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Uptake and Disposition of Select Pharmaceuticals by Bluegill Exposed at Constant Concentrations in a Flow-Through Aquatic Exposure System Jian-Liang Zhao, Edward T. Furlong, Heiko L. Schoenfuss, Dana Ward Kolpin, Kyle L. Bird, David J. Feifarek, Eric A Schwab, and Guang-Guo Ying Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00604 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

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Environmental Science & Technology

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Uptake and Disposition of Select Pharmaceuticals by Bluegill Exposed at Constant

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Concentrations in a Flow-Through Aquatic Exposure System

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Jian-Liang Zhao,†,‡ Edward T. Furlong,*,§ Heiko L. Schoenfuss,|| Dana W. Kolpin,

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David J. Feifarek,|| Eric A. Schwab,§ Guang-Guo Ying†,‡

⊥

Kyle L. Bird,||

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†

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Chemistry, South China Normal University, Guangzhou 510006, China

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‡

The Environmental Research Institute, MOE Key Laboratory of Environmental Theoretical

State Key Laboratory of Organic Geochemistry, Guangzhou institute of geochemistry, Chinese

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Academy of Sciences, Guangzhou 510640, P R China

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§

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||

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⊥

National Water Quality Laboratory, U.S. Geological Survey, Denver, CO 80225, United States

Aquatic Toxicology Laboratory, St. Cloud State University, St. Cloud, MN 56301, United States U.S. Geological Survey, Iowa City, IA 52240, United States

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Preliminary citation: Zhao, J.-L., Furlong, E.T., Schoenfuss, H.L., Kolpin, D.W., Bird, K.L., Feifarek,

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D.J., Schwab, E.A., and Ying, G.-G., 2016, Uptake and Disposition of Select Pharmaceuticals by

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Bluegill Exposed at Constant Concentrations in a Flow-Through Aquatic Exposure System, planned

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for submission to Environmental Science and Technology.

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DISCLAIMER: This draft manuscript is distributed solely for purposes of scientific peer review. Its

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content is deliberative and predecisional, so it must not be disclosed or released by reviewers. 1

ACS Paragon Plus Environment


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Because the manuscript has not yet been approved for publication by the U.S. Geological Survey

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(USGS), it does not represent any official USGS finding or policy.

2


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ABSTRACT: The increasing use of pharmaceuticals has led to their subsequent input into and

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release from wastewater treatment plants, with corresponding discharge into surface waters that may

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subsequently exert adverse effects upon aquatic organisms. Although the distribution of

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pharmaceuticals in surface water has been extensively studied, the details of uptake, internal

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distribution, and kinetic processing of pharmaceuticals in exposed fish have received less attention.

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For this research, we investigated the uptake, disposition, and toxicokinetics of five pharmaceuticals

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(diclofenac, methocarbamol, rosuvastatin, sulfamethoxazole, and temazepam) in bluegill sunfish

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(Lepomis macrochirus) exposed to environmentally relevant concentrations (1,000–4,000 ng L−1) in

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a flow-through exposure system. Temazepam and methocarbamol were consistently detected in

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bluegill biological samples with the highest concentrations in bile of 4, 940 and 180 ng g−1,

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respectively, while sulfamethoxazole, diclofenac, and rosuvastatin were only infrequently detected.

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Over 30-day exposures, the relative magnitude of mean concentrations of temazepam and

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methocarbamol in biological samples generally followed the order: bile ≫ gut > liver and brain >

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muscle, plasma, and gill. Ranges of bioconcentration factors (BCFs) in different biological samples

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were 0.71–3,959 and 0.13–48.6 for temazepam and methocarbamol, respectively. Log BCFs were

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statistically positively correlated to pH adjusted log Kow (that is, log Dow), with the strongest relations

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for liver and brain (r2 = 0.92 and 0.99, respectively), implying that bioconcentration patterns of

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ionizable pharmaceuticals depend on molecular status, that is, whether a pharmaceutical is unionized

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or ionized at ambient tissue pH. Methocarbamol and temazepam underwent rapid uptake and

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elimination in bluegill biological compartments with uptake rate constants (Ku) and elimination rate

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constants (Ke) at 0.0066–0.0330 h−1 and 0.0075–0.0384 h−1, respectively, and half-lives at 18.1–92.4 3



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h. Exposure to mixtures of diclofenac, methocarbamol, sulfamethoxazole, and temazepam had little

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or no influence on the uptake and elimination rates, suggesting independent multiple uptake and

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disposition behaviors of pharmaceuticals by fish would occur when exposed to effluent-influenced

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surface waters.

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KEYWORDS: Pharmaceuticals; Bluegill sunfish; Uptake; Toxicokinetics; Chemical mixtures

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INTRODUCTION

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Pharmaceuticals are one class of contaminants of emerging concern (CECs) that have received

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specific widespread attention from scientists and the public over the past twenty years.1−3

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Pharmaceuticals are extensively used worldwide for disease treatment for both human and veterinary

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purposes.4 After use, the parent pharmaceuticals and their metabolites are primarily excreted in urine

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or feces, which can enter into the sewer lines and be treated in municipal sewage treatment systems

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or other wastewater treatment facilities (WWTPs).5 However, most pharmaceuticals are not

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completely removed in conventional municipal sewage treatment systems6 or in animal wastewater

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treatment facilities,7 and typically enter into surface water with the release of treated effluents.6,7

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Hence, pharmaceuticals and other CECs have been detected ubiquitously in aquatic environments

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worldwide.2 The occurrence of pharmaceuticals in WWTPs, surface water and sediments has been

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extensively reviewed.8−10 Subsequently, organisms inhabiting those environments were assessed for

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the presence of pharmaceuticals.11−22 In the United States, a pilot study by the U.S. Environmental

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Protection Agency documented that select pharmaceutical compounds can be found in fish muscle

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and livers.16 Among the range of antidepressants present in wastewater-influenced surface water,

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several, such as fluoxetine and sertraline, were found in wild fish bile, brain, liver, and muscle

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samples.17,22 The antihistamine diphenhydramine was found in muscle from German fish bank

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samples.18 Nonsteroidal anti-inflammatory drugs (NSAIDs), psychotropic agents and hypertensive

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agent were frequently detected in brain, liver, kidney, muscle, and gills of wild fish from

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wastewater-impacted streams in Japan.11 Antibiotics were also detected in bile, plasma, liver, muscle

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samples of wild fish from urbanized areas in South China.19 5



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The ecotoxicological effects of pharmaceuticals have been previously reviewed,23,24 noting that

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pharmaceuticals in single or mixtures could pose adverse effects to organisms in the environment. So

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far, most of the adverse effects of pharmaceuticals were expressed as the external concentrations of

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pharmaceuticals, e.g. pharmaceuticals in the water phase.25 However, the response of an organism to

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a toxic substance is determined by the quantity of the substance that reaches an organ or tissue.14,25–27

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Recent research has demonstrated the uptake, internal distribution, and metabolism of

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pharmaceuticals in fish from wastewater impacted water or controlled laboratory

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exposure.11−13,19,28,29 A fish plasma model was used to explain the uptake and effects of active

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pharmaceuticals in fish compartments.11,30,31 Most of pharmaceuticals studied were ionizable in the

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aqueous phase; previous studies suggested that ionizable pharmaceuticals usually display different

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bioaccumulation patterns compared with nonpolar organic compounds.19,32 Hence, a more detailed

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study on the tissue-specific uptake and disposition of various pharmaceuticals is warranted.

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The purpose of the research described herein was to develop a more detailed understanding of

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how ionizable pharmaceuticals of varying physicochemical properties are taken up by a model fish

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species and distributed to different organs and compartments under conditions of constant exposure

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at concentrations within the range previously observed in aquatic environments. Representative

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pharmaceuticals were chosen based on existing literature on water and fish concentrations, patterns

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of pharmaceutical use worldwide, prior analytical method reports for compounds, and diversity of

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physicochemical properties. To constrain the experiment to a manageable framework, five target

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pharmaceuticals were selected: diclofenac (DCF), methocarbamol (MET), rosuvastatin (RSV),

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sulfamethoxazole (SMX), and temazepam (TMZ). In addition, a binary mix (TMZ and MET) and a 6


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four pharmaceutical mixture (DCF, MET, TMZ, and SMX) at their single exposure concentrations

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were included to evaluate possible interactions among the compounds during uptake and tissue

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disposition. DCF is a nonsteroidal anti-inflammatory drug (NSAID) and is widely consumed

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globally.33 MET is a muscle relaxant used to treat skeletal muscle spasms. RSV is a drug used to

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lower cholesterol levels. SMX is a sulfonamide antibiotic commonly used for human and veterinary

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purposes,34,35 and has been shown to induce mortality and alter the composition of exposed

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groundwater microbial populations.36 TMZ is a psychoactive drug frequently used for the short-term

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treatment of insomnia.37 SMX and TMZ were among the top 200 drugs prescribed in U.S. as listed in

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RxList (2015),33 while MET was listed as among the top 20 produced drugs in U.S.38

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Previously reported concentrations of these five pharmaceuticals in effluents usually ranged

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from tens of nanograms per liter (ng L−1) to thousands of ng L−1.39 DCF was frequently detected in

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effluents around the world,40 and was present at concentrations of up to 5450 ng L−1 in effluent from

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Italy.41 RSV also was found in effluent from Canada at 552 ng L−1 and from Europe at 979 ng

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L−1.42,43 SMX from different effluents has been widely reported, with maximum concentrations in

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effluents from Europe up to 1,690 ng L−1.43 TMZ concentrations in effluents from Netherlands were

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up to 1,020 ng L−1.44 Recently published research for MET in effluents shows concentrations ranging

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between 638 to 1560 ng L−1.45 The five pharmaceuticals chosen also exhibit toxicity to organisms,

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such as daphnia,46 mussel47 and rats.48 These five compounds have also been implicated in a range of

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other adverse environmental effects, such as promoting antibiotic resistance transmission.36

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Toxicokinetics were used to evaluate the time course of the uptake of elimination of toxicants in organisms.49 It is important to understand the fate of pharmaceuticals in the environment, and 7



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potential effects to aquatic organisms, in order to improve pharmaceutical risk assessments. Recent

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studies on the uptake and elimination kinetics of pharmaceuticals in aquatic organisms indicate that

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pharmaceuticals are rapidly taken up and subsequently eliminated from specific compartments or

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whole organisms.27,31,50,51 One- or two-compartment first-order kinetics models were used to explain

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the uptake and elimination processes, suggesting common pharmaceutical metabolism mechanisms

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were occurring for these pharmaceuticals. However, reports on the uptake and elimination kinetics in

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adult fish exposed to diverse classes of pharmaceuticals and pharmaceutical mixtures are much more

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limited, and this information is essential for a more realistic understanding of the effects of

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pharmaceuticals on fish and other aquatic organisms.

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Hence, in the research presented herein, we conducted the following studies: (1) tissue-specific

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uptake and bioconcentration of five target pharmaceuticals with different physicochemical properties

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in bluegill sunfish (Lepomis macrochirus) exposed to a constant flow-through system; (2) uptake and

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elimination toxicokinetics modeling of pharmaceuticals exposed to environmentally relevant

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concentrations with single and binary exposures of TMZ and MET, followed by exposure to a

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mixture of four compounds (DCF, MET, TMZ, and SMX). These results provide quantitative

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information that allow for new insights into the within-organism absorption, disposition, and

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excretion of these ubiquitous aquatic contaminants, and supports formulation of ecological risk and

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human exposure assessments for pharmaceuticals in fish.

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MATERIALS AND METHODS Chemicals. High purity standards of TMZ, SMX, and DCF were purchased from 8


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Sigma-Aldrich. MET was purchased from Grace Alltech Associates. RSV was obtained from

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Alsachim. Isotopically-labeled compounds were used as internal standards. Temazepam-D5 was

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obtained from Cambridge Isotope Laboratories. Sulfamethoxazole-13C6 was obtained from

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Sigma-Aldrich. Methocarbamol-D3 and diclofenac-D4 were purchased from CDN Isotopes.

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Rosuvastatin-13C-D4 was purchased from Alsachim. The physicochemical properties of the five

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target compounds are summarized in Table S1 of Supporting Information.

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For fish exposure, stock solutions were prepared in molecular biology grade absolute ethanol

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(Fisher Scientific) with ethanol alone as a solvent blank control. For instrumental analysis, single

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compound stock solutions were prepared in methanol at a concentration of 1000 mg L−1 and stored at

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−20ºC. Stock mixtures of 10 mg L−1 were prepared in methanol and stored at the same conditions.

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Working standard solutions of individual and mixed pharmaceuticals with corresponding internal

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standards (ISs) (1 mg L−1), as well as standard solutions for calibration curves were prepared in

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methanol/water (3:7, v/v) before each analytical run.

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Fish Exposure. In this study, bluegill sunfish was selected as the model species because it is a

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common species in freshwater environments throughout North America and has been used for

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exposure both in laboratory and in field studies.52 Experiments were conducted in a flow-through

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system previously used for other studies.53 Juvenile bluegill (15–20 g) were purchased from a

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commercial hatchery (10,000 Lakes Aquaculture, Osakis, MN, USA) and allowed to acclimatize to

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laboratory conditions at least one week prior to use. Fish were fed with commercial pellets at a rate

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of 1–2% bodyweight per day. Three exposure experiments of increasing complexity were conducted

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from February through October of 2014. The first experiment was a bioconcentration exposure at 9



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lower water-phase concentrations with the aim of establishing appropriate exposure concentrations.

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The second exposure was a bioconcentration experiment with higher water-phase concentrations, and

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the third exposure experiment used the same concentrations as the second experiment but included

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uptake and elimination phases to identify toxicokinetic properties. Exposure concentrations were

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initially based upon reported environmental and effluent concentrations for the five

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pharmaceuticals.39–45

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First (Lower Concentration) Exposure. Fish were exposed to single pharmaceuticals at single

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target constant exposure concentration (1,000 ng L−1 for each analyte) in replicate flow-through

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aquaria. Each pharmaceutical solution was prepared in a stock tank, and pumped into a mixing tank

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and mixed with oxygenated water to produce a nominal concentration of 1,000 ng L−1. To confirm

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the aqueous stability of analytes, water samples were periodically collected from each tank during

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the exposure period, shipped to the USGS National Water Quality Laboratory, and analyzed by direct

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aqueous injection HPLC-MS/MS. The concentration of dissolved oxygen, pH, and temperature were

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also monitored daily in all tanks (Data see Table S2). Fish were fed every other day for the duration

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of the uptake exposure. On days 0, 10, 20, and 30, fish were euthanized with tricaine

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methanesulfonate (MS-222, Western Chemical) according to euthanasia guidelines.54 Immediately

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after the fish had been euthanized, the muscle, liver, brain, gill, gastrointestinal tract (GI tract), bile,

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and whole blood were excised and weighed. Blood was stored in a vial pre-rinsed with heparin to

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avoid blood coagulation and centrifuged at 10000 g; the supernatant (plasma) was transferred to a

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plastic vial. For this study, subsamples were shipped on dry ice from St. Cloud State University to

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the U.S. Geological Survey National Water Quality Laboratory, where they were stored at −70 °C 10


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prior to pharmaceuticals analysis. The cold chain was maintained throughout all steps. Second Exposure (high water concentrations exposure). As in the first exposure experiment,

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fish were exposed to pharmaceuticals at single constant water phase concentrations. Concentrations

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were adjusted to improve the detection of the five compounds: the nominal concentrations of SMX,

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TMZ and DCF were doubled to 2,000 ng L−1; the nominal concentrations of MET and RSV were

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quadrupled to 4,000 ng L−1. The duration of exposure and the harvest of tissues were the same as the

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first exposure.

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Third Exposure (Toxicokinetics study). In the third exposure experiment, concentrations

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remained the same as the second exposure, but RSV was dropped because of rare detection in

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biological samples. Instead, two additional exposures were conducted: one with a binary mix of

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TMZ and MET, and one with a mixture of four compounds (DCF, MET, TMZ, and SMX). The

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experimental design for all exposures also included an exposure/elimination cycle – 14 days uptake

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and 14 days elimination. Sampling frequency was increased to better describe uptake/elimination

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rates (days 0, 1, 2, 3, 5, 8, 13, 14 for uptake, and days 14, 15, 16, 17, 20, 22, 28 for elimination) and a

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third replicate exposure aquarium was added to each treatment to maintain comparable fish densities

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to experiments one and two. The harvest of tissues was the same as the first two exposures.

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Sample Preparation and Instrumental Conditions. A QuEChERS (Quick, Easy, Cheap,

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Effective, Rugged, and Safe)-based method55 was developed to analyze five pharmaceuticals in fish

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brain, gill, gut, liver and muscles biological samples. Samples were first conducted with enzyme

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hydrolysis in order to release the potential glucuronide and sulfate metabolites to free form.56 Then,

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an Agilent extraction kit was used for salt extraction and the Supelco Z-Sep/C18 kit was selected as 11



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dispersive solid phase extraction (d-SPE) for purification. Pharmaceuticals in bile s and in plasma

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derived from whole blood after centrifugation, were isolated using a solid-phase extraction (SPE)

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step (Phree SPE, tube format, obtained from Phenomenex Inc.) that removed phospholipids from the

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sample liquid. For bile, an additional Z-Sep/C18 kit was used as a d-SPE step in order to remove

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additional green-color interferences. Final extracts were analyzed using an Agilent 1200

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High-performance liquid chromatography (HPLC) system coupled with 6410 Triple Quadrupole

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mass spectrometer (MS/MS) with an ESI source. Concentrations of the five pharmaceutical

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compounds in samples were quantified using the internal standard method. Addition details for

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extraction and instrumental analysis can be found in text of Supporting Information and Tables S3–

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S5.

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Bioconcentration Factor Calculation and Toxicokinetic Modeling. The bioconcentration

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factors (BCFs) for each pharmaceutical in bile, brain, gill, gut, liver, muscle, and plasma were

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calculated based on the average concentration in each fish biological compartment and the

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corresponding actual concentration in the water phase.57 The BCFs in fish biological compartments

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were calculated by the equation BCF = 1000×C/Cw, where BCF is the bioconcentration factor with

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units of L kg−1, and Cw is chemical concentration in the water phase with units of ng L−1, and C is the

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chemical concentration in fish biological compartment with units of ng g−1 wet weight (w/w).

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Uptake and elimination toxicokinetic analysis was performed on the basis of average chemical

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concentrations in each sample at each sample collection time point using the classical

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pseudo-first-order toxicokinetics model.58 The elimination rate constant (Ke) was obtained by fitting

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a nonlinear regression to the equation Ct = C0 e-Ke t , where C0 is the concentration of chemical in the 12


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biological sample at the beginning of the experiment, Ct is the concentration at time t, and t is time

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(h). The uptake rate constant (Ku) was calculated by fitting a nonlinear regression to the equation

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Ct = (Ku /Ke )C0 (1-e

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biological samples were estimated according to the equation t1/2 = −ln 0.5/Ke. The Systat Software

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SigmaPlot program (version 11.0) was used for model fitting.

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-Ke t

), using the values of Ke obtained above. The half-lives (t1/2) of chemicals in

Statistical Analysis. Differences of pharmaceutical concentrations between experiment batches,

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sample types, and enzyme hydrolysis experiments were demonstrated using statistical analysis. The

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IBM SPSS statistics program (version 16.0) was used for all statistical analysis, and a p value of
liver > brain >

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plasma > muscle ≈ gill. Fish liver is an important metabolism compartments which metabolizes

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pharmaceuticals and excretes them to bile. TMZ concentration in bile were approximately 100 times

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higher than those in other biological samples. High concentration of TMZ in bile suggests that the

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hepatic metabolism pathway and biliary excretion are important in bluegill. High internal

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concentrations in fish biological compartments probably pose adverse effects to fish.14,60 When the

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concentrations of fish exposed to low and high water phase concentrations of TMZ were compared,

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no statistically significant increase in muscle (p = 0.142), liver (p = 0.534), brain (p = 0.968), or bile

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(p = 0.432) was observed. This suggests that TMZ uptake reached a plateau concentration in tissues

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when exposed to 1,000 ng L−1 water phase concentrations according to the principles of drug

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action.66 In the human body, TMZ is eliminated mainly through urine, accounting for 80% of the

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dose, of which 88% was the direct glucuronide. In rats, urinary excretion accounted for only 15% of

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dose; the majority of dose was eliminated through biliary excretion (85%).67 The high detection of

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TMZ in bile in the current study implies that the biliary excretion mechanism is probably important 16


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in bluegill sunfish. MET was intermittently detected, or detected at a concentration near the LOQ when exposed at

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1,000 ng L−1 water phase concentrations (Table 1). The detected concentrations were around LOQ

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values for muscle, liver, brain, and bile samples, suggesting that limited uptake of parent MET at low

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water concentrations. However, detection frequency increased to 100% in all samples and all sample

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types when the water phase concentration was quadrupled to 4,000 ng L−1. Mean MET

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concentrations in biological samples decreased in the order: bile ≫ gut > liver ≈ brain > muscle ≈

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plasma ≈ gill. This progression, similar to TMZ, suggests similar modes of action for the two drugs.

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Bile showed high potential to accumulate MET compared to other biological compartments; the

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maximum concentration was 180 ng g−1 ww, while the maximum concentrations in other biological

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compartments did not exceed 4.0 ng g−1 ww.

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The other three pharmaceuticals, DCF, RSV, and SMX, were generally not detected or detected

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infrequently at low concentrations in fish tissues, with measured concentrations below or near their

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respective LOQs whether exposed to either low or high water phase concentrations (Table 1). In the

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second exposure, SMX was present in 3 out of 3 samples, in gut with a mean concentration of 4.43 ±

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2.88 ng g−1 ww, but it was not detected in plasma, suggesting that uptake from the intestine was

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limited and would perhaps be eventually excreted through feces. These results from our exposures

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for SMX were similar to a previous study, where no metabolism of SMX was found in rainbow trout

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fish liver S9 fractions;68 but different from the finding in human plasma, where methyl, hydroxyl,

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and glucuronide metabolites of the SMX were detected.63 DCF was consistently detected in muscle

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from the first exposure and infrequently detected in the second exposure, and almost never detected 17



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in liver, brain, bile, plasma, or gut samples (Table 1), but was found in gill (5.97 ± 0.79 ng g−1 ww)

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during the second exposure. Gill is an important uptake tissue that can transfer pharmaceuticals to

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blood. No parent DCF was found in plasma, suggesting a rapid biotransformation in plasma. The

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concentrations of DCF in bluegill were quite different from other studies, which found that high

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uptake rate of DCF in liver, bile and kidney tissues of rainbow trout (Oncorhynchus mykiss).11,45,65,69

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But our result for DCF are similar to the report by Huerta et al. (2013) in whole fish of Barbus

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graellsii in from Mediterranean rivers.70 Hence, these observed differences may be due to

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interspecies variations in uptake and metabolic capacity for DCF. RSV was also seldom found in

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muscle, liver, brain, bile, and gill samples, but found in all gut and plasma samples with mean

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concentrations at 3.84 and 1.85 ng g−1 w/w, respectively. Potential reasons were probably due to

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rapid biotransformation to other metabolites but not glucuronide forms in bluegill samples for these

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pharmaceuticals.

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Pharmaceutical concentrations in bluegill biological samples during the 14-day uptake phase of

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the third exposure are illustrated in Figure 1 and Table S8. The number of detections and

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concentrations of TEM, MET, SMX, and DCF in single, binary component, or four-component

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exposures were generally similar to those in the second exposure experiment. Overall, the uptake

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varied with different pharmaceuticals, and varied with sample types. Increasing the exposure

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concentrations resulted in more increased detection frequency for MET, but not for SMX, RSV, and

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DCF. Detection frequency was most consistent for TEM and MET in all sample matrixes, whereas

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for DCF, SMX, and RSV, the frequency of detection for any individual pharmaceutical was

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dependent upon sample type and exposure concentration. 18


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Bioconcentration Patterns. The ranges of BCFs in different biological samples are provided in

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Table 1. Bluegill bile clearly showed the greatest bioconcentration for both of TMZ and MET. The

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maximum BCFs for TMZ and MET in bile were up to 3,960 L kg−1 and 48.6 L kg−1, respectively. In

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comparison, BCFs in other biological samples were generally below 100 L kg−1 for TMZ, and below

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1 L kg−1 for MET. The bile results suggest removal by a biliary excretion mechanism for TMZ in

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bluegill was likely similar to that in rats, but different from that in humans.71 BCFs of SMX, DCF

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and RSV in bluegill biological samples were generally at the range from zero to several L kg−1 (Table

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1), suggesting the poor bioconcentration of the parent compounds for these three pharmaceuticals in

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bluegill biological samples. The BCFs of DCF in bluegill were similar to those in a study by

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Togunde et al. (2012),15 in which DCF was not detected in muscle and < LOQ in bile of rainbow

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trout. Our DCF results were also lower than other reported values, where BCFs ranging between 320

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and 950 L kg−1 in bile of rainbow trout were observed.65 The BCFs of SMX in muscle and liver of

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common carp (Cyprinus carpio) were in the range of 1.65–119.32 L kg−1,51 approximately 25 times

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higher than the BCFs in bluegill observed in this study. Similar to our results, SMX was infrequently

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detected in wild fish.16,18,19,72 Taken together, our study and published results suggest that the

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bioconcentration patterns of pharmaceuticals vary greatly with fish species and parent

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pharmaceutical.

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BCFs for nonpolar lipophilic organic compounds are thought to be correlated with compound

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hydrophobicity, represented by log Kow values from empirical models.73 However, pharmaceuticals

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are usually ionizable compounds; thus their bioconcentration by fish tissues may also reflect pH

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dependence. Bioconcentration can correlate poorly with log Kow values for ionizable 19



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pharmaceuticals.11,31,74,75 Molecular dissociation leads to reduced bioconcentration because ionic

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organic species cross biomembranes at a slower rate than their corresponding neutral molecules.

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Bioaccumulation of weak acids or bases under environmental pH conditions is influenced by the

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fraction of neutral molecule (fn).76 Hence, the pH-adjusted octanol–water partition coefficient (log

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Dow) is more suitable to evaluate bioconcentration for ionizable compounds by correcting the log Kow

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for fn.29,30,77 Calculation of fn and log Dow for the five pharmaceuticals is described in Supporting

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Information and the results are listed in Table S9. Correlation of log BCFs in different biological

373

samples plotted against log Dow and log Kow for the five pharmaceuticals are shown in Figure 2 and

374

Figure S3, respectively. Mean values of BCFs calculated from first and second exposures were used.

375

No significant positive correlations were found between log BCFs and log Kow (p > 0.05 for each

376

single biological sample (Figure S3A–S3G) and all biological samples (Figure S3H). In contrast, log

377

BCFs were positively correlated with log Dow values for several types of bluegill biological samples.

378

Liver (Figure 2B), brain (Figure 2C), and all biological samples (Figure 2H) had statistically

379

significant correlations between log BCFs and log Dow values (p < 0.01). These positive correlations

380

strongly suggest that the bioconcentration of ionizable pharmaceuticals in bluegill biological samples

381

was a function of the fraction of pharmaceutical ionized and that log Dow better reflects tissue uptake,

382

as noted in several previous studies.11,74

383

Uptake and Elimination Toxicokinetics. In light of the consistent detection of TMZ and MET

384

in bluegill biological samples, exposure to these two compounds was studied in greater detail by

385

determining their uptake and elimination toxicokinetics, exposing fish to single concentrations of

386

each compound, a binary mixture of both compounds, and as a mixture of TMZ, MET, SMX, and 20


Page 21 of 44


387

DCF. The time course for uptake and elimination of TMZ and MET from biological samples of

388

bluegill is shown in Figure 1. Both TMZ and MET followed similar patterns of increasing exposure,

389

reaching a concentration plateau within seven days of exposure and dropping to nearly undetectable

390

concentrations between days 17 and 30 after exposure was stopped at day 14. This same overall

391

pattern was observed regardless of whether fish were exposed to each pharmaceutical singly, as a

392

binary mixture of TMZ and MET, or as part of the full mixture of all four compounds.

393

DCF and SMX were only infrequently detected during uptake and elimination courses in the

394

mixture of four compounds exposure, which was consistent with the findings from the previous two

395

exposures. Hence, only the toxicokinetics of TMZ and MET are discussed.

396

The toxicokinetic processes were simulated by a pseudo-first-order model.58 Elimination rate

397

constants (Ke) and uptake rate constants (Ku) of TMZ and MET are listed in Table 2. Following

398

accumulation and reaching a plateau in seven days, accumulated pharmaceuticals in muscle, liver,

399

brain, and bile were rapidly eliminated during the depuration period, showing relatively large

400

elimination rate constants and short half-lives (Table 2). The elimination rate constants (Ke) for TMZ

401

and MET were 0.0075–0.0384 h−1 and 0.0082–0.0203 h−1, respectively, with corresponding

402

half-lives of 18.1–92.4 h and 34.1–84.5 h, respectively. As noted, uptake of TMZ and MET rapidly

403

reached a plateau, with the uptake rate constants (Ku) for TMZ and MET of 0.0066–0.0330 h−1 and

404

0.0070–0.0257 h−1, respectively. Bile had the lowest uptake and elimination rates compared with

405

muscle, liver, and brain samples. The Ku values in muscle, liver, and brain samples were 2.25–5.00

406

and 1.76–3.67 times higher compared to those in bile for TMZ and MET, respectively. The Ke values

407

in muscle, liver, and brain samples were 1.09–4.00 and 0.88–2.15 times higher to those in bile for 21



Page 22 of 44

408

TMZ and MET, respectively. This may reflect the relative accessibility of each biological

409

compartments in the distribution of pharmaceuticals into the fish wherein exposure of muscle and

410

liver is more direct, particularly the liver as the primary detoxification organ, while the brain is more

411

isolated from direct exposure through the blood-brain barrier, and bile may reflect it’s role in the

412

final elimination of contaminants. The uptake and elimination rates of TMZ in our study were similar

413

to results reported previously for caffeine, diphenhydramine, diltiazem, carbamazepine, and

414

ibuprofen in mosquito fish, with half-lives of 32–141 h;50 but substantially faster than results

415

observed for SMX and sulfadiazine in common carp with half-lives of 8.1–15.1 d.51

416

TMZ and MET displayed different uptake and elimination rates when exposed as

417

single-compounds, a binary mixture of these two compounds, and as a mixture of four compounds

418

(Figure S4A–D). The Ku and Ke values of TMZ decreased somewhat in the order of single exposure >

419

binary mixture > the mixture of four compounds in all types of samples (Figure S4A and B). In

420

comparison, the Ku and Ke values of MET were similar at single exposure, binary mixture, and as a

421

mixture of four compounds in all types of samples (Figure S4C and D). In effluent-influenced rivers,

422

pharmaceuticals are likely present as dynamic equilibrium mixtures and would undergo uptake

423

simultaneously by resident fish. These results, particularly lower Ke values, suggest that

424

pharmaceutical mixtures could have greater potential for adverse effects in wild fish78.79 compared to

425

single compound exposures.

426

Environmental Significance. This study provides a comprehensive assessment of the uptake

427

and elimination of pharmaceuticals in bluegill sunfish that is consistent with the dynamic equilibrium

428

concentration regime anticipated to be the real-world conditions for most fish species living 22


Page 23 of 44


429

downstream from a continuously discharging source (e.g. WWTP). By examination of multiple

430

pharmaceuticals, singly and in mixtures, we were able to assess the relative importance of uptake

431

between different pharmaceutical classes. It is clear from this study that bioconcentration varied

432

between parent pharmaceuticals with TMZ > MET > SMX, DCF, and RSV, and with

433

pharmaceuticals TMZ and MET being the most consistently detected across sample types of blue gill.

434

These two pharmaceuticals also demonstrated relatively rapid uptake and elimination kinetics,

435

suggesting that concentrations in biological samples are driven by external concentrations and that

436

persistence within biological samples is likely to be low in the absence of a substantial external

437

contaminant source or gradient.

438

The results from this study suggest that, in most cases, pharmaceutical uptake is the same either

439

singly or in mixtures. While this fact does not rule out synergistic effects, our data suggest that

440

uptake mechanisms are independent of pharmaceutical class, or that the physicochemical properties,

441

and thus conditions for uptake, are similar for the five compounds measured in this study. It is

442

important to note that only parent pharmaceuticals were determined in this study; efforts to evaluate

443

potential metabolites are underway for the samples collected, and such metabolite determination is

444

critical for better evaluating the overall effects of pharmaceutical exposure. Furthermore, we

445

evaluated five pharmaceuticals that represent five therapeutic actions. It is possible that multiple

446

pharmaceuticals of similar therapeutic action may interact in ways not represented in the current

447

study. This work provides a context for establishing uptake and elimination studies for larger

448

numbers of pharmaceuticals, particularly the complex mixtures that most aquatic organisms are

449

exposed to under typical ambient environmental conditions. Future work should expand the number 23



450

and classes of pharmaceuticals exposed, and in particular assess uptake and elimination for

451

pharmaceuticals encompassing the widest array of physicochemical properties, and include

452

corresponding pharmaceutical degradation products. Recently published works showing that

453

metformin, fexofenadine, venlafaxine, carbamazepine, etc. are frequently detected in rivers of the

454

United States,80,81 suggesting that these compounds might be appropriate for future uptake and

455

elimination assessment. Development of methods that permit concurrent determination of both

456

parent pharmaceuticals and metabolites in an exposure study similar to ours would provide a

457

comprehensive assessment of pharmaceutical fate and behavior.14 However the results from the

458

current study demonstrate that more generalized modeling approaches can be used to predict likely

459

compound uptake and elimination in the absence of laboratory data, provide a means for designing

460

more sophisticated and realistic experimental exposure scenarios, and thus develop more realistic

461

ecosystem and human exposure assessments.

Page 24 of 44

462 463

ASSOCIATED CONTENT

464

Supporting Information

465

Additional information about sample preparation, instrumental analysis, calculation of Log Dow, and

466

additional tables and figures are given in Supporting Information. This material is available free of

467

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

468 469

AUTHOR INFORMATION

470

Corresponding Author 24


Page 25 of 44

471


*Phone/fax: 303-236-3941/303-236-3499. Email address: [email protected] (Edward T. Furlong).

472 473

ACKNOWLEDGMENTS

474

The authors would like to acknowledge support from the USGS National Water Quality Laboratory

475

and the Toxic Substances Hydrology Program’s Contaminants of Emerging Concern in the

476

Environment Project. The first author (JLZ) gratefully acknowledges support from China

477

Scholarship Council and National Natural Science Foundation of China (NSFC No. 41101462) as a

478

visiting researcher at USGS National Water Quality Laboratory. Funding to one of us (HLS) was

479

provided by the National Science Foundation (CBET 1336062). Undergraduate and graduate

480

students in the St. Cloud State University Aquatic Toxicology Laboratory are acknowledged for their

481

help with all aspects of the fish exposure experiments. Use of trade or product names is for

482

identification purposes only and does not imply endorsement by the U.S. Geological Survey or the

483

U.S. Government.

484

25



485 486 487 488

Page 26 of 44

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FIGURE CAPTIONS:

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Figure 1. Kinetics of uptake (0–14 d) and elimination (14–28 d) of temazepam and methocarbamol in

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tissues of bluegill exposed to temazepam and methocarbamol individually, as a binary mixture of

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temazepam and methocarbamol, and as a mixture of all four pharmaceuticals. The nominal water

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phase concentrations were 2,000 ng L−1 for temazepam and 4,000 ng L−1 for methocarbamol. A:

728

muscle; B: liver; C: brain; D: bile. Open circles are experiment data, and lines are fitting of kinetics.

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Figure 2. Correlations between mean values of bioconcentration factors (log BCFs) and the pH

731

adjusted octanol–water partition coefficient (log Dow). A: muscle; B: liver; C: brain; D: bile; E:

732

plasma; F: gut; G: gill; H: all biological samples together.

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38


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Figure 1.

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39



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Figure 2.

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40


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Table 1. Uptake of temazepam, methocarbamol, sulfamethoxazole, diclofenac, and rosuvastatin in different sample types from bluegill exposed to pharmaceuticals using a flow-through system for up to 30 daysa. first exposure second exposure −1 sample concentration (ng g−1 ww) detectio concentration (ng g ww) BCF (L kg−1) detection BCF (L kg−1) n range mean ± SD range mean ± SD temazepam muscle 3/3 0.93–1.97 1.50 ± 0.53 0.94–1.99 9/9 1.08–5.69 2.84 ± 1.39 0.71–3.47 liver 3/3 11.2–32.6 19.7 ± 11.4 10.4–32.9 9/9 9.17–25.5 16.7 ± 5.45 5.21–20.5 brain 3/3 5.85–14.0 8.64 ± 4.65 5.63–14.1 9/9 4.42–15.1 8.74 ± 3.50 2.70–9.30 bile 3/3 2,350–4,170 3,010 ± 1,010 2,370–3,870 9/9 2,350–4,940 3,540 ± 959 1,340–3,960 plasma 3/3 3.85–7.92 5.23 ± 2.33 2.19–4.50 gut 9/9 23.9–139.5 69.2 ± 41.1 15.6–95.4 gill 3/3 2.56–5.81 3.89 ± 1.70 1.45–3.30 methocarbamol muscle 1/2 ND–

Uptake and Disposition of Select Pharmaceuticals ... - ACS Publications

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