Tracing the Biotransformation of PCBs and PBDEs in Common Carp

Subscriber access provided by Fudan University

Article

Tracing the Biotransformation of PCBs and PBDEs in Common Carp (Cyprinus carpio) Using Compound-specific and Enantiomer-specific Stable Carbon Isotope Analysis Bin Tang, Xiaojun Luo, Yan-Hong Zeng, and Bi Xian Mai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05130 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Environmental Science & Technology

Tracing the Biotransformation of PCBs and PBDEs in Common Carp (Cyprinus carpio) Using Compound-specific and Enantiomer-specific Stable Carbon Isotope Analysis Bin Tang †, ‡, Xiao-Jun Luo †,*, Yan-Hong Zeng †, Bi-Xian Mai †

†

State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of

Environmental Resources Utilization and Protection, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, P. R. China ‡

University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

* Corresponding author Phone: +86-20-85297622; Fax: 86-20-85290706; E-mail address: [email protected] .

1

ACS Paragon Plus Environment


Table of Contents/ Abstract Art

(6.00cm × 4.50cm)

2


Page 2 of 34

Page 3 of 34


1

ABSTRACT

2

Compound-specific and enantiomer-specific carbon isotope composition was

3

investigated in terms of biotransformation of polychlorinated biphenyls (PCBs) and

4

polybrominated diphenyl ethers (PBDEs) as well as atropisomers of chiral PCB

5

congeners in fish by exposing common carp (Cyprinus carpio) to certain PCB and

6

PBDE congeners. The calculated carbon isotope enrichment factors (εC) for PCBs 8,

7

18, and 45 were -1.99‰, -1.84‰, and -1.70‰, respectively, providing evidence for

8

the metabolism of these congeners in fish. The stable carbon isotopic compositions of

9

PBDE congeners clearly reflect the debromination of PBDEs in carp. Significant

10

isotopic fractionation was also observed during the debromination process of BDE

11

153 (εC = -0.86‰). Stereoselective elimination of the chiral PCB congeners 45, 91,

12

and 95 was observed, indicating a stereoselective biotransformation process. The

13

similar εC for E1- (-1.63‰) and E2-PCB 45 (-1.74‰) indicated that both atropisomers

14

were metabolized by the same reaction mechanisms and stereoselection did not occur

15

at carbon bond cleavage. However, the εC values of (+)-PCB 91 (-1.5‰) and (-)-PCB

16

95 (-0.77‰) were significantly different from those of (-)-PCB91 and (+) PCB95,

17

respectively. In the latter, no significant isotopic fractionations were observed,

18

indicating that the stereoselective elimination of PCBs 91 and 95 could be caused by a

19

different reaction mechanism in the two atropisomers.

20

3



21

Page 4 of 34

INTRODUCTION

22

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs)

23

are two kinds of anthropogenic organic compounds, which are well known for their

24

persistence, bioaccumulation, long-range transport potential, and toxicity1, 2. Because

25

of these characteristics, PCBs and components of the penta-BDE and octa-BDE

26

technical mixtures have been regulated by the Stockholm Convention on persistent

27

organic pollutants (POPs)3, 4.

28

Despite their persistence in the environment, PCBs and PBDEs can undergo

29

biotransformation in wildlife and humans, and generate metabolites that may be more

30

toxic than their parent compounds, resulting in a serious threat to the biota5, 6. To date,

31

several studies have demonstrated the biotransformation of PCBs in fish7-9, although

32

fish are considered inefficient in the biotransformation of PCBs compared to birds and

33

mammals10,

34

(MeSO2-PCBs), two kinds of PCB metabolites, have been detected in fish7,

35

Additionally, biotransformation of PBDEs in fish was reported in previous studies13-15.

36

Fish have not demonstrated much ability to form hydroxylated PBDEs (OH-PBDEs);

37

however, they have reductively debrominated PBDEs both in vivo and in vitro, and

38

species-specific differences in metabolic rates and products were observed14, 15.

11

.

Hydroxylated

PCBs

(OH-PCBs)

and

methylsulfone

PCBs 12

.

39

During recent decades, compound-specific isotope analysis (CSIA) has

40

undergone rapid development, which has led to important applications for the

41

assessment of the origin and degradation of organic compounds in the environment16.

42

The concept of isotope fractionation relies on the observation of shifts in ratios of

4


Page 5 of 34


43

stable isotopes caused by the breakage of chemical bonds during bio-/chemical

44

transformations, which can lead to the enrichment of heavier isotopes in the residual

45

fractions and their depletion in the degradation products16-18. The extent of stable

46

isotope fractionation allows for the qualitative and quantitative assessment of

47

pollutant biodegradation, as well as the elucidation of the reaction mechanism19. The

48

use of CSIA for environmental investigation has been applied to study the

49

biotransformation and trophic dynamics of PCBs and PBDEs in fish in our previous

50

studies17, 20-22.

51

Of the 209 PCB congeners, a group of 19 congeners are axially chiral and form

52

stable atropisomers under ambient conditions 23. The stereoisomeric patterns of chiral

53

PCBs (described as enantiomeric fractions, EFs) is a useful tool for characterizing

54

biochemical processes23 because biological reactivity would lead to a preferential

55

biotransformation of individual atropisomers. Stereoselective biotransformation of

56

chiral PCBs has been observed in fish7, 9, 20. However, little information is available

57

regarding the mechanisms of stereoselective biotransformation. In recent years,

58

enantiomer-specific isotope analysis (ESIA) has become a promising new approach

59

that could provide insight into stereoselective fate and source apportionment of

60

environmental

61

biochemical enzymatic reaction mechanisms during biotransformation19. The ESIA

62

approach has been applied in the study of stereoselective biodegradation of

63

α-hexachlorocyclohexane (α-HCH), polar herbicides (phenoxy acids), and galaxolide

64

and phenoxy alkanoic methyl herbicides24. Currently, no study has been conducted to

organic

contaminants24,

and

provide

5


information

regarding


65

investigate the changes in stable isotope signatures of atropisomers of chiral PCB

66

congeners during the biotransformation processes.

67

In the present study, a dose of certain PCB and PBDE congeners was

68

administered to common carp (Cyprinus carpio) via their diet for 28 days. This was

69

followed by a depuration period of 84 days, during which the carp were fed

70

unfortified, non-spiked food. The primary objective of this study was to trace the

71

biotransformation of PCBs and PBDEs in fish using CSIA. We further investigated

72

the changes in atropisomeric composition of chiral PCB congeners and the carbon

73

stable isotope fractionation of individual atropisomers to verify the stereoselective

74

biotransformation and its mechanism using ESIA.

75

EXPERIMENTAL SECTION

76

Standards and Reagents. Nineteen PCB congeners ( PCBs 8, 18, 20, 31, 44, 45,

77

49, 52, 91, 95, 101, 132, 138, 149, 174, 177, 180, 183, and 187) and five PBDE

78

congeners (BDEs 85, 99, 100, 153, and 154) were obtained from AccuStandard (New

79

Haven, CT, USA). MeSO2-PCB, OH-PCB, OH-PBDE, methoxy PCB (MeO-PCB),

80

and methoxy PBDE (MeO-PBDE) standards (given in details in supporting

81

information, SI) were obtained from Wellington Laboratories (Guelph, ON, Canada).

82

Pesticide grade acetone (Ace), dichloromethane (DCM), and n-hexane (Hex) were

83

purchased from CNW Technologies GmbH (Dusseldorf, Germany). Guaranteed

84

reagent grade concentrated sulfuric acid (H2SO4) and anhydrous sodium sulfate were

85

acquired from Guangzhou Chemical Reagent Factory (Guangzhou City, China).

6


Page 6 of 34

Page 7 of 34


86

Food Preparation. Commercial fish food (protein > 40% and crude fat > 4.5%)

87

was obtained from Zhongshan President Enterprise Co., Ltd. (Guangdong, China).

88

Approximately 400 – 450 µg of each PCB congener (PCBs 8, 18, 20, 31, 44, 45, 49,

89

52, 91, 95, 101, 132, 138, 149, 174, 177, 180, 183, and 187) and 1.8 mg of each

90

PBDE congener (BDEs 85, 99, 100, 153, and 154) were first dissolved in 10 g of cod

91

liver oil (Peter Moller, Norway), which was then mixed with 140 g of fish food pellets.

92

The initial concentrations of each PCB and PBDE congener in the food were 2.8–3.2

93

µg g-1 and 12.9 µg g-1 dry weight (dw), respectively. Non-spiked food, which was

94

used for the depuration phase and control group, was treated in an identical manner

95

but without addition of PCBs and PBDEs. Food was homogenized by mixing in a

96

shaking incubator (24 h, 20°C), and then air-dried for 24 h and stored in the dark at

97

-20°C in amber stopper-sealed jars throughout their use. Spiked food samples were

98

collected at the beginning and end of the feeding intervals to confirm the associated

99

PCB and PBDE concentrations.

100

Exposure and Sampling. Sixty-four common carp with average initial weights

101

and lengths of 17.1 ± 2.7 g (mean ± SD, similarly hereafter) and 10.3 ± 0.5 cm,

102

respectively, were purchased from a local aquarium market in Guangzhou, China. At

103

the beginning of the experiment, six fish were removed as background samples. The

104

remaining fish (n = 58) were randomly distributed between two rectangular glass

105

aquariums (150 cm × 45 cm × 100 cm). One was designated as control group (n = 18),

106

in which fish were fed non-spiked food throughout the experiment. The other

107

aquarium was the treated group (n = 40). Each tank was filled with filtered 7



108

dechlorinated tap water, which was maintained at a temperature of 22 ± 1°C and

109

circulated using a submerged pump at a rate of 2.5 L min-1. The water in each

110

aquarium was gently aerated to maintain oxygen saturation under a 12-h light and

111

dark photoperiod cycle. Fish were acclimated to the non-spiked diet in the laboratory

112

for two weeks prior to exposure, and were fed food at a rate of 1% of their average

113

body weight per day.

114

After 28 days of exposure (uptake period), the fish were fed non-spiked food for

115

84 days (depuration period). Fish were sampled on days 0, 7, 21, and 28 of the uptake

116

period, and on days 14, 28, 42, 56, 70, and 84 of the depuration period. On each

117

sampling day, four fish were randomly chosen from the exposed group to determine

118

their fork length and weight. Blood samples were obtained from the dorsal aorta using

119

syringes, transferred into 5-mL Teflon tubes, and centrifuged at 3000 rpm for 30 min

120

to obtain the serum. Then, the fish were dissected and separated into the gill, liver,

121

gonad, gastrointestinal tract (GI, including the stomach and intestines with undigested

122

food removed, spleen, kidney, heart and adipose fat associated with these organs), and

123

carcass (whole fish minus gill, gonad, liver and GI tract). The gill, liver, gonad, and

124

GI of fish sampled on the same day were weighed and respectively pooled to form

125

two composite samples, the sera were pooled into one sample, whereas carcass was

126

combined correspondingly to form two samples prior to extraction for CSIA. All

127

samples were freeze-dried, ground into powder, weighed, and stored at -20°C prior to

128

being analyzed. Similarly, two fish were randomly collected from the control group

129

on each sampling day, and treated in the same way as those in the exposure group. 8


Page 8 of 34

Page 9 of 34


130

Sample Preparation and Extraction. The gill, liver, gonad, GI, and one-tenth

131

of the carcass were used for PCB, PBDE, and MeSO2-PCB quantification analysis,

132

and the sera were analyzed for PCBs, PBDEs, OH-PCBs, and OH-PBDEs. The

133

remainder of the carcass material sampled on the same day was respectively

134

combined into two samples and used for compound-specific stable carbon isotope

135

analysis.

136

The extraction and quantification analysis procedures used for fish tissues (serum,

137

gill, liver, gonad, GI, and carcass) were similar to those described in previous

138

studies12, 25, with minor modifications, and given in details in the SI.

139

Approximately 10 g dry weight for each sample was used for stable carbon

140

isotope analysis. The method for purification of PCBs and PBDEs in fish for CSIA

141

was based on our previous studies20,

142

descriptions of the sample extraction and cleanup procedures are given in the SI. No

143

significant isotope fractionation of the target compounds was observed during the

144

purification process20, 21.

21

, with minor modifications. Detailed

145

Instrumental Analysis. Determination of PCBs was performed using an Agilent

146

7890A gas chromatography (GC) coupled with a 5975C mass selective detector (MS)

147

in an electron impact (EI) ion source mode. A DB-5 MS column (60 m × 0.25-mm i.d.

148

× 0.25-µm film thickness) was used for PCB separation. A Chirasil-Dex column (25

149

m × 0.25-mm i.d. × 0.25-µm film thickness) was used to separate PCB 91, 95, 132,

150

149, and 174 atropisomers. A BGB-172 column (30 m × 0.25-mm i.d. × 0.18-µm film

9



151

thickness) was used to separate PCB 183 atropisomers, and a Cyclosil-B column (30

152

m × 0.25-mm i.d. × 0.25-µm film thickness) was used to separate PCB 45

153

atropisomers. Chiral PCB compositions were expressed as EFs, which were defined

154

as follows: EF =

155

A A+B

156

where A and B represent the areas of the (+)- and the (-)-atropisomer peaks in the

157

stereoselective chromatograph column, respectively, for PCBs 91, 95, 132, 149, and

158

for the first-eluting (E1) and second-eluting (E2) atropisomers, respectively, for PCBs

159

45, 174 and 183. The (-)-atropisomer elutes first for PCBs 95, 132, 136, and 149, and

160

the (+)-atropisomer elutes first for PCB 9126, 27, whereas the eluting orders for PCBs

161

45, 174 and 183 atropisomers were unknown. The oven temperature programs are

162

given in detail in the SI.

163

PBDEs, MeSO2-PCBs, OH-PCBs, and OH-PBDEs (OH-PCBs and OH-PBDEs

164

were derivatized to their methoxy analogues by diazomthane before instrumental

165

analysis, details in SI) were analyzed using an Agilent 6890N GC coupled with a

166

5975B MS in the electron capture negative ionization (ECNI) mode, and were

167

separated with a DB-XLB capillary column (30 m × 0.25-mm i.d. × 0.25-µm film

168

thickness). Details of the GC conditions and oven temperature programs are given in

169

the SI. Quantification was based on internal calibration curves constructed from

170

standard solutions at eight concentrations.

171

GC-C-Isotope Ratio Mass Spectrometry (IRMS) Analysis. The purities of the

172

extracts used for CSIA were first checked using an Agilent 7890A GC-5975B MS 10


Page 10 of 34

Page 11 of 34


173

system with the EI ion source in the full scan mode. A secondary Aroclor mixture

174

1242/1248/1254/1260 (1:1:1:1) and penta-BDE mixture (DE-71) were used as

175

standards for the qualitative analysis of PCBs and PBDEs, respectively. The

176

individual congeners of the PCBs and PBDEs were identified by comparing the mass

177

spectrum and the retention time of the target compounds with the calibration

178

standards.

179

CSIA measurements of PCBs and PBDEs were performed using a method

180

similar to that used in our previous study22, with minor modifications. The same

181

columns were used for CSIA as those used for quantification analysis. Detailed

182

descriptions of the CSIA procedures are given in the SI. A co-injected standard,

183

2,4,6-trichlorobiphenyl (PCB 30), whose δ13C value was first determined offline with

184

a Flash 2000 EA-Delta V Plus IRMS (Thermo-Fisher Scientific, USA) and was

185

spiked into the extract before conducting the CSIA. The online-measured δ13C value

186

for PCB30 (-29.14‰ to -28.86‰) was close to the offline-measured value (-28.80‰),

187

indicating data reliability.

188

Bioaccumulation Parameters. All concentrations in fish tissues and serum were

189

lipid-based. The bioaccumulation parameters, including assimilation efficiencies (αa),

190

depuration rate constants (kd), half-lives (t1/2), and biomagnification factors (BMFs) of

191

PCB and PBDE congeners were calculated according to equations similar to those

192

described in a previous study8, and given in detail in the SI.

193

Carbon Stable Isotope Calculations. The carbon isotope ratios were reported in 11



Page 12 of 34

194

δ notation in parts per thousand (‰) relative to the international carbon isotope

195

standard Vienna Pee Dee Belemnite (V-PDB), according to the following equation

196

(Eqn. (1)):

δ13C =

197

R sample − R standard R standard

×1000

(1),

198

where Rsample and Rstandard represent the 13C/12C ratios of the sample and the V-PDB

199

standard for the carbon isotopic analysis, respectively.

200 201

202

The Rayleigh equation was used to quantify isotope fractionation upon biodegradation:

R  C  ln  t  = ( α-1) ln  t   R0   C0 

(2), 13

C/12C) of the target

203

where Rt and R0 are the isotopic compositions (ratio of

204

compounds at time t and time 0 of the depuration period, α is the carbon isotope

205

fractionation factor, and Ct and C0 are the concentrations of the substrate at time t and

206

time 0 of the depuration period, respectively. The carbon isotope enrichment factor ε

207

was calculated according to Eqn. (3):

208

ε = ( α-1) × 1000

(3)

209

Statistical Analysis. All data are presented as means ± standard deviations

210

unless otherwise specified. Statistical analyses were performed using the SPSS 21

211

software for Windows (SPSS). The level of significance was set at p = 0.05

212

throughout the study. The statistical differences in the EFs of chiral PCBs and δ13C

213

values between different groups of samples were determined by one-way analysis of

214

variance (ANOVA) with Tukey’s post-hoc test.

12


Page 13 of 34


215

RESULTS AND DISCUSSIONS

216

Background Levels and Quality Control. No natural mortality was observed

217

throughout the experiment period. BDEs 47, 100, 153, and 154 were detected in

218

background common carp at concentrations ranging from 5.8 ± 1.2 to 11.0 ± 2.3 ng

219

g-1 lipid weight (lw), 7.2 ± 1.5 to 23.2 ± 1.6 ng g-1 lw, 1.2 ± 0.52 to 15.4 ± 0.08 ng g-1

220

lw, and 2.9 ± 0.75 to 9.5 ± 0.97 ng g-1 lw in fish tissues, respectively. All 19 PCB

221

congeners

222

concentrations of PCB congeners in the carcass, liver, gonad, GI, gills, and serum

223

ranged from 1.3 ± 0.68 to 14.3 ± 2.8 ng g-1 lw, 2.3 ± 1.3 to 23.4 ± 4.5 ng g-1 lw, 1.4 ±

224

0.72 to 12.1 ± 2.4 ng g-1 lw, 1.3 ± 0.70 to 11.6 ± 2.3 ng g-1 lw, 1.2 ± 0.62 to 5.8 ± 1.1

225

ng g-1 lw, and 2.5 ± 0.78 to 8.1 ± 0.05 ng g-1 lw, respectively. No OH-PCBs,

226

OH-PBDEs, or MeSO2-PCBs were detected in the background or control samples at

227

the beginning or end of the experiment. The PCB and PBDE levels in the background

228

samples were two to three orders of magnitude lower than those in the exposed fish.

229

And spiking test using these background fish confirmed that the influence of

230

background PCBs and PBDEs on the isotopic composition of PCBs and PBDEs in the

231

exposure group was negligible (Table S1). The concentrations of PCB and PBDE

232

congeners in the spiked food pellet homogenate were ranged from 2.9 ± 0.06 to 3.3 ±

233

0.13 µg g-1 dw and 12.8 ± 0.20 to 12.9 ± 0.29 µg g-1 dw, respectively, which were

234

very close to the nominal concentrations. More details regarding quality assurance

235

and control are given in the SI.

236

were

detected

in

background

common

carp.

The

background

Bioaccumulation Parameters of PCB and PBDE. The uptake curves were 13



237

similar for most PCB congeners. All congeners reached their highest concentrations at

238

the end of the uptake period (28 d) (Figure S1), and no steady state was observed

239

during the 28-d exposure. The elimination of PCB congeners in all tissues followed

240

first order depuration kinetics. The uptake and depuration kinetics constants,

241

including assimilation efficiencies (αa), depuration rate constants (kd), half-lives (t1/2),

242

and biomagnification factors (BMFs), of PCB congeners in the carcass, liver, gonad,

243

GI, gill, and serum were calculated (Table S2).

244

The calculated bioaccumulation parameters of PCBs in the present study were

245

quite similar to those reported by Fisk et al.8 in juvenile rainbow trout (Oncorhynchus

246

mykiss). Assimilation efficiencies of PCB congeners in liver (63–86%) and GI

247

(43–57%) were much higher than those in the carcass (28–41%), gonad (26–33%),

248

gill (15–23%), and serum (21–36%). The liver exhibited the highest assimilation

249

efficiencies (Table S2), which could be related to the fact that liver is the first organ in

250

which contaminants deposit after absorption from the GI28. The depuration rates were

251

fastest in the liver and slowest in the carcass. The rapid elimination rate in the liver

252

resulted because it is a rich blood-perfused organ and the main organ for the

253

metabolism of xenobiotic chemicals. The poor blood-perfusion in muscle could be

254

partly responsible for the low-elimination rate of chemicals in the carcass. The

255

depuration rates of PCBs 8, 18, and 45 were 2 or 3 times that of other PCB congeners

256

and the BMF of these three congeners was less than 1 (for other congeners >1). This

257

result indicated that there was another elimination pathway, such as metabolism, for

258

these chemicals (discussed below, this section). 14


Page 14 of 34

Page 15 of 34


259

Exclusive of the five spiked PBDE congeners, five metabolic debromination

260

congeners (BDEs 28, 47, 49, 66, and 101) were also detected in fish tissues (Figure

261

S2). Previous studies have demonstrated that BDE 85, BDE 99, and BDE 153 can

262

debrominate to lower brominated congeners, such as BDE 101, BDE 47, BDE 49, and

263

BDE 6614, 15, and BDE 66 can be further debrominated to BDE 2829, whereas BDE

264

100 and BDE 154 were resistant to metabolic debromination in the common carp14, 15.

265

Our results further supported this finding. Although the five spiked PBDE congeners

266

had similar concentrations in the spiked food, the levels of BDE 100 and BDE 154

267

were more than two times those of BDE 153 and were 2 to 3 orders of magnitude

268

higher than those of BDE 85 and BDE 99. Conversely, BDE 47, the potential

269

debrominated product of BDEs 85, 99, and 15315, had the highest concentration in

270

carp tissues. After a 14-d depuration, BDE 85 could not be detected and BDE 99 was

271

at a very low concentration (< 32 ng g-1 lw) in carp.

272

The assimilation efficiencies and BMFs for BDEs 100, 153, and 154, and the

273

depuration rates and half-lives for all PBDE congeners, except for BDEs 85 and 99,

274

were calculated for all target tissues. The absence of BDEs 85 and 99 was caused by

275

their rapid metabolism in carp and they were generally lower than the detection limits

276

during the depuration phase. BDE 100 and BDE 154 showed similar assimilation

277

efficiencies and depuration rates in fish tissues. Thus, it was reasonable to assume that

278

all five BDE congeners would exhibit similar bioaccumulation behavior if no

279

debromination occurred for BDEs 85, 99 and 153. A ratio of the concentration of

280

(BDE 85 + BDE 99 + BDE 153 + all debrominated congeners) to that of (BDE 100 + 15



281

BDE 154) would be predicted to be 1.5 based on the similar concentrations of the five

282

congeners in food. Indeed, the ratio was 1.5 after 7 d exposure. However, the ratio

283

decreased from 1.5 after day 7 of exposure to 0.9 at the end of experiment. Two

284

possible explanations can be proposed for the decrease. Firstly, hydroxylation of the

285

lower brominated BDE congeners could be possible because several OH-BDE

286

chemicals (details in Figure S3) were detected in the fish. Secondly, the excretive rate

287

of BDE 28, 47, and 66 may be faster than that of BDE 154 and BDE 100. The

288

assimilation efficiencies and BMFs of BDEs 100 and 154 were significantly higher,

289

whereas the depuration rates were significantly lower than those of BDE 153 (Table

290

S3), which could attributed to the metabolism of BDE 153. The depuration rates and

291

half-lives of the metabolic debromination congeners (i.e., BDEs 28, 47, 49, 66, and

292

101) were also calculated, but these values cannot be interpreted as the actual

293

depuration rates because congeners are formed continuously during the entire

294

experiment.

295

The half-lives (t1/2) and BMFs of the exposed PCB and PBDE congeners in fish

296

carcasses are plotted versus log Kow in Figure 1. The t1/2 and BMFs increased with

297

increasing Kow at log Kow < 7 and then decreased with increasing Kow. This trend was

298

also observed and explained in previous studies, in which rainbow trout was exposed

299

to organic compounds, including PCBs7, 8. However, the half-lives and BMFs of PCBs

300

18, 45, 91 and 95, and BDE 153 were generally lower than their expected values

301

according to the log Kows in fish, indicating a metabolic process occurred for these

302

chemicals7. 16


Page 16 of 34

Page 17 of 34


303

Biotransformation

and

Compound-specific

Stable

Carbon

Isotope

304

Signatures of PCB and PBDE. The bioaccumulation parameters provided clues to

305

the possible biotransformation of chemicals. To elucidate the metabolism of PCBs and

306

PBDEs in carp, compound-specific stable carbon isotope signatures of PCB and

307

PBDE were measured. All 19 PCB congeners in the fish samples were available for

308

isotope analysis, except PCBs 8 and 18 at the last two sampling points, and PCB 45 at

309

the last sampling point (Figure S4). No significant isotopic fractionation was observed

310

throughout the experiment for PCBs 20, 28, 44, 49, 52, 91, 95, 101, 132, 138, 149,

311

174, 177, 180, 183, and 187 (F8, 53 = 0.608–1.959 , p = 0.074-0.766)20, 24, indicating

312

that no biotransformation occurred or the biotransformation occurred with no

313

detectable isotope fractionation.

314

The stable carbon isotope compositions of PCBs 8, 18, and 45 were the same as

315

those in the spiked food during the exposure period (Figure 2). However, a heavy

316

isotope enrichment trend with depuration time was observed for these three congeners

317

(Figure 2). The carbon isotope composition of PCBs 8, 18, and 45 increased from

318

-29.5 ± 0.2‰, -32.4 ± 0.1‰, and 30.1 ± 0.2‰ at the beginning of depuration to -25.0

319

± 0.1‰, -26.5 ± 0.2‰, and -24.43 ± 0.2‰ at the end of the experiment, respectively.

320

These results affirmed that biotransformation occurred in the fish22,

321

neither OH-PCBs nor MeSO2-PCBs were detected in the sera or fish tissues of the

322

exposed carp throughout the experiment. This likely occurred because other kinds of

323

metabolites or conjugates were generated, but they could not be detected by the

324

methods used the present studies20, 31. 17


30

. However,


325

Stable carbon isotope ratios were precisely measured for four PBDE congeners

326

(BDEs 47, 100, 153, and 154) (Figure 3). No obvious isotope fractionation was

327

observed for BDEs 100 (F8, 53 = 1.75, p = 0.113) and 154 (F8, 53= 1.185, p = 0.329)

328

(Figure 3), indicating that these two congeners were accumulated by common carp

329

with or without undetectable biotransformation, which is in accordance with the

330

results of laboratory exposure experiments14,

331

increased from -29.4 ± 0.3‰ in the spiked food to -26.1 ± 0.1‰ in the carp at the end

332

of the experiment (Figure 3). BDE 153 could undergo metabolic debromination to

333

form BDE 47 and BDE 101 at similar rates14. The observed heavy isotope enrichment

334

indicated isotope fractionation during the metabolic debromination of BDE 153 in

335

common carp. The stable carbon isotopic fractionation of BDE 99 in tiger barbs

336

(Puntigrus tetrazona) was similarly observed17. A previous study demonstrated that

337

reductive dechlorination of PCBs during the microbial degradation process did not

338

produce stable carbon isotopic fractionation32. No remarkable isotopic fractionations

339

for long chain n-alkanes or polycyclic aromatic hydrocarbons were reported during

340

microbial degradation33. It remains unknown whether stable carbon isotopic

341

fractionation occurs during microbial degradation of PBDEs. The isotopic signature

342

might allow for the differentiation of debromination by fish from debromination by

343

microbial degradation in future studies.

21

. The δ13C values of BDE 153

344

Of the five metabolic debromination congeners, only BDE 47 occurred in a

345

sufficient amount for CSIA. The isotope ratio for BDE 47 ranged from -28.9 ± 0.3‰

346

to -28.1 ± 0.1‰ (Figure 3). This was consistent with the hypothesis that BDE 47 18


Page 18 of 34

Page 19 of 34


347

primarily originates from BDE 85, BDE 99, and BDE 153. Using the sample from 7 d

348

exposure as an example, the concentrations of BDE 154 and BDE 100 in carp

349

carcasses were 3,700 ng g-1 and 3,780 ng g-1 lw, respectively. The concentrations of

350

BDEs 85, 99, and 153 would approximately equal to 3,700 ng g-1 lw if no

351

debromination occurred. However, the concentration of BDEs 85, 99, and 153 were

352

only 5 ng g-1, 50 ng g-1, and 1730 ng g-1, respectively. The concentration of BDE 101

353

was 660 ng g-1 lw. As a rough estimate, we calculated that 42.5%, 42.5%, and 15% of

354

BDE 47 in the carcasses was derived respectively from BDE 85, BDE 99, and BDE

355

153. The calculated δ13C value was -28.49‰, which was close to the measured value

356

(-28.33‰). In most cases, the measured δ13C value of BDE 47 was slightly higher

357

than the calculated δ13C value, which occurred because a small fraction of BDE 47

358

had been metabolized to form OH-PBDE. In fact, hydroxylated BDE 47 was

359

frequently detected (Figure S3) in the sera of fish.

360

The isotopic enrichment factors (εC) calculated according to the Rayleigh

361

equation were -1.99‰, -1.84‰, -1.70‰, and -0.86‰ for PCBs 8, 18, and 45, and

362

BDE 153, respectively (Figure 4). To the best of our knowledge, the only reported

363

carbon enrichment factor for hydrodebromination of PBDEs was determined to be

364

-2.11‰ during UV irradiation of BDE 4734, whereas no carbon isotope enrichment

365

factors were available for the metabolic debromination of PBDEs and metabolism of

366

PCBs in fish prior to this study.

367

Enantiomer-specific Stable Carbon Isotope Analysis. Of the seven chiral PCB

19



368

congeners (PCBs 45, 91, 95, 132, 149, 174, and 183), PCBs 132, 149, 174, and 183

369

were found to be racemic (EF = 0.5) throughout the course of the experiment (Figure

370

5), which implies that either no metabolism occurred or their biotransformation was

371

achiral7, 20. PCBs 45, 91, and 95, on the other hand, were racemic during the uptake

372

phase but non-racemic during the depuration period (Figure 5). No significant

373

differences were found for the EF values of each chiral PCB congeners among

374

different tissues (F5, 116 = 0.02–1.504, p = 0.149–0.99). The relative abundances of the

375

E1 - atropisomer of PCB 45, (-)-atropisomer of PCB 91, and (+)-atropisomer of PCB

376

95, increased with time, indicating the preferential metabolism of E2-PCB 45,

377

(+)-PCB 91, and (-)-PCB 95 in carp (Figure 5), which was in agreement with our

378

previous study20. Buckman et al.7 observed that (+)-PCB 91 (E1-PCB 91) and

379

(+)-PCB 136 were preferentially biotransformed by rainbow trout, whereas PCB95

380

was racemic. This was slightly different from our results. Species-specific metabolism

381

of chiral PCBs has been suggested as a potential reason for this difference 20.

382

Carbon isotope compositions were obtained for each atropisomer of PCBs 45, 91,

383

95, 132, and 149 (Figure 6), whereas they were absent for atropisomers of PCBs 174

384

and 183 because of their relatively poorly resolved chromatograph on chiral columns.

385

The initial δ13C values were the same for the pair of atropisomers of each chiral PCB

386

congener (Figure 6). For the congeners with no EF changes (PCBs 132 and 149),

387

isotopic fractionation was also not observed in either atropisomer, which implied no

388

biotransformation occurred for these congeners in common carp.

20


Page 20 of 34

Page 21 of 34


389

Regarding the congeners with EF changes, different situations were exhibited for

390

different congeners. A shift in carbon isotope composition of E1- and E2-PCB 45

391

toward a more positive δ13C with similar extent (from -29.9 ± 0.3‰ to -24.4 ± 0.2‰

392

for E1-PCB 45 and from -30.0 ± 0.3‰ to -23.4 ± 0.4‰ for E2-PCB 45) was observed

393

during the depuration period (Figure 6). This indicated that both atropisomers were

394

involved in metabolic processes, but the E2-atropisomer was metabolized faster than

395

the E1-atropisomer because the EF values increased with depuration time. The εC of

396

E1-PCB 45 (-1.63‰) was similar to that of E2-PCB 45 (-1.74‰) (Figure S6),

397

indicating both atropisomers were metabolized by similar reaction mechanisms.

398

However, because E2 was biotransformed faster than E1, other steps, such as

399

substrate uptake into the cell and binding of the substrate to enzyme, than isotope

400

sensitive carbon bond cleavage might be rate limiting for the metabolism of PCB 45,

401

and those steps were stereoselective.

402

A significant heavy isotope enrichment was observed for (+)-PCB 91 (from

403

-31.65 ± 0.03‰ to -30.36 ± 0.17‰) during the depuration period (F8, 53 = 27.867, p

Tracing the Biotransformation of PCBs and PBDEs in Common Carp

Recommend Documents