Long-Chain Perfluoroalkyl acids (PFAAs) Affect the Bioconcentration

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Long-chain perfluoroalkyl acids (PFAAs) affect the bioconcentration and tissue distribution of short-chain PFAAs in zebrafish (Danio rerio) Wu Wen, Xinghui Xia, Diexuan Hu, Dong Zhou, Haotian Wang, Yawei Zhai, and hui Lin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03647 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 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

1

Long-chain perfluoroalkyl acids (PFAAs) affect the bioconcentration and

2

tissue distribution of short-chain PFAAs in zebrafish (Danio rerio)

3

Wu Wen, Xinghui Xia*, Diexuan Hu, Dong Zhou, Haotian Wang, Yawei Zhai, Hui Lin

4 5

School of Environment, Beijing Normal University, State Key Laboratory of Water Environment

6

Simulation, Beijing 100875, China

7

*

Corresponding author. Tel./fax: +86 10 58805314.

E-mail address: [email protected] (X. Xia) 1 / 34

ACS Paragon Plus Environment


Page 2 of 34

8

Abstract

9

Short- and long-chain perfluoroalkyl acids (PFAAs), ubiquitously co-existing in the environment,

10

can be accumulated in organisms by binding with proteins and their binding affinities generally

11

increase with their chain length. Therefore, we hypothesized that long-chain PFAAs will affect the

12

bioconcentration of short-chain PFAAs in organisms. To testify this hypothesis, the bioconcentration

13

and tissue distribution of five short-chain PFAAs (linear C-F=3-6) were investigated in zebrafish in

14

the absence and presence of six long-chain PFAAs (linear C-F=7-11). The results showed that the

15

concentrations of the short-chain PFAAs in zebrafish tissues increased with exposure time till steady

16

states reached in the absence of long-chain PFAAs. However, in the presence of long-chain PFAAs,

17

these short-chain PFAAs in tissues increased till peak values reached and then decreased till steady

18

states, and the uptake and elimination rate constants of short-chain PFAAs declined in all tissues and

19

their BCFss decreased by 24-89%. The inhibitive effect of long-chain PFAAs may be attributed to

20

their competition for transporters and binding sites of proteins in zebrafish with short-chain PFAAs.

21

These results suggest that the effect of long-chain PFAAs on the bioconcentration of short-chain

22

PFAAs should be taken into account in assessing the ecological and environmental effects of short-

23

chain PFAAs.

24 25

Key words: Short-chain perfluoroalkyl acids (PFAAs); Bioconcentration; Tissue distribution;

26

Zebrafish (Danio rerio); Perfluorinated substances (PFASs).

27

2 / 34


Page 3 of 34

28


Abstract Art I’m a long-chain PFAA. Protein

Concentration/(ng·g-1, ww)

I’m a short-chain PFAA.

Liver

700 600 500 400 300 200 100 0 0

5

10

15

Time/(d-1)

Protein

29 30

3 / 34


20

25

30


31

Page 4 of 34

1 Introduction

32

Perfluoroalkyl acids (PFAAs) are a class of synthetic organic compounds characterized by a

33

hydrophobic perfluoroalkyl chain and a hydrophilic carboxylate or a sulfonate functional group.

34

Because of their special physical and chemical properties (thermal and chemical stability,

35

hydrophobicity and lipophobicity), the long-chain PFAAs (no less than seven fluorinated carbons)

36

have been manufactured and used in numerous industrial and commercial applications for over half

37

century,1 which leads to their widespread distribution in the environment.2-4 Additionally, some of

38

these pollutants have been testified to be bioaccumulative5, 6 and toxic.7 From 2000, 3M Company

39

gradually discontinued the manufacture of some long-chain perfluoroalkyl substances. In 2002, the

40

U.S. Environmental Protection Agency published a rule to regulate the use of perfluorooctanoic

41

sulfonate(PFOS) and related compounds.8 In 2009, at the 4th Conference of the Parties in the

42

Stockholm Convention, PFOS and related compounds were registered under Annex B of the

43

Stockholm Convention on Persistent Organic Pollutants. Meanwhile, some short-chain PFAAs (less

44

than seven fluorinated carbons),9 which have the similar physiochemical properties to long-chain

45

PFAAs, have been synthesized and used as substitutes for long-chain PFAAs to satisfy the

46

application demand.9-11

47

With their extensive use around the world, the short-chain PFAAs have been detected in water,

48

such as rivers and lakes.4, 9, 12, 13 Studies show that perfluorobutyric acid (PFBA) and perfluorobutane

49

sulfonate (PFBS) are the most abundant short-chain PFAAs in water,9, 14, 15 and their concentrations

50

are sometimes up to μg·L-1.9 In addition, the short-chain PFAAs have also been detected in aquatic

51

animals, and even in humans.12, 16-18 Numerous investigations show that some short-chain PFAAs

52

have increasing temporal shifting trends in water and aquatic organisms. 19-21 For example, a

53

significant increasing trend of PFBS has been found in cetacean and dolphin liver samples from 2002

54

to 2014 due to the increasing usage of C4 based alternatives.21 It suggests that some short-chain

55

PFAAs show a potential bioconcentration in some kinds of organisms. However, the information

56

about the distribution and bioconcentration of short-chain PFAAs in different tissues of aquatic biota,

57

such as fish, is limited.5, 22 4 / 34


Page 5 of 34


58

Numerous studies suggest that the interaction of PFAAs and proteins plays a key role in the

59

PFAA bioaccumulation.23-25 Researchers find that some types of proteins can bind with PFAAs,

60

including serum albumin, liver fatty acid-binding protein, some transport proteins, etc.26-30

61

According to the result of the molecular docking analysis, hydrophobic fluorinated carbon chain can

62

extend to the interior of the binding pocket to make the maximum hydrophobic contact while

63

hydrophilic acidic group faces towards the entrance of the pocket.7, 27, 28 Therefore, their binding

64

affinities increase with the number of fluorinated carbons.27, 28 For the binding of short-chain PFAAs

65

with proteins, the stability may be weaker than long-chain PFAAs. Additionally, the binding sites of

66

proteins are reported to be limited for PFAAs.31, 32 Therefore, we hypothesized that the long-chain

67

PFAAs will affect the bioconcentration and tissue distribution of short-chain PFAAs in fish.

68

To test this hypothesis, the effects of multiple long-chain PFAAs on the bioconcentration and

69

tissue distribution of multiple short-chain PFAAs in zebrafish (Danio rerio) were studied in the

70

present research, considering that many PFAAs often co-exist in the environment.4, 12, 33 The tested

71

short-chain PFAAs included PFBA, PFBS, perfluoropentanoic acid (PFPeA), perfluorohexanoic

72

acid (PFHxA), and perfluoroheptanoic acid (PFHpA) which are widely detected in environmental

73

media, and the long-chain PFAAs included perfluorooctanoic acid (PFOA), PFOS,

74

perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid

75

(PFUnA), and perfluorododecanoic acid (PFDoA). The bioconcentration kinetics, tissue distribution

76

including blood, brain, gill, intestine, liver, muscle and ovary, and bioconcentration factors (BCFss,

77

at steady state) of the short-chain PFAAs were investigated in the absence and presence of the long-

78

chain PFAAs. The effect of PFAA properties and tissue types on the bioconcentration was also

79

investigated. Furthermore, the effect mechanisms of long-chain PFAAs on the bioconcentration

80

kinetics, tissue distribution, and BCFss of short-chain PFAAs were also analyzed.

81

2. Materials and Methods

82

2.1 Chemicals and reagents

5 / 34



Page 6 of 34

83

PFBA (99%) were obtained from Matrix Scientific Trade Co. (Columbia, USA). PFPeA (98%)

84

and PFHpA (98%) were supplied by Tokyo Chemical Industries (Tokyo, Japan). PFBS (98%) and

85

PFHxA (98%), PFOA (96%), PFOS (98%), PFNA (97%), PFDA (98%), PFUnA (95%), and PFDoA

86

(95%) were purchased from Acros Organics (New Jersey, USA). Isotopically labeled standards,

87

including 13C4-PFBA (MPFBA, 99%), 13C4-PFOA (MPFOA, 99%), 13C4-PFBA (MPFOS, 99%) and

88

13

89

Wellington Laboratories (Guelph, Canada). Two stock solutions were prepared for fish exposure.

90

The first one contained five short-chain PFAAs at a total concentration of 1000 mg·L-1 in methanol

91

(200 mg·L-1 for each PFAA). The second one contained six long-chain PFAAs at a total

92

concentration of 1200 mg·L-1 in methanol (200 mg·L-1 for each PFAA). Chromatography grade

93

methanol and acetonitrile were supplied by J.T. Baker of Phillipsburg (NJ, USA). Methyl-tert-butyl

94

ether (MTBE, 99.5%) was purchased from Acros Organics (New Jersey, USA). Ammonium acetate

95

(98%) and tetrabutylammonium hydrogen sulfate (TBA, 99%) were obtained from Amethyst

96

Chemicals of J&K Scientific Ltd. (Beijing, China). Any water used in this study was produced by a

97

Milli-Q purification system (Millipore, Germany).

98

2.2 Zebrafish maintenance and experimental design

C4-PFDoA (MPFDoA, 99%), which were used as recovery indicators, were supplied by

99

Given that some PFAAs can be transferred from mother into egg cells and cause toxic effects

100

on offspring,34-36 adult female zebrafish were chose as the test fish. They were purchased from a

101

local fish farm in Beijing and were allowed to acclimate to laboratory conditions in dechlorinated

102

tap water for two weeks prior to PFAA exposure. The acclimation and following exposure

103

experiments were conducted with a 14:10 hour light/dark photoperiod at a temperature of 23±2oC

104

and the zebrafish were fed with commercial fodder daily.

105

All exposure experiments were conducted in polypropylene (PP) tanks. These tanks were

106

divided into three groups. The first group was used as the control without any PFAAs. The second

107

group was used for the five short-chain PFAA exposure at a total concentration of 50 μg·L-1 (10

108

μg·L-1 for each PFAA). The third group was used for the five short-chain and six long-chain PFAA

109

exposure at a total concentration of 110 μg·L-1 (10 μg·L-1 for each PFAA). Each tank contained 50 6 / 34


Page 7 of 34


110

randomly selected zebrafish. The bioconcentration experiment lasted 28 days. Throughout the

111

exposure period, the water in each tank was replaced daily. Uneaten food was siphoned from the

112

containers after feeding (30 minutes). The faeces of zebrafish was also siphoned twice a day. The

113

control group without any PFAAs was conducted the same as the PFAA-exposed groups. Three

114

replicate tanks were available for the control and PFAA-exposed groups.

115

To check for the PFAA concentration of the exposure water, fifty-milliliter water from each

116

tank was collected at the beginning and end of the experiment, as well as after 24 hours’ exposure.

117

Five fish (pooled as a sample) from each tank were sampled at 1, 2, 3, 5, 9, 14, 21, and 28 d for

118

PFAA analysis. Sampled fish were anesthetized and rinsed with Milli-Q water, and then dried with

119

filter paper. A blood sample was drawn from the caudal vein, and fish were subsequently killed by

120

cervical dislocation. Fish tissues (blood, brain, gill, intestine, liver, muscle, and ovary) were collected

121

separately and the wet weight (ww) was obtained. All samples were homogenized for the analysis

122

of PFAA concentration. All zebrafish were treated humanely with the aim of alleviating any

123

suffering according to the OECD Guideline for the Testing of Chemicals.37

124

2.3 Sample preparation and PFAA extraction

125

For the determination of the short- and long-chain PFAAs in water, all water samples were

126

pretreated as previously reported by Taniyasu et al.38 and Zhou et al.9 with some modifications.

127

Briefly, the Oasis WAX cartridges (Waters®, 6 cc, 150 mg) were preconditioned with 4 mL of

128

methanol containing 0.1% ammonium hydroxide (in methanol), 4 mL of methanol, and 4mL of

129

Milli-Q water, sequentially. Then, 4 mL of water sample (without filtering) spiked with 10 ng of

130

each recovery indicator was passed through the cartridge at a rate of one drop per second. The

131

cartridge was washed with 4 mL of buffer (25 mM acetic acid in Milli-Q water), and then the residual

132

water was removed with vacuum pump. The PFAAs were eluted sequentially with 4 mL of methanol

133

and 4 mL of methanol with 0.1% ammonium hydroxide. The eluate was evaporated to near dryness

134

under a gentle nitrogen stream and reconstituted with 1 mL of methanol and filtered with 0.2-μm

135

nylon membranes into PP autosamper vials with PP caps for injection. 7 / 34



Page 8 of 34

136

For the determination of the short- and long-chain PFAAs in blood, samples (obtained from 5

137

pooled fish) were extracted by the method reported by Yeung et al.39 with some modifications.

138

Briefly, the blood sample was spiked with 10 ng of each recovery indicator. A total of 2 mL of

139

acetonitrile was added and extracted via sonication for 30 min, followed by centrifugation at 3000

140

rpm at 4 oC for 15 min. The supernatant was transferred to another clean tube. The extraction was

141

repeated with a further 2 mL of acetonitrile twice. The extracts were combined and evaporated under

142

a gentle nitrogen stream to about 0.5 mL and then diluted with 50 mL of Milli-Q water. The diluent

143

was then loaded onto a preconditioned Oasis WAX cartridge. The subsequent procedures were the

144

same as the water samples. The eluate was reconstituted with 0.4-0.8 mL methanol and filtered for

145

injection.

146

For the determination of the short- and long-chain PFAAs in muscle, samples (obtained from 5

147

pooled fish) were extracted with the method reported by So et al.40 and Taniyasu et al.38 with some

148

modifications. Briefly, the homogenized sample spiked with 10 ng of each recovery indicator was

149

sonicated in 5 mL of 10 mM KOH (in methanol) at 60 °C for 30 min, and then vortexed, shaken

150

(300 rpm, 25 oC, 16 h), and centrifuged (4000 rpm, 15 min). The supernatant was transferred to

151

another clean tube. The extraction repeated with a further 5 mL of 10 mM KOH (in methanol) twice.

152

The extracts were combined and the subsequent procedures were the same as blood samples. The

153

eluate was reconstituted with 0.5-1 mL methanol and filtered for injection.

154

For the determination of the short- and long-chain PFAAs in gill, brain, liver, intestine, and

155

ovary, samples (obtained from 5 pooled fish) were extracted by iron-pairing method.41 In brief, the

156

homogenized tissue sample spiked with 10 ng of each recovery indicator was shaken (250 rpm, 30

157

min, 25±0.5 oC) in the iron-pairing solution containing 0.5 mL of 0.5 M TBA (pH=10), 1 mL of 0.25

158

M sodium carbonate buffer (pH=10), and 2.5 mL MTBE. The organic layer was separated by

159

centrifuging (4000 rmp, 15 min) and transferred to a clean tube. The extraction was repeated with a

160

further 2.5 mL of MTBE twice. All extracts were combined in 10-mL PP centrifuge tube. The

161

subsequent procedures were the same as blood samples. The eluate was reconstituted with 0.2-1 mL

162

of methanol and filtered for injection. 8 / 34


Page 9 of 34

163


2.4 Instrumental analysis of PFAAs

164

The short- and long-chain PFAAs were analyzed by liquid chromatography-tandem mass

165

spectrometry (LC-MS/MS; Dionex Ultimate 3000 and Applied Biosystems API 3200) in

166

electrospray negative ionization mode. Chromatographic separation was in combination with a

167

binary pump, an autosampler and a column oven equipped with a Waters Sunfire C18 Column (5

168

μm pore size, 4.6×150 mm). The injection volume was 10 μL. Water (5 mM ammonium acetate)

169

and methanol were used as mobile phase. At a flow rate of 1 mL·min-1, the mobile phase gradient

170

started with a 1 min isocratic step at 70% of methanol, and then ramped to 95% of methanol in 6.5

171

min, held at 95% of methanol for 3 min, and then ramped down to 70% of methanol in 0.5 min. For

172

quantitative analysis, analyte ions were monitored using multiple reaction monitoring mode. The

173

details of instrument parameters are shown in Tables S1 and S2.

174

2.5 Data analysis

175

All statistical analyses were performed using SPSS 18.0 for windows (SPSS Inc., Chicago

176

(IL)., USA) and Microsoft Excel 2016. Bioconcentration data were dynamically fit by Origin 2016

177

to obtain the kinetic parameters. Analysis of variance (ANOVA, one factor) was carried out to test

178

differences between each two compared groups, and difference was considered significant when

179

the significance level was smaller than 0.05.

180

2.6 Quality assurance and quality control (QA/QC)

181

Throughout the experiments, disposable PP beakers, bottles, tubes, and pipettes were used to

182

prevent target compounds adsorption and contamination from glassware and other vessels

183

containing fluorochemicals. All vessels were cleaned by methanol three times before use.

184

The method detection limits (MDLs) and method quantitation limits(MQLs) for the target

185

compounds were defined as tree and ten times of the signal to noise ratio, respectively, and the

186

details of the MDLs/MQLs for water and tissues are listed in Table S3. All the target chemicals in

187

samples were quantified by external standard calibration. Calibration standards were prepared in 9 / 34



Page 10 of 34

188

methanol with the concentrations for each PFAA ranging from 0.2 to 200 μg·L-1. The correlation

189

coefficients of the standard curves were higher than 0.99. To ensure the extraction efficiency and

190

accuracy of determination, spike recovery experiments for target PFAAs in water and biological

191

samples were conducted, and the recoveries ranged from 75.2±3.2% to 105±14.8% and from 73.3

192

±1.9% to 104±10.0% (Table S4), respectively. Procedural blanks and calibration verification

193

standards were run after every 10 samples to check the background contamination and the validity

194

of the calibration. A new calibration curve was run if the quality-control standard was not measured

195

within ±20% of its theoretical value. The variations of measured PFAA concentration of the

196

exposure solution were lower than 5% (Table S5), which indicated that the aqueous PFAA

197

concentration did not change significantly during the exposure experiment. In addition, PFAAs in

198

tissues of zebrafish in the control group ranged from no detection to 11.69 ng·gww-1 (PFHpA in

199

liver) but they were at least 50 times lower than that in the exposure group. The concentrations of

200

target compounds in fish food were tested. Their concentrations were lower than MDLs, except for

201

PFOS, which was higher than MDL but lower than MQL.

202

3 Results and Discussion

203

3.1 Body condition of zebrafish

204

Brain-somatic index (BSI), hepatic-somatic index (HSI), and condition factor (K-factor) were

205

monitored to assess any toxicological effects on zebrafish.42 As shown in Table S6, no significant

206

differences in the length and weight of zebrafish were observed among different groups. No

207

significant differences were observed in the BSI, HSI, and K-factor between PFAA-exposed and

208

control groups. No mortality was observed in all the control and PFAA-exposed groups. These

209

results suggest that the exposure condition did not exert significant toxicological effects on zebrafish.

210

3.2 Bioconcentration of the short-chain PFAAs in zebrafish in the absence of the long-

211

chain PFAAs

212

3.2.1 Bioconcentration kinetic parameters in the absence of the long-chain PFAAs. 10 / 34


Page 11 of 34


213

All target compounds were detected in tissues of zebrafish except PFBA in brain and ovary.

214

According to the bioconcentration curves (Figure 1), the concentration of the short-chain PFAAs in

215

tissues (liver, blood, intestine, gill, ovary, brain, and muscle) increased with exposure time at the

216

first few days, and a steady state reached after 5 days’ exposure. Bioconcentration kinetic parameters

217

for the short-chain PFAAs in tissues were obtained by fitting the curves with the two-compartment

218

kinetic model: 43 k

Cb =Cw ku0 ·(1-e-ke0·t )

219

e0

(1)

220

Where Cb is the PFAA concentration in a tissue (ng·gww-1) at time t (d); Cw is the PFAA

221

concentration in the water (ng·mL-1); ku0 (mL·gww-1·d-1) and ke0 (d-1) are the uptake and elimination

222

rate constants of PFAAs in a tissue, respectively; the subscript of “0” refers to the kinetic parameters

223

in the absence of the long-chain PFAA exposure. The results are shown in Table 1. The ku0 values

224

were influenced by the PFAA properties and the tissue types. Firstly, the ku0 values increased with

225

the fluorinated carbons in any tissue, which was consist with the bioconcentration of long-chain

226

PFAAs (C-F≥7) in aquatic animals.5, 44 Take the liver for example, the ku0 values for PFBA (C-F=3),

227

PFPeA (C-F=4), PFHxA (C-F=5), and PFHpA (C-F=6) were 5.1, 13, 18, and 36 mL·gww-1·d-1

228

respectively. Secondly, the acidic headgroup had an influence on ku0 values. Although both PFBS

229

and PFPeA had four fluorinated carbons, the ku0 value of PFBS in each tissue was significantly

230

higher than that of PFPeA. The ku0 values of PFBS in tissues ranged from0.64 to 16 mL·gww-1·d-1,

231

while the ku0 value of PFPeA ranged from 0.23 to 13 mL·gww-1·d-1. Thirdly, the ku0 value of each

232

PFAA was different among various tissues, which was highest in liver and blood, followed by

233

intestine, gill and ovary, and lowest in brain and muscle. This is similar with the previous research

234

that studied the bioconcentration of long-chain PFAAs in rainbow trout (Oncorhynchus mykiss).5 As

235

for the ke0, fluorinated carbons or acidic headgroup had no obvious influence on ke0 values of the

236

short-chain PFAAs. This is consistent with the study that reported the bioconcentration kinetic

237

parameters of three short-chain PFAAs (PFHxA, PFBA, and PFBS) in zebrafish tissues (plasma,

238

liver, muscle and Ovary ).22

11 / 34



Page 12 of 34

239

3.2.2 Bioconcentration and tissue distribution factors of the short-chain PFAAs at steady state

240

in the absence of the long-chain PFAAs.

241

The values of BCFss (L·kg-1) for the short-chain PFAAs at steady state (day 28) (Table 1) were

242

calculated by the equation: BCFss =Cb/Cw, where Cb is the concentration of PFAA in the tissue of

243

zebrafish, ng·gww-1, and Cw is PFAA concentration in water, ng·mL-1. According to the results (Table

244

1, Figures 2 and S1), it is similar to the uptake rate constants that the concentrations and BCFss of

245

PFAAs in tissues of zebrafish were affected by PFAA properties and tissue types. Initially, the

246

concentrations and BCFss of PFAAs increased with fluorinated carbons in each tissue and significant

247

positive relationships were observed between logBCFss and fluorinated carbon numbers (P

Long-Chain Perfluoroalkyl acids (PFAAs) Affect the Bioconcentration

Recommend Documents