How Does Predation Affect the Bioaccumulation of Hydrophobic


Mar 20, 2015 - compounds (HOCs) increases with the trophic level of aquatic ... HOC, in low-to-high aquatic trophic levels under constant freely disso...
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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

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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

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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

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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

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water-based

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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

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1 INTRODUCTION

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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

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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

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

49

gastrointestinal tract as a result of predation/ingestion. 4

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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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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2 MATERIALS AND METHODS

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2.1 Chemicals and Materials

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

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

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

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

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

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

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

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

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

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

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

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

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2.2 Preparation and Characterization of Passive Dosing Dishes

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

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

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

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

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

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

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

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

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

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

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(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)

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

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

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

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

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K MeOH :AFW =

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

(3)

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

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

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

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

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

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MeOH and vice versa.

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2.3 Establishment of Passive Dosing Exposure Systems

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

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

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

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

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

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

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

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

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

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

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

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2.4 Cultivation of D. magna, Zebrafish, and Cichlid

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

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

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

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

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

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

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

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

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2.5 PAH Bioaccumulation in Zebrafish that Prey on D. magna

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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triplicate.

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

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

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

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

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daphnids per fish per day.

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2.6 PAH Bioaccumulation in Cichlids that Prey on Zebrafish

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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triplicate.

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2.7 Extraction and Analysis of PAHs and Fish Lipids

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

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

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

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

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

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

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

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

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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

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

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

224

(n=5).34

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2.8 Data Analysis

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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

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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

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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

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mortality was observed in any group throughout the experiment.

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3. RESULTS AND DISCUSSION

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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,

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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.

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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

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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

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

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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,

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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<0.01). These findings indicate that predation on D.

276

magna does not change the final bioaccumulation equilibrium of PAHs in zebrafish; the

277

equilibrium body burden of PAHs in fish only depends on the freely dissolved concentrations

278

in water.

279

3.2 Influence of Predation on the Bioaccumulation Kinetics of PAHs in Zebrafish

280 281 282

To determine the bioaccumulation kinetics of PAHs, the following two-compartment kinetic model was applied in the present study:38

dCb = Cw ku − Cb ke dt

(4)

283

where Cb is the PAH concentration in the test organism (µg kg-1); t is the exposure time (d); Cw

284

is the PAH concentration in the water phase (µg L-1); ku is the PAH uptake rate constant to the

285

test organism (L kg-1 d-1); and ke is the PAH elimination rate constant from the test organism

286

(d-1). The integration form of the above equation was obtained as follows:

287

Cw ku − Cb ke = e − ke (t − A)

(5)

288

where A is an integral constant. According to the bioaccumulation curves of PAHs (Figure 1),

289

uptake dominated at the beginning of exposure (t<2 d), and elimination dominated after the

290

peak value was achieved. Therefore, in the initial 2 days, Cw ku1 − Cb ke1 > 0 . Considering this

291

initial integral condition, Cb was obtained as follows:

292 293

Cb = Cw

(

)

ku 1 ⋅ 1 − e − ke1 ⋅t , t < 2 ke1

(6)

After exposure for 2 days, C w ku 2 − Cb ke 2 < 0 . With this initial integral condition, Cb was 12

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obtained as follows:

Cb = Cw

ku 2  k  +  C peak − Cw u 2  ⋅ e − ke 2 ⋅(t − 2 ) , t ≥ 2 ke 2  ke 2 

(7)

296

where ku1 and ke1 are the uptake and elimination rate constants of PAHs during the initial 2 days,

297

respectively; ku2 and ke2 are those after exposure for 2 days; Cpeak and Cw

298

value and the final equilibrium value of body burden in the test organism, respectively.

ku 2 refer to the peak ke 2

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All of the blank values were deducted before dynamic fitting to obtain ku1, ke1, ku2, and ke2

300

for each PAH. As shown in Table 1 and Table 2, for each bioaccumulation curve, ku1 and ku2

301

were almost identical, suggesting that the uptake rate constant was stable for the whole

302

exposure period. However, the elimination rate constant ke2 was remarkably higher than ke1,

303

with the increase ranging from 78.2% to 113% for all PAHs at 1µg L-1, 5µg L-1 or 10µg L-1,

304

indicating that elimination rate constant increased after the peak. This increase might be

305

because enzymes in fish that participate in PAH metabolism are activated as PAH

306

concentrations increase in organisms.39-41

307

According to the results shown in Table 1, with or without predation, no significant

308

change in kinetic constant values, including both ku and ke, was observed with increased freely

309

dissolved PAH concentration. However, compared to the groups in the absence of predation,

310

both the uptake rate constants (ku1 and ku2) and elimination rate constants (ke1 and ke2) of PAHs

311

in zebrafish were elevated with predation on D. Magna, with the increase ranging from 20.8%

312

to 39.4% for ku1, 20.4% to 38.1% for ku2, 5.9% to 17.7% for ke1, and 25.3% to 38.6% for ke2 at

313

10 µg L-1 PAH exposure. Furthermore, both the uptake and elimination rate constants of PAHs

314

increased with the amount of predation (Table 2).

315 316 317

To analyze the mechanism by which predation influences the bioaccumulation kinetics of PAHs in zebrafish, equation (4) was modified as follows by considering the predation process:

dCb = Cw ⋅ k1 + C f ⋅ k2 − Cb ⋅ ke dt

(8)

318

where k1 (L kg-1 d-1) and k2 (d-1) are the uptake rate constants of PAHs from the water and prey,

319

respectively; Cf is the PAH concentration in the prey (µg kg-1 dry weight); and the other 13

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320

parameters are the same as in equation (4). Because the prey (D. magna) reached

321

bioaccumulation equilibrium under the same freely dissolved PAH concentrations as the

322

predator (zebrafish) before predation, the PAH concentrations in D. magna (Cf) can be

323

calculated as BAFD·CW, where BAFD is the water-based bioaccumulation factor of PAHs in D.

324

magna (L kg-1). Then, C f ⋅ k2 can be calculated as n ⋅ BAFD ⋅ Cw ⋅

325

of predation of D. magna per fish per day (d-1); WD and WZ are the dry weight of one D. magna

326

and zebrafish (kg-1), respectively. Thus, k2 is equal to n ⋅

327

transformed as follows:

WD , where n is the amount WZ

WD , and equation (8) can be WZ

 dCb W  = Cw ⋅  k1 + n ⋅ BAFD ⋅ D  − Cb ⋅ ke dt WZ  

328

329

It is apparent that the ku value in equation (4) is equal to k1 + n ⋅ BAFD ⋅

330

D. magna, and n ⋅ BAFD ⋅

(9)

WD with predation on WZ

WD is the contribution of predation to the ku value. WZ

331

Because the dry weight of each test D. magna or zebrafish was almost identical, it can be

332

deduced that the increase of uptake rate constants for PAHs in zebrafish caused by predation

333

would be positively correlated with the amount of predation on D. magna and the BAFD values

334

of PAHs. These theoretical linear correlations were demonstrated by the results shown in

335

Figure S2 and Figure S3. The increase in the ku value for each PAH caused by predation

336

increased linearly with the amount of predation on D. magna, and the increase in ku for PAHs

337

increased linearly with their BAFD values when the amount of predation was 5 or 10 daphnids

338

per fish per day. Furthermore, as shown in Table S5, the measured increase in the values of

339

PAH uptake rate constants in zebrafish caused by predation on D. magna was consistent with

340

the increase predicted by equation (9). These data indicate that zebrafish can accumulate PAHs

341

from D. magna by ingestion, causing an additional increase in the PAH uptake rate. In addition,

342

as shown in Figure 2, the peak values of PAHs in zebrafish increased remarkably in the

343

presence of predation on D. magna, with the increase ranging from 30.1% to 44.1%, from 14

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20.4% to 30.1%, from 17.2% to 23.9%, and from 37.7% to 45.0% for phenanthrene,

345

anthracene, fluoranthene, and pyrene, respectively (Table S6), and the peak value of each PAH

346

in zebrafish increased linearly with the amount of predation on D. magna (Table S7).

347

Meanwhile, as shown in Table 2, the elimination rate constants ke1 and ke2 both increased

348

with amount of predation as well. Because the final BAF value of each PAH did not change

349

with the variation in amount of predation, it can be deduced that the PAH elimination rates in

350

zebrafish were also elevated to maintain the bioaccumulation balance under predation

351

conditions as a type of self-adaptive mechanism. Therefore, the increase in the uptake and

352

elimination rate constants suggests that PAH metabolism in zebrafish, including assimilation

353

and dissimilation, might be enhanced by predation.

354

3.3 Influence of Predation on PAH Bioaccumulation in Cichlids

355

The bioaccumulation curves of PAHs in cichlids were similar to those in zebrafish. As

356

shown in Figure 3, the body burden of PAHs in cichlids increased with time in the initial 2 days,

357

then decreased and reached bioaccumulation equilibrium after exposure for 14 days.

358

Furthermore, similar to the effect of predation on PAH bioaccumulation in zebrafish, predation

359

elevated the uptake and elimination rate constants of PAHs as well as the peak body burden of

360

PAHs, while it did not affect the final bioaccumulation equilibrium in cichlids (Table 3 and

361

Table S8). For example, the BAF values of phenanthrene in cichlids were 675±90 L kg-1 and

362

685±99 L kg-1 in the presence and absence of predation on zebrafish, respectively.

363

In addition, the lipid-normalized bioaccumulation factors (BAFlip= BAF/lipid content) of

364

each PAH in the fish of two different trophic levels were almost identical (Table 3); this finding

365

confirms that the final bioaccumulation equilibrium of PAHs is related to a partition between

366

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

367

bioaccumulation equilibrium in the presence of stable freely dissolved PAH concentrations.

368

This conclusion is in agreement with the speculation proposed by Chiou14 and it is also

369

supported by the positive correlation between the logBAF and logkow values of HOCs reported

370

by many researchers.15,42-44

371 372

Therefore, the bioaccumulation kinetics of HOCs in aquatic organisms can be described with the following model when considering the predation process: 15

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W   dCb = Cw ⋅  k w + n ⋅ BAFprey ⋅ prey  − Cb ⋅ ke dt W  

374

where Cb is the HOC concentration in the test organism (µg kg-1); Cw is the HOC concentration

375

in the water phase (µg L-1); t is the exposure time (d); kw is the HOC uptake rate constant from

376

the water phase (L kg-1 d-1); n is the number of prey per organism per day; BAFprey is the

377

water-based bioaccumulation factor of HOC in prey (L kg-1); Wprey and W are dry weight of one

378

prey and test organism (kg-1), respectively; and ke is the HOC elimination rate constant from

379

the test organism (d-1). In addition, if a predator feeds on several types of prey, their

380

contribution to the uptake rates of HOCs in this predator can be combined. The cause of the

381

reported phenomena of HOC biomagnification along the food chain of aquatic ecosystems can

382

be analyzed as follows. If HOCs have not reached bioaccumulation equilibrium,

383

biomagnification occurs due to the enhanced uptake rates caused by predation in higher trophic

384

organisms in addition to higher lipid contents. If HOCs have reached bioaccumulation

385

equilibrium, the biomagnification is only due to the higher lipid contents in organisms at higher

386

trophic levels. Besides, the equilibrium body burden of HOCs in aquatic organisms may not

387

increase with trophic level. For instance, the equilibrium body burden of PAHs in D. magna

388

(lower trophic level) was higher than those in zebrafish and cichlids (higher trophic level) in

389

the present study (Table S9).

(10)

390

According to the findings obtained in the present study, in natural aquatic ecosystems, if

391

HOC bioaccumulation in organisms reaches equilibrium, the body burden of HOCs will be

392

dependent on the freely dissolved concentrations of HOCs in water phase; predation will not

393

affect the bioaccumulation factor. However, because predation enhances the uptake rates of

394

HOCs, the body burden of HOCs in predators before reaching equilibrium will be positively

395

correlated with their amount of predation, the BAF values of HOCs in the prey, and the body

396

weight of the prey. The peak values of HOCs in predators will be significantly elevated due to

397

the predation. In this case, predation will increase the ecological risk of HOCs. In addition, the

398

bioavailable concentrations of HOCs (e.g., freely dissolved concentrations) are affected by

399

environmental water conditions including dissolved organic matter (DOM), colloids,

400

suspended sediments, cations and so on,29,45,46 and HOCs associated with DOM, colloids, and 16

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401

suspended sediments might also be absorbed by organisms through the ingestion of DOM and

402

particles. Therefore, further research should be carried out to study the comprehensive effects

403

of environmental conditions and predation as well as ingestion on HOC bioaccumulation in

404

aquatic organisms.

405

Acknowledgements

406

The study was supported by the Fund for Innovative Research Group of the National

407

Natural Science Foundation of China (51421065), National Science Foundation for

408

Distinguished Young Scholars (51325902), and National Science Foundation of China

409

(51279010).

410

References

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Table 1 Bioaccumulation kinetic parameters of PAHs in zebrafish with and without predation on D. Manga.

PAHs

Phenanthrene

Anthracene

Fluoranthene

Pyrene

Exposure

ku1(L kg-1 d-1)

ku2(L kg-1 d-1)

ke1(d-1)

BAF (L kg-1)

ke2(d-1)

P

NP

P

NP

P

NP

P

NP

P

NP

10µg/L

365±27

286±26

0.31±0.01

0.25±0.01

377±35

273±48

0.66±0.02

0.47±0.01

574±59

576±33

5µg/L

381±35

275±31

0.30±0.01

0.25±0.01

366±47

262±14

0.65±0.02

0.45±0.01

564±44

583±43

1µg/L

373±41

268±53

0.29±0.01

0.23±0.01

353±37

280±37

0.66±0.01

0.48±0.02

543±82

579±37

10µg/L

435±56

312±73

0.31±0.03

0.27±0.02

429±32

332±31

0.68±0.02

0.51±0.02

627±66

657±58

5µg/L

423±65

346±35

0.30±0.02

0.28±0.01

419±38

333±36

0.70±0.01

0.52±0.02

596±95

635±91

1µg/L

451±62

334±47

0.30±0.02

0.28±0.02

438±46

319±46

0.70±0.02

0.50±0.01

629±51

641±102

10µg/L

645±85

534±35

0.38±0.031

0.34±0.02

666±25

553±56

0.76±0.01

0.58±0.02

875±96

928±91

5µg/L

679±71

483±43

0.38±0.02

0.32±0.01

648±78

497±37

0.74±0.02

0.57±0.01

881±87

867±89

1µg/L

685±63

495±45

0.37±0.02

0.32±0.01

702±41

483±59

0.76±0.01

0.57±0.02

923±92

849±83

3

10µg/L

914±54

753±85

0.36±0.021

0.33±0.01

908±72

699±68

0.75±0.02

0.60±0.01

1.21×10 ±79

1.17×103±103

5µg/L

898±71

794±85

0.38±0.012

0.33±0.01

898±63

737±58

0.77±0.03

0.61±0.02

1.17×103±95

1.21×103±92

1µg/L

905±57

766±53

0.38±0.016

0.35±0.01

928±77

712±67

0.76±0.02

0.60±0.02

1.22×103±101

1.20×103±72

* P = with predation; NP = without predation * The initial part 1 (0~2d) and the subsequent part 2 (2~16d) of the PAH bioaccumulation curves were fitted by equations (3) and (4), respectively.

51

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Table 2 Bioaccumulation kinetic parameters of PAHs in zebrafish with different amounts of predation on Daphnia Manga (daphnids per fish per day)

54

at 10 µg L-1 PAH exposure

55 PAH

log kow

56

57

Phenanthrene

4.54

58

59

Anthracene

4.57

60 Fluoranthene

5.18

61

62

63

64

Pyrene

5.98

Amount of predation

ku1(L kg-1 d-1)

Ke1(d-1)

0

288±15

0.27±0.01

275±18

0.47±0.01

586±39

5

361±26

0.31±0.01

380±26

0.65±0.02

574±67

10

432±37

0.33±0.02

474±35

0.73±0.03

585±54

0

317±20

0.27±0.01

338±29

0.51±0.02

627±53

5

429±22

0.31±0.01

423±44

0.68±0.03

644±91

10

489±19

0.33±0.02

510±25

0.80±0.02

630±79

0

533±31

0.35±0.01

551±51

0.57±0.02

901±74

5

641±34

0.37±0.02

672±43

0.77±0.02

846±91

10

753±40

0.39±0.02

778±67

0.85±0.04

877±77

0

749±55

0.31±0.01

693±64

0.59±0.02

1.18×103±72

5

920±83

0.37±0.02

914±71

0.75±0.02 1.26×103±117

ku2(L kg-1 d-1) Ke2(d-1)

BAF (L kg-1)

1.10×103±89 0.38±0.02 1.06×103±69 0.86±0.03 1.19×103±102

10 * log kow values of PAHs are from Neely.32

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Table 3 Bioaccumulation kinetic parameters of PAHs in cichlids with and without predation on zebrafish at 10 µg L-1 PAH exposure

68

69

70

PAH

71

Phenanthrene

72 Anthracene

73 Fluoranthene

74

75

76 77

Pyrene

ku1(L kg-1d-1) (cichlids)

Ke1(d-1) (cichlids)

ku2(L kg-1d-1) (cichlids)

Ke2(d-1) (cichlids)

BAF (L kg-1) (cichlids)

BAF (L kg-1) (zebrafish)

With

349±21

0.22±0.01

358±23

0.53±0.01

675±90

576±13

3.89±0.003 3.88±0.003

without

312±34

0.20±0.01

303±18

0.44±0.01

685±99

581±26

3.89±0.007 3.89±0.002

With

453±23

0.25±0.01

445±30

0.59±0.01

761±53

635±37

3.94±0.005 3.92±0.008

without

374±18

0.22±0.01

361±19

0.48±0.01

743±66

640±29

3.93±0.007 3.93±0.006

Predation

3

logBAFlip (cichlid)

logBAFlip (zebrafish)

With

731±42

0.35±0.02

727±69

0.65±0.02

1.12×10 ±85

894±49

4.11±0.012 4.07±0.007

without

625±62

0.31±0.01

605±54

0.59±0.01 1.10×103±102

902±51

4.10±0.008 4.08±0.009

3

3

With

924±61

0.35±0.02

908±87

0.67±0.02 1.36×10 ±105 1.24×10 ±66 4.19±0.020 4.21±0.012

without

755±59

0.31±0.01

739±57

0.55±0.02 1.33×103±110 1.24×103±93 4.18±0.011 4.22±0.021

* On average, the lipid content in dry weight is 8.77±0.15% for cichlid, and 7.56±0.11% for zebrafish.

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Figure captions

580 581 582 583

Figure 1 Bioaccumulation curves of PAHs in zebrafish with and without predation on Daphnia

584

magna at different PAH exposure levels (Mean±standard deviation, n=3)

585 586

Figure 2 Bioaccumulation curves of PAHs in zebrafish with different amounts of predation on

587

Daphnia magna (daphnids per fish per day) at 10 µg L-1 PAH exposure (Mean±standard

588

deviation, n=3).

589 590

Figure 3 Bioaccumulation curves of PAHs in cichlids with and without predation on zebrafish

591

at 10 µg L-1 PAH exposure (Mean±standard deviation, n=3).

592 593 594 595 596 597 598 599 600 601 602 603 26

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

12000

15000

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

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, predation

12000

9000

6000 6000 3000 3000

0

0 0

3

6

18000

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

Anthracene

9

12

15

12000

3

6

24000

Fluoranthene 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 predation

15000

0

9000

9

12

15

Pyrene 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 predation

18000

12000

6000 6000 3000

0

0 0

3

6

9

12

0

15

3

6

9

12

15

Exposure time (d)

Exposure time (d)

604

Figure 1 Bioaccumulation curves of PAHs in zebrafish with and without predation on Daphnia

605

magna at different PAH exposure levels (Mean±standard deviation, n=3)

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15000

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

Phenanthrene

Anthracene

18000

0 D.magna per fish per day

12000

5 D.magna per fish per day 10 D.magna per fish per day

9000

0 D.magna per fish per day 15000

5 D.magna per fish per day 10 D.magna per fish per day

12000

9000 6000 6000 3000 3000

0

0 0

3

6

9

12

0

15

3

6

9

12

15

21000 30000

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

Pyrene

Fluoranthene

18000

0 D.magna per fish per day

15000

0 D.magna per fish per day

24000

5 D.magna per fish per day

5 D.magna per fish per day

10 D.magna per fish per day

12000

10 D.magna per fish per day

18000

9000 12000 6000 6000 3000 0

0 0

3

6

9

12

0

15

3

6

Exposure time (d)

9

12

15

Exposure time (d)

614 615

Figure 2 Bioaccumulation curves of PAHs in zebrafish with different amounts of predation on

616

Daphnia magna (daphnids per fish per day) at 10 µg L-1 PAH exposure (Mean±standard

617

deviation, n=3)

618 619 620 621 622 623

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24000

18000

Anthracene

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

Phenanthrene 20000

15000

without predation

without predation

with predation 12000

16000

9000

12000

6000

8000

3000

4000

0

0 0

3

6

9

12

25000

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

with predation

15

18

3

6

9

12

15

18

21

30000

Fluoranthene

Pyrene

without predation

20000

0

21

without predation

24000

with predation

with predation

15000

18000

10000

12000

5000

6000

0

0 0

3

6

9

12

15

18

0

21

3

6

Exposure time (d)

9

12

15

18

21

Eexposure time (d)

624

Figure 3 Bioaccumulation curves of PAHs in cichlids with and without predation on zebrafish

625

at 10 µg L-1 PAH exposure (Mean±standard deviation, n=3)

626 627 628 629 630 631 632 633 29

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