Bioavailability of Pyrene Associated with Different Types of Protein

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Environmental Processes

Bioavailability of Pyrene Associated with Different Types of Protein Compounds: Direct Evidence for Its Uptake by Daphnia magna Hui Lin, Xinghui Xia, Xiaoman Jiang, Siqi Bi, Haotian Wang, Yawei Zhai, Wu Wen, and Xuejun Guo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03349 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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Environmental Science & Technology

Bioavailability of Pyrene Associated with Different Types of Protein

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Compounds: Direct Evidence for Its Uptake by Daphnia magna

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Hui Lin, Xinghui Xia*, Xiaoman Jiang, Siqi Bi, Haotian Wang, Yawei Zhai, Wu Wen, Xuejun Guo

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

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100875, China

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*

Corresponding author. Phone: +86 10 58805314.

E-mail: [email protected] 1

ACS Paragon Plus Environment


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Abstract The protein-like dissolved organic matter (DOM) is ubiquitous in aquatic environments.

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However, the bioavailability of protein-like DOM-associated hydrophobic organic compounds (HOCs)

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is not well understood, and in particular, the direct evidence of their uptake by organisms is scarce. In

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the present work, the tryptone (2 000 Da), bovine serum albumin (BSA, 66 000 Da), and phycocyanin

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(120 000 Da) were chosen as model protein-like DOM, which were labelled by commercial

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fluorescein (cy5) to investigate the uptake mechanisms of protein compound-associated pyrene (a

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typical HOC) by D. magna. The pyrene concentration in the tissues except gut and immobilization of

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D. magna were detected to calculate the bioavailable fraction of protein compound-associated pyrene

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when the freely dissolved pyrene concentration was controlled through passive dosing devices. The

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results demonstrated that the tryptone could permeate cellular membrane and directly enter into the

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tissues of D. magna from the exposure solutions, whereas the BSA and phycocyanin might indirectly

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enter into the tissues from the gut. A part of pyrene associated with protein compounds was

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bioavailable to D. magna; the order of their bioavailable fractions was trypone (54.6−58.1%) >

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phycocyanin (21.6−32.8%) > BSA (17.7−26.8%); the difference was principally related to the uptake

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mechanisms of pyrene associated with different types of protein. This work suggests that the protein

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compound-associated

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eco-environmental hazard of HOCs in natural waters.

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Key words: Bioavailability; Uptake mechanisms; Protein compounds; Passive dosing devices;

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Immobilization; Freely dissolved concentration

HOCs

should

be

considered

to

evaluate

46 47 48 49 50 2


the

bioavailability

and

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TOC Graphic

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1. INTRODUCTION

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Hydrophobic organic compound (HOC) pollution in waters is a great environmental concern and

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can pose a threat to organisms as well as human health. HOCs can be associated with protein

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compounds which contain a high content of hydrophobic groups through hydrophobic interaction.1–3

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In natural aquatic environments, protein compounds are important components of dissolved organic

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matter (DOM) in addition to fulvic-like and humic-like substances.4–6 Protein-like DOM is widely

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distributed in natural waters,7–9 and its components as well as structures have been extensively studied

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by fluorescence and UV absorption spectrum.10,11 Protein-like DOM is a key carrier of HOCs and the

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association of HOCs with them can greatly control the distribution, fate, and bioavailability of

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HOCs.12,13

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Some previous studies showed that the addition of DOM to the exposure systems could decrease

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the bioavailability of HOCs through reducing the concentrations of freely dissolved HOCs,14–17 while

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on the other hand it could elevate the bioavailability of HOCs through the effect of DOM-promoted

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diffusive mass transfer.18–20 Recent research demonstrated that a part of pyrene associated with fulvic

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and humic acid was bioavailable to D. magna, which was indirectly proved by controlling the freely

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dissolved pyrene concentration with PDMS devices.21 However, whether the DOM-associated HOCs

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can pass through the membrane and be directly absorbed by organisms is not clear, and it needs to be

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verified with direct evidence.

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So far, the related studies were mainly focused on investigating the effect of nonprotein-like

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DOM (such as humic and fulvic acid) on the bioavailability of HOCs;22,23 the report concerning the

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influence of protein-like DOM on the bioavailability of HOCs was scarce. Only a few studies have

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found that the bioaccumulation of perfluoroalkyl substances by D. magna was elevated by low

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concentrations of protein compounds and decreased by high concentrations of protein

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compounds.3,12,24 Hence, the influencing mechanisms of protein-like DOM on the bioavailability of 4


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HOCs are not well-understood, especially the direct evidence on the bioavailability and uptake of HOCs associated with protein-like DOM to organisms is lacked.

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It has been found that the sorption ability of DOM to HOCs was related to DOM molecular

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weights,25,26 and the bioaccumulation of HOCs by organisms could be affected by the types and

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molecular weights of protein compounds in water.12 Furthermore, the substances with small molecular

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weight were easier to pass through cell membrane compared to that with big molecular weight.27 For

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example, because the molecular weights of organic fluorescent dyes including fluorescein, rhodamine,

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and cyanine are lower than 1000 Da, they could pass through cell membrane freely and have been

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applied to bioimaging field.28–30 Accordingly, we hypothesize that the effect of protein-like DOM on

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the bioavailability of HOCs might depend on the types and molecular weights of protein-like DOM.

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To test this hypothesis, we chose pyrene (one kind of polycyclic aromatic hydrocarbon, PAHs) as

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a representative HOC to investigate the uptake mechanisms and the bioavailability (chemical activity

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and accessibility) of HOCs associated with different types of protein compounds to model organism D.

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magna. Tryptone, bovine serum albumin (BSA), and phycocyanin, which are abundant in natural

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environments, were chosen as model protein-like DOM. The passive dosing systems31,32 were used to

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hold the freely dissolved concentration (Cfree) of pyrene during the experiment; the concentrations of

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total and protein compound-associated pyrene were changed with the concentrations and types of

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protein compounds. The D. magna immobilization and the pyrene concentration in the tissues without

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gut of D. magna were investigated to calculate the bioavailability of pyrene associated with three

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types of protein compounds. Furthermore, because protein compounds could be labelled with

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fluorescent probes for biological imaging,33–36 tryptone and BSA labelled with fluorescein cy5 as well

120

as fluorescent phycocyanin were used to provide direct evidence for the uptake mechanisms of pyrene

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associated with protein compounds by D. magna through analyzing the distribution of protein

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compounds in D. magna with a laser confocal microscope.

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2. MATERIALS AND METHODS

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2.1. Test Chemicals

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Pyrene (>98%), meta-terphenyl (>98%, internal standard), and 2-fluorobiphenyl (>97%,

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surrogate standard, CAS no. 321-60-8) in solid phase were obtained from J&K Scientific, Ltd.

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(Beijing, China). Tryptone (2 000 Da on average) and BSA (66 000 Da on average) in solid phase

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were obtained from Sigma-Aldrich, Ltd. (U.S.A.), and the fluorescent phycocyanin (120 000 Da on

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average, λex = 647 nm) in solid phase were bought from Taizhou Binmei Biotechnology Co., Ltd

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(Taizhou, China) (Table S1). The tryptone and BSA labelled with fluorescent dye cy5 were purchased

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from Biosynthesis Biotechnology Co., Ltd. (Beijing, China), and the cy5 (792 Da, λex = 649 nm, λem =

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670 nm) was purchased from Sigma-Aldrich, Ltd. (U.S.A.). Poly-(dimethylsiloxane) (PDMS)

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elastomer was bought from Dow Corning Co. Ltd. (Shanghai, China). PAHs standard solution was

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acquired from AccuStandard, Inc. (New Haven, CT, U.S.A.). The methanol, n-hexane, and

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dichloromethane, which are HPLC grade, were obtained from J&K Scientific Ltd. (Beijing, China).

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The ultrapure water (Millipore, USA) was used in the present work.

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2.2. Establishment of Passive Dosing Dishes and Preparation of Exposure Solutions

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The passive dosing dishes, which were prepared on the basis of our previous study (details are in

139

Supporting Information),37,38 were used to hold the Cfree of pyrene constant during the experiment.

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Based on the partition coefficient (Kmethanol:AFW, 8.07 × 104) of pyrene between the methanol loading

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solution and artificial freshwater (AFW) reported in our previous work,21,39,40 the pyrene

142

concentration in methanol (3.6 g L–1) was prepared to obtain the constant Cfree of pyrene (45 µg L–1)

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in the present work. The PDMS dishes were placed into this methanol containing pyrene which were

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renewed every 24 h. After 72 h, each PDMS dish was washed by ultrapure water and then transferred

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to the protein solutions of different concentrations and types. Then, they were used as exposure

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solutions for the next biological experiment.

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2.3. Experiments on Immobilization of D. magna and Pyrene Concentration in D. magna

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To investigate the influence of protein compounds on D. magna, the protein solutions (1.0, 10.0,

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and 30.0 mg L–1) in the absence of pyrene were prepared with AFW, and 30 D. magna (6-24 h) were

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chosen and put in each beaker containing protein solution. Then, the beakers were put in a RXZ-500B

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temperature-monitored artificial climate incubator (Beijing, China), which was controlled at a

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condition of 23 ± 1 °C, photoperiod 16:8 (light/dark), and light intensity of 2300 lx. The D. magna,

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which did not move after 15 seconds, was monitored every 12 h until the end of experiment (48 h).

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Then, the immobilization (%) of D. magna was acquired. The control experiments were conducted in

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the AFW without protein compounds. Each experiment was carried out in triplicate.

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To investigate the influence of pyrene associated with protein compounds on D. magna

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immobilization in the solutions with pyrene, a series of 1, 10, and 30 mg L–1 protein solution (500 mL)

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were prepared in 1 L beaker (Figure S1). The Cfree of pyrene was held at 45.0 µg L–1 constantly by

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placing two PDMS dishes loaded with pyrene into each beaker. The parafilm being punched by

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syringe was used to cover each experimental beaker. These beakers were transferred to the shaking

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incubator (85 rpm, 24 °C, dark). After 3 days (reaching equilibrium), a part of solution (200 mL) was

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taken out from the beaker to analyze Cfree of pyrene and the total pyrene concentration (The detailed

163

analytical method is depicted in Supporting Information), and 300 mL of exposure solution was

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remained in the beaker. Then, a total of 30 D. magna (6−24 h old) were put in this beaker. The D.

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magna immobilization (%) was monitored every 12 h until the end of experiment (48 h). All of the D.

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magna were taken out from the beaker with glass pipette at the end of the immobilization experiment.

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Subsequently, 20 D. magna (14 days old) with similar size were put in this beaker. Then, the D.

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magna alive were collected from each exposure solution after exposure for 5 h and 48 h, respectively. 7



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To eliminate the effect of pyrene associated with protein compounds in the gut on the determination

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of pyrene concentration absorbed by D. magna, the collected D. magna were dissected under a

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stereomicroscope of Tech XTS30-HDMI (Beijing, China) to obtain the tissues and gut of D. magna,

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respectively. Then, they were placed into refrigerator (−20 °C) until the analysis of pyrene content in

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D. magna. For both the immobilization and pyrene content experiments, the control experiments were

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carried out in the pyrene solutions without protein compounds. The blank experiments were

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conducted in the AFW and in the protein solutions without pyrene, respectively.

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Meanwhile, to obtain the uptake curves of pyrene by the D. magna, the pyrene concentration in

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the tissues in the control systems containing 45 µg L–1 pyrene and the test systems containing 45 µg

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L–1 pyrene and 10 mg L–1 protein compound was respectively detected after exposure for 1, 3, 7, 11,

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24, 36, and 48 h (The detailed analytical method is depicted in Supporting Information). To determine

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the difference in lipid content of D. magna between the groups with and without protein compounds,

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another experiment was conducted by adding 120 D. magna (14 days old) into the control group

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containing 45 µg L–1 pyrene and the test group containing 45 µg L–1 pyrene and 10 mg L–1 protein

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compounds, respectively based on the method reported by Leslie et al.41 (The detailed analytical

184

method is depicted in Supporting Information). Each experiment was carried out in triplicate.

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To obtain the correlation curves between the Cfree of pyrene and the D. magna immobilization in

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the solutions only with pyrene, a series of 30, 45, and 60 µg L–1 pyrene solutions in AFW were

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prepared by PDMS device, the preparing method of which was similar to that mentioned above

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(section 2.2). Then, 30 D. magna (6−24 h old) were put in each pyrene solution. The D. magna

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immobilization (%) was monitored every 12 h until the end of experiment (48 h). Each experiment

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was carried out in triplicate. All the immobilization experiments of D. magna at various conditions

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above were conducted at the same time.

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2.4. Uptake of Protein Compounds by D. magna Investigated by Laser Scanning Confocal 8


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Microscopy

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To determine whether the protein compound-associated pyrene can be absorbed by D. magna

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directly, the uptake of protein compounds by D. magna was investigated. The solutions of 83.0 mg L–

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1

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according to the results of the fluorescent protein standard curve through fluorescence spectrum

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experiments of these fluorescent protein compounds (Figures S2 and S3, the detailed analytical

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method is depicted in Supporting Information). A total of 30 D. magna (6−24 h) were collected and

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transferred to 1 L AFW at least 12 h to empty their gut, and then 10 D. magna were placed in each

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100 mL fluorescent protein solutions. Subsequently, three D. magna were randomly taken out and

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detected by laser scanning confocal microscopy at 5 h, 24 h, and 48 h, respectively. The luminescence

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emission of D. magna was collected at 600−700 nm as detection signals and the laser irradiation was

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chosen as 635 nm. The blank experiments were carried out in the AFW without protein compounds.

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2.5. Chemical and Biochemical Analysis

tryptone-cy5, 99.2 mg L–1 BSA-cy5, and 72.1 mg L–1 phycocyanin were prepared, respectively

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The PDMS fiber and liquid-liquid extraction techniques were respectively applied to analyze the

207

concentrations of freely dissolved pyrene and total pyrene in solutions during the whole experiments

208

based on the previous study.42–44 In brief, a piece of 1 cm PDMS fiber (Polymicro Tech) was put in the

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exposure solution to detect the freely dissolved pyrene concentration, and the exposure solution was

210

transferred into the separating funnel to analyze the total pyrene concentration by the liquid-liquid

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extraction techniques. Then, the protein compound-associated pyrene concentration could be obtained

212

from subtracting the freely dissolved pyrene concentration from the total pyrene concentration. The

213

details for these procedures are depicted in Supporting Information. The analysis method of pyrene

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concentration in the tissues and gut of D. magna (ng/one D. magna) was on the basis of the previous

215

investigation;21 the detailed analytical method is depicted in Supporting Information.

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2.6. Quality Assurance and Quality Control

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The pyrene standard curve (5−1000 µg L−1) detected with GC-MS was corrected with 100 µg L–1

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meta-terphenyl solution (internal standard), and the correlation coefficient for this curve was higher

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than 0.99. The pyrene detection limit of GC-MS was 0.05 µg L−1. The recoveries of pyrene in the

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solutions containing protein compounds and D. magna were 88.9 ± 7.8% and 89.6 ± 9.8%,

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respectively. The Cfree of pyrene was kept at 45 µg L−1, and the variation was ≤2% during the

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exposure experiment (Table S2). The dissolved oxygen was 8.10 ± 0.12 mg L–1 and the pH was 7.00 ±

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0.05, which was stable during the whole experiments. The pyrene content in D. magna incubated in

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solutions of protein compounds or AFW without pyrene could not be detected by GC-MS. The toxic

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sensitivity of D. magna to potassium dichromate (standard compound) was detected, and the EC50 at

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24 h was 0.6 mg L–1, which was within the range (0.50 mg L–1 and 1.2 mg L–1) suitable for toxicity

227

test.45

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3. RESULTS AND DISCUSSION

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3.1. Effect of Protein Compounds Itself without Pyrene on the Immobilization of D. magna

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The D. magna immobilization in the protein solutions without pyrene was lower than that in the

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control only containing AFW (Figure S4). For example, the D. magna immobilization in the control

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reached 100% at 120 h; however, it was only 16.7−46.7 % and 16.7−40.0% in the 10 and 30 mg L–1

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protein solutions at 120 h, respectively. These results indicated that the food effect of tryptone, BSA,

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and phycocyanin (10, 30 mg L–1) on D. magna was significant, and it might reduce the

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immobilization of D. magna. It might be attributed to that the protein compounds could provide

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nutrient substances to D. magna, which was similar to nonprotein-like DOM.46 In addition, the order

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of protein compound effect on D. magna immobilization was tryptone (2 000 Da) > BSA (66 000 Da) >

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phycocyanin (120 000 Da). For example, the D. magna immobilization was 46.7%, 56.7%, and 75%

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in the systems with 10 mg L–1 tryptone, BSA, and phycocyanin, respectively after exposure for 144 h.

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The results suggested that the protein with smaller molecular weight exerted stronger food effect to D.

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magna.

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When the concentration of protein compounds ranged from 0 to 10 mg L–1, the D. magna

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immobilization decreased significantly (Figure 1); however, when the concentrations of protein

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compounds ranged from 10 to 30 mg L–1, the decease of D. magna immobilization was not significant

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which indicated that the food effect of protein compounds at different levels (10 and 30 mg L–1) was

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identical. The reason was that the protein compounds at low levels were insufficient to provide

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essential food energy for D. magna, but 10 mg L–1 protein compounds could provide enough food

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energy for D. magna. The food effect of protein compounds with different concentrations found in

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this study was similar to the earlier reported results that the food effect of 10 mg C L–1 humic acid

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equaled that of 30 mg C L–1 humic acid, a kind of nonprotein-like DOM.21

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3.2. Effect of Protein Compounds on the Immobilization of D. magna in the Presence of Pyrene

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Because the freely dissolved concentration of pyrene was kept at 45 µg L–1 with PDMS devices

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for both systems in the presence and absence of protein compounds, the pyrene associated with

254

protein compounds existed in the exposure solutions in addition to freely dissolved pyrene. As shown

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in Table S3, the concentrations of protein compound-associated pyrene in the exposure solutions and

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the partition coefficients (Kp, L Kg–1) of pyrene between the protein compounds and water were

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ordered as phycocyanin > BSA > tryptone. For example, in the 10 mg L–1 protein systems, the

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concentrations of pyrene associated with tryptone, BSA, and phycocyanin were 31.2, 37.8, and 45.3

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µg L–1, respectively, and the logKp of pyrene were 4.65, 4.92, and 5.00 in the tryptone, BSA, and

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phycocyanin systems, respectively. These results were comparable to the previous studies that the

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logKBSA/W (BSA-water partition coefficient, L Kg–1) of pyrene was 4.76,47 the logKp (soy

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peptone-water partition coefficients, L Kg–1) of perfluoroalkyl substances was 4.11−5.49,12 and the 11



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logKdoc (organic carbon normalized partition coefficient, L Kg–1 C–1) of pyrene between the humic

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acid and water was 3.92−5.53.21,48,49

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As shown in Figure 2, after exposure for 48 h, the D. magna immobilization was 48.3% in the

266

control systems without protein compounds, which was comparable to the results that 48 h D. magna

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immobilization at 28 µg L–1 freely dissolved pyrene was about 50%50 as well as that 48 h D. magna

268

immobilization at 40 µg L–1 freely dissolved pyrene was about 36%.37 Compared to the control

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systems without protein compounds, the immobilization of D. magna in the protein systems was

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elevated at 24, 36, and 48 h of exposure. For example, after exposure for 36 h, the immobilization of

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D. magna was enhanced by 25.2%, 46.2%, and 55.2% in the systems containing tryptone, BSA, and

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phycocyanin (10 mg L–1), respectively in comparison with the control systems without protein

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compounds. These results suggested that pyrene associated with protein compounds could induce

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extra toxicity in comparison with the control systems only containing pyrene. As shown in Figure S5,

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the enhancement of immobilization caused by the pyrene associated with protein compounds

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increased with increasing concentrations of protein compounds. For example, compared to the control

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systems without protein compounds, the immobilization of D. magna increased by 6.7%, 25.2%, and

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49.2% in the systems containing 1, 10, and 30 mg L–1 tryptone, respectively, and increased by 10.2%,

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45.0%, and 70.3% in the systems containing 1, 10, and 30 mg L–1 BSA, respectively, and increased by

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15.0%, 55.2%, and 85.3% in the systems containing 1, 10, and 30 mg L–1 phycocyanin, respectively

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after exposure for 36 h. These results demonstrated that the increase of D. magna immobilization

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caused by adding the protein compounds into the pyrene solution was related to the concentration and

283

types (molecular weights) of protein compounds.

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It should be noted that at a 12 h of exposure, the D. magna immobilization in the systems with 1

285

or 10 mg L–1 protein compounds was lower than the control systems without protein compounds. This

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might be attributed to that the pyrene accumulation by D. magna didn’t reach steady state at 12 h

287

(Figure S6); the food effect of protein compounds exceeded the toxicity caused by pyrene associated 12


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with protein compounds. In addition, lack of food in the control group might affect the behavior of D.

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magna more subtle before being immobile at the beginning of exposure, and thus influence the

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bioaccumulation of pyrene by D. magna as well as the toxicity to D. magna. The results were similar

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to that the humic acid could decrease the toxicity of PAHs to D. magna.51

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3.3. Effect of Protein Compounds on the Pyrene Concentration in the Tissues of D. magna in the

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Presence of Pyrene

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The lipid contents of D. magna in the control group only containing pyrene and the test group

295

containing pyrene and protein compounds were 21.7% ± 0.7% and 22.9% ± 0.6% (dry weight),

296

respectively, and there was no significant difference between these two groups, suggesting that the

297

presence of protein compounds would not exert significant influence on the constitution of D. magna.

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As shown in Figure 3, the concentration of pyrene accumulated in the tissues without gut of D. magna

299

in the protein systems was higher than that in the control systems without protein compounds when

300

the Cfree of pyrene was controlled to be 45 µg L–1 for both systems. For example, after exposure for 5

301

h, the pyrene concentration in the tissues of D. magna was enhanced by 19.9%, 34.2%, and 46.0% in

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the systems containing 10 mg L–1 tryptone, BSA, and phycocyanin, respectively in comparison with

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the control systems. After exposure for 48 h, the pyrene concentration in the D. magna has reached

304

steady state (Figure S6); the pyrene concentration in the tissues was enhanced by 136.8%, 53.6%, and

305

78.4%, respectively in the 10 mg L–1 protein compound systems. The enhancement caused by the

306

presence of protein compound was the highest for the tryptone, and followed by the phycocyanin and

307

BSA. With the increasing concentration of protein compounds (1−30 mg L–1), the enhancement of

308

pyrene concentration in the tissues in the protein systems compared to the control systems increased.

309

For instance, compared to the control systems without protein compounds, the pyrene concentration

310

in the tissues increased from 51.8% to 228.0% in the tryptone systems, from 23.7% to 120 % in the

311

BSA systems, and from 38.9% to 169.6% in the phycocyanin systems when the protein compound

312

concentration ranged from 1 to 30 mg L–1. In addition, the enhancement caused by tryptone was 13



313

obviously higher than that caused by BSA and phycocyanin. This observation indicated that the

314

enhanced pyrene accumulation in the tissues caused by protein compounds was related to the

315

concentrations and molecular weights of protein compounds. Because the steady-state concentration

316

of PAHs accumulated in organisms was controlled by the chemical activity of PAHs.52,53

317

Consequently, the difference of pyrene concentration in the tissues between the protein systems and

318

the control systems inferred that the pyrene associated with protein compounds might be an

319

independent chemical complex, which could also reach a steady-state concentration in the tissues of D.

320

magna. The results suggested that the pyrene accumulation by the tissues of D. magna might be

321

enhanced through the direct absorption of protein compound-associated pyrene by the tissues. Li et al.

322

reported that the presence of DOM elevated the bioaccumulation of PAHs in zebrafish, suggesting

323

that DOM-associated PAHs could be accumulated in zebrafish as a new complex.54

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3.4. Direct Evidence for the Uptake of Pyrene Associated with Protein Compounds by D. magna

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To prove the pyrene associated with protein compounds can enter into the tissues of D. magna,

326

the uptake of tryptone-cy5, BSA-cy5, and fluorescent phycocyanin by D. magna was investigated

327

with laser scanning microscopy. As shown in Figure 4, there was no luminescence signal in D. magna

328

for the blank systems without protein compounds after exposure for 5, 24, and 48 h, indicating that

329

the D. magna itself incubated in AFW would not produce luminescence disturbance in this study. For

330

the systems of tryptone-cy5, the intense luminescence was observed in the gut of D. magna after

331

exposure for 5, 24, and 48 h, and the luminescence was observed in the tissues of D. magna after

332

exposure for 48 h. This observation indicated that tryptone (2 000 Da) could enter into the gut through

333

ingestion, and a part of tryptone could be absorbed by tissues through direct uptake, which included

334

transmembrane transport and endocytosis happening on the skin and gut wall of D. magna.21 For the

335

systems of BSA-cy5, the luminescence was observed in the gut of D. magna after exposure for 5, 24,

336

and 48 h; for the systems of fluorescent phycocyanin, the luminescence was observed in the gut of D. 14


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magna after exposure for 48 h. This observation indicated that both BSA and phycocyanin could enter

338

into the gut through ingestion. In addition, the food effect of tryptone, BSA, and phycocyanin

339

indicated that they could be digested in the gut and the new protein fragments with smaller molecular

340

weights might enter into the tissues through direct uptake.

341

These results indicated that a part of tryptone could enter into the tissues of D. magna from

342

exposure solutions and intestinal digestive juice through direct uptake, and the new fragments of

343

tryptone, BSA, and phycocyanin produced in the gut also might enter into the tissues of D. magna

344

through direct uptake. Hence, the pyrene associated with protein compounds could also enter into the

345

tissues of D. magna through direct uptake. Accordingly, the uptake of pyrene associated with protein

346

compounds into the tissues of D. magna was mainly through three routes (Figure 5): (1) pyrene

347

associated with protein of small molecular weight could be considered to be a new kind of substance,

348

which could permeate the cell membrane and enter into the tissues from the exposure solutions and

349

intestinal digestive juice directly, leading to the elevation of steady-state concentration of total pyrene

350

in D. magna tissues; (2) the pyrene associated with new protein fragments of small molecular weight

351

produced in the gut could pass through the gut wall of D. magna and enter into the tissues, resulting in

352

the enhancement of pyrene accumulation in D. magna tissues; (3) the pyrene desorbed from the

353

protein compounds in the gut could pass through the gut wall of D. magna. In addition to route (1),

354

both route (2) and (3) could increase chemical activity of pyrene (including freely dissolved and

355

protein compounds-associated pyrene) in the gut microenvironment as well as the uptake rate of

356

pyrene, and then elevate the steady-state concentration of pyrene accumulated in the tissues of D.

357

magna in the systems with protein compounds compared to that without protein compounds. It should

358

be noted that because both the uptake and elimination rates of HOCs in organisms in natural waters

359

might be elevated by the DOM-promoted diffusive mass effect simultaneously,55 the steady-state

360

concentration of pyrene accumulated in D. magna might not be affected by the DOM-promoted

361

diffusive mass effect. For the three kinds of protein compounds studied in this research, the 15



Page 16 of 32

362

tryptone-associated pyrene could get into the D. magna tissues through route (1), (2), and (3); the

363

BSA-associated and phycocyanin-associated pyrene could get into the D. magna tissues through route

364

(2) and (3).

365

3.5. Bioavailable Fraction of Pyrene Associated with Different Types of Protein Compounds

366

The bioavailable fraction of protein compound-associated pyrene can be calculated on the basis

367

of the pyrene concentration in the D. magna tissues. In this study, the pyrene bioaccumulation factor

368

was supposed to be an invariant constant in D. magna. The value through dividing the pyrene

369

concentration in the tissues by the concentration of freely dissolved pyrene (Cfree, µg L–1) in the

370

control systems in the absence of protein compounds was equivalent to that through dividing the

371

pyrene concentration in the tissues by the concentration of effective pyrene (Ceffective, µg L–1) in the

372

protein systems. Hence, the Ceffective could be derived. The bioavailable fraction of pyrene associated

373

with protein compounds (Fprotein, %) could be calculated on the basis of the pyrene concentration in

374

tissues of D. magna in the protein systems by equation (1):

375

=

× 100%

(1)

376

where Cprotein-associated pyrene (µg L–1) was the concentration of protein compound-associated pyrene

377

during the experiment. As shown in Table 1, after exposure for 48 h (steady state), the bioavailable

378

fractions of tryptone-associated, BSA-associated, and phycocyanin-associated pyrene were 55.7%,

379

18.5%, and 23.4%, respectively for the 30 mg L–1 protein systems, and 54.6, 17.7%, and 21.6%,

380

respectively for the 10 mg L–1 protein systems. The data indicated that the bioavailable fraction of

381

protein compound-associated pyrene was the highest in the tryptone systems, and the bioavailable

382

fraction of phycocyanin-associated pyrene was a little higher than that of BSA-associated pyrene.

383

Similarly, on the basis of relationship between the freely dissolved pyrene concentration (Cfree,

384

µg L−1) and the D. magna immobilization in the control systems without protein compounds (Figure

385

S7), the concentration of effective pyrene (C'effective, µg L−1) could be derived for the systems 16


Page 17 of 32


386

containing protein compounds. As mentioned in section 3.1, the food effect caused by 30 mg L–1

387

protein compounds was almost equivalent to that caused by 10 mg L–1 protein compounds.

388

Consequently, to eliminate the influence of protein food effect on the bioavailability quantification,

389

the difference between the D. magna immobilization in the systems of 30 mg L–1 and 10 mg L–1

390

protein compounds was used to calculate the bioavailable fraction of protein compound-associated

391

pyrene (F'protein, %). Therefore, the value of F'protein on the basis of D. magna immobilization could be

392

calculated by equation (2):

′ =

393

! "# ! $#

"# $#

× 100%

(2)

394

where C'effective30 and C'effective10 (µg L–1) were the concentrations of effective pyrene according to the

395

D. magna immobilization in the 30 mg L–1 and 10 mg L–1 protein systems, respectively;

396

Cprotein-associated

397

compound-associated pyrene in the 30 mg L–1 and 10 mg L–1 protein exposure solutions, respectively.

398

As shown in Table 1, for the 30 mg L–1 protein systems, the bioavailable fractions of

399

tryptone-associated pyrene, BSA-associated pyrene, and phycocyanin-associated pyrene were 57.9%,

400

26.7%, and 31.9%, respectively after exposure for 36 h, and 58.1%, 26.8%, and 32.8%, respectively

401

after exposure for 48 h. The bioavailable fraction of tryptone-associated pyrene was far higher than

402

that of phycocyanin-associated and BSA-associated pyrene. These bioavailable fraction values

403

calculated on the basis of D. magna immobilization were higher than that calculated by the pyrene

404

concentration in tissues of D. magna, which indicated that protein-like DOM-promoted diffusive mass

405

transfer effect could enhance the accessibility of pyrene to D. magna, leading to the increase of D.

406

magna immobilization.

pyrene30

and

Cprotein-associated

pyrene10

were

the

concentrations

of

protein

407

As mentioned in section 3.4, the tryptone belongs to small molecular weight protein-like DOM,

408

which could pass through the skin of D. magna from the exposure solutions directly. Therefore, the

409

tryptone-associated pyrene could enter into the tissues of D. magna through route (1), (2), and (3).

17



410

However, both the BSA (66 000 Da) and phycocyanin (120 000 Da) belongs to the big molecular

411

weight protein-like DOM, which could not pass through the skin of D. magna from the exposure

412

solutions directly; the BSA-associated and phycocyanin-associated pyrene could enter into the tissues

413

of D. magna only through route (2) and (3). In addition, the faster digestion of small molecular weight

414

protein compounds than big molecular weight protein compounds in the intestinal tracts might result

415

in a greater increase of pyrene concentration in the tissue. Hence, the bioavailable fraction of

416

tryptone-associated pyrene was the highest among the three types of protein compounds. Because the

417

concentration of phycocyanin-associated pyrene in the gut of D. magna was higher than that of

418

BSA-associated pyrene (Figure S8), and the amount of pyrene sorbed on phycocyanin was higher

419

than that sorbed on BSA (Table S3), the bioavailable fraction of phycocyanin-associated pyrene was a

420

little higher than that of BSA-associated pyrene.

421

3.6. Environmental Implications

422

In this study, the pyrene associated with protein compounds was directly proved to be partly

423

bioavailable to D. magna through fluorescence labelling method as well as the confocal laser

424

scanning microscopy; the bioavailability of pyrene associated with protein compounds was related to

425

the types and molecular weights of protein compounds. The pyrene concentration in the tissues of D.

426

magna in the 10 mg L–1 protein systems was enhanced by 53.6−136.8% in comparison with that in the

427

control systems without protein compounds when the Cfree of pyrene was controlled at 45 µg L–1. The

428

protein compound-associated pyrene contributed to 34.9−57.8% of the total pyrene accumulated in

429

the tissues when the protein compound concentration was 10 mg L–1 in the exposure solutions.

430

Furthermore, the D. magna immobilization was enhanced by 25.2−55.2% in the 10 mg L–1 protein

431

compound systems compared to that without protein compounds after exposure for 36 h. The results

432

indicated that the pyrene associated with protein compounds made a great contribution to the total

433

pyrene toxicity in waters. In addition, the results suggested that the biomagnification of pyrene is 18


Page 18 of 32

Page 19 of 32

434


more favorable when it is associated with a more readily digested organic matter.

435

Protein compounds are widely distributed in aquatic environments, and the molecular weight,

436

component, and structure of protein-like DOM in waters were different in various natural waters.

437

Consequently, we should not only consider the bioavailability of freely dissolved HOCs but also

438

consider the bioavailability of pyrene associated with protein-like DOM of various molecular weights,

439

components and structures to evaluate the bioavailability and eco-environmental hazard of HOCs in

440

natural waters. In addition, protein-like DOM, as food for organisms, might be utilized by various

441

organisms in different ways. Hence, more organisms should be taken into account when the

442

bioavailability of HOCs was investigated in natural waters. In particular, in order to investigate the

443

bioavailability of HOCs associated with DOM directly, some new fluorescent dye having higher

444

fluorescence quantum yield should be designed for different types of DOM and organisms.

445

Supporting Information

446

The Supporting Information is available free of charge on the ACS Publications website at DOI:

447

Additional details on the establishment of passive dosing dishes, determination of freely dissolved

448

pyrene concentration, analysis of protein compound-associated pyrene concentration, fluorescence

449

spectrum experiments, uptake curves of pyrene by D. magna, analysis of lipid content of D. magna

450

and analysis of pyrene concentration in the tissues and gut of D. magna. Tables showing

451

physico-chemical characteristics of protein compounds, pyrene concentrations in the exposure

452

solutions, and partition coefficients. Figures showing experimental schematic, fluorescence spectra of

453

protein compounds, relationship between fluorescence intensity and protein compound concentrations,

454

effects of protein compounds on the immobilization of D. magna, uptake curves of pyrene by D.

455

magna, relationship between the immobilization of D. magna and the freely dissolved pyrene

456

concentration, and the concentration of pyrene accumulated in the gut of D. magna.

19



457

Acknowledgements

458

This work was supported by the National Key R&D Program of China (grant no.

459

2017YFA0605001), the National Natural Science Foundation of China (grant no. 91547207), and the

460

fund for Innovative Research Group of the National Natural Science Foundation of China (grant no.

461

51721093).

462

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620

621

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26


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Table 1 Bioavailable fraction of protein compound-associated pyrene to D. magna

Protein compounds

Cfree of pyrene (µg L–1)

Bioavailable fraction of pyrene associated with protein compounds (%) According to the According to the pyrene concentration immobilization of in the tissues of D. magna D. magna Cprotein = Cprotein = Cprotein = Cprotein = –1 –1 –1 30 mg L 30 mg L 30 mg L 10 mg L–1 36 h

48 h

48 h

48 h

Tryptone

45.0

57.9±5.0

58.1±3.2

55.7±2.4

54.6±2.1

BSA

45.0

26.7±2.3

26.8±1.7

18.5±0.8

17.7±0.9

Phycocyanin

45.0

31.9±2.1

32.8±2.5

23.4±1.1

21.6±0.8

629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 27


Immobilization (%)


100 Tryptone Control -1 1 mg L 80 -1 10 mg L -1 30 mg L 60 40 20 0 0

48

72

96 120 144 168 192 216 240

BSA

100

Immobilization (%)

24

Control -1 1 mg L -1 10 mg L -1 30 mg L

80 60 40 20 0 0

48

72

96 120 144 168 192 216 240

Phycocyanin

100

Immobilization (%)

24


80 60 40 20 0 0

24

48

72

96 120 144 168 192 216 240

Time (h)

648 649

Figure 1. Effects of protein compounds at different concentrations on the immobilization of D. magna

650

after exposure for 240 h in the absence of pyrene (mean ± standard deviation, n = 3). Control means

651

without protein compounds.

652

28


Page 28 of 32

Page 29 of 32


90 1 mg L-1 protein compounds -1 45 µg L pyrene

Immobilization (%)

75

Control Tryptone BSA Phycocyanin

60 45 30 15 0 0

12

24

36

48

36

48

36

48

90 10 mg L-1 protein compounds

Immobilization (%)

75

-1

45 µg L pyrene Control Tryptone BSA Phycocyanin

60 45 30 15 0 0

12

24

90 30 mg L-1 protein compounds -1

Immobilization (%)

75

45 µg L pyrene Control Tryptone BSA Phycocyanin

60 45 30 15 0 0

653

12

24

Time (h)

654

Figure 2. Effects of protein compounds (1, 10, and 30 mg L–1) with different types on the

655

immobilization of D. magna after exposure for 48 h in the systems with 45 µg L–1 freely dissolved

656

pyrene (mean ± standard deviation, n = 3). Control means without protein compounds. The

657

immobilization of D. magna was zero in the protein solutions without pyrene after exposure for 48 h.

658 659 660 661 662 663 664 29



Page 30 of 32

50 Control -1 1 mg L -1 10 mg L -1 30 mg L

(ng/one D. magna)

Cpyrene in the tissues

5h 40 30 20 10 0 Tryptone

BSA

50


(ng/one D. magna)

48 h

Cpyrene in the tissues

Phycocyanin

40 30 20 10 0 Tryptone

BSA

Phycocyanin

Protein compounds of different types

665 666

Figure 3. Concentration of pyrene accumulated in the tissues excluding gut of D. magna in the

667

systems containing 45 µg L–1 pyrene and protein compounds of different concentrations after

668

exposure for 5 h and 48 h. (mean ± standard deviation, n = 3). Control means without protein

669

compounds.

670 671 672 673 674 30


Page 31 of 32


675 676

Figure 4. Luminescence and overlay images of D. magna incubated in 83.0 mg L–1 tryptone-cy5, 99.2

677

mg L–1 BSA-cy5, and 72.1 mg L–1 phycocyanin solutions after exposure for 5, 24, and 48 h. λex = 635

678

nm, λem = 600-700 nm. Control means without protein compounds.

679 31



680 681

Figure 5. Scheme of uptake routes of protein compound-associated pyrene into the tissues of D.

682

magna. Route① pyrene associated with protein compound enters into the tissues from exposure

683

solutions and gut, Route② pyrene associated with new protein compound fragments enters into the

684

tissues from gut, Route③ pyrene desorbed from the protein enters into the tissues from gut.

685 686 687 688 689 690 691 692 693 694 695 696 697 698 32


Page 32 of 32

Bioavailability of Pyrene Associated with Different Types of Protein

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