Bioaccumulation behavior of pharmaceuticals and personal care

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Bioaccumulation behavior of pharmaceuticals and personal care products in adult zebrafish (Danio Rerio): influence of physical-chemical properties and biotransformation Fangfang Chen, Zhiyuan Gong, and Barry C. Kelly Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02918 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Environmental Science & Technology

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Bioaccumulation behavior of pharmaceuticals and personal care products in adult

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zebrafish (Danio Rerio): influence of physical-chemical properties and biotransformation

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Fangfang Chen1, Zhiyuan Gong1, 2, Barry C. Kelly3*

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Graduate School of Integrated Sciences and Engineering (NGS), National University of Singapore, Singapore 117456, Singapore

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Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore 3

Department of Civil and Environmental Engineering, National University of Singapore, Singapore 117576, Singapore

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* Corresponding author. Present address: Department of Civil and Environmental Engineering,

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National University of Singapore, Block E1A, #07-03, No.1 Engineering Drive 2, Singapore

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117576. Tel. (+65-6516 3764); Fax (+65-6779 1635); Email: [email protected]

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Graphical Abstract for TOC

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ABSTRACT

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The factors influencing bioaccumulation of pharmaceuticals and personal care products (PPCPs) in

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aquatic organisms are not well understood. The present study involved a comprehensive laboratory

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investigation to assess the bioaccumulation behavior of several PPCPs in adult zebrafish (Danio

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rerio). The studied PPCPs included several ionogenic organic compounds (IOCs) such as weak acids

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and weak bases. Experiments involved two exposure groups (high and low) and a control group, with

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a 6 day aqueous exposure, followed by a 7 day depuration phase under flow-through conditions.

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Uptake rate constants, (ku) ranged between 0.19 and 8,610 L⋅kg-1⋅d-1, while depuration rate constants

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(kd) ranged between 0.14 and 5.14 d-1 in different fish tissues. Steady-state bioconcentration factor

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(BCFss) values varied widely among the studied PPCPs, ranging from 0.09 to 6,460. In many cases,

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BCFss values of individual PPCPs differed substantially among different fish tissues. Positive linear

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relationships were observed between log BCFss values and physical-chemical properties such as

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octanol-water distribution coefficients (log Dow) membrane-water distribution coefficients (log Dmw),

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albumin-water distribution coefficients (log DBSAw) and muscle protein-water distribution coefficients

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(log Dmpw), indicating the importance of lipid-, phospholipid- and protein-water partitioning. The

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results also showed that for many PPCPs, the estimated whole-body metabolism rate constant (km)

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values were comparable to the observed depuration rate (kd), indicating metabolism plays a major

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role in the overall elimination of these compounds in zebrafish. An exception was sertraline, which

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exhibited a kd value (0.4-0.5 d-1) that was much higher than the estimated whole-body km (0.03 d-1).

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Overall, the results help to better understand the influence of physical-chemical properties and

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biotransformation on bioaccumulation behavior of these contaminants of concern in aquatic

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organisms.

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Keywords: pharmaceuticals, personal care products, zebrafish, Dmw, Dpw, bioconcentration

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INTRODUCTION

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Concern regarding the occurrence of pharmaceutical and personal care products (PPCPs) in the

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environment has increased in recent years. Many PPCPs are considered high production volume

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substances.1-4 Discharge of these compounds to the environment typically occurs via wastewater

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associated with manufacturing facilities, hospitals, households and agriculture.5-7 PPCP residues

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have been detected in municipal wastewater treatment plant (WWTP) effluents,8-11 surface

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water,12-16 as well as tap water.17, 18 Concentrations of PPCPs in WWTP effluents and surface

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waters are typically in the range of µg⋅L-1 (ppb) to ng⋅L-1 (ppt).19

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PPCPs have been frequently detected in aquatic organisms, including various invertebrates and

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fish, indicating these compounds can bioaccumulate.20-26 Nallani et al. reported a very low

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bioconcentration factor (BCF) of ibuprofen (BCFs, 0.08–1.4 L⋅kg-1), following a 28 day

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bioconcentration study in fathead minnow and channel catfish.27 Carbamazepine

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metabolites also exhibited relatively low bioaccumulation factors (BAFs) in Jenynsia

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multidentata, ranging between 5 and 9 L⋅kg-1.28 Other studies have suggested more hydrophobic

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pharmaceuticals may exhibit a much greater bioaccumulation potential, especially those

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compounds with low biotransformation rates.29, 30

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The majority of PPCPs are classified as ionogenic organic compounds (IOCs), including weak

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acids and weak bases. The factors governing the bioaccumulation behavior of these compounds

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are not well understood. BCFs of neutral organic chemicals can be effectively predicted by the

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octanol-water partition coefficients (Kow), assuming metabolism is negligible and lipid-water

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exchange is the controlling process. For assessing IOC bioaccumulation potential, Armitage et al.


and its

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recently proposed a mechanistic mass balance bioconcentration model that utilizes pH dependent

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distribution coefficients, including the chemical’s octanol-water distribution coefficient (Dow),

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membrane-water distribution coefficient (Dmw), as well as the estimated chemical distribution

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between water and non-lipid organic matter (NLOM), which in fish is comprised of mainly

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proteins.31, 32 Log Dow values (calculated at pH 7) range widely for common PPCPs. For example,

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log Dow of Ibuprofen is 1.5, which is three orders of magnitude lower than that of triclosan (log

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Dow = 4.7).

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It is well understood that pharmaceuticals are efficiently metabolized in humans.33-36 Studies

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investigating the biotransformation of PPCPs in fish have reported varying degrees of

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metabolism of these compounds.37-39 In vitro xenobiotic metabolism assays using subcellular

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fractions (e.g. microsomes, cytosols or S9 fractions), cellular organelles (e.g. hepatocytes) or

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metabolizing recombinant enzymes can provide a reasonable estimate of in vivo

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biotransformation rate constant values (km) for a given fish species.38, 39 Quantitative Structure-

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Activity Relationship (QSAR) models such as BCFWIN™ can also provide size- and

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temperature-specific estimates of whole-body km and biotransformation half-lives of different

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classes of compounds in fish. Knowledge of km can help improve bioaccumulation models and

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chemical bioaccumulation assessments.40-43 This is particularly true for metabolizable substances,

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which include many of the PPCPs.

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The objective of the present study was to conduct a controlled laboratory experiment to evaluate

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the uptake and elimination kinetics and BCFs of several PPCPs in adult zebrafish (Danio rerio).

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The study provides important compound specific bioaccumulation metrics for several PPCPs.

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The generated bioaccumulation metrics include uptake rate constants (ku, L⋅kg-1⋅d-1), depuration 5 ACS Paragon Plus Environment


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rate constants (kd, d-1), as well as kinetically derived BCFs (BCFk) and observed steady-state

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BCFs (BCFss). The extent of biotransformation of individual PPCPs was assessed by comparing

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estimated whole-body km values with overall elimination rate constants. For one compound,

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fluoxetine, metabolism was further evaluated by assessing the behavior of its primary metabolite,

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norfluoxetine. As the studied PPCPs varied widely in therapeutic class and physical-chemical

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properties, we were able to assess relationships between the observed bioaccumulation behavior

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and key properties such as Dow, Dmw, as well as bovine serum albumin-water distribution

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coefficients (DBSAw) and muscle protein-water distribution coefficients (Dmpw). The findings will

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be useful for future biomonitoring and bioaccumulation assessment initiatives of these important

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contaminants of concern.

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MATERIALS AND METHODS

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Standards and Reagents. High purity (> 97%) standards of diclofenac, naproxen, triclosan,

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ibuprofen, diphenhydramine, gemfibrozil, sertraline, risperidone and simvastatin were purchased

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from Sigma-Aldrich Co. LLC. (St Louis, USA). Bisphenol A (BPA) was purchased from Merck

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(Hohenbrunn, Germany). Carbamazepine was purchased from TCI-EP (Tokyo, Japan), and

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fluoxetine was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Details

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regarding the various isotopically labeled surrogate standards used for isotope dilution

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quantification of test compounds are provided in the Supporting Information (SI).

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Experimental setup and collection of samples. Adult female zebrafish were purchased from a

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local supplier, Mainland Tropical Fish Farm Pte Ltd. Fish were acclimated to laboratory

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conditions for two weeks prior to the experiments. The experiments generally followed OECD

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guidelines for a flow-through chemical bioconcentration test.44 The adult female zebrafish 6 ACS Paragon Plus Environment

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weighed approximately 1 g each. The fish were maintained in dechlorinated water at a

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temperature of 25 ± 2°C, with a 16: 8 hour light/dark cycle. The pH of water in the exposure

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tanks was monitored every two days. The average pH was 7.6. Further details pertaining to

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experimental design and setup are described in the SI.

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Water samples were collected daily to determine the exposure concentrations of the various test

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compounds. Time-weighted average concentrations in fish tissues were used to calculate BCFs.

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At six time-points during uptake phase (i.e., 4 h, 8 h, 1 d, 2 d, 3 d, and 6 d) and four time-points

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during the depuration phase (i.e., 7 d, 8 d, 10 d and 13 d) fish (three replicates of 5 pooled

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individuals) from the treatment groups and control group were sacrificed, at which time liver,

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ovary, muscle and plasma were collected and immediately frozen and stored at -20°C.

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Chemical Analysis. Water samples (15 mL) collected from the flow-through system were mixed

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with 1.5 mL of 1 % formic acid, and spiked with isotopically labeled surrogate compounds prior

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to solid phase extraction (SPE). SPE was performed using Phenomenex X-33u 200 mg cartridges.

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The conditioning of the SPE cartridges was performed with 5 mL of methanol, followed by 5 mL

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of Milli-Q water at a flow rate of 5 mL·min-1 under vacuum. The 15 mL water samples were then

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passed through the cartridges at a flow rate of 5 mL·min-1. The cartridges were washed with 5

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mL of 5 % methanol in water and eluted with 3 mL of acetone, followed by 5 mL of methanol.

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SPE extracts were collected in centrifuge tubes and then evaporated to dryness under a gentle

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nitrogen stream. 120 µL of methanol: Milli-Q water (1:4, v/v) was added to rinse the tube before

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transferring to LC vials, which were equipped with 200 µL glass inserts. Lastly, 25 ng of the

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injection internal standard (TCPAA-13C6) was spiked into the extracts prior to instrumental

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Extraction of plasma samples was conducted using our previously developed method involving

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liquid-liquid extraction of fish plasma micro-aliquots.45 Briefly, 20 µL of plasma was added to a

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2 mL of centrifuge tube, which was then spiked with isotopically labeled surrogate compounds.

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Samples were sonicated with 1 mL of acetone for 3 min and then centrifuged at 12,000 rpm at

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4°C for 5 min. Then extraction was repeated a second time with another 1 mL of acetone. The

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combined supernatant was evaporated to dryness under a gentle nitrogen stream. Extracts were

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reconstituted and transferred into LC vials and then spiked with the injection internal standard

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(TCPAA-13C6). For tissues, frozen samples (liver ~0.1g; muscle ~0.5g; ovary ~0.5g) were

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thawed and weighed directly in 15 mL polypropylene centrifuge tubes. Milli-Q water (0.5 mL)

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was added to the samples. Tissues were then homogenized via sonication. 2 mL of 1 % of formic

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acid was added, along with the isotopically labeled surrogate compounds. The spiked tissue

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samples were then extracted with 5 mL of methanol via sequential sonication for 30 min (twice).

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Extracts were then centrifuged at 10000 rpm at 4oC for 5 min. The supernatant was collected and

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diluted to 100 mL with Milli-Q water prior to SPE, which followed the same protocol as for

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waters samples (above). The injection internal standard (TCPAA-13C6) was spiked just prior to

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instrumental analysis.

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Liquid chromatography was performed with a DIONEX Ultimate 3000 HPLC system.

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Compounds were separated chromatographically using a high efficiency Agilent Poroshell 120

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SB-C18 column (2.1×50 mm, 2.7 µm). The column was maintained at a temperature of 40°C.

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The sample volume injected was 10 µL. Chromatographic separations were carried out using the

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following mobile phase gradient: solvent (A) 5 mM ammonium acetate in Milli-Q water, solvent

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(B) methanol: acetonitrile (1:1, v/v) at a flow rate of 0.3 mL·min-1. The gradient elution was as


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follows: initial conditions, 20% B; 0.5-4 min, increase linearly to 100% B; 4-6.5 min, 100% B;

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6.5-6.7 min, followed by a return to initial conditions; 6.7-9 min, 20% B.

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The HPLC instrument was coupled to a QTRAP 5500 hybrid triple quadrupole-linear ion trap

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mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with a turbo Ion Spray source,

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capable of fast polarity switching and analysis of positive and negative ions. Source-dependent

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parameters for compounds analyzed in ESI positive mode were: curtain gas, 30 psi; nitrogen

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collision gas, high; source temperature was 550 °C; ion spray voltage was 5500 V; ion source

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gases GS1 and GS2 were set at 55 and 60 psi, respectively. For compounds analyzed in negative

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mode, parameters were as follows: curtain gas, 15 psi; ion spray voltage was −4500 V; while all

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other parameters were the same as in ESI positive mode. Other compound specific parameters

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are summarized in Table S1 of the SI. Target analytes were quantified using an isotope dilution

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approach, based on relative response to corresponding isotopically labeled surrogate compounds.

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As quantification was conducted using isotope dilution, all concentrations are recovery corrected.

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More information, including spike-recovery test results, as well as method detection limits

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(MDLs) and method quantification limits (MQLs) are provided in Table S2-S3 of the SI.

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Data analysis. Growth-corrected concentrations of the individual PPCPs were used to determine

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uptake rate constants (ku), depuration rate constants (kd), half-life (t1/2) and BCFk values using

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Equation S2 and S3 of the SI. The measured chemical concentrations observed in fish tissues at

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the end of the uptake phase were used to determine the apparent BCFss. Following Martin et al.

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(2003) an exponential growth model was utilized to determine growth corrected chemical

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concentrations in zebrafish tissues.46 Analysis of variance (ANOVA) was used to assess



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significant differences in chemical concentrations between time points, employing a significance

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level (α) of 0.05. More details are provided in the SI.

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Physical-chemical properties including molecular structure, acid dissociation constants (pKa),

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Kow, Dow, Dmw, DBSAw, and Dmpw were compiled from various sources (Table 1). Dmw values were

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calculated based on the approach proposed for IOCs by Armitage et al.32 DBSAw and Dmpw values

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were estimated following the approach outlined by Henneberger et al. using polyparameter linear

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free energy relationship (PP-LFER) models.47, 48 See Table S4 in the SI for more details regarding

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these calculations.

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RESULTS AND DISCUSSION

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Toxicological effects of PPCPs on zebrafish. For the high dose group only 2 fish (0.56 %) died

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during the uptake phase and no fish died during depuration phase. No mortality was observed in

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the control and low dose groups. No statistically significant differences were observed in fish

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growth rate between the exposed fish and those in the control group (p > 0.46), (Table S5 of the

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SI). All three groups of zebrafish exhibited normal growth throughout the experiment (Table S5

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of the SI). No obvious abnormality in fish swimming pattern was observed. Some difference in

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feeding behavior was observed between the high dose group and the low exposure and control

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groups. Fish in the high exposure group were comparatively slower in response to supplied feed.

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The fish in the high exposure group generally exhibited a lower degree of feeding, as denoted by

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excess feed in the bottom of tank. No statistically significant difference was found between the

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liver somatic index (LSI) of exposed groups and control fish. However, the trend for LSI in the

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high exposure group fish was higher than other groups, indicating elevated LSI beyond day 6.

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The data indicate that fish in the high exposure group may have suffered sub-lethal effects due to 10 ACS Paragon Plus Environment

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continuous high PPCP exposure during the experiment. Karlsson et al. showed that LSI in rats

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increased significantly when exposed to BPA (0.025, 0.25 and 2.5 mg/L dosage, respectively),49

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which was one of the test chemicals in the present study. Previous studies have reported renal

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lesions and alternations of the gills in rainbow trout chronically exposed (28 d exposure) to

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diclofenac at 1 µg⋅L-1.50 Also, chronic exposure (28 d) of sertraline at 3 µg⋅L-1 been to shown

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decreases serotonin reuptake transporter binding and shelter-seeking behavior in adult male

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fathead minnows.51

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Concentrations of PPCPs in water and zebrafish. During the uptake phase, PPCP

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concentrations in water ranged between 0.03 ± 0.005 (risperidone) and 4.48 ± 0.43 µg⋅L-1

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(gemfibrozil) in the low exposure tank and between 0.010 ± 0.008 (simvastatin) and 12.50 ± 1.40

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(gemfibrozil) in the high exposure tank (Table 1). Concentrations were stable throughout the

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exposure phase. Thus, the mean water concentrations were substituted as Cw (µg⋅L-1) in Equation

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S2 of the SI for determination of ku values. PPCPs were not detectable in control tanks or in

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exposure tanks during the depuration phase (> day 6).

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Figure S1 of the SI illustrates the growth-corrected uptake and depuration phase concentrations

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of individual PPCPs in liver, muscle, ovary and plasma of exposed zebrafish. All test chemicals

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were detected in plasma and tissues, with the exception of diclofenac in ovary (low exposure

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group), BPA in plasma (low exposure group) and simvastatin in plasma (both groups).

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Uptake kinetics. Uptake rate constants (ku, L⋅kg-1⋅d-1) of the various PPCPs in different tissues

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ranged between 0.19 to 8,610 (Table S6 of the SI). For the majority of the studied PPCPs uptake

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was relatively fast. In some cases (e.g. ibuprofen and sertraline in liver), the time to the apparent



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steady-state concentration was within 1 d. For other compounds such as triclosan, uptake into

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tissues was comparatively slower. In some cases, uptake differed substantially for different

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tissues. For example, the ku for triclosan in liver, muscle and ovary was determined as 3,850, 946

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and 0.56 L⋅kg-1⋅d-1, respectively. For some compounds, internal concentrations did not appear to

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reach a steady-state plateau by the end of the exposure phase (day 6), indicated by concentrations

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on day 6 that were still significantly higher (p < 0.05) than the previous time point. However,

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based on the observed data, the exposure phase duration (6 d) was sufficient for these

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compounds to achieve approximate (95%) steady-state (t95%), which is estimated to be about 4

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half-lives. Specifically, the t1/2 values of the studied PPCPs were typically < 1.5 d, which

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corresponds to t95% values generally ≤ the 6 d exposure period.

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The nonlinear regression technique was not suitable for determination of ku in some cases. The

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nonlinear regression model worked well for determination of ku for triclosan, BPA and

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simvastatin. However, determination of ku using this approach was problematic for other

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compounds such as carbamazepine, naproxen and diclofenac. For those compounds, the model

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fitting failed, with relatively low r2 values (r2 < 0.3). For example, during the uptake phase,

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concentrations of naproxen and gemfibrozil in liver and diclofenac, gemfibrozil, ibuprofen,

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naproxen and carbamazepine in plasma were highly variable, exhibiting a fluctuating trend prior

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to the apparent steady-state concentration. The variability in tissue residue concentrations may be

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due to in vivo biotransformation of these compounds.52

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The observed ku values are comparable to those previously reported for PPCPs in fish. For

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example, Wang and Gardinali (2013) investigated uptake of PPCPs in mosquito fish tissues


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(Gambusia holbrooki), reporting uptake rate constants of 0.2, 5.1 and 15.4 L⋅kg-1⋅d-1 for

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carbamazepine, diphenhydramine and ibuprofen, respectively.53

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Karlsson (2013) reported that uptake of diclofenac and fluoxetine is highly sensitive to changes

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in pH, as demonstrated by BCFs varying by two orders of magnitude (diclofenac) and four

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orders of magnitude (fluoxetine) across three pH units in worms.54 Nakamura et al. observed

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BCFs of fluoxetine in Japanese medaka (Oryzias latipes) increased significantly with increasing

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pH near the pKa values.55 In the present study, pH of water in exposure tanks was monitored

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every two days, with an average pH of 7.6. While pH undoubtedly plays a major role in the

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uptake of ionizable PPCPs, a full assessment of this is beyond the scope of the present study.

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Elimination kinetics. Observed depuration rate constants (kd) for the various PPCPs in different

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tissues and ∑tissues ranged between 0.14 and 5.14 d-1, with corresponding t1/2 in the range of

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0.13 to 5.06 days (Table S6 of the SI). The t1/2 values tended to be lower in liver compared to

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ovary and muscle, likely due to higher rates of biotransformation and clearance in liver. t1/2

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values for ∑tissues ranged between approximately 0.2 to 2 days for the studied PPCPs. The

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observed elimination kinetics of the studied PPCPs are generally comparable to previous

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observations. For example, whole-body t1/2 values of 1.3, and 1.4 days were previously reported

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for ibuprofen and diphenhydramine in mosquito fish.53 In the present study, the ∑tissues t1/2

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values for ibuprofen, diphenhydramine in zebrafish in the low exposure group were determined

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to be 1.05 ± 0.40 and 0.53 ± 0.03 days, respectively. However, t1/2 for some compounds was

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much shorter than those previously reported. For example, the t1/2 of fluoxetine in ∑tissues in the

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present study (approximately 2 days) was substantially lower than the 9.4 ± 1.1 day whole-body

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t1/2 reported for fluoxetine in Japanese medaka. Conversely, the observed depuration of PPCPs 13 ACS Paragon Plus Environment


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was much faster than our recent observations for perfluoroalkyl substances (PFASs) in adult

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zebrafish,56 which exhibited t1/2 values on the order of weeks. The faster depuration of PPCPs

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may be, in part, due to higher rates of biotransformation compared to PFASs, which are highly

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resistant to biotransformation in fish.46, 57

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Bioconcentration factors. BCFss values of individual PPCPs in plasma, liver, muscle, ovary and

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∑tissues of zebrafish in the low and high exposure groups are shown in Table 2. There were no

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significant differences between BCFs in the low and high exposure groups, indicating no

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concentration dependent bioaccumulation behavior. BCFss values in different tissues ranged from

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0.09 to 6,460 L⋅kg-1. BCFss values of the various PPCPs for ∑tissues, which is representative of

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whole-body BCFss, ranged between 0.2 and 465. The observed BCFss values in these adult

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zebrafish are generally comparable to previously reported BCF values of PPCPs in fish.28, 30, 59-61

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Ibuprofen, naproxen, diclofenac, risperidone, gemfibrozil, sertraline and diphenhydramine

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exhibited relatively low BCFss values, ranging between 0.1 and 200, indicating low

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bioconcentration potential of those compounds. Also, BCFss values of these compounds were

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similar in plasma, liver, muscle and ovary. Triclosan, BPA and simvastatin exhibited the highest

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BCFss values of the studied PPCPs. Bioconcentration potential of triclosan, BPA and simvastatin

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differed among different tissues with BCFs ordered liver > ovary > muscle > plasma. The BCFss

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of triclosan in liver was approximately 50 times greater than those in muscle and plasma.

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Figure S2 of the SI illustrates the relationship between apparent log BCFss and log BCFk of

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individual PPCPs in different tissues. The two different BCF estimates are generally comparable.

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However, BCFss values for some compounds in muscle of fish from the low exposure group were


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substantially higher (10-100 times higher) than calculated BCFk values. Conversely, BCFss

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values for some compounds in ovary of fish from the low exposure group were significantly

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lower (10-100 times) than calculated BCFk values. In many cases, determination of the BCFk was

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not possible due to high variability of tissue residue concentrations during the uptake phase. For

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those compounds, we can only utilize the apparent BCFss for assessing bioconcentration potential.

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Influence of physical-chemical properties. Figure 1 illustrates plots of log BCFss values of

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individual PPCPs in zebrafish tissues versus log DBSAw, log Dmw, log Dmpw, which represent the

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anticipated chemical distribution at equilibrium at defined pH between water and key biological

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constituents such as albumin, phospholipids and structural proteins. The data show positive

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relationships between BCF and increasing membrane-water and protein-water distribution

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coefficients. The data in Figure 1 also indicate that steady-state bioconcentration of PPCPs in

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plasma may be more influenced by sorption to plasma proteins, compared to phospholipids, as

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the log DBSAw-log BCFss relationship is much stronger than the log Dmw-log BCFss.

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Positive correlations were observed between physical-chemical properties and BCFs of

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individual PPCPs for ∑tissues (see Figure S3 of the SI). The increasing trend of BCFs with

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increasing hydrophobicity is consistent with previous measurements and models for hydrophobic

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organic chemicals, as well as ionizable organic compounds. At steady-state, the chemical BCF in

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fish is typically represented as the ratio of uptake and depuration rate constants (i.e., BCF = ku /

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[k2 + km + ke]), where k2 is the gill elimination rate constant, km is the biotransformation rate

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constant and ke is the fecal egestion rate constant.62, 63 The overall depuration rate constant, kd, is

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the sum of k2, km, and ke, assuming negligible growth dilution. For neutral hydrophobic organic

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compounds, fecal egestion is expected to be orders of magnitude lower than depuration via 15 ACS Paragon Plus Environment


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respiratory elimination in fish (k2 >> ke), especially for less hydrophobic compounds (log Kow
> km). The authors reported similar results for propranolol and

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norethindrone.67 Also, Paterson and Metcalfe reported a relatively long t1/2 (9.4 ± 1.1 d) for

416

fluoxetine in Japanese medaka,37 which is much higher than the observed t1/2 in zebrafish from

417

the present study. The apparent differences may be due to interspecies variation of

418

biotransformation capacity and/or differences with experimental conditions, exposure

419

concentrations, concomitant analyte effects and/or water quality parameters (e.g. pH).

420

Overall, this study provides new bioaccumulation metrics data for several PPCPs, including ku,

421

kd, t1/2, BCFk and BCFss. The results highlight the importance of physical-chemical properties

422

such as DBSAw, Dmpw and Dmw on bioaccumulation potential of these contaminants. The results also

423

demonstrate that the biotransformation rate constant (km) is a key parameter influencing the

424

bioaccumulation potential of these compounds. Further studies of partitioning behavior and

425

biotransformation of these compounds may help to develop more effective models and improve

426

our understanding of bioaccumulation behaviour of these compounds in aquatic organisms and

427

food chains.

428

ACKNOWLEDGMENTS

429

This work was supported by NUS Cross-Faculty Research Grant for Interdisciplinary Research

430

grant to B.C. Kelly and the Singapore National Research Foundation under its Environmental &

431

Water Technologies Strategic Research Programme and administered by the Environment & 21 ACS Paragon Plus Environment


432

Water Industry Programme Office (EWI) of the PUB, grant number R-154-000-328-272. We

433

thank the NUS Environmental Research Institute (NERI) for facility use and technique support.

434

ASSOCIATED CONTENT

435

Supporting Information. Additional information includes details regarding experimental setup,

436

chemical analyses, data analyses, as well as supplemental tables (Tables S1-S8), supplemental

437

figures (Figures S1-S5) and supplemental references. This information is available free of charge

438

via the Internet at http://pubs.acs.org


Page 22 of 34

Page 23 of 34


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Table 1. Test compounds, physicochemical properties of selected PPCPs compounds, and mean aqueous exposure concentration (Mean ± SD, n=18). Waterborne Concentration (µ µg⋅⋅L-1)

Naproxen Ibuprofen Diclofenac

Analgesic Analgesic Analgesic

acidic acidic acidic

Fraction Ionized Molecules at pH 7 (%) 99.3 99.7 99.9

Gemfibrozil

Antihyperlipidemic

acidic

99.5

4.75

4.77

2.57

3.11

3.6

1.70

4.48 ± 0.43

Triclosan Risperidone Diphenhydramine Fluoxetine Sertraline Carbamazepine Bisphenol A

Antiseptic Antipsychotic Antihistamine Antidepressant Antidepressant Antiepileptic Plastic Monomer

acidic basic basic basic basic neutral neutral

13.7 92.2 98.3 99.9 99.7 1 × 10-7 5× 10-4

7.80 8.07 8.76 10.1 9.47 13.9 10.3

4.76 3.49 3.27 4.05 5.29 2.45 3.32

4.70 2.39 1.52 1.25 2.91 2.45 3.32

5.47 2.78 2.74 3.71 4.33 3.80 4.41

7.09 NA g 0.97 2.37 2.42 2.32 2.77

4.29 NA g 1.27 2.18 2.56 1.58 2.05

0.04 ± 0.01 0.03 ± 0.005 0.89 ± 0.08 0.88 ± 0.05 0.20 ± 0.02 1.36 ± 0.07 1.94 ± 1.39

Simvastatin

Antihyperlipidemic

neutral

3× 10-7

13.5

4.68

4.68

4.85

3.27

3.02

< MDL

Chemical

Class

Acidic/Basic

pKa a

4.84 4.41 4.18

Log Kow,N b

Log Log Log Log Dow Dmw DBSAw Dmpw (pH=7) (pH=7) (pH=7.4) (pH=7)

Low Dose

High Dose

9.48 ± 0.46 5.04 ± 0.71 8.00 ± 0.96 12.50 ± 1.40 0.23 ± 0.07 0.20 ± 0.04 4.35 ± 0.38 3.60 ± 0.34 0.76 ± 0.11 5.35 ± 0.46 5.72 ± 2.80 0.01 ± 0.008 ND

c

d

e

f

3.18 3.97 4.51

1.06 1.50 1.87

1.46 1.81 2.66

3.45 3.37 4.12

1.35 1.35 1.99

3.89 ± 0.54 1.03 ± 0.11 1.54 ± 0.51

Antidepressant basic 99.1 9.05 4.18 2.16 4.05 1.64 1.44 ND Norfluoxetine h a Estimated values, obtained from SCIfinder. b Obtained from EPI suite (Experimental database or KOWWIN v1.67). c Dow (pH 7, L⋅kg-1) values were estimated following the approach outlined by Armitage et al..32 Specifically, the octanol-water partition coefficient for ionic species was determined as Log Kow, I = Log Kow,N - ∆ow. Subsequently, Dow was determined as, Dow (pH 7) = fN × Kow, N + fI × Kow, I where fN is the fraction of chemical in neutral form and fI is the fraction of chemical in charged form at pH 7, 28 ACS Paragon Plus Environment

Page 29 of 34


as predicted by the Henderson-Hasselbalch equation. d Dmw (pH 7, L⋅kg-1) values were estimated following the approach outlined by Armitage et al..32 Specifically, the liposome-water partition coefficient for neutral species was determined as Log Kmw, N = 1.01 × Log Kow + 0.12, the liposome-water partition coefficient for ionic species was determined as Log Kmw, I = Log Kmw,N - ∆mw. Subsequently, Dmw was determined as, Dmw (pH 7) = fN × Kmw, N + fI × Kmw, I where fN is the fraction of chemical in neutral form and fI is the fraction of chemical in charged form at pH 7. e Bovine serum albumin-water distribution coefficient, DBSAw (pH 7.4, L⋅kg-1) values were estimated following the approach outlined by Henneberger et al. using polyparameter linear free energy relationship (PP-LFER) models.68 The detailed method was described in the SI and substance descriptors listed in Table S5 of the SI. f Muscle protein-water distribution coefficient, Dmpw (pH 7, L⋅kg-1 dry weight) values were estimated using PP-LFER models as described by Henneberger et al..48 The detailed method was described in the SI and substance descriptors listed in Table S5 of the SI. g NA: not available h Norfluoxetine was not spiked in the exposure system which is the active metabolite of fluoxetine.



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Table 2. Observed steady-state bioconcentration factor (BCFss ± standard deviation) of individual PPCPs in zebrafish plasma, liver, muscle, ovary and ∑Tissues. BCFss Plasma BCFss Liver BCFss Muscle BCFss Ovary BCFss ∑Tissues -1 -1 -1 (dimensionless) (L⋅⋅kg-1) b (L⋅⋅kg ) (L⋅⋅kg ) (L⋅⋅kg ) Low High Low High Low High Low High Low High Exposure Exposure Exposure Exposure Exposure Exposure Exposure Exposure Exposure Exposure Group Group Group Group Group Group Group Group Group Group (L) (H) (L) (H) (L) (H) (L) (H) (L) (H) Naproxen 4.36±0.68 8.95±0.72 3.82±0.98 6.12±4.26 0.73±0.11 1.12±0.13 0.42±0.09 1.06±0.05 0.80±4.11 1.41±8.11 Ibuprofen 12.6±4.65 11.2±1.83 2.69±0.35 2.26±0.37 0.53±0.08 0.65±0.11 0.90±0.32 1.43±0.48 0.88±9.11 1.11±4.17 Diclofenac 2.82±0.94 2.52±0.39 1.11±0.41 0.51±0.07 0.18±0.09 0.09±0.03 NA 0.17±0.11 0.18±7.46 0.16±2.23 Gemfibrozil 7.30±1.48 14.6±1.75 22.5±2.17 19.1±3.99 1.43±0.38 2.62±0.47 0.20±0.05 1.35±0.45 2.05±3.22 3.07±9.37 Risperidone 3.74±1.49 3.99±1.03 15.9±5.59 14.0 ±3.86 6.50±2.10 5.01±0.90 53.1±21.62 28.2±6.78 23.4±5.21 13.6±2.58 Diphenhydramine 0.59±0.16 1.06±0.20 3.38±1.71 4.67±1.05 1.12±0.27 1.12±0.10 13.8±5.61 12.4±1.09 5.69±1.96 5.25±9.46 Fluoxetine 4.83±0.29 4.26±0.41 41.6±2.52 43.0±4.11 2.25±0.40 2.10±0.23 48.4±2.93 47.2±4.51 20.4±1.24 20.0±1.91 Sertraline 3.95±0.40 3.67±0.85 64.7±10.7 79.4±11.8 5.32±1.10 8.72±1.81 126 ±12.6 171±25.3 50.9±5.15 69.4±17.26 Carbamazepine 0.83±0.26 1.45±0.14 1.24±0.63 1.85±0.71 1.21±0.21 0.43±0.10 2.55±0.65 3.00±0.30 1.68±5.14 1.41±7.13 Bisphenol A ND 5.32±2.57 183±134 183±91.1 1.57±1.13 2.43±1.24 12.1±8.65 21.9±10.5 13.8±9.91 17.9±8.62 Simvastatin ND ND 57.1±47.3a 151±136 329±116 143±124 897±164a 641±544 514±38.0a 318±256 Triclosan 51.3±12.4 46.6±14.1 6460±1560 4730±1500 136±36.7 121±41.3 2261±127 104±37.6 465±113 332±191 a -1 Calculated by concentration at steady-state divided by MDL (0.010 ng⋅mL ) as the chemical concentration in water is lower than MDL. b ∑Tissues is an estimate of whole-body BCF, based on the observed concentrations in plasma and analyzed tissues, along with the corresponding total weight of the compartment/tissue in these adult female zebrafish. Specifically, BCFss ∑Tissues = (Cplasma*0.0045 + Cliver*0.02 + Cmuscle*0.25 + Covary*0.15) / (0.0045 + 0.02 + 0.25 + 0.15) / Cw. The weight of the different compartment/tissue were measured in the same fish from the present study, with average values determined as 0.0045 g, 0.02g, 0.25g and 0.15 g for plasma, liver, muscle and ovary, respectively.


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Table 3. Observed uptake and elimination rate constants (ku and kd) and steady-state bioconcentration factor (BCFss) of fluoxetine, as well as those of pseudo-BCFss of norfluoxetine and total fluoxetine (fluoxetine + norfluoxetine) in zebrafish liver (L⋅kg-1). ku (L⋅⋅kg-1⋅d-1)

kd (d-1)

Half-life (t1/2, d)

BCFss (L⋅⋅kg-1)

Fluoxetine

79.1 ± 30.6

0.90 ± 0.26

0.77 ± 0.22

41.6 ± 2.52

Norfluoxetine

36.4 ± 4.5

0.17 ± 0.09

4.09 ± 2.22

112 ± 43.4

Total Fluoxetine (Fluoxetine + Norfluoxetine)

53.6 ± 6.5

0.21 ± 0.09

3.28 ± 1.32

153 ± 61.2

-

-

-

73%

Fluoxetine

83.3 ± 28.2

1.29 ± 0.18

0.54 ± 0.07

43.0 ± 4.1

Norfluoxetine

35.1 ± 5.2

0.20 ± 0.05

3.52 ± 0.83

99.6 ± 13.2

Total Fluoxetine (Fluoxetine + Norfluoxetine)

57.9 ± 7.1

0.28 ± 0.04

2.50 ± 0.35

137 ± 13.1

-

-

-

72%

Low Exposure Group

% Reduction in BCFss due to biotransformation High Exposure Group

% Reduction in BCFss due to biotransformation



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Table 4. Estimated biotransformation rate constant (km, d-1) and biotransformation half-life (d) of test chemicals, along with the observed total depuration rate constant (kd, d-1) and depuration half-life (d) for the studied compounds and the estimated contribution of metabolism (%). The estimated km and whole-body biotransformation half-life values were derived from EPI Suite v4.11, calculated for a 1 g fish at 25 °C. Compound

Observed Depuration Rate Constant for ∑Tissues (kd, d-1) a

Observed Total Depuration Half-life for ∑Tissues (d) a

Naproxen

2.54 ± 0.24

0.27 ± 0.03

Estimated Estimated Estimated Estimated % Whole-body Whole-body Biotranformation Contribution of Biotransformation Biotransformation rate constant/ Rate Constant Half-life (d) Observed Metabolism (km, d-1) Depuration Rate (km/kd × 100) Constant (km/kd) 0.783

0.885

0.31

31 %

Ibuprofen 2.98 ± 0.30 0.23 ± 0.02 0.726 0.954 b b Diclofenac NA NA 0.220 3.15 Gemfibrozil 2.59 ± 0.54 0.27 ± 0.06 0.524 1.32 Risperidone 1.33 ± 0.19 0.52 ± 0.08 12.7 0.054 Diphenhydramine 1.03 ± 0.10 0.68 ± 0.07 28.2 0.025 Fluoxetine 0.37 ± 0.03 1.85 ± 0.17 0.298 2.33 Sertraline 0.43 ± 0.07 1.62 ± 0.27 0.030 23.3 Carbamazepine 1.43 ± 0.55 0.48 ± 0.19 9.09 0.076 Bisphenol A 0.92 ± 0.08 0.75 ± 0.06 5.18 0.133 b b Simvastatin NA NA 3.70 0.187 Triclosan 0.68 ± 0.05 1.02 ± 0.07 0.414 1.67 a Observed kd and t1/2 data are for high exposure group. b NA= not available. Depuration rate constant value determinations for these compounds.

0.25 NA b 0.20 9.60 c 27.3 c 0.81 0.07 6.35 c 5.63 c NA b 0.61

25% NA b 20 % 100 % 100 % 81 % 7% 100 % 100 % NA b 61 %


Page 33 of 34


Figure Captions Figure 1. Relationship between steady-state bioconcentration factors (log BCFss) of individual test chemicals versus physicalchemical properties (log Dmw, log Dmpw and log DBSAw) for plasma (A, E), liver (B, F),) muscle (C, G) and ovary (D, H). Plotted data include observations in fish from both the low and high exposure groups.




Page 34 of 34

Bioaccumulation behavior of pharmaceuticals and personal care

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