Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester- and

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Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester- and Ether-linked Bound Residues in an Oxic Sandy Soil Fangjie Li, Jiajia Wang, Bingqi Jiang, Xue Yang, Boris Kolvenbach, LianHong Wang, Yini Ma, Philippe F.-X. Corvini, and Rong Ji Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01900 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 7, 2015

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Environmental Science & Technology

1

Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester-

2

and Ether-linked Bound Residues in an Oxic Sandy Soil

3 4

Fangjie Li,1 Jiajia Wang,1 Bingqi Jiang,1 Xue Yang,1 Peter Nastold,2 Boris

5

Kolvenbach, 2 Lianhong Wang,1 Yini Ma,1 Philippe François-Xavier Corvini,1,2 Rong

6

Ji1,*

7

1

8

Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, China

9

2

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

Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts

10

Northwestern Switzerland, Gründenstrasse 40, Muttenz CH-4132, Switzerland

11

*: corresponding author, Tel: +86-25-8968 0581; E-Mail: [email protected]

12

13

Abstract

14

Bound-residue formation is a major dissipation process of most organic xenobiotics in soil.

15

However, both the formation and nature of bound residues of tetrabromobisphenol A (TBBPA)

16

in soil are unclear. Using a 14C-tracer, we studied the fate of TBBPA in an oxic soil during 143

17

days of incubation. TBBPA dissipated with a half-life of 14.7 days; at the end of incubation,

18

19.6% mineralized and 66.5% formed bound residues. Eight extractable metabolites were

19

detected, including TBBPA methyl ethers, single-ring bromophenols, and their methyl ethers.

20

Bound residues (mostly bound to humin) rapidly formed during the first 35 days. The amount

21

of those humin-bound residues then quickly decreased, whereas total bound residues

22

decreased slowly. By contrast, residues bound to humic acids and fulvic acids increased

23

continuously until a plateau was reached. Ester- and ether-linked residues accounted for

24

9.6−27.0% of total bound residues during the incubation, with ester linkages being

25

predominant. Residues bound via ester linkages consisted of TBBPA, TBBPA monomethyl

26

ether, and an unknown polar compound. Our results indicated that bound-residue formation is

27

the major pathway of TBBPA dissipation in oxic soil and provide first insights into the

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28

chemical structure of the reversibly ester-linked bound residues of TBBPA and its

29

metabolites.

30

1. Introduction

31

Tetrabromobisphenol A [4,4’-isopropylidenebis(2,6-dibromophenol), TBBPA] is used as

32

a reactive intermediate in the production of epoxy and polycarbonate resins and as an additive

33

in acrylonitrile-butadiene-styrene resin and high-impact polystyrene.1 It accounts for

34

approximately 60% of all applied brominated flame retardants.2 The yearly TBBPA global

35

market was over 200,000 tons in 2013.3 TBBPA is characterized by low water solubility

36

(0.17–4.16 mg L−1 at 25°C) and high octanol/water partition coefficient (log KOW = 4.5–6.5 at

37

25°C) at neutral pH.4 It is widely distributed in soils, water, and sediments2,5–7 as well as in

38

biota.8 A growing body of studies has shown that TBBPA has thyroid hormonal activity9,10,11

39

and leads to immunological effects12,13,14 and inhibition of synaptic neurotransmitter

40

uptake.15,16 At environmentally relevant concentrations, TBBPA is particularly toxic to fish.

41

17,18

42

Soil is one of the major sinks of TBBPA in the environment. TBBPA concentrations have

43

been reported to be as high as 450 mg/kg in contaminated soil.19 TBBPA can be biodegraded

44

in soil under both anoxic and oxic conditions.20–25 Under anoxic conditions, TBBPA is mainly

45

reduced to less-brominated intermediates and finally to bisphenol A (BPA).19,22 Under oxic

46

condition, TBBPA can be O-methylated to form its mono- and di-methyl ethers (MeO-TBBPA

47

and diMeO-TBBPA, respectively) by microorganisms.21,26 In oxic soil slurry with nutrient

48

amendment, TBBPA can be degraded to a variety of products, including polar single-ring

49

compounds,

50

ipso-substitution, debromination, and O-methylation.23, In soil with oxic–anoxic interfaces,

51

such as a submerged soil–plant system in wetlands, MeO-TBBPA and diMeO-TBBPA are the

52

major products of TBBPA and are persistent in the system25. It is reported that diMeO-TBBPA

53

can be also toxic to aquatic organism.27 Both aerobic and anaerobic transformation of TBBPA

54

in soil lead to formation of large amounts of bound residues, corresponding to a TBBPA loss

55

of 28–62%.22,23,25

MeO-TBBPA,

and

diMeO-TBBPA via

oxidative

2 / 26


cleavage,

type

II

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56

Bound-residue formation is considered as a xenobiotic detoxification process in soil, as

57

they are regarded as stable and recalcitrant toward microbial attack and transport.28,29 It is

58

usually assumed that the residues consist of the parent compounds and their transformation

59

products. However, atoms of the metabolized compounds incorporated into soil biomass, e.g.,

60

bacteria, are also regarded as bound residues.30 The parent compounds and their

61

transformation products may be associated with soil organic matter through physico-chemical

62

enclosure or covalent bonding with ether, ester, or C–C linkages.31,32 These mechanisms for

63

binding of residues to soil organic matter impact the stability of the residues in soil, which in

64

turn determines the mobility, bioaccumulation, and biodegradation of the xenobiotics in

65

environmental compartments.22,30,33

66

Selective chemical cleavage of covalent bonds, followed by identification of the released

67

products may provide information about the nature of the bound residues. In this manner,

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Richnow et al.29,34 found ester- and ether-linked residues of polycyclic aromatic hydrocarbons

69

in soil, Martens35 quantified the composition and concentration of ester- and ether-linked

70

phenolic acids in plants and soils, and Riefer et al.36 revealed that reversible ester (amine)

71

linkages are responsible for binding of nonylphenol residues to soil humic substances.

72

Recently, Liu et al.22 reported that bound residues of TBBPA formed in anoxic soil may be

73

released to a large extent when the soil is exposed to air. The released residues consisted of

74

the parent compound TBBPA and its debrominated degradation products.22 The complexity of

75

the mixture of degradation products of TBBPA formed under oxic conditions23 suggests that

76

bound-residue formation of TBBPA in oxic soil can originate from the binding of a variety of

77

molecules to soil organic matter. TBBPA and most of its identified metabolites are phenolic

78

compounds,22,23,25,37,38 which tend to bind to humic substances via ester and ether

79

linkages.30,36,39,40 Detailed investigation about the bonding characteristics and the chemical

80

structures are needed to understand the environmental fate of TBBPA and to assess the risk of

81

its bound residues, However, little effort has been made to characterize the bound residues of

82

TBBPA that are formed under oxic conditions, and the mechanisms for the bound-residue

83

formation are still unclear.

84 85

In the present study, we used a

14

C-tracer (1) to investigate the fate of TBBPA in soil

under oxic conditions, (2) to quantify the distribution of bound residues of TBBPA within 3 / 26



86

different humic fractions, and (3) to quantify the contribution of ester and ether linkages to

87

bound-residue formation of TBBPA and its metabolites by using sequential alkaline

88

hydrolysis according to Martens.

89

2. Materials and Methods

90

2.1

14

C-Labeled TBBPA

Uniformly

91

35

14

C-ring-labeled TBBPA (14C-TBBPA) was synthesized from uniformly

92

14

93

with acetone using sulphuric acid as catalyst to form

94

brominated by N-bromosuccinimide with the catalyst BF3 to form 14C-TBBPA. The product

95

was purified by preparative thin layer chromatography on silica gel. The obtained 14C-TBBPA

96

had a specific radioactivity of 1.48 × 109 Bq mmol−1, a chemical purity of 97% (as analysed

97

by liquid chromatography-mass spectrometry), and a radiochemical purity of 99% (as

98

analysed by high performance liquid chromatography coupled to a radio flow detector, see

99

below).23

100

C-ring-labeled phenol (14C-phenol) in our laboratory.23 Briefly, 14C-phenol was condensed 14

C-ring-labelled BPA, which was

2.2 Soil Incubation Experiments

101

The test soil was a loamy sand, taken from the topsoil (0−20 cm) of an agricultural field

102

near the town of Lalden, located in the upper Rhȏne valley in Switzerland, containing 1.4%

103

total organic carbon, 4.2% clay, 19.5% silt, and 76.3% sand (determined by using pipette

104

method),41 with a pH of 7.0 (0.01 M CaCl2). The soil was gently air-dried and passed through

105

a 2-mm sieve prior to use.

106

Aliquots of a methanolic solution of

14

C-TBBPA (33.3 KBq) were added using a

107

microsyringe to 0.2 g of soil (dry weight). After evaporation of the methanol under a gentle

108

stream of nitrogen gas, the spiked soil was thoroughly mixed with another 2.3 g soil (dry

109

weight) in a 50-mL serum flask. The TBBPA concentration in the flasks was 5.0 mg per kg

110

soil (dry weight). The homogeneity of 14C-TBBPA distribution within the soil was verified by

111

determining the radioactivity of soil subsamples (0.02–0.05 g) from the flask (recovery = 98 ± 4 / 26


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112

2.0%, n = 3). The soil moisture was adjusted to 70% of the maximal water-holding capacity

113

by

114

polytetrafluoroethylene-wrapped rubber stopper and incubated at 20 ± 1 °C in the dark. The

115

14

116

mL NaOH (1 M). The trap was suspended from the bottom of the stopper. Controls containing

117

sterilized soil (autoclaved at 120 °C for 30 min twice on two consecutive days) were prepared

118

to evaluate abiotic transformation of TBBPA in the soil. All experiments were performed in

119

triplicate.

adding

0.70

mL

sterile

H2O.

The

flask

was

then

closed

with

a

CO2 released from the soil during the incubation was trapped in a 6-mL vial containing 1.0

120

The flasks were opened for 30 s everyday to allow oxygen in the headspace. Water loss

121

from the soil during incubation was compensated by adding the same amount of distilled

122

sterile water to the soil. At each sampling time, three flasks from each treatment were

123

sacrificed for analyses of radioactivity in the NaOH trap; amounts of 14C-TBBPA, metabolites,

124

and bound residues; and characterization of the specific linkages between bound residues and

125

soil humic substances (see below).

126

2.3 Extraction and Fractionation of Soil

127

At each sampling time, soil samples (2.5 g) were freeze-dried and extracted with

128

methanol (15 mL) once and ethyl acetate (15 mL) twice by repeated shaking (150 rpm, 1 h)

129

and centrifugation (3,500g, 10 min). The supernatants (extracts) were combined, and

130

radioactivity in aliquots was quantified by liquid scintillation counting (LSC, see below). The

131

radioactivity in organic solvent extracts was considered as the extractable residues fraction.

132

The organic extracts were then evaporated to dryness using a rotary evaporator and

133

resuspended in 1 mL methanol for analyses by means of high-performance liquid

134

chromatography (HPLC) coupled to a radio flow detector (HPLC−14C-LSC, see below).

135

The radioactivity remaining in the soil after exhaustive extraction with organic solvent

136

was defined as the radioactivity assignable to bound residues (non-extractable residues) and

137

was measured by combustion and subsequent LSC (see below). The bound residues were

138

further fractionated into residues bound to fulvic acids, humic acids, and humin according to

139

their solubility in alkaline solution.42 Briefly, aliquots (0.4 g) of air-dried soil pellets

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14

140

containing bound residues of

C-TBBPA and its metabolites were extracted with 0.1 M

141

oxygen-free NaOH (1.6 mL) for 36 h by horizontal shaking at 200 rpm. After centrifugation

142

at 16,000 g for 30 min, the supernatants (i.e., alkaline soluble humic substances) were

143

separated from the pellets (i.e., humin fraction) and acidified to pH 1 with 6 M HCl. The

144

precipitates containing humic acids were separated from the supernatant containing fulvic

145

acids by centrifugation at 10,000 g for 30 min. The radioactivity in the fractions of humin,

146

alkaline soluble humic substances, and fulvic acids was determined by LSC (see below) and

147

all the detected radioactivity in the sub-samples were significantly high (> 50 Bq).

148

2.4 Cleavage of Ester- and Ether-Linked Bound Residues

149

Ester and ether linkages of bound residues were specifically cleaved by alkaline

150

hydrolysis according to Martens.35 The procedures are summarized in Supporting Information

151

(SI) Figure S1.

152

Ester cleavage. For organic solvent extraction, soil (1 g) was extracted with 4 mL of 1 M

153

NaOH for 4 h on a reciprocal shaker at 90 °C. After extraction, the sample was centrifuged

154

(5,000 g, 15 min), and the pellet was washed with distilled water (3 mL) once. The

155

supernatants (hydrolysates) were combined, and radioactivity in an aliquot (100 µL) was

156

quantified by LSC (see below). The alkaline supernatants (hydrolysates) were acidified with 4

157

M HCl to pH 1−2 and extracted with dichloromethane (CH2Cl2). The acidic aqueous

158

suspension was reserved for analysis of ether linkages (see below). The CH2Cl2 extracts were

159

dried with anhydrous sodium sulfate and then evaporated to dryness in a rotary evaporator

160

and resuspended in 1 mL methanol. Radioactivity in an aliquot (100 µL) was quantified by

161

LSC (see below), and the rest of the methanolic solution was analyzed by HPLC−14C-LSC

162

(see below).

163

Ether cleavage. The soil pellet remaining after ester cleavage was suspended in 4 mL of 4 M

164

NaOH. The mixture was vortexed for 1 min and then heated for 15 min at 120 °C. After

165

cooling, the reaction mixture was centrifuged (5,000 g, 15 min), and the pellet was washed

166

with distilled water (2 mL) once. Supernatants (hydrolysates) were combined. The acidic

167

aqueous suspension from the ester cleavage step (see above) was alkalized by adding solid 6 / 26


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NaOH to a final concentration of 4 M. The mixture was heated for 15 min at 120 °C and then

169

centrifuged. All alkaline supernatants (4 M NaOH) (hydrolysates) were combined, and

170

radioactivity in an aliquot (100 µL) was quantified by LSC (see below). The rest was acidified

171

with 4 M HCl and extracted with CH2Cl2 as described above. Radioactivity in an aliquot (100

172

µL) of the CH2Cl2 extract was quantified by LSC (see below). The rest of the organic extract

173

was concentrated and analyzed by HPLC−14C-LSC (see below).

174

2.5 Analyses

175

HPLC analysis. HPLC was performed on a Nucleosil C18 column (250 mm × 4 mm) at 40 °C

176

with an Agilent HPLC Series 1100 system (Agilent Technologies, Germany) equipped with an

177

auto-injector, a degasser, a diode array detector, and an online radio flow liquid scintillation

178

detector (HPLC-14C-LSC).23

179

Purification of metabolites. Aliquots (200 µL) of soil organic extracts were injected

180

repeatedly into the HPLC system, and radioactive fractions were collected peak by peak. The

181

fractions were evaporated to dryness under a gentle stream of nitrogen gas and resuspended in

182

200 µL methanol for further analysis by gas chromatography−mass spectrometry (GC−MS)

183

after derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (BSTFA).23

184

Determination of radioactivity. Radioactivity was quantified using an LSC (Tri-Carb 2800 TR;

185

Perkin Elmer, USA). To determine radioactivity of

186

solution was mixed with 15 mL of the scintillation cocktail Lumasafe (PerkinElmer); to

187

determine radioactivity in organic extracts, 1 mL extract was mixed with 15 mL Lumasafe. To

188

determine bound radioactivity in soil residues, about 100 mg of the extracted and air-dried

189

soil particles was combusted with an oxidizer (Sample Oxidizer Model 307; PerkinElmer).

190

The generated

191

PerkinElmer), mixed with 10 mL scintillation cocktail Permafluor (PerkinElmer), and then

192

quantified by LSC. To determine radioactivity in alkaline extracts and the fulvic acid fraction,

193

1 mL alkaline extract or 0.8 mL fulvic acid solution was mixed with 15 mL Lumasafe. The

194

radioactivity of the humic acid fraction was calculated by subtracting the radioactivity of the

195

fulvic acid fraction from that of the alkaline extract. The humin fraction was dried at 65 °C

14

14

CO2 trapped in NaOH, 1 mL NaOH

CO2 was absorbed by 10 mL carbon dioxide absorber (Carbon-Sorb E;

7 / 26



Page 8 of 26

196

for 2 days and then ground to powder; radioactivity in an aliquot of 50 mg was determined by

197

combustion as described above. To determine radioactivity in the alkaline hydrolysates, a 100

198

µL aliquot was mixed with 15 mL Lumasafe.

199

Data analysis. Data on TBBPA degradation were fitted to the first-order kinetics Ct = C0 e−kt,

200

where C0 is the initial concentration, Ct is the concentration at time t, and k is the degradation

201

rate constant. CO2 formation was fitted to the linear kinetics. The regression was carried out

202

using SigmaPlot 12.0. The half-life (t1/2) was calculated using the equation t1/2 = ln 2/k.

203

Significance was analyzed using Student’s t-test; a statistical probability of p < 0.05 was

204

considered significant.

205

3. Results and Discussion

206

3.1 Fate of TBBPA in Soil

207

We measured the relative amounts of radioactivity of 14CO2 and of extractable residues

208

and bound residues from 14C-TBBPA during 143 days of incubation in non-sterilized (active)

209

and sterilized (control) soil (Figure 1). About 19.6 ± 0.2% of the initially applied

210

mineralized at a linear rate of 0.16 ± 0.01% day−1 during 143 days of incubation in active soil.

211

The amount of the extractable radioactivity decreased rapidly to about 20% within the first 35

212

days, followed by a continuous decrease at lower rates to less than 15% by the end of the

213

incubation. During this phase, bound residues were formed without a lag phase and accounted

214

for 66.5% of the initially applied radioactivity at the end of incubation (Figure 1a). After the

215

rapid increase at the beginning of incubation, bound residues declined slightly over time

216

(Figure 1a), which indicated that their formation was partly reversible in soil under the

217

incubation conditions used and that bound residues were released, probably attributable to

218

microbial activity.

219

significantly higher (p < 0.05) than those of TBBPA in oxic soil slurry with addition of easily

220

degradable carbon source (peptone, yeast extract, and glucose),

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degradable carbon source might reduce degradation of TBBPA in soil. However, the

222

mineralization rate was similar to that of TBBPA in flooded soil,

30,33,43

14

C was

The rate of mineralization and bound-residue formation were

8 / 26


25

23

indicating that easily

though the bound-residue

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223

formation in the submerged soil was much slower. 25 The rapid formation of bound residues of

224

TBBPA was characteristic in the oxic natural soil (Figure 1a). In sterilized soil, mineralization of

225

14

C-TBBPA was negligible (Figure 1b), which 14

226

indicated that microbial activity was responsible for mineralization of

C-TBBPA in active

227

soil. However, significant but lower amounts of bound residues of TBBPA (about 15%, Figure

228

1b) rapidly formed in sterilized soil, i.e., abiotically, within the first 17 days of incubation,

229

and the amount remained almost constant during the remaining incubation (Figure 1b). It was

230

previously observed that a similar amount of bound residues of TBBPA form under abiotic

231

conditions in soil incubated under anoxic conditions.22 The abiotic formation may be

232

attributed to the strong adsorption of 14C-TBBPA onto soil matrices and enclosure in pores of

233

soil organic matter, organo-clay complexes, and interlayers of clay minerals.36,44 It is worthy

234

to note that sterilization process may affect soil structure and properties, which could

235

influence the abiotic formation of bound residues observed in the sterilized soil. The

236

extractable radioactivity from sterilized soil was fully recovered as 14C-TBBPA (SI Figure S2),

237

which indicated that TBBPA was not abiotically transformed. All these findings underline that

238

microbial transformation, i.e., microbial metabolic processes, crucially impact bound-residue

239

formation in active soil.

240

3.2 Metabolites of TBBPA in Soil

241

HPLC−14C-LSC analysis of the soil organic extracts showed that TBBPA was

242

transformed into several metabolites (Figure 2). Eight metabolites were detected, including

243

single-ring brominated phenols and TBBPA methyl ethers (Figure 2; SI Table S1). The

244

identification of the metabolites was based on comparison with authentic compounds or on

245

bromine isotope patterns and characteristic mass fragments of the metabolites.

246

metabolites profile was similar to that previously found in oxic soil slurry with nutrient source

247

addition;

248

soil (SI Figure S3). The metabolites diMeO-TBBPA was detected after 143 days of incubation

249

in the present study, but was not observed in the oxic soil slurry during 20 days of incubation,

250

23

23

23

The

based on these results, we propose three pathways for TBBPA transformation in

which suggested that the formation of the dimethyl ether derivative of TBBPA is slow in the

9 / 26



251

oxic soil environment. However, in soil planted with the common reed Phragmites australis,

252

diMeO-TBBPA may form quite rapidly (< 10 days incubation).

25

253

The kinetics of TBBPA transformation and metabolites formation during incubation

254

(Figure 3) showed that transformation of TBBPA in active soil did not have a lag period; it

255

followed first-order decay kinetics with a kinetic constant of 0.047 ± 0.004% day−1 (half-life

256

t1/2 = 14.7 ± 1.25 days) (Figure 3a), which is significantly (p < 0.05) higher than that in oxic

257

soil slurry with nutrient amendment (t1/2 = 40.8 ± 10.4 days)23 and in flooded soil (t1/2 = 20.8 ±

258

0.1 days).25 This indicated that in the previous study, easily degradable carbon contained in

259

the nutrient amendment was preferentially utilized by TBBPA-degrading microorganisms.

260

TBBPA methyl ethers were continuously formed, while single-ring metabolites accumulated

261

to their maximum amounts on day 27 and completely disappeared after 143 days of

262

incubation (Figure 3b). Their decay was probably the result of further mineralization and

263

binding to soil humic substances, i.e., bound-residue formation (Figure 1a).

264

3.3 Distribution of Bound Residues of TBBPA in Humic Fractions We determined the distribution of bound residues of

265

14

C-TBBPA in different soil

266

fractions (fulvic acid, humic acid, and humin) of the soil organic matrix according to their

267

solubility in alkaline and acidic solution. During the first 35 days of incubation in active soil,

268

the highest amount (46.4 ± 4.5%) of radioactivity was located in the humin fraction, followed

269

by humic acid and fulvic acid fractions (Figure 4a). Humin-bound radioactivity was

270

considerably lower on day 55 (28.2 ± 2.9%) and remained at this level throughout the

271

remaining incubation (Figure 4a). By contrast, bound residues in sterilized soil were mainly

272

associated to the humic acid fraction (Figure 4b). We conclude from a comparison of the

273

distribution of residues in active and sterilized soils (Figure 4) that microbial transformation

274

of 14C-TBBPA increases the formation of residues that are mainly bound to humin and humic

275

acid.

276

The increase in the amount of bound residues from 14C-TBBPA in microbially active soil

277

may be attributed to (1) stimulated physical entrapment of TBBPA and its metabolites in soil

278

organic and inorganic matrices by microorganisms that contribute significantly to the

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formation of organo-clay complexes and aggregates of soil, (2) assimilation of

280

14

C-TBBPA-derived carbon into microbial biomass, as significant mineralization (20%) of

281

14

C-TBBPA occurred in the soil (Figure 1a) and mineralization is accompanied by formation

282

of biogenic bound residues,30 and (3) chemical binding of TBBPA and its metabolites to soil

283

humic substances via ester, ether, and C–C linkages. TBBPA and many of its metabolites have

284

phenolic groups (SI Figure S3); compounds with phenolic structure likely react with soil

285

humic substances via coupling reactions and have a great tendency to form bound residues.31

286

The distribution of bound residues within humic fractions (Figure 4a) suggested that in the

287

early phase of the incubation, humin is the preferential reaction partner for the formation of

288

bound residues over humic acid and fulvic acid. This may be attributed to the stronger

289

adsorption of TBBPA and its metabolites to humin, which consists of more organic carbon

290

than humic acid and fulvic acid45 and which provides more adsorption sites, as adsorption is

291

the first step in the formation of bound residues via covalent linkages.31,46 Humin fraction may

292

contain black carbon and kerogen,47 which strongly sorb hydrophobic organic pollutants, and

293

therefore could influence their bound-residue formation behavior. Preferential binding of

294

14

295

mineralization of

296

plants, where mineralization of 14C-TBBPA accounts for 3−12%.25

C-TBBPA to humin has also been observed during incubation in anoxic soil, where 14

C-TBBPA is negligible,22 and in submerged soil with growing wetland

297

In active soil, after the rapid initial increase in radioactivity attributed to humin-bound

298

residues, the percentage decreased from 46.4% on day 35 to 28.2% on day 55 and remained

299

almost constant until the end of the incubation (Figure 4a). This decrease suggested that the

300

initially formed humin-bound residues are partially reversibly bound and are bioavailable.

301

The observed decrease in total bound residues after 35 days of incubation (Figure 1) was

302

mainly attributed to the decrease in humin-bound residues, as residues bound to humic acids

303

and fulvic acids increased during the first 55 days of incubation and their levels remained

304

constant thereafter (27−29% and 9−10% for residues bound to humic acids and fulvic acids,

305

respectively, Figure 4a). It has been reported that the release of bound residues from soil is

306

due to microbial activity or alteration of environmental conditions, such as pH, redox

307

potential, and nutrient availability.22,30,33 The decrease of bound residues in active soil after

308

long incubation may be due to microbial activity, which initiates the release of humin-bound 11 / 26



309

residues via attacks by enzymes or desorption by biosurfactants.

310

By contrast, in sterilized soil, the amounts of bound residues in the different humic

311

fractions increased to maximal amounts within 17−35 days and remained constant until the

312

end of the incubation (Figure 4b). The formation of these residues was initiated by abiotic

313

ageing processes and could occur in two phases: initial fast adsorption onto soil matrices,

314

followed by slower diffusion into remote microsites of organic and inorganic soil

315

aggregates.32

316

3.4 Ester- and Ether-Linked Bound Residues

317

The ability of soil to incorporate xenobiotics is based on various mechanisms ranging

318

from adsorption to sequestration to chemical reactions; the latter result in strong covalent

319

bonds.46 The binding mechanisms involve oxidation of xenobiotics to free radicals or

320

quinones that subsequently couple to surrounding soil humic substances via C-O, C-C, and

321

C-N bondings.30,40,48 Phenoxyl radicals generated by birnessite prefer to react with humic

322

acids and are incorporated into humic acids mainly via ester or ether linkages.40 We expected

323

ester- and ether-linked bound residues as TBBPA and most of its metabolites (SI Figure S3)

324

possess phenol moieties.

325

The sequential alkaline hydrolysis of bound residues released the residues that were

326

formed via ester or ether linkages (Figure 5). This indicated that the formation of ester- and

327

ether-linked bound residues of TBBPA and its metabolites within humic substances occurred

328

rapidly and without a lag phase, and ester linkages contributed much more than ether linkages

329

to bound-residue formation. At the end of the incubation, ester- and ether-linked bound

330

residues accounted for 5.0% and 1.3% of the initially applied TBBPA, respectively (Figure 5).

331

The increase in the amount of the ester-linked bound residues during the first 27 days is

332

consistent with the rapid increase in total bound residues in soil (Figure 1a) and can be

333

attributed to microbial activity. By contrast, the amount of ether-linked bound residues

334

increased slowly during the entire incubation (Figure 5). After 27 days of incubation,

335

ester-linked bound residues decreased in active soil (Figure 5), which suggested that they

336

were released due to microbial activity. It has also been observed for other xenobiotics, such

12 / 26


Page 12 of 26

Page 13 of 26


337

as nonylphenol and polycyclic aromatic hydrocarbons, that the proportion of bound residues

338

linked via ester linkages is higher than that of residues linked via ether linkages.34,36 The

339

contribution of ester linkages (0.26−0.61% of residues bound to humic acids) was also higher

340

than that of ether linkages (0.05−0.25%) to bound residues of phenanthrene, anthracene, and

341

pyrene in soil humic acids.34

342

The radioactivity attributed to residues bound via ester and ether linkages accounted for

343

9.6−27.0% of the total bound residues during the incubation (SI Figure S4). This indicated

344

that other mechanisms, such as covalent bonds (e.g., C–C or C–N bonds, or metal bridges

345

between compounds and humic macromolecules via ligand-exchange mechanisms) or

346

sequestration into interlays of clay minerals, play important roles in bound-residue formation.

347

Further investigations are needed to explore the roles of other mechanisms.

348

To obtain further information about the ester-linked bound residues, we analyzed the

349

residues released by alkaline hydrolysis by HPLC−14C-LSC. TBBPA and two metabolites

350

were present in the hydrolysate (Figure 6). The released metabolites consisted of one

351

compound more polar than TBBPA [retention time (tR) 3 min] and one compound less polar

352

than TBBPA (tR 27 min) (Figure 6). Most of the ester-linked bound residues involved the

353

parent compound TBBPA and the polar compound. The less polar metabolite had the same tR

354

as MeO-TBBPA (Figure 2), and the polar compound was not detected in the organic extract

355

of the soil, which indicated rapid binding of the polar compound to humic substances in soil.

356

By contrast, none of the single-ring metabolites identified in the organic extracts (Figure 2)

357

were detected in the hydrolysate. Considering the mineralization (Figure 1), this absence of

358

single-ring metabolites supports the degradation pathway in which TBBPA is mineralized via

359

these metabolites in soil (SI Figure S3). The mineralization suggests that these metabolites

360

may be incorporated into microbial biomass.30 It should be noted that the absence of

361

single-ring metabolites in the ester-linked bound residues does not rule out the formation of

362

their bound residues via other bonds, such as C–C and C–N. During incubation, the amount of

363

ester-linked bound TBBPA increased during the first 27 days of incubation and then

364

decreased slowly (Figure 7). This is in agreement with the dynamics of the total ester-linked

365

bound residues (Figure 5). The amount of bound residues assignable to the more polar

366

compound via ester linkages increased continuously and finally reached a plateau (Figure 7). 13 / 26



367

By contrast, the amount of the ester-linked compound MeO-TBBPA was small and became

368

minor at the end of the incubation (Figure 7), which suggested that hydrophilic compounds

369

are more easily incorporated into soil humic substances via ester linkages.

370

3.5 Environmental Implications

371

This study provides comprehensive information on the fate of TBBPA in native oxic soil

372

and the first chemical structures of bound residues of TBBPA. The results indicated that

373

bound-residue formation is the major mechanism for TBBPA dissipation in oxic soil.

374

Therefore, detailed investigation on their nature is essential to assess environmental risks

375

related to this pollutant. As humin is the humic fraction with the lowest water solubility,

376

formation of residues bound to humin could provide the highest stability for pollutants and

377

could inhibit their transport, biotransformation, and bioaccumulation, thereby posing the

378

lowest risk to the environment. However, the decrease in humin-bound residues during

379

incubation (Figure 4a) and the formation of considerable amounts of hydrolysable and

380

therefore reversible ester-linked bound residues observed in our study (Figure 5) strongly hint

381

at the potential release of bound residues of TBBPA and its metabolites in soil environments.

382

The release of bound residues may pose a threat to the environment because the residues

383

become available to biota and may be accumulated in soil organisms, plants, and animals. The

384

nature of the majority of bound residues of TBBPA and its metabolites is still unclear; further

385

characterization of their binding mechanisms, molecular structure, and stability against

386

microbial activity and environmental condition changes are needed.

387

Acknowledgements

388

This work was supported by National Natural Science Foundation of China (NSFC)

389

(Grant Nos. 21237001, 21477052, 21177057, and 21407075) and Sino-Swiss Science and

390

Technology Cooperation (SSSTC) (Grant No. EG 06-032010), which provided scholarships

391

for F.J. Li, J.J. Wang, B.Q. Jiang, and X. Yang to carry out experiments in Switzerland.

14 / 26


Page 14 of 26

Page 15 of 26


392

Supporting Information

393

Figures and table showing the proposed degradation pathway of TBBPA, identification of

394

metabolites, and amounts of ester- and ether-linked bound residues relative to amounts of

395

total bound residues can be found in the Supporting Information. This material is available

396

free of charge via the Internet at http://pubs.acs.org/.

397

398

References

399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428

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tetrabromobisphenol A dimethyl ether disrupts normal zebrafish (Danio rerio) development and matrix metalloproteinase expression. Aquat. Toxicol. 2010, 100, 255-262. Bollag, J. M.; Myers, C. J.; Minard, R. D. Biological and chemical interactions of pesticides with soil organic matter. Sci. Total Environ. 1992, 123, 205-217. Richnow, H. H.; Seifert, R.; Hefter, J.; Kästner, M.; Mahro, B.; Michaelis, W. Metabolites of xenobiotica and mineral oil constituents linked to macromolecular organic matter in polluted environments. Org. Geochem. 1994, 22, 671-681. Kästner, M.; Nowak, K. M.; Miltner, A.; Trapp, S.; Schäffer, A. Classification and modelling of non-extractable residue (NER) formation of xenobiotics in soil - a synthesis. Crit. Rev. Environ. Sci. Technol. 2014, 44, 1-65. Kästner, M.; Streibich, S.; Bezrer, M.; Richnow, H. H.; Fritsche, W. Formation of bound residues during microbial degradation of [C-14]anthracene in soil. Appl. Environ. Microbiol. 1999, 65, 1834-1842. Gevao, B.; Semple, K. T.; Jones, K. C. Bound pesticide residues in soils: a review. Environ. Pollut. 2000, 108, 3-14. Barriuso, E.; Benoit, P.; Dubus, I. G. Formation of pesticide nonextractable (bound) residues in soil: magnitude, controlling factors and reversibility. Environ. Sci. Technol. 2008, 42, 1845-1854. Richnow, H. H.; Seifert, R.; Hefter, J.; Link, M.; Francke, W.; Schäfer, G.; Michaelis, W. Organic pollutants associated with macromolecular soil organic matter: Mode of binding.Org. Geochem. 1997, 26, 745-758. Martens, D. A. Identification of phenolic acid composition of alkali-extracted plants and soils. Soil Sci. Soc. Am. J. 2002, 66, 1240-1248. Riefer, P.; Klausmeyer, T.; Adams, A.; Schmidt, B.; Schäffer, A.; Schwarzbauer, Z. Incorporation mechanisms of a branched nonylphenol isomer in soil-derived organo-clay complexes during a 180-day experiment. Environ. Sci. Technol. 2013, 47, 7155-7162. An, T. C.; Zu, L.; Li, G. Y.; Wan, S. G.; Mai, B. X.; Wong, P. K. One-step process for debromination and aerobic mineralization of tetrabromobisphenol-A by a novel Ochrobactrum sp. T isolated from an e-waste recycling site. Bioresour. Technol. 2011, 102, 9148-9154. Li, F. J.; Jiang, B. Q.; Nastold, P.; Kolvenbach, B. A.; Chen, J. Q.; Wang, L. H.; Guo, H. Y.; Corvini, P. F. X.; Ji, R. Enhanced transformation of tetrabromobisphenol A by nitrifiers in nitrifying activated sludge. Environ. Sci. Technol. 2015, 49, 4283-4292. Li, C. L.; Ji, R.; Vinken, R.; Hommes, G.; Bertmer, M.; Schäffer, A.; Corvini, P. F. X. Role of dissolved humic acids in the biodegradation of a single isomer of nonylphenol by Sphingomonas sp. Chemosphere 2007, 68, 2172-2180. Li, C. L.; Zhang, B.; Ertunc, T.; Schäffer, A.; Ji, R. Birnessite-induced binding of phenolic monomers to soil humic substances and nature of the bound residues. Environ. Sci. Technol. 2012, 46, 8843-8850. FAL, RAC, FAW. Extraction von Schwermetallen mit Natriumnitrat (1:2.5). Schweizerische Referenzmethoden der Eidgenössischen landwirtschaftlichen Forschungsanstalten. . Eidgenössischen Forschungsanstalt FAL, RAC, FAW.

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[42] Shan, J.; Brune, A.; Ji, R. Selective digestion of the proteinaceous component of humic substances by the geophagous earthworms Metaphire guillelmi and Amynthas corrugatus. Soil Biol. Biochem. 2010, 42, 1455-1462. [43] Gevao, B.; Jones, K. C.; Semple, K. T. Formation and release of non-extractable 14 C-dicamba residues in soil under sterile and non-sterile regimes. Environ. Pollut. 2005, 133, 17-24. [44] Semple, K. T.; Morriss, A. W. J.; Paton, G. I. Bioavailability of hydrophobic organic contaminants in soils: fundamental concepts and techniques for analysis. Eur. J. Soil Biol. 2003, 54, 809-818. [45] Kohl, S. D.; Rice, J. A. The binding of contaminants to humin: a mass balance. Chemosphere 1998, 36, 251-261. [46] Riefer, P.; Klausmeyer, T.; Schäffer, A., Schwarzbauer, J.; Schmidt, B. Distribution, fate and formation of non-extractable residues of nonylphenol isomer in soil with special emphasis on soil derived organo-clay complexes. J. Environ. Sci. Health B 2011, 46, 394-403. [47] Xiao, B. H.; Yu, Z. Q.; Huang, W. L.; Song, J. Z.; Peng, A. P. Black carbon and kerogen in soils and sediments. 2. Their roles in equilibrium sorption of less-polar organic pollutants. Environ. Sci. Technol. 2004, 38, 5842-5852. [48] Dec, J.; Bollag, J.-M. Phenoloxidase-mediated interactions of phenols and anilines with humic materials. J. Environ. Qual. 2000, 29, 665-676.

536

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537

Extractable residues Bound residues 14 CO2 Total recovery

a

Radioactivity of extractable, bound residues and CO2 (% of initial amount)

100 80 60 40 20 0 0

20 40 60 80 100 120 140

b

100 80 60 40 20 0 0

538

20 40 60 80 100 120 140

Incubation time (day)

539

Figure 1. Radioactivity recovered from organic-extractable and bound residues (left y-axis)

540

and 14CO2 (right y-axis) during incubation of 14C-TBBPA in active (a) and sterilized (b) soil.

541

Data are mean values of three individual experiments; bars indicate standard deviation.

542

19 / 26



Page 20 of 26

Radioactivity

TBBPA

day 35

day 143

0

543

5

10

15 20 25 Retention time (min)

30

35

40

544

Figure 2. Radiochromatograms of organic extracts of active soil incubated with 14C-TBBPA

545

for 35 or 143 days.

546 547

20 / 26



Radioactivity of metabolites in extracts (% of initial amount)

Radioactivity of TBBPA in extracts (% of initial amount)

Page 21 of 26

a

100 80 60

Active soil Sterilized soil

40 20 0 0

20 40 60 80 100 120 140

14 TBBPA methyl ether(s) Single-ring metabolites

12

b

10 8 6 4 2 0 0

20 40 60 80 100 120 140


548 549

Figure 3. Degradation of TBBPA in sterilized and active soil during incubation (a), and

550

formation of metabolites in active soil (b). No metabolite was detected in sterilized soil (SI

551

Figure S2). Data are mean values of three individual experiments; bars indicate standard

552

deviation.

553

21 / 26



554 555

Radioactivity in humic fractions (% of initial amount)

50

a

40 30 20 10 0 0

20 40 60 80 100 120 140

50

b

40

Fulvic acids Humic acids Humin

30 20 10 0 0

556

20 40 60 80 100 120 140

Incubation time (day) 14

557

Figure 4. Amounts of bound residues of

C-TBBPA within humic fractions (fulvic acids,

558

humic acids, and humin) during 143 days of incubation in active (a) and sterilized (b) soil.

559

Data are mean values of three individual experiments; bars indicate standard deviation.

560

22 / 26


Page 22 of 26

Page 23 of 26


561

Radioactivity in ester- or ether-linked bound residues (% of initial amount)

562 Ester linkages in active soil Ether linkages in active soil Ester linkages in sterilized soil Ether linkages in sterilized soil

8 6 4 2 0 0

20 40 60 80 100 120 140 Incubation time (day)

563 564

Figure 5. Released radioactivity assignable to ester- and ether-linked bound residues from

565

active and sterilized soils after alkaline treatments. Data are mean values of three individual

566

experiments; bars indicate standard deviation.

567

23 / 26



Active soil Active soil Sterilized soil

TBBPA

Radioactivity

More polar compound MeO-TBBPA

0

5

10 15 20 25 30 35

Retention time (min)

568 569

Figure 6. Radiochromatograms of organic extracts of alkaline hydrolysates of bound residues

570

after incubation of 14C-TBBPA in active soil (red line) and in sterilized soil (blue line) for 27

571

days.

572

24 / 26


Page 24 of 26

Page 25 of 26


Radioactivity of ester-linked bound residues (% of initial amount)

573

574

10 TBBPA More polar compound MeO-TBBPA

8 6 4 2 0 0

20 40 60 80 100 120 140 Incubation time (day)

575

Figure 7. Amounts of TBBPA, the unidentified more polar compound (shown in Figure 6),

576

and MeO-TBBPA that were bound via ester linkages to soil matrices during incubation of

577

14

C-TBBPA in active soil under oxic conditions.

578

25 / 26



Page 26 of 26

579

580

Graphic abstract

581

CO2

80

TBBPA methyl ethers

TBBPA

60 100

40

Bound residues

20

Humic substances

0 0

20

40

60

80 100 120 140


Distribution in ester-linked bound residues (%)

Radioactivity distribution (%)

100

80

More polar compound

60 40 20

TBBPA

0 20

40

60

80 100 120 140


582

26 / 26


Fate of Tetrabromobisphenol A (TBBPA) and Formation of Ester- and

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