How Does Predation Affect the Bioaccumulation of Hydrophobic


Mar 20, 2015 - Long-Chain Perfluoroalkyl acids (PFAAs) Affect the Bioconcentration and Tissue Distribution of Short-Chain PFAAs in Zebrafish (Danio re...
0 downloads 10 Views 1MB Size


Subscriber access provided by SUNY DOWNSTATE

Article

How Does Predation Affect the Bioaccumulation of Hydrophobic Organic Compounds in Aquatic Organisms? Xinghui Xia, Husheng Li, Zhifeng Yang, Xiaotian Zhang, and Haotian Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00071 • Publication Date (Web): 20 Mar 2015 Downloaded from http://pubs.acs.org on March 25, 2015

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 29

Environmental Science & Technology

How Does Predation Affect the Bioaccumulation of Hydrophobic

1

Organic Compounds in Aquatic Organisms?

2

Xinghui Xia∗, Husheng Li, Zhifeng Yang, Xiaotian Zhang, Haotian Wang

3 4

State Key Laboratory of Water Environment Simulation, School of Environment, Beijing

5

Normal University, Beijing 100875, China

6 7 8



Corresponding author phone: +86-10-58805314; fax: +86-10-58805314; e-mail: [email protected] 1

ACS Paragon Plus Environment

Environmental Science & Technology

Page 2 of 29

Abstract

9 10 11

It is well known that the body burden of hydrophobic organic compounds (HOCs)

12

increases with the trophic level of aquatic organisms. However, the mechanism of HOC

13

biomagnification is not fully understood. To fill this gap, this study investigated the effect of

14

predation on the bioaccumulation of polycyclic aromatic hydrocarbons (PAHs), one type of

15

HOC, in low-to-high aquatic trophic levels under constant freely dissolved PAH concentrations

16

(1, 5 or 10 µg L-1) maintained by passive dosing systems. The tested PAHs included

17

phenanthrene, anthracene, fluoranthene, and pyrene. The test organisms included zebrafish,

18

which prey on Daphnia magna, and cichlids, which prey on zebrafish. The results revealed that

19

for both zebrafish and cichlids, predation elevated the uptake and elimination rates of PAHs.

20

The increase of uptake rate constant ranged from 20.8% to 39.4% in zebrafish with the amount

21

of predation of 5 daphnids per fish per day, and the PAH ;luptake rate constant increased with

22

the amount of predation. However, predation did not change the final bioaccumulation

23

equilibrium; the equilibrium concentrations of PAHs in fish only depended on the freely

24

dissolved

25

bioaccumulation factor of each PAH was constant for fish at different trophic levels. These

26

findings infer that the final bioaccumulation equilibrium of PAHs is related to a partition

27

between water and lipids in aquatic organisms, and predation between trophic levels does not

28

change bioaccumulation equilibrium but bioaccumulation kinetics at stable freely dissolved

29

PAH concentrations. This study suggests that if HOCs have not reached bioaccumulation

30

equilibrium, biomagnification occurs due to enhanced uptake rates caused by predation in

31

addition to higher lipid contents in higher trophic organisms. Otherwise, it is only due to the

32

higher lipid contents in higher trophic organisms.

33

Key words: Biomagnification, Bioaccumulation, Bioavailability, HOCs, Predation, Freely

34

dissolved concentration, Passive dosing system

concentration

in

water.

Furthermore,

the

lipid-normalized

35 36 37 38 2

ACS Paragon Plus Environment

water-based

Page 3 of 29

Environmental Science & Technology

TOC Art

39 40

CPAH in zebrafish (ng g-1 d wt.)

12000

Phenanthrene 10µg/L PAH, predation 10µg/L PAH, no predation 5µg/L PAH, predation 5µg/L PAH, no predation 1µg/L PAH, predation 1µg/L PAH, no predaton

9000

Freely dissolved PAHs by passive dosing

6000

Loading 3000

PDMS Paintcoat 0 0

3

6

9

12

15

D. magna

Zebrafish

Cichlid

Predation

41 42

Freely Dissolved Exposure

3

ACS Paragon Plus Environment

Dosing

Environmental Science & Technology

43

1 INTRODUCTION

44

Hydrophobic organic compounds (HOCs) have attracted attention around the world for a

45

long time because most of them are toxic to organisms and are liable to accumulate in

46

organisms due to their lipophilicity.1-3 In general, there are two main routes for HOC

47

bioaccumulation in aquatic organisms. One is membrane absorption from freely dissolved

48

HOCs through their gills and skin; the other is intestinal wall absorption through their

49

gastrointestinal tract as a result of predation/ingestion. 4

50

Trophic structure and HOC bioaccumulation in aquatic food webs have been modeled by

51

the utility of delta-15 N,5 and many studies have shown that most HOCs are capable of

52

biomagnification along the food chain of aquatic ecosystems, indicating that the HOC body

53

burden, i.e. HOC concentrations in organisms, would increase with the trophic level.4,6,7 The

54

traditional explanation for this phenomenon is that high trophic organisms predate lower ones,

55

leading to the enhancement of HOC body burden in the high trophic organisms.8,9 However,

56

many researchers have reservations. Research on trout from Lake Ontario did not find a

57

correlation between PCB (polychlorinated biphenyl) concentrations and fish trophic level.10

58

Olsson et al.11 found out that the trophic level of perch (one type of fish) was positively

59

correlated with their length, but the observed oral contraceptive concentrations in perch with

60

length < 20 cm did not show any increase with trophic level. Wang and Wang12 demonstrated

61

that exposure to dissolved dichlorodiphenyltrichloroethane (DDT) is a more significant route

62

than dietary intake for their bioaccumulation in Lutjanus argentimaculatus. Previously,

63

Chiou13,14 suggested that at least a near partition equilibrium of solute exists between water and

64

fish lipids. Furthermore, organisms at a higher trophic level generally have larger bodies and

65

higher lipid contents.15 In this case, we can suppose that it might not be predation that causes

66

higher HOC concentrations at high trophic levels but rather the higher lipid content.

67

However, no consensus has been reached regarding the mechanism of HOC

68

biomagnification along the food chain of aquatic ecosystems because minimal laboratory and

69

direct evidence is available to demonstrate whether and how freely dissolved HOC exposure

70

and predation along the food chain influence the equilibrium and kinetics of HOC

71

bioaccumulation in aquatic organisms. This absence is mainly due to the lack of well-defined 4

ACS Paragon Plus Environment

Page 4 of 29

Page 5 of 29

Environmental Science & Technology

72

and stable freely dissolved test substances for bioaccumulation tests, especially for the

73

long-time tests.16 Traditional experiments are inevitably compromised by test compound

74

losses,17 complex effects of the cosolvents on test organisms,18 and adverse impacts of the

75

excessive renewal of exposure media on test organisms. In recent years, the passive dosing

76

system19-21 has enabled defined and constant freely dissolved HOC exposure in a simpler and

77

less materially costly way than flow-through systems.22 Passive dosing vials have been made to

78

maintain stable dissolved exposure of HOCs in toxicity experiments for aquatic organisms such

79

as Daphnia magna23 (D. magna) and fish embryos24.

80

In the present study, polycyclic aromatic hydrocarbons (PAHs), which are widely present

81

in aquatic environments,25,26 were chosen as model HOCs to investigate the effect of freely

82

dissolved HOC concentrations and predation between trophic levels on the equilibrium and

83

kinetics of HOC bioaccumulation in organisms at different trophic levels. Four PAHs including

84

phenanthrene, anthracene, fluoranthene, and pyrene were studied, which represent three-ring

85

and four-ring PAHs with logarithmic octanol-water partition coefficients (logkow) ranging from

86

4.54 to 5.98. A passive dosing system was scaled up and modified to maintain stable freely

87

dissolved concentrations of PAHs in the exposure media. D. magna, zebrafish (Danio rerio),

88

and cichlids (Dimidiochromis kiwinge) were chosen as test organisms due to the simple and

89

clear predator/prey relations among them, where cichlids prey on zebrafish, and zebrafish prey

90

on D. magna. The effect of freely dissolved PAH concentrations and amount of predation on

91

the equilibrium and kinetics of PAH bioaccumulation in zebrafish and cichlids were studied.

92

2 MATERIALS AND METHODS

93

2.1 Chemicals and Materials

94

Phenanthrene, anthracene, fluoranthene, and pyrene in solid phase were purchased from

95

the Johnson Matthey Company (Alfa Aesar), with purity >98% for each PAH. The standard

96

solution mixture of the PAHs at a certified concentration of 200.0 µg/mL for each compound in

97

dichloromethane and methanol solution (v:v, 1:1) was purchased from AccuStandard. Surrogate

98

standard 2-fluorobiphenyl was purchased from J&K Chemical Ltd. with purity >97%. The

99

internal standard substance m-terphenyl was purchased from AccuStandard with purity >98%.

100

The poly-(dimethyl siloxane) (PDMS) elastomer was prepared from a Silastic MDX4-4210 5

ACS Paragon Plus Environment

Environmental Science & Technology

Page 6 of 29

101

BioMedical grade Elastomer kit (Dow Corning) purchased from Baili (Shanghai) Medicinal

102

materials trade Inc. of China. High-performance liquid chromatography grade methanol,

103

dichloromethane, hexane, and acetone were purchased from J.T. Baker; all other

104

analytical-grade reagents and chemicals were from Beijing Chemical Reagents. Milli-Q water

105

(Super Q-treated, Millipore) was used in the present study.

106

2.2 Preparation and Characterization of Passive Dosing Dishes

107

Passive dosing dishes were prepared by casting PDMS into 60 mm-diameter glass culture

108

dishes. A total of 15±0.15 g mixture of PDMS prepolymer and the matched catalyst (10:1,

109

weight) were added to each culture dish, which was considered to be sufficient to hold 1000 ml

110

water.27 These dishes were then vacuumed to eliminate trapped air and subsequently placed in

111

an oven at 110°C for 48 h to complete the curing. Cured dishes were soaked in methanol for 72

112

h to remove impurities and oligomers and were then rinsed with Milli-Q water three times

113

before loading. Different concentrations of PAH solutions in methanol were prepared as

114

loading solutions. Dishes cured in triplicate were immersed in these solutions for at least 72 h

115

(100 ml solution per dish) with loading solutions refreshed every 24 h. After that, loaded dishes

116

were rinsed with Milli-Q water three times and then placed in 1000 ml artificial freshwater

117

(AFW) for at least 24 h to complete the dosing procedure.

118 119 120 121 122 123

For the loading procedure, the partition coefficients of PAHs between PDMS and methanol (MeOH) (KPDMS:MeOH, L kg-1) were calculated as follows: 27 K PDMS :MeOH =

C PDMS CMeOH

(1)

For the dosing procedure, the partition coefficients of PAHs between AFW and methanol (KAFW:PDMS, kg L-1) were calculated as follows: 27

K AFW :PDMS =

C AFW CPDMS

(2)

124

where CMeOH is the PAH concentration in the methanol loading solution (µg L-1); CPDMS

125

PAH concentration in PDMS paint coats (µg kg-1); CAFW is the PAH concentration in dosed

126

artificial freshwater (µg L-1). Thus, the partition coefficients of PAHs between MeOH and

127

AFW (KMeOH:AFW) could be calculated with the following equation: 6

ACS Paragon Plus Environment

is the

Page 7 of 29

128

Environmental Science & Technology

K MeOH :AFW =

CMeOH 1 = C AFW K PDMS:MeOH ⋅ K AFW :PDMS

(3)

129

The partition coefficients (KMeOH:AFW) were obtained by linear regression between the

130

PAH concentration in MeOH loading solution and AFW. According to the results shown in

131

Figure S1 (in Supporting information), the KMeOH:AFW values for phenanthrene, anthracene,

132

fluoranthene, and pyrene were 3.80×104, 5.56×104, 9.10×104 and 1.29×105, respectively. Based

133

on the value of KMeOH:AFW, the concentrations of PAHs in AFW can be deduced from that in

134

MeOH and vice versa.

135

2.3 Establishment of Passive Dosing Exposure Systems

136

Based on the value of KMeOH:AFW, the PDMS loading solution was prepared by dissolving

137

0.3797 g phenanthrene, 0.5558 g anthracene, 0.9103 g fluoranthene, and 1.2864 g pyrene per

138

liter methanol. Part of this solution was diluted at 2:1 or 10:1, and the PDMS dishes were

139

individually loaded with the three solutions described above and then the freely dissolved

140

concentration of each PAH in the water was maintained at 10 µg L-1, 5µg L-1, and 1µg L-1,

141

respectively. After loading, these dishes were rinsed with Milli-Q water and individually placed

142

into 2 L tanks with 1 L AFW to prepare the passive dosing exposure systems. They were

143

allowed to stand for 24 h before the start of the experiment, which was long enough for the

144

PAHs to equilibrate between PDMS and water. The results showed that the measured freely

145

dissolved PAH concentrations in AFW were consistent with the calculated values based on

146

KMeOH:AFW (Table S1 and S2 in Supporting Information).

147

2.4 Cultivation of D. magna, Zebrafish, and Cichlid

148

D. magna were cultured in the laboratory under the conditions described in the guidelines

149

of the Organization for Economic Cooperation and Development for the testing of chemicals.28

150

In brief, D. magna were cultured in AFW and maintained at 21±0.5°C under a 14: 10 (light:

151

dark) photoperiod, meanwhile daphnids were fed a suspension of Scenedesmus subspicatus

152

twice daily. The detailed culture procedures were described in our previous study.29 Zebrafish

153

and cichlids were cultured in AFW at 23±0.5°C during the day and 21±0.5°C at night under a

154

14: 10 (light: dark) photoperiod and fed commercial fodder daily. The fish were placed in AFW 7

ACS Paragon Plus Environment

Environmental Science & Technology

155

without feeding for a week before the start of the exposure experiments.

156

2.5 PAH Bioaccumulation in Zebrafish that Prey on D. magna

157

The bioaccumulation experiments were conducted in 15 cm×10 cm×20 cm glass tanks. A

158

total of 20 cultured zebrafish of similar size and the same generation were placed in each tank

159

with 1 L AFW containing 1 µg L-1, 5 µg L-1 or 10 µg L-1 PAH, which was maintained by the

160

PDMS passive dosing system as mentioned above. They were cultured at 23±0.5°C during the

161

day and 21±0.5°C at night under a 14: 10 (light: dark) photoperiod. The fish were fed D.

162

magna every day (5 daphnids per fish per day, 3±0.3% of the zebrafish wet biomass per day).

163

The D. magna were correspondingly exposed to 1 µg L-1, 5 µg L-1 or 10 µg L-1 PAH in the

164

media for at least 24 h before feeding, which was long enough for the PAHs to reach

165

bioaccumulation equilibrium in D. magna,29 and the PAH concentrations in D. magna from

166

each exposure system were also measured. To make up for the evaporation loss of water during

167

the exposure tests, sterilized de-ionized water prepared by the corresponding passive dosing

168

procedure was added to the tanks every 3 days to keep the medium volume constant at 1 L. The

169

zebrafish were sampled at 0, 1, 2, 3, 5, 7, 9, 12, and 16 d, and they were sampled 24 h after the

170

last feeding to ensure that all daphnids were digested. At each time point, 2 fish were

171

transferred from each tank by fish net to a glass beaker and rinsed with Milli-Q water. Then the

172

heads of the zebrafish were cut down; the zebrafish were dried with filter paper and put on

173

aluminum foils, and the wet weight was obtained (Table S3). They were then stored at -20°C

174

until subsequent processing. A control group was used to study the bioaccumulation of PAHs in

175

zebrafish in the absence of predation on D. magna, and a blank experiment without spiking

176

with PAHs and predation was also conducted. Each experimental variation was tested in

177

triplicate.

178

To study the effect of amount of predation of D. magna on PAH bioaccumulation in

179

zebrafish, a total of 20 zebrafish were placed in each prepared tank containing 10 µg L-1 freely

180

dissolved PAH maintained by PDMS passive dosing as mentioned above. The bioaccumulation

181

experiment was performed as described above except that the zebrafish were fed 0, 5 or 10

182

daphnids per fish per day.

8

ACS Paragon Plus Environment

Page 8 of 29

Page 9 of 29

183

Environmental Science & Technology

2.6 PAH Bioaccumulation in Cichlids that Prey on Zebrafish

184

The PAH bioaccumulation experiments in cichlids were conducted in 30 cm×25 cm×20

185

cm glass tanks. A total of 10 cultured cichlids of similar size and the same generation were

186

placed in each tank with 8 L AFW containing 10 µg L-1 freely dissolved PAHs, which was

187

maintained by a larger-scale PDMS passive dosing as described above. They were cultured at

188

23±0.5°C during the day and 21±0.5°C at night under a 14: 10 (light: dark) photoperiod, and

189

aeration pumps were used to maintain a saturated dissolved oxygen level. The cichlids were

190

fed zebrafish every 3 days (1 zebrafish per fish per 3 days, 3±0.3 % of the cichlid biomass per

191

day). The zebrafish were correspondingly exposed to 10 µg L-1 PAH media for at least 12 d

192

before feeding, which was long enough for PAHs to reach bioaccumulation equilibrium (Figure

193

1). As described in section 2.5, new exposure medium was added periodically to make up for

194

the evaporation loss of water. The cichlids were sampled at 0, 1, 2, 5, 8, 14, and 20 d, and they

195

were sampled 48 h after the last feeding to ensure that all zebrafish were digested. At each time

196

point, 1 cichlid was transferred from each tank by fish net to a glass beaker and rinsed with

197

Milli-Q water. The heads of the fish were cut down; then the fish was dried with filter paper

198

and placed on aluminum foil, and the wet weight was obtained. They were stored at -20°C until

199

subsequent processing. A control group was used to study the bioaccumulation of PAHs in

200

cichlids that preyed on zebrafish not contaminated with PAHs, and a blank experiment without

201

PAH spiking and predation was also conducted. Each experimental variation was tested in

202

triplicate.

203

2.7 Extraction and Analysis of PAHs and Fish Lipids

204

Solid phase extraction (SPE) was used to extract PAHs from water samples; the procedure

205

was described in our previous study.26 D. mangna, zebrafish, and cichlid samples were freeze

206

dried for 72 h, and fish samples were ground in a ceramic mortar. The PAHs in fish powder and

207

daphnids were extracted by a modified ultrasound-assisted extraction method.30,31 Briefly, 8 ml

208

of extraction agent (n-hexane: dichloromethane=1:1, v:v) and 100 µl (50 ng) of

209

2-fluorobiphenyl solution, the surrogate standard, were added to each 10 ml glass scale test

210

tube containing weighed fish powder or daphnids. Those tubes were sealed and stored at -4°C

211

for a 24 h before being sonicated. After a 30 min ultrasonic bath in a KQ5200DE ultrasonic 9

ACS Paragon Plus Environment

Environmental Science & Technology

212

machine, the extract was transferred into 15 ml glass tubes, and another 8 ml extraction agent

213

was added to the original test tube for another 30 min ultrasonic bath. Extracts of two

214

replications were placed in 15 ml glass tubes and concentrated to less than 2 ml by gentle

215

nitrogen blowing. The concentrated extracts were filtered with 0.45 µm Teflon membranes;

216

filtrates were subsequently dried with nitrogen to less than 0.5 ml. They were then

217

homogenized with 50 µl m-terphenyl (1 mg L-1) solution, the internal standard, and transferred

218

into 2-ml sample vials provided by Agilent and diluted with n-hexane to 1 ml. All of the vials

219

were sealed and kept at -4°C until PAH analyses. The PAH concentrations were analyzed using

220

a Varian 3800 gas chromatography-4000 ion trap mass spectrometry system equipped with a

221

VF-5ms column; the detailed conditions were described in our previous study.32,33 To test the

222

lipid contents of zebrafish and cichlids, 0.5 g fish powder was processed as described for PAH

223

extraction. The lipid content in dry weight (%) was determined by the mass differential method

224

(n=5).34

225

2.8 Data Analysis

226

All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA).

227

The bioaccumulation data were dynamically fit by SigmaPlot 10.0 (Systat Software, Inc.) to

228

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

229

test the differences between each pair of compared groups. The difference was considered

230

significant when the significance level was less than 0.05. The Pearson correlation coefficient

231

was calculated and used to test the significance of the correlation between each pair of

232

variables.

233

2.9 Quality Assurance and Quality Control

234

The determined limits of quantification (LOQs) (S/N=10) for GC-MS analysis of the

235

target analytes were in the range of 0.05−0.10 µg L-1. The internal standard calibration curves

236

(5, 10, 20, 50, 100, 200, 500, 800, and 1000 µg L-1) with correlation coefficients all higher than

237

0.99. Method blank samples free of D. magna, zebrafish or cichlid were processed using the

238

same pretreatment procedure used for organism samples, and the target contaminants were

239

below the detection limits. The recoveries of phenanthrene, anthracene, fluoranthene, and 10

ACS Paragon Plus Environment

Page 10 of 29

Page 11 of 29

Environmental Science & Technology

240

pyrene were 78.7±7.92%, 82.3±6.97%,95.9±10.8%, and 99.9±11.6% in the organisms, and

241

92.3±5.3%, 88.5±6.92%,101±10.4%, and 94.2±10.8% in the exposure media, respectively

242

(n=5).

243

For the bioaccumulation tests, the freely dissolved PAH concentrations of the exposure

244

media were measured at the beginning and end of all tests, and the results indicated that their

245

variations were ≤ 3% (Table S1 and S2). No significant differences in whole-body growth rates

246

were found between treated and control fish, indicating that PAHs and a relatively low food

247

intake did not have any obvious effect on zebrafish and cichlid growth (Table S3 and S4). No

248

mortality was observed in any group throughout the experiment.

249

3. RESULTS AND DISCUSSION

250

3.1 Influence of Predation on the PAH Bioaccumulation Equilibrium in Zebrafish

251

According to the results shown in Figure 1 and Figure 2, the body burden of PAHs in

252

zebrafish initially increased rapidly and reached the peak value, and then the body burden

253

started to decrease with time. Finally, bioaccumulation equilibrium was achieved after

254

exposure for 12 d. Similar bioaccumulation curves of PAHs have been observed by other

255

researchers.35,36 For example, Djomo et al. 35 observed rapid PAH uptake during the first 24 h

256

before the decrease-to-equilibrium process in a zebrafish experiment. Sun et al.36 observed a

257

similar phenomenon for phenanthrene bioaccumulation in Carassius auratus. Interestingly,

258

regardless of the exposure level, the body burden of each PAH in zebrafish was almost the

259

same in the presence and absence of predation when bioaccumulation equilibrium was reached

260

(Figure 1). In addition, the bioaccumulation factor (BAF, L kg-1), calculated as CB/Cw (where

261

CB is the PAH concentration in the organism in the equilibrium state, µg kg-1dry weight, and Cw is

262

the freely dissolved PAH concentration in the water phase, µg L-1), was obtained for each PAH.

263

As shown in Table 1, no significant difference in the BAF of each PAH was observed between

264

the groups in the presence and absence of predation at 1, 5, or 10 µg L-1 exposure. For example,

265

the BAF values of anthracene were 657±58 and 627±66 L kg-1 in the absence and presence of

266

predation, respectively, at 10 µg L-1 PAH exposure. Furthermore, as shown in Figure 2, the

267

amount of D. magna predation did not have a significant influence on the body burden of PAHs 11

ACS Paragon Plus Environment

Environmental Science & Technology

Page 12 of 29

268

in zebrafish after bioaccumulation equilibrium was reached, and no significant difference in

269

BAF value existed among the groups with different amounts of predation for each PAH (Table

270

2).

271

Upon equilibrium, the body burden of each PAH in zebrafish increased with the freely

272

dissolved concentration (Figure 1). However, as shown in Table 1, with or without predation,

273

the BAF value of each PAH in zebrafish was constant for each freely dissolved concentration,

274

and the logarithmic values of BAF (logBAF) of the four types of PAHs were positively

275

correlated with their logkow ( r>0.99, p