S and Nine Novel Analogs in Biological

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Tetrabromobisphenol-A/S and Nine Novel Analogs in Biological Samples from the Chinese Bohai Sea: Implications for Trophic Transfer Aifeng Liu, Guangbo Qu, Miao Yu, Yanwei Liu, Jianbo Shi, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06378 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 23, 2016

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Page 1 of 33

Environmental Science & Technology

Tetrabromobisphenol-A/S and Nine Novel Analogs in Biological

Samples

from

the

Chinese

Bohai

Sea:

Implications for Trophic Transfer

Ai-feng Liua, Guang-bo Qua, Miao Yua, Yan-wei Liua, Jian-bo Shia,b,*, Gui-bin Jianga,c

a

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China b

c

Institute of Environment and Health, Jianghan University, Wuhan 430056, China

College of Resources and Environment, University of Chinese Academy of Sciences,

Beijing 100049, China

* Corresponding author Tel/fax: +86-10-62849129 E-mail: [email protected] (Jian-bo Shi)

1

ACS Paragon Plus Environment


ABSTRACT GRAPHICS

2


Page 2 of 33

Page 3 of 33


Abstract

1 2 3

Tetrabromobisphenol-A/S (TBBPA/S) analogs have raised substantial concern

4

because of their adverse effects and potential bioaccumulative properties, such as

5

TBBPA bis(allyl ether) (TBBPA-BAE) and TBBPA bis(2,3-dibromopropyl ether)

6

(TBBPA-BDBPE). In this study, a comprehensive method for simultaneous

7

determination of TBBPA/S and nine novel analogs, including TBBPA-BAE,

8

TBBPA-BDBPE, TBBPS-BDBPE, TBBPA mono(allyl ether) (TBBPA-MAE),

9

TBBPA

mono(2-bromoallyl

ether)

(TBBPA-MBAE),

TBBPA

10

mono(2,3-dibromopropyl ether) (TBBPA-MDBPE), TBBPS-MAE, TBBPS-MBAE

11

and TBBPS-MDBPE in biological samples was developed. The distribution patterns

12

and trophic transfer properties of TBBPA/S and analogs in various biological samples

13

collected from the Chinese Bohai Sea were then studied in detail. For the first time,

14

TBBPA-MBAE and TBBPS-BDBPE were detected in biological samples and

15

TBBPA-MBAE was identified as a byproduct. The concentrations of TBBPA and

16

analogs ranged from ND (not detected or below the method detection limit) to 2782.8

17

ng/g lipid weight (lw), and for TBBPS and analogs ranged from ND to 927.8 ng/g lw.

18

High detection frequencies (>86%) for TBBPA, TBBPS and TBBPA-MAE,

19

TBBPA-MDBPE, TBBPS-MAE, TBBPS-MBAE and TBBPS-MDBPE were obtained.

20

Meanwhile, TBBPA, TBBPS and these five analogs displayed trophic dilution

21

tendencies due to significantly negative correlations between trophic levels and

22

lipid-corrected concentrations together with the trophic magnification factors (from

23

0.31 to 0.55). The results also indicated the novel TBBPA-MAE, TBBPA-MBAE,

24

TBBPA-MDBPE, TBBPS-MAE, TBBPS-MBAE and TBBPS-MDBPE could be

25

generated not only as byproducts, but also as the probable transformation products of

26

commercial TBBPA/S derivatives.

3



27 28

Page 4 of 33

Introduction Brominated flame retardants (BFRs) are the subject of widespread concern due

29

to their extensive use and potential environmental and health risks.1,

30

Tetrabromobisphenol-A (TBBPA) is one of the most important BFRs, and represents

31

about 60% of the total BFR market. TBBPA and commercially available analogs,

32

such as tetrabromobisphenol-S (TBBPS), TBBPA bis(allyl ether) (TBBPA-BAE),

33

TBBPA

34

bis(2,3-dibromopropyl ether) (TBBPS-BDBPE), are used as additive and reactive

35

BFRs in print circuit boards, plastics, etc.1 The EU risk assessment confirmed the

36

environmental risk of using TBBPA as an additive in BFRs due to their increased

37

leakage potential.3 TBBPA-BAE, TBBPA-BDBPE and TBBPS-BDBPE1, 2 have been

38

found in soil, dust, sewage sludge, and eggs, with the highest observed concentration

39

being 9960 ng/g dry weight (dw).4-8 Since TBBPA has been extensively evaluated for

40

its endocrine disruption effects, neurotoxicity and reproductive-development toxicity,

41

the recently reported toxic assessments for TBBPA derivatives mainly focused on the

42

potential neurotoxic effects of TBBPA-BAE,9 maternal transfer properties of

43

TBBPA-BDBPE10 and the slow liver elimination rate of TBBPA-BDBPE in rats.11

bis(2,3-dibromopropyl

ether)

(TBBPA-BDBPE)

and

2

TBBPS

44

The wide application of TBBPA/S in consumer products has resulted in the

45

occurrence of byproducts or degradation products in the environment as emerging

46

contaminants.12,

47

TBBPS-BDBPE have been identified as novel contaminants and these byproducts

48

have been shown to pose more severe environmental risks than TBBPA-BAE,

49

TBBPA-BDBPE and TBBPS-BDBPE,

50

(TBBPA-MAE),

51

TBBPS mono(allyl ether) (TBBPS-MAE), TBBPS mono(2-bromoallyl ether)

13

Several byproducts of TBBPA-BAE, TBBPA-BDBPE and

TBBPA

such as

mono(2,3-dibromopropyl

TBBPA mono(allyl ether) ether)

4


(TBBPA-MDBPE),

Page 5 of 33


52

(TBBPS-MBAE),

and

TBBPS

mono(2,3-dibromopropyl

ether)

53

(TBBPS-MDBPE).13-15 TBBPA-BAE, TBBPA-BDBPE and TBBPS-BDBPE are

54

linked by the C(aryl)-O-C(alkyl) bond, and cleavage of the O-C(alkyl) bond is quite

55

sensitive to the bacterial biodegradation, UV irradiation and super-reduced

56

conditions.16-18 TBBPA-BDBPE has been observed to transform into TBBPA through

57

hydrolysis in aquatic mesocosms.19 TBBPA/S derivatives have the potential for ether

58

bond cleavage and formation of the mono-modified degradation products or

59

byproducts.13, 14 Based on their physical-chemical properities and primary toxicities,

60

these byproducts or degradation products have been predicted to be more

61

bioaccumulative and toxic than TBBPA/S derivatives.15, 20

62

Dietary intake, especially seafood consumption, is a major pathway of human

63

exposure to some BFRs, metabolites of which have also been found in marine

64

organisms and human breast milk.20-22 Currently, TBBPA/S and analogs have mainly

65

been analyzed in abiotic matrices and river organisms, and only a few studies have

66

been conducted in marine environments.22 Although its concentration is low, TBBPA

67

has been detected in different marine organisms including mollusks, crab, fish and

68

porpoises collected from the North Sea.23 The novel TBBPA/S analogs,

69

TBBPA-MAE,

70

TBBPS-MDBPE were identified in mollusk samples with concentrations ranging

71

from ND (not detected or below the method detection limit) to 4.1 ng/g dw.14, 15

72

However, the bioaccumulation and trophic transfer properties of these novel

73

TBBPA/S analogs in the aquatic food web have never been studied and much about

74

them remains unknown. Moreover, further study of novel TBBPA/S analogs in

75

complex biota matrices has also been impeded by a lack of available methods and

76

pure standards.

TBBPA-MDBPE,

TBBPS-MAE,

5


TBBPS-MBAE

and


Page 6 of 33

77

Various methods have been developed for the detection of TBBPA/S and their

78

derivatives. TBBPA-BDBPE was first detected using a high pressure liquid

79

chromatography–diode array detector (HPLC–DAD); in this research, the high

80

detection limit revealed the need for more sensitive method.24 Gas chromatography

81

coupled with mass spectrometry (GC–MS) was also applied in the analysis of

82

TBBPA-BDBPE;6 however, this method was controversial because of its farfetched

83

mass spectrum explanation.5 The thermolability could induce the decomposition of

84

TBBPA/S and their derivatives in the process of programming temperature increase in

85

GC–MS.1,

86

mass spectrometry (EESI–MS) provided an alternative approach for sensitive analysis

87

of TBBPA-BAE, but this method was only applied for simple matrix analysis, such as

88

water samples.25, 26 Atmospheric pressure photoionization mass spectrometry (APPI–

89

MS)

90

TBBPS-BDBPE detection,5 but the dopant agent and unpopularity of the APPI source

91

made this method be not widely adopted for the further study of TBBPA and TBBPS

92

derivatives.2 With respect to sample extraction and purification, accelerated solvent

93

extraction (ASE),24 Soxhlet extraction,6 liquid-liquid extraction,25,

94

extraction (SPE)5 and gel permeation chromatography (GPC)10 have been used for the

95

analysis of several specific TBBPA/S derivatives. However, the simultaneous

96

determination of TBBPA, TBBPS, and their derivatives and byproducts is still a

97

challenge because their polar diversity increases difficulties in extraction, purification

98

and instrumental analysis. Moreover, there is no analytical method available for

99

determination of some novel TBBPA analogs, such as TBBPA diol, TBBPA

was

2

Recently developed reactive extractive electrospray ionization tandem

sensitive

and

100

mono(2,3-dihydroxylpropyl

101

(TBBPA-MBAE).13

specific

ether)

for TBBPA-BAE,

and

TBBPA

TBBPA-BDBPE

26

solid phase

mono(2-bromoallyl

6


and

ether)

Page 7 of 33


102

In this study, a comprehensive method for simultaneous determination of

103

TBBPA/S and nine analogs in biological samples was developed using a single

104

extract. Using the proposed method, the distribution patterns and tropic transfer

105

properties of these compounds in various biological samples from the Chinese Bohai

106

Sea were studied in detail. For the first time, TBBPA-MBAE and TBBPS-BDBPE

107

were detected in biological samples and TBBPA-MBAE was identified as a byproduct.

108

In addition, the trophic dilution tendencies of TBBPA/S analogs are being presented

109

for the first time.

110 111 112

Materials and Methods Chemicals and Materials. The structures of TBBPA/S analogs are shown in

113

Table

1.

The

byproducts,

114

TBBPA-MDBPE

115

TBBPS-MDBPE (96%), were synthesized in our laboratory and were further

116

characterized by 1H nuclear magnetic resonance (1HNMR) and high resolution mass

117

spectrometry (HRMS).14,

118

HRMS spectra of TBBPA-MBAE are shown in the Figure S1 and S2 (Supporting

119

Information). TBBPA (50 µg/mL in methanol),

120

methanol) and D10-labeled TBBPA (100 µg/mL in acetonitrile) were purchased from

121

Cambridge Isotope Laboratories, Inc. (Massachusetts, USA). TBBPA-BAE (99%)

122

and TBBPA-BDBPE (99%) were purchased from Sigma-Aldrich. TBBPS (98%) and

123

TBBPS-BDBPE (99%) were purchased from Beijing Apis Biotechnology Co., Ltd.

124

All solid standards were dissolved in methanol at the concentration of 100 µg/mL and

125

stored in a refrigerator at 4°C. Methanol, acetone, hexane and methylene dichloride

126

(DCM) were all HPLC grade. Deionized water was generated by a Milli-Q advantage

(98%),

15

TBBPA-MAE

TBBPS-MAE

(99%),

(99%),

TBBPA-MBAE

TBBPS-MBAE

(98%),

(98%)

and

The synthesis procedures, as well as the 1HNMR and

13

C12-labeled TBBPA (50 µg/mL in

7



Page 8 of 33

127

A10 system. Three solid commercial products of TBBPA-BAE and TBBPA-BDBPE

128

were purchased from three BFR factories located in the south coastal area of the

129

Chinese Bohai Sea to test the existence status of the mono-modified byproducts.

130 131

Instrumental Parameters of HPLC–MS/MS and HPLC–DAD. HPLC–ESI–

132

MS/MS (2695 HPLC, Quattro Ultima triple quadrupole mass spectrometer, Waters,

133

Milford, MA) was used for determination of TBBPA, TBBPS, TBBPS-BDBPE,

134

TBBPA-MAE, TBBPA-MBAE, TBBPA-MDBPE, TBBPS-MAE, TBBPS-MBAE

135

and TBBPS-MDBPE under negative ionization mode, and the injection volume was

136

20 µL. HPLC−DAD (Thermo Ultimate 3000, UV wavelength 214 nm) was optimized

137

for detection of TBBPA-BAE and TBBPA-BDBPE, and the injection volume was 30

138

µL. The detailed instrument parameters are described in Table 2 and Supporting

139

Information.

140 141

Sample Collection. The Chinese Bohai Sea, whose total area is 77.3×103 Km2,

142

is located in the northeast China and surrounded by 17 densely populated coastal

143

cities. The coastal region surrounding the Chinese Bohai Sea has convenient

144

transportation, intensive industry and a well-developed economy, and is famous for

145

the Bohai Economic Rim. However, due to anthropogenic activities in the

146

surrounding area, environmental quality has declined with increasing economic

147

development over the past few decades. Relatively high concentrations of persistent

148

organic pollutants and metals have been reported in different mollusks,

149

phytoplankton/seston, zooplankton, invertebrate, fish and seabird species of the

150

Chinese

151

polychlorinated

Bohai

Sea,

such

biphenyls

as (PCB),

polycyclic

aromatic

polybrominated

hydrocarbons

diphenylethers

8


(PAHs), (PBDEs),

Page 9 of 33


152

organochlorine pesticides and mercury.14, 27-32

153

In this study, a total of 97 biological samples (including 5 phycophyta species, 2

154

zooplankton species, 14 invertebrate species and 13 fish species) were collected from

155

the coastal areas of the Chinese Bohai Sea near five cities, Dalian (DL), Huludao

156

(HLD), Tianjin (TJ), Penglai (PL) and Yantai (YT), in July and November of 2012.

157

Detailed information for each sample is presented in Table S1 (Supporting

158

Information). The collected samples were transported to the laboratory on ice and

159

cleaned with tap water and deionized water in the laboratory. The viscera and skin

160

were excised and discarded. Approximately 100−500 g of wet soft tissue of each

161

species collected at a single sampling site was homogenized in a blender to form a

162

composite sample. The samples were freeze-dried, ground and stored at −20 °C before

163

analysis.

164

Sample Preparation. Each sample (1.0 g) was mixed with anhydrous sodium 13

165

sulfate (5g), spiked with

C12-labeled TBBPA (10 ng) surrogate standards, and

166

extracted with 100 mL DCM on an accelerated solvent extractor (Dionex ASE 350) at

167

100 °C and 1500 psi for 12 min with 3 cycles. The extraction solvent was then

168

removed using rotary evaporator and solvent-exchanged to 0.5 mL of DCM/hexane

169

(1/1) and purified using GPC (DCM/hexane, 1/1; 38 g S-X3 biobeads). The first 90

170

mL fraction of GPC obtained after loading the sample was discarded after which 100

171

mL of elution solvent was collected and concentrated to 0.5 mL with a rotary

172

evaporator and a gentle stream of nitrogen. The sample was loaded onto a Supelclean

173

LC-Si cartridge (500 mg, 6 mL) and washed with 6 mL DCM/hexane (1/9, V/V, F1),

174

eluted with 10 mL DCM/hexane (1/3, V/V, F2), 10 mL DCM/hexane (1/1, V/V, F3)

175

and 10 mL NH3·H2O/acetone (0.5%, V/V, F4). TBBPA-BAE and TBBPA-BDBPE

176

existed in fraction F2; TBBPS-BDBPE and a trace amount of TBBPA, TBBPA-MAE,

9



Page 10 of 33

177

TBBPA-MBAE and TBBPA-MDBPE existed in fraction F3; most of TBBPA, TBBPS

178

and the mono-modified byproducts existed in fraction F4. F2, F3 and F4 were each

179

concentrated with a gentle stream of nitrogen. F2 was solvent-changed to 50 µL

180

methanol and analyzed with HPLC-DAD. F4 was solvent-changed to 1 mL

181

DCM/hexane (1/1, V/V), further purified with ENVI-Carb cartridge (500 mg, 6 mL)

182

and eluted with 10 mL NH3·H2O/acetone (0.5%, V/V) which combined with F3 from

183

the LC-Si cartridge. After concentration with a stream of nitrogen, this fraction was

184

solvent changed to 200 µL methanol, spiked with internal standard D10-labeled

185

TBBPA (10 ng) and analyzed with HPLC-ESI-MS/MS.

186 187

Trophic Level (TL) and Trophic Magnification Factor (TMF) Determination.

188

The TL and TMF were calculated based on the stable isotope analysis for δ15N which

189

was determined with a Thermo DELTA V Advantage isotope ratio mass spectrometer

190

interfaced to a Flash EA1112 HT elemental analyzer (Thermo Fisher, USA). The

191

specific details are described in Supporting Information.

192 193

Results and Discussion

194

Optimization of Instrumental Parameters. For ESI−MS/MS parameters’

195

optimization, individual standard solution for each target compound was injected to

196

obtain mass spectra under the negative ionization mode. For TBBPA, TBBPA-MAE,

197

TBBPA-MBAE, TBBPA-MDBPE, TBBPS, TBBPS-MAE, TBBPS-MBAE and

198

TBBPS-MDBPE generated [M-H]- ions as the parent ions. For TBBPS-BDBPE, the

199

parent ion 764.5 ([M-C3H5Br2]-) was generated from ether bond fragmentation. The

200

optimized quantitative and qualitative ions are shown in Table 2.

201

Using

HPLC–ESI–MS/MS,

TBBPA,

TBBPA-MAE,

10


TBBPA-MBAE,

Page 11 of 33


202

TBBPA-MDBPE, TBBPS, TBBPS-MAE, TBBPS-MBAE, TBBPS-MDBPE and

203

TBBPS-BDBPE could be determined simultaneously (Figure 1). Herein, we tried to

204

analyze TBBPA-BAE and TBBPA-BDBPE by APPI-MS of Agilent Technologies

205

(6460 Triple Quadrupole MS/MS) and Waters (Quattro Ultima Triple Quadrupole

206

MS/MS), but the MS signals were very low. After optimization of pretreatment

207

method, HPLC coupled with a UV detector was previously used for the analysis of

208

TBBPA-BAE and TBBPA-BDBPE.33 In this study, by using HPLC-DAD,

209

TBBPA-BAE and TBBPA-BDBPE could be separated completely with other

210

interference peaks specifically and they were quantified by the peak area (UV 214 nm,

211

Figure 1).

212 213

Optimization of the SPE Method for TBBPA/S Analogs by Single Extract.

214

Three kinds of SPE cartridges (SupelcleanTM ENVI-CarbTM, LC-Florisil@, LC-Si,

215

500 mg, 6 mL) were used to evaluate the cleaning and concentration efficiencies

216

after GPC purification. A variety of eluents, including hexane, different proportions

217

of hexane/DCM, DCM, acetone, 0.5% NH3•H2O/acetone were evaluated for the

218

elution of the target compounds from these three cartridges. TBBPA-BAE and

219

TBBPA-BDBPE showed the lowest absorption capacity with the LC-Si cartridges,

220

followed by TBBPS-BDBPE, TBBPA-MDBPE, TBBPA-MBAE and TBBPA-MAE.

221

TBBPA, TBBPS, TBBPS-MAE, TBBPS-MBAE and TBBPS-MDBPE showed the

222

greatest absorption capacity with LC-Si cartridges and could only be eluted using

223

acetone. We minimized the matrix interference that could affect HPLC–DAD signals

224

by using a weak polar eluent, DCM/hexane (1/3, V/V), for the elution of

225

TBBPA-BAE and TBBPA-BDBPE. It was found that 10 mL 0.5% NH3•H2O/acetone

226

could elute the other loaded compounds efficiently, but interferences were also

11



227

eluted from LC-Si cartridges. Therefore, ENVI-CarbTM cartridge was subsequently

228

applied for the concentration and purification of high polar TBBPA, TBBPS and

229

their byproducts. Meanwhile, we found that 20 mL acetone was better than 10 mL as

230

an eluent for the recovery of TBBPA, TBBPS and byproducts. Because phenols are

231

weak acids, a small amount of a weak basic can be helpful for the elution of the

232

target compounds. Thus 10 mL 0.5% NH3•H2O/acetone was sufficient for

233

completely eluting TBBPA, TBBPS and their byproducts. The residue obtained from

234

F3 using LC-Si cartridge could be further purified with an ENVI-CarbTM cartridge to

235

eliminate most pigment and interference. The elution of TBBPS-BDBPE from LC-Si

236

cartridges was better than from ENVI-CarbTM cartridges. The combination of LC-Si

237

and ENVI-CarbTM cartridges provided an efficient pretreatment method for all of the

238

11 compounds. Finally, the purification and concentration for these 11 diversely

239

polar compounds were effectively completed by single ASE extraction.

240 241

Method Performance. TBBPA, TBBPS, TBBPS-BDBPE and their byproducts

242

in samples were identified by retention time, quantitative ions and qualitative ions

243

comparison with the corresponding standards. Quantification of TBBPA,

244

TBBPA-MAE, TBBPA-MBAE and TBBPA-MDBPE, TBBPS, TBBPS-MAE,

245

TBBPS-MBAE, TBBPS-MDBPE and TBBPS-BDBPE in the environmental samples

246

was performed using the internal standard method and corrected using surrogate

247

recovery. The standard curve was established between the peak area ratios of the

248

compounds/internal standard and the concentrations, and was used to quantify the

249

target compounds in collected samples. Procedural blanks, standard solution (10

250

ppb), replicate sample and methanol were tested with each batch of samples to check

251

for potential interference and cross-contamination. The selection of the quantitative

12


Page 12 of 33

Page 13 of 33


252

and qualitative ions of 13C12-labeled TBBPA and D10-labeled TBBPA could avoid the

253

interference between these two substances in MS analysis (detailed discussion in

254

Supporting Information). The instrument detection limits (IDLs) and instrument

255

quantification limits (IQLs) were determined by 3/1 and 10/1 signal to noise value

256

(S/N) separately. The method quantification limits (MQLs) and method detection

257

limits (MDLs) were based on replicate analyses (n = 8) of blank river fish sample

258

spiked at concentrations of 3-5 times of the IQLs and the IDLs, respectively. MQLs

259

were defined as the minimum amount of analyte producing a peak with S/N = 10/1

260

and MDLs were defined as S/N = 3/1. MDLs, MQLs, recoveries and matrix effects

261

are all shown in Table S2. The IDLs of TBBPA, TBBPA-MAE, TBBPA-MBAE,

262


263

TBBPS-BDBPE ranged from 0.02 to 2 pg, and the IQLs ranged from 0.07 to 7 pg

264

(Table

265


266

TBBPS-BDBPE ranged from 0.6 pg/g dw to 100 pg/g dw, and the MQLs ranged

267

from 2.0 pg/g dw to 300 pg/g dw (Table S2).

S2).

The

MDLs

of

TBBPA,

TBBPA-MAE,

TBBPA-MBAE,

268

TBBPA-BAE and TBBPA-BDBPE were quantified with an external method,

269

and the results were not corrected using surrogate recovery. The instrument detection

270

limits (IDLs) of TBBPA-BAE and TBBPA-BDBPE were 300 and 3000 pg, and the

271

instrument quantification limits (IQLs) were 900 and 9000 pg. The MDL and MQL

272

for TBBPA-BAE were 700 pg/g dw and 2,300 pg/g dw which were much better than

273

those of APCI-MS/MS (MDL 20,000 pg/g dw and MQL 50,000 pg/g dw) applied

274

for the analysis of biological samples.14 Meanwhile, the MDL and MQL for

275

TBBPA-BDBPE in biological samples, which benefiting from the optimized

276

pretreatment method, were 6,000 pg/g dw and 20,000 pg/g dw, much lower than the

13



277

previously reported values of 80,000 pg/g dw and 180,000 pg/g dw.14 Thus, it

278

appears optimized pretreatment method can compensate for insufficient sensitivity of

279

the detector.

280

The recoveries ranged from 75% to 106% for Paralichthys olivaceus (Par)

281

samples (n=4) at different spiking amounts (1, 10, 100 and 1000 ng/g dw). The

282

method was further evaluated with five different matrices including Mugil soiuy

283

(MugS), Crassostrea talienwhanensis (Ost), Portunus trituberculatus (Por),

284

Metapenaeus ensis (Met) and Ulva pertusa (Ulv) at a spiking amount of 10 ng/g dw

285

(for TBBPA-BAE and TBBPA-BDBPE were 100 ng/g dw). The recoveries ranged

286

from 65% to 108% and the standard deviations were all less than 10%. The matrix

287

effects ranged from 0.88 to 1.15 at different spiking concentrations (1, 10, 100 and

288

1000 ng/mL), which indicated that signal enhancement or signal suppression for the

289

target compounds could be ignored. These results indicated that we had developed

290

an effective method for the simultaneous analysis of TBBPA, TBBPS, and their

291

derivatives and byproducts in various biological samples. The single extract

292

pretreatment procedure can reduce the amount of sample required for analysis and

293

improve analysis efficiency. This method could also be widely applied for further

294

study of the transportation and transformation of TBBPA/S analogs in the

295

environment.

296 297

TBBPA/S Analogs in Commercial Products. Since they are produced with

298

TBBPA/S as raw materials, the commercial products of BFRs contain hypothetical

299

mono-modified compounds as impurities when the reactions of TBBPA/S with

300

corresponding reagents are incomplete.13 In our previous studies, TBBPA-MAE and

301

TBBPA-MDBPE were identified as the byproducts of commercial TBBPA-BAE and

14


Page 14 of 33

Page 15 of 33


302

TBBPA-BDBPE;14 TBBPS-MAE, TBBPS-MBAE and TBBPS-MDBPE were also

303

identified as byproducts of commercial TBBPS-BDBPE.15 In this study,

304

TBBPA-MAE and TBBPA-MDBPE were also detectable in the three commercial

305

TBBPA-BAE and TBBPA-BDBPE products with average concentrations of 9.2 g/kg

306

and 5.4 g/kg, respectively. For the first time, TBBPA-MBAE was detectable in the

307

three commercial TBBPA-BAE and TBBPA-BDBPE, with average concentrations of

308

10 mg/kg and 5 mg/kg, respectively, concentrations that were much lower than those

309

of TBBPA-MAE and TBBPA-MDBPE in commercial products. The results indicated

310

TBBPA-MBAE was also a byproduct of TBBPA derivatives.

311 312

TBBPA/S Analogs in Biological Samples Collected from the Chinese Bohai

313

Sea. Using the proposed method, 11 target compounds in the collected marine

314

biological samples were determined and reported on a lipid weight (lw) basis (Table

315

S1 and Table 3). The concentrations of individual compound, TBBPA and its analogs,

316

ranged from ND to 2782.8 ng/g lw; for TBBPS and its analogs concentrations ranged

317

from ND to 927.8 ng/g lw. Relative high concentrations of TBBPS were observed,

318

with a concentration range of ND to 927.8 ng/g lw. The concentration ranges of

319

TBBPA-MAE, TBBPA-MBAE, TBBPA-MDBPE, TBBPS-MAE, TBBPS-MBAE and

320

TBBPS-MDBPE were ND to 252.1 ng/g lw, ND to 19.7 ng/g lw, ND to 49.2 ng/g lw,

321

ND to 108.3 ng/g lw, 0.1 to 151.1 ng/g lw and ND to 68.2 ng/g lw, respectively.

322

Except for TBBPA-MBAE, whose detection frequency is 58%, the detection

323

frequencies for TBBPA, TBBPS and the mono-modified byproducts were all higher

324

than

325

concentrations ranged from ND to 898.4 ng/g lw, ND to 2782.8 ng/g lw and ND to

326

55.5

86%.

ng/g

For

lw,

TBBPA-BAE,

respectively.

TBBPA-BDBPE

The

detection

and

TBBPS-BDBPE,

frequencies

15


for

the

TBBPA-BAE,


327

TBBPA-BDBPE and TBBPS-BDBPE were 26%, 4% and 31%, respectively. The

328

lower detection frequencies for these three derivatives, which are directly produced by

329

factories, may be due to two factors: first, the rapid degradation or elimination rates of

330

the derivatives, which have been reported for TBBPA-BDBPE when tested with

331

zebrafish,34 and second, the low detection sensitivity of TBBPA-BAE and

332

TBBPA-BDBPE. The concentration levels and detection frequencies of TBBPA-MAE,

333

TBBPA-MDBPE, TBBPS-MAE, TBBPS-MBAE and TBBPS-MDBPE were higher in

334

this study than those in mollusk samples collected from coastal areas of the Chinese

335

Bohai Sea.14, 15 The detection frequencies of TBBPA, TBBPS and the mono-modified

336

byproducts were comparable to other BFRs that were detected in the same area, such

337

as PBDEs and hexabromocyclododecane (HBCD).31 With the wide use of TBBPA/S,

338

these byproducts have become the prevailing contaminants in this regions and their

339

concentrations are gradually increasing.

340 341

Trophic Transfer of TBBPA, TBBPS and Byproducts. Regression and

342

correlation analysis between the lipid-corrected concentrations and the TLs were

343

conducted for TBBPA, TBBPS, TBBPA-MAE, TBBPA-MDBPE, TBBPS-MAE,

344

TBBPS-MBAE and TBBPS-MDBPE (detection frequency >86%) using 86 samples

345

with more than 2 replicate samples. In case of the values lower than MDLs and

346

between MDLs and MQLs, the concentrations were treated as half of MDLs and half

347

of MQLs, respectively. In Figure 2, negative correlations were observed between the

348

concentrations of TBBPA, the total concentration of TBBPA byproducts, TBBPS, the

349

total concentration of TBBPS byproducts and the TLs. The total concentration of

350

TBBPA-BAE and TBBPA-BDBPE increased with the trophic level between TL 0.7

351

to 2.7, but decreased between TL 2.7 to 3.2. The concentration of TBBPS-BDBPE

16


Page 16 of 33

Page 17 of 33


352

remained stable and no significant correlation between TL was observed. The levels

353

of target compounds detected in mollusk and plant samples were higher than those

354

found in fish samples. As shown in Figure 3, logarithmic lipid-normalized

355

concentrations of all target compounds decreased significantly with increasing TLs

356

and the correlations between these two factors were significant (p < 0.05, Table S3.).

357

The TMF values for the target compounds ranged from 0.31 to 0.55, as shown in

358

Figure 3. These results indicated that TBBPA, TBBPS and their byproducts undergo

359

trophic dilution in the Chinese Bohai Sea food web, which is similar to a number of

360

other organic pollutants including tris(2,3-dibromopropyl) isocyanurate, HBCD,

361

PAHs, dibenzo-p-dioxin (PCDD), hexachlorocyclohexane and PBDEs.28-31

362

The biomagnification potential of pollutants is often evaluated using the

363

octanol-water partition coefficient (log Kow), which is estimated with the Estimation

364

Programs Interface (EPI) Suite software.8, 13, 30, 31 Generally, a compound with log

365

Kow >5 is considered to be potentially bioaccumulative.8, 13, 35, 36 Although the log Kow

366

values of the mono-modified byproducts were lower than the corresponding

367

derivatives, they all showed log Kow >5, indicating their potential bioaccumulative

368

abilities.8 For TBBPA, TBBPS and the byproducts, the log Kow ranged from 5.21 to

369

9.3615 and no significant relationship was observed between log Kow values and TMFs.

370

However, some other POPs with comparable log Kow values, such as DDT,

371

hexachlorobenzene and non- and mono-ortho PCBs, exhibited positive correlation

372

with TLs in the Chinese Bohai Sea marine food web: the TMF values of these

373

substances were all higher than 1.27, 28 These results indicate that the log Kow is not the

374

only factor influencing trophic transfer behavior of TBBPA, TBBPS and their

375

byproducts, and that there are other determining factors at work, such as assimilation

376

efficiencies, elimination rate, degradation rate and metabolic transformation.

17



377 378

Environmental Significance. The correlation analyses between TBBPA, TBBPS

379

and the byproducts revealed that these similarly structured compounds have similar

380

sources and similar environmental fates. The Spearman correlation analyses for

381

TBBPA, TBBPA-MAE, TBBPA-MDBPE, TBBPS, TBBPS-MAE, TBBPS-MBAE

382

and TBBPS-MDBPE were conducted and the p values of Spearman’s rank correlation

383

test were shown in Table S3. Concentrations of TBBPA were significantly correlated

384

with that of TBBPA-MAE (p 424.6

45

550.6->422.6

45

TBBPA- C12

45

554.6->430.6

45

556.6->432.6

45

TBBPS

50

564.6->249.8

47

564.6->78.9

65

TBBPA-MAE

35

582.7->526.6

43

580.7->524.6

43

TBBPA-MBAE

40

660.7->526.6

45

662.7->526.6

45

TBBPA-MDBPE

50

742.6->526.6

50

740.6->524.6

50

TBBPS-MAE

35

604.7->563.6

38

602.7->561.6

38

TBBPS-MBAE

35

682.6->563.6

30

684.6->563.6

30

TBBPS-MDBPE

60

764.5->563.6

35

762.6->561.6

35

TBBPS-BDBPE

60

764.5->563.6

35

762.6->561.6

35

Compounds

TBBPA TBBPA-D10 13

Cone (V)

629

28


Page 29 of 33


630

Table 3. Descriptive statistics of the target TBBPA/S and analogs in the collected

631

marine biological samples.

632 633 634

TBBPA

Con. Range (ng/g lw) ND-207.3

Media (ng/g lw) 7.3

Geometric Mean (ng/g lw) 6.9

Mean (ng/g lw) 24.2

Detection Frequency 87%

TBBPA-MAE

ND-252.1

4.6

4.8

4.8

98%

TBBPA-MBAE

ND-19.7

0.1

0.1

0.1

58%

TBBPA-MDBPE

ND-49.2

3.1

2.9

2.9

95%

TBBPS

ND-927.8

21.8

15.8

15.8

97%

TBBPS-MAE

ND-108.3

2.9

2.1

2.1

87%

TBBPS-MBAE

0.1-151.1

5.6

5.5

5.5

100%

TBBPS-MDBPE

ND-68.2

2.5

2.7

2.7

97%

TBBPA-BAE

ND-898.4

NQ

8.9

8.9

26%

TBBPA-BDBPE

ND-2782.8

NQ

39.1

39.1

4%

TBBPS-BDBPE

ND-55.5

NQ

0.3

0.3

31%

NQ: not quantified. For the statistical analysis, ND was treated as half value of MDL and NQ was treated as half value of MQL.

29



635

Figure Captions

636 637

Figure 1. HPLC-ESI--MS/MS (A, B) and HPLC-UV (C, D, 214 nm) chromatograms

638

of TBBPA/S and nine analogs. A: standard solution (2 ng/mL). B: Grateloupia

639

ramosissima sample collected from Huludao. C: standard solution (1 µg/mL). D:

640

Portunus trituberculatus sample collected from Dalian.

641 642

Figure 2. Concentrations (ng/g lw) distribution for TBBPA/S analogs with trophic

643

levels. ∑TBBPA-MXXX is the total concentration of TBBPA-MAE, TBBPA-MBAE

644

and TBBPA-MDBPE; ∑TBBPS-MXXX is the total concentration of TBBPS-MAE,

645

TBBPS-MBAE and TBBPS-MDBPE; ∑TBBPA-BXXX is the total concentration of

646

TBBPA-BAE and TBBPA-BDBPE.

647 648

Figure 3. Logarithmic concentrations (ng/g lw) of target compounds and trophic level

649

relationships for marine biological samples from the Chinese Bohai Sea. Linear

650

regression lines were used for TMF calculation.

30


Page 30 of 33

Page 31 of 33


A

TBBPS-MDBPE TBBPA-MBAE TBBPS-MBAE TBBPS-MAE

TBBPA-MDBPE TBBPS-BDBPE TBBPA-MAE

TBBPS TBBPA

B TBBPS-MDBPE

TBBPS TBBPS-MBAE

TBBPA-MBAE TBBPA-MDBPE TBBPA-MAE

TBBPS-MAE TBBPA

1

3

5

7

9

11

13

C

TBBPA-BAE TBBPA-BDBPE

TBBPA-BAE TBBPA-BDBPE

3 651

4

5

6

7

D

8

9

10

Time (min)

652

Figure 1. HPLC-ESI--MS/MS (A, B) and HPLC-UV (C, D, 214 nm) chromatograms

653

of TBBPA/S and nine analogs. A: standard solution (2 ng/mL). B: Grateloupia

654

ramosissima sample collected from Huludao. C: standard solution (1 µg/mL). D:

655

Portunus trituberculatus sample collected from Dalian.

31



Page 32 of 33

656

920 TBBPA ∑TBBPA-MXXX TBBPS ∑TBBPS-MXXX ∑TBBPA-BXXX TBBPS-BDBPE

Concentration (ng/g lw)

910

900 400

200

0 0.7 - 1.2

1.2 - 1.7

1.7 - 2.2

2.2 - 2.7

2.7 - 3.2

3.2 - 4.2

Trophic Level 657 658

Figure 2. Concentration (ng/g lw) distribution for TBBPA/S analogs with trophic

659

levels. ∑TBBPA-MXXX is the total concentration of TBBPA-MAE, TBBPA-MBAE

660

and TBBPA-MDBPE; ∑TBBPS-MXXX is the total concentration of TBBPS-MAE,

661

TBBPS-MBAE and TBBPS-MDBPE; ∑TBBPA-BXXX is the total concentration of

662

TBBPA-BAE and TBBPA-BDBPE.

32


Page 33 of 33


Y=4.61-1.10X, r2=0.31, TMF=0.33

Y: Ln Concentration (ng/g lw)

6

Y=4.35-1.10X, r2=0.37, TMF=0.33

6

4

4

4

2

2

2

0

0

0

-2

TBBPA 1

-2 2

3

Y=4.99-0.86X, TMF=0.42

6

4

r2=0.36,

TBBPA-MAE 1

2

-2 3

Y=3.80-1.17X, TMF=0.31

6

Y=3.01-0.59X, r2=0.36, TMF=0.55

6

TBBPA-MDBPE 1

4

r2=0.33,

2

3

Y=3.74-0.77X, TMF=0.46

4

r2=0.33,

Y=2.60-0.65X, r2=0.44, TMF=0.52

4

4

4

2

4

2

2

0

2 0 0

-2

0

-2 -2

-4 TBBPS-MAE

TBBPS 1

2

3

4

1

2

-2

3

1

4

TBBPS-MDBPE

TBBPS-MBAE 2

3

4

1

2

3

4

X: Trophic Level 663 664

Figure 3. Logarithmic concentrations (ng/g lw) of target compounds and trophic level relationships for marine biological samples from the

665

Chinese Bohai Sea. Linear regression lines were used for TMF calculation.

33


S and Nine Novel Analogs in Biological

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