+ on ryegrass growth

372 The toxicity of [C mim]

4

+ towards ryegrass was also investigated. After a 15-day

373 exposure, plants that had been treated with 10 mg/L [C mim]

4

+ or more showed

374 severe toxicity symptoms, i.e., yellow leaves, brown roots, and less fibrous roots

375 (Figure 2). A high concentration (e.g., 10 and 15 mg/L) of [C mim]

4

+ caused a

376 significant growth inhibition of ryegrass. However, the plant root, leaf elongation

377 rates and the total plant biomass increase rate were not significantly affected by

378 dosing [C mim]

4

+ at low (e.g., 1 and 2 mg/L) concentrations (Figure 3). Although the

379 total root and leaf length and biomass increased over cultivation time in all

380 treatments, the growth rate of the total plant length decreased from 0.10 cm/day to

381 0.04 cm/day as [C mim]

4

+ concentrations increased from 0 to 15 mg/L. After a

382 15-day exposure to 15 mg/L of [C mim]

4

+ , the plants only increased approximately

383 119% in terms of fresh weight, but the total fresh weight of control plants without

19

ACCEPTED MANUSCRIPT

384

[C mim]

4

+

exposure increased approximately 460%. The inhibition of [C mim]

4

+

on

385 ryegrass growth was also described using half maximal effective concentrations

386 (EC

50 ), which was calculated as 8.29 mg/L (Figure 3d).

387 The chlorophyll content of ryegrass decreased slightly after a 15-day exposure

388

389

390

to low concentrations of [C mim]

4

+ ; however, the content significantly decreased

with increasing [C mim]

4

+ concentration (Figure 4a). The oxidative stress in the

different parts of ryegrass after exposure to [C mim]

4

+ was assessed by measuring the

391 levels of CAT, SOD and POD (Figure 4b-4d). Compared to the control plants

392

393

without exposure to [C mim]

4

+ , the antioxidant enzyme activities in different parts of

the ryegrass did not change after exposure to 1 mg/L of [C mim]

4

+ , consistent with

394 the low ROS level in the plant tissues (Figure 4e). However, the CAT, SOD and POD

395

activities progressively increased with increasing [C mim]

4

+ concentration, which

396 increased approximately 122%, 43% and 105% in the roots and approximately 23%,

397

398

60% and 181% in the leaves, with an increase in [C mim]

4

+ concentration from 0 to

15 mg/L. Meanwhile, exposure to 15 mg/L of [C mim]

4

+ resulted in a significant

399 increase in ROS level in the different ryegrass tissues (Figure 4e).

400 3.5 [C 4 mim]

+ toxicity to ryegrass

401 The toxicity test is useful to understand plant tolerance to toxic chemicals and

402 identify appropriate phytoremediation plants. Ionic liquids can exhibit toxicities to

403 living organisms (Thuy Pham et al. 2010), such as inhibiting the plant growth rate,

404 reducing photosynthesis, and inducing excessive ROS generation and membrane

20

ACCEPTED MANUSCRIPT

405 deterioration as a consequence of oxidative stress. The inhibition of plant growth

406 under high [C 4 mim] + concentration exposure decreased the ryegrass [C 4 mim] +

407 removal efficiency. The EC

50

value of [C mim]

4

+ for ryegrass was lower than that for

408 duckweed obtained by Matzke et al., (Matzke et al. 2007) indicating that the toxicity

409 of [C mim]

4

+ to ryegrass was relatively higher. The color of ryegrass leaves after

410 exposure to a high concentration of [C mim]

4

+ changed from green to pale white,

411 suggesting

that the

toxic

effects

of

[C 4 mim] +

resulted

in

damage

to

the

412 photosynthetic apparatus. Plant photosynthesis is inhibited as the plant chlorophyll

413 content decreases because chlorophyll is responsible for the capture of sunlight. This

414 could be confirmed in this study by decreasing the chlorophyll content in the plant

415 tissues due to excessive ROS generation under exposure to a high [C mim]

4

416 concentration (Figure 4).

+

417 Due to the toxicity of ROS, plants would re-upgrade the primarily antioxidant

418 system to overcome ROS mediated oxidative stress. As biomarkers of the enzymatic

419 antioxidant defense system of plants, the SOD, CAT and POD activities in ryegrass

420 were found to increase markedly under various [C mim]

4

+ stresses, suggesting that

421 the antioxidative system might play an important role in protecting the plant cells

422 from oxidative damage caused by [C mim]

4

+ . The excess ROS can’t be scavenge

423 effectively by damaged antioxidant system in the ryegrass, which could further

424 disrupt to cellular function. As a result, the plant growth rate decreased. With respect

425 to the design of an inherently safe [C mim]

4

+ concentration for phytoremediation, the

426 results of this study suggested that ryegrass could tolerate [C mim]

4

+

at

a high

21

ACCEPTED MANUSCRIPT

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

concentration (e.g., 5 mg/L) in an aquatic environment without significant influences

on plant growth and uptake.

Conclusions

The

current

studies

demonstrated

that

ionic

liquids

are

not

so-called

“environmentally friendly” chemicals. They might threaten the natural environment

and should be removed or degraded after they are discharged into the environment.

In this study, ryegrass was used for imidazolium removal from water. Results show

that [C 4 mim]

+ could be taken up, accumulated and metabolized by plants in vivo

with a high efficiency. The phytoaccumulation and consequent metabolization of

ionic

liquid

in

the

different

parts

of

the

plants

were

also

confirmed.

Two

hydroxylated metabolites and two secondary metabolites were detected in the

ryegrass after [C mim]

4

+ uptake. These results may have important implications for

the potential application of phytoremediation for removing ionic liquids from

contaminated water or soil. To validate this potential application, field experiments

will be needed to investigate the long-term phytoaccumulation and phytodegradation

of ionic liquids

in

plants

under different

environmental

conditions.

From

an

application perspective, after remediation, the ionic liquid accumulated plants could

be harvested and incinerated and then disposed safely such as landfill. In future

studies, the disposal of the ionic liquid-contaminated plants and economic and life

cycle analyses should be studied.

22

ACCEPTED MANUSCRIPT

449

450

Acknowledgements

 

451

The authors wish to thank National Natural Science Foundation of China

452

(51738012, 518250804, 51608465, 21607003 and 51821006) for the partial support

453

of this work. The numerical calculations were performed on the supercomputing

454

system at the Supercomputing Center at the University of Science and Technology of

455

China, China.

456

457

Reference

458

Aibibu, N.a., Liu, Y., Zeng, G., Wang, X., Chen, B., Song, H. and Xu, L. (2010)

459

Cadmium accumulation in vetiveria zizanioides and its effects on growth,

460

physiological and biochemical characters. Bioresource Technology 101(16),

461

6297-6303.

 

462

Amde, M., Liu, F.J., Pang, L. (2015) Environmental Application, Fate, Effects, and

463

Concerns of Ionic Liquids: A Review. Environmental Science & Technology, 49

464

(21), 12611–12627.

 

465

Arnold, K., Bordoli, L., Kopp, J. and Schwede, T. (2006) The SWISS-MODEL

466

workspace: a web-based environment for protein structure homology modelling.

467

Bioinformatics 22(2), 195-201.

 

468

Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J. and Schwede, T. (2009)

469

Protein

structure

homology

modeling

using

SWISS-MODEL

workspace.

470

Nature Protocols 4(1), 1-13.

 

23

ACCEPTED MANUSCRIPT

471 Brix, H., DyhrJensen, K. and Lorenzen, B. (2002) Rootzone acidity and

472 nitrogen source affects

Typha latifolia L. growth and uptake kinetics of

473 ammonium and nitrate. Journal of Experimental Botany 53(379), 2441-2450.

474 Calza P., Noe G., Fabbri D., Santoro V., Minero C., Vione D., Medana C. (2017)

475 Photoinduced

transformation

of

pyridinium-based ionic liquids,

and

476 implications for their photochemical behavior in surface waters. Water Research,

477 122, 194-206

478 Carter, L.J., Harris, E., Williams, M., Ryan, J.J., Kookana, R.S. and Boxall, A.B.A.

479 (2014) Fate and uptake of pharmaceuticals in soil–plant systems. Journal of

480 Agricultural and Food Chemistry 62(4), 816-825.

481 Czerwicka, M., Stolte, S., Müller, A., Siedlecka, E.M., Gołębiowski, M., Kumirska, J.

482 and Stepnowski, P. (2009) Identification of ionic l iquid breakdown products in

483 an advanced oxidation system. Journal of Hazardous Materials 171(1–3),

484 478-483.

485 Delley, B. (2000) From molecules to solids with the DMol

486 Chemical Physics 113(18), 7756-7764.

3 approach. Journal of

487 Domey, J., Bergemann, C., Bremer-Streck, S., Krumbein, I., Reichenbach, J.R.,

488 Teichgraber, U. and Hilger, I. (2015) Long-term prevalence of NIRF-labeled

489 magnetic

nanoparticles

for

the

diagnostic

and

intraoperative

imaging

of

490 inflammation. Nanotoxicology 10(1), 20-31.

491 Duan, J.J., He, S.Y., Feng, Y.F., Yu, Y.L., Xue L.H., and Yang, L.Z. (2017) Floating

492 ryegrass

mat

for

the

treatment

of

24

low-pollution

wastwater.

Ecological

ACCEPTED MANUSCRIPT

493 Engineering 108, 172-178.

494 Eggen, T., Asp, T.N., Grave, K. and Hormazabal, V. (2011) Uptake and translocation

495

of

metformin,

ciprofloxacin

and

narasin

in

forage-

496 Chemosphere 85(1), 26-33.

and

crop

plants.

497 Felizeter, S., McLachlan, M.S. and de Voogt, P. (20 12) Uptake of perfluorinated

498 alkyl acids by hydroponically grown lettuce (Lactuca sativa). Environmental

499 Science & Technology 46(21), 11735-11743.

500 Fu, Z.Q., Wang, Y., Chen, J.W., Wang, Z.Y. and Wang, X.B. (2016) How PBDEs are

501 transformed into dihydroxylated and dioxin metabolites catalyzed by the active

502 center

of

cytochrome

P450s:

A

DFT

study.

503 Technology 50(15), 8155-8163.

Environmental

Science

&

504 Gallego, S.M., Benavídes, M.P. and Tomaro, M.L. (1996) Effect of heavy metal ion

505 excess on sunflower leaves: evidence for involvement of oxidative stress. Plant

506 Science 121(2), 151-159.

507 Giannopolitis, C.N. and Ries, S.K. (1977) Superoxide dismutases: I. Occurrence in

508 higher plants. Plant Physiology 59(2), 309-314.

509 Girvan, H.M., Toogood, H.S., Littleford, R.E., Seward, H.E., Smith, W.E., Ekanem,

510

I.S.,

Leys,

D.,

Cheesman,

M.R.

and

Munro,

A.W.

(2009)

Novel

haem

511 co-ordination variants of flavocytochrome P450BM3. Biochemical Journal

512 417(1), 65-76.

513 Grimme, S. (2006) Semiempirical GGA-type density functional constructed with a

514 long-range dispersion correction.

Journal of Computational Chemistry 27(15),

25

ACCEPTED MANUSCRIPT

515 1787-1799.

516 Halgren, T.A. and Lipscomb, W.N. (1977) The synchronous-transit method for

517 determining

reaction

pathways

and

locating

molecular

transition

518 states. Chemical Physics Letters 49(2), 225-232.

519 Hsu, F.C., Marxmiller, R.L. and Yang, A.Y.S. (1990) Study of root uptake and xylem

520 translocation of cinmethylin and related compounds in detopped soybean roots

521 using a pressure chamber technique. Plant Physiology 93(4), 1573-1578.

522 Jastorff, B., Störmann, R., Ranke, J., Mölter, K., Stock, F., Oberheitmann, B.,

523 Hoffmann, W., Hoffmann, J., Nüchter, M., Ondruschka, B. and Filser, J. (2003)

524 How hazardous are ionic liquids? Structure–activity relationships and biological

525 testing as important elements for sustainability evaluation. Green Chemistry

526 5(2), 136-142.

527 Klamt, A. and Schuurmann, G. (1993) COSMO: a new approach to dielectric

528 screening in solvents with explicit expressions for the screening energy and its

529 gradient. Journal of the Chemical Society, Perkin Transactions. 2 (5), 799-805.

530 Markiewicz, M., Piszora, M., Caicedo, N., Jungnickel, C. and Stolte, S. (2013)

531 Toxicity of ionic liquid cations and anions towards activated sewage sludge

532 organisms from different sources - Consequences for biodegradation testing and

533 wastewater treatment plant operation. Water Research, 47, 2921-2928.

534 Matzke, M., Stolte, S., Arning, J., Uebers, U. and Filser, J. (2008) Imidazolium based

535 ionic liquids in soils: effects of the side chain length on wheat (Triticum

536 aestivum) and cress (Lepidium sativum) as affected by different clays and

26

ACCEPTED MANUSCRIPT

537 organic matter. Green Chemistry 10(5), 584-591.

538 Matzke,

M.,

Stolte,

S.,

Thiele,

K.,

Juffernholz,

T.,

Arning,

J.,

Ranke,

J.,

539 Welz-Biermann, U. and Jastorff, B. (2007) The influence of anion species on

540 the toxicity of

1-alkyl-3-methylimidazolium

ionic liquids

observed

in

an

541 (eco)toxicological test battery. Green Chemistry 9(11), 1198-1207.

542 MuthusaravananN, S., Sivarajasekar, N., Vivek, J.S., Paramasivan, T., Naushad, Mu.,

543 Prakashmaran, J., Gayathri, V., Al-Duaij, Omar K. (2018) Phytoremediation of

544 heavy

metals:

mechanisms,

methods

545 Chemistry Letters 16(4), 1339-1359.

and

enhancements

Envritonmental

546 Neumann, J., Grundmann, O., Thoming, J., Schulte, M. and Stolte, S. (2010)

547 Anaerobic biodegradability of ionic liquid cations under denitrifying conditions.

548 Green Chemistry 12(4), 620-627.

549 OECD. (1984) OECD Guideline for Testing of Chemicals 201: Alga, growth

550 inhibition test., Paris.

551 Ortiz de Montellano, P.R. (2010) Hydrocarbon hydroxylation by cytochrome P450

552 enzymes. Chemical Review 110(2), 932-948.

553 Pan, J.Y., Fu, J., Deng, S.G., Lu, X.Y. (2014) Microwave-Assisted Degradation of

554 Lignin Model Compounds in Imidazolium-Based Ionic Liquids. Energy &

555 Fuels 28, 1380-1386.

556 Perdew,

J.P.,

Burke,

K.

and

Ernzerhof,

M.

(1996)

Generalized

gradient

557 approximation made simple. Chemical Physics Letters 77(18), 3865-3868.

558 Perdew, J.P., Chevary, J.A., Vosko, S.H., Jackson, K.A., Pederson, M.R., Singh, D.J.

27

ACCEPTED MANUSCRIPT

559 and Fiolhais, C. (1992) Atoms, molecules, solids, and surfaces-applications of

560 the generalized gradient approximation for exchange and correlation. Physical

561 Review B 46(11), 6671-6687.

562 Petkovic, M., Seddon, K.R., Rebelo, L.P.N. and Silva Pereira, C. (2011) Ionic liquids:

563 a pathway to environmental acceptability. Chemical Society Review 40(3),

564 1383-1403.

565 Pham, T.P.T., Cho, C. W. and Yun, Y. S. (2010) Environmental fate and toxicity of

566 ionic liquids: A review. Water Research 44(2), 352-372.

567

Rittle,

J.

and

Green,

568 characterization,

569 933-937.

M.T.

(2010)

Cytochrome

and

C-H

bond

activation

P450

Compound

kinetics.

Science

I:

Capture,

330(6006),

570 Schnoor, J.L., Licht, L.A., McCutcheon, S.C., Wolfe, N.L. and Carreira, L.H. (1995)

571 Phytoremediation of organic and nutrient contaminants. Environmental Science

572 & Technology 29(7), 318A-323A.

573 Sicbaldi, F., Sacchi, G.A., Trevisan, M. and Del Re, A.A.M. (1997) Root uptake and

574 xylem translocation of pesticides from different chemical classes. Journal of

575 Pesticide Science 50(2), 111-119.

576 Spasiano, D., Siciliano, A., Race, M., Marotta, R., Guida, M., Andreozzi, R. and

577 Pirozzi, F. (2016) Biodegradation, ecotoxicity and UV

254

/H 2 O 2

treatment of

578 imidazole, 1-methyl-imidazole and N,N '-alkyl-imidazolium chlorides in water.

579 Water Research, 106, 450-460

580 Stolte, S., Abdulkarim, S., Arning, J., Blomeyer-Nienstedt, A. K., Bottin-Weber, U.,

28

ACCEPTED MANUSCRIPT

581 Matzke,

M.,

Ranke,

J.,

Jastorff,

B.

and

Thoming,

J.

(2008)

Primary

582 biodegradation of ionic liquid cations, identification of degradation products of

583 1-methyl-3-octylimidazolium

chloride

and

electrochemical

wastewater

584 treatment

of

poorly

biodegradable

compounds.

Green

Chemistry

10(2),

585 214-224.

586 Thamke, V.R., Tapase, S.R. and Kodam, K.M. (2017) Evaluation of risk assessment

587 of new industrial pollutant, ionic liquids on environmental living systems.

588 Water Research, 125, 237-248.

589 van Aken, B., Yoon, J.M., Just, C.L. and Schnoor, J.L. (2004) Metabolism and

590 Mineralization of Hexahydro-1,3,5-trinitro-1,3,5-triazine Inside Poplar Tissues

591 (Populus deltoides × nigra DN-34). Environmental Science & Technology

592 38(17), 4572-4579.

593 Yin, X.L., Jiang, L., Song, N.H. and Yang, H. (2008) Toxic reactivity of wheat

594 (Triticum aestivum) plants to herbicide isoproturon. Journal Agricultural and

595 Food Chemistry 56(12), 4825-4831.

596 Yu, M., Wang, S.H., Luo, Y.R., Han, Y.W., Li, X.Y., Zhang, B.J. and Wang, J.J.

597 (2009) Effects of the 1-alkyl-3-methylimidazolium bromide ionic liquids on the

598 antioxidant

defense

system

of

Daphnia

magna.

Ecotoxicology

and

599 Environmental Safety 72(6), 1798-1804.

600 Zhou, H., Lv, P., Shen, Y., Wang, J. and Fan, J. (2013) Identification of degradation

601 products of ionic liquids in an ultrasound assisted zero-valent iron activated

602 carbon micro-electrolysis system and their degradation mechanism. Water

29

ACCEPTED MANUSCRIPT

603 Research 47(10), 3514-3522.

30

ACCEPTED MANUSCRIPT

Table 1 [C 4 mim] + concentrations in water and phytoaccumulation in different control

treatments

samples

exposure time

(days)

initial [C 4 mim] +

conc.

residual [C 4 mim] +

conc.

 

(mg/L)

(mg/L)

untreated plants

Roots

10

-

-

Water

-

dead ryegrass

Roots

10

5

0.22±0.03

Water

4.62±0.01

excised ryegrass

Roots

10

5

0.35±0.01

Water

4.49±0.02

blank control

Roots

10

5

-

Water

4.92±0.03

604

31

ACCEPTED MANUSCRIPT

605

606

Table 2 Removal of [C mim]

4

+ by ryegrass and phytoaccumulation at various initial

[C

4 mim]

+ concentrations

[C

4 mim] +

root

leaf

accumulated

removal

TF

RCF

initial conc.

(mg/g/FW)

(mg/g/FW)

fraction in plant

*

efficiency

(mg/L)

 

(%)

(%)

 

1

0.025±0.001

0.020±0.001

89.1

100

0.81

4.9

2

0.091±0.015

0.032±0.004

84.9

100

0.35

45.3

5

0.160±0.008

0.089±0.003

77.8

90.7

0.55

32.1

10

0.430±0.029

0.100±0.010

55.9

71.5

0.23

42.9

15

0.773±0.071

0.120±0.001

51.6

66.2

0.15

51.5

607

* defined as (plant accumulated [C mim]

4

+

) /(total dosed [C mim]

4

+

)×100%)

608 FW: fresh weight

609

32

ACCEPTED MANUSCRIPT

610

611

Table 3 Detected m/z, theoretical mass, relativity, intensity of signals in the mass

spectra of the intermediates in ryegrass

No.

molecular

m/z

theoretical

intensity

relativity

composition

mass

M1

C

8 H 15 N

2

139.12257

139.12298

5.7×10

4

16.33

M2

C 8

H 15 N 2

O

155.11769

155.11789

7.8×10

3

2.26

M3

C 8 H 12 N 2 O 2

168.08890